DNA vaccines for eliciting t cell immunity in the lung

Abstract

Provided herein are nucleic acid vaccine constructs comprising synthetic polynucleotides encoding a severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) protein, or a functional portion, fragment, or variant thereof, conjugated to a macrophage inflammatory protein-3 alpha (MIP-3a) or other chemokine that binds to a chemokine receptor 6 (CCR6), or a functional portion, fragment, or variant thereof, or to an antibody, or antigen binding portion thereof, that binds to a CCR6. Methods for making the vaccine constructs and their use in prophylaxis and treatment of SARS-CoV-2 infections are also provided.

Description

DNA VACCINES FOR ELICITING T CELL IMMUNITY IN THE LUNGCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. Provisional Patent Application Ser. No. 63 / 516,200, filed July 28, 2023, the disclosure of which is incorporated herein by reference.REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0002] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on July 24, 2024, is named “0184.274-PCT_SL.xml” and is 7,478 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.BACKGROUND OF THE INVENTION

[0003] The SARS-CoV-2 pandemic has provided yet one more example of how the mutability of RNA viruses hinders efforts to develop effective immunoprophylactic strategies. Emerging variants have been uniformly less susceptible to neutralizing antibodies elicited by exposure to earlier vaccines or prior infection. Resistance of new variants to preexisting neutralizing antibody is not absolute but varies with both the extent of mutation in vaccine targeted proteins and the concentration of neutralizing antibody targeting previous viral variants. While repeated immunization targeting variants of the SARS-CoV-2 has proceeded as a strategy for addressing waning protective immunity, there has been sustained resistance to repeated immunization, as evidenced by surveys of current attitudes toward vaccination and the small proportion of Americans who received the most recent vaccine booster.

[0004] Although declining antibody concentrations increases the risk of acquiring SARS- CoV-2 infection, evidence from clinical and non-human primate studies has indicated that the currently available vaccines may elicit T-cell mediated immune responses that are less susceptible than neutralizing antibody responses to the immune escape associated with SARS-CoV-2 variants. These T-cell responses may at least partially explain how vaccines that elicit relatively short-lived neutralizing antibodies can still attenuate disease severity without preventing infection.

[0005] These findings indicate that foci of vaccine development efforts to counter ongoing emergence of variants should include development of vaccines that elicit both more sustained antibody concentrations and T-cell responses with activity at the site of infection. Unlike the situation for circulating neutralizing antibodies elicited by immunization or previous infection that are readily accessible at the time of pathogen exposure, T-cell immunity is most effective if pathogen-specific T cells pre-exist within the targeted organ. To counter respiratory pathogens, multiple studies have indicated that intranasal (IN) immunization is more effective than systemic immunization at eliciting T-cell responses in the lungs.

[0006] As such, there exists an unmet need for continued SARS-CoV-2 vaccine development to therapeutically and / or prophylactically address SARS-CoV-2 infections.SUMMARY OF THE INVENTION

[0007] This disclosure describes compositions, methods, kits, and related aspects for providing prophylaxis and / or reducing treatment times for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections. In some aspects, for example, the present disclosure provides nucleic acid vaccine constructs involving fusion of the gene encoding a SARS-CoV- 2 protein with the gene encoding the immature dendritic cell-targeting chemokine MIP- 3a / CCL20 (Af / E-Az SAR.S-CoV-2 receptor-binding domain (RBD) protein or sometimes referred to as a “fusion vaccine”). In some embodiments, the present disclosure provides nucleic acid vaccine constructs involving fusion of the gene encoding RBD with a gene encoding another chemokine that binds to a chemokine receptor 6 (CCR6) or with a gene encoding an antibody, or antigen binding portion thereof, that binds to a CCR6. In some embodiments, intranasal immunization with these nucleic acid vaccines expressing MIP- A / . RBD generate robust, immune responses and enhance antiviral activity when combined with antiviral agents. These and other aspects will be apparent upon complete review of the present disclosure, including the accompanying figures.

[0008] In one aspect, the present disclosure provides a nucleic acid vaccine (e.g., a DNA vaccine, an RNA vaccine (e.g., an mRNA vaccine), etc.) composition comprising a synthetic polynucleotide encoding a severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) protein, or a functional portion, fragment, or variant thereof, conjugated to a macrophage inflammatory protein-3 alpha (MIP-3a) or other chemokine that binds to a chemokine receptor 6 (CCR6), or a functional portion, fragment, or variant thereof, or to an antibody, orantigen binding portion thereof, that binds to a CCR6. In some embodiments, the SARS- CoV-2 protein comprises a SARS-CoV-2 spike protein, or a functional portion, fragment, or variant thereof. In some embodiments, the synthetic polynucleotide comprises the nucleotide sequence of SEQ ID. NOS: 1 and / or 2. In some embodiments, the synthetic polynucleotide comprises the nucleotide sequence of SEQ ID. NO: 3. In some embodiments, the MIP-3a is murine or human. In some embodiments, the synthetic polynucleotide is codon-optimized for expression in a mammalian cell. In some embodiments, the mammalian cell is a human cell.

[0009] In some embodiments, a recombinant nucleic acid vector encoding the nucleic acid vaccine compositions is provided. In some embodiments, the vector is a pSecTag2B plasmid or a pVaxl plasmid (e.g., comprising an IgE signal peptide). Other vectors are optionally utilized. In some embodiments, a pharmaceutical composition comprising a recombinant nucleic acid vector as disclosed herein is provided. In some embodiments, the pharmaceutical composition lacks a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier comprises a lipid nanoparticle (LNP), a polymeric nanoparticle, a lipidoid, a liposome, a lipoplex, a peptide carrier, a nanoparticle mimic, or a conjugate thereof. In some embodiments, the pharmaceutical composition does not comprise an additional biologically active agent. In some embodiments, the pharmaceutical compositions further include at least one additional biologically active agent (e.g., an antiviral agent or the like).

[0010] In another aspect, the present disclosure provides a method of providing prophylaxis to, and / or treating a SARS-CoV-2 infection in, a subject in need thereof comprising administering to the subject an effective amount of a pharmaceutical composition disclosed herein. In some embodiments, the composition is administered to the subject prior to, concurrent with, and / or after administering at least one antiviral agent to the subject. In some embodiments, the composition is administered as one or more boost doses after an initial administration of the composition to the subject. In some embodiments, the composition is administered intramuscularly and / or intranasally to the subject.

[0011] In another aspect, the present disclosure provides a vaccine composition, comprising a polypeptide that comprises a severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) protein, or a functional portion, fragment, or variant thereof, conjugated to a macrophage inflammatory protein-3 alpha (MIP-3a) or other chemokine that binds to a chemokine receptor 6 (CCR6), or a functional portion, fragment, or variant thereof, or to an antibody, or antigen binding portion thereof, that binds to a CCR6. In some embodiments, thepolypeptide comprises a SARS-CoV-2 spike protein, or a functional portion, fragment, or variant thereof. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID. NOS: 4 and / or 5. In some embodiments, the present disclosure provides a pharmaceutical composition comprising a vaccine composition disclosed herein. In some of these embodiments, the pharmaceutical composition does not comprise an additional biologically active agent. In some of these embodiments, the pharmaceutical composition further comprises at least one additional biologically active agent. In some embodiments, the pharmaceutical composition lacks a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0012] In some of these embodiments, the present disclosure provides a method of providing prophylaxis to, and / or treating a SARS-CoV-2 infection in a subject in need thereof comprising administering to the subject an effective amount of the vaccine composition. In some embodiments, the composition is administered to the subject prior to, concurrent with, and / or after administering at least one antiviral agent to the subject. In some of these embodiments, the composition is administered as one or more boost doses after an initial administration of the composition to the subject. In some of these embodiments, the composition is administered intramuscularly and / or intranasally to the subject.

[0013] In another aspect, the present disclosure provides a method of providing prophylaxis to, and / or treating a respiratory viral infection in, a subject in need thereof. The method comprises intranasally administering to the subject an effective amount of a pharmaceutical composition that comprises a synthetic polynucleotide encoding a respiratory viral protein, or a functional portion, fragment, or variant thereof, conjugated to a macrophage inflammatory protein-3 alpha (MIP-3a) or other chemokine that binds to a chemokine receptor 6 (CCR6), or a functional portion, fragment, or variant thereof, or to an antibody, or antigen binding portion thereof, that binds to a CCR6. In some embodiments, the pharmaceutical composition lacks a pharmaceutically acceptable carrier (e.g., the synthetic polynucleotide is administered to the subject as, for example, as naked DNA). In some embodiments, the method further comprises a pharmaceutically acceptable carrier (e.g., a liposome or the like).BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1A. Schematically depicts an exemplary expression cassette according to some embodiments. Figure discloses SEQ ID NO: 6.

[0015] Figure IB. Schematically depicts an exemplary recombinant nucleic acid vector according to some embodiments. Figure discloses SEQ ID NO: 6.

[0016] Figures 2A-2D. Effect of vaccine formulation on concentration of specific antibody maintained over 12 months, a) Vaccination and serum sampling schedule for Balb / c mice, with primary vaccination initiated at 6-8 weeks of age. Mice were immunized IM with 10 pg of plasmid DNA encoding either MipRBD, RBD or PBS. Immediately following vaccination each mouse received in vivo electroporation as described in Methods. Twenty -four hours after immunization each mouse received 50 pg CpG at the immunization site. Tail vein bleeds were obtained at the indicated time points post the initial immunization, b) Reciprocal antibody titers, determined by ELISA, indicates the highest dilution of serum at which the absorbance was twice the average value of the negative control wells (average of no peptide, no primary antibody, no secondary antibody, and no substrate wells in duplicate). Antibody titer was logic transformed for analysis and Area Under the Curve (AUC) was conducted to compare differences and significance. Error bars represent standard error of the mean (SEM). 95% confidence intervals for AUC were PBS (33-33); RBD (56.40-58.87); and MipRBD (60.57-63.92). Comparison of MipRBD vs RBD reciprocal antibody titers at individual time points using one-way ANOVA demonstrated significant differences at the 10 month and 12 month time points (p<0.05). c) Reciprocal antibody titers in BAL fluid 12 months post the primary immunization, with significance determined by one-way ANOVA. d) Reciprocal titers of neutralizing antibody at different points over the 12 months post primary immunization. Significance reported was determined by one-way ANOVA and indicates differences between RBD and MipRBD groups. ****p<0.0001, ***p=0.0002, *p<0.05.

[0017] Figure 3A and 3B. Effect of route of administration and vaccine formulation on serum antibody concentrations, a) Vaccination and serum or tissue sampling schedule for 6-8 week old Balb / c mice immunized with 50 pg MipRBD IM followed by electroporation and CpG administration's as described for Figure lb, or IN with 100 pg of plasmid DNA encoding either MipRBD or RBD in a volume of 50 pl of PBS in each nostril. 50 pl PBS alone in each nostril was used as a control IN immunization. Electroporation was not employed for intranasal immunization and 50 pg CpG, 25 pg in each nostril in 50 pl PBS,was only administered one day after the final immunization, b) Serum antibody titers were determined by ELISA as described for Figure lb. Significance of differences in titer was determined using one way ANOVA. ****p<0.0001. Effect of route of administration and vaccine formulation on T cell recruitment to the lungs.

[0018] Figure 4. Effect of route of administration and vaccine formulation on T cell recruitment to the lungs. Vaccination and tissue harvesting schedule was as described in Figure 3a. Three weeks after the final vaccination, the recruitment of T-cell sub-populations to the lungs elicited by the different immunization protocols was determined by flow cytometric analysis. Differences between groups were analyzed using one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001.

[0019] Figures 5A-5C. Effect of administration route and vaccine formulation on IFNy responses by T-cells from lung and spleen. Vaccination and tissue harvesting schedule was as described in Figure 3a. Lymphocytes harvested from lungs or spleen were incubated in vitro with 1 pg RBD protein for 16 hours, with the final 4 hours accumulating intracellular cytokines. Flow cytometric analysis was then performed to evaluate T-cell ZFNy expression associated with different immunization regimens. *p<0.05.

[0020] Figure 6. Effect of administration route and vaccine formulation onTNFa responses by T-cells from lung and spleen. Vaccination and tissue harvesting schedule was as described in Figure 3a. Lymphocytes harvested from lungs or spleen were incubated in vitro with 1 pg RBD protein for 16 hours, with the final 4 hours accumulating intracellular cytokines. Flow cytometric analysis was then performed to evaluate T-cell TNFa expression associated with different immunization regimens. *p<0.05.DETAILED DESCRIPTION OF THE INVENTION

[0021] In some aspects, the present disclosure provides a COVID vaccine that incorporates the chemokine macrophage inflammatory protein-3 alpha (MIP-3a or CCL20). In some embodiments, for example, the simple intranasal administration of a plasmid DNA encoding the MIP-3a component fused to a portion of the SARS-CoV-2 spike DNA elicits a potent T cell response in the lungs of subjects. In contrast, intranasal administration of the vaccine construct without the MIP-3a component or systemic, intramuscular immunization with the MIP-3a containing construct generally elicits effectively no lung T cell response. Moreover, this plasmid DNA typically needs no encapsulation in a lipid or viral particle andno use of adjuvants. These and other attributes of the present disclosure will be apparent upon a complete review of this specification, including the accompanying figures.

[0022] In accordance with an embodiment, the present disclosure provides a nucleic acid vaccine composition comprising a synthetic polynucleotide encoding the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, or a functional portion, fragment, or variant thereof, conjugated (e.g., directly or via a linker or spacer nucleotide sequence) to a synthetic polynucleotide encoding a macrophage inflammatory protein-3 alpha (MIP-3a) or other chemokine that binds (e.g., specifically binds) to a chemokine receptor 6 (CCR6), or a functional portion, fragment, or variant thereof, or to a synthetic polynucleotide encoding an antibody, or antigen binding portion thereof, that binds (e.g., specifically binds) to a CCR6. As used herein, the term “antibody” refers to an immunoglobulin or an antigen-binding domain thereof. The term includes but is not limited to polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, human, canonized, canine, felinized, feline, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies. The antibody can include a constant region, or a portion thereof, such as the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes. For example, heavy chain constant regions of the various isotypes can be used, including: IgGi, IgG2, IgGs, IgG4, IgM, IgAi, IgA2, IgD, and IgE. By way of example, the light chain constant region can be kappa or lambda. The term “monoclonal antibody” refers to an antibody that displays a single binding specificity and affinity for a particular target, e.g., epitope. As used herein, the term “antigen binding portion” refers to a portion of an antibody that binds to a chemokine receptor 6 (CCR6), e.g., a molecule in which one or more immunoglobulin chains is not full length, but which binds to CCR6. Examples of binding portions encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VLC, VHC, CL and CHI domains: (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VHC and CHI domains; (iv) a Fv fragment consisting of the VLC and VHC domains of a single arm of an antibody, (v) a dAb fragment, which consists of a VHC domain; and (vi) an isolated complementarity determining region (CDR) having sufficient framework to bind, e.g., an antigen binding portion of a variable region. An antigen binding portion of a light chain variable region and an antigen binding portion of a heavy chain variable region, e.g., the two domains of the Fv fragment, VLC and VHC, can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VLC and VHC regionspair to form monovalent molecules (known as single chain Fv (scFV). Such single chain antibodies are also encompassed within the term “antigen binding portion” of an antibody. The term “antigen binding portion” encompasses a single-domain antibody (sdAb), also known as a “nanobody” or “VHH antibody,” which is an antibody fragment consisting of a single monomeric variable antibody domain. These antibody binding portions are obtained using conventional techniques known to those with skill in the art, and the portions are screened for utility in the same manner as are intact antibodies.

[0023] In some embodiments, the synthetic polynucleotide comprises the nucleotide sequence of SEQ ID. NOS: 1, 2, or 3 (shown below in Tables 1 and 2) or comprises a polynucleotide having at least 80%, 85%, 90%, 95%, 99% sequence identity with SEQ ID NOS: 1, 2, or 3. The phrase “functional portion, fragment, or variant thereof’ in the context of the proteins described herein, refers to a portion or fragment of the full-length protein or a non-wild-type form of the protein (full-length, portion, or fragment thereof) that retains a desired property or function, such as targeting a cell type (e.g., an iDC), improving immunogenicity, effectuating vaccination, or the like. In some embodiments, vaccine compositions of the present disclosure comprise polypeptides that comprises a SARS-CoV-2 protein (e.g., an RBD protein), or a functional portion, or fragment or variant thereof, conjugated to MIP-3a, or a functional portion or fragment or variant thereof (e.g., MIP- fusion protein).TABLE 1TABLE 2

[0024] In some embodiments, synthetic polynucleotide encoding a SARS-CoV-2 protein, or a functional portion, fragment, or variant thereof, conjugated to MIP-3a, or a functional portion, fragment, or variant thereof include other nucleic acid elements, such as leader sequences, spacers or linkers, tags, and / or the like. An example of such a synthetic polynucleotide configuration is shown in Figure 1 A. In some embodiments, the linker comprises a polynucleotide which encodes one or more amino acids. The linker can be a polynucleotide encoding 1 to 50 amino acids, including, for example, 2, 3, 4, 5, 10, 15, 20, 30, 40, up to 50 amino acids. In an embodiment, the linker is a dipeptide. In another embodiment, the linker is the dipeptide Glu-Phe. Typically, the synthetic polynucleotides of the present disclosure are included as expression cassettes in a recombinant nucleic acid vector, such as a plasmid (e.g., a pSectag2B plasmid (see, FIG. IB), a pVAXl plasmid, etc.) or the like. Exemplary expression vectors are described further herein.

[0025] As used herein, the term “SARS-CoV-2 spike protein” refers to a glycoprotein that assembles into trimers that form structures, called peplomers or spikes, that project from the surface of the SARS-CoV-2 virion. The function of the spike glycoprotein is to mediate viral entry into host cells by interacting with molecules on the exterior cell surfaces and fusing the viral and cellular membranes. Spike glycoprotein is a class I fusion protein that contains two regions, known as SI and S2. The SI region comprises the receptor-binding domain (RBD) that binds to receptors on the cell surface. SARS-CoV-2 interacts with angiotensin-converting enzyme 2 (ACE2). The S2 region comprises the fusion peptide and other fusion infrastructure needed for membrane fusion with host cells, a necessary step for infection and viral replication.

[0026] As used herein, the term “macrophage inflammatory protein 3a” or “MIP-3a” (also known as chemokine (C-C motif) ligand 20 (CCL20) or liver activation regulated chemokine (LARC)) refers to a small cytokine belonging to the CC chemokine family. The protein attracts memory T cells and natural killer cells to sites of inflammation, as well as immature dendritic cells. MIP-3a is implicated in the formation and function of mucosal lymphoid tissues via chemoattraction of lymphocytes and dendritic cells towards the epithelial cells surrounding these tissues. In addition, MIP-3a elicits its effects on its target cells by binding and activating the chemokine receptor CCR6. In some embodiments, the MIP-3a can be murine, porcine, ovine, bovine, human, or combinations thereof. It will be understood by those of ordinary skill in the art that many different isoforms of these exist andare optionally used in the compositions disclosed herein. Examples of some of these include homo sapiens (human) Gene ID: 6364 and Mus musculus (house mouse) Gene ID: 20297.

[0027] The synthetic polynucleotides of the nucleic acid vaccine (e.g., DNA vaccines, mRNA vaccines, etc.) compositions of the present disclosure can be comprised within an expression cassette. The term “expression cassette” or “expression vector” as used herein refers to a nucleotide sequence, which is capable of affecting expression of a protein coding sequence in a host compatible with such sequences. Expression cassettes typically include at least a promoter operably linked with the polypeptide coding sequence; and, optionally, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression may also be included, e.g., enhancers. “Operably linked”, refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription of the DNA sequence. Thus, expression cassettes include plasmids, recombinant viruses, any form of a recombinant “naked DNA” vector, and the like. In some embodiments, expression cassettes include elements that have been codon optimized for expression in the intended host.

[0028] The term “immunogen” or “immunogenic composition” is synonymous with “antigen or antigenic” and refers to a compound or composition comprising a peptide, polypeptide or protein which is “immunogenic,” i.e., capable of eliciting, augmenting or boosting a cellular and / or humoral immune response, either alone or in combination or linked or fused to another substance. An immunogenic composition can be a peptide of at least about 5 amino acids, a peptide of 10 amino acids in length, a fragment 15 amino acids in length, a fragment 20 amino acids in length or greater; smaller immunogens may require presence of a “carrier” polypeptide e.g., as a fusion protein, aggregate, conjugate or mixture, preferably linked (chemically or otherwise) to the immunogen. The immunogen can be recombinantly expressed from a vaccine vector, which can be naked DNA comprising the immunogen’s coding sequence operably linked to a promoter, e.g., an expression cassette. The immunogen includes one or more antigenic determinants or epitopes, which may vary in size from about 3 to about 15 amino acids. In some embodiments, the immunogen or antigen is a polypeptide comprising a A7 / E-A / S AR.S-CoV-2 fusion protein as described herein.

[0029] In accordance with some embodiments, the present disclosure provides a recombinant vector encoding the nucleic acid vaccine compositions described herein. By “nucleic acid” as used herein includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and / or purified) from natural sources,which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered intemucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide. It is generally preferred that the nucleic acid does not comprise any insertions, deletions, inversions, and / or substitutions. However, it may be suitable in some instances, as discussed herein, for the nucleic acid to comprise one or more insertions, deletions, inversions, and / or substitutions, such as when a given polynucleotide encodes a functional portion, fragment, or variant of a SARS-CoV-2 protein (e.g., a SARS- CoV-2 spike protein) and / or MIP-3a.

[0030] Preferably, the nucleic acids of the present disclosure are recombinant. As used herein, the term “recombinant” refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above. For purposes herein, the replication can be in vitro replication or in vivo replication.

[0031] The nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. For example, a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine-substituted nucleotides). Examples of modified nucleotides that can be used to generate the nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3 -methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio- N6-isopentenyladenine, uracil-5-oxyacetic acid, wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the invention can be purchased from companies, such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0032] In some embodiments, the substituted nucleic acid sequence may be optimized. Without being bound to a particular theory, it is believed that optimization of the nucleic acid sequence increases the translation efficiency of the mRNA transcripts. Optimization of the nucleic acid sequence may involve substituting a native codon for another codon that encodes the same amino acid, but can be translated by tRNA that is more readily available within a cell, thus increasing translation efficiency. Optimization of the nucleic acid sequence may also reduce secondary mRNA structures that would interfere with translation, thus increasing translation efficiency. In some embodiments, codon optimization is performed using Genscript.

[0033] In some embodiments, the present disclosure also provides an isolated or purified nucleic acid comprising a nucleotide sequence which is complementary to the nucleotide sequence of any of the nucleic acids described herein or a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.

[0034] The nucleic acids of the present disclosure can be incorporated into a recombinant expression vector. In this regard, the invention provides recombinant expression vectors comprising any of the nucleic acids of the invention. For purposes herein, the term “recombinant expression vector” means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. The vectors of the invention are not naturally occurring as a whole. However, parts of the vectors can be naturally occurring. The recombinant expression vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be singlestranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The recombinant expression vectors can comprise naturally occurring, non-naturally occurring intemucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or altered nucleotides or intemucleotide linkages does not hinder the transcription or replication of the vector.

[0035] In some embodiments, the nucleic acid vaccine composition or expression cassette will be inserted into a DNA vector or plasmid. The recombinant expression vector of the present disclosure can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host. Suitable vectors include those designed forpropagation and expansion or for expression or both, such as plasmids and viruses. The vector can be selected from the group consisting of the pSectag2B or pVAXl series (ThermoFisher Scientific, Carlsbad, CA), the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), pcDNA3 family of plasmids, the pNGVL4a plasmid, and the pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors, such as XGT10, XGT11, XZapII (Stratagene), XEMBL4, and XNM1149, also can be used. Examples of plant expression vectors include pBIOl, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). The recombinant expression vectors of the present disclosure can be prepared using standard recombinant DNA techniques well known to persons having ordinary skill in the art. Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived, e.g., from ColEl, 2 p plasmid, X, SV40, bovine papilloma virus, and the like.

[0036] Desirably, the recombinant expression vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., mammalian, bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA-based.

[0037] The recombinant expression vector can include one or more marker genes, which allow for selection of transformed or transfected hosts. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. Suitable marker genes for the expression vectors disclosed herein may include, for instance, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes, among others.

[0038] Recombinant expression vectors can comprise a native or nonnative promoter operably linked to the nucleotide sequence encoding the fusion proteins, polypeptide, or protein (including functional portions and functional variants thereof), or to the nucleotide sequence which is complementary to or which hybridizes to the nucleotide sequence encoding the fusion proteins, polypeptide, or protein. The selection of promoters, e.g., strong, weak, inducible, tissue-specific and developmental-specific, is within the ordinary skill of the artisan. Similarly, the combining of a nucleotide sequence with a promoter is also within theskill of the artisan. The promoter can be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long-terminal repeat of the murine stem cell virus.

[0039] In accordance with an embodiment, the present invention provides a composition comprising a polypeptide encoding a A / / / J-3a SARS-CoV-2 fusion protein, or a functional portion, fragment, variant thereof. It will be understood by persons of ordinary skill in the art that the polypeptide encoding a MIP-3a / SARS-CoV-2 fusion protein, or a functional portion, fragment, variant thereof is a fusion polypeptide which acts as an immunogen to the immune system and is expressed in the cells of the subject that have taken up the nucleic acid vaccine of the present invention. In some embodiments, the synthetic polypeptide comprises the amino acid sequence of SEQ ID. NOS: 4 or 5 (shown below in Table 3 or comprises a polypeptide having at least 80%, 85%, 90%, 95%, 99% sequence identity with SEQ ID NOS: 4 or 5.TABLE 3

[0040] In some embodiments, the present disclosure provides a synthetic polypeptide molecule comprising at least one of the polypeptides described herein along with at least one other polypeptide. The other polypeptide can exist as a separate polypeptide of the fusion protein, or can exist as a polypeptide, which is expressed in frame (in tandem) with one of the polypeptides described herein. The other polypeptide can encode any peptidic or proteinaceous molecule, or a portion thereof. Suitable methods of making fusion proteins are known in the art, and include, for example, recombinant methods. See, for instance, Choi et al., Mol. Biotechnol. 31 : 193-202 (2005), which is incorporated by reference.

[0041] Included in the scope of the present disclosure are functional variants of the fusion proteins, and polypeptides, and proteins described herein. Functional variants can include fusion proteins, polypeptides, or proteins having substantial or significant sequence identity or similarity to a parent fusion proteins, polypeptides, or proteins, which functional variant retains the biological activity of the fusion proteins, polypeptides, or proteins of which it is a variant. In reference to the parent fusion proteins, polypeptides, or proteins, the functionalvariant can, for instance, be at least about 30%, 50%, 75%, 80%, 90%, 98% or more identical in amino acid sequence to the parent fusion proteins, polypeptide, or protein.

[0100] The functional variant can, for example, comprise the amino acid sequence of the parent fusion proteins, polypeptide, or protein with at least one conservative amino acid substitution. Conservative amino acid substitutions are known in the art, and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is exchanged for another amino acid that has the same chemical or physical properties. For instance, the conservative amino acid substitution can be an acidic amino acid substituted for another acidic amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Vai, He, Leu, Met, Phe, Pro, Trp, Vai, etc.), a basic amino acid substituted for another basic amino acid (Lys, Arg, etc.), an amino acid with a polar side chain substituted for another amino acid with a polar side chain (Asn, Cys, Gin, Ser, Thr, Tyr, etc.), etc.

[0101] Alternatively or additionally, the functional variants can comprise the amino acid sequence of the parent fusion proteins, polypeptide, or protein with at least one nonconservative amino acid substitution. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with or inhibit the biological activity of the functional variant. Preferably, the non-conservative amino acid substitution enhances the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent fusion proteins, polypeptide, or protein.

[0102] The fusion polypeptides, and / or proteins of the invention (including functional portions and functional variants thereof) can be obtained by methods known in the art. Suitable methods of de novo synthesizing polypeptides and proteins are described in references, such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2005; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2000; and U.S. Patent No. 5,449,752, which are each incorporated by reference. Also, polypeptides and proteins can be recombinantly produced using the nucleic acids described herein using standard recombinant methods. See, for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Further, some of the fusion proteins, polypeptides, and proteins of the present disclosure (including functional portions and functional variants thereof) can be isolated and / or purified from a source, such as a plant,a bacterium, an insect, a mammal, e.g., a rat, a mouse, a human, etc. Methods of isolation and purification are well-known in the art. Alternatively, the fusion proteins, polypeptides, and / or proteins described herein (including functional portions and functional variants thereof) can be commercially synthesized by companies, such as Synpep (Dublin, CA), Peptide Technologies Corp. (Gaithersburg, MD), and Multiple Peptide Systems (San Diego, CA). In this respect, the fusion proteins, polypeptides, and proteins can be synthetic, recombinant, isolated, and / or purified.

[0103] In accordance with some embodiments, the present disclosure provides various pharmaceutical compositions comprising the DNA constructs or polypeptide compositions described herein for use as a vaccine. Thus, in further embodiments, the present disclosure provides the use of a pharmaceutical composition comprising a vaccine, and a pharmaceutically acceptable carrier, as a medicament, preferably as a medicament for the treatment of a SARS-CoV-2 infection in a subject.

[0104] In a further embodiment, the present invention provides a method for treating a SARS-CoV-2 infection in a subject in need thereof comprising administering to the subject an effective amount of the nucleic acid vaccine compositions and / or the synthetic polypeptide compositions described herein.

[0105] In some embodiments, the present disclosure provides methods of providing prophylaxis to, and / or treating a SARS-CoV-2 infection in, a subject in need thereof comprising administering to the subject an effective amount of a composition disclosed herein. In some embodiments, the composition is administered to the subject prior to, concurrent with, and / or after administering at least one antibiotic agent to the subject. In some embodiments, the composition is administered as one or more boost doses after an initial administration of the composition to the subject.

[0106] In some embodiments, the term “administering” means that the compositions of the present disclosure are introduced into a subject, preferably a subject receiving treatment for a SARS-CoV-2 infection, and the compounds are allowed to come in contact with the one or more infected cells or population of cells in vivo. In some embodiments, the composition is administered intramuscularly and / or intranasally to the subject.

[0107] It will be understood to persons having ordinary skill in the art that the nucleic acid vaccine or polypeptide vaccine compositions described herein can be administered in a regimen where there is a first or priming dose of vaccine composition administered to the subject, then after a period of time (e.g., 5 to 180 or more days), a second, third or more boostdose of vaccine is then administered to the subject. In some embodiments, the boost dose is administered 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 up to 50 days apart.

[0108] In some embodiments, the carrier is a pharmaceutically acceptable carrier. With respect to pharmaceutical compositions, the carrier can be any of those conventionally used and is limited only by chemi co-phy si cal considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration. The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use. In some embodiments, the pharmaceutical compositions of the present disclosure further include at least one additional biologically active agent (e.g., an antibiotic agent or the like). In some embodiments, the pharmaceutical compositions of the present disclosure lack a pharmaceutically acceptable carrier.

[0109] The choice of carrier will be determined in part by the chemical properties of the vaccines as well as by the particular method used to administer the vaccines. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the invention. The following formulations for intranasal, parenteral, subcutaneous, intravenous, intramuscular, intradermal, intraarterial, intrathecal and intraperitoneal administration are exemplary and are in no way limiting. More than one route can be used to administer the first and second vaccine, and in certain instances, a particular route can provide an immediate and more effective response than another route.

[0110] Injectable formulations are in accordance with the present disclosure according to some embodiments. Formulations for effective pharmaceutical carriers for injectable compositions are well-known to persons having ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Trissei, 14th ed., (2007)).[OHl] In accordance with some embodiments, the vaccines of the present invention can be administered other ways known in the art. For example, the vaccines can be administered via use of electroporation techniques. Suitable electroporation techniques are disclosed in U.S. Pat. Nos. 6,010,613, 6,603,998, and 6,713,291, all of which are incorporated herein by reference. Other physical approaches can include needle-free injection systems (NFIS) (e.g., as disclosed in U.S. Pat. No. 9,333,300, which is incorporated herein by reference), gene gun,biojector, ultrasound, and hydrodynamic delivery, all of which employ a physical force that permeates the cell membrane and facilitates intracellular gene transfer. Chemical vaccination approaches typically use synthetic or naturally occurring compounds (e.g., cationic lipids, cationic polymers, lipid-polymer hybrid systems) as carriers to deliver the nucleic acid into the cells.

[0112] In some other embodiments, intramuscular administration of the vaccines of the present invention may be achieved by the use of a needless injection device to administer a virus or plasmid DNA suspension (using, e.g., Biojector™) or a freeze-dried powder containing the vaccine (e.g., in accordance with techniques and products of Powdeiject).

[0113] In some aspects the vaccines disclosed herein are formulated in a lipid nanoparticle (LNP). The use of LNPs enables the effective delivery of chemically vaccines. Both modified and unmodified LNP formulated vaccines are optionally utilized. In some embodiments the vaccines disclosed herein are superior to conventional vaccines by a factor of at least 10 fold, 20 fold, 40 fold, 50 fold, 100 fold, 500 fold or 1,000 fold.

[0114] In one set of embodiments, lipid nanoparticles (LNPs) are provided. In one embodiment, a lipid nanoparticle comprises lipids including an ionizable lipid (such as an ionizable cationic lipid), a structural lipid, a phospholipid, and the nucleic acid vaccine. Each of the LNPs described herein or otherwise known to persons having ordinary skill in the art may be used as a formulation for the vaccines described herein. In some embodiments, the LNP comprises an ionizable lipid, a PEG-modified lipid, a phospholipid and a structural lipid. In some embodiments, the LNP has a molar ratio of about 20-60% ionizable lipid: about 5-25% phospholipid: about 25-55% structural lipid; and about 0.5-15% PEG-modified lipid. In some embodiments, the LNP comprises a molar ratio of about 50% ionizable lipid, about 1.5% PEG-modified lipid, about 38.5% structural lipid and about 10% phospholipid. In some embodiments, the LNP comprises a molar ratio of about 55% ionizable lipid, about 2.5% PEG lipid, about 32.5% structural lipid and about 10% phospholipid. In some embodiments, the ionizable lipid is an ionizable amino or cationic lipid and the phospholipid is a neutral lipid, and the structural lipid is a cholesterol. In some embodiments, the LNP has a molar ratio of 50:38.5: 10: 1.5 of ionizable lipid: cholesterol :DSPC: PEG2000-DMG.Additional details regarding LNPs and other carriers that are optionally adapted for use with the vaccines of the present disclosure are also described in, for example, U.S. Patent Application Publication No. US 20200254086, which is incorporated by reference in its entirety.

[0115] For purposes of the present disclosure, the amount or dose of the vaccine administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the subject over a selected time frame. The dose will typically be determined by the efficacy of the first and second vaccine and the condition of the given subject, as well as the body weight of that subject to be treated.

[0116] Typically, the attending physician will decide the dosage of first and second vaccine with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, to be administered, route of administration, and the severity of the condition being treated. By way of example and not intending to limit the invention, the dose of the vaccine is about 1 to 10,000 pg of vaccine to the subject being treated. In some embodiments, the dosage range of the vaccine is about 500 pg-6,000 pg of vaccine. In a preferred embodiment, the dosage of the vaccine is about 3,000 Pg-10117] In accordance with some embodiments, the present disclosure provides pharmaceutical compositions comprising the nucleic acid vaccine compositions and / or the polypeptide compositions described herein in combination with at least one additional biologically active agent. An “active agent” and a “biologically active agent” are used interchangeably herein to refer to a chemical or biological compound that induces a desired pharmacological and / or physiological effect in which the effect may be prophylactic or therapeutic. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of those active agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the terms “active agent,” “pharmacologically active agent” and “drug” are used, then, it is to be understood that the invention includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, antibiotics, etc. The active agent can be a biological entity, such as a virus or cell, whether naturally occurring or manipulated, such as transformed. In some embodiments, the pharmaceutical compositions of the present disclosure do not comprise an additional biologically active agent.

[0118] The biologically active agent may vary widely with the intended purpose for the composition. The term active is art-recognized and refers to any moiety that is a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject. Examples of biologically active agents, that may be referred to as “drugs”, are described in well-known literature references such as the Merck Index, the Physicians’ DeskReference, and The Pharmacological Basis of Therapeutics, and they include, without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances which affect the structure or function of the body; or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment.

[0119] Non-limiting examples of biologically active agents include following: antiinflammatory agents such as steroids, non-steroidal anti-inflammatory agents, anti-pyretic and analgesic agents, antigenic materials, antibiotics, and anti-viral drugs.

[0120] Various forms of the biologically active agents may be used. These include, without limitation, such forms as uncharged molecules, molecular complexes, salts, ethers, esters, amides, prodrug forms and the like, which are biologically activated when implanted, injected or otherwise placed into a subject.

[0121] It will be understood that the methods of treatment using an effective amount of the nucleic acid vaccine compositions in combination with an effective amount of one or more additional biologically active agents, the combination can occur either simultaneously or serially with at least one other. The dosing regimens of the above methods can also comprise a first dose of vaccine an additional biologically active agent, followed by a second or more dose of vaccine and optionally an additional biologically active agent as needed.

[0122] As used herein, the term “subject” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.

[0123] In accordance with an embodiment, the present invention provides a cell or population of cells expressing the synthetic polypeptide compositions described herein. It will be understood that the cells or population of cells expressing the synthetic polypeptide compositions were in contact with the nucleic acid vaccine compositions and / or the synthetic polypeptide compositions in vitro or in vivo.

[0124] As defined herein, in another embodiment, the term “contacting” means that the one or more compounds of the present disclosure are introduced into a sample having at least one cell and appropriate enzymes or reagents, in a test tube, flask, tissue culture, chip, array,plate, microplate, capillary, or the like, and incubated at a temperature and time sufficient to permit binding and uptake of the at least one compound to the cell. Methods for contacting the samples with the compounds, and other specific binding components are known to those skilled in the art, and may be selected depending on the type of assay protocol to be run. Incubation methods are also standard and are known to those skilled in the art.

[0125] In accordance with an embodiment, the present disclosure provides kits that contain the compositions or pharmaceutical compositions used with the methods, as described above, to practice the methods of the invention. The kits can contain various combinations of vaccines and the like. The kit can contain instructional material teaching methodologies, e.g., means to administer the compositions used to practice the methods, means to inject or infect cells, patients or animals with vaccines of the present disclosure, means to monitor the resultant immune response and assess the reaction of the individual to which the compositions have been administered, and the like.

[0126] Following long-standing patent law convention, the terms “a,” “an,” and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a subject” includes a plurality of subjects, unless the context clearly is to the contrary (e.g., a plurality of subjects), and so forth.

[0127] Throughout this specification and the claims, the terms “comprise,” “comprises,” and “comprising” are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the term “include” and its grammatical variants are intended to be nonlimiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.

[0128] For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, parameters, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about” even though the term “about” may not expressly appear with the value, amount or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact, but may be approximate and / or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art depending on the desired properties sought to be obtained by the presently disclosed subject matter. For example, the term “about,” when referring to a value can be meant to encompass variations of, in some embodiments, ± 100% in someembodiments ± 50%, in some embodiments ± 20%, in some embodiments ± 10%, in some embodiments ± 5%, in some embodiments ±1%, in some embodiments ± 0.5%, and in some embodiments ± 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

[0129] Further, the term “about” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

[0130] The following examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are only intended for the purposes of illustration, and are not to be construed as limiting in any manner to make compounds of the disclosure by other methods.EXAMPLE

[0131] Introduction

[0132] In murine vaccine model systems analyzing protective or therapeutic efficacy against malaria, tuberculosis, and melanoma, we have studied the impact of fusing vaccine antigen to the chemokine macrophage inflammatory protein 3a (MIP-3a), also known as Chemokine (C-C motif) ligand 20 (CCL20). This chemokine binds C-C Motif Chemokine Receptor 6 (CCR6), a member of the G protein-coupled receptor family found on immature, but not mature dendritic cells (DC). Targeting immature DC (iDC) distinguishes this vaccine construct from other dendritic cell-targeting vaccines, which engage receptors expressed by more differentiated DC to enhance vaccine efficacy. Employment of this vaccine platform has generated greater and, in some model systems, more sustained responses than those observed when the vaccine antigen is not fused to the chemokine. While MIP-3a serves as a chemoattract for iDC, previous studies have demonstrated the requirement of fusing thevaccine antigen to chemokine to achieve maximum efficacy. In our recent study in a mouse challenge model using a therapeutic vaccine targeting the M. tuberculosis stringent response, we found that intranasal immunization elicited significantly greater antibacterial activity than intramuscular immunization and that intranasal vaccination with the MIP-3a-fused vaccine was also significantly more effective than immunization with vaccine antigen alone. Optimal immune responses were observed with a DNA vaccine construct that required no encapsulation of the DNA nor use of adjuvant to achieve the observed therapeutic efficacy.

[0133] In the current example, we have examined in a mouse model the comparative immunogenicity of a DNA vaccine incorporating the receptor binding domain (RBD) of the SARS-CoV-2 spike protein with or without fusion to MIP-3a. Our results indicate that over a year of observation the fusion vaccine elicited and sustained significantly higher antibody concentrations compared to vaccine only incorporating RBD. Of particular note was the persistence in the recipients of the fusion vaccine of neutralizing antibody responses for at least four months after they were no longer detectable in the recipients of vaccine only encoding RBD. Further, we found that IN immunization yielded significantly greater T-cell responses in the lung than those elicited by IM immunization, but only when RBD was fused to MIP-3a.

[0134] Methods

[0135] Vaccine: pUC57 plasmid containing DNA encoding codon-optimized RBD(Spike amino acids 319-541 of Wuhan-Hu-1 isolate) was purchased from GenScript (Piscataway, NJ). DNA was extracted and ligated into a previously generated pSecTag2b plasmid by Hindlll and BamHI to generate RBD alone and also by KasI and BamHI to generate Mip3a-RBD (restriction enzymes from ThermoFisher, Waltham, MA) containing DNA encoding MIP3a. Proper insertion was confirmed by agarose gel electrophoresis and sequencing, and the expression of target genes was confirmed by immunoblots of cell lysates and supernatants following transfection of HEK293T cells (American Type Culture Collection, Manassas, VA) utilizing the Trans-IT 293 transfection system (Minis Bio, Madison, WI). Vaccination plasmids were selected by ampicillin (100 mg / mL) and Qiagen® Endo-Free® Plasmid Series kits were used to extract the ligation product from DH5-a E. coli (Invitrogen™ ThermoFisher Scientific, Waltham, MA) and plasmid DNA was diluted with endotoxin-free IxPBS. Nanodrop spectrophotometry was used to test the purity of the extracted DNA.

[0136] Mice: Six- to eight-week-old female BALB / c mice were purchased from Charles River Laboratories, Inc, Wilmington, MA. All mice were kept in a pathogen-free micro-isolation facility in accordance with the National Institutes of Health guidelines for the humane use of laboratory animals. All experimental procedures involving mice were approved by the Institutional Animal Care and Use Committee of the Johns Hopkins University.

[0137] IM Vaccination'. . For IM vaccination (Figure 2), the DNA vaccine construct was diluted in lx endotoxin-free PBS to a 10-pg dose (DNA concentration of 143 ng / pL), and each mouse received a volume of 70 pL by injection into the right gastrocnemius muscle. Immediately following injection, the muscle was pulsed using an ECM 830 Electro Square Porator with 2-Needle Array Electrode (BTX Harvard Apparatus; Holliston, MA) under the following parameters: 106 V; 20 ms pulse length; 200 ms pulse interval; 8 total pulses. CpG Type B (ODN1826) (InvivoGen, San Diego, CA) was used as the adjuvant for IM immunization and was diluted in lx endotoxin-free PBS at Img / mL. The adjuvant was injected intramuscularly in the right gastrocnemius muscle at a volume of 50pL. Three doses of the vaccine were given, each two weeks apart, and the adjuvant was given 1 day after every vaccination to allow for expression of protein from the DNA vaccine.

[0138] IN vaccination', the DNA vaccine construct was diluted in lx endotoxin-free PBS to a concentration of 2 pg / pL for all groups. Mice were anesthetized before vaccination by inhalation of vaporized isoflurane. For each IN dose, 100 pg of vaccine was administered in each nostril in a volume of 50pL. Each mouse was monitored in an upright position until complete recovery and vaccine absorption was confirmed by lack of nasal discharge. The IM immunization used for comparison purposes in the IN immunization studies employed the procedure described above for the long-term antibody studies except that a dose of 50 pg of plasmid DNA was used. Previous studies have shown that this dose was optimal for the MIP-3 a vaccine formulations for eliciting cell-mediated immune responses by IM immunization (29).

[0139] Lymphocyte Isolation: Under sterile conditions, mouse spleens and lungs were harvested and placed in IxPBS on ice. Lungs were transferred to wells containing 1 ml of digestion buffer (RPMI media, 167pg / mL Liberase, and lOOpg / mL DNase), minced with scissors, and incubated at 37°C for 30 minutes. Lungs and spleens were ground gently with a pestle over a 70-pM mesh filter into 50-mL conical tubes and immediately centrifuged at 300g for 10 minutes at 4°C. The supernatant was removed and the pellet was then fully resuspended using 1 mL ACK lysis buffer (Quality Biological, Gaithersburg, MD) and incubated at room temperature (RT) for 3-4 minutes. To stop cell lysis, cells were dilutedwith 20-30 mL of cold IxPBS, and were then pelleted at 300g for 10 minutes 4°C. After another resuspension in 10 mL cold IxPBS and centrifugation under the same conditions, the supernatant was removed and the pellet was resuspended in 1 mL (lungs) or 4 mL (spleens) freezing medium (90% FBS, 10% DMSO) and aliquoted into 2 (lungs) and 4 (spleens) tubes for cryostorage using isopropanol cooling containers (Mr. Frosty, ThermoFisher Scientific, Waltham, MA) at -80°C for at least 4 hours and then moved to -150°C.

[0140] Enzyme Linked Immunosorbent Assay (ELISA ) For Determining Serum AntibodyConcentration: At the indicated time points post vaccination (Figure 2a) approximately 100 pl of mouse blood was collected in heparin coated 1 ml microcentrifuge tubes by tail vein nicking and spun at 2200g. The plasma supernatant was frozen and stored at -20°C until used. Humoral immune responses to RBD protein were measured using ELISA.I g / microwell of S-RBD His-tag recombinant protein (Invitrogen, Rockford, IL) diluted in sterile lx PBS was used to coat the ELISA plates overnight at 4°C. Each well of the plate was washed three times using 250 pL of washing solution (0.05% Tween 20 diluted in lx PBS). Plates were emptied and residual liquid was discarded before blocking each well with 250 pL blocking solution (1% BSA in lx PBS) for 30 minutes at RT. lOOpL of mouse serum samples serially diluted in triplicate in blocking solution were then added to each well and the plate was incubated at RT for 2 hours. lOOpL of HRP Goat Anti -Mouse IgG (H+L) secondary antibody (Biotium, Fremont) diluted 1 : 1000 in blocking solution was added to each well and incubated at RT for 1 hour after washing the plates six times with 250pL / well washing solution. The plates were washed six times using the same amount of washing solution again after the secondary antibody incubation, and lOOpL of KPL ABTS® Peroxidase Substrate (SeraCare Life Sciences, Inc., Milford, MA) was then added into each well and the plates were incubated at RT in the dark for 1 hour. Data were collected using the Synergy HT at O.D.405nm (BioTek Instruments, Inc., Winooski, VT). The endpoint ELISA titer was determined as the highest dilution of serum at which the absorbance was twice the average value of the negative control wells (either no substrate, no primary antibody, or no secondary antibody).

[0141] SARS-CoV-2 virus and neutralization assay: SARS-CoV-2 infectious virus titers were determined via a 50% tissue culture infectious dose as previously described. An early SARS-CoV-2 isolate containing the Spike D614G mutation, was used for all neutralization assays as previously described. All serum was diluted 2-fold (final dilutions of plasma range from 1 :20 to 1 :2560), infectious virus was added at a final concentration of 1 x 103TCID50 / mL to the serial dilutions, incubated for 1 h at room temperature. One hundred microliters of virus-serum mixture (containing 100 TCID50 units) was transferred to a 96- well plate of VeroE6-TMPRSS2 cells in sextuplets, and then incubated until a cytopathic effect was observed in the controls and the highest sera dilutions. The cells were fixed and stained, and the NTs were calculated as the highest serum dilutions that eliminated the cytopathic effect in 50% of the wells (NT50). A positive threshold was defined as NT > 20. An area under the curve analysis then converted the endpoint titer to a continuous variable.

[0142] Broncho-alveolar lavage. Wash fluid { IxPBS, lOOuM EDTA from 0.5M pH 8 liquid stock (Corning, Glendate, AZ), Protease inhibitors (lx PMSF from 200x stock [Cell Signaling Technology, Danvers, MA])} was prepared the day before BAL and stored at 4° C. Following mouse euthanasia and cardiac puncture, the trachea was exposed and a mouse endotracheal tube (20Gxl”, Kent Scientific) was inserted into the trachea to a point just above the carina. The guide needle was removed and a syringe was attached to the endotracheal tube, which was manually held in place during syringe attachment. Then, 0.5 ml of the wash fluid was slowly injected into the lungs with visual confirmation that the lobes were inflated and that there was no leaking. The injected liquid was aspirated with mild pressure applied to the inflated lobes, as necessary. The aspirated fluid was transferred to a 1.5 ml. vial on ice and then transferred to a new tube for centrifugation at 400g for 7 minutes at 4° C. The supernatant was stored at either 20°C or -80°C. depending on subsequent plans for use.

[0143] Flow Cytometry: Cells were recovered by thawing briefly in a water bath at 37°C and diluted slowly to 10 mL with warm complete media (RPMI, 10%FBS, lx antibiotics, 20mM HEPES, 1% sodium pyruvate, 1% non-essential amino acids, and 1% L-glutamine). Cells were spun for 7 minutes at 250g at RT and then resuspended in a smaller volume of warm media to obtain a final concentration of 5 x 105- 1 x 106viable cells / well in 200 pL complete media. The cells were then rested in 5%CO2 incubator at 37°C for 2-3 hours prior to stimulation with 1 pg SARS-CoV-2 S protein RBD (Invitrogen, Rockford, IL) at 37°C for 16 hours. During the final four hours, the cells were incubated with Brefeldin-A and costimulatory antibodies anti-CD28 and anti-CD49d (Biolegend Cat. Nos 420601, 102116, and 103629). After stimulation, 20 pL of EDTA (20 mM EDTA diluted in IxPBS at pH7.4) was added to each well to ensure cells were in suspension and then transferred to a 96-well V-bottom plate. After centrifugation at 300g for 5 minutes at RT, cells were washed with 150 pL FACS buffer (0.5% Bovine serum albumin (Sigma-Aldrich, St. Louis, MO) in lxsterile PBS) and pelleted again. Cells were stained with 100 pL / well Live / Dead (L / D) stain (1 : 1000 dilution in lx sterile PBS) for 30 minutes at RT in the dark (LIVE / DEAD Fixable Near-IR Dead Cell Stain Kit, ThermoFisher Scientific). Cells were pelleted and resuspended in 150 pL FACS buffer and washed. Following the L / D stain, 50 pL 2% Fc block (TruStain FcX, Biolegend Cat. No 101320) were added to each well and incubated in the dark for 15 minutes on ice. After centrifugation, cells were stained in the dark for 20 minutes with an anti-mouse monoclonal -antibody (mAb) cocktail (50pL per well, diluted in FACS buffer), including 1 :500 FITC conjugated anti-CD4 (Biolegend Cat. No 100405), 1 :200 PercPCy5.5 conjugated anti-CD3 (Biolegend Cat. No 100217), and 1 :200 Alexa Fluor 700 conjugated anti-CD8 (Biolegend Cat. No. 155022). After centrifuging and resuspending in 50pL Fixation buffer (Cyto-Fast Fix / Perm Buffer Set, Biolegend, Cat No 426803), cells were then incubated in the dark either at RT for 30 minutes or at 4°C overnight.

[0144] For intracellular cytokine staining, intracellular anti-mouse mAb cocktail (50pL per well, diluted in lx Cyto-Fast Perm buffer) was used to stain the cells in the dark at RT for 20 minutes. The cocktail includes 1 :500 PECy7-conjugated anti-TNFa (Biolegend Cat. No. 506323) and 1 : 100 APC-conjugated anti-LFN-y (Biolegend Cat. No. 505809). lOOpL Cyto- Fast Perm buffer was added to each well for centrifugation at 400g at RT for 5 minutes.Cells were then washed and resuspended with 150 pL FACS buffer and read on the Attune™ NxT flow cytometer (Thermo Fisher Scientific, Waltham, MA). Flow data were analyzed using Flow Jo software (FlowJo 10.8.1, LLC Ashland, OR).

[0145] Statistics: Anti-RBD IgG titers were logio-transformed. Oneway analysis of variance with Tukey correction was used for comparisons of multiple groups following nasal immunization and multiple unpaired t-tests and area under the curve analyses were used to test for significance of differences in the long-term antibody studies. For all tests, p < 0.05 was considered to be significant. Prism Graphpad 9 (San Diego, CA) was used for all statistical analyses and figure generation.

[0146] Results

[0147] Effect of fusion of vaccine antigen to MIP-3a on maintaining sustained antibody concentrations. To evaluate maintenance of antibody concentrations following vaccination, 6-8 week-old BALB / c mice were vaccinated intramuscularly three times at two-week intervals with saline, or with 10 pg of plasmid DNA encoding either codon-optimized MIP- 3a-RBD or codon-optimized RBD using the electroporation procedure described in Methods. One day following plasmid inoculation, 50 pg of the adjuvant CpG was injected into the immunization site. Blood for serum IgG or neutralizing antibody concentrations wasobtained before each vaccination, two weeks after the final vaccination, at monthly intervals through six months post-vaccination and at two monthly intervals thereafter through 12 months (Figure 2a). Antibody titers at all time points evaluated were greater in recipients of the fusion vaccine compared to recipients of vaccine encoding only RBD (mean difference = 2.77+ 0.9 fold), with the difference assuming significance at the later time points post vaccination (p=0.05 at 10 months, p=0.005 at 12 months). The area under the curve analysis of the antibody concentrations over time for the two vaccination protocols also showed nonoverlapping 95% confidence intervals for RBD (57.64; 56.40-58.87) and for MIP-3a-RBD (62.25; 60.57-63.92). Compared to IM immunization with the RBD construct alone, IM immunization with the MIP-3a-RBD vaccine construct yielded significantly higher IgG concentrations in BAL 12 months after initiation of vaccination.

[0148] Neutralization results

[0149] Impact of intranasal vaccination route and use of the fusion vaccine construct on recruitment of T cells to the lungs

[0150] Because of the potential role of T-cell responses in attenuating the severity of SARS-CoV-2 disease, we examined the impact of route of administration, as well as the role of MIP-3a fusion, in eliciting lung T-cell responses, as well as serum antibody responses. These studies evaluated immunogenicity within a shorter time frame and did not analyze duration of the observed immune responses (Figure 4a). The intranasal vaccination regimen employed was identical to that successfully employed in our previously described TB vaccine formulation, in which higher DNA plasmid doses were used to compensate for the lack of electroporation. Mice were immunized on four occasions at three-week intervals and received either IN immunization with 200 pg of vaccine (100 pg in each nostril in 50 pl PBS) or IM immunization with 50 pg of vaccine administered with electroporation and use of the CpG adjuvant, as described. For the first three IN immunizations, plasmid DNA vaccine was administered without adjuvant or electroporation. Because interim antibody analyses indicated no serum antibody response after three IN immunizations, a fourth round of immunizations was undertaken, using the CpG adjuvant (25 pg in 50 pl PBS in each nostril) for the IN, as well as the IM vaccination. Addition of CpG to the IN vaccine failed to elicit a significant antibody response (Figure 4b). As indicated (Figure 4a), despite receipt of four immunizations, serum IgG concentrations remained significantly below those observed with a temporally identical IM immunization protocol. The MIP-3a fusion vaccine offered no advantage in eliciting systemic antibody responses following intranasal immunization.Similarly, IN immunization failed to elicit serum IgA antibody responses (data not shown).

[0151] The inclusion of MIP-3a in the vaccine formulation did, however, have a highly significant impact on the ability to recruit antigen specific T cells to the lung. At 12 weeks after initial vaccine administration (six weeks after final vaccination), recipients of the MIP- 3a fusion vaccine had an approximately four-fold increase in the number of CD8+T cells in the lungs compared to recipients of saline, RBD only vaccine, or, importantly, IM immunization with the MIP-3a-fusion vaccine followed by electroporation and use of the CpG adjuvant (Figure 5). In fact, IM immunization or IN immunization with the RBD alone construct did not elicit T-cell responses in the lung that exceeded that of control mice receiving only PBS.

[0152] To examine antigen-specific T-cell responses elicited in the lungs and spleen by IM vs. IN immunization, we evaluated by flow cytometry IFN-y expression by T cells recovered at the 12-week time point following ex vivo stimulation with RBD (Figure 6). In a pattern similar to that observed with T-cell recruitment to the lungs, only the MIP-3a fusion vaccine elicited a significantly increased number of RBD-responsive T cells in the lungs compared to the other experimental and control groups (Figure 6a). Conversely, no significant enhancement of IFNy-expressing T cells was observed in the spleen of either IN or IM immunized mice. A trend towards a greater percentage of IFNy-expressing T cells in the spleen of mice immunized IM with the MIP-3a-RBD fusion construct compared to the PBS control was observed, but due to considerable variation in the control group receiving PBS by the IN route, this trend did not achieve statistical significance (p=0.24).

[0153] Similar results were observed in patterns of TNFa expression by T cells in the lungs and spleen following either IN or IM immunization (Figure 7). IN immunization with MIP-3a-RBD fusion vaccine resulted in significant enhancement of the number of TNFa- expressing T cells in the lungs compared to vaccine encoding only RBD. IM immunization with the MIP-3a-RBD fusion vaccine failed to elicit a similar number of TNFa-expressing T cells in the lungs as following IN immunization, a difference that bordered on statistical significance.

[0154] Discussion

[0155] The current studies have demonstrated that, compared to a DNA vaccine encoding only the SARS-CoV-2 RBD antigen, fusion of the chemokine MIP-3a to the RBD antigen in the vaccine construct resulted in persistently higher antibody concentrations in serum over a 12-month observation period (Figures 2b). An area under the curve analysis over that time frame indicated that the difference between the antibody responses to the two vaccine constructs was significant. At two individual time points, 10 and 12 months followinginitiation of vaccination, the concentrations of antibody maintained was also significantly different between the two vaccine formulations, indicating the potential for a more extended period of protection against viral infection or severe disease provided by the fusion construct. Importantly, significantly higher antibody concentrations among recipients of the MIP-3a fusion construct were also observed in BAL at the 12-month time point.

[0156] The results also indicate that fusion of vaccine antigen to the chemokine was critical to the recruitment of T cells to the lungs following IN immunization. Upon in vitro stimulation with the RBD protein, only mice immunized IN with the MIP-3a fusion vaccine construct elicited an fFNy+ T-cell response that was significantly above control levels observed in unimmunized mice. IM immunization with the fusion vaccine construct failed to elicit that T-cell response in the lungs.

[0157] Importantly, previous studies have indicated that, when the species-appropriate MIP3a fusion product is used, no immune response is generated to the autologous MIP3a component, even while markedly elevated responses are observed to the targeted antigen. While fusing vaccine antigens to DC-targeting ligands is not novel per se, studies using that approach have typically employed ligands for receptors found only on mature DC, missing the opportunity to enhance antigen uptake in iDC and modify antigen processing during the earliest stages of the adaptive immune response.

[0158] Particularly relevant to the current work, studies by others examining IN immunization determined that MIP-3a played a unique role in eliciting immune responses at that site. MIP-3a has been demonstrated to drive DC recruitment to the nasal mucosa and further promoted the development of transepithelial dendrites in these cells. This effect resulted in enhanced antigen uptake and rapid migration of DC into the draining cervical lymph nodes.

[0159] Our studies indicate that both inclusion of MIP-3a in the vaccine construct and intranasal immunization was critical in the current model system for eliciting effector T-cell responses in the lungs. Multiple studies in the clinical setting have indicated that currently employed IM immunization regimens have not been effective in reducing nasal shedding of virus in the setting of breakthrough infections and studies in non-human primates have indicated the importance of T-cell immunity in preventing nasal shedding. It has been demonstrated in non-human primates the ability of intratracheal / intranasal immunization with a parainfluenza virus vectored pre-fusion stabilized spike protein vaccine to elicit protective T-cell and antibody responses that included prevention of viral shedding in the upper and lower airways. It has also been noted the dependence on T-cell immunity for prevention ofviral shedding. In addition, it has also been demonstrated the ability of IN immunization with an experimental polyethyleneimine adjuvanted SARS-CoV-2 spike protein to sustain systemic and BAL antibody levels, as well as lung T-cell responses. Potential disadvantages of these approaches are the reduced likelihood that a viral -vectored vaccine could be used for booster immunizations, the production issues associated with protein vaccines and the untested clinical safety and efficacy associated with polyethyleneimine used as a vaccine adjuvant.

[0160] The current example identifies novel approaches for potentially eliciting both more durable antibody-mediated protection, as well as enhancing T-cell responses within the lungs. Both of these outcomes offer the potential for more effective protection against emerging viral variants and for reduction in viral shedding by vaccinated individuals with breakthrough infections. The results also provide an experimental system in which the individual protective contributions of humoral and cell-mediated immunity can be studied. While the current vaccine was studied as an easily constructed DNA formulation, the conclusions on the role of MIP-3a on the observed responses should be applicable across formulations that avoid issues around the use of viral-vectored or protein vaccines. Our results do suggest that a dual vaccination approach for this construct, IN and IM, would be most effective in providing optimal protection, warranting further study. These studies represent a preliminary analysis of an approach that will require currently planned future studies that define the duration of IN immunization elicited T-cell mediated protection, the roles of different T-cell subsets, the efficacy of this vaccine construct in murine and nonhuman primate challenge models, and the ability of mRNA formulations of this vaccine construct to elicit similar immune responses. While DNA vaccines have historically elicited poor responses in human studies, recent clinical studies demonstrating the efficacy of a DNA- formulated SARS-CoV-2 vaccine indicate the potential utility of stable, properly formulated DNA vaccines in the clinical setting.

[0161] Some further aspects are also defined in the following clauses:

[0162] Clause 1 : A nucleic acid vaccine composition comprising a synthetic polynucleotide encoding a severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) protein, or a functional portion, fragment, or variant thereof, conjugated to a macrophage inflammatory protein-3 alpha (MIP-3a) or other chemokine that binds to a chemokine receptor 6 (CCR6), or a functional portion, fragment, or variant thereof, or to an antibody, or antigen binding portion thereof, that binds to a CCR6.

[0163] Clause 2: The nucleic acid vaccine composition of Clause 1, wherein the SARS- CoV-2 protein comprises a SARS-CoV-2 spike protein, or a functional portion, fragment, or variant thereof.

[0164] Clause 3 : The nucleic acid vaccine composition of Clause 1 or Clause 2, wherein the synthetic polynucleotide comprises DNA.

[0165] Clause 4: The nucleic acid vaccine composition of any of Clauses 1-3, wherein the synthetic polynucleotide comprises RNA.

[0166] Clause 5: The nucleic acid vaccine composition of any of Clauses 1-4, wherein the synthetic polynucleotide comprises the nucleotide sequence of SEQ ID. NOS: 1 and / or 2.

[0167] Clause 6: The nucleic acid vaccine composition of any of Clauses 1-5, wherein the synthetic polynucleotide comprises the nucleotide sequence of SEQ ID. NO: 3.

[0168] Clause 7: The nucleic acid vaccine composition of any of Clauses 1-6, wherein the MIP-3a is murine or human.

[0169] Clause 8: The nucleic acid vaccine composition of any of Clauses 1-7, wherein the synthetic polynucleotide is codon-optimized for expression in a mammalian cell.

[0170] Clause 9: The nucleic acid vaccine composition of any of Clauses 1-8, wherein the mammalian cell is a human cell.

[0171] Clause 10: A recombinant nucleic acid vector encoding the nucleic acid vaccine compositions of any of Clauses 1-9.

[0172] Clause 11 : The vector of any of Clauses 1-10, wherein the vector is a pSecTag2B plasmid or a pVaxl plasmid.

[0173] Clause 12: A pharmaceutical composition comprising the recombinant nucleic acid vector of any of Clauses 1-11.

[0174] Clause 13: The pharmaceutical composition of any of Clauses 1-12, wherein the pharmaceutical composition does not comprise an additional biologically active agent.

[0175] Clause 14: The pharmaceutical composition of any of Clauses 1-13, further comprising at least one additional biologically active agent.

[0176] Clause 15: The pharmaceutical composition of any of Clauses 1-14, wherein the pharmaceutical composition lacks a pharmaceutically acceptable carrier.

[0177] Clause 16: The pharmaceutical composition of any of Clauses 1-15, further comprising a pharmaceutically acceptable carrier.

[0178] Clause 17: The pharmaceutical composition of any of Clauses 1-16, wherein the pharmaceutically acceptable carrier comprises a lipid nanoparticle (LNP), a polymericnanoparticle, a lipidoid, a liposome, a lipoplex, a peptide carrier, a nanoparticle mimic, or a conjugate thereof.

[0179] Clause 18: A method of providing prophylaxis to, and / or treating a SARS-CoV-2 infection in, a subject in need thereof comprising administering to the subject an effective amount of the pharmaceutical composition of any of Clauses 1-17.

[0180] Clause 19: The method of any of Clauses 1-18, wherein the composition is administered to the subject prior to, concurrent with, and / or after administering at least one antiviral agent to the subject.

[0181] Clause 20: The method of any of Clauses 1-19, wherein the composition is administered as one or more boost doses after an initial administration of the composition to the subject.

[0182] Clause 21 : The method of any of Clauses 1-20, wherein the composition is administered intramuscularly and / or intranasally to the subject.

[0183] Clause 22: A vaccine composition, comprising a polypeptide that comprises a severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) protein, or a functional portion, fragment, or variant thereof, conjugated to a macrophage inflammatory protein-3 alpha (MIP-3a) or other chemokine that binds to a chemokine receptor 6 (CCR6), or a functional portion, fragment, or variant thereof, or to an antibody, or antigen binding portion thereof, that binds to a CCR6.

[0184] Clause 23 : The vaccine composition of Clause 22, wherein the polypeptide comprises a SARS-CoV-2 spike protein, or a functional portion, fragment, or variant thereof.

[0185] Clause 24: The vaccine composition of Clause 22 or Clause 23, wherein the polypeptide comprises the amino acid sequence of SEQ ID. NOS: 4 and / or 5.

[0186] Clause 25: A pharmaceutical composition comprising the vaccine composition of any of Clauses 22-24.

[0187] Clause 26: The pharmaceutical composition of any of Clauses 22-25, wherein the pharmaceutical composition does not comprise an additional biologically active agent.

[0188] Clause 27: The pharmaceutical composition of any of Clauses 22-26, further comprising at least one additional biologically active agent.

[0189] Clause 28: The pharmaceutical composition of any of Clauses 22-27, wherein the pharmaceutical composition lacks a pharmaceutically acceptable carrier.

[0190] Clause 29: The pharmaceutical composition of any of Clauses 22-28, further comprising a pharmaceutically acceptable carrier.

[0191] Clause 30: A method of providing prophylaxis to, and / or treating a SARS-CoV-2 infection in a subject in need thereof comprising administering to the subject an effective amount of the composition of any of Clauses 22-29.

[0192] Clause 31 : The method of any of Clauses 22-30, wherein the composition is administered to the subject prior to, concurrent with, and / or after administering at least one antiviral agent to the subject.

[0193] Clause 32: The method of any of Clauses 22-31, wherein the composition is administered as one or more boost doses after an initial administration of the composition to the subject.

[0194] Clause 33: The method of any of Clauses 22-32, wherein the composition is administered intramuscularly and / or intranasally to the subject.

[0195] Clause 34: A method of providing prophylaxis to, and / or treating a respiratory viral infection in, a subject in need thereof comprising intranasally administering to the subject an effective amount of a pharmaceutical composition that comprises a synthetic polynucleotide encoding a respiratory viral protein, or a functional portion, fragment, or variant thereof, conjugated to a macrophage inflammatory protein-3 alpha (MIP-3a) or other chemokine that binds to a chemokine receptor 6 (CCR6), or a functional portion, fragment, or variant thereof, or to an antibody, or antigen binding portion thereof, that binds to a CCR6.

[0196] Clause 35: The method of Clause 34, wherein the pharmaceutical composition lacks a pharmaceutically acceptable carrier.

[0197] Clause 36: The method of Clause 34 or Clause 35, further comprising a pharmaceutically acceptable carrier.

[0198] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0199] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value isincorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0200] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

Claims:

1. A nucleic acid vaccine composition comprising a synthetic polynucleotide encoding a severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) protein, or a functional portion, fragment, or variant thereof, conjugated to a macrophage inflammatory protein-3 alpha (MIP-3a) or other chemokine that binds to a chemokine receptor 6 (CCR6), or a functional portion, fragment, or variant thereof, or to an antibody, or antigen binding portion thereof, that binds to a CCR6.

2. The nucleic acid vaccine composition of claim 1, wherein the SARS-CoV-2 protein comprises a SARS-CoV-2 spike protein, or a functional portion, fragment, or variant thereof.

3. The nucleic acid vaccine composition of claim 1, wherein the synthetic polynucleotide comprises DNA.

4. The nucleic acid vaccine composition of claim 1, wherein the synthetic polynucleotide comprises RNA.

5. The nucleic acid vaccine composition of claim 1, wherein the synthetic polynucleotide comprises the nucleotide sequence of SEQ ID. NOS: 1 and / or 2.

6. The nucleic acid vaccine composition of claim 1, wherein the synthetic polynucleotide comprises the nucleotide sequence of SEQ ID. NO: 3.

7. The nucleic acid vaccine composition of claim 1, wherein the MIP-3a is murine or human.

8. The nucleic acid vaccine composition of claim 1, wherein the synthetic polynucleotide is codon-optimized for expression in a mammalian cell.

9. The nucleic acid vaccine composition of claim 8, wherein the mammalian cell is a human cell.

10. A recombinant nucleic acid vector encoding the nucleic acid vaccine compositions of claim 1.

11. The vector of claim 10, wherein the vector is a pSecTag2B plasmid or a pVaxl plasmid.

12. A pharmaceutical composition comprising the recombinant nucleic acid vector of claim 10.

13. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition does not comprise an additional biologically active agent.

14. The pharmaceutical composition of claim 12, further comprising at least one additional biologically active agent.

15. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition lacks a pharmaceutically acceptable carrier.

16. The pharmaceutical composition of claim 12, further comprising a pharmaceutically acceptable carrier.

17. The pharmaceutical composition of claim 16, wherein the pharmaceutically acceptable carrier comprises a lipid nanoparticle (LNP), a polymeric nanoparticle, a lipidoid, a liposome, a lipoplex, a peptide carrier, a nanoparticle mimic, or a conjugate thereof.

18. A method of providing prophylaxis to, and / or treating a SARS-CoV-2 infection in, a subject in need thereof comprising administering to the subject an effective amount of the pharmaceutical composition of claim 12.

19. The method of claim 18, wherein the composition is administered to the subject prior to, concurrent with, and / or after administering at least one antiviral agent to the subject.

20. The method of claim 18, wherein the composition is administered as one or more boost doses after an initial administration of the composition to the subject.

21. The method of claim 18, wherein the composition is administered intramuscularly and / or intranasally to the subject.

22. A vaccine composition, comprising a polypeptide that comprises a severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) protein, or a functional portion, fragment, or variant thereof, conjugated to a macrophage inflammatory protein-3 alpha (MIP- 3a) or other chemokine that binds to a chemokine receptor 6 (CCR6), or a functional portion, fragment, or variant thereof, or to an antibody, or antigen binding portion thereof, that binds to a CCR6.

23. The vaccine composition of claim 22, wherein the polypeptide comprises a SARS- CoV-2 spike protein, or a functional portion, fragment, or variant thereof.

24. The vaccine composition of claim 22, wherein the polypeptide comprises the amino acid sequence of SEQ ID. NOS: 4 and / or 5.

25. A pharmaceutical composition comprising the vaccine composition of claim 22.

26. The pharmaceutical composition of claim 25, wherein the pharmaceutical composition does not comprise an additional biologically active agent.

27. The pharmaceutical composition of claim 25, further comprising at least one additional biologically active agent.

28. The pharmaceutical composition of claim 25, wherein the pharmaceutical composition lacks a pharmaceutically acceptable carrier.

29. The pharmaceutical composition of claim 25, further comprising a pharmaceutically acceptable carrier.

30. A method of providing prophylaxis to, and / or treating a SARS-CoV-2 infection in a subject in need thereof comprising administering to the subject an effective amount of the composition of claim 22.

31. The method of claim 30, wherein the composition is administered to the subject prior to, concurrent with, and / or after administering at least one antiviral agent to the subject.

32. The method of claim 30, wherein the composition is administered as one or more boost doses after an initial administration of the composition to the subject.

33. The method of claim 30, wherein the composition is administered intramuscularly and / or intranasally to the subject.

34. A method of providing prophylaxis to, and / or treating a respiratory viral infection in, a subject in need thereof comprising intranasally administering to the subject an effective amount of a pharmaceutical composition that comprises a synthetic polynucleotide encoding a respiratory viral protein, or a functional portion, fragment, or variant thereof, conjugated to a macrophage inflammatory protein-3 alpha (MIP-3a) or other chemokine that binds to achemokine receptor 6 (CCR6), or a functional portion, fragment, or variant thereof, or to an antibody, or antigen binding portion thereof, that binds to a CCR6.

35. The method of claim 34, wherein the pharmaceutical composition lacks a pharmaceutically acceptable carrier.

36. The method of claim 34, further comprising a pharmaceutically acceptable carrier.

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