WO2025175124A1 - Recombinant herpes simplex virus glycoprotein b for treatment and prevention of herpes simplex virus infections - Google Patents

Abstract

Compositions comprising herpes simplex virus glycoprotein B (gB) lacking the C-terminal membrane proximal region and/or mRNA encoding same are provided herein. Methods of vaccinating, immunizing and/or treating a subject against a herpes simplex virus-2 (HSV-2) infection, a herpes simplex virus-1 (HSV-1) infection, or a HSV-1 and HSV-2 co-infection, the methods comprising administering to the subject an effective amount of pharmaceutical composition comprising the recombinant gB and/or the recombinant gB mRNA.

Description

RECOMBINANT HERPES SIMPLEX VIRUS GLYCOPROTEIN B FOR TREATMENT

AND PREVENTION OF HERPES SIMPLEX VIRUS INFECTIONS

SEQUENCE LISTING

The Instant Application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on February 14, 2025, is named “EIC0076PCT” and is 11,549 bytes in size. The Sequence Listing does not go beyond the disclosure in the application as filed.

FEDERAL RESEARCH STATEMENT

[0001] This invention was made with government support under grant number AI77673 awarded by the National Institutes of Health, NIAID. The government has certain rights in the invention.

BACKGROUND

[0002] Herpes simplex virus 1 (HSV-1) and Herpes simplex virus 2 (HSV-2) are prevalent human pathogens. HSV- 1 infects approximately 67% of the population by 49 years of age and is the primary cause of oral and ocular disease, a leading cause of infectious corneal blindness and fatal infectious encephalitis, and has emerged as the more common cause of genital disease in the developed world. HSV-2 is estimated to over 400 million people worldwide, is the primary cause of genital disease in the developing world and a major risk factor for HIV acquisition and transmission.

[0003] The enormous global health burden of these two related viruses has resulted in extensive vaccine development efforts, which have primarily focused on the generation of neutralizing antibodies (nAbs) targeting the viral envelope glycoproteins D (gD) as the correlate of immune protection. Unfortunately, these vaccines including a Chiron vaccine comprised of gD and gB combined with MF59, a GSK vaccine comprised of gD adjuvanted with AS04 and a replication defective virus deleted in two genes involved in viral replication, but expressing gD (dl529), met with little clinical success. Moreover, the animal models, which primarily relied on vaginal challenges of females with laboratory adapted viral strains, did not predict the clinical outcomes.

[0004] Additional strategies for preventing and treating infection and disease caused by HSV-1 and/or HSV-2 would be beneficial. BRIEF DESCRIPTION

[0005] To address these failures, the inventors modified the mouse models by challenging both male and female mice via multiple different routes with high doses of low passage clinical isolates. These modifications recapitulated clinical experiences and identified a central role for glycoprotein B (gB) and for polyfunctional antibodies that mediate antibodydependent cellular cytolysis (ADCC), antibody-dependent cell mediated phagocytosis (ADCP), complement dependent cytolysis (CDC) as well as cytolytic T cell responses in HSV prevention. Subsequent studies showed that the Chiron gD/gB vaccine and the GSK gD protein vaccines elicited exclusively neutralizing Ab responses primarily targeting only gD and failed to elicit ADCC responses in humans. The inventors hypothesized that gD might prevent ADCC responses as an immune evasion strategy and engineered a single-cycle virus deleted in HSV-2 gD (AgD-2). The AgD-2 vaccine strain is grown on gD-1 expressing complementing cells to yield phenotypically gD+ but genetically gD null virions. The virus is thus restricted to a single round of infection because gD is required for further spread. Preclinical studies demonstrated that AgD-2 was safe and provided significantly greater protection than prior failed vaccine candidates in our optimized mouse models. It completely protected mice challenged with 10- lOOx the lethal dose of clinical isolates of HSV-1 or HSV-2 and passive transfer studies determined that the protection was mediated by ADCC. Transfer of immune serum from AgD-2- vaccinated mice into naive wild-type (WT) completely protected against viral challenge but protection was lost when serum was transferred into FcyRI V ^/_ mice, FcyRIV is the mouse receptor mediating ADCC. Monoclonal antibodies (mAbs) isolated from AgD-2 vaccinated mice were isolated and characterized. The most potent ADCC-mediating mAb, BMPC-23, protected WT mice when configured as mouse IgG2c and mice expressing human FcyRIII when engineered as a human IgGl. Cryo-EM identified the target of BMPC-23 to be domain IV of glycoprotein B (gB).

[0006] Glycoprotein B (gB) is a membrane-bound glycoprotein and corresponds to gene U39 in the HSV genome. gB is structurally highly conserved between herpesviruses and appears to play a role in virus cell fusion and cellular spread of virus infection.

[0007] Unexpectedly and surprisingly, the inventors discovered that recombinant glycoprotein B (gB) when administered to a subject could elicit protective immune responses and is effective as a monovalent vaccine for the prevention of herpes simplex virus (HSV) infections. The use of gB as a monovalent vaccine has not been shown to provide protection against HSV infections before. [0008] Therefore, described herein is an isolated recombinant HSV glycoprotein B (gB) polypeptide for use in a pharmaceutical composition, a vaccine composition, or an immune composition, for treatment and/or prevention of HSV-1 and/or HSV-2 infection.

[0009] In one aspect, recombinant gB polypeptide is an HSV-1 gB (gB-1) or an HSV-2 gB (gB-2).

[0010] In another aspect, the recombinant gB polypeptide lacks only the C-terminal membrane proximal region of gB. In yet another aspect, the recombinant polypeptide is gB - 1 and comprises amino acid residues 31 to 730 identified in SEQ ID NO:1 and encoded by the nucleic acid sequence identified in SEQ ID NO:2. In another aspect, a variant of the gB-1 polypeptide that comprises at least one mutation (e.g., insertion, substitution or deletion) and may have at least 85% identity to SEQ ID NO:1.

[0011] In another aspect, the recombinant polypeptide is gB-2 and lacks only the C- terminal membrane proximal region of gB. The recombinant gB-2 comprises amino acid residues identified in SEQ ID NO:3 and encoded by the nucleic acid sequence identified in SEQ ID NO:4. In another aspect, a variant of gB-2 that comprises at least one mutation (e.g., insertion, substitution or deletion) and may have at least 85% identity to SEQ ID NO:3.

[0012] The vaccine composition can comprise both either recombinant gB-1 or recombinant gB2, or both recombinant gB-1 and recombinant gB2. The vaccine composition can further comprise an adjuvant or an adjuvant component. The vaccine composition or immunogenic composition may further include a pharmaceutically acceptable carrier and/or other components in a formulation suitable for application to a subject in need thereof. The vaccine or immunogenic composition may be formulated for delivery d by intramuscularly, intradermal delivery, subcutaneously, intravaginally, intraperitoneally, oral delivery, nasal delivery, buccal delivery, or rectal delivery.

[0013] The vaccine composition can further comprise at least one RNA vaccine comprising at least one RNA or mRNA which codes for the recombinant gB, the RNA or mRNA comprising at least one open reading frame coding for at least recombinant HSV-1 or HSV-2 glycoprotein B.

[0014] The invention also relates to method of vaccinating a subject against a herpes simplex virus-2 (HSV-2) infection, the method comprising administering to the subject an amount of recombinant gB effective to vaccinate the subject for an HSV-2 infection.

[0015] A method of vaccinating a subject against a herpes simplex virus-1 (HSV-1) infection, the method comprising administering to the subject an amount of a recombinant gB and/or an mRNA that codes for recombinant gB effective to vaccinate the subject for an HSV-1 infection. [0016] A method of vaccinating a subject against a HSV-1 and HSV-2 co-infection, the method comprising administering to the subject an amount of a recombinant gB and/or an mRNA that codes for recombinant gB effective to vaccinate the subject for an HSV- 1 and HSV- 2 co-infection.

[0017] A method of immunizing a subject against herpes simplex virus-1 (HSV-1) infection, herpes simplex virus-2 (HSV-2) infection, or an HSV-1 and HSV-2 co-infection, comprising administering to the subject recombinant gB and/or an mRNA that codes for recombinant gB in an amount effective to immunize the subject for the HSV-2 infection, the HSV-1 infection, or the HSV-1 and HSV-2 co-infection.

[0018] A method of treating or preventing an HSV-2 infection in a subject or treating a disease caused by an HSV-2 infection in a subject, comprising administering to the subject an amount of recombinant gB and/or an mRNA that codes for recombinant gB effective to treat the subject for the HSV-2 infection.

[0019] A method of treating or preventing an HSV-1 infection in a subject or treating a disease caused by an HSV-1 infection in a subject, comprising administering to the subject an amount of recombinant gB and/or an mRNA that codes for recombinant gB effective to treat the subject for the HSV-1 infection.

[0020] A method of treating or preventing an HSV-1 and HSV-2 coinfection in a subject or treating a disease caused by an HSV-1 and HSV-2 coinfection in a subject, comprising administering to the subject an amount of a recombinant HSV-2 glycoprotein B and/or an mRNA that codes for recombinant gB effective to treat the subject for the HSV-1 and HSV-2 coinfection.

[0021] In an aspect, the subject is human, an elderly subject or a pediatric subject. The subject may be a healthy individual, an individual at risk for exposure to HSV, a subject who has been treated for HSV, or a subject who is currently infected with HSV and whose condition may be improved by the immune response elicited by the herein described gB composition.

[0022] The above described and other features are exemplified by the following figures and detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The following figures are exemplary embodiments wherein the like elements are numbered alike.

[0024] FIG. 1 shows recombinant glycoprotein B provides significantly greater protection than gD in skin challenge model. Mice were prime-boost vaccinated with the indicated proteins (combined with Allum-MPL), or as control AgD-2 and then challenged on the skin with 10xLD90 of HSV-lB3xl. 1 (top panel), n=5 mice/group. HSV-2(4675) (bottom panel) (n=10/group for gB and ovalbumin and n-5 for gD or HSV-2(SD90) (middle panel) and monitored for 14 days. Survival curves are shown.

[0025] FIG. 2 shows recombinant glycoprotein B provides significant protection compared to gD against vaginal challenges. Prime-boost vaccinated mice (n=5 per group) were challenged intravaginally with 10xLD90 of HSV-lB³xl. l (top panel) or HSV-2(SD90) (bottom panel) and monitored for 14 days. Survival curves are shown.

[0026] FIG. 3 shows recombinant gB vaccination prevents establishment of latency following genital skin challenge of mice with HSV-2(4674) (n=5 mice/group).

[0027] FIG. 4 shows amino acid sequence alignment of the translated HSV-1 gB (gB-1) (SEQ ID NO:1) and HSV-2 gB (gB-2) (SEQ ID NO:3) expression constructs. A gap is indicated by a dash (-) to show where an amino acid is missing in one sequence as compared to the other in the alignment. An asterisk (*) indicates a position where all sequences have the same amino acid. A colon (:) represents a position where most sequences have similar amino acids with similar chemical properties. A period (.) indicates a conserved substitution.

DETAILED DESCRIPTION

[0028] It has been surprisingly discovered that HS V gB is a dominant target of protective ADCC responses, and administration of recombinant gB, or recombinant gB adjuvanted protein or mRNA could elicit protective immune responses against infection with HSV-1 and/or HSV-2.

[0029] As used herein, "therapeutically effective amount" or “effective amount” or “amount effective” refers to a quantity of a specific substance sufficient to achieve a desired effect in a subject.

[0030] “Treat” or “treating,” means to administer a vaccine of the disclosure or a product of the disclosure to a subject or patient having one or more disease symptoms, or being suspected of having a disease, for which the vaccine or product has therapeutic activity or prophylactic activity. The vaccine or product can be administered in an amount effective to alleviate one or more disease symptoms in the treated subject, whether by inducing the regression of or inhibiting the progression of such symptom(s) by any clinically measurable degree. The terms further include a postponement of development of the symptoms associated with a disorder and/or a reduction in the severity of the symptoms of such disorder. The terms further include ameliorating existing uncontrolled or unwanted symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. [0031] “Preventing” means administering an amount of a vaccine of the disclosure or a product of the disclosure sufficient to significantly reduce the likelihood of a disease from occurring in a subject who may he predisposed to the disease but who does not have it. In the context of viral infection “preventing” includes administering an amount of the vaccine or an immune product resulting from administration of the vaccine to a subject known to be at enhanced risk of viral infection.

[0032] Terms such as “including,” “containing,” and “having” should be interpreted openly herein, e.g., as meaning “including, but not limited to,” “including, without limitation,” or “comprising,” unless the description clearly states otherwise. Comprising means including any detectable amount of an element or including any detectable performance of a step.

[0033] Disclosed herein are methods of making recombinant gB polypeptide for use in the methods and composition of the present invention. The recombinant gB polypeptide lacks only the C-terminal membrane proximal region of gB. Recombinant gB-1 comprises amino acid residues 31 to 730 identified in SEQ ID NO:1, and is encoded by the nucleic acid sequence identified in SEQ ID NO:2. Recombinant gB-2 is set forth in SEQ ID NO:3 and encoded by the nucleic acid set forth in SEQ ID NO:4. A homolog or variant of gB-1 or gB-2 may differ from gB-1 or gB-2 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. The recombinant gB polypeptides described herein are effective for vaccinating, immunizing and/or treating a subject against a herpes simplex virus-2 (HSV-2) infection, a herpes simplex virus- 1 (HSV-1) infection, or a HSV-1 and HSV-2 co-infection. The methods of the invention comprise administering to the subject recombinant glycoprotein B (gB). In one aspect, recombinant gB polypeptide is an HSV- 1 gB. In another aspect, recombinant gB is an HSV-2 gB.

[0034] SEQ ID NO: 1 - HSV1 gB Amino Acid sequence: MGVHECPAWLWLLLSLLSLPLGLPVLGAPSSPGTPGVAAATQAANGGPATPAPPAPGA PPTGDPKPKKNKKPKPPKPPRPAGDNATVAAGHATLREHLRDIKAENTDANFYVCPPPT GATVVQFEQPRRCPTRPEGQNYTEGIAVVFKENIAPYKFKATMYYKDVTVSQVWFGHR YSQFMGIFEDRAPVPFEEVIDKINAKGVCRSTAKYVRNNLETTAFHRDDHETDMELKPA NAATRTSRGWHTTDLKYNPSRVEAFHRYGTTVNCIVEEVDARSVYPYDEFVLATGDFV YMSPFYGYREGSHTEHTSYAADRFKQVDGFYARDLTTKARATAPTTRNLLTTPKFTVA WDWVPKRPSVCTMTKWQEVDEMLRSEYGGSFRFSSDAISTTFTTNLTEYPLSRVDLGD CIGKDARDAMDRIFARRYNATHIKVGQPQYYLANGGFLIAYQPLLSNTLAELYVREHLR EQSRKPPNPTPPPPGASANASVERIKTTSSIEFARLQFTYNHIQRHVNDMLGRVAIAWCE LQNHELTLWNEARKLNPNAIASATVGRRVSARMLGDVMAVSTCVPVAADNVIVQNSM RISSRPGACYSRPLVSFRYEDQGPLVEGQLGENNELRLTRDAIEPCTVGHRRYFTFGGGY VYFEEYAYSHQLSRADITTVSTFIDLNITMLEDHEFVPLEVYTRHEIKDSGLLDYTEVQR

RNQLHDLRFADIDTVIHADANAAGGGSENLYFQSGGSHHHHHHHHHH*

[0035] HSV-1 gB nucleic acid sequence of expression construct:

ATGGGCGTGCACGAGTGCCCCGCCTGGCTGTGGCTGCTGCTGAGCCTGCTGAGTCTA

CCTCTCGGCCTGCCTGTGCTAGGCGCTCCGAGTTCCCCCGGCACGCCTGGGGTCGCG

GCCGCGACCCAGGCGGCGAACGGGGGCCCTGCCACTCCGGCGCCGCCCGCCCCTGG

CGCCCCCCCAACGGGGGACCCGAAACCGAAGAAGAACAAAAAACCGAAACCCCCA

AAGCCGCCGCGCCCCGCCGGCGACAACGCGACCGTCGCCGCGGGCCACGCCACCCT

GCGCGAGCACCTGCGGGACATCAAGGCGGAGAACACCGATGCAAACTTTTACGTGT

GCCCACCCCCCACGGGCGCCACGGTGGTGCAGTTCGAGCAGCCGCGCCGCTGCCCG

ACCCGGCCCGAGGGTCAGAACTACACGGAGGGCATCGCGGTGGTCTTCAAGGAGA

ACATCGCCCCGTACAAGTTCAAGGCCACCATGTACTACAAAGACGTCACCGTTTCG

CAGGTGTGGTTCGGCCACCGCTACTCCCAGTTTATGGGGATCTTTGAGGACCGCGCC

CCCGTCCCCTTCGAGGAGGTGATCGACAAGATCAACGCCAAGGGGGTCTGTCGGTC

CACGGCCAAGTACGTGCGCAACAACCTGGAGACCACCGCGTTTCACCGGGACGACC

ACGAGACCGACATGGAGCTGAAACCGGCCAACGCCGCGACCCGCACGAGCCGGGG

CTGGCACACCACCGACCTCAAGTACAACCCCTCGCGGGTGGAGGCGTTCCACCGGT

ACGGGACGACGGTAAACTGCATCGTCGAGGAGGTGGACGCGCGCTCGGTGTACCCG

TACGACGAGTTTGTGCTGGCGACTGGCGACTTTGTGTACATGTCCCCGTTTTACGGC

TACCGGGAGGGGTCGCACACCGAACACACCAGCTACGCCGCCGACCGCTTCAAGCA

GGTCGACGGCTTCTACGCGCGCGACCTCACCACCAAGGCCCGGGCCACGGCGCCGA

CCACCCGGAACCTGCTCACGACCCCCAAGTTCACCGTGGCCTGGGACTGGGTGCCA

AAGCGCCCGTCGGTCTGCACCATGACCAAGTGGCAGGAGGTGGACGAGATGCTGCG

CTCCGAGTACGGCGGCTCCTTCCGATTCTCCTCCGACGCCATATCCACCACCTTCAC

CACCAACCTGACCGAGTACCCGCTCTCGCGCGTGGACCTGGGGGACTGCATCGGCA

AGGACGCCCGCGACGCCATGGACCGCATCTTCGCCCGCAGGTACAACGCGACGCAC

ATCAAGGTGGGCCAGCCGCAGTACTACCTGGCCAATGGGGGCTTTCTGATCGCGTA

CCAGCCCCTTCTCAGCAACACGCTCGCGGAGCTGTACGTGCGGGAACACCTCCGAG

AGCAGAGCCGCAAGCCCCCAAACCCCACGCCCCCGCCGCCCGGGGCCAGCGCCAAC

GCGTCCGTGGAGCGCATCAAGACCACCTCCTCCATCGAGTTCGCCCGGCTGCAGTTT

ACGTACAACCACATACAGCGCCATGTCAACGATATGTTGGGCCGCGTTGCCATCGC

GTGGTGCGAGCTGCAGAATCACGAGCTGACCCTGTGGAACGAGGCCCGCAAGCTGA

ACCCCAACGCCATCGCCTCGGCCACCGTGGGCCGGCGGGTGAGCGCGCGGATGCTC

GGCGACGTGATGGCCGTCTCCACGTGCGTGCCGGTCGCCGCGGACAACGTGATCGT

CCAAAACTCGATGCGCATCAGCTCGCGGCCCGGGGCCTGCTACAGCCGCCCCCTGG TCAGCTTTCGGTACGAAGACCAGGGCCCGTTGGTCGAGGGGCAGCTGGGGGAGAAC AACGAGCTGCGGCTGACGCGCGATGCGATCGAGCCGTGCACCGTGGGACACCGGCG CTACTTCACCTTCGGCGGGGGCTACGTGTACTTCGAGGAGTACGCGTACTCCCACCA GCTGAGCCGCGCCGACATCACCACCGTCAGCACCTTCATCGACCTCAACATCACCAT GCTGGAGGATCACGAGTTTGTCCCCCTGGAGGTGTACACCCGCCACGAGATCAAGG ACAGCGGCCTGCTGGACTACACGGAGGTCCAGCGCCGCAACCAGCTGCACGACCTG CGCTTCGCCGACATCGACACGGTCATCCACGCCGACGCCAACGCCGCCGGTGGCGG AAGCGAGAACCTGTACTTCCAGTCAGGAGGCAGTCATCACCATCACCATCACCATC ACCATCACTGA

[0036] SEQ ID NO: 3 - HSV-2 gB Amino Acid sequence:

MGVHECPAWLWLLLSLLSLPLGLPVLGSGGVAATVAANGGPASRPPPVPSPATTKARK RKTKKPPKRPEATPPPDANATVAAGHATLRAHLREIKVENADAQFYVCPPPTGATVVQ

FEQPRRCPTRPEGQNYTEGIAVVFKENIAPYKFKATMYYKDVTVSQVWFGHRYSQFMG IFEDRAPVPFEEVIDKINTKGVCRSTAKYVRNNMETTAFHRDDHETDMELKPAKVATRT SRGWHTTDLKYNPSRVEAFHRYGTTVNCIVEEVDARSVYPYDEFVLATGDFVYMSPFY GYREGSHTEHTSYAADRFKQVDGFYARDLTTKARATSPTTRNLLTTPKFTVAWDWVP

KRPAVCTMTKWQEVDEMLRAEYGGSFRFSSDAISTTFTTNLTEYSLSRVDLGDCIGRDA REAIDRMFARKYNATHIKVGQPQYYLATGGFLIAYQPLLSNTLAELYVREYMREQDRK PRNATPAPLREAPSANASVERIKTTSSIEFARLQFTYNHIQRHVNDMLGRIAVAWCELQN HELTLWNEARKLNPNAIASATVGRRVSARMLGDVMAVSTCVPVAPDNVIVQNSMRVS SRPGTCYSRPLVSFRYEDQGPLIEGQLGENNELRLTRDALEPCTVGHRRYFIFGGGYVYF EEYAYSHQLSRADVTTVSTFIDLNITMLEDHEFVPLEVYTRHEIKDSGLLDYTEVQRRN QLHDLRFADIDTVIRADANAAGGGSENLYFQSGGSHHHHHHHHHH*

[0037] SEQ ID NO:4, HSV-2 gB nucleic acid sequence of expression construct:

ATGGGCGTGCACGAGTGCCCCGCCTGGCTGTGGCTGCTGCTGAGCCTGCTGAGTCTA CCTCTCGGCCTGCCTGTGCTAGGCtcgggcggcgtggccgcgaccgtcgcggcgaacgggggtcccgcctcccg gccgccccccgtcccgagccccgcgaccaccaaggcccggaagcggaaaaccaaaaagccgcccaagcggcccgaggcgacccc gccccccgacgccaacgcgaccgtcgccgccggccacgccacgctgcgcgcgcacctgcgggaaatcaaggtcgagaacgccgatg cccagttttacgtgtgcccgcccccgacgggcgccacggtggtgcagtttgagcagccgcgccgctgcccgacgcgcccggaggggc agaactacacggagggcatcgcggtggtcttcaaggagaacatcgccccgtacaaattcaaggccaccatgtactacaaagacgtgacc gtgtcgcaggtgtggttcggccaccgctactcccagtttatggggatattcgaggaccgcgcccccgttcccttcgaggaggtgatcgaca agattaacaccaagggggtctgccgctccacggccaagtacgtgcggaacaacatggagaccaccgcgtttcaccgggacgaccacga gaccgacatggagctcaagccggcgaaggtcgccacgcgcacgagccgggggtggcacaccaccgacctcaagtacaacccctcgc gggtggaggcgttccatcggtacggcacgacggtcaactgcatcgtcgaggaggtggacgcgcggtcggtgtacccgtacgatgagttt gtgctggcgacgggcgactttgtgtacatgtccccgttttacggctaccgggaggggtcgcacaccgagcacaccagctacgccgccga ccgcttcaagcaggtcgacggcttctacgcgcgcgacctcaccacgaaggcccgggccacgtcgccgacgacccgcaacttgctgacg acccccaagtttaccgtggcctgggactgggtgccgaagcgaccggcggtctgcaccatgaccaagtggcaggaggtggacgagatgc tccgcgccgagtacggcggctccttccgcttctcctccgacgccatctcgaccaccttcaccaccaacctgaccgagtactcgctctcgcg cgtcgacctgggcgactgcatcggccgggatgcccgcgaggccatcgaccgcatgtttgcgcgcaagtacaacgccacgcacatcaag gtgggccagccgcagtactacctggccacggggggcttcctcatcgcgtaccagcccctcctcagcaacacgctcgccgagctgtacgt gcgggagtacatgcgggagcaggaccgcaagccccggaatgccacgcccgcgccactgcgggaggcgcccagcgccaacgcgtcc gtggagcgcatcaagaccacctcctcgatcgagttcgcccggctgcagtttacgtataaccacatacagcgccacgtgaatgacatgctgg ggcgcatcgccgtcgcgtggtgcgagctgcagaaccacgagctgactctctggaacgaggcccgcaagctcaaccccaacgccatcgc ctccgccaccgtcggccggcgggtgagcgcgcgcatgctcggagacgtcatggccgtctccacgtgcgtgcccgtcgccccggacaac gtgatcgtgcagaactcgatgcgcgtcagctcgcggccggggacgtgctacagccgccccctggtcagctttcggtacgaagaccaggg cccgctgatcgaggggcagctgggcgagaacaacgagctgcgcctcacccgcgacgcgctcgagccgtgcaccgtgggccaccggc gctacttcatcttcggcgggggctacgtgtacttcgaggagtacgcgtactctcaccagctgagtcgcgccgacgtcaccaccgtcagcac cttcatcgacctgaacatcaccatgctggaggaccacgagtttgtgcccctggaggtctacacgcgccacgagatcaaggacagcggcct gctggactacacggaggtccagcgccgcaaccagctgcacgacctgcgctttgccgacatcgacacggtcatccgcgccgacgccaac gccgccGGTGGCGGAAGCGAGAACCTGTACTTCCAGTCAGGAGGCAGTCATCACCATC ACCATCACCATCACCATCACTGA

[0038] The recombinant gB polypeptides of the present invention may have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or sequence similarity with SEQ ID NO: 1 or SEQ ID NO: 3. As known in the art “similarity” between two polypeptides or polynucleotides is determined by comparing the amino acid or nucleotide sequence and its conserved nucleotide or amino acid substitutes of one polynucleotide or polypeptide to the sequence of a second polynucleotide or polypeptide. Also known in the art is “identity” which means the degree of sequence relatedness between two polypeptides or two polynucleotide sequences as determined by the identity of the match between two strings of such sequences. Both identity and similarity can be readily calculated (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exist a number of methods to measure identity and similarity between two polynucleotide or polypeptide sequences, the terms “identity” and “similarity” are well known to skilled artisans (Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods commonly employed to determine identity or similarity between two sequences include but are not limited to those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipman, D., SIAM J. Applied Math. 48:1073 (1988).

[0039] Variants of gB are included in the present invention. Variants may have amino acid insertions, substitutions and/or deletions that have minimal to no effect on the activity, function or shape of the isolated polypeptide. Examples of such substitutions include the substitution of one non-polar residue for another, the substitution of one polar residue for another, the substitution of one basic residue for another, or the substitution of one acidic residue for another. Those skilled in the art will recognize non-natural amino acids may also be used. Non-natural amino acids include, for example, beta-alanine (beta-Ala), or other omega-amino acids, such as 3-amino propionic, 2,3-diamino propionic (2,3-diaP), 4-amino butyric and so forth, alpha-aminisobutyric acid (Aib), sarcosine (Sat), ornithine (Om), citrulline (Cit), t- butylalanine (t-BuA), t-butylglycine (t-BuG), N-methylisoleucine (N-Melle), phenylglycine (Phg), and cyclohexylalanine (Cha), norleucine (Nle), cysteic acid (Cya) 2-naphthylalanine (2- Nal); 1 ,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); beta-2-thienylalanine (Thi); and methionine sulfoxide (MSO).

[0040] The nucleotide sequences encoding recombinant gB polypeptides of the present invention may be codon optimized to enhance expression in varying host cells. Codon optimization refers to modifying the nucleotide sequence in order to enhance protein expression in a host cell of interest by replacing one or more codons of the native sequence with codons that are more frequently used in the genes of that host cell or in the genes of the host the cell was derived from. Various species exhibit particular bias for certain codons of a particular amino acid.

[0041] The recombinant gB isolated polypeptides of the present invention may be prepared by any known techniques. For example, the isolated polypeptides may be expressed through genetic engineering. By way of example, the translation of recombinant DNA. The gB isolated polypeptides may also be prepared synthetically. By way of example, the gB isolated polypeptides may be synthesized using the solid-phase synthetic technique initially described by Merrifield (J. Am Chem. Soc. 85:2149-2154), which is incorporated herein by reference. Other polypeptide synthesis techniques may be found, for example, Kent el al. (1985) in Synthetic Peptides in Biology and Medicine, eds. Alitalo et al. , Elsevier Science Publishers, 295-358.

[0042] The gB isolated polypeptides of the present invention may be isolated or obtained in substantially pure form. Substantially pure means that the proteins and/or polypeptides and/or peptides are essentially free of other substances with which they may be found in nature or in vivo systems to an extent practical and appropriate for their intended use. In particular, gB polypeptides are sufficiently pure and are sufficiently free from other biological constituents of their host cells so as to be useful in, for example, generating antibodies, sequencing, or producing pharmaceutical preparations. By techniques well known in the art, substantially pure polypeptides may be produced in light of the nucleic acid and amino acid sequences disclosed herein. Because a substantially purified isolated polypeptide of the invention may be admixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the isolated polypeptide may comprise only a certain percentage by weight of the preparation. The isolated polypeptide is nonetheless substantially pure in that it has been substantially separated from the substances with which it may be associated in living systems.

[0043] The present invention further provides isolated gB polypeptides comprising additional polypeptides. The additional polypeptides may be fragments of a larger polypeptide. In one embodiment, there are one, two, three, four, or more additional polypeptides fused to the gB isolated polypeptides. In some embodiments, the additional polypeptides are fused toward the amino terminus of the gB isolated polypeptides. In other embodiments, the additional polypeptides are fused toward the carboxyl terminus of the gB isolated polypeptides. In further embodiments, the additional polypeptides flank the gB isolated polypeptides.

[0044] In some embodiments, the additional polypeptides aid in directing the secretion or subcellular localization of the gB isolated polypeptides. Such polypeptides are referred to as a “signal sequence.” A secretory signal is described, for example U.S. Pat. Nos. 6,291,212 and 5,547,871, both of which are herein incorporated by reference in their entirety. Secretory signal sequence encodes secretory peptides. A secretory peptide is an amino acid sequence that acts to direct the secretion of gB from a cell. Secretory peptides are generally characterized by a core of hydrophobic amino acids and are typically (but not exclusively) found at the amino termini of newly synthesized proteins. The secretory peptide may be cleaved gB isolated polypeptide during secretion. Secretory peptides may contain processing sites that allow cleavage of the signal peptide from the mature protein as it passes through the secretory pathway. Processing sites may be encoded within the signal peptide or may be added to the signal peptide by, for example, in vitro mutagenesis. Secretory signal sequences may be required for a complex series of post- translational processing steps to allow for secretion of gB. The signal sequence may immediately follow the initiation codon and encodes a signal peptide at the amino-terminal end of gB. The signal sequence may precede the stop codon and encodes a signal peptide at the carboxy-terminal end of gB. In most cases, the signal sequence is cleaved off by a specific protease, called a signal peptidase. Examples of a secretory signal sequences include, but are not limited to ompA, pelB, and ST pre-pro. [0045] In some embodiments, the additional polypeptides aid the stabilization, structure and/or the purification of the gB isolated polypeptides. In some embodiments the additional polypeptides may comprise an epitope. In other embodiments, the additional polypeptides may comprise an affinity tag. By way of example, fusion of a polypeptide comprising an epitope and/or an affinity tag to the gB isolated polypeptide may aid purification and/or identification of the polypeptide. By way of example, the polypeptide segment may be a His-tag, a myc-tag, an S-peptide tag, a MBP tag (maltose binding protein), a GST tag (glutathione S -transferase), a FLAG tag, a thioredoxin tag, a GFP tag (green fluorescent protein), a BCCP (biotin carboxyl carrier protein), a calmodulin tag, a Strep tag, an HSV-epitope tag, a V5-epitope tag, and a CBP tag. The use of such epitopes and affinity tags is known to those skilled in the art.

[0046] In further embodiments, the additional polypeptides may provide a gB isolated polypeptide comprising sites for cleavage of the polypeptide. As an example, a polypeptide may be cleaved by hydrolysis of the peptide bond. In some embodiments, the cleavage is performed by an enzyme. In some embodiments, cleavage occurs in the cell. In other embodiments, cleavage occurs through artificial manipulation and/or artificial introduction of a cleaving enzyme. By way of example, cleavage enzymes may include pepsin, trypsin, chymotrypsin, thrombin, and/or Factor Xa. Cleavage allows ease of isolating the gB isolated polypeptides from the polypeptides. Cleavage may further allow for the separation of the toxin A portion from the toxin B portion. Cleavage may also allow isolation of the gB isolated polypeptide fused to polypeptides from other polypeptides, such as through cleavage of an epitope utilized to purify the expressed protein.

[0047] The gB isolated polypeptides may further possess additional structural modifications not shared with the same organically synthesized peptide, such as adenylation, carboxylation, glycosylation, hydroxylation, methylation, phosphorylation or myristylation. These added structural modifications may be further be selected or preferred by the appropriate choice of recombinant expression system. On the other hand, fusion polypeptides may have its sequence extended by the principles and practice of organic synthesis.

[0048] The present invention also provides nucleic acids encoding the gB isolated polypeptides, gB-1 polypeptide encoded by the nucleic acid sequence set forth in SEQ ID NO:2, and gB-2 polypeptide encoded by nucleic acid sequence set forth in SEQ ID NO:4. Nucleic acids may include single or double stranded forms of deoxyribonucleotides or ribonucleotides or polymers thereof. The present invention provides ribonucleic acids encoding the gB isolated polypeptides. The present invention also provides for nucleic acids that hybridize under stringent conditions to a nucleic acid encoding the gB-1 and gB-2 isolated polypeptide and the complement thereof. Stringent conditions refer to the degree of homology between a probe and a filter-bound nucleic acid; the higher the stringency, the higher percent homology between the probe and filter bound nucleic acid. The temperature for a stringent wash may be determined based on the Tm of the nucleic acid (based on G/C content). Stringent conditions may further be affected by the concentration of salt in a buffer, such as standard sodium citrate (SSC). The present invention provides for nucleic acids having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence similarity or sequence identity with SEQ ID NO: 2 or SEQ ID NO:4.

[0049] Preferred methods to determine identity are designed to give the largest match between the two sequences tested. Methods to determine identity and similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, et al. , Nucl. Acid Res. 12(1):387 (1984)), BLASTP, BLASTN, FASTA (Altschul, et al., J. Mol. Biol. 215:403 (1990)). The degree of similarity or identity referred to above is determined as the degree of identity between the two sequences, often indicating a derivation of the first sequence from the second. The degree of identity between two nucleic acids may be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Needleman and Wunsch J. Mol. Biol. 48:443-453 (1970)). For purposes of determining the degree of identity between two nucleic acids for the present invention, GAP is used with the following settings: GAP creation penalty of 5.0 and GAP extension penalty of 0.3.

[0050] The present invention also provides a vector comprising a nucleic acid encoding for the gB- 1 or gB-2 isolated polypeptide. A vector may be any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA, although RNA vectors are also available. Vectors include, but are not limited to, plasmids and phagemids. A cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase.

[0051] Vectors may further contain a promoter sequence. A promoter may include an untranslated nucleic acid usually located upstream of the coding region that contains the site for initiating transcription of the nucleic acid. The promoter region may also include other elements that act as regulators of gene expression. In further embodiments of the invention, the expression vector contains an additional region to aid in selection of cells that have the expression vector incorporated. The promoter sequence is often bounded (inclusively) at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes.

[0052] Vectors may further contain one or more marker sequences suitable for use in the identification and selection of cells which have been transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., O-galactosidase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques. Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.

[0053] An expression vector is one into which a desired nucleic acid may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Expression refers to the transcription and/or translation of an endogenous gene, transgene or coding region in a cell.

[0054] A coding sequence and regulatory sequences are operably joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.

[0055] The gB isolated polypeptides of the present invention may be produced by expressing the encoding nucleic acid in host cells. The nucleic acid may be transformed or transfected into host cells. Accordingly, some aspects of the present invention include the transformation and/or transfection of nucleic acid encoding the gB isolated polypeptides. Transformation is the introduction of exogenous or heterologous nucleic acid to the interior of a prokaryotic cell. Transfection is the introduction of exogenous or heterologous nucleic acid to the interior of a eukaryotic cell. The transforming or transfecting nucleic acid may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In prokaryotes, for example, the transforming nucleic acid may be maintained on an episomal element such as a plasmid or viral vector. With respect to eukaryotic cells, a stably transfected cell is one in which the transfecting nucleic acid has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transfected nucleic acid.

[0056] Higher eukaryotic cell cultures may be used to express the proteins of the present invention, whether from vertebrate or invertebrate cells, including insects, and the procedures of propagation thereof are known (see, for example, Kruse et al. (1973) Tissue Culture, Academic Press).

[0057] Host cells and vectors for replicating the nucleic acids and for expressing the encoded gB isolated polypeptides are also provided. Any vectors or host cells may be used, whether prokaryotic or eukaryotic. Many vectors and host cells are known in the art for such purposes. It is well within the skill of the art to select an appropriate set for the desired application. Suitable host cells for expressing the polypeptides of the present invention in higher eukaryotes include: yeasts such as Saccharomyces (e.g., S. cerevisiae); 293 (human embryonic kidney) (ATCC CRL-1573); 293F (Invitrogen, Carlsbad Calif.); 293T and variant 293T/17 (293tsA1609neo and variant ATCC CRL- 11268) (human embryonic kidney transformed by SV40 T antigen); COS-7 (monkey kidney CVI line transformed by SV40)(ATCC CRL1651); BHK (baby hamster kidney cells) (ATCC CRL 10); CHO (Chinese hamster ovary cells); mouse Sertoli cells; CVI (monkey kidney cells) (ATCC CCL70); VERO76 (African green monkey kidney cells) (ATCC CRL1587); HeLa (human cervical carcinoma cells) (ATCC CCL2); MDCK (canine kidney cells) (ATCC CCL34); BRL3A (buffalo rat liver cells) (ATCC CRL1442); W138 (human lung cells) (ATCC CCL75); HepG2 (human liver cells) (HB8065); and MMT 060652 (mouse mammary tumor) (ATCC CCL51).

[0058] Another aspect of the invention is directed to the generation of antibodies. Examples of antibodies encompassed by the present invention, include, but are not limited to, antibodies produced by immunizing a subject with the gB-1 and/or gB-2 isolated polypeptide. Antibodies generated by immunizing with gB-1 and/or gB-2 isolated polypeptide may be characterized using methods well known in the art. The antibodies produced by using the isolated gB polypeptide(s) of the present invention can encompass monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heavy chain only antibodies, heteroconjugate antibodies, single chain (ScFv), single domain antibodies, variants thereof, isolated polypeptides comprising an antibody portion, humanized antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Preferred antibodies are derived from murine, rat, human, rabbit, canine, porcine, dromedary, camel, llama, feline, primate, or any other origin (including chimeric, fragment and/or humanized antibodies).

[0059] In other embodiments, the antibodies produced by immunizing with the gB polypeptide(s) are then humanized by methods known in the art. A humanized antibody is an immunoglobulin molecule that contains minimal sequence derived from non-human immunoglobulin. In yet other embodiments, fully human antibodies are obtained by using commercially available mice that have been engineered to express specific human immunoglobulin proteins. In other embodiments, the antibodies are chimeric. A chimeric antibody is an antibody that combines characteristics from two different antibodies. Methods of preparing chimeric antibodies are known in the art.

[0060] In other embodiments, the nucleotide sequence that encodes the antibodies is obtained and then cloned into a vector for expression or propagation. In another embodiment, antibodies are made recombinantly and expressed using methods known in the art. By way of example, the gB isolated polypeptide may be used as an antigen for the purposes of isolating recombinant antibodies by these techniques. Antibodies can be made recombinantly by using the gene sequence to express the antibody recombinantly in host cells. Methods for making variants of antibodies and recombinant antibodies are known in the art.

[0061] In the context of the present invention recombinant gB is an antigen. The term ‘antigen” refers typically to a substance which may be recognized by the immune system, preferably by the adaptive immune system, and is capable of triggering an antigen-specific immune response, e.g. by formation of antibodies and/or antigen-specific T cells as part of an adaptive immune response. Typically, an antigen may be or may comprise a peptide or protein which comprises at least one epitope and which may be presented by the MHC to T cells. In the sense of the present invention an antigen may be the product of translation of a provided RNA, preferably an mRNA as defined herein. In this context, also fragments, variants and derivatives of recombinant gB peptides and proteins comprising at least one epitope are understood as antigens.

[0062] Epitopes (also called ‘antigen determinant’) can be distinguished in T cell epitopes and B cell epitopes. T cell epitopes or parts of the proteins in the context of the present invention may comprise fragments preferably having a length of about 6 to about 20 or even more amino acids, e.g. fragments as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 11, or 12 amino acids), or fragments as processed and presented by MHC class II molecules, preferably having a length of about 13 or more amino acids, e.g. 13, 14, 15, 16, 17, 18, 19, 20 or even more amino acids, wherein these fragments may be selected from any part of the amino acid sequence. These fragments are typically recognized by T cells in the form of a complex consisting of the peptide fragment and an MHC molecule, i.e. the fragments are typically not recognized in their native form. B cell epitopes are typically fragments located on the outer surface of (native) protein or peptide antigens as defined herein, preferably having 5 to 15 amino acids, more preferably having 5 to 12 amino acids, even more preferably having 6 to 9 amino acids, which may he recognized by antibodies, i.e. in their native form.

[0063] Such epitopes of proteins or peptides may furthermore be selected from any of the herein mentioned variants of such proteins or peptides. In this context antigenic determinants can be conformational or discontinuous epitopes which are composed of segments of the proteins or peptides as defined herein that are discontinuous in the amino acid sequence of the proteins or peptides as defined herein but are brought together in the three-dimensional structure or continuous or linear epitopes which are composed of a single polypeptide chain.

[0064] A vaccine is typically understood to be a prophylactic or therapeutic material providing at least one antigen, preferably an immunogen. “Providing at least one antigen” means, for example, that the vaccine comprises the antigen, e.g. recombinant gB, or that the vaccine comprises a molecule that codes for the antigen or a molecule comprising the antigen. For example, the vaccine may comprise a nucleic acid, such as an RNA or mRNA (e.g. RNA vaccine), which codes for a peptide or protein that comprises the recombinant gB antigen.

[0065] An RNA vaccine is defined herein as a vaccine comprising at least one RNA molecule comprising at least one open reading frame (ORF) coding for at least one antigen, e.g. recombinant gB. In the context of the present invention, the at least one RNA molecule comprised by the vaccine is preferably an isolated RNA molecule encoding recombinant gB or a fragment thereof. This at least one RNA is preferably viral RNA, self-replicating RNA (replicon) or most preferably mRNA. Also included herein are RNA/DNA hybrids which means that the at least one RNA molecule of the RNA vaccine consists partially of ribonucleotides and partially of deoxyribonucleotides. In this context, the at least one RNA of the RNA vaccine consists of at least 50% of ribonucleotides, more preferably to at least 60%, 70%, 80%, 90% and most preferably to 100%. In this context, the at least one RNA of the RNA vaccine can also be provided as complexed RNA or mRNA, as virus particle and as replicon particle.

[0066] A stabilized nucleic acid, typically, may be essentially resistant to in vivo degradation (e.g. degradation by an exo- or endo-nuclease) and/or ex vivo degradation (e.g. by the manufacturing process prior to vaccine administration, e.g. in the course of the preparation of the RNA vaccine solution to be administered). Stabilization of RNA, particularly mRNA can, e.g., be achieved by providing a 5'-Cap structure, a Poly(A) tail, a poly (C) tail, and/or any other UTR modification. It can also be achieved by backbone modification, sugar modification, base modification, and/or modification of the G/C-content of the nucleic acid. Various other methods are known in the art and conceivable in the context of the invention.

[0067] Nucleic acid molecules used according to the invention as defined herein may be prepared using any method known in the art, including synthetic methods such as e.g. solid phase synthesis, in vivo propagation (e.g. in vivo propagation of viruses), as well as in vitro methods, such as in vitro transcription reactions.

[0068] For preparation of a nucleic acid molecule, especially if the nucleic acid is in the form of an RNA or mRNA, a corresponding DNA molecule may e.g. be transcribed in vitro. This DNA template preferably comprises a suitable promoter, e.g. a T7 or SP6 promoter, for in vitro transcription, which is followed by the desired nucleotide sequence coding for the nucleic acid molecule, e.g. mRNA, to be prepared and a termination signal for in vitro transcription. The DNA molecule, for example identified in SEQ ID NO:2 or SEQ ID NON, which forms the template of the at least one RNA of interest, may be prepared by fermentative proliferation and subsequent isolation as part of a plasmid which can be replicated in bacteria. Plasmids which may be mentioned as suitable for the present invention are e.g. the plasmids pT7 Ts (GenBank accession number U26404; Lai et al., Development 1995, 121: 2349 to 2360), pGEM® series, e.g. pGEM®-l (GenBank accession number X65300; from Promega) and pSP64 (GenBank accession number X65327); cf. also Mezei and Storts, Purification of PCR Products, in: Griffin and Griffin (ed.), PCR Technology: Current Innovation, CRC Press, Boca Raton, Fla., 2001.

[0069] In aspects, the gB vaccine comprising a recombinant gB polypeptide composition and/or mRNA encoding the recombinant gB polypeptide (recombinant gB mRNA) is combined with an adjuvant, and the adjuvanted composition is administered to the subject.

[0070] The type of adjuvant is not limited, and can be any adjuvant capable of augmenting, enhancing and/or boosting the immune response of the subject to the recombinant HSV gB antigen relative to administration of non-adjuvanted recombinant HSV gB. Non- limiting examples of adjuvants include alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide, squalene, Quil A, MPL, Freund's adjuvant, oil in water emulsions (such as squalene or peanut oil), CpG. Adjuvants can be used with or without other specific immunostimulating agents such as MPL or 3-DMP, QS21, polymeric or monomeric amino acids such as polyglutamic acid or polylysine, or other immunopotentiating agents. Adjuvants can be used with or without other specific immunostimulating agents such as MPL or 3-DMP, QS21, polymeric or monomeric amino acids such as polyglutamic acid or polylysine, or other immunopotentiating agents.

[0071] Formulations will contain an effective amount of the active ingredients (e.g., drug, antigen and adjuvant) together with carrier or suitable amounts of vehicle in order to provide pharmaceutically-acceptable compositions suitable for administration to a human or animal.

[0072] The relative amounts of active ingredients, such as amounts of the recombinant gB polypeptide, within a dose and the dosing schedule may be adjusted appropriately for efficacious administration to a subject (e.g., animal or human). This adjustment may also depend on the subject's particular disease or condition, and whether treatment or prophylaxis is intended. To simplify administration of the formulation to the subject, each unit dose contains the active ingredients in predetermined amounts for a single round of immunization.

[0073] In another embodiment, the vaccine or the therapeutic composition formulation comprising the gB antigen and/or adjuvant may be applied separately but along with other therapeutic agents, such e.g., anesthetics, analgesics, anti-inflammatories, steroids, antibiotics, antiarthritics, anorectics, antihistamines, and antineoplastics.

[0074] Dosage schedule of administration and efficacy of the vaccine can be determined by methods known in the art. The amount of the vaccine and the immunization regimen may depend on the particular antigen and the adjuvant employed, the mode and frequency of administration, and the desired effect (e.g., protection and/or treatment). In general, the vaccine of the invention may be administered in amounts ranging between 1 pg and 100 mg, such as e.g. between 60 pg and 600 pg. A single dose of the vaccine comprising the gB-1 and/or gB-2 polypeptide may be in a range from about 1 pg to about 1 mg, preferably from about 5 pg to about 500 pg, more preferably from about 20 pg to about 200 pg.

[0075] The immunization regimen can be determined by methods known in the art. Administration of the vaccine can be repeated as is determined to be necessary by one skilled in the art. For example, a priming dose may be followed by 1, 2, 3 or more booster doses at weekly, bi-weekly or monthly intervals. In an embodiment of the present invention, the priming dose is followed by one or two booster administration in intervals from about 7 to about 14 days such as e.g. after 7 days and 21 days after first prime. In a preferred embodiment, the therapeutically effective amount of the vaccine is administered two or three times in intervals of 14 days+/-l , 2 or 3 days (bi-weekly) to a subject. Tn an embodiment of the present invention, the therapeutically effective amount of the vaccine is administered once.

[0076] In various aspects, a method of vaccinating a subject against herpes simplex virus-2 (HSV-2) infection comprises administering to the subject an amount of the recombinant gB-1 and/or gB-2 and/or recombinant gB-1 and/or gB-2 mRNA effective to vaccinate the subject for an HSV-2 infection.

[0077] In various aspects, a method of vaccinating a subject against herpes simplex virus-1 (HSV-1) infection comprises administering to the subject of the recombinant HSV gB-1 and/or gB-2 and/or recombinant gB-1 and/or gB-2 mRNA effective to vaccinate the subject for an HSV-1 infection.

[0078] In various aspects, a method of vaccinating a subject against an HSV-1 and HSV- 2 co-infection comprises administering to the subject an amount of the recombinant HSV gB-1 and/or gB-2 and/or recombinant gB-1 and/or gB-2 mRNA effective to vaccinate the subject for an HSV-2 infection.

[0079] Also disclosed herein are methods of immunizing a subject against herpes simplex virus-1 (HSV-1) infection, herpes simplex virus-2 (HSV-2) infection, or an HSV-1 and HSV-2 co-infection. A method of immunizing a subject against a herpes simplex virus-1 (HSV- 1) infection, a herpes simplex virus-2 (HSV-2) infection, or an HSV-1 and HSV-2 co-infection, comprises administering to the subject an amount of the recombinant gB-1 and/or gB-2 and/or recombinant gB-1 and/or gB-2 mRNA effective to immunize the subject for the HSV-2 infection, the HSV-1 infection, or the HSV-1 and HSV-2 co-infection.

[0080] Disclosed herein also are methods of preventing or treating an HSV-2 infection, an HSV-1 infection, or an HSV-2 and HSV-1 co-infection in a subject. Also disclosed are methods of preventing or treating a disease caused by an HSV-2 infection, an HSV-1 infection, or an HSV-2 and HSV-1 co-infection in a subject.

[0081] A method of preventing or treating an HSV-2 infection, an HSV-1 infection, or an HSV-2 and HSV-1 co-infection in a subject comprises administering to the subject an amount of the recombinant HSV gB-1 and/or gB-2 and/or recombinant gB-1 and/or gB-2 mRNA effective to treat the subject for the HSV-2 infection.

[0082] A method of preventing or treating a disease cause by an HSV-2 infection, an HSV-1 infection, or an HSV-2 and HSV-1 co-infection in a subject comprises administering to the subject an amount of the recombinant HSV gB-1 and/or gB-2 and/or recombinant gB-1 and/or gB-2 mRNA effective to treat the subject for the HSV-2 disease caused by the HSV-2 infection, the HSV-1 infection, or the HSV-2 and HSV-1 co-infection.

[0083] In aspects, the disclosed methods of vaccinating, immunizing and/or treating a subject comprise administering an amount of a pharmaceutical formulation comprising the recombinant HSV gB-1 and/or gB-2 and/or recombinant gB-1 and/or gB-2 mRNA.

[0084] A composition or pharmaceutical composition or vaccine comprising the recombinant HSV gB-1 and/or gB-2 and/or recombinant gB-1 and/or gB-2 mRNA, or a combination thereof can be administered to the subject. The composition or pharmaceutical composition or vaccine comprising the recombinant HSV gB-1 and/or gB-2 and/or recombinant gB-1 and/or gB-2 mRNA is formulated for administration to a subject.

[0085] In aspects, a composition or pharmaceutical composition or vaccine comprising the recombinant HSV gB and/or recombinant gB mRNA is formulated so that it is suitable for administration to a human subject. In particular, the composition or pharmaceutical formulation or vaccine is formulated to be suitable for the intended route of administration to a subject. In an embodiment, the composition or pharmaceutical composition or vaccine is formulated so that it is suitable for subcutaneous, intramuscular, intradermal, or intravaginal administration to a subject. In embodiments of the methods herein, and embodiments of the composition or pharmaceutical composition or vaccine formulations herein, administration can be auricular, buccal, conjunctival, cutaneous, subcutaneous, endocervical, endosinusial, endotracheal, enteral, epidural, via hemodialysis, interstitial, intrabdominal, intraamnio tic, intra-arterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intracistemal, intracorneal, intracoronary, intradermal, intradiscal, intraductal, intraepidermal, intraesophagus, intragastric, intravaginal, intragingival, intraileal, intraluminal, intralesional, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intraepicardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intravenous, intraventricular, intravesical, intravitreal, laryngeal, nasal, nasogastric, ophthalmic, oral, oropharyngeal, parenteral, percutaneous, periarticular, peridural, rectal, inhalationally, retrobulbar, subarachnoid, subconjuctival, sublingual, submucosal, topically, transdermal, transmucosal, transplacental, transtracheal, ureteral, urethral, or vaginal. A combination comprising at least one of the foregoing routes of administration can also be used.

[0086] In the present disclosure, the subject is a subject in need of treatment or prevention of infection with HSV-2, HSV-1, or HSV-2 and HSV-1 co-infection. The subject can also be a subject in need of treatment or prevention of a disease caused by infection with HSV-2, HSV-1, or HSV-2 and HSV-1 co-infection. The subject is a mammalian subject. For example, the subject is a human subject. The recombinant HSV gB-1 and/or gB-2 and/or recombinant gB- 1 and/or gB-2 mRNA can be formulated for administration to a human subject.

[0087] All references, including publications, patent applications, and patents, cited herein, are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. The disclosure of such documents relating to compositions and methods can be combined with the teachings provided herein to provide additional useful compositions and applications. However, the citation and incorporation of patent documents herein is limited to the technical disclosure of such patent documents and does not reflect any view of the validity, patentability, and/or enforceability of any claims thereof. Moreover, in the event of any conflict between this disclosure and the teachings of such documents, the content of this disclosure will control with respect to properly understanding the various aspects of the invention. Numerous references have been included in this disclosure to incorporate information available from other sources that illustrate the scope of the invention or aid in putting aspects of it into practice. While efforts have been made to include the most relevant references for such purposes, readers will understand that not every aspect of every cited reference will be applicable to the practice of the invention.

[0088] Numerous examples of aspects are provided in this disclosure to illuminate such parts of the invention. The breadth and scope of the invention should not be limited by any of the exemplary embodiments. No language in the specification should be construed as indicating any element is essential to the practice of the invention unless such a requirement is explicitly stated.

[0089] This disclosure is further illustrated by the following examples, which are nonlimiting.

EXAMPLE

MATERIALS AND METHODS

[0090] The sequence encoding glycoprotein B (gB) from HSV 1 (accession AFH41177, residues 31 to 730, lacking the signal sequence and the C-terminal membrane proximal region, as described in Maurer, Ulrike E, Tzviya Zeev-Ben-Mordehai, Arun Prasad Pandurangan, Tina M Cairns, Brian P Hannah, J Charles Whitbeck, Roselyn J Eisenberg, and others. "The Structure of Herpesvirus Fusion Glycoprotein B-bilayer Complex Reveals the Protein-membrane and Lateral Protein-protein Interaction." Structure 21, no. 8 (2013) 1396-1405: doi:10. 1016/j .str.2013.05.018.) was synthesized by Gen9Bio and cloned into a modified version of pIRES-acGFP with the human erythropoietin signal peptide, a C-terminal TEV-cleavable deca-His tag and a neo resistance marker. Suspension HEK293 freestyle cells (IL, Life Technologies™) were transfected with the expression plasmid using linear PEI. Valproic acid (VP A) was added to 3mM 24 hours post-transfection, and culture supernatant was harvested by centrifugation (10 minutes, 1000g) 6 days post-transfection. Alternatively, a 30ml culture of HEK293F cells was transfected with the expression plasmid using PEI as above, and a stable pool of transfectants selected by addition of G418 to 800 g/ml. Stable transfectants were expanded to a volume of up to 3L, VPA added to a concentration of 3mM, and culture supernatant harvested by centrifugation 6 days post VPA addition, or when cell viability was reduced to 70%. Protein was affinity purified with nickel resin (HIS60, Clontech) and gel filtration chromatography (Superdex™ S200 26/60, GE) in PBS, or in PBS with the addition of 0.1M arginine and 2 - 10% glycerol. Endotoxin level was determined as <lEU/mg by LAL assay (QCL™, Lonza).

[0091] Mice immunized with HSV-1 glycoprotein B. The protein is comprised of residues 31 to 730, lacking only the C-terminal membrane proximal region and was produced in HEK293 cells as described above, and affinity purified with nickel resin. Mice were vaccinated intramuscularly (2 doses administered 3 weeks apart) with gB or the same concentration of gD (also produced in HEK293 cells). Both proteins were administered in combination with Alum and MPL. Controls included mice vaccinated with ovalbumin (negative control) or mice vaccinated with AgD-2 (positive control). AgD-2 is an engineered single-cycle virus deleted in HSV-2 gD. The AgD-2 vaccine strain is grown on gD-1 expressing complementing cells to yield phenotypically gD+ but genetically gD null virions. The virus is thus restricted to a single round of infection because gD is required for further spread.

[0092] Mice were then challenged either on the skin with lethal doses of clinical isolates of HSV-1 (1.1) or HSV-2 (4674 or SD90) (Fig. 1) or intravaginally (Fig. 2). Note that SD90 is a low passage isolate from Africa previously shown to be resistant to protection by gD protein vaccines and dl529. Results indicate that recombinant gB provides significantly greater protection than gD in a skin challenge model and provides significant protection compared to gD against vaginal challenges.

[0093] Neuronal tissue was harvested at the time of demise (or Day 14 in surviving mice) and analyzed by quantitative PCR for presence of viral DNA as a biomarker of latency. [0094] Results show that recombinant gB vaccination prevents establishment of latency following genital skin challenge of mice with HSV-2(4674).

[0095] Additionally, the vaccine elicits high titer anti-gB Abs, but the Abs have significantly less neutralizing activity compared to the gD vaccine, suggesting that protection is mediated either by non-neutralizing (ADCC, ADCP or complement activating antibody responses) and/or T cell responses.

[0096] In conclusion, recombinant gB used as a subunit vaccine is highly effective for preventing or controlling HSV infections, including the development of viral latency. A vaccine comprising recombinant gB either as a recombinant protein or as an mRNA encoding the recombinant gB in combination with an adjuvant can be a widely used vaccine.

[0097] The compositions, methods, and articles can alternatively comprise, consist of, or consist essentially of, any appropriate materials, steps, or components herein disclosed. The compositions, methods, and articles can additionally, or alternatively, be formulated so as to be devoid, or substantially free, of any materials (or species), steps, or components, that are otherwise not necessary to the achievement of the function or objectives of the compositions, methods, and articles.

[0098] The terms “first,” “second,” and the like, do not denote any order, quantity, or importance, but rather are used to distinguish one element from another. The terms “a” and “an” and “the” do not denote a limitation of quantity and are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. “Or” means “and/or” unless clearly stated otherwise. Reference throughout the specification to “some embodiments”, “an embodiment”, and so forth, means that a particular element described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments. A “combination thereof’ is open and includes any combination comprising at least one of the listed components or properties optionally together with a like or equivalent component or property not listed.

[0099] Unless specified to the contrary herein, all test standards are the most recent standard in effect as of the filing date of this application, or, if priority is claimed, the filing date of the earliest priority application in which the test standard appears.

[0100] Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this application belongs. All cited patents, patent applications, and other references are incorporated herein by reference in their entirety. However, if a term in the present application contradicts or conflicts with a term in the incorporated reference, the term from the present application takes precedence over the conflicting term from the incorporated reference.

[0101 ] While particular embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents that are or may be presently unforeseen may arise to applicants or others skilled in the art. Accordingly, the appended claims as filed and as they may be amended are intended to embrace all such alternatives, modifications variations, improvements, and substantial equivalents.

REFERENCES

1. Corey L, Langenberg AG, Ashley R, et al. Recombinant glycoprotein vaccine for the prevention of genital HSV-2 infection: two randomized controlled trials. Chiron HSV Vaccine Study Group. JAMA. Jul 28 1999;282(4):331-40. doi:10.1001/jama.282.4.331

2. Belshe RB, Leone PA, Bernstein DI, et al. Efficacy results of a trial of a herpes simplex vaccine. N Engl J Med. Jan 5 2012;366(l):34-43. doi:10.1056/NEJMoal l03151

3. Dropulic LK, Oestreich MC, Pietz HL, et al. A Randomized, Double-Blinded, Placebo- Controlled, Phase 1 Study of a Replication-Defective Herpes Simplex Virus (HSV) Type 2 Vaccine, HSV529, in Adults With or Without HSV Infection. J Infect Dis. Aug 9 2019;220(6):990-1000. doi: 10.1093/infdis/jiz225

4. Burn Aschner C, Knipe DM, Herold BC. Model of vaccine efficacy against HSV-2 superinfection of HSV-1 seropositive mice demonstrates protection by antibodies mediating cellular cytotoxicity. NPJ Vaccines. May 7 2020;5(l):35. doi: 10.1038/s41541-020-0184-7

5. Burn Aschner C, Pierce C, Knipe DM, Herold BC. Vaccination Route as a Determinant of Protective Antibody Responses against Herpes Simplex Virus. Vaccines (Basel). Jun 5 2020;8(2)doi:10.3390/vaccines8020277

6. Burn C, Ramsey N, Garforth SJ, Almo S, Jacobs WR, Jr., Herold BC. A Herpes Simplex Virus (HSV)-2 Single-Cycle Candidate Vaccine Deleted in Glycoprotein D Protects Male Mice From Lethal Skin Challenge With Clinical Isolates of HSV-1 and HSV-2. J Infect Dis. Feb 14 2018;217(5):754-758. doi: 10.1093/infdis/jix628

7. Kao CM, Goymer J, Loh LN, Mahant A, Bum Aschner C, Herold BC. Murine Model of Maternal Immunization Demonstrates Protective Role for Antibodies That Mediate Antibody- Dependent Cellular Cytotoxicity in Protecting Neonates From Herpes Simplex Virus Type 1 and Type 2. J Infect Dis. Feb 18 2020;221(5):729-738. doi: 10.1093/infdis/jiz521 8. Petro C, Gonzalez PA, Cheshenko N, et al. Herpes simplex type 2 virus deleted in glycoprotein D protects against vaginal, skin and neural disease. eLife.

2015;4doi: 10.7554/eLife.06054

9. Petro CD, Weinrick B, Khajoueinejad N, et al. HSV-2 DeltagD elicits FcgammaR- effector antibodies that protect against clinical isolates. JCI Insight. Aug 04 2016;l(12)doi:10.1172/jci.insight.88529

10. Kohl S, Charlebois ED, Sigouroudinia M, et al. Limited antibody-dependent cellular cytotoxicity antibody response induced by a herpes simplex virus type 2 subunit vaccine. J Infect Dis. Ian 2000;181(l):335-9. doi: 10.1086/315208

11. Mahant A, Guerguis S, Blevins TP, et al. Herpes Simplex Virus Glycoprotein D Antibodies Fail to Elicit Antibody-Dependent Cell-Mediated Cytotoxicity: Implications for Future Vaccines. I Infect Dis. lul 14 2022;doi:10.1093/infdis/jiac284

12. Burn Aschner C, Loh LN, Galen B, et al. HVEM signaling promotes protective antibody -dependent cellular cytotoxicity (ADCC) vaccine responses to herpes simplex viruses. Sci Immunol. Aug 14 2020;5(50)doi:10.1126/sciimmunol.aax2454

13. Kuraoka M, Aschner CB, Windsor IW, et al. A non-neutralizing glycoprotein B monoclonal antibody protects against herpes simplex virus disease in mice. J Clin Invest. Feb 1 2023; 133(3)doi: 10.1172/JCI161968

Claims

CLAIMS What is claimed is:

1. A recombinant HSV glycoprotein B (gB) polypeptide wherein the HSV polypeptide is an HSV-1 gB (gB-1) or an HSV-2 gB (gB-2), or a combination thereof.

2. The gB-1 polypeptide of Claim 1, wherein gB-1 comprises amino acid residues 31 to 730 of herpes simplex virus (HSV) type 1 glycoprotein B, the polypeptide identified in SEQ ID NO:1, or a sequence with at least 85% identity to SEQ ID NO: 1.

3. The gB-1 polypeptide of Claim 1, wherein gB-1 is encoded by the nucleic acid sequence identified in SEQ ID NO:2, or a sequence with 85% identity to SEQ ID NO:2.

4. The gB-2 polypeptide of Claim 1, wherein gB-2 has the sequence set forth in SEQ ID NO:3, or a sequence with at least 85% identity to SEQ ID NO:3.

5. The gB-2 polypeptide of Claim 4, wherein gB-2 is encoded by the nucleic acid sequence identified in SEQ ID NO:4, or a sequence with at least 85% identity to SEQ ID NO:4.

6. A pharmaceutical composition comprising the recombinant HSV gB of Claim 1, wherein gB is gB-1 and/or gB-2, or a combination thereof, and a pharmaceutically acceptable carrier or excipient.

7. The pharmaceutical composition of Claim 6, further comprising at least one RNA which codes for gB-1 and/or gB-2, or a combination thereof, and a pharmaceutically acceptable carrier or excipient.

8. The pharmaceutical composition of any one of Claims 6 or 7, further comprising an adjuvant, wherein the adjuvant comprises alum, MPL, or a combination thereof.

9. A method for treating or preventing herpes simplex virus infections in a subject, the method comprising administering the composition according to any one of Claims 6-8 to said subject in an amount effective to treat or prevent said infections.

10. The method according to Claim 9, wherein the herpes simplex virus infection is a herpes simplex virus type 1 (HSV-1) infection, a herpes simplex virus type 2 (HSV-2) infection, or a HSV-1 and HSV-2 co-infection.

11. A method for immunizing a subject against a herpes simplex virus infection, the method comprising administering to the subject the composition according to any one of Claims 6-8 in an amount effective to immunize the subject for a herpes virus infection.

12. The method of Claim 11, wherein the herpes simplex virus infection is a herpes simplex virus type 1 (HSV-1) infection, a herpes simplex virus type 2 (HSV-2) infection, or a HSV-1 and HSV-2 co-infection.

13. The method of any one of Claims 9-12, wherein the pharmaceutical composition is formulated for subcutaneous, intramuscular, intradermal, mucosal, or intravaginal administration.