T cell-mediated protection against lethal 2009 pandemic H1N1 influenza virus infection in a mouse model

Abstract

Genetic mutation and reassortment of influenza virus gene segments, in particular those of hemagglutinin (HA) and neuraminidase (NA), that lead to antigenic drift and shift are the major strategies for influenza virus to escape preexisting immunity. The most recent example of such phenomena is the first pandemic of H1N1 influenza of the 21st century, which started in 2009. Cross-reactive antibodies raised against H1N1 viruses circulating before 1930 show protective activity against the 2009 pandemic virus. Cross-reactive T-cell responses can also contribute to protection, but in vivo support of this view is lacking. To explore the protection mechanisms in vivo, we primed mice with H1 and H3 influenza virus isolates and rechallenged them with a virus derived from the 2009 H1N1 A/CA/04/09 virus, named CA/E3/09. We found that priming with influenza viruses of both H1 and H3 homo- and heterosubtypes protected against lethal CA/E3/09 virus challenge. Convalescent-phase sera from these primed mice conferred no neutralization activity in vitro and no protection in vivo. However, T-cell depletion studies suggested that both CD4 and CD8 T cells contributed to the protection. Taken together, these results indicate that cross-reactive T cells established after initial priming with distally related viruses can be a vital component for prevention of disease and control of pandemic H1N1 influenza virus infection. Our results highlight the importance of establishing cross-reactive T-cell responses for protecting against existing or newly emerging pandemic influenza viruses.

FIG. 1.

Dose-dependent infection of mice with CA/E3/09 virus. As indicated in the figure, different doses of CA/E3/09 virus were inoculated intranasally into C57BL/6 mice. Body weight loss (A) and survival (B) were monitored daily. Data presented are average values ± standard deviations (SD) for at least 5 mice for each group and are representative of two independent experiments.

FIG. 2.

Prior priming with different influenza virus strains provides protection against lethal infection with CA/E3/09. (A) Cohorts of C57BL/6 mice were primed with PR8 (5 PFU/mouse), X31 (3 × 10⁵ EID₅₀/mouse), NC/99 (9 × 10⁴ EID₅₀/mouse), or CA/E3/09 (30 PFU/mouse) virus, and infections were confirmed by body weight loss for up to 14 days. Naïve or primed mice were rested for a total of 42 days and rechallenged with a predetermined lethal dose of CA/E3/09 virus (3 × 10³ PFU/mouse). Body weight loss (B) and survival (C) were monitored daily. (D) On day 3 after rechallenge, lung samples were collected from PR8-, X31-, and NC/99-primed or nonprimed mice, and viruses were titrated by MDCK cell-based plaque assay. Data presented are average values ± SD for at least 10 mice for each group and are representative of at least two independent experiments.

FIG. 3.

Immune sera from primed mice were not protective. Mice were infected with PR8 (5 PFU), X31 (3 × 10⁵ EID₅₀), NC/99 (9 × 10⁴ EID₅₀), or CA/E3/09 (30 PFU) virus, and sera were collected 3 and 5 weeks after infection. Serum samples from each infected group were pooled and injected into mice before infection with a lethal dose of CA/E3/09 virus. Body weight loss (A and B) and survival (C and D) were monitored daily. Data presented are average values ± SD for at least 6 mice for each group.

FIG. 4.

CD4 and CD8 cells contributed to protection against CA/E3/09 virus infection. Mice were primed with X31 virus (3 × 10⁵ EID₅₀) and rested for 42 days. Anti-CD4 (GK1.5), anti-CD8 (2.43), both CD4 and CD8 antibodies, or isotype control antibodies were injected into mice before and after rechallenge with lethal CA/E3/09 virus infection (3,000 PFU). Weight loss (A) and survival (B) were monitored. Values shown are average percentages of initial weight ± SD for 5 mice in each group.