Identification and Development of Therapeutics for COVID-19

Abstract

The COVID-19 pandemic is a rapidly evolving crisis. With the worldwide scientific community shifting focus onto the SARS-CoV-2 virus and COVID-19, a large number of possible pharmaceutical approaches for treatment and prevention have been proposed.

Full text

Identification and Development of Therapeutics for COVID-19
Halie M. Rando,
a
,
b
,
c
Nils Wellhausen,
a
Soumita Ghosh,
d
Alexandra J. Lee,
a
Anna Ada Dattoli,
e
Fengling Hu,
f
James Brian Byrd,
g
Diane N. Rafizadeh,
h
,
i
Ronan Lordan,
j
Yanjun Qi,
k
Yuchen Sun,
k
Christian Brueffer,
l
Jeffrey M. Field,
e
Marouen Ben Guebila,
m
Nafisa M. Jadavji,
n
,
o
Ashwin N. Skelly,
h
,
p
Bharath Ramsundar,
q
Jinhui Wang,
r
Rishi Raj Goel,
p
YoSon Park,
a
COVID-19 Review Consortium, Simina M. Boca,
s
,
t
Anthony Gitter,
u
,
v
Casey S. Greene
b
,
c
,
e
,
w
a
Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, Pennsylvania, USA
b
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado, USA
c
Center for Health AI, University of Colorado School of Medicine, Aurora, Colorado, USA
d
Institute of Translational Medicine and Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
e
Department of Systems Pharmacology & Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
f
Department of Biostatistics, Epidemiology and Informatics, University of Pennsylvania, Philadelphia, Pennsylvania, USA
g
University of Michigan School of Medicine, Ann Arbor, Michigan, USA
h
Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
i
Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania, USA
j
Institute for Translational Medicine and Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
k
Department of Computer Science, University of Virginia, Charlottesville, Virginia, USA
l
Department of Clinical Sciences, Lund University, Lund, Sweden
m
Department of Biostatistics, Harvard School of Public Health, Boston, Massachusetts, USA
n
Biomedical Science, Midwestern University, Glendale, Arizona, USA
o
Department of Neuroscience, Carleton University, Ottawa, Ontario, Canada
p
Institute for Immunology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA
q
The DeepChem Project
r
Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
s
Innovation Center for Biomedical Informatics, Georgetown University Medical Center, Washington, DC, USA
t
Early Biometrics & Statistical Innovation, Data Science & Artificial Intelligence, R & D, AstraZeneca, Gaithersburg,
Maryland, USA
u
Department of Biostatistics and Medical Informatics, University of Wisconsin—Madison, Madison, Wisconsin, USA
v
Morgridge Institute for Research, Madison, Wisconsin, USA
w
Childhood Cancer Data Lab, Alex’s Lemonade Stand Foundation, Philadelphia, Pennsylvania, USA
ABSTRACT After emerging in China in late 2019, the novel coronavirus severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2) spread worldwide, and as of mid-
2021, it remains a significant threat globally. Only a few coronaviruses are known to
infect humans, and only two cause infections similar in severity to SARS-CoV-2: Severe
acute respiratory syndrome-related coronavirus, a species closely related to SARS-CoV-2
that emerged in 2002, and Middle East respiratory syndrome-related coronavirus,which
emerged in 2012. Unlike the current pandemic, previous epidemics were controlled
rapidly through public health measures, but the body of research investigating severe
acute respiratory syndrome and Middle East respiratory syndrome has proven valuable
for identifying approaches to treating and preventing novel coronavirus disease 2019
(COVID-19). Building on this research, the medical and scientific communities have
responded rapidly to the COVID-19 crisis and identified many candidate therapeutics.
The approaches used to identify candidates fall into four main categories: adaptation
of clinical approaches to diseases with related pathologies, adaptation based on viro-
logical properties, adaptation based on host response, and data-driven identification
(ID) of candidates based on physical properties or on pharmacological compendia. To
date, a small number of therapeutics have already been authorized by regulatory
agencies such as the Food and Drug Administration (FDA), while most remain under
Editor Jack A. Gilbert, University of California
San Diego
Copyright © 2021 Rando et al. This is an open-
access article distributed under the terms of
the Creative Commons Attribution 4.0
International license.
Address correspondence to Casey S. Greene,
casey.s.greene@cuanschutz.edu.
This represents one section of a larger evolving
review on SARS-CoV-2 and COVID-19, which is
regularly updated and available at https://
greenelab.github.io/covid19-review/.
This is a review paper that is authored by
scientists for an audience of scientists to
discuss research that is in progress. If you are
interested in guidelines on testing, therapies,
or other issues related to your health, you
should not use this document. Instead, you
should collect information from your local
health department, the CDC’s guidance, or
your own government.
Published
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REVIEW
2 November 2021
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investigation. The scale of the COVID-19 crisis offers a rare opportunity to collect data
on the effects of candidate therapeutics. This information provides insight not only
into the management of coronavirus diseases but also into the relative success of dif-
ferent approaches to identifying candidate therapeutics against an emerging disease.
IMPORTANCE The COVID-19 pandemic is a rapidly evolving crisis. With the worldwide sci-
entific community shifting focus onto the SARS-CoV-2 virus and COVID-19, a large num-
ber of possible pharmaceutical approaches for treatment and prevention have been pro-
posed. What was known about each of these potential interventions evolved rapidly
throughout 2020 and 2021. This fast-paced 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. As illustrated by the continued threat of viral epi-
demics during the current millennium, a rapid and strategic response to emerging viral
threats can save lives. In this review, we explore how different modes of identifying can-
didate therapeutics have borne out during COVID-19.
KEYWORDS COVID-19, review, therapeutics
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). COVID-19 is associated with symptoms
ranging from mild or even asymptomatic to severe, and up to 2% of patients diag-
nosed with COVID-19 die from COVID-19-related complications such as acute respira-
tory disease syndrome (ARDS) (1). As a result, public health efforts have been critical to
mitigating the spread of the virus. However, as of mid-2021, COVID-19 remains a signif-
icant worldwide concern (Fig. 1), with the cases in some regions in 2021 surging far
above the numbers reported during the initial outbreak in early 2020. While a number
of vaccines have been developed and approved in different countries starting in late
2020 (2), vaccination efforts have not proceeded at the same pace throughout the
world and are not yet close to ending the pandemic.
Due to the continued threat of the virus and the severity of the disease, the identifi-
cation and development of therapeutic interventions have emerged as significant
international priorities. Prior developments during other recent outbreaks of emerging
diseases, especially those caused by human coronaviruses (HCoVs), have guided bio-
medical research into the behavior and treatment of this novel coronavirus infection.
However, previous emerging HCoV-related disease threats were controlled much more
quickly than SARS-CoV-2 through public health efforts (Fig. 1). The scale of the COVID-
19 pandemic has made the repurposing and development of pharmaceuticals more
urgent than in previous coronavirus epidemics.
LESSONS FROM PRIOR HCoV OUTBREAKS
At first, SARS-CoV-2’s rapid shift from an unknown virus to a significant worldwide
threat closely paralleled the emergence of Severe acute respiratory syndrome-related co-
ronavirus 1 (SARS-CoV-1), which was responsible for the 200222003 SARS epidemic.
The first documented case of COVID-19 was reported in Wuhan, China, in November
2019, and the disease quickly spread worldwide in the early months of 2020. In com-
parison, the first case of SARS was reported in November 2002 in the Guangdong
Province of China, and it spread within China and then into several countries across
continents during the first half of 2003 (3, 8, 9). In fact, genome sequencing quickly
revealed the virus causing COVID-19 to be a novel betacoronavirus closely related to
SARS-CoV-1 (10).
While similarities between these two viruses are unsurprising given their close phy-
logenetic relationship, there are also some differences in how the viruses affect
humans. SARS-CoV-1 infection is severe, with an estimated case fatality rate (CFR) for
SARS of 9.5% (8), while estimates of the CFR associated with COVID-19 are much lower,
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at up to 2% (1). SARS-CoV-1 is highly contagious and spread primarily by droplet trans-
mission, with a basic reproduction number (R
0
) of 4 (i.e., each person infected was esti-
mated to infect four other people) (8). There is still some controversy whether SARS-
CoV-2 is primarily spread by droplets or is primarily airborne (11–14). Most estimates of
its R
0
fall between 2.5 and 3 (1). Therefore, SARS is thought to be a deadlier and more
transmissible disease than COVID-19.
With the 17-year difference between these two outbreaks, there were major differ-
ences in the tools available to efforts to organize international responses. At the time
that SARS-CoV-1 emerged, no new HCoV had been identified in almost 40 years (9).
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 World Health Organization (WHO) (9). In contrast, the SARS-
CoV-2 genomic sequence was released on 3 January 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 HCoVs known at the time of its discovery (8), SARS-CoV-2 is closely related to
SARS-CoV-1 and is a more distant relative of another HCoV characterized in 2012,
Middle East respiratory syndrome-related coronavirus (MERS-CoV) (15, 16). Significant
efforts had been dedicated toward understanding SARS-CoV-1 and MERS-CoV and
how they interact with human hosts. Therefore, SARS-CoV-2 emerged under very dif-
ferent circumstances than SARS-CoV-1 in terms of scientific knowledge about HCoVs
and the tools available to characterize them.
Despite the apparent advantages for responding to SARS-CoV-2 infections, COVID-
19 has caused many orders of magnitude more deaths than SARS did (Fig. 1). The SARS
FIG 1 Cumulative global incidence of COVID-19 and SARS. As of 8 September 2021, 222,559,803 COVID-19 cases and 4,596,394 COVID-19 deaths had been
reported worldwide since 22 January 2020. A total of 8,432 cases and 813 deaths were reported for SARS from 17 March 2003 to 11 July 2003. SARS-CoV-1
was officially contained on 5 July 2003, within 9 months of its appearance (3). In contrast, SARS-CoV-2 remains a significant global threat nearly 2 years
after its emergence. COVID-19 data are from the COVID-19 Data Repository by the Center for Systems Science and Engineering at Johns Hopkins University
(4, 5). SARS data are from the WHO (6) and were obtained from a data set on GitHub (7). See https://greenelab.github.io/covid19-review/ for the most
recent version of this figure, which is updated daily.
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outbreak was officially determined to be under control in July 2003, with the success
credited to infection management practices such as mask wearing (9). MERS-CoV is still
circulating and remains a concern; although the fatality rate is very high at almost
35%, the disease is much less easily transmitted, as its R
0
has been estimated to be 1
(8). The low R
0
in combination with public health practices allowed for its spread to be
contained (8). Neither of these trajectories are comparable to that of SARS-CoV-2,
which remains a serious threat worldwide over a year and a half after the first cases of
COVID-19 emerged (Fig. 1).
POTENTIAL APPROACHES TO THE TREATMENT OF COVID-19
Therapeutic interventions can utilize two approaches: they can mitigate the effects
of an infection that harms an infected person, or they can hinder the spread of infec-
tion within a host by disrupting the viral life cycle. The goal of the former strategy is to
reduce the severity and risks of an active infection, while for the latter, it is to inhibit
the replication of a virus once an individual is infected, potentially freezing disease pro-
gression. Additionally, two major approaches can be used to identify interventions
that might be relevant to managing an emerging disease or a novel virus: drug repur-
posing and drug development. Drug repurposing involves identifying an existing com-
pound that may provide benefits in the context of interest (17). This strategy can focus
on either approved or investigational drugs, for which there may be applicable preclin-
ical or safety information (17). Drug development, on the other hand, provides an op-
portunity to identify or develop a compound specifically relevant to a particular need,
but it is often a lengthy and expensive process characterized by repeated failure (18).
Drug repurposing therefore tends to be emphasized in a situation like the COVID-19
pandemic due to the potential for a more rapid response.
Even from the early months of the pandemic, studies began releasing results from
analyses of approved and investigational drugs in the context of COVID-19. The rapid
timescale of this response meant that, initially, most evidence came from observational
studies, which compare groups of patients who did and did not receive a treatment to
determine whether it may have had an effect. This type of study can be conducted rap-
idly but is subject to confounding. In contrast, randomized controlled trials (RCTs) are
the gold standard method for assessing the effects of an intervention. Here, patients
are prospectively and randomly assigned to treatment or control conditions, allowing
for much stronger interpretations to be drawn; however, data from these trials take
much longer to collect. Both approaches have proven to be important sources of infor-
mation in the development of a rapid response to the COVID-19 crisis, but as the pan-
demic draws on and more results become available from RCTs, more definitive answers
are becoming available about proposed therapeutics. Interventional clinical trials are
currently investigating or have investigated a large number of possible therapeutics
and combinations of therapeutics for the treatment of COVID-19 (Fig. 2).
The purpose of this review is to provide an evolving resource tracking the status of
efforts to repurpose and develop drugs for the treatment of COVID-19. We highlight
four strategies that provide different paradigms for the identification of potential phar-
maceutical treatments. The WHO guidelines (20) and a systematic review (21) are com-
plementary living documents that summarize COVID-19 therapeutics.
REPURPOSING DRUGS FOR SYMPTOM MANAGEMENT
A variety of symptom profiles with a range of severity are associated with COVID-19
(1). In many cases, COVID-19 is not life-threatening. A study of COVID-19 patients in a
hospital in Berlin, Germany, reported that the highest risk of death was associated with
infection-related symptoms, such as sepsis, respiratory symptoms such as ARDS, and
cardiovascular failure or pulmonary embolism (22). Similarly, an analysis in Wuhan,
China, reported that respiratory failure (associated with ARDS) and sepsis/multiorgan
failure accounted for 69.5% and 28.0% of deaths, respectively, among 82 deceased
patients (23). COVID-19 is characterized by two phases. The first is the acute response,
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where an adaptive immune response to the virus is established and in many cases can
mitigate viral damage to organs (24). The second phase characterizes more severe
cases of COVID-19. Here, patients experience a cytokine storm, whereby excessive pro-
duction of cytokines floods into circulation, leading to systemic inflammation, immune
dysregulation, and multiorgan dysfunction that can cause multiorgan failure and death
if untreated (25). ARDS-associated respiratory failure can occur during this phase.
Cytokine dysregulation was also identified in patients with SARS (26, 27).
In the early days of the COVID-19 pandemic, physicians sought to identify potential
treatments that could benefit patients, and in some cases shared their experiences and
advice with the medical community on social media sites such as Twitter (28). These on-
the-ground treatment strategies could later be analyzed retrospectively in observational
studies or investigated in an interventional paradigm through RCTs. Several notable cases
involved the use of small-molecule drugs, which are synthesized compounds of low molec-
ular weight, typically less than 1 kDa (29). Small-molecule pharmaceutical agents have been
a backbone of drug development since the discovery of penicillin in the early twentieth
century (30). 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 (PPIs) have facilitated advances in precise targeting of spe-
cific structures using small molecules (30). Small-molecule drugs today encompass a wide
range of therapeutics beyond antibiotics, including antivirals, protein inhibitors, and many
broad-spectrum pharmaceuticals.
FIG 2 COVID-19 clinical trials. Trial data are from the University of Oxford Evidence-Based Medicine Data Lab’s COVID-19 TrialsTracker (19). As of 31
December 2020, there were 6,987 COVID-19 clinical trials of which 3,962 were interventional. The study types include only types used in at least five
trials. Only interventional trials are analyzed in the figures depicting status, phase, and intervention. Of the interventional trials, 98 trials had reported
results as of 31 December 31 2020. Recruitment status and trial phase are shown only for interventional trials in which the status or phase is recorded.
Common interventions refers to interventions used in at least 10 trials. Combinations of interventions, such as hydroxychloroquine with azithromycin,
are tallied separately from the individual interventions. See https://greenelab.github.io/covid19-review/ for the most recent version of this figure, which
is updated daily.
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Many treatments considered for COVID-19 have relied on a broad-spectrum
approach. These treatments do not specifically target a virus or particular host receptor
but rather induce broad shifts in host biology that are hypothesized to be potential
inhibitors of the virus. This approach relies on the fact that when a virus enters a host,
the host becomes the virus’s environment. Therefore, the state of the host can also
influence the virus’s ability to replicate and spread. The administration and assessment
of broad-spectrum small-molecule drugs on a rapid time course were feasible because
they are often available in hospitals, or in some cases, they may also be prescribed to a
large number of outpatients. One of the other advantages is that these well-estab-
lished compounds, if found to be beneficial, are often widely available, in contrast to
boutique experimental drugs.
In some cases, prior data were available from experiments examining the response
of other HCoVs or HCoV infections to a candidate drug. In addition to nonpharmaceuti-
cal interventions such as encouraging nonintubated patients to adopt a prone position
(31), knowledge about interactions between HCoVs and the human body, much of
which emerged from SARS and MERS research over the past 2 decades, led to the sug-
gestion that a number of common drugs might benefit COVID-19 patients. However,
the short duration and low case numbers of prior outbreaks were less well suited to
the large-scale study of clinical applications than the COVID-19 pandemic is. As a result,
COVID-19 has presented the first opportunity to robustly evaluate treatments that
were common during prior HCoV outbreaks to determine their clinical efficacy. The
first year of the COVID-19 pandemic demonstrated that there are several different tra-
jectories that these clinically suggested, widely available candidates can follow when
assessed against a widespread, novel viral threat.
One approach to identifying candidate small-molecule drugs was to look at the
approaches used to treat SARS and MERS. Treatment of SARS and MERS patients priori-
tized supportive care and symptom management (8). Among the clinical treatments
for SARS and MERS that were explored, there was generally a lack of evidence indicat-
ing whether they were effective. Most of the supportive treatments for SARS were
found inconclusive in meta-analysis (32), and a 2004 review reported that not enough
evidence was available to make conclusions about most treatments (33). However, one
strategy adopted from prior HCoV outbreaks is currently the best-known treatment for
severe cases of COVID-19. Corticosteroids are broad-spectrum treatments and are a
well-known, widely available treatment for pneumonia (34–39) that have also been
debated as a possible treatment for ARDS (40–45). Corticosteroids were also used and
subsequently evaluated as possible supportive care for SARS and MERS. In general,
studies and meta-analyses did not identify support for corticosteroids to prevent mor-
tality in these HCoV infections (46–48); however, one study found that the effects
might be masked by variability in treatment protocols, such as dosage and timing (33).
While the corticosteroids most often used to treat SARS were methylprednisolone and
hydrocortisone, availability issues for these drugs at the time led to dexamethasone
also being used in North America (49).
Dexamethasone (9
a
-fluoro-16
a
-methylprednisolone) is a synthetic corticosteroid that
binds to glucocorticoid receptors (50, 51). It functions as an anti-inflammatory agent by
binding to glucocorticoid receptors with higher affinity than endogenous cortisol (52).
Dexamethasone and other steroids are widely available and affordable, and they are often
used to treat community-acquired pneumonia (53) as well as chronic inflammatory condi-
tions such as asthma, allergies, and rheumatoid arthritis (54–56). Immunosuppressive
drugs such as steroids are typically contraindicated in the setting of infection (57), but
because COVID-19 results in hyperinflammation that appears to contribute to mortality via
lung damage, immunosuppression may be a helpful approach to treatment (58). A clinical
trial that began in 2012 recently reported that dexamethasone may improve outcomes for
patients with ARDS (40), but a meta-analysis of a small amount of available data about
dexamethasone as a treatment for SARS suggested that it may, in fact, be associated with
patient harm (59). However, the findings for SARS may have been biased by the fact that
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allofthestudiesexaminedwereobservationalandalargenumberofinconclusivestudies
were not included (60). The questions of whether and when to counter hyperinflammation
with immunosuppression in the setting of COVID-19 (as in SARS [27]) was an area of
intense debate, as the risks of inhibiting antiviral immunity needed to be weighed against
the beneficial anti-inflammatory effects (61). As a result, guidelines early in the pandemic
typically recommended avoiding treating COVID-19 patients with corticosteroids such as
dexamethasone (59).
Despite this initial concern, dexamethasone was evaluated as a potential treatment for
COVID-19 (Appendix). Dexamethasone treatment comprised one arm of the multisite
Randomized Evaluation of COVID-19 Therapy (RECOVERY) trial in the United Kingdom (62).
This study found that the 28-day mortality rate was lower in patients receiving dexametha-
sone than in those receiving the standard of care (SOC). 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 the SOC in patients who were ventilated (29.3% versus
41.4%) and among those who were receiving oxygen supplementation (23.3% versus
26.2%) at randomization, but not in patients who were breathing independently (17.8%
versus 14.0%). These findings also suggested that dexamethasone may have reduced pro-
gression to mechanical ventilation, especially among patients who were receiving oxygen
support at randomization. Other analyses have supported the importance of disease course
in determining the efficacy of dexamethasone: additional results suggest greater potential
for patients who have experienced symptoms for at least 7 days and patients who were
not breathing independently (63). A meta-analysis that evaluated the results of the
RECOVERY trial alongside trials of other corticosteroids, such as hydrocortisone, similarly
concluded that corticosteroids may be beneficial to patients with severe COVID-19 who are
receiving oxygen supplementation (64). Thus, it seems likely that dexamethasone is useful
for treating inflammation associated with immunopathy or cytokine release syndrome
(CRS), which is a condition caused by detrimental overactivation of the immune system (1).
In fact, corticosteroids such as dexamethasone are sometimes used to treat CRS (65).
Guidelines were quickly updated to encourage the use of dexamethasone in severe cases
(66), and this affordable and widely available treatment rapidly became a valuable tool
against COVID-19 (67), with demand surging within days of the preprint’s release (68).
APPROACHES TARGETING THE VIRUS
Therapeutics that directly target the virus itself hold the potential to prevent people
infected with SARS-CoV-2 from developing potentially damaging symptoms (Fig. 3).
Such drugs 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.
This process requires inhibiting the replication cycle of a virus by disrupting one of six
fundamental steps (69). In the first of these steps, the virus attaches to and enters the
host cell through endocytosis. Then the virus undergoes uncoating, which is classically
defined as the release of viral contents into the host cell. Next, the viral genetic mate-
rial enters the nucleus where it is replicated during the biosynthesis stage. During
the assembly stage, viral proteins are translated, allowing new viral particles to be
assembled. In the final step, new viruses are released into the extracellular environ-
ment. Although antivirals are designed to target a virus, they can also impact other
processes in the host and may have unintended effects. Therefore, these therapeutics
must be evaluated for both efficacy and safety. As the technology to respond to
emerging viral threats has also evolved over the past 2 decades, a number of candidate
treatments have been identified for prior viruses that may be relevant to the treatment
of COVID-19.
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,
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the RNA-dependent RNA polymerase (RdRP), for replication (71, 72). RdRP is therefore a
potential target for antivirals against RNA viruses. Disruption of RdRP is the proposed
mechanism underlying the treatment of SARS and MERS with ribavirin (73). Ribavirin is
an antiviral drug effective against other viral infections that was often used in combina-
tion with corticosteroids and sometimes interferon (IFN) medications to treat SARS and
MERS (9). However, analyses of its effects in retrospective and in vitro analyses of SARS
and the SARS-CoV-1 virus, respectively, have been inconclusive (9). While IFNs and riba-
virin have shown promise in in vitro analyses of MERS, their clinical effectiveness remains
unknown (9). The current COVID-19 pandemic has provided an opportunity to assess the
clinical effects of these treatments. As one example, ribivarin was also used in the early
days of COVID-19, but a retrospective cohort study comparing patients who did and did
not receive ribivarin revealed no effect on the mortality rate (74).
Since nucleotides and nucleosides are the natural building blocks for RNA synthesis,
an alternative approach has been to explore nucleoside and nucleotide analogs for
their potential to inhibit viral replication. Analogs containing modifications to nucleo-
tides or nucleosides can disrupt key processes, including replication (75). A single
incorporation does not influence RNA transcription; however, multiple events of incor-
poration lead to the arrest of RNA synthesis (76). One candidate antiviral considered
for the treatment of COVID-19 is favipiravir (Avigan), also known as T-705, which was
discovered by Toyama Chemical Co., Ltd. (77). It was previously found to be effective
at blocking viral amplification in several influenza virus subtypes as well as other RNA
FIG 3 Mechanisms of action for potential therapeutics. Potential therapeutics currently being studied can target the SARS-CoV-2 virus or modify the host
environment through many different mechanisms. Here, the relationships between the virus, host cells, and several therapeutics are visualized. Drug names
are color coded according to the grade assigned to them by the Center for Cytokine Storm Treatment & Laboratory’s CORONA Project (70) (green for
grade A, lime for grade B, orange for grade C, and red for grade D).
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viruses, such as Flaviviridae and Picornaviridae, through a reduction in plaque forma-
tion (78) and viral replication in Madin-Darby canine kidney cells (79). Favipiravir (6-flu-
oro-3-hydroxy-2-pyrazinecarboxamide) acts as a purine and purine nucleoside analog
that inhibits viral RNA polymerase in a dose-dependent manner across a range of RNA
viruses, including influenza viruses (80–84). Biochemical experiments showed that favi-
piravir was recognized as a purine nucleoside analog and incorporated into the viral
RNA template. In 2014, the drug was approved in Japan for the treatment of influenza
that was resistant to conventional treatments like neuraminidase inhibitors (85).
Though initial analyses of favipiravir in observational studies of its effects on COVID-19
patients were promising, recent results of two small RCTs suggest that it is unlikely to
affect COVID-19 outcomes (Appendix).
In contrast, another nucleoside analog, remdesivir, is one of the few treatments
against COVID-19 that has received FDA approval. Remdesivir (GS-5734) is an intrave-
nous antiviral that was proposed by Gilead Sciences as a possible treatment for Ebola
virus disease. It is metabolized to GS-441524, an adenosine analog that inhibits a broad
range of polymerases and then evades exonuclease repair, causing chain termination
(86–88). Gilead received an emergency use authorization (EUA) for remdesivir from the
FDA early in the pandemic (May 2020) and was later found to reduce mortality and re-
covery time in a double-blind, placebo-controlled, phase 3 clinical trial performed at
60 trial sites, 45 of which were in the United States (89–92). Subsequently, the WHO
Solidarity trial, a large-scale, open-label trial enrolling 11,330 adult inpatients at 405
hospitals in 30 countries around the world, reported no effect of remdesivir on in-hos-
pital mortality, duration of hospitalization, or progression to mechanical ventilation
(93). Therefore, additional clinical trials of remdesivir in different patient pools and in
combination with other therapies may be needed to refine its use in the clinic and
determine the forces driving these differing results. Remdesivir offers proof of principle
that SARS-CoV-2 can be targeted at the level of viral replication, since remdesivir tar-
gets the viral RNA polymerase at high potency. Identification of such candidates
depends on knowledge about the virological properties of a novel threat. However,
the success and relative lack of success, respectively, of remdesivir and favipiravir
underscore the fact that drugs with similar mechanisms will not always produce similar
results in clinical trials.
DISRUPTING HOST-VIRUS INTERACTIONS
Interrupting viral colonization of cells. Some of the most widely publicized exam-
ples of efforts to repurpose drugs for COVID-19 are broad-spectrum, small-molecule
drugs where the mechanism of action made it seem that the drug might disrupt inter-
actions between SARS-CoV-2 and human host cells (Fig. 3). However, the exact out-
comes of such treatments are difficult to predict a priori, and there are several exam-
ples where early enthusiasm was not borne out in subsequent trials. One of the most
famous examples of an analysis of whether a well-known medication could provide
benefits to COVID-19 patients came from the assessment of chloroquine (CQ) and
hydroxychloroquine (HCQ), which are used for the treatment and prophylaxis of
malaria as well as the treatment of lupus erythematosus and rheumatoid arthritis in
adults (94). These drugs are lysosomotropic agents, meaning they are weak bases that
can pass through the plasma membrane. It was thought that they might provide bene-
fits against SARS-CoV-2 by interfering with the digestion of antigens within the lyso-
some and inhibiting CD4 T-cell stimulation while promoting the stimulation of CD8 T
cells (95). These compounds also have anti-inflammatory properties (95) and can
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 (TLRs)
and TLR signaling (95).
In vitro analyses reported that CQ inhibited cell entry of SARS-CoV-1 (96) and that
both CQ and HCQ inhibited viral replication within cultured cells (97), leading to early
hope that it might provide similar therapeutic or protective effects in patients.
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However, while the first publication on the clinical application of these compounds to
the inpatient treatment of COVID-19 was very positive (98), it was quickly discredited
(99). Over the following months, extensive evidence emerged demonstrating that CQ
and HCQ offered no benefits for COVID-19 patients and, in fact, carried the risk of dan-
gerous side effects (Appendix). The nail in the coffin came when findings from the
large-scale RECOVERY trial were released on 8 October 2020. This study enrolled
11,197 hospitalized patients whose physicians believed it would not harm them to par-
ticipate and used a randomized, open-label design to study the effects of HCQ com-
pared to the standard of care (SOC) at 176 hospitals in the United Kingdom (100).
Rates of COVID-19-related mortality did not differ between the control and HCQ arms,
but patients receiving HCQ were slightly more likely to die due to cardiac events.
Patients who received HCQ also had a longer duration of hospitalization than patients
receiving usual care and were more likely to progress to mechanical ventilation or
death (as a combined outcome). As a result, enrollment in the HCQ arm of the
RECOVERY trial was terminated early (101). The story of CQ/HCQ therefore illustrates
how initial promising in vitro analyses can fail to translate to clinical usefulness.
A similar story has arisen with the broad-spectrum, small-molecule anthelmintic
ivermectin, which is a synthetic analog of avermectin, a bioactive compound produced
by a microorganism known as Streptomyces avermectinius and Streptomyces avermitilis
(102, 103). Avermectin disrupts the ability of parasites to avoid the host immune
response by blocking glutamate-gated chloride ion channels in the peripheral nervous
system from closing, leading to hyperpolarization of neuronal membranes, disruption
of neural transmission, and paralysis (102, 104, 105). Ivermectin has been used since
the early 1980s to treat endo- and ectoparasitic infections by helminths, insects, and
arachnids in veterinary contexts (102, 106) and since the late 1980s to treat human par-
asitic infections as well (102, 104). More recent research has indicated that ivermectin
might function as a broad-spectrum antiviral by disrupting the trafficking of viral pro-
teins by both RNA and DNA viruses (105, 107, 108), although most of these studies
have demonstrated this effect in vitro (108). The potential for antiviral effects on SARS-
CoV-2 were investigated in vitro, and ivermectin was found to inhibit viral replication
in a cell line derived from Vero cells (Vero-hSLAM) (109). However, inhibition of viral
replication was achieved at concentrations that were much higher than that explored
by existing dosage guidelines (110, 111), which are likely to be associated with signifi-
cant side effects due to the increased potential that the compound could cross the
mammalian blood-brain barrier (112, 113).
Retrospective studies and small RCTs began investigating the effects of standard
doses of this low-cost, widely available drug. One retrospective study reported that
ivermectin reduced all-cause mortality (114), while another reported no difference in
clinical outcomes or viral clearance (115). Small RCTs enrolling less than 50 patients
per arm have also reported a wide array of positive (116–120) and negative results
(121, 122). A slightly larger RCT enrolling 115 patients in two arms reported inconclu-
sive results (123). Hope for the potential of ivermectin peaked with the release of a
preprint reporting results of a multicenter, double-blind RCT where a 4-day course of
ivermectin was associated with clinical improvement and earlier viral clearance in
400 symptomatic patients and 200 close contacts (124); however, concerns were
raised about both the integrity of the data and the paper itself (125, 126), and this
study was removed by the preprint server Research Square (127). A similarly sized
RCT suggested no effect on the duration of symptoms among 400 patients split
evenly across the intervention and control arms (128), and although meta-analyses
have reported both null (129, 130) and beneficial (131–138) effects of ivermectin on
COVID-19 outcomes, the certainty is likely to be low (132). These findings are poten-
tially biased by a small number of low-quality studies, including the preprint that has
been taken down (139), and the authors of one (140) have issued a notice (131) that
they will revise their study with the withdrawn study removed. Thus, much like HCQ/
CQ, enthusiasm for research that either has not or should not have passed peer
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review has led to large numbers of patients worldwide receiving treatments that
might not have any effect or could even be harmful. Additionally, comments on the
now-removed preprint include inquiries into how best to self-administer veterinary
ivermectin as a prophylactic (127), and the FDA has posted information explaining
why veterinary ivermectin should not be taken by humans concerned about COVID-
19 (141). Ivermectin is now one of several candidate therapeutics being investigated
in the large-scale TOGETHER (142) and PRINCIPLE (143) clinical trials. The TOGETHER
trial, which previously demonstrated no effect of HCQ and lopinavir-ritonavir (144),
released preliminary results in early August 2021 suggesting that ivermectin also has
no effect on COVID-19 outcomes (145).
While CQ/HCQ and ivermectin are well-known medications that have long been
prescribed in certain contexts, investigation of another well-established type of phar-
maceutical was facilitated by the fact that it was already being taken by a large number
of COVID-19 patients. Angiotensin-converting enzyme inhibitors (ACEIs) and angioten-
sin II receptor blockers (ARBs) are among today’s most commonly prescribed medica-
tions, often being used to control blood pressure (146, 147). In the United States, for
example, they are prescribed well over 100,000,000 times annually (148). Prior to the
COVID-19 pandemic, the relationship between ACE2, ACEIs, and SARS had been con-
sidered as possible evidence that ACE2 could serve as a therapeutic target (149), and
the connection had been explored through in vitro and molecular docking analysis
(150) but ultimately was not pursued clinically (151). Data from some animal models
suggest that several, but not all, ACEIs and several ARBs increase ACE2 expression in
the cells of some organs (152), but clinical studies have not established whether
plasma ACE2 expression is increased in humans treated with these medications (153).
In this case, rather than introducing ARBs/ACEIs, a number of analyses have investi-
gated whether discontinuing use affects COVID-19 outcomes. An initial observational
study of the association of exposure to ACEIs or ARBs with outcomes in COVID-19 (154)
was retracted from the New England Journal of Medicine due to concerns related to
data availability (155). As RCTs have become available, they have demonstrated no
effect of continuing versus discontinuing ARBs/ACEIs on patient outcomes (156, 157)
(Appendix). Thus, once again, despite a potential mechanistic association with the pa-
thology of SARS-CoV-2 infection, these medications were not found to influence the
trajectory of COVID-19 illness.
For medications that are widely known and common, clinical research into their effi-
cacy against a novel threat can be developed very quickly. This feasibility can present a
double-edged sword. For example, HCQ and CQ were incorporated into the SOC in
many countries early in the pandemic and had to be discontinued once their potential
to harm COVID-19 patients became apparent (158, 159). Dexamethasone remains the
major success story from this category of repurposed drugs and is likely to have saved
a large number of lives since summer 2020 (67).
Manipulating the host immune response. Treatments based on understanding a
virus and/or how a virus interacts with the human immune system can fall into two
categories: they can interact with the innate immune response, which is likely to be a
similar response across viruses, or they can be specifically designed to imitate the
adaptive immune response to a particular virus. In the latter case, conservation of
structure or behavior across viruses determines interest in whether drugs developed
for one virus can treat another. During the COVID-19 pandemic, a number of candidate
therapeutics have been explored in these categories, with varied success.
Knowledge gained from characterizing SARS-CoV-1 and MERS-CoV from a funda-
mental biological perspective along with their interactions with the human immune
system provides a theoretical basis for identifying candidate therapies. Biologics are a
particularly important class of drugs for efforts to address HCoV through this paradigm.
They are produced from components of living organisms or viruses, historically primar-
ily from animal tissues (160). Biologics have become increasingly feasible to produce as
recombinant DNA technologies have advanced (160).
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There are many differences on the development side between biologics and syn-
thesized pharmaceuticals, such as small-molecule drugs. Typically, biologics are orders
of magnitude larger than small-molecule drugs and are catabolized by the body to
their amino acid components (161). They are often heat sensitive, and their toxicity can
vary, as it is not directly associated with the primary effects of the drug; in general,
their physiochemical properties are much less understood compared to small mole-
cules (161). Biologics include significant medical breakthroughs such as insulin for the
management of diabetes and vaccines and monoclonal antibodies (MAbs) and interfer-
ons (IFNs), which can be used to target the host immune response after infection.
MAbs have revolutionized the way we treat human diseases and have become some
of the best-selling drugs in the pharmaceutical market in recent years (162). There are
currently 79 FDA approved MAbs on the market, including antibodies for viral infections
(e.g., ibalizumab for human immunodeficiency virus and palivizumab for respiratory syn-
cytial virus) (162, 163). Virus-specific neutralizing antibodies commonly target viral sur-
face glycoproteins or host structures, thereby inhibiting viral entry through receptor
binding interference (164, 165). This interference is predicted to reduce the viral load,
mitigate disease, and reduce overall hospitalization. MAbs can be designed for a particu-
lar virus, and significant advances have been made in the speed at which new MAbs can
be identified and produced. At the time of the SARS and MERS epidemics, interest in
MAbs to reduce infection was never realized (166, 167), but this allowed for MAbs to
quickly be considered among the top candidates against COVID-19.
(i) Biologics and the innate immune response. Deaths from COVID-19 often occur
when inflammation becomes dysregulated following an immune response to the SARS-
CoV-2 virus. Therefore, one potential approach to reducing COVID-19 mortality rates is to
manage the inflammatory response in severely ill patients. One candidate therapeutic
identified that uses this mechanism is tocilizumab (TCZ). TCZ is a MAb that was developed
to manage chronic inflammation caused by the continuous synthesis of the cytokine IL-6
(168). IL-6 is a proinflammatory cytokine belonging to the interleukin family, which is com-
prised by immune system regulators that are primarily responsible for immune cell differ-
entiation. Often used to treat chronic inflammatory conditions such as rheumatoid arthritis
(168), TCZ has become a pharmaceutical of interest for the treatment of COVID-19 because
of the role IL-6 plays in this disease. It has also been approved to treat CRS caused by chi-
meric antigen receptor T-cell therapy (CAR-T) treatments (169). While the secretion of IL-6
can be associated with chronic conditions, IL-6 is a key player in the innate immune
response and is secreted by macrophages in response to the detection of pathogen-asso-
ciated molecular patterns and damage-associated molecular patterns (168). An analysis of
191 inpatients 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
died had higher IL-6 levels at admission than those who recovered (170). Additionally, IL-6
levels remained higher throughout the course of hospitalization in the patients who ulti-
mately died (170).
Currently, TCZ is being administered either as a monotherapy or in combination
with other treatments in 73 interventional COVID-19 clinical trials (Fig. 2). A number of
retrospective studies have been conducted in several countries (171–176). In general,
these studies have reported a positive effect of TCZ on reducing mortality in COVID-19
patients, although due to their retrospective designs, significant limitations are present
in all of them (Appendix). It was not until 11 February 2021 that a preprint describing
preliminary results of the first RCT of TCZ was released as part of the RECOVERY trial
(177). TCZ was found to reduce 28-day mortality from 33% in patients receiving the
SOC alone to 29% in those receiving TCZ. Combined analysis of the RECOVERY trial
data with data from smaller RCTs suggested a 13% reduction in 28-day mortality (177).
While this initial report did not include the full results expected from the RECOVERY
trial, this large-scale RCT provides strong evidence that TCZ may offer benefits for
COVID-19 patients. The RECOVERY trial along with results from several other RCTs
(178–182) were cited as support for the EUA issued for TCZ in June 2021 (183).
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However, the fact that TCZ suppresses the immune response means that it does carry
risks for patients, especially a potential risk of secondary infection (Appendix).
TCZ is just one example of a candidate drug targeting the host immune response
and specifically excessive inflammation. For example, interferons (IFNs) have also been
investigated; these are a family of cytokines critical to activating the innate immune
response against viral infections. Synairgen has been investigating a candidate drug,
SNG001, which is an IFN-
b
-1a formulation to be delivered to the lungs via inhalation
(184) that they reported reduced progression to ventilation in a double-blind, placebo-
controlled, multicenter study of 101 patients with an average age in the late 50s (185,
186). However, these findings were not supported by the large-scale WHO Solidarity
trial, which reported no significant effect of IFN-
b
-1a on patient survival during hospi-
talization (93), although differences in the designs of the two studies, and specifically
the severity of illness among enrolled patients, may have influenced their divergent
outcomes (Appendix). Other biologics influencing inflammation are also being
explored (Appendix). It is also important that studies focused on inflammation as a
possible therapeutic target consider the potential differences in baseline inflammation
among patients from different backgrounds, which may be caused by differing life
experiences (see reference 187).
(ii) Biologics and the adaptive immune response. While TCZ is an example of an
MAb focused on managing the innate immune response, other treatments are more
specific, targeting the adaptive immune response after an infection. In some cases,
treatments can utilize biologics obtained directly from recovered individuals. From
the very early days of the COVID-19 pandemic, polyclonal antibodies from convales-
cent plasma were investigated as a potential treatment for COVID-19 (188, 189).
Convalescent plasma was used in prior epidemics, including SARS, Ebola virus disease,
and even the 1918 Spanish influenza (188, 190). Use of convalescent plasma transfu-
sion (CPT) over more than a century has aimed to reduce symptoms and improve
mortality in infected people (190), possibly by accelerating viral clearance (188).
However, it seems unlikely that this classic treatment confers any benefit for COVID-
19 patients. Several systematic reviews have investigated whether CPT reduced mor-
tality in COVID-19 patients, and although findings from early in the pandemic (up to
19 April 2020) did support the use of CPT (190), the tide has shifted as the body of
available literature has grown (191). While titer levels were suggested as a possible
determining factor in the success of CPT against COVID-19 (192), the large-scale
RECOVERY trial evaluated the effect of administering high-titer plasma specifically and
found no effect on mortality or hospital discharge over a 28-day period (193). These
results thus suggest that, despite initial optimism and an EUA from the FDA, CPT is
unlikely to be an effective therapeutic for COVID-19.
A different narrative is shaping up around the use of MAbs specifically targeting
SARS-CoV-2. During the first SARS epidemic in 2002, neutralizing antibodies (nAbs)
were found in SARS-CoV-1-infected patients (194, 195). Several studies following up on
these findings identified various S-glycoprotein epitopes as the major targets of nAbs
against SARS-CoV-1 (196). Coronaviruses use trimeric spike (S) glycoproteins on their
surface to bind to the host cell, allowing for cell entry (197, 198). Each S-glycoprotein
protomer is comprised of an S1 domain, also called the receptor binding domain
(RBD), and an S2 domain. The S1 domain binds to the host cell, while the S2 domain
facilitates the fusion between the viral envelope and host cell membranes (196). The
genomic identity between the RBD of SARS-CoV-1 and SARS-CoV-2 is around 74%
(199). Due to this high degree of similarity, preexisting antibodies against SARS-CoV-1
were initially considered candidates for neutralizing activity against SARS-CoV-2. While
some antibodies developed against the SARS-CoV-1 spike protein showed cross-neu-
tralization activity with SARS-CoV-2 (200, 201), others failed to bind to SARS-CoV-2
spike protein at relevant concentrations (202). Cross-neutralizing activities were de-
pendent on whether the epitope recognized by the antibodies were conserved
between SARS-CoV-1 and SARS-CoV-2 (200).
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Technological advances in antibody drug design as well as in structural biology mas-
sively accelerated the discovery of novel antibody candidates and the mechanisms by
which they interact with the target structure. Within just a year of the structure of the
SARS-CoV-2 spike protein being published, an impressive pipeline of monoclonal anti-
bodies targeting SARS-CoV-2 entered clinical trials, with hundreds more candidates in
preclinical stages. The first human monoclonal neutralizing antibody specifically against
the SARS-CoV-2 S glycoprotein was developed using hybridoma technology (203), where
antibody-producing B cells developed by mice are inserted into myeloma cells to pro-
duce a hybrid cell line (the hybridoma) that is grown in culture. The 47D11 antibody
clone was able to cross-neutralize SARS-CoV-1 and SARS-CoV-2. This antibody (now ABVV-
47D11) has recently entered clinical trials in collaboration with AbbVie. Additionally, an
extensive monoclonal neutralizing antibody pipeline has been developed to combat the
ongoing pandemic, with over 50 different antibodies in clinical trials (204). Thus far, the
monotherapy sotrovimab and two antibody cocktails (bamlanivimab/estesevimab and
casirivimab/imdevimab) have been granted EUAs by the FDA.
One of the studied antibody cocktails consists of bamlanivimab and estesevimab.
Bamlanivimab (Ly-CoV555) is a human MAb that was derived from convalescent
plasma donated by a recovered COVID-19 patient, evaluated in research by the
National Institute of Allergy and Infectious Diseases (NIAID), and subsequently devel-
oped by AbCellera and Eli Lilly. The neutralizing activity of bamlanivimab was initially
demonstrated in vivo using a nonhuman primate model (205). On the basis of these
positive preclinical data, Eli Lilly initiated the first human clinical trial for a monoclonal
antibody against SARS-CoV-2. The phase 1 trial, which was conducted in hospitalized
COVID-19 patients, was completed in August 2020 (206). Estesevimab (LY-CoV016 or
JS-016) is also a monoclonal neutralizing antibody against the spike protein of SARS-
CoV-2. It was initially developed by Junshi Biosciences and later licensed and devel-
oped through Eli Lilly. A phase 1 clinical trial to assess the safety of etesevimab was
completed in October 2020 (207). Etesevimab was shown to bind an epitope on the
spike protein different from that of bamlanivimab, suggesting that the two antibodies
used as a combination therapy would further enhance their clinical use compared to a
monotherapy (208). To assess the efficacy and safety of bamlanivimab alone or in com-
bination with etesevimab for the treatment of COVID-19, a phase 2/3 trial (BLAZE-1)
(209) was initiated. The interim analysis of the phase 2 portion suggested that bamlani-
vimab alone was able to accelerate the reduction in viral load (210). However, more
recent data suggest that only the bamlanivimab/etesevimab combination therapy is
able to reduce viral load in COVID-19 patients (208). Based on these data, the combina-
tion therapy received an EUA for COVID-19 from the FDA in February 2021 (211).
A second therapy is comprised of casirivimab and imdevimab (REGN-COV2).
Casirivimab (REGN10933) and imdevimab (REGN10987) are two monoclonal antibodies
against the SARS-CoV-2 spike protein. They were both developed by Regeneron in a
parallel high-throughput screening (HTS) to identify neutralizing antibodies from either
humanized mice or patient-derived convalescent plasma (212). In these efforts, multi-
ple antibodies were characterized for their ability to bind and neutralize the SARS-CoV-
2 spike protein. The investigators hypothesized that an antibody cocktail, rather than
each individual antibody, could increase the therapeutic efficacy while minimizing the
risk for virus escape. Therefore, the authors tested pairs of individual antibodies for
their ability to simultaneously bind the RBD of the spike protein. Based on these data,
casirivimab and imdevimab were identified as the lead antibody pair, resulting in the
initiation of two clinical trials (213, 214). Data from this phase 1 to 3 trial published in
the New England Journal of Medicine shows that the REGN-COV2 antibody cocktail
reduced viral load, particularly in patients with high viral load or whose endogenous
immune response had not yet been initiated (215). However, in patients who already
initiated an immune response, exogenous addition of REGN-COV2 did not improve the
endogenous immune response. Both doses were well tolerated with no serious events
related to the antibody cocktail. Based on these data, the FDA granted an EUA for
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REGN-COV2 in patients with mild to moderate COVID-19 who are at risk of developing
severe disease (216). Ongoing efforts are trying to evaluate the efficacy of REGN-COV2
to improve clinical outcomes in hospitalized patients (213).
Sotrovimab is the most recent MAb to receive an EUA. It was identified in the mem-
ory B cells of a 2003 survivor of SARS (217) and was found to be cross-reactive with
SARS-CoV-2 (201). This cross-reactivity is likely attributable to conservation within the
epitope, with 17 out of 22 residues conserved between the two viruses, four conserva-
tively substituted, and one semiconservatively substituted (201). In fact, these residues
are highly conserved among sarbecoviruses, a clade that includes SARS-CoV-1 and
SARS-CoV-2 (201). This versatility has led to it being characterized as a “super-antibody”
(218), a potent, broadly neutralizing antibody (219). Interim analysis of data from a clin-
ical trial (220) reported high safety and efficacy of this MAb in 583 COVID-19 patients
(221). Compared to placebo, sotrovimab was found to be 85% more effective in reduc-
ing progression to the primary endpoint, which was the proportion of patients who,
within 29 days, were either hospitalized for more than 24 h or died. Additionally, rates
of adverse events were comparable, and in some cases lower, among patients receiv-
ing sotrovimab compared to patients receiving a placebo. Sotrovimab therefore repre-
sents a MAb therapeutic that is effective against SARS-CoV-2 and may also be effective
against other sarbecoviruses.
Several potential limitations remain in the application of MAbs to the treatment of
COVID-19. One of the biggest challenges is identifying antibodies that not only bind to
their target but also prove to be beneficial for disease management. Currently, use of
MAbs is limited to people with mild to moderate disease that are not hospitalized, and
it has yet to be determined whether they can be used as a successful treatment option
for severe COVID-19 patients. While preventing people from developing severe illness
provides significant benefits, patients with severe illness are at the greatest risk of
death, and therefore therapeutics that provide benefits against severe illness are par-
ticularly desirable. It remains to be seen whether MAbs confer any benefits for patients
in this category.
Another concern about therapeutics designed to amplify the response to a specific
viral target is that they may need to be modified as the virus evolves. With the ongoing
global spread of new SARS-CoV-2 variants, there is a growing concern that mutations
in the SARS-CoV-2 spike protein could escape antibody neutralization, thereby reduc-
ing the efficacy of monoclonal antibody therapeutics and vaccines. A comprehensive
mutagenesis screen recently identified several amino acid substitutions in the SARS-
CoV-2 spike protein that can prevent antibody neutralization (222). While some muta-
tions result in resistance to only one antibody, others confer broad resistance to multi-
ple MAbs as well as polyclonal human sera, suggesting that some amino acids are “hot
spots”for antibody resistance. However, it was not investigated whether the resistance
mutations identified result in a fitness advantage. Accordingly, an impact on neutraliz-
ing efficiency has been reported for the B.1.1.7 (Alpha) variant first identified in the
United Kingdom and the B.1.351 (Beta) variant first identified in in South Africa (223–
225). As of 25 June 2021, the CDC recommended a pause in the use of bamlanivimab
and etesevimab due to decreased efficacy against the P.1 (Gamma) and B.1.351 (Beta)
variants of SARS-CoV-2 (226). While the reported impact on antibody neutralization
needs to be confirmed in vivo, it suggests that some adjustments to therapeutic anti-
body treatments may be necessary to maintain the efficacy that was reported in previ-
ous clinical trials.
Several strategies have been employed to try to mitigate the risk of diminished anti-
body neutralization. Antibody cocktails such as those already holding an EUA may help
overcome the risk for attenuation of the neutralizing activity of a single monoclonal
antibody. These cocktails consist of antibodies that recognize different epitopes on the
spike protein, decreasing the likelihood that a single amino acid change can cause re-
sistance to all antibodies in the cocktail. However, neutralizing resistance can emerge
even against an antibody cocktail if the individual antibodies target subdominant
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epitopes (224). Another strategy is to develop broadly neutralizing antibodies that tar-
get structures that are highly conserved, as these are less likely to mutate (227, 228) or
to target epitopes that are insensitive to mutations (229). Sotrovimab, one such
“super-antibody,”is thought to be somewhat robust to neutralization escape (230) and
has been found to be effective against all variants assessed as of 12 August 2021 (231).
Another antibody (ADG-2) targets a highly conserved epitope that overlaps the human
angiotensin-converting enzyme 2 (hACE2) binding site of all clade 1 sarbecoviruses
(232). Prophylactic administration of ADG-2 in an immunocompetent mouse model of
COVID-19 resulted in protection against viral replication in the lungs and respiratory
burden. Since the epitope targeted by ADG-2 represents an Achilles’heel for clade 1
sarbecoviruses, this antibody, like sotrovimab, might be a promising candidate against
all circulating variants as well as emerging SARS-related coronaviruses. To date, it has
fared well against the Alpha, Beta, Gamma, and Delta variants (231).
The development of MAbs against SARS-CoV-2 has made it clear that this technol-
ogy is rapidly adaptable and offers great potential for the response to emerging viral
threats. However, additional investigation may be needed to adapt MAb treatments to
SARS-CoV-2 as it evolves and potentially to pursue designs that confer benefits for
patients at the greatest risk of death. While polyclonal antibodies from convalescent
plasma have been evaluated as a treatment for COVID-19, these studies have sug-
gested fewer potential benefits against SARS-CoV-2 than MAbs; convalescent plasma
therapy has been thoroughly reviewed elsewhere (188, 189). Thus, advances in biolog-
ics for COVID-19 illustrate that an understanding of how the host and virus interact can
guide therapeutic approaches. The FDA authorization of two combination MAb thera-
pies, in particular, underscores the potential for this strategy to allow for a rapid
response to a novel pathogen. Additionally, while TCZ is not yet as established, this
therapy suggests that the strategy of using biologics to counteract the cytokine storm
response may provide therapies for the highest-risk patients.
HIGH-THROUGHPUT SCREENING FOR DRUG REPURPOSING
The drug development process is slow and costly, and developing compounds specif-
ically targeted to an emerging viral threat is not a practical short-term solution.
Screening existing drug compounds for alternative indications is a popular alternative
(233–236). HTS has been a goal of pharmaceutical development since at least the mid-
1980s (237). Traditionally, phenotypic screens were used to test which compounds
would induce a desired change in in vitro or in vivo models, focusing on empirical, func-
tion-oriented exploration naive to molecular mechanism (238–240). In many cases, these
screens utilize large libraries that encompass a diverse set of agents varying in many
pharmacologically relevant properties (e.g., reference 241). The compounds inducing a
desired effect could then be followed up on. Around the turn of the millennium, advan-
ces in molecular biology allowed for HTS to shift toward screening for compounds inter-
acting with a specific molecular target under the hypothesis that modulating that target
would have a desired effect. These approaches both offer pros and cons, and today a
popular view is that they are most effective in combination (238, 240, 242).
Today, some efforts to screen compounds for potential repurposing opportunities are
experimental, but others use computational HTS approaches (233, 243). Computational
drug repurposing screens can take advantage of big data in biology (17) and as a result
aremuchmorefeasibletodaythanduringtheheightoftheSARSandMERSoutbreaksin
the early 2000s and early 2010s, respectively. Advancements in robotics also facilitate the
experimental component of HTS (235). For viral diseases, the goal of drug repurposing is
typically to identify existing drugs that have an antiviral effect likely to impede the virus of
interest. While both small molecules and biologics can be candidates for repurposing, the
significantly lower price of many small-molecule drugs means that they are typically more
appealing candidates (244).
Depending on the study design, screens vary in how closely they are tied to a hy-
pothesis. As with the candidate therapeutics described above, high-throughput
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experimental or computational screens can proceed based on a hypothesis. Just as
remdesivir was selected as a candidate antiviral because it is a nucleoside analog (245),
so too can high-throughput screens select libraries of compounds based on a molecu-
lar hypothesis. Likewise, when the library of drugs is selected without basis in a poten-
tial mechanism, a screen can be considered hypothesis free (245). Today, both types of
analyses are common both experimentally and computationally. Both strategies have
been applied to identifying candidate therapeutics against SARS-CoV-2.
Hypothesis-driven screening. Hypothesis-driven screens often select drugs likely
to interact with specific viral or host targets or drugs with desired clinical effects, such
as immunosuppressants. There are several properties that might identify a compound
as a candidate for an emerging viral disease. Drugs that interact with a target that is
shared between pathogens (i.e., a viral protease or a polymerase) or between a viral
pathogen and another illness (i.e., a cancer drug with antiviral potential) are potential
candidates, as are drugs that are thought to interact with additional molecular targets
beyond those they were developed for (243). Such research can be driven by in vitro or
in silico experimentation. Computational analyses depend on identifying compounds
that modulate preselected proteins in the virus or host. As a result, they build on ex-
perimental research characterizing the molecular features of the virus, host, and candi-
date compounds (236).
One example of the application of this approach to COVID-19 research comes from
work on protease inhibitors. Studies have shown that viral proteases play an important
role in the life cycle of viruses, including coronaviruses, by modulating the cleavage of vi-
ral polyprotein precursors (246). Several FDA-approved drugs target proteases, such as
lopinavir and ritonavir for human immunodeficiency virus (HIV) infection and simeprevir
for hepatitis C virus infection. Serine protease inhibitors were previously suggested as
possible treatments for SARS and MERS (247). One early study (197) suggested that
camostat mesylate, a protease inhibitor, could block the entry of SARS-CoV-2 into lung
cells in vitro. Two polyproteins encoded by the SARS-CoV-2 replicase gene, pp1a and
pp1ab, are critical for viral replication and transcription (248). These polyproteins must
undergo proteolytic processing, which is usually conducted by the main protease (M
Pro
),
a 33.8-kDa SARS-CoV-2 protease that is therefore fundamental to viral replication and
transcription. Therefore, it was hypothesized that compounds targeting M
Pro
could be
used to prevent or slow the replication of the SARS-CoV-2 virus.
Both computational and experimental approaches facilitated the identification of
compounds that might inhibit SARS-CoV-2 M
Pro
. In 2005, computer-aided design facili-
tated the development of a Michael acceptor inhibitor, now known as N3, to target M
Pro
of SARS-like coronaviruses (249). N3 binds in the substrate binding pocket of M
Pro
in sev-
eral human CoVs (HCoVs) (249–252). The structure of N3-bound SARS-CoV-2 M
Pro
has
been solved, confirming the computational prediction that N3 would similarly bind in
the substrate binding pocket of SARS-CoV-2 (248). 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 (248). A library of
approximately 10,000 compounds was screened in a fluorescence resonance energy
transfer assay constructed using SARS-CoV-2 M
Pro
expressed in Escherichia coli (248).
Six leads were identified in this hypothesis-driven screen. In vitro analysis revealed
that ebselen had the strongest potency in reducing the viral load in SARS-CoV-2-
infected Vero cells (248). Ebselen is an organoselenium compound with anti-inflamma-
tory and antioxidant properties (253). Molecular dynamics analysis further demon-
strated the potential for ebselen to bind to M
Pro
and disrupt the protease’s enzymatic
functions (254). However, ebselen is likely to be a promiscuous binder, which could di-
minish its therapeutic potential (248, 255), and compounds with higher specificity may
be needed to translate this mechanism effectively to clinical trials. In July 2020, phase
2 clinical trials commenced to assess the effects of SPI-1005, an investigational drug
from Sound Pharmaceuticals that contains ebselen (256), on 60 adults presenting with
each of moderate (257) and severe (258) COVID-19. Other M
Pro
inhibitors are also being
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evaluated in clinical trials (259, 260). Pending the results of clinical trials, N3 remains a
computationally interesting compound based on both computational and experimen-
tal data, but whether these potential effects will translate to the clinic remains
unknown.
Hypothesis-free screening. Hypothesis-free screens use a discovery-driven
approach, where screens are not targeted to specific viral proteins, host proteins, or
desired clinical modulation. Hypothesis-free drug screening began 20 years ago with
the testing of libraries of drugs experimentally. Today, like many other areas of biology,
in silico analyses have become increasingly popular and feasible through advances in
biological big data (245, 261). Many efforts have collected data about interactions
between drugs and SARS-CoV-2 and about the host genomic response to SARS-CoV-2
exposure, allowing for hypothesis-free computational screens that seek to identify new
candidate therapeutics. Thus, they utilize a systems biology paradigm to extrapolate
the effect of a drug against a virus based on the host interactions with both the virus
and the drug (236).
Resources such as the COVID-19 Drug and Gene Set Library, which at the time of its
publication contained 1,620 drugs sourced from 173 experimental and computational
drug sets and 18,676 human genes sourced from 444 gene sets (262), facilitate such
discovery-driven approaches. Analysis of these databases indicated that some drugs
had been identified as candidates across multiple independent analyses, including
high-profile candidates such as CQ/HCQ and remdesivir (262). Computational screen-
ing efforts can then mine databases and other resources to identify potential PPIs
among the host, virus, and established and/or experimental drugs (263). Subject mat-
ter expertise from human users may be integrated to various extents depending on
the platform (e.g., references 263 and 264). These resources have allowed studies to
identify potential therapeutics for COVID-19 without an a priori reason for selecting
them.
One example of a hypothesis-free screen for COVID-19 drugs comes from a PPI net-
work-based analysis that was published early in the pandemic (265). Here, researchers
cloned the proteins expressed by SARS-CoV-2 in vitro and quantified 332 virus-host PPIs
using affinity purification mass spectrometry (265). They identified two SARS-CoV-2 pro-
teins (Nsp6 and Orf9c) that interacted with host Sigma-1 and Sigma-2 receptors. Sigma
receptors are located in the endoplasmic reticulum of many cell types, and type 1 and 2
Sigma receptors have overlapping but distinct affinities for a variety of ligands (266).
Molecules interacting with the Sigma receptors were then analyzed and found to have
an effect on viral infectivity in vitro (265). A follow-up study evaluated the effect of per-
turbing these 332 proteins in two cell lines, A549 and Caco-2, using knockdown and
knockout methods, respectively, and found that the replication of SARS-CoV-2 in cells
from both lines was dependent on the expression of SIGMAR1, which is the gene that
encodes the Sigma-1 receptor (267). Following these results, drugs interacting with
Sigma receptors were suggested as candidates for repurposing for COVID-19 (e.g., refer-
ence 268). Because many well-known and affordable drugs interact with the Sigma
receptors (265, 269), they became a major focus of drug repurposing efforts. Some of
the drugs suggested by the apparent success of Sigma receptor-targeting drugs were al-
ready being investigated at the time. HCQ, for example, forms ligands with both Sigma-1
and Sigma-2 receptors and was already being explored as a candidate therapeutic for
COVID-19 (265). Thus, this computational approach yielded interest in drugs whose anti-
viral activity was supported by initial in vitro analyses.
Follow-up research, however, called into question whether the emphasis on drugs
interacting with Sigma receptors might be based on a spurious association (270). This
study built on the prior work by examining whether antiviral activity among com-
pounds correlated with their affinity for the Sigma receptors and found that it did not.
The study further demonstrated that cationic amphiphilicity was a shared property
among many of the candidate drugs identified through both computational and phe-
notypic screens and that it was likely to be the source of many compounds’proposed
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antiviral activity (270). Cationic amphiphilicity is associated with the induction of phos-
pholipidosis, which is when phospholipids accumulate in the lysosome (271).
Phospholipidosis can disrupt viral replication by inhibiting lipid processing (272) (see
the discussion of HCQ in the Appendix). However, phospholipidosis is known to trans-
late poorly from in vitro models to in vivo models or clinical applications. Thus, this
finding suggested that these screens were identifying compounds that shared a physi-
ochemical property rather than a specific target (270). The authors further demon-
strated that antiviral activity against SARS-CoV-2 in vitro was correlated with the induc-
tion of phospholipidosis for drugs both with and without cationic amphiphilicity (270).
This finding supports the idea that the property of cationic amphicility was being
detected as a proxy for the shared effect of phospholipidosis (270). They demonstrated
that phospholipidosis-inducing drugs were not effective at preventing viral propaga-
tion in vivo in a murine model of COVID-19 (270). Therefore, removing hits that induce
phospholipidosis from computational and in vitro experimental repurposing screens
(e.g., reference 273) may help emphasize those that are more likely to provide clinical
benefits. This work illustrates the importance of considering confounding variables in
computational screens, a principle that has been incorporated into more traditional
approaches to drug development (274).
One drug that acts on Sigma receptors does, however, remain a candidate for the
treatment of COVID-19. Several psychotropic drugs target Sigma receptors in the central
nervous system and thus attracted interest as potential COVID-19 therapeutics following
the findings of two host-virus PPI studies (275). For several of these drugs, the in vitro
antiviral activity (267) was not correlated with their affinity for the Sigma-1 receptor (270,
275) but was correlated with phospholipidosis (270). However, fluvoxamine, a selective
serotonin reuptake inhibitor that is a particularly potent Sigma-1 receptor agonist (275),
has shown promise as a preventative of severe COVID-19 in a preliminary analysis of
data from the large-scale TOGETHER trial (145). As of 6 August 2021, this trial had col-
lected data from over 1,400 patients in the fluvoxamine arm of their study, half of whom
received a placebo (145). Only 74 patients in the fluvoxamine group had progressed to
hospitalization for COVID-19 compared to 107 in the placebo group, corresponding to a
relative risk of 0.69; additionally, the relative risk of mortality between the two groups
was calculated to be 0.71. These findings support the results of small clinical trials that
have found fluvoxamine to reduce clinical deterioration relative to a placebo (276, 277).
However, the ongoing therapeutic potential of fluvoxamine does not contradict the find-
ing that hypothesis-free screening hits can be driven by confounding factors. The
authors point out that its relevance would not just be antiviral as it has a potential
immunomodulatory mechanism (276). It has been found to be protective against septic
shock in an in vivo mouse model (278). It is possible that fluvoxamine also exerts an anti-
viral effect (279). Thus, Sigma-1 receptor activity may contribute to fluvoxamine’s poten-
tial effects in treating COVID-19, but it is not the only mechanism by which this drug can
interfere with disease progression.
Potential and limitations of high-throughput analyses. Computational screening
allows for a large number of compounds to be evaluated to identify those most likely
to display a desired behavior or function. This approach can be guided by a hypothesis
or can aim to discover underlying characteristics that produce new hypotheses about
the relationship between a host, a virus, and candidate pharmaceuticals. The examples
outlined above illustrate that HTS-based evaluations of drug repurposing can potentially
provide valuable insights. Computational techniques were used to design compounds
targeting M
Pro
based on an understanding of how this protease aids viral replication, and
M
Pro
inhibitors remain promising candidates (235), although the clinical trial data are not
yet available. Similarly, computational analysis correctly identified the Sigma-1 receptor
as a protein of interest. Although the process of identifying which drugs might modulate
the interaction led to an emphasis on candidates that ultimately have not been sup-
ported, fluvoxamine remains an appealing candidate. The difference between the pre-
liminary evidence for fluvoxamine compared to other drugs that interact with Sigma
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receptors underscores a major critique of hypothesis-free HTS in particular: while these
approaches allow for brute force comparison of a large number of compounds against a
virus of interest, they lose the element of expertise that is associated with most successes
in drug repurposing (245).
There are also practical limitations to these methods. One concern is that computa-
tional analyses inherently depend on the quality of the data being evaluated. The ur-
gency of the COVID-19 pandemic led many research groups to pivot toward computa-
tional HTS research without familiarity with best practices in this area (235). As a result,
there is an excessive amount of information available from computational studies
(280), but not all of it is high quality. Additionally, the literature used to identify and
validate targets can be difficult to reproduce (281), which may pose challenges to tar-
get-based experimental screening and to in silico screens. Some efforts to repurpose
antivirals have focused on host, rather than viral, proteins (236), which might be
expected to translate poorly in vivo if the targeted proteins serve essential functions in
the host. Concerns about the practicality of hypothesis-free screens to gain novel
insights are underscored by the fact that very few or possibly no success stories have
emerged from hypothesis-free screens over the past 20 years (245). These findings sug-
gest that data-driven research can be an important component of the drug repurpos-
ing ecosystem, but that drug repurposing efforts that proceed without a hypothesis,
an emphasis on biological mechanisms, or an understanding of confounding effects
may not produce viable candidates.
CONSIDERATIONS IN BALANCING DIFFERENT APPROACHES
The approaches described here offer a variety of advantages and limitations in
responding to a novel viral threat and building on existing bodies of knowledge in dif-
ferent ways. Medicine, pharmacology, basic science (especially virology and immunol-
ogy), and biological data science can all provide different insights and perspectives for
addressing the challenging question of which existing drugs might provide benefits
against an emerging viral threat. A symptom management-driven approach allows
clinicians to apply experience with related diseases or related symptoms to organize a
rapid response aimed at saving the lives of patients already infected with a new dis-
ease. Oftentimes, the pharmaceutical agents that are applied are small-molecule,
broad-spectrum pharmaceuticals that are widely available and affordable to produce,
and they may already be available for other purposes, allowing clinicians to administer
them to patients quickly either with an EUA or off-label. In this vein, dexamethasone
has emerged as the strongest treatment against severe COVID-19 (Table 1).
Alternatively, many efforts to repurpose drugs for COVID-19 have built on information
gained through basic scientific research of HCoV. Understanding how related viruses
function has allowed researchers to identify possible pharmacological strategies to dis-
rupt pathogenesis (Fig. 3). Some of the compounds identified through these methods
include small-molecule antivirals, which can be boutique and experimental medications
like remdesivir (Table 1). Other candidate drugs that intercept host-pathogen interac-
tions include biologics, which imitate the function of endogenous host compounds.
Most notably, several MAbs that have been developed (casirivimab, imdevimab, bamla-
nivimab, and etesevimab) or repurposed (sotrovimab and tocilizumab) have now been
granted EUAs (Table 1). Although not discussed here, several vaccine development pro-
grams have also met huge success using a range of strategies (2).
All of the small-molecule drugs evaluated and most of the biologics are repurposed,
and thus hinge on a theoretical understanding of how the virus interacts with a human
host and how pharmaceuticals can be used to modify those interactions rather than
being designed specifically against SARS-CoV-2 or COVID-19. As a result, significant
attention has been paid to computational approaches that automate the identification
of potentially desirable interactions. However, work in COVID-19 has made it clear that
relevant compounds can also be masked by confounding factors, and spurious associa-
tions can drive investment in candidate therapeutics that are unlikely to translate to
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TABLE 1 Summary table of candidate therapeutics examined in this paper
Treatment Grade
a
Category FDA status
b
Evidence available
c
Suggested effectiveness
d
Dexamethasone A Small molecule, broad
spectrum
Used off-label RCT Supported: RCT shows improved outcomes over the SOC,
especially in severe cases such as CRS
Remdesivir A Small molecule, antiviral,
adenosine analog
Approved for
COVID-19 (and
EUA for
combination with
baricitinib)
RCT Mixed: Conflicting evidence from large WHO-led Solidarity
trial vs U.S.-focused RCT and other studies
Tocilizumab A Biologic, monoclonal
antibody
EUA RCT Mixed: It appears that TCZ may work well in combination
with dexamethasone in severe cases, but not as
monotherapy
Sotrovimab NA Biologic, monoclonal
antibody
EUA RCT Supported: Phase 2/3 clinical trial showed reduced
hospitalization/death
Bamlanivimab and etesevimab B and NA Biologic, monoclonal
antibodies
EUA RCT Supported: Phase 2 clinical trial showed reduction in viral
load, but FDA pause recommended because it may be
less effective against the Delta variant
Casirivimab and imdevimab NA Biologic, monoclonal
antibodies
EUA RCT Supported: Reduced viral load at interim analysis
Fluvoxamine B Small-molecule, Sigma-1
receptor agonist
NA RCT Supported: Support from two small RCTs and preliminary
support from interim analysis of TOGETHER trial
SNG001 B Biologic, interferon None RCT Mixed: Support from initial RCT but no effect found in
WHO’s Solidarity trial
M
Pro
protease inhibitors NA Small molecule,
protease inhibitor
None Computational prediction,
in vitro studies
Unknown
ARBs and ACEIs C Small molecule, broad
spectrum
None Observational studies and
some RCTs
Not supported: Observational study retracted, RCTs suggest
no association
Favipiravir D Small molecule, antiviral,
nucleoside analog
None RCT Not supported: RCTs do not show significant improvements
for individuals taking this treatment, good safety profile
HCQ/CQ D Small molecule, broad
spectrum
None RCT Not supported, possibly harmful: Nonblinded RCTs showed
no improvement over the SOC, safety profile may be
problematic
Convalescent plasma transfusion D Biologic, polyclonal
antibodies
EUA RCT Mixed: Supported in small trials but not in large-scale
RECOVERY trial
Ivermectin D Small molecule, broad
spectrum
None RCT Mixed: Mixed results from small RCTs, major supporting RCT
now withdrawn, Preliminary results of large RCT
(TOGETHER trial) suggest no effect on emergency room
visits or hospitalization for COVID-19
a
“Grade”is the rating given to each treatment by the Systematic Tracker of Off-label/Repurposed Medicines Grades (STORM) maintained by the Center for Cytokine Storm Treatment & Laboratory (CSTL) at the University of
Pennsylvania (70). A grade of A indicates that a treatment is considered effective, a grade of B indicates that all or most RCTs have shown positive results, a grade of C indicates that RCT data are not yet available, and a grade of D
indicates that multiple RCTs have produced negative results. Treatments not in the STORM database are indicated as NA for not available.
b
FDA status is also provided where available.
c
The evidence available is based on the progression of the therapeutic through the pharmaceutical development pipeline, with RCTs as the most informative source of evidence.
d
The effectiveness is summarized based on the current available evidence; large trials such as RECOVERY and Solidarity trial are weighted heavily in this summary. This table was last updated on 20 August 2021. This table was last
updated on 20 August 2021.
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the clinic. Such spurious hits are especially likely to impact hypothesis-free screens.
However, hypothesis-free screens may still be able to contribute to the drug discovery
or repurposing ecosystem, assuming the computational arm of HTS follows the same
trends seen in its experimental arm. In 2011, a landmark study in drug discovery dem-
onstrated that although more new drugs were discovered using target-based rather
than phenotypic approaches, the majority of drugs with a novel molecular mechanism
of action (MMOA) were identified in phenotypic screens (282). This pattern applied
only to first-in-class drugs, with most follower drugs produced by target-based screen-
ing (239). These findings suggest that target-based drug discovery is more successful
when building on a known MMOA and that modulating a target is most valuable when
the target is part of a valuable MMOA (240). Building on this, many within the field sug-
gested that mechanism-informed phenotypic investigations may be the most useful
approach to drug discovery (238, 240, 242). As it stands, data-driven efforts to identify
patterns in the results of computational screens allowed researchers to notice the
shared property of cationic amphicility among many of the hits from computational
screening analyses (270). While easier said than done, efforts to fill in the black box
underlying computational HTS and recognize patterns among the identified com-
pounds aid in moving data-oriented drug repurposing efforts in this direction.
The unpredictable nature of success and failure in drug repurposing for COVID-19 thus
highlights one of the tenets of phenotypic screening: there are a lot of “unknown
unknowns,”and a promising mechanism at the level of an MMOA will not necessarily prop-
agate up to the pathway, cellular, or organismal level (238). Despite the fact that apparently
mechanistically relevant drugs may exist, identifying effective treatments for a new viral dis-
ease is extremely challenging. Targets of repurposed drugs are often nonspecific, meaning
that the MMOA can appear to be relevant to COVID-19 without a therapeutic or prophylac-
tic effect being observed in clinical trials. The difference in the current status of remdesivir
and favipiravir as treatments for COVID-19 (Table 1) underscores how difficult it is to predict
whether a specific compound will produce a desired effect, even when the mechanisms are
similar. Furthermore, the fact that many candidate COVID-19 therapeutics were ultimately
identified because of their shared propensity to induce phospholipidosis underscores how
challenging it can be to identify a mechanism in silico or in vitro that will translate to a suc-
cessful treatment. While significant progress has been made thus far in the pandemic, the
therapeutic landscape is likely to continue to evolve as more results become available from
clinical trials and as efforts to develop novel therapeutics for COVID-19 progress.
TOWARDS THE NEXT HCOV THREAT
Only very limited testing of candidate therapies was feasible during the SARS and
MERS epidemics, and as a result, few treatments were available at the outset of the
COVID-19 pandemic. Even corticosteroids, which were used to treat SARS patients,
were a controversial therapeutic prior to the release of the results of the large
RECOVERY trial. The scale and duration of the COVID-19 pandemic have made it possi-
ble to conduct large, rigorous RCTs such as RECOVERY, Solidarity, TOGETHER, and
others. As results from these trials have continued to emerge, it has become clear that
small clinical trials often produce spurious results. In the case of HCQ/CQ, the therapeu-
tic had already attracted so much attention based on small, preliminary (and in some
cases, methodologically concerning) studies that it took the results of multiple large
studies before attention began to be redirected to more promising candidates (283). In
fact, most COVID-19 clinical trials lack the statistical power to reliably test their hypoth-
eses (284, 285). In the face of an urgent crisis like COVID-19, the desire to act quickly is
understandable, but it is imperative that studies maintain strict standards of scientific
rigor (235, 274), especially given the potential dangers of politicization, as illustrated
by HCQ/CQ (286). Potential innovations in clinical trial structure, such as adaptable clin-
ical trials with master protocols (287) or the sharing of data among small clinical trials
(285) may help to address future crises and to bolster the results from smaller studies,
respectively.
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In the long term, new drugs specific for treatment of COVID-19 may also enter de-
velopment. 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. Crystal structures of the SARS-CoV-2
main protease have been resolved (248, 288). Much work remains to be done to deter-
mine 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. Such efforts are likely to prove valuable in managing future emergent
HCoV, just as research from the SARS and MERS pandemic has provided a basis for the
COVID-19 response.
APPENDIX
Dexamethasone. In order to understand how dexamethasone reduces inflammation,
it is necessary to consider the stress response 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
(289, 290). 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 (291). 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 (289). 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 alpha (TNF-
a
) and the stimulation of anti-inflammatory
cytokines such as IL-10, meaning that the stress response can regulate inflammatory
immune activity (290). Administration of dexamethasone has been found to correspond
to dose-dependent inhibition of IL-12 production, but not to affect IL-10 (292); the fact
that this relationship could be disrupted by administration of a glucocorticoid receptor
antagonist suggests that it is regulated by the receptor itself (292). 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 biological mechanism that reduces inflammation following a threat such
as immune challenge.
Initial support for dexamethasone as a treatment for COVID-19 came from the United
Kingdom’s RECOVERY trial (62), which assigned over 6,000 hospitalized COVID-19
patients to the standard of care (SOC) or treatment (dexamethasone) arms of the trial at a
2:1 ratio. At the time of randomization, some patients were ventilated (16%), others were
on noninvasive 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 endpoint was the patient’sstatusat28days
postrandomization (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 SOC group (21.6% versus 24.6%; P,0.001). However, the effect was driven
by improvements in patients receiving mechanical ventilation or supplementary oxygen.
One possible confounding factor 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 the conclusions, although the duration of symptoms was found to be
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significantly associated with the effect of dexamethasone administration. Thus, the
results of this large, randomized, and multisite, 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.
It is not surprising that administration of an immunosuppressant would be most
beneficial in severe cases where the immune system was dysregulated toward
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 (293). Additionally, the drug could potentially
slow viral clearance and inhibit patients’ability to develop antibodies to SARS-CoV-2 (59,
293), with the lack of data about viral clearance being put forward as a major limitation of
the RECOVERY trial (294). Furthermore, dexamethasone has been associated with side
effects that include psychosis, glucocorticoid-induced diabetes, and avascular necrosis
(59), and the RECOVERY trial did not report outcomes with enough detail to be able to
determine whether they observed similar complications. The effects of dexamethasone
have also been found to differ among populations, especially in high-income versus
middle- or low-income countries (295). However, since the RECOVERY trial’s results were
released, strategies have been proposed for administering dexamethasone alongside
more targeted treatments to minimize the likelihood of negative side effects (293). Given
the available evidence, dexamethasone is currently the most promising treatment for
severe COVID-19.
Favipiravir. The effectiveness of favipiravir for treating patients with COVID-19 is
under investigation. Evidence for the drug inhibiting viral RNA polymerase is based on
time-of-drug addition studies that found that viral loads were reduced with the addition
of favipiravir in early times postinfection (80, 83, 84). An open-label, nonrandomized,
before-after controlled study for COVID-19 was recently conducted (296). 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 although the
control patients received antivirals, two subsequent large-scale analyses, the WHO
Solidarity trial and the Randomized Evaluation of COVID-19 Therapy (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 (93, 297, 298). Treatment
was applied on days 2 to 14; treatment stopped either when viral clearance was
confirmed or on day 14. The efficacy of the treatment was measured first by the time
until viral clearance using Kaplan-Meier survival curves and second by 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 reverse transcription-PCR (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%) (P= 0.004). 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. Despite the study reporting clinical
improvement in favipiravir-treated patients, several study design issues are problematic
and lower confidence in the overall conclusions. For example, the study was neither
randomized nor carried out in a blind manner. Moreover, the selection of patients did not
take into consideration important factors such as previous clinical conditions or sex, and
there was no age categorization. Additionally, it should be noted that this study was
temporarily retracted and then restored without explanation (299).
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In late 2020 and early 2021, the first randomized controlled trials of favipiravir for the
treatment of COVID-19 released results (300–302). The first (300) used a randomized,
controlled, open-label design to compare two drugs, favipiravir and blaver marboxil, to
the SOC alone. Here, the SOC included antivirals such as lopinavir/ritonavir and was
administered to all patients. The primary endpoint analyzed was viral clearance at day
14. The sample size for this study was very small, with 29 total patients enrolled, and no
significant effect of the treatments was found for the primary outcome or any of the
secondary outcomes analyzed, which included mortality. The second study (301) was
larger, with 96 patients enrolled, and included only individuals with mild to moderate
symptoms who were randomized into two groups: one receiving chloroquine (CQ) in
addition to the SOC, and the other receiving favipiravir in addition to the SOC. This
study reported a nonsignificant trend for patients receiving favipiravir to have a shorter
hospital stay (13.29 days compared to 15.89 for CQ; P= 0.06) and less likelihood of
progressing to mechanical ventilation (P= 0.118) or to an oxygen saturation of ,90%
(P= 0.129). These results, combined with the fact that favipiravir was being compared
to CQ, which is now widely understood to be ineffective for treating COVID-19, thus do
not suggest that favipiravir was likely to have had a strong effect on these outcomes.
On the other hand, another trial of 60 patients reported a significant effect of favipiravir
on viral clearance at 4 days (a secondary endpoint), but not at 10 days (the primary
endpoint) (302). This study, as well as a prior study of favipiravir (303), also reported that
the drug was generally well tolerated. Thus, in combination, these small studies suggest
that the effects of favipiravir as a treatment for COVID-19 cannot be determined based
on the available evidence, but additionally, none raise major concerns about the safety
profile of the drug.
Remdesivir. At the outset of the COVID-19 pandemic, remdesivir did not have any
have any FDA-approved use. A clinical trial in the Democratic Republic of Congo found
some evidence of effectiveness against Ebola virus disease (EVD), but two antibody
preparations were found to be more effective, and remdesivir was not pursued (304).
Remdesivir also inhibits polymerase and replication of the coronaviruses MERS-CoV and
SARS-CoV-1 in cell culture assays with submicromolar 50% inhibitory concentrations (IC
50
s)
(305). It has also been found to inhibit SARS-CoV-2, showing synergy with CQ in vitro (88).
Remdesivir was first used on some COVID-19 patients under compassionate use
guidelines (306–308). All were in late stages of COVID-19 infection, and initial reports
were inconclusive about the drug’sefficacy. Gilead Sciences, the maker of remdesivir,
led a recent study 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 (92).
There were significant issues with the study design, or lack thereof. There was no
randomized control group. The inclusion criteria were variable: some patients required
only 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.
Eight patients were excluded from the analysis mainly due to missing postbaseline
information; thus, their health was 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
(309). These studies did not alter the understanding of the efficacy of remdesivir in
treating COVID-19, but the encouraging results provided motivation for placebo-
controlled studies.
The double-blind placebo-controlled ACTT-1 trial (89, 90) 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
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presentation at baseline (89). The treatment was 200 mg on day 1, followed by 100 mg
on days 2 through 10. Data were analyzed from a total of 1,059 patients who completed
the 29-day course of the trial, with 517 assigned to remdesivir and 508 to placebo (89).
The two groups were well matched demographically and clinically at baseline. Those
who received remdesivir had a median recovery time of 10 days, compared with
15 days in those who received placebo (rate ratio for recovery, 1.29; 95% confidence
interval [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 noninvasive
and invasive ventilation at randomization (89). Largely on the results of this trial, the
FDA reissued and expanded the EUA for remdesivir for the treatment of hospitalized
COVID-19 patients ages 12 and older (310). Additional clinical trials (88, 311–314) are
under way to evaluate the use of remdesivir to treat COVID-19 patients at both early
and late stages of infection and in combination with other drugs (Fig. 2). As of 22
October 2020, remdesivir received FDA approval based on three clinical trials (315).
However, results suggesting no effect of remdesivir on survival were reported by the
WHO Solidarity trial (93). Patients were randomized in equal proportions into four
experimental conditions and a control condition, corresponding to four candidate
treatments for COVID-19 and the SOC, respectively; no placebo was administered. The
2,750 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 assessed for in-hospital
death (primary endpoint), duration of hospitalization, and progression to mechanical
ventilation. There were also 2,708 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 died during initial hospitalization, with 301 in the
remdesivir group and 303 in the control group. The rate ratio of death between these
two groups was therefore not significant (0.95; 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 reference 89) stratified
by supplemental oxygen status. A meta-analysis of the four studies yielded an overall rate
ratio for death of 0.91 (P= 0.20). 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 Solidaritytrial, Gilead, which manufactures remdesivir,
released a statement pointing to the fact that the Solidarity trial was not placebo
controlled or double blind and at the time of release, the statement had not been peer
reviewed (316); these sentiments have been echoed elsewhere (317). 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 cases
rather than severe cases (298, 318). However, the publication associated with the trial
sponsored by Gilead did purport 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) (89). Additionally, a smaller analysis of 598 patients, of whom two-thirds were
randomized to receive remdesivir for either 5 or 10 days, reported a small effect of
treatment with remdesivir for 5 days relative to the standard of care in patients with
moderate COVID-19 (319). These results suggest that remdesivir could improve outcomes
for patients with moderate COVID-19 but that additional information would be needed
to understand the effects of different durations of treatment. Therefore, the Solidarity trial
may point to limitations in the generalizability of other research on remdesivir, especially
since the broad international nature of the Solidarity clinical trial, which included
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countries with a wide range of economic profiles and a variety of health care systems,
provides a much-needed global perspective in a pandemic (298). On the other hand, only
62% of patients in the Solidarity trial were randomized on the day of admission or 1 day
afterwards (93), and concerns have been raised that differences in disease progression
could influence the effectiveness of remdesivir (298). Despite the findings of the
Solidarity trial, remdesivir remains available for the treatment of COVID-19 in many
places. Remdesivir has also been investigated in combination with other drugs, such as
baricitinib, which is an inhibitor of Janus kinase 1 and 2 (320); the FDA has issued an EUA
for the combination of remdesivir and baricitinib in adult and pediatric patients (321).
Follow-up studies are needed and, in many cases, are under way to further investigate
remdesivir-related outcomes.
Similarly, the extent to which the remdesivir dosing regimen could influence outcomes
continues to be under consideration. A randomized, open-label trial compared the effect of
remdesivir on 397 patients with severe COVID-19 over 5 versus 10 days (91, 309),
complementing the study that found that a 5-day course of remdesivir improved outcomes
for patients with moderate COVID-19 but a 10-day course did not (319). Patients in the two
groups were administered 200 mg of remdesivir intravenously on the first day, followed by
100 mg on the subsequent 4 or 9 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. It reported no significant differences across
several outcomes for patients receiving a 5-day or 10-day course, when correcting for
baseline clinical status. The data did suggest 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 (309). Thus, additional research is also
required to determine whether the dosage and duration of remdesivir administration
influences outcomes.
In summary, remdesivir is the first FDA-approved antiviral against SARS-CoV-2 as
well as the first FDA-approved COVID-19 treatment. Early investigations of this drug
established proof of principle that drugs targeting the virus can benefit COVID-19
patients. 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 human immunodeficiency
virus (HIV) and herpes. Notably, the HIV and herpesvirus 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. Remdesivir provides evidence that a related approach may
be beneficial for the treatment of COVID-19.
Hydroxychloroquine and chloroquine. Chloroquine (CQ) and hydroxychloroquine
(HCQ) increase cellular pH by accumulating in their protonated form inside lysosomes
(95, 322). This shift in pH inhibits the breakdown of proteins and peptides by the
lysosomes during the process of proteolysis (95). 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 ACE2 (96). 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 (97). Additionally, an early case study of three COVID-19
patients reported the presence of antiphospholipid antibodies in all three patients
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(323). Antiphospholipid antibodies are central to the diagnosis of the antiphospholipid
syndrome, a disorder that HCQ has often been used to treat (324–326). Because the
90% effective concentration (EC
90
) of CQ in Vero E6 cells (6.90
m
M) can be achieved in
and tolerated by rheumatoid arthritis (RA) patients, it was hypothesized that it might
also be possible to achieve the effective concentration in COVID-19 patients (327).
Additionally, clinical trials have reported HCQ to be effective in treating HIV (328) and
chronic hepatitis C (329). 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, and
trials often investigated these drugs in combination with azithromycin (AZ) and/or zinc
supplementation. However, as more evidence has emerged, it has become clear that
HCQ/CQ offer no benefits against SARS-CoV-2 or COVID-19.
(i) Trials assessing therapeutic administration of HCQ/CQ. The initial study
evaluating HCQ as a treatment for COVID-19 patients was published on 20 March 2020
by Gautret et al. (98). This nonrandomized, nonblinded, nonplacebo clinical trial
compared HCQ to the SOC in 42 hospitalized patients in southern France. It reported
that patients who received HCQ showed higher rates of virological clearance by
nasopharyngeal swab on days 3 to 6 compared to the SOC. This study also treated six
patients with both HCQ plus 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 small sample size and the
lack of the following: randomization, blinding, placebo (no “placebo pill”given to the
SOC group), intention-to-Treat analysis, correction for sequential multiple comparisons,
and trial preregistration. Furthermore, the trial arms were entirely confounded by the
hospital, and there were false-negative outcome measurements (see reference 330).
Two of these weaknesses are due to inappropriate data analysis and can therefore be
corrected post hoc by recalculating the Pvalues (lack of intention-to-treat analysis and
multiple comparisons). However, all other weaknesses are fundamental design flaws
and cannot be corrected for. Thus, the conclusions cannot be generalized outside the
study. The International Society of Antimicrobial Chemotherapy, the scientific
organization that publishes the journal where the article appeared, subsequently
announced that the article did not meet its expected standard for publications (99),
although it has not been officially retracted.
Because of the preliminary data presented in this study, HCQ treatment was
subsequently explored by other researchers. About 1 week later, a follow-up case study
reported that 11 consecutive patients were treated with HCQ plus AZ using the same
dosing regimen (331). One patient died, two were transferred to the intensive care unit
(ICU), and one developed a prolonged QT interval, leading to discontinuation of HCQ-
plus-AZ administration. As in the Gautret et al. study (98), the outcome assessed was
virological clearance at day 6 posttreatment, as measured from nasopharyngeal swabs.
Of the 10 living patients on day 6, 8 remained positive for SARS-CoV-2 RNA. As 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 plus AZ tested negative for SARS-
CoV-2 RNA by day 6 posttreatment. This case study illustrated the need for further
investigation using robust study design to evaluate the efficacy of HCQ and/or CQ.
On 10 April 2020, the results of a randomized, nonplacebo trial of 62 COVID-19
patients at the Renmin Hospital of Wuhan University were released (332). This study
investigated whether HCQ decreased time to fever break or time to cough relief
compared to the SOC (332). 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 the study of Gautret et al. (98) 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
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sufficient detail to understand analysis, drastic disparities between preregistration (333)
and published protocol (including differences in the inclusion and exclusion criteria,
number of experimental groups, number of patients enrolled, and outcome analyzed),
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 nonplacebo 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 Pvalues, but all of the other issues
were design and statistical weaknesses that cannot be corrected for. Additionally,
disparities between the preregistered and published protocols raise concerns about
experimental design. The design limitations mean that the conclusions cannot be
generalized outside 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 the SOC (334). 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. The small sample size is one weakness, 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 also have issues with data integrity and/or
provenance. A Letter to the Editor published in BioScience Trends on 16 March 2020
claimed that numerous clinical trials have shown that HCQ is superior to control
treatment in inhibiting the exacerbation of COVID-19 pneumonia (335). This letter has
been cited by numerous papers in the primary literature, review articles, and media
alike (336, 337). However, the letter referred to 15 preregistration 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 preregistrations. 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 (338) 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 and
HCQ with and without a macrolide). They concluded that treatment with CQ or 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 (339). These studies demonstrate that increased skepticism in evaluation of
the HCQ/CQ and COVID-19 literature may be warranted, possibly because of the
significant attention HCQ and CQ have received as possible treatments for COVID-19 and
the politicization of these drugs.
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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 (340). A prolonged QT interval is associated with
ventricular arrhythmia (341). Furthermore, a separate study (342) 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 plus AZ. The results indicated that short-term use of HCQ was not associated
with additional risk but that HCQ plus AZ was associated with an enhanced risk of
cardiovascular complications (such as a 15% increased risk of chest pain, calibrated
HR of 1.15 and 95% CI of 1.05 to 1.26) and a twofold increased 30-day risk of
cardiovascular mortality (calibrated HR of 2.19; 95% CI, 1.22 to 3.94). 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 have led to the removal of CQ/HCQ from the SOC practices in
several countries (343, 344). As of 25 May 2020, WHO had suspended administration of
HCQ as part of the worldwide Solidarity Trial (345), 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).
Additional research has emerged largely identifying HCQ/CQ to be ineffective
against COVID-19 while simultaneously revealing a number of significant side effects. A
randomized, open-label, nonplacebo 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 (346). The trial compared treatment with HCQ in conjunction with
the SOC to the 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 (estimated difference between SOC plus HCQ and
SOC, 4.1%; 95% CI, –10.3% to 18.5%). The secondary endpoints were an amelioration of
the symptoms of the disease such as axillary temperature #36.6°C, pulse oximeter
blood oxygen saturation reading (SpO
2
)of.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 on HCQ plus SOC
versus 21 days on SOC; P= 0.97). Additionally, 30% of the patients receiving the SOC
plus HCQ reported adverse outcomes compared to 8.8% of patients receiving only the
SOC, with the most common adverse outcome in the SOC-plus-HCQ group being
diarrhea (10% versus 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 years. The SOC in this
study included the use of antivirals (lopinavir-ritonavir, arbidol, oseltamivir, virazole,
entecavir, ganciclovir, and interferon alfa), which the authors note could influence the
results. Thus, they note that an ideal SOC would need to exclude the use of antivirals,
but that ceasing antiviral treatment raised ethical concerns at the time that the study
was conducted. 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-negative results (e.g.,
reference 347). 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
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against COVID-19. This study provided no support for HCQ against COVID-19, as there
was no difference between the two groups in either negative seroconversion at 28 days
or symptom alleviation, and in fact, more severe adverse outcomes were reported in
the group receiving HCQ.
Additional evidence comes from a retrospective analysis (348) that examined data
from 368 COVID-19 patients across all U.S. Veteran Health Administration medical
centers. The study retrospectively investigated the effect of the administration of HCQ
(n= 97), HCQ plus 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 analysis included only men, and the median age was
65 years. The rate of death was 27.8% in the HCQ-only treatment group, 22.1% in the
HCQ-plus-AZ treatment group, and 14.1% in the no-HCQ group. 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-plus-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 of 1.43 [P= 0.48] [HCQ] and
0.43 [P= 0.09] [HCQ plus 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 plus AZ is safer than HCQ contradicts the findings
of the previous large-scale analysis of 20 years of records that found HCQ plus AZ to be
more frequently associated with cardiac arrhythmia than HCQ alone (342); 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 RECOVERY trial were released on 8 October 2020. This
study used a randomized, open-label design to study the effects of HCQ compared to
the SOC in 11,197 patients at 176 hospitals in the United Kingdom (100). 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, and 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 preexisting conditions (56%
versus 57% overall). In the HCQ arm of the study, patients received 800 mg at baseline
and again after 6 h and then 400 mg at 12 h and every subsequent 12 h. 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.
COVID-19-related mortality was not affected by HCQ in the RECOVERY trial (rate ratio,
1.09; 95% CI, 0.97 to 1.23; P= 0.15), but cardiac events were increased in the HCQ arm
(0.4 percentage points), as was the duration of hospitalization (rate ratio for discharge
alive within 28 days, 0.90; 95% CI, 0.83 to 0.98) and likelihood of progression to
mechanical ventilation or death (risk ratio, 1.14; 95% CI, 1.03 to 1.27). 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. Therefore, though none of the studies have been conducted in a blind
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manner, examining them together makes it clear that the available evidence points to
significant dangers associated with the administration of HCQ to hospitalized COVID-19
patients, without providing any support for its efficacy.
(ii) HCQ for the treatment of mild cases. One additional possible therapeutic
application of HCQ considered was the treatment of mild COVID-19 cases in otherwise
healthy individuals. This possibility was assessed in a randomized, open-label, multicenter
analysis conducted in Catalonia (Spain) (349). This analysis enrolled adults 18 and older
who had been experiencing mild symptoms of COVID-19 for fewer than 5 days.
Participants were randomized into an HCQ arm (n=136)andacontrolarm(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 6 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. No significant differences between the two groups were found: the difference
in viral load between the HCQ and control groups was 0.01 (95% CI, 20.28 to 0.29) at day
3and20.07 (95% CI, 20.44 to 0.29) at day 7, the relative risk of hospitalization was 0.75
(95% CI, 0.32 to 1.77), and the difference in time to complete resolution of symptoms was
22days(P= 0.38). This study thus suggests that HCQ does not improve recovery from
COVID-19, even in otherwise healthy adult patients with mild symptoms.
(iii) 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 (350). Asymptomatic adults
in the United States and Canada who had been exposed to SARS-CoV-2 within the past 4
days were enrolled in an online study to evaluate whether administration of HCQ over 5
days influenced the probability of developing COVID-19 symptoms over a 14-day period.
Of the participants, 414 received HCQ and 407 received a placebo. No significant
difference in the rate of symptomatic illness was observed between the two groups
(11.8% for HCQ, 14.3% for placebo; P= 0.35). The HCQ condition was associated withside
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 health care 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
confounding factor 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, 4 of the 20 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 years, 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 overrepresented 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 prevention after SARS-CoV-2 exposure 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, once again no
evidence was found suggesting an effect of HCQ against COVID-19.
A second study (351) examined the effect of administering HCQ to health care
workers as a preexposure 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 a randomized, double-blind, and placebo-controlled
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study, and it sought to address some of the limitations of the first prophylactic study.
The goal was to enroll 200 health care 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 years) and made up largely of people who were white, women, and without
preexisting conditions. At the second interim analysis, more individuals in the
treatment group than the control group had contracted COVID-19 (four versus three),
causing the estimated z-score to fall below the preestablished threshold for futility. As a
result, the trial was terminated early, offering additional evidence against the use of
HCQ for prophylaxis.
(iv) 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
significant media attention and public interest in its potential as both a therapeutic and
prophylactic. Initially, it was hypothesized that CQ/HCQ might be effective against
SARS-CoV-2 in part because 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 proinflammatory cytokines (352). 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, research has not found support for a positive
effect of HCQ on COVID-19 patients. 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-positive results than false-
negative results), initial optimism about HCQ has largely dissipated. The most
methodologically rigorous analysis of HCQ as a prophylactic (350) 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 (93). Thus, HCQ/CQ are not likely to
be effective therapeutic or prophylactic agents against COVID-19. One case study
identified drug-induced phospholipidosis as the cause of death for a COVID-19 patient
treated with HCQ (272), suggesting that in some cases, the proposed mechanism of
action may ultimately be harmful. Additionally, one study identified an increased risk of
mortality in older men receiving HCQ, and administration of HCQ and HCQ plus AZ did
not decrease the use of mechanical ventilation in these patients (348). HCQ use for
COVID-19 could also lead to shortages for antimalarial or antirheumatic use, where it
has documented efficacy. Despite significant early attention, these drugs appear to be
ineffective against COVID-19. Several countries have now removed CQ/HCQ from their
SOC for COVID-19 due to the lack of evidence of efficacy and the frequency of adverse
effects.
ACE inhibitors and angiotensin II receptor blockers. Several clinical trials testing
the effects of ACEIs or ARBs on COVID-19 outcomes are ongoing (353–359). Clinical
trials are needed because the findings of the various observational studies bearing on
this topic cannot be interpreted as indicating a protective effect of the drug (360, 361).
Two analyses (353, 359) have reported no effect of continuing or discontinuing ARBs
and ACEIs on patients admitted to the hospital for COVID-19. The first, known as
REPLACE COVID (156), was a randomized, open-label study that enrolled patients who
were admitted to the hospital for COVID-19 and were taking an ACEI at the time of
admission. They enrolled 152 patients at 20 hospitals in seven countries and
randomized them into two arms, continuation (n= 75) and discontinuation (n= 77).
The primary outcome evaluated was a global rank score that integrated several
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dimensions of illness. The components of this global rank score, such as time to death
and length of mechanical ventilation, were evaluated as secondary endpoints. This
study reported no differences between the two groups in the primary outcome or any
of the secondary outcomes.
Similarly, a second study (157) used a randomized, open-label design to examine the
effects of continuing versus discontinuing ARBs and ACEIs on patients hospitalized for
mild to moderate COVID-19 at 29 hospitals in Brazil. This study enrolled 740 patients
but had to exclude one trial site from all analyses due to the discovery of violations of
Good Clinical Trial practice and data falsification. After this exclusion, 659 patients
remained, with 334 randomized to discontinuation and 325 to continuation. In this
study, the primary endpoint analyzed was the number of days that patients were alive
and not hospitalized within 30 days of enrollment. The secondary outcomes included
death (including in-hospital death separately), number of days hospitalized, and
specific clinical outcomes such as heart failure or stroke. Once again, no significant
differences were found between the two groups. Initial studies of randomized
interventions therefore suggest that ACEIs and ARBs are unlikely to affect COVID-19
outcomes. These results are also consistent with findings from observational studies
(summarized in reference 156). Additional information about ACE2, observational studies
of ACEIs and ARBs in COVID-19, and clinical trials on this topic have been summarized
(362). Therefore, despite the promising potential mechanism, initial results have not
provided support for ACEIs and ARBs as therapies for COVID-19.
Tocilizumab. 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-6 receptor alpha [IL-6R
a
] or glycoprotein 80 [gp80]) that
exists in both membrane-bound (IL-6R
a
) and soluble (sIL-6R
a
) forms (363). 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-6R
b
or gp130
(363, 364). Unlike membrane-bound IL-6R
a
, which is found only on hepatocytes and
some types of leukocytes, gp130 is found on most cells (365). When IL-6 binds to sIL-
6R
a
, the complex can then bind to a gp130 protein on any cell (365). The binding of IL-
6 to IL-6R
a
is termed classical signaling, while its binding to sIL-6R
a
is termed trans-
signaling (365–367). 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 (365). Similarly, IL-6 is
known to play a role in Crohn’s disease via trans-signaling, but not classical signaling
(365). Both classical and trans-signaling can occur through three independent
pathways: the Janus-activated kinase-STAT3 pathway, the Ras/mitogen-activated
protein kinase pathway and the phosphoinositol-3 kinase/Akt pathway (363). These
signaling pathways are involved in a variety of different functions, including cell type
differentiation, immunoglobulin synthesis, and cellular survival signaling pathways,
respectively (363). The ultimate result of the IL-6 cascade is to direct transcriptional
activity of various promoters of proinflammatory cytokines, such as IL-1, TFN, and even
IL-6 itself, through the activity of NF-
k
B (363). IL-6 synthesis is tightly regulated both
transcriptionally and posttranscriptionally, 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 (368).
Therefore, drugs inhibiting the binding of IL-6 to IL-6R
a
or sIL-6R
a
are of interest for
combating the hyperactive inflammatory response characteristic of cytokine release
syndrome (CRS) and cytokine storm syndrome (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. Interest in TCZ as a possible treatment for
COVID-19 was piqued by early evidence indicating that COVID-19 deaths may be
induced by the hyperactive immune response, often referred to as CRS or CSS (170), as
IL-6 plays a key role in this response (369). The observation of elevated IL-6 in patients
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who died relative to those who recovered (170) could reflect an overproduction of
proinflammatory interleukins, suggesting that TCZ could potentially palliate some of
the most severe symptoms of COVID-19 associated with increased cytokine production.
This early interest in TCZ as a possible treatment for COVID-19 was bolstered by 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 (176). 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 analyses released thus far (171), consisting of 1,351
patients admitted to several care centers in Italy, compared the rates at which patients
who received TCZ died or progressed to invasive medical ventilation over a 14-day
period compared to patients receiving only the SOC. Under this definition, the SOC
could include other drugs such as HCQ, azithromycin, lopinavir-ritonavir, 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 (sequential organ failure assessment) score, they found that patients
treated with TCZ were less likely to progress to invasive medical ventilation and/or
death (adjusted HR = 0.61, 95% CI 0.40 to 0.92, P= 0.020); the results of analysis of
death and ventilation separately suggest that this effect may have been driven by
differences in the death rate (20% of control versus 7% of TCZ-treated patients). The
study reported particular benefits for patients whose PaO
2
/FiO
2
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, United States, where 64% of all 239 COVID-19
patients in the study period were administered TCZ based on assignment by a
standardized algorithm (172). 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 (173). This study compared COVID-19 patients admitted to the hospital
before and after 13 March 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 average age
of 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, 95% CI = 0.004
to 0.347, P= 0.004). The study reported improvement of survival outcomes after the
addition of TCZ to the SOC regime, with 11 of 23 patients (47.8%) admitted prior to
March 13th dying compared to 2 of 62 (3.2%) admitted afterwards (HR = 0.035, 95% CI,
0.004 to 0.347, P= 0.004). 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
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disease likely improved over this time frame 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.
Other retrospective analyses have utilized a 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 intensive care unit (ICU) admission or death (174). 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 the 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 the 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 (175). 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. In general, caution
must be exercised when interpreting subgroup analyses after a primary combined
endpoint analysis. These last findings highlight the need to search for a balance
between impairing a harmful immune response, such as the one generated during CRS/
CSS, and preventing the worsening of the clinical picture of the patients by potential
new viral infections. Early meta-analyses and systematic reviews have investigated the
available data about TCZ for COVID-19. One meta-analysis (370) evaluated 19 studies
published or released as preprints prior to 1 July 2020 and found that the overall trends
were supportive of the frequent conclusion that TCZ does improve survivorship, with a
significant HR of 0.41 (P,0.001). This trend improved when they excluded studies that
administered a steroid alongside TCZ, with a significant HR of 0.04 (P,0.001). They
also found some evidence for reduced invasive ventilation or ICU admission, but only
when excluding all studies except a small number whose estimates were adjusted for
the possible bias introduced by the challenges of stringency during the enrollment
process. A systematic analysis of 16 case-control studies of TCZ estimated an odds ratio
of mortality of 0.453 (95% CI, 0.376 to 0.547;, P,0.001), suggesting possible benefits
associated with TCZ treatment (371). 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 potential biases in publication. A different systematic
review of studies investigating TCZ treatment for COVID-19 analyzed 31 studies that
had been published or released as preprints and reported that none carried a low risk of
bias (372). Therefore, the present evidence is not likely to be sufficient for conclusions
about the efficacy of TCZ.
On 11 February 2021, a preprint describing the first randomized control trial of TCZ
was released as part of the RECOVERY trial (177). Of the 21,550 patients enrolled in the
RECOVERY trial at the time, 4,116 adults hospitalized with COVID-19 across the 131 sites
in the United Kingdom were assigned to the arm of the trial evaluating the effect of
TCZ. Of these patients, 2,022 were randomized to receive TCZ and 2,094 were
randomized to receive the SOC, with 79% of patients in each group available for
analysis at the time that the initial report was released. The primary outcome measured
was 28-day mortality, and TCZ was found to reduce 28-day mortality from 33% of
patients receiving the SOC alone to 29% of those receiving TCZ, corresponding to a rate
ratio of 0.86 (95% CI, 0.77 to 0.96; P= 0.007). TCZ was also significantly associated with
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the probability of hospital discharge within 28 days for living patients, which was 47%
in the SOC group and 54% in the TCZ group (rate ratio, 1.22; 95% CI, 1.12 to 1.34; P,
0.0001). A potential statistical interaction between TCZ and corticosteroids was
observed, with the combination providing greater mortality benefits than TCZ alone,
but the authors note that caution is advisable in light of the number of statistical tests
conducted. Combining the RECOVERY trial data with data from seven smaller
randomized control trials indicates that TCZ is associated with a 13% reduction in 28-
day mortality (rate ratio, 0.87; 95% CI, 0.79 to 0.96; P= 0.005) (177).
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 (RA) (373), and a recent
study found the drug to be safe for pregnant and breastfeeding women (374).
However, TCZ may increase the risk of developing infections (373), and RA 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 (375). As a result, TCZ is
contraindicated in patients with active infections such as tuberculosis (376). Previous
studies have investigated, with various results, a possible increased risk of infection in
RA patients administered TCZ (377, 378), although another study reported that the
incidence rate of infections was higher in clinical practice RA patients treated with TCZ
than in the rates reported by clinical trials (379). 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 (P,0.0001) (171).
Reactivation of hepatitis B virus 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 (380), 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 (172). An increased risk of bacterial
infection was also identified in a systematic review of the literature, based on the
unadjusted estimates reported (370). In the RECOVERY trial, however, only 3 out of
2,022 participants in the group receiving TCZ developed adverse reactions determined
to be associated with the intervention, and no excess deaths were reported (177). TCZ
administration to COVID-19 patients is not without risks and may introduce additional
risk of developing secondary infections; however, while caution may be prudent when
treating patients who have latent viral infections, the results of the RECOVERY trial
indicate that adverse reactions to TCZ are very rare among COVID-19 patients broadly.
In summary, approximately 33% of hospitalized COVID-19 patients develop ARDS
(381), which is caused by an excessive early response of the immune system which
can be a component of CRS/CSS (172, 376). This overwhelming inflammation is
triggered by IL-6. TCZ is an inhibitor of IL-6 and therefore may neutralize the
inflammatory pathway that leads to the cytokine storm. The mechanism suggests
TCZ could be beneficial for the treatment of COVID-19 patients experiencing
excessive immune activity, and the RECOVERY trial reported a reduction in 28-day
mortality. Interest in TCZ as a treatment for COVID-19 was also supported by two
meta-analyses (370, 382), but a third meta-analysis found that all of the available
literature at that time carried a risk of bias (372). Additionally, different studies used
different dosages, number of doses, and methods of administration. Ongoing
research may be needed to optimize administration of TCZ (383), although similar
results were reported by one study for intravenous and subcutaneous administration
(171). 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.
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Interferons. IFNs are a family of cytokines critical to activating the innate immune
response against viral infections. Interferons are classified into three categories based
on their receptor specificity: types I, II, and III (369). Specifically, IFNs I (alpha interferon
[IFN-
a
] and beta interferon [IFN-
b
]) and II (gamma interferon [IFN-
g
]) induce the
expression of antiviral proteins (384). Among these IFNs, IFN-
b
has already been found
to strongly inhibit the replication of other coronaviruses, such as SARS-CoV-1, in cell
culture, while IFN-
a
and -
g
were shown to be less effective in this context (384). There is
evidence that patients with higher susceptibility to ARDS indeed show deficiency in
IFN-
b
. For instance, infection with other coronaviruses impairs IFN-
b
expression and
synthesis, allowing the virus to escape the innate immune response (385). On 18 March
2020, Synairgen plc received approval to start a phase 2 trial for SNG001, an IFN-
b
-1a
formulation to be delivered to the lungs via inhalation (184). SNG001, which contains
recombinant interferon
b
-1a, was previously shown to be effective in reducing viral
load in an in vivo model of swine flu and in vitro models of other coronavirus infections
(386). In July 2020, a press release from Synairgen stated that SNG001 reduced
progression to ventilation in a double-blind, placebo-controlled, multicenter study of
101 patients with an average age in the late 50s (185). These results were subsequently
published in November 2020 (186). The study reports that the participants were
assigned at a ratio of 1:1 to receive either SNG001 or a placebo that lacked the active
compound, by inhalation for up to 14 days. The primary outcome they assessed was the
change in patients’score on the WHO Ordinal Scale for Clinical Improvement (OSCI) on
trial day 15 or 16. SNG001 was associated with an odds ratio of improvement on the
OSCI scale of 2.32 (95% CI, 1.07 to 5.04; P= 0.033) in the intention-to-treat analysis and
2.80 (95% CI, 1.21 to 6.52; P= 0.017) in the per-protocol analysis, corresponding to
significant improvement in the SNG001 group on the OSCI on day 15 or 16. Some of the
secondary endpoints analyzed also showed differences: on day 28, the odds ratio (OR)
for clinical improvement on the OSCI was 3.15 (95% CI, 1.39 to 7.14; P= 0.006), and the
odds of recovery on day 15/16 and on day 28 were also significant between the two
groups. Thus, this study suggested that IFN-
b
1 administered via SNG001 may improve
clinical outcomes.
In contrast, the WHO Solidarity trial reported no significant effect of IFN-
b
-1a on
patient survival during hospitalization (93). Here, the primary outcome analyzed was in-
hospital mortality, and the rate ratio for the two groups was 1.16 (95% CI, 0.96 to 1.39; P=
0.11) administering IFN-
b
-1a to 2,050 patients and comparing their response to 2,050
controls. However, there are a few reasons that the different findings of the two trials
might not speak to the underlying efficacy of this treatment strategy. One important
consideration is the stage of COVID-19 infection analyzed in each study. The Synairgen
trial enrolled only patients who were not receiving invasive ventilation, corresponding to
a less severe stage of disease than many patients enrolled in the Solidarity trial, as well as
a lower overall rate of mortality (387). Additionally, the method of administration differed
between the two trials, with the Solidarity trial administering IFN-
b
-1a subcutaneously
(387). The differences in findings between the studies suggest that the method of
administration might be relevant to outcomes, with nebulized IFN-
b
-1a more directly
targeting receptors in the lungs. A trial that analyzed the effect of subcutaneously
administered IFN-
b
-1a on patients with ARDS between 2015 and 2017 had also reported
no effect on 28-day mortality (388), while a smaller study analyzing the effect of
subcutaneous IFN administration did find a significant improvement in 28-day mortality for
COVID-19 (389). At present, several ongoing clinical trials are investigating the potential
effects of IFN-
b
-1a, including in combination with therapeutics such as remdesivir (390)
and administered via inhalation (184). Thus, as additional information becomes available, a
more detailed understanding of whether and under which circumstances IFN-
b
-1a is
beneficial to COVID-19 patients should develop.
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
Rando et al.
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benefit of dexamethasone and the ongoing potential of tocilizumab for treatment of
COVID-19 suggest that other anti-inflammatory agents might also hold value for the
treatment of COVID-19. Current evidence supporting the treatment of severe COVID-19
with dexamethasone suggests that the need to curtail the cytokine storm inflammatory
response transcends the risks of immunosuppression, and other anti-inflammatory
agents may therefore benefit patients in this phase of the disease. 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 RA patients, the cost for tocilizumab ranges from
$179.20 to $896 per dose for the intravenous (IV) form and $355 for the prefilled syring
(391). Several other anti-inflammatory agents used for the treatment of autoimmune
diseases may also be able to counter the effects of the cytokine storm induced by the
virus, and some of these, such as cyclosporine, are likely to be more cost-effective and
readily available than biologics (392). While tocilizumab targets IL-6, several other
inflammatory markers could be potential targets, including TNF-
a
. Inhibition of TNF-
a
by
a compound such as etanercept was previously suggested for treatment of SARS-CoV-1
(393) and may be relevant for SARS-CoV-2 as well. Another anti-IL-6 antibody, sarilumab,
is also being investigated (394, 395). 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 (396). Baricitinib, in particular,
may be able to reduce the ability of SARS-CoV-2 to infect lung cells (397). Clinical trials
studying baricitinib in COVID-19 have already begun in the United States and in Italy
(398, 399). 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, which are most beneficial late in
disease progression, much research is focused on identifying therapeutics for early
stage patients. For example, although studies of HCQ 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 (400). 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
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 (401). 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 (402). 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.
ID and Development of Therapeutics for COVID-19
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Some research is also investigating potential therapeutics and prophylactics that
would interact with components of the innate immune response. For example, TLRs are
pattern recognition receptors that recognize pathogen- and damage-associated molecular
patterns and contribute to innate immune recognition and, more generally, promotion of both
the innate and adaptive immune responses (403). 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 (404). Therefore, TLR agonists hold some
potential for broad-spectrum prophylaxis.
ACKNOWLEDGMENTS
James Brian Byrd was funded by FastGrants to conduct a COVID-19-related clinical
trial. Christian Brueffer is an employee and shareholder of SAGA Diagnostics AB. YoSon
Park is affiliated with Pfizer Worldwide Research. The author has no financial interests to
declare and contributed as an author prior to joining Pfizer, and the work was not part
of a Pfizer collaboration nor was it funded by Pfizer. Simina M. Boca is currently an
employee at AstraZeneca, Gaithersburg, MD, USA, and may own stock or stock options;
work was initially conducted at Georgetown University Medical Center, with writing,
reviewing, and editing continued while working at AstraZeneca. Anthony Gitter filed a
patent application with the Wisconsin Alumni Research Foundation related to classifying
activated T cells. The other authors declare no conflicts of interest.
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. We are also
very grateful to James Fraser for suggestions about successes and limitations in the
area of computational screening for drug repurposing. We are grateful to the following
contributors for reviewing pieces of the text: Nadia Danilova, James Eberwine and Ipsita
Krishnan.
The members of the COVID-19 Review Consortium include the following: Vikas
Bansal, John P. Barton, Simina M. Boca, Joel D Boerckel, 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,
Daniel S. Himmelstein, Fengling Hu, Nafisa M. Jadavji, Jeremy P. Kamil, 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,
Amruta Naik, YoSon Park, Dimitri Perrin, Yanjun Qi, Diane N. Rafizadeh, Bharath
Ramsundar, Halie M. Rando, Sandipan Ray, Michael P. Robson, Vincent Rubinetti,
Elizabeth Sell, Lamonica Shinholster, Ashwin N. Skelly, Yuchen Sun, Yusha Sun, Gregory
L Szeto, Ryan Velazquez, Jinhui Wang, and Nils Wellhausen.
Authors with similar contributions are ordered alphabetically.
Halie M. Rando contributed to Project Administration, Software, Visualization, Writing -
Original Draft, and Writing - Review & Editing. Nils Wellhausen contributed to Project
Administration, Visualization, Writing - Original Draft, and Writing - Review & Editing.
Soumita Ghosh, Anna Ada Dattoli, and Marouen Ben Guebila contributed to Writing - Original
Draft. Alexandra J. Lee, Fengling Hu, James Brian Byrd, and Jeffrey M. Field contributed to
Writing - Original Draft and Writing - Review & Editing. Diane N. Rafizadeh and Ronan Lordan
contributed to Project Administration, Writing - Original Draft, and Writing - Review & Editing.
Yanjun Qi and Yuchen Sun contributed to Visualization. Christian Brueffer contributed to
Project Administration and Writing - Review & Editing. Nafisa M. Jadavji contributed to
Supervision, Writing - Original Draft, and Writing - Review & Editing. Ashwin N. Skelly, Bharath
Ramsundar, Rishi Raj Goel, and YoSon Park contributed to Writing - Review & Editing. Jinhui
Wang contributed to Writing - Original Draft. The COVID-19 Review Consortium contributed to
Project Administration. Simina M. Boca and Casey S. Greene contributed to Project
Administration and Writing - Review & Editing. Anthony Gitter contributed to Project
Administration, Software, Visualization, and Writing - Review & Editing.
Rando et al.
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