Significant impact of sequence variations in the nucleoprotein on CD8 T cell-mediated cross-protection against influenza A virus infections

Abstract

Background: Memory CD8 T cells to influenza A viruses are widely detectable in healthy human subjects and broadly cross-reactive for serologically distinct influenza A virus subtypes. However, it is not clear to what extent such pre-existing cellular immunity can provide cross-subtype protection against novel emerging influenza A viruses.

Methodology/principal findings: We show in the mouse model that naturally occurring sequence variations of the conserved nucleoprotein of the virus significantly impact cross-protection against lethal disease in vivo. When priming and challenge viruses shared identical sequences of the immunodominant, protective NP(366)/D(b) epitope, strong cross-subtype protection was observed. However, when they did not share complete sequence identity in this epitope, cross-protection was considerably reduced. Contributions of virus-specific antibodies appeared to be minimal under these circumstances. Detailed analysis revealed that the magnitude of the memory CD8 T cell response triggered by the NP(366)/D(b) variants was significantly lower than those triggered by the homologous NP(366)/D(b) ligand. It appears that strict specificity of a dominant public TCR to the original NP(366)/D(b) ligand may limit the expansion of cross-reactive memory CD8 T cells to the NP(366)/D(b) variants.

Conclusions/significance: Pre-existing CD8 T cell immunity may provide substantial cross-protection against heterosubtypic influenza A viruses, provided that the priming and the subsequent challenge viruses share the identical sequences of the immunodominant, protective CTL epitopes.

Figure 1. Body weight loss and survival after heterosubtypic influenza virus infections.

Five to eight B6 mice per group were primed intranasally with X31 or Memphis H3N2 influenza A viruses as indicated or equal volume of allantoic fluid as control. 35 to 45 days after priming, the animals were challenged with lethal doses of heterosubtypic H1N1 influenza viruses as indicated. Body weight loss (left panel) and survival (right panel) of the animals were monitored until day 19 after lethal challenge. Animals that lost 25% of their initial body weight were considered moribund and sacrificed according the animal protocol. The results were expressed as body weight loss of individual mice per group. The numbers on the lower right corner of each graph indicate survival rate of each group (the number of animals survived/total number of animals tested).

Figure 2. Strain-specific and cross-reactive antibodies following lethal challenge of primed mice with heterosubtypic influenza A virus.

Five B6 mice per group were primed intranasally as described in figure 1. Serum, lung and nasal washes were sampled from individual animals at day 37 after priming and tested for serum HI titers (A), nasal wash IgA titers (B), lung wash IgG titers (C) against homotypic as well as heterosubtypic influenza A viruses as indicated. Serum IgG antibody titers to M2 protein was determined by an ELISA using synthetic M2e peptide as antigen (D).

Figure 3. Effect of CD8- or CD4 T cell depletion on heterosubtypic immunity.

Five B6 mice per group were primed intranasally as described in figure 1. Mock-primed (A) or Memphis virus-primed (B) animals were depleted of either CD8- or CD4 T cell subset in vivo as described in Materials and Methods. Monitoring of body weight loss after lethal challenge with Taiwan virus and expression of the results were described in detail in figure 1. One representative result is shown from two independent studies with similar results. Asterisk (*) indicates a statistically significant difference when CD8-depleted group was compared with PBS control group.

Figure 4. Magnitude and composition of specific and cross-reactive CD8 T cells to wild-type and variant NP₃₆₆/D^b epitope after heterosubtypic influenza virus infections.

Five to ten B6 mice per group were primed and challenged with lethal doses of heterosubtypic influenza A viruses as described in figure 1. Lung tissues were collected from individual animals five days after the lethal challenge and examined for the magnitude of the CD8 T cell response to the wild-type and variant NP₃₆₆/D^b epitope (A). The mean percentages of the cell subsets from the same set of the data were used to assess the composition of the NP₃₆₆/D^b response specific for the original NP₃₆₆ priming sequence or cross-reactive to the NP₃₆₆ challenge variants as indicated (B).

Figure 5. Plasticity of a dominant public TCR to NP₃₆₆/D^b variants.

Serially titrated amount of synthetic NP₃₆₆/D^b peptides identified in table 1 were used to stabilize D^b expression on the surface of a TAP-deficient RMA-S cells in an RMA-S assay (A). The results were expressed as mean fluorescence intensity (MFI). The ability of the NP₃₆₆/D^b variants to trigger activation of a T cell transfectant (clone A3-4) expressing the dominant public TCR specific for the PR8-NP₃₆₆/D^b ligand, was assessed by an IL-2 assay (B). The data are representative of two independent experiments.