Influenza vaccine for cattle

Abstract

The present disclosure is directed to recombinant influenza hemagglutinin (HA) polypeptides, which are useful in the preparation of immunogenic compositions and vaccines for treating and preventing disease in cattle.

Description

Agent Ref. No. P14876WO00 TITLE: INFLUENZA VACCINE FOR CATTLE CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to provisional application U.S. Serial No. 63 / 669,600, filed July 10, 2024, which is incorporated herein by reference in its entirety. SEQUENCE LISTING XML

[0002] The instant application contains a sequence listing, which has been submitted in XML file format by electronic submission and is hereby incorporated by reference in its entirety. The XML file, created on July 9, 2025, is named P14876WO00.xml and is 27,391 bytes in size. TECHNICAL FIELD

[0003] This disclosure relates generally to vaccine compositions useful for stimulating immune responses against influenza. BACKGROUND

[0004] Highly pathogenic avian influenza (HPAI) A (H5N1) virus belonging to clade 2.3.4.4b was first detected in wild birds in the United States in late 2021 and spread widely across North America. In early 2022, HPAI H5N1 virus began causing outbreaks in commercial and backyard poultry flocks. The widespread distribution of HPAI H5N1 virus eventually spilled into multiple mammal species within those areas. By the end of 2023, HPAI H5N1 had been reported in more than 20 different mammal species in the United States. Dairy cattle were affected in the spring of 2024, when multiple dairy herds were determined to be positive for HPAI H5N1 clade 2.3.4.4b virus. The rapid spread of the virus across herds in more than a dozen states has prompted the need for an efficacious and rapidly deployable vaccine. SUMMARY

[0005] Recombinant influenza hemagglutinin (HA) polypeptides comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO: 1 are provided. ImmunogenicAgent Ref. No. P14876WO00 fragments of the recombinant HA polypeptide of the disclosure are provided. Multimeric polypeptides comprising at least two of the recombinant HA polypeptides of the disclosure are also provided.

[0006] Immunogenic compositions comprising a recombinant HA polypeptide of the disclosure and a pharmaceutically acceptable carrier are provided. Methods for inducing an immune response against an influenza virus in a subject comprising administering to the subject an immunogenic composition of the disclosure are also provided.

[0007] Vaccine compositions comprising a recombinant HA polypeptide of the disclosure and a pharmaceutically acceptable carrier are provided. Methods of treating or preventing disease caused by an influenza virus comprising administering to the subject a vaccine composition of the disclosure are also provided.

[0008] Polynucleotides encoding a recombinant HA polypeptide of the disclosure are provided. Vectors comprising a polynucleotide of the disclosure are provided. Cells comprising a polynucleotide of the disclosure are provided. Methods for producing a recombinant HA polypeptide comprising expressing a polynucleotide of the disclosure in a cell are also provided.

[0009] While multiple embodiments are disclosed, still other embodiments of the present disclosure will become apparent based on the detailed description, which shows and describes illustrative embodiments of the disclosure. Accordingly, the figures and detailed description are to be regarded as illustrative in nature and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The following drawings form part of the specification and are included to further demonstrate certain embodiments. In some instances, embodiments can be best understood by referring to the accompanying figures in combination with the detailed description presented herein. The description and accompanying figures may highlight a certain specific example, or a certain embodiment. However, one skilled in the art will understand that portions of the example or embodiment may be used in combination with other examples or embodiments.

[0011] FIG.1 shows mammary gland lesions in cattle in study of highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus infection in domestic dairy cattle and cats, United States, 2024. A, B) Mammary gland tissue sections stained with hematoxylin and eosin. A)Agent Ref. No. P14876WO00 Arrowheads indicate segmental loss within open secretory mammary alveoli. Original magnification ×40. B) Arrowheads indicate epithelial degeneration and necrosis lining alveoli with intraluminal sloughing. Asterisk indicates intraluminal neutrophilic inflammation. Original magnification ×400. C, D) Mammary gland tissue sections stained by using avian influenza A immunohistochemistry. C) Staining indicates lobular distribution of avian influenza A virus. Original magnification ×40. D) Staining indicates strong nuclear and intracytoplasmic immunoreactivity of intact and sloughed epithelial cells within mammary alveoli. Original magnification ×400.

[0012] FIG.2 shows lesions in cat tissues in study of highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus infection in domestic dairy cattle and cats, United States, 2024. Tissue sections were stained with hematoxylin and eosin; insets show staining of avian influenza A viruses via immunohistochemistry by using the chromogen 3,3′- diaminobenzidine tetrahydrochloride. Original magnification ×200 for all images and insets. A) Section from cerebral tissue. Arrowheads show perivascular lymphocytic encephalitis, gliosis, and neuronal necrosis. Inset shows neurons. B) Section of lung tissue showing lymphocytic and fibrinous interstitial pneumonia with septal necrosis and alveolar edema; arrowheads indicate lymphocytes. Inset shows bronchiolar epithelium, necrotic cells, and intraseptal mononuclear cells. C) Section of heart tissue. Arrowhead shows interstitial lymphocytic myocarditis and focal peracute myocardial coagulative necrosis. Inset shows cardiomyocytes. D) Section of retinal tissue. Arrowheads show perivascular lymphocytic retinitis with segmental neuronal loss and rarefaction in the ganglion cell layer. Asterisks indicate attenuation of the inner plexiform and nuclear layers with artifactual retinal detachment. Insets shows all layers of the retina segmentally within affected areas have strong cytoplasmic and nuclear immunoreactivity to influenza A virus.

[0013] FIG.3 shows microscopic mammary lesions of an index case in US dairy cattle naturally infected with highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b. A) Mammary gland alveoli show epithelial attenuation and vacuolation (arrows), leading to degeneration with intraluminal sloughing and neutrophilic intraluminal inflammation (asterisk). Hematoxylin and eosin stain. B) Mammary gland alveoli show degenerative epithelial cells (asterisks) and strong intracytoplasmic and nuclear immunoreactivity to influenza A virus nucleoprotein. C) Cuboidal epithelium lining of the interlobular duct was markedly attenuated (arrowheads) with abundant intraluminal sloughing and neutrophilicAgent Ref. No. P14876WO00 inflammation (asterisk). Hematoxylin and eosin stain. D) Interlobular duct shows of attenuating and degenerative epithelium by intranuclear and cytoplasmic immunoreactivity with immunopositive intraluminal debris and inflammation (asterisk). Scale bars indicate 200 μm. E, F) Modified Wright's stained representative cytology images of milk from a dairy cow with highly pathogenic avian influenza A(H5N1) virus infection, demonstrating moderate to marked neutrophilic inflammation (arrows) with low numbers of vacuolated macrophages (arrows) among large numbers of lipid vacuoles. A 100-cell count was performed, and nucleated cells were found to consist of 83% neutrophils, 12% macrophages, and 5% lymphocytes. Original magnifications ×500 for panel E and ×1,000 for panel F.

[0014] FIG.4 shows respiratory tract tissues from a US dairy cow infected with highly pathogenic avian influenza A(H5N1) virus, showing IAV-Np (teal chromogen), individually duplexed with MAL-I (magenta chromogen), MAL-II (magenta chromogen), and SNA (magenta chromogen) using chromogenic staining. Representative images are shown for IAV-Np / MAL-I (A, D, G, J), IAV-Np / MAL-II (B, E, H, K), and IAV-Np / SNA (C, F, I, L) are shown. No IAV-Np was observed in the unaffected respiratory tissue sections. Intense granular to punctate labeling for MAL-I (A) and MAL-II (B) were observed within goblet cells (arrowheads), along the apical ciliated margin (arrows), and glands of the trachea. SNA labeling (C) was confined to intraepithelial round cells (arrows), endothelium, and glands of the trachea. Similar labeling for MAL-I (D) and MALII (E) was observed within the bronchial lumen within goblet cells (arrowheads) and along the apical cell margin (arrows). SNA (F) labeling was only observed in rare goblet cells (arrowheads) and lamina propria round cells (arrows) in the bronchus. Bronchioles had diffuse, fine, fibrillary to apical membranous labeling (arrows) MAL-I (G) and MALII (H). No substantial labeling was detected within the mucosal epithelial cells of bronchioles with SNA (I). Diffuse, fine, apical membranous labeling of pneumocytes lining alveoli was observed with MAL-I (J) and MAL-II (K) (arrows). SNA (L) labeling within the alveolar portions of the lung was confined to endothelium and interstitial round cells. Scale bars indicate 200 μm. IAV-Np, influenza A virus nucleoprotein; MAL, Maackia amurensis lectin; SNA, Sambucus nigra lectin.

[0015] FIG.5 shows respiratory tract tissues from a US dairy cow infected with highly pathogenic avian influenza A(H5N1) virus, labeled with IAV-Np (DyLight 594),Agent Ref. No. P14876WO00 individually duplexed with MAL-I (Alexa Fluor 647), MAL-II (Alexa Fluor 647), and SNA (Alexa Fluor 647) using fluorescent staining. Representative merged images are shown for IAV-Np and MAL-I (A, D, G, J), IAV-Np and MAL-II (B, E, H, K), and IAV- Np and SNA (C, F, I, L). IAV-Np labeling was not detected within the respiratory tissue sections. Intense, granular to punctate, cytoplasmic MAL-I, MAL-II, and SNA labeling was observed in goblet cells (arrowheads) and glands of the trachea (A–C). Similar goblet cell labeling (arrowheads) for MAL-I (D) and MAL-II (E) was observed in the bronchi with weak SNA labeling (F). Multifocal, moderate, fibrillar, apical, membranous MAL-I (A) and MAL-II (B) labeling (arrows) was observed on the tracheal epithelium. The respiratory epithelium of the bronchi, bronchioles, and alveoli had diffuse, moderate to intense, apical, fibrillar MAL-I labeling (arrows) (D, G, J). The respiratory epithelium of the bronchi had multifocal, moderate, fibrillar, apical MAL-II labeling (arrows) (E). The respiratory epithelium of the bronchioles and alveoli had diffuse MAL-II labeling (arrows) (H, K). Intraluminal secretory material (asterisks) in the bronchi and bronchioles were intensely labeled with MAL-I and MAL-II (G, H). Membranous, granular SNA labeling (arrows) was observed in intraepithelial and lamina proprial round cells in the trachea and bronchi (C, F). Scale bars indicate 50 μm. IAV-Np, influenza A virus nucleoprotein; MAL, Maackia amurensis lectin; SNA, Sambucus nigra lectin.

[0016] FIG.6 shows unaffected region of the mammary gland from a US dairy cow infected with highly pathogenic avian influenza A(H5N1) virus, showing IAV-Np (teal chromogen), individually duplexed with MAL-I (magenta chromogen), MAL-II (magenta chromogen), and SNA (magenta chromogen) using chromogenic staining. Representative images of IAV-Np / MAL-I (A, B), IAV-Np / MAL-II (C, D), and IAV-Np / SNA (E, F) showing no IAV-Np labeling within unaffected mammary gland tissue sections. No MAL-I was detected in the mammary glandular (A) or interlobular duct epithelium (B). Within the alveolar gland epithelium, intense, granular, fibrillar labeling (arrows) of the apical portion labeling of MAL-II (C) was noted, with no epithelial labeling within the interlobular duct (D). Multifocal, strong, punctate, apical labeling (arrows) with SNA was observed within the mammary glandular epithelium (E). Scant apical labeling (arrow) was observed within the interlobular ductal epithelium with SNA (F). Scale bars indicate 200 μm. IAV-Np, influenza A virus nucleoprotein; MAL, Maackia amurensis lectin; SNA, Sambucus nigra lectin.Agent Ref. No. P14876WO00

[0017] FIG.7 shows unaffected region of the mammary gland from a US dairy cow infected with highly pathogenic avian influenza A(H5N1) virus, labeled with IAV-Np (DyLight 594), individually duplexed with MAL-I (Alexa Fluor 647), MAL-II (Alexa Fluor 647), and SNA (Alexa Fluor 647) using fluorescent staining. Representative merged images of IAV-Np / MAL-I (A, B), IAV-Np / MAL-II (C, D), and IAV-Np / SNA (E, F). IAV- Np labeling was not detected within unaffected mammary gland tissue sections. MAL-I labeling was neither observed in the epithelial cells (arrows) lining the secretory alveoli (A) nor on the epithelial cells (arrowheads) lining the interlobular ducts (B). Intense, apical, fibrillary MAL-II labeling was observed in the epithelial cells (arrows) lining the secretory alveoli of unaffected mammary glands (C). MAL-II labeling was not observed in the epithelial cells (arrowheads) lining the interlobular ducts (D). Intense, apical, granular, membranous to cytoplasmic SNA labeling in the epithelial cells lining (arrows) the secretory alveoli (E) and lining the interlobular ducts (arrowheads) (F) was observed. Scale bars indicate 50 μm. IAV-Np, influenza A virus nucleoprotein; MAL, Maackia amurensis lectin; SNA, Sambucus nigra lectin.

[0018] FIG.8 shows infected region of the mammary gland from a US dairy cow infected with highly pathogenic avian influenza A(H5N1) virus, labeled with IAV-Np (DyLight 594), individually duplexed either with epithelial marker pan-cytokeratin (Alexa Fluor 647) (B, C) or macrophage or monocytic marker ionized calcium binding adaptor molecule 1 (Iba1) (Alexa Fluor 647) (D) using fluorescent labeling. A representative image from a hematoxylin and eosin stain section highlights the cellular architecture of the affected mammary gland (A). Unaffected secretory alveoli were observed in the section (solid black outline). Secretory alveoli variably contain eosinophilic proteinaceous material and corpora amylacea. The secretory alveoli were lined by cuboidal epithelial cells (arrows) that were variably vacuolated. A few secretory alveoli were disrupted by inflammation (macrophages and neutrophils) and epithelial necrosis (dashed outline). A single interlobular duct (asterisk) lined by a bilayer of low cuboidal epithelial cells (arrows) was observed. The duct contains eosinophilic proteinaceous fluid with scattered inflammatory cells (macrophages and neutrophils) and sloughed epithelium (A). Epithelial cells lining secretory alveoli (B) and interlobular ducts (C) were labeled with pancytokeratin as expected. IAV-Np intranuclear co-labeling (arrows) in epithelial cells lining the secretory alveoli (B) and ducts (C). Intraluminal cells within secretory alveoli had intranuclear IAV-Agent Ref. No. P14876WO00 Np labeling (arrows) that variably co-labeled with pan-cytokeratin. Likewise, Iba1 labeling (arrows) was observed in the lumens of secretory alveoli or interlobular (alveolus labeling highlighted by white dotted outline) and interstitium (highlighted by the solid white outline) (D). Iba1 labeling was intense, diffuse, and cytoplasmic. IAV-Np intranuclear and intracytoplasmic labeling was less commonly co-labeled within Iba1 labeled cells (arrows) (D). IAV-Np labeling was not observed in Iba1-labeled cells in the interstitium solid white outline). Insets highlight intranuclear labeling in panels C, B, and D, and the white boxes in the corresponding images represent the origin of the inset image. Scale bars indicate 200 μm (A), 50 μm (B, C, D), and 20 μm (insets). IAV-Np, influenza A virus nucleoprotein; Iba1, ionized calcium binding adaptor molecule 1.

[0019] FIG.9 shows infected region of the mammary gland from a US dairy cow infected with highly pathogenic avian influenza A(H5N1) virus, labeled with IAV-Np (DyLight 594), individually duplexed with MAL-I (Alexa Fluor 647), MAL-II (Alexa Fluor 647), and SNA (Alexa Fluor 647) using fluorescent staining. Representative merged images of IAV-Np / MAL-I (A, B), IAV-Np / MAL-II (C, D), and IAV-Np / SNA (E, F) are shown. Moderate to intense intranuclear and intracytoplasmic IAV-Np labeling (arrows) was observed in epithelial cells lining the secretory alveolus of the mammary gland; however, no MAL-I labeling was detected (A). Rare, moderate intranuclear IAV-Np labeling (arrows), but no MAL-I labeling was observed in epithelial cells lining the interlobular duct (solid outline) (B). Intense, apical, fibrillary MAL-II labeling (arrows) was observed in the epithelial cells lining the secretory alveoli of infected mammary glands (C). IAV-Np labeling was co-labeled with MAL-II in individual epithelial cells (arrows). IAV-Np labeling (arrows) was observed in epithelial cells of interlobular ducts (solid outline) (D). MAL-II labeling was not observed in interlobular ducts but was detected in the unaffected secretory alveoli (dashed outline) (D). Intraluminal cells with cytoplasmic MAL-II labeling were observed (arrow) (D). Intense, apical, granular, membranous to cytoplasmic SNA labeling (arrows) was observed in the epithelial cells lining the secretory alveoli of infected mammary glands (E). IAV-Np intranuclear labeling was co-labeled with SNA in individual epithelial cells (arrows) in the secretory alveoli (E). SNA (arrows) and IAV-Np (arrows) were observed in epithelial cells of the interlobular ducts (dashed or solid white outline) and were co-labeled to individual ductal epithelial cells (F). Sloughed intraluminal cells had membranous SNA labeling (arrows). Adjacent secretory alveoli had prominent SNAAgent Ref. No. P14876WO00 labeling (dashed white outline). The dotted white outline highlights a severely affected secretory gland (F). Insets highlight white boxed areas in panels B, D, and F. Scale bar indicates 20 μm (A, C, E), 50 μm (B, D, F), and 20 μm (insets). IAV-Np, influenza A virus nucleoprotein; MAL, Maackia amurensis lectin; SNA, Sambucus nigra lectin.

[0020] FIG.10 shows infected region of the mammary gland from a US dairy cow infected with highly pathogenic avian influenza A(H5N1) virus, showing IAV-Np (teal chromogen), individually duplexed with MAL-I (magenta chromogen), MAL-II (magenta chromogen), and SNA (magenta chromogen) using chromogenic staining. Representative images IAV-Np / MAL-I (A, B), IAV-Np / MAL-II (C, D), and IAV-Np / SNA (E, F) in infected mammary gland and interlobular duct are shown. Strong intracytoplasmic and intranuclear immunoreactivity with IAV-Np was observed within the glandular epithelium of the mammary alveolus and interlobular ducts (arrowheads) with no substantial MAL-I labeling (A, B). Scant, multifocal, apical, punctate MAL-II labeling was observed in degenerative mammary gland epithelial cells (arrows) with strong, intranuclear, and intracytoplasmic immunoreactivity for IAV-Np (arrowheads) (C). Interlobular ductal epithelium exhibits strong, multifocal intranuclear and intracytoplasmic immunoreactivity to IAV-Np in attenuated and sloughed cells (arrowheads) with no substantial epithelial MAL-II detected (D). Strong, multifocal, punctate, apical labeling with SNA in attenuated or degenerative epithelial cells (arrows) with strong, intranuclear, and intracytoplasmic immunoreactivity for IAV-Np (arrows) was observed within mammary gland epithelium (E). Scant, multifocal, delicate, apical labeling for SNA (arrows) within ductal epithelium with strong, intranuclear, and intracytoplasmic immunoreactivity within sloughed, intraluminal epithelial cells for IAV-Np (arrowheads) was observed in an interlobular duct (F). Scale bars indicate 200 μm. IAV-Np, influenza A virus nucleoprotein; MAL, Maackia amurensis lectin; SNA, Sambucus nigra lectin.

[0021] FIG.11A-B shows evolutionary history of H5N12.3.4.4b in North America before and during the introduction and emergence in US cattle. FIG.11A shows a Bayesian time- scaled phylogeny of the HA gene between December 2021 and April 2024, demonstrating the single introduction of the virus from wild birds to dairy cattle with an estimated date of November 2023 (95% credible interval: October 2023 to January 2024). B3.13 strains inherited PB1, PA, HA, NA, MP, and NS genes from a B3.6 ancestor and acquired different PB2 and NP genes from North American LPAI viruses. The Bayesian phylogenyAgent Ref. No. P14876WO00 of the hemagglutinin (HA) gene was paraphyletically subsampled to maintain tree topology and demonstrate the 16 subsequent spillovers from cattle to domestic cats, poultry, and peridomestic animals. FIG.11B shows a simplified visualization of a hypothesis on the evolutionary history of the H5N12.3.4.4b B3.13 genotype that emerged in cattle and a range of other mammalian and avian hosts. This time-scaled phylogeny was inferred with maximum likelihood methods from concatenated genomes: Inferred dates are presented at nodes for the most recent common ancestor, and these are congruent with those estimated using Bayesian methods on the HA gene tree presented in FIG.11A. The monophyletic clade of B3.13 genotype viruses was collapsed, and the group was presented as a single triangle. BRIEF DESCRIPTION OF THE SEQUENCES

[0022] SEQ ID NO: 1 is the amino acid sequence of the ECD of HA of the highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus from cattle.

[0023] SEQ ID NO: 2 is the nucleotide sequence encoding the ECD of HA codon optimized for sf9 cells.

[0024] SEQ ID NO: 3 is the amino acid sequence of the ECD of HA and the T4 foldon trimerization domain.

[0025] SEQ ID NO: 4 is the nucleotide sequence encoding the ECD of HA and the T4 foldon trimerization domain codon optimized for sf9 cells.

[0026] SEQ ID NO: 5 is the amino acid sequence of the HA signal peptide and the ECD of HA.

[0027] SEQ ID NO: 6 is the nucleotide sequence encoding the HA signal peptide and the ECD of HA codon optimized for sf9 cells.

[0028] SEQ ID NO: 7 is the amino acid sequence of the HA signal peptide, the ECD of HA, and the T4 foldon trimerization domain.

[0029] SEQ ID NO: 8 is the nucleotide sequence encoding the HA signal peptide, the ECD of HA, and the T4 foldon trimerization domain codon optimized for sf9 cells.

[0030] SEQ ID NO: 9 is the amino acid sequence of the HA signal peptide, the ECD of HA, the T4 foldon trimerization domain, and a cleavable 7x His purification tag.Agent Ref. No. P14876WO00

[0031] SEQ ID NO: 10 is the nucleotide sequence encoding the HA signal peptide, the ECD of HA, the T4 foldon trimerization domain, and a cleavable 7x His purification tag codon optimized for sf9 cells.

[0032] SEQ ID NO: 11 is the amino acid sequence of the T4 foldon trimerization domain.

[0033] SEQ ID NO: 12 is the nucleotide sequence encoding the T4 foldon trimerization domain codon optimized for sf9 cells.

[0034] SEQ ID NO: 13 is the amino acid sequence of the HA signal peptide.

[0035] SEQ ID NO: 14 is the nucleotide sequence encoding the HA signal peptide codon optimized for sf9 cells.

[0036] SEQ ID NO: 15 is the amino acid sequence of the enzymatic cleavage site.

[0037] SEQ ID NO: 16 is the nucleotide sequence encoding enzymatic cleavage site codon optimized for sf9 cells.

[0038] SEQ ID NO: 17 is the amino acid sequence of the 7x His purification tag.

[0039] SEQ ID NO: 18 is the nucleotide sequence encoding 7x His purification tag codon optimized for sf9 cells. DETAILED DESCRIPTION

[0040] So that the present disclosure may be more readily understood, certain terms are first defined. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the disclosure pertain. Many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of the embodiments of the present disclosure without undue experimentation, the preferred materials and methods are described herein. In describing and claiming the embodiments of the present disclosure, the following terminology will be used in accordance with the definitions set out below.

[0041] It is to be understood that all terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting in any manner or scope. For example, as used in this specification and the appended claims, the singular forms “a,” “an” and “the” can include plural referents unless the content clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The word “or” means any one member of a particular list and alsoAgent Ref. No. P14876WO00 includes any combination of members of that list. Further, all units, prefixes, and symbols may be denoted in its SI accepted form.

[0042] Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range. Throughout this disclosure, various embodiments of this disclosure are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges, fractions, and individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6, and decimals and fractions, for example, 1.2, 3.8, 1½, and 4¾. This applies regardless of the breadth of the range.

[0043] The term “adjuvant” refers to a compound that enhances the effectiveness of the vaccine and may be added to the formulation that includes the immunizing agent. “Adjuvanted” refers to a composition that incorporates or is combined with an adjuvant.

[0044] An “immunogenic composition” refers to a composition of matter that comprises at least one antigen, which elicits an immunological response in the host of a cellular and / or antibody-mediated immune response to the composition or vaccine of interest. Usually, an “immunological response” includes but is not limited to one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells and / or gamma-delta T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. In certain embodiments, the host will display either a therapeutic or protective immunological response such that resistance to new infection will be enhanced and / or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction or lack of clinical signs normally displayed by an infected host, a quicker recovery time and / or a lowered duration or bacterial titer in the tissues or body fluids or excretions of the infected host compared to a healthy control. In certain embodiments, the reduction in symptoms is statistically significant when compared to a control.Agent Ref. No. P14876WO00

[0045] The term “immunogenic fragment” as used herein refers to a polypeptide or a fragment of a polypeptide, or a nucleotide sequence encoding the same which comprises an allele-specific motif, an epitope or other sequence such that the polypeptide or the fragment will bind an MHC molecule and induce a cytotoxic T lymphocyte (“CTL”) response, and / or a B cell response (for example, antibody production), and / or T-helper lymphocyte response, and / or a delayed type hypersensitivity (DTH) response against the antigen from which the immunogenic polypeptide or the immunogenic fragment is derived. A DTH response is an immune reaction in which T cell-dependent macrophage activation and inflammation cause tissue injury. A DTH reaction to the subcutaneous injection of antigen is often used as an assay for cell-mediated immunity.

[0046] The term “isolated” is used to indicate that a cell, peptide, or nucleic acid is separated from its native environment. Isolated peptides and nucleic acids may be substantially pure, i.e., essentially free of other substances with which they may be bound in nature. It is furthermore to be understood that the isolated polynucleotide molecules and the isolated RNA molecules of the present disclosure include both synthetic molecules and molecules obtained through recombinant techniques, such as by in vitro cloning and transcription.

[0047] As used herein, “a pharmaceutically acceptable carrier” or “pharmaceutical carrier” includes any and all excipients, solvents, growth media, dispersion media, coatings, stabilizing agents, diluents, preservatives, inactivating agents, antimicrobial, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. Such ingredients include those that are safe and appropriate for use in veterinary applications. Pharmaceutically acceptable carriers are typically non-toxic, inert, solid or liquid carriers.

[0048] As used herein, “subject” refers to any animal for which the administration of a composition is desired. It includes mammals and non-mammals, including primates, livestock, companion animals, laboratory test animals, captive wild animals, aves, reptiles, and fish. Thus, this term includes but is not limited to monkeys, humans, swine, bovines, cattle, sheep, goats, equines, mice, rats, guinea pigs, hamsters, rabbits, felines, canines, chickens, turkeys, ducks, other poultry, frogs, and lizards. In certain embodiments, the subject is a bovine. The term “bovine” refers to members of the biological subfamily Bovinae which includes medium- to large-sized ungulates such as domestic dairy and beef cattle, bison, African buffalo, the water buffalo, etc. In certain embodiments, the subject isAgent Ref. No. P14876WO00 a dairy cow. In certain embodiments, the subject is a calf. In certain embodiments, the subject is at risk of being infected by an influenza virus (e.g., a highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus).

[0049] The term “vaccine” refers to an antigenic preparation used to produce immunity to a disease, in order to prevent or ameliorate the effects of infection. Vaccines are typically prepared using a combination of an immunologically effective amount of an immunogen together with an adjuvant effective for enhancing the immune response of the vaccinated subject against the immunogen.

[0050] Vaccine formulations will contain a “therapeutically effective amount” of the active ingredient, that is, an amount capable of eliciting an induction of an immunoprotective response in a subject to which the composition is administered. In the treatment and prevention of influenza, for example, a “therapeutically effective amount” would be an amount that enhances resistance of the vaccinated subject to new infection and / or reduces the clinical severity of the disease. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by a subject infected with influenza, a quicker recovery time and / or a lowered count of virus particles. Vaccines can be administered prior to infection, as a preventative measure against influenza. Alternatively, vaccines can be administered after the subject already has contracted a disease. Vaccines given after exposure to influenza virus may be able to attenuate the disease, triggering a superior immune response than the natural infection itself.

[0051] The present disclosure provides for reduction of the incidence of and / or severity of clinical symptoms associated with an influenza virus (e.g., highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus) infection. In certain embodiments, the severity and / or incidence of clinical symptoms in subjects receiving the immunogenic composition of the present disclosure are reduced at least 10% in comparison to subjects not receiving such an administration when both groups (subjects receiving and subjects not receiving the composition) are challenged with or exposed to infection by an influenza virus. In certain embodiments, the incidence or severity is reduced at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100%, wherein the subjects receiving the composition of the present disclosure exhibit no clinical symptoms, or alternatively exhibit clinical symptoms of reduced severity.Agent Ref. No. P14876WO00

[0052] The terms “sequence identity” or “percent identity” are used interchangeably herein. For the purpose of this disclosure, it is defined here that in order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid for optimal alignment with a second amino or nucleic acid sequence). The amino acid or nucleotide residues at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical positions / total number of positions (i.e., overlapping positions)×100). Preferably, the two sequences are the same length. When sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” A sequence comparison may be carried out over the entire lengths of the two sequences being compared or over fragment of the two sequences. Typically, the comparison will be carried out over the full length of the two sequences being compared. However, sequence identity may be carried out over a region of, for example, twenty, fifty, one hundred or more contiguous amino acid residues. The skilled person will be aware of the fact that several different computer programs are available to determine the homology between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. Recombinant Influenza Virus Hemagglutinin Polypeptides

[0053] Provided herein is a recombinant influenza virus hemagglutinin polypeptide comprising or consisting of the extracellular domain of HA (ectodomain, ECD) or anAgent Ref. No. P14876WO00 immunogenic portion thereof, and a heterologous trimerization domain. A recombinant HA polypeptide comprises or is encoded by one or more nucleic acids that are derived from a nucleic acid which was artificially constructed. For example, the nucleic acid can comprise, or be encoded by, a cloned nucleic acid formed by joining heterologous nucleic acids.

[0054] The recombinant HA polypeptide includes hemagglutinin-derived sequences (such as the ECD) and may include other non-hemagglutinin derived sequences, for example a non-hemagglutinin derived heterologous trimerization domain. Typically, the hemagglutinin-derived sequences are in the order that they appear in naturally derived hemagglutinin and the trimerization domain occurs towards, or at the C-terminal, for example in the C-terminal half of the recombinant HA polypeptide. The recombinant HA polypeptide of the disclosure may in particular consist of or comprise the ECD of HA or an immunogenic portion thereof followed by a heterologous trimerization domain, in that order, wherein the trimerization domain is in the C-terminal half of the recombinant HA polypeptide.

[0055] The recombinant HA polypeptide of the disclosure may be fused to or contain further polypeptide other than ECD and trimerization domain. The sequence encoding the further polypeptide optionally includes additional features such as a flexible linker between the HA derived sequences and other heterologous amino acid sequences. The linkers can facilitate the independent folding of the HA domains and other heterologous sequences. The linker may be an amino acid sequence that is synthesized as part of a recombinant fusion protein. In certain embodiments, a chemical linker is used to connect synthetically or recombinantly produced subsequences. Such flexible linkers are known to those skilled in the art.

[0056] In addition to flexible linkers, or alternatively, the fusion proteins optionally include polypeptide subsequences from proteins which are unrelated to hemagglutinin, e.g., a sequence with affinity to a known antibody to facilitate affinity purification and / or detection. Such detection and purification-facilitating domains include, but are not limited to, metal chelating peptides such as polyhistidine tracts and histidine-tryptophan modules that allow purification on immobilized metals and protein A domains that allow purification on immobilized immunoglobulin. Examples include heterologous fusion sequences encoding gD tags, c-Myc epitopes, poly-histidine tags, fluorescent proteins (e.g.,Agent Ref. No. P14876WO00 GFP), beta-galactosidase protein or glutathione S transferase or any other sequence useful for detection or purification of the fusion protein expressed in or on a cell. In certain embodiments, the further polypeptide sequence is a polyhistidine tag. The inclusion of a cleavable linker sequence between the purification domain (e.g., polyhistidine tag) and rHA antigen may be useful to facilitate purification. For example an enzyme cleavage site, such as a thrombin cleavage site, may be included between the further polypeptide and the rest of the recombinant HA polypeptide sequences. Hence, the recombinant HA polypeptide of the disclosure may consist of or comprise (in order) the ECD of HA or an immunogenic portion thereof, a heterologous trimerization domain, a purification tag (e.g., polyhistidine tag) and optionally a cleavable linker sequence between the purification tag and the rest of the recombinant HA polypeptide. A cleavable linker sequence, for example an enzyme cleavage site, may alternatively or additionally be included between the trimerization domain and the rest of the recombinant HA polypeptide sequences. This can allow the trimerization domain to be removed in the final recombinant HA polypeptide.

[0057] The polynucleotide or construct encoding recombinant HA polypeptide may include a signal peptide. In certain embodiments, the natural signal peptide sequence in hemagglutinin is used. Typically, the signal peptide is appropriate for the host cell in which the recombinant HA is expressed. In certain embodiments, the natural signal peptide sequence in hemagglutinin is deleted and replaced with a baculovirus signal peptide, for example secretion signal gp67 (Whitford et al 1989, J. Virol.63, 1393-1399), for expression in insect cells. The gene containing recombinant HA polypeptide and baculovirus signal peptide can be introduced into a baculovirus expression vector so that the baculovirus promoter directs the transcription of the fusion proteins in infected insect cells. The signal peptide directs the translation of the recombinant HA polypeptide into the insect cell glycosylation pathway and is not present on the mature protein.

[0058] Accordingly, a polynucleotide encoding the recombinant HA polypeptide of the disclosure may consist of or comprise sequence encoding the ECD of HA or an immunogenic portion thereof, a heterologous trimerization domain (e.g., foldon), a purification tag (e.g., polyhistidine tag) and a signal peptide, such as in the order: a signal peptide, the ECD of HA or an immunogenic portion thereof, a heterologous trimerization domain (e.g., foldon), a purification tag (e.g., polyhistidine tag). As another example, the nucleic acid encoding the rHA antigen of the invention may consist of or compriseAgent Ref. No. P14876WO00 sequence encoding (in order): a signal peptide, the ECD of HA or an immunogenic portion thereof, a heterologous trimerization domain (e.g., foldon), a cleavable linker sequence, and a purification tag (e.g., polyhistidine tag).

[0059] For example, the recombinant HA polypeptide of the disclosure may comprise an amino acid sequence comprising the ECD of HA (e.g., SEQ ID NO: 1) or an immunogenic portion or derivative thereof, the heterologous trimerization domain (foldon) shown in SEQ ID NO: 11 or a derivative of this sequence that maintains the ability to induce the recombinant HA monomers to form trimers. For example, the recombinant HA polypeptide may comprise SEQ ID NO: 3, 5, 7, or 9, or an immunogenic portion or derivative thereof that maintains the immunogenicity of HA.

[0060] In certain embodiments, the recombinant HA polypeptide of the disclosure lacks the transmembrane domain of hemagglutinin. In certain embodiments, the recombinant HA polypeptide of the disclosure lacks the intracellular domain of hemagglutinin. Extracellular Domain (ECD) or Immunogenic Portion Thereof

[0061] The extracellular domain (or ectodomain, ECD) component of HA is present in wild-type HA protein at the cell surface. The recombinant HA polypeptide of the disclosure may comprise a full length ECD, or an immunogenic portion thereof. The ECD or immunogenic portion thereof, may in certain embodiments be a variant or derivative of wild-type HA protein (for example containing amino acid substitutions, deletions or additions).

[0062] The immunogenic portion thereof, may include one or more regions of HA for which it is desired to direct an immune response. Such regions may include known conserved and / or variable epitopes of hemagglutinin that elicit neutralizing antibodies upon vaccination. In certain embodiments, the portion thereof is capable of proper folding to interact in a hemagglutination assay.

[0063] For instance, the ECD may consist or comprise the HA1 and / or HA2 region of HA. Alternatively, the ECD may consist or comprise the head and / or stalk region of HA. An ECD may consist or comprise the HA2 subunit and a portion of the HA1 subunit, that together form the stalk region of HA. The ECD may consist of the stalk region of HA, a so-called “headless” form of HA. In particular, in certain embodiments, the HA antigen may lack the HA head, or part of the head, such as more than 25% e.g., more than 50%,Agent Ref. No. P14876WO00 such as more than 75% of the amino acid residues of the head, or lack the HA1 part of the head. Alternatively, the HA sequence may lack the HA stalk, or part of the stalk, such as more than 25%, e.g., more than 50%, such as more than 75% of the amino acid residues of the stalk, or lack the HA2 part of the stalk.

[0064] The terms “HA1” refers to the region of the HA protein including amino acid residues from approximately 1-330 of the extracellular domain of HA protein. HA1 comprises all residues that are N-terminal to the HA1 / HA2 cleavage peptide of the precursor HA0 protein, including the receptor binding domain of the HA protein.

[0065] The term “HA2” refers to the region of the HA protein including amino acid residues from approximately 331-504 of the HA0 hemagglutinin polypeptide. Of note, these residues within the HA2 chain are commonly numbered independently of those in HA1, such that HA2 residues may be numbered consecutively 1-174. The HA2 chain comprises all residues that are C-terminal to the HA1 / HA2 cleavage peptide of the precursor HA0 protein, including the hydrophobic peptide responsible for insertion within the host cell membrane during the process of membrane fusion.

[0066] The term “HA stalk” refers to the region of the HA protein including residues from approximately 1-42 and 274-330 of the HA1 chain as well as residues (1-174) of the HA2 chain. The stalk is located in the membrane-proximal region of the HA, directly beneath the vestigial esterase domain of the HA1 globular head.

[0067] The term “HA head” refers to a globular head region of the HA protein excluding the transmembrane domain and any intracellular region, which is composed of part of HA1 and that contains a sialic acid binding pocket that mediates virus attachment to the host cell. See for example Hai et al (J. Virol, 201286(10): 5774-5781). Trimerization Domain

[0068] A suitable trimerization domain is one that induces recombinant HA monomers to form trimers. In certain embodiments, the trimerization domain is or is derived from the natural trimerization domain of T4 phage fibritin “foldon”. A 27 amino acid foldon sequence may be used which forms a β-propeller structure comprising the C terminus of the fibritin domain of the T4 bacteriophage. Other suitable trimerization domains include chloramphenicol acetyl transferase (CAT) and a leucine zipper trimerization motif derived from the yeast transcription activator GCN4. In certain embodiments, the trimerizationAgent Ref. No. P14876WO00 domain, such as foldon, is placed at the C terminus of the HA polypeptide. In certain embodiments, the trimerization domain is fused via a short linker region to the HA sequence. The region between the trimerization domain and HA sequence may optionally include a cleavable linker sequence, so it is possible to isolate the HA sequence from the trimerization at later stages. Thus, the HA sequence may be linked (e.g., in order), optionally via a linker sequence, to heterologous sequence comprising a protease cleavage site, the trimerization domain and a purification tag such as histidine tag to aid in purification. Such heterologous trimerization domains may be linked to HA sequences by techniques known in the art, such as molecular cloning.

[0069] For example, the trimerization domain may comprise or consist of the amino acid sequence shown in SEQ ID NO: 11 or a derivative of this sequence that maintains the ability to induce recombinant HA monomers to form trimers. Polynucleotides encoding the trimerization domain may consist of or comprise SEQ ID NO: 12 or a derivative of this sequence that maintains the ability to induce expressed recombinant HA monomers to form trimers. Methods of Preparing Recombinant HA

[0070] Use of recombinant DNA technology to produce influenza vaccines offers several advantages. These include avoiding the steps of adaptation and passage of infectious viruses in eggs and production of more highly purified protein under safer and more stringently controlled conditions. Moreover, no virus inactivation step has to be included. Any suitable cloning and expression system may be used to recombinantly produce the recombinant HA polypeptide.

[0071] Nucleotide sequences encoding the recombinant HA polypeptide of the disclosure may be synthesized, and / or cloned and expressed according to techniques well known to those in the art. See for example, Sambrook, et al, Molecular Cloning, A Laboratory Manual, Vols.1-3, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989). In certain embodiments, the polynucleotide sequences will be codon optimized for a particular recipient host cell using standard methodologies. For example, a DNA construct encoding a hemagglutinin sequence can be codon optimized for expression in other hosts, e.g., bacteria, mammalian or insect cells. Suitable host cells may include bacterial cells such asAgent Ref. No. P14876WO00 E. coli, fungal cells such as yeast, insect cells such as Drosophila S2, Spodoptera Sf9, Sf00+ or Hi-5, and animal cells such as CHO.

[0072] Hemagglutinin sequences may be produced by standard recombinant methods known in the art, such as polymerase chain reaction (PCR) or reverse transcriptase PCR, reverse engineering or the DNA can be synthesized. For PCR, primers can be prepared using hemagglutinin nucleotide sequences that are available in publicly available databases. Polynucleotide constructs may be assembled from PCR cassettes and sequentially cloned into a vector containing a selectable marker for propagation in a host cell.

[0073] A recombinant vector can then be introduced into the host cell by injection, transfection or electroporation or other methods (for example, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation). Commercial transfection reagents such as Lipofectamine (Invitrogen) are also available.

[0074] The recombinant HA polypeptide can be recovered and purified from recombinant cell cultures by methods known in the art, including anion and / or cation exchange chromatography, affinity chromatography. Techniques such as SDS-PAGE can be used to analyze fractions of protein eluted from these separation / purification techniques. Such methods are well known to those skilled in the art and will not be presented in detail here.

[0075] Proper folding of the recombinant HA polypeptide can be determined, for example by using the red blood cell hemagglutination assay, by the ability of the protein to bind an influenza receptor, by immunogenicity testing in a host animal and / or determination of the ability of the protein to assume an appropriate quaternary structure such as rosette formation.

[0076] In certain embodiments, a baculovirus expression system is used, which is described below. Baculovirus Expression System

[0077] When using a baculovirus expression system, the recombinant HA polypeptide of the disclosure together with any heterologous sequence (for example HA sequences together with the trimerization domain) can be inserted into a baculovirus expression vector. Recombinant baculovirus that express foreign genes can be made by homologousAgent Ref. No. P14876WO00 recombination between baculovirus DNA and plasmids containing the insert, using well known techniques. The insertion may, for example be made so that the insert is under the transcription control of the polyhedron promoter, the baculovirus promoter.

[0078] Example of baculovirus expression vectors including a vector derived from the well characterized Autographa californica Nuclear Polyhedrosis Virus (AcNPV) which replicates efficiently in susceptible cultured insect cells.

[0079] Any suitable insect host cell can be used to produce the recombinant HA polypeptide, including but not limited to Sf900+, Sf9 or Hi-5 cells. In certain embodiments, the insect host cell is Sf9 or Hi-5. Cells may be propagated with culture medium and culture conditions known to be suitable for the selected host cell. Cells may be propagated for example in monolayer or in free suspension culture.

[0080] The recombinant HA polypeptide may then be isolated from the host cells using methods well known in the art. For example, the cell culture may be centrifuged, the supernatant collected and run through appropriate anionic and / or cationic exchange columns to purify the protein. Techniques such as SDS-PAGE and / or immunoblotting may be used to check the identity and integrity of proteins. A fraction of interest containing recombinant HA polypeptide may then be further purified, for example by passing through a nickel column so that rHA displaying a histidine tag binds the nickel in the column.

[0081] The extent of trimerization may be tested for example by crosslinking of HA using a suitable crosslinking agent and then use of gel migration techniques, as well known in the art. Other techniques include electron microscopy or spectroscopy-based techniques that are also well known to those skilled in the art. Vaccine and Immunogenic Compositions

[0082] The disclosure also relates to an immunogenic composition, suitable to be used as a vaccine, which comprises a recombinant HA polypeptide of the disclosure. In certain embodiments, the immunogenic compositions according to the disclosure elicit a specific humoral immune response toward the influenza virus comprising neutralizing antibodies.

[0083] In certain embodiments, the immunogenic compositions comprise an HA polypeptide of the disclosure and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be, e.g., water, a stabilizer, a preservative, culture medium, or a buffer. Immunogenic and vaccine compositions comprising the recombinantAgent Ref. No. P14876WO00 HA polypeptide of the disclosure can be prepared in the form of a suspension or in a lyophilized form or, alternatively, in a frozen form. If frozen, glycerol or other similar agents may be added to enhance stability when frozen.

[0084] The vaccine or immunogenic compositions of the present disclosure can be formulated following accepted convention to include acceptable carriers for subjects, such as standard buffers, stabilizers, diluents, preservatives, and / or solubilizers and can also be formulated to facilitate sustained release. Diluents include water, saline, dextrose, ethanol, glycerol, and the like. Additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin, among others. Other suitable vaccine vehicles and additives, including those that are particularly useful in formulating subunit vaccines, are known or will be apparent to those skilled in the art. See, e.g., Remington's Pharmaceutical Science, 18th ed., 1990, Mack Publishing, which is incorporated herein by reference.

[0085] In certain embodiments, the composition further comprises an adjuvant. Suitable adjuvants include, for example, vitamin E acetate solubilisate, aluminum hydroxide, aluminum phosphate or aluminum oxide, (mineral) oil emulsions, non-ionic detergents, squalene and saponins. Other adjuvants which may be used include an oil based adjuvants such as Freund's complete adjuvant (FCA), and Freund's incomplete adjuvant (FIA). It has been found that cross-linked olefinically unsaturated carboxylic acid polymers, such as CARBOPOL® 971 polymer, are particularly suitable adjuvants for use in the present compositions.

[0086] In certain embodiments, the adjuvant is an oil-in-water emulsion. Examples for suitable oil-in water emulsions are EMULSIGEN® based adjuvants, such as EMULSIGEN® (an oil-in-water emulsion), EMULSIGEN-D® (an oil-in-water) with dimethyldioctadecylammonium bromide (DDA)), EMULSIGEN-P® (an oil-in-water) with a proprietary immunostimulant), EMULSIGEN-75® (a double adjuvant comprised of an oil-in-water) with a cross-linked polymer), and EMULSIGEN®-BCL (an oil-in-water emulsion that is free of animal origin components). (MVP Technologies, Inc. Omaha, Nebr., USA). Pharmaceutical / vaccine compositions that comprise inactivated PEDV or recombinant PEDV proteins, have been effectively adjuvanted with oil-in water emulsions, preferably with such EMULSIGEN®-based adjuvants, more preferably with EMULSIGEN® (an oil-in-water emulsion that is free of animal origin components)Agent Ref. No. P14876WO00 and EMULSIGEN®-BCL (an oil-in-water emulsion that is free of animal origin components).

[0087] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as Mercury((o-carboxyphenyl)thio)ethyl sodium salt, octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG), TWEEN®or PLURONICS®.

[0088] Embodiments herein relating to “vaccine compositions” of the disclosure are also applicable to embodiments relating to “immunogenic compositions” of the disclosure, and vice versa. Methods of Treatment

[0089] In certain embodiments, the recombinant HA polypeptide or composition comprising the recombinant HA polypeptide is for use in medicine, such as for use in the prevention of, or vaccination against, influenza e.g., administered to a subject (e.g., a bovine) at risk for influenza infection (e.g., highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus infection).

[0090] The compositions described herein can be administered to a subject at therapeutically effective doses to prevent influenza. The dosage will depend upon the subject receiving the composition as well as factors such as the size, weight, and age of the host.

[0091] The precise amount of composition to be employed in a formulation will depend on the route of administration and the nature of the subject (e.g., age, size, stage / level ofAgent Ref. No. P14876WO00 disease), and should be decided according to the judgment of the practitioner and each subject’s circumstances according to standard clinical techniques. An effective immunizing amount is that amount sufficient to treat or prevent influenza in a subject.

[0092] Immunogenicity of a composition can be determined by monitoring the immune response of test subjects following immunization with the composition by use of any immunoassay known in the art. Generation of a humoral (antibody) response and / or cell- mediated immunity may be taken as an indication of an immune response. Test subjects may include animals such as pigs, mice, hamsters, dogs, cats, rabbits, cows, horses, sheep, poultry (e.g., chickens, ducks, geese, and turkeys), and humans.

[0093] The immune response of the test subjects can be analyzed by various approaches such as: the reactivity of the resultant immune serum to the immunogenic conjugate, as assayed by known techniques, e.g., enzyme linked immunosorbent assay (ELISA), immunoblots, immunoprecipitations, virus neutralization, etc.; or, by protection of immunized hosts from infection by the pathogen and / or attenuation of symptoms due to infection by the pathogen in immunized hosts as determined by any method known in the art, for assaying the levels of an infectious disease agent, e.g., the viral levels (for example, by culturing of a sample from the subject), or other technique known in the art. The levels of the infectious disease agent may also be determined by measuring the levels of the antigen against which the immunoglobulin was directed. A decrease in the levels of the infectious disease agent or an amelioration of the symptoms of the infectious disease indicates that the composition is effective.

[0094] The composition can be tested in vitro for the desired therapeutic or prophylactic activity, prior to in vivo use in animals. For example, in vitro assays that can be used to determine whether administration of a specific composition is indicated include in vitro cell culture assays in which appropriate cells from a cell line or cells cultured from a subject having a particular disease or disorder are exposed to or otherwise administered a composition, and the effect of the composition on the cells is observed.

[0095] Alternatively, the composition may be assayed by contacting the composition to cells (either cultured from a subject or from a cultured cell line) that are susceptible to infection by the infectious disease agent but that are not infected with the infectious disease agent, exposing the cells to the infectious disease agent, and then determining whether the infection rate of cells contacted with the composition was lower than the infection rate ofAgent Ref. No. P14876WO00 cells not contacted with the composition. Infection of cells with an infectious disease agent may be assayed by any method known in the art.

[0096] In addition, the composition can be assessed by measuring the level of the molecule against which the antibody is directed in the animal model or subject at suitable time intervals before, during, or after therapy. Any change or absence of change in the amount of the molecule can be identified and correlated with the effect of the treatment on the subject. The level of the molecule can be determined by any method known in the art.

[0097] After vaccination of an animal using the methods and compositions of the present disclosure, any binding assay known in the art can be used to assess the binding between the resulting antibody and the particular molecule. These assays may also be performed to select antibodies that exhibit a higher affinity or specificity for the particular antigen.

[0098] Suitable routes of administration include but are not limited to intranasal, oral, intradermal, intramuscular, and subcutaneous. The skilled artisan will recognize that compositions of the disclosure may also be administered in one, two or more doses, as well as, by other routes of administration. For example, such other routes include intracutaneously, intravenously, intravascularly, intraarterially, intraperitoneally, intrathecally, intratracheally, intracutaneously, intracardially, intralobally, intramedullarly, intrapulmonarily, and intravaginally. Depending on the desired duration and effectiveness of the treatment, the compositions of the disclosure may be administered once or several times, also intermittently, for instance on a daily basis for several days, weeks or months and in different dosages.

[0099] Any suitable device may be used to administer the composition, including syringes, droppers, needleless injection devices, patches, and the like. The route and device selected for use will depend on the composition of the adjuvant, the antigen, and the subject, and such are well known to the skilled artisan. Oral administration may be direct, via water, or via feed (solid or liquid feed). When provided in liquid form, the vaccine may be lyophilized with reconstitution, or provided as a paste, for direct addition to feed (mix in or top dress) or otherwise added to water or liquid feed. In certain embodiments, the compositions are administered to greater than one subject at a time through means known in the art, for example, through mass intranasal administration of a group of animals.

[0100] In certain embodiments, the administration route is the subcutaneous route. Any suitable device may be used for subcutaneous delivery, for example classical needle. InAgent Ref. No. P14876WO00 certain embodiments, a needle-free jet injector service is used. Such devices are well known in the art. In certain embodiments, the device is pre-filled with a liquid vaccine formulation. Embodiments

[0101] The following numbered embodiments also form part of the present disclosure:

[0102] 1. A recombinant influenza hemagglutinin (HA) polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO: 1.

[0103] 2. The recombinant HA polypeptide of embodiment 1, wherein the recombinant HA polypeptide lacks a transmembrane domain.

[0104] 3. The recombinant HA polypeptide of embodiment 1 or embodiment 2, further comprising a trimerization domain.

[0105] 4. The recombinant HA polypeptide of embodiment 3, wherein the trimerization domain has the amino acid sequence set forth in SEQ ID NO: 11.

[0106] 5. The recombinant HA polypeptide of any one of embodiments 1-4, further comprising a purification tag.

[0107] 6. The recombinant HA polypeptide of any one of embodiments 1-5, wherein the recombinant HA polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO: 3, 5, 7, or 9.

[0108] 7. The recombinant HA polypeptide of any one of embodiments 1-6, wherein the recombinant HA polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7, or 9.

[0109] 8. A multimeric polypeptide comprising at least two recombinant HA polypeptides of any one of embodiments 1-7.

[0110] 9. The multimeric polypeptide of embodiment 8, wherein the multimeric polypeptide is trimeric and comprises three recombinant HA polypeptides.

[0111] 10. An immunogenic composition comprising the recombinant HA polypeptide of any one of embodiments 1-7 or the multimeric polypeptide of embodiment 8 or embodiment 9; and a pharmaceutically acceptable carrier.

[0112] 11. A method for inducing an immune response against an influenza virus in a subject, the method comprising: administering to the subject the immunogenic compositionAgent Ref. No. P14876WO00 of embodiment 10, optionally wherein the influenza virus is a highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus.

[0113] 12. The method of embodiment 11, wherein the subject is a bovine.

[0114] 13. The method of embodiment 12, wherein the bovine is a cow, bull, or calf.

[0115] 14. The method of any one of embodiments 11-13, wherein the immunogenic composition is administered intranasally, parenterally, subcutaneously, intramuscularly, or intradermally.

[0116] 15. A vaccine composition comprising the recombinant HA polypeptide of any one of embodiments 1-7 or the multimeric polypeptide of embodiment 8 or embodiment 9; and a pharmaceutically acceptable carrier.

[0117] 16. The vaccine composition of embodiment 15, wherein the composition further comprises an adjuvant.

[0118] 17. The vaccine composition of embodiment 16, wherein the adjuvant is an oil-in- water emulsion adjuvant.

[0119] 18. A method of treating or preventing disease caused by an influenza virus comprising: administering to the subject the vaccine composition of any one of embodiments 15-17, optionally wherein the influenza virus is a highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus.

[0120] 19. The method of embodiment 18, wherein the subject is a bovine.

[0121] 20. The method of embodiment 19, wherein the bovine is a cow, bull, or calf.

[0122] 21. The method of any one of embodiments 18-20, wherein the vaccine composition is administered intranasally, parenterally, subcutaneously, intramuscularly, or intradermally.

[0123] 22. The method of any one of embodiments 18-21, wherein the composition is administered to the subject twice.

[0124] 23. An immunogenic fragment of the recombinant HA polypeptide of any one of embodiment 1-7.

[0125] 24. A polynucleotide encoding the recombinant HA polypeptide of any one of embodiment 1-7.

[0126] 25. The polynucleotide of embodiment 24, wherein the polynucleotide comprises a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO: 2, 4, 6, 8, or 10.Agent Ref. No. P14876WO00

[0127] 26. The polynucleotide of embodiment 24 or embodiment 25, wherein the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10.

[0128] 27. A vector comprising the polynucleotide of any one of embodiments 24-26.

[0129] 28. A cell comprising the polynucleotide of any one of embodiments 24-26 or the vector of embodiment 27.

[0130] 29. The cell of embodiment 28, wherein the cell is an insect cell.

[0131] 30. A method for producing a recombinant HA polypeptide, the method comprising: expressing the polynucleotide of any one of embodiments 24-26 or the vector of embodiment 27 in a cell.

[0132] 31. The method of embodiment 30, further comprising isolating the recombinant HA polypeptide from the cell.

[0133] 32. The method of embodiment 30 or embodiment 31, wherein the cell is an insect cell.

[0134] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0135] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended embodiments.

[0136] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES Example 1: Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus Infection in Domestic Dairy Cattle and Cats, United States, 2024

[0137] This example reports highly pathogenic avian influenza A(H5N1) virus in dairy cattle and cats in Kansas and Texas, United States, which reflects the continued spread of clade 2.3.4.4b viruses that entered the country in late 2021. Infected cattle experienced nonspecific illness, reduced feed intake and rumination, and an abrupt drop in milkAgent Ref. No. P14876WO00 production, but fatal systemic influenza infection developed in domestic cats fed raw (unpasteurized) colostrum and milk from affected cows. Cow-to-cow transmission appears to have occurred because infections were observed in cattle on Michigan, Idaho, and Ohio farms where avian influenza virus–infected cows were transported. Although the US Food and Drug Administration has indicated the commercial milk supply remains safe, the detection of influenza virus in unpasteurized bovine milk is a concern because of potential cross-species transmission. Continued surveillance of highly pathogenic avian influenza viruses in domestic production animals is needed to prevent cross-species and mammal-to- mammal transmission. Materials and Methods

[0138] Milk samples (cases 2–5) and fresh and formalin-fixed tissues (cases 1, 3–5) from dairy cattle were received at the ISUVDL from Texas on March 21 and from Kansas on March 22, 2024. The cattle exhibited nonspecific illness and reduced lactation, as described previously. The tissue samples for diagnostic testing came from 3 cows that were euthanized and 3 that died naturally; all postmortem examinations were performed on the premises of affected farms.

[0139] The bodies of two adult domestic shorthaired cats from a north Texas dairy farm were received at the ISUVDL for a complete postmortem examination on March 21, 2024. The cats were found dead with no apparent signs of injury and were from a resident population of ≈24 domestic cats that had been fed milk from sick cows. Clinical disease in cows on that farm was first noted on March 16; the cats became sick on March 17, and several cats died in a cluster during March 19–20. In total, >50% of the cats at that dairy became ill and died. Cerebrum, cerebellum, eye, lung, heart, spleen, liver, lymph node, and kidney tissue samples were collected from the cats and placed in 10% neutral-buffered formalin for histopathology.

[0140] At ISUVDL, formalin-fixed tissues from affected cattle and cats were trimmed, embedded in paraffin, and processed for hematoxylin / eosin staining and histologic evaluation. For immunohistochemistry (IHC), 4-µm–thick sections were prepared from paraffin-embedded tissues, placed on Superfrost Plus slides (VWR), and dried for 20 minutes at 60°C. A Ventana Discovery Ultra IHC / ISH research platform (Roche) was used for deparaffinization until and including counterstaining. All products except the primary antibody were obtained from Roche. Automated deparaffination was followed byAgent Ref. No. P14876WO00 enzymatic digestion with protease 1 for 8 minutes at 37°C and endogenous peroxidase blocking. The primary influenza A virus antibody was obtained from the hybridoma cell line H16-L10–4R5 (ATCC) and diluted at 1:100 in Discovery PSS diluent; sections were incubated with antibody for 32 minutes at room temperature. Next, the sections were incubated with a hapten-labeled conjugate, Discovery anti-mouse HQ, for 16 minutes at 37°C, followed by a 16-minute incubation with the horseradish peroxidase conjugate, Discovery anti-HQ HRP. A ChromoMap DAB kit was used for antigen visualization, followed by counterstaining with hematoxylin and then bluing. Positive controls were sections of IAV-positive swine lung. Negative controls were sections of brain, lung, and eyes from cats not infected with IAV.

[0141] Milk samples were diluted 1:3 vol / vol in phosphate buffered saline, pH 7.4 (Gibco / Thermo Fisher Scientific), by mixing 1 unit volume of milk with 3 unit volumes of phosphate buffered saline. Ten percent homogenates of mammary glands, brains, lungs, spleens, and lymph nodes were prepared in Earle’s balanced salt solution (Sigma-Aldrich). Processing was not necessary for ocular fluid, rumen content, or serum samples. After processing, samples were extracted according to a National Animal Health Laboratory Network (NAHLN) protocol that had 2 NAHLN-approved deviations for ISUVDL, consisting of the MagMax Viral RNA Isolation Kit for 100 µL sample volumes and a Kingfisher Flex instrument (both Thermo Fisher Scientific).

[0142] Real-time reverse transcription PCR (rRT-PCR) was performed using an NAHLN- approved assay with 1 deviation, which was the VetMAX-Gold SIV Detection kit (Thermo Fisher Scientific), to screen for the presence of IAV RNA. Samples were tested along with the VetMAX XENO Internal Positive Control to monitor the possible presence of PCR inhibitors. Each rRT-PCR 96-well plate included 2 positive amplification controls, 2 negative amplification controls, 1 positive extraction control, and 1 negative extraction control. The rRT-PCR was run on an ABI 7500 Fast thermocycler and data were analyzed with Design and Analysis Software 2.7.0 (both Thermo Fisher Scientific). Samples with cycle threshold (Ct) values <40.0 were considered positive for virus.

[0143] After the screening rRT-PCR, IAV RNA–positive samples were analyzed for the H5 subtype and H5 clade 2.3.4.4b by using the same RNA extraction and NAHLN- approved rRT-PCR protocols as described previously, according to standard operating procedures. PCR was performed on the ABI 7500 Fast thermocycler using appropriateAgent Ref. No. P14876WO00 controls to detect H5-specific IAV. Samples with Ct values <40.0 were considered positive for the IAV H5 subtype.

[0144] Genomic sequencing was conducted on 2 milk samples from infected dairy cattle in Texas and 2 tissue samples (lung and brain) from cats that died at a different Texas dairy. The whole-genome sequencing data were subjected to bioinformatics analysis to assemble the 8 different IAV segment sequences according to previously described methods. The hemagglutinin (HA) and neuraminidase (NA) sequences were used for phylogenetic analysis. Reference sequences for the HA and NA segments of IAV H5 clade 2.3.4.4 were obtained from publicly available databases, including GISAID (gisaid.org) and GenBank. The sequences were aligned using MAFFT version 7.520 software (mafft.cbrc.jp / alignment / server / ) to create multiple sequence alignments for subsequent phylogenetic analysis. IQTree2 (github.com / iqtree / iqtree2) was used to construct the phylogenetic tree from the aligned sequences. The software was configured to automatically identify the optimal substitution model by using the ModelFinder Plus option, ensuring the selection of the most suitable model for the dataset and, thereby, improving the accuracy of the reconstructed tree. The resulting phylogenetic tree was visualized by using iTOL (itol.embl.de), a web-based platform for interactive tree exploration and annotation. Results Gross Lesions in Cows and Cats

[0145] All cows were in good body condition with adequate rumen fill and no external indications of disease. Postmortem examinations of the affected dairy cows revealed firm mammary glands typical of mastitis; however, mammary gland lesions were not consistent. Two cows that were acutely ill before postmortem examination had grossly normal milk and no abnormal mammary gland lesions. The gastrointestinal tract of some cows had small abomasal ulcers and shallow linear erosions of the intestines, but those observations were also not consistent in all animals. The colon contents were brown and sticky, suggesting moderate dehydration. The feces contained feed particles that appeared to have undergone minimal ruminal fermentation. The rumen contents had normal color and appearance but appeared to have undergone minimal fermentation.

[0146] The 2 adult cats (1 intact male, 1 intact female) received at the ISUVDL were in adequate body and postmortem condition. External examination was unremarkable. MildAgent Ref. No. P14876WO00 hemorrhages were observed in the subcutaneous tissues over the dorsal skull, and multifocal meningeal hemorrhages were observed in the cerebrums of both cats. The gastrointestinal tracts were empty, and no other gross lesions were observed. Microscopic Lesions in Cows and Cats

[0147] The chief microscopic lesion observed in affected cows was moderate acute multifocal neutrophilic mastitis (FIG.1); however, mammary glands were not received from every cow. Three cows had mild neutrophilic or lymphocytic hepatitis. Because they were adult cattle, other observed microscopic lesions (e.g., mild lymphoplasmacytic interstitial nephritis and mild to moderate lymphocytic abomasitis) were presumed to be nonspecific, age-related changes. No major lesions were observed in the other evaluated tissues. IHC for IAV antigen was performed on all evaluated tissues; the only tissues with positive immunoreactivity were mastitic mammary glands from 2 cows, which showed nuclear and cytoplasmic labeling of alveolar epithelial cells and cells within lumina (FIG. 1), and multifocal germinal centers within a lymph node from 1 cow (TABLE 1).

[0148] Both cats had microscopic lesions consistent with severe systemic virus infection, including severe subacute multifocal necrotizing and lymphocytic meningoencephalitis with vasculitis and neuronal necrosis, moderate subacute multifocal necrotizing and lymphocytic interstitial pneumonia, moderate to severe subacute multifocal necrotizing and lymphohistiocytic myocarditis, and moderate subacute multifocal lymphoplasmacytic chorioretinitis with ganglion cell necrosis and attenuation of the internal plexiform and nuclear layers (TABLE 2; FIG.2). IHC for IAV antigen was performed on multiple tissues (brain, eye, lung, heart, spleen, liver, and kidney). Positive IAV immunoreactivity was detected in brain (intracytoplasmic, intranuclear, and axonal immunolabeling of neurons), lung, and heart, as well as multifocal and segmental immunoreactivity within all layers of the retina (FIG.2).

[0149] When available, tissue homogenates (e.g., lung, spleen, and lymph nodes), ocular fluid, and rumen contents from 6 cows were also tested by IAV and H5 subtype-specific PCR (TABLE 3). However, the PCR findings were not consistent. For example, the tissue homogenates and ocular fluid tested positive in some but not all cows. In case 5, cow 1, the milk sample tested negative by IAV screening PCR, but the spleen homogenate tested positive by IAV screening, H5 subtype, and H52.3.4.4 PCR. For 2 cows (case 3, cow 1; and case 4, cow 1) that had both milk and rumen contents available, both samples testedAgent Ref. No. P14876WO00 positive for IAV. Nevertheless, all IAV-positive nonmammary gland tissue homogenates, ocular fluid, and rumen contents had markedly elevated Ct values in contrast to the low Ct values for milk and mammary gland homogenate samples.

[0150] Brain and lung samples from the 2 cats (case 6, cats 1 and 2) were tested by IAV screening and H5 subtype-specific PCR (TABLE 3). Both sample types were positive by IAV screening PCR; Ct values were 9.9–13.5 for brain and 17.4–24.4 for lung samples, indicating high amounts of virus nucleic acid in those samples. The H5 subtype and H5 2.3.4.4 PCR results were also positive for the brain and lung samples; Ct values were consistent with the IAV screening PCR (TABLE 3).

[0151] TABLE 1. Microscopic lesions observed in cattle in study of highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus infection in domestic dairy cattle and cats, United States, 2024.Agent Ref. No. P14876WO00

[0152] TABLE 2. Microscopic lesions observed in cats in study of highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus infection in domestic dairy cattle and cats, United States, 2024.

[0153] TABLE 3. PCR results from various specimens in study of highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus infection in domestic dairy cattle and cats, United States, 2024.Agent Ref. No. P14876WO00Agent Ref. No. P14876WO00 Phylogenetic Analyses

[0154] The sequences of all 8 segments of the HPAI viruses from both cow milk and cat tissue samples were assembled. The hemagglutinin (HA) and neuraminidase (NA) sequences were used specifically for phylogenetic analysis to delineate the clade of the HA gene and subtype of the NA gene.

[0155] For HA gene analysis, both HA sequences derived from cow milk samples exhibited a high degree of similarity, sharing 99.88% nucleotide identity, whereas the 2 HA sequences from cat tissue samples showed complete identity at 100%. The HA sequences from the milk samples had 99.94% nucleotide identities with HA sequences from the cat tissues, resulting in a distinct subcluster comprising all 4 HA sequences, which clustered together with other H5N1 viruses belonging to clade 2.3.4.4b. The HA sequences were deposited in GenBank (accession nos. PP599465 [case 2, cow 1], PP599473 [case 2, cow 2], PP692142 [case 6, cat 1], and PP692195 [case 6, cat 2]).

[0156] For NA gene analysis, the 2 NA sequences obtained from cow milk samples showed 99.93% nucleotide identity. Moreover, the NA sequences derived from the milk samples exhibited complete nucleotide identities (100%) with those from the cat tissues. The 4 NA sequences were grouped within the N1 subtype of HPAI viruses. The NA sequences were deposited in GenBank (accession nos. PP599467 [case 2, cow 1], PP599475 [case 2, cow 2], PP692144 [case 6, cat 1], and PP692197 [case 6, cat 2]). Discussion

[0157] This case series differs from most previous reports of IAV infection in bovids, which indicated cattle were inapparently infected or resistant to infection. An H5N1 strain of IAV in dairy cattle was described that resulted in apparent systemic illness, reduced milk production, and abundant virus shedding in milk. The magnitude of this finding is further emphasized by the high death rate (≈50%) of cats on farm premises that were fed raw colostrum and milk from affected cows; clinical disease and lesions developed that were consistent with previous reports of H5N1 infection in cats presumably derived from consuming infected wild birds. Although exposure to and consumption of dead wild birds cannot be completely ruled out for the cats described in this report, the known consumption of unpasteurized milk and colostrum from infected cows and the high amount of virus nucleic acid within the milk make milk and colostrum consumption a likely route of exposure. Therefore, these findings suggest cross-species mammal-to-mammalAgent Ref. No. P14876WO00 transmission of HPAI H5N1 virus and raise new concerns regarding the potential for virus spread within mammal populations. Horizontal transmission of HPAI H5N1 virus has been previously demonstrated in experimentally infected cats and ferrets and is suspected to account for large dieoffs observed during natural outbreaks in mink and sea lions. Future experimental studies of HPAI H5N1 virus in dairy cattle should seek to confirm cross- species transmission to cats and potentially other mammals.

[0158] Clinical IAV infection in cattle has been infrequently reported in the published literature. The first report occurred in Japan in 1949, where a short course of disease with pyrexia, anorexia, nasal discharge, pneumonia, and decreased lactation developed in cattle. In 1997, a similar condition occurred in dairy cows in southwest England leading to a sporadic drop in milk production, and IAV seroconversion was later associated with reduced milk yield and respiratory disease. Rising antibody titers against human-origin influenza A viruses (H1N1 and H3N2) were later again reported in dairy cattle in England, which led to an acute fall in milk production during October 2005–March 2006. Limited reports of IAV isolation from cattle exist; most reports occurred during the 1960s and 1970s in Hungary and in the former Soviet Union, where H3N2 was recovered from cattle experiencing respiratory disease. Direct detection of IAV in milk and the potential transmission from cattle to cats through feeding of unpasteurized milk has not been previously reported.

[0159] An IAV-associated drop in milk production in dairy cattle appears to have occurred during >4 distinct periods and within 3 widely separated geographic areas: 1949 in Japan, 1997–1998 and 2005–2006 in Europe, and 2024 in the United States (this report). The sporadic occurrence of clinical disease in dairy cattle worldwide might be the result of changes in subclinical infection rates and the presence or absence of sufficient baseline IAV antibodies in cattle to prevent infection. Milk IgG, lactoferrin, and conglutinin have also been suggested as host factors that might reduce susceptibility of bovids to IAV infection. Contemporary estimates of the seroprevalence of IAV antibodies in US cattle are not well described in the published literature. One retrospective serologic survey in the United States in the late 1990s showed 27% of serum samples had positive antibody titers and 31% had low-positive titers for IAV H1 subtype-specific antigen in cattle with no evidence of clinical infections. Antibody titers for H5 subtype-specific antigen have not been reported in US cattle.Agent Ref. No. P14876WO00

[0160] The susceptibility of domestic cats to HPAI H5N1 is well-documented globally, and infection often results in neurologic signs in affected felids and other terrestrial mammals. Most cases in cats result from consuming infected wild birds or contaminated poultry products. The incubation period in cats is short; clinical disease is often observed 2–3 days after infection. Brain tissue has been suggested as the best diagnostic sample to confirm HPAI virus infection in cats, and these results support that finding. One unique finding in the cats from this report is the presence of blindness and microscopic lesions of chorioretinitis. Those results suggest that further investigation into potential ocular manifestations of HPAI H5N1 virus infection in cats might be warranted.

[0161] The genomic sequencing and subsequent analysis of clinical samples from both bovine and feline sources provided considerable insights. The HA and NA sequences derived from both bovine milk and cat tissue samples from different Texas farms had a notable degree of similarity. Those findings strongly suggest a shared origin for the viruses detected in the dairy cattle and cat tissues. Further research, case series investigations, and surveillance data are needed to better understand and inform measures to curtail the clinical effects, shedding, and spread of HPAI viruses among mammals. Although pasteurization of commercial milk mitigates risks for transmission to humans, a 2019 US consumer study showed that 4.4% of adults consumed raw milk >1 time during the previous year, indicating a need for public awareness of the potential presence of HPAI H5N1 viruses in raw milk.

[0162] Ingestion of feed contaminated with feces from wild birds infected with HPAI virus is presumed to be the most likely initial source of infection in the dairy farms. Although the exact source of the virus is unknown, migratory birds (Anseriformes and Charadriiformes) are likely sources because the Texas panhandle region lies in the Central Flyway, and those birds are the main natural reservoir for avian influenza viruses. HPAI H5N1 viruses are well adapted to domestic ducks and geese, and ducks appear to be a major reservoir; however, terns have also emerged as an important source of virus spread. The mode of transmission among infected cattle is also unknown; however, horizontal transmission has been suggested because disease developed in resident cattle herds in Michigan, Idaho, and Ohio farms that received infected cattle from the affected regions, and those cattle tested positive for HPAI H5N1. Experimental studies are needed to decipher the transmissionAgent Ref. No. P14876WO00 routes and pathogenesis (e.g., replication sites and movement) of the virus within infected cattle.

[0163] In conclusion, it was shown that dairy cattle are susceptible to infection with HPAI H5N1 virus and can shed virus in milk and, therefore, might potentially transmit infection to other mammals via unpasteurized milk. A reduction in milk production and vague systemic illness were the most commonly reported clinical signs in affected cows, but neurologic signs and death rapidly developed in affected domestic cats. HPAI virus infection should be considered in dairy cattle when an unexpected and unexplained abrupt drop in feed intake and milk production occurs and for cats when rapid onset of neurologic signs and blindness develop. The recurring nature of global HPAI H5N1 virus outbreaks and detection of spillover events in a broad host range is concerning and suggests increasing virus adaptation in mammals. Surveillance of HPAI viruses in domestic production animals, including cattle, is needed to elucidate influenza virus evolution and ecology and prevent cross-species transmission. Example 2: Sialic Acid Receptor Specificity in Mammary Gland of Dairy Cattle Infected with Highly Pathogenic Avian Influenza A(H5N1) Virus

[0164] In March 2024, the US Department of Agriculture's Animal and Plant Health Inspection Service reported detection of highly pathogenic avian influenza (HPAI) A(H5N1) virus in dairy cattle in the United States for the first time. One factor that determines susceptibility to HPAI H5N1 infection is the presence of specific virus receptors on host cells; however, little is known about the distribution of the sialic acid (SA) receptors in dairy cattle, particularly in mammary glands. The distribution of SA receptors in the respiratory tract and mammary gland of dairy cattle naturally infected with HPAI H5N1 was compared. The respiratory and mammary glands of HPAI H5N1-infected dairy cattle are rich in SA, particularly avian influenza virus-specific SA α2,3-gal. Mammary gland tissues co-stained with sialic acids and influenza A virus nucleoprotein showed predominant co-localization with the virus and SA α2,3-gal. HPAI H5N1 exhibited epitheliotropism within the mammary gland, and rare immunolabeling was observed within macrophages.Agent Ref. No. P14876WO00 Materials and Methods Sample Collection

[0165] Formalin-fixed and paraffin-embedded sections of trachea, lung, and mammary gland tissues, as well as milk in EDTA tubes, from 2 adult Holstein dairy cows in Texas, USA, diagnosed with HPAI H5N1 virus infection at the Iowa State University Veterinary Diagnostic Laboratory (Ames, IA, USA) in March 2024, were used. The cows reportedly exhibited a nonspecific illness, including reduced lactation and thickened, yellow milk with a similar appearance to colostrum. The diagnosis was based on detection of HPAI H5N1 IAV by real-time reverse transcription PCR (rRT-PCR) in the mammary gland and lung, as well as detection of IAV nucleoprotein by immunohistochemistry in the mammary glands. In addition, affected tissue sections were confirmed to be positive for IAV matrix gene nucleic acid by RNAscope in situ hybridization assay. Testing of macroscopic and microscopic lesions, including IAV chromogenic immunohistochemistry and rRT-PCR, was previously described. The mammary glands from the cows had a multifocal lesion pattern, which enabled the dissection of affected and unaffected regions of the mammary gland. Unaffected regions were used as control tissue. To be classified as unaffected, the sections could not have any inflammation or epithelial changes and were required to have a negative IAV immunohistochemistry result. Cytologic Evaluation of Milk Sample

[0166] Milk from EDTA tubes was used to make direct smears onto cytocentrifuged slides. Cytocentrifugation of milk was performed using a Shandon Cytospin3 (ThermoFisher Scientific) at 72 g for 10 minutes (low acceleration). Air-dried slides were prepared and stained with modified Wright stain on an automated stainer (Siemens Healthineers). Differential cell counts were determined under ×1,000 original magnification by counting 100 nucleated cells. Fluorescently Labeled Dual Lectin and Immunochemistry Staining

[0167] Mammary tissues were characterized for sialic acids by using an automated adaptation of a lectin histochemistry assay previously described combined with influenza A nucleoprotein (IAV-Np) staining. Formalin-fixed, paraffin-embedded tissue sections of the bovine mammary gland were sectioned at 4 μm with placement on Superfrost Plus slides (VWR International). Slides were dried at 60°C for 20 minutes before deparaffinization and staining on the Ventana Discovery Ultra research platform (RocheAgent Ref. No. P14876WO00 Diagnostics). Heat retrieval was accomplished by using cell conditioning solution at 100°C for 24 minutes (Roche Diagnostics). Slides were blocked with 1X Carbo-Free blocking solution (Vector Laboratories) for 32 minutes, then treated with a Streptavidin / Biotin Blocking Kit (Vector Laboratories) with a separate application each of 12 minutes. Sections were incubated for 4 hours at room temperature with 1 of the 3 lectins (SNA, MAL-I, MAL-II) from Vector Laboratories at the specified concentrations, where SNA is specific for α2,6-gal / GalNAc, MAL-I is specific for N-linked or O-linked glycans with SAα2,3-gal-β (1–4) GlcNAc, and MAL-II is specific for O-linked glycans with SA α2,3- gal-β (1–3) GalNAc. After lectin incubation, streptavidin conjugated with Alexa Fluor 647 (ThermoFisher Scientific) was applied and sections were incubated for 2 hours at room temperature. Immunostaining with IAV-Np was performed by incubating sections with rabbit recombinant monoclonal anti-nucleoprotein for IAV labeled with DyLight 594 (Novus Biologicals) for 4 hours at room temperature. Counterstaining and mounting were manually performed with Prolong Gold antifade mountant with DAPI (ThermoFisher Scientific). Negative assay controls consisted of primary lectin or antibody omission. Nonspecific lectin labeling was evaluated by using sialidase-A treated sections. Positive lectin assay controls were performed on porcine tissues because lectin labeling has been previously established. Positive IAV-Np controls were also performed on porcine lung tissue with known IAV infection status (data not shown). Multicolor fluorescent staining by using lectin and IAV-Np assay was performed to aid co-labeling.

[0168] Multicolor immunofluorescent staining was conducted by using mouse monoclonal anti-human cytokeratin, Clone AE1 / AE3 (Agilent Technologies), and rabbit monoclonal anti-ionized calcium binding adaptor molecule 1 (Iba1) (Abcam), along with the primary IAV-Np antibody previously described. Modifications to this lectin-antibody procedure include heat retrieval with cell conditioning solution for 48 minutes at 100°C, replacing the lectin with an antibody incubation at 37°C (Iba1 at 60 minutes and cytokeratin at 32 minutes), as well as incubation of a Biotinylated Link from a Universal LSAB2 Kit (Agilent Technologies) for 1 hour before application of the streptavidin conjugate. Slides were then examined and imaged by using a BX-53 Olympus trinocular microscope equipped with an Olympus DP23 camera, Excelitas X-Cite mini+ compact illumination system, and CellSyns Dimension software. ResultsAgent Ref. No. P14876WO00

[0169] Retrospectively collected respiratory tract and mammary gland samples from 2 naturally infected lactating Holstein dairy cows with HPAI H5N1 virus infections were selected for evaluation. The clinicopathologic manifestations, detection methods, and sequencing data pertaining to the dairy cows in this study have been previously reported.

[0170] In brief, the HPAI H5N1 virus–infected mammary gland had acute multifocal moderate mastitis with epithelial attenuation lining the secretory alveoli and interlobular ducts, as well as intraluminal neutrophilic inflammation (FIG.3). Immunohistochemistry for IAV-Np on the affected mammary gland showed intranuclear and intracytoplasmic immunoreactivity in alveolar and interlobular ductal epithelial cells. The milk from the affected gland was examined cytologically, which had not previously been reported. Routine cytologic assessment identified moderate to marked neutrophilic and mild macrophagic inflammation consistent with mastitis.

[0171] The evaluation of bovine tissue distribution for SA α2–3 and SA α2–6 receptors was considered to be warranted, given the detection of HPAI H5N1 virus and lesion development in the dairy cows. The distribution of SA α2–3 and SA α2–6 receptors in the respiratory and mammary tissues from the cows was evaluated through fluorescent and chromogenic-based lectin histochemistry.

[0172] Both fluorescent and chromogenic lectin histochemistry methodologies concur with the distribution of lectins (TABLE 4; FIG.4, FIG.5). SNA labeling was observed only in goblet cells, submucosal glands, and intraepithelial and lamina proprial immune cells (suspected to be lymphocytes) of the trachea and bronchi. MAL-I and MAL-II labeling was multifocal, weak to moderate, apical, and membranous in the respiratory epithelium of the trachea. MAL-I and MAL-II labeling in the tracheal and bronchial goblet cells and submucosal glandular epithelial cells was intense, granular, and cytoplasmic. However, MAL-I and MAL-II labeling differed in the bronchial respiratory epithelium, where MAL- I labeling was more diffuse, whereas MAL-II labeling was multifocal. MAL-I and MAL-II labeling of the respiratory epithelium was similar in the bronchioles and alveoli, where labeling was diffuse.

[0173] In the mammary gland, MAL-II and SNA labeling were expressed in unaffected secretory alveoli (FIG.6, FIG.7). Unaffected interlobular ducts were the only structures observed to have SNA labeling, and no detectable MAL-I or IAV-Np labeling was observed in unaffected mammary gland sections. SNA labeling was moderate to intense,Agent Ref. No. P14876WO00 granular to fibrillary, membranous to cytoplasmic, and predominately apically located within the alveolar lining epithelium, and the expression was abundant in the epithelial cells lining the interlobular ducts. MAL-II labeling was intense, fibrillary, membranous, and exclusively apical, no MAL-II labeling was observed in interlobular duct epithelium.

[0174] Overall, lectin histochemistry results within unaffected (i.e., respiratory tract and mammary gland) and affected (i.e., mammary gland) tissues closely mirrored fluorescent microscopic findings (TABLE 4). Not surprisingly, the sensitivity and localization of labeling with chromogenic, lectin-based assays were not as definitive as fluorescent labeling. No MAL-I labeling was detected in the mammary gland with lectin histochemistry.

[0175] As described previously, the HPAI H5N1 virus–infected mammary gland had multifocal acute moderate mastitis with prominent epithelial changes in the secretory alveoli and ducts with sloughed intraluminal epithelial cells, macrophages, and neutrophils (FIG.8). Pancytokeratin (epithelial marker) and Iba1 (macrophage marker) were used to evaluate the intracellular distribution of IAV-Np. Co-labeling of IAV-Np with pan- cytokeratin was observed in cells lining the secretory alveoli and interlobular ducts. IAV- Np labeling was more widely distributed and intense in the secretory alveolar epithelium than in the ductal epithelium. Some intraluminal cells within secretory alveoli and ducts had both intranuclear and cytoplasmic IAV-Np expression. Intraluminal cells are commonly labeled with pancytokeratin (sloughed epithelial cells) and rare Iba1-positive cells (macrophages). Interstitial Iba1-labeled cells did not have IAV-Np co-labeling. Co- labeling with intranuclear IAV-Np labeling and MAL-II and SNA was observed in epithelial cells lining the secretory alveoli (FIG.9, FIG.10). Only co-labeling with intranuclear IAV-Np labeling and SNA was observed in the ductal epithelial cells, given that no MAL-II labeling was observed in the ducts. MAL-I labeling could not be detected with IAV-Np because MAL-I labeling was not observed in the secretory alveoli or ducts.Agent Ref. No. P14876WO00

[0176] TABLE 4. Distribution of α2,3 and α2,6 receptors with influenza A virus nucleoprotein in the respiratory tract and mammary gland of US dairy cattle naturally infected with highly pathogenic avian influenza A(H5N1) virus.Discussion

[0177] The presence of HPAI H5N1 virus in dairy cattle, more so in the mammary gland and milk, once again highlights the importance of IAV adaptability to other nontraditional species and cross-species transmission. This finding reiterates the need for active IAV surveillance efforts in animal species. Like coronaviruses, IAVs have a broad host range involving avian and mammal species. RNA viruses are inherently error-prone, and infection of new host species gives the virus additional opportunities to replicate and subsequently mutate to be better adapt to novel hosts. Another contributing factor to the broad host range of IAV is the presence of virus SA receptors. Many RNA and DNA viruses, including SARS-CoV-2 (co-receptor), use SA as host receptors for initial attachment and entry into the cells.Agent Ref. No. P14876WO00

[0178] In general, IAVs originating from humans and swine preferentially bind to SA linked to galactose through α2,6 linkage, whereas the IAVs of avian and equine species preferentially bind to SA linked to galactose through α2,3 linkage. The susceptibility of a host to IAV infection is determined by the type of SA receptor present on the host cell surface, along with other host factors. Previous studies indicated that the HPAI H5N1 virus preferentially binds to α2,3-linked SA receptors and is expressed abundantly in avian upper airways and their gastrointestinal tracts. IAV strains established in mammals, particularly in humans and swine, exhibit a higher tropism or affinity for α2,6-linked SA receptors, which are predominantly expressed on the epithelial lining of their upper airways.

[0179] SAs are 9-carbon carboxylated monosaccharides synthesized in animals but not in plants. The most common forms of SAs are N-acetylneuraminic acid (Neu5Ac) or N- glycoloylneuraminic acid (Neu5Gc). Although IAVs bind to both Neu5Ac and Neu5Gc, the role of Neu5Gc was reported to be nonfunctional. SAs are also a major component of milk. Cattle and goat milk predominantly contain Neu5Gc versus Neu5Ac, and their content decreases with the progression of the lactation stage.

[0180] This study explores the expression and distribution of SAs in the respiratory tract and mammary glands of Holstein dairy cows naturally infected with HPAI H5N1 virus. The lectins such as SNA, MAL-I, and MAL-II used in this study specifically detect the Neu5Ac form of SA. These findings suggest that SAs are widely expressed in these tissues with predominant SA α2,3-gal-β (1–3) GalNAc (MAL-II), followed by α2,6-linked SA (SNA) and SAα2,3-gal-β (1–4) GlcNAc (MAL-I). However, the expression and distribution varied across tissues. The lectin binding specificity was further validated in a normal mammary gland of a biobanked slide, in which, after removing SAs (90%) by using sialidase A, lectin expression was negative, and so was low pathogenicity H5 virus binding affinity in those tissues. Although HPAI H5N1 and low pathogenicity avian influenza H5N9 viruses were of completely different clades, the findings suggest that no limitations would exist regarding receptor availability and distribution in the bovine mammary gland for IAVs to bind.

[0181] In the respiratory tract, the dairy cows expressed both SAα2,3-gal and SAα2,6-gal. Unlike in pigs and humans, the mammal-specific SAα2,6-gal in cattle is mainly confined to the subepithelial region of the trachea, occasionally in the goblet cells and subepithelial glands. The expression of SAα2,3-gal continued on the epithelium of bronchi to alveolarAgent Ref. No. P14876WO00 pneumocytes. The findings suggest that avian IAVs have the potential binding affinity to the bovine respiratory tract. In the mammary gland, both SA α2,3-gal-β (1–3) GalNAc (MAL-II) and SAα2,6-gal (SNA) were expressed, and co-localization was mainly observed in the alveolar gland and intralobular duct epithelium, whereas SA α2,3-gal-β (1–4) GlcNAc was minimal and confined to interstitial regions. Mammalian- and avian-origin IAVs might bind to cells in the mammary gland. These findings were further supported by the co-localization of intranuclear and cytoplasmic IAV-Np of HPAI H5N1 virus in some intraluminal cells within secretory alveoli and ducts, suggesting possible virus replication.

[0182] The exact pathogenesis of mastitis caused by HPAI H5N1 virus in dairy cattle remains to be elucidated. The initial evaluation of IAV mastitis in dairy cattle identified strong epitheliotropism on the basis of on histologic, immunohistochemical, and FA evaluation. The intramacrophagic viral IAV-Np could be the result of phagocytosis or active infection with virus replication. Intranuclear localization observed in this case may suggest viral replication in macrophages. Previous studies have shown that H5N1 virus replicates efficiently in human macrophages. Even in pigs, pulmonary alveolar macrophages expressing SA receptors have been shown to be susceptible to IAVs and undergo rapid apoptosis. Phagocytosis of viral antigen also is probably occurring given that only intraluminal macrophages mixed with inflammatory exudate contained viral antigen, whereas the interstitial macrophages did not. Overall, resident or infiltrating macrophages in the bovine mammary gland may be susceptible to infection, but further studies are required.

[0183] IAV infections are typically respiratory tract infections. Severe infections can result in a systemic inflammatory response, but the spread of IAV to tissues outside of the respiratory tract is rare. HPAI H5N1 and H5N8 viruses been reported to cause lesions outside of the respiratory tract, including encephalitis and myocarditis, in humans and wild mammals. The multifocal random pattern of viral distribution and lesion development in dairy cattle may suggest either a hematogenous spread (viremia or lymphohistiocytic trafficking) or ascending infection from the teat sinus. Although it is beyond the scope of this article, it will be interesting to explore other suspected routes of infection. The involvement of other organ systems (e.g., the gastrointestinal tract through pancreatic tropism), have been observed in naturally infected poultry and cats with systemic disease.Agent Ref. No. P14876WO00

[0184] Virus attachment through hemagglutinin to host cell SA and the association between the attachment pattern, disease pathogenesis, and transmission efficiency is complex. Slight changes in the receptor-binding activity, as occurs with changes in pH, can influence the functional balance of the IAV infection process. The pH required for the fusion of many avian-adapted IAVs is less acidic than human IAVs. The pH of normal cow milk is mildly acidic (6.3–6.9), and the pH of mastitis milk often increases; however, pH was not measured in the submitted milk sample. It is possible that the mildly acidic environment in the bovine mammary gland, coupled with the presence of SA receptors across the lacteal ducts, can be some of the predisposing factors for HPAI H5N1 virus infection in dairy cattle. As hypothesized earlier, imbalances in pH-associated hemagglutinin and neuraminidase conformational changes may lead to inefficient viral progeny release. However, those observations require additional studies, and the pH of milk from experimentally infected cows should be assessed temporally to assess how these imbalances may affect viral replication.

[0185] Sporadic human cases of H5N1 virus infection have occurred when humans are in close and prolonged contact with birds. Humans have some α2,3-linked SA receptors deep within their lungs, and prolonged close contact with infected birds is postulated to cause infection attributable to inhaling large amounts of virus from those birds with introduction into the deeper recesses of the lungs. Even in the current HPAI H5N1 outbreak, there was a reported case of a dairy farm worker who had direct and close exposure to dairy cows that had onset of bilateral conjunctivitis and was later confirmed positive for H5N1 clade 2.3.4.4b virus through rRT-PCR and sequence analysis. Sequence analysis of the hemagglutinin gene in HPAI H5N1 virus samples from cattle and humans reportedly lacked changes in the receptor-binding affinity (i.e., the virus still preferentially binds to SA α2–3–linked receptors). However, the sample size tested in both dairy cattle (2 cows) and humans (1 human) is relatively small. Continued sequence analysis of identified cases is important to assess viral mutations and potential adaptation to other species. This monitoring will assist preparedness and decision making.

[0186] From a public health standpoint, there is an urgent need to understand why these avian influenza viruses are now infecting so many mammal species. There is also a need to understand why and how often these viruses infect humans. This study is an initial foray into answering those questions. Ongoing surveillance and sequence comparisons of HPAIAgent Ref. No. P14876WO00 viruses in various mammals are needed to better understand spillover events and the underlying pathogenic mechanisms. A better understanding of viral host range will help inform public health decisions and guide research to help prevent future influenza epidemics. Example 3: Genomic characterization of highly pathogenic avian influenza A H5N1 virus newly emerged in dairy cattle

[0187] In March 2024, the emergence of highly pathogenic avian influenza (HPAI) A (H5N1) infections in dairy cattle was detected in the United Sates for the first time. This example describes the genetic characterization of HPAI viruses from dairy cattle showing an abrupt drop in milk production, as well as from two cats, six wild birds, and one skunk. They share nearly identical genome sequences, forming a new genotype B3.13 within the 2.3.4.4b clade. B3.13 viruses underwent two reassortment events since 2023 and exhibit critical mutations in HA, M1, and NS genes but lack critical mutations in PB2 and PB1 genes, which enhance virulence or adaptation to mammals. The PB2 E627 K mutation in a human case associated with cattle underscores the potential for rapid evolution post infection, highlighting the need for continued surveillance to monitor public health threats. Materials and methods Samples

[0188] Two bovine milk samples from dairy cattle from Texas were received at the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) on March 21, 2024. These milk samples were from cattle reportedly exhibiting a nonspecific illness and reduced milk production. Two adult domestic shorthaired cats from a north Texas dairy farm were received at the ISU VDL for a postmortem examination on March 21, 2024. The cats, part of a population of about 24 domestic cats fed milk from sick cows, were found dead with no visible injuries. Clinical disease in the cows started on March 16, and the cats became sick on March 17, with several dying between March 19 and 20. Over 50% of the cats at the dairy fell ill and died. Whole genome sequencing (WGS)

[0189] WGS was conducted on two milk samples and lung and brain tissue samples from two cats. Viral RNA was extracted using the MagMAX pathogen RNA kit (ThermoFisher Scientific, Waltham, Massachusetts) and a KingFisher Flex system. Sequencing librariesAgent Ref. No. P14876WO00 were prepared (ThermoFisher Scientific, Waltham, Massachusetts) as described previously. Next-generation sequencing was performed on an Illumina MiSeq platform following standard protocols at the ISU VDL. Approximately 2,000,000 raw sequencing reads per sample underwent preprocessing with Trimmomatic v0.36 and quality checking with FastQC. Quality-trimmed reads were aligned to reference sequences obtained from the NCBI Influenza Sequence Database using BWA-MEM. Mapped reads were then extracted using SAMtools and used for de novo assembly. Contigs for each segment were assembled using ABySS and SPAdes. Manual curation in SeqMan Pro was conducted to eliminate extraneous sequences and trim chimeric contigs, resulting in a consensus sequence for each segment. Phylogenetic analysis

[0190] Reference sequences were obtained from NCBI Influenza Virus database (ncbi.nlm.nih.gov / genomes / FLU / FLU.html) and Global Initiative on Sharing Avian Influenza Data (GISAID) EpiFlu database (platform.gisaid.org). The coding regions of these sequences were aligned using MAFFT v7.475 with default settings. Sequences containing duplicates, omissions, significant frameshift errors, or with less than 80% length integrity were excluded. The remaining sequences were randomly subsampled to ensure no more than 10 sequences per location at any given time. The GTR + F + G4 substitution model was selected using the Bayesian information criterion by ModelFinder in IQ-Tree v1.6.12, and maximum likelihood (ML) trees of eight gene segments of the isolates were constructed with 1,000 bootstraps. Genotype was determined using GenoFLU (github.com / USDA-VS / GenoFLU).

[0191] A maximum clade credibility (MCC) time-scaled phylogenetic tree of HA sequences from clade 2.3.4.4b H5N1 viruses was generated using the Markov Chain Monte Carlo (MCMC) method with BEAST v1.10.4. A preliminary check using TempEst (v1.5.1) confirmed a significant temporal signal in the dataset, supported by linear regression of root-to-tip distance against sampling date (R2 = 0.8812, Correlation Coefficient = 0.9387). An uncorrelated lognormal relaxed molecular clock and a SRD06 nucleotide substitution model were implemented using the MCMC method run for 100 million generations and sampled every 10,000 generations. Runs were assessed in Tracer v1.7.2 for sufficient convergence [effective sample size (ESS) > 200], and a MCC tree was generated inAgent Ref. No. P14876WO00 TreeAnnotator v1.10.4 after removing the first 10% of runs as burn-in. The obtained MCC tree was edited using FigTree v1.4.4. Results Whole genome sequencing of clade 2.3.4.4b HPAI viruses from dairy cattle and cats in Texas in March, 2024

[0192] In February 2024, veterinarians in the Texas panhandle region observed lactating dairy cattle showing reduced feed intake, decreased milk production, and thickened yellow milk resembling colostrum. The syndrome peaked 4–6 days after onset and subsided within 10–14 days, mainly affecting older cows in mid to late lactation. By early March 2024, similar cases were reported in southwestern Kansas and northeastern New Mexico, with mortalities observed in wild birds and domestic cats near the affected areas. On March 21, 2024, milk samples from dairy cattle and fresh tissues from cats in Texas were received at the ISU VDL. The most significant lesion observed in tissues from affected dairy cows was necrotizing and suppurative mastitis, while two domestic cats exhibited severe necrotizing and lymphocytic encephalitis and necrotizing myocarditis. Real-time reverse transcription PCR testing yielded positive results for influenza A virus H5 clade 2.3.4.4b in the milk samples from the affected dairy cows along with brain and lung tissue from two domestic cats that reportedly consumed raw colostrum and milk at a dairy in Texas.

[0193] The two milk samples from two cows and brain and lung samples from two cats, which tested positive for IAV, underwent next-generation sequencing (NGS) and full genome sequences were successfully obtained on March 23, 2024 for subtyping and other further analyses. NGS analyses confirmed that all four individual samples were positive for HPAI A (H5N1). These sequences have been deposited in GenBank with the BioProject number PRJNA1092030. By March 31, the National Veterinary Service Laboratories (NVSL) had determined HPAI genome sequences from six wild birds, one skunk, and four additional dairy cattle from Texas. These sequences are available in the GISAID database (gisaid.org) and were included in this study for analysis. Phylogenetic and reassortment analysis

[0194] To track the genetically most closely related strains, a comprehensive search was conducted within the GISAID database. The results indicated that the viruses isolated from wild birds, cows, cats, and a skunk in Texas during March 2024 shared a common ancestor with nearly 100 percent homology. To elucidate the phylogeny of these HPAI A (H5N1)Agent Ref. No. P14876WO00 viruses, individual phylogenetic analyses were conducted for each genome segment, including a subset of HPAI A (H5N1) reference sequences obtained from avian and mammalian sources in the USA submitted to GISAID since January 1, 2021. The analysis revealed that the 8 segment sequences of the four HPAI H5N1 viruses in this study, along with others from dairy cattle, wild birds, and a skunk during the 2024 outbreak, closely aligned and formed a new cluster within the HPAI A (H5N1). According to GenoFlu (github.com / USDA-VS / GenoFlu), the HA genes of the clade 2.3.4.4b H5N1 in America since 2021 are divided into three lineages: ea1, ea2, and ea3. The HA genes of the four viruses and others from the 2024 outbreak, were grouped under lineage ea1. Additionally, the NA genes were clustered within ea1, PB2 in am2.2, PB1 in am4, PA in ea1, NP in am8, M in ea1, and NS in am1.1 lineages. Furthermore, the time to the most recent common ancestor (tMRCA) of all available H5N1 viruses in Texas in 2024 was evaluated by constructing the MCC tree of the HA gene using BEAST v1.10.4. It is speculated that this new strain is a descendant of wild birds-originated viruses that emerged in 2023 (node ages as 2023.819).

[0195] The automated data pipeline available at github.com / USDA-VS / GenoFlu was further applied to define their genotype. The four HPAI H5N1 viruses, along with others from dairy cattle, wild birds, and a skunk during this outbreak period in 2024 belonged to genotype B3.13, resulting from a reassortment event involving genotype B3.7 and a low pathogenic avian influenza (LPAI) virus. The B3.7 genotype, which emerged in 2023, contributed seven gene segments, including PB2, PB1, PA, HA, NA, M, and NS, while the NP gene of B3.13 was originating from an LPAI virus resembling A / mallard / Alberta / 567 / 2021 (H11N9)-like strains. According to the GenoFlu analysis, the B3.7 genotype represents a 4 + 4 reassortant strain, with the HA, NA, PA, and MP genes originating from the H5N1 virus strain A1 in 2020, while the remaining segments (PB2, PB1, NP, and NS) are closely related to LPAI viruses. These findings provide compelling evidence that the HPAI H5N1 viruses during this outbreak period in 2024 underwent reassortment events involving both HPAI and LPAI viruses. Critical amino acid substitution analysis

[0196] A comprehensive analysis of amino acid mutations was conducted, closely scrutinizing them to identify any changes potentially associated with increased affinity to human-type receptors, heightened virulence, transmission or adaptation to mammalianAgent Ref. No. P14876WO00 hosts, and the mutants for antiviral resistance. Critical sites among eight HPAI virus isolates originating from dairy cattle and two from cats were compared with those from terrestrial and marine mammals in the public source. This included an extensive dataset comprising 173 strains from Canidae, 39 strains from Felidae, 53 isolates from Mustelidae, six strains from Ursidae, three strains from other species of Bovidae, two strains from Procyonidae, as well as 68 marine mammal isolates, comprising 38 strains from Phocidae, 16 strains from Otariidae, and 14 strains from Delphinidae (TABLE 5). All 8 HPAI H5N1 isolates derived from dairy cattle and two cats demonstrated the presence of residues 137A, 158N, and 160A within their HA segments, which may increase binding affinity to the human-type receptor, while none contained residues 192I, 225D, or 228S. This consistent pattern mirrors that observed in the majority of HPAI isolates from both terrestrial and marine mammals. Furthermore, all eight HPAI H5N1 isolates originating from dairy cattle and two cats exhibited residues 30D, 43M, and 215A in M1, as well as 42S, 103F, and 106M in NS1. Once again, this pattern is aligned with the prevalent composition observed across HPAI isolates from terrestrial and marine mammals, these mutants may increase the viral virulence in mammals. It is noteworthy that mutations 271A, 292 V, 591 K, 627 K / V / A, or 701N in PB2, previously associated with mammalian host adaptation and enhanced transmission, were absent in all eight HPAI H5N1 isolates originating from dairy cattle and two cats, while the HPAI virus from the human case exhibited E627 K mutation in PB2. Conversely, these mutations displayed a high frequency of occurrence in strains from Felidae and a lower frequency in strains from Canidae, Mustelidae, Phocidae, Otariidae, and Delphinidae. Additionally, no critical site mutations associated with increased influenza antiviral resistance have been identified in the virus.

[0197] TABLE 5. Mutations detected in the clade 2.3.4.4b H5N1 viruses have contributed to increased binding to human-type receptors and virulence in mammal.Agent Ref. No. P14876WO00 M6 307 8 311 3 5 6 8 3 28361411SN30F 37 8 26 8 3 28 6141ep 422 S / / / / / / / / / / yyat- mnta)amr1 e2h2 D / / / / / / / / / / t uhbAom tuHyn 88Ia / / / / / / / / / 3ti 5 1nisndiifHf(r6537 3cao 1 A71 3 5 6 8 3 2836141aethponteiece 45N 37 5335 6 8 3 2836141msar 1 1Aercn3 3i31A717335 6 8 3 2836141.o)s)N( n9i3 ea )6 e(lt ) et3(adeiadta e(srotae dileac ea neesdi ) aoadet adiyrdi ydiaicdi niih.tHfona37ilsu)3s i)v co o)ra )pl ) nC1(eFM5( rUaD8( oBr)P2(h8P3( t 6O1( e4D1( atulylaramoirthcmi gese euntarrnirsa oA CeTMaNAgent Ref. No. P14876WO00 Discussion

[0198] The widespread outbreaks of HPAI A (H5N1) clade 2.3.4.4b virus, since October 2020, have raised significant concerns regarding its impact on various mammalian species globally. Recent data reveal that, as of the latest assessment, 37 new mammal species have been afflicted since 2021. The majority of these cases involve wild terrestrial mammals such as foxes, skunks, bears, bobcats, and raccoons. Intriguingly, there have been sporadic infections among domestic pets like domestic cats and dogs, as well as marine mammals, including dolphins and sea lions. Moreover, from January 2022 to April 2023, eight documented human cases of H5N1 influenza from clade 2.3.4.4b have been recorded, several of which were severe or fatal, underlining the gravity of this situation. Adding to this growing list of affected species, an H5N1 influenza virus strain from clade 2.3.4.4b infecting dairy cattle associated with a sudden drop in milk production is now characterized. The detection of this virus in bovine milk raises a potential public health concern related to the risk to dairy workers in close contact with potentially infected cattle, zoonotic transmission through unpasteurized milk, and subsequent human-to-human transmission. This underscores the need for public awareness, pasteurization of milk to maintain adequate food safety, outbreak management, and a holistic approach to human health management.

[0199] In addition to being the first documented occurrence of HPAI A (H5N1) clade 2.3.4.4b virus infection in domestic dairy cattle, pathology observations in this outbreak revealed an apparent tissue tropism for mammary gland in lactating domestic dairy cattle. Prior to this incident, the clade 2.3.4.4b IAV has typically caused systemic and respiratory diseases in wild mammals. Gross and microscopic lesions in wild mammals were frequently observed in organs such as the lung, heart, liver, spleen, and kidney, with some cases resulting in lesions in the brain leading to neurological signs. Furthermore, while it is widely recognized that certain strains of HPAI H5N1 clade 2.3.4.4b virus can breach the blood–brain barrier, this is the first instance where the virus may penetrate the blood-milk barrier and be present in milk, with its underlying mechanisms remains unknown.

[0200] During this outbreak, HPAI virus strains from various sources such as wild birds, dairy cattle, cats, and a skunk, along with a human, displayed remarkably high nucleotide identities in their genome sequences, forming a distinct phylogenetic subcluster. These findings suggest a single introduction of the clade 2.3.4.4b strain into Texas andAgent Ref. No. P14876WO00 neighbouring regions by wild birds. The widespread detection of this HPAI virus strain across diverse regions and species underscores the complexity of its transmission pathways. Given the established role of migratory birds as reservoirs for avian influenza viruses, it is important to highlight Texas's location within the Central flyway. Texas also has overlap in bird migratory patterns with neighboring states that are part of the Mississippi Flyway. Considering these factors, a highly plausible transmission route is hypothesized: wild birds may spread the virus through direct contact or contamination of water sources or feed staffs utilized by dairy cattle or other animals such as skunks. Consequently, other cattle in the herd, workers and domestic felids on dairy farms may contract the virus through direct contact with infected cattle or after consuming raw colostrum and milk from infected cattle. The detection of the same strain of HPAI viruses in various wild bird species, such as blackbirds and common grackles in Texas and Canada geese in Wyoming (Central Flyway), provides further support for this hypothesis. Another potential transmission scenario involves bovine-to-bovine spread. Recently, the USDA has verified the presence of this HPAI virus strain in dairy herds located in Idaho, Michigan, Ohio, North Carolina, and South Dakota. In these cases, a documented history exists of cattle introduction from farms in the initial outbreak area, further supporting the hypothesis that lateral transmission can occur among cattle.

[0201] The thorough examination of mutation adaptations, particularly those linked to human receptor binding affinity, increased virulence, transmission, or adaptation to mammalian hosts, offers critical insights into the risks posed by this specific strain of HPAI viruses. Notably, all HPAI viruses originating from dairy cattle and cats exhibit consistent amino acid residues in the HA gene, including 137A, 158N, and 160A, which have been documented to enhance the affinity of avian influenza viruses for human-type receptors. Additionally, these dairy cattle-derived and cat-derived HPAI viruses harbour key virulence-increasing amino acid residues, such as 30D, 43M, and 215A in M1, as well as 42S, 103F, and 106M in NS1. The presence of these amino acid mutations raises legitimate concerns regarding the potential for cross-species transmission to humans and other mammalian species. It is noteworthy that crucial mutations associated with mammalian host adaptation and enhanced transmission, specifically residues 271A, 292 V, 591 K, 627 K / V / A, 701N, in PB2, and 228S, along with the virulence-increasing residue 66S in PB1-F2, were conspicuously absent in all HPAI virus strains derived from dairyAgent Ref. No. P14876WO00 cattle and cats. There are no amino acid substitutions in the studied cattle sequences that indicate selection for better replication in or transmission among mammals compared to the sequences in other mammals infected with this clade. This observation suggests that the current overall risk to human health is relatively low. However, it is imperative to recognize that influenza viruses have the capacity for rapid evolution within their host environments post infection. A recent human case with direct contact with infected dairy cattle revealed a genetic change (PB2 E627 K), indicating the potential for adaptation or transmission events. This underscores the dynamic nature of influenza viruses and the importance of continued surveillance and vigilance in monitoring potential threats to human health. Example 4: Emergence and interstate spread of highly pathogenic avian influenza A(H5N1) in dairy cattle in the United States

[0202] Highly pathogenic avian influenza (HPAI) viruses have critical consequences for animal health and the agricultural economy and may have pandemic potential. HPAI related to the goose / Guangdong 2.3.4.4 hemagglutinin (HA) H5NX phylogenetic clade has spread to nearly 100 countries, causing infections resulting in mortality events, and is recognized as a panzootic, crossing multiple species barriers. HPAI virus circulation is ongoing in Europe and North America, with recent data indicating a shift in biology and transmission. After an initial trans-Atlantic incursion in late 2021, the HPAI H5N1 clade 2.3.4.4b virus caused widespread outbreaks across North America. The outbreaks resulted in extensive mortality events in wild bird and mammal species, mortality and culling of poultry when detected in agricultural systems, a large number of interspecies transmission events into wild mammals, and human infections.

[0203] The frequent and recent transmission of HPAI clade 2.3.4.4 between avian species in North America has resulted in the emergence of substantial genetic diversity. Experimental studies on some viruses from the HPAI clade 2.3.4.4 have shown that they can bind to both human α2,6-linked and avian α2,3-linked sialic acid receptors. Genomic analyses have documented that approximately half of the sequences from mammals globally within the HPAI H5N1 clade 2.3.4.4b have amino acid signatures in the polymerase basic 2 (PB2) protein that have been associated with mammalian adaptation through enhanced viral replication, host-specific polymerase activity, and temperatureAgent Ref. No. P14876WO00 sensitivity [E627K (E627→K), D701N, and / or T271A)]. Additionally, the introduction of HPAI H5N1 into farmed mink in Europe in 2022 provided evidence that transmission to, and within, a population of mammalian hosts could result in mutations in the HA associated with human receptor recognition and in the neuraminidase (NA) protein that affected sialic acid binding in a manner similar to human influenza A viruses. These results are particularly important because from January 2022 to 1 April 2024, there were 13 reported human cases of H5N1 from the HPAI clade 2.3.4.4b worldwide, with some having severe consequences, including mortality. Consequently, it is critical to determine how evolution of the HPAI clade 2.3.4.4b in wild birds and the associated spillovers and transmission in mammals impacts genomic and phenotype features that alter the potential for human infection and transmission.

[0204] On 25 March 2024, HPAI H5N1 clade 2.3.4.4b was confirmed in dairy cattle in Texas after reports of decreased milk yields in Texas, Kansas, and New Mexico. Shortly thereafter, the virus was identified in cattle in eight other United States (US) states by members of the National Animal Health Laboratory Network (NAHLN). The virus was found in mammary tissue and milk; these samples were collected to determine whether bacterial or viral pathogens were driving milk yield changes. It was also detected in cats and peridomestic animals on affected premises. Overall, the detection of influenza A virus (IAV) in cattle has been rarely documented, but there is prior evidence for its replication within the mammary gland, as well as association with a reduction in milk yield, and experimental studies have shown that bovine calves are susceptible to infection and may asymptomatically shed virus. The goal of this study was to analyze genetic sequence data collected after the introduction of HPAI H5N1 in late 2021 into the Atlantic flyway of North America and its onward circulation and reassortment with North American wild bird–origin, low-pathogenicity viruses that resulted in more than 100 distinct genotypes in the US. These data were combined with newly generated whole-genome sequence data and epidemiological information from the outbreak among US dairy cattle to help us understand when the interspecies transmission event occurred and the consequences of animal movement for the persistence of the virus. Phylodynamic analysis of the US HPAI H5N1 viruses detected in dairy cattle was performed alongside epidemiologically linked wild bird, poultry, and peridomestic animal data. A within-host evolutionary assessment of the virus was also developed to determine how transmission in dairy cattle affects genomicAgent Ref. No. P14876WO00 diversity and whether this increases the potential for this host to serve as a reservoir for zoonotic IAV. H5 clade 2.3.4.4b introduction into the US

[0205] The H5N1 clade 2.3.4.4b genotype A1 was first identified in American wigeon (Mareca americana) and blue-winged teal (Spatula discors) collected in December 2021. More than 100 genotypes representing different gene constellations have been characterized within the US, but 70% of all viruses fall into only seven genotypes. Despite genetic differences, spillover events have been associated with the predominance of a genotype rather than any specific link between genotype and host specificity. The introduction of genotype A3 was identified in April 2022 likely via the Pacific flyway and was followed by genotype A4. Another two introductions were identified to have entered the US via the Atlantic flyway, including an A6 Eurasian virus that had a reassorted Eurasian NA (H5N5). Widespread detections in wild bird populations continue to result in point source spillovers to poultry, but there is limited evidence for lateral transmission among poultry flocks. Viruses of multiple genotypes have also been detected in ~20 mammal species in the US, often related to mortality or severe neurologic signs, but these appear to be dead end hosts. In early March 2024, HPAI H5N1 was identified in neurologic goat kids on a farm where poultry had recently been depopulated for HPAI control measures; this event was unrelated to the dairy event and involved a different virus genotype. Detection of HPAI H5N1 in dairy cattle

[0206] In the US, nearly 10 million dairy cattle are raised in farms across all 50 states. Dairy production is concentrated in California, Wisconsin, New York, Idaho, and Pennsylvania but these locations are within a network of cow-calf operations that are most frequently located in Texas, Oklahoma, Missouri, Nebraska, South Dakota, and Kansas. Of the more than 60,000 dairy premises, ~30% house more than 2000 animals. Farming is characterized by extensive animal movement within and between farms to facilitate production; for example, nonlactating young stock may move off-farm for rearing with the heifers, returning to the operation by calving or before. Nearly all dairy operations report animal replacement in the herd, with 40% of replacements raised off-site.

[0207] In late January 2024, production veterinarians observed dairy cattle displaying reductions in milk production, decreased feed intake, and changes in milk quality. On 20Agent Ref. No. P14876WO00 March 2024, members of the NAHLN identified IAV in milk and in a few nasal swabs from a Texas dairy and forwarded samples to the National Veterinary Services Laboratories (NVSL) for confirmatory testing on 25 March 2024. Testing revealed the presence of H5N1 clade 2.3.4.4b genotype B3.13. Shortly after the identification of HPAI H5N1 genotype B3.13 in Texas, it was confirmed in additional Texas herds and herds in other states. Samples collected in the initial outbreak between 7 March 2024 and 8 April 2024 have virus characterized as genotype B3.13 that included 26 dairy cattle premises across eight states and six poultry premises in three states. The NVSL conducted whole- genome sequencing and analysis, using a custom software program that identified transmission chains on the basis of genomic similarity to provide rapid feedback in support of epidemiologic investigations. The phylogenetic and available epidemiologic data derived from questionnaires with dairy producers indicated that the genotype B3.13 virus was moved between dairy cattle premises, as well as poultry premises, via multiple transmission routes. Detection of the B3.13 genotype in cattle locations that have no known epidemiologic links to confirmed premises indicates there are affected herds that have not yet been identified. A single interspecies transmission event

[0208] To determine when the HPAI H5 clade 2.3.4.4b virus was introduced into cattle in the US, a phylogenetic analysis was conducted using whole-genome sequence data collected from poultry, wild birds, and mammals (FIG.11). From 2022 to the present, clade 2.3.4.4b viruses have been reported in more than 9000 wild birds in at least 163 species across 49 states and Washington, DC, in more than 200 mammals in at least 20 species across 29 states and Washington, DC, and more than 1120 poultry flocks across 48 states. In the time-scaled Bayesian HA gene phylogeny, the H5N12.3.4.4b sequences isolated from cattle clustered within a single monophyletic clade, which supports a single spillover event followed by lateral transmission. Introduction of HPAI from unsampled poultry populations represents an alternate hypothesis, but a single spillover event was also supported by the maximum likelihood phylogeny reconstructed from concatenated whole- genome sequences of B3.13 genotype strains (FIG.11B), and this pattern was evident in the gene segment maximum likelihood phylogenies: polymerase basic 2 (PB2) protein, polymerase acidic (PA) protein, nucleoprotein (NP), NA, matrix protein (MP), and nonstructural (NS) protein. For the polymerase basic 1 (PB1) gene segment, >97% ofAgent Ref. No. P14876WO00 sequences from cattle isolates formed a single monophyletic clade; the remaining sequences were placed in a parental clade owing to the high genetic similarity. The single- introduction hypothesis was further supported by an analysis of single-nucleotide polymorphisms (SNPs). A subsequent maximum likelihood phylogenetic analysis on n = 1156 genomes of B3.13 genotype strains further supported the proposition of a single origin from an avian reservoir host. The inferred evolutionary rate for the HA gene tree was 6.19 × 10−3substitutions per site / year [95% highest posterior density (HPD), 5.21 × 10−3to 7.19 × 10−3]; the estimated evolutionary rate for the cattle clade was not plausible (3.96 × 10−2, 95% HPD, 2.13 × 10−2to 6.05 × 10−2) and likely requires a different dataset or analytical technique. The time to the most recent common ancestor (TMRCA) for the HA segment estimated with Bayesian methods for the cattle clade was estimated as 10 November 2023 (95% HPD, 4 October 2023 to 22 January 2024). A clade 2.3.4.4b genotype B3.13 infection in a dairy worker was diagnosed at the end of March 2024 and was similar to the HPAIV from cattle. The TMRCA estimated with Bayesian methods for the cattle group and the human virus was estimated as 27 September 2023 (95% HPD, 18 August 2023 to 9 December 2023). These two groups shared a TMRCA that was estimated as 14 August 2023 (95% HPD, 23 July 2023 to 19 October 2023). These are broad TMRCA estimates and suggest that after the B3.13 introduction into cattle, there was limited local circulation for >4 months. The Bayesian estimates were congruent with TMRCA inferred by using maximum likelihood methods (FIG.11B); the TMRCA for each of the gene segments [the TMRCA ranged from 4 October 2023 to 13 February 13 2024] is feasible given epidemiological information and is consistent with independent estimations. Cattle transmission to peridomestic species

[0209] The phylogenetic tree topology indicated that after introduction, the virus persisted in cattle populations, with subsequent evidence for transmission from cattle into poultry and peridomestic animal species. There were as many as nine cattle to poultry, one cattle to raccoon, two cattle to cats, and three cattle to wild bird transmission events. The wild bird transmission events were restricted to the common grackle (Quiscalus quiscula), blackbirds, and pigeons. These animals were collected from premises with cattle where genotype B3.13 HPAIV was identified. The Bayesian discrete state analysis that quantified the movement of HPAIV between six different host categories (poultry, wild bird, cattle,Agent Ref. No. P14876WO00 wild mammals, cat, and humans) provided sufficient evidence to verify that HPAI in cattle resulted in infections in other hosts. The possibility that this genotype is circulating in unsampled locations and unknown hosts cannot be excluded, because the existing analysis suggests that data are missing and because incomplete surveillance may obscure transmission inferred with phylogenetic methods. The gap in data is highlighted by the human infection with genotype B3.13 HPAIV, in which the HA gene sequence did not nest within the virus HA gene sequences from cattle. This could indicate that HPAIV in unsampled cows was the source of infection or that within-host evolution resulted in divergence sufficient to result in a different phylogenetic grouping. It is most likely, however, that asymptomatic transmission and lack of surveillance in epidemiologically important populations drove this pattern. The analysis of transmission chains within the cattle B3.13 clade using a phylogenomic maximum likelihood approach suggested unsampled transmission in late 2023 and early 2024 (fig. S8), and the TMRCA indicates that there was >4 months of circulation before confirmation. However, given the decline in milk production in monitored dairy herds, it is unlikely that the spillover occurred outside of the described TMRCA ranges. Reassortment in migratory bird populations

[0210] From January 2022 to April 2024, more than 482 commercial and 645 backyard flocks tested positive for HPAI. A major component in the dissemination of the 2.3.4.4b viruses is movement across four migratory flyways and the infection of nearly 200 wild avian species. Extensive genetic reassortment with existing North American wild bird LPAIVs is a consequence of the host breadth and the geographic breadth. In the analysis, reassortment generated a spectrum of different genotypes, but the HA, NA, and M genes had preferential pairings in a manner that also occurs in mammalian adapted IAVs. There was transient detection of reassorted viruses with different combinations of PB2, PB1, PA, NP, and NS genes. A total of 243 putative reassortment events were detected across the HA phylogeny. These events were associated with PB2 (137 / 243), PB1 (78 / 243), PA (36 / 243), NP (126 / 243), and NS (52 / 243) gene segments. With current surveillance data, there was no evidence that most of these events persisted. However, there were 24 reassortment events that resulted in new genotypes with more than 20 downstream detections. The spillover into cattle was preceded by a reassortment event(s) involving different PB2 and NP genes, likely derived from wild bird LPAI in late 2023. ThisAgent Ref. No. P14876WO00 reassortment resulted in the B3.13 genotype, which is maintained across the clade of epidemiologically linked cattle samples and shows no evidence for further reassortment after the spillover. The NP gene acquired by the reassortment event may have resulted in a phenotype change that mediated the emergence of this virus genotype in cattle. There are no comparative phenotypic data to support this assertion, but the NP gene is associated with multiple processes in the virus life cycle and was implicated in increasing the transmission efficiency of IAV in the swine host. An alternate explanation is that the new reassorted virus was a passenger on an overlapping set of ecological parameters (e.g., environmental contamination from infected wild birds) that resulted in the HPAI H5N1 spillover. HPAI H5N1 dispersal with cattle movements

[0211] Epidemiological records documented dairy cattle movement from a Texas herd (at shipment, HPAI status unknown) to North Carolina and Idaho. Records also indicated that asymptomatic or presymptomatic dairy cattle—some that were subsequently diagnosed as HPAIV positive—were moved from a Texas herd to Michigan and to Ohio and that a Kansas herd was moved to South Dakota. An asymmetric discrete-trait model with Bayesian stochastic search variable selection was also applied to reconstruct how clade 2.3.4.4b HPAIV H5 isolated in cattle moved among the eight US states (Idaho, Texas, Kansas, Michigan, New Mexico, North Carolina, Ohio, and South Dakota). The interstate HPAIV movement, which is inferred by these phylodynamic techniques, showed that after the first confirmed case in Texas, HPAIV moved rapidly across the US. The phylogenetic signal within the HA gene of B3.13 genes in cattle was relatively low, and state transitions were conservatively identified when a Bayes factor >3 with an associated posterior probability >75%. Using these thresholds, there was phylogenetic evidence for the movement of the B3.13 genotype from Texas to Kansas, Michigan, and New Mexico. Evidence in the phylodynamic analyses indicated that after its introduction into Michigan, B3.13 was moved into North Carolina. However, it was more likely that a confirmed Texas-to-North Carolina animal movement moved the virus, because no link between Michigan and North Carolina dairy herds could be confirmed. In a phylogenomic analysis, there were cohesive groups of sequences isolated from each US state; this suggested that after mixing and movement of animals in March and April 2024, a spatially structured host and virus population emerged. These data support the proposition that a single genotypeAgent Ref. No. P14876WO00 was introduced and that direct movement of cattle—based upon production practices— created opportunities for virus dissemination that have since been reduced. The movement of viruses from one region to another will provide opportunities for reassortment and subsequent changes in genomic diversity. Areas that received large shipments of cattle and their viruses will provide additional opportunities for reassortment and the emergence of IAV strains with phenotypes that may have increased zoonotic potential. Low-frequency genomic variants and within-host evolution

[0212] A total of 227 cattle viral whole genomes collected during the first phase of the outbreak between 7 March and 3 April 2024 were analyzed, along with data from an additional 1,250 viral genomes collected from cattle between 4 April and 29 October 2024. Within-host sequence variants across the genome that were present in >0.5% of whole- genome sequencing reads were identified, matching to the Influenza Research Database Sequence Feature Variant Tool and a literature search. If a sequence variant were to arise that provided a selective advantage, it could increase in frequency through transmission and potentially alter the virus phenotype. The analyses were restricted to field-collected specimens, with no ability to assess whether single-nucleotide variants (SNVs) affected virus attributes. In addition, most SNVs were present only at low frequencies. In the cattle sequence data, there were 822 amino acid sites with nonsynonymous amino acid changes; of the variable amino acid sites, 406 nonsynonymous mutations occurred at sites associated with functional changes (mean 44.32 ± 5.06 potential functional changes per sample; range: 24 to 64). An additional 605 synonymous mutations were detected in the dataset. Variants were associated with changes in virulence in HA, MP, NP, and PB2 (e.g., in TABLE 6: Q134K, Q154R, Q234K, and P337L in HA; S207G in MP; D701N in PB2). SNVs previously associated with host-range specificity (e.g., E229K in NS) were also detected. In PB2, variants associated with mammalian adaptation (an increase in E627K, D701N, and V495I) were identified, with E627K present at a frequency of 33% in a single animal. The mammalian adaptation PB2271A was not detected in cattle, even at low frequencies; however, one mammalian sample contained the mutation in the consensus gene, and mutations on HA affecting receptor binding affinity were detected (TABLE 6). Within the PB2, there was the detection of 631L (one strain had 631P), an amino acid position that has been associated with mammalian transmission. When analyzing an additional 1250 cattle samples collected between 4 April and 29 October 2024, 10 low-Agent Ref. No. P14876WO00 frequency SNVs increased to higher frequencies; these included virulence markers in HA (position 336) and NS (position 229) and a mammalian adaptation marker in NA (position 430). Twelve other nonsynonymous SNVs with unknown functions were observed to increase in frequency, with 21 SNVs increasing to >5% of the total cattle samples: These may reflect a founder effect or evolution associated with phenotypic change. Determining the biological relevance of these sequence variants requires additional experimental study and should be monitored because some have been associated with transmission efficiency and mammalian adaptation. Synonymous (πS) and nonsynonymous (πN) site ratios were also calculated to assess natural selection; πN / πS<1 is suggestive of purifying selection, and πN / πS>1 is suggestive of positive selection. When diversity estimates were combined across genes, all cattle strains exhibited πN / πS <1; the gene πN / πS estimates varied, with none having >1 (HA, πN / πS = 0.042; MP, πN / πS = 0.566; NA, πN / πS = 0.188; NP, πN / πS = 0.032; NS, πN / πS= 0.355; PA, πN / πS= 0.160; PB1, πN / πS= 0.048; PB2, πN / πS= 0.039), suggesting that the within-host virus populations tended to exhibit weak purifying selection when compared with an ancestral reference.Agent Ref. No. P14876WO00

[0213] TABLE 6. Sequence variants detected in sites within H5N12.3.4.4b strains isolated in cattle that have been associated with host adaptation and virulence. Raw read data from cattle samples were processed, and high- and low-frequency single-nucleotide variants (SNVs) were identified relative to the most recent common ancestor of the phylogenetic group in this study. The SNVs that induced a coding region change were screened against a database of positions associated with functional change, with a relevant selection shown here. The number of cattle samples with the SNVs were enumerated: The first number represents data from March and April 2024, and the number in parentheses represents data from April to October 2024. The mean allele frequency was calculated, the presence of the mutation within the consensus gene sequence was determined, and the variants detected at low frequencies were counted. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. adapt., adaptation; resist., resistance.Agent Ref. No. P14876WO00 Discussion

[0214] The HPAI H5N1 genotype B3.13 viruses circulating in cattle represent a pandemic threat, given the evidence of cattle-to-human transmission events and persistence across the US. On the basis of current information, once infected, a cow may shed virus for 2 to 3 weeks, increasing the window in which zoonotic transmission may occur. Additionally, in the field data, some amino acid mutations at sites associated with mammalian adaptation were detected that had already become fixed in the virus population. This result likely reflects the ~4 months of evolution in dairy cattle. Notably, low-frequency sequence variants within cattle were also detected, even within the limited time after the first spillover. The majority of these sequence variants have not increased in detection frequency; however, some changes increased in prevalence over time. The potential remains for these viruses to acquire other genetic markers that mediate the probability of transmission in mammalian hosts. Experimental studies have demonstrated limited airborne transmission efficiency in cattle, the B3.13 genotype has rarely been detected in migratory birds, and the majority of cases are restricted to affected dairy or poultry premises. These findings suggest that this remains a distinctive spillover with potential for control. However, turkeys and pigs may be colocated on dairy premises, each species may be infected with IAV, and coinfection could result in reassortment and the emergence of new strains with elevated zoonotic potential. Monitoring of cattle and other agricultural animals will inform future risk assessments and provide an early warning for whether this interspecies transmission event and dissemination of the viruses represents a future threat to human health.

[0215] In countries with endemic HPAI H5N1 in poultry, there may be low levels of immunity against H5N1 viruses, but there is no evidence for this in the US. This is concerning because the immunological landscape in humans affects disease severity, and clade 2.3.4.4b B3.13 virus have demonstrated the capacity to cross the species barrier. The existing prepandemic candidate vaccine viruses (CVVs) do retain cross-reactivity with currently circulating clade 2.3.4.4b HPAI H5N1. These CVVs are coordinated and shared among the WHO Global Influenza Surveillance and Response Network for use by academic, government, and industry partners for R&D. However, recent viruses collected in the US had reduced reactivity with the A / Astrakhan / 3212 / 2020 CVV, and on the basis of these data and other genetic and epidemiologic measures, a new clade 2.3.4.4b CVV hasAgent Ref. No. P14876WO00 been proposed. An outbreak of HPAI H5N1 B3.13 genotype in humans may be detected— given passive and active surveillance at human-animal interfaces—and minimized through deployable medical countermeasures. However, preemptive control of animal IAV in wild and domestic populations before zoonoses is ideal. Testing of cattle before movement between states will reduce HPAI H5N1 dissemination. In addition, control strategies may be refined through passive IAV surveillance of cattle, swine, and wild birds, alongside a National Milk Testing Strategy to identify IAV transmission hotspots. After HPAI detection, poultry locations are depopulated and other farm locations can implement enhanced biosecurity efforts to control wild birds on premises, reduce shared personnel and equipment between locations, apply vaccine interventions to target circulating diversity, and decrease mixed-species farming.

[0216] In this study, it is suggested that the B3.13 genotype had limited local circulation for >4 months. This suggests a lack of surveillance for IAV in nontarget hosts and wildlife species, reducing the potential to detect and subsequently control new viruses before zoonotic transmission. This problem may be rectified through surveillance strategies derived from alternate sample types (e.g., wastewater, bulk milk) alongside better support for passive surveillance of wild mammals and birds. This information will enable detection and identification of outbreak sources and provide the data necessary to monitor virus spread and evolution. Additionally, although genomic surveillance has increased, there has not been a concomitant increase in the number of phenotypically characterized animal IAV or ecological studies that systematically describe features associated with IAV interspecies transmission. This deficiency is highlighted by detection of hundreds of reassorted IAVs, yet whether these viruses possess altered attributes could not be determined. Ideally, genomic surveillance data can be shared and linked with phenotypic data to facilitate the identification of IAV for characterization and risk assessment. More generally, this study demonstrates that IAV is a transboundary pathogen that requires coordination across regulatory agencies and between animal and public health organizations to improve the health of hosts and reduce pandemic risk. Materials and methods Sample isolation, whole genome sequencing and assembly

[0217] IAV extraction and reverse transcription real-time PCR (RT-rtPCR) were performed at the NAHLN member labs and the US Department of Agriculture (USDA),Agent Ref. No. P14876WO00 NVSL, according to the standard operating procedures. Influenza A virus RNA from samples was amplified and after amplification was completed, cDNA libraries for iSeq were generated by using the Illumina DNA Sample Preparation Kit, (M) Tagmentation (Illumina) and the 300-cycle iSeq Reagent Kit v2 (Illumina) according to manufacturer instructions. Reference guided assembly of genome sequences was performed using IRMA v1.1.4. A total of 237 whole genomes collected between 7 March 2024 and 3 April 2024 were generated (10 samples did not have sufficient depth for the SNV analysis). Public data from 4 April 2024 to 29 October 2024 were subsequently analyzed to confirm initial patterns; this information encompass an additional 1250 whole genomes. All raw read data are provided within the NCBI Sequence Read Archive with consensus genomes assembled and submitted to NCBI GenBank. Data and code used in this manuscript are archived at a GitHub repository. Field epidemiological investigations

[0218] Dairy herds were identified following reporting of clinically sick cattle to local animal health officials and confirmatory testing at the USDA, NVSL. Following confirmation of a positive detection of HPAI H5N1, information was collected by the administration of the USDA Dairy Cattle Emerging Health Event: Epidemiological Questionnaire. Completion of this document was voluntary and reliant upon the operation and the willingness of the producer. Information included details on animal movements, status and source of animals on the farm, handling of waste milk, manure disposition, the presence of other animal species on the premise, whether locations shared personnel and equipment, the number of visitors to an operation, and whether the location had observed wild bird mortality. The focus of these investigations was to identify potential transmission routes for pathogen spread between affected dairy premises and affected poultry premises. Putative transmission routes were also determined through an analysis of SNPs in the clade 2.3.4.4b consensus sequences that were collected from each cattle or poultry location. SNPs were annotated and compared using a pipeline called vSNP. The pipeline infers phylogenetic trees using RAxML and generates tables of SNPs relative to a reference composed of two North American wild bird origin segments (PB2, NP) and six segments from a H5N1 clade 2.3.4.4b clade virus. Epidemiological data from the questionnaires were paired with the genomic data to determine viral genome sequence similarity and to identify links between premises. Links were grouped as animal links, non-animal links thatAgent Ref. No. P14876WO00 included movement distinct from cattle or poultry (e.g., shared farm personnel), and genomic epidemiological links derived from the vSNP analysis. Data sources and curation for Bayesian phylodynamic analyses

[0219] To generate a reference dataset of relevant HPAI H5N1 strains, Global Initiative on Sharing All Influenza Data (GISAID) was queried for all H5 genomes with eight complete gene segments available collected between 1 January 2020 and 29 March 2024 in the US. This query provided 23,322 sequences from 2,915 genomes; the HA gene sequence was extracted and the data were aligned using MAFFT v7.526 with default parameters. A maximum likelihood phylogenetic tree was subsequently inferred with IQ-Tree v2.2.2 following automatic model selection. Based on this tree, all data were classified using a custom python clade identifier called GenoFLU (github.com / USDA-VS / GenoFLU) and filtered to only those strains from the HPAI clade 2.3.4.4b. Identical HA sequences were then removed, ensuring retention of the cattle strains published in GISAID and the associated human case (A / Texas / 37 / 2024, GISAID accession EPI_ISL_19027114). This process resulted in a reference gene dataset of 1,393 strains. For computational efficiency, these were subsampled using smot v.1.0.0, maintaining representation by sampling 20% of the tips within each monophyletic clade based on an HA gene phylogeny inferred using FastTree v2.1.11. To complement and provide support for Bayesian phylogenetic analyses, the resultant dataset of n = 964 strains was used in maximum likelihood phylogenetic analyses. For these, ancestral sequences of North American 2.3.4.4b strains for each gene segment were inferred by using a maximum likelihood tree inferred with IQ-Tree v2.2.2 for each gene segment as input for TreeTime v0.9.4 ancestral reconstruction functionality. The ancestral sequences were reconstructed at the deepest nodes on each gene tree that comprised at least 99% of all strains in the subsampled dataset. Bayesian evolutionary inference and discrete phylogeography

[0220] HPAI H5N1 clade 2.3.4.4b introduction into dairy cattle was estimated under a Bayesian framework in BEAST v.1.10.4 with the BEAGLE library. For this analysis, the representative dataset of n = 964 strains was used, all sequences associated with the dairy cattle clade were maintained, and taxa outside of this monophyletic group were randomly sampled, resulting in an aligned HA dataset of n = 587 HA genes. A generalized time reversible (GTR) nucleotide substitution model with gamma-distributed site heterogeneity, an uncorrelated relaxed clock with lognormal distribution, and a Gaussian Markov randomAgent Ref. No. P14876WO00 field (GMRF) Bayesian skyride with time aware-smoothing for the coalescent model were implemented. Ten independent Markov chain Monte Carlo (MCMC) sampling runs were conducted with 50 million iterations, sampling every 5,000 iterations. A similar process was conducted on the remaining seven IAV gene segments: the representative dataset of n = 964 strains was used, all sequences associated with the dairy cattle clade were maintained, and taxa outside of this monophyletic group were randomly sampled with a goal to have aligned datasets of n ~750 for each gene. For these seven genes, a GTR nucleotide substitution model with gamma-distributed site heterogeneity, a strict molecular clock, and a GMRF Bayesian skyride with time aware-smoothing for the coalescent model were implemented. For these seven genes, five MCMC chains were run for 50 million iterations each, sampling parameters and trees every 5,000 iterations. The results were analyzed using the GMRF skyride reconstruction in Tracer v1.7.2, convergence and mixing was assessed, and runs were combined to ensure an effective sample size of more than 200. A time-scaled maximum clade credibility (MCC) tree was generated using TreeAnnotator v1.8.4 using median node heights and ~20% burn-in. Dates of time to the most recent common ancestor (TMRCA) were inferred from the nodes between clades, using the 95% HPD as the range of uncertainty. The TMRCA dates inferred from the Bayesian analyses in BEAST were supported through a phylogenomic analysis on concatenated gene segments and gene segment analyses using the TreeTime suite.

[0221] To reconstruct the spatial diffusion of the H5N1 virus across states within the US and different hosts (poultry, wild bird, cattle, mammal, domestic mammal, and humans), an asymmetric discrete-trait phylogeographic analyses with Bayesian stochastic search variable selection, a strict molecular clock, and an exponential growth model in BEAST v.1.10.4 was applied. This analysis was conducted on the subsampled and aligned HA dataset and the extracted monophyletic clade of cattle HA clade 2.3.4.4b genes with any host category (host) or cattle only (location). In this approach, each HA gene had a US state and host group designated as a trait, and transitions from one category to another (e.g., from Texas to New Mexico) were inferred along the internal branches representing the evolutionary history of the virus. These transitions are termed Markov jumps, and state change counts for the HA clade trait were reconstructed in BEAST v1.10.4. Five independent MCMC chains were conducted with 50 million iterations, sampling every 5,000 iterations. Convergence and mixing were assessed in Tracer v1.7.2, as describedAgent Ref. No. P14876WO00 above, and runs were combined to ensure an effective sampling size of more than 200. The resulting posterior trees were used to provide estimates of the ancestral region and host for each internal node. SpreaD3 v.0.9.6 was then used to estimate the Bayes Factor (BF) from the estimated state transition rates and used as statistical support for spatial movements; thresholds used in previously published studies were imposed, i.e., a definitive spatial movement had a BF > 100, and sufficient evidence was assessed as 100 > BF > 3. Reassortment analyses

[0222] A separate dataset for reassortment analysis was assembled that comprehensively included all H5N1 whole genomes collected from January 2020 to 19 April 2024 by downloading n = 27,177 HPAI H5N1 gene sequences from GISAID [accessed 19 April 2024]. This search included all data from North America, and only those strains with complete sequences for each of the eight gene segments were maintained, resulting in n = 2,893 whole genomes. These data were merged with the assembled whole genomes from this study and alignments for each gene were generated using MAFFT v7.526 with default parameters. Phylogenetic trees for each gene segment were subsequently inferred using IQ- Tree v2.2.2 under the GTR model of nucleotide substitutions, with stationary probabilities estimated from the empirical base frequencies and five free-rate categories. To identify reassortment events on the HA phylogeny, TreeSort v.0.1.1 was applied with a maximum molecular clock deviation parameter of 2.5. TreeSort uses TreeTime to estimate the substitution rates for each gene segment and identifies branches on a tree with a high signal of reassortment. n = 35 strains that were molecular clock outliers in the HA gene and were not related to the outbreak in dairy cattle were then removed, and the TreeSort analysis was repeated. Phylogenomic analyses over concatenated B3.13 genomes

[0223] The coding regions of all gene segments were concatenated into whole genome sequences for all reassortment-free B3.13 strains from the reassortment analysis above. This resulted in n = 256 aligned whole genome sequences, including n = 220 cattle strains. A phylogenetic tree from these sequences was inferred using an edge-linked partition model in IQ-Tree v.2.2.2, where every partition corresponded to an individual gene segment and was associated with a different GTR model with empirical base frequencies and five free-rate categories. IQ-Tree was run with ultrafast bootstrap with 1000 replicates. The resulting tree topology was then used as input to TreeTime v.0.9.4 to infer a time-Agent Ref. No. P14876WO00 scaled tree of the B3.13 genomes. TreeTime was executed with an option to account for covariation in tip dates due to shared ancestry. To validate the initial phylogenomic analysis, this procedure was repeated on 29 October 2024 with an updated comprehensive dataset of B3.13 genomes collected up to 29 October 2024. The updated analysis was conducted over n = 1156 whole genomes, n = 1113 of which were cattle. Within-host genetic diversity analysis

[0224] A bioinformatics pipeline was developed for processing, calling SNVs, and analyzing Illumina short read data for influenza A virus called “Flumina” (github.com / flu- crew / Flumina). The pipeline uses Python v3.10, R v4.4, and SnakeMake to organize programs and script execution. A custom Python script organizes raw reads for SnakeMake that subsequently executes other programs for variant calling. The pipeline cleans the raw reads of adapter contamination, low complexity sequences, and other sequencing artifacts using the program FASTP. IRMA v1.1.4 was used on the processed reads to generate consensus contigs that were used for phylogenetic and phylodynamic analyses and other summary statistics and graphs. Following these steps, the pipeline maps reads to an ancestral reference strain, by indexing using BWA [bwa index -a bwtsw function]. Then it uses SAMtools to create an fai index with the faidx function and generate a sequence dictionary for GATK v4.4 using the CreateSequenceDictionary function.

[0225] For calling of high frequency SNVs, GATK v4.4 was used following best practices for discovering and calling SNVs. The pipeline processes the cleaned reads through the GATK4 functions FastqToSam, RevertSam, and AddorReplaceReadGroups. Next, the BAM of the reads are converted back to Fastq and mapped with BWA using the function bwa mem -M. The mapped reads BAM file are merged with the unmapped reads BAM, and duplicate reads are marked after sorting the BAM file by coordinate with the functions SortSam and MarkDuplicates. Finally, HaplotypeCaller is used (parameters: -ERC GVCF -ploidy 1) to call haplotypes, and GenotypeGVCFs is used to genotype the sample. Specific variants were selected using the SelectVariants function for the SNP type of variant. Using VariantFiltration, a filtered set of variants were created by applying the following filters: Qual < 30 (Quality), QD < 2 (Quality by Depth), SOR < 3 (StrandOddsRatio), FS < 60 (FisherStrand), MQ < 40 (MapQuality), MQRankSum < 12.5 (MapQuality RankSumTest), and ReadPosRankSum < 8 (ReadPosRankSumTest). To call low frequency SNVs, the program LoFreq was applied, with the similar preprocessing steps where the same BAMAgent Ref. No. P14876WO00 was used as input from HaplotypeCaller. A database was generated using the Sequence Feature Variant Types tool from the Influenza Research Database for all eight genes, and SNVs associated with nonsynonymous changes in each gene were screened to determine whether these had previously been associated with phenotypic change. The numbering in this database uses the full coding region for each gene segment and H5 numbering for the HA gene. In the NA gene, amino acid changes in the stalk region were excluded due to uncertainty on phenotype impact. To estimate genome-wide estimates of natural selection, the program SNPGenie was used on the VCF files. Inference of B3.13 transmission chains in dairy cattle

[0226] Inference of transmission chains between hosts, sampled diversity, sampling time, and generation time were estimated from the temporally scaled genome tree using the TransPhylo R package. As input, the B3.13 tree generated in the phylogenomic analysis was used. Multifurcations in the tree were suppressed to bifurcations and all edge weights were rounded to at least 1 × 10−9. The prior generation time and sampling density were gamma-distributed parameters extracted from published studies resulting in a mean of 5 days and a variance of 2 days. Protocol 3 was adapted ensuring an Effective Sample Size (ESS) >200 at the end of the simulations. The MCMC simulations were run for 1 × 107iterations, discarding the first 5% as burn-in. After the simulations converged, the medoid tree from the posterior set of inferred trees was used as the final inferred transmission tree. Transmissions of HPAIV from host to host (e.g., wild bird to cattle) were inferred by TransPhylo and the distribution of incident cases (sampled and unsampled) was generated by adapting protocol 4. Example 5: Recombinant HA5 Protein Vaccine

[0227] A novel recombinant HA5 protein vaccine using a trimerization domain was developed in baculovirus. The HA5 sequence was codon optimized for sf9 cells and modified to remove the transmembrane domain, add a T4 foldon domain, and add a cleavable 7x His purification tag (SEQ ID NO: 10). Example 6: Evaluating efficacy of recombinant H5 Hemagglutinin subunit vaccine in calvesAgent Ref. No. P14876WO00

[0228] The objective of this example is to evaluate the immunogenicity of an H5 HA subunit vaccine in calves.

[0229] A total of eight, 7-8 week old, weaned Holstein bull calves are enrolled in the experiment. Prior to enrollment, the calves are confirmed for seronegative status against H5 by hemagglutination inhibition (HI) testing. Calves are transported to the ISU Animal Resource Station outside of Ames, IA. They are group housed in a dry lot for the duration of the study. Calves are divided into two treatment groups: three serve as nonvaccinated controls, and five receive the experimental vaccine. Calves are immunized subcutaneously in the neck with 250 µg of H5 adjuvanted with EMULSIGEN® oil-in-water adjuvant on day 0. Control calves receive the adjuvant plus saline. A booster vaccine (or adjuvant only) is administered once approximately 3 weeks later.

[0230] Serum samples are collected weekly via the jugular vein, starting on day 0 (prior to the first vaccination, through 4 weeks post vaccination. A total of 7 serum samples are collected over the course of the study.

[0231] The immunogenicity of the vaccine is confirmed using two common serologic assays: 1) indirect ELISAs using recombinant H5 or heat-inactivated H5N1 virus as the antigen; and 2) HI assays using recombinant H5. If successful, additional serum samples are evaluated using microneutralization assays against viable H5N1. This work requires a BSL3 facility and is performed only after initial assays confirming immunogenicity.

Claims

Agent Ref. No. P14876WO00 What is claimed is:

1. A recombinant influenza hemagglutinin (HA) polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO:

1.

2. The recombinant HA polypeptide of claim 1, wherein the recombinant HA polypeptide lacks a transmembrane domain.

3. The recombinant HA polypeptide of claim 1, further comprising a trimerization domain.

4. The recombinant HA polypeptide of claim 3, wherein the trimerization domain has the amino acid sequence set forth in SEQ ID NO:

11.

5. The recombinant HA polypeptide of claim 1, further comprising a purification tag.

6. The recombinant HA polypeptide of claim 1, wherein the recombinant HA polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO: 3, 5, 7, or 9.

7. The recombinant HA polypeptide of claim 1, wherein the recombinant HA polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7, or 9.

8. A multimeric polypeptide comprising at least two recombinant HA polypeptides of any one of claims 1-7.

9. The multimeric polypeptide of claim 8, wherein the multimeric polypeptide is trimeric and comprises three recombinant HA polypeptides.

10. An immunogenic composition comprising the recombinant HA polypeptide of any one of claims 1-7; and a pharmaceutically acceptable carrier.Agent Ref. No. P14876WO00 11. A method for inducing an immune response against an influenza virus in a subject, the method comprising: administering to the subject the immunogenic composition of claim 10.

12. The method of claim 11, wherein the subject is a bovine.

13. The method of claim 12, wherein the bovine is a cow, bull, or calf.

14. The method of claim 11, wherein the immunogenic composition is administered intranasally, parenterally, subcutaneously, intramuscularly, or intradermally.

15. A vaccine composition comprising the recombinant HA polypeptide of any one of claims 1-7; and a pharmaceutically acceptable carrier.

16. The vaccine composition of claim 15, wherein the composition further comprises an adjuvant.

17. The vaccine composition of claim 16, wherein the adjuvant is an oil-in-water emulsion adjuvant.

18. A method of treating or preventing disease caused by an influenza virus comprising: administering to the subject the vaccine composition of claim 15.

19. The method of claim 18, wherein the subject is a bovine.

20. The method of claim 19, wherein the bovine is a cow, bull, or calf.

21. The method of claim 18, wherein the vaccine composition is administered intranasally, parenterally, subcutaneously, intramuscularly, or intradermally.Agent Ref. No. P14876WO00 22. The method of claim 18, wherein the composition is administered to the subject twice.

23. An immunogenic fragment of the recombinant HA polypeptide of any one of claims 1-7.

24. A polynucleotide encoding the recombinant HA polypeptide of any one of claims 1-7.

25. The polynucleotide of claim 24, wherein the polynucleotide comprises a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO: 2, 4, 6, 8, or 10.

26. The polynucleotide of claim 24, wherein the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10.

27. A vector comprising the polynucleotide of claim 24.

28. A cell comprising the polynucleotide of claim 24.

29. The cell of claim 28, wherein the cell is an insect cell.

30. A method for producing a recombinant HA polypeptide, the method comprising: expressing the polynucleotide of claim 24 in a cell.

31. The method of claim 30, further comprising isolating the recombinant HA polypeptide from the cell.

32. The method of claim 30, wherein the cell is an insect cell.

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