[0001] This application claims benefit of United States provisional patent application number 62/196,600, filed July 24, 2015, the entire contents of which are incorporated by reference into this application.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grant number AI094019 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA EFS-WEB

[0003] The content of the ASCII text file of the sequence listing named "UW60WOU1_SL", which is 23 kb in size, was created on July 22, 2016, and electronically submitted via EFS-Web herewith the application. The sequence listing is incorporated herein by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

[0004] The invention relates to molecules, compositions and methods that can be used for the treatment and prevention of viral infection and other diseases. More particularly, the invention identifies epitopes of herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) proteins that can be used for methods involving molecules and compositions having the antigenic specificity of HSV-specific T cells. In addition, the invention relates to methods for detecting, treating and preventing HSV infection, as well as methods for inducing an immune response to HSV. The epitopes described herein are also useful in the development of diagnostic and therapeutic agents for detecting, preventing and treating viral infection and other diseases.

BACKGROUND OF THE INVENTION

[0005] HSV-1 and HSV-2 are genetically related viruses. Each has about 77 open reading frames that encode proteins. The level of identity varies between pairs of homologous proteins. HSV gene names are complex but for the purposes of this document, UL is used as the standard shorthand for unique long, referring to the largest region of the HSV genome that is both haploid (unique, not present in repeats) and long. HSV encode an enzyme with ribonucleotide reductase activity. The final enzyme is composed of both the UL40 gene product and the UL39 gene product. The UL39 gene product has a higher molecular weight and is therefore termed ribonucleotide reductase large subunit or RR1 . Hereinafter, it will just be called UL39 while the italicized UL39 will be used for the DNA gene.

[0006] CD8 T cells are an important part of the host defense against HSV-1 and HSV-2. HSV- specific CD8 T cells permanently localize to sites of recurrent skin or eye infection even after recurrent infections heal, and per animal models provide local protection against recurrence. HSV-specific CD8 T cells also localize to the trigeminal ganglia (TG) in HSV-1 -infected humans, as well as experimental animals. The TG is the site of long-term latency and persistent infection in humans. Vaccines that elicit CD8 T cells alone in the absence of antibody or CD4 T cells can protect animals for HSV challenge, albeit only if the CD8 T cells are at quite high levels. The Genocea bivalent vaccine containing glycoprotein D of HSV-2 and a truncated version of HSV-2 protein ICP4 has shown significant clinical activity in proof of concept clinical trials for the treatment of recurrent genital herpes (GH). ICP4 was chosen on the basis of a high population prevalence of cellular immune responses (CD8 and CD4), particularly in persons with milder or asymptomatic infection.

[0007] HSV-specific CD8 resident memory T cells localize to human genital skin. HSV-specific tissue resident memory or "pulled" CD8 T-cells show antiviral activity in animal models. HSV- specific CD8 T-cells have been difficult to study due to low responses and HLA class I down- regulation.

[0008] There remains a need for effective vaccine for the treatment and prevention of HSV infection, particularly one that will elicit a robust immune response in a broad population of subjects.

SUMMARY OF THE INVENTION

[0009] The invention meets these needs and others by providing methods and compositions that have been shown to elicit both CD4 and CD8 T cell responses in subjects expressing a variety of HLA types that represent a broad population. In one embodiment, the invention provides an isolated polynucleotide encoding the amino acid sequence of HSV UL39 (SEQ ID NO: 1). In one embodiment, the invention provides an isolated polynucleotide encoding an alphaherpesvirus polypeptide comprising up to 80% of the amino acid sequence of HSV UL39 (SEQ ID NO: 1), wherein the polypeptide comprises amino acids 421 -445 and 733-753 of SEQ ID NO: 1 . In one embodiment, the polypeptide comprises a plurality of epitopes listed in Table 1/Figure 2, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 epitopes listed in Table 1/Figure 2, and/or in Table 3. The epitopes may be selected based on cross- reactivity between HSV-1 and HSV-2, as shown in Figure 1 and/or Table 1/Figure 2, and/or based on the HLA binding motif indicated herein. In one embodiment, the polypeptide comprises one or more of the regions indicated in Figure 1 , such as amino acids 213-225, 309- 321 , 421 -433, 433-445, 461 -473, 509-521 , 517-529, 581 -593, 649-661 , 677-689, 709-721 , 733- 745, 741 -753, 793-805, and 901 -913 of HSV UL39 (SEQ ID NO: 1). In another embodiment, the multi-epitope polypeptide comprises one or more contiguous portions of the UL39 protein shown in SEQ ID NO: 1 that encompasses multiple epitopes. Representative examples of such contiguous portions include, but are not limited to, amino acids 213-913, 421-913, 421 -529, 649-913, 421 -445, 733-753, and 421 -713 of SEQ ID NO: 1 .

[0010] In some embodiments, the polynucleotide further encodes an epitope of an HSV protein other than UL39, such as UL46. Representative additional HSV proteins from which epitopes may be selected include, but are not limited to, those listed in Table 2 as recognized by CD4 T cells in a large percentage of subjects tested: RS1 (ICP4), UL27, US6, UL23, UL19, and US8, as well as those recognized by CD8 T cells in a large percentage of subjects tested: UL25, UL27 (gB2), UL29 (ICP8), UL40 (RR2), and UL47 (VP13/14). In an alternative embodiment, the invention provides an HSV polypeptide, or polynucleotide encoding same, selected from any combination of one or more of RS1 (ICP4), US6, US8, UL19, UL23, UL25, UL27 (gB2), UL29 (ICP8), UL39, UL40 (RR2), and UL47 (VP13/14), including immunogenic fragments thereof.

[001 1 ] In some embodiments, the encoded polypeptide is full-length. In another embodiment, the polypeptide is up to 900, or optionally, 1000, amino acids in length. In other embodiments, the polypeptide is up to 800, 700, 600, 500, 400, 300, or 200 amino acids in length. In some embodiments, the polynucleotide additionally encodes a heterologous peptide.

[0012] The invention additionally provides a vector comprising a polynucleotide as described herein, as well as a host cell transformed with the vector of the invention. Also provided is a recombinant alphaherpesvirus polypeptide encoded by a polynucleotide as described herein.

[0013] The invention further provides a pharmaceutical composition comprising a

polynucleotide as described herein, and, optionally, a pharmaceutically acceptable carrier. The polynucleotide may be naked DNA, part of a vector, or incorporated into a recombinant virus. In some embodiments, the pharmaceutical composition further comprises an adjuvant. Also provided is a pharmaceutical composition comprising an isolated HSV polypeptide as described herein, and, optionally, a pharmaceutically acceptable carrier. Typically, the polypeptide comprises up to 80% of the amino acid sequence of HSV UL39 protein shown in SEQ ID NO: 1. In some embodiments, the polypeptide comprises amino acids 421 -445 and 733-753 of SEQ ID NO: 1 . Optionally, the pharmaceutical composition further comprises an adjuvant.

[0014] In addition, the invention provides a method of treating a herpes simplex virus type 1 (HSV-1) or a herpes simplex virus type 2 (HSV-2) infection in a subject, which method comprises administering a composition of the invention to the subject. Also provided is a method of killing an alphaherpesvirus infected cell, a method of enhancing secretion of antiviral or immunomodulatory lymphokines, amethod of enhancing production of HSV-specific antibody, a method of enhancing proliferation of HSV-specific T cells, or a method of inhibiting HSV replication. These methods comprise contacting the infected cell with a polypeptide of the invention. The invention further provides a method of inducing an immune response to an HSV infection in a subject comprising administering a composition of the invention to the subject. Also provided are kits comprising materials for use in carrying out these methods.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Fig. 1 Human CD8 and CD4 T-cell epitopes in HSV-1 UL39 (RR1 ) discovered using HSV-1 (+) HSV-2 (-) subjects and HSV-2(+) subjects. This schematic diagram illustrates the positions of epitopes along the UL39 protein (of HSV- 1 and HSV-2) that are recognized by both CD4 and CD8 T cells from persons infected with either viral strain. The thick lines represent the length of UL39 from amino acids 1 to 1 143, with short bars marking locations of noted epitopes, some of which are HSV-1 type-specific (darkest 3 bars) due to disparate sequence in HSV-2 (versus HSV-1 ) at residues 213-225, 309-321 , and 581 -593. The remaining epitopes are type- common, as the 10 marked with medium-toned bars have identical sequence in HSV-1 and HSV-2 (at residues 433-445, 509-521 , 517-529, 649-661 , 677-689, 901 -913, 693-705, 697-709, 777-789, and 961 -973), and the 12 marked with lighter-toned bars have similar sequence in HSV-2 as HSV-1 (residues 421 -433, 461 -473, 709-721 , 733-745, 741 -753, 793-805, 445-457, 465-477, 469-481 , 593-603, 965-977, and 965-981 ) . The short bars in the top figure indicates proven CD8 T cell epitopes, validated using artificial antigen presenting cells expressing only the single HLA class I molecule shown above the top horizontal bar. The amino acid numbers of biologically active 13 amino acid long peptides are shown below the top horizontal bar. The lower horizontal bar indicates proven CD4 T cell epitopes in UL39 of HSV- 1 .

[0016] Fig. 2 (also labeled Table 1 ) shows an analysis of a CD8 T cell epitopes detected in UL39 of HSV- 1 (columns 3-5) and homologous regions of UL39 of HSV-2 (columns 6-8).

Column 2 shows the HLA class I restriction element empirically proven to restrict the peptide. The sequences in column 3 are the predicted minimal epitopes within the empirically proven epitopes shown in Figure 1 , upper stick figure. The peptides listed correspond, from top to bottom, to SEQ ID NO: 3-29. Predictions were performed using the IEDB website tools. In column 6, "nothing good" indicates that the HSV-2 homolog region does not contain a peptide predicted to bind the HLA molecule in column 2. "ident" means the HSV-2 homolog peptide is identical to the proven HSV-1 peptide epitope. If a sequence is shown in Column 6, the sequence is for the homologous region in HSV-2 UL39 with amino acid residues divergent from HSV-1 in lighter font. Columns 4 and 7 indicate the predicted binding affinities of the HSV-1 and HSV-2 peptides shown, respectively. Low numbers indicate predicted good binding and are predictive of T cell responses.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The invention provides HSV antigens that are useful for the prevention and treatment of HSV infection, and more particularly, specific epitopes that elicit immune responses in a high proportion of the population, several of which are cross-reactive between HSV- 1 and HSV-2. Disclosed herein are antigens and/or their constituent epitopes. In some embodiments, T-cells having specificity for antigens of the invention have demonstrated cytotoxic activity against virally infected cells and/or whole virus. The identification of immunogenic antigens responsible for T-cell specificity facilitates the development of improved anti-viral therapeutic and prophylactic strategies. Compositions containing epitopes or polynucleotides encoding epitopes of the invention provide effectively targeted vaccines for prevention and treatment of alphaherpesvirus infection.

Overview

[0018] We have studied the CD4 and CD8 T cell response to each of the known 77 HSV-1 proteins in 42 persons (for CD4 responses) infected with HSV-1 or 37 persons (for CD8 responses) infected with HSV-1 . We have studied the CD8 T cell response to each of the known 77 HSV-2 proteins in 26 persons infected with HSV-2. In each case, every HSV-1 or HSV-2 protein were studied as full length proteins.

[0019] In each of the 3 genome-wide screens, UL39 showed excellent population prevalence of T cell responses. More specifically, for CD4 responses to HSV-1 , responses to full length UL39 of HSV- 1 were present in 54.8% of persons. Other leading HSV- 1 ORFs in these CD4 assays included ICP4, UL26, UL27, and US6. For CD8 responses to HSV- 1 , responses to full length UL39 of HSV- 1 were present in 78.4% of persons. Other leading HSV-1 ORFs in these CD8 assays included UL27, UL46, UL48, and ICP4. For CD8 responses to HSV-2, responses to full length UL39 of HSV-2 were present in 38.5% of persons. Other leading HSV-2 ORFs in these CD8 assays included UL1 , UL19, UL29, and UL46.

[0020] The finding that UL39 is a population prevalent T cell antigen for both CD4 and CD8 T cells responses for HSv- 1 and HSV-2 could not be predicted from immunologic principles or prior data. HSV encodes 77 proteins and there is no known reason why any one protein should be so immunodominant. Taken together, these proteome-wide surveys of CD4 and CD8 T-cell responses to every known HSV-1 and HSV-2 protein show that T cell responses to UL39 are prevalent in the human population. Therefore, UL39 is an excellent candidate for therapeutic vaccines to treat HSV infections that seek to stimulate HSV-reactive T cell responses. Definitions

[0021 ] All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified.

[0022] As used herein, "polypeptide" includes proteins, fragments of proteins, and peptides, whether isolated from natural sources, produced by recombinant techniques or chemically synthesized. Polypeptides of the invention typically comprise at least about 6 amino acids, and can be at least about 15 amino acids. Typically, optimal immunological potency for peptide epitopes is obtained with lengths of 8-10 amino acids. Those skilled in the art also recognize that additional adjacent sequence from the original (native) protein can be included, and is often desired, in an immunologically effective polypeptide suitable for use as a vaccine. This adjacent sequence can be from 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length to as much as 15, 20, 25, 30, 35, 40, 45, 50, 75 or 100 amino acids in length or more.

[0023] As used herein, particularly in the context of polypeptides of the invention, "consisting essentially of means the polypeptide consists of the recited amino acid sequence and, optionally, adjacent amino acid sequence. The adjacent sequence typically consists of additional, adjacent amino acid sequence found in the full length antigen, but variations from the native antigen can be tolerated in this adjacent sequence while still providing an

immunologically active polypeptide.

[0024] As used herein, "multi-epitope polypeptide" means a polypeptide comprising two or more non-identical epitopes. The epitopes can be from the same or different proteins, and/or from the same or different organism. Optionally, the polypeptide may comprise more than one copy of a particular epitope, and/or more than one variant of a particular epitope. The multi- epitope polypeptide is 12 to 1200 amino acids in length. In some embodiments, the multi- epitope polypeptide is up to 600 amino acids in length.

[0025] In some embodiments, the multi-epitope polypeptide is not conjugated to and is devoid of a carrier fusion protein. In other embodiments, the multi-epitope polypeptide further comprises a carrier sequence, whereby the peptide epitopes are inserted within a sequence of a carrier polypeptide or are coupled to a carrier sequence. In some embodiments, the multi- epitope polypeptide is produced as a recombinant fusion protein comprising a carrier sequence.

[0026] As used herein, a "spacer" refers to a bond, an amino acid, or a peptide comprising at least two amino acids. A spacer is typically not more than 25 amino acids in length. In some embodiments, the spacer comprises 1 to 4 neutral amino acids. In some embodiments, the spacer comprises adjacent native sequence of the epitope's sequence of origin, where, for example, the native sequence facilitates presentation of epitope for correct processing. [0027] As used herein, "epitope" refers to a molecular region of an antigen capable of eliciting an immune response and of being specifically recognized by the specific immune T-cell produced by such a response. Another term for "epitope" is "determinant" or "antigenic determinant". Those skilled in the art often use the terms epitope and antigen interchangeably in the context of referring to the determinant against which an immune response is directed.

[0028] As used herein, "HSV polypeptide" includes HSV-1 and HSV-2, unless otherwise indicated. References to amino acids of HSV-1 proteins or polypeptides are based on the genomic sequence information regarding HSV-1 (strain 17+) as described in McGeoch et al., 1988, J. Gen. Virol. 69: 1531 -1574; GenBank Accession No. JN555585.1 . References to amino acids of HSV-2 proteins or polypeptides are based on the genomic sequence information regarding HSV-2 as described in A. Dolan et al., 1998, J. Virol. 72(3) :2010-2021 ; GenBank Accession No. JN561323.2.

[0029] As used herein, "substitutional variant" refers to a molecule having one or more amino acid substitutions or deletions in the indicated amino acid sequence, yet retaining the ability to be "immunologically active", or specifically recognized by an immune cell. The amino acid sequence of a substitutional variant is preferably at least 80% identical to the native amino acid sequence, or more preferably, at least 90% identical to the native amino acid sequence.

Typically, the substitution is a conservative substitution.

[0030] One method for determining whether a molecule is "immunologically active",

"immunologically effective", or can be specifically recognized by an immune cell, is the cytotoxicity assay described in D.M. Koelle et al., 1997, Human Immunol. 53: 195-205. Other methods for determining whether a molecule can be specifically recognized by an immune cell are described in the examples provided hereinbelow, including the ability to stimulate secretion of interferon-gamma or the ability to lyse cells presenting the molecule. An immune cell will specifically recognize a molecule when, for example, stimulation with the molecule results in secretion of greater interferon-gamma than stimulation with control molecules. For example, the molecule may stimulate greater than 5 pg/ml, or preferably greater than 10 pg/ml, interferon-gamma secretion, whereas a control molecule will stimulate less than 5 pg/ml interferon-gamma. Proliferation assays for confirming CD4 T-cell epitopes are described in Laing, et al., 2015, J. infect. Dis. Doi: 10.1093/infdis/jiv165.

[0031 ] As used herein, "vector" means a construct, which is capable of delivering, and preferably expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0032] As used herein, "expression control sequence" means a nucleic acid sequence that directs transcription of a nucleic acid. An expression control sequence can be a promoter, such as a constitutive or an inducible promoter, or an enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

[0033] As used herein, the term "nucleic acid" or "polynucleotide" refers to a

deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.

[0034] As used herein, "antigen-presenting cell" or "APC" means a cell capable of handling and presenting antigen to a lymphocyte. Examples of APCs include, but are not limited to, macrophages, Langerhans-dendritic cells, follicular dendritic cells, B cells, monocytes, fibroblasts and fibrocytes. Dendritic cells (also referred to as "DCs") are a preferred type of antigen presenting cell. Dendritic cells are found in many non-lymphoid tissues but can migrate via the afferent lymph or the blood stream to the T-dependent areas of lymphoid organs. In non-lymphoid organs, dendritic cells include Langerhans cells and interstitial dendritic cells. In the lymph and blood, they include afferent lymph veiled cells and blood dendritic cells, respectively. In lymphoid organs, they include lymphoid dendritic cells and interdigitating cells.

[0035] As used herein, "modified" to present an epitope refers to antigen-presenting cells

(APCs) that have been manipulated to present an epitope by natural or recombinant methods. For example, the APCs can be modified by exposure to the isolated antigen, alone or as part of a mixture, peptide loading, or by genetically modifying the APC to express a polypeptide that includes one or more epitopes.

[0036] As used herein, "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include, but are not limited to, (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, furmaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonic acid; (b) salts with polyvalent metal cations such as zinc, calcium, bismuth, barium,

magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; or (c) salts formed with an organic cation formed from Ν,Ν'-dibenzylethylenediamine or ethylenediamine; or (d) combinations of (a) and (b) or (c), e.g., a zinc tannate salt; and the like. The preferred acid addition salts are the trifluoroacetate salt and the acetate salt.

[0037] As used herein, "pharmaceutically acceptable carrier" includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline.

[0038] Compositions comprising such carriers are formulated by well known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990).

[0039] As used herein, "adjuvant" includes adjuvants commonly used in the art to facilitate the stimulation of an immune response. Examples of adjuvants include, but are not limited to, helper peptide; aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Ml); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (Smith-Kline Beecham); QS-21 (Aquilla); MPL or 3d-MPL (Corixa Corporation, Hamilton, MT); LEIF; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A; muramyl tripeptide phosphatidyl ethanolamine or an immunostimulating complex, including cytokines (e.g., GM-CSF or interleukin-2, -7 or -12) and immunostimulatory DNA sequences. In some embodiments, an adjuvant such as a helper peptide or cytokine can be provided via a polynucleotide encoding the adjuvant. Representative examples of such adjuvants for use in polynucleotide vaccines include, but are not limited to, ubiquitin and toll-like receptor (TLR) ligands.

[0040] As used herein, "a" or "an" means at least one, unless clearly indicated otherwise.

[0041 ] As used herein, to "prevent" or "protect against" a condition or disease means to hinder, reduce or delay the onset or progression of the condition or disease.

[0042] As used herein, "corresponding position" refers to an amino acid residue that is present in a second sequence at a position corresponding to a specified amino acid residue in a first sequence which is the same position as the position in the first sequence when the two sequences are aligned to allow for maximum sequence identity between the two sequences.

[0043] As used herein, a "heterologous" sequence or a "heterologous" molecule refers to a moiety not naturally occurring in conjunction with a recited sequence or molecule. Representative examples of the heterologous molecule include, but are not limited to, a polypeptide, antibody, epitope, polynucleotide, small molecule or drug. Such heterologous moieties can be useful for improving solubility, delivery, immunogenicity, efficacy, detection, or identification of the recited sequence or molecule. In some embodiments, the heterologous sequence is inert or an unrelated sequence.

[0044] As used herein, the terms "comprise" or "include", or variations such as "comprises" or "comprising", "includes" or "including" mean the inclusion of a recited item or group of items, but not the exclusion of any other item or group of items.

Constructs and Compositions of the Invention

[0045] The invention provides, in one embodiment, a polynucleotide construct encoding a multi-epitope polypeptide. The multi-epitope polypeptide comprises one or more immunogenic portions of HSV UL39, and, in some embodiments, less than the full length of UL39. By omitting portions of UL39 not necessary for immunogenicity, it is possible to incorporate additional desired sequence, including epitopes of other HSV proteins, such as UL46, as one example. Representative additional HSV proteins from which epitopes may be selected include, but are not limited to, those listed in Table 2 as recognized by CD4 T cells in a large percentage of subjects tested: RS1 (ICP4), UL27, US6, UL23, UL19, and US8, as well as those recognized by CD8 T cells in a large percentage of subjects tested: UL25, UL27 (gB2), UL29 (ICP8), UL40 (RR2), and UL47 (VP13/14). In an alternative embodiment, the invention provides an HSV polypeptide, or polynucleotide encoding same, selected from any combination of one or more of RS1 (ICP4), US6, US8, UL19, UL23, UL25, UL27 (gB2), UL29 (ICP8), UL39, UL40 (RR2), and UL47 (VP13/14), including immunogenic fragments thereof. In one embodiment, the polynucleotide construct comprises nucleic acid sequences encoding, in operable linkage, a plurality of epitopes as described herein. In some embodiments, the construct further comprises a heterologous sequence. In one embodiment, the invention provides a recombinant alphaherpesvirus multi-epitope polypeptide, such as a polypeptide encoded by a polynucleotide described herein. Also provided is a multi-epitope polypeptide produced by a recombinant virus genetically modified to express an alphaherpesvirus multi-epitope polypeptide described herein.

[0046] UL39/RR1 (SEQ ID NO:1)

1 masrpaassp vearapvggq eaggpsaatq geaagaplah ghhvycqrvn gvmvlsdktp

61 gsasyrisdn nfvqcgsnct miidgdvvrg rpqdpgaaas papfvavtni gagsdggtav

121 vafggtprrs agtstgtqta dvptealggp pppprftlgg gccscrdtrr rsavfggegd

181 pvgpaefvsd drssdsdsdd sedtdsetls hassdvsgga tyddaldsds ssddslqidg

241 pvcrpwsndt apldvcpgtp gpgadaggps avdphaptpe agaglaadpa varddaegls 301 dprprlgtgt aypvpleltp enaeavarfl gdavnrepal mleyfcrcar eetkrvpprt 361 fgspprlted dfgllnyalv emqrlcldvp pvppnaympy ylreyvtrlv ngfkplvsrs

421 arlyrilgvl vhlrirtrea sfeewlrske valdfglter lreheaqlvi laqaldhydc

481 lihstphtlv erglqsalky eefylkrfgg hymesvfqmy triagflacr atrgmrhial

541 gregswwemf kfffhrlydh qivpstpaml nlgtrnyyts scylvnpqat tnkatlrait 601 snvsailarn ggiglcvqaf ndsgpgtasv mpalkvldsl vaahnkesar ptgacvylep

661 whtdvravlr mkgvlageea qrcdnifsal mpdlffkrl irhldgeknv twtlfdrdts

721 msladfhgee feklyqhlev mgfgeqipiq elaygivrsa attgspfvmf kdavnrhyiy

781 dtqgaaiags nlcteivhpa skrssgvcnl gsvnlarcvs rqtfdfgrlr davqacvlmv

841 nimidstlqp tpqctrgndn lrsmgigmqg lhtaclklgl dlesaefqdl nkhiaevmll 901 samktsnalc vrgarpfnhf krsmyragrf hwerfpdarp ryegewemlr qsmmkhglrn

961 sqfvalmpta asaqisdvse gfaplftnlf skvtrdgetl rpntlllkel ertfsgkrll

1021 evmdsldakq wsvaqalpcl epthplrrfk tafdydqkll idlcadrapy vdhsqsmtly

1081 vtekadgtlp astlvrllvh aykrglktgm yyckvrkatn sgvfggddni vcmscal

[0047] UL46/VP1 1/12 (SEQ ID NO:2)

1 mqrrtrgass lrlarcltpa nlirgdnagv perrifggcl lptpegllsa avgalrqrsd

61 daqpafltct drsvrlaarq hntvpesliv dglasdphye yirhyasaat qalgevelpg 121 gqlsrailtq ywkylqtvvp sgldvpedpv gdcdpslhvl lrptlapkll artpfksgav 181 aakyaatvag lrdalhriqq ymffmrpadp srpstdtalr lnellayvsv lyrwaswmlw 241 ttdkhvchrl spsnrrflpl ggspeapaet farhldrgps gttgsmqcma lraavsdvlg 301 hltrlanlwq tgkrsggtyg tvdtvvstve vlsivhhhaq yiinatltgy gvwatdslnn

361 eylraavdsq erfcrttapl fptmtapswa rmelsikawf gaalaadllr ngapslhyes 421 ilrlvasrrt twsagpppdd masgpgghra gggtcrekiq rarrdneppp lprprlhstp 481 astrrfrrrr adgagpplpd andpvaeppa aatqpatyyt hmgevpprlp arnvagpdrr 541 ppaatcpllv rraslgsldr prvwgpapeg epdqmeatyl taddddddar rkathaasar 601 erhapyedde siyetvsedg grvyeeipwm rvyenvcvnt anaapaspyi eaenplydwg

661 gsalfsppgr tgppppplsp spvlarhran altndgptnv aalsalltkl kregrrsr

[0048] The alphaherpesvirus multi-epitope polypeptide comprises a plurality of

alphaherpesvirus peptide epitopes linked in a series, wherein each epitope in the series is linked to an adjacent epitope by a spacer. The spacer comprises a bond, an amino acid, or a peptide comprising at least two amino acids. The spacer can be selected to facilitate epitope processing and/or cleavage between two or more epitopes. A spacer is typically not more than 25 amino acids in length. In some embodiments, the spacer comprises 1 to 4 neutral amino acids. In some embodiments, the spacer comprises adjacent native sequence of the epitope's sequence of origin, where, for example, the native sequence facilitates presentation of epitope for correct processing. Optimization of poly-epitope immunogens is described, for example, in Reguzova et al., 2015, PLoS One 10(3):e01 16412 (PMC4364888). In some embodiments, the spacer comprises a cleavable sequence. In one embodiment, a cleavable spacer is cleaved by intracellular enzymes. In a more specific embodiment, the cleavable spacer comprises a protease specific cleavable sequence.

[0049] The plurality of alphaherpesvirus peptide epitopes comprises at least one epitope described herein, and typically comprises at least one epitope selected from Table 1. In one embodiment, the plurality of alphaherpesvirus peptide epitopes comprises epitopes that elicit T- cell reactivity to HSV-1 and/or HSV-2. In one embodiment, the plurality of peptide epitopes comprises at least two epitopes selected from Table 1. In another embodiment, the plurality of peptides epitopes comprises 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 15, or 18 epitopes selected from Table 1. In one embodiment, the plurality of peptide epitopes comprises no more than 20 epitopes described herein. In another embodiment, the plurality of peptide epitopes comprises no more than 15 epitopes described herein. In another embodiment, the plurality of peptide epitopes comprises no more than 10 epitopes described herein.

[0050] In one embodiment, the epitopes are selected from the group consisting of:

SSDVSGGATY (SEQ ID NO: 3), YMESVFQMY (SEQ ID NO: 4), RLYRILGVLV (SEQ ID NO: 5), RLYRILGVIVHL (SEQ ID NO: 6), LVNPQATTNK (SEQ ID NO: 7), AMKTSNALCVR (SEQ ID NO: 8), RIRTREASF (SEQ ID NO: 9), EIVHPASKR (SEQ ID NO: 10), EIVHPSCKR (SEQ ID NO: 1 1), RPTGACVYL (SEQ ID NO: 12), GTAYPVPL (SEQ ID NO: 13), RLYRILGVLVHL (SEQ ID NO: 14), RLYRILGVIVH (SEQ ID NO: 15), TLFDRDTSM (SEQ ID NO: 16), SLFDRDTSM (SEQ ID NO: 17), QHLEVMGF (SEQ ID NO: 18), EHLEAMGF (SEQ ID NO: 19),

FQMYTRIAGFL (SEQ ID NO: 20), TRIAGFLAC (SEQ ID NO: 21), QRCDNIFSA (SEQ ID NO: 22), QRCDNIFSAL (SEQ ID NO: 23), REHEAQLVIL (SEQ ID NO: 24), REHEAQLMIL (SEQ ID NO: 25), GEQIPIQEL (SEQ ID NO: 26), GETIPIQDL (SEQ ID NO: 27), RTREASFEEW (SEQ ID NO: 28), REASFEEW (SEQ ID NO: 29), WLRSKEVALDFGL (SEQ ID NO: 30),

EAQLVILAQALDH (SEQ ID NO: 31), VILAQALDHYDCL (SEQ ID NO: 32), KATLRAITSNVSA (SEQ ID NO: 33), PDLFFKRLIRHLD (SEQ ID NO: 34), FKRLIRHLDGEKN (SEQ ID NO: 35),

HYIYDTQGAAIAG (SEQ ID NO: 36), SQFVALMPTAASA (SEQ ID NO: 37), ALMPTAASAQISD (SEQ ID NO: 38), and TAASAQISDVSEG (SEQ ID NO: 39). In one embodiment, two of the above-listed peptides are included in the multi-epitope peptide. In other embodiments, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the above-listed peptides are included in the multi-epitope polypeptide. In one embodiment, the multi-epitope polypeptide includes one peptide from each HLA allele type listed in Table 1/Figure 2. In another embodiment, the plurality of peptide epitopes comprises at least one epitope identified herein as an HLA A*0201- restricted epitope, at least one epitope identified herein as an HLA B*0702-restricted epitope, and, optionally, at least one epitope identified herein as an HLA A*0101 -restricted, and, optionally, at least one epitope identified herein as an HLA A*0301 -restricted epitope. These HLA restricting alleles are desirable as they are prevalent genetic variants that are present at allele frequencies of 10-50% in many ethnically diverse populations. For example, between 30- 60% of persons from many ethnicities have the HLA A*0201 allele. Specifically, the epitope RLYRILGVLHL (SEQ ID NO: 14) has been proven in our lab to restricted by HLA A*0201 , the epitope RPTGACVYL (SEQ ID NO: 12) has been proven in our lab to be restricted by HLA B*0702, both epitopes SSDVSGGATY (SEQ ID NO: 3) and YMESVFQMY (SEQ ID NO: 4) have been proven in our lab to be restricted by HLA A*0101 , and the epitope LVNPQATTNK (SEQ ID NO: 7) has been proven in our lab to be restricted by HLA A*0301. The HLA allelic variants B*0702, A*0101 , and A*0301 are each present in 10-20% of people in several major ethnic groups. By using HLA based prioritization and combining these epitopes or regions of UL39 that contain these epitopes in combination, one can create a vaccine candidate that would be expected to induce or boost CD8 responses in a large proportion of individuals in diverse ethnic groups.

[0051] In one embodiment, the epitopes are selected from the group consisting of the following portions identified by amino acid residues of UL39 (SEQ ID NO: 1): 421-433, 433, 445, 461- 473, 509-521 , 517-529, 649-661 , 677-689, 709-721 , 733-745, 741-753, 793-805, and 901-913. In another embodiment, the multi-epitope polypeptide comprises one or more contiguous portions of the UL39 protein shown in SEQ ID NO: 1 that encompasses multiple epitopes. Representative examples of such contiguous portions include, but are not limited to, amino acids 213-913, 421-913, 421-529, 649-913, 421-445, 733-753, and 421-713.

[0052] A fragment of a polypeptide of the invention consists of less than the complete amino acid sequence of the corresponding protein, but includes the recited epitope or antigenic region. As is understood in the art and confirmed by assays conducted using fragments of widely varying lengths, additional sequence beyond the recited epitope can be included without hindering the immunological response. A fragment of the invention can be as few as 8 amino acids in length, or can encompass 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the full length of the protein.

[0053] The optimal length for the polypeptide of the invention will vary with the context and objective of the particular use, as is understood by those in the art. In some vaccine contexts, a full-length protein or large portion of the protein (e.g., up to 100 amino acids, 150 amino acids, 200 amino acids, 250 amino acids or more) provides optimal immunological stimulation, while in others, a short polypeptide (e.g., less than 50 amino acids, 40 amino acids, 30 amino acids, 20 amino acids, 15 amino acids or fewer) comprising the minimal epitope and/or a small region of adjacent sequence facilitates delivery and/or eases formation of a fusion protein or other means of combining the polypeptide with another molecule or adjuvant. HSV UL39 is 1 137 amino acids in length, making it desirable to identify portions of the full protein that can be used in a vaccine. Often use of the full-length protein in a vaccine is impractical due to exceeding the payload capacity and/or toxicity concerns.

[0054] A polypeptide for use in a composition of the invention comprises an HSV polypeptide that contains an epitope or minimal stretch of amino acids sufficient to elicit an immune response. These polypeptides typically consist of such an epitope and, optionally, adjacent sequence. Those skilled in the art are aware that the HSV epitope can still be immunologically effective with a small portion of adjacent HSV or other amino acid sequence present.

Accordingly, some polypeptides of the invention will consist essentially of the recited epitope and have a total length of up to 15, 20, 25 or 30 amino acids.

[0055] A typical embodiment of the invention is directed to a polypeptide consisting essentially of a plurality of epitopes listed in Table 1. More specifically, a polypeptide consisting of an epitope listed in Table 1 and, optionally, up to 15 amino acids of adjacent native sequence. In some embodiments, the polypeptide is fused with or co-administered with a heterologous peptide. The heterologous peptide can be another epitope or unrelated sequence. The unrelated sequence may be inert or it may facilitate the immune response. In typical embodiments, the epitope is part of a multi-epitopic vaccine, in which numerous epitopes are combined in one polypeptide.

[0056] In general, polypeptides (including fusion proteins) and polynucleotides as described herein are isolated. An "isolated" polypeptide or polynucleotide is one that is removed from its original environment. The isolated molecule can then be of particular use because multiple copies are available in a substantially purified preparation, enabling utilization of the molecule in ways not possible without isolation and/or recombinant production. For example, a naturally occurring protein is isolated if it is separated from some or all of the coexisting materials in the natural system. An isolated HSV polypeptide of the invention is one that has been isolated, produced or synthesized such that it is separate from a complete, native herpes simplex virus, although the isolated polypeptide may subsequently be introduced into a recombinant virus. A recombinant virus that comprises an isolated polypeptide or polynucleotide of the invention is an example of subject matter provided by the invention. Preferably, such isolated polypeptides are at least about 90% pure, more preferably at least about 95% pure and most preferably at least about 99% pure. A polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not part of the natural environment.

[0057] The polypeptide can be isolated from its naturally occurring form, produced by recombinant means or synthesized chemically. Recombinant polypeptides encoded by DNA sequences described herein can be readily prepared from the DNA sequences using any of a variety of expression vectors known to those of ordinary skill in the art. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide.

Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Preferably the host cells employed are E. coli, yeast or a mammalian cell line such as Cos or CHO. Supernatants from the soluble host/vector systems that secrete recombinant protein or polypeptide into culture media may be first concentrated using a commercially available filter. Following concentration, the concentrate may be applied to a suitable purification matrix such as an affinity matrix or an ion exchange resin. Finally, one or more reverse phase HPLC steps can be employed to further purify a recombinant polypeptide.

[0058] Fragments and other variants having less than about 100 amino acids, and generally less than about 50 amino acids, may also be generated by synthetic means, using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, wherein amino acids are sequentially added to a growing amino acid chain (Merrifield, 1963, J. Am. Chem. Soc. 85:2146-2149). Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied BioSystems Division (Foster City, CA), and may be operated according to the manufacturer's instructions.

[0059] Variants of the polypeptide for use in accordance with the invention can have one or more amino acid substitutions, deletions, additions and/or insertions in the amino acid sequence indicated that result in a polypeptide that retains the ability to elicit an immune response to alphaherpesvirus-infected cells. Typically, such alterations of the sequence are made to areas outside the minimal epitopes identified herein, and may be selected to minimize risk of autoimmune problems or toxicity. For example, UL39 has an enzymatically active site that would not be desirable to include in a vaccine. Such variants may generally be identified by modifying one of the polypeptide sequences described herein and evaluating the reactivity of the modified polypeptide using a known assay such as a T cell assay described herein.

Polypeptide variants preferably exhibit at least about 70%, more preferably at least about 90%, and most preferably at least about 95% identity to the identified polypeptides. These amino acid substitutions include, but are not necessarily limited to, amino acid substitutions known in the art as "conservative". Those skilled in the art recognize that any substitutions are preferably made in amino acids outside of the minimal epitope identified herein.

[0060] A "conservative" substitution is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine,

phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also, or alternatively, contain nonconservative changes. In a preferred embodiment, variant polypeptides differ from a native sequence by substitution, deletion or addition of five amino acids or fewer. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide.

[0061] One can readily confirm the suitability of a particular variant by assaying the ability of the variant polypeptide to elicit an immune response. The ability of the variant to elicit an immune response can be compared to the response elicited by the parent polypeptide assayed under identical circumstances. One example of an immune response is a cellular immune response. The assaying can comprise performing an assay that measures T cell stimulation or activation. Examples of T cells include CD4 and CD8 T cells.

[0062] One example of a T cell stimulation assay is a cytotoxicity assay, such as that described in Koelle, DM et al., Human Immunol. 1997, 53;195-205. In one example, the cytotoxicity assay comprises contacting a cell that presents the antigenic viral peptide in the context of the appropriate HLA molecule with a T cell, and detecting the ability of the T cell to kill the antigen presenting cell. Cell killing can be detected by measuring the release of radioactive ⁵¹ Cr from the antigen presenting cell. Release of ⁵¹ Cr into the medium from the antigen presenting cell is indicative of cell killing. An exemplary criterion for increased killing is a statistically significant increase in counts per minute (cpm) based on counting of ⁵¹ Cr radiation in media collected from antigen presenting cells admixed with T cells as compared to control media collected from antigen presenting cells admixed with media.

Fusion Proteins

[0063] The polypeptide can be a fusion protein. In one embodiment, the fusion protein is soluble. A soluble fusion protein of the invention can be suitable for injection into a subject and for eliciting an immune response. Within certain embodiments, a polypeptide can be a fusion protein that comprises multiple polypeptides as described herein, or that comprises at least one polypeptide as described herein and an unrelated sequence. In one example, the fusion protein comprises a HSV epitope described herein (with or without flanking adjacent native sequence) fused with non-native sequence. A fusion partner may, for example, assist in providing T helper epitopes (an immunological fusion partner), preferably T helper epitopes recognized by humans, or may assist in expressing the protein (an expression enhancer) at higher yields than the native recombinant protein. Certain preferred fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners may be selected so as to increase the solubility of the protein or to enable the protein to be targeted to desired intracellular compartments. Still further fusion partners include affinity tags, which facilitate purification of the protein.

[0064] Fusion proteins may generally be prepared using standard techniques, including chemical conjugation. Preferably, a fusion protein is expressed as a recombinant protein, allowing the production of increased levels, relative to a non-fused protein, in an expression system. Briefly, DNA sequences encoding the polypeptide components may be assembled separately, and ligated into an appropriate expression vector. The 3' end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion protein that retains the biological activity of both component polypeptides.

[0065] A peptide linker sequence may be employed to separate the first and the second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., 1985, Gene 40:39-46; Murphy et al., 1986, Proc. Natl. Acad. Sci. USA 83:8258-8262; U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751 ,180. The linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference. [0066] The ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements. The regulatory elements responsible for expression of DNA are located 5' to the DNA sequence encoding the first polypeptides. Similarly, stop codons required to end translation and transcription termination signals are present 3' to the DNA sequence encoding the second polypeptide.

[0067] Fusion proteins are also provided that comprise a polypeptide of the present invention together with an unrelated immunogenic protein. Preferably the immunogenic protein is capable of eliciting a recall response. Examples of such proteins include tetanus, tuberculosis and hepatitis proteins (see, for example, Stoute et al., 1997, New Engl. J. Med., 336:86-9).

[0068] Within preferred embodiments, an immunological fusion partner is derived from protein D, a surface protein of the gram-negative bacterium Haemophilus influenza B (WO 91/18926). Preferably, a protein D derivative comprises approximately the first third of the protein (e.g., the first N-terminal 100-1 10 amino acids), and a protein D derivative may be lipidated. Within certain preferred embodiments, the first 109 residues of a Lipoprotein D fusion partner is included on the N-terminus to provide the polypeptide with additional exogenous T-cell epitopes and to increase the expression level in E. coli (thus functioning as an expression enhancer). The lipid tail ensures optimal presentation of the antigen to antigen presenting cells. Other fusion partners include the non-structural protein from influenza virus, NS1 (hemaglutinin). Typically, the N-terminal 81 amino acids are used, although different fragments that include T- helper epitopes may be used.

[0069] In another embodiment, the immunological fusion partner is the protein known as LYTA, or a portion thereof (preferably a C-terminal portion). LYTA is derived from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytA gene; Gene 43:265-292, 1986). LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE. This property has been exploited for the development of E. coli C-LYTA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C-LYTA fragment at the amino terminus has been described (see Biotechnology 10:795-798, 1992). Within a preferred embodiment, a repeat portion of LYTA may be incorporated into a fusion protein. A repeat portion is found in the C-terminal region starting at residue 178. A particularly preferred repeat portion incorporates residues 188-305.

[0070] In some embodiments, it may be desirable to couple a therapeutic agent and a polypeptide of the invention, or to couple more than one polypeptide of the invention. For example, more than one agent or polypeptide may be coupled directly to a first polypeptide of the invention, or linkers that provide multiple sites for attachment can be used. Alternatively, a carrier can be used. Some molecules are particularly suitable for intercellular trafficking and protein delivery, including, but not limited to, VP22 (Elliott and O'Hare, 1997, Cell 88:223-233; see also Kim et al., 1997, J. Immunol. 159:1666-1668; Rojas et al., 1998, Nature Biotechnology 16:370; Kato et al., 1998, FEBS Lett. 427(2):203-208; Nagahara et al., 1998, Nature Med. 4(12):1449-1452).

[0071] A carrier may bear the agents or polypeptides in a variety of ways, including covalent bonding either directly or via a linker group. Suitable carriers include proteins such as albumins (e.g., U.S. Patent No. 4,507,234, to Kato et al.), peptides and polysaccharides such as aminodextran (e.g., U.S. Patent No. 4,699,784, to Shih et al.). A carrier may also bear an agent by noncovalent bonding or by encapsulation, such as within a liposome vesicle (e.g., U.S. Patent Nos. 4,429,008 and 4,873,088).

Polynucleotides, Vectors, Host Cells and Recombinant Viruses

[0072] The invention provides polynucleotides that encode one or more polypeptides of the invention. The polynucleotide can be included in a vector. The vector can further comprise an expression control sequence operably linked to the polynucleotide of the invention. In some embodiments, the vector includes one or more polynucleotides encoding other molecules of interest. In one embodiment, the polynucleotide of the invention and an additional

polynucleotide can be linked so as to encode a fusion protein.

[0073] Within certain embodiments, polynucleotides may be formulated so to permit entry into a cell of a mammal, and expression therein. Such formulations are particularly useful for therapeutic purposes, as described below. Those of ordinary skill in the art will appreciate that there are many ways to achieve expression of a polynucleotide in a target cell, and any suitable method may be employed. For example, a polynucleotide may be incorporated into a viral vector such as, but not limited to, adenovirus, adeno-associated virus, retrovirus, vaccinia or a pox virus (e.g., avian pox virus). Techniques for incorporating DNA into such vectors are well known to those of ordinary skill in the art. A retroviral vector may additionally transfer or incorporate a gene for a selectable marker (to aid in the identification or selection of transduced cells) and/or a targeting moiety, such as a gene that encodes a ligand for a receptor on a specific target cell, to render the vector target specific. Targeting may also be accomplished using an antibody, by methods known to those of ordinary skill in the art.

[0074] The invention also provides a host cell transformed with a vector of the invention. The transformed host cell can be used in a method of producing a polypeptide of the invention. The method comprises culturing the host cell and recovering the polypeptide so produced. The recovered polypeptide can be purified from culture supernatant. [0075] Vectors of the invention can be used to genetically modify a cell, either in vivo, ex vivo or in vitro. Several ways of genetically modifying cells are known, including transduction or infection with a viral vector either directly or via a retroviral producer cell, calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes or microspheres containing the DNA, DEAE dextran, receptor-mediated endocytosis, electroporation, micro-injection, and many other techniques known to those of skill. See, e.g., Sambrook et al. Molecular Cloning - A Laboratory Manual (2nd ed.) 1 -3, 1989; and Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994 Supplement).

[0076] Examples of viral vectors include, but are not limited to retroviral vectors based on, e.g., HIV, SIV, and murine retroviruses, gibbon ape leukemia virus and other viruses such as adeno- associated viruses (AAVs) and adenoviruses. (Miller et al. 1990, Mol. Cell Biol. 10:4239; J. Kolberg 1992, NIH Res. 4:43, and Cornetta et al. 1991 , Hum. Gene Ther. 2:215). Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), ecotropic retroviruses, simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations. See, e.g. Buchscher et al. 1992, J. Virol. 66(5):2731 -2739; Johann et al. 1992, J. Virol. 66(5): 1635-1640; Sommerfelt et al. 1990, Virol. 176:58-59; Wilson et al. 1989, J. Virol. 63:2374-2378; Miller et al. 1991 , J. Virol. 65:2220-2224, and Rosenberg and Fauci 1993 in Fundamental Immunology, Third Edition, W.E. Paul (ed.) Raven Press, Ltd., New York and the references therein; Miller et al. 1990, Mol. Cell. Biol.

10:4239; R. Kolberg 1992, J. NIH Res. 4:43; and Cornetta et al. 1991 , Hum. Gene Ther. 2:215.

[0077] In vitro amplification techniques suitable for amplifying sequences to be subcloned into an expression vector are known. Examples of such in vitro amplification methods, including the polymerase chain reaction (PCR), ligase chain reaction (LCR), Qp-replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA), are found in Sambrook et al. 1989, Molecular Cloning - A Laboratory Manual (2nd Ed) 1^3; and U.S. Patent No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al. eds.) Academic Press Inc. San Diego, CA 1990. Improved methods of cloning in vitro amplified nucleic acids are described in U.S. Patent No. 5,426,039.

[0078] The invention additionally provides a recombinant microorganism genetically modified to express a polynucleotide of the invention. The recombinant microorganism can be useful as a vaccine, and can be prepared using techniques known in the art for the preparation of live attenuated vaccines. Examples of microorganisms for use as live vaccines include, but are not limited to, viruses and bacteria. In a preferred embodiment, the recombinant microorganism is a virus. Examples of suitable viruses include, but are not limited to, vaccinia virus and other poxviruses.

Compositions

[0079] In one embodiment, the composition is a pharmaceutical composition. The composition can comprise a therapeutically or prophylactically effective amount of construct, recombinant virus, or polypeptide, of the invention. An effective amount is an amount sufficient to elicit or augment an immune response, e.g., by activating T cells. One measure of the activation of T cells is a cytotoxicity assay, as described in D.M. Koelle et al., 1997, Human Immunol. 53:195- 205. In some embodiments, the composition is a vaccine. In some embodiments, the composition of the invention further comprises a carrier. The carrier can be a pharmaceutically acceptable carrier, or other carrier that facilitates use of the composition.

[0080] While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. Compositions of the present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed.

[0081] Such compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives. Alternatively, compositions of the present invention may be formulated as a lyophilizate.

Compounds may also be encapsulated within liposomes via known methods.

[0082] The composition of the invention can further comprise one or more adjuvants. Examples of adjuvants include, but are not limited to, helper peptide, alum, Freund's, muramyl tripeptide phosphatidyl ethanolamine or an immunostimulating complex, including cytokines. In some embodiments, such as with the use of a polynucleotide vaccine, an adjuvant such as a helper peptide or cytokine can be provided via a polynucleotide encoding the adjuvant. Vaccine preparation is generally described in, for example, M.F. Powell and M.J. Newman, eds., "Vaccine Design (the subunit and adjuvant approach)," Plenum Press (NY, 1995).

Pharmaceutical compositions and vaccines within the scope of the present invention may also contain other compounds, which may be biologically active or inactive. For example, one or more immunogenic portions of other viral antigens may be present, either incorporated into a fusion polypeptide or as a separate compound, within the composition or vaccine. Additional information about peptide vaccines can be found in Li et al. , 2014, Vaccines 2: 515-536, and about adjuvant use with a Herpes zoster vaccine in Lai et al. , 2015, New Engl J Med DOI : 10.1056/ NEJMoal 501 184.

[0083] A pharmaceutical composition or vaccine may contain DNA encoding one or more of the polypeptides of the invention, such that the polypeptide is generated in situ. As noted above, the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacteria and viral expression systems. Numerous gene delivery techniques are well known in the art, such as those described by Rolland, 1998, Crit. Rev. Therap. Drug Carrier Systems 15: 143-198, and references cited therein. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal) . Bacterial delivery systems involve the administration of a bacterium (such as Bacillus- Calmette-Guerrin) that expresses an immunogenic portion of the polypeptide on its cell surface or secretes such an epitope. In a preferred embodiment, the DNA may be introduced using a viral expression system (e.g. , vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus. Suitable systems are disclosed, for example, in Fisher-Hoch et al. , 1989, Proc. Natl. Acad. Sci. USA 86:317-321 ; Flexner et al. , 1989, Ann. My Acad. Sci. 569:86-103; Flexner et al. , 1990, Vaccine 8: 17-21 ; U.S. Patent Nos.4,603, 1 12, 4,769,330, and 5,017,487; WO 89/01973; U.S. Patent No. 4,777, 127; GB 2,200,651 ; EP 0,345,242; WO 91 102805; Berkner, 1988, Biotechniques 6:616-627;

Rosenfeld et al. , 1991 , Science 252:431 -434; Kolls et al. , 1994, Proc. Natl. Acad. Sci. USA 91 :215-219; Kass-Eisler et al. , 1993, Proc. Natl. Acad. Sci. USA 90: 1 1498-1 1502; Guzman et al. , 1993, Circulation 88:2838-2848; and Guzman et al. , 1993, Cir. Res. 73: 1202-1207.

Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be "naked," as described, for example, in Ulmer et al. , 1993, Science 259: 1745-1749 and reviewed by Cohen, 1993, Science 259: 1691 -1692. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.

Administration of the Compositions

[0084] Treatment includes prophylaxis and therapy. Prophylaxis or treatment can be accomplished by a single direct injection at a single time point or multiple time points.

Administration can also be nearly simultaneous to multiple sites. Patients or subjects include mammals, such as human, bovine, equine, canine, feline, porcine, and ovine animals as well as other veterinary subjects. Typical patients or subjects are human.

[0085] Compositions are typically administered in vivo via parenteral (e.g. intravenous, subcutaneous, and intramuscular) or other traditional direct routes, such as buccal/sublingual, rectal, oral, nasal, topical, (such as transdermal and ophthalmic), vaginal, pulmonary, intraarterial, intraperitoneal, intraocular, or intranasal routes or directly into a specific tissue, such as by implantation into a target organ, injection into a target tissue, or introduction of a scaffold to a target site.

[0086] The compositions are administered in any suitable manner, often with pharmaceutically acceptable carriers. Suitable methods of administering cells in the context of the present invention to a patient are available, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.

[0087] A variety of delivery vehicles may be employed within pharmaceutical compositions and vaccines to facilitate production of an antigen-specific immune response that targets HSV- infected cells. Delivery vehicles include antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells engineered to be efficient APCs. Such cells may, but need not, be genetically modified to increase the capacity for presenting antigen, to improve activation and/or maintenance of the T cell response, to have antiviral effects per se and/or to be immunologically compatible with the receiver (i.e. , matched HLA haplotype). APCs may generally be isolated from any of a variety of biological fluids and organs, including tumor and peritumoral tissues, and may be autologous, allogeneic, syngeneic or xenogeneic cells.

[0088] The dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial therapeutic response in the patient over time, or to inhibit infection or disease due to infection. Thus, the composition is administered to a patient in an amount sufficient to alleviate, reduce, cure or at least partially arrest symptoms and/or complications from the disease or infection. An amount adequate to accomplish this is defined as a "therapeutically effective dose."

[0089] The dose will be determined by the activity of the composition produced and the condition of the patient, as well as the body weight or surface areas of the patient to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse side effects that accompany the administration of a particular composition in a particular patient. In determining the effective amount of the composition to be administered in the treatment or prophylaxis of diseases, the physician needs to evaluate the progression of the disease, and any treatment-related toxicity. [0090] For example, a vaccine or other composition containing a subunit HSV protein can include 1 -10,000 micrograms of HSV protein per dose. In a preferred embodiment, 10-1000 micrograms of HSV protein is included in each dose in a more preferred embodiment 10-100 micrograms of HSV protein dose. Preferably, a dosage is selected such that a single dose will suffice or, alternatively, several doses are administered over the course of several months. For compositions containing HSV polynucleotides or peptides, similar quantities are administered per dose.

[0091 ] In one embodiment, between 1 and 10 doses may be administered over a 52 week period. Preferably, 6 doses are administered, at intervals of 1 month, and booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual patients. A suitable dose is an amount of a compound that, when administered as described above, is capable of promoting an antiviral immune response, and is at least 10-50% above the basal (i.e., untreated) level. Such vaccines should also be capable of causing an immune response that leads to an improved clinical outcome in vaccinated patients as compared to non-vaccinated patients. In general, for pharmaceutical compositions and vaccines comprising one or more polypeptides, the amount of each polypeptide present in a dose ranges from about 0.1 μg to about 5 mg per kg of host. Preferably, the amount ranges from about 10 to about 1000 μg per dose. Suitable volumes for administration will vary with the size, age and immune status of the patient, but will typically range from about 0.1 mL to about 5 mL, with volumes less than about 1 mL being most common.

[0092] Compositions comprising immune cells are preferably prepared from immune cells obtained from the subject to whom the composition will be administered. Alternatively, the immune cells can be prepared from an HLA-compatible donor. The immune cells are obtained from the subject or donor using conventional techniques known in the art, exposed to APCs modified to present an epitope of the invention, expanded ex vivo, and administered to the subject. Protocols for ex vivo therapy are described in Rosenberg et al., 1990, New England J. Med. 9:570-578. In addition, compositions can comprise APCs modified to present an epitope of the invention.

In Vivo Testing of Identified Antigens

[0093] Conventional techniques can be used to confirm the in vivo efficacy of the identified HSV antigens. For example, one technique makes use of a mouse challenge model. Those skilled in the art, however, will appreciate that these methods are routine, and that other models can be used.

[0094] Once a compound or composition to be tested has been prepared, the mouse or other subject is immunized with a series of injections. For example up to 10 injections can be administered over the course of several months, typically with one to 4 weeks elapsing between doses. Following the last injection of the series, the subject is challenged with a dose of virus established to be a uniformly lethal dose. A control group receives placebo, while the experimental group is actively vaccinated. Alternatively, a study can be designed using sublethal doses. Optionally, a dose-response study can be included. The end points to be measured in this study include death and severe neurological impairment, as evidenced, for example, by spinal cord gait. Survivors can also be sacrificed for quantitative viral cultures of key organs including spinal cord, brain, and the site of injection. The quantity of virus present in ground up tissue samples can be measured. Compositions can also be tested in previously infected animals for reduction in recurrence to confirm efficacy as a therapeutic vaccine.

[0095] Efficacy can be determined by calculating the IC50, which indicates the micrograms of vaccine per kilogram body weight required for protection of 50% of subjects from death. The IC50 will depend on the challenge dose employed. In addition, one can calculate the LD50, indicating how many infectious units are required to kill one half of the subjects receiving a particular dose of vaccine. Determination of post mortem viral titer provides confirmation that viral replication was limited by the immune system.

[0096] A subsequent stage of testing would be a vaginal inoculation challenge. For acute protection studies, mice can be used. Because they can be studied for both acute protection and prevention of recurrence, guinea pigs provide a more physiologically relevant subject for extrapolation to humans. In this type of challenge, a non-lethal dose is administered, the guinea pig subjects develop lesions that heal and recur. Measures can include both acute disease amelioration and recurrence of lesions. The intervention with vaccine or other composition can be provided before or after the inoculation, depending on whether one wishes to study prevention versus therapy. Methods and Uses of the Invention

[0097] The invention provides a method for treatment and/or prevention of an alphaherpesvirus infection, such as an HSV infection, in a subject. The method comprises administering to the subject a composition, polynucleotide, or polypeptide of the invention. The composition, polynucleotide or polypeptide can be used as a therapeutic or prophylactic vaccine. In one embodiment, the HSV is HSV-1 . Alternatively, the HSV is HSV-2. The invention additionally provides a method for inhibiting alphaherpesvirus replication, for killing alphaherpesvirus - infected cells, for increasing secretion of lymphokines having antiviral and/or

immunomodulatory activity, and for enhancing production of herpes-specific antibodies. The method comprises contacting an HSV-infected cell with an immune cell directed against an antigen of the invention, for example, as described in the Examples presented herein. The contacting can be performed in vitro or in vivo. In a preferred embodiment, the immune cell is a T cell. T cells include CD4 and CD8 T cells. Alternatively, the methods for inhibiting alphaherpesvirus replication, for killing alphaherpesvirus -infected cells, for increasing secretion of lymphokines having antiviral and/or immunomodulatory activity, and for enhancing production of herpes-specific antibodies can be achieved by administering a composition, polynucleotide or polypeptide of the invention to a subject. Compositions of the invention can also be used as a tolerizing agent against immunopathologic disease.

[0098] In addition, the invention provides a method of producing immune cells directed against an alphaherpesvirus, such as HSV. The method comprises contacting an immune cell with an alphaherpesvirus polypeptide of the invention. The immune cell can be contacted with the polypeptide via an antigen-presenting cell, wherein the antigen-presenting cell is modified to present an antigen included in a polypeptide of the invention. Preferably, the antigen- presenting cell is a dendritic cell. The cell can be modified by, for example, peptide loading or genetic modification with a nucleic acid sequence encoding the polypeptide. In one

embodiment, the immune cell is a T cell. T cells include CD4 and CD8 T cells. Also provided are immune cells produced by the method. The immune cells can be used to inhibit HSV replication, to kill HSV-infected cells, in vitro or in vivo, to increase secretion of lymphokines having antiviral and/or immunomodulatory activity, to enhance production of herpes-specific antibodies, or in the treatment or prevention of HSV infection in a subject.

[0099] The invention also provides a diagnostic assay. The diagnostic assay can be used to identify the immunological responsiveness of a patient suspected of having a herpetic infection and to predict responsiveness of a subject to a particular course of therapy. The assay comprises exposing T cells of a subject to an antigen of the invention, in the context of an appropriate APC, and testing for immunoreactivity by, for example, measuring IFNv, proliferation or cytotoxicity. Suitable assays are known in the art.

Kits

[0100] For use in the methods described herein, kits are also within the scope of the invention. Such kits can comprise a package or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements (e.g. , constructs, polynucleotides, polypeptides, recombinant virus) to be used in the method. Typically, the kit comprises one or more constructs or polypeptides of the invention. The kit further comprises one or more containers, with one or more constructs or polypeptides stored in the containers. The kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In addition, a label can be provided on the container to indicate that the composition is used for a specific therapeutic or non-therapeutic application, and can also indicate directions for use. Directions and or other information can also be included on an insert which is included with the kit. EXAMPLES

[0101 ] The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the invention.

Example 1 : Screening for CD8 Reactivity to ORFs of HSV-1 and HSV-2

[0102] This Example demonstrates use of DC cross-presentation, CD137-based sorting, and a library of each HSV-1 ORF, to screen each HLA A and B allele in 37 HSV- 1 -infected, HSV-2 seronegative donors for CD8 reactivity to each HSV-1 ORF. The median number of HSV-1 proteins recognized per person by CD8 T-cells was 12 (range 1 to 24) . Overall, 55 of 76 HSV-1 proteins were recognized by at least one person, and 34 of the 37 HLA alleles tested could restrict CD8 responses. The UL46 tegument protein and ribonucleotide reductase large subunit (UL39) were recognized by 85% and 81 % of subjects, respectively. UL39 had greater HLA diversity, with responses for 19 of 37 (51 %) of HLA alleles, in comparison to 14 (38%) for UL46. A minimum of 17 novel peptide epitopes, using conservative criteria, were defined in HSV-1 UL39 via 13-mer peptides as shown in Fig. 1 and Fig. 2. For most 13-mer epitopes, aAPC assays allowed proof of HLA restriction, and software algorithms confirmed at least one predicted high-affinity HLA-binding internal peptide. Amongst the 17 UL39 epitopes, 5 were sequence-identical in HSV-2 186, and 9 others retained a predicted high HLA- binding internal sequence. Thus, this data predicts that UL39 of HSV-2 will also be a very strong stimulator of CD8 T-cell responses. UL39 is a rational candidate for CD8-oriented HSV vaccines, especially for HSV- 1 , but also for diverse HSV-2 genotypes.

[0103] The inventors have studied 37 HSV-1 infected persons using blood samples and determined the presence or absence of CD8 T-cell responses to every HSV- 1 protein. The proteins UL39 and UL46 are the most frequently recognized protein in this large data set. The sequences of the proteins used are from HSV-1 strain 17+. We have also studied 26 HSV-2- infected persons using a similar technology in which the CD8 T cell response to each HSV-2 protein is determined. In this group, UL39 is also very frequently recognized. Other HSV proteins frequently recognized include UL25, UL27 (gB2) , UL29 (ICP8) , UL40 (RR2) , and UL47 (VP13/14) . [0104] Table 1 (Figure 2) shows a condensed and curated version of newly documented fine CD8 T-cell epitopes in UL39 of HSV-1 . The experimental data in Table 1 comes from a subset of the subjects. For some of the row listings in Table 1 , lab data was obtained from multiple persons that shared the same genetic variant at the HLA A or B locus. The indicated 13-mer peptides in HSV-1 are from strain 17+ in Genbank and are proven to be reactive in our lab tests. These 13-mer peptides are shown in Figure 1 . The indicated HLA class I allelic variant molecules shown in Figure 1 and in Table 1 are proven to be physically required to present these 13-mer peptides to the human CD8 T-cells. The indicated shorter HSV-1 peptides in Table 1 are predicted to be the approximate minimal antigenic units within the Figure 1 proven 13-mer peptides, but have not yet been experimentally proven to be recognized. The indicated IC50 binding numbers (lower numbers correspond to higher affinity binding) indicate the computer-predicted binding of the short predicted UL39 peptide to the indicated HLA allelic variant. The computer algorithm is available from the Immune Epitope Database (IEDB) Analysis Resource at http://tools.immuneepitope.org/mhci/. Values of < 5000 nanomolar are generally felt to reflect usually high affinity binding. These numbers are provided as

independent "virtual" verification that the combinations of 13mer peptides and recombinant HLA molecules that we studied in the wet lab are very likely to be correct. Finally, the data in Table 1 and Figure 1 concerning HSV-2 are predictions. The HSV-2 UL39 molecule from strain 186 has partial sequence identity to the HSV- 1 UL39 molecule. If the HSV-2 sequence is identical in the 13mer peptide region, it is extremely likely that the CD8 T-cells. If the HSV-2 sequence is divergent, we used tools at the IEDB Analysis Resource (http://tools. immuneepitope.org/mhci/) to study the HSV-2 13mer sequence homolog of the proven, antigenically positive HSV-1 sequence. The entry "nothing good" in Table 1 indicates that all peptides within the HSV-2 13mer had predicted IC50 values >5000 nanomolar and are thus less likely to be antigenic for CD8 T-cells. If there is variation in the HSV-2 13mer, but the variant maintains good predicted binding (IC50 < 5000 nanomolar), the sequence of the best predicted peptide is provided. The overall interpretation of Table 1 is that the high population prevalence of responses to UL39 of HSV-1 shown in Table 1 is supported by many discrete fine epitopes restricted by diverse but also population prevalent HLA allelic variants. In addition, many of the CD8 T cells defined using HSV- 1 infected persons are likely to cross-react with HSV-2.

[0105] We then studied the CD8 T cell response to each of the known 77 HSV-2 proteins in 26 persons infected with HSV-2. The methods used were those described in Jing et al. J. Clin. Invest. 122, 654-673, 2012. On the basis of the data presented herein, the HSV UL39 is a suitable subunit vaccine candidate for both HSV- 1 and HSV-2 therapeutic vaccine applications. Example 2: Screening for CD4 Reactivity to ORFs of HSV-1

[0106] HSV epitopes were screened for reactivity in CD4 T cell assays. Again, UL39 stood out as reactive in samples from 23 out of 42 subjects tested, or 54.8% of subjects positive. These data further support the value of UL39 as a vaccine candidate. In addition, the following Table 2 lists additional HSV genes that were recognized by CD4 T cells in a large percentage of subjects.

Table 2 n=42 subjects

gene percent recognizing virology

RS1 (ICP4) 88.1 regulatory, virion

UL27 88.1 gBl, env, fusion

US06 83.3 gDl, env, binding

tegument, abundant

UL46 71.4 virion

UL23 57.1 thymidine kinase

UL19 54.8 major capsid

US08 31.0 gEl, HZ/Su homolog

[0107] In brief, peripheral blood T cells from persons who were HSV-1 infected, some of whom had HSV-2 co-infection, were stimulated with whole HSV-1 virus and reactive cells were enriched and expanded and then tested for CD4 T cell responses to every HSV-1 protein. Six subjects with HSV-1 infection were studied in detail, all of whom were HSV-1 infected and some of whom were HSV-2 co-infected. Among samples that were positive for full-length UL39 protein of HSV-1 , we then tested these samples with peptides 13 amino acids in length and overlapping by 9 amino acids. The peptide tests were done in 3 stages: first in cell proliferation assays with pools of peptides, secondly in cell proliferation assays with single peptides that were constituents of pools that showed positive responses in the initial pool assays, and thirdly in interferon gamma intracellular cytokine secretion assays using the same single peptides that were positive in the second stage assays. The assays technologies have been published in or are referred to in Jing et al. J Immunol 2016 196:2205-2218. Thus, the peptide epitopes listed herein have been independently confirmed in three assays and in two different functional assays. While these assays used peptides from HSV-1 , note is made that the sequences are generally highly similar or in many cases completely identical in HSV-2, and that CD4 T cells generally tolerate some degree of amino acid substitution and are able to retain cross- recognition of related peptide sequences. Similarly, the CD8 epitopes mentioned above are from HSV-1 , but in many cases are highly similar or identical in HSV-2. Therefore, we propose that the use of UL39-based compositions from either HSV-1 or HSV-2, or a combination of both, could have a therapeutic immune boosting effect on antiviral T cell responses to either or both HSV type. Example 3: T Cells Derived from Human Trigeminal Ganglia Are Reactive to UL39 Epitopes

[0108] We have investigated whether or not CD8 T cells that are present in the human TG recognize HSV-1 UL39. HSV-1 established a life-long persistent infection in TG neurons, associated with intermittent reactivation resulting in asymptomatic infection of the innervating mucosal tissues, and on occasion symptomatic infection of variable severity ranging from mild herpes labialis to potentially sigh-threatening ocular infections. T cells were cultured from human TG, obtained shortly after death of HSV-1 infected individuals, and the percentage CD4 and CD8 T-cells reactive to HSV-1 determined by interferon gamma intracellular cytokine secretion assays using autologous cells productively infected with HSV-1. The assays technologies have been published in or are referred to in Van Velzen M, et al. (Plos Pathog. 2013; 9: e1003547). Subsequently, HSV-1 proteome-wide scans revealed that human intra-TG T-cell responses included both CD4 and CD8 T-cells directed to one to three HSV-1 proteins per person. Notably, HSV-1 UL39 was targeted by CD8 T-cells in 4 of 8 HLA-discordant donors (Velzen M, et al. Plos Pathog. 2013; 9: e1003547). The data from human TG-derived CD8 T- cells confirm and extend the blood-derived T-cell findings that HSV-1 UL39 is an

immunoprevalent CD8 T-cell target in HSV-1 infected individuals. Specifically, we identified that the UL39 reactive CD8 T-cells recovered from two different human TG donors are directed to the following HSV-1 UL39 peptides presented by specific HLA-I alleles: HSV-1 UL39 peptide 741-753/HLA-B 001 , 901-913/HLA-A*3101 , 461-473/HLA-B 001. In the Van Velzen M, et al. (Plos Pathog. 2013; 9: e1003547) paper, we showed that human TG-resident CD8 T cells expressed cytolytic effector molecules such as perforin and granzymeB and antiviral cytokines like interferon gamma and tumor necrosis factor alpha, that together imply that these T cells monitor for viral reactivation and restrict viral replication. Based on our published TG UL39 data and these new TG UL39 data, showing that UL39 peptides identified by systemic (blood) CD8 T-cells are also recognized by TG-resident CD8 T-cells in unrelated HSV-1 infected individuals, we conclude that at the physiologically relevant anatomic site of persistent HSV-1 infection (human TG), HSV-1 UL39-specific CD8 T-cells are frequently present and are highly likely to assist in the control of viral replication. Therefore, there is a rational for prevention and treatment of HSV infections with immune boosting modalities that target UL39.

Table 3: Newly discovered CD4 T cell epitopes in HSV-1 UL39 protein.

Peptid e Sequence SEQ ID NO:

HSV1_ UL39. 445-457 WLRSKEVALDFGL 30

HSV1_ UL39. .465-477 EAQLVI LAQALDH 31

HSV1_ UL39. .469-481 VI LAQALDHYDCL 32

HSV1_ UL39. .593-605 KATLRAITSNVSA 33

HSV1_ UL39. .693-705 PDLFFKRLI RHLD 34

HSV1 UL39 697-709 FKRLI RHLDGEKN 35 HSVl_UL39_777-789 HYIYDTQGAAIAG* 36

HSVl_UL39_961-973 SQFVALMPTAASA** 37

HSVl_UL39_965-977 ALMPTAASAQISD 38

HSV1_UL39_969-981 TAASAQISDVSEG 39

* positive responses in 2 of 6 subjects

** positive responses in 3 of 6 subjects

[0109] Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this invention pertains.

[01 10] From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.