# Population-level toggling of T cell immune escape at human leukocyte antigen anchor residues in SARS-CoV-2 Spike proteins, in an ethnically diverse population region

## TL;DR

This study explores how SARS-CoV-2 evades T cell immunity by mutating at key HLA binding sites in South Africa's diverse population.

## Contribution

The paper introduces a novel method to detect population-specific immune escape mutations at HLA anchor motifs in SARS-CoV-2 Spike proteins.

## Key findings

- Seventeen Spike peptides showed immune escape at HLA anchor motifs, with 16/17 being confirmed T cell epitopes.
- Eight peptides overlapped with both HLA-I and HLA-II escape mutations, indicating complex immune evasion strategies.
- Immune escape mutations were under directional evolution and linked to zoonotic adaptation.

## Abstract

There is currently limited understanding of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) adaptation to the human leukocyte antigen (HLA) proteins which mediate CD8 (HLA-I) and CD4 (HLA-II) T cell immune responses. We investigated population-level T cell immune escape in SARS-CoV-2 Spike protein at amino acid binding positions (the anchor motifs) preferred by the highly restrictive peptide binding grooves of the HLA. SARS-CoV-2 Spike protein sequences isolated in South Africa from January 2020 until June 2022, were used. All possible 9-mer and 15-mer peptides in the sequence alignment were scanned for matches to HLA-I and HLA-II anchor motifs, respectively. Peptide positions with matched anchor motifs and ≥1% mismatched sequences were investigated for immune escape using immunoinformatic prediction methods and directional evolution along the phylogenetic tree. Toggling of short-lived immune escape mutations at HLA-I anchor motifs was observed in 17 peptides across Spike. Eight of these overlapped with HLA-II escape mutations. Six mutations were related to zoonotic adaptation. All 17 sites were under significant directional evolution along the phylogenetic tree, and 16/17 are within published confirmed or inferred T cell epitopes. Immune escape predictions for HLA- A*66:01/A*68:01 were common (n = 7/17). HLA- A*02:05, A*03:01, B*07:02, B*08:01, B*58:01, DRB1*04:01 and DQA1*01:02-DQB1*06:02 were each associated with at least two escape mutations. This immunoinformatic prediction of T cell immune escape at HLA anchor motifs: (i)shortlisted potentially understudied population-specific HLA and immune escape (ii)revealed a footprint of underlying toggling of short-lived immune escape mutations, and (iii)has potential to cost-effectively guide pre-clinical research questions on the inclusion of partially conserved but dominant epitopes in vaccine immunogens.

During a T cell immune response, protein molecules known as Human leukocyte antigens (HLA) bind to foreign pathogen peptides and present these to T cells. The genetic diversity of HLA within and between populations enables immune responses against a wide range of infecting pathogens. Each HLA has pockets where binding affinity to the pathogen peptide is strongest and only certain unique amino acids (known as an ‘anchor motifs’) are required. Viruses can mutate amino acids at the anchor motifs as a mechanism to prevent HLA binding and hence escape attack by the T cell immune response (i.e., immune escape). Here we investigated immune escape at anchor motifs in SARS-CoV-2 Spike peptides of viruses circulating in South Africa during 2020–2022. We found seventeen Spike peptides with immune escape at anchor motifs for HLA which are present in high frequency in at least one major ethnic group in South Africa. Short-lived but repeated episodes (i.e., ‘toggling’) of immune escape were observed. All mutations are known variants of concern, and 16/17 peptides are previously confirmed T cell targets, affirming the importance of this computational immunology analysis in revealing and short-listing important immune responses in a defined population, for further investigation in pre-clinical research.