key: cord-0725262-ix5piha1
authors: Nersisyan, Stepan; Zhiyanov, Anton; Zakharova, Maria; Ishina, Irina; Kurbatskaia, Inna; Mamedov, Azad; Galatenko, Alexey; Shkurnikov, Maxim; Gabibov, Alexander; Tonevitsky, Alexander
title: Alterations in SARS-CoV-2 Omicron and Delta peptides presentation by HLA molecules
date: 2022-02-22
journal: bioRxiv
DOI: 10.1101/2022.02.21.481175
sha: c8424a6ef79e106f265f1348f6e583cc1b301557
doc_id: 725262
cord_uid: ix5piha1

The T-cell immune response is a major determinant of effective SARS-CoV-2 clearance. Here, using the recently developed T-CoV bioinformatics pipeline (https://t-cov.hse.ru) we analyzed the peculiarities of the viral peptide presentation for the Omicron, Delta and Wuhan variants of SARS-CoV-2. First, we showed the absence of significant differences in the presentation of SARS-CoV-2-derived peptides by the most frequent HLA class I/II alleles and the corresponding HLA haplotypes. Then, the analysis was limited to the set of peptides originating from the Spike proteins of the considered SARS-CoV-2 variants. The major finding was the destructive effect of the Omicron mutations on PINLVRDLPQGFSAL peptide, which was the only tight binder from the Spike protein for HLA-DRB1*03:01 allele and some associated haplotypes. Specifically, we predicted a dramatical decline in binding affinity of HLA-DRB1*03:01 and this peptide after N211 deletion, L212I substitution and EPE 212-214 insertion. The computational prediction was experimentally validated by ELISA with the use of corresponding thioredoxin-fused peptides and recombinant HLA-DR molecules. Another finding was the significant reduction in the number of tightly binding Spike peptides for HLA-B*07:02 HLA class I allele (both for Omicron and Delta variants). Overall, the majority of HLA alleles and haplotypes was not significantly affected by the mutations, suggesting the maintenance of effective T-cell immunity against the Omicron and Delta variants. Finally, we introduced the Omicron variant to T-CoV portal and added the functionality of haplotype-level analysis to it.

T-cell immune response plays a pivotal role in the pathogenesis of COVID-19 (Sekine et al., 41 2020; Nelde et al., 2021; Shkurnikov et al., 2021) . Cytotoxic (CD8) T-cells become activated 42 through recognition of viral peptides presented by HLA class I molecules on the surface of 43 antigen-presenting cells (APCs). The same mechanism based on recognition of HLA-I/peptide 44 complex is further used to identify and destroy infected cells. Unlike cytotoxic T-cells, helper 45 (CD4) T-cells become activated through interaction between their T-cell receptors (TCR) and 46 viral peptides presented by HLA class II proteins. One of the main effector functions of helper T-47 cells consists in delivering the second activation signal to B-cells, which is necessary for the 48 initiation of antibody production (Kumar, Connors & Farber, 2018) .

Recently emerged SARS-CoV-2 variants effectively escape neutralization by antibodies directed 51 to the Spike protein of the base Wuhan variant. According to Nextstrain project, at the beginning 52 of February 2022 most of new COVID-19 cases were driven by Omicron and Delta variants. 53 While replication and antibody-mediated neutralization of these variants were studied 54 extensively (Shiehzadegan et al., 2021; Cameroni et al., 2021; Zhao et al., 2022) , the role of T-55 cell immune response and possibility of T-cell immunity evasion are to be uncovered. In several 56 recent studies preservation of robust T-cell immunity against the Omicron variant was suggested 57 (GeurtsvanKessel et al., 2021; Keeton et al., 2021; May et al., 2021; Liu et al., 2022; Mazzoni et 58 al., 2022) . However, a more elaborative analysis should be conducted to address the population-59 level diversity of HLA molecules.

We recently developed T-cell COVID-19 Atlas (T-CoV) -the computational pipeline and web 62 portal for evaluation of impact of SARS-CoV-2 mutations on HLA-peptide interactions 63 (Nersisyan et al., 2022) . Here we used T-CoV to compare viral peptide presentation for the three 64 variants: Wuhan, Delta and Omicron. Since the major part of the existing vaccines are based on 65 the Spike protein of the reference variant (Kyriakidis et al., 2021) , the comparisons were 66 separately conducted for the whole virus and Spike protein peptidomes. At the beginning, the 67 analysis was performed on a single allele-level; 64 HLA class I (HLA-A, HLA-B, HLA-C) and 68 105 HLA class II (HLA-DR, HLA-DQ, HLA-DP) abundant alleles were screened. Then, the 69 most relevant findings (differential HLA-peptide interactions) were validated experimentally 70 with ELISA. Next, the considered alleles were combined into the theoretical library of all 71 possible haplotypes, and the peptide presentation analysis was conducted at the level of 72 haplotypes. Finally, Allele Frequency Net Database was utilized to highlight the most frequent 73 haplotypes with altered Omicron/Delta peptide presentation (Gonzalez-Galarza et al., 2020) . The 74 workflow of the study is presented in Figure 1 .

Aside from the conducted analysis, we made two updates to T-CoV portal. First, we added the 77 Omicron variant to the list of available variants. Second, the haplotype-level analysis was 78 introduced to the portal in a form of user-friendly web tool (https://t-cov.hse.ru/haplotypes). were predicted with netMHCpan 4.1 and netMHCIIpan 4.0 (Reynisson et al., 2020) . Then, 90 predicted affinities were compared between the reference Wuhan variant and Omicron/Delta.

HLA/peptide pairs whose affinities were altered by at least two folds were used in the 92 downstream analysis. 

The substrate construct, carrying only thioredoxin with the linker, was used as a negative control. In addition to the worldwide frequency data, we used our previously described dataset of 428 132 volunteers to assess the HLA alleles distribution in Moscow, Russia (Shkurnikov et al., 2021) . 

With the use of T-CoV database we obtained the lists of viral peptides which were predicted to 142 be tight binders (affinity 50 nM) for the worldwide prevalent HLA class I and class II alleles.

The analysis was conducted for three SARS-CoV-2 variants: Wuhan, Delta and Omicron. Since 144 the immune system of many individuals was exposed only to Spike protein of the reference relative increase in the number of tight binders).

Much more alleles (ten) demonstrated significantly altered peptide presentation ( -25% or 

There was another HLA class II allele with differential peptide presentation. Specifically, P681R and a whole HLA haplotype is inherited from each patient (Choo, 2007) . Given that, a single 225 "weakened" allele in a set of "strong" alleles will not affect much total peptide presentation by We also updated our T-CoV portal to allow users to perform the analysis with their set of HLA 232 alleles ( Figure 4) .

For HLA class II we considered frequencies of HLA-DRB1/DPB1/DQB1 haplotypes since Omicron/Delta peptide presentation at the level of the whole SARS-CoV-2 proteome (Table S2) .

As expected, all these genotypes contained HLA-B*35:03 allele, which was the only selected Significant alterations in peptide presentation for HLA class II haplotypes were found only for 258 the Spike protein case (Table S3) . Surprisingly, these differences were completely opposite for There were only four frequent HLA class II haplotypes affected by the Delta mutations and six 266 haplotypes for the Omicron variant (Table S3) 

Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron 334 antigenic shift

The HLA System: Genetics, Immunology, Clinical Testing, and Clinical

Data, disease and diplomacy: GISAID's innovative 338 contribution to global health

Omicron-specific T-and B-cell responses in COVID-19 vaccine recipients

Allele frequency net database (AFND) 2020 update: Gold-standard data 348 classification, open access genotype data and new query tools

The mutational landscape of SARS-CoV-2 variants diversifies T cell targets 353 in an HLA-supertype-dependent manner

SARS-CoV-2 spike

T cell responses induced upon vaccination or infection remain robust against Omicron

SARS-CoV-2-specific T cells are 367 rapidly expanded for therapeutic use and target conserved regions of the membrane protein

Human T Cell Development, Localization, and 370 Function throughout Life

SARS-CoV-2 372 vaccines strategies: a comprehensive review of phase 3 candidates. npj Vaccines 6:28

Vaccines Elicit Highly Cross-Reactive 376

Cellular Immunity to the SARS-CoV-2 Omicron Variant

Allele for Multiple Sclerosis HLA-DRB1*01:01 Provides Kinetic Discrimination of Myelin 382 and Exogenous Antigenic Peptides

Immunosequencing and epitope mapping reveal substantial preservation 386 of the T cell immune response to Omicron generated by SARS-CoV-2 vaccines

Spike-Specific CD4+ T Cell Response Is Conserved Against Variants of Concern

Including Omicron

SARS-CoV-2-derived peptides 399 define heterologous and COVID-19-induced T cell recognition

T-CoV: a comprehensive portal 402 of HLA-peptide interactions affected by SARS-CoV-2 mutations

0: improved predictions of MHC antigen presentation by concurrent motif 406 deconvolution and integration of MS MHC eluted ligand data

Hapl-o-Mat: open-source software for HLA haplotype 409 frequency estimation from ambiguous and heterogeneous data

Robust T Cell Immunity in Convalescent Individuals with Asymptomatic or Mild 423 COVID-19

Analysis of the Delta Variant 425

1.617.2 COVID-19

Association of HLA Class I Genotypes With Severity of Coronavirus Disease-19

Human CD4+ T cells specific for dominant epitopes of SARS-CoV-2

Spike and Nucleocapsid proteins with therapeutic potential

SARS-CoV-2 Omicron variant shows less 435 efficient replication and fusion activity when compared with Delta variant in TMPRSS2-436 expressed cells