US 20140193460A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0193460 A1 Spector et al. (43) Pub. Date: Jul. 10, 2014

(54) HERPES VIRUS VACCNE AND METHODS OF (60) Provisional application No. 61/503.945, filed on Jul. 1, USE 2011. (71) Applicant: The Regents of the University of California, Oakland, CA (US) Publication Classification (72) Inventors: Deborah H. Spector, La Jolla, CA (US); Christopher S. Morello, Carlsbad, CA (51) Int. Cl. (US); Kimberly A. Kraynyak, A639/245 (2006.01) Encinitas, CA (US) (52) U.S. Cl. CPC ...... A61K 39/245 (2013.01) (73) Assignee: The Regents of the University of California, Oakland, CA (US) USPC ...... 424/231.1; 424/229.1 (21) Appl. No.: 14/141,251 (57) ABSTRACT (22) Filed: Dec. 26, 2013 Related U.S. Application Data Provided herein are, interalia, vaccines and methods of using (63) Continuation of application No. PCT/US2012/ the same for the treatment or prevention of Herpesvirus infec 045290, filed on Jul. 2, 2012. tions. Patent Application Publication Jul. 10, 2014 Sheet 1 of 30 US 2014/0193460 A1

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HERPES VIRUS VACCNE AND METHODS OF lates with an increase in HSV-2 shedding (Schacker, T. et al., USE J. Infect. Dis., 178:1616-1622 (1998)). In addition, a prophy lactic vaccine eliciting neutralizing antibody responses did CROSS-REFERENCES TO RELATED not result in Sterilizing immunity (Corey, L. et al., J. Amer: APPLICATIONS Med. Assoc., 282:331-340 (1999)). Human studies have also 0001. This application is a continuation of International unveiled the role of CD8+ cytotoxic T cells in reducing Patent Application No. PCT/US2012/045290, filed Jul. 2, HSV-2 replication and shedding. During primary infection, 2012, which claims the benefit of U.S. Provisional Patent CD8+ T cells may prevent acute ganglion infection (Koelle, Application No. 61/503.945, filed Jul. 1, 2011, each of which D. M., and L. Corey, Annu. Rev. Med., 59:381-395 (2008)). In is incorporated herein by reference in its entirety and for all addition, CD8+ T cells have been found to infiltrate genital purposes. lesions, correlating with viral clearance (Koelle, D. M. et al., J. Clin. Invest., 101:1500-1508 (1998)), and to accumulate at STATEMENT AS TO RIGHTS TO INVENTIONS nerve endings ingenital skin during HSV-2 reactivation (Zhu, MADE UNDER FEDERALLY SPONSORED J. et al., J. Exp. Med., 204:595-603 (2007)). In the commonly RESEARCH AND DEVELOPMENT utilized HSV-2 mouse and guinea pig models, the roles of cellular immunity have been less defined or the data have 0002 This invention was made with government support been ambiguous. T cell depletion studies in the mouse have under 1R21AI073585 awarded by the National Institutes of suggested a role for both CD4+ and CD8+ T in protection Health. The Government has certain rights in the invention. against intravaginal (i.vag.) HSV-2 challenge (Milligan, G. N., and D. I. Bernstein, Virology, 229:259-268 (1997); Milli REFERENCE TO A “SEQUENCE LISTING. A gan, G. N. D. I. Bernstein, and N. Bourne, J. Immunol., TABLE, ORACOMPUTER PROGRAM LISTING 160:6093-6100 (1998); Parr, M. B., and E. L. Parr, J. Virol., APPENDIX SUBMITTED ON A COMPACT DISK 72:2677-2685 (1998)). The T-cell mediated clearance of virus 0003. The Sequence Listing written in file 88654 from the mucosa is largely dependent upon IFN-Y (Milligan, O0671OPC-844147 ST25.TXT, created on Jul. 2, 2012, G. N., and D.I. Bernstein, Virology, 229:259-268 (1997)), and 4,077 bytes, machine format IBM-PC, MS-Windows operat T cells activated in the absence of IFN-Y are not protective ing system, is hereby incorporated by reference in its entirety against HSV-2 infection (Johnson, A. J. et al., J. Reprod. for all purposes. Immunol., 84:8-15 (2010)). 0006 Although many strategies have been tried, up to the BACKGROUND OF THE INVENTION present disclosure no successful HSV-2 vaccine has been 0004. In the United States, Herpes Simplex Virus type 2 developed (Koelle, D. M., and L. Corey, Annu. Rev. Med., (HSV-2) is one of the most common sexually transmitted 59:381-395 (2008)). The glycoproteinsgD and gE have been infections, affecting 16.2% of adults (Xu, F. et al., Seropreva widely used as vaccine antigens in order to generate neutral lence of Herpes Simplex Virus Tipe 2 Among Persons Aged izing antibodies (Corey, L. et al., J. Amer: Med. Assoc., 282: 14-49 Years-United States, 2005-2008, Centers for Disease 331-340 (1999); Straus, S. E. et al., J. Infect. Dis., 176:1129 Control and Prevention (2010)). HSV-2 infection can cause 1134 (1997)). Tested in two clinical trials in HSV-2 significant morbidity and mortality in neonates (Kimberlin, discordant couples, the most promising vaccine candidate, D. W., Herpes, 14:11-16 (2007)) and immune compromised manufactured by GlaxoSmithKline (GSK), was composed of hosts (Dupuis, S. et al., Nat. Genet., 33:388-391 (2003)). In a secreted gld protein formulated with MPL and alum; how addition, HSV-2 infection is the primary cause of genital ever, it was efficacious only in HSV-1 and -2 seronegative lesions and has been linked to an increased risk of human women (Stanberry, L. R. et al., N. Engl. J. Med., 347: 1652 immunodeficiency virus (HIV) acquisition (Freeman, E. E. et 1661 (2002)). A subsequent Phase 3 study in 8.323 HSV-1 al., AIDS, 20:73-83 (2006); Wald, A., and K. Link, J. Infect. and -2 seronegative women (Herpevac trial) was not able to Dis., 185:45-52 (2002)). HSV-2 replicates in the genital epi show significant protection against HSV-2 in this cohort. The thelium, followed by retrograde axonal transport to the dorsal secreted form of go2 is truncated as a result of the deletion of root ganglia where virus can remain and establish a lifelong, the transmembrane domain (Higgins, T. J. et al., J. Infect. latent infection with periodic reactivation. What is striking, Dis., 182:1311-1320 (2000)). A previous study by Strasseret however, is that most HSV-2 infections are asymptomatic, al. showed that, when compared to the full-length or cytosolic and yet these individuals shed virus with high frequency. As a portion ofg)2, the secreted form provided the best protection result, HSV-2 is commonly transmitted from individuals who from acute disease in both mice and guinea pigs (Strasser, J. have no history of genital lesions (Mertz, G.J. J. Infect. Dis., E. et al., J. Infect. Dis., 182:1304-1310 (2000)). 198: 1098-1100 (2008)). HSV-1 infection is also a significant 0007 Replication-defective virus vaccines for HSV-2 are cause of primary genital herpes disease and its incidence has also in the clinical trial pipeline as they have been Successful been increasing in the U.S. and Europe (Wald, A. 2006. Geni in animal models. UL5 (DNA helicase), UL29 (single tal HSV-1 infections. Sex Transm. Infect 82:189-190). Nota Stranded DNA binding protein) are among the many proteins bly, in the negative control group of the Herpevac vaccine required for HSV-2 replication (Challberg, M.D., Proc. Natl. trial, HSV-1 had a higher attack rate for infection than did Acad. Sci. USA, 83:9094-9098 (1986); Knipe, D. M., Virus HSV-2 (3.2% vs. 1.5%, respectively) (Belshe, R. B., et al., Res., 37:85-123 (1989)). A mutant virus lacking UL5 and Efficacy results of a trial of a herpes simplex vaccine. The UL29 has exhibited promising immunogenicity and Subse New England Journal of Medicine, 2012. 3.66(1): p. 34-43.) quent protection in the mouse (Da Costa, X. et al., J. Virol., 0005 Without wishing to be bound by any theory, it is 74:7963-7971 (2000); DaCosta, X.J. et al., Proc. Natl. Acad. believed that the correlates of protective immunity against Sci. USA, 96:6994-6998 (1999)) and guinea pig models HSV-2 are unknown. Studies in HSV-2/HIV-1 co-infected (Hoshino, Y. et al., J. Virol.., 79:410-418 (2005); Hoshino, Yet individuals have shown that the loss of CD4+ T cells corre al., J. Infect. Dis., 200:1088-1095 (2009); Hoshino, Y. et al., US 2014/0193460 A1 Jul. 10, 2014

Vaccine, 26:4034-4040 (2008)). In the past, inactivated ug DNA) together with either 25ug of pVAX DNA (-g|D2t) HSV-2 vaccines also were tested in humans, but subsequently org)2t DNA (+gD2t) and then given a lethal, i.vag. challenge dismissed due to a lack of controls required for accurate data of 10xLDs of HSV-2 strain G. FIG. 2B. Disease severity in interpretation (reviewed in (Whitley, R. J., Herpes Simplex the immunized mice after i.vag. challenge. Symbols shown in Viruses, p. 2461-2509. In D. M. Knipe and P. M. Howley the legend show the group mean disease score for each vac (ed.), Fields Virology, Fourth ed, vol. 2. Lippincott Williams cine group using the disease severity Scale described in Mate & Wilkins, Philadelphia (2001))). Provided herein are solu rials and Methods and error bars represent standard error of tions to these and other problems in the art. the mean (SEM). FIG. 2C. Vaginal virus shedding following i.Vag. challenge. On the days postchallenge shown, each BRIEF SUMMARY OF THE INVENTION mouse was Swabbed intravaginally and Swabs were placed in 1 ml of DNG and frozen until standard plaque assay on Vero 0008 Provided in a first aspect is a Herpesvirus (HV) cells as described in Materials and Methods. Symbols repre vaccine including inactivated HV, a lipopolysaccharide sent the group meanLog 10 PFU, bars represent SEM, and the (LPS)-derived adjuvant and an aluminum-based mineral salt dotted line shows the limit of sensitivity of the assay. Assays adjuvant. yielding no plaques were assigned a titer of one-half the 0009 Provided in a second aspect is a method of preparing detection limit (or 5 PFU per Swab for this experiment) for a Herpesvirus (HV) vaccine, the method including: contact graphing and statistical analysis. For clarity, brackets are ing an HV-cell mixture with a sulfated or sulfonated polysac shown for the groups that were coimmunized with or without charide, wherein the HSV-cell mixture includes HV particles, gD2t DNA. FIG. 2D. Experiment 2: Mice (n=8) were coim cells and portions of cells; separating the HSV particles from munized with UL5+UL30 with or without g|D2t DNA. Mice the cells thereby forming isolated HV particles; inactivating received 20 ug each of UL5 and UL30 together with 20 g of the isolated HV particles thereby forming inactivated HV either pVAX org)2t DNA. I. vag. challenge and survival for particles; combining the inactivated HV particles with a 21 days is shown as in A. FIG. 2E. Disease severity following lipopolysaccharide-derived adjuvant and an aluminum-based challenge as in FIG. 2B. FIG. 2F. Vaginal virus shedding mineral salt adjuvant thereby forming an HV Vaccine. following challenge as in FIG. 2C. 0010 Provided in a third aspect is a method of treating or 0015 FIG. 3A to FIG.3C. DNA immunization with con preventing an HV infection in a patient in need of the treat served essential genes elicits antigen specific CD8+ T cells. ment or prevention. The method including administering a FIG. 3A. In two separate experiments, splenocytes were iso therapeutically or prophylactically effective amount of an HV lated from BALB/c mice that were either naive (Naive), vaccine as described herein (including embodiments). HSV-2 infected in the footpad 7 days prior to harvest (HSV 0011 Provided in a fourth aspect is a method of treating or 2), or i.d. immunized with pVAX (pVAX). Splenocytes were preventing a disease in a patient in need of the treatment or stimulated with either ICP27 plasmid transfected BALB prevention. The method including administering a therapeu SV40 cells (ICP27 Cells:gray bars) or an ICP27 peptide of a tically or prophylactically effective amount of an HV vaccine known H-2" CD8+ T cell epitope (ICP27 Peptide, blackbars) as described herein (including embodiments). and CD8+ T cells producing IFN-Y were analyzed by intrac 0012 Provided in a fifth aspect is a kit including an HV ellular cytokine staining FIG. 3B and FIG.3C. Splenocytes vaccine as described herein (including embodiments) and from individual BALB/c mice (n=4) immunized with pVAX, instructions for administering the HV Vaccine to a patient. UL5, or UL30 DNA plasmids either alone or in the combi nations shown were stimulated with UL5 or UL30 plasmid BRIEF DESCRIPTION OF THE DRAWINGS transfected BALB SV40 cells in two separate experiments. 0013 FIG. 1A to FIG. 1C.. Expression of HSV-2 ORFs One mouse that was infected with HSV-2 in the footpad 7 from plasmid vaccines and purification of gl)2t protein. FIG. days prior to harvest was also analyzed (HSV-2). Plotted are 1A. 293FT cells were transiently transfected with each the values from each mouse after subtraction of the back HSV-2 ORF plasmid as shown or empty parent vector ground staining values from stimulation with pcDNA3-trans (pVAX) and cells were harvested at 48 h post transfection for fected SV40 BALB cells. Lines represent group means and SDS-PAGE and Western blot analysis using anti-FLAG M2 closed symbols indicate values for individual mice. monoclonal antibody. FIG. 1B. 293FT cells were transfected (0016 FIG. 4A to FIG. 4C. Protection against HSV-2 after with gl)2t plasmid and cells and media were harvested on the immunization with FI-HSV2 as compared with g|D2t protein. days post transfection shown. Untransfected 293FT cells and Mice (n=8) were s.c. immunized twice with either 107 PFU media were harvested on day 2 of the experiment (293). equivalents of FI-HSV2, and equal volume of a FI-Mock Media were subjected to anti-FLAG M2 affinity chromatog virus preparation, or 5ug of purified g|D2t protein, with each raphy and the bound material was solubilized in SDS-PAGE immunization together with MPL and alum adjuvant as buffer and analyzed together with the corresponding cell described in Materials and Methods. FIG. 4A. Twenty-one lysates using the type common anti-g|D monoclonal antibody day postchallenge Survival of each vaccine group. FIG. 4B. DL6. FIG. 1C. The g|D2t protein from media from pc3Aneo Disease severity following challenge as in FIG. 2B. FIG. 4C. gD2t transfected 293FT cells was purified by anti-FLAG M2 Vaginal virus shedding following challenge as in FIG. 2C and column and eluted with 3xFLAG peptide, the gl)2t-contain Vaginal virus titers of each mouse on day 2 postchallenge. ing fractions combined, and the Volumes shown were ana Each symbol represents the Logo PFU titer of each mouse, lyzed by SDS-PAGE and Coomassie blue staining. horizontal lines represent the group means, and the dottedline 0014 FIG. 2A to FIG. 2F. Protection of BALB/c mice shows the assay detection limit (equivalent to 2 PFU per against HSV-2 after immunization with UL5 and/or UL30 swab). plasmids with or without g|D2t plasmid co-immunization. (0017 FIG. 5A to FIG.5D. Protection against HSV-2 after FIG. 2A. Experiment 1. Twenty-one day survival of mice prime-boost immunization. FIG. 5A. Timeline of prime (n=8) that were immunized with the HSV-2 genes shown (25 boost immunization, challenge, and postchallenge outcome US 2014/0193460 A1 Jul. 10, 2014

measurements. Mice were i.d. primed with either pVAX, FI-HSV2 used in the other groups. Four weeks later, mice gD2t DNA, or a cocktail of UL5, UL30, and g|D2t DNAs on were given an additional immunization, rested for 3 weeks, the weeks shown. Eight mice from each DNA prime group and then i.vag. challenged with HSV-2 as above. A. Vaginal were s.c. boosted twice on the weeks shown with MPL/Alum virus shedding on day 2 (Left panel) or day 4 (Right panel) together with either 107 PFU equivalents of FI-HSV2, an postchallenge as in FIG. 4C except that the limit of assay equal Volume of FI-Mock virus preparation, or 5 uggD2t sensitivity was 10 PFU per swab. Data points below the protein subunit (gD2tSU). FIG. 5B. Postchallenge survival of sensitivity limit(s) were staggered for increased visibility in mice given the prime-boost combinations. One mouse in the the panels for both FIG. 7A and FIG. 7C. FIG. 7B and FIG. UL5, UL30, gD2t DNAS-g)2t protein group developed a 7C. Mice surviving the HSV-2 challenge were medrox severe ear infection and was sacrificed prior to the challenge yprogesterone treated as before, and 17 weeks after chal day. FIG.5C. Disease severity in the prime-boost immunized lenge, these mice were re-challenged and a naive group of mice following i.vag. challenge as in FIG. 2B. Each panel mice was challenged i.vag. with 10xLDs of HSV-2. Mice shows the immunization groups given the same boost type, were swabbed for vaginal HSV-2 shedding on day 2 and day with either Mock (FI-Mock) (Left), gD2t protein (Middle), or 4 as above. Vaginal HSV-2 titers for the naive group are FI-HSV2 (Right) boosts. FIG. 5D. Vaginal virus shedding shown (FIG. 7B). Vaginal HSV-2 shedding in each mouse following i.vag. challenge as in FIG. 2C, with immunization following challenge and re-challenge is plotted as the vaginal groups given the same boost type shown together and the HSV-2 titer on day 2 following HSV-2 challenge on the x-axis pVAX-FI-Mock group shown in each panel for comparative and on day 2 following re-challenge on the y-axis (FIG. 7C). purposes. Each FI-HSV2 immunization group is shown in a separate 0018 FIG. 6A to FIG. 6D. Protection against HSV-2 shed panel for clarity, and the limit of detection for both assays was ding after a single formulation of FI-HSV2 and DNA plas 10 PFU per Swab. The numbers of overlapping data points on mids in MPL plus Adju-Phos: FIG. 6A and FIG. 6B. Groups each graph are shown. Significance scores of 'P-0.05;" of mice were immunized with 107 PFU equivalents of FI p-0.01; and "P<0.001 were determined by Kruskal-Wallis HSV2, 12.5ug of MPL, and 54 ug of Adju-Phos (AdP). Some plus Dunn's Multiple Comparison Tests. groups received 20 or 50 ug of each plasmid UL5, UL30, and 0020 FIG. 8A to FIG. 8C. Immunization with FI-HSV2 gD2t. In addition, a negative control group received 60 ug of formulated in MPL/Alhydrogel is required for protection pVAX DNA mixed with MPL, Adju-Phos, and a volume of against HSV-2 replication, disease, and latent viral load in the FI-Mock equal to the FI-HSV2 used in the other groups. Four DRG. Groups of mice (n=8) were immunized s.c. ori.m. with weeks later, mice were given an additional immunization, dextran sulfate-derived FI-HSV2 (1 g of protein) in either rested for either 3 weeks (FIG. 6A) or 9 weeks (FIG. 6B), and MPL and Alhydrogel (equivalent to 54 ug of Al) or DPBS. In then i.vag. challenged with HSV-2 as above. Mice were addition, a negative control group received a Volume of FI swabbed for vaginal HSV-2 shedding on day 2 and day 4 and Mock equal to the FI-HSV2 used in the other groups in MPL data are shown similarly as in the FIG. 4C legend except that and Alhydrogel. Two injections were given 4 weeks apart, and the limits of assay sensitivity were 4 PFU (FIG. 6A) and 2.22 mice were challenged 3 weeks later. FIG. 8A. Vaginal virus PFU (FIG. 6B) per swab. FIG. 6C and FIG. 6D Virus specific shedding on day 2 (left panel) or day 4 (right panel) postch antibody levels in immunized mice pre-challenge. Dashed allenge as in FIG. 6A and FIG. 6B except that the limit of lines represent the assay limits of sensitivity, and individual assay sensitivity was 2.2 PFU per Swab. FIG. 8B. Disease titers below the assay limit were assigned a value of one-half scores (using the scale described in FIG. 4A to FIG. 4C) from the assay limit for calculation and graphing purposes. (FIG. days 1-14 postchallenge were Summed for individual mice 6C) IgG titers measured by an ELISA against dextran sulfate (each symbol shows the value for one mouse). FIG. 8C. derived HSV-2 (2.35x10° PFU per well) before short-term Protection against HSV-2 latent DNA load in the DRG. Four (left panel) or long-term (right panel) challenge. The endpoint weeks postchallenge the lumbosacral DRG from each surviv titer was defined as the highest, Fit Spline interpolated recip ing mouse and 4 naive mice were removed, pooled, frozen, rocal dilution of serum at which the Aaos of the virion-coated and the DRG DNA from each pool was extracted and quan well was equal to twice the Aaos of the same serum dilution in tified by spectrophotometry. Cross-contamination safe a well coated with an equal mass of Vero cell lysate. (FIG. guards, sample storage, DRG DNA extraction, and HSV-2 6D.) Virus neutralizing antibody titers prior to long-term copy number determined by TaqMan quantitative PCR (Ap challenge. Endpoint neutralization titers were calculated as plied Biosystems, Inc.) using primers and a probe specific for the highest reciprocal serum dilution that resulted in a 50% gG2 were as previously described 19. Each reaction con reduction in the number of input PFU (50 PFU of dextran tained 300ng of DRG DNA and the DNA load for each mouse sulfate-derived HSV-2) using Fit Spline interpolation as is expressed as HSV-2 DNA copy number per 300 ng of DRG above. Data points representing titers below the sensitivity DNA. The line represents the limit of detection for the assay limit were staggered for increased visibility. Significance (2 copies of HSV-2 DNA per 300 ng of DRG DNA). Unifor scores of "P<0.05: "P<0.01; and "P<0.001 were deter mity of each template was ensured by TaqMan quantification mined by Kruskal-Wallis plus Dunn's Multiple Comparison of the mouse adipsin gene (GenBank accession no. X04673. Tests. 1): sense primer (TGT GGC AAT GGC AAAAAGC) (SEQ 0019 FIG. 7A to FIG.7C. Protection against HSV-2 chal IDNO:14), antisense primer (TGTTAC CATTTGTGATGT lenge and re-challenge after immunization with FI-HSV2 in TTT CGAT) (SEQ ID NO:15), and probe (6-FAM-CGT MPL plus Alhydrogel or Adju-Phos. Groups of mice were CTA TAC-ZEN-CCG AGT GTCATC CTA CCG GA-Iowa i.m. or s.c. immunized as indicated with 107 PFU equivalents Black F Quencher) (SEQID NO:16). of FI-HSV2, 12.5ug of MPL, and either 54 ug of Alhydrogel (0021 FIG. 9A to FIG.9C. Virus specific antibody and T (Alh) or 450 ug of Adju-Phos (AdP). In addition, a negative cell levels in mice immunized s.c. or i.m. with FI-HSV2 in control group received 60 lug of pVAX DNA mixed with MPL/Alhydrogel or DPBS. FIG.9A and FIG.9B. Virus spe MPL, Adju-Phos, and a volume of FI-Mock equal to the cific antibody levels in immunized mice. FIG. 9A. ELISA US 2014/0193460 A1 Jul. 10, 2014 against HSV-2 virion was used to measure prechallenge, total significances by Kruskal-Wallis and Dunn's multiple com IgG (left panel) or IgG1 and IgG2a isotypes (right panel) in parison tests are indicated by asterisks with significance lev the Sera of immunized mice. The mean endpoint reciprocal els shown in the legend. Dotted lines indicate the assay limit titers and SD are shown for each immunization group as in of detection, and the box indicates nearly complete clearance FIG. 6C. FIG. 9B. Virus neutralizing antibody titers were of virus in this immunization group on day 7. measured against HSV-2 virion as in FIG. 6D. FIG.9C. The (0029 FIG. 17. HSV-2 virion specific IgG as measured by means and standard errors of virus specific CD4+ T cells (left ELISA using dextran sulfate wash derived HSV-2. Each sym panel) and CD8+ T cells (right panel) producing IFN-y were bol represents the group mean Log 10 Endpoint titer and bars measured by intracellular cytokine staining Splenocytes from indicate SEM. Arrows show weeks of immunization (Weeks immunized and then in vivo restimulated mice (n=4) were 0 and 4) or week of challenge (Week 7). collected and assayed 3 weeks following the second injection 0030 FIG. 18. Protection against anogenital disease of as described in Materials and Methods. immunized guinea pigs following re-challenge with HSV-2. 0022 FIG. 10. Immunization groups and timeline for Each symbol represents the Summed disease score of an indi mouse experiment evaluating protective efficacy against vidual (Indiv.) guinea pig from days 0 through 14 post intra HSV-2 challenge or re-challenge. Adjuvants Adju-Phos vaginal re-challenge (given 18 weeks following the first chal (AdP) or Alhydrogel (AlH) were each tested in formulations lenge) and bars represent group means. Symbols of guinea with formalin inactivated HSV-2 (FI-HSV2) and monophos pigs that showed at least one lesion day are indicated (Les), phoryl lipid A. Vaccines were administered by intramuscular with all of the animals in the Naive group showing lesions (i.m.) or Subcutaneous (s.c.) routes in the adjuvants and routes (All Les). Statistical significances by Kruskal-Wallis and shown. Syringes indicate weeks of vaccination, the blood Dunn's multiple comparison tests are indicated by asterisks drop indicates the week of blood sampling, and dotted arrows with significance levels as follows: *P<0.05; **P<0.01; indicate weeks of lethal HSV-2 challenge. Resulting data are ***P<0.001; NS, not significant. represented by FIG. 7A to FIG.7C. 0031 FIG. 19. Vaginal HSV-2 shedding levels on days 2 0023 FIG. 11. Total virion-specific IgG, and neutraliza and 7 post re-challenge. Intravaginal Swabs were performed tion titers. and stored for quantification of infectious virus by plaque 0024 FIG. 12. Immunization groups and timeline for assay. Each symbol represents the Log PFU/swab of an indi mouse experiment evaluating the adjuvant effect of mono vidual (Indiv.) guinea pig and bars represent group means. phosphoryl lipid A (MPL) together with Alhydrogel Alum Dotted lines represent the assay limit of detection. Statistical (AlH) for intramuscular (i.m.) or subcutaneous (s.c.) immu significances by Kruskal-Wallis and Dunn's multiple com nization when formulated with formalin inactivated HSV-2 parison tests are indicated by asterisks with significance lev (FI-HSV2). Syringes indicate weeks of vaccination, the els as follows *P<0.05; **P<0.01: ***P<0.001. blood drop indicates the week of blood sampling, blue arrows 0032 FIG. 20. XYplot of the vaginal HSV-2 shedding for indicate weeks of in vivo restimulation (Week 6.5) or harvest individual (Indiv.) guinea pigs on day 2 post challenge (Week 7) for intracellular cytokine staining (ICS) assay, and (X-axis) and day 2 post re-challenge (y-axis), with values the dotted arrow indicate the week of lethal HSV-2 challenge. calculated as the Log 10 PFU per Swab. Dotted lines indicate DPBS, Dulbecco's phosphate buffered saline. Resulting data assay limits of sensitivity. are represented by FIG. 8A to FIG. 8C and FIG. 9A to FIG. 0033 FIG. 21. Experimental scheme and timeline for test 9.C. ing the efficacy of formalin inactivated HSV-2 (FI-HSV2) to 0025 FIG. 13. Experimental method for intracellular protect against the homologous (Strain G) or heterologous cytokine staining assay. Data shown corresponds to FIG. 9C, (Strain 186syn+-1 and Strain 89-390). HSV-2 strains isolated with the figure also showing the background staining levels. from the U.S. Syringes indicate weeks of vaccination, the 0026 FIG. 14. Immunization groups and timeline for blood drop indicate the week of blood sampling, and dotted guinea pig experiment evaluating protective efficacy against arrows indicate weeks of sublethal HSV-2 challenge (Week 7) challenge or rechallenge with HSV-2. Formalin inactivated or repeat (Week 9). Note that repeat challenge was performed (FI-)HSV-2 or Mock virion (FI-Mock) were formulated with only on animals that had no detectable HSV-2 from intrav monophosphoryl lipid A and Alhydrogel and then adminis aginal swabs on day 2 postchallenge. PFU eq., PFU equiva tered by intramuscular (i.m.) or Subcutaneous (s.c.) routes. lents; MPL, monophosphoryl lipid A. Alum, Alhydrogel; Syringes indicate weeks of vaccination, blood drops indicate High, 5x10 PFU challenge: Low, 2.5x10 PFU challenge: weeks of blood sampling, and dotted arrows indicate weeks FI-Mock, formalin inactivated mock virion preparation; s.c., of sublethal HSV-2 challenge. PFU equiv., PFU equivalents. Subcutaneous 0027 FIG. 15. Protection against anogenital disease of 0034 FIG. 22. Protection against anogenital disease of immunized guinea pigs during primary (acute phase) HSV-2 immunized guinea pigs, with challenge and repeat challenge infection. Each symbol represents the Summed disease score data combined. Each symbol represents the Summed disease of an individual (Indiv.) guinea pig from days 0 through 14 score of an individual (Indiv.) guinea pig from days 0 through post intravaginal challenge and bars represent group means. 14 post intravaginal challenge and bars represent group Symbols for the guinea pigs that showed at least one lesion means. Disease scoring criteria are listed in the legend. Sym day are bracketed. Statistical significances by Kruskal-Wallis bols of guinea pigs that showed at least one lesion day are and Dunn's multiple comparison tests are indicated by aster bracketed. Statistical significances by Kruskal-Wallis and isks with significance levels shown in the legend. Dunn's multiple comparison tests are indicated by asterisks 0028 FIG. 16. Vaginal HSV-2 shedding levels on days 2 with significance levels indicated in the legend. No FI-HSV2 and 7 postchallenge. Intravaginal Swabs were performed and immunized animals developed lesions. stored for quantification of infectious virus by plaque assay. 0035 FIG. 23. Vaginal HSV-2 shedding levels on days 2 Each symbol represents the Log PFU/swab of an individual and 7 post challenge with the homologous (Strain G) or with (Indiv.) guinea pig and bars represent group means. Statistical heterologous (186syn+-1 and 89-390) strains of HSV-2. US 2014/0193460 A1 Jul. 10, 2014

Intravaginal Swabs were performed and stored for quantifica refers to any indicia of Success in protecting a Subject or tion of infectious virus by plaque assay. Each symbol repre patient (e.g. a subject or patient at risk of developing a disease sents the Log PFU/swab of an individual (Indiv.) guinea pig or condition) from developing, contracting, or having a dis and bars represent group means. Dotted lines represent the ease or condition (e.g. HV (e.g. an HV selected from the assay limit of detection. Boxes indicate nearly complete virus group consisting of HHV-1, HHV-2, HHV-3, HHV-4, HHV clearance by day 7. Statistical significances by Kruskal-Wal 5, HHV-6, HHV-7, and HHV-8) infection), including prevent lis and Dunn's multiple comparison tests with significance ing one or more symptoms of a disease or condition or dimin levels indicated in the legend. ishing the occurrence, severity, or duration of any symptoms of a disease or condition following administration of a pro DETAILED DESCRIPTION OF THE INVENTION phylactic or preventative composition as described herein. 0040. An "effective amount” is an amount sufficient for a I. Definitions compound to accomplish a stated purpose relative to the 0036. The abbreviations used herein have their conven absence of the compound (e.g. achieve the effect for which it tional meaning within the chemical and biological arts. The is administered, treat a disease, reduce spread of HV (e.g. an chemical structures and formulae set forth herein are con HV selected from the group consisting of HHV-1, HHV-2, structed according to the standard rules of chemical Valency HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, and HHV-8), known in the chemical arts. reduce one or more symptoms of a disease or condition (e.g. 0037. The terms “a” or “an,” as used in herein means one lesions, virus production, lytic cycle)). An example of an or more. In addition, the phrase “substituted with an as “effective amount' is an amount sufficient to contribute to the used herein, means the specified group may be substituted treatment, prevention, or reduction of a symptom or symp with one or more of any or all of the named substituents. toms of a disease, which could also be referred to as a “thera 0038. Description of compounds of the present invention peutically effective amount.” A “reduction of a symptom or is limited by principles of chemical bonding known to those symptoms (and grammatical equivalents of this phrase) skilled in the art. Accordingly, where a group may be substi means decreasing of the severity or frequency of the symptom tuted by one or more of a number of substituents, such sub (S), or elimination of the symptom(s). A "prophylactically stitutions are selected so as to comply with principles of effective amount of a composition (vaccine) is an amount of chemical bonding and to give compounds which are not a composition that, when administered to a subject, will have inherently unstable and/or would be known to one of ordinary the intended prophylactic effect, e.g., preventing or delaying skill in the art as likely to be unstable under ambient condi the onset (or reoccurrence) of an injury, disease (e.g. HV (e.g. tions, such as aqueous, neutral, and several known physi an HV selected from the group consisting of HHV-1, HHV-2, ological conditions. HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, and HHV-8) infec 0039. The terms “treating” or “treatment” refers to any tion), pathology or condition, or reducing the likelihood of the indicia of Success in the treatment or amelioration of an onset (or reoccurrence) of an injury, disease, pathology, or injury, disease, pathology or condition, including any objec condition, or their symptoms. The full prophylactic effect tive or Subjective parameter Such as abatement; remission; does not necessarily occur by administration of one dose, and diminishing of symptoms or making the injury, pathology or may occur only after administration of a series of doses (e.g. condition more tolerable to the patient; slowing in the rate of prime-boost). Thus, a prophylactically effective amount may degeneration or decline; making the final point of degenera be administered in one or more administrations. The exact tion less debilitating; improving a patient's physical or men amounts will depend on the purpose of the treatment, and will tal well-being. The treatment or amelioration of symptoms be ascertainable by one skilled in the art using known tech can be based on objective or subjective parameters; including niques (see, e.g., Lieberman, Pharmaceutical Dosage Forms the results of a physical examination, neuropsychiatric (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of exams, and/or a psychiatric evaluation. For example, the cer Pharmaceutical Compounding (1999); Pickar, Dosage Cal tain methods presented herein Successfully treat HV (e.g. an culations (1999); and Remington. The Science and Practice HV selected from the group consisting of HHV-1, HHV-2, of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, and HHV-8) infec Williams & Wilkins). tion by decreasing the incidence of HV (e.g. an HV selected 0041) “Control or “control experiment' is used in accor from the group consisting of HHV-1, HHV-2, HHV-3, HHV dance with its plain ordinary meaning and refers to an experi 4, HHV-5, HHV-6, HHV-7, and HHV-8) infection, reducing ment in which the Subjects or reagents of the experiment are one or more symptoms of HV (e.g. an HV selected from the treated as in a parallel experiment except for omission of a group consisting of HHV-1, HHV-2, HHV-3, HHV-4, HHV procedure, reagent, or variable of the experiment. In some 5, HHV-6, HHV-7, and HHV-8) infection, or preventing the instances, the control is used as a standard of comparison in spread of HV (e.g. an HV selected from the group consisting evaluating experimental effects. In some embodiments, a of HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, control is the measurement of HV (e.g. an HV selected from and HHV-8). In some embodiments of the compositions or the group consisting of HHV-1, HHV-2, HHV-3, HHV-4, methods described herein, treating HV (e.g. an HV selected HHV-5, HHV-6, HHV-7, and HHV-8) infection or one or from the group consisting of HHV-1, HHV-2, HHV-3, HHV more symptoms of HV (e.g. an HV selected from the group 4, HHV-5, HHV-6, HHV-7, and HHV-8) infection includes consisting of HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, slowing the rate of growth or spread of HV (e.g. an HV HHV-6, HHV-7, and HHV-8) infection in the absence of a selected from the group consisting of HHV-1, HHV-2, HHV composition (e.g. Vaccine) as described herein (including 3, HHV-4, HHV-5, HHV-6, HHV-7, and HHV-8) or reducing embodiments). the occurrence of lesions. The term “treating and conjuga 0042 “Contacting is used in accordance with its plain tions thereof, include prevention of an injury, pathology, con ordinary meaning and refers to the process of allowing at least dition, or disease. The term “preventing or “prevention' two distinct species (e.g. compositions, vaccines, virus, bio US 2014/0193460 A1 Jul. 10, 2014 molecules, or cells) to become Sufficiently proximal to react, in need thereof, refers to a living organism (e.g. human) at risk interact or physically touch. It should be appreciated; how of developing, contracting, or having a disease or condition ever, the resulting reaction product can be produced directly (e.g. HV (e.g. an HV Selected from the group consisting of from a reaction between the added reagents or from an inter HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, mediate from one or more of the added reagents which can be and HHV-8) infection or disease associated with an HV (e.g. produced in the reaction mixture. In some embodiments, an HV selected from the group consisting of HHV-1, HHV-2, contacting refers to allowing radiation (e.g. UV, gamma) to HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, and HHV-8)). interact with matter (e.g. virus, virus component, virus pro 0048 “Disease' or “condition” refer to a state of being or tein, virus nucleic acid). health status of a patient or Subject capable of being treated 0043. The term “contacting may include allowing two with the compositions (e.g. Vaccines) or methods provided species to react, interact, or physically touch, wherein the two herein. In some embodiments, the disease is a disease related species may be a composition (e.g. Vaccine) as described to (e.g. caused by) HV (e.g. an HV selected from the group herein and a cell, virus, virus particle, protein, enzyme, or consisting of HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, patient. In some embodiments contacting includes allowing a HHV-6, HHV-7, and HHV-8). Examples of diseases, disor composition described herein to interact with a protein or ders, or conditions include, but are not limited to herpetic enzyme that is involved in a signaling pathway. In some gingivostomatitis, herpes labialis, herpes genitalis, herpetic embodiments contacting includes allowing a composition whitlow, herpes gladiatorum, herpesviral encephalitis, herp described herein to interact with an HV (e.g. an HV selected esviral meningitis, herpes esophagitis, herpes keratitis, Bell's from the group consisting of HHV-1, HHV-2, HHV-3, HHV palsy, Mollaret's meningitis, herpes rugbeiorum, eczema her 4, HHV-5, HHV-6, HHV-7, and HHV-8). In some embodi peticum, herpetic neuralgia, or post-herpetic neuralgia. In ments contacting includes allowing an agent described herein some instances, “disease' or “condition” refers to HV (e.g. an to interact with an HV (e.g. an HV selected from the group HV selected from the group consisting of HHV-1, HHV-2, consisting of HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, and HHV-8) infec HHV-6, HHV-7, and HHV-8) and inactivate the HV (e.g. an tion. In some instances, “disease' or “condition” refers to HV selected from the group consisting of HHV-1, HHV-2, herpetic gingivostomatitis. In some instances, “disease' or HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, and HHV-8). “condition” refers to herpes labialis. In some instances, "dis 0044 As defined herein, the term “inhibition”, “inhibit, ease' or “condition” refers to herpes genitalis. In some “inhibiting and the like in reference to a protein-inhibitor or instances, “disease' or “condition” refers to herpetic whitlow. interaction means negatively affecting (e.g. decreasing) the In some instances, “disease' or “condition” refers to herpes activity or function of the protein. In some embodiments gladiatorum. In some instances, “disease' or “condition” inhibition refers to reduction of a disease or symptoms of refers to herpesviral encephalitis. In some instances, "dis disease. In some embodiments inhibition refers to reduction ease' or “condition” refers to herpesviral meningitis. In some of the growth, proliferation, or spread of a virus (e.g. HV (e.g. instances, “disease' or “condition” refers to herpes esophagi an HV selected from the group consisting of HHV-1, HHV-2, tis. In some instances, “disease' or “condition” refers to her HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, and HHV-8)). In pes keratitis. In some instances, “disease' or “condition some embodiments inhibition refers to preventing the infec refers to Bell's palsy. In some instances, “disease' or “con tion of a subject by a virus (e.g. HV (e.g. an HV selected from dition” refers to Mollaret's meningitis. In some instances, the group consisting of HHV-1, HHV-2, HHV-3, HHV-4, “disease' or “condition” refers to herpes rugbeiorum. In some HHV-5, HHV-6, HHV-7, and HHV-8)). In some embodi instances, “disease' or “condition” refers to eczema herpeti ments, inhibition refers to a reduction in the activity of a cum. In some instances, “disease' or “condition” refers to signal transduction pathway or signaling pathway. Thus, inhi herpetic neuralgia. In some instances, “disease' or “condi bition includes, at least in part, partially or totally blocking tion” refers to post-herpetic neuralgia. In some instances, stimulation, decreasing, preventing, or delaying activation, or “disease' or “condition” refers to HSV1 infection. In some inactivating, desensitizing, or down-regulating the signaling instances, “disease' or “condition” refers to HSV2 infection. pathway or enzymatic activity or the amount of a protein. In some instances, “disease' or “condition” refers to HSV1 0045. The term “modulator” refers to a composition that and HSV2 infection. In some instances, “disease' or “condi increases or decreases the level of a target (e.g. molecule, cell, tion” refers to HHV-3 infection. In some instances, “disease' virus particle, protein) or the function of a target or the physi or “condition” refers to HHV-4 infection. In some instances, cal state of the target. “disease' or “condition” refers to HHV-5 infection. In some 0046. The term “modulate” is used in accordance with its instances, “disease' or “condition” refers to HHV-6 infection. plain ordinary meaning and refers to the act of changing or In some instances, “disease' or “condition” refers to HHV-7 varying one or more properties. “Modulation” refers to the infection. In some instances, “disease' or “condition” refers process of changing or varying one or more properties. For to HHV-8 infection. In some instances, “disease' or “condi example, as applied to the effects of a modulator on a target, tion” refers to an HV (e.g. HHV-1, HHV-2, HHV-3, HHV-4, to modulate means to change by increasing or decreasing a HHV-5, HHV-6, HHV-7, or HHV-8) infection. property or function of the target or the amount of the target. 0049. “Pharmaceutically acceptable excipient' and “phar 0047. “Patient” or “subject in need thereof refers to a maceutically acceptable carrier” refer to a substance that aids living organism Suffering from or prone to a disease or con the administration of an active agent to and absorption by a dition that can be treated by administration of a pharmaceu Subject and can be included in the compositions of the present tical composition as provided herein. Non-limiting examples invention without causing a significant adverse toxicological include humans, other mammals, bovines, rats, mice, dogs, effect on the patient. Non-limiting examples of pharmaceu monkeys, goat, sheep, cows, deer, and other non-mammalian tically acceptable excipients include water, NaCl, normal animals. In some embodiments, a patient is human. In some saline solutions, lactated Ringers, normal Sucrose, normal embodiments, a patient or subject in need thereof or a patient glucose, binders, fillers, disintegrants, lubricants, coatings, US 2014/0193460 A1 Jul. 10, 2014

Sweeteners, flavors, salt solutions (such as Ringer's Solution), to induce an immune response in the Subject or to reduce one alcohols, oils, gelatins, carbohydrates such as lactose, amy or more symptoms of a disease (e.g. HHV-1, HHV-2, HHV-3, lose or starch, fatty acid esters, hydroxymethycellulose, poly HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8 infection). vinyl pyrrolidine, and colors, and the like. Such preparations 0053. It should be noted that throughout the application can be sterilized and, if desired, mixed with auxiliary agents that alternatives are written in Markush groups, for example, Such as lubricants, preservatives, stabilizers, wetting agents, each amino acid position that contains more than one possible emulsifiers, salts for influencing osmotic pressure, buffers, amino acid. It is specifically contemplated that each member coloring, and/or aromatic Substances and the like that do not of the Markush group should be considered separately, deleteriously react with the compounds of the invention. One thereby comprising another embodiment, and the Markush of skill in the art will recognize that other pharmaceutical group is not to be read as a single unit. excipients are useful in the present invention. 0054) The terms “peptide,” “polypeptide,” and “protein' 0050. The term “preparation” is intended to include the are used interchangeably hereinto refer to a polymer of amino formulation of the active compound with encapsulating mate acid residues. rial as a carrier providing a capsule in which the active com 0055. The term “peptidyl and “peptidyl moiety” means a ponent with or without other carriers, is surrounded by a monovalent peptide. carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, 0056. The term “amino acid refers to naturally occurring cachets, and lozenges can be used as Solid dosage forms and synthetic amino acids, as well as amino acid analogs. suitable for oral administration. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later 0051. As used herein, the term “administering means oral modified, e.g., hydroxyproline, Y-carboxyglutamate, and administration, administration as a Suppository, topical con O-phosphoserine. Amino acid analogs refers to compounds tact, intravenous, intraperitoneal, intramuscular, intrale that have the same basic chemical structure as a naturally sional, intrathecal, intranasal, intradermal, mucosal, intrarec occurring amino acid, i.e., an O-carbon that is bound to a tal, intravaginal, topical, transcutaneous (e.g. as in hydrogen, a carboxyl group, an amino group, and an R group, Combadiere, PLoS ONE 5(5): e10818), or subcutaneous e.g., homoserine, norleucine, methionine Sulfoxide, methion administration, or the implantation of a slow-release device, ine methyl Sulfonium. Such analogs have modified R groups e.g., a mini-osmotic pump, to a Subject. Administration is by (e.g., norleucine) or modified peptide backbones, but retain any route, including parenteral and transmucosal (e.g., buc the same basic chemical structure as a naturally occurring cal, Sublingual, palatal, gingival, nasal, Vaginal, rectal, or amino acid. Amino acid mimetics refers to chemical com transdermal). Parenteral administration includes, e.g., intra pounds that have a structure that is different from the general venous, intramuscular, intra-arteriole, intradermal, Subcuta chemical structure of an amino acid, but that functions in a neous, intraperitoneal, intraventricular, and intracranial. manner similar to a naturally occurring amino acid. An oli Other modes of delivery include, but are not limited to, the use gomer comprising amino acid mimetics is a peptidomimetic. of liposomal formulations, intravenous infusion, transdermal patches, etc. By “co-administer it is meant that a composi A peptidomimetic moiety is a monovalent peptidomimetic. tion described herein is administered at the same time, just 0057 Amino acids may be referred to herein by either prior to, or just after the administration of one or more addi their commonly known three letter symbols or by the one tional therapies, for example HV (e.g. HHV-1, HHV-2, HHV letter symbols recommended by the IUPAC-IUB Biochemi 3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) infection cal Nomenclature Commission. Nucleotides, likewise, may therapies such as antiviral drugs (e.g. acyclovir, famciclovir, be referred to by their commonly accepted single-letter codes. valacyclovir) or a different HV (e.g. HHV-1, HHV-2, HHV-3, 0.058 An amino acid or nucleotide base “position' is HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vaccine (e.g. denoted by a number that sequentially identifies each amino DNA vaccine, DNA vaccine including different genes, iso acid (or nucleotide base) in the reference sequence based on lated protein vaccine, different inactivated virus vaccine). its position relative to the N-terminus (or 5'-end). Due to The compositions (e.g. Vaccines) of the invention can be deletions, insertions, truncations, fusions, and the like that administered alone or can be coadministered to the patient. must be taken into account when determining an optimal Coadministration is meant to include simultaneous or alignment, in general the amino acid residue number in a test sequential administration of the compounds individually or in sequence determined by simply counting from the N-termi combination (more than one composition) and includes vac nus will not necessarily be the same as the number of its cine administrationina prime-boost method. Thus, the prepa corresponding position in the reference sequence. For rations can also be combined, when desired, with other active example, in a case where a variant has a deletion relative to an Substances (e.g. to reduce metabolic degradation, increase aligned reference sequence, there will be no amino acid in the immune response (e.g. adjuvant)). The compositions of the variant that corresponds to a position in the reference present invention can be delivered by transdermally, by a sequence at the site of deletion. Where there is an insertion in topical route, transcutaneously, formulated as applicator an aligned reference sequence, that insertion will not corre Sticks, Solutions, Suspensions, emulsions, gels, creams, oint spond to a numbered amino acid position in the reference ments, pastes, jellies, paints, powders, and aerosols. sequence. In the case of truncations or fusions there can be 0052. The term “administer (or administering) an HV vac stretches of amino acids in either the reference or aligned cine” means administering a composition that prevents or sequence that do not correspond to any amino acid in the treats an HV (e.g. HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, corresponding sequence. HHV-6, HHV-7, or HHV-8) infection in a subject. Adminis 0059. The terms “numbered with reference to or “corre tration may include, without being limited by mechanism, sponding to when used in the context of the numbering of a allowing sufficient time for the HV (e.g. HHV-1, HHV-2, given amino acid or polynucleotide sequence, refers to the HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vaccine numbering of the residues of a specified reference sequence US 2014/0193460 A1 Jul. 10, 2014 when the given amino acid or polynucleotide sequence is length. Nucleic acids and polynucleotides are a polymers of compared to the reference sequence. any length, including longer lengths, e.g., 200, 300, 500, 0060 A “conservative substitution' as used with respect 1000, 2000, 3000, 5000, 7000, 10,000, etc. In certain embodi to amino acids, refers to the Substitution of an amino acid with ments, the nucleic acids herein contain phosphodiester bonds. a chemically similar amino acid. Amino acid Substitutions In other embodiments, nucleic acid analogs are included that which often preserve the structural and/or functional proper may have alternate backbones, comprising, e.g., phosphora ties of the polypeptide in which the substitution is made are midate, phosphorothioate, phosphorodithioate, or O-meth known in the art and are described, for example, by H. ylphosphoroamidite linkages (see Eckstein, Oligonucle Neurath and R. L. Hill, 1979, in “The Proteins. Academic otides and Analogues: A Practical Approach, Oxford Press, New York. The most commonly occurring exchanges University Press); and peptide nucleic acid backbones and are isoleucine/valine, tyrosine? phenylalanine, aspartic acid/ linkages. Other analog nucleic acids include those with posi glutamic acid, lysinefarginine, methionine/leucine, aspartic tive backbones; non-ionic backbones, and non-ribose back acid/asparagine, glutamic acid/glutamine, leucine/isoleu bones, including those described in U.S. Pat. Nos. 5.235,033 cine, methioninetisoleucine, threonine?serine, tryptophan/ and 5,034,506, and Chapters 6 and 7, ASC Symposium Series phenylalanine, tyrosine? histidine, tyrosine?tryptophan, 580, Carbohydrate Modifications in Antisense Research, glutamine/arginine, histidine/asparagine, histidine/ Sanghui & Cook, eds. Nucleic acids containing one or more glutamine, lysine?.asparagine, lysine? glutamine, lysine? carbocyclic Sugars are also included within one definition of glutamic acid, phenylalanine/leucine, phenylalanine/me nucleic acids. Modifications of the ribose-phosphate back thionine, serine/alanine, serine/asparagine, Valine/leucine, bone may be done for a variety of reasons, e.g., to increase the and Valine/methionine. The following eight groups each con stability and half-life of such molecules in physiological envi tain amino acids that are conservative Substitutions for one ronments or as probes on a biochip. Mixtures of naturally another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), occurring nucleic acids and analogs can be made; alterna Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) tively, mixtures of different nucleic acid analogs, and mix Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), tures of naturally occurring nucleic acids and analogs may be Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine made. (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) 0065. A particular nucleic acid sequence also encom Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins passes 'splice variants. Similarly, a particular protein (1984)). In some embodiments, there may beat least 1, at least encoded by a nucleic acid encompasses any protein encoded 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least by a splice variant of that nucleic acid. “Splice variants, as 8, at least 9, at least 10, at least 15, at least 20, at least 25, at the name Suggests, are products of alternative splicing of a least 30, at least 35, or at least 40 conservative substitutions. gene. After transcription, an initial nucleic acid transcript In some embodiments, there may be 1,2,3,4,5,6,7,8,9, 10. may be spliced such that different (alternate) nucleic acid 15, 20, 25, 30, 35, or 40 conservative substitutions. splice products encode different polypeptides. Mechanisms 0061. The term “amino acid substitution set or “substitu for the production of splice variants vary, but include alternate tion set' refers to a group of amino acid substitutions. A splicing of exons. Alternate polypeptides derived from the substitution set can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, same nucleic acid by read-through transcription are also 14, 15, or more amino acid Substitutions. encompassed by this definition. Any products of a splicing 0062. The term "isolated refers to a nucleic acid, poly reaction, including recombinant forms of the splice products, nucleotide, polypeptide, protein, or other component that is are included in this definition. partially or completely separated from components with 0.066 Nucleic acid is “operably linked when it is placed which it is normally associated (other proteins, nucleic acids, into a functional relationship with another nucleic acid cells, etc.). In some embodiments, an isolated polypeptide or sequence. For example, DNA for a presequence or secretory protein is a recombinant polypeptide or protein. leader is operably linked to DNA for a polypeptide if it is 0063 A nucleic acid (such as a polynucleotide), a expressed as a preprotein that participates in the Secretion of polypeptide, or a cell is “recombinant' when it is artificial or the polypeptide; a promoter or enhancer is operably linked to engineered, or derived from or contains an artificial or engi a coding sequence if it affects the transcription of the neered protein or nucleic acid (e.g. non-natural or not wild sequence; or a ribosome binding site is operably linked to a type). For example, a polynucleotide that is inserted into a coding sequence if it is positioned so as to facilitate transla vector or any otherheterologous location, e.g., in a genome of tion. Generally, “operably linked' means that the DNA a recombinant organism, Such that it is not associated with sequences being linked are near each other, and, in the case of nucleotide sequences that normally flank the polynucleotide a secretory leader, contiguous and in reading phase. However, as it is found in nature is a recombinant polynucleotide. A enhancers do not have to be contiguous. Linking is accom protein expressed in vitro or in vivo from a recombinant plished by ligation at convenient restriction sites. If such sites polynucleotide is an example of a recombinant polypeptide. do not exist, then synthetic oligonucleotide adaptors or link Likewise, a polynucleotide sequence that does not appear in ers are used in accordance with conventional practice. nature, for example a variant of a naturally occurring gene, is 0067. “Identity” or “percent identity,” in the context of two recombinant. or more polypeptide sequences, refers to two or more 0064 “Nucleic acid' or "oligonucleotide' or “polynucle sequences or Subsequences that are the same or have a speci otide' or grammatical equivalents used herein means at least fied percentage of amino acid residues that are the same (e.g., two nucleotides covalently linked together. The term “nucleic share at least about 70%, at least about 75%, at least about acid' includes single-, double-, or multiple-stranded DNA, 80%, at least about 85%, at least about 88% identity, at least RNA and analogs (derivatives) thereof. Oligonucleotides are about 89%, at least about 90%, at least about 91%, at least typically from about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50 or about 92%, at least about 93%, at least about 94%, at least more nucleotides in length, up to about 100 nucleotides in about 95%, at least about 96%, at least about 97%, at least US 2014/0193460 A1 Jul. 10, 2014

about 98%, or at least about 99% identity) over a specified tion; size and tolerance of the individual; severity of the region to a reference sequence, when compared and aligned condition; risk of side effects; and the route of administration. for maximum correspondence over a comparison window, or One of skill will recognize that the dose can be modified designated region as measured using a sequence comparison depending on the above factors or based on therapeutic algorithms or by manual alignment and visual inspection. progress. The term “dosage form” refers to the particular 0068. Optimal alignment of sequences for comparison format of the pharmaceutical orpharmaceutical composition, and determination of sequence identity can be determined by and depends on the route of administration. For example, a a sequence comparison algorithm or by visual inspection dosage form can be in a liquid form for nebulization, e.g., for (see, generally, Ausubel et al., infra). When optimally align inhalants, in a tablet or liquid, e.g., for oral delivery, or a saline ing sequences and determining sequence identity by visual Solution, e.g., for injection. inspection, percent sequence identity is calculated as the (0073. The term “herpesvirus' or “herpes virus' or “HV” number of residues of the test sequence that are identical to refers to human herpesviruses and may be used, depending on the reference sequence divided by the number of non-gap the context, to refer to one, more, or all of the human herpes positions and multiplied by 100. When using a sequence viruses, including Human Herpesvirus-1 (HHV-1. Herpes comparison algorithm, test and reference sequences are Simplex Virus-1, HSV1, HSV-1), HHV-2 (Herpes Simplex entered into a computer, Subsequence coordinates and Virus-2, HSV2, HSV-2), HHV-3 (Varicella Zoster Virus, sequence algorithm program parameters are designated. The VZV), HHV-4 (Epstein-Barr Virus, EBV), HHV-5 (Cytome sequence comparison algorithm then calculates the percent galovirus, CMV, HCMV), HHV-6, HHV-7, HHV-8 (Kaposi's sequence identities for the test sequences relative to the ref Sarcoma-associated Herpesvirus, KSHV). In some embodi erence sequence, based on the program parameters as known ments, a herpesvirus is HHV-1. In some embodiments, a in the art, for example BLAST or BLAST 2.0. For example, herpesvirus is HHV-2. In some embodiments, aherpesvirus is comparison can be conducted, e.g., by the local homology HHV-3. In some embodiments, a herpesvirus is HHV-4. In algorithm of Smith & Waterman, 1981, Adv. Appl. Math. some embodiments, a herpesvirus is HHV-5. In some 2:482, by the homology alignment algorithm of Needleman embodiments, a herpesvirus is HHV-6. In some embodi & Wunsch, 1970, J. Mol. Biol. 48:443, by the search for ments, a herpesvirus is HHV-7. In some embodiments, a similarity method of Pearson & Lipman, 1988, Proc. Natl. herpesvirus is HHV-8. In some embodiments, each of the Acad. Sci. USA 85:2444, or by computerized implementa terms HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, tions of these algorithms (GAP BESTFIT. FASTA, and HHV-7, and HHV-8 may refer to all strains of each respective TFASTA in the Wisconsin Genetics Software Package, HHV. In some embodiments, each of the terms HHV-1, HHV Genetics Computer Group, 575 Science Dr. Madison, Wis.). 2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, and HHV-8 may Thus alignment can be carried out for sequences that have refer to a single strain of that HHV. In some embodiments, deletions and/or additions, as well as those that have substi each of the terms HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, tutions, as well as naturally occurring, e.g., polymorphic or HHV-6, HHV-7, and HHV-8 may include mutants of that allelic variants, and man-made variants. particular HHV. In some embodiments HV is HSV (HHV-1 0069. The phrase “substantial sequence identity” or “sub and/or HHV-2). stantial identity, in the context of two polypeptide sequences, (0074 The terms “Herpes Simplex Virus' and “HSV are refers to a sequence that has at least 70% identity to a refer used according to their common meaning and refer to the ence sequence. Percent identity can be any integer from 70% double stranded DNA viruses Herpes Simplex Virus 1 to 100%. Two polypeptide sequences that have 100% (HSV1, HSV-1, HHV-1) and Herpes Simplex Virus 2 (HSV2, sequence identity are said to be “identical.” A polypeptide HSV-2, HHV-2). In some embodiments, HSV includes all sequence is said to have “substantial sequence identity” to a strains of HSV1 and HSV2. In some embodiments, HSV may reference sequence when the sequences have at least about refer to a single strain of HSV1 or HSV2. In some embodi 70%, at least about 75%, at least 80%, at least 85%, at least ments, HSV includes multiple strains of HSV1 and/or HSV2. 90%, at least 91%, at least 92%, at least 93%, at least 94%, at In some embodiments, HSV includes mutants of HSV1 and/ least 95%, at least 96%, at least 97%, at least 98%, or at least or HSV2. The terms “HSV1 and “HSV2 are used according 99% or greater sequence identity as determined using the to their plain ordinary meaning. In some embodiments, HSV1 methods described herein, such as BLAST using standard includes all strains or HSV1. In some embodiments, HSV1 parameters as described above. includes mutants of HSV1. In some embodiments, HSV2 0070 The term “expression' includes any step involved in includes all strains or HSV2. In some embodiments, HSV2 the production of the polypeptide including, but not limited includes mutants of HSV2. to, transcription, post-transcriptional modification, transla 0075. The terms “inactivate”, “inactivating, and other tion, post-translational modification, and secretion. verb forms thereof, when describing a virus or the process of 0071. An amino acid or peptide is "heterologous' to modifying a virus, refer to a virus that is incapable of estab another sequence with which it is operably linked if the two lishing a viable infection in a human, replicating, and gener sequences are not associated in nature. ating virus particles capable of infecting a second human, and 0072 The terms “dose' and “dosage' are used inter the process of generating Such a virus. In some embodiments, changeably herein. A dose refers to the amount of active an inactivated virus comprises a mutation that prevents the ingredient given to an individual at each administration. For virus from replicating. In some embodiments, an inactivated the present methods and compositions provided herein, the virus comprises a mutation that prevents the virus from dose may generally refer to the amount of disease (e.g. HV infecting a Subject (e.g. human). In some embodiments, an (e.g. HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, inactivated virus comprises a mutation that prevents the virus HHV-7, or HHV-8) infection) treatment. The dose will vary from infecting a cell (e.g. human cell). In some embodiments, depending on a number of factors, including the range of inactivating a virus includes contacting the virus with an normal doses for a given therapy, frequency of administra agent that causes the virus to become incapable of establish US 2014/0193460 A1 Jul. 10, 2014

ing a viable infection in a human and generating virus par aluminum hydroxide. In some embodiments, an aluminum ticles capable of infecting a second human. In some embodi based mineral salt adjuvant is aluminum hydroxide. In some ments, inactivating a virus includes contacting the virus with embodiments, an aluminum-based mineral Salt adjuvant a cross-linking agent. In some embodiments, inactivating a includes aluminum phosphate. In some embodiments, an alu virus includes contacting the virus with an oxidizing agent. In minum-based mineral salt adjuvant is aluminum phosphate. Some embodiments, inactivating a virus includes contacting In some embodiments, an aluminum-based mineral salt adju the virus with a reducing agent. In some embodiments, inac vant includes potassium aluminum Sulfate. In some embodi tivating a virus includes contacting the virus with heat. In ments, an aluminum-based mineral salt adjuvant is potassium Some embodiments, inactivating a virus includes contacting the virus with radiation. In some embodiments, inactivating a aluminum sulfate. In some embodiments, an aluminum virus includes contacting the virus with a detergent. In some based mineral salt adjuvant is aluminum hydroxide adjuvant. embodiments, inactivating a virus includes contacting the In some embodiments, an aluminum-based mineral salt adju virus with a pH changing agent. In some embodiments, inac vant is aluminum phosphate adjuvant. In some embodiments, tivating a virus includes contacting the virus with a chemical an aluminum-based mineral salt adjuvant is potassium alu agent. In some embodiments, inactivating a virus includes minum sulfate adjuvant. In some embodiments, an alumi contacting the virus with a furocoumarin. In some embodi num-based mineral salt adjuvant is Alum. In some embodi ments, inactivating a virus includes contacting the virus with ments, an aluminum-based mineral salt adjuvant is CAS no. aziridine. In some embodiments, inactivating a virus includes 21645-51-2. In some embodiments, an aluminum-based min contacting the virus with ethylenimine. In some embodi eral salt adjuvant is aluminum hydroxide gel. In some ments, inactivating a virus includes contacting the virus with embodiments, an aluminum-based mineral salt adjuvant is a binary ethylenimine. In some embodiments, inactivating a aluminum hydroxide gel in the form of a white gelatinous virus includes contacting the virus with beta-propiolactone. precipitate. In some embodiments, an aluminum-based min eral salt adjuvant is CAS no. 7784-30-7. In some embodi 0076. The terms "lipopolysaccharide-derived adjuvant” ments, an aluminum-based mineral salt adjuvant is aluminum and “LPS-derived adjuvant” refer to lipopolysaccharides or phosphate gel. In some embodiments, an aluminum-based molecules derived from lipopolysaccharides commonly mineral salt adjuvant is aluminum phosphate gel in the form employed as part of a vaccine formulation. The LPS-derived of a white gelatinous precipitate. In some embodiments, an adjuvant may be designed to increase the immune response of aluminum-based mineral salt adjuvant is not Imject Alum a subject when administered to the Subject as a component of AdjuvantTM. In some embodiments, an aluminum-based min a vaccine or method of vaccination relative to the absence of eral salt adjuvant is aluminum hydroxide without magnesium the lipopolysaccharide molecule. In some embodiments, an hydroxide. In some embodiments, an aluminum-based min LPS-derived adjuvant includes a portion of an LPS. In some eral salt adjuvant is AlhydrogelTM. In some embodiments, an embodiments, an LPS-derived adjuvant includes an LPS. In aluminum-based mineral salt adjuvant is Adju-phosTM In some embodiments, an LPS-derived adjuvant includes a Some embodiments, an aluminum-based mineral salt adju modified portion of an LPS. In some embodiments, an LPS vant is AdjuphosTM. In some embodiments, an aluminum derived adjuvant includes a component that is a portion of an based mineral salt adjuvant is amorphous aluminum hydrox LPS and another component that is not a portion of an LPS. In ide and not crystalline aluminum hydroxide. In some some embodiments, an LPS-derived adjuvant includes a embodiments, an aluminum-based mineral Salt adjuvant component that corresponds to a portion of an LPS, but which includes amorphous aluminum and not crystalline aluminum. is chemically synthesized. The term "Lipopolysaccharides’ In some embodiments, an aluminum-based mineral salt adju and “LPS are used according to their plain meaning in Biol vant is crystalline aluminum hydroxide and not amorphous ogy, Biochemistry, and Immunology and refer to molecules aluminum hydroxide. In some embodiments, an aluminum comprising one or more lipids and one or more polysaccha based mineral salt adjuvant includes crystalline aluminum rides covalently bonded together. In some embodiments, LPS and not amorphous aluminum. In some embodiments, an are components of the outer membrane of Gram-negative aluminum-based mineral salt adjuvant includes crystalline bacteria. In some embodiments, and LPS-derived adjuvant is aluminum oxyhydroxide. In some embodiments, an alumi a monophorphoryl lipid A (MPL) adjuvant. num-based mineral salt adjuvant is crystalline aluminum 0077. The term “adjuvant” is used in accordance with its oxyhydroxide. In some embodiments, an aluminum-based plain ordinary meaning within Immunology and refers to a mineral salt adjuvant includes amorphous aluminum Substance that is commonly used as a component of a vaccine. hydroxyphosphate. In some embodiments, an aluminum Adjuvants may increase an antigen specific immune response based mineral salt adjuvant is amorphous aluminum in a subject when administered to the subject with one or more hydroxyphosphate. In some embodiments, an aluminum specific antigens as part of a vaccine. In some embodiments, based mineral salt adjuvant includes aluminum oxyhydroxide an adjuvant accelerates an immune response to an antigen. In and not aluminum hydroxycarbonate. In some embodiments, Some embodiments, an adjuvant prolongs an immune an aluminum-based mineral salt adjuvant is aluminum oxy response to an antigen. In some embodiments, an adjuvant hydroxide and not aluminum hydroxycarbonate. In some enhances an immune response to an antigen. In some embodi embodiments, an aluminum-based mineral Salt adjuvant ments, an adjuvant is selected from the group consisting of includes aluminum oxyhydroxide and not magnesium aluminum-based mineral salt adjuvant, squalene, hydroxide. In some embodiments, an aluminum-based min lipopolysaccharide-derived adjuvant, bacterial cell wall com eral salt adjuvant is aluminum oxyhydroxide and not magne ponents, molecular cages, nucleic acid, oil, Virosome, QS21, sium hydroxide. In some embodiments, an aluminum-based and MF59. mineral salt adjuvant does not include amorphous aluminum 0078. The term “aluminum-based mineral salt adjuvant” hydroxide in which some hydroxyls are replaced by sulfate refers to an adjuvant including aluminum. In some embodi anions. In some embodiments, an aluminum-based mineral ments, an aluminum-based mineral salt adjuvant includes salt adjuvant includes aluminum oxyhydroxide in a Boeh US 2014/0193460 A1 Jul. 10, 2014

mite-like pattern. In some embodiments, an aluminum-based AdjuphosTM. In some embodiments, an aluminum phosphate mineral salt adjuvant is aluminum oxyhydroxide in a Boeh adjuvant includes amorphous aluminum hydroxyphosphate. mite-like pattern. In some embodiments of an aluminum In some embodiments of an aluminum phosphate adjuvant based mineral salt adjuvant described above, the description described above, the description is of the aluminum phos is of the aluminum-based mineral salt adjuvant prior to inclu phate adjuvant prior to inclusion in a vaccine. sion in a vaccine. In some embodiments, an aluminum-based I0081. The term “lipid A adjuvant” refers to an adjuvant mineral salt adjuvant is an aluminum containing adjuvant including the lipid A portion of lipopolysaccharide isolated approved by the FDA for administration to humans. In some from the remainder of a lipopolysaccharide molecule. A lipid embodiments, an aluminum-based mineral salt adjuvant is an A adjuvant is an example of an LPS-derived adjuvant. In aluminum hydroxide adjuvant approved for administration to Some embodiments, lipid A includes two glucosamine mol humans by the FDA. In some embodiments, an aluminum ecules covalently bonded to fatty acid molecules and includ based mineral salt adjuvant is an aluminum phosphate adju ing one phosphate molecule covalently bonded to each glu vant approved for administration to humans by the FDA. cosamine molecule. In some embodiments, lipid A includes 007.9 The term “aluminum hydroxide adjuvant” as used six fatty acid molecules. In some embodiments, lipid A herein refers to the aluminum hydroxide adjuvant that includes six fatty acid molecules each comprising 10 to 16 includes aluminum hydroxide and is currently used in carbon atoms. licensed human vaccines. In some embodiments, "aluminum I0082. The terms “TLR4” and “Toll-like receptor 4” refer hydroxide adjuvant” as used herein refers to the aluminum to the human gene, mRNA, and/or protein associated with hydroxide adjuvant that is currently used in licensed human UniProt O00206, Entrez 7099, RefSeq NM 003266.3, and/ vaccines and is used in accordance with the use of that term in or NP 003257.1. Hem S. L., Vaccine 23 (2007) 4985-4986. In some embodi I0083. The terms “bind”, “bound”, “binding, and other ments, an aluminum hydroxide adjuvant includes CAS no. verb forms thereof are used in accordance with their plain 21645-51-2. In some embodiments, an aluminum hydroxide ordinary meaning within Enzymology and Biochemistry and adjuvant is aluminum hydroxide gel. In some embodiments, refer to the formation of one or more interactions or contacts an aluminum hydroxide adjuvant is aluminum hydroxide gel between two compositions that may optionally interact. in the form of a white gelatinous precipitate. In some embodi Binding may be intermolecular or intramolecular. ments, an aluminum hydroxide adjuvant includes aluminum I0084. The term “potassium aluminum sulfate adjuvant” hydroxide and does not include magnesium hydroxide. In refers to an adjuvant that includes potassium aluminum Sul some embodiments, an aluminum hydroxide adjuvant is fate. AlhydrogelTM. In some embodiments, an aluminum hydrox I0085. The term “vaccine” is used according to its plain ide adjuvant includes crystalline aluminum hydroxide and ordinary meaning within medicine and Immunology and not amorphous aluminum hydroxide. In some embodiments, refers to a composition including an antigenic component for an aluminum hydroxide adjuvant includes crystalline alumi administration to a subject (e.g. human), which elicits an numand notamorphous aluminum. In some embodiments, an immune response to the antigenic component. In some aluminum hydroxide adjuvant includes crystalline aluminum embodiments a vaccine is a therapeutic. In some embodi oxyhydroxide. In some embodiments, an aluminum hydrox ments, a vaccine is prophylactic. In some embodiments a ide is crystalline aluminum oxyhydroxide. In some embodi vaccine includes one or more adjuvants. ments, an aluminum hydroxide adjuvant includes aluminum I0086. The term “replication decreasing DNA mutation” as oxyhydroxide and not aluminum hydroxycarbonate. In some applied to a virus, refers to a mutation of the viral genome embodiments, an aluminum hydroxide adjuvant is aluminum resulting in a decrease in replication of the virus relative to the oxyhydroxide and not aluminum hydroxycarbonate. In some identical virus not including the replication decreasing DNA embodiments, an aluminum hydroxide adjuvant does not mutation. In some embodiments, a replication decreasing include amorphous aluminum hydroxide in which some DNA mutation prevents viral replication. hydroxyls are replaced by Sulfate anions. In some embodi I0087. The term “chemical inactivation” as applied to inac ments, aluminum hydroxide adjuvant includes aluminum tivation of a virus (e.g. HV. HSV. HHV-1, HHV-2, HHV-3, oxyhydroxide in a Boehmite-like pattern. In some embodi HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8), refers to a ments of an aluminum hydroxide adjuvant described above, method of inactivating (as defined herein) a virus including the description is of the aluminum hydroxide adjuvant prior to contacting the virus with a chemical agent, wherein the con inclusion in a vaccine. tacting causes the inactivation of the virus. 0080. The term “aluminum phosphate adjuvant” as used I0088. The term “cross-linking agent' is used in accor herein refers to the aluminum phosphate adjuvant that dance with its plain meaning within Chemistry and Biochem includes aluminum phosphate and is currently used in istry and refers to an agent (e.g. chemical, biologic) capable licensed human vaccines. In some embodiments, "aluminum of causing a covalent bond (e.g. irreversible, reversible) to phosphate adjuvant’ as used herein refers to the aluminum form between two or more chemical species (e.g. proteins, phosphate adjuvant that is currently used in licensed human nucleic acids, Sugars, cells, compound, any combination vaccines and is used in accordance with the use of that term in thereof) and connecting two or more compositions. In some Hem S. L., Vaccine 23 (2007) 4985-4986. In some embodi embodiments, the two or more compositions are directly con ments, an aluminum phosphate adjuvant includes CAS no. nected through covalent bonds. In some embodiments, the 7784-30-7. In some embodiments, an aluminum phosphate two or more compositions are connected through covalent adjuvant is aluminum phosphate gel. In some embodiments, bonds to one or more cross-linker molecules or portions of an aluminum phosphate adjuvant is aluminum phosphate gel one or more cross-linker molecules. In some embodiments, in the form of a white gelatinous precipitate. In some embodi the compositions are connected through covalent bonds to ments, an aluminum phosphate adjuvant is Adju-phosTM. In intermediate molecules or atoms that are not crosslinkers or Some embodiments, an aluminum phosphate adjuvant is portions of the crosslinker. US 2014/0193460 A1 Jul. 10, 2014

0089. The term “oxidizing agent' is used according to its 0099. The term “plaque forming units” is used according plain ordinary meaning in Chemistry and refers to a Substance to its plain ordinary meaning in Virology and refers to a unit that removes electrons from a second Substance in a redox of measurement based on the number of plaques per unit reaction. Volume of a sample. In some embodiments the units are based 0090 The term “reducing agent' is used according to its on the number of plaques that could form when infecting a plain ordinary meaning in Chemistry and refers to a Substance monolayer of Susceptible cells. Plaque forming unit equiva that donates electrons to a second Substance in a redox reac lents are units of measure of inactivated virus. In some tion. embodiments, plaque forming unit equivalents are derived 0091. The term “radiation' is used according to its plain from plaque forming units for a sample prior to inactivation. ordinary meaning in physics and includes both ionizing and 0100. The term “prime-boost' or “prime boost’ as applied non-ionizing radiation. Some examples of radiation include, to a methodology of administering vaccines is used according but are not limited to, UV, infrared, alpha, beta, gamma, to its plain ordinary meaning in Virology and Immunology X-ray, visible, microwave. and refers to a method of vaccine administration in which a 0092. The term "aldehyde cross-linking agent' is used first dose of a vaccine or vaccine component is administered according to its plain ordinary meaning in Chemistry and to a subject or patient to begin the administration (prime) and refers to a cross-linking agent as defined herein, wherein one at a later time (e.g. hours, days, weeks, months later) a second or more of the cross-linker moieties involved in forming vaccine is administered to the same patient or subject (boost). covalent bonds is an aldehyde. In some embodiments, all (e.g. The first and second vaccines may be the same or different but two) of the reactive moieties are aldehydes. In some embodi are intended to both elicit an immune response useful in ments, one reactive moiety is an aldehyde and one or more treating or preventing the same disease or condition (e.g. reactive moieties are not aldehydes. infection by HV, HSV, HHV-1, HHV-2, HHV-3, HHV-4, 0093. The term “formalin' is used according to its plain HHV-5, HHV-6, HHV-7, or HHV-8). In some embodiments ordinary meaning in Chemistry and refers to an aqueous the prime is a DNA vaccine including one or more viral genes solution including formaldehyde (e.g. 37% by mass). The or portions thereof and the boost is a DNA vaccine including components of formalin are typically designed to be in Suffi one or more viral genes or portions thereof, one or more viral cient quantities to inactivate a virus. In some embodiments, proteins or portions thereof, or one or more inactivated or formalin may include methanol. In some embodiments, for attenuated viruses. In some embodiments, the prime is one or malin does not include methanol. more attenuated or inactivated viruses and the boost is a DNA 0094. The term “viral shedding is used according to its vaccine including one or more viral genes orportions thereof, plain ordinary meaning in Medicine and Virology and refers one or more viral proteins or portions thereof, or one or more to the production and release of virus from an infected cell. In inactivated or attenuated viruses. In some embodiments the some embodiments, the virus is released from a cell of a prime is one or more viral proteins orportions thereof and the Subject. In some embodiments virus is released into the envi boost is a DNA vaccine including one or more viral genes or ronment from an infected Subject. In some embodiments the portions thereof, one or more viral proteins or portions virus is released from a cell within a subject. thereof, or one or more inactivated or attenuated viruses. In 0095. The term “lesion' is used according to its plain Some embodiments, the prime is a vaccine including an inac ordinary meaning within medicine and refers to an abnormal tivated virus (e.g. HV, HSV, HHV-1, HHV-2, HHV-3, HHV-4, ity or damage to the tissue of a Subject. HHV-5, HHV-6, HHV-7, or HHV-8) and the boost is a second 0096. The term "sulfated polysaccharide' is used accord dose of the same vaccine as the prime. ing to its plain ordinary meaning in Biochemistry and Gly 0101 The term “associated” or “associated with as used cobiology and refers to two or more covalently bonded herein to describe a disease (e.g. a virus associated disease, monosaccharides (including nitrogen containing monosac HV associated disease, HSV associated disease, HHV-1 asso charides), wherein one or more of the monosaccharides is ciated disease, HHV-2 associated disease, HHV-3 associated Sulfated. In some embodiments, a Sulfated polysaccharide disease, HHV-4 associated disease, HHV-5 associated dis may be a synthetically made or modified Sulfated polysac ease, HHV-6 associated disease, HHV-7 associated disease, charide or a synthetically sulfated polysaccharide. Examples or HHV-8 associated disease) means that the disease is caused include, but are not limited to, heparin, heparan Sulfate, and by, or a symptom of the disease is caused by, what is described dextran sulfate. as disease associated or what is described as associated with 0097. The term “sulfonated polysaccharide” is used the disease. For example, a disease associated with HSV2 or according to its plain ordinary meaning in Biochemistry and HSV2 infection may be a disease that results (entirely or Glycobiology and refers to two or more covalently bonded partially) from HSV2 or HSV2 infection. As used herein, monosaccharides (including nitrogen containing monosac what is described as being associated with a disease, if a charides), wherein one or more of the monosaccharides is causative agent, could be a target for treatment of the disease. Sulfonated. In some embodiments, a Sulfonated polysaccha For example, a disease associated with HSV2 or HSV2 infec ride may be a synthetically made or modified sulfonated tion, may be treated with an HSV2 vaccine as described polysaccharide or a synthetically Sulfonated polysaccharide. herein (including embodiments). For example, a disease 0098. The term “virus particle' is used according to its associated with HSV2 may be a disease that a subject with plain ordinary meaning within Virology and refers to a virion HSV2 or HSV2 infection is at higher risk of developing as including the viral genome (e.g. DNA, RNA, single strand, compared to a subject without HSV2 or HSV2 infection. double strand), viral capsid and associated proteins, and in the 0102 The term "isolated nucleic acid sequence formula case of enveloped viruses (e.g. HV, HSV. HHV-1, HHV-2, tion” refers to a formulation having a nucleic acid in the HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8), an absence of protein (e.g. viral protein). A person of ordinary envelope including lipids and optionally components of host skill in the art will understand that trace amounts of protein cell membranes, and/or viral proteins. may be present in the isolated nucleic acid sequence formu US 2014/0193460 A1 Jul. 10, 2014

lation. In some embodiments, an isolated nucleic acid 26, 27, 28, 29, 30, 31, 32,33, 34, 35,36, 37,38, 39, 40, 41, 42, sequence formulation is a nucleic acid described in 43, 44, 45,46, 47, 48,49, 50, 51, 52,53,54, 55,56, 57,58, 59, WO2007 106404 (e.g. p)NA). In some embodiments, the 60, 61, 62,63, 64, 65, 66, 67,68, 69,70, 71,72, 73,74, 75,76, description above applies to the isolated nucleic acid 77,78, 79,80, 81, 82, 83, 84,85, 86, 87, 88,89,90,91, 92,93, sequence formation prior to incorporation into a vaccine. 94, 95, 96, 97,98, 99, or 100%. Unless indicated otherwise, 0103. The term “nucleic acid vaccine” refers to a vaccine the term “about in the context of a numeric value indicates in which the antigenic component is an isolated nucleic acid the nominal valuet 10% of the nominal value. In some sequence formulation. The term “DNA vaccine” refers to a embodiments, “about may be the nominal value. vaccine in which the antigenic component is a DNA sequence (“DNA antigenic component'). The isolated nucleic acid II. Compositions sequence formulation antigenic component (e.g. DNA anti 0106 Provided in a first aspect is a Herpesvirus (HV) (e.g. genic component) may be an isolated nucleic acid sequence HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, formulation (e.g. "DNA sequence') that encodes an antigenic HHV-7, or HHV-8)) vaccine including inactivated HV (e.g. protein Such that when the isolated nucleic acid sequence HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, formulation antigenic component (e.g. DNA antigenic com HHV-7, or HHV-8), a lipopolysaccharide (LPS)-derived ponent) is expressed, an antigenic protein is produced. The adjuvant and an aluminum-based mineral Salt adjuvant. DNA antigenic component may be a DNA plasmid (e.g. 0107. In some embodiments of the HV (e.g. HSV. HHV-1, pVAX), optionally including a DNA sequence capable of HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV being translated to a corresponding protein (coding 8) vaccine, the LPS-derived adjuvant is MPL: the aluminum sequence), wherein the coding sequence is translated into a based mineral salt adjuvant is aluminum hydroxide adjuvant protein capable of causing an immune response specific to the or aluminum phosphate adjuvant; and the vaccine does not protein, when administered to a Subject (e.g. human). In some include an isolated nucleic acid sequence formulation. In embodiments a DNA vaccine includes the vaccine DNA plas some embodiments of the vaccine, the HV is a Herpes Sim mid and DNA plasmids including coding sequences plex Virus (HSV). In some embodiments of the vaccine, the described in WO2007 106404. In some embodiments, a DNA HSV is HSV1. In some embodiments of the vaccine, the HSV vaccine is the pVAX plasmid not ligated to a coding sequence. is HSV2. In some embodiments of the vaccine, the HSV is In some embodiments, a DNA vaccine is the pVAX plasmid HSV1 and HSV2. In some embodiments of the vaccine, the ligated to a coding sequence. In some embodiments, a DNA HV is Human Cytomegalovirus (HCMV). In some embodi vaccine is the pVAX plasmid ligated to a coding sequence ments of an HV vaccine, the HV is HHV-1. In some embodi from the HSV2 genome. In some embodiments, a DNA vac ments of an HV vaccine, the HV is HHV-2. In some embodi cine is the pVAX plasmid ligated to a coding sequence from ments of an HV vaccine, the HV is HHV-3. In some the HCMV genome. In some embodiments, a DNA vaccine is embodiments of an HV vaccine, the HV is HHV-4. In some the pVAX plasmid ligated to a coding sequence from the embodiments of an HV vaccine, the HV is HHV-5. In some HSV1 genome. In some embodiments, a DNA vaccine is a embodiments of an HV vaccine, the HV is HHV-6. In some plasmid capable of expressing an antigen in a human, wherein embodiments of an HV vaccine, the HV is HHV-7. In some the antigen is a portion of an HSV2 protein. In some embodi embodiments of an HV vaccine, the HV is HHV-8. In some ments, a DNA vaccine is a plasmid capable of expressing an embodiments of an HV (e.g. HSV. HHV-1, HHV-2, HHV-3, antigen in a human, wherein the antigen is a portion of an HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vaccine, the HSV1 protein. In some embodiments, a DNA vaccine is a lipopolysaccharide-derived adjuvant is derived from the Sal plasmid capable of expressing an antigen in a human, wherein monella minnesota LPS. In some embodiments of an HV (e.g. the antigen is a portion of an HSV protein. In some embodi HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, ments, a DNA vaccine is a plasmid capable of expressing an HHV-7, or HHV-8) vaccine, the lipopolysaccharide-derived antigen in a human, wherein the antigen is a portion of an HV adjuvant is derived from the Salmonella minnesota Re595 (e.g. HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, LPS. In some embodiments of an HV (e.g. HSV. HHV-1, HHV-7, or HHV-8) protein. HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV 0104. The term “vaccinate’, or additional verb forms 8) vaccine, the lipopolysaccharide-derived adjuvant is thereof, refers to administering a vaccine to a subject (e.g. derived from the R595 LPS. In some embodiments of an HV human) and eliciting an antigen specific immune response, (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, wherein the antigen is included in the vaccine. The term HHV-7, or HHV-8) vaccine, the lipopolysaccharide-derived “vaccinate' may also refer to eliciting an antigen specific adjuvant is a lipid Aadjuvant. In some embodiments of an HV immune response against an administered antigen. In some (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, embodiments, vaccinate is to provide prophylaxis against a HHV-7, or HHV-8) vaccine, the lipopolysaccharide-derived disease or infectious agent (e.g. HV. HSV. HHV-1, HHV-2, adjuvant is a lipid A adjuvant without an (R)-3-hydroxytet HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8). radecanoyl moiety. In some embodiments of an HV (e.g. 0105. The term “portion” refers to a subset of a whole, HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, which may also be the whole. In some embodiments, a por HHV-7, or HHV-8) vaccine, the lipopolysaccharide-derived tion is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, adjuvant is a lipid A adjuvant without a 1-phosphate moiety. 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, In some embodiments of an HV (e.g. HSV. HHV-1, HHV-2, 34,35,36, 37,38, 39, 40, 41,42, 43,44, 45,46, 47, 48,49, 50, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vac 51, 52,53,54, 55,56, 57,58, 59, 60, 61, 62,63, 64, 65,66, 67, cine, the lipopolysaccharide-derived adjuvant is monophos 68, 69,70, 71,72, 73,74, 75,76, 77,78, 79,80, 81, 82, 83, 84, phoryl lipid A. In some embodiments of an HV (e.g. HSV. 85, 86, 87, 88, 89,90, 91, 92,93, 94, 95, 96, 97,98, 99, or HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or 100%. In some embodiments, a portion is 1, 2, 3, 4, 5, 6, 7, 8, HHV-8) vaccine, the lipopolysaccharide-derived adjuvant is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 3-O-desacyl-4'-monophosphoryl lipid A. In some embodi US 2014/0193460 A1 Jul. 10, 2014

ments of an HV (e.g. HSV. HHV-1, HHV-2, HHV-3, HHV-4, 2. In some embodiments, the detergent is Triton-X-100. In HHV-5, HHV-6, HHV-7, or HHV-8) vaccine, the some embodiments the detergent is NP-40. In some embodi lipopolysaccharide-derived adjuvant is capable of binding the ments, the detergent is Tween-20. In some embodiments, the TLR4 protein. In some embodiments of an HV (e.g. HSV. inactivated HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-5, HHV-6, HHV-7, or HHV-8) is formed by contacting HHV-8) vaccine, the lipopolysaccharide-derived adjuvant is the HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, a synthetic MPL analogue adjuvant. In some embodiments of HHV-6, HHV-7, or HHV-8) with one or more agents includ an HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, ing UV radiation and a furocoumarin. In some embodiments, HHV-6, HHV-7, or HHV-8) vaccine, the aluminum-based the furocoumarin is psoralen. In some embodiments, the furo mineral salt adjuvant is aluminum hydroxide adjuvant. In coumarin is 4'-aminomethyl-4.5".8-trimethylpsoralen. In some embodiments of an HV (e.g. HSV. HHV-1, HHV-2, Some embodiments, the furocoumarin is angelicin. In some HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vac embodiments, the furocoumarin is Xanthotoxin. In some cine, the aluminum-based mineral salt adjuvant is aluminum embodiments, the furocoumarin is bergapten. In some phosphate adjuvant. In some embodiments of an HV (e.g. embodiments, the furocoumarin is nodakenetin. In some HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, embodiments of an HV (e.g. HSV. HHV-1, HHV-2, HHV-3, HHV-7, or HHV-8) vaccine, the aluminum-based mineral salt HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vaccine, the adjuvant is a potassium aluminum sulfate adjuvant. In some inactivated HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, embodiments of an HV (e.g. HSV. HHV-1, HHV-2, HHV-3, HHV-5, HHV-6, HHV-7, or HHV-8) is an inactivated single HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vaccine, the strain of the HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, aluminum-based mineral salt adjuvant includes crystalline HHV-5, HHV-6, HHV-7, or HHV-8). In some embodiments aluminum hydroxide and not amorphous aluminum hydrox of an HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV ide or aluminum hydroxycarbonate or magnesium hydroxide. 5, HHV-6, HHV-7, or HHV-8) vaccine, the inactivated HV In some embodiments of an HV (e.g. HSV. HHV-1, HHV-2, (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vac HHV-7, or HHV-8) is a combination of two or more inacti cine, the aluminum-based mineral salt adjuvant includes alu vated strains of the HV (e.g. HSV. HHV-1, HHV-2, HHV-3, minum phosphate gel in the form of a white gelatinous pre HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8). In some cipitate. In some embodiments of an HV (e.g. HSV. HHV-1, embodiments, the inactivated HV is an inactivated single HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV strain of HSV1. In some embodiments, the inactivated HV is 8) vaccine, the aluminum-based mineral salt adjuvant an inactivated single strain of HSV2. In some embodiments, includes aluminum hydroxide gel in the form of a white the inactivated HV is a combination of two or more inacti gelatinous precipitate. In some embodiments of an HV (e.g. vated strains of HSV 1. In some embodiments, the inactivated HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HV is a combination of two or more inactivated strains of HHV-7, or HHV-8) vaccine, the inactivated HV (e.g. HSV. HSV2. In some embodiments, the inactivated HV is a com HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or bination of one or more inactivated strains or HSV1 and one HHV-8) includes a replication decreasing DNA mutation. In or more inactivated strains of HSV2. In some embodiments of some embodiments of an HV (e.g. HSV. HHV-1, HHV-2, an HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vac HHV-6, HHV-7, or HHV-8) vaccine, the HV (e.g. HSV, HHV cine, the inactivated HV (e.g. HSV. HHV-1, HHV-2, HHV-3, 1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) is formed by HHV-8) vaccine is formulated for intramuscular administra chemical inactivation. In some embodiments of an HV (e.g. tion. In some embodiments of an HV (e.g. HSV. HHV-1, HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV HHV-7, or HHV-8) vaccine, the inactivated HV (e.g. HSV. 8) vaccine, the HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or 4, HHV-5, HHV-6, HHV-7, or HHV-8) vaccine is formulated HHV-8) is formed by contacting the HV (e.g. HSV. HHV-1, for intradermal administration. In some embodiments of an HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, 8), with one or more agents selected from a cross-linking HHV-6, HHV-7, or HHV-8) vaccine, the HV (e.g. HSV, HHV agent, oxidizing agent, reducing agent, heat, radiation, deter 1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or gent, a pH changing agent, and a chemical agent selected HHV-8) vaccine is formulated for mucosal administration. In from a furocoumarin, aziridine, ethylenimine, binary ethyl some embodiments of an HV (e.g. HSV. HHV-1, HHV-2, enimine, and beta-propiolactone. In some embodiments, the HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vac chemical agentis aziridine. In some embodiments, the chemi cine, the HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, cal agent is ethylenimine. In some embodiments, the chemi HHV-5, HHV-6, HHV-7, or HHV-8) vaccine is formulated for cal agent is binary ethylenimine. In some embodiments, the intranasal administration. In some embodiments of an HV chemical agent is beta-propiolactone. In some embodiments, (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, the radiation is UV radiation. In some embodiments, the HHV-7, or HHV-8) vaccine, the HV (e.g. HSV, HHV-1, HHV radiation is electron beam radiation. In some embodiments, 2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vac the radiation is infrared radiation. In some embodiments, the cine is formulated for intrarectal administration. In some radiation is gamma radiation. In some embodiments, the embodiments of an HV (e.g. HSV. HHV-1, HHV-2, HHV-3, cross-linking agent is an aldehyde cross-linking agent. In HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vaccine, the HV Some embodiments, the cross-linking agent is formaldehyde. (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, In some embodiments, the cross-linking agent is formalin. In HHV-7, or HHV-8) vaccine is formulated for intravaginal Some embodiments, the oxidizing agent is sodium periodate. administration. In some embodiments of an HV (e.g. HSV. In some embodiments, the oxidizing agent is hydrogen per HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or oxide. In some embodiments, the reducing agent is aldrithiol HHV-8) vaccine, the HV (e.g. HSV, HHV-1, HHV-2, HHV-3, US 2014/0193460 A1 Jul. 10, 2014

HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vaccine is for HHV-7, or HHV-8) vaccine, the isolated nucleic acid mulated for topical administration. In some embodiments of sequence formulation is a DNA vaccine. In some embodi an HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, ments of an HV (e.g. HSV. HHV-1, HHV-2, HHV-3, HHV-4, HHV-6, HHV-7, or HHV-8) vaccine, the HV (e.g. HSV, HHV HHV-5, HHV-6, HHV-7, or HHV-8) vaccine, the DNA vac 1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or cine is an antigenic isolated nucleic acid sequence formula HHV-8) vaccine is formulated for subcutaneous administra tion. tion. In some embodiments of an HV (e.g. HSV. HHV-1, 0.108 Provided in another aspect is a Herpes Simplex HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV Virus (HSV) vaccine including inactivated HSV, a 8) vaccine, the HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV lipopolysaccharide (LPS)-derived adjuvant and an alumi 4, HHV-5, HHV-6, HHV-7, or HHV-8) vaccine is formulated num-based mineral salt adjuvant. for transcutaneous administration. In some embodiments of 0109. In some embodiments of the HSV vaccine, the HSV an HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, is HSV1. In some embodiments of the HSV vaccine, the HSV HHV-6, HHV-7, or HHV-8) vaccine, the isolated nucleic acid is HSV2. In some embodiments of the HSV vaccine, the HSV sequence formulation is an antigenic isolated nucleic acid is HSV1 and HSV2. In some embodiments of the HSV vac sequence formulation. In some embodiments of an HV (e.g. cine, the lipopolysaccharide-derived adjuvant is derived from HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, the Salmonella minnesota LPS. In some embodiments of the HHV-7, or HHV-8) vaccine, the isolated nucleic acid HSV vaccine, the lipopolysaccharide-derived adjuvant is sequence formulation is a DNA vaccine. In some embodi derived from the Salmonella minnesota Re595 LPS. In some ments of an HV (e.g. HSV. HHV-1, HHV-2, HHV-3, HHV-4, embodiments of the HSV vaccine, the lipopolysaccharide HHV-5, HHV-6, HHV-7, or HHV-8) vaccine, the DNA vac derived adjuvant is derived from the R595 LPS. In some cine is an antigenic isolated nucleic acid sequence formula embodiments of the HSV vaccine, the lipopolysaccharide tion. In some embodiments of an HV (e.g. HSV. HHV-1, derived adjuvant is a lipid A adjuvant. In some embodiments HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV of the HSV vaccine, the lipopolysaccharide-derived adjuvant 8) vaccine, the vaccine vaccinates a recipient of the vaccine is a lipid A adjuvant without an (R)-3-hydroxytetradecanoyl against HV (e.g. HSV. HHV-1, HHV-2, HHV-3, HHV-4, moiety. In some embodiments of the HSV vaccine, the HHV-5, HHV-6, HHV-7, or HHV-8) infection for up to 1, 2, lipopolysaccharide-derived adjuvant is a lipid Aadjuvant that 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or does not include an (R)-3-hydroxytetradecanoyl moiety. In more years. In some embodiments of an HV (e.g. HSV. HHV some embodiments of the HSV vaccine, the lipopolysaccha 1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or ride-derived adjuvant is a lipid A adjuvant without a 1-phos HHV-8) vaccine, the vaccine protects a patient administered phate moiety. In some embodiments of the HSV vaccine, the the vaccine against one or more strains of the HV (e.g. HSV. lipopolysaccharide-derived adjuvant is a lipid Aadjuvant that HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or does not include a 1-phosphate moiety. In some embodiments HHV-8). In some embodiments of an HV (e.g. HSV. HHV-1, of the HSV vaccine, the lipopolysaccharide-derived adjuvant HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV is monophosphoryl lipid A (MPL). In some embodiments of 8) vaccine, the vaccine treats a patient administered the vac the HSV vaccine, the lipopolysaccharide-derived adjuvant is cine for one or more strains of the HV (e.g. HSV. HHV-1, 3-O-desacyl-4'-monophosphoryl lipid A. In some embodi HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV ments of the HSV vaccine, the lipopolysaccharide-derived 8). In some embodiments of an HV (e.g. HSV. HHV-1, HHV adjuvant is capable of binding the TLR4 protein. In some 2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vac embodiments of the HSV vaccine, the lipopolysaccharide cine, the vaccine prevents a patient administered the vaccine derived adjuvant is a synthetic MPL analogue adjuvant. In from being infected by, contracting, getting, or having one or some embodiments of the HSV vaccine, the aluminum-based more strains of the HV (e.g. HSV. HHV-1, HHV-2, HHV-3, mineral salt adjuvant is an aluminum hydroxide adjuvant. In HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8). In some some embodiments of the HSV vaccine, the aluminum-based embodiments of an HV (e.g. HSV. HHV-1, HHV-2, HHV-3, mineral salt adjuvant is an aluminum phosphate adjuvant. In HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vaccine, the some embodiments of the HSV vaccine, the aluminum-based vaccine prevents a patient administered the vaccine from mineral salt adjuvant is a potassium aluminum Sulfate adju being infected by, contracting, getting, or having more than vant. In some embodiments of the HSV vaccine, the inacti one strain of the HV (e.g. HSV. HHV-1, HHV-2, HHV-3, vated HSV includes a replication decreasing DNA mutation. HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8). In some In some embodiments of the HSV vaccine, the inactivated embodiments, the Herpesvirus (HV) (e.g. HSV. HHV-1, HSV includes a replication decreasing DNA mutation that HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV prevents viral replication. In some embodiments of the HSV 8)) vaccine includes inactivated HV (e.g. HSV. HHV-1, HHV vaccine, the inactivated HSV is formed by chemical inacti 2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8), a vation. In some embodiments of the HSV vaccine, the inac lipopolysaccharide (LPS)-derived adjuvant and an alumi tivated HSV is formed by contacting the HSV with one or num-based mineral salt adjuvant and the LPS-derived adju more agents selected from a cross-linking agent, oxidizing vant is MPL: the aluminum-based mineral salt adjuvant is agent, reducing agent, heat, radiation, detergent, a pH chang aluminum hydroxide adjuvant or aluminum phosphate adju ing agent, and a chemical agent selected from a furocou vant; and the vaccine does not include an isolated nucleic acid marin, aziridine, ethylenimine, binary ethylenimine, and sequence formulation. In some embodiments of an HV (e.g. beta-propiolactone. In some embodiments of the HSV vac HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, cine, the inactivated HSV is formed by contacting the HSV HHV-7, or HHV-8) vaccine, the isolated nucleic acid with aziridine. In some embodiments of the HSV vaccine, the sequence formulation is an antigenic isolated nucleic acid inactivated HSV is formed by contacting the HSV with eth sequence formulation. In some embodiments of an HV (e.g. ylenimine. In some embodiments of the HSV vaccine, the HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, inactivated HSV is formed by contacting the HSV with binary US 2014/0193460 A1 Jul. 10, 2014

ethylenimine. In some embodiments of the HSV vaccine, the istration. In some embodiments of the HSV vaccine, the inactivated HSV is formed by contacting the HSV with beta vaccine is formulated for intravaginal administration. In some propiolactone. In some embodiments of the HSV vaccine, the embodiments of the HSV vaccine, the vaccine is formulated inactivated HSV is formed by contacting the HSV with UV for topical administration. In some embodiments of the HSV radiation. In some embodiments of the HSV vaccine, the vaccine, the vaccine is formulated for Subcutaneous admin inactivated HSV is formed by contacting the HSV with elec istration. In some embodiments of the HSV vaccine, the tron beam radiation. In some embodiments of the HSV vac vaccine is formulated for parenteral administration. In some cine, the inactivated HSV is formed by contacting the HSV embodiments of the HSV vaccine, the vaccine is formulated with infrared radiation. In some embodiments of the HSV for oral administration. In some embodiments of the HSV vaccine, the inactivated HSV is formed by contacting the vaccine, the vaccine is formulated for transcutaneous admin HSV with gamma radiation. In some embodiments of the istration. HSV vaccine, the inactivated HSV is formed by contacting 0110. In some embodiments, a chemical agent for inacti the HSV with an aldehyde cross-linking agent. In some Vating HSV is aziridine. In some embodiments, a chemical embodiments of the HSV vaccine, the inactivated HSV is agent for inactivating HSV is ethylenimine. In some embodi formed by contacting the HSV with formaldehyde. In some ments, a chemical agent for inactivating HSV is binary eth embodiments of the HSV vaccine, the inactivated HSV is ylenimine. In some embodiments, a chemical agent for inac formed by contacting the HSV with formalin. In some tivating HSV is beta-propiolactone. In some embodiments, a embodiments of the HSV vaccine, the inactivated HSV is radiation for inactivating HSV is UV radiation. In some formed by contacting the HSV with sodium periodate. In embodiments, a radiation for inactivating HSV is electron some embodiments of the HSV vaccine, the inactivated HSV beam radiation. In some embodiments, a radiation for inacti is formed by contacting the HSV with hydrogen peroxide. In vating HSV is infrared radiation. In some embodiments, a some embodiments of the HSV vaccine, the inactivated HSV radiation for inactivating HSV is gamma radiation. In some is formed by contacting the HSV with aldrithiol-2. In some embodiments, a cross-linking agent for inactivating HSV is embodiments of the HSV vaccine, the inactivated HSV is an aldehyde cross-linking agent. In some embodiments, a formed by contacting the HSV with Triton-X-100. In some cross-linking agent for inactivating HSV is formaldehyde. In embodiments of the HSV vaccine, the inactivated HSV is Some embodiments, a cross-linking agent for inactivating formed by contacting the HSV with NP-40. In some embodi HSV is formalin. In some embodiments, across-linking agent ments of the HSV vaccine, the inactivated HSV is formed by for inactivating HSV is sodium periodate. In some embodi contacting the HSV with Tween-20. In some embodiments of ments, an oxidizing agent for inactivating HSV is hydrogen the HSV vaccine, the inactivated HSV is formed by contact peroxide. In some embodiments, a reducing agent for inacti ing the HSV with two or more agents including UV radiation vating HSV is aldrithiol-2. In some embodiments, a detergent and a furocoumarin. In some embodiments of the HSV vac for inactivating HSV is Triton-X-100. In some embodiments, cine, the inactivated HSV is formed by contacting the HSV a detergent for inactivating HSV is NP-40. In some embodi with UV radiation and psoralen. In some embodiments of the ments, a detergent for inactivating HSV is Tween-20. In some HSV vaccine, the inactivated HSV is formed by contacting embodiments, a furocoumarin for inactivating HSV is psor the HSV with UV radiation and 4'-aminomethyl-4,5".8-trim alen. In some embodiments, a furocoumarin for inactivating ethylpsoralen. In some embodiments of the HSV vaccine, the HSV is 4'-aminomethyl-4,5'.8-trimethylpsoralen. In some inactivated HSV is formed by contacting the HSV with UV embodiments, a furocoumarin for inactivating HSV is angeli radiation and angelicin. In some embodiments of the HSV cin. In some embodiments, a furocoumarin for inactivating vaccine, the inactivated HSV is formed by contacting the HSV is xanthotoxin. In some embodiments, a furocoumarin HSV with UV radiation and Xanthotoxin. In some embodi for inactivating HSV is bergapten. In some embodiments, a ments of the HSV vaccine, the inactivated HSV is formed by furocoumarin for inactivating HSV is nodakenetin. contacting the HSV with UV radiation and bergapten. In 0111. In some embodiments of the HSV vaccine, the LPS some embodiments of the HSV vaccine, the inactivated HSV derived adjuvant is MPL: the aluminum-based mineral salt is formed by contacting the HSV with UV radiation and adjuvant is aluminum hydroxide adjuvant or aluminum phos nodakenetin. In some embodiments of the HSV vaccine, the phate adjuvant; and the vaccine does not include an isolated inactivated HSV is an inactivated single strain of HSV1. In nucleic acid sequence formulation. In some embodiments of some embodiments of the HSV vaccine, the inactivated HSV the HSV vaccine, the isolated nucleic acid sequence formu is an inactivated single strain of HSV2. In some embodiments lation is an antigenic isolated nucleic acid sequence formu of the HSV vaccine, the inactivated HSV is a combination of lation. In some embodiments of the HSV vaccine, the isolated two or more inactivated strains of HSV 1. In some embodi nucleic acid sequence formulation is a DNA vaccine. In some ments of the HSV vaccine, the inactivated HSV is a combi embodiments of the HSV vaccine, the DNA vaccine is an nation of two or more inactivated strains of HSV2. In some antigenic isolated nucleic acid sequence formulation. In some embodiments of the HSV vaccine, the inactivated HSV is a embodiments of the HSV vaccine, the vaccine vaccinates a combination of one or more inactivated strains or HSV1 and recipient of the vaccine against HSV infection for up to 1, 2, one or more inactivated strains of HSV2. In some embodi 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or ments of the HSV vaccine, the vaccine is formulated for more years. In some embodiments of the HSV1 vaccine, the intramuscular administration. In some embodiments of the LPS-derived adjuvant is MPL: the aluminum-based mineral HSV vaccine, the vaccine is formulated for intradermal salt adjuvant is aluminum hydroxide adjuvant or aluminum administration. In some embodiments of the HSV vaccine, phosphate adjuvant; and the vaccine does not include an the vaccine is formulated for mucosal administration. In some isolated nucleic acid sequence formulation. In some embodi embodiments of the HSV vaccine, the vaccine is formulated ments of the HSV1 vaccine, the isolated nucleic acid for intranasal administration. In some embodiments of the sequence formulation is an antigenic isolated nucleic acid HSV vaccine, the vaccine is formulated for intrarectal admin sequence formulation. In some embodiments of the HSV1 US 2014/0193460 A1 Jul. 10, 2014

vaccine, the isolated nucleic acid sequence formulation is a sulfated or sulfonated polysaccharide is selected from dext DNA vaccine. In some embodiments of the HSV1 vaccine, ran Sulfate, heparin, and heparan Sulfate. In some embodi the DNA vaccine is an antigenic isolated nucleic acid ments of the method of preparing an HV (e.g. HSV. HHV-1, sequence formulation. In some embodiments of the HSV 1 HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV vaccine, the vaccine vaccinates a recipient of the vaccine 8) vaccine, the HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV against HSV1 infection for up to 1,2,3,4,5,6,7,8,9, 10, 11, 4, HHV-5, HHV-6, HHV-7, or HHV-8) particles are isolated 12, 13, 14, 15, 16, 17, 18, 19, 20, or more years. In some at a concentration of 107 to 10" (pfu/microgram protein). In embodiments of the HSV2 vaccine, the LPS-derived adjuvant Some embodiments of the method of preparing an HV (e.g. is MPL: the aluminum-based mineral salt adjuvant is alumi num hydroxide adjuvant or aluminum phosphate adjuvant; HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, and the vaccine does not include an isolated nucleic acid HHV-7, or HHV-8) vaccine, the HV (e.g. HSV, HHV-1, HHV sequence formulation. In some embodiments of the HSV2 2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vac vaccine, the isolated nucleic acid sequence formulation is an cine comprises 107 to 10" (pfu equivalents/microgram pro antigenic isolated nucleic acid sequence formulation. In some tein) of inactivated HV (e.g. HSV. HHV-1, HHV-2, HHV-3, embodiments of the HSV2 vaccine, the isolated nucleic acid HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8). In some sequence formulation is a DNA vaccine. In some embodi embodiments of the method of preparing an HV (e.g. HSV. HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or ments of the HSV2 vaccine, the DNA vaccine is an antigenic HHV-8) vaccine, the separating includes centrifugation, isolated nucleic acid sequence formulation. In some embodi polyethylene glycol precipitation, filtration, gel filtration, ments of the HSV2 vaccine, the vaccine vaccinates a recipient ultra-filtration, tangential flow ultra-filtration, or affinity of the vaccine against HSV2 infection for up to 1, 2, 3, 4, 5, 6, chromatography. In some embodiments of the method of 7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more years. preparing an HV (e.g. HSV. HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vaccine, the III. Pharmaceutical Compositions and Methods lipopolysaccharide-derived adjuvant is derived from the Sal 0112 Provided in a second aspect is a method of preparing monella minnesota LPS. In some embodiments of the method a Herpesvirus (HV (e.g. HSV. HHV-1, HHV-2, HHV-3, HHV of preparing an HV (e.g. HSV. HHV-1, HHV-2, HHV-3, 4, HHV-5, HHV-6, HHV-7, or HHV-8)) vaccine, the method HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vaccine, the including: contacting an HV (e.g. HSV. HHV-1, HHV-2, lipopolysaccharide-derived adjuvant is derived from the Sal HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8)-cell monella minnesota Re595 LPS. In some embodiments of the mixture with a sulfated or sulfonated polysaccharide, wherein method of preparing an HV (e.g. HSV. HHV-1, HHV-2, HHV the HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, 3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vaccine, the HHV-6, HHV-7, or HHV-8)-cell mixture comprises HV par lipopolysaccharide-derived adjuvant is derived from the ticles, cells and portions of cells; separating the HV (e.g. R595 LPS. In some embodiments of the method of preparing HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, an HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-7, or HHV-8) particles from the cells thereby forming HHV-6, HHV-7, or HHV-8) vaccine, the lipopolysaccharide isolated HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, derived adjuvant is a lipid A adjuvant. In some embodiments HHV-5, HHV-6, HHV-7, or HHV-8) particles; inactivating of the method of preparing an HV (e.g. HSV. HHV-1, HHV-2, the isolated HV particles thereby forming inactivated HV HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vac (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, cine, the lipopolysaccharide-derived adjuvant is a lipid A HHV-7, or HHV-8) particles; combining the inactivated HV adjuvant without a (R)-3-hydroxytetradecanoyl moiety. In (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, Some embodiments of the method of preparing an HV (e.g. HHV-7, or HHV-8) particles with a lipopolysaccharide-de HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, rived adjuvant and an aluminum-based mineral salt adjuvant HHV-7, or HHV-8) vaccine, the lipopolysaccharide-derived thereby forming an HV (e.g. HSV. HHV-1, HHV-2, HHV-3, adjuvant is a lipid A adjuvant without a 1-phosphate moiety HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vaccine. (MPL). In some embodiments of the method of preparing an 0113. In some embodiments of the method of preparing an HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HV vaccine, the HV is HSV. In some embodiments of the HHV-6, HHV-7, or HHV-8) vaccine, the lipopolysaccharide method of preparing an HV vaccine, the HSV is HSV1. In derived adjuvant is 3-O-desacyl-4'-monophosphoryl lipid A. some embodiments of the method of preparing an HV vac In Some embodiments of the method of preparing an HV (e.g. cine, the HSV is HSV2. In some embodiments of the method HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, of preparing an HV vaccine, the HSV is HSV1 and HSV2. In HHV-7, or HHV-8) vaccine, the lipopolysaccharide-derived some embodiments of the method of preparing an HV vac adjuvant is monophosphoryllipid A (MPL). In some embodi cine, the HV is HHV-1. In some embodiments of the method ments of the method of preparing an HV (e.g. HSV. HHV-1, of preparing an HV vaccine, the HV is HHV-2. In some HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV embodiments of the method of preparing an HV vaccine, the 8) vaccine, the lipopolysaccharide-derived adjuvant is a syn HV is HHV-3. In some embodiments of the method of pre thetic MPL analogue adjuvant. In some embodiments of the paring an HV vaccine, the HV is HHV-4. In some embodi method of preparing an HV (e.g. HSV. HHV-1, HHV-2, HHV ments of the method of preparing an HV vaccine, the HV is 3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vaccine, the HHV-5. In some embodiments of the method of preparing an lipopolysaccharide-derived adjuvant is capable of binding the HV vaccine, the HV is HHV-6. In some embodiments of the TLR4 protein. In some embodiments of the method of pre method of preparing an HV vaccine, the HV is HHV-7. In paring an HV (e.g. HSV. HHV-1, HHV-2, HHV-3, HHV-4, some embodiments of the method of preparing an HV vac HHV-5, HHV-6, HHV-7, or HHV-8) vaccine, the aluminum cine, the HV is HHV-8. In some embodiments of the method based mineral salt adjuvant is an aluminum hydroxide adju of preparing an HV (e.g. HSV. HHV-1, HHV-2, HHV-3, vant. In some embodiments of the method of preparing an HV HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vaccine, the (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, US 2014/0193460 A1 Jul. 10, 2014

HHV-7, or HHV-8) vaccine, the aluminum-based mineral salt 0114 Provided in a third aspect is a method of treating or adjuvant is an aluminum phosphate adjuvant. In some preventing an HV (e.g. HSV. HHV-1, HHV-2, HHV-3, HHV embodiments of the method of preparing an HV (e.g. HSV. 4, HHV-5, HHV-6, HHV-7, or HHV-8) infection in a patient HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or in need of the treatment or prevention, the method including HHV-8) vaccine, the aluminum-based mineral salt adjuvant is administering a therapeutically or prophylactically effective a potassium aluminum Sulfate adjuvant. In some embodi amount of any one of the HV (e.g. HSV. HHV-1, HHV-2, ments of the method of preparing an HV (e.g. HSV. HHV-1, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vac HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV cines described herein (including embodiments). 8) vaccine, the aluminum-based mineral salt adjuvant 0.115. In some embodiments of the method, the HV is includes crystalline aluminum hydroxide and not amorphous HSV. In some embodiments of the method, the HSV is HSV1. aluminum hydroxide or aluminum hydroxycarbonate or mag In some embodiments of the method, the HSV is HSV2. In nesium hydroxide. In some embodiments of the method of some embodiments of the method, the HSV is HSV1 and preparing an HV (e.g. HSV. HHV-1, HHV-2, HHV-3, HHV-4, HSV2. In some embodiments of the method, the HV is HHV HHV-5, HHV-6, HHV-7, or HHV-8) vaccine, the aluminum 1. In some embodiments of the method, the HV is HHV-2. In based mineral salt adjuvant includes aluminum phosphate gel some embodiments of the method, the HV is HHV-3. In some in the form of a white gelatinous precipitate. In some embodi embodiments of the method, the HV is HHV-4. In some ments of the method of preparing an HV (e.g. HSV. HHV-1, embodiments of the method, the HV is HHV-5. In some HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV embodiments of the method, the HV is HHV-6. In some 8) vaccine, the aluminum-based mineral salt adjuvant embodiments of the method, the HV is HHV-7. In some includes aluminum hydroxide gel in the form of a white embodiments of the method, the HV is HHV-8. In some embodiments of the method, the HV infection causes a dis gelatinous precipitate. In some embodiments of the method of ease selected from herpetic gingivostomatitis, herpes labialis, preparing an HV (e.g. HSV. HHV-1, HHV-2, HHV-3, HHV-4, herpes genitalis, herpetic whitlow, herpes gladiatorum, herp HHV-5, HHV-6, HHV-7, or HHV-8) vaccine, the inactivating esviral encephalitis, herpesviral meningitis, herpes esophagi includes contacting the HV (e.g. HSV. HHV-1, HHV-2, HHV tis, herpes keratitis, Bell’s palsy, Mollaret's meningitis, her 3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) with one or pes rugbeiorum, eczema herpeticum, herpetic neuralgia, and more agents selected from a cross-linking agent, oxidizing post-herpetic neuralgia. In some embodiments of the method, agent, reducing agent, heat, radiation, detergent, a pH chang the method is a method of treating. In some embodiments of ing agent, and a chemical agent selected from a furocou the method, the method is a method of preventing. In some marin, aziridine, ethylenimine, binary ethylenimine, and embodiments of the method, the method includes a reduction beta-propiolactone. In some embodiments of the method, the in viral shedding. In some embodiments of the method, the chemical agent is aziridine. In some embodiments of the method includes a reduction in the frequency of lesion occur method, the chemical agent is ethylenimine. In some embodi rence. In some embodiments of the method, the method ments of the method, the chemical agent is binary ethylen includes a reduction in the duration of lesion occurrence. In imine. In some embodiments of the method, the chemical some embodiments of the method, the method includes intra agent is beta-propiolactone. In some embodiments of the muscular administration. In some embodiments of the method, the radiation is UV radiation. In some embodiments method, the method includes intradermal administration. In of the method, the radiation is electron beam radiation. In some embodiments of the method, the method includes some embodiments of the method, the radiation is infrared mucosal administration. In some embodiments of the radiation. In some embodiments of the method, the radiation method, the method includes intranasal administration. In is gamma radiation. In some embodiments of the method, the some embodiments of the method, the method includes cross-linking agent is an aldehyde cross-linking agent. In intrarectal administration. In some embodiments of the Some embodiments of the method, the cross-linking agent is method, the method includes intravaginal administration. In formaldehyde. In some embodiments of the method, the some embodiments of the method, the method includes topi cross-linking agent is formalin. In some embodiments of the cal administration. In some embodiments of the method, the method, the oxidizing agent is sodium periodate. In some method includes transcutaneous administration. In some embodiments of the method, the oxidizing agent is hydrogen embodiments of the method, the method includes subcutane peroxide. In some embodiments of the method, the reducing ous administration. In some embodiments of the method, an agent is aldrithiol-2. In some embodiments of the method, the isolated nucleic acid sequence formulation is not adminis detergent is Triton-X-100. In some embodiments of the tered to the patient. In some embodiments of the method, the method, the detergent is NP-40. In some embodiments of the isolated nucleic acid sequence formulation is a DNA method, the detergent is Tween-20. In some embodiments of sequence. In some embodiments of the method, the isolated the method, the inactivating comprises contacting the HV nucleic acid sequence formulation is a nucleic acid vaccine. (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, In some embodiments of the method, the isolated nucleic acid HHV-7, or HHV-8) with one or more agents including UV sequence formulation is a DNA vaccine. In some embodi radiation and a furocoumarin. In some embodiments of the ments of the method, the method does not include adminis method, the furocoumarin is psoralen. In some embodiments tration of a prime HV (e.g. HSV. HHV-1, HHV-2, HHV-3, of the method, the furocoumarin is 4'-aminomethyl-4,5'.8- HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) DNA vaccine. trimethylpsoralen. In some embodiments of the method, the In some embodiments of the method, the method does not furocoumarin is angelicin. In some embodiments of the include administration of an HV (e.g. HSV. HHV-1, HHV-2, method, the furocoumarin is Xanthotoxin. In some embodi HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) DNA ments of the method, the furocoumarin is bergapten. In some vaccine. In some embodiments of the method, the method embodiments of the method, the furocoumarin is nodakene does not include administration of a DNA vaccine including tin. a gene selected from HSVUL30, UL5, gD2, gD2t, orportions US 2014/0193460 A1 Jul. 10, 2014

thereof. In some embodiments of the method, the method herpes esophagitis. In some embodiments of the method, the administration consists of a single administration of the HV disease is herpes keratitis. In some embodiments of the (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, method, the disease is Bell's palsy. In some embodiments of HHV-7, or HHV-8) vaccine. In some embodiments of the the method, the disease is Mollaret's meningitis. In some method, the method administration consists of a prime-boost embodiments of the method, the disease is herpes rug administration of the HV (e.g. HSV. HHV-1, HHV-2, HHV-3, beiorum. In some embodiments of the method, the disease is HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vaccine. In eczema herpeticum. In some embodiments of the method, the some embodiments of the method, the method administration disease is herpetic neuralgia. In some embodiments of the consists of a prime-boost-boost administration of the HV method, the disease is post-herpetic neuralgia. In some (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vaccine. In some embodiments of the embodiments of the method, the disease is Alzheimer's dis method of treating or preventing a disease, the method ease. In some embodiments of the method, the method is a includes treating the disease. In some embodiments of the method of treating. In some embodiments of the method, the method of treating or preventing a disease, the method method is a method of preventing. In some embodiments of includes preventing the disease. In some embodiments of the the method, the method includes a reduction in viral shed method of treating or preventing a disease, the method is ding. In some embodiments of the method, the method treating the disease. In some embodiments of the method of includes a reduction in the frequency of lesion occurrence. In treating or preventing a disease, the method is preventing the some embodiments of the method, the method includes a disease. In some embodiments of the method of treating or reduction in the duration of lesion occurrence. In some preventing a disease, the method includes administering a embodiments of the method, the method includes intramus therapeutically effective amount of an HV vaccine as cular administration. In some embodiments of the method, described herein (including embodiments). In some embodi the method includes intradermal administration. In some ments of the method of treating or preventing a disease, the embodiments of the method, the method includes mucosal administration. In some embodiments of the method, the method includes administering a prophylactically effective method includes intranasal administration. In some embodi amount of an HV Vaccine as described herein (including ments of the method, the method includes intrarectal admin embodiments). istration. In some embodiments of the method, the method 0116 Provided in a fourth aspect is a method of treating or includes intravaginal administration. In some embodiments preventing a disease in a patient in need of the treatment or of the method, the method includes topical administration. In prevention, the method including administering a therapeuti some embodiments of the method, the method includes tran cally or prophylactically effective amount of any one of the Scutaneous administration. In some embodiments of the HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, method, the method includes Subcutaneous administration. In HHV-6, HHV-7, or HHV-8) vaccines described herein (in Some embodiments of the method, an isolated nucleic acid cluding embodiments). sequence formulation is not administered to the patient. In 0117. In some embodiments of the method, the disease is some embodiments of the method, the isolated nucleic acid an HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, sequence formulation is a DNA sequence. In some embodi HHV-6, HHV-7, or HHV-8) associated disease. In some ments of the method, the isolated nucleic acid sequence for embodiments of the method, the HV is HSV. In some embodi mulation is a nucleic acid vaccine. In some embodiments of ments of the method, the HSV is HSV1. In some embodi the method, the isolated nucleic acid sequence formulation is ments of the method, the HSV is HSV2. In some embodi a DNA vaccine. In some embodiments of the method, the ments of the method, the HSV is HSV1 and HSV2. In some method does not include administration of a prime HV (e.g. embodiments of the method, the HV is HHV-1. In some HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, embodiments of the method, the HV is HHV-2. In some HHV-7, or HHV-8) DNA vaccine. In some embodiments of embodiments of the method, the HV is HHV-3. In some the method, the method does not include administration of an embodiments of the method, the HV is HHV-4. In some HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, embodiments of the method, the HV is HHV-5. In some HHV-6, HHV-7, or HHV-8) DNA vaccine. In some embodi embodiments of the method, the HV is HHV-6. In some ments of the method, the method does not include adminis embodiments of the method, the HV is HHV-7. In some tration of a DNA vaccine including a gene selected from HSV embodiments of the method, the HV is HHV-8. In some UL30, UL5, gD2, gD2t, or portions thereof. In some embodi embodiments of the method, the disease is selected from the ments of the method, the method administration consists of a group consisting of herpetic gingivostomatitis, herpes labia single administration of the HV (e.g. HSV. HHV-1, HHV-2, lis, herpes genitalis, herpetic whitlow, herpes gladiatorum, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vac herpesviral encephalitis, herpesviral meningitis, herpes cine. In some embodiments of the method, the method admin esophagitis, herpes keratitis, Bell’s palsy, Mollaret's menin istration consists of a prime-boost administration of the HV gitis, herpes rugbeiorum, eczema herpeticum, herpetic neu (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, ralgia, and post-herpetic neuralgia. In some embodiments of HHV-7, or HHV-8) vaccine. In some embodiments of the the method, the disease is herpetic gingivostomatitis. In some method, the method administration consists of a prime-boost embodiments of the method, the disease is herpes labialis. In boost administration of the HV (e.g. HSV. HHV-1, HHV-2, Some embodiments of the method, the disease is herpes geni HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vac talis. In some embodiments of the method, the disease is cine. In some embodiments of the method, a patient is pro herpetic whitlow. In some embodiments of the method, the tected against one or more strains of an HV (e.g. HSV. HHV disease is herpes gladiatorum. In some embodiments of the 1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or method, the disease is herpesviral encephalitis. In some HHV-8) following administration of the HV (e.g. HSV. HHV embodiments of the method, the disease is herpesviral men 1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or ingitis. In some embodiments of the method, the disease is HHV-8) vaccine. In some embodiments of the method, a US 2014/0193460 A1 Jul. 10, 2014 20 patient is prevented from being infected by, contracting, hav treating or preventing an HSV infection, the HSV infection ing, or getting one or more strains of an HV (e.g. HSV. causes a disease selected from the group consisting of her HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or petic gingivostomatitis, herpes labialis, herpes genitalis, her HHV-8) following administration of the HV (e.g. HSV. HHV petic whitlow, herpes gladiatorum, herpesviral encephalitis, 1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or herpesviral meningitis, herpes esophagitis, herpes keratitis, HHV-8) vaccine. In some embodiments of the method, a Bell’s palsy, Mollaret's meningitis, herpes rugbeiorum, patient is prevented from being infected by, contracting, hav eczema herpeticum, herpetic neuralgia, and post-herpetic ing, or getting more than one strain of an HV (e.g. HSV. neuralgia. In some embodiments of the method of treating or HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or preventing an HSV infection, the method includes treating HHV-8) following administration of the HV (e.g. HSV. HHV the HSV infection. In some embodiments of the method of 1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or treating or preventing an HSV infection, the method includes HHV-8) vaccine. In some embodiments of the method, a preventing the HSV infection. In some embodiments of the patient treated for one or more strains of an HV (e.g. HSV. method of treating or preventing an HSV infection, the HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or method is treating the HSV infection. In some embodiments HHV-8) following administration of the HV (e.g. HSV. HHV of the method of treating or preventing an HSV infection, the 1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or method is preventing the HSV infection. In some embodi HHV-8) vaccine. In some embodiments of the method of ments of the method of treating or preventing an HSV infec treating or preventing a disease, the method includes treating tion, the method includes administering a therapeutically the disease. In some embodiments of the method of treating or effective amount of an HSV vaccine as described herein preventing a disease, the method includes preventing the dis (including embodiments). In some embodiments of the ease. In some embodiments of the method of treating or method of treating or preventing an HSV infection, the preventing a disease, the method is treating the disease. In method includes administering a prophylactically effective Some embodiments of the method of treating or preventing a amount of an HSV vaccine as described herein (including disease, the method is preventing the disease. In some embodiments). In some embodiments of the method of treat embodiments of the method of treating or preventing a dis ing or preventing an HSV infection, the method includes a ease, the method includes administering a therapeutically reduction in viral shedding. In some embodiments of the effective amount of an HV vaccine as described herein (in method of treating or preventing an HSV infection, the cluding embodiments). In some embodiments of the method method includes a reduction in the frequency of lesion occur of treating or preventing a disease, the method includes rence. In some embodiments of the method of treating or administering a prophylactically effective amount of an HV preventing an HSV infection, the method includes a reduction vaccine as described herein (including embodiments). in the duration of lesion occurrence. In some embodiments of 0118 Provided in a fifth aspect is a kit including any one of the method of treating or preventing an HSV infection, the the HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, method includes intramuscular administration of an HSV HHV-6, HHV-7, or HHV-8) vaccines as described herein vaccine as described herein (including embodiments). In (including embodiments) and instructions for administering Some embodiments of the method of treating or preventing an the HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HSV infection, the method includes intradermal administra HHV-6, HHV-7, or HHV-8) vaccine to a patient. In some tion of an HSV vaccine as described herein (including embodiments of the kit, the HV (e.g. HSV. HHV-1, HHV-2, embodiments). In some embodiments of the method of treat HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vaccine ing or preventing an HSV infection, the method includes is administered in a prime-boost administration. In some mucosal administration of an HSV vaccine as described embodiments of the kit, the HV is HHV-1. In some embodi herein (including embodiments). In some embodiments of ments of the kit, the HV is HHV-2. In some embodiments of the method of treating or preventing an HSV infection, the the kit, the HV is HHV-3. In some embodiments of the kit, the method includes intranasal administration of an HSV vaccine HV is HHV-4. In some embodiments of the kit, the HV is as described herein (including embodiments). In some HHV-5. In some embodiments of the kit, the HV is HHV-6. In embodiments of the method of treating or preventing an HSV some embodiments of the kit, the HV is HHV-7. In some infection, the method includes intrarectal administration of embodiments of the kit, the HV is HHV-8. In some embodi an HSV vaccine as described herein (including embodi ments of the kit, the HV (e.g. HSV. HHV-1, HHV-2, HHV-3, ments). In some embodiments of the method of treating or HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vaccine treats a preventing an HSV infection, the method includes intravagi patient for one or more strains of the HV (e.g. HSV. HHV-1, nal administration of an HSV vaccine as described herein HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV (including embodiments). In some embodiments of the 8). HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, method of treating or preventing an HSV infection, the HHV-6, HHV-7, or HHV-8) method includes topical administration of an HSV vaccine as 0119 Provided in another aspect is a method of treating or described herein (including embodiments). In some embodi preventing an HSV infection in a patient in need of the treat ments of the method of treating or preventing an HSV infec ment or prevention. The method including administering a tion, the method includes Subcutaneous administration of an therapeutically or prophylactically effective amount of an HSV vaccine as described herein (including embodiments). HSV vaccine described herein (including embodiments). In some embodiments of the method of treating or preventing 0120 In some embodiments of the method of treating or an HSV infection, the method includes transcutaneous preventing an HSV infection, the HSV is HSV1. In some administration of an HSV vaccine as described herein (in embodiments of the method of treating or preventing an HSV cluding embodiments). infection, the HSV is HSV2. In some embodiments of the I0121. In some embodiments of the method of treating or method of treating or preventing an HSV infection, the HSV preventing an HSV infection, the method does include is HSV1 and HSV2. In some embodiments of the method of administration of an HSV DNA vaccine (e.g. HSV1 DNA US 2014/0193460 A1 Jul. 10, 2014

vaccine, HSV2 DNA vaccine, HSV1 and HSV2 DNA vac wherein a trace amount of a nucleic acid is an amount less cine). In some embodiments of the method of treating or thananamount necessary to elicitan antigen specific immune preventing an HSV infection, the method does include response to the nucleic acid or a protein expressed from or by administration of prime HSV DNA vaccine (e.g. HSV1 DNA the nucleic acid. In some embodiments, a trace amount of a vaccine, HSV2 DNA vaccine, HSV1 and HSV2 DNA vac nucleic acid is less than about 12.5, 10, 9, 8.5, 8, 7, 6, 5, 4, 3, cine). In some embodiments of the method of treating or 2, 1, 0.9, 0.8, 0.7, 0.6,0.5,0.4, 0.3, 0.2, 0.1, 0.009, 0.008, preventing an HSV infection, the method does include 0.007, 0.006, 0.005, 0.004, 0.003, 0.002, 0.001 micrograms administration of an HSV DNA vaccine including a UL30 of the nucleic acid. In some embodiments, a trace amount of gene or portion of the gene. In some embodiments of the nucleic acid is less than about 12.5, 10, 9, 8.5, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6,0.5,0.4, 0.3, 0.2, 0.1, 0.009, 0.008, method of treating or preventing an HSV infection, the 0.007, 0.006, 0.005, 0.004, 0.003, 0.002, 0.001 micrograms method does include administration of an HSV DNA vaccine of the nucleic acid per dose of vaccine. In some embodiments, including a UL5 gene orportion of the gene. In some embodi a trace amount of nucleic acid is less than about 12.5, 10, 9, ments of the method of treating or preventing an HSV infec 8.5, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6,0.5,0.4,0.3, 0.2,0.1, tion, the method does include administration of an HSV DNA 0.009, 0.008, 0.007, 0.006, 0.005, 0.004, 0.003, 0.002, 0.001 vaccine including a gC2 gene orportion of the gene. In some micrograms of the nucleic acid per dose of vaccine adminis embodiments of the method of treating or preventing an HSV tered i.m. In some embodiments, a trace amount of nucleic infection, the method does include administration of an HSV acid is less than 12.5, 10, 9, 8.5, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, DNA vaccine including ag|D2t gene orportion of the gene. In 0.7, 0.6,0.5,0.4, 0.3, 0.2, 0.1, 0.009, 0.008, 0.007, 0.006, Some embodiments of the method of treating or preventing an 0.005, 0.004, 0.003, 0.002, 0.001 micrograms of the nucleic HSV infection, the method does include administration of an acid. In some embodiments, a trace amount of nucleic acid is HSV protein vaccine (e.g. HSV1 protein vaccine, HSV2 pro less than 12.5, 10, 9, 8.5, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, tein vaccine, HSV1 and HSV2 protein vaccine). In some 0.5,0.4,0.3, 0.2,0.1, 0.009, 0.008, 0.007, 0.006, 0.005, 0.004, embodiments of the method of treating or preventing an HSV 0.003, 0.002, 0.001 micrograms of the nucleic acid per dose infection, the method does include administration of an HSV of vaccine. In some embodiments, a trace amount of nucleic protein vaccine including a gC2t protein. In some embodi acid is less than 12.5, 10, 9, 8.5, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, ments of the method of treating or preventing an HSV infec 0.7, 0.6,0.5,0.4, 0.3, 0.2, 0.1, 0.009, 0.008, 0.007, 0.006, tion, the method does not include administration of an HSV 0.005, 0.004, 0.003, 0.002, 0.001 micrograms of the nucleic DNA vaccine (e.g. HSV1 DNA vaccine, HSV2 DNA vaccine, acid per dose of vaccine administered im. In some embodi HSV1 and HSV2 DNA vaccine). In some embodiments of the ments, a trace amount of nucleic acid is 1 microgram. In some method of treating or preventing an HSV infection, the embodiments, a trace amount of nucleic acid is 0.5 micro method does not include administration of prime HSV DNA grams. In some embodiments, a trace amount of nucleic acid vaccine (e.g. HSV1 DNA vaccine, HSV2 DNA vaccine, is an amountless than an amount set forth in DNA vaccines in HSV1 and HSV2 DNA vaccine). In some embodiments of the WO2007 106404 or administered to elicit an immune method of treating or preventing an HSV infection, the method does not include administration of an HSV DNA response in WO2007 106404. In some embodiments of the vaccine including a UL30 gene orportion of the gene. In some method, an isolated nucleic acid sequence formulation is not embodiments of the method of treating or preventing an HSV administered to the patient. In some embodiments of the infection, the method does not include administration of an method, the isolated nucleic acid sequence formulation is a HSV DNA vaccine including a UL5 gene or portion of the DNA sequence. In some embodiments of the method, the gene. In some embodiments of the method of treating or isolated nucleic acid sequence formulation is a nucleic acid preventing an HSV infection, the method does not include vaccine. In some embodiments of the method, the isolated administration of an HSV DNA vaccine including a g|D2 gene nucleic acid sequence formulation is a DNA vaccine. orportion of the gene. In some embodiments of the method of 0.122 Provided in another aspect is a method of preparing treating or preventing an HSV infection, the method does not a Herpes Simplex Virus (HSV) vaccine, the method includ include administration of an HSV DNA vaccine including a ing: contacting an HSV-cell mixture with a sulfated or sul gD2t gene orportion of the gene. In some embodiments of the fonated polysaccharide, wherein the HSV-cell mixture method of treating or preventing an HSV infection, the includes HSV particles, cells and portions of cells; separating method does not include administration of an HSV protein the HSV particles from the cells thereby forming isolated vaccine (e.g. HSV1 protein vaccine, HSV2 protein vaccine, HSV particles; inactivating the isolated HSV particles HSV1 and HSV2 protein vaccine). In some embodiments of thereby forming inactivated HSV particles; combining the the method of treating or preventing an HSV infection, the inactivated HSV particles with a lipopolysaccharide-derived method does not include administration of an HSV protein adjuvant and an aluminum-based mineral salt adjuvant vaccine including a g|D2t protein. It will be understood by a thereby forming an HSV vaccine. person of ordinary skill in the art that when a composition or I0123. In some embodiments of the method of preparing an method that does not include a DNA vaccine or a nucleic acid HSV vaccine, the HSV is HSV1. In some embodiments of the sequence is recited, it is intended that such composition or method of preparing an HSV vaccine, the HSV is HSV2. In method does not include the intentional addition of a compo some embodiments of the method of preparing an HSV vac sition comprising a nucleic acid composition (e.g. DNA vac cine, the HSV is HSV1 and HSV2. In some embodiments of cine, nucleic acid vaccine, antigenic nucleic acid, nucleic acid the method of preparing an HSV vaccine, the sulfated or sequence, or other nucleic acid composition) described as Sulfonated polysaccharide is selected from a group consisting being excluded or lacking In some embodiments a composi of dextran Sulfate, heparin, heparan Sulfate. In some embodi tion or method that does not include a nucleic acid composi ments of the method of preparing an HSV vaccine, the HSV tion may include a nucleic acid composition in trace amounts particles are isolated at a concentration of 107 to 10' (pfu/ or as included in another component of the composition, microgram protein). In some embodiments of the method of US 2014/0193460 A1 Jul. 10, 2014 22 preparing an HSV vaccine, the HSV vaccine includes 10 to ide oraluminum hydroxycarbonate or magnesium hydroxide. 10' (pful equivalents/microgram protein) of inactivated HSV. In some embodiments of the method of preparing an HSV In some embodiments of the method of preparing an HSV vaccine, the aluminum-based mineral salt adjuvant includes vaccine, the separating is selected from a group consisting of aluminum phosphate gel in the form of a white gelatinous centrifugation, polyethylene glycol precipitation, filtration, precipitate. In some embodiments of the method of preparing gel filtration, ultra-filtration, tangential flow ultra-filtration, an HSV vaccine, the aluminum-based mineral salt adjuvant or affinity chromatography. In some embodiments of the includes aluminum hydroxide gel in the form of a white method of preparing an HSV vaccine, the lipopolysaccha gelatinous precipitate. In some embodiments of the method of ride-derived adjuvant is derived from the Salmonella minine preparing an HSV vaccine, the inactivating including contact sota LPS. In some embodiments of the method of preparing ing the HSV with one or more agents selected from the group an HSV vaccine, the lipopolysaccharide-derived adjuvant is consisting of a cross-linking agent, oxidizing agent, reducing derived from the Salmonella minnesota Re595 LPS. In some agent, heat, radiation, detergent, a pH changing agent, and a embodiments of the method of preparing an HSV vaccine, the chemical agent selected from a furocoumarin, aziridine, eth lipopolysaccharide-derived adjuvant is derived from the ylenimine, binary ethylenimine, and beta-propiolactone. In R595 LPS. In some embodiments of the method of preparing some embodiments of the method of preparing an HSV vac an HSV vaccine, the lipopolysaccharide-derived adjuvant is a cine, the inactivating includes contacting the HSV with aziri lipid A adjuvant. In some embodiments of the method of dine. In some embodiments of the method of preparing an preparing an HSV vaccine, the lipopolysaccharide-derived HSV vaccine, the inactivating includes contacting the HSV adjuvant is a lipid A adjuvant without a (R)-3-hydroxytet with ethylenimine. In some embodiments of the method of radecanoyl moiety. In some embodiments of the method of preparing an HSV vaccine, the inactivating includes contact preparing an HSV vaccine, the lipopolysaccharide-derived ing the HSV with binary ethylenimine. In some embodiments adjuvant is a lipid A adjuvant without a 1-phosphate moiety. of the method of preparing an HSV vaccine, the inactivating In some embodiments of the method of preparing an HSV includes contacting the HSV with beta-propiolactone. In vaccine, the lipopolysaccharide-derived adjuvant is a lipid A some embodiments of the method of preparing an HSV vac adjuvant that does not include a (R)-3-hydroxytetradecanoyl cine, the inactivating includes contacting the HSV with UV moiety. In some embodiments of the method of preparing an radiation. In some embodiments of the method of preparing HSV vaccine, the lipopolysaccharide-derived adjuvant is a an HSV vaccine, the inactivating includes contacting the lipid A adjuvant that does not include a 1-phosphate moiety. HSV with electronbeam radiation. In some embodiments of In some embodiments of the method of preparing an HSV the method of preparing an HSV vaccine, the inactivating vaccine, the lipopolysaccharide-derived adjuvant is mono includes contacting the HSV with infrared radiation. In some phosphoryl lipid A (MPL). In some embodiments of the embodiments of the method of preparing an HSV vaccine, the method of preparing an HSV vaccine, the lipopolysaccha inactivating includes contacting the HSV with gamma radia ride-derived adjuvant is 3-O-desacyl-4'-monophosphoryl tion. In some embodiments of the method of preparing an lipid A. In some embodiments of the method of preparing an HSV vaccine, the inactivating includes contacting the HSV HSV vaccine, the lipopolysaccharide-derived adjuvant is a with aldehyde cross-linking agent. In some embodiments of synthetic MPL analogue adjuvant. In some embodiments of the method of preparing an HSV vaccine, the inactivating the method of preparing an HSV vaccine, the lipopolysaccha includes contacting the HSV with formaldehyde. In some ride-derived adjuvant is capable of binding the TLR4 protein. embodiments of the method of preparing an HSV vaccine, the In some embodiments of the method of preparing an HSV inactivating includes contacting the HSV with formalin. In vaccine, the aluminum-based mineral salt adjuvant is an alu some embodiments of the method of preparing an HSV vac minum hydroxide adjuvant. In some embodiments of the cine, the inactivating includes contacting the HSV with method of preparing an HSV vaccine, the aluminum-based sodium periodate. In some embodiments of the method of mineral salt adjuvant is an aluminum phosphate adjuvant. In preparing an HSV vaccine, the inactivating includes contact some embodiments of the method of preparing an HSV vac ing the HSV with hydrogen peroxide. In some embodiments cine, the aluminum-based mineral salt adjuvant is a potas of the method of preparing an HSV vaccine, the inactivating sium aluminum Sulfate adjuvant. In some embodiments of the includes contacting the HSV with aldrithiol-2. In some method of preparing an HSV vaccine, the aluminum-based embodiments of the method of preparing an HSV vaccine, the mineral salt adjuvant includes an aluminum hydroxide adju inactivating includes contacting the HSV with Triton-X-100. vant. In some embodiments of the method of preparing an In some embodiments of the method of preparing an HSV HSV vaccine, the aluminum-based mineral salt adjuvant vaccine, the inactivating includes contacting the HSV with includes an aluminum phosphate adjuvant. In some embodi NP-40. In some embodiments of the method of preparing an ments of the method of preparing an HSV vaccine, the alu HSV vaccine, the inactivating includes contacting the HSV minum-based mineral salt adjuvant includes a potassium alu with Tween-20. In some embodiments of the method of pre minum sulfate adjuvant. In some embodiments of the method paring an HSV vaccine, the inactivating includes contacting of preparing an HSV vaccine, the aluminum-based mineral the HSV with UV radiation and a furocoumarin. In some salt adjuvant is an aluminum hydroxide adjuvant. In some embodiments of the method of preparing an HSV vaccine, the embodiments of the method of preparing an HSV vaccine, the inactivating includes contacting the HSV with UV radiation aluminum-based mineral salt adjuvant is an aluminum phos and psoralen. In some embodiments of the method of prepar phate adjuvant. In some embodiments of the method of pre ing an HSV vaccine, the inactivating includes contacting the paring an HSV vaccine, the aluminum-based mineral salt HSV with UV radiation and 4'-aminomethyl-4,5'.8-trimeth adjuvant is a potassium aluminum sulfate adjuvant. In some ylpsoralen. In some embodiments of the method of preparing embodiments of the method of preparing an HSV vaccine, the an HSV vaccine, the inactivating includes contacting the aluminum-based mineral salt adjuvant includes crystalline HSV with UV radiation and angelicin. In some embodiments aluminum hydroxide and not amorphous aluminum hydrox of the method of preparing an HSV vaccine, the inactivating US 2014/0193460 A1 Jul. 10, 2014

includes contacting the HSV with UV radiation and xantho ments, a chemical agent for inactivating HSV is binary eth toxin. In some embodiments of the method of preparing an ylenimine. In some embodiments, a chemical agent for inac HSV vaccine, the inactivating includes contacting the HSV tivating HSV is beta-propiolactone. In some embodiments, a with UV radiation and bergapten. In some embodiments of radiation for inactivating HSV is UV radiation. In some the method of preparing an HSV vaccine, the inactivating embodiments, a radiation for inactivating HSV is electron includes contacting the HSV with UV radiation and nodak beam radiation. In some embodiments, a radiation for inacti enetin. In some embodiments of the method of preparing an vating HSV is infrared radiation. In some embodiments, a HSV vaccine, the inactivating is contacting the HSV with radiation for inactivating HSV is gamma radiation. In some aziridine. In some embodiments of the method of preparing embodiments, a cross-linking agent for inactivating HSV is an HSV vaccine, the inactivating is contacting the HSV with an aldehyde cross-linking agent. In some embodiments, a ethylenimine. In some embodiments of the method of prepar cross-linking agent for inactivating HSV is formaldehyde. In ing an HSV vaccine, the inactivating is contacting the HSV Some embodiments, a cross-linking agent for inactivating with binary ethylenimine. In some embodiments of the HSV is formalin. In some embodiments, across-linking agent method of preparing an HSV vaccine, the inactivating is for inactivating HSV is sodium periodate. In some embodi contacting the HSV with beta-propiolactone. In some ments, an oxidizing agent for inactivating HSV is hydrogen embodiments of the method of preparing an HSV vaccine, the peroxide. In some embodiments, a reducing agent for inacti inactivating is contacting the HSV with UV radiation. In vating HSV is aldrithiol-2. In some embodiments, a detergent some embodiments of the method of preparing an HSV vac for inactivating HSV is Triton-X-100. In some embodiments, cine, the inactivating is contacting the HSV with electron a detergent for inactivating HSV is NP-40. In some embodi beam radiation. In some embodiments of the method of pre ments, a detergent for inactivating HSV is Tween-20. In some paring an HSV vaccine, the inactivating is contacting the embodiments, a furocoumarin for inactivating HSV is psor HSV with infrared radiation. In some embodiments of the alen. In some embodiments, a furocoumarin for inactivating method of preparing an HSV vaccine, the inactivating is HSV is 4'-aminomethyl-4,5'.8-trimethylpsoralen. In some contacting the HSV with gamma radiation. In some embodi embodiments, a furocoumarin for inactivating HSV is angeli ments of the method of preparing an HSV vaccine, the inac cin. In some embodiments, a furocoumarin for inactivating tivating is contacting the HSV with aldehyde cross-linking HSV is xanthotoxin. In some embodiments, a furocoumarin agent. In some embodiments of the method of preparing an for inactivating HSV is bergapten. In some embodiments, a HSV vaccine, the inactivating is contacting the HSV with furocoumarin for inactivating HSV is nodakenetin. formaldehyde. In some embodiments of the method of pre 0.125 Provided in another aspect is a method of treating or paring an HSV vaccine, the inactivating is contacting the preventing a disease in a patient in need of the treatment or HSV with formalin. In some embodiments of the method of prevention. The method including administering a therapeu preparing an HSV vaccine, the inactivating is contacting the tically or prophylactically effective amount of an HSV vac HSV with sodium periodate. In some embodiments of the cine as described herein (including embodiments). In some method of preparing an HSV vaccine, the inactivating is embodiments, a patient in need of preventing a disease is a contacting the HSV with hydrogen peroxide. In some patient at risk of developing, contracting, getting, or having embodiments of the method of preparing an HSV vaccine, the the disease. inactivating is contacting the HSV with aldrithiol-2. In some I0126. In some embodiments of the method of treating or embodiments of the method of preparing an HSV vaccine, the preventing a disease, the disease is associated with HSV 1. In inactivating is contacting the HSV with Triton-X-100. In Some embodiments of the method of treating or preventing a some embodiments of the method of preparing an HSV vac disease, the disease is caused by HSV1. In some embodi cine, the inactivating is contacting the HSV with NP-40. In ments of the method of treating or preventing a disease, the some embodiments of the method of preparing an HSV vac disease is associated with HSV2. In some embodiments of the cine, the inactivating is contacting the HSV with Tween-20. method of treating or preventing a disease, the disease is In some embodiments of the method of preparing an HSV caused by HSV2. In some embodiments of the method of vaccine, the inactivating is contacting the HSV with UV treating or preventing a disease, the disease is selected from radiation and a furocoumarin. In some embodiments of the the group consisting of herpetic gingivostomatitis, herpes method of preparing an HSV vaccine, the inactivating is labialis, herpes genitalis, herpetic whitlow, herpes gladi contacting the HSV with UV radiation and psoralen. In some atorum, herpesviral encephalitis, herpesviral meningitis, her embodiments of the method of preparing an HSV vaccine, the pes esophagitis, herpes keratitis, Bell's palsy, Mollaret's inactivating is contacting the HSV with UV radiation and meningitis, herpes rugbeiorum, eczema herpeticum, herpetic 4'-aminomethyl-4,5".8-trimethylpsoralen. In some embodi neuralgia, and post-herpetic neuralgia. In some embodiments ments of the method of preparing an HSV vaccine, the inac of the method of treating or preventing a disease, the disease tivating is contacting the HSV with UV radiation and angeli is herpetic gingivostomatitis. In some embodiments of the cin. In some embodiments of the method of preparing an HSV method of treating or preventing a disease, the disease is vaccine, the inactivating is contacting the HSV with UV herpes labialis. In some embodiments of the method of treat radiation and Xanthotoxin. In some embodiments of the ing or preventing a disease, the disease is herpes genitalis. In method of preparing an HSV vaccine, the inactivating is Some embodiments of the method of treating or preventing a contacting the HSV with UV radiation and bergapten. In disease, the disease is herpetic whitlow. In some embodi some embodiments of the method of preparing an HSV vac ments of the method of treating or preventing a disease, the cine, the inactivating is contacting the HSV with UV radiation disease is herpes gladiatorum. In some embodiments of the and nodakenetin. method of treating or preventing a disease, the disease is 0124. In some embodiments, a chemical agent for inacti herpesviralencephalitis. In some embodiments of the method Vating HSV is aziridine. In some embodiments, a chemical of treating or preventing a disease, the disease is herpesviral agent for inactivating HSV is ethylenimine. In some embodi meningitis. In some embodiments of the method of treating or US 2014/0193460 A1 Jul. 10, 2014 24 preventing a disease, the disease is herpes esophagitis. In includes transcutaneous administration of an HSV vaccine as Some embodiments of the method of treating or preventing a described herein (including embodiments). disease, the disease is herpes keratitis. In some embodiments I0127. In some embodiments of the method of treating or of the method of treating or preventing a disease, the disease preventing a disease, the method does include administration is Bell's palsy. In some embodiments of the method of treat of an HSV DNA vaccine (e.g. HSV1 DNA vaccine, HSV2 ing or preventing a disease, the disease is Mollaret's menin DNA vaccine, HSV1 and HSV2 DNA vaccine). In some gitis. In some embodiments of the method of treating or embodiments of the method of treating or preventing a dis preventing a disease, the disease is herpes rugbeiorum. In ease, the method does include administration of prime HSV Some embodiments of the method of treating or preventing a DNA vaccine (e.g. HSV1 DNA vaccine, HSV2 DNA vaccine, disease, the disease is eczema herpeticum. In some embodi HSV1 and HSV2 DNA vaccine). In some embodiments of the ments of the method of treating or preventing a disease, the method of treating or preventing a disease, the method does disease is herpetic neuralgia. In some embodiments of the include administration of an HSV DNA vaccine including a method of treating or preventing a disease, the disease is UL30 gene or portion of the gene. In some embodiments of post-herpetic neuralgia. In some embodiments of the method the method of treating or preventing a disease, the method of treating or preventing a disease, the disease is Alzheimer's does include administration of an HSV DNA vaccine includ disease. In some embodiments of the method of treating or ing a UL5 gene or portion of the gene. In some embodiments preventing a disease, the method includes treating the disease. of the method of treating or preventing a disease, the method In some embodiments of the method of treating or preventing does include administration of an HSV DNA vaccine includ a disease, the method includes preventing the disease. In ing a g|D2 gene or portion of the gene. In some embodiments of the method of treating or preventing a disease, the method Some embodiments of the method of treating or preventing a does include administration of an HSV DNA vaccine includ disease, the method is treating the disease. In some embodi ing a g|D2t gene orportion of the gene. In some embodiments ments of the method of treating or preventing a disease, the of the method of treating or preventing a disease, the method method is preventing the disease. In some embodiments of the does include administration of an HSV protein vaccine (e.g. method of treating or preventing a disease, the method HSV1 protein vaccine, HSV2 protein vaccine, HSV1 and includes administering a therapeutically effective amount of HSV2 protein vaccine). In some embodiments of the method an HSV vaccine as described herein (including embodi of treating or preventing a disease, the method does include ments). In some embodiments of the method of treating or administration of an HSV protein vaccine including a g|D2t preventing a disease, the method includes administering a protein. In some embodiments of the method of treating or prophylactically effective amount of an HSV vaccine as preventing a disease, the method does not include adminis described herein (including embodiments). In some embodi tration of an HSV DNA vaccine (e.g. HSV1 DNA vaccine, ments of the method of treating or preventing a disease, the HSV2 DNA vaccine, HSV1 and HSV2 DNA vaccine). In method includes a reduction in viral shedding. In some Some embodiments of the method of treating or preventing a embodiments of the method of treating or preventing a dis disease, the method does not include administration of prime ease, the method includes a reduction in the frequency of HSV DNA vaccine (e.g. HSV1 DNA vaccine, HSV2 DNA lesion occurrence. In some embodiments of the method of vaccine, HSV1 and HSV2 DNA vaccine). In some embodi treating or preventing a disease, the method includes a reduc ments of the method of treating or preventing a disease, the tion in the duration of lesion occurrence. In some embodi method does not include administration of an HSV DNA ments of the method of treating or preventing a disease, the vaccine including a UL30 gene orportion of the gene. In some method includes intramuscular administration of an HSV embodiments of the method of treating or preventing a dis vaccine as described herein (including embodiments). In ease, the method does not include administration of an HSV Some embodiments of the method of treating or preventing a DNA vaccine including a UL5 gene or portion of the gene. In disease, the method includes intradermal administration of an Some embodiments of the method of treating or preventing a HSV vaccine as described herein (including embodiments). disease, the method does not include administration of an In some embodiments of the method of treating or preventing HSV DNA vaccine including a gC2 gene or portion of the a disease, the method includes mucosal administration of an gene. In some embodiments of the method of treating or HSV vaccine as described herein (including embodiments). preventing a disease, the method does not include adminis In some embodiments of the method of treating or preventing tration of an HSV DNA vaccine including a gl)2t gene or a disease, the method includes intranasal administration of an portion of the gene. In some embodiments of the method of HSV vaccine as described herein (including embodiments). treating or preventing a disease, the method does not include In some embodiments of the method of treating or preventing administration of an HSV protein vaccine (e.g. HSV1 protein a disease, the method includes intrarectal administration of an vaccine, HSV2 protein vaccine, HSV1 and HSV2 protein HSV vaccine as described herein (including embodiments). vaccine). In some embodiments of the method of treating or In some embodiments of the method of treating or preventing preventing a disease, the method does not include adminis a disease, the method includes intravaginal administration of tration of an HSV protein vaccine including a gC2t protein. It an HSV vaccine as described herein (including embodi will be understood by a person of ordinary skill in the art that ments). In some embodiments of the method of treating or when a composition or method that does not include a DNA preventing a disease, the method includes topical administra vaccine or a nucleic acid sequence is recited, it is intended that tion of an HSV vaccine as described herein (including Such composition or method does not include the intentional embodiments). In some embodiments of the method of treat addition of a composition comprising a nucleic acid compo ing or preventing a disease, the method includes Subcutane sition (e.g. DNA vaccine, nucleic acid vaccine, antigenic ous administration of an HSV vaccine as described herein nucleic acid, nucleic acid sequence, or other nucleic acid (including embodiments). In some embodiments of the composition) described as being excluded or lacking In some method of treating or preventing a disease, the method embodiments a composition or method that does not include US 2014/0193460 A1 Jul. 10, 2014 a nucleic acid composition may include a nucleic acid com able for ingestion by the patient. The compositions of the position in trace amounts or as included in another compo present invention can also be administered by injection, that nent of the composition, wherein a trace amount of a nucleic is, intravenously, intramuscularly, intracutaneously, Subcuta acid is an amount less than an amount necessary to elicit an neously, intraduodenally, or intraperitoneally. Also, the com antigen specific immune response to the nucleic acid or a positions described herein can be administered by inhalation, protein expressed from or by the nucleic acid. In some for example, intranasally. Additionally, the compositions of embodiments, a trace amount of a nucleic acid is less than the present invention can be administered transdermally. It is about 12.5, 10,9,8.5, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6,0.5, also envisioned that multiple routes of administration (e.g., 0.4, 0.3, 0.2, 0.1, 0.009, 0.008, 0.007, 0.006, 0.005, 0.004, intramuscular, oral, transdermal, mucosal, intranasal, 0.003, 0.002, 0.001 micrograms of the nucleic acid. In some intrarectal, intravaginal, Subcutaneous, transcutaneous, topi embodiments, a trace amount of nucleic acid is less than cal, intradermal) can be used to administer the compositions about 12.5, 10,9,8.5, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6,0.5, of the invention. Accordingly, the present invention also pro 0.4, 0.3, 0.2, 0.1, 0.009, 0.008, 0.007, 0.006, 0.005, 0.004, vides pharmaceutical compositions including a pharmaceu 0.003, 0.002, 0.001 micrograms of the nucleic acid per dose tically acceptable excipient and one or more compositions of vaccine. In some embodiments, a trace amount of nucleic described herein (including embodiments). acid is less than about 12.5, 10, 9, 8.5, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, I0131 For preparing pharmaceutical compositions from 0.8, 0.7, 0.6,0.5,0.4,0.3, 0.2, 0.1, 0.009, 0.008, 0.007, 0.006, the compositions of the present invention, pharmaceutically 0.005, 0.004, 0.003, 0.002, 0.001 micrograms of the nucleic acceptable carriers can be either solid or liquid. Solid form acid per dose of vaccine administered im. In some embodi preparations include powders, tablets, pills, capsules, ments, a trace amount of nucleic acid is less than 12.5, 10, 9, cachets, Suppositories, and dispersible granules. A Solid car 8.5, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6,0.5,0.4,0.3, 0.2,0.1, rier can be one or more Substances, which may also act as 0.009, 0.008, 0.007, 0.006, 0.005, 0.004, 0.003, 0.002, 0.001 diluents, flavoring agents, binders, preservatives, tablet dis micrograms of the nucleic acid. In some embodiments, a trace integrating agents, or an encapsulating material. amount of nucleic acid is less than 12.5, 10, 9, 8.5, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6,0.5,0.4,0.3, 0.2,0.1, 0.009, 0.008, 0.132. In powders, the carrier is a finely divided solid in a 0.007, 0.006, 0.005, 0.004, 0.003, 0.002, 0.001 micrograms mixture with the finely divided active component (e.g. a com of the nucleic acid perdose of vaccine. In some embodiments, position provided herein). In tablets, the active component is a trace amount of nucleic acid is less than 12.5, 10, 9, 8.5, 8, mixed with the carrier having the necessary binding proper 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6,0.5,0.4,0.3, 0.2,0.1, 0.009, ties in Suitable proportions and compacted in the shape and 0.008, 0.007, 0.006, 0.005, 0.004, 0.003, 0.002, 0.001 micro size desired. The powders and tablets preferably contain from grams of the nucleic acid per dose of vaccine administered 5% to 70% of the active compound. i.m. In some embodiments, a trace amount of nucleic acid is 0.133 Suitable solid excipients include, but are not limited 1 microgram. In some embodiments, a trace amount of to, magnesium carbonate; magnesium Stearate; talc; pectin; nucleic acid is 0.5 micrograms. In some embodiments, a trace dextrin; starch; tragacanth; a low melting wax, cocoa butter; amount of nucleic acid is an amount less than an amount set carbohydrates; Sugars including, but not limited to, lactose, forth in DNA vaccines in WO2007 106404 or administered to Sucrose, mannitol, or Sorbitol, starch from corn, wheat, rice, elicit an immune response in WO2007 106404. In some potato, or other plants; cellulose Such as methyl cellulose, embodiments of the method, an isolated nucleic acid hydroxypropylmethyl-cellulose, or sodium carboxymethyl sequence formulation is not administered to the patient. In cellulose; and gums including arabic and tragacanth; as well some embodiments of the method, the isolated nucleic acid as proteins including, but not limited to, gelatin and collagen. sequence formulation is a DNA sequence. In some embodi If desired, disintegrating or solubilizing agents may be added, ments of the method, the isolated nucleic acid sequence for Such as the cross-linked polyvinyl pyrrolidone, agar, alginic mulation is a nucleic acid vaccine. In some embodiments of acid, or a salt thereof. Such as sodium alginate. the method, the isolated nucleic acid sequence formulation is a DNA vaccine. 0.134 Dragee cores are provided with suitable coatings Such as concentrated Sugar Solutions, which may also contain 0128 Provided in another aspect is a pharmaceutical com gum arabic, talc, polyvinylpyrrolidone, carbopol gel, poly position including a pharmaceutically acceptable excipient ethylene glycol, and/or titanium dioxide, lacquer Solutions, and any of the compositions (e.g. Vaccines) described herein and Suitable organic solvents or solvent mixtures. Dyestuffs (including embodiment). or pigments may be added to the tablets or dragee coatings for 0129. The compositions described herein (including product identification or to characterize the quantity of active embodiments and examples) can be administered alone or composition (i.e., dosage). Pharmaceutical preparations of can be coadministered to the patient. Coadministration is the invention can also be used orally using, for example, meant to include simultaneous or sequential administration of push-fit capsules made of gelatin, as well as Soft, sealed the compositions individually or in combination (more than capsules made of gelatin and a coating Such as glycerol or one composition). Thus, the preparations can also be com sorbitol. bined, when desired, with other active Substances (e.g. to reduce metabolic degradation, increase immune response 0.135 For preparing suppositories, a low melting wax, (e.g. adjuvants)). An example of coadministration of vaccine Such as a mixture of fatty acid glycerides or cocoa butter, is compositions is a prime-boost method of administration. first melted and the active composition is dispersed homoge 0130. The compositions of the present invention can be neously therein, as by stirring. The molten homogeneous prepared and administered in a wide variety of oral, mixture is then poured into convenient sized molds, allowed parenteral and topical dosage forms. Oral preparations to cool, and thereby to solidify. include tablets, pills, powder, dragees, capsules, liquids, loz 0.136 Liquid form preparations include solutions, Suspen enges, cachets, gels, syrups, slurries, Suspensions, etc., Suit sions, and emulsions, for example, water or water/propylene US 2014/0193460 A1 Jul. 10, 2014 26 glycol solutions. For parenteral injection, liquid preparations occurring phosphatides, such as soybean lecithin, esters or can be formulated in solution in aqueous polyethylene glycol partial esters derived from fatty acids and hexitol anhydrides, Solution. Such as Sorbitan mono-oleate, and condensation products of 0.137 When parenteral application is needed or desired, these partial esters with ethylene oxide. Such as polyoxyeth particularly suitable admixtures for the compounds of the ylene Sorbitan mono-oleate. The emulsion can also contain invention are injectable, sterile solutions, preferably oily or Sweetening agents and flavoring agents, as in the formulation aqueous solutions, as well as Suspensions, emulsions, or of syrups and elixirs. Such formulations can also contain a implants, including Suppositories. In particular, carriers for demulcent, a preservative, or a coloring agent. parenteral administration include aqueous Solutions of dex 0.141. The pharmaceutical preparation is preferably in unit trose, Saline, pure water, ethanol, glycerol, propylene glycol, dosage form. In Such form the preparation is Subdivided into peanut oil, Sesame oil, polyoxyethylene-block polymers, and unit doses containing appropriate quantities of the active the like. Ampules are convenient unit dosages. The composi component. The unit dosage form can be a packaged prepa tions of the invention can also be incorporated into liposomes ration, the package containing discrete quantities of prepara or administered via transdermal pumps or patches. Pharma tion, Such as packeted tablets, capsules, and powders in vials ceutical admixtures Suitable for use in the present invention or ampoules. Also, the unit dosage form can be a capsule, are well-known to those of skill in the art and are described, tablet, cachet, or lozenge itself, or it can be the appropriate for example, in Pharmaceutical Sciences (17th Ed., Mack number of any of these in packaged form. Pub.Co., Easton, Pa.) and WO 96/05309, the teachings of 0142. Some compositions may have limited solubility in both of which are hereby incorporated by reference. water and therefore may require a surfactant or other appro 0138 Aqueous solutions suitable for oral use can be pre priate co-solvent in the composition. Such co-solvents pared by dissolving the active component (e.g. compositions include: Polysorbate 20, 60 and 80; Pluronic F-68, F-84 and described herein, including embodiments, examples) in water P-103; cyclodextrin; polyoxyl 35 castor oil; or other agents and adding Suitable colorants, flavors, stabilizers, and thick known to those skilled in the art. Such co-solvents are typi ening agents as desired. Aqueous Suspensions Suitable for cally employed at a level between about 0.01% and about 2% oral use can be made by dispersing the finely divided active by weight. component in water with viscous material. Such as natural or 0.143 Viscosity greater than that of simple aqueous solu synthetic gums, resins, methylcellulose, sodium carboxym tions may be desirable to decrease variability in dispensing ethylcellulose, hydroxypropylmethylcellulose, sodium algi the formulations, to decrease physical separation of compo nate, polyvinylpyrrolidone, gum tragacanth and gum acacia, nents of a suspension or emulsion of formulation and/or oth and dispersing or wetting agents such as a naturally occurring erwise to improve the formulation. Such viscosity building phosphatide (e.g., lecithin), a condensation product of an agents include, for example, polyvinyl alcohol, polyvinyl alkylene oxide with a fatty acid (e.g., polyoxyethylene Stear pyrrolidone, methyl cellulose, hydroxy propyl methylcellu ate), a condensation product of ethylene oxide with a long lose, hydroxyethyl cellulose, carboxymethyl cellulose, chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), hydroxy propyl cellulose, chondroitin Sulfate and salts a condensation product of ethylene oxide with a partial ester thereof, hyaluronic acid and salts thereof, combinations of the derived from a fatty acid and a hexitol (e.g., polyoxyethylene foregoing, and other agents known to those skilled in the art. Sorbitol mono-oleate), or a condensation product of ethylene Such agents are typically employed at a level between about oxide with a partial ester derived from fatty acid and a hexitol 0.01% and about 2% by weight. Determination of acceptable anhydride (e.g., polyoxyethylene Sorbitan mono-oleate). The amounts of any of the above adjuvants is readily ascertained aqueous Suspension can also contain one or more preserva by one skilled in the art. tives such as ethyl or n-propyl p-hydroxybenzoate, one or 0144. The compositions of the present invention may more coloring agents, one or more flavoring agents and one or additionally include components to provide Sustained release more Sweetening agents, such as Sucrose, aspartame or sac and/or comfort. Such components include high molecular charin. Formulations can be adjusted for osmolarity. weight, anionic mucomimetic polymers, gelling polysaccha 0.139. Also included are solid form preparations that are rides and finely-divided drug carrier substrates. These com intended to be converted, shortly before use, to liquid form ponents are discussed in greater detail in U.S. Pat. Nos. 4,911, preparations for oral administration. Such liquid forms 920; 5,403,841; 5,212,162; and 4,861,760. The entire include Solutions, Suspensions, and emulsions. These prepa contents of these patents are incorporated herein by reference rations may contain, in addition to the active component, in their entirety for all purposes. colorants, flavors, stabilizers, buffers, artificial and natural 0145 Pharmaceutical compositions provided by the Sweeteners, dispersants, thickeners, solubilizing agents, and present invention include compositions wherein the active the like. ingredient (e.g. compositions described herein, including 0140. Oil Suspensions can contain a thickening agent, embodiments) is contained in a therapeutically or prophylac Such as beeswax, hard paraffin or cetyl alcohol. Sweetening tically effective amount, i.e., in an amount effective to achieve agents can be added to provide a palatable oral preparation, its intended purpose. The actual amount effective for a par Such as glycerol, Sorbitol or Sucrose. These formulations can ticular application will depend, inter alia, on the condition be preserved by the addition of an antioxidant Such as ascor being treated. When administered in methods to treat a dis bic acid. As an example of an injectable oil vehicle, see Minto, ease, Such compositions will contain an amount of active J. Pharmacol. Exp. Ther. 281:93-102, 1997. The pharmaceu ingredient effective to achieve the desired result, e.g., prevent tical formulations of the invention can also be in the form of HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, oil-in-water emulsions. The oily phase can be a vegetable oil HHV-6, HHV-7, or HHV-8) infection, prevent HSV (e.g. or a mineral oil, described above, or a mixture of these. HSV1 and/or HSV2) infection, and/or reducing, eliminating, Suitable emulsifying agents include naturally-occurring or slowing the progression of disease symptoms (e.g. HV gums, such as gum acacia and gum tragacanth, naturally (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, US 2014/0193460 A1 Jul. 10, 2014 27

HHV-7, or HHV-8) infection, HSV (e.g. HSV1 and/or HSV2) presence and severity of adverse side effects, preferred mode infection). Determination of a therapeutically or prophylac of administration and the toxicity profile of the selected agent. tically effective amount of a composition of the invention is 0152 The ratio between toxicity and therapeutic effect for well within the capabilities of those skilled in the art, espe a particular composition is its therapeutic index and can be cially in light of the detailed disclosure herein. expressed as the ratio between LDs (the amount of compo 0146 The dosage and frequency (single or multiple doses) sition lethal in 50% of the population) and EDs (the amount administered to a mammal can vary depending upon a variety of composition effective in 50% of the population). Compo of factors, for example, whether the mammal suffers from sitions that exhibit high therapeutic indices are preferred. another disease, and its route of administration; size, age, sex, Therapeutic index data obtained from cell culture assays and/ health, body weight, body mass index, and diet of the recipi or animal studies can be used in formulating a range of dos ent; nature and extent of symptoms of the disease being ages for use in humans. The dosage of Such compositions treated (e.g. HV (e.g. HSV. HHV-1, HHV-2, HHV-3, HHV-4, preferably lies within a range of plasma concentrations that HHV-5, HHV-6, HHV-7, or HHV-8) infection, HSV (e.g. include the EDs with little or no toxicity. The dosage may HSV1 and/or HSV2) infection), kind of concurrent treatment, vary within this range depending upon the dosage form complications from the disease being treated or other health employed and the route of administration utilized. See, e.g. related problems. Other therapeutic regimens or agents can be Finglet al., In: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, used in conjunction with the methods and compositions of Ch. 1, p. 1, 1975. The exact formulation, route of administra Applicants invention. Adjustment and manipulation of tion and dosage can be chosen by the individual physician in established dosages (e.g., frequency and duration) are well view of the patient’s condition and the particular method in within the ability of those skilled in the art. which the composition is used. 0147 For any compositions described herein, the thera peutically effective amount can be initially determined from IV. Administration cell culture assays. Target concentrations will be those con 0153. The compositions of the present invention can be centrations of active composition(s) that are capable of delivered by transdermally, by a topical route, transcutane achieving the methods described herein, as measured using ously, formulated as applicator Sticks, solutions, Suspensions, the methods described herein or known in the art. emulsions, gels, creams, ointments, pastes, jellies, paints, 0148. As is well known in the art, therapeutically or pro powders, and aerosols. The terms "compositions of the phylactically effective amounts for use in humans can also be present invention”, “compositions as described herein (in determined from animal models. For example, a dose for cluding embodiments)”, “compositions described herein (in humans can beformulated to achieve a concentration that has cluding embodiments)”, “composition provided herein', and been found to be effective in animals. The dosage in humans the like are used interchangeably. can be adjusted by monitoring compositions effectiveness 0154 The pharmaceutical compositions of the present and adjusting the dosage upwards or downwards, as invention can be provided as a salt and can be formed with described above. Adjusting the dose to achieve maximal effi many acids, including but not limited to hydrochloric, Sulfu cacy in humans based on the methods described above and ric, acetic, lactic, tartaric, malic. Succinic, etc. Pharmaceuti other methods is well within the capabilities of the ordinarily cal compositions described herein may be salts of a com skilled artisan. pound or composition which are prepared with relatively 0149 Dosages may be varied depending upon the require nontoxic acids or bases, depending on the particular substitu ments of the patient and the composition being employed. ents found on the compositions described herein. When com The dose administered to a patient, in the context of the positions of the present invention contain relatively acidic present invention should be sufficient to effect a beneficial functionalities, base addition salts can be obtained by con therapeutic or prophylactic response in the patient over time. tacting the neutral form of such compositions with a sufficient The size of the dose also will be determined by the existence, amount of the desired base, either neat or in a suitable inert nature, and extent of any adverse side-effects. Determination Solvent. Examples of pharmaceutically acceptable base addi of the proper dosage for a particular situation is within the tion salts include Sodium, potassium, calcium, ammonium, skill of the practitioner. Generally, treatment is initiated with organic amino, or magnesium salt, or a similar salt. When Smaller dosages which are less than the optimum dose of the compositions of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contact compound. Thereafter, the dosage is increased by Small incre ing the neutral form of Such compositions with a sufficient ments until the optimum effect under circumstances is amount of the desired acid, either neat or in a suitable inert reached. Solvent. Examples of pharmaceutically acceptable acid addi 0150 Dosage amounts and intervals can be adjusted indi tion salts include those derived from inorganic acids like vidually to provide levels of the administered composition hydrochloric, hydrobromic, nitric, carbonic, monohydrogen effective for the particular clinical indication being treated or carbonic, phosphoric, monohydrogenphosphoric, dihydro prevented. This will provide a therapeutic or prophylactic genphosphoric, Sulfuric, monohydrogensulfuric, hydriodic, regimen that is commensurate with the severity of the indi orphosphorous acids and the like, as well as the salts derived vidual’s disease state. from relatively nontoxic organic acids like acetic, propionic, 0151. Utilizing the teachings provided herein, an effective isobutyric, maleic, malonic, benzoic, Succinic, Suberic, prophylactic ortherapeutic treatment regimen can be planned fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolyl that does not cause substantial toxicity and yet is effective to Sulfonic, citric, tartaric, methanesulfonic, and the like. Also treat the clinical symptoms demonstrated by the particular included are salts of amino acids such as arginate and the like, patient. This planning should involve the careful choice of and salts of organic acids like glucuronic or galactunoric active composition by considering factors such as composi acids and the like (see, e.g., Berge et al., Journal of Pharma tion potency, relative bioavailability, patient body weight, ceutical Science 66:1-19 (1977)). Certain specific composi US 2014/0193460 A1 Jul. 10, 2014 28 tions of the present invention contain both basic and acidic embodiments, the active agents can be formulated separately. functionalities that allow the compositions to be converted In another embodiment, the active and/or adjunctive agents into either base or acid addition salts. Other pharmaceutically may be linked or conjugated to one another. In some embodi acceptable carriers known to those of skill in the art are ments, co-administration includes administering vaccines in suitable for the present invention. Salts tend to be more a prime-boost method within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or soluble in aqueous or other protonic solvents that are the 12 weeks of each other, or within 1,2,3,4,5,6,7,8,9, 10, 11, corresponding free base forms. 12 months of each other. In some embodiments, the prime 0155 The neutral forms of the compositions may be boost administration is 4 weeks apart. regenerated by contacting the salt with a base or acid and 0159. As a non-limiting example, the compositions isolating the parent compositions in the conventional manner. described herein can be co-administered (e.g. prime-boost) The parent form of the compositions differs from the various with another HSV vaccine including, but not limited to, HSV salt forms in certain physical properties, such as solubility in DNA vaccines, HSV DNA vaccine comprising one or more polar solvents, but otherwise the salts are equivalent to the portions of one or more of the HSV (e.g. HSV1 or HSV2) parent form of the compositions for the purposes of the genes (e.g. UL1, UL2, UL3, UL4, UL5, UL6, UL7, UL8, present invention. UL9, UL10, UL11, UL12, UL13, UL14, UL15, UL16, UL17, 0156. In another embodiment, the compositions of the UL18, UL19, UL20, UL21, UL22, UL23, UL24, UL25, present invention are useful for parenteral administration, UL26, UL27, UL28, UL29, UL30, UL31, UL32, UL33, Such as intravenous (IV) administration or administration UL34, UL35, UL36, UL37, UL38, UL39, UL40, UL41, into a body cavity or lumen of an organ. The formulations for UL42, UL43, UL44, UL45, UL46, UL47, UL48, UL49, administration will commonly comprise a solution of the UL50, UL51, UL52, UL53, UL54, UL55, UL56, US1, US2, compositions of the present invention dissolved in a pharma US3, US4, US5, US6, US7, US8, US9, US10, US11, US12, ceutically acceptable carrier. Among the acceptable vehicles RS1, ICP0, LRP1, LRP2, RL1, or LAT), HSV protein vac and solvents that can be employed are water and Ringer's cine, HSV protein vaccine comprising one or more portions Solution, an isotonic sodium chloride. In addition, Sterile of one or more of the HSV (e.g. HSV1 or HSV2) proteins (e.g. fixed oils can conventionally be employed as a solvent or protein expressed from the HSV gene UL1, UL2, UL3, UL4. Suspending medium. For this purpose any bland fixed oil can UL5, UL6, UL7, UL8, UL9, UL10, UL11, UL12, UL13, be employed including synthetic mono- or diglycerides. In UL14, UL15, UL16, UL17, UL18, UL19, UL20, UL21, addition, fatty acids such as oleic acid can likewise be used in UL22, UL23, UL24, UL25, UL26, UL27, UL28, UL29, the preparation of injectables. These solutions are sterile and UL30, UL31, UL32, UL33, UL34, UL35, UL36, UL37, generally free of undesirable matter. These formulations may UL38, UL39, UL40, UL41, UL42, UL43, UL44, UL45, be sterilized by conventional, well known sterilization tech UL46, UL47, UL48, UL49, UL50, UL51, UL52, UL53, niques. The formulations may contain pharmaceutically UL54, UL55, UL56, US1, US2, US3, US4, US5, US6, US7, acceptable auxiliary Substances as required to approximate US8, US9, US10, US11, US12, RS1, ICP0, LRP1, LRP2, physiological conditions such as pH adjusting and buffering RL1, or LAT), an inactivated HSV, and the like. In some agents, toxicity adjusting agents, e.g., Sodium acetate, sodium embodiments, the compositions described herein are not co chloride, potassium chloride, calcium chloride, sodium lac administered (e.g. prime-boost) with another HSV vaccine tate and the like. The concentration of the compositions of the selected from the group consisting of HSV DNA vaccines, present invention in these formulations can vary widely, and HSV DNA vaccine comprising one or more portions of one or will be selected primarily based on fluid volumes, viscosities, more of the HSV (e.g. HSV1 or HSV2) genes (e.g. UL1, UL2, body weight, and the like, in accordance with the particular UL3, UL4, UL5, UL6, UL7, UL8, UL9, UL10, UL11, UL12, mode of administration selected and the patient’s needs. For UL13, UL14, UL15, UL16, UL17, UL18, UL19, UL20, IV administration, the formulation can be a sterile injectable UL21, UL22, UL23, UL24, UL25, UL26, UL27, UL28, preparation, such as a sterile injectable aqueous or oleaginous UL29, UL30, UL31, UL32, UL33, UL34, UL35, UL36, Suspension. This Suspension can be formulated according to UL37, UL38, UL39, UL40, UL41, UL42, UL43, UL44, the known art using those Suitable dispersing or wetting UL45, UL46, UL47, UL48, UL49, UL50, UL51, UL52, agents and Suspending agents. UL53, UL54, UL55, UL56, US1, US2, US3, US4, US5, US6, 0157. The compositions described herein can be used in US7, US8, US9, US10, US11, US12, RS1, ICP0, LRP1, combination with one another, with other active agents LRP2, RL1, or LAT), HSV protein vaccine, HSV protein known to be useful in treating a disease associated with HV vaccine comprising one or more portions of one or more of (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, the HSV (e.g. HSV1 or HSV2) proteins (e.g. protein HHV-7, or HHV-8) or HSV (e.g. HSV1 and/or HSV2) infec expressed from the HSV gene UL1, UL2, UL3, UL4, UL5, tion, or with adjunctive agents that may not be effective alone, UL6, UL7, UL8, UL9, UL10, UL11, UL12, UL13, UL14, but may contribute to the efficacy of the active agent (e.g. UL15, UL16, UL17, UL18, UL19, UL20, UL21, UL22, LPS-derived adjuvants, aluminum-based mineral salt adju UL23, UL24, UL25, UL26, UL27, UL28, UL29, UL30, vants). UL31, UL32, UL33, UL34, UL35, UL36, UL37, UL38, 0158. In some embodiments, co-administration includes UL39, UL40, UL41, UL42, UL43, UL44, UL45, UL46, administering one active agent within 0.5, 1, 2, 4, 6, 8, 10, 12. UL47, UL48, UL49, UL50, UL51, UL52, UL53, UL54, 16, 20, or 24 hours of a second active agent. Co-administra UL55, UL56, US1, US2, US3, US4, US5, US6, US7, US8, tion includes administering two active agents simultaneously, US9, US 10, US 11, US 12, RS1, ICP0, LRP1, LRP2, RL1, or approximately simultaneously (e.g., within about 1, 5, 10, 15. LAT), an inactivated HSV, and the like. 20, or 30 minutes of each other), or sequentially in any order. 0160 The pharmaceutical compositions of the present In some embodiments, co-administration can be accom invention may besterilized by conventional, well-known ster plished by co-formulation, i.e., preparing a single pharma ilization techniques or may be produced under sterile condi ceutical composition including both active agents. In other tions. Aqueous solutions can be packaged for use or filtered US 2014/0193460 A1 Jul. 10, 2014 29 under aseptic conditions and lyophilized, the lyophilized the blood of the intended recipient, and aqueous and non preparation being combined with a sterile aqueous Solution aqueous sterile Suspensions that can include Suspending prior to administration. The compositions can contain phar agents, solubilizers, thickening agents, stabilizers, and pre maceutically acceptable auxiliary Substances as required to servatives. Injection solutions and Suspensions can also be approximate physiological conditions, such as pH adjusting prepared from sterile powders, granules, and tablets. In the and buffering agents, tonicity adjusting agents, wetting practice of the present invention, compositions can be admin agents, and the like, e.g., sodium acetate, Sodium lactate, istered, for example, by intravenous infusion, orally, topi Sodium chloride, potassium chloride, calcium chloride, Sor cally, intraperitoneally, intravesically, or intrathecally. bitan monolaurate, and triethanolamine oleate. Parenteral administration, nasal administration, oral admin 0161 Formulations suitable for oral administration can istration, and intravenous administration are the preferred comprise: (a) liquid Solutions, such as an effective amount of methods of administration. The formulations of compositions a packaged compound or drug suspended in diluents, e.g., can be presented in unit-dose or multi-dose sealed containers, water, saline, or PEG 400; (b) capsules, sachets, or tablets, Such as ampoules and vials. each containing a predetermined amount of an HSV (e.g. 0.165. The pharmaceutical preparation is preferably in unit HSV1 and/or HSV2) vaccine composition, as liquids, solids, dosage form. In Such form the preparation is Subdivided into granules or gelatin; (c) Suspensions in an appropriate liquid; unit doses containing appropriate quantities of the active and (d) suitable emulsions. Tablet forms can include one or component, e.g., an HV (e.g. HSV. HHV-1, HHV-2, HHV-3, more of lactose, Sucrose, mannitol, Sorbitol, calcium phos HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vaccine com phates, corn starch, potato starch, microcrystalline cellulose, position or HSV (e.g. HSV1 and/or HSV2) vaccine compo gelatin, colloidal silicon dioxide, talc, magnesium Stearate, sition. The unit dosage form can be a packaged preparation, Stearic acid, and other excipients, colorants, fillers, binders, the package containing discrete quantities of preparation, diluents, buffering agents, moistening agents, preservatives, Such as packeted tablets, capsules, and powders in vials or flavoring agents, dyes, disintegrating agents, and pharmaceu ampoules. Also, the unit dosage form can be a capsule, tablet, tically compatible carriers. Lozenge forms can comprise an cachet, or lozenge itself, or it can be the appropriate number HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, of any of these in packaged form. The composition can, if HHV-6, HHV-7, or HHV-8) vaccine composition or HSV desired, also contain other compatible therapeutic agents. (e.g. HSV1 and/or HSV2) vaccine composition in a flavor, 0166 In therapeutic use for the treatment of HV (e.g. HSV. e.g., Sucrose, as well as pastilles comprising for example, an HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-8) infection or a disease related to HV (e.g. HSV. HHV HHV-6, HHV-7, or HHV-8) vaccine composition or HSV 1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or (e.g. HSV1 and/or HSV2) vaccine composition in an inert HHV-8) infection, the HV (e.g. HSV, HHV-1, HHV-2, HHV base. Such as gelatin and glycerin or Sucrose and acacia emul 3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vaccine com sions, gels, and the like, containing, in addition to for position may be varied depending upon the requirements of example, an HV (e.g. HSV. HHV-1, HHV-2, HHV-3, HHV-4, the patient, the severity of the condition being treated, and the HHV-5, HHV-6, HHV-7, or HHV-8) vaccine composition or HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HSV (e.g. HSV1 and/or HSV2) vaccine composition, carriers HHV-6, HHV-7, or HHV-8) vaccine composition being known in the art. employed. For example, dosages can be empirically deter 0162 The composition of choice, alone or in combination mined considering the type and stage of HV (e.g. HSV. HHV with other Suitable components, can be made into aerosol 1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or formulations (i.e., they can be “nebulized') to be adminis HHV-8) related disease diagnosed in a particular patient. The tered via inhalation. Aerosol formulations can be placed into dose administered to a patient, in the context of the present pressurized acceptable propellants, such as dichlorodifluo invention, should be sufficient to affect a beneficial therapeu romethane, propane, nitrogen, and the like. tic or prophylactic response in the patient over time. The size 0163 Suitable formulations for rectal administration of the dose will also be determined by the existence, nature, include, for example, Suppositories, which comprises an and extent of any adverse side-effects that accompany the effective amount of a packaged HV (e.g. HSV. HHV-1, HHV administration of a composition in a particular patient. Deter 2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, or HHV-8) vac mination of the proper dosage for a particular situation is cine composition or HSV (e.g. HSV1 and/or HSV2) vaccine within the skill of the practitioner. Generally, treatment is composition, with a Suppository base. Suitable Suppository initiated with Smaller dosages which are less than the opti bases include natural or synthetic triglycerides or paraffin mum dose of the composition. Thereafter, the dosage is hydrocarbons. In addition, it is also possible to use gelatin increased by small increments until the optimum effect under rectal capsules which contain a combination of for example, circumstances is reached. For convenience, the total daily an HV (e.g. HSV, HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, dosage may be divided and administered in portions during HHV-6, HHV-7, or HHV-8) vaccine composition or HSV the day, if desired. (e.g. HSV1 and/or HSV2) vaccine composition of choice, 0167. In some embodiments, there is provided a method of with a base, including, for example, liquid triglycerides, poly preventing or treating a Herpes Simplex Virus Type 2 (HSV ethylene glycols, and paraffin hydrocarbons. 2) infection in a Subject. The method includes administering 0164. Formulations suitable for parenteral administration, to a subject in need thereof a DNA vaccine, wherein the DNA Such as, for example, by intraarticular (in the joints), intrav vaccine includes a DNA sequence encoding a portion of at aginal, intrarectal intravenous, intramuscular, intratumoral, least one conserved HSV-2 gene required for virus replica intradermal, intraperitoneal, and Subcutaneous routes, tion. include aqueous and non-aqueous, isotonic sterile injection (0168. In some embodiments, the DNA vaccine further Solutions, which can contain antioxidants, buffers, bacteri includes a monophosphoryl lipid A (MPL) adjuvant. In some ostats, and solutes that render the formulation isotonic with embodiments, the DNA vaccine further includes an alum US 2014/0193460 A1 Jul. 10, 2014 30 adjuvant. In some embodiments, the DNA vaccine includes is not a well-defined form of alum suitable for clinical trans whole, inactivated HSV-2. In some embodiments, the DNA lation. Therefore we tested the protective efficacy of vaccines vaccine includes formalin-inactivated HSV-2 (Fl-HSV-2). In that were formulated with MPL and aluminum hydroxide Some embodiments, the DNA sequence encodes at least a (Alhydrogel) or aluminum phosphate (Adju-Phos) adjuvants, portion of a DNA polymerase from HSV-2. In some embodi both of which have been licensed for use in clinical vaccines. ments, the DNA polymerase is HSV-2 UL30 DNA poly We also tested various routes of immunization. The test merase. In some embodiments, the DNA vaccine includes a immunization groups were: 1) FI-HSV2/MPL/Alhydrogel DNA molecule encoding at least a portion of HSV-2 secreted given subcutaneously; 2) FI-HSV2/MPL/Alhydrogel given glycoprotein D2 (gD2t). In some embodiments, the DNA intramuscularly; and 3) FI-HSV2/MPL/Adju-Phos given vaccine further includes a DNA molecule encoding at least a intramuscularly. portion of HSV-2 secreted glycoprotein D2 (gD2t). In some 0.175. Following intravaginal challenge with HSV-2, the embodiments, the DNA sequence encodes at least a portion of meantiters in the groups receiving FI-HSV2 were reduced by a helicase from HSV-2. In some embodiments, the helicase is 3 Logs. In addition, half or more of the 8 mice in each HSV-2 UL5 helicase. In some embodiments, the administra FI-HSV2 groups had reductions in virus shedding to levels at tion step of the method is prime-boost immunization. or below the detection limit. Taken together, immunization 0169. In another embodiment, there is provided a DNA with FI-HSV2 and MPL together with the aluminum contain vaccine which includes a DNA sequence encoding a portion ing adjuvants Alhydrogel or Adju-Phos resulted in protection of at least one conserved HSV-2 gene required for virus against HSV-2 disease and shedding that was at leastas robust replication. as that afforded by Imject alum. In this experiment, several of 0170. In some embodiments, the DNA vaccine further the FI-HSV2 immunized mice in each group had no detect includes a monophosphoryl lipid A (MPL) adjuvant. In some able shedding (<10 PFU) on either day tested. embodiments, the DNA vaccine further includes an alum 0176 Without wishing to be bound by any theory, it is adjuvant. In some embodiments, the DNA vaccine includes believed important to question whether the high level protec whole, inactivated HSV-2. In some embodiments, the DNA tion against challenge that was provided by the FI-HSV2/ vaccine includes formalin-inactivated HSV-2 (Fl-HSV-2). In MPL/Alum would also be effective against a long-term re Some embodiments, the DNA sequence encodes at least a challenge. To this end, the FI-HSV2 immunized mice were portion of a DNA polymerase from HSV-2. In some embodi re-challenged 17 weeks after the first challenge with the same ments, the DNA polymers is HSV-2 UL30 DNA polymerase. 10xLDso dose of HSV-2). Strikingly, only 2 of the rechal In some embodiments, the DNA vaccine includes a DNA lenged mice in the FI-HSV2 groups had a detectable, low molecule encoding at least a portion of HSV-2 secreted gly level of virus, with the mouse in the FI-HSV2/Alhydrogels.c. coprotein D2 (gD2t). In some embodiments, the DNA vac group showing detectable virus on day 2 later dying on day cine further includes a DNA molecule encoding at least a 21. It is particularly notable that in the FI-HSV2/Alhydrogel portion of HSV-2 secreted glycoprotein D2 (gD2t). In some i.m. group, 5 of the 8 mice had no detectable virus on day 2 embodiments, the DNA sequence encodes at least a portion of postchallenge or post-rechallenge, and 2 additional mice in a helicase from HSV-2. In some embodiments, the helicase is this group had very low level shedding postchallenge (10'- HSV-2 UL5 helicase. 10' PFU) and no detectable virus post-rechallenge. 0171 Accordingly, in some embodiments of the method 0177 Taken together, we found that immunization with and vaccine described herein, there is provided formalin FI-HSV2 that was formulated with MPL and aluminum con inactivated herpesvirus mixed with an aluminum-based min taining adjuvants elicits nearly complete protection against eral salt adjuvant (alhydrogel or adju-phos) and monophos HSV-2 disease. Moreover, these vaccines provide strong and phoryl lipid A (MPL) as a vaccine to generate protective durable protection against vaginal HSV-2 shedding, with immune responses. Some animals showing sterilizing immunity, following chal 0172. In some embodiments, dextran sulfate was lenge or long-term rechallenge. employed to release high titers of herpesviruses from infected 0.178 We have also evaluated the formalin-inactivated cells, greatly reducing the amount of cellular material in the HSV-2 (FI-HSV2)/MPL/Alhydrogel vaccine in the guinea preparation. The resulting virus is Subsequently purified and pig model of acute and recurrent HSV-2 genital disease and inactivated with formaldehyde. compared it with the gl)2t subunit protein vaccine in MPL/ 0173 We have developed and tested several vaccine com Alum. After challenge, both groups showed highly significant binations against herpes simplex virus type 2 (HSV-2) in both protection against acute viral disease and viral shedding com mouse and guinea pig models of genital disease. Included in pared to the mock-immunized animals. Only 1 of 10 guinea these vaccines were the adjuvant monophosphoryl lipid A pigs immunized with FI-HSV2/MPL/Alhydrogel had an (MPL) and various formulations of Alum. We asked whether acute-phase lesion and virus shedding that persisted to day 9 a multivalent vaccine consisting of FI-HSV2 together with postchallenge. Only this animal developed recurrent lesions MPL/Alum could be more protective than gl)2 protein sub over a 100 day period, whereas 3 out of 10 animals in the g|D2t unit/MPL/Alum vaccination. subunit protein/MPL/Alhydrogel developed recurrent 0.174. Accordingly, we have found that immunization with lesions. To determine whether the protection against recur FI-HSV2/MPL/Alum provided significantly higher levels of rent lesions or vaginal HSV-2 disease was associated with protection against both disease and virus shedding compared differences in the number of latent viral genomes in the DRG with g)2t protein immunization in the mouse model of geni or in postreactivation immunity, the lumbosacral DRG from tal disease. In fact, in one experiment, 2 of the 8 mice that each Surviving guinea pig were harvested. The levels of were immunized only with FI-HSV-2 did not shed any detect HSV-2 DNA were quantified by real-time quantitative PCR. able virus on any day post challenge. All of the early experi Only the single guinea pig immunized with FI-HSV2/MPL/ ments were done with Imject Alum from Pierce, which con Alhydrogel that had both acute and recurrent lesions had sists of both crystalline and amorphous aluminum phases and detectable HSV-2 DNA (84 copies). In contrast, 4 of the 10 US 2014/0193460 A1 Jul. 10, 2014

gD2t Subunit MPL/Alhydrogel-immunized guinea pigs had Embodiment 11 detectable HSV-2 DNA levels. Taken together, these studies showed when the FI-HSV2/MPL alhydrogel vaccine was 0189 The vaccine of any one of Embodiments 1 to 8 and given to the guinea pigs, with the exception of one “nonre 10, wherein said lipopolysaccharide-derived adjuvant is a sponder animal in the group, there was complete protection lipid A adjuvant without a 1-phosphate moiety. against both acute and recurrent lesion development, unde tectable levels of HSV-2 DNA in the DRG, lower cumulative Embodiment 12 acute disease scores, and lower numbers of recurrent disease 0190. The vaccine of any one of Embodiments 1 to 8 and days (Morello, C. S. et al., 2011, Virol, 85:3461-3472). 10 to 11, wherein said lipopolysaccharide-derived adjuvant is monophosphoryl lipid A (MPL). V. Selected Embodiments Embodiment 13 Embodiment 1 0191 The vaccine of any one of Embodiments 1 to 8 and 0179 A Herpes Simplex Virus (HSV) vaccine comprising 10 to 11, wherein said lipopolysaccharide-derived adjuvant is inactivated HSV, a lipopolysaccharide (LPS)-derived adju 3-O-desacyl-4'-monophosphoryl lipid A. vant and an aluminum-based mineral salt adjuvant. Embodiment 2 Embodiment 14 0180. The vaccine of Embodiment 1, wherein said LPS 0.192 The vaccine of any one of Embodiments 1 to 13, derived adjuvant is MPL: said aluminum-based mineral salt wherein said lipopolysaccharide-derived adjuvant is capable adjuvant is aluminum hydroxide adjuvant or aluminum phos of binding the TLR4 protein. phate adjuvant; and said vaccine does not comprise an iso lated nucleic acid sequence formulation. Embodiment 15 0193 The vaccine of any one of Embodiments 1 to 14, Embodiment 3 wherein said lipopolysaccharide-derived adjuvant is a syn 0181. The vaccine of any one of Embodiments 1 to 2, thetic MPL analogue adjuvant. wherein said HSV is HSV1. Embodiment 16 Embodiment 4 0194 The vaccine of any one of Embodiments 1 to 15, 0182. The vaccine of any one of Embodiments 1 to 2, wherein said aluminum-based mineral salt adjuvant is an wherein said HSV is HSV2. aluminum hydroxide adjuvant. Embodiment 5 Embodiment 17 0183 The vaccine of any one of Embodiments 1 to 2, (0195 The vaccine of any one of Embodiments 1 to 15, wherein said HSV is HSV1 and HSV2. wherein said aluminum-based mineral salt adjuvant is an aluminum phosphate adjuvant. Embodiment 6 0184 The vaccine of any one of Embodiments 1 to 2, Embodiment 18 wherein said lipopolysaccharide-derived adjuvant is derived 0196. The vaccine of any one of Embodiments 1 to 15, from the Salmonella Minnesota LPS. wherein said aluminum-based mineral salt adjuvant includes crystalline aluminum hydroxide and not amorphous alumi Embodiment 7 num hydroxide or aluminum hydroxycarbonate or magne 0185. The vaccine of any one of Embodiments 1 to 2, sium hydroxide. wherein said lipopolysaccharide-derived adjuvant is derived from the Salmonella Minnesota Re595 LPS. Embodiment 19 (0197) The vaccine of any one of Embodiments 1 to 15, Embodiment 8 wherein said aluminum-based mineral salt adjuvant includes 0186 The vaccine of any one of Embodiments 1 to 8, aluminum phosphate gel in the form of a white gelatinous wherein said lipopolysaccharide-derived adjuvant is derived precipitate. from the R595 LPS. Embodiment 20 Embodiment 9 0198 The vaccine of any one of Embodiments 1 to 15, 0187. The vaccine of any one of Embodiments 1 to 8, wherein said aluminum-based mineral salt adjuvant includes wherein said lipopolysaccharide-derived adjuvant is a lipid A aluminum hydroxide gel in the form of a white gelatinous adjuvant. precipitate. Embodiment 10 Embodiment 21 0188 The vaccine of any one of Embodiments 1 to 8, (0199 The vaccine of any one of Embodiments 1 to 15, wherein said lipopolysaccharide-derived adjuvant is a lipid A wherein said aluminum-based mineral salt adjuvant is a adjuvant without an (R)-3-hydroxytetradecanoyl moiety. potassium aluminum sulfate adjuvant. US 2014/0193460 A1 Jul. 10, 2014 32

Embodiment 22 Embodiment 35 0200. The vaccine of any one of Embodiments 1 to 21, 0213. The vaccine of any one of Embodiments 24 to 32, wherein said inactivated HSV comprises a replication wherein said cross-linking agent is formalin. decreasing DNA mutation. Embodiment 36 Embodiment 23 0214. The vaccine of any one of Embodiments 24 to 35, 0201 The vaccine of any one of Embodiments 1 to 21, wherein said oxidizing agent is sodium periodate. wherein said inactivated HSV is formed by chemical inacti Vation. Embodiment 37 0215. The vaccine of any one of Embodiments 24 to 35, Embodiment 24 wherein said oxidizing agent is hydrogen peroxide. 0202 The vaccine of any one of Embodiments 1 to 21, wherein said inactivated HSV is formed by contacting said Embodiment 38 HSV with one or more agents selected from a cross-linking agent, oxidizing agent, reducing agent, heat, radiation, deter 0216. The vaccine of any one of Embodiments 24 to 37, gent, a pH changing agent, and a chemical agent selected wherein said reducing agent is aldrithiol-2. from a furocoumarin, aziridine, ethylenimine, binary ethyl Embodiment 39 enimine, and beta-propiolactone. 0217. The vaccine of any one of Embodiments 24 to 38, Embodiment 25 wherein said detergent is Triton-X-100. 0203 The vaccine of Embodiment 24, wherein said Embodiment 40 chemical agent is aziridine. 0218. The vaccine of any one of Embodiments 24 to 38, Embodiment 26 wherein said detergent is NP-40. 0204. The vaccine of Embodiment 24, wherein said chemical agent is ethylenimine. Embodiment 41 0219. The vaccine of any one of Embodiments 24 to 38, Embodiment 27 wherein said detergent is Tween-20. 0205 The vaccine of Embodiment 24, wherein said chemical agent is binary ethylenimine. Embodiment 42 0220. The vaccine of any one of Embodiments 1 to 41, Embodiment 28 wherein said inactivated HSV is formed by contacting said 0206. The vaccine of Embodiment 24, wherein said HSV with one or more agents comprising UV radiation and a chemical agent is beta-propiolactone. furocoumarin. Embodiment 29 Embodiment 43 0207. The vaccine of any one of Embodiments 24 to 28, 0221) The vaccine of any one of Embodiments 1 to 42, wherein said radiation is UV radiation. wherein said furocoumarin is psoralen. Embodiment 30 Embodiment 44 0208. The vaccine of any one of Embodiments 24 to 28, 0222. The vaccine of any one of Embodiments 1 to 42, wherein said radiation is electron beam radiation. wherein said furocoumarin is 4'-aminomethyl-4,5'.8-trimeth ylpsoralen. Embodiment 31 Embodiment 45 0209. The vaccine of any one of Embodiments 24 to 28, wherein said radiation is infrared radiation. 0223) The vaccine of any one of Embodiments 1 to 42, wherein said furocoumarin is angelicin. Embodiment 32 0210. The vaccine of any one of Embodiments 24 to 28, Embodiment 46 wherein said radiation is gamma radiation. 0224. The vaccine of any one of Embodiments 1 to 42, wherein said furocoumarin is Xanthotoxin. Embodiment 33 Embodiment 47 0211. The vaccine of any one of Embodiments 24 to 32, wherein said cross-linking agent is an aldehyde cross-linking 0225. The vaccine of any one of Embodiments 1 to 42, agent. wherein said furocoumarin is bergapten. Embodiment 34 Embodiment 48 0212. The vaccine of any one of Embodiments 24 to 32, 0226. The vaccine of any one of Embodiments 1 to 42, wherein said cross-linking agent is formaldehyde. wherein said furocoumarin is nodakenetin. US 2014/0193460 A1 Jul. 10, 2014

Embodiment 49 Embodiment 62 0227. The vaccine of any one of Embodiments 1 to 48, 0240. The vaccine of any one of Embodiments 1 to 53, wherein said inactivated HSV is an inactivated single strain of formulated for Subcutaneous administration. HSV1. Embodiment 63 Embodiment 50 0241 The vaccine of any one of Embodiments 2 to 62, 0228. The vaccine of any one of Embodiments 1 to 48, wherein said isolated nucleic acid sequence formulation is an wherein said inactivated HSV is an inactivated single strain of antigenic isolated nucleic acid sequence formulation. HSV2. Embodiment 64 Embodiment 51 0242. The HSV vaccine of any one of Embodiments 2 to 0229. The vaccine of any one of Embodiments 1 to 48, 62, wherein said isolated nucleic acid sequence formulation is wherein said inactivated HSV is a combination of two or more a DNA vaccine. inactivated strains of HSV 1. Embodiment 65 Embodiment 52 0230. The vaccine of any one of Embodiments 1 to 48, 0243 The HSV vaccine of Embodiment 64, wherein said wherein said inactivated HSV is a combination of two or more DNA vaccine is an antigenic isolated nucleic acid sequence inactivated strains of HSV2. formulation.

Embodiment 53 Embodiment 66 0231. The vaccine of any one of Embodiments 1 to 48, 0244. The HSV vaccine of any one of Embodiments 1 to wherein said inactivated HSV is a combination of one or more 65, wherein said vaccine vaccinates a recipient of said vac inactivated strains or HSV1 and one or more inactivated cine against HSV infection for up to 1,2,3,4,5,6,7,8,9, 10. Strains of HSV2. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more years.

Embodiment 54 Embodiment 67 0232. The vaccine of any one of Embodiments 1 to 53, 0245. A method of treating or preventing HSV infection in a patient in need of such treatment or prevention, said method formulated for intramuscular administration. comprising administering a therapeutically or prophylacti Embodiment 55 cally effective amount of the HSV vaccine of any one of Embodiments 1 to 66. 0233. The vaccine of any one of Embodiments 1 to 53, formulated for intradermal administration. Embodiment 68 Embodiment 56 0246 The method of Embodiment 67, wherein said HSV is HSV1. 0234. The vaccine of any one of Embodiments 1 to 53, formulated for mucosal administration. Embodiment 69 Embodiment 57 0247 The method of Embodiment 67, wherein said HSV is HSV2. 0235. The vaccine of any one of Embodiments 1 to 53, formulated for intranasal administration. Embodiment 70 Embodiment 58 0248. The method of Embodiment 67, wherein said HSV is HSV1 and HSV2. 0236. The vaccine of any one of Embodiments 1 to 53, formulated for intrarectal administration. Embodiment 71 Embodiment 59 0249. The method of any one of Embodiments 67 to 70, wherein said HSV infection causes a disease selected from 0237. The vaccine of any one of Embodiments 1 to 53, herpetic gingivostomatitis, herpes labialis, herpes genitalis, formulated for intravaginal administration. herpetic whitlow, herpes gladiatorum, herpesviral encephali tis, herpesviral meningitis, herpes esophagitis, herpes kerati Embodiment 60 tis, Bell’s palsy, Mollaret's meningitis, herpes rugbeiorum, 0238. The vaccine of any one of Embodiments 1 to 53, eczema herpeticum, herpetic neuralgia, and post-herpetic formulated for topical administration. neuralgia. Embodiment 61 Embodiment 72 0239. The vaccine of any one of Embodiments 1 to 53, (0250. The method of any one of Embodiments 67 to 71, formulated for transcutaneous administration. comprising a therapeutically effective amount. US 2014/0193460 A1 Jul. 10, 2014 34

Embodiment 73 Embodiment 87 0251. The method of any one of Embodiments 67 to 71, 0265. The method of any one of Embodiments 67 to 78, comprising a prophylactically effective amount. comprising Subcutaneous administration. Embodiment 74 Embodiment 88 0252) The method of any one of Embodiments 67 to 73, 0266. A method of preparing a Herpes Simplex Virus comprising treating said disease. (HSV) vaccine, said method comprising: contacting an HSV-cell mixture with a sulfated or sulfonated Embodiment 75 polysaccharide, wherein said HSV-cell mixture comprises 0253) The method of any one of Embodiments 67 to 73, HSV particles, cells and portions of cells; comprising preventing said disease. separating said HSV particles from said cells thereby forming isolated HSV particles: Embodiment 76 inactivating said isolated HSV particles thereby forming inactivated HSV particles: 0254 The method of any one of Embodiments 67 to 75, comprising a reduction in viral shedding. combining said inactivated HSV particles with a lipopolysac charide-derived adjuvant and an aluminum-based mineral Embodiment 77 salt adjuvant thereby forming an HSV vaccine. 0255. The method of any one of Embodiments 67 to 76, Embodiment 89 comprising a reduction in the frequency of lesion occurrence. 0267 The method of Embodiment 88, wherein said HSV Embodiment 78 is HSV1. 0256 The method of any one of Embodiments 67 to 77, Embodiment 90 comprising a reduction in the duration of lesion occurrence. 0268 The method of Embodiment 88, wherein said HSV Embodiment 79 is HSV2. 0257. The method of any one of Embodiments 67 to 78, Embodiment 91 comprising intramuscular administration. 0269. The method of Embodiment 88, wherein said HSV Embodiment 80 is HSV1 and HSV2.

0258. The method of any one of Embodiments 67 to 78, Embodiment 92 comprising intradermal administration. (0270. The method of any one of Embodiments 88 to 91, Embodiment 81 wherein said sulfated or sulfonated polysaccharide is selected from dextran Sulfate, heparin, and heparan Sulfate. 0259. The method of any one of Embodiments 67 to 78, comprising mucosal administration. Embodiment 93 Embodiment 82 (0271 The method of any one of Embodiments 88 to 92, wherein said HSV particles are isolated at a concentration of 0260. The method of any one of Embodiments 67 to 78, 107 to 10' (pfu/microgram protein). comprising intranasal administration. Embodiment 94 Embodiment 83 (0272. The method of any one of Embodiments 88 to 93, 0261 The method of any one of Embodiments 67 to 78, wherein said HSV vaccine comprises 107 to 10" (pfu equiva comprising intrarectal administration. lents/microgram protein) of inactivated HSV. Embodiment 84 Embodiment 95 0262 The method of any one of Embodiments 67 to 78, (0273. The method of any one of Embodiments 88 to 94, comprising intravaginal administration. wherein said separating comprises centrifugation, polyethyl ene glycol precipitation, filtration, gel filtration, ultra-filtra Embodiment 85 tion, tangential flow ultra-filtration, or affinity chromatogra 0263. The method of any one of Embodiments 67 to 78, phy. comprising topical administration. Embodiment 96 Embodiment 86 (0274 The method of any one of Embodiments 88 to 95, 0264. The method of any one of Embodiments 67 to 78, wherein said lipopolysaccharide-derived adjuvant is derived comprising transcutaneous administration. from the Salmonella Minnesota LPS. US 2014/0193460 A1 Jul. 10, 2014

Embodiment 97 crystalline aluminum hydroxide and not amorphous alumi num hydroxide or aluminum hydroxycarbonate or magne (0275. The method of any one of Embodiments 88 to 95, wherein said lipopolysaccharide-derived adjuvant is derived sium hydroxide. from the Salmonella Minnesota Re595 LPS. Embodiment 109 Embodiment 98 (0287. The method of any one of Embodiments 88 to 105, (0276. The method of any one of Embodiments 88 to 95, wherein said aluminum-based mineral salt adjuvant includes wherein said lipopolysaccharide-derived adjuvant is derived aluminum phosphate gel in the form of a white gelatinous from the R595 LPS. precipitate.

Embodiment 99 Embodiment 110 (0277. The method of any one of Embodiments 88 to 98, (0288. The method of any one of Embodiments 88 to 105, wherein said lipopolysaccharide-derived adjuvant is a lipid A wherein said aluminum-based mineral salt adjuvant includes adjuvant. aluminum hydroxide gel in the form of a white gelatinous precipitate. Embodiment 100 Embodiment 111 (0278. The method of any one of Embodiments 88 to 98, wherein said lipopolysaccharide-derived adjuvant is a lipid A (0289. The method of any one of Embodiments 88 to 105, adjuvant without a (R)-3-hydroxytetradecanoyl moiety. wherein said aluminum-based mineral salt adjuvant is a potassium aluminum sulfate adjuvant. Embodiment 101 (0279. The method of any one of Embodiments 88 to 98 Embodiment 112 and 100, wherein said lipopolysaccharide-derived adjuvant is 0290. The method of any one of Embodiments 88 to 111, monophosphoryl lipid A. wherein said inactivating comprises contacting said HSV with one or more agents selected from a cross-linking agent, Embodiment 102 oxidizing agent, reducing agent, heat, radiation, detergent, a 0280. The method of any one of Embodiments 88 to 98 pH changing agent, and a chemical agent selected from a and 100, wherein said lipopolysaccharide-derived adjuvant is furocoumarin, aziridine, ethylenimine, binary ethylenimine, a lipid A adjuvant without a 1-phosphate moiety. and beta-propiolactone. Embodiment 103 Embodiment 113 (0281. The method of any one of Embodiments 88 to 98, 0291. The method of Embodiment 112, wherein said 100, and 102, wherein said lipopolysaccharide-derived adju chemical agent is aziridine. vant is 3-O-desacyl-4'-monophosphoryl lipid A. Embodiment 114 Embodiment 104 0292. The method of Embodiment 112, wherein said 0282. The method of any one of Embodiments 88 to 98 chemical agent is ethylenimine. and 100 to 103, wherein said lipopolysaccharide-derived adjuvant is a synthetic MPL analogue adjuvant. Embodiment 115 0293. The method of Embodiment 112, wherein said Embodiment 105 chemical agent is binary ethylenimine. (0283. The method of any one of Embodiments 88 to 104, wherein said lipopolysaccharide-derived adjuvant is capable Embodiment 116 of binding the TLR4 protein. 0294 The method of Embodiment 112, wherein said Embodiment 106 chemical agent is beta-propiolactone. 0284. The method of any one of Embodiments 88 to 105, Embodiment 117 wherein said aluminum-based mineral salt adjuvant is an aluminum hydroxide adjuvant. 0295) The method of any one of Embodiments 112 to 116, wherein said radiation is UV radiation. Embodiment 107 Embodiment 118 0285. The method of any one of Embodiments 88 to 105, wherein said aluminum-based mineral salt adjuvant is an 0296. The method of any one of Embodiments 112 to 116, aluminum phosphate adjuvant. wherein said radiation is electron beam radiation. Embodiment 108 Embodiment 119 0286. The method of any one of Embodiments 88 to 105, 0297. The method of any one of Embodiments 112 to 116, wherein said aluminum-based mineral salt adjuvant includes wherein said radiation is infrared radiation. US 2014/0193460 A1 Jul. 10, 2014 36

Embodiment 120 Embodiment 133 0298. The method of any one of Embodiments 112 to 116, 0311. The method of any one of Embodiments 112 to 130, wherein said radiation is gamma radiation. wherein said furocoumarin is angelicin. Embodiment 121 Embodiment 134 0299. The method of any one of Embodiments 112 to 120, 0312 The method of any one of Embodiments 112 to 130, wherein said cross-linking agent is an aldehyde cross-linking wherein said furocoumarin is Xanthotoxin. agent. Embodiment 135 Embodiment 122 0313. The method of any one of Embodiments 112 to 130, 0300. The method of any one of Embodiments 112 to 120, wherein said furocoumarin is bergapten. wherein said cross-linking agent is formaldehyde. Embodiment 136 Embodiment 123 0314. The method of any one of Embodiments 112 to 130, wherein said furocoumarin is nodakenetin. 0301 The method of any one of Embodiments 112 to 120, wherein said cross-linking agent is formalin. Embodiment 137 Embodiment 124 0315. A method of preventing or treating a Herpes Sim plex Virus (HSV) infection in a subject, comprising admin 0302) The method of any one of Embodiments 112 to 123, istering to a subject in need thereof a DNA vaccine, said DNA wherein said oxidizing agent is sodium periodate. vaccine comprising a DNA sequence encoding a portion of at least one conserved HSV gene required for virus replication. Embodiment 125 0303. The method of any one of Embodiments 112 to 123, Embodiment 138 wherein said oxidizing agent is hydrogen peroxide. 0316. The method of Embodiment 137, wherein said HSV is HSV1. Embodiment 126 0304. The method of any one of Embodiments 112 to 125, Embodiment 139 wherein said reducing agent is aldrithiol-2. 0317. The method of Embodiment 137, wherein said HSV is HSV2. Embodiment 127 Embodiment 140 0305. The method of any one of Embodiments 112 to 126, wherein said detergent is Triton-X-100. 0318. The method of any one of Embodiments 137 to 139, said DNA vaccine further comprising a monophosphoryl Embodiment 128 lipid A (MPL) adjuvant. 0306 The method of any one of Embodiments 112 to 126, Embodiment 141 wherein said detergent is NP-40. 0319. The method of any one of Embodiments 137 to 140, Embodiment 129 said DNA vaccine further comprising an alum adjuvant. 0307. The method of any one of Embodiments 112 to 126, Embodiment 142 wherein said detergent is Tween-20. 0320. The method of any one of Embodiments 137 to 141, Embodiment 130 said DNA vaccine comprising whole, inactivated HSV-2. 0308. The method of any one of Embodiments 112 to 129, Embodiment 143 wherein said inactivating comprises contacting said HSV 0321. The method of any one of Embodiments 137 to 141, with one or more agents comprising UV radiation and a said DNA vaccine comprising formalin-inactivated HSV-2 furocoumarin. (F1-HSV-2). Embodiment 131 Embodiment 144 0309 The method of any one of Embodiments 112 to 130, 0322 The method of any one of Embodiments 137 to 141, wherein said furocoumarin is psoralen. said DNA sequence encoding at least a portion of a DNA polymerase from HSV-2. Embodiment 132 Embodiment 145 0310. The method of any one of Embodiments 112 to 130, wherein said furocoumarin is 4'-aminomethyl-4,5".8-trimeth 0323. The method of Embodiment 144, wherein said DNA ylpsoralen. polymerase is HSV-2 UL30 DNA polymerase. US 2014/0193460 A1 Jul. 10, 2014 37

Embodiment 146 Embodiment 159 0324. The method of Embodiment 144, said DNA vaccine 0337 The DNA vaccine of Embodiment 157, said DNA further comprising a DNA molecule encoding at least a por vaccine further comprising a DNA molecule encoding at least tion of HSV-2 secreted glycoprotein D2 (gD2t). a portion of HSV-2 secreted glycoprotein D2 (gD2t). Embodiment 147 Embodiment 160 0325 The method of any one of Embodiments 137 to 146, 0338. The DNA vaccine of any one of Embodiments 150 said DNA sequence encoding at least a portion of a helicase to 159, said DNA sequence encoding at least a portion of a from HSV-2. helicase from HSV-2.

Embodiment 148 Embodiment 161 0326. The method of Embodiment 147, wherein said heli 0339. The DNA vaccine of Embodiment 160, wherein case is HSV-2 UL5 helicase. said helicase is HSV-2 UL5 helicase.

Embodiment 149 Embodiment 162 0340. A method of treating or preventing a disease in a 0327. The method of any one of Embodiments 137 to 148, patient in need of Such treatment or prevention, said method wherein said administration is prime-boost immunization. comprising administering a therapeutically or prophylacti Embodiment 150 cally effective amount of the HSV vaccine of any one of Embodiments 1 to 66. 0328. A DNA vaccine, comprising a DNA sequence encoding a portion of at least one conserved HSV gene Embodiment 163 required for virus replication. 0341 The method of Embodiment 162, wherein said HSV is HSV1. Embodiment 151 0329. The DNA vaccine of Embodiment 150, wherein Embodiment 164 said HSV is HSV1. 0342. The method of Embodiment 162, wherein said HSV is HSV2. Embodiment 152 0330. The DNA vaccine of Embodiment 150, wherein Embodiment 165 Said HSV is HSV2. 0343. The method of Embodiment 162, wherein said HSV is HSV1 and HSV2. Embodiment 153 0331. The DNA vaccine of any one of Embodiments 150 Embodiment 166 to 152, further comprising a monophosphoryl lipid A (MPL) 0344) The method of any one of Embodiments 162 to 165, adjuvant. wherein said disease is selected from the group consisting of herpetic gingivostomatitis, herpes labialis, herpes genitalis, Embodiment 154 herpetic whitlow, herpes gladiatorum, herpesviral encephali tis, herpesviral meningitis, herpes esophagitis, herpes kerati 0332 The DNA vaccine of any one of Embodiments 150 tis, Bell’s palsy, Mollaret's meningitis, herpes rugbeiorum, to 153, further comprising an alum adjuvant. eczema herpeticum, herpetic neuralgia, and post-herpetic neuralgia. Embodiment 155 0333. The DNA vaccine of any one of Embodiments 150 Embodiment 167 to 154, comprising whole, inactivated HSV-2. (0345. The method of Embodiment 166, wherein said dis ease is herpetic gingivostomatitis. Embodiment 156 0334) The DNA vaccine of any one of Embodiments 150 Embodiment 168 to 154, comprising formalin-inactivated HSV-2 (Fl-HSV-2). 0346. The method of Embodiment 166, wherein said dis ease is herpes labialis. Embodiment 157 0335. The DNA vaccine of any one of Embodiments 150 Embodiment 169 to 154, said DNA sequence encoding at least a portion of a 0347 The method of Embodiment 166, wherein said dis DNA polymerase from HSV-2. ease is herpes genitalis. Embodiment 158 Embodiment 170 0336. The DNA vaccine of Embodiment 157, wherein 0348. The method of Embodiment 166, wherein said dis said DNA sequence is HSV-2 UL30 DNA polymerase. ease is herpetic whitlow. US 2014/0193460 A1 Jul. 10, 2014

Embodiment 171 Embodiment 185 0349 The method of Embodiment 166, wherein said dis 0363. The method of any one of Embodiments 162 to 183, ease is herpes gladiatorum. comprising treating said disease Embodiment 172 Embodiment 186 0350. The method of Embodiment 166, wherein said dis 0364 The method of any one of Embodiments 162 to 184, ease is herpesviral encephalitis. comprising preventing said disease.

Embodiment 173 Embodiment 187 0351. The method of Embodiment 166, wherein said dis 0365. The method of any one of Embodiments 162 to 186, ease is herpesviral meningitis. comprising a reduction in viral shedding.

Embodiment 174 Embodiment 188 0352. The method of Embodiment 166, wherein said dis ease is herpes esophagitis. 0366. The method of any one of Embodiments 162 to 187, comprising a reduction in the frequency of lesion occurrence. Embodiment 175 Embodiment 189 0353. The method of Embodiment 166, wherein said dis ease is herpes keratitis. 0367 The method of any one of Embodiments 162 to 188, comprising a reduction in the duration of lesion occurrence. Embodiment 176 Embodiment 190 0354. The method of Embodiment 166, wherein said dis ease is Bell's palsy. 0368. The method of any one of Embodiments 162 to 189, comprising intramuscular administration. Embodiment 177 0355 The method of Embodiment 166, wherein said dis Embodiment 191 ease is Mollaret's meningitis. 0369. The method of any one of Embodiments 162 to 189, comprising intradermal administration. Embodiment 178 0356. The method of Embodiment 166, wherein said dis Embodiment 192 ease is herpes rugbeiorum. 0370. The method of any one of Embodiments 162 to 189, Embodiment 179 comprising mucosal administration. 0357 The method of Embodiment 166, wherein said dis Embodiment 193 ease is eczema herpeticum. 0371. The method of any one of Embodiments 162 to 189, Embodiment 180 comprising intranasal administration. 0358. The method of Embodiment 166, wherein said dis Embodiment 194 ease is herpetic neuralgia. 0372. The method of any one of Embodiments 162 to 189, Embodiment 181 comprising intrarectal administration. 0359. The method of Embodiment 166, wherein said dis Embodiment 195 ease is post-herpetic neuralgia. 0373 The method of any one of Embodiments 162 to 189, Embodiment 182 comprising intravaginal administration. 0360. The method of Embodiment 166, wherein said dis Embodiment 196 ease is Alzheimer's disease. 0374. The method of any one of Embodiments 162 to 189, Embodiment 183 comprising topical administration. 0361. The method of any one of Embodiments 162 to 182, Embodiment 197 comprising a therapeutically effective amount of the HSV vaccine. 0375. The method of any one of Embodiments 162 to 189, comprising transcutaneous administration. Embodiment 184 Embodiment 198 0362. The method of any one of Embodiments 162 to 182, comprising a prophylactically effective amount of the HSV 0376. The method of any one of Embodiments 162 to 189, vaccine. comprising Subcutaneous administration. US 2014/0193460 A1 Jul. 10, 2014 39

Embodiment 199 Embodiment 211 0377 The method of any one of Embodiments 162 to 198, 0389. The vaccine of Embodiment 210, wherein said HSV wherein said method does not comprise administration of a is HSV1. prime HSV DNA vaccine. Embodiment 212 Embodiment 200 0390 The vaccine of Embodiment 210, wherein said HSV 0378. The method of any one of Embodiments 162 to 199, is HSV2. wherein said method does not comprise administration of an HSV DNA vaccine. Embodiment 213 Embodiment 201 0391 The vaccine of Embodiment 210, wherein said HSV is HSV1 and HSV2. 0379 The method of any one of Embodiments 162 to 198, wherein said method does not comprise administration of a Embodiment 214 DNA vaccine comprising a gene selected from UL30, UL5, D2, gD2t, or portions thereof. 0392 The vaccine of any one of Embodiments 202 to 213, wherein said lipopolysaccharide-derived adjuvant is derived Embodiment 202 from the Salmonella minnesota LPS. 0380 A Herpesvirus (HV) vaccine comprising inactivated Embodiment 215 HV, a lipopolysaccharide (LPS)-derived adjuvant and an alu minum-based mineral salt adjuvant. 0393. The vaccine of any one of Embodiments 202 to 213, wherein said lipopolysaccharide-derived adjuvant is derived Embodiment 203 from the Salmonella minnesota Re595 LPS. 0381. The vaccine of Embodiment 202, Embodiment 216 wherein said LPS-derived adjuvant is MPL: said aluminum-based mineral salt adjuvant is aluminum 0394 The vaccine of any one of Embodiments 202 to 213, hydroxide adjuvant or aluminum phosphate adjuvant; and wherein said lipopolysaccharide-derived adjuvant is derived said vaccine does not comprise an isolated nucleic acid from the R595 LPS. sequence formulation. Embodiment 217 Embodiment 204 0395. The vaccine of any one of Embodiments 202 to 216, wherein said lipopolysaccharide-derived adjuvant is a lipid A 0382. The vaccine of any one of Embodiments 202 to 203, adjuvant. wherein said HV is HHV-3. Embodiment 218 Embodiment 205 0396 The vaccine of any one of Embodiments 202 to 216, 0383. The vaccine of any one of Embodiments 202 to 203, wherein said lipopolysaccharide-derived adjuvant is a lipid A wherein said HV is HHV-4. adjuvant without an (R)-3-hydroxytetradecanoyl moiety.

Embodiment 206 Embodiment 219 0384 The vaccine of any one of Embodiments 202 to 203, 0397) The vaccine of any one of Embodiments 202 to 216, wherein said HV is HHV-5. wherein said lipopolysaccharide-derived adjuvant is mono phosphoryl lipid A. Embodiment 207 0385. The vaccine of any one of Embodiments 202 to 203, Embodiment 220 wherein said HV is HHV-6. 0398. The vaccine of any one of Embodiments 202 to 216 and 218, wherein said lipopolysaccharide-derived adjuvant is Embodiment 208 a lipid A adjuvant without a 1-phosphate moiety 0386 The vaccine of any one of Embodiments 202 to 203, wherein said HV is HHV-7. Embodiment 221 0399. The vaccine of any one of Embodiments 202 to 216 Embodiment 209 and 218 to 220, wherein said lipopolysaccharide-derived (0387. The vaccine of any one of Embodiments 202 to 203, adjuvant is 3-O-desacyl-4'-monophosphoryl lipid A. wherein said HV is HHV-8. Embodiment 222 Embodiment 210 0400. The vaccine of any one of Embodiments 202 to 221, 0388. The vaccine of any one of Embodiments 202 to 203, wherein said lipopolysaccharide-derived adjuvant is capable wherein said HV is a Herpes Simplex Virus (HSV). of binding the TLR4 protein. US 2014/0193460 A1 Jul. 10, 2014 40

Embodiment 223 Embodiment 234 04.01 The vaccine of any one of Embodiments 202 to 222, 0412. The vaccine of any one of Embodiments 202 to 231, wherein said lipopolysaccharide-derived adjuvant is a syn wherein said inactivated HV is formed by contacting said HV thetic MPL analogue adjuvant. with one or more agents selected from a cross-linking agent, oxidizing agent, reducing agent, heat, radiation, detergent, a Embodiment 224 pH changing agent, and a chemical agent selected from a furocoumarin, aziridine, ethylenimine, binary ethylenimine, 0402. The vaccine of any one of Embodiments 202 to 223, and beta-propiolactone. wherein said aluminum-based mineral Salt adjuvant is alumi num hydroxide adjuvant. Embodiment 235 Embodiment 225 0413. The vaccine of Embodiment 234, wherein said chemical agent is aziridine. 0403. The vaccine of any one of Embodiments 202 to 223, wherein said aluminum-based mineral Salt adjuvant is alumi Embodiment 236 num phosphate adjuvant. 0414. The vaccine of Embodiment 234, wherein said Embodiment 226 chemical agent is ethylenimine. 04.04 The vaccine of any one of Embodiments 202 to 223, Embodiment 237 wherein said aluminum-based mineral salt adjuvant is adju phos. 0415. The vaccine of Embodiment 234, wherein said chemical agent is binary ethylenimine. Embodiment 227 Embodiment 238 04.05 The vaccine of any one of Embodiments 202 to 223, wherein said aluminum-based mineral salt adjuvant is alhy 0416) The vaccine of Embodiment 234, wherein said drogel. chemical agent is beta-propiolactone.

Embodiment 228 Embodiment 239 0406. The vaccine of any one of Embodiments 202 to 223, 0417. The vaccine of any one of Embodiments 234 to 238, wherein said aluminum-based mineral salt adjuvant is a wherein said radiation is UV radiation. potassium aluminum sulfate adjuvant. Embodiment 240 Embodiment 229 0418. The vaccine of any one of Embodiments 234 to 238, 0407. The vaccine of any one of Embodiments 202 to 223, wherein said radiation is electron beam radiation. wherein said aluminum-based mineral salt adjuvant includes crystalline aluminum hydroxide and not amorphous alumi Embodiment 241 num hydroxide or aluminum hydroxycarbonate or magne 0419. The vaccine of any one of Embodiments 234 to 238, sium hydroxide. wherein said radiation is infrared radiation.

Embodiment 230 Embodiment 242 0408. The vaccine of any one of Embodiments 202 to 223, wherein said aluminum-based mineral salt adjuvant includes 0420. The vaccine of any one of Embodiments 234 to 238, aluminum phosphate gel in the form of a white gelatinous wherein said radiation is gamma radiation. precipitate. Embodiment 243 Embodiment 231 0421. The vaccine of any one of Embodiments 234 to 242, wherein said cross-linking agent is an aldehyde cross-linking 04.09. The vaccine of any one of Embodiments 202 to 223, agent. wherein said aluminum-based mineral salt adjuvant includes aluminum hydroxide gel in the form of a white gelatinous precipitate. Embodiment 244 0422 The vaccine of any one of Embodiments 234 to 242, Embodiment 232 wherein said cross-linking agent is formaldehyde. 0410 The vaccine of any one of Embodiments 202 to 231, Embodiment 245 wherein said inactivated HV comprises a replication decreas ing DNA mutation. 0423. The vaccine of any one of Embodiments 234 to 242, wherein said cross-linking agent is formalin. Embodiment 233 Embodiment 246 0411. The vaccine of any one of Embodiments 202 to 231, wherein said inactivated HV is formed by chemical inactiva 0424 The vaccine of any one of Embodiments 234 to 245, tion. wherein said oxidizing agent is sodium periodate. US 2014/0193460 A1 Jul. 10, 2014 41

Embodiment 247 Embodiment 260 0425 The vaccine of any one of Embodiments 234 to 245, 0438. The vaccine of any one of Embodiments 202 to 258, wherein said oxidizing agent is hydrogen peroxide. wherein said inactivated HV is a combination of two or more inactivated strains of the HV. Embodiment 248 Embodiment 261 0426. The vaccine of any one of Embodiments 234 to 245, wherein said reducing agent is aldrithiol-2. 0439. The vaccine of Embodiment 259, wherein said inac tivated HV is an inactivated single strain of HSV1. Embodiment 249 Embodiment 262 0427. The vaccine of any one of Embodiments 234 to 246, wherein said detergent is Triton-X-100. 0440 The vaccine of Embodiment 259, wherein said inac tivated HV is an inactivated single strain of HSV2. Embodiment 250 Embodiment 263 0428 The vaccine of any one of Embodiments 234 to 246, 0441. The vaccine of Embodiment 260, wherein said inac wherein said detergent is NP-40. tivated HV is a combination of two or more inactivated strains Embodiment 251 of HSV 1. 0429. The vaccine of any one of Embodiments 234 to 246, Embodiment 264 wherein said detergent is Tween-20. 0442. The vaccine of Embodiment 260, wherein said inac tivated HV is a combination of two or more inactivated strains Embodiment 252 of HSV2. 0430. The vaccine of any one of Embodiments 234 to 251, wherein said inactivated HV is formed by contacting said HV Embodiment 265 with one or more agents comprising UV radiation and a furocoumarin. 0443) The vaccine of any one of Embodiments 202 to 258, wherein said inactivated HV is a combination of one or more inactivated strains or HSV 1 and one or more inactivated Embodiment 253 Strains of HSV2. 0431. The vaccine of any one of Embodiments 234 to 252, wherein said furocoumarin is psoralen. Embodiment 266 0444 The vaccine of any one of Embodiments 202 to 265, Embodiment 254 formulated for intramuscular administration. 0432. The vaccine of any one of Embodiments 234 to 252, wherein said furocoumarin is 4'-aminomethyl-4,5".8-trimeth Embodiment 267 ylpsoralen. 0445. The vaccine of any one of Embodiments 202 to 265, formulated for intradermal administration. Embodiment 255 0433. The vaccine of any one of Embodiments 234 to 252, Embodiment 268 wherein said furocoumarin is angelicin. 0446. The vaccine of any one of Embodiments 202 to 265, formulated for mucosal administration. Embodiment 256 0434. The vaccine of any one of Embodiments 234 to 252, Embodiment 269 wherein said furocoumarin is Xanthotoxin. 0447 The vaccine of any one of Embodiments 202 to 265, formulated for intranasal administration. Embodiment 257 0435 The vaccine of any one of Embodiments 234 to 252, Embodiment 270 wherein said furocoumarin is bergapten. 0448. The vaccine of any one of Embodiments 202 to 265, formulated for intrarectal administration. Embodiment 258 Embodiment 271 0436 The vaccine of any one of Embodiments 234 to 252, wherein said furocoumarin is nodakenetin. 0449 The vaccine of any one of Embodiments 202 to 265, formulated for intravaginal administration. Embodiment 259 Embodiment 272 0437. The vaccine of any one of Embodiments 202 to 258, wherein said inactivated HV is an inactivated single strain of 0450. The vaccine of any one of Embodiments 202 to 265, the HV. formulated for topical administration. US 2014/0193460 A1 Jul. 10, 2014 42

Embodiment 273 Embodiment 284 0451. The vaccine of any one of Embodiments 202 to 265, 0462. The method of Embodiment 279, wherein said HV formulated for transcutaneous administration. is HHV-5.

Embodiment 274 Embodiment 285 0452. The vaccine of any one of Embodiments 202 to 265, 0463. The method of Embodiment 279, wherein said HV formulated for Subcutaneous administration. is HHV-6.

Embodiment 275 Embodiment 286 0453 The vaccine of any one of Embodiments 203 to 274, 0464. The method of Embodiment 279, wherein said HV wherein said isolated nucleic acid sequence formulation is an is HHV-7. antigenic isolated nucleic acid sequence formulation. Embodiment 287 Embodiment 276 0465. The method of Embodiment 279, wherein said HV 0454. The HV vaccine of any one of Embodiments 203 to is HHV-8. 275, wherein said isolated nucleic acid sequence formulation is a DNA vaccine. Embodiment 288

Embodiment 277 0466. The method of Embodiment 279, wherein said HV is an HSV. 0455 The HV vaccine of Embodiment 276, wherein said DNA vaccine is an antigenic isolated nucleic acid sequence Embodiment 289 formulation. 0467. The method of Embodiment 288, wherein said HSV is HSV1. Embodiment 278 0456. The HV vaccine of any one of Embodiments 202 to Embodiment 290 277, wherein said vaccine vaccinates a recipient of said vac 0468. The method of Embodiment 288, wherein said HSV cine against HV infection for up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. is HSV2. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more years. Embodiment 291 Embodiment 279 0469. The method of Embodiment 288, wherein said HSV 0457. A method of preparing a Herpesvirus (HV) vaccine, is HSV1 and HSV2. said method comprising: contacting an HV-cell mixture with a sulfated or sulfonated Embodiment 292 polysaccharide, wherein said HV-cell mixture comprises HV particles, cells and portions of cells; 0470 The method of any one of Embodiments 279 to 291, separating said HV particles from said cells thereby forming wherein said sulfated or sulfonated polysaccharide is selected isolated HV particles: from dextran Sulfate, heparin, and heparan Sulfate. inactivating said isolated HV particles thereby forming inac tivated HV particles: Embodiment 293 combining said inactivated HV particles with a lipopolysac 0471. The method of any one of Embodiments 279 to 292, charide-derived adjuvant and an aluminum-based mineral wherein said HV particles are isolated at a concentration of salt adjuvant thereby forming an HV vaccine. 10 to 10" (pfu/microgram protein). Embodiment 280 Embodiment 294 0458. The method of Embodiment 279, wherein said HV 0472. The method of any one of Embodiments 279 to 293, is HHV-1. wherein said HV vaccine comprises 107 to 10" (pfu equiva lents/microgram protein) of inactivated HV. Embodiment 281 Embodiment 295 0459. The method of Embodiment 279, wherein said HV 0473. The method of any one of Embodiments 279 to 294, is HHV-2. wherein said separating comprises centrifugation, polyethyl Embodiment 282 ene glycol precipitation, filtration, gel filtration, ultra-filtra tion, tangential flow ultra-filtration, or affinity chromatogra 0460. The method of Embodiment 279, wherein said HV phy. is HHV-3. Embodiment 296 Embodiment 283 0474. The method of any one of Embodiments 279 to 295, 0461 The method of Embodiment 279, wherein said HV wherein said lipopolysaccharide-derived adjuvant is derived is HHV-4. from the Salmonella minnesota LPS. US 2014/0193460 A1 Jul. 10, 2014

Embodiment 297 crystalline aluminum hydroxide and not amorphous alumi num hydroxide or aluminum hydroxycarbonate or magne 0475. The method of any one of Embodiments 279 to 295, wherein said lipopolysaccharide-derived adjuvant is derived sium hydroxide. from the Salmonella minnesota Re595 LPS. Embodiment 309 Embodiment 298 0487. The method of any one of Embodiments 279 to 305, 0476. The method of any one of Embodiments 279 to 295, wherein said aluminum-based mineral salt adjuvant includes wherein said lipopolysaccharide-derived adjuvant is derived aluminum phosphate gel in the form of a white gelatinous from the R595 LPS. precipitate.

Embodiment 299 Embodiment 310 0477. The method of any one of Embodiments 279 to 298, 0488. The method of any one of Embodiments 279 to 305, wherein said lipopolysaccharide-derived adjuvant is a lipid A wherein said aluminum-based mineral salt adjuvant includes adjuvant. aluminum hydroxide gel in the form of a white gelatinous precipitate. Embodiment 300 Embodiment 311 0478. The method of any one of Embodiments 279 to 298, wherein said lipopolysaccharide-derived adjuvant is a lipid A 0489. The method of any one of Embodiments 279 to 305, adjuvant without a (R)-3-hydroxytetradecanoyl moiety. wherein said aluminum-based mineral salt adjuvant is a potassium aluminum sulfate adjuvant. Embodiment 301 0479. The method of any one of Embodiments 279 to 298 Embodiment 312 and 300, wherein said lipopolysaccharide-derived adjuvant is 0490 The method of any one of Embodiments 279 to 311, monophosphoryl lipid A. wherein said inactivating comprises contacting said HV with one or more agents selected from a cross-linking agent, oxi Embodiment 302 dizing agent, reducing agent, heat, radiation, detergent, a pH 0480. The method of any one of Embodiments 279 to 298 changing agent, and a chemical agent selected from a furo and 300, wherein said lipopolysaccharide-derived adjuvant is coumarin, aziridine, ethylenimine, binary ethylenimine, and a lipid A adjuvant without a 1-phosphate moiety. beta-propiolactone. Embodiment 303 Embodiment 313 0481. The method of any one of Embodiments 279 to 298 0491. The method of Embodiment 312, wherein said and 300 to 302, wherein said lipopolysaccharide-derived chemical agent is aziridine. adjuvant is 3-O-desacyl-4'-monophosphoryl lipid A. Embodiment 314 Embodiment 304 0492. The method of Embodiment 312, wherein said 0482. The method of any one of Embodiments 279 to 298 chemical agent is ethylenimine. and 300 to 303, wherein said lipopolysaccharide-derived adjuvant is a synthetic MPL analogue adjuvant. Embodiment 315 0493. The method of Embodiment 312, wherein said Embodiment 305 chemical agent is binary ethylenimine. 0483. The method of any one of Embodiments 279 to 304, wherein said lipopolysaccharide-derived adjuvant is capable Embodiment 316 of binding the TLR4 protein. 0494. The method of Embodiment 312, wherein said Embodiment 306 chemical agent is beta-propiolactone. 0484. The method of any one of Embodiments 279 to 305, Embodiment 317 wherein said aluminum-based mineral salt adjuvant is an aluminum hydroxide adjuvant. 0495. The method of any one of Embodiments 312 to 316, wherein said radiation is UV radiation. Embodiment 307 Embodiment 318 0485 The method of any one of Embodiments 279 to 305, wherein said aluminum-based mineral salt adjuvant is an 0496 The method of any one of Embodiments 312 to 316, aluminum phosphate adjuvant. wherein said radiation is electron beam radiation. Embodiment 308 Embodiment 319 0486 The method of any one of Embodiments 279 to 305, 0497. The method of any one of Embodiments 312 to 316, wherein said aluminum-based mineral salt adjuvant includes wherein said radiation is infrared radiation. US 2014/0193460 A1 Jul. 10, 2014 44

Embodiment 320 Embodiment 333 0498. The method of any one of Embodiments 312 to 316, 0511. The method of any one of Embodiments 312 to 330, wherein said radiation is gamma radiation. wherein said furocoumarin is angelicin. Embodiment 321 Embodiment 334 0499. The method of any one of Embodiments 312 to 320, 0512. The method of any one of Embodiments 312 to 330, wherein said cross-linking agent is an aldehyde cross-linking wherein said furocoumarin is Xanthotoxin. agent. Embodiment 335 Embodiment 322 0513. The method of any one of Embodiments 312 to 330, wherein said furocoumarin is bergapten. (0500. The method of any one of Embodiments 312 to 320, wherein said cross-linking agent is formaldehyde. Embodiment 336 Embodiment 323 0514. The method of any one of Embodiments 312 to 330, wherein said furocoumarin is nodakenetin. 0501) The method of any one of Embodiments 312 to 320, wherein said cross-linking agent is formalin. Embodiment 337 0515. A method of treating or preventing HV infection in Embodiment 324 a patient in need of such treatment or prevention, said method 0502. The method of any one of Embodiments 312 to 323, comprising administering a therapeutically or prophylacti wherein said oxidizing agent is sodium periodate. cally effective amount of the HV vaccine of any one of Embodiments 202 to 278. Embodiment 325 Embodiment 338 0503. The method of any one of Embodiments 312 to 323, 0516. The method of Embodiment 337, wherein said HV wherein said oxidizing agent is hydrogen peroxide. is HHV-1.

Embodiment 326 Embodiment 339 0504. The method of any one of Embodiments 312 to 325, 0517. The method of Embodiment 337, wherein said HV wherein said reducing agent is aldrithiol-2. is HHV-2.

Embodiment 327 Embodiment 340 0505. The method of any one of Embodiments 312 to 326, 0518. The method of Embodiment 337, wherein said HV wherein said detergent is Triton-X-100. is HHV-3.

Embodiment 328 Embodiment 341 0506. The method of any one of Embodiments 312 to 326, 0519. The method of Embodiment 337, wherein said HV wherein said detergent is NP-40. is HHV-4. Embodiment 329 Embodiment 342 0520. The method of Embodiment 337, wherein said HV 0507. The method of any one of Embodiments 312 to 326, is HHV-5. wherein said detergent is Tween-20. Embodiment 343 Embodiment 330 0521. The method of Embodiment 337, wherein said HV 0508. The method of any one of Embodiments 312 to 329, is HHV-6. wherein said inactivating comprises contacting said HV with one or more agents comprising UV radiation and a furocou Embodiment 344 marin. 0522 The method of Embodiment 337, wherein said HV Embodiment 331 is HHV-7. 0509. The method of any one of Embodiments 312 to 330, Embodiment 345 wherein said furocoumarin is psoralen. 0523 The method of Embodiment 337, wherein said HV is HHV-8. Embodiment 332 Embodiment 346 0510. The method of any one of Embodiments 312 to 330, wherein said furocoumarin is 4'-aminomethyl-4,5".8-trimeth 0524. The method of Embodiment 337, wherein said HV ylpsoralen. is an HSV. US 2014/0193460 A1 Jul. 10, 2014

Embodiment 347 Embodiment 358 0525. The method of Embodiment 346, wherein said HSV 0536. The method of any one of Embodiments 350 or 357, is HSV1. wherein said disease is herpetic gingivostomatitis. Embodiment 348 Embodiment 359 0526. The method of Embodiment 346, wherein said HSV 0537. The method of any one of Embodiments 350 or 357, is HSV2. wherein said disease is herpes labialis. Embodiment 349 Embodiment 360 0527. The method of Embodiment 346, wherein said HSV 0538. The method of any one of Embodiments 350 or 357, is HSV1 and HSV2. wherein said disease is herpes genitalis. Embodiment 350 Embodiment 361 0528. The method of any one of Embodiments 337 to 349, 0539. The method of any one of Embodiments 350 or 357, wherein said HV infection causes a disease selected from wherein said disease is herpetic whitlow. herpetic gingivostomatitis, herpes labialis, herpes genitalis, herpetic whitlow, herpes gladiatorum, herpesviral encephali Embodiment 362 tis, herpesviral meningitis, herpes esophagitis, herpes kerati tis, Bell’s palsy, Mollaret's meningitis, herpes rugbeiorum, (0540. The method of any one of Embodiments 350 or 357, eczema herpeticum, herpetic neuralgia, and post-herpetic wherein said disease is herpes gladiatorum. neuralgia. Embodiment 363 Embodiment 351 (0541. The method of any one of Embodiments 350 or 357, 0529. A method of treating or preventing a disease in a wherein said disease is herpesviral encephalitis. patient in need of Such treatment or prevention, said method comprising administering a therapeutically or prophylacti Embodiment 364 cally effective amount of the HV vaccine of any one of Embodiments 202 to 278. (0542. The method of any one of Embodiments 350 or 357, wherein said disease is herpesviral meningitis. Embodiment 352 Embodiment 365 0530. The method of Embodiment 351, wherein said dis ease is an HV associated disease. (0543. The method of any one of Embodiments 350 or 357, wherein said disease is herpes esophagitis. Embodiment 353 Embodiment 366 0531. The method any one of Embodiments 351 to 352, wherein said HV is an HSV. (0544 The method of any one of Embodiments 350 or 357, wherein said disease is herpes keratitis. Embodiment 354 Embodiment 367 0532. The method of Embodiment 353, wherein said HSV is HSV1. (0545. The method of any one of Embodiments 350 or 357, wherein said disease is Bell's palsy. Embodiment 355 Embodiment 368 0533. The method of Embodiment 353, wherein said HSV is HSV2. (0546. The method of any one of Embodiments 350 or 357, wherein said disease is Mollaret's meningitis. Embodiment 356 0534. The method of Embodiment 353, wherein said HSV Embodiment 369 is HSV1 and HSV2. (0547. The method of any one of Embodiments 350 or 357, wherein said disease is herpes rugbeiorum. Embodiment 357 0535. The method of any one of Embodiments 351 to 356, Embodiment 370 wherein said disease is selected from the group consisting of herpetic gingivostomatitis, herpes labialis, herpes genitalis, (0548. The method of any one of Embodiments 350 or 357, herpetic whitlow, herpes gladiatorum, herpesviral encephali wherein said disease is eczema herpeticum. tis, herpesviral meningitis, herpes esophagitis, herpes kerati Embodiment 371 tis, Bell’s palsy, Mollaret's meningitis, herpes rugbeiorum, eczema herpeticum, herpetic neuralgia, and post-herpetic (0549. The method of any one of Embodiments 350 or 357, neuralgia. wherein said disease is herpetic neuralgia. US 2014/0193460 A1 Jul. 10, 2014 46

Embodiment 372 Embodiment 386 0550. The method of any one of Embodiments 350 or 357, 0564) The method of any one of Embodiments 337 to 380, wherein said disease is post-herpetic neuralgia. comprising intravaginal administration. Embodiment 373 Embodiment 387 0551. The method of any one of Embodiments 350 or 357, 0565. The method of any one of Embodiments 337 to 380, wherein said disease is Alzheimer's disease. comprising topical administration. Embodiment 374 Embodiment 388 0552. The method of any one of Embodiments 337 to 373, 0566. The method of any one of Embodiments 337 to 380, wherein said method is a method of treating. comprising transcutaneous administration. Embodiment 375 Embodiment 389 0553. The method of any one of Embodiments 337 to 373, 0567. The method of any one of Embodiments 337 to 380, wherein said method is a method of preventing. comprising Subcutaneous administration. Embodiment 376 Embodiment 390 0554. The method of any one of Embodiments 337 to 375, 0568. The method of any one of Embodiments 337 to 389, wherein said method includes a therapeutically effective wherein an isolated nucleic acid sequence formulation is not amount of the HV vaccine. administered to said patient. Embodiment 377 Embodiment 391 0555. The method of any one of Embodiments 337 to 375, 0569. The method of Embodiment 390, wherein said iso wherein said method includes a prophylactically effective lated nucleic acid sequence formulation is a DNA sequence. amount of the HV vaccine. Embodiment 392 Embodiment 378 0570. The method of Embodiment 390, wherein said iso 0556. The method of any one of Embodiments 337 to 377, lated nucleic acid sequence formulation is a nucleic acid comprising a reduction in viral shedding. vaccine. Embodiment 379 Embodiment 393 0557. The method of any one of Embodiments 337 to 378, 0571. The method of Embodiment 390, wherein said iso comprising a reduction in the frequency of lesion occurrence. lated nucleic acid sequence formulation is a DNA vaccine. Embodiment 380 Embodiment 394 0558. The method of any one of Embodiments 337 to 379, (0572. The method of any one of Embodiments 337 to 389, comprising a reduction in the duration of lesion occurrence. wherein said method does not comprise administration of a prime HV DNA vaccine. Embodiment 381 Embodiment 395 0559 The method of any one of Embodiments 337 to 380, comprising intramuscular administration. (0573. The method of any one of Embodiments 337 to 389, wherein said method does not comprise administration of an Embodiment 382 HV DNA vaccine. 0560. The method of any one of Embodiments 337 to 380, Embodiment 396 comprising intradermal administration. (0574. The method of any one of Embodiments 337 to 389, Embodiment 383 wherein said method does not comprise administration of a DNA vaccine comprising a gene selected from an HSV 0561. The method of any one of Embodiments 337 to 380, UL30, UL5, gD2, gD2t, or portions thereof. comprising mucosal administration. Embodiment 397 Embodiment 384 (0575. The method of any one of Embodiments 337 to 396, 0562. The method of any one of Embodiments 337 to 380, consisting of a single administration of said HV Vaccine. comprising intranasal administration. Embodiment 398 Embodiment 385 (0576. The method of any one of Embodiments 337 to 396, 0563. The method of any one of Embodiments 337 to 380, consisting of a prime-boost administration of said HV Vac comprising intrarectal administration. cine. US 2014/0193460 A1 Jul. 10, 2014 47

Embodiment 399 Embodiment 410 0577. The method of any one of Embodiments 337 to 396, 0588. The kit of any one of Embodiments 400 to 401, consisting of a prime-boost-boost administration of said HV wherein said HV vaccine treats a subject administered said vaccine. HV vaccine for multiple strains of said HV. Embodiment 400 VI. Examples 0578. A kit comprising the HV vaccine of any one of 0589. There are provided, inter alia, methods of use of Embodiments 202 to 278 and instructions for administering DNA vaccines encoding conserved HSV-2 genes that are said HV vaccine to a patient. necessary for virus replication as well as a vaccine consisting of whole, inactivated HSV-2 together with monophosphoryl Embodiment 401 lipid A (MPL) and aluminum-based mineral salt adjuvants. 0579. The kit of Embodiment 400, wherein said HV vac Immunization with the DNA polymerase (UL30) or helicase cine is administered in a prime-boost administration. (UL5) genes of HSV-2 was found to elicit CD8+ T cell responses and coimmunization of UL30 with a DNA encod Embodiment 402 ing secreted glycoprotein D2 (gD2t) resulted in slightly increased protection against disease after lethal, intravaginal 0580. The HV vaccine of any one of Embodiments 1 to 66, (i.Vag.) challenge when compared to immunization with g|D2t 150 to 161, and 202 to 278, wherein said HV vaccine protects DNA alone. Protection against disease, death, and virus shed a subject administered said HV vaccine from multiple strains ding was greater in mice immunized with whole, formalin of Said HV. inactivated HSV-2 (FI-HSV2) when compared to a gl)2t pro tein subunit vaccine. Prime-boost immunization with UL5, Embodiment 403 UL30, gD2t DNAs and FI-HSV2 resulted in nearly complete 0581. The HV vaccine of any one of Embodiments 1 to 66, protection against vaginal disease. Single formulation immu 150 to 161, and 202 to 278, wherein said HV vaccine vacci nization with DNA vaccines, FI-HSV2, and MPL in an alu nates a subject administered said HV vaccine from multiple minum phosphate (Adju-Phos) adjuvant did not give an strains of said HV. enhancement in protection relative to FI-HSV2/MPL/Adju PhoS alone. Most strikingly, intramuscular immunization Embodiment 404 with FI-HSV2 together with MPL and Alhydrogel adjuvants resulted in undetectable levels of HSV-2 shedding (<10 PFU) 0582. The HV vaccine of any one of Embodiments 1 to 66, in the majority of mice after both a challenge and a Subse 150 to 161, and 202 to 278, wherein said HV vaccine treats a quent long-term (17 weeks) re-challenge. subject administered said HV vaccine for multiple strains of 0590. Without wishing to be bound by any theory, it is said HV. believed that genes that are expressed early in the viral life cycle, essential for viral replication, and evolutionarily con Embodiment 405 served may be useful vaccine targets for a cellular immune 0583. The method of any one of Embodiments 67 to 87, response. Using another herpesvirus challenge model, 137 to 149, 162 to 201, and 337 to 399, wherein said patient murine cytomegalovirus (MCMV), we demonstrated that the is vaccinated against one or more strains of said HV following CD8+ T cell responses generated against DNA vaccines said administration. encoding the conserved, essential genes DNA polymerase and helicase were protective (Morello, C. S. et al., J. Virol., Embodiment 406 81:7766-7775 (2007)). When DNA immunization was com bined with an inactivated virus plus adjuvant boost, mice 0584) The method of any one of Embodiments 67 to 87, were completely protected against virus replication in the 137 to 149, 162 to 201, and 337 to 399, wherein said patient target organs following systemic challenge (Morello, C. S. et is protected against one or more strains of said HV following al., J. Virol. 79:159-175 (2005); Morello, C.S., M.Ye, and D. said administration. H. Spector. J. Virol. 76:4822-4835 (2002)). Importantly, mice were almost completely protected against a mucosal Embodiment 407 challenge after the systemic prime-boost immunization (Mo 0585. The method of any one of Embodiments 67 to 87, rello, C. S. et al., J. Virol. 79:159-175 (2005)). 137 to 149, 162 to 201, and 337 to 399, wherein said patient 0591 Based on the above studies, we tested whether is treated for one or more strains of said HV following said homologous HSV-2 vaccine targets as well as a similar prime administration. boost strategy could be efficacious against an i.vag. HSV-2 challenge in mice. DNA vaccines encoding genes required for Embodiment 408 viral DNA replication, UL30 (HSV-2 DNA polymerase) and UL5 (helicase), were tested either alone or together with the 0586. The kit of any one of Embodiments 400 to 401, truncated glycoprotein D2 (gD2t) plasmid. The DNA primed wherein said HV vaccine vaccinates a subject administered animals were subsequently boosted with either g|D2t protein said HV vaccine from multiple strains of said HV. with MPL/Alum or formalin inactivated HSV-2 (FI-HSV2) Embodiment 409 together with MPL/Alum. For comparison, an additional group received a vaccine similar to that of the one adminis 0587. The kit of any one of Embodiments 400 to 401, tered in the GSK trials, a purified g|D2t protein formulated wherein said HV vaccine protects a subject administered said with MPL/alum alone. We also tested single formulation HV vaccine from multiple strains of said HV. vaccines consisting of adjuvant plus FI-HSV2 in the presence US 2014/0193460 A1 Jul. 10, 2014 48 or absence of DNA. Following i.vag. HSV-2 challenge, the adjuvant did not increase protection relative to FI-HSV2/ protective efficacies of the vaccines were compared with MPL/Adju-Phos alone; and 5) addition of MPL/Alum to the respect to outcomes of acute disease and virus shedding, and FI-HSV2 was required for optimal protection against disease, protection against a Subsequent challenge. viral replication, and latent virus load in the dorsal root gan 0592 Our results revealed the following: 1) When DNA glia (DRG). Most notably, an optimized vaccine formulation encoding HSV-2 genes was solely used as the vaccine, the of FI-HSV2MPL/Alhydrogel given i.m. completely pro inclusion of the g|D2t plasmid in the mix was critical for tected against detectable vaginal HSV-2 shedding in the providing protection against lethality and disease as well as majority of animals and HSV-2 latent DNA in the DRG of all reduction in viral shedding resulting from HSV-2 challenge. animals. 2) Mice immunized with gl)2t protein with MPL/alum had more deaths, higher disease scores, and increased levels of A. MATERIALS AND METHODS viral shedding compared to mice that received FI-HSV-2 plus MPL/alum. 3) When mice were first immunized with a DNA 0595 Cells and Viruses. vaccine containing gC2t, UL5 and UL30 DNA, a boost of 0596 Vero cells (ATCC CLL-81) were purchased from FI-HSV-2 in MPL/Alum provided greater protection against ATCC and propagated in Dulbecco's Modified Eagle disease and viral shedding following an i.vag. HSV-2 chal Medium (DMEM) supplemented with 5% heat-inactivated lenge than did a boost of glo2t protein in MPL/alum. 4) An fetal bovine serum (HI-FBS), 5% heat-inactivated newborn optimized vaccine containing only FI-HSV-2 in MPL/Alum calf serum (HI-NCS), 100 U penicillin and 100 ug strepto provided nearly complete protection against HSV-2 viral mycin per ml, and 2 mMadditional L-glutamine. 293FT cells shedding after a lethal mucosal (i.vag.) short term challenge (Invitrogen) were propagated in DMEM (4.5 g glucose per and long-term re-challenge. And 5) addition of either empty liter) supplemented with 10% HI-FBS, 1xMEM nonessential vector DNA or DNA encoding HSV-2 genes to FI-HSV-2 in amino acids, and penicillin, Streptomycin, and additional MPL/Alum reduced the efficacy of a single formulation vac glutamine as above. Sera and Supplements were from Invit C1G. rogen Life Technologies. 0593. Initial work focused on developing a vaccine against 0597 HSV-2 strain G (Ejercito, P. M. et al., J. Gen. Virol., cytomegalovirus using the mouse model. We began to explore 2:357-364 (1968)) was a gift from David M. Knipe and was the possibility that increased protection against mouse propagated by infection of confluent Vero monolayers at an cytomegalovirus (MCMV) replication could be achieved by MOI of 0.1, incubation at 33°C. for 72 h, and then harvest of expanding the CD8+-T-cell-mediated immune responses and the cell-associated virus as previously described (Dudek, T. et protective antibody responses against multiple epitopes with al., Virology, 372:165-175 (2008)). This HSV-2 stock had a a vaccine that included killed whole virus (formalin-inacti titer on Vero cells of 2.08x10 PFU permland an approximate vated) in a strong adjuvant, such as Alum. Our results showed LDso of 5x10 PFU in medroxyprogesterone acetate-treated that prior vaccination with formalin-inactivated murine (see below) BALB/c mice following i.vag. infection. cytomegalovirus (FI-MCMV) in alum provided greater 0598 FI-HSV2that was prepared from cell associated and reduction in titers of virus in the spleen and particularly in the extracellular virus was obtained following the infection of salivary glands than did a comparative vaccination with vari confluent Vero cell monolayers at an MOI of 0.05 to 0.1 and ous DNAs encoding MCMV genes when the mice were chal incubation at 33°C. for 3 days. Cells were shaken off flasks, lenged with a high dose of virulent virus (Morello et al., 2002: pelleted, resuspended in 117" of the volume of infected cul Morello et al. 2005) ture media, and Sonicated in a Misonix cuphorn Sonicator. 0594. Herpes Simplex Virus Type 2 (HSV-2) infection can The sonicate was clarified by centrifugation at 500xg for 5 result in life-long recurrent genital disease, asymptomatic min. The resulting pellet was resuspended in Tris-buffered virus shedding, and transmission. No vaccine to date has saline (TBS) and homogenized in a Tenbroeck homogenizer. shown significant protection clinically. Here, we used a The homogenate was clarified by centrifugation as above and mouse model of genital HSV-2 infection to test the efficacy of the resulting Supernatant combined with the first Supernatant. a vaccine consisting of whole, formalin-inactivated HSV-2 The combined supernatants were clarified again at 4000xg (FI-HSV2) formulated with monophosphoryl lipid A (MPL) for 15 min and the virus in the Supernatant was concentrated and alum adjuvants. Vaccine components were administered by ultracentrifugation through a cushion of 25% (w/v) sorbi alone or as a prime-boost immunization together with DNA tol in Tris-buffered saline (TBS) in an SW-27 rotor at 25,000 vaccines encoding a truncated glycoprotein D2 (gD2t) and RPM for 1 h. Pellets were resuspended overnight in TBS, the two conserved HSV-2 genes necessary for virus replication, virus was repelleted by ultracentrifugation as above, and the UL5 (DNA helicase) and UL30 (DNA polymerase). Our pellets were resuspended in TBS. The resulting preparation results show: 1) Compared with mock immunized controls, was analyzed for infectious virus by plaque assay on Vero mice immunized with FI-HSV2 plus MPL/Alum consistently cells and for protein content by Bradford protein assay (Bio showed protection against disease burden and total viral shed Rad) with BSA (Pierce) standard. For inactivation, a 37% ding while the mice immunized with gl)2t protein with MPL/ (w/v) formaldehyde stock solution was diluted 2000-fold in alum did not; 2) Protection against genital disease and viral Dulbecco's phosphate buffered saline (DPBS, Invitrogen) replication correlated with the type of boost in a prime-boost and an equal Volume was added to the virus (final formalde immunization with little advantage afforded by a DNA prime; hyde dilution of 1:4000 or 0.009%). After 72 h incubation at 3) Intramuscular (i.m.) immunization with FI-HSV2 in MPL/ 37°C. with end-over-end mixing, equimolar sodium bisulfite Alhydrogel adjuvant provided nearly complete protection was added to quench residual formaldehyde and 0.1 ml of the against vaginal HSV-2 shedding after a lethal intravaginal formalin-treated virus was used to infect Vero cells to confirm (i.Vag.) short-term challenge and long-term rechallenge; 4) the absence of detectable infectivity. Inactivated virus was Single formulation immunization with DNA vaccines, FI stored at -80° C. in aliquots. The preparation had a titer of HSV2, and MPL in an aluminum phosphate (Adju-Phos) 7x107 PFU equivalents per ml and a pre-inactivation protein US 2014/0193460 A1 Jul. 10, 2014 49

content of 4x10" PFU perug of protein. FI-Mock was pre TABLE 2 - Continued pared in parallel from mock infected Vero cells and media as described above. Primers used for construction of vaccine plasmids 0599 FI-HSV2 was prepared from the virus-containing Seculence (5' - 3 media of Vero cells that were infected and incubated as for the HSV-2 ORF FI-HSV2 above except that media was harvested on day 5 Cloned or pWAX postinfection. Media was twice clarified by centrifugation, Mutagenesis Forward Primer Reverse Primer first at 500xg for 5 min and subsequently at 4000xg for 15 min. The virus in the resultant Supernatant was concentrated GCCACCATGTTTTG TGCTAGAGTATCAAAGG by ultracentrifugation as above except through a cushion of TGCCGCGGGCGGC CTCTATGCAACATTCGA 25% (w/v) sucrose in DPBS. Virus pellets were resuspended (SEQ ID NO : 3) CG (SEQ ID NO : 4) in DPBS on ice overnight and the combined virus was titered, US6 (gD2t) GCCACCATGGGGCG GACGTCCTGGATCGACG analyzed for protein content, inactivated, quenched, con TTTGACCTCC G (SEQ ID NO: 6) firmed for lack of infectivity, and stored in aliquots as above. (SEO ID NO; 5) The extracellular FI-HSV2 had a final titer of 3x10 PFU UL54 (ICP27) GCCACCATGGCTAC AAATAGGGAGTTGCAGT equivalents per ml, and prior to inactivation, a protein content CGACATTGATATG AGAAGTATTTGCCGTG of 10 PFU per ug of protein. FI-Mock was prepared in (SEO ID NO: 7) (SEQ ID NO: 8) parallel from the media of mock infected Vero cells as described above. HincII site GCCAGATATACGCCA CAATAATCAATGTCATG 0600 FI-HSV2 was also prepared by releasing virus from deletion in TGACATTGATTATTG GCGTATATCTGGC infected Vero cells by washing them with dextran sulfate, pVAX1 (SEO ID NO: 9) (SEQ ID NO: 1.O) clarifying the wash by low-speed centrifugation, and pellet HincII site + GGTACCGAGCTCGGA GAATTCCACCACACTGG ing the virus through a cushion by ultracentrifugation as FLAG tag TCCACGTCGACTACA ACTATTATCACTTGTCA previously described (Morello, C. S. et al., 2011, Id.). addition in AGGATGACGATGACA TCGTCATCCTTGTAGTC 0601 Plasmid Construction and Expression. pVAX1.1 AGTGATAATAGTCCA GACGTGGATCCGAGCTC 0602 Genomic DNA was prepared from HSV-2 strain GTGTGGTGGAATTC GGTACC G-infected Vero cells using the DNeasy Blood and Tissue Kit (SEQ ID NO: 11) (SEQ ID NO: 12) (Qiagen) and used as a template in PCR reactions using KOD Hot-Start DNA Polymerase (Novagen/CN Biosciences). The following ORFs were amplified: DNA polymerase (UL30), 0603 The pVAX1 plasmid vector that was designed for helicase-primase (UL5), truncated gl)2 (gD2t) correspond DNA vaccine development in accordance with FDA guide ing to the extracellular portion of the US6 gene (aa 1-327), lines was purchased from Invitrogen. Expression of the ORFs and ICP27 (UL54) (see Table 1 for complete list). HSV-2- in this is driven by the strong, constitutive HCMV major IE specific primers were designed based on the strain HG52 promoter/enhancer. This vector was modified by the genome sequence (Genbank accession no. Z86099.2) and QuikChange mutagenesis technique (Stratagene) in 2 steps. sense primers were designed to introduce a Kozak consensus First, a point mutation was created to remove the single sequence (GCC ACC) proximal to the initiating AUG (see HincII restriction site in the vector (to yield intermediate Table 2 for the sequences of all primers used in the study). vectorpVAX1.1). Second, the multicloning site was modified to contain a unique HincII site followed by a FLAG tag TABLE 1. coding sequence and 3 in-frame stop codons (see Table 2 for mutagenesis primer sequences used). The final vector, desig HSV-2 genes used for DNA immunization or intracellular nated pVAX1.2 and abbreviated below as pVAX, was the cytokine staining basis for the cloning and expression of carboxyterminally HSV-2 HCMV FastA Overlap FLAG-tagged HSV-2 ORFs. ORF Gene Product Homolog Identity (%) (aa) 0604 PCR products were ligated to HincII-digested UL5 Helicase-primase subunit UL105 39 753 UL30 DNA polymerase catalytic UL54 38 955 pVAX1.2 vector, and resulting clones were screened by Subunit restriction digestion and sequencing of the HSV-2 ORFs US6 glycoprotein D2t (aa 1-327) — (Eton Biosciences, San Diego, Calif.). For purification of gl)2 ULS4 ICP27, gene regulator UL69 37 52 protein subunit vaccine, the glo2t-FLAG ORF was subcloned ORF, open reading frame, into the pcDNA3-based vector pc3Aneo (Ye, M. C. S. 'aa, amino acids, Morello, and D. H. Spector. J. Virol. 76:2100-2112 (2002)). ssDNA, single-stranded DNA The UL5, UL30, and ICP27 ORFs were also subcloned into pc3Aneo for use in the cell-based CD8+ T cell assay below. TABLE 2 0605 Compared to the HSV-2 HG52 reference sequence, Primers used for construction of vaccine plasmids the cloned UL5 (helicase-primase) from strain G contained 3 Sedulence (5'-3') silent mutations, and the cloned gL)2t contained a single silent mutation. The cloned UL30 sequence contained 3 base dif HSV-2 ORF Cloned or pWAX ferences from the reference strain with the resultant putative Mutagenesis Forward Primer Reverse Primer amino acid changes, G25-A (Ala-Thr)., C43-A (Pro-Ser), and T179-C (Leu-Pro). To distinguish between HSV-2 strain UL5 GCCACCATGGCGGC ATAGACAATGACCACAT GTCCGGCGGGGAG TCGGATCGCGTAGAGC polymorphisms and PCR cloning artifacts, 1.4 kb of the 5' end (SEQ ID NO: 1) (SEQ ID NO: 2) of the UL30 ORF was amplified from the HSV-2 strain G infected Vero cell DNA and the PCR product was directly sequenced. All 3 differences in the UL30 clone were found in US 2014/0193460 A1 Jul. 10, 2014 50 the strain GPCR product, indicating that these are present in ware (NIH), a standard curve of BSA concentrations was HSV-2 strain G. The ICP27 clone contained one strain differ generated by optical density and the g|D2 protein concentra ence, A492-C (Lys-Asn). tions were interpolated. Protein yields were approximately 5 0606 Plasmids were purified by Qiagen Endo-Free Mega ug of g|D2t per ml of media. Interestingly, we also found that or Giga anion exchange columns. DNAS were resuspended to the majority of the secreted gl)2t protein had a carboxy ~2.5-3 mg per ml of endotoxin-free Tris-HCl (pH 8), and terminal fragment cleaved off. The resulting protein had lost stored at -20°C. in aliquots. Immediately prior to injections, the FLAG tag but showed no shift in migration on a gel. These DNAs are thawed and diluted in endotoxin-free Tris buffered findings were similar to those of Murata et al., who showed saline, pH 8. that the media of Hep2, but not Vero, cells infected with 0607 Expression of full-length ORF-FLAG fusion pro HSV-2 contained a secreted, but fully glycosylated, form of teins was confirmed by transient transfection into 293FT cells gD2 (Murata, T. et al., J. Gen. Virol.., 83:2791-2795 (2002)). (Invitrogen) using Lipofectamine-2000 (Invitrogen) fol For our g|D2t protein-based immunizations, only the lowed by Western blot. Blots were probed with anti-FLAG uncleaved, FLAG-tag purified protein was used. mouse monoclonal antibody (M2, Sigma) that was directed 0611 Immunization and Challenge of Mice. against the carboxy-terminal FLAG tag fused onto each 0612 Specific pathogen free and Helicobacter spp. PCR HSV-2 ORF. Blots containing lysates from pVAX1.2-g|D2t negative BALB/c female mice were purchased from Charles transfected cells or media were probed with anti-FLAG or River Laboratories at 3-5 weeks of age and housed in anti-gld mouse monoclonal antibody DL6 (Santa Cruz, Bio microisolator cages. Mice were allowed to acclimate for at technology, Inc.). DL6 binds an HSV-1/2 common, con least 2 weeks prior to immunization or infection. served linear epitope of gld (aa 272-279.) FIG. 1A shows that 0613 For DNA-based immunizations, mice were intrad the resultant specific protein band for each HSV-2 ORF ermally (i.d.) injected into the tail 3 times in 2 weeks with 30 migrated to the expected position, with expected molecular ul of DNA in endotoxin free TBS (see Results for DNA masses as follows: UL5, 99 kDa; UL30, 137 kDa, gD2t, 45 doses). For coimmunization with plasmids, plasmid DNAS kDa, and ICP27, 55.9 kDa. were mixed together prior to injection. 0608 Preparation of g|D2 Protein. 0614 For protein-based vaccination, MPL from Salmo 0609. 293FT cells in 10 cm tissue culture dishes were nella minnesota Re 595 (Salmonella minnesota Re595) transiently transfected as above. The next day, cells were fed (MPL) was purchased from Sigma, resuspended to 1 mg per with media containing 0.4% (v/v) of mammalian protease ml of 0.5% triethanolamine, emulsified by sonication as pre inhibitor cocktail (Sigma P 1860), and on d 3 posttransfec viously described (Baldridge, J. R., and R.T. Crane, Methods, tion, the media was collected, a protease inhibitor cocktail 19:103-107 (1999)), and stored at 4°C. untiluse. FI-HSV2 or (Sigma P8340) was added to a 1% final concentration, and the purified go2 subunit stocks were thawed and diluted in either media was clarified by centrifugation. A time course of DPBS (protein vaccine only experiment) or endotoxin-free expression of the pVAX-g|D2t vector was performed in order ddH2O (DNA prime protein vaccine boost experiment) and to characterize the secretory efficiency of the gl)2t ORF, and MPL was added. Imject alum (Pierce) was added dropwise both whole cell lysates and anti-FLAG immunoaffinity puri while mixing and then additionally mixed 30 min at room fied media were subjected to Western blot analysis (FIG. 1B). temperature before injection. Using the gld-specific monoclonal antibody DL6, we found 0615. For the protein-based vaccine only experiment, that the g)2t protein was detectable in the media as early as mice received 2 subcutaneous (s.c.) injections at approxi day 2 posttransfection, with peak levels in the media occur mately mid-back and 3 weeks apart with either 1) 107 PFU ring after day 2. equivalents of the cell-associated and extracellular FI-HSV2 0610 For preparation of a gl)2t protein subunit vaccine, (approximately 250 g protein), 2) an equal Volume of FI we Subsequently performed large-scale transfections of Mock, or 3) 5uggl)2 subunit. Each dose also contained 12.5 293FT cells with the SV40 ori-containing vector pc3Aneo ug of MPL and Imject alum (Pierce) equivalent to 725 ug Al. gD2t and then Subjected the g)2t-containing media to anti Mice were challenged 4 weeks following the last protein FLAG immunoaffinity chromatography. After clarification of based vaccination. the media containing secreted, truncated g|D2, binding buffer 0616) For the DNA prime protein-based vaccine boost was added Such that the following concentrations were experiment, mice were primed by 3 i.d. injections in the tail, obtained prior to loading onto a column of anti-FLAG M2 2 weeks apart, with either 1) 50 ug pVAX, 2) 50 luggD2t slurry: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.1% DNA, or 3) a cocktail of 20 ug each of the UL5, UL30, and (v/v) TritonX-100. The column was thenwashed with 50 mM gD2t DNAs. Five weeks after the last DNA injection, mice Tris-buffered Saline--0.1% Triton X-100 and then 50 mM from each DNA prime group were boosted s.c. with either 1) Tris-buffered saline prior to elution with 100 ug 3xFLAG 107 PFU equivalents of extracellular FI-HSV2 (containing peptide (Sigma) per ml of 50 mM Tris-buffered saline fol 8.3 ug protein), 2) an equal Volume of FI-Mock, or 3) 5ug lowing manufacturer's guidelines. The fractions containing gD2 subunit. Each dose also contained 12.5ug of MPL and truncated gl)2 protein were identified by SDS-PAGE and Imject alum equivalent to 125 ug Al(OH)3 and 43.3 ug of Al. overnight Coomassie blue staining (GelCode Blue, Pierce). Mice received a second boost 3 weeks after the first and then After overnight storage on ice, the fractions containing g|D2 were challenged 4 weeks later. protein were mixed and stored in aliquots at -80° C. Purity 0.617 For the experiment in which DNA vaccines were and gl)2 protein concentration were determined by SDS combined with FI-HSV2 in Adju-Phos adjuvant, mice were PAGE and staining as above with bovine serum albumin given bilateral intramuscular (i.m.) injections of 40 ul into standards run on the same gel for quantification purposes. each quadriceps muscles with dextran Sulfate wash-derived FIG.1C shows a representative Coomassie blue stained SDS FI-HSV2 (or dextran sulfatewash-derived FI-Mock) in MPL/ PAGE gel of the g|D2t-containing eluate and the BSA stan Adju-Phos adjuvant as follows. Each immunization con dard used for quantification purposes. Using Image-J Soft tained a total of 12.5 lug of MPL, Adju-Phos (Accurate US 2014/0193460 A1 Jul. 10, 2014

Chemical & Scientific, Westbury, N.Y.) equivalent to 90 ug in 24-well plates were infected for 1 h, 37° C., on a 50-60 Al, and either 1) 107 PFU equivalents of FI-HSV2 (0.34 pug of RPM orbit shaker with an infection volume of 0.25 ml DNG protein), 2) 60 ug of pVAX DNA plus a volume of FI-Mock per well. After adsorption, the inocula were removed and the equal to that of the FI-HSV2 above, 3) 60 g of pVAX DNA monolayers were overlaid with 0.5 ml of DMEM with 1% plus 107 PFU equivalents of FI-HSV2, 4) 20 g each of UL5, HI-NCS and 0.35% sterileagarose. After 2d incubation at 37° UL30, and g|D2t DNAs plus 107 PFU equivalents of C. and 7% CO2, formalin in DPBS was added to a 4% for FI-HSV2, or 5) 50 g each of UL5, UL30, and g|D2t DNAs malin final concentration and the plates were fixed at 25°C. plus 107 PFU equivalents FI-HSV2. Adju-Phos was first con for at least 3 h. Agarose overlays were aspirated and mono centrated 2-fold by centrifugation at 500xg for 5 min fol layers stained with crystal violet. The assay limit of sensitiv lowed by the removal of buffer supernatant equivalent to one ity of each assay is denoted below, and assays yielding no half the total volume. FI-HSV2 or FI-Mock was adsorbed to plaques were assigned a titer of one-half the detection limit the concentrated Adju-Phos for 1 h with mixing prior to the for graphing and calculation purposes. addition of MPL and, if added, the DNA(s). Mice were given 0622 Intracellular Cytokine Staining. an additional injection after 4 weeks. A short-term i.vag. 0623 Two weeks following the final injection, levels of challenge of mice from groups 1, 2, and 5 above was given 3 antigen specific CD8+ T cells elicited by DNA immuniza weeks after the second injection and the remaining mice were tions were measured as previously described (Morello, C. S. given a long-term i.vag. challenge 9 weeks after the second et al., J. Virol. 81:7766-7775 (2007)). BALB SV40 (H-2) injection. cells were grown in 96-well plates to 80% confluence and 0618. For the experiment comparing the Alhydrogel or transfected with 0.5ug of plasmid DNA and 1.25ul of Fugene Adju-Phos adjuvants, injection routes, and protection against 6 (Roche) per well. challenge and re-challenge, 107 PFU equivalents (9.5 or 13.5 0624 Forty-eight hours post transfection, splenocytes ug of protein for the first or second immunizations, respec from immunized mice were harvested and erythrocytes were tively) of extracellular FI-HSV2 (or FI-Mock) and 12.5ug of lysed (BD Pharm Lyse). 8x10 splenocytes were added to MPL was given. As above, FI-HSV2 or FI-Mock was first duplicate wells of transfected cells in the presence of brefel adsorbed to Alhydrogel (Accurate Chemical & Scientific, din A (GolgiPlug: BD) and stimulated for 8 hours at 37° C. Westbury, N.Y.) or 2-fold concentrated Adju-Phos equivalent and 7% CO. As a positive control, 2x10° splenocytes were to 54 ug or 450 ug of Al, respectively, prior to addition of MPL stimulated with 50 ng PMA and 1 lug ionomycin per ml. and DNA, if used. Groups of mice were immunized with the Duplicate wells of 2x10° splenocytes were also stimulated following components and immunization routes: 1) 60 lug with 0.1 M of a previously published H-2" CD8+ T cell pVAX DNA and FI-Mock in Adju-Phos given i.m., 2) FI epitope of ICP27 (HGPSLYRTF (SEQ ID NO:13): Gen HSV2 in Alhydrogel given s.c., 3) FI-HSV2 in Alhydrogel Script, NJ (Haynes, J. R. et al., Vaccine, 24:5016-5026 given i.m., or 4) FI-HSV2 in Adju-Phos given i.m. Mice (2006)). received an identical immunization after 4 weeks and then 0625 For staining, duplicate wells were combined into i.vag. challenged 3 weeks later. Mice that survived the HSV-2 one well of a 96-well round bottom plate and washed once challenge were medroxyprogesterone treated and re-chal with PBS. To differentiate live versus dead cells, a viability lenged 17 weeks after the first challenge as above. A group of dye (50LA per well) was added to cells for 10 minutes at 37° 8 naive mice was challenged at the same time to demonstrate C. (LIVE/DEAD fixable violet; Molecular Probes, Invitro the infectivity of the re-challenge virus. gen). Fc block (CD16/32: BD) was added (2 ug per well), and 0619 Intravaginal virus challenge, disease scoring, and splenocytes were incubated for 10 min at 4°C. to prevent quantification of viral shedding. On dayS-7 and -1 relative to nonspecific binding to Fc receptors. Splenocytes were stained challenge, mice received a Subcutaneous injection of 3 mg of for surface markers CD8-AX488 (Clone 53-6.7; BD) and medroxyprogesterone acetate (Depo-Provera, Sigma CD4-Ax647 (clone RM4-5; BD) for 30 minutes at 4° C. and M1629) in order to synchronize the estrus cycle and to then permeabilized and fixed using the BDCytofix/Cytoperm increase susceptibility to vaginal HSV infection (Parr, M. B. kit. CD3-PE-Cy5 (Clone 145-2C11: BD) and IFN-y-PE et al., Lab. Invest., 70:369-380 (1994)). Mice were swabbed (Clone XMG1.2: BD) were included in the intracellular stain with a DPBS-moistened polyester tipped swab (MicroPur for 45 minutes at 4°C. Data from 50,000 live CD3+ T cells 1001D, PurFybr Solon, Rhinelander, Wis.) prior to i.vag. were collected on a BD FACSCanto flow cytometer and ana challenge with 5x10" PFU of HSV-2 strain G (ca. 10 LDso lyzed with BD FACSDiva software at the Research Flow doses) by micropipette. Mice were scored daily for 21 days Cytometry Core Facility of the San Diego Center for AIDS for the severity of vaginal inflammation from 0 (no vaginitis), Research and the Veterans Medical Research Foundation and 1 (mild Swelling or redness only), 2 (moderate Swelling or VA San Diego Healthcare System, La Jolla, Calif. erosions), 3 (severe genital maceration), or 4 (central nervous system involvement or death), with half scores given to inter B. DNA IMMUNIZATION WITH THE mediate disease levels. Mice discovered with hind limb CONSERVED, ESSENTIAL HSV-2 GENES paralysis were given a score of 4 and immediately sacrificed. TOGETHER WITH g|D2t DNA IS PROTECTIVE 0620. To measure vaginal virus shedding, intravaginal AGAINST LETHAL INTRAVAGINAL. (I.VAG.) swab samples were collected on days 1 through 5 (or 6) CHALLENGE postchallenge using polyester tipped Swabs (above) that were 0626. Our previous DNA immunization studies using the pre-moistened with DPBS+1% HI-NCS+0.1% (w/v) glucose MCMV model showed that vaccination with plasmids (DNG). Swabs were stored in 1 ml of DNG at -80°C. in until expressing two essential genes that are conserved among titration by plaque assay on Vero cell monolayers. herpesviruses, DNA polymerase (MCMV M54) and a heli 0621 For plaque assays, Swabs were quickly thawed in a case/primase subunit (MCMV M105), elicited protection 37°C. water bath and Vortex mixed at full-speed for 30s prior against virus replication in the spleen. This protection was as to serial dilution in DNG. Confluent monolayers of Vero cells high as that afforded by the immunodominant gene IE1-pp 89 US 2014/0193460 A1 Jul. 10, 2014 52

(Morello, C. S. et al., J. Virol., 81:7766-7775 (2007)). DNA immunized with either pVAX, UL5, or UL30 had mean immunization with these two MCMV genes also resulted in HSV-2 titers of 10 PFU per swab, while the meantiters in primary and postchallenge CD8+ T cell levels that were the gl)2t DNA immunized groups were reduced between 1 to higher than those elicited by MCMV infection (Morello, C.S. 1.4 Logs (FIG. 2C). Titers peaked in all of the immunization et al., J. Virol., 81:7766-7775 (2007)). We also showed that groups on day 2 postchallenge, with the pVAX immunized mice immunized with a mixture of plasmids encoding group shedding 10 PFU and the other immunization groups MCMV glycoprotein B and two nonstructural proteins fol having titer reductions of 0.5 to 1.5 logs. The UL30+g|D2t lowed by a boost with formalin-inactivated MCMV in alum group had the lowest titers on both days 1 and 2. By day 3 showed long-term complete protection against a systemic postchallenge, all groups including the pVAX control had challenge with MCMV and almost complete protection similartiters of 10 PFU. After this time, the shedding in all of against a mucosal challenge (Morello, C. S. et al., J. Virol., the groups receiving g|D2t DNA showed increased clearance 79:159-175 (2005)). kinetics relative to the groups not receiving g)2t. Taken 0627 Because protective effects have been described for together, immunization with UL30+g)2t DNAs resulted in the truncated, secreted g|D2 DNA in both the mouse and both the lowest disease and the lowest shedding levels early in guinea pig models (Bernstein, D. I. et al., Vaccine, 17:1964 infection, but shedding from days 3 to 6 was identical to that 1969 (1999); Fotouhi, F. et al., Fems Immunol. Med. Micro in mice receiving only gl)2t DNA. biol., 54:18-26 (2008); Higgins, T. J. et al., J. Infect. Dis., 0631. A second immunization experiment was performed 182:1311-1320 (2000); Hoshino, Y. et al., J. Virol. 79:410 to test whether enhanced protection against disease or virus 418 (2005); Strasser, J. E. et al., J. Infect. Dis., 182:1304-1310 shedding could be achieved through coimmunization with (2000)), we were interested in determining whether con UL5 and UL30 DNAs. Groups of 8 mice were immunized served, essential HSV-2 genes could be used together with the with either 50 lug of pVAX (pVAX group) or 25 uggD2t+25 gD2t DNA to augment the level of protection. In view of the ug pVAX DNA (gD2t group). For the UL5+UL30 groups, 20 limited success of DNA only vaccines in the human trials to ug each of UL5 and UL30 DNAs were immunized with either date, we additionally sought to assess whether DNA immu 20 ug of pVAX (UL5+UL30 group) or 20 ug of go2t DNA nization could augment the protection and immune responses (UL5+UL30+g|D2t group). Immunized mice were treated elicited by a whole, killed HSV-2 vaccine together with MPL with medroxyprogesterone acetate and i.vag. challenged 3 and alum adjuvants. weeks after the last immunization as above. Death, disease, 0628. In our initial experiment we examined the protective and vaginal HSV-2 shedding were assessed. efficacy of the corresponding conserved HSV-2 genes for the 0632 FIG. 2D illustrates that none of the mice immunized DNA polymerase (UL30) and helicase/primase (UL5) in the with pVAX survived through 21 days postchallenge. Com presence and absence of the DNA encoding the truncated bining UL5 and UL30 DNA in the vaccine without the addi HSV-2 glycoprotein D (gD2t). Eight mice per group were i.d. tion of g)2t DNA also did not increase protection against immunized with either 50 ug of pVAX (pVAX group), 25 ug death (FIG. 2D), disease (FIG. 2E), or reduce HSV-2 shed each of pVAX and g|D2t DNA (gD2t group), 25ug of pVAX ding (FIG. 2F), relative to that observed in groups immunized plus 25 ug of UL5 or UL30 DNA (UL5 or UL30 groups, with pVAX, UL5, or UL30 DNA alone. WhengD2t DNA was respectively), or 25 ug UL5 or UL30 DNA plus 25 uggl)2t added to the UL5 plus UL30 vaccine, resultant viral titers DNA (UL5+g|D2t and UL30+g|D2t groups, respectively). were similar to that of the group immunized with glo2t DNA Mice were treated with medroxyprogesterone acetate to alone (FIG. 2F). increase susceptibility to i.vag. HSV-2 infection, and 3 weeks after the last DNA immunization, they were given a lethal, C. DNA IMMUNIZATION WITH CONSERVED i.vag. challenge of 5x10" PFU (approximately 10xLDso) of ESSENTIAL GENES ELICITS ANTIGEN HSV-2 strain G. Mice were monitored for survival through SPECIFIC CD8+ T CELLS day 21 postchallenge and scored for disease severity using a 0633. In view of the lack of protection elicited by the UL5 previously described severity scale as described in the Mate and UL30 DNAs in the absence of g|D2t DNA, it was impor rials and Methods through day 12 postchallenge. Intravaginal tant to determine whether vaccination elicited CD8+ T cells. Swabs were collected on days 1-6 postchallenge to measure Mice were immunized three times in two weeks with the UL5 HSV-2 shedding. or UL30 plasmids. Two weeks following the final immuniza 0629. We found that immunization with either UL5 or tion, mice were sacrificed and their spleens were harvested. UL30 DNA alone did not increase 21-day survival relative to Splenocytes were stimulated for 8 hours in the presence of the pVAX controls, but these DNAS together with gl)2t DNA BALB SV40 stimulator cells that had been transiently trans resulted in protection, as measured by Survival, that was simi fected with the respective antigen-encoding plasmid or empty lar to that generated by glC2t DNA alone (FIG. 2A). Similarly, pcDNA3 plasmid, and an intracellular cytokine staining anogenital disease severity in the mice immunized with UL5 assay was performed. or UL30 alone were similar to that in the pVAX group (FIG. 0634. We initially performed two independent experi 2B). Mean disease severity in the groups receiving gC2t DNA ments to validate the use of antigen-expressing BALB SV40 was reduced relative to the pVAX controls, with disease transfectants to stimulate CD8+ T cells to produce IFN-y. For severity peaking at 1.2-1.7 on days 4 or 5 postchallenge and these studies, we infected BALB/c mice with 2x10 PFU of then decreasing to day 11. On day 11, one mouse died in the HSV-2 Strain G in the footpad, and 7 days later isolated gD2t group and in the UL5+g|D2t group (FIG. 2B). Of all the splenocytes from the infected and control naive mice. As vaccine groups, immunization with UL30+gl)2t DNAS another control, splenocytes were collected from mice that resulted in the lowest peak disease (mean of 1.2) and total were immunized intradermally with the backbone vector disease burden through day 12, and no deaths. DNA (pVAX). The splenocytes were stimulated either with 0630 Vaginal HSV-2 shedding was measured by plaque BALB SV40 cells transfected with pcDNA3-ICP27 (ICP27 assay on Vero cell monolayers. On day 1 postchallenge, mice cells) or an ICP27 peptide corresponding to a known CD8+ T US 2014/0193460 A1 Jul. 10, 2014

cell epitope in BALB/c mice (Haynes, J. R. et al., Vaccine, ease score in the mice immunized with the gL)2t protein was 24:5016-5026 (2006)). FIG.3A shows that a mean of 0.6% of approximately 1.0 until day 12 postchallenge, and then rose the CD8+ T cells from HSV-2 infected mice were IFN-y slightly (FIG. 4B). In contrast, the peak mean disease score in positive when stimulated with ICP27 cells as compared to a the FI-HSV2 immunized group was 0.5 on day 5 postchal mean of 1.0% of the CD8+ T cells when stimulated with the lenge, and there were only 2 additional days of 0.3 to 0.4 mean ICP27 peptide. Only background levels of IFN-Y positive scores. The maximum disease score for individual mice in CD8+ T cells were detected in the splenocytes from the con this group was 0.5, except for one mouse with a score of 1.0 trol naive mice or pVAX DNA immunized mice regardless of for one day only. Thus, only a transient, low-level inflamma the type of stimulation. tion in the anogenital region after lethal HSV-2 challenge 0635. In the next experiment, 2 groups of mice (n=4) were occurred in FI-HSV2 immunization animals. given intradermal injections of 60 ug total plasmid DNA: 0640 Levels of vaginal virus shedding on days 1 through pVAX, UL5, or UL30. FIG. 3B shows that mice immunized 6 postchallenge are shown in FIG. 4C. Peak mean virus titers with either UL5 or UL30 had varying levels of CD8+ T cell were observed on day 2 postchallenge for the FI-Mock group responses to UL5 (mean=0.875%) or UL30 (mean=0.825%). and day 3 for the glo2t protein group. Mean virus titers in the These numbers are likely an underestimate since a lower FI-HSV2 group were reduced 2.1 to 3.7 Logs compared with frequency of ICP27 specific CD8+ T cells was detected when the FI-Mock group. Although on day 1 postchallenge, the the splenocytes from HSV-2 infected mice were stimulated gD2t protein group had a mean titer reduction of 27-fold with ICP27 cells than with the ICP27 peptide (FIG. 3A). relative to the FI-Mock group, by day 3 postchallenge, these Nevertheless, UL5 and UL30 represent novel vaccine target groups showed similar levels of virus shedding (FIG. 4C). antigens since IFN-y+CD8+ T cells specific for either protein (0641 Compared with the peak mean virus titers of 107 were also not observed in HSV-2 infected mice. and 10 PFU observed in the FI-Mock group and the g|D2t 0636. In a repeat experiment, a combination group with 60 protein group, respectively, mean titers in the FI-HSV2 ug each of UL5, and UL30 was included (FIG. 3C). The immunized mice ranged between 10' and 10° PFU CD8+ T cell responses in mice immunized with UL5 or UL30 throughout the observation period. On day 2 postchallenge, alone were lower than the first experiment (mean=0.600% the mean level of viral shedding in the FI-HSV2 immunized and 0.325%, respectively). The variability between the mice was 3600-fold lower than that in the FI-Mock group. experiments is likely due to the inconsistent nature of the However, as shown in FIG. 4D, protection against Vaginal transfected stimulator cells, since a third experiment resulted HSV-2 shedding in individual mice in the gl)2t protein and in mean UL5 and UL30 responses of 0.875% and 0.750%, FI-HSV2 groups did show considerable variability. Viral respectively. When mice were co-immunized with UL5 plus titers in individual FI-HSV2 immunized mice ranged from UL30 DNA, CD8+ T cell responses to both UL5 and UL30 approximately 10 PFU to below the limit of assay protection were detectable (mean=0.375% and 0.425%, respectively). (2 PFU per swab). Two of the FI-HSV2 immunized mice had In Summary, the conserved, essential HSV-2 gene plasmids no detectable virus on any day through day 6 postchallenge, UL5 and UL30, elicit modest CD8+ T cell responses, as although they did show low-level inflammation (severity measured by IFN-Y production. scores of 0.5) between days 3 and 6 postchallenge. Taken D. IMMUNIZATION WITH FI-HSV2 TOGETHER together, immunization with FI-HSV2/MPL/Alum provided WITH MPLAALUMISHIGHLY PROTECTIVE high levels of protection against both disease and virus shed AGAINST HSV-2 DISEASE AND SHEDDING ding compared with g)2t protein immunization. 0637 Because subunit vaccination with HSV-2 envelope E. PRIME-BOOST IMMUNIZATION WITH UL5, glycoproteins, including the GSK g|D2 Vaccine containing an UL30, AND GD2T DNAS-FI-HSV2 PROVIDES MPL/Alum adjuvant, has not shown consistent levels of pro NEARLY COMPLETE PROTECTION AGAINST tection in clinical trials, we asked whether a multivalent vac HSV-2 GENITAL DISEASE cine consisting of FI-HSV2 together with MPL/Alum could be more protective than go2 protein subunit/MPL/Alum vac 0642 We had successfully used a DNA prime inacti cination. For the FI-HSV2 vaccine, HSV-2 was harvested vated whole virus boost strategy against Systemic or mucosal from infected Vero cells and extracellular supernatants, pel MCMV challenge in mice, with undetectable levels of virus leted through a sorbitol cushion, washed with DPBS, and in the target organs after challenge (Morello, C. S. et al., J. inactivated with formalinas described in Materials and Meth Virol. 79:159-175 (2005); Morello, C. S., M. Ye, and D. H. ods. In addition, an FI-Mock virion preparation was made in Spector. J. Virol.., 76:4822-4835 (2002)). We therefore pro parallel using mock-infected Vero cells. Secreted gl)2t pro ceeded to test whether this strategy would also provide high tein was prepared using anti-FLAG immunoaffinity chroma level protection and to compare the various efficacies of tography on the extracellular supernatants of 293FT cells that immunization with DNA, protein subunit, or FI-HSV2 alone were transiently transfected with pc3Aneo-gD2t vector. or in combination. 0638 Mice (n=8) were subcutaneously (s.c.) immunized (0643 Mice (n=8) were DNA primed with one of the fol and then boosted 3 weeks later with one of the following: 107 lowing: 1) 50 lug of pVAX, 2) 50 g of gl)2t DNA, or 3) a PFU equivalents of FI-HSV2, a volume of FI-Mock equal to cocktail of 20 g each of the UL5, UL30, and g|D2t DNAS the FI-HSV2 above, or 5ug of gl)2t protein, with each vac using the schedule shown in FIG. 5A. Five weeks after the last cine including MPL/Alum. Mice were challenged as above 4 DNA injection, mice from each DNA prime group were s.c. weeks following the last vaccination. boosted with one the following formulated with MPL/Alum 0639 We found that by 21 days postchallenge, all 8 of the 1) 107 PFU equivalents of FI-HSV2, 2) an equal volume of FI-Mock immunized mice had died, whereas all 8 of the FI-Mock, or 3) 5 luggl)2 subunit. Mice received a second FI-HSV2 immunized mice survived (FIG. 4A). In the g|D2t boost 3 weeks after the first and then were lethally i.vag. protein immunized group, 5 mice Survived. Peak mean dis challenged 4 weeks later as above. US 2014/0193460 A1 Jul. 10, 2014 54

0644 Survival to 21 days postchallenge is summarized in 0650 Boosting withg)2t protein resulted in greater initial FIG. 5B. In the mice primed with pVAX, boosting with the decreases in HSV-2 shedding of 1.5 to 2 Logs relative to the FI-Mock preparation gave no protection against death. In the pVAX-FI-Mock group, while only the gl)2t DNA-g|D2t pro FI-Mock boosted mice, prior immunization with gl)2t DNA tein group maintained decreased levels of virus shedding on resulted in 5 of 8 mice surviving, and the addition of UL5 and days 3 through 5 postchallenge. In the latter group, viral UL30 DNAs to the g|D2t DNA prime resulted in fewer survi infection was more rapidly cleared, and virus levels were at or vors (3 of 8). Boosting with gl)2t protein was completely below the limit of detection in 7 of 8 mice by day 6 postch protective against death in the pVAX and gL)2t DNA primed allenge (FIG. 5D, middle). Of note, 2 of the 8 mice in this groups. One mouse in the UL5, UL30, gD2t DNA primed group had no detectable virus in any of the Swabs (<10 PFU group that was boosted with g)2t protein died postchallenge per Swab). (and one died of an ear infection before challenge), leaving 0651 Boosting with FI-HSV2 was found to result in the only 6 survivors. Boosting with FI-HSV2 gave similar pro greatest initial decreases of virus shedding, from 2-2.25 Logs tection against death when compared with the g|D2t protein, on day 1 postchallenge in each of the DNA primed groups with one mouse in each of the pVAX and UL5, UL30, and relative to the pVAX-FI-Mock controls (FIG.5D, right). On gD2t DNAS groups succumbing to challenge. day 2 postchallenge, mean HSV-2 shedding levels in the 0645. The severity of disease in the mice with each prime pVAX-FI-HSV2 group were approximately 3.5 Logs lower boost combination was scored through day 12 postchallenge than controls, and virus was rapidly cleared in this group to a (FIG. 5C). Of the mice boosted with FI-Mock, all of the mean level at the detection limit by day 6. Peak HSV-2 shed pVAX primed mice were dead by day 11 postchallenge, while ding was similar in the g|D2t DNA-FI-HSV2 group, although priming with gl)2t DNA or a combination of UL5, UL30, and the peak occurred earlier in the infection and viral shedding gD2t DNAs gave similar peak mean disease scores of 0.9-1 on persisting one additional day. However, 3 of 8 mice in gl)2t day 7 postchallenge and then near resolution to below a mean DNA-FI-HSV2 group had no detectable shedding throughout of 0.25-0.5 by day 10 (FIG.5C, left). However, late deaths in the entire time course. Finally, a peak titer of 10 PFU on day the g|D2t DNA (3 mice) and UL5, UL30, and g|D2t DNAs (5 3 in the UL5, UL30, gD2t DNAS-FI-HSV2 group was slightly mice) resulted in the increasing mean scores shown. higher than in the other FI-HSV2 groups, and virus in this 0646 Peak mean disease scores in the mice boosted with grouppersisted at a levelofapproximately 10° PFU until days gD2t protein were lowest in the gl)2t DNA primed mice (0.3), 6-7 postchallenge. with disease at this level lasting between days 3 to 7 postch allenge (FIG. 5C, middle). The peak disease in the groups F. CO-IMMUNIZATION WITH PLASMID DNA primed with pVAX or a combination of UL5, UL30, and gL)2t AND FI-HSV2 IN ADJU-PHOSASA SINGLE DNAs and then boosted with g|D2t protein had similar levels FORMULATION IS LESS EFFECTIVE THAN of 0.9 and 0.7. FI-HSV2 IN ADJU-PHOSALONE (0647. The groups boosted with FI-HSV2 all showed a 0652 While the prime-boost strategy proved highly pro peakin disease at day 4 postchallenge, with the lowest peak of tective, co-injection of the DNA and FI-HSV2 in a single 0.3 in the UL5, UL30, gD2t DNAS-FI-HSV2 group (FIG.5C, formulation would be highly advantageous for both manufac right). The highest level of disease observed in the mice in this turing vaccine and for clinical application. In addition, com latter group was a score of 1 for one day. The remaining mice bination of the two platforms may result in cooperative pro had disease scores of 0 or 0.5 until disease was completely tective responses to the vaccine components. Adju-Phos is a resolved by day 8 postchallenge. However, one mouse in this clinically approved aluminum phosphate adjuvant that when group that had no observable vaginal disease except for a delivered together with a hepatitis B viral antigen encoding single 0.5 score on day 2 postchallenge died on day 20. plasmid DNA and protein, elicited both a Th1 and Th2 type 0648. In general, protection against vaginal disease immune response against the DNA and protein, respectively seemed to be most closely related to the type of boost. Groups (Kwissa, M. et al., J. Mol. Med., 81:502-510 (2003)). Accord that were boosted with gl)2t protein showed more protection ingly, groups of mice (n=8) were given intramuscular injec than the groups that received HSV-2 DNA and the FI-Mock tions with MPL/Adju-Phos formulated with 107 PFU equiva boost, and groups receiving the FI-HSV-2 boost showed the lents FI-HSV2+/-DNA plasmids encoding UL5, UL30, and highest level of protection. The best protection was afforded gD2t (50 ug each). The negative control group received MPL/ in the prime boost groups given g|D2t DNA-gD2t protein or Adju-Phosformulated pVAX and FI-Mock. Mice were given UL5, UL30, gD2t DNAS-FI-HSV2, with similar peak levels a second injection 4 weeks after the first and then medroX of disease but lower total disease burden in the latter group. yprogesterone treated and challenged as previously 0649 Vaginal HSV-2 shedding was measured from intra described. vaginal Swabs on days 1 through 10 postchallenge by plaque 0653. On day 2 postchallenge, all of the negative control assay. Viral shedding in the pVAX primed-Mock boosted mice (pVAX+FI-Mock) were shedding HSV-2 and the mean groups peaked at 10 PFU on day 2 postchallenge, with titer was 10° PFU (FIG. 6A, left panel). The groups of mice virus still detectable in all of these mice on day 7 postchal that received the FI-HSV2 had significantly reduced mean lenge (FIG. 5D, left). Mock boosted mice that were primed titers (approximately 3 Logs, P-0.001 and P-0.01, Kruskal with glo2t DNA or a combination of UL5, UL30, and gL)2t Wallis, plus Dunn's Multiple Comparisons Test). In addition DNAs initially had approximately 10-fold lower titer on day half of the mice in the group that received FI-HSV2 (no DNA) 1 postchallenge, but only the latter group showed this had levels of HSV-2 shedding that were below the detection decrease on day 2. Decreases in vaginal virus shedding rela limit. By Day 4, 7 out of 8 of these animals, and half of the tive to pVAX primed mice were more evident by day 5 postch UL5, UL30, gD2t+FI-HSV2 immunized group had levels of allenge, and by day 7, virus was detectable in only 1 of the shedding that were at or below the limit of detection (FIG. 6A, gD2t DNA primed mice and in none of the UL5, UL30, gD2t right panel). In contrast, the mean titers of the pVAX--FI DNAs primed mice. Mock immunized animals remained elevated at 10 PFU. US 2014/0193460 A1 Jul. 10, 2014

Immunization with UL5, UL30, gD2t DNA and 0657 To this end, groups of 8 mice were immunized with FI-HSV2MPL/Adju-Phos was not significantly better than 107 PFU equivalents of FI-HSV2 and MPL as above, except FI-HSV2MPL/Adju-Phos alone. that the types of alum and the injection routes were as follows: 0654. In another experiment, the durability of the immune 1) FI-HSV2/MPL/Alhydrogel given s.c.; 2) FI-HSV2/MPL/ response elicited by the single formulation was examined by Alhydrogel given i.m.; or 3) FI-HSV2/MPL/Adju-Phos given waiting 9 weeks after the second immunization before chal i.m. A control group received FI-Mock/MPL/Adju-Phos lenge. In addition to the previous 3 groups included in the given i.m. Mice were given a second injection 4 weeks after short-term challenge (pVAX--FI-Mock, FI-HSV2, and FI the first and then medroxyprogesterone treated and chal HSV2+50 ug each UL5, UL30, gD2t), 2 more groups formu lenged as above. lated in MPL/Adju-Phos were added. 1) FI-HSV2+20 ug 0658. Two mice in the pVAX FI-Mock group died prior to each UL5, UL30, gD2t DNA plasmids in order to test the dose challenge, and the remaining mice in this group Succumbed to used in the earlier DNA experiments and 2) FI-HSV2+60 ug HSV-2 disease by day 12 postchallenge. No deaths occurred pVAX DNA plasmid to serve as a control. Two days postch in any of the FI-HSV2 immunized groups. For the few ani allenge, the pVAX+FI-Mock group had mean titers of 107 mals in this group that showed signs of infection, only mild PFU whereas the group that received FI-HSV2 alone (no erythema or Swelling was observed. Mean disease scores in DNA) had almost a 4 Log reduction in titer, 10' PFU (FIG. the FI-HSV2 groups peaked on day 7 postchallenge to similar 6B. left panel). Compared to the negative control animals, the levels of 0.2-0.25, and complete resolution of disease was groups that received UL5, UL30, gD2t DNA, either 20 or 50 observed in 7 of 8 mice in each group by day 11. ug, had a similar approximate 3 Log reduction, 10' and 0659 On day 2 postchallenge, the mean HSV-2 titer in the 10', respectively. By Day 4, 6 out of 8 mice in the FI-HSV2 pVAX FI-Mock control group was 10' PFU, and the mean only group had virus titers that were below the limit of detec titers in the groups receiving FI-HSV2 were reduced by 3 tion, whereas only 1 or 2 out of 8 animals in the 20 g and 50 Logs (FIG. 7A, left panel). In addition, half or more of the ug UL5, UL30, gD2t DNA--FI-HSV2 groups, respectively, mice in each FI-HSV2 groups had reductions in virus shed were shedding virus below the limit of detection (FIG. 6B, ding to levels at or below the detection limit. While HSV-2 right panel). The reduction in virus shedding on Days 2 and 4 titers in the pVAX FI-Mock control group remained at the 10 for each of the groups that received antigenic DNA--FI-HSV2 PFU level on day 4 postchallenge, no or only very low levels were statistically significant compared to the pVAX FI-Mock of virus was detectable in any of the FI-HSV2 immunized group (P<0.001 to P-0.05, Kruskal-Wallis plus Dunn's Mul mice (FIG. 7A, right panel). The reduction in virus shedding tiple Comparison Tests). However, the group that received the for each of the FI-HSV2 groups, relative to the pVAX FI pVAX--FI-HSV2, did not have a statistically significant Mock group, was statistical significant on each day postchal reduction in virus shedding on either day, with meantiters of lenge (P<0.05 to P-0.001, Kruskal-Wallis plus Dunn's Mul 10°7 and 10°, respectively. tiple Comparison Tests). No significant differences, however, 0655 Taken together, these data suggest that the combi were seen between the protected groups (FIG. 7A). Taken nation of FI-HSV2 and plasmid DNA into a single formula together, immunization with FI-HSV2 and MPL together tion with MPL/Adju-Phos is not as efficacious in preventing with the aluminum containing adjuvants Alhydrogel or Adju HSV-2 shedding as FI-HSV2 alone. These results were also Phos resulted in protection against HSV-2 disease and shed observed in the HSV-2 disease scores in the animals that ding that was at least as robust as that afforded by Imject alum. received the same type of immunization and the long-term 0660. In this experiment, several of the FI-HSV2 immu challenge. Of greater significance, the FI-HSV2 immunized nized mice in each group had no detectable shedding (<10 animals had the best protection against both viral shedding PFU) on either day tested. An important question was and disease in both a short- and long-term challenge, Suggest whether the high level protection against challenge that was ing that FI-HSV2 elicits a durable and highly protective provided by the FI-HSV2/MPL/Alum would also be effective immune response. against a long-term re-challenge. To this end, the FI-HSV2 immunized mice were medroxyprogesterone treated as G. IMMUNIZATION WITH FI-HSV2 IN before and then re-challenged 17 weeks after the first chal ALHYDROGEL OR ADJU-PHOSADJUVANTS IS lenge with the same 10xLDso dose of HSV-2. As a control for HIGHLY PROTECTIVE AGAINST HSV-2 the re-challenge, a naive group of 8 mice was infected con REPLICATION FOLLOWING CHALLENGE AND currently with the same HSV-2 inoculum as the rest of the RE-CHALLENGE 1CC. 0656. In the experiment above, we found that the best 0661. One of the FI-HSV2/Alhydrogel/s.c. mice died after protection against virus shedding tested was provided by the first medroxyprogesterone treatment, and thus there were immunization with FI-HSV2 alone. We next sought to con 7 mice remaining in this group for re-challenge. All of the firm these results and to determine whether the FI-HSV2 naive mice died by day 14 postchallenge, and one of the 7 immunization could be improved. One of the first consider mice of the FI-HSV2/Alhydrogel/s.c. mice died on day 21 ations was that Imject alum consists of both crystalline and after the re-challenge. None of the other mice showed HSV-2 amorphous aluminum phases and is not a well-defined form disease beyond mild erythema or Swelling through day 21 of alum suitable for clinical translation. Since both aluminum postchallenge. hydroxide and aluminum phosphate adjuvants have been 0662. The re-challenge dose of HSV-2 given to the naive licensed for use in vaccines, it was important to test the more group resulted in virus shedding levels of approximately 10 clinically relevant forms of alum, Such as Alhydrogel or Adju PFU on both days 2 and 4 postchallenge (FIG. 7B), which Phos, to eliminate the possibility that an undefined aspect of were similar to those in the pVAX FI-Mock group on the the Imject alum contributed to the protection. We also sought respective days postchallenge (FIG. 7A left and right). Strik to test whether the route of immunization could affect the ingly, only 2 of the re-challenged mice in the FI-HSV2 groups resultant protection. (n=23) had a detectable, low level of virus. The mouse in the US 2014/0193460 A1 Jul. 10, 2014 56

FI-HSV2/Alhydrogel/s.c. group that died on day 21 had gD2t protein-immunized mice during the first week post detectable virus on day 2 (see e.g., FIG.7C). It is particularly challenge. When we combined the DNA prime with the FI notable that in the FI-HSV2/Alhydrogelfi.m. group, 5 of the 8 HSV2 or glo2t protein boost, we found that the combination mice had no detectable virus on day 2 postchallenge or post of the UL5, UL30, gD2t DNA prime and FI-HSV2 boost gave re-challenge, and 2 additional mice in this group had very low nearly complete protection against HSV-2 genital disease. level shedding postchallenge (10-10' PFU) and no detect 0666. The high level of protection observed in the group able virus post re-challenge (FIG. 7C, middle). No virus was that was primed with empty vector and then boosted with detectable in any of the FI-HSV2 groups on day 4 post re FI-HSV-2/MPL/Alum prompted us to determine whether challenge. Taken together, we found that immunization with modification of the composition of the inactivated virus vac FI-HSV2 that was formulated with MPL and any of a variety cine could improve its efficacy as a single vaccine. After of alum adjuvants elicits nearly complete protection against completion of the prime-boost studies, we learned that that HSV-2 disease. Moreover, these vaccines provide strong and Imject alum consists of both crystalline and amorphous alu durable protection against Vaginal HSV-2 shedding, with minum phases and is not a well-defined form of alum Suitable Some animals showing sterilizing immunity, following chal for clinical translation (Hem, S. L. et al., Vaccine, 25:4985 lenge or long-term re-challenge. 4986 (2007)). This raised the concern that an undefined aspect of the Imject alum contributed to the protection, and H. ANALYSIS thus it was essential that we test the more well-defined alum 0663. In this study, we tested the protective properties of adjuvants, Alhydrogel and Adju-Phos, that are used in clinical two vaccine components against HSV-2 genital infection vaccines. when each component was given either alone or as a prime 0667 Adju-Phos has been previously shown to be a suc boost vaccine. The prime component consisted of DNA cessful formulation for the co-delivery of DNA plasmid and encoding the immunogenic g)2t protein, which has been protein antigens in that immune responses were elicited to shown to provide protective effects in both the mouse and both vaccine components (Kwissa, M. et al., J. Mol. Med., guinea pig models (Bourne, N. et al., Vaccine, 14:1230-1234 81:502-510 (2003)). We found that when we combined plas (1996); Bourne, N. et al., J. Infect. Dis., 173:800-807 (1996): mid DNAs encoding UL5, UL30, and g|D2t with the FI-HSV2 Higgins, T. J. et al., J. Infect. Dis., 182:1311-1320 (2000); in Adju-Phos in a single injection or gave FI-HSV2 in Adju Strasser, J. E. et al., J. Infect. Dis., 182:1304-1310 (2000)). Phos alone, mice were significantly protected from HSV-2 We also tested DNA encoding the conserved essential pro shedding after a short-term challenge (3 weeks following the teins HSV-2 DNA polymerase (UL30) and helicase/primase last immunization). The addition of the DNAs did not further (UL5), as previous studies had shown that their counterparts enhance the protection relative to that elicited by FI-HSV2 encoded by MCMV were individually protective against a alone and Surprisingly may have decreased it. This effect was systemic challenge of MCMV in mice (Morello, C. S. et al., more apparent following a long-term challenge (9 weeks post J. Virol., 81:7766-7775 (2007)). The boost component con boost), regardless of whether the DNA was empty vector or tained either formalin-inactivated HSV-2 (FI-HSV-2) formu encoded viral antigen. Kwissa, et al. showed that although an lated with one of several alum compounds and MPL orgD2t aluminum phosphate adjuvant formulated with a plasmid protein plus MPL and alum. DNA that expresses the hepatitis B virus surface antigen 0664 Although DNA immunization with both the UL5 (HBSAg) can elicit both HBSAg-specific IFN-y secreting and and UL30 genes elicited a CD8+ T-cell response as measured cytotoxic CD8+ T cells, coadministration of this formulation by IFN-Y production, neither gene was protective against with either of 2 heterologous proteins abrogated the CD8+ T disease or viral shedding unless the DNA encoding g|D2t was cytolytic activity without affecting the levels of IFN-Y posi also included. Since we and others have shown that g|D2t tive cells (Kwissa, M. et al., J. Mol. Med., 81:502-510 DNA immunization generates a high level of neutralizing (2003)). While addition of pVAX DNA to the FI-HSV2/MPL/ antibody specific for go2 (Flo, J., Vaccine, 21:1239-1245 Adju-Phos vaccine resulted in decreased protective efficacy, (2003); Hoshino.Y. et al., J. Virol.., 79:410-418 (2005)), it may total IgG, IgG1, and IgG2a levels were not affected. This be that this antibody was more protective against the genital Suggests that a different immune correlate of protection elic challenge, at least locally, than CD8 T cells directed against ited by the FI-HSV2 may have been decreased by pVAX UL5 or UL30. However, relative to mice immunized with DNA addition and it is possible that coadministration of the gD2t DNA alone or g|D2t plus UL5, the group immunized HSV-2 antigen expressing plasmids (UL5, UL30, and g2t) with g|D2t DNA plus UL30 had the lowest peak disease (mean may have compensated partially for the decrease in protec of 1.2) and disease burden through day 12 and the greatest tion. In any event, the FI-HSV2MPL/Adju-Phosformulation reduction in viral shedding on days 1 and 2. The combination alone provided impressive levels of protection, with 4 out of of UL5 and UL30 DNAs did not appear to further augment the the 8 animals at day 2 and 6 out of the 8 animals at day 4 protection elicited by either plasmid alone or when given with shedding virus at or below the limit of detection. or without gl)2t DNA co-administration. 0668. Accordingly, we compared the efficacy of vaccines 0665. In the mice receiving prime-boost immunizations, containing FI-HSV2 formulated with MPL and Adju-Phos protection against HSV-2 vaginal disease was most closely that was given intramuscularly with FI-HSV-2 formulated related to the type of boost, with groups receiving the with MPL and Alhydrogel that was given either intramuscu FI-HSV-2/MPL/Alum boost showing a higher level of pro larly or subcutaneously. No deaths occurred in any of the tection than groups that received gl)2t protein in MPL/Alum, FI-HSV2 groups and genital disease was minimal following and both boosts providing more protection than immuniza challenge with HSV-2. In fact, the mild erythema and swell tion with HSV-2 DNA alone. Boosting with FI-HSV-2/MPL/ ing seen in a few mice may have been due to the inflammatory Alum also provided the greatest decreases of peak virus shed response, since half or more of the mice in each group had ding. The mean virus titer in the FI-HSV2 group was 3.5-4.2 reductions in virus shedding to levels at or below the detection Logs lower than mock group and 2.8-3.5 Logs lower than limit, Suggesting that these animals may have sterilizing US 2014/0193460 A1 Jul. 10, 2014 57 immunity. To determine if the protection was durable and 97% reduction in recurrent lesion days compared with the mice were resistant to a second challenge, they were re mock controls, had the highest reduction in days with recur challenged 17 weeks after the first challenge with the same rent disease, and contained the lowest mean HSV-2 DNA load 10xLDso dose of HSV-2 given for the first challenge. It was in the dorsal root ganglia. However, the FI-HSV2/MPL/Al particularly noteworthy that only 2 of the re-challenged mice hydrogel component alone (the pVAX empty vector DNA in the FI-HSV2 groups had a detectable, low level of virus. FI-HSV2 group), with the exception of one “nonresponder Overall, the best protection was seen in the FI-HSV2/Alhy animal, elicited complete protection against both acute and drogel/im. group, where 5 of the 8 mice had no detectable recurrent lesions and detectable HSV-2 DNA in the DRG. virus after the first challenge or after the re-challenge given Cumulative acute disease scores and numbers of recurrent over 4 months later, and 2 additional mice in this group had disease days were also comparable to the UL5, UL30, gD2t very low level shedding after the first challenge and no detect DNA-FI-HSV2 group. able virus after the re-challenge. These results are also in 0671 Disclosed herein is use of the mouse model to fur sharp contrast with our results in mice that were immunized ther characterize the immunity and protection afforded by the with glo2t DNA or protein in which the mice that were most DNA, FI-HSV2, and adjuvant. We also tested single formu protected from virus shedding after the first challenge subse lation vaccines in order to simplify and expedite administra quently showed the highest shedding levels after the re-chal tion and to increase protective responses. lenge. These exciting results force further consideration of an inactivated virus vaccine that is formulated with appropriate I. MATERIALS AND METHODS adjuvants. 0669 Recently, we reported the results of experiments in Vaccines which we evaluated a similar prime-boost strategy for its 0672 FI-HSV2 and a formalin inactivated mock prepara protective efficacy against i.vag. HSV-2 challenge in guinea tion (FI-Mock) were prepared from HSV-2 infected or unin pigs, a model in which both acute and recurrent disease could fected cells, respectively. Extracellular virus was purified and be studied (Morello, C. S. et al., J. Virol.., 85:3461-3472 inactivated as described above 19, and dextran sulfate wash (2011)). Guinea pigs were primed with 3 plasmid DNAs: the derived virus was purified as described above for ELISA gD2t DNA plus UL5 and UL30 or two additional conserved, antigen 19 and then formalin inactivated. Cell associated essential genes of HSV-2 (UL29 and UL52). Both of these virus was obtained from the clarified supernatant of HSV-2 groups were Subsequently boosted with formalin-inactivated infected Vero cells that were Sonicated in virus containing HSV-2 (FI-HSV2) in MPL/Alhydrogel. As a control for the extracellular media. The virus was subsequently pelleted and contribution of the FI-HSV2/MPL/alum alone to the protec inactivated as described above 19. No infectious virus was tion, a third group was primed with plasmid backbone DNA detected after inactivation of any preparation. The prepara (pVAX) and boosted with FI-HSV-2. For comparative pur tion used for each experiment is denoted in each figure leg poses, a fourth vaccine group received purified gL)2t protein end. All FI-HSV2 immunizations contained 107 PFU equiva plus MPL/Alhydrogel. The UL5, UL30, gD2t DNA-FI lents and 12.5 ug of MPL (Sigma L6895). Alum was HSV2 group showed a 97% reduction in recurrentlesion days purchased from Thermo Pierce (Imject alum) or Accurate compared with the mock controls. Relative to the other immu Chemical & Scientific (Adju-Phos and Alhydrogel), with Al nized groups, this group also had the highest reduction in days doses described in figure legends. Plasmids expressing with recurrent disease, and contained the lowest mean HSV-2 HSV-2 strain GUL5, UL30, andg|D2t (aa 1-327) and the g|D2t DNA load in the dorsal root ganglia. We noted, however, that protein subunit vaccine were constructed and prepared as when the FI-HSV2-MPL-Alhydrogel vaccine was given to described above 19. the pVAX-primed guinea pigs, with the exception of one “nonresponder animal in the group, there was complete pro Mice and Ethics Statement tection against both acute and recurrent lesion development. This group also had undetectable levels of HSV-2 DNA in the 0673 Female BALB/c mice were purchased from Charles DRG, lower cumulative acute disease scores, and lower num River Laboratories, housed in microisolator cages, and accli bers of recurrent disease days that were comparable to those mated for at least 2 weeks prior to use. Studies were carried for the UL5, UL30, gD2t DNA-FI-HSV2 group. Based upon out in strict accordance with the recommendations in the the success of the FI-HSV2/MPL/Alhydrogel in protecting Guide for the Care and Use of Laboratory Animals of the against virus shedding in mice after challenge and long-term National Institutes of Health. All use of vertebrate animals re-challenge and against acute and recurrent disease in the was approved by the Institutional Animal Care and Use Com guinea pig, it is possible that this vaccination strategy could mittee, University of California, San Diego. provide broad and durable immunity against HSV-2 that is at least as effective as that engendered by attenuated or replica Intravaginal HSV-2 Challenge Model tion-defective HSV-2 vaccines in animal models. The use of 0674) Mice were treated with medroxyprogesterone inactivated HSV-2 also eliminates the safety concerns of a acetate (Depo-Provera, Sigma M1629) 20 and i.vag. attenuated virus vaccine that has the potential for the estab swabbed with a DPBS-moistened polyester tipped swab (Mi lishment of latency or recombination with a wild-type virus. croPur 1001D, PurFybr Solon, Rhinelander, Wis.) immedi 0670. We recently evaluated a DNA prime-inactivated ately prior to instillation of 5x10 PFU of HSV-2 strain G (ca. virus boost strategy for its efficacy against intravaginal 10 LDso doses) by micropipette. The HSV-2 preparation used (i.Vag.) HSV-2 challenge in guinea pigs, a model for studying for challenge was described above 19. both acute and recurrent disease 19. Guinea pigs were 0675 Anogenital disease was scored as described in the primed with plasmids encoding g|D2t, UL5, and UL30, and FIG. 4A to FIG. 4C legend. Vaginal virus shedding of infec then boosted with formalin-inactivated HSV-2 (FI-HSV2) in tious virus was measured by plaque assay 19, and the limit MPL/Alhydrogel. After i.vag. challenge, this group showed a of sensitivity of each assay is denoted in each figure. US 2014/0193460 A1 Jul. 10, 2014

Antibody Quantification: HSV-2 Virion Specific IgG significant reductions in total disease burden (P<0.01 for the pVAX primed group and P-0.001 for the others) (FIG.5C) 0676 ELISAs were as described above 19 except that and total virus shedding (P<0.001 for pVAX, P<0.01 forgD2t alkaline phosphatase goat anti-mouse conjugates specific for DNA, and P<0.05 for UL5, UL30, and gL)2t DNAs) (FIG. IgG (Sigma), IgG1 or IgG2a (Southern Biotech) were used, 5D). The gl)2t DNA-g|D2t protein group also showed signifi and endpoint titers were Fit Spline interpolated (GraphPad cantly reduced disease burden and total virus shedding (P<0. Prism 5.0d). 001). Total shedding was lower in FI-HSV2 boosted mice compared to go2t protein, but the differences were not sta Neutralizing Antibody Assay tistically significant. 0677 Complement-dependent HSV-2 neutralizing anti bodies were measured by plaque reduction assay as described K. CO-IMMUNIZATION WITH TESTED previously 21. PLASMID DNA AND FI-HSV2 IN MPLFADJU-PHOSASA SINGLE Intracellular Cytokine Staining FORMULATION DID NOT ENHANCE FI-HSV2 0678 Eleven days following the second injection, mice MEDIATED PROTECTION were in vivo restimulated by HSV-2 injection in the footpad. 0683. We next tested whether the vaccine components Four days later, splenocytes were stimulated in vitro with 10 could be combined into a single formulation, as immune PFU per cell of HSV-2 (or an equivalent volume of a mock responses were shown to be elicited against both plasmid preparation) for 2 hours. Brefeldin A was added for an addi DNA and protein following codelivery in aluminum phos tional 8 hours. phate (Adju-Phos) 22. Mice were injected with FI-HSV2 0679. For staining, a viability dye (LIVE/DEAD fixable and MPL/Adju-Phos with or without the UL5, UL30, and violet; Molecular Probes, Invitrogen) and Fc block (CD16/ gD2t DNAs. Two days post short-term challenge, both vac 32: BD) was added for 30 minutes at 4°C. Surface markers cinated groups had significantly reduced titers compared to CD8-AX488 (Clone 53-6.7; BD) and CD4-Ax647 (clone the negative control group (P<0.01 and P-0.001, respec RM4-5; BD) were added for 30 minutes at 4°C. and then cells tively) (FIG. 6A, left). In the FI-HSV2 alone group, no virus permeabilized and fixed using the BDCytofix/Cytoperm kit. was detected in 4 of the animals on day 2 and in 6 animals on CD3-PE-Cy5 (Clone 145-2C11; BD) and IFN-y-PE (Clone day 4 (FIG. 6A, right). XMG 1.2: BD) were included in the intracellular stain for 45 0684. We next tested the durability of responses, and 2 minutes at 4°C. Data from 50,000-100,000 live CD3+ T cells days post long-term challenge, the FI-HSV2 alone group had were collected on a BD FACSCanto flow cytometer and ana a significant 4 Log reduction in titers relative to Mock (P<0. lyzed with BD FACSDiva software at the Research Flow 001), with rates of undetectable virus similar to those follow Cytometry Core Facility of the San Diego Center for AIDS ing short-term challenge (FIG. 6B). On day 4, all of the pVAX Research and the Veterans Medical Research Foundation and FI-HSV2 mice had detectable virus. VA San Diego Healthcare System, La Jolla, Calif. 0685 Levels of HSV-2 specific IgG prior to short-term challenge were similarly high in both vaccinated groups, and Statistical Analysis decreased by 1.5-2.5-fold in the long-term challenge animals 0680 Kruskal-Wallis analysis determined statistical sig (FIG. 6C left versus right). Surprisingly, neutralizing anti nificance for all data groups and Dunn's multiple comparison body titers prior to long-term challenge were only signifi tests (GraphPad Prism 5.0d) compared all pairs of vaccine cantly above Mock in animals that received antigenic DNA groups. Significance scores, *P-0.05; **P<0.01: *** (P<0.001 compared to pVAX FI-Mock) (FIG. 6C). P<0.001; and (ns), not significant. L. IMMUNIZATION WITH FI-HSV2 IN J. FI-HSV2 PROVIDES MORE CONSISTENT ALHYDROGEL OR ADJU-PHOSADJUVANTS IS PROTECTION AGAINST HSV-2 GENITAL HIGHLY PROTECTIVE AGAINST HSV-2 DISEASE AND SHEDDING THAN d2 REPLICATION FOLLOWING CHALLENGE AND SUBUNIT RECHALLENGE 0681 To examine the protective efficacy of the protein 0686. The best protection against virus shedding was based "boost' vaccine components, mice were immunized observed in FI-HSV2MPL/Adju-Phos immunized mice. We twice with FI-HSV2, FI-Mock, or gl)2t protein (each plus next compared two immunization routes, intramuscular MPL/Imject Alum), and i.vag. challenged. FI-HSV2 was (i.m.) and Subcutaneous (s.c.), in addition to the adjuvants completely protective against death (FIG. 4A), with both Adju-Phos and Alhydrogel (a well-defined aluminum anogenital disease (FIG. 4B) and vaginal virus shedding hydroxide compared to Imject), and assessed the Subsequent (FIG. 4C) significantly reduced below FI-Mock controls protection against a second challenge. (P<0.001). On day 2, vaginal virus titer reductions in the 0687. On day 2 postchallenge, virus shedding in all the FI-HSV2 mice were reduced 3.6 Logs compared with FI FI-HSV2 groups was significantly reduced by 3 Logs com Mock (P<0.001), although FI-HSV2 and g2t-mediated pro pared to mock controls (P<0.01 or P-0.05) (FIG. 7A, left). tection were variable (FIG. 4C). Half or more of the mice in each FI-HSV2 group had shed 0682 Next, we examined the protective efficacy of DNA ding levels at or below the detection limit. By day 4 postch priming (gD2t DNA alone or with UL5 and UL30 DNAs) allenge, no or very low level virus was detectable in any of the followed by the above boost types in MPL/Alum (see FIG.5A FI-HSV2 immunized mice (FIG. 7A, right). for timeline). Boosting with gl)2t or FI-HSV2 gave similar 0688 Mice immunized im. with FI-HSV2 in Alhydrogel protection against death (FIG. 5B), and compared with or Adju-Phos had similar high levels of virus-specific IgG that pVAX-FI-Mock, all 3 of the FI-HSV2 boosted groups had were statistically greater than Mock (P<0.001 and P-0.01, US 2014/0193460 A1 Jul. 10, 2014 59 respectively) (FIG. 11, left). FI-HSV2 in Alhydrogel i.m. response. Addition of MPL/Alhydrogel to the FI-HSV2 elicited significantly higher IgG compared to s.c. (P<0.05). groups gave significantly increased IgG1 responses com Although all groups were significantly protected against pared to their respective DPBS groups (i.m.: P-0.01 and s.c.: shedding, FI-HSV2giveni.m., but not s.c., elicited significant P<0.05), suggesting a Th2 bias (FIG.9A, right). neutralizing antibody titers (P<0.01) (FIG. 11, right). 0694 FIG.9B shows that the highest levels of neutralizing 0689 Nearly half of the animals receiving FI-HSV2 had antibody titers resulted from i.m. vaccination with FI-HSV2 no detectable shedding, so we next tested whether this high (P<0.001 compared to FI-Mock). Addition of MPL/Alhydro level protection would also be effective against re-challenge. gel i.m. gave a 2.4-fold increase (not significant) in neutral After long-term rechallenge, only 2 of 23 mice immunized ization titers compared to the DPBS i.m. group, and neither with FI-HSV2 had detectable vaginal virus (FIG. 7C y-axis) s.c. group was statistically different than FI-Mock. with levels that were low compared with naive controls (FIG. 0695 Finally, an ICS assay was performed to analyze 7B). Strikingly, 5 of 8 FI-HSV2/Alhydrogel/i.m. mice had no IFN-y+ T cell responses to the inactivated virus (FIG. 9C). detectable virus on day 2 postchallenge or post-rechallenge Low levels of HSV-2 specific CD4+ IFN-y+ T cells were (FIG.7C, middle). detected in the splenocytes of vaccinated and in vivo restimu lated mice (net mean range from 0.15 to 0.43%), compared to M. THE ADDITION OF LPS-DERIVED the control group (net mean=0.05%). However, CD8+ T cell ADJUVANTAALUMINUM-BASED MINERAL responses were undetectable above background. Stimulation SALT ADJUVANT (E.G. MPL/ALHYDROGEL) of splenocytes with a pool of overlapping peptides spanning TO THE FI-HSV2 VACCINE IS REQUIRED FOR gD2 did not result in any CD4+ or CD8+ T cell responses PROTECTION AGAINST HSV-2 REPLICATION, above background. DISEASE, AND LATENTVIRAL LOAD IN THE DRG N. ANALYSIS 0690 We next determined whether formulation of 0696. In this study, it was determined that the best protec FI-HSV2 in MPL/Alhydrogel was necessary for optimal pro tion against HSV-2 disease and shedding was obtained by i.m. tection and confirmed the above effect of immunization route injection of inactivated HSV2/LPS-derived adjuvant/alumi on protection. On day 2 postchallenge, mice immunized with num-based mineral salt adjuvant (e.g. FI-HSV2/MPL/Alhy FI-HSV2 in MPL/Alhydrogel had statistically significant ca. drogel). This vaccine prevented HSV-2 disease other than 4 Log reductions in mean virus titer relative to Mock (P<0. mild inflammation, reduced virus shedding by 3 to 4 Logs, 001), with 5 or 2 of 8 animals immunized i.m. or s.c. having protected the majority of animals from detectable vaginal no detectable virus, respectively (FIG. 8A, left). By contrast, virus even after two challenges, and provided complete pro shedding levels following immunization with FI-HSV2 in tection against HSV-2 DNA in DRG. The high level efficacy DPBS were not significantly different than Mock. In the i.m. in these experiments was dependent upon formulation with injection groups, MPL/Alhydrogel provided significantly MPL/Alhydrogel or MPL/Adju-Phos. The long-term chal lower virus levels than did DPBS (P<0.05). On day 4, the lenge experiment with FI-HSV2/MPL/Adju-Phos demon FI-HSV2 MPL/Alhydrogel groups had very similar mean strated that responses were durable, as similar high level titers and 6 of 8 animals each with no detectable virus (FIG. protection was observed at 3 and 9 weeks post-boost. 8A, right). (0697 Immunization with FI-HSV2/MPL/Alhydrogel by (0691 Mice immunizedi.m. with FI-HSV2MPL/Alhydro different routes affected the immune response, but not the gel were completely protected against any disease beyond resulting high level protection (FIGS. 7-9, 11). In 2 indepen very slight erythema (P<0.001) (FIG. 8B). While the FI dent experiments, i.m. immunization elicited significantly HSV2 MPL/Alhydrogel s.c. and DPBS i.m. groups were also higher levels of virus neutralizing antibodies compared to the significantly protected from disease relative to controls (P<0. s.c. and mock groups (FIGS. 11 and 9B). Clinical studies of 01), the DPBS s.c. group was not. Two mice died in each of vaccines for influenza 23), diphtheria and tetanus (DT) 24, the DPBS groups. and others 25, 26 have compared i.m. Versus s.c. adminis 0692 Four weeks postchallenge, DRG from the surviving tration. In general, similar levels of antibody titers were mice and 4 naive mice were harvested to quantify latent attained with either route, but overall fewer adverse events HSV-2 DNA levels by real-time qPCR (FIG. 8C). Interest (local and/or systemic) were observed following i.m. injec ingly, animals immunized i.m. with FI-HSV2 in MPL/Alhy tion. drogel had no detectable HSV-2 DNA. Only two mice from 0698 Similar to a recent study comparing HSV-2 subunit the FI-HSV2MPL/Alhydrogel s.c. group had detectable vaccines 27, we found that no single measure of immunity HSV-2 DNA, with the mouse with 14 genome copies having correlated with protection, including neutralizing antibody, the highest titers on day 2 and the mouse with 18 copies total IgG, IgG subtypes, or T cell responses. We found that having undetectable virus. FI-HSV2/MPL/Alhydrogel im. elicited levels of virus spe (0693 I.m. immunization with FI-HSV2MPL/Alhydrogel cific CD4+ IFN-y+ T cells similar to those observed in elicited the highest titers of virus specific total IgG (FIG.9A, BALB/c mice immunized with replication-defective HSV-2 left) and neutralizing antibodies (FIG. 9B), with IgG titers vaccines as measured with a similar assay 21, 28. In general, approximately 10-fold higher than in the FI-HSV2/DPBS FI-HSV2/MPL/Alhydrogel immunization consistently i.m. group (FIG.9A, left). FI-HSV2 DPBS given im, but not resulted in ca. 4 Log reductions in peak virus replication, s.c., elicited significant IgG levels relative to controls (P<0. reductions similar to the those in mice immunized with rep 05). Addition of MPL/Alhydrogel to the FI-HSV2 s.c. immu lication-defective virus vaccines 28, subunit vaccines with nization increased IgG levels by 7.6-fold and to a significant experimental adjuvants 29, and live attenuated vaccines 30, level over Mock (P<0.01). We tested whether the injection 31. Most notably, the majority of FI-HSV2/MPL/Alhydro route or addition of MPL/Alhydrogel biased the HSV-2 spe gel i.m. immunized mice had no detectable virus shedding, cific antibody response toward a Th1 (IgG2a) or Th2 (IgG1) Suggesting they may have had sterilizing immunity. US 2014/0193460 A1 Jul. 10, 2014 60

0699 Compared with a single glycoprotein subunit, a tion: two randomized controlled trials. Chiron HSV Vac potential advantage of a whole virus vaccine is that it delivers cine Study Group. J Amer Med Assoc 1999 Jul. 28; 282 abroad diversity of antibody targets in the context of the virus (4):331-40. particle. We considered the possibility that FI-HSV2 medi (0709) 9 Koelle DM, Corey L. Herpes simplex: insights ated protection was largely due to responses against the FI on pathogenesis and possible vaccines. Annu Rev Med HSV2 derived g|D2. However, the cell associated FI-HSV2 2008: 59:381-95. used in FIG.4A to FIG.4C contained 2.91g ofg)2 compared 0710 10 Zhu J, Koelle DM, Cao J. Vazquez J. Huang M to 5ug in the gL)2t protein group, indicating that g|D2 was not L., Hladik F. et al. Virus-specific CD8+ T cells accumulate the key to protection. Subsequent FI-HSV2 preparations near sensory nerve endings in genital skin during Subclini derived from dextran sulfate washes contained only 20 ng gD2 per dose and elicited the highest protection. Thus, the cal HSV-2 reactivation. J Exp Med 2007 Mar. 19; 204(3): optimal protection from FI-HSV2 was not likely due to the 595-603. nanogram quantities of g)2 protein but rather the total 0711 11 Koelle D M. Posavad C M, Barnum G R, response to the virion-associated antigens. Johnson ML, Frank J M. Corey L. Clearance of HSV-2 (0700 We have now shown that FI-HSV2/MPL/Alhydro from recurrent genital lesions correlates with infiltration of gel protects mice against virus shedding after challenge and HSV-specific cytotoxic T lymphocytes. J Clin Invest 1998 long-term rechallenge, and guinea pigs against acute and Apr. 1; 101(7): 1500-8. recurrent disease 19. The protection in animal models 0712 12 Belshe RB. Leone PA, Bernstein DI, Wald A, appears to be at least as high as that engendered by attenuated Levin MJ. Stapleton JT, et al. Efficacy results of a trial of or replication-defective HSV-2 vaccines, strategies that have a herpes simplex vaccine. N Engl J Med 2012 Jan. 5; the potential for the establishment of latency or recombina 3.66(1):34-43. tion with a wild-type virus. While vaccination with inacti 0713 13 Da Costa X, Kramer MF, Zhu J, Brockman M. vated virus eliminates many safety concerns, a major criti A. Knipe D M. Construction, phenotypic analysis, and cism of the first-generation inactivated HSV-2 vaccines has immunogenicity of a UL5/UL29 double deletion mutant of been the poor durability of elicited responses. However, we herpes simplex virus 2. J Virol 2000 September: 74(17): show that formulation with adjuvants such as MPL/Alhydro 7963-71. gel challenges this dogma. These exciting results demand 0714 14 Da Costa XJ, Jones CA, Knipe DM. Immu further consideration of inactivated HSV-2 vaccines formu nization against genital herpes with a vaccine virus that has lated with the modern, clinically approved adjuvants. defects in productive and latent infection. Proc Natl Acad Sci USA 1999 Jun. 8: 96(12):6994-8. O. REFERENCES 0715 15 Hoshino Y. Dalai SK, Wang K, Pesnicak L. Lau (0701 1 Xu F, Sternberg MR, Gottlieb SL, Berman SM, TY, Knipe DM, et al. Comparative efficacy and immuno Markowitz L. E. Forhan SE, et al. Seroprevalence of Her genicity of replication-defective, recombinant glycopro pes Simplex Virus Type 2 Among Persons Aged 14-49 tein, and DNA vaccines for herpes simplex virus 2 infec Years United States, 2005-2008. Atlanta, Ga.: Centers tions in mice and guinea pigs. JVirol 2005 January; 79(1): for Disease Control and Prevention: 2010 Apr. 23, 2010. 410-8. (0702 (2 Kimberlin D W. Management of HSV encepha 071.6 16 Hoshino Y. Pesnicak L. Dowdell KC. Burbelo litis in adults and neonates: diagnosis, prognosis and treat PD, Knipe DM, Straus SE, et al. Protection from herpes ment. Herpes 2007 June; 14(1): 11-6. simplex virus (HSV)-2 infection with replication-defective 0703 3 Dupuis S. Jouanguy E. Al-Hajjar S. Fieschi C. HSV-2 or glycoprotein D2 vaccines in HSV-1-seropositive Al-Mohsen I Z. Al-Jumaah S, et al. Impaired response to and HSV-1-seronegative guinea pigs. J Infect Dis 2009 interferon-alpha/beta and lethal viral disease in human Oct. 1; 200(7): 1088-95. STAT1 deficiency. Nat Genet. 2003 March; 33(3):388-91. 07.17 17 Hoshino Y. Pesnicak L. Dowdell KC, Lacayo J. (0704) 4 Freeman E. E. Weiss HA, Glynn J. R. Cross PL, Dudek T, Knipe DM, et al. Comparison of immunogenic Whitworth JA, Hayes R.J. Herpes simplex virus 2 infection ity and protective efficacy of genital herpes vaccine candi increases HIV acquisition in men and women: systematic dates herpes simplex virus 2 d15-29 and d15-29-41L in review and meta-analysis of longitudinal studies. AIDS mice and guinea pigs. Vaccine 2008 Jul. 29; 26(32):4034 2006 Jan. 2: 20(1):73-83. 40. 0705 5 Wald A. Link K. Risk of human immunodefi ciency virus infection in herpes simplex virus type 2-se 0718I 18. Roizman B, Knipe D M, Whitley R.J. Herpes ropositive persons: a meta-analysis. J Infect Dis 2002 Jan. Simplex Viruses. In: Knipe D M. Howley PM, editors. 1; 185(1):45-52. Fields Virology. 5th ed. Philadelphia: Lippincott Williams (0706 (6 Schiffer J.T. Wald A. Selke S. Corey L, Magaret & Wilkins, 2007: 2501-601. A. The kinetics of mucosal herpes simplex virus-2 infec 0719 19 Morello CS, Levinson M. S. Kraynyak KA, tion in humans: evidence for rapid viral-host interactions. J Spector DH. Immunization with Herpes Simplex Virus 2 Inf Dis 2011 Aug. 15; 204(4):554-61. (HSV-2) genes plus inactivated HSV-2 Is highly protective (0707 (7 Schacker T. Zeh J. Hu H L, Hill E. Corey L. against acute and recurrent HSV-2 disease. J Virol 2011 Frequency of symptomatic and asymptomatic herpes sim April; 85(7):3461-72. plex virus type 2 reactivations among human immunode 0720 20 Parr MB, Kepple L, McDermott MR, Drew M ficiency virus-infected men. J Infect Dis 1998 December; D, Bozzola J. J. Parr E L. A mouse model for studies of 178(6):1616-22. mucosal immunity to vaginal infection by herpes simplex 0708) 8 Corey L. Langenberg AG, Ashley R. Sekulovich virus type 2. Lab Invest 1994 March; 70(3):369-80. RE, Izu A E, Douglas J. M. Jr., et al. Recombinant glyco 0721 21 Dudek T. Mathews LC. Knipe DM. Disruption protein vaccine for the prevention of genital HSV-2 infec of the U(L)41 gene in the herpes simplex virus 2 d15-29 US 2014/0193460 A1 Jul. 10, 2014 61

mutant increases its immunogenicity and protective capac 0728 (28 Dudek T. E., Torres-Lopez E. Crumpacker C, ity in a murine model of genital herpes. Virology 2008 Mar. Knipe DM. Evidence for differences in immunologic and 1:372(1):165-75. pathogenesis properties of herpes simplex virus 2 strains 0722 22 Kwissa M, Lindblad E. B. Schirmbeck R, from the United States and South Africa. J Inf Dis 2011 Reimann J. Codelivery of a DNA vaccine and a protein May 15; 203(10): 1434-41. vaccine with aluminum phosphate stimulates a potent and 0729 (29 Awasthi S, Lubinski J M, Shaw CE, Barrett S multivalent immune response. J Mol Med 2003 August; M. Cai M. Wang F, et al. Immunization with a vaccine 81 (8):502-10. combining herpes simplex virus 2 (HSV-2)glycoprotein C 0723 23 Cook IF, Barr I, Hartel G, Pond D, Hampson A (gC) and g) subunits improves the protection of dorsal W. Reactogenicity and immunogenicity of an inactivated root ganglia in mice and reduces the frequency of recurrent influenza vaccine administered by intramuscular or Subcu vaginal shedding of HSV-2 DNA in guinea pigs compared taneous injection in elderly adults. Vaccine 2006 Mar. 20; to immunization with gld alone. J Virol 2011 October; 24(13):2395-402. 85(20): 10472-86. 0724 24 Mark A, Carlsson RM, Granstrom M. Subcu (0730 30 Awasthi S, Zumbrun E. E., Si H. Wang F, Shaw taneous versus intramuscular injection for booster DT C E. Cai M. et al. Live attenuated herpes simplex virus 2 vaccination of adolescents. Vaccine 1999 Apr. 9; 17(15 glycoprotein E deletion mutant as a vaccine candidate 16):2067-72. defective in neuronal spread. J Virol 2012 April; 86(8): 0725 25 Pittman P R. Aluminum-containing vaccine 4586-98. associated adverse events: role of route of administration and gender. Vaccine 2002 May 31; 20 Suppl3:S48-50. (0731 31 Halford W. P. Puschel R. Gershburg E, Wilber 0726 (26 Ruben FL. Froeschle J E. Meschievitz C, Chen A, Gershburg S, Rakowski B. A live-attenuated HSV-2 K. George J. Reeves-Hoche MK, et al. Choosing a route of ICP0 virus elicits 10 to 100 times greater protection against administration for quadrivalent meningococcal polysac genital herpes thanaglycoprotein D Subunit vaccine. PLoS charide vaccine: intramuscular versus Subcutaneous. Clin One 2011; 6(3):e 17748. Infect Dis 2001 January; 32(1): 170-2. 0732. It is understood that the examples and embodiments 0727 27 Khodai T, Chappell D, Christy C, Cockle P. described herein are for illustrative purposes only and that Eyles J. Hammond D. et al. Single and combination herpes various modifications or changes in light thereofwill be Sug simplex virus type 2 glycoprotein vaccines adjuvanted gested to persons skilled in the art and are to be included with CpG oligodeoxynucleotides or monophosphoryllipid within the spirit and purview of this application and scope of A exhibit differential immunity that is not correlated to the appended claims. All publications, patents, and patent protection in animal models. Clin Vaccine 1 mm 2011 applications cited herein are hereby incorporated by refer October; 18(10): 1702-9. ence in their entirety for all purposes.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 16

<21 Oc SEO ID NO 1 <211 LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <22 Os FEATURE; OTHER INFORMATION: Synthetic oligonucleotide primer.

<4 OOs SEQUENCE: 1

27

SEO ID NO 2 LENGTH: 33 TYPE: DNA ORGANISM: Artificial sequence FEATURE; OTHER INFORMATION: Synthetic oligonucleotide primer.

<4 OOs SEQUENCE: 2

ataga caatg accacatt.cg gatcgcgtag agc 33

SEO ID NO 3 LENGTH: 27 TYPE: DNA ORGANISM: Artificial sequence FEATURE; OTHER INFORMATION: Synthetic oligonucleotide primer. US 2014/0193460 A1 Jul. 10, 2014 62

- Continued <4 OOs, SEQUENCE: 3 gccaccatgt tttgttgcc.gc gggcggc 27

<210s, SEQ ID NO 4 &211s LENGTH: 36 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide primer.

<4 OOs, SEQUENCE: 4 tgctagagta t calaaggctic tatgcaac at t cacg 36

<210s, SEQ ID NO 5 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide primer.

<4 OOs, SEQUENCE: 5 gccaccatgg ggcgtttgac CtcC 24

<210s, SEQ ID NO 6 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide primer.

<4 OOs, SEQUENCE: 6 gacgt.cctgg atcgacgg 18

<210s, SEQ ID NO 7 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide primer.

<4 OO > SEQUENCE: 7 gccaccatgg ct accgacat tatatg 27

<210s, SEQ ID NO 8 &211s LENGTH: 33 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide primer.

<4 OOs, SEQUENCE: 8 aaat agggag ttgcagtaga agt atttgcc gtg 33

<210s, SEQ ID NO 9 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide primer.

<4 OOs, SEQUENCE: 9 gccagatata cqc catgaca ttgattattg 3 O US 2014/0193460 A1 Jul. 10, 2014 63

- Continued <210s, SEQ ID NO 10 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide primer.

<4 OOs, SEQUENCE: 10 caataatcaa tdt catggcg tatat ctdgc 3 O

<210s, SEQ ID NO 11 &211s LENGTH: 74 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide primer.

<4 OOs, SEQUENCE: 11 ggtaccgagc ticggat CCaC gtcgactaca aggatgacga tigacaagtga taatagt cca 6 O gtgtggtgga attic 74

<210s, SEQ ID NO 12 &211s LENGTH: 74 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide primer.

<4 OOs, SEQUENCE: 12 gaattic cacc acactggact attat cactt gtcatcgtca toctitgtagt cqacgtggat 6 O cc.gagctcgg tacc 74

<210s, SEQ ID NO 13 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic polypeptide.

<4 OOs, SEQUENCE: 13 His Gly Pro Ser Lieu. Tyr Arg Thr Phe 1. 5

<210s, SEQ ID NO 14 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide primer.

<4 OOs, SEQUENCE: 14 tgtggcaatg gcaaaaagc 19

<210s, SEQ ID NO 15 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide primer.

<4 OOs, SEQUENCE: 15 tgttaccatt tdtgatgttt togat 25 US 2014/0193460 A1 Jul. 10, 2014 64

- Continued <210s, SEQ ID NO 16 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide primer. 22 Os. FEATURE: <221 > NAMEAKEY: modified base <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: 5' c modified with cqtctatac oligonucleotide modified at 5' c with 6-FAM oligonucleotide and linked through Zen (TM) linker. 22 Os. FEATURE: <221 > NAMEAKEY: modified base <222s. LOCATION: (2O) . . (2O) <223> OTHER INFORMATION: 3" a modified with Iowa Black F Quencher

<4 OOs, SEQUENCE: 16 cc.gagtgtca t c ctaccgga

1. A Herpesvirus (HV) vaccine comprising inactivated HV. (iv) combining said inactivated HV particles with a a lipopolysaccharide (LPS)-derived adjuvant and an alumi lipopolysaccharide-derived adjuvant and an aluminum num-based mineral salt adjuvant. based mineral salt adjuvant thereby forming an HV vac 2. The vaccine of claim 1, cine. wherein said LPS-derived adjuvant is MPL: 35. The method of claim 34, wherein said HV is an HSV. said aluminum-based mineral salt adjuvant is aluminum 36.-80. (canceled) hydroxide adjuvant or aluminum phosphate adjuvant; 81. The method of claim 35, wherein said sulfated or sul and fonated polysaccharide is selected from dextran Sulfate, hep said vaccine does not comprise an isolated nucleic acid arin, and heparan sulfate. sequence formulation. 82.-100. (canceled) 3.-8. (canceled) 101. The method of claim 35, wherein said inactivating 9. The vaccine of claim 1, wherein said HV is a Herpes comprises contacting said HV with one or more agents Simplex Virus (HSV). selected from across-linking agent, oxidizing agent, reducing 10-17. (canceled) agent, heat, radiation, detergent, a pH changing agent, and a 18. The vaccine of claim 9, wherein said lipopolysaccha chemical agent selected from a furocoumarin, aziridine, eth ride-derived adjuvant is monophosphoryl lipid A (MPL) ylenimine, binary ethylenimine, and beta-propiolactone. 19.-20. (canceled) 102.-125. (canceled) 21. The vaccine of claim 9, wherein said lipopolysaccha 126. A method of treating or preventing HV infection in a ride-derived adjuvant is capable of binding the TLR4 protein. patient in need of Such treatment or prevention, said method 22. The vaccine of claim 9, wherein said lipopolysaccha comprising administering a therapeutically or prophylacti ride-derived adjuvant is a synthetic MPL analogue adjuvant. cally effective amount of the HV vaccine of claims 9. 23.-28. (canceled) 127-131. (canceled) 29. The vaccine of claim 9, wherein said inactivated HV 132. A method of treating or preventing a disease in a comprises a replication decreasing DNA mutation. patient in need of Such treatment or prevention, said method 30. The vaccine of claim 9, wherein said inactivated HV is comprising administering a therapeutically or prophylacti formed by chemical inactivation. cally effective amount of the HV vaccine of claim 9. 31. The vaccine of claim 9, wherein said inactivated HV is formed by contacting said HV with one or more agents 133. (canceled) selected from across-linking agent, oxidizing agent, reducing 134. The method claim 132, wherein said HV is an HSV. agent, heat, radiation, detergent, a pH changing agent, and a 135.-137. (canceled) chemical agent selected from a furocoumarin, aziridine, eth 138. The method of claim 134, wherein said disease is ylenimine, binary ethylenimine, and beta-propiolactone. selected from the group consisting of herpetic gingivostoma 32-33. (canceled) titis, herpes labialis, herpes genitalis, herpetic whitlow, her 34. A method of preparing a Herpesvirus (HV) vaccine, pes gladiatorum, herpesviral encephalitis, herpesviral men said method comprising: ingitis, herpes esophagitis, herpes keratitis, Bell's palsy, (i) contacting an HV-cell mixture with a sulfated or sul Mollaret's meningitis, herpes rugbeiorum, eczema herpeti fonated polysaccharide, wherein said HV-cell mixture cum, herpetic neuralgia, and post-herpetic neuralgia. comprises HV particles, cells and portions of cells; 139.-178. (canceled) (ii) separating said HV particles from said cells thereby 179. The method of claim 138, consisting of a prime-boost forming isolated HV particles: administration of said HSV vaccine. (iii) inactivating said isolated HV particles thereby forming 180. The method of claim 138, consisting of a prime-boost inactivated HV particles: boost administration of said HSV vaccine. US 2014/0193460 A1 Jul. 10, 2014 65

181. A kit comprising the HV vaccine of claim 9 and instructions for administering said HV Vaccine to a patient. 182. (canceled)