Dairy cattle herds mount a characteristic antibody response to highly pathogenic H5N1 avian influenza viruses

Abstract

An unprecedented outbreak of a highly pathogenic avian influenza virus, H5 clade 2.3.4.4b, was reported in U.S. dairy cattle during the spring of 2024. It has now spread to hundreds of herds across multiple states. In humans, antibodies to the hemagglutinin (HA) protein confer the strongest protection against infection. Human herd immunity limits viral spread but also drives the emergence of antigenic variants that escape dominant antibody responses. We used store-bought milk to profile the collective H5N1 antibody response of dairy cattle herds. We detected HA binding antibodies in specific samples from states with recent/ongoing outbreaks. These antibodies present in milk neutralized replicating virus expressing dairy cattle HA and neuraminidase (NA). Despite originating from independent vendors, dairies/plants, geographic regions, and time, antibodies present in these samples are remarkably similar in activity and HA binding specificity. The dominant antibody response was clade 2.3.4.4b HA specific, followed by cross-reactivity with other H5s. Whether the uniformity of the response is a pathway to achieve herd immunity or an avenue for antigenic variants to rapidly escape remains to be seen.IMPORTANCEEstablishing human herd immunity ends pandemics. For influenza viruses, this immunity drives continued antigenic evolution that enables viruses to infect once-immune individuals. An outbreak of highly pathogenic avian influenza virus was detected in dairy cattle in 2024 and has spread rapidly across herds and states. We report approaches to assess dairy cattle herd immunity using store-bought milk samples. Across samples separated by geography and time, we find dairy cattle mount a strikingly similar antibody response that is strongest to the dairy cattle virus. Benchmarking immunity at this phase of the outbreak is important to understand either eradication or the emergence of antigenic variants that enable reinfection.

Keywords: humoral immunity; immunology; surveillance studies; veterinary immunology; virology.

Fig 1

Antibodies in milk have antiviral activity. (A) Dilution at which each milk sample (Table 1) completely inhibited infection by rVSV-H5N1dc2024, and IC₅₀ neutralizing titers for each milk sample. (B) Representative images of rVSV-H5N1dc2024-infected GFP-expressing cells at 1 and 2 days post-infection in the presence of each milk sample at a 1:10 dilution. (C) Neutralizing titers of purified IgG and mAbs (21, 24–26) for rVSV-H5N1dc2024. IgG sample numbers match the milk sample from which they were purified. (D) Representative images of rVSV-H5N1dc2024-infected cells in the presence of subneutralizing concentrations of purified IgG over 5 days. IgG concentration for each is indicated. Wells that have reached complete viral spread are outlined in red. Images taken at half-day intervals are not shown. (E) Protection from viral spread by purified milk IgG. Spread of rVSV-H5N1dc2024 infection in the presence of purified IgG was assessed twice per day over 5 days for each concentration of IgG. “Survival” was defined as conditions where infection had not spread throughout the well.

Fig 2

Breadth of HA reactivity of milk antibodies. Binding of antibodies in milk to recombinant full-length secreted ectodomain HA trimers from 83 unique isolates was assessed by ELISA. Nipah virus F protein (NiV-F) was included as a negative control. Bound antibody was detected using horseradish peroxidase-conjugated protein A/G. Bars are colored based on HA subtype, with H5 clade 2.3.4.4.b H5 in darker red for emphasis. The identity of each HA is provided in Table S2.

Fig 3

Purified IgG from milk reacts most strongly to clade 2.3.4.4.b H5 HAs. ELISA titrations were performed to determine the EC₅₀ values for each purified IgG sample to H5, H2, H1, and H3 HAs. A/dairy cow/Texas/24-008749-001/2024 and A/Astrakhan/3212/2020 both belong to clade 2.3.4.4b. mAb FluA-20 (27) was included as a broadly binding control antibody.