Differential Recognition of Influenza A Viruses by M1_58–66Epitope-Specific CD8⁺T Cells Is Determined by Extraepitopic Amino Acid Residues

Abstract

Natural influenza A virus infections elicit both virus-specific antibody and CD4؉ and CD8 ؉ T cell responses. Influenza A virusspecific CD8؉ cytotoxic T lymphocytes (CTLs) contribute to clearance of influenza virus infections. Viral CTL epitopes can display variation, allowing influenza A viruses to evade recognition by epitope-specific CTLs. Due to functional constraints, some epitopes, like the immunodominant HLA-A*0201-restricted matrix protein 1 (M1 58 -66 ) epitope, are highly conserved between influenza A viruses regardless of their subtype or host species of origin. We hypothesized that human influenza A viruses evade recognition of this epitope by impairing antigen processing and presentation by extraepitopic amino acid substitutions. Activation of specific T cells was used as an indication of antigen presentation. Here, we show that the M1 58 -66 epitope in the M1 protein derived from human influenza A virus was poorly recognized compared to the M1 protein derived from avian influenza A virus. Furthermore, we demonstrate that naturally occurring variations at extraepitopic amino acid residues affect CD8 ؉ T cell recognition of the M1 58 -66 epitope. These data indicate that human influenza A viruses can impair recognition by M1 58 -66 -specific CTLs while retaining the conserved amino acid sequence of the epitope, which may represent a yet-unknown immune evasion strategy for influenza A viruses. This difference in recognition may have implications for the viral replication kinetics in HLA-A*0201 individuals and spread of influenza A viruses in the human population. The findings may aid the rational design of universal influenza vaccines that aim at the induction of cross-reactive virus-specific CTL responses. IMPORTANCE Influenza viruses are an important cause of acute respiratory tract infections. Natural influenza A virus infections elicit both humoral and cellular immunity. CD8؉ cytotoxic T lymphocytes (CTLs) are directed predominantly against conserved internal proteins and confer cross-protection, even against influenza A viruses of various subtypes. In some CTL epitopes, mutations occur that allow influenza A viruses to evade recognition by CTLs. However, the immunodominant HLA-A*0201-restricted M1 58 -66 epitope does not tolerate mutations without loss of viral fitness. Here, we describe naturally occurring variations in amino acid residues outside the M1 58 -66 epitope that influence the recognition of the epitope. These results provide novel insights into the epidemiology of influenza A viruses and their pathogenicity and may aid rational design of vaccines that aim at the induction of CTL responses.

Variation at Extra-epitopic Amino Acid Residues Influences Suppression of Influenza Virus Replication by M1 58-66 Epitope-Specific CD8 + T Lymphocytes

“…The use of this in vitro coculture system and isogenic influenza viruses with different M1 protein-encoding gene segments allowed us to detect differences in influenza virus replication in the presence of M1 58-66 -specific CD8 ϩ T lymphocytes. M1 58-66 -specific CD8 ϩ T lymphocytes reduced replication of a virus with extra-epitopic amino acid residues of the human signature to a lesser extent than that with an M1 protein with an avian signature, in concordance with the impaired kinetics and extent of M1 58-66specific CD8 ϩ T lymphocyte activation shown previously (27).…”

“…First, in the ViroSpot microneutralization assay, virus replication is monitored in Madin-Darby canine kidney (MDCK) cells (28), which do not express any HLA molecules. Therefore, adherent A549, A549-HLA-A*0201 ϩ (A549A2), and A549-HLA-B*2705 ϩ (A549B27) cells were used to study virus replication in the presence of human CD8 ϩ T lymphocytes, as A549 cells are highly susceptible to infection with influenza viruses (27) and the HLA-transgenic cells are capable of presenting the HLA-A*0201-restricted M1 58-66 and HLA-B*2705-restricted NP 174-184 epitopes, respectively.…”

“…We recently demonstrated that preferred extra-epitopic amino acid residues in human influenza viruses (15V, 27R, 101R, 115I, and 121A; human signature) surrounding the M1 58-66 epitope delay activation and reduce the killing capacity of M1 58-66 -specific CD8 ϩ T lymphocytes, possibly by influencing the intracellular processing and presentation kinetics of the epitope (27). However, it is not clear if these extra-epitopic amino acids favor virus replication in the presence of M1 58-66 -specific CD8 ϩ T lymphocytes.…”

Polyanhydride Nanovaccine Induces Robust Pulmonary B and T Cell Immunity and Confers Protection Against Homologous and Heterologous Influenza A Virus Infections

“…This protection against homologous and heterologous virus appears to be long lasting as IAV-nanovax vaccination also conferred protection in mice challenged at 100 days post vaccination (Figure 6). While the IAV-nanovax formulation tested herein contained only IAV HA and NP proteins, studies have demonstrated that immunity directed against additional IAV proteins such as NA and M1 can enhance protection (47,48). One of the benefits of our nanovaccine platform is the ability to easily "plug and play" new antigens within the formulation.…”

Influenza A surface glycosylation and vaccine design

“…Recent development of universal influenza vaccines is focused on the use of conserved peptides or proteins as antigens with different adjuvants and administration methods to induce immune response (26)(27)(28)(29). Expression of the conserved regions of IAV proteins by using viral vector systems can induce CD8 + T cells against lethal IAV challenge in animals, but it still only recognizes one target (30).…”