key: cord-1035817-023h20vk
authors: Faiza, Muniba; Abdullah, Tariq; Calderon-Tantalean, Jose Franklin; Upadhyay, Manish Ravindranath; Abdelmoneim, Abdelrahman H.; Akram, Fareeha; Thakur, Bhupender Singh; Abdulaziz, Ibrahim; Ononamadu, Chimaobi James; Ghoraba, Dina Abdelazim; Munawar, Saba; Faruque, MD Fakhrul Islam; Kigen, Collins; Sharma, Abhishek; Kumar, Ashwani; Khalid, Aqsa; Gharip, Ali; Gupta, Ankit; Manikumar, Manne; Chaudhary, Uma
title: In silico multi-epitope vaccine against covid19 showing effective interaction with HLA-B*15:03
date: 2020-06-14
journal: bioRxiv
DOI: 10.1101/2020.06.10.143545
sha: bdad47079700e7b9aae9bf35b79f6f170c1c93eb
doc_id: 1035817
cord_uid: 023h20vk

The recent outbreak of severe acute respiratory syndrome (SARS) coronavirus (CoV)-2 (SARS-CoV-2) causing coronavirus disease (covid19) has posed a great threat to human health. Previous outbreaks of SARS-CoV and Middle East respiratory Syndrome CoV (MERS-CoV) from the same CoV family had posed similar threat to human health and economic growth. To date, not even a single drug specific to any of these CoVs has been developed nor any anti-viral vaccine is available for the treatment of diseases caused by CoVs. Subunits present in spike glycoproteins of SARS-CoV and SARS-CoV-2 are involved in binding to human ACE2 Receptor which is the primary method of viral invasion. As it has been observed in the previous studies that there are very minor differences in the spike glycoproteins of SARS-CoV and SARS-CoV-2. SARS-CoV-2 has an additional furin cleavage site that makes it different from SARS-CoV (Walls et al., 2020). In this study, we have analyzed spike glycoproteins of SARS-CoV-2 and SARS-CoV phylogenetically and subjected them to selection pressure analysis. Selection pressure analysis has revealed some important sites in SARS-CoV-2 and SARS-CoV spike glycoproteins that might be involved in their pathogenicity. Further, we have developed a potential multi-epitope vaccine candidate against SARS-CoV-2 by analyzing its interactions with HLA-B*15:03 subtype. This vaccine consists of multiple T-helper (TH) cells, B-cells, and Cytotoxic T-cells (CTL) epitopes joined by linkers and an adjuvant to increase its immunogenicity. Conservation of selected epitopes in SARS, MERS, and human hosts, suggests that the designed vaccine could provide cross-protection. The vaccine is designed in silico by following a reverse vaccinology method acknowledging its antigenicity, immunogenicity, toxicity, and allergenicity. The vaccine candidate that we have designed as a result of this work shows promising result indicating its potential capability of simulating an immune response.

Phylogenetic analysis 112 The constructed ML tree is shown in Figure 1 . The phylogenetic tree of spike 113 glycoproteins of SARS-CoV and SARS-CoV-2 showed distinct clades of these protein groups. The selection pressure analysis of spike glycoprotein sequences of SARS-CoV-2 showed 123 positive selection on a few sites. However, no single site has been identified which has 124 experienced negative selection. Besides, BUSTED found no evidence of gene-wide episodic 125 diversifying/positive selection. Three positively selected sites were identified by MEME at a 126 p-value threshold of 0.05 and four such sites were identified by FUBAR (two sites coinciding 127 with MEME) with a posterior probability of 0.9 (Additional file 1). Out of these four unique 128 identified sites, two sites were found to be potentially relevant. These two sites, Cys538 and 129 Thr549, were mapped on the structure of SARS-CoV-2 spike glycoprotein (PDB ID: 6VXX) 130 ( Figure 2 ). As evident from the results shown in Figure 2 , these sites were found to be 131 present on β-sheets. The intermolecular interactions among β-sheets in a folded structure 132 are considered important and have long been recognized (Nowick, 2008) . They are involved 133 in protein-protein interaction, protein quaternary structure, and protein aggregation. β-134 sheet interactions in some biological processes have been considered as potential targets for 135 the treatment of diseases including cancer (J. Wang, Li, & Jasti, 2018) and AIDS (Li et al., 136 2013). Therefore, the two sites identified as positively selected sites may be relevant in spike 137 glycoprotein structure stability and formation. In silico design of multi-epitope vaccine 155 A complete scheme of in silico vaccine design is shown in Figure 4 and explained in the 156 following sections.

Antigenic sequences from SARS-CoV-2 spike glycoproteins 158 15 antigenic high scoring (threshold >0.5) sequences were obtained out of 23 sequences 159 of SARS-CoV-2 spike glycoprotein submitted to the VaxiJen server. These selected sequences 160 were used to identify T-cell, B-cell, and CTL epitopes.

Epitopes prediction from antigenic sequences of SARS-CoV-2 spike glycoproteins 162 The total number of T-cell, B-cell, and CTL predicted epitopes were 94, 89, and 47 163 respectively. The predicted epitopes were further analyzed for their immunogenicity. This 164 provided 24, 15, and 7 T-cell, B-cell, and CTL epitopes with potential immunogenicity 165 respectively. These epitope sequences were further subjected to toxicity analysis. All these 166 resultant sequences showed non-toxic behavior and were subjected to further analyses. the values indicate the predicted structure of the HLA-B*15:03 subtype is of high quality. The 175 predicted structure was validated by plotting the Ramachandran plot. The plot showed 94.2% residues in most favored regions, 5% in additionally allowed regions, 0.8% in generously allowed regions, and 0% in disallowed regions ( Figure 5B ). This structure was 178 used for molecular docking of the selected epitopes possessing potential immunogenicity.

Screening of potential epitopes for multi-epitope vaccine development 180 The selected epitope sequences were docked with HLA-B*15:03 subtype. The top five 181 epitopes showing the lowest binding affinity were selected for further analyses. The binding 182 affinities of the selected potential epitopes are shown in Table 1 along with their homology 183 searched for MERS, SARS, and human host. According to the obtained docking results, these 184 epitopes bound to the receptor effectively. This method was performed for each type of 185 epitope (T-cell, B-cell, and CTL epitopes).

In silico design of multi-epitope vaccine 187 The designed multi-epitope vaccine consisted of 699 amino acid residues including 188 linkers, adjuvant, and a 6x-His tag added at the C-terminus for purification purposes ( Figure   189 6). This potential vaccine is non-allergen. Molecular weight is 75143.19, theoretical pI is I-Tasser predicted five models, out of which model 1 was selected having a C-score of -197 2.03 ( Figure 9A ). The predicted structure was validated by plotting the Ramachandran plot.

The plot showed 69% residues in most favored regions, 25.2% residues in additionally 199 allowed regions, 3.6% residues in generously allowed regions, and 2.1% residues in 200 disallowed regions ( Figure 9B ). Table 2 . Amongst them, the closely interacting residues include Ile90, 212 Lys92, Glu100, Ser155, Ala174, Gln179, Arg181, and Arg194 ( Figure 10B ). The molecular 213 docking of candidate multi-epitope vaccine with HLA-B*15:03 suggests that this designed 214 vaccine may be a good candidate against covid19.

In silico Immune simulation of multi-epitope vaccine shows a significant immune 

The total B-cell number increased from 0 on day 1 to near 700 on day 21 before slowly 240 falling again ( Figure 13A ). Moreover, antibodies production was noticed to occur almost five 241 days after the beginning of infection with an earlier rise in Immunoglobulin-M (IgM) followed by the rise of Immunoglobulin-G (IgG). Both were noticed to fall until day 20, before 243 rising again after the second injection at day 21 ( Figure 13B ).

Cytokines, Interleukins, and Natural killer (NK) cells: 245 Cytokines are necessary for successful vaccination. Here, for our designed vaccine 246 candidate, all cytokines started to increase after day 0 to reach a maximum level at day 5 and 247 7, then started to fall, only to rise again after the second vaccine injection at day 21 to reach 248 similar levels except for Interferon-ɣ (IFN-ɣ) whose subsequent rise is only up to 1250 in 249 comparison to 420000 in the first injection reaction. Special focus was on the response of between 79% to 89% as tested on three different datasets (Doytchinova & Flower, 2007) . Gly199. Active residues for multi-epitope vaccine identified using CPORT were Tyr18, 375 Phe281, Asp292, Ile377, Gly380, Leu385, Ser389, Leu390, Gly538, Gly542, Ala545, Glu546, 376 Leu549, Ala550, Pro553, Gly554, Pro555, Asn559, Val560, Phe561, Ala562, Pro564, Gly566, 377 Pro567, Gly568, Pro569, Gly570, Val571, Val572, Val573, Ala576, Asn594, His597, Ala599, 378 Ser601, Phe604, Ala635, Cys639, Pro640, Phe641, Glu643, Val644, Phe645, Ala646, Ala647, 379 Tyr648, Phe649, Gly650, Asp651, Asp652, Thr653, Val654, Ile655, Glu656, Val657, Ala658, 380 Ala659, Tyr660, Phe661, Ser662, Tyr663, Phe664, Ala665, Val666, His667, Phe668, Ile669, 381 vaccine. An adjuvant is added at the amino-terminal with the help of the EAAAK linker (grey).

Th cell epitopes and B-cell epitopes were joined using GPGPG linkers (red) and CTL epitopes 563 were joined using AAY linkers (Yellow). TH cell, B-cell, and CTL epitopes are depicted with 564 light blue, light green, and sky blue colors respectively. Table 3 Sequence details of spike glycoprotein sequences of SARS-CoV-2 that were 593 obtained from phylogenetic analysis and were utilized in further analyses. vaccine. An adjuvant is added at the amino-terminal with the help of the EAAAK linker (grey).

Th cell epitopes and B-cell epitopes were joined using GPGPG linkers (red) and CTL epitopes 627 were joined using AAY linkers (Yellow). TH cell, B-cell, and CTL epitopes are depicted with 628 light blue, light green, and sky blue colors respectively. 

SARS-CoV spike glycoprotein (PDB ID: 5X58). Different colors on the structure depict 607 different regions. NTD: N-terminal domain

Subdomain1, SD2: Subdomain2, SD3: Subdomain3, CH: Central helix, BH: β-hairpin

Upstream helix, FP: Fusion protein, HR1: Heptad repeat1

Figure 7 Predicted secondary structure of the multi-epitope vaccine

Figure 9 A) Predicted three-dimensional structure of the multi-epitope vaccine

Ramachandran plot of the predicted structure of the multi-epitope vaccine