Immunomic analysis of the repertoire of T-cell specificities for influenza A virus in humans

Abstract

Continuing antigenic drift allows influenza viruses to escape antibody-mediated recognition, and as a consequence, the vaccine currently in use needs to be altered annually. Highly conserved epitopes recognized by effector T cells may represent an alternative approach for the generation of a more universal influenza virus vaccine. Relatively few highly conserved epitopes are currently known in humans, and relatively few epitopes have been identified from proteins other than hemagglutinin and nucleoprotein. This prompted us to perform a study aimed at identifying a set of human T-cell epitopes that would provide broad coverage against different virus strains and subtypes. To provide coverage across different ethnicities, seven different HLA supertypes were considered. More than 4,000 peptides were selected from a panel of 23 influenza A virus strains based on predicted high-affinity binding to HLA class I or class II and high conservancy levels. Peripheral blood mononuclear cells from 44 healthy human blood donors were tested for reactivity against HLA-matched peptides by using gamma interferon enzyme-linked immunospot assays. Interestingly, we found that PB1 was the major target for both CD4(+) and CD8(+) T-cell responses. The 54 nonredundant epitopes (38 class I and 16 class II) identified herein provided high coverage among different ethnicities, were conserved in the majority of the strains analyzed, and were consistently recognized in multiple individuals. These results enable further functional studies of T-cell responses during influenza virus infection and provide a potential base for the development of a universal influenza vaccine.

FIG. 1.

PB1 and M1 are major targets for both CD4⁺ and CD8⁺ T-cell responses. Bars represent the fraction (%) of the total number of epitopes from the current study derived from each of the viral proteins (A); the recognition frequency (%) for the different viral proteins (number of epitopes recognized/number of peptides tested) (B); and the fraction (%) of the total number of epitopes from previously published studies derived from each of the viral proteins (C).

FIG. 2.

Identified T-cell epitopes are highly conserved among various influenza virus strains and subtypes. (A) The conservancy levels among the selected set of 23 influenza A virus strains for each epitope identified in previously published studies, or in the current study, and the conservancy level among influenza A virus strains of the H5N1, H6N1, H7N7, and H9N2 avian subtypes for each epitope identified in the current study are shown. (B) The conservancy levels among the more recent (2005 to 2007) influenza A virus strains of the H1N1, H3N2, or H5N1 subtypes are shown for each epitope identified in the current study.

FIG. 3.

The identified epitopes provide broad population coverage. Based on the binding data for each class I-restricted (A) or class II-restricted (B) epitope, theoretical population coverage was calculated. The number of possible epitope-HLA allele combinations as a function of the fraction of each population (%) is shown. Horizontal dashed line indicates the number of possible epitope-HLA alleles presented to at least 80% of each population.

FIG. 4.

The majority of the individuals tested responded to both the HLA class I- and class II-restricted epitope sets. (A) PBMCs from 20 individuals were tested for the recognition of pools of the set of class I-restricted (A) and class II-restricted (B) epitopes in IFN-γ ELISPOT assays. The average SI (specific response/nonspecific response) from three independent experiments is plotted for each donor. Gray bars, significantly positive responses; open bars, negative responses.