WAYS OF MAKING EFFECTIVE AND SAFE VACCINES AGAINST SARS-CoV -2

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WAYS OF MAKING EFFECTIVE AND SAFE VACCINES AGAINST SARS-CoV -2
Md. Selim Reza*1,2, Farzana Mim2, Dr. Mohammad Rezaul Quader3,Dr. Mohammad Jahidur Rahman Khan4,
Md. Sabir Hossain2, Kazi Rasel Uddin2, Salina Akter2 and Dr. Sharmin Rahman5
1COVID-19 RT-PCR LAB, Bangabandhu Sheikh Mujib Medical College, Faridpur, Bangladesh.
2Department of Biochemistry and Molecular Biology, Jahangirnagar University, Savar, Dhaka, Bangladesh.
3Department of Biochemistry, Bangabandhu Sheikh Mujib Medical College, Faridpur, Bangladesh.
4Department of Microbiology, Shaheed Suhrawardy Medical College, Bangladesh.
5Department of Pharmacology, Ibrahim Medical College, Dhaka, Bangladesh.
Article Received on 10/07/2021 Article Revised on 31/07/2021 Article Accepted on 20/08/2021
1. INTRODUCTION
Coronaviruses (CoVs) are a group of related viruses that
can cause respiratory tract infection in humans ranging
from mild symptoms to lethal outcomes. In the past 18
years, three novel coronaviruses have crossed the species
barrier to infect humans and cause human-to-human
transmission. The first lethal coronavirus SARS-CoV
emerged in 2002 in Guangdong Province, China. During
the 2002–2004 outbreak, SARS-CoV had infected 8,098
people and resulted in 774 SARS-associated deaths (~
10% mortality rate) across 29 countries before it
disappeared. Despite efforts from the scientific
community, no vaccine became commercially available
(WHO, 2003). In September 2012, the world
experienced the emergence of the Middle East
respiratory syndrome (MERS) coronavirus, originated in
Saudi Arabia. The disease has affected 27 countries,
resulting in 2494 cases and 858 deaths (~ 35% mortality
rate). MERS cases are still being reported but no major
outbreak has been declared since 2015. As in the case of
SARS, no commercial vaccine is available for MERS
(WHO, 2020).
Explanations behind the absence of commercial and
effective vaccines for SARS and MERS are fluctuated.
On account of MERS, almost certainly, the vaccine
development was postponed due to the shortage of
reasonable and cost-effective small animal models
during pre-clinical experimentation. Also, it is likely that
a vaccine has not been conveyed on account of the low
interest in putting resources into a vaccine for a disease
that has delivered moderately low and geographically
concentrated cases. This last factor may have
additionally added to the absence of a vaccine for SARS,
as in it was viewed as inconsequential to keep putting
resources into a vaccine for an infection whose cases
stopped to be accounted for in 2004.
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European Journal of Biomedical
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Volume: 8
Issue: 9
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*Corresponding Author: Md. Selim Reza
COVID-19 RT-PCR LAB, Bangabandhu Sheikh Mujib Medical College, Faridpur, Bangladesh.
ABSTRACT
Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by SARS–associated
coronavirus (SARS-CoV). Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is another new type
of coronavirus that causes the Coronavirus Disease 2019 (COVID-19), which has been the most challenging
pandemic in this century. SARS-CoV-2 which is genetically similar to SARS-CoV and Middle East respiratory
syndrome coronavirus (MERS-CoV) is an enveloped, single and positive-stranded RNA virus. Considering its
high mortality and rapid spread, an effective vaccine is urgently needed to control this pandemic. Vaccines, such
as inactivated vaccines, attenuated vaccines, nucleic acid-based vaccines, and vector vaccines, which have already
been demonstrated their prophylactic efficacy against MERS-CoV and SARS-CoV, so these candidates could be
used as a potential tool for the development of a safe and effective vaccine against SARS-CoV-2. The inactivated
SARS-CoV vaccine may be the first one available for clinical use because it is easy to generate; however, safety is
the main concern. The spike (S) protein of SARS-CoV is the major inducer of neutralizing antibodies, and the
receptor-binding domain (RBD) in the S1 subunit of S protein contains multiple conformational neutralizing
epitopes. This suggests that recombinant proteins containing RBD and vectors encoding the RBD sequence can be
used to develop safe and effective SARS-CoV-2 vaccines.
KEYWORDS: Corona virus; Pandemic; SARS-CoV-2; COVID-19; MERS-CoV; Vaccine.

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Graphical Abstract
Coronavirus disease 2019 (COVID-19) is a current
pandemic caused by the severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2). The first cases
were reported from Wuhan, China, in December 2019
(Del Rio et al., 2020). Currently there is no specific
antiviral drug against SARS-CoV-2, finding a vaccine
for this virus therefore remains a high priority. Vaccines
are the most effective and economical means to prevent
and control infectious diseases. The development of an
effective vaccine against SARS-CoV-2 infection is
urgently required. Although no vaccines are
commercially available for SARS and MERS, past and
current vaccine development efforts against these
diseases might be of high value for the development of
an effective vaccine for COVID-19. So far, many
pharmaceutical companies and academic institutions
worldwide have launched their programs on vaccine
development against SARS-CoV-2.
2. THE GENETICS OF SARS-COV
Coronaviruses are the largest group of RNA viruses from
the subfamily Orthocoronavirinae or Coronavirinae in
the family Coronaviridae, in the order Nidovirales. They
are enveloped viruses with a positive-sense single-
stranded RNA genome and a nucleocapsid of helical
symmetry (Cherry et al., 2017). They have characteristic

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club-shaped spikes that project from their surface, which
in electron micrographs create an image reminiscent of
the solar corona, from which their name derives
(Almeida JD et al., 1968). Their genome size is relatively
large for RNA viruses, between 27 and 34 kB (de Groot
et al., 2011). Coronaviruses infect mammals and birds
causing varied symptoms such as respiratory tract
disease and diarrhea. In humans, coronavirus infections
have been shown to be potentially lethal. Until now,
there are seven genera of CoVs that are known to infect
humans. Four of these genera, including Human
Coronavirus 229E (HCoV-229E), Human Coronavirus
OC43 (HCoV-OC43), Human Coronavirus NL63
(HCoVNL63), and Human Coronavirus HKU1 (HCoV-
HKU1) have been identified as causing up to a third of
community-acquired upper respiratory tract infections.
The other three CoVs, Severe Acute Respiratory
Syndrome Coronavirus (SARS-CoV), Middle East
Respiratory Syndrome Coronavirus (MERSCoV), and
Severe Acute Respiratory Syndrome Coronavirus 2
(SARS-CoV-2), are highly pathogenic and can lead to
severe respiratory diseases and fatal outcome in infected
patients (CDC, 2020).
SARS-CoV-2 is a positive-strand RNA virus that
belongs to the group of Betacoronaviruses. The genome
of SARS-CoV-2 is approximately 29,700 nucleotides
long and shares 79.5% sequence identity with SARS-
CoV and 50% with MERS-CoV (Lu R. et al., 2020). The
six functional open reading frames (ORFs) are arranged
in order from 5′ to 3′: replicase (ORF1a/ORF1b), spike
(S), envelope (E), membrane (M) and nucleocapsid (N)
(Figure 1). In addition, seven putative ORFs encoding
accessory proteins are interspersed between the structural
genes (Chan J. F. et al., 2020). Most of the proteins
encoded by SARS-CoV-2 have a similar length to the
corresponding proteins in SARS-CoV. Of the four
structural genes, SARS-CoV-2 shares more than 90%
amino acid identity with SARS-CoV except for the S
gene, which diverges (Lu R. et al., 2020). The replicase
gene covers two thirds of the 5′ genome, and encodes a
large polyprotein (pp1ab), which is proteolytically
cleaved into 16 non-structural proteins that are involved
in transcription and virus replication. Most of these
SARS-CoV-2 non-structural proteins have greater than
85% amino acid sequence identity with SARS-CoV
(Chan J. F. et al., 2020).
The SARS-CoV–like virus that exists in animals does
not cause typical SARS-like disease in the natural hosts
and is not transmitted from animals to humans. Under
certain conditions, the virus may have evolved into the
early human SARS-CoV, with the ability to be
transmitted from animals to humans or even from
humans to humans, resulting in localized or even global
outbreaks and mild to severe human disease. An early
report by Zhou et al. identified a closely related SARSr-
CoV genome sequence, RaTG13, which shared a 96%
whole-genome sequence identity with SARS-CoV-2,
indicating a probable bat origin of SARS-CoV-2 (Zhou
et al., 2020). Since then, more SARS-CoV-2-related viral
genome sequences from bats have been reported from
Eastern China and Japan, and from pangolins in China.
However, the immediate animal ancestor or progenitor
virus, the equivalent of the >99% identical SARS-CoV
sequences identified in civets during the SARS outbreak
in 2003, remains elusive for SARS-CoV-2. Identification
of the origin and immediate progenitor viruses are not
only important academically, but also critical for public
health measures to prevent future outbreaks caused by
SARS-CoV-2 or closely related viruses.
Figure 1: The genome and virion structure of coronaviruses (CoVs). a) The genome structure of SARS-CoV,
MERS-CoV, and SARS-CoV-2. b) The virion structure of SARS-CoV.

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3. ANTIGEN SELECTION FOR SARS-COV
VACCINES
Many viral proteins are essential for the life cycle of
CoVs. For entering target cells, S protein first binds to
cellular receptors through its receptor-binding domain
(RBD), and the receptor-virus complex is subsequently
translocated to endosomes (Figure 2) (Du L et al., 2009).
Both SARS-CoV and SARS-CoV-2 S proteins bind to
angiotensin- converting enzyme 2 (ACE2), while the S
protein of MERS-CoV uses dipeptidyl peptidase-4
(DPP4) as its cellular receptor (Wang N et al., 2020). At
the endosome, S protein is further cleaved into S1 (RBD-
containing) and S2 (non-RBD-containing) subunits, and
the S2 subunit mediates fusion between the viral
envelope and the host cell membrane (Du L et al., 2009).
After entering the cell, several Nsps, particularly
RNA‐dependent RNA polymerase (Nsp12) and helicase
(Nsp13), mediate the replication of the CoV genome and
the transcription of CoV mRNA (Snijder EJ et al., 2016).
The CoV mRNA is further translated into different
nonstructural and structural proteins. The N proteins bind
to CoV genomic RNA to form viral nucleocapsids, and
S, E, M proteins form the envelope of CoV. After
assembly, viral particles bud through an endoplasmic
reticulum (ER)-Golgi pathway and exit the cells by
exocytosis (Du L et al., 2009).
Figure 2: The putative life cycle of SARS-CoV-2.
3.1 Spike protein (S protein)
The S protein of coronaviruses (CoVs) is responsible for virus binding, fusion, and entry and is a major inducer of
neutralizing antibodies. S protein is currently the most promising antigen formulation for SARS-CoV-2 vaccine
research. First, it is surface exposure and thus is able to be directly recognized by host immune system. Second, it
mediates the interaction with host cell by binding to the receptor ACE2, which is essential for subsequent virus entry to
target cells and causing subsequent pathogenicity (Wrapp D et al., 2020). Finally, the homologue proteins were already
used for vaccine development against SARS-CoV and MERS-CoV, and were proved to be effective (Zhou Y et al.,
2018; Du L et al., 2009). Studies have shown that antibodies generated against the S protein are long-lasting and
immunodominant in recovered SARS patients. In addition, the anti-S antibody can neutralize SARS-CoV and MERS-
CoV and provides protective effects in animals and humans. Moreover, many S protein-based vaccines against SARS-
CoV and MERS-CoV have been shown to elicit potent immune responses and protective effects in preclinical models
(Coleman CM et al., 2014; Muthumani K et al., 2015). These results corroborate that CoV S protein serves as an ideal
vaccine target to induce neutralizing antibodies and protective immunity.
The monomer of S protein from SARS-CoV-2 contains 1273 amino acids, with a molecular weight of about 140 kDa.
Self-association naturally assembles the S protein into a homo-trimer, typically similar to the first class of membrane
fusion protein (Class I viral fusion protein). The S protein contains two subunits (S1 and S2) (Figure 3). The S1 subunit
can be further defined with two domains termed the N-terminal domain (NTD) and the C-terminal domain (CTD). The
receptor binding domain (RBD) is located in the CTD. S2 subunit contains the basic elements required for membrane
fusion, including an internal membrane fusion peptide (FP), two 7-peptide repeats (HR), a membrane proximal external
region (MPER), and a trans-membrane domain (TM) (Li F, 2016). Recently, the structure of the SARS-CoV-2 S trimer
in the pre-fusion conformation and the RBD domain in complex with ACE2 has been successfully determined, which
has provided valuable information for vaccine design based on this protein (Wrapp D et al., 2020). So far, the potential
fragments of S protein for use as antigens in vaccine development include the full-length S protein, the RBD domain,
the S1 subunit, NTD, and FP.

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Figure 3: Strategies for designing vaccines for severe acute respiratory syndrome (SARS) using spike (S)
protein.
i. The full-length S protein
Full-length proteins are likely to maintain the correct
conformation of the protein, capable of providing more
epitopes and exhibiting higher immunogenicity.
Muthumani et al. reported that DNA vaccine encoding
MERS-CoV S protein was immunogenic in mice,
camels, and rhesus macaques. Animals immunized with
the DNA vaccine show reduced typical clinical
symptoms including pneumonia during the infection
(Muthumani et al., 2015).
ii. RBD
RBD, a fragment (193 aa residues) in the middle of S1
subunit of S protein, is responsible for virus binding to
the receptor on target cells (Figure 4). Since the RBD of
S protein directly interacts with the ACE2 receptor on
host cells, RBD immunization induced specific
antibodies may block this recognition and thus
effectively prevent the invasion of the virus. RBD
domain is relatively conserved as compared with S1
subunit and was reported to contain multiple
conformational neutralizing epitopes, making it more
suitable for vaccine development (Jiang S et al., 2005).
As a matter of fact, most of SARS-CoV-2 subunit
vaccines currently under development use RBD as the
antigen. Moreover, the RBD domain was also used in the
development of SARS-CoV and MERS-CoV vaccines.
For example, studies have demonstrated that
recombinant RBD consists of multiple conformational
neutralizing epitopes that can induce high titer of
neutralizing antibodies against SARS-CoV (Zhu X et al.,
2013). Lan et al. reported that Rhesus macaques
immunized with the recombinant RBD formulated with
alum adjuvant could produce neutralizing antibodies, in
association with observed mitigation of the clinical
symptoms during MERS-CoV infection (Lan J et al.,
2015).
Figure 4: Interactions between SARS-CoV-2-RBD and ACE2. (A) The PD of ACE2 mainly engages the α1 helix
in the recognition of the RBD. The α2 helix and the linker between β3 and β4 also contribute to the interaction.
Only one RBD-ACE2 is shown. (B to D) Detailed analysis of the interface between SARS-CoV-2-RBD and
ACE2. Polar interactions are indicated by red dashed lines. NAG, N-acetylglucosamine.

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iii. NTD
Like RBD, the N-terminal domains (NTD) of S protein
from several coronaviruses were reported to show
carbohydrate receptor-binding activity. One study
reported that rNTD of S protein from MERS-CoV
induced potent cellular immunity and antigen-specific
neutralizing antibodies in mice and was protective
against the viral challenge (Jiaming L et al., 2017).
However, as the genomes of coronaviruses are highly
variable, it is better to use antibodies targeting different
epitopes to avoid immune escape of the virus. Although
the function of S1-NTD of SARS-CoV-2 has not been
elucidated, it may also be involved in the binding of
certain receptors and can also serve as a candidate
antigen.
iv. S1 subunit
The S1 subunit, which contains both RBD and NTD, is
mainly involved in the S protein binding to the host
receptor. It is also widely used in vaccine development.
Wang et al. reported that MERS-CoV S1 protein
formulated with MF59 adjuvant protected hDPP4
transgenic mice against lethal virus challenge, and the
protection correlated well with the neutralizing antibody
titer (Wang Y et al., 2017).
v. FP
The FP domain of the S2 subunit is involved in the
membrane fusion of the virus, which is also a key step in
viral pathogenicity. Therefore, it may also serve as a
vaccine candidate antigen (Alsaadi EAJ et al., 2019).
3.2 Nucleocapsid protein (N protein)
The N protein is the most abundant protein in
coronavirus, and it is normally highly conserved, with a
molecular weight of about 50 kDa. N protein has
multiple functions including formation of nucleocapsids,
signal transduction virus budding, RNA replication, and
mRNA transcription (McBride R et al., 2014). This
protein was reported to be highly antigenic, 89% of
patients who developed SARS, produced antibodies to
this antigen (Leung DT et al., 2004). N protein-based
vaccines usually cannot induce neutralizing antibodies,
likely due to the fact that N protein is not displayed on
the CoV surface. However, N protein has the advantage
of being more conserved across CoV species than S
protein, making it a potential target for a T-cell inducing,
universal CoV vaccine (Wang N et al., 2020). One recent
study has shown that a viral vector vaccine expressing N
protein can induce CD4+ T cell-dependent protection
against SARS-CoV and MERSCoV, suggesting the
feasibility of N protein-based T-cell inducing CoV
vaccines (Zhao J et al., 2016).
3.3 Membrane protein (M protein)
The M protein of coronavirus plays a central role in virus
assembly, turning cellular membranes into workshops
where virus and host factors come together to make new
virus particles. It is a trans-membrane glycoprotein with
a molecular weight of about 25 kDa (Neuman BW et al.,
2011). It was reported that immunization with the full
length of M protein is able to elicit efficient neutralizing
antibodies in SARS patients. Immunogenic and structural
analysis also indicated that the trans-membrane domain
of the M protein contains a T cell epitope cluster that is
able to induce a strong cellular immune response (Liu J
et al., 2010). M protein is also highly conserved in
evolution among different species; therefore, it may be
used as a candidate antigen for developing SARS-CoV-2
vaccine.
3.4 Envelope protein (E protein)
E gene encodes a small multifunctional protein that
possesses ion channel (IC) activity, an important
function in virus-host interaction. SARS-CoV E protein
is a 76-amino acid transmembrane protein actively
synthesized during viral infection, that mainly localizes
at the ERGIC region of the cell, where virus budding and
morphogenesis take place. Different requirements of E
protein during the virus cycle have been described
among CoVs (Pervushin K et al., 2009). Compared with
S, N, and M protein, E protein is not suitable for use as
an immunogen. Studies have shown that SARS-CoV E
protein is an important virulence factor, and the secretion
of inflammatory factors IL-1, TNF, and IL-6 are
significantly reduced after knocking out E protein
(Nieto-Torres JL et al., 2014).
4. VACCINE DEVELOPMENT STRATEGIES AND
PLATFORMS
Viral zoonosis had come about into numerous disease
epidemics in recent years and development of new
strains from their zoonotic hosts makes them
exceptionally hard to foresee (Lau SK et al., 2005). Prior,
CoV were thought as powerless pathogen for people
causing mild influenza like sickness but with consistent
episodes like SARS in 2002, MERS in 2012 and now
COVID-19 their pathogenicity is very grounded
internationally (Chan JF et al., 2015). Such repeated
transmission prompting worldwide economy misfortunes
makes CoV vaccines profoundly desirable, as of now
there are no antiviral medications avilable against CoV.
Much effort is being made to develop vaccine against
SARS-CoV2 on accounts to tackle the current
coronavirus pandemic. Different regions investigated for
the inquiry of an ideal immunization against SARS-CoV,
includes inactivated virus vaccines, recombinant viral
vaccines, protein subunit vaccine, DNA vaccines, RNA
vaccines, non-replicating viral vector, replicating viral
vector, and live-attenuated vaccines (Figure 5)
(Kyriakidis NC et al., 2021). Each approach has
advantages and disadvantages (Table 1) (Li YD et al.,
2020). As the SARS-CoV-2 shares similarities in the
genetic makeup of two deadly coronaviruses, i.e. SARS
and MERS, vaccine strategies used to combat SARS and
MERS viruses are being adopted to guide the
formulation and development of new SARS-CoV-2
vaccines (Giovanni Salvatori et al., 2020).

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Figure 5: Overview of the strategies used for vaccine development and delivery.
Table 1: Advantages and disadvantages of different vaccine platforms (Li YD et al., 2020).
Vaccine platform
Advantages
Disadvantages
Whole inactivated
virus vaccine
Stronger immune response; Safer
than live attenuated virus
Potential epitope alteration by inactivation
process
Live attenuated
virus vaccine
Stronger immune response;
Preservation of native antigen;
Mimicking natural infection
Risk of residual virulence, especially for
immunocompromised people
Viral vector
vaccine
Stronger immune response;
Preservation of native antigen;
Mimicking natural infection
More complicated manufacturing process;
Risk of genomic integration; Response
dampened by pre-existing immunity
against vector
Subunit vaccine
Safe and well-tolerated
Lower immunogenicity; Requirement of
adjuvant or conjugate to increase
immunogenicity
Viral-like particle
vaccine
Safe and well-tolerated; mimicking
native virus conformation
Lower immunogenicity; More complicated
manufacturing process
DNA vaccine
Safe and well-tolerated; Stable
under room temperature; Highly
adaptable to new pathogen; Native
antigen expression
Lower immunogenicity; Difficult
administration route; Risk of genomic
integration
RNA vaccine
Safe and well-tolerated; Highly
adaptable to new pathogen; Native
antigen expression
Lower immunogenicity; Requirement of
low temperature storage and transportation;
Potential risk of RNA-induced interferon
response

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4.1 Live-attenuated vaccines
Live attenuated vaccines are produced by generating a
genetically weakened version of the virus that replicates
to a limited extent, causing no disease but inducing
immune responses that are similar to that induced by
natural infection (Figure 6). This type of vaccines usually
elicits robust and long-term memory immune responses
after a single dose. Attenuation can be achieved by
adapting the virus to unfavourable conditions (for
example, growth at lower temperature, growth in non-
human cells) or by rational modification of the virus (for
example, by codon de-optimization or by deleting genes
that are responsible for counteracting innate immune
recognition) (Broadbent AJ et al., 2016). An important
advantage of these vaccines is that they can be given
intranasally, after which they induce mucosal immune
responses that can protect the upper respiratory tract—
the major entry portal of the virus. In addition, because
the virus is replicating in the vaccinated individual, the
immune response is likely to target both structural and
non-structural viral proteins by way of antibodies and
cellular immune responses. However, disadvantages to
these vaccines include safety concerns and the need to
modify the virus, which is time-consuming if carried out
by traditional methods and technically challenging when
reverse genetics is used. Deletion of the structural E gene
may prove to be the first step in the field of developing a
live-attenuated vaccine against SARS‑CoV or MERS-
CoV (DeDiego ML et al., 2007). Two attenuated SARS-
CoV-2 virus vaccine candidates are in pre-clinical
development currently – the first a joint effort by US
company Codagenix and the Serum Institute of India,
and the second run by Indian Immunologicals Ltd and
Australia‘s Griffith University. They use viral
deoptimization to synthesize ―rationally designed‖ live-
attenuated vaccines (WHO, 2020).
Figure 6: The way live attenuated virus vaccines act.
4.2 Inactivated virus vaccines
Inactivated virus vaccines also known as the WKV
(whole Killed Virus) vaccines represent a pathogen
whose ability to infect and replicate has been ceased,
consequently making it sterile but retaining its ability to
act as an immunogen, so that the immune system could
still work if such a pathogen is injected into a host
(Figure 7). Inactivated vaccines are prepared by
neutralizing the pathogen as a whole by chemicals or by
heat and radiation. It is thought that inactivated vaccines
can be prepared with much less effort which makes them
one of the attractive types of vaccines prepared in the
market today. These vaccines work by exposing the same
epitopes which a virus otherwise would have presented,
thus eliciting an immune response. Inactivated SARS-
CoV has been tested in humans and found to be safe and
elicited SARS-CoV specific neutralizing antibodies,
however the efficiency the vaccine in humans yet to be
reported (Lin JT et al., 2007). These data suggested that
inactivated vaccines are safe and they are able to induce
SARS-CoV specific neutralizing antibodies so
inactivated viral vaccines could also be evaluated as
potential vaccine candidate against SARS-CoV-2.
Several inactivated vaccine candidates have entered
clinical trials, with three candidates from China in phase
III trials, and one from India, one from Kazakhstan and
two from China in phase I or II clinical trials (WHO,
2020).
Figure 7: The way inactivated (killed) virus vaccines
act.
4.3 Viral-vector based vaccines
Viral vector vaccines are recombinant viruses that
encode antigens of interest in an unrelated modified
virus. They deliver antigen into the cells mimicking
natural infection, so they induce strong antigen-specific
cellular and humoral immune responses per se, thereby
obviating the need for additional adjuvants. In addition,

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viral vectors are able to accept large insertions in their
genome, providing a flexible platform for antigen design.
Despite these advantages, there are several drawbacks.
The manufacturing process for viral vector vaccines is
more complicated than other approaches, including the
optimization of cellular systems and the exclusion of
contaminants, which can greatly affect the efficiency of
viral vectors (Rauch S et al., 2018). Moreover,
recombinant viruses carry the risk of integrating their
genome into the human host, so additional biosafety
assessment will be required before entering clinical
trials. Finally, if the chosen viral vector can infect the
general populations, the pre-existing immunity on the
viral vector could dampen the induced immune response,
which has been seen in adenovirus and measle virus-
based vaccines (Fausther-Bovendo H et al., 2014).
There are two broad viral vector groups used for vaccine
production, namely replication-competent and
replication-incompetent viral vectored vaccines (Figure
8). Replication-competent vectors need lower dose to
elicit strong responses as the multiplying vector can
result in enhanced antigen presentation. Conversely,
replication- incompetent vectors should be administered
in higher dosages since they are devoid of a self-
propagation capacity. However, this last characteristic
allows translating into safer platforms.
Most viral vector coronavirus vaccines target the S
antigen. Several attempts have been made in the
direction of the development of SARS coronavirus
(SARS-CoV) vaccine for which various viral vectors are
genetically engineered to express SARS-CoV proteins in
them (Table 2). Adenovirus and modified vaccinia virus
Ankara (MVA) are the two most common viral vectors
used in the development of SARS-CoV and MERS-CoV
vaccines. Related SARS-CoV-2 vaccine research has
been carried out by the following institutions. Houston-
based Greffex Inc. has completed the construction of
SARS-CoV-2 adenovirus vector vaccine with Greffex
Vector Platform and should have now moved to animal
testing. Tonix Pharmaceuticals announced research to
develop a potential SARS-CoV-2 vaccine based on
Horsepox Virus (TNX-1800). Johnson & Johnson has
adopted the AdVac® adenoviral vector platform for
vaccine development (Gonzalez-Nicolini V et al., 2006).
Figure 8: The way replication-competent and replication-incompetent viral vector vaccines act.
Table 2: Replication-competent and replication-incompetent viral vectors investigated as vaccine candidates
against SARS-CoV-2 (Malik JA et al., 2021).
Replication-competent vectors
Replication-incompetent vectors
YF17D Vector
Measles Vector
Horsepox vector
LVVV based on attenuated influenza virus
backbone
Influenza vector
Replication-competent VSV chimeric virus
technology
Newcastle disease virus vector
Avian paramyxovirus vector
Sendai virus vector
Adenovirus-based
MVA encoded VLP
Replication defective Simian
Adenovirus
adenovirus-based NasoVAX
adenovirus-based + HLA-matched peptides
Inactivated Flu-based SARS-CoV2 vaccine
+ Adjuvant
Influenza A H1N1 vector
parainfluenza virus 5 -based vaccine
Recombinant deactivated rabies virus
Dendritic cell-based vaccine

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4.4 Subunit vaccines
The principle underlying the development of subunit
vaccines was based upon the observation that do not
need to administer the entire pathogen to elicit strong
immune responses, but merely an immunogenic fragment
(Figure 9). Subunit vaccines are prepared either from
antigen purification of pathogens replicated in cell
cultures or from recombinantly expressed antigens.
These vaccines commonly require adjuvant addition in
order to deliver danger signals to antigen-presenting cells
and provoke robust immune responses. Since there is no
live fragment involve, there is no danger of prompting a
disease (B. Sarkar et al., 2019). Subunit antibodies can
be additionally classified into protein-based subunit
vaccines, polysaccharide vaccines, and conjugate subunit
vaccines. Protein subunit vaccines are more steady and
safer than live attenuated and inactivated/killed vaccines.
They can be manufactured in a more cost-efficient
manner as compared to other types of vaccines. A
shortcoming of this strategy is that if isolated proteins get
denatured, may bind to different antibodies than the
targeted protein of the pathogen (Wang M et al., 2016).
Figure 9: The way subunit vaccines act.
Protein subunits comprising spike (S), envelope (E),
membrane (M), and nucleocapsid (N) that are expressed
by SARS-CoV-2 are viral antigens actuate neutralizing
antibodies and generate a protective immune response
(Pandey SC et al., 2020). To develop vaccines based on
CoV subunit, S proteins are the preferred candidate,
because S proteins have sites for binding of receptor as
well as for membrane fusion; vaccines based on S
protein are likely to activate antibodies that prevent the
binding of virus and later fusion of membranes as well
thereby counteracting infection of virus. S-ectodomain
proteins of full length merged with/ without S or S fold
on i.e. fold on trimeric motif, may induce precise
antibody response and neutralizing antibody thus causing
protection of the vaccinated mice to SARS-CoV
infection (Li J et al., 2013). Besides this, S protein
associated with SARS-CoV is also shown to be
responsible for eliciting responses by T-cell (CD4+ and
CD8+) (Huang J et al., 2007).
RBD is a segment in the centre of S1 subunit of S protein
which is ~193 aa residues and binds to receptors present
in the target cells. It has been illustrated that antisera of
SARS infected person and that of animals inoculated
with inactivated SARS-CoV, effectively responded with
RBD (He Y et al., 2004a). As compared to S protein of
full length, high number of neutralizing antibodies were
induced with RBD because in contrast to S protein with
full length, no immunodominant regions are present in
RBD that elicits antibodies that are not neutralizing (He
Y et al., 2004b). Thus, the generation of antibodies
targeting the RBD subunit of SARS-CoV-2 would be an
important preventive and treatment strategy that can be
tested further in suitable models before clinical trials.
So far, several institutions have initiated programs on the
SARS-CoV-2 subunit vaccine. For example, Intravacc.in
collaboration with EpiVax are working on the outer
membrane vesicle (OMV) delivery platform with
synthetically produced SARS-CoV2 epitopes. It is one of
the diverse platforms that are being investigated to
produce a subunit vaccine. The candidate is currently
under pre-clinical evaluation. Other diverse novel
platforms that are being investigated under this strategy
include GP-96 backbone and li-key peptide. Besides,
Johnson & Johnson, Pasteur Institute, and Chongqing
Zhifei Biological Products Co., Ltd. also started subunit
vaccine development against SARS-CoV-2 (WHO,
2020).
4.5 Virus-like-particle vaccines
Virus-like particles (VLPs) are self-assembled viral
structural proteins that mimic the conformation of native
viruses but lack the viral genome. Compared with protein
subunit vaccines, VLP vaccines present epitope in
conformation that is more similar to the native virus,
leading to better immunization responses (Figure 10). In
addition, compared to whole virus vaccines, the
production of VLP vaccines does not involve live virus
or inactivation steps, which makes them safer vaccine
candidates. The highly repetitive antigenic surface of
VLP vaccines also help induce stronger antibody
response by efficiently cross-linking B-cell surface
receptors (Hill BD et al., 2018). VLP‘s are the newest
vaccine development platform. They are further
classified as enveloped and non-enveloped VLP‘s based
on their structure. Enveloped VLP‘s consist of the cell
membrane of the host cell called an envelope and this
envelope contains integrated target antigens displayed on
the surface (B. Sarkar et al., 2019).

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Figure 10: The way virus-like-particle vaccines act.
Few SARS-CoV and MERS-CoV VLP vaccines have
been reported so far. For SARS-CoV, Lokugamage et al.
have demonstrated that chimeric VLPs composed of
SARS-CoV S protein and mouse hepatitis virus E, M and
N proteins can induce neutralizing antibody responses
and reduce SARS-CoV virus titer in mice lung after viral
challenge (Lokugamage KG et al., 2008). For MERS-
CoV VLP vaccines, Wang et al. have shown that VLPs
containing MERS-CoV S, E and M proteins can induce
specific antibody response and Th1-mediated cellular
immunity in rhesus macaques (Wang C et al., 2017a).
The same research group developed another chimeric
VLP vaccine containing the fusion of the receptor-
binding domain (RBD) of MERS CoV S protein and the
canine parvovirus (CPV) VP2 structural protein. They
showed that this VLP vaccine induces MERS-CoV-
specific antibody response and T-cell immunity in mice
(Wang C et al., 2017b). These studies suggested that
VLP vaccines hold the potential for clinically effective
coronavirus vaccines.
Up to now, there have been 13 SARS-CoV-2 protein
subunit vaccines entering clinical trials (WHO, 2020).
Among these vaccines, a leading company Novavax,
with its NVX-CoV2373 vaccine, has entered a phase IIb
trial in South Africa (NCT04533399) and a phase III trial
in the UK (2020-004123-16). NVX-CoV2373 contains a
prefusion stabilized full-length spike protein adjuvanted
with their proprietary saponin-based adjuvant (Novavax,
2020).
4.6 Nucleic acid vaccines
Nucleic acid (DNA and mRNA) vaccines are very quick
to produce, yet were untested as successful human
vaccine strategies. Antigen that encodes either plasmid
DNA or RNA i.e. mRNA or viral replicon, are used in
the nucleic acid based approaches. After being taken and
expressed by a cell, these antigens, which are encoded by
nucleic acid, induce antibody and cell -mediated
response as well. Owing to the simplicity in the
alteration of antigen they permit, both the approaches are
tremendously adaptable. Antigen production in the target
cells suggests the benefit of imitating synthesis of protein
throughout infectivity. Prominently, they allow any
preference antigen delivery, despite of the fact that it was
either isolated from bacteria, virus or any parasite
thereby permitting development of vaccine against broad
pathogen group. Again vaccine characteristics are not
dependent on encoded proteins so, there is no need to set
up new production, purification, validation methods and
manufacturing services for production of nucleic acid-
based vaccines in large scale. Furthermore, these
vaccines are predicted to have minor safety issues as
nucleic acid is swiftly degraded within the human body.
4.6.a DNA vaccines
DNA vaccines contain genes encoding viral antigenic
components that are expressed by plasmid vectors and
delivered into cells through electroporation. Compared
with other vaccine technologies, DNA vaccines offer a
fast and flexible platform for vaccine development and
production, making it an attractive technology to combat
emerging epidemics like SARS-CoV-2. In addition,
antigen production of DNA vaccines happens in the
target cells, which helps recapitulate the native
conformation and post-translational modification of viral
antigens (Figure 11). DNA molecules are generally quite
stable, permitting the storage of DNA vaccines at +4 °C,
thereby simplifying the distribution of this type of
vaccines. However, an important drawback of DNA
vaccines is their limited immunogenicity due to their
inability to spread and amplify in vivo. Therefore, it is
important to consider strategies that can enhance the
potency of DNA vaccines, such as adding adjuvant or
using a prime-boost regimen. Besides, the genomic
integration of DNA vaccines into the host chromosome
is another biosafety concern, which may lead to
mutagenesis and oncogenesis (Rauch S et al., 2018).
Several DNA vaccine candidates have been reported for
SARS-CoV, including the S-, M-, and N protein-based
vaccines (Wang Z et al., 2005). Although all of them can
generate a certain level of antibody and cell-immune
responses, only S protein-based DNA vaccine has been
shown to induce protective effect against SARS-CoV
infection, probably due to the indispensable role of S
protein in receptor binding. Yang et al. has demonstrated
that immunization with DNA encoding full-length S
protein, S protein lacking part of cytoplasmic domain, S
protein lacking both cytoplasmic and transmembrane
domains can all induce neutralizing antibodies and T-cell
immune responses, as well as providing protective effect
in mice (Yang ZY et al., 2004). This promising result
leads to a following phase I clinical trial based on SARS-
CoV full-length S protein DNA vaccine, which showed
that the vaccine was well-tolerated in patients and can

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induce neutralizing antibodies and T cell responses in
healthy adults (Martin JE et al., 2004).
Figure 11: The way DNA vaccines act.
Similar to SARS-CoV, several studies on MERS-CoV
DNA vaccines have demonstrated optimistic results.
Muthumani et al. reported that a full-length S protein-
based MERS-CoV DNA vaccine can induce potent
cellular immunity and antigen-specific neutralizing
antibodies in mice, macaques, and camels, and macaques
vaccinated with this DNA vaccine were protected against
MERS-CoV challenge without demonstrating any
clinical or radiographic signs of pneumonia (Muthumani
K et al., 2015). Building on these encouraging data, a
phase I clinical trial based on this MERS-CoV DNA
vaccine (GLS-5300, or INO-4700) has been completed.
The results showed that GLS-5300 is well tolerated with
no vaccine-associated serious adverse events, and
immunization with GLS-5300 induces durable immune
responses in 85% of participants after two vaccinations
(Modjarrad K et al., 2019). These data support further
development of the GLS-5300 vaccine. Notably, a
SARS-CoV-2 DNA vaccine candidate, INO-4800, is
based on the same design as GLS-5300, and this vaccine
is now in phase I/II clinical trial (NCT04447781 and
NCT04336410) (Smith TRF et al., 2020).
Taken together, DNA vaccines encoding full-length S or
S1 protein have demonstrated encouraging results to
fight against SARS-CoV and MERS-CoV. The same
strategy is likely to be generalizable to SARS-CoV-2
DNA vaccine considering the biological similarity. So
far, two SARS-CoV-2 DNA vaccines are under
development. Inovio Pharmaceuticals developed a DNA
vaccine candidate termed INO-4800, which is in
preclinical studies and will soon enter phase I clinical
trials. Applied DNA Sciences Subsidiary, LineaRx, and
Takis Biotech collaborated for the development of a
linear DNA vaccine candidate against SARS-CoV-2,
which is now in preclinical studies (WHO, 2020).
4.6.b RNA vaccines
RNA vaccines consist of viral antigen-encoding
messenger RNAs that can be translated by human cells to
produce antigenic proteins and stimulate the immune
system. RNA vaccines are usually delivered in complex
with additional agents, such as protamine or lipid- and
polymer-based nanoparticles, to increase its efficacy
(Kauffman KJ et al., 2016). Similar to DNA vaccines,
RNA vaccines have the advantages of being highly
adaptable to new pathogens and being able to
recapitulate the native conformation and modifications of
antigenic proteins (Figure 12). Furthermore, compared
with DNA vaccines, RNA vaccines have some additional
benefits. Unlike DNA, RNA does not interact with host-
cell DNA and therefore obviate the risks of genomic
integration. Besides, RNA vaccines can be given through
multiple routes including traditional intravenous
injection, whereas DNA vaccines need to be
administered via special devices like electroporation or
gene gun. Nevertheless, RNA vaccines do have some
drawbacks. Exogenous RNA can activate interferon-
mediated antiviral immune response and lead to stalled
translation and mRNA degradation, which suppress the
efficacy of RNA vaccines (Sahin U et al., 2004). In
addition, interferon signaling is associated with
inflammation and potential autoimmunity (Pardi N et al.,
2018). Even though there have not been severe cases of
RNA vaccine-induced autoimmune diseases, it is
important to carefully evaluate this potential adverse
effect.
Although there were no RNA vaccine studies for SARS-
CoV or MERS-CoV in the past two decades, there have
already been 6 novel RNA vaccines reaching clinical
trials for SARS-CoV-2 since the outbreak of COVID-19.
So far, a SARS-CoV-2 mRNA vaccine (mRNA-1273,
encoding S protein) developed by Moderna, has been
launched in animal experiments and clinical batch
production. It is expected that clinical trials will be
conducted on 20–25 healthy volunteers by the end of
April. Fudan University is in collaboration with
Shanghai Jiaotong University and Bluebird
Biopharmaceutical Company to develop a SARS-CoV-2
mRNA vaccine using two different strategies. The first is
to use mRNA to express the SARS-CoV-2 S protein and
RBD domain, the efficacy of this vaccine is now under
evaluation in mice. The second is the use of mRNA to
express virus-like particles in vivo. In addition, German
biopharmaceutical company CureVac AG, Stermirna
Therapeutics, BDGENE Therapeutics, Guanhao Biotech,
ZY Therapeutics Inc., CanSino Biologics Inc., Baylor
College of Medicine, University of Texas, Tongji
university also announced their progress on mRNA
vaccine development against SARS-CoV-2 (WHO,
2020).

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Figure 12: The way RNA vaccines act.
4.7 Adjuvant
Adjuvants incorporated in prophylactic and/or
therapeutic vaccine formulations impact vaccine efficacy
by enhancing, modulating, and/or prolonging the
immune response (Figure 13). In addition, they reduce
antigen concentration and the number of immunizations
required for protective efficacy, therefore contributing to
making vaccines more cost effective. Adjuvants are
critical components of both subunit and certain
inactivated vaccines because they induce specific
immune responses that are more robust and long-lasting.
In addition, for live attenuated vaccines and live vector
vaccines, adjuvants are required to enhance the immune
response in the development of vaccines (Bonam SR et
al., 2017). A review of the history of coronavirus vaccine
development demonstrates that only a few adjuvants,
including aluminum salts, emulsions, and TLR agonists,
have been formulated for SARS-CoV and MERS-CoV
vaccines in experimental and pre-clinical studies.
Vaccine development utilizing various platforms is one
of the strategies that has been proposed to address
COVID-19 pandemic. In order to accelerate the
development of a SARS-CoV-2 vaccine, the preferred
adjuvant should be those have been widely used in other
marketable vaccines, including classic aluminum
adjuvant, aluminum adjuvants enhance the immune
response by facilitating phagocytosis and slowing the
diffusion of antigens from the injection site. It can
efficiently stimulate Th2 immune response upon
injection (Hogenesch H, 2013). In addition, MF59,
MF59 is an oil-in-water emulsion composed of Tween
80, sorbitol trioleate, and squalene, and it has already
been used in flu vaccines in Europe and the United
States. The mechanism of MF59 is to create a transient
immune environment at the injection site, then to recruit
immune cells to induce antigen-specific immune
responses (Tsai TF, 2013). Currently, GSK announced
that they would make its established pandemic vaccine
adjuvant platform technology available to enhance the
development of an effective vaccine against SARS-CoV-
2, and agreements have been reached with Clover
Biopharmaceutical Inc. and the University of
Queensland, Australia. Because adjuvants were able to
regulate the type of immune response, the optimal
adjuvant should be selected according to the design of
the vaccine. In order to induce a more efficient immune
response, a combination of different types of adjuvants
could be applied to improve the immune efficacy.
Figure 13: Characteristic Properties of Vaccine Adjuvants.

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5. MECHANISM OF ACTION OF VARIOUS
VACCINE CANDIDATES
The most effective licensed vaccines elicit long-term
antigen-specific antibody responses by plasma cells in
addition to the development of persisting T cell and B
cell memory. In case of SARS-CoV infection both
humoral and cellular immune responses are crucial for
the clearance of infection (Figure 14). Recombinant virus
vectors work in a similar manner like an endogenous
pathogen, by expressing axenic target protein in
cytoplasm of the host cell. After, processing of such
endogenous antigen, MHC class 1 molecules present
them to CD8+ T-lymphocytes, which causes production
of T-cytotoxic cells. This pathway, leads to
establishment of cell-mediated immunity, which is
crucial in getting rid of virus infected cells. Sub unit
vaccine candidate particularly RBD of S protein of
SARS-CoV contains major antigenic determinants that
can induce neutralizing antibodies (Bonavia A et al.,
2003). The SARS-CoV S protein can also induce CD8+
T-cell responses. The RBD of S protein contains multiple
conformation-dependent epitopes and is the main domain
that induces neutralizing antibody and T-cell immune
responses against SARS-CoV infection making it an
important target for vaccine development (He Y et al.,
2006). The approaches for developing RBD-based
vaccines against SARS-CoV have provided useful
information for designing safe and effective vaccines
against SARS-CoV-2 since RBDs of SARS-CoV-2 also
contain similar epitopes. Similarly, Adenoviral vectors
are able to induce potent antibody as well as T cell
responses with variations in the immune response
depending on the serotype employed. Replication
deficient Ad5, one of the most widely used adenoviral
vectors, is able to induce exceptionally potent CD8+ T
cell as well as antibody responses (Humphreys IR et al.,
2018).
Furthermore, DNA vaccination is also able to elicit both
humoral and cellular immune responses, through
activation of CD8+ cytotoxic and CD4+ helper T cells,
respectively. Upon entry in the cell, DNA vaccines are
sensed by a variety of innate immune receptors i.e.
STING/TBK1/IRF3 pathways and the AIM2
inflammasome and many other factors are involved in
DNA vaccine mode of action but the exact mechanism of
action is yet to be evaluated. However, immunization
with S protein encoding DNA vaccine elicited protective
immunity against SARS-CoV infection in a mouse
model by inducing T cell and neutralizing antibody
responses (Yang ZY et al., 2004). Another nucleotide-
based vaccine i.e. Exogenous mRNA is also
immunostimulatory, as it is recognized by a variety of
cell surface, endosomal and cytosolic innate immune
receptors. Mammalian cells can sense foreign RNA via
Pattern recognition receptors (PRRs) such as TLR3,
TLR7 and TLR8 located in the endosomes and RIG-I,
MDA-5 and PKR located in the cytoplasm as well as
NLRP3 and NOD2 (Chen N et al., 2017). Activation of
the PRRs by mRNA vaccines results in a robust innate
immune response including production of chemokines
and cytokines such as IL-12 and TNF at the inoculation
site, which are innate factors crucial for the induction of
an effective adaptive immune response against the
encoded antigen. The mRNA vaccines can also induce an
immunological repertoire associated with the generation
of high magnitude long-lived antibodies (Edwards DK et
al., 2017).
Figure 14: Mechanism of action of various vaccine candidates.

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6. PREVIOUS PROGRESS OF SARS‑COV AND
MERS‑COV IMMUNIZATION STRATEGIES
6.1 Vaccines for SARS‑CoV
After the SARS epidemic in 2002–2003, several
laboratories around the world started to conduct vaccine
development studies for preventing the disease (Table 3).
The majority of the subunit vaccines targeted the spike
(S) glycoprotein of the virus. SARS-CoV uses this
glycoprotein to bind and enter the host cells (Du L et al.,
2009). Therefore, a vaccine that induces strong immune
responses against this protein will have a significant
effect on the deterrence of virus entry to the host cells
during natural infection.
Vaccines based on a live-attenuated or inactivated virus,
recombinant viral vectors, DNA, virus-like particles
(VLPs) and soluble proteins were studied, mainly in pre-
clinical studies. Live-attenuated and inactivated viruses
are based on the use of the whole SARS-CoV as a
vaccine. The virus has been rendered nonreplicating, and
infectivity has been greatly reduced by means of deleting
components of the virus genome or by using physical or
chemical methods (Petrovsky N et al., 2004). In the case
of recombinant viral vectors, viruses different from the
SARS-CoV that are capable of host cell infection have
been genetically engineered to express components of
the SARS-CoV (Lauer KB et al., 2017). VLPs are non-
infectious multiprotein structures formed from viral
proteins that self-assemble into virus-like structures
(Urakami A et al., 2017). So far, only vaccines based on
an inactivated SARS virus, DNA and soluble proteins
based on the SARS S glycoprotein reached a clinical
stage (phase I) (Lin JT et al., 2007; Martin JE et al.,
2008; NIH, 2013).
6.2 Vaccines for MERS‑CoV
Several vaccines have been developed for MERS
coronavirus since its emergence in 2012 (Table 3). As in
the case of the SARS vaccines, most of the subunit
vaccines for MERS are based on the S glycoprotein.
Vaccines based on inactivated and live attenuated
viruses, recombinant viral vectors, nanoparticles, DNA
and soluble proteins have been developed and tested
predominantly in animal models. Up to date, only a
DNA-based vaccine has already been tested in clinical
trials (phase I) (Modjarrad K et al., 2019) with other
vaccines such as MVA (modified vaccinia virus Ankara)
and adenoviruses being currently under study at that
clinical stage (NIH, 2019).
Table 3: Different types of vaccination strategies against SARS-CoV and MERS-CoV (Pandey SC et al., 2020).
Vaccines strategies
Vaccine candidates
Phase
Nucleotide based
DNA vaccines
S, M and N genes
Phase I, II
(NCT03721718)
mRNA vaccines
mRNA -1273 and
BNT162 encoding S protein
Phase I
(mRNA-1273)
and Preclinical
(BNT162)
Subunit vaccine
Spike glycoproteins (S), Membrane proteins (M),
Nucleoproteins (N)
Preclinical
Recombinant Vector Vaccines
Coronavirus proteins/glycoproteins expressed by
attenuated adenovirus/poxvirus/newcastle disease
virus
Phase I
(NCT03399578,
NCT03615911
Attenuated vaccines
Gene deletion of various essential genes (S,N,E
genes),
Nonstructural proteins (nsp) encoding genes
Preclinical
Inactivated virus vaccines
Inactivated or whole killed virus (WKV)
Preclinical
7. RECENT PROGRESS ON SARS‑COV‑2
VACCINE DEVELOPMENT
Since the publication of the genome sequence of SARS-
CoV-2, on January 11th, 2020, an endeavor of
unprecedented speed and magnitude set out to develop a
vaccine against the disease (Table 4). Early scientific
opinions predicted that it would take at least a year to a
year and a half to get a SARS-CoV-2 vaccine approved
for use in the United States. Still, recent advances on the
field have made possible the issuing of emergency use
authorizations (EUAs) by several national and
international drug regulation agencies for different
vaccine candidates against SARS-CoV-2 in less than a
year since the virus genome sequence was released. An
ideal SARS-CoV-2 vaccine should meet the following
requirements: protect not only from severe disease but
also thwart infection in all vaccinated populations,
including less immunocompromised individuals, elicit
long term memory immune responses after a minimal
number of immunizations or booster doses, the
manufacturing company should be able to ramp up
production to produce billions of doses annually and
have the potential to make it easily accessible for
worldwide vaccination campaigns at an affordable cost
and at limited time (AEP, 2020).

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Table 4: COVID-19 vaccine in phase I and II clinical trials (Poland GA et al., 2020).
Vaccine Developers
Vaccine type
Location
Trial number
Phase 1 trials only
Inovio
DNA (INO-4800)
USA
NCT04336410
Genexine
DNA (GX-19)
South Korea
NCT04445389
Academy of Military Sciences; Suzhou Abogen
Biosciences; Walvax Biotechnology
mRNA (ARCoV)
China
∙∙
ReiThera; Lazzaro Spallanzani National
Institute for Infectious Diseases
Gorilla adenovirus vector
(GRAd-CoV2)
Italy
NCT04
Clover Pharmaceuticals; Dynavax Technologies
Protein (SCB-2019)
∙∙
NCT04405908
Vaxine
Protein
Australia
NCT04453852
Medicago; GSK; Dynavax Technologies
Virus-like particle
USA
NCT04450004
University of Queensland; CSL
Proteins
Australia
NCT04495933
Kentucky Bioprocessing
Plant
USA
NCT04473690
Medigen; Dynavax Technologies
Protein (MVC-COV1901)
Taiwan
NCT04487210
Adimmune
Protein (AdimrSC-2f)
Taiwan
NCT04522089
West China Hospital of Sichuan University
Protein
China
NCT04470609
Sanofi; GSK
Protein
∙∙
NCT04537208
Merck; Pasteur Institute
Measles vector
France
NCT04497298
Research Institute for Biological Safety
Problems
Inactivated virus
(QazCovid)
Kazakhstan
NCT04530357
Themis; Merck; University of
Pittsburgh Center for Vaccine Research
Vesicular stomatitis virus
vectored (COVID-19–101)
Belgium;
France
NCT04497298
Symvivo
Oral (bacTRL-Spike)
USA; Canada
NCT04334980
Phase 1 and phase 2 trials
Imperial College London; Morningside Ventures
Self-amplifying RNA
UK
..
AnGes; Osaka University; Takara Bio
DNA (AG0302-COVID19)
Japan
NCT0452708;
NCT04463472
Arcturus; Duke-NUS Medical School
mRNA (LUNAR-COV19)
Singapore
NCT04480957
Johnson & Johnson; Beth Israel
Deaconess Medical Center
Adenovirus serotype 26 vector
(Ad26.COV2-S)
USA
NCT04436276
Novavax
Nanoparticle
(NVX-CoV2373)
USA; South
Africa
NCT04533399
Finlay Vaccine Institute
Protein (Soberana 1)
Cuba
∙∙
Vector Institute
Peptide (EpiVacCorona)
Russia
NCT04527575
Bharat Biotech; Indian Council of Medical
Research; National Institute of Virology
Inactivated virus (Covaxin)
India
NCT04471519
Anhui Zhifei Longcom Biopharmaceutical;
Institute of Microbiology of the Chinese
Academy of Sciences
Protein
China
∙∙
Zydus Cadila
DNA (ZyCoV-D)
India
..
Curevac
mRNA (CVnCoV)
Germany,
Belgium
NCT04449276,
NCT04515147
7.1 Vaccine candidates against SARS-CoV-2 in phase
3 clinical trial
To date, the FDA has issued an Emergency Use
Authorization (EUA) for the Moderna, Pfizer-BioNTech,
and Janssen COVID-19 vaccines. Several other COVID-
19 vaccine candidates remain in development (Table 5).

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Table 5: COVID-19 vaccine in phase III clinical trials (Poland GA et al., 2020).
Vaccine Developers
Vaccine type
Location
Trial number
AstraZeneca; University of Oxford (30
000 participants)
Chimpanzee adenovirus
(ChAdOx1/AXD1222)
UK; India; Brazil,
South Africa; USA
Moderna; National Institutes of Health
(30 000 participants)
RNA (mRNA-1273)
USA
NCT04470427
Pfizer; BioNTech
(44,000 participants)
RNA (BNT162b1 and
BNT162b2)
USA
NCT04368728
The Janssen Pharmaceutical
Companies of Johnson & Johnson (60
000 participants)
Adenovirus serotype 26
vector (Ad26.COV2.S)
USA; Argentina;
Brazil; Chile;
Columbia;
Mexico; Peru;
Philippines;
South Africa;
Ukraine
NCT04505722
The Gamaleya National Research
Centre for Epidemiology and
Microbiology; Academy of Military
Medical Sciences (40,000 participants)
Adenovirus serotype 5
vector and adenovirus
serotype 26 vector
(Sputnik V)
Russia
NCT04530396
CanSino Biologics; Academy of
Military Medical Sciences (40,000
participants)
Adenovirus serotype 5
vector (Ad5CoV)
China; Pakistan
NCT04526990
Sinovac Biotech
(9000 participants)
Inactivated virus
(CoronaVac)
Brazil; Indonesia
..
Sinopharm; Wuhan Institute of
Biological Products
(21,000 participants)
Inactivated virus
The United Arab
Emirates;
Bahrain; Peru;
Morocco;
Argentina;
Jordan
..
Sinopharm; Beijing Institute of
Biological Products
(5000 participants)
Inactivated virus
(BBIBP-CorV)
The United Arab
Emirates
..
7.1.1 Inactivated virus vaccines
i. CoronaVac (Sinovac Research and Development
Co.)
This vaccine (CoronaVac) is a chemically inactivated,
whole-virus preparation administered in a two-dose
regimen (at day 0 and day 28) and was granted an
emergency use authorisation by Chinese authorities in
July, 2020, before the initiation of phase 3 studies. This
authorisation reportedly resulted in nearly 90% of
company employees being immunised with the vaccine
(Wee S-L et al., 2020). No serious adverse events were
reported. The vaccine elicited anti-RBD antibodies, as
measured by ELISA, and neutralising antibodies 14 days
after the second dose of vaccine in 92·4% of individuals
receiving the vaccine at 0 and 14 days, and in 97·4% of
those receiving the vaccine at 0 and 28 days.
Importantly, neutralising antibody responses were
significantly higher in younger adults (aged 18–39 years)
than in older adults (aged 40–59 years), and stronger
responses were noted in participants given the second
dose on day 28 than in those given the second dose on
day 14. A phase 3 trial has been launched in Brazil and
Indonesia, with the trial in Brazil aiming to enrol 9000
health-care personnel.
ii. BBIBP-CorV (Beijing Institute of
Biotechnology/China National Biotech Group-
Sinopharm)
The inactivated virus vaccine candidate developed by
Sinopharm is the result of their collaboration with the
Beijing Institute of Biological Products. 2.3.3. BBIBP-
CorV was developed by β-propiolactone-mediated
inactivation of the 19nCoV-CDC-Tan-HB02 strain
SARSCoV-2 that was replicated in Vero cells and
adjuvanted with aluminium hydroxide (Xia S et al.,
2021). Aluminium hydroxide activates the NLRP3
receptor subunit of the inflammasome and promotes the
secretion of high-levels of inflammasome-derived IL-1β
and IL-18, thus activating proinflammatory mechanisms
of the immune system (He P et al., 2015). Preclinical
studies on animal models showed that the aluminium
hydroxide-adjuvanted vaccine candidate induced the
production of high levels of neutralizing antibodies titers
against SARS-CoV-2 as calculated by microtitration
experiments. A phase 3 clinical trial began in July, 2020,
and plans to enrol 21,000 participants in the United Arab
Emirates, Bahrain, Peru, Morocco, Argentina, and
Jordan. In late August, 2020, Sinopharm researchers
revealed that they had already begun to administer the
vaccine to health-care personnel and groups at high risk
of becoming infected.

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7.1.2 Protein subunit vaccines
i. NVX-CoV2373 (Novavax)
Similar to inactivated pathogen vaccines, protein subunit
candidates usually exhibit an extremely favorable safety
profile but require multiple boost doses and elicit low
grade cellular responses. Maryland-based Novavax has
developed a prefusion full-length recombinant SARS-
CoV-2 S glycoprotein nanoparticle expressed in a
baculovirus-Sf9 system and is administered with an
adjuvant named Matrix M1. Saponin based Matrix M1
adjuvant is used precisely to tackle the absence of cell
mediated immune responses that characterize protein
subunit vaccines (He P at al., 2015). Matrix M-
adjuvanted NVX-CoV2373 was first investigated in
animal models, such as rats and baboons to assess
immunogenicity. Indeed, addition of the adjuvant was
found to significantly enhance antibody production in
immunized BALB/c mice and induce strong T-cell
responses that exhibited a Th1-skewed phenotype.
Administration of a two-dose regimen of Matrix M
adjuvanted NVX-CoV2373 elicited high titer antibodies
that were shown to efficiently neutralize in vitro the
cytopathic effects of SARS-CoV-2 on Vero E6 cells and
also to prevent the infection of mice transfected to
express the human ACE2 receptor with SARSCoV-2.
Moreover, these results were replicated in olive baboons
receiving intramuscularly two doses of Matrix M-
adjuvanted NVXCoV2373 with an interval of 3 weeks
(Tian JH et al., 2021). On September 23rd, Novavax
launched a Phase 3 trial that aims to enrol up to 9000
volunteers in the United Kingdom and is planning to
expand it in the US, India, and other countries.
ii. ZF2001 (Anhui Zhifei Longcom
Biopharmaceutical/Chinese Academy of Medical
Sciences)
The latest subunit vaccine candidate to enter Phase 3
clinical studies is the adjuvanted RBD-dimeric antigen
designed by Anhui Zhifei Longcom Biopharmaceutical
and the Institute of Microbiology of the Chinese
Academy of Medical Sciences. Phase 3 clinical study
was launched on December and will be initially carried
out in China and Uzbekistan while Indonesia, Pakistan
and Ecuador will follow as study sites (Clinical Trial
Identifier: NCT04646590 and Registration Number:
ChiCTR2000040153). The design of the study involves
recruitment of 22,000 volunteers from China and 7000
subjects outside China for a total of 29,000 volunteers.
There are still no published results on this candidate,
however data from its Phase 2 placebo-controlled clinical
trial (Clinical Trial Identifier: NCT04466085) conducted
on a total of 900 participants ranging from 18 to 59 years
old suggest that a 2 or 3 dose regimen is evaluated. Each
immunization will be separated by the next by 4 weeks
(China Daily, 2020).
7.1.3 Nucleic acid vaccines
7.1.3.a mRNA vaccines
i. mRNA-1273 (Moderna/US NIAID)
Moderna‘s COVID-19 vaccine is an mRNA vaccine that
has been shown to be highly effective in preventing
symptomatic COVID-19 disease. The vaccine, mRNA-
1273, received emergency use authorization (EUA) from
FDA in December 2020 for use in individuals 18 years
of age and older, making it the second COVID-19
vaccine authorized in the United States. The vaccine is
based on an mRNA molecule that contains the
information for the synthesis of the stabilized prefusion
form of the SARS-CoV-2 S protein encapsulated in a
lipid nanoparticle vector that enhances uptake by host
immune cells. The administered mRNA uses the host cell
transcription and translation machinery to produce the
viral antigen that is afterward presented in T
lymphocytes and is also directly recognized by B cells of
the host, thereby initiating an adaptive immune response
directed against the S protein of the virus (Corbett KS et
al., 2020). The Moderna vaccination series consists of 2
intramuscular doses, 0.5 mL each, given 4 weeks apart.
Second doses administered within a grace period of ≤4
days from the recommended date for the second dose are
considered valid; however, doses administered earlier do
not need to be repeated. One potential issue for vaccine
deployment is that a storage temperature of –20°C is
required (Jackson LA et al., 2020).
ii. mRNA-BNT162b2/Comirnaty
(Pfizer/BioNTech/Fosun Pharma)
Pfizer-BioNTech‘s COVID-19 vaccine is an mRNA
vaccine that has been shown to be highly effective in
preventing symptomatic COVID-19 disease. The
vaccine, BNT162b2, received emergency use
authorization (EUA) from the U.S. Food and Drug
Administration in December 2020 for use in individuals
16 years of age and older, making it the first COVID-19
vaccine authorized in the United States. Preliminary data
in non-human primate models revealed that
immunization of BALB/c mice with candidate
BNT162b2 induced strong humoral and cellular anti-
SARS-CoV-2 responses characterized by high titers of
specific neutralizing antibodies and activation of CD8+
and CD4+ T lymphocytes that exhibited a Th1 skewed
phenotype. Neutralizing antibody levels were assessed
with a VSV-based GFP-encoding vector that had been
pseudo-typed to present the SARS-CoV-2 S protein on
its envelope. Results from Phase 1 randomized placebo-
controlled clinical trials showed that BNT162b2
generates minimum side effects both in younger and
older participants (Annette B. et al., 2020). Also, two
different candidates were evaluated in these trials,
namely BNT162b1 and BNT162b2. Both candidates
induced the production of similarly high dose-dependent
neutralizing antibody titers against SARS-CoV-2 in the
inoculated participants. Indeed, the neutralizing antibody
titers were higher or equal to SARS-CoV-2 convalescent
sera. The Pfizer-BioNTech COVID-19 vaccination series
consists of 2 intramuscular doses given 3 weeks apart.

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Second doses administered within a grace period of ≤4
days from the recommended date for the second dose are
considered valid; however, doses administered earlier do
not need to be repeated. BNT162b2 requires storage at –
80°C, a fact that could pose logistical problems
(Mulligan MJ et al., 2020).
7.1.3.b DNA vaccines
i. INO-4800 (Inovio/International Vaccine Institute)
Although Pennsylvania-based company Inovio has not
yet entered officially Phase 3 trials their candidate is the
most advanced SARS-CoV-2 DNA vaccine so far.
Inovio Pharmaceuticals has developed several
experimental DNA-based vaccines which are
administered intradermally with the aid of a portable
device called ‗Cellectra 2000‘ that delivers a small
electric pulse allowing for efficient cellular and nuclear
uptake of the DNA molecules through an electroporation
mechanism. Their candidate is a two-dose vaccine
(INOVIO, 2020).
7.1.4 Replication-defective viral vector vaccines
i. Ad5-nCoV (CanSino Biological/Beijing Institute of
Biotechnology/Academy of Military Medical
Sciences)
The Chinese company CanSino Biologics in
collaboration with the Institute of Biology of China‘s
Academy of Military Medical Sciences developed a
candidate using human adenovirus serotype 5 vector
(Ad5) to deliver the information that codifies for SARS-
CoV-2 full-length S protein into host cells. Ad5 is the
main adenoviral serotype in humans, meaning that a
significant percentage of individuals may have recent
contact, and thus, pre-existing immunity against the viral
vector that could hamper robust immune responses
against the presented antigen as well.
This candidate vaccine was tested in a phase 1 clinical
trial of 108 healthy adults aged 18–60 years. Neutralising
antibody titres increased by at least four times from
baseline in 11 (31%) of 36 participants in the middle
dose group at day 14 and in 18 (50%) at day 28, and in
15 (42%) of 36 participants in the high-dose group at day
14 and in 27 (75%) at day 28 (Zhu FC et al.,2020). The
phase 3 trial includes 40 000 participants aged 18 years
and older and is underway in Pakistan and China.
Information on storage conditions has not yet been
released for this vaccine, but storage conditions are likely
to be similar to those of other vaccines based on
adenovirus vectors and might involve either refrigeration
or storage at –20°C.
ii. AZD1222 (AstraZeneca/Oxford University)
Oxford University (Oxford, UK) and AstraZeneca have
developed a chimpanzee adenovirus-vectored
investigational vaccine (ChAdOx1/AZD1222) encoding
the spike glycoprotein of SARS-CoV-2 (van Doremalen
N et al., 2020). The vaccine showed both
immunogenicity and protective efficacy in non-human
primates given a prime-boost vaccination schedule. A
phase 1/2 trial with 543 individuals receiving the
AZD1222 vaccine tested a prime (5·0 × 10¹⁰ viral
particles) and a prime-boost (2·5 × 10¹⁰ or 5·0 × 10¹⁰
viral particles) schedule (Pedro M Folegatti et al., 2020).
The study showed the induction of humoral responses,
characterised by anti-spike glycoprotein IgG and
neutralizing antibodies, and IFNγ T-cell responses in
most recipients after the first dose of vaccine and an
additional increase in humoral immune outcomes after
the second dose of vaccine. Humoral immune outcomes
in vaccine recipients were similar to those observed in
convalescent plasma from patients who had recovered
from COVID-19. A significant benefit of Oxford-
AstraZeneca‘s COVID-19 vaccine over the Moderna and
Pfizer COVID-19 vaccines is that it can be stored and
distributed at 2-8°C.
iii. Gam-COVID-Vac/Sputnik V (Gamaleya Research
Institute/Health Ministry of the Russian
Federation/Acellena Contract Drug Research and
Development)
The Gamaleya National Research Centre for
Epidemiology and Microbiology have published the
results of two phase 1/2 clinical trials of their COVID-19
vaccine consisting of recombinant adenovirus serotype
26 (rAd26) vector and recombinant adenovirus serotype
5 (rAd5) vector, both carrying the gene for the SARS-
CoV-2 spike glycoprotein (rAd26-S and rAd5-S)
(Logunov DY et al., 2020). These candidate vaccines
(1·0 × 10¹¹ viral particles per vaccine dose) were tested
in 76 healthy individuals aged 18–60 years (38
participants in each study). Local and systemic reactions
were mild, and 100% of recipients in seroconverted, with
RBD ELISA titres and neutralizing antibody titres equal
to or more than titres observed in convalescent plasma
from patients who had recovered from COVID-19. CD4+
and CD8+ Th cell immune responses were detected in all
volunteers and peaked at day 28 after vaccination. A
phase 3 safety and efficacy trial will recruit 40,000
participants from different age and risk groups (Bucci E,
2020).
iv. JNJ-78436735/Ad26.COV2.S (Janssen and Beth
Israel Deaconess Medical Center)
Janssen Pharmaceuticals is the vaccine development
branch of Johnson & Johnson pharmaceutic. Their
candidate is a replicating-defective adenovirus 26 based
vector expressing the stabilized pre-fusion S protein of
SARS-CoV-2, a method developed a decade ago by
researchers of the Beth Israel Deaconess Medical Center.
Their main difference from the CanSino vaccine
candidate is the adenovirus serotype. As opposed to the
ubiquitous Ad5 serotype, very few people have been
exposed to the rare Ad26 serotype, therefore, pre-
existing immunity against the vector reducing this
candidate‘s immunogenicity is not expected to be a
major concern. The second advantage of this candidate is
that the dosing schedule involves a single immunization.
This candidate vaccine requires storage at 2–8°C (Loftus
P, 2020). On November 15th, Janssen informed that they

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will initiate a second Phase 3 randomized, double-blind,
placebo-controlled clinical trial studying the safety and
efficacy of a two-dose regimen of their candidate. The
study will involve 30,000 adult participants from
Belgium, Colombia, France, Germany, the Philippines,
South Africa, Spain, the United Kingdom and the United
States that will receive either two doses of the
Ad26.COV2. S vaccine candidate or a placebo with a 57-
day interval (Johnson & Johnson, 2020).
8. CHALLENGES IN VACCINE DEVELOPMENT
An ideal vaccine should be safe, even in
immunocompromised people, should be highly effective
and optimally induce ‗sterilizing‘ immunity, should retail
immunogenicity despite adverse storage, should be
inexpensive, free from toxicity and adverse effects,
should give long term protection, should have high
thermal stability. Vaccine development is a lengthy,
expensive process and many challenges arise during the
development, manufacturing, and mass distribution (Ada
GL, 1991).
8.1 Time
The major challenge in developing SARS-CoV2 vaccine
is the fast tracking of every step in the discovery,
development, and evaluation process. Traditional vaccine
development can take 15 years or more, starting with a
lengthy discovery phase in which vaccines are designed
and exploratory preclinical experiments are conducted
(Figure 15). This is usually followed by a phase in which
more formal preclinical experiments and toxicology
studies are performed and in which production processes
are developed. During this process an investigational
new drug (IND) application is filed and the vaccine
candidate then enters phase I, II and III trials. If, when
phase III trials are completed, the predetermined end
points have been met, a biologics licence application
(BLA) is filed, reviewed by regulatory agencies and
finally the vaccine is licensed. After that point, large-
scale production begins (Krammer F, 2020). However,
due to an expedited increase in the number of COVID-19
cases worldwide, vaccine regulatory authorities both at
international and national levels are forced to fast track
every process of development to meet the world‘s
immediate vaccine requirement. The scientific
communities are using multiple approaches to shorten
development phase including overlapping clinical phase
and using advanced computer-aided and biotechnological
tools. Vaccine development for SARS-CoV-2 is
following an accelerated timeline. Because of knowledge
gained from the initial development of vaccines for
SARS-CoV and MERS-CoV, the discovery phase was
omitted. Existing processes were adopted, and phase I/II
trials were started. Phase III trials were initiated after the
interim analysis of phase I/II results, with several clinical
trial stages running in parallel. In the meantime, vaccine
producers have started the large-scale production of
several vaccine candidates, at risk.
Figure 15: Traditional and accelerated vaccine-development pipelines.

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8.2 Toxicity and adverse effects
The fast-tracking of vaccine development processes
highten the risk of increased side effects. There is an
immense possibility that some essential data might go
missing or unnoticed at this accelerated speed of
development. Researchers are concerned about the risk
to public health if any important evaluation goes under
notice. The use of animal models as a requirement of
preclinical evaluation is usually needed to assess the
safety and access risk associated with the new vaccine
candidates or combination vaccines before starting first-
in-human trials. Thorough pre-clinical and laboratory
testing have to go in compliance with good laboratory
practice guidelines and with national guidelines on
animal experimentations. These studies are also
necessary to establish characteristics (physical, chemical,
and biological) of the vaccine candidates. Some research
groups have omitted these essential animal testing
studies in the face of global health crisis while others are
running parallel pre-clinical and first-in-human trials.
This break from the usual protocol is worrisome and a
challenge that requires prime attention. Note that the
translation of animal studies to humans may not 100%
with regards to toxicity studies. However, some animal
models such as the mice, ferrets, syrian hamster, and
rhesus macaques are demonstrating symptoms similar to
humans upon exposure to SARS-CoV-2 (Deb B et al.,
2020).
Safety assessment of very recent technologies like DNA,
RNA, VLP‘s is to be given more importance as even if
there is evidence of safety and efficacy, there are very
few/no vaccine so far licensed and used in a large
population. Serious adverse events and allergies were
previously reported due to protein, non-protein
impurities found in vaccines therefore thorough quality
check during the manufacturing process is mandatory
before distribution. A lamentable occurrence had
occurred on account of polio immunization in 1955
because the cycle of inactivating the live virus was
flawed. There were reports of paralysis and within a
month the mass immunization program against polio was
deserted. It was later uncovered that the vaccine had
resulted in causing 40,000 instances of polio, leaving 200
youngsters with differing degrees of paralysis and
several deaths (Fitzpatrick M, 2006). Therefore
manufacturing methodology has to be extremely audited
and validated; manufacturers should be extremely
meticulous in producing large quantities of vaccine
doses. This will be challenging during the present race to
license the first SARS-CoV2 vaccine and mass-produce
a large number of doses.
8.3 Mutations
In the early pandemic situation, there was concern
among the scientific community over mutations arising
in SARS-CoV-2. However recent studies have indicated
no cause for concern. The outcomes of phylogenetic
examination of various SARS-CoV-2 strains procured
from various nations showed that all the glycoproteins of
various strains of SARS-CoV-2, obtained from various
nations were strongly related to each other; hence
antibody structured against one strain would be
successful against the various strains of SARS-CoV-2
from various nations. Nevertheless, it is essential to
continuously monitor genomic sequence given the
knowledge of previous experience on virus mutation rate
(Duffy S, 2018).
8.4 Long term protection
Ideally, vaccination should provide long term protection.
However, immunization induced resistance blurs after
some time and the loss of protection varies with every
disease (John Cohen, 2019). Two doses of inactivated
polio antibody (IPV) are 90 % effective or more against
polio and three doses are 99 %–100 % effective and the
duration of protection lasts for several years to decades
(CDC, 2018). Most promising SARS-CoV-2 vaccine
candidates in clinical trials require booster doses. It is too
early to say any of it provides long term protection.
Reinfection is another major aspect affecting the
protection period. A very recent study has confirmed
reinfection with genomic evidence. It was concluded that
SARS-CoV-2 might flow among the human populace
regardless of crowd insusceptibility on account of
general infection or immunization. Additional
monitoring of patients with reinfection will help
optimized vaccine design against SARS-CoV-2 (To KK
et al., 2020).
8.5 Antibody-dependent enhancement (ADE)
Vaccination induces humoral and cellular immune
response in immunized individuals. In the normal
condition, when the homologous virus enters an
immunized body, it will be neutralized or cleared by
vaccine- induced neutralizing antibodies (Abs) or
specific T cells, respectively. In the context of vaccine-
associated disease enhancement, vaccines mainly induce
non- neutralizing Abs or low titres of neutralizing Abs
(suboptimal concentration) or type 2 T helper cell (TH2
cell)- biased T cell responses (Figure 16). When these
vaccinated individuals are challenged by homotypic or
heterotypic serotype viruses, the antibodies will
immediately recognize the viruses and mediate antibody-
dependent disease exacerbation in two ways. First, virus–
antibody complexes might enter Fc receptor (FcR)-
bearing cells, such as dendritic cells and monocytes, by
FcR- mediated internalization, which is termed
‗antibody- dependent enhancement‘ (ADE). For viruses
with innate tropism for FcR- bearing cells, such as
dengue virus, ADE will result in higher viral loads than
in conditions without antibodies. After entry, the virus,
no matter whether it replicates or does not replicate, may
activate a harmful immune response, resulting in the
release of proinflammatory cytokines (Tirado SM et al.,
2003).
ADE of ailment is an overall worry for the development
of immunizations and treatments since it possibly
intensifies the infection or triggers dangerous

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immunopathology (Arvin AM et al., 2020). A serious
ADE was seen in the case of the dengue vaccine. The
rate of hospitalization was increased in vaccinated
children than in non-vaccinated children. It was
discovered that the antibody imitated the primary
infection and that a reduction in the immunity presented
a few youngsters to the danger of ADE in case of a
subsequent secondary infection. ADE is believed to be
liable for causing COVID-19. So far there are none in-
vitro, in-vivo, or clinical proof of ADE happening in
COVID-19 patients nonetheless, ADE may represent
some serious results during the regular course of the
illness (Negro F, 2020). Careful monitoring of ADE over
several years post-vaccination is necessary especially
during the mass vaccination program as ADE is evident
when enough people have been vaccinated.
Figure 16: Mechanisms of vaccine-associated disease enhancement. a | ADE causes an increased viral replication
or b | may activate the release of proinflammatory cytokines. c | Aside from ADE, antibody–antigen complexes
can stimulate the complement pathway through activation of the C1q pathway, thus further strengthening the
inflammatory responses. d | Vaccine- associated disease enhancement can also involve a TH2 cell- biased
immune response. The activated TH2 cells contribute to the activation of antibody production. However, they
release interleukin-4 (IL-4), IL-13 and IL-5, as well as eosinophil chemoattractant, thus resulting in eosinophil
infiltration and proinflammatory cytokine production in the lung. Natural killer (NK) cells and CD8+ cytotoxic
T lymphocytes (CTLs) are poorly stimulated in TH2 cell- skewed immune responses. The exaggerated cytokine
release, activation of the complement pathway and the excessive mobilization of eosinophils all contribute to the
infiltration of the lung by eosinophils, neutrophils and lymphocytes, and production of inflammatory cytokines,
leading to acute lung injury or acute respiratory distress syndrome.
8.6 Cost
Even if a single vaccine is proven safe and efficacious,
large scale manufacturing and distribution will be
challenging especially if vaccine candidate involves
novel technologies as very few manufacturing plant have
previous experiences in mass production. The
establishment will have to comply with the GLP
guidelines for the particular vaccine candidate. Setting
up new premises and infrastructures for vaccine
production which is meeting complete quality guidelines
will have cost involving. Also, in the current global rush
to develop a vaccine, there is a possibility that this very
crucial compliance step might miss adequate attention;
posing a potential danger. The challenge is to vaccinate

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the entire world population. Experts worry that this
might be physically difficult to achieve owing to
resource scarcity. Also, there should be production
balanced against the need for other vaccines. The kind of
infrastructure needed for production will depend on the
type of vaccine (To KK et al., 2020). The world‘s
governments and companies need to invest enough
money so that vaccines can be made quickly available.
Financial losses may also occur if the COVID-19
pandemic is ended before development phases are
completed as experienced from previous epidemics like
SARS and MERS.
9. CONCLUSIONS AND PROSPECTS
The rapid spread of severe acute respiratory syndrome
coronavirus 2 (SARS- CoV-2) has elicited an equally
rapid response aiming to develop a COVID-19 vaccine.
There are more questions than answers for the newly
identified virus, including the etiology, epidemiology,
structural basis, mechanism of pathogenesis, pathological
immune response, etc. Particularly, the cellular and
humoral immune response of the host in response to the
virus infection that are essential for vaccine design,
remains unclear. All these aspects need to be addressed
by basic research in the near future for successful
vaccine development. Countries all over the world,
regardless of political ideologies, can unite and work
together to achieve fast and successful COVID-19
vaccine development in the near future. More studies are
required urgently to reach the most successful vaccine
candidate in order to minimize the growing number of
COVID-19 cases. Recently, more and more countries
and R&D institutions announced their program on
vaccine development against SARS-CoV-2.
However, vaccine development has its own rules. After
vaccine design and preparation, it will undergo efficacy
and safety evaluation, quality standard establishment
before entering clinical trials. Post-licensure vaccine
safety assessment is a long-term assessment of adverse
event occurring post-vaccination. Keeping entire hope on
a vaccine to end the pandemic should not be encouraged.
Since the vaccine alone cannot combat the pandemic,
modern preventive social strategies are needed so that
the world can battle the present pandemic and be able to
face another pandemic if it occurs in the future.
According to WHO, world leaders and the public must
follow and come up with a novel social measure to
reduce viral concentrates within the human populace.
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