# Selective pressure mediated by influenza virus M158–66 epitope-specific CD8+T cells promotes accumulation of extra-epitopic amino acid substitutions associated with viral resistance to these T cells

## TL;DR

This study shows that T cells targeting a specific influenza virus protein can drive mutations in other parts of the protein, helping the virus evade immune detection.

## Contribution

Demonstrates in vitro that selective pressure from M158–66-specific CD8+T cells promotes extra-epitopic mutations in influenza M1 protein.

## Key findings

- Extra-epitopic amino acid substitutions associated with reduced T cell recognition accumulate under selective pressure.
- Avian influenza viruses showed higher mutation rates at these sites when exposed to M158–66-specific T cells.
- Mutant variants emerged even without T cell pressure, but were more prevalent under it.

## Abstract

•Use of a unique co-culture system to study influenza virus evolution in HLA-A*02:01 transgenic A549 cells under selective pressure by cloned human T cells specific for the conserved M158–66 CTL epitope.•Upon serial co-culture passages, virus evolution was assessed by next generation sequencing using molecularly cloned recombinant viruses containing the M1 gene of an avian or a human influenza A virus.•Accumulation of previously described extra-epitopic amino acid substitutions associated with reduced recognition by M158–66 –specific CD8+ T cells is promoted by selective pressure mediated by these cells.

Use of a unique co-culture system to study influenza virus evolution in HLA-A*02:01 transgenic A549 cells under selective pressure by cloned human T cells specific for the conserved M158–66 CTL epitope.

Upon serial co-culture passages, virus evolution was assessed by next generation sequencing using molecularly cloned recombinant viruses containing the M1 gene of an avian or a human influenza A virus.

Accumulation of previously described extra-epitopic amino acid substitutions associated with reduced recognition by M158–66 –specific CD8+ T cells is promoted by selective pressure mediated by these cells.

Influenza viruses are notorious for their capacity to evade host immunity. Not only can they evade recognition by virus-neutralizing antibodies, there is also evidence that they accumulate mutations in epitopes recognized by virus-specific CD8+T cells. In addition, we have shown previously that human influenza A viruses were less well recognized than avian influenza viruses by CD8+T cells directed to the highly conserved, HLA-A*02:01 restricted M158–66 epitope located in the Matrix 1 (M1) protein. Amino acid differences at residues outside the epitope were responsible for the differential recognition, and it was hypothesized that this reflected immune adaptation of human influenza viruses to selective pressure exerted by M158–66-specific CD8+T cells in the human population. In the present study, we tested this hypothesis and investigated if selective pressure exerted by M158–66 epitope-specific CD8+T cells could drive mutations at the extra-epitopic residues in vitro. To this end, isogenic influenza A viruses with the M1 gene of a human or an avian influenza virus were serially passaged in human lung epithelial A549 cells that transgenically express the HLA-A*02:01 molecule or not, in the presence or absence of M158–66 epitope-specific CD8+T cells. Especially in the virus with the M1 gene of an avian influenza virus, variants emerged with mutations at the extra-epitopic residues associated with reduced recognition by M158–66-specific T cells as detected by Next Generation Sequencing. Although the emergence of these variants was observed in the absence of selective pressure exerted by M158–66 epitope-specific CD8+T cells, their proportion was much larger in the presence of this selective pressure.