key: cord-340432-vm6m0kb4 authors: Srivastava, Sukrit; Verma, Sonia; Kamthania, Mohit; Agarwal, Deepa; Saxena, Ajay Kumar; Kolbe, Michael; Singh, Sarman; Kotnis, Ashwin; Rathi, Brijesh; Nayar, Seema. A.; Shin, Ho-Joon; Vashisht, Kapil; Pandey, Kailash C title: Computationally validated SARS-CoV-2 CTL and HTL Multi-Patch Vaccines designed by reverse epitomics approach, shows potential to cover large ethnically distributed human population worldwide date: 2020-09-06 journal: bioRxiv DOI: 10.1101/2020.09.06.284992 sha: doc_id: 340432 cord_uid: vm6m0kb4 Background The SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a positive-sense single-stranded RNA coronavirus responsible for the ongoing 2019-2020 COVID-19 outbreak. The highly contagious COVID-19 disease has spread to 216 countries in less than six months. Though several vaccine candidates are being claimed, an effective vaccine is yet to come. In present study we have designed and theoretically validated novel Multi-Patch Vaccines against SARS-CoV-2. Methodology A novel reverse epitomics approach, “overlapping-epitope-clusters-to-patches” method is utilized to identify multiple antigenic regions from the SARS-CoV-2 proteome. These antigenic regions are here termed as “Ag-Patch or Ag-Patches”, for Antigenic Patch or Patches. The identification of Ag-Patches is based on clusters of overlapping epitopes rising from a particular region of SARS-CoV-2 protein. Further, we have utilized the identified Ag-Patches to design Multi-Patch Vaccines (MPVs), proposing a novel methodology for vaccine design and development. The designed MPVs were analyzed for immunologically crucial parameters, physiochemical properties and cDNA constructs. Results We identified 73 CTL (Cytotoxic T-Lymphocyte), 49 HTL (Helper T-Lymphocyte) novel Ag-Patches from the proteome of SARS-CoV-2. The identified Ag-Patches utilized to design MPVs cover 768 (518 CTL and 250 HTL) overlapping epitopes targeting different HLA alleles. Such large number of epitope coverage is not possible for multi-epitope vaccines. The large number of epitopes covered implies large number of HLA alleles targeted, and hence large ethnically distributed human population coverage. The MPVs:Toll-Like Receptor ectodomain complex shows stable nature with numerous hydrogen bond formation and acceptable root mean square deviation and fluctuation. Further, the cDNA analysis favors high expression of the MPVs constructs in human cell line. Conclusion Highly immunogenic novel Ag-Patches are identified from the entire proteome of SARS CoV-2 by a novel reverse epitomics approach. We conclude that the novel Multi-Patch Vaccines could be a highly potential novel approach to combat SARS-CoV-2, with greater effectiveness, high specificity and large human population coverage worldwide. ABSTRACT FIGURE: A Multi-Patch Vaccine design to combat SARS-CoV-2 and a method to prepare thereof. Multi-Patch Vaccine designing to combat SARS-CoV-2 infection by reverse epitomics approach, “Overlapping-epitope-clusters-to-patches” method. Immunogenicity of all the screened CTL epitopes was also obtained by 279 using "MHC I Immunogenicity" tool of IEDB with all the parameters set to default 280 analyzing 1st, 2nd, and C-terminus amino acids of the given epitope (Calis et al., 281 2013 (Sievers et al., 2011) . The Patches of the SARS-312 CoV-2 protein sequences showing consensus with the clusters of overlapping 313 epitopes were chosen and shortlisted as antigenic patches (Ag-Patches). This 314 approach of search and identification of antigenic patches from source protein in 315 a reverse epitomics manner, i.e. from epitopes to antigenic patches of source 316 protein, is here defined as "Overlapping-epitope-clusters-to-patches" method 317 ( Figure 1A, 1B) . The here provided reverse epitomics approach, "Overlapping-318 epitope-clusters-to-patches" method to identify Antigenic Patches (Ag-Patches) 319 from pathogen's (here SARS-CoV-2) protein is included in filed Patent No: 320 worldwide. In this way, the population coverage by an epitope-MHC pair could be 333 determined (Sturniolo et al., 1999) . Deviation) is the deviation between templet residues and query residues that are 408 structurally aligned by TM-align. 409 The refinement of all the generated three CTL and two HTL MPV models The RMSD value of the refined model shows the conformational deviation from 415 the initial input protein model. 416 The GalaxyRefine tool refines the input tertiary structure by repeated 417 structure perturbation as well as by subsequent structural relaxation and 418 molecular dynamics simulation. The tool GalaxyRefine generates reliable core 419 structures from multiple templates and then re-builds the loops or termini by 420 Cell epitope from the given tertiary structure based on the location of residues in 438 the proteins 3D structure. The farthest residue to be considered was limited to 6 439 Å. The residues lying outside of an ellipsoid covering 90% of the inner core 440 residues of the protein score highest Protrusion Index (PI) of 0.9; and so on. The 441 discontinuous epitopes predicted by the ElliPro tool are clustered based on the 442 distance "R" in Å between two residues centers of mass lying outside of the 443 largest possible ellipsoid of the protein tertiary structure. The larger the value of 444 R, greater would be the distance between the residues (residue discontinuity) of 445 ToxinPred study of all the screened CTL and HTL epitopes revealed that all the 528 screened epitopes were Non-Toxic (Supplementary table S1 Patches from CTL and a total of 49 Ag-Patches from HTL high scoring 768 533 overlapping epitopes (518 CTL and 250 HTL epitopes) were identified (Table 1 & Multi-Epitope Vaccines. The MEVs would also face challenge to give raise to the 544 epitopes in their intact form upon chop down processing by proteasome or 545 lysosome, for presentation by APC ( Figure 1A, 1B, 2, 3 & 4) highly immunogenic Ag-Patches were utilized to design two HTL (HTL-MPV-1 & 563 HTL-MPV-2) Multi-Patch Vaccines. The identified Ag-Patches were highly 564 conserved in nature and were identified on the basis of significant number of 565 overlapping epitope forming clusters (Figure 2, 3 & 4) . further analyzed for their amino acid sequence conservancy across the source 729 protein sequence library retrieved from NCBI protein database, by the "Epitope 730 Conservancy Analysis" tool of IEDB. We found that all the identified 731 immunogenic Ag-Patches are highly conserved and both the CTL & HTL Ag-732 Patches are significantly conserved with their 100% amino acid residues largely 733 conserved amongst the protein sequences of SARS-CoV-2 retrieved from NCBI 734 Protein database (CTL epitopes cluster Ag-Patches were 47.50% to 100% 735 conserved (largely above 91.23%) and HTL epitopes cluster Ag-Patches were 736 91.83% to 100% conserved (Table 1 & 2) . (Table 3) . (Supplementary table S10 , S11, S12, and S13). In the present study, we have reported a novel method to design a 1170 vaccine against SARS-CoV-2 by utilizing multiple antigenic patches from the viral 1171 proteins. The Ag-Patches used have been identified by the clusters of 1172 overlapping epitopes. The identification of these Ag-Patches was performed by 1173 reverse epitomics analysis, of the high scoring CTL and HTL epitopes screened 1174 from all the ORF proteins of the SARS-CoV-2 virus. The method is here termed 1175 as "Overlapping-epitope-clusters-to-patches" method. All the screened epitopes 1176 were well characterized for their conservancy, immunogenicity, non-toxicity and 1177 large population coverage. The clusters of the overlapping epitopes lead us to 1178 identify the Ag-Patches. These Ag-Patches from all the ORF proteins of the 1179 SARS-CoV-2 proteome were utilized further to design Multi-Patch Vaccine (MPV) 1180 candidate against the SARS-CoV-2 infection. 1181 The designed MPVs from the antigenic patches of SARS-CoV-2 proteins 1182 have several advantages over to the subunit and multi-epitope based vaccines. 1183 The Ag-Patches utilized were identified and collected from the entire proteome of 1184 the SARS-CoV-2. This would enhance the efficiency of the vaccines and lead the 1185 vaccine to be more effective. The MPVs consists of the identified Ag-Patches will 1186 have potential to raise multiple epitopes in clusters upon the chop down 1187 processing by proteasome and lysosome in the Antigen Presenting Cell (APC). 1188 The identified Ag-Patches will also provide larger chance of the epitopes raised 1189 after proteasome and lysosomal processing to get presentation by the APC and 1190 elicit immune response. Since the Ag-Patches were identified by the large 1191 number of epitopes forming clusters, the MPVs designed would have potential to 1192 raise larger number of epitopes upon proteasome and lysosomal processing, 1193 hence larger number of HLA alleles could be targeted and hence larger ethnic 1194 human population could be covered by the MPVs, in comparison to the limited 1195 number of epitope used in multi-epitope vaccines. For instance, the three MPVs 1196 designed in the present study used the Ag-Patches which were identified by 768 1197 Two patents have been filed from the report. 1246 GLN325:SER342, GLY423:THR391, GLY425:THR391, SER427:THR400 Supplementary figure S3. 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