Effect of Seasonal Influenza Vaccines on Avian Influenza A(H5N1) Clade 2.3.4.4b Virus Infection in Ferrets

Abstract

Highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b viruses have infected >1,000 herds of dairy cattle and hundreds of poultry flocks in the United States since the beginning of 2024. Seventy human cases have been reported during that period, mainly through occupational exposure. Although prior influenza A(H1N1)pdm09 virus infection has been shown to confer protection against influenza A(H5N1) clade 2.3.4.4b virus infection in the ferret model, it remains unclear if influenza vaccines, known to elicit a less potent and narrower cross-reactive immune response, can achieve a similar effect. In this article, we demonstrate that immunization with commercially available human seasonal influenza vaccines also confers partial protection against disease caused by H5N1 clade 2.3.4.4b virus in ferrets, which is partially associated with the presence of cross-reactive antibodies targeting H5N1 virus antigens.

Keywords: H5N1; Influenza; United States; avian influenza; clade 2.3.4.4b; ferrets; vaccines; viruses.

Figure 1

Hemagglutinin-inhibiting antibody titers measured by using HAI in study of the effect of seasonal influenza vaccines on influenza A(H5N1) clade 2.3.4.4b virus infection in ferrets. n = 6 ferrets per vaccinated group, n = 12 ferrets in the mock vaccinated group. Colored dots indicate individual values, horizontal lines within boxes represent geometric mean titers, box tops and bottoms indicate interquartile ranges, and error bars indicate limits of the distribution. Dotted gray horizontal line indicates limit of detection. H1, A/Victoria/2570/2019(H1N1)pdm09; H3, A/Delaware/01/2021(H3N2); H5, A/Michigan/90/2024/(H5N1); HAI, hemagglutination inhibition assay; IIV3, Fluarix trivalent inactivated influenza vaccine (GlaxoSmithKline Biologicals, https://www.gsk.com); LAIV, FluMist live attenuated influenza vaccine (AstraZeneca, https://www.astrazeneca.com); RIV, Flublok recombinant influenza vaccine (Sanofi, https://www.sanofi.com).

Figure 2

Neuraminidase-inhibiting antibody titers measured by using ELLA in study of the effect of seasonal influenza vaccines on influenza A(H5N1) clade 2.3.4.4b virus infection in ferrets. n = 6 ferrets per the vaccinated group, n = 12 ferrets in the mock vaccinated group. Colored dots indicate individual values, horizontal lines within boxes represent geometric mean titers, box tops and bottoms indicate interquartile ranges, and error bars indicate limits of the distribution. Dotted gray horizontal line indicates limit of detection. ELLA, enzyme-linked lectin assay; IIV3, Fluarix trivalent inactivated influenza vaccine (GlaxoSmithKline Biologicals, https://www.gsk.com); LAIV, FluMist live attenuated influenza vaccine (AstraZeneca, https://www.astrazeneca.com); MA/22, A/Massachusetts/18/2022(H3N2); RIV, Flublok recombinant influenza vaccine (Sanofi, https://www.sanofi.com); TX/24, A/Texas/37/2024(H5N1); WI/22, A/Wisconsin/67/2022(H1N1pdm09).

Figure 3

Hemagglutinin-binding antibody titers measured by using multiplex influenza antibody detection assay in study of the effect of seasonal influenza vaccines on influenza A(H5N1) clade 2.3.4.4b virus infection in ferrets. n = 6 ferrets per vaccinated group, n = 12 ferrets in the mock vaccinated group. A) Globular head of hemagglutinin H1 from influenza A/Wisconsin/67/2022(H1N1pdm09). B) Globular head of hemagglutinin H3 from A/Massachusetts/18/2022(H3N2). C) Globular head of hemagglutinin H5 from A/Texas/37/2024(H5N1). D) Ectodomain of hemagglutinin H5 from A/Texas/37/2024(H5N1). E) Stalk of hemagglutinin H1 from A/Michigan/45/2015(H1N1pdm09). F) Stalk of hemagglutinin H3 from A/Singapore/INFIMH-16-0019/2016(H3N2). Colored dots indicate individual values, horizontal lines within boxes represent geometric mean titers, box tops and bottoms indicate interquartile ranges, and error bars indicate limits of the distribution. Dotted gray horizontal line indicates limit of detection. IIV3, Fluarix trivalent inactivated influenza vaccine (GlaxoSmithKline Biologicals, https://www.gsk.com); LAIV, FluMist live attenuated influenza vaccine (AstraZeneca, https://www.astrazeneca.com); RIV, Flublok recombinant influenza vaccine (Sanofi, https://www.sanofi.com).

Figure 4

Neuraminidase antibody titers measured by using multiplex influenza antibody detection assay in study of the effect of seasonal influenza vaccines on influenza A(H5N1) clade 2.3.4.4b virus infection in ferrets. n = 6 ferrets per vaccinated group, n = 12 ferrets in the mock vaccinated group. A) Neuraminidase N1 from A/Wisconsin/67/2022(H1N1pdm09). B) Neuraminidase N1 from A/Texas/37/2024(H5N1). C) Neuraminidase N2 from A/Massachusetts/18/2022(H3N2). D) Expanded view of neuraminidase N1 from A/Wisconsin/67/2022(H1N1pdm09) to more clearly show the cross-reactive responses postvaccination. E) Expanded view of neuraminidase N1 from A/Texas/37/2024(H5N1) to more clearly show the cross-reactive responses postvaccination. Colored dots indicate individual values, horizontal lines within boxes represent geometric mean titers, box tops and bottoms indicate interquartile ranges, and error bars indicate limits of the distribution. Dotted gray horizontal line indicates limit of detection. IIV3, Fluarix trivalent inactivated influenza vaccine (GlaxoSmithKline Biologicals, https://www.gsk.com); LAIV, FluMist live attenuated influenza vaccine (AstraZeneca, https://www.astrazeneca.com); RIV, Flublok recombinant influenza vaccine (Sanofi, https://www.sanofi.com).

Figure 5

Nucleoprotein antibody titers for A/Brisbane/10/2007(H3N2) virus measured by using multiplex influenza antibody detection assay in study of the effect of seasonal influenza vaccines on influenza A(H5N1) clade 2.3.4.4b virus infection in ferrets. n = 6 ferrets per vaccinated group, n = 12 ferrets in the mock vaccinated group. Colored dots indicate individual values, horizontal lines within boxes represent geometric mean titers, box tops and bottoms indicate interquartile ranges, and error bars indicate limits of the distribution. Dotted gray horizontal line indicates limit of detection. IIV3, Fluarix trivalent inactivated influenza vaccine (GlaxoSmithKline Biologicals, https://www.gsk.com); LAIV, FluMist live attenuated influenza vaccine (AstraZeneca, https://www.astrazeneca.com); RIV, Flublok recombinant influenza vaccine (Sanofi, https://www.sanofi.com).

Figure 6

Body weight and influenza A(H5N1) clade 2.3.4.4b viral titers from nasal wash and tissue samples collected during study of the effect of seasonal influenza vaccines on clade 2.3.4.4b H5N1 virus infection in ferrets. n = 6 ferrets per vaccinated group, n = 12 ferrets in the mock vaccinated group. A) Body weights were monitored for 14 days postinfection and scaled to each ferret’s weight on day 0. We assessed p values relative to the mock-infected group by using a linear mixed model with repeated measures in R with lmerTest version 3.1.3 (The R Project for Statistical Computing, https://www.r-project.org); p values are indicated in colors matching the relevant body weight traces. Lines are plotted as means ± SDs. B) We collected nasal washes with phosphate buffered saline and measured the amount of virus by standard plaque formation assays. We assessed p values by using a linear mixed model with repeated measures in R with lmerTest version 3.1.3. C) Three days after infection, we euthanized 3 ferrets per immunized group and 6 ferrets in the mock control group. We measured the amount of virus present in each tissue sample by standard plaque formation assay and assessed p values relative to the mock-infected group. In panels B and C, colored dots indicate individual values, horizontal lines within boxes represent geometric mean titers, box tops and bottoms indicate interquartile ranges, and error bars indicate limits of the distribution. Dotted gray horizontal line indicates limit of detection. IIV3, Fluarix trivalent inactivated influenza vaccine (GlaxoSmithKline Biologicals, https://www.gsk.com); LAIV, FluMist live attenuated influenza vaccine (AstraZeneca, https://www.astrazeneca.com); RIV, Flublok recombinant influenza vaccine (Sanofi, https://www.sanofi.com).