Influenza A(H5) clade 2.3.4.4b viruses have spread widely among wild birds and poultry and caused infections in mammals, including humans, raising public health concerns (1). A case of human infection with H5N5 clade 2.3.4.4b virus was reported in Washington, USA during 2025; the patient subsequently died, representing the second fatal A(H5) case in the United States (1,2). A(H1) and A(H5) hemagglutinins (HAs) belong to influenza A virus phylogenetic group 1. Most antibodies targeting the HA stalk, a conserved, membrane-proximal region of HA, are cross-reactive with HAs within the same phylogenetic group (3). Studies have demonstrated that previous influenza A(H1N1)pdm09 virus infection can provide some protection against H5N1 clade 2.3.4.4b viruses (4,5). The emergence of H5N5 clade 2.3.4.4b virus raises questions about whether previous A(H1N1)pdm09 immunity confers similar protection against A(H5) viruses bearing antigenically distinct neuraminidases (NAs). We used a preimmune ferret model to compare cross-protection conferred by previous A(H1N1)pdm09 infection against A(H5) viruses with homologous or heterologous NA subtypes. We conducted all experiments with H5N5 and H5N1 viruses in a Biosafety Level 3 enhanced facility with approval from the Centers for Disease Control and Prevention Institutional Animal Care and Use Committee.

We intranasally inoculated 12 ferrets serologically naive to seasonal influenza viruses with 10³ PFUs of A(H1N1)pdm09 A/Nebraska/14/2019 virus. We confirmed seroconversion 3 months postinfection by using a hemagglutination inhibition (HAI) assay, which detects antibodies that inhibit HA-mediated hemagglutination, with a geometric mean titer of 320 (95% CI 245.3–417.3) (Appendix Figure, panel A). Using previously described multiplex binding assays (6), we detected high levels of antibodies binding to the head of A(H1N1)pdm09, but not HA head or ectodomain from H5N1 or H5N5 viruses, and antibodies binding to N1 NAs from A(H1N1)pdm09 and H5N1 clade 2.3.4.4b viruses, but not N5 NA from H5N5 virus. In addition, we detected antibodies binding to the group 1 HA stalk derived from A(H1N1)pdm09 virus (Appendix Figure, panels B, C). We then intranasally challenged those animals, referred to as preimmune ferrets, with 10⁴ PFU of H5N1 clade 2.3.4.4b A/Washington/239/2024 or H5N5 A/Washington/2148/2025 virus (6 ferrets per virus), alongside naive control groups without previous influenza immunity. We euthanized 3 ferrets per group on day 4 postinfection to assess viral dissemination; we monitored the remaining animals for clinical signs, weight loss, viral shedding in nasal washes, and virus presence in throat and rectal swabs.

Figure 1

Figure 1. Pathogenicity of influenza A(H5N1) and A(H5N5) clade 2.3.4.4b viruses from study of cross-protection after A(H1N1)pdm09 infection. We challenged ferrets previously intranasally inoculated with A/Nebraska/14/2019 (H1N1) virus (A[H1N1]pdm09 (preimmune group) 3...

Severe disease, characterized by lethargy, fever, and substantial weight loss (maximum 13.8% in H5N1 virus inoculated ferrets and 18.4% in H5N5 virus inoculated ferrets), developed in naive ferrets inoculated with H5N1 or H5N5 viruses. We euthanized all animals on postinfection days 5–6 for H5N1 virus–inoculated ferrets and days 6–10 for H5N5 virus–inoculated ferrets after they reached predefined humane endpoints, consistent with previous reports (7,8). In contrast, preimmune ferrets challenged with H5N1 virus exhibited minimal disease, a mean maximum weight loss of 2.4%, and 100% survival (Figure 1, panels A–C). Preimmune ferrets challenged with H5N5 also showed reduced disease severity (mean maximum weight loss 7.6%); however, 1 animal reached a humane endpoint on day 7 postinfection (Figure 1, panels B, C).

Figure 2

Figure 2. Viral titers after A(H5N1) and A(H5N5) challenge in ferrets in a study on cross-protection against influenza A(H5) clade 2.3.4.4b viruses after A(H1N1)pdm09 infection. A, B) Mean viral titers in nasal...

Preimmune ferrets challenged with either H5N1 or H5N5 virus shed similar levels of virus in nasal wash samples during days 1–4 postinfection (range of mean peak titers 10^3.8–10^5.2 PFU/mL) as naive controls but exhibited reduced titers in throat swab samples on day 4 for H5N5 virus and days 2 and 3 for H5N1 virus (Figure 2, panels A, B). Viral clearance from nasal washes occurred 2–4 days earlier in preimmune ferrets. We detected H5N1 virus less frequently in rectal swab samples from preimmune animals than from naive controls, whereas we did not observe that reduction for ferrets infected with H5N5 virus (Figure 2, panels A, B).

Influenza A(H1N1)pdm09 virus preimmunity did not substantially reduce viral titers in the upper respiratory tract (nasal turbinates) or soft palate after H5N1 or H5N5 virus challenge (Figure 2, panels C, D). However, A(H1N1)pdm09 preimmunity largely prevented viral dissemination to the lower respiratory tract (trachea and lungs) and extrapulmonary tissues for both viruses, with only sporadic low-level viral detection. However, intestinal viral titers were comparable between preimmune and naive ferrets challenged with H5N5 virus (Figure 2, panels C, D).

Detection of group 1 HA stalk antibodies after A(H1N1)pdm09 infection (Appendix Figure), together with previous evidence of HA stalk-mediated cross-protection against disease caused by the influenza A viruses within the same phylogenetic group (3,9), suggests that cross-reactive HA stalk antibodies might contribute to protection against A(H5) viruses. In addition, N1 cross-reactivity might further enhance protection against H5N1 virus. Consistent with our findings, limited N5 cross-reactivity has been reported in humans despite detectable N1 responses (10). Further studies using H5-specific HA stalk antigens and functional assays are needed to determine the contribution of cross-reactive antibodies and cellular immunity to protection against zoonotic influenza A viruses. Our findings showed that previous A(H1N1)pdm09 viral infection conferred partial cross-protection against H5N1 and H5N5 viruses in ferrets, reducing disease severity, lethality, and viral dissemination.

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Suggested citation for this article: Sun X, Belser JA, Li Z-N, Brock N, Pulit-Penaloza JA, Kieran TJ, et al. Cross-protection against influenza A(H5) clade 2.3.4.4b viruses after A(H1N1)pdm09 infection. Emerg Infect Dis. 2026 Dec [date cited]. https://doi.org/10.3201/eid3212.261032