MHC-II constrains the natural neutralizing antibody response to the SARS-CoV-2 spike RBM in humans

SARS-CoV-2 antibodies develop within two weeks of infection, but wane relatively rapidly post-infection, raising concerns about whether antibody responses will provide protection upon re-exposure. Here we revisit T-B cooperation as a prerequisite for effective and durable neutralizing antibody responses centered on a mutationally constrained RBM B cell epitope. T-B cooperation requires co-processing of B and T cell epitopes by the same B cell and is subject to MHC-II restriction. We evaluated MHC-II constraints relevant to the neutralizing antibody response to a mutationally-constrained B cell epitope in the receptor binding motif (RBM) of the spike protein. Examining common MHC-II alleles, we found that peptides surrounding this key B cell epitope are predicted to bind poorly, suggesting a lack MHC-II support in T-B cooperation, impacting generation of high-potency neutralizing antibodies in the general population. Additionally, we found that multiple microbial peptides had potential for RBM cross-reactivity, supporting previous exposures as a possible source of T cell memory.


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Upon infection with SARS-CoV-2 the individual undergoes seroconversion. In mildly 35 symptomatic patients, seroconversion occurs between day 7 and 14, includes IgM and IgG, and 36 outlasts virus detection with generally higher IgG levels in symptomatic than asymptomatic 37 groups in the early convalescent phase (1). Alarmingly, the IgG levels in both asymptomatic and 38 symptomatic patients decline during the early convalescent phase, with a median decrease of 39 ~75% within 2-3 months after infection (2). This suggests that the systemic antibody response 40 which follows natural infection with SARS-CoV-2 is rapid but short-lived, with the possibility of 41 no residual immunity after 6-12 months (3) affecting primarily neutralizing antibodies in plasma 42 (4). 43 The generation of an antibody response requires cooperation between a B cell producing  cooperative interaction among lymphocytes as cooperation exists between CD4 T and CD8 T 66 cells (23) and between two CD4 T cells responding to distinct epitopes on the same antigen (24).

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A model based on coprocessing of T and B epitopes also led to the suggestion that 68 preferential T-B pairing could be based on topological proximity (25-29) so that during BCR- 69 mediated internalization the T cell epitope is protected by the paratope of the BCR. Indeed, a 70 more recent study showed that not only is CD4 T cell help a limiting factor in the development 71 of antibodies to smallpox (vaccinia virus), but that there also exists a deterministic epitope CoV-2 patients whose paratope is specific for sites outside the RBD (34). 3) RBD antibodies, 87 including NAbs, derived from SARS-CoV-2 patients that map to a restricted site in the RBD 88 (35-41). Cryo-EM of this third antibody category shows that they bind to residues in or around 89 the four amino acids Phe-Asp-Cys-Tyr (FNCY) in the receptor binding motif (RBM) (residues 90 437-508) which is inside the larger RBD (residues 319-541) at the virus:ACE2 interface (36).

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Although the RBD has been shown to be an immunodominant target of serum antibodies in 92 COVID-19 patients (42), high potency NAbs are directed against a conserved portion of the 93 RBM on or around the FNCY patch, a sequence only found in the RBD of SARS-CoV-2 and not 94 in other coronaviruses. Indeed while the RBD is mutationally tolerant, the RBM is constrained to 95 the wild-type amino acids (43), implying that the B cell epitope included in this region of the 96 virus:ACE2 interface is resistant to antigenic drift. Thus, we may refer to this site as a key RBM

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As mounting evidence suggests that the NAb response in COVID-19 patients is relatively 106 short-lived, we decided to test the hypothesis that associative recognition of the key RBM B cell 107 epitope and proximal MHC-II-restricted epitopes may be defective with detrimental effects on 108 preferential T-B pairing. Therefore, to quantify the potential effects of T-B cooperation in vivo, 109 we analyzed all 15mer putative MHC-II epitopes (+/-50 amino acid residues) relative to the key 110 RBM B cell epitope for coverage by all known 5,620 human MHC-II alleles and predicted 111 binding affinity. The analysis shows that there exists in general less availability of effective T 112 cell epitopes in close proximity to the key RBM B cell epitope in the human population.

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Topology of a key RBM B cell epitope 116 Within the 222 amino acid long RBD of the spike protein (residues 319-541), the RBM (residues 117 437-508) is the portion of the spike protein that establishes contact with the ACE2 receptor (Fig   118   1A). The contact residues span a relatively large surface involving approximately 17 residues 119 (36), among them residues F486, N487, Y489 form a loop, which we term the FNCY patch, 120 which is surface exposed and protrudes up towards the ACE2 receptor from the bulge of the 121 RBD (Fig 1B-C). F486 forms hydrophobic interactions with three ACE2 residues (L79, M82, 122 W83). N487 forms hydrogen bonds with Q24 and W83, and Y489 is linked with K31 via a 123 hydrophobic interaction. This makes the amino acid residues in or around the FNCY patch a 124 logical B cell epitope target for antibodies blocking the virus:receptor interaction. In addition, 125 these core residues are mutationally constrained by the ACE2 contact surface (43). Not  (green) (PDB: 6M0J) with contact residues highlighted in blue and the FNCY patch highlighted in red.

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(B-C) Spike protein RBD with ACE2 contact residues and FNCY patch residues labeled in two 141 orientations (front and back). (D) Heatmap of neutralizing antibody contact residues (purple) on the spike 142 protein RBM region (positions 437-508). Black dots indicate ACE2 contact residues and the FNCY patch 143 is highlighted in red. Source data available in Supplemental Table 1. where median affinity is calculated across the 1911 most common MHC-II alleles (Fig 2A), 155 which was highly correlated with scores across all 5620 MHC-II alleles (Fig 2B; Fig 1). Interestingly, the RBM region containing the FNCY patch was free of 160 glycans that could potentially mask the epitope (Fig 2D). We further evaluated the distributions 161 of binding affinities for the 20 best-ranked peptides across all sites in the spike protein (Fig 2E), 162 and in comparison, the distributions for the best 20 peptides overlapping positions within +/-50 163 residues of the FNCY patch (Fig 2F). In the best case, less than half of the considered MHC-II 164 alleles bound a shared peptide close to the FNCY patch, whereas at other sites there were 165 multiple peptides that could be bound by nearly all of the MHC-II alleles (Fig 2E). This  To further assess whether population variation in MHC-II MHC alleles might contribute 186 to heterogeneity in potential to generate neutralizing antibodies, we also evaluated the potential 187 of MHC-II supertypes to restrict peptides from neighboring the FNCY patch. Greenbaum et al. 188 previously defined 7 supertypes that group MHC-II alleles based on shared binding repertoire.

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These 7 supertypes account for between 46%-77% of haplotypes and cover over 98% of 190 individuals when all four loci are considered together (50). We revisited our analysis of peptide 191 restriction proximal to the FNCY patch treating each supertype separately. There was 192 considerable variability in potential to effectively present FNCY patch proximal sequences 193 across supertypes (Fig 3A-B, Fig 2). Only 3 supertypes 194 (DP2, main DP and DR4) commonly presented peptides overlapping the FNCY patch (Fig 3B). 195 We were able to obtain population allele frequencies for four populations from the Be The Match only a rough approximation. In general, DP and DR haplotypes were able to restrict more FNCY 202 patch proximal sequences (Fig 3D).  (Fig 4A) or spot forming cells (Fig 4B), we noted relatively few 224 responses proximal to the FNCY patch in the RBM. Accordingly, few other coronaviruses had 225 limited homology to the FNCY region, and none fully included the FNCY patch (Fig 5A). individuals. Data from Table S1 from (46).

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A notable exception in Mateus' results is peptide 486FNCYFPLQSYGFQPT500, which 234 was reported to induce a CD4 T cell response in an unexposed individual. In this case, the 235 peptide was restricted by HLA-DRB1*0101 or HLA-DQA1*0101/DQB1*0501. We found that 236 the peptide sequence had greater in silico predicted affinity to HLA-DRB1*0101. To explain the 237 conundrum, we blasted this peptide against the "refseq_protein" database excluding SARS-CoV-238 2 (Methods). Surprisingly, the sequences with the best homology for this query were not from 239 coronaviruses but rather from common pathogens, first among them parasites of the 240 Cryptosporidium genus of apicomplexan parasitic alveolates. These sequences included 241 conserved anchor positions for the HLA-DRB*0101 allele making it plausible that a prior 242 exposure could account for the formation of a memory CD4 T cell response (Fig 5B-C). To  Table 2). We found peptides associated with multiple microbial 247 organisms that may meet the criteria to potentially generate CD4 T cell memory relevant to the 248 RBM of SARS-CoV-2 (Fig 5D).  Remarkably, however, a BLAST analysis revealed a 10 amino acid sequence match with 304 proteins from pathogens including those from the Cryptosporidium genus, with identity in 305 binding motif and anchor residues (agretope) for the restricting MHC-II allele strongly 306 suggesting peptide cross-reactivity. Cryptosporidium hominis is a parasite that causes watery 307 diarrhea that can last up to 3 weeks in immunocompetent patients (58). Additional possibilities 308 for cross-reactivity to the RBM, albeit of a lesser stringency, involve antigens from 309 Micromonospora, Pseudomonas, Blastococcus, Lactobacillus, and Bacteroides (Fig 5D). Thus,