Diverse Heterologous Primary Infections Radically Alter Immunodominance Hierarchies and Clinical Outcomes Following H7N9 Influenza Challenge in Mice

Abstract

The recent emergence of a novel H7N9 influenza A virus (IAV) causing severe human infections in China raises concerns about a possible pandemic. The lack of pre-existing neutralizing antibodies in the broader population highlights the potential protective role of IAV-specific CD8+ cytotoxic T lymphocyte (CTL) memory specific for epitopes conserved between H7N9 and previously encountered IAVs. In the present study, the heterosubtypic immunity generated by prior H9N2 or H1N1 infections significantly, but variably, reduced morbidity and mortality, pulmonary virus load and time to clearance in mice challenged with the H7N9 virus. In all cases, the recall of established CTL memory was characterized by earlier, greater airway infiltration of effectors targeting the conserved or cross-reactive H7N9 IAV peptides; though, depending on the priming IAV, each case was accompanied by distinct CTL epitope immunodominance hierarchies for the prominent K ^bPB1 ₇₀₃, D ^bPA ₂₂₄, and D ^bNP ₃₆₆ epitopes. While the presence of conserved, variable, or cross-reactive epitopes between the priming H9N2 and H1N1 and the challenge H7N9 IAVs clearly influenced any change in the immunodominance hierarchy, the changing patterns were not tied solely to epitope conservation. Furthermore, the total size of the IAV-specific memory CTL pool after priming was a better predictor of favorable outcomes than the extent of epitope conservation or secondary CTL expansion. Modifying the size of the memory CTL pool significantly altered its subsequent protective efficacy on disease severity or virus clearance, confirming the important role of heterologous priming. These findings establish that both the protective efficacy of heterosubtypic immunity and CTL immunodominance hierarchies are reflective of the immunological history of the host, a finding that has implications for understanding human CTL responses and the rational design of CTL-mediated vaccines.

Author Summary

The emergence of human infections with a novel strain of avian-origin H7N9 virus in China raises a pandemic concern. The introduction of a new subtype in humans makes people at all ages susceptible due to the lack of population-wide neutralizing antibodies. However, cross-subtype protection from existing host immunity might provide important protection that can limit severe disease. Our study found that previous infection with non-H7N9 subtype viruses such as H9N2 viruses or H1N1 viruses could provide protection against lethal H7N9 challenge to varying degrees in mice. The virus-specific memory CD8+ T cells generated by the previous infection but targeting conserved or related portions of the internal proteins (epitopes) of the H7N9 viruses were selectively expanded and recruited at very early time points after H7N9 challenge, contributing to protective efficacy. The magnitude of the priming virus-generated memory CD8+ T cells was the best predictor of the protective efficacy of the heterosubtypic immunity against subsequent H7N9 challenge in these animals, when compared to the magnitude of the challenge response or the degree of epitope conservation between the priming and challenge strains. These results demonstrate the complexity of cross-reactive CD8+ T cell dynamics and suggest that significant protective immunity can be present even when few epitopes are conserved during heterosubtypic influenza infection.