Multi-antigen herpes simplex vaccine

Abstract

Herpes simplex virus (HSV) is a significant concern for the global health community due to its morbidity and often-asymptomatic nature. Infected individuals may develop conditions such as ocular disease or genital herpes as characteristic manifestations of the infection. The complexity of HSV's infectious mechanisms has led to adaptations in existing treatment options, but despite these advancements, no definitive or highly effective vaccine has yet been found. Several promising vaccine candidates have been developed using recombinant technology, genetic engineering, and advanced methods. Over the past decade, vaccine development has increasingly favored viral vector-based vaccines, which often elicit a stronger immune response than other approved vaccines that may require boosters. The protective efficacy of five recombinant adenovirus-based therapeutic vaccines is described herein, focusing on their impact on the frequency and function of DRG- and VM-resident CD4+ and CD8+ T cells and their effect on the frequency and severity of recurrent genital herpes.

Description

MULTI-ANTIGEN HERPES SIMPLEX VACCINECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 721,830 filed November 18, 2024 and U.S. Provisional Application No. 63 / 560,455 filed March 1, 2024, the specifications of which are incorporated herein in their entirety by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant Nos. AI150091, EY019896. and AI143348 awarded by National Institute of Health. The government has certain rights in the invention.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (UCI 24_05 PCT.xml; Size: 46,288 bytes; and Date of Creation: March 3, 2025) is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0004] The present invention relates to therapeutic vaccines, for example, viral vaccines, such as those directed to herpes, e.g., recurrent herpes. The therapeutic vaccine strategy described herein comprises priming T cells (subunit HSV antigen) and pulling primed T cells (using T-cell attracting chemokines) to reduce recurrent herpes more effectively than vaccination with the subunit HSV antigen alone.BACKGROUND OF THE INVENTION

[0005] Microbes occupy all of our body surfaces and a vast majority of microbes establish themselves as persistent “colonists,” thriving in complex communities within and on our bodies. Viral diseases make a very significant contribution to the burden of human disease. Genital herpes is a recurrent, often painful sexually transmitted disease, caused by herpes simplex viruses type 1 (HSV-1) or type 2 (HSV-2). Most cases of genital herpes are caused by HSV-2. HSV-1 manifests as blisters or cold sores in and around the mouth. Herpes simplex virus type 2 (HSV-2) affects both women and men; however, women are more susceptible to the infection. Approximately 491 million people are living v\i th HSV type 2 infection in those up to 49 years of age, showing that HSV-2 has a substantial effect on the health of millions of people worldwide. After exposure of HSV-2 virus to mucosal genital surfaces, the virus replicates in the mucosalepithelial cells, causing the development of acute genital herpetic lesions. After the initial infection is resolved, a latent virus established in the infected ganglia; the virus enters the nerve termini innervating peripheral vaginal tissues and is subsequently transported by retrograde to the nucleus of the sensory neurons of dorsal root ganglia (DRG), where it establishes a dormant state within the neuronal cells. The activation of the latent virus causes recurrent disease. The first-in-line treatment of HSV infections is acyclovir, but the recent emergence of ACV-resistant strains emphasizes the need for an effective vaccine against HSV.

[0006] Vaccination is very effective, producing a high level of protection against illness and death that probably lasts a lifetime, although the degree of immunity can wane after 5-10 years. Four main vaccine approaches have been tested in the past four decades to fight herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infections and diseases: (1) Inactivated ‘"killed” HSV vaccines; (2) Live-attenuated HSV vaccines; (3) Replication-defective HSV vaccines; and (4) Subunit HSV vaccines. Over the last two decades, only a single subunit protein vaccine strategy, based on the HSV-2 glycoprotein D (gD), delivered with or without gB, has been tested and retested in clinical trials. Despite inducing strong neutralizing antibodies, this subunit vaccine strategy proved unsuccessful in clinical trials.BRIEF SUMMARY OF THE INVENTION

[0007] It is an objective of the present invention to provide compositions and methods for multi-antigen herpes simplex vaccines, that allow for the treatment of herpes, e.g., recurrent herpes (e.g., genital, oro-facial, dermal or ocular), as specified in the independent claims. Embodiments of the invention are given in the dependent claims. Embodiments of the present invention can be freely combined with each other if they are not mutually exclusive.

[0008] In some embodiments, the present invention features a multi-antigen herpes vaccine composition. In some embodiments, the composition comprises a sequence encoding or comprises two or more different herpes antigens selected from a group consisting of RR1, RR2, gD, or VP22. In other embodiments, the composition comprises a sequence encoding or comprises three or more different herpes antigens selected from a group consisting of RR1, RR2, gD. or VP22. In some embodiments, the antigens may be encoded according to specific sequences, such as RR1 (SEQ ID NO: 1 or SEQ ID NO: 2), RR2 (SEQ ID NO: 3 or SEQ ID NO: 4), gD (SEQ ID NO: 7 or SEQ ID NO: 8), or VP 16 (SEQ ID NO: 5 or SEQ ID NO: 6). Alternatively, the antigens may be selected from the group consisting of RR1, RR2, gD, or VP22, VP11 / 12. The aforementioned compositions may further comprise a T cell attracting chemokine(e.g., CCL5, CXCL9, CXCL10, CXCL11, or a combination thereof) and / or a composition that promotes T cell proliferation and T-cell memory (e g., IL-7, IL-2, or IL-15).

[0009] In some embodiments, the present invention features a multi-antigen herpes vaccine composition. In some embodiments, the composition comprises a delivery- system encoding two or more different herpes antigens selected from a group consisting of RR1, RR2, gD, or VP22. In other embodiments, the composition comprises a delivery system encoding three or more different herpes antigens selected from a group consisting of RR1, RR2, gD, or VP16. The antigens may be encoded according to specific sequences, such as RR1 (SEQ ID NO: 1 or SEQ ID NO: 2). RR2 (SEQ ID NO: 3 or SEQ ID NO: 4). gD (SEQ ID NO: 7 or SEQ ID NO: 8), or VP 16 (SEQ ID NO: 5 or SEQ ID NO: 6). The delivery system may further include an adjuvant, a pharmaceutical carrier, or both an adjuvant and a pharmaceutical carrier. In certain embodiments, the delivery system is a single system, while in others, it comprises two or more delivery systems. The delivery system may- utilize an adeno-associated viral (AAV) vector, such as AAV8 or AAV9, or a vesicular stomatitis virus (VSV) vector. The antigens may be operatively linked to a generic promoter, such as a CMV or CAG promoter, to enhance expression. Additionally, the composition may encode a T cell-attracting chemokine, including but not limited to CCL5, CXCL9, CXCL10, or CXCL1 L which may also be operatively linked to a generic promoter. In further embodiments, the delivery system encodes a composition that promotes T cell proliferation and memory-, such as IL-7, IL-2, or IL-15, which may be driven by the same promoter as the T cell-attracting chemokine. These features collectively enhance the immunogenicity and efficacy of the vaccine composition by facilitating robust and sustained immune responses.

[0010] Likewise, in some embodiments, the composition may comprise a delivery- system (e.g., a first delivery system) encoding two or more different herpes antigens selected from a group consisting of RR1, RR2. or VP22. In some embodiments, the delivery system may further include an adjuvant, a pharmaceutical carrier, or both an adjuvant and a pharmaceutical carrier. In some embodiments, the composition may further comprise a delivery- system (e.g., a second delivery- system) encoding a gD herpes antigen. In certain embodiments, the antigens may be encoded according to specific sequences, such as RR1 (SEQ ID NO: 1 or SEQ ID NO: 2). RR2 (SEQ ID NO: 3 or SEQ ID NO: 4), gD (SEQ ID NO: 7 or SEQ ID NO: 8), or VP16 (SEQ ID NO: 5 or SEQ ID NO: 6). The delivery system (e.g., a second delivery- system) encoding a gD herpes antigen may further include an adjuvant, a pharmaceutical carrier, or both an adjuvant anda pharmaceutical carrier. In some embodiments, either delivery system (e.g., either the first delivery system or the second delivery system) may utilize an adeno-associated viral (AAV) vector, such as AAV8 or AAV9, or a vesicular stomatitis virus (VSV) vector. The antigens may be operatively linked to a generic promoter, such as a CMV or CAG promoter, to enhance expression. In some embodiments, either delivery system may additionally encode a T cell-attracting chemokine (e.g., CCL5, CXCL9, CXCL10, CXCL11, or a combination thereof) and / or a component that enhances T cell proliferation and memory (e.g., IL-7, IL-2, or IL-15). These elements may be operatively linked to a generic promoter, ensuring coordinated expression. In certain embodiments, both the T cell-attracting chemokine and the T cell-proliferation component are driven by the same promoter, optimizing immune activation and response.

[0011] In some embodiments, the present invention features a composition comprising two or more different messenger ribonucleic acids (mRNAs), each mRNA comprising an open reading frame encoding herpes antigens selected from a group consisting of RR1, RR2, gD, or VP22. In some embodiments, the two or more mRNAs are formulated in a lipid nanoparticle. Alternatively, in other embodiments, the present invention features a composition comprising three or more different messenger ribonucleic acids (mRNAs). each mRNA comprising an open reading frame encoding herpes antigens selected from a group consisting of RR1, RR2, gD, or VP22. In some embodiments, the three or more mRNAs are formulated in a lipid nanoparticle. In some embodiments, the lipid nanoparticle includes a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid. Alternatively, the antigens may be selected from the group consisting of RR1, RR2. gD, or VP22. VPI 1 / 12. Additionally, in some embodiments, each mRNA may comprise a 5’ untranslated region (UTR), a 3’ UTR, a poly(A) tail, a 5’ cap, or a combination thereof. In certain embodiments, the mRNAs incorporate N1 -methylpseudouridine (ml\| / ) modifications to enhance stability and translational efficiency.

[0012] Likewise, in some embodiments, the present invention may feature a pharmaceutical composition comprising a first lipid nanoparticle and a second lipid nanoparticle. In some embodiments, the first lipid nanoparticle comprises two or more different messenger ribonucleic acids (mRNAs) encapsulated therein, each mRNA comprising an open reading frame encoding a herpes antigen selected from a group consisting of RR1, RR2, or VP22. Additionally, in some embodiments, the second lipid nanoparticle comprises a gD herpes antigen encapsulated therein. In some embodiments, the lipid nanoparticle includes a cationic lipid, a PEG-modified lipid, asterol, and a non-cationic lipid. Additionally, in some embodiments, each mRNA may comprise a 5’ untranslated region (UTR), a 3’ UTR, a poly(A) tail, a 5’ cap, or a combination thereof. In certain embodiments, the mRNAs incorporate N1 -methylpseudouridine (ml v| / ) modifications to enhance stability and translational efficiency.

[0013] In further embodiments, the present invention features a multi-antigen herpes vaccine composition. In some embodiments, the composition comprises a sequence encoding or comprising two or more different herpes antigens selected from a group consisting of RR1, RR2, gD. or VP22; wherein the sequence is formulated in a lipid nanoparticle. In other embodiments, the composition comprises a sequence encoding or comprising three or more different herpes antigens selected from a group consisting of RR1, RR2, gD, or VP22; wherein the sequence is formulated in a lipid nanoparticle. In some embodiments, the lipid nanoparticle includes a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid.

[0014] In some embodiments, the vaccine compositions described herein provide protection against initial infection or reinfection by one or more herpesviruses. The targeted herpesviruses may include Herpes simplex virus type 1 (HSV-1) and / or Herpes simplex virus type 2 (HSV-2). In certain embodiments, the vaccine elicits a robust and durable immune response characterized by the induction of antibodies (Abs), CD4+T helper (Thl) cells, and / or CD8+cytotoxic T lymphocytes (CTLs), thereby enhancing long-term immunity.

[0015] Additionally, in some embodiments, the present invention features a method of eliciting a T-cell response, an IFN-y response, or reducing viral titers in an individual, the method comprising administering to said individual the vaccine composition as described herein.

[0016] One of the unique and inventive technical features of the present invention is the incorporation of both B cell and T cell antigens within a single composition (e.g., a vaccine or pharmaceutical composition). Specifically, the composition includes glycoprotein D (gD) as a B cell antigen, along with ribonucleotide reductase subunits 1 and 2 (RR1 and RR2) and VP22 as T cell antigens. Without wishing to limit the invention to any theory or mechanism, it is believed that the technical feature of the present invention advantageously provides enhanced immune protection against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), which can cause ocular herpes, cold sores, or genital herpes. None of the presently known prior references or works have the unique inventive technical feature of the present invention.[00171 Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0018] The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which:

[0019] FIG. 1A, IB, 1C, ID, and IE shows protection against genital herpes infection in HSV-2 infected guinea pigs following therapeutic immunization with five Adenovirus based vaccine candidates. FIG. 1A shows a timeline of HSV-2 infection, virological and immunological analysis. Guinea pigs were infected intravaginally on day 0 with 5 X 10' PFU of HSV-2 (MS strain). Once the acute infection was resolved latently infected animals were divided into 7 groups and then immunized intramuscularly on day 15 with 1010genomic copies of AD-5 expressing HSV-2 antigens (RR2, RR1, gD, VP22 and Vpl6). The replication defective dl529 mutant vaccine was used as positive control. From day 25 to 56 post-infection, the guinea pigs were monitored daily for the severity of genital herpes lesion, scored on a scale of 0-4 and vaginal swabs were collected to detect virus shedding and to quantify HSV-2 DNA copy numbers. FIG. IB shows the cumulative scoring of vaginal lesions observed during recunent infection. FIG. 1C shows the cumulative positive days with recurrent genital lesions. FIG. ID shows HSV-2 DNA copy numbers detected in the VM of vaccinated and mock-vaccinated guinea pig groups. FIG. IE shows HSV-2 DNA copy numbers detected in the DRG of vaccinated and mock- vaccinated guinea pig groups.

[0020] FIG. 2A. 2B, and 2C shows formation of antibodies in HSV-2 infected guinea pigs following therapeutic immunization with five Adenovirus-based vaccine candidates. FIG. 2A shows the levels of antigen-specific IgG detected in the guinea pigs vaccinated with RR2, RR1, gD, VP22 and VP 16 AD5 expressing antigens by ELISA. The sera was evaluated at 1 :1000 dilution. FIG. 2B shows the binding affinity of IgG from the indicated AD5 HSV-2 antigen vaccinated groups compared to the mock and dl529 controls to the native proteins (FIG. 2C; left panel) Representative ELISpot images showing average frequencies of IFN-g producing cell spots from mononuclear cells from VM tissue (1 x 10scells per well) of HSV-2 infected guineapigs treated with different HSV-2 proteins namely RR2, RR1, VP22, gD, VP 16. dl529 was used as a positive control. (FIG. 2C; right panel The bar diagrams show the average / mean numbers (+ SD) of IFN-g-spot forming cells (SFCs) after stimulation with HSV-2 proteins in VM tissues of different groups of guinea pigs. A strong response is defined for mean SFCs > 25 per 1 x 105stimulated mononuclear cells.

[0021] FIG. 3 A and 3B shows frequency of CD4 and CD8 T cells in the vaginal mucosa of HSV-2 infected guinea pigs following therapeutic immunization with five Adenovirus based vaccine candidates. Fifty-six days post-infection, guinea pigs were euthanized, and single-cell suspensions from the DRG, vaginal mucosa (VM), and spleen were obtained after collagenase treatment. The DRG, VM, and spleen cells were stained for CD8+and CD4+expressing T cells and then analyzed by FACS. Representative FACS data (FIG. 3A) and average frequencies (FIG. 3B) of CD8+CD4+T cells detected in the DRG (top panel), VM (middle panel) and Spleen (lower panel) of vaccinated animals, dl529 vaccinated and mock vaccinated animals. Cells were analyzed using a BD LSR Fortessa Flow Cytometry system with 1 x 10bevents. The indicated P values performed by one-way ANOVA for significance show statistical significance between vaccinated versus mock-vaccinated control groups.

[0022] FIG. 4A and 4B shows increased frequencies of CD4+CD44+T cells and CD8+CD44+T cells in the DRG and VM of HSV-2 infected guinea pigs following therapeutic immunization with five Adenovirus based vaccine candidates: FIG. 4A shows representative FACS data (left panel) and average frequencies (right panel) of CD4+CD44+T cells detected in the DRG, VM of vaccinated and mock vaccinated animals. FIG. 4B shows representative FACS data (left panel) and average frequencies (right panel) of CD8+CD44+T cells detected in the DRG, and VM of vaccinated and mock vaccinated animals.

[0023] FIG. 5A and 5B shows Increased frequencies of CD4+CD69+T cells and CD8+CD69+T cells in the DRG and VM of HSV-2 infected guinea pigs following therapeutic immunization with five Adenovirus-based vaccine candidates: FIG. 5 A shows representative FACS data (left panel) and average frequencies (right panel) of CD4+CD69+T cells detected in the DRG, and VM of vaccinated and mock vaccinated animals. FIG. 5B shows representative FACS data (left panel) and average frequencies (right panel) of CD8+CD69+T cells detected in the DRG, and VM of vaccinated and mock vaccinated animals.

[0024] FIG. 6A and 6B shows increased frequencies of CD8+CD103+T cells and CD8+CRTAM T cells in the DRG and VM of HSV-2 infected guinea pigs following therapeutic immunization with five Adenovirus based vaccine candidates: FIG. 6 A sho srepresentative FACS data (left panel) and average frequencies (right panel) of CD8 CD1031T cells detected in the DRG, and VM of vaccinated and mock vaccinated animals. FIG. 6B shows representative FACS data (left panel) and average frequencies (right panel) of CD8+CRTAM T cells detected in the DRG, and VM of vaccinated and mock vaccinated animals.

[0025] FIG. 7A and 7B shows increased frequencies of CD4+Ki-67 T cells and CD8+Ki-67 T cells in the DRG and VM of HSV-2 infected guinea pigs following therapeutic immunization with five Adenovirus based vaccine candidates: FIG. 7A shows representative FACS data (left panel) and average frequencies (right panel) of CD4+Ki-67 T cells detected in the DRG, and VM of vaccinated and mock vaccinated animals. FIG. 7B shows representative FACS data (left panel) and average frequencies (right panel) of CD8+Ki-67 T cells detected in the DRG, and VM of vaccinated and mock vaccinated animals.

[0026] FIG. 8A and 8B shows increased frequencies of CD4+IFN-g T cells and CD8+IFN-g T cells in the DRG and VM of HSV-2 infected guinea pigs following therapeutic immunization with five Adenovirus based vaccine candidates: FIG. 8 A shows representative FACS data (left panel) and average frequencies (right panel) of CD4+IFN-g T cells detected in the DRG, and VM of vaccinated and mock vaccinated animals. FIG. 8B shows representative FACS data (left panel) and average frequencies (right panel) of CD8+IFN-g T cells detected in the DRG, and VM of vaccinated and mock vaccinated animals.

[0027] FIG. 9A and 9B shows increased frequencies of CD4+IFN-g T cells and CD8+IFN-g T cells in the DRG and VM of HSV-2 infected guinea pigs following therapeutic immunization with five Adenovirus based vaccine candidates: FIG. 9A shows representative FACS data (left panel) and average frequencies (right panel) of CD4+TNF-a T cells detected in the DRG, and VM of vaccinated and mock vaccinated animals. FIG. 9B shows representative FACS data (left panel) and average frequencies (right panel) of CD8+TNF-a T cells detected in the DRG, and VM of vaccinated and mock vaccinated animals.

[0028] FIG. 10 shows non-limiting examples of how the antigens of the compositions described herein may be arranged. In some embodiments, the vaccine compositions may be delivered as an mRNA / LNP vaccine, an adenovirus vaccine, or an adeno-associated virus (AAV), e.g., AAV8

[0029] vaccine or (6) as prime / pull vaccine i.e. Antigen (s) + Chemokine CXCL11

[0030] FIG. 11 shows schematic views of non-limiting examples of vaccine compositions showing an optional molecular adjuvant, T cell attracting chemokine, and / or composition for promoting T cell proliferation, as well as non-limiting examples of orientations of said optional molecular adjuvant, T cell attracting chemokine, and / or composition for promoting T cellproliferation.

[0031] FIG. 12A, 12B and 12C shows protection against genital herpes infection in HSV-2 infected guinea pigs following therapeutic immunization with mRNA-LNP expressing HSv-2 vaccine candidates gD, RR1, and RR2. FIG. 12A shows non-limiting examples of nucleoside-modified gD, RR1 and RR2 mRNAs encapsulated in lipid nanoparticles (LNP) vaccine (i.e., mRNA / LNP therapeutic vaccine). FIG. 12B shows a timeline of HSV-2 infection, mRNA-LNP immunization, and virological and immunological analysis. Guinea pigs were infected intravaginally on day 0 with 1 X 106PFU of HSV-2 (MS strain). Once the acute infection was resolved, latently infected animals were divided into 9 groups and then immunized intramuscularly on days 14 and 28 with lOug of mRNA-LNP expressing HSV-2 antigens (RR2, RR1, and gD). From day 35 to 56 post-infection, the guinea pigs were monitored daily for the severity of genital herpes lesion, scored on a scale of 0-4 and vaginal swabs were collected to detect virus shedding and to quantify HSV-2 DNA copy numbers. FIG. 12C shows cumulative scoring of vaginal lesions observed during recurrent infectionDETAILED DESCRIPTION OF THE INVENTION

[0032] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which a disclosed invention belongs. The singular terms “a,” “an,” and "the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The term "comprising" means that other elements can also be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation. Stated another way, the term "comprising" means "including principally, but not necessary solely". Furthermore, variation of the word "comprising", such as "comprise" and "comprises", have correspondingly the same meanings. In one respect, the technology described herein related to the herein described compositions, methods, and respective component(s) thereof, as essential to the invention, yet open to the inclusion of unspecified elements, essential or not ("comprising").

[0033] Suitable methods and materials for the practice and / or testing of embodiments of the disclosure are described below. Such methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar or equivalent to those described herein can be used. For example, conventional methods well known in the art to which the disclosure pertains are described in various general and more specific references, including, forexample. Sambrook et al.. Molecular Cloning: A Laboratory Manual, 2d ed.. Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology7, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al.. Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory7Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory7Manual, Cold Spring Harbor Laboratory Press, 1999, Gene Expression Technology7(Methods in Enzymology. Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, Calif), ‘‘Guide to Protein Purification” in Methods in Enzymology (M. P. Deutshcer, ed.. (1990) Academic Press, Inc ); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray. The Humana Press Inc.. Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, Tex.), the disclosures of which are incorporated in their entirety herein by reference.

[0034] Although methods and materials similar or equivalent to those described herein can be used to practice or test the disclosed technology, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0035] As used herein, the terms "immunogenic protein, polypeptide, or peptide" or “antigen” refer to polypeptides or other molecules (or combinations of polypeptides and other molecules) that are immunologically active in the sense that once administered to the host, it is able to evoke an immune response of the humoral and / or cellular ty pe directed against the protein. In embodiments, the protein fragment has substantially the same immunological activity as the total protein. Thus, a protein fragment according to the disclosure can comprises or consists essentially of or consists of at least one epitope or antigenic determinant. An "immunogenic" protein or polypeptide, as used herein, may include the full-length sequence of the protein, analogs thereof, or immunogenic fragments thereof. "Immunogenic fragment" refers to a fragment of a protein that includes one or more epitopes and thus elicits the immunological response described above.

[0036] Synthetic antigens are also included within the definition, for example, poly-epitopes, flanking epitopes, and other recombinant or synthetically derived antigens. Immunogenic iofragments for purposes of the disclosure may feature at least about 1 amino acid, at least about 3 amino acids, at least about 5 amino acids, at least about 10-15 amino acids, or about 15-25 amino acids or more amino acids, of the molecule. There is no critical upper limit to the length of the fragment, which could comprise nearly the full-length of the protein sequence, or the full-length of the protein sequence, or even a fusion protein comprising at least one epitope of the protein.

[0037] As used herein, the term "immunological response" to a composition or vaccine refers to the development in the host of a cellular and / or antibody-mediated immune response to a composition or vaccine of interest. Usually, an "immunological response" includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, and / or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. The host may display either a therapeutic or protective immunological response so resistance to new infection will be enhanced and / or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host, a quicker recovery time and / or a lowered viral titer in the infected host.

[0038] As used herein, the term "variant" refers to a substantially similar sequence. For polynucleotides, a variant comprises a deletion and / or addition and / or change of one or more nucleotides at one or more sites within the native polynucleotide and / or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a "native" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or an amino acid sequence, respectively. Variants of a particular polynucleotide of the disclosure (e.g., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. "Variant" protein is intended to mean a protein derived from the native protein by deletion or addition of one or more amino acids at one or more sites in the native protein and / or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present disclosure are biologically active, that is they have the abi li ty to elicit an immune response.

[0039] A “subject’’ is an individual and includes, but is not limited to, a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig, or rodent), a fish, a bird, a reptile or an amphibian. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included. A“patient” is a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects.

[0040] As used herein, the terms "treat" or "treatment" or "treating" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow the development of the disease, such as slow down the development of a disorder, or reducing at least one adverse effect or symptom of a condition, disease or disorder, e.g., any disorder characterized by insufficient or undesired organ or tissue function. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced as that term is defined herein. Alternatively, a treatment is "effective" if the progression of a disease is reduced or halted. That is, "treatment" includes not just the improvement of symptoms or decrease of markers of the disease, but also a cessation or slowing of progress or worsening of a symptom that would be expected in absence of treatment. Beneficial or desired clinical results include, but are not limited to. alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (e.g., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. “Treatment” also includes ameliorating a disease, lessening the severity of its complications, preventing it from manifesting, preventing it from recurring, merely preventing it from worsening, mitigating an inflammatory response included therein, or a therapeutic effort to affect any of the aforementioned, even if such therapeutic effort is ultimately unsuccessful.

[0041] A “disease” is a state of health of a subject, wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated, then the subject's health continues to deteriorate. In contrast, a “disorder” in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject's state of health.

[0042] As used herein, the term “carrier” or “pharmaceutically acceptable carrier” or “pharmaceutically acceptable vehicle” refers to any appropriate or useful carrier or vehicle for introducing a composition to a subject. Pharmaceutically acceptable carriers or vehicles may be conventional but are not limited to conventional vehicles. For example, E. W. Martin, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 15th Edition (1975)and D. B. Troy, ed. Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, Baltimore MD and Philadelphia, PA, 21st Edition (2006) describe compositions and formulations suitable for pharmaceutical delivery' of one or more therapeutic compounds or molecules. Carriers (e.g., pharmaceutical carriers, pharmaceutical vehicles, pharmaceutical compositions, pharmaceutical molecules, etc.) are materials generally known to deliver molecules, proteins, cells and / or drugs and / or other appropriate material into the body. In general, the nature of the carrier will depend on the nature of the composition being delivered as well as the particular mode of administration being employed. In addition to biologically -neutral carriers, pharmaceutical compositions administered may contain minor amounts of non- toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like. Patents that describe pharmaceutical carriers include, but are not limited to: U.S. Patent No. 6,667,371; U.S. Patent No. 6,613,355; U.S. Patent No. 6,596,296; U.S. Patent No. 6,413,536; U.S. Patent No. 5,968,543; U.S. Patent No. 4,079, 038; U.S. Patent No. 4,093,709; U.S. Patent No. 4,131,648; U.S. Patent No. 4,138,344; U.S. Patent No. 4,180.646; U.S. Patent No. 4.304,767; U.S. Patent No. 4,946,931, the disclosures of which are incorporated in their entirety by reference herein. The carrier may, for example, be solid, liquid (e.g., a solution), foam, a gel, the like, or a combination thereof. In some embodiments, the carrier comprises a biological matrix (e.g., biological fibers, etc.). In some embodiments, the carrier comprises a synthetic matrix (e.g., synthetic fibers, etc.). In certain embodiments, a portion of the carrier may comprise a biological matrix and a portion may comprise synthetic matrix.

[0043] The terms “administering”, and “administration” refer to methods of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, administering the compositions orally, parenterally (e.g., intravenously and subcutaneously), by intramuscular injection, by intraperitoneal injection, intrathecally, transdermally, extracorporeally, topically or the like.

[0044] A composition can also be administered by topical intranasal administration (intranasally) or administration by inhalant. As used herein, “topical intranasal administration” means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism (device) or droplet mechanism (device), or through aerosolization of the composition. Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. As used herein, “an inhaler” can be a spraying device or a droplet device for delivering a composition comprising the vaccinecomposition, in a pharmaceutically acceptable carrier, to the nasal passages and the upper and / or lower respiratory tracts of a subject. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intratracheal intubation. The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the disorder being treated, the particular composition used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

[0045] A composition can also be administered by buccal delivery or by sublingual delivery. As used herein “buccal deliver} ” may refer to a method of administration in which the compound is delivered through the mucosal membranes lining the cheeks. In some embodiment, for a buccal delivery the vaccine composition is placed between the gum and the cheek of a patient. As used herein “sublingual delivery” may refer to a method of administration in which the compound is delivered through the mucosal membrane under the tongue. In some embodiments, for a sublingual delivery' the vaccine composition is administered under the tongue of a patient.

[0046] Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained.

[0047] Referring now to FIGs. 1A-12C, the present invention features therapeutic vaccines, for example, viral vaccines, such as those directed to herpes, e.g., recurrent herpes, including genital, oro-facial, dermal, or ocular. The therapeutic vaccine strategy described herein comprises priming T cells (subunit HSV antigen) and pulling primed T cells (using T-cell attracting chemokines) to reduce recurrent genital herpes more effectively than vaccination with the subunit HSV antigen alone.

[0048] The present invention features multi-antigen herpes vaccines, methods of use. and methods of producing said vaccines, methods of preventing herpes infections, etc. The present invention also provides methods of testing said vaccines, e.g., using particular animal models and clinical trials. The vaccine compositions herein can induce efficient and powerful protection against herpes disease or infection, e.g., by inducing the production of antibodies (Abs). CD4+ Thelper (Thl) cells, and CD8+ cytotoxic T-cells (CTL).

[0049] Vaccine Composition

[0050] In some embodiments, the compositions described herein may be used to prevent a herpes infection prophy lactically in a subject. In some embodiments, the compositions described herein may elicit an immune response in a subject.

[0051] In some embodiments, the vaccine composition comprises one antigen. In some embodiments, the vaccine composition comprises one or more antigens. In some embodiments, the vaccine composition comprises two antigens. In some embodiments, the vaccine composition comprises two or more antigens. In some embodiments, the vaccine composition comprises three antigens. In some embodiments, the vaccine composition comprises three or more antigens. In some embodiments, the vaccine composition comprises four antigens. In some embodiments, the vaccine composition comprises four or more antigens. In some embodiments, the vaccine composition comprises five or more antigens.

[0052] Table 1 shows Below are specific nucleotide and codon optimized sequences of envelope glycoprotein B (gB), envelope glycoprotein D (gD). ribonucleotide reductase subunit 1 (RR1), ribonucleotide reductase subunit 2 (RR2), VP16, VP22, gC2, gD2, and gE2 antigens. In some embodiments, the aforementioned antigens may be incorporated as single or multi-antigen vaccine for herpes simplex virus type 1 and Type 2. Note the gB, gD, RR1, RR2, VP16, and VP22 antigen-based herpes simplex vaccine may be administered as 1) nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNP) (i.e., combined mRNA / LNP-based herpes vaccine; 2) or as adenovirus vaccine or as adeno-associated vaccine; 3) or as a DNA vaccine; or 4) as a protein + adjuvant vaccine.

[0053] In some embodiments, the aforementioned antigens comprise a sequence that is at least 50%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to any of SEQ ID NOs: 1-18. The sequences may include modifications such as substitutions, deletions, or truncations, provided that the resulting antigen retains the ability to elicit an immune response. For instance, truncations may remove non-essential regions while preserving key epitopes necessary for immunogenicity. Similarly, conservative ammo acid substitutions may be introduced to enhance stability, solubility, or manufacturability without compromising antigenic function. These variations may allow for optimization of the composition while maintaining or even improving its immunogenic potential.

[0054] In some embodiments, the present invention features a multi-antigen herpes vaccine composition. In some embodiments, the composition comprising a sequence encoding or comprising two or more different herpes antigens selected from a group consisting of RR1, RR2, gD. or VP22. In other embodiments, the composition comprising a sequence encoding or comprising three or more different herpes antigens selected from a group consisting of RR1, RR2, gD, or VP22. The antigens may be encoded according to specific sequences, such as RR1 (SEQ ID NO: 1 or SEQ ID NO: 2), RR2 (SEQ ID NO: 3 or SEQ ID NO: 4), gD (SEQ ID NO: 7 or SEQ ID NO: 8), or VP16 (SEQ ID NO: 5 or SEQ ID NO: 6). Alternatively, the antigens may be selected from the group consisting of RR1, RR2, gD, or VP22, VP 11 / 12.

[0055] Molecular Adjuvants and T Cell Enhancements

[0056] In certain embodiments, the vaccine composition comprises a molecular adjuvant and / or one or more T Cell enhancement compositions. The adjuvant and / or enhancement compositions may help improve the immunogenicity and / or long-term memory of the vaccine composition. Non-limiting examples of molecular adjuvants include CpG, such as a CpG polymer, and flagellin.

[0057] In some embodiments, the vaccine composition comprises a T cell attracting chemokine. The T cell attracting chemokine helps pull the T cells from the circulation to the appropriate tissues. Non-limiting examples of T cell attracting chemokines include CCL5, CXCL9, CXCL10, CXCL11, CCL25, CCL28. CXCL14, CXCL17, or a combination thereof.

[0058] In some embodiments, the vaccine composition comprises a composition that promotes T cell proliferation. Non-limiting examples of compositions that promote T cell proliferation include IL-7, IL- 15, IL-2, or a combination thereof.

[0059] In some embodiments, the vaccine composition comprises a composition that promotes T cell homing in the genitals. Non-limiting examples of compositions that promote T cell homing include CCL25, CCL28, CXCL14, CXCL17, or a combination thereof.

[0060] In certain embodiments, the molecular adjuvant and / or the T cell attracting chemokine and / or the composition that promotes T cell proliferation are delivered with a separate antigen delivery' system from the large sequences.

[0061] In preferred embodiments, the T-cell enhancement compositions described herein (e.g., CXCL9, CXCL10, IL-7, IL-2) may be integrated into a separate delivery system from the vaccine compositions. In some embodiments, the T-cell enhancement compositions described herein (e g., CXCL9, CXCL10, IL-7. IL-2) may be integrated into the same delivery system as the vaccine compositions.

[0062] In certain embodiments, the vaccine composition comprises a tag. For example, in some embodiments, the vaccine composition comprises a His tag. The present invention is not limited to a His tag and includes other tags such as those known to one of ordinary skill in the art. such as a fluorescent tag (e g., GFP, YFP, etc.), etc.

[0063] Antigen Delivery System

[0064] The present invention also features vaccine compositions in the form of an antigen delivery system. Any appropriate antigen delivery system may be considered for the delivery’ of the antigens described herein. The present invention is not limited to the antigen delivery systems described herein.

[0065] In certain embodiments, the antigen delivery' system is for targeted delivery of the vaccine composition, e.g., for targeting to the tissues of the body where the virus replicates.

[0066] In certain embodiments, the antigen delivery system comprises adenoviruses such as but not limited to Ad5, Ad26, Ad35, etc., as well as carriers such as lipid nanoparticles, polymers, peptides, etc. In other embodiments, the antigen delivery system comprises a vesicular stomatitis virus (V SV) vector.

[0067] The present invention is not limited to adenovirus vector-based antigen delivery systems. In certain embodiments, the antigen delivery system comprises an adeno-associated virus vector-based antigen delivery system, such as but not limited to the adeno-associated virus vector type 9 (AAV9 serotype), AAV type 8 (AAV8 serotype), etc.

[0068] In the antigen delivery system, the one or more antigens are operatively linked to a promoter. In certain embodiments, the one or more antigens are operatively linked to a generic promoter. For example, in certain embodiments, the one or more antigens are operatively linked to a CMV promoter. In certain embodiments, the one or more antigens are operatively linked to a CAG, EFIA, EFS, CBh, SFFV, MSCV, mPGK, hPGK, SV40, UBC, or another appropriate promoter. In some embodiments, the one or more antigens are operatively linked to a tissue-specific promoter.

[0069] In certain embodiments, the vaccine composition comprises a molecular adjuvant. In certain embodiments, the molecular adjuvant is operatively linked to a generic promoter, e.g., as described above. In certain embodiments, the molecular adjuvant is operatively linked to a tissue-specific promoter.

[0070] As discussed, in certain embodiments, the vaccine composition comprises a T cell attracting chemokine. In certain embodiments, the T cell attracting chemokine is operatively linked to a generic promoter, e.g., as described above. In certain embodiments, the T cell attracting chemokine is operatively linked to a tissue-specific promoter.

[0071] As discussed, in certain embodiments, the vaccine composition comprises a composition for promoting T cell proliferation. In certain embodiments, the composition for promoting T cell proliferation is operatively linked to a generic promoter, e.g., as described above. In certain embodiments, the composition for promoting T cell proliferation is operatively linked to a tissue-specific promoter.

[0072] In certain embodiments, the T cell attracting chemokine and the composition that promotes T cell proliferation are driven by the same promoter (e.g., the T cell attractingchemokine and the composition that promotes T cell proliferation are synthesized as a peptide). In certain embodiments, the T cell attracting chemokine and the composition that promotes T cell proliferation are driven by different promoters. In certain embodiments, the antigen, the T cell attracting chemokine, and the composition that promotes T cell proliferation are driven by the same promoter. In certain embodiments, the antigen, the T cell attracting chemokine, and the composition that promotes T cell proliferation are driven by the different promoters. In certain embodiments, the T cell attracting chemokine and the composition that promotes T cell proliferation are driven by the same promoter, and the one or more antigens are driven by a different promoter.

[0073] In some embodiments, the antigen delivery system comprises one or more linkers between the T cell attracting chemokine and the composition that promotes T cell proliferation. In certain embodiments, linkers are used between one or more of the epitopes. The linkers may allow for cleavage of the separate molecules (e.g.. chemokine). For example, in some embodiments, a linker is positioned between IL-7 (or IL-2) and CCL5, CXCL9, CXCL10, CXCL11, CCL25, CCL28, CXCL14, CXCL17, etc. In some embodiments, a linker is positioned between IL-15 and CCL5, CXCL9, CXCL10, CXCL11, CCL25, CCL28, CXCL14, CXCL17, etc. In some embodiments, a linker is positioned between the antigen or large sequence and another composition, e.g., IL- 15, IL-7, CCL5, CXCL9, CXCL10, CXCL11, CCL25, CCL28, CXCL14, CXCL17, etc. A non-limiting example of a linker is T2A, E2A, P2A, or the like. The composition may feature a different linker between each open reading frame.

[0074] The present invention includes mRNA sequences encoding any of the vaccine compositions or portions thereof herein, e.g., a molecular adjuvant, a T cell enhancement, etc. The present invention also includes modified mRNA sequences encoding any of the vaccine compositions or portions thereof herein. The present invention also includes DNA sequence encoding any of the vaccine compositions or portions thereof herein.

[0075] In certain embodiments, nucleic acids of a vaccine composition herein are chemically modified. In some embodiments, the nucleic acids of a vaccine composition therein are unmodified. In some embodiments, all or a portion of the uracil in the open reading frame has a chemical modification. In some embodiments, a chemical modification is in the 5-position of the uracil. In some embodiments, a chemical modification is a N1 -methyl pseudouridine. In some embodiments, all or a portion of the uracil in the open reading frame has an N1 -methyl pseudouridine in the 5-position of the uracil.

[0076] In certain embodiments, an open reading frame of a vaccine composition herein encodes one antigen. In some embodiments, an open reading frame of a vaccine composition herein encodes two or more antigens. In some embodiments, an open reading frame of a vaccine composition herein encodes three or more antigens. In some embodiments, an open reading frame of a vaccine composition herein encodes four or more antigens. In some embodiments, an open reading frame of a vaccine composition herein encodes five or more antigens.

[0077] Methods

[0078] The compositions described herein, e.g., the antigens, the vaccine compositions, the antigen delivery systems, the chemokines, the adjuvants, etc., may be used to prevent herpes in a subject. In some embodiments, the compositions described herein, e.g., the antigens, the vaccine compositions, the antigen delivery systems, the chemokines, the adjuvants, etc., may be used to prevent a herpes infection prophy lactically in a subject. In some embodiments, the compositions described herein, e.g.. the antigens, the vaccine compositions, the antigen delivery systems, the chemokines, the adjuvants, etc., may elicit an immune response in a subject. In some embodiments, the compositions described herein, e.g., the antigens, the vaccine compositions, the antigen delivery systems, the chemokines, the adjuvants, etc., may prolong an immune response induced by the multi-antigen herpes vaccine composition and increases T-cell migration.

[0079] Methods for preventing herpes in a subject may include administering to the subject a therapeutically effective amount of a multi-antigen herpes composition according to the present invention. In some embodiments, the composition elicits an immune response in the subject. In some embodiments, the composition induces memory B and T cells. In some embodiments, the composition induces resident memory T cells (Trm). In some embodiments, the composition prevents virus replication, e.g., in the areas where the vims normally replicates. In some embodiments, the composition prevents a cytokine storm, e.g., in the areas where the virus normally replicates. In some embodiments, the composition prevents inflammation or an inflammatory response, e.g., in the areas where the vims normally replicates. In some embodiments, the composition improves homing and retention of T cells, e.g., in the areas where the virus normally replicates.

[0080] Methods for preventing a herpes infection prophylactically in a subject may comprise administering to the subject a prophylactically effective amount of a multi-antigen herpes vaccine composition according to the present invention. In some embodiments, the composition elicits an immune response in the subject. In some embodiments, the composition induces memory B andT cells. In some embodiments, the composition induces resident memory T cells (Trm). In some embodiments, the composition prevents virus replication, e.g., in the areas where the virus normally replicates. In some embodiments, the composition prevents a cytokine storm, e.g., in the areas where the virus normally replicates. In some embodiments, the composition prevents inflammation or an inflammatory response, e.g., in the areas where the virus normally replicates. In some embodiments, the composition improves homing and retention of T cells, e.g., in the areas where the virus normally replicates.

[0081] Methods for eliciting an immune response in a subject may include administering to the subject a vaccine composition according to the present invention, wherein the composition elicits an immune response in the subject. In some embodiments, the composition induces memory B and T cells. In some embodiments, the composition induces resident memory' T cells (Trm). In some embodiments, the composition prevents virus replication, e.g., in the areas where the virus normally replicates. In some embodiments, the composition prevents a cytokine storm, e.g., in the areas where the virus normally replicates. In some embodiments, the composition prevents inflammation or an inflammatory response, e.g., in the areas where the vims normally replicates. In some embodiments, the composition improves homing and retention of T cells, e.g., in the areas where the virus normally replicates.

[0082] Methods for prolonging an immune response induced by a vaccine composition of the present invention and increasing T cell migration to particular tissues may comprise co-expressing a T-cell attracting chemokine. a composition that promotes T cell proliferation, and a vaccine composition (e.g., antigen) according to the present invention.

[0083] Methods for prolonging the retention of memory T-cell into the lungs induced by a vaccine composition of the present invention and increasing virus-specific tissue resident memory T-cells (TRM cells) may comprise co-expressing a T-cell attracting chemokine, a composition that promotes T cell proliferation, and a vaccine composition (e.g., antigen) according to the present invention.

[0084] The vaccine composition may be administered through standard means, e.g., through an intravenous route (i.v.), an intranasal route (i n.), or a sublingual route (s.l.).

[0085] In certain embodiments, the method comprises administering to the subject a second (e.g., booster) dose. The second dose may comprise the same vaccine composition or a differentvaccine composition. Additional doses of one or more vaccine compositions may be administered.

[0086] Sequential Vaccine Delivery Methodology

[0087] In some embodiments, the present invention features a method of delivering the vaccine to induce heterologous immunity in a subject. In some embodiments, the method comprises administering a first multi-antigen herpes vaccine composition dose using a first delivery system. In further embodiments, the method comprises administering a second vaccine composition dose using a second delivery system. In some embodiments, the second composition is administered 8 days after administration of the first composition. In some embodiments, the second composition is administered 9 days after administration of the first composition. In some embodiments, the second composition is administered 10 days after administration of the first composition. In some embodiments, the second composition is administered 11 days after administration of the first composition. In some embodiments, the second composition is administered 12 days after administration of the first composition. In some embodiments, the second composition is administered 13 days after administration of the first composition. In some embodiments, the second composition is administered 14 days after administration of the first composition. In some embodiments, the second composition is administered from 14 to 30 days after administration of the first composition. In some embodiments, the second composition is administered from 30 to 60 days after administration of the first composition. In other embodiments, the first delivery system and the second delivery system are different. In some embodiments, the peptide vaccine composition is administered 14-days after the administration of the first vaccine composition dose. In some embodiments, the peptide vaccine composition is administered 30 or 60 days after the administration of the first vaccine composition dose.

[0088] In some embodiments, the first delivery system or the second delivery system comprises an mRNA. a modified mRNA. or a peptide vector. In other embodiments, the peptide vector comprises adenovirus or an adeno-associated virus vector.

[0089] In some embodiments, the present invention features a method of delivering the vaccine to induce heterologous immunity in a subject (i.e., prime / pull). In some embodiments, the method comprises administering a multi-antigen herpes vaccine composition. In further embodiments, the method comprises administering at least one T-cell attracting chemokine after administering the multi-antigen herpes vaccine composition. In some embodiments, the T-cell attracting chemokine is administered 8 days after the vaccine composition is administered. Insome embodiments, the T-cell attracting chemokine is administered 9 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered 10 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered 11 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered 12 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered 13 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered 14 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered from 14 to 30 days after administration of the vaccine composition. In some embodiments, the T-cell attracting chemokine is administered from 30 to 60 days after administration of the vaccine composition. In some embodiments, the T cell-attracting chemokine composition is administered 8 to 14-days after the administration of the final vaccine composition dose. In some embodiments, the cell-attracting chemokine composition is administered 30 or 60 days after the administration of the final vaccine composition dose.

[0090] The present invention also features a novel “prime, pull, and boost” strategy. In other embodiments, the present invention features a method to increase the size and maintenance of resident B-cells, CD4+ T cells, and CD8+ T cells to protect against herpes. In some embodiments, the method comprises administering a multi-antigen herpes vaccine composition. In other embodiments, the method comprises administering at least one T-cell attracting chemokine after administering the multi-antigen herpes vaccine composition. In further embodiments, the method comprises administering at least one cytokine after administering the T-cell attracting chemokine. In some embodiments, the T-cell attracting chemokine is administered 14 days after administering the multi-antigen herpes composition. In other embodiments, the cytokine is administered 10 days after administering the T-cell attracting chemokine. In some embodiments, the T-cell attracting chemokine is administered 8 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered 9 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered 10 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered 11 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered 12 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered 13 days after the vaccinecomposition is administered. In some embodiments, the T-cell attracting chemokine is administered 14 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered from 14 to 30 days after administration of the vaccine composition. In some embodiments, the T-cell attracting chemokine is administered from 30 to 60 days after administration of the vaccine composition. In some embodiments, the cytokine is administered 8 days after administering the T-cell attracting chemokine. In some embodiments, the cytokine is administered 9 days after administering the T-cell attracting chemokine. In some embodiments, the cytokine is administered 10 days after administering the T-cell attracting chemokine. In some embodiments, the cytokine is administered 11 days after administering the T-cell attracting chemokine. In some embodiments, the cytokine is administered 12 days after administering the T-cell attracting chemokine. In some embodiments, the cytokine is administered 13 days after administering the T-cell attracting chemokine. In some embodiments, the cytokine is administered 14 days after administering the T-cell attracting chemokine. In some embodiments, the cytokine is administered from 14 to 30 days after administering the T-cell attracting chemokine. In some embodiments, the cytokine is administered from 30 to 60 days after administering the T-cell attracting chemokine. In some embodiments, the cytokine composition is administered 8 to 14-days after the administration of the T cell-attracting chemokine. In some embodiments, the cytokine composition is administered 30 or 60 days after the administration of the T cell-attracting chemokine.

[0091] The present invention further features a novel “prime, pull, and keep” strategy . In further embodiments, the present invention features a method to increase the size and maintenance of resident B-cells, CD4+ T cells, and CD8+ T cells to protect against herpes. In some embodiments, the method comprises administering a multi-antigen herpes vaccine composition. In other embodiments, the method comprises administering at least one T-cell attracting chemokine after administering the multi-antigen herpes vaccine composition. In further embodiments, the method comprises administering at least one mucosal chemokine after administering the T-cell attracting chemokine. In some embodiments, the T-cell attracting chemokine is administered 14 days after administering the multi-antigen herpes composition. In other embodiments, the mucosal chemokines are administered 10 days after administering the T-cell attracting chemokine. In some embodiments, the T-cell attracting chemokine is administered 8 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered 9 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered 10 days after the vaccinecomposition is administered. In some embodiments, the T-cell attracting chemokine is administered 11 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered 12 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered 13 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered 14 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered from 14 to 30 days after administration of the vaccine composition. In some embodiments, the T-cell attracting chemokine is administered from 30 to 60 days after administration of the vaccine composition. In some embodiments, the mucosal chemokine is administered 8 days after administering the T-cell attracting chemokine.

[0092] In some embodiments, the mucosal chemokine is administered 9 days after administering the T-cell attracting chemokine. In some embodiments, the mucosal chemokine is administered 10 days after administering the T-cell attracting chemokine. In some embodiments, the mucosal chemokine is administered 11 days after administering the T-cell attracting chemokine. In some embodiments, the mucosal chemokine is administered 12 days after administering the T-cell attracting chemokine. In some embodiments, the mucosal chemokine is administered 13 days after administering the T-cell attracting chemokine. In some embodiments, the mucosal chemokine is administered 14 days after administering the T-cell attracting chemokine. In some embodiments, the mucosal chemokine is administered from 14 to 30 days after administering the T-cell attracting chemokine. In some embodiments, the mucosal chemokine is administered from 30 to 60 days after administering the T-cell attracting chemokine. In some embodiments, the mucosal chemokine composition is administered 8 to 14-days after the administration of the T cell-attracting chemokine. In some embodiments, the mucosal cytokine composition is administered 30 or 60 days after the administration of the T cell -attracting chemokine.

[0093] In some embodiments, the mucosal chemokines may comprise CCL25, CCL28, CXCL14, CXCL17, or a combination thereof. In some embodiments, the T-cell attracting chemokines may comprise CCL5, CXCL9, CXCL10. CXCL11, or a combination thereof. In some embodiments, the cytokines may comprise IL- 15, IL-2, IL-7 or a combination thereof.

[0094] In some embodiments, the efficacy (or effectiveness) of a vaccine composition herein is greater than 60%. In some embodiments, the efficacy (or effectiveness) of a vaccine composition herein is greater than 70%. In some embodiments, the efficacy (or effectiveness) of a vaccinecomposition herein is greater than 80%. In some embodiments, the efficacy (or effectiveness) of a vaccine composition herein is greater than 90%. In some embodiments, the efficacy (or effectiveness) of a vaccine composition herein is greater than 95%.

[0095] Vaccine efficacy may be assessed using standard analyses (see. e.g., Weinberg et al., J Infect Dis. 2010 Jun. 1; 201(11): 1607-10). For example, vaccine efficacy may be measured by double-blind, randomized, clinical controlled trials. Vaccine efficacy may be expressed as a proportionate reduction in disease attack rate (AR) between the unvaccinated (ARU) and vaccinated (ARV) study cohorts and can be calculated from the relative risk (RR) of disease among the vaccinated group with use of the following formulas: Efficacy=(ARU-ARV) / ARUxl00; and Efficacy=(l-RR)xl00.

[0096] Likewise, vaccine effectiveness may be assessed using standard analyses (see, e.g., Weinberg et al.. J Infect Dis. 2010 Jun. 1; 201(11): 1607-10). Vaccine effectiveness is an assessment of how a vaccine (which may have already proven to have high vaccine efficacy) reduces disease in a population. This measure can assess the net balance of benefits and adverse effects of a vaccination program, not just the vaccine itself, under natural field conditions rather than in a controlled clinical trial. Vaccine effectiveness is proportional to vaccine efficacy (potency) but is also affected by how well target groups in the population are immunized, as well as by other non-vaccine-related factors that influence the ‘real-world’ outcomes of hospitalizations, ambulatory visits, or costs. For example, a retrospective case-control analysis may be used, in which the rates of vaccination among a set of infected cases and appropriate controls are compared. Vaccine effectiveness may be expressed as a rate difference, with use of the odds ratio (OR) for developing infection despite vaccination: Effectiveness=(l-OR)xl00.

[0097] In some embodiments, the vaccine immunizes the subject against herpes for up to 1 year. In some embodiments, the vaccine immunizes the subject against herpes for up to 2 years. In some embodiments, the vaccine immunizes the subject against herpes for more than 1 year, more than 2 years, more than 3 years, more than 4 years, or for 5-10 years.

[0098] Pharmaceutical Carriers

[0099] In certain embodiments, the vaccine composition further comprises a pharmaceutical carrier. Pharmaceutical carriers are well known to one of ordinary skill in the art. For example, in certain embodiments, the pharmaceutical carrier is selected from the group consisting of w ater, an alcohol, a natural or hardened oil, a natural or hardened wax, a calcium carbonate, a sodiumcarbonate, a calcium phosphate, kaolin, talc, lactose and combinations thereof. In some embodiments, the pharmaceutical carrier may comprise a lipid nanoparticle, an adenovirus vector, or an adeno-associated virus vector. In some embodiments, the vaccine composition is constructed using an adeno-associated virus vectors-based antigen delivery system.

[0100] Also provided herein is vaccine of any one of the foregoing paragraphs, formulated in a nanoparticle (e.g., a lipid nanoparticle). In some embodiments, the nanoparticle has a mean diameter of 50-200 nm. In some embodiments, the nanoparticle is a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of about 20-60% cationic lipid, 0.5-15% PEG-modified lipid, 25-55% sterol, and 25% non-cationic lipid. In some embodiments, the cationic lipid is an ionizable cationic lipid, and the non-cationic lipid is a neutral lipid, and the sterol is a cholesterol. In some embodiments, the cationic lipid is selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[ l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9-((4-(dimethy lamino)butanoyl)oxy)heptadecanedioate (L319).

[0101] EXAMPLE

[0102] The following is a non-limiting example of the present invention. It is to be understood that said example is not intended to limit the present invention in any way. Equivalents or substitutes are within the scope of the present invention.

[0103] Adenovirus vectors were engineered to cany' these genes that encode five different antigens from the herpes virus. These five antigens were RR1(UL39), RR2(UL40), gD(gly coprotein D), VP16(UL48), and VP22(UL49) viral antigens. These antigens demonstrated moderate to significant protection as subunit vaccines. HSV-2 latent-infected guinea pigs were immunized intramuscularly with these rAd-Ags expressing five different adenoviral antigens. Recombinant adenovirus-based vaccines showed higher reductions in genital lesions and stronger antigen-specific T-cell responses. Moreover, the severity of genital lesions in mice immunized with rAd-Ags was significantly decreased compared to Mock. The rAd-RR2 and rAd-gD displayed superior protection compared to other antigens in inducing T-cell responses, IFN-y response, or reducing viral titers. In addition, the rAd-RR2 and rAd-gD were also comparable to dl-529 in preventing recurrence, hence posing a potential therapeutic vaccine against recurrent infections.

[0104] Animals: Female guinea pigs (Hartley strain, Charles River Laboratories, San Diego, CA) weighing 275-350 g (5-6 weeks old) were housed at the University of California, Irvine vivarium. The Institutional Animal Care and Use Committee of the University of California, Irvine, reviewed and approved the protocol for these studies (IACUC # AUP -22-086). A group size of 10 had 90% power to detect a difference of two-fold or higher between experimental group means with a significance level of 0.05.

[0105] Virus generation: Adenovirus type 5 (rAd5) vaccines were generated based on the published antigen sequences of HSV-2, variants. These sequences were inserted into a recombinant plasmid containing rAd5 sequence that is deleted in El and E3 genes. The respective transgenes were cloned into the El region that additionally contains a downstream molecular dsRNA adjuvant in the gene cassette and is expressed together with the transgene in the target cell. rAd5 particles were generated by transfection of transgene-containing DNA into Expi293F cells (ThermoFisher Scientific) to generate rAd5 virions which were purified by CsCl density centrifugation.

[0106] Infection and Immunization of Guinea Pigs: Throughout this study, the MS strain of HSV-2 was used. Guinea pigs (n = 42) were infected intravaginally with 5 x 10’ pfu of HSV-2 (strain MS). The virus was diluted to a final volume of lOOpl using dPBS and administered using a pipette. The viral delivery was made possible using a butterfly infusion tubing (BD Vacutainer-368656) attached to the pipette tip. Once the acute infection was resolved, latently infected animals were separated into 7 groups (6 animals each) and vaccinated intramuscularly in the right hind calf muscle on day 15 post-infection. BD 1 ml Tuberculin (TB) syringe 25G was used for intramuscular injection of Adenovirus type 5 expressing RR2, RR1, gD, VP22 and VP16 proteins (rAd-RR2, rAd-RRl, rAd-gD, rAd-VP22,and rAd-VP16). The adenovirus was diluted to a final 100 pl / guinea pig volume using dPBS. D1529 treated animals were used as positive control while the Mock group was treated with adenovirus vector alone.

[0107] Monitoring of recurrent HSV-2 disease in guinea pigs. To study genital HSV-2 infection, female guinea pigs were infected intravaginally with HSV-2. The acute (primary) genital lesions resolve by two weeks, followed by spontaneous reoccurrence over the ensuing months (recurrent phase). Viral shedding may be evidenced without even visible lesions by qPCR. Guinea pigs were examined for vaginal lesions and were recorded for each animal every alternate day starting from day 25 until day 52 post-challenge.

[0108] Real-time qPCR for HSV-2 Quantification from Vaginal Swabs and dorsal root ganglia: Vaginal swabs were collected every alternate day using a Dacron swab (type 1; Spectrum Laboratories, Los Angeles, CA) starting from day 25 until day 52 post-challenge. Individual swabs were transferred to a 2 mL sterile cryogenic vial containing 500ul culture medium and stored at -80°C until use. On day 52 post-challenge, VM and twelve lower lumbar and sacral dorsal root ganglia (DRG) per guinea pig were collected by cutting through the lumbar end of the spine. DNA was isolated from the collected vaginal swab, VM and DRG of guinea pigs by using DNeasy blood and tissue kits (Qiagen). The presence of HSV-2 DNA was quantified by real-time PCR with 100 ng DNA. HSV-2 DNA copy number was determined using purified HSV-2 DNA (Advanced Biotechnologies, Columbia, MD). Primer sequences for HSV-2 Us9 were: primer forward, 5'-GGCAGAAGCCTACTACTCGGAAA-3', and reverse 5 -CCATGCGCACGAGGAAGT-3'.

[0109] Splenocyte isolation: Spleens were harvested from guinea pigs at 52 days post-infection. Spleens were placed in 10 ml of cold PBS with 10% fetal bovine serum (FBS) and 2X antibiotic-antimycotic (Life Technologies, Carlsbad, CA). Spleens were minced finely and sequentially passed through a 100 pm mesh and a 70 pm mesh (BD Biosciences, San Jose, CA). Cells were then pelleted via centrifugation at 400 x g for 10 minutes at 4°C. Red blood cells were lysed using a lysis buffer and washed again. Isolated splenocytes were diluted to 1 * 106viable cells per ml in RPMI media with 10% (v / v) FBS and 2 x antibiotic-antimycotic. Viability was determined by Trypan blue staining.

[0110] Isolation of lymphocytes from the guinea pig's vaginal mucosa and dorsal root ganglia. Vaginal mucosa and DRG were minced into fine pieces on a Petri Dish using a fine scalpel. The tissue was subjected to collagenase (7mg / ml) treatment and allowed to digest at 37°C for one hour on a rocker. After one hour, the digested tissue suspension was passed through a 100 pm cell strainer, followed by centrifugation and washing with RPMI media containing 10% FBS. Lymphocytes in the cell pellets were then suspended in 40% percoll, layered on top of 70% percoll, and centrifuged at 900 x g at room temperature for 30 minutes with the brake-off. The lymphocytes at the interface layer between 40% and 70% Percoll layers were harvested, washed with three volumes of RPMI, and spun down at 740 x g.

[0111] Enzyme-Linked Immunosorbent Assay (ELISA). Blood (5 ml) was drawn from each guinea pig into yellow-top Vacutainer tubes (Becton, Dickinson). Sera were isolated by centrifugation for 10 min at 800 x g. For measuring antigen-specific antibody titers in the sera ofimmunized guinea pigs, ELISA plates were coated with 50 ng of individual HSV-2 proteins and incubated with guinea pig serum at a 1 : 1,000 dilution, followed by horseradish peroxidase (HRP)-conjugated anti-guinea pig IgG.

[0112] Flow cytometry analysis: Vaginal mucosa cells and splenocytes were analyzed by flow cytometry using the following antibodies: mouse anti-guinea pig CD8 (clone MCA752F, Bio-Rad Laboratories, Hercules, CA), mouse anti-guinea pig CD4 (clone MCA749PE, Bio-Rad Laboratories), anti -mouse CRTAM (clone 11-5, Biolegend, San Diego, CA), anti-mouse / human CD44 (clone IM7, Biolegend), anti-mouse CD69 (clone H1.2F3, BD Biosciences, San Jose, CA), and anti-mouse CD103 (clone 2E7. Biolegend). For surface staining, mAbs against various cell markers were added to a total of 1X106cells in phosphate-buffered saline containing 1% FBS and 0.1% sodium azide (fluorescence-activated cell sorter [FACS] buffer) and left for 45 minutes at 4°C. At the end of the incubation period, the cells were washed twice with FACS buffer. A total of 100,000 events were acquired by the LSRII (Becton Dickinson, Mountain View, CA), followed by analysis using FlowJo version 10 software (TreeStar, Ashland, OR).

[0113] Statistical analyses: Data for each assay were compared by analysis of variance (ANOVA) using GraphPad Prism version 10.1.0. Differences between the groups were identified by ANOVA and multiple comparison procedures. Data are expressed as the mean + SD. Results were considered statistically significant at P < 0.05.

[0114] Therapeutic immunization of HSV-2 infected guinea pigs with AD5 expressing RR2 and gD protects against recurrent genital disease: Guinea pigs (n = 42) were infected intravaginally with 5 x 105pfu of HSV-2 (strain MS) (FIG. 1A). Once acute infection was resolved, latently infected animals were randomly divided into seven groups (n = 6) and then vaccinated intramuscularly on days 15 post-infection with Ad5 expressing RR2, RR1, gD, Vp22 and VP16 proteins (rAd-RR2. rAd-RRl, rAd-gD, rAd-VP22.and rAd-VP16). D1529 treated animals were used as positive control while Mock-vaccinated guinea pigs (n = 6), who received adenovirus alone, were used as negative controls. Starting on day 25 until day 52, the guinea pigs were observed and scored regularly for genital lesions. The vaccinated animals exhibited significantly lower cumulative vaginal lesions (FIG. IB) and an overall significant reduction in cumulative positive days of recurrence compared to the mock-vaccinated controls (FIG. 1C). rAd-RR2, and, rAd-gD vaccinated animals displayed lowest cumulative vaginal lesions and an overall reduced cumulative positive days of recurrence compared to the other vaccinated animals(FIG. IB and 1C)

[0115] On day 52 post-infection, the vaccinated guinea pigs exhibited lower HSV-2 DNA copy numbers in the DRG and VM than did mock-vaccinated controls (FIG. ID & IE), which was associated with a significant reduction in cumulative virus vaginal shedding in the vaccinated group as compared to the mock vaccinated control group. rAd-RR2, and . rAd-gD vaccinated animals displayed lowest HSV-2 DNA copy numbers in the DRG and VM which was comparable to dl529 (FIG. ID & IE). The severity of genital herpetic lesions scored on a scale of 0 to 4, also confirmed the cure of recurrent disease in rAd-RR2, and, rAd-gD vaccinated guinea pigs (FIG. IF). The lowest genital lesions were observed in guinea pigs vaccinated with rAd-RR2 (FIG. IF). The other vaccinated groups were moderately protective against genital lesions (FIG. IF). However, the mock vaccinated group showed no significant protection against recurrent genital herpes lesions (FIG. IF, right panel). Altogether, these results indicate that therapeutic immunization with AD-RR2 and AD-gD protected HSV-2-seropositive guinea pigs against recurrent genital herpes infection and disease.

[0116] Therapeutic immunization of HSV-2 infected guinea pigs with AD5-antigen produced higher antibody titer against the corresponding antigen: Five-weeks after the Adenovirus immunization, immune sera was collected and tested for Antigen specific IgG by ELISA. Significantly higher levels of IgG specific to immunizing antigens were detected by ELISA in the serum of guinea pigs that were vaccinated with AD5-antigen (FIG. 2A). Sera from Ad-5 vaccinated animals evaluated at 1 :1000 dilution recognized their corresponding coating antigens (FIG. 2B). Immune sera from guinea pigs that were vaccinated with rAd-RR2, rAd-VP22, and rAd-gD had IgG that bound with high affinity to corresponding coated antigen (FIG. 2B). Sera from dl529 vaccinated animals evaluated at 1 : 1000 dilution also recognized RR2, Vp22 and gD as coating antigens (FIG. 2B). However, sera from mock vaccinated animals displayed less titer (FIG. 2B). Furthermore, the highest frequencies of IFN-y-producing cells measured by ELISpot were detected in the VM cell suspensions of guinea pigs that were vaccinated with rAd-RR2. rAd-RRl, and rAd-gD while lesser spot forming cells were observed in VM cell suspension of guinea pigs that were immunized with rAd-VP22, and rAd-VP16 (FIG. 2C & 2D). Mock displayed the lowest spot forming cells(FIG. 2C & 2D).

[0117] Therapeutic vaccination of HSV-2 infected guinea pigs with rAd-RR2, rAd-RRl and rAd-gD increased the frequencies of tissue-resident CD4+and CD8+T cells in the VM and DRG: Guinea pigs (n = 42) were infected intravaginally with 5 \ I (f pfu of HSV-2 (strain MS). Once the acute infection was resolved, latently infected animals were immunizedwith IO10adenovirus antigen viral particles (VP) on day 15 post-infection. One of the groups was immunized with dl529 (positive control) while another group was treated with vector alone (adenovirus alone) and referred to as mock. On day 52 post-infection, guinea pigs were euthanized, single-cell suspensions from the VM tissue were obtained, and the DRG, VM, and Spleen resident CD4+and CD8+T cells were analyzed. A significantly higher frequencies of CD4+and CD8+T cells in the rAd-RR2, rAd-RRl, rAd-gD, and rAd-VP16 vaccinated groups compared to those with the mock-vaccinated group (i.e., adjuvant alone) in the DRG tissues (FIG. 3A & 3B: top panel). rAd-VP22 didn’t show significantly higher frequency of CD4+and CD8+T cells compared to mock- vaccinated group (FIG. 3A & 3B: top panel). Also, rAd-VP16 had less frequency of CD8+T cells compared to mock-vaccinated group (FIG. 3A & 3B: top panel). A significantly higher frequencies of CD4+cells in the rAd-RR2 vaccinated groups compared to those with the mock-vaccinated group (i.e., adjuvant alone) in the VM tissues was observed (FIG. 3A & 3B: middle panel). rAd-RRl, rAd-gD, and rAd-VP16 showed relatively but not significant higher frequencies of CD4+cells compared to Mock (FIG. 3A & 3B: middle panel). A significantly higher frequencies of CD8 T cells in the rAd-RR2, rAd-VP22, and rAd-VP16) vaccinated groups compared mock was obsereved (FIG. 3A & 3B: middle panel). The spleen showed a significantly higher frequencies of CD4+T cells in the rAd-RR2, rAd-RRl, rAd-VP22, rAd-gD, and rAd-VP16) vaccinated groups compared to Mock (FIG. 3A & 3B: lower panel). A significantly higher frequency of CD8+T cells was observed in the rAd-RR2, rAd-VP22, and rAd-VP16 in vaccinated groups compared to mock (FIG. 3A & 3B: lower panel).

[0118] Therapeutic vaccination of HSV-2 infected guinea pigs with rAd-RR2, rAd-RRl and rAd-gD efficiently generates total memory T cells in the VM and DRG: The generation of total memory cells in the VM and DRG tissue of HSV-2-infected and vaccinated guinea pigs was subsequently examined.. The total memory cells in the VM and DRG were analyzed by observing the expression of CD44 memory cell markers on CD4 T and CD81T cells by FACS. Significantly higher frequencies of CD4+CD44+T cells were induced in the DRG by rAd-RR2, rAd-RRl, followed by rAd-gD, rAd-VP22 and rAd-VP16 vaccinated groups compared to Mock (FIG. 4A: top panel). However, higher frequencies of CD4+CD44+T cells were induced in the VM of rAd-RR2. rAd-RRl and rAd-gD followed by rAd-VP16 vaccinated groups compared to Mock (FIG. 4A: bottom panel). rAd-RR2, rAd-RRl, rAd-gD, rAd-VP22 and rAd-VP16 vaccinated groups showed higher frequencies of CD8+CD44+T cells compared to Mock in the DRG of guinea pigs (FIG. 4B: top panel). However, only rAd-RR2, rAd-RRl, andrAd-gD showed higher frequencies of CD8 CD441T cells compared to Mock in the VM of guinea pigs (FIG. 4A: bottom panel).

[0119] Therapeutic vaccination of HSV-2 infected guinea pigs with rAd-antigen generates activated T cells in the VM and DRG: CD69 and CRTAM are rapidly expressed on the cell surface following activation and are used to identify activated immune cells. To assess this, the activation status of T cells in the VM and DRG tissue of HSV-2-infected and vaccinated guinea pigs was examined. The activation status in the VM and DRG was analyzed by observing the expression of CD69 markers on CD4+T and CD8+T cells by FACS. Significantly higher frequencies of CD4+CD69+T cells were induced in the DRG by rAd-RR2, rAd-RRl. rAd-gD, rAd-VP22 and rAd-VP16 vaccinated groups compared to Mock (FIG. 5A: top panel). However, higher frequencies of CD4+CD69+T cells were induced in the VM of rAd-RR2, rAd-RRl and rAd-gD followed by rAd-VP16 vaccinated groups compared to Mock (FIG. 5A: bottom panel). rAd-RR2, rAd-RRl. rAd-gD. and rAd-VP22 vaccinated groups showed higher frequencies of CD8+CD69+T cells compared to Mock in the DRG of guinea pigs (FIG. 5B: top panel). However, only rAd-RR2, and rAd-gD followed by rAd-RRl, and rAd-VP22 showed higher frequencies of CD8+CD69+T cells compared to Mock in the VM of guinea pigs (FIG. 5B: bottom panel).

[0120] Therapeutic vaccination of HSV-2 infected guinea pigs with rAd-antigen efficiently generates tissue-resident memory in the VM and DRG: Next, the association of various protection parameters (i.e., virus shedding and severity and frequency of recurrent genital herpes lesions) with tissue-resident memory that resides at the vaginal mucocutaneous and DRG tissue of HSV-2-infected and vaccinated guinea pigs was determined. CD 103 is a marker for tissue-resident memory7T-cells and binds to E-cadherin to facilitate the retention of T cells within epithelial cells. This allows the resident immune cells to provide rapid and localized immune response upon virus entry or reactivation. The resident T cells in the VM and DRG were analyzed by observing the expression of CD103 on CD8+T by FACS. CRTAM marker was also analyzed to determine the activation status of on CD8+T cells. Significantly higher frequencies of CD8 CD I 03 T cells were induced in the DRG by vaccination of rAd-RR2, rAd-RRl, rAd-gD, and rAd-VP22 compared to Mock (FIG. 6A: top panel). However, higher frequencies of CD8 CD 103 T cells were induced only in the VM of rAd-RR2 and rAd-gD vaccinated groups compared to Mock (FIG. 5A: bottom panel). rAd-RR2, rAd-RRl, and rAd-VP16 vaccinated groups showed higher frequencies of CD8+CRTAM+T cells compared to Mock in the DRG ofguinea pigs (FIG. 6B: top panel). However, rAd-RR2, rAd-RRl, rAd-gD, and rAd-VP showed higher frequencies of CD8+CRTAM+T cells compared to Mock in the VM of guinea pigs (FIG.6B: bottom panel).

[0121] Induced protection from HSV-2 infection following therapeutic rAd-antigen vaccination is associated with more functional tissue-resident IFN-y+TNFa+Ki67 CD4 and CD8+ T cells: Next, the function of CD44 and CD8+ T cells was compared in the DRG, and VM, of HSV-2-infected guinea pigs following rAd-antigen vaccination. On day 52, after the immunization guinea pigs were euthanized, and single-cell suspensions from the DRG and VM tissues were obtained, and the function of DRG-resident and VM-resident T cells was analyzed by production of Ki67, IFN-y and TNF-a expression by FACS.

[0122] Significantly higher frequencies of CD4+Ki67+T cells were induced in the DRG of rAd-RR2 and rAd-gD, vaccinated groups compared to Mock (FIG. 7A: top panel). However, higher frequencies of CD4 I<i67 T cells were induced in the VM of rAd-RR2 and rAd-gD followed by rAd-VP 16 , rAd-RRl and rAd-VP22 vaccinated groups compared to Mock (FIG. 7A: bottom panel). Similarly, rAd-RR2, rAd-RRl, rAd-gD, and rAd-VP16 vaccinated groups showed higher frequencies of CDS' Ki67' T cells compared to Mock in the DRG of guinea pigs (FIG. 7B: top panel). However, rAd-RR2, rAd-RRl, rAd-gD, and rAd-VP22 showed higher frequencies of CD8+Ki67+T cells compared to Mock in the VM of guinea pigs (FIG. 7B: bottom panel). Conversely. rAd-VP 16 did not show any higher frequencies of CD8+Ki67+T cells compared to Mock in the VM of guinea pigs (FIG. 7B: bottom panel).

[0123] rAd-RR2, rAd-RRl, rAd-gD, and rAd-VP22 vaccinated groups displayed high frequencies of CD4+IFNg+T cells in the DRG (FIG. 8A: top panel). While rAd-VP16, and rAd-VP22 vaccinated groups displayed low frequencies of CD4+IFNg+T cells in the VM (FIG. 8A: bottom panel). However, all rAD-antigen groups displayed high frequencies of CD8+lFNg+T cells in the DRG (FIG. SB: top panel). While rAd-VP16, showed low frequencies of CD8+IFNg+T cells in the VM (FIG. 8B: bottom panel). Similarly CD4+TNF-a was exhibited by all rAD-antigen groups in the DRG (FIG. 9A: top panel). While only rAd-RR2, and rAd-gD displayed higher frequencies of CD4+TNF-a in the VM (FIG. 9B: bottom panel). Likewise, all rAD-antigen groups except rAd-VP16 displayed high frequency of CD8+TNFa+T cells in the DRG and VM (FIG. 9B).

[0124] HSV-2 infection affects more than 500 million people worldwide and increasesthe risk of HIV acquisition and transmission. The health and socioeconomic burden concerning genital herpes highlights the need for a therapeutic herpes vaccine that can alleviate the disease impact. Therapeutic vaccines targeted towards HSV-2 infected people will reduce both symptomatic and asymptomatic genital HSV-2 shedding, and reduce the risk of transmission of both HSV and HSV-associated viral diseases like HIV. Extensive research in both animal models (mouse, guinea pig) and human studies have led to an improved but incomplete understanding of the immune responses that need to be stimulated by a vaccine. These include HSV antigen-specific neutralizing antibody responses, which are thought to be particularly important for a prophylactic vaccine. Adaptive cellular immune responses have been shown to be important for clearance of viruses in people with HSV infection, and stimulation of these responses are essential for therapeutic vaccines. In addition, stimulation of mucosal immune responses at the site of infection is important for both prophylactic and therapeutic vaccines.

[0125] Vaccine strategies for therapeutic as well as prophylactic vaccination include subunit vaccines, attenuated or replication deficient virus, and DNA-based vaccines. Immunizing guinea pigs with an immunogenic HSV-2 protein RR2 and treatment with adenovirus-expressing chemokine provide better protection against recurrent genital herpes. This strategy helped to prevent the migration of HSV-2 from mucosa to neurons leading to decreased reactivation and viral shedding. Subunit vaccines target HSV surface glycoproteins and generate strong neutralizing antibody responses. These glycoproteins are commonly targeted by the immune system, however, they did not provide sufficient protection against recurrent herpetic disease when included in the vaccine. HSV-2-based glycoprotein-subunit vaccines failed to provide adequate antiviral protection in large-scale clinical tnals as a result of the virus developing strategies to evade immune responses. Although these strategies have not produced a licensed vaccine, they have provided important information about correlates of protection and proof of concept to direct future HSV vaccine studies. Given that glycoprotein subunit vaccines have failed to prevent or reduce the symptoms of HSV-2 genital herpes in trials, perhaps it is time to consider the logical alternative of an adenovirus based vaccine.

[0126] An increased number and function of DRG-resident CD4+and CD8+T cells is associated with a reduction of HSV virus in the DRG, reduced severity, and rate of recurrent genital herpes in latently infected vaccinated guinea pigs. The presence of T cells near infected neurons affects the viral replication and / or reduced recurrences of HSV-2 from latency, which in turn reduces the severity of recurrent genital herpes. Moreover, the presence and increasednumber of functional CD41and CD81T cells in vaginal mucosa also affects viral replication at the entry site. To address this, the therapeutic efficacy of recombinant adenoviruses was evaluated in a guinea pig model, which is capable of expressing full length envelope glycoprotein D (gD), the tegument protein VP22 (encoded by the UL49 gene) and VP 16 (encoded by the UL48 gene), and ribonucleotide reductase subunit 1& 2 protein (RR1; encoded by the UL39 gene & RR2; encoded by the UL40 gene). Recombinant adenovirus-based vaccines exhibited protection against HSV infection and generated strong antigen-specific T-cell responses. Moreover, the severity' of genital lesions in mice immunized with rAd antigens was significantly decreased. Compared with rAd-gd and rAd-RR2; rAd-VP22 and rAd-VP16 showed lesser protection against the intravaginal HSV-2 challenge.

[0127] The present study demonstrates that intramuscular vaccination of guinea pigs with rAD-antigen increased the number of functional tissue-resident IFN-y-producing CD69+CD44+CD4+and CRTAM+CD103+CD8+TRMcells in the vaginal mucosa, and protected against spontaneous recurrent genital herpes in the infected guinea pigs. These robust local B- and T-cell responses were associated with a significant reduction in both virus shedding and the severity7and frequency of recurrent genital herpes lesions. Interestingly, therapeutic immunization with rAdVP22 and rAd-VP16 did not result in a more robust protection than with rAd-RR2, rAd-RRl and rAd-gD. These prechnical findings demonstrate rAd-RR2, rAd-RRl and rAd-gD as viable vaccine candidates to be incorporated in the next genital herpes therapeutic mucosal vaccine to be clinically tested. Nevertheless, it is interesting that the protective efficacy of rAd-RR2 and rAd-gD vaccine was comparable to that induced with the dl5-29. The rAd-RR2 and rAd-gD demonstrate a viable B- and T-cell candidate antigen to be incorporated in future genital herpes therapeutic mucosal vaccines.

[0128] In conclusion, this study demonstrates that immunizing guinea pigs with an immunogenic rAd-RR2 and rAd-gD vaccine provides better protection against recunent genital herpes. The use of recombinant adenovirus seems to produce a required immune response that leads to decreased reactivation and viral shedding. Recombinant adenovirus elicits a robust cellular and humoral immunity' against the encoded antigen and vaccination with rAd can produce effective anti-viral immunity. To date, no study has reported the role of rAd-RR2 and rAd-gD vaccine in attracting more functional DRG-resident CD4+and CD8+T cells which in turn affects recurrent genital herpes infection and disease. In the present study, an increased number and function of DRG-resident CD4+and CD8+T cells were associated with a reductionin the HSV-2 DNA copy numbers in the DRG. reduced severity, and rate of recurrent genital herpes in latently infected vaccinated guinea pigs. The presence of T cells near infected neurons may likely affect the viral replication and / or reduced recurrences of HSV-2 from latency, which in turn reduces the severity of recurrent genital herpes. Moreover, the presence and increased number of functional CD4+and CD8+T cells in vaginal mucosa may also affect viral replication at the entry site. However, providing mechanistic evidence of the direct implication of DRG- and VM-resident CD4+and CD8+T cells in reduction of spontaneous active replication would require extensive in vivo and in vitro studies, and this will be the subject of future reports.

[0129] As used herein, the term ‘’about” refers to plus or minus 10% of the referenced number.

[0130] Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting essentially of or “consisting of’, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting essentially of’ or “consisting of’ is met.

Claims

WHAT IS CLAIMED IS:

1. A multi-antigen herpes vaccine composition, the composition comprising a sequence encoding or comprising two or more different herpes antigens selected from a group consisting of RR1, RR2, gD. or VP22.

2. A multi-antigen herpes vaccine composition, the composition comprising a sequence encoding or comprising three or more different herpes antigens selected from a group consisting of RR1, RR2, gD, or VP22.

3. The composition of claim 1 or claim 2, wherein the herpes antigens are further selected from the group consisting of RR1. RR2. gD. or VP22. VP 11 / 12.

4. The composition of any one of claims 1-3, further comprising a T cell attracting chemokine.

5. The composition of claim 4, wherein the T cell attracting chemokine is CCL5, CXCL9, CXCL10, CXCL11, or a combination thereof.

6. The composition of any one of claims 1-5, further comprising a composition that promotes T cell proliferation and T-cell memory'.

7. The composition of claim 6, wherein the composition that promotes T cell proliferation and T-cell memory is IL-7. IL-2, or IL-15.

8. The composition of any one of claims 1-7, wherein the vaccine composition protects against infection or reinfection of one or more herpes viruses.

9. The composition of claim 8, wherein the herpes virus comprises Herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2).

10. The composition of any one of claims 1-9. wherein the vaccine composition induces strong and long-lasting protection mediated by antibodies (Abs), CD4+ T helper (Thl) cells, and / or CD8+ cytotoxic T-cells (CTL).I L A method of eliciting a T-cell response, an IFN-y response, or reducing viral titers in an individual, the method comprising administering to said individual the vaccine composition according to any one of claims 1-10.

12. A multi-antigen herpes vaccine composition, the composition comprising a delivery system encoding two or more different herpes antigens selected from a group consisting of RR1, RR2, gD, or VP22.

13. A multi-antigen herpes vaccine composition, the composition comprising a delivery system encoding three or more different herpes antigens selected from a group consisting of RRl, RR2, gD, or VP 16.

14. The composition of claim 12 or claim 13, further comprising an adjuvant.

15. The composition of any one of claims 12-14, further comprising a pharmaceutical carrier.

16. A multi-antigen herpes vaccine composition, the composition comprising a pharmaceutical carrier, an adjuvant, and delivery system encoding two or more different herpes antigens selected from a group consisting of RR1, RR2, gD, or VP22.

17. A multi-antigen herpes vaccine composition, the composition comprising a pharmaceutical carrier, an adjuvant, and delivery system encoding three or more different herpes antigens selected from a group consisting of RR1, RR2, gD, or VP22.

18. The composition of any one of claims 12-17, wherein the herpes antigens are further selected from the group consisting of RR1, RR2, gD, or VP22, VP11 / 12.

19. The composition of any one of claims 12-18, wherein the delivery system is a single delivery system.

20. The composition of any one of claims 12-19, wherein the delivery system comprises two or more delivery systems.

21. The composition of any one of claims 12-20, wherein the delivery system is an adeno-associated viral vector antigen delivery system.

22. The composition of claim 21, wherein the adeno-associated viral vector is an adeno-associated virus vector type 8 (AAV8 serotype) or an adeno-associated virus vector type 9 (AAV9 serotype).

23. The composition of claim 21 or claim 22, wherein the adeno-associated viral vector is an adeno-associated virus vector type 8 (AAV8 seroty pe).

24. The composition of claim 21 or claim 22. wherein the adeno-associated viral vector is an adeno-associated virus vector type 9 (AAV9 serotype).

25. The composition of any one of claims 12-24, wherein the delivery system is a vesicular stomatitis virus (VSV) vector.

26. The composition of any one of claims 12-25, wherein antigens are operatively linked to a generic promoter.

27. The composition of claim 26, wherein the generic promoter is a CMV or a CAG promoter.

28. The composition of any one of claims 12-27, wherein the delivery7system further encodes a T cell attracting chemokine.

29. The composition of claim 28, wherein the T cell attracting chemokine is CCL5. CXCL9, CXCL10, CXCL 11 , or a combination thereof.

30. The composition of claim 28 or claim 29, wherein the T cell atracting chemokine is operatively linked to a generic promoter.

31. The composition of any one of claims 12-30, wherein the delivery system further encodes a composition that promotes T cell proliferation and T-cell memory.

32. The composition of claim 31, wherein the composition that promotes T cell proliferation and T-cell memory is IL-7, IL-2, or IL-15.

33. The composition of claim 31 or claim 32, wherein the composition that promotes T cell proliferation and T-cell memory is operatively linked to a generic promoter.

34. The composition of any one of claim 28-33, wherein the T cell attracting chemokine and the composition that promotes T cell proliferation and T-cell memory are driven by the same promoter.

35. A multi-antigen herpes vaccine composition, the composition comprising a delivery system encoding two or more different herpes antigens selected from a group consisting of RRl, RR2, or VP22.

36. The composition of claim 35, further comprising an adjuvant.

37. The composition of claim 35 or claim 36, further comprising a pharmaceutical carrier.

38. A multi-antigen herpes vaccine composition, the composition comprising a pharmaceutical carrier, an adjuvant, and delivery system encoding two or more different herpes antigens selected from a group consisting of RR1, RR2, or VP22.

39. The composition of any one of claims 35-38, further comprising a delivery system encoding a gD herpes antigen.

40. The composition of any one of claims 35-39, wherein the delivery system is an adeno-associated viral vector antigen delivery system.

41. The composition of claim 40, wherein the adeno-associated viral vector is an adeno-associated virus vector type 8 (AAV8 serotype) or an adeno-associated virus vector type 9 (AAV9 serotype).

42. The composition of claim 40 or claim 41. wherein the adeno-associated viral vector is an adeno-associated virus vector type 8 (AAV8 serotype).

43. The composition of claim 40 or claim 41, wherein the adeno-associated viral vector is an adeno-associated virus vector type 9 (AAV9 seroty pe).

44. The composition of any one of claims 35-43, wherein the delivery system is a vesicular stomatitis virus (VSV) vector.

45. The composition of any one of claims 35-44, wherein antigens are operatively linked to ageneric promoter.

46. The composition of claim 45, wherein the generic promoter is a CMV or a CAG promoter.

47. The composition of any one of claims 35-46, wherein the delivery' system further encodes a T cell attracting chemokine.

48. The composition of claim 47, wherein the T cell attracting chemokine is CCL5, CXCL9, CXCL10, CXCL11, or a combination thereof.

49. The composition of claim 47 or claim 48, wherein the T cell attracting chemokine is operatively linked to a generic promoter.

50. The composition of any one of claims 35-49, wherein the delivery system further encodes a composition that promotes T cell proliferation and T-cell memory.

51. The composition of claim 50, wherein the composition that promotes T cell proliferation and T-cell memory is IL-7, IL-2, or IL-15.

52. The composition of claim 50 or claim 51, wherein the composition that promotes T cell proliferation and T-cell memory is operatively linked to a generic promoter.

53. The composition of any one of claim 35-52, wherein the T cell attracting chemokine and the composition that promotes T cell proliferation and T-cell memory are driven by the same promoter.

54. A composition, comprising two or more different messenger ribonucleic acids (mRNAs), each mRNA comprising an open reading frame encoding a herpes antigens selected from a group consisting of RR1, RR2, gD, or VP22; wherein the two or more mRNAs are formulated in a lipid nanoparticle.

55. A composition, comprising three or more different messenger ribonucleic acids (mRNAs), each mRNA comprising an open reading frame encoding a herpes antigens selected from a group consisting of RR1, RR2, gD, or VP22; wherein the three or more mRNAs are formulated in a lipid nanoparticle.

56. The composition of claim 54 or claim 55, wherein the lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid.

57. A composition, comprising two or more different messenger ribonucleic acids (mRNAs), each mRNA comprising an open reading frame encoding a herpes antigens selected from a group consisting of RR1, RR2, gD, or VP22; wherein the two or more mRNAs are formulated in a lipid nanoparticle, wherein the lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid.

58. A composition, comprising three or more different messenger ribonucleic acids (mRNAs), each mRNA comprising an open reading frame encoding a herpes antigens selected from a group consisting of RR1, RR2, gD, or VP22; wherein the three or more mRNAs are formulated in a lipid nanoparticle, wherein the lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid.

59. The composition of any one of claims 54-58, wherein the herpes antigens are further selected from the group consisting of RR1, RR2, gD, or VP22, VP 11 / 12.

60. The composition of any one of claims 54-59, wherein each mRNA further comprises a 5’ untranslated region (UTR) and a 3’UTR.

61. The composition of any one of claims 54-60, wherein each mRNA further comprises a poly (A) tail and a 5’ cap.

62. The composition of any one of claims 54-60, wherein each mRNA further comprises a poly (A) tail and a 5’ cap analog.

63. The composition of any one of claims 54-62, herein the mRNAs comprise N1 -methylpseudouridine (mly) modified mRNAs.

64. A pharmaceutical composition comprising a first lipid nanoparticle and a second lipid nanoparticle, wherein the first lipid nanoparticle comprises two or more different messenger ribonucleic acids (mRNAs) encapsulated therein, each mRNA comprising an open reading frame encoding a herpes antigens selected from a group consisting of RR1, RR2, or VP22; wherein the second lipid nanoparticle comprises a gD herpes antigen encapsulated therein.

65. The composition of claim 64, wherein the lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid.

66. The composition of claim 64 or claim 65, wherein each mRNA further comprises a 5’ untranslated region (UTR) and a 3’UTR.

67. The composition of any one of claims 64-66, wherein each mRNA further comprises a poly (A) tail and a 5’ cap.

68. The composition of any one of claims 64-66, wherein each mRNA further comprises a poly (A) tail and a 5’ cap analog.

69. The composition of any one of claims 64-68, herein the mRNAs comprise N 1 -methylpseudouridine (ml\| / ) modified mRNAs.

70. A multi-antigen herpes vaccine composition, the composition comprising a sequenceencoding or comprising two or more different herpes antigens selected from a group consisting of RR1, RR2, gD, or VP22; wherein the sequence is formulated in a lipid nanoparticle.

71. A multi-antigen herpes vaccine composition, the composition comprising a sequence encoding or comprising three or more different herpes antigens selected from a group consisting of RR1, RR2, gD, or VP22; wherein the sequence is formulated in a lipid nanoparticle.

72. The composition of claim 70 or claim 71, wherein the sequence is formulated in a lipid nanoparticle comprising a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid.

73. The composition of any one of claims 12-72, wherein the vaccine composition protects against infection or reinfection of one or more herpes viruses.

74. The composition of claim 73, wherein the herpes virus comprises Herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2).

75. The composition of any one of claims 12-72, wherein the vaccine composition induces strong and long-lasting protection mediated by antibodies (Abs), CD4+ T helper (Thl) cells, and / or CD8+ cytotoxic T-cells (CTL).

76. A method of eliciting a T-cell response, an IFN-y response, or reducing viral titers in an individual, the method comprising administering to said individual the vaccine composition according to any one of claims 12-72.

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