A Mutation in the HLA-B * 2705-Restricted NP 383-391 Epitope Affects the Human Influenza A Virus-Specific Cytotoxic T-Lymphocyte Response In Vitro

Abstract

ABSTRACT Viruses can exploit a variety of strategies to evade immune surveillance by cytotoxic T lymphocytes (CTL), including the acquisition of mutations in or adjacent to CTL epitopes. Recently, an amino acid substitution (R384G) in an HLA-B * 2705-restricted CTL epitope in the influenza A virus nucleoprotein (nucleoprotein containing residues 383 to 391 [NP 383-391 ]; S R YWAIRTR, where R is the residue that was mutated) was associated with escape from CTL-mediated immunity. The effect of this mutation on the in vitro influenza A virus-specific CTL response was studied. To this end, two influenza A viruses, one with and one without the NP 383-391 epitope, were constructed by reverse genetics and designated influenza viruses A/NL/94-384R and A/NL/94-384G, respectively. The absence of the HLA-B * 2705-restricted CTL epitope in influenza virus A/NL/94-384G was confirmed by using 51 Cr release assays with a T-cell clone specific for the NP 383-391 epitope. In addition, peripheral blood mononuclear cells (PBMC) stimulated with influenza virus A/NL/94-384G failed to recognize HLA-B * 2705-positive target cells pulsed with the original NP 383-391 peptide. The proportion of virus-specific CD8 + gamma interferon (IFN-γ)-positive T cells in in vitro-stimulated PBMC was determined by intracellular IFN-γ staining after restimulation with virus-infected autologous B-lymphoblastoid cell lines and C1R cell lines expressing only HLA-B * 2705. The proportion of virus-specific CD8 + T cells was lower in PBMC stimulated in vitro with influenza virus A/NL/94-384G obtained from several HLA-B * 2705-positive donors than in PBMC stimulated with influenza virus A/NL/94-384R. This finding indicated that amino acid variations in CTL epitopes can affect the virus-specific CTL response and that the NP 383-391 epitope is the most important HLA-B * 2705-restricted epitope in the nucleoprotein of influenza A viruses.