WO2025184356A1 - Vaccines and compositions against gamma herpesviruses - Google Patents

Abstract

The disclosure provides compositions, methods of treatment, and methods of making and using compositions to deliver a nucleic acid to a subject. Compositions described herein include lipid carriers, optionally including an inorganic particle, capable of admixing with nucleic acids. Methods of using these compositions as a vaccine for treatment of a gamma herpesvirus or a cancer are also provided.

Description

VACCINES AND COMPOSITIONS AGAINST GAMMA HERPESVIRUSES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63/558,943, filed February 28, 2024, and U.S. Provisional Patent Application No. 63/560,834, filed March 4, 2024, the contents of each of which is incorporated herein by reference in their entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ST .26 xml format and is hereby incorporated by reference in its entirety. Said xml copy, created on February 20, 2025, is named 201953-737601_SL.xml and is 58, 140 bytes in size.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0003] This invention was made with government support under AI147846 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0004] Gamma herpesviruses have been linked to cancer, Kaposi's sarcomas, and autoimmune conditions. Initial infection is asymptomatic, which prevents early detection of gamma herpesvirus infections. Vaccinations can provide prophylactic protection against infectious diseases, including, but not limited to, viral, bacterial, and/or parasitic diseases. Vaccination can also rapidly prevent the spread of virus responsible for causing a disease. The gamma herpesviruses can evolve resistance to vaccines and avoid detection, which is a major challenge for preventing cancers and autoimmune conditions cause by these viruses.

BRIEF SUMMARY

[0005] Provided herein are compositions, wherein the compositions comprise: a lipid earner, wherein the lipid carrier comprises: a surface comprising cationic lipids; and a hydrophobic core, wherein the hydrophobic core comprises liquid oil, wherein lipids present in the hydrophobic core are in liquid phase at 25 degrees Celsius; and at least one nucleic acid encoding for a gH-gL viral protein antigen sequence or a functional variant thereof, wherein the at least one nucleic acid is complexed to the surface of the lipid carrier. Further provided herein are compositions, wherein the gH-gL viral protein antigen sequence or the functional variant thereof are from an Epstein-Barr Virus (EBV). Further provided herein are compositions, wherein the gH-gL viral protein antigen sequence or the functional variant thereof are from a Kaposi’s sarcoma virus. Further provided herein are compositions, wherein the gH-gL viral protein antigen sequence comprises a sequence encoding for a recombinant protein, wherein the recombinant protein comprises a gH region and a gL region. The gH region can be at the N- terminus of the recombinant protein or the C-terminus of the recombinant protein. The gL region can be at the N-terminus of the recombinant protein or the C-terminus of the recombinant protein. [0006] Provided herein are compositions, wherein the compositions comprise: a lipid carrier, wherein the lipid carrier comprises: cationic lipids; surfactants; and liquid oil; and at least one nucleic acid encoding for a gH-gL viral protein antigen sequence or a functional variant thereof. Further provided herein are compositions, wherein the gH-gL viral protein antigen sequence or the functional variant thereof are from an Epstein-Barr Virus (EBV). Further provided herein are compositions, wherein the gH-gL viral protein antigen sequence or the functional variant thereof are from a Kaposi's sarcoma virus. Further provided herein are compositions, wherein the gH-gL viral protein antigen sequence comprises a sequence encoding for a recombinant protein, wherein the recombinant protein comprises a gH region and a gL region. The gH region can be at the N-terminus of the recombinant protein or the C-terminus of the recombinant protein. The gL region can be at the N-terminus of the recombinant protein or the C-terminus of the recombinant protein.

[0007] Provided herein are compositions, wherein the compositions comprise: a nucleic acid comprising: a first region encoding for an RNA-dependent RNA polymerase complex from a virus; and a second region encoding for a gH-gL viral protein antigen sequence or a functional variant thereof.

[0008] Provided herein are compositions, wherein the compositions comprise: a first nucleic acid encoding for an RNA-dependent RNA polymerase complex from a virus; and a second nucleic acid encoding for a gH-gL viral protein antigen sequence or a functional variant thereof.

[0009] Provided herein are compositions, wherein the compositions comprise: a nucleic acid comprising: a first region for an RNA-dependent RNA polymerase complex from a virus; and a second region encoding for a gH or a functional variant thereof; a self-cleaving peptide; and a gL or a functional variant thereof.

[0010] Provided herein are compositions, wherein the compositions comprise: a first nucleic acid encoding for an RNA-dependent RNA polymerase complex from a virus; and a second nucleic acid encoding for a gL or a functional variant thereof; a self-cleaving peptide; and a gH or a functional variant thereof.

[0011] Provided herein are compositions, wherein the compositions comprise: a nucleic acid comprising: a first region encoding for an RNA-dependent RNA polymerase complex from a virus; and a second region encoding for: (1) a gH-gL viral protein antigen sequence or a functional variant thereof; and (2) a viral protein antigen.

[0012] Provided herein are compositions, wherein the compositions comprise: a first nucleic acid encoding for an RNA-dependent RNA polymerase complex from a virus; and a second nucleic acid encoding for (1) a gH-gL viral protein antigen sequence or a functional variant thereof; and (2) a viral protein antigen.

[0013] Provided herein are compositions, wherein the compositions comprise: a first nucleic acid encoding for an RNA-dependent RNA polymerase complex from a virus; a second nucleic acid encoding for a gH-gL viral protein antigen sequence or a functional variant thereof; and a third nucleic acid sequence encoding for an additional viral protein antigen.

[0014] Provided herein are suspensions, wherein the suspensions comprise: a composition provided herein.

[0015] Provided herein are pharmaceutical compositions, wherein the pharmaceutical compositions comprise: a composition provided herein; and a pharmaceutically acceptable salt, excipient, or carrier.

[0016] Provided herein are methods of generating an immune response in a subject, wherein the methods comprise: administering to a subject a composition provided herein, thereby generating an immune response to the gH-gL viral protein antigen or a functional fragment thereof.

[0017] Provided herein are methods of treating an infection in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby treating the infection.

[0018] Provided herein are methods of treating cancer in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby treating the cancer.

Provided herein are kits, wherein the kits comprise: a composition provided herein, packaging, and materials therefor.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The novel features of the compositions and methods provided herein are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present compositions and methods provided herein will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the compositions and methods provided herein are utilized, and the accompanying drawings of which:

[0020] FIGURES 1A-1D show schematics and graphs of development and initial immunogenicity studies of repRNAs encoding EBV gH-gL. FIG. 1A shows a schematic of the repRNA inserts encoding gH and gL separated by a P2A peptide. FIG. IB shows a schematic of the immunization schedule in C57BL/6 mice. FIG. 1C shows a graph of gH-gL binding titers as measured in plasma by ELISA. Each dot represents an individual mouse (n = 4) at weeks 0 and 2, or pooled plasma from the same 4 mice at week 6. The lines connect the mean (or pooled plasma) across the tested timepoints. X-Axis: time (weeks), Y-Axis: reciprocal endpoint titer. FIG. ID shows a graph of neutralizing titer in B cells following treatment with gH-P2A-gL or gL-P2A-gL. The ability of plasma pooled from the 4 mice in FIG. 1C to neutralize EBV infection of B cells is reported as the reciprocal dilution to reduce infectivity by 50% (IDso). The lines connect the mean (or pooled plasma) across the tested timepoints in FIGs. 1C and ID. X-Axis: time (weeks) , Y-Axis: reciprocal endpoint titer.

[0021] FIGURES 2A-2F show graphs for dose optimization of gH-gL repRNA immunizations. FIG. 2A shows a graph of the kinetics of reciprocal gH-gL endpoint binding titers following 2 matched doses of repRNA encoded gH-gL delivered at weeks 0 and 8 as indicated by arrows, FIG. 2C shows a graph of the kinetics of half-maximal neutralizing titers against EBV infection of epithelial cells following 2 matched doses of repRNA encoded gH-gL delivered at weeks 0 and 8 as indicated. FIG. 2E shows a graph of the kinetics of half-maximal neutralizing titers against EBV infection of B cells following two matched doses of repRNA encoded gH-gL delivered at weeks 0 and 8 as indicated. X-Axis of FIG. 2A: time (weeks), Y- Axis of FIG. 2A: reciprocal endpoint titer, X- Axis of FIGs. 2C, 2E: time (weeks), Y- Axis of FIGs. 2C, 2E: reciprocal IDso. FIG. 2B shows a graph of the binding titers following 2 mismatched doses of repRNA encoded gH-gL delivered at weeks 0 and 8 as indicated. FIG. 2D shows a graph of the EBV neutralizing titers measured in epithelial cell infection assays following 2 mis-matched doses of repRNA encoded gH-gL delivered at weeks 0 and 8 as indicated. FIG. 2F shows a graph of EBV neutralizing titers measured in B cell infection assays following 2 mis-matched doses of repRNA encoded gH-gL delivered at weeks 0 and 8 as indicated. Each dot represents an individual mouse at each timepoint (n=4 per group for mirrored prime/boost and n=5 per group for mixed dose prime/boost) and lines connect the means. Arrows indicate the time of immunization. X-Axis of FIG. 2B: time (weeks). Y- Axis of FIG. 2B: reciprocal endpoint titer, X- Axis of FIGs. 2D, 2F: time (weeks), Y- Axis of FIGs. 2D, 2F: reciprocal IDso

[0022] FIGURES 3A-3J show graphs showing the relative neutralizing titers elicited from full-length repRNA encoded gH-gL compared to the gH-gL ectodomain (amino acids 1 to 679). FIG. 3A shows a graph of reciprocal gH-gL endpoint binding titers elicited by 2 matched doses of full length and gH-gL ectodomain delivered at weeks 0 and 8 as indicated by arrows, measured by ELISA as indicated. X-Axis: time (weeks); Y-Axis: reciprocal endpoint titer. FIGs. 3B-3C show graphs of EBV neutralizing titers elicited by full length and gH-gL ectodomain repRNA immunization. FIG. 3B shows epithelial cells neutralizing titers elicited by full length and gH- gL ectodomain repRNA immunization. FIG. 3C shows B cell neutralizing titers elicited by full length and gH-gL ectodomain repRNA immunization. X-Axis of FIGs. 3B-3C: time (weeks); Y-Axis: reciprocal IDso. Titers from full length gH-gL immunization replicated from FIGS. 2A- 2F are shown in FIGs. 3A-3C for comparison. Each dot represents an individual mouse at each timepoint (n=4 for full length gH-gL and n = 5 for gH-gL ectodomain) and the lines connect the means. The arrows indicate the time of immunization. FIGs. 3D-3J show immune plasma from mice vaccinated with full length and gH-gL repRNA were evaluated for their ability to compete for binding to EBV gH-gL with the indicated monoclonal antibodies at their half-maximal binding concentration by competitive ELISA. Each dot represents a technical replicate with a line connecting the mean. X-Axis of FIGs. 3D-3J: dilution ; Y-Axis: % max binding.

[0023] FIGURES 4A-4C show graphs comparing the response of 2 matched doses of repRNA encoded full-length monomeric gH-gL to gH-gL multimers delivered at weeks 0 and 8 as indicated by arrows. FIG. 4A shows a graph of reciprocal endpoint gH-gL binding titers from mice immunized with full-length gH-gL, gH-gL 4-mer, gH-gL 7-mer and gH-gL 60-mer repRNAs were measured by ELISA. FIGs. 4B-4C show that EBV neutralizing titers in the plasma from the mice in FIG. 4A FIG. 4B shows a graph measuring EBV neutralizing titers in the plasma from the mice in FIG. 4A as measured in epithelial cell infection assays. FIG. 4C shows a graph measuring EBV neutralizing titers in the plasma from the mice in FIG. 4A as measured in B cell infection assays. In FIG. 4A, titers from full length 10 ug prime and boost gH-gL immunizations at weeks 0 and 8 are replicated from FIGS. 2A-2F for comparison. Each dot represents an individual mouse at each timepoint (n=5 per group in multimer construct vaccinations, n=8 for full length gH-gL) and the lines connect the means. The arrows indicate the time of immunization. X- Axis of FIGs. 4A-4C: time (weeks). Y- Axis of FIG. 4A: endpoint titer. Y- Axis of FIGs. 4B-4C: reciprocal IDso. [0024] FIGURES 5A- 5J show schematics, plots, and graphs indicating an immune response in humanized mice after EBV challenge. FIG. 5A shows a schematic indicating that 0.5 mg IgG harvested from plasma of gH-gL protein (ectodomain) immunized mice, full-length gH-gL repRNA immunized mice, control IgG from naive animals, or PBS was delivered to humanized mice via intraperitoneal injection. 24 hr after transfer, mice were bled to ensure antibody transfer and infected with 33,000 Raji-infectious units of EBV. Mice were then bled weekly starting at week 2 post challenge to monitor for signs of infection, all mice were euthanized at week 12 or earlier if humane endpoints were met. FIG. 5B shows a plot of reciprocal endpoint titers of total IgG binding 24 hours post transfer measured by ELISA. FIG. 5C shows a plot of reciprocal endpoint titers of gH-gL binding 24 hours post transfer measured by ELISA. Y-axis of FIGs. 5B-5C: reciprocal endpoint titer. X- axis of FIGs. 5B-5C: experimental group. FIG. 5D shows a graph of survival of mice after challenge. Significant differences were determined using a log-rank Mantel-Cox test. X-Axis: time (day), Y-Axis: probability of survival. FIGs. 5E-5H show graphs indicating viral DNA in the peripheral blood of negative control mice as well as mice that received IgG from repRNA immunized, protein immunized, and control IgG groups was measured by qPCR as indicated. FIG. 51 shows a plot of the viral DNA copy number quantified in splenic DNA extracts at necropsy. Each dot represents an individual mouse, the bar represents the median copy number, and the dashed line indicates the limit of detection. FIG. 5J shows a plot of the spleen weights at necropsy, each dot represents an individual mouse, and bar represents the median weight. Significant differences in FIG. 51 and FIG. 5J were determined using Mann- Whitney tests (*p < 0.05).

[0025] FIGURES 6A-6B show plots indicating T cell response to repRNA immunization. FIG. 6A shows a plot of the T cell response to IFNy CDS in gH-gL stimulated splenocytes using an intercellular staining assay. FIG. 6B shows a plot of IFN\⁺ CD8⁺ T cell responses in gH-gL-stimulated splenocytes using an intercellular staining assay. Each dot represents an individual mouse, and the bars represent the means. Significant differences were determined by Mann Whitney U test. X-Axis of FIG. 6A and FIG. 6B: repRNA and protein; Y- Axis of FIG. 6A: % of IFNy CD8⁺ T cells, Y- axis of FIG. 6B: % of IFNy⁺ CD4⁺ cells.

[0026] FIGURE 7 shows a graph of gH-gL binding of purified IgG after two immunizations with full length gH-gL encoded by repRNA or the recombinant gH-gL ectodomain as indicated. X-Axis: pg/mL IgG; Y-Axis: A₄₅₀.

[0027] FIGURES 8A-8B show plots of cell engraftment in humanized mice (from FIGS. 5A-5J). FIG. 8A shows a plot of the frequency of human CD45⁺ engraftment in the mice assigned to the various treatment groups. FIG. 8B shows a plot of the frequency of human CD 19⁺ B cells in the mice assigned to the various treatment groups. X-Axis of FIGs. 8A-8B : uninfected control, repRNA IgG, protein IgG. control IgG; Y-Axis of FIG. 8A: % of lymphocytes; Y- Axis of FIG. 8B: % of human CD45⁺.

[0028] FIGURES 9A-9D show graphs of mouse weights after challenge. FIG. 9A shows a graph of the percent of starting weight over time for uninfected control mice treated with PBS. FIG. 9B shows a graph of the percent of the starting weight over time for mice given IgG elicited by repRNA prior to EBV challenge. FIG. 9C shows a graph of the percent of the starting weight over time for mice given IgG elicited by protein vaccination prior to challenge. FIG. 9D shows a graph of the percent of the starting weight over time for mice given control IgG prior to challenge (positive control). Each dashed line indicates threshold for humane endpoint. X-Axis of FIGs. 9A-9D: time (days post infection; Y-Axis of FIGs. 9A-9D: % starting weight.

[0029] FIGURE 10 shows spleens from individual animals collected at the time of euthanasia.

[0030] FIGURES 11A-11I show schematic representations of nanoparticle (NP) carriers. FIG. 11A shows an oil-in-water emulsion and nucleic acids. FIG. 11B shows a nanostructured lipid carrier and nucleic acids. FIG. 11C shows a lipid inorganic nanoparticle and nucleic acids. FIGs. 11D shows a nanoparticle comprising a cationic lipid membrane, a hydrophobic core, and nucleic acids. FIG. 11E shows a nanoparticle comprising a cationic lipid membrane, a hydrophobic core, inorganic nanoparticles within the membrane of the nanoparticle, and a plurality of nucleic acids. FIG. 11F shows a nanoparticle having a cationic lipid membrane, a liquid oil core (e.g, squalene), and two or more RNA or DNA molecules. FIG. 11G show's a nanoparticle having a cationic lipid membrane, inorganic particles, a liquid oil core, and two or more RNA or DNA molecules. FIG. 11H shows a nanoparticle having a cationic lipid membrane, a solid core (e.g., glyceryl trimyristate-dynasan), and two or more RNA or DNA molecules. FIG. HI shows a nanoparticle having a cationic lipid membrane (e.g.. phospholipids, PEG-lipid), a solid core (e.g., cholesterol, ionizable cationic lipid), and two or more RNA or DNA molecules. Schematics are not to scale.

[0031] FIGURES 12A-12C show' graphs of the immunogenicity of repRNAs encoding EBV gH/gL compared to recombinant gp350. FIG. 12A shows a graph of the ability’ of sera from mice immunized with repRNA encoding gH/gL, repRNA encoding gH/gL/gp42 repRNA encoding gH/gL/gp42 + repRNA encoding gp50, or recombinant gp350 + Adjuvant (comparable to Alum MPLA used in GSK gp350 Phase II trial) to bind recombinant gp350. Y-axis: Endpoint titers (dilutions). X-axis: Week (4 or 7). FIG. 12B- FIG. 12C show graphs of the neutralization of EBV in serum from mice immunized with repRNA encoding gH/gL. or recombinant gp350 + Adjuvant (from A). FIG. 12B shows a graph of epithelial cell neutralization of EBV. Y-axis: Reciprocal ID50. X-axis: Week (4 or 7). FIG. 12C shows a graph of B cell neutralization of EBV. Y-axis: Reciprocal ID50. X-axis: Week (4 or 7). Each dot represents an individual mouse (n=5), the horizontal bars represent the means, and the error bars represent the standard deviation. [0032] Various aspects now will be described more fully hereinafter. Such aspects may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey its scope to those skilled in the art.

DETAILED DESCRIPTION

[0033] Provided herein are compositions, kits, methods, and uses thereof for inducing an immune response to an infectious microorganism, wherein the infectious microorganism is a cancer-associated microorganism. As an alternative to protein-based vaccines, the compositions provided herein leverage nucleic acid-based delivery' of glycoprotein immunogens from a cancer- associated virus. An attenuated variant of an alphavirus was used to generate self-amplifying replicon RNA (repRNA) vaccines where the viral RNA replication complex is intact, but the structural genes are replaced with a gene of interest (for example, a viral glycoprotein construct). Delivery of repRNA conjugated to a lipid carrier provided herein promoted the synthesis of antigen-encoding RNA in the host cell that self-adjuvants by triggering innate immune responses and promoting antigen cross-priming which enhances humoral and cellular immune responses compared to conventional mRNA. The compositions provided here also elicit superior humoral responses to a microorganism, including the induction of vaccine specific CD8+ T cell responses and neutralizing B cells upon virus challenge; and increase antibody titers relative to untreated subjects or subjects treated with alternative compositions described herein. The compositions provided herein also limit the dissemination of RNA to the injection site which induces antigenspecific adaptive immunity while avoiding systemic inflammation.

[0034] Briefly, further described herein are (1) nucleic acids encoding for a gH-gL and viral protein antigens; (2) RNA polymerases; (3) carriers; (4) combination compositions; (5) thermally stable, dried, and lyophilized vaccines; (6) pharmaceutical compositions; (7) dosing; (8) administration; (9) therapeutic applications; and (10) kits.

Definitions

[0035] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

[0036] All references, patents and patent applications disclosed herein are incorporated byreference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document. All references disclosed herein, including patent references and non-patent references, are hereby incorporated by reference in their entirety⁷ as if each was incorporated individually. However, where a patent, patent application, or publication containing express definitions is incorporated by reference, those express definitions should be understood to apply to the incorporated patent, patent application, or publication in which they are found, and not necessarily to the text of this application, in particular the claims of this application, in which instance, the definitions provided herein are meant to supersede.

[0037] The indefinite articles “a” and "an.'’ as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” [0038] The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by⁷ the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as anon-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0039] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law .

[0040] As used herein, “optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0041] As used herein, the term “about” or “approximately” means a range of up to ± 20 % of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” is implicit and in this context means within an acceptable error range for the particular value.

[0042] The term “effective amount” or “therapeutically effective amount” refers to an amount that is sufficient to achieve or at least partially achieve the desired effect.

(1) Nucleic Acids Encoding for gH-gL, gL-gH, and Viral Protein Antigens

[0043] Provided herein are compositions, wherein the compositions comprise: a nucleic acid encoding a gH-gL viral protein antigen sequence, a gL-gH viral protein antigen sequence, or a functional fragment thereof for use in the treatment or the prevention of a viral infection, cancer, or a viral infection associated with cancer. A gL-gH viral protein antigen is a heterodimer composed of glycoproteins gL and gH that are found in viruses. In some embodiments, the compositions provided herein modulate an immune response in a subject. Provided herein are compositions comprising at least one nucleic acid. In some embodiments, the at least one nucleic acid comprises deoxyribonucleic acid (DNA). In some embodiments, the at least one nucleic acid comprises ribonucleic acid (RNA). In some embodiments, the at least one nucleic acid comprises DNA, RNA. or a combination thereof. In some embodiments, compositions provided herein comprise one or more nucleic acids. In some embodiments, compositions provided herein comprise two or more nucleic acids. In some embodiments, compositions provided herein comprise at least one DNA. In some embodiments, compositions provided herein comprise at least one RNA. In some embodiments, compositions provided herein comprise at least one DNA and at least one RNA. The nucleic acids provided herein can encode for one or more viral protein antigens provided herein. For example, a nucleic acid can encode for a first sequence encoding for a gH-gL provided herein or a gL-gH provided herein; and a second sequence encoding for an additional viral protein antigen. As another example, a composition provided herein can comprise a first nucleic acid encoding for a gH-gL provided herein or a gL-gH provided herein. The gH and gL sequences encoded by the at least one nucleic acid provided herein can be in any order or configuration on the same nucleic acid.

[0044] In some embodiments, a gH-gL viral protein antigen sequence provided herein comprises a sequence encoding for a recombinant protein, wherein the recombinant protein comprises a gH region and a gL region. The gH region can be at the N-terminus of the recombinant protein or the C-terminus of the recombinant protein. The gL region can be at the N-terminus of the recombinant protein or the C-terminus of the recombinant protein. In some embodiments, a gH-gL viral protein antigen sequence provided herein comprises a functional variant of the gH or gL. A functional variant of a gH or a gL can be any protein fragment, ortholog, homolog, mutant protein, or recombination form of the gH or the gL that produces desired function when administered to a cell, a tissue, or a subject. In some embodiments, the functional variant modulates in an immune response in a subject to a gamma herpesvirus or a viral protein antigen provided herein.

[0045] The nucleic acids provided herein may be linear or include a secondary⁷ structure (e.g., a hairpin). In some embodiments, the nucleic acid is a polynucleotide comprising modified nucleotides or bases, and/or their analogs. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of compositions provided herein. Modified nucleobases which can be incorporated into modified nucleosides and nucleotides and be present in the RNA molecules include: m5C (5 -methylcytidine), m5U (5-methyluridine), m6A (N6- methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), ml A (1 -methyladenosine); m2A (2-methyladenosine); Am (2-1-O-methyladenosine); ms2m6A (2-methylthio-N6- methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio- N6isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2- methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A (N6-glycinylcarbamoyladenosine); t6A (N6-threonyl carbamoyladenosine); ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine); m6t6A (N6-methyl-N6-threonylcarbamoyladenosine); hn6A (N6- hydroxynorv alyl carbamoyl adenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2'-O-ribosyladenosine (phosphate)); I (inosine); mil (1- methylinosine); m'lm (l,2'-O-dimethylinosine); m3C (3-methylcytidine); Cm (2T-O- methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcylidine): f5C (5-fonnylcytidine); m5Cm (5,2-O-dimethylcytidine); ac4Cm (N4acetyl2TOmethylcytidine); k2C (lysidine); mlG (1- methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2'-O- methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2'-O-dimethylguanosine); m22Gm (N2,N2,2'-O-trimethylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); yW (wybutosine): o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galtactosyl-queuosine); manQ (mannosyl-queuosine); preQo (7- cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G* (archaeosine); D (dihydrouridine); m5Um (5,2'-O-dimethyluridine); s4U (4-thiouridine); m5s2U (5-methyl-2- thiouridine); s2Um (2-thio-2'-O-methyluridine); acp3U (3-(3-amino-3-carboxypropyl)uridine); hoSU (5-hydroxyuridine); moSU (5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5-(carboxyhydroxymethyl)uridine)); mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5 -methoxy carbonyl methyluridine); mcm5Um (S-methoxycarbonylmethyl-2-O-methyluridine); mcm5s2U (5- methoxycarbonylmethyl-2-thiouridine); nm5s2U (5-aminomethyl-2-thiouridine); mnm5U (5- methyl aminomethyluridine); mnm5s2U (5-methylaminomethyl-2 -thiouridine); mnm5se2U (5- methylaminomethyl-2-selenouridine); ncm5U (5 -carbamoylmethyl uridine); ncm5Um (5- carbamoylmethyl-2'-O-methyluridine); cmnm5U (5-carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethylaminomethyl-2-L-Omethyluridine); cmnm5s2U (5- carboxymethylaminomethyl-2-thiouridine); m62A (N6,N6-dimethyladenosine); Tm (2'-O- methylinosine); m4C (N4-methylcytidine); m4Cm (N4,2-O-dimethylcytidine); hm5C (5- hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5-carboxymethyluridine); m6Am (N6,T-O-dimethyladenosine); m62Am (N6.N6.O-2-trimethyladenosine); m2'7G (N2,7- dimethylguanosine); m2'2'7G (N2,N2,7-trimethylguanosine); m3Um (3,2T-O-dimethyluridine); m5D (5-methyldihydrouridine); f5Cm (5-formyl-2'-O-methylcytidine); mlGm (l,2'-O- dimethylguanosine); m'Am (1,2-O-dimethyl adenosine) irinomethyluridine); tm5s2U (S- taurinomethyl-2-thiouridine)); imG-14 (4-demethyl guanosine); imG2 (isoguanosine); ac6A (N6-acetyladenosine), hypoxanthine, inosine. 8-oxo-adenine. 7-substituted derivatives thereof, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(Ci-C6)-alkyluracil, 5- methyluracil, 5-(C2-Ce)-alkenyluracil, 5-(C2-C'6)-alkynyluracil. 5 -(hydroxy methyl)uracil, 5- chlorouracil, 5 -fluorouracil. 5-bromouracil, 5-hydroxycytosine, 5-(Ci-C6)-alkylcytosine, 5- methylcytosine, 5-(C2-C6)-alkenylcytosine. 5-(C2-C6)-alkynylcytosine. 5 -chlorocytosine, 5- fluorocytosine, 5-bromocytosine, N²-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza- 7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine, 7-deaza-8-substituted guanine, 8- hydroxyguanine, 6-thioguanine, 8-oxoguanine. 2-aminopurine, 2-amino-6-chloropurine, 2,4- diaminopurine. 2,6-diaminopurine, 8-azapurine. substituted 7-deazapurine, 7-deaza-7- substituted purine, 7-deaza-8-substituted purine, hydrogen (a basic residue), m5C, m5U, m6A, s2U, W, or 2'-O-methyl-U. Any one or any combination of these modified nucleobases may be included in the self-repli eating RNA of the compositions provided herein. Many of these modified nucleobases and their corresponding ribonucleosides are available from commercial suppliers. If desired, the nucleic acid can contain phosphoramidite, phosphorothioate. and/or methylphosphonate linkages. The RNA sequence can be modified with respect to its codon usage, for example, to increase translation efficacy and half-life of the RNA. A poly A tail (e.g., of about 30 adenosine residues or more) may be attached to the 3' end of the RNA to increase its half-life. The 5' end of the RNA may be capped with a modified ribonucleotide with the structure m7G (5') ppp (5') N (cap 0 structure) or a derivative thereof, which can be incorporated during RNA synthesis or can be enzymatically engineered after RNA transcription (e g., by using Vaccinia Virus Capping Enzyme (VCE) consisting of mRNA triphosphatase, guanylyl- transferase and guanine-7-methyltransferase, which catalyzes the construction of N7- monomethylated cap 0 structures). Cap structure can provide stability and translational efficacy to the RNA molecule. The 5' cap of the RNA molecule may be further modified by a 2'-O- Methyltransferase which results in the generation of a cap 1 structure (m7Gppp [m2'-O] N), which may further increase translation efficacy. A cap 1 structure may also increase in vivo potency.

[0046] Compositions provided herein can comprise a plurality of nucleic acids that are present in an amount of about 5 nanograms up to about 1 milligram. In some embodiments, the plurality of nucleic acids nucleic acids provided herein are present in an amount of up to about 25, 50, 75, 100, 150, 175 ng. In some embodiments, nucleic acids provided herein are present in an amount of up to about 1 mg. In some embodiments, nucleic acids provided herein are present in an amount of about 0.05 pg, 0.1 pg, 0.2 pg, 0.5, pg 1 pg, 5 pg, 10 pg, 12.5 pg, 15 pg, 25 pg, 40 pg, 50 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg. 600 pg, 700 pg, 800 pg. 900 pg, 1 mg. In some embodiments, nucleic acids provided herein are present in an amount of 0.05 pg. 0. 1 pg. 0.2 pg, 0.5 pg, 1 pg, 5 pg, 10 pg, 12.5 pg, 15 pg, 25 pg, 40 pg, 50 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 mg. In some embodiments, nucleic acids provided herein are present in an amount of about 5 pg. about 10 pg, about 25 pg, about 50 pg, or about 100 pg. In some embodiments, nucleic acids provided herein are present in an amount of up to about 5 pg, about 10 pg, about 25 pg, about 50 pg, or 100 pg. In some embodiments, the nucleic acid is at least about 200, 250, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000. 9000, 10,000, 11,000, 12,000, 13,000. 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 nucleotides in length. In some embodiments, the nucleic acid is up to about 7000, 8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 nucleotides in length. In some embodiments, the nucleic acid is about 7500, 10,000, 15,000, or 20,000 nucleotides in length.

Gamma Herpesviruses and Viral Protein Antigens [0047] Provided herein are infectious disease antigens for recognition by hosts. In some embodiments, compositions provided herein comprise at least one nucleic acid sequence that encodes for a protein antigen that is derived from a microorganism. In some embodiments, compositions provided herein comprise at least one nucleic acid sequence that encodes for a viral protein antigen that is derived from a virus, for example, a gamma herpesvirus provided herein. In some embodiments, the antigen is a surface protein or a transmembrane protein expressed on the surface of a microbial organism. In some embodiments, the viral protein antigen is a glycoprotein B, a glycoprotein E, a glycoprotein L, a glycoprotein H, a glycoprotein M, a glycoprotein I. a glycoprotein N, a glycoprotein (gp) 42, a gp350, or a K8. 1 protein.

[0048] The compositions provided herein comprise at least one nucleic acid encoding for a protein antigen from a microorganism that is an infectious microorganism. In some embodiments, the infectious microorganism causes hyperproliferation of a population of cells in a tissue in a subject. In some embodiments, protein antigens provided herein comprise a protein from a microorganism that causes a cancer in a subject. In some embodiments, the microorganism is a herpesvirus. In some embodiments, the herpesvirus is a gamma herpesvirus. In some embodiments, the microorganism is a rhadinovirus. In some embodiments, the microorganism is an Epstein-Barr Virus (EBV), a Kaposi’s sarcoma-associated herpesvirus, a herpesvirus saimiri, a herpesvirus ateles, a murine herpesvirus 68, or a functional variant of any of the foregoing.

[0049] EBV is a ubiquitous gamma herpesvirus. While primary infection typically is asymptomatic, infection of mammalian hosts can result in infectious mononucleosis and has been found to cause cancer and autoimmune conditions such as rheumatoid arthritis and multiple sclerosis. EBV primarily infects B cells and epithelial cells and has distinct attachment and entry pathways for each cell type. For example, the viral fusion machinery for EBV and other gamma herpesviruses include the gH, gL, and gB proteins. gH and gL form a 1 : 1 heterodimeric complex that acts as a regulator of membrane fusion, relaying a triggering signal to the fusogen gB, after binding one or more host cell surface receptors.

[0050] Provided herein are compositions, wherein the compositions comprise: at least one nucleic acid that encodes for a gH-gL viral protein antigen. The compositions provided herein are for use modulating an immune response in a subject to a viral protein antigen provided herein. The gH-gL viral protein antigen can be derived from any gamma herpesvirus provided herein. In some embodiments, the gH and gL are encoded by an RNA or a DNA. In some embodiments, the gH and gL are encoded by a single self-replicating RNA or a DNA encoding the selfreplicating RNA. In some embodiments, the gH is encoded upstream of the gL (e.g., at the 5' end of the nucleic acid). In some embodiments, the gL is encoded upstream of the gH (e.g., at the 5' end of the nucleic acid). In some embodiments, the gH-gL viral protein antigen further comprises a self-cleaving protein. In some embodiments, compositions provided herein comprise a gH, a self-cleaving protein, and a gL. In some embodiments, compositions provided herein comprise a gL, a self-cleaving protein, and a gH. Exemplar} nucleic acid construct configurations are provided in FIG. 1A.

[0051] In some embodiments, nucleic acids provided herein encode for gH and gL are listed in Table 1 or a fragment thereof. In some embodiments, the nucleic acid comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence which specifically binds a viral protein antigen listed in Table 1. In some embodiments, the nucleic acid provided herein comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence similarity to an RNA sequence listed in Table 1. Percent (%) sequence identity for a given sequence relative to a reference sequence is defined as the percentage of identical residues identified after aligning the two sequences and introducing gaps if necessary, to achieve the maximum percent sequence identity. Percent identity can be calculated using alignment methods known in the art, for instance alignment of the sequences can be conducted using publicly available software such as BLAST, Align, ClustalW2. Those skilled in the art can determine the appropriate parameters for alignment, but the default parameters for BLAST are specifically contemplated. Exemplar}' nucleic acid sequences encoding for exemplar ' viral protein antigens are listed in Table 1.

Attorney Docket No.: 201953-737601

Table 1: Viral Protein Antigen Sequences.

Attorney Docket No.: 201953-737601

Attorney Docket No.: 201953-737601

[0052] In some embodiments, compositions provided herein comprise a nucleic acid sequence encoding for the amino acid sequence of any one of SEQ ID NOS: 1-6. In some embodiments, compositions provided herein comprise a nucleic acid sequence comprising any one of SEQ ID NOS: 7-12. In some embodiments, compositions provided herein comprise a DNA sequence that has complementarity to any one of SEQ ID NOS: 7-12. In some embodiments, the nucleic acid sequence comprises an RNA or DNA sequence that encodes for an antigen of SEQ ID NO: 1. SEQ ID NO: 2, SEQ ID NO: 3. SEQ ID NO: 4, or any combination thereof. In some embodiments, the nucleic acid sequence comprises an RNA or DNA sequence that encodes for a self-cleaving peptide comprising SEQ ID NO: 13. In some embodiments, the self-cleaving peptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 13. In some embodiments, the self-cleaving peptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 13. In some embodiments, the self-cleaving peptide comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 13. In some embodiments, the self-cleaving peptide comprises SEQ ID NO: 13.

(2) Self-Replicating Nucleic Acids

[0053] Provided herein are compositions comprising a self-replicating nucleic acid. The viral protein antigens provided herein or fragment thereof can be encoded as part of a selfreplicating nucleic acid construct. In some embodiments, the self-replicating nucleic acid molecule comprises at least one or more genes selected from the group consisting of viral replicases, viral proteases, viral helicases and other nonstructural viral proteins, and also comprises 5'- and 3'-end cis-active replication sequences, and an antigenic sequence encoding an antigen protein. A subgenomic promoter that directs expression of the heterologous sequence(s) can be included in the self-replicating nucleotide sequence. If desired, a heterologous sequence may be fused in frame to other coding regions in the self-replicating RNA and/or may be under the control of an internal ribosome entry site (IRES).

[0054] In some embodiments, the self-replicating nucleotide sequence is a self-replicating RNA molecule. Self-replicating RNA molecules are designed so that the self-replicating RNA molecule cannot induce production of infectious viral particles. This can be achieved, for example, by omitting one or more viral genes encoding for structural proteins that are necessary for the production of viral particles in the self-repli eating RNA. For example, when the selfreplicating RNA molecule is based on an alpha virus, such as Sindbis virus (SIN), Semliki forest virus and Venezuelan equine encephalitis virus (VEE), one or more genes encoding for viral structural proteins, such as capsid and/or envelope glycoproteins, can be omitted. If desired, self- replicating RNA molecules of the compositions provided herein can be designed to induce production of infectious viral particles that are attenuated or virulent, or to produce viral particles that are capable of a single round of subsequent infection.

[0055] A self-replicating RNA molecule can, when delivered to a vertebrate cell even without any proteins, lead to the production of multiple daughter RNAs by transcription from itself (or from an antisense copy of itself). The self-replicating RNA can be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase which then produces transcripts from the delivered RNA. Thus, the delivered RNA leads to the production of multiple daughter RNAs. These transcripts are antisense relative to the delivered RNA and may be translated themselves to provide in situ expression of encoded antigens, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the encoded antigen(s).

[0056] The self-replicating RNA molecules provided herein can contain one or more modified nucleotides and therefore have improved stability and be resistant to degradation and clearance in vivo, and other advantages. In some embodiments, self-replicating RNA molecules that contain modified nucleotides avoid or reduce stimulation of endosomal and cytoplasmic immune receptors when the self-replicating RNA is delivered into a cell. This permits selfreplication, amplification and expression of protein to occur. This also reduces safety concerns relative to self-replicating RNA that does not contain modified nucleotides, because the selfreplicating RNA that contains modified nucleotides reduce activation of the innate immune system and subsequent undesired consequences (e.g.. inflammation at injection site, irritation at injection site, pain, and the like). RNA molecules produced as a result of self-replication are recognized as foreign nucleic acids by the cytoplasmic immune receptors. Thus, self-replicating RNA molecules that contain modified nucleotides provide for efficient amplification of the RNA in a host cell and expression of viral protein antigens provided herein, as well as adjuvant effects. [0057] In some embodiments, self-replicating RNA molecules provided herein contain at least one modified nucleotide. Modified nucleotides that are not part of the 5' cap (e.g., in addition to the modification that are part of the 5" cap) can be used. Accordingly, the selfreplicating RNA molecule can contain a modified nucleotide at a single position, can contain a particular modified nucleotide (e.g, pseudouridine, N6-methyladenosine, 5 -methylcytidine, 5- methyluridine) at two or more positions, or can contain two, three, four, five, six, seven, eight, nine, ten or more modified nucleotides (e.g., each at one or more positions). Preferably, the selfreplicating RNA molecules comprise modified nucleotides that contain a modification on or in the nitrogenous base, but do not contain modified sugar or phosphate moieties. In some examples, between 0.001% and 99% or 100% of the nucleotides in a self-replicating RNA molecule are modified nucleotides. For example, 0.001%-25%, 0.01%-25%, 0. l%-25%, or 1%- 25% of the nucleotides in a self-replicating RNA molecule are modified nucleotides. In other examples, between 0.001% and 99% or 100% of a particular unmodified nucleotide in a selfreplicating RNA molecule is replaced with a modified nucleotide. For example, about 1% of the nucleotides in the self-replicating RNA molecule that contain uridine can be modified, such as by replacement of uridine with pseudouridine. In other examples, the desired amount (percentage) of two, three, or four particular nucleotides (nucleotides that contain uridine, cytidine, guanosine, or adenine) in a self-replicating RNA molecule are modified nucleotides. For example, 0.001%-25%, 0.01%-25%, 0.1%-25%, or l%-25% of a particular nucleotide in a self-replicating RNA molecule are modified nucleotides. In other examples, 0.001%-20%, 0.001%-15%, 0.001%-10%. 0.01%-20%. 0.01%-15%, 0.1%-25, 0.01%-10%, l%-20%. 1%- 15%, 1 %-l 0%, or about 5%, about 10%, about 15%, about 20% of a particular nucleotide in a self-replicating RNA molecule are modified nucleotides. In some embodiments, less than 100% of the nucleotides in a self-replicating RNA molecule are modified nucleotides. In some embodiments, less than 100% of a particular nucleotide in a self-replicating RNA molecule are modified nucleotides. In some embodiments, self-replicating RNA molecules comprise at least some unmodified nucleotides.

[0058] Self-replicating RNA molecules that comprise at least one modified nucleotide can be prepared using any suitable method. Several suitable methods are known in the art for producing RNA molecules that contain modified nucleotides. For example, a self-replicating RNA molecule that contains modified nucleotides can be prepared by transcribing (e.g, in vitro transcription) a DNA that encodes the self-replicating RNA molecule using a suitable DNA- dependent RNA polymerase, such as T7 phage RNA polymerase, SP6 phage RNA polymerase, T3 phage RNA polymerase, and the like, or mutants of these polymerases which allow efficient incorporation of modified nucleotides into RNA molecules. The transcription reaction will contain nucleotides and modified nucleotides, and other components that support the activity of the selected polymerase, such as a suitable buffer, and suitable salts. The incorporation of nucleotide analogs into a self-replicating RNA may be engineered, for example, to alter the stability of such RNA molecules, to increase resistance against RNases, to establish replication after introduction into appropriate host cells (“infectivity” of the RNA), and/or to induce or reduce innate and adaptive immune responses. Suitable synthetic methods can be used alone, or in combination with one or more other methods (e.g., recombinant DNA or RNA technology), to produce a self-replicating RNA molecule that contain one or more modified nucleotides. [0059] Nucleic acid synthesis can also be performed using suitable recombinant methods that are well-known and conventional in the art, including cloning, processing, and/or expression of polynucleotides and gene products encoded by such polynucleotides. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic polynucleotides are examples of known techniques that can be used to design and engineer polynucleotide sequences. Site-directed mutagenesis can be used to alter nucleic acids and the encoded proteins, for example, to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations and the like.

[0060] In some embodiments, nucleic acids provided herein code for an RNA polymerase. In some embodiments, nucleic acids provided herein code for a viral RNA polymerase. In some embodiments, nucleic acids provided herein code for: (1) a viral RNA polymerase; and (2) a protein or functional fragment thereof. In some embodiments, compositions provided herein comprise a first nucleic acid encoding for a viral RNA polymerase; and a second nucleic acid encoding for a protein or functional fragment thereof.

[0061] Provided herein are compositions comprising a self-replicating RNA. A selfreplicating RNA (also called a replicon) includes any genetic element, for example, a plasmid, cosmid, bacmid, phage or virus that is capable of replication largely under its own control. Selfreplication provides a system for self-amplification of the nucleic acids provided herein in mammalian cells. In some embodiments, the self-replicating RNA is single stranded. In some embodiments, the self-replicating RNA is double stranded.

[0062] Provided herein are compositions comprising a nucleic acid sequence that encodes for an RNA polymerase complex. An RNA polymerase complex provided herein can include but is not limited to: an alphavirus RNA polymerase, an Eastern equine encephalitis virus (EEEV) RNA polymerase, a Western equine encephalitis virus (WEEV) RNA polymerase, Venezuelan equine encephalitis virus (VEEV) RNA polymerase, a Chikungunya virus (CHIK.V) RNA polymerase, a Semliki Forest virus (SFV) RNA polymerase, or a Sindbis virus (SINV) RNA polymerase. In some embodiments, the RNA polymerase is a VEEV RNA polymerase. In some embodiments, the nucleic acid sequence that encodes for the RNA polymerase complex comprises at least 85% identity to the nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the nucleic acid encoding for the RNA polymerase comprises at least 90% identity to the nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the nucleic acid encoding for the RNA polymerase comprises at least 95% identity' to the nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the nucleic acid encoding for the RNA polymerase comprises at least 99% identity to the nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the nucleic acid encoding for the RNA polymerase is SEQ ID NO: 14.

[0063] In some embodiments, the amino acid sequence for VEEV RNA polymerase comprises at least 85% identity to

RELPVLDSAAFNVECFKKYACNNEYWETFKENPIRLTEENVVNYITKLKGP (SEQ ID NO: 15), TQMRELPVLDSAAFNVECFKKYACNNEYWETFKENPIRLTE (SEQ ID NO: 16), or SEQ ID NO: 17. In some embodiments, the amino acid sequence for VEEV RNA polymerase comprises at least 90% identity to SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In some embodiments, the amino acid sequence for VEEV RNA polymerase comprises at least 95% identity' SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In some embodiments, the amino acid sequence for VEEV RNA polymerase comprises at least 99% identity to SEQ ID NO: 15. SEQ ID NO: 16, or SEQ ID NO: 17. In some embodiments, the amino acid sequence for VEEV RNA polymerase comprises to SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17

[0064] Provided herein are compositions and methods comprising replicon RNA (repRNA) encoding for one or more structural proteins from a non-enveloped virus. In some embodiments, the repRNA encodes a protease. In some embodiments, the repRNA encodes the 3CD protease. In some embodiments, the structural protein and the protease are co-expressed. In further embodiments, the repRNA comprises one or more open reading frames. In some embodiments, the open reading frames are separated by an internal ribosomal entry’ site (IRES). In some embodiments, the open reading frames are separated by a ribosomal skipping peptide sequence. In some embodiments, the ribosomal skipping peptide sequence is from Thosea asigna virus (T2A). In some embodiments the T2A comprises an amino acid sequence comprises SEQ ID NO: 18. In some embodiments, a nucleic acid provided herein further comprises a sequence encoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide comprises a 2A self-cleaving peptide (P2A, SEQ ID NO: 13 or SEQ ID NO: 19). In some embodiments, a nucleic acid provided herein further comprises a sequence encoding a furin cleavage site (SEQ ID NO: 20)

(3) Carriers

[0065] Provided herein are various compositions comprising a nanoparticle (also referred to herein as carriers) or a plurality of nanoparticles. Nanoparticles are also referred to herein as carriers or abbreviated as NPs. Nanoparticles provided herein may be an organic, inorganic, or a combination of inorganic and organic materials that are less than about I micrometer (pm) in diameter. In some embodiments, nanoparticles provided herein are lipid carriers for a nucleic acid provided herein. In some embodiments, nanoparticles provided herein are used as a delivery system. In some embodiments, nucleic acids provided herein are in complex with the nanoparticle. In some embodiments, the nucleic acids provided herein are in complex with the membrane of the nanoparticle. In some embodiments, nucleic acids provided herein are in complex with the hydrophilic surface of the nanoparticle. In some embodiments, nucleic acids provided herein are within of encapsulated within the core of the nanoparticle. In some embodiments, nucleic acids provide herein are within the hydrophobic core.

[0066] Provided herein are various compositions comprising lipid carrier complexes or nanoparticle-complexes, wherein a plurality⁷ of lipid carriers or a plurality⁷ of nanoparticles interact physically, chemically, and/or covalently with a nucleic acid provided herein and/or other nanoparticles. The specific type of interaction between lipid carriers or between nanoparticles will depend upon the characteristic shapes, sizes, chemical compositions, physical properties, and physiologic properties. Nanoparticles provided herein can include but are not limited to: oil in water emulsions, nanostructured lipid carriers (NLCs), cationic nanoemulsions (CNEs), vesicular phospholipid gels (VPG), polymeric nanoparticles, cationic lipid nanoparticles, liposomes, gold nanoparticles, solid lipid nanoparticles (LNPs or SLNs), mixed phase core NLCs, ionizable lipid carriers, magnetic carriers, polyethylene glycol (PEG)- functionalized carriers, cholesterol-functionalized carriers, poly lactic acid (PLA)-functionalized earners, and polylactic-co-gly colic acid (PLGA)-functionalized lipid carriers.

[0067] Various nanoparticles and formulations of nanoparticles (i.e.. nanoemulsions) are employed. Exemplary nanoparticles are illustrated in FIGS. 11A-11I. Oil in water emulsions, as illustrated in FIGs. 11A (not to scale), are stable, immiscible fluids containing an oil droplet dispersed in water or aqueous phase. FIG. 11B (not to scale) illustrates a nanostructured lipid earner (NLCs) which can comprise a blend of solid organic lipids (e.g., trimyristin) and liquid oil (e.g., squalene). In NLCs, the solid lipid is dispersed in the liquid oil. The entire nanodroplet is dispersed in the aqueous (water) phase. In some embodiments, the nanoparticle comprises inorganic nanoparticles, as illustrated in FIG. 11C (not to scale), as solid inorganic nanoparticles (e.g, iron oxide nanoparticles) dispersed in liquid oil. The entire nanodroplet is then dispersed as a colloid in the aqueous (water) phase. FIG. 11D (not to scale), illustrates a nanostructured lipid carrier (NLCs) comprising cationic lipids, hydrophobic surfactants, hydrophilic surfactants forming a hydrophobic core. A surface of the NLC forms a complex with a plurality of nucleic acids (nucleic acid-nanoparticle complexes and). The entire nanodroplet is dispersed in the aqueous (water) phase. FIG. HE (not to scale), illustrates NLCs of FIG. HD comprising solid inorganic nanoparticles within the hydrophobic core. FIG. 11F (not to scale), illustrates a nanoparticle comprising a cationic lipid membrane (e.g., DOTAP), a liquid oil core (e.g, squalene) without an inorganic particle, and one or more nucleic acids, wherein the one or more nucleic acids are in complex with the membrane. In some embodiments, nanoparticle of FIGs. 11F further comprises iron oxide nanoparticles within the core as shown in FIG. 11G (not to scale). In some embodiments, a nanoparticle provided herein comprises a solid core comprising glyceryl trimyristate-dynasan (FIG. 11H). In some embodiments, a nanoparticle provided herein comprises a solid core comprising an ionizable cationic lipid and cholesterol (FIG. 111). In some embodiments, the nanoparticles provided herein are dispersed in an aqueous solution. Nonlimiting examples of aqueous solutions include water (e.g., sterilized, distilled, deionized, ultra- pure, RNAse-free, etc.), saline solutions (e.g, Kreb's, Ascaris, Dent’s, Tet’s saline), or 1% (w/v) dimethyl sulfoxide (DMSO) in water.

[0068] In some embodiments, the nanoparticles provided herein comprise a hydrophilic surface. In some embodiments, the hydrophilic surface comprises a cationic lipid. In some embodiments, the hydrophilic surface comprises an ionizable lipid. In some embodiments, the nanoparticle comprises a membrane. In some embodiments, the membrane comprises a cationic lipid. In some embodiments, the nanoparticles provided herein comprise a cationic lipid. Exemplary cationic lipids for inclusion in the hydrophilic surface include, without limitation: 1,2- dioleoyloxy-3 (trimethylammonium)propane (DOTAP), 3( -| N — (N',N'-dimethylaminoethane) carbamoyl] cholesterol (DC Cholesterol), dimethyldioctadecylammonium (DDA); 1,2-dimyristoyl 3-trimethylammoniumpropane(DMTAP).dipalmitoyl(C 16:0)trimethyl ammonium propane (DPTAP), distearoyltrimethylammonium propane (DSTAP), N-[l-(2,3- dioleyloxy)propyl]N,N,Ntrimethylammonium, chloride (DOTMA), N,N-dioleoyl-N,N- dimethylammonium chloride (DODAC), 1.2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), l,2-dioleoyl-3-dimethylammonium-propane (DODAP), and 1,2- dilinoleyloxy-3- dimethylaminopropane (DLinDMA),l,r-((2-(4-(2-((2-(bis(2 -hydroxy dodecyl)amino)ethyl)(2- hydroxydodecyl)amino)ethyl)piperazin-l -yl)ethyl)azanediyl)bis(dodecan-2-ol) (C 12-200), 306OH0, tetrakis(8-methylnonyl) 3,3',3'',3"'-(((methylazanediyl) bis(propane-3,l diyl))bis (azanetriyl))tetrapropionate, 9A1P9, decyl (2-(dioctylammonio)ethyl) phosphate; A2-Iso5- 2DC18, ethyl 5,5-di((Z)-heptadec-8-en-l-yl)-l-(3-(pyrrolidin-l-yl)propyl)-2,5-dihydro-lH- imidazole-2-carboxylate; ALC-0315, ((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2- hexyldecanoate); ALC-0159, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide; 0- sitosterol, (3S,8S,9S,10R.13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13- dimethyl-2,3,4.7.8.9.10,l l,12.13,14.15,16,17-tetradecahydro-IH-cyclopenta[a]phenanthren-3-ol; BAME-016B, bis(2-(dodecyldisulfanyl)ethyl) 3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6- diazahexacosyl)azanediyl)dipropionate; BHEM-Cholesterol, 2-(((((3 S,8S,9S, 1 OR, 13R, 14S, 17R)- 10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,l l,12,13,14,15,16,17- tetradecahydro-lH-cyclopenta[a]phenanthren-3-yl)oxy)carbonyl)amino)-N,N-bis(2- hydroxyethyl)-N-methylethan-l-aminium bromide; cKK-E12, 3,6-bis(4-(bis(2- hydroxydodecyl)amino)butyl)piperazine-2, 5-dione; DC-Cholesterol, 3P-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol; DLin-MC3-DMA, (6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino) butanoate; DOPE, 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine; DOSPA, 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]- N,N-dimethyl-l-propanaminium trifluoroacetate; DSPC, l,2-distearoyl-sn-glycero-3- phosphocholine; ePC, ethylphosphatidylcholine; FTT5, hexa(octan-3-yl) 9,9', 9", 9"', 9"", 9"'"- ((((benzene-l,3,5-tricarbonyl)yris(azanediyl)) tris (propane-3,1 -diyl)) tris(azanetriyl))hexanonanoate; Lipid H (SM-102), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6- (undecyloxy)hexyl)amino) octanoate; OF-Deg-Lin, (((3,6-dioxopiperazine-2,5-diyl)bis(butane- 4, 1 -diy l))bis(azanetriy l))tetrakis(ethane-2, 1 -diyl) (9Z,9'Z,9 "Z,9"'Z, 12Z, 12'Z, 12"Z, 12"'Z)-tetrakis (octadeca-9,12-dienoate); PEG2000-DMG, (R)-2,3-bis(myristoyloxy)propyl-l -(methoxy poly(ethylene glycol)2000) carbamate; TT3, or Nl,N3,N5-tris(3- (didodecylamino)propyl)benzene-l,3,5-tricarboxamide. Other examples for suitable classes of lipids include, but are not limited to, the phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylglycerol (PGs); and PEGylated lipids including PEGylated version of any of the above lipids (e.g.. DSPE-PEGs). In some embodiments, the nanoparticle provided herein comprises DOTAP.

[0069] In some embodiments, the nanoparticle provided herein comprises an oil. In some embodiments, the oil is in liquid phase. Non-limiting examples of oils that can be used include a- tocopherol. coconut oil, grapeseed oil, lauroyl polyoxylglyceride, mineral oil, monoacylglycerol, palm kernel oil, olive oil, paraffin oil, peanut oil, propolis, squalene, squalane, solanesol, soy lecithin, soybean oil, sunflower oil, a triglyceride, or vitamin E. In some embodiments, the nanoparticle provided herein comprises a triglyceride. Exemplary triglycerides include but are not limited to: capric triglycerides, caprylic triglycerides, a caprylic and capric triglycerides, triglyceride esters, and myristic acid tri glycerins. In some embodiments, the oil is in solid phase. In some embodiments, the oil comprises solanesol.

[0070] In some embodiments, the nanoparticles provided herein comprise a liquid organic material and a solid inorganic material. In some embodiments, the nanoparticle provided herein comprises an inorganic particle. In some embodiments, the inorganic particle is a solid inorganic particle. In some embodiments, the nanoparticle provided herein comprises the inorganic particle within the hydrophobic core.

[0071] In some embodiments, the nanoparticle provided herein comprises a metal. In some embodiments, the nanoparticle provided herein comprises a metal within the hydrophobic core. The metal can be without limitation, a metal salt, a metal oxide, a metal hydroxide, or a metal phosphate. In some embodiments, the nanoparticle provided herein comprises aluminum oxide (AI2O3),, aluminum oxyhydroxide, iron oxide (Fe₃O₄, Fe₂O₃, FeO, or combinations thereof), titanium dioxide, silicon dioxide (S1O2). aluminum hydroxyphosphate (Al(OH)_x(PO4)_y), calcium phosphate (Ca₃(PO ₄)₂). calcium hydroxyapatite (Ca₁₀(P0₄)₆(OH)₂), iron gluconate, or iron sulfate. The inorganic particles may be formed from one or more same or different metals (any metals including transition metal). In some embodiments, the inorganic particle is a transition metal oxide. In some embodiments, the transition metal is magnetite (Fe₃O₄), maghemite (y-Fe2O3), wü stite (FeO), or hematite (alpha (a)- Fe₂O₃). In some embodiments, the metal is aluminum hydroxide or aluminum oxyhydroxide, and a phosphate-terminated lipid or a surfactant, such as oleic acid, oleylamine, SDS, TOPO or DSPA is used to coat the inorganic solid nanoparticle before it is mixed with the liquid oil to form the hydrophobic core.

[0072] In some embodiments, the metal can comprise a paramagnetic, a superparamagnetic, a ferrimagnetic or a ferromagnetic compound. In some embodiments, the metal is a superparamagnetic iron oxide (Fe₃O₄).

[0073] In some embodiments, the nanoparticle provided herein comprises a cationic lipid, an oil, and an inorganic particle. In some embodiments, the nanoparticle provided herein comprises DOTAP; squalene and/or glyceryl trimyristate-dynasan; and iron oxide. In some embodiments, the nanoparticle provided herein further comprises a surfactant. Thus, in some embodiments, the nanoparticles provided herein comprise a cationic lipid, an oil, an inorganic particle, and a surfactant.

[0074] Surfactants are compounds that lower the surface tension between two liquids or between a liquid and a solid component of the nanoparticles provided herein. Surfactants can be hydrophobic, hydrophilic, or amphiphilic. In some embodiments, the nanoparticle provided herein comprises a hydrophobic surfactant. Exemplary hydrophobic surfactants that can be employed include but are not limited to: sorbitan monolaurate (SPAN® 20), sorbitan monopalmitate (SPAN® 40), sorbitan monostearate (SPAN® 60), sorbitan tristearate (SPAN® 65), sorbitan monooleate (SPAN® 80), and sorbitan trioleate (SPAN® 85). Suitable hydrophobic surfactants include those having a hydrophilic-lipophilic balance (HLB) value of 10 or less, for instance, 5 or less, from 1 to 5. or from 4 to 5. For instance, the hydrophobic surfactant can be a sorbitan ester having an HLB value from 1 to 5, or from 4 to 5. In some embodiments, the nanoparticle provided herein comprises a hydrophilic surfactant, also called an emulsifier.

[0075] In some embodiments, the nanoparticle provided herein comprises polysorbate. Polysorbates are oily liquids derived from ethoxylated sorbitan (a derivative of sorbitol) esterified with fatty’ acids. In some embodiments, the nanoparticle or lipid carrier provided herein comprises a hydrophilic surfactant. Exemplary hydrophilic surfactants that can be employed include but are not limited to: polysorbates such as TWEEN®, Kolliphor, Scathes, Alkest. or Canarcel; polyoxyethylene sorbitan ester (polysorbate); polysorbate 80 (polyoxyethylene sorbitan monooleate, or TWEEN® 80); polysorbate 60 (polyoxyethylene sorbitan monostearate, or TWEEN® 60); polysorbate 40 (polyoxyethylene sorbitan monopalmitate, or TWEEN® 40); and polysorbate 20 (polyoxyethylene sorbitan monolaurate, or TWEEN® 20). In one embodiment, the hydrophilic surfactant is polysorbate 80.

[0076] Nanoparticles provided herein comprises a hydrophobic core surrounded by a lipid membrane (e .g., a cationic lipid such as DOTAP). In some embodiments, the hydrophobic core comprises: one or more inorganic particles; a phosphate-terminated lipid; and a surfactant.

[0077] Inorganic solid nanoparticles described herein may be surface modified before mixing with the liquid oil. For instance, if the surface of the inorganic solid nanoparticle is hydrophilic, the inorganic solid nanoparticle may be coated with hydrophobic molecules (or surfactants) to facilitate the miscibility of the inorganic solid nanoparticle with the liquid oil in the “oil’' phase of the nanoemulsion particle. In some embodiments, the inorganic particle is coated with a capping ligand, the phosphate-terminated hpid, and/or the surfactant. In some embodiments the hydrophobic core comprises a phosphate-terminated lipid. Exemplary phosphate-terminated lipids that can be employed include but are not limited to: trioctylphosphine oxide (TOPO) or distearyl phosphatidic acid (DSPA). In some embodiments, the hydrophobic core comprises surfactant is a phosphorous-terminated surfactant, a carboxylate-terminated surfactant, a sulfate- terminated surfactant, or an amine-terminated surfactant. Typical carboxylate-terminated surfactants include oleic acid. Typical amine terminated surfactants include oleylamine. In some embodiments, the surfactant is distearyl phosphatidic acid (DSPA), oleic acid, oleylamine or sodium dodecyl sulfate (SDS). In some embodiments, the inorganic solid nanoparticle is a metal oxide such as an iron oxide, and a surfactant, such as oleic acid, oleylamine, SDS, DSPA, or TOPO, is used to coat the inorganic solid nanoparticle before it is mixed with the liquid oil to form the hydrophobic core.

[0078] In some embodiments, the hydrophobic core comprises: one or more inorganic particles containing at least one metal hydroxide or oxyhydroxide particle optionally coated with a phosphate- terminated lipid, a phosphorous-terminated surfactant, a carboxylate- terminated surfactant, a sulfate-terminated surfactant, or an amine-terminated surfactant; and a liquid oil containing naturally occurring or synthetic squalene; a cationic lipid comprising DOTAP; a hydrophobic surfactant comprising a sorbitan ester selected from the group consisting of: sorbitan monostearate, sorbitan monooleate, and sorbitan trioleate; and a hydrophilic surfactant comprising a polysorbate.

[0079] In some embodiments, the hydrophobic core comprises: one or more inorganic nanoparticles containing aluminum hydroxide or aluminum oxyhydroxide nanoparticles optionally coated with TOPO, and a liquid oil containing naturally occurring or synthetic squalene; the cationic lipid DOTAP; a hydrophobic surfactant comprising sorbitan monostearate; and a hydrophilic surfactant comprising polysorbate 80.

[0080] In some embodiments, the hydrophobic core consists of: one or more inorganic particles containing at least one metal hydroxide or oxyhydroxide particle optionally coated with a phosphate- terminated lipid, a phosphorous-terminated surfactant, a carboxylate- terminated surfactant, a sulfate-terminated surfactant, or an amine-terminated surfactant; and a liquid oil containing naturally occurring or synthetic squalene; a cationic lipid comprising DOTAP; a hydrophobic surfactant comprising a sorbitan ester selected from the group consisting of: sorbitan monostearate, sorbitan monooleate, and sorbitan trioleate; and a hydrophilic surfactant comprising a polysorbate. In some embodiments, the hydrophobic core consists of: one or more inorganic nanoparticles containing aluminum hydroxide or aluminum oxyhydroxide nanoparticles optionally coated with TOPO, and a liquid oil containing naturally occurring or synthetic squalene; the cationic lipid DOTAP; a hydrophobic surfactant comprising sorbitan monostearate; and a hydrophilic surfactant comprising polysorbate 80. In some embodiments, the nanoparticle provided herein can comprise from about 0.2% to about 40% w/v squalene, from about 0.001% to about 10% w/v iron oxide nanoparticles, from about 0.2% to about 10 % w/v DOTAP, from about 0.25% to about 5% w/v sorbitan monostearate, and from about 0.5% to about 10% w/v polysorbate 80. In some embodiments the nanoparticle provided herein from about 2% to about 6% w/v squalene, from about 0.01% to about 1% w/v iron oxide nanoparticles, from about 0.2% to about 1 % w/v DOTAP, from about 0.25% to about 1% w/v sorbitan monostearate, and from about 0.5%) to about 5% w/v polysorbate 80. In some embodiments, the nanoparticle provided herein can comprise from about 0.2% to about 40% w/v squalene, from about 0.001% to about 10% w/v aluminum hydroxide or aluminum oxyhydroxide nanoparticles, from about 0.2% to about 10 % w/v DOTAP. from about 0.25% to about 5% w/v sorbitan monostearate, and from about 0.5% to about 10% w/v polysorbate 80. In some embodiments, the nanoparticle provided herein can comprise from about 2% to about 6% w/v squalene, from about 0.01% to about 1% w/v aluminum hydroxide or aluminum oxyhydroxide nanoparticles, from about 0.2% to about 1 % w/v DOTAP, from about 0.25% to about 1% w/v sorbitan monostearate, and from about 0.5%) to about 5% w/v polysorbate 80.

[0081] In some embodiments, a composition provided herein comprises at least one nanoparticle formulation as described in Table 2. In some embodiments, a composition provided herein comprises any one of NP-1 to NP-31. In some embodiments, a composition provided herein comprises any one of NP-1 to NP-42.

Table 2. Nanoparticle Formulations.

[0082] In some embodiments, nanoparticles provided herein comprise: sorbitan monostearate (e.g., SPAN® 60), polysorbate 80 (e.g., TWEEN® 80), DOTAP, squalene, and no solid particles. In some embodiments, nanoparticles provided herein comprise: sorbitan monostearate (e.g., SPAN® 60), polysorbate 80 (e.g., TWEEN® 80), DOTAP. squalene, and iron oxide particles. In some embodiments, nanoparticles provided herein comprise an immune stimulant. In some embodiments, the immune stimulant is squalene. In some embodiments, the immune stimulant is a medium chain triglyceride. In some embodiments, the immune stimulant is Miglyol 810 or Miglyol 812. Miglyol 810 is a triglyceride ester of saturated caprylic and capric fatty acids and glycerol. Miglyol 812 is a triglyceride ester of saturated coconut/palm kernel oil derived caprylic and capric fatty acids and plant derived glycerol. In some embodiments, the immune stimulant can decrease the total amount of protein produced, but can increase the immune response to a composition provided herein (<?.g., when delivered as a vaccine). In some embodiments, the immune stimulant can increase the total amount of protein produced, but can decrease the immune response to a composition provided herein.

[0083] Nanoparticles provided herein can be of various average diameters in size. In some embodiments, nanoparticles provided herein have an average diameter (z- average hydrodynamic diameter, measured by dynamic light scattering) ranging from about 20 nanometers (nm) to about 200 nm. In some embodiments, the z-average diameter of the nanoparticle ranges from about 20 nm to about 150 nm. from about 20 nm to about 100 nm, from about 20 nm to about 80 nm, from about 20 nm to about 60 nm. In some embodiments, the z-average diameter of the nanoparticle) ranges from about 40 nm to about 200 nm, from about 40 nm to about 150 nm, from about 40 nm to about 100 nm. from about 40 nm to about 90 nm, from about 40 nm to about 80 nm, or from about 40 nm to about 60 nm. In one embodiment, the z- average diameter of the nanoparticle is from about 40 nm to about 80 nm. In some embodiments, the z-average diameter of the nanoparticle is from about 40 nm to about 60 nm. In some embodiments, the nanoparticle is up to 100 nm in diameter. In some embodiments, the nanoparticle is 50 to 70 nm in diameter. In some embodiments, the nanoparticle is 40 to 80 nm in diameter. In some embodiments, the inorganic particle (e.g., iron oxide) within the hydrophobic core of the nanoparticle can be an average diameter (number weighted average diameter) ranging from about 3 nm to about 50 nm. For instance, the inorganic particle can have an average diameter of about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, or about 50 nm.

[0084] Nanoparticles provided herein may be characterized by the polydispersity index (PDI), which is an indication of their quality with respect to size distribution. In some embodiments, average poly dispersity index (PDI) of the nanoparticles provided herein ranges from about 0. 1 to about 0.5. In some embodiments, the average PDI of the nanoparticles can range from about 0.2 to about 0.5, from about 0. 1 to about 0.4, from about 0.2 to about 0.4, from about 0.2 to about 0.3, or from about 0.1 to about 0.3.

[0085] In some embodiments, the nanoparticles provided herein comprise an oil-to- surfactant molar ratio ranging from about 0. 1 : 1 to about 20: 1, from about 0.5: 1 to about 12: 1, from about 0.5: 1 to about 9: 1, from about 0.5: 1 to about 5: 1, from about 0.5: 1 to about 3: 1. or from about 0.5: 1 to about 1 : 1. In some embodiments, the nanoparticles provided herein comprise a hydrophilic surfactant-to-lipid ratio ranging from about 0.1: 1 to about 2: 1, from about 0.2: 1 to about 1.5: 1, from about 0.3: 1 to about 1: 1, from about 0.5: 1 to about 1 : 1, or from about 0.6: 1 to about 1: 1. In some embodiments, the nanoparticles provided herein comprise a hydrophobic surfactant-to-lipid ratio ranging from about 0.1 : 1 to about 5:1, from about 0.2: 1 to about 3: 1, from about 0.3: 1 to about 2:1, from about 0.5: 1 to about 2:1, or from about 1 :1 to about 2: 1. In some embodiments, the nanoparticles provided herein comprise from about 0.2% to about 40% w/v liquid oil, from about 0.001% to about 10% w/v inorganic solid nanoparticle, from about 0.2% to about 10% w/v lipid, from about 0.25% to about 5% w/v hydrophobic surfactant, and from about 0.5% to about 10% w/v hydrophilic surfactant. In some embodiments, the lipid comprises a cationic lipid, and the oil comprises squalene, and/or the hydrophobic surfactant comprises sorbitan ester. In some embodiments, nanoparticles provided herein comprise a ratio of the esters that yields a hydrophilic-lipophilic balance between 8 and 1 1. In some embodiments, nucleic acids provided herein are incorporated, associated with, or complexed a lipid carrier provided herein to form a lipid carrier-nucleic acid complex. In some embodiments, the lipid carrier-nucleic acid complex is formed via non-covalent interactions or via reversible covalent interactions.

(4) Combination Compositions

[0086] Provided herein are compositions comprising a nanoparticle described herein and at least one nucleic acid encoding for a gH-gL. In some embodiments, the compositions comprise an RNA polymerase complex region. Further provided herein is a nanoemulsion comprising a plurality of nanoparticles provided herein. In some embodiments, the nucleic acid further encodes for a self-replicating RNA polymerase. In some embodiments, the nucleic acid further encodes for a self-replicating RNA-dependent RNA polymerase. In some embodiments, the nucleic acid encoding for the self-replicating RNA polymerase is on the same nucleic acid strand as the nucleic acid sequence encoding for the protein (e.g, cis). In some embodiments, the nucleic acid encoding the self-replicating RNA polymerase is on a different nucleic acid strand as the nucleic acid sequence encoding for the protein (e.g., trans). In some embodiments, the nucleic acid encoding the self-replicating RNA polymerase is a DNA molecule. In some embodiments, nucleic acid sequences encoding for a viral protein antigen provided herein are DNA or RNA molecules. In some embodiments, viral protein antigens provided herein are encoded by DNA. Nanoparticles for inclusion include, without limitation, any one of NP-1 to NP-30, or any one of NP-1 to NP-31. In some embodiments, the nanoparticle comprises NP-1. In some embodiments, the nanoparticle comprises NP-2. In some embodiments, the nanoparticle comprises NP-3. In some embodiments, the nanoparticle comprises NP-4. In some embodiments, the nanoparticle comprises NP-5. In some embodiments, the nanoparticle comprises NP-6. In some embodiments, the nanoparticle comprises NP-7. In some embodiments, the nanoparticle comprises NP-8. In some embodiments, the nanoparticle comprises NP-9. In some embodiments, the nanoparticle comprises NP-10. In some embodiments, the nanoparticle comprises NP-11. In some embodiments, the nanoparticle comprises NP-12. In some embodiments, the nanoparticle comprises NP-13. In some embodiments, the nanoparticle comprises NP-14. In some embodiments, the nanoparticle comprises NP-15. In some embodiments, the nanoparticle comprises NP-16. In some embodiments, the nanoparticle comprises NP-17. In some embodiments, the nanoparticle comprises NP-18. In some embodiments, the nanoparticle comprises NP-18. In some embodiments, the nanoparticle comprises NP-19. In some embodiments, the nanoparticle comprises NP-20. In some embodiments, the nanoparticle comprises NP-21. In some embodiments, the nanoparticle comprises NP-22. In some embodiments, the nanoparticle comprises NP-23 In some embodiments, the nanoparticle comprises NP-24. In some embodiments, the nanoparticle comprises NP-25. In some embodiments, the nanoparticle comprises NP-26. In some embodiments, the nanoparticle comprises NP-27. In some embodiments, the nanoparticle comprises NP-28. In some embodiments, the nanoparticle comprises NP-28. In some embodiments, the nanoparticle comprises NP-29. In some embodiments, the nanoparticle comprises NP-30. In some embodiments, the nanoparticle comprises NP-31. In some embodiments, the nanoparticle comprises any of NP-1 to NP-31 and a cryoprotectant. In some embodiments, the cryoprotectant is a sugar described herein. In some embodiments, nucleic acids for inclusion include, without limitation, comprise a region comprising any one of, or a plurality of, SEQ ID NOS: 7-12, or encoding an amino acid sequence of any one of SEQ ID NOS: 1-6. In some embodiments, nucleic acids for inclusion include, without limitation, comprise a region complementary to any one SEQ ID NOS: 7-12, or encoding an amino acid sequence of any one of SEQ ID NOS: 1-6. In some instances, the nucleic acids further comprise a region encoding for an RNA polymerase, e.g., a region comprising a sequence of SEQ ID NO: 13.

[0087] In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 7 or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 8 or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 9 or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 10 or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 11 or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 12 or a functional fragment thereof. In some embodiments, a nucleic acid described herein comprises a sequence encoded for a viral protein antigen described here and for an RNA-dependent RNA polymerase. In some embodiments, the RNA-dependent RNA polymerase is a VEEV RNA polymerase. In some embodiments, the two nucleic acid coding elements are present in separate nucleic acids. In some embodiments, the two nucleic acid coding elements are present on the same nucleic acid. [0088] In some embodiments, compositions provided herein comprise NP-30 and a nucleic acid comprising a sequence that is at least 85% identical to SEQ ID NO: 7 or a functional fragment thereof. In some embodiments, compositions provided herein comprise NP-30 and a nucleic acid comprising a sequence that is at least 85% identical to SEQ ID NO: 8 or a functional fragment thereof. In some embodiments, compositions provided herein comprise NP-30 and a nucleic acid comprising a sequence that is at least 85% identical to SEQ ID NO: 9 or a functional fragment thereof. In some embodiments, compositions provided herein comprise NP-30 and a nucleic acid comprising a sequence that is at least 85% identical to SEQ ID NO: 10 or a functional fragment thereof. In some embodiments, compositions provided herein comprise NP- 30 and a nucleic acid comprising a sequence that is at least 85% identical to SEQ ID NO: 11 or a functional fragment thereof. In some embodiments, compositions provided herein comprise NP-30 and a nucleic acid comprising a sequence that is at least 85% identical to SEQ ID NO: 12 or a functional fragment thereof.

[0089] In some embodiments, compositions provided herein comprise NP-1 and a nucleic acid comprising a sequence that is at least 85% identical to SEQ ID NO: 7 or a functional fragment thereof. In some embodiments, compositions provided herein comprise NP-1 and a nucleic acid comprising a sequence that is at least 85% identical to SEQ ID NO: 8 or a functional fragment thereof. In some embodiments, compositions provided herein comprise NP-30 and a nucleic acid comprising a sequence that is at least 85% identical to SEQ ID NO: 9 or a functional fragment thereof. In some embodiments, compositions provided herein comprise NP-1 and a nucleic acid comprising a sequence that is at least 85% identical to SEQ ID NO: 10 or a functional fragment thereof. In some embodiments, compositions provided herein comprise NP- 1 and a nucleic acid comprising a sequence that is at least 85% identical to SEQ ID NO: 11 or a functional fragment thereof. In some embodiments, compositions provided herein comprise NP- 1 and a nucleic acid comprising a sequence that is at least 85% identical to SEQ ID NO: 12 or a functional fragment thereof. [0090] Compositions provided herein can be characterized by an nitrogen: phosphate (N:P) molar ratio. The N:P ratio is determined by the amount of cationic lipid in the nanoparticle which contain nitrogen and the amount of nucleic acid used in the composition which contain negatively charged phosphates. A molar ratio of the lipid carrier to the nucleic acid can be chosen to increase the delivery efficiency of the nucleic acid, increase the ability of the nucleic acid-carrying nanoemulsion composition to elicit an immune response to the viral protein antigen, increase the ability of the nucleic acid-carrying nanoemulsion composition to elicit the production of antibody titers to the viral protein antigen in a subject. In some embodiments, compositions provided herein have a molar ratio of the lipid carrier to the nucleic acid can be characterized by the nitrogen-to-phosphate molar ratio, which can range from about 0.01: 1 to about 1000: 1, for instance, from about 0.2: 1 to about 500: 1, from about 0.5: 1 to about 150: 1. from about 1 : 1 to about 150: 1, from about 1 : 1 to about 125: 1, from about 1 : 1 to about 100: 1. from about 1 : 1 to about 50: 1 , from about 1 : 1 to about 50: 1, from about 5 : 1 to about 50:1 , from about 5 : 1 to about 25: 1, or from about 10: 1 to about 20: 1 In some embodiments, the molar ratio of the lipid carrier to the nucleic acid, characterized by the nitrogen-to-phosphate (N:P) molar ratio, ranges from about 1: 1 to about 150: 1. from about 5: 1 to about 25: 1, or from about 10: 1 to about 20: 1. In some embodiments, the N:P molar ratio of the nanoemulsion composition is about 15: 1. In some embodiments, the nanoparticle comprises a nucleic acid provided herein covalently attached to the membrane.

[0091] Compositions provided herein can be characterized by an oil-to-surfactant molar ratio. In some embodiments, the oil-to-surfactant ratio is the molar ratio of squalene: cationic lipid, hydrophobic surfactant, and hydrophilic surfactant. In some embodiments, the oil-to- surfactant ratio is the molar ratio of squalene: DOTAP, hydrophobic surfactant, and hydrophilic surfactant. In some embodiments, the oil-to-surfactant ratio is the molar ratio of squalene: DOTAP, sorbitan monostearate, and polysorbate 80. In some embodiments, the oil-to surfactant molar ratio ranges from about 0.1: 1 to about 20: 1, from about 0.5: 1 to about 12: 1, from about 0.5: 1 to about 9: 1, from about 0.5: 1 to about 5:1, from about 0.5: 1 to about 3:1, or from about 0.5: 1 to aboutl: 1. In some embodiments, the oil-to-surfactant molar ratio is at least about 0.1 : 1, at least about 0.2: 1, at least about 0.3: 1. at least about 0.4: 1, at least about 0.5: 1, at least about 0.6: 1, at least about 0.7: 1. In some embodiments, the oil-to surfactant molar ratio is at least about 0.4: 1 up to 1: 1.

[0092] Compositions provided herein can be characterized by hydrophilic surfactant-to- cationic lipid ratio. In some embodiments, the hydrophilic surfactant-to-cationic lipid ratio ranges from about 0.1: 1 to about 2: 1, from about 0.2: 1 to about 1.5: 1, from about 0.3: 1 to about 1: 1, from about 0.5:1 to about 1 : 1, or from about 0.6:1 to about 1:1. Compositions provided herein can be characterized by hydrophobic surfactant-to-lipid (e.g., cationic lipid) ratio. In some embodiments, the hydrophobic surfactant-to-lipid ratio ranges from about 0.1 : 1 to about 5: 1, from about 0.2: 1 to about 3: 1, from about 0.3: 1 to about 2:1, from about 0.5: 1 to about 2: 1, or from about 1 : 1 to about 2: 1. In some embodiments, the cationic lipid is DOTAP.

[0093] Further provided herein is a dried composition comprising a sorbitan fatty acid ester, an ethoxylated sorbitan ester, a cationic lipid, an immune stimulant, and an RNA. Further provided herein are dried compositions, wherein the dried composition comprises sorbitan monostearate (e.g., SPAN® 60), polysorbate 80 (e.g., TWEEN® 80), DOTAP, an immune stimulant, and an RNA.

(5) Thermally Stable, Dried, and Lyophilized Infectious Diseases Vaccines

[0094] Provided herein are dried or lyophilized compositions and vaccines. Further provided herein are pharmaceutical compositions comprising a dried or lyophilized composition provided herein that is reconstituted in a suitable diluent and a pharmaceutically acceptable earner. In some embodiments, the diluent is aqueous. In some embodiments, the diluent is water. [0095] In some embodiments, a lyophilized composition is generated by a low temperature dehydration process involving the freezing of the composition, followed by a lowering of pressure, and removal of ice by sublimation. In some cases, lyophilisation also involves the removal of bound water molecules through a desorption process. In some embodiments, compositions and vaccines provided herein are spray dried. Spray drying is a process by which a solution is fed through an atomizer to create a spray, which is thereafter exposed to a heated gas stream to promote rapid evaporation. When sufficient liquid mass has evaporated, the remaining solid material in the droplet forms particles which are then separated from the gas stream (e.g., using a filter or a cyclone). Drying aids in the storage of the compositions and vaccines provided herein at higher temperatures (e.g, greater than 4 degrees Celsius) as compared to the sub-zero temperatures needed for the storage of existing mRNA vaccines. In some embodiments, dried compositions and lyophilized compositions provided herein comprise (a) a lipid carrier, wherein the lipid carrier is a nanoemulsion comprising: (i) a hydrophobic core; (ii) one or more inorganic nanoparticles; (iii) and one or more lipids; (b) one or more nucleic acids; and (c) at least one cryoprotectant. In some embodiments, the cryoprotectant is selected from the group consisting of: sucrose, maltose, trehalose, mannitol, glucose, and any combinations thereof. Additional examples of cryoprotectants include but are not limited to: dimethyl sulfoxide (DMSO). glycerol, propylene glycol, ethylene glycol, 3-O-methyl-D- glucopyranose (3-OMG), polyethylene glycol (PEG), 1,2-propanediol, acetamide, trehalose, formamide, sugars, proteins, and carbohydrates. In some embodiments, compositions and methods provided herein comprise at least one cryoprotectant. Exemplary cryoprotectants for inclusion are, but not limited to, sucrose, maltose, trehalose, mannitol, or glucose, and any combinations thereof. In some embodiments, additional or alternative cryoprotectant for inclusion is sorbitol, ribitol, erythritol, threitol, ethylene glycol, or fructose. In some embodiments, additional or alternative cryoprotectant for inclusion is dimethyl sulfoxide (DMSO), glycerol, propylene glycol, ethylene glycol, 3-O-methyl-D-glucopyranose (3-OMG), polyethylene glycol (PEG), 1,2-propanediol, acetamide, trehalose, formamide, sugars, proteins, and carbohydrates. In some embodiments, the cry oprotectant is present at about 1% w/v to at about 20% w/v, preferably about 10% w/v to at about 20% w/v. and more preferably at about 10% w/v. In some aspects of the disclosure, the cryoprotectant is sucrose. In some aspects of the disclosure, the cryoprotectant is maltose. In some aspects of the disclosure, the cryoprotectant is trehalose. In some aspects of the disclosure, the cryoprotectant is mannitol. In some aspects of the disclosure, the cryoprotectant is glucose. In some embodiments, the cry oprotectant is present in an amount of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140. 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 325, 350, 375, 400, 450, 500 or more mg. In some embodiments, the cryoprotectant is present in an amount of about 50 to about 500 mg. In some embodiments, the cryoprotectant is present in an amount of about 200 to about 300 mg. In some embodiments, the cryoprotectant is present in an amount of about 250 mg. In some embodiments, the cryoprotectant is present in amount of a lyophilized composition by weight of at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more percent. In some embodiments, the cryoprotectant is present in amount of a lyophilized composition by weight of about 95%. In some embodiments, the cryoprotectant is present in amount of a lyophilized composition by weight of 80 to 98%. 85 to 98%, 90 to 98%, or 94 to 96%. In some embodiments, the cryoprotectant is a sugar. In some embodiments, the sugar is sucrose, maltose, trehalose, mannitol, or glucose. In some embodiments, the sugar is sucrose. In some embodiments, the sucrose is present in an amount of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150. 160, 170, 180. 190, 200, 210, 220, 230. 240, 250, 260. 270, 280, 290, 300, 325. 350, 375, 400, 450, 500 or more mg. In some embodiments, the sucrose is present in an amount of about 50 to about 500 mg. In some embodiments, the sucrose is present in an amount of about 200 to about 300 mg. In some embodiments, the sucrose is present in an amount of about 250 mg. In some embodiments, the sucrose is present in amount of a lyophilized composition by weight of at least 50. 55. 60. 65. 70. 75. 80. 85, 90, 95 or more percent. In some embodiments, the sucrose is present in amount of a lyophilized composition by weight of about 95%. In some embodiments, the sucrose is present in amount of a lyophilized composition by weight of 80 to 98%. 85 to 98%, 90 to 98%, or 94 to 96%.

[0096] In some embodiments, the cryoprotectant is sucrose. In some embodiments, the cryoprotectant is at a concentration of at least about 0.1% w/v. In some embodiments, the cryoprotectant is at a concentration of about 1% w/v to at about 20% w/v. In some embodiments, the cryoprotectant is at a concentration of about 10% w/v to at about 20% w/v. In some embodiments, the cryoprotectant is at a concentration of about 10% w/v.

[0097] In some embodiments, compositions and vaccines provided herein are thermally stable. A composition is considered thermally stable when the composition resists the action of heat or cold and maintains its properties, such as the ability to protect a nucleic acid molecule from degradation at given temperature. In some embodiments, compositions and vaccines provided herein are thermally stable at about 25 °C or standard room temperature. In some embodiments, compositions and vaccines provided herein are thermally stable at about 45 °C. In some embodiments, compositions and vaccines provided herein are thermally stable at about - 20 °C. In some embodiments, compositions and vaccines provided herein are thermally stable at about 2 °C to about 8 °C. In some embodiments, compositions and vaccines provided herein are thermally stable at a temperature of at least about -80 °C, at least about- 20 °C, at least about 0 °C, at least about 2 °C, at least about 4 °C, at least about 6 °C, at least about 8 °C, at least about 10 °C, at least about 20 °C, at least about 25 °C, at least about 30 °C, at least about 37 °C, up to 45 °C. In some embodiments, compositions and vaccines provided herein are thermally stable for at least about 5 day, at least about 1 week, at least about 2 weeks, at least about 1 month, up to 3 months. In some embodiments, compositions and vaccines provided herein are stored at a temperature of at least about 4° C up to 37 °C for at least about 5 day, at least about 1 week, at least about 2 weeks, at least about 1 month, up to 3 months. In some embodiments, compositions and vaccines provided herein are stored at a temperature of at least about 20 °C up to 25 °C for at least about 5 day, at least about 1 week, at least about 2 weeks, at least about 1 month, up to 3 months.

[0098] Also provided herein are methods for preparing a lyophilized composition comprising obtaining a lipid carrier, wherein the lipid carrier is a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles and one or more lipids; incorporating one or more nucleic acid into the lipid carrier to form a lipid carrier- nucleic acid complex; adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; and lyophilizing the formulation to form a lyophilized composition. [0099] Further provided herein are methods for preparing a spray-dried composition comprising obtaining a lipid carrier, wherein the lipid carrier is a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles and one or more lipids; incorporating one or more nucleic acid into the lipid carrier to form a lipid carrier- nucleic acid complex; adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; and spray drying the formulation to form a spray-dried composition.

[00100] Further provided herein are methods for reconstituting a lyophilized composition comprising: obtaining a lipid carrier, wherein the lipid carrier is a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles, and one or more lipids; incorporating one or more nucleic acid into the said lipid carrier to form a lipid carrier-nucleic acid complex; adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; lyophilizing the formulation to form a lyophilized composition; and reconstituting the lyophilized composition in a suitable diluent.

[00101] Further provided herein are methods for reconstituting a spray-dried composition comprising: obtaining a lipid carrier, wherein the lipid carrier is a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles, and one or more lipids, incorporating one or more nucleic acid into the said lipid carrier to form a lipid carrier-nucleic acid complex; adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; spray drying the formulation to form a spray-dried composition; and reconstituting the spray- dried composition in a suitable diluent.

(6) Pharmaceutical Compositions

[00102] Provided herein are pharmaceutical compositions, wherein the pharmaceutical compositions comprise: a composition provided herein or a nucleic acid provided herein. In some embodiments, compositions provided herein are combined with pharmaceutically acceptable salts, excipients, and/or earners to form a pharmaceutical composition. Pharmaceutical salts, excipients, and carriers may be chosen based on the route of administration, the location of the target issue, and the time course of delivery of the drug. A pharmaceutically acceptable carrier or excipient may include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., compatible with pharmaceutical administration.

[00103] In some embodiments, the pharmaceutical composition is a suspension comprising a composition provided herein. In some embodiments, suspensions provided herein comprise a plurality of nanoparticles or compositions provided herein. In some embodiments, compositions provided herein are in a suspension, optionally a homogeneous suspension. In some embodiments, compositions provided herein are in an emulsion form.

[00104] In some embodiments, the pharmaceutical composition is in the form of a solid, semi-solid, liquid or gas (aerosol). Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile waler or other sterile injectable medium prior to use.

[00105] Solid dosage forms for oral administration include capsules, tablets, pills, pow ders, and granules. In such solid dosage forms, the encapsulated or unencapsulated conjugate is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone. sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, some silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also comprise buffering agents.

(7) Dosing

[00106] Compositions provided herein may be formulated in dosage unit form for ease of administration and uniformity of dosage. A dosage unit form is a physically discrete unit of a composition provided herein appropriate for a subject to be treated. It will be understood, however, that the total usage of compositions provided herein will be decided by the attending physician within the scope of sound medical judgment. For any composition provided herein the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, such as mice, rabbits, dogs, pigs, or non-human primates. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. Therapeutic efficacy and toxicity' of compositions provided herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose is therapeutically effective in 50% of the population) and LD₅₀ (the dose is lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions which exhibit large therapeutic indices may be useful in some embodiments. The data obtained from cell culture assays and animal studies may be used in formulating a range of dosage for human use. Exemplary amounts of total nucleic acid for incorporation in a composition described herein includes about 1, 2, 2.5, 5, 7.5, 10, 12.5, 15, 20, 25, 30, 35, 40, 45, 50 micrograms (pg) or more.

(8) Administration

[00107] Provided herein are compositions and pharmaceutical compositions for administering to a subject in need thereof. In some embodiments, pharmaceutical compositions provided here are in a form which allows for compositions provided herein to be administered to a subject.

[00108] In some embodiments, the administering is local administration or systemic administration. In some embodiments, a composition described herein is formulated for administration / for use in administration via a subcutaneous, intradermal, intramuscular, intranasal inhalation, intravenous, intraperitoneal, intracranial, sublingual, oral, or intrathecal route. In some embodiments, the administering is every 1, 2. 4, 6, 8, 12. 24, 36, 48, 60, 72, 84, 96, 108, or 120 hours. In some embodiments, the administering is daily (every 24 hours), weekly (every 7 days), or monthly (every 28, 29, 30, or 31 days). In some embodiments, the administering is repeated at least about every' 7 days (168 hours), every' 10 days (240 hours), every 12 days, every 13 days, every 14 days, every 15 days, every 16 days, every 17 days, every 18 days, every 19 days, every 20 days, every 21 days, every 22 days, every 23 days, every 24 days, every 25 days, every 26 days, every 27 days, 28 days, every 29 days, every 30 days, every 35 days, every' 40 days, every' 42 days, every' 45 days, every' 50 days, every' 52 days, every' 55 days, or every 56 days. In some embodiments, the administering is repeated at least about every 14 days up to about every 56 days. In some embodiments, the administering is repeated at least about every 14 days up to about every' 28 days. In some embodiments, the administering is repeated at least about every 28 days to every 56 days.

[00109] In some embodiments, a single dose of a composition provided herein is administered to a subject. In some embodiments, a composition or pharmaceutical composition provided herein is administered to the subject by two doses. In some embodiments, a second dose of a composition or pharmaceutical composition provided herein is administered about 28 days or 56 days after the first dose. In some embodiments, a first dose is administered, and a second dose is administered about 14 days later, or about 21 days later, or about 28 days later, or about 35 days later, or about 42 days later, or about 49 days later, or about 56 days later, or about 63 days later, or about 70 days later, or about 77 days later, or about 84 days later. In some embodiments, the second dose is administered about 10-90 days following administration of the first dose, or about 15-85 days following administration of the first dose, or about 20-80 days following administration of the first dose, or about 25-75 days following administration of the first dose, or about 30-70 days following administration of the first dose, or about 35-65 days following administration of the first dose, or about 40-60 days following administration of the first dose.

[00110] In some embodiments, a third dose of a composition or pharmaceutical composition provided herein is administered to a subject. In some embodiments, the third dose is administered about 1 month following administration of the second dose, about 2 months following administration of the second dose, about 3 months following administration of the second dose, about 4 months following administration of the second dose, about 5 months following administration of the second dose, about 6 months following administration of the second dose, about 7 months following administration of the second dose, about 8 months following administration of the second dose, about 9 months following administration of the second dose, about 10 months following administration of the second dose, about 11 months following administration of the second dose, about 12 months following administration of the second dose, about 13 months following administration of the second dose, about 14 months following administration of the second dose, about 15 months following administration of the second dose, about 16 months following administration of the second dose, about 17 months following administration of the second dose, or about 18 months following administration of the second dose.

(9) Therapeutic Applications [00111] Provided herein are methods for modulating an immune response in a subject, wherein the methods comprise: administering to a subject a composition provided herein. In some embodiments, the immune response comprises increasing the titer of neutralizing antibodies to the antigen as compared to a subject that has not been administered the composition. In some embodiments, the immune response comprises increasing an amount of CD4+ and/or CD8+ positive T-cells as compared to a subject that has not been administered the composition. In some embodiments, the immune response comprises increasing an amount of neutralizing epithelial cells as compared to a subject that has not been administered the composition. In some embodiments, the immune response comprises increasing an amount of neutralizing B cells as compared to a subject that has not been administered the composition. Provided herein are methods of generating an immune response in a subject, wherein the methods comprise: administering to a subject a composition provided herein, thereby generating an immune response to a gH-gL viral protein antigen or a functional fragment thereof.

[00112] Provided herein are methods of treating a disease or a disorder in a subject. In some embodiments, the disease or disorder is an infection. In some embodiments, the infection is a viral infection, a bacterial infection, a parasitic infection, a fungal infection, or a yeast infection. In some embodiments, the subject has, is suspected of having, or is at risk of developing a viral infection. In some embodiments, the viral infection is a herpesvirus infection. In some embodiments, the subject is at risk of developing an infectious disease or disorder. In some embodiments, the subject has contracted an infectious disease by way of contact with another infected subject. In some embodiments, the subject has contracted an infectious disease from contaminated drinking water. In some embodiments, the subject has contracted the infectious disease from a different species carrying the microorganism. In some embodiments, the subject has, is suspected of having, or is diagnosed as having a gamma herpesvirus infection. In some embodiments, the subject has or is diagnosed with a gamma herpesvirus-associated cancer. In some embodiments, the subject has, is suspected of having, or is diagnosed as having an Epstein- Barr virus. In some embodiments, the gamma herpesvirus and the Epstein-Barr virus are associated with cancer.

[00113] Provided herein are methods of treating a cancer in a subject. In some embodiments, the subject has, is at risk of developing, or is diagnosed with cancer. In some embodiments, the subject is immunocompromised or immunosuppressed. In some embodiments, the cancer is a solid cancer, an abdominal cancer or a blood cancer. In some embodiments, the blood cancer is lymphoma or leukemia. In some embodiments, the blood cancer is a plasmablastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, a B- lymphoproliferative disease, diffuse large B-cell lymphoma, Burkit's lymphoma, natural killer (NK) cell lymphoma, a Hodgkin's disease, or a T cell lymphoma. In some embodiments, the subject has, is at risk for developing, or is suspected of having a skin cancer. In some embodiments, the skin cancer is a basal cell cancer, a melanoma, a Merkel cell cancer, a squamous cell carcinoma, a cutaneous lymphoma, a Kaposi sarcoma, or a skin adnexal cancer. In some embodiments, the subject has, is at risk for developing, or is suspected of having a pancreatic cancer. In some embodiments, the pancreatic cancer is a pancreatic adenocarcinoma, a pancreatic exocrine cancer, a pancreatic neuroendocrine cancer, an islet cell cancer, or a pancreatic endocrine cancer. In some embodiments, the subject has, is at risk for developing, or is suspected of having a colon cancer, a prostate cancer, an ovarian cancer, or a breast cancer.

[00114] Provided herein are methods of treating a subject, wherein the subject has, is suspected of having, is at risk of developing, or is diagnosed with an autoimmune disease. In some embodiments, the autoimmune disease comprises autoimmune disease comprises systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, type 1 diabetes, arthritis, a neurodegenerative disease, multiple sclerosis, or celiac disease.

[00115] A subject provided herein can be an animal or a human. The compositions provided herein can be administered as a treatment for a human disease or used in veterinary practice. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a domesticated animal or livestock.

(10) Kits

[00116] Provided herein is a kit comprising a composition provided herein, a pharmaceutical composition provided herein; and optionally, a delivery system for administration to a subject. In some embodiments, the kit further comprises one or more surfactants. In some embodiments, a formulation of a composition described herein is prepared in a single container for administration. In some embodiments, a formulation of a composition described herein is prepared two containers for administration, separating the nucleic acid from the nanoparticle earner. As used herein, ’container" includes vessel, vial, ampule, tube, cup, box, bottle, flask, jar, dish, well of a single-well or multi-well apparatus, reservoir, tank, or the like, or other device in which the herein disclosed compositions may be placed, stored and/or transported, and accessed to remove the contents. Examples of such containers include glass and/or plastic sealed or re-sealable tubes and ampules, including those having a rubber septum or other sealing means that is compatible with withdrawal of the contents using a needle and syringe. In some implementations, the containers are RNase free.

[00117] In some embodiments, the kit comprises: a lipid carrier and at least one nucleic acid. In some embodiments, the kit comprises: a first nucleic acid sequence encoding for an RNA- dependent RNA polymerase complex from a virus. In some embodiments, the kit comprises: a first nucleic acid sequence encoding for a gH-gL viral protein antigen sequence or a functional variant thereof. In some embodiments, the kit comprises: a composition comprising: a first nucleic acid sequence encoding for an RNA-dependent RNA polymerase complex from a virus; and a second nucleic acid sequence encoding for a gH-gL viral protein antigen sequence or a functional variant thereof. In some embodiments, the kit comprises: a composition comprising: a lipid carrier; a first nucleic acid sequence encoding for an RNA-dependent RNA polymerase complex from a virus; and a second nucleic acid sequence encoding for a gH-gL viral protein antigen sequence or a functional variant thereof.

Exemplary Embodiments

[00118] Provided herein are compositions, wherein the compositions comprise: a lipid earner, wherein the lipid carrier comprises: cationic lipids; surfactants; and liquid oil; and at least one nucleic acid encoding for a gH-gL viral protein antigen sequence or a functional variant thereof. Further provided herein are compositions, wherein the gH-gL viral protein antigen sequence is from a rhadinovirus. Further provided herein are compositions, wherein the gH-gL viral protein antigen sequence is from an Epstein-Ban Virus (EBV). a Kaposi’s sarcoma- associated herpesvirus, a herpesvirus saimiri, a herpesvirus ateles, a murine herpesvirus 68, or a functional variant of any of the foregoing. Further provided herein are compositions, wherein the gH-gL viral protein antigen sequence comprises a sequence encoding for a recombinant protein. Further provided herein are compositions, wherein the at least one nucleic acid encoding for a gH-gL viral protein antigen sequence or a functional variant thereof comprises: (1) a gH region; and (2) a gL region. Further provided herein are compositions, wherein the recombinant protein comprises SEQ ID NO: 1 (gH), SEQ ID NO: 2 (gL), SEQ ID NO: 3 (gH-gL), or SEQ ID NO: 4 (gL-gH). Further provided herein are compositions, wherein the nucleic acid further encodes for an RNA polymerase complex region. Further provided herein are compositions, wherein the RNA polymerase complex region comprises a Venezuelan equine encephalitis virus (VEEV) RNA polymerase. Further provided herein are compositions, wherein the nucleic acid encoding for the RNA polymerase complex region comprises SEQ ID NO: 14 (VEEV RNA sequence). Further provided herein are compositions, wherein the liquid oil comprises a-tocopherol, coconut oil, grapeseed oil, lauroyl polyoxylglyceride, mineral oil, monoacylglycerol, palm kernel oil, olive oil, paraffin oil, peanut oil, propolis, squalene, squalane, soy lecithin, soybean oil, sunflower oil, a triglyceride, or vitamin E. Further provided herein are compositions, wherein the triglyceride is capric triglyceride, caprylic triglyceride, a caprylic and capric triglyceride, a triglyceride ester, or myristic acid triglycerin. Further provided herein are compositions, wherein the cationic lipids comprise: l,2-dioleoyloxy-3 (trimethylammonium)propane, 30-[N — (N'.N'- dimethylaminoethane) carbamoyl] cholesterol, dimethyldi octadecylammonium, 1,2-dimyristoyl 3 -trimethyl ammoniumpropane, dipalmitoyl(C16:0)trimethyl ammonium propane, distearoyltrimethylammonium propane, N-[l-(2,3- dioleyloxy)propyl]N,N,Ntrimethylammonium, chloride, N,N-dioleoyl-N,N- dimethylammonium chloride. l,2-dioleoyl-sn-glycero-3-ethylphosphocholine, l,2-dioleoyl-3- dimethylammonium-propane, 1,2- dilinoleyloxy-3-dimethylaminopropane,l,r-((2-(4-(2-((2- (bis(2 -hydroxy dodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-l - yl)ethyl)azanediyl)bis(dodecan-2-ol), tetrakis(8-methylnonyl) 3,3',3",3"'-(((methylazanediyl) bis(propane-3,l diyl))bis (azanetriyl))tetrapropionate, decyl (2-(dioctylammonio)ethyl) phosphate, ethyl 5,5-di((Z)-heptadec-8-en-l-yl)-l-(3-(pyrrolidin-l-yl)propyl)-2,5-dihydro-lH- imidazole-2-carboxylate, ((4-hydroxybutyl)azanediyl)bis(hexane-6, 1 -diy l)bis (2- hexyldecanoate, 2-[(poly ethylene glycol)-2000]-N,N-ditetradecylacetamide,

(3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl- 2,3,4.7.8.9.10,l l,12,13,14.15,16,17-tetradecahydro-lH-cyclopenta[a]phenanthren-3-ol, bis(2- (dodecyldisulfanyl)ethyl) 3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6- diazahexacosyl)azanediyl)dipropionate, 2-(((((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17- ((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,l l,12,13,14,15,16,17-tetradecahydro-lH- cyclopenta[a]phenanthren-3-yl)oxy)carbonyl)amino)-N,N-bis(2-hydroxyethyl)-N-methylethan- 1-aminium bromide, 3.6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2.5-dione, 3β- [N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, (6Z,9Z,28Z,31Z)-heptatriaconta- 6,9,28,31-tetraen- 19-yl 4-(dimethylamino) butanoate, l,2-dioleoyl-sn-glycero-3- phosphoethanolamine, 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-l- propanaminium trifluoroacetate, 1 ,2-distearoyl-sn-gly cero-3 -phosphocholine, ethylphosphatidylcholine, hexa(octan-3-yl) 9,9',9'',9"',9"",9"'"- ((((benzene- 1,3,5- tricarbonyl)yris(azanediyl)) tris (propane-3,1 -diyl)) tris(azanetriyl))hexanonanoate, heptadecan- 9-yl 8-((2 -hydroxy ethyl)(6-oxo-6- (undecyloxy )hexyl)amino) octanoate, (((3,6-dioxopiperazine- 2,5-diyl)bis(butane-4, l-diyl))bis(azanetriyl))tetrakis(ethane-2,l-diyl)

(9Z,9'Z,9"Z,9"'Z.12Z,12'Z.12"Z.12"'Z)-tetrakis (octadeca-9,12-dienoate), Nl,N3,N5-tris(3- (didodecylamino)propyl)benzene-l,3,5-tricarboxamide. Further provided herein are compositions, wherein the surfactant comprise a hydrophobic surfactant and a hydrophilic surfactant. Further provided herein are compositions, wherein the lipid carrier comprises an inorganic particle. Further provided herein are compositions, wherein the inorganic particle is within the hydrophobic core of the lipid carrier. Further provided herein are compositions, wherein the inorganic particle comprises a metal. Further provided herein are compositions, wherein the metal comprises a metal salt, a metal oxide, a metal hydroxide, or a metal phosphate. Further provided herein are compositions, wherein the metal oxide comprises aluminum oxide, aluminum oxyhydroxide, iron oxide, titanium dioxide, or silicon dioxide. Further provided herein are compositions, wherein the lipid carrier further comprises a hydrophobic surfactant. Further provided herein are compositions, wherein the hydrophobic surfactant comprises sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, or sorbitan trioleate. Further provided herein are compositions, wherein the lipid carrier further comprises a hydrophilic surfactant. Further provided herein are compositions, wherein the hydrophilic surfactant comprises a polysorbate. Further provided herein are compositions, wherein the lipid earner is characterized as having a z-average diameter particle size measurement of about 20 nm to about 80 nm when measured using dynamic light scattering. Further provided herein are compositions, wherein the lipid carrier is characterized as having a z-average diameter particle size measurement of about 20 nm to about 60 nm when measured using dynamic light scattering. Further provided herein are compositions, wherein the nucleic acid comprises DNA. Further provided herein are compositions, wherein the nucleic acid comprises RNA. Further provided herein are compositions, wherein the compositions further comprise: a nucleic acid that modulates an innate immune response in a subject. Further provided herein are compositions, wherein the compositions further comprise sodium citrate. Further provided herein are compositions, wherein the compositions further comprise sucrose, optionally, wherein the sucrose is present in an amount of about 50 mg.

[00119] Provided herein are compositions, wherein the compositions comprise: a lipid carrier, wherein the lipid carrier comprises: a surface comprising cationic lipids; and a hydrophobic core, wherein the hydrophobic core comprises liquid oil, wherein lipids present in the hydrophobic core are in liquid phase at 25 degrees Celsius, at least one nucleic acid encoding for a gH-gL viral protein antigen sequence or a functional variant thereof, wherein the at least one nucleic acid is complexed to the surface of the lipid carrier. Further provided herein are compositions, wherein the gH-gL viral protein antigen sequence is from a rhadinovirus. Further provided herein are compositions, wherein the gH-gL viral protein antigen sequence is from an Epstein-Barr Virus (EBV), a Kaposi’s sarcoma-associated herpesvirus, a herpesvirus saimiri, a herpesvirus ateles, a murine herpesvirus 68, or a functional variant of any of the foregoing. Further provided herein are compositions, wherein the gH-gL viral protein antigen sequence comprises a sequence encoding for a recombinant protein. Further provided herein are compositions, wherein the at least one nucleic acid encoding for a gH-gL viral protein antigen sequence or a functional variant thereof comprises: (1) a gH region; and (2) a gL region. Further provided herein are compositions, wherein the recombinant protein comprises SEQ ID NO: 1 (gH), SEQ ID NO: 2 (gL), SEQ ID NO: 3 (gH-gL), or SEQ ID NO: 4 (gL-gH). Further provided herein are compositions, wherein the nucleic acid further encodes for an RNA polymerase complex region. Further provided herein are compositions, wherein the RNA polymerase complex region comprises a Venezuelan equine encephalitis virus (VEEV) RNA polymerase. Further provided herein are compositions, wherein the nucleic acid encoding for the RNA polymerase complex region comprises SEQ ID NO: 14 (VEEV RNA sequence). Further provided herein are compositions, wherein the liquid oil comprises a-tocopherol, coconut oil, grapeseed oil, lauroyl polyoxylglyceride, mineral oil, monoacylglycerol, palm kernel oil, olive oil, paraffin oil, peanut oil. propolis, squalene, squalane, soy lecithin, soybean oil, sunflower oil, a triglyceride, or vitamin E. Further provided herein are compositions, wherein the triglyceride is capric triglyceride, caprylic triglyceride, a caprylic and capric triglyceride, a triglyceride ester, or myristic acid triglycerin. Further provided herein are compositions, wherein the cationic lipids comprise: l,2-dioleoyloxy-3 (trimethylammonium)propane. 30-[N — (N'.N¹- dimethylaminoethane) carbamoyl] cholesterol, dimethyldioctadecylammonium, 1,2-dimyristoyl 3 -trimethyl ammoniumpropane, dipalmitoyl(C16:0)trimethyl ammonium propane, distearoyltrimethylammonium propane, N-[l-(2,3- dioleyloxy)propyl]N,N,Ntrimethylammonium, chloride, N.N-dioleoyl-N,N- dimethylammonium chloride. l,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 1.2-dioleoyl-3- dimethylammonium-propane, 1,2- dilinoleyloxy-3-dimethylaminopropane,I,r-((2-(4-(2-((2- (bis(2 -hydroxy dodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-l- yl)ethyl)azanediyl)bis(dodecan-2-ol), tetrakis(8-methylnonyl) 3,3',3",3"'-(((methylazanediyl) bis(propane-3,l diyl))bis (azanetriyl))tetrapropionate, decyl (2-(dioctylammonio)ethyl) phosphate, ethyl 5,5-di((Z)-heptadec-8-en-l-yl)-l-(3-(pyrrolidin-l-yl)propyl)-2,5-dihydro-lH- imidazole-2-carboxylate, ((4-hydroxybutj l)azanediyl)bis(hexane-6, 1 -diy l)bis (2- hexyldecanoate, 2-[(poly ethylene glycol)-2000]-N,N-ditetradecylacetamide,

(3S,8S.9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl- 2,3,4.7.8.9.10,l l,12.13,14.15,16,17-tetradecahydro-IH-cyclopenta[a]phenanthren-3-ol, bis(2- (dodecyldisulfanyl)ethyl) 3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6- diazahexacosyl)azanediyl)dipropionate, 2-(((((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17- ((R)-6-methylheptan-2-y l)-2,3,4,7.8.9.10, 11 , 12.13, 14.15, 16, 17-tetradecahydro- 1H- cyclopenta[a]phenanthren-3-yl)oxy)carbonyl)amino)-N,N-bis(2-hydroxyethyl)-N-methylethan- 1-aminium bromide, 3, 6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2, 5-dione, 3|3- [N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol, (6Z,9Z,28Z,31Z)-heptatriaconta- 6,9,28,3 l-tetraen-19-yl 4-(dimethylamino) butanoate. 1.2-dioleoyl-sn-glycero-3- phosphoethanolamine, 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-l- propanaminium trifluoroacetate, l,2-distearoyl-sn-glycero-3-phosphocholine, ethylphosphatidylcholine, hexa(octan-3-yl) 9, 9', 9", 9"', 9"", 9"'"- ((((benzene- 1,3,5 - tricarbonyl)yris(azanediyl)) tris (propane-3,1 -diyl)) tris(azanetriyl))hexanonanoate, heptadecan- 9-yl 8-((2-hydroxyethyl)(6-oxo-6- (undecyloxy )hexyl)amino) octanoate, (((3.6-dioxopiperazine- 2,5-diyl)bis(butane-4, 1 -diyl))bis(azanetriyl))tetrakis(ethane-2,l -diyl)

(9Z,9'Z,9"Z,9"'Z,12Z,12'Z,12''Z,12'"Z)-tetrakis (octadeca-9,12-dienoate), Nl,N3,N5-tris(3- (didodecylamino)propyl)benzene-l,3,5-tricarboxamide. Further provided herein are compositions, wherein the lipid carrier further comprises surfactants. Further provided herein are compositions, wherein the surfactants comprise a hydrophilic surfactant and a hydrophobic surfactant. Further provided herein are compositions, wherein the lipid carrier comprises an inorganic particle. Further provided herein are compositions, wherein the inorganic particle is within the hydrophobic core of the lipid carrier. Further provided herein are compositions, wherein the inorganic particle comprises a metal. Further provided herein are compositions, wherein the metal comprises a metal salt, a metal oxide, a metal hydroxide, or a metal phosphate. Further provided herein are compositions, wherein the metal oxide comprises aluminum oxide, aluminum oxyhydroxide, iron oxide, titanium dioxide, or silicon dioxide. Further provided herein are compositions, wherein the lipid carrier further comprises a hydrophobic surfactant. Further provided herein are compositions, wherein the hydrophobic surfactant comprises sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, or sorbitan trioleate. Further provided herein are compositions, wherein the lipid carrier further comprises a hydrophilic surfactant. Further provided herein are compositions, wherein the hydrophilic surfactant comprises a polysorbate. Further provided herein are compositions, wherein the lipid carrier is characterized as having a z-average diameter particle size measurement of about 20 nm to about 80 nm when measured using dynamic light scattering. Further provided herein are compositions, wherein the lipid carrier is characterized as having a z-average diameter particle size measurement of about 20 nm to about 60 nm when measured using dynamic light scattering. Further provided herein are compositions, wherein the nucleic acid comprises DNA. Further provided herein are compositions, wherein the nucleic acid comprises RNA. Further provided herein are compositions, wherein the compositions further comprise: a nucleic acid that modulates an innate immune response in a subject. Further provided herein are compositions, wherein the compositions further comprise sodium citrate. Further provided herein are compositions, wherein the compositions further comprise sucrose, optionally, wherein the sucrose is present in an amount of about 50 mg.

[00120] Provided herein are compositions, wherein the compositions comprise: a nucleic acid comprising: a first region encoding for an RNA-dependent RNA polymerase complex from a virus; and a second region encoding for a gH-gL viral protein antigen sequence or a functional variant thereof. Further provided herein are compositions, wherein the first region or the second region comprises RNA, DNA, or a combination thereof. Further provided herein are compositions, wherein the second region further comprises a sequence encoding a self-cleaving peptide. Further provided herein are compositions, wherein the gH-gL viral protein antigen sequence is from a rhadinovirus. Further provided herein are compositions, the gH-gL viral protein antigen sequence is from an Epstein-Barr Virus (EBV), a Kaposi’s sarcoma-associated herpesvirus, a herpesvirus saimiri, a herpesvirus ateles, a murine herpesvirus 68, or a functional variant of any of the foregoing. Further provided herein are compositions, wherein the gH-gL viral protein antigen sequence comprises a sequence encoding for a recombinant protein. Further provided herein are compositions, wherein the recombinant protein comprises SEQ ID NO: 1 (gH). SEQ ID NO: 2 (gL), SEQ ID NO: 3 (gH-gL), or SEQ ID NO: 4 (gL-gH). Further provided herein are compositions, w herein the RNA polymerase complex region comprises a Venezuelan equine encephalitis virus (VEEV) RNA polymerase. Further provided herein are compositions, wherein the nucleic acid encoding for the RNA-dependent RNA polymerase complex comprises SEQ ID NO: 14. Further provided herein are compositions, wherein the first nucleic acid or the second nucleic acid is present in an amount of up to about 100 micrograms (pg). Further provided herein are compositions, wherein the first nucleic acid or the second nucleic acid is present in an amount of up to about 25 pg. Further provided herein are compositions, wherein the composition further comprises a lipid carrier. Further provided herein are compositions, wherein the lipid earner is in complex with the first nucleic acid or the second nucleic acid. Further provided herein are compositions, a surface of the lipid carrier is in complex with the first nucleic acid and the second nucleic acid. Further provided herein are compositions, wherein the composition further comprises an additional nucleic acid encoding for a viral protein antigen or a cancer-associated protein antigen. Further provided herein are compositions, wherein the additional nucleic acid is in complex with a lipid carrier. Further provided herein are compositions, wherein the composition is lyophilized.

[00121] Provided herein are suspensions, wherein the suspensions comprise: any one of the compositions provided herein.

[00122] Provided herein are pharmaceutical compositions, wherein the pharmaceutical compositions comprise: any one of the compositions provided herein; and a pharmaceutically acceptable excipient. Further provided herein are pharmaceutical compositions, wherein the excipient comprises a sugar. Further provided herein are pharmaceutical compositions, wherein the sugar comprises sucrose.

[00123] Provided herein are methods of generating an immune response in a subject, wherein the methods comprise: administering to a subject the composition provided herein, thereby generating an immune response to the gH-gL viral protein antigen or the functional fragment thereof. Further provided herein are methods, wherein the antigen is a viral protein antigen. Further provided herein are methods, the antigen is a cancer-associated antigen. Further provided herein are methods, wherein the subject has, is suspected of having, is at risk of developing, or is diagnosed with a gamma herpesvirus infection. Further provided herein are methods, wherein the subject has, is suspected of having, is at risk of developing, or is diagnosed with a cancer. Further provided herein are methods, wherein the cancer is a carcinoma, a sarcoma, a lymphoma, or a solid cancer. Further provided herein are methods, wherein the cancer comprises a nasopharyngeal cancer, an abdominal cancer, or a blood cancer. Further provided herein are methods, wherein the blood cancer is a plasmablastic lymphoma, a primary central nervous system lymphoma, a primary effusion lymphoma, a B-lymphoproliferative disease, a diffuse large B-cell lymphoma, a Burkit's lymphoma, a natural killer (NK) cell lymphoma, a Hodgkin's disease, or a T cell lymphoma. Further provided herein are methods, wherein the subject is immunocompromised or immunosuppressed. Further provided herein are methods, wherein the subject has, is suspected of having, is at risk of developing, or is diagnosed with an autoimmune disease. Further provided herein are methods, wherein the autoimmune disease comprises: systemic lupus ery thematosus, rheumatoid arthritis, inflammatory' bowel disease, ulcerative colitis, Crohn's disease, type 1 diabetes, arthritis, a neurodegenerative disease, multiple sclerosis, and celiac disease. Further provided herein are methods, wherein the composition is administered to the subject by⁷ two doses. Further provided herein are methods, wherein the administering comprises administering a second dose of the composition at about 28 days to 56 days after a first dose of the composition. Further provided herein are methods, wherein the methods further comprise: administering a third dose of the composition to said subject. Further provided herein are methods, wherein the composition is administered intramuscularly, subcutaneously, or intranasally. Further provided herein are methods, wherein the methods further comprise: administering to the subject a second composition comprising: a cancer-associated antigen or a nucleic acid encoding for the cancer-associated antigen. Further provided herein are methods, wherein the methods further comprise: administering to the subject a second composition comprising: a viral protein antigen or a nucleic acid encoding the viral protein antigen. Further provided herein are methods, wherein the immune response comprises increasing a titer of neutralizing antibodies to the gH-gL viral protein antigen as compared to a subject that has not been administered the composition. Further provided herein are methods, wherein the immune response comprises increasing an amount of vaccine-specific CD4⁺ and/or CD8⁺ positive T-cells as compared to a subject that has not been administered the composition. Further provided herein are methods, wherein the immune response comprises increasing an amount of neutralizing epithelial cells in the subject relative to a subject that has not been administered the composition. Further provided herein are methods, wherein the immune response comprises increasing an amount of neutralizing B cells in the subject relative to a subject that has not been administered the composition. Further provided herein are methods, wherein the immune response comprises increasing an amount of neutralizing epithelial cells in the subject relative to a subject that has not been administered the composition. Further provided herein are methods, wherein the immune response comprises increasing an amount of neutralizing B cells in the subject relative to a subject that has not been administered the composition.

[00124] Further provided herein are methods, wherein the subject is a human subject. Further provided herein are methods, wherein the subject has or is diagnosed with a gamma herpesvirus-associated cancer.

[00125] Provided herein are methods of treating or preventing an infection in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby treating the infection. Further provided herein are methods, wherein the subject is a human subject.

[00126] Provided herein are methods of treating cancer in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby treating the cancer. Further provided herein are methods, wherein the subject is a human subject. Further provided herein are methods, wherein the subj ect has or is diagnosed with a gamma herpesvirus-associated cancer. [00127] Provided herein are kits, wherein the kits comprise: a composition provided herein, packaging, and materials therefor.

[00128] The following examples are set forth to illustrate more clearly the principle and practice of embodiments disclosed herein to those skilled in the art and are not to be construed as limiting the scope of any claimed embodiments. Unless otherwise stated, all parts and percentages are on a weight basis.

EXAMPLES

Example 1: Generation of a gH-gL repRNA vaccination.

[00129] Plasmids were generated by the following methods. Codon optimized cDNA encoding EBV gH (GenBank AFY97969.1) and gL (GenBank: AFY97944.1) separated by a furin cleavage site (SEQ ID NO: 20) and a P2A self-cleaving peptide in both orientations (gH- furin-P2A-gL or gL-furin-P2A-gH, SEQ ID NO: 13) with a 5’ Kozak consensus sequence were synthesized by Twist Biosciences and cloned into p658 encoding the 5' and 3' untranslated regions and the nonstructural open reading frame of Venezuelan equine encephalitis virus, strain TC-83, between Pfl FI and Sac II sites creating p658-gH-gL and p658-gL-gH. P658-gL-gH-Ecto was created by introducing a stop codon at AA 170 in gH using the QuikChange® II (Agilent Technologies, Inc., Delaware. USA)) Site-directed mutagenesis kit. P658-gL-gH-MDT1100, P658-gL-gH-C4b, and P658-gL-gH-I3 were produced by amplifying the entire P658-gL-gH- Ectodomain plasmid using gene-specific pnmers and Platinum™ SuperFi™ II DNA Polymerase (Life Technologies Corporation, Delaware, USA) according to the manufacturer’s instructions. Separate sets of gene specific primers with overlapping homology to the 5’ and 3’ ends of the amplified linear P658-gL-gH-Ecto fragment were used to amplify the MDT1100, C4b and 13 multimerization domains from pTT3-gH-IMX313, pCVL-UCOE0.7-SFFV-gH-C153T- cTRP(6)ss-IRES-GFP, and pCVL-UCOE0.7-SFFV-gH-C153T-I3-IRES-GFP plasmids, respectively. The linear P658-gL-gH-Ecto fragment was fused to each multimerization domain fragment using the In-Fusion® Snap Assembly Master Mix (Takara Bio USA, Inc., Delaware, USA) according to the manufacturer’s instructions.

[00130] The variable regions corresponding to 769B10, 1D8, and 770F7 heavy and light chains were synthesized by Integrated DNA Technologies and cloned into pTT3-AMM01-HC and pTT3-AMM01LC (for lambda) or pTT3-E!DlLC (for Kappa). All plasmids were confirmed by Sanger sequencing. [00131] repRNA production: Template p658 DNAs were linearized by enzymatic digestion with Not I followed by phenol-chloroform treatment and ethanol precipitation. Linearized template was transcribed using the MEGAscript® T7 Transcription Kit (Ambion, Inc., Delaware, USA) followed by capping with New England Biolabs Vaccinia Capping System. Capped transcripts were then precipitated in lithium chloride and resuspended in nuclease-free water to a final concentration of 1 mg/ml and analyzed by agarose-gel electrophoresis. All RNA was aliquoted and stored at -80 degrees C.

[00132] Nanoparticle production: NP-30 was prepared. Briefly, the oil phase (squalene. Span 60, and DOTAP) was sonicated for 30 min in a 65° C water bath. Separately, the aqueous phase, containing Tween 80 and sodium citrate dihydrate solution in water, was prepared with continuous stirring until all components were dissolved. The oil and aqueous phases were then mixed and emulsified using a VWR 200 homogenizer (VWR International), and the crude colloid was subsequently processed by passaging through a microfluidizer at 137895 kPa with an LM10 microfluidizer equipped with an Hl 0Z 100-pm ceramic interaction chamber (Microfluidics) until the Z-average hydrodynamic diameter, measured by dynamic light scattering (Malvern Zetasizer Nano S). reached 50 ± 5 nm with a 0.2 polydispersity index. The microfluidized NP-30 was terminally filtered with a 200-nm pore-size poly ethersulfone filter and stored at 2° to 8° C. The gH-gL constructs shown in FIG. 1A were complexed with NP-30.

Example 2: Initial characterization of the gH-gL and gL-gH repRNAs.

[00133] To enable co-delivery of both gH and gL on a single repRNA, two constructs were designed. The first encodes gL and then full length gH (including the transmembrane and cytoplasmic domains) in tandem separated by a P2A self-cleaving peptide and the second encodes gH and then gL separated by P2A (FIG. 1A). Both constructs included the native signal peptide and a furin cleavage site upstream of P2A to facilitate removal of residual P2A residues self- N-terminal protein following self-cleavage. 10 pg of each repRNA constmct were transcribed in vitro via T7 polymerase, formulated with NP-30, and delivered to groups of 4 C57BL/6 mice at weeks 0 and 4. Blood was collected at the time of the first immunization and 2 weeks after each immunization for serological analyses. Both constructs were immunogenic and elicited reciprocal endpoint binding titers on the order of 1x10⁴ as soon as 2 weeks after the first immunization that reached IxlO⁵ by week 6. Both constructs also elicited low, but detectable titers of antibodies that could neutralize EBV infection of B cells by week 2 that were not boosted by a second immunization at week 4. Example 3: Methods and Assays for Evaluating the Effects of gH-gL repRNA vaccination in vivo.

[00134] The following methods and assays were carried out to determine the dose for vaccination needed to modulate an immune response to the gH and gL antigen in cells and in animals.

[00135] Recombinant Proteins: The recombinant gH-gL ectodomain was produced by transfecting pTT3-gH-HIS-AVI and pTT3-gL in 293 6E cells using PEI Max according to the manufacturer’s instructions and purified using NiNTA affinity chromatography followed by size exclusion chromatography as previously described. Recombinant monoclonal antibodies were produced by co-transfecting heavy and light chain plasmids into 293 6E cells using PEI Max according to the manufacturer's instructions and purified using Protein A Agarose (Gold Bio).

[00136] Preparation of EBP Reporter Viruses: To produce B-cell tropic GFP reporter viruses (B95-8/F), 5 10⁶ 293-2089 cells were seeded on a 100 mm tissue culture dish in cRPMI containing 100 pg/ml hygromycin. 48 hr later the cells were washed with PBS, the media was replaced with cRPMI without hygromycin, and cells were transfected with 6 pg each of p509 and p2670 expressing BZLF1 and BALF4, respectively, using GeneJuice® transfection reagent (Merck KGAA, Germany). 72 hr post transfection, the cell supernatant was collected and centrifuged at 500 x g for 3 min to pellet any cell debris, and passed through a 0.8 pm filter. Virions were concentrated 25-50-fold by centrifugation at 25,000 × g for 2 hr and re-suspended in PBS. Vims was stored at -80 degrees C and thawed immediately before use.

[00137] Epithelial cell tropic virus was produced from Akata-GFP EBV cells suspended at 4x l0⁶ cells/ml in RPMI containing 1% FBS by adding goat anti-human IgG (Southern Biotech) to a final concentration of 100 pg/ml, and the culture was incubated at 37 degrees C for 4 hr. Cells were then diluted to 2x l0⁶ cells/ml in RPMI containing 1% FBS and cultured for 72 hr. Cultures were centrifuged at 300 x g for 10 min to pellet cells and supernatant was passed through a 0.8 pm filter. Bacitracin was added to a final concentration of 100 pg/ml. Virions were concentrated 25 x by centrifugation at 25,000 x g for 2 hr and re-suspended in RPMI containing 100 pg/ml bacitracin. Virus was stored at -80 degrees C and thawed immediately before use.

[00138] EBV neutralization assay in B cells: EBV neutralization assays were carried out in

Raji cells as previously described. In short, plasma from mice was serially diluted in 25 pl cRPMI in triplicate in 96-well round-bottom plates. 12.5 pl of diluted of B95-8/F virus diluted to achieve an infection frequency of approximately 1-5% was added and plates were incubated for 1 hour at 37 degrees C. Following the incubation, 12.5 pl of Raji cells at 4xl0⁶ cells/ml was added to each well and incubated for an hour at 37 degrees C. Cells were then washed once in cRPMI, re-suspended in fresh cRPMI at 37°C. 72 hr later, cells were fixed in 10% formalin and the percentage of GFP Raji cells was determined using Luminex Guava HT, a BD FACS symphony or BD FACS Celesta.

[00139] To account for any false positive cells due to auto-fluorescence in the GFP channel, the average %GFP⁺ cells in negative control wells (n=5-10) was subtracted from each well. The infectivity' (%GFP⁺) for each well was plotted as a function of the logio of the plasma dilution. Plasma dilution is reported relative to the final assay volume (50 pl). The neutralization curve was fit using the log (inhibitor) vs response-variable slope (four parameters) analysis in GraphPad Prism ® 10.0.2 (GraphPad Software, LLC., California, USA).

[00140] EBV Neutralization Assay in Epithelial Cells: SVKCR2 cells were seeded at a density of 1.5 x 10⁴ cells per well in a 96 well flat-bottom tissue culture plate. The next day plasma was serially diluted in duplicate wells of a 96 well plate, then Akata-GFP virus was added to each well and incubated for 15 min. The media was then aspirated from the SVKCR2 cells and replaced by the antibody-virus mixture. The plates were incubated at 37 degrees C for 48 hr, then cells w ere detached from the plate using 0.25% try psin, transferred to a 96 well round bottom plate, washed twice with PBS, and fixed with 10% formalin. The percentage of GFP⁺ cells were determined on a BD FACS Symphony or BD FACS Celesta and percent neutralization was determined as in the B cell neutralization assay.

[00141] Mice: NOD-SCID I12rgnuii (NSG) mice were housed in a pathogen-free facility. All NSG mice used in this Example were female and 6 weeks old when experiments were initiated.

[00142] Immunizations in C57BL 6 mice: Comparative immunogenicity studies were performed in groups of 4 or 5 C57BL/6 mice between 7 and 10 w eeks of age. Each group included both male and female mice. Blood was collected retro-orbitally before immunization for a baseline measurement. Mice were immunized at weeks 0 and 8. Injections of repRNA were formulated by diluting RNA to the desired concentration in PBS and sterile filtering. Sterile RNA was then mixed as a 1 :1 volumetric ratio with sterile NP-30, 40% sucrose, 100 mM citrate, and RNAse free water. Final repRNA concentrations were 1 pg/ml, 10 pg/ml, 100 pg/ml. immunizations were delivered via split dose intramuscular injection consisting of two 50 pL doses delivered to each rear leg. Blood was collected retro-orbitally every' 2 weeks after the first and second immunizations or via cardiac puncture at indicated timepoints. Blood was collected in citrate coated tubes. Plasma was separated from whole blood via centrifugation, then heat inactivated at 56 degrees C for 30 min. [00143] To generate IgG for passive transfer experiments, immunizations were performed in groups of 25 C57BL/6 mice (12 or 13 male and female, varied per group) between 7 and 10 weeks of age. After collecting a pre-bleed, mice were immunized at weeks 0 and 8 with 5 pg of gH-gL monomer in PBS with 50% (v/v) Sigma Adjuvant System (SAS) (Sigma) for a total volume of 100 pL per immunization. Mice were immunized with 10 pg repRNA via intramuscular injection as above. Blood was collected retro-orbitally at week 8 and via cardiac puncture at week 12. Plasma was separated from whole blood via centrifugation and collected plasma was heat inactivated at 56 degrees Celsius for 30 min.

[00144] Measurement of plasma antibody endpoint binding titers by anti-His capture ELISA: Microplates were prepared by pipetting 30 pl/well of rabbit anti-His tag antibody (Sigma Aldrich) at a concentration of 0.5 pg/ml into 384 well microplates at 4 degrees Celsius for 16 hr in a solution of 0.1 M NaHCO₃ pH 9.4-9.6 (coating buffer). The following day, plates were washed 4 times with lx PBS and 0.02% Tween 20 (ELISA wash buffer) prior to blocking for 1 hour with 90 pl/well of PBS containing 10% non-fat milk and 0.02% Tween 20 (blocking buffer). After blocking, plates were washed 4 times and 30 pl/well of 2 pg/ml monomeric His-tagged gH-gL diluted in blocking buffer was added to the plate and incubated for 1 hour. Plates were washed and plasma diluted in blocking buffer was added to the top row of the plate. Three-fold serial dilutions were performed in duplicate followed by a 1 hour incubation at 37 degrees Celsius. Additional control wells containing immobilized gH-gL but no immune plasma were included. After washing, a 1 :4,000 dilution of goat anti-mouse IgG-HRP (SouthemBiotech) in blocking buffer was added to each well and incubated at 37 degrees Celsius for 1 hr. After four washes, 30 pl/well of SureBlue Reserve™ TMB Microwell Peroxidase substrate (SeraCare) was added. After 5 min, 30 pl/well of IN sulfuric acid was added and the A₄₅₀ of each well was read on a Molecular Devices SpectraMax ® M2 (Molecular Devices, LLC., Delaware, USA) plate reader. The binding threshold was defined as the average plus 10 times the SD of the determined by calculating the average of A₄₅₀ values of the control wells. Endpoint titers were interpolated from the point of the curve that intercepted the binding threshold using the Prism 9.2.0 package. [00145] Competitive binding titers as measured by ELISA: Coating, blocking, and gH-gL immobilization steps were performed. Following capture of monomeric gH-gL, equal amounts of plasma from each mouse in a group were pooled and diluted in blocking buffer and 2-fold serial dilutions were performed, followed by a 1 hr incubation at 37 degrees Celsius. After washing, monoclonal antibodies AMM01, CL40, CL59, E1D1, 769B10, 770F7 , and 1D8 were added at a concentration that achieves half-maximal binding (EC50; pre-determined in the same assay in the absence of competing plasma) to each well containing serially diluted pooled plasma from each group, followed by a 1 hr incubation at 37 degrees Celsius. After four washes with ELISA washing buffer, a 1:20,000 dilution of goat anti-human IgG-HRP (Jackson ImmunoResearch) in blocking buffer was added to each well and incubated at 37 degrees Celsius for 1 h followed by four washes with ELISA wash buffer. Addition of SureBlue Reserve™ TMB Microwell Peroxidase substrate, addition of IN sulfuric acid, and reading of plates was performed as described above. The average A₄₅₀ values of buffer only control wells were subtracted from each mAb containing well and plotted in GraphPad Prism ® 9.2.0. A₄₅₀ values were plotted as a function of the logio of the plasma dilution. A binding curve was fit using the Sigmoidal, 4PL, X is log(concentration) least squares fit function. Maximum binding was defined as the best-fit value for the top of each curve computed in Prism. A₄₅₀ values at each dilution on the curve were divided by the maximum binding and multiplied by 100 to calculate the % of max binding ([A₄₅₀ at each dilution/ max binding] xlOO). The titer at which half- maximal binding was observed was interpolated from the binding curve using the GraphPad Prism ® 9.2.0 package (GraphPad Software).

[00146] Measurement of total plasma IgG: Plasma was serially diluted in ELISA coating buffer in duplicate and incubated on 384-well microplates at 4 degrees Celsius for 16 hours. At least 10 additional control wells were included that contained only coating buffer and no plasma. The next day, plates were w ashed 4 times with ELISA w ash buffer prior to blocking for 1 hour with 100 pl/well of ELISA blocking buffer. After blocking, plates were washed and a 1:4000 dilution of goat anti-mouse IgG Human ads-HRP in ELISA blocking buffer was added to each ell and incubated 1 hour at 37 degrees Celsius. Plates were washed and addition of SureBlue Reserve™ TMB Microwell Peroxidase substrate, addition of IN sulfuric acid, and reading of plates w as performed as described above. The average A₄₅₀ values of buffer only control wells were subtracted from each plasma containing well and plotted in Prism 9.2.0. A₄₅₀ values were plotted as a function of the logio of the plasma dilution. A binding curve was fit using the Sigmoidal, 4PL, X is log(concentration) least squares fit function. The binding threshold was determined as provided above.

[00147] IgG purification from murine plasma: Terminal plasma from each group was pooled, diluted in protein G binding buffer, and passed over a column containing 1 ml of protein A/G resin. The column was then washed 3 times with five column volumes of binding buffer. Finally, IgG was eluted from the resin in 1 ml fractions using IgG elution buffer. Fractions were buffer exchanged into PBS, concentrated, passed through a 0.2 pm filter, and quantified by measuring the absorbance at 280 nm using a Nanodrop® One (Nanodrop Technologies LLC). Example 4: Dose optimization for gH-gL repRNA vaccination.

[00148] A time and dosing schedule for administration of the gH-gL repRNA vaccine are described herein. The gL-P2A-gH construct was selected, which elicited higher binding titers than the gH-P2A-gL construct (FIG. 1A).

[00149] Groups of 4 C57BL/6 mice received 0.1 pg, 1 pg, and 10 pg repRNA formulated with NP-30 at week 0. The animals were bled biweekly and the gH-gL-binding endpoint titer was monitored in near-real time. Mice were immunized as shown in FIG. IB. Reciprocal endpoint gH/gL binding titers were measured in plasma by ELISA (FIG. 1C). The abil ity of plasma pooled from the 4 mice in FIG. 1C to neutralize EBV infection of B cells reported as the reciprocal dilution to reduce infectivity by 50% (ID50). The lines connect the mean (or pooled plasma) across the tested timepoints in FIGS. 1C and ID. Following the priming immunization, dose-dependent increase in the binding titers was observed until week 6, followed by a slight waning by week 8 (FIG. 2A), at which point they received a second immunization with the same dose. The endpoint binding titers were boosted in the animals that received a second 10 pg dose and promoted durability in the immune response up to 2 months. A slight boost was also observed in the animals that received a 1 pg dose, but not in the animals that received a 0.1 pg second dose (FIG. 2A)

[00150] The ability of the immune plasma were examined to neutralize EBV infection of epithelial and B cells in vitro. The trends observed in the endpoint binding titer kinetics were mirrored in the neutralizing titers against epithelial cell infection. Following the prime, there was a dose-dependent increase in epithelial-cell neutralizing titers until week 6 that waned slightly by week 8 and were boosted by a second dose in the 10 pg and 1 pg but not the 0.1 pg groups (FIG. 2C). Only the 10 pg dose elicited antibodies capable of neutralizing B cell infection after a single immunization (FIG. 2E). Two weeks after a second immunization with a 10 pg dose, these titers were boosted over 10-fold to a peak reciprocal half-maximal inhibitory dilution (IDso) near IxlO³' Weak transient neutralizing titers were observed 6 weeks after a second dose with 1 pg of repRNA, while the 0.1 pg dose failed to elicit B-cell neutralizing antibodies at any timepoint (FIG. 2E).

[00151] It was then examined how varying the dose of the prime and boost impacted the immunogenicity of gH-gL repRNA. Two de-escalating dose regimens, 10 pg/ 1 pg and 10 pg/ 0.1 pg, and one escalating dose 0.1 pg/ 10 pg regimen were evaluated. Animals that received a 10 pg prime had comparable binding and B cell neutralizing titers as the original cohort of mice from the 10 pg/ 10 pg group at week 8 (FIGs. 2A-2B, 2E-2F), while the epithelial cell neutralizing titers were lower in the mice in the 10 pg/ 1 pg and 10 pg/ 0. 1 pg at week 8 (FIGs. 2C-2D). None of the binding or neutralizing titers were boosted when the second dose was lower (FIGs. 2B, 2D, 2F,). After the initial dose, the 0.1 pg/ 10 pg group had low gH-gL binding and epithelial cell neutralizing titers (FIGs. 2B and 2D), but no measurable B cell neutralizing titers (FIG. 2F). However, after the boost immunization of 10 pg at week 8, there were slightly higher gH-gL binding titers and epithelial cell neutralizing titers than the other mixed dose groups (FIGs. 2B and 2D), but similar B cell neutralizing titers (FIG. 2F). Across all prime/boost regimens evaluated, a 10 pg prime followed by a 10 pg boost elicited superior binding and neutralizing titers.

Example 5: Anchored gH-gL monomer and gH-gL ectodomain immunogenicity.

[00152] After determining an effective dose regimen for gH-gL repRNA/NP-30 vaccination, the immunogenicity of a secreted gH-gL ectodomain with full-length gH-gL was compared. To produce the ectodomain, gH was truncated at amino acid 679 in the gL-P2A-gH construct. 10 pg of repRNA encoding the ectodomain formulated with NP-30 was delivered at weeks 0 and 8 and plasma samples were collected through week 18. Following a single immunization, the ectodomain elicited gH-gL binding titers that were nearly identical to full length gH-gL (FIG. 3 A), however the epithelial cell neutralizing antibody titers were ~ 10-fold lower than full length gH-gL and this repRNA failed to elicit B cell neutralizing titers by week 8 (FIG. 3B-3C). A second immunization with the ectodomain boosted the epithelial cell neutralizing titers to similar levels achieved with full-length gH-gL, but the B cell neutralizing titers were ~5-fold lower and the neutralizing titers decayed more rapidly in both assays (FIG.

3A-3C).

[00153] Immune plasma from mice vaccinated wi th full length and gH/gL repRNA were evaluated for their ability- to compete for binding to EBV gH/gL with the indicated monoclonal antibodies by competitive ELISA. (FIG. 3D-3J).

[00154] Due to the observed similar overall binding titers but differences in neutralization, it was investigated whether there were qualitative differences in gH-gL epitope-recognition between the plasma the antibodies elicited by these two constructs. Plasma collected at week 12 from both groups was pooled and evaluated for its ability to compete for binding with monoclonal antibodies (mAbs) with defined epitopes on gH-gL, including AMMOL 769B10, E1D1 and CL40, CL59, 1D8, and 770F7 binding. With the exception of E1D1, which binds an epitope entirely on gL, plasma elicited by full length gH-gL competed the binding of all mAbs as more potently than plasma elicited by the gH-gL ectodomain. Full length gH-gL elicits a qualitatively different antibody response than the gH-gL ectodomain as shown by the full-length gH-gL having higher neutralizing titers.

Example 6: Membrane retained gH-gL monomer repRNA immunogenicity relative to secreted gH-gL multimers.

[00155] Multimerization of gH-gL through genetic fusion to self-assembling nanoparticles can improve the immunogenicity of gH-gL. Therefore, the immunogenicity of multimeric gH- gL was evaluated when delivered by repRNA with and without NP-30. Mice were immunized with 10 pg of repRNA encoding the gH-gL ectodomain presented as a different multimeric constructs, a 4-mer, 7-mer, and a 60-mer. Similar gH-gL binding titers were elicited by the gH- gL monomer, the 4-mer. and the 7-mer. while those elicited by the 60-mer were slightly lower (FIG. 4A). In the epithelial cell neutralizing assay, the 7-mer and 60-mer elicited similar titers that were lower than those elicited by the 4-mer (FIG. 4B). In the B cell neutralizing assay, the 4-mer elicited the highest titers, followed by the 7-mer, and then the 60-mer. None of repRNA delivered nanoparticles elicited epithelial or B cell neutralizing antibodies that were as potent as full-length gH-gL (FIG. 4B and 4C). Raw data is shown in Example 11.

Example 7: Methods for the passive transfer of IgG

[00156] The following methods were used to determine whether the gH-gL repRNA vaccination protects humanized mice from lethal EBV challenge.

[00157] EBV infection in humanized mice: Twenty- five six week old NSG mice were irradiated (275R of total body irradiation) and received IxlO⁶ CD34+ huPBSC in 200 pl PBS through intravenous (i.v.) injection. Eight weeks later, successful human cell engraftment was confirmed by the presence of human CD45+ cells in peripheral blood by flow cytometry (FIG. 8A and FIG. 8B). Using 50 pl blood. RBCs were lysed and cells were stained using a BV510 viability dye, and the following antibodies at a 1: 100 dilution unless otherwise noted: hCD45 FITC (eBioscience), mCD45 APC (eBioscience) (1:500 dilution), hCD33 PE (BD Bioscience), hCD19 BV711 (Biolegend), hCD4 AF700 (eBioscience) and hCD8 BV421 (BD Bioscience). Cells were stained for 30 min on ice, washed twice in FACS buffer, fixed in 200 pl of 10% formalin 15 min on ice, washed and resuspended in 200 pl FACS buffer for acquisition and analyzed on a BDFACS Celesta. 10 w eeks post-engrafiment, 500 pg of experimental or control antibodies were injected per humanized NSG mouse via intraperitoneal injection (i.p.). 24 hr later, mice were bled in the left eye to confirm passive transfer of IgG, and received a dose of EBV B95.8/F67 equivalent to 33.000 Raji infectious units as determined by infection of Raji cells via retro-orbital injection in the right eye. Each group of mice receiving the same IgG preparation and/or EBV were housed separately from unchallenged mice to avoid the potential for contamination. Mice were weighed three times weekly. Beginning at two weeks postinfection, peripheral blood samples were collected to measure the presence of EBV DNA in whole blood. Twelve weeks post-challenge, or until mice lost 20% of their starting weight, mice were euthanized. Spleens were photographed (FIG. 10) and weighed (FIG. 5J), then DNA was extracted from splenocytes utilizing the DNeasy ® Blood & Tissue Kit (Qiagen GmbH, Germany) and according to the manufacturer’s instructions for subsequent viral load analysis (FIG. 51)

[00158] Stimulation of splenocytes: Spleens were harvested from 5 male and 5 female mice immunized with repRNA gH-gL or protein monomer gH-gL at week 12 post immunization. Splenocytes were isolated by mechanical dissociation in RBC lysis buffer (ThermoFisher) using a 100 pm fdter. After dissociation and lysis, cells were washed in FACS buffer once and resuspended in 5 ml FACS buffer. In 96-well plates, splenocytes were plated at a concentration of 2x10⁶ cells/well in cRPMI. Cells were stimulated with either cRPMI alone (negative control), 50 pg/ml gH-gL in cRPMI, or 0.5 pg/ml anti-CD3 (ThermoFisher) and 0.25 pg/ml anti-CD28 (ThermoFisher) (positive control). Cells were incubated at 37 degrees Celsius 5% CO2 for 72 hr prior to start of intracellular staining. Five hours before the end of restimulation 20 pl of brefeldin A (eBioscience) at 10 ng/ml and 20 pl lOOOx monensin (eBioscience)was added to each well.

Quantitative PCR analysis of human cells in huCD34 engrafted mice: A primer-probe mix specific for the EBV BALF5 gene was used to quantify EBV in DNA extracted from blood or spleen in hCD34 engrafted NSG recipient mice at the time points described. Each 25 pl qPCR reaction contained 12.5 pl QuantiTect ® Probe PCR Master Mix (Qiagen GmbH, Germany), 600 nM of each primer and 300 nM of FAM-labeled probe (IDT), 1.25 pl of a TaqMan® VIC-labeled RNase- P primer probe mix (Roche Molecular Systems, Inc., Delaware, USA). For analysis of splenocytes, reactions contained 1 pg DNA extracted from splenocytes as template. To analyze EBV in peripheral blood, 50 pl of blood collected via cardiac puncture or retro-orbital bleed DNA extracted using the DNeasy ® Blood and Tissue Kit (QIAGEN) and eluted in 50 pl of Buffer AE (QIAGEN). 10 pl of extracted DNA was used as template in qPCR. Reactions were heated to 95 degrees Celsius for 15 minutes to activate DNA polymerase followed by 50 cycles of 95 degrees Celsius for 15 s 60 degrees Celsius for 60 s, on an Applied Biosystems QuantStudio® 7 Flex Real Time PCR System (Life Technologies Corporation, Delaware, USA). Synthetic DNA fragments containing the BALF5 target gene as well as flanking genomic regions were synthesized as double stranded DNA gBlocks (IDT), and were used to generate a standard curve with known gene copy numbers ranging from 10⁷-10° copies/pl. The copy number in extracted DNA was determined by interpolating from the standard curve. Serial dilutions of reference standard were used to experimentally determine a limit of detection of 6.25 copies, which corresponds to the amount of template that can be detected in > 95% of reactions. For graphical purposes, samples with no amplification or those yielding values below the limit of detection were assigned a value of 0.625 copies.

[00159] Intracellular staining (ICS): After stimulation, plates were centrifuged at 400 x g for 5 min at 8 degrees Celsius and supernatants were transferred to a new plate and frozen at -20 degrees Celsius. Cell pellets were resuspended in 200 pl FACS buffer, centrifuged at 400 x g for 5 min, and resuspended in 50 pl viability staining mix: 1:500 BV510 live-dead dye (eBioscience) and 1:500 Fc Block (Biolegend) in PBS. Cells were stained on ice in dark for 15 min. 150 pl FACS buffer was added to each well, plates were centrifuged 400 x g 5 min. and supernatant removed. Cell pellets were then resuspended in surface staining mix: a 1 :200 dilution of the following anti- mouse CD45 BUV805 (BD Bioscience), CD3 BUV395 (BD Bioscience), CD8 BUV737 (BD Bioscience), and CD4 PerCPCy5.5 (Thermofisher) antibodies in FACS buffer. Cells were stained on ice in dark 30 min. After staining, cells were resuspended in 150 pl FACS buffer and washed once in 200 pl FACS buffer. Cells were then fixed and permeabilized for 20 min on ice using 100 pl IX CytoFix solution (BD Bioscience). Plates then washed twice in IX CytoPerm Wash Buffer (BD Bioscience). ICS was then done by resuspension in 50 pl/well ICS mix: in CytoPerm wash buffer, a 1:200 dilution anti-mouse IFN-y AF488 (Biolegend). Cells were stained on ice in dark 30 min. Cells washed twice in CytoPerm ® wash buffer (Thermo Electron LED GmbH, Germany) and resuspended in FACS buffer for acquisition. Samples were acquired on BD Fortessa X50 cytometer. The frequency of IFNy⁺ cells in the Lymphocyte/Singlet/Live/CD45⁺/CD4⁺ or CD8⁺ population was determined for each sample. The frequency of CD4⁺ or CD8⁺ T cells expressing IFNy from baseline cRPMI stimulation was subtracted from the final reported values.

Example 8: Passive transfer of IgG elicited by gH-gL repRNA vaccination protects humanized mice from lethal EBV challenge

[00160] Having established that two 10 pg doses of repRNA-encoded full length gH-gL delivered at weeks 0 and 8 displayed superior immunogenicity to all other dosing regimens and constructs tested, it was evaluated whether antibodies elicited by this regimen are protective in vivo. To do this, a passive transfer and challenge assay was performed in humanized mice. Use of humanized mice as a small animal model of EBV infection is well established. In short, NSG mice are irradiated, engrafted with human CD34⁺ peripheral blood stem cells, which then reconstitute the human hematopoietic compartment in the mouse. This allows for EBV infection of human B cells in vivo. Humanized mice generate poor antibody responses, therefore it was decided to passively transfer IgG from immunized C57BL/6 mice into humanized mice prior to EBV challenge. This approach has been used for evaluation of mAbs and vaccine elicited antibodies.

[00161] Twenty-five C57BL/6 mice were immunized with 10 pg of monomeric gH-gL repRNA at weeks 0 and 8. To compare this to a more conventional recombinant vaccine, another 25 C57BL/6 mice were given two doses of 5 pg of purified monomeric gH-gL ectodomain formulated with Sigma Adjuvant System at weeks 0 and 8 (FIG. 5A). At week 12, mice were euthanized and IgG was harvested from pooled plasma. IgG purified from mice immunized with repRNA showed stronger binding to gH-gL than IgG purified from protein vaccinated mice (FIG. 7)

[00162] After verifying successful engraftment of human CD45⁺ cells in humanized mice (FIG. 7). 500 pg of purified IgG from mice immunized with gH-gL encoded by repRNA or protein was delivered to groups of 4 humanized mice. An additional 5 humanized mice received 500 pg of IgG purified from unimmunized mice. The next day, mice were bled to confirm IgG transfer and challenged with 33,000 Raji infectious units of EBV. Five mice that did not receive IgG transfer remained unchallenged and served as an uninfected control group (FIG. 5A). No animals had plasma IgG prior to transfer, but all had similar levels at the time of challenge (FIG. 5B). The repRNA group had higher gH-gL specific ELISA titers compared to the protein group (FIG. 5C) consistent with the higher activity of the purified IgG (FIG. 7).

[00163] Starting 2 weeks post challenge and continuing weekly for 10 weeks, mice were weighed three times a week (FIG. 9A-D)) and bled weekly. To monitor for infection, DNA was extracted from whole blood and qPCR was used to measure viral DNA. At week 12, or sooner if humane endpoints were met, mice were euthanized and spleens were examined for splenomegaly (FIG. 5J), tumorigenesis (FIG. 10) and presence of viral DNA (FIG. 51).

[00164] Following challenge, 100% of the mice in the uninfected control group survived (FIG. 5D) and lacked detectable viral DNA in the blood (FIG. 5E) and the spleen (FIG. 51). In contrast, none of the mice in the group that received control IgG survived beyond 8 weeks (FIG. 5D). All were viremic (FIG. 5F) and had high levels of viral DNA in the spleen (FIG. 51). These mice also developed splenomegaly (FIG. 5J) and had splenic tumors (FIG. 10). Three of the mice in the protein group developed viremia by w eek 8 (FIG. 5G) and two required euthanasia at weeks 8 and 9 (FIG. 5D). and the other three survived until week 12 (60% survival). Four of five mice in the protein group had elevated levels of viral DNA in the spleen (FIG. 51), three developed splenomegaly (FIG. 5 J), and two developed splenic tumors (FIG. 10). In the repRNA group, only one mouse exhibited transient low-level viremia (FIG. 5H) and 100% of the mice survived for 12 weeks following challenge (FIG. 5D). At week, 12 the spleen weights were comparable to the uninfected controls (FIG. 5J) and free of viral DNA (FIG. 51) and tumors (FIG. 10). Raw data is shown in Example 11.

[00165] In sum. immunization with gH-gL repRNA elicited higher gH-gL IgG titers that provided superior protection from lethal EBV challenge, as compared to a conventional proteinbased vaccine formulation in a humanized mouse model.

Example 9: Prime and boost immunization with gH-gL monomer repRNA elicited better cellular responses than immunization with gH-gL monomer protein

[00166] The challenge experiments demonstrated that antibodies elicited by repRNA immunization conferred superior antibody-mediated protection than antibodies elicited by protein. To compare cellular immunity elicited by both vaccines, splenocytes were collected from ten animals used to generate IgG for transfer experiments, stimulated with recombinant gH-gL ex vivo, CD4⁺ and CD8⁺ T cells were analyzed for production of IFNy and/or IL-2. Mice vaccinated with repRNA had higher amounts of vaccine specific CD8⁺ T cells, as defined by the frequency of IFN\⁺ CD8⁺ T cells after splenocyte exposure to gH-gL as compared to a media only baseline (FIG. 6A). No significant difference in CD4⁺ T cell vaccine responses were observed (FIG. 6B).

Example 10. Immunogenicity of NP-30/repRNAs encoding EBV gH/gL.

[00167] Mice were immunized with NP-30 formulated with a repRNA encoding for gH/gL and gH/gL/gp42 (See FIG. 1 A) were compared with mice immunized with a repRNA encoding gp50, or recombinant gp350 + Adjuvant. The recombinant gp350 adjuvant is comparable to Alum MPLA used in a Phase II clinical trial). Blood was draw n and serum was isolated 4 weeks and 7 weeks following immunization. Serum protein binding to gp350 was evaluated for off- target effects. Mice immunized with repRNA gH/gL and repRNA gH/gL/gp42 did not have gp350 binding in serum relative to mice immunized with repRNA gH/gL/gp42 + repRNA gp350 and recombinant gp350+ adjuvant (FIG. 12A).

[00168] Serum from mice immunized with repRNA encoding gH/gL, or recombinant gp350 + Adjuvant (from FIG. 12A) was evaluated for its ability to neutralize EBV infection epithelial cells (FIG. 12B) or B cells (FIG. 12C) as indicated. Mice immunized with repRNA encoding gH/gL had both epithelial cell and B cell neutralization of EBV infection at week 4 and week 7 relative to gp350-immunized animals. The data indicates that the EBV gH-gL repRNA conjugated to a nanoparticle (NP-30) effectively induces an immune response to the virus in vivo.

Example 11. Statistical Analysis of Reciprocal Endpoint Titers.

[00169] The tables below provided additional raw data for reciprocal end point titers described in Example 6 and the results of an EBV challenge assay of humanized mice described in Example 8.

[00170] Titers at individual timepoints were compared to the 10 pg/10 pg NP-30 /repRNA encoded full length gH/gL group and summarized below. Significance was calculated by Mann-Whitney test. Week 22 post-immunization of the 10 pg/lOpg NP-30/repRNA encoded full-length gH/gL was compared to week 23 of the multimer groups, and week 32 compared to week 34. Two-tailed p values shown in table.

[00171] Table 3. Statistical analyses for data shown in FIG. 4A-FIG. 4C.

ND - Not Done. Two-tailed p values shown in table

[00172] Table 4. Summary of results from EBV challenge of humanized mice in FIG.

10 after passive transfer of vaccine elicited IgG. Data is shown in FIG. 5B-FIG. 5J.

N/A - Not Applicable.

SEQUENCES

SEQ ID NOS: 1-6: See Table 1

SEQ ID NO: 7 - RNA sequence encoding for EBV glycoprotein H

AUGCAGCUGCUGUGUGUCUUUUGCUUGGUGCUUCUCUGGGAGGUGGGAGCGGCCUCCCUUAGUGAGGUGAAGCUUCAC CUUGAUAUUGAAGGUCAUGCUAGUCAUUAUACAAUAC CUUGGACAGAGCUGAUGGCAAAGGUUC CAGGUUUGAGUC CU GAGGCGCUCUGGAGGGAAGCCAAUGUUACCGAAGACCUGGCCUCAAUGCUCAAUAGAUACAAACUUAUUUACAAGACU UCAGGGACUCUGGGUAUAGCAUUGGCGGAACCAGUAGACAUUCCAGCGGUUUCUGAAGGUUCUAUGCAAGUAGACGCU UCUAAAGUUCACCCUGGUGUUAUAUCCGGGCUUAAUAGCCCGGCUUGUAUGCUGAGCGCACCCUUGGAAAAACAGCUG UUUUACUACAUCGGUACGAUGCUCCCAAACACGCGCCCUCAUUCCUAUGUGUUCUAUCAACUCCGCUGCCAUCUCUCU UAUGUAGCCUUGAGCAUAAAUGGAGACAAGUUUCAGUAUACCGGUGCAAUGACAUCAAAGUUUCUCAUGGGGACUUAC AAGAGGGUAACCGAAAAGGGGGACGAACAUGUCCUUAGUCUGGUGUUUGGGAAGACUAAAGACCUUCCGGAUCUCAGA GGACCGUUUUCUUAUCCAAGCUUGACUUCCGCACAGUCCGGUGAUUACUCCCUGGUCAUUGUUACUACAUUUGUUCAC UAUGCGAACUUCCACAACUAUUUUGUUCCGAAUCUCAAGGAUAUGUUCUCUCGCGCUGUUACUAUGACAGCCGCAAGU UACGCGCGGUAUGUCUUGCAAAAGUUGGUCUUGCUCGAAAUGAAGGGAGGCUGUAGGGAACCGGAACUUGACACCGAA ACACUGACUACGAUGUUCGAAGUUAGUGUAGCUUUUUUCAAGGUCGGCCACGCUGUUGGCGAAACGGGAAACGGUUGC GUUGAUCUGCGCUGGCUGGCAAAAUCCUUUUUUGAAUUGACAGUUCUCAAAGACAUAAUCGGAAUCUGCUAUGGGGCU ACUGUAAAAGGAAUGCAGAGCUACGGACUGGAGAGACUUGCUGCUAUGCUUAUGGCUACAGUUAAGAUGGAGGAGCUU GGACAUCUUACAACCGAGAAGCAAGAAUAUGCGUUGCGAUUGGCAACUGUGGGGUAUCCUAAGGCAGGGGUAUACAGU GGACUGAUAGGAGGAGCUACCAGUGUUCUGCUGAGUGCGUACAACCGGCAUCCGUUGUUCCAACCGCUUCACACAGUA AUGCGAGAGACGCUCUUCAUCGGAAGUCACGUAGUGCUGCGGGAACUGCGACUCAAUGUGACUACGCAGGGCCCGAAU CUCGCGCUGUAUCAGUUGCUGUCUACGGCCUUGUGUAGUGCCUUGGAAAUCGGUGAAGUCUUGAGAGGGUUGGCGCUC GGGACCGAGUCAGGACUCUUUUCCCCAUGCUACCUGAGUCUGAGAUUUGAUCUCACAAGAGAUAAGCUCCUGUCAAUG GCUCCUCAGGAGGCUACUCUUGAUCAGGCGGCCGUCAGUUACGCGGUUGACGGAUUCCUUGGUCGGCUCAGUUUGGAG AGAGAAGAUAGAGAUGCCUGGCAUCUUCCUGCCUACAAAUGUGUCGACAGACUCGAUAAGGUUCUGAUGAUCAUCCCG UUGAUUAAUGUAACCUUCAUAAUCUCCAGUGACAGAGAGGUACGGGGUUCCGCACUCUACGAAGCAUCUACGACAUAC CUCUCCUCAAGUCUUUUUCUUAGUCCUGUCAUCAUGAACAAAUGCUCACAGGGAGCCGUGGCCGGGGAACCACGACAA AUACCGAAGAUCCAGAAUUUUACCAGAACACAGAAGUCCUGCAUCUUCUGCGGCUUUGCUCUCUUGUCAUAUGACGAG AAAGAGGGUUUGGAAACCACCACCUAUAUCACUUCUCAGGAAGUCCAGAAUAGCAUCCUUUCCUCAAACUACUUCGAU UUUGAUAACCUUCACGUC CAUUAC CUGCUC CUCACAACGAACGGAACGGUUAUGGAAAUAGCGGGC CUUUACGAGGAA CGCGCUCAUGUGGUGUUGGCUAUCAUCCUCUACUUUAUAGCCUUUGCCUUGGGCAUAUUCCUGGUACAUAAGAUCGUG AUGUUCUUUCUUCGCAGAAAACGCGGGUCCGGC

SEQ ID NO: 8 - RNA sequence encoding for EBV glycoprotein L

AUGCGGGCGGUGGGAGUAUUCCUCGCAACGUGUCUGGUCACAAUAUUUGUACUCCCUACAUGGGGUAACUGGGCGUAU CCCUGCUGCCAUGUGACACAGCUUCGCGCACAACAUCUCCUUGCACUUGAGAACAUUAGCGACAUAUAUCUUGUAUCA AACCAGACCUGUGACGGAUUCUCCCUUGCCAGUUUGAAUUCACCGAAAAAUGGCUCAAACCAGCUCGUAAUAUCUAGA UGUGCGAAUGGACUCAAUGUGGUCAGCUUCUUUAUAUCUAUUCUCAAGAGAUCUAGCUCUGCUCUUACCGGGCACUUG AGGGAAUUGCUUACCACUCUCGAGACUUUGUACGGAUCAUUCUCAGUCGAAGAUCUUUUUGGGGCCAAUCUGAACAGG UAUGCCUGGCAUCGCGGAGGAUGA

SEQ ID NO: 9 - RNA sequence encoding for EBV gH-P2A-gL gauaggcggcgcaugagagaagcccagaccaauuaccuacccaaaAUGGAGAAAGUUCACGUUGACAUCGAGGAAGAC AGCCCAUUCCUCAGAGCUUUGCAGCGGAGCUUCCCGCAGUUUGAGGUAGAAGCCAAGCAGGUCACUGAUAAUGACCAU GCUAAUGC CAGAGCGUUUUCGCAUCUGGCUUCAAAACUGAUCGAAACGGAGGUGGACC CAUC CGACACGAUC CUUGAC AUUGGAAGUGCGCCCGCCCGCAGAAUGUAUUCUAAGCACAAGUAUCAUUGUAUCUGUCCGAUGAGAUGUGCGGAAGAU CCGGACAGAUUGUAUAAGUAUGCAACUAAGCUGAAGAAAAACUGUAAGGAAAUAACUGAUAAGGAAUUGGACAAGAAA AUGAAGGAGCUGGCCGCCGUCAUGAGCGACCCUGACCUGGAAACUGAGACUAUGUGCCUCCACGACGACGAGUCGUGU CGCUACGAAGGGCAAGUCGCUGUUUACCAGGAUGUAUACGCGGUUGACGGACCGACAAGUCUCUAUCACCAAGCCAAU AAGGGAGUUAGAGUCGCCUACUGGAUAGGCUUUGACACCACCCCUUUUAUGUUUAAGAACUUGGCUGGAGCAUAUCCA UCAUACUCUACCAACUGGGCCGACGAAACCGUGUUAACGGCUCGUAACAUAGGCCUAUGCAGCUCUGACGUUAUGGAG CGGUCACGUAGAGGGAUGUCCAUUCUUAGAAAGAAGUAUUUGAAACCAUCCAACAAUGUUCUAUUCUCUGUUGGCUCG ACCAUCUACCACGAGAAGAGGGACUUACUGAGGAGCUGGCACCUGCCGUCUGUAUUUCACUUACGUGGCAAGCAAAAU UACACAUGUCGGUGUGAGACUAUAGUUAGUUGCGACGGGUACGUCGUUAAAAGAAUAGCUAUCAGUCCAGGCCUGUAU GGGAAGCCUUCAGGCUAUGCUGCUACGAUGCACCGCGAGGGAUUCUUGUGCUGCAAAGUGACAGACACAUUGAACGGG GAGAGGGUCUCUUUUCCCGUGUGCACGUAUGUGCCAGCUACAUUGUGUGACCAAAUGACUGGCAUACUGGCAACAGAU GUGAGUGCGGACGACGCGCAAAAACUGCUGGUUGGGCUCAACCAGCGUAUAGUCGUCAACGGUCGCACCCAGAGAAAC ACCAAUACCAUGAAAAAUUACCUUUUGCCCGUAGUGGCCCAGGCAUUUGCUAGGUGGGCAAAGGAAUAUAAGGAAGAU CAAGAAGAUGAAAGGCCACUAGGACUACGAGAUAGACAGUUAGUCAUGGGGUGUUGUUGGGCUUUUAGAAGGCACAAG AUAACAUCUAUUUAUAAGCGCCCGGAUACCCAAACCAUCAUCAAAGUGAACAGCGAUUUCCACUCAUUCGUGCUGCCC

AGGAUAGGCAGUAACACAUUGGAGAUCGGGCUGAGAACAAGAAUCAGGAAAAUGUUAGAGGAGCACAAGGAGCCGUCA CCUCUCAUUACCGCCGAGGACGUACAAGAAGCUAAGUGCGCAGCCGAUGAGGCUAAGGAGGUGCGUGAAGCCGAGGAG UUGCGCGCAGCUCUACCACCUUUGGCAGCUGAUGUUGAGGAGCCCACUCUGGAGGCAGACGUCGACUUGAUGUUACAA GAGGCUGGGGCCGGCUCAGUGGAGACACCUCGUGGCUUGAUAAAGGUUACCAGCUACGAUGGCGAGGACAAGAUCGGC UCUUACGCUGUGCUUUCUCCGCAGGCUGUACUCAAGAGUGAAAAAUUAUCUUGCAUCCACCCUCUCGCUGAACAAGUC AUAGUGAUAACACACUGUGGCCGAAAAGGGCGUUAUGCCGUGGAACCAUACCAUGGUAAAGUAGUGGUGCCAGAGGGA CAUGCAAUACCCGUCCAGGACUUUCAAGCUCUGAGUGAAAGUGCCACCAUUGUGUACAACGAACGUGAGUUCGUAAAC AGGUACCUGCACCAUAUUGCCACACAUGGAGGAGCGCUGAACACUGAUGAAGAAUAUUACAAAACUGUCAAGCCCAGC GAGCACGACGGCGAAUACCUGUACGACAUCGACAGGAAACAGUGCGUCAAGAAAGAACUAGUCACUGGGCUAGGGCUC ACAGGCGAGCUGGUGGAUCCUCCCUUCCAUGAAUUCGCCUACGAGAGUCUGAGAACACGACCAGCCGCUCCUUACCAA GUAC CAAC CAUAGGGGUGUAUGGCGUGC CAGGAUCAGGCAAGUCUGGCAUCAUUAAAAGCGCAGUCAC CAAAAAAGAU CUAGUGGUGAGCGCCAAGAAAGAAAACUGUGCAGAAAUUAUAAGGGACGUCAAGAAAAUGAAAGGGCUGGACGUCAAU GCCAGAACUGUGGACUCAGUGCUCUUGAAUGGAUGCAAACACCCCGUAGAGACCCUGUAUAUUGACGAAGCUUUUGCU UGUCAUGCAGGUACUCUCAGAGCGCUCAUAGCCAUUAUAAGACCUAAAAAGGCAGUGCUCUGCGGGGAUCCCAAACAG UGCGGUUUUUUUAACAUGAUGUGCCUGAAAGUGCAUUUUAACCACGAGAUUUGCACACAAGUCUUCCACAAAAGCAUC UCUCGC CGUUGCACUAAAUCUGUGACUUCGGUCGUCUCAAC CUUGUUUUACGACAAAAAAAUGAGAACGACGAAUC CG AAAGAGACUAAGAUUGUGAUUGACACUACCGGCAGUACCAAACCUAAGCAGGACGAUCUCAUUCUCACUUGUUUCAGA GGGUGGGUGAAGCAGUUGCAAAUAGAUUACAAAGGCAACGAAAUAAUGACGGCAGCUGCCUCUCAAGGGCUGACCCGU

AAAGGUGUGUAUGCCGUUCGGUACAAGGUGAAUGAAAAUCCUCUGUACGCACCCACCUCAGAACAUGUGAACGUCCUA CUGACCCGCACGGAGGACCGCAUCGUGUGGAAAACACUAGCCGGCGACCCAUGGAUAAAAACACUGACUGCCAAGUAC CCUGGGAAUUUCACUGCCACGAUAGAGGAGUGGCAAGCAGAGCAUGAUGCCAUCAUGAGGCACAUCUUGGAGAGACCG GACCCUACCGACGUCUUCCAGAAUAAGGCAAACGUGUGUUGGGCCAAGGCUUUAGUGCCGGUGCUGAAGACCGCUGGC AUAGACAUGACCACUGAACAAUGGAACACUGUGGAUUAUUUUGAAACGGACAAAGCUCACUCAGCAGAGAUAGUAUUG AACCAACUAUGCGUGAGGUUCUUUGGACUCGAUCUGGACUCCGGUCUAUUUUCUGCACCCACUGUUCCGUUAUCCAUU AGGAAUAAUCACUGGGAUAACUCCCCGUCGCCUAACAUGUACGGGCUGAAUAAAGAAGUGGUCCGUCAGCUCUCUCGC AGGUACCCACAACUGCCUCGGGCAGUUGCCACUGGAAGAGUCUAUGACAUGAACACUGGUACACUGCGCAAUUAUGAU CCGCGCAUAAACCUAGUACCUGUAAACAGAAGACUGCCUCAUGCUUUAGUCCUCCACCAUAAUGAACACCCACAGAGU GACUUUUCUUCAUUCGUCAGCAAAUUGAAGGGCAGAACUGUCCUGGUGGUCGGGGAAAAGUUGUCCGUCCCAGGCAAA AUGGUUGACUGGUUGUCAGACCGGCCUGAGGCUACCUUCAGAGCUCGGCUGGAUUUAGGCAUCCCAGGUGAUGUGCCC AAAUAUGACAUAAUAUUUGUUAAUGUGAGGACCCCAUAUAAAUACCAUCACUAUCAGCAGUGUGAAGACCAUGCCAUU AAGCUUAGCAUGUUGACCAAGAAAGCUUGUCUGCAUCUGAAUCCCGGCGGAACCUGUGUCAGCAUAGGUUAUGGUUAC GCUGACAGGGCCAGCGAAAGCAUCAUUGGUGCUAUAGCGCGGCAGUUCAAGUUUUCCCGGGUAUGCAAACCGAAAUCC UCACUUGAAGAGACGGAAGUUCUGUUUGUAUUCAUUGGGUACGAUCGCAAGGCCCGUACGCACAAUCCUUACAAGCUU UCAUCAAC CUUGAC CAACAUUUAUACAGGUUCCAGACUC CACGAAGC CGGAUGUGCAC CCUCAUAUCAUGUGGUGCGA GGGGAUAUUGCCACGGCCACCGAAGGAGUGAUUAUAAAUGCUGCUAACAGCAAAGGACAACCUGGCGGAGGGGUGUGC GGAGCGCUGUAUAAGAAAUUCCCGGAAAGCUUCGAUUUACAGCCGAUCGAAGUAGGAAAAGCGCGACUGGUCAAAGGU GCAGCUAAACAUAUCAUUCAUGCCGUAGGACCAAACUUCAACAAAGUUUCGGAGGUUGAAGGUGACAAACAGUUGGCA GAGGCUUAUGAGUCCAUCGCUAAGAUUGUCAACGAUAACAAUUACAAGUCAGUAGCGAUUCCACUGUUGUCCACCGGC AUCUUUUCCGGGAACAAAGAUCGACUAACCCAAUCAUUGAACCAUUUGCUGACAGCUUUAGACACCACUGAUGCAGAU GUAGCCAUAUACUGCAGGGACAAGAAAUGGGAAAUGACUCUCAAGGAAGCAGUGGCUAGGAGAGAAGCAGUGGAGGAG AUAUGCAUAUCCGACGACUCUUCAGUGACAGAACCUGAUGCAGAGCUGGUGAGGGUGCAUCCGAAGAGUUCUUUGGCU GGAAGGAAGGGCUACAGCACAAGCGAUGGCAAAACUUUCUCAUAUUUGGAAGGGACCAAGUUUCACCAGGCGGCCAAG GAUAUAGCAGAAAUUAAUGCCAUGUGGCCCGUUGCAACGGAGGCCAAUGAGCAGGUAUGCAUGUAUAUCCUCGGAGAA AGCAUGAGCAGUAUUAGGUCGAAAUGCCCCGUCGAAGAGUCGGAAGCCUCCACACCACCUAGCACGCUGCCUUGCUUG UGCAUCCAUGCCAUGACUCCAGAAAGAGUACAGCGCCUAAAAGCCUCACGUCCAGAACAAAUUACUGUGUGCUCAUCC UUUCCAUUGCCGAAGUAUAGAAUCACUGGUGUGCAGAAGAUCCAAUGCUCCCAGCCUAUAUUGUUCUCACCGAAAGUG CCUGCGUAUAUUCAUCCAAGGAAGUAUCUCGUGGAAACACCACCGGUAGACGAGACUCCGGAGCCAUCGGCAGAGAAC CAAUCCACAGAGGGGACACCUGAACAACCACCACUUAUAACCGAGGAUGAGACCAGGACUAGAACGCCUGAGCCGAUC AUCAUCGAAGAGGAAGAAGAGGAUAGCAUAAGUUUGCUGUCAGAUGGCCCGACCCACCAGGUGCUGCAAGUCGAGGCA GACAUUCACGGGCCGCCCUCUGUAUCUAGCUCAUCCUGGUCCAUUCCUCAUGCAUCCGACUUUGAUGUGGACAGUUUA UC CAUACUUGACAC CCUGGAGGGAGCUAGCGUGAC CAGCGGGGCAACGUCAGC CGAGACUAACUCUUACUUCGCAAAG AGUAUGGAGUUUCUGGCGCGACCGGUGCCUGCGCCUCGAACAGUAUUCAGGAACCCUCCACAUCCCGCUCCGCGCACA AGAACACCGUCACUUGCACCCAGCAGGGCCUGCUCGAGAACCAGCCUAGUUUCCACCCCGCCAGGCGUGAAUAGGGUG AUCACUAGAGAGGAGCUCGAGGCGCUUACCCCGUCACGCACUCCUAGCAGGUCGGUCUCGAGAACCAGCCUGGUCUCC AACCCGCCAGGCGUAAAUAGGGUGAUUACAAGAGAGGAGUUUGAGGCGUUCGUAGCACAACAACAAUGACGGUUUGAU GCGGGUGCAUACAUCUUUUCCUCCGACACCGGUCAAGGGCAUUUACAACAAAAAUCAGUAAGGCAAACGGUGCUAUCC GAAGUGGUGUUGGAGAGGACCGAAUUGGAGAUUUCGUAUGCCCCGCGCCUCGACCAAGAAAAAGAAGAAUUACUACGC AAGAAAUUACAGUUAAAUCCCACACCUGCUAACAGAAGCAGAUACCAGUCCAGGAAGGUGGAGAACAUGAAAGCCAUA ACAGCUAGACGUAUUCUGCAAGGCCUAGGGCAUUAUUUGAAGGCAGAAGGAAAAGUGGAGUGCUACCGAACCCUGCAU CCUGUUCCUUUGUAUUCAUCUAGUGUGAACCGUGCCUUUUCAAGCCCCAAGGUCGCAGUGGAAGCCUGUAACGCCAUG UUGAAAGAGAACUUUCCGACUGUGGCUUCUUACUGUAUUAUUCCAGAGUACGAUGCCUAUUUGGACAUGGUUGACGGA GCUUCAUGCUGCUUAGACACUGCCAGUUUUUGCCCUGCAAAGCUGCGCAGCUUUCCAAAGAAACACUCCUAUUUGGAA CCCACAAUACGAUCGGCAGUGCCUUCAGCGAUCCAGAACACGCUCCAGAACGUCCUGGCAGCUGCCACAAAAAGAAAU UGCAAUGUCACGCAAAUGAGAGAAUUGCCCGUAUUGGAUUCGGCGGCCUUUAAUGUGGAAUGCUUCAAGAAAUAUGCG UGUAAUAAUGAAUAUUGGGAAACGUUUAAAGAAAACCCCAUCAGGCUUACUGAAGAAAACGUGGUAAAUUACAUUACC AAAUUAAAAGGACCAAAAGCUGCUGCUCUUUUUGCGAAGACACAUAAUUUGAAUAUGUUGCAGGACAUACCAAUGGAC AGGUUUGUAAUGGACUUAAAGAGAGACGUGAAAGUGACUCCAGGAACAAAACAUACUGAAGAACGGCCCAAGGUACAG

GUGAUCCAGGCUGCCGAUCCGCUAGCAACAGCGUAUCUGUGCGGAAUCCACCGAGAGCUGGUUAGGAGAUUAAAUGCG

GUCCUGCUUCCGAACAUUCAUACACUGUUUGAUAUGUCGGCUGAAGACUUUGACGCUAUUAUAGCCGAGCACUUCCAG

CCUGGGGAUUGUGUUCUGGAAACUGACAUCGCGUCGUUUGAUAAAAGUGAGGACGACGCCAUGGCUCUGACCGCGUUA

AUGAUUCUGGAAGACUUAGGUGUGGACGCAGAGCUGUUGACGCUGAUUGAGGCGGCUUUGGGCGAAAUUUCAUCAAUA

CAUUUGCCCACUAAAACUAAAUUUAAAUUCGGAGCCAUGAUGAAAUCUGGAAUGUUCCUCACACUGUUUGUGAACACA

GUCAUUAACAUUGUAAUCGCAAGCAGAGUGUUGAGAGAACGGCUAACCGGAUCACCAUGUGCAGCAUUCAUUGGAGAU

GACAAUAUCGUGAAAGGAGUCAAAUCGGACAAAUUAAUGGCAGACAGGUGCGCCACCUGGUUGAAUAUGGAAGUCAAG

AUUAUAGAUGCUGUGGUGGGCGAGAAAGCGCCUUAUUUCUGUGGAGGGUUUAUUUUGUGUGACUCCGUGACCGGCACA

GCGUGCCGUGUGGCAGACCCCCUAAAAAGGCUGUUUAAGCUUGGCAAACCUCUGGCAGCAGACGAUGAACAUGAUGAU

GACAGGAGAAGGGCAUUGCAUGAAGAGUCAACACGCUGGAACCGAGUGGGUAUUCUUUCAGAGCUGUGCAAGGCAGUA

GAAUCAAGGUAUGAAACCGUAGGAACUUCCAUCAUAGUUAUGGCCAUGACUACUCUAGCUAGCAGUGUUAAAUCAUUC

AGCUACCUGAGAGGGGCCCCUAUAACUCUCUACGGCUAAccugaauggacuacgacauagucuaguccgccgccaccA

UGCAGCUGCUGUGUGUCUUUUGCUUGGUGCUUCUCUGGGAGGUGGGAGCGGCCUCCCUUAGUGAGGUGAAGCUUCACC

UUGAUAUUGAAGGUCAUGCUAGUCAUUAUACAAUACCUUGGACAGAGCUGAUGGCAAAGGUUCCAGGUUUGAGUCCUG

AGGCGCUCUGGAGGGAAGCCAAUGUUACCGAAGACCUGGCCUCAAUGCUCAAUAGAUACAAACUUAUUUACAAGACUU

CAGGGACUCUGGGUAUAGCAUUGGCGGAACCAGUAGACAUUCCAGCGGUUUCUGAAGGUUCUAUGCAAGUAGACGCUU

CUAAAGUUCACCCUGGUGUUAUAUCCGGGCUUAAUAGCCCGGCUUGUAUGCUGAGCGCACCCUUGGAAAAACAGCUGU

UUUACUACAUCGGUACGAUGCUCCCAAACACGCGCCCUCAUUCCUAUGUGUUCUAUCAACUCCGCUGCCAUCUCUCUU

AUGUAGCCUUGAGCAUAAAUGGAGACAAGUUUCAGUAUACCGGUGCAAUGACAUCAAAGUUUCUCAUGGGGACUUACA

AGAGGGUAACCGAAAAGGGGGACGAACAUGUCCUUAGUCUGGUGUUUGGGAAGACUAAAGACCUUCCGGAUCUCAGAG

GACCGUUUUCUUAUCCAAGCUUGACUUCCGCACAGUCCGGUGAUUACUCCCUGGUCAUUGUUACUACAUUUGUUCACU

AUGCGAACUUCCACAACUAUUUUGUUCCGAAUCUCAAGGAUAUGUUCUCUCGCGCUGUUACUAUGACAGCCGCAAGUU

ACGCGCGGUAUGUCUUGCAAAAGUUGGUCUUGCUCGAAAUGAAGGGAGGCUGUAGGGAACCGGAACUUGACACCGAAA

CACUGACUACGAUGUUCGAAGUUAGUGUAGCUUUUUUCAAGGUCGGCCACGCUGUUGGCGAAACGGGAAACGGUUGCG

UUGAUCUGCGCUGGCUGGCAAAAUCCUUUUUUGAAUUGACAGUUCUCAAAGACAUAAUCGGAAUCUGCUAUGGGGCUA

CUGUAAAAGGAAUGCAGAGCUACGGACUGGAGAGACUUGCUGCUAUGCUUAUGGCUACAGUUAAGAUGGAGGAGCUUG

GACAUCUUACAACCGAGAAGCAAGAAUAUGCGUUGCGAUUGGCAACUGUGGGGUAUCCUAAGGCAGGGGUAUACAGUG

GACUGAUAGGAGGAGCUACCAGUGUUCUGCUGAGUGCGUACAACCGGCAUCCGUUGUUCCAACCGCUUCACACAGUAA

UGCGAGAGACGCUCUUCAUCGGAAGUCACGUAGUGCUGCGGGAACUGCGACUCAAUGUGACUACGCAGGGCCCGAAUC

UCGCGCUGUAUCAGUUGCUGUCUACGGCCUUGUGUAGUGCCUUGGAAAUCGGUGAAGUCUUGAGAGGGUUGGCGCUCG

GGACCGAGUCAGGACUCUUUUCCCCAUGCUACCUGAGUCUGAGAUUUGAUCUCACAAGAGAUAAGCUCCUGUCAAUGG

CUCCUCAGGAGGCUACUCUUGAUCAGGCGGCCGUCAGUUACGCGGUUGACGGAUUCCUUGGUCGGCUCAGUUUGGAGA

GAGAAGAUAGAGAUGCCUGGCAUCUUCCUGCCUACAAAUGUGUCGACAGACUCGAUAAGGUUCUGAUGAUCAUCCCGU

UGAUUAAUGUAACCUUCAUAAUCUCCAGUGACAGAGAGGUACGGGGUUCCGCACUCUACGAAGCAUCUACGACAUACC

UCUCCUCAAGUCUUUUUCUUAGUCCUGUCAUCAUGAACAAAUGCUCACAGGGAGCCGUGGCCGGGGAACCACGACAAA

UACCGAAGAUCCAGAAUUUUACCAGAACACAGAAGUCCUGCAUCUUCUGCGGCUUUGCUCUCUUGUCAUAUGACGAGA

AAGAGGGUUUGGAAACCACCACCUAUAUCACUUCUCAGGAAGUCCAGAAUAGCAUCCUUUCCUCAAACUACUUCGAUU

UUGAUAACCUUCACGUCCAUUACCUGCUCCUCACAACGAACGGAACGGUUAUGGAAAUAGCGGGCCUUUACGAGGAAC

GCGCUCAUGUGGUGUUGGCUAUCAUCCUCUACUUUAUAGCCUUUGCCUUGGGCAUAUUCCUGGUACAUAAGAUCGUGA

UGUUCUUUCUUCGCAGAAAACGCGGGUCCGGCgcgaccaauuuuucacugcuuaaacaagccggagacguugaggaaa acccaggaccgAUGCGGGCGGUGGGAGUAUUCCUCGCAACGUGUCUGGUCACAAUAUUUGUACUCCCUACAUGGGGUA ACUGGGCGUAUCCCUGCUGCCAUGUGACACAGCUUCGCGCACAACAUCUCCUUGCACUUGAGAACAUUAGCGACAUAU AUCUUGUAUCAAACCAGACCUGUGACGGAUUCUCCCUUGCCAGUUUGAAUUCACCGAAAAAUGGCUCAAACCAGCUCG UAAUAUCUAGAUGUGCGAAUGGACUCAAUGUGGUCAGCUUCUUUAUAUCUAUUCUCAAGAGAUCUAGCUCUGCUCUUA CCGGGCACUUGAGGGAAUUGCUUACCACUCUCGAGACUUUGUACGGAUCAUUCUCAGUCGAAGAUCUUUUUGGGGCCA AUCUGAACAGGUAUGCCUGGCAUCGCGGAGGAUGAuaaccgcggugucaaaaaccgcguggacgugguuaacaucccu gcugggaggaucagccguaauuauuauaauuggcuuggugcuggcuacuauuguggccauguacgugcugaccaacca gaaacauaauugaauacagcagcaauuggcaagcugcuuacauagaacucgcggcgauuggcaugccgccuuaaaauu uuuauuuuauuuuuucuuuucuuuuccgaaucggauuuuguuuuuaauauuucaaaaaaaaaaaaaaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaa

SEQ ID NO: 10 - RNA sequence encoding for EBV gL-P2A-gH gauaggcggcgcaugagagaagcccagaccaauuaccuacccaaaAUGGAGAAAGUUCACGUUGACAUCGAGGAAGAC AGCCCAUUCCUCAGAGCUUUGCAGCGGAGCUUCCCGCAGUUUGAGGUAGAAGCCAAGCAGGUCACUGAUAAUGACCAU GCUAAUGC CAGAGCGUUUUCGCAUCUGGCUUCAAAACUGAUCGAAACGGAGGUGGACC CAUC CGACACGAUC CUUGAC AUUGGAAGUGCGCCCGCCCGCAGAAUGUAUUCUAAGCACAAGUAUCAUUGUAUCUGUCCGAUGAGAUGUGCGGAAGAU CCGGACAGAUUGUAUAAGUAUGCAACUAAGCUGAAGAAAAACUGUAAGGAAAUAACUGAUAAGGAAUUGGACAAGAAA AUGAAGGAGCUGGCCGCCGUCAUGAGCGACCCUGACCUGGAAACUGAGACUAUGUGCCUCCACGACGACGAGUCGUGU CGCUACGAAGGGCAAGUCGCUGUUUACCAGGAUGUAUACGCGGUUGACGGACCGACAAGUCUCUAUCACCAAGCCAAU AAGGGAGUUAGAGUCGCCUACUGGAUAGGCUUUGACACCACCCCUUUUAUGUUUAAGAACUUGGCUGGAGCAUAUCCA UCAUACUCUACCAACUGGGCCGACGAAACCGUGUUAACGGCUCGUAACAUAGGCCUAUGCAGCUCUGACGUUAUGGAG CGGUCACGUAGAGGGAUGUCCAUUCUUAGAAAGAAGUAUUUGAAACCAUCCAACAAUGUUCUAUUCUCUGUUGGCUCG ACCAUCUACCACGAGAAGAGGGACUUACUGAGGAGCUGGCACCUGCCGUCUGUAUUUCACUUACGUGGCAAGCAAAAU UACACAUGUCGGUGUGAGACUAUAGUUAGUUGCGACGGGUACGUCGUUAAAAGAAUAGCUAUCAGUCCAGGCCUGUAU GGGAAGCCUUCAGGCUAUGCUGCUACGAUGCACCGCGAGGGAUUCUUGUGCUGCAAAGUGACAGACACAUUGAACGGG GAGAGGGUCUCUUUUCCCGUGUGCACGUAUGUGCCAGCUACAUUGUGUGACCAAAUGACUGGCAUACUGGCAACAGAU GUCAGUGCGGACGACGCGCAAAAACUGCUGGUUGGGCUCAACCAGCGUAUAGUCGUCAACGGUCGCACCCAGAGAAAC ACCAAUACCAUGAAAAAUUACCUUUUGCCCGUAGUGGCCCAGGCAUUUGCUAGGUGGGCAAAGGAAUAUAAGGAAGAU CAAGAAGAUGAAAGGCCACUAGGACUACGAGAUAGACAGUUAGUCAUGGGGUGUUGUUGGGCUUUUAGAAGGCACAAG AUAACAUCUAUUUAUAAGCGCCCGGAUACCCAAACCAUCAUCAAAGUGAACAGCGAUUUCCACUCAUUCGUGCUGCCC AGGAUAGGCAGUAACACAUUGGAGAUCGGGCUGAGAACAAGAAUCAGGAAAAUGUUAGAGGAGCACAAGGAGCCGUCA CCUCUCAUUACCGCCGAGGACGUACAAGAAGCUAAGUGCGCAGCCGAUGAGGCUAAGGAGGUGCGUGAAGCCGAGGAG UUGCGCGCAGCUCUACCACCUUUGGCAGCUGAUGUUGAGGAGCCCACUCUGGAGGCAGACGUCGACUUGAUGUUACAA GAGGCUGGGGCCGGCUCAGUGGAGACACCUCGUGGCUUGAUAAAGGUUACCAGCUACGAUGGCGAGGACAAGAUCGGC UCUUACGCUGUGCUUUCUCCGCAGGCUGUACUCAAGAGUGAAAAAUUAUCUUGCAUCCACCCUCUCGCUGAACAAGUC AUAGUGAUAACACACUCUGGCCGAAAAGGGCGUUAUGCCGUGGAACCAUACCAUGGUAAAGUAGUGGUGCCAGAGGGA CAUGCAAUACCCGUCCAGGACUUUCAAGCUCUGAGUGAAAGUGCCACCAUUGUGUACAACGAACGUGAGUUCGUAAAC AGGUACCUGCACCAUAUUGCCACACAUGGAGGAGCGCUGAACACUGAUGAAGAAUAUUACAAAACUGUCAAGCCCAGC GAGCACGACGGCGAAUACCUGUACGACAUCGACAGGAAACAGUGCGUCAAGAAAGAACUAGUCACUGGGCUAGGGCUC ACAGGCGAGCUGGUGGAUCCUCCCUUCCAUGAAUUCGCCUACGAGAGUCUGAGAACACGACCAGCCGCUCCUUACCAA GUAC CAAC CAUAGGGGUGUAUGGCGUGC CAGGAUCAGGCAAGUCUGGCAUCAUUAAAAGCGCAGUCAC CAAAAAAGAU CUAGUGGUGAGCGCCAAGAAAGAAAACUGUGCAGAAAUUAUAAGGGACGUCAAGAAAAUGAAAGGGCUGGACGUCAAU

GCCAGAACUGUGGACUCAGUGCUCUUGAAUGGAUGCAAACACCCCGUAGAGACCCUGUAUAUUGACGAAGCUUUUGCU UGUCAUGCAGGUACUCUCAGAGCGCUCAUAGCCAUUAUAAGACCUAAAAAGGCAGUGCUCUGCGGGGAUCCCAAACAG UGCGGUUUUUUUAACAUGAUGUGCCUGAAAGUGCAUUUUAACCACGAGAUUUGCACACAAGUCUUCCACAAAAGCAUC UCUCGC CGUUGCACUAAAUCUGUGACUUCGGUCGUCUCAAC CUUGUUUUACGACAAAAAAAUGAGAACGACGAAUC CG AAAGAGACUAAGAUUGUGAUUGACACUACCGGCAGUACCAAACCUAAGCAGGACGAUCUCAUUCUCACUUGUUUCAGA GGGUGGGUGAAGCAGUUGCAAAUAGAUUACAAAGGCAACGAAAUAAUGACGGCAGCUGCCUCUCAAGGGCUGACCCGU AAAGGUGUGUAUGCCGUUCGGUACAAGGUGAAUGAAAAUCCUCUGUACGCACCCACCUCAGAACAUGUGAACGUCCUA CUGACCCGCACGGAGGACCGCAUCGUGUGGAAAACACUAGCCGGCGACCCAUGGAUAAAAACACUGACUGCCAAGUAC CCUGGGAAUUUCACUGCCACGAUAGAGGAGUGGCAAGCAGAGCAUGAUGCCAUCAUGAGGCACAUCUUGGAGAGACCG GACCCUACCGACGUCUUCCAGAAUAAGGCAAACGUGUGUUGGGCCAAGGCUUUAGUGCCGGUGCUGAAGACCGCUGGC AUAGACAUGACCACUGAACAAUGGAACACUGUGGAUUAUUUUGAAACGGACAAAGCUCACUCAGCAGAGAUAGUAUUG AACCAACUAUGCGUGAGGUUCUUUGGACUCGAUCUGGACUCCGGUCUAUUUUCUGCACCCACUGUUCCGUUAUCCAUU AGGAAUAAUCACUGGGAUAACUCCCCGUCGCCUAACAUGUACGGGCUGAAUAAAGAAGUGGUCCGUCAGCUCUCUCGC AGGUACCCACAACUGCCUCGGGCAGUUGCCACUGGAAGAGUCUAUGACAUGAACACUGGUACACUGCGCAAUUAUGAU CCGCGCAUAAACCUAGUACCUGUAAACAGAAGACUGCCUCAUGCUUUAGUCCUCCACCAUAAUGAACACCCACAGAGU GACUUUUCUUCAUUCGUCAGCAAAUUGAAGGGCAGAACUGUCCUGGUGGUGGGGGAAAAGUUGUGCGUCCCAGGCAAA AUGGUUGACUGGUUGUCAGACCGGCCUGAGGCUACCUUCAGAGCUCGGCUGGAUUUAGGCAUCCCAGGUGAUGUGCCC AAAUAUGACAUAAUAUUUGUUAAUGUGAGGACCCCAUAUAAAUACCAUCACUAUCAGCAGUGUGAAGACCAUGCCAUU AAGCUUAGCAUGUUGACCAAGAAAGCUUGUCUGCAUCUGAAUCCCGGCGGAACCUGUGUCAGCAUAGGUUAUGGUUAC GCUGACAGGGCCAGCGAAAGCAUCAUUGGUGCUAUAGCGCGGCAGUUCAAGUUUUCCCGGGUAUGCAAACCGAAAUCC UCACUUGAAGAGACGGAAGUUCUGUUUGUAUUCAUUGGGUACGAUCGCAAGGCCCGUACGCACAAUCCUUACAAGCUU UCAUCAAC CUUGAC CAACAUUUAUACAGGUUCCAGACUC CACGAAGC CGGAUGUGCAC CCUCAUAUCAUGUGGUGCGA GGGGAUAUUGCCACGGCCACCGAAGGAGUGAUUAUAAAUGCUGCUAACAGCAAAGGACAACCUGGCGGAGGGGUGUGC GGAGCGCUGUAUAAGAAAUUCCCGGAAAGCUUCGAUUUACAGCCGAUCGAAGUAGGAAAAGCGCGACUGGUCAAAGGU GCAGCUAAACAUAUCAUUCAUGCCGUAGGACCAAACUUCAACAAAGUUUCGGAGGUUGAAGGUGACAAACAGUUGGCA GAGGCUUAUGAGUCCAUCGCUAAGAUUGUCAACGAUAACAAUUACAAGUCAGUAGCGAUUCCACUGUUGUCCACCGGC AUCUUUUCCGGGAACAAAGAUCGACUAACCCAAUCAUUGAACCAUUUGCUGACAGCUUUAGACACCACUGAUGCAGAU GUAGCCAUAUACUGCAGGGACAAGAAAUGGGAAAUGACUCUCAAGGAAGCAGUGGCUAGGAGAGAAGCAGUGGAGGAG AUAUGCAUAUCCGACGACUCUUCAGUGACAGAACCUGAUGCAGAGCUGGUGAGGGUGCAUCCGAAGAGUUCUUUGGCU GGAAGGAAGGGCUACAGCACAAGCGAUGGCAAAACUUUCUCAUAUUUGGAAGGGACCAAGUUUCACCAGGCGGCCAAG GAUAUAGCAGAAAUUAAUGCCAUGUGGCCCGUUGCAACGGAGGCCAAUGAGCAGGUAUGCAUGUAUAUCCUCGGAGAA AGCAUGAGCAGUAUUAGGUCGAAAUGCCCCGUCGAAGAGUCGGAAGCCUCCACACCACCUAGCACGCUGCCUUGCUUG UGCAUCCAUGCCAUGACUCCAGAAAGAGUACAGCGCCUAAAAGCCUCACGUCCAGAACAAAUUACUGUGUGCUCAUCC UUUCCAUUGCCGAAGUAUAGAAUCACUGGUGUGCAGAAGAUCCAAUGCUCCCAGCCUAUAUUGUUCUCACCGAAAGUG CCUGCGUAUAUUCAUCCAAGGAAGUAUCUCGUGGAAACACCACCGGUAGACGAGACUCCGGAGCCAUCGGCAGAGAAC CAAUCCACAGAGGGGACACCUGAACAACCACCACUUAUAACCGAGGAUGAGACCAGGACUAGAACGCCUGAGCCGAUC AUCAUCGAAGAGGAAGAAGAGGAUAGCAUAAGUUUGCUGUCAGAUGGCCCGACCCACCAGGUGCUGCAAGUCGAGGCA GACAUUCACGGGCCGCCCUCUGUAUCUAGCUCAUCCUGGUCCAUUCCUCAUGCAUCCGACUUUGAUGUGGACAGUUUA UC CAUACUUGACAC CCUGGAGGGAGCUAGCGUGAC CAGCGGGGCAACGUCAGC CGAGACUAACUCUUACUUCGCAAAG AGUAUGGAGUUUCUGGCGCGACCGGUGCCUGCGCCUCGAACAGUAUUCAGGAACCCUCCACAUCCCGCUCCGCGCACA AGAACACCGUCACUUGCACCCAGCAGGGCCUGCUCGAGAACCAGCCUAGUUUCCACCCCGCCAGGCGUGAAUAGGGUG AUCACUAGAGAGGAGCUCGAGGCGCUUACCCCGUCACGCACUCCUAGCAGGUCGGUCUCGAGAACCAGCCUGGUCUCC AACCCGCCAGGCGUAAAUAGGGUGAUUACAAGAGAGGAGUUUGAGGCGUUCGUAGCACAACAACAAUGACGGUUUGAU GCGGGUGCAUACAUCUUUUCCUCCGACACCGGUCAAGGGCAUUUACAACAAAAAUCAGUAAGGCAAACGGUGCUAUCC

GAAGUGGUGUUGGAGAGGACCGAAUUGGAGAUUUCGUAUGCCCCGCGCCUCGACCAAGAAAAAGAAGAAUUACUACGC

AAGAAAUUACAGUUAAAUCCCACACCUGCUAACAGAAGCAGAUACCAGUCCAGGAAGGUGGAGAACAUGAAAGCCAUA

ACAGCUAGACGUAUUCUGCAAGGCCUAGGGCAUUAUUUGAAGGCAGAAGGAAAAGUGGAGUGCUACCGAACCCUGCAU

CCUGUUCCUUUGUAUUCAUCUAGUGUGAACCGUGCCUUUUCAAGCCCCAAGGUCGCAGUGGAAGCCUGUAACGCCAUG

UUGAAAGAGAACUUUCCGACUGUGGCUUCUUACUGUAUUAUUCCAGAGUACGAUGCCUAUUUGGACAUGGUUGACGGA

GCUUCAUGCUGCUUAGACACUGCCAGUUUUUGCCCUGCAAAGCUGCGCAGCUUUCCAAAGAAACACUCCUAUUUGGAA

CCCACAAUACGAUCGGCAGUGCCUUCAGCGAUCCAGAACACGCUCCAGAACGUCCUGGCAGCUGCCACAAAAAGAAAU

UGCAAUGUCACGCAAAUGAGAGAAUUGCCCGUAUUGGAUUCGGCGGCCUUUAAUGUGGAAUGCUUCAAGAAAUAUGCG

UGUAAUAAUGAAUAUUGGGAAACGUUUAAAGAAAACCCCAUCAGGCUUACUGAAGAAAACGUGGUAAAUUACAUUACC

AAAUUAAAAGGACCAAAAGCUGCUGCUCUUUUUGCGAAGACACAUAAUUUGAAUAUGUUGCAGGACAUACCAAUGGAC

AGGUUUGUAAUGGACUUAAAGAGAGACGUGAAAGUGACUCCAGGAACAAAACAUACUGAAGAACGGCCCAAGGUACAG

GUGAUCCAGGCUGCCGAUCCGCUAGCAACAGCGUAUCUGUGCGGAAUCCACCGAGAGCUGGUUAGGAGAUUAAAUGCG

GUCCUGCUUCCGAACAUUCAUACACUGUUUGAUAUGUCGGCUGAAGACUUUGACGCUAUUAUAGCCGAGCACUUCCAG

CCUGGGGAUUGUGUUCUGGAAACUGACAUCGCGUCGUUUGAUAAAAGUGAGGACGACGCCAUGGCUCUGACCGCGUUA

AUGAUUCUGGAAGACUUAGGUGUGGACGCAGAGCUGUUGACGCUGAUUGAGGCGGCUUUCGGCGAAAUUUCAUCAAUA

CAUUUGCCCACUAAAACUAAAUUUAAAUUCGGAGCCAUGAUGAAAUCUGGAAUGUUCCUCACACUGUUUGUGAACACA

GUCAUUAACAUUGUAAUCGCAAGCAGAGUGUUGAGAGAACGGCUAACCGGAUCACCAUGUGCAGCAUUCAUUGGAGAU

GACAAUAUCGUGAAAGGAGUCAAAUCGGACAAAUUAAUGGCAGACAGGUGCGCCACCUGGUUGAAUAUGGAAGUCAAG

AUUAUAGAUGCUGUGGUGGGCGAGAAAGCGCCUUAUUUCUGUGGAGGGUUUAUUUUGUGUGACUCCGUGACCGGCACA

GCGUGCCGUGUGGCAGACCCCCUAAAAAGGCUGUUUAAGCUUGGCAAACCUCUGGCAGCAGACGAUGAACAUGAUGAU

GACAGGAGAAGGGCAUUGCAUGAAGAGUCAACACGCUGGAACCGAGUGGGUAUUCUUUCAGAGCUGUGCAAGGCAGUA

GAAUCAAGGUAUGAAACCGUAGGAACUUCCAUCAUAGUUAUGGCCAUGACUACUCUAGCUAGCAGUGUUAAAUCAUUC

AGCUACCUGAGAGGGGCCCCUAUAACUCUCUACGGCUAAccugaauggacuacgacauagucuaguccgccgccaccA

UGCGGGCGGUGGGAGUAUUCCUCGCAACGUGUCUGGUCACAAUAUUUGUACUCCCUACAUGGGGUAACUGGGCGUAUC

CCUGCUGCCAUGUGACACAGCUUCGCGCACAACAUCUCCUUGCACUUGAGAACAUUAGCGACAUAUAUCUUGUAUCAA

ACCAGACCUGUGACGGAUUCUCCCUUGCCAGUUUGAAUUCACCGAAAAAUGGCUCAAACCAGCUCGUAAUAUCUAGAU

GUGCGAAUGGACUCAAUGUGGUCAGCUUCUUUAUAUCUAUUCUCAAGAGAUCUAGCUCUGCUCUUACCGGGCACUUGA

GGGAAUUGCUUACCACUCUCgagacuuuguacggaucauucucagucgaagaucuuuuuggggccaaucugaacaggu augccuggcaucgcggaggacgcagaaaacgcggguccggcgcgaccaauuuuucacugcuuaaacaagccggagacg uugaggaaaacccaggaccgAUGCAGCUGCUGUGUGUCUUUUGCUUGGUGCUUCUCUGGGAGGUGGGAGCGGCCUCCC

UUAGUGAGGUGAAGCUUCACCUUGAUAUUGAAGGUCAUGCUAGUCAUUAUACAAUACCUUGGACAGAGCUGAUGGCAA

AGGUUCCAGGUUUGAGUCCUGAGGCGCUCUGGAGGGAAGCCAAUGUUACCGAAGACCUGGCCUCAAUGCUCAAUAGAU

ACAAACUUAUUUACAAGACUUCAGGGACUCUGGGUAUAGCAUUGGCGGAACCAGUAGACAUUCCAGCGGUUUCUGAAG

GUUCUAUGCAAGUAGACGCUUCUAAAGUUCACCCUGGUGUUAUAUCCGGGCUUAAUAGCCCGGCUUGUAUGCUGAGCG

CACCCUUGGAAAAACAGCUGUUUUACUACAUCGGUACGAUGCUCCCAAACACGCGCCCUCAUUCCUAUGUGUUCUAUC

AACUCCGCUGCCAUCUCUCUUAUGUAGCCUUGAGCAUAAAUGGAGACAAGUUUCAGUAUACCGGUGCAAUGACAUCAA

AGUUUCUCAUGGGGACUUACAAGAGGGUAACCGAAAAGGGGGACGAACAUGUCCUUAGUCUGGUGUUUGGGAAGACUA

AAGACCUUCCGGAUCUCAGAGGACCGUUUUCUUAUCCAAGCUUGACUUCCGCACAGUCCGGUGAUUACUCCCUGGUCA

UUGUUACUACAUUUGUUCACUAUGCGAACUUCCACAACUAUUUUGUUCCGAAUCUCAAGGAUAUGUUCUCUCGCGCUG

UUACUAUGACAGCCGCAAGUUACGCGCGGUAUGUCUUGCAAAAGUUGGUCUUGCUCGAAAUGAAGGGAGGCUGUAGGG

AACCGGAACUUGACACCGAAACACUGACUACGAUGUUCGAAGUUAGUGUAGCUUUUUUCAAGGUCGGCCACGCUGUUG GCGAAACGGGAAACGGUUGCGUUGAUCUGCGCUGGCUGGCAAAAUCCUUUUUUGAAUUGACAGUUCUCAAAGACAUAA

UCGGAAUCUGCUAUGGGGCUACUGUAAAAGGAAUGCAGAGCUACGGACUGGAGAGACUUGCUGCUAUGCUUAUGGCUA

CAGUUAAGAUGGAGGAGCUUGGACAUCUUACAACCGAGAAGCAAGAAUAUGCGUUGCGAUUGGCAACUGUGGGGUAUC

CUAAGGCAGGGGUAUACAGUGGACUGAUAGGAGGAGCUACCAGUGUUCUGCUGAGUGCGUACAACCGGCAUCCGUUGU

UCCAACCGCUUCACACAGUAAUGCGAGAGACGCUCUUCAUCGGAAGUCACGUAGUGCUGCGGGAACUGCGACUCAAUG

UGACUACGCAGGGCCCGAAUCUCGCGCUGUAUCAGUUGCUGUCUACGGCCUUGUGUAGUGCCUUGGAAAUCGGUGAAG

UCUUGAGAGGGUUGGCGCUCGGGACCGAGUCAGGACUCUUUUCCCCAUGCUACCUGAGUCUGAGAUUUGAUCUCACAA

GAGAUAAGCUCCUGUCAAUGGCUCCUCAGGAGGCUACUCUUGAUCAGGCGGCCGUCAGUUACGCGGUUGACGGAUUCC

UUGGUCGGCUCAGUUUGGAGAGAGAAGAUAGAGAUGCCUGGCAUCUUCCUGCCUACAAAUGUGUCGACAGACUCGAUA

AGGUUCUGAUGAUCAUCCCGUUGAUUAAUGUAACCUUCAUAAUCUCCAGUGACAGAGAGGUACGGGGUUCCGCACUCU

ACGAAGCAUCUACGACAUACCUCUCCUCAAGUCUUUUUCUUAGUCCUGUCAUCAUGAACAAAUGCUCACAGGGAGCCG

UGGCCGGGGAACCACGACAAAUACCGAAGAUCCAGAAUUUUACCAGAACACAGAAGUCCUGCAUCUUCUGCGGCUUUG

CUCUCUUGUCAUAUGACGAGAAAGAGGGUUUGGAAACCACCACCUAUAUCACUUCUCAGGAAGUCCAGAAUAGCAUCC

UUUCCUCAAACUACUUCGAUUUUGAUAACCUUCACGUCCAUUACCUGCUCCUCACAACGAACGGAACGGUUAUGGAAA

UAGCGGGCCUUUACGAGGAACGCGCUCAUGUGGUGUUGGCUAUCAUCCUCUACUUUAUAGCCUUUGCCUUGGGCAUAU

UCCUGGUACAUAAGAUCGUGAUGUUCUUUCUUUGAuaaccgcggugucaaaaaccgcguggacgugguuaacaucccu gcugggaggaucagccguaauuauuauaauuggcuuggugcuggcuacuauuguggccauguacgugcugaccaacca gaaacauaauugaauacagcagcaauuggcaagcugcuuacauagaacucgcggcgauuggcaugccgccuuaaaauu uuuauuuuauuuuuucuuuucuuuuccgaaucggauuuuguuuuuaauauuucaaaaaaaaaaaaaaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaa

SEQ ID NO: 11 -RNA sequence encoding for Kaposi’s sarcoma virus glycoprotein H - Based on ORF22 NCBI Reference: NC_009333.1:37212-39416 Human herpesvirus 8 strain GK18, complete genome

AUGCAGGGUCUAGCCUUCUUGGCGGCCCUUGCAUGCUGGCGAUGCAUAUCGUUGACAUGUGGAGCCACUGGCGCGUUG

CCGACAACGGCGACGACAAUAACCCGCUCCGCCACGCAGCUCAUCAAUGGGAGAACCAACCUCUCCAUAGAACUGGAA

UUCAACGGCACUAGUUUUUUUCUAAAUUGGCAAAAUCUGUUGAAUGUGAUCACGGAGCCGGCCCUGACAGAGUUGUGG

ACCUCCGCCGAAGUCGCCGAGGACCUCAGGGUAACUCUGAAAAAGAGGCAAAGUCUUUUUUUCCCCAACAAGACAGUU

GUGAUCUCUGGAGACGGCCAUCGCUAUACGUGCGAGGUGCCGACGUCGUCGCAAACUUAUAACAUCACCAAGGGCUUU

AACUAUAGCGCUCUGCCCGGGCACCUUGGCGGAUUUGGGAUCAACGCGCGUCUGGUACUGGGUGAUAUCUUCGCAUCA

AAAUGGUCGCUAUUCGCGAGGGACACCCCAGAGUAUCGGGUGUUUUACCCAAUGAAUGUCAUGGCCGUCAAGUUUUCC

AUAUCCAUUGGCAACAACGAGUCCGGCGUAGCGCUCUAUGGAGUGGUGUCGGAAGAUUUCGUGGUCGUCACGCUCCAC

AACAGGUCCAAAGAGGCUAACGAGACGGCGUCCCAUCUUCUGUUCGGUCUCCCGGAUUCACUGCCAUCUCUGAAGGGC

CAUGCCACCUAUGAUGAACUCACGUUCGCCCGAAACGCAAAAUAUGCGCUAGUGGCGAUCCUGCCUAAAGAUUCUUAC

CAGACACUCCUUACAGAGAAUUACACUCGCAUAUUUCUGAACAUGACGGAGUCGACGCCCCUCGAGUUCACGCGGACG

AUCCAGACCAGGAUCGUAUCAAUCGAGGCCAGGCGCGCCUGCGCAGCUCAAGAGGCGGCGCCGGACAUAUUCUUGGUG

UUGUUUCAGAUGUUGGUGGCACACUUUCUUGUUGCGCGGGGCAUUGCCGAGCACCGAUUUGUGGAGGUGGACUGCGUG

UGUCGGCAGUAUGCGGAACUGUAUUUUCUCCGCCGCAUCUCGCGUCUGUGCAUGCCCACGUUCACCACUGUCGGGUAU

AACCACACCACCCUUGGCGCUGUGGCCGCCACACAAAUAGCUCGCGUGUCCGCCACGAAGUUGGCCAGUUUGCCCCGC

UCUUCCCAGGAAACAGUGCUGGCCAUGGUCCAGCUUGGCGCCCGUGAUGGCGCCGUCCCUUCCUCCAUUCUGGAGGGC

AUUGCUAUGGUCGUCGAACAUAUGUAUACCGCCUACACUUAUGUGUACACACUCGGCGAUACUGAAAGAAAAUUAAUG

UUGGACAUACACACGGUCCUCACCGACAGCUGCCCGCCCAAAGACUCCGGAGUAUCAGAAAAGCUACUGAGAACAUAU

UUGAUGUUCACAUCAAUGUGUACCAACAUAGAGCUGGGCGAAAUGAUCGCCCGCUUUUCCAAACCGGACAGCCUUAAC AUCUAUAGGGCAUUCUCCCCCUGCUUUCUAGGACUAAGGUACGAUUUGCAUCCAGCCAAGUUGCGCGCCGAGGCGCCG

CAGUCGUCCGCUCUGACGCGGACUGCCGUUGCCAGAGGAACAUCGGGAUUCGCAGAAUUGCUCCACGCGCUGCACCUC

GAUAGCUUAAAUUUAAUUCCGGCGAUUAACUGUUCAAAGAUUACAGCCGACAAGAUAAUAGCUACGGUACCCUUGCCU

CACGUCACGUAUAUCAUCAGUUCCGAAGCACUCUCGAACGCUGUUGUCUACGAGGUGUCGGAGAUCUUCCUCAAGAGU

GCCAUGUUUAUAUCUGCUAUCAAACCCGAUUGCUCCGGCUUUAACUUUUCUCAGAUUGAUAGGCACAUUCCCAUAGUC

UACAACAUCAGCACACCAAGAAGAGGUUGCCCCCUUUGUGACUCUGUAAUCAUGAGCUACGAUGAGAGCGAUGGCCUG

CAGUCUCUCAUGUAUGUCACUAAUGAAAGGGUGCAGACCAACCUCUUUUUAGAUAAGUCACCUUUCUUUGAUAAUAAC

AACCUACACAUUCAUUAUUUGUGGCUGAGGGACAACGGGACCGUAGUGGAGAUAAGGGGCAUGUAUAGAAGACGCGCA

GCCAGUGCUUUGUUUCUAAUUCUCUCUUUUAUUGGGUUCUCGGGGGUUAUCUACUUUCUUUACAGACUGUUUUCCAUC CUUUAUUAGACGGUCAAUAAA

SEQ ID NO: 12 - RNA sequence encoding for Kaposi’s sarcoma virus glycoprotein L - Based on ORF47 NCBI Reference: NC_009333.1:c70014-67444 Human herpesvirus 8 strain GK18, complete genome

AUGGGGAUCUUUGCGCUAUUUGCCGUCCUGUGGACCACCCUAUUGGUCACAUCUCACGCAUACGUCGCCUUACCAUGU

UGCGCAAUUCAGGCAUCGGCAGCCUCUACCCUGCCGUUGUUCUUUGCGGUCCACUCUAUCCACUUCGCCGAUCCGAAU

CACUGCAACGGGGUCUGUAUAGCCAAGCUGCGAAGCAAAACAGGCGACAUUACCGUGGAAACAUGCGUGAAUGGGUUU

AAUCUGAGGUCAUUUUUAGUCGCGGUCGUUCGAAGAUUGGGGUCCUGGGCGUCGCAGGAAAACCUGAGGUUGUUGUGG

UAUUUACAACGAAGUUUGACGGCCUAUACUGUAGGUUUUAACGCGACCACUGCAGAUAGCUCUAUUCACAACGUAAAC

AUAAUUAUAAUAAGCGUGGGAAAGGCCAUGAACCGGACAGGUUCUGUUAGCGGAAGUCAGACUCGGGCUAAAAGCAGC

AGCCGGAGAGCGCACGCAGGUCAAAAGGGAAAAUAAGUUCAACAUGGACGCAUGGUUGCAACAGACGGUCUUUAGGGG

CACCCUAUCCAUCAGUCAGGGGGUGGACGACCGGGAUCUGUUACUGGCACCUAAGUGGAUUUCCUUUCUGAGCCUCUC

AUCAUUUCUGAAACAGAAACUGCUCUCGCUGCUCAGACAGAUUCGGGAACUUAGGCUAACCACCACAGUGUAUCCCCC

ACAGGACAAGCUGAUGUGGUGGUCCCACUGCUGCGAUCCAGAGGAUAUUAAAGUGGUGAUCUUAGGCCAGGACCCGUA

CCACAAGGGCCAAGCUACUGGCCUGGCGUUUAGUGUGGAUCCGCAAUGUCAGGUUCCACCCAGUUUGAGAAGCAUCUU

UAGAGAGCUAGAGGCUUCCGUCCCCAAUUUCAGUACUCCUUCCCACGGGUGCCUCGACAGCUGGGCUCGCCAGGGUGU

GUUGCUACUAAACACAGUUUUGACGGUGGAGAAGGGGAGGGCCGGCUCACACGAGGGACUUGGCUGGGAUUGGUUCAC

GAGUUUCAUCAUCAGUAGCAUAUCCUCAAAGUUAGAACAUUGCGUUUUUCUCCUGUGGGGGCGCAAGGCCAUUGACAG

AACUCCGCUCAUAAACGCACAGAAACACCUGGUGCUUACGGCCCAGCAUCCAUCUCCGCUGGCCUCUCUUGGUGGCCG

ACACUCGCGAUGGCCUCGGUUCCAGGGCUGUAAUCACUUUAACCUAGCCAACGACUAUUUGACUCGCCACCGGCGUGA

GACUGUGGACUGGGGCCUGUUGGAGCAGUAAAGGCAAUAACUCGUGUGCUUUGUAAAUUUCCGCCCCUAGCGGUCAAC

CCCGUACAAGGCCAUGGCGAUGUUUGUGAGGACCUCGUCUAGCACACACGAUGAAGAGAGAAUGCUUCCAAUUGAAGG

AGCGCCUCGCAGACGACCCCCCGUGAAGUUCAUAUUCCCACCUCCACCUCUUUCAUCACUUCCAGGAUUUGGCAGGCC

GCGCGGCUAUGCUGGACCCACGGUGAUAGAUAUGUCUGCCCCAGACGACGUCUUCGCCGAGGACACGCCAUCGCCGCC

AGCAACCCCUCUGGAUCUACAGAUAUCCCCGGAUCAGUCGAGCGGCGAAUCUGAAUAUGACGAGGAUGAGGAAGAUGA

AGAUGAAGAAGAAAAUGACGAUGUUCAGGAGGAAGACGAGCCAGAGGGGUACCCUGCAGACUUUUUUCAACCUUUAUC

UCACUUGCGCCCGAGGCCUCUGGCCAGACGGGCCCAUACGCCCAAACCGGUAGCAGUGGUAGCGGGCCGCGUGCGCAG

UUCAACGGACACGGCGGAGUCCGAGGCGUCCAUGGGAUGGGUUAGUCAGGAUGACGGAUUUUCCCCUGCUGGGCUCUC

ACCUUCAGACGACGAGGGGGUUGCUAUCCUGGAACCGAUGGCGGCAUACACUGGGACCGGGGCAUACGGACUUUCACC

UGCUUCCAGAAAUAGUGUACCUGGAACACAAAGUUCACCAUACAGCGACCCUGAUGAAGGGCCCUCGUGGCGCCCCCU

GCGCGCCGCACCCACCGCGAUCGUCGACCUGACAUCGGACUCUGAUAGCGAUGACAGUUCCAACUCUCCGGACGUGAA

CAAUGAGGCCGCGUUUACCGACGCGCGCCAUUUUUCCCACCAGCCACCCUGGUCCGAGGAGGACGGAGAAGACCAAGG

GGAAGUAUUGAGUCAGAGAAUCGGGCUCAUGGACGUGGGCCAGAAGCGCAAAAGGCAGUCUACCGCCUCCUCUGGUAG CGAGGAUGUGGUGCGCUGCCAGAGACAACCAAACUUAAGCCGCAAAGCAGUGGCGUCUGUGAUAAUUAUAUCCUCGGG

GAGUGACACAGACGAGGAGCCCUCGUCCGCCGUGAGCGUGAUCGUGUCUCCGUCGAGCACAAAGGGUCACCUCCCAAC

CCAAUCUCCCAGUACUUCCGCCCACUCGAUUUCAUCAGGAAGCACAACUACCGCGGGGUCCAGGUGCAGCGACCCAAC

CCGCAUCCUGGCCUCCACGCCACCCCUGUGUGGAAACGGUGCAUAUAACUGGCCGUGGCUGGACUGAUAAAUAAA

SEQ ID NO: 13 - P2A sequence

ATNFSLLKQAGDVEENPGP

SEQ ID NO: 14 VEEV RNA Sequence

AUAGGCGGCGCAUGAGAGAAGCCCAGACCAAUUACCUACCCAAAAUGGAGAAAGUUCACGUUGACAUCGAGGAAGACA

GCCCAUUCCUCAGAGCUUUGCAGCGGAGCUUCCCGCAGUUUGAGGUAGAAGCCAAGCAGGUCACUGAUAAUGACCAUG

CUAAUGCCAGAGCGUUUUCGCAUCUGGCUUCAAAACUGAUCGAAACGGAGGUGGACCCAUCCGACACGAUCCUUGACA

UUGGAAGUGCGCCCGCCCGCAGAAUGUAUUCUAAGCACAAGUAUCAUUGUAUCUGUCCGAUGAGAUGUGCGGAAGAUC

CGGACAGAUUGUAUAAGUAUGCAACUAAGCUGAAGAAAAACUGUAAGGAAAUAACUGAUAAGGAAUUGGACAAGAAAA

UGAAGGAGCUGGCCGCCGUCAUGAGCGACCCUGACCUGGAAACUGAGACUAUGUGCCUCCACGACGACGAGUCGUGUC

GCUACGAAGGGCAAGUCGCUGUUUACCAGGAUGUAUACGCGGUUGACGGACCGACAAGUCUCUAUCACCAAGCCAAUA

AGGGAGUUAGAGUCGCCUACUGGAUAGGCUUUGACACCACCCCUUUUAUGUUUAAGAACUUGGCUGGAGCAUAUCCAU

CAUACUCUACCAACUGGGCCGACGAAACCGUGUUAACGGCUCGUAACAUAGGCCUAUGCAGCUCUGACGUUAUGGAGC

GGUCACGUAGAGGGAUGUCCAUUCUUAGAAAGAAGUAUUUGAAACCAUCCAACAAUGUUCUAUUCUCUGUUGGCUCGA

CCAUCUACCACGAGAAGAGGGACUUACUGAGGAGCUGGCACCUGCCGUCUGUAUUUCACUUACGUGGCAAGCAAAAUU

ACACAUGUCGGUGUGAGACUAUAGUUAGUUGCGACGGGUACGUCGUUAAAAGAAUAGCUAUCAGUCCAGGCCUGUAUG

GGAAGCCUUCAGGCUAUGCUGCUACGAUGCACCGCGAGGGAUUCUUGUGCUGCAAAGUGACAGACACAUUGAACGGGG

AGAGGGUCUCUUUUCCCGUGUGCACGUAUGUGCCAGCUACAUUGUGUGACCAAAUGACUGGCAUACUGGCAACAGAUG

UCAGUGCGGACGACGCGCAAAAACUGCUGGUUGGGCUCAACCAGCGUAUAGUCGUCAACGGUCGCACCCAGAGAAACA

CCAAUACCAUGAAAAAUUACCUUUUGCCCGUAGUGGCCCAGGCAUUUGCUAGGUGGGCAAAGGAAUAUAAGGAAGAUC

AAGAAGAUGAAAGGCCACUAGGACUACGAGAUAGACAGUUAGUCAUGGGGUGUUGUUGGGCUUUUAGAAGGCACAAGA

UAACAUCUAUUUAUAAGCGCCCGGAUACCCAAACCAUCAUCAAAGUGAACAGCGAUUUCCACUCAUUCGUGCUGCCCA

GGAUAGGCAGUAACACAUUGGAGAUCGGGCUGAGAACAAGAAUCAGGAAAAUGUUAGAGGAGCACAAGGAGCCGUCAC

CUCUCAUUACCGCCGAGGACGUACAAGAAGCUAAGUGCGCAGCCGAUGAGGCUAAGGAGGUGCGUGAAGCCGAGGAGU

UGCGCGCAGCUCUACCACCUUUGGCAGCUGAUGUUGAGGAGCCCACUCUGGAGGCAGACGUCGACUUGAUGUUACAAG

AGGCUGGGGCCGGCUCAGUGGAGACACCUCGUGGCUUGAUAAAGGUUACCAGCUACGAUGGCGAGGACAAGAUCGGCU

CUUACGCUGUGCUUUCUCCGCAGGCUGUACUCAAGAGUGAAAAAUUAUCUUGCAUCCACCCUCUCGCUGAACAAGUCA

UAGUGAUAACACACUCUGGCCGAAAAGGGCGUUAUGCCGUGGAACCAUACCAUGGUAAAGUAGUGGUGCCAGAGGGAC

AUGCAAUACCCGUCCAGGACUUUCAAGCUCUGAGUGAAAGUGCCACCAUUGUGUACAACGAACGUGAGUUCGUAAACA

GGUACCUGCACCAUAUUGCCACACAUGGAGGAGCGCUGAACACUGAUGAAGAAUAUUACAAAACUGUCAAGCCCAGCG

AGCACGACGGCGAAUACCUGUACGACAUCGACAGGAAACAGUGCGUCAAGAAAGAACUAGUCACUGGGCUAGGGCUCA

CAGGCGAGCUGGUGGAUCCUCCCUUCCAUGAAUUCGCCUACGAGAGUCUGAGAACACGACCAGCCGCUCCUUACCAAG

UACCAACCAUAGGGGUGUAUGGCGUGCCAGGAUCAGGCAAGUCUGGCAUCAUUAAAAGCGCAGUCACCAAAAAAGAUC

UAGUGGUGAGCGCCAAGAAAGAAAACUGUGCAGAAAUUAUAAGGGACGUCAAGAAAAUGAAAGGGCUGGACGUCAAUG

CCAGAACUGUGGACUCAGUGCUCUUGAAUGGAUGCAAACACCCCGUAGAGACCCUGUAUAUUGACGAAGCUUUUGCUU

GUCAUGCAGGUACUCUCAGAGCGCUCAUAGCCAUUAUAAGACCUAAAAAGGCAGUGCUCUGCGGGGAUCCCAAACAGU

GCGGUUUUUUUAACAUGAUGUGCCUGAAAGUGCAUUUUAACCACGAGAUUUGCACACAAGUCUUCCACAAAAGCAUCU CUCGCCGUUGCACUAAAUCUGUGACUUCGGUCGUCUCAACCUUGUUUUACGACAAAAAAAUGAGAACGACGAAUCCGA AAGAGACUAAGAUUGUGAUUGACACUACCGGCAGUACCAAACCUAAGCAGGACGAUCUCAUUCUCACUUGUUUCAGAG GGUGGGUGAAGCAGUUGCAAAUAGAUUACAAAGGCAACGAAAUAAUGACGGCAGCUGCCUCUCAAGGGCUGACCCGUA AAGGUGUGUAUGCCGUUCGGUACAAGGUGAAUGAAAAUCCUCUGUACGCACCCACCUCAGAACAUGUGAACGUCCUAC UGACCCGCACGGAGGACCGCAUCGUGUGGAAAACACUAGCCGGCGACCCAUGGAUAAAAACACUGACUGCCAAGUACC CUGGGAAUUUCACUGCCACGAUAGAGGAGUGGCAAGCAGAGCAUGAUGCCAUCAUGAGGCACAUCUUGGAGAGACCGG ACCCUACCGACGUCUUCCAGAAUAAGGCAAACGUGUGUUGGGCCAAGGCUUUAGUGCCGGUGCUGAAGACCGCUGGCA UAGACAUGACCACUGAACAAUGGAACACUGUGGAUUAUUUUGAAACGGACAAAGCUCACUCAGCAGAGAUAGUAUUGA ACCAACUAUGCGUGAGGUUCUUUGGACUCGAUCUGGACUCCGGUCUAUUUUCUGCACCCACUGUUCCGUUAUCCAUUA GGAAUAAUCACUGGGAUAACUCCCCGUCGCCUAACAUGUACGGGCUGAAUAAAGAAGUGGUCCGUCAGCUCUCUCGCA GGUACCCACAACUGCCUCGGGCAGUUGCCACUGGAAGAGUCUAUGACAUGAACACUGGUACACUGCGCAAUUAUGAUC CGCGCAUAAACCUAGUACCUGUAAACAGAAGACUGCCUCAUGCUUUAGUCCUCCACCAUAAUGAACACCCACAGAGUG ACUUUUCUUCAUUCGUCAGCAAAUUGAAGGGCAGAACUGUCCUGGUGGUCGGGGAAAAGUUGUCCGUCCCAGGCAAAA UGGUUGACUGGUUGUCAGACCGGCCUGAGGCUACCUUCAGAGCUCGGCUGGAUUUAGGCAUCCCAGGUGAUGUGCCCA AAUAUGACAUAAUAUUUGUUAAUGUGAGGACCCCAUAUAAAUACCAUCACUAUCAGCAGUGUGAAGACCAUGCCAUUA AGCUUAGCAUGUUGACCAAGAAAGCUUGUCUGCAUCUGAAUCCCGGCGGAACCUGUGUCAGCAUAGGUUAUGGUUACG CUGACAGGGCCAGCGAAAGCAUCAUUGGUGCUAUAGCGCGGCAGUUCAAGUUUUCCCGGGUAUGCAAACCGAAAUCCU CACUUGAAGAGACGGAAGUUCUGUUUGUAUUCAUUGGGUACGAUCGCAAGGCCCGUACGCACAAUCCUUACAAGCUUU CAUCAACCUUGACCAACAUUUAUACAGGUUCCAGACUCCACGAAGCCGGAUGUGCACCCUCAUAUCAUGUGGUGCGAG GGGAUAUUGCCACGGCCACCGAAGGAGUGAUUAUAAAUGCUGCUAACAGCAAAGGACAACCUGGCGGAGGGGUGUGCG GAGCGCUGUAUAAGAAAUUGCCGGAAAGCUUCGAUUUACAGCCGAUCGAAGUAGGAAAAGCGCGACUGGUCAAAGGUG CAGCUAAACAUAUCAUUCAUGCCGUAGGACCAAACUUCAACAAAGUUUCGGAGGUUGAAGGUGACAAACAGUUGGCAG AGGCUUAUGAGUCCAUCGCUAAGAUUGUCAACGAUAACAAUUACAAGUCAGUAGCGAUUCCACUGUUGUCCACCGGCA UCUUUUCCGGGAACAAAGAUCGACUAACCCAAUCAUUGAACCAUUUGCUGACAGCUUUAGACACCACUGAUGCAGAUG UAGCCAUAUACUGCAGGGACAAGAAAUGGGAAAUGACUCUCAAGGAAGCAGUGGCUAGGAGAGAAGCAGUGGAGGAGA

UAUGCAUAUCCGACGACUCUUCAGUGACAGAACCUGAUGCAGAGCUGGUGAGGGUGCAUCCGAAGAGUUCUUUGGCUG GAAGGAAGGGCUACAGCACAAGCGAUGGCAAAACUUUCUCAUAUUUGGAAGGGACCAAGUUUCACCAGGCGGCCAAGG AUAUAGCAGAAAUUAAUGCCAUGUGGCCCGUUGCAACGGAGGCCAAUGAGCAGGUAUGCAUGUAUAUCCUCGGAGAAA GCAUGAGCAGUAUUAGGUCGAAAUGCCCCGUCGAAGAGUCGGAAGCCUCCACACCACCUAGCACGCUGCCUUGCUUGU GCAUCCAUGC CAUGACUC CAGAAAGAGUACAGCGC CUAAAAGCCUCACGUC CAGAACAAAUUACUGUGUGCUCAUC CU UUCCAUUGCCGAAGUAUAGAAUCACUGGUGUGCAGAAGAUCCAAUGCUCCCAGCCUAUAUUGUUCUCACCGAAAGUGC CUGCGUAUAUUCAUCCAAGGAAGUAUCUCGUGGAAACACCACCGGUAGACGAGACUCCGGAGCCAUCGGCAGAGAACC AAUC CACAGAGGGGACAC CUGAACAACCAC CACUUAUAACCGAGGAUGAGACCAGGACUAGAACGC CUGAGC CGAUCA UCAUCGAAGAGGAAGAAGAGGAUAGCAUAAGUUUGCUGUCAGAUGGCCCGACCCACCAGGUGCUGCAAGUCGAGGCAG ACAUUCACGGGCCGCCCUCUGUAUCUAGCUCAUCCUGGUCCAUUCCUCAUGCAUCCGACUUUGAUGUGGACAGUUUAU CCAUACUUGACACCCUGGAGGGAGCUAGCGUGACCAGCGGGGCAACGUCAGCCGAGACUAACUCUUACUUCGCAAAGA GUAUGGAGUUUCUGGCGCGACCGGUGCCUGCGCCUCGAACAGUAUUCAGGAACCCUCCACAUCCCGCUCCGCGCACAA GAACACCGUCACUUGCACCCAGCAGGGCCUGCUCGAGAACCAGCCUAGUUUCCACCCCGCCAGGCGUGAAUAGGGUGA UCACUAGAGAGGAGCUCGAGGCGCUUACCCCGUCACGCACUCCUAGCAGGUCGGUCUCGAGAACCAGCCUGGUCUCCA ACCCGCCAGGCGUAAAUAGGGUGAUUACAAGAGAGGAGUUUGAGGCGUUCGUAGCACAACAACAAUGACGGUUUGAUG CGGGUGCAUACAUCUUUUCCUCCGACACCGGUCAAGGGCAUUUACAACAAAAAUCAGUAAGGCAAACGGUGCUAUCCG AAGUGGUGUUGGAGAGGACCGAAUUGGAGAUUUCGUAUGCCCCGCGCCUCGACCAAGAAAAAGAAGAAUUACUACGCA AGAAAUUACAGUUAAAUCCCACACCUGCUAACAGAAGCAGAUACCAGUCCAGGAAGGUGGAGAACAUGAAAGCCAUAA

CAGCUAGACGUAUUCUGCAAGGCCUAGGGCAUUAUUUGAAGGCAGAAGGAAAAGUGGAGUGCUACCGAACCCUGCAUC CUGUUCCUUUGUAUUCAUCUAGUGUGAACCGUGCCUUUUCAAGCCCCAAGGUCGCAGUGGAAGCCUGUAACGCCAUGU UGAAAGAGAACUUUCCGACUGUGGCUUCUUACUGUAUUAUUCCAGAGUACGAUGCCUAUUUGGACAUGGUUGACGGAG CUUCAUGCUGCUUAGACACUGCCAGUUUUUGCCCUGCAAAGCUGCGCAGCUUUCCAAAGAAACACUCCUAUUUGGAAC CCACAAUACGAUCGGCAGUGCCUUCAGCGAUCCAGAACACGCUCCAGAACGUCCUGGCAGCUGCCACAAAAAGAAAUU GCAAUGUCACGCAAAUGAGAGAAUUGCCCGUAUUGGAUUCGGCGGCCUUUAAUGUGGAAUGCUUCAAGAAAUAUGCGU

GUAAUAAUGAAUAUUGGGAAACGUUUAAAGAAAAC CC CAUCAGGCUUACUGAAGAAAACGUGGUAAAUUACAUUAC CA AAUUAAAAGGACCAAAAGCUGCUGCUCUUUUUGCGAAGACACAUAAUUUGAAUAUGUUGCAGGACAUACCAAUGGACA GGUUUGUAAUGGACUUAAAGAGAGACGUGAAAGUGACUCCAGGAACAAAACAUACUGAAGAACGGCCCAAGGUACAGG UGAUCCAGGCUGCCGAUCCGCUAGCAACAGCGUAUCUGUGCGGAAUCCACCGAGAGCUGGUUAGGAGAUUAAAUGCGG UCCUGCUUCCGAACAUUCAUACACUGUUUGAUAUGUCGGCUGAAGACUUUGACGCUAUUAUAGCCGAGCACUUCCAGC CUGGGGAUUGUGUUCUGGAAACUGACAUCGCGUCGUUUGAUAAAAGUGAGGACGACGCCAUGGCUCUGACCGCGUUAA

UGAUUCUGGAAGACUUAGGUGUGGACGCAGAGCUGUUGACGCUGAUUGAGGCGGCUUUCGGCGAAAUUUCAUCAAUAC

AUUUGCCCACUAAAACUAAAUUUAAAUUCGGAGCCAUGAUGAAAUCUGGAAUGUUCCUCACACUGUUUGUGAACACAG UCAUUAACAUUGUAAUCGCAAGCAGAGUGUUGAGAGAACGGCUAACCGGAUCACCAUGUGCAGCAUUCAUUGGAGAUG

ACAAUAUCGUGAAAGGAGUCAAAUCGGACAAAUUAAUGGCAGACAGGUGCGCCACCUGGUUGAAUAUGGAAGUCAAGA UUAUAGAUGCUGUGGUGGGCGAGAAAGCGC CUUAUUUCUGUGGAGGGUUUAUUUUGUGUGACUC CGUGAC CGGCACAG CGUGCCGUGUGGCAGACCCCCUAAAAAGGCUGUUUAAGCUUGGCAAACCUCUGGCAGCAGACGAUGAACAUGAUGAUG ACAGGAGAAGGGCAUUGCAUGAAGAGUCAACACGCUGGAACCGAGUGGGUAUUCUUUCAGAGCUGUGCAAGGCAGUAG AAUCAAGGUAUGAAACCGUAGGAACUUCCAUCAUAGUUAUGGCCAUGACUACUCUAGCUAGCAGUGUUAAAUCAUUCA GCUACCUGAGAGGGGCCCCUAUAACUCUCUACGGCUAACCUGAAUGGACUACGACAUAGUCUAGUCCGCCAAG

SEQ ID NO: 15 - VEEV RNA polymerase Amino Acid Sequence (NCBI Accession: AXP98866.1)

RELPVLDSTXAFNVECFKKYACNNEYWETFKENPIRLTEENWNYITKLKGP

SEQ ID NO: 16 - VEEV RNA polymerase Amino Acid Sequence (NCBI Accession: AXP98867.1)

TQMREL PVLD SAAFNVE C FKKYACNNE YWE T FKENP I RLTE

SEQ ID NO: 17 - Polyprotein Amino Acid Sequence [Venezuelan equine encephalitis virus] (GenBank: ALE15116.1)

MKAITARRILQGLGHYLKAEGKVECYRTLHPVPLYSSSVNRAFSSPKVAVEACNAMLKENFPTVASYCI I

PEYDAYLDMIDGASCCLDTASFCPAKLRSFPKKHSYLEPTIRSAVPSAIQNTLQNVLAAATKRNCNVTQM

RELPVLDSAAFNVECFKKYACNNEYWKTFKENPIRLTEEWINYITKLKGPKAAALYAKTHNLNMLQDIP

MDRFVMDLKRDVKVTPGTKHTEERPKVQVIQAADPLATAYLCGIHRELVRRLNAVLLPNIHTLFDMSAED

FDAI IAEHFQPGDCVLETDIASFDKSEDDAMALTAMMILEDLGVDAELLTLIEAAFGEISSIHLPTKTKF KFGAMMKSGMFLTLFVNTVINIVIASRVLRERLTGSPCAAFIGDDNIVKGVKSDKLMADRCATWLNMEVK I IDAWGEKAPYFCGGFILCDSVTGTACRVADPLKRLFKLGKPLAADDEHDDDRRRALHEESTRWNRVGI LPELCKAVESRYETVGTSVIVMAMATLASSVKSFSYLRGAPITLYG

SEQ ID NO: 18 - T2A Amino Acid Sequence

EGRGSLLTCGDVEENPGP SEQ ID NO: 19 - P2A Amino Acid Sequence - with GSG on the N terminus for stability.

GSGATNFSLLKQAGDVEENPGP

SEQ ID NO: 20 - Furin Cleavage Site

RRKRGSG

Claims

CLAIMS What is claimed is:

1. A composition comprising: a lipid carrier, wherein the lipid carrier comprises: a surface comprising cationic lipids; and a hydrophobic core, wherein the hydrophobic core comprises liquid oil, wherein lipids present in the hydrophobic core are in liquid phase at 25 degrees Celsius; and at least one nucleic acid encoding for a gH-gL viral protein antigen sequence or a functional variant thereof, wherein the at least one nucleic acid is complexed to the surface of the lipid carrier.

2. The composition of claim 1 , wherein the gH-gL viral protein antigen sequence is from a rhadinovirus .

3. The composition of claim 1, wherein the gH-gL viral protein antigen sequence is from an Epstein-Barr Virus (EBV), a Kaposi's sarcoma-associated herpesvirus, a herpesvirus saimiri, a herpesvirus ateles, a murine herpesvirus 68, or a functional variant of any of the foregoing.

4. The composition of claim 1, wherein the gH-gL viral protein antigen sequence comprises a sequence encoding for a recombinant protein.

5. The composition of claim 1. wherein the at least one nucleic acid encoding for a gH-gL viral protein antigen sequence or a functional variant thereof comprises: (1) a gH region; and (2) a gL region.

6. The composition of claim 4, wherein the recombinant protein comprises SEQ ID NO: 1 (gH), SEQ ID NO: 2 (gL), SEQ ID NO: 3 (gH-gL), or SEQ ID NO: 4 (gL-gH).

7. The composition of claim 1, wherein the nucleic acid further encodes for an RNA polymerase complex region.

8. The composition of claim 7, wherein the RNA polymerase complex region comprises a Venezuelan equine encephalitis virus (VEEV) RNA polymerase.

9. The composition of claim 8, wherein the nucleic acid encoding for the RNA polymerase complex region comprises SEQ ID NO: 14 (VEEV RNA sequence).

10. The composition of claim 1, wherein the liquid oil comprises a-tocopherol, coconut oil, grapeseed oil, lauroyl polyoxylglyceride, mineral oil, monoacylglycerol, palm kernel oil, olive oil. paraffin oil, peanut oil. propolis, squalene, squalane, soy lecithin, soybean oil, sunflower oil. a triglyceride, or vitamin E.

11. The composition of claim 10, wherein the triglyceride is capric triglyceride, capry lic triglyceride, a caprylic and capric triglyceride, a triglyceride ester, or myristic acid triglycerin.

12. The composition of claim 1, wherein the lipid carrier comprises an inorganic particle.

13. The composition of claim 12, wherein the inorganic particle is within the hydrophobic core of the lipid carrier.

14. The composition of claim 12, wherein the inorganic particle comprises a metal.

15. The composition of claim 14, wherein the metal comprises a metal salt, a metal oxide, a metal hydroxide, or a metal phosphate.

16. The composition of claim 15, wherein the metal oxide comprises aluminum oxide, aluminum oxyhydroxide, iron oxide, titanium dioxide, or silicon dioxide.

17. The composition of claim 1, wherein the lipid carrier further comprises a hydrophobic surfactant.

18. The composition of claim 17, wherein the hydrophobic surfactant comprises sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, or sorbitan trioleate.

19. The composition of claim 1, wherein the lipid carrier further comprises a hydrophilic surfactant.

20. The composition of claim 19, wherein the hydrophilic surfactant comprises a polysorbate.

21. The composition of claim 1, wherein the lipid carrier is characterized as having a z- average diameter particle size measurement of about 20 nm to about 80 nm when measured using dynamic light scattering.

22. The composition of claim 1, wherein the lipid carrier is characterized as having a z- average diameter particle size measurement of about 20 nm to about 60 nm when measured using dynamic light scattering.

23. The composition of claim 1, wherein the nucleic acid comprises DNA.

24. The composition of claim 1, wherein the nucleic acid comprises RNA.

25. The composition of claim 1 , further comprising a nucleic acid that modulates innate immune response.

26. The composition of claim 1, further comprising sodium citrate.

27. The composition of claim 1, further comprising sucrose, optionally, wherein the sucrose is present in an amount of about 50 mg.

28. The composition of claim 1, wherein the cationic lipids comprise: 1,2-dioleoyloxy-

3 (trimethylammonium)propane, 3 -[N — (N',N'-dimethylaminoethane) carbamoyl] cholesterol, dimethyldioctadecylammonium. 1,2-dimyristoyl 3-trimethylammoniumpropane, dipalmitoyl(C16:0)trimethyl ammonium propane, distearoyltrimethylammonium propane, N-[l- (2,3- dioleyloxy)propyl]N,N,Ntrimethylammonium, chloride, N,N-dioleoyl-N,N- dimethylammonium chloride, l,2-dioleoyl-sn-glycero-3-ethylphosphocholine, l,2-dioleoyl-3- dimethylammonium-propane, 1.2- dilinoleyloxy-3-dimethylaminopropane,l,l’-((2-(4-(2-((2- (bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-l- yl)ethyl)azanediyl)bis(dodecan-2-ol), tetrakis(8-methylnonyl) 3,3',3",3"'-(((methylazanediyl) bis(propane-3,l diyl))bis (azanetriyl))tetrapropi onate, decyl (2-(dioctylammonio)ethyl) phosphate, ethyl 5.5-di((Z)-heptadec-8-en-l-yl)-l-(3-(pyrrolidin-l-yl)propyl)-2,5-dihydro-lH-imidazole-2- carboxylate, ((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2-hexyldecanoate, 2-

[(poly ethylene glycol)-2000]-N,N-ditetradecylacetamide, (3S,8S,9S,10R,l 3R,14S, 17R)-17-

((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,l l,12,13,14,15,16,17- tetradecahydro-lH-cyclopenta[a]phenanthren-3-ol, bis(2-(dodecyldisulfanyl)ethyl) 3,3'-((3- methyl-9-oxo- 10-oxa- 13, 14-dithia-3,6-diazahexacosy l)azanediyl)dipropi onate. 2-

(((((3 S,8 S,9S , 1 OR, 13R, 14S, 17R)-10, 13-dimethy 1- 17-((R)-6-methylheptan-2-yl)- 2,3,4,7,8,9,10,11,12, 13, 14, 15, 16, 17-tetradecahydro-lH-cyclopenta[a]phenanthren-3- yl)oxy)carbonyl)amino)-N,N-bis(2-hydroxyethyl)-N-methylethan-l-aminium bromide, 3,6-bis(4- (bis(2-hydroxydodecyl)amino)butyl)piperazine-2.5-dione, 3β-[N-(N'.N'-dimethylaminoethane)- carbamoyl]cholesterol. (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28.31-tetraen-19-yl 4- (dimethylamino) butanoate, l,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 2,3-dioleyloxy-N- [2-(sperminecarboxamido)ethyl]-N,N-dimethyl-l -propanaminium trifluoroacetate, 1 ,2-distearoyl- sn-glycero-3-phosphocholine, ethylphosphatidylcholine, hexa(octan-3-yl) 9, 9', 9", 9"', 9''", 9"'"- ((((benzene-l,3,5-tricarbonyl)yris(azanediyl)) tris (propane-3,1 -diyl)) tris(azanetriyl))hexanonanoate, heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-

(undecyloxy)hexyl)amino) octanoate, (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4, 1- diyl))bis(azanetriyl))tetrakis(ethane-2,l-diyl) (9Z,9'Z,9''Z.9"'Z,12Z,12'Z,12"Z,12"'Z)-tetrakis (octadeca-9,12-di enoate), Nl.N3,N5-tris(3-(didodecylamino)propyl)benzene- 1,3,5- tricarboxamide.

29. A composition comprising: a nucleic acid comprising: a first region encoding for an RNA-dependent RNA polymerase complex from a virus; and a second region encoding for a gH-gL viral protein antigen sequence or a functional variant thereof.

30. The composition of claim 29, wherein the first region or the second region comprises RNA, DNA, or a combination thereof.

31. The composition of claim 29, wherein the second region further comprises a sequence encoding a self-cleaving peptide.

32. The composition of claim 29, wherein the gH-gL viral protein antigen sequence is from a rhadinovirus .

33. The composition of claim 29, wherein the gH-gL viral protein antigen sequence is from an Epstein-Barr Virus (EBV), a Kaposi's sarcoma-associated herpesvirus, a herpesvirus saimiri, a herpesvirus ateles, a murine herpesvirus 68, or a functional variant of any of the foregoing.

34. The composition of claim 29, wherein the gH-gL viral protein antigen sequence comprises a sequence encoding for a recombinant protein.

35. The composition of claim 34, wherein the recombinant protein comprises SEQ ID NO: 1 (gH), SEQ ID NO: 2 (gL), SEQ ID NO: 3 (gH-gL), or SEQ ID NO: 4 (gL-gH).

36. The composition of claim 29, wherein the RNA polymerase complex region comprises a Venezuelan equine encephalitis virus (VEEV) RNA polymerase.

37. The composition of claim 36, wherein the nucleic acid encoding for the RNA- dependent RNA polymerase complex comprises SEQ ID NO: 14.

38. The composition of claim 29, wherein the first nucleic acid or the second nucleic acid is present in an amount of up to about 100 micrograms (pg).

39. The composition of claim 29, wherein the first nucleic acid or the second nucleic acid is present in an amount of up to about 25 pg.

40. The composition of claim 29, wherein the composition further comprises a lipid carrier.

41. The composition of claim 40, wherein the lipid carrier is in complex with the first nucleic acid or the second nucleic acid.

42. The composition of claim 40, wherein a surface of the lipid carrier is in complex with the first nucleic acid and the second nucleic acid.

43. The composition of claim 29, wherein the composition further comprises an additional nucleic acid encoding for a viral protein antigen or a cancer-associated protein antigen.

44. The composition of claim 43, wherein the additional nucleic acid is in complex with a lipid carrier.

45. The composition of any one of claims 1 to 43, wherein the composition is lyophilized.

46. A suspension comprising the composition of any one of claims 1 to 45.

47. A pharmaceutical composition comprising the composition of any one of claims 1 to 45; and a pharmaceutically acceptable excipient.

48. The pharmaceutical composition of claim 47, wherein the excipient comprises a sugar.

49. The pharmaceutical composition of claim 48, wherein the sugar comprises sucrose.

50. A method of generating an immune response in a subject, the method comprising: administering to a subject the composition of any one of claims 1 to 45, thereby generating an immune response to the gH-gL viral protein antigen or a functional fragment thereof.

51. The method of claim 50, wherein the antigen is a viral protein antigen.

52. The method of claim 50, wherein the antigen is a cancer-associated antigen.

53. The method of any one of claims 50 to 52, wherein the subject has, is suspected of having, is at risk of developing, or is diagnosed with a gamma herpesvirus infection.

54. The method of any one of claims 50 to 52. wherein the subject has, is suspected of having, is at risk of developing, or is diagnosed with a cancer.

55. The method of claim 54, wherein the cancer is a carcinoma, a sarcoma, a lymphoma, or a solid cancer.

56. The method of claim 54, wherein the cancer comprises a nasopharyngeal cancer, an abdominal cancer, or a blood cancer.

57. The method of claim 56, wherein the blood cancer is a plasmablastic lymphoma, a primary central nerv ous system lymphoma, a primary effusion lymphoma, a B-lymphoproliferative disease, a diffuse large B-cell lymphoma, a Burkit's lymphoma, a natural killer (NK) cell lymphoma, a Hodgkin's disease, or a T cell lymphoma.

58. The method of any one of claims 50 to 57, wherein the subject is immunocompromised or immunosuppressed.

59. The method of any one of claims 50 to 58, wherein the subject has, is suspected of having, is at risk of developing, or is diagnosed with an autoimmune disease.

60. The method of claim 59, wherein the autoimmune disease comprises: systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, ty pe 1 diabetes, arthritis, a neurodegenerative disease, multiple sclerosis, and celiac disease.

61. The method of any one of claims 50 to 60, wherein the composition is administered to the subject by two doses.

62. The method of claim 61, wherein the administering comprises administering a second dose of the composition at about 28 days to 56 days after a first dose of the composition.

63. The method of claim 61, further comprising administering a third dose of the composition to said subject.

64. The method of any one of claims 50 to 63, wherein the composition is administered intramuscularly, subcutaneously, or intranasally.

65. The method of any one of claims 50 to 64, further comprising, administering to the subject a second composition comprising: a cancer-associated antigen or a nucleic acid encoding for the cancer-associated antigen.

66. The method of any one of claims 50 to 65. further comprising, administering to the subject a second composition comprising: a viral protein antigen or a nucleic acid encoding the viral protein antigen.

67. The method of any one of claims 50 to 66, wherein the immune response comprises increasing a titer of neutralizing antibodies to the gH-gL viral protein antigen as compared to a subject that has not been administered the composition.

68. The method of any one of claims 50 to 67, wherein the immune response comprises increasing an amount of vaccine-specific CD4+ and/or CD8+ positive T-cells as compared to a subject that has not been administered the composition.

69. The method of any one of claims 50 to 68, wherein the immune response comprises increasing an amount of neutralizing epithelial cells in the subject relative to a subject that has not been administered the composition.

70. The method of any one of claims 50 to 69, wherein the immune response comprises increasing an amount of neutralizing B cells in the subject relative to a subject that has not been administered the composition.

71. The method of any one of claims 50 to 70, wherein the immune response comprises increasing an amount of antibody binding titers to an antigen in the subject relative to a subject that has not been administered the composition.

72. A method of treating an infection in a subject, the method comprising: administering to the subject the composition of any one of claims 1 to 45, thereby treating the infection.

73. A method of treating cancer in a subject, the method comprising: administering to the subject the composition of any one of claims 1 to 45, thereby treating the cancer.

74. The method of any one of claims 50 to 73, wherein the subject is a human subject.

75. The method of any one of claims 50 to 74. wherein the subject has or is diagnosed with a gamma herpesvirus-associated cancer.

76. A kit comprising the composition of any one of claims 1 to 45, packaging, and materials therefor.