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v1

Review

SARS-CoV-2/COVID-19: Viral Genomics,

Epidemiology, Vaccines, and Therapeutic
Interventions
Mohammed Uddin 1,2#, Farah Mustafa 3#, Tahir A. Rizvi 4, Tom Loney 1, Hanan Al Suwaidi 1,
Ahmad Al Marzouqi 3, Afaf Kamal Eldin 5, Nabeel Alsabeeha 6, Thomas E. Adrian 1, Cesare
Stefanini 7, Norbert Nowotny 1,8, Alawi Alsheikh-Ali 1* and Abiola C. Senok 1*
1
College of Medicine, Mohammed Bin Rashid University of Medicine and Health Sciences, Dubai, United
Arab Emirates; Mohammed.Uddin@mbru.ac.ae (M.U.); tom.loney@mbru.ac.ae (T.L.);
Hanan.Alsuwaidi@mbru.ac.ae (H.A.S.); Thomas.Adrian@mbru.ac.ae (T.A.); Norbert.Nowotny@mbru.ac.ae
(N.N.); Alawi.Alsheikhali@mbru.ac.ae (A.A.); abiola.senok@mbru.ac.ae (A.C.S.)
2
The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, ON, Canada
3
Department of Biochemistry, College of Medicine and Health Sciences, United Arab Emirates University, Al
Ain, United Arab Emirates; fmustafa@uaeu.ac.ae (F.M.); ahmedh@uaeu.ac.ae (A.A.M.)
4
Department of Microbiology & Immunology, College of Medicine and Health Sciences, United Arab
Emirates University, Al Ain, United Arab Emirates; tarizvi@uaeu.ac.ae (T.A.R.)
5
Department of Food, Nutrition and Health, United Arab Emirates University, Al Ain, United Arab Emirates;
afaf.kamal@uaeu.ac.ae (A.K.E.)
6
Ministry of Health and Prevention, Dubai, United Arab Emirates; dr.nabeel@yahoo.com (N.A.)
7
Department of Biomedical Engineering, Healthcare Engineering Innovation Center (HEIC), Khalifa
University, Abu Dhabi, United Arab Emirates; cesare.stefanini@ku.ac.ae (C.S.)
8
Viral Zoonoses, Emerging and Vector-Borne Infections Group, Institute of Virology, University of
Veterinary Medicine Vienna, Vienna, Austria; Norbert.Nowotny@vetmeduni.ac.at (N.N.)

#M.U and F.M contributed equally to this work.

* Correspondence: Alawi.Alsheikhali@mbru.ac.ae (A.A.); abiola.senok@mbru.ac.ae (A.C.S)

Received: date; Accepted: date; Published: date

Abstract: The COVID-19 pandemic is due to infection caused by the novel SARS-CoV-2 that
impacts the lower respiratory tract. The spectrum of symptoms ranges from asymptomatic
infections to mild respiratory symptoms to the lethal form of COVID-19 which is associated with
severe pneumonia, acute respiratory distress and fatality. At present, the global case fatality rate of
COVID-19 laboratory confirmed cases is ~4.7% ranging from ~0.3-0.4% in Chile and Israel to
~10.8% in Italy. To address this global crisis, up-to-date information on the viral genomics and
transcriptomics is crucial for understanding the origins and global dispersal of the virus,
providing insight into viral pathogenicity, transmission and epidemiology, and enabling strategies
for therapeutic interventions, drug discovery and vaccine development. Therefore, this review
provides a comprehensive overview of COVID-19 epidemiology, genomic etiology, findings from
recent transcriptomic map analysis, viral-human protein interactions, molecular diagnostics, and
the current status of vaccine and novel therapeutic intervention development. Moreover, we
provide an extensive list of resources that will help the scientific community access numerous
types of databases related to SARS-CoV-2 OMICs and approaches to therapeutics related to
COVID-19 treatment.

Keywords: SARS-CoV-2; COVID-19; Coronavirus; Pandemic; Viral Genomics

J. Clin. Med. 2020, 9, x; doi: FOR PEER REVIEW www.mdpi.com/journal/jcm

© 2020 by the author(s). Distributed under a Creative Commons CC BY license.

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1. Introduction
In December 2019, several cases of a new respiratory illness were described in Wuhan, Hubei
Province, China. By January 2020, it was confirmed that these infections were caused by a novel
coronavirus which was subsequently named SARS-CoV-2, while the disease it caused COVID-19
[1-3]. This novel coronavirus is closely related to the previously described SARS-CoV identified in
the 2002-2003 outbreak [4]. The World Health Organization (WHO) recently declared the ongoing
SARS-CoV-2 outbreak as a pandemic [1]. To contain the spread of the virus, we are witnessing the
implementation of strict measures unprecedented in modern times. Major cities and entire nations
have been placed under lockdown with restrictions on travel and gatherings as well as closure of
schools and businesses. These measures, along with the closure of international borders and
restrictions on international travel have had significant economic impact, resulting in a sharp
decline in major financial indices and prompting fears of a global recession.
As the number of confirmed infections and fatalities continue to increase daily, it is crucial to
further our understanding of the virus transmission patterns and epidemiology. Despite only ~3
months into the outbreak, there is a wealth of information emerging on the virus genomic makeup
& evolution, and its transcriptomic mapping, including virus-human protein interactions. Such
information is urgently needed for the identification of therapeutic targets for intervention and
vaccine development, in addition to informing preventive policies and patient care decisions. The
primary purpose of this review is to provide an update on the epidemiology, modes of
transmission, a summary of the genomics and transcriptomics of SARS-CoV-2, as well as
therapeutic interventions in the absence of a vaccine. Furthermore, we examine how the viral
genomics and molecular epidemiology informs therapeutic and vaccine development as well as
public health strategies. We have also compiled a resource table outlining the numerous databases
related to SARS-CoV-2 whole genome sequencing, transcriptomic map, strain tracing,
SARS-CoV-2-human protein-protein interactions, and clinical trials for repurposed drugs and
vaccines (Table).

Table: SARS-CoV-2 Resources Related to Genomics, Transcriptomics and Phenotypes

Category Data Type Database

SARS-CoV-2 Genome DNA Sequencing Data https://www.ncbi.nlm.nih.gov/gen

Sequencing Data bank/sars-cov-2-seqs/

SARS-CoV-2 Transcriptomic RNA Sequencing Data Open Science Framework:

Map accession number
doi:10.17605/OSF.IO/8F6N9

SARS-CoV-2 and Human Mass Spectrometry Raw http://proteomecentral.proteomexc

Protein Interactions Data hange.org/cgi/GetDataset?ID=PXD
018117

List of Clinical Trials Clinical Trial Related https://clinicaltrials.gov/ct2/home

Information
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2. Epidemiology and Transmission of SARS-CoV-2

To date (March 29, 2020), over 480,000 laboratory-confirmed cases of COVID-19 have been
reported worldwide with more than ~31,000 deaths in more than 175 countries [5]. In most
countries, increases in the number of confirmed cases are following an exponential growth
trajectory during the early and peak stages of the outbreak. At present, the global case fatality rate
of COVID-19 laboratory confirmed cases is ~4.7% ranging from ~0.3-0.4% in Chile and Israel to
~10.8% in Italy [6]. Whilst it is difficult to compare case fatality rates between countries when they
are at different stages of the outbreak, variations are most likely due to the scope of population
testing, the age structure and health status of the population, and the health systems within each
country [6]. Clinical characteristics of SARS-CoV-2 patients from China [7,8], South Korea [9], and
the United States [6] have recently been reported with fever, dry cough and shortness of breath
being the most common clinical presentations. Although the outbreak is evolving, the current
global data suggests that the number of cases is doubling every four days, with ~20% of confirmed
COVID-19 patients requiring hospitalization (median hospital stay of 12 days), and 25% of
hospitalized patients (~5% of all cases) needing intensive critical care [6,7,9]. The severity and
outcome of the disease seem to be highly correlated with the late age of onset where more severe
forms of COVID-19 were observed for adults ≥55 years [6,7,9]. Additionally, an age-dependent
fatality rate has been demonstrated with the lowest risk observed among those under the age of 19
(0-0.1%) and 20-54 years (0.1-0.8%); however, the risk of mortality increases incrementally, affecting
1.4-4.9% in the 55-74-year age group, 4.3-10.5% among those aged 75-84 years, with the highest
fatality rate of 10.4-27.3% in those aged ≥85 years [6,7,9,10]. Individuals with underlying health
issues, such as cardiovascular disorders, diabetes, liver & kidney disease, malignant tumours, or a
suppressed immune system, seem to have the severe form of the disease and increased fatality rate
[6,7,9-11].
SARS-CoV-2 has a natural origin [12] and is primarily transmitted via inhalation of droplets
expelled when an infected patient coughs. Fomite-mediated transmission is another important
source of transmission when hands which have touched surfaces contaminated by droplets are
used to touch the face, eyes, or nose. Current modeling of SARS-CoV-2 spread estimates a basic
reproduction number (R0) of 2.2 [13] (see Box). This reported R0 is higher than seasonal influenza,
indicating the potential for sustained human-to-human transmission within a population unless
strict containment and public health measures are implemented and sustained. As a new
coronavirus, there is currently insufficient data to reach a consensus on the potential for seasonality
of SARS-CoV-2 transmission. However, two major factors that may have an influence on
seasonality are changes in environmental parameters and human behavior [14]. Specifically,
outdoor (e.g., temperature, humidity, sunlight/vitamin D status) and indoor environmental factors
(e.g., temperature, humidity, air change rate, etc.) influence both virus transmission parameters (e.g.
virus viability, airborne aerosolization, droplet spray and direct contact) and host defenses (e.g.,
airway antiviral immune defense and efficiency of nasal and bronchial mucociliary clearance).
Although the seasonality has not been confirmed for SARS-CoV-2, there are now accumulating
evidence that high temperature and humidity might play a significant role in transmission [15,16].
To evaluate the stability of SARS-CoV-2 in aerosols and on different surfaces [17], a series of
well-controlled experiments revealed that the virus remained viable in aerosols throughout the
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duration of the experiment (3 hours; median half-time of 1.1-1.2 hours) [17]. SARS-CoV-2 was
found to be most stable on plastic and stainless steel with viable virus detected up to 72 hours
post-application and no viable virus was found on copper or cardboard after 4 and 24 hours,
respectively [17]. In this experimental model, SARS-CoV-2 exhibited similar stability to SARS-CoV.
Therefore, the differences

Box: SARS CoV-2 Related Definitions

 SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2
 COVID-19 or Covid-19: Corona virus disease, 2019. COVID-19 is the official name of the
disease manifested by the SARS-CoV-2.
 R0: Reproduction number that defines the number of secondary cases that will be produced
by a single infectious index case in a population that is fully susceptible to the disease. For
example, a R0 of 2 means that, on average, one primary index case would infect two other
people generating two secondary cases. Continuous horizontal (human-to-human)
transmission will occur if R0 is above the critical threshold of one.
 Fomite Transmission: A fomite is any inanimate object (i.e. surface) when contaminated with
or exposed to infectious agent, can serve as a source to transmit the agent into a new host.
 Non-Pharmacological Interventions (NPIs): NPIs are evidence based non-invasive, mostly
policy/regulation driven interventions on human health. NPIs (i.e. Physical [“Social”]
distancing) can be very effective to contain viral shedding.

in the epidemiological trends of the 2002-2003 SARS-CoV outbreak and the ongoing SARS-CoV-2
pandemic are more likely due to other factors such as high viral loads in the upper respiratory tract
and the potential for individuals infected with SARS-CoV-2 to shed and transmit the virus whilst
asymptomatic [17-19]. Overall, the findings indicate that continued aerosol and fomite transmission
(see Box) of SARS-CoV-2 is highly plausible as the virus remains viable and infectious in droplets
for numerous hours and on surfaces for up to three days [17]. This has now raised the concern that
airborne transmission might be occurring [17], though epidemiological evidence rules out the
relevance of such transmission to disease spread [20,21].

3. Genomics of SARS CoV-2

SARS-CoV-2 is a β-coronavirus similar to the viruses that cause SARS (severe acute respiratory
syndrome) and MERS (Middle East respiratory syndrome). Human coronaviruses are not new
and have been identified in the population since the late 1960s, causing mild symptoms similar to
common colds [22]. Of the seven strains known, four infect the upper respiratory tract and cause
mild symptoms, while three are associated with the lower respiratory tract, causing severe disease,
including SARS-CoV, MERS-CoV, and now SARS-CoV-2. Like other coronaviruses, SARS-CoV-2
is an enveloped, single-stranded, positive-sense RNA virus with a non-segmented genome ~30 kb
in size [12,23] (Figure 1). The viral genome codes for 16 non-structural proteins (Nsps) required for
virus replication and pathogenesis, four structural proteins, including envelope (E), membrane (M),
nucleocapsid (N), and spike (S) glycoprotein important for virus subtyping & response to vaccines,
and nine other accessory factors [23,24] (Figure 1). The first SARS-CoV-2 genome was published on
24th January 2020, just a few weeks into the outbreak [25] and exhibited genomic and phylogenetic
similarity to SARS-CoV, particularly in the S gene and receptor-binding domain (RBD), indicating
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the capability of direct human-to-human transmission. The genomic sequence of SARS-CoV-2

shows that although it is 75-80% identical to SARS-CoV [4,12], it is even more closely related to
several bat coronaviruses, in particular the Bat SARS-related coronavirus SARSr-CoV RaTG13 [25].
Phylogenetic analyses of SARS-CoV-2 genomes have identified bats as the primary reservoir of
SARS-like coronaviruses [26] displaying high sequence similarity (96.2%) between BatCoV and
SARS-CoV-2

Figure 1. (a) Illustration of the full-length genome of SARS-CoV-2 showing the location of open
reading frames 1a and 1b encoding the Non-structural proteins, Nsp (blue), structural proteins
(brown), and accessory factors (green). The numbers on top refer to the genomic RNA. (b)
Schematic representation of the SARS-CoV-2 virus particle and its interaction with its host cellular
receptor, ACE2. The infection pathway is shown where after docking of the virus particle on cell
surface, the TMPRRSS2 cellular protease activates the viral protein S allowing entry of SARS-CoV-2
into human cells. The protein coded by the viral genes and some of the notable interactions
(dashed line) with other host proteins are shown that can potentially be targeted by drugs (blue
circles).

genomes. [27] Sequence analysis of the viral spike protein further suggests new mutations in its
RBD determine not only the host range but also the cellular tropism of the virus [2,28-30].
Interestingly, a similar observation was made in viruses from pangolin SARSr-CoVs, one of the
putative intermediate host species that may have been used by SARS-CoV-2 for its species jump
into humans [31]. A few months prior to the emergence of SARS-CoV-2, the Pangolin-CoV whole
genome was sequenced from a dead Malayan Pangolin (Manis javanica) that showed 91.02% and
90.55% identical genome sequences to SARS-CoV-2 and BatCoV RaTG13, respectively [32]. The
sequence analysis also revealed that the S1 protein of Pangolin-CoV was much more closely related
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to SARS-CoV-2 than to RaTG13. Whilst these findings suggest Pangolin species as a reservoir of
coronaviruses, the analysis does not prove the potential of Pangolin as the intermediate host of
SARS-CoV-2.
Sequences of SARS-CoV-2 have now been reported from many parts of the world and this data
has proved useful in tracking the global spread of the virus [33] (see Table for resources related to
genomics). For example, a recent analysis of 103 SARS-CoV-2 genomes has identified two major
subtypes (designated L and S) that are well-defined by two different single nucleotide
polymorphisms (SNPs) [31]. This rapid evolution of SARS-CoV-2 is not surprising as coronaviruses
habour error-prone RNA-dependent RNA polymerases which make the occurrence of mutations
and recombination events rather frequent [34-37]. Within Wuhan, China, the L type was found in
∼70% of cases and was observed to be the more aggressive and contagious form compared to the
original S type [31]. The virus has further mutated and expanded into numerous strains. The
geographical diversity of different strains may help correlate COVID-19-related severity, mortality
rate, and treatment options. Genomic epidemiology of SARS-CoV-2 should also shed light on the
origins of regional outbreaks, global dispersal, and epidemiological history of the virus [38]. More
importantly, in case of an inability to diagnose infections empirically due to the speed of epidemics
or lack of test kits, such as the case with COVID-19, genomic epidemiology could be used to
estimate virus rate of replication in the population as well as burden of infection, allowing
healthcare professionals to make urgent policy decisions appropriately. Thus, there is ongoing
work geared towards mapping the spread of different SARS-CoV-2 strains across the world.

4. Transcriptomic Map and SARS CoV-2-Human Protein-Protein Interactions to Identify Drug

Targets
The transcriptome profile of SARS-CoV-2 isolated from COVID-19 patients has recently been
constructed using both ”long read DNA/RNA (Nanopore) sequencing” and “sequencing by
synthesis” techniques [23] (see Table for SARS-CoV-2 sequencing and OMICs related resources).
Direct RNA sequencing (without requiring reverse transcription) has further allowed detection of
RNA modifications on the genomic RNA (Table). By combining both sequencing and RNA
modification data, scientists in South Korea have identified 41 potential RNA modification sites that
could be important for virus replication and its associated pathogenesis [23]. Thus, transcriptomic
insights should further provide a better understanding of the viral life cycle and its virulence.
After cell entry, the virus RNA transcript produces nonstructure proteins (Nsp1 through
Nsp16) using two open reading frames (ORF1a and 1b, Figure B). A recent study on
protein-protein interaction mapping using mass spectrometry identified 332 SARS-CoV-2-human
protein interactions, including 69 interactions that can be targeted by existing FDA-approved drugs
[24] (Table). We observed one interesting similarity in both the transcriptomic and proteomic
studies, where the last reading frame (ORF10) expression was extremely low. Although the
transcriptomic study questioned the annotation of ORF10 due to extremely low RNA expression,
the proteomic analysis identified strong interaction of ORF10 with CUL2 complex, an E3
ubiquitin-protein ligase complex that mediates ubiquitination of target proteins [23,24]. This
suggests that the virus may be able to subvert this complex and use it for degradation of host
restriction factors that limit virus replication, making it a good target for drug development against
the virus.
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In humans, the ACE2 gene encodes the angiotensin-converting enzyme-2. Evidence from
recent studies suggests that ACE2 is the host receptor for the novel SARS-CoV-2 similar to
SARS-CoV [39,40],. The binding of SARS-CoV-2 to the ACE2 receptor (via the S protein) [40] is 10-20
folds higher compared to SARS-CoV, which may be one of the reasons for the higher
human-to-human transmission of SARS-CoV-2. The binding between SARS-CoV-2 and ACE2 has
been confirmed by multiple recent independent studies [24,39]. ACE2 is found in the lower
respiratory tract of humans on epithelial cells lining the lung alveoli and bronchioles as well as the
endothelial cells and myocytes of pulmonary blood vessels, partly explaining the severe respiratory
syndrome associated with these viruses [41]. ACE2 is also found on the enterocytes in the small
intestines, that may further explain the gastrointestinal symptoms associated with the viral
infection as well as its detection in faeces [42]. In a recent study, it has been shown that the ACE2
gene displays single nucleotide polymorphims with differential allele frequency accross the globe
[43]. The allele frequency for the host gene was also shown to be different between males and
females.
The viral spike (S) protein is responsible for viral entry into susceptible cells by interacting with
the ACE2 receptor25. This process requires “priming” of the S protein by the host transmembrane
serine protease 2 (TMPRSS2) which cleaves the S protein into two functional subunits: S1 and S2.
The S1 subunit then is able to interact with the ACE2 receptor, while the S2 subunit facilitates
viral fusion with the host cell membrane, allowing virus entry into the target cell [39] (Figure A).
The current knowledge of the cellular infection pathway involving ACE2 and TMPRSS2 thus
provide good candidates for therapeutics, such as antibodies that can interfere with virus
attachment and fusion with target cells (such as protease inhibitors).

5. Molecular Diagnosis of COVID-19

As the COVID-19 pandemic continues to spread rapidly, there is a growing demand for rapid
point-of-care testing of the virus. The current gold standard for diagnosing COVID-19 depends
upon detection of the viral genetic material (RNA) in a nasopharyngeal swab or sputum sample.
While this technique is sensitive and can detect the virus earlier in the infection, it requires
polymerase chain reaction (PCR), a technology that amplifies the amount of genetic material to
detectable levels and takes several hours to perform [44]. In recent weeks, rapid molecular tests
using automated platforms have received fast-track approvals from regulatory authorities. These
are high throughput automated tests with a turnaround time of 45-60 minutes.
To detect newer mutated strains, it is essential to apply next generation sequencing to identify
the viral genome with specific mutations. Currently, ‘sequencing by synthesis’ technique (by
Illumina Inc.) and ‘long read sequencing’ (by Oxford Nanopore Technology) are being used to
identify viral genomes at single-base resolution levels [12,23,31]. Nanopore sequencing technology
might have an advantage over other sequencing platforms due to its small and compact size,
allowing flexibility of conducting DNA/RNA sequencing in the field in remote locations that lack
full-fledged accredited reference laboratories.
Besides targeting the genome, another diagnostic approach for SARS-CoV-2 aims at detecting
antibodies produced by the patient’s immune system against the virus. Several such “antibody”
tests have been reported over the past few weeks for SARS-CoV-2 [45]. Although the antibody
associated tests are faster, their use for diagnosis is limited by the fact that it usually takes several
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days and up to two weeks after an infection takes place for antibodies to be detectable. Therefore,
antibody-based testing is not a reliable method to diagnose COVID-19; however, they may be
useful for population-based testing to estimate the proportion of the population with immunity and
identifying susceptible individuals. Such information may also be useful for public health measures,
including return-to-work protocols and social segregation of susceptible individuals. A third type
of testing relies on detecting viral proteins (antigens) [46] likely to be useful since they do not
depend on a detectable rise in patient-produced antibodies. Globally, several companies are
working on developing such rapid antigen-antibody-based and CRISPR-Cas12 based assays which
have received rapid emergency use authorization by respective regulatory agencies [47].
A comprehensive list of SARS-CoV-2 diagnostic assays (both molecular and immunological)
that have been commercialized and those under developments globally can be found at:
https://www.finddx.org/covid-19/pipeline/.

6. Vaccine Development for SARS-CoV-2

No vaccines currently exist (March 29th, 2020- date of manuscript submission) to prevent
SARS-CoV-2 infection. However, many efforts are in progress, including the classical inactivated
and attenuated vaccines, the subunit and viral vector-based vaccines, as well as the newer DNA-
and RNA-based vaccines. Each approach has its own advantages and disadvantages and all
approaches are being developed simultaneously to come up with an effective vaccine.
Among the four structured proteins, the spike protein is considered the most promising for
vaccine development since i) it is common to different coronaviruses encountered, and ii) is
exposed to an individual’s immune system, allowing the body to make an immune response
against it and remember it for future protection. Furthermore, such a vaccine can prevent infection
since it would inhibit virus entry into susceptible cells. Due to their previous experience with
vaccine development for SARS in 2003 (against SARS-CoV), scientists have had a head start in
using the S protein for vaccine development and one such vaccine has already entered human
clinical trials, while others are on their way.
So far, the first vaccine to go into clinical trials is the mRNA-1273 vaccine. It is a novel
RNA-based vaccine which uses part of the genetic code of the spike protein embedded in special
lipid-based nanoparticles for injection into the body [48]. It has been developed by the biotech firm
Moderna Therapeutics who was already working on SARS-CoV and MERS-CoV vaccines which
were adapted to SARS-CoV-2. The RNA part of the vaccine instructs cells to express the S protein to
elicit the immune response. After having shown potential in animal testing, the first Phase I
clinical trial of this vaccine started on March 16, 2020 in collaboration with the NIH on 45 healthy
individuals between the ages of 18-55 years. Several other mRNA-based vaccines (e.g. by CureVac,
BNT162 by BioNTech & Prifzer) are in different stages of development. The BioNTech mRNA
vaccine (Mainz, Germany) encapsulates the nucleic acid in special 80 nm ionizable, glycol-lipid
nanoparticles. Clinical testing is expected to start in April, 2020 [48].
Another vaccine that is ready for clinical trials in China has been developed by CanSino
Biologics (Tianjin, China), the company that also has developed and marked a vaccine for Ebola.
The vaccine based on their adenovirus vaccine platform, is named Ad5-nCoV. It is entering phase
I clinical trials in healthy individuals between 18-60 years of age in Wuhan, China. According to
the Chinese Registry of Clinical Trials: (http://www.chictr.org.cn/showproj.aspx?proj=51154)
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The Precision Vaccines Program (PVP) at Boston Children’s Hospital is developing a novel
vaccine designed especially for the older population who are at the greatest risk of developing
severe complications (https://discoveries.childrenshospital.org/coronavirus-vaccine/). The study is
funded by the US National Institutes of Health and the vaccine is testing efficacy of different
adjuvants (small molecules added to vaccines to boost immune response) along with the S protein.
Since older adults have weakened immune systems, the goal is to generate a robust, longer lasting,
and broader immune response against the S protein in the older aged population. Towards this end,
the vaccine is currently being tested using age-specific human in vitro systems to expedite the
process.
Other than these, there has been an acceleration in developing other novel vaccine approaches
and therapeutic interventions to combat viral infection [48]. Some of these approaches in advanced
stages of development and/or testing are summarized below.

Contemporary and Novel Approaches to Vaccine Development

 INO-4800: a DNA-based vaccine by Inovio Pharmaceuticals, a Pennsylvania Biotech company.
Phase I clinical trials are expected to start in April in the US followed by China and South Korea.
Funding from Bill and Melinda Gates Foundation is accelerating its testing and scale-up for
intradermal delivery using electroporation along with collaboration with the Beijing Advaccine
Biotechnology.
 Codagenix Biotech: Is developing a computationally designed and re-coded live-attenuated
SARS-CoV-2 vaccine in which sequence of the target gene of interest has been changed by
swapping its optimized codons with non-optimized ones. Codagenix is collaborating with
Serum Institute of India to scale-up manufacturing of this vaccine.
 Clover (Sichuan) Biopharmaceuticals: Is developing a recombinant SARS-CoV-2 S protein
trimer subunit vaccine and is in preclinical testing with GlaxoSmithKline’s Pandemic Adjuvant
Technology.
 Sanofi Pharmaceuticals: Is proceeding with preclinical and clinical trials using a recombinant
vaccine of undisclosed SARS-CoV-2 protein(s) expressed in baculovirus system to mount
immune response.
 Johnson & Johnson (Jansen): In partnership with Biomedical Advanced Research and
Development Authority (BARDA) is also developing single-dose, intranasal, recombinant
adenovirus-based vaccine by expressing an undisclosed viral protein to stimulate the immune
system using human retinal cells. The vaccine is currently being tested in animals.
 Altimmune, Inc.: Is currently testing a one-dose, intranasal, replication-defective,
adenovirus-vector-based vaccine incorporating the SARS-CoV-2 S protein. The vaccine is
currently being tested in animals.
 Novavax: Is developing a nanoparticle-based vaccine that displays the S protein with
saponin-based Matrix (M) protein adjuvant. This vaccine stimulates entry of
antigen-presenting cell into the injection site and enhances antigen presentation in local lymph
nodes to boost the immune response.

Other Experimental Therapeutic Interventions

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 AbCellera: This Canadian biotech has discovered >500 unique antibodies from sera of a
convalescent COVID-19 patient and in partnership with Eli Lilly is developing purely human
IgG1 mAbs-based treatments for coronavirus infection.
 Alnylam Pharmaceuticals has developed technology for delivering aerosolized siRNAs against
SARS-CoV2 to deliver directly to lungs which is being tested both in vitro and in vivo.
 Apeiron Biologics is employing recombinant protein strategy by designing recombinant ACE2
enzyme (APN01) allowing it to bind to virus particles in circulation thus making them
refractory for entering into the cells.
 InflaRx and Beijing Defengrei Biotechnology are using human IgG1 mAbs against
complement factor 5a. Antibodies from both companies have been approved for clinical trials
in China.
 NanoViricides are being created in another approach in which the S protein is chemically
attached to “virucidal nanomicelles”. Testing is in progress in culture only.

An extensive list of vaccines under developments, their current status, and other therapeutic
interventions can be found at: https://www.nature.com/articles/d41587-020-00005-z.

7. Drug Repurposing for COVID-19

Given the need to find effective treatment for symptomatic patients, the approach of
repurposing old drugs with antiviral properties and agents approved or under investigation for
other viral infections has been adopted. In the abscence of a vaccine, the World Health Organization
recently launched the SOLIDARITY trial which is an international clinical trial to address this
challenge. The drugs included in this trial are lopinavir and ritonavir, lopinavir and ritonavir plus
interferon beta as well as chloroquine, and remdesivir. The role of existing antiretroviral drugs and
pathways are as follows:
 Lopinavir-Ritonavir combination (Kaletra): This is an FDA-approved drug for HIV treatment.
Lopinavir is a protease inhibitor that inhibits a late step in viral replication, while ritonavir
helps boost the activity of lopinavir by inhibiting CYP3A liver enzymes that slows down the
rate at which lopinavir is broken down in the liver. Findings from in vitro work indicates its
potential for COVID-19 treatment. In China and South Korea, Lopinavir-Ritonavir has been
used either on its own or in combination with either alpha interferon (China) or
chloroquine/hydroxychloroquine (South Korea) for COVID-19 treatment with some success.
However, new data from China reported in the New England Journal of Medicine on
Wednesday 18th March 2020, casts doubt on the beneficial effect in seriously ill COVID-19
patients [49].
 Favipiravir (Favilavir or Avigan): This is an RNA-dependent RNA polymerase inhibitor
developed by Fujifilm Toyama Chemical’s in Japan that is safe and has been effective in other
viral infections, including influenza. It has now been found to be useful against SARS-CoV-2 as
tested in clinical trials in Wuhan and Shenzhen involving 340 patients. In addition to speeding
up viral clearance from the body from 11 to 4 days, it ameliorates lung injury.
 Chloroquine/Hydroxychloroquine: Chloroquine is an inexpensive drug for the treatment of
malaria and features on the WHO list of essential medicines. It is also used as an
anti-inflammatory agent for the treatment of autoimmune diseases. Chloroquine is thought to
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inhibit virus replication by increasing endosomal pH as many viruses such as Ebola and
Marburg that require the acidic environment of the endosome for successful replication.
However, a recent study showed that the anti-inflammatory effects of chloroquine are mediated
by upregulation of the cyclin-dependent kinase inhibitor, p21 [50]. In vitro studies have shown
its potent antiviral effect against the SARS-CoV-2 [51]. A multicenter clinical trial in China has
reported efficacy with amelioration of exacerbation of pneumonia and acceptable safety margin
with use of chloroquine for treatment of COVID-19 [11]. Hydroxychloroquine is an analogue of
chloroquine which is more stable with better clinical safety profile and has anti-SARS-CoV-2
activity. It has been shown to quicken recovery and clearance of the virus in COVID-19 patients
and used successfully in combination with the macrolide antibiotic azithromycin [52]. Larger
studies with controlled design are needed before conclusive recommendations for chloroquine
in the treatment of COVID-19 can be made.
 Remdesivir: Remdesivir is a nucleotide analogue with broad spectrum antiviral activity
against many RNA viruses. Like Favipiravir, it blocks RNA-dependent RNA polymerase, an
enzyme that replicates the viral genome, inhibiting an early step in virus replication, compared
to protease inhibitors that target the late steps of virus replication. This is an investigational
drug developed by Gilead Sciences for the treatment of Ebola, MERS-CoV, and SARS-CoV2,
and other RNA viruses. Currently, it is not approved for any of the diseases.
SNG001: SNG001 is an inhaled experimental drug developed by the UK biotech firm Synairgen.
The ability to inhale the drug will allow the patients to “self-administer” it by using a small
hand-held nebulizer. It was developed for the severe lung disease chronic-obstructive
pulmonary disorder (COPD), but due to the current COVID-19 crisis, it has been fast-tracked
for use in a 100-patient clinical trial that will be starting soon.
 Tocilizumab: Tocilizumab was developed by the Tokyo-based Chugai and Roche of Basel,
Switzerland. It is an immunosuppressive drug (antibody) against IL-6 receptor that blocks IL-6
production during the cytokine storm observed in severely ill COVID-19 patients.
 Kinases: p21-activated protein kinases (PAKs) are cytosolic serine/threonine protein kinases
downstream of small (p21) GTPases, including members of the Cdc42 and Rac families.
Multiple studies have shown that the major pathogenic kinase in this group, PAK1, plays a
major role in the entry, replication and spread of several important viruses, including influenza
and HIV [53,54]. Coronaviruses exploit macropinocytosis to gain entry into cells and this
process has been shown to be dependent on PAK1 activity [55,56]. Targeting of PAK1 to
prevent micropinocytosis has been implicated for therapeutic intervention [57]. This strongly
suggests that PAK1-inhibitors could be valuable for the treatment of COVID-19 infection.
PAK-1 inhibitors include caffeic acid and its ester, propolis, ketorolac and triptolide.
Unfortunately, all these have problems with solubility and cell penetration. However, newer
PAK-1 inhibitors, such as 15K (the 1,2,3-triazolyl ester of ketorolac, that is 500 times more
potent at inhibiting PAK1 than the parent compound [58], minnelide (in which a hydroxyl
group of triptolide is phosphorylated, boosting its water-solubility over 3000 times [59], and
frondoside A [60] are much more potent and may be of value in suppressing the effects of this
virus.

8. Non-Pharmacological Interventions
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J. Clin. Med. 2020, 9, x FOR PEER REVIEW 12 of 17

At present, there are no vaccines or specific pharmacological interventions available to contain

the horizontal transmission of SARS-CoV-2. Moreover, effective COVID-19-specific pharmaceutical
interventions and vaccines are not expected to be available for 3-12 months [3]. Therefore, the most
effective public health response to the ongoing outbreak is to follow non-pharmacological
interventions (NPI) such as early case identification and isolation, vigilant contact tracing of
potential secondary cases, travel restrictions & bans, stringent contact reductions, physical (“social”)
distancing, improved hygiene and continuous hand wash. Such an approach requires closure of
non-essential public spaces, services and facilities, a transition to digital learning modalities for
educational institutions, and self-isolation/work from home initiatives for businesses. Modelling
estimates indicate that integrated NPIs are likely to achieve the strongest and most rapid effect, if
implemented early in the outbreak [61]. These NPIs are interim measures as the quest for better
understanding of the viral genomics continues and the information garnered unlocks the doors for
development of effective therapeutic interventions and vaccines.
9. Future Directions for COVID-19 Research
The global efforts to contain the COVID-19 pandemic are primarily aimed to reduce the
number of infected persons, minimize the excessive burden on healthcare systems, and reduce the
social and economic impact of the pandemic. These efforts will provide the much-needed respite
during the period required for the development, testing and approval of an effective vaccine. Until
vaccines are available, it is likely that non-pharmacological interventions will remain the primary
line of defense to contain this pandemic. Therefore, accurate and up-to-date data on the daily
number of new cases and the case characteristics can inform modeling of future projections of new
cases and planning for anticipated healthcare capacity. Timely and accurate national data on
hospital bed and intensive care capacity along with daily census is essential for such planning.
COVID-19 will have a significant global impact on the social, cultural and economic infrastructures
that are envisaged to be long lasting and may take many years to recover. Healthcare systems
should consider integrating effective regulatory measures to tackle future pandemics. This crucial
lesson was learnt by countries which experienced the previous SARS-CoV outbreak and informed
the response to this pandemic in Hong Kong, Singapore, and Taiwan, for example. Genomic
characterization will have implications related to pathogenicity, transmissibility and response to
therapy of the viral isolates for local and global populations. The understanding of the genetic
makeup of the viral strains is also critical for drug discovery and designing of effective vaccines. To
better prepare for the next global pandemic, application of artificial intelligence (AI) should be
evaluated to track infections before the outbreak happens. Bluedot Inc., a Canadian AI company for
infectious diseases, flagged unusual infection related activity in Wuhan, China and reported the
spread nine days before WHO officially declared the outbreak [62]. In this era of emerging viral
infections, the global community must work together and deploy the very best of its technological
resources to address the current pandemic and ensure preparedness for future outbreaks.

Author Contributions: Conceptualization, A.A., M.U. and A.C.S; Preparation of draft manuscript
by M.U, F.M. T.A.R, T.L and A.C.S; Figure was prepared by M.U and F.M. All authors contributed
to critical review and editing of the manuscript and approved the submitted manuscript.

Funding: This research received no external funding.

Acknowledgments: None
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J. Clin. Med. 2020, 9, x FOR PEER REVIEW 13 of 17

Conflicts of Interest: The authors declare no conflict of interest

References

© 2020 by the authors. Submitted for possible open access publication under the terms
and conditions of the Creative Commons Attribution (CC BY) license
(http://creativecommons.org/licenses/by/4.0/).

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