The Hypervariable Immunodominant NP_418-426Epitope from the Influenza A Virus Nucleoprotein Is Recognized by Cytotoxic T Lymphocytes with High Functional Avidity

Abstract

Recently it was shown that influenza A viruses can accumulate mutations in epitopes associated with escape from recognition by human virus-specific cytotoxic T lymphocytes (CTL). It is unclear what drives diversification of CTL epitopes and why certain epitopes are variable and others remain conserved. It has been shown that simian immunodeficiency virus-specific CTL that recognize their epitope with high functional avidity eliminate virus-infected cells efficiently and drive diversification of CTL epitopes. T-cell functional avidity is defined by the density of major histocompatibility complex class I peptide complexes required to activate specific CTL. We hypothesized that functional avidity of CTL contributes to epitope diversification and escape from CTL also for influenza viruses. To test this hypothesis, the functional avidity of polyclonal CTL populations specific for nine individual epitopes was determined. To this end, peripheral blood mononuclear cells from HLA-A-and -B-genotyped individuals were stimulated in vitro with influenza virus-infected cells to allow expansion of virus-specific CTL, which were used to determine the functional avidity of CTL specific for nine individual epitopes in enzyme-linked immunospot assays. We found that the functional avidity for the respective epitopes varied widely. Furthermore, the functional avidity of CTL specific for the hypervariable NP 418-426 epitope was significantly higher than that of CTL recognizing other epitopes (P < 0.01). It was speculated that the high functional avidity of NP 418-426 -specific CTL was responsible for the diversification of this influenza A virus CTL epitope.

HLA class I molecules consistently present internal influenza epitopes

“…Synthetic versions of the PB1, HA, and the 3 M1 HLA-B*0702 peptides identified here have not been tested for immune recognition in other studies. Also consistent with our direct epitope discovery and supertype data, NP 418 -426 has been identified as a HLA-B*3501 CTL epitope during human inf luenza infection (6,24). The immunogenicity testing of NP 418 -426 with HLA-B*0702 and ϪB*3501 by others is consistent with our direct discovery approach showing that NP 418 -426 is presented by both members of the B7 supertype and recognized by CTL following infection.…”

“…Of the 7 directly discovered influenza A virus ligands, only the NP 418-426 peptide contains the I-A b binding motif. However, it is unlikely that the NP 418-426 IFN-␥ response generated by lymphocytes isolated from PR8 infected HLA-B*0702 transgenic mice is largely because of CD4 T cell reactivity given that the NP 418-426 epitope elicits CD8 T cell reactivity during in vitro stimulation of human peripheral blood mononuclear cells (PBMC) isolated from healthy HLA-B*0702 and -B*3501 adults (13,(22)(23)(24).…”

Differential Recognition of Influenza A Viruses by M158–66Epitope-Specific CD8+T Cells Is Determined by Extraepitopic Amino Acid Residues

“…1) (6 -8, 33, 34). Most likely, the selective pressure against the M1 58 -66 epitope is high, considering the immunodominant nature of the epitope (35) and the high prevalence of the HLA-A*0201 allele in the Caucasian population (Ͼ40%) (36). However, mutations at TCR contact or anchor residues were not tolerated in this epitope without loss of viral fitness (21,37), which coincides with the presence of a highly conserved nuclear export signal overlapping the M1 58 -66 epitope (38).…”

“…2). The NP 383-391 epitope was included as a control, since CTLs specific for this epitope have functional avidity similar to that of CTLs directed to the M1 58 -66 epitope (35). These plasmids were used in the FATT-CTL assay to monitor the lytic activity by M1 58 -66 -specific CD8 ϩ T cells (Fig.…”

Immunogenicity of novel consensus-based DNA vaccines against avian influenza

“…In addition, the functional constraints placed on internal structural genes, inaccessible to antibodies, limit their ability to mutate. These functional constraints, linked to immunodominant epitopes, have been well documented for the internal influenza proteins [12][13][14]. In addition, the presence of preexisting cross-reactive CD4+ helper cells allows for the more rapid generation of a typespecific antibody response, which can aid in neutralizing remaining virus [15].…”