Influenza vaccination accelerates recovery of ferrets from lymphopenia

Abstract

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.

Figure 1. Flow cytometry of ferret peripheral blood leukocytes.

Representative stains for (A) unstained, unpermeablized cells after gating out doublets; (B) CD3 vs CD79a; (C) CD11b; (D) CD3 vs CD11b; (E) CD3 vs CD4; (F) CD3 vs CD8; (G) CD4 vs CD8 (ungated cells); (H) CD4 vs CD8 (CD3 gated cells); (I) CD3 vs CD8 vs CD79a (B cells: Green; CD8+ve T cells: Red; CD8-ve T cells: Blue; Granulocytes/NK cells/Monocytes/DC: Gray); (J) FSC vs SSC vs CD11b (Monocytes/DC: Red; Lymphocytes: Blue; Granulocytes: Gray).

Figure 2. Hemagglutination inhibition (HI) titers.

(A–C) Ferrets were immunized with commercial human TIV on days 0 and 21, and challenged with Perth/16 on day 35. Blood samples were collected on days 0, 21, 35, 42, and 49. (D–F) Ferrets were challenged with Perth/16 and NY/21 on day 0. Blood samples were collected on days 0, 9 and 14. HI assays were performed using (A, D) A/Victoria/210/2009 (H3N2), (B, E) A/California/07/2009 (H1N1pdm), and (C, F) B/Brisbane/60/2008 antigen controls. “Mock”, unvaccinated mock-infected animals; “Naïve”, unvaccinated animals challenged with Perth/16; “TIV”, vaccinated animals challenged with Perth/16; “H3N2”, unvaccinated animals challenged with Perth/16; “H1N1pdm”, unvaccinated animals challenged with NY/21. † indicates p<0.0001; * indicates 0.0001<p<0.05. Error bars represent 95% confidence intervals, calculated using compound symmetry covariance to pool variability across groups.

Figure 3. Clinical responses to infection.

(A–C) Two weeks after booster vaccination, ferrets were challenged intranasally with 10⁶ PFU of Perth/16. (D–F) Unvaccinated ferrets were challenged intranasally with 1×10⁶ PFU of NY/21 or Perth/16. (A, D) Body weight and (B, E) temperature were measured daily. Data shown are normalized to the individual animals’ weight or temperature on the day of challenge (day 0). (C, F) Nasal washes were obtained on days 0–9 and virus titers in the nasal washes were measured. Error bars represent 95% confidence intervals. “Mock”, unvaccinated mock-infected animals; “Naïve”, unvaccinated animals challenged with Perth/16; “TIV”, vaccinated animals challenged with Perth/16. “H3N2”, unvaccinated animals challenged with Perth/16; “H1N1pdm”, unvaccinated animals challenged with NY/21. † indicates p<0.0001; * indicates 0.0001<p<0.05. “MvN”, comparing mock-infected ferrets to naïve ferrets; “MvT”, comparing mock-infected ferrets to ferrets vaccinated with TIV; “NvT”, comparing naïve ferrets to ferrets vaccinated with TIV; “MvH3”, comparing mock-infected ferrets to ferrets infected with H3N2; “MvH1”, comparing mock-infected ferrets to ferrets infected with H1N1pdm09; “H1vH3”, comparing H1N1pdm09 infected ferrets to ferrets infected with H3N2.

Figure 4. Peripheral blood leukocyte subsets following infection of vaccinated and naïve animals with Perth/16.

Ferrets were bled on days 0–7, 9, 11, and 13 relative to the day of viral challenge, and cells were stained and analyzed by flow cytometry as described in the text. (A) Total white blood cell counts were obtained using a Hemavet apparatus. Percentages of (B) CD3-positive cells (T cells), (C) CD8-positive cells (cytotoxic T lymphocytes), (D) CD3-positive cells (T cells) and CD8-negative cells, (E) CD79a-positive cells (B cells), and (F) CD11b-positive cells excluding CD11b-high/FSC-high cells (granulocytes) were measured. For each animal the frequencies were normalized to the ferret’s values on day 0; the Y axis represents percent of values on day 0. “Mock”, unvaccinated mock-infected animals; “Naïve”, unvaccinated animals challenged with Perth/16; “TIV”, vaccinated animals challenged with Perth/16. † indicates p<0.0001; * indicates 0.0001<p<0.05. “MvN”, comparing mock-infected ferrets to naïve ferrets; “MvT”, comparing mock-infected ferrets to ferrets vaccinated with TIV; “NvT”, comparing naïve ferrets to ferrets vaccinated with TIV.

Figure 5. Peripheral blood leukocyte subsets following infection of naïve animals with Perth/16 and NY/21.

Ferrets were bled on days 0–7, 9, 11, and 13 relative to the day of viral challenge, and cells were stained and analyzed by flow cytometry as described in the text. (A) Total white blood cell counts were obtained using a Hemavet apparatus. Percentages of (B) CD3-positive cells (T cells), (C) CD8-positive cells (cytotoxic T lymphocytes), (D) TH cells (CD3+ and CD4+), (E) CD79a-positive cells (B cells), and (F) CD11b-positive cells excluding CD11b-high/FSC-high cells (granulocytes) were measured. For each animal the frequencies were normalized to the ferret’s values on day 0; the Y axis represents percent of values on day 0. “Mock”, unvaccinated mock-infected animals; “H3N2”, unvaccinated animals challenged with Perth/16; “H1N1pdm”, unvaccinated animals challenged with NY/21. † indicates p<0.0001; * indicates 0.0001<p<0.05. “MvH3”, comparing mock-infected ferrets to ferrets infected with H3N2; “MvH1”, comparing mock-infected ferrets to ferrets infected with H1N1pdm09; “H1vH3”, comparing H1N1pdm09 infected ferrets to ferrets infected with H3N2.