Differences in Presentation of SARS-CoV-2 Omicron Strain Variant BA.1–BA.5 Peptides by HLA Molecules

Abstract

In this work, we analyzed the binding affinities of mutated peptides of Omicron strain variants BA.1–BA.5 and the worldwide prevalent HLA alleles. Bioinformatics analysis was conducted with the use of T-CoV web portal. We showed that, for all five viral variants, mutations cause a significant reduction in the number of tightly binding peptides for HLA-B*07:02 and HLA-C*01:02 molecules. At the same time, there were novel potential mutant epitopes (binding affinity less than 50 nM) in case of HLA-A*32:01 allele. Interestingly, mutations caused multidirectional effect on the binding affinities of the viral peptides and HLA-DRB1*03:01. Specifically, Spike protein mutations in the BA.1 variant caused more than 100-fold decrease in PINLVRDLPQGFSAL binding affinity, 10-fold decrease in affinity in the case of BA.2, BA.4, and BA.5 variants, and 30% increase in affinity for the BA.3 variant.

The T-cell immune response plays a key role in the pathogenesis of COVID-19 [1, 2]. Two main classes of T cells perform critical functions in fighting infection. Cytotoxic T lymphocytes (CD8 T cells) destroy virus-infected cells. One of the main functions of T helper cells (CD4 T cells) is to deliver the second activating signal to B cells, which is required for the production of antibodies. The key step in T-cell activation is the interaction of the T-cell receptor with the viral peptide in the major histocompatibility complex (MHC, or HLA). There are a large number of different alleles that encode HLA molecules, with each of the respective HLA variants having its own, specific set of peptides to which it can bind with high affinity. HLA alleles associated with the severity of COVID-19 have been previously identified [3–5].

It is known that mutations in the SARS-CoV-2 virus can lead to significant changes in the affinities of the interactions of the corresponding peptides with HLA molecules [6, 7]. Previously, we developed a bioinformatic algorithm and a T-CoV web portal (https://t-cov.hse.ru), which makes it possible to perform an exhaustive analysis of the effect of mutations on the efficiency of presentation of linear SARS-CoV-2 peptides by HLA molecules [8]. In addition to the analysis of individual alleles, the portal provides the possibility of analysis at the level of haplotypes [9].

According to the Nextstrain portal (https://nextstrain.org) data, the dominant variant of SARS-CoV-2 in 2022 is the Omicron strain. It is known that a large number of mutations in the Spike protein allows Omicron to effectively evade antibodies against the Spike protein of the base strain [10]. In addition, the Spike protein of this variant lost the ability to interact with the serine protease TMPRSS2, as a result of which the efficiency of virus replication significantly decreased [11]. Earlier, we have shown that mutations in the BA.1 and BA.2 variants of the Omicron strain lead to a critical decrease in the affinity of the interaction between HLA-DRB1*03:01 and the only epitope of the Spike protein corresponding to this HLA molecule (bioinformatic analysis and ELISA) [9]. Interestingly, two different mutations affecting a single region led to a decrease in affinity: the N211 deletion, L212I replacement, and the EPE 212–214 insert in case of the BA.1 variant and the V213G replacement in the case of the BA.2 variant. As far as we know, such analysis has not yet been performed for the BA.3, BA.4, and BA.5 variants.

The aim of this work was to perform a comparative analysis of the efficiency of the presentation of viral peptides of Omicron strain variants BA.1–BA.5 by the most common variants of HLA molecules.

The primary protein sequences of various SARS-CoV-2 variants were retrieved from the GISAID portal [12] in FASTA format using the following identifiers:

• Wuhan base strain: EPI_ISL_402125;

• Omicron BA.1: EPI_ISL_6699752;

• Omicron BA.2: EPI_ISL_9884589;

• Omicron BA.3: EPI_ISL_9854919;

• Omicron BA.4: EPI_ISL_11873073;

• Omicron BA.5: EPI_ISL_13302233.

The retrieved amino acid sequences were divided into various overlapping linear peptides 8–14 aa long in the case of HLA class I (HLA-I) and 15–20 aa long in the case of HLA class II (HLA-II). The affinities of interactions between viral peptides and the most common HLA molecules in the world population (64 HLA-I alleles and 105 HLA-II alleles) were predicted  using  the netMHCpan version 4.1 and netMHCIIpan version 4.0 software [13] integrated into the T-CoV algorithm [8]. The scale of the predicted affinities was divided into three groups: ≤50 nM (high binding affinity), 50 to 500 nM (medium affinity), and more than 500 nM (low affinity or no binding). A significant increase or decrease in the binding affinity of a peptide was defined as a situation where the predicted affinity was in or out of the high binding affinity range, respectively.

Data were processed using Python version 3.8. The construction of heat maps with hierarchical clustering of rows/columns was performed using the seaborn library version 0.11.2 [14].

Figure 1 shows the mutation diagram for the Omicron BA.1–BA.5 variants of the SARS-CoV-2 virus (89 unique mutations). Many mutations were common to several variants: 24 mutations (27%) were common to all variants, 9 mutations (10%) were common to four variants, 13 mutations (15%) were common to three variants, and 11 mutations (12%) were common to two variants. The rest of the changes (36%) were specific to different Omicron variants. More than half of all changes (46 out of 89 mutations (52%)) were in the Spike protein. It is important to note that specific mutations were evenly distributed in all SARS-CoV-2 proteins (Fisher’s exact test was used for each protein separately, p > 0.3 in all cases).

Fig. 1.

Distribution of mutations in five variants (BA.1–BA.5) of the SARS-CoV-2 Omicron strain. The labels include the name of the viral protein and the mutation identifier (in local coordinates of the corresponding protein). The colors in the vertical bar on the left correspond to the viral proteins.

We have previously shown that a large number of viral epitopes throughout the entire virus proteome (including the auxiliary and non-structural proteins) limits the ability of the virus to evade antigen presentation due to several or dozens of mutations [9]. As a result, our further analysis was focused on the SARS-CoV-2 Spike protein, because a significant number of COVID-19 vaccines are based on the use of this protein. In the case of the analysis of HLA class I, consistent changes in the affinities of the mutant peptides of various variants of Omicron were obtained. In particular, a significant decrease in the affinities of interactions of viral peptides from the Spike protein with HLA molecules encoded by the HLA-B*07:02 and HLA-C*01:02 alleles was found for all five variants. In the first case (HLA-B*07:02), a 1000-fold decrease in affinity was due to the P681H amino acid substitution (original epitope SPRRARSVA); in the second case (HLA-C*01:02), a five-fold decrease in affinity was due to the Y505H substitution, which fell into the YQPYRVVVL epitope (both substitutions P681H and Y505H were present in all Omicron variants, see Fig. 1). Mutations that significantly increased the binding affinity of some peptides to HLA-I molecules were also found. For example, three adjacent substitutions at positions 371, 373, and 375 of the Spike protein led to the appearance of epitopes with an affinity of less than 50 nM for HLA-A*32:01: VLYNLAPFF (BA.1) and VLYNFAPFF (BA.2–BA.5). Positions 373 and 375 corresponded to the substitutions S373P and S375F present in variants BA.1–BA.5, and position 371 corresponded to the S371L substitution in the case of the BA.1 variant and S371F for other viruses.

A less consistent pattern was observed for HLA class II alleles. For the aforementioned HLA-DRB1*03:01 allele, there is a single Spike protein peptide with a predicted binding affinity of less than 50 nM (PINLVRDLPQGFSAL, 27 nM). Notably, the affinity is reduced by two orders of magnitude due to mutations in BA.1 (N211 deletion, L212I substitution, insert 212–214 EPE) and by one order of magnitude in the case of BA.2 (V213G substitution). The changes predicted using bioinformatics methods were previously validated by us experimentally using ELISA [9]. Interestingly, the sequences of the Omicron variants BA.4 and BA.5 in this region were identical to BA.2 (V213G substitution), whereas in the case of BA.3, the N211 deletion and L212I substitution were detected, resulting in an unexpected increase in the binding affinity by 30% (Fig. 2). Thus, the effect of the observed mutations in the case of the HLA-DRB1*03:01 allele can be divided into three classes: the 212–214 EPE insert leads to a complete loss of interaction, the V213G substitution reduces the interaction affinity by an order of magnitude, and the L212I substitution doubles the binding affinity (the N211 deletion is not relevant since it corresponds to the flank part of the peptide).

Fig. 2.

Mutations in the PINLVRDLPQGFSAL epitope (highlighted in italics) lead to a change in the affinity of the interaction with HLA-DRB1*03: 01. Mutations are shown in bold.

A similar effect was observed for the HLA-DRB1*15 allele family (HLA-DRB1*15:01, HLA-DRB1*15:02, and HLA-DRB1*15:03). In the case of BA.2, BA.4, and BA.5 viruses, a new epitope YSVLYNFAPFFAFKC (44 nM) evolved as a result of four colocalized mutations S371F, S373P, S375F, andT376A. In the case of the BA.1 and BA.3 viruses, which lack the T376A mutation, there was no increase in the predicted binding affinity.

Thus, it was shown that the peptides of the five variants of the SARS-CoV-2 Omicron strain have different binding affinity profiles for the major histocompatibility complex molecules. The results obtained indicate the need to study the T-cell immune response to each of the Omicron BA.1–BA.5 variants separately. The results of the analysis of BA.3, BA.4, and BA.5 strains were added to the T-CoV portal.

Abbreviations:

HLA

human leukocyte antigen system (MHC, major histocompatibility complex)

ELISA

enzyme-linked immunosorbent assay

FUNDING

The study was supported by a grant from the Ministry of Science and Higher Education of the Russian Federation (agreement no. 075-15-2021-1049).

COMPLIANCE WITH ETHICAL STANDARDS

The authors declare that they have no conflicts of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.