Vaccination against seasonal influenza A/H3N2 virus reduces the induction of heterosubtypic immunity against influenza A/H5N1 virus infection in ferrets

Abstract

Infection with seasonal influenza viruses induces a certain extent of protective immunity against potentially pandemic viruses of novel subtypes, also known as heterosubtypic immunity. Here we demonstrate that infection with a recent influenza A/H3N2 virus strain induces robust protection in ferrets against infection with a highly pathogenic avian influenza virus of the H5N1 subtype. Prior H3N2 virus infection reduced H5N1 virus replication in the upper respiratory tract, as well as clinical signs, mortality, and histopathological changes associated with virus replication in the brain. This protective immunity correlated with the induction of T cells that cross-reacted with H5N1 viral antigen. We also demonstrated that prior vaccination against influenza A/H3N2 virus reduced the induction of heterosubtypic immunity otherwise induced by infection with the influenza A/H3N2 virus. The implications of these findings are discussed in the context of vaccination strategies and vaccine development aiming at the induction of immunity to pandemic influenza.

FIG. 1.

Antibody responses after vaccination and outcomes of infection with influenza A/H3N2 virus. (A) Mean virus-specific antibody responses ± standard deviations (SD), as measured with the hemagglutination inhibition assay 28 days after the first vaccination and 28 days after the second vaccination for unvaccinated animals (shaded bars) and vaccinated animals (open bars). (B) Mean weight loss of vaccinated (open circles) and unvaccinated (shaded circles) animals after inoculation with influenza A/H3N2 virus. (C and D) Virus titers of nasal (C) and pharyngeal (D) swabs collected at 2 and 4 dpi with influenza A/H3N2 virus. Shaded bars represent mean results ± SD for unvaccinated animals, and open bars represent mean results ± SD for vaccinated animals. **, P < 0.01 compared with unvaccinated animals. The gray area indicates the detection limit of the assay.

FIG. 2.

T-cell immune responses 28 days after infection with influenza A/H3N2 virus. Proliferation of CD3⁺ CD8⁻ cells was measured upon stimulation with whole inactivated influenza A/Brisbane/010/07 (H3N2) virus antigen (A) and upon stimulation with whole inactivated influenza A/Indonesia/5/2005 (H5N1) virus antigen (B). *, P < 0.05 compared with unvaccinated, mock-infected animals.

FIG. 3.

Weight loss after inoculation with influenza A/Indonesia/5/05 virus. After inoculation with influenza A/H5N1 virus, ferrets were weighed, and the relative weight loss compared to the body weight at the day of inoculation was calculated. The data shown are relative weight losses for all individual ferrets for unvaccinated, H3N2 virus-primed animals of group 1 (A), vaccinated, H3N2 virus-primed animals of group 2 (B), vaccinated, unprimed animals of group 3 (C), and unvaccinated and unprimed animals of group 4 (D). +, ferrets that died or had to be euthanized due to the presence of severe clinical symptoms.

FIG. 4.

Virus titers in nasal and pharyngeal swabs collected after inoculation with influenza A/H5N1 virus. Nasal (A) and pharyngeal (B) swabs were collected at 2, 4, 6, and 7 dpi with influenza A/H5N1 virus, and virus titers were determined. The data shown are mean virus titers ± SD for unvaccinated, H3N2 virus-infected ferrets of group 1 (white bars), vaccinated, H3N2 virus-infected ferrets of group 2 (dark shaded bars), vaccinated, mock-infected ferrets of group 3 (light shaded bars), and unvaccinated, mock-infected ferrets of group 4 (black bars). *, P < 0.05 between groups; **, P < 0.01 between groups. The gray area indicates the detection limit of the assay.

FIG. 5.

Examples of histologic findings in tissues of ferrets inoculated with influenza A/H5N1 virus. Seven days after inoculation, histopathologic changes associated with the expression of influenza virus antigen were observed in brains and lungs of ferrets without heterosubtypic immunity, i.e., in three ferrets of group 2 (vaccinated, H3N2 virus-infected group) and in all ferrets of groups 3 (vaccinated, mock-infected group) and 4 (mock-vaccinated, mock-infected group) (A, C, E, and G). In the cerebrum, there is necrosis of neurons and infiltration with inflammatory cells (A) associated with expression of influenza virus antigen in neurons (C). (E) In the lungs, cell debris and erythrocytes are present in the alveolar lumens and neutrophils are present in the alveolar walls. (G) This is associated with the expression of influenza virus antigen in type II pneumocytes. Neither histopathological changes (B and F) nor influenza virus expression (D and H) were present in lungs and brains of five ferrets of group 2 (vaccinated, H3N2 virus-infected group) and all ferrets of group 1 (mock-vaccinated, H3N2 virus-infected group).