Immunogenic composition and uses thereof

Abstract

The disclosure provides a composition comprising a nucleic acid encoding a human alpha coronavirus (HCoV) receptor binding domain (RBD) peptide and a severe acute respiratory syndrome coronavirus 2 (SCoV2) RBD peptide.

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: 59799_SeqListing.xml, Size: 115,886 bytes; Created: October 24, 2024.SUMMARY

[0003] In one aspect, the disclosure provides a composition comprising a nucleic acid encoding a human alpha coronavirus (HCoV) receptor binding domain (RBD) peptide and a severe acute respiratory syndrome coronavirus 2 (SCoV2) RBD peptide.

[0004] In another aspect, the disclosure provides methods of stimulating an immune response in a subject in need there comprising administering a composition comprising a nucleic acid encoding a human alpha coronavirus (HCoV) receptor binding domain (RBD) peptide and a severe acute respiratory syndrome coronavirus 2 (SCoV2) RBD peptide.DETAILED DESCRIPTION

[0005] The present disclosure is based, in part, on the discovery of compositions comprising a nucleic acid encoding a human alpha coronavirus (HCoV) receptor binding domain (RBD) peptide and a severe acute respiratory syndrome coronavirus 2 (SCoV2) RBD peptide for the prevention and / or treatment of SCoV2 coronavirus infections.

[0006] 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.

[0007] Coronavirus

[0008] 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 respiratorycoronavirus, 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.

[0009] In some or any aspects, a coronavirus described herein is in the genus of alpha coronavirus, and the coronavirus antigens can be of or derived from any species or strains in the genus of alpha coronavirus. In some embodiments, the alpha-coronavirus is a human alpha coronavirus (HCaCoV). Exemplary human alpha-coronaviruses included, but are not limited to, HCaCoV-229E and HCaCoV-NL63. In some embodiments, the human alpha coronavirus is HCaCoV-229E. In some embodiments, the human alpha coronavirus is HCaCoV-NL63.

[0010] In some or any aspects, a coronavirus described herein is in the genus of Betacoronavirus and the coronavirus antigens can be of or derived from any species or strains in the genus of Beta-coronavirus. 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.

[0011] In some or any aspects, the compositions described herein comprises nucleic acid encoding a human alpha coronavirus (HCaCoV) receptor binding domain (RBD) peptide and a severe acute respiratory syndrome (SARS) coronavirus 2 (SCoV2) RBD peptide.

[0012] In some or any aspects, the HCaCoV RBD peptide is a HCoV-229E RBD peptide. In some or any aspects, the nucleic acid encodes a HCoV-229E RBD peptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 5. In some or any aspects, the nucleic acid encodes a HCaCoV-229E RBD peptide comprising 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: 1 or SEQ ID NO: 5. In some orany aspects, the nucleic acid encodes a HCaCoV-229E RBD peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 5.

[0013] In some or any aspects, the HCaCoV RBD peptide is a HCaCoV-NL63 RBD peptide. In some or any aspects, the nucleic acid encodes a HCaCoV-NL63 RBD peptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 6. In some or any aspects, the nucleic acid encodes a HCaCoV-NL63 RBD peptide comprising 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: 2 or SEQ ID NO: 6. In some or any aspects, the nucleic acid encodes a HCaCoV-NL63 RBD peptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 6.

[0014] In some or any aspects, the nucleic acid encodes a SCoV2 RBD peptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 7 (Wuhan RBD). In some or any aspects, the composition comprises a SCoV2 RBD peptide comprising 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:3 or SEQ ID NO: 7. In some or any aspects, the nucleic acid encodes a SCoV2 RBD peptide comprising the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 7.

[0015] In some or any aspects, the nucleic acid encodes a SCoV2 RBD peptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 8 (Omicron XBB1 .5). In some or any aspects, the nucleic acid encodes a SCoV2 RBD peptide comprising 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:4 or SEQ ID NO: 8. In some or any aspects, the nucleic acid encodes a SCoV2 RBD peptide comprising the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 8.

[0016] In some or any aspects, the nucleic acid encodes a HCaCoV-229E RBD domain peptide comprising 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: 1 or SEQ ID NO: 5 and a SCoV2 RBD peptide comprising 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: 3 or SEQ ID NO: 7. In some or any aspects, the nucleic acid encodes a HCaCoV-229E RBD peptide comprising the amino acid sequence set forth in SEQ ID NO: 1or SEQ ID NO: 5 and a SCoV2 RBD peptide comprising the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 7.

[0017] In some or any aspects, the nucleic acid encodes a HCaCoV-229E RBD domain peptide comprising 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: 1 or SEQ ID NO: 5 and a SCoV2 RBD peptide comprising 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: 4 or SEQ ID NO: 8. In some or any aspects, the nucleic acid encodes a HCaCoV-229E RBD peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 5 and a SCoV2 RBD peptide comprising the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 8.

[0018] In some or any aspects, the nucleic acid encodes a HCaCoV-NL63 RBD domain peptide comprising 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: 2 or SEQ ID NO: 6 and a SCoV2 RBD peptide comprising 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: 3 or SEQ ID NO: 7. In some or any aspects, the nucleic acid encodes a HCaCoV-NL63 RBD peptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 6 and a SCoV2 RBD peptide comprising the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 7.

[0019] In some or any aspects, the nucleic acid encodes a HCaCoV-NL63 RBD domain peptide comprising 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: 2 or SEQ ID NO: 6 and a SCoV2 RBD peptide comprising 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: 4 or SEQ ID NO: 8. In some or any aspects, the nucleic acid encodes a HCaCoV-NL63 RBD peptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 6 and a SCoV2 RBD peptide comprising the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 8.

[0020] 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 inSEQ ID NO: 55. In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence set forth in SEQ ID NO: 52.

[0021] 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: 55. In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence set forth in SEQ ID NO: 53.

[0022] 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: 55. In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence set forth in SEQ ID NO: 54.

[0023] 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: 55. In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence set forth in SEQ ID NO: 55.

[0024] 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: 56. In some or any aspects, the composition comprises an amino acid sequence set forth in SEQ ID NO: 56.

[0025] 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: 57. In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence set forth in SEQ ID NO: 57.

[0026] 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: 58. In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence set forth in SEQ ID NO: 105.

[0027] 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: 59. In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence set forth in SEQ ID NO: 106.

[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: 60. In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence set forth in SEQ ID NO: 107.

[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: 108. In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence set forth in SEQ ID NO: 108.

[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: 109. In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence set forth in SEQ ID NO: 109.

[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: 110. In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence set forth in SEQ ID NO: 110.

[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: 111. In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence set forth in SEQ ID NO: 111.

[0033] 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: 112. In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence set forth in SEQ ID NO: 112.

[0034] 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: 113. In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence set forth in SEQ ID NO: 113.

[0035] 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: 114. In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence set forth in SEQ ID NO: 114.

[0036] 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: 115. In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence set forth in SEQ ID NO: 115.

[0037] 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: 116. In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence set forth in SEQ ID NO: 116.

[0038] 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: 117. In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence set forth in SEQ ID NO: 117.

[0039] 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: 118. In some or any aspects, the composition comprises a nucleic acid that encodes an amino acid sequence set forth in SEQ ID NO: 118.

[0040] A sequence which is not 100% identical to the particular sequences described herein 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 andstereochemical 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.

[0041] Immunogenic epitope

[0042] 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.

[0043] 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.

[0044] 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 thatinhibits 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 immune response 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).

[0045] In some or any aspects, the immunogen comprises a nucleic acid that encodes a conserved SCoV2 CD8+ T cell epitope peptide. 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: 9-44.

[0046] 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: 9- 15.

[0047] In some or any aspects, the immunogen comprises a nucleic acid that encodes a conserved HCaCoV-NL63 CD8+ T cell epitope peptide. In some or any aspects, the conserved HCaCoV-NL63 CD8+ T cell epitope peptide is an amino acid sequence set forth in any one of SEQ ID NOs: 58-85.

[0048] In some or any aspects, the conserved CD8+ T cell epitope peptide is a HCaCoV- NL63 polymerase enzyme epitope peptide. In some or any aspects, the HCaCoV-NL63polymerase enzyme epitope peptide is an amino acid sequence set forth in any one of SEQ ID NOs: 58-64.

[0049] In some or any aspects, the immunogen comprises a nucleic acid that encodes a conserved HCaCoV-229E CD8+ T cell epitope peptide. In some or any aspects, the conserved HCaCoV-229E CD8+ T cell epitope peptide is an amino acid sequence set forth in any one of SEQ ID NOs: 86-96.

[0050] In some or any aspects, the conserved CD8+ T cell epitope peptide is a HCaCoV- NL229E polymerase enzyme epitope peptide. In some or any aspects, the HCaCoV-NL229E polymerase enzyme epitope peptide is an amino acid sequence set forth in any one of SEQ ID NOs: 86-95.

[0051] 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: 36-44.

[0052] 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: 36-44, optionally conjugated together via a linker (e.g., a cathepsin S receptor linker, “KVSVR,” (SEQ ID NO: 45), or “RVSVR” (SEQ ID NO: 104). In some or any aspects, the immunogen comprises a nucleic acid encoding an amino acid sequence set forth in SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101 , SEQ ID NO: 101 , SEQ ID NO: 102, or SEQ ID NO: 103.

[0053] Nucleic acids, Vectors

[0054] Nucleic acid molecules comprising nucleotide sequences encoding the HCaCoV and SCoV2 RBD peptides (and optionally the conserved SCoV2 CD8+ T cell epitope immunogen peptides) 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.

[0055] 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).

[0056] 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.

[0057] In some or any aspects, nucleic acids encoding the HCaCoV 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 HCaCoV and / or SCoV2 CD8+ T cell epitope peptides are present on the same vector as the nucleic acids encoding the HCaCoV and SCoV2 RBD peptides. In some or any aspects, nucleic acids encoding the HCaCoV 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.

[0058] 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.

[0059] Formulations and Routes of Administration

[0060] In some or any aspects, the composition comprises a HCaCoV RBD peptide and a SCoV2 RBD peptide and a pharmaceutically acceptable carrier or excipient. 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 ofstabilizing agents also include poloxamer (e.g. poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338 and poloxamer 407). The pharmaceutically 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

[0061] 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).

[0062] 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 an antigen presenting cell. In some embodiments, the antigen presenting cell is a T cell. In some or any aspects, the antigen presenting cell is a dendritic cell. In some or any aspects, the antigen presenting cell is a macrophage. In some or any aspects, the nanoparticle is specific for both a dendritic cell and a macrophage.

[0063] The compositions described herein can be administered by any route, including a systemic or mucosal route. Exemplary administrations may include injection via the intranasal (IN), intramuscular (IM), intraperitoneal (IP), intradermal (ID) or subcutaneous (SC) routes; or via mucosal administration to the oral / alimentary, respiratory, genitourinary tracts. Following an initial administration, subjects may receive one or several booster administrations.Methods of use

[0064] The disclosure provides a method of treating or preventing a coronavirus infection, the method comprising administering an 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 effectiveamount 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 SCoV2, Alternative or in addition, administering the composition optionally induces a CD8+ and / or CD4+ T cell response against SARS-CoV2.

[0065] In some or any aspects, the method comprises administering a first composition comprising a nucleic acid encoding a HCaCoV RBD peptide described herein and a SCoV2 RBD peptide described herein and a second composition comprising one or more nucleic acids encoding one or more SCoV2 CD8+ T cell epitope peptides described herein.

[0066] 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.

[0067] 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 be diagnosed 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.EXAMPLESMaterials and Methods

[0072] l-TASSER Homology Model Analyses for B-cell Construct. I-TASSER monomer homology modeling

[0011] was used to identify the best structural conformation of the B-cell constructs for SCoV2 Wuhan, SCoV2 Omicron XBB1 .5, NL63, 92 229E, FCoV1 LICD1 , and FCoV2 79-1146. I-TASSER composes the structure from the submitted sequence based on the Protein Data Bank (PDB) database, which currently has over 213,000 PDB files

[0012] , These PDB files are the production of X-ray crystallography, NMR spectroscopy, and cryoelectron microscopy, submitted by researchers to determine the 3-dimensional structure of the protein. I-TASSER also provides the PDBs of the top ten structures from which the final five structures were configured from the submitted sequence. Only one sequence submission at a time is permitted to I-TASSER. The best structure is chosen from the five structures provided by I-TASSER. Each chosen structure is further refined by removing additional amino acid (aa) residues in the N-terminal end of the RBD of the aCoVs, without affecting the main RBD structure consisting of p-sheet conformations from which the RBM extends out. Such removal is readily determined using the Schrodinger PyMOL 2.5.5 program

[0013] , which provides the structure based on amino acid sequence. The RBD is connected with a short CTD sequence (16 amino acids), identical between Wuhan and Omicron. The short CTD sequence directly follows the HR1 sequence. The HR1 sequence is connected directly to the remaining HR2-TM-CT sequence at the SH. The ideal HR1 / HR2 stem for the RBD must provide two helixes similar to the native HR1 and HR2 on the S2 protein of the full-length spike (S) protein. I-TASSER was used to determine the best HR1 and HR2 helixes, mimicking the native HR1 / HR2 structures with minimal segmentation.Since the B-cell construct is not the full-length sequence of the S protein, 111 the I-TASSER confidence score (C-score) will be between -1 and -5 instead of 2 and -5, with a higher number corresponding to better confidence in the structure.

[0073] Production of CTL / TH epitope chains. The pairwise sequence alignment was performed on RdRp sequences of SCoV2 and aCoVs using the JustBio server

[0014] , The JustBio server provided the amino acid (aa) sequence similarity and identity between the two RdRp sequences to evaluate the level of epitope conservation existing between them. The CTL epitopes on the RdRp sequence were determined by the NetMHCpan 4.1 server

[0015] and further confirmed as CTL epitopes by the NetCTL 1 .2 server

[0016] . The 9mer CTL epitopes were mapped on the pairwise alignment of SCoV2 / aCoV RdRp sequences. The selection of the best CTL epitopes is described in the result section. The TH epitope on the RdRp sequence of the SCoV2 and aCoV were identified by NetMHClIpan 4.0

[0017] and NetMHCll 2.3

[0018] servers. The TH epitope was selected based on its ability to overlap the CTL epitope, enabling the TH epitope to be expressed in proximity to the adjacent CTL epitope. A single TH epitope overlap was chosen only when the TH epitope did not connect to any of the selected CTL epitopes in the same sequence chain. Both ends of each CTL epitope, TH epitope, CTL epitope overlap, or CTL / TH overlap are connected with cathepsin- S linker (RVSVR). This linker is susceptible to cleavage by the enzyme cathepsin S, which is ubiquitously present but most concentrated in the endosome of the antigen-presenting cells

[0019] ,

[0074] The selected human leukocyte antigen (HLA) class-l alleles are more commonly present in the U.S. population, and these include HLA-A1 , HLA-A2, HLA-A3, HLA-B7, HLA- B40 / B44, and HLA-B27

[0020] . The supertype HLA-A1 includes A01 :01 (more commonly found in Caucasians) and A30:01 (more commonly found in African Americans). The supertype HLA-A2 includes A02:01 (both populations) and A68:02 (African Americans), and the supertype HLA-A3 includes only A03:01 (both populations). The supertype HLA-B7 includes B07:02 (both populations) and B53:01 (African Americans), and supertype HLA-B27 includes B027:05 (Caucasians) and B15:03 (African Americans). The supertype HLA- B40 / B44 includes B40:01 and B44:02 for Caucasians, B44:03 for both populations, and B45:01 for African Americans. The common class-ll HLA alleles include DRB1 *0701 , DRB1 *1501 , DRB1 *0301 , DRB1 *1503, DRB1 *0901 , and DRB1 *0101

[0021] . The racial ranking for the HLA-DRB1 alleles is based on the bone marrow donor bank. Our analyses determined that TH epitopes for DRB1 *0701 (rank-1 for Hispanic and rank-2 for European, African, and Asian), DRB1 *0301 (rank-2 for Hispanic, rank-3 for European, rank-4 for African, and rank-7 for Asian), and DRB1 *0901 (rank-1 for Asian) were frequently found together. Similarly, the TH epitopes of DRB1 *1501 (rank-1 for European and rank-4 for Asian and Hispanic), DRB1 *1503 (rank-1 for African), and DRB1 *0101 (rank-4 for European) were often on the same epitope(s). More importantly, DRB1 *0701 covered all four races inthe top two rankings. We aimed to include TH 1 epitopes of all predominant DRB1 allotypes for human and feline CTL / TH epitope chains.

[0075] The feline leukocyte antigen (FLA) algorithm server is not currently available. Domestic cats can recognize HLA-A2 peptides and HLA-27 peptides

[0022] , Hence, the servers for human HLA alleles have been used to characterize the surrogate allotypes of the CoV peptides recognized by cats. Such peptide recognition can be determined by running CoV stimulants in IFNy and IL2 ELISpot analyses with peptide-vaccinated cats.

[0076] Production of pDNA-LNP-based pan-CoV vaccine. Plasmid concentration was made to 50pg / mL in aqueous buffer. LipidFlexTM (PreciGenome LLC, San Jose, CA) was mixed with 4-(dimethylamino)-butanoic acid, (10Z,13Z)-1 -(9Z,12Z)-9,12-octadecadien-1 -yl- 10,13-nonadecadien-1 -yl ester (DLin-MC3-DMA) in ethanol to generate the lipid mixture. The ratio of LipidFlex to DLin-MC3-DMA was predetermined by the testing of several ratios to test DNA-encapsulation ability by gel retardation assay. The lipid nanoparticles containing plasmid (pDNA-LNP) were constructed using NanoGenerator Flex-S (PreciGenome LLC, San Jose, CA). The formulated pDNA-LNPs were dialyzed against DNAse-free water and resuspended in PBS. The size and uniformity of the pDNA-LNP were tested using nanoPartica SZ-100V2 (Horiba, Ltd; Kyoto, Japan), which was uniformly 150nm in size. The pDNA-LNP was made fresh for each vaccination and injected within six hours of manufacturing.

[0077] Construction of plasmids with minimalized SCoV2 and FCoV1 spike glycoproteins (pCI-H2B-GFP-SCoV2Wuhan-W1 F11w and pCI-H2B-GFP-FCoV1UCDi-Ori). The pCI-H2B- GFP-SCoV2Wuhan-W1 F11w is our second-generation minimalized SCoV2Wuhan spike glycoprotein. This construct differs from the pCI-H2B-GFP-SCoV2Wuhan-Ori by the exchange in the SCoV2wUhan HRT1 and HRT2 with FCOVI UCDI HRT1 and HRT2. This exchange will remove the potential of developing bullous pemphigus (BP) lesion caused by the SCoV2 SH- HRT2 having identical sequence with BP sequence, which FCoV1 SH-HRT2 does not. However, SCoV2 SH induces neutralizing antibodies (NAbs) to human p-coronaviruses and therefore remains on the SCoV1Wuhan-W1 F11w. The resulting final structure is configured as SCoVI wuhan RBD-FCoV1 / HRT1 -SH-FCoV1 / HRT2-TM-CT. The pCI-H2B-GFP-FCoV1UCDi- Ori has expressed strongly in Fc9 cells in our previous in vitro study and is used as the positive control for the plasmid construct expression. These sequences were individually cloned at Nhel and Pstl sites of pCI-H2B-GFP to derive pCI-H2B-GFP-SCoV1Wuhan-W1 F11w and pCI-H2B-GFP-FCoV1 ucDi-Ori. Both construct plasmids have green fluorescent protein (GFP) sequence insert which was used to assess the level of in vitro plasmid expression in species-specific cell line.

[0078] The full-length SCoV2Wuhan spike (Genbank Accession No. YP009724390.1) has been decreased from 1484 amino acids (aa) to 438 aa for SCoV2Wuhan-W1 F11w. Potential inflammatory (ICAM-1 & superantigen motifs) and adverse reaction (neurotoxins & BP motifs) regions have been deleted to prevent adverse reactions in humans. However, this minimalized construct was designed to retain the native structure that will combine into a trimeric structure when expressed.

[0079] GFP Expression of pCI-H2B-GFP-SCoV2 construct by Flow Cytometry. In order to determine if such plasmid will express in feline cells, feline embryonic-9 (Fc9) cells at 70- 90% cell confluency in 25cm2flasks in 3-mL of fresh Corning EMEM culture media were transfected with various amounts of either pCI-H2B-GFP-SCoV2wUhan-W1 F11w or pCI-H2B- GFP-FCoV1 ucDi-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% CO2 at 37°C, the spent plasmid-containing culture media was decanted and replaced with 3-mL of fresh culture media and incubated for plasmid 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 (see 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.

[0080] 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 intomultiple 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 were evaluated and diagrammed with PPT. The immunoblot strips for the SCoV2 RBD used the identical procedure described previously (Yamamoto JK, et al. Viruses 15(4), 914, 2023) on the production and band development of the SCoV2 RBD immunoblot strips.Example 1 - Minimalized SCoV2 Spike Protein that is devoid of Adverse Epitope Regions

[0081] SCoV2 spike (S) has three robust neutralizing antibody (NAb) epitope regions, which include the RBD, receptor binding motif (RBM), and SH [6,24-26]. A weak NAb epitope region, which overlaps the fusion peptide (FP) and resides immediately next to a neurotoxin motif was deleted [27,28]. The entire spike 2 (S2) regions from S1 -S2 cleavage site-to-HR1 and HR1-to-SH were deleted. Only RBD (320-545 aa) with the RBM, HR1 , SH, 424 HR2, transmembrane (TM), and cytoplasmic tail (CT) were retained in the B-cell construct, with the final structure of RBD-HR1 -SH-HR2-TM-CT.

[0082] l-TASSER homology modeling was used to confirm the structural integrity of the B- cell constructs for SCoV2 Wuhan, SCoV2 Omicron XBB1 .5, HCCaCoV-NL63, and HCCaCoV-229E. These B-cell constructs served as immunogen components for the human multivalent pan-coronavirus (CoV) vaccine to be delivered in lipid nanoparticles (LNP). Similarly, the feline pan-CoV vaccine was composed of B-cell constructs for FCoV1 , FCoV2, SCoV2 Wuhan, and SCoV2 Omicron XBB1 .5. The amino acid sequence of the RBD-HR1- SH-HR2-TM-CT construct for SCoV2- Wuhan (411 aa) and FCoV1 (496 aa) was inputted individually to the l-TASSER server to obtain the homology model first for SCoV2 Wuhan and subsequently for FCoV1 UCD1 .

[0083] The SCoV2 Wuhan RBD-HR1-SH-HR2-TM-CT construct provided an outstanding structure exposing the RBM on RBD, HR1 with alpha helix, and HR2 with another alpha helix, as previously described for a full-length SCoV2 S protein and SCoV2 RBD-HR1 -HR2 [29,30]. The amino acid sequence for FCoV1-UCD1 RBD-HR1-SH-HR2-TM-CT construct was submitted and also resulted in an extending RBM from the RBD. The RBD had an extremely long N-terminal end, and its C-terminal end had an alpha helix each for HR1 and HR2. Using these two constructs as the foundation structures, the remaining B-cell constructs were developed for the human and feline pan-CoV vaccines.Example 2 - Developing the B-cell constructs for the human pan-CoV vaccine

[0084] In order to serve as a pan-CoV vaccine with multiple cross-protective potentials, our goal was to produce a multivalent vaccine that confers protection against SCoV2 variants and also against HCCaCoVs. The target HCCaCoVs are the NL63 and 229E, and their cell receptor and receptor contact sites on the virus are well documented [31 ,32], Hence, the B-cell constructs of these two HCCaCoVs have been constructed by l-TASSER to ensure proper extension of the RBM, exposing its contact sites to the cell receptor.Example 3 - B-Cell Constructs of HCCaCoV-NL63 and HCCaCoV-229E

[0085] The NL63 RBD was shortened at the N-terminal end by 113 amino acid residues into an NL63 sRBD of the final NL63 construct sRBD- HR1 -SH-HR2-TM-CT. The removal of the long N-terminal sequence of the NL63 RBD did not affect the sRBD structure with the p- sheet configuration, which extends out the RBM with the three contact sites for the host’s hACE2

[0029] . The contact sites are shown in the left, right, and top views of the NL63 homology model with a confidence score (C-score) of -1 .84 by l-TASSER

[0031] . I-TASSER used the full-length CoV spike (S) sequence as the template, and the C-score ranges from 2 to -5. The total aa residues for the full-length NL63 spike (S) protein is 1356 aa residues.Hence, the NL63 B-cell construct is only 397 aa residues, which is 29.3% of the full-length S structure. The 299E RBD was similarly shortened by 123 aa residues into an sRBD and integrated into a 229E sRBD-HR1-SH-HR2-TM-CT construct. The three 229E RBM contact sites for the human aminopeptidase-N (hAPN) receptor are extending out from the RBD p- sheet panel

[0031] , and the two N-glycosylation sites on the RBD are located away from the RBM contact sites. Lastly, the HR1 and HR2 helixes are intact, and the total structure has a reasonable C-score of -1 .84.Example 4 - Selection of recombinant B-cell constructs for human pan-CoV vaccine

[0086] The following Example describes the generation of B-cell constructs of Wuhan and Omicron XBB1 .5, where the HR2 sequence of both Wuhan and Omicron was exchanged with HR2 from an aCoV.

[0087] The SCoV2 Wuhan RBD-HR1-SH-HR2-TM-CT construct was used as the foundation B-cell construct for the three recombinant Wuhan constructs. The SCoV2 Wuhan RBD-FCoV1 / HR1 -SH-FCoVI (HR2-TM-CT), RBD-FCoV1 / HR1 -SH-FCoV1 / HR2-TMf-CT with FCoV1 adaptor for TM, and RBD-FCoV1 / HR1 -SH- FCoV1 / HR2-TMg-CT with SCoV2 adaptor for SCoV2 TM are hereby called Wuhan Recombinant-1 , Recombinant-2, and Recombinant-3 constructs. These three are recombinants of Wuhan RBD and SH with FCoV1 HR1 and HR2. Wuhan Recombinant 1 also has FCoV1 TM and CT. Remarkably, the FCoV1 TM has a small helix of 1 .5-turn at the C-terminal juncture of TM overlapping into CT, whereas the Wuhan TM has a small helix of 2-turns in the middle of TM.

[0088] Recombinant 1 has two HR1 helixes of 4-turns, each divided by a half-turn helix. Its HR2 has a long helix with 9-turns and an extremely short helix with a 0.75-turn on the N- terminal end. The Wuhan Recombinant 2 has two segmented HR1 helixes of 4.5-turns and 6-turns, and two segmented HR2 helixes of 1-turn and 6-turns. Finally, the Wuhan Recombinant 3 has three segmented HR1 helixes of two 1 .5-turns followed by 6-turns and two segmented HR2 helixes of 0.35-turn and 10-turns. Since the stem consists of FCoV1 HR1 and HR2, the helical composition of FCoV1 HR1 and HR2 must be evaluated. Unlike the long RBD of the foundation FCoV1 construct, FCoV1 with a shorter RBD had two segmented HR1s of a 0.5-turn and 8-turns, and 649 tightly connected four segmented HR2 helixes of 2-turns, a 0.5-turn, a 0.35-turn, and 5- 650 turns.

[0089] The resulting constructs are as follows:

[0090] (1) SCoV2 Wuhan (Wuhan Recombinant 3; Seq-W1 F11w) RBD-FCoV1 / HR1-SH- FCoV1 / HR2-TMg-CT, SCoV2;

[0091] (2) Omicron XBB1.5 (Omicron Recombinant 2; Seq-OM1C1w) RBD-FCoV1 / HR1 -SH-FCoV1 / HR2-TMg-CT, HCCaCoV-NL63 (Seq-sR3) sRBD-HR1-SH-HR2-TM-CT, and

[0092] (3) HCCaCoV-229E (Seq-5sR) sRBD-HRI -SH-HR2-TM-CT.

[0093] Discussion:

[0094] The foregoing Examples demonstrate that constructs that do not include adverse epitopes (e.g., ICAM-1 and superantigen motifs) can retain the protective B-cell epitopes on RBD, RBM, and SH. Complete NTD and the majority of CTD of the S1 protein were removed, leaving the RBM within the RBD and the adjacent 16 aa of CTD as the binding sites for NAbs and other protective antibodies. All of the S2 proteins, except for HR1 , SH, HR2, TM, and CT, were also deleted. The resulting SCoV2, NL63, and 229E B-cell constructs of RBD-HR1-SH-HR2-TM-CT were produced, reconstructed, and refined using the l-TASSER monomer homology modeling [11 ,36,37], and the Expasy program of theSwiss Institute of Bioinformatics, for biochemical and biophysical analyses

[0040] . The most difficult reconstruction was the exchange of Wuhan and Omicron HR2s with HR2 from an aCoV. The HR2 sequences of aCoVs were considerably different from aa sequences of SCoV2 HR2 and did not possess the BP sequence.

[0095] Table 2. Number of amino acid and pl distributions of the CoV construct*RBD-HR1-SH-HR2-TM-CT is the B-cell construct (BC), whereby the RBD includes a short C-terminal domain (sCTD) in the final construct. Abbreviations: Wuhan (W), Omicron XBB1 .5 (O), Not applicable (NA).

[0096] All four aCoV HR2 exchanges resulted in the destruction of the SCoV2 HR1 helix . Shortening of the FCoV2 HR2 (sHR2) to match the SCoV2 HR1 helix caused the destruction of SCoV2 HR1 and a short FCoV2 sHR2 helix in the Wuhan RBD-HR1 -SH-FCoV2 / sHR2- TM-CT. The exchange with the FCoV1 sHR2 resulted in a major shortening of the FCoV1 sHR2 helix in the Wuhan RBD-HR1 -SH-FCoV1 / sHR2-TM-CT. As a last exchange, both SCoV2 HR1 and HR2 were exchanged with those of either 229E or FCoV1 . Exchanging with HR1 / HR2 of 229E caused total destruction of 229E HR1 helix in both constructs with or without 229E TM-CT. Surprisingly, the exchanges with FCoV1 HR1 / HR2 retained the FCoV1 HR1 and HR2 helixes for all three.

[0097] The B-cell constructs produced are 34.4% and 34.5% of the full-length SCoV2 S protein with 1273 amino acid residues for Wuhan and 1269 amino acid residues for Omicron XBB1 .5, respectively. Consequently, about three times more B-cell constructs can be added to pDNA or mRNA. Since the RBDs of SCoV2s and aCoVs cross-react, such cross-reactive epitopes may strengthen the anti-CoV immunity against CoVs as the pan-CoV vaccine.

[0098] References:1 . Yamamoto, et al., Viruses 2023, 15, 914.2. Nair, S. et al., lnterferon-y / IL-2 and mRNA responses to the SARS-CoV2, feline coronavirus serotypes 1 (FCoV1), and FCoV2 receptor binding domains by the T cells from COVID-19-vaccinated humans and FCoV1 -infected cats. In Handbook of ELISPOT - Methods and Protocols, 4th ed.; Kalyuzhny, A.E. Ed.; Springer Nature, in press.3. Goddardet al., Vaccine. 2022, 40, 5153-5159.4. Centers for Disease Control and Prevention, Vaccines & Immunizations: Pfizer- BioNTech COVID- 19 vaccine reactions & adverse events. https: / / www.cdc.gOv / vaccines / covid-19 / info-by-product / pfizer / reactogenicity.html#print (accessed on 12 Dec 2023)5. Centers for Disease Control and Prevention, Vaccines & Immunizations: the Moderna COVID-19 Vaccine’s local reactions, systemic reactions, adverse events, and serious adverse events, www.cdc.gov / vaccines / covid-19 / info-by- product / moderna / reactogenicity.html (accessed on 12 Dec 2023)6. 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Claims

What is claimed is:1 . A composition comprising a nucleic acid encoding a human alpha coronavirus (HCaCoV) receptor binding domain (RBD) peptide and a severe acute respiratory syndrome (SARS) coronavirus 2 (SCoV2) RBD peptide.

2. The composition of claim 1 , wherein the human alpha coronavirus is HCaCoV-229E or HCaCoV-NL63.

3. The composition of claim 2, wherein the HCaCoV RBD peptide comprises an amino acid sequence that is at least 70% identical to the amino acid sequence set forth in any one of SEQ ID NO: 1 , 2, 5 or 6.

4. The composition of any one of claims 1-3, wherein the HCaCoV RBD peptide comprises an amino acid sequence that is at least 70% identical to the amino acid sequence set forth in any one of SEQ ID NO: 1 , 2, 5, or 6.

5. The composition of claim 1 , wherein the SCoV2 RBD peptide comprises an amino acid sequence that is at least 70% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, 7, or 8.

6. The composition of any one of claims 1-4, wherein the SCoV2 RBD peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, 7, or 8.

7. The composition of any one of claims 1 -10, comprising a HCaCoV RBD peptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1 , 2, 5, or 6 and a SCoV2 RBD peptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, 7, or 8.

8. The composition of any one of claims 1 -10, comprising a nucleic acid encoding an amino acid sequence set forth in any one of SEQ ID NOs: 52-57, and 105-118.

9. The composition of any one of claims 1-8, 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).

10. The composition of claim 9, wherein the composition is formulated in a lipid nanoparticle.11 . The composition of claim 10, wherein the lipid nanoparticle is T-cell specific.

12. The composition of claim 11 , wherein the lipid nanoparticle is specific for a dendritic cell.

13. The composition of claim 10, wherein the lipid nanoparticle is specific for a macrophage.

14. A vector comprising a nucleic acid encoding a human alpha coronavirus (HCaCoV) receptor binding domain (RBD) peptide and a severe acute respiratory syndrome coronavirus 2 (SCoV2) RBD peptide.

15. The vector of claim 14, wherein the human alpha coronavirus is HCaCoV- 229E or HCaCoV-NL63.

16. The vector of claim 14 or claim 15, wherein the HCaCoV RBD peptide comprises an amino acid sequence that is at least 70% identical to the amino acid sequence set forth in any one of SEQ ID NO: 1 , 2, 5, or 6.

17. The vector of any one of claims 14-16, wherein the HCaCoV RBD peptide comprises an amino acid sequence that is at least 70% identical to the amino acid sequence set forth in any one of SEQ ID NO: 1 , 2, 5, or 6.

18. The vector of claim 17, wherein the SCoV2 RBD peptide comprises an amino acid sequence that is at least 70% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, 7, or 8.

19. The vector of any one of claims 14-18, wherein the SCoV2 RBD peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, 7, or 8.

20. The vector of any one of claims 14-19, comprising a HCaCoV RBD peptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1 , 2, 5, or 6 and a SCoV2 RBD peptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, 7, or 8.21 . The vector of any one of claims 14-20, comprising a nucleic acid encoding an amino acid sequence set forth in any one of SEQ ID NOs: SEQ ID NOs: 52-57, and 105- 118.

22. A method for treating or preventing a coronavirus infection in a subject in need thereof, the method comprising administering to the subject an effective amount composition of any one of claims 1 -21 .

23. A method for inducing an immune response in a subject, the method comprising administering to a subject in need thereof an effective amount of the method comprising administering to the subject an effective amount composition of any one of claims 1-21 .

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