WO2022115471A1

WO2022115471A1 - Vaccination by heterologous boost immunization

Info

Publication number: WO2022115471A1
Application number: PCT/US2021/060606
Authority: WO
Inventors: Jasdave CHAHAL; Magnus RUEPING; Jorg EPPINGER; Ram Karan; Christian Mandl; Dominik RENN; Justine MCPARTLAN; Poulami TALUKDER
Original assignee: Tiba Biotech Llc; King Abdullah University Of Science And Technology
Priority date: 2020-11-24
Filing date: 2021-11-23
Publication date: 2022-06-02

Abstract

Vaccine combinations for protecting a subject against a Coronavirus disease comprising a priming nucleic acid vaccines and boost protein vaccines or multi-modal nucleic acid/protein vaccines are described. Methods of preventing, reducing, inhibiting, or delaying the symptoms of an infection caused by a Coronavirus, or of inducing an immune response against a viral pathogen in a subject are provided.

Description

VACCINATION BY HETEROLOGOUS BOOST IMMUNIZATION

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. provisional application No. 63/117,762, filed November 24, 2020, which is incorporated by reference as if fully set forth.

[0002] The sequence listing electronically filed with this application titled “Sequence Listing,” which was created on November 23, 2021 and had a size of 68,562 bytes is incorporated by reference herein as if fully set forth.

FIELD OF INVENTION

[0003] The present disclosure relates to methods of conferring immunity in an organism against disease by vaccination and the composition of vaccines used for this purpose.

BACKGROUND

[0004] Classically, vaccines are administered as a single drug substance consisting of recombinant subunits, inactivated pathogens, live attenuated pathogens, nucleic acid, or viral vector formulations. They are not administered as combinations of the different modalities, and boosts are typically performed with the identical form of vaccine as homologous boost. The superior method of immunization is required in clinical practice. The method must be rapid to adapt and manufacture for swift deployment in response to novel outbreaks. This need was clearly demonstrated by the 2019/2020 COVID-19 pandemic that ravaged the global economy and led to hundreds of thousands of deaths worldwide.

[0005] Additionally, Middle East respiratory syndrome (MERS), a viral respiratory infection endemic on the Arabian Peninsula, has a case fatality rate reaching 34%, globally. This is caused by the MERS-coronavirus (MERS-CoV), a member of the same betacoronavirus genera as SARS-CoV and SARS-CoV-2. First isolated in 2012, this is a recurring zoonotic disease in which the primary reservoir is dromedary camels. MERS remains an endemic risk across the region of origin, with seroprevalence in camels ranging from 70% upwards to 95%, and continued transmission to the human population to this day. The superior method of immunization is required to prevent or treat diseases caused by coronaviruses.

SUMMARY

[0006] In an aspect, the invention relates to a method of preventing, reducing, inhibiting, or delaying the symptoms of an infection caused by a Coronavirus, or of inducing an immune response against the Coronavirus in a subject. The method comprises administering to the subject a prime vaccine comprising an effective amount of a polynucleotide encoding a first antigenic protein or a subunit thereof, and a first carrier. The method also comprises administering to the subject a boost vaccine comprising an effective amount of a second antigenic protein, or a subunit thereof, and a second carrier. The second antigenic protein is a subunit of the first antigenic protein.

[0007] In an aspect, the invention relates to a vaccine combination for protecting a subject against a Coronavirus disease comprising a prime vaccine and a boost vaccine. The prime vaccine comprises an effective amount of a polynucleotide encoding a first antigenic protein or a subunit thereof, and a first carrier. The boost vaccine comprises an effective amount of a second antigenic protein, or a subunit thereof, and a second carrier. The second antigenic protein is a subunit of the first antigenic protein.

[0008] In an aspect, the invention relates to a kit comprising any one of the prime and boost vaccines disclosed herein. The kit is used for sequential administration of the prime and boost vaccines.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The following detailed description of preferred embodiments of the present invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, particular embodiments are shown in the drawings. It is understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. In the drawings:

[0010] FIG. 1 are schematic drawings illustrating rationale of a focusing boost approach to driving potent immunization against a neutralization target on a pathogen antigen.

[0011] FIG. 2 illustrates result of RNA prime/RBD protein focusing boost immunization in mice developing superior antibody titers in comparison to RNA prime or RBD protein prime alone.

[0012] FIG. 3 illustrates ELISpot assay using splenocytes isolated from vaccinated animals.

[0013] FIG. 4 illustrates the focusing-boost approach.

[0014] FIG. 5 illustrates the Day 76 ELISA data.

[0015] FIG. 6 illustrates the Day 60 ELISA data.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Certain terminology is used in the following description for convenience only and is not limiting. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0017] The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The phrase “at least one” followed by a list of two or more items, such as “A, B, or C,” means any individual one of A, B or C as well as any combination thereof.

[0018] The words "right," "left," "top," and "bottom" designate directions in the drawings to which reference is made.

[0019] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. [0020] The present disclosure relates to a heterologous prime/boost vaccine platform that focuses an immune response to effectively neutralize a pathogen of interest in a mammalian host organism.

[0021] As used herein, the term “coronavirus” refers to single-stranded, positive- sense RNA viruses that belong to the Coronaviridae family. The Coronaviridae family includes but is not limited to SARS-Cov, MERS-Cov and SARS-CoV-2 (COVID-19) viruses, and their mutants. The coronavirus genome encodes numerous non-structural proteins and four major structural proteins including the spike (S), nucleocapsid (N), membrane (M) and small envelope (E). Spike (S) protein, a large envelope glycoprotein, is composed of S1 and S2 subunits. The “Receptor-binding domain (RBD)” is located in the S1 subunit. Preferable example of the structural protein used herein is coronavirus RBD. Spike protein, S1 and S2 subunits of the spike protein and RBD of COVID-19 and its mutants have been identified and published (Wrapp et al., 2020 Mar 13;367(6483):1260-1263, which is incorporated herein by reference as if fully set forth).

[0022] The terms “subject” and “individual” are used interchangeably herein, and mean a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents. In an embodiment, the subject may be a mammal, e.g., a primate, e.g., a human. The terms, “patient” and “subject” are used interchangeably herein. The terms, “patient” and “subject” are used interchangeably herein.

[0023] Preferably, the subject is a mammal. The mammal may be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. In addition, the methods described herein may be used to treat domesticated animals and/or pets. A subject may be male or female. A subject may be a child or adult.

[0024] The present disclosure relates to heterologous immunization methods by prime and boost vaccines, which reduce off-target effects and enhance the beneficial immune response against functional domains of viral proteins. A booster vaccine targets functionally relevant pathogen B cell epitopes to focus a patient’s secondary adaptive antibody response to components of a pathogen that ensure its neutralization. FIG. 1 illustrates the heterologous immunization method referred to herein as the “Focusing Boost” principle. This principle is applied by conscientious design of both, the prime vaccine and focusing boost vaccine to ensure maximal induction of the immune system against the desired target. The prime and boost vaccines are selected to operate by fundamentally different mechanisms (e.g., nucleic acid for one, and protein for the other) in order to benefit from the heterologous mechanism that induces the strongest responses. The booster immunogen is designed to be a subunit of the antigen delivered in the prime dose, to promote a high-affinity neutralizing IgG response and the induction of long-lasting antibody-producing plasma cells and memory B cells. [0025] A prime-boost regimen may be deemed heterologous based on several factors, including selection of antigen, type of vector, delivery route, and adjuvant. As used herein, the term “heterologous” and “heterologous prime-boost” refer to an immunization regimen that uses two different vaccine modalities targeting the same or related antigen target, one being an RNA-based vaccine and the other being a protein-based vaccine. The heterologous regime may extend to a multimodal vaccine. The term “multi-modal” refers to a single vaccine candidate consisting of two or more components, here RNA and protein components, each component playing a dominant role at different periods of an immunization schedule.

[0026] “Antigen” as used herein is defined as a molecule that triggers an immune response. The immune response may involve either antibody production, or the activation of specific immunologically active cells, or both. The antigen may refer to any molecule capable of stimulating an immune response, including macromolecules such as proteins, peptides, or polypeptides. The antigen may be a structural component of a pathogen, ora cancer cell. The antigen may be synthesized, produced recombinantly in a host, or may be derived from a biological sample, including but not limited to a tissue sample, cell, or a biological fluid. [0027] The term antigen herein may refer to the “first antigenic protein” or the “second antigenic protein.” For example, the first antigenic protein or the second antigenic protein may be a protein comprising one or more antigenic determinants of the viral pathogen described herein. The first antigenic protein may be the same protein as the second antigenic protein. The second antigenic protein may be a subunit or an epitope of the first antigenic protein. The second antigenic protein may be a subunit or an epitope of the antigenic protein that is similar to the first antigenic protein.

[0028] An embodiment provides a heterologous vaccine combination for protecting a subject against a disease comprising a prime vaccine and a boost vaccine. The prime vaccine may comprise an effective amount of a polynucleotide encoding a first antigenic protein or a subunit thereof, and a first carrier. The boost vaccine may comprise an effective amount of a second antigenic protein, or a subunit thereof, and a second carrier. The first and second antigenic proteins may be of identical sequence. Each one of the prime and boost may be included in a formulation with carrier. Adjuvant may be formulated with either or both prime and boost vaccine compositions. The prime and boost vaccine combination may be formulated without adjuvant.

[0029] The first antigenic protein or the second antigenic protein may be isolated or derived from a viral pathogen. The viral pathogen may be adenovirus, rhinovirus, rotavirus, West Nile virus, Zika virus, herpes, or coronavirus (CoV). The coronavirus may be one of MERS CoV, SARS-CoV and SARS-CoV-2 viruses.

[0030] The prime vaccine

[0031] The prime vaccine, also referred to herein as a prime vaccine composition, or a nucleic acid vaccine, relies on cell processes to produce an antigen. The prime vaccine may be designed such that the full-length form of the antigen is expressed, ensuring its appropriate post-translational processing and display to the adaptive cellular immune system via MHC class I and II presentation. [0032] In an embodiment, the prime vaccine may comprise a polynucleotide encoding a full- length antigenic protein or only minimally truncated disease-related protein or protein domain. The minimally truncated disease-related protein or protein domain may be a protein that retains the overall antigenic tertiary structure. The polynucleotide may be a molecule that encodes the full-sized form of a pathogen antigen. For example, the polynucleotide may be the complete coding sequence of a virion surface protein.

[0033] In an embodiment, the polynucleotide may encode the first antigenic protein or the second antigenic protein. The first antigenic protein or the second antigenic protein may be a smaller subunit or specific epitope or combination of shorter epitopes derived from that initial prime antigen sequence. The term “epitope” refers to a portion of an antigen that is recognized by the immune system. The term “protein subunit” refers to a protein subunit of the pathogen expressed in a recombinant expression system and purified for use in a vaccine formulation.

[0034] The polynucleotide may encode the first antigenic protein or the second antigenic protein that are naturally-occurring proteins, or naturally-occurring variant proteins having amino acid deletions, substitutions, or additions, or a fusion of two more different naturally occurring proteins. The polynucleotide may encode antigenic proteins encompassing polypeptides that comprise one or more amino acid analogs, derivatives, or stereoisomers. The polynucleotide may encode the protein subunit comprising a receptor binding domain (RBD). The RBD may be derived from a virus, e.g., may be a virion surface protein.

[0035] The polynucleotide may encode the first antigenic protein or the second antigenic protein that comprises a viral Spike protein, or a fragment thereof. The viral Spike protein may be a peptide or polypeptide corresponding to one or more antigenic determinants of the receptor binding domain of the SARS-CoV Spike protein, SARS-CoV-2 Spike protein, or MERS-CoV Spike protein. The receptor binding domain of the SARS-CoV spike protein, SARS-CoV-2 Spike protein, or MERS-CoV Spike protein may be 10-20 amino acid residues in length, and may contain more than one peptide determinants of up to about 30-50 residues or more.

[0036] The polynucleotide may encode a Spike SARS-CoV protein. The Spike SARS-CoV protein may be the full-length protein. The full-length SARS-CoV protein may have an amino acid sequence as set forth in SEQ ID NO: 1. The polynucleotide may encode a fragment of the Spike SARS-CoV protein. The fragment may be of 10 amino acids or longer. The fragment may comprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, or any number of amino acids in a range between any two of the foregoing (endpoints inclusive). The polynucleotide may encode the SARS-CoV protein comprising an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 1 . The polynucleotide may encode the S1 portion of the viral Spike protein, or Spike S1 SARS-CoV. The polynucleotide may encode the Spike S1 SARS-CoV comprising an amino acid sequence as set forth in SEQ ID NO: 4. The polynucleotide may encode the Spike S1 SARS-CoV comprising an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 4. The polynucleotide may encode the Spike S1 SARS-CoV that is a fragment of a Spike S1 SARS-CoV of 10 amino acids or longer.

[0037] The polynucleotide may encode a Spike SARS-CoV-2 protein. The polynucleotide may encode the full-length Spike SARS-CoV-2 protein. The full-length Spike SARS-CoV-2 may have an amino acid sequence as set forth in SEQ ID NO: 2. The polynucleotide may encode a fragment of the Spike SARS-CoV-2 protein. The polynucleotide may encode the fragment that is 10 amino acids or longer. The polynucleotide may encode the fragment comprising 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, or any number of amino acids in a range between any two of the foregoing (endpoints inclusive). The polynucleotide may encode the Spike SARS-CoV-2 comprising an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 2. The polynucleotide may encode the S1 portion of the viral Spike protein, or Spike S1 SARS-CoV-2. The polynucleotide may encode the Spike S1 SARS-CoV-2 comprising an amino acid sequence as set forth in SEQ ID NO: 5. The polynucleotide may encode the Spike S1 SARS-CoV-2 comprising an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 5. The polynucleotide may encode the fragment of a Spike S1 SARS-CoV-2 of 10 amino acids or longer. [0038] The polynucleotide may encode a Spike MERS-CoV protein. The polynucleotide may encode the full-length Spike MERS-CoV protein. The full-length Spike MERS-CoV protein may have an amino acid sequence as set forth in SEQ ID NO: 3. The polynucleotide may encode a fragment of the Spike MERS-CoV protein. The polynucleotide may encode the fragment of 10 amino acids or longer. The polynucleotide may encode the fragment comprising 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, or any number of amino acids in a range between any two of the foregoing (endpoints inclusive). The polynucleotide may encode the Spike MERS-CoV protein comprising an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 3. The polynucleotide may encode the S1 portion of the viral Spike protein, or Spike S1 MERS-CoV protein. The polynucleotide may encode the Spike S1 MERS- CoV comprising an amino acid sequence as set forth in SEQ ID NO: 6. The polynucleotide may encode the Spike S1 MERS-CoV comprising an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 6. The polynucleotide may encode a fragment of a Spike S1 MERS-CoV of 10 amino acids or longer.

[0039] Determining percent identity of two amino acid sequences or two nucleic acid sequences may include aligning and comparing the amino acid residues or nucleotides at corresponding positions in the two sequences. If all positions in two sequences are occupied by identical amino acid residues or nucleotides then the sequences are said to be 100% identical. Percent identity is measured by the Smith Waterman algorithm (Smith TF, Waterman MS 1981 “Identification of Common Molecular Subsequences,” J Mol Biol 147: 195 -197, which is incorporated herein by reference as if fully set forth).

[0040] The polynucleotide may be a DNA or RNA molecule. The term “DNA” or “DNA molecule” or ‘‘deoxyribonucleic acid molecule” refers to a polymer of deoxyribonucleotides. The DNA molecule may be a polynucleotide, oligonucleotide, DNA, or cDNA molecule. The DNA molecule may encode wild-type or engineered proteins, peptides or polypeptides. The term "RNA" or "RNA molecule" or "ribonucleic acid molecule" refers to a polymer of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, or more ribonucleotides).

[0041] The RNA molecule may be a messenger RNA (mRNA) or replicon RNA (repRNA). The RNA molecule may be a monocistronic or polycistronic mRNA.

[0042] A monocistronic mRNA refers to an mRNA comprising only one sequence encoding a protein, polypeptide or peptide. A polycistronic mRNA typically refers to two or more sequences encoding two or more proteins, polypeptides or peptides. An mRNA may encode a protein, polypeptide, or peptide that acts as an antigen. The messenger RNAs (mRNAs) are single-stranded RNAs that define the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis when ribosomes bind to the mRNA. The DNA or RNA molecules may be chemically modified.

[0043] The replicon RNA (repRNA) refers to a replication-competent, progeny-defective RNA virus genome that is incapable of producing infectious progeny virions. Viral genomes that are typically modified for use as repRNAs include ‘‘positive strand” RNA viruses. The modified viral genomes function as both mRNA and templates for replication. The repRNA molecule may be derived from an RNA virus or a retrovirus. The RNA virus may be an alphavirus, for example, Venezuelan equine encephalitis virus (VEE). An alphavirus-based ‘‘replicon” expression vectors may be used in embodiments herein (Pushko et al. (1997) Virology 239:389-401 ; which is incorporated by reference herein as if fully set forth).

[0044] The repRNA of replicon expression vectors may contain viral genes that include but not limited to replicases, proteases, helicases, 5'- and 3'-end cis-active replication sequences, and other nonstructural viral proteins. The repRNA may also comprise heterologous sequences that encode amino acid sequence, for example, of the first or second antigenic proteins.

[0045] The repRNA may include a subgenomic promoter that directs expression of the heterologous sequence. The heterologous sequence, for example, encoding the first or second antigenic proteins, may be fused in frame to other coding regions in the repRNA. The heterologous sequences may also may be under the control of an internal ribosome entry site (IRES). Overexpression of proteins or antigens by repRNA molecules may lead to stimulation of toll-like receptors (TLR) and non TLR pathways by the products of RNA replication and amplification.

[0046] The repRNA molecules of the embodiments herein may be within a range of 4 kb to 15 kb. For example, the repRNA may be about 4 kb, about 5 kb, about 6 kb, about 7 kb, about 8 kb, about 9 kb, about 10 kb, about 11 kb, about 12 kb, about 13 kb, about 14 kb, or about 15 kb. The repRNA may be 4 kb to 15kb, 4 kb to 14 kb, 4 kb to 13 kb, 4 kb to 12 kb, 4 kb to 11 kb, 4 kb to 10 kb, 5 kb to 15 kb, 5 kb to 14 kb, 5 kb to 13 kb, 5 kb to 12 kb, 5 kb to 11 kb, 5 kb to 10 kb, 6 kb to 15 kb, 6 kb to 14 kb, 6 kb to 13 kb, 6 kb to 12 kb, 6 kb to 11 kb, 6 kb to 10 kb, 5 kb to 8 kb, 5 kb to 6 kb, 4 kb to 6 kb, 7 kb to 10 kb, 9 kb to 11 kb, or any length in a range between any two of the foregoing (endpoints inclusive).

[0047] The repRNA molecules described herein may comprise one or more modified nucleotides. The modified nucleotides may be 5-methyluridine, 5-methylcytidine, pseudouridine, or N6-methyladenosine.

[0048] The repRNA molecule may encode one or more antigenic proteins. The proteins or polypeptides generated from the repRNA may be expressed as a fusion polypeptide. The proteins may be expressed as separate proteins, polypeptide or peptide sequences. The repRNA of the invention may encode one or more antigenic proteins that contain one or more epitopes capable of eliciting an immune response, e.g., a helper T-cell response or a cytotoxic T-cell response. The repRNA may encode antigenic proteins together with cytokines or other immunomodulators which may be useful for production of bivalent or multivalent vaccine.

[0049] The repRNA molecules may be screened or analyzed to confirm their therapeutic and prophylactic properties using known in vitro or in vivo assays. Vaccines comprising repRNA molecule may be assessed for their effect on induction of proliferation or effector function of the particular lymphocyte type of interest. For example, spleen cells from immunized mice may be isolated and the capacity of cytotoxic T lymphocytes to lyse autologous target cells that contain a repRNA molecule that encodes a polypeptide antigen may be assessed. T helper cell differentiation may be analyzed by measuring proliferation or production of TH1 (IL-2 and IFN-y) and/or TH2 (IL-4 and IL-5) cytokines by ELISA or directly in CD4+ T cells by cytoplasmic cytokine staining and flow cytometry.

[0050] RepRNA molecules that encode a polypeptide antigen may also be tested for ability to induce humoral immune responses, as evidenced, for example, by induction of B cell production of antibodies specific for an antigen of interest. These assays may be performed by using peripheral B lymphocytes from immunized individuals. Such assay methods are known to those of skill in the art. Other assays may be used characterize the repRNA molecules of the embodiments herein. [0051] In an embodiment, the polynucleotide may be an mRNA or repRNA encoding the viral Spike protein, or a fragment thereof. The mRNA or repRNA may encode a Spike SARS-CoV protein, Spike SARS-CoV-2 protein or Spike MERS-CoV. The polynucleotide encoding the Spike SARS-CoV protein may be an RNA sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence set forth in SEQ ID NO: 7. The polynucleotide encoding the Spike SARS-CoV-2 protein may be an RNA sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 8. The polynucleotide encoding the Spike MERS-CoV. protein may be an RNA sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 9.

[0052] The polynucleotide may be encapsulated in a first carrier system for in vivo administration. The first carrier may be, but is not limited to, an anionic liposome, a cationic liposome, or a dendrimer.

[0053] The term “anionic liposomes” refers to liposomes that include lipids comprising an anionic group. Anionic liposomes may be formed by anionic phospholipids. The phospholipids may include but are not be limited to 1 ,2-Didecanoyl-sn-Glycero-3-phosphatidylcholine (DDPC); 1 ,2-Dierucoyl-sn- Glycero-3-Phosphate (DEPA); 1 ,2-Erucoyl-sn-Glycero-3-phosphatidylcholine (DEPC); 1 ,2-Dierucoyl- sn-Glycero-3-phosphatidylethanolamine (DEPE); 1 ,2-Dierucoyl-sn-Glycero-3[Phosphatidyl-rac-(1- glycerol . . .) (DEPG); 1 ,2-Linoleoyl-sn-Glycero-3-phosphatidylcholine (DLOPC); 1 ,2-Dilauroyl-sn- Glycero-3-Phosphate (DLPA); 1 ,2-Dilauroyl-sn-Glycero-3-phosphatidylcholine (DLPC); 1 ,2-Dilauroyl- sn-Glycero-3-phosphatidylethanolamine (DLPE); 1 ,2-Dilauroyl-sn-Glycero-3[Phosphatidyl-rac-(1- glycerol . . .) (DLPG); 1 ,2-Dilauroyl-sn-Glycero-3-phosphatidylserine (DLPS); 1 ,2-Dimyristoyl-sn- glycero-3-phosphoethanolamine (DMG); 1 ,2-Dimyristoyl-sn-Glycero-3-Phosphate (DMPA); 1 ,2- Dimyristoyl-sn-Glycero-3-phosphatidylcholine (DMPC); 1 ,2-Dimyristoyl-sn-Glycero-3- phosphatidylethanolamine (DMPE); 1 ,2-Myristoyl-sn-Glycero-3[Phosphatidyl-rac-(1 -glycerol . . .) (DMPG); 1 ,2-Dimyristoyl-sn-Glycero-3-phosphatidylserine (DMPS); 1 ,2-Dioleoyl-sn-Glycero-3- Phosphate (DOPA); 1 ,2-Dioleoyl-sn-Glycero- 3-phosphatidylcholine (DOPC); 1 ,2-Dioleoyl-sn- Glycero-3-phosphatidylethanolamine (DOPE); 1,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac-(1- glycerol . . .) (DOPG); 1 ,2-Dioleoyl-sn-Glycero-3-phosphatidylserine (DOPS); 1 ,2-Dipalmitoyl-sn- Glycero-3-Phosphate DPPC 1 ,2-Dipalmitoyl-sn-Glycero-3-phosphatidylcholine (DPPA); 1 ,2- Dipalmitoyl-sn-Glycero-3-phosphatidylethanolamine (DPPE) 1 ,2-Dipalmitoyl-sn-Glycero- 3[Phosphatidyl-rac-(1 -glycerol . . .) (DPPG); 1 ,2-Dipalmitoyl-sn-Glycero-3-phosphatidylserine (DPPS); 1 ,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE); 1 ,2-Distearoyl-sn-Glycero-3- Phosphate (DSPA); 1 ,2-Distearoyl-sn-Glycero-3-phosphatidylcholine (DSPC); 1 ,2-Distearpyl-sn- Glycero-3-phosphatidylethanolamine (DSPE); 1 ,2-Distearoyl-sn-Glycero-3[Phosphatidyl-rac-(1- glycerol . . .) (DSPG); 1 ,2-Distearoyl-sn-Glycero-3-phosphatidylserine (DSPS); Egg-PC HEPC Hydrogenated Egg PC (EPC); High purity Hydrogenated Soy PC HSPC Hydrogenated Soy PC (HSPC); 1-Myristoyl-sn-Glycero-3-phosphatidylcholine (LYSOPC MYRISTIC); 1-Palmitoyl-sn- Glycero-3-phosphatidylcholine (LYSOPC PALMITIC); 1-Stearoyl-sn-Glycero-3-phosphatidylcholine Milk Sphingomyelin (LYSOPC STEARIC); 1-Myristoyl,2-palmitoyl-sn-Glycero 3-phosphatidylcholine (MPPC); 1-Myristoyl,2-stearoyl-sn-Glycero-3-phosphatidylcholine (MSPC); 1-Palmitoyl,2-myristoyl- sn-Glycero-3-phosphatidylcholine (PMPC); 1-Palmitoyl,2-oleoyl-sn-Glycero-3-phosphatidylcholine (POPC); 1-Palmitoyl-2-oleoyl-sn-Glycero-3-phosphatidylethanolamine (POPE); 1 ,2-Dioleoyl-sn- Glycero-3[Phosphatidyl-rac-(1-glycerol) . . .] (POPG); 1-Palmitoyl,2-stearoyl-sn-Glycero-3- phosphatidylcholine (PSPC); 1-Stearoyl,2-myristoyl-sn-Glycero-3-phosphatidylcholine (SMPC); 1- Stearoyl,2-oleoyl-sn-Glycero- 3-phosphatidylcholine (SOPC); and 1 -Stearoyl,2-palmitoyl-sn-Glycero- 3-phosphatidylcholine (SPPC). Useful phospholipids may include, but are not limited to, phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines, and phosphatidylglycerols. [0054] The term “cationic liposomes” refers to liposomes that are made in whole or part from positively charged lipids, or more specifically a lipid that comprises both a cationic group and a lipophilic portion. The positively charged moieties of cationic lipids used in cationic liposomes provide advantageous structural features. For instance, the lipophilic portion of the cationic lipid is hydrophobic and thus may direct itself away from the aqueous interior of the liposome and associate with other nonpolar and hydrophobic species, or conversely, the cationic moiety may associate with polar molecules and species with which it can complex in the aqueous interior of the cationic liposome. The positively charged liposomes may interact with the negatively charged nucleic acid molecules to form a stable complex. Cationic lipids may include but are not be limited to, dioleoyl trimethylammonium propane (DOTAP), 1 ,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1 ,2-dioleyloxy- N,Ndimethyl-3-aminopropane (DODMA), 1 ,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA). Examples of cationic liposomes include lipofectin, lipofectamine, and lipofectace.

[0055] Liposomes may include zwitterionic lipids. As used herein the term “zwitterionic” refers to a molecule that contains both positive and negative charges, but have a net neutral charge, Zwitterionic lipids, also referred to herein as neutral lipids, may include but not be limited to acyl zwitterionic lipids and ether zwitterionic lipids. Zwitterionic lipids may include DPPC, DOPC and dodecylphosphocholine.

[0056] Liposomes may be formed from a single lipid or from a mixture of lipids. The hydrophilic portion of a lipid can be PEGylated, i.e., modified by covalent attachment of a polyethylene glycol to increase stability and prevent non-specific adsorption of the liposomes (Heyes et al. (2005) J Controlled Release 107:276-87, which is incorporated herein by reference as if fully set forth).

[0057] The term dendrimer” refers to a highly branched macromolecule with a spherical shape. The surface of the dendrimer molecule may be modified in many ways, and many of the properties of the resulting construct may be determined by its surface. The dendrimers may be modified to have a positive surface charge, i.e., to be cationic dendrimers. The cationic dendrimers may form temporary association with the nucleic acids. Upon reaching its destination the dendrimer-nucleic acid complex may be then taken into the cell via endocytosis.

[0058] An exemplary size for a single dendrimer-nucleic acid complex, also referred to herein as modified dendrimer nanoparticles (MDNPs), may be in the range of 30 nm to 1 ,000 nm in the longest dimension. MDNPs may have an average size from 30 nm to 450 nm, inclusive, from 50 nm to 300 nm, inclusive, or more from 60 nm to 250 nm, inclusive. MDNPS may be alkyl-modified dendrimer nanoparticles. Nanoparticle size may be influenced by the length of the alkyl chain that substitutes the core dendrimer. Methods of making and formulating modified dendrimer nanoparticles are described in WO2021 207020, published October 14, 2021 ; US 20210330600, published October 28, 2021; and US 20210338789, published November 4, 2021 ; all of which are incorporated herein by reference as if fully set forth.

[0059] The polynucleotide may be non-covalently bound or covalently bound to the first carrier. The polynucleotide may be electrostatically bound to the charged carrier molecule through an ionic bond.

[0060] Boost Vaccine

[0061 ] In an embodiment, the boost vaccine may be a protein vaccine. The protein vaccine may comprise the first antigenic protein or the second antigenic protein.

[0062] In an embodiment, the first antigenic protein or the second antigenic protein may be identical to the proteins encoded by a polynucleotide included in the prime vaccine disclosed herein. In an embodiment, the first antigenic protein or the second antigenic protein may differ from the proteins encoded by a polynucleotide included in the prime vaccine disclosed herein.

[0063] In an embodiment, the first antigenic protein or the second antigenic protein may be a viral Spike protein, or a fragment thereof. The viral Spike protein may be a peptide or polypeptide corresponding to one or more antigenic determinants of the receptor binding domain (RBD) of the SARS-CoV Spike protein, SARS-CoV-2 Spike protein, or MERS-CoV Spike protein. The receptor binding domain of the SARS-CoV spike protein, SARS-CoV-2 Spike protein, or MERS-CoV Spike protein may be 10-20 amino acid residues in length, and may contain more than one peptide determinants or up to about 30-50 residues or more. [0064] The viral Spike protein may be a Spike SARS-CoV protein. The Spike SARS-CoV protein may be the full-length protein. The full-length SARS-CoV protein may have an amino acid sequence as set forth in SEQ ID NO: 1. The viral Spike protein may be a fragment of the Spike SARS-CoV protein. The fragment may be of 10 amino acids or longer. The fragment may comprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, or any number of amino acids in a range between any two of the foregoing (endpoints inclusive). The SARS-CoV protein may have an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 1. The SARS-CoV protein may be the S1 portion of the viral Spike protein, or Spike S1 SARS-CoV. The Spike S1 SARS-CoV may have an amino acid sequence as set forth in SEQ ID NO: 4. The Spike S1 SARS-CoV may have an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 4. The Spike S1 SARS-CoV may be a fragment of a Spike S1 SARS-CoV of 10 amino acids or longer.

[0065] The viral Spike protein may be a Spike SARS-CoV-2 protein. The Spike SARS-CoV-2 protein may be the full-length protein. The full-length Spike SARS-CoV-2 may have an amino acid sequence as set forth in SEQ ID NO: . The viral Spike protein may be a fragment of the Spike SARS- CoV-2 protein. The fragment may be of 10 amino acids or longer. The fragment may comprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, or any number of amino acids in a range between any two of the foregoing (endpoints inclusive). The Spike SARS-CoV-2 may have an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 2. The Spike SARS-CoV-2 may be the S1 portion of the viral Spike protein, or Spike S1 SARS-CoV-2. The Spike S1 SARS-CoV-2 may have an amino acid sequence as set forth in SEQ ID NO: 5. The Spike S1 SARS-CoV-2 may have an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 5. The Spike S1 SARS-CoV-2 may be a fragment of a Spike S1 SARS-CoV-2 of 10 amino acids or longer.

[0066] The viral Spike protein may be a Spike MERS-CoV protein. The Spike MERS-CoV protein may be the full-length protein. The full-length Spike MERS-CoV protein may have an amino acid sequence as set forth in SEQ ID NO: 3. The viral Spike protein may be a fragment of the Spike MERS- CoV protein. The fragment may be of 10 amino acids or longer. The fragment may comprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, or any number of amino acids in a range between any two of the foregoing (endpoints inclusive). The Spike MERS-CoV protein may have an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 3. The Spike MERS-CoV protein may be the S1 portion of the viral Spike protein, or Spike S1 MERS-CoV protein. The Spike S1 MERS-CoV may have an amino acid sequence as set forth in SEQ ID NO: 6. The Spike S1 MERS-CoV may have an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 6. The Spike S1 MERS-CoV may be a fragment of a Spike S1 MERS-CoV of 10 amino acids or longer.

[0067] In an embodiment the first antigenic protein, the second antigenic protein or subunit (i.e., RBD) may be fused to a suitable second carrier. The second carrier may include but may not be limited to GvpA-protein of gas vesicle nanoparticles (GVNP), such as those produced by halophilic archaea; GvpC-protein which is binding to the surface of gas vesicle nanoparticles (GVNP); a protein forming a self-assembling nanocage, e.g., ferritin and ferritin-like proteins; a protein forming solid self- assembled structures, e.g., keyhole limpet hemocyanin (KLH), and Concholepas concholepas hemocyanin (CCH); and proteins forming virus-like particles (VLPs) (Pfeifer, 2012 Nat Rev Microbiol 10, 705-715; and Dutta et al., 2015 Malar J 14, 406; both of which are incorporated herein by reference as if fully set forth).

[0068] Immunoregulatory agents

[0069] In an embodiment, the first antigenic protein, the second antigenic protein may be fused with an adjuvanting protein, an immune-stimulating protein, or a peptide moiety. The adjuvanting protein may comprise a keyhole limpet hemocyanin (KLH), or Concholepas concholepas hemocyanin (CCH). The peptide moiety may comprise a CD4+ T cell-activating helper peptide.

[0070] In an embodiment, any boost vaccine described herein may be used as the prime vaccine.

[0071] In an embodiment, any prime vaccine described herein may be used as the boost vaccine. The prime vaccine may be the same vaccine as used as the boost vaccine.

[0072] In an embodiment, the prime vaccine may be a multi-modal vaccine. The multi-modal vaccine may be a vaccine comprising two or more components. The first component may be an RNA component as described herein. The second component may be a protein component described herein. Each component of the multi-modal vaccine may play a dominant role at different periods of an immunization schedule.

[0073] Method of Immunization

[0074] An embodiment provides a method of preventing, reducing, inhibiting, or delaying the symptoms of an infection caused by a viral pathogen, or of inducing an immune response against a viral pathogen in a subject, such as a vertebrate, preferably a mammal, is provided. The viral pathogens may be but are not limited to SARS-Cov, MERS-Cov and SARS-CoV-2 (COVID-19) viruses, and mutants thereof. The method may comprise administering to the subject a prime vaccine comprising an effective amount of any one of polynucleotides encoding a first antigenic protein or a subunit thereof, and a first carrier. The prime vaccine may be any of prime vaccines described herein. The method may also comprise administering to the subject a boost vaccine comprising an effective amount of a second antigenic protein, or a subunit thereof, and a second carrier. The second antigenic protein may the first antigenic protein or a subunit of the first antigenic protein. The boost vaccine may be anyone of boost vaccines described herein. The boost vaccine may be administered subsequently to the prime vaccine. The boost vaccine may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40,

41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 or 60, or any number of weeks in a range between any two of the foregoing (endpoints inclusive) after the prime vaccine is administered.

[0075] In an embodiment, a method of immunization is provided. The method may involve “priming” and “boosting” immunization regimes, in which the immune response induced by a prime vaccine may be boosted by a boost vaccine. For example, following priming (at least once) with any one of polynucleotides encoding a first antigenic protein or a subunit thereof, a boost vaccine comprising an effective amount of a second antigenic protein, or a subunit thereof, may be administered to boost the immune response in the primed host. The second antigenic protein may be the first antigenic protein or a subunit of the first antigenic protein.

[0076] The subject may be primed and/or boosted more than once. For example, the immunization strategy may be prime, prime, boost; or prime, boost, boost. In an embodiment, the prime vaccine may be administered as least twice, at least 3 times, at least 4 times, at least 5 times, or at least 6 times. In an embodiment, the boost vaccine may be administered as least twice, at least 3 times, at least 4 times, at least 5 times, or at least 6 times.

[0077] In an embodiment, administration of the boosting may be performed weeks or months following administration of the prime vaccine. For example, the boost vaccine may be administered 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 35 weeks, 40 weeks, 45 weeks, 50 weeks, 55 weeks, 60 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years or any period of time in a range between any two of the foregoing (endpoints inclusive) after the priming composition is administered. [0078] In an embodiment of the method, prime and boost vaccines may be administered as a multi-modal vaccine. The multi-modal vaccine may comprise an RNA component and a protein component. The RNA component may comprise an effective amount of any one of polynucleotides encoding a first antigenic protein or a subunit thereof and the protein component may comprise an effective amount of a second antigenic protein, or a subunit thereof. The second antigenic protein may be the first antigenic protein or a subunit of the first antigenic protein.

[0079] When the RNA component and the protein component are co-administered, it may be desirable to package the protein component and RNA component separately. The two components may be combined prior to administration, for example, within 72 hours, 70 hours, 65 hours, 60 hours, 55 hours, 50 hours, 45 hours, 40 hours, 36 hours, 35 hours, 30 hours, 24 hours, 20 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes or any period with a range between any two of the foregoing (endpoints inclusive).

[0080] As used herein, the terms "treat," treating," "treatment," refer to reducing or ameliorating a disorder and/or symptoms associated therewith. Treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

[0081] As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

[0082] The vaccine may be administered in an effective amount. The efficacy of the therapeutic treatment may involve monitoring pathogen infection following administration of prime and boost vaccines disclosed herein. The efficacy of prophylactic treatment may involve monitoring immune responses systemically and/or mucosally against the antigen. Systemic monitoring of the immune responses may involve monitoring the levels of lgG1 and lgG2a production whereas mucosal monitoring may involve assessing the level of IgA production. Antigen-specific serum antibody responses may be determined post-immunization but pre-challenge. Antigen-specific mucosal antibody responses may be determined post-immunization and post-challenge.

[0083] Assessing the immunogenicity of the prime vaccine disclosed herein where the nucleic acid encodes an antigenic protein may involve expressing the antigen recombinantly for screening patient sera or mucosal secretions by immunoblot and/or microarrays. A positive reaction between the protein and the patient sample may indicate that the patient has mounted an immune response to the assessed protein.

[0084] The efficacy of the prime and boost vaccines may also be determined in vivo by challenging appropriate animal models of the pathogen infection. [0085] In an embodiment, the prime vaccine or the boost vaccine may be administered according to an appropriate dosage schedule. Dosage may involve administration of a single dose schedule or multiple doses. Multiple doses may be used for administration of prime vaccine or boost vaccine. Multiple doses of the vaccines may be administered by the same or different routes. For example, the prime vaccine may be administered parenterally and the boost vaccine may be administered via mucosal route. Alternatively, the prime vaccine may be administered via mucosal route, and the boost vaccine may be administered parenterally. Multiple doses may be administered at least 1 week apart. [0086] A satisfactory effect may be obtained by systemic administration, e.g., intramuscular administration, subcutaneous administration or intravenous administration 1 -4 times at the amount of 10³-10¹⁰ Infectious Unit (IU) or 0.01-500 pg per time, preferably 10⁵-10¹⁰ IU or 0.1 -100 pg per time, for example 10⁷- 10⁹ IU or 1-50 pg per one time. The replicon may preferably be formulated in a vaccine composition suitable for administration in a conventional manner.

[0087] In an embodiment, routes of administration may include, but not be limited to, intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, and intraocular injection. Oral and transdermal administration, as well as administration by inhalation or suppository may also be used. The preferred routes of administration may include intramuscular, intradermal and subcutaneous injection.

[0088] Method of making

[0089] An embodiment provides a method of preparing a vaccine combination for protecting a subject against a disease.

[0090] In an embodiment, the method may comprise preparing the prime vaccine described herein. The method may comprise combining the polynucleotide encoding the first antigenic protein with the first carrier. The method may comprise combining the polynucleotide with alkyl-modified dendrimer-based materials (modified dendrimer nanoparticle, MDNP), by self-assembly of a polynucleotides and alkyl-modified dendrimers, by self-assembly of polynucleotides and cationic and neutral lipids, or by self-assembly of polynucleotides, alkyl-modified dendrimers and appropriately charged lipids.

[0091] In an embodiment, the method may comprise preparing the boost vaccine described herein. The boost vaccine may be manufactured in vitro, and may allow the facile production of stable truncations of that full-sized antigen (i.e., functional subunits and/or epitopes), which will serve to focus the body’s humoral response. The protein vaccine may be manufactured or formulated in such a way as to promote strong B cell induction, which will lead to high IgG affinity and specificity for the focusing boost immunogen. [0092] In an embodiment, the first antigenic protein, the second antigenic protein or its subunit (i.e., RBD) may be engineered in such a way to be arrayed in 3D space in a regular, repeated fashion in order to promote B cell receptor engagement, clustering, and activation.

[0093] In an embodiment, the first antigenic protein, the second antigenic protein or its subunit (i.e., RBD) may be arrayed by tandem genetic fusion to create a repeated RBD domain construct expressed as a single polypeptide chain.

[0094] In an embodiment, the first antigenic protein, the second antigenic protein or its subunit (i.e., RBD) may be arrayed or presented on a suitable carrier through a chemical conjugation reaction including but not limited to the following methods (Boutureira and Bernardes, 2015 Chem Rev 115, 2174-2195; Hoyt et al., Nat Rev Chem 3, 147-171 ; De Gruyter et al., 2017 Biochemistry 30, 3863— 3873; and Brune and Howarth, 2018 Front. Immunol. 9:1432, all of which are incorporated herein by reference as if fully set forth): disulfide-bond formation between the thiol functionality of an antigen’s native or engineered cysteine and a thiol moiety present on the carrier’s surface; covalent bond formation between the thiol functionality of an antigen’s native or engineered cysteine and a cysteine-reactive functionality present on the carrier’s surface including but not limited to maleimide, haloacteamide, alkene (for radical initiator or photosensitizer promoted thiol-ene reaction) and alkyne (for radical initiator or photosensitizer promoted thiol-yne reaction) linkers; amide bond formation through native or non-native chemical ligation between an antigen’s native or engineered N-terminal cysteine and a thioester present on the carrier’s surface; covalent bond formation between the nucleophilic amine functionality of an antigen’s lysine or N-terminus and an electrophilic functionality present on the carrier’s surface including but not limited to activated acids (e.g. acyl halides, NHS esters, sulfo-NHS esters, O-acylisourea from carbodiimide-mediated activation of carboxylic acid moieties, and mixed anhydrides or acylimidazols from N,N’-carbonyldiimidazole or N,N’-disuccinimidyl carbonate mediated activation of carboxylic acid moieties) activated carbamates (e.g., from N,N’-carbonyldiimidazole or N,N’-disuccinimidyl carbonate mediated activation of amine moieties), activated carbonates (e.g., from N,N’-carbonyldiimidazole or N,N’-disuccinimidyl carbonate mediated activation of hydroxy moieties), vinyl sulfones, isocyanates, isothiocyanates, and squaric acids; covalent bond formation through imine formation or reductive amination reactions (e.g., in presence of sodium cyanoborohydride) between the nucleophilic amine functionality of an antigen’s lysine or N-terminus and the carbonyl moiety (e.g., of an aldehyde or ketone) present on the carrier’s surface; amide bond formation between the carboxylate functionality of an antigen’s aspartate, glutamate or C-terminus activated by conversion with a carbodiimide (e.g., EDC) or phosgene derived (e.g., CDI or DSC) reagent and a nucleophilic functionality present on the carrier’s surface including but not limited to amine and alcohol moieties; ester bond formation between the carboxylate functionality of an antigen’s aspartate, glutamate or C-terminus and a diazoalkane or diazoacetyl functionality present on the carrier’s surface; bioorthogonal conjugation (e.g., carbonyl condensation, Staudinger ligation, strain-promoted [3+2] cycloaddition, dipolar cycloaddition reactions, inverse electron demand Diels-Alder cycloadditions, transition metal catalyzed cycloadditions, 1,3-photoclick cycloadditions, and transition metal catalyzed C-C coupling reactions) between a reactive group on the antigen introduced by prior chemical conjugation or incorporation of an respective UAA and the complementary group present on the carrier’s surface.

[0095] In an embodiment, the first antigenic protein, the second antigenic protein or its subunit (i.e., RBD) may be expressed comprising an engineered peptide sequence facilitating enzymatic conjugation to a suitable carrier presenting the complementary peptide sequence, on which it is arrayed or presented by a method including but not limited to the following: Esterase-, Sortase-, subtiligase- and SpyLigase-catalyzed transpeptidation; transglutaminase-catalyzed amide-bond formation; and lipoic acid ligase-catalyze acylation.

[0096] In an embodiment, the first antigenic protein, the second antigenic protein or its subunit (i.e., RBD) may be expressed comprising an engineered peptide sequence facilitating arraying on a suitable carrier through complexation of transition metals, e.g., through poly-his-tag coordination to Nickel(ll)-ions presented through a chelate on the carriers’ surface.

[0097] In an embodiment, the first antigenic protein, the second antigenic protein or its subunit (i.e., RBD) may be arrayed by genetic fusion to a gene coding for a suitable self-assembling carrier protein, including but not limited to: e.g. GvpA-protein of gas vesicle nanoparticles (GVNP), such as those produced by halophilic archaea GvpC-protein which is binding to the surface of gas vesicle nanoparticles (GVNP); a protein forming a self-assembling nanocage, e.g. ferritin and ferritin-like proteins; a protein forming solid self-assembled structures, e.g., keyhole limpet hemocyanin (KLH), and Concholepas concholepas hemocyanin (CCH); and proteins forming virus-like particles (VLPs) (Pfeifer, 2012 Nat Rev Microbiol 10, 705; and Dutta et al., 2015, Malar J 14, 406, both of which are incorporated herein by reference as if fully set forth).

[0098] In an embodiment, the first antigenic protein, the second antigenic protein or its subunit (i.e., RBD) may be arrayed or presented on a suitable carrier through electrostatic attraction including but not limited to the following: electrostatic immobilization of an antigen with a positive charge in the applied buffer or with a genetically fused tag coding for a highly basic peptide sequence on a negatively charged carrier (e.g., anionic liposome, dendrimer, polynucleotide or synthetic nanoparticle); and electrostatic immobilization of an antigen with a negative charge in the applied buffer or with a genetically fused tag coding for an acidic peptide sequence on a positively charged carrier (e.g., cationic liposome, dendrimer or synthetic nanoparticle). [0099] In an embodiment, the first antigenic protein, the second antigenic or its subunit (i.e., RBD) may be arrayed or presented on a suitable carrier through reaction with a bi- or multifunctional cross-linker including, but not limited to glutaraldehyde, formaldehyde, CDI, and di- or oligo-NHS- esters.

[00100] In an embodiment, the first antigenic protein, the second antigenic protein or its subunit (i.e., RBD) may be conjugated through the utilization of a SpyTag and SpyCatcher-type interaction. [00101] In an embodiment, the carrier’s surface on which the first antigenic protein, the second antigenic protein or its subunit (i.e., RBD) is arrayed or presented by one of the methods specified above may be that of a synthetic nanoparticle, such as those produced by alkyl-modified dendrimer- based materials (modified dendrimer nanoparticle, MDNP), by self-assembly of a polynucleotides and alkyl-modified dendrimers, by self-assembly of polynucleotides and cationic and neutral lipids, or by self-assembly of polynucleotides, alkyl-modified dendrimers and appropriately charged lipids. Conjugation may result in the linkage of the first antigenic protein, the second antigenic protein or its subunit (i.e., RBD) to one or several of the following moieties: an anionic, neutral or cationic lipid; a PEG moiety anchored to an anionic, neutral or cationic lipid; the backbone or functional group (e.g., amines) of the dendron.

[00102] In an embodiment, the carrier’s surface on which the first antigenic protein, the second antigenic protein or its subunit (i.e., RBD) is arrayed or presented by one of the methods specified above may be that of a liposome consisting of anionic, neutral or cationic lipids or a mixture of these. Conjugation may result in the linkage of the first antigenic protein, the second antigenic protein or its subunit (i.e., RBD) to one or several of the following moieties: the polar head group or the aliphatic chain of a phospholipid; the core or the hydroxyl group of a sterol derived lipid; the polar head group of a saccharolipid, and the polar head group of a sphingolipid.

[00103] In an embodiment, the carrier on which the first antigenic protein, the second antigenic protein or its subunit (i.e., RBD) is arrayed or presented by one of the methods specified above may contain one or several moieties with adjuvanting or other immune-stimulating properties including, but not limited to: incorporation of an adjuvanting lipid like monophosphoryl lipid A and its derivatives, D- (+)-trehalose 6,6'-dibehenate, and cationic lipids like dimethyldioctadecylammonium into a liposome or a modified dendrimer nanoparticle; incorporation of a CpG-oligonucleotide or a RNA molecule in a liposome or a modified dendrimer nanoparticle; and conjugation of a CD4+ T cell-activating helper peptide (e.g. PADRE sequence AKFVAAWTLKAAA; SEQ ID NO: 10) to a self-assembling carrier protein, a liposome-forming lipid or a modified dendrimer nanoparticle.

[00104] In an embodiment, the first antigenic protein, the second antigenic protein or its subunit (i.e., RBD) may be expressed as a genetic fusion product with an adjuvanting or otherwise immune- stimulating protein or peptide moiety, including but not limited to: a CD4+ T cell-activating helper peptide (e.g., PADRE sequence AKFVAAWTLKAAA; SEQ ID NO: 10); and a protein with proven adjuvanting properties, e.g., keyhole limpet hemocyanin (KLH), and Concholepas hemocyanin (CCH). [00105] In an embodiment, the first antigenic protein, the second antigenic protein or its subunit (i.e., RBD) may be conjugated to one or several lipid anchor moieties prior to mixing with a in a liposome or a modified dendrimer nanoparticle.

[00106] In an embodiment, the first antigenic protein, the second antigenic protein or its subunit (i.e., RBD) may be tetramerized through the utilization of a biotin/streptavidin-type interaction, including but not limited to: chemical conjugation of biotin with the antigen protein and its binding to a tetrameric streptavidin or streptavidin-like protein; incorporation of a non-canonic amino acid with a biotin side chain into the antigen protein and its binding to a tetrameric streptavidin or streptavidin-like protein; BirA-catalyzed enzymatic conjugation of biotin with the AviTag of an correspondingly engineered antigen protein and its binding to a tetrameric streptavidin or streptavidin-like protein; and genetic fusion of a strep-tag type sequence (e.g., WSHPQFEK; SEQ ID NO: 11) with an antigen protein and its binding to a tetrameric streptavidin or streptavidin-like protein.

[00107] In an embodiment, the first antigenic protein, the second antigenic protein or its subunit (i.e., RBD) may be arrayed by conjugation to a microgel or hydrogel using the above described conjugation methods.

[00108] In an embodiment, the first antigenic protein, the second antigenic protein or its subunit (i.e., RBD) may be arrayed by conjugation to a DNA origami nanostructure.

[00109] In an embodiment, the first antigenic protein, the second antigenic protein or its subunit (i.e., RBD) may be used without conjugation to a carrier moiety.

[00110] In an embodiment, prior to its application the first antigen proteinic, the second antigenic protein or its subunit (i.e., RBD) containing formulation may be mixed with specific depot-forming adjuvants such as squalene/water and other nanoparticulate delivery systems.

[00111] The following list includes particular embodiments of the present invention. But the list is not limiting and does not exclude alternate embodiments, or embodiments otherwise described herein. Percent identity described in the following embodiments list refers to the identity of the recited sequence along the entire length of the reference sequence.

[00112] EMBODIMENTS

1. A vaccine combination for protecting a subject against a Coronavirus disease comprising a prime vaccine and a boost vaccine, wherein the prime vaccine comprises an effective amount of a polynucleotide encoding a first antigenic protein or a subunit thereof, and a first carrier; and the boost vaccine comprises an effective amount of a second antigenic protein, or a subunit thereof, and a second carrier, wherein the second antigenic protein is a subunit of the first antigenic protein.

2. The vaccine combination of embodiment 1 , wherein the Coronavirus disease is caused by a viral pathogen selected from the group consisting of SARS-CoV, MERS-Cov and SARS- CoV-2 viruses.

3. The vaccine combination of one or both embodiments 1 and 2, wherein the subunit of the first antigenic protein or the second antigenic protein comprises a receptor binding domain.

4. The vaccine combination of any one or more of embodiments 1 - 3, wherein the first antigenic protein or subunit thereof is a viral Spike protein selected from the group consisting of Spike SARS-CoV, Spike SARS-CoV-2, and Spike MERS-CoV.

5. The vaccine combination of any one or more of embodiments 1 - 4, wherein the polynucleotide is selected from the group consisting of an mRNA, a repRNA and a DNA.

6. The vaccine combination of any one or more of embodiments, wherein the viral pathogen is SARS-CoV, and the polynucleotide encodes a Spike SARS-CoV protein, or a fragment thereof.

7. The vaccine combination of any one or more of embodiments 1 - 6, wherein the polynucleotide encodes the Spike SARS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 1.

8. The vaccine combination of any one or more of embodiments 1 - 7, wherein the polynucleotide encodes a Spike S1 SARS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 4.

9. The vaccine combination of any one or more of embodiments 1 - 8, the polynucleotide encodes the Spike S1 SARS-CoV protein, and comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 7.

10. The vaccine combination of any one or more of embodiments 1 -6, wherein the viral pathogen is SARS-CoV-2, and the polynucleotide encodes a Spike SARS-CoV-2 protein, or a fragment thereof.

11. The vaccine combination of any one or more of embodiments 1 - 6 and 10, wherein the polynucleotide encodes the Spike SARS-CoV-2 protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 2.

12. The vaccine combination of any one or more of embodiments 1 - 6 and 10 - 11, wherein the polynucleotide encodes a Spike S1 SARS-CoV-2 protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 5. 13. The vaccine combination of any one or more of embodiments 1 - 6 and 10 - 12, the polynucleotide encodes the Spike S1 SARS-CoV-2 protein, and comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 8.

14. The vaccine combination of any one or more of embodiments 1 - 6, wherein the viral pathogen is MERS-CoV, and the polynucleotide encodes a Spike MERS-CoV protein, or a fragment thereof.

15. The vaccine combination of any one or more of embodiments 1 - 6 and 14, wherein the polynucleotide encodes the Spike MERS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 3.

16. The vaccine combination of any one or more of embodiments 1 - 6 and 14 - 15, wherein the polynucleotide encodes a Spike S1 MERS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 6.

17. The vaccine combination of any one or more of embodiments 1 - 6 and 14 - 16, wherein the polynucleotide encodes the Spike S1 MERS-CoV protein, and comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 9.

18. The vaccine combination of any one or more of embodiments 1 - 17, wherein the second antigenic protein or subunit thereof is a viral Spike protein selected from the group consisting of Spike SARS-CoV, Spike SARS-CoV-2, and Spike MERS-CoV.

19. The vaccine combination of any one or more of embodiments 1 - 18, wherein the viral Spike protein is a Spike SARS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 1 .

20. The vaccine combination of any one or more of embodiments 1 - 19, wherein the Spike SARS-CoV protein comprises a Spike S1 SARS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 4.

21. The vaccine combination of any one or more of embodiments 1 - 18, wherein the viral Spike protein is a SARS-CoV-2 protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 2, or a fragment thereof.

22. The vaccine combination of any one or more of embodiments 1 - 18, and 21 , wherein the SARS-CoV-2 protein comprises a Spike S1 SARS-CoV-2 protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 5.

23. The vaccine combination of any one or more of embodiments 1 - 18, and 21 - 22, wherein the viral Spike protein is a Spike MERS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 3. 24. The vaccine combination of any one or more of embodiments 1 - 18, and 21 - 23, wherein the Spike MERS-CoV protein comprises a Spike S1 MERS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 6.

25. The vaccine combination of any one or more of embodiments 1 - 24, wherein the first carrier is selected from the group consisting of: an anionic liposome, a cationic liposome, and a dendrimer.

26. The vaccine combination of any one or more of embodiments 1 - 25, wherein the second carrier is selected from the group consisting of: a gas vesicle nanoparticle (GVNP), ferritin, ferritin-like proteins, keyhole limpet hemocyanin (KLH), Concholepas concholepas hemocyanin (CCH), and virus-like particles.

27. The vaccine combination of any one or more of embodiments 1 - 26, wherein the second antigenic protein is fused with an adjuvanting protein, an immune-stimulating protein, or a peptide moiety.

28. The vaccine combination of any one or more of embodiments 1 - 27, wherein the adjuvanting protein comprises a compound selected from the group consisting of: a keyhole limpet hemocyanin (KLH), and Concholepas concholepas hemocyanin (CCH).

29. The vaccine combination of any one or more of embodiments 1 - 28, wherein the peptide moiety comprises a CD4+ T cell-activating helper peptide.

30. The vaccine combination of any one or more of embodiments 1 - 29, wherein the prime vaccine further comprises an effective amount of a second antigenic protein, or a subunit thereof, wherein the second antigenic protein is a subunit of the first antigenic protein.

31. A method of preventing, reducing, inhibiting, or delaying the symptoms of an infection caused by a Coronavirus, or of inducing an immune response against the Coronavirus in a subject the method comprising: administering to the subject a prime vaccine of any one or more of embodiments 1 - 30; and subsequently administering to the subject a boost vaccine of any one or more of embodiments

1 - 30.

32. A method of preventing, reducing, inhibiting, or delaying the symptoms of an infection caused by a Coronavirus, or of inducing an immune response against the Coronavirus in a subject the method comprising: administering to the subject a prime vaccine comprising an effective amount of a polynucleotide encoding a first antigenic protein or a subunit thereof, and a first carrier; and subsequently administering to the subject a boost vaccine comprising an effective amount of a second antigenic protein, or a subunit thereof, and a second carrier, wherein the second antigenic protein is a subunit of the first antigenic protein.

33. The method of embodiment 32, wherein the Coronavirus is selected from the group consisting of SARS-CoV, MERS-Cov and SARS-CoV-2 viruses.

34. The method of one or both embodiments 32 and 33, wherein the subunit of the first antigenic protein or the second antigenic protein comprises a receptor binding domain.

35. The method of any one or more of embodiments 32 - 34, wherein the first antigenic protein or subunit thereof is a viral Spike protein selected from the group consisting of Spike SARS- CoV, Spike SARS-CoV-2, and Spike MERS-CoV.

36. The method of any one or more of embodiments 32 - 35, wherein the polynucleotide is selected from the group consisting of an mRNA, a repRNA and a DNA.

37. The method of any one or more of embodiments 32 - 36, wherein the viral pathogen is SARS-CoV, and the polynucleotide encodes a Spike SARS-CoV protein, or a fragment thereof.

38. The method of any one or more of embodiments 32 - 37, wherein the polynucleotide encodes the Spike SARS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 1.

39. The method of any one or more of embodiments 32 - 38, wherein the polynucleotide encodes a Spike S1 SARS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 4.

40. The method of any one or more of embodiments 32 - 39, wherein the polynucleotide encodes the Spike S1 SARS-CoV protein, and comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 7.

41 . The method of any one or more of embodiments 32 - 36, wherein the viral pathogen is SARS-CoV-2, and the polynucleotide encodes a Spike SARS-CoV-2 protein, or a fragment thereof.

42. The method of any one or more of embodiments 32 - 36 and 41 , wherein the polynucleotide encodes the Spike SARS-CoV-2 protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 2.

43. The method of any one or more of embodiments 32 - 36 and 41 - 42, wherein the polynucleotide encodes a Spike S1 SARS-CoV-2 protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 5.

44. The method of any one or more of embodiments 32 - 36 and 41 - 43, the polynucleotide encodes the Spike S1 SARS-CoV-2 protein, and comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 8. 45. The method of any one or more of embodiments 32 - 36, wherein the viral pathogen is MERS-CoV, and the polynucleotide encodes a Spike MERS-CoV protein, or a fragment thereof.

46. The method of any one or more of embodiments 32 - 36 and 45, wherein the polynucleotide encodes the Spike MERS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 3.

47. The method of any one or more of embodiments 32 - 36, and 45 - 46, wherein the polynucleotide encodes a Spike S1 MERS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 6.

48. The method of any one or more of embodiments 32 - 36 and 45 -47, the polynucleotide encodes the Spike S1 MERS-CoV protein, and comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 9.

49. The method any one or more of embodiments 32 - 48, wherein the second antigenic protein or subunit thereof is a viral Spike protein selected from the group consisting of Spike SARS- CoV, Spike SARS-CoV-2, and Spike MERS-CoV proteins.

50. The method any one or more of embodiments 32 -49, wherein the viral Spike protein is a Spike SARS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 1.

51. The method any one or more of embodiments 32 - 50, wherein the Spike SARS-CoV protein comprises a Spike S1 SARS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 4.

52. The method of any one or more of embodiments 32 - 49, wherein the viral Spike protein is a SARS-CoV-2 protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 2.

53. The method any one or more of embodiments 32 - 49, and 52, wherein the SARS- CoV-2 protein comprises a Spike S1 SARS-CoV-2 protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 5.

54. The method any one or more of embodiments 32 -49, wherein the viral Spike protein is a Spike MERS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 3.

55. The method any one or more of embodiments 32 - 49, and 54, wherein the Spike MERS-CoV protein comprises a Spike S1 MERS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 6.

56. The method any one or more of embodiments 32 - 55, wherein the first carrier is selected from the group consisting of: an anionic liposome, a cationic liposome, and a dendrimer. 57. The method any one or more of embodiments 32 - 56, wherein the second carrier is selected from the group consisting of: a gas vesicle nanoparticle (GVNP), ferritin, ferritin-like proteins, keyhole limpet hemocyanin (KLH), Concholepas concholepas hemocyanin (CCH), and virus-like particles.

58. The method any one or more of embodiments 32 - 57, wherein the second antigenic protein is fused with an adjuvanting protein, an immune-stimulating protein, or a peptide moiety.

59. The method any one or more of embodiments 32 - 58, wherein the adjuvanting protein comprises a compound selected from the group consisting of: a keyhole limpet hemocyanin (KLH), and Concholepas concholepas hemocyanin (CCH).

60. The method of any one or more of embodiments 32 - 59, wherein the peptide moiety comprises a CD4+ T cell-activating helper peptide.

61. The method any one or more of embodiments 32 - 60, wherein the prime vaccine further comprises an effective amount of a second antigenic protein, ora subunit thereof, wherein the second antigenic protein is a subunit of the first antigenic protein

62. The method any one or more of embodiments 32 - 36, wherein the boost vaccine is administered in a period of time ranging from 1 week to 60 weeks after the prime vaccine is administered.

63. A kit comprising the prime and boost vaccines of any one or more of embodiments 1 - 30, wherein the kit is used for sequential administration of the prime and boost vaccines.

64. The kit of embodiment 63, wherein the prime and boost vaccines are included in separate containers.

65. The kit of embodiment 63, wherein the prime and boost vaccines are included in the same container.

66. The kit any one or more of embodiments 63 - 65, wherein the prime and boost vaccines are in liquid or solid form.

67. A method of preparing a vaccine combination of any one or more of embodiments 1 - 30 for protecting a subject against a Coronavirus disease comprising: preparing the prime vaccine by combining the polynucleotide encoding the first antigenic protein or the second antigenic protein with the first carrier; and preparing the boost vaccine by combining the first antigenic protein or the second antigenic protein with the second carrier.

68. The method of embodiment 67, wherein the step of combining comprises the step of self-assembly of the polynucleotide and an alkyl-modified dendrimer. 69. The method of embodiment 67, wherein the step of combining comprises the step of self-assembly of the polynucleotide and cationic or neutral lipids.

70. The method of embodiment 67, wherein the step of combining comprises the step of self-assembly of the polynucleotide, an alkyl-modified dendrimer and cationic or neutral lipids.

71. The method of any one or embodiments 67 - 70, wherein preparing the prime vaccine comprises incorporation of an adjuvanting lipid into the first carrier, wherein the adjuvanting lipid is selected from the group consisting of: monophosphoryl lipid A, and a derivative thereof, D-(+)- trehalose 6,6’-dibehenate.

72. The method of embodiment 67, wherein the step of combining comprises the step of chemical conjugation of the first antigenic protein or the second antigenic protein with the second carrier.

73. The method of embodiment 72, wherein the chemical conjugation comprises the reaction selected from the group consisting of: disulfide-bond formation between a thiol moiety of the first antigenic protein or the second antigenic protein and a thiol moiety present on the surface of the second carrier; covalent bond formation between a first thiol moiety of the first antigenic protein or the second antigenic protein and a cysteine-reactive moiety present on the surface of the second carrier; amide bond formation through chemical ligation between an N-terminal cysteine of the first antigenic protein or the second antigenic protein and a thioester present on the surface of the second carrier; covalent bond formation between the nucleophilic amine group of lysine or N-terminus of the first antigenic protein or the second antigenic protein and an electrophilic moiety present on the surface of the second carrier; amide bond formation between the carboxylate group of aspartate or C- terminus activated by conversion with a carbodiimide or phosgene derived reagent of the first antigenic protein or the second antigenic protein and a nucleophilic moiety present on the surface of the second carrier; ester bond formation between the carboxylate moiety of aspartate, glutamate or C-terminus of the first antigenic protein or the second antigenic protein and a diazoalkane or diazoacetyl moiety present on the surface of the second carrier; and biorthogonal conjugation between a reactive group of the first antigenic protein or the second antigenic protein and the complementary group present on the surface of the second carrier.

74. The method of any one or more of embodiments 71 - 73, wherein, preparing the boost vaccine comprises expressing the first antigenic protein or the second antigenic protein as a fusion with an adjuvanting protein, immune stimulating protein or peptide moiety.

75. The method of any one or more of embodiments 71 - 74, wherein the adjuvanting protein comprises a keyhole limpet hemocyanin (KLH), or Concholepasconcholpeas hemocyanin (CCH). 76. The method of any one or more of embodiments 71 - 75, wherein the immune stimulating protein comprises a CD4+ T cell-activating helper peptide.

77. The method of any one or more of embodiments 71 - 76, wherein CD4+ T cellactivating helper peptide comprises an amino acid sequence of SEQ ID NO: 10.

78. The method of any one or more of embodiments 67 - 77, wherein prior to the step of combining with the second carrier the first antigenic protein or the second antigenic protein is tetramerized through biotin/streptavid in-type reaction selected from the following group consisting of: chemical conjugation with biotin or tetrameric streptavidin or streptavidin-like protein; incorporation of a non-cationic amino acid with a biotin side chain for binding to a tetrameric streptavidin or streptavidin-like protein; Bir-A -catalyzed enzymatic conjugation of biotin with AviTag for binding to a tetrameric streptavidin or streptavidin-like protein; and genetic fusion with a strep-tag sequence for binding to a tetrameric streptavidin or streptavidin-like protein.

79. The method of embodiment 78, wherein the strep-tag sequence is set forth in SEQ ID NO: 11.

[00113] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[00114] Further embodiments herein may be formed by supplementing an embodiment with one or more elements from any one or more other embodiments herein, and/or substituting one or more elements from one embodiment with one or more elements from one or more other embodiments EXAMPLES

[00115] The following non-limiting examples are provided to illustrate particular embodiments. The embodiments throughout may be supplemented with one or more details from one or more examples below, and/or one or more elements from an embodiment may be substituted with one or more details from one or more examples below.

[00116] Example 1. Heterologous Vaccination Approaches

[00117] A successful vaccine generates potent and long-term protection against a pathogen of interest. While a single-dose vaccine is convenient and cost-effective, in many instances a subsequent “booster” immunization against the pathogen is required to ensure persistent cellular and humoral immunity. Studies suggest that heterologous prime-boost immunization, where the booster delivers antigen and induces immune responses by a mechanism distinct from the prime, can be more effective than comparable homologous prime-boost regimens of the same vaccine construct. Heterologous prime-boost regimens could reduce the quantity and number of requisite doses, allowing vaccine stockpiles to serve a greater population. Heterologous immunization is believed to induce a balanced and thorough immune response against a target pathogen, ensuring greater establishment of immune memory (Lu S., 2009, Curr Opin Immunol, 21(3):346— 51 , which is incorporated by reference herein as if fully set forth).

[00118] A heterologous prime-boost vaccination strategy, incorporating a nucleic acid vaccine and traditional protein vaccine, was reported to improve immune protection and durability. A nucleic acid (RNA or DNA) prime followed by a protein boost was used in these studies (Zhao et al., 2009 Vet Immunol Immunopathol, 131(3— 4):158— 66; Borhani et al., 2015 Arch Iran Med, (4):223-7l; and Banerjee et al., 2012 Retrovirology, 9(S2): P312, all of which are incorporated by reference herein as if fully set forth).

[00119] Cellular immune responses have been induced with a DNA prime followed by one or more viral vector boosts. Such viral vectors include adenovirus, vaccinia, fowlpox, and vesicular stomatitis virus (Banerjee et al., 2012 Retrovirology, 9(S2): P312; and Fioretti et al. 2010, J Biomed Biotechnol, 2010:1-16, both ofwhich are incorporated by reference herein as if fully setforth). Primary vaccinations with DNA followed by boosting with a recombinant poxvirus vector encoding the same immunogen could elicit a protective CD8+ T cell response in animal models against various diseases such as HIV, malaria, and even cancer (Alekseeva et al., 2009 Genet Vaccines Ther, 7(1):7, which is incorporated by reference herein as if fully set forth).

[00120] A DNA prime followed by a protein or peptide boost has been shown to induce both humoral and cellular immune responses (Lu S., 2009, Curr Opin Immunol, 21 (3):346— 51 , which is incorporated by reference herein as if fully set forth). This approach has been demonstrated against different viral diseases, such as: HCV, HSV, HIV, and HPV (Kardani et al., 2016 Vaccine, 34(4):413— 23). Alone, proteins and peptides are relatively non-immunogenic, requiring additional engineering or adjuvants to provide the necessary innate immune stimulation to drive the desired adaptive response. The immunogenicity of protein subunits can be vastly increased by structuring the proteins in a regular, patterned arrangement in three-dimensional space; i.e., in an arrayed ultrastructure that mimics the appearance of many pathogen surface structural antigens. This ensures engagement and clustering of target-specific immunoglobulin receptor (Ig) receptors on B cell surfaces, and promotion of antibodies targeting the specific structural epitopes present on the target antigen (Akkaya et al., 2020 Nat Rev Immunol 20, 229-238; and Slifka et al., 2019 Front. Immunol. 10:956, both of which are incorporated by reference herein as if fully set forth).

[00121] Traditional viral vector approaches employ repurposed inherently immunogenic virus as the carrier system for antigen expression. This poses a risk of "anti-vector" immune responses, either due to prior natural infections or prior immunizations using the same or similar viral vector. This has been shown to reduce the effectiveness of such vaccines, particularly in homologous prime-boost regimens. For example, the adenovirus-vectored COVID-19 vaccine ChAdOxl nCoV-10 leads to substantially reduced effectiveness of the second dose, apparently due to antibodies against the adenovirus vector induced by the prime immunization.

[00122] An optimized heterologous vaccine formula must be able to promote specificity of the intense immune responses it engenders against the most important, functionally relevant epitopes and/or subunits that will ensure protection. This feature would prevent off-target effects such as antibody-dependent enhancement of disease (ADE), which can arise if non-neutralizing immune responses are induced, for example in Dengue virus infections (Halstead, 2015, Dengue Antibody- Dependent Enhancement: Knowns and Unknowns. In: Antibodies for Infectious Diseases, In American Society of Microbiology, p. 249-71 , which is incorporated by reference herein as if fully set forth).

[00123] Traditional viral vector approaches employ repurposed inherently immunogenic virus as the carrier system for antigen expression. This poses a risk of "anti-vector" immune responses, either due to prior natural infections or prior immunizations using the same or similar viral vector. This has been shown to reduce the effectiveness of such vaccines, particularly in homologous prime-boost regimens. For example, the adenovirus-vectored COVID-19 vaccine ChAdOxl nCoV-10 leads to substantially reduced effectiveness of the second dose, apparently due to antibodies against the adenovirus vector induced by the prime immunization.

[00124] An optimized heterologous vaccine formula must be able to promote specificity of the intense immune responses it engenders against the most important, functionally relevant epitopes and/or subunits that will ensure protection. This feature would prevent off-target effects such as antibody-dependent enhancement of disease (ADE), which can arise if non-neutralizing immune responses are induced, for example in Dengue virus infections (Halstead, 2015, Dengue Antibody- Dependent Enhancement: Knowns and Unknowns. In: Antibodies for Infectious Diseases, In American Society of Microbiology, p. 249-71 ; which is incorporated by reference as if fully set forth). [00125] The approach described herein avoids the risk of such anti-vector immune responses. Even in the case of a multi-modal vaccine, as defined below, the risk of such potential anti-vector immune response is completely averted as no viral vector is present in the product, so all immune responses at prime and boost are directed against only the desired antigen.

[00126] A heterologous prime/boost vaccine platform focuses on an immune response to effectively neutralize a pathogen of interest in a mammalian host organism.

[00127] The component vaccines and their schedule of administration are based on two complimentary immunization types: 1) a nucleic acid vaccine which encodes an antigen of interest paired with 2) a subunit or epitope of the same or similar antigen in the form of a protein formulated with or without adjuvant. The nucleic acid vaccine, which relies on cell processes to produce an antigen, is best designed such that the full-length form of the antigen is expressed, ensuring its appropriate post-translational processing and display to the adaptive cellular immune system via MHC class I and II presentation. The protein component, which can be manufactured in vitro, allows the facile production of stable truncations of that full-sized antigen (i.e., functional subunits and/or epitopes), which will serve to focus the body’s humoral response. The protein component is ideally manufactured or formulated in such a way as to promote strong B cell induction, which will lead to high IgG affinity and specificity for the focusing boost immunogen. Examples of such a protein preparation include patterned arraying of the protein in a rigid 3D structure to cause B cell receptor clustering, and formulation with adjuvants that induce strong B cell activation and infiltration via depot effects.

[00128] Example 2. COVID-19 Spike protein immunization via RNA prime/RBD protein focusing boost compared to prime-only immunization

[00129] COVID-19 Spike protein immunization via RNA prime/RBD protein focusing boost compared to prime-only immunizations. Fresh mice or mice previously primed with COVID-19 S mRNA (5 months prior; SEQ ID NO: 8) were injected with a protein COVID-19 vaccine candidate (protein sequence matching UNIPROT database reference P0DTC2, positions 319-541) formulated with aluminum hydroxide, a common adjuvant. After 9 days, serum was collected and anti-S1 domain titer was determined by ELISA. [00130] FIG. 2 illustrates result of RNA prime/RBD protein focusing boost immunization (mRNA prime + RBD boost) in mice developing superior antibody titers in comparison to RNA prime (mRNA prime (pre-boost)) or RBD protein prime alone (RBD protein vaccinated only). In FIG. 2, the lower limit of detection (LOD) and upper limit of quantification (LOQ) are indicated. Antibody titers in the protein- boosted group were higher than the upper limit of this assay. Compared to immunization with RNA or RBD protein alone, antibody titers generated by RNA prime/RBD protein focusing boost were at least ~8-fold higher.

[00131] Example 3. COVID-19 Spike protein immunization via RNA prime/RBD protein focusing boost compared to homologous boost immunizations

[00132] COVID-19 Spike protein immunization via RNA prime/RBD protein focusing boost boost using the same constructs depicted in Example 2, compared to homologous boost immunizations prime induces strong COVID-19 Spike protein-specific T cell responses. RNA is an optimal modality for the first public immunization campaign in response to an outbreak because it is rapid to manufacture compared to other technologies (e.g., protein, virus) and induces T cell responses, which mount protective action in a patient far more quickly (within days) than antibody titers, which take longer (weeks) to arise. To demonstrate that strong T cell responses occur against COVID-19 when using the RNA-based vaccine, mice were injected with either 10 pg of mRNA Spike nanoparticles, or were left untreated. 11 days post injection, mice were sacrificed and splenocytes were isolated. 1/100th of a spleen was plated per well in a PVDF membrane 96 well plate coated with AN18 anti- IFN gamma antibody. Cells were stimulated with 0.25 pg/mL CD28 and 0.25 pg/mL CD49d. For samples stimulated with spike peptide 4 (GYLQPRTFL; SEQ ID NO: 12), final concentration in the well was 2 pg/mL. For samples stimulated with the Pepmix (JPT Peptide Technologies), final concentration in the well was 2 pg/mL. For positive control, BD T cell activation cocktail was used. 16 hrs post-stimulation, cells were removed from the wells and anti-mouse IFN-gamma R6-4A2 biotinylated antibody was used to detect the presence of IFN-gamma. Streptavidin-HRP (BioLegend) was added and plates were developed with AEC Chromagen kit (Sigma Aldrich). FIG. 3 illustrates ELISpot assay using splenocytes isolated from vaccinated animals. In this figure, wells to the left show data for mice stimulated with stimulated with spike peptide 4 (PEP 4), wells in the middle show data for mice stimulated with the Pepmix (PEPMIX), and wells to the right show data for mice treated with BD T cell activation cocktail (positive control; POS CTL).

[00133] Referring to FIG. 3, the distinctive dots were visible on the membranes after development with AEC Chromagen which is indicative of IFN-gamma positive secreting splenocytes, with the number of spots indicating the number of IFN-gamma secreting cells. Multiple spots were observed for ex-vivo re-stimulated splenocytes from mRNA Spike vaccinated mice (top panel) stimulated with PEP 4 comparable with the positive control treatment; and some spots were observed for mice stimulated with PEPMIX. No spots were observed for ex vivo re-stimulated splenocytes from unvaccinated mice in both PEP4 and PEPMIX treatment.

[00134] FIG. 4 shows the focusing-boost approach. The focusing-boost approach improves RBD- specific IgG titers. Two groups of 5 mice each (Cage 1 and Cage 2) were immunized with an RNA prime and bled 18 days later (pre-boost samples, first and third sets on each graph). Animals were boosted with the indicated vaccine (RNA boost or RBD protein (Protein boost)) and serum sampled 18 days later. Serum IgG titers specific for the full Spike protein trimer (Anti-Spike IgG ELISA; left panel) or the RBD specifically (Anti-RBD IgG ELISA; right panel) were determined by ELISA on immobilized recombinant Spike trimer or RBD. It was observed that RNA immunization alone induces strong Spike antibodies, however RBD specific responses are lacking. RBD protein boosting confers a strong RBD-specific response, indicating greater neutralizing potential and potentially less off-target antibody-mediated effects than RNA vaccination alone. The lower and upper limits of detection/quantification are displayed as gray lines.

[00135] As illustrated in FIG. 4, the data clearly show the that the focusing boost technology (box marked as Focusing boost) can achieve superior immune response compared to the conventional boost regimen applied for other COVID-19 vaccine candidates (box marked as Conventional boost). While full-length S mRNA induces good anti-Spike humoral responses, replacing the mRNA boost with focusing RBD protein boost is necessary for strong neutralizing antibody titers. Referring to FIG. 4, it was observed that full-length S RNA appears to require two shots to induce RBD antibody titers significantly above the baseline level. Boosting with the specific desired protein subdomain to 'focus' enhances these titers by over an order of magnitude compared to just using mRNA.

[00136] Example 4. Camelid vaccination

[00137] Eradication of MERS, persistent but localized zoonotic disease, requires addressing the primary domestic reservoir. Over 80% of dromedary camels are infected with MERS-CoV during the first year of life, suggesting early immunization of calves prior to exposure is necessary for an effective eradication campaign. An early RNA prime immunization has particular utility as such vaccines provide rapid protective cellular immunity, with a heterologous focusing boost providing more complete and long lasting humoral sterilizing immunity. In addition, a repeat dosing of the same two- part RNA/protein boost, defined below as a multi-modal vaccine, has notably enhanced ability to induce neutralizing antibody-mediated immunity. Rather than two heterologous products, only a single vaccine candidate, given at two different stages of an immunization, need be designed, produced and regulated, while still triggering both the cellular and humoral branches of the immune system. As a related betacoronavirus, SARS-CoV-2 can serve as a model for MERS-CoV vaccination in camelid animals.

[00138] An RNA vaccine candidate against SARS-CoV-2 was used, as a prime dose, that encodes the full-length Spike protein. Briefly, DNA fragments were synthesized commercially, ligated into template plasmids, and linearized by restriction digest. The mRNA was synthesized by in vitro transcription (IVT) using commercially available IVT (ThermoFisher) and enzymatic Cap1 capping kits (CellScript). The RNA payload was then formulated with modified dendrimer delivery material to form spherical lamellar nanoparticles ~150 nm in diameter. Particles were sterile-filtered and characterized by dynamic light scattering (DLS) and RNA encapsulation efficiency verified by agarose gel electrophoresis using standard laboratory practices.

[00139] As a boost, the full-length SARS-CoV-2 Spike trimeric protein complex was expressed in human suspension cell culture (Expi293T). The C-terminus was modified with a hydrophilic glycine/serine linker and terminal purification tag. The protein was expressed for 5 days in suspension cultures, and purified by affinity chromatography. Integrity was then confirmed by immunoblot and size-exclusion chromatography using standard laboratory practices.

[00140] After taking a baseline pre-bleed, a healthy camelid (llama) was immunized with a heterologous prime/boost strategy targeting the SARS-CoV-2 Spike protein, using the vaccine materials described above, through intramuscular injection. The Day 0 prime consisted of 5 ml of RNA vaccine encoding the S protein at 0.2 mg/ml for a total 1 mg dose. Animals were allowed to rest. At Day 29 reactivity to the S protein was detected at the lower end of detection by ELISA against trimeric S protein or RBD domain alone as shown in Table 1.

Table 1. Prime-Boost Immunization Schedule.

[00141] The Day 33 boost was delivered by intramuscular injection, consisting of both 4.5 mL of the same RNA delivered as the prime and 1.5 mL of the above-mentioned protein. This contained 500 pg of protein along with an emulsified oil adjuvant. Reactivity to the S protein was confirmed by ELISA against the trimeric S protein or RBD domain alone, exhibiting titers at a serum dilution of 1 :300 at Day 60 (FIG. 6). FIG. 5 illustrates the Day 76 ELISA data. In this figure, light gray bars (left) show optical density for Pre-Vaccination titers (Day 29), dark gray bars (middle) show Post-Prime titers, and clear bars (right) Post-Boost titers (day 76). The logarithm of the serum dilution is shown on the x-axis (i.e., from left to right, increasing dilution of serum).

[00142] The same RNA/protein boost was delivered again by intramuscular injection on Day 65. FIG. 6 illustrates the Day 60 ELISA data that preceded this boost (FIG. 6). In this figure, optical density is shown for Pre-Vaccination- RBD protein (bar to the left), Pre-Vaccination - Spike protein (middle bar, left), Post-Vaccination - RBD protein (middle bar, right), and Post-Vaccination - Spike (bar to the right) titers for each dilution point. The second boost led to markedly improved reactivity against the viral Spike protein, with signal discerned at a dilution of <1 :30,000 at Day 76 (FIG. 5).

[00143] The experimental results go beyond the original observations of the findings described herein, to suggest that a repeat dosing of a “multi-modal” vaccine candidate has enhanced potential to induce sterilizing immunity. Here the term “multi-modal” is used to mean a single vaccine candidate consisting of two or more_components, here RNA/protein components, each component playing a dominant role at different periods of an immunization schedule. This will have practical manufacturing value in that a single multi-modal vaccine product, given at two different stages of an immunization regimen, can in total effectively mobilize the cellular and humoral branches of the immune system.

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[00144] The references cited throughout this application, are incorporated for all purposes apparent herein and in the references themselves as if each reference was fully set forth. For the sake of presentation, specific ones of these references are cited at particular locations herein. A citation of a reference at a particular location indicates a manner(s) in which the teachings of the reference are incorporated. However, a citation of a reference at a particular location does not limit the manner in which all of the teachings of the cited reference are incorporated for all purposes. [00145] It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but is intended to cover all modifications which are within the spirit and scope of the invention as defined by the appended claims; the above description; and/or shown in the attached drawings.

Claims

CLAIMS What is claimed is:

1. A method of preventing, reducing, inhibiting, or delaying the symptoms of an infection caused by a Coronavirus, or of inducing an immune response against the Coronavirus in a subject the method comprising: administering to the subject a prime vaccine comprising an effective amount of a polynucleotide encoding a first antigenic protein or a subunit thereof, and a first carrier; and subsequently administering to the subject a boost vaccine comprising an effective amount of a second antigenic protein, or a subunit thereof, and a second carrier, wherein the second antigenic protein is a subunit of the first antigenic protein.

2. The method of claim 1 , wherein the Coronavirus is selected from the group consisting of SARS-CoV, MERS-Cov and SARS-CoV-2 viruses.

3. The method of claim 1 , wherein the subunit of the first antigenic protein or the second antigenic protein comprises a receptor binding domain.

4. The method of claim 1 , wherein the first antigenic protein or subunit thereof is a viral Spike protein selected from the group consisting of Spike SARS-CoV, Spike SARS-CoV-2, and Spike MERS-CoV.

5. The method of claim 1, wherein the polynucleotide is selected from the group consisting of an mRNA, a repRNA and a DNA.

6. The method of any one of claims 1 - 5, wherein the viral pathogen is SARS-CoV, and the polynucleotide encodes a Spike SARS-CoV protein, or a fragment thereof.

7. The method of claim 6, wherein the polynucleotide encodes the Spike SARS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 1.

8. The method of claim 7, wherein the polynucleotide encodes a Spike S1 SARS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 4.

9. The method of claim 8, wherein the polynucleotide encodes the Spike S1 SARS-CoV protein, and comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 7.

10. The method of any one of claims 1 - 5, wherein the viral pathogen is SARS-CoV-2, and the polynucleotide encodes a Spike SARS-CoV-2 protein, or a fragment thereof.

11 . The method of claim 10, wherein the polynucleotide encodes the Spike SARS-CoV- 2 protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 2.

12. The method of claim 11 , wherein the polynucleotide encodes a Spike S1 SARS-CoV- 2 protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 5.

13. The method of claim 12, the polynucleotide encodes the Spike S1 SARS-CoV-2 protein, and comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 8.

14. The method of any one of claims 1 - 5, wherein the viral pathogen is MERS-CoV, and the polynucleotide encodes a Spike MERS-CoV protein, or a fragment thereof.

15. The method of claim 14, wherein the polynucleotide encodes the Spike MERS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 3.

16. The method of claim 15, wherein the polynucleotide encodes a Spike S1 MERS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 6.

17. The method of claim 16, the polynucleotide encodes the Spike S1 MERS-CoV protein and comprised an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 9.

18. The method of claim 1 , wherein the second antigenic protein or subunit thereof is a viral Spike protein selected from the group consisting of Spike SARS-CoV, Spike SARS-CoV-2, and Spike MERS-CoV.

19. The method of claim 18, wherein the viral Spike protein is a Spike SARS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 1.

20. The method of claim 19, wherein the Spike SARS-CoV protein comprises a Spike S1 SARS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 4.

21. The method of claim 18, wherein the viral Spike protein is a SARS-CoV-2 protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 2.

22. The method of claim 21 , wherein the SARS-CoV-2 protein comprises a Spike S1 SARS-CoV-2 protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 5.

23. The method of claim 18, wherein the viral Spike protein is a Spike MERS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 3.

24. The method of claim 23, wherein the Spike MERS-CoV protein comprises a Spike S1 MERS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 6.

25. The method of claim 1 , wherein the first carrier is selected from the group consisting of: an anionic liposome, a cationic liposome, and a dendrimer.

26. The method of claim 1 , wherein the second carrier is selected from the group consisting of: a gas vesicle nanoparticle (GVNP), ferritin, ferritin-like proteins, keyhole limpet hemocyanin (KLH), Concholepas hemocyanin (CCH), and virus-like particles.

27. The method of claim 1 , wherein the second antigenic protein is fused with an adjuvanting protein, an immune-stimulating protein, or a peptide moiety.

28. The method of claim 27, wherein the adjuvanting protein comprises a compound selected from the group consisting of: a keyhole limpet hemocyanin (KLH), and Concholepas hemocyanin (CCH).

29. The method of claim 27, wherein the peptide moiety comprises a CD4+ T cellactivating helper peptide.

30. The method of claim 1 , wherein the prime vaccine further comprises an effective amount of a second antigenic protein, or a subunit thereof, wherein the second antigenic protein is a subunit of the first antigenic protein

31 . The method of claim 1 , wherein the boost vaccine is administered in a period of time ranging from 1 week to 60 weeks after the prime vaccine is administered.

32. A vaccine combination for protecting a subject against a Coronavirus disease comprising a prime vaccine and a boost vaccine, wherein the prime vaccine comprises an effective amount of a polynucleotide encoding a first antigenic protein or a subunit thereof, and a first carrier; and the boost vaccine comprises an effective amount of a second antigenic protein, or a subunit thereof, and a second carrier, wherein the second antigenic protein is a subunit of the first antigenic protein.

33. The vaccine combination of claim 32, wherein the Coronavirus disease is caused by a viral pathogen selected from the group consisting of SARS-CoV, MERS-Cov and SARS-CoV-2 viruses.

34. The vaccine combination of claim 32, wherein the subunit of the first antigenic protein or the second antigenic protein comprises a receptor binding domain.

35. The vaccine combination of claim 32, wherein the first antigenic protein or subunit thereof is a viral Spike protein selected from the group consisting of Spike SARS-CoV, Spike SARS- CoV-2, and Spike MERS-CoV.

36. The vaccine combination of claim 32, wherein the polynucleotide is selected from the group consisting of an mRNA, a repRNA and a DNA.

37. The vaccine combination of claims 33, wherein the viral pathogen is SARS-CoV, and the polynucleotide encodes a Spike SARS-CoV protein, or a fragment thereof.

38. The vaccine combination of claim 37, wherein the polynucleotide encodes the Spike SARS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 1.

39. The vaccine combination of claim 38, wherein the polynucleotide encodes a Spike S1 SARS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 4.

40. The vaccine combination of claim 32, the polynucleotide encodes the Spike S1 SARS-CoV protein, and comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 7.

41. The vaccine combination of claim 33, wherein the viral pathogen is SARS-CoV-2, and the polynucleotide encodes a Spike SARS-CoV-2 protein, or a fragment thereof.

42. The vaccine combination of claim 41 , wherein the polynucleotide encodes the Spike SARS-CoV-2 protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 2.

43. The vaccine combination of claim 42, wherein the polynucleotide encodes a Spike S1 SARS-CoV-2 protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 5.

44. The vaccine combination of claim 32, the polynucleotide encodes the Spike S1 SARS-CoV-2 protein, and comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 8.

45. The vaccine combination of claim 33, wherein the viral pathogen is MERS-CoV, and the polynucleotide encodes a Spike MERS-CoV protein, or a fragment thereof.

46. The vaccine combination of claim 45, wherein the polynucleotide encodes the Spike MERS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 3.

47. The vaccine combination of claim 46, wherein the polynucleotide encodes a Spike S1 MERS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 6.

48. The vaccine combination of claim 33, the polynucleotide encodes the Spike S1 MERS-CoV protein, and comprises an RNA with at least 90% identity to the sequence set forth in SEQ ID NO: 9.

49. The vaccine combination of claim 32, wherein the second antigenic protein or subunit thereof is a viral Spike protein selected from the group consisting of Spike SARS-CoV, Spike SARS- CoV-2, and Spike MERS-CoV.

50. The vaccine combination of claim 32, wherein the viral Spike protein is a Spike SARS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 1.

51. The vaccine combination of claim 50, wherein the Spike SARS-CoV protein comprises a Spike S1 SARS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 4.

52. The vaccine combination of claim 32, wherein the viral Spike protein is a SARS-CoV-2 protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 2, or a fragment thereof.

53. The vaccine combination of claim 52, wherein the SARS-CoV-2 protein comprises a Spike S1 SARS-CoV-2 protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 5.

54. The vaccine combination of claim 32, wherein the viral Spike protein is a Spike MERS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 3.

55. The vaccine combination of claim 54, wherein the Spike MERS-CoV protein comprises a Spike S1 MERS-CoV protein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 6.

56. The vaccine combination of claim 32, wherein the first carrier is selected from the group consisting of: an anionic liposome, a cationic liposome, and a dendrimer.

57. The vaccine combination of claim 32, wherein the second carrier is selected from the group consisting of: a gas vesicle nanoparticle (GVNP), ferritin, ferritin-like proteins, keyhole limpet hemocyanin (KLH), Concholepas hemocyanin (CCH), and virus-like particles.

58. The vaccine combination of claim 32, wherein the second antigenic protein is fused with an adjuvanting protein, an immune-stimulating protein, or a peptide moiety.

59. The vaccine combination of claim 58, wherein the adjuvanting protein comprises a compound selected from the group consisting of: a keyhole limpet hemocyanin (KLH), and

Concholepas hemocyanin (CCH).

60. The vaccine combination of claim 58, wherein the peptide moiety comprises a CD4+ T cell-activating helper peptide.

61. The vaccine combination of claim 32, wherein the prime vaccine further comprises an effective amount of a second antigenic protein, or a subunit thereof, wherein the second antigenic protein is a subunit of the first antigenic protein.

62. A kit comprising the prime and boost vaccines included in the vaccine combination of claim 32, wherein the kit is used for sequential administration of the prime and boost vaccines.

63. The kit of claim 62 further comprising an immunoregulatory agent.

64. The kit of claim 63, wherein the immunoregulatory agent is an adjuvant.

65. The kit of claim 62, wherein the prime and boost vaccines are included in the same container, or separate containers.

66. The kit of claim 62, wherein the prime and boost vaccines are in liquid form or solid form.