key: cord-0733738-kfxgiu6c authors: Pacheco-Olvera, Diana Laura; Remy-Hernández, Stephanie Saint; García-Valeriano, María Guadalupe; Rivera-Hernández, Tania; López-Macías, Constantino title: Bioinformatic analysis of B and T cell epitopes from SARS-CoV-2 Spike, Membrane and Nucleocapsid proteins as a strategy to assess possible cross-reactivity between emerging variants, including Omicron, and other human coronaviruses date: 2022-02-17 journal: bioRxiv DOI: 10.1101/2022.02.16.480759 sha: c75bd844f692ec19481c7d510543f727af6ad476 doc_id: 733738 cord_uid: kfxgiu6c The COVID-19 pandemic caused by SARS-CoV-2 produced a global health emergency since December 2019, that up to the end of January 2022 had caused the death of more than 5.6 million people worldwide. Despite emergence of new variants of concern, vaccination remains one of the most important tools to control the pandemic. All approved vaccines and most of the vaccine candidates use the spike protein of the virus as a target antigen to induce protective immune responses. Several variants of the virus present key mutations in this protein which render the virus, at different rates, to evade the neutralizing antibody response. Although experimental evidence suggests that cross-reactive responses between coronaviruses are present in the population, it is unknown which potential antigens shared between different coronaviruses could be responsible for these responses. This study provides predictions of new potential B and T cell epitopes within SARS-CoV-2 Spike (S), Membrane (M) and Nucleocapsid (N) proteins together with a review of the reported B epitopes of these proteins. We also analyse amino acid changes present in the epitopes of variants of concern (VOC) and variants being monitored (VBM), and how these might affect the immune response, as these changes may alter the peptides’ immunogenicity index and the antigen presentation by related HLA alleles. Finally, given these observations, we performed an identity analysis between the repertoire of potential epitopes of SARS-CoV-2 and other human coronaviruses to identify which are conserved among them. The results shown here together with the published experimental evidence, allow us to support the hypothesis that antibody and T cell cross-reactive responses to common coronaviruses epitopes, could contribute to broaden the protective response to SARS-CoV-2 and its variants. This evidence could help not only to understand cross-reactive responses among coronaviruses but also contribute to elucidate their role in immunity to SARS-CoV-2 induced by infection and/or vaccination. Finally, these findings could promote targeted analysis of antigen-specific immune responses and might orient and drive the rational development of new SARS-CoV-2 vaccines including candidates that ideally provide “universal” protection against other coronaviruses relevant to human health. The alleles were used for the prediction given that they are present with a high frequency in 167 the Mexican population and also have a significant frequency worldwide 34 Figure 1C ), we were able to locate and 252 visualize the position of each of the ERIS in magenta, EPITS in pink, and the ECE in purple. 254 In total, we identified for S protein 128 CD8 and CD4 T cells epitopes ( Table 2 ). And finally for N protein we identified 11 EPITS, of which 2 were NER restricted 265 to MCH-I, one NER was classified as promiscuous, and none were MHC-II restricted. For 266 the visualization of epitopes, we used an optimized 3D model of the three proteins ( Figure 267 2), the ERIS are depicted in blue, while EPITS are shown in green and finally CD4+ or CD8+ T cells ECE appear in red. The epitopes described in the literature can be found throughout 269 the structure of spike protein, the predicted epitopes are present throughout the structure the N protein 3D model shows that the distribution of the epitopes is across the entire protein, while the ECE were mapped mainly in the RNA-binding domain, the binding site and within 275 the dimerization domain ( Figure 2C ). To map the mutations reported for the Alpha, Beta, Gamma, Delta and Omicron variants of 280 concern, we used an optimized 3D model of the S protein, where the mutations that have Figure 4D ). Supplementary Table 8 shows the conserved peptide sequences with more than 50% identity 327 between the SARS-CoV-2 sequence and the other six coronaviruses. For epitopes restricted 328 to MHC-II (red), we identified 9 conserved epitopes, following the same distribution pattern Figure 4F ). As expected, epitopes between SARS-CoV and SARS-CoV-2 shared the highest identity percentages among coronaviruses. In the case of epitopes that are considered as promiscuous (that can be 338 recognized by both CD8+ and CD4+ T cells) 14 epitopes were found in S protein, of which 339 2 (cyan) have more than 50% identity. For M protein, 6 promiscuous epitopes were identified, while for the N protein, 7 promiscuous epitopes were found (Supplementary Table 9 ). Table 364 1), predicted in our study, have also been described by Polyiam and collaborators, within the 365 polypeptide (SYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSN). These epitopes are located at the incision site of the S1/S2 (Figure 1A) , where the RRAR sequence is 367 recognized and cleaved by the furin protease, resulting in the separation of the S1 and S2 368 domains during virus assembly. Antibodies directed to this immunodominant region could 369 block the cleavage of the S protein during viral invasion process 57 . Alpha and Beta SARS- CoV-2 variants exhibit amino acid substitutions in these epitopes, we could also identify 371 these changes in sequences from virus isolated in several countries such as Estonia, South Africa and Singapore (Supplementary Table 5 conserved between SARS-CoV and SARS-CoV-2 in these regions that could explain cross-395 neutralization 88 . We could not find any conserved linear B cell epitopes that had at least 50% identity between the two coronaviruses, suggesting that neutralization at these sites is mainly 397 mediated by conformational epitopes. The main limitation of this study is that the B cell 398 epitopes prediction tools we used does not predict conformational epitopes. As well as the case of S protein, immunodominant epitopes have also been reported for Table 421 9). In vitro studies have characterized that specific CD4+ and CD8+ lymphocytes of vaccination 95 . These cross-reactive epitopes are located in the S2 region, which, compared to 432 S1 and RBD in particular, is less polymorphic (Supplementary Table 9 ), supporting the use 433 of these epitopes for the design of broad spectrum vaccines. In our study, we were able to detect several CD4 and CD8 T cell epitopes, as well as 436 promiscuous epitopes, most of which are located in the S2 region (Supplementary Table 2 ), the study of epitopes of this region could be also relevant to study the memory T cell An important concern, is if immunity developed by infection or vaccination by SARS-CoV- 2 Wuhan strain and its S protein respectively, could protect against infection, symptomatic 470 mild or severe disease or dead caused by the variants of concern of this virus. Mapping the 471 amino acid changes within S protein from the variants of concern together with B and T cell 472 epitopes reported here, revealed that RBD, NTD, and S1 incision sites (Figure 3 In conclusion, we found new B and T cell potential epitopes in SARS-CoV-2 S, M and N 506 proteins, and we identified T and B cell epitopes with high percentage of identity with other 507 human coronaviruses. We observe that T cell epitopes have higher identity percentages 508 compared to B cell epitopes. In addition, we mapped the mutations that are within the S 509 protein of SARS-CoV-2 and its variants and observed that most epitopes changes are located 510 on the S1 region. Furthermore, we found that the numbers of changes located within epitopes Acknowledgments: We would like to thank Guillermo Ramón Torres for kindly facilitating 526 the use of PyMOL®️ software in order to build the 3D models presented in this manuscript. 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T cell epitopes reported by in silico studies found in the literature are shown in blue color, epitopes predicted in this study are shown in green, and predicted epitopes that have been experimentally confirmed to be immunogenic are shown in red