Immunogenic composition and uses thereof

Abstract

The disclosure provides a composition comprising at least one nucleic acid encoding an amino acid sequence set forth in SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, or SEQ ID NO: 103 and uses thereof.

Description

IMMUNOGENIC COMPOSITION AND USES THEREOFFIELD

[0001] The present disclosure is in the field of vaccines and the treatment or prevention of coronavirus infections.INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002] This application contains, as a separate part of the disclosure, a Sequence Listing in computer-readable form which is incorporated by reference in its entirety and identified as follows: 59798_SeqListing.xml, Size: Size: 258,351 bytes; Created: November 12, 2024.BACKGROUND

[0003] Coronaviruses (CoVs) are a highly diverse family of enveloped positive-sense, single-stranded RNA viruses with a lipid envelope. CoVs infect humans, other mammals including livestock and companion animals and avian species like domestic and free-living birds. Feline (FCoV) and canine (CCoV) coronaviruses are widespread among dog and cat populations. FCoV belongs to the alphacoronavirus genus and two phylogenetic lineages are currently known, serotype 1 and 2 (FCoV1 , FCoV2). Recombination is a common behavior for CoVs that may generate new variants able to infect species other than their natural reservoir.1For example, FCoV2 originated by a double recombination event between the FCoV1 and CCoV.2

[0004] Both FCoV serotypes infect predominantly epithelial cells of the gastrointestinal tract and cause a mild gastrointestinal disease (e.g., diarrhea, vomiting, and transient weight loss) in domestic cats and especially in kittens. Often, upon chronic infection, these viruses can also mutate into pathogenic and fatal variants. These variants, called feline infectious peritonitis viruses (FIPVs), infect monocytes and macrophages, leading to systemic infection3-5. No successful FIPV treatments have been available for decades; cats with FIPV either die or have to be euthanized.6The median survival time of untreated cats is only eight to nine days.6Unfortunately, FIPV vaccine development has been hampered by antibody dependent enhancement (ADE), whereby vaccine candidates that induce neutralizing antibodies make the infection worse.7-9

[0005] For humans, three highly pathogenic and deadly human coronaviruses have emerged within the last two decades, namely SARS-CoV1 (SCoV1 ), MERS-CoV and the virus causing the current COVID-19 pandemic, SARS-CoV-2 (SCoV2).10Unlike the FCoVs, SCoV1 and SCoV2 belong to the betacoronavirus genus. SCoV2 is thought to have originated from recombination events between CoVs of other species, possibly through one or more intermediate hosts.11 12The COVID-19 pandemic has been partially controlledusing SARS-CoV-2 vaccines. However, while the vaccines prevent severe and fatal disease, their efficacy wanes over time, necessitating booster doses.13Vaccination has also been unable to prevent "breakthrough" infections in fully vaccinated individuals, allowing subsequent transmission of the disease to others.13Moreover, immunity following natural infection with SARS-CoV-2, combined with vaccine- induced immunity, has so far not prevented the emergence and rapid spread of "variants of concern," such as the highly transmissible delta (B.1 .617.2) variant and the omicron (B.1 .1 .529) variant.13

[0006] There is now evidence of SARS-CoV-2 spillover from humans to animals reported in dogs, cats, tigers, lions, gorillas and minks.14-16SARS-CoV-2 infection in domestic dogs and cats has been reported in 22 countries in America, Europe and Asia.17 18Cats are susceptible to SARS-CoV-2 infection and can show mild-to-moderate respiratory symptoms, while dogs developed no or mild respiratory symptoms.14’1920There remains a need in the art for a vaccine that is protective across multiple species.SUMMARY

[0007] In one aspect, the disclosure provides a composition comprising at least one nucleic acid encoding an amino acid sequence set forth in SEQ ID NO: 99 [W2F12], SEQ ID NO: 100 [W1 F11], SEQ ID NO: 101 [0m1C], SEQ ID NO: 102 [2B], or SEQ ID NO: 103 [6B],

[0008] In another aspect, the disclosure provides methods of stimulating an immune response in a subject in need there comprising administering to the subject a composition comprising at least one nucleic acid encoding an amino acid sequence set forth in SEQ ID NO: 99 [W2F12], SEQ ID NO: 100 [W1 F11 ], SEQ ID NO: 101 [Om1 C], SEQ ID NO: 102 [2B], or SEQ ID NO: 103 [6B],BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 top panel provides the UF-RBD SCoV2 sequence (SEQ ID NO: 1). The middle panel provides the MB-RBD SCoV2 sequence (SEQ ID NO: 3). The bottom panel provides a sequence alignment of both sequences showing that the MB-RBD sequence is longer by 12 aa on the carboxyl-end.

[0010] Figure 2A is a bar graph of FCoV2 antibody titers generated by ELISA using FCoV2 whole-virus as the antigen, illustrating FCoV2 seropositivity of four naturally infected laboratory queens (UGAQ1 , UGAQ2, UGAQ3, UGAQ4) and weak FCoV2 seropositivity of sera obtained from three previously SPF toms (5HQT 1 , HOJT2, HOGT3) after they mated with the positive queens. The bars filled with the slashed lines depict the mating pair, UGAQ3 and 5HQT1 and the resulting juvenile cat, Y2B. The bars filled with small dots represent the mating pair UGAQ4 and HOJT2, and their litter D4A, D4D, and D4F. Theunfilled bars show two queens (UGAQ1 , UGAQ2) and a tom (HOGT3) that did not successfully mate. The serum from SPF cat, HOE, was used as a negative control. Figure 2B is a bar graph of antibody titers generated by ELISA using SCoV2 RBD illustrating crossreactivity of the FCoV2 anti-sera obtained from the queens and juvenile cats with SCoV2 RBD. Serum from SPF cat, was used as a negative control. The month(s) post UF arrival when the blood collection were performed on the queens.

[0011] Figure 3A is a bar graph showing antibody titers, generated under stringent ELISA conditions, against FCoV2 (SEQ ID NO: 5), SCoV2-UF-RBD (SEQ ID NO: 1 ), SCoV2-MB- RBD (SEQ ID NO: 3) and bovine serum albumin (BSA) using anti-sera from FCoV positive Queens. Serum from HOE SPF was used as a negative control. The ages of the queens is indicated.

[0012] Figure 3B is a bar graph showing antibody titers, generated under stringent ELISA conditions, against FCoV2, UF-RBD, and MB-RBD using sera obtained from toms before and after mating with FCoV positive queens.

[0013] Figure 4 is a bar graph showing lack of bovine serum albumin (BSA) antibodies in sera from the three toms pre-mating and at two time points post mating with the FCoV positive queens.

[0014] Figure 5A is a bar graph whose first three sets of bars show antibody titers obtained under stringent ELISA conditions, against FCoV2 whole virus antigen, SCoV2 RBD, and bovine serum albumin (BSA) using sera obtained from the juvenile cats. The dark bars indicate titers from sera obtained at 12 weeks of age for Y2B and at 16 weeks of age for D4A, D4D, and D4F. The light bars indicate titers from sera obtained at 16 weeks of age for Y2B and at 20 weeks of age for the three D4 cats. Serum from HOE SPF was used as a negative control while serum from FCoV positive queen (UGAQ4) was used as a positive control.

[0015] Figures 5B1 and B2 are images showing immunoblot strips results for each antigen antibody titer group described in (Figure 5A) with the exception of the BSA antigen group which was not performed. The serum from SPF cat HOF was used as negative controls for all three antigens.

[0016] Figures 6A-6D provide immunoblot analyses results of the sera from FCoV+ queens and SCoV2 RBD-positive toms using the SCoV2 UF-RBD (Figure 6A), the proposed FCoV2 RBD (Figure 6B), the cross-reactive FCoV2-wv (Figure 6C), and the proposed (Figure 6D) FCoV1 RBD immunoblot strips.

[0017] Figures 7A and 7B are graphs showing the anti-CoV RBD IFNy and IL-2 ELISpot responses of the peripheral blood mononuclear cells (PBMC) from a COVID-19 vaccinated subjects.

[0018] Figure 8 shows that chronically FCoV1 -infected cats (G1 ,G2,G7) have antibodies to FCoV whole-virus and live together, causing constant reinfection.

[0019] Figure 9 is an amino acid sequence comparison of the full-length spike protein of SCoV2- Wuhan (SEQ ID NO: 104) and SCoV2 Omicron XBB1.5 (SEQ ID NO: 105).DETAILED DESCRIPTION

[0020] The present disclosure is based, in part, on the discovery of compositions comprising a nucleic acid encoding a feline coronavirus (FCoV) receptor binding domain (RBD) peptide and a human SARS coronavirus serotype 2 (SCoV2) RBD peptide for the prevention and / or treatment of FCoV1 , FCoV2, CCoV and SCoV2 coronavirus infections. As shown in the Examples, the disclosed composition induced strong immune responses in subjects immunized with the compositions.

[0021] In some or any aspects, the composition comprising a nucleic acid or peptide disclosed herein elicits an immune response in the subject when administered to the subject. In some embodiments, the compositions may prevent, ameliorate, palliate, or eliminate disease from the host.

[0022] Coronavirus

[0023] The term “coronavirus” refers to a virus in the family Coronaviridae, which is in turn classified within the order Nidovirales. The coronaviruses are large, enveloped, positive- stranded RNA viruses. The coronaviruses have the largest genomes of the RNA viruses known in the art and replicate by a unique mechanism that results in a high frequency of recombination. The coronaviruses include antigenic groups I, II, and III. Nonlimiting examples of coronaviruses include SARS coronavirus (e.g., SARS-CoV and SARS-CoV-2), MERS coronavirus, transmissible gastroenteritis virus (TGEV), human respiratory coronavirus, porcine respiratory coronavirus, canine coronavirus, feline enteric coronavirus, feline infectious peritonitis virus, rabbit coronavirus, murine hepatitis virus, sialodacryoadenitis virus, porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, avian infectious bronchitis virus, and turkey coronavirus, as well as chimeras thereof. Additional information related to coronavirus including classification, virion structure, genome structure, genetics and pathology is described, for example, in KV Holmes, Encyclopedia of Virology, 1999: 291-298, the content of which is incorporated herein by reference.

[0024] In some or any aspects, a coronavirus described herein is in the genus of Alphacoronavirus, and the coronavirus antigens can be of or derived from any species or strains in the genus of Alpha-coronavirus. In some or any aspects, a coronavirus described herein is in the genus of Beta-coronavirus and the coronavirus antigens can be of or derived from any species or strains in the genus of Beta-coronavirus. Member viruses in the genus of Alphacoronavirus and Beta-coronavirus are enveloped, positive-strand RNA viruses that can infect mammals.

[0025] A coronavirus described herein is optionally in the subgenus Tegacovirus of Alphacoronavirus genus, including but not limited to feline coronavirus serotype 1 (FCoV1 ), feline coronavirus serotype 2 (FCoV2) and canine coronavirus (CCoV).

[0026] Within the genus Beta-coronavirus, five subgenera or lineages have been recognized, including Embecovirus (lineage A), Sarbecovirus (lineage B), Merbecovirus (lineage C), Nobecovirus (lineage D), and Hibecovirus. Accordingly, in some or any aspects, the coronavirus described herein can be any strain or species in any of the subgenera or lineages of Beta-coronavirus. For example, a coronavirus peptide can be of or derived from any species or strains in the subgenus of Sarbecovirus, including but not limited to human SARS-CoV and SARS-CoV2.

[0027] In some or any aspects, the compositions described herein comprises comprising at least one nucleic acid encoding an amino acid sequence at least 70% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence identical to an amino acid sequence set forth in SEQ ID NO: 99 [W2F12], SEQ ID NO: 100 [W1 F11 ], SEQ ID NO: 101 [Om1 C], SEQ ID NO: 102 [2B], or SEQ ID NO: 103 [6B],

[0028] In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence that is at least 70% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence set forth in SEQ ID NO: 99. In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence set forth in SEQ ID NO: 99.

[0029] In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence that is at least 70% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence set forth in SEQ ID NO: 100. In some or any aspects, the composition comprises an amino acid sequence set forth in SEQ ID NO: 100.

[0030] In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence that is at least 70% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence set forth in SEQ ID NO: 101. In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence set forth in SEQ ID NO: 101 .

[0031] In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence that is at least 70% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence set forth in SEQ ID NO: 102. In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence set forth in SEQ ID NO: 102.

[0032] In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence that is at least 70% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence set forth in SEQ ID NO: 103. In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence set forth in SEQ ID NO: 103.

[0033] A sequence which is not 100% identical to the particular sequences described herein (e.g., SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101 , SEQ ID NO: 102 or SEQ ID NO: 103) may comprise one or more conservative substitutions. Conservative substitutions generally correspond to substitution of a reference amino acid with a functionally equivalent reside with similar physiochemical properties. A functionally equivalent residue of an amino acid typically can refer to other amino acid residues having physiochemical and stereochemical characteristics substantially similar to the original amino acid. The physiochemical properties include water solubility (hydrophobicity or hydrophilicity), dielectric and electrochemical properties, physiological pH, partial charge of side chains (positive, negative or neutral) and other properties identifiable to one of skill in the art. The stereochemical characteristics include spatial and conformational arrangement of the amino acids and their chirality. For example, glutamic acid is considered to be a functionally equivalent residue to aspartic acid in the sense of the current disclosure.Tyrosine and tryptophan are considered as functionally equivalent residues to phenylalanine. Arginine and lysine are considered as functionally equivalent residues to histidine.

[0034] Immunogen

[0035] In some or any aspects, the composition described herein comprises (or is administered with) an immunogenic epitope The term "immunogenic epitope as used herein refers to smallest component of an antigen that induces a subject to mount an immune response, and when epitopes are combined they serve as the vaccine immunogen. An antigen is composed of multiple epitopes to induce strong immune response. The terms “immunogenic epitope” and “immunogen” are used synonymously herein.

[0036] In some or any aspects, the “epitope” refers to a portion of antigen that an immunoglobulin or antibody binds to, e.g., at least s, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more consecutive or non-consecutive amino acids in a unique steric conformation, which may be “linear” or “conformational”. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996). In a linear epitope, all the interaction sites between a protein and an interaction molecule (e.g., an antibody) are present linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interaction sites span over amino acid residues that are not necessarily adjacent in the primary sequence but from a three dimensional structure recognized by an antibody.

[0037] An "immune response" is a response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus, such as an antigen (e.g., formulated as an antigenic composition or a vaccine). An immune response can be a B cell response, which results in the production of specific antibodies, such as antigen specific neutralizing antibodies. An immune response can also be a T cell response, such as a CD4+ response or a CD8+ response. B cell and T cell responses are aspects of a "cellular" immune response. An immune response can also be a "humoral" immune response, which is mediated by antibodies. In some cases, the response is specific for a particular antigen (that is, an "antigen-specific response"). A "protective immune response" is an immune response that inhibits a detrimental function or activity of an antigen, or decreases symptoms (including death) that result from the antigen. A protective immune response can be measured, for example, by immune assays using a serum sample from an immunized subject for testing the ability of serum antibodies for inhibition of tumor cell expansion, such as: ELISA- neutralization assay, antibody dependent cell-mediated cytotoxicity assay (ADCC), complement-dependent cytotoxicity (CDC), antibody dependent cell-mediated phagocytosis (ADCP), enzyme-linked immunospot (ELISpot). In addition, vaccine efficacy can be tested by measuring the T cell response CD4+ and CD8+ after immunization, using flow cytometry (FACS) analysis or ELISpot assay. The protective immune response can be tested by measuring resistance to antigen challenge in vivo in an animal model. In humans, a protective immune response can be demonstrated in a population study, comparing measurements of symptoms, morbidity, mortality, etc. in treated subjects compared to untreated controls. Exposure of a subject to an immunogenic stimulus, such as an antigen (e.g., formulated as an antigenic composition or vaccine), elicits a primary immune response specific for the stimulus, that is, the exposure "primes" the immune response. A subsequent exposure, e.g., by immunization, to the stimulus can increase or "boost" the magnitude (or duration, or both) of the specific immune response. Thus, "boosting" a preexisting immuneresponse by administering an antigenic composition increases the magnitude of an antigenspecific response, (e.g., by increasing antibody titer and / or affinity, by increasing the frequency of antigen specific B or T cells, by inducing maturation effector function, or a combination thereof).

[0038] In some or any aspects, the immunogen comprises a nucleic acid that encodes conserved FCoV1 CD8+ T cell epitope peptide. In some or any aspects, the immunogen comprises a nucleic acid that encodes a conserved SCoV2 CD8+ T cell epitope peptide.

[0039] In some or any aspects, the conserved FCoV1 CD8+ T cell epitope peptide is an amino acid sequence set forth in any one of SEQ ID NOs: 47-66. In some or any aspects, the conserved SCoV2 CD8+ T cell epitope peptide is an amino acid sequence set forth in any one of SEQ ID NOs: 19-46.

[0040] In some or any aspects, the conserved CD8+ T cell epitope peptide is a SCoV2 polymerase enzyme epitope peptide. In some or any aspects, the SCoV2 polymerase enzyme epitope peptide is an amino acid sequence set forth in any one of SEQ ID NOs: 19- 24.

[0041] In some or any aspects, the conserved CD8+ T cell epitope peptide is a SCoV2 M- protease peptide. In some or any aspect, the SCoV2 M-protease peptide is an amino acid sequence set forth in any one of SEQ ID NOs: 68-75.

[0042] In some or any aspects, the conserved CD8+ T cell epitope peptide is a FCoV1 M- protease peptide. In some or any aspect, the FCoV1 M-protease peptide is an amino acid sequence set forth in any one of SEQ ID NOs: 76-83.

[0043] In some or any aspects, the immunogen comprises a nucleic acid encoding an amino acid sequence composed of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, or 16 M- protease T cell epitope peptides set forth in SEQ ID NOs: 68-83, optionally conjugated together via a linker (e.g., a cathepsin S receptor linker, “KVSVR,” (SEQ ID NO: 84)). In some or any aspects, the adjuvant comprises a nucleic acid encoding an amino acid sequence composed of 2, 3, or 4 M-protease T cell epitope peptides set forth in SEQ ID NOs: 68-71 , optionally conjugated together via a linker (e.g., a cathepsin S receptor linker, “KVSVR,” (SEQ ID NO: 84)). In some or any aspects, the adjuvant comprises a nucleic acid encoding an amino acid sequence set forth in SEQ ID NO: 89.

[0044] In some or any aspects, the immunogen comprises a nucleic acid encoding an amino acid sequence composed of 2, 3, or 4 M-protease T cell epitope peptides set forth in SEQ ID NOs: 72-75, optionally conjugated together via a linker (e.g., a cathepsin S receptorlinker, “KVSVR,” (SEQ ID NO: 84)). In some or any aspects, the adjuvant comprises a nucleic acid encoding an amino acid sequence set forth in SEQ ID NO: 90.

[0045] In some or any aspects, the immunogen comprises a nucleic acid encoding an amino acid sequence composed of 2, 3, or 4 M-protease T cell epitope peptides set forth in SEQ ID NOs: 76-79, optionally conjugated together via a linker (e.g., a cathepsin S receptor linker, “KVSVR,” (SEQ ID NO: 84)). In some or any aspects, the adjuvant comprises a nucleic acid encoding an amino acid sequence set forth in SEQ ID NO: 91 .

[0046] In some or any aspects, the immunogen comprises a nucleic acid encoding an amino acid sequence composed of 2, 3, or 4 M-protease T cell epitope peptides set forth in SEQ ID NOs: 80-83, optionally conjugated together via a linker (e.g., a cathepsin S receptor linker, “KVSVR,” (SEQ ID NO: 84)). In some or any aspects, the adjuvant comprises a nucleic acid encoding an amino acid sequence set forth in SEQ ID NO: 92.

[0047] In some or any aspects, the immunogen comprises a nucleic acid encoding an amino acid sequence comprising 2, 3, or 4 M-protease T cell epitopes and a Th1 peptide. Exemplary Th1 peptides include, but are not limited to, amino acid sequence set forth in SEQ ID NO: 85-88.

[0048] In some or any aspects, the immunogen comprises one or more nucleic acids encoding one or more M-protease amino acid sequences set forth in SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91 or SEQ ID NO: 92. In some or any aspects, the immunogen comprises one or more nucleic acids encoding one or more amino acid sequences set forth in SEQ ID NO: 89 and SEQ ID NO: 90. In some or any aspects, the immunogen comprises one or more nucleic acids encoding one or more amino acid sequences set forth in SEQ ID NO: 89 and SEQ ID NO: 91 . In some or any aspects, the immunogen comprises one or more nucleic acids encoding one or more amino acid sequences set forth in SEQ ID NO: 89 and SEQ ID NO: 92. In some or any aspects, the immunogen comprises one or more nucleic acids encoding one or more amino acid sequences set forth in SEQ ID NO: 90 and SEQ ID NO: 91 . In some or any aspects, the immunogen comprises amino acid sequences set forth in SEQ ID NO: 90 and SEQ ID NO: 92.

[0049] Nucleic acids, Vectors

[0050] Nucleic acid molecules comprising nucleotide sequences encoding the FCoV and SCoV2 RBD peptides (and optionally the conserved FCOV and / or SCoV2 CD8+ T cell epitope adjuvant peptides) are also contemplated. Nucleic acid molecules encoding the amino acid sequences set forth in any one of SEQ ID NOs: 93-103 are also contemplated. Optionally, the nucleic acid molecule(s) are inserted into a “vector.” As used herein, the term “vector” refers to any element, such as a plasmid, phage, transposon, cosmid, chromosome,virus, virus capsid, virion, etc., which is capable of transferring and / or transporting a nucleic acid to a host cell and / or that allows or facilitates the manipulation of a nucleic acid molecule. The disclosure contemplates naked or complexed nucleic acid molecules, as well as cloning vectors and expression vectors comprising any of the nucleic acid molecules described herein. The cloning or expression vector may be a viral vector or a non-viral vector.

[0051] In some or any aspects, the vector is a recombinant viral vector. In some embodiments, the recombinant viral vector is a recombinant adeno-associated vector (rAAV Vector).

[0052] An rAAV vector typically includes a serotype capsid protein that encapsulates a recombinant genome. The AAV genome typically includes functional 5' and 3' inverted terminal repeats sequences (ITR sequences). The ITR sequences are often flanked by exogenous nucleotide sequences that replace rep or cap genes found in wild-type AAVs. ITR sequences provide functional rescue, replication, and packaging to rAAVs. In some examples, the ITR sequences are from AAV2. In some or any aspects, the AAV is of the AAV1 , AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV 12, AAV13, AAVPHP.B, or AAVrh74 serotype.

[0053] In some or any aspects, nucleic acids encoding the FCoV (e.g., FCoV1 and / or FCoV2) and SCoV2 RBD peptides are present on separate vectors, although the nucleic acid sequences encoding all of the RBD peptides may be present on the same vector. In some or any aspects, nucleic acids encoding FCoV and / or SCoV2 CD8+ T cell epitope peptides are present on the same vector as the nucleic acids encoding the FCoV and SCoV2 RBD peptides. In some or any aspects, nucleic acids encoding the FCoV and SCoV2 RBD peptides are present on a separate (different) vector. In this regard, the disclosure provides a system comprising multiple different expression vectors comprising nucleic acids encoding different components of the composition disclosed herein.

[0054] Expression vectors typically comprise “expression regulatory elements,” which are generally a collection of promoter sequences, upstream regulatory regions, and transcriptional regulatory elements, which jointly drive replication, transcription and translation of coding region sequences.

[0055] Formulations and Routes of Administration

[0056] In some or any aspects, the composition comprises a FCoV RBD peptide and a SCoV2 RBD peptide and a pharmaceutically acceptable carrier or excipient. In some or any aspects, the composition comprises a nucleic acid encoding an amino acid sequence set forth in any one of SEQ ID NOs: 99-103.

[0057] Exemplary pharmaceutically acceptable excipients or carriers can include a buffer, such as Tris (trimethamine), phosphate (e.g. sodium phosphate), acetate, borate (e.g. sodium borate), citrate, glycine, histidine and succinate (e.g. sodium succinate), suitably sodium chloride, histidine, sodium phosphate or sodium succinate. The pharmaceutically acceptable excipient may include a salt, for example sodium chloride, potassium chloride or magnesium chloride. Optionally, the pharmaceutically acceptable excipient contains at least one component that stabilizes solubility and / or stability. Examples of solubilizing / stabilizing agents include detergents, for example, laurel sarcosine and / or polysorbate (e.g. Tween™80). Examples of stabilizing agents also include poloxamer (e.g. poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338 and poloxamer 407). The phamaceutically acceptable excipient may include a non-ionic surfactant, for example polyoxyethylene sorbitan fatty acid esters, Polysorbate-80 (Tween™80), Polysorbate-60 (Tween™60), Polysorbate-40 (Tween™40) and Polysorbate-20 (Tween™20), or polyoxyethylene alkyl ethers (suitably polysorbate-80). Alternative solubilizing / stabilizing agents include arginine, and glass forming polyols (such as sucrose, trehalose and the like). The pharmaceutically excipient may be a preservative, for example phenol, 2-phenoxyethanol, or thiomersal. Other pharmaceutically acceptable excipients include sugars (e.g. lactose, sucrose), and proteins (e.g. gelatine and albumin). Pharmaceutically acceptable carriers include water, saline solutions, aqueous dextrose and glycerol solutions

[0058] In some or any aspects, the composition is formulated in a nanoparticle, a liposome, a microparticle, a microsphere, a nanosphere, a unilamellar vesicle, a multilamellar vesicle, or a virus-like particle (VLP).

[0059] In some or any aspects, the composition is formulated in a nanoparticle (e.g., a lipid nanoparticle). Optionally, the nanoparticle has a mean diameter of 50-200 nm. In some or any aspects, the lipid nanoparticle comprises DLin-MC3-DMA ionizable lipid and four structural lipids: 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1 ,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE), and 1 ,2-dimyristoyl-sn-glycero-3- methoxypolyethyleneglycol 2000 (DMG-Peg), and cholesterol. In some or any aspects, the nanoparticle is specific for a T cell. In some or any aspects, the nanoparticle is specific for a dendritic cell. In some or any aspects, the nanoparticle is specific for a macrophage. In some or any aspects, the nanoparticle is specific for both a dendritic cell and a macrophage.

[0060] The compositions described herein can be administered by any route, including a systemic or mucosal route. Exemplary administrations may include injection via the intramuscular (IM), intraperitoneal (IP), intradermal (ID), intranasal (IN), or subcutaneous (SC) routes; or via mucosal administration to the oral / alimentary, respiratory, genitourinarytracts. Following an initial administration, subjects may receive one or several booster administrations.Methods of use

[0061] The disclosure provides a method of treating or preventing a coronavirus infection, the method comprising administering a composition described herein to a subject in need thereof. The disclosure further provides a method of stimulating an immune response in a subject in need thereof. The method comprises administering to the subject an effective amount of the composition, thereby stimulating an immune response in the subject. In some or any aspects, administering the composition induces a B cell response against (i) SARS- CoV-2, (ii) FCoV1 , and (iii) FCoV2. Alternative or in addition, administering the composition optionally induces a CD8+ and / or CD4+ T cell response against (i) SARS-CoV-2, (ii) FCoV1 , and (iii) FCoV2.

[0062] In some or any aspects, the method comprises administering a first composition comprising a nucleic acid encoding an amino acid sequence set forth in any one of SEQ ID NOs: 99-103 and a second composition comprising one or more nucleic acids encoding one or more FCoV and / or SCoV2 CD8+ T cell epitope peptides.

[0063] In some or any aspects, the first composition comprises a nucleic acid encoding an amino acid sequence that is at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 99. In some or any aspects, the first composition comprises a nucleic acid encoding an amino acid sequence that is at least 70% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence set forth in SEQ ID NO: 99. In some or any aspects, the first composition comprises a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 99.

[0064] In some or any aspects, the first composition comprises a nucleic acid encoding an amino acid sequence that is at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 100. In some or any aspects, the first composition comprises a nucleic acid encoding an amino acid sequence that is at least 70% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence set forth in SEQ ID NO: 100. In some or any aspects, the first composition comprises a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 100.

[0065] In some or any aspects, the first composition comprises a nucleic acid encoding an amino acid sequence that is at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 101. In some or any aspects, the first composition comprises a nucleic acid encoding an amino acid sequence that is at least 70% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequenceset forth in SEQ ID NO: 101. In some or any aspects, the first composition comprises a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 101 .

[0066] In some or any aspects, the first composition comprises a nucleic acid encoding an amino acid sequence that is at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 102. In some or any aspects, the first composition comprises a nucleic acid encoding an amino acid sequence that is at least 70% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence set forth in SEQ ID NO: 102.

[0067] In some or any aspects, the first composition comprises a nucleic acid encoding an amino acid sequence that is at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 103. In some or any aspects, the first composition comprises a nucleic acid encoding an amino acid sequence that is at least 70% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence set forth in SEQ ID NO: 103.

[0068] In some or any aspects, the second composition comprises a nucleic acid encoding a SCoV2 M-protease peptide comprising amino acid sequence set forth in any one of SEQ ID NOs: 68-75, a FCoV1 M-protease peptide is an amino acid sequence set forth in any one of SEQ ID NOs: 76-83, or combinations thereof.

[0069] In some or any aspects, the second composition comprises a nucleic acid encoding an amino acid sequence composed of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, or 16 M-protease T cell epitope peptides set forth in SEQ ID NOs: 68-83, optionally conjugated together via a linker (e.g., a cathepsin S receptor linker, “KVSVR,” (SEQ ID NO: 84)). In some or any aspects, the second composition comprises a nucleic acid encoding an amino acid sequence composed of 2, 3, or 4 M-protease T cell epitope peptides set forth in SEQ ID NOs: 68-71 , optionally conjugated together via a linker (e.g., a cathepsin S receptor linker, “KVSVR,” (SEQ ID NO: 84)). In some or any aspects, the second composition comprises a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 89.

[0070] In some or any aspects, the second composition comprises a nucleic acid encoding an amino acid sequence composed of 2, 3, or 4 M-protease T cell epitope peptides set forth in SEQ ID NOs: 72-75, optionally conjugated together via a linker (e.g., a cathepsin S receptor linker, “KVSVR,” (SEQ ID NO: 84)). In some or any aspects, the second composition comprises a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 90.

[0071] In some or any aspects, the second composition comprises a nucleic acid encoding an amino acid sequence composed of 2, 3, or 4 M-protease T cell epitope peptidesset forth in SEQ ID NOs: 76-79, optionally conjugated together via a linker (e.g., a cathepsin S receptor linker, “KVSVR,” (SEQ ID NO: 84)). In some or any aspects, the second composition comprises a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 91.

[0072] In some or any aspects, the second composition comprises a nucleic acid encoding an amino acid sequence composed of 2, 3, or 4 M-protease T cell epitope peptides set forth in SEQ ID NOs: 80-83, optionally conjugated together via a linker (e.g., a cathepsin S receptor linker, “KVSVR,” (SEQ ID NO: 84)). In some or any aspects, the second composition comprises a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 92.

[0073] In some or any aspects, the nucleic acid encoding an amino acid sequence comprising 2, 3, or 4 M-protease T cell epitope peptides further comprises a Th2 peptide. Exemplary Th2 peptides include, but are not limited to, amino acid sequences set forth in SEQ ID NO: 85-88.

[0074] In some or any aspects, the second composition comprises one or more nucleic acids encoding M-protease amino acid sequences set forth in SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91 or SEQ ID NO: 92. In some or any aspects, the second composition comprises one or more nucleic acids encoding M-protease amino acid sequences set forth in SEQ ID NO: 89 and SEQ ID NO: 90. In some or any aspects, the second composition comprises one or more nucleic acids encoding amino acid sequences set forth in SEQ ID NO: 89 and SEQ ID NO: 91 . In some or any aspects, the second composition comprises amino acid sequences set forth in SEQ ID NO: 89 and SEQ ID NO: 92. In some or any aspects, the second compositions comprises one or more nucleic acids encoding M- protease amino acid sequences set forth in SEQ ID NO: 90 and SEQ ID NO: 91 . In some or any aspects, the second composition comprises one or more nucleic acids encoding M- protease amino acid sequences set forth in SEQ ID NO: 90 and SEQ ID NO: 92.

[0075] In some or any aspects, the first composition and second composition are formulated in a lipid nanoparticle. In some or any aspects, the second composition is formulation is a different (separate) lipid nanoparticle. In some or any aspects, the lipid nanoparticle is T cell specific. In some or any aspects, the lipid nanoparticle is specific for a dendritic cell. In some or any aspects, the lipid nanoparticle is specific for a dendritic cell. In some or any aspects, the nanoparticle is specific for both a dendritic cell and a macrophage.

[0076] The terms "subject", "subject in need", and "individual" refer to an animal, in particular vertebrates, such as mammals. The subject is, in various aspects, a cat, a dog, a hamster, or a human, although other animals also are contemplated. The subject may not bediagnosed with coronavirus infection and / or experiencing symptoms; alternatively, the subject may be suffering from a coronavirus infection and / or suffering from symptoms of a coronavirus infection and / or diagnosed with a coronavirus infection. In some or any aspects, the subject has been exposed to a coronavirus (e.g., the subject has come in contact with a person or an animal that is infected with a coronavirus). Alternatively, the subject may be at risk of being exposed to a coronavirus.

[0077] The term "treatment" or "treat" refers to an intervention made in response to a disease, disorder or physiological condition (e.g., a coronavirus infection or symptoms associated with the infection) manifested by the subject. Treatment does not require the complete curing of a disorder; “treatment” encompasses reduction of symptoms associated with a coronavirus infection. Exemplary symptoms of a coronavirus infection include, but are not limited to, fever, cough, tiredness, a loss of taste or smell, shortness of breath or difficulty breathing, muscle aches, chills, sore throat, runny nose, headache, chest pain, pink eye (conjunctivitis), nausea, vomiting, diarrhea, rash, pneumonia and acute respiratory distress syndrome.

[0078] As used herein, the term "prevention" refer to a reduced likelihood that a subject will be susceptible to a coronavirus infection, or less susceptible to symptoms associated with a coronavirus infection. Rather, it denotes that the likelihood of the occurrence of the event has been reduced by the instant method.

[0079] The phrase "effective amount" as used herein means that amount of the composition disclosed herein which is effective for inducing an immune response in the subject. In some embodiments, an effective amount is necessary to inhibit coronavirus replication or to measurably alleviate outward symptoms of the viral infection. In some embodiments, a therapeutically effective amount is an amount that prevents one or more signs or symptoms that can be caused by a coronavirus infection.

[0080] In some embodiments, the determination of an effective amount of the vaccine composition can be measured by measuring the titer of antibodies produced against a coronavirus. Methods of determining antibody titers are described in the example section of the present disclosure (see, for example, Examples 1 , 4, and 5).EXAMPLESMaterials and Methods

[0081] Animal populations: The studies used laboratory cats that were bred and cared for under the University of Florida (UF) IACUC protocols 201801838, 201101838, 201401838, 201701838, and 202001838, for over a decade. The specific pathogen-free (SPF) toms wereinbred from SPF cats initially derived from intact female (Vendor - Harlan Sprague Dawley, Inc., Indianapolis, IN) and male SPF cats (Vendor - Cedar River Laboratories, Mason City, IA). Four queens were donated by the University of Georgia (UGA) in fall 2019, which were purchased by UGA from Liberty Research Inc (Waverly, NY). Juvenile cats were generated by accidental mating of the donated queens with the three SPF toms at a UF laboratory, with the goal to outbreed the inbred SPF colony for higher fertility with larger litter size. The eight group-housed laboratory cats were initially purchased from Liberty Research, Inc. However, their animal code did not show the same lineages as the UGA queens and were considered different cat lineages (i.e., different origin), unrelated to both UGA queens and UF toms. All cats purchased from Liberty Research, Inc. were vaccinated against rabies, using the RABVAC3 vaccine, and against feline panleukopenia (distemper), calici, and rhinotracheitis (herpes) viruses, along with hemorrhagic feline calicivirus strain, using the Fel-O-Vax PCT + Calicivax vaccine.

[0082] Table 1 : the study populationSARS-CoV-2 RBD peptides

[0083] Two different versions of SCoV2, University of Florida-RBD (UF-RBD, SEQ ID NO. 1 ) and MassBiologic-RBD (MB-RBD, SEQ ID NO: 3) were used. The UF-RBD was produced using Harvard Wuhan RBD plasmid21and expressed in EXPI293F cells. An SCoV2 RBD set forth in SEQ ID NO: 9 and SEQ ID NO: 10 was also used.

[0084] Feline cell lines: Crandell feline kidney (CrFK) fibroblast, felis catus 9 (Fc9), and felis catus whole fetus-4 (Fcwf-4) cells were provided by Dr. N.C. Pederson of the University of California, Davis. All of these feline cell lines were maintained on Eagle MEM media with 1 .5 g / L sodium bicarbonate, NEAA, L-glutamine, and sodium pyruvate supplemented with 10% fetal bovine serum (FBS) with 50 pg / mL gentamycin. These cells were maintained at 37°C with 5% CO2 and passaged every 2-3 days using 0.25% Trypsin-EDTA (Gibco-LifeTechnologies, Grand Island, NY). In order to prevent changes in growth patterns, these cells were culture amplified and then cryopreserved in liquid nitrogen tanks in multiple vials.

[0085] Production and partial purification of FCoV2 whole-virus: The CrFK cells were infected with FCoV2 WSU79-1146, purchased from ATCC to produce the stock of crude FCoV2 inoculum for in vitro infection studies, and partially purified FCoV2 whole-virus (WV) stock for ELISA and immunoblots. The infected culture fluids directly from the culture flask (175 cm) were pooled and clarified free of cell debris by low-speed centrifugation at 2800- 3000 rpm for 45 min at 5°C. In addition, the freeze-thawed culture fluids from the frozen flasks with residual media were pooled and then clarified free of cell debris by low-speed centrifugation at 2800-3000 rpm for 45 min at 5°C. They were then combined with a portion of the clarified culture fluid from above, at one part clarified direct culture fluid to two parts of clarified cell-debris fluid. Both direct and combined clarified fluids were partially purified by the following three methods. In the first method, the clarified fluid was purified by ultracentrifugation at 16,000 rpm for 2 h at 5°C to obtain pooled virus pellets. The pellets then underwent multiple washes with PBS, while being concentrated by Vivaspin 20 Centrifugal Concentrators (Satorius, Gottingen, Germany) with PES membrane of 100k molecular weight cut off (MWCO). In the second method, the combined clarified fluid was directly concentrated by Vivaspin 20 Centrifugal Concentrators by 10-50-fold, while the concentrated virus fluid underwent additional 3-5 washes with PBS using the Centrifugal Concentrator. The multiple washes with PBS using the Centrifugal Concentrator 100k were performed until the red color of the phenol red was not present in the preparation from the first method. This method resulted in a FCoV2-WV stock preparation with no detectable residual BSA (67 kDa), but the virus titer (670 pg / mL) was lower than the second method and had undetectable 180 kDa S glycoprotein. The second method resulted in a preparation with an extremely light pink color from the phenol red of the media, but a reasonably high FCoV2 load (1 .0 mg / mL) with detectable levels of 180 kDa S glycoprotein. This method was used the most for immunoblot production. The last method consisted of direct concentration of direct-clarified fluid using multiple Centrifugal Concentrators with minimal PBS washes. As a result, this preparation retained the phenol red color and about 5% residual BSA from FBS. This method provided a virus preparation at a high concentration (150 mg / mL), needed for use in screening a large number of cat sera with FCoV2-WV ELISA.

[0086] Transfection and expression of Expi293F Cells with SCoV2, FCoV1 , and FCoV2 RBD plasmids and purification of RBD proteins: The plasmids pVRC containing RBD constructs were transiently transfected into Expi293F cells using the ExpiFectamine 293 Transfection Kit (Thermo Fisher, Waltham, MA). Briefly, the cell density was adjusted to 3x106cells / mL in a final volume of 100 mL of Expi293 expression media and allowed to grow24 h to reach a final density of 5.5x106cells / mL. The plasmid DNA (1 pg / mL) and ExpiFectamine 293 reagent were individually diluted with Opti-MEM Reduced Serum Medium (Thermo Fisher, Waltham, MA), incubated 5 minutes at room temperature, and then mixed together. The ExpiFectamine 293 / plasmid DNA mixture was incubated at room temperature for 20 minutes and mixed with Expi293F cells. The cells were incubated on an orbital shaker in a 37°C incubator with 8% CO2. After 24 h of incubation, transfection enhancers-1 and 2 were added and incubated for three days. Then, the cell culture was centrifuged at 1800 x g for 30 min to collect the supernatant for protein purification. The culture supernatant was concentrated to a final volume of 5 mL using a Macrosep Omega Advance Centrifugal Device with a cutoff of 10 kDa (PALL Laboratory, Port Washington, NY). The concentrated supernatant was passed through equilibrated TALON Metal Affinity Resin (Takara Bio Inc, Shiga, Japan). The column was washed with 10 volume of PBS containing NaCI (300 mM) and imidazole (20 mM) to remove all contaminants.Subsequently, the protein was eluted from the column using PBS containing 250 mM imidazole. The eluted fractions were concentrated using a Nanosep Advance Centrifugal Device with 10K Omega (PALL Laboratory). Estimation of protein purity and quantity were achieved with SDS-PAGE and a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL), respectively.Enzyme-linked immunosorbent assay (ELISA)

[0087] FCoV whole-virus and SCoV2 RBD ELISAs with overnight serum incubation: ELISA plate (Costa, Corning, NY, USA) wells were coated with 100pL of 100 pg / mL FCoV2 whole virus antigen or 100 pg / mL SCoV2 RBD or FCoV RBD antigen in sodium bicarbonate ELISA coating buffer, pH 9.5 (BioLegend, San Diego, CA) and incubated overnight at 4 °C. The next day, the plates were washed three times with phosphate-buffered saline tween (PBST). Non-specific binding sites were blocked with 100 pL per well of blocking solution (5% skimmed milk in sterile PBST- 0.5% Tween-20) for 1 h at 37 °C. After washing with PBST three times, 50 pL of cat sera was diluted to 1 :100 with the blocking solution and incubated at room temperature overnight. After washing with PBST three times using the BioTek ELx-405 plate washer, horseradish peroxidase-conjugated goat anti-cat IgG (HRP- anti-cat-IgG) diluted 1 :4,000 (SouthernBioTech, Birmingham, AL) in PBST was added and incubated at room temperature (RT) for 2 h. After washing, 100 pL of 3, 3,5,5- tetramethylbenzidine (TMB) substrate solution (BioLegend) was added to the wells and incubated at RT for 15 min, the reaction was stopped by adding 100 pL of 1 N HCI in sterile water as stop solution.The ELISA titer was measured at OD450 using BioTek’s Synergy HTX Multi-Mode Microplate Reader (BioTeK, Winooski, VT, USA).

[0088] Stringent FCoV whole-virus and SCoV2 RBD ELISAs with one hour serum incubation: To ensure that the serum ELISA reactivity was specific to the FCoV2 wholevirus, FCoV-RBD or SCoV2-RBD antigen, sera from all four LIGA queens and three toms were incubated individually at the same dilution for only 1 h, instead of overnight. PBS was used instead of bicarbonate buffer for coating of the antigen on the ELISA plates. In addition, a bovine serum albumin (BSA) antigen control was included since veterinary vaccines used at the time the UGA queens were contained contaminating BSA, most likely from the cell cultures used during the manufacture of the viral vaccines. The BSA control was also important because the FCoV2 whole-virus preparation contained about 5% BSA, whereas the RBDs were highly purified and devoid of BSA.

[0089] Immunoblot analysis using FCoV2-WV, SCoV2 UF-RBD, FCoV1 RBD, and FCoV2 RBD as antigen substrate: The purified proteins were analyzed by SDS-PAGE and Western blot, also called immunoblot. Briefly, the proteins (100 pg) were boiled at 95°C for 5 min in a sample buffer (bromophenol blue, glycerol, reducing agent p-mercaptoethanol, and SDS in Tris-HCL buffer). They were loaded evenly into a single, 7-cm wide well of the stacking gel, with one 4-mm wide well at the end for pre-stained marker, and separated by 10% Tris-HCL gel with 30% or 40% acrylamide / bis. The proteins were transferred to nitrocellulose membrane for immunoblot analysis. Each nitrocellulose blot was cut vertically into 263.2- mm width strips using a Novex Model NZ-1 CIS membrane strip cutter (Novel Experimental Technology, San Diego, CA). Each mini-gel provided 21-22 strips of the antigen (RBD or FCoV2 whole-virus), with two strips on one end containing the Precision Plus Protein All Blue Pre-stained Standards (Bio-Rad). Two strips on the other end were discarded for lack of or uneven antigen presence. Each immunoblot strip was incubated individually with FCoV- infected cat serum or plasma at a dilution of 1 :50 or 1 :100 in a blocking buffer containing 5% non-fat dry milk in PBST and incubated overnight at RT on a rocker. After three washes, the strips were then incubated with alkaline phosphatase (AP)-conjugated goat anti-cat IgG (1 :1 ,000) (SouthernBiotech) for 2 h at RT. Subsequently after three washes, the reactive bands were visualized with freshly mixed AP substrate reagents from AP-Conjugate Substrate Kit (Bio-Rad). Upon drying, the strips were aligned and scanned at a designated brightness and contrast.

[0090] FCo V2 neutralizing antibody (NAb) assay and RBD blocking assay against FCo V2:The Fc9 cells were used for both FCoV2 neutralizing antibody studies and FCoV2 / SCoV2 RBD blocking studies against live FCoV2 infection. The FCoV2 NAb assay was a modification of FCoV2 NAb assay described previously.23The modification consisted of a 96-well round-bottom microculture plate system where the diluted FCoV2 preparation (EMEM culture media with 5% heat-inactivated FBS) at 2 TCID50 were incubated with equalvolume of serially three-fold diluted cat serum (EMEM culture media at 5% heat-inactivated FBS). The plates were incubated at 37°C in a 5% CO2 incubator for 45 min. Note that the first row of the 12 wells were not used in order to prevent the drying effect on the 12 edge wells with the most concentrated serum dilution. Thus, the most concentrated serum dilutions of 1 :6 with the virus preparation (i.e., 1 :3 serum dilution with 1 :2 virus dilution) were in the second rows. The remaining serum dilutions in the wells were as follows: third rows with 1 :18, fourth rows with 1 :54, fifth rows with 1 : 162, sixth rows with 1 :486, seventh rows with 1 :1458, and eighth rows with 1 :4374). Subsequently, 0.1 mL of the mixture of each well was transferred to the flat-bottom wells of Fc9 cell monolayer with 95-97% confluency and incubated for 24 h at 37°C and 5% CO2. The spent culture fluids were discarded. We then added 0.1 mL of 0.25% sterile methyl cellulose in EMEM with 5% heat-inactivated FBS to each well of the plate, and incubated at 37°C in 5% CO2 for 18 h or until the 35-50 virus plaques per well were observed in the duplicate virus control wells. The FCoV2 plaques in the wells were inactivated and stained with 0.1 mL / well of 1% crystal violet in 100% methanol at RT for 10 min. We then added 0.15 mL of 1% crystal violet in 20% methanol in each well, incubated at RT for 24 h, decanted, and removed the stain with water. The RBD blocking assay against FCoV2 is a modification of the above FCoV2 NAb assay. This assay differes from NAb assay by the following three features: 1) A set amount of RBD is used instead of cat serum. 2) FCoV2 dose was 4 TCID50 instead of 2 TCID50 to assure 100% cytopathic effect (CPE) on the Fc9 cells. 3) The flat-bottom plates with Fc9 cells at 98-100% confluency instead of 95-97% confluency were used at the time of adding the virus mixture.

[0091] Molecular Weights of RBDs: Based on the gel analysis using Coomassie Blue staining and anti-penta His monoclonal antibody (MAb)-treated RBD-immunoblot (data not shown), the approximate molecular weights of the coronavirus RBDs have been determined and confirmed also by the banding patterns of the immunoblots at 10% Tris-HCL gel with 30% acrylamide / bis compared to 40% acrylamide / bis. Both the gel staining and immunoblot results contained the RBDs with histidine (His)-tag sequence. These results demonstrate that the SCoV2 RBDs with tag (SEQ ID NO: 2), FCoV1 (SEQ ID NO: 6), and FCoV2 (SEQ ID NO: 8) are all highly glycosylated (Fig G1 B, glycan MW is subtraction of two MW). All of these RBD pDNA, except for MB-RBD plasmid, expressed 70 aa tag residues (7,454 kDa) resulting from the HRV cleavage site, 8x His-Tag, and streptavidin-binding peptide (SBP)- Tag. Hence, approximately 7,454 kDa should be subtracted from all RBDs that used the same plasmid for expression. The approximate molecular weight (MW) of the RBD alone (minus tag sequences) are the following for SCoV2 UF (gp30; SEQ ID NO: 1 ), FCoV1 (gp52; SEQ ID NO: 5), and FCoV2 (gp59; SEQ ID NO: 7) .

[0092] For example, SCoV2 Wuhan LIF-RBD without 70 aa tag residues will result in 211 aa residues of 23,750 kDa without glycosylation, and with glycosylation about 29,564 kDa (round up to gp30) (SEQ ID NO: 1 with tag and SEQ ID NO: 1 without tag). The SCoV2 Wuhan MB-RBD without two tags of 16 aa residues and 6x His) will result in a peptide of 223 aa residues which has a MW of 25,099 kDa without glycosylation and with glycosylation of about 30,992 kDa (gp31 ; SEQ ID NO: 3). Although the number of aa residues is higher with SCoV2 MB-RBD, the IIF-RBD has higher MW on the gel and immunoblot (data not shown) due to the 70 aa sequence of the tags. The FCoV1 RBD has a solid band separately above the heavily glycosylated thick band as shown with a red arrow (data not shown).

[0093] Construction of plasmid with minimalized FCoV1 spike glycoproteins (pCI-H2B- GFP-FCoV1 Wuhan-W1F11w and pCI-H2B-GFP-FCoV1UCD1-Ori). The pCI-H2B-GFP- FCoV1 LICD1 -Ori is the first generation minimalized FCoV1 LICD1 spike, consisting of only essential proteins RBD-HRT1 -SH-HRT2-TM-CT, whereas the FCoV1 UCD1 -2BN has additional shortening of the N-terminal end of its RBD consisting of sRBD-HRT1-SH-HRT2- TM-CT. These sequences were individually cloned at Nhel and Pstl sites of pCI-H2B-GFP to obtain pCI-H2B-GFP-SCoV1 UCD1-2BN and pCI-H2B-GFP-FCoV1 UCD1-Ori. Both construct plasmids have green fluorescent protein (GFP) sequence insert which will be used to assess the level of in vitro plasmid expression in species-specific cell line.

[0094] The full-length FCoV1 LICD1 spike (Genbank Accession No. AB088222) has been decreased from 1457 amino acids (aa) to 496 aa for FCoV1 UCD1 -Ori and to 394 aa for FCoV1 LICD1 -2BN. Furthermore, additional 102 aa has been deleted predominantly from the N-terminal end of the RBD from the FCoV1 LICD1 -Ori structure to obtain FCoV1 LICD1 - 2BN structure. Such major aa deletions were conducted to remove potential inflammatory and adverse reaction regions, but these minimalized constructs were designed to retain the native structure that will combine into a trimeric structure when expressed. The native trimeric structure, as a B-cell immunogen, is essential in inducing the broadly neutralizing antibodies (bNAbs) against all FCoV1 strains.

[0095] GFP Expression of pCI-H2B-GFP-FCoV1 constructs by Flow Cytometry. In order to determine if such plasmids will express in feline cells, Fells catis embryonic-9 (Fc9) cells at 70-85% cell confluency in 25cm2flasks in 3-mL of fresh Corning EMEM culture media were transfected with various amounts of either pCI-H2B-GFP-FCoV1 LICD1 -2BN or pCI- H2B-GFP-FCoV1 UCD1-Ori in Takara’s Xfect Polymer. Transfection was performed using Takara’s Xfect Transfection Reagent (Cat. #631317) according to their protocol. The EMEM culture media contained 5% heat-inactivated fetal calf serum (FCS) and 50 pg / mL of gentamycin. After 4 hr of incubation in 5% CO2at 37°C, the spent plasmid-containing culture media was decanted and replaced with 3-mL of fresh culture media and incubated forplasmid expression. The plasmid expression was stopped upon 48 hr of incubation. The expressed fluids were individually collected in sterile 15-mL tubes to be further processed by Immunoblot Analyses. The cells were washed thrice with sterile PBS and treated with EDTA-trypsin to remove the adherent cells from the flask at 37°C for 2 min. Trypsinization reaction was stopped with equal amount of culture media, and the content pipetted into sterile 15-mL tube. The cell suspension was centrifuged at 1700 rpm for 3.5 min to obtain a cell pellet. Upon discarding the fluid, the cell pellet was resuspended in 1-mL of PBS and pipetted into a sterile 1 ,8-mL microfuge tube to perform a rapid (30 sec) centrifugation in a microfuge (first wash). One more PBS wash was performed before the cells were suspended in a fresh PBS containing 4% paraformaldehyde and stored at 5°C for 20 min. Subsequently, the cells were washed twice and resuspended in PBS to be stored at 5°C until the next day when the cells were evaluated for GFP expression using flow cytometer. The GFP in the plasmid will determine the level of transfection and replication of transfected cells.

[0096] Immunoblot Analyses of the Culture Fluid from pDNA-FCoV1 Transfected Fc9 Cells. The fluids containing the plasmid-expressed proteins were centrifuged at 2000 rpm at 5°C for 45 min to remove the cell pellet from each fluid preparation. The cell-free fluid was individually concentrated with Sartorius Vivaspin 6-mL column (15-mL tube column) of molecular weight cut off (MWCO) of 30K by centrifugation at 1800 rpm in 5°C for 25 min. Each concentrate was transferred to individual sterile 1 ,8-mL microfuge and frozen immediately at -80°C in ultra-low freezer. After first thaw, the samples were aliquoted into multiple sterile microfuge tubes of smaller quantities to be store at -80°C, while the concentration of one vial from each sample was measured by Qubit 4 Fluorometer. Eight pg of each sample was loaded into each well of the 10-well SDS-PAGE mini-gel (30% acrylamide, under reducing condition). The gels were electrophoresed at 80 volts until the gel front of the 25kDa red band were at the bottom edge of the gel. The gels were then transferred onto the commercial mini-gel nitrocellulose membrane for 2.0-2.5 hr at 120 volts for 2 hr or until all Precision Plus Protein (Dual Color 25kDa & 75kDa red) Standards transferred completely. The immunoblots in the individual sterile square petri-plate were incubated on a rocker at room temperature (RT) for overnight with 12-mL of reacting serum at the concentration described on the figures (1 :50 or 1 :30 dilution in 5% skin milk blocking buffer). The plates were washed with washing buffer and incubated with Serotec’s goat alkaline phosphatase (AP)-conjugated anti-human IgG or goat AP-conjugated anti-cat IgG at 1 :500 dilution in blocking buffer for 2 hr on a rocker at RT. Upon washing thrice with washing buffer, each blot was reacted with Bio-Rad AP substrate for 7-10 min. The stained immunoblots are pressed dried before scanning the blots into 1200 dpi TIFF files which wereevaluated and diagrammed with PPT. The immunoblot strips for whole-virus FCoV1 used the identical procedure described in our previous publication (Yamamoto JK, et al. Viruses 15(4), 914, 2023) on the production and band development of the whole-virus FCoV2 RBD immunoblot strips.Example 1 - Crossreactivitv of FCOV2 antisera with SCOV2 RBD peptides

[0097] The following example describes serological assays performed to detect FCoV2 seroconversion in FCoV naturally transmitted laboratory cats and cross reactivity of FCoV2 antisera with SCoV2 RBD (SEQ ID NO: 1 , 3, 9 and 10).

[0098] FCoV2 whole-virus and SCoV2 RBD ELISA: Studies on FCoV were initiated when three SPF inbred toms at a UF laboratory developed minor episode(s) of diarrhea after accidentally mating with four laboratory queens from UGA. The four juvenile laboratory cats born from two UGA queens (UGAQ3, UGAQ4) had FCoV antibodies at 12 and 16 weeks of age based on FCoV2 whole-virus ELISA. The UGA queens were all seropositive for FCoV2 (Fig. 2A) and the sera from the toms, after mating, were weakly seropositive for FCoV2 (Fig. 2B). Anti-sera from three of the four juvenile cats cross-reacted moderately with the SCoV2 RBD tested by SCoV2 RBD ELISA (Figure 2B).

[0099] Stringent FCoV whole-virus ELISA and SCoV2 RBD ELISA: Two UGA queens(UGAQ1 , UGAQ4) had high levels of serum reactivity with FCoV but no reactivity with BSA and SCoV2 RBDs (SEQ ID NOs: 1 , 2, and 5), while the other two queens (UGAQ2, UGAQ3) had high serum reactivity with FCoV, which was slightly higher than the serum reactivity with BSA (Figure 3A). The sera from UGAQ2 also had substantial cross-reactivity with both SCoV2 RBDs, whereas the sera from UGAQ3 had modest cross-reactivity with MB-RBD and below the threshold cross-reactivity with UF-RBD.

[0100] As expected, the sera from all toms before mating had no reactivity with FCoV, both UF / MB-RBDs (Figure 3B), and BSA (BSA, Figure. 4). However, post-mating sera from all toms had significant reactivity with SCoV2 RBDs but not with FCoV, when compared to the corresponding pre-mating control results. The highest titers to SCoV2 RBDs were observed at the earliest time point of serum collection, closest to the first exposure to the FCoV-positive queen. Subsequent sera showed declines which were still significantly different from the pre-mating-serum titers (p<0.05). The decline suggested that the development of cross-reactive antibodies to SCoV2 RBD could have occurred during active FCoV infection. A slight conflict was observed between the substantial reactivity with FCoV for the toms (Figure 2A) and no reactivity with FCoV for the toms (Figure 3B). Overall, the sera from all three toms cross-reacted strongly with SCoV2 RBD by a stringent ELISA. Onlyone long-term FCoV infected queen UGAQ2 also had high titer of cross-reactive antibodies to SCoV2 RBD (Figure 3A).

[0101] Juvenile cat studies: The juvenile cats were most likely seropositive for FCoV either by passive maternal FCoV antibodies from their FCoV-infected mothers (UGAQ3, LIGAQ4) or became actively infected with FCoV transmitted from their mother. It has been reported that kittens weaned away from their FCoV-infected mothers at 4-5 weeks of age remain free of FCoV infection, but not those after 5 weeks of age.24. The sera from one juvenile cat (Y2B), born from LIGAQ3, and three juvenile cats (D4A, D4D, D4F), born from LIGAQ4 (Table 1), were monitored for FCoV and SCoV2 RBD antibodies at 12-16 weeks and 16-20 weeks, respectively. Only D4D showed a sign of active FCoV infection based on the increase in antibodies cross-reacting with SCoV2 RBD by ELISA (Figure 5A). Immunoblot analysis results for serum reactivity to SCoV2 RBD from D4D demonstrates stronger cross-reactivity to SCoV2 RBD, when compared to those of the remaining three juvenile cats (Figure 5B).

[0102] Example 2 - Identification of FCoV1 and FCoV2 RBD Peptides

[0103] The following example describes the identification, selection and production of the FCoV1 and FCoV2 RBD peptides that were used in the serological assays.

[0104] FCoV1 and FCoV2 RBDs: The location for FCoV1 and FCoV2 RBDs has recently been predicted to be around residues 526-676, based on RBD sequence locations of porcine enteric diarrhea virus (PEDV) RBD at B residues 510-640 and testicular gastrointestinal enteric virus (TGEV) RBD at D3 residues 500-651 .25-27The TGEV RBD has been reported to bind to the porcine APN as its primary host cell receptor,27 28but whether PEDV RBD binds to pAPN as its host cell receptor is still controversial.2629The RBD sequence prediction of both PEDV and TGEV was based on monoclonal antibody (MAb) studies, identifying the most potent neutralizing MAb(s) to these porcine alphacoronaviruses reacting to B and D3 residue regions, respectively.2627

[0105] Since species-specific ACE2 is used by SCoV2 to infect cats, dogs, and humans, we reasoned that the FCoV1 and FCoV2 RBDs could be identified by aligning the LIF-RBD and MB-RBD sequences against four known FCoV1 S1 sequences and against four known FCoV2 S1 sequences. The Wuhan SCoV2 RBD sequence is distinctly different in aa sequence from the RBD sequences of FCoV1 (11 .5% & 12.3% identity; 31 .8% & 33.6% similarity) and FCoV2 (12.2% & 12.3% identity; 36.5% & 37.7% similarity), the first value is based on IIF-RBD, and the second value is based on MB-RBD. Hence, the locations for our proposed FCoV2 and FCoV1 RBDs may be in the vicinity exposed to the effect of immune pressure.

[0106] The full length aa sequence comparison of S glycoproteins of the SCoV2 Wuhan strain and FCoV2 WSU79-1146 strain displays the S1 / S2 cleavage site for SCoV2 at a location different from the S1 / S2 cleavage site for FCoV2.30-32In addition, the SCoV2 RBD sequence alignment pattern with single FCoV2 RBD sequence of the full length S protein sequence differs slightly from the one aligned with four FCoV2 RBD sequences (data not shown), even though both analyses used the JusBio alignment server. The full-length S sequences between SCoV2 and FCoV2 show the RBD has the least sequence similarity followed by the NTD and then C-terminal domain with the most similarity or conservation among two S1 domains (Table 3). This pattern was the same whether the S1 / S2 cleavage site for SCoV2 or FCoV2 was used. Furthermore, S2 sequence had the most aa sequence conservation between SCoV2 Wuhan and FCoV2 WSU79-1 146.

[0107] Table 3: Summary of amino acid (aa) sequence identity and similarity between SCoV2 and FCoV2.

[0108] Next, the full-length S sequences of FCoV1 UCD-1 strain were compared to the S sequence of the SCoV2 Wuhan strain (Table 4) to determine if similar changes occur as those observed when comparing the SCoV2 and FCoV2 (Table 3). Major changes in gap location are observed on the SCoV2 sequence when compared to the single FCoV1 sequence or to the four FCoV1 sequences (data not shown). The S1 / S2 cleavage site for SCoV2 is only 46 aa plus five gaps away from the counterpart S1 / S2 cleavage site for FCoV1.30’31Conversely, the FCoV1 S1 / S2 cleavage site is 37 aa plus 14 gaps from the counterpart SCoV2 cleavage site.30Thus, the S1 / S2 cleavage sites are in closer proximity between SCoV2 and FCoV1 than those between SCoV2 and FCoV2. The most aa sequence conservation is observed at the S2 sequence, followed by S1 CTD, S1 NTD, and then S1 RBD, which is identical in pattern to those observed between SCoV2 and FCoV2. These results suggest that NTD between SCoV2 and FCoV1 / FCoV2, with the second least similarity (Table 4), may be structurally also exposed in the trimeric configuration allowing for more changes due to host immune pressure.33 34

[0109] Table 4: Summary of amino acid (aa) sequence identity and similarity between SCoV2 and FCoV1 .

[0110] Overall, the above results from sequence analyses suggest that the RBD for SCoV2 infection of cats may reside at a similar site as the RBD for SCoV2 infection of humans, with possibly similar contact aa residues in the receptor binding motif (RBM) to react to fACE2 and hACE2, respectively.Example 3 - Detection of cross reactivity of cat anti-sera with FCoV2 RBD by immunoblottinq.

[0111] FCoV2 RBD (SEQ ID NO. 7), with the most cross-reactive sectional regions overlapping with SCoV2 RBD (SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 9 and SEQ ID NO: 10) was produced in the same expression (EXPI293F cells) system as SCoV-2 IIF-RBD (SEQ ID NO: 1), purified similarly as SCoV2 IIF-RBD (SEQ ID NO: 1 ) in PBS, and used to develop the immunoblot strips. Two sera (UGAQ2, UGAQ4) from the four queens and the serum from all three toms strongly cross-reacted with the SCoV2 UF-RBD (Figure 6a) but none of them reacted with FCoV2 RBD (Figure 6B). All sera from the queens reacted with FCoV2 whole-virus immunoblot strips strongly at the membrane (M, 28-32kDa), nucleocapsid (NC, 43kDa), degraded spike S2 (80-90kDa), and whole spike S (190kDa) (Figure 6C), with weaker bands at 20kDa, 22kDa, 40kDa, and 55kDa except of UGAQ1 . The single serum available from LIGAQ1 was collected when she was severely sick from uteritis and on antibiotics. Her serum only reacted to FCoV2 whole-virus at 90kd and higher (Figure 6C) but not to SCoV2 (Figure 6A), FCoV2 (Figure 6B), and FCoV1 (Figure 6D) RBDs. The toms had reactivity to NC, M, and proteins at 10kDa, 20kDa and 22kDa but no reactivity to whole-virus S proteins and bands above 90 kDa except for HOGT3 at 2 months post-mating. The same sera from the toms did not react to FCoV2 RBD except for one tom (HOGT3) which reacted weakly with FCoV2 RBD (Figure 6B) and without FCoV2 NAb titer (Figure 2A) but strongly with FCoV1 RBD (Figure 6D). Preliminary results demonstrate that the serum from three UGA queens (UGAQ2, UGAQ3, UGAQ4) and all three toms reacted with FCoV1 RBD (Figure 6D). The nil-to-weak cross-reactivity to FCoV2 RBD and strong reactivity toFCoV1 RBD suggest that our toms and queens were infected with FCoV1 which is the most common serotype in the U.S.

[0112] The observation that FCoV1 infected cats cross-reacting with SCoV2 RBD was unexpected, since the amino acid sequence similarity is slightly more conserved between SCoV2 and FCoV2 than with FCoV1 (data not shown). Their sera also had nil-to-minimum titers of FCoV2 neutralizing antibodies which further supports FCoV1 infection of our queens and toms (Figure 2A).

[0113] The sera from all four queens and three toms were evaluated for their crossreactivity to SCoV2 UF-RBD immunoblot strips. Only sera from UGAQ2 and all three toms had strong cross-reactivity to SCoV2 RBD. Note that UGAQ4 was moderately cross-reactive to SCoV2 RBD only at the first blood collection (10mo post-mating). The fact that the sera from UGAQ2 and all three toms strongly reacted with SCoV2 UF-RBD further confirms that their sera do cross-react with SCoV2 UF-RBD (SEQ ID NO: 1 ) and MB-RBD (SEQ ID NO: 3) even under stringent ELISA (Figure 3A: UGAQ2; Figure 3B: 5HQT1 , HOJT2, HOGT3). The cross-reactive antibodies reacted to SCoV2 RBD immunoblot strips produced under a reducing condition, which suggests that these antibodies are reacting to linear peptide(s), glycan, or both.Example 4 -In vitro FCoV2 infection blocking activity by FCoV2 RBD and SCoV2 RBDs

[0114] The following Example determines whether the FCoV2 RBD is capable of blocking the FCoV2 infection of the feline cell line (Fc9 cells). Duplicate wells, starting at 2.86 pg / mL, were serially diluted three-fold for each well, up to the titer of 0.106 pg / mL (fourth duplicate wells) and presented <50% cytopathic effect (CPE), whereas all wells for virus control had 100% CPE including the PBS controls. Thus, one TCID50 titer, the titer with 50% CPE, is between 0.035-0.106 pg / mL (data not shown). Another assay demonstrated 1 TCID50 titer of 50% blocking observed at about 0.100 pg / mL of FCoV2 RBD (data not shown). The SCoV2 RBD at 64.2 pg / mL blocked 100% of FCoV2 infection (first duplicate wells), whereas 42.8 pg / mL blocked 30% CPE. Both FCoV2 and SCoV2 RBDs caused no cellular toxicity (data not shown). All plates were photographed at 100% brightness and 65% contrast. Overall, 0.1 -1.0 pg / mL of FCoV2 RBD blocked 50%-100% of FCoV2 infection, respectively; whereas 48-64 pg / mL of SCoV2 RBD cross-blocked 50%-100% of FCoV2 infection, respectively, at doses without any cellular toxicity. The data provided in this Example confirms that FCoV2 RBD sequence (SEQ ID NO: 8) includes the RBD site associated with FCoV2 infection.Discussion

[0115] Cross-reactive antibodies to SCoV2 RBD developing in cats during active FCoV infection was totally unexpected. The peak sera from UGAQ2 and all three toms cross-reacted strongly with sensitive SCoV2 RBD ELISA and immunoblot, but not with FCoV2 RBD (Figures 6A and 6B). This observation indicated that the cats were most likely infected with FCoV1 . In addition, all toms lacked FCoV2 whole-virus ELISA titer except for one tom (HOGT3) at post-9 months FCoV contact infection (Figure 3). This observation supported the almost nil-to-low cross-reactivity with FCoV2 whole-virus antigen in the less stringent FCoV2 ELISA, using overnight serum incubation at 1 :100 dilution (Figure 2A). At 1 :50 serum dilution of FCoV2 whole-virus immunoblot analysis, the sera from the toms also reacted to FCoV2, but only minimally-to-moderately at NC and M but not at S glycoproteins. This suggests that FCoV infection was most likely low in titer in these toms. In addition, it has been reported that M glycoproteins are expressed most during in vitro infection followed by NC proteins and then S glycoproteins3536. Hence, the FCoV2 antigen levels on the immunoblot may also reflect such antigen distribution.

[0116] Perhaps the most striking observation was the sera from the toms cross-reacting to SCoV2 RBD (SEQ ID NO: 1) without reacting to FCoV2 S glycoprotein on the FCoV2 whole-virus immunoblot (Figures 6A and 6C). These results indicate that cross-reactive antibodies to SCoV2 RBD appeared without major development of cross-reactive antibodies to FCoV2 S glycoprotein, including S2 glycoprotein at most timepoints, if they were indeed infected with more common FCoV1 . None of the cats (queens, toms, and their first- generation kittens) developed major neutralizing antibodies to FCoV2 based on FCoV2 WSU79-1146 neutralization assay on feline Fc9 cell line (Figure 6D). This result further supports that the cats were infected with FCoV1 . The fact that the immunoblots used in the experiments described herein were developed under reducing conditions suggests that the cross-reacting epitopes on SCoV2 RBD are either linear amino acid epitopes and / or glycosylated epitope(s).

[0117] The lack of serum cross-reactivity with FCoV2 Spike glycoproteins by the toms was unexpected since all four queens, including UGAQ2, reacted with FCoV2 S glycoprotein (Figure 6C). Since S2 glycoprotein has high amino acid sequence identity and similarity, the likelihood of the cross-reactivity with S2 of the S glycoprotein may be a strong possibility based on the study by Zhao et al. 2007.33Table 5 below provides a summary of similarity and sequence identity of SCoV1 and SCoV2 S1 and S2 spike glycoproteins.

[0118] Table 5: Summary of Amino Acid (AA) Sequence Identity and Similarity between SCoV2 and SCoV1 S1 or S2 spike glycoproteins.

[0119] The analysis herein shows the NTD as the least conserved domain with 80.4% similarity, and the S2 glycoprotein as the most conserved domain with 90% identity and 98.5% similarity. Our comparative analysis also shows RBD to be slightly more conserved than NTD but still less conserved than the CTD. Therefore, our finding that FCoV-infected cats develop cross-reacting antibodies to SCoV2 RBD was greatly unexpected when their study showed no cross-reactivity to SCoV1 S1 glycoprotein, since SCoV1 and SCoV2 have 64.8% identity and 87.9% similarity (Table 5).

[0120] Remarkably, the study demonstrated that SCoV2 UF-RBD at a high concentration of 48 pg / mL was able to cross-block or cross-protect against FCoV2 infection of feline cell line at 1 TCID50 dose without any cellular toxicity. The ability of FCoV2 and SCoV2 RBDs to block in vitro FCoV2 infection also suggest that these RBDs may be important for developing an effective pan-coronavirus vaccine for pet animals such as cats, dogs, and hamsters. SCoV2 infected hamsters from Europe imported to pet shops in Hong Kong have been reported as a source of two separate hamster-to-human transmissions and subsequent human-to-human transmission of SCoV2 Delta variant, with the sequence found predominantly in Europe.37Inoculation of laboratory Syrian golden hamsters resulted in infection of the hamsters with major loss in weight, lung infection, and respiratory disease.38-42

[0121] Profound acute SCoV2 infection of humans causes gastrointestinal (Gl) manifestation (diarrhea, vomiting, nausea, abdominal pain), including longer fecal shedding than those detected in the nasopharyngeal samples.43-46Both FCoV and CCoV also cause Gl tract disease in their respective animal hosts and clinically affect the kittens and puppies more than adults, with the exception of FIPV disease.5’4447FCoV2 RBD sequence alignment comparison of CCoV serotype 2 (CCoV2) and FCoV2 RBDs shows 95.9% amino acid sequence similarity and 87.7% amino acid sequence identity (Table 6).

[0122] Table 6: Summary of Amino Acid (AA) Sequence Identity and Similarity between CCoV2 and FCoV2.

[0123] The high sequence similarity between FCoV2 and CCoV2 RBDs may explain why CCoV2 can infect fAPN expressing feline cells28. Although FCoV1 and CCoV1 RBDs possess only amino acid sequence identity of 55.5%, their sequence similarity of 80.8% is remarkably high (Table 7), suggesting that they have a common lineage with evolutionary changes and perhaps also by sectional recombination.34’4348

[0124] Table 7: Summary of Amino Acid (AA) Sequence Identity and Similarity between CCoV1 and FCoV1

[0125] The most dissimilar sequence region is in the NTD of the S1 sequences between the FCoV1 and CCoV1 and between the FCoV2 and CCoV2, while the most conserved region is found beyond RBD at the carboxyl-end of S1 and with the S2 sequences (Tables 6 and 7). These findings suggest that a composition developed by combining FCoV1 RBD (SEQ ID NO: 5) and FCoV2 RBD (SEQ ID NO: 7) together with SCoV2 RBD (SEQ ID NO: 1 , SEQ ID NO: 3 and SEQ ID NO: 10), optionally including the addition of CCoV1 / CCoV2 (SEQ ID NOs: 11 and 12, respectively) RBDs, is effective in inhibiting SCoV2 infection in cats, dogs, and hamsters.Example 5 - Anti-CoV RBD IFNy and IL-2 ELISpot responses

[0126] Anti-CoV RBD IFNy and IL-2 ELISpot responses of the peripheral blood mononuclear cells (PBMC) from a COVID-19 vaccinated subject Y3. Subject Y3 has been vaccinated five-times with COVID-19 vaccine since 2020-2022. The last vaccination consisted of Moderna bivalent COVID-19 vaccine but those prior to this vaccine were all monovalent Pfizer COVID-19 vaccine. The IFNy and IL-2 responses to human (SCoV2) and feline (FCoV1 , FCoV2) receptor binding domains (RBDs) are shown with the months postlast vaccination, when the blood was collected for PBMC isolation. Subject Y3 received five vaccination and therefore this subject’s IFNy responses to all RBDs appeared to be hyperreactive at 4mo post 5th vaccination (p5v) and completely gone by 9mo p5v. However, the IL-2 ELISpot responses suggest that vaccine-induced IL-2 responses still remain at 10mo p5v. The cytokine IL-2 is known as a strong T-cell immune mediator and not known as an inflammatory cytokine. The red dotted line represents the threshold of the average PBS control response. Since IFNy is well known to be inflammatory cytokine as well as a moderate dose to be important T cell immune modulator. Current results suggest that COVID19 vaccine- induced IFNy for this subject is responding as inflammatory cytokine that crashes in response by 9mo p5v. Those responses to RBD(s) substantially above this threshold would indicate a substantial or significant PBMC response. The statistical significant difference between the PBS control and the respective responses to the RBDs is shown as p<0.025 (*) and p<0.05 (**), based on two-tailed paired T-Test. This subject has never been infected with SCoV2. The description of this subject’s profile is shown in the table below.

[0127] Three COVID-19 vaccinated subjects (Y2,Y8,Y10) were reported to be free of SCoV2 exposure, whereas one subject Y9 was infected with SCoV2 one year after the second vaccination which is also about three month before the third vaccination. The blood was collected from Y9 at 12 months post last 3rd vaccination or 1 .5 years post SCoV2 infection. Y2 and Y10 have multiple FCoV1 infected cats which may have altered their COVID-19 vaccination results by constant shedding of FCoV1 in their household. In contrast, vaccinated / infected Y9 has no FCoV1 infected cats at household but has significant IFNy responses to FCoV1 and FCoV2 RBD but not to SCoV2 RBD when his last vaccination was 3.5mo since blood collection for the PBMC used in the assay. Subject Y8 has no cats and has not been infected with SCoV2. This subject who is same age as Y9 (48 years-old) hadno responses to any RBDs even though he was last vaccinated 9mo ago with Pfizer bivalent. In contrast subject Y10, who was vaccinated with Pfizer monovalent 9mo before the blood collection for PBMC shown, had significant IFNy responses to SCoV2 and FCoV1 RBDs and substantial IFNy response to FCoV2 RBD. Y10, at the time of bleeding was 21 years-old, has retained the CoV RBD responses far better than Y8, Y9, and Y2, and even Y3 at 9mo p5v (Fig 7). Surprisingly, IFNy responses to SCoV2 RBD are slightly-to-substantially lower levels than those to FCoV1 RBD for Y2, Y10, and Y9, and even Y3 at 10mo p5v with IL-2 response. Such results suggest immunogen-specific downregulation of immune responses.

[0128] As shown in Figures 7A and 7B, COVID-19 vaccines induce FCoV1 and FCoV2 RBD responses more strongly than those to SCoV2 RBD. The dotted line for each subject represents the threshold of the average PBS control response. Reponses to RBD(s) that are substantially above this threshold indicates a substantial or significant PBMC response. The statistically significant difference between the PBS control and the respective responses to the RBD(s) are shown as p<0.05 (*), based on two-tailed paired T-Test.

[0129] Figure 8 shows that chronically FCoV1 -infected cats (G1 ,G2,G7) have antibodies to FCoV whole-virus and live together, causing constant reinfection. FCoV1 -infection-cleared cats (4GA, Y2E) do not have antibodies to FCoV by living individually in single housing unit to prevent reinfection that occurs when housed together with one or more FCoV1 -infected cats. The dotted line for each cat represents the threshold of the average PBS control response. These results suggest that downregulation of IFNy responses to FCoV1 RBD in the infecting coronavirus FCoV1 occurs with cats G1 , G2, and 4GA. This observation may be similar to the downregulation of IFNy responses to SCoV2 RBD of Y2, Y10 and especially with subject Y9. Those responses to RBD(s) substantially above this threshold would indicate a substantial or significant PBMC response. The statistically significant difference between the PBS control and the respective responses to the RBD(s) are shown as p<0.05 (*) and p<0.025 (**), based on two-tailed paired T-Test.Example 6 - Alternative FCoV vaccine constructs

[0130] Compared to the B-cell constructs of SCoV2-Wuhan and FCoVs (FCoV1 and FCoV2) described in the previous Examples, the sequences have been modified to decrease adverse epitopes and to decrease the size of the construct to allow more RBD to be included in the lipid nanoparticles (see amino acid sequences SEQ ID NO: 102 and SEQ ID NO: 103). As to the SCoV2- Wuhan and the additional SCoV2-Omicron XBB1 .5 RBD sequences, both the heptad repeat 1 (HR1) and 2 (HR2) have been replaced with FCoV1 HR1 and HR2-TM-CT, while retaining the SCoV2 stem helix (SH) (see the amino acid sequences set forth in SEQ ID NO: 99, SEQ ID NO: 100, and SEQ ID NO: 101 ). BothWuhan and XBB1 .5 are identical in amino acid sequences at C-Terminal Spike 1 (S1 RBDE,16 amino acids) section and SH-HR2-TM-CT, and only one closely similar aa change in HR1 (see Figure 9).

[0131] HR1-SH-HR2-TM-CT of the B-cell construct: The adverse epitopes of the SCoV2 HR2 are caused by the high amino acid sequence identity to the bullous pemphigus (BP) antigen-1 e. BP antigen causes skin lesion which is caused by the auto-antibodies to the skin protein. Consequently, SCoV2 HR2 must be exchanged with other coronavirus HR2 with minimal sequence similarity to BP antigen. Additionally, we have determined that HR1 and HR2 must match with the coronavirus strain used. The only strain that so far has worked was the FCoV1 HR1 and HR2 combination with SCoV2 SH. Furthermore, the FCoV1 RBD of the RBD-HR1 -SH-HR2-TM-CT construct has been shortened by 92 aa and is called FCoV1 sRBD-HR1 -SH-HR2-TM-CT. Similarly, FCoV2 RBD of the previous RBD- HR1-SH-HR2-TM-CT construct has been shorted by over 90 aa and in addition FCoV2 HR2 was replaced with FCoV1 HR2 to provide better alpha helix structural stability. These shortening and changes in few basic amino acids provide additional structural stability and solubility, and proper acidic pl of the construct. The acidic pl will allow the construct to express on Golgi apparatus and endoplasmic reticulum membranes for proper expression of the RBD and SH in the cells. RBD and SH have the epitopes that induce the production of neutralizing antibodies against SCoV2s and FCoVs. Exemplary “HR1-SH-HR2-TM-CT” constructs include those constructs comprising an amino acid sequence set forth in SEQ ID NOs: 97-103.Example 7 - Immunoassay of culture fluid from Fc9 cells transfected with pDNA-FCoV1 inserts reacted with serum from FCoV1 -infected or uninfected SPF cat

[0132] The pDNA-FCoV1 UCD1-2BN represents the plasmid containing the insert FCoV1 strain LICD1 with sequence 2BN. The pDNA-FCoV1 UCD1 -Ori represents the plasmid containing the insert FCoV1 UCD1 with the sequence Ori, which was initially called “FCoV1 - sequence LE”. The pDNA used for both constructs is pCI-H2B-GFP. The 25cm2flasks with 70-90% confluent Fc9 cells were exchanged with 3-mL of 5% heat-inactivated fetal calf serum (FCS) in Corning EMEM media with 50 pg / mL of gentamycin before adding the various amounts of plasmid and incubating (5% CO2 at 37°C) for 4 hr before removing the spent fluid and replacing with 3-mL of fresh culture media. The plasmid expression was stopped upon 48 hr of incubation (5% CO2 at 37°C). The individual collected fluids were concentrated with Sartorius Vivaspin 6-mL column (15-mL centricon) of molecular weight cut off (MWCO) of 30K at 1800 rpm for 25 min at 5°C refrigerated centrifuge. Each concentrate was collected in individual sterile 1.8-mL microfuge and frozen immediately at -80°C ultralow freezer. After first thaw, the samples were aliquoted into multiple sterile microfuge ofsmaller quantities and the concentration measure by Qubit 4 Fluorometer to load onto the 10-well SDS-PAGE mini-gel (30% acrylamide, under reducing condition) and ran until the gel front reached the 25kDa red band at the edge of the gel. The gels were transferred onto the commercial mini-gel nitrocellulose membrane for 2.0-2.5 hr at a voltage of 120 until all Precision Plus Protein (Dual Color 25kDa & 75kDa red) Standards transferred completely. The immunoblots in individual square petri-plate were incubated on a rocker at room temperature (RT) for overnight with 12-mL of reacting serum at the concentration described on the figures (1 :50 or 1 :30 dilution in blocking buffer). The plates were washed with washing buffer and incubated with goat AP-conjugated anti-human IgG or goat AP-conjugated anticat IgG at 1 :500 dilution in blocking buffer for two hr on a rocker at RT. Upon washing thrice with washing buffer, each blot was treated with Bio-Rad substrate for alkaline phosphate (AP) and reaction allowed for 7-10 min.

[0133] Serum from an SPF cat 2FS infected for 7 weeks with FCoV1 -strain UCD-2 incubated at 1 :30 dilution on a immunoblot. The AP substrate reaction was allowed for 8 min and no adjustments were made for brightness and contrast. A single strong band was observed for the expressed FCoV1 -2BN protein at 70 killodalton (kDa) and FCoV1 -UCD1 - Ori based on molecular weight (MW) marker, indicating FCoV1 antigen bands immediately below the MW red band at 75 kDa. A single band with antigen from FCoV1 -2BN transfected with 5 ug / flask was weak at 70 kDa. A thick band for BSA was observed at 55kDa in all samples, indicating this FCoV1 -infected cat has anti-BSA antibodies. The MWCO used to concentrate the expressed viral RBD-HR1-SH-HR2-TM-CT proteins from the pDNAs include the BSA (65 kDa) and degraded BSA from the FCS used for culturing.

[0134] The whole-virus FCOV1 UCD2 immunoblot strips consist of concentrated whole virus using Vivaspin 20-mL column with MWCO of 100K and thus has minimal BSA reactivity. All reacting serum were performed at serum dilution of 1 :50. The separated FCoV1 proteins were identified on an immunoblot (not shown) as spike 1 (S1 at 120 kDa), S2 (76 kDa), nucleocapsid (NC at 43 kDa), membrane (M at 30 kDa) with the FCoV1 Positive Control serum (FCoV1+ Control). The same FCoV1 ucD2-infected cat 2FS serum was used and its reaction is shown in the middle as the second whole-virus immunoblot strip with strong S2 and NC bands, weak M band, and additional solid strong thin band at 100 kDa, which is projected to be unglycosylated viral polymerase RdRp. The negative control serum from SPF HOE was used for the third whole-virus immunoblot strip and has no reaction. The strips were dark due to the 10min treatment with AP substrate and therefore, this figure was adjusted to 3% brightness but 0% contrast. The FCoV1 infecting cat 2FS and the whole virus used for the immunoblot strip is the same virus FCOV1 UCD2. In contrast, the expressed pDNA FCoV1 construct insert is based on FCOVI UCDI which is a different strain from theFCoV1 ucD2to be used as a heterologous challenge virus against pDNA-FCoV1 ucoi construct-LNP vaccinated cats.

[0135] The negative control serum from SPF HOE at 1 :30 dilution showed a single degraded BSA band at 55 kDa. Thus, the degraded BSA observed at 55 kDa with FCoV1 - infected cat serum was also observed with SPF control cat HOE.

[0136] Mouse monoclonal antibody (mMAb) to BSA was reacted with a 8-lane immunoblot transferred from a 8-well gel with samples from FCoV1 -2BN transfection at 3 pg, 4 pg, and 5 pg and with samples from FCoV1 UCD1 -Ori transfection at 3 pg, 4 pg, and 5 pg, including MW and Fc9 transfection reagent. The anti-BSA mMAb reacted to two glycosylated bands at 58-65 kDa and 50-55 kDa, having no other bands detected. This observation supports the findings that the band 55 kDa is most likely degraded BSA. The MW standard was loaded at the commercially recommended 7 pL per well instead of 8 pL. Since the MW markers are also soaked with serum and all reacting reagents the increase to 8 L was essential to visualize the MW markers.

[0137] Summary:

[0138] The serum from an SPF cat 2FS infected for 7 weeks with FCoV1 -strain UCD-2 reacted to bands at 70 kDa and 55 kDa, which are most likely corresponding to the expressed FCoV1 construct and degraded bovine serum albumin (BSA), respectively. The predicted molecular weight (MW) for FCoV1 LICD1 -2BN (394 aa) is 44,920 Da without glycosylation and 70-77 kDa with eight N-glycosylation. The predicted MW for FCoV1 LICD1 - Ori (496 aa) is 56,249 without glycosylation and 77-80 kDa with seven N-glycosylation. O- glycosylation algorithm did not predict any O-glycosylation for these constructs. Since their predicted MWs are very close and our gels were not gradient gels, thus our immunoblot provided a band at 70 kDa for both FCoV1 LICD1 -2BN and FCoV1 LICD1 -Ori with serum from infected cat 2FS. In support of this observation, the negative control serum from SPF cat HOE reacted to only one band at 55 kDa, further suggesting that band 55 kDa may be a degraded BSA. Furthermore, the mouse monoclonal Ab to BSA (mNAb anti-BSA) reacted to two thick glycosylated bands at 58-65 kDa and 50-55 kDa, without having any other bands. Hence, the band 55 kDa observed with sera from infected cat 2FS and SPF cat HOE are most likely reacting to a degraded BSA. The same sera from both cats 2FS and HOE were reacted with whole-virus (WV) FCOV1 UCD2 immunoblot strips. As expected, the infected cat 2FS serum reacted with 100, 76, 43, and 30 kDa bands most likely corresponding to FCoV1 RdRp polymerase, spike 2 (S2), nucleocapsid (NC), and membrane (M) proteins. In contrast, SPF cat HOE serum had no reactivity to any FCoV1 proteins. Since the serum used from 2FS was collected at 7 wk post-FCoV1 ucD2 infection (wpi), the serum to becollected at 11 wpi should have stronger reactivity with S2 glycoprotein and potentially develop Abs to S1 glycoprotein. Such serum should help confirm our current results. Please note that the MWCO of 30kDa for the column used to concentrate the expressed viral constructs also concentrated both BSA and degraded BSA. However, the WV- FCoV1 UCD2 immunoblot strips were free of contaminated BSA and degraded BSA because the column with MWCO of 100 kDa was used to purify and concentrate the WV-FCoV1 , removing all BSA and degraded BSA from the WV-FCoV1 preparation.Example 8 - Immunoassay of the culture fluid from pDNA-construct transfected Fc9 cells reacted with serum from SCoV2-infected or COVID-2 vaccinated human subject

[0139] The same procedure as described in Example 7 was used to load, run the gel, and then transfer gel contents to the nitrocellulose membrane. The development of the immunoblots was different by reacting to human serum at 1 :50 dilution and Serotech’s goat anti-human IgG at 1 :500. The blot antigens are a comparison between pDNA-SCoV2- W1 F11w construct versus pDNA-FCoV1 UCD1 -Ori construct. The pDNA-SCoV2-W1 F11w represents the plasmid containing the insert Wuhan W1 F11w which was replaced at S2 with FCoV1 HR1 and HR2, showing the following construct Wuhan RBD-FCoV1 / HR1-SH- FCoV1 / HR2-TMg-CT. All immunoblot reaction was stopped at 7 min of substrate reaction and made no change in brightness and contrast adjustments. In addition, two MW standard lanes were run at lane 1 and another at lane 4 or 6 to evaluate the level of alignment of expressed protein(s) or BSA in each lane. Furthermore, MW standard were loaded at 8 piper lane.

[0140] The serum collected at pre-COVID-2 timepoint from human subject Y3 at 64 years- old was reacted with immunoblot containing samples in each lane as shown immediately below the immunoblot. The 200 kDa band appears to be reacting most likely to Fc9 cellular debris. The 65 kDa band for BSA and the 55 kDa band was identified as the degraded BSA.

[0141] The serum collected from subject Y3 at 70 years of age was reacted with immunoblot. Y3 had serum reactivity to only three bands with highest band of 200 kDa to most likely cellular band and two lower bands at 65 kDa for BSA and 55 kDa for degraded BSA. This subject had a total of four COVID-2 vaccinations (two in 2021 and two in 2022) by the time of serum collected on 12 / 08 / 2023, which represent 14 months post-last vaccination in October 2022. No serum reactivity to the SCoV2-W1 F11w construct or to the FCoV1 UCD1 -Ori construct were detected, but the same serum reacted to SCoV2 RBD.

[0142] The serum collected from human subject Y5, who was 27 years-old, was reacted with immunoblot. The Y5 serum collected on 10 / 13 / 22 represents 2 months post-SCoV2 infection with extremely mild illness, which should be around when high levels of anti-SCoV2antibodies with peak SCoV2 neutralizing antibodies develop. Three viral protein bands were observed at 270 kDa, 80-90 kDa, and 70 kDa. The 270 kDa bands were most likely to be the trimeric SCoV2-WF11w proteins. In contrast, the 80-90 kDa band were detected strongly in two of three SCov2-W1 F11 w lanes and weakly in three FCoV1 LICD1 -Ori lanes. Furthermore, the extremely weak 70 kDa band was detected in all lanes loaded with viral proteins suggesting a reaction with a degraded SCoV2 protein and cross-reaction with FCoV1 protein. The 200 kDa band appears to be FFc9 cellular protein. Finally, the 50 kDa may be degraded BSA for the range of degraded BSA band to be 50-55 kDa.

[0143] The same human sera for Y3’s pre-COVID-2, Y3 at 14 months post-last COVID-2 vaccination, Y5 at 2 months post-SCoV2 infection were reacted to a immunoblot strip with SCoV2 RBD. The Y3’s pre-CoVID-2 serum had no reaction to the SCoV2 RBD, whereas Y3’s post-4th vaccination and Y5’s post-infection had strong antibodies to SCoV2 RBD. Upon monitoring the serum reaction time of the band development SCoV2 RBD band, Y5’s band developed earlier and slightly darker than the post-vaccination Y3’s band, suggesting that the antibody quality of Y3’s serum collected at 2 months post-SCoV2 infection has better reaction to SCoV2 protein than Y3’s 14 months post-last vaccination serum. In addition, the antibody quality and strength may be better from a youthful 27 years-old subject than a 70 years-old subject.

[0144] Summary of results:

[0145] The pre-COVID-2 and the COVID-2-vaccinated sera from human subject Y3 reacted with bands at 200 kDa, 65 kDa, and 55 kDa which are most likely corresponding to cell antigen, BSA, and degraded BSA, respectively. Since COVID-2 vaccinated serum was collected at 14mo post-last vaccination, consequently, its SCoV2 Ab titer may be too low to react with the expressed pDNA SCoV2 construct. Furthermore, the same serum reacted with SCoV2 RBD in SCoV2 RBD-immunoblot strip when her pre-COVID-2 serum had no reactivity to SCoV2 RBD-immunoblot strip. In contrast, the serum from post-SCoV2-infected subject Y5 collected at 2mo post-infection reacted moderately-to-strongly with bands at 270 and 80-90 kDa and weakly with bands at 70 kDa and 55 kDa in immunoblot and strongly with SCoV2 RBD-immunoblot strip (panel D). The 70 kDa, 80-90 kDa, and 270 kDa bands are projected to be those of viral antigens, and the 65 kDa and 55 kDa bands are projected to be those of BSA and degraded BSA, respectively. The predicted MW for SCoV2- W1 F11w (438 aa) is 49,152 without glycosylation and 70-78 kDa with seven N-glycosylation. The predicted MW for FCoV1 ucoi-Ori (496 aa) is 56,249 without glycosylation and 77-80 kDa with seven N-glycosylation. Since their predicted MWs are very close and our gels were not gradient gels, therefore, our immunoblot provided a band at 70 kDa and 80 kDa for both SCoV2Wuhan-W1 F11w and FCoV1 ucDi-Ori. More importantly, our human data demonstratethat many sera from SCoV2-infected human subjects cross-react moderately-to-strongly to FCoV S2 by immunoblot strip analyses. The 270, 80-90, and 70 kDa bands on lanes with SCoV2-W1 F11w samples may be bands to trimeric SCoV2 clump, monomeric SCoV2- W1 F11w construct, and glycosylation-degraded SCoV2 construct, respectively. The bands at 200 and 50 kDa are prevalent in all sample lanes, except for transfection-reagent sample lane, and may be corresponding to the cellular antigens or latter to the degraded BSA. The comparison of all three immunoblot reactivities indicate that Y5 has lower anti-BSA Abs compared to those of Y3.References1. Holmes et al, Philos. Trans. R. Soc. Lond. B. Biol. Sci. 2004;359(1447):1059-1065.2. Herrewegh, et al. 1998. J. Virol. 72 (5), 4508-4514.3. Horzinek MC, Osterhaus AD. The virology and pathogenesis of feline infectious peritonitis. Brief review. Arch Virol. 1979;59(1 — 2) :1 — 15.4. Vennema Het al. 1998. Virology 1998; 243(1 ):150-157.5. Pedersen, N.C. Vet J 2014; 201 :123-132.6. Krentz et al. Viruses. 2021 ;13(11):2228. doi:10.3390 / v131122287. Weiss et al. Comp Immunol Microbiol Infect Dis. 1981 ;4(2) : 175-189.8. Pedersen et al. (1984) American journal of veterinary research, 45(12), 2580-2585.9. Hohdatsu et al. Arch Virol. 1991 ;120(3-4):207-217.10. Zhu et al. Respir Res. 2020;21 (1):224.11. Li et al.. Sci Adv. 2020;6(27):eabb9153. Published 2020 Jul 1.12. Wardeh et al. Nat Commun. 2021 ;12(1 ):780. Published 2021 Feb 16.13. Morens et al. N Engl J Med. 2022;386(4):297-299.14. McAloose et al.. mBio. 2020;11 (5):e02220-20. Published 2020 Oct 13.15. Newman et al. New York, March-April 2020. MMWR Morb Mortal Wkly Rep. 2020;69(23):710-713. Published 2020 Jun 12.16. Sit et al. Nature. 2020;586(7831):776-778.17. Decaro et al. Front Vet Sci. 2021 ;8:619207. Published 2021 Feb 10.18. OIE . (2021b). SARS-CoV-2 in animals. Situation report 1. OIE.19. Sailleau et al. Transbound Emerg Dis. 2020;67(6):2324-2328.20. Segales et al. Proc Natl Acad Sci U S A. 2020;117(40):24790-24793.21. Nanishi et al. Sci Transl Med. 2022; Jan 26; 14(629) :eabj5305.22. Yates JL, et al., Cell Rep Med. 2021 , e1 -4.23. Kida K, et al., J Vet Med Sci. 1999; 61 :935-938.24. Aillo SE, Moses MA. Feline Infectious Peritonitis: Management of Multicat Households, In: The Merck Veterinary Manual; Chapter: Generalized Conditions, 1 1th Edition, 2016. p788.25. Regan et al. Virology 2012; 430:90-99.26. Li et al. J Virol 2017; 91 (12) e00273-17.27. Reguera et al. J Gen Virol 2011 ; 92:1 117-112628. Tusell et al. J Virology, 2007; 81 :1261 -127329. Shirato et al. J Gen Virol 2016; 97:2528-2539.30. Coutard et al. Antiviral Res 2020; Apr;176:104742.31 . Millet et al. Virus Res 2015; Apr 16;202:120-34.32. Follis et al., Virology. 2006 Jul 5;350(2):358-69.33. Zhao et al. Viruses, 2019, 11 , 743.34. Jaimes et al. Viruses 2020, 12, 83.35. Arya et al., J Mol Biol 2021 ; Jan 22; 233(2)166725.36. Yan et al., Signal Transduct Target Ther 2022; Jan 27; 7(1 ):26.37. World Organization for Animal Health: Global cases of SARS-CoV-2 in animals. / www.oie.int / en / what-we-offer / emergency-and-resilience / covid-19 / #ui-id-3 (accessed 3 / 27 / 2022) (Report ID FUR_154029).38. Sia et al., Nature. 2020 Jul;583(7818):834-838.39. Abdelnabi et al., EBioMedicine. 2021 Jun;68:103403.40. Osterrieder et al., Viruses 2020; Jul 20;12(7):779.41 . Mohandas et al., Viruses 2021 ; Sep 5;13(9):1773.42. Rogers et al., Science. 2020;369(6506):956-963.43. Vlasova et al., Emerg Microbes Infect 2022; Dec;11 (1 ):699-702.Informal Sequence Listing

Claims

What is claimed is:1 . A composition comprising at least one nucleic acid encoding an amino acid sequence set forth in SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101 , SEQ ID NO: 102, or SEQ ID NO: 103.

2. The composition of claim 1 , wherein the nucleic acid encodes the amino acid sequence set forth in SEQ ID NO: 99.

3. The composition of claim 1 , wherein the nucleic acid encodes the amino acid sequence set forth in SEQ ID NO: 100.

4. The composition of claim 1 , wherein the nucleic acid encodes the amino acid sequence set forth in SEQ ID NO: 101.

5. The composition of claim 1 , wherein the nucleic acid encodes the amino acid sequence set forth in SEQ ID NO: 102.

6. The composition of claim 1 , wherein the nucleic acid encodes the amino acid sequence set forth in SEQ ID NO: 103.

7. The composition of any one of claims 1-6, wherein the composition is formulated in a nanoparticle, a liposome, a microparticle, a microsphere, a nanosphere, a unilamellar vesicle, a multilamellar vesicle, or a virus-like particle (VLP).

8. The composition of any one of claims 1-7, wherein the composition is formulated in a lipid nanoparticle.

9. The composition of claim 8, wherein the lipid nanoparticle is T-cell specific.

10. The composition of claim 9, wherein the lipid nanoparticle is specific for a dendritic cell.11 . The composition of claim 8, wherein the lipid nanoparticle is specific for a macrophage.

12. The composition of claim 8, wherein the lipid nanoparticle is specific for a dendritic cell and a macrophage.

13. A vector comprising a nucleic acid encoding an amino acid sequence set forth in ID NO:

99. SEQ ID NO: 100, SEQ ID NO: 101 , SEQ ID NO: 102, or SEQ ID NO: 103.

14. A method for inducing an immune response in a subject, the method comprising administering the composition of any one of claims 1-12 in an amount effective to induce the immune response in the subject.

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