Multi-platform feed-based antiviral system for highly pathogenic avian influenza (HPAI) with extended applications to zoonotic diseases
Patent Information
- Application Number
- PCT/US2026/023491
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-04-14
- Filing Date
- 2026-04-13
- Publication Date
- 2026-10-22
Smart Images
Figure US2026023491_22102026_PF_FP_ABST
Abstract
Description
MULTI-PLATFORM FEED-BASED ANTIVIRAL SYSTEM FOR HIGHLY PATHOGENIC AVIAN INFLUENZA (HPAI) WITH EXTENDED APPLICATIONS TO ZOONOTIC DISEASESCROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] This application claims priority to U.S. provisional patent application no.63 / 788,715, filed April 14, 2025 and now pending. This application is incorporated herein by reference.TECHNICAL FIELD
[0002] This application relates to compositions and methods for preventing and treating highly pathogenic avian influenza (HPAI; H5N1, H7N9) in poultry through feed-delivered antiviral agents utilizing genetically engineered diatoms. This application also relates to modular adaptations for USDA Tier 1 zoonotic pathogens including Rift Valley fever virus and Nipah virus, as well as the use of phage adjuvants to treat secondary infections.BACKGROUND
[0003] Highly pathogenic avian influenza (HPAI), including certain H5 and H7 subtype viruses such as H5N1 and H7N9, presents a significant threat to human and animal health. In poultry, HPAI outbreaks can spread rapidly through flocks and are associated with high mortality. This high mortality necessitates extensive culling, movement restrictions, and other control measures that disrupt food production and trade. Public health authorities have also identified certain avian influenza viruses as zoonotic threats because they can infect humans following exposure to infected birds or contaminated environments and, in some cases, cause severe disease with a high fatality rate among reported human cases. These features have driven sustained surveillance and response efforts by international and national health agencies.
[0004] In the United States and globally, HPAI outbreaks have resulted in the loss of large numbers of commercial and backyard birds, supply chain disruption, increased egg and poultry prices, and substantial expenditures for containment, depopulation, disposal,-1- 186631122.1cleaning, disinfection, indemnity, and surveillance. At the same time, the continued geographic spread and host range expansion of HPAI viruses has increased concern regarding persistence in wild birds, transmission into domestic poultry, and spillover into mammals.
[0005] A further basis for demand is the pandemic potential associated with avian influenza A viruses. H5N1 has circulated in birds for decades and caused sporadic severe human infections, while H7N9 emerged as a major concern after causing human infections linked to poultry exposure. Even where sustained human-to-human transmission has not been established, the ability of influenza viruses to evolve through mutation and reassortment supports continued concern that avian strains could acquire properties that increase transmissibility or alter host range. As a result, government agencies, producers, and public health stakeholders continue to seek technologies that improve prevention, reduce viral spread, protect animal welfare and food security, and mitigate zoonotic risk.
[0006] The risk of secondary infections following primary viral increases demand for proactive HPAI treatments still further. Secondary infections, particularly bacterial infections that arise following a primary viral infection, can increase morbidity, mortality, and treatment complexity. In many settings, these infections are caused by opportunistic bacterial pathogens, including strains that are resistant to multiple antibiotics or capable of forming biofilms that reduce antibiotic susceptibility. World Health Organization, “Antimicrobial resistance”; Centers for Disease Control and Prevention, “About Antimicrobial Resistance.”
[0007] Nevertheless, prevention of HPAI and subsequent secondary infections in poultry remains challenging to implement at scale because available vaccination strategies must be integrated with surveillance, outbreak response, and trade-sensitive disease monitoring programs. In particular, some whole-virus or broadly immunogenic vaccine approaches may complicate Differentiating Infected from Vaccinated Animals (DIVA) testing unless paired with suitable marker-vaccine designs and validated companion diagnostics, thereby creating operational challenges for flock surveillance and regulatory control programs. At the same time, injectable vaccination programs can be labor-intensive, timesensitive, and difficult to deploy efficiently across large commercial flocks, especially where repeated handling of birds is required. These practical constraints have sustained demand-2- 186631122.1for vaccination platforms and administration methods that preserve surveillance utility while reducing handling burden and improving scalability in poultry production systems. Because existing biosecurity, surveillance, and outbreak-control measures may not fully prevent introduction or reintroduction of relevant viruses, there remains a strong need for scalable compositions, methods, and platforms for prevention, detection, treatment, and containment of HPAI in poultry populations and at the animal-human interface. Capua I and Marangon S, “Control of avian influenza in poultry,” Emerging Infectious Diseases, 2006.
[0008] Antiviral proteins have emerged as a potential strategy for preventing and treating a variety of human and animal diseases. These proteins generally act by interfering with one or more stages of a viral life cycle, including viral attachment and entry, uncoating, genome replication, transcription, translation, assembly, and release, or by enhancing host innate and adaptive immune responses that suppress viral propagation. Some antiviral proteins are naturally occurring components of host defense, such as interferons and interferon-stimulated gene products, whereas others may be engineered, recombinant, or derived from antibodies, lectins, or other binding proteins that recognize viral or host targets. By engaging specific viral proteins, viral nucleic acids, or host pathways required for infection, antiviral proteins can reduce viral burden, limit tissue damage, and alter disease progression. Samuel, CE, “Antiviral actions of interferons,” Clinical Microbiology Reviews, 2001; Schneider, W.M., Chevillotte, M.D., Rice, C.M., “Interferon-stimulated genes: a complex web of host defenses,” Annual Review of Immunology, 2014; De Clercq, E., Li, G., “Approved antiviral drugs over the past 50 years,” Clinical Microbiology Reviews, 2016.
[0009] Among the best-known antiviral proteins used clinically in human medicine are interferon-alpha and related cytokine-based products, which have been used in the treatment of chronic viral infections such as hepatitis B and hepatitis C. Monoclonal antibodies and antibody-derived proteins have also been developed to neutralize viral pathogens or block viral entry, including products for respiratory syncytial virus and, more recently, emerging viral diseases in outbreak settings. Recombinant antiviral proteins and protein biologies have attracted continuing interest because they can offer high target specificity, can sometimes be adapted rapidly to new pathogens, and may be used alone or in combination with small-molecule antivirals or vaccines. In veterinary and animal health-3- 186631122.1settings, antiviral proteins have likewise been investigated or applied to address viral disease in companion animals, livestock, aquaculture, and other managed animal populations. Interferons, species-specific cytokines, antiviral peptides, and antibody-based biologies have been explored for use against a range of viral pathogens, including infections affecting respiratory, enteric, systemic, and economically important production traits. Although translation to routine field use may be affected by cost, dosing, host specificity, stability, and route-of-administration considerations, the need for additional antiviral countermeasures in both human and animal populations continues to support development of improved antiviral proteins with greater potency, broader applicability, and more practical delivery formats. Pestka, S., “The interferons: 50 years after their discovery, there is much more to learn,” Journal of Biological Chemistry, 2007; “Health Topics: Biologicals,” World Health Organization, 2026.
[0010] Meanwhile, bacteriophages have emerged as a useful strategy for treating secondary infections because lytic phages can selectively infect and kill bacterial pathogens while potentially reducing disruption of commensal microbiota relative to broad-spectrum antibacterial agents. Advances in phage isolation, genomic screening, characterization of host range, and formulation of multi-phage cocktails have further supported the development of phage-based approaches for difficult-to-treat bacterial infections. Interest in phage therapy for secondary infections has also grown because conventional antibiotics may be less effective in the face of antimicrobial resistance. Phages may be administered alone, in cocktails, or in combination with antibiotics, and such combinations have been investigated fortheir potential to broaden bacterial coverage, suppress resistance, and improve bacterial clearance. Kortright KE et al., “Phage Therapy: A Renewed Approach to Combat Antibiotic-Resistant Bacteria,” Cell Host & Microbe, 2019.SUMMARY
[0011] The present technology provides a feed-compatible delivery system of HPAI-targeting antiviral proteins, with extended applications to zoonotic disease including Rift Valley fever virus and Nipah Virus. The present technology leverages HPAI-specific constructs that incorporate NS1 and PB2 truncations. Particular emphasis is placed on the development of DIVA-compliant formulations that avoid trade restrictions. The present -4- 186631122.1technology also leverages a delivery system that centers on diatom-phage coencapsulation, thus permitting the tandem delivery of antiviral proteins to prevent and treat HPIA, as well as phage adjuvants for the prevention and treatment of secondary infections. Modular adaptations for USDA Tier 1 zoonotic pathogens such as Rift Valley fever virus and Nipah Virus are available that utilize alternative expression platforms such as yeast and Bacillus subtilis.BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Fig. 1 shows a schematic illustrating the prevention and treatment of highly pathogenic avian influenza (HPAI) in poultry through the use of diatom-phage coencapsulation that can be added to animal feed.
[0013] Fig. 2 shows the viral load of highly pathogenic avian influenza (HPAI) in control flocks, versus flocks that have been treated with diatom-phage co-encapsulations.DETAILED DESCRIPTION
[0014] Embodiments provided herein relate to compositions that contain one or more proteins derived from natural vectors such diatoms, yeast, or bacteria. Embodiments provided herein also relate to methods in which one or more proteins produced using one or more of these products are used to treat animals such as poultry.
[0015] Embodiments provided herein also relate to methods in which one or more proteins from vectors including diatoms, yeast, or bacteria are used to feed animals such as poultry. The one or more proteins may be derived from a microbe and may be exogenously expressed in the vector.
[0016] In some embodiments, one or more proteins function as one or more of vaccines, antiviral, and functional feeds. The present protein(s) thus may be considered to "treat" an animal when administered (e.g., fed) to the animal in a "therapeutically effective amount," that is, an amount which is effective to act as a vaccine, antiviral, and / or functional feed.-5- 186631122.1
[0017] In some embodiments, one or more proteins comprise one or more genetically engineered proteins. In some embodiments, the genetically engineered protein(s) function as any one or more of vaccines, antivirals, and / or functional feeds.
[0018] In some embodiments, any one or more of the subunits within the genetically engineered protein(s) function as any one or more of vaccines, antivirals, and / or functional feeds.
[0019] In some embodiments, the bioengineered protein comprises one or more adjuvants. In some embodiments, the adjuvant comprises one or more bacteriophages. Terms
[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. The use of the term "including" as well as other forms, such as "include," "includes," and "included," is not limiting. The use of the term "having" as well as other forms, such as "have," "has," and "had," is not limiting. As used in this specification, whether in a transitional phrase or in the body of the claim, the terms "comprise(s)" and "comprising" are to be interpreted as having an open-ended meaning. That is, the above terms are to be interpreted synonymously with the phrases "having at least" or "including at least." For example, when used in the context of a process, the term "comprising" means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound, composition, or device, the term "comprising" means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components.
[0021] As used in the description and the appended claims, the singular forms "a", "an" and "the" are used interchangeably and intended to include the plural forms as well and fall within each meaning, unless the context clearly indicates otherwise. Also, as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the listed items, as well as the lack of combinations when interpreted in the alternative ("or").
[0022] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by-6- 186631122.1increments of 0.1. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term "about". The term "about" also includes the exact value "X" in addition to minor increments of "X" such as "X + 0.1" or "X- 0.1." It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.
[0023] As used herein, "expression", "expressed", or "encodes" refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. Expression may include splicing of the mRNA in a diatom cell or other forms of post-transcriptional modification or post-translational modification.
[0024] As used herein, the term "about" will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, "about" will mean up to plus or minus 10% of the particular term.
[0025] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but not excluding others.
[0026] The terms "administration of' or "administering" an active agent should be understood to mean providing an active agent to the animal in need of treatment in a form that can be introduced into that animal's body in a therapeutically effective amount.
[0027] An animal "in need of treatment" can be any animal (e.g., poultry) that is in need of an improvement to its immune system, so as to increase the animal's chance of survival.
[0028] As used herein, the term "diatom" refers to algae or microalgae belonging to the Phylum Bacillariophyta that contain cell walls (frustules) made up of two overlapping halfshells, the epitheca and hypotheca. Frustules are composed of nanopattemed biogenic or opaline silica. Nonlimiting examples of diatoms include classes Bacillariophyceae (Raphid, pennate diatoms) and Coscinodiscophyceae (centric diatoms). Nonlimiting examples of genera of centric diatoms include Thalassiosira, Cyclotella, Conticribra, and Chaetoceros. Nonlimiting examples of centric diatoms of the genus Thalassiosira include Thalassiosira pseudonana, Thalassiosira weissflogii. Nonlimiting examples of centric diatoms of the genus-7- 186631122.1Conticribra include Conticribra weissflogii. Nonlimiting examples of centric diatoms of the genus Cyclotella include Cyclotella cryptica. Nonlimiting examples of centric diatoms of the genus Chaetoceros include Chaetoceros muelleri. Nonlimiting examples of pennate diatoms of the genus Cylindrotheca include Cylindrotheca fusiformis. Nonlimiting examples of pennate diatoms of the genus Phaeodactylum include Phaeodactylum tricornutum.
[0029] As used herein, the term “bacteriophage” or “phage” refers to a lytic bacteriophage capable of infecting a bacterial host and causing bacterial cell lysis, thereby reducing, eliminating, or otherwise controlling a target bacterial population. Unless otherwise indicated, the term excludes temperate phages used primarily for lysogenic integration. Lytic therapeutic phages may include wild-type phages, adapted phages, selected phages, purified phages, enriched phage preparations, recombinant phages, engineered phages, host-range variants, and biologically active derivatives thereof, provided that such phages retain lytic activity against a target bacterium. In some embodiments, lytic therapeutic phages may be selected from morphologically distinct groups commonly recognized in the art, including tailed phages having contractile, noncontractile, or short tails, as well as other lytic phages suitable for antibacterial applications. Nonlimiting examples of lytic therapeutic phages include phages active against bacterial genera including Escherichia, Salmonella, Staphylococcus, Streptococcus, Pseudomonas, Klebsiella, Acinetobacter, Enterococcus, Clostridium, Campylobacter, and Listeria. Nonlimiting examples include lytic phages active against Escherichia coli, Salmonella enterica, Staphylococcus aureus, Pseudomonas aeruginosa, and Listeria monocytogenes. The term bacteriophage further includes individual lytic phages and phage cocktails comprising two or more distinct lytic phages, as well as formulations comprising one or more such lytic phages together with carriers, stabilizers, excipients, matrices, silica-containing materials, diatom-derived materials, or other delivery components suitable for therapeutic use.
[0030] As used herein, reference to a protein being "from" an organism, such as microalgae, is intended to encompass either a protein that is endogenously expressed in the organism or a protein that is exogenously expressed in the organism. A protein that is exogenously expressed in the organism can derive from the same organism in which the-8- 186631122.1protein is expressed or it can derive from a different organism in which the protein is expressed.
[0031] As used herein, the phrase "adjuvantic phage" refers to any phage that is used to enhance the immune response of an antigen.
[0032] As used herein, the term "microbe" refers to any microorganism, for example, a bacterium such as Salmonella enterica, Bacillus subtilis, Vibrio anguillarum, Pseudomonas aeruginosa, Salmonella typhimurium, Serratia marcescens, Edwardsiella tarda, Proteus mirabilis, Escherichia coli, Shigella flexneri, Listeria monocytogenes, Bartonella bacilliformis, Rhizobium meliloti, Campylobacter jejuni, Campylobacter coli, Helicobacter pylori, Helicobacter felis, Helicobacter hepaticus, Legionella pneumophila, Salmonella marcescens, or Wolinella succinogenes.
[0033] As used herein, the term "variant" may also be referred to herein as analog or variation. A variant refers to any substitution, deletion, or addition to a nucleotide sequence or an amino acid sequence.
[0034] As used herein, the phrase "therapeutically effective amount" refers to a sufficient quantity of the active agents of the present disclosure, in a suitable composition, and in a suitable dosage form to treat or prevent the symptoms, or onset of complications from a given ailment, disease or condition. An example of ailments, disease, or conditions includes an insufficient immune response by an organism to external / environmental stimuli that can result in death of the organism.
[0035] As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and / or bodily fluids of animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.
[0036] As used herein, the phrase "poultry feed" refers to food that poultry eat in their natural habitat. The food can be ground or mixed together. The food can be processed and include additives. The food can include any one or more of algae, algae-derived products, vegetable proteins, and wheat. The food can be ground or mixed together to form pellets of-9- 186631122.1various sizes. The food can be processed and include additives other than the food from the natural habitat.
[0037] As used herein, when referring to a protein being "derived from" a vector, it is meant that the protein is obtained by expressing the vector in a cell.Production of Antiviral Proteins Using Bioengineered Diatoms
[0038] In certain embodiments, diatom species are selected on the basis of known production performance, environmental tolerance, and compatibility with downstream stabilization and delivery systems. By way of example, Phaeodactylum tricornutum is widely recognized in the art as a tractable diatom platform for biotechnological production and has been reported, under selected conditions, to achieve yields of a target product of up to about 2.5 mg / g dry weight. Such reported productivity makes P. tricornutum a suitable organism in embodiments directed to the generation of antiviral proteins, antiviral peptides, antiviral metabolites, or other antiviral products of interest. In some embodiments, the use of P. tricornutum may be advantageous due to one or more attributes including manageable cultivation, established use as a model diatom, and suitability for integration into production systems in which harvested biomass, cellular fractions, secreted products, or extracted actives are further processed for stabilization or formulation.
[0039] In further embodiments, Thalassiosira pseudonana may be employed as an additional or alternative production organism where adaptation to saline or brackish water cultivation conditions is desirable. The art recognizes T. pseudonana as a model centric diatom with utility in controlled cultivation systems, and its suitability for growth in marine or brackish environments may provide flexibility in relation to water sourcing, salinity management, and environmental operating conditions. Accordingly, a production platform may select between P. tricornutum, T. pseudonana, or combinations with one or more additional diatom species based on criteria including product yield, growth rate, salinity tolerance, robustness under culture conditions, and compatibility with recovery or formulation objectives. In certain embodiments, a brackish-water-adapted or brackish-water-compatible cultivation regime may be used to support production efficiency, to reduce freshwater dependence, or to align manufacturing with local environmental resources.-10- 186631122.1
[0040] In certain embodiments, diatoms may be engineered or otherwise configured to produce one or more antiviral proteins, protein fragments, dominant-negative viral polypeptides, decoy proteins, binding proteins, or inhibitory derivatives that target highly pathogenic avian influenza (HPAI) or one or more components of the influenza viral replication machinery. Byway of example, and without limitation, such antiviral products may include modified NS1 -derived polypeptides, including N-terminally truncated forms such as NS1 (A1-73), PB2-derived truncations, and other influenza-targeting constructs selected to interfere with viral replication, host-pathogen interactions, immune evasion, polymerase function, intracellular trafficking, or assembly-related processes. In some embodiments, the expressed antiviral product may be present in diatom biomass, cellular compartments, extracellular fractions, processed lysates, or purified or partially purified preparations, and may subsequently be incorporated into a stabilization system, including silica-associated formulations, feed-compatible compositions, or other delivery formats contemplated herein. The disclosure therefore contemplates the use of engineered diatom platforms as biological producers of HPAI-relevant antiviral agents, while permitting variation in the identity, length, truncation pattern, sequence optimization, expression context, and downstream presentation of the antiviral product according to the intended application.
[0041] In some embodiments, antiviral products obtained from diatom biomass, diatom lysates, diatom-derived fractions, or purified diatom-expressed materials may be stabilized by association with silica-containing structures. The silica may comprise native diatom biosilica, isolated frustules, processed frustule-derived material, synthetic silica, modified silica, or silica-containing composite matrices. Without being bound by theory, silica encapsulation or silica-associated immobilization may reduce loss of antiviral activity by shielding the antiviral product from one or more destabilizing conditions, including moisture fluctuation, oxidation, proteolysis, pH variation, thermal stress, mechanical stress, ultraviolet exposure, or premature release in a target environment. In certain embodiments, the antiviral product may be entrapped within, adsorbed onto, coated by, embedded in, or otherwise associated with a silica-based architecture that functions as a protective carrier and, optionally, as a release-modulating structure. The extent of stabilization may depend on one or more variables including silica porosity, particle morphology, surface chemistry,-11- 186631122.1hydration behavior, loading interaction, and the physicochemical characteristics of the antiviral product.
[0042] In certain implementations, silica encapsulation may be used to preserve the integrity or bioactivity of antiviral proteins, peptides, glycoproteins, nucleoprotein complexes, or small-molecule antiviral actives derived from or produced by the diatom. In some embodiments, the silica-containing material may permit storage in dry or semi-dry formats, incorporation into orally administrable compositions, or delivery in a system requiring resistance to handling and environmental stress prior to use. In further embodiments, silica-associated stabilization may be combined with one or more additional excipients, coatings, or matrix-forming materials selected to further modulate stability, release behavior, carrier compatibility, or manufacturability. Thus, the disclosed systems contemplate that diatom-derived antiviral products may be generated in a first biological platform and thereafter maintained in a more stable and application-suitable form through silica-based encapsulation or silica-mediated protective association.
[0043] In certain embodiments, diatom-based production systems may be implemented at laboratory, pilot, demonstration, or commercial scale, including in closed or semi-closed photobioreactor systems having working volumes of up to about 10,000 L, or more, where appropriate for the selected organism, product, and process configuration. Such scale-up may enable commercially relevant production of diatom biomass, diatom-derived biosilica, recombinant products, antiviral agents, nutritional components, or other biologically derived materials described herein. In some implementations, operation at larger photobioreactor volumes may support improved manufacturing efficiency, batch consistency, process control, containment, and integration with downstream harvesting, separation, stabilization, and formulation workflows. Accordingly, the disclosure contemplates that diatom cultivation need not be limited to bench-scale or experimental formats, and may instead be adapted to industrially relevant phototrophic manufacturing environments, including photobioreactor platforms sized to support pilot and commercial deployment.Production of Antiviral Proteins Using Alternative Bioengineered Platforms-12- 186631122.1
[0044] In certain embodiments, alternative biological production platforms may be used in place of genetically engineered diatoms for generation of the antiviral product, provided that the selected host is capable of producing the desired molecule with suitable yield, activity, and downstream formulation compatibility. By way of example, yeast-based systems, including Saccharomyces cerevisiae, Pichia spp., or Komagataella phaffii, as well as bacterial systems such as Bacillus subtilis, may be employed as recombinant expression hosts for production of antiviral proteins, peptides, subunits, or other biologically active agents contemplated herein. Such alternative hosts may be advantageous in certain implementations due to established fermentation performance, scalability, genetic tractability, secretion capability, reduced dependence on phototrophic cultivation, or compatibility with existing industrial manufacturing infrastructure. In some embodiments, the antiviral product produced in yeast or Bacillus subtilis may be recovered in purified, partially purified, secreted, intracellular, or biomass-associated form and thereafter incorporated into a stabilization system of the kind described herein, including silica encapsulation, silica-associated immobilization, or formulation with silica-containing matrices. Accordingly, the disclosure contemplates that the benefits attributed herein to diatom-based production and silica-mediated stabilization may also be achieved, in whole or in part, using non-diatom host platforms selected according to the intended product, manufacturing environment, and end-use application.Production of Bacteriophage Adjuvants
[0045] In certain embodiments, the disclosure relates to bacteriophage-based compositions for use in the prevention, reduction, or treatment of infections secondary to a viral infection. The secondary infection may comprise a bacterial infection arising during or following a primary viral disease, including infections associated with respiratory tract compromise, epithelial barrier disruption, immune dysregulation, altered microbiota, hospitalization, mechanical ventilation, or other conditions that increase susceptibility to opportunistic bacterial colonization or overgrowth. In some embodiments, the bacteriophage component may comprise one or more lytic bacteriophages selected for activity against one or more bacterial species associated with such secondary infections. The bacteriophage may be used alone or in combination with one or more additional therapeutic agents,-13- 186631122.1including antibiotics, anti-inflammatory agents, supportive care agents, or other anti-infective compositions.
[0046] In certain embodiments, the bacteriophage composition may comprise naturally occurring phages, adapted phages, selected phages, host-range variants, engineered phages, recombinant phages, phage cocktails, or combinations thereof. The phage may be configured to infect bacterial organisms implicated in secondary infections associated with viral disease, including but not limited to Staphylococcus, Streptococcus, Pseudomonas, Klebsiella, Acinetobacter, Escherichia, Haemophilus, or other clinically relevant genera. In some embodiments, the phage may be selected on the basis of one or more properties including lytic activity, host range, resistance suppression potential, biofilm activity, compatibility with other agents, manufacturability, or suitability for administration to a subject in need thereof. In certain implementations, the disclosure contemplates phage preparations that are enriched, purified, concentrated, buffered, stabilized, or otherwise formulated into pharmaceutically or veterinarily acceptable compositions.
[0047] In some embodiments, a bacteriophage useful in the disclosed methods may be generated, amplified, propagated, recovered, or otherwise produced in association with a bacterial host susceptible to infection by the selected phage. In certain embodiments, production may involve a host system, culture regime, recovery workflow, and purification framework selected to provide a phage preparation having desired infectivity, purity, stability, and safety characteristics. The disclosure contemplates that such production may be carried out in liquid culture, semisolid systems, continuous systems, batch systems, or other suitable manufacturing formats, and may optionally include one or more downstream processing operations selected to reduce host-cell impurities, cellular debris, endotoxin burden, unwanted proteins, nucleic acids, or other process-related contaminants. The particular production architecture may vary depending on phage identity, intended route of administration, subject species, regulatory framework, and scale of manufacture.
[0048] In further embodiments, the produced bacteriophage may be converted into a dry or substantially dry form to facilitate storage, shipping, handling, or incorporation into a final dosage format. In some embodiments, the phage may be provided as a lyophilized composition. The lyophilized composition may comprise one or more excipients selected to-14- 186631122.1preserve phage viability or infectivity during freezing, drying, storage, and reconstitution. Such excipients may include, without limitation, sugars, polyols, amino acids, proteins, polymers, salts, buffering agents, surfactants, antioxidants, or combinations thereof, provided that the selected excipient system is compatible with the intended phage and use context. In certain embodiments, the phage may be dried as a bulk material, as aliquoted units, as particles, as a coated preparation, as a matrix-associated preparation, or as part of a multi-component formulation.
[0049] Lyophilization may enhance the practical utility of a bacteriophage composition by reducing hydrolytic degradation, limiting loss of activity during extended storage, improving transport stability, and enabling reconstitution or direct incorporation into a delivery vehicle at or near the point of use. In some embodiments, the lyophilized phage may be formulated for reconstitution in a liquid carrier prior to administration. In other embodiments, the lyophilized phage may be incorporated into a powder, capsule, tablet, sachet, aerosolizable composition, nebulizable composition, intranasal composition, oral composition, or other delivery format appropriate to the target indication. In certain embodiments, the lyophilized preparation may further comprise one or more protective coatings, matrix materials, or packaging configurations selected to reduce exposure to moisture, oxygen, heat, shear, or other destabilizing conditions encountered during storage and distribution.
[0050] In certain implementations, a disclosed phage composition may be administered to a subject having, suspected of having, or at risk of developing a bacterial infection secondary to a viral infection, including a respiratory viral infection. In some embodiments, administration may occur during the primary viral illness, after resolution of the primary viral illness, upon detection of bacterial colonization, upon emergence of clinical signs consistent with secondary bacterial infection, or in relation to another defined treatment window. The disclosed compositions may be designed to reduce bacterial burden, limit progression of secondary infection, reduce reliance on broad-spectrum antibiotics, or improve outcomes in subjects susceptible to post-viral bacterial complications. All such embodiments in which bacteriophage compositions are configured for use against-15- 186631122.1secondary bacterial infections associated with viral disease are contemplated within the scope of the disclosure.Extended Zoonotic Applications
[0051] In certain embodiments, the disclosed production and stabilization platforms may be extended to zoonotic disease applications involving viral targets of agricultural, veterinary, and public health significance. By way of example, recombinant yeast platforms, including Pichia spp. or Komagataella phaffii, may be employed to produce one or more antiviral proteins, binding proteins, decoy constructs, immunogenic fragments, or other biologically active agents directed to Rift Valley fever virus (RVFV) targets, including, without limitation, NSs-associated target proteins or NSs-directed therapeutic constructs. Such yeast-based systems may be selected in certain implementations due to their established scalability, secretion capability, and compatibility with industrial biomanufacturing workflows. In some embodiments, the resulting antiviral product may be recovered in secreted, intracellular, partially purified, or purified form and incorporated into a stabilization or delivery format suitable for veterinary or zoonotic disease countermeasure development. The disclosure therefore contemplates that the expression platforms described herein may be adapted for use against USDA Tier 1 or other high-concern zoonotic pathogens, while varying the nature of the expressed product, the host platform, and the downstream formulation according to the intended use.
[0052] In further embodiments, diatom-based platforms may be used for expression of antiviral products relevant to other zoonotic viruses, including Nipah virus, for example where the target of interest comprises the V protein or a V-protein-associated function, interaction, or antagonism pathway. In some implementations, a diatom host may be selected to generate a recombinant antiviral product, inhibitory construct, binding moiety, or immunologically relevant composition directed to one or more Nipah virus-associated targets, and the resulting material may optionally be stabilized using silica-associated formulations, biomass-based preparations, or other delivery-compatible formats described herein. More broadly, the disclosure contemplates that distinct biological platforms, including yeast and diatoms, may be matched to different zoonotic target classes based on manufacturing, stability, environmental, and end-use considerations, thereby supporting a-16- 186631122.1flexible framework for development of antiviral products directed to multiple viral families without limiting the disclosure to any single host organism, target protein, or disease context.Formulation of Feed-Compatible Diatom-Phage Co-Encapsulation Formulations
[0053] In certain embodiments, the disclosure relates to compositions comprising one or more bacteriophages and one or more diatom-derived materials configured for inclusion in an poultry feed composition. The diatom-derived material may comprise whole diatom biomass, fragmented diatom biomass, purified frustules, partially purified biosilica, modified biosilica, or combinations thereof. The bacteriophage component may comprise one or more lytic bacteriophages selected for activity against one or more bacterial species of interest in poultry production systems. In some embodiments, the bacteriophage and the diatom-derived material are associated within a common particulate, matrix, granule, agglomerate, bead, pellet, coating, or composite structure such that the resulting composition is suitable for admixture with, deposition onto, or incorporation into feed, feed premix, mash, crumble, pellet, granule, or other orally delivered poultry feed format.
[0054] In some embodiments, co-encapsulation refers to an arrangement in which the bacteriophage component and the diatom-derived material are present within, on, around, or otherwise associated with a common carrier architecture. The association may be direct or indirect and may be achieved through physical entrapment, adsorption, absorption, surface association, embedding within a polymeric or nonpolymeric matrix, enrobement, multilayer deposition, or combinations thereof. In certain embodiments, the diatom-derived material may function as a structural scaffold, protective carrier, adsorptive substrate, nutrient-bearing component, flow aid, moisture-management component, or releasemodulating component. In some implementations, the diatom-derived material may be used in native form, processed form, calcined form, chemically treated form, size-reduced form, fractionated form, or surface-functionalized form, provided that the selected form remains compatible with intended phage performance and feed use.
[0055] In various embodiments, the common carrier architecture may further comprise one or more additional encapsulating materials selected to improve manufacturability, storage stability, compatibility with feed processing, or persistence during gastrointestinal-17- 186631122.1transit. Such materials may include edible binders, film-forming agents, polysaccharides, proteins, lipids, waxes, starches, gums, cellulose-derived materials, mineral excipients, buffering agents, cryo- or lyoprotective agents, anti-caking agents, antioxidants, moisture scavengers, or combinations thereof. In some embodiments, the bacteriophage may be associated with the diatom-derived material prior to incorporation of one or more outer protective layers. In other embodiments, the diatom-derived material and one or more optional excipients may define an interior or intermediate region, and the bacteriophage may be present in the same or a different region of the encapsulated structure. In still other embodiments, the composition may comprise a heterogeneous population of particles in which some particles are enriched for diatom-derived material and others are enriched for bacteriophage, while the overall formulation remains a co-formulated poultry feed additive.
[0056] In certain embodiments, the disclosed compositions may be configured to preserve bacteriophage viability to a greater extent than an otherwise comparable unencapsulated bacteriophage preparation during one or more conditions encountered in manufacture, transport, storage, feed handling, or administration. Without limiting mechanism, the diatom-derived material and optional matrix components may reduce exposure of the bacteriophage to moisture fluctuation, oxygen, shear, compression, dust generation, thermal stress, pH variation, enzymatic degradation, or other destabilizing conditions. In some embodiments, the composition may be selected to provide immediate release, delayed release, sustained release, site-preferential release, condition-responsive release, or multi-phase release of bacteriophage after ingestion by a poultry subject. In certain implementations, release behavior may be influenced by one or more variables including particle size, porosity, surface chemistry, layer composition, matrix density, hydration properties, or interactions between the bacteriophage and the diatom-derived material.
[0057] The disclosed compositions may be formulated for administration to any suitable poultry subject, including broilers, layers, breeders, turkeys, ducks, geese, game birds, or other domesticated or managed birds. In some embodiments, the composition may be incorporated into complete feed, top-dressed onto feed, included in a supplement, included in a starter ration, grower ration, finisher ration, breeder ration, or layer ration, or-18- 186631122.1administered in conjunction with another orally acceptable composition. In various embodiments, the feed composition may further comprise one or more vitamins, minerals, amino acids, probiotics, prebiotics, postbiotics, enzymes, organic acids, phytogenic components, immunomodulatory agents, anticaking agents, pellet binders, or other feed additives. The bacteriophage component may be selected to target one or more bacterial genera relevant to poultry health, performance, food safety, or environmental burden, including but not limited to Salmonella, Campylobacter, Escherichia, Clostridium, Enterococcus, Staphylococcus, or combinations thereof.
[0058] In some embodiments, the compositions described herein may be provided as a dry powder, particulate blend, coated particulate, premix, concentrate, suspension for deposition onto feed, or other manufacturable intermediate. In certain embodiments, the physical format may be selected such that the composition remains compatible with commercial feed processing equipment and desired flock-scale administration practices. In some embodiments, the composition may be adapted for use before, during, or after one or more stressors associated with increased bacterial burden or susceptibility, including hatch transition, transport, dietary transition, environmental challenge, vaccination, viral infection, antimicrobial withdrawal, or co-housing changes. The disclosed co-encapsulated systems may thereby support one or more objectives selected from bacterial reduction, microbiome management, bird performance support, flock uniformity, reduced pathogen shedding, reduced contamination pressure, or combinations thereof.
[0059] In certain embodiments, the disclosure further contemplates kits, premixes, and multi-component systems comprising the bacteriophage component, the diatom-derived material, and instructions or specifications for combining the components into a feedcompatible delivery format. In some embodiments, the components may be pre-associated before distribution. In other embodiments, one or more components may be maintained separately until a later manufacturing stage. The relative amounts, identities, and presentation formats of the bacteriophage component and the diatom-derived material may be varied according to the target bird species, target bacterial organism, feed type, production stage, storage profile, or regulatory environment. All such variations in which-19- 186631122.1bacteriophages and diatom-derived materials are jointly configured for oral delivery in poultry feed are contemplated within the scope of the disclosure.
[0060] In certain embodiments, formulations comprising lyophilized bacteriophages in combination with silica-containing diatom materials, including whole diatoms, diatom-derived biosilica, frustule-containing preparations, or silica-enriched diatom fractions, may be configured to provide extended storage stability, including a shelf life of at least about 12 months under suitable storage conditions. Without being bound by theory, the combination of lyophilization and silica-associated stabilization may reduce moisture-driven degradation, preserve phage infectivity, limit activity loss during storage and transport, and improve resistance to environmental stressors encountered during handling and distribution. In some implementations, such formulations may retain functional suitability for intended use following prolonged storage in dry form, thereby supporting manufacture, packaging, stockpiling, field deployment, and commercial distribution. Accordingly, the disclosure contemplates storage-stable phage-diatom formulations in which the structural and moisture-management properties of silica-containing diatom material cooperate with a lyophilized phage presentation to support shelf-life periods of up to about 12 months, or longer, as determined under the relevant stability protocol and storage environment.
[0061] In certain embodiments, the disclosed formulations may be configured to remain compatible with Differentiating Infected from Vaccinated Animals (DIVA) testing strategies, which may be important in veterinary, zoonotic, and agricultural settings where surveillance, outbreak control, and trade-related disease status determinations depend on the ability to distinguish vaccinated populations from naturally infected populations. DIVA-compatible formulations may provide substantial downstream advantages by permitting continued serologic or molecular monitoring following administration, supporting targeted culling or movement-control decisions, reducing diagnostic ambiguity during outbreak investigations, and facilitating regulatory reporting, compartmentalization, and continuity-of-commerce objectives. In some implementations, compatibility with DIVA testing may also improve adoption by commercial producers and animal health authorities because it may enable use of prophylactic or therapeutic interventions without materially compromising established surveillance frameworks or the interpretability of herd- or flock-level testing data.-20- 186631122.1Accordingly, in certain aspects, formulations designed to preserve DIVA discrimination may contribute not only to clinical or production objectives, but also to broader disease management infrastructure, including epidemiologic tracing, vaccination policy implementation, certification programs, export assurance, and coordinated regional or national response measures.Dosing
[0062] In certain embodiments, co-encapsulated diatom-phage formulations may be configured for veterinary administration to poultry populations for use in programs directed to reducing susceptibility to, limiting complications associated with, or otherwise managing avian influenza and related secondary infectious conditions. The formulation may be presented in a form suitable for flock-level or individual administration, including incorporation into feed-compatible, water-compatible, particulate, dry-powder, or unit-dose compositions, and may be selected to support handling, storage, environmental robustness, and compatibility with routine poultry management practices. In some embodiments, the diatom component may function as a silica-containing carrier, protective matrix, nutritional support, or delivery architecture for one or more biologically active phage-containing components, while the overall composition is designed to preserve activity until use and to enable exposure of the subject animal to the intended active material under field-relevant conditions. The disclosure further contemplates administration in a single-dose format or in a multidose regimen, including initial and follow-on administrations, repeated exposures, maintenance administrations, or programmatic use across one or more stages of poultry production, provided that the selected regimen is appropriate to the intended prophylactic or therapeutic objective. Accordingly, the disclosure contemplates prophylactic and therapeutic applications in which co-encapsulated diatom-phage systems are adapted for use in poultry production environments, including hatchery, broiler, layer, breeder, backyard, or other poultry management settings, while permitting variation in presentation format, route of administration, number of administrations, relative timing of administrations, and diseasemanagement objective according to the intended implementation.
[0063] In certain embodiments, administration of a co-encapsulated diatom-phage formulation to a subject, population, environment, material, or other treatment context-21- 186631122.1associated with highly pathogenic avian influenza (HPAI) may be sufficient to reduce viral burden, viral presence, viral detection level, viral shedding, or another measure of HPAI-associated load relative to a suitable control, baseline, pretreatment value, or comparator condition. In some embodiments, the reduction may be at least about 1 %, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or about 100%, inclusive of values therebetween. In certain aspects, such reduction may be determined based on any suitable biological, molecular, virological, or epidemiological readout, including reduction in detectable viral material, reduction in infective burden, reduction in transmission-associated burden, reduction in persistence, or improvement in a related disease-management indicator. The disclosure further contemplates that the observed degree of reduction may vary depending on formulation characteristics, administration format, treatment timing, target setting, measurement methodology, comparator selection, and other implementation-specific variables.
[0064] In certain embodiments, administration of a co-encapsulated diatom-associated formulation may be sufficient to reduce viral burden, viral presence, viral detection level, viral shedding, persistence, transmission-associated burden, or another measure of virus-associated load relative to a suitable control, baseline, pretreatment value, or comparator condition in connection with zoonotic pathogen applications. In some embodiments, the reduction may be at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or about 100%, inclusive of values therebetween. In certain aspects, such reduction may be evaluated using any suitable biological, molecular, virological, or epidemiological readout, including reduction in detectable viral material, reduction in infective burden, reduction in persistence, or improvement in a related disease-management indicator. The disclosure further contemplates application of such formulations in relation to zoonotic pathogens of agricultural, veterinary, or public health interest, including, by way of nonlimiting example, Rift Valley fever virus, Nipah virus, and other viral agents, while recognizing that the degree -22- 186631122.1of observed reduction may vary depending on formulation characteristics, implementation context, comparator selection, and measurement methodology.
[0065] From the foregoing, it will be appreciated that specific embodiments of the invention have been described herein for purposes of illustration, but that various modifications may be made without deviating from the scope of the invention. Accordingly, the invention is not limited except as by the appended claims.-23- 186631122.1
Claims
CLAIMSI / We claim:
1. A poultry feed composition comprising diatoms selected from the group consisting of Phaeodactylum tricornutum, Thalassiosira pseudonana, and Cyclotella cryptica, wherein said diatoms express highly pathogenic avian influenza (HPAI) protein NS1 (A1-73) and are silica-encapsulated.
2. The composition of claim 1, wherein the HPAI antiviral protein further comprises PB2 truncations.
3. The composition of claim 1, further comprising B. subtilis spores expressing PB1-F2 inhibitors.
4. The composition of claim 1, wherein said diatoms express Rift Valley fever virus (RVFV) NSs protein.
5. The composition of claim 1, wherein said diatoms express Nipah Virus V protein.
6. The composition of claim 1, wherein said composition comprises lytic bacteriophages co-encapsulated with the diatoms, wherein the lytic bacteriophages are-24- 186631122.1active against bacterial pathogens selected from the group consisting of Salmonella species and Escherichia coli.
7. The composition of claim 1, wherein the poultry is selected from the group consisting of Gallus gallus domesticus, Meleagris gallopavo, and Anas platyrhynchos domesticus.
8. The composition of claim 1 , wherein the lytic bacteriophages are lyophilized.
9. The composition of claim 1 , wherein the composition has a shelf life of at least 12 months under suitable storage conditions.
10. The composition of claim 1 , wherein the composition is DIVA-com pliant.
11. A feed composition for animals, comprising: genetically engineered microorganisms selected from the group consisting of yeast and Bacillus subtllis, wherein the microorganisms express a zoonotic pathogen antiviral protein; silica-containing encapsulation surrounding the genetically engineered microorganisms; and lytic bacteriophages co-encapsulated with the microorganisms.
12. The composition of claim 11, wherein the yeast is selected from the group consisting of Saccharomyces cerevisiae, Pichia species, and Komagataella phaffii.
13. The composition of claim 11 , wherein the zoonotic pathogen antiviral protein is selected from the group consisting of Rift Valley fever virus NSs protein and Nipah Virus V protein.
14. A method of treating highly pathogenic avian influenza (HPAI) in poultry, comprising: administering to poultry a therapeutically effective amount of a feed composition comprising: genetically engineered diatoms selected from the group consisting of-25- 186631122.1Phaeodactylum tricornutum, Thalassiosira pseudonana, and Cyclotella cryptica, wherein the diatoms express NS1 (A1-73); silica encapsulation surrounding the genetically engineered diatoms; and lytic bacteriophages co-encapsulated with the diatoms.
15. The method of claim 14, wherein the administering reduces viral burden by at least 50% compared to untreated controls.
16. The method of claim 14, wherein the administering reduces viral burden by at least 90% compared to untreated controls.
17. The method of claim 14, wherein the lytic bacteriophages prevent or treat secondary bacterial infections.
18. The method of claim 14, wherein the feed composition is incorporated into complete feed, top-dressed onto feed, or included in a supplement.
19. The method of claim 14, wherein the diatoms are produced in photobioreactor systems.
20. The method of claim 14, wherein the method maintains compatibility with Differentiating Infected from Vaccinated Animals (DIVA) testing strategies.-26- 186631122.1