Experimental protocol.

<p>Male Fitch ferrets (Mustela putorius furo), approximately 6 months of age were used in this study. Groups of 8 ferrets were infected intranasally with 1 x 10⁶ pfu of A/Perth/16/09, or groups of 4 to 6 ferrets were mock infected. Baseline weights and temperatures were obtained for the three consecutive days prior to challenge and on day 0 (the day of challenge). Following challenge, ferrets were monitored for change in body weight and temperature as well as clinical signs of illness on a daily basis for 5 days. Blood samples were collected for Flow Cytometry (days 0–5) and serum collected (days 0, 2 and 5) to asses antibody responses (HI assays). All ferrets were euthanized on day 2 (4 Perth/16-infected ferrets) or day 5 post-challenge (4 Perth/16-infected and all mock-infected ferrets). Viral titers were determined from nasal washes (days 0–5) nasal turbinates (days 2 and 5), BALF (days 2 and 5) and trachea (upper and lower regions; days 2 and 5). Leukocyte purification was performed from peripheral blood (days 0–5), from lymph nodes (days 2 and 5) and from BALF (days 2 and 5).</p

Lymph node leukocyte subsets following infection.

<p>For screening of immune cell migration in response to influenza infection, MRLN, MdLN and MsLN were collected. Purified cell subsets from MRLN (A-B), MdLN (C) and MsLN (D) were stained and analyzed by flow cytometry. In MRLN, percentage (A) and absolute number (B) of T_H (CD3+ and CD4+), Tc lymphocytes (CD3+ and CD8+), B cells (CD79a+) and CD11b-positive cells were measured. In MdLN and MsLN, only percentages (C-D) of T_H (CD3+ and CD4+), Tc lymphocytes (CD3+ and CD8+), B cells (CD79a+) and CD11b-positive cells were measured. For mock infected animals (M), LNs were screened at day 5 post-challenge and for Perth/16 infected animals at days 2 and 5 post-challenge. A p value of 0.05 was used as the cutoff for statistical significance (* p ≤ 0.05; ** p ≤ 0.01; † p ≤ 0.001). Error bars represent SEM.</p

Peripheral blood leukocyte subsets following infection.

<p>Ferrets were bled on days 0–5 relative to the day of viral challenge, and cells were stained and analyzed by flow cytometry as described in the text. Percentage and absolute number of T_H (CD3+ and CD4+), Tc lymphocytes (CD3+ and CD8+), B cells (CD79a+) and CD11b-positive cells were measured (A-H). 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. Group averages are reported here. “Mock” ferrets were infected intranasally with sterile egg allantoic fluid; “Perth/16” ferrets infected intranasally with 1 x 10⁶ pfu of A/Perth/16/09. For mock animals (M), absolute number of same cell subsets were measured at day 5 post-challenge and for the infected animals at days 2 and 5 post-challenge. A p value of 0.05 was used as the cutoff for statistical significance (* p ≤ 0.05; ** p ≤ 0.01; † p ≤ 0.001). Error bars represent SEM.</p

Tissue leukocyte cell counts following infection.

<p>For evaluation of cellularity, the total cell numbers were calculated in spleen, MRLN, BALF (A), and in peripheral blood (PB) (B). Group averages are reported. For the mock-infected animals (M), all data are collected on day 5 post-challenge, and for the virus-infected animals on days 2 and 5 post-challenge (d2 and d5). A p value of 0.05 was used as the cutoff for statistical significance (* p ≤ 0.05; ** p ≤ 0.01; † p ≤ 0.001). Error bars represent SEM.</p

Clinical responses to infection.

<p>To measure morbidity the temperature (A) and body weight (B) readings of infected and control ferrets were recorded daily (days -3 to 5). Data shown are normalized to the individual animals’ weight or temperature on the day of challenge (day 0), and group averages are reported. “Mock” ferrets were infected intranasally with sterile egg allantoic fluid; “Perth/16” ferrets infected intranasally with 1 x 10⁶ pfu of A/Perth/16/09. The nasal cavities of all ferrets were washed daily with 1ml of PBS on days 0–5; and viral titers in the nasal washes (C) were determined. In addition, viral titers were determined in tissues (D); BALF, nasal turbinates (NT), upper trachea (UTr), and lower trachea (LTr). A p value of 0.05 was used as the cutoff for statistical significance (* p ≤ 0.05; ** p ≤ 0.01; † p ≤ 0.001). Error bars represent SEM.</p

BALF and Spleen leukocyte subsets following infection.

<p>For screening of immune cell migration in response to influenza infection, BALF and Spleen were collected. Purified cell subsets from BALF (A-B) and Spleen (C-D) were stained and analyzed by flow cytometry. Percentages (A, C) and absolute number (B, D) of T_H (CD3+ and CD4+), Tc lymphocytes (CD3+ and CD8+), B cells (CD79a+) and CD11b-positive cells were measured. For mock infected animals (M), BALF and spleen were screened at day 5 post-challenge and for Perth/16 infected animals, at days 2 and 5 post-challenge. A p value of 0.05 was used as the cutoff for statistical significance (* p ≤ 0.05; ** p ≤ 0.01; † p ≤ 0.001). Error bars represent SEM.</p

Influenza Vaccination Accelerates Recovery of Ferrets from Lymphopenia

<div><p>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.</p></div

Flow cytometry of ferret peripheral blood leukocytes.

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

Antibodies used in this study.

<p>Antibodies used in this study.</p

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

<p>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. (<b>A</b>) Total white blood cell counts were obtained using a Hemavet apparatus. Percentages of (<b>B</b>) CD3-positive cells (T cells), (<b>C</b>) CD8-positive cells (cytotoxic T lymphocytes), (<b>D</b>) CD3-positive cells (T cells) and CD8-negative cells, (<b>E</b>) CD79a-positive cells (B cells), and (<b>F</b>) 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