The SARS-CoV-2 S protein interacts with the host cell receptor, ACE2, to mediate virus infection. For this rea- son, the S protein, or its RBD domain, have been used to develop most of the anti-COVID-19 vaccines that are currently in use around the globe. As the pandemic pro- gressed, several mutations within this protein have been identified in different SARS-CoV-2 variants of concern. At different levels, these mutations render the virus resistant to neutralization mediated by antibodies generated from a previous infections or vaccination, resulting in a reduced immunity against infection with these variants. However, the level of protection against serious disease, hospitaliza- tion and death has remained constant.
Through the literature review and epitope prediction ex- ercises carried out in this work, we were able to compile 116 linear B-cell epitopes within the S protein, reporting 7 new epitopes, most of them located at the RBD, the junc- tion of S1/S2 and the furin site ( Figure 1 A). Recent studies identified neutralizing antibodies directed at these sites. In that sense, the new epitopes reported here are potential tar- gets for neutralizing antibodies ( 78 , 104 ). Several regions of the S protein have been shown to be targets for neutral- izing antibodies. Of these, the RBD is one of the most important binding sites It has been reported that the epitope GDEVRQIAPGQTGKIADYNYKLPDD, which overlaps with one of the epitopes predicted in this work (Supplementary Table 3), generates neutralizing anti- bodies in mice ( 105 ). In addition, other studies have shown that different monoclonal antibodies that interact with multiple residues at this site, exhibit neutralizing activ- ity ( 96 , 106 ). The epitopes ASYQTQTNSPRRARSVASQ and IIAYTMSLGAENSVAYSNN (Supplementary Table 3), predicted in our study, have also been described by Polyiam et al. within the SYQTQTNSPRRARSVASQSI- 
IAYTMSLGAENSVAYSN polypeptide. These epitopes are located at the incision site of S1/S2 where the RRAR sequence is recognized and cleaved by the fu- rin protease, resulting in the separation of the S1 and S2 domains during virus assembly. Antibodies directed at this immunodominant region could block the cleavage of the S protein during the viral invasion process ( 78 ). Both the Alpha and Beta SARS-CoV-2 variants exhibit amino acid substitutions in these epitopes, and we could also iden- tify these changes in sequences from viruses isolated in several countries such as Estonia, South Africa and Singa- pore (Supplementary Table 8). Of particular importance, the Beta variant presents a change in the IIAYTMSL- GAENSVAYSNN peptide, where the A701V substitution affects this epitope at the cleavage site of cathepsin L ( 107 ). The peptides ILPDPSKPSKRS, FIEDLLFNKVT- LADAGFFIKQYGDCLG and PSKPSKRSFIEDLLFNKV are neutralizing epitopes located at the S2 excision site (residues 805-842). Antibody binding at this region results in the inhibition of the molecule excision. Additionally, we located an epitope at the cytoplasmic site of the S protein (CKFDEDDSEPVLKGVKLHYT-1234-1273) ((Supplementary Table 3), which has been identified as an immunodominant epitope ( 94 , 108 ). This epitope has not been shown to generate neutralizing antibodies against the SARS-CoV-2 virus. However, a study with the porcine epidemic diarrhea virus (PEDV) (an alpha coronavirus) reported B-cell epitopes for neutralizing antibodies located at the cytoplasmic region of the S protein ( 109 ). These results suggest that this region could be another important targets for neutralizing antibodies within the SARS-CoV-2 S protein. Recent studies have reported individuals who recov- ered from SARS-CoV infection that have neutralizing an- tibodies against the virus but are not able to neutralize SARS-CoV-2. However, when these subjects were vacci- nated with one or two doses of the BNT162b2 mRNA vaccine, they produced high-level, broad-spectrum anti- body responses that could effectively neutralize all of the SARS-CoV-2 variants of interest and seasonal human coro- naviruses ( 28 , 110 ). Given that the NTD and RBD are the main targets for neutralization, we searched for epitopes conserved between SARS-CoV and SARS-CoV-2 in these regions that could explain cross-neutralization ( 29 ). We could not find any conserved linear B-cell epitopes that had at least 50% shared identity between the two coro- naviruses, suggesting that neutralization at these sites is mainly mediated by conformational epitopes. As was the case for the S protein, immuno-dominant epitopes have also been reported for the SARS-CoV-2 M and N proteins. In patients with severe disease treated in the Intensive Care Unit (ICU), high IgG titers specific to the S protein linear B-cell epitopes TESNKKFLPFQQF- GRDIA, PSKPSKRSFIEDLLFNK and to the N protein epitope NNAAIVLQLPQGTTLPKG have been found ( 80 ). Additionally, the N protein epitope mentioned above has been associated with lymphopenia in patients with COVID- 19 ( 14 ). These S and N epitopes have a low mutation rate ( < 2%) and could be used as markers for COVID-19 in- duced immunopathology ( 14 ). Even though neutralizing antibody responses are in- volved in protection against COVID-19 induced by SARS- CoV-2 infection or vaccination, T-cell immune responses have also been identified as an extremely important com- ponent of the immunity against COVID-19. A study in patients with mild and severe COVID-19 showed the pres- ence of effector and central memory SARS-CoV-2-specific T-cells. In particular, mild cases generated higher lev- els of cytokine-producing CD8 + T-cells ( 111 ). Strong memory-specific T-cell responses to SARS-CoV-2 have also been detected in individuals who had mild and asymp- tomatic infections, in some cases even in the absence of an antibody response ( 112 ). Another study reported T-cell responses specific to SARS-CoV-2 peptide stim- ulation in pre-pandemic samples, which suggests T-cell cross-reactivity with seasonal coronaviruses ( 56 ). Although pending experimental confirmation, our work provides a panel of 33 T-cell epitopes that could potentially be in-
volved in cross-reactive T-cell responses to different coro- naviruses (Supplementary Table 12). Even though ElliPro and DiscoTope yielded the pre- diction of epitopes in similar regions of the spike glyco- protein, there are limitations in the accuracy of these pre- dictions given the nature of conformational epitopes. As more experimental data is generated, predictions of con- formational epitopes for the SARS-CoV-2 spike glycopro- tein will become more refined and precise. Despite the limitations, we believe that investigating potential neutral- izing antibodies against the predicted residues should be pursued. Given that coronaviruses have a latent pandemic potential, we were interested to see if amongst the epitopes compiled in this study there were conserved epitopes be- tween the SARS-CoV-2 spike glycoprotein and other rel- evant human coronaviruses. We identified only 7 linear B-cell epitopes that shared a certain identity between the SARS-CoV-2 spike glycoprotein and that of the other coro- naviruses. The percentage of shared identity between pep- tides ranged from 19–100%, with only one epitope with at least 60% shared identity between SARS-CoV-2 and all the other coronaviruses. Recent studies have shown that anti-spike antibodies generated in response to SARS-CoV infection recognize the spike glycoprotein of SARS-CoV- 2 and vice versa, suggesting antigenic similarities between the spike glycoprotein of these two viruses. However, these cross-reactive antibodies did not show any neutralizing ac- tivity against any other virus except the one that caused the infection ( 113 ). Given that SARS-CoV and SARS-CoV-2 share the highest similarity, it is highly unlikely that these antibodies could have neutralizing activity against any of the other human coronaviruses. Another study showed that memory B-cells from convalescent patients once in- fected with SARS-CoV, produce a repertoire of mono- clonal antibodies that cross-neutralize SARS-CoV-2, while they showed no binding affinity for the OC43 or MERS spike glycoproteins ( 114 ). A different study showed that antibody responses against infection with seasonal human coronaviruses elicits neutralizing antibodies against SARS- CoV-2. However, the responses were measured shortly af- ter infection occurred and therefore there is no informa- tion about the potential longevity of such responses ( 115 ). All these observations show that cross-reactive humoral re- sponses between coronaviruses are plausible. However, it is still not clear whether they are robust enough to provide protection against infection and if they are long-lasting. In vitro studies have shown that specific CD4 + and CD8 + lymphocytes from convalescent patients are acti- vated by S protein peptides ( 37 , 116 , 117 ). In addition, a study with the BNT162b2 vaccine reported that before vaccination, SARS-CoV-2 S protein specific T-cells were found in some unexposed individuals, suggesting these clones were induced by exposure to seasonal coronaviruses ( 110 ). The presence of cross-reactive T-cells has been as- sociated with better humoral and cellular responses to vac-cination, and the authors also reported the presence of Th1 CD4 + and polyclonal CD8 + T-cells as well as cen- tral memory CD4 + and CD8 + T-cells for 6 months after immunization, suggesting long-lasting T-cell responses in- duced by vaccination ( 118 ). These cross-reactive epitopes are located in the S2 region, which, compared to the S1 and the RBD in particular, is less polymorphic (Supple- mentary Table 12), supporting the use of these epitopes for the design of broad-spectrum vaccines ( Figures 7 A- 7 F, and Supplementary Table 13). In our study, we were able to detect several CD4 + and CD8 + T-cell epitopes, as well as promiscuous epitopes, most of which are located in the S2 region (Supplementary Table 7). The study of epitopes of this region could be also relevant to study the memory T-cell compartment induced by SARS-CoV-2 infection or vaccination. In addition to the S protein epitopes reported here, we found 83 and 105 epitopes within the M and N proteins, respectively. In the case of the M protein, only one MHC-I and one MHC- II epitope were found; whereas for the N protein, only two MHC-I epitopes were found ( Figures 4 B and 4 C). In a previous study, 34 participants with severe and mild COVID-19 responded to M protein peptides, 11 responded to GAVILRLRGHLRIAGHHLGR, 16 to TSRTLSYYKL- GASQRVA and 3 to LLESELVIGAVILRGHLR (Supple- mentary Table 7). The first two epitopes activated CD4 + T-cells and the later CD8 + T-cells ( 83 ). These peptides
were also found by our analysis. Furthermore, the M pro- tein epitope LRGHLRIAGHHLGRCDIKDL has previously been described as a highly conserved epitope. A study con- ducted by Heide et al. reported that this peptide was recog- nized by 12 out of 34 patients with COVID-19, inducing CD4 + T-cells to polarize to an effector memory pheno- type. These data suggest that M protein epitopes could also be relevant in the induction of immunity against SARS- CoV-2 ( 119 ). As for the N protein, a study reported that in 19 out of 37 donors of peripheral blood cells who had not been exposed to SARS-CoV or to SARS-CoV-2, the presence of SARS-CoV-2-specific CD4 + IFN γT-cells was detected ( 119 ), again suggesting potential cross-reactive responses with seasonal coronaviruses. In addition, T-cell responses against NSP7 and NSP13 non-structural proteins have been identified in donors with no previous exposure to SARS- CoV or SARS-CoV-2 ( 111 ), whereas donor samples from COVID-19 and SARS recovered patients reacted preferen- tially to the N protein. In addition, the characterization of specific responses to the N protein in a donor without prior exposure to SARS-CoV and SARS-CoV-2, identified the CD4 + and CD8 + T-cell epitope MKDLSPRWFYYLGT- GPEAG, considered to be a promiscuous epitope by Peng et al.; this epitope was also recognized by 12 out of 34 patients who recovered from mild and severe COVID-19 ( 83 ). Part of this epitope, from position 104–113 (Supple-mentary Table 7), was also found by our analysis, and is located within an N-protein region with a high degree of similarity to the MERS-CoV, OC43, and HKU1 N proteins. Therefore, we consider this epitope could be relevant for broadly protective vaccines. An important issue is whether the immunity gener- ated by infection or vaccination with the SARS-CoV- 2 Wuhan strain, and with its S protein, respectively, could provide protection against infection, symptomatic mild or severe disease or death caused by the vari- ants of concern of this virus. Mapping the amino acid changes within the S protein from the variants of con- cern together with the B- and T-cell epitopes reported here, revealed that the RBD, NTD and S1 incision site ( Figure 5 ), are the main regions of the protein that presented amino acid deletions and substitutions. These changes were found in B- and T-cell epitopes and might alter their antigenicity. Such is the case with several B-cell epitopes (GDEVRQIAPGQTGKIADYNYKLPDD, YQAGSTPCNGV, and YGFQPTNGVGYQ) and T-cells epitopes (NATRFASVYAWNRK, CVADYSVLYNSASF- SKCYGVSPTKLN, DLCFTNVYADSFVI, RQIAPGQT- GKIA, TPCNGVEGFNCY, LQSYGFQPTNGVG, and YGYQPYRVVLSF) reported in this study. Additionally, epitope DPFLGVYYHKNNKSWMESEFRVYSSANNCT- FEYVSQPFLM is recognized by the monoclonal antibody 4A8 ( 120 ), where a deletion in the alpha variant Y144/14 and a substitution in the gamma variant R190S have also been reported (Supplementary Table 8). Data gathered involving the Omicron variant has re- vealed that the N440K, G446S, G496S, and Q498R mu- tations confer the ability to escape antibody responses. It was also found that the Q498S and N501S mutations are involved in immune evasion mechanisms through improv- ing the binding to the ACE2 receptor ( 121 ) ( Figure 5 ) (Supplementary Tables 8-10). This might have an impact on vaccine effectiveness, since it has been reported that the sera from patients recovered from infection with the Wuhan strain, or vaccinated with the original strain S pro- tein vaccine, present a reduction on neutralizing antibody titres against the variants of concern. The neutralizing ca- pacity of antibodies against Omicron is importantly re- duced in healthy and COVID-19 convalescent individuals who completed their vaccination scheme with BNT162b2 (Pfizer/BioNTech) or mRNA-1273 (Moderna-mRNA) vac- cines, as well as in COVID-19 convalescent individuals (not vaccinated). This suggests that, despite the vaccina- tion status, protection could also be affected. In particu- lar, epidemiological data shows that previously acquired immunity is not highly protective against symptomatic in- fection with other variants ( 122–124 ). However, we found multiple cross-reactive B- and T-cell epitopes that are not altered within the different variants, suggesting that protec- tion against symptomatic or severe disease could be main- tained in these individuals although the protection against
infection may be compromised. It has been noted that the majority of patients with a third vaccine dose did not present severe symptoms against different variants includ- ing Omicron, suggesting that vaccine boosters do amplify the neutralizing capacity of antibodies. In addition, we hy- pothesize that conserved epitopes could be involved in pro- tection against severe disease or death with novel variants ( 122 , 124 ). In conclusion, the results of this work and the experi- mental evidence that emerges daily, support the theory that cross-reactive B- and T-cell responses to coronaviruses’ common epitopes, could play a key role in the immu- nity to SARS-CoV-2 and its variants. We found new B- and T-cell potential epitopes in SARS-CoV-2 S, M and N proteins, and we identified T- and B-cell epitopes with a high percentage of shared identity with other human coro- naviruses. We noted that T-cell epitopes have higher shared identity percentages compared to B-cell epitopes. In addi- tion, we analyzed the mutations present within the S pro- tein of SARS-CoV-2 and its variants, and observed that most epitope changes are located on the S1 region. Fur- thermore, we found that the greater number of changes located within total epitopes are found in Delta, followed by Beta, Gamma, Omicron and Alpha. Nevertheless, an important number of epitopes remained unchanged among these variants, suggesting that these conserved S, M and N protein epitopes could mediate the cross-protection induced by infection and might be involved in the protection against new SARS-CoV-2 variants. Taken together, this knowledge could be useful for the rational design of new and broad- spectrum SARS-CoV-2 vaccines.
