Selective pressure mediated by influenza virus M1 58-66 epitope-specific CD8 + T cells promotes accumulation of extra-epitopic amino acid substitutions associated with viral resistance to these T 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.