Scrutinizing the SARS-CoV-2 protein information for the designing an effective vaccine
encompassing both the T-cell and B-cell epitopes
Neha Jain
¶
, Uma Shankar
¶
, Prativa Majee
¶
, Amit Kumar*
Discipline of Biosciences and Biomedical Engineering, Indian Institute of Technology Indore, Indore, Simrol,
Indore, 453552, India
¶
These authors contributed equally to this work
*To whom correspondence should be addressed.
Amit Kumar, Discipline of Biosciences and Biomedical Engineering, Indian Institute of Technology-Indore, Indore,
Simrol - 453 552, India, Contact - +91-732-430-6771, Email: amitk@iiti.ac.in
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ABSTRACT
Novel SARS coronavirus (SARS-CoV-2) has caused a pandemic condition world-wide and
has been declared as public health emergency of International concern by WHO in a very
short span of time. The community transmission of this highly infectious virus has severely
affected various parts of China, Italy, Spain and USA among others. The prophylactic
solution against SARS-CoV-2 infection is challenging due to the high mutation rate of its
RNA genome. Herein, we exploited a next generation vaccinology approach to construct a
multi-epitope vaccine candidate against SARS-CoV-2 with high antigenicity, safety and
efficacy to combat this deadly infectious agent. The whole proteome was scrutinized for the
screening of highly conserved, antigenic, non-allergen and non-toxic epitopes having high
population coverage that can elicit both humoral and cellular mediated immune response
against COVID-19 infection. These epitopes along with four different adjuvants were utilized
to construct a multi-epitope vaccine candidate that can generate strong immunological
memory response having high efficacy in humans. Various physiochemical analyses revealed
the formation of a stable vaccine product having a high propensity to form a protective
solution against the detrimental SARS-CoV-2 strain with high efficacy. The vaccine
candidate interacted with immunological receptor TLR3 with high affinity depicting the
generation of innate immunity. Further, the codon optimization and in silico expression show
the plausibility of the high expression and easy purification of the vaccine product. Thus, this
present study provides an initial platform of the rapid generation of an efficacious protective
vaccine for combating COVID-19.
Keywords: Protective vaccine; COVID-19; SARS-CoV-2; multi-epitope vaccine; Next
generation vaccinology; Antigenic epitopes
INTRODUCTION:
The most disastrous outbreak of recent days which have put the world into a pandemic threat
involves the novel Severe Acute Respiratory Syndrome Coronavirus 2 or the SARS-CoV-2 or
the 2019-nCoV causing the COVID-19 outbreak. According to the situation report published
by World Health Organization on March 25th, 2020, 413,467 confirmed cases have been
reported globally along with 18,433 deaths affecting over 197 countries world-wide
(https://www.who.int/emergencies/diseases/novel-coronavirus-2019). Due to easy and
efficient transportation and migration throughout the world, the virus has spread to 197
countries or areas from its epicenter, i.e. the city of Wuhan, China[1]. The SARS-CoV-2
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belongs to the Coronaviridae family being a close relative to the earlier reported Severe
Acute Respiratory Syndrome (SARS) virus and Middle East Respiratory Syndrome (MERS)
virus. This virus majorly affects the lower respiratory tract causing pneumonia, fever, dry
cough, sore throat, dyspnea like symptoms which resolve spontaneously but certain cases
result in fatal complications like severe pneumonia, acute respiratory distress syndrome
(ARDS), organ failure, septic shock or pulmonary edema often leading to death[2]. The
present situation is getting out of control as we don’t have any drug or vaccine to treat this
recently emerged virus and supportive treatment and preventive measures are the only ways
to restrict COVID-19 outcome[3].
Surfacing of SARS-CoV-2 in China was reported recently on December, 2019 and
therefore very limited and naïve information is available regarding its genomic constituency
and pathogenesis. Hence, the researchers rely on its close cousin viruses, SARS-CoV and
MERS-CoV for better understanding of its mode of infection. Phylogenetic analysis have
shown that bat-CoV-RaTG13 displays 96.3% sequence similarity while bat-SL-CoVZC45
and bat-SL-CoVZXC21 exhibit about 88% identity with the SARS-CoV-2 virus but the other
two pathogenic coronaviruses, SARS-CoV and MERS-CoV have less sequence identity
being merely 79% and 50% respectively[4, 5]. Intriguingly, the basic genomic structure for
all these coronaviruses remains the same. The single-stranded positive sense RNA genome of
SARS-CoV-2 basically encodes 9860 amino acids translating into several non-structural
proteins like replicase (orf1a/b), nsp2, nsp3 and accessory proteins (orf3a,orf7a/b) along with
structural proteins including Spike (S), Envelope (E), Membrane (M), and Nucleocapsid (N)
proteins[4]. The Spike glycoprotein aids in receptor binding and thereby, helps in the entry of
the virus in the host cells, while the Envelope protein and the Membrane protein assist in the
virus assembly and packaging of the new virions. On the other hand, the Nucleocapsid
protein is essential for the genomic RNA synthesis required for the viral survival and
proliferation. The non-structural viral proteins including viral enzymes and other accessory
proteins help in the SARS-CoV-2 genome replication and infection[6].
The lessons learnt from the SARS-CoV and MERS-CoV outbreak have helped in
better understanding of the situation of the new coronavirus epidemic and pushed researchers
to develop vaccine or therapeutic solution to succumb the current situation. Previously
several animal models including mouse, Syrian hamster, ferrets as well as non-human
primate models like rhesus macaque, common marmoset have been used to study SARS-CoV
and MERS-CoV and also for the purpose of vaccine development[7-9]. An effective vaccine
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should robustly activate both the humoral and the cell-mediated immunity to establish strong
protection against the pathogen[10]. The antibody generation by B-cell activation as well as
acute viral clearance by T-cells along with virus-specific memory generation by CD8+ T-
cells are equally important to develop immunity against the coronavirus[11]. In case of
respiratory virus like coronaviruses, the mucosal immunity plays an essential role and
therefore route of administration of the vaccine is also important in the context of vaccine
development. Vaccines designed against previously reported SARS-CoV and MERS-CoV
majorly focuses on the Spike protein of virus which consists of S1 and S2 subunits bearing
the receptor binding domain (RBD) of the virus and cell fusion machinery respectively.
Mutations in the spike protein have also been reported to be responsible for the change in
host cell tropism[12]. The S protein is considered most antigenic and thereby can evoke
immune responses and generate neutralizing antibodies that can block the virus attachment to
the host cells[13]. Other viral proteins which were explored for vaccine development include
N protein, E protein and the NSP16 proteins[14-18]. Almost all the platforms for vaccine
development for SARS-CoV and MERS-CoV have been investigated including the life-
attenuated ones, recombinant viruses, sub-unit protein vaccines, DNA vaccines, Viral vector-
based vaccines, nanoparticle-based vaccines, etc. which may form the base for the vaccine
designing against the newly emerged SARS-CoV-2[10, 19]. Here we have designed a multi-
epitope-based vaccine for the SARS-CoV-2 using next generation vaccinology approach
where the recently available genome and proteome of the SARS-CoV-2 were maneuvered
and a potential vaccine candidate was conceived. Similar strategy was employed previously
for SARS-CoV and MERS-CoV[20-25] as well as certain findings are reported for the newly
emerged SARS-CoV-2[26-28]. While immunoinformatics techniques was utilized by groups
to predict the B-cell and cytotoxic T-cell epitopes in the SARS-CoV-2 surface glycoprotein,
N protein[27-29], others have utilized the information to design epitope-based vaccine based
on the SARS-CoV-2 spike glycoprotein[26]. Along with the structural proteins, utilizing the
non-structural and accessory proteins for the vaccine development can aid in better
development of an efficacious vaccine for long term by neutralizing the mutation rate of this
RNA virus. In this study, we explored the whole proteome of SARS-CoV-2 to scrutinize the
highly conserved antigenic epitopes for the construction of a multi-epitope vaccine candidate
that can effectively elicit both humoral and cellular mediated immune response against
COVID-19. The constructed vaccine product has high population coverage and along with
adaptive immunity, can as well lead to initiation of innate immune response further
enhancing the generation of memory immunity.
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With the continuous transmission of the virus across borders and increasing health
burden on the global scale, SARS-CoV-2 demands an urgent immunization therapy. As
depicted by Shan Lu, the Community Acquired Coronavirus Infection (CACI) caused by this
virus can shatter the socio-economic condition worldwide and the development of vaccine
against the SARS-CoV-2 should be encouraged to manage the present situation as well as it
can serve as a prototype for other coronaviruses[30, 31]. Thereby, our analysis provides a
platform for the development of a protective vaccine candidate that can be tested in-vitro and
in-vivo and may lead to faster development of an efficient vaccine against the SARS-CoV-2
infection.
MATERIAL AND METHOD
1. Retrieval of SARS-CoV-2 proteome
The complete proteome of latest reported novel Wuhan strain of SARS Coronavirus
(SARS-CoV-2) was downloaded from the Nucleotide database available at National
Center for Biotechnology Information (NCBI). The database was also thoroughly
searched for the genomes of other available human infecting strains of Coronavirus
till date and was downloaded for further analysis.
2. Antigenicity prediction in the Coronavirus proteome
For the antigenic analysis of the proteome of the COVID-19 strain, VaxiJen v2.0
server available at http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html [32].
For antigenicity prediction of the Proteins of SARS-CoV-2 with higher accuracy,
Virus model available at the VaxiJen server with a threshold of 0.4 was utilized.
Proteins with a VaxiJen score ≥ 0.4 were taken for epitope prediction analysis.
3. Selection of MHC I and MHC II alleles
MHC class I and II alleles were selected on the basis of their occurrence worldwide.
We focused specially on the countries which are severely affected by the deadly
SARS-CoV-2 strain. For MHC class I, following 14 HLA alleles HLA-A01:01, HLA-
A02:01, HLA-A02:03, HLA-A02:06, HLA-A03:01, HLA-A11:01, HLA-A24:02,
HLA-A31:01, HLA-A33:01, HLA-B07:02, HLA-B35:01, HLA-B51:01, HLA-
B54:01, HLA-B58:01 were used that cover 89.84 % of the total world population as
predicted by The Allele frequency Net Database [33]. For MHC II, DRB1_0101,
DRB1_0301, DRB1_0401, DRB1_0405, DRB1_0701, DRB1_0802, DRB1_0901,
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