Porcine astrovirus 4 spike proteins and methods of use

Abstract

Provided are porcine astrovirus 4 (PoAstV4) capsid spike proteins. In some embodiments, the PoAstV4 capsid spike proteins comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO: 1. Also provided are variants thereof comprising 85% or greater identity to 150 or greater consecutive positions of the amino acid sequence set forth in SEQ ID NO: 1, and / or functional fragments thereof. Also provided are immunogenic compositions comprising a PoAstV4 capsid spike protein of the present disclosure, and methods of eliciting an immune response to porcine astrovirus 4 in a mammalian subject (e.g., a porcine animal) using such compositions. Methods of assessing a mammalian subject for a porcine astrovirus 4 (PoAstV4) capsid antigen exposure (e.g., exposure via natural PoAstV4 infection or vaccination with a PoAstV4 capsid antigen) using the PoAstV4 capsid spike proteins of the present disclosure are also provided.

Description

[0001] Atty. Docket: UCSC-413WO (2025-913-1 )

[0002] PORCINE ASTROVIRUS 4 SPIKE PROTEINS AND METHODS OF USE

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 701 ,446, filed September 30, 2024, which application is incorporated herein by reference in its entirety.

[0005] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0006] The contents of the electronic sequence listing (UCSC-413WO_SEQLIST; Size: 9,058 bytes; and Date of Creating: September 26, 2025) is herein incorporated by reference in its entirety.

[0007] INTRODUCTION

[0008] Astroviruses infect avian and mammalian species. Human astrovirus is a leading cause of viral gastroenteritis in children [1 ,2] and in rare cases causes encephalitis [3]. Despite over 90% of adults having antibodies to at least one serotype [4,5], there are no vaccines or treatments available. Astroviruses are nonenveloped, positive-sense, single-stranded RNA viruses that belong to the Astroviridae family [1 ,6,7] and are named after their ‘star-like’ appearance when observed by electron microscopy (EM) [8]. Their genomes vary from 6 to 8 kb and contain three ORFs called ORF1 a, ORF1 b, and ORF2 [1 ,6, 7, 9]. ORF1a and 1 b encode non-structural polyproteins, while ORF2 encodes the viral capsid structural protein that is the target for host antibodies [9]. The capsid core domain forms the icosahedral shell around the virus genome, whereas the capsid spike domain is thought to be responsible for the attachment and entry of the virus [10-12], The capsid spike domain is the primary target of serum antibodies, is the target of all known neutralizing monoclonal antibodies, and is being evaluated as a subunit vaccine antigen [10-15],

[0009] Porcine astrovirus (PoAstV) was first detected by EM in feces of piglets with diarrhea in 1980 [16,17], There are five known genotypes of PoAstV [18,19], which are thought to be more closely related to other species than to each other [20,21], This divergence among genotypes suggests different ancestral origin of PoAstVs. PoAstVs have been detected across the globe including South Africa [7,24] Canada

[0021] , China

[0022] , Colombia [7,23], and Chile

[0024] , and all five genotypes are present in the US with high incidence [17,20], Therefore, PoAstV is thought to have a wide geographical distribution [7] and to be endemic in commercial swine in the US

[0025] , One study of fecal samples from 509 pigs from 255 farms across 19 US states showed PoAstV4 had the highest prevalence

[0020] at 62% (317 / 509), and 64% (326 / 509) had at least one of the PoAstV Atty. Docket: UCSC-413WO (2025-913-1 ) genotypes. Multiple astroviruses have been detected in a single pig at once, [7,17,20,21] which could provide opportunity for recombination to occur and lead to the emergence of new strains [7]-

[0010] Multiple studies have connected PoAstV to a range of disease manifestations, with the virus frequently detected in feces of pigs displaying diarrheal symptoms as well as asymptomatic pigs [7,17,21], PoAstV5 is a cause of clinical enteritis

[0026] , while PoAstV3 has been identified and characterized in the central nervous system of pigs with neurologic signs and nonsuppurative polioencephalomyelitis

[0027] , Additionally, PoAstV4 has been identified in nasal samples from pigs with respiratory disease

[0017] ,

[0011] Recently, researchers investigated cases of bronchitis and / or tracheitis in pigs where PCR results were negative for influenza virus and other known causes of respiratory virus infection in pigs

[0025] . Next generation sequencing revealed reads of PoAstV4, leading to the hypothesis that the respiratory disease in these pigs was associated with PoAstV4. In a retrospective study of cases of tracheitis and / or bronchitis of unknown etiology, RNA in situ hybridization (ISH) was used to detect PoAstV4 RNA in airway epithelium (trachea, bronchi, or bronchioles), revealing PoAstV4 RNA in 73% (85 / 117) of cases

[0025] . This reveals that PoAstV4 is strongly associated with lesions of epitheliotropic viral infection in young pigs with clinical respiratory disease.

[0012] SUMMARY

[0013] Provided are porcine astrovirus 4 (PoAstV4) capsid spike proteins. In some embodiments, the PoAstV4 capsid spike proteins comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO: 1 . Also provided are variants thereof comprising 85% or greater identity to 150 or greater consecutive positions of the amino acid sequence set forth in SEQ ID NO: 1 , and / or functional fragments thereof. Also provided are immunogenic compositions comprising a PoAstV4 capsid spike protein of the present disclosure, and methods of eliciting an immune response to porcine astrovirus 4 in a mammalian subject (e.g., a porcine animal) using such compositions. Methods of assessing a mammalian subject for a porcine astrovirus 4 (PoAstV4) capsid antigen exposure (e.g., exposure via natural PoAstV4 infection or vaccination with a PoAstV4 capsid antigen) using the PoAstV4 capsid spike proteins of the present disclosure are also provided.

[0014] BRIEF DESCRIPTION OF THE FIGURES

[0015] FIG. 1A-1 C: Predicting the boundaries of the PoAstV4 capsid spike domain. (A) Phylogenetic analysis of human and animal astroviruses was performed using MEGA X with a Atty. Docket: UCSC-413WO (2025-913-1 )

[0016] MUSCLE (EMBL-EBI) alignment of full-length ORF2 capsid protein sequences and the maximumlikelihood method and a JTT matrix-based model. The tree with the highest log likelihood (- 38204.70) is shown. The PoAstV4 capsid sequences are labeled with their accession number. (B) Pairwise amino acid sequence identity between PoAstV4 spike and other astrovirus spikes, generated using MUSCLE (EMBL-EBI) alignment. (C) Top: AlphaFold2 prediction of the full- length PoAstV4 capsid, with the predicted spike domain colored in cyan, and the rest of the sequence in gray. Bottom: Design of the recombinant PoAstV4 spike expression construct showing the predicted residues for the PoAstV4 spike domain.

[0017] FIG. 2A-2B: Purification of recombinant PoAstV4 capsid spike protein. (A) SDS-PAGE of affinity purification elution fractions of recombinant PoAstV4 capsid spike protein. Lane 1 , BioRad Precision Plus molecular weight markers; Lane 2, final wash; Lane 3-6, purified PoAstV4 spike elutions. (B) Size exclusion chromatography traces on a Superdex 200 column of PoAstV4 spike in cyan and BioRad gel filtration standards in gray.

[0018] FIG. 3A-3D: Structure of the porcine astrovirus 4 capsid spike protein. (A) PoAstV4 spike dimer, with individual protomers colored in magenta and cyan. (B) Electron density maps contoured at 1 ® around residues 591 -599. (C) Overlay of the AlphaFold2 predicted model (dark gray) with the experimentally-determined crystal structure. (D) Overlay of the AlphaFold3

[0027] predicted model (light gray) with the crystal structure. Figures were generated in PyMOL.

[0019] FIG. 4: Comparison of the PoAstV4 spike to other astrovirus spikes. Structures are shown as cartoons, with one protomer colored rainbow and the other protomer colored gray. PDB codes are noted. TM alignment scores between the PoAstV4 spike and the respective astrovirus spike is reported below its structure. Figures were generated in PyMOL.

[0020] FIG. 5A-5B: Anti-PoAstV4 spike IgG ELISA. (A) ELISA data showing a dose-dependent response towards recombinant PoAstV4 spike antigen in two presumed seropositive (+) pig serum samples. Low-no reactivity was observed in the presumed seronegative (-) pig serum samples from two CDCD piglets. Each sample was measured in triplicate, with the average reported, and error bars represent the standard deviation. (B) A matching negative control ELISA using an ELISA plate not coated with an antigen showed low or no reactivity (<0.197 absorbance at 450 nm), supporting the specificity of the ELISA.

[0021] FIG. 6: PoAstV4 spike IgM and IgG seroconversion. The 5 and 8 days post-challenge (DPC) results are from pigs that were euthanized on those days. Results from 10 to 21 DPC are from the same animals. Data are presented as means with standard error mean error bars. Atty. Docket: UCSC-413WO (2025-913-1 )

[0022] DETAILED DESCRIPTION

[0023] Before the proteins, compositions and methods of the present disclosure are described in greater detail, it is to be understood that the proteins, compositions and methods are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the proteins, compositions and methods will be limited only by the appended claims.

[0024] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the proteins, compositions and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the proteins, compositions and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the proteins, compositions and methods.

[0025] Certain ranges are presented herein with numerical values being preceded by the term “about." The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0026] 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 the proteins, compositions and methods belong. Although any proteins, compositions and methods similar or equivalent to those described herein can also be used in the practice or testing of the proteins, compositions and methods, representative illustrative proteins, compositions and methods are now described.

[0027] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and / or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present proteins, compositions and methods are not entitled to antedate such publication, Atty. Docket: UCSC-413WO (2025-913-1 ) as the date of publication provided may be different from the actual publication date which may need to be independently confirmed.

[0028] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0029] It is appreciated that certain features of the proteins, compositions and methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the proteins, compositions and methods, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present proteins, compositions and methods and are disclosed herein just as if each and every such subcombination was individually and explicitly disclosed herein.

[0030] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0031] PORCINE AST OVIRUS 4 (POASTV4) CAPSID SPIKE PROTEINS

[0032] Porcine astrovirus 4 (PoAstV4) has been recently associated with respiratory disease in pigs. Described herein is the design and production of a PoAstV4 capsid spike protein for use as an antigen in serological assays and vaccination. Structural prediction of the full-length PoAstV4 capsid protein guided the design of a recombinant PoAstV4 capsid spike domain expression plasmid. The recombinant PoAstV4 capsid spike was expressed in Escherichia coli, purified by affinity and size-exclusion chromatography, and its crystal structure was determined at 1.85 A resolution, enabling structural comparisons to other animal and human astrovirus capsid spike structures. The recombinant PoAstV4 capsid spike protein was also used as an antigen for the Atty. Docket: UCSC-413WO (2025-913-1 ) successful development of a serological assay to detect PoAstV4 antibodies, demonstrating that the recombinant PoAstV4 capsid spike retains antigenic epitopes found on the native PoAstV4 capsid.

[0033] Accordingly, aspects of the present disclosure include porcine astrovirus 4 (PoAstV4) capsid spike proteins. The terms “protein”, “polypeptide”, or “peptide” are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The amino acids may include the 20 “standard” genetically encodable amino acids, amino acid analogs, or a combination thereof.

[0034] In certain embodiments, provided is a PoAstV4 capsid spike protein comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 1 , or a variant thereof comprising 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater identity to 150 or greater, 175 or greater, 200 or greater, 225 or greater, or 230 or greater consecutive positions of the amino acid sequence set forth in SEQ ID NO: 1. In some instances, the percent identity is to the entire length of the amino acid sequence of SEQ ID NO:1 . A variant may be a functional fragment comprising 235 or fewer amino acids, but 150 or greater, 175 or greater, 200 or greater, 225 or greater, or 230 or greater consecutive amino acids of the amino acid sequence set forth in SEQ ID NO: 1 , with 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater identity, or 100% identity to the amino acid sequence.

[0035] Table 1 - Amino Acid Sequences of PoAstV4 Capsid Spike Proteins Atty. Docket: UCSC-413WO (2025-913-1 )

[0036] ** M at position 1 = start codon; G at position 2 = linker; AAELA (SEQ ID NO: 5) = linker;

[0037] LVPRGSSA (SEQ ID NO: 6) = protease cleavage site for removal of his-tag; HHHHHHHHHH (SEQ ID NO: 7) = his-tag

[0038] In certain embodiments, a variant PoAstV4 capsid spike protein comprises one or more amino acid substitutions, deletions, or truncations (e.g., a truncation at the N-terminus and / or C- terminus of the domain). Conservative substitutions are shown in Table 2 under the heading of "preferred substitutions." More substantial changes are provided in Table 2 under the heading of "exemplary substitutions," and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into a polypeptide and the products screened for a desired activity, e.g., retained / improved immunogenicity, retained / improved performance in an assay for anti-PoAstV4 capsid spike protein antibodies, and / or the like.

[0039] Table 2 - Amino Acid Substitutions Atty. Docket: UCSC-413WO (2025-913-1 )

[0040] Amino acids may be grouped according to common side-chain properties:

[0041] (1 ) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;

[0042] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu;

[0043] (4) basic: His, Lys, Arg;

[0044] (5) residues that influence chain orientation: Gly, Pro;

[0045] (6) aromatic: Trp, Tyr, Phe.

[0046] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0047] In some embodiments, the PoAstV4 capsid spike protein is fused to a heterologous sequence of amino acids. By “heterologous" in this context is meant the sequence of amino acids Atty. Docket: UCSC-413WO (2025-913-1 ) is not present in a polypeptide comprising the PoAstV4 capsid spike protein in nature. Non-limiting examples of heterologous amino acid sequences include one or more tags (e.g., his-tag and / or the like) or a fragment thereof, a linker or a fragment thereof, one or more peptide adjuvants, among others.

[0048] As used herein, a PoAstV4 capsid spike protein “consists essentially of” the amino acid sequence set forth in SEQ ID NO: 1 when the protein is a fragment in which one, two, three, four, five, six, seven, eight, nine or ten amino acids is independently absent from the N-terminus and / or C-terminus relative to SEQ ID NO: 1 , or when the protein has one, two, three, four, five, six, seven, eight, nine or ten additional amino acids independently at the N-terminus and / or C-terminus relative to SEQ ID NO: 1 .

[0049] The PoAstV4 capsid spike proteins of the present disclosure may be produced using any convenient approach. Where a protein is chemically synthesized, the synthesis may proceed via liquid-phase or solid-phase. Various forms of solid-phase synthesis (SPPS), such as Fmoc and Boc, are available for synthesizing the PoAstV4 capsid spike proteins of the present disclosure. Details of the chemical synthesis are known in the art (e.g., Ganesan A. 2006 Mini Rev. Med Chem. 6:3-10 and Camarero JA et al. 2005 Protein Pept Lett. 12:723-8). Briefly, small insoluble, porous beads are treated with functional units on which peptide chains are built. After repeated cycling of coupling / deprotection, the free N-terminal amine of a solid-phase attached peptide or amino acid is coupled to a single N-protected amino acid unit. This unit is then deprotected, revealing a new N-terminal amine to which a further amino acid may be attached. The peptide remains immobilized on the solid-phase and may undergo a filtration process before being cleaved off.

[0050] PoAstV4 capsid spike proteins of the present disclosure may also be produced by recombinant methods. Where the PoAstV4 capsid spike proteins are produced using recombinant techniques, the PoAstV4 capsid spike proteins may be produced as an intracellular protein or as a secreted protein, using any suitable construct and any suitable host cell, which can be a prokaryotic or eukaryotic cell, such as a bacterial (e.g., E. coli, including but not limited to E. coli strains engineered for incorporation of non-natural amino acids) or a yeast host cell, respectively. Other examples of eukaryotic cells that may be used as host cells include insect cells, mammalian cells, and / or plant cells. Where mammalian host cells are used, the cells may include one or more of the following: human cells (e.g. HeLa, 293, H9, and Jurkat cells); mouse cells (e.g., X3, NIH3T3, pancreatic ductal adenocarcinoma 2.1 , L cells, and C127 cells); primate cells (e.g. Cos 1 , Cos 7, and CV1 ) and hamster cells (e.g., Chinese hamster ovary (CHO) cells). Atty. Docket: UCSC-413WO (2025-913-1 )

[0051] Accordingly, a PoAstV4 capsid spike protein of the present disclosure may be a recombinant PoAstV4 capsid spike protein. Such a PoAstV4 capsid spike protein may be recombinant by virtue of having been expressed in a recombinant host cell. In the context of the present disclosure, the terms “recombinant host cells,” “host cells,” “cell lines,” “cell cultures,” and other such terms refer to cells which have been used as recipients for a recombinant vector or other transferred recombinant DNA encoding the PoAstV4 capsid spike protein (e.g., a vector or other transferred DNA comprising a domain encoding the PoAstV4 capsid spike protein operably linked to a heterologous promoter), and include the progeny of the cell which has been transformed or transfected. According to some embodiments, the recombinant host cell that expresses the PoAstV4 capsid spike protein is a bacterial host cell, e.g. an E. coli host cell. As will be appreciated, a PoAstV4 capsid spike protein expressed in a recombinant bacterial host cell (e.g., an E. coli host cell) will exhibit a glycosylation pattern that differs from a glycosylation pattern of naturally occurring PoAstV4 capsid spike proteins, e.g., PoAstV4 capsid spike proteins expressed in mammalian cells. For example, the recombinant PoAstV4 capsid spike protein may exhibit no glycosylation or a lesser extent of glycosylation as compared to naturally occurring PoAstV4 capsid spike proteins expressed by a porcine animal infected with PoAstV4. In some embodiments, a recombinant PoAstV4 capsid spike protein is expressed in a host cell (e.g., an E. Coli host cell) lacking glycosyltransferases.

[0052] A wide range of host-vector systems suitable for the expression of the PoAstV4 capsid spike proteins may be employed according to standard procedures known in the art. See, e.g., Sambrook et al. 1989 Current Protocols in Molecular Biology Cold Spring Harbor Press, New York and Ausubel et al. 1995 Current Protocols in Molecular Biology, Eds. Wiley and Sons. Methods for introduction of genetic material into host cells include, for example, transformation, electroporation, conjugation, calcium phosphate methods and the like. The method for transfer can be selected so as to provide for stable expression of the PoAstV4 capsid spike proteinencoding nucleic acid. The PoAstV4 capsid spike protein-encoding nucleic acid can be provided as an inheritable episomal element (e.g., a plasmid) or can be genomically integrated. A variety of appropriate vectors for use in production of a polypeptide of interest are available commercially.

[0053] Vectors can provide for extrachromosomal maintenance in a host cell or can provide for integration into the host cell genome. The expression vector provides transcriptional and translational regulatory sequences, and may provide for inducible or constitutive expression, where the coding region is operably linked under the transcriptional control of the transcriptional initiation region, and a transcriptional and translational termination region. In general, the Atty. Docket: UCSC-413WO (2025-913-1 ) transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. Promoters can be either constitutive or inducible, and can be a strong constitutive promoter (e.g., T7, and the like). Expression constructs generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding proteins of interest. A selectable marker operative in the expression host may be present to facilitate selection of cells containing the vector. In addition, the expression construct may include additional elements. For example, the expression vector may have one or two replication systems, thus allowing it to be maintained in organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification. In addition the expression construct may contain a selectable marker gene to allow the selection of transformed host cells. Selectable genes are well known in the art and will vary with the host cell used.

[0054] Isolation and purification of the PoAstV4 capsid spike proteins can be accomplished according to methods known in the art. For example, a PoAstV4 capsid spike protein can be isolated from a lysate of cells genetically modified to express the protein constitutively and / or upon induction, or from a synthetic reaction mixture, by immunoaffinity purification, which generally involves contacting the sample with an anti-PoAstV4 capsid spike protein antibody (or in the case of a tagged polypeptide, an anti-tag antibody), washing to remove non-specifically bound material, and eluting the specifically bound polypeptide. The isolated PoAstV4 capsid spike proteins can be further purified by dialysis and other methods normally employed in protein purification methods. In one embodiment, the PoAstV4 capsid spike proteins may be isolated using metal chelate chromatography methods. PoAstV4 capsid spike proteins of the present disclosure may contain modifications (e.g., affinity tags or the like) to facilitate isolation.

[0055] The PoAstV4 capsid spike proteins may be prepared in substantially pure or isolated form (e.g., free from other polypeptides). The PoAstV4 capsid spike proteins (or complexes thereof) can be present in a composition that is enriched for the PoAstV4 capsid spike proteins relative to other components that may be present (e.g., other polypeptides or other host cell components). Purified PoAstV4 capsid spike proteins may be provided such that the PoAstV4 capsid spike proteins are present in a composition that is substantially free of other expressed proteins, e.g., less than 98%, less than 95%, less than 90%, less than 80%, less than 60%, or less than 50%, of the composition is made up of other expressed proteins. Atty. Docket: UCSC-413WO (2025-913-1 )

[0056] NUCLEIC ACIDS, EXPRESSION CONSTRUCTS AND CELLS

[0057] Nucleic Acids and Expression Constructs

[0058] Aspects of the present disclosure further include nucleic acids and expression constructs. For example, provided are nucleic acids encoding any of the PoAstV4 capsid spike proteins of the present disclosure. The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to describe a polymer of any length composed of nucleotides, e.g., deoxyribonucleotides, and may be produced enzymatically or synthetically. The term “nucleotide” is intended to include those moieties that contain not only the naturally occurring purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated riboses or other heterocycles. Modified nucleosides or nucleotides also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen atoms or aliphatic groups, or are functionalized as ethers, amines, or the like.

[0059] Because of the knowledge of the codons corresponding to the various amino acids, availability of an amino acid sequence of a PoAstV4 capsid spike protein of interest provides a description of all the polynucleotides capable of encoding a PoAstV4 capsid spike protein of interest. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons allows an extremely large number of nucleic acids to be made, all of which encode the polypeptides and domains disclosed herein. Thus, having identified a particular amino acid sequence, those of ordinary skill in the art could make any number of different nucleic acids by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the polypeptide of interest. In this regard, the present disclosure specifically contemplates each and every possible variation of polynucleotides that could be made by selecting combinations based upon the possible codon choices, and all such variations are to be considered specifically disclosed for any polypeptide disclosed herein, including the amino acid sequences of the PoAstV4 capsid spike proteins set forth in Table 1. Non-limiting examples of nucleotide sequences encoding the PoAstV4 capsid spike proteins set forth in Table 1 are provided in Table 3 below.

[0060] Table 3 - Nucleotide Sequences Encoding PoAstV4 Capsid Spike Proteins Atty. Docket: UCSC-413WO (2025-913-1) Atty. Docket: UCSC-413WO (2025-913-1 )

[0061] The nucleotide sequences of the nucleic acids of the present disclosure may be codon- optimized. “Codon-optimized” refers to changes in the codons of the polynucleotide encoding a polypeptide to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, a nucleic acid of the present disclosure encoding a polypeptide may be codon- optimized for optimal production from the host organism selected for expression, e.g., bacterial cells, such as E. coli cells.

[0062] Also provided are expression constructs comprising any of the nucleic acids of the present disclosure. As used herein, an “expression construct” is a circular or linear polynucleotide (a polymer composed of naturally occurring and / or non-naturally occurring nucleotides) comprising a region that encodes a PoAstV4 capsid spike protein of the present disclosure, operably linked to a suitable promoter, e.g., a constitutive or inducible promoter. In some embodiments, expression of the PoAstV4 capsid spike protein is under the control of one or more exogenous (including heterologous) regulatory elements, e.g., promoter, enhancer, etc., present in the expression construct. By “heterologous” in this context is meant the regulatory element (e.g., promoter) to which the nucleic acid is operably linked is a regulatory element which is not operably linked to the nucleic acid in nature.

[0063] In some embodiments, expression of the PoAstV4 capsid spike protein may be controlled by one or more endogenous regulatory elements, e.g., promoter, enhancer, etc., at or near a genomic locus into which the expression construct is inserted or knocked in.

[0064] The expression constructs (e.g., vectors) can be suitable for replication and integration in prokaryotes, eukaryotes, or both. The expression constructs may contain functionally appropriately oriented transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid encoding the first subunit, the Atty. Docket: UCSC-413WO (2025-913-1 ) second subunit, or both. The expression constructs optionally contain generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in both eukaryotes and prokaryotes, e.g., as found in shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems.

[0065] To obtain high levels of expression of a cloned nucleic acid it is common to construct expression constructs which typically contain a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription / translation terminator, each in functional orientation to each other and to the protein-encoding sequence. Examples of regulatory regions suitable for this purpose in E. coli are the promoter and operator region of the E. coli tryptophan biosynthetic pathway, the leftward promoter of phage lambda (PL), and the L-arabinose (araBAD) operon. The inclusion of selection markers in DNA vectors transformed in E. coli is also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol. Expression systems for expressing the polypeptide are available using, for example, E. coli, Bacillus sp. and Salmonella. E. co / / systems may also be used. Transducing cells with nucleic acids (e.g., expression constructs) can involve, for example, incubating lipidic microparticles containing nucleic acids with cells or incubating viral vectors containing nucleic acids with cells within the host range of the vector.

[0066] In certain embodiments, upon delivery of an expression construct to cells, one or more of the expression constructs are episomal (e.g., extra-chromosomal), where by “episome” or “episomal” is meant a polynucleotide that replicates independently of the cell’s chromosomal DNA. A non-limiting example of an episome that may be employed is a plasmid.

[0067] According to some embodiments, upon delivery of an expression construct to cells, the expression construct integrates (e.g., by insertion or knock-in) into the genome of the cell. In certain embodiments, the expression construct is adapted for site-specific integration into the genome. For example, an expression construct may be adapted for site-specific integration into the genome, where the site-specific integration inactivates a gene within the genome of the cell. Functional integration of an expression construct may be achieved through various means, including through the use of integrating vectors, including viral and non-viral vectors. In some instances, a retroviral vector, e.g., a lentiviral vector, may be employed. In some instances, a non- retroviral integrating vector may be employed. An integrating vector may be contacted with the cells in a suitable transduction medium, at a suitable concentration (or multiplicity of infection), and for a suitable time for the vector to infect the target cells, facilitating functional integration of the expression construct. Non-limiting examples of useful viral vectors include retroviral vectors, Atty. Docket: UCSC-413WO (2025-913-1 ) lentiviral vectors, adenoviral (Ad) vectors, adeno-associated virus (AAV) vectors, hybrid Ad-AAV vector systems, and the like.

[0068] Cells and Compositions

[0069] Aspects of the present disclosure further include cells comprising a nucleic acid of the present disclosure, as well as cells comprising an expression construct of the present disclosure. In certain embodiments, the cells are prokaryotic cells (e.g., bacteria), a yeast cells, insect (e.g., drosophila) cells, amphibian (e.g., frog, e.g., Xenopus) cells, plant cells, etc. According to some embodiments, the cells are mammalian cells. Mammalian cells of interest include human cells, rodent cells, and the like.

[0070] Approaches for introducing the nucleic acid or expression construct into cells of interest are known and may include contacting a population of cells with the nucleic acid or expression construct under conditions in which the nucleic acid or expression construct is delivered to cells of the population of cells. The contacting step may comprise contacting the population of cells with the nucleic acid or expression construct, e.g., by combining the cells and the nucleic acid or expression construct in a single mixture under conditions suitable for delivery (e.g., transfection, transduction, etc.) of the nucleic acid or expression construct into cells of the population of cells.

[0071] A variety of suitable approaches and conditions for the delivery of nucleic acids and expression constructs to cells are known. According to some embodiments, delivery is carried out by microinjection, transfection, lipofection, heat-shock, electroporation, transduction, gene gun, DEAE-dextran-mediated transfer, and / or the like.

[0072] IMMUNOGENIC COMPOSITIONS

[0073] Aspects of the present disclosure further include immunogenic compositions. The immunogenic compositions comprise a PoAstV4 capsid spike protein of the present disclosure. Such compositions are suitable for administration to a mammalian subject, e.g., a porcine animal.

[0074] The immunogenic compositions may comprise at least one adjuvant. In some embodiments, the compositions comprise two adjuvants. The purpose of the adjuvant(s) is to increase or stimulate the immune response in the subject. Adjuvants that may be employed include, but are not limited to, aluminum phosphate, aluminum hydroxide, and / or CpG amongst others. Any suitable CpG known to those skilled in the art can be used in view of the present disclosure. Examples of such CpG oligonucleotides include, but are not limited to CpG2006 (also known as CpG 7909), CpG 1018, CpG2395, CpG2216 or CpG2336. A CpG can be lipidated using methods known in the art in view of the present disclosure. In some embodiments, 3' terminus or Atty. Docket: UCSC-413WO (2025-913-1 )

[0075] 5’ terminus of a CpG oligonucleotide is covalently linked to a cholesterol molecule through a phosphate bond, optionally via a PEG linker. Thus, in some instances, the adjuvant is CpG-chol (cholesterol).

[0076] The immunogenic compositions may be administered to the subject by any appropriate route of administration. In some instances, a composition is suitable for administration by intramuscular, subcutaneous, intranasal or oral administration. In addition, immunogenic compositions may be incorporated into sustained release matrices such as biodegradable polymers, the polymers being implanted in the vicinity of, or in close proximity to, where delivery is desired.

[0077] METHODS OF USE

[0078] Aspects of the present disclosure further include methods of eliciting an immune response to porcine astrovirus 4 in a mammalian subject (e.g., a porcine animal), the method comprising administering an immunogenic composition of the present disclosure to the mammalian subject in an amount effective to elicit an immune response to porcine astrovirus 4 in the mammalian subject.

[0079] The phrase “an amount effective to elicit an immune response” means that there is a detectable difference between an immune response indicator measured before and after administration of a particular antigen preparation. Immune response indicators include but are not limited to: antibody titer or specificity, as detected by an assay such as enzyme-linked immunosorbent assay (ELISA), bactericidal assay, flow cytometry, immunoprecipitation, Ouchterlony immunodiffusion; binding detection assays of, for example, spot, Western blot or antigen arrays; cytotoxicity assays, etc.

[0080] In some embodiments, the methods of eliciting an immune response of the present disclosure comprise administering the immunogenic composition intramuscularly, subcutaneously, intranasally or orally.

[0081] METHODS OF ASSESSING MAMMALIAN SUBJECTS FOR A PORCINE ASTROVIRUS 4 (POASTV4) CAPSID ANTIGEN EXPOSURE

[0082] Aspects of the present disclosure further include methods of assessing a mammalian subject for a porcine astrovirus 4 (PoAstV4) capsid antigen exposure. Such methods may include contacting a substrate having attached to the surface thereof PoAstV4 capsid spike proteins of the present disclosure with a biological sample obtained from the mammalian subject, e.g., a Atty. Docket: UCSC-413WO (2025-913-1 ) porcine animal. The methods further include assessing for binding of antibodies to the PoAstV4 capsid spike proteins.

[0083] In certain embodiments, the capsid antigen exposure is via natural infection with PoAstV4. Such methods find use, e.g., for determining whether the mammalian subject has a PoAstV4 infection. According to other embodiments, the capsid antigen exposure is via vaccination with a PoAstV4 capsid antigen or a vaccine comprising a nucleic acid encoding a PoAstV4 capsid antigen, e.g., a PoAstV4 spike protein antigen or a vaccine comprising a nucleic acid encoding a PoAstV4 spike protein antigen. Such methods find use, e.g., for assessing the effectiveness of vaccination of the mammalian subject with a PoAstV4 capsid antigen or a vaccine comprising a nucleic acid encoding a PoAstV4 capsid antigen. In certain embodiments, a vaccine comprising a nucleic acid encoding a PoAstV4 capsid antigen (e.g., a PoAstV4 spike protein antigen) is an mRNA, DNA, or viral vector vaccine. In some instances, the biological sample is whole blood obtained from the mammalian subject or a fraction thereof, optionally wherein the biological sample is serum.

[0084] The assessing may comprise contacting antibodies bound to the PoAstV4 capsid spike proteins, if present in the biological sample, with a secondary antibody specific for IgG of the type produced by the mammalian subject (e.g., anti-swine IgG secondary antibody when the mammalian subject is a porcine animal), removing unbound secondary antibody, and assessing for the presence of secondary antibody bound to IgG of the type produced by the mammalian subject.

[0085] In some instances, the secondary antibody comprises a detectable label, and assessing for the presence of the secondary antibody comprises assessing for the detectable label. In certain embodiments, the secondary antibody is linked to an enzyme, and the assessing is by enzyme-linked immunosorbent assay (ELISA). Non-limiting examples of enzymes that may be employed include horseradish peroxidase (HRP), alkaline phosphatase (AP), p-galactosidase, acetylcholinesterase, and catalase.

[0086] The following examples are offered by way of illustration and not by way of limitation.

[0087] EXPERIMENTAL

[0088] To further understand PoAstV4 infection and prevalence, described in the examples below is the generation of a recombinant PoAstV4 capsid spike protein for structural and antigenic studies. The crystal structure of the PoAstV4 capsid spike was determined at 1 .85 A resolution, validating its three-dimensional folding and enabling structural comparisons to the capsid spikes Atty. Docket: UCSC-413WO (2025-913-1 ) from other astroviruses. The PoAstV4 capsid spike protein was also used as an antigen for the development of an enzyme-linked immunosorbent assay, demonstrating that the recombinant PoAstV4 capsid spike retains antigenic epitopes found on the native PoAstV4 capsid. The examples herein provide the basis for a PoAstV4 capsid spike subunit vaccine to prevent PoAstV4 disease.

[0089] Example 1 - Delineation and production of recombinant PoAstV4 capsid spike domain

[0090] The protein sequence of PoAstV4 capsid spike has low sequence identity to the capsid spikes of other astrovirus species (FIG. 1 A,B). Therefore, we could not determine the spike domain boundaries based on sequence alignment alone, and instead determined the spike domain boundaries by predicting the structure of the full PoAstV4 capsid protein using AlphaFold2

[0032] , We designed a PoAstV4 spike construct containing residues 420 to 655 (SEQ ID NO: 1 ) from the full-length capsid protein of PoAstV4 (FIG. 1C). The recombinant PoAstV4 spike protein expressed in E. coli resulted in high yields of soluble protein (>12 mg / Liter E. coli). The calculated molecular weight of the PoAstV4 spike is 27.8 kD (+1.6 kD cleavable histidine affinity tag), and this was confirmed using SDS-PAGE analysis of purified PoAstV4 spike (FIG. 2A). Size exclusion chromatography showed purified PoAstV4 spike protein eluted at the approximate size of a homodimer (FIG. 2B), which suggests that the recombinant protein is folded correctly.

[0091] Example 2 - Crystal structure of PoAstV4 soike and similarity to other astroviruses

[0092] To further substantiate the folding of the recombinant PoAstV4 spike, we used X-ray crystallography to determine the structure of the PoAstV4 spike to 1 .85 A resolution (FIG. 3, Table 1 ). A trimmed AlphaFold2 model was used for molecular replacement. The high R factors are likely due to the highly anisotropic data, however the electron density maps are clear (FIG. 3B). Structural alignment of the crystal structure of the PoAstV4 spike dimer with an AlphaFold2 predicted model using TM-align

[0037] reveals an RMSD of 0.72 A and a TM alignment score of 0.886 (FIG. 3C), whereas an alignment with an AlphaFold3

[0030] predicted model reveals an RMSD of 0.49 A and a TM alignment score of 0.992 (FIG. 3D), demonstrating the accuracy of these structure prediction programs and improvements from AlphaFold2 to AlphaFold3.

[0093] The PoAstV4 capsid spike crystal structure reveals a homodimeric protein formed of mainly beta-strands (FIG. 3), similar to other animal and human astrovirus capsid spikes, and consistent with in-solution studies with size-exclusion chromatography (FIG. 2B). The structure confirms that the predicted PoAstV4 spike residues 420 to 655 form the structural domain. Atty. Docket: UCSC-413WO (2025-913-1 )

[0094] Interface analysis using the PDBePISA server reveals 4660 A buried at the dimer interface, with 64-65 interacting residues in each chain.

[0095] Compared to other experimentally solved astrovirus spike structures [13,38-41 ] PoAstV4 spike has the highest structural similarity to murine astrovirus (MuAstV) (FIG. 4), as seen visually with a notable beta sheet cleft at the top of both spikes and by their high TM alignment score

[0037] of 0.809 (FIG. 4). This TM alignment score between PoAstV4 and MuAstV spike is the highest compared to other astrovirus spikes assessed (FIG. 4). At first, it may be surprising that the PoAstVI spike is not the most structurally similar to PoAstV4 spike, however phylogenetic analyses suggests that PoAstV4 is more evolutionarily related to MuAstV (FIG. 1A).

[0096] Table 1 - Data collection and refinement statistics. Atty. Docket: UCSC-413WO (2025-913-1 )

[0097] Example 3 - Development of an ELISA to detect PoAstV4 antibodies in pig sera

[0098] To determine if the recombinant PoAstV4 spike can be used as an antigen to detect PoAstV antibodies, we developed an enzyme-linked immunosorbent assay (ELISA). Serum samples were collected from pigs in Iowa with suspected exposure during an outbreak of PoAstV4, making them presumed to be seropositive. A strong dose-dependent IgG reactivity towards the PoAstV4 spike antigen was observed in sera from presumed seropositive PoAstV4 adult pigs (FIG. 5A). Sera from presumed seronegative cesarean-derived, colostrum-deprived (CDCD) pigs, which do not receive maternal antibodies, exhibited minimal reactivity (450nm absorbance values <0.187) and all sera had low signal in the negative control plate not coated with antigen (< 0.197 nm at 450 nm) (FIG. 5B), validating the ELISA specificity. Atty. Docket: UCSC-413WO (2025-913-1 )

[0099] 4 - PoAstV4 IqM and IgG seroconversion

[0100] PoAstV4 IgM and IgG seroconversion were evaluated using a PoAstV4 capsid spike ELISA (20). Antibodies against IgM became detectable at 8 DPC with a peak of response at 14 DPC and contraction thereafter. IgG antibodies against PoAstV4 were first detectable at 10 DPC with a steady increase in quantity up to the end of the study at 21 DPC. All sera were negative for anti-PoAstV4 IgM and IgG on 0 DPC (FIG. 6).

[0101] Discussion

[0102] PoAstVs represent a significant concern within swine populations, posing health risks to individual pigs in addition to financial burdens on swine farms. While PoAstVs are traditionally associated with diarrheal disease, PoAstV4 has recently been associated with respiratory disease in young pigs

[0027] . Here, we investigated the structural and antigenic features of the PoAstV4 capsid spike.

[0103] Phylogenetic analyses highlight the divergence of PoAstV lineages, indicating likely distinct ancestral origins. In particular, the PoAstV4 capsid exhibits sequence similarity with the murine astrovirus capsid more than with other known PoAstV capsids, with an amino acid sequence identity of -30% and clustering together in phylogenetic trees. The structural studies here further support this evolutionary relationship, with the PoAstV4 spike sharing structural similarities and a higher alignment score with the murine astrovirus spike structure than with the PoAstVI spike structure. Future structural studies on other PoAstV spikes may further illuminate their evolutionary relationship with other mammalian astroviruses.

[0104] In human astroviruses, the capsid spike is the primary target of host antibodies [10-15]. As such, recombinant human astrovirus capsid spikes have been used as antigens in immunoassays for evaluating seroprevalence in human populations [4], To determine if the PoAstV4 spike is also targeted by host antibodies, we developed an ELISA to detect PoAstV spike-reactive IgG in pig sera. We observed dose-dependent reactivity for the PoAstV spike antigen by two presumed seropositive samples, suggesting previous infection by PoAstV4.

[0105] Materials and Methods

[0106] Phylogenetic analysis of astroviruses with MEGA X

[0107] Amino acid sequences of the full length astrovirus capsid ORF2 were aligned using MUSCLE (EMBL-EBI)

[0028] , The following ORF2 sequences were used: human astrovirus 1 , GenBank #AAC34717.1 ; human astrovirus 2, UniProt #Q82446.1 ; human astrovirus 3, UniProt #Q9WFZ0.1 ; human astrovirus 4, UniProt #Q3ZN05.1 ; human astrovirus 5, UniProt #Q4TWH7.1 ; Atty. Docket: UCSC-413WO (2025-913-1 ) human astrovirus 6, UniProt #Q67815.1 ; human astrovirus 7, UniProt #Q96818.2; human astrovirus 8, UniProt #Q9IFX1 .2; human astrovirus VA1 , GenBank #YP_003090288.1 ; human astrovirus VA2, NCBI #ACX83591.2; human astrovirus VA3, NCBI #YP_006905860.1 ; human astrovirus VA4, NCBI #YP_006905857.1 ; human astrovirus VA5; human astrovirus MLB1 , NCBI #YP_002290968.1 ; human astrovirus MLB2, GenBank #YP_004934010.1 ; human astrovirus MLB3, GenBank #YP 006905854.1 ; murine astrovirus, GenBank #QBQ83077.1 ; turkey astrovirus 1 , UniProt #Q9JH68; turkey astrovirus 2, UniProt #Q9Q3G5; turkey astrovirus 3, GenBank # AAV37187.1 ; porcine astrovirus 1 , GenBank #UZG75482.1 porcine astrovirus 2, GenBank #AZB49326.1 ; porcine astrovirus 3, NCBI #YP_007003832.1 ; porcine astrovirus 4, GenBank #AMN16564; porcine astrovirus 4, GenBank #AMN16570.1 , porcine astrovirus 4, GenBank #QDZ38040.1 ; porcine astrovirus 4, GenBank #PP806170.1 ; porcine astrovirus 5, GenBank #UXD79156.1 ; mink astrovirus (mamastrovirus 10), GenBank #AAO32083.; ovine astrovirus (mamastrovirus 13), GenBank #QDP38704.1. The evolutionary history of astrovirus species was inferred by using the maximum-likelihood method and a Jones-Taylor-Thornton (JTT) matrix-based model

[0029] . The tree shown has the highest log likelihood (-38204.70). The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying neighbor-joining and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The tree was rooted using the turkey astroviruses as an outgroup. There were 30 amino acid sequences in this dataset and 1008 sites. Evolutionary analyses were conducted in MEGA X

[0030] .

[0108] Comparison of pairwise identity between PoAstV4 spike and HAstV spikes

[0109] MUSCLE (EMBL-EBI) alignment was used to perform pairwise identity between astrovirus spike sequences. The same accession numbers utilized in the phylogenetic analysis section were used for the human astrovirus spikes, murine astrovirus spike, and for the porcine astrovirus spikes. AlphaFold3

[0031] was used to predict the spike domain from the full-length capsid for PoAstV2, PoAstV3, and PoAstV5. The subsequent amino acids corresponding to spike domains in the ORF2 capsid protein were used: PoAstVI , 420-669; PoAstV2, 409-648; PoAstV3, 480-736; PoAstV4 (GenBank #PP806170.1 ), 420-655; PoAstV5, 470-689; HAstVI , 431-644; HAstV2,429- 644; HAstV3, 432-645; HAstV4, 430-644; HAstV5, 429-641 ; HAstV6, 430-642; HAstV7, 431- 644; HAstV8, 490-705; HAstV-MLB1 , 420-646; HAstV-MLB2, 417-643; HAstV-MLB3, 417-643; Atty. Docket: UCSC-413WO (2025-913-1 )

[0110] HAstV-VA1 , 408-682; HAstV-VA2, 404-688; HAstV-VA3, 388-691 ; HAstV-VA4, 408-685;

[0111] HAstV-VA5, 406-678, MuAstV, 428-676.

[0112] Design and production of recombinant PoAstV4 capsid spike protein

[0113] The full-length PoAstV4 capsid protein sequence derived from an infected pig in 2022 was retrieved from GenBank (GenBank #PP806170.1 ) and its structure was predicted using the AlphaFold2 server ColabFold

[0032] , The predicted structure was used to delineate the termini of the PoAstV4 capsid spike domain as amino acids (420 - 655). An E. coli-codon-optimized synthetic gene encoding the PoAstV4 spike was cloned into the pET52b expression plasmid inframe with an N-terminal methionine and a C-terminal thrombin protease cleavage site and a 10- histidine tag by GenScript. The plasmid was transformed into T7 Express E. coli (New England Biolabs) and grown at 37°C in Luria Broth with 50 pg / mL ampicillin. Expression of recombinant PoAstV4 capsid spike was induced with 1 mM isopropyl-p-d-thiogalactopyranoside (IPTG), and the cultures were shaken overnight at 18°C. E. coli cultures were centrifuged and pellets were resuspended in Buffer A (20 mM Tris-CI pH 8.0, 500 mM NaCI, 20 mM imidazole) containing 1 X EDTA-free protease inhibitors (Millipore), benzonase (Millipore), and 2.5 mM MgCI2. E. coli was lysed with ultrasonication and the lysate was centrifuged at 40,000 g for 30 min. The supernatant was 0.22pm-filtered and then incubated with TALON beads prewashed with Buffer A for 1 hr at 4°C with rotation. The beads were washed 12 times with Buffer A, and recombinant PoAstV4 spike was eluted in Buffer B (20 mM Tris-CI pH 8.0, 500 mM NaCI, 500 mM imidazole). Approximately 73 mg of recombinant PoAstV4 spike protein was obtained from a 6 L expression. The 10x-histidine tag was removed from a portion of the PoAstV4 spike protein by incubation with bovine thrombin protease (Millipore 60-516-01 KU) overnight at 4°C during dialysis into TBS (10 mM Tris-HCI pH 8.0, 150 mM NaCI). Remaining PoAstV4 spike protein with 10x-histidine tag intact was buffer exchanged into TBS using a desalting column (Cytiva #170851 ). The PoAstV4 spike proteins with or without the 10x-hisidine tag were purified further by size exclusion chromatography using a Superdex 200 16 / 600 column in TBS pH 8.0.

[0114] Structural determination of PoAstV4 Spike

[0115] Purified PoAstV4 spike was concentrated to 23 mg / mL in TBS. PoAstV4 spike protein crystals were grown in 2 pL drops consisting of a 1 :1 ratio of protein solution to well solution containing 0.2 M calcium acetate hydrate, 0.1 M Tris-CI pH 7, and 12% PEG 3000, using hanging drop vapor diffusion at 22°C. One crystal was transferred into a cryoprotectant solution containing the well solution with 25% PEG 400 before being flash frozen into liquid nitrogen. The Advanced Atty. Docket: UCSC-413WO (2025-913-1 )

[0116] Light Source Beamline 5.0.1 was used to collect X-ray diffraction data with a wavelength 0.97 A at cryogenic temperatures. Diffraction data was highly anisotropic and a number of frames (75 / 720) had to be omitted due to poorly defined spots. This data set was processed and scaled with DIALS (ccp4i2) using a low resolution cutoff of 40 A and a high resolution cutoff of 1 .85 A. A model generated by AlphaFold2 / ColabFold

[0031] was trimmed to remove residues residues 432- 435, 625-628, and 640-655, and this model was used for molecular replacement with Phaser. The molecular replacement phase data was used for the first round of refinement to build an initial model, which was modeled manually using Coot

[0033] and refined in Phenix

[0034] to create an improved model in chain A. Chain A was used for molecular replacement on the other chains, and AutoBuild was then applied to correct incorrectly placed residues. The model was polished using Coot

[0033] and refined in Phenix

[0034] to generate the final structure. Calcium ions were modeled, as it fit the CheckMyMetal

[0035] parameters best in comparison to other ions of similar size or charge (Mg, K, Mn, Na) that were modeled and tested for coordination chemistry, agreement of experimental B-factors, occupancy, and the metal binding environmental motif. There was calcium acetate in the crystallography condition, which is the likely source of the calcium ion.

[0117] Development of an enzyme-linked immunosorbent assay to evaluate PoAstV antibodies in pig sera

[0118] Serum samples from pigs with known or suspected exposure to PoAstV4 were used as presumed seropositive serum samples. Serum from cesarean-derived, colostrum-deprived (CDCD) pigs were used as presumed seronegative serum samples

[0036] . A ninety-six well medium-binding ELISA plate (Corning #9017) was coated with 50 pL of 10 pg / mL purified PoAstV4 spike in phosphate buffered saline (PBS), or PBS alone as a “no antigen” control, covered with microplate sealing tape (Corning #6575) and incubated overnight at 4°C. The plate was washed three times (200 pl each) with PBS-T (PBS+0.1% Tween). Then 200 pl of blocking solution (PBS-T+5% milk) was added to all wells of the plate and incubated for 2 h at room temperature. After the incubation, the blocking solution was thrown off the plate and tapped on a kimwipe to dry. Next, 80 pL of blocking buffer was added to all wells and an extra 64 pL were added to Row A. 16 pL of a prediluted 1 :10 sera sample was added to row A, making an initial 1 :100 dilution on the plate. Each serum sample was added in triplicate, with three columns dedicated to an individual sample. A multichannel pipette was used to pipette up and down 4-6 times in row A and to transfer 80 pL to row B. This was repeated through row G (no sera in last row), and the last 80 pL were discarded. The plate was incubated for 2 h at room temperature, Atty. Docket: UCSC-413WO (2025-913-1 ) and then washed 3 times with 200 pL PBS-T. Next, 50 pL of Anti-swine IgG-HRP secondary antibody (Jackson ImmunoResearch Laboratories #114-035-003) diluted at 1 :10,000 in PBS-T + 1% milk was added to each well, and the plate was incubated for 1 h at room temperature. The plate was then washed three times with 200 pL PBS-T. To develop the plate, 100 pL of TMB substrate (Sigma #T0440) was added to all wells and incubated for 9 min at room temperature, followed immediately by quenching with 100 pL 1 N H2SO4. The 450 nm absorbance in each well was measured with a plate reader.

[0119] PoAstV4 IgM and IgG ELISAs

[0120] Serum Enzyme-linked immunosorbent assays (ELISAs) were performed to evaluate both serum IgM and IgG responses against the recombinant PoAstV4 capsid spike antigen (GenBank #PP806170.1 , amino acids 420-655), using an adapted protocol (20). Three 96-well mediumbinding ELISA plates (Corning #9017) were coated with 50 pL of 10 pg / mL purified recombinant PoAstV4 capsid spike in phosphate buffered saline pH 7.4 (PBS), or PBS alone as a “no antigen” control, covered with microplate sealing tape (Corning #6575) and incubated overnight at 4°C. The plate was washed three times (200 pl each) with PBS-T (PBS+0.1% Tween). Then 200 pl of blocking solution (PBS-T+5% milk) was added to all wells of the plate and incubated for 2 h at room temperature. After the incubation, the blocking solution was thrown off the plate and tapped on a kimwipe to dry. Next, 396 pL of blocking buffer and 4 pL of challenged or non-challenged porcine serum was added to a 96-well mixing block and mixed with a multichannel pipette. Then, 120 pL of the diluted serum was transferred from its corresponding well in the fraction collector to the same well on the ELISA plate, making a 1 :100 dilution on the plate. Each serum sample was added in triplicate, with three columns dedicated to an individual sample. The plate was incubated for 2 h at room temperature, and then washed 3 times with 200 pL PBS-T. Next, 50 pL of either Anti-swine IgG-HRP secondary antibody (Jackson ImmunoResearch Laboratories #1 14-035-003) diluted at 1 :10,000 in PBS-T + 1 % milk or Anti-swine IgM-HRP secondary antibody (BioRad #AAI48P) diluted at 1 :3,000 in PBS-T + 1% milk was added to each well, and the plate was incubated for 1 h at room temperature. The plate was then washed three times with 200 pL PBS- T. To develop the plate, 100 pL of TMB substrate (Sigma #T0440) was added to all wells and incubated for 8 min at room temperature, followed immediately by quenching with 100 pL 1 N H2SO4. The 450 nm absorbance in each well was measured with a plate reader.

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[0170] Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

Atty. Docket: UCSC-413WO (2025-913-1 )WHAT IS CLAIMED IS:1 . A porcine astrovirus 4 (PoAstV4) capsid spike protein comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 1 , a variant thereof comprising 85% or greater identity to 150 or greater consecutive positions of the amino acid sequence set forth in SEQ ID NO: 1 , and / or a functional fragment thereof.

2. The PoAstV4 capsid spike protein of claim 1 fused to a heterologous amino acid sequence.

3. The PoAstV4 capsid spike protein of claim 1 or 2, wherein the PoAstV4 capsid spike protein is a recombinant PoAstV4 capsid spike protein expressed in a bacterial cell.

4. The PoAstV4 capsid spike protein of claim 3, wherein the bacterial cell is an E. coli cell.

5. An immunogenic composition comprising the PoAstV4 capsid spike protein of any one of claims 1 -4, wherein the composition is suitable for administration to a mammalian subject.

6. The immunogenic composition of claim 5, wherein the composition comprises an adjuvant.

7. A method of eliciting an immune response to porcine astrovirus 4 in a mammalian subject, the method comprising administering the immunogenic composition of claim 5 or claim 6 to the mammalian subject in an amount effective to elicit an immune response to porcine astrovirus 4 in the mammalian subject.

8. The method of claim 7, wherein the mammalian subject is a porcine animal.

9. The method of claim 7 or 8, wherein the immunogenic composition is administered intramuscularly, subcutaneously, intranasally or orally.

10. A substrate having attached to the surface thereof PoAstV4 capsid spike proteins as defined in any one of claims 1 -4.Atty. Docket: UCSC-413WO (2025-913-1 )11 . The substrate of claim 10, wherein the substrate is a well of a plate.

12. The substrate of claim 11 , wherein the plate is a multi-well plate.

13. A method of assessing a mammalian subject for a porcine astrovirus 4 (PoAstV4) capsid antigen exposure, the method comprising: contacting the substrate of any one of claims 10-12 with a biological sample obtained from the mammalian subject; and assessing for binding of antibodies to the PoAstV4 capsid spike proteins.

14. The method of claim 13, wherein the capsid antigen exposure is via natural infection with PoAstV4.

15. The method of claim 13, wherein the capsid antigen exposure is via vaccination with a PoAstV4 capsid antigen or a vaccine comprising a nucleic acid encoding a PoAstV4 capsid antigen.

16. The method of any one of claims 13-15, wherein the mammalian subject is a porcine animal.

17. The method of any one of claims 13-16, wherein the biological sample is whole blood obtained from the mammalian subject or a fraction thereof, optionally wherein the biological sample is serum.

18. The method of any one of claims 13-17, wherein the assessing comprises: contacting antibodies bound to the PoAstV4 capsid spike proteins, if present in the biological sample, with a secondary antibody specific for IgG of the type produced by the mammalian subject; removing unbound secondary antibody; and assessing for the presence of secondary antibody bound to IgG of the type produced by the mammalian subject.Atty. Docket: UCSC-413WO (2025-913-1 )19. The method of claim 18, wherein the secondary antibody comprises a detectable label, and wherein assessing for the presence of the secondary antibody comprises assessing for the detectable label.

20. The method of claim 18, wherein the secondary antibody is linked to an enzyme, and wherein the assessing is by enzyme-linked immunosorbent assay (ELISA).21 . The method of claim 20, wherein the enzyme is horseradish peroxidase (HRP), alkaline phosphatase (AP), p-galactosidase, acetylcholinesterase, or catalase.

22. A nucleic acid encoding the PoAstV4 capsid spike protein of claim 1 or 2 operably linked to a heterologous promoter.

23. A cell comprising the nucleic acid of claim 22.

24. The cell of claim 23, wherein the cell is a bacterial cell.

25. The cell of claim 24, wherein the bacterial cell is an E. coli cell.

26. A method of producing the PoAstV4 capsid spike protein of claim 1 or 2, the method comprising culturing the cell of any one of claims 23-25 under conditions suitable for expression of the PoAstV4 capsid spike protein, wherein the PoAstV4 capsid spike protein is produced.

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