Structural basis to design multi-epitope vaccines against Novel Coronavirus 19 (COVID19) infection, the ongoing pandemic emergency: an in silico approach

The 2019 novel coronavirus (COVID19 / Wuhan coronavirus), officially named as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is a positive-sense single-stranded RNA coronavirus. SARS-CoV-2 causes the contagious COVID19 disease also known as 2019-nCoV acute respiratory disease and has led to the ongoing 2019–20 pandemic COVID19 outbreak. The effective counter measures against SARS-CoV-2 infection require the design and development of specific and effective vaccine candidate. In the present study, we have screened and shortlisted 38 CTL, 33 HTL and 12 B cell epitopes from the eleven Protein sequences of SARS-CoV-2 by utilizing different in silico tools. The screened epitopes were further validated for their binding with their respective HLA allele binders and TAP (Transporter associated with antigen processing) molecule by molecular docking. The shortlisted screened epitopes were further utilized to design novel two multi-epitope vaccines (MEVs) composed of CTL, HTL and B cell epitopes overlaps with potential to elicit humoral as well as cellular immune response against SARS-CoV-2. To enhance the immune response for our vaccine design, truncated (residues 10-153) Onchocerca volvulus activation-associated secreted protein-1 (Ov-ASP-1) has been utilized as an adjuvant at N terminal of both the MEVs. Further molecular models for both the MEVs were prepared and validated for their stable molecular interactions with Toll-Like Receptor 3 (TLR 3). The codon-optimized cDNA of both the MEVs were further analyzed for their potential of high level of expression in a human cell line. The present study is very significant in terms of molecular designing of prospective CTL and HTL vaccine against SARS-CoV-2 infection with the potential to elicit cellular as well as humoral immune response. (SARS-CoV-2), Coronavirus, Human Transporter associated with antigen processing (TAP), Toll-Like Receptor (TLR), Epitope, Immunoinformatics, Molecular Docking, Molecular dynamics simulation, Multi-epitope Vaccine Graphical abstract The designed CTL (Cytotoxic T lymphocyte) and HTL (Helper T lymphocyte) multi-epitope vaccines (MEV) against COVID19 infection. Both the CTL and HTL MEV models show a very stable and well fit conformational complex formation tendency with the Toll like receptor 3. CTL and HTL MEVs: ribbon; Toll like receptor 3: gray cartoon; Adjuvant [truncated (residues 10-153) Onchocerca volvulus activation-associated secreted protein-1]: orange ribbon regions; Epitopes: cyan ribbons regions; 6xHis Tag: magenta ribbon regions.


Introduction 136
The novel coronavirus (COVID19), officially named as Severe Acute Respiratory NetMHCIIpan) and generate "Percentile rank" and a "total score" respectively. 273 The screening is based on the total amount of cleavage sites in the Immunogenicity of all the screened CTL epitopes was also obtained by 282 using "MHC I Immunogenicity" tool of IEDB 283 (http://tools.iedb.org/immunogenicity/) with all the parameters set to default 284 analyzing 1st, 2nd, and C-terminus amino acids of the given screened epitope 285  of IEDB (http://tools.iedb.org/population/) was used to elucidate the world human 303 population coverage by the shortlisted 38 CTL and 33 HTL epitopes derived from 304 nine SARS-CoV-2 proteins (Bui et al., 2006). T cells recognize the complex 305 between a specific major MHC molecule and a particular pathogen-derived 306 epitope. The given epitope will elicit a response only in an individual that express 307 an MHC molecule, which is capable of binding that particular epitope. This 308 denominated MHC restriction of T cell responses and the MHC polymorphism 309 provides the basis for population coverage study. The MHC types are expressed 310 14 at dramatically different frequencies in different ethnicities. Hence a vaccine with 311 larger population coverage could be of greater importance (Sturniolo et al., 312 1999). Clinical administration of multiple-epitopes involving both the CTL and the 313 HTL epitopes are predicted here to have a greater probability of larger human 314 population coverage worldwide. 315

B Cell Epitope Prediction 317
Sequence-based B Cell epitope prediction. Protein sequence-based method 318 "Bepipred Linear Epitope Prediction" was utilized to screen linear B cell epitopes 319 from eleven different SARS-CoV-2 proteins. The tool "B Cell Epitope Prediction 320 Tools" of IEDB server (http://tools.iedb.org/bcell/) was utilized. In this screening, 321 the parameters such as hydrophilicity, flexibility, accessibility, turns, exposed 322 surface, polarity and the antigenic propensity of the polypeptides is correlated 323 with its location in the protein. This allows the search for continuous epitopes 324 prediction from protein sequence. The prediction is base on the propensity scales 325 for each of the 20 amino acids. For a window size n, the i -(n-1)/2 neighboring 326 residues on each side of residue i are used to compute the score for the residue 327 i. The method "Bepipred Linear Epitope Prediction" utilized here is based on the 328 propensity scale method as well as the physiochemical properties of the given 329 antigenic sequence to screen potential epitopes (Larsen et al., 2006). 330 331

Characterization of potential epitopes 332
Epitope conservation analysis. The shortlisted CTL, HTL and B cell epitopes 333 screened from eleven SARS-CoV-2 proteins were analyzed for the conservancy 334 of their amino acid sequence by "Epitope Conservancy Analysis" tool 335  of templet proteins is based on TM-score of the structural alignment between the 508 query structure model and known structures. The RMSD is the RMSD between 509 templet residues and query residues that are structurally aligned by TM-align. 510 The refinement of both the generated MEV models was performed by 511

ModRefiner
(https://zhanglab.ccmb.med.umich.edu/ModRefiner/) and 512 GalaxyRefine tool (http://galaxy.seoklab.org/cgi-bin/submit.cgi?type=REFINE) 513 The GalaxyRefine tool refines the query tertiary structure by repeated 519 structure perturbation as well as by utilizing the subsequent structural relaxation 520 by the molecular dynamics simulation. The tool GalaxyRefine generates reliable 521 core structures from multiple templates and then re-builds unreliable loops or 522 termini by using an optimization-based refinement method (Ko et

Molecular interaction analysis of chosen CTL and HTL epitopes with HLA alleles. 786
The molecular docking study of chosen CTL and HTL epitopes with their 787 respective HLA class I and II allele binders was performed by PatchDock tool. 788 The study revealed a significant molecular interaction between all the chosen 789 epitopes and their HLA allele binders showing multiple hydrogen bond formations 790 (Fig.3). Furthermore, the B-factor analysis of all the epitope-HLA allele 791 complexes has also shown the epitope ligand to have stable (blue) binding The molecular docking interaction analysis of the chosen CTL epitopes with the 894 TAP transporter cavity has shown a significantly strong molecular interaction with 895 several hydrogen bonds formation at different sites of the TAP transporter cavity. 896 Two sites of interaction were of particular interest, one closer to the cytoplasmic 897 end and another closer to the ER lumen (Fig.5). This study confirms the 898 feasibility of transportation of chosen CTL epitopes from the cytoplasm to the ER 899 lumen which is an essential event for the representation of epitope by the HLA 900 allele molecules on the surface of antigen-presenting cells.  (Table 5).   The epitopes are shown in cyan. The adjuvant (Ov-ASP-1) is shown in orange. 992 The linkers are shown in gray and 6xHis tag is shown in magenta. Both the 993 cartoon and surface presentation of both the MEVs are shown. 994