4  Discussion
In this study, SARS-COV-2 virus proteins were analyzed by using In-silico methods, and can be further utilized for vaccine trials as per earlier successes in the case of similar SARS-COV studies, and later observed in the development of polyclonal antibodies [27]. Here we obtained two epitopes ITLCFTLKR and VYQLRARSV after successful docking and molecular dynamics simulation; furthermore, these two epitopes were subjected to population coverage and toxicity analysis. Similarly, in another study, for MERS-COV, nucleocapsid peptides were used for T-cell epitope prediction, and found to be successful [36]. The IEDB and NCBI-GenBank database were fully deployed to analyze sequence homology, to predict targets for COV-2 in case of viral protein identification as per the related studies [14], as ViPR (Virus Pathogen database analysis resource) are also dependent on IEDB and GenBank primarily [30]. We analyzed five different proteins in SARS-COV-2 for the present study (because of their availability in the NCBI-GenBank database and importance in a structural role in SARS-COV-2 [14] and finally revealed T–Cell epitopes that can be used for wet lab considerations and time savings. In a very recent study, different epitopes were found for SARS-COV-2, based on In-silico approaches and focused on only surface glycoprotein [3], but in our research study there are many differences as we analyzed a different group of proteins from SARS-COV-2 to sort out short length T-Cell epitopes specific to MHC I as well as MHC II diversified HLA-Alleles.
It is reported for SARS-CoV HLA-B*4601, HLA-B*0703, HLA-DR B1*1202 are activated [26], interaction with different MHC I and II allelic forms namely HLA-A*11:01, HLA-A*68:01, HLA-DRB1*01:01 and HLA-DRB1*07:01. CD4+ and CD8+ memory T cells. Based on prior literature, it is anticipated that it can persist for four years as in the case of SARS-CoV recovered individuals, show T-cell proliferation, DTH response, and production of IFN-γ [12]. We surmise that our screen can be more effective and useful. Primarily molecular docking reveals three Epitopes, but as we proceed to Molecular dynamic simulations, it reveals best interactions for two epitopes i.e., ITLCFTLKR and VYQLRARSV, with acceptable stability analyzed with the help of MDWeb and identified by using best available tools with easy-to-apply methods. One recent study was found to be focused on developing monoclonal antibodies like CR-3022 against the Spike protein of SARS-COV-2 that also exhibits interaction with ACE (Angiotensin Converting Enzyme) enzyme of the Human respiratory epithelium and requires complex neutralizing mechanisms for several binding domains [35], whereas in our study the putative T-cell epitopes can directly interact with MHC-Allelic sets that can be useful for developing immunization against SARS-COV-2. ProtParam [42] analysis further reveals the stability of the considered epitopes, and final revelation of one epitope ITLCFTLKR is screened out. This particular Epitope shows an instability index of 35.68 with a grand average of hydropathicity (GRAVY) calculated as 0.844, and the estimated half-life for this peptide was determined to be 20 h for mammalian reticulocytes.
Satisfactory population coverage was observed for targeted epitopes - HLA allelic complexes at the worldwide, South Asia, and India level. The biochemical integrity in epitope structure was further evident by deploying Ramachandran plot analysis. Both epitopes were non-toxic, non-allergenic, and possess good antigenicity. In a similar study of the preliminary analysis of COVID-19 vaccine targets [1] the investigators tried to use the spike protein and nucleo-capsid protein sequences of SARS-COV that are homologous to some extant with SARS-COV-2 proteins to determine multiple different epitopes for Vaccine prediction, but in our study out of five two proteins namely ORF-3a and ORF-7a specific to SARS-COV-2 were found to be putative T-cell epitope determinants that create useful information; these proteins are also important for viral replication [41]. Both biochemical parameters, as well as an advanced HMM and ANN based algorithm in selected Immuno-informatics tools, were very useful to present a clear picture of predicted epitopes for crafting vaccine against SARS-COV-2. The only limitation that can be considered as future scope is that these easily synthesized peptides should be tested with In-vitro study for more practical validation.