Ferrets are a useful animal model for human influenza virus infections, since they closely mimic the pathogenesis of influenza viruses observed in humans. However, a lack of reagents, especially for flow cytometry of immune cell subsets, has limited research in this model. Here we use a panel of primarily species cross-reactive antibodies to identify ferret T cells, cytotoxic T lymphocytes (CTL), B cells, and granulocytes in peripheral blood. Following infection with seasonal H3N2 or H1N1pdm09 influenza viruses, these cell types showed rapid and dramatic changes in frequency, even though clinically the infections were mild. The loss of B cells and CD4 and CD8 T cells, and the increase in neutrophils, were especially marked 1–2 days after infection, when about 90% of CD8+ T cells disappeared from the peripheral blood. The different virus strains led to different kinetics of leukocyte subset alterations. Vaccination with homologous vaccine reduced clinical symptoms slightly, but led to a much more rapid return to normal leukocyte parameters. Assessment of clinical symptoms may underestimate the effectiveness of influenza vaccine in restoring homeostasis.
BackgroundVaccines against avian influenza viruses often require high hemagglutinin (HA) doses or adjuvants to achieve serological titers associated with protection against disease. In particular, viruses of the H7 subtype frequently do not induce strong antibody responses following immunization.ObjectivesTo evaluate whether poor immunogenicity of H7 viruses is an intrinsic property of the H7 hemagglutinin.MethodsWe compared the immunogenicity, in naïve mice, of purified recombinant HA from two H7 viruses [A/Netherlands/219/2003(H7N7) and A/New York/107/2003(H7N2)] to that of HA from human pandemic [A/California/07/2009(H1N1pdm09)] and seasonal [A/Perth16/2009(H3N2)] viruses.ResultsAfter two intramuscular injections with purified hemagglutinin, mice produced antibodies to all HAs, but the response to the human virus HAs was greater than to H7 HAs. The difference was relatively minor when measured by ELISA, greater when measured by hemagglutination inhibition assays, and more marked still by microneutralization assays. H7 HAs induced little or no neutralizing antibody response in mice at either dose tested. Antibodies induced by H7 were of significantly lower avidity than for H3 or H1N1pdm09.ConclusionsWe conclude that H7 HAs may be intrinsically less immunogenic than HA from seasonal human influenza viruses.
Here we define the epitopes on HA that are targeted by a group of 9 recombinant monoclonal antibodies (rmAbs) isolated from memory B cells of mice, immunized by infection with A(H1N1)pdm09 virus followed by a seasonal TIV boost. These rmAbs were all reactive against the HA1 region of HA, but display 7 distinct binding footprints, targeting each of the 4 known antigenic sites. Although the rmAbs were not broadly cross-reactive, a group showed subtype-specific cross-reactivity with the HA of A/South Carolina/1/18. Screening these rmAbs with a panel of human A(H1N1)pdm09 virus isolates indicated that naturally-occurring changes in HA could reduce rmAb binding, HI activity, and/or virus neutralization activity by rmAb, without showing changes in recognition by polyclonal antiserum. In some instances, virus neutralization was lost while both ELISA binding and HI activity were retained, demonstrating a discordance between the two serological assays traditionally used to detect antigenic drift.
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