key: cord-0959037-d2j9wpqk
authors: Kalita, Parismita; Padhi, AdityaK.; Zhang, Kam Y.J.; Tripathi, Timir
title: Design of a peptide-based subunit vaccine against novel coronavirus SARS-CoV-2
date: 2020-05-04
journal: Microb Pathog
DOI: 10.1016/j.micpath.2020.104236
sha: f89d5c37625138c6d8520506adcc5d258efb044e
doc_id: 959037
cord_uid: d2j9wpqk

Abstract Coronavirus disease 2019 (COVID-19) is an emerging infectious disease that was first reported in Wuhan, China, and has subsequently spread worldwide. In the absence of any antiviral or immunomodulatory therapies, the disease is spreading at an alarming rate. A possibility of a resurgence of COVID-19 in places where lockdowns have already worked is also developing. Thus, for controlling COVID-19, vaccines may be a better option than drugs. An mRNA-based anti-COVID-19 candidate vaccine has entered a phase 1 clinical trial. However, its efficacy and potency have to be evaluated and validated. Since vaccines have high failure rates, as an alternative, we are presenting a new, designed multi-peptide subunit-based epitope vaccine against COVID-19. The recombinant vaccine construct comprises an adjuvant, cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and B-cell epitopes joined by linkers. The computational data suggest that the vaccine is non-toxic, non-allergenic, thermostable, with the capability to elicit a humoral and cell-mediated immune response. The stabilization of the vaccine construct is validated with molecular dynamics simulation studies. This unique vaccine is made up of 33 highly antigenic epitopes from three proteins that have a prominent role in host-receptor recognition, viral entry, and pathogenicity. We advocate this vaccine must be synthesized and tested urgently as a public health priority.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belongs to the genus Betacoronavirus of the Coronaviridae family and is identified as the pathogen of Coronavirus disease 2019 (COVID-19) [1] . The epicenter of the COVID-19 coronavirus outbreak was the central Chinese city of Wuhan, from where it spread globally. On 30 th January 2020, the World Health Organization officially declared the COVID-19 epidemic as a public health emergency of international concern. Human to human transmission occurs through droplets, contact, and fomites. People with COVID-19 show symptoms of fever, cough, muscle aches, headache, and diarrhea. The principal feature of the severe disease is acute onset of hypoxemic respiratory failure with bilateral infiltrates.

The virus genome has been sequenced that allowed the development of diagnostic tests and research into vaccines and therapeutics [1, 2] . A specific RT-PCR-based test has been developed that is in use for clinical diagnoses [3] . The abundance of publications in the first three months of 2020 indicates the intensive scientific effort to address both molecular mechanisms and therapeutic routes for treating COVID-19 [4] . More than 200 clinical trials are currently underway to test novel and repurposed compounds against SARS-CoV-2 [5, 6] . Certain drugs, including hydroxychloroquine, chloroquine, and remdesivir, are being tested in clinical trials [7] [8] [9] . One small study reported that combination therapy of hydroxychloroquine with azithromycin reduced the detection of viral RNA compared to control [10, 11] . A recent openlabel trial with two protease inhibitors, lopinavir, and ritonavir, failed [12] . Several inactivated vaccines, viral vectored vaccines (adenovirus vector, ankara vector), nanoparticle-based vaccines, fusion-protein based vaccines, adjuvanted vaccines, recombinant protein, and DNA vaccines, as well as live-attenuated vaccines, are also being developed and tested, but these vaccines are many months away from the market [13] [14] [15] [16] . A phase 1 clinical trial of Moderna's mRNA-based SARS-CoV-2 candidate vaccine, mRNA-1273, has started on March 16, 2020 [17] [18] [19] . However, this is the first of several steps in the clinical trial process for evaluating the potential benefits of the vaccine.

The SARS-CoV-2 consists of single, positive-stranded RNA and four structural proteins: a spike glycoprotein (S), a membrane glycoprotein (M), an envelope protein (E), and a nucleocapsid protein (N) [20] . To enter the host cells, the virus uses a densely glycosylated spike protein that binds to the angiotensin-converting enzyme 2 (ACE2) receptor with high affinity 4 [21, 22] . Structural and biochemical studies suggest that the RBD has an ultra-high binding affinity to the human ACE2 receptor [23] . Few groups have designed subunit vaccines against SARS-CoV-2; however, their workflow involved either use of single protein for vaccine design [24, 25] or used only CTL epitopes without considering the importance of B-cell or HTL epitopes [26] . Some subunit-vaccines are also in preclinical trials [27, 28] . Here, we focused on designing a multi-epitope-based subunit vaccine against SARS-CoV-2 using 33 highly antigenic epitopes. We believe that experimental evaluation may result in a novel and immunogenic vaccine that may confer protection against SARS-CoV-2 infection.

The protein sequences of SARS-CoV-2 were retrieved from the NCBI database (https://www.ncbi.nlm.nih.gov/nuccore/MN996531.1/) for subunit vaccine development (Table   1 ) [29] . Each of these proteins was screened for their average antigenic propensity using the antigenic peptides prediction tool (http://imed.med.ucm.es/Tools/antigenic.pl). Proteins with an antigenic probability score of greater than 0.8 were considered for vaccine construction.

The helper T-lymphocyte (HTL) epitopes for the selected SARS-CoV-2 proteins were predicted using the MHC-II epitope prediction tool from the Immune Epitope Database (IEDB, http://tools.iedb.org/mhcii/). Selected epitopes had the lowest percentile rank and IC 50 values.

Additionally, these epitopes were checked by the IFN epitope server (http://crdd.osdd.net/raghava/ifnepitope/) for the capability to induce Th1 type immune response accompanied by IFN-ϒ production. Cytotoxic T-lymphocyte (CTL) epitopes for the screened proteins were predicted using the NetCTL1.2 server (http://www.cbs.dtu.dk/services/NetCTL/). B-cell epitopes for the screened SARS-CoV-2 proteins were predicted using the ABCPred server (http://crdd.osdd.net/raghava/abcpred/). The prediction of the toxic/non-toxic nature of all the selected HTL, CTL, and B-cell epitopes was checked using the ToxinPred module (http://crdd.osdd.net/raghava/toxinpred/multi_submit.php).

The vaccine subunit was designed by adding an adjuvant, HTL, CTL, and B-cell epitopes connected by specific linkers to provide adequate separation of epitopes in vivo. EAAAK linker was used to join the adjuvant and HTL. Intra HTL, Intra CTL, and B-cell epitopes were joined using GPGPG, AAY, and KK, respectively. To enhance the immunogenicity of the vaccine construct, the TLR-3 agonist, human β-defensin 1 (Uniprot ID: P60022), was used as the adjuvant.

The immunogenicity of the vaccine was determined using the VaxiJen server (http://www.ddgpharmfac.net/vaxijen/VaxiJen/VaxiJen.html) and ANTIGENpro module of SCRATCH protein predictor (http://scratch.proteomics.ics.uci.edu/). The allergenicity of the vaccine was checked using AllerTOP v2.0 (http://www.ddg-pharmfac.net/AllerTOP/) and AlgPred Server (http://crdd.osdd.net/raghava/algpred/).

The physiochemical characteristics of the vaccine were determined using the ProtParam tool of the ExPASy database server (http://web.expasy.org/protparam/).

The secondary structure of the subunit vaccine construct was predicted using PSIPred 4.0 Protein Sequence Analysis Workbench (http://bioinf.cs.ucl.ac.uk/psipred/), while the tertiary structure was predicted by de novo structure prediction-based trRosetta modeling suite. trRosetta uses a deep residual neural network to predict the inter-residue distance and orientation distributions of the input sequence. Then it converts predicted distance and orientation distributions into smooth restraints to build 3D structure models-based on direct energy minimization. The model of the vaccine construct with the best TM-score was validated by PROCHEK v.3.5 structures were saved at every 10 ps for structural and dynamic analysis.

The analysis from MD simulation was performed as described earlier [30] . Briefly, the backbone 

Java Codon Adaptation Tool (JCAT) (http://www.jcat.de/) was used for codon optimization of the vaccine sequence to test high-level expression of the vaccine in E. coli strain K12. NEBcutter (http://nc2.neb.com/NEBcutter2/) was used for the selection of restriction enzyme cleavage sites, and the expression vector pET28a(+) was selected. In silico clone of the vaccine was designed using the SnapGene 1.1.3 restriction cloning tool.

The amino acid sequence of the three SARS-CoV-2 proteins, namely, nucleocapsid protein, membrane glycoprotein, and surface spike glycoprotein, were retrieved from the NCBI database (Table 1) . These proteins are known to have a prominent role in host receptor recognition, viral entry, and pathogenicity. The proteins with an antigenic score of greater than 0.8 (Table 1) were used further for the prediction of epitopes for subunit vaccine designing. A schematic representation of the methodology for the construction of the subunit vaccine candidate is shown in Figure 1 .

Helper T-lymphocytes are the key players of the adaptive immune response. They are involved in the activation of B-cells and cytotoxic T cells for antibody production and killing infected target cells, respectively. All three proteins were subjected to the IEDB MHC-II epitope prediction module for HTL prediction. A total of six highest immunogenic epitopes of 15-mer 8 were selected based on their percentile rank and IC 50 values. Also, all these epitopes showed positive scores on IFNepitope server output (Table 2) . B-cells are the main components of humoral immunity during the adaptive immune response that produces antibodies, which recognize antigens. Therefore, it was necessary to predict B-cell epitopes before vaccine designing. ABCpred was performed for predicting B-cell epitope, and a total of 9 epitopes with top scores from the three proteins were considered for the vaccine (Table 3) .

CTL epitopes are essential for inducing MHC-I cellular immune response by neutralizing virus-infected cells and damaged cells via releasing cytotoxic proteins like granzymes, perforins, etc. The CTL epitopes were predicted for all selected proteins using the NetCTL 1.2 server.

Here, A2, A3, and B7 supertypes were considered for prediction as they cover at least 88.3% of the total ethnic population. Eighteen epitopes with a combined score of >0.75 were finally considered for the vaccine (Table 4 ). All the selected HTL, CTL, and B-cell epitopes were subjected to the ToxinPred module to screen for their toxicity. Supplementary Table 1 shows that all epitopes chosen for the vaccine were non-toxic.

A total of 6 HTLs, 18 CTLs, and 9 B-cell epitopes derived from the three proteins were used to design the subunit vaccine (566 amino acid residues) against SARS-CoV-2 (Supplementary Figure 1) . The human β-defensin 1(68 amino acid residues) sequence was added as an adjuvant followed by the HTL, CTL, and B-cell epitopes and linked by specific linkers.

An important parameter of vaccine designing is ensuring that the constructed vaccine is immunogenic to induce a humoral and/or cell-mediated immune response against the targeted virus. The computational data suggest that our vaccine is antigenic with a probability score of 0.513 and 0.732 predicted by VaxiJen v2.0 and ANTIGENPro servers, respectively. The allergenicity score was found to be −0.658 in AlgPRED prediction module. Additionally, the vaccine was also found to be non-allergic using AllerTOP v2.0. respectively, suggesting that the vaccine is thermostable and hydrophilic, respectively.

The secondary structure was predicted using the PSIPRED 4.0 server (Supplementary Figure 2) .

The tertiary structure of the vaccine was predicted using the trRosetta modeling suite. The 3D model generated by trRosetta modeling was subjected to the PROCHECK server, where

Ramachandran plot statistics were generated. The output showed 98.4% residues were present in the favored region, 1.0% residues in the generously allowed region, and 0.6% residues in disallowed regions. Further, the Z-score plot and energy plot was generated by the ProSA web server. The calculated Z-score (−8.46) lies within the X-ray crystal structure range. The energy plot suggested that all the residues have low energy value in the modeled structure ( Figure 2 ).

Vaccine-receptor docking was performed to evaluate the binding energy of the vaccine with its TLR-3 receptor. ClusPro analysis provided 30 vaccine-receptor complexes with respective energy scores. The lowest energy complex with a binding energy of about −1491 kJ.mol -1 was selected and subjected to MD simulation ( Figure 3 ).

The binding modes, dynamics, and stability of the vaccine-TLR3 complex were evaluated using a 40 ns MD simulation study (Figure 4) . The atomic-level interaction between vaccine and TLR3 was determined, and root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), hydrogen bond, and contact energy were calculated.

The RMSD data suggests that the receptor-vaccine complex was stabilized after about 20

ns until the end of the simulation ( Figure 4A ). All the calculations were then done for the 20-40 ns MD simulation trajectory. Next, RMSF calculation, which gives information about the residue wise dynamics of a protein with respect to its initial position, was done. An average RMSF value of 0.39 nm was observed for the complex ( Figure 4B ). Our subsequent analysis of the changes in the Rg for the vaccine-TLR3 complex during the simulation was also determined, and the average Rg was found to be 1.47 nm, demonstrating the compactness of the TLR3 receptor with vaccine subunit during the simulations ( Figure 4C ). We further analyzed the hydrogen bonds that Typically, the largest associated eigenvalues define the essential subspace in which most of the protein dynamics occur. For this, the clusters of stable states of PCA for the TLR3-vaccine complex were visualized and analyzed. The trace value calculated from the covariance matrix of the TLR3-vaccine was found to be 2.18 nm 2 , suggesting that the complex exhibited compact behavior during the simulation (Supplementary Figure 3) . Lastly, the detailed interactions between TLR3 and the vaccine protein were computed from the starting structure of MD simulations and the stabilized structure of the complex extracted from MD simulated trajectory ( Figure 5 and Table 5 ). The higher total number of interactions in the stabilized complex suggests the stability and tighter binding of the vaccine with TLR3.

The codon optimization index ensures the relationship between codon usage and gene expression in a heterologous system. The JCAT output was further analyzed in NEBcutter, and at the N-and 11 C-terminal ends of the optimized vaccine sequence, BamHI and NdeI restriction sites were added that are non-cutters for the vaccine construct but are present in the multiple cloning site of the selected expression vector pET28a(+). In silico clone was generated using the SnapGene 1.1.3 restriction cloning tool that resulted in a cloned product of 7034 bp ( Figure 6 ).

Some reports suggest that 5 to 10% of recovered patients in Wuhan test positive again; this indicates a possibility of a resurgence of COVID-19 in places where lockdowns have already worked. As a consequence, the spread can also be caused by asymptomatic carriers [31] [32] [33] . A positive re-test, however, may also be because the original test was false-negative, and the patient was not actually COVID-negative. Whatever may be the case, a vaccine is a better option for coronavirus management than drugs.

The efforts to produce a vaccine against coronavirus are moving at a rapid pace. Two candidate vaccines are in Phase I clinical trials: i) An adenovirus type-5 vector-based vaccine, and ii) an LNP-encapsulated mRNA vaccine. Studies evaluating the safety and immunogenicity of these vaccines are underway. Additionally, several vaccine candidates are under preclinical evaluation [34] . Though these trials are underway, there are known situations that vaccines have failed.

Recently few groups have tried designing subunit vaccines against SARS-CoV-2; however, their workflow involved either use of single protein for vaccine design [24, 25] or used only CTL epitopes without considering the importance of B-cell or HTL epitopes [35] . We considered all of these points while designing the vaccine. Based on extensive bioinformatics analysis, we used three proteins to design a multi-epitope subunit vaccine against novel coronavirus SARS-CoV-2.

These proteins are nucleocapsid protein (N), membrane glycoprotein (M), and the surface spike glycoprotein (S). The N protein is involved in packaging the viral genome into a helical ribonucleocapsid, and it plays a fundamental role during viral self-assembly [36] . The M protein is responsible for the assembly and immunogenicity of virus particles. The S protein mediates the entrance of the virus to human respiratory epithelial cells by interacting with cell surface receptor ACE2. The S protein has two regions: S1, for host cell receptor binding; and S2, for membrane fusion. The S protein is a key target for the development of vaccines, therapeutic antibodies, and diagnostics for coronavirus [15, 37, 38] . Although the S protein is a promising immunogen for protection, optimizing antigen design is critical to ensure an optimal immune response. Our vaccine contains a suitable adjuvant, HTL, CTL, and B-cell epitopes that are joined by suitable linkers. Furthermore, the epitopes were screened for their toxicity potential.

The subunit vaccine was found to be thermostable, antigenic, and non-allergenic. Molecular docking and MD simulation provided insights about the interaction, stability, and dynamics of the vaccine-receptor complex. The data suggest constructive intermolecular interactions between the vaccine protein and the TLR-3 receptor. Also, the in-silico cloning suggests the potential expression of the vaccine in a microbial expression system, thereby making it a potential vaccine against SARS-CoV-2 infection.

The development of a vaccine is a lengthy and expensive process, with high failure rates, and it typically takes multiple candidates and several years to produce a commercial vaccine.

Upon optimization of the production process, the subunit vaccines can be rapidly tested and released in the market. They consist of only the antigenic portion of the pathogens that may directly elicit an immune response. Additionally, the vaccine does not utilize live pathogen, thus, reducing the risk of pathogenicity reversal. Hence, it can be used in immune-suppressed patients as well and elicit long-lived immunity. Computational studies suggest that our multi-epitope based subunit vaccine has a probability of showing good protective efficacy and safety against SARS-CoV-2 infection in humans. We suggest the synthesis and experimental evaluation of this vaccine to determine its immunogenic potency.

The authors acknowledge RIKEN ACCC for the Hokusai supercomputing resources. 

A pneumonia outbreak associated with a new coronavirus of probable bat origin

The proximal origin of SARS-CoV-2

Early diagnosis of SARS coronavirus infection by real time RT-PCR

Research and Development on Therapeutic Agents and Vaccines for COVID-19 and Related Human Coronavirus Diseases

Network-based drug repurposing for novel coronavirus 2019-nCoV/SARS-CoV-2

Ongoing Clinical Trials for the Management of the COVID-19

Compounds with therapeutic potential against novel respiratory 2019 coronavirus

Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro

Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro

Information for clinicians on therapeutic options for COVID-19 patients

Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial

A Trial of Lopinavir-Ritonavir in Adults Hospitalized with Severe Covid-19

The SARS-CoV-2 vaccine pipeline: an overview

SARS vaccines: where are we?

Immune responses in COVID-19 and potential vaccines: Lessons learned from SARS and MERS epidemic

The COVID-19 vaccine development landscape

NIH clinical trial of investigational vaccine for COVID-19 begins

The pandemic pipeline

Coronaviruses: an overview of their replication and pathogenesis

Structure of SARS coronavirus spike receptor-binding domain complexed with receptor

Structural basis for the recognition of the SARS-CoV-2 by full-length human ACE2

Characterization of the receptorbinding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine

Design of multi epitope-based peptide vaccine against E protein of human 2019-nCoV: An immunoinformatics approach

Development of epitope-based peptide vaccine against novel coronavirus 2019 (SARS-COV-2): Immunoinformatics approach

T Cell Epitope-Based Vaccine Design for Pandemic Novel Coronavirus 2019-nCoV

The first coronavirus drug candidate is set for testing in China

Clover initiates development of recombinant subunit-trimer vaccine for Wuhan coronavirus

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Development of multi-epitope driven subunit vaccine against Fasciola gigantica using immunoinformatics approach

Mystery In Wuhan: Recovered coronavirus patients test negative then positive

Some recovered coronavirus patients in Wuhan are testing positive again. SlashdotMedia

We need to be alert: Scientists fear second coronavirus wave as China's lockdowns ease

WHO. DRAFT landscape of COVID-19 candidate vaccines -21

T Cell Epitope-Based Vaccine Design for Pandemic Novel Coronavirus 2019-nCoV

Transient oligomerization of the SARS-CoV N protein--implication for virus ribonucleoprotein packaging

The spike protein of SARS-CoV--a target for vaccine and therapeutic development

Immunogenicity of candidate MERS-CoV DNA vaccines based on the spike protein

The authors have declared no competing interest.

PK and AKP carried out the experiments. PK and TT conceived the study and participated in its design and coordination. PK, AKP, KYJZ, and TT analyzed the data and drafted the manuscript.All authors read and approved the final manuscript. Figure 1 : Schematic representation of the multi-epitope subunit vaccine candidate designing using B-cell, CTL, and HTL epitopes.

• We present a multi-epitope subunit-based vaccine designed using an integrated immunoinformatics approach. • Our vaccine is made up of 33 highly antigenic epitopes from three vital pathogen proteins.• Computational data predict that the vaccine is non-toxic, non-allergenic, and immunogenic.• An experimental evaluation of this vaccine is required to determine its practical immunogenic potency.

The authors have declared no conflict of interest.