SARS-CoV-2 and COVID-19: An Evolving Review of Diagnostics and Therapeutics

This manuscript (permalink) was automatically generated from greenelab/covid19-review@bd8f0ff on January 21, 2021.

Authors

COVID-19 Review Consortium: Vikas Bansal, John P. Barton, Simina M. Boca, Christian Brueffer, James Brian Byrd, Stephen Capone, Shikta Das, Anna Ada Dattoli, John J. Dziak, Jeffrey M. Field, Soumita Ghosh, Anthony Gitter, Rishi Raj Goel, Casey S. Greene, Marouen Ben Guebila, Fengling Hu, Nafisa M. Jadavji, Sergey Knyazev, Likhitha Kolla, Alexandra J. Lee, Ronan Lordan, Tiago Lubiana, Temitayo Lukan, Adam L. MacLean, David Mai, Serghei Mangul, David Manheim, Lucy D'Agostino McGowan, YoSon Park, Dimitri Perrin, Yanjun Qi, Diane N. Rafizadeh, Bharath Ramsundar, Halie M. Rando, Sandipan Ray, Michael P. Robson, Elizabeth Sell, Lamonica Shinholster, Ashwin N. Skelly, Yuchen Sun, Gregory L Szeto, Ryan Velazquez, Jinhui Wang, Nils Wellhausen

Abstract

Since late 2019, Coronavirus disease 2019 (COVID-19) has spread around the world, resulting in the declaration of a pandemic by the World Health Organization (WHO). This infectious disease is caused by the newly identified Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). Research on the SARS-CoV-2 virus and the disease it causes is emerging rapidly through global scientific efforts. Short-term mitigation of viral impacts will require public health interventions, and long-term mitigation will require new diagnostic and therapeutic technologies. The urgency of the pandemic has led to a rapidly emerging scientific literature on SARS-CoV-2 and COVID-19. This manuscript represents a collaborative effort to organize and consolidate this body of literature. We present information about the virus in the context of what is known about related viruses, describe the pathogenesis of COVID-19, and synthesize studies emerging about the diagnosis and treatment of COVID-19 alongside literature about related illnesses. We summarize this emerging literature with an eye towards discussing elements of the disease that will be fundamental to efforts to develop interventions. Our review is a collaboratively-authored, evolving document into which we seek to incorporate the ever-expanding body of information on the topic. This document provides a snapshot as of October, 2020. We continue to accept new contributions and anticipate future snapshots until technologies to mitigate the pandemic are widely deployed.

How to Contribute

We invite potential contributors to introduce themselves through GitHub: https://github.com/greenelab/covid19-review/issues/17

We have established a community chat room on a service called Gitter: https://gitter.im/covid19-review/community

More information about how to contribute is available in a README document on GitHub: https://github.com/greenelab/covid19-review#sars-cov-2-and-covid-19-an-evolving-review-of-diagnostics-and-therapeutics

1 Introduction

On January 21, 2020, the World Health Organization (WHO) released its first report concerning what is now known as the Coronavirus Disease 2019 (COVID-19) [1]. This infectious disease came to international attention on December 31, 2019 following an announcement by national officials in China describing 44 cases of a respiratory infection of unknown cause. The first known cases were located in Wuhan City within the Hubei province of China, but the disease spread rapidly throughout China and subsequently around the world. At the time of the WHO’s first situation report [1], 282 confirmed cases had been identified. Most of these cases were in China, but one to two exported cases had also been identified in each of several neighboring countries (Thailand, Japan, and the Republic of Korea). One week later, 4,593 confirmed cases had been identified, spanning not only Asia, but also Australia, North America, and Europe [2]. On March 11, 2020, the WHO formally classified the situation as a pandemic [3]. On April 4, 2020, the WHO reported that the global number of confirmed cases had surpassed one million [4].

As international attention remains focused on the ongoing public health crisis, the scientific community has responded by mobilizing resources and turning much of its attention to the virus and disease. This rapid influx of information is disseminated by traditional publishing mechanisms, preprint servers, and press releases, which provide a venue for scientists to release findings without undergoing the formal publication process. While having information available is valuable to efforts to understand and combat COVID-19, many contributions come from researchers across a wide range of fields who have varying degrees of experience working on coronaviruses and related topics. The volume of information available, much of which has not gone through rigorous peer review, presents a significant challenge to individual efforts to keep abreast of the state of COVID-19 research [5]. However, research on these topics is proceeding so rapidly that any static review is likely to quickly become dated. Our goal as a community is to consolidate information about the virus in the context of related viruses and to synthesize rapidly emerging literature centered on the diagnosis and treatment of COVID-19. We used an open publishing framework, Manubot [6], to manage hundreds of contributions from the community to create a living, scholarly document. We designed software to generate figures, such as , that automatically update using external data sources. Our primary goal is to sort and distill informative content out of the overwhelming flood of information [5] and help the broader scientific community become more conversant on this critical subject. Thus, our approach has been to develop a real-time, collaborative effort that welcomes submissions from scientists worldwide into this ongoing effort. This document represents the first snapshot, which aims to reflect the state of the field as of October, 2020. We plan to refine and expand this document until technologies to mitigate the pandemic are widely available.

1.1 Interdisciplinary Context

Collaboration across several broad areas of research is critical, as different areas provide different information and context necessary to understanding the virus and disease. This review provides a biological perspective on the virus Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and efforts to develop diagnostic, prophylactic, and therapeutic responses to COVID-19. We provide only brief summaries of two other important perspectives on this pandemic: epidemiology and public health. Research in these areas often seeks to anticipate, model, and prevent outbreaks of infectious disease or to understand and manage human behavior relevant to health and disease. Their insights are critical to mounting a global response to the pandemic. Epidemiological analyses have investigated patterns of transmission within and between communities, the symptoms associated with and the duration of infection and/or contagiousness, and how the virus propagates, among other characteristics [7]. Epidemiology also has a close relationship to public policy because it provides model-based insights into how preventative measures and public response can shift outcomes [8]. Public health addresses social and human factors influencing individuals’ exposure and susceptibility to pathogens, such as resource availability, inequality, human behavior, and access to accurate information. Strategies from public health and epidemiology for managing the current epidemic have included the promotion of hand hygiene, social distancing, and personal protective equipment such as masks to mitigate spread, as well as containment approaches such as test, trace, and isolate, which depends on widespread testing, contact tracing, and quarantining. An effective public health management strategy involves response coordination, disease surveillance, intervention monitoring, risk communication, and health education (including the containment of “infodemics” of false information) [9]. Epidemiology and public health intersect with the topics addressed in this manuscript because they both inform and benefit from relevant biotechnological developments. For example, the development of accurate and fast diagnostic testing is relevant to test, trace, and isolate strategies for containment, and public education will be critical to deploying vaccines once they become available. The present analysis focuses less on human and social factors and more on the basic biology of infection, diagnosis, and recovery, but these areas are inextricable in understanding and responding to the COVID-19 pandemic.

1.2 Initial Characterization of SARS-CoV-2

The first genome sequence of the virus was released on January 3, 2020 and revealed that the cluster of pneumonia cases seen in Wuhan were caused by a novel coronavirus [10]. Multiple research groups have drafted the genome sequence of SARS-CoV-2 based on sequences developed from clinical samples collected from the lower respiratory tract, namely bronchoalveolar lavage fluid (BALF), and the upper respiratory tract, in the form of throat swabs [11,12,13]. Analysis of the SARS-CoV-2 genome revealed significant sequence homology with two coronaviruses known to infect humans, with about 79% identity to SARS-CoV-1 and 50% to MERS-CoV [13]. However, the highest degree of similarity was observed between SARS-CoV-2 and bat-derived SARS-like coronaviruses (bat-SL-CoVZC45 and bat-SL-CoVZXC21) [12,13], with identity between SARS-CoV-2 and RATG13 as high as 96.2% [12,14]. This evidence therefore suggests the SARS-CoV-2 virus is the result of zoonotic transfer of a virus from bats to humans. Nevertheless, some fragments between SARS-CoV-2 and RATG13 differ by up to 17%, suggesting a complex natural selection process during zoonotic transfer. While the S region is highly similar to that of viruses found in pangolins [15], there is no consensus about the origin of S in SARS-CoV-2, as it could potentially be the result either of recombination or coevolution [14,16]. Though the intermediate host serving as the source for the zoonotic introduction of SARS-CoV-2 to human populations has not yet been identified, the SARS-CoV-2 virus has been placed within the coronavirus phylogeny through comparative genomic analyses. Genomic analyses and comparisons to other known coronaviruses suggest that SARS-CoV-2 is unlikely to have originated in a laboratory – either purposely engineered and released, or escaped – and instead evolved naturally in an animal host [17]. While the position of the SARS-CoV-2 virus within the coronavirus phylogeny has been largely resolved, the functional consequences of molecular variation between this virus and other viruses, such as its bat and pangolin sister taxa or SARS-CoV-1, remain unknown [17]. Fortunately, the basic genome structure of coronaviruses is highly conserved, and insight into the mechanisms the virus uses to enter cells, replicate, and spread is likely to be available from prior research in coronaviruses.

1.3 Coronaviruses and Humans Hosts

Coronaviruses have long been known to infect animals and have been the subject of veterinary medical investigations and vaccine development efforts due to their effect on the health of companion and agricultural animals [18]. Most coronaviruses show little to no transmission in humans. However, today it is thought that approximately one-third of common cold infections are caused by four human coronaviruses (HCoV): Human coronavirus 229E (HCoV-229E), Human coronavirus NL63 (HCoV-NL63), Human coronavirus OC43 (HCoV-OC43), and Human coronavirus HKU1 (HCoV-HKU1) [19,20]. The first HCoV were identified in the 1960s: HCoV-229E in 1965 [21] and HCoV-OC43 in 1967 [22]. Both of these viruses cause cold-like symptoms [23,24]. Two additional HCoV were subsequently identified [25,26]. In 2003, HCoV-NL63 [25] was first identified in a 7-month-old infant and then in clinical specimens collected from seven additional patients, five of whom were infants younger than 1 year old and the remainder of whom were adults. CoV-HKU1 was identified in samples collected from a 71-year-old pneumonia patient in 2004 and then found in samples collected from a second adult patient [26]. These viruses are associated with respiratory diseases of varying severity, ranging from common cold to severe pneumonia, with severe symptoms mostly observed in immunocompromised individuals [27]. In addition to these relatively mild HCoV, however, highly pathogenic human coronaviruses have been identified, including Severe acute respiratory syndrome-related coronavirus (SARS-CoV or SARS-CoV-1) and Middle East respiratory syndrome-related coronavirus (MERS-CoV) [19,28,29].

At the time that SARS-CoV-1 emerged in the early 2000s, no HCoV had been identified in almost 40 years [28]. The first case of SARS was reported in November 2002 in the Guangdong Province of China, and over the following month, the disease spread more widely within China and then into several countries across multiple continents [28,30]. Unlike previously identified HCoV, SARS was much more severe, with an estimated death rate of 9.5% [30]. It was also highly contagious via droplet transmission, with a basic reproduction number (R0) of 4 (i.e., each person infected was estimated to infect four other people) [30]. However, the identity of the virus behind the infection remained unknown until April of 2003, when the SARS-CoV-1 virus was identified through a worldwide scientific effort spearheaded by the WHO [28]. SARS-CoV-1 belonged to a distinct lineage from the two other HCoV known at the time [30]. By July 2003, the SARS outbreak was officially determined to be under control, with the success credited to infection management practices [28]. A decade later, a second outbreak of severe respiratory illness associated with a coronavirus emerged, this time in the Arabian Peninsula. This disease, known as Middle East respiratory syndrome (MERS), was linked to another novel coronavirus, MERS-CoV. The fatality rate associated with MERS is much higher than that of SARS, at almost 35%, but the disease is much less easily transmitted, with an R0 of 1 [30]. Although MERS is still circulating, its low reproduction number has allowed for its spread to be contained [30]. The COVID-19 pandemic is thus associated with the seventh HCoV to be identified and the fifth since the turn of the millennium, though additional HCoVs may be in circulation but remain undetected.

SARS-CoV-1 and MERS-CoV were ultimately managed largely through infection management practices (e.g., mask wearing) and properties of the virus itself (i.e., low rate of transmission), respectively [28,30]. Vaccines were not used to control either virus, although vaccine development programs were established for SARS-CoV-1 [31]. In general, care for SARS and MERS patients focuses on supportive care and symptom management [30]. Clinical treatments for SARS and MERS developed during the outbreaks generally do not have strong evidence supporting their use. Common treatments included Ribavirin, an antiviral, often in combination with corticosteroids or sometimes interferon (IFN) medications, which would both be expected to have immunomodulatory effects [28]. However, retrospective and in vitro analyses have reported inconclusive results of these treatments on SARS and the SARS-CoV-1 virus, respectively [28]. IFNs and Ribavirin have shown promise in in vitro analyses of MERS, but their clinical effectiveness remains unknown [28]. Therefore, only limited strategy for the pharmaceutical management of COVID-19 can be adopted from previous severe HCoV infections. Research in response to prior outbreaks of HCoV-borne infections, such as SARS and MERS, have, however, provided a strong foundation for hypotheses about the pathogenesis of SARS-CoV-2 as well as potential diagnostic and therapeutic approaches.

1.3.1 Human Immune Response to Viral Threats

Understanding the fundamental organization of the human immune response to viral threats is critical to understanding the varied response to SARS-CoV-2. The human immune system utilizes a variety of innate and adaptive responses to protect against the pathogens it encounters. The innate immune system consists of barriers, such as the skin, mucous secretions, neutrophils, macrophages, and dendritic cells. It also includes cell-surface receptors that can recognize the molecular patterns of pathogens. The adaptive immune system utilizes antigen-specific receptors that are expressed on B and T lymphocytes. These components of the immune system typically act together; the innate response acts first, and the adaptive response begins to act several days after initial infection following the clonal expansion of T and B cells [32]. After a virus enters into a host cell, its antigen is presented by major histocompatibility complex 1 (MHC 1) molecules and is then recognized by cytotoxic T lymphocytes.

In the case of COVID-19, there is also concern about the immune system becoming over-active. One of the main immune responses contributing to the onset of acute respiratory distress syndrome (ARDS) in COVID-19 patients is cytokine storm syndrome (CSS), which causes an extreme inflammatory response due to a release of pro-inflammatory cytokines and chemokines by immune effector cells. In addition to respiratory distress, this mechanism can lead to organ failure and death in severe COVID-19 cases [33]. Details of how the human body responds to SARS-CoV-2, both in healthy and pathological ways, and how these mechanisms can inform the identification of diagnostic, prophylactic, and therapeutic responses are explored in detail throughout this manuscript.

1.3.2 Clinical Presentation of COVID-19

A great diversity of symptom profiles has been observed for COVID-19, although a large study from Wuhan, China suggests fever and cough as the two most common symptoms on admission [34]. One early retrospective study in China described the clinical presentations of patients infected with SARS-CoV-2 as including lower respiratory tract infection with fever, dry cough, and dyspnea [35]. This study [35] noted that upper respiratory tract symptoms were less common, which suggests that the virus targets cells located in the lower respiratory tract. However, data from the New York City region [36,37] showed variable rates of fever as a presenting symptom, suggesting that symptoms may not be consistent across samples. These differences are present when comparing both between institutions in similar locations and between different regions experiencing COVID-19 outbreaks, leading to conflicting reports of the frequency of fever as a presenting symptom for patients upon hospital admission. For example, even within New York City, one study [36] identified low oxygen saturation (<90% without the use of supplemental oxygen or ventilation support) in a significant percentage of patients upon presentation, while another study [37] reported cough, fever, and dyspnea as the most common presenting symptoms. The variability of both which symptoms present and their severity makes it difficult for public health agencies to provide clear recommendations for citizens regarding what symptoms indicate SARS-CoV-2 infection and should prompt isolation.

1.4 Role of the COVID-19 Review

Several review articles on aspects of COVID-19 have already been published. These have included reviews on the disease epidemiology [38], immunological response [39], diagnostics [40], and pharmacological treatments [39,41]. Others [42,43] provide narrative reviews of progress on some important ongoing COVID-19 research questions. With the worldwide scientific community uniting during 2020 to investigate SARS-CoV-2 and COVID-19 from a wide range of perspectives, findings from many disciplines are relevant on a rapid timescale to a broad scientific audience. Additionally, many findings are published as preprints, which are available prior to going through the peer review process. As a result, centralizing, summarizing, and critiquing new literature broadly relevant to COVID-19 can help to expedite the interdisciplinary scientific process that is currently happening at an advanced pace. We are particularly interested in providing background to the development of diagnostic, prophylactic, and therapeutic approaches to COVID-19. Two major concerns within diagnosis include the detection of current infections in individuals with and without symptoms, and the detection of past exposure without an active infection. In the latter category, identifying whether individuals can develop or have developed sustained immunity is also a major consideration. The development of high-throughput, affordable methods for detecting active infections and sustained immunity will be critical to understanding and controlling the disease. The identification of interventions that can mitigate the effect of the virus on exposed and infected individuals is a significant research priority. Some possible approaches include the identification of existing pharmaceuticals that reduce the severity of infection, either by reducing the virus’ virulence (e.g., antivirals) or managing the most severe symptoms of infection. Due to the long timeline for the development of novel pharmaceuticals, in most cases, research surrounding possible pharmaceutical interventions focuses on the identification and investigation of existing compounds whose mechanisms may be relevant to COVID-19. Other foci of current research include the identification of antibodies produced by survivors of COVID-19 and the development of vaccines. Understanding the mechanisms describing host-virus interactions between humans and SARS-CoV-2 is thus critical to identifying candidate therapeutics. An overview of the topics covered is visualized in Thus, in this review, we seek to consolidate information about efforts to develop strategies for diagnosis and therapeutics as new information is released by the scientific community. We include information from both traditional peer-reviewed scientific literature and from preprints, which typically have not undergone peer review but have been critically evaluated by the scientists involved in this effort. The goal of this manuscript is to present preliminary findings within the broader context of COVID-19 research and to identify the broad interpretations of new research, as well as limitations to interpretability.

2 Genomic and Viral Structure of SARS-CoV-2 in the Context of Pathogenesis, Symptomology, and Transmission

2.1 Abstract

The novel coronavirus SARS-CoV-2, which emerged in late 2019, has been a major force shaping the year 2020 as it spread around the world infecting tens of millions of people with coronavirus disease 2019 (COVID-19). While the viral species was unknown prior to January 2020, its similarity to other coronaviruses that infect humans has allowed for rapid insight into the mechanisms that it uses to infect humans, as well as the ways in which the human immune system can respond. Here, we contextualize SARS-CoV-2 among other coronaviruses and identify what is known and what can be inferred about its behavior once inside a human host. Because the genomic content of coronaviruses, which specifies the virus’s structure, is highly conserved, early genomic analysis provided a significant head start in predicting viral pathogenesis. The pathogenesis of the virus offers insights into symptomology, transmission, and individual susceptibility. Additionally, prior research into interactions between the human immune system and coronaviruses identified how these viruses can evade the immune system’s protective mechanisms. We also explore systems-level research into the regulatory and proteomic effects of SARS-CoV-2 infection and the immune response. Understanding the structure and behavior of the virus serves to contextualize the many facets of the COVID-19 pandemic and can influence efforts to control the virus and treat the disease.

2.2 Importance

COVID-19 involves a number of organ systems and can present with a wide range of symptoms. However, understanding how the virus infects epithelial cells can contextualize how these systems connect. Similarly, the modes of viral transmission have been under debate throughout much of 2020, yet the available research suggests that these patterns are very similar to those observed in closely related viruses like SARS-CoV-1 and possibly MERS-CoV. Exploring the structure, phylogeny, and pathogenesis of the virus therefore helps to guide interpretation of the broader impacts of the virus on the human body and on human populations. For this reason, an in-depth exploration of viral mechanisms is critical to a robust understanding of the COVID-19 pandemic.

2.3 Introduction

The current coronavirus disease 2019 (COVID-19) pandemic, caused by the Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) virus, represents an acute global health crisis. Symptoms of the disease can range from mild to severe to fatal [44] and can affect a variety of organs and systems [45]. Outcomes of the disease can include acute respiratory distress (ARDS) and acute lung injury, as well as damage to other organ systems [45,46]. Understanding the progression of the disease, including these diverse symptoms, depends on understanding how the virus interacts with the host. Additionally, the fundamental biology of the virus can provide insights into how it is transmitted among people, which can, in turn, inform efforts to control its spread. As a result, a thorough understanding of the pathogenesis of SARS-CoV-2 is a critical foundation on which to build an understanding of COVID-19 and the pandemic as a whole.

The rapid identification and release of the genomic sequence of the virus in January 2020 [10] provided early insight into the virus in a comparative genomic context. The viral genomic sequence clusters with known coronaviruses (order Nidovirales, family Coronaviridae, subfamily Orthocoronavirinae). Phylogenetic analysis of the coronaviruses reveals four major subclades, each corresponding to a genus: the alpha, beta, delta, and gamma coronaviruses. Among them, alpha- and betacoronaviruses infect mammalian species, gammacoronaviruses infect avian species, and deltacoronaviruses infect both mammalian and avian species [47]. The novel virus now known as SARS-CoV-2 was identified as betacoronavirus belonging to the B lineage based on phylogenetic analysis of a polymerase chain reaction (PCR) amplicon fragment from five patients along with the full genomic sequence [48]. This lineage also includes the Severe acute respiratory syndrome-related coronavirus (SARS-CoV-1) that caused the 2002-2003 outbreak of Severe Acute Respiratory Syndrome (SARS) in humans [48]. Because viral structure and mechanisms of pathogenicity are highly conserved within the order, this phylogenetic analysis provides a basis for forming hypotheses about how the virus interacts with hosts, including which tissues, organs, and systems would be most susceptible to SARS-CoV-2 infection. Coronaviruses that infect humans (HCoV) are not common, but prior research into other HCoV such as SARS-CoV-1 and Middle East respiratory syndrome-related coronavirus (MERS-CoV), as well as other viruses infecting humans such as a variety of influenza species, established a strong foundation that accelerated the pace of SARS-CoV-2 research.

Coronaviruses are large viruses that can be identified by their distinctive “crown-like” shape (Figure 1). Their spherical virions are made from lipid envelopes ranging from 100 to 160 nanometers (nm) in which peplomers of two to three spike (S) glycoproteins are anchored, creating the crown [49,50]. These spikes, which are critical to both viral pathogenesis and to the response by the host immune response, have been visualized using cryo-electron microscopy [51]. Because they induce the human immune response, they are also the target of many proposed therapeutic agents. Viral pathogenesis is typically broken down into three major components: entry, replication, and spread [52]. However, in order to draw a more complete picture of pathogenesis, it is also necessary to examine how infection manifests clinically, identify systems-level interactions between the virus and the human body, and consider the possible effects of variation or evolutionary change on pathogenesis and virulence. In this way, both biomedicine and genomics are important pieces of the puzzle of SARS-CoV-2 presentation and pathogenesis.

2.4 Coronavirus Structure and Pathogenesis

2.4.1 Structure of Coronaviruses

Genome structure is highly conserved among coronaviruses, meaning that the relationship between the SARS-CoV-2 genome and its pathogenesis can be inferred from prior research in related viral species. The genomes of viruses in the Nidovirales order share several fundamental characteristics. They are non-segmented, which means the viral genome is contained in a single capsid, and are enveloped, which means that the genome and capsid are encased by a lipid bilayer. Coronaviruses have large positive-sense RNA (ssRNA+) genomes ranging from 27 to 32 kilobases in length [13,53]. The SARS-CoV-2 genome lies in the middle of this range at 29,903 bp [13]. Genome organization is highly conserved within the order [53]. There are three major regions: one containing the replicase gene and one containing the genes encoding structural proteins [53] (Figure 1). The replicase gene comprises about two-thirds of the genome and consists of two open reading frames that are translated with ribosomal frameshifting [53]. This polypeptide is then translated into 16 non-structural proteins (nsp), except in Gammacoronaviruses where nsp1 is absent, that form replication machinery used to synthesize viral RNA [54]. The remaining third of the genome encodes structural proteins, including the spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins. Additional accessory genes are sometimes present between these two regions, depending on the species or strain. Much attention has been focused on the S protein, which is a critical structure involved in cell entry.

Figure 1: Structure of SARS-CoV-2 capsid and genome. A) The genomic structure of coronaviruses is highly conserved, including the order and organization of genes such as the spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins. B) The physical structure of the coronavirus virion, including the components encoded by conserved genes S, E, M and N.

2.4.2 Pathogenic Mechanisms of Coronaviruses

While, like most viruses, it is possible that SARS-CoV-1 and SARS-CoV-2 can enter cells through endocytosis, coronaviruses are able to target cells for entry through fusion with the plasma membrane [55,56]. This process is conserved among coronaviruses and is closely associated with the content of their genomes. Cell entry proceeds in three steps: binding, cleavage, and fusion. First, the viral spike protein binds to a host cell via a recognized receptor. Coronaviruses can bind to a range of host receptors [57,58], with binding conserved only at the genus level [47]. Viruses in the betacoronavirus genus, to which SARS-CoV-2 belongs, are known to bind to the CEACAM1 protein, 5-N-acetyl-9-O-acetyl neuraminic acid (Neu 5,9 Ac2), and to the angiotensin-converting enzyme 2 (ACE2) [57]. SARS-CoV-2 has a high affinity for the human ACE2 receptor, which is expressed in the vascular epithelium, other epithelial cells, and cardiovascular and renal tissues [59,60]. The binding process is guided by the molecular structure of the spike protein, which is structured in three segments: an ectodomain, a transmembrane anchor, and an intracellular tail [61]. The ectodomain forms the crown-like structures on the viral membrane and contains two subdomains known as the S1 and S2 subunits [62]. The S1 (N-terminal) domain forms the head of the crown and contains the receptor binding motif, and the S2 (C-terminal) domain forms the stalk that supports the head [62]. The S1 subunit guides the binding of the virus to a host cell receptor, and the S2 subunit guides the fusion process [61].

After the binding of the S1 subunit to a receptor, the spike protein is often cleaved at the S1-S2 boundary by a host protease [63,64,65]. Similar to SARS-CoV, SARS-CoV-2 exhibits redundancy in which host proteases can cleave the S protein [66]. Specifically, both transmembrane protease serine protease-2 (TMPRSS2) and cathepsins B/L have been shown to mediate SARS-CoV-2 S protein proteolytic priming, and small molecule inhibition of these enzymes fully inhibited viral entry in vitro [66,67]. Proteolytic priming prepares the S protein for fusion [64,65]. The two subunits remain bound by van der Waals forces, with the S1 subunit stabilizing the S2 subunit during the membrane fusion process [63]. Electron microscopy suggests that in some coronaviruses, including SARS-CoV-1 and MERS-CoV, a six-helix bundle separates the two subunits in the postfusion conformation, and the unusual length of this bundle facilitates membrane fusion through the release of additional energy [47]. Cleavage at a second site within S2 by these same proteases activates S for fusion by inducing conformational changes [63]. The viral membrane can then fuse with the endosomal membrane to release the viral genome into the host cytoplasm. Once the virus enters a host cell, the replicase gene is translated and assembled into the viral replicase complex. This complex then synthesizes the double-stranded RNA (dsRNA) genome from the genomic ssRNA(+). The dsRNA genome is transcribed and replicated to create viral mRNAs and new ssRNA(+) genomes [53,68]. From there, the virus can spread into other cells. In this way, the genome of SARS-CoV-2 provides insight into the pathogenic behavior of the virus.

2.4.3 Immune Evasion Strategies

Research in other HCoV provides some indication of how SARS-CoV-2 infection proceeds in spite of the human immune response. By infecting the epithelium, viruses such as SARS-CoV-1 are known to bypass the physical barriers such as skin and mucus that comprise the immune system’s first line of defense [69]. Once the virus infiltrates host cells, it is adept at evading detection. CD163+ and CD68+ macrophage cells are especially crucial for the establishment of SARS-CoV-1 in the body [69]. These cells most likely serve as viral reservoirs that help shield SARS-CoV-1 from the innate immune response. According to a study on the viral dissemination of SARS-CoV-1 in Chinese macaques, viral RNA could be detected in some monocytes throughout the process of differentiation into dendritic cells [69]. Thus lack of active viral replication allows SARS-CoV-1 to escape innate immunity because reduced levels of detectable viral RNA allow the virus to avoid both natural killer (NK) cells and Toll-like receptors [69]. Even during replication, SARS-CoV-1 is able to mask its dsRNA from detection by the immune system. Although dsRNA is a pathogen-associated molecular pattern (PAMP) that would typically initiate a response from the innate immune system [70], in vitro analysis of nidoviruses including SARS-CoV-1 suggests that these viruses can induce the development of double-membrane vesicles that protect the dsRNA signature from being detected by the host immune system [71]. This protective envelope can therefore insulate these coronaviruses from the innate immune response’s detection mechanism [33].

HCoV are also known to interfere with the host immune response, rather than just evade it. For example, the virulence of SARS-CoV is increased by nsp1, which can suppress host gene expression by stalling mRNA translation and inducing endonucleolytic cleavage and mRNA degradation [72]. SARS-CoV-1 also evades the immune response by interfering with type I IFN induction signaling, which is a mechanism that leads to cellular resistance to viral infections. SARS-CoV-1 employs methods such as ubiquitination and degradation of RNA sensor adaptor molecules MAVS and TRAF3/6 [73]. Also, MERS-CoV downregulates antigen presentation via MHC class I and MHC class II, which leads to a reduction in T cell activation [73]. These evasion mechanisms, in turn, can lead to systemic infection. Coronaviruses such as SARS-CoV-1 are also able to evade the humoral immune response through other mechanisms, such as inhibiting certain cytokine pathways or down-regulating antigen presentation by the cells [71].

2.4.4 Host Cell Susceptibility

ACE2 and TMPRSS2 have been identified as the primary entry portal and as a critical protease, respectively, in facilitating the entry of SARS-CoV-1 and SARS-CoV-2 into a target cell [51,66,74,75,76]. This finding has led to a hypothesized role for ACE2 and TMPRSS2 expression in determining which cells, tissues, and organs are most likely to be infected by SARS-CoV-2. The ACE2 receptor is expressed in numerous organs, such as the heart, kidney, and intestine, but it is most prominently expressed in alveolar epithelial cells; this pattern of expression is expected to contribute to the virus’ association with lung pathology [59,77]. Clinical investigations of COVID-19 patients have detected SARS-CoV-2 transcripts in bronchoalveolar lavage fluid (BALF) (93% of specimens), sputum (72%), nasal swabs (63%), fibrobronchoscopy brush biopsies (46%), pharyngeal swabs (32%), feces (29%) and blood (1%) [78]. Two studies reported that SARS-CoV-2 could not be detected in the urine specimens [78,79]; however, a third study identified four urine samples (out of 58) that were positive for SARS-CoV-2 nucleic acids [80]. Although respiratory failure remains the leading cause of death for COVID-19 patients [81], SARS-CoV-2 infection can damage many other organ systems including the heart [82], kidneys [83,84], liver [85], and gastrointestinal tract [86,87]. As it becomes clear that SARS-CoV-2 infection can damage multiple organs, the scientific community is pursuing multiple avenues of investigation in order to build a consensus about how the virus affects the human body.

2.5 Clinical Presentation of COVID-19

Pathogenesis is closely linked with the clinical presentation of the disease caused by the virus. Reports have described diverse symptom profiles associated with COVID-19. Differences in the frequency of symptoms are found when comparing both between institutions in similar locations and between different regions. A large study from Wuhan, China conducted early in the pandemic identified fever and cough as the two most common symptoms that patients reported at hospital admission [34], while a retrospective study in China described the clinical presentations of patients infected with SARS-CoV-2 as including lower respiratory tract infection with fever, dry cough, and dyspnea [35]. This study [35] noted that upper respiratory tract symptoms were less common, suggesting that the virus preferentially targets cells located in the lower respiratory tract. However, data from the New York City region [36,37] revealed variable rates of fever as a presenting symptom, suggesting that symptoms may not be consistent across samples. For example, even within New York City, one study [36] identified low oxygen saturation (<90% without the use of supplemental oxygen or ventilation support) in a significant percentage of patients upon presentation, while another study [37] reported cough, fever, and dyspnea as the most common presenting symptoms. The variability of both which symptoms present and their severity has presented challenges for public health agencies to provide clear recommendations for citizens regarding what symptoms indicate SARS-CoV-2 infection and should prompt isolation. Patients may also experience loss of smell, myalgias (muscle aches), fatigue, or headache. Gastrointestinal symptoms can also present [88], and the CDC includes nausea and vomiting, as well congestion and runny nose, on its list of symptoms consistent with COVID-19 [44]. A recent preprint using data from an app-based survey of 500,000 individuals in the US found that among those tested for SARS-CoV-2, a loss of taste or smell, fever, and a cough were significant predictors of a positive test result [89]. It is important to note that in this study, the predictive value of symptoms may be underestimated if they are not specific to COVID-19 because the outcome measured was a positive, as opposed to a negative, COVID-19 test result. At the time the surveys were conducted, due to limits in US testing infrastructure, respondents typically needed to have some symptoms known to be specific to COVID-19 in order to qualify for testing in the first place. Widespread testing of asymptomatic individuals may therefore provide additional insight into the range of symptoms associated with COVID-19.

Consistent with the wide range of symptoms observed, COVID-19 can affect diverse body systems in addition to causing respiratory problems [90]. For example, COVID-19 can lead to acute kidney injury, especially in patients with severe respiratory symptoms or certain preexisting conditions [91]. It can also cause neurological complications [92,93], potentially including stroke, seizures or meningitis [94,95]. COVID-19 has also been associated with an increased incidence of large vessel stroke, particularly in patients under the age of 40 [96], and other thrombotic events including pulmonary embolism and deep vein thrombosis [97]. The mechanism behind these complications has been suggested to be related to coagulopathy, with reports indicating the presence of antiphospholipid antibodies [98] and elevated levels of d-dimer and fibrinogen degradation products in deceased patients [99]. Other viral infections have been associated with coagulation defects and changes to the coagulation cascade; notably SARS was also found to lead to disseminated intravascular coagulation and was associated with both pulmonary embolism and deep vein thrombosis [100]. The mechanism behind these insults has been suggested to be related to inflammation-induced increases in the von Willebrand factor clotting protein, leading to a pro-coagulative state [100]. Abnormal clotting (thromboinflammation or coagulopathy) has been increasingly discussed recently as a possible key mechanism in many cases of severe COVID-19, and may be associated with the high d-dimer levels often observed in severe cases [101,102,103]. This excessive clotting in lung capillaries has been suggested to be related to a dysregulated activation of the complement system, part of the innate immune system [104,105].

2.5.1 Cytokine Release Syndrome

Symptoms of a disease can be caused by a pathogen, but they can also be caused by the immune system’s reaction to the pathogen. A dysregulated immune response can cause significant damage to the host [106,107,108]. The inflammatory response has received particular attention for its role in both a healthy response to infection and a pathogenic one. Inflammation is one of the most visible components of the immune response, as it is responsible for the hallmarks of injury, such as pain, heat, and swelling [109]. In response to injury or to signaling by pattern recognition receptors (PRRs) indicating the detection of a molecular pattern associated with a pathogen or foreign body, the immune system stimulates leukocytes that travel to the site of the threat, where they then produce cytokines [109]. Cytokines are a diverse group of small proteins that play an important role in intercellular signaling [110]. Cytokines can be both pro- and anti-inflammatory, which means they can either stimulate or inhibit the production of additional cytokines [110,111]. Some notable pro-inflammatory cytokines include the interleukins: IL-1β and IL-6 and tumor necrosis factor α (TNF-α) [111]. Anti-inflammatory cytokines play an immunoregulatory role complementary to the cascading effect of pro-inflammatory cytokines [110,111]. A number of interleukins and interferons play anti-inflammatory roles, and receptors or receptor antagonists for inflammatory cytokines are also important for regulating inflammation [111]. IL-10 is an anti-inflammatory cytokine of particular note because it regulates the expression of TNF-α, IL-1, and IL-6 [111]. When the pro- and anti-inflammatory responses are both commensurate with the threat posed, the immune system drives a shift back to homeostasis [112]. However, when the responses are disproportionate, the cytokine response can become dysregulated. Too low of an inflammatory response will not eliminate the immune threat [112]. In contrast, if the response is dysregulated towards excessive pro-inflammatory cytokine activity, inflammation can cascade [113] and cause cell damage, among other problems [109]. Elevated levels of inflammation over the long-term are associated with many chronic health conditions, including type 2 diabetes, dementia and Alzheimer’s, and arthritis, among others [114]. On a shorter timescale, dysregulated systemic inflammation can cause sepsis, which can lead to multi-organ failure and death [110,115].

Cytokines have been investigated for their role in the immune response to lung infections long before the COVID-19 pandemic. Dysregulation of the inflammatory response, including elevated levels of pro-inflammatory cytokines, is found in patients with ARDS, which is a severe condition that can arise from pneumonia, SARS, and COVID-19 [113]. One study of patients with and at risk for ARDS, specifically those who were intubated for medical ventilation, found that shortly after the onset of ARDS, anti-inflammatory cytokine concentration in BALF increased relative to the concentration of pro-inflammatory cytokines [116]. The results suggest that an increase in pro-inflammatory cytokines such as IL-6 may signal the onset of ARDS, but recovery depends on an increased anti-inflammatory response [116]. However, patients with severe ARDS were excluded from this study. Acute phase response to an infection can also cause damage to the capillary endothelium, allowing leaks that disrupt the balance between pro-inflammatory cytokines and their regulators [116]. Hyperactivity of the pro-inflammatory response due to lung infection is commonly associated with acute lung injury and more rarely with the more severe manifestation, ARDS [110]. The heightened inflammatory response in the lungs can also serve as a source for systemic inflammation, or sepsis, which can lead to multi-organ failure [110]. The shift from local to systemic inflammation is a phenomenon often referred to broadly as a cytokine storm [110] or, more precisely, as cytokine release syndrome (CRS) [117]. Sepsis is a known possible complication of pneumonia, and in an analysis of over 1,400 US pneumonia patients, IL-6, tumor necrosis factor (TNF), and IL-10 were found to be elevated at intake in patients who developed severe sepsis and/or ultimately deceased [118]. IL-6 and TNF are pro-inflammatory cytokines, while IL-10; is anti-inflammatory [118]. However, this study reported that unbalanced pro-/anti-inflammatory cytokine profiles were rare, although they measured only the three cytokines listed above. Prior work therefore made it clear that pulmonary infection and injury were associated with systemic inflammation through sepsis. While IL-6 is a biomarker sometimes used to assess cytokine storm activity in sepsis [110], the relationship between cytokine profiles and the risks associated with sepsis may be more complex. IL-6 is a pleiotropic cytokine that plays an integral role in both the inflammatory and anti-inflammatory responses and is associated with both healthy and pathological responses to viral threat [119].

The inflammatory response was identified early on as a potential driver of COVID-19 outcomes due to existing research in SARS and emerging research in COVID-19. In addition to the known role of cytokines in ARDS and lung infection more broadly, immunohistological analysis at autopsy of patients who deceased from SARS revealed that ACE2-expressing cells that were infected by SARS-CoV-1 showed elevated expression of IL-6, IL-1β, and TNF-α [120]. Similarly, the introduction of the S protein from SARS-CoV-1 to mouse macrophages was found to increase production of IL-6 and TNF-α [121]. For SARS-CoV-2 infection leading to COVID-19, early reports described a cytokine storm syndrome-like response in patients with particularly severe infections [77,122,123]. Among patients hospitalized with COVID-19 in Wuhan, China, 112 out of 191 (59%) developed sepsis, including all 54 of the non-survivors [35]. However, the argument has been made that while the cytokine levels observed in COVID-19 patients fall outside of the normal range, they are not as high as typically found in patients with ARDS [124]. Regardless, inflammation has received significant interest both in regards to the pathology of COVID-19 as well as potential avenues for treatment, as the similarities between the cytokine storm and the pathophysiology of COVID-19 has led to the suggestion that a number of immunomodulatory pharmaceutical interventions could hold therapeutic value for the treatment of COVID-19 [125].

2.5.2 Pediatric Presentation

The presentation of COVID-19 infection can vary greatly among pediatric patients and, in some cases, manifests in distinct ways from COVID-19 in adults. Evidence suggests that while children and adolescents tend to have mostly asymptomatic infections, those that are symptomatic exhibit a mild illness [126,127,128,129]. A review examined symptoms reported in 17 studies of children infected with COVID-19 during the early months of the COVID-19 epidemic in China and one study from Singapore [130]. Of the more than a thousand cases described, the most common reports were for mild symptoms such as fever, dry cough, fatigue, nasal congestion and/or runny nose, while three children were reported to be asymptomatic. Severe lower respiratory infection was described in only one of the pediatric cases reviewed. Gastrointestinal symptoms such as vomiting or diarrhea were occasionally reported. Radiologic findings were not always reported in the case studies reviewed, but when they were mentioned they included bronchial thickening, ground-glass opacities, and/or inflammatory lesions [130]. Neurological symptoms have also been reported [131].

These analyses indicate that most pediatric cases of COVID-19 are not severe. Indeed, it is estimated that less than 1% of pediatric cases result in critical illness [128,132]. However, serious complications and, in rare cases, deaths have occurred [133]. Of particular interest, children have occasionally experienced a serious inflammatory syndrome, multisystem inflammatory syndrome in children (MIS-C), following COVID-19 infection. This syndrome is similar in some respects to Kawasaki disease or to Kawasaki disease shock syndrome [134,135,136] and is thought to be a distinct clinical manifestation of SARS-CoV-2 due to its distinct cytokine profile and the presence of burr cells in peripheral blood smears [137,138]. MIS-C has been associated with heart failure in some cases [139]. One case study [140] described an adult who appeared to show symptoms similar to MIS-C after exposure to COVID-19, but cautioned against broad conclusions; a second possible adult case has also been reported [141]. The presentation of SARS-CoV-2 infection is therefore likely to be largely distinct between adult and pediatric populations.

2.6 Systems-Level Effects

Systems biology provides a cross-disciplinary analytical paradigm through which the host response to an infection can be analyzed. This field integrates the “omics” (genomics, transcriptomics, proteomics, metabolomics, etc.) with bioinformatics and other computational approaches. These cutting-edge research paradigms hold enormous potential for the study of the complexity of biological systems and human diseases [142]. Over the last decade, systems biology approaches have been used widely to study the pathogenesis of diverse types of life-threatening acute and chronic infectious diseases [143]. Omics-based studies have also provided meaningful information regarding host immune responses and surrogate protein markers in several viral, bacterial and protozoan infections [144]. Though the complex pathogenesis and clinical manifestations of SARS-CoV-2 infection are not yet fully understood, omics technologies offer the opportunity for discovery-driven analysis of biological changes associated with SARS-CoV-2 infection. For example, previous studies suggest that infection by coronaviruses, such as SARS-CoV-1 and MERS-CoV, and other viruses is associated with the upregulation of ACE2. In several preliminary assays and an analysis of previous microarray data, ACE2 expression was reported to be significantly upregulated following infection of human embryonic kidney cells and human airway epithelial cells [77]. This study also reported that direct stimulation with inflammatory cytokines such as type I interferons (e.g., IFNβ) resulted in the upregulation of ACE2 in human bronchial epithelial cells, with treated groups showing four-fold higher ACE2 expression than control groups at 18 hours post-treatment [77]. Whether SARS-CoV-2 facilitates the positive regulation of its own transmission between host cells is still unclear, the host immune response itself likely plays a key role in mediating infection-associated pathologies. A systems-biology approach allows for analyses such as these to identify possible phenotypic and endophenotypic responses to SARS-CoV-2 infection and to develop new hypotheses about how pathogenesis proceeds.

2.6.1 Transcriptomics

In addition to the study described above, two other studies have profiled expression following SARS-CoV-2 infection using human cell lines. The first study [145] compared transcriptional responses to SARS-CoV-2 and to other respiratory viruses, including MERS-CoV, SARS-CoV, human parainfluenza virus 3 (HPIV3), respiratory syncytial virus (RSV), and influenza A virus (IAV). The authors analyzed the responses of three human cell lines: A549 (adenocarcinomic human alveolar basal epithelial cells), Calu-3 (human airway epithelial cells derived from human bronchial submucosal glands), and MRC-5 (human fetal lung fibroblast cells). As the viral receptor ACE2 has low expression in A549 cells, they supplemented the A549 cells with adenovirus (AdV)-based vectors expressing either mCherry (a fluorescent protein used as a control) or ACE2 (A549-ACE2). The authors also measured host transcriptional responses to SARS-CoV-2 in primary normal human bronchial epithelial cells (HBEC or NHBE cells), nasal washes from an animal model (ferret), and lung samples from two COVID-19 patients. Differential expression (DE) analysis was then carried out to compare infected cells with control cells that underwent only a mock treatment. In the hosts where SARS-CoV-2 was able to replicate efficiently, DE analysis revealed that the transcriptional response was significantly different from the response to all of the other viruses tested. A unique proinflammatory cytokine signature associated with SARS-CoV-2 was present in cells exposed to both high and low doses of the virus, with the cytokines IL-6 and IL1RA uniquely elevated in response to SARS-CoV-2 relative to other viruses. However, the A549-ACE2 cells showed significant IFN-I or IFN-III expression when exposed to high, but not low, doses of SARS-CoV-2. This finding suggests that IFN induction is dependent on the extent of exposure. Similarly, in cells from the NHBE line, ferrets, and COVID-19 patients, chemokine signaling was significantly enriched, but there was no significant induction of IFN-I or IFN-III. Together, these results suggest that SARS-CoV-2 induces a limited antiviral state with low IFN-I or IFN-III expression and a moderate IFN-stimulated gene response, in contrast to other viruses. However, in ACE2-expressing A549 cells, this state could be overcome by using a 10-fold increase in SARS-CoV-2 exposure. This finding suggests that the SARS-CoV-2 interferon antagonist is insufficient for large doses of the virus [145]. This hypothesis was further supported by a recent study [146] that showed that the SARS-CoV-2 ORF3b gene suppresses IFNB1 promoter activity (IFN-I induction) more efficiently than the SARS-CoV-1 ORF3b gene. Taken together, these findings suggest that a unique cytokine profile is associated with the response to the SARS-CoV-2 virus and that this response differs depending on the extent of exposure.

Another study [147] analyzed cells’ transcriptional response to SARS-CoV-2 and SARS-CoV-1 over time. They characterized the response of three human cell lines, H1299 (human non-small cell lung carcinoma cell line), Calu-3, and Caco-2 (human epithelial colorectal adenocarcinoma cell line), at 4 to 36 hours post infection (hpi). Using poly(A) bulk RNA-seq, the authors found negligible susceptibility of H1299 cells (< 0.08 viral read percentage of total reads) compared to Caco-2 and Calu-3 cells (>10% of viral reads). This finding suggests that the risk of infection varies among cell types, and that cell type could influence which hosts are more or less susceptible. Based on visual inspection of microscopy images alongside transcriptional profiling, the authors also showed distinct responses among the host cell lines evaluated. In contrast to Caco-2, Calu-3 cells infected with SARS-CoV-2 showed signs of impaired growth and cell death at 24 hpi, as well as moderate IFN induction with a strong up-regulation of IFN-stimulated genes. Interestingly, the results were similar to those reported in Calu-3 cells exposed to much higher levels of SARS-CoV-2 [145], as described above. This finding suggests that IFN induction in Calu-3 cells is not dependent on the level of exposure, in contrast to A549-ACE2 cells. The discrepancy could be explained by the observations that Calu-3 cells are highly susceptible to SARS-CoV-2 and show rapid viral replication [67], whereas A549 cells are incompatible with SARS-CoV-2 infection [148]. This discrepancy raises the concern that in vitro models may vary in their similarity to the human response, underscoring the importance of follow-up studies in additional models.

2.6.2 Proteomics

One early proteomics study investigated changes associated with in vitro SARS-CoV-2 infection using Caco-2 cells [149]. This study reported that SARS-CoV-2 induced alterations in multiple vital physiological pathways, including translation, splicing, carbon metabolism and nucleic acid metabolism in the host cells. Another area of interest is whether SARS-CoV-2 is likely to induce similar changes to other HCoV. For example, because of the high level of sequence homology between SARS-CoV-2 and SARS-CoV-1, it has been hypothesized that sera from convalescent SARS-CoV-1 patients might show some efficacy in cross-neutralizing SARS-CoV-2-S-driven entry [66]. However, despite the high level of sequence homology, certain protein structures might be immunologically distinct, which would be likely to prohibit effective cross-neutralization across different SARS species [150]. Consequently, proteomic analyses of SARS-CoV-1 might also provide some essential information regarding the new pathogen [151,152].

Considering the paucity of omics-level big data sets for SARS-CoV-2 currently available, existing data hubs that contain information for other coronaviruses such as UniProt, NCBI Genome Database, The Immune Epitope Database and Analysis Resource (IEDB), and The Virus Pathogen Resource (ViPR) will serve as useful resources for computational and bioinformatics research on SARS-CoV-2. Using such databases, the systems-level reconstruction of the protein-protein interaction (PPI) will enable the generation of hypotheses about the mechanism of action of SARS-CoV-2 and suggest potential drug targets. In an initial study [153], 26 of the 29 SARS-CoV-2 proteins were cloned and expressed in HEK293T kidney cells, allowing for the identification of 332 high-confidence human proteins interacting with them. Notably, this study suggested that SARS-CoV-2 interacts with innate immunity pathways. Ranking pathogens by the similarity between their interactomes and that of SARS-CoV-2 suggested West Nile virus, Mycobacterium tuberculosis, and human papillomavirus infections as the top three hits. Therefore, given the lung symptoms associated with COVID-19, the Mycobacterium tuberculosis host-pathogen interactome in particular might provide new insights to the mechanism of SARS-CoV-2 infection. Additionally, it was suggested that the envelope protein, E, could disrupt host bromodomain-containing proteins, i.e., BRD2 and BRD4, that bind to histones, and the spike protein could likely intervene in viral fusion by modulating the GOLGA7-ZDHHC5 acyl-transferase complex to increase palmitoylation, which is a post-translational modification that affects how proteins interact with membranes [154].

Another study [155] used patient-derived peripheral blood mononuclear cells (PBMCs) to identify 251 host proteins targeted by SARS-CoV-2. This study also reported that more than 200 host proteins were disrupted following infection. In particular, a network analysis showed that nsp9 and nsp10 interacted with NF-Kappa-B-Repressing Factor (NKRF), which encodes a transcriptional repressor that mediates repression of genes responsive to Nuclear Factor kappa-light-chain-enhancer of activated B-cells (NF-kB). These genes are important to pro-, and potentially also anti-, inflammatory signaling [156]. This finding could explain the exacerbation of the immune response that shapes the pathology and the high cytokine levels characteristic of COVID-19, possibly due to the chemotaxis of neutrophils mediated by IL-8 and IL-6. Finally, it was suggested [157] that the E protein of both SARS-CoV-1 and SARS-CoV-2 has a conserved Bcl-2 Homology 3 (BH3)-like motif, which could inhibit anti-apoptosis proteins, e.g., BCL2, and trigger the apoptosis of T cells. Several compounds are known to disrupt the host-pathogen protein interactome, largely through the inhibition of host proteins. Therefore, preliminary research in the proteomics of SARS-CoV-2 infection suggests that drugs modulating the protein-level interactions between virus and host might be worth investigating.

2.7 Viral Evolution and Virulence

Like that of SARS-CoV-1, the entry of SARS-CoV-2 into host cells is mediated by interactions between the viral spike glycoprotein, S, and human ACE2 (hACE2) [63,66,158,159,160,161,162,163]. Differences in how the S proteins of the two viruses interact with the hACE2 receptor could also partially account for the increased transmissibility of SARS-CoV-2. Recent studies have reported conflicting binding constants for the S-hACE2 interaction, though they have agreed that the SARS-CoV-2 S protein binds with equal, if not greater, affinity than the SARS-CoV-1 S protein does [51,63,161]. The C-terminal domain of the SARS-CoV-2 S protein in particular was identified as the key region of the virus that interacts with hACE2, and the crystal structure of the C-terminal domain of the SARS-CoV-2 S protein in complex with hACE2 reveals stronger interaction and a higher affinity for receptor binding than that of SARS-CoV-1 [162]. Among the 14 key binding residues identified in the SARS-CoV-1 S protein, eight are conserved in SARS-CoV-2, and the remaining six are semi-conservatively substituted, potentially explaining variation in binding affinity [63,161]. Recent crystal structures have shown that the receptor-binding domain (RBD) of the SARS-CoV-2 S protein, like that of other coronaviruses, undergoes stochastic hinge-like movement that flips it from a “closed” conformation, in which key binding residues are hidden at the interface between protomers, to an “open” one [51,63]. Because the RBD plays such a critical role in viral entry, blocking its interaction with ACE2 could represent a promising therapeutic approach. Nevertheless, despite the high structural homology between the SARS-CoV-2 RBD and that of SARS-CoV-1, monoclonal antibodies targeting SARS-CoV-1-RBD failed to bind to SARS-CoV-2-RBD [51]. Promisingly, though, sera from convalescent SARS patients inhibited SARS-CoV-2 viral entry in vitro, albeit with lower efficiency than it inhibited SARS-CoV-1 [66].

Comparative genomic analysis reveals that several regions of the coronavirus genome are likely critical to virulence. The S1 domain of the spike protein, which contains the receptor binding motif, evolves more rapidly than S’s S2 domain [57,58]. However, even within the S1 domain, some regions are more conserved than others, with the receptors in S1’s N-terminal domain (S1-NTD) evolving more rapidly than those in its C-terminal domain (S1-CTD) [58]. Both S1-NTD and S1-CTD are involved in receptor binding and can function as RBDs to bind proteins and sugars [57], but RBDs in the S1-NTD typically bind to sugars, while those in the S1-CTD recognize protein receptors [47]. Viral receptors show higher affinity with protein receptors than sugar receptors [47], which suggests that positive selection on or relaxed conservation of the S1-NTD might reduce the risk of a deleterious mutation that would prevent binding. The SARS-CoV-2 S protein also contains an RRAR furin recognition site at the S1/S2 junction [51,63], setting it apart from both bat coronavirus RaTG13, with which it shares 96% genome sequence identity, and SARS-CoV-1 [12]. Such furin cleavage sites are commonly found in highly virulent influenza viruses, and as such may contribute to the heightened pathogenicity of SARS-CoV-2 [164,165]. Effective cell entry is a critical component to pathogenesis and therefore an important process to understand when examining possible therapeutics.

2.8 Mechanism of Transmission

Once a human host is infected with a virus, person-to-person viral transmission of a virus can occur through several possible mechanisms. The primary mechanisms associated with respiratory viruses are contact, droplet, and aerosol transmission [166]. Contact transmission can occur through either direct contact with a contagious person or indirect contact with active viral particles on a contaminated surface [167]. This latter mode of transmission is also called fomite transmission [168]. Viral particles can enter the body if they then come in contact with the oral, nasal, eye, or other mucus membranes [167]. Droplet transmission occurs when a contagious individual sneezes, coughs, or exhales and produces respiratory droplets that can contain a large number of viral particles [167]. Contact with these droplets can occur either through direct exposure to the droplets, such as breathing in droplets produced by a sneeze, or exposure to particles that have settled on a surface [167]. Aerosol transmission refers to much smaller particles (less than 5 micrometers) that are also produced by sneezing, coughing, or exhaling [166,167]. The small size of these particles allows them to remain suspended over a longer period of time and potentially to be moved by air currents [167]. Additionally, viral particles deposited on surfaces via large respiratory droplets can also later be aerosolized [167]. Droplet and/or contact transmission are both well-accepted modes of transmission for many viruses associated with common human illnesses, including influenza and rhinovirus [167]. The extent to which aerosol transmission contributes to the spread of respiratory viruses is less clear. In influenza A, for example, viral particles can be detected in aerosols produced by infected individuals, but the extent to which these particles drive the spread of influenza A infection remains under debate [166,167,169,170,171]. Regardless of its role in the spread of influenza A, however, aerosol transmission likely played a role in outbreaks such as the 1918 Spanish Influenza (H1N1) and 2009 “swine flu” (pH1N1) [171]. Contact, droplet, and aerosol transmission are therefore all worth evaluating when considering possible modes of transmission for a respiratory virus like SARS-CoV-2.

2.8.1 Transmission of HCoV

All three of these mechanisms have been identified as contributors to the transmission of HCoV [167], including the highly pathogenic coronaviruses SARS-CoV-1 and MERS-CoV [28,172]. Transmission of SARS-CoV-1 is thought to proceed primarily through droplet transmission, but aerosol transmission is also considered possible [167], and fomite transmission may have also played an important role in some outbreaks [173]. Similarly, the primary mechanism of MERS transmission is thought to be droplets because inter-individual transmission appears to be associated with close interpersonal contact (e.g., household or healthcare settings), but aerosolized particles of the MERS virus have been reported to persist much more robustly than influenza A under a range of environmental conditions [174,175]. While droplet-based and contact transmission were initially considered to be the primary modes by which SARS-CoV-2 spread [176], as additional information has emerged, the possibility of aerosol transmission has also been raised [177,178,179]. For example, the detection of SARS-CoV-2 viral particles in air samples taken from hospitals treating COVID-19 patients led to the concern that the virus could be spreading via aerosols [180]. The stability of the virus both in aerosols and on a variety of surfaces appeared similar to that of SARS-CoV-1 [178], and fomite transmission could also play a role in transmission (e.g., [181]). However, while the possibility of aerosol transmission seems plausible, the evidence suggests that droplet and contact transmission are the dominant mechanisms driving the spread of the virus [182], and the risk of fomite transmission under real-world conditions is likely to be substantially lower than the conditions used for experimental analyses [183]. These mechanisms may differ in their relevance to different types of transmission events, such as transmission within households, nosocomial transmissions, and transmission in indoor versus outdoor spaces.

2.8.2 Symptoms and Viral Spread

Other aspects of pathogenesis are also important to understanding how the virus spreads, especially the relationship between symptoms, viral shedding, and contagiousness. Symptoms associated with reported cases of COVID-19 range from mild to severe [44], but some individuals who contract COVID-19 remain asymptomatic throughout the duration of the illness [184]. The incubation period, or time period between exposure and the onset of symptoms, has been estimated at five to eight days (4.91 and 7.54 in two different cities) [185,186], and estimates suggest that viral shedding may begin long before the onset of symptoms (12.3 days with a 95% CI of 5.9 - 17.0) and peak around the onset of symptoms [187]. As these trends became apparent, concerns arose due to the potential for individuals who did not or did not yet show symptoms to transmit the virus [188]. Recovered individuals may also be able to transmit the virus after their symptoms cease. Estimates of the communicable period based on twenty-four individuals who tested positive for SARS-CoV-2 prior to or without developing symptoms estimated that individuals may be contagious for one to twenty-one days, but they note that this estimate may be low [184]. Initially, viral nucleic acids were reported to remain at observable levels in the respiratory specimens of recovering hospitalized COVID-19 patients for a median of 20 days and with a maximum observed duration through 37 days, when data collection for the study ceased [35]. As more estimates of the duration of viral shedding are released, they are beginning to converge around approximately three weeks from first positive PCR test and/or onset of symptoms (which, if present, are usually identified within three days of the initial PCR test). For example, viral shedding was reported for up to 28 days following symptom onset by a second study [189] and for one to 24 days from first positive PCR test with a median of 12 days [79]. On the other hand, almost 70% of patients were reported to still have symptoms at the time that viral shedding ceased, although all symptoms reduced in prevalence between onset and cessation of viral shedding (CVS) [190]. They also reported that the median time that elapsed between the onset of symptoms and CVS was 23 days and between first positive PCR test and CVS was 17 days [190]. The fact that this study reported symptom onset to predate the first positive PCR test by an average of three days, however, suggests that there may be some methodological differences between it and related studies. Furthermore, an analysis of residents of a nursing home with a known SARS-CoV-2 case measured similar viral load in residents who were asymptomatic regardless of whether they later developed symptoms, and the load in the asymptomatic residents was comparable to that of residents who displayed either typical of atypical symptoms [191]. Taken together, these results suggest that the presence or absence of symptoms are not reliable predictors of viral shedding or of SARS-CoV-2 status (e.g, [192]). However, viral shedding is not necessarily indicative of contagiousness. The risk of spreading the infection was low after ten days from the onset of symptoms, as viral load in sputum was found to be unlikely to pose a significant risk based on their efforts to culture samples in vitro [189].

The extent to which asymptomatic (or presymptomatic) individuals are able to transmit SARS-CoV-2 has been a question of both scientific and community interest. Early reports (February and March 2020) described transmission from presymptomatic SARS-CoV-2-positive individuals to close family contacts [193,194]. One of these reports [194] also included a description of an individual who tested positive for SARS-CoV-2 but never developed symptoms. Later analyses also sought to estimate the proportion of infections that could be traced back to a presymptomatic or asymptomatic individual (e.g., [195]). Estimates of the proportion of individuals with asymptomatic infections have varied widely. The proportion of asymptomatic individuals on board the Diamond Princess cruise ship, which was the site of an early COVID-19 outbreak, was estimated at 17.9% [196]. In contrast, a model using the prevalence of antibodies among residents of Wuhan, China estimated a much higher of asymptomatic cases, at approximately 7 in 8, or 87.5% [197]. An estimate of the population of care homes in London found that, among the residents (median age 85), the rate of asymptomatic infection was 43.8%, and among the caretakers (median age 47), the rate was 49.1% [198]. The duration of viral shedding may also be longer in individuals with asymptomatic cases of COVID-19 compared to those who do show symptoms [199]. As a result, the potential for individuals who do not know they have COVID-19 to spread the virus raises significant concerns. In Singapore and Tianjin, two cities studied to estimate incubation period, an estimated 40-50% and 60-80% of cases, respectively, were estimated to be caused by contact with an asymptomatic individuals [185]. An analysis of viral spread in the Italian town of Vo’, which was the site of an early COVID-19 outbreak, revealed that 42.5% of cases were asymptomatic and that the rate was similar across age groups [200]. They argued that the town’s lockdown was imperative for controlling the spread of COVID-19 because it isolated the asymptomatic individuals. While more models are likely to emerge to better explore the effect of asymptomatic individuals on SARS-CoV-2 transmission, these results suggest that strategies for identifying and containing asymptomatic, but contagious individuals are important for managing community spread.

2.8.3 Estimating the Fatality Rate

Estimating the occurrence of asymptomatic and mild COVID-19 cases is important to identifying the mortality rate associated with COVID-19. The mortality rate of greatest interest would be the total number of fatalities as a fraction of the total number of people infected. One metric reported is often case fatality rate (CFR), which simply compares the number of COVID-19 related deaths to the number of confirmed or suspected cases. However, in locations without universal testing protocols, it is impossible to identify all exposed or all infected individuals because so many asymptomatic or mild cases go undetected. Therefore, a more informative metric is the infection fatality rate (IFR), which compares the known deaths to the estimated number of cases. Meta-analyses have produced estimates of global IFR ranging from as low as 0.1% to as high as 1.04% [201,201,202,203], and this estimate was also supported by a repeated cross-sectional serosurvey conducted in New York City that revealed an estimated IFR of 0.97% [204]. All of these estimates note that IFR varies widely around the world. Estimates of infection rates are becoming more feasible as more data becomes available for modeling and will be bolstered as serological testing becomes more common and available.

2.9 Dynamics of Transmission

Disease spread dynamics can be estimated using R0, the basic reproduction number, and Rt, the effective reproduction number. Thus, accurate estimates are crucial to understanding the dynamics of infection and to predicting the effects of different interventions. R0 and the timescale of infection (measured by the infectious period and the exposed period) govern population-level epidemic dynamics, with R0 being one of the most important epidemiological parameters [205]. R0 is the average number of new (secondary) infections caused by one infected person, assuming a wholly susceptible population [206]. A simple mechanistic model used to describe infectious disease dynamics is a susceptible-infected-recovered (SIR) compartmental model. In this formulation individuals move through three states: susceptible, infected, and recovered; two parameters, \(\gamma\) and \(\beta\), specify the rate at which the infectious recover, and the infection transmission rate, respectively. In this simple formulation, R0 is estimated as the ratio of \(\beta\) and \(\gamma\).[205,207]. A pathogen can invade a susceptible population only if R0 > 1 [205,208]. The spread of an infectious disease at a particular time t can be quantified by Rt, the effective reproduction number, which assumes that part of the population has already recovered (and thus gained immunity to reinfection) or that mitigating interventions were put into place. For example, if only a fraction St of the population is still susceptible, Rt = St x R0. When Rt is greater than 1, an epidemic grows (i.e., the proportion of the population that is infectious increases); when Rt is less than 1, the proportion of the population that is infectious decreases. R0 and Rt can be estimated directly from epidemiological data or inferred using mathematical modeling. Modeling approaches are typically based upon a classic epidemiological model structure: the SIR model and its extensions [209,210]. In the context of SARS-CoV-2, more complex modified susceptible-exposed-infectious-recovered (SEIR) models are commonly used.

Estimates of R0 for COVID-19 lie in the range R0=1.4-6.5 [211,212,213]. Variation in R0 is expected between different populations, and the estimated values of R0 discussed below are for specific populations in specific environments. The different estimates of R0 should not necessarily be interpreted as a range of estimates of the same underlying parameter. In one study of international cases, the predicted value was R0=1.7 [214]. In China (both Hubei province and nationwide), the value was predicted to lie in the range R0=2.0-3.6 [211,215,216]. Another estimate based on a cruise ship where an outbreak occurred predicted R0=2.28 [217]. SEIR model-derived estimates of R0 range from 2.0 - 6.5 in China [218,219,220,221] to R0=4.8 in France [222]. Using the same model as for the French population, a study estimated R0=2.6 in South Korea [222], which is consistent with other studies [223]. From a meta-analysis of studies estimating R0, [212] predict the median as R0=2.79.

Inference of the effective reproduction number can provide insight into how populations respond to an infection and the effectiveness of interventions. In China, Rt was predicted to lie in the range 1.6-2.6 in January 2020, before travel restrictions [224]. Rt decreased from 2.35 one week before travel restrictions were imposed (January 23, 2020), to 1.05 one week after. Using their model, the authors also estimated the probability of new outbreaks occurring: the probability of a single individual exporting the virus and causing a large outbreak is 17-25%, assuming MERS-like or SARS-like transmission, and the probability of a large outbreak occurring after ≥4 infections exist at a new location is greater than 50%. An independent study came to similar conclusions, finding Rt=2.38 in the two-week period before January 23 with a decrease to Rt = 1.34 (using data from January 24 to February 3) or Rt=0.98 (using data from January 24 to February 8) [213]. In South Korea, Rt was inferred for February through March 2020 in two cities, Daegu (the center of the outbreak) and Seoul [223]. Metro data was also analyzed to estimate the effects of social distancing measures. Rt decreased in Daegu from around 3 to <1 over the period that social distancing measures were introduced. In Seoul, Rt decreased slightly, but remained close to 1 (and larger than Rt in Daegu). These findings indicate that social distancing measures appeared to be effective in containing the infection in Daegu, but in Seoul, Rt remained above 1, meaning secondary outbreaks remained possible. The study also shows the importance of region-specific analysis: the large decline in case load nationwide was mainly due to the Daegu region and could mask persistence of the epidemic in other regions, such as Seoul and Gyeonggi-do. In Iran, estimates of Rt declined from 4.86 in the first week to 2.1 by the fourth week after the first cases were reported [225]. In Europe, analysis of 11 countries inferred the dynamics of Rt over a time range from the beginning of the outbreak until March 28, 2020, by which point most countries had implemented major interventions (such as school closures, public gathering bans, and stay-at-home orders) [226]. Across all countries, the mean Rt before interventions began was estimated as 3.87; Rt varied considerably, from below 3 in Norway to above 4.5 in Spain. After interventions, Rt decreased by an average of 64% across all countries, with mean Rt=1.43. The lowest predicted value was 0.97 for Norway and the highest was 2.64 for Sweden, which may be in part because Sweden did not implement social distancing measures on the same scale as other countries. The study concludes that while large changes in Rt are observed, it is too early to tell whether the interventions put into place are sufficient to decrease Rt below 1.

More generally, population-level epidemic dynamics can be both observed and modelled [207]. Data and empirically determined biological mechanisms inform models, while models can be used to try to understand data and systems of interest or to make predictions about possible future dynamics, such as the estimation of capacity needs [227] or the comparison of predicted outcomes among prevention and control strategies [228,229]. Many current efforts to model Rt have led to tools that assist the visualization of estimates in real time or over recent intervals [230,231]. While these may be valuable resources, it is important to note that the estimates arise from models containing many assumptions and are dependent on the quality of the data they use, which varies widely by region.

2.10 Molecular Signatures and Transmission

Genetic variation in SARS-CoV-2 has been used to elucidate patterns over time and space. Mutations observed in individual SARS-CoV-2 genome sequences can be used to trace transmission patterns and have provided insights during outbreak investigations [14,232,233]. Similar mutations observed in several patients may indicate that the patients belong to the same transmission group. The tracking of SARS-CoV-2 mutations is recognized as an essential tool for controlling outbreaks that may facilitate tracing the paths of SARS-CoV-2’s spread [234]. Several studies used phylogenetic analysis to determine the source of local COVID-19 outbreaks in Connecticut (USA), [235], the New York City area (USA) [236], and Iceland [237]. There is an ongoing effort to collect SARS-CoV-2 genomes throughout the COVID-19 outbreak, and as of January 18, 2021 more than 381,000 genome sequences have been collected from patients. The sequencing data can be found at GISAID [238], NCBI [239], and COVID-19 data portal [240].

2.11 Conclusions

The novel coronavirus SARS-CoV-2 is the third HCoV to emerge in the 21st century, and research into previous HCoVs has provided a strong foundation for characterizing the pathogenesis and transmission of SARS-CoV-2. Critical insights into how the virus interacts with human cells have been gained from previous research in HCoV and other viral infections. As with other HCoV, the immune response to SARS-CoV-2 is likely driven by detection of its spike protein, which allows it to enter cells through the ACE2 receptor. Epithelial cells have also emerged as the major cellular target of the virus, contextualizing the respiratory and gastrointestinal symptoms that are frequently observed in COVID-19. Many of the mechanisms that facilitate the pathogenesis of SARS-CoV-2 are currently under consideration as possible targets for the treatment or prevention of COVID-19. Research in other viruses also provides a foundation for understanding the transmission of SARS-CoV-2 among people and can therefore inform efforts to control the virus’s spread. Though it remains a question whether aerosol and fomite transmission contribute to the spread of SARS-CoV-2 in real-world settings, in general, much like SARS-CoV-1 and MERS-CoV, this virus seems to be spread primarily by droplet transmission. Asymptomatic transmission was also a concern in the SARS outbreak of 2002-03 and, as in current pandemic, presented challenges for estimating rates of infection [241]. However, in 2021, we are fortunate to be able to build on top of 18 years of SARS-CoV research in order to rapidly ascertain the identity and behavior of the virus.

COVID-19 is a complex disease with many possible presentations that appear to vary across the lifespan. Variability in presentation, including cases with no respiratory symptoms or with no symptoms altogether, were also reported during the SARS epidemic at the beginning of the 21st century [241]. The variability of both which symptoms present and their severity have presented challenges for public health agencies seeking to provide clear recommendations regarding which symptoms indicate SARS-CoV-2 infection and should prompt isolation. Additionally, asymptomatic cases add complexity both to efforts to estimate critical statistics such as R0 and Rt, which are critical to understanding the transmission of the virus, and IFR, which is an important component of understanding its impact on a given population. The development of diagnostic technologies over the course of the pandemic have facilitated more accurate identification, including of asymptomatic cases. As more cases have been diagnosed, the health conditions and patient characteristics associated with more severe infection have also become more clear, although there are likely to be significant sociocultural elements that also influence these outcomes. While many efforts have focused on adults, and especially older adults because of the susceptibility of this demographic, additional research is needed to understand the presentation of COVID-19 and MIS-C in pediatric patients. As more information is uncovered about the pathogenesis of HCoV and SARS-CoV-2 specifically, the diverse symptomology of COVID-19 has and likely will continue to conform with the ever-broadening understanding of how SARS-CoV-2 functions within a human host.

While the SARS-CoV-2 virus is very similar to other HCoV in several ways, including in its genomic structure and the structure of the virus itself, there are also some differences that may account for differences in the COVID-19 pandemic compared to the SARS and MERS of the past two decades. The R0 of SARS-CoV-2 has been estimated to be similar to SARS-CoV-1 but much higher than that of MERS-CoV [30,30]. While the structures of the viruses are very similar, evolution among these species may account for differences in their transmissibility and virulence. For example, the acquisition of a furin cleavage site the S1/S2 boundary within the SARS-CoV-2 S protein may be associated with increased virulence. Additionally, concerns have been raised about the accumulation of mutations within the SARS-CoV-2 species itself, and whether these could influence virulence. The coming of age of genomic technologies has made these types of analyses feasible, and genomics research characterizing changes in SARS-CoV-2 along with temporal and spatial movement is likely to provide additional insights into whether within-species evolution influences the effect of the virus on the human host.

Additionally, omics technologies have made it feasible to characterize the host response to the virus. Although at present, much of the omics research has utilized in vitro models, such systems-level approaches represent a promising opportunity to characterize the host response to the SARS-CoV-2 virus. For example, analysis of PPI using publicly available data identified some similarities between COVID-19 and tuberculosis infection [153]. This finding suggests that insights into COVID-19 may be gained by systems-level analysis of a wide array of infections, even those that are not causes by viruses, as is the case with the bacterium M. tuberculosis. As more data is collected, large-scale, omics-based analyses will become more feasible.

Thus, though the COVID-19 crisis is still evolving, the insights acquired over the past 20 years of HCoV research have provided a solid foundation for understanding the SARS-CoV-2 virus and the disease it causes. As the scientific community continues to respond to COVID-19 and to elucidate more of the relationships between viral pathogenesis, transmission, and symptomology, and as more data about the regulatory shifts associated with COVID-19 becomes available, this understanding will no doubt continue to develop to reveal additional interdisciplinary connections among virology, pathogenesis, and health. As additional information becomes available, this review will be updated to reflect the changing state of research in this area. At present, understanding the SARS-CoV-2 virus and its pathogenesis is critical to a holistic understanding of the COVID-19 pandemic.

3 Evolutionary and Genomic Analysis of SARS-CoV-2

3.1 Abstract

3.2 Introduction

3.3 Initial Characterization of SARS-CoV-2

The first genome sequence of the virus was released on January 3, 2020 and revealed that the cluster of pneumonia cases seen in Wuhan were caused by a novel coronavirus [10]. Multiple research groups have drafted the genome sequence of SARS-CoV-2 based on sequences developed from clinical samples collected from the lower respiratory tract, namely bronchoalveolar lavage fluid (BALF), and the upper respiratory tract, in the form of throat swabs [11,12,13]. Analysis of the SARS-CoV-2 genome revealed significant sequence homology with two coronaviruses known to infect humans, with about 79% identity to SARS-CoV-1 and 50% to MERS-CoV [13]. However, in this analysis, the highest degree of similarity was observed between SARS-CoV-2 and bat-derived SARS-like coronaviruses (bat-SL-CoVZC45 and bat-SL-CoVZXC21) [13]. Other analyses have reported even greater similarity between SARS-CoV-2 and the bat coronavirus BatCoV-RaTG13, with identity as high as 96.2% [12,14], and the closely related pangolin coronavirus [15]. This evidence therefore suggests the SARS-CoV-2 virus is the result of zoonotic transfer of a virus from bats to humans. Nevertheless, some fragments between SARS-CoV-2 and RATG13 differ by up to 17%, suggesting a complex natural selection process during zoonotic transfer. While the S region is highly similar to that of viruses found in pangolins [15], there is no consensus about the origin of S in SARS-CoV-2, as it could potentially be the result either of recombination or coevolution [14,16]. Though the intermediate host serving as the source for the zoonotic introduction of SARS-CoV-2 to human populations has not yet been identified, the SARS-CoV-2 virus has been placed within the coronavirus phylogeny through comparative genomic analyses. Genomic analyses and comparisons to other known coronaviruses suggest that SARS-CoV-2 is unlikely to have originated in a laboratory – either purposely engineered and released, or escaped – and instead evolved naturally in an animal host [17]. While the position of the SARS-CoV-2 virus within the coronavirus phylogeny has been largely resolved, the functional consequences of molecular variation between this virus and other viruses, such as its bat and pangolin sister taxa or SARS-CoV-1, remain unknown [17]. Fortunately, the basic genome structure of coronaviruses is highly conserved, and insight into the mechanisms the virus uses to enter cells, replicate, and spread is likely to be available from prior research in coronaviruses.

Additionally, worldwide sequencing of viral samples has provided some preliminary insights into possible mechanisms of adaptation in the virus, and omics-based analysis of patient samples has elucidated some of the biological changes the virus induces in its human hosts.

3.4 Coronaviruses and Animal Hosts

Coronaviruses have long been known to infect animals and have been the subject of veterinary medical investigations and vaccine development efforts due to their effect on the health of companion and agricultural animals [18].

Most coronaviruses show little to no transmission in humans. However, today it is thought that approximately one-third of common cold infections are caused by four human coronaviruses (HCoV): Human coronavirus 229E (HCoV-229E), Human coronavirus NL63 (HCoV-NL63), Human coronavirus OC43 (HCoV-OC43), and Human coronavirus HKU1 (HCoV-HKU1) [19,20]. The first HCoV were identified in the 1960s: HCoV-229E in 1965 [21] and HCoV-OC43 in 1967 [22]. Both of these viruses typically cause cold-like symptoms, including upper and lower respiratory infections [23,24,242], but they have also been associated with gastrointestinal symptoms [243]. Two additional HCoV were subsequently identified [25,26]. In 2003, HCoV-NL63 [25] was first identified in a 7-month-old infant and then in clinical specimens collected from seven additional patients, five of whom were infants younger than 1 year old and the remainder of whom were adults. CoV-HKU1 was identified in samples collected from a 71-year-old pneumonia patient in 2004 and then found in samples collected from a second adult patient [26]. These viruses are associated with respiratory diseases of varying severity, ranging from common cold to severe pneumonia, with severe symptoms mostly observed in immunocompromised individuals [27], and also have gastrointestinal involvement in some cases [243]. In addition to these relatively mild HCoV, however, highly pathogenic human coronaviruses have been identified, including Severe acute respiratory syndrome-related coronavirus (SARS-CoV or SARS-CoV-1) and Middle East respiratory syndrome-related coronavirus (MERS-CoV) [19,28,29].

At the time that SARS-CoV-1 emerged in the early 2000s, no HCoV had been identified in almost 40 years [28]. The first case of SARS was reported in November 2002 in the Guangdong Province of China, and over the following month, the disease spread more widely within China and then into several countries across multiple continents [28,30]. Unlike previously identified HCoV, SARS was much more severe, with an estimated death rate of 9.5% [30]. It was also highly contagious via droplet transmission, with a basic reproduction number (R0) of 4 (i.e., each person infected was estimated to infect four other people) [30]. However, the identity of the virus behind the infection remained unknown until April of 2003, when the SARS-CoV-1 virus was identified through a worldwide scientific effort spearheaded by the WHO [28]. SARS-CoV-1 belonged to a distinct lineage from the two other HCoV known at the time [30]. By July 2003, the SARS outbreak was officially determined to be under control, with the success credited to infection management practices [28]. A decade later, a second outbreak of severe respiratory illness associated with a coronavirus emerged, this time in the Arabian Peninsula. This disease, known as Middle East respiratory syndrome (MERS), was linked to another novel coronavirus, MERS-CoV. The fatality rate associated with MERS is much higher than that of SARS, at almost 35%, but the disease is much less easily transmitted, with an R0 of 1 [30]. Although MERS is still circulating, its low reproduction number has allowed for its spread to be contained [30]. The COVID-19 pandemic is thus associated with the seventh HCoV to be identified and the fifth since the turn of the millennium, though additional HCoVs may be in circulation but remain undetected.

SARS-CoV-1 and MERS-CoV were ultimately managed largely through infection management practices (e.g., mask wearing) and properties of the virus itself (i.e., low rate of transmission), respectively [28,30]. Vaccines were not used to control either virus, although vaccine development programs were established for SARS-CoV-1 [31]. In general, care for SARS and MERS patients focuses on supportive care and symptom management [30]. Clinical treatments for SARS and MERS developed during the outbreaks generally do not have strong evidence supporting their use. Common treatments included Ribavirin, an antiviral, often in combination with corticosteroids or sometimes interferon (IFN) medications, which would both be expected to have immunomodulatory effects [28]. However, retrospective and in vitro analyses have reported inconclusive results of these treatments on SARS and the SARS-CoV-1 virus, respectively [28]. IFNs and Ribavirin have shown promise in in vitro analyses of MERS, but their clinical effectiveness remains unknown [28]. Therefore, only limited strategy for the pharmaceutical management of COVID-19 can be adopted from previous severe HCoV infections. Research in response to prior outbreaks of HCoV-borne infections, such as SARS and MERS, have, however, provided a strong foundation for hypotheses about the pathogenesis of SARS-CoV-2 as well as potential diagnostic and therapeutic approaches.

3.5 Zoonotic Transfer of Coronaviruses

3.6 Evolution of the SARS-CoV-2 Virus

3.6.1 Emergence of SARS-CoV-2

3.6.2 Evolution of SARS-CoV-2 Variants

Evolution in SARS-CoV-2 has also been observed over a short timescale. After zoonotic transfer, SARS-CoV-2 continued evolving in the human population [232]. The SARS-CoV-2 mutation rate is moderate compared to other RNA viruses [233], which likely restricts the pace of evolution in SARS-CoV-2. Nevertheless, genomic analyses have yielded statistical evidence of ongoing evolution. There are two known variants of the spike protein that differ by a single amino acid at position 614 (G614 and D614), and there is evidence that G614 had become more prevalent than D614 by June 2020 [244]. While there is a hypothesis that this genomic change increased the SARS-CoV-2 infectivity and virulence, this hypothesis has not yet been tested due to a lack of data [245]. Another study [233] identified 198 recurrent mutations in a dataset of 7,666 curated sequences. This pattern of convergent evolution at some sites could indicate that certain mutations confer an adaptive advantage. While it is evident that SARS-CoV-2 exhibits moderate potential for ongoing and future evolution, the relationship between mutations and pathogenicity is not yet known. Additional data is needed in order to understand patterns of evolutionary change and whether they are likely to affect virulence.

3.7 Conclusions

As of October 2020 the SARS-CoV-2 virus remains a serious worldwide threat. The scientific community has responded by rapidly collecting and disseminating information about the SARS-CoV-2 virus and the associated illness, COVID-19. The rapid identification of the genomic sequence of the virus allowed for early contextualization of SARS-CoV-2 among other known respiratory viruses. The pathogen is a coronavirus that is closely related to SARS-CoV-1, which caused the SARS pandemics of the early 2000s. Knowing the phylogenetic context and genomic sequence of the virus then allowed for rapid insights into its structure and pathogenesis.

4 Diagnostics

4.1 Abstract

4.2 Introduction

Identifying individuals who have contracted COVID-19 is crucial to slowing down the global pandemic. Given the high transmissibility of SARS-CoV-2, the development of reliable assays to detect SARS-CoV-2 infection even in asymptomatic carriers is vitally important. For instance, the deployment of wide-scale diagnostic testing followed by the isolation of infected people has been a key factor in South Korea’s successful strategy for controlling the spread of the virus. Following the first release of the genetic sequence of the virus by Chinese officials on January 10, 2020, the first test was released about 13 days later [246]. Diagnostic approaches utilizing a variety of methods are currently or have been developed. There are two main classes of diagnostic tests: molecular tests, which can diagnose an active infection by identifying the presence of SARS-CoV-2, and serological tests, which can assess whether an individual was infected in the past via the presence or absence of antibodies against SARS-CoV-2. Molecular tests are essential for identifying individuals for treatment and alerting their contacts to quarantine and be alert for possible symptoms. While serological tests may be of interest to individuals who wish to confirm they were infected with SARS-CoV-2 in the past, their potential for false positives means that they are not currently recommended for this use. However, serological tests provide population-level information for epidemiological analysis, as they can be used to estimate the extent of the infection in a given area. Thus, they may be useful in efforts to better understand the percent of cases that manifest as severe versus mild and for guiding public health and economic decisions regarding resource allocation and counter-disease measures.

Understanding the fundamental organization of the human immune response to viral threats is critical to understanding the varied response to SARS-CoV-2. The human immune system utilizes a variety of innate and adaptive responses to protect against the pathogens it encounters. The innate immune system consists of barriers, such as the skin, mucous secretions, neutrophils, macrophages, and dendritic cells. It also includes cell-surface receptors that can recognize the molecular patterns of pathogens. The adaptive immune system utilizes antigen-specific receptors that are expressed on B and T lymphocytes. These components of the immune system typically act together; the innate response acts first, and the adaptive response begins to act several days after initial infection following the clonal expansion of T and B cells [32]. After a virus enters into a host cell, its antigen is presented by major histocompatibility complex 1 (MHC 1) molecules and is then recognized by cytotoxic T lymphocytes.

In the case of COVID-19, there is also concern about the immune system becoming over-active. One of the main immune responses contributing to the onset of acute respiratory distress syndrome (ARDS) in COVID-19 patients is cytokine storm syndrome (CSS), which causes an extreme inflammatory response due to a release of pro-inflammatory cytokines and chemokines by immune effector cells. In addition to respiratory distress, this mechanism can lead to organ failure and death in severe COVID-19 cases [33].

4.3 Molecular Tests

Molecular tests are used to identify distinct genomic subsequences of a viral molecule in a sample and thus to diagnose an active viral infection. This first requires identifying which biospecimens are likely to contain the virus during infection and then acquiring these samples from the patient(s) to be tested. Common sampling sources for molecular tests include nasopharyngeal cavity samples, such as throat wash and saliva [247], and stool samples [248]. Once a sample is acquired from a patient, molecular testing will utilize a number of steps, described below, to analyze a sample and identify whether evidence of SARS-CoV-2 is present. When testing for RNA viruses like SARS-CoV-2, pre-processing is necessary in order to create DNA from the RNA sample. The DNA can then be amplified with PCR. Some tests use the results of the PCR itself to determine whether the pathogen is present, but in other cases, it may be necessary to sequence the amplified DNA. Sequencing requires an additional pre-processing step: library preparation. Library preparation is the process of preparing the sample for sequencing, typically by fragmenting the sequences and adding adapters [249]. In some cases, library preparation can involve other modifications of the sample, such as adding “barcoding” to identify a particular sample in the sequence data, which is useful for pooling samples from multiple sources. There are different reagents used for library preparation that are specific to identifying one or more target sections with PCR [250]. Sequential pattern matching is then used to identify unique subsequences of the virus that identify it in specific. If sufficient subsequences are found, the test is considered positive.

4.3.1 RT-PCR

Real-Time Polymerase Chain Reaction (RT-PCR) tests determine whether a target is present by measuring the rate of amplification during PCR compared to a standard. When the target is RNA, such as in the case of RNA viruses, the RNA must be converted into complementary DNA during pre-processing. The Drosten Lab, from Germany, was the first lab to establish and validate a diagnostic test to detect SARS-CoV-2. This test uses RT-PCR with reverse transcription [246] to detect several regions of the viral genome: the ORF1b of the RNA-dependent RNA polymerase (RdRP), the Envelope protein gene (E), and the Nucleocapsid protein gene (N). In collaboration with several other labs in Europe and in China, the researchers confirmed the specificity of this test with respect to other coronaviruses against specimens from 297 patients infected with a broad range of respiratory agents. Specifically this test utilizes two probes against RdRP, one of which is specific to SARS-CoV-2 [246]. Importantly, this assay was not found to return false positive results.

4.3.2 qRT-PCR

Chinese researchers developed a quantitative real-time reverse transcription PCR (qRT-PCR) test to identify two gene regions of the viral genome, ORF1b and N [251]. This assay was tested on samples coming two COVID-19 patients and a panel of positive and negative controls consisting of RNA extracted from several cultured viruses. The assay uses the N gene to screen patients, while the ORF1b gene region is used to confirm the infection [251]. In this case the test was designed to detect sequences conserved across sarbecoviruses, or viruses within the same subgenus as SARS-CoV-2. Considering that SARS-CoV-1 and SARS-CoV-2 are the only sarbecoviruses currently known to infect humans, a positive test can be assumed to indicate that the patient is infected with SARS-CoV-2. However, this test is not able to discriminate the genetics of viruses within the sarbecovirus clade.

4.3.2.1 dPCR

Digital PCR (dPCR) is a new generation of PCR technologies offering an alternative to traditional real-time quantitative PCR. In dPCR, a sample is partitioned into thousands of compartments, such as nanodroplets (droplet dPCR or ddPCR) or nanowells, and a PCR reaction takes place in each compartment. This design allows for a digital read-out where each partition is either positive or negative for the nucleic acid sequence being tested for, allowing for much higher throughput. While dPCR equipment is not yet as common as that for RT-PCR, dPCR for DNA targets generally achieves higher sensitivity than other PCR technologies while maintaining high specificity, though sensitivity is slightly lower for RNA targets [252]. High sensitivity is particularly relevant for SARS-CoV-2 detection, since low viral load in clinical samples can lead to false negatives. Suo et al. [253] performed a double-blind evaluation of ddPCR for SARS-CoV-2 detection on 57 samples, comprised by 43 samples from suspected positive patients and 14 from supposed convalescents, that had all tested negative for SARS-CoV-2 using RT-PCR. Despite the initial negative results, 33 out of 35 (94.3%) patients were later clinically confirmed positive. All of these individuals tested positive using ddPCR. Additionally, of 14 supposed convalescents who had received two consecutive negative RT-PCR tests, nine (64.2%) tested positive for SARS-CoV-2 using ddPCR. Two symptomatic patients tested negative with both RT-PCR and ddPCR, but were later clinically diagnosed positive, and 5 of the 14 suspected convalescents tested negative by ddPCR. While this study did not provide a complete head-to-head comparison to RT-PCR in all aspects, e.g., no samples testing positive using RT-PCR were evaluated by ddPCR, the study shows the potential of dPCR for viral detection even in highly diluted samples. In a second study, Dong et al. [254] compared the results of qRT-PCR and ddPCR testing for SARS-CoV-2 in 194 samples, including 103 samples from suspected patients, 75 from contacts and close contacts, and 16 from suspected convalescents. Of the 103 suspected patient samples, 29 were reported as positive, 25 as negative, and 49 as suspected by qRT-PCR; all patients were later confirmed to be SARS-CoV-2 positive. Of the qRT-PCR negative or suspected samples, a total of 61 (17 negative and 44 suspected) were later confirmed to be positive by ddPCR, improving the overall detection rate among these patients from 28.2% to 87.4%. Of 75 patient samples from contacts and close contacts, 48 tested negative with both methods, and these patients were observed to remain healthy. Within the remaining 27 patient samples, 10 tested positive, 1 negative, and 16 suspect with qRT-PCR. Fifteen out of 16 suspect samples and the negative test results were overturned by ddPCR, decreasing the rate of suspect cases from 21% to 1%. Importantly, all samples that tested positive using qRT-PCR also tested positive using ddPCR. Among the 16 convalescent patients, qRT-PCR identified 12 as positive, three as suspect, and one as negative, but RT-dPCR identified all 16 as positive. This evidence further indicates that the lower limit of detection made possible by ddPCR may be useful for identifying when COVID-19 patients are cleared of the virus. Overall, these studies suggest that dPCR is a promising tool for overcoming the problem of false-negative SARS-CoV-2 testing.

4.3.3 Pooled and Automated PCR Testing

Due to limited supplies and the need for more tests, several labs have found ways to pool or otherwise strategically design tests to increase throughput. The first such result came from Yelin et al. [255], who found they could pool up to 32 samples in a single qPCR run. This was followed by larger-scale pooling with slightly different methods [256]. Although these approaches are also PCR based, they allow for more rapid scaling and higher efficiency for testing than the initial PCR-based methods developed.

4.3.3.1 CRISPR-based Detection

Two CRISPR-associated nucleases, Cas12 and Cas13, have been used for nucleic acid detection. Multiple assays exploiting these nucleases have emerged as potential diagnostic tools for the rapid detection of SARS-CoV-2 genetic material and therefore SARS-CoV-2 infection. The SHERLOCK method (Specific High-sensitivity Enzymatic Reporter unLOCKing) from Sherlock Biosciences relies on Cas13a to discriminate between inputs that differ by a single nucleotide at very low concentrations [257]. The target RNA is amplified by RT-RPA and T7 transcription, and the amplified product activates Cas13a. The nuclease then cleaves a reporter RNA, which liberates a fluorescent dye from a quencher. Several groups have used the SHERLOCK method to detect SARS-CoV-2 viral RNA. An early study reported that the method could detect 7.5 copies of viral RNA in all 10 replicates, 2.5 copies in 6 out of 10, and 1.25 copies in 2 out of 10 runs [258]. It also reported 100% specificity and sensitivity on 114 RNA samples from clinical respiratory samples (61 suspected cases, among which 52 were confirmed and nine were ruled out by metagenomic next-generation sequencing, 17 nCoV-/HCoV+ cases and 36 samples from healthy subjects), and a reaction turnaround time of 40 minutes. A separate study screened four designs of SHERLOCK and extensively tested the best-performing assay. They determined the limit of detection to be 10 copies/μl using both fluorescent and lateral flow detection [259]. Lateral flow test strips are simple to use and read, but there are limitations in terms of availability and cost per test. Another group therefore proposed the CREST protocol (Cas13-based, Rugged, Equitable, Scalable Testing), which uses a P51 cardboard fluorescence visualizer, powered by a 9V battery, for the detection of Cas13 activity instead of immunochromatography [260]. CREST can be run, from RNA sample to result, with no need for AC power or a dedicated facility, with minimal handling in approximately 2 hours. Testing was performed on 14 nasopharyngeal swabs. CREST picked up the same positives as the CDC-recommended TaqMan assay with the exception of one borderline sample that displayed low-quality RNA.

The DETECTR method (DNA Endonuclease-Targeted CRISPR Trans Reporter) from Mammoth Biosciences involves purification of RNA extracted from patient specimens, amplification of extracted RNAs by loop-mediated amplification, which is a rapid, isothermal nucleic acid amplification technique, and application of their Cas12-based technology. In their assay, guide RNAs were designed to recognize portions of sequences corresponding to the SARS-CoV-2 genome, specifically the N2 and E regions [261]. In the presence of SARS-CoV-2 genetic material, sequence recognition by the guide RNAs results in double-stranded DNA cleavage by Cas12, as well as cleavage of a single-stranded DNA molecular beacon. The cleavage of this molecular beacon acts as a colorimetric reporter that is subsequently read out in a lateral flow assay and indicates the positive presence of SARS-CoV-2 genetic material and therefore SARS-CoV-2 infection. The 40-minute assay is considered positive if there is detection of both the E and N genes or presumptive positive if there is detection of either of them. The assay had 95% positive predictive agreement and 100% negative predictive agreement with the US Centers for Disease Control and Prevention SARS-CoV-2 real-time RT–PCR assay. The estimated limit of detection was 10 copies per μl reaction, versus 1 copy per μl reaction for the CDC assay. These results have been confirmed by other DETECTR approaches. Using RTRPA for amplification, another group detected 10 copies of synthetic SARS-CoV-2 RNA per μl of input within 60 minutes of RNA sample preparation in a proof-of-principle evaluation [262]. The DETECTR protocol was improved by combining RT-RPA and CRISPR-based detection in a one-pot reaction that incubates at a single temperature, and by using dual crRNAs (which increases sensitivity). This new assay, known as All-In-One Dual CRISPR-Cas12a (AIOD-CRISPR), detected 4.6 copies of SARS-CoV-2 RNA per μl of input in 40 minutes [263]. Another single-tube, constant-temperature approach using Cas12b instead of Cas12a achieved a detection limit of 5 copies/μl in 40-60 minutes [264]. It was also reported that that electric field gradients can be used to control and accelerate CRISPR assays by co-focusing Cas12-gRNA, reporters, and target [265]. The authors generated an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. They also used ITP for automated purification of target RNA from raw nasopharyngeal swab samples. Combining this ITP purification with loop-mediated isothermal amplification, their ITP-enhanced assay to achieved detection of SARS-CoV-2 RNA (from raw sample to result) in 30 minutes.

There is an increasing body of evidence that CRISPR-based assays offer a practical solution for rapid, low-barrier testing in areas that are at greater risk of infection, such as airports and local community hospitals. In the largest study to date, DETECTR was compared to qRT-PCR on 378 patient samples [266]. The authors reported a 95% reproducibility. Both techniques were equally sensitive in detecting SARS-CoV-2. Lateral flow strips showed a 100% correlation to the high-throughput DETECTR assay. Importantly, DETECTR was 100% specific for SARS-CoV-2 and did not detect other human coronaviruses.

4.3.4 Limitations of Molecular Tests

Tests that identify SARS-CoV-2 using nucleic-acid-based technologies will identify only individuals with current infections and are not appropriate for identifying individuals who have recovered from a previous infection. Within this category, different types of tests have different limitations. For example, PCR-based test can be highly sensitive, but in high-throughput settings they can show several problems:

  1. False-negative responses, which can present a significant problem to large-scale testing. To reduce occurrence of false negatives, correct execution of the analysis is crucial [267].
  2. Uncertainty surrounding the SARS-CoV-2 viral shedding kinetics, which could affect the result of a test depending on when it was taken [267].
  3. Type of specimen, as it is not clear which clinical samples are best for detecting the virus [267].
  4. Expensive machinery, which might be present in major hospitals and/or diagnostic centers but is often not available to smaller facilities [268].
  5. Timing of the test, which might take up to 4 days to return results [268].
  6. The availability of supplies for testing, including swabs and testing media, has been limited [269].
  7. Because the guide RNA can recognize other interspersed sequences on the patient’s genome, false positives and a loss of specificity can occur.

Similarly, in tests that use CRISPR, false positives can occur due to the specificity of the technique, as the guide RNA can recognize other interspersed sequences on the patient’s genome. As noted above, false negatives are a significant concern for several reason. Importantly, clinical reports indicate that it is imperative to exercise caution when interpreting the results of molecular tests for SARS-CoV-2 because negative results do not necessarily mean a patient is virus-free [270].

4.4 Serological Tests

Although diagnostic tests based on the detection of genetic material can be quite sensitive, they cannot provide information about the extent of the disease over time. Most importantly, they would not work on a patient who has fully recovered from the virus at the time of sample collection. In this context, serological tests, which use serum to test for the presence of antibodies against SARS-CoV-2, are significantly more informative. Additionally, serological tests can help scientists to understand why the disease has a different course among patients, as well as which strategies might work to manage the spread of the infection. Furthermore, serological tests hold significant interest because of the possibility that they could provide information relevant to advancing economic recovery and allowing reopenings. For instance, people that had developed antibodies might be able to return to work, assuming (still-unproven) protective immunity [271]. Some infectious agents can be controlled through “herd immunity”, which is when a critical mass within the population acquires immunity through vaccination and/or infection, preventing an infectious agent from spreading widely. A simple SIR model predicts that to achieve the required level of exposure for herd immunity to be effective, at least (1-(1/R0)) fraction of the population must be immune or, equivalently, less than (1/R0) fraction of the population susceptible [208]. However, for SARS-CoV-2 and COVID-19, the R0 and mortality rates that have been observed suggest that relying on herd immunity without some combination of vaccines, proven treatment options, and strong non-pharmaceutical measures of prevention and control would likely result in a significant loss of life.

4.4.1 Sustained Immunity to COVID-19

In the process of mounting a response to a pathogen, the immune system produces antibodies specific to the pathogen. Understanding the acquisition and retention of antibodies is important both to the diagnosis of prior (inactive) infections and to the development of vaccines. The two immunoglobulin classes most pertinent to these goals are immunoglobulin M (IgM), which are the first antibodies produced in response to an infection, and immunoglobulin G (IgG), which are the most abundant antibodies found in the blood. Following SARS infection, IgM and IgG antibodies were detected in the second week post-infection. IgM titers peaked by the first month post-infection, and then declined to undetectable levels after day 180. IgG titers peaked by day 60, and persisted in all donors through the two-year duration of study [272]. A two-year longitudinal study following convalesced SARS patients with a mean age of 29 found that IgG antibodies were detectable in all 56 patients surveyed for at least 16 months, and remained detectable in all but 4 (11.8%) of patients through the full two-year study period [273]. These results suggest that immunity to SARS-CoV-1 is sustained for at least a year.

The persistence of antibodies to SARS-CoV-2 remains under investigation. Circulating antibody titers to other coronaviruses have been reported to decline significantly after 1 year [274]. Autopsies of lymph nodes and spleens from severe acute COVID-19 patients showed a loss of T follicular helper cells and germinal centers that may explain some of the impaired development of antibody responses [275]. An early study (initially released on medRxiv on February 25, 2020) presented a chemiluminescence immunoassay to a synthetic peptide derived from the amino acid sequence of the SARS_CoV-2 S protein [276]. This method was highly specific to SARS-CoV-2 and detected IgM in 57.2% and IgG in 71.4% and 57.2% of sera samples from 276 confirmed COVID-19 patients. They reported that they could detect IgG within two days of the onset of fever and were not able to detect IgM any earlier, a pattern they compared to findings in MERS. The duration over which these antibodies persist remains unknown.

4.4.2 Current Approaches

Several countries are now focused on implementing antibody tests, and in the United States, the FDA recently approved a serological test by Cellex for use under emergency conditions [277]. Specifically, the Cellex qSARS-CoV-2 IgG/IgM Rapid Test is a chromatographic immunoassay designed to qualitatively detect IgM and IgG antibodies against SARS-CoV-2 in the plasma (from a blood sample) of patients suspected to have developed the SARS-CoV-2 infection [277]. Such tests illuminate the progression of viral disease, as IgM are the first antibodies produced by the body and indicate that the infection is active. Once the body has responded to the infection, IgG are produced and gradually replace IgM, indicating that the body has developed immunogenic memory [278]. The test cassette contains a pad of SARS-CoV-2 antigens and a nitrocellulose strip with lines for each of IgG and IgM, as well as a control (goat IgG) [277]. In a specimen that contains antibodies against the SARS-CoV-2 antigen, the antibodies will bind to the strip and be captured by the IgM and/or IgG line(s), resulting in a change of color [277]. With this particular assay, results can be read within 15-20 minutes [277]. Other research groups, such as the Krammer lab of the Icahn School of Medicine at Mount Sinai, proposed an ELISA test that detects IgG and IgM that react against the receptor binding domain (RBD) of the spike proteins (S) of the virus [279]. The authors are now working to get the assay into clinical use [280].

4.4.3 Limitations of Serological Tests

Importantly, false-positives can occur due to the cross-reactivity with other antibodies according to the clinical condition of the patient [277]. Therefore, this test should be used in combination with RNA detection tests [277]. Due to the long incubation times and delayed immune responses of infected patients, serological tests are insufficiently sensitive for a diagnosis in the early stages of an infection. The limitations due to timing make serological tests far less useful for enabling test-and-trace strategies.

4.5 Possible Alternatives to Current Practices for Identifying Active Cases

Clinical symptoms are too similar to other types of pneumonia to be sufficient as a sole diagnostics criterion. In addition, as noted above, identifying asymptomatic cases is critical. Even among mildly symptomatic patients, a predictive model based on clinical symptoms had a sensitivity of only 56% and a specificity of 91% [281]. More problematic is that clinical symptom-based tests are only able to identify already symptomatic cases, not presymptomatic or asymptomatic cases. They may still be important for clinical practice, and for reducing tests needed for patients deemed unlikely to have COVID-19.

X-ray diagnostics have been reported to have high sensitivity but low specificity in some studies [282]. Other studies have shown that specificity varies between radiologists [283], though the sensitivity reported here was lower than that published in the previous paper. However, preliminary machine-learning results have shown far higher sensitivity and specificity from analyzing chest X-rays than was possible with clinical examination [284]. X-ray tests with machine learning can potentially detect asymptomatic or presymptomatic infections that show lung manifestations. This approach would still not recognize entirely asymptomatic cases. Given the above, the widespread use of X-ray tests on otherwise healthy adults is likely inadvisable.

4.6 Strategies and Considerations for Determining Whom to Test

Early in the COVID-19 pandemic, testing was typically limited to individuals considered high risk for developing serious illness [285]. This approach often involved limiting testing to people with severe symptoms and people showing mild symptoms that had been in contact with a person who had tested positive. Individuals who are asymptomatic (i.e., potential spreaders) and individuals who are able to recover at home are therefore often unaware of their status. However, this method of testing administration misses a high proportion of infections and does not allow for test-and-trace methods to be used. For instance, a recent study from Imperial College estimates that in Italy, the true number of infections was around 5.9 million in a total population ~60 million, compared to the 70,000 detected as of March 28th [226]. Another analysis, which examined New York state, indicated that as of May, 2020, approximately 300,000 cases had been reported in a total population of approximately 20 million [286]. This corresponded to ~1.5% of the population, but ~12% of individuals sampled statewide were estimated as positive through antibody tests (along with indications of spatial heterogeneity at higher resolution) [286]. Technological advancements that facilitate widespread, rapid testing will therefore improve the potential to accurately assess the rate of infection and aid in controlling the virus’ spread.

4.7 Conclusions

Major advancements have been made in identifying diagnostic approaches. The development of diagnostic technologies have been rapid, beginning with the release of the SARS-CoV-2 viral genome sequence in January. As of October 2020, a range of diagnostic tests have become available. One class of tests uses PCR (RT-PCR or qRT-PCR) to assess the presence of SARS-CoV-2 RNA, while another typically uses ELISA to test for the presence of antibodies to SARS-CoV-2. The former approach is useful for identifying active infections, while the latter measures hallmarks of the immune response and therefore can detect either active infections or immunity gained from prior infection. Combining these tests leads to extremely accurate detection of SARS-CoV-2 infection (98.6%), but when used alone, PCR-based tests are recommended before 5.5 days after the onset of the illness and antibody tests after 5.5 days [287]. Other strategies for testing can also influence the tests’ accuracy, such as the use of nasopharyngeal swabs versus BALF [287], which allow for trade-offs between patient’s comfort and test sensitivity. Additionally, technologies such as digital PCR may allow for scale-up in the throughput of diagnostic testing, facilitating widespread testing. One major question that remains is whether people who recover from SARS-CoV-2 develop sustained immunity, and over what period this immunity is expected to last. Some reports have suggested that some patients may develop COVID-19 reinfections (e.g., [288]), but the rates of reinfection are currently unknown. Serologic testing combined with PCR testing will be critical to confirming purported cases of reinfection and to identifying the duration over which immunity is retained and to understanding reinfection risks.

5 Identification and Development of Prophylactics and Therapeutics for COVID-19

5.1 Abstract

The novel coronavirus SARS-CoV-2 emerged in late 2019 and went on to significantly impact most countries worldwide. Only a small number of coronaviruses are known to infect humans, and only two are associated with the severe outcomes associated with SARS-CoV-2: SARS-CoV-1, a sister species that emerged in 2002, and MERS-CoV, which emerged in 2012. Although both of these previous pandemics were rapidly controlled, no vaccines or robust therapeutic interventions were identified. However, previous insights into the immune response to coronaviruses, many made in the context of better understanding SARS and MERS, have been beneficial in identifying approaches to the treatment and prophylaxis of COVID-19. A number of potential therapeutics were rapidly identified, leading to a large number of clinical trials investigating a variety of possible therapeutic approaches being initiated early on in the pandemic. Similarly, previous advances in vaccine development allowed for the rapid design and testing of candidate prophylactics. As a result, by the end of 2020, a small number of therapeutics and vaccines have been authorized by regulatory agencies such as the Food and Drugs Administration in the United States, and many more continue to be under investigation. Here, we describe a range of approaches for the treatment and prevention of COVID-19 along with their proposed mechanisms of action and the current status of clinical investigation into each candidate. The status of these investigations will continue to evolve, and this review will be updated as progress is made.

5.2 Importance

The COVID-19 pandemic is a rapidly evolving crisis. When the worldwide scientific community shifted its focus onto the SARS-CoV-2 virus and the disease it causes, a large number of possible pharmaceutical approaches for treatment and prevention were proposed. What is known about each of these potential interventions has evolved rapidly throughout 2020. In March 2020, we began monitoring a range of candidates and have continued to update this manuscript as new information became available throughout 2020. Some have been supported, others have been revealed to be unlikely to have any therapeutic benefits, and most require more data before a conclusion can be drawn. This rapidly changing area of research provides important insight into how the ongoing pandemic can be managed and also demonstrates the power of interdisciplinary collaboration to rapidly understand a virus and match its characteristics with existing or novel pharmaceuticals.

5.3 Introduction

The novel coronavirus Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) emerged in late 2019 and quickly precipitated the worldwide spread of novel coronavirus disease 2019 (COVID-19), the disease caused by its infection. COVID-19 is associated with symptoms ranging from none (asymptomatic) to mild to severe, with approximately 2% of patients dying from COVID-19-related complicates such as acute respiratory disease syndrome (ARDS) [cite pathogenesis/transmission]. The virus is likely spread between people primarily by droplets, with the role of contact and aerosol transmission still in question [182,183]. As a result, public health guidelines have been critical to efforts to control the spread of the virus. However, as of December 2020, COVID-19 remains a significant worldwide concern (Figure 2), with cases in some places surging far above the numbers reported in early 2020. Due to the continued threat of the virus and the severity of the disease, the identification and development of prophylactic and therapeutic interventions have emerged as significant international priorities. Both approaches hold valuable potential for controlling the impact of the disease. Prophylactics induce immunity to prevent an individual from contracting a disease whereas therapeutics treat a disease in individuals who have already been infected. For a highly infectious virus like SARS-CoV-2, a vaccine would hold particular value because it could bolster the immune response to the virus of the population broadly, thereby driving a lower rate of infection and likely significantly reducing fatalities. However, the vaccine development process has historically been slow, and vaccines fail to provide immediate prophylactic protection or treat ongoing infections [289]. Thus, there is also an immediate need for treatments that palliate symptoms and prevent the most severe outcomes from infection. Fortunately, prior developments during other recent pandemics, especially those caused by human coronaviruses (HCoV), has provided a number of hypotheses guiding a biomedical approach to the novel coronavirus infection.

2,057,215 COVID-19 deaths had been reported worldwide as of January 19, 2021 (Figure 2).

Figure 2: Cumulative global COVID-19 deaths since January 22, 2020. Data are from the COVID-19 Data Repository by the Center for Systems Science and Engineering at Johns Hopkins University [290].

5.3.1 Lessons from Prior HCoV Outbreaks

SARS-CoV-2’s rapid shift from an unknown virus to a significant worldwide threat closely parallels the emergence of Severe acute respiratory syndrome-related coronavirus (SARS-CoV-1). The first documented case of COVID-19 was reported in Wuhan, China in November 2019, and the disease quickly spread worldwide during the early months of 2020. Similarly, the first case of SARS was reported in November 2002 in the Guangdong Province of China and spread within China and then into several countries across continents over the following months [28,30]. In fact, the virus causing COVID-19 was quickly identified through genome sequencing as a novel betacoronavirus closely related to SARS-CoV-1 10]. There are other similarities but also some differences in characteristics of the two viruses relevant to their spread. SARS infection is severe, with an estimated death rate of 9.5% [30], while estimates of the death rate associated with COVID-19 are much lower, at approximately 2% [cite pathogenesis/transmission section] . SARS-CoV-1 is highly contagious via droplet transmission and has a basic reproduction number (R0) of 4 (i.e., each person infected was estimated to infect four other people) [30]. SARS-CoV-2 also appears to be spread primarily by droplet transmission [182,183], and most estimates of its R0 fall between 2.5 and 3 [cite transmission/pathogenesis]. Furthermore, the 17-year difference in the timing of these two outbreaks has led to some major differences in the tools available for the international community’s response. At the time that SARS-CoV-1 emerged, no new HCoV had been identified in almost 40 years [28]. The identity of the virus underlying the SARS disease remained unknown until April of 2003, when the SARS-CoV-1 virus was characterized through a worldwide scientific effort spearheaded by the WHO [28]. In contrast, the SARS-CoV-2 genomic sequence was released on January 3, 2020 [10], only days after the international community became aware of the novel pneumonia-like illness now known as COVID-19. While SARS-CoV-1 belonged to a distinct lineage from the two other HCoV known at the time of its discovery [30], SARS-CoV-2 is closely related to SARS-CoV-1 and a more distant relative of another HCoV characterized in 2012, Middle East respiratory syndrome-related coronavirus [13,291]. Thus, despite their phylogenetic similarity, the viruses have different characteristics relevant to their spread, and the SARS-CoV-2 virus emerged under very different circumstances than SARS-CoV-1 in terms of scientific knowledge about HCoV.

The trajectories of the pandemics associated with each of the viruses have also diverged significantly. By July 2003, the SARS outbreak was officially determined to be under control, with the success credited to infection management practices such as mask wearing [28]. In contrast, MERS is still circulating and remains a concern because the fatality rate is very high at almost 35%, but the disease is much less easily transmitted, with an R0 of 1 [30]. The low R0 in combination with public health practices allowed for its spread to be contained [30]. Neither of these trajectories are comparable to that of SARS-CoV-2, which remains a serious threat worldwide a full year after the first known cases of COVID-19. However, early results suggest that pharmaceutical interventions for COVID-19 may be more successful than previous efforts to develop prophylactics and therapeutics for SARS and MERS. Vaccines were not used to control either virus, although vaccine development programs were established for SARS-CoV-1 [31]. Care for SARS and MERS patients prioritized supportive care and symptom management [30]. To the extent that clinical treatments for SARS and MERS were explored, there is generally a lack of evidence supporting their efficacy. For example, Ribavirin is an antiviral that was often used in combination with corticosteroids and sometimes interferon (IFN) medications to treat SARS and MERS [28], but its effects have been found to be inconclusive in retrospective and in vitro analyses of SARS and the SARS-CoV-1 virus, respectively [28]. IFNs and Ribavirin have shown promise in in vitro analyses of MERS, but their clinical effectiveness remains unknown [28]. Therefore, only limited pharmaceutical advances from prior HCoV outbreaks can be adopted to COVID-19. Importantly, though, prior analyses of the virological and pathogenic properties of SARS-CoV-1 and MERS-CoV provide a strong foundation for the development of hypotheses about SARS-CoV-2 that have served to accelerated the development and identification of potential prophylactic and therapeutic approaches.

5.3.2 Therapeutic Approaches

Therapeutic approaches to the current pandemic can utilize two potential avenues: they can reduce the symptoms that are harmful to COVID-19 patients, or they can directly target the virus to hinder the spread of infection. The goal of the former is to reduce the severity and risks of an active infection while for the latter, it is to inhibit the development of the virus once an individual is infected. A variety of symptom profiles with a range of severity are associated with COVID-19, many of which are not life-threatening. A study of COVID-19 patients in a hospital in Berlin, Germany found that the symptoms associated with the highest risk of death included infection-related symptoms, such as sepsis, respiratory symptoms such as ARDS, and cardiovascular failure or pulmonary embolism [292]. Therefore, therapeutics that reduce risk associated with these severe outcomes are of particular interest to reducing the pandemic death toll. Therapeutics that directly target the virus itself typically fall into the broad category of antivirals. Antiviral therapies hinder the spread of a virus within the host, rather than destroying existing copies of the virus, and these drugs can vary in their specificity to a narrow or broad range of viral targets. For both categories, uncertainty often surrounds treatments’ exact mechanisms of action, as most therapies have secondary or off-target effects.

In this review, we describe some treatments and prophylactics that have been considered for COVID-19 and classify them according to their biological properties, specifically whether they are biologics (produced from components of organisms) or small molecules. Small molecule drugs include drugs targeted at viral particles, drugs targeted at host proteins, and broad spectrum pharmaceuticals, while biologics include antibodies, interferons, and vaccines. A large number of clinical trials investigating a wide range of possible therapeutics and prophylactics for COVID-19 are currently in progress or have already been completed (Figure 3). The purpose of this review is to critically appraise the literature surrounding a subset of clinical trials and to evaluate a range of approaches to repurpose existing or develop novel approaches to the prevention, mitigation, and treatment of coronavirus infections. The identity of the virus underlying the SARS disease remained unknown until April of 2003, when the SARS-CoV-1 virus was characterized through a worldwide scientific effort spearheaded by the WHO [28]. In contrast, the SARS-CoV-2 genomic sequence was released on January 3, 2020 [10], only days after the international community became aware of the novel pneumonia-like illness now known as COVID-19. While SARS-CoV-1 belonged to a distinct lineage from the two other HCoV known at the time of its discovery [30], SARS-CoV-2 is closely related to SARS-CoV-1 and a more distant relative of another HCoV characterized in 2012, Middle East respiratory syndrome-related coronavirus [13,291]. Thus, despite their phylogenetic similarity, the viruses have different characteristics relevant to their spread, and the SARS-CoV-2 virus emerged under very different circumstances than SARS-CoV-1 in terms of scientific knowledge about HCoV.

Figure 3: COVID-19 clinical trials. There are 6,417 COVID-19 clinical trials and 168 trials with results as of November 9, 2020. The recruitment statuses and trial phases are shown only for trials in which the status or phase is recorded. The study types include only types used in at least five trials. The common interventions are all interventions used in at least ten trials. Combinations of interventions, such as Hydroxychloroquine + Azithromycin, are tallied separately from the individual interventions. Trials data are from the University of Oxford Evidence-Based Medicine Data Lab’s COVID-19 TrialsTracker [293].

5.4 Small Molecule Drugs

Small molecules are synthesized compounds of low molecular weight, typically less than 1 kilodalton (kDa) [294]. Small-molecule pharmaceutical agents have been a backbone of drug development since the discovery of penicillin in the early twentieth century [295]. It and other antibiotics have long been among the best known applications of small molecules to therapeutics, but biotechnological developments such as the prediction of protein-protein interactions have facilitated advances in precise targeting of specific structures using small molecules [295]. Small molecule drugs today encompass a wide range of therapeutics beyond antibiotics, including antivirals, protein inhibitors, and many broad-spectrum pharmaceuticals.

5.4.1 Small Molecule Antivirals

Antiviral drugs against SARS-CoV-2 are designed to inhibit replication of a virus within an epithelial host cell. This process requires inhibiting the replication cycle of a virus by disrupting one of six fundamental steps [296]. In the first step, the virus attaches to the host cell, which it next penetrates through endocytosis. Then the virus undergoes uncoating, which is classically defined as the release of viral contents into the host cell, and next the viral genetic material enters the nucleus where it gets replicated during the biosynthesis stage. During the assembly stage, viral proteins are translated, allowing new viral particles to be assembled, and during the final step, release, the new viruses are released into the extracellular environment. Many antiviral drugs are designed to inhibit the replication of viral genetic material during the biosynthesis step. Unlike DNA viruses, which can use the host enzymes to propagate themselves, RNA viruses like SARS-CoV-2 depend on their own polymerase, the RNA-dependent RNA polymerase (RdRP), for replication [297,298]. Targeting RdRP is therefore an effective strategy for antivirals against RNA viruses and is the proposed mechanism underlying the treatment of SARS and MERS with Ribavirin [299]. However, although antivirals are designed to target a virus, they can also impact other processes in the host and may have unintended effects.

5.4.1.1 Nucleoside and Nucleotide Analogs

5.4.1.1.1 Favipiravir

Favipiravir (Avigan), also known as T-705, was discovered by Toyama Chemical Co., Ltd. [300]. The drug was found to be effective at blocking viral amplification in several influenza subtypes as well as other RNA viruses, such as Flaviviridae and Picornaviridae, through a reduction in plaque formation [301] and viral replication in Madin-Darby Canine Kidney (MDCK) cells [302]. Furthermore, inoculation of mice with favipiravir was shown to increase survival of influenza infections [301,302]. In 2014, the drug was approved in Japan for the treatment of patients infected with influenza that was resistant to conventional treatments like neuraminidase inhibitors [303]. Favipiravir (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) acts as a purine and purine nucleoside analogue that inhibits viral RNA polymerase in a dose-dependent manner across a range of RNA viruses, including Influenza virus [304,305,306,307,308]. Nucleotides and nucleosides are the natural building blocks for RNA synthesis. Because of this, modifications to nucleotides and nucleosides can disrupt key processes including replication [309]. Biochemical experiments showed that favipiravir was recognized as a purine nucleoside analogue and incorporated into the viral RNA template. A single incorporation does not influence RNA transcription; however, multiple events of incorporation lead to the arrest of RNA synthesis [310]. Evidence for T-705 inhibiting viral RNA polymerase are based on time-of-drug addition studies that found that viral loads were reduced with the addition of favipiravir in early times post-infection [304,307,308].

The effectiveness of favipiravir for treating patients with COVID-19 is currently under investigation. An open-label, nonrandomized, before-after controlled study was recently conducted [311]. The study included 80 COVID-19 patients (35 treated with favipiravir, 45 control) from the isolation ward of the National Clinical Research Center for Infectious Diseases (The Third People’s Hospital of Shenzhen), Shenzhen, China. The patients in the control group were treated with other antivirals, such as lopinavir and ritonavir. It should be noted that subsequent, large-scale analyses, the WHO Solidarity trial and the RECOVERY trial, identified no effect of lopinavir or of a lopinavir-ritonavir combination, respectively, on the metrics of COVID-19-related mortality that each assessed [???,312,313]. Treatment was applied on days 2-14; treatment stopped either when viral clearance was confirmed or at day 14. The efficacy of the treatment was measured by 1) the time until viral clearance using Kaplan-Meier survival curves, and 2) the improvement rate of chest computed tomography (CT) scans on day 14 after treatment. The study found that favipiravir increased the speed of recovery, measured as viral clearance from the patient by RT-PCR, with patients receiving favipiravir recovering in 4 days compared to 11 days for patients receiving antivirals such as lopinavir and ritonavir. Additionally, the lung CT scans of patients treated with favipiravir showed significantly higher improvement rates (91%) on day 14 compared to control patients (62%). However, there were adverse side effects in 4 (11%) favipiravir-treated patients and 25 (56%) control patients. The adverse side effects included diarrhea, vomiting, nausea, rash, and liver and kidney injury. Overall, despite the study reporting clinical improvement in favipiravir-treated patients, due to some issues with study design, it cannot be determined whether treatment with favipiravir had an effect or whether these patients would have recovered regardless of any treatment. For example, despite the significant differences observed between the two treatment groups, follow-up analysis is necessary due to the small sample size. The selection of patients did not take into consideration important factors such as previous clinical conditions or sex, and there was no age categorization. The study was neither randomized nor blinded, and the baseline control group was another antiviral instead of a placebo. Therefore, randomized controlled trials are still required.

5.4.1.1.2 Remdesivir

Remdesivir (GS-5734) is an intravenous antiviral that was developed by Gilead Sciences to treat Ebola Virus Disease (EVD). At the outset of the COVID-19 pandemic, it did not have any have any FDA-approved use. However, on May 1, 2020, the FDA issued an Emergency Use Authorization (EUA) for remdesivir for the treatment of hospitalized COVID-19 patients [314]. The EUA was based on information from two clinical trials, NCT04280705 and NCT04292899 [315,316,317,318]. Remdesivir is metabolized to GS-441524, an adenosine analog that inhibits a broad range of polymerases and then evades exonuclease repair, causing chain termination [319,320,321]. A clinical trial in the Democratic Republic of Congo found some evidence of effectiveness against EVD, but two antibody preparations were found to be more effective, and remdesivir was not pursued [322]. Although it was developed against EVD, remdesivir also inhibits polymerase and replication of the coronaviruses MERS-CoV and SARS-CoV-1 in cell culture assays with submicromolar IC50s [323]. It has also been found to inhibit SARS-CoV-2, showing synergy with chloroquine in vitro [321]. The effectiveness of remdesivir for treating patients with COVID-19 is currently under investigation.

Remdesivir was first used on some COVID-19 patients under compassionate use guidelines [324,325]. All were in late stages of COVID-19 infection, and these reports were inconclusive about the drug’s efficacy. Gilead Sciences, the maker of remdesivir, led a recent publication that reported outcomes for compassionate use of the drug in 61 patients hospitalized with confirmed COVID-19. Here, 200 mg of remdesivir was administered intravenously on day 1, followed by a further 100 mg/day for 9 days [318]. There were significant issues with the study design, or lack thereof. There was no randomized control group. The inclusion criteria were variable: some patients only required low doses of oxygen, while others required ventilation. The study included many sites, potentially with variable inclusion criteria and treatment protocols. The patients analyzed had mixed demographics. There was a short follow-up period of investigation. Some patients worsened, some patients died, and eight were excluded from the analysis mainly due to missing post-baseline information; thus, their health is unaccounted for. Therefore, even though the study reported clinical improvement in 68% of the 53 patients ultimately evaluated, due to the significant issues with study design, it could not be determined whether treatment with remdesivir had an effect or whether these patients would have recovered regardless of treatment. Another study comparing 5- and 10-day treatment regimens reported similar results but was also limited because of the lack of a placebo control [327]. The study did not alter our understanding of the efficacy of remdesivir in treating COVID-19, but the encouraging results provided motivation for placebo-controlled studies.

Remdesivir was later tested in a double-blind placebo-controlled phase 3 clinical trial performed at 60 trial sites, 45 of which were in the United states [316,317]. The trial recruited 1,062 patients and randomly assigned them to placebo treatment or treatment with remdesivir. Patients were stratified for randomization based on site and the severity of disease presentation at baseline [317]. The treatment was 200 mg on day 1, followed by 100 mg on days 2 through 10. Data was analyzed from a total of 1,059 patients who completed the 29 day course of the trial (517 assigned to remdesivir and 508 to placebo) [317]. The two groups were well matched demographically and clinically at baseline. Those who received remdesivir had a median recovery time of 10 days, as compared with 15 days in those who received placebo (rate ratio for recovery, 1.29; 95% CI, 1.12 to 1.49; p < 0.001). The Kaplan-Meier estimates of mortality by 14 days were 6.7% with remdesivir and 11.9% with placebo, with a hazard ratio (HR) for death of 0.55 and a 95% CI of 0.36 to 0.83, and at day 29, remdesivir corresponded to 11.4% and the placebo to 15.2% (HR: 0.73; 95% CI: 0.52 to 1.03). Serious adverse events were reported in 131 of the 532 patients who received remdesivir (24.6%) and in 163 of the 516 patients in the placebo group (31.6%). This study also reported an association between remdesivir administration and both clinical improvement and a lack of progression to more invasive respiratory intervention in patients receiving non-invasive and invasive ventilation at randomization [317]. Largely on the results of this trial, the FDA issued an Emergency Use Authorization (EUA) for remdesivir for the treatment of hospitalized COVID-19 patients. Clinical trials are currently underway to evaluate the use of remdesivir to treat COVID-19 patients at both early and late stages of infection and in combinations with other drugs (Figure 3). These trials include [321,328,329,330,331]. As of October 22, 2020, remdesivir received FDA approval based on three clinical trials [332].

However, results suggesting no effect of remdesivir on survival were reported by the WHO Solidarity trial [???]. This large-scale, open-label trial enrolled 11,330 adult in-patients at 405 hospitals in 30 countries around the world [???]. Patients were randomized in equal proportions into four experimental and a control conditions, corresponding to four candidate treatments for COVID-19 and SOC, respectively; no placebo was administered. The 2750 patients in the remdesivir group were administered 200 mg intravenously on the first day and 100 mg on each subsequent day until day 10 and assess for in-hospital death (primary endpoint), duration of hospitalization, and progression to mechanical ventilation. There were also 2708 control patients who would have been eligible and able to receive remdesivir were they not assigned to the control group. A total of 604 patients among these two cohorts deceased during initial hospitalization. The rate ratio of death between these two groups was therefore not significant (p = 0.50), suggesting that the administration of remdesivir did not affect survival. The two secondary analyses similarly did not find any effect of remdesivir. Additionally, the authors compared data from their study with data from three other studies of remdesivir (including [317]) stratified by supplemental oxygen status. The adjusted rate ratio for death based on this meta-analysis was 0.91. These results thus do not support the previous findings that remdesivir reduced median recovery time and mortality risk in COVID-19 patients.

In response to the results of the Solidarity trial, Gilead, which manufactures remdesivir under the name Veklury^R, released a statement pointing to the fact that the Solidarity trial was not placebo-controlled or double-blind and at the time of the statement had not been peer reviewed [333]; these sentiments have been echoed elsewhere [334]. Other critiques of this study have noted that antivirals are not typically targeted at patients with severe illness, and therefore remdesivir could be more beneficial for patients with mild rather than severe cases [313,335]. However, the publication associated with the trial sponsored by Gilead did suggest an effect of remdesivir on patients with severe disease, identifying an 11 versus 18 day recovery period (rate ratio for recovery: 1.31, 95% CI 1.12 to 1.52), although the results of a significance test were not provided [317]. Additionally, a smaller analysis comparing treatment with remdesivir to standard of care in patients with moderate COVID-19 reported a small effect size, corresponding to minimal differences between these groups at day 11 [336]. Therefore, some of the findings from this trial do seem to contradict the findings of the Solidarity trial. On the other hand, only 62% of patients in the Solidarity trial were randomized on the day of admission or one day afterwards [???], and differences in disease progression may influence the effect of remdesivir [313], so there could be be additional confounding factors in one or both trials. The broad international nature of the Solidarity clinical trial, which included countries with a wide range of economic profiles and a variety of healthcare systems, provides a much-needed global perspective in a pandemic [313]. Despite the findings of the Solidarity trial, remdesivir remains available for the treatment of COVID-19 in many places. Follow-up studies are needed and in many cases are underway to further investigate remdesivir-related outcomes, with possibilities including combinations of remdesivir with other drugs such as baricitinib, which is an inhibitor of Janus kinase 1 and 2 [337]. Similarly, the extent to which the remdesivir dosing regimen could influence outcomes is still under consideration. A randomized, open-label trial compared the effect of remdesivir on 397 patients with severe COVID-19 over 5 versus 10 days [315,327]. Patients in the two groups were administered 200 mg of remdesivir intravenously on the first day, followed by 100 mg on the subsequent four or nine days, respectively. The two groups differed significantly in their clinical status, with patients assigned to the 10-day group having more severe illness. This study also differed from most because it included not only adults, but also pediatric patients as young as 12 years old. This study found no significant differences across several outcomes for patients receiving a 5-day or 10-day course, when correcting for baseline clinical status. They did find evidence suggesting that the 10-day course might reduce mortality in the most severe patients at day 14, but the representation of this group in the study population was too low to justify any conclusions [327]. Thus, additional research is also required to determine whether the dosage and duration of remdesivir administration influences outcomes.

In summary, remdesivir is a first in class drug due to its FDA approval. Early investigations of this drug establish proof of principle that drugs targeting the virus can benefit COVID-19 patients. It also shows proof of principle that SARS-CoV-2 can be targeted at the level of viral replication, since remdesivir targets the viral RNA polymerase at high potency. Moreover, one of the most successful strategies for developing therapeutics for viral diseases is to target the viral replication machinery, which are typically virally encoded polymerases. Small molecule drugs targeting viral polymerases are the backbones of treatments for other viral diseases including HIV and Herpes. Notably, the HIV and herpes polymerases are a reverse transcriptase and a DNA polymerase, respectively, whereas SARS-CoV-2 encodes an RdRP, so most of the commonly used polymerase inhibitors are not likely to be active against SARS-CoV-2. In clinical use, polymerase inhibitors show short term benefits for HIV patients but for long term benefits they must be part of combination regimens. They are typically combined with protease inhibitors, integrase inhibitors, and even other polymerase inhibitors. Additional clinical trials of remdesivir in different patient pools and in combination with other therapies will refine its use in the clinic.

5.4.1.2 Protease Inhibitors

Several studies showed that viral proteases play an important role in the life cycle of viruses, including coronaviruses, by modulating the cleavage of viral polyprotein precursors [338]. Several FDA-approved drugs target proteases, including lopinavir and ritonavir for HIV infection and simeprevir for hepatitis C virus infection. In particular, serine protease inhibitors were suggested for the treatment of SARS and MERS viruses [339]. Recently, a study [66] suggested that camostat mesylate, an FDA-approved protease inhibitor (PI) could block the entry of SARS-CoV-2 into lung cells in vitro. However, to test the efficacy of PIs in patients, randomized clinical trials will need to be conducted on patients and healthy volunteers. Investigation of possible PIs that would work against SARS-CoV-2 has been driven by computational predictions, leading to the computer-aided design of a Michael acceptor inhibitor, N3, to target a protease critical to SARS-CoV-2 replication.

Discovery of the N3 mechanism arose from interest in the two polyproteins encoded by the SARS-CoV-2 replicase gene, pp1a and pp1ab, that are critical for viral replication and transcription [340]. These polyproteins must undergo proteolytic processing. This processing is usually conducted by Mpro, a 33.8-kDa SARS-CoV-2 protease that is therefore fundamental to viral replication and transcription. N3 was designed computationally [341] to bind in the substrate binding pocket of the Mpro protease of SARS-like coronaviruses [342], therefore inhibiting proteolytic processing. Subsequently, the structure of N3-bound SARS-CoV-2 Mpro was solved [340], confirming the computational prediction. N3 was tested in vitro on SARS-CoV-2-infected Vero cells, which belong to a line of cells established from the kidney epithelial cells of an African green monkey, and was found to inhibit SARS-CoV-2 [340].

Although N3 is a strong inhibitor of SARS-CoV-2 in vitro, its safety and efficacy still need to be tested in healthy volunteers and patients. After the design and confirmation of N3 as a highly potent Michael acceptor inhibitor and the identification of Mpro’s structure [340,343], 10,000 compounds were screened for their in vitro anti-Mpro activity. The six leads that were identified were ebselen, disulfiram, tideglusib, carmofur, and PX-12. In vitro analysis revealed that Ebselen had the strongest potency in reducing the viral load in SARS-CoV-2-infected Vero cells [340]. Ebselen is an organoselenium compound with anti-inflammatory and antioxidant properties [344] It has been proposed as a possible treatment for conditions ranging from bipolar disorder to diabetes to heart disease [344], and a preliminary investigation of ebselen as a treatment for noise-induced hearing loss provided promising reports of its safety [345]. For COVID-19, the NSP5 in SARS-CoV-2 contains a cysteine at the active site of Mpro, and ebselen is able to inactivate the protease by bonding covalently with this cysteine to form a selenosulfide [344,346]. Interestingly there has been some argument that selenium deficiency may be associated with more severe COVID-19 outcomes [347,348,349], possibly indicating that its antioxidative properties are protective [346]. On the other hand, ebselen and the other compounds identified are likely to be promiscuous binders, which could diminish their therapeutic potential [340]. While there is clear computational and in vitro support for ebselen’s potential as a COVID-19 therapeutic, results from clinical trials are not yet available for this compound.

In summary, N3 is a computationally designed molecule that inhibits the viral transcription through inhibiting Mpro. Ebselen is both a strong Mpro inhibitor and strong inhibitor of viral replication in vitro that was found to reduce SARS-CoV-2 viral load even more effectively than N3. Ebselen is a very promising compound since its safety has been demonstrated in other indications. However, Ebselen is likely a false positive since it is a promiscuous compound that can have many targets [350]. Therefore, compounds with higher specificity may be required to effectively translate to clinical trials.

5.4.2 Broad-Spectrum Pharmaceuticals

When a virus enters a host, the host becomes the virus’ environment. Therefore, the state of the host can also influence the virus’s ability to replicate and spread. Traditionally, viral targets have been favored for pharmaceutical interventions because altering host processes is likely to be less specific than targeting the virus directly [351]. On the other hand, targeting the host offers potential for a complementary strategy to antivirals that could broadly limit the ability of viruses to replicate [351]. As a result, therapeutic approaches that target host proteins have become an area of interest for SARS-CoV-2. Viral entry receptors in particular have emerged as a target of interest. Entry of SARS-CoV-2 into the cell depends on binding to the Angiotensin-converting enzyme 2 (ACE2) receptor, which is catalyzed by the enzyme encoded by TMPRSS2 [66]. In principle, drugs that reduce the expression of these proteins or sterically hinder viral interactions with them might reduce viral entry into cells.

Due to the urgent nature of the COVID-19 pandemic, many of the pharmaceutical agents that have been widely publicized as having possible therapeutic or prophylactic effects are broad-spectrum pharmaceuticals that pre-date the COVID-19 pandemic. These treatments are not specifically targeted at the virus itself or at the host receptors it relies on, but rather induce broad shifts in host biology that are hypothesized to be potential inhibitors of the virus. In most cases, interest in particular candidate medications arises because they are already available for other purposes. However, the fact that the targets of these agents are non-specific means that the mechanism of action can appear to be relevant to COVID-19 without a therapeutic or prophylactic effect being observed in rigorous testing. This category of drugs has also received significant attention from the media and general public, often before rigorous testing has been able to determine their effectiveness against SARS-CoV-2.

5.4.2.1 ACEi and ARB

Angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor blockers (ARBs) are among today’s most commonly prescribed medications [352,353]. In the United States, for example, they are prescribed well over 100,000,000 times annually. Data from some animal models suggest that several, but not all, ACE inhibitors and several ARBs increase ACE2 expression in the cells of some organs [354]. Clinical studies have not established whether plasma ACE2 expression is increased in humans treated with these medications [355]. While randomized clinical trials are ongoing, a variety of observational studies have examined the relationship between exposure to ACE inhibitors or ARBs and outcomes in patients with COVID-19. An observational study of the association of exposure to ACE inhibitors or ARB with outcomes in COVID-19 was retracted from the New England Journal of Medicine [356]. Moreover, because observational studies are subject to confounding, randomized controlled trials are the standard means of assessing the effects of medications, and the findings of the various observational studies bearing on this topic cannot be interpreted as indicating a protective effect of the drug [357,358]. Several clinical trials testing the effects of ACE inhibitors or ARBs on COVID-19 outcomes are ongoing [359,360,361,362,363,364]. These studies of randomized intervention will provide important data for understanding whether exposure to ACEis or ARBs is associated with COVID-19 outcomes. Additional information about ACE2, observational studies of ACE inhibitors and ARBs in COVID-19, and clinical trials on this topic have been summarized [365].

5.4.2.2 Hydroxychloroquine and Chloroquine

Chloroquine (CQ) and hydroxychloroquine (HCQ) are lysosomotropic agents, meaning they are weak bases that can pass through the plasma membrane. Both drugs increase cellular pH by accumulating in their protonated form inside lysosomes [366,367]. This shift in pH inhibits the breakdown of proteins and peptides by the lysosomes during the process of proteolysis [367]. A number of mechanisms have been proposed through which these drugs could influence the immune response to pathogen challenge. For example, CQ/HCQ can interfere with digestion of antigens within the lysosome and inhibit CD4 T-cell stimulation while promoting the stimulation of CD8 T-cells [367]. CQ/HCQ can also decrease the production of certain key cytokines involved in the immune response, including interleukin-6 (IL-6), and inhibit the stimulation of toll-like receptors (TLR) and TLR signaling [367]. The drugs also have anti-inflammatory and photoprotective effects and may also affect rates of cell death, blood clotting, glucose tolerance, and cholesterol levels [367].

Interest in CQ and HCQ for treating COVID-19 was catalyzed by a mechanism observed in in vitro studies of both SARS-CoV-1 and SARS-CoV-2. In one study, CQ inhibited viral entry of SARS-CoV-1 into Vero E6 cells, a cell line that was derived from Vero cells in 1968, through the elevation of endosomal pH and the terminal glycosylation of the cellular entry receptor, ACE2 [368]. Increased pH within the cell, as discussed above, inhibits proteolysis, and terminal glycosylation of ACE2 is thought to interfere with virus-receptor binding. An in vitro study of SARS-CoV-2 infection of Vero cells found both HCQ and CQ to be effective in inhibiting viral replication, with HCQ being more potent [369]. Additionally, an early case study of three COVID-19 patients reported the presence of antiphospholipid antibodies in all three patients [98]. Antiphospholipid antibodies are central to the diagnosis of the antiphospholipid syndrome, a disorder that HCQ has often been used to treat [370,371,372]. Because the 90% effective concentration (EC90) of CQ in Vero E6 cells (6.90 μM) can be achieved in and tolerated by rheumatoid arthritis patients, it was hypothesized that it might also be possible to achieve the effective concentration in COVID-19 patients [373]. Additionally, HCQ has been found to be effective in treating HIV [374] and chronic Hepatitis C [375]. Together, these studies triggered initial enthusiasm about the therapeutic potential for HCQ and CQ against COVID-19. HCQ/CQ has been proposed both as a treatment for COVID-19 and a prophylaxis against SARS-CoV-2 exposure. HCQ/CQ are sometimes administered with azithromycin (AZ) and/or zinc supplementation.

5.4.2.2.1 Therapeutic Administration of HCQ/CQ

The initial study evaluating HCQ as a treatment for COVID-19 patients was published on March 20, 2020 by Gautret et al. [376]. This non-randomized, non-blinded, non-placebo clinical trial compared HCQ to standard of care (SOC) in 42 hospitalized patients in southern France. They reported that patients who received HCQ showed higher rates of virological clearance by nasopharyngeal swab on Days 3-6 when compared to standard care. This study also treated six patients with both HCQ + AZ and found this combination therapy to be more effective than HCQ alone. However, the design and analyses used showed weaknesses that severely limit interpretability of results, including the lack of randomization, lack of blinding, lack of placebo, lack of Intention-To-Treat analysis, lack of correction for sequential multiple comparisons, trial arms entirely confounded by hospital, false negatives in outcome measurements, lack of trial pre-registration, and small sample size. Two of these weaknesses are due to inappropriate data analysis and can therefore be corrected post hoc by recalculating the p-values (lack of Intention-To-Treat analysis and multiple comparisons). However, all other weaknesses are fundamental design flaws and cannot be corrected for. Thus, conclusions cannot be generalized outside of the study. The International Society of Antimicrobial Chemotherapy, the scientific organization that publishes International Journal of Antimicrobial Agents where the article appeared, has announced that the article does not meet its expected standard for publications [377], although it has not been officially retracted. Because of the preliminary data presented in this study, the use of HCQ in COVID-19 treatment was subsequently explored by other researchers. About one week later, a follow-up case study reported that 11 consecutive patients were treated with HCQ + AZ using the same dosing regimen [378]. One patient died, two were transferred to the ICU, and one developed a prolonged QT interval, leading to discontinuation of HCQ + AZ. As in the Gautret et al. study, the outcome assessed was virological clearance at Day 6 post-treatment, as measured in nasopharyngeal swabs. Of the ten living patients on Day 6, eight remained positive for SARS-CoV-2 RNA. Like in the original study, interpretability was severely limited by the lack of a comparison group and the small sample size. However, these results stand in contrast to the claims by Gautret et al. that all six patients treated with HCQ + AZ tested negative for SARS-CoV-2 RNA by Day 6 post-treatment. This case study illustrated the need for further investigation using robust study design to evaluate the efficacy of HCQ and/or CQ.

On April 10, 2020, a randomized, non-placebo trial of 62 COVID-19 patients at the Renmin Hospital of Wuhan University was released [379]. This study investigated whether HCQ decreased time to fever break or time to cough relief when compared to SOC [379]. This trial found HCQ decreased both average time to fever break and average time to cough relief, defined as mild or no cough. While this study improved on some of the methodological flaws in Gautret et al. by randomizing patients, it also had several flaws in trial design and data analysis that prevent generalization of the results. These weaknesses include the lack of placebo, lack of correction for multiple primary outcomes, inappropriate choice of outcomes, lack of sufficient detail to understand analysis, drastic disparities between pre-registration and published protocol, and small sample size. The choice of outcomes may be inappropriate as both fevers and cough may break periodically without resolution of illness. Additionally, for these outcomes, the authors reported that 23 of 62 patients did not have a fever and 25 of 62 patients did not have a cough at the start of the study, but the authors failed to describe how these patients were included in a study assessing time to fever break and time to cough relief. It is important to note here that the authors claimed “neither the research performers nor the patients were aware of the treatment assignments.” This blinding seems impossible in a non-placebo trial because at the very least, providers would know whether they were administering a medication or not, and this knowledge could lead to systematic differences in the administration of care. Correction for multiple primary outcomes can be adjusted post hoc by recalculating p-values, but all of the other issues were design and statistical weaknesses that cannot be corrected for. Additionally, the observation of drastic disparities between pre-registration and published protocol could indicate p-hacking [380]. The design limitations mean that the conclusions cannot be generalized outside of the study. A second randomized trial, conducted by the Shanghai Public Health Clinical Center, analyzed whether HCQ increased rates of virological clearance at day 7 in respiratory pharyngeal swabs compared to standard care [381]. This trial was published in Chinese along with an abstract in English, and only the English abstract was read and interpreted for this review. The trial found comparable outcomes in virological clearance rate, time to virological clearance, and time to body temperature normalization between the treatment and control groups. A known weakness is small sample size, with only 30 patients enrolled and 15 in each arm. This problem suggests the study is underpowered to detect potentially useful differences and precludes interpretation of results. Additionally, because only the abstract could be read, other design and analysis issues could be present. Thus, though these studies added randomization to their assessment of HCQ, their conclusions should be interpreted very cautiously. These two studies assessed different outcomes and reached differing conclusions about the efficacy of HCQ for treating COVID-19; the designs of both studies, especially with respect to sample size, meant that no general conclusions can be made about the efficacy of the drug.

Several widely reported studies on HCQ have issues with data integrity and/or provenance. A Letter to the Editor published in BioScience Trends on March 16, 2020 claimed that numerous clinical trials have shown that HCQ is superior to control treatment in inhibiting the exacerbation of COVID-19 pneumonia [382]. This letter has been cited by numerous primary literature, review articles, and media alike [383,384]. However, the letter referred to 15 pre-registration identifiers from the Chinese Clinical Trial Registry. When these identifiers are followed back to the registry, most trials claim they are not yet recruiting patients or are currently recruiting patients. For all of these 15 identifiers, no data uploads or links to publications could be located on the pre-registrations. At the very least, the lack of availability of the primary data means the claim that HCQ is efficacious against COVID-19 pneumonia cannot be verified. Similarly, a recent multinational registry analysis [385] analyzed the efficacy of CQ and HCQ with and without a macrolide, which is a class of antibiotics that includes Azithromycin, for the treatment of COVID-19. The study observed 96,032 patients split into a control and four treatment conditions (CQ with and without a macrolide; HCQ with and without a macrolide). They concluded that treatment with CQ and HCQ was associated with increased risk of de novo ventricular arrhythmia during hospitalization. However, this study has since been retracted by The Lancet due to an inability to validate the data used [386]. These studies demonstrate that increased skepticism in evaluation of the HCQ/CQ and COVID-19 literature may be warranted, possible because of the significant attention HCQ and CQ have received as possible treatments for COVID-19 and the politicization of these drugs.

Despite the fact that the study suggesting that CQ/HCQ increased risk of ventricular arrhythmia in COVID-19 patients has now been retracted, previous studies have identified risks associated with HCQ/CQ. A patient with systemic lupus erythematosus developed a prolonged QT interval that was likely exacerbated by use of HCQ in combination with renal failure [387]. A prolonged QT interval is associated with ventricular arrhythmia [388]. Furthermore, a separate study [389] investigated the safety associated with the use of HCQ with and without macrolides between 2000 and 2020. The study involved 900,000 cases treated with HCQ and 300,000 cases treated with HCQ + AZ. The results indicated that short-term use of HCQ was not associated with additional risk, but that HCQ + AZ was associated with an enhanced risk of cardiovascular complications (15-20% increased risk of chest pain) and a two-fold increased risk of mortality. Therefore, whether studies utilize HCQ alone or HCQ in combination with a macrolide may be an important consideration in assessing risk. As results from initial investigations of these drug combinations have emerged, concerns about the efficacy and risks of treating COVID-19 with HCQ and CQ has led to the removal of CQ/HCQ from SOC practices in several countries [390,391]. As of May 25, 2020, WHO had suspended administration of HCQ as part of the worldwide Solidarity Trial [392], and later the final results of this large-scale trial that compared 947 patients administered HCQ to 906 controls revealed no effect on the primary outcome, mortality during hospitalization (rate ratio: 1.19; P=0.23)

As additional research has emerged, HCQ/CQ have increasingly been demonstrated to be ineffective against COVID-19 and to carry a number of significant side effects. A randomized, open-label, non-placebo trial of 150 COVID-19 patients was conducted in parallel at 16 government-designated COVID-19 centers in China to assess the safety and efficacy of HCQ [393]. The trial compared treatment with HCQ in conjunction with SOC to SOC alone in 150 infected patients who were assigned randomly to the two groups (75 per group). The primary endpoint of the study was the negative conversion rate of SARS-CoV-2 in 28 days, and the investigators found no difference in this parameter between the groups. The secondary endpoints were an amelioration of the symptoms of the disease such as axillary temperature ≤36.6°C, SpO2 >94% on room air, and disappearance of symptoms like shortness of breath, cough, and sore throat. The median time to symptom alleviation was similar across different conditions (19 days in HCQ+SOC vs. 21 days in SOC). Additionally, 30% of the patients receiving SOC+HCQ reported adverse outcomes compared to 8.8% of patients receiving only SOC, with the most common adverse outcome in the SOC+HCQ group being diarrhea (10% vs. 0% in the SOC group, p=0.004). However, there are several factors that limit the interpretability of this study. Most of the enrolled patients had mild-to-moderate symptoms (98%), and the average age was 46. SOC in this study included the use of antivirals (Lopinavir-Ritonavir, Arbidol, Oseltamivir, Virazole, Entecavir, Ganciclovir, and Interferon alfa), which appeared to introduce confounding effects. Thus, to isolate the effect of HCQ, SOC would need to exclude the use of antivirals. In this trial, the samples used to test for the presence of the SARS-CoV-2 virus were collected from the upper respiratory tract, and the authors indicated that the use of upper respiratory samples may have introduced false negatives (e.g., [78]). Another limitation of the study that the authors acknowledge was that the HCQ treatment began, on average, at a 16-day delay from the symptom onset. The fact that this study was open-label and lacked a placebo limits interpretation, and additional analysis is required to determine whether HCQ reduces inflammatory response. Therefore, despite some potential areas of investigation identified in post hoc analysis, this study cannot be interpreted as providing support for HCQ as a therapeutic against COVID-19.

Additional evidence comes from a retrospective analysis [394] that examined data from 368 COVID-19 patients across all United States Veteran Health Administration medical centers. The study retrospectively investigated the effect of the administration of HCQ (n=97), HCQ + AZ (n=113), and no HCQ (n=158) on 368 patients. The primary outcomes assessed were death and the need for mechanical ventilation. Standard supportive care was rendered to all patients. Due to the low representation of women (N=17) in the available data, the study included only men, and the median age was 65 years. The rate of death in the HCQ-only treatment condition was 27.8% and in the HCQ + AZ treatment condition, it was 22.1%. In comparison to the 14.1% rate of death in the no-HCQ cohort, these data indicated a statistically significant elevation in the risk of death for the HCQ-only group compared to the no-HCQ group (adjusted HR: 2.61, p=0.03), but not for the HCQ + AZ group compared to the no-HCQ group (adjusted HR: 1.14; p=0.72). Further, the risk of ventilation was similar across all three groups (adjusted HR: 1.43, p=0.48 (HCQ) and 0.43, p=0.09 (HCQ + AZ) compared to no HCQ). The study thus showed evidence of an association between increased mortality and HCQ in this cohort of COVID-19 patients but no change in rates of mechanical ventilation among the treatment conditions. The study had a few limitations: it was not randomized, and the baseline vital signs, laboratory tests, and prescription drug use were significantly different among the three groups. All of these factors could potentially influence treatment outcome. Furthermore, the authors acknowledge that the effect of the drugs might be different in females and pediatric subjects, since these subjects were not part of the study. The reported result that HCQ + AZ is safer than HCQ contradicts the findings of the previous large-scale analysis of twenty years of records that found HCQ + AZ to be more frequently associated with cardiac arrhythmia than HCQ alone [389]; whether this discrepancy is caused by the pathology of COVID-19, is influenced by age or sex, or is a statistical artifact is not presently known.

Finally, findings from the Randomized Evaluation of COVID-19 Therapy (RECOVERY) trial were released on October 8, 2020. This study used a randomized, open-label design to study the effects of HCQ compared to standard care at 176 hospitals in the United Kingdom [395]. This large study enrolled 11,197 hospitalized patients whose physicians believed it would not harm them to participate. Patients were randomized into either the control group or one of the treatment arms, with twice as many patients enrolled in the control group as any treatment group. Of the patients eligible to receive HCQ, 1,561 were randomized into the HCQ arm while 3,155 were randomized into the control arm. The demographics of the HCQ and control groups were similar in terms of average age (65 years), proportion female (approximately 38%), ethnic make-up (73% versus 76% white), and prevalence of pre-existing conditions (56% versus 57% overall). In the HCQ arm of the study, patients received 800 mg at baseline and again after 6 hours, then 400 mg at 12 hours and every subsequent 12 hours. The primary outcome analyzed was all-cause mortality, and patient vital statistics were reported by physicians upon discharge or death, or else at 28 days following HCQ administration if they remained hospitalized. The secondary outcome assessed was the combined risk of progression to invasive mechanical ventilation or death within 28 days. By the advice of an external data monitoring committee, the HCQ arm of the study was reviewed early, leading to it being closed due a lack of support for HCQ as a treatment for COVID-19. Patients who received HCQ had a longer duration of hospitalization than patients receiving usual care, were less likely to be discharged alive within 28 days, and were more likely to progress to mechanical ventilation. This large-scale study thus builds upon studies in the United States and China to suggest that HCQ is not an effective treatment, and in fact may negatively impact COVID-19 patients due to its side effects. The rates of COVID-19-related reported in the RECOVERY trial did not differ between the control and HCQ arms, but patients receiving HCQ were more likely to die due to cardiac events. Therefore, though none of the studies have been blinded, taken together it becomes clear that all of the available evidence points to significant concerns about the administration of HCQ to hospitalized COVID-19 patients, without providing any support for its efficacy.

5.4.2.2.2 HCQ for the Treatment of Mild Cases

One additional possible therapeutic application considered was the treatment of mild COVID-19 cases in otherwise healthy individuals. This possibility was assessed in a randomized, open-label, multi-center analysis conducted in Catalonia (Spain) [396]. This analysis enrolled adults 18 and older who had been experiencing mild symptoms of COVID-19 for fewer than five days. Participants were randomized into an HCQ arm (N=136) and a control arm (N=157), and those in the treatment arm were administered 800 mg of HCQ on the first day of treatment followed by 400 mg on each of the subsequent six days. The primary outcome assessed was viral clearance at days 3 and 7 following the onset of treatment, and secondary outcomes were clinical progression and time to complete resolution of symptoms. They found no significant differences between the two groups. This study thus suggests that HCQ does not improve recovery from COVID-19, even in otherwise healthy adult patients with mild symptoms.

5.4.2.2.3 Prophylactic Administration of HCQ

An initial study of the possible prophylactic application of HCQ utilized a randomized, double-blind, placebo-controlled design to analyze the administration of HCQ prophylactically [397]. Asymptomatic adults in the United States and Canada who had been exposed to SARS-CoV-2 within the past four days were enrolled in an online study to see whether administration of HCQ over five days would influence the probability of developing COVID-19 symptoms over a 14-day period. Of the participants, 414 received HCQ and 407 received a placebo. The participants averaged 40 years of age and 51.6% were women. They found no significant difference in the rate of symptomatic illness between the two groups (11.8% HCQ, 14.3% placebo, p=0.35). The HCQ condition was associated with side effects, with 40.1% of patients reporting side effects compared to 16.8% in the control group (p<0.001). However, likely due to the high enrollment of healthcare workers (66% of participants) and the well-known side effects associated with HCQ, a large number of participants were able to correctly identify whether they were receiving HCQ or a placebo (46.5% and 35.7%, respectively). Furthermore, due to a lack of availability of diagnostic testing, only 20 of the 107 cases were confirmed with a PCR-based test to be positive for SARS-CoV-2. The rest were categorized as “probable” or “possible” cases by a panel of four physicians who were blind to the treatment status. One possible confounder is that a patient presenting one or more symptoms, which included diarrhea, was defined as a “possible” case, but diarrhea is also a common side effect of HCQ. Additionally, four of the twenty PCR-confirmed cases did not develop symptoms until after the observation period had completed, suggesting that the 14-day trial period may not have been long enough or that some participants also encountered secondary exposure events. Finally, in addition to the young age of the participants in this study, which ranged from 32 to 51, there were possible impediments to generalization introduced by the selection process, as 2,237 patients who were eligible but had already developed symptoms by day 4 were enrolled in a separate study. It is therefore likely that asymptomatic cases were over-represented in this sample, which would not have been detected based on the diagnostic criteria used. Therefore, while this study does represent the first effort to conduct a randomized, double-blind, placebo-controlled investigation of HCQ’s effect on COVID-19 symptoms in a large sample, the lack of PCR tests and several other design flaws significantly impede interpretation of the results. However, in line with the results from therapeutic studies, there is no evidence that HCQ provides protection against COVID-19.

A second study [398] examined the effect of administering HCQ to healthcare workers as a pre-exposure prophylactic. The primary outcome assessed was the conversion from SARS-CoV-2 negative to SARS-CoV-2 positive status over the 8 week study period. This study was also randomized, double-blind, and placebo-controlled and sought to address some of the limitations of the first prophylactic study. They aimed to enroll 200 healthcare workers, preferentially those working with COVID-19 patients, at two hospitals within the University of Pennsylvania hospital system in Philadelphia, PA. Participants were randomized 1:1 to receive either 600 mg of HCQ daily or a placebo, and their SARS-CoV-2 infection status and antibody status were assessed using RT-PCR and serological testing, respectively, at baseline, 4 weeks, and 8 weeks following the beginning of the treatment period. The statistical design of the study accounted for interim analyses at 50 and 100 participants in case efficacy or futility of HCQ for prophylaxis became clear earlier than completion of enrollment. The 139 individuals enrolled comprised a study population that was fairly young (average age 33) and made of largely of people who were white, women, and without pre-existing conditions. At the second interim analysis, more individuals in the treatment group than the control group had contracted COVID-19 (4 versus 3), causing the estimate z-score to fall below the pre-established threshold for futility. As a result, the trial was terminated early, offering additional evidence against the use of HCQ for prophylaxis.

5.4.2.2.4 Summary of HCQ/CQ Research Findings

Early in vitro evidence indicated that HCQ could be an effective therapeutic against SARS-CoV-2 and COVID-19, leading to early interest in its potential as both a therapeutic and prophylaxis. Initially it was hypothesized that CQ/HCQ might be effective against SARS-CoV-2, especially since CQ and HCQ have both been found to inhibit the expression of CD154 in T-cells and to reduce TLR signaling that leads to the production of pro-inflammatory cytokines [399]. Clinical trials for COVID-19 have more often used HCQ rather than CQ because it offers the advantages of being cheaper and having fewer side effects than CQ. However, a large analysis of patients receiving HCQ from January 2000 through March 2020 reported that the combination of HCQ and azithromycin, but not other macrolides, was associated with an elevated risk of cardiovascular complications and mortality. Multiple clinical studies have already been carried out to assess HCQ as a therapeutic agent for COVID-19, and many more are in progress. To date, none of these studies have used randomized, double-blind, placebo-controlled designs with a large sample size, which would be the gold standard. Despite the design limitations (which would be more likely to produce false positives than false negatives), initial optimism about HCQ has not been substantiated by these studies. The most methodologically rigorous HCQ-focused analysis to date [397] evaluated the prophylactic potential of HCQ and found no significant differences between the treatment and control groups, and the WHO’s global Solidarity trial similarly reported no effect of HCQ on mortality. Thus, HCQ/CQ are not likely to be effective therapeutic or prophylactic agents against COVID-19. Additionally, one study identified an increased risk of mortality in older men receiving HCQ, and administration of HCQ and HCQ+AZ did not decrease the use of mechanical ventilation in these patients [394]. HCQ use for COVID-19 could also lead to shortages for anti-malarial or anti-rheumatic use, where it has been definitively proven to be effective. Despite significant attention, these drugs appear to be ineffective against COVID-19. Several countries have now removed CQ from their SOC for COVID-19 due to the lack of evidence of efficacy and the frequency of adverse effects.

5.4.2.3 Dexamethasone

Dexamethasone (9α-fluoro-16α-methylprednisolone) is a synthetic corticosteroid that binds to glucocorticoid receptors [400,401]. It was first synthesized in the late 1950s as an anti-inflammatory and has been used to treat rheumatoid arthritis and other inflammatory conditions [402,403]. Steroids such as dexamethasone are widely available and affordable, and they are often used to treat community-acquired pneumonia [404]. A clinical trial that began in 2012 recently reported that dexamethasone may improve outcomes for patients with ARDS [405]. However, meta-analysis of a small amount of available data regarding the use of dexamethasone to treat SARS suggested that it may, in fact, be associated with patient harm [406], although these findings may have been biased by the fact that all of the studies examined were observational and a large number of inconclusive studies were not included [407]. Dexamethasone works as an anti-inflammatory by binding to glucocorticoid receptors with higher affinity than endogenous cortisol [408]. In order to understand how dexamethasone works to reduce inflammation, it is necessary to consider the stress response more broadly. In response to stress, corticotropin‐releasing hormone stimulates the release of neurotransmitters known as catecholamines, such as epinephrine, and steroid hormones known as glucocorticoids, such as cortisol [409,410]. While catecholamines are often associated with the fight-or-flight response, the specific role that glucocorticoids play is less clear, although they are thought to be important to restoring homeostasis [411]. Immune challenge is a stressor that is known to interact closely with the stress response. The immune system can therefore interact with the central nervous system; for example, macrophages can both respond to and produce catecholamines [409]. Additionally, the production of both catecholamines and glucocorticoids is associated with inhibition of proinflammatory cytokines such as IL-6, IL-12, and tumor necrosis factor-α (TNF‐α) and the stimulation of anti-inflammatory cytokines such as IL-10, meaning that the stress response can regulate inflammatory immune activity [410]. Administration of dexamethasone has been found to correspond to dose-dependent inhibition of IL-12 production, but not to affect IL-10 [412]; the fact that this relationship could be disrupted by administration of a glucocorticoid-receptor antagonist suggests that it is regulated by the receptor itself [412]. Thus, the administration of dexamethasone for COVID-19 is likely to simulate the release of glucocorticoids endogenously during stress, resulting in binding of the synthetic steroid to the glucocorticoid receptor and the associated inhibition of the production of proinflammatory cytokines. In this model, dexamethasone reduces inflammation by stimulating the mechanism that reduces inflammation following a threat such as immune challenge. Immunosuppressive drugs such as steroids are typically contraindicated in the setting of infection [413], but because COVID-19 results in hyperinflammation that appears to contribute to mortality via lung damage, immunosuppression may be a helpful approach to treatment [122]. The decision of whether and/or when to counter hyperinflammation with immunosuppression in the setting of COVID-19 was an area of intense debate, as the risks of inhibiting antiviral immunity needed to be weighed against the beneficial anti-inflammatory effects [414]. As a result, guidelines early in the pandemic typically recommended avoiding treating COVID-19 patients with corticosteroids such as dexamethasone [406].

The application of dexamethasone for the treatment of COVID-19 was evaluated as part of the multi-site RECOVERY trial in the United Kingdom [415]. Over 6,000 hospitalized COVID-19 patients were assigned into standard care or treatment (dexamethasone) arms of the trial with a 2:1 ratio. At the time of randomization, some patients were ventilated (16%), others were on non-invasive oxygen (60%), and others were breathing independently (24%). Patients in the treatment arm were administered dexamethasone either orally or intravenously at 6 mg per day for up to 10 days. The primary end-point was the patient’s status at 28-days post-randomization (mortality, discharge, or continued hospitalization), and secondary outcomes analyzed included the progression to invasive mechanical ventilation over the same period. The 28-day mortality rate was found to be lower in the treatment group than in the standard care group (21.6% vs 24.6%, p <0.001). However, this finding was driven by differences in mortality among patients who were receiving mechanical ventilation or supplementary oxygen at the start of the study. The report indicated that dexamethasone reduced 28-day mortality relative to standard care in patients who were ventilated (29.3% vs. 41.4%) and among those who were receiving oxygen supplementation (23.3% vs. 26.2%) at randomization, but not in patients who were breathing independently (17.8% vs. 14.0%). One possible confounder is that patients receiving mechanical ventilation tended to be younger than patients who were not receiving respiratory support (by 10 years on average) and to have had symptoms for a longer period. However, adjusting for age did not change their conclusions, although the duration of symptoms was found to be significantly associated with the effect of dexamethasone administration. These findings also suggested that dexamethasone may have reduced progression to mechanical ventilation, especially among patients who were receiving oxygen support at randomization. Thus, this large, randomized, and multi-site, albeit not placebo-controlled, study suggests that administration of dexamethasone to patients who are unable to breathe independently may significantly improve survival outcomes. Additionally, dexamethasone is a widely available and affordable medication, raising the hope that it could be made available to COVID-19 patients globally.

The results of the RECOVERY trial’s analysis of dexamethasone suggest that this therapeutic is effective primarily in patients who had been experiencing symptoms for at least seven days and patients who were not breathing independently [416]. Thus, it seems likely that dexamethasone is useful for treating inflammation associated with immunopathy or cytokine release syndrome. In fact, corticosteroids such as dexamethasone are sometimes used to treat CRS [417]. It is not surprising that administration of an immunosuppressant would be most beneficial when the immune system was dysregulated towards inflammation. However, it is also unsurprising that care must be taken in administering an immunosuppressant to patients fighting a viral infection. In particular, the concern has been raised that treatment with dexamethasone might increase patient susceptibility to concurrent (e.g., nosocomial) infections [418]. Additionally, the drug could potentially slow viral clearance and inhibit patients’ ability to develop antibodies to SARS-CoV-2 [406,418]. Furthermore, dexamethasone has been associated with side effects that include psychosis, glucocorticoid-induced diabetes, and avascular necrosis [406], and the RECOVERY trial did not report outcomes with enough detail to be able to determine whether they observed similar complications. However, since these results were released, strategies have been proposed for administering dexamethasone alongside more targeted treatments to minimize the likelihood of negative side effects [418]. Given the available evidence, dexamethasone is currently the most promising treatment for severe COVID-19.

5.5 Biologics

Biologics are produced from components of living organisms or viruses. They include treatments such as humanized monoclonal antibodies (mAb) tocilizumab (TCZ), and neutralizing antibodies (nAbs), as well as prophylactics such as vaccines. Historically produced from animal tissue, biologics have become increasingly feasible to produce as recombinant DNA technologies have advanced [419]. Often, they are glycoproteins or peptides [420], but whole viruses can also be used therapeutically or prophylactically, not only for vaccines but also as vectors for gene therapy or therapeutic proteins or for oncolytic virotherapy [421]. They are typically catabolized by the body to their amino acid components [420]. There are many differences on the development side between biologics and synthesized pharmaceuticals, such as small molecule drugs. Biologics are typically orders of magnitude larger than small molecule drugs and their physiochemical properties are often much less understood [420]. They are often heat sensitive, and their toxicity can vary, as it is not directly associated with the primary effects of the drug [420]. However, this class includes some extremely significant medical breakthroughs, including insulin for the management of diabetes and the smallpox vaccine. As a result, it is another possible avenue through which the pharmacological management of SARS-CoV-2 infection can be explored.

5.5.1 Tocilizumab

TCZ is a receptor antibody that was developed to manage chronic inflammation caused by the continuous synthesis of the cytokine IL-6 [422]. IL-6 is a pro-inflammatory cytokine belonging to the interleukin family, which is comprised by immune system regulators that are primarily responsible for immune cell differentiation. Often used to treat conditions such as rheumatoid arthritis [422], TCZ has become a pharmaceutical of interest for the treatment of COVID-19 because of the role IL-6 plays in this disease. While secretion of IL-6 can be associated with chronic conditions, it is a key player in the innate immune response and is secreted by macrophages in response to the detection of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) [422]. An analysis of 191 in-patients at two Wuhan hospitals revealed that blood concentrations of IL-6 differed between patients who did and did not recover from COVID-19. Patients who ultimately deceased had higher IL-6 levels at admission than those who recovered [35]. Additionally, IL-6 levels remained higher throughout the course of hospitalization in the patients who ultimately deceased [35]. This finding provided some early evidence that COVID-19 deaths may be induced by the hyperactive immune response, often referred to as cytokine release syndrome (CRS) or cytokine storm syndrome (CSS), as IL-6 plays a key role in this response [110]. In this context, the observation of elevated IL-6 in patients who died may reflect an over-production of proinflammatory interleukins, suggesting that TCZ may palliate some of the most severe symptoms of COVID-19 associated with increased cytokine production.

Human IL-6 is a 26-kDa glycoprotein that consists of 184 amino acids and contains two potential N-glycosylation sites and four cysteine residues. It binds to a type I cytokine receptor (IL-6Rα or glycoprotein 80) that exists in both membrane-bound (IL-6Rα) and soluble (sIL-6Rα) forms [423]. It is not the binding of IL-6 to the receptor that initiates pro- and/or anti-inflammatory signaling, but rather the binding of the complex to another subunit, known as IL-6Rβ or glycoprotein 130 (gp13) [423,424]. Unlike membrane-bound IL-6Rα, which is only found on hepatocytes and some types of leukocytes, gp130 is found on most cells [425]. When IL-6 binds to sIL-6Rα, the complex can then bind to a gp130 protein on any cell [425]. The binding of IL-6 to IL-6Rα is termed classical signaling, while its binding to sIL-6Rα is termed trans-signaling [425,426,427]. These two signaling processes are thought to play different roles in health and illness. For example, trans-signaling may play a role in the proliferation of mucosal T-helper TH2 cells associated with asthma, while an earlier step in this proliferation process may be regulated by classical signaling [425]. Similarly, IL-6 is known to play a role in Crohn’s Disease via trans-, but not classical, signaling [425]. Both classical and trans-signaling can occur through three independent pathways: the Janus-activated kinase-STAT3 pathway, the Ras/Mitogen-Activated Protein Kinases (MAPK) pathway and the Phosphoinositol-3 Kinase/Akt pathway [423]. These signaling pathways are involved in a variety of different functions, including cell type differentiation, immunoglobulin synthesis, and cellular survival signaling pathways, respectively [423]. The ultimate result of the IL-6 cascade is to direct transcriptional activity of various promoters of pro-inflammatory cytokines, such as IL-1, TFN, and even IL-6 itself, through the activity of NF-κB [423]. IL-6 synthesis is tightly regulated both transcriptionally and post-transcriptionally, and it has been shown that viral proteins can enhance transcription of the IL-6 gene by strengthening the DNA-binding activity between several transcription factors and IL-6 gene-cis-regulatory elements [428]. Therefore, drugs inhibiting the binding of IL-6 to IL-6Rα or sIL-6Rα are of interest for combating the hyperactive inflammatory response characteristic of CRS and CSS. TCZ is a humanized monoclonal antibody that binds both to the insoluble and soluble receptor of IL-6, providing de facto inhibition of the IL-6 immune cascade.

Tocilizumab is being administered either as an intervention or as concomitant medication in 84 COVID-19 clinical trials (Figure 3). No randomized, placebo-controlled studies of TCZ have currently released results. Therefore, no conclusions can be drawn about its efficacy for the treatment of COVID-19. However, early interest in TCZ as a possible treatment for COVID-19 emerged from a very small retrospective study in China that examined 20 patients with severe symptoms in early February 2020 and reported rapid improvement in symptoms following treatment with TCZ [429]. Subsequently, a number of retrospective studies have been conducted in several countries. Many studies use a retrospective, observational design, where they compare outcomes for COVID-19 patients who received TCZ to those who did not over a set period of time. For example, one of the largest retrospective, observational analysis released to date [430] compared the rates at which patients who received TCZ deceased or progressed to invasive medical ventilation over a 14-day period compared to patients receiving only SOC. Under this definition, SOC could include other drugs such as HCQ, azithromycin, lopinavir-ritonavir or darunavir-cobicistat, or heparin. While this study was not randomized, a subset of patients who were eligible to receive TCZ were unable to obtain it due to shortages; however, these groups were not directly compared in the analysis. After adjusting for variables such as age, sex, and SOFA score, they found that patients treated with TCZ were less likely to progress to invasive medical ventilation and/or death (adjusted HR = 0.61, CI 0.40-0.92, p=0.020), although analysis of death and ventilation separately suggests that this effect may have been driven by differences in the death rate (20% of control versus 7% of TCZ-treated patients). They reported particular benefits for patients whose PaO2/FiO2 ratio, also known as the Horowitz Index for Lung Function, fell below a 150 mm Hg threshold. They found no differences between groups administered subcutaneous versus intravenous TCZ.

Another retrospective observational analysis of interest examined the charts of patients at a hospital in Connecticut, USA where 64% of all 239 COVID-19 patients in the study period were administered TCZ based on assignment by a standardized algorithm [431]. They found that TCZ administration was associated with more similar rates of survivorship in patients with severe versus nonsevere COVID-19 at intake, defined based on the amount of supplemental oxygen needed. They therefore proposed that their algorithm was able to identify patients presenting with or likely to develop CRS as good candidates for TCZ. This study also reported higher survivorship in Black and Hispanic patients compared to white patients when adjusted for age. The major limitation with interpretation for these studies is that there may be clinical characteristics that influenced medical practitioners decisions to administer TCZ to some patients and not others. One interesting example therefore comes from an analysis of patients at a single hospital in Brescia, Italy, where TCZ was not available for a period of time [432]. This study compared COVID-19 patients admitted to the hospital before and after March 13, 2020, when the hospital received TCZ. Therefore, patients who would have been eligible for TCZ prior to this arbitrary date did not receive it as treatment, making this retrospective analysis something of a natural experiment. Despite this design, demographic factors did not appear to be consistent between the two groups, and the average age of the control group was older than the TCZ group. The control group also had a higher percentage of males and a higher incidence of comorbidities such as diabetes and heart disease. All the same, the multivariate HR, which adjusted for these clinical and demographic factors, found a significant difference between survival in the two groups (HR=0.035, CI=0.004-0.347, p = 0.004). They reported improvement of survival outcomes after the addition of TCZ to their SOC regime, with 11 of 23 patients (47.8%) admitted prior to March 13th dying compared to 2 of 62 (3.2%) admitted afterwards. They also reported a reduced progression to mechanical ventilation in the TCZ group. However, this study also holds a significant limitation: the time delay between the two groups means that knowledge about how to treat the disease likely improved over this timeframe as well. All the same, the results of these observational retrospective studies provide support for TCZ as a pharmaceutical of interest for follow-up in clinical trials.

In addition to the retrospective observational studies, other analysis have utilized a retrospective case-control design to match pairs of patients with similar baseline characteristics, only one of whom received TCZ for COVID-19. In one such study, TCZ was significantly associated with a reduced risk of progression to ICU admission or death [433]. This study examined only 20 patients treated with TCZ (all but one of the patients treated with TCZ in the hospital during the study period) and compared them to 25 patients receiving SOC. For the combined primary endpoint of death and/or ICU admission, only 25% of patients receiving TCZ progressed to an endpoint compared to 72% in the SOC group (p = 0.002, presumably based on a chi-square test based on the information provided in the text). When the two endpoints were examined separately, progression to invasive medical ventilation remained significant (32% SOC compared to 0% TCZ, p = 0.006) but not for mortality (48% SOC compared to 25% TCZ, p = 0.066). In contrast, a study that compared 96 patients treated with TCZ to 97 patients treated with SOC only in New York City found that differences in mortality did not differ between the two groups, but that this difference did become significant when intubated patients were excluded from the analysis [434]. Taken together, these findings suggest that future clinical trials of TCZ may want to include intubation as an endpoint. However, these studies should be approached with caution, not only because of the small number of patients enrolled and the retrospective design, but also because they performed a large number of statistical tests and did not account for multiple hypothesis testing. These last findings highlight the need to search for a balance between impairing a harmful immune response, such as the one generated during CRS and CSS, and preventing the worsening of the clinical picture of the patients by potential new viral infections.

Though data about TCZ for COVID-19 is still only just emerging, some meta-analyses and systematic reviews have investigated the available data. One meta-analysis [435] evaluated 19 studies published or released as preprints prior to July 1, 2020 and found that the overall trends supportive of the frequent conclusion that TCZ does improve survivorship, with a significant HR of 0.41. This trend improved when they excluded studies that administered a steroid alongside TCZ, with a significant HR of 0.04. They also found some evidence for reduced IMV or ICU admission, but only when excluding all studies except a small number reporting estimates that were adjusted for possible bias introduced by the challenges of stringency during the enrollment process. A systematic analysis of nine case-control studies estimated a HR of 0.482, which was also significant [436]. Although these estimates are similar, it is important to note that they are drawing from the same literature and are therefore likely to be affected by the same biases in publication. A second systematic review of studies investigating TCZ treatment for COVID-19 analyzed 31 studies that had been published or released as pre-prints and reported that none carried a low risk of bias (RoB) [437]. Therefore, the present evidence is not likely to be sufficient for conclusions about the efficacy of TCZ.

Additionally, there are possible risks associated with the administration of TCZ for COVID-19. TCZ has been used for over a decade to treat rheumatoid arthritis [438], and a recent study found the drug to be safe for pregnant and breastfeeding women [439]. However, TCZ may increase the risk of developing infections [438], and rheumatoid arthritis patients with chronic hepatitis B infections had a high risk of hepatitis B virus reactivation when TCZ was administered in combination with other RA drugs [440]. As a result, TCZ is contraindicated in patients with active infections such as tuberculosis [441]. Previous studies have investigated, with varying results, a possible increased risk of infection in rheumatoid arthritis patients administered TCZ [442,443], although another study reported that the incidence rate of infections was higher in clinical practice rheumatoid arthritis patients treated with TCZ than in the rates reported by clinical trials [444]. In the investigation of 544 Italian COVID-19 patients, the group treated with TCZ was found to be more likely to develop secondary infections, with 24% compared to 4% in the control group [430]. Reactivation of hepatitis B and herpes simplex virus 1 was also reported in a small number of patients in this study, all of whom were receiving TCZ. A July 2020 case report described negative outcomes of two COVID-19 patients after receiving TCZ, including one death; however, both patients were intubated and had entered septic shock prior to receiving TCZ [445], likely indicating a severe level of cytokine production. Additionally, D-dimer and sIL2R levels were reported by one study to increase in patients treated with TCZ, which raised concerns because of the potential association between elevated D-dimer levels and thrombosis and between sIL2R and diseases where T-cell regulation is compromised [431]. An increased risk of bacterial infection was also identified in a systematic review of the literature, based on the unadjusted estimates reported [435]. In summary, TCZ administration to COVID-19 patients is not without risks, may introduce additional risk of developing secondary infections, and should be approached especially cautiously for patients who have latent viral infections.

In summary, approximately 25% of coronavirus patients develop ARDS, which is caused by an excessive early response of the immune system which can be a component of cytokine release syndrome [431] and cytokine storm syndrome [441]. This overwhelming inflammation is triggered by IL-6. TCZ is an inhibitor of IL-6 and therefore may be able to neutralize the inflammatory pathway that leads to the cytokine storm. While the mechanism suggests TCZ may be beneficial for the treatment of COVID-19 patients experiencing excessive immune activity, no randomized controlled trials are available assessing its effect. However, small initial studies have found preliminary indications that TCZ may reduce progression to invasive medical ventilation and/or death. It should be noted that SOC varied widely across retrospective studies, with one study administering HCQ, lopinavir-ritonavir, antibiotics, and/or heparin as part of standard care. Interest in TCZ as a treatment for COVID-19 was supported by two meta-analyses that converged on a HR estimate of approximately 0.45 [435,436], but a third meta-analysis found that all of the available literature carries a risk of bias, with even the largest available TCZ studies to date carrying a moderate risk of bias under the ROBINS-I criteria [437]. Additionally, different studies used different dosages, number of doses, and methods of administration; ongoing research may be needed to optimize administration of TCZ [446], although similar results were reported by one study for intravenous and subcutaneous administration [430]. Clinical trials that are in progress are likely to provide additional insight into the effectiveness of this drug for the treatment of COVID-19 along with how it should be administered.

5.5.2 Neutralizing Antibodies

Monoclonal antibodies have revolutionized the way we treat human diseases. They have become some of the best-selling drugs in the pharmaceutical market in recent years [447]. There are currently 79 FDA approved mAbs on the market including antibodies for viral infections (e.g. Ibalizumab for HIV and Palivizumab for RSV) [447,448]. Virus-specific neutralizing antibodies commonly target viral surface glycoproteins or host structures, thereby inhibiting viral entry through receptor binding interference [449,450]. This section discusses current efforts in developing neutralizing antibodies against SARS-CoV-2 and how expertise gained from previous approaches for MERS-CoV and SARS-CoV-1 may benefit antibody development.

5.5.2.1 Spike (S) Neutralizing Antibody

During the first SARS epidemic in 2002, nAbs were found in SARS-CoV-1-infected patients [451,452]. Several studies following up on these findings identified various S-glycoprotein epitopes as the major targets of neutralizing antibodies against SARS-CoV-1 [453]. The passive transfer of immune serum containing nAbs from SARS-CoV-1-infected mice resulted in protection of naïve mice from viral lower respiratory tract infection upon intranasal challenge [454]. Similarly, a meta-analysis suggested that administration of plasma from recovered SARS-CoV-1 patients reduced mortality upon SARS-CoV-1 infection [455]. Similar results were observed in MERS-CoV infection during the second coronavirus-related epidemic of the 21st century. In these cases, neutralizing antibodies were identified against various epitopes of the receptor binding domain (RBD) of the S glycoprotein [456,457]. Coronaviruses use trimeric spike (S) glycoproteins on their surface to bind to host cell receptors, such as ACE2, allowing for cell entry [63,66]. Each S glycoprotein protomer is comprised of an S1 domain, also called the RBD, and an S2 domain. The S1 domain binds to host cell receptors while the S2 domain facilitates the fusion between the viral envelope and host cell membranes [453]. Although targeting of the host cell receptor ACE2 shows efficacy in inhibiting SARS-CoV-2 infection [74], given the physiological relevance of ACE2 [458], it would be favorable to target virus-specific structures rather than host receptors. This forms the rationale of developing neutralizing antibodies against the S glycoprotein, disrupting its interaction with ACE2 and other receptors and thereby inhibiting viral entry.

The first human neutralizing antibody against SARS-CoV-2 targeting the trimeric spike (S) glycoproteins was developed using hybridoma technology [459], where antibody-producing B-cells developed by mice can be inserted into myeloma cells to produce a hybrid cell line (the hybridoma) that is grown in culture. The 47D11 clone was able to cross-neutralize SARS-CoV-1 and SARS-CoV-2 by a mechanism that is different from receptor binding interference. The exact mechanism of how this clone neutralizes SARS-CoV-2 and inhibits infection in vitro remains unknown, but a potential mechanism might be antibody-induced destabilization of the membrane prefusion structure [459,460]. The ability of this antibody to prevent infection at a feasible dose needs to be validated in vivo, especially since in vitro neutralization effects have been shown to not be reflective of in vivo efficacy [461]. Only a week later, a different group successfully isolated multiple nAbs targeting the RBD of the S glycoprotein from blood samples taken from COVID-19 patients in China [150]. Interestingly, the patient-isolated antibodies did not cross-react with RBDs from SARS-CoV-1 and MERS-CoV, although cross-reactivity to the trimeric spike proteins of SARS-CoV-1 and MERS-CoV was observed. This finding suggests that the RBDs between the three coronavirus species are immunologically distinct and that the isolated nAbs targeting the RBD of SARS-CoV-2 are species specific. While this specificity is desirable, it also raises the question of whether these antibodies are more susceptible to viral escape mechanisms. Viral escape is a common resistance mechanism to nAb therapy due to selective pressure from neutralizing antibodies [462,463]. For HIV, broadly neutralizing antibodies (bnAbs) targeting the CD4 binding site (CD4bs) show greater neutralization breadth than monoclonal antibodies, which target only specific HIV strains [464]. For MERS-CoV, a combination of multiple neutralizing antibodies targeting different antigenic sites prevented neutralization escape [465]. It was found that the different antibody isolates did not target the same epitopes, suggesting that using them in combination might produce a synergistic effect that prevents viral escape [150]. It was also demonstrated that binding affinity of the antibodies does not reflect their capability to compete with ACE2 binding. Furthermore, no conclusions about correlations between the severity of disease and the ability to produce neutralizing antibodies can be drawn at this point. Rather, higher neutralizing antibody titers were more frequently found in patients with severe disease. Correspondingly, higher levels of anti-spike IgG were observed in patients that deceased from infection compared to patient that recovered [466].

Results from the SARS and MERS epidemics thus provide valuable lessons for the design of neutralizing antibodies for the current outbreak. The findings for SARS-CoV-1 and MERS-CoV can aid in identifying which structures constitute suitable targets for nAbs, despite the fact that the RBD appears to be distinct between the three coronavirus species. These studies also suggest that a combination of nAbs targeting distinct antigens might be necessary to provide protection [465]. The biggest challenge remains identifying antibodies that not only bind to their target, but also prove to be beneficial for disease management. On that note, a recently published study indicates that anti-spike antibodies could make the disease worse rather than eliminating the virus [466]. These findings underscores our current lack of understanding the full immune response to SARS-CoV-2.

5.5.3 Interferons

IFNs are a family of cytokines critical to activating the innate immune system response against viral infections. Interferons are classified into three categories based on their receptor specificity: types I, II and III [110]. Specifically, IFNs I (IFN-𝛼 and 𝛽) and II (IFN-𝛾) induce the expression of antiviral proteins that bring the viral RNA to degradation [467]. Among these IFNs, IFN-𝛽 has already been found to strongly inhibit the replication of other coronaviruses, such as SARS-CoV-1, in cell culture, while IFN-𝛼 and 𝛾 were shown to be less effective in this context [467]. There is evidence that patients with higher susceptibility to ARDS indeed show deficiency in IFN-𝛽. For instance, infection with other coronaviruses impairs IFN-𝛽 expression and synthesis, allowing the virus to escape the innate immune response [468]. On March 18 2020, Synairgen plc received approval to start a phase II trial for SNG001, an IFN-𝛽-1a formulation to be delivered to the lungs via inhalation. SNG001 was already shown to be effective in reducing viral load in an in vivo model of swine flu and in vitro models of other coronavirus infections [469]. In July, a press release from Synairgen stated that SNG001 reduced progression to ventilation in a double-blind, placebo-controlled, multi-center study of 101 patients with an average age in the late 50s [470]. They also reported that patients in the treatment group showed greater recovery and lower breathlessness. However, given that this information was released in a press release rather than in a manuscript and thus cannot be thoroughly reviewed, these findings should be considered preliminary. Additionally, the WHO Solidarity trial reported no significant effect of IFN-𝛽1a on patient survival during hospitalization [???].

5.5.4 Vaccines

5.5.4.1 Vaccine Development

Flu-like illnesses caused by viruses are a common target of vaccine development programs, and influenza vaccine technology in particular has made many strides. During the H1N1 influenza outbreak, vaccine development was accelerated because of the existing infrastructure, along with the fact that regulatory agencies had already decided that vaccines produced using egg- and cell-based platforms could be licensed under the regulations used for a strain change. Critiques of the production and distribution of the H1N1 vaccine have stressed the need for alternative development-and-manufacturing platforms that can be readily adapted to new pathogens. Although a monovalent H1N1 vaccine was not available before the pandemic peaked in the United States and Europe, it was available soon afterward as a stand-alone vaccine that was eventually incorporated into commercially available seasonal influenza vaccines [471]. If H1N1 vaccine development provides any indication, considering developing and manufacturing platforms for promising COVID-19 vaccine trials early could hasten the emergence of an effective prophylactic vaccine against SARS-CoV-2.

The first critical step towards developing a vaccine against SARS-CoV-2 was characterizing the target, which fortunately happened early in the COVID-19 outbreak with the sequencing and dissemination of the viral genome [472]. The Coalition for Epidemic Preparedness Innovations (CEPI) is coordinating global health agencies and pharmaceutical companies to develop vaccines against SARS-CoV-2. As of September 2020, there were over 180 vaccine candidates against SARS-CoV-2 in development [473]. Unlike many global vaccine development programs previously, such as with H1N1, the vaccine development landscape for COVID-19 includes vaccines produced by a wide array of technologies. Experience in the field of oncology is encouraging COVID-19 vaccine developers to use next-generation approaches to vaccine development, which have led to the great diversity of vaccine development programs [474]. These diverse technology platforms include DNA, RNA, virus-like particle, recombinant protein, both replicating and non-replicating viral vectors, live attenuated virus, and inactivated virus approaches (Figure 4). Given the wide range of vaccines under development, it is possible that some vaccine products may eventually be shown to be more effective in certain subpopulations, such as children, pregnant women, immunocompromised patients, the elderly, etc. The requirements for a successful vaccine trial and deployment are complex and may require coordination between government, industry, academia, and philanthropic entities [475]. While little is currently known about immunity to SARS-CoV-2, vaccine development typically tests for serum neutralizing activity, as this has been established as a biomarker for adaptive immunity in other respiratory illnesses [476].

Figure 4: Vaccine Development Strategies. Several different strategies can and are being employed for the development of vaccines. Each approach capitalizes on different features of the SARS-CoV-2 virus and delivery through a different platform.
5.5.4.1.1 DNA Vaccines

This vaccination method involves the direct introduction of a plasmid containing a DNA sequence encoding the antigen(s) against which an immune response is sought into appropriate tissues [477]. This approach may offer several advantages over traditional vaccination approaches, such as the stimulation of both B- as well as T-cell responses and the absence of any infectious agent. Currently, a Phase I safety and immunogenicity clinical trial of INO-4800, a prophylactic vaccine against SARS-CoV-2, is underway [478]. The vaccine developer Inovio Pharmaceuticals Technology is overseeing administration of INO-4800 by intradermal injection followed by electroporation with the CELLECTRA® device to healthy volunteers. Electroporation is the application of brief electric pulses to tissues in order to permeabilize cell membranes in a transient and reversible manner. It has been shown that electroporation can enhance vaccine efficacy by up to 100-fold, as measured by increases in antigen-specific antibody titers [479]. The safety of the CELLECTRA® device has been studied for over seven years, and these studies support the further development of electroporation as a safe vaccine delivery method [480]. The temporary formation of pores through electroporation facilitates the successful transportation of macromolecules into cells, allowing cells to robustly take up INO-4800 for the production of an antibody response. Approved by the U.S. FDA on April 6, 2020, the Phase I study is enrolling up to 40 healthy adult volunteers in Philadelphia, PA at the Perelman School of Medicine and at the Center for Pharmaceutical Research in Kansas City, MO. The trial has two experimental arms corresponding to the two locations. Participants in Experimental Group 1 will receive one intradermal injection of 1.0 milligram (mg) of INO-4800 followed by electroporation using the CELLECTRA® 2000 device twice, administered at Day 0 and Week 4. Participants in Experimental Group 2 will receive two intradermal injections of 1.0 mg (total 2.0 mg per dosing visit) of INO-4800 followed by electroporation using the CELLECTRA® 2000 device, administered at Day 0 and Week 4. Safety data and the initial immune responses of participants from the trial are expected by the end of the summer of 2021. The development of a DNA vaccine against SARS-CoV-2 by Inovio could be an important step forward in the world’s search for a COVID-19 vaccine. Although exciting, the cost of vaccine manufacturing and electroporation may make scaling the use of this technology for prophylactic use for the general public difficult.

5.5.4.1.2 RNA Vaccines

RNA vaccines are nucleic-acid based modalities that code for viral antigens against which the human body elicits a humoral and cellular immune response. The mRNA technology is transcribed in vitro and delivered to cells via lipid nanoparticles (LNP) [481]. They are recognized by ribosomes in vivo and then translated and modified into functional proteins [482]. The resulting intracellular viral proteins are displayed on surface MHC proteins, provoking a strong CD8+ T cell response as well as a CD4+ T cell and B cell-associated antibody responses [482]. Naturally, mRNA is not very stable and can degrade quickly in the extracellular environment or the cytoplasm. The LNP covering protects the mRNA from enzymatic degradation outside of the cell [483]. Codon optimization to prevent secondary structure formation and modifications of the poly-A tail as well as the 5’ untranslated region to promote ribosomal complex binding can increase mRNA expression in cells. Furthermore, purifying out dsRNA and immature RNA with FPLC (fast performance liquid chromatography) and HPLC (high performance liquid chromatography) technology will improve translation of the mRNA in the cell [482,484]. Vaccines based on mRNA delivery confer many advantages over traditional viral vectored vaccines and DNA vaccines. In comparison to live attenuated viruses, mRNA vaccines are non-infectious and can be synthetically produced in an egg-free, cell-free environment, thereby reducing the risk of a detrimental immune response in the host [485]. Unlike DNA vaccines, mRNA technologies are naturally degradable and non-integrating, and they do not need to cross the nuclear membrane in addition to the plasma membrane for their effects to be seen [482]. Furthermore, mRNA vaccines are easily, affordably, and rapidly scalable.

Although mRNA vaccines have been developed for therapeutic and prophylactic purposes, none have previously been licensed or commercialized. Nevertheless, they have shown promise in animal models and preliminary clinical trials for several indications, including rabies, coronavirus, influenza, and cytomegalovirus [486]. Preclinical data previously identified effective antibody generation against full-length FPLC-purified influenza hemagglutinin stalk-encoding mRNA in mice, rabbits, and ferrets [487]. Similar immunological responses for mRNA vaccines were observed in humans in Phase I and II clinical trials operated by the pharmaceutical-development companies Curevac and Moderna for rabies, flu, and zika [484]. Positively charged bilayer LNPs carrying the mRNA attract negatively charged cell membranes, endocytose into the cytoplasm [483], and facilitate endosomal escape. LNPs can be coated with modalities recognized and engulfed by specific cell types. LNPs that are 150nm or less effectively enter into lymphatic vessels.

There are three types of RNA vaccines: non-replicating, in vivo self-replicating, and in vitro dendritic cell non-replicating [488]. Non-replicating mRNA vaccines consist of a simple open reading frame (ORF) for the viral antigen flanked by the 5’ UTR and 3’ poly-A tail. In vivo self-replicating vaccines encode a modified viral genome derived from single-stranded, positive sense RNA alphaviruses [482,484]. The RNA genome encodes the viral antigen along with proteins of the genome replication machinery, including an RNA polymerase. Structural proteins required for viral assembly are not included in the engineered genome [482]. Self-replicating vaccines produce more viral antigens over a longer period of time, thereby evoking a more robust immune response [488]. Finally, in vitro dendritic cell non-replicating RNA vaccines limit transfection to dendritic cells. Dendritic cells are potent antigen-presenting immune cells that easily take up mRNA and present fragments of the translated peptide on their MHC proteins, which can then interact with T cell receptors. Ultimately, primed T follicular helper cells can stimulate germinal center B cells that also present the viral antigen to produce antibodies against the virus [489]. These cells are isolated from the patient, grown and transfected ex vivo, and reintroduced to the patient [490].

mRNA-1273 was the first COVID-19 vaccine to enter a phase I clinical in the United States. ModernaTX, Inc. is currently spearheading an investigation on the immunogenicity and reactogenicity of mRNA-1273, a conventional lipid nanoparticle encapsulated RNA encoding a full-length prefusion stabilized spike (S) protein for SARS-CoV-2 [491]. In a study that is ongoing, forty-five participants were enrolled and given intramuscular injections of mRNA-1273 in their deltoid muscle on day 1 and day 29, and then followed for the next twelve months. Healthy males and non-pregnant females aged 18-55 years were recruited for this study and divided into three groups receiving 25, 100, or 250 micrograms (μg) of mRNA-1273. IgG ELISA assays on patient serology samples are being used to examine the immunogenicity of the vaccine [491].

A preliminary report describing initial safety and immunogenicity findings is now available [476]. Binding antibodies were observed at two weeks after the first dose at all concentrations. At the time point one week after the second dose was administered on day 29, the pseudotyped lentivirus reporter single-round-of-infection neutralization assay (PsVNA), which was used to assess neutralizing activity, reached a median level similar to the median observed in convalescent plasma samples. Participants reported mild and moderate systemic adverse events after the day 1 injection, and one severe local event was observed in each of the two highest dose levels. The second injection led to severe systemic adverse events for three of the participants at the highest dose levels, with one participant in the group being evaluated at an urgent care center on the day after the second dose. The reported localized adverse events from the second dose were similar to those from the first.

In summary, mRNA vaccines are promising tools in the prevention and control of pandemics because they may sidestep many challenges associated with traditional vaccine design and manufacturing. As of August 2020, placebo-blinded and randomized results for mRNA vaccines are not yet available, but the initial results are promising. In the dose ranging study, even the lowest dose of 25 μg of mRNA-1273 appeared to be sufficient to induce an immunogenicity profile comparable to that observed in convalescent plasma and appeared to be well tolerated among the population of healthy, non-pregnant participants from 18-55 years of age. Based on the preliminary results from mRNA-1273 [476], a new trial aims to enroll 30,000 participants who will be randomized for two injections of 100 μg of mRNA-1273 or placebo, again spaced 28 days apart [492].

5.5.4.2 Adjuvants for Vaccines

Adjuvants include a variety of molecules or larger microbial-related products that have an effect on the immune system or an immune response of interest. They can either be comprised of or contain immunostimulants or immunomodulators. Adjuvants are sometimes included within vaccines, especially vaccines other than live-attenuated and inactivated viruses, in order to enhance the immune response. A review on the development of SARS-CoV-2 vaccines [493] also included a brief summary of the potential of adjuvants for these vaccines, including a brief description of some already commonly used adjuvants. Different adjuvants can regulate different types of immune responses, so the type or combination of adjuvants used in a vaccine will depend on both the type of vaccine and concern related to efficacy and safety. A variety of possible mechanisms for adjuvants have been researched [494,495,496], including the following: induction of DAMPs that can be recognized by certain PRRs of the innate immune system; functioning as PAMP that can also be recognized by certain PRRs; and more generally enhancing the humoral or cellular immune responses. Selection of one or more adjuvants requires considering how to promote the advantageous effects of the components and/or immune response and, likewise, to inhibit possible deleterious effects. There are also considerations related to the method of delivering (or co-delivering) the adjuvant and antigen components of a vaccine.

5.5.4.3 Trained Immunity

Another approach that is being investigated explores the potential for vaccines that are not made from the SARS-CoV-2 virus to confer what has been termed trained immunity. In a recent review [497], trained immunity was defined as forms of memory that are temporary (e.g., months or years) and reversible. It is induced by exposure to whole-microorganism vaccines or other microbial stimuli that generates heterologous protective effects. Trained immunity can be displayed by innate immune cells or innate immune features of other cells, and it is characterized by alterations to immune responsiveness to future immune challenges due to epigenetic and metabolic mechanisms. These alterations can take the form of either an increased or decreased response to immune challenge by a pathogen. Trained immunity elicited by non-SARS-CoV-2 whole-microorganism vaccines could potentially improve SARS-CoV-2 susceptibility or severity [498].

One type of stimulus which research indicates can induce trained immunity is bacillus Calmette-Guerin (BCG) vaccination. BCG is an attenuated form of bacteria Mycobacterium bovis. The vaccine is most commonly administered for the prevention of tuberculosis in humans. Clinical trials in non-SARS-CoV-2-infected adults have been designed to assess whether BCG vaccination could have prophylactic effects against SARS-CoV-2 by reducing susceptibility, preventing infection, or reducing disease severity. A number of trials are now evaluating the effects of the BCG vaccine or the related vaccine VPM1002 [498,499,500,501,502,503,504,505,506,507,508,509,510,511,512].

The ongoing trials are using a number of different approaches. Some trials enroll healthcare workers, other trials hospitalized elderly adults without immunosuppression who get vaccinated with placebo or BCG at hospital discharge, and yet another set of trials older adults (>50 years) under chronic care for conditions like hypertension and diabetes. One set of trials, for example, uses time until first infection as the primary study endpoint; more generally, outcomes measured in some of these trials are related to incidence of disease and disease severity or symptoms. Some analyses have suggested a possible correlation at the country level between the frequency of BCG vaccination (or BCG vaccination policies) and the severity of COVID-19 [498]. Currently it is unclear whether this correlation has any connection to trained immunity. Many possible confounding factors are also likely to vary among countries, such as age distribution, detection efficiency, stochastic epidemic dynamic effects, differences in healthcare capacity over time in relation to epidemic dynamics, and these have not been adequately accounted for in current analyses. It is unclear whether there is an effect of the timing of BCG vaccination, both during an individual’s life cycle and relative to the COVID-19 pandemic. Additionally, given that severe SARS-CoV-2 may be associated with a dysregulated immune response, it is unclear what alterations to the immune response would be most likely to be protective versus pathogenic (e.g., [108,498,513,514]). The article [498] proposes that trained immunity might lead to an earlier and stronger response, which could in turn reduce viremia and the risk of later, detrimental immunopathology. While trained immunity is an interesting possible avenue to complement vaccine development efforts through the use of an existing vaccine, additional research is required to assess whether the BCG vaccine is likely to confer trained immunity in the case of SARS-CoV-2.

5.6 Discussion

With the emergence of the COVID-19 pandemic caused by the coronavirus SARS-CoV-2, the development and/or identification of therapeutic and prophylactic interventions became an issue of international urgency. In previous outbreaks of HCoV, namely SARS and MERS, the development of these interventions was very limited. As research has progressed, several potential approaches to treatment have emerged (Figure 5).. Most notably, remdesivir has been approved by the FDA for the treatment of COVID-19, and dexamethasone, which was approved by the FDA in 1958, has been found to improve outcomes for patients with severe COVID-19. Other potential therapies are being still being explored and require additional data (Figure 3). Additionally, major advances in vaccines using mRNA and adenoviruses that have led to three vaccines becoming available or close to becoming available in late 2020 (Figure 5). As more evidence becomes available, the potential for existing and novel therapies to improve outcomes for COVID-19 patients will become better understood.

Figure 5: Mechanism of Action for Potential Therapeutics Potential therapeutics currently being studied target the SARS-CoV-2 or modify the host environment through many different mechanisms. Here, the relationship between the virus and several therapeutics described above are visualized.

Insights into the pathogenesis and immune response to SARS-CoV-2 have also provided some insights into prophylactics [cite therapeutics]. Though some concerns remain about the duration of sustained immunity for convalescents, vaccine development efforts are ongoing and show initial promising results. The Moderna trial, for example, reported that the neutralizing activity in participants who received two doses of the vaccine was similar to that observed in convalescent plasma. Insights from the immune response to SARS-CoV-2 have also guided the identification of potential therapeutics. As cases have become better characterized, it has become evident that many patients experience an initial immune response to the virus that is typically characterized by fever, cough, dyspnea, and related symptoms. However, the most serious concern is cytokine release syndrome, when the body’s immune response becomes dysregulated, resulting in an extreme inflammatory response. The RECOVERY trial, a large-scale, multi-arm trial enrolling about 15% of all COVID-19 patients in the United Kingdom, was the first to identify that the widely available steroid dexamethasone seems to be beneficial for patients suffering from this immune dysregulation [415]. The results of efforts to identify therapeutic treatments to treat patients early in the course of infection have been more ambiguous. Early interest in the drugs hydroxychloroquine and chloroquine yielded no promising results from studies with robust experimental designs. On the other hand, the experimental drug remdesivir, which was developed for Ebola, has received enough support from early analyses to receive FDA approval, but results have been mixed. The potential for other drugs, such as tocilizumab, to reduce recovery time remains unclear, but some early results were promising.

One concern is that the presentation of COVID-19 appears to be heterogeneous across the lifespan. Many adult cases, especially in younger adults, present with mild symptoms or even asymptomatically, while others, especially in older adults, can be severe or fatal. In children, the SARS-CoV-2 virus can present as two distinct diseases, COVID-19 or MIS-C. The therapeutics and prophylactics discussed here were all tested in adults. One of the two mRNA vaccines, Pfizer and BioNTech’s BNT162b2, has been issued an EUA for patients as young as 16 [515], while ModernaTX has begun a clinical trial to assess its mRNA vaccine in adolescents ages 12 to 18 [516]. However, additional research is needed to identify therapeutics that address the symptoms characteristic of pediatric COVID-19 and MIS-C cases.

5.6.1 Potential Avenues of Interest for Therapeutic Development

Given what is currently known about these therapeutics for COVID-19, a number of related therapies beyond those explored above may also prove to be of interest. For example, the demonstrated benefit of dexamethasone and the ongoing potential of tocilizumab for treatment of COVID-19 suggests that other anti-inflammatory agents might also hold value for the treatment of COVID-19. Given that current evidence about treating COVID-19 with dexamethasone suggests that the need to curtail the cytokine storm inflammatory response to the virus can transcend the risks of immunosuppression, exploration of more anti-inflammatory agents may be warranted. While dexamethasone is considered widely available and generally affordable, the high costs of biologics such as tocilizumab therapy may present obstacles to wide-scale distribution of this drug if it proves of value. At the doses used for rheumatoid arthritis patients, the cost for tocilizumab ranges from $179.20 to $896 per dose for the IV form and $355 for the pre-filled syringe [517]. There are several anti-inflammatory agents used for the treatment of autoimmune diseases that may also be able to counter the effects of the cytokine storm induced by the virus, some of which, such as cyclosporine, are likely to be more cost-effective and readily available than biologics [518]. While tocilizumab targets IL-6, several other inflammatory markers could be potential targets, including TNF-alpha. Inhibition of TNF-alpha by an inhibitor such as Etanercept has been previously suggested for treatment of SARS-CoV-1 [519] and may be relevant for SARS-CoV-2 as well. Another anti-IL-6 antibody, sarilumab, is also being investigated [520,521]. Baricitinib and other small molecule inhibitors of the Janus-activated kinase pathway also curtail the inflammatory response and have been suggested as potential options for SARS-CoV-2 infections [522]. Baricitinib in particular may be able to reduce the ability of SARS-CoV-2 to infect lung cells [523]. Clinical trials studying baricitinib in COVID-19 have already begun in the US and in Italy [524,525]. Identification and targeting of further inflammatory markers that are relevant in SARS-CoV-2 infection may be of value for curtailing the inflammatory response and lung damage.

In addition to immunosuppressive treatments that are most beneficial late in disease progression, much research is focused on identifying treatments would be likely to benefit early-stage patients. For example, although studies of hydroxychloroquine have not supported the early theory-driven interest in this antiviral treatment, alternative compounds with related mechanisms may still have potential. Hydroxyferroquine derivatives of HCQ have been described as a class of bioorganometallic compounds that exert antiviral effects with some selectivity for SARS-CoV-1 in vitro [526]. Future work could explore whether such compounds exert antiviral effects against SARS-CoV-2 and whether they would be safer for use in COVID-19. Another potential approach is the development of antivirals, which could be broad-spectrum, specific to coronaviruses, or targeted to SARS-CoV-2. Development of new antivirals is complicated by the fact that none have yet been approved for human coronaviruses. Intriguing new options are emerging, however. Beta-D-N4-hydroxycytidine (NHC) is an orally bioavailable ribonucleotide analog showing broad-spectrum activity against RNA viruses, which may inhibit SARS-CoV-2 replication in vitro and in vivo in mouse models of HCoVs [527]. A range of other antivirals are also in development. Development of antivirals will be further facilitated as research reveals more information about the interaction of SARS-CoV-2 with the host cell and host cell genome, mechanisms of viral replication, mechanisms of viral assembly, and mechanisms of viral release to other cells; this can allow researchers to target specific stages and structures of the viral life cycle. Finally, antibodies against viruses, also known as antiviral monoclonal antibodies, could be an alternative as well and are described in detail in an above section. The goal of antiviral antibodies is to neutralize viruses through either cell-killing activity or blocking of viral replication [528]. They may also engage the host immune response, encouraging the immune system to hone in on the virus. Given the cytokine storm that results from immune system activation in response to the virus, which has been implicated in worsening of the disease, a neutralizing antibody (nAb) may be preferable. Upcoming work may explore the specificity of nAbs for their target, mechanisms by which the nAbs impede the virus, and improvements to antibody structure that may enhance the ability of the antibody to block viral activity.

Some research is also investigating potential therapeutics and prophylactics that would interact with components of the innate immune response. For example, there are a variety of TLRs, PRRs that recognize PAMPs and DAMPs. TLRs form a part of innate immune recognition and can more generally contribute to promoting both innate and adaptive responses [106]. In mouse models, poly(I:C) and CpG, which are agonists of toll-like receptors TLR3 and TLR9, respectively, showed protective effects when administered prior to SARS-CoV-1 infection [529]. Therefore, TLR agonists hold some potential for broad-spectrum prophylaxis.

Given that a large number of clinical trials are currently in progress, more information about the potential of these and other therapeutics should become available over time. This information, combined with advances in understanding the molecular structure and viral pathogenesis of SARS-CoV-2, may lead to a more complete understanding of how the virus affects the human host and what strategies can improve outcomes. To date, investigations of potential therapeutics for COVID-19 have focused primarily on repurposing existing drugs. This approach is necessary given the urgency of the situation as well as the extensive time required for developing and testing new therapies. However, in the long-term, new drugs specific for treatment of COVID-19 may also enter development. Development of novel drugs is likely to be guided by what is known about the pathogenesis and molecular structure of SARS-CoV-2. For example, understanding the various structural components of SARS-CoV-2 may allow for the development of small molecule inhibitors of those components. Currently, crystal structures of the SARS-CoV-2 main protease have recently been resolved [340,530], and efforts are already in place to perform screens for small molecule inhibitors of the main protease, which have yielded potential hits [340]. Much work remains to be done to determine further crystal structures of other viral components, understand the relative utility of targeting different viral components, perform additional small molecule inhibitor screens, and determine the safety and efficacy of the potential inhibitors. While still nascent, work in this area is promising. Over the longer term, this approach and others may lead to the development of novel therapeutics specifically for COVID-19 and SARS-CoV-2.

5.6.2 Conclusions of the Present Analysis

Due to the large number of clinical trials currently under examination (Figure 3), not all candidates are examined here. Instead, this review seeks to provide an overview of the range of mechanisms that have been explored and to examine some prominent candidates in the context of the pathogenesis of and immune response to SARS-CoV-2. As more research becomes available, this review will be updated to include additional therapeutics that emerge and to include new findings that are released about those discussed here. While no therapeutics or vaccines were developed for SARS-CoV-1 or MERS-CoV, the current state of COVID-19 research suggests that the body of literature produced before and after the emergence of these viruses has prepared the biomedical community for a rapid response to novel HCoV like SARS-CoV-2. At present, two pharmaceutical therapeutics and one vaccine have been authorized for the treatment and prevention, respectively, of COVID-19. As the COVID-19 pandemic continues to be a topic of significant worldwide concern, more information is expected to become available about pharmaceutical mechanisms that can be used to combat this, and possibly other, HCoV. These advances therefore not only benefit the international community’s ability to respond to the current crisis, but are also likely to shape responses to future viral threats.

6 Dietary Supplements and Nutraceuticals Under Investigation for COVID-19 Prevention and Treatment

6.1 Abstract

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused global disruption and a significant loss of life. Scientists and physicians around the world have worked to discover and develop novel treatments or repurpose existing treatments in order to identify potential prophylactic and therapeutic agents to stem the spread of SARS-CoV-2 and reduce the devastation of this pandemic. Dietary supplements and nutraceuticals are also being investigated in this context. Despite a lack of evidence to support their efficacy or safety for the treatment or prevention of COVID-19, the beginning of the pandemic led to increased sales of vitamins, supplements, nutraceuticals, and various other products purporting ‘immune-boosting’ effects. Considerable interest amongst the scientific community and emerging evidence of potential utility in other therapeutic contexts has led to further investigation of these products. In this review, we critically appraise the evidence surrounding dietary supplements and nutraceuticals for the prophylaxis and treatment of COVID-19. These include vitamin C, vitamin D, omega 3 polyunsaturated fatty acids (PUFA), probiotics, and zinc. These compounds were selected as they are currently under clinical investigation. For example, there is increasing evidence to suggest that vitamin D supplementation may be beneficial due to plausible associations between vitamin D deficiency with COVID-19 incidence and possibly disease severity. A growing number of scientific organizations are considering or advising for increased vitamin D supplementation to those at high risk of COVID-19. Overall, though, caution is warranted as further evidence and clinical trials are required before conclusive evidence-based recommendations can be formulated for any specific dietary supplements or nutraceuticals. Consumers should also be aware of misinformation and false promises surrounding some supplement products, which may lack safety and efficacy evidence due to poor regulation by authorities. However, considering that nutritional status plays a significant role in patient outcomes, it would be prudent to advise the general public to follow a healthy diet and lifestyle to prevent nutrient deficiencies and to maintain a healthy immune system.

6.2 Importance

Sales of dietary supplements and nutraceuticals have increased since the start of the pandemic due to their perceived health benefits. These include vitamin C, vitamin D, and zinc, which many consider synonymous with the treatment of respiratory infections and support of immune health. However, little is known about the efficacy of these dietary supplements and nutraceuticals against the novel coronavirus (SARS-CoV-2) or the disease it causes, COVID-19. This review provides a critical overview of the potential prophylactic and therapeutic value of various dietary supplements and nutraceuticals from the evidence available to date. Evidence indicates that vitamin D deficiency may be associated with greater incidence of infection and severe COVID-19 and that vitamin D supplementation may be of prophylactic and therapeutic value. However, considerably more research is required to determine whether dietary supplements and nutraceuticals exhibit prophylactic and therapeutic value against SARS-CoV-2 infection and COVID-19.

6.3 Introduction

Scientists and the medical community are scrambling to repurpose or discover novel host-directed therapies against the coronavirus diseases 2019 (COVID-19) pandemic caused by the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Some successful therapies such as remdesivir and dexamethasone have been identified for hospitalized patients. Furthermore, most societies have adopted the non-pharmacological preventative public health strategies that reduce the transmission of SARS-CoV-2. However, many individuals have sought additional protections via the consumption of various dietary supplements and nutraceuticals with purported beneficial effects. Indeed, a patient’s nutritional status seems to play a role in COVID-19 susceptibility and outcomes [531,532,533,534,535].

Nutraceuticals and dietary supplements are related but distinct non-pharmaceutical products. Nutraceuticals are classified as supplements with health benefits beyond their basic nutritional value [536,537]. The key difference between a dietary supplement and a nutraceutical is that nutraceuticals should not only supplement the diet, but also aid in the prophylaxis and/or treatment of a disorder or disease [538]. Due to the significant interest from the general public in these dietary additives, whether and to what extent nutraceuticals or dietary supplements can provide any prophylactic or therapeutic benefit remains a topic of interest for the scientific community. Despite the lack of any evidence to support their use, the beginning of the pandemic led sales of these products to soar. In the United States, dietary supplement and nutraceutical sales were worth $345 million for the entirety of 2019, whereas sales reached $435 million during the six-week period of the outbreak that ended on April 5 of 2020, which grew by a further $151 million by May 17 2020 [539]. In France, New Zealand, India, and China similar trends in sales were reported [540,541,542,543]. The increase in sales was driven by a consumer perception that dietary supplements and nutraceuticals would protect consumers from infection and/or mitigate the impact of infection due to the various “immune-boosting” claims of these products [544,545]. However, dietary supplements and nutraceuticals, unlike pharmaceuticals, are not subject to the same regulatory protocols that protect consumers of medicines. Indeed, nutraceuticals do not entirely fall under the responsibility of the Food and Drug Administration (FDA), but they are monitored as dietary supplements according to the Dietary Supplement, Health and Education Act 1994 (DSHEA) [546] and the Food and Drug Administration Modernization Act 1997 (FDAMA) [547]. Due to the increased sales of dietary supplements and nutraceuticals, in 1996 the FDA established the Office of Dietary Supplement Programs (ODSP) to increase surveillance. Novel products or nutraceuticals must now submit a new dietary ingredient notification to the ODSP for review.
There are significant concern that these legislations do not adequately protect the consumer as they ascribe responsibility to the manufacturers to ensure the safety of the product before manufacturing or marketing [548]. Manufacturers are not required to register or even seek approval from the FDA to produce or sell food supplements or nutraceuticals. Health or nutrient content claims for labeling purposes are approved based on an authoritative statement from the Academy of Sciences or relevant federal authorities once the FDA has been notified and on the basis that the information is known to be true and not deceptive [548].

In Europe, the safety of supplements are monitored by the European Union (EU) under Directive 2002/46/EC [549]. However, nutraceuticals are not directly mentioned. Consequently, nutraceuticals can be generally described as either a medicinal product under Directive 2004/27/EC [550] or as a ‘foodstuff’ under Directive 2002/46/EC of the European council. In order to synchronize the various existing legislations, Regulation EC 1924/2006 on nutrition and health claims was put into effect to assure customers of safety and efficacy of products and to deliver understandable information to consumers. However, specific legislation for nutraceuticals is still elusive. Health claims are permitted on a product label only following compliance and authorization according to the European Food Safety Authority (EFSA) guidelines on nutrition and health claims [551]. EFSA does not currently distinguish between food supplements and nutraceuticals for health claim applications of new products, as claim authorization is dependent on the availability of clinical data in order to substantiate efficacy [552]. However, these guidelines seem to provide more protection to consumers than the FDA regulations, but potentially at the cost of innovation in the sector [553]. Looking further afield, the situation becomes even more complicated at a global level where countries such as China and India have existing regulatory frameworks for traditional medicines and phytomedicines not commonly consumed by Western society [554]. Currently, there is a debate among scientists and regulatory authorities surrounding the development of a widespread regulatory framework to deal with the challenges of safety and health claim substantiation for nutraceuticals [548,552] as these products do not necessarily follow the same rigorous clinical trial frameworks used to approve the use of pharmaceuticals. Such regulatory disparities have been highlighted by the pandemic as many individuals and companies have attempted to profit from the vulnerabilities of others by overstating claims in relation to the treatment of COVID-19 using supplements and nutraceuticals. The FDA has written several letters to prevent companies marketing or selling products based on false hyperbolic promises to prevent SARS-CoV-2 infection or treat COVID-19 [555,556,557]. These letters came in response to efforts to market nutraceutical prophylactics against COVID-19, some of which charged the consumer as much as $23,000 [558]. There have even been some incidents highlighted in the media because of their potentially life threatening consequences; for example, the use of oleandrin was touted as a potential “cure” by individuals close to the former President of the United States despite its high toxicity [559]. Thus, heterogeneous and at times relaxed regulatory standards have permitted high-profile cases of the sale of nutraceuticals and dietary supplements that are purported to provide protection against COVID-19, despite a lack of research into these compounds.

Notwithstanding the issues of poor safety, efficacy, and regulatory oversight, some dietary supplements and nutraceuticals have exhibited therapeutic and prophylactic potential. Some have been linked with reduced immunopathology, antiviral and anti-inflammatory activities, or even the prevention of ARDS [544,560,561]. A host of potential candidates have been highlighted in the literature that target various aspects of the COVID-19 viral pathology, while others are thought to prime the host immune system. These candidates include vitamins and minerals along with extracts and omega-3 polyunsaturated fatty acids (n-3 PUFA) [562]. In vitro and in vivo studies suggest that nutraceuticals containing phycocyanobilin, N-acetylcysteine, glucosamine, selenium or phase 2 inductive nutraceuticals (e.g. ferulic acid, lipoic acid, or sulforaphane) can prevent or modulate RNA virus infections via amplification of the signaling activity of mitochondrial antiviral-signaling protein (MAVS) and activation of toll-like receptor 7 (TLR7) [563]. While promising, further animal and human studies are required to assess the therapeutic potential of these various nutrients and nutraceuticals against COVID-19. For the purpose of this review, we have highlighted some of the main dietary supplements and nutraceuticals that are currently under investigation for their potential prophylactic and therapeutic applications. These include n-3 PUFA, zinc, vitamins C and D, and probiotics.

6.4 n-3 PUFA

One supplement that has been explored for beneficial effects against various viral infections is n-3 PUFA [562], such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). EPA and DHA intake can come from a diet high in fish or through dietary supplementation with fish oils or purified oils [564]. Other more sustainable sources of EPA and DHA include algae [565], which can also be exploited for their rich abundance of other bioactive compounds such as ACE inhibitor peptides and antiviral agents including phycobiliproteins, sulfated polysaccharides, and calcium-spirulan [566]. They can mediate inflammation and therefore may have the capacity to modulate the adaptive immune response [537,564,567]. Another potential mechanism that has led to interest in n-3 PUFA for viral infections is its potential as a precursor molecule for the biosynthesis of endogenous specialized proresolving mediators (SPM), such as protectins and resolvins, that actively resolve inflammation and infection [568]. Finally, some COVID-19 patients, particularly those with comorbidities, are at a significant risk of thrombotic complications including arterial and venous thrombosis [103,569]. Therefore, the use of prophylactic and therapeutic anticoagulants and antithrombotic agents is under consideration [570,571], which could potentially include n-3 PUFA.

SPM have exhibited beneficial effects against a variety of lung infections, including some caused by RNA viruses [572,573]. Indeed, protectin D1 has been shown to increase survival from H1N1 viral infection in mice by affecting the viral replication machinery [574]. Several mechanisms for SPM have been proposed, including preventing the release of pro-inflammatory cytokines and chemokines or increasing phagocytosis of cellular debris by macrophages [575]. In influenza, SPM promote antiviral B lymphocytic activities [576], and protectin D1 has been shown to increase survival from H1N1 viral infection in mice by affecting the viral replication machinery [574]. It is hypothesized that SPM could aid in the resolution of the cytokine storm and pulmonary inflammation associated with COVID-19 [577,578]. Another theory is that some comorbidities such as obesity could lead to deficiencies of SPM, which could in turn be related to the occurrence of adverse outcomes for COVID-19 [???]. However, not all studies are in agreement that n-3 PUFA or their resulting SPM are effective against infections [579]. At a minimum, the effectiveness of n-3 PUFA against infections would be dependent on the dosage, timing, and the specific pathogens responsible [580]. On another level, there is still the question of whether fish oils can raise the levels of SPM levels upon ingestion and in response to acute inflammation in humans [581]. Currently, Karolinska University Hospital is running a trial that will measure the levels of SPM as a secondary outcome following intravenous supplementation of n-3 PUFA in hospitalized COVID-19 patients to determine whether n-3 PUFA provides therapeutic value [582].

The increased risk of thrombotic complications in COVID-19 infected patients was reported relatively late in comparison to other manifestations of COVID-19 [97,569]. Considering that there is significant evidence that n-3 fatty acids and other fish oil-derived lipids possess antithrombotic properties and anti-inflammatory properties [583,584], they may have therapeutic value against the prothrombotic complications of COVID-19. Based on concern among the medical community that the use of investigational therapeutics on COVID-19 patients already on antiplatelet therapies due to a pre-existing comorbidities may lead to issues with dosing and drug choice, and/or negative drug-drug interactions [570], this supplementation holds particular interest for the treatment of patients already receiving pharmaceutical antiplatelet therapies. As a result, the use of other therapeutics, such as dietary sources of n-3 fatty acids or nutraceuticals with antiplatelet activities, may be beneficial and warrant further investigation. A new clinical trial [585] is currently recruiting COVID-19 positive patients to investigate the anti-inflammatory activity of a recently developed, highly purified nutraceutical derivative of EPA known as icosapent ethyl (VascepaTM) [586]. Other randomized controlled trials are in the preparatory stages with the intention of investigating the administration of EPA and other bioactive compounds to COVID-19 positive patients to determine whether anti-inflammatory effects or disease state improvements are observed [587,588]. Finally, while there have been studies investigating the therapeutic value of n-3 fatty acids against ARDS in humans, there is still limited evidence of their effectiveness [589]. It should be noted that the overall lack of human studies in this area means there is limited evidence as to whether these supplements could affect COVID-19 infection. Consequently, clinical trials have been proposed and many are in the preparatory stages. These trials will investigate whether the anti-inflammatory potential of n-3 PUFA and their derivatives is beneficial in the treatment of COVID-19. Therefore, the evidence is not present to draw conclusions about whether n-3 PUFA will be useful in treating COVID-19, but as there is likely little harm associated with a diet rich in fish oils, interest in these products by the general public is unlikely to have negative effects.

6.5 Zinc

There is evidence that certain nutrient supplements may exhibit some benefit against RNA viral infections. Zinc is a trace metal obtained from dietary sources or supplementation that is important for the maintenance of immune cells involved in adaptive and innate immunity [590]. Zinc supplements can be administered orally as a tablet or as a lozenge and they are available in many forms, such as zinc picolinate, zinc acetate, and zinc citrate. Zinc is also available from dietary sources including meat, seafood, nuts, seeds, legumes, and dairy. The role of zinc in immune function has been extensively reviewed [590]. Zinc is an important signaling molecule and zinc levels can alter host defense systems. In inflammatory situations such as an infection, zinc can regulate leukocyte immune responses and it can activate the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), thus altering cytokine production [591,592]. In particular, zinc supplementation can increase natural killer cell levels, which are important cells for host defense against viral infections [590,593].

Adequate zinc intake has been associated with reduced incidence of infection [594] and antiviral immunity [595]. A randomized, double-blind, placebo-controlled trial that administered zinc supplementation to elderly subjects over the course of a year found zinc deficiency to be associated with increased susceptibility to infection and that zinc deficiency could be prevented through supplementation [594]. Clinical trial data supports the utility of zinc to diminish the duration and severity of symptoms associated with common colds when it is provided within 24 hours of the onset of symptoms [596,597]. An observational study has shown that COVID-19 patients had significantly lower zinc levels in comparison to healthy controls, they also tended to have more complications (70.4% vs 30.0%, p = 0.009), and potentially prolonged hospital stays (7.9 vs 5.7 days, p = 0.048) [598].
In coronaviruses specifically, in vitro evidence demonstrates that the combination of zinc (Zn2+) and zinc ionophores (pyrithione) can interrupt the replication mechanisms of SARS-CoV-GFP (a fluorescently tagged SARS-CoV-1) and a variety of other RNA viruses [599,600]. Currently, there are over twenty clinical trials registered with the intention to use zinc in a preventative or therapeutic manner. However, many of these trials proposed the use of zinc in conjunction with hydroxychloroquine and azithromycin [601,602,603,604], and it is not known how the lack of evidence supporting the use of hydroxychloroquine will affect investigation of zinc. One retrospective observational study of New York University Langone hospitals in New York compared outcomes among hospitalized COVID-19 patients administered hydroxychloroquine and azithromycin with zinc sulfate (n = 411) versus hydroxychloroquine and azithromycin alone (n = 521). Notably, zinc is the only treatment that was used in this trial that is still under consideration as a therapeutic agent due to to the lack of efficacy and potential adverse events associated with hydroxychloroquine and azithromycin against COVID-19 [605,606,607]. While the addition of zinc sulfate did not affect the duration of hospitalization, the length of ICU stays or patient ventilation duration, univariate analyses demonstrated that zinc did increase the frequency of patients discharged and decreased the requirement for ventilation and referrals to the ICU, and reduced mortality [608]. However, a smaller retrospective study at Hoboken University Medical Center New Jersey failed to observe an association between zinc supplementation and survival of hospitalized patients [609].

Other trials, however, are investigating zinc in conjunction with other supplements such as vitamin C or n-3 PUFA [588,610]. Though there is, overall, encouraging data for zinc supplementation against the common cold and viral infections, there is currently limited evidence to suggest zinc supplementation has any beneficial effects against the current novel COVID-19; thus, the clinical trials that are currently underway will provide vital information on the efficacious use of zinc in COVID-19 prevention and/or treatment. However, given the limited risk, maintaining a healthy diet to ensure an adequate zinc status may be advisable for individuals seeking to reduce their likelihood of infection.

6.6 Vitamin C

Vitamins B, C, D, and E have also been suggested as potential nutrient supplement interventions for COVID-19 [562,611]. In particular vitamin C has been proposed as a potential therapeutic agent against COVID-19 due to its long history of use against the common cold and other respiratory infections [612,613]. Vitamin C can be obtained via dietary sources such as fruits and vegetables or via supplementation. Vitamin C plays a significant role in promoting immune function due to its effects on various immune cells. It affects inflammation by modulating cytokine production, decreasing histamine levels, enhancing the differentiation and proliferation of T- and B-lymphocytes, increasing antibody levels, and protecting against the negative effects of reactive oxygen species amongst other effects related to COVID-19 pathology [614,615,616]. Vitamin C is utilized by the body during viral infections, as evinced by lower concentrations in leukocytes and lower concentrations of urinary vitamin C. Post-infection, these levels return to baseline ranges [617,618,619,620,621]. Indeed, COVID-19 patients appear to be deficient in vitamin C [622,623]. COVID-19 is also associated with the formation of microthrombi and coagulopathy [105] that contributes to the observed lung pathology [624], which can be ameliorated by early infusions of vitamin C by inhibiting endothelial surface P-selectin expression and platelet-endothelial adhesion [625]. Indeed, intravenous vitamin C also reduced D-dimer levels in a case study of 17 COVID-19 patients [626], which are notably elevated in COVID-19 patients [???,102].

A recent meta-analysis found consistent support for regular vitamin C supplementation reducing the duration of the common cold, but that supplementation with vitamin C (> 200 mg) failed to reduce the incidence of colds [627]. Individual studies have found Vitamin C to reduce the susceptibility of patients to lower respiratory tract infections such as pneumonia [628]. Another meta-analysis has demonstrated in twelve trials that vitamin C supplementation reduced the length of stay of patients in intensive care units (ICUs) by 7.8% (95% CI: 4.2% to 11.2%; p = 0.00003). Furthermore, high doses (1-3 g/day) significantly reduced the length of an ICU stay by 8.6% in six trials (p = 0.003). Vitamin C also shortened the duration of mechanical ventilation by 18.2% in three trials in which patients required intervention for over 24 hours (95% CI 7.7% to 27%; p = 0.001) [629]. Despite these findings, the CITRUS ALI study failed to show a benefit of a 96-hour infusion of vitamin C to treat ARDS, which is a severe complication of COVID-19 infection [630]. Nevertheless, clinical trials have begun, as highlighted by Carr et al. [613], the first of which was initiated in Wuhan, China. Others will be conducted in Canada, China, Iran, and the USA. These trials intend to investigate the use of intravenous vitamin C in hospitalized COVID-19 patients. The first trial established in Wuhan, China [631] has reported initial results indicating that 12 g/12 hr of intravenous vitamin C for 7 days in 56 critically ill COVID-19 patients resulted in a promising reduction of 28-day mortality (p = 0.06) in univariate survival analysis [632]. Indeed, the same study reported a significant decrease in IL-6 levels by day 7 of vitamin C infusion (p = 0.04) [633].

Even though evidence supporting the use of vitamin C is beginning to emerge, we will not know how effective vitamin C is as a therapeutic for quite some time. Currently (as of December 2020) over fifteen trials are registered with clinicaltrials.gov that are either recruiting, active or are currently in preparation. When completed, these trials will provide crucial evidence on the efficacy of vitamin C as a therapeutic for COVID-19 infection. However, the majority of supplementation studies investigate the intravenous infusion of vitamin C in severe patients. Therefore, there is a lack of studies investigating the potential prophylactic administration of vitamin C via oral supplementation for healthy individuals or potentially asymptomatic SARS-CoV-2 positive patients. Once again, vitamin C intake is part of a healthy diet and the vitamin likely presents minimal risk, but its potential prophylactic or therapeutic effects against COVID-19 are yet to be determined.

6.7 Vitamin D

Of all of the supplements currently under investigation, vitamin D has become a leading prophylactic and therapeutic candidate against SARS-CoV-2. Vitamin D can modulate both the adaptive and innate immune system and is associated with various aspects of immune health and antiviral defense [634,635,636,637,638]. Vitamin D can be sourced through diet or supplementation, but it is mainly biosynthesized by the body on exposure to ultraviolet light (UVB) from sunlight. Vitamin D deficiency is associated with an increased susceptibility to infection [639]. In particular, vitamin D deficient patients are at risk of developing acute respiratory infections [640] and ARDS [640]. 1,25-dihydroxyvitamin D3 is the active form of vitamin D that is involved in adaptive and innate responses; however, due to its low concentration and a short half life of a few hours, it is measured by its longer lasting and more abundant precursor 25-hydroxyvitamin D. The vitamin D receptor is expressed in various immune cells, and vitamin D is an immunomodulator of antigen presenting cells, dendritic cells, macrophages, monocytes, and T- and B-lymphocytes [639,641]. Due to its potential immunomodulating properties, vitamin D supplementation may be advantageous to maintain a healthy immune system.

Early in the pandemic it was postulated that an individual’s vitamin D status may significantly affect their risk of developing COVID-19 [642]. This hypothesis was derived from the fact that the current pandemic emerged in winter in Wuhan China when 25-hydroxyvitamin D concentrations are at their lowest due to a lack of sunlight, whereas in the Southern Hemisphere, where it was nearing the end of the summer and higher 25-hydroxyvitamin D concentrations would be higher, the number of cases was low. This led researchers to question whether there was a seasonal component to the SARS-CoV-2 pandemic and whether vitamin D levels might play a role [642,643,644,645]. Though it is assumed that COVID-19 is seasonal, multiple other factors that can affect vitamin D levels should also be considered. These factors include an individual’s nutritional status, their age, their occupation, skin pigmentation, potential comorbidities, and the variation of exposure to sunlight due to latitude amongst others. Indeed, it has been estimated that each degree of latitude north of 28 degrees corresponded to a 4.4% increase of COVID-19 mortality, indirectly linking a persons vitamin D levels via exposure to UVB light to COVID-19 mortality [643].

As the pandemic evolves, further research of varying quality has investigated some of the potential links identified early in the pandemic [642] between vitamin D and COVID-19. Indeed, studies are beginning to investigate whether there is any prophylactic and/or therapeutic relationship between vitamin D and COVID-19. A study in Switzerland demonstrated that 27 SARS-CoV-2 positive patients exhibited 25-hydroxyvitamin D plasma concentrations that were significantly lower (11.1 ng/ml) than those of SARS-CoV-2 negative patients (24.6 ng/ml; p = 0.004), an association that held when stratifying for patients greater than 70 years old [646]. These findings seem to be supported by a Belgian observational study of 186 SARS-CoV-2 positive patients exhibiting symptoms of pneumonia, where 25-hydroxyvitamin D plasma concentrations were measured and a CT scan of the lungs was obtained upon hospitalization [647]. A significant difference in 25-hydroxyvitamin D levels was observed between the SARS-CoV-2 patients and 2,717 season-matched diseased controls. Both female and male patients possessed lower median 25-hydroxyvitamin D concentrations than the control group (18.6 ng/ml versus 21.5 ng/ml; p = 0.0016) and a higher rate of vitamin D deficiency (58.6% versus 42.5%). Evidence of sexual dimorphism was also apparent, as female patients had equivalent levels of 25-hydroxyvitamin D to the control group, whereas male patients were deficient in 25-hydroxyvitamin D relative to male controls (67% versus 49%; p = 0.0006). Notably, vitamin D deficiency was progressively lower in males with advancing radiological disease stages (p = 0.001). These studies are supported by several others that indicate that vitamin D status may be an independent risk factor for the severity of COVID-19 [648,649,650,651] and in COVID-19 patients relative to population-based controls [652]. Indeed, serum concentrations of 25-hydroxyvitamin D above 30 ng/ml, which indicate vitamin D sufficiency, seems to be associated with a reduction in serum C-reactive protein (CRP), an inflammatory marker, along with increased lymphocyte levels, which suggests that vitamin D levels may modulate the immune response by reducing risk for cytokine storm in response to SARS-CoV-2 infection [652]. A study in India has determined that COVID-19 fatality is higher in patients with severe COVID-19 and low serum 25-hydroxyvitamin D (mean level 6.2 ng/ml; 97% vitamin D deficient) levels versus asymptomatic non-severe patients with higher levels of vitamin D (mean level 27.9 ng/ml; 33% vitamin D deficient) [653]. In the same study, vitamin D deficiency was associated with higher levels of inflammatory markers including IL-6, ferritin, and tumor necrosis factor (TNF)-α. Collectively, these studies add to a multitude of observational studies reporting potential associations between low levels of 25-hydroxyvitamin D and COVID-19 incidence and severity [646,651,652,654,655,656,657,658,659,660].

Despite these studies establishing a link between vitamin D status and COVID-19 severity, an examination of data from the UK Biobank did not support this thesis [661,662]. These analyses examined 25-hydroxyvitamin D concentrations alongside SARS-CoV-2 positivity and COVID-19 mortality in over 340,000 UK Biobank participants. However, these studies have caused considerable debate that will likely be settled following further studies [663,664]. Overall, while the evidence suggest that there is likely an association between low serum 25-hydroxyvitamin D and COVID-19 incidence, these studies must be interpreted with caution as there is the potential for reverse causality, bias, and other confounding factors including that vitamin D deficiency is also associated with numerous pre-existing conditions and risk factors that can increase the risk for severe COVID-19 [531,643,665,666].

While these studies inform us of the potential importance of vitamin D sufficiency and the risk of SARS-CoV-2 infection and severe COVID-19, they fail to conclusively determine whether vitamin D supplementation can therapeutically affect the clinical course of COVID-19. In one study, 40 vitamin D deficient asymptomatic or mildly symptomatic participants patients were either randomized to receive 60,000 IU of cholecalciferol daily for at least 7 days (n = 16) or a placebo (n = 24) with a target serum 25-hydroxyvitamin D level >50 ng/ml. At day 7, 10 patients achieved >50 ng/ml, and another 2 by day 14. By the end of the study, the treatment group had a greater proportion of vitamin D-deficient participants that tested negative for SARS-CoV-2 RNA, and they had a significantly lower fibrinogen levels, potentially indicating a beneficial effect [667]. A pilot study in Spain determined that early high dose calcifediol (~21,000 IU days 1-2 and ~11,000 IU days 3-7 of hospital admission) with hydroxychloroquine and azithromycin to 50 hospitalized COVID-19 patients significantly reduced ICU admissions and may have reduced disease severity versus hydroxychloroquine and azithromycin alone [668].
While this study received significant criticism the National Institute for Health and Care Excellence (NICE) in the UK [669], an independent follow-up statistical analysis supported the findings of the study as a result of cholecalciferol treatment [670]. Another trial of 986 COVID-19 hospitalized patients in three UK hospitals administered cholecalciferol supplementation (≥ 280,000 IU in a time period of 7 weeks) to 151 patients and found an association with a reduced risk of COVID-19 mortality, regardless of baseline 25-hydroxyvitamin D levels [671]. Indeed, a double-blind, randomized, placebo-controlled trial of 240 hospitalized COVID-19 patients in Sao Paulo, Brazil administered a single 200,000 IU oral dose of vitamin D. While levels of 25-hydroxyvitamin D did increase from 21% to ~27% (p = 0.001) and were well tolerated, there was no reduction in the length of hospital stay, mortality, and no change to any other relevant secondary outcomes [672]. These early findings are still inconclusive with regards to the therapeutic value of vitamin D supplementation. However, other trials are underway including one trial that is investigating the utility of vitamin D as an immune-modulating agent by monitoring whether administration of vitamin D precipitates an improvement of health status in non-severe symptomatic COVID-19 patients and whether vitamin D prevents patient deterioration [673]. Other trials are examining various factors including mortality, symptom recovery, severity of disease, rates of ventilation, inflammatory markers such as CRP and IL-6, blood cell counts, and the prophylactic capacity of vitamin D administration [673,674,675,676]. Concomitant administration of vitamin D with pharmaceuticals such as aspirin [677] and bioactive molecules such as resveratrol [678] are also under investigation.

The effectiveness of vitamin D supplementation against COVID-19 remains open for debate. Yet, there is no doubt that vitamin D deficiency is a widespread issue and should be mitigated for not only because of its potential link to SARS-CoV-2 incidence [679], but also due to its importance for overall health. There is a possibility that safe exposure to sunlight could improve endogenous synthesis of vitamin D, potentially strengthening the immune system. However, sun exposure is not sufficient on its own, particularly in the winter months. Indeed, preemptive supplementation of vitamin D and encouraging people to maintain a healthy diet for optimum vitamin D status is likely to raise serum levels of 25-hydroxyvitamin D while being unlikely to carry major health risks while the possible link between vitamin D status and COVID-19 is further investigated. These principles seem to be the basis of a number of guidelines issued by some countries and scientific organizations that have advised supplementation of vitamin D during the pandemic.
The Académie Nationale de Médecine in France recommends rapid testing of 25-hydroxyvitamin D for people over 60 years old to identify those most at risk of vitamin D deficiency and advising them to obtain a bolus dose of 50,000 to 100,000 IU vitamin D to limit respiratory complications. They have also recommended that those under 60 years old should take 800 to 1,000 IU daily if they receive a SARS-CoV-2 positive test [680]. Both Public Health England and Public Health Scotland have advised members of the Black, Asian, and minority ethnic (BAME) communities to supplement for vitamin D in light of evidence that they may be at higher risk for vitamin D deficiency and other COVID-19 risk factors. Indeed, African Americans may have up to a 6-fold higher COVID-19 mortality rate than white populations [681,682,683]. However, other UK scientific bodies including the NICE recommends that individuals supplement for vitamin D as per usual UK government advice, but they warn that people should not supplement for vitamin D solely to prevent COVID-19. However, the NICE have provided guidelines for research to investigate the supplementation of vitamin D in the context of COVID-19 [684]. In Slovenia, doctors have been advised to provide nursing home patients with vitamin D [685]. Despite vitamin D deficiency being a widespread issue in the United States [686] the National Institutes of Health have stated that there is “insufficient data to recommend either for or against the use of vitamin D for the prevention or treatment of COVID-19” [687]. These are just some examples of how public health guidance has responded to the emerging evidence with regards to vitamin D and COVID-19. In contrast, some companies have used the emerging evidence surrounding vitamin D to sell products that claim to prevent and treat COVID-19, which in one incident required a federal court to intervene and issue an injunction barring the sale of vitamin-D-related products, due to a lack of clinical data to support their claims [688] It it clear that further studies and clinical trials are required to conclusively determine the prophylactic and therapeutic potential of vitamin D supplementation against COVID-19. Until such time that sufficient evidence emerges, individuals should follow their national guidelines surrounding vitamin D intake to achieve vitamin D sufficiency.

6.8 Probiotics

Probiotics are “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” [689]. Some studies suggest that probiotics are beneficial against common viral infections and there is modest evidence to suggest that they can modulate the immune response [690,691], and as a result it has been hypothesized that probiotics may have therapeutic value worthy of investigation against SARS-CoV-2 [692]. Probiotics and next-generation probiotics, which are more akin to pharmacological-grade supplements, have been associated with multiple potential beneficial effects for allergies, digestive tract disorders, and even metabolic diseases through their anti-inflammatory and immunomodulatory effects [693,694]. However, the mechanisms by which probiotics affect these various conditions would likely differ among strains, with the ultimate effect of the probiotic depending on the heterogeneous set of bacteria present [694]. Some of the beneficial effects of probiotics include reducing inflammation by promoting the expression of anti-inflammatory mediators, inhibiting toll-like receptors (TLR) 2 and 4, direct competition with pathogens, the synthesis of antimicrobial substances or other metabolites, improving intestinal barrier function, and/or favorably altering the gut microbiota and the brain-gut axis [694,695,696]. It is also thought that lactobacilli such as Lactobacillus paracasei, Lactobacillus plantarum and Lactobacillus rhamnosus have the capacity to bind to and inactivate some viruses via adsorptive and/or trapping mechanisms [697]. Other probiotic lactobacilli and even non-viable bacterium-like particles have been shown to reduce both viral attachment to host cells and viral titers, along with reducing cytokine synthesis, enhancing the antiviral IFN-α response, and inducing various other antiviral mechanisms [???,697,698,699,700,701,702,703,704,705]. These antiviral and immunobiotic mechanisms and others have been reviewed in detail elsewhere [561,692,706]. However, there is also a bi-directional relationship between the lungs and gut microbiota known as the gut-lung axis [707], whereby gut microbial metabolites and endotoxins may affect the lungs via the circulatory system and the lung microbiota in return may affect the gut [708]. Therefore, the gut-lung axis may play role in our future understanding of COVID-19 pathogenesis and become a target for probiotic treatments [709]. Moreover, as microbial dysbiosis of the respiratory tract and gut may play a role in some viral infections, it has been suggested that SARS-CoV-2 may interact with our commensal microbiota [561; 710; 10.3389/fmicb.2020.01840] and that the lung microbiome could play a role in developing immunity to viral infections [711]. These postulations, if correct, could lead to the development of novel probiotic and prebiotic treatments. However, significant research is required to confirm these associations and their relevance to patient care, if any.

Probiotics have tentatively been associated with the reduction of risk and duration of viral upper respiratory tract infections [712,713,714]. Some meta-analyses that have assessed the efficacy of probiotics in viral respiratory infections have reported moderate reductions in the incidence and duration of infection [713,715]. Indeed, randomized controlled trials have shown that administering Bacillus subtilis and Enterococcus faecalis [716], Lactobacillus rhamnosus GG [717], or Lactobacillus casei and Bifidobacterium breve with galactooligosaccharides [718] via the nasogastric tube to ventilated patients reduced the occurrence of ventilator-associated pneumonia in comparison to the respective control groups in studies of viral infections and sepsis. These findings are supported by a recently published meta-analysis [719]. There is a significant risk of ventilator-associated bacterial pneumonia in COVID-19 patients [720], but it can be challenging for clinicians to diagnose this infection due to the fact that severe COVID-19 infection presents with the symptoms of pneumonia [721]. Therefore, an effective prophylactic therapy for ventilator-associated pneumonia in severe COVID-19 patients would be of significant therapeutic value.

Probiotics are generally synonymous with the treatment of gastrointestinal issues due to their supposed anti-inflammatory and immunomodulatory effects [722]. Notably, gastrointestinal symptoms commonly occur in COVID-19 patients [723], and the ACE2 receptor is highly expressed in enterocytes of the ileum and colon, suggesting that these organs may be a potential route of infection [724,725]. Indeed, SARS-CoV-2 viral RNA has been detected in human feces [86,726], and fecal-oral transmission of the virus has not yet been ruled out [727]. Rectal swabs of some SARS-CoV-2 positive pediatric patients persistently tested positive for several days despite negative nasopharyngeal tests, indicating the potential for fecal viral shedding [728]. However, there is conflicting evidence for the therapeutic value of various probiotics against the incidence or severity of gastrointestinal symptoms in viral or bacterial infections such as gastroenteritis [729,730]. Nevertheless, it has been proposed that the administration of probiotics to COVID-19 patients and healthcare workers may prevent or ameliorate the gastrointestinal symptoms of COVID-19, a hypothesis that several clinical trials are now preparing to investigate [731,732]. Other studies are investigating whether probiotics may affect patient outcomes following SARS-CoV-2 infection [733].

Generally, the efficacy of probiotic use is a controversial topic among scientists. In Europe, EFSA has banned the term probiotics on products labels, which has elicited either criticism for EFSA or support for probiotics from researchers in the field [689,734,735]. This is due to the hyperbolic claims placed on the labels of various probiotic products, which lack rigorous scientific data to support their efficacy. Overall, the data supporting probiotics in the treatment or prevention of many different disorders and diseases is not conclusive as the quality of the evidence is generally considered low [712]. However, in the case of probiotics and respiratory infections, the evidence seems to be supportive of their potential therapeutic value. Consequently, several investigations are underway to investigate the prophylactic and therapeutic potential of probiotics for COVID-19. However, blind use of conventional probiotics for COVID-19 is cautioned against until the pathogenesis of SARS-CoV-2 is further established [736]. Until clinical trials investigating the prophylactic and therapeutic potential of probiotics for COVID-19 are complete, it is not possible to provide an evidence-based recommendation for their use. Despite these concerns, complementary use of probiotics as an adjuvant therapeutic has been proposed by the Chinese National Health Commission and National Administration of Traditional Chinese Medicine [87].

6.9 Discussion

The purported prophylactic and therapeutic benefits of dietary supplements and nutraceuticals for multiple disorders, diseases, and infections has been the subject of significant research and debate for the last few decades. Inevitably, scientists are also investigating the potential for these various products to treat or prevent COVID-19. This interest also extends to consumers, which led to a remarkable increase of sales of dietary supplements and nutraceuticals throughout the pandemic due to a desire to obtain additional protections from infection and disease. It is pertinent to clarify that the nutraceuticals discussed in this review, namely vitamin C, vitamin D, n-3 PUFA, zinc, and probiotics, have been selected because of their possible relevance to the biological mechanisms that can beneficially affect viral and respiratory infections and because they are currently under clinical investigation. However, there are various other products and molecules that have garnered scientific interest and require further investigation. These include polyphenols, lipid extracts, and tomato-based nutraceuticals among others, which have all been suggested for the prevention of potential cardiovascular complications of COVID-19 such as thrombosis [561,571]. Melatonin is another supplement that has been identified as a potential antiviral agent against SARS-CoV-2 using computational methods [737], and it has also been highlighted as a potential therapeutic agent for COVID-19 due to its documented antioxidant, anti-apoptotic, immunomodulatory, and anti-inflammatory effects [571,738,739]. Notably, melatonin, vitamin D and zinc were included in the treatment plan of the Former President of the United States upon hospitalization due to COVID-19 [740]. These are just some of the many substances and supplements under investigation that currently have limited evidence to support their use or supplementation with the aim to prevent or treat COVID-19. While there is plenty speculation surrounding the use of supplements, a survey of 327,720 users of the COVID Symptom Study App found that the consumption of n-3 PUFA supplements, probiotics, multivitamins, and vitamin D was associated with a lower risk of SARS-CoV-2 infection in women but not men after adjusting for potential confounders [741]. According to the authors, the sexual dimorphism observed may in part be because supplements may better support females due to known differences between the male and female immune systems, or it could be due to behavioral and health consciousness differences between the sexes [741]. Certainly, randomized controlled trials are required to investigate these findings further.

In this review, we report the findings to date for several dietary supplements and nutraceuticals. While existing evidence suggests potential benefits of n-3 PUFA and probiotic supplementation for COVID-19 treatment and prophylaxis, clinical data is lacking but trials are underway. Both zinc and vitamin C supplementation in hospitalized patients seem to be associated with positive outcomes; however, further clinical trials are required. In any case, both vitamin C and zinc intake are part of a healthy diet and both likely presents minimal risk when supplemented for, but their potential prophylactic or therapeutic effects against COVID-19 are yet to be determined. On the other hand, mounting evidence from observational studies indicates that there is an association between vitamin D deficiency and COVID-19 incidence has also been supported by meta-analysis [742]. Indeed, scientists are working to confirm these findings and to determine whether a patient’s serum 25-hydroxyvitamin D levels are also associated with COVID-19 severity. Clinical trials are required to determine whether preemptive vitamin D supplementation may mitigate against severe COVID-19. In terms of the therapeutic potential of vitamin D, initial evidence from clinical trials are conflicting, but seem to indicate that vitamin D supplementation may reduce COVID-19 severity [668]. The various clinical trials currently underway will be imperative to provide information on the efficacious use of vitamin D supplementation for COVID-19 prevention and/or treatment.

Finally, it is known that a patient’s nutritional status affects health outcomes in various infectious diseases [535], and COVID-19 is no different [533,743,744]. Some of the main risk factors for severe COVID-19, which also happen to be linked to poor nutritional status includes obesity, hypertension, cardiovascular diseases, type II diabetes mellitus, and indeed age-related malnutrition [531,533,745]. Although not the main focus of this review, it is important to consider the nutritional challenges associated with severe COVID-19 patients. Hospitalized COVID-19 patients tend to report an unusually high loss of appetite preceding admission, some suffer diarrhea and gastrointestinal symptoms that result in significantly lower food intake, and patients with poorer nutritional status were more likely to have worse outcomes and require nutrition therapy [746]. Dysphagia also seems to be a significant problem in pediatric patients that suffered multisystem inflammatory syndrome (MIS-C) [747] and rehabilitating COVID-19 patients, potentially contributing to poor nutritional status [748]. Almost two-thirds of discharged COVID-19 ICU patients exhibit significant weight loss, of which 26% had weight loss greater than 10% [744]. As investigated in this review, hospitalized patients also tend to exhibit vitamin D deficiency or insufficiency, which may be associated with greater disease severity [742]. Therefore, further research is required to determine how dietary supplements and nutraceuticals may contribute to the treatment of severely ill and rehabilitating patients who often rely on enteral nutrition.

6.10 Conclusions

Despite all the potential benefits of nutraceutical and dietary supplement interventions presented, currently there is a paucity of clinical evidence to support their use for the prevention or mitigation of COVID-19 infection. Nevertheless, optimal nutritional status can prime an individual’s immune system to protect against the effects of acute respiratory viral infections by supporting normal maintenance of the immune system [531,535]. Nutritional strategies can also play a role in the treatment of hospitalized patients, as malnutrition is a risk to COVID-19 patients [748]. Overall, supplementation of vitamin C, vitamin D, and zinc may be an effective method of ensuring their adequate intake to maintain optimal immune function, which may also convey beneficial effects against viral infections due to their immunomodulatory effects. Individuals should pay attention to their nutritional status, particularly their intake of vitamin D, considering its deficiency tends to be widespread. The prevailing evidence seems to indicate an association between vitamin D deficiency with COVID-19 incidence and, potentially, severity [643]. As a result, some international authorities have advised the general public, particularly those at high risk of infection, to consider vitamin supplementation. However, further well-controlled clinical trials are required to confirm the observations presented in this review. Many supplements and nutraceuticals designed for various ailments are available in the United States and beyond that are not strictly regulated [749]. Consequently, there can be safety and efficacy concerns associated with many of these products. Often, the vulnerable members of society can be exploited in this regard and unfortunately the COVID-19 pandemic is no different. As mentioned above, the FDA has issued warnings to several companies for advertising falsified claims in relation to the preventative and therapeutic capabilities of their products against COVID-19 [750]. Further intensive investigation is required to establish the effects of these nutraceuticals, if any, against COVID-19. Until more effective therapeutics are established, the most effective mitigation strategies consist of encouraging standard public health practices such as regular hand washing with soap, wearing a face mask, and covering a cough with your elbow [751], along with following social distancing measures, “shelter in place” guidelines, expansive testing, and contact tracing [752,753]. Indeed, in light of this review, it would also be pertinent to adopt a healthy diet and lifestyle following national guidelines in order to maintain optimal immune health.

7 Social Factors Influencing COVID-19 Exposure and Outcomes

7.1 Social Factors Influencing COVID-19 Outcomes

In addition to understanding the fundamental biology of the SARS-CoV-2 virus and COVID-19, it is critical to consider how the broader environment can influence both COVID-19 outcomes and efforts to develop and implement treatments for the disease. The evidence clearly indicates that social environmental factors are critical determinants of individuals’ and communities’ risks related to COVID-19. There are distinct components to COVID-19 susceptibility, and an individual’s risk can be elevated at one or all stages from exposure to recovery/mortality: an individual may be more likely to be exposed to the virus, more likely to get infected once exposed, more likely to have serious complications once infected, and be less likely to receive adequate care once they are seriously ill. The fact that differences in survival between Black and white patients were no longer significant after controlling for comorbidities and socioeconomic status (type of insurance, neighborhood deprivation score, and hospital where treatment was received) in addition to sex and age [754] underscores the relevance of social factors to understanding mortality differences between racial and ethnic groups. Moreover, the Black patients were younger and more likely to be female than white patients, yet still had a higher mortality rate without correction for the other variables [754]. Here, we outline a few systemic reasons that may exacerbate the COVID-19 pandemic in communities of color.

7.2 Factors Observed to be Associated with Susceptibility

As COVID-19 has spread into communities around the globe, it has become clear that the risks associated with this disease are not equally shared by all individuals or all communities. Significant disparities in outcomes have led to interest in the demographic, biomedical, and social factors that influence COVID-19 severity. Untangling the factors influencing COVID-19 susceptibility is a complex undertaking. Among patients who are admitted to the hospital, outcomes have generally been poor, with rates of admission to the intensive care unit (ICU) upwards of 15% in both Wuhan, China and Italy [34,755,756]. However, hospitalization rates vary by location [757]. This variation may be influenced by demographic (e.g., average age in the area), medical (e.g., the prevalence of comorbid conditions such as diabetes), and social (e.g., income or healthcare availability) factors that vary geographically. Additionally, some of the same factors may influence an individual’s probability of exposure to SARS-CoV-2, their risk of developing a more serious case of COVID-19 that would require hospitalization, and their access to medical support. As a result, quantifying or comparing susceptibility among individuals, communities, or other groups requires consideration of a number of complex phenomena that intersect across many disciplines of research. In this section, the term “risk factor” is used to refer to variables that are statistically associated with more severe COVID-19 outcomes. Some are intrinsic characteristics that have been observed to carry an association with variation in outcomes, whereas others may be more functionally linked to the pathophysiology of COVID-19.

7.2.1 Patient Traits Associated with Increased Risk

Two traits that have been consistently associated with more severe COVID-19 outcomes are male sex and advanced age (typically defined as 60 or older, with the greatest risk among those 85 and older [758]). In the United States, males and older individuals diagnosed with COVID-19 were found to be more likely to require hospitalization [759,760]. A retrospective study of hospitalized Chinese patients [35] found that a higher probability of mortality was associated with older age, and world-wide, population age structure has been found to be an important variable for explaining differences in outbreak severity [761]. The CFR for adults over 80 has been estimated upwards of 14% or even 20% [762]. Male sex has also been identified as a risk factor for severe COVID-19 outcomes, including death [763,764,765]. Early reports from China and Europe indicated that even though the case rates were similar across males and females, males were at elevated risk for hospital admission, ICU admission, and death [764], although data from some US states indicates more cases among females, potentially due to gender representation in care-taking professions [766]. In older age groups (e.g., age 60 and older), comparable absolute numbers of male and female cases actually suggests a higher rate of occurrence in males, due to increased skew in the sex ratio [764]. Current estimates based on worldwide data suggest that, compared to females, males may be 30% more likely to be hospitalized, 80% more likely to be admitted to the ICU, and 40% more likely to die as a result of COVID-19 [765]. There also may be a compounding effect of advanced age and male sex, with differences time to recovery worst for males over 60 years old relative to female members of their age cohort [767].

Both of these risk factors can be approached through the lens of biology. The biological basis for greater susceptibility with age is likely linked to the prevalence of extenuating health conditions such as heart failure or diabetes [762]. Several hypotheses have been proposed to account for differences in severity between males and females. For example, some evidence suggests that female sex hormones may be protective [764,766]. ACE2 expression in the kidneys of male mice was observed to be twice as high as that of females, and a regulatory effect of estradiol on ACE2 expression was demonstrated by removing the gonads and then supplementing with estradiol [766,768]. Other work in mice has shown an inverse association between mortality due to SARS-CoV-1 and estradiol, suggesting a protective role for the sex hormone [766]. Similarly, evidence suggests that similar patterns might be found in other tissues. A preliminary analysis identified higher levels of ACE2 expression in the myocardium of male patients with aortic valve stenosis showed than female patients, although this pattern was not found in controls [764]. Additionally, research has indicated that females respond to lower doses than males of heart medications that act on the Renin angiotensin aldosterone system (RAAS) pathway, which is shared with ACE2 [764]. Additionally, several components of the immune response, including the inflammatory response, may differ in intensity and timing between males and females [766,768]. This hypothesis is supported by some preliminary evidence showing that female patients who recovered from severe COVID-19 had higher antibody titers than males [766]. Sex steroids can also bind to immune cell receptors to influence cytokine production [764]. Additionally, social factors may influence risks related to both age and sex: for example, older adults are more likely to live in care facilities, which have been a source for a large number of outbreaks [769], and gender roles may also influence exposure and/or susceptibility due to differences in care-taking and/or risky behaviors (e.g., caring for elder relatives and smoking, respectively) [764] among men and women (however, it should be noted that both transgender men and women are suspected to be at heightened risk [770].)

7.2.2 Comorbid Health Conditions

A number of pre-existing or comorbid conditions have repeatedly been identified as risk factors for more severe COVID-19 outcomes. Several underlying health conditions were identified at high prevalence among hospitalized patients, including obesity, diabetes, hypertension, lung disease, and cardiovascular disease [757]. Higher Sequential Organ Failure Assessment (SOFA) scores have been associated with a higher probability of mortality [35], and comorbid conditions such as cardiovascular and lung disease as well as obesity were also associated with an increased risk of hospitalization and death, even when correcting for age and sex [763]. Diabetes may increase the risk of lengthy hospitalization [771] or of death [771,772]. [773] and [774] discuss possible ways in which COVID-19 and diabetes may interact. Obesity also appears to be associated with higher risk of severe outcomes from SARS-CoV-2 [775,776]. Obesity is considered an underlying risk factor for other health problems, and the mechanism for its contributions to COVID-19 hospitalization or mortality is not yet clear [777]. Dementia and cancer were also associated with the risk of death in an analysis of a large number (more than 20,000) COVID-19 patients in the United Kingdom [763]. It should be noted that comorbid conditions are inextricably tied to age, as conditions tend to be accumulated over time, but that the prevalence of individual comorbidities or of population health overall can vary regionally [778]. Several comorbidities that are highly prevalent in older adults, such as COPD, hypertension, cardiovascular disease, and diabetes, have been associated with CFRs upwards of 8% compared to an estimate of 1.4% in people without comorbidities [762,779]. Therefore, both age and health are important considerations when predicting the impact of COVID-19 on a population [778]. However, other associations may exist, such as patients with sepsis having higher SOFA scores – in fact, SOFA was developed for the assessment of organ failure in the context of sepsis, and the acronym originally stood for Sepsis-Related Organ Failure Assessment [780,781]. Additionally, certain conditions are likely to be more prevalent under or exacerbated by social conditions, especially poverty, as is discussed further below.

7.2.3 Ancestry

A number of studies have suggested associations between individuals’ racial and ethnic backgrounds and their COVID-19 risk. In particular, Black Americans are consistently identified as carrying a higher burden of COVID-19 than white Americans [759,760], with differences in the rates of kidney complications from COVID-19 particularly pronounced [91]. Statistics from a number of cities indicate significant discrepancies between the proportion of COVID-19 cases and deaths in Black Americans relative to their representation in the general population [681]. In addition to Black Americans, disproportionate harm and mortality from COVID-19 has also been noted in Latino/Hispanic Americans and in Native American and Alaskan Native communities, including the Navajo nation [782,783,784,785,786,787]. In Brazil, indigenous communities likewise carry an increased burden of COVID-19 [788]. In the United Kingdom, nonwhite ethnicity (principally Black or South Asian) was one of several factors found to be associated with a higher risk of death from COVID-19 [789].

From a genetic standpoint, it is highly unlikely that ancestry itself predisposes individuals to contracting COVID-19 or to experiencing severe COVID-19 outcomes. Examining human genetic diversity indicates variation over a geographic continuum, and that most human genetic variation is associated with the African continent [790]. African-Americans are also a more genetically diverse group relative to European-Americans, with a large number of rare alleles and a much smaller fraction of common alleles identified in African-Americans [791]. Therefore, the idea that African ancestry (at the continent level) might convey some sort of genetic risk for severe COVID-19 contrasts with what is known about worldwide human genetic diversity [792]. The possibility for genetic variants that confer some risk or some protection remains possible, but has not been widely explored, especially at a global level. Research in Beijing of a small number (n=80) hospitalized COVID-19 patients revealed an association between severe COVID-19 outcomes and homozygosity for an allele in the interferon-induced transmembrane protein 3 (IFITM3) gene, which was selected as a candidate because it was previously found to be associated with influenza outcomes in Chinese patients [793]. Genetic factors may also play a role in the risk of respiratory failure for COVID-19 [794,795,796]. However, genetic variants associated with outcomes within ancestral groups are far less surprising than genetic variants explaining outcomes between groups. Alleles in ACE2 and TMPRSS2 have been identified that vary in frequency among ancestral groups [797], but whether these variants are associated with COVID-19 susceptibility has not been explored.

Instead, examining patterns of COVID-19 susceptibility on a global scale that suggest that social factors are of primary importance in predicting mortality. Reports from several sub-Saharan African countries have indicated that the effects of the COVID-19 pandemic have been less severe than expected based on the outbreaks in China and Italy. In Kenya, for example, estimates of national prevalence based on testing blood donors for SARS-CoV-2 antibodies were consistent with 5% of Kenyan adults having recovered from COVID-19 [798]. This high seroprevalence of antibodies lies in sharp contrast to the low number of COVID-19 fatalities in Kenya, which at the time was 71 out of 2093 known cases [798]. Likewise, a serosurvey of health care workers in Blantyre City, Malawi reported an adjusted antibody prevalence of 12.3%, suggesting that the virus had been circulating more widely than thought and that the death rate was up eight times lower than models had predicted [799]. While several possible hypotheses for the apparent reduced impact of COVID-19 on the African continent are being explored, such as young demographics in many places [800], these reports present a stark contrast to the severity of COVID-19 in Americans and Europeans of African descent. Additionally, ethnic minorities in the United Kingdom also tend to be younger than white British living in the same areas, yet the burden of COVID-19 is still more serious for minorities, especially people of Black Caribbean ancestry, both in absolute numbers and when controlling for age and location [801]. Furthermore, the groups in the United States and United Kingdom that have been identified as carrying elevated COVID-19 burden, namely Black American, indigenous American, and Black and South Asian British, are quite distinct in their position on the human ancestral tree. What is shared across these groups is instead a history of disenfranchisement under colonialism and ongoing systematic racism. A large analysis of over 11,000 COVID-19 patients hospitalized in 92 hospitals across U.S. states revealed that Black patients were younger, more often female, more likely to be on Medicaid, more likely to have comorbidities, and came from neighborhoods identified as more economically deprived than white patients [754]. This study reported that when these factors were accounted for, the differences in mortality between Black and white patients were no longer significant. Thus, the current evidence suggests that the apparent correlations between ancestry and health outcomes must be examined in the appropriate social context.

7.3 Environmental Influences on Susceptibility

7.3.1 Exposure to COVID-19

Social distancing has emerged as one of the main social policies used to manage the COVID-19 epidemic in many countries. Many governments issued stay-at-home orders, especially in the initial months of the crisis. However, data clearly indicates that these orders impacted different socioeconomic groups differently. In U.S. counties with and without stay-at-home orders, smartphone tracking indicated a significant decrease in the general population’s mobility in April relative to February through March of 2020 (-52.3% and -60.8%, respectively) [802]. A linear relationship was observed between counties’ reduction in mobility and their wealth and health, as measured by access to health care, food security, income, space, and other factors [802]. Counties with greater reductions in mobility were also found to have much lower child poverty and household crowding and to be more racially segregated, and to have fewer youth and more elderly residents [802]. Similar associations between wealth and decreased mobility were observed in cellphone GPS data from Colombia, Indonesia, and Mexico collected between January and May 2020 [803], as well as in a very large data set from several US cities [804]. These disparities in mobility are likely to be related to the role that essential workers have played during the pandemic. Essential workers are disproportionately likely to be female, people of color, immigrants, and to have an income below 200% of the poverty line [805]. Black Americans in particular are over-represented among front-line workers and in professions where social distancing is infeasible [806]. Health care work in particular presents an increased risk of exposure to SARS-CoV-2 [806,807,808,809,810]. In the United Kingdom, (South) Asians are more likely than their white counterparts to be medical professionals [801], although BAME medical professionals are still disproportionately represented in the proportion of National Health Service staff deaths [811]. Similar trends have been reported for nurses, especially nurses of color, in the United States [812]. Furthermore, beyond the risks associated with work itself, use of public transportation may also impact COVID-19 risk [813]. The socioeconomic and racial/ethnic gaps in who is working on the front lines of the pandemic make it clear that socioeconomic privilege is likely to decrease the probability of exposure to SARS-CoV-2.

Increased risk of exposure can also arise outside the workplace. Nursing homes and skilled nursing facilities received attention early on as high-risk locations for COVID-19 outbreaks [814]. Prisons and detention centers also confer a high risk of exposure or infection [815,816]. Populations in care facilities are largely older adults, and in the United States, incarcerated people are more likely to be male and persons of color, especially Black [817]. Additionally, multi-generational households are less common among non-Hispanic white Americans than people of other racial and ethnic backgrounds [818], increasing the risk of exposure for more susceptible family members. Analysis suggests that household crowding may also be associated with increased risk of COVID-19 exposure [802], and household crowding is associated with poverty [819]. Forms of economic insecurity like housing insecurity, which is associated with poverty and more pronounced in communities subjected to racism [820,821], would be likely to increase household crowding and other possible sources of exposure. As a result, facets of systemic inequality such as mass incarceration of Black Americans and poverty are likely to increase the risk of exposure outside of the workplace.

7.3.2 Severity of COVID-19 Following Exposure

Following exposure to SARS-CoV-2, the likelihood that an individual develops COVID-19 and the severity of the disease presentation can be influenced by a number of social factors. As discussed above, a number of patient characteristics are associated with the likelihood of severe COVID-19 symptoms. In some cases, these trends run counter to those expected given rates of exposure: for example, although women are more likely to be exposed, men are more likely to be diagnosed with, hospitalized from, or die from COVID-19 [766]. In the case of comorbid conditions and racial/ethnic demographics, however, social factors are highly likely to modulate or at least influence the apparent association between these traits and the increased risk from COVID-19. In particular, the comorbidities and racial/ethnic correlates of severe COVID-19 outcomes suggest that poverty confers additional risk for COVID-19.

In order to explore the relationship between poverty and COVID-19 outcomes, it is necessary to consider how poverty impacts biology. In particular, we focus on the United States and the United Kingdom. Comorbidities that increase risk for COVID-19, including obesity, type II diabetes, hypertension, and cardiovascular disease, are known to be intercorrelated [822]. Metabolic conditions related to heightened inflammation, like obesity, type II diabetes, and hypertension, are more strongly associated with negative COVID-19 outcomes than other comorbid conditions, such as chronic heart disease [823]. As discussed above, dysregulated inflammation characteristic of cytokine release syndrome is one of the greatest concerns for COVID-19-related death. Therefore, it is possible that chronic inflammation characteristic of these metabolic conditions predisposes patients to COVID-19-related death [823]. The association between these diseases and severe COVID-19 outcomes is a concern from a health equity perspective because poverty exposes people to “obesogenic” conditions [824] and is therefore unsurprisingly associated with higher incidence of obesity and associated disorders [825]. Furthermore, cell phone GPS data suggests that lower socioeconomic status may also be associated with decreased access to healthy food choices during the COVID-19 pandemic [826,827], suggesting that health-related risk factors for COVID-19 may be exacerbated as the pandemic continues [828]. Chronic inflammation is a known outcome of chronic stress (e.g., [829,830,831,832]). Therefore, the chronic stress of poverty is likely to influence health broadly (as summarized in [833]) and especially during the stress of the ongoing pandemic.

A preprint [834] provided observational evidence that geographical areas in the United States that suffer from worse air pollution by fine particulate matter have also suffered more COVID-19 deaths per capita, after adjusting for demographic covariates. Although lack of individual-level exposure data and the impossibility of randomization make it difficult to elucidate the exact causal mechanism, this finding would be consistent with similar findings for all-cause mortality (e.g., [835]). Exposure to air pollution is associated with both poverty (e.g., [836]) and chronic inflammation [837]. Other outcomes of environmental racism, such as the proximity of abandoned uranium mines to Navajo land, can also cause respiratory illnesses and other health issues [787]. Similarly, preliminary findings indicate that nutritional status (e.g., vitamin D deficiency [652]) may be associated with COVID-19 outcomes, and reduced access to grocery stores and fresh food often co-occurs with environmental racism [787,838]. Taken together, the evidence suggests that low-income workers who face greater exposure to SARS-CoV-2 due to their home or work conditions are also more likely to face environmental and social stressors associated with increased inflammation, and therefore with increased risk from COVID-19. In particular, structural racism can play an important role on disease severity after SARS-CoV-2 exposure, due to consequences of racism which include an increased likelihood of poverty and its associated food and housing instability. COVID-19 can thus be considered a “syndemic”, or a synergistic interaction between several epidemics [839]. As a result, it is not surprising that people from minoritized backgrounds and/or with certain pre-existing conditions are more likely to suffer severe effects of COVID-19, but these “risk factors” are likely to be causally linked to poverty.

7.3.3 Access to Treatment

Finally, COVID-19 outcomes can be influenced by access to healthcare. Receiving care for COVID-19 can, but does not always, include receiving a positive test for the SARS-CoV-2 virus. For example, it is common to see treatment guidelines for suspected cases regardless of whether the presence of SARS-CoV-2 has been confirmed (e.g., [840]). Whether and where a patient is diagnosed can depend on their access to testing, which can vary both between and within countries. In the United States, it is not always clear whether an individual will have access to free testing [841,842]. The concern has been raised that more economic privilege is likely to correspond to increased access to testing, at least within the United States [843]. This is supported by the fact that African Americans seem to be more likely to be diagnosed in the hospital, while individuals from other groups were more likely to have been diagnosed in ambulatory settings in the community [759]. Any delays in treatment are a cause for concern [843], which could potentially be increased by an inability to acquire testing because in the United States, insurance coverage for care received can depend on a positive test [844].

Another important question is whether patients with moderate to severe cases are able to access hospital facilities and treatments, to the extent that they have been identified. Early findings from China as of February 2020 suggested the COVID-19 mortality rate to be much lower in the most developed regions of the country [845], although reported mortality is generally an estimate of CFR, which is dependent on rates of testing. Efforts to make treatment accessible for all confirmed and suspected cases of COVID-19 in China are credited with expanding care to people with fewer economic resources [846]. In the United States, access to healthcare varies widely, with certain sectors of the workforce less likely to have health insurance; many essential workers in transportation, food service, and other frontline fields are among those likely to be uninsured or underinsured [843]. As of 2018, Hispanic Americans of all races were much less likely to have health insurance than people from non-Hispanic backgrounds [847]. Therefore, access to diagnostics and care prior to the development of severe COVID-19 is likely to vary depending on socioeconomic and social factors, many of which overlap with the risks of exposure and of developing more severe COVID-19 symptoms. This discrepancy ties into concerns about broad infrastructural challenges imposed by COVID-19. A major concern in many countries has been the saturation of healthcare systems due to the volume of COVID-19 hospitalizations (e.g., [227]). Similarly, there have been shortages of supplies such as ventilators that are critical to the survival of many COVID-19 patients, leading to extensive ethical discussions about how to allocate limited resources among patients [848,849,850,851]. Although it is generally considered unethical to consider demographic factors such as age, sex, race, or ethnicity while making such decisions, and ideally this information would not be shared with triage teams tasked with allocating limited resources among patients [852], there are substantial concerns about implicit and explicit biases against older adults [853], premature infants [854], and people with disabilities or comorbidities [852,855,856]. Because of the greater burden of chronic disease in populations subjected to systemic racism, algorithms intended to be blind to race and ethnicity could, in fact, reinforce systemic inequalities caused by structural racism [857,858,859]. Because of this inequality, it has been argued that groups facing health disparities should be prioritized by these algorithms [860]. This approach would carry its own ethical concerns, including the fact that many resources that need to be distributed do not have well-established risks and benefits [860].

As the pandemic has progressed, it has become clear that ICU beds and ventilators are not the only limited resources that needs to be allocated, and, in fact, the survival rate for patients who receive mechanical ventilation is lower than these discussions would suggest [861]. Allocation of interventions that may reduce suffering, including palliative care, has become critically important [861,862]. The ambiguities surrounding the risks and benefits associated with therapeutics that have been approved under emergency use authorizations also present ethical concerns related to the distribution of resources [860]. For example, remdesivir, discussed above, is currently available for the treatment of COVID-19 under compassionate use guidelines and through expanded access programs, and in many cases has been donated to hospitals by Gilead [863,864]. Regulations guiding the distribution of drugs in situations like these typically do not address how to determine which patients receive them [864]. Prioritizing marginalized groups for treatment with a drug like remdesivir would also be unethical because it would entail disproportionately exposing these groups to a therapeutic that may or not be beneficial [860]. On the other hand, given that the drug is one of the most promising treatments available for many patients, using a framework that tacitly feeds into structural biases would also be unethical. At present, the report prepared for the Director of the CDC by Ethics Subcommittee of the CDC fails to address the complexity of this ethical question given the state of structural racism in the United States, instead stating that “prioritizing individuals according to their chances for short-term survival also avoids ethically irrelevant considerations, such as race or socioeconomic status” [865]. In many cases, experimental therapeutics are made available only through participation in clinical trials [866]. However, given the history of medical trials abusing minority communities, especially Black Americans, there is a history of unequal representation in clinical trial enrollment [866]. As a result, the standard practice of requiring enrollment in a clinical trial in order to receive experimental treatment may also reinforce patterns established by systemic racism.

7.3.4 Access to and Representation in Clinical Trials

Experimental treatments are often made available to patients primarily or even exclusively through clinical trials. The advantage of this approach is that clinical trials are designed to collect rigorous data about the effects of a treatment on patients. The disadvantage is that access to clinical trials is not equal among all people who suffer from a disease. Two important considerations that can impact an individual’s access to clinical trials are geography and social perceptions of clinical trials. For the first, the geographic distribution of trial recruitment efforts are typically bounded and can vary widely among difference locations, and for the second, the social context of medical interactions can impact strategies for and the success of outreach to different communities. Differential access to clinical trials raises concerns because it introduces biases that can influence scientific and medical research on therapeutics and prophylactics broadly. Concerns about bias in clinical trials need to address both trial recruitment and operation. In the present crisis, such biases are particularly salient because COVID-19 is a disease of global concern. Treatment is needed by people all over the world, and clinical research that characterizes treatment outcomes in a variety of populations is critically important.

Global representation in clinical trials is important to ensuring that experimental treatments are available equally to COVID-19 patients who may need them. The advantage to a patient of participation in a clinical trial is that they may receive an experimental treatment they would not have been able to access otherwise. The potential downsides of participation include that the efficacy and side effects of such treatments are often poorly characterized and that patients who enroll in clinical trials will in some cases run the risk of being assigned to a placebo condition where they do not receive the treatment but miss out on opportunities to receive other treatments. The benefits and burdens of clinical trials therefore need to be weighed carefully to ensure that they don’t reinforce existing health disparities. The WHO Director‐General Tedros Adhanom Ghebreyesus stated his condemnation of utilizing low and middle income countries as test subjects for clinical trials, yet having highly developed countries as the majority of clinical trial representation is also not the answer [867]. Figure 6 showcases two choropleths detailing COVID-19 clinical trial recruitment by country. China, the United States, and France are among the countries with the most clinical trial recruiting for trials with single-country enrollment. Many countries have little to no clinical trial recruiting, with the continents of Africa and South America much less represented than Asia, Europe, and North America. Trials that recruit across multiple countries do appear to broaden geographic representation, but these trials seem to be heavily dominated by the United States and European Union.

Figure 6: Geographic distribution of COVID-19 clinical trials. The density of clinical trials is reported at the country level. As of November 9, 2020, there are 6,417 trials in the University of Oxford Evidence-Based Medicine Data Lab’s COVID-19 TrialsTracker [293]. The top figure demonstrates the density of trials recruiting only from a singular country, while the bottom shows the distribution of recruitment for trials that involve more than one country.

A few different concerns arise from this skewed geographic representation in clinical trial recruitment. First, treatments such as remdesivir that are promising but primarily available to clinical trial participants are unlikely to be accessible by people in many countries. Second, it raises the concern that the findings of clinical trials will be based on participants from many of the wealthiest countries, which may lead to ambiguity in whether the findings can be extrapolated to COVID-19 patients elsewhere. Especially with the global nature of COVID-19, equitable access to therapeutics and vaccines has been a concern at the forefront of many discussions about policy (e.g., [868], yet data like that shown in Figure 6 demonstrates that accessibility is likely to be a significant issue. Another concern with the heterogeneous international distribution of clinical trials is that the governments of countries leading these clinical trials might prioritize their own populations once vaccines are developed, causing unequal health outcomes [869]. Additionally, even within a single state in the United States (Maryland), geography was found to influence the likelihood of being recruited into or enrolled in a clinical trial, with patients in under-served rural areas less likely to enroll [870]. Thus, geography both on the global and local levels may influence when treatments and vaccines are available and who is able to access them. Efforts such as the African Union’s efforts to coordinate and promote vaccine development [871] are therefore critical to promoting equity in the COVID-19 response.

Even when patients are located within the geographic recruitment area of clinical trials, however, there can still be demographic inequalities in enrollment. When efforts are made to ensure equal opportunity to participate in clinical trials, there is no significant difference in participation among racial/ethnic groups [872]. However, within the United States, real clinical trial recruitment numbers have indicated for many years that racial minorities, especially African-Americans, tend to be under-represented (e.g., [873,874,875,876]). This trend is especially concerning given the disproportionate impact of COVID-19 on African-Americans. Early evidence suggests that the proportion of Black, Latinx, and Native American participants in clinical trials for drugs such as remdesivir is much lower than the representation of these groups among COVID-19 patients [877].

One proposed explanation for differences among racial and ethnic groups in clinical trial enrollment refers to different experiences in healthcare settings. While some plausible reasons for the disparity in communication between physicians and patients could be a lack of awareness and education, mistrust in healthcare professionals, and a lack of health insurance [872], a major concern is that patients from certain racial and ethnic groups are marginalized even while seeking healthcare. In the United States, many patients experience “othering” from physicians and other medical professionals due to their race or other external characteristics such as gender (e.g., [878]). Many studies have sought to characterize implicit biases in healthcare providers and whether they affect their perceptions or treatment of patients. A systematic review that examined 37 such studies reported that most (31) identified racial and/or ethnic biases in healthcare providers in many different roles, although the evidence about whether these biases translated to different attitudes towards patients was mixed [879], with similar findings reported by a second systematic review [880]. However, data about real-world patient outcomes are very limited, with most studies relying on clinical vignette-based exercises [879], and other analyses suggest that physician implicit bias could impact the patient’s perception of the negativity/positivity of the interaction regardless of the physician’s explicit behavior towards the patient [881]. Because racism is a common factor in both, negative patient experiences with medical professionals are likely to compound other issues of systemic inequality, such as a lack of access to adequate care, a lack of insurance, or increased exposure to SARS-CoV-2 [882]. Furthermore, the experience of being othered is not only expected to impact patients’ trust in and comfort with their provider, but also may directly impact whether or not the patient is offered the opportunity to participate in a clinical trial at all. Some studies suggest communication between physicians and patients impacts whether or not a physician offers a patient participation in a clinical trial. For example, researchers utilized a linguistic analysis to assess mean word count of phrases related to clinical trial enrollment, such as voluntary participation, clinical trial, etc. [872]. The data indicated that the mean word count of the entire visit was 1.5 times more for white patients in comparison to Black patients. In addition, the greatest disparity between white and Black patients’ experience was the discussion of risks, with over 2 times as many risk-related words spoken with white patients than Black patients [872]. The trends observed for other clinical trials raise the concern that COVID-19 clinical trial information may not be discussed as thoroughly or as often with Black patients compared to white patients.

These discrepancies are especially concerning given that many COVID-19 treatments are being or are considered being made available to patients prior to FDA approval through Emergency Use Authorizations. In the past, African-Americans have been over-represented relative to national demographics in use of the FDA’s Exception From Informed Consent (EFIC) pathway [883]. Through this pathway, people who are incapacitated can receive an experimental treatment even if they are not able to consent and there is not sufficient time to seek approval from an authorized representative. This pathway presents concerns, however, when it is considered in the context of a long history of systematic abuses in medical experimentation where informed consent was not obtained from people of color, such as the Tuskegee syphilis experiments [884]. While the goal of EFIC approval is to provide treatment to patients who urgently need it, the combination of the ongoing legacy of racism in medicine renders this trend concerning. With COVID-19, efforts to prioritize people who suffer from systemic racism are often designed with the goal of righting some of these inequalities (e.g., [885]), but particular attention to informed consent will be imperative in ensuring these trials are ethical given that the benefits and risks of emerging treatments are still poorly characterized. Making a substantial effort to run inclusive clinical trials is also important because of the possibility that racism could impact how a patient responds to a treatment. For example, as discussed above, dexamethasone has been identified as a promising treatment for patients experiencing cytokine release syndrome, but the mechanism of action is tied to the stress response. A study from 2005 reported that Black asthma patients showed reduced responsiveness to dexamethasone in comparison to white patients and suggested Black patients might therefore require higher doses of the drug [886]. In the context of chronic stress caused by systemic racism, this result is not surprising: chronic stress is associated with dysregulated production of glucocorticoids [887] and glucocorticoid receptor resistance [888]. However, it underscores the critical need for treatment guidelines to take into account differences in life experience, which would be facilitated by the recruitment of patients from a wide range of backgrounds. Attention to the social aspects of clinical trial enrollment must therefore be an essential component of the medical research community’s response to COVID-19.

7.4 Conclusions and Future Directions

As the COVID-19 pandemic evolves, the scientific community’s response will be critical for identifying potential pharmacological and biotechnological developments that may aid in combating the virus and the disease it causes. However, this global crisis highlights the importance of mounting a response based on collaboration among a wide variety of disciplines. Understanding the basic science of the virus and its pathogenesis is imperative for identifying and envisioning possible diagnostic and therapeutic approaches; understanding how social factors can influence outcomes and shape implementation of a response is critical to disseminating any scientific advancements. Summarizing such a complex and ever-changing topic presents a number of challenges. This review represents the effort of over 50 contributors to distill and interpret the available information. However, this text represents a dynamic and evolving document, and we welcome continued contributions from all researchers who have insights into how these topics intersect. A multidisciplinary perspective is critical to understanding this evolving crisis, and in this review we seek to use open science tools to coordinate a response among a variety of researchers. We intend to publish additional updates as the situation evolves.

8 Discussion

As of October 2020 the SARS-CoV-2 virus remains a serious worldwide threat. The scientific community has responded by rapidly collecting and disseminating information about the SARS-CoV-2 virus and the associated illness, COVID-19. The rapid identification of the genomic sequence of the virus allowed for early contextualization of SARS-CoV-2 among other known respiratory viruses. The pathogen is a coronavirus that is closely related to SARS-CoV-1, which caused the SARS pandemics of the early 2000s. Knowing the phylogenetic context and genomic sequence of the virus then allowed for rapid insights into its structure and pathogenesis. As with other HCoV, the immune response to SARS-CoV-2 is likely driven by detection of its spike protein, which allows it to enter cells through the ACE2 receptor. Epithelial cells have also emerged as the major cellular target of the virus, contextualizing the respiratory and gastrointestinal symptoms that are frequently observed in COVID-19. However, as COVID-19 cases have been more widely characterized, it has become clear that the disease presentation is highly heterogeneous. Many cases, especially in younger adults, present with mild symptoms or even asymptomatically, while others, especially in older adults, can be severe or fatal. In children, the SARS-CoV-2 virus can present as two distinct diseases, COVID-19 or MIS-C. While the overall infection fatality rate remains unknown, estimates suggest that it is not more than 1%; however, the severity of many non-lethal cases makes COVID-19 an ongoing, significant concern.

Characterizing the rate of infection and fatality rates hinges on the availability of rapid and accurate diagnostic testing. Major advancements have been made in identifying diagnostic approaches. The development of diagnostic technologies have been rapid, beginning with the release of the SARS-CoV-2 viral genome sequence in January. As of October 2020, a range of diagnostic tests have become available. One class of tests uses PCR (RT-PCR or qRT-PCR) to assess the presence of SARS-CoV-2 RNA, while another typically uses ELISA to test for the presence of antibodies to SARS-CoV-2. The former approach is useful for identifying active infections, while the latter measures hallmarks of the immune response and therefore can detect either active infections or immunity gained from prior infection. Combining these tests leads to extremely accurate detection of SARS-CoV-2 infection (98.6%), but when used alone, PCR-based tests are recommended before 5.5 days after the onset of the illness and antibody tests after 5.5 days [287]. Other strategies for testing can also influence the tests’ accuracy, such as the use of nasopharyngeal swabs versus BALF [287], which allow for trade-offs between patient’s comfort and test sensitivity. Additionally, technologies such as digital PCR may allow for scale-up in the throughput of diagnostic testing, facilitating widespread testing. One major question that remains is whether people who recover from SARS-CoV-2 develop sustained immunity, and over what period this immunity is expected to last. Some reports have suggested that some patients may develop COVID-19 reinfections (e.g., [288]), but the rates of reinfection are currently unknown. Serologic testing combined with PCR testing will be critical to confirming purported cases of reinfection and to identifying the duration over which immunity is retained and to understanding reinfection risks.

One of the goals of characterizing the immune response is to identify strategies for the prophylactic enhancements of immunity. Though some concerns remain about the duration of sustained immunity for convalescents, vaccine development efforts are ongoing and show initial promising results. The Moderna trial, for example, reported that the neutralizing activity in participants who received two doses of the vaccine was similar to that observed in convalescent plasma. Vaccine development for COVID-19 is progressing rapidly compared to typical timelines, but vaccine development is still a lengthy process. In the meantime, some advances have also been made in the treatment of patients with COVID-19. As cases have become better characterized, it has become evident that many patients experience an initial immune response to the virus that is typically characterized by fever, cough, dyspnea, and related symptoms. However, the most serious concern is cytokine release syndrome, when the body’s immune response becomes dysregulated, resulting in an extreme inflammatory response. The RECOVERY trial, a large-scale, multi-arm trial enrolling about 15% of all COVID-19 patients in the United Kingdom, was the first to identify that the widely available steroid dexamethasone seems to be beneficial for patients suffering from this immune dysregulation [415]. Efforts to identify therapeutic treatments to treat patients early in the course of infection have been more ambiguous. Early interest in the drugs hydroxychloroquine and chloroquine yielded no promising results from studies with robust experimental designs. The experimental drug remdesivir, which was developed for Ebola, has received enough support from early analyses to receive FDA approval, but results have been mixed. The potential for other drugs, such as tocilizumab, to reduce recovery time remains unclear, but some early results were promising.

8.1 Additional Therapeutics of Interest

Given what is currently known about these therapeutics for COVID-19, a number of related therapies beyond those explored above may also prove to be of interest. For example, the demonstrated benefit of dexamethasone and the ongoing potential of tocilizumab for treatment of COVID-19 suggests that other anti-inflammatory agents might also hold value for the treatment of COVID-19. Given that current evidence about treating COVID-19 with dexamethasone suggests that the need to curtail the cytokine storm inflammatory response to the virus can transcend the risks of immunosuppression, exploration of more anti-inflammatory agents may be warranted. While dexamethasone is considered widely available and generally affordable, the high costs of biologics such as tocilizumab therapy may present obstacles to wide-scale distribution of this drug if it proves of value. At the doses used for rheumatoid arthritis patients, the cost for tocilizumab ranges from $179.20 to $896 per dose for the IV form and $355 for the pre-filled syringe [517]. There are several anti-inflammatory agents used for the treatment of autoimmune diseases that may also be able to counter the effects of the cytokine storm induced by the virus, some of which, such as cyclosporine, are likely to be more cost-effective and readily available than biologics [518]. While tocilizumab targets IL-6, several other inflammatory markers could be potential targets, including TNF-alpha. Inhibition of TNF-alpha by an inhibitor such as Etanercept has been previously suggested for treatment of SARS-CoV-1 [519] and may be relevant for SARS-CoV-2 as well. Another anti-IL-6 antibody, sarilumab, is also being investigated [520,521]. Baricitinib and other small molecule inhibitors of the Janus-activated kinase pathway also curtail the inflammatory response and have been suggested as potential options for SARS-CoV-2 infections [522]. Baricitinib in particular may be able to reduce the ability of SARS-CoV-2 to infect lung cells [523]. Clinical trials studying baricitinib in COVID-19 have already begun in the US and in Italy [524,525]. Identification and targeting of further inflammatory markers that are relevant in SARS-CoV-2 infection may be of value for curtailing the inflammatory response and lung damage.

In addition to immunosuppressive treatments that are most beneficial late in disease progression, much research is focused on identifying treatments would be likely to benefit early-stage patients. For example, although studies of hydroxychloroquine have not supported the early theory-driven interest in this antiviral treatment, alternative compounds with related mechanisms may still have potential. Hydroxyferroquine derivatives of HCQ have been described as a class of bioorganometallic compounds that exert antiviral effects with some selectivity for SARS-CoV-1 in vitro [526]. Future work could explore whether such compounds exert antiviral effects against SARS-CoV-2 and whether they would be safer for use in COVID-19. Another potential approach is the development of antivirals, which could be broad-spectrum, specific to coronaviruses, or targeted to SARS-CoV-2. Development of new antivirals is complicated by the fact that none have yet been approved for human coronaviruses. Intriguing new options are emerging, however. Beta-D-N4-hydroxycytidine (NHC) is an orally bioavailable ribonucleotide analog showing broad-spectrum activity against RNA viruses, which may inhibit SARS-CoV-2 replication in vitro and in vivo in mouse models of HCoVs [527]. A range of other antivirals are also in development. Development of antivirals will be further facilitated as research reveals more information about the interaction of SARS-CoV-2 with the host cell and host cell genome, mechanisms of viral replication, mechanisms of viral assembly, and mechanisms of viral release to other cells; this can allow researchers to target specific stages and structures of the viral life cycle. Many researchers have also focused their attention to the potential use of dietary supplements and nutraceuticals. Indeed, there has been recent interest for the potential use of vitamin D as a prophylactic and therapeutic agent against COVID-19 as several observational studies have linked low vitamin D status to its incidence [643,651]. These associations have yet to be confirmed and rigorous trials are required before considering supplementation recommendations. However, the nutritional status and general health of a patient can affect their outcomes in various diseases, thus it would be pertinent to advise people to follow a healthy diet and life style to the best of their ability to prevent nutrient deficiencies and insufficiencies and to maintain a healthy immune system [533]. Finally, antibodies against viruses, also known as antiviral monoclonal antibodies, could be an alternative as well and are described in detail in an above section. The goal of antiviral antibodies is to neutralize viruses through either cell-killing activity or blocking of viral replication [528]. They may also engage the host immune response, encouraging the immune system to hone in on the virus. Given the cytokine storm that results from immune system activation in response to the virus, which has been implicated in worsening of the disease, a neutralizing antibody (nAb) may be preferable. Upcoming work may explore the specificity of nAbs for their target, mechanisms by which the nAbs impede the virus, and improvements to antibody structure that may enhance the ability of the antibody to block viral activity.

Some research is also investigating potential therapeutics and prophylactics that would interact with components of the innate immune response. For example, there are a variety of TLRs, PRRs that recognize PAMPs and DAMPs. TLRs form a part of innate immune recognition and can more generally contribute to promoting both innate and adaptive responses [106]. In mouse models, poly(I:C) and CpG, which are agonists of toll-like receptors TLR3 and TLR9, respectively, showed protective effects when administered prior to SARS-CoV-1 infection [529]. Therefore, TLR agonists hold some potential for broad-spectrum prophylaxis.

Given that a large number of clinical trials are currently in progress, more information about the potential of these and other therapeutics should become available over time. This information, combined with advances in understanding the molecular structure and viral pathogenesis of SARS-CoV-2, may lead to a more complete understanding of how the virus affects the human host and what strategies can improve outcomes. To date, investigations of potential therapeutics for COVID-19 have focused primarily on repurposing existing drugs. This approach is necessary given the urgency of the situation as well as the extensive time required for developing and testing new therapies. However, in the long-term, new drugs specific for treatment of COVID-19 may also enter development. Development of novel drugs is likely to be guided by what is known about the pathogenesis and molecular structure of SARS-CoV-2. For example, understanding the various structural components of SARS-CoV-2 may allow for the development of small molecule inhibitors of those components. Currently, crystal structures of the SARS-CoV-2 main protease have recently been resolved [340,530], and efforts are already in place to perform screens for small molecule inhibitors of the main protease, which have yielded potential hits [340]. Much work remains to be done to determine further crystal structures of other viral components, understand the relative utility of targeting different viral components, perform additional small molecule inhibitor screens, and determine the safety and efficacy of the potential inhibitors. While still nascent, work in this area is promising. Over the longer term, this approach and others may lead to the development of novel therapeutics specifically for COVID-19 and SARS-CoV-2.

9 Application of an Open Publishing Framework to an Emerging Public health Crisis

9.1 Abstract

9.2 Introduction

As international attention remains focused on the ongoing public health crisis, the scientific community has responded by mobilizing resources and turning much of its attention to the virus and disease. This rapid influx of information is disseminated by traditional publishing mechanisms, preprint servers, and press releases, which provide a venue for scientists to release findings without undergoing the formal publication process. While having information available is valuable to efforts to understand and combat COVID-19, many contributions come from researchers across a wide range of fields who have varying degrees of experience working on coronaviruses and related topics. The volume of information available, much of which has not gone through rigorous peer review, presents a significant challenge to individual efforts to keep abreast of the state of COVID-19 research [5]. However, research on these topics is proceeding so rapidly that any static review is likely to quickly become dated. Our goal as a community is to consolidate information about the virus in the context of related viruses and to synthesize rapidly emerging literature centered on the diagnosis and treatment of COVID-19. We used an open publishing framework, Manubot [6], to manage hundreds of contributions from the community to create a living, scholarly document. We designed software to generate figures that automatically update using external data sources. Our primary goal is to sort and distill informative content out of the overwhelming flood of information [5] and help the broader scientific community become more conversant on this critical subject. Thus, our approach has been to develop a real-time, collaborative effort that welcomes submissions from scientists worldwide into this ongoing effort. This document represents the first snapshot, which aims to reflect the state of the field as of October, 2020. We plan to refine and expand this document until technologies to mitigate the pandemic are widely available.

In an effort to keep pace as new information about COVID-19 and SARS-CoV-2 becomes available, this project is an open, collaborative effort that invited contributions from the scientific community broadly, similar to previous efforts to develop collaborative reviews [889,890]. Contributors were recruited by word of mouth and on Twitter. Existing efforts to train early-career scientists were also integrated: Appendix A contains summaries written by the students, post-docs, and faculty of the Immunology Institute at the Mount Sinai School of Medicine [891,892], and two of the authors were recruited through the American Physician Scientist Association’s Virtual Summer Research Program [893]. The project was managed through GitHub [894] using Manubot [6] to continuously generate a version of the manuscript online [895]. Contributors developed text that was proposed through GitHub’s pull request system and then reviewed and approved by at least one other author. While this document reflects the current version of record, the online version will continue to be developed as information about the pandemic emerges. Below, we will describe the processes used to synthesize the literature.

9.3 Technical Infrastructure

9.3.1 Collaborative Writing and Manuscript Generation

Manubot [6] is a collaborative framework developed to adapt open-source software development techniques and version control for manuscript writing. Here, Manubot was used to generate a manuscript from text maintained using GitHub, a popular, online version control interface. The GitHub implementation allowed users to contribute either using git on the command line or using the GitHub user interface, and we developed documentation for users with less experience with this platform. Manubot also provides a functionality to create a bibliography using digital object identifiers (DOIs), website URLs, or other identifiers such as PubMed identifiers and arXiv IDs. Due to the needs of this project, project contributors also implemented new features in Manubot and Zotero, which Manubot uses to extract metadata for some types of citations. These features support directly citing clinical trial identifiers such as clinicaltrials:NCT04292899 [315] and generating the complete review manuscript along with the individual manuscripts reviewing specific topics. Finally, Manubot and GitHub Actions continuous integration allowed for scripted updates to be run each time the manuscript was generated. These scripts were used to check that the manuscript was built correctly, run spellchecking, and cross-reference the manuscripts cited in this review, summarized in Appendix A, and discussed in the project’s issues and pull requests.

9.3.2 Data Analysis and Visualization

The combination of Manubot and GitHub Actions also made it possible to dynamically update information such as statistics and visualizations in the manuscript. Data about worldwide cases and deaths from the COVID-19 Data Repository by the Center for Systems Science and Engineering at Johns Hopkins University [290] were read using a Python script. Similarly, the clinical trials statistics and figure were generated based on data from the University of Oxford Evidence-Based Medicine Data Lab’s COVID-19 TrialsTracker [293]. In both cases, frequency data were plotted using Matplotlib [896] in Python. The figure showing the geographic distribution of COVID-19 clinical trials was generated using the countries associated with the trials listed in the COVID-19 TrialsTracker, converting the country names to 3-letter ISO codes using pycountry or manual adjustment when necessary, and visualizing the geographic distribution of trial recruitment using geopandas.

GitHub Actions runs a nightly workflow to update these external data and regenerate the statistics and figures for the manuscript. The workflow uses the GitHub API to detect and save the latest commit of the external data sources, which are both GitHub repositories. It then downloads versioned data from that snapshot of the external repositories and runs bash and Python scripts to calculate the desired statistics and produce the summary figures. The statistics are stored in JSON files that are accessed by Manubot to populate the values of placeholder template variables dynamically every time the manuscript is built. For instance, the template variable {{ebm_trials_results}} in the manuscript is replaced by the actual number of clinical trials with results, 168. The template variables also include versioned URLs to the dynamically updated figures. The JSON files and figures are stored in the external-resources branch of the manuscript’s GitHub repository, which acts as versioned storage. The GitHub Actions workflow automatically adds and commits the new JSON files and figures to the external-resources branch every time it runs, and Manubot uses the latest version of these resources when it builds the manuscript.

The workflow file is available from https://github.com/greenelab/covid19-review/blob/master/.github/workflows/update-external-resources.yaml and the scripts are available from https://github.com/greenelab/covid19-review/tree/external-resources. The Python package versions are available in https://github.com/greenelab/covid19-review/blob/external-resources/environment.yml.

9.4 Article Selection and Evaluation

Relevant articles were identified and submitted as issues on GitHub for review. Articles were classified as diagnostic, therapeutic, or other, and a template was developed to guide the review of papers and preprints in each category. Following a framework often used for assessing medical literature, the review consisted of examining methods used in each relevant article, assignment (whether the study was observational or randomized), assessment, results, interpretation, and how well the study extrapolates [897]. For examples of each template, please see Appendices B-D.

9.4.1 Diagnostic Papers

9.4.1.1 Methods

Reviewers began by describing the study question(s) being investigated by the article. They then described the study population, the sample size, the prevalence of the disease in the study population, countries / regions considered in case of human subjects, demographics of participants, the setting, and any remaining inclusion / exclusion criteria considered. They then described the reference test or “gold standard,” if one was utilized.

9.4.1.2 Assignment

Reviewers described how new and reference tests were assigned, including additional relevant details about the study design. For example, reviewers were asked whether the diagnostic test resulted in rigorous assignments of case status or was biased towards sicker or healthier individuals.

9.4.1.3 Assessment

Reviewers described how the test was performed. For example, for both standard and reference tests, reviewers described technical details of assays used, when measurements were taken and by whom. Subsequently, they described how individuals were classified as positive or negative cases and whether results were precise and reproducible with repeated tests. Reviewers described whether there were any missing data, whether some participants underwent only one test, or whether there were individuals with inconclusive results.

9.4.1.4 Results

Reviewers reported the estimated sensitivity, specificity, positive predictive value (PPV), and negative predicted value (NPV), as well as confidence bounds around these measures, if provided.

9.4.1.5 Interpretation

Reviewers reported how well the test ruled in or ruled out disease based on the population, if there were identified side effects, and patient adherence.

9.4.1.6 Extrapolation

Reviewers described how well this test will extrapolate outside the measured population.

9.4.2 Therapeutic Papers

9.4.2.1 Methods

Reviewers began by describing the study question(s) being investigated by the article. They then described the study population, the sample size, the prevalence of the disease in the study population, countries / regions considered in case of human subjects, demographics of participants, the setting, and any remaining inclusion / exclusion criteria considered.

9.4.2.2 Assignment

Reviewers described how the treatment is assigned, whether it was an interventional or observational study, whether randomization took place, etc.

9.4.2.3 Assessment

9.4.2.3.1 Outcome Assessment

Reviewers described the outcome that was assessed and evaluated whether it was appropriate given the underlying study question. They described whether there were any missing data such as whether there were individuals lost to follow up. They then describe whether there were any potential sources of bias such as lack of blinding in a randomized controlled trial.

9.4.2.3.2 Statistical Methods Assessment

Reviewers described which statistical methods were used for inference and whether applied methods were appropriate for the study. They then described whether adjustments were made for possible confounders.

9.4.2.4 Results

Reviewers described the estimated association between the treatment and outcome. They described measures of confidence or statistical significance, if provided.

9.4.2.5 Interpretation

Reviewers described whether a causal claim could be made. They described whether any side effects or interactions with other drugs were identified, as well as any subgroup findings.

9.4.2.6 Extrapolation

Reviewers describe how the study may extrapolate to a different species or population.

9.5 Conclusions

Figure 7: Summary of the relationships among topics covered in this review.

Several review articles on aspects of COVID-19 have already been published. These have included reviews on the disease epidemiology [38], immunological response [39], diagnostics [40], and pharmacological treatments [39,41]. Others [42,43] provide narrative reviews of progress on some important ongoing COVID-19 research questions. With the worldwide scientific community uniting during 2020 to investigate SARS-CoV-2 and COVID-19 from a wide range of perspectives, findings from many disciplines are relevant on a rapid timescale to a broad scientific audience. Additionally, many findings are published as preprints, which are available prior to going through the peer review process. As a result, centralizing, summarizing, and critiquing new literature broadly relevant to COVID-19 can help to expedite the interdisciplinary scientific process that is currently happening at an advanced pace. We are particularly interested in providing background to the development of diagnostic, prophylactic, and therapeutic approaches to COVID-19. Two major concerns within diagnosis include the detection of current infections in individuals with and without symptoms, and the detection of past exposure without an active infection. In the latter category, identifying whether individuals can develop or have developed sustained immunity is also a major consideration. The development of high-throughput, affordable methods for detecting active infections and sustained immunity will be critical to understanding and controlling the disease. The identification of interventions that can mitigate the effect of the virus on exposed and infected individuals is a significant research priority. Some possible approaches include the identification of existing pharmaceuticals that reduce the severity of infection, either by reducing the virus’ virulence (e.g., antivirals) or managing the most severe symptoms of infection. Due to the long timeline for the development of novel pharmaceuticals, in most cases, research surrounding possible pharmaceutical interventions focuses on the identification and investigation of existing compounds whose mechanisms may be relevant to COVID-19. Other foci of current research include the identification of antibodies produced by survivors of COVID-19 and the development of vaccines. Understanding the mechanisms describing host-virus interactions between humans and SARS-CoV-2 is thus critical to identifying candidate therapeutics. An overview of the topics covered is visualized in Figure 7. Thus, in this review, we seek to consolidate information about efforts to develop strategies for diagnosis and therapeutics as new information is released by the scientific community. We include information from both traditional peer-reviewed scientific literature and from preprints, which typically have not undergone peer review but have been critically evaluated by the scientists involved in this effort. The goal of this manuscript is to present preliminary findings within the broader context of COVID-19 research and to identify the broad interpretations of new research, as well as limitations to interpretability.

10 Additional Items

10.1 Competing Interests

Author Competing Interests Last Reviewed
Halie M. Rando None 2020-03-22
Casey S. Greene None 2020-01-20
Michael P. Robson None 2020-11-12
Simina M. Boca None 2020-11-07
Nils Wellhausen None 2020-11-03
Ronan Lordan None 2020-11-03
Christian Brueffer Employee and shareholder of SAGA Diagnostics AB. 2020-11-11
Sandipan Ray None 2020-11-11
Lucy D'Agostino McGowan Received consulting fees from Acelity and Sanofi in the past five years 2020-11-10
Anthony Gitter Filed a patent application with the Wisconsin Alumni Research Foundation related to classifying activated T cells 2020-11-10
Anna Ada Dattoli None 2020-03-26
Ryan Velazquez None 2020-11-10
John P. Barton None 2020-11-11
Jeffrey M. Field None 2020-11-12
Bharath Ramsundar None 2020-11-11
Adam L. MacLean None 2020-11-11
Alexandra J. Lee None 2020-11-09
Immunology Institute of the Icahn School of Medicine None 2020-04-07
Fengling Hu None 2020-04-08
Nafisa M. Jadavji None 2020-11-11
Elizabeth Sell None 2020-11-11
Jinhui Wang None 2020-04-13
Diane N. Rafizadeh None 2020-11-11
Ashwin N. Skelly None 2020-11-11
Marouen Ben Guebila None 2020-11-11
Likhitha Kolla None 2020-11-16
David Manheim None 2020-11-11
Soumita Ghosh None 2020-11-09
James Brian Byrd Funded by FastGrants to conduct a COVID-19-related clinical trial 2020-11-12
YoSon Park None 2020-11-11
Vikas Bansal None 2020-05-26
Stephen Capone None 2020-11-11
John J. Dziak None 2020-11-11
Yuchen Sun None 2020-11-11
Yanjun Qi None 2020-07-09
Lamonica Shinholster None 2020-11-11
Temitayo Lukan None 2020-11-10
Sergey Knyazev None 2020-11-11
Dimitri Perrin None 2020-11-11
Serghei Mangul None 2020-11-11
Shikta Das None 2020-08-13
Gregory L Szeto None 2020-11-16
Tiago Lubiana None 2020-11-11
David Mai None 2021-01-08
COVID-19 Review Consortium None 2021-01-16
Rishi Raj Goel None 2021-01-20

10.2 Author Contributions

Author Contributions
Halie M. Rando A, B, C, D, E
Casey S. Greene X, Y, Z
Michael P. Robson Software
Simina M. Boca MISSING
Nils Wellhausen MISSING
Ronan Lordan MISSING
Christian Brueffer MISSING
Sandipan Ray MISSING
Lucy D'Agostino McGowan MISSING
Anthony Gitter MISSING
Anna Ada Dattoli Writing - Original Draft
Ryan Velazquez MISSING
John P. Barton MISSING
Jeffrey M. Field Writing - Original Draft
Bharath Ramsundar Investigation, Writing - Review & Editing
Adam L. MacLean MISSING
Alexandra J. Lee MISSING
Immunology Institute of the Icahn School of Medicine Data Curation
Fengling Hu Writing - Original Draft, Writing - Review & Editing
Nafisa M. Jadavji Writing - Original Draft, Writing - Review & Editing
Elizabeth Sell MISSING
Jinhui Wang MISSING
Diane N. Rafizadeh Writing - Original Draft, Writing - Review & Editing
Ashwin N. Skelly MISSING
Marouen Ben Guebila MISSING
Likhitha Kolla Writing - Original Draft
David Manheim Investigation, Writing - Original Draft
Soumita Ghosh Writing - Original Draft
James Brian Byrd MISSING
YoSon Park MISSING
Vikas Bansal MISSING
Stephen Capone Writing - Original Draft, Writing - Review & Editing
John J. Dziak MISSING
Yuchen Sun Visualization
Yanjun Qi Visualization
Lamonica Shinholster MISSING
Temitayo Lukan Investigation, Writing - Original Draft
Sergey Knyazev MISSING
Dimitri Perrin Writing - Original Draft, Writing - Review & Editing
Serghei Mangul MISSING
Shikta Das Writing - Review & Editing
Gregory L Szeto MISSING
Tiago Lubiana MISSING
David Mai MISSING
COVID-19 Review Consortium Project Administration
Rishi Raj Goel Writing - Original Draft, Writing - Review & Editing

10.3 Acknowledgements

We thank Nick DeVito for assistance with the Evidence-Based Medicine Data Lab COVID-19 TrialsTracker data. We thank Yael Evelyn Marshall who contributed writing (original draft) as well as reviewing and editing of pieces of the text but who did not formally approve the manuscript, as well as Ronnie Russell, who contributed text to and helped develop the structure of the manuscript early in the writing process and Matthias Fax who helped with writing and editing text related to diagnostics. We are grateful to the following contributors for reviewing pieces of the text: Nadia Danilova, James Eberwine and Ipsita Krishnan.

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510. Reducing Hospital Admission of Elderly in SARS-CoV-2 Pandemic Via the Induction of Trained Immunity by Bacillus Calmette-Guérin Vaccination, a Randomized Controlled Trial
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558. FTC Sues California Marketer of $23,000 COVID-19 “Treatment” Plan
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562. Potential interventions for novel coronavirus in China: A systematic review
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563. Nutraceuticals have potential for boosting the type 1 interferon response to RNA viruses including influenza and coronavirus
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565. The Potential Beneficial Effect of EPA and DHA Supplementation Managing Cytokine Storm in Coronavirus Disease
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570. COVID-19 and Thrombotic or Thromboembolic Disease: Implications for Prevention, Antithrombotic Therapy, and Follow-Up
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574. The Lipid Mediator Protectin D1 Inhibits Influenza Virus Replication and Improves Severe Influenza
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576. The Specialized Proresolving Mediator 17-HDHA Enhances the Antibody-Mediated Immune Response against Influenza Virus: A New Class of Adjuvant?
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588. Anti-inflammatory/Antioxidant Oral Nutrition Supplementation on the Cytokine Storm and Progression of COVID-19: A Randomized Controlled Trial
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589. Functional Role of Dietary Intervention to Improve the Outcome of COVID-19: A Hypothesis of Work
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596. Efficacy of Zinc Against Common Cold Viruses: An Overview
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599. Zn2+ Inhibits Coronavirus and Arterivirus RNA Polymerase Activity In Vitro and Zinc Ionophores Block the Replication of These Viruses in Cell Culture
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600. The SARS-coronavirus papain-like protease: Structure, function and inhibition by designed antiviral compounds
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601. A Randomized Study Evaluating the Safety and Efficacy of Hydroxychloroquine and Zinc in Combination With Either Azithromycin or Doxycycline for the Treatment of COVID-19 in the Outpatient Setting
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603. Therapies to Prevent Progression of COVID-19, Including Hydroxychloroquine, Azithromycin, Zinc, Vitamin D, Vitamin B12 With or Without Vitamin C, a Multi-centre, International, Randomized Trial: The International ALLIANCE Study
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604. Early Intervention in COVID-19: Favipiravir Verses Standard Care - Full Text View - ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT04373733

605. Hydroxychloroquine with or without Azithromycin in Mild-to-Moderate Covid-19
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606. The efficacy and safety of hydroxychloroquine for COVID-19 prophylaxis: A systematic review and meta-analysis of randomized trials
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607. Effect of hydroxychloroquine with or without azithromycin on the mortality of coronavirus disease 2019 (COVID-19) patients: a systematic review and meta-analysis
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608. Zinc sulfate in combination with a zinc ionophore may improve outcomes in hospitalized COVID-19 patients
Philip M. Carlucci, Tania Ahuja, Christopher Petrilli, Harish Rajagopalan, Simon Jones, Joseph Rahimian
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609. The Minimal Effect of Zinc on the Survival of Hospitalized Patients With COVID-19
Jasper Seth Yao, Joseph Alexander Paguio, Edward Christopher Dee, Hanna Clementine Tan, Achintya Moulick, Carmelo Milazzo, Jerry Jurado, Nicolás Della Penna, Leo Anthony Celi
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610. Coronavirus Disease 2019- Using Ascorbic Acid and Zinc Supplementation (COVIDAtoZ) Research Study A Randomized, Open Label Single Center Study
Milind Desai
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611. Vitamin B12 May Inhibit RNA-Dependent-RNA Polymerase Activity of nsp12 from the COVID-19 Virus
Naveen Narayanan, Deepak T. Nair
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612. The Long History of Vitamin C: From Prevention of the Common Cold to Potential Aid in the Treatment of COVID-19
Giuseppe Cerullo, Massimo Negro, Mauro Parimbelli, Michela Pecoraro, Simone Perna, Giorgio Liguori, Mariangela Rondanelli, Hellas Cena, Giuseppe D’Antona
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613. The Emerging Role of Vitamin C in the Prevention and Treatment of COVID-19
Anitra C. Carr, Sam Rowe
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614. Vitamin C Mitigates Oxidative Stress and Tumor Necrosis Factor-Alpha in Severe Community-Acquired Pneumonia and LPS-Induced Macrophages
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615. Intravenous infusion of ascorbic acid decreases serum histamine concentrations in patients with allergic and non-allergic diseases
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616. Vitamin C and Immune Function
Anitra Carr, Silvia Maggini
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617. Changes in Leucocyte Ascorbic Acid during the Common Cold
R. Hume, Elspeth Weyers
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618. ASCORBIC ACID FUNCTION AND METABOLISM DURING COLDS
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619. Metabolism of ascorbic acid (vitamin C) in subjects infected with common cold viruses
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620. Vitamin C and Infections
Harri Hemilä
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621. Vitamin C and the common cold
Harri Hemilä
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622. Serum Levels of Vitamin C and Vitamin D in a Cohort of Critically Ill COVID-19 Patients of a North American Community Hospital Intensive Care Unit in May 2020: A Pilot Study
Cristian Arvinte, Maharaj Singh, Paul E. Marik
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623. Vitamin C levels in patients with SARS-CoV-2-associated acute respiratory distress syndrome
Luis Chiscano-Camón, Juan Carlos Ruiz-Rodriguez, Adolf Ruiz-Sanmartin, Oriol Roca, Ricard Ferrer
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624. Targeting coagulation activation in severe COVID-19 pneumonia: lessons from bacterial pneumonia and sepsis
Ricardo J. José, Andrew Williams, Ari Manuel, Jeremy S. Brown, Rachel C. Chambers
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625. Vitamin C and Microvascular Dysfunction in Systemic Inflammation
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626. The use of IV vitamin C for patients with COVID-19: a case series
Raul Hiedra, Kevin Bryan Lo, Mohammad Elbashabsheh, Fahad Gul, Robert Matthew Wright, Jeri Albano, Zurab Azmaiparashvili, Gabriel Patarroyo Aponte
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627. Vitamin C for preventing and treating the common cold
Harri Hemilä, Elizabeth Chalker
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628. Vitamin C intake and susceptibility to pneumonia
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629. Vitamin C Can Shorten the Length of Stay in the ICU: A Meta-Analysis
Harri Hemilä, Elizabeth Chalker
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630. Effect of Vitamin C Infusion on Organ Failure and Biomarkers of Inflammation and Vascular Injury in Patients With Sepsis and Severe Acute Respiratory Failure
Alpha A. Fowler, Jonathon D. Truwit, R. Duncan Hite, Peter E. Morris, Christine DeWilde, Anna Priday, Bernard Fisher, Leroy R. Thacker, Ramesh Natarajan, Donald F. Brophy, … Matthew Halquist
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631. Intravenous high-dose vitamin C for the treatment of severe COVID-19: study protocol for a multicentre randomised controlled trial
Fang Liu, Yuan Zhu, Jing Zhang, Yiming Li, Zhiyong Peng
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632. Pilot Trial of High-dose vitamin C in critically ill COVID-19 patients
Jing Zhang, Xin Rao, Yiming Li, Yuan Zhu, Fang Liu, Guangling Guo, Guoshi Luo, Zhongji Meng, Daniel De Backer, Hui Xiang, Zhi-Yong Peng
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633. High-dose vitamin C infusion for the treatment of critically ill COVID-19
Jing Zhang, Xin Rao, Yiming Li, Yuan Zhu, Fang Liu, Guangling Guo, Guoshi Luo, Zhongji Meng, Daniel De Backer, Hui Xiang, Zhi-Yong Peng
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634. Vitamin D and Infectious Diseases: Simple Bystander or Contributing Factor?
Pedro Gois, Daniela Ferreira, Simon Olenski, Antonio Seguro
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635. Vitamin D and Influenza—Prevention or Therapy?
Beata M. Gruber–Bzura
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636. Immunologic Effects of Vitamin D on Human Health and Disease
Nipith Charoenngam, Michael F. Holick
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637. Vitamin D and respiratory health
D. A. Hughes, R. Norton
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638. Regulation of Immune Function by Vitamin D and Its Use in Diseases of Immunity
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639. Vitamin D and the Immune System
Cynthia Aranow
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640. Vitamin D in the prevention of acute respiratory infection: Systematic review of clinical studies
David A. Jolliffe, Christopher J. Griffiths, Adrian R. Martineau
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641. Vitamin D: modulator of the immune system
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642. Evidence that Vitamin D Supplementation Could Reduce Risk of Influenza and COVID-19 Infections and Deaths
William B. Grant, Henry Lahore, Sharon L. McDonnell, Carole A. Baggerly, Christine B. French, Jennifer L. Aliano, Harjit P. Bhattoa
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643. Perspective: Vitamin D deficiency and COVID‐19 severity – plausibly linked by latitude, ethnicity, impacts on cytokines, ACE2 and thrombosis
J. M. Rhodes, S. Subramanian, E. Laird, G. Griffin, R. A. Kenny
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644. COVID-19 fatalities, latitude, sunlight, and vitamin D
Paul B. Whittemore
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645. Editorial: low population mortality from COVID-19 in countries south of latitude 35 degrees North supports vitamin D as a factor determining severity
Jonathan M. Rhodes, Sreedhar Subramanian, Eamon Laird, Rose A. Kenny
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646. 25-Hydroxyvitamin D Concentrations Are Lower in Patients with Positive PCR for SARS-CoV-2
Antonio D’Avolio, Valeria Avataneo, Alessandra Manca, Jessica Cusato, Amedeo De Nicolò, Renzo Lucchini, Franco Keller, Marco Cantù
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647. Vitamin D deficiency as risk factor for severe COVID-19: a convergence of two pandemics
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648. Vitamin D sufficiency, a serum 25-hydroxyvitamin D at least 30 ng/mL reduced risk for adverse clinical outcomes in patients with COVID-19 infection
Zhila Maghbooli, Mohammad Ali Sahraian, Mehdi Ebrahimi, Marzieh Pazoki, Samira Kafan, Hedieh Moradi Tabriz, Azar Hadadi, Mahnaz Montazeri, Mehrad Nasiri, Arash Shirvani, Michael F. Holick
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649. Role of vitamin D in preventing of COVID-19 infection, progression and severity
Nurshad Ali
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650. Low plasma 25(OH) vitamin D level is associated with increased risk of COVID‐19 infection: an Israeli population‐based study
Eugene Merzon, Dmitry Tworowski, Alessandro Gorohovski, Shlomo Vinker, Avivit Golan Cohen, Ilan Green, Milana Frenkel‐Morgenstern
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651. Association of Vitamin D Status and Other Clinical Characteristics With COVID-19 Test Results
David O. Meltzer, Thomas J. Best, Hui Zhang, Tamara Vokes, Vineet Arora, Julian Solway
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652. Vitamin D Status in Hospitalized Patients with SARS-CoV-2 Infection
José L Hernández, Daniel Nan, Marta Fernandez-Ayala, Mayte García-Unzueta, Miguel A Hernández-Hernández, Marcos López-Hoyos, Pedro Muñoz-Cacho, José M Olmos, Manuel Gutiérrez-Cuadra, Juan J Ruiz-Cubillán, … Víctor M Martínez-Taboada
The Journal of Clinical Endocrinology & Metabolism (2020-10-27) https://doi.org/ghh737
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653. Analysis of vitamin D level among asymptomatic and critically ill COVID-19 patients and its correlation with inflammatory markers
Anshul Jain, Rachna Chaurasia, Narendra Singh Sengar, Mayank Singh, Sachin Mahor, Sumit Narain
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654. Low 25-Hydroxyvitamin D Levels on Admission to the Intensive Care Unit May Predispose COVID-19 Pneumonia Patients to a Higher 28-Day Mortality Risk: A Pilot Study on a Greek ICU Cohort
Alice G. Vassiliou, Edison Jahaj, Maria Pratikaki, Stylianos E. Orfanos, Ioanna Dimopoulou, Anastasia Kotanidou
Nutrients (2020-12-09) https://doi.org/ghr95d
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655. Vitamin D deficiency as a predictor of poor prognosis in patients with acute respiratory failure due to COVID-19
G. E. Carpagnano, V. Di Lecce, V. N. Quaranta, A. Zito, E. Buonamico, E. Capozza, A. Palumbo, G. Di Gioia, V. N. Valerio, O. Resta
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656. Vitamin D Deficiency and Outcome of COVID-19 Patients
Aleksandar Radujkovic, Theresa Hippchen, Shilpa Tiwari-Heckler, Saida Dreher, Monica Boxberger, Uta Merle
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657. Impact of Vitamin D Deficiency on COVID-19—A Prospective Analysis from the CovILD Registry
Alex Pizzini, Magdalena Aichner, Sabina Sahanic, Anna Böhm, Alexander Egger, Gregor Hoermann, Katharina Kurz, Gerlig Widmann, Rosa Bellmann-Weiler, Günter Weiss, … Judith Löffler-Ragg
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658. Does Serum Vitamin D Level Affect COVID-19 Infection and Its Severity?-A Case-Control Study
Kun Ye, Fen Tang, Xin Liao, Benjamin A. Shaw, Meiqiu Deng, Guangyi Huang, Zhiqiang Qin, Xiaomei Peng, Hewei Xiao, Chunxia Chen, … Jianrong Yang
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659. Lower levels of vitamin D are associated with SARS-CoV-2 infection and mortality in the Indian population: An observational study
Sunali Padhi, Subham Suvankar, Venketesh K. Panda, Abhijit Pati, Aditya K. Panda
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660. Vitamin D Deficiency Is Inversely Associated with COVID-19 Incidence and Disease Severity in Chinese People
Xia Luo, Qing Liao, Ying Shen, Huijun Li, Liming Cheng
The Journal of Nutrition (2021-01) https://doi.org/ghr939
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661. Vitamin D concentrations and COVID-19 infection in UK Biobank
Claire E. Hastie, Daniel F. Mackay, Frederick Ho, Carlos A. Celis-Morales, Srinivasa Vittal Katikireddi, Claire L. Niedzwiedz, Bhautesh D. Jani, Paul Welsh, Frances S. Mair, Stuart R. Gray, … Jill P. Pell
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662. Vitamin D and COVID-19 infection and mortality in UK Biobank
Claire E. Hastie, Jill P. Pell, Naveed Sattar
European Journal of Nutrition (2020-08-26) https://doi.org/ghr93p
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663. Low serum 25‐hydroxyvitamin D (25[OH]D) levels in patients hospitalized with COVID‐19 are associated with greater disease severity
Grigorios Panagiotou, Su Ann Tee, Yasir Ihsan, Waseem Athar, Gabriella Marchitelli, Donna Kelly, Christopher S. Boot, Nadia Stock, James Macfarlane, Adrian R. Martineau, … Richard Quinton
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664. Letter in response to the article: Vitamin D concentrations and COVID-19 infection in UK biobank (Hastie et al.)
W. B. Grant, S. L. McDonnell
Diabetes & Metabolic Syndrome: Clinical Research & Reviews (2020-09) https://doi.org/ghc7p4
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665. Vitamin D deficiency in African Americans is associated with a high risk of severe disease and mortality by SARS-CoV-2
Virna Margarita Martín Giménez, Felipe Inserra, León Ferder, Joxel García, Walter Manucha
Journal of Human Hypertension (2020-08-13) https://doi.org/ghr933
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666. Evidence for possible association of vitamin D status with cytokine storm and unregulated inflammation in COVID-19 patients
Ali Daneshkhah, Vasundhara Agrawal, Adam Eshein, Hariharan Subramanian, Hemant Kumar Roy, Vadim Backman
Aging Clinical and Experimental Research (2020-09-02) https://doi.org/ghr93q
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667. Short term, high-dose vitamin D supplementation for COVID-19 disease: a randomised, placebo-controlled, study (SHADE study)
Ashu Rastogi, Anil Bhansali, Niranjan Khare, Vikas Suri, Narayana Yaddanapudi, Naresh Sachdeva, GD Puri, Pankaj Malhotra
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668. “Effect of calcifediol treatment and best available therapy versus best available therapy on intensive care unit admission and mortality among patients hospitalized for COVID-19: A pilot randomized clinical study”
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670. Mathematical analysis of Córdoba calcifediol trial suggests strong role for Vitamin D in reducing ICU admissions of hospitalized COVID-19 patients
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672. Effect of Vitamin D 3 Supplementation vs Placebo on Hospital Length of Stay in Patients with Severe COVID-19: A Multicenter, Double-blind, Randomized Controlled Trial
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677. The LEAD COVID-19 Trial: Low-risk, Early Aspirin and Vitamin D to Reduce COVID-19 Hospitalizations
Louisiana State University Health Sciences Center in New Orleans
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678. Randomized Double-Blind Placebo-Controlled Proof-of-Concept Trial of a Plant Polyphenol for the Outpatient Treatment of Mild Coronavirus Disease (COVID-19)
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679. Current vitamin D status in European and Middle East countries and strategies to prevent vitamin D deficiency: a position statement of the European Calcified Tissue Society
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689. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic
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701. Antiviral Activity of Exopolysaccharides Produced by Lactic Acid Bacteria of the Genera Pediococcus, Leuconostoc and Lactobacillus against Human Adenovirus Type 5
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702. Prevention of respiratory syncytial virus infection with probiotic lactic acid bacterium Lactobacillus gasseri SBT2055
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703. Effect of probiotic on innate inflammatory response and viral shedding in experimental rhinovirus infection – a randomised controlled trial
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704. Immunobiotic lactobacilli reduce viral-associated pulmonary damage through the modulation of inflammation–coagulation interactions
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705. Nasal priming with immunobiotic lactobacilli improves the adaptive immune response against influenza virus
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706. The potential application of probiotics and prebiotics for the prevention and treatment of COVID-19
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707. Pulmonary-intestinal cross-talk in mucosal inflammatory disease
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708. The role of the lung microbiota and the gut-lung axis in respiratory infectious diseases
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710. Oral Microbiome and SARS-CoV-2: Beware of Lung Co-infection
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715. Effectiveness of probiotics on the duration of illness in healthy children and adults who develop common acute respiratory infectious conditions: a systematic review and meta-analysis
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716. Effect of probiotics on the incidence of ventilator-associated pneumonia in critically ill patients: a randomized controlled multicenter trial
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717. Probiotic Prophylaxis of Ventilator-associated Pneumonia
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718. Synbiotics modulate gut microbiota and reduce enteritis and ventilator-associated pneumonia in patients with sepsis: a randomized controlled trial
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719. Probiotics for the Prevention of Ventilator-Associated Pneumonia: A Meta-Analysis of Randomized Controlled Trials
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720. COVID-19: An Alert to Ventilator-Associated Bacterial Pneumonia
Helvécio Cardoso Corrêa Póvoa, Gabriela Ceccon Chianca, Natalia Lopes Pontes Póvoa Iorio
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721. The challenge of ventilator-associated pneumonia diagnosis in COVID-19 patients
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722. Prophylactic use of probiotics for gastrointestinal disorders in children
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723. Effect of Gastrointestinal Symptoms in Patients With COVID-19
Zili Zhou, Ning Zhao, Yan Shu, Shengbo Han, Bin Chen, Xiaogang Shu
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724. The digestive system is a potential route of 2019-nCov infection: a bioinformatics analysis based on single-cell transcriptomes
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725. Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein reveal a prerequisite conformational state for receptor binding
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726. Prolonged presence of SARS-CoV-2 viral RNA in faecal samples
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727. Enteric involvement of coronaviruses: is faecal–oral transmission of SARS-CoV-2 possible?
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728. Characteristics of pediatric SARS-CoV-2 infection and potential evidence for persistent fecal viral shedding
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729. Modulation of rotavirus severe gastroenteritis by the combination of probiotics and prebiotics
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730. Multicenter Trial of a Combination Probiotic for Children with Gastroenteritis
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731. Synbiotic Therapy of Gastrointestinal Symptoms During Covid-19 Infection: A Randomized, Double-blind, Placebo Controlled, Telemedicine Study (SynCov Study)
Medical University of Graz
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732. Multicentric Study to Assess the Effect of Consumption of Lactobacillus Coryniformis K8 on Healthcare Personnel Exposed to COVID-19
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733. The Intestinal Microbiota as a Therapeutic Target in Hospitalized Patients With COVID-19 Infection
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734. Probiotics: definition, scope and mechanisms of action
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735. Health benefits and health claims of probiotics: bridging science and marketing
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736. Probiotics and COVID-19: one size does not fit all
Joyce WY Mak, Francis KL Chan, Siew C Ng
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737. Network-based drug repurposing for novel coronavirus 2019-nCoV/SARS-CoV-2
Yadi Zhou, Yuan Hou, Jiayu Shen, Yin Huang, William Martin, Feixiong Cheng
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738. Role of Melatonin on Virus-Induced Neuropathogenesis—A Concomitant Therapeutic Strategy to Understand SARS-CoV-2 Infection
Prapimpun Wongchitrat, Mayuri Shukla, Ramaswamy Sharma, Piyarat Govitrapong, Russel J. Reiter
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739. Nutraceutical Strategies for Suppressing NLRP3 Inflammasome Activation: Pertinence to the Management of COVID-19 and Beyond
Mark F. McCarty, Simon Bernard Iloki Assanga, Lidianys Lewis Luján, James H. O’Keefe, James J. DiNicolantonio
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740. Update: Here’s what is known about Trump’s COVID-19 treatment
Jon Cohen
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741. Dietary supplements during the COVID-19 pandemic: insights from 1.4M users of the COVID Symptom Study app - a longitudinal app-based community survey
Panayiotis Louca, Benjamin Murray, Kerstin Klaser, Mark S Graham, Mohsen Mazidi, Emily R Leeming, Ellen Thompson, Ruth Bowyer, David A Drew, Long H Nguyen, … Cristina Menni
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742. Vitamin D deficiency aggravates COVID-19: systematic review and meta-analysis
Marcos Pereira, Alialdo Dantas Damascena, Laylla Mirella Galvão Azevedo, Tarcio de Almeida Oliveira, Jerusa da Mota Santana
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743. ESPEN expert statements and practical guidance for nutritional management of individuals with SARS-CoV-2 infection
Rocco Barazzoni, Stephan C. Bischoff, Joao Breda, Kremlin Wickramasinghe, Zeljko Krznaric, Dorit Nitzan, Matthias Pirlich, Pierre Singer
Clinical Nutrition (2020-06) https://doi.org/ggtzjq
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744. Nutritional status assessment in patients with Covid-19 after discharge from the intensive care unit
Nassim Essabah Haraj, Siham El Aziz, Asma Chadli, Asma Dafir, Amal Mjabber, Ouissal Aissaoui, Lhoucine Barrou, Chafik El Kettani El Hamidi, Afak Nsiri, Rachid AL Harrar, … Moulay Hicham Afif
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745. Nutrition Status Affects COVID‐19 Patient Outcomes
Mette M Berger
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748. Nutritional management of COVID-19 patients in a rehabilitation unit
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750. Coronavirus Update: FDA and FTC Warn Seven Companies Selling Fraudulent Products that Claim to Treat or Prevent COVID-19
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751. Coronavirus Disease 2019 (COVID-19) – Prevention & Treatment
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752. Potential roles of social distancing in mitigating the spread of coronavirus disease 2019 (COVID-19) in South Korea
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753. Evaluating the Effectiveness of Social Distancing Interventions to Delay or Flatten the Epidemic Curve of Coronavirus Disease
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754. Association of Race With Mortality Among Patients Hospitalized With Coronavirus Disease 2019 (COVID-19) at 92 US Hospitals
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755. Characteristics of and Important Lessons From the Coronavirus Disease 2019 (COVID-19) Outbreak in China
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756. Critical Care Utilization for the COVID-19 Outbreak in Lombardy, Italy
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757. Hospitalization Rates and Characteristics of Patients Hospitalized with Laboratory-Confirmed Coronavirus Disease 2019 — COVID-NET, 14 States, March 1–30, 2020
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758. COVID-19 and Your Health
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759. Disparities In Outcomes Among COVID-19 Patients In A Large Health Care System In California
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760. Characteristics Associated with Hospitalization Among Patients with COVID-19 — Metropolitan Atlanta, Georgia, March–April 2020
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761. Demographic science aids in understanding the spread and fatality rates of COVID-19
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762. ‐19 and Older Adults: What We Know
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768. Sex differences in renal angiotensin converting enzyme 2 (ACE2) activity are 17β-oestradiol-dependent and sex chromosome-independent
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773. COVID-19 infection may cause ketosis and ketoacidosis
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774. COVID-19 pandemic, coronaviruses, and diabetes mellitus
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775. Severe obesity, increasing age and male sex are independently associated with worse in-hospital outcomes, and higher in-hospital mortality, in a cohort of patients with COVID-19 in the Bronx, New York
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778. Besides population age structure, health and other demographic factors can contribute to understanding the COVID-19 burden
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783. Covid-19: Black people and other minorities are hardest hit in US
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785. The Fullest Look Yet at the Racial Inequity of Coronavirus
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787. Historical Environmental Racism, Structural Inequalities, and Dik’os Ntsaaígíí-19 (COVID-19) on Navajo Nation
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788. Protect Indigenous peoples from COVID-19
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789. Factors associated with COVID-19-related death using OpenSAFELY
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792. NIH must confront the use of race in science
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793. Interferon-Induced Transmembrane Protein 3 Genetic Variant rs12252-C Associated With Disease Severity in Coronavirus Disease 2019
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794. Genomewide Association Study of Severe Covid-19 with Respiratory Failure
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795. APOE e4 Genotype Predicts Severe COVID-19 in the UK Biobank Community Cohort
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796. Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection
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797. New insights into genetic susceptibility of COVID-19: an ACE2 and TMPRSS2 polymorphism analysis
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798. Seroprevalence of anti-SARS-CoV-2 IgG antibodies in Kenyan blood donors
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799. High SARS-CoV-2 seroprevalence in Health Care Workers but relatively low numbers of deaths in urban Malawi
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800. Africa’s pandemic puzzle: why so few cases and deaths?
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801. Are some ethnic groups more vulnerable to COVID-19 than others? https://www.ifs.org.uk/inequality/chapter/are-some-ethnic-groups-more-vulnerable-to-covid-19-than-others/

802. Quantifying the social distancing privilege gap: a longitudinal study of smartphone movement
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803. Uncovering socioeconomic gaps in mobility reduction during the COVID-19 pandemic using location data
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804. Mobility network models of COVID-19 explain inequities and inform reopening
Serina Chang, Emma Pierson, Pang Wei Koh, Jaline Gerardin, Beth Redbird, David Grusky, Jure Leskovec
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805. A Basic Demographic Profile of Workers in Frontline Industries
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806. Differential occupational risk for COVID‐19 and other infection exposure according to race and ethnicity
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807. Estimating the burden of United States workers exposed to infection or disease: A key factor in containing risk of COVID-19 infection
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808. Coronavirus (COVID-19) related deaths by occupation, England and Wales: deaths registered up to and including 20 April 2020
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809. Which occupations have the highest potential exposure to the coronavirus (COVID-19)?
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810. Disparities in the risk and outcomes from COVID-19
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811. Exclusive: deaths of NHS staff from covid-19 analysed
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813. Racial Disparity in COVID-19 Deaths: Seeking Economic Roots with Census data.
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814. Mortality, Admissions, and Patient Census at SNFs in 3 US Cities During the COVID-19 Pandemic
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815. COVID-19 in Prisons and Jails in the United States
Laura Hawks, Steffie Woolhandler, Danny McCormick
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816. COVID-19 Cases and Deaths in Federal and State Prisons
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