An effective universal vaccine for influenza will likely need to induce virus-specific T-cells, which are the major mediator of heterosubtypic cross-protection between different subtypes of influenza A virus. In this study we characterise the cell-mediated immune response in ferrets during heterosubtypic protection induced by low-dose H1N1 virus infection against an H3N2 virus challenge, given 4 weeks later. Although the ferrets were not protected against the infection by H3N2 virus, the duration of virus shedding was shortened, and clinical disease was markedly reduced. No cross-reactive neutralizing antibodies were detected, but cross-reactive interferon-gamma-secreting T cells were detected in the circulation prior to H3N2 challenge. These T-cells peaked at 11 days post-H1N1 infection, and were strongly induced in blood and in lung following H3N2 infection. The rapid induction of interferon-gamma-secreting cells in ferrets previously infected with H1N1 virus, but not in naïve ferrets, suggests induction of memory T-cells. These results are in accord with the observations that pre-existing cross-reactive T-cells correlate with protection in humans and have implications for outbreak modelling and universal vaccine design.
Seasonal influenza virus infections cause yearly epidemics which are the source of a significant public health burden worldwide. The ferret model for human influenza A virus (IAV) is widely used and has several advantages over other animal models such as comparable symptomology, similar receptor distribution in the respiratory tract to humans and the ability to be infected with human isolates without the need for adaptation. However, a major disadvantage of the model has been a paucity of reagents for the evaluation of the cellular immune response. Investigation of T-cell mediated immunity in ferrets is crucial to vaccine development and efficacy studies. In this study we have used commercially produced antibodies to ferret interferon gamma (IFN-?) allowing us to reliably measure influenza-specific IFN-? as a marker of the cellular immune response using both enzyme-linked immunospot (ELISpot) and enzyme-linked immunosorbent (ELISA) techniques. Here we demonstrate the application of these tools to evaluate cellular immunity in ferrets infected with clinically relevant seasonal H1N1 and H3N2 IAV subtypes at equivalent doses. Using small heparinised blood samples we were able to observe the longitudinal influenza-specific IFN-? responses of ferrets infected with both seasonal subtypes of IAV and found a notable increase in influenza-specific IFN-? responses in circulating peripheral blood within 8 days post-infection. Both seasonal strains caused a well-defined pattern of influenza-specific IFN-? responses in infected ferrets when compared to naïve animals. Additionally, we found that while the influenza specific IFN-? responses found in peripheral circulating blood were comparable between subtypes, the influenza specific IFN-? responses found in lung lymphocytes significantly differed. Our results suggest that there is a distinct difference between the ability of the two seasonal influenza strains to establish an infection in the lung of ferrets associated with distinct signatures of acquired immunity.
