WO2023245159A1 - Recombinant herpes simplex virus 2 (hsv-2) vectors and engineered transgenic vero cell lines - Google Patents

Abstract

A recombinant herpes simplex virus 2 (HSV-2) vaccine vector including a complete deletion of the gene encoding glycoprotein D and its promoter is described, and methods for producing virus and virions of the recombinant HSV-2 in transgenic Vero cells expressing HSV-1 glycoprotein D are provided. Compositions including the recombinant HSV-2 and methods of using the compositions are also provided.

Description

RECOMBINANT HERPES SIMPLEX VIRUS 2 (HSV-2) VECTORS AND ENGINEERED

TRANSGENIC VERO CELL LINES

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from Provisional Application No. 63/491,448 filed on March 21, 2023 and Provisional Application No. 63/352,826 filed on June 16, 2022, incorporated in their entirety herein.

INCORPORATION-BY-REFERENCE OF ELECTRONICALLY FILED MATERIAL

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 June 15, 2023 is named “XVS0014PCT -2 XML seq list 6-15- 23.XML” and is 49,152 bytes in size. The Sequence Listing does not go beyond the disclosure in the application as filed.

FEDERAL RESEARCH STATEMENT

This invention was made with government support under R01 AH 17321 and AI26170 awarded by the National Institute of Allergy and Infectious Diseases. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0001] Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) persist as significant health problems globally, disproportionally impacting developing countries and poor communities around the world and fueling the HIV epidemic. Vaccines are urgently needed for these infections as currently there is no effective vaccine for HSV-1, HSV-2 or HIV. HSV-1 is the primary cause of infectious blindness, while HSV-2 is the primary cause of genital ulcers globally, although HSV-1 is now more commonly identified in association with genital tract disease in developed countries. Genital herpes is a recurrent, lifelong disease that can stigmatize and psychologically impact those affected. Infection with HSV-2 significantly increases the likelihood of acquiring and transmitting HIV, while vertical transmission of either serotype often leads to severe infant morbidity or death. Recent clinical trials of HSV-2 vaccines based on sub-unit formulations using viral glycoprotein D (gD) alone or in combination with glycoprotein B (gD and gB) have failed, despite inducing systemic neutralizing antibodies. Surprisingly, an HSV-2 gD subunit (gD-2) vaccine provided partial protection against HSV-1, but no protection against HSV-2. Several attenuated viruses been evaluated pre-clinically, but clinical studies to date have been limited to therapeutic applications (reducing frequency of recurrences) and have also failed to show efficacy. Thus, novel vaccine strategies must be engineered and evaluated.

[0002] The present invention addresses this need for new and improved HSV-1 and HSV-2 vaccines.

SUMMARY OF THE INVENTION

[0003] Herpesvirus genomes readily undergo homologous recombination during replication, often leading to reversion to wild-type during complementation of attenuated or replication-defective viruses via homologous recombination with a helper gene provided in trans, which is a major problem in efforts to generate a vaccine strain. In order to avoid reversion, the attenuated HSV-2 vaccine strain provided herein comprises a precise and complete deletion of the HSV-2 glycoprotein D open reading frame and its promoter from the HSV-2 genome. In one aspect, the recombinant HSV-2 virus with a complete deletion of the HSV-2 glycoprotein D open reading frame and its promoter from the HSV-2 genome is referred to as AgD2P and has a deletion of the whole intergenic region between the U_S5 and U_S6 coding sequence containing the gD promoter. When grown in a complementing cell which expresses a herpes simplex virus type 1 glycoprotein D, e.g., HSV-1 gD or gD-1, such that the AgD2P virus can undergo productive replication, homologous recombination between the attenuated HSV-2 viral genome and sequences in the complementing cell does not result in reversion of the attenuated HSV-2 into an infective virus.

[0004] Additionally, removal of sequences in the complementing cell that are homologous to the AgD2P virus sequences further ensures that a homologous recombination event between the AgD2P virus and the complementing cell does not occur, thereby eliminating the possibility that a replication competent HSV-2 virus is generated.

[0005] Therefore, herein provided is a recombinantly engineered Vero cell expressing an HSV-1 gD encoding gene that is able to complement a AgD2P virus such that the virus can replicate, however, the minimal HSV-1 gD sequences comprising a minimal promoter, the HSV-1 gD encoding gene, and a polyadenylation signal are devoid of homologous HSV-2 gD gene sequences. In one aspect, the cell is a recombinantly engineered cell comprising a heterologous nucleic acid encoding HSV-1 glycoprotein D and devoid of DNA sequences homologous to sequences present in the recombinant HSV-2 having a deletion of an HSV-2 glycoprotein D-encoding gene and its promoter. In another aspect, the engineered cell has minimally required DNA sequences for gD complementation permitting replication of the recombinant HSV-2. In yet another aspect, minimally required DNA sequences for gD complementation include the HSV-1 gD protein coding sequences, minimal promoter sequences, and a poly adenylation signal. In one aspect, the minimal promoter sequence comprises or is the sequence 5’ATCCCCTAAGGGGGAGGGGCCATTTTACGAGGAGGAGGGGTATAACAAAGTCT GTCTTTAAAAAGCAGGGGTTAGGGAGTTGTTCGGTCATAAGCTTCAGCGCGAAC GACCAACTACCCCGATCATCAGTTATCCTTAAGGTCTCTTTTGTGTGGTGCGTTCC GGT 3’, identified in SEQ ID NO: 1, based on the HSV-1 17+ strain, and described in Roger, J. Watson, 1983, Gene 26, 307-312. gD promoter sequences from other strains of HSV-1 can be used. In another aspect, the cell is a Vero cell. In yet another aspect, the engineered complementing cell is VerB::gDl, a Vero cell containing minimally required DNA sequences for gD complementation. In yet another aspect, the engineered complementing cell is VerB::gD1.6C, a Vero cell containing a minimal promoter having SEQ ID NO: 32, and minimally required DNA sequences for gD complementation.

[0006] Therefore, herein provided is an isolated, recombinant herpes simplex virus-2 (HSV-2), having a complete deletion of an HSV-2 glycoprotein D-encoding gene (U_S6) and its promoter in the genome thereof. In one aspect, an isolated, recombinant herpes simplex virus-2 (HSV-2) having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof is provided for treating or preventing an HSV-1, HSV-2, or HSV-1 and HSV-2 co-infection in a subject.

[0007] Also provided is a virion of an isolated, recombinant HSV-2 having a complete deletion of an HSV-2 glycoprotein D-encoding gene (U_S6) and its promoter in the genome thereof. In one aspect, a virion of an isolated, recombinant HSV-2 having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof is provided for treating or preventing an HSV-1, HSV-2, or HSV-1 and HSV-2 co-infection in a subject.

[0008] An isolated cell is provided comprising therein a recombinant HSV-2 genome as described herein or a recombinant HSV-1 gene as described herein, wherein the cell is not present in a human.

[0009] Also provided is a vaccine composition comprising the recombinant HSV-2 virus as described herein, or the virion as described herein. [0010] Also provided is a composition comprising the recombinant HSV-2 virus as described herein, or the virion as described herein, wherein the genome of the virus or virion comprises at least a deletion of a second gene, wherein the second gene is necessary for HSV-2 viral replication or virulence.

[0011] Also provided is a pharmaceutical composition comprising the recombinant HSV-2 virus as described herein, or the virion as described herein, and a pharmaceutically acceptable carrier.

[0012] Also provided is a method of eliciting an immune response in a subject comprising administering to the subject an amount of (i) the recombinant HSV-2 virus as described herein; (ii) a virion thereof as described herein, (iii) the vaccine as described herein; (iv) a composition as described herein; or (v) a pharmaceutical composition as described herein, in an amount effective to elicit an immune response in a subject.

[0013] Also provided is a method of treating an HSV-1, HSV-2, or HSV-1 and HSV- 2 co-infection in a subject or treating a disease caused by an HSV-1, HSV-2 or co-infection in a subject comprising administering to the subject an amount of (i) the recombinant HSV-2 virus as described herein; (ii) a virion thereof as described herein, (iii) a vaccine as described herein; (iv) a composition as described herein; or (v) a pharmaceutical composition as described herein, in an amount effective to treat an HSV-1, HSV-2, or a co-infection or treat a disease caused by an HSV-1, HSV-2, or a co-infection in a subject.

[0014] Also provided is a method of vaccinating a subject for HSV-1, HSV-2, or HSV-1 and HSV-2 co-infection comprising administering to the subject an amount of (i) the recombinant HSV-2 virus as described herein; (ii) a virion thereof as described herein, (iii) a vaccine as described herein; (iv) a composition as described herein; or (v) a pharmaceutical composition as described herein, in an amount effective to vaccinate a subject for HSV-1, HSV-2, or a co-infection.

[0015] Also provided is a method of immunizing a subject against HSV-1, HSV-2, or HSV-1 and HSV-2 co-infection comprising administering to the subject an amount of (i) the recombinant HSV-2 virus as described herein; (ii) a virion thereof as described herein, (iii) a vaccine as described herein; (iv) a composition as described herein; or (v) a pharmaceutical composition as described herein, in an amount effective to immunize a subject against HSV- 1, HSV-2, or a co-infection.

[0016] In an embodiment of the vaccines, compositions and pharmaceutical compositions, and of the methods of use thereof, the amount of recombinant HSV-2 is an amount of pfu (plaque forming unit) of recombinant HSV-2 effective to achieve the stated aim.

[0017] Also provided is a method of producing a virion of a recombinant herpes simplex virus-2 (HSV-2), having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof and comprising a HSV-1 glycoprotein D on a lipid bilayer thereof, comprising infecting a cell comprising a heterologous nucleic acid encoding a HSV-1 glycoprotein D with a recombinant herpes simplex virus-2 (HSV-2) having the complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof under conditions permitting replication of the recombinant herpes simplex virus-2 (HSV-2) and recovering a HSV-2 virion produced by the cells. In one aspect, the cell is a recombinantly engineered cell comprising a heterologous nucleic acid encoding HSV-1 glycoprotein D and devoid of DNA sequences homologous to sequences present in the recombinant HSV-2 having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter. In another aspect, the engineered cell has minimally required DNA sequences for gD complementation permitting replication of the recombinant HSV-2. In yet another aspect, minimally required DNA sequences for gD complementation include the HSV-1 gD protein coding sequences, minimal promoter sequences, and a polyadenylation signal. In one aspect, the minimal promoter sequences based on the HSV-1 17+ strain comprises or is the sequence identified in SEQ ID NO: 1. gD promoter sequences from other strains of HSV-1 can be used. In another aspect, the cell is a Vero cell. In yet another aspect, the engineered complementing cell is VerB::gDl, a Vero cell containing minimally required DNA sequences for gD complementation. In yet another aspect, the engineered complementing cell is VerB::gD1.6C, a Vero cell comprising minimally required gDl sequences for complementation, under control of a minimal promoter with a 5’ 3 -nucleotide modification having the sequence identified in SEQ ID NO:32.

[0018] Also provided is a recombinant nucleic acid having the same sequence as a genome of a wild-type HSV-2 except that the recombinant nucleic acid does not comprise a sequence encoding an HSV-2 glycoprotein D open reading frame and does not comprise a glycoprotein D promoter.

[0019] Also provided is an isolated, recombinant herpes simplex virus-2 (HSV-2) having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof and further comprising a heterologous antigen of a pathogen. In one aspect, the pathogen is a virus, a bacterium, a parasite. [0020] Also provided is a method of inducing antibody dependent cell mediated cytotoxicity (ADCC) against an antigenic target in a subject comprising administering to the subject an isolated, recombinant herpes simplex virus-2 (HSV-2) having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof and further comprising a heterologous antigen on a lipid bilayer thereof in an amount effective to induce antibody dependent cell mediated cytotoxicity (ADCC) against an antigenic target. In one aspect, the recombinant HSV-2 is produced in a complementing cell engineered to express an HSV-1 gD protein, comprising a nucleic acid encoding HSV-1 glycoprotein D and devoid of DNA sequences homologous to those present in the recombinant HSV-2 having a deletion of an HSV-2 glycoprotein D-encoding gene and its promoter. In one aspect, the complementing cell further comprises heterologous sequences for expressing the heterologous antigen. In one aspect, the heterologous antigen is an HSV-1 antigen, an HSV- 2 antigen, a bacterial antigen, a parasite antigen, a viral antigen, or a combination thereof.

[0021] A method is provided for eliciting an immune response in a first subject against an HSV-2 and/or HSV-1 infection, comprising effectuating passive transfer to the first subject of an amount of a product from a pregnant female immunized with HSV-2 having a complete deletion of the entire HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof and wherein said HSV-2 is phenotypically complemented with a herpes simplex virus- 1 (HSV-1) glycoprotein D by propagating said HSV-2 in a complementing cell expressing said HSV-1 glycoprotein D, wherein the product comprises antibodies induced thereby, effective to elicit an immune response against an HSV-2 and/or HSV-1 infection in the first subject, wherein the first subject is a fetus or neonate. In one aspect, the complementing cell is engineered to express heterologous HSV-1 gD protein, comprising a heterologous nucleic acid encoding HSV-1 glycoprotein D and devoid of DNA sequences homologous to DNA sequences present in the recombinant HSV-2 having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter. Also provided is a method of inhibiting a perinatal HSV-1 and/or HSV-2 infection in a neonate comprising administering to a female pregnant with a fetus which will become the neonate an amount of a recombinant HSV-2 virus having a complete deletion in glycoprotein D- encoding gene and its promoter in the genome thereof and wherein said HSV-2 is phenotypically complemented with a herpes simplex virus- 1 (HSV-1) glycoprotein D by propagating said HSV-2 in a complementing cell expressing said HSV-1 glycoprotein D, effective to inhibit a perinatal HSV-1 and/or HSV-2 infection in a neonate. In one aspect, the complementing cell is engineered to express heterologous HSV-1 gD protein, comprising a heterologous nucleic acid encoding HSV-1 glycoprotein D and devoid of homologous DNA sequences present in the recombinant HSV-2 having a deletion of an HSV-2 glycoprotein D- encoding gene and its promoter. In one aspect, the complementing cell is a VerB::gDl cell, a Vero cell containing minimally required DNA sequences for gD complementation. In another aspect, the complementing cell is VerB::gD1.6C, a Vero cell comprising gDl sequences for complementation, under control of a minimal promoter with a 5’ 3 -nucleotide modification having the sequence identified in SEQ ID NO:32.

[0022] Also provided is a method of inhibiting HSV-1 and/or HSV-2 viral dissemination from a mother to her neonate comprising administering to the mother an amount of an HSV-2 having a complete deletion of the HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof and wherein said HSV-2 is phenotypically complemented with a herpes simplex virus- 1 (HSV-1) glycoprotein D by propagating said HSV-2 in a complementing cell expressing said HSV-1 glycoprotein D, effective to inhibit HSV-1 and/or HSV-2 viral dissemination from a mother to her neonate. In one aspect, the complementing cell is engineered to express heterologous HSV-1 gD protein, comprising a heterologous nucleic acid encoding HSV-1 glycoprotein D and devoid of homologous DNA sequences present in the recombinant HSV-2 having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter. In one aspect, the complementing cell is a VerB::gDl cell, a Vero cell containing minimally required DNA sequences for gD complementation. In another aspect, the complementing cell is VerB::gD1.6C, a Vero cell comprising gDl sequences for complementation, under control of a minimal promoter with a 5’ 3-nucleotide modification having the sequence identified in SEQ ID NO:32.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIGs. 1A and B show cosmid substrates for generating deletions in the Usi- Usi2 regions of HSV-2 G DNA. 1A) The cosmid pYUB2156 was generated by making large Sau3A partial digests and cosmid cloning them into pYUB 328 double cos vector. The pYUB 328 contains a single Bglll site (compatible with Sau3A) flanked by 2 Pad sites (TTAAT/TAA). Since the Pad sequence is not found in high G-C DNA like herpesvirus or Mycobacteria genome, Pad digestion of either pYUB 2156 or pBRLl 171 releases an E. coli vector and a large linear fragment that is ideal for homologous recombination with HSV-2 DNA. IB) The cosmid pBRLl 171 is a derivative of pYUB2156 in which the gD-2 gene and its promoter are deleted and replaced by a Swal site (TTAAT/TA). The Swal site provides a useful genetic marker as it is not found in HSV DNA. [0024] FIG. 2 shows a DNA gel fractionation confirming deletion of gD-2 by PCR.

[0025] FIG. 3 shows the steps in construction of VerB::gDl (VerB::gD-l in the figure) cell line. Step 1. Vero cell lines were recombinantly engineered to contain a sitespecific recombination sequence, attB (attachment site) derived from Mycobacterium smegmatis, which allows the site-specific integration of any exogenous DNA that contains the bacteriophage attachment site (attP) mediated by the Bxbl integrase. Two parental cell lines were used as starting materials, VerB-ocl and VCI B-G2 where the attB site was placed in a safe harbor site within the AAVS1 locus. Step 2. The attP-shuttle plasmid containing the attP site, the gD-1 expression cassette and the histidinol dehydrogenase gene (hisD) selection marker was constructed and used to integrate the gD-1 expression cassette into the Vero-attB site in the presence of Bxbl integrase mRNA.

[0026] FIGs. 4A-D show schematic maps for pMAECL20 and intermediate plasmids used for preparation of attP-shuttle plasmid pMAECL20. 4A) pMAE7; 4B) pMAECLl; 4C) pMAECL7; and 4D) pMAECL20.

[0027] FIG. 5 shows the expression of gD-1 in Vero cells transfected with pMAECL20 and co-infected with HSV-2 AgD2::RFP virus. Vero cells were transfected with pMAECL20 and 24 h post-transfection, cells were infected with AgD2::RFP virus at MOI 0.1 PFU or mock infected. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton™ X-100, and stained with an antibody against HSV-1 gD (Santa Cruz, sc sc69802) in 3% BSA-PBS for 16 h at 4°C. Then, cells were washed with PBS and incubated with AF488 goat anti-mouse IgG secondary antibody (Invitrogen, cat #A32723) for 1 h. Images were captured using an EVOS™ 5000 fluorescence microscope.

[0028] FIG. 6 shows results of screening of VerB::gDl cell clones. VerB::gDl cells were infected with HSV-2 AgD2::RFP virus at an MOI of 0.01 PFU. Infected cells were imaged and cytopathic effect (CPE) characteristic of HSV infection was recorded daily using the EVOS™ 5000 fluorescence microscope.

[0029] FIGs. 7A and B show growth kinetics. VerB::gDl cell clones and VD60 cells were infected with AgD2P virus at MOI 0.1 PFU. 7A) Infected cells were imaged at 8, 24, and 72 hpi using an EVOS™ 5000 Imaging System. Infected cells were harvested at different time points after infection (18, 24, 72 hpi), and the AgD2P virus produced was titrated by plaque assay in VD60 cells (7B).

[0030] FIG. 8 shows productivity. VerB::gDl cell clones and VD60 were infected with AgD2P virus at different MOIs and incubated at different temperatures (34, 35, 37°C). Infected cells were harvested at 90% CPE. Produced AgD2P virus were titrated by plaque assay in VD60 cells.

[0031] FIG. 9 shows results of a phenotype assay. VerB::gDl cells were infected with AgD2P virus at MOI 0.01 PFU, incubated at 37°C and harvested at 4-8 dpi. AgD2P virus produced in VerB::gDl cells was used to infect Vero cells in serial passages. Infected cells were imaged and harvested at 8 dpi.

[0032] FIG. 10 is a schematic of pMAECE41. The pMAECE20 plasmid was modified by site directed mutagenesis to insert 3 nucleotides upstream the gD-1 promoter sequence, thus generating the attP-shuttle plasmid pMAECE41.

[0033] FIG. 11 shows the expression of gD-1 in Vero cells transfected with pMAECE41 and co-infected with HSV-2 AgD2P. Vero cells were transfected with pMAECE41 and 24 h post-transfection, cells were infected with AgD2P virus at MOI 0.1 PFU or mock infected (non-infected). Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton™ X-100, and stained with an antibody against HSV-1 gD (Santa Cruz, sc69802) in 3% BSA-PBS for 16 h at 4°C. Then, cells were washed with PBS and incubated with AF488 goat anti-mouse IgG secondary antibody (Invitrogen, cat #A32723) for 1 h. Images were captured using EVOS™ 5000 fluorescence microscope.

[0034] FIG. 12 shows the expression of gD-1 in VerB::gD1.6C infected with AgD2P virus by immunofluorescence assay. VerB::gD1.6C and VD60 cells were infected with AgD2P virus at MOI 0.1 PFU or mock infected. Cells were fixed 48 hpi, permeabilized and stained with an antibody against HSV-1 gD (Santa Cruz, sc69802) followed by incubation with secondary antibody AF488 goat anti-mouse IgG (Invitrogen, cat #A32723). Images were captured using EVOS™ 5000 fluorescence microscope.

[0035] FIG. 13A-C show gD-1 protein expression analysis in VerB::gD1.6C cells infected with AgD2P virus by western blot assay. VerB::gD1.6C (13A), VD60 (13B) and Vero cells (13C) were infected (referred to as cc) with AgD2P, AgD2 or HSV-2 MS virus at MOI 1 PFU or mock infected. Viral protein expression was analyzed at different time points post-infection (6, 8, 12, 20 hpi) in a 7% SDS-PAGE, HSV-1 gD protein was detected using a mouse IgG anti-HSV gD primary antibody (Santa Cruz, sc sc69802) followed by a secondary antibody goat anti-mouse IgG conjugated with HRP (Invitrogen, cat #31430) for 1 h. Target proteins were visualized by enhanced chemiluminescence (ECE Western blotting analysis system; GE Healthcare) and imaged on the iBright™ 1500 Western Blot Imaging System (Invitrogen). [0036] FIG. 14A and B show productivity. (14A) Productivity comparison. VerB::gD1.6C, VD60 and Vero cells were infected with AgD2P virus at MOI 0.01 and incubated at 37°C. (14B) Large production. VerB::gD1.6C cells were infected with AgD2P virus at MOI 0.01 and incubated at 37°C. Produced AgD2P virus were titrated by plaque assay in VerB::gD1.6C and VD60 cells.

[0037] FIG. 15A and B show survival curve and disease scores curve of immunized mice after challenge with HSV-2 strain MS. Mice were primed and boosted after 3 weeks with AgD2P (referred to as AgD2P/6C produced on VerB::gD1.6C cells) or AgD2 (AgD2/VD60, produced on VD60 cells) at 5E+05 PFU or 5E+06 PFU of vaccine viruses. Two weeks after the boost, mice were challenged with IxLDgo dose of HSV-2 strain MS by a skin scarification model. Animals were evaluated daily for (15A) survival and (15B) epithelial and neurological disease scores. Survival was analyzed using a Gehan-Breslow- Wilcoxon test ****P<0.0001.

[0038] FIG. 16A and B show results of FcyRIV activation assay of immunized mice. Mice were primed and boosted after 3 weeks with AgD2P (referred to as AgD2P/6C in the figure legend produced on VerB::gD1.6C cells) or AgD2 (referred to as AgD2/VD60, produced on VD60 cells) at 5E+05 PFU or 5E+06 PFU of vaccine viruses and sera collected 2 weeks after the boost. Serum of immunized or control non-immunized mice was analyzed for mFcyRIV activation by luciferase effector cell reporter assay (mouse FcyRIV activation assay, Promega) using HSV-2 strain MS-infected target cells (data represented as mean +SD values of 10 mice/group). Results are shown as fold induction reciprocal to serial serum dilution (n = 10 mice/pool; data represented as mean, SD obtained from replicates) (A) or to a single dilution for individual mouse (bars represent mean). For 16A, *P < 0.05, **P < 0.01, AgD2P vaccinated groups vs. control-vaccinated group via 2-way ANOVA. For 16B, *P < 0.01, **P < 0.005; AUCs were generated for each group and then analyzed via one-way ANOVA comparing AgD2P and AgD2 groups with control values or against each other.

DETAILED DESCRIPTION OF THE INVENTION

[0039] Throughout this application various publications are referred to, including by number in square brackets. Full citations for these references may be found at the end of the specification. The disclosures of these publications, and all patents, patent application publications and books referred to herein, are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.

[0040] Disclosed herein is an isolated, recombinant herpes simplex virus-2 (HSV-2) having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof. Also disclosed are genetically modified complementing cells capable of producing HSV-1 glycoprotein D, such that HSV-2 virions are produced comprising a non- HSV-2 surface glycoprotein D on a lipid bilayer thereof. The recombinant HSV-2 and complementing cell do not share homologous sequences, thereby preventing reversion of the recombinant HSV-2 to wild-type during virus propagation, increasing the safety of the resulting HSV-2 vaccine composition. Methods of making and using the recombinant HSV-2 and complementing cells are disclosed.

[0041] The term "locus" refers to a specific location on a chromosome. A known locus can contain known genetic information, such as one or more polymorphic marker sites.

[0042] A "target locus" is a region of DNA into which a gene or polynucleotide of interest is integrated, e.g., a region of chromosomal or mitochondrial DNA in a cell.

[0043] A "nucleic acid construct" or “heterologous nucleic acid” or “vector” as used herein, refers to a nucleic acid sequence, and in particular a DNA sequence, that originates from a source foreign to the particular host cell, or, if from the same source, is modified from its original form. The nucleic acid construct or heterologous nucleic acid is constructed to comprise one or more functional units not found together in nature and is designed to transfer a nucleic acid (or nucleic acids) to a host cell. Examples include circular, double- stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes comprising heterologous (non-native) nucleic acid sequences, and the like. The heterologous nucleic acid can be a DNA sequence. The heterologous nucleic acid includes a DNA sequence of a transgene or heterologous gene. A host cell including the heterologous nucleic acid expresses the heterologous gene. The nucleic acid construct can also be referred to as a vector.

[0044] A “transcription unit”, “expression unit” or “expression cassette” defines a region within a vector, construct or polynucleotide sequence that contains one or more genes to be transcribed, wherein the genes contained within the segment are operably linked to each other. They are transcribed from a single promoter and transcription is terminated by at least one polyadenylation signal. As a result, the different genes are at least transcriptionally linked. More than one protein or product can be transcribed and expressed from each transcription unit (multicistronic transcription unit). Each transcription unit will comprise the regulatory elements necessary for the transcription and translation of any of the selected sequence that are contained within the unit. And each transcription unit may contain the same or different regulatory elements. For example, each transcription unit may contain the same terminator. IRES element or introns may be used for the functional linking of the genes within a transcription unit. A vector or polynucleotide sequence may contain more than one transcription unit.

[0045] A “heterologous nucleic acid” refers to a nucleic acid sequence or polynucleotide, and in particular a DNA sequence, that originates from a source foreign to the particular host genome, or, if from the same source, is modified from its original form. The heterologous nucleic acid is constructed to comprise one or more functional units not found together in nature and is designed to transfer a nucleic acid (or nucleic acids) to a host genome. Examples include circular, double-stranded, extrachromosomal DNA molecules (plasmid, shuttle plasmid), cosmids (plasmids containing cos sequences from lambda phage), viral genomes comprising heterologous (non-native) nucleic acid sequences, and the like.

[0046] The term "gene" refers to a nucleotide sequence associated with a biological function. Thus, a gene includes a coding sequence and/or the regulatory sequence required for its expression. A gene can also include non-coding DNA segments such as regulatory elements that, for example, form recognition sequences for other proteins. A gene can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters. A “transgene” or “heterologous gene” refers to a gene that originates from a source foreign to the host cell or, if from the same source, is modified from its original form. Thus, the terms refer to a DNA segment which is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. A heterologous gene is expressed to yield a heterologous polypeptide. The term "stably integrated" refers to a heterologous nucleic acid that is incorporated into a host genome, replicates as the cell replicates, and is transferred to progeny. In the present disclosure, the host cell is a Vero cell, and the heterologous nucleic acid is integrated into the Vero cell genome and passed to progeny cells.

[0047] A DNA segment (nucleotide sequence) is "operably linked" when placed into a functional relationship with another DNA segment. For example, DNA for a signal sequence is operably linked to DNA for a gene encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it stimulates the transcription of the sequence. In general, DNA sequences that are operably linked are contiguous, and in the case of a signal sequence both contiguous and in reading phase. However, enhancers, for example, need not be contiguous with the coding sequences whose transcription they control. Linking is accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof.

[0048] The term "recombinant" when used with reference to a cell indicates that the cell replicates a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid. Recombinant cells can contain polynucleotides that are not found within the native (non-recombinant) form of the cell.

[0049] A cell has been "transformed" or "transfected" by exogenous or heterologous DNA, e.g., a DNA construct, when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell.

[0050] The term "transgenic" refers to a cell that includes a specific genetic modification that was introduced into the cell, or into an ancestor of the cell. Such modifications can include one or more point mutations, deletions, insertions, or combinations thereof. In the present disclosure, transgenic refers to a cell comprising a heterologous nucleic acid introduced into the cell.

[0051] "Recombination sites" are specific polynucleotide sequences that are recognized by the recombinase enzymes described herein. Typically, two different sites are involved (termed "complementary sites"), one present in the target nucleic acid (e.g., a chromosome or episome of a eukaryote) and another on the nucleic acid that is to be integrated at the target recombination site. The terms "attB" and "attP," which refer to attachment (or recombination) sites originally from a bacterial target and a phage donor, respectively, are used herein. The attB site referred to herein is specifically the Bxbl attB site. The recombination sites can include left and right arms separated by a core or spacer region. Upon recombination between the attB and attP sites, and concomitant integration of a nucleic acid at the target, the recombination sites that flank the integrated DNA are referred to as "attL" and "attR."

[0052] The term "promoter" refers to a region of DNA that initiates transcription of a particular gene. The promoter includes the core promoter, which is the minimal portion of the promoter required to properly initiate transcription and can also include regulatory elements such as transcription factor binding sites. The regulatory elements may promote transcription or inhibit transcription. Regulatory elements in the promoter can be binding sites for transcriptional activators or transcriptional repressors. A promoter can be constitutive or inducible. A constitutive promoter refers to one that is always active and/or constantly directs transcription of a gene above a basal level of transcription. An inducible promoter is one which is capable of being induced by a molecule or a factor added to the cell or expressed in the cell. An inducible promoter may still produce a basal level of transcription in the absence of induction, but induction typically leads to significantly more production of the protein.

[0053] The term “polyadenylation signal” or “polyadenylation signal sequence” as used herein refers to a sequence contain the AAUAAA sequence which when recognized by transcription factors in the cell results in the addition of a poly(A) tail to an RNA transcript, normally at the 3’ end, of a messenger RNA (mRNA). The poly(A) tail consists of multiple adenosine monophosphates or a stretch of adenine bases. In eukaryotes, polyadenylation is part of the process that produces mature mRNA and is important for the nuclear export, translation and stability of mRNA.

[0054] The term "transactivation," as used herein refers to the activation of a gene sequence by factors encoded by a regulatory gene, and which is not necessarily contiguous with the gene sequence to which it binds and activates.

[0055] Provided herein is an isolated, recombinant herpes simplex virus-2 (HSV-2) having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof. As used herein, a complete deletion of an HSV-2 glycoprotein D- encoding gene and its promoter in the genome thereof is equivalent to deletion of the entire HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof, such that no HSV-2 glycoprotein D or fragment thereof is produced in the recombinant HSV-2.

[0056] In an embodiment, the HSV-2 glycoprotein D gene, or the Us6 gene, comprises the nucleic acid sequence set forth in SEQ ID NO:2: 5’ATGGGGCGTTTGACCTCCGGCGTCGGGACGGCGGCCCTGCTAGTTGTCGCGGT GGGACTCCGCGTCGTCTGCGCCAAATACGCCTTAGCAGACCCCTCGCTTAAGATG GCCGATCCCAATCGATTTCGCGGGAAGAACCTTCCGGTTTTGGACCAGCTGACCG ACCCCCCCGGGGTGAAGCGTGTTTACCACATTCAGCCGAGCCTGGAGGACCCGTT CCAGCCCCCCAGCATCCCGATCACTGTGTACTACGCAGTGCTGGAACGTGCCTGC CGCAGCGTGCTCCTACATGCCCCATCGGAGGCCCCCCAGATCGTGCGCGGGGCTT CGGACGAGGCCCGAAAGCACACGTACAACCTGACCATCGCCTGGTATCGCATGG GAGACAATTGCGCTATCCCCATCACGGTTATGGAATACACCGAGTGCCCCTACAA CAAGTCGTTGGGGGTCTGCCCCATCCGAACGCAGCCCCGCTGGAGCTACTATGAC AGCTTTAGCGCCGTCAGCGAGGATAACCTGGGATTCCTGATGCACGCCCCCGCCT TCGAGACCGCGGGTACGTACCTGCGGCTAGTGAAGATAAACGACTGGACGGAGA TCACACAATTTATCCTGGAGCACCGGGCCCGCGCCTCCTGCAAGTACGCTCTCCC CCTGCGCATCCCCCCGGCAGCGTGCCTCACCTCGAAGGCCTACCAACAGGGCGT GACGGTCGACAGCATCGGGATGCTACCCCGCTTTATCCCCGAAAACCAGCGCAC CGTCGCCCTATACAGCTTAAAAATCGCCGGGTGGCACGGCCCCAAGCCCCCGTA CACCAGCACCCTGCTGCCGCCGGAGCTGTCCGACACCACCAACGCCACGCAACC CGAACTCGTTCCGGAAGACCCCGAGGACTCGGCCCTCTTAGAGGATCCCGCCGG GACGGTGTCTTCGCAGATCCCCCCAAACTGGCACATCCCGTCGATCCAGGACGTC GCGCCGCACCACGCCCCCGCCGCCCCCAGCAACCCGGGCCTGATCATCGGCGCG CTGGCCGGCAGTACCCTGGCGGTGCTGGTCATCGGCGGTATTGCGTTTTGGGTAC GCCGCCGCGCTCAGATGGCCCCCAAGCGCCTACGTCTCCCCCACATCCGGGATGA CGACGCGCCCCCCTCGCACCAGCCATTGTTTTACTAG 3’ (HSV-2 reference strain G).

[0057] In one embodiment, the complete HSV-2 glycoprotein sequence is deleted from the recombinant HSV-2 genome. In an embodiment, the HSV-2 glycoprotein D- encoding gene is an HSV-2 Us6 gene. (For example, see Dolan et al. J Virol. 1998 March; 72(3): 2010-2021. (PMCID: PMC109494) “The Genome Sequence of Herpes Simplex Virus Type 2” for HSV-2 genome and Us6 gene, hereby incorporated by reference in its entirety).

[0058] In one embodiment, the HSV-2 glycoprotein D amino acid sequence comprises or is the sequence identified as MGRLTSGVGTAALLVVAVGLRVVCAKYALADPSLKMADPNRFRGKNLPVLDQLTD PPGVKRVYHIQPSLEDPFQPPSIPITVYYAVLERACRSVLLHAPSEAPQIVRGASDEAR KHTYNLTIAWYRMGDNCAIPITVMEYTECPYNKSLGVCPIRTQPRWSYYDSFSAVSE DNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRARASCKYALPLRIPPAACLT SKAYQQGVTVDSIGMLPRFIPENQRTVALYSLKIAGWHGPKPPYTSTLLPPELSDTTN ATQPELVPEDPEDSALLEDPAGTVSSQIPPNWHIPSIQDVAPHHAPAAPSNPGLIIGAL AGSTLAALVIGGIAFWVRRRAQMAPKRLRLPHIRDDDAPPSHQPLFY (HSV-2 reference strain G) (SEQ ID NOG).

[0059] In an embodiment, the HSV-2 glycoprotein D promoter sequence comprises the nucleic acid sequence set forth in SEQ ID NO:4:

5 ’ TTTTTCGCCTTTCTGGCCTTGCCCCCACCCCATCGCCCCGATTGTGTGTCGGGTG CCCGGGGTACAGCAGCTATGGAGCGGTCGGTAATATAACTTTGGTTGTCGCCACA CGCCCCGTGCCGGGCATGGGTTGTGCGGGAAGGACGAAATAATCCGGCGATCCC CAAGCGTACCAACTGGGGGGGGGGGGGGGGGGGAAAAGAAACTAAAAACACAT CAAGCCCACAACCCATCCCACAATGGGGGTTATGGCGGACCCACCGCACCACCA TACTCCGATTCGACCACATATGCAACCAAATCACCCCCAGAGGGGAGGTTCCATT TTTACGAGGAGGAGGAGTATAATAGAGTCTTTGTGTTTAAAACCCGGGGTCGGTG TGGTGTTCGGTCATAAGCTGCATTGCGAACGACTAGTCGCCGTTTTTCGTGTGCA TCGCGTATCACGGC 3’(HSV-2 reference strain G).

[0060] In one embodiment, the recombinant HSV-2 virus with a complete deletion of the glycoprotein D gene comprises a deletion of the glycoprotein D promoter set forth in SEQ ID NO:4.

[0061] In one embodiment, the HSV-2 glycoprotein D comprises the amino acid sequence set forth in SEQ ID NO:5: MGRLTSGVGTAALLVVAVGLRVVCAKYALADPSLKMADPNRFRGKNLPVLDQLTD PPGVKRVYHIQPSLEDPFQPPSIPITVYYAVLERACRSVLLHAPSEAPQIVRGASDEAR KHTYNLTIAWYRMGDNCAIPITVMEYTECPYNKSLGVCPIRTQPRWSYYDSFSAVSE DNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRARASCKYALPLRIPPAACLT SKAYQQGVTVDSIGMLPRFIPENQRTVALYSLKIAGWHGPKPPYTSTLLPPELSDTTN ATQPELVPEDPEDSALLEDPAGTVSSQIPPNWHIPSIQDVAPHHAPAAPSNPGLIIGAL AGSTLAVLVIGGIAFWVRRRAQMAPKRLRLPHIRDDDAPPSHQPLFY (HSV-2 reference strain HG52).

[0062] In an embodiment, the isolated, recombinant HSV-2 further comprises a herpes simplex virus- 1 (HSV-1) glycoprotein D on a lipid bilayer thereof.

[0063] In an embodiment, the HSV-1 glycoprotein D comprises the amino acid sequence set forth in SEQ ID NO:6: MGGAAARLGAVILFVVIVGLHGVRGKYALADASLKMADPNRFRGKDLPVLDQLTD PPGVRRVYHIQAGLPDPFQPPSLPITVYYAVLERACRSVLLNAPSEAPQIVRGASEDV RKQPYNLTIAWFRMGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNYYDSFSAVS EDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRAKGSCKYALPLRIPPSACL SPQAYQQGVTVDSIGMLPRFIPENQRTVAVYSLKIAGWHGPKAPYTSTLLPPELSETP NATQPELAPEDPEDSALLEDPVGTVAPQIPPNWHIPSIQDAATPYHPPATPNNMGLIA GAVGGSLLAALVICGIVYWMRRRTQKAPKRIRLPHIREDDQPSSHQPLFY (HS V- 1 reference strain F).

[0064] In an embodiment, the HSV-2 in which the HSV-2 glycoprotein D-encoding gene and its promoter are completely deleted is an HSV-2 having a genome (prior to the deletion) as set forth in one of the following Genbank listed sequences: HSV-2(G) (KU310668), HSV-2(4674) (KU310667), B3x2.1 (KU310662), B3x2.2 (KU310663), B3x2.3 (KU310664), B3x2.4 (KU310665), B3x2.5 (KU310666). [0065] Also provided is a virion of an isolated, recombinant HSV-2 having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof. In an embodiment, the HSV-2 glycoprotein D-encoding gene is an HSV-2 Us6 gene.

[0066] Also provided is a recombinant nucleic acid having the same sequence as a genome of an HSV-2 except that the sequence does not comprise a sequence encoding an HSV-2 glycoprotein D and its promoter.

[0067] In an embodiment, the virion further comprises an HSV-1 glycoprotein D on a lipid bilayer thereof. In an embodiment, the virus further comprises an HSV-1 on a lipid bilayer thereof.

[0068] An isolated cell is provided comprising therein a recombinant HSV-2 genome which does not comprise an HSV-2 Us6 gene or its promoter. In one aspect, the recombinant HSV-2 genome is recombinant by virtue of having had an HSV-2 glycoprotein D gene and its promoter deleted therefrom. In an embodiment, the cell is a complementing cell which provides expressed HSV-1 glycoprotein not encoded for by the recombinant HSV-2 genome. In an embodiment, the complementing cell comprises a heterologous nucleic acid encoding an HSV-1 glycoprotein D. In an embodiment, the cell expresses HSV-1 glycoprotein D on a membrane thereof. In an embodiment of the cell, the HSV-1 glycoprotein D is encoded by a heterologous nucleic acid, which heterologous nucleic acid is an HSV-1 glycoprotein D gene, or is a nucleic acid having a sequence identical to an HSV-1 glycoprotein D gene. In another embodiment, the cell is devoid of DNA sequences that can cause recombination between the cell and the recombinant HSV-2 virus. In another embodiment, the complementing cell has minimal gD-1 sequences for expressing gD-1. In one aspect, the gD-1 sequences are configured for expression in the Vero cell, or are in an expression cassette. In one aspect, the minimal gD-1 sequences comprise a gD-1 encoding gene, a minimal promoter, and a polyadenylation signal. In one aspect, the minimal promoter sequences comprises or is the sequence

5 ’ ATCCCCTAAGGGGGAGGGGCC ATTTTACGAGGAGGAGGGGTATAAC AAAGTCT GTCTTTAAAAAGCAGGGGTTAGGGAGTTGTTCGGTCATAAGCTTCAGCGCGAAC GACCAACTACCCCGATCATCAGTTATCCTTAAGGTCTCTTTTGTGTGGTGCGTTCC GGT 3’, identified in SEQ ID NO: 1, based on the HSV-1 17+ strain, and described in Roger, J. Watson, 1983, Gene 26, 307-312. In another aspect, the minimal gD-1 sequences comprise a gD- 1 encoding gene, a modified minimal promoter having an additional 3 nucleotides at the 5’ end, and a poly adenylation signal, the modified minimal promoter sequence comprises or is the sequence 5’CGAATCCCCTAAGGGGGAGGGGCCATTTTACGAGGAGGAGGGGTATAACAAA GTCTGTCTTTAAAAAGCAGGGGTTAGGGAGTTGTTCGGTCATAAGCTTCAGCGCG AACGACCAACTACCCCGATCATCAGTTATCCTTAAGGTCTCTTTTGTGTGGTGCG TTCCGGT 3’, identified in SEQ ID NO:32, based on the HSV-1 17+ strain.

[0069] Since the identity of gD among HSV-1 strains is 99-100%, gD promoter sequences from other strains of HSV-1 can be used. In one aspect the complementing cell comprises an attB attachment site from Mycobacterium smegmatis. In another aspect the attB site is a 38 bp attB site (also referred to herein interchangeably as “attB sequence”) from Mycobacterium smegmatis: GGCTTGTCGACGACGGCGGTCTCCGTCGTCAGGATCAT- 3’, SEQ ID NO:7).

[0070] In another aspect, the cell is a Vero cell. In one aspect, the attB site is inserted into the Vero cell genome between positions 1529 and 1530 of the adeno-associated virus integration site 1 (AAVS1) locus on chromosome 6 (Osada, N. et al, 2014, DNA Research 21, 673-683; Kotin and Berns, 1992, EMBO J 11:5071-5078; AAVS1 sequence on chromosome 19 of human genome accession no. AC010327.8), alternatively, between positions 2155 and 2156 of the AAVS1 locus, between positions 2408 and 2409 of the AAVS1 locus, or a combination thereof. In one aspect, the Vero cell comprising the attB site is a parental cell. In one aspect the gD-1 expression cassette, or gD-1 cassette, comprising minimal gD-1 sequences for expressing gD-1 in a cell are inserted at an attB site inserted into the parental Vero cell genome between positions 1529 and 1530 of the AAVS1 locus on chromosome 6 resulting in a Vero cell comprising a gD-1 transgene. In an aspect, the AAVS1 locus comprising the attB recombination sequence has the 4 kilobase sequence. The gD-1 expression cassette can be part of a plasmid designed to comprise the Bxbl attP attachment sequence site (5’- GGTTTGTCTGGTCAACCACCGCGGTCTCAGTGGTGTACGGTACAAACC-3’, SEQ ID NO: 8). Homologous recombination between the attB and attP sites assisted by Bxbl integrase introduced into the cell along with the gD-1 expression cassette results in specific integration of the gD-1 cassette at the target locus of the Vero cell genome. The recombination sites flanking the integrated DNA, for example the gD-1 cassette, are referred to as attL at one end and attR at the second end. In an aspect, the plasmid comprising the gD- 1 cassette does not comprise or contain an antibiotic resistance marker, a simian vacuolating virus (SV40) sequence, or a cytomegalovirus (CMV) sequence. In one aspect, the plasmid comprising the gD-1 cassette contains or comprises a histidinol dehydrogenase gene operably linked to a eukaryotic elongation factor- 1 alpha (EFloc) promoter derived from the human EEF1A1 gene and/or the prokaryotic EM7 promoter, a synthetic promoter based on the bacteriophage T7 promoter.

[0071] Also provided is a method of producing the recombinantly engineered complementing cell or cell line comprising a gD-1 transgene comprising minimal gD-1 sequences, stably inserted at a defined position in the cellular genome such that the gD-1 is expressed, for use in producing an HSV-2 vaccine, wherein the HSV-2 vaccine is produced by infecting the complementing cell with a recombinant HSV-2 virus having a complete deletion of a gD encoding gene and its promoter, wherein the cell expresses gD-1 from minimal gD-1 sequences inserted in the cell’s genome, the minimal sequences comprising gD-1 encoding gene, a minimal promoter, and a polyadenylation signal, such that the recombinant HSV-2 virus is produced comprising gD-1 on its lipid bilayer. Methods for producing similar complementing cells is described in published US Patent Application No. US2022-0090146. All references cited herein are hereby incorporated by reference in their entirety.

[0072] In one aspect, the method of producing a transgenic Vero cell comprising a heterologous gD-1 minimal sequence stably integrated in a target locus on a chromosome of the Vero cell genome comprises contacting the parental genetically modified Vero cell with a heterologous nucleic acid comprising a gD-1 encoding sequence, a minimal promoter or a modified minimal promoter, and a polyadenylation site, such that recombination and insertion of the gD-1 nucleic acid at the target locus occurs. In one aspect, the parental modified Vero cell comprises an attB sequence or attachment site at the target locus. A gD-1 sequence comprising an attP sequence or attachment site from a bacteriophage Bxbl, and an mRNA encoding a bacteriophage Bxbl integrase and a nuclear localization sequence are introduced such that the heterologous gD-1 nucleic acid is inserted at the target locus in the parental genetically modified Vero cell by sequence specific recombination between the Bxbl attB sequence in the genetically modified Vero cell and the attP sequence in the heterologous nucleic acid mediated by the Bxbl integrase, to produce the transgenic Vero cell. In one aspect, the Bxbl attP attachment site sequence is a 48 bp nucleic acid identified in SEQ ID NO:8. In one aspect, the parental modified Vero cell is a VerB::gDl.l clone 1529 generated from Vero cell line CCL81 which contains the attB sequence inserted into the Vero genome between positions 1529 and 1530 of the AAVS1 locus on chromosome 6. In another aspect, the parental modified Vero cell is a VerB::gD1.4 clone PPP1R12C-M12 generated from Vero cell line CCL81 which contains the attB sequence inserted into the Vero genome AAVS1 intron 1 of the protein phosphatase 1 regulatory subunit 12C (PPP1R12C). In another aspect, the parental modified Vero cell is a VerB::gD1.6C clone generated from Vero cell line CCL81. In another aspect, the gD-1 is the Use gene open reading frame from HSV-1 strain 17+, comprises or is the sequence identified as SEQ ID NO:9: 5’ATGGGGGGGGCTGCCGCCAGGTTGGGGGCCGTGATTTTGTTTGTCGTCATAGTG GGCCTCCATGGGGTCCGCGGCAAATATGCCTTGGTGGATGCCTCTCTCAAGATGG CCGACCCCAATCGCTTTCGCGGCAAAGACCTTCCGGTCCTGGACCAGCTGACCGA CCCTCCGGGGGTCCGGCGCGTGTACCACATCCAGGCGGGCCTACCGGACCCGTTC CAGCCCCCCAGCCTCCCGATCACGGTTTACTACGCCGTGTTGGAGCGCGCCTGCC GCAGCGTGCTCCTAAACGCACCGTCGGAGGCCCCCCAGATTGTCCGCGGGGCCT CCGAAGACGTCCGGAAACAACCCTACAACCTGACCATCGCTTGGTTTCGGATGG GAGGCAACTGTGCTATCCCCATCACGGTCATGGAGTACACCGAATGCTCCTACAA CAAGTCTCTGGGGGCCTGTCCCATCCGAACGCAGCCCCGCTGGAACTACTATGAC AGCTTCAGCGCCGTCAGCGAGGATAACCTGGGGTTCCTGATGCACGCCCCCGCGT TTGAGACCGCCGGCACGTACCTGCGGCTCGTGAAGATAAACGACTGGACGGAGA TTACACAGTTTATCCTGGAGCACCGAGCCAAGGGCTCCTGTAAGTACGCCCTCCC GCTGCGCATCCCCCCGTCAGCCTGCCTCTCCCCCCAGGCCTACCAGCAGGGGGTG ACGGTGGACAGCATCGGGATGCTGCCCCGCTTCATCCCCGAGAACCAGCGCACC GTCGCCGTATACAGCTTGAAGATCGCCGGGTGGCACGGGCCCAAGGCCCCATAC ACGAGCACCCTGCTGCCCCCGGAGCTGTCCGAGACCCCCAACGCCACGCAGCCA GAACTCGCCCCGGAAGACCCCGAGGATTCGGCCCTCTTGGAGGACCCCGTGGGG ACGGTGGCGCCGCAAATCCCACCAAACTGGCACATACCGTCGATCCAGGACGCC GCGACGCCTTACCATCCCCCGGCCACCCCGAACAACATGGGCCTGATCGCCGGC GCGGTGGGCGGCAGTCTCCTGGCAGCCCTGGTCATTTGCGGAATTGTGTACTGGA TGCGCCGCCACACTCAAAAAGCCCCAAAGCGCATACGCCTCCCCCACATCCGGG AAGACGACCAGCCGTCCTCGC ACC AGCCCTTGTTTTACTAG 3 ’

[0073] The complete HSV-1 genome has NCBI accession no. NC_001806.2. gD-1 encoding nucleic acids from other HSV-1 strains can be used, such as KOS GenBank accession # KT887224), B3xl.l (KU310657), B3xl.2 (KU310658), B3xl.3 (KU310659), B3xl.4(KU310660), B3xl.5 (KU310661), to name a few. HSV-1 gD and HSV-2 gD have 86% identity. In order to overcome this high identity, a gD sequence that is codon optimized for expression in human cells can be used. This procedure is based on Computer assisted algorithms that redistribute codons in a helper gene, thereby eliminating regions of homology, while enabling manipulation of factors such as codon-pair bias and CpG content to effectively titrate helper-gene protein levels (Li et al., 2017 -DOI: 10.1038/srep44404; Coleman et al., 2008 - doi: 10.1126/science.l 155761).

[0074] In another aspect, the minimal promoter is from HSV-1 strain 17+, having a sequence identified in SEQ ID NO: 1, or a modified minimal promoter having a sequence identified in SEQ ID NO:32. gD-1 minimal promoter encoding nucleic acids from other HSV-1 strains can be used, such as KOS GenBank accession # KT887224), B3xl.l (KU310657), B3xl.2 (KU310658), B3xl.3 (KU310659), B3xl.4(KU310660), B3xl.5 (KU310661), to name a few. The minimal promoter sequence (5’ non-transcribed gD region) contains: cap sites specifying the gD-1 mRNA 5’ terminus for cap-dependent initiation of mRNA translation), putative CCAAT and TATA boxes which play a role in determining the level and initiation points of transcription and the sequences necessary for early promoter activation which lie within 83 bp of the RNA cap-sites (Watson, 1983 Gene, 26:307-312; Everett, 1983 Nucleic Acid Research 11 (19):6647-6666; Jogger et al., 2004 Virology 318 (doi: 10.1016/j.virol.2003.10.004); Fan et al., 2017 (DOI: 10.1038/srep43712). Any part of the promoter can be altered or removed as long as gD-1 expression in Vero cells is retained.

[0075] Also provided is a method of producing a virion of a recombinant herpes simplex virus-2 (HSV-2), having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof and comprising an HSV-1 glycoprotein D on a lipid bilayer thereof, comprising infecting a cell comprising a heterologous nucleic acid encoding a HSV-1 glycoprotein D with a recombinant herpes simplex virus-2 (HSV-2) having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof under conditions permitting replication of the recombinant herpes simplex virus-2 (HSV-2) and recovering a HSV-2 virion comprising HSV-1 glycoprotein D on a lipid bilayer thereof produced by the cell.

[0076] In an embodiment, the cell expresses HSV-1 glycoprotein D on a membrane thereof. In one aspect, the cell is a transgenic cell comprising a gene encoding HSV-1 glycoprotein D. In one aspect, the cell is a recombinantly engineered cell comprising a heterologous nucleic acid encoding HSV-1 glycoprotein D and devoid of DNA sequences homologous to sequences present in the recombinant HSV-2 having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter. In another aspect, the engineered cell has minimally required DNA sequences for gD complementation permitting replication of the recombinant HSV-2. In yet another aspect, minimally required DNA sequences for gD complementation include the HSV-1 gD protein coding sequences, minimal promoter sequences or modified minimal promoter sequences, and a poly adenylation signal. In one aspect, the minimal promoter sequences comprises or is the sequence 5’ATCCCCTAAGGGGGAGGGGCCATTTTACGAGGAGGAGGGGTATAACAAAGTCT GTCTTTAAAAAGCAGGGGTTAGGGAGTTGTTCGGTCATAAGCTTCAGCGCGAAC GACCAACTACCCCGATCATCAGTTATCCTTAAGGTCTCTTTTGTGTGGTGCGTTCC GGT 3’, identified in SEQ ID NO: 1, based on the HSV-1 17+ strain, and described in Roger, J. Watson, 1983, Gene 26, 307-312. gD promoter sequences from other strains of HSV-1 can be used. In another aspect, the cell is a Vero cell. In yet another aspect, the engineered complementing cell is VerB::gDl, a Vero cell containing minimally required DNA sequences for gD complementation.

[0077] In another aspect, minimally required DNA sequences for gD complementation include the HSV-1 gD protein coding sequences, modified minimal promoter sequences, and a poly adenylation signal. In one aspect, the modified minimal promoter sequences comprises or is the sequence 5’CGAATCCCCTAAGGGGGAGGGGCCATTTTACGAGGAGGAGGGGTATAACAAA GTCTGTCTTTAAAAAGCAGGGGTTAGGGAGTTGTTCGGTCATAAGCTTCAGCGCG AACGACCAACTACCCCGATCATCAGTTATCCTTAAGGTCTCTTTTGTGTGGTGCG TTCCGGT 3’, identified in SEQ ID NO:32, based on the HSV-1 17+ strain. gD promoter sequences from other strains of HSV-1 can be used. In another aspect, the cell is a Vero cell. In yet another aspect, the engineered complementing cell is VerB::gD1.6C, a Vero cell containing minimally required DNA sequences for gD complementation with a modified minimal promoter.

[0078] Also provided is a recombinant nucleic acid having the same sequence as a genome of a wild-type HSV-2 except that the recombinant nucleic acid does not comprise a sequence encoding an HSV-2 glycoprotein D or its promoter. In an embodiment, the recombinant nucleic acid is a DNA. In an embodiment, the recombinant nucleic acid is an RNA.

[0079] HSV-2 gD is required for viral entry and cell-to-cell spread, deletion of gD-2 results in a single-cycle virus, unable to spread to other cells. This disclosure provides a method of propagating or producing a recombinant HSV-2 virus comprising a genome having a complete deletion of a glycoprotein D gene and its promoter, the method comprising: providing a transgenic Vero cell comprising at least one copy of a nucleic acid encoding HSV-1 glycoprotein, gD-1, a minimal promoter and a polyadenylation signal inserted in a target locus on a chromosome of the Vero cell genome, wherein the transgenic Vero cell expresses gD-1; contacting the transgenic Vero cell with the recombinant HSV-2 virus; and phenotypically complementing the recombinant HSV-2 virus with the gD-1 expressed by the transgenic Vero cell to propagate the single cycle HSV-2 virus. In one aspect the recombinant HSV-2 virus with a complete deletion of the gene encoding glycoprotein D and its promoter is AgD2P virus. In another aspect the transgenic Vero cell is a VerB::gDl cell. In yet another aspect, the transgenic Vero cell is VerB::gD1.6C. In one aspect, a method comprising VerB::gDl transgenic cells for propagating AgD2P virus for use in a pharmaceutical composition for preventing, treating, or eliciting an immune response in a subject against an infection with HSV-2, HSV-1 or a co-inf ection with HSV-1 and HSV-2. In another aspect, a method comprising VerB::gD1.6C transgenic cells for propagating AgD2P virus for use in a pharmaceutical composition for preventing, treating, or eliciting an immune response in a subject against an infection with HSV-2, HSV-1 or a co-infection with HSV-1 and HSV-2.

[0080] Also provided is a vaccine composition comprising the recombinant HSV-2 virus as described herein, or the virion as described herein. In one aspect, the recombinant HSV-2 virus or virion is AgD2P produced in VerB::gDl or VerB::gD1.6C complementing cell line. In an embodiment, the vaccine comprises an immunological adjuvant. In an embodiment, the vaccine does not comprise an immunological adjuvant. In an embodiment of the vaccine, compositions or pharmaceutical compositions described herein comprising a recombinant HSV-2, the HSV-2 is live.

[0081] Also provided is a composition comprising the recombinant HSV-2 virus as described herein, or the virion as described herein, wherein the genome of the virus or virion comprises at least a deletion of a second gene, wherein the second gene is necessary for HSV-2 viral replication or virulence.

[0082] A pharmaceutical composition comprising the recombinant HSV-2 virus as described herein, or the virion as described herein, and a pharmaceutically acceptable carrier.

[0083] In an embodiment, the composition or pharmaceutical composition or vaccine is formulated so that it is suitable for subcutaneous administration to a human subject. In an embodiment, the composition or pharmaceutical composition or vaccine is formulated so that it is suitable for intravaginal administration to a human subject. In an embodiment, the composition or pharmaceutical composition or vaccine is formulated so that it is suitable for intra-muscular, intra-nasal, or mucosal administration to a human subject. [0084] Also provided is a method of eliciting an immune response in a subject comprising administering to the subject an amount of (i) the recombinant HSV-2 virus as described herein; (ii) a virion thereof as described herein, (iii) a vaccine as described herein; (iv) a composition as described herein; or (v) a pharmaceutical composition as described herein, in an amount effective to elicit an immune response in a subject.

[0085] Also provided is a method of treating an HSV-2 infection in a subject or treating a disease caused by an HSV-1, HSV-2 or an HSV-1 and HSV-2 co-infection in a subject comprising administering to the subject an amount of (i) the recombinant HSV-2 virus as described herein; (ii) a virion thereof as described herein, (iii) the vaccine as described herein; (iv) a composition as described herein; or (v) a pharmaceutical composition as described herein, in an amount effective to treat an HSV-1, HSV-2 or co-infection or treat a disease caused by an HSV-1, HSV-2 or co-infection in a subject. In an embodiment, the methods comprise treating an HSV-1 or HSV-2 pathology caused by an HSV-1, HSV-2 or co-infection. In an embodiment of the methods, the disease caused by an HSV-1, HSV-2 or co-infection is a genital ulcer. In an embodiment of the methods, the disease caused by an HSV-1, HSV-2 or co-infection is herpes, oral herpes, herpes whitlow, genital herpes, eczema herpeticum, herpes gladiatorum, HSV keratitis, HSV retinitis, HSV encephalitis or HSV meningitis.

[0086] In an embodiment of the methods herein regarding treating, or vaccinating for, an HSV-1, HSV-2 or co-infection (i.e., infection with both HSV-1 and HSV-2), separate, individual, embodiments of treating an HSV-1 infection, treating an HSV-2 infection, treating a co-infection, vaccinating against an HSV-1 infection, vaccinating against an HSV-2 infection, and vaccinating against a co-infection, are each provided.

[0087] Also provided is a method of vaccinating a subject for HSV-1, HSV-2 or coinfection comprising administering to the subject an amount of (i) the recombinant HSV-2 virus as described herein; (ii) a virion thereof as described herein, (iii) the vaccine as described herein; (iv) a composition as described herein; or (v) a pharmaceutical composition as described herein, in an amount effective to vaccinate a subject for HSV-1, HSV-2 or coinfection.

[0088] Also provided is a method of immunizing a subject against HSV-1, HSV-2 or co-infection comprising administering to the subject an amount of (i) the recombinant HSV-2 virus as described herein; (ii) a virion thereof as described herein, (iii) a vaccine as described herein; (iv) a composition as described herein; or (v) a pharmaceutical composition as described herein, in an amount effective to immunize a subject against HSV-1, HSV-2 or coinfection.

[0089] In an embodiment of the methods, the subject is administered a subcutaneous or intravaginal priming dose and is administered a second dose subcutaneously or intravaginally. In an embodiment of the methods, the subject is administered as many subcutaneous or intravaginal priming doses to elicit anti-HSV antibodies and T cells.

[0090] Also provided is an isolated, recombinant herpes simplex virus-2 (HSV-2) having a deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof for treating or preventing an HSV-1, HSV-2 or co-inf ection in a subject. In an embodiment, the isolated, recombinant HSV-2 further comprises a herpes simplex virus- 1 (HSV-1) on a lipid bilayer thereof. In an embodiment of the isolated, recombinant HSV-2, the HSV-2 glycoprotein D-encoding gene is an HSV-2 Us6 gene. In one aspect, the recombinant HSV-2 virus is AgD2P propagated in a VerB::gDl complementing cell, e.g. VerB::gDl.l, VerB::gD1.4 or VerB::gD1.6C.

[0091] Also provided is a virion of an isolated, recombinant HSV-2 having a deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof for treating or preventing an HSV-1, HSV-2 or co-inf ection in a subject. In an embodiment, the virion further comprises an HSV-1 glycoprotein D on a lipid bilayer thereof. In an embodiment, the HSV-2 glycoprotein D-encoding gene is an HSV-2 Us6 gene. In one aspect, the HSV-2 virion is AgD2P propagated in a VerB::gDl complementing cell, e.g. VerB::gDl.l, VerB::gD1.4 or VerB::gD1.6C.

[0092] In an embodiment, of the virus or virion as described, the HSV-1, HSV-2 or co-infection causes a genital ulcer.

[0093] An isolated, recombinant herpes simplex virus-2 (HSV-2) is provided having a deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof. In one aspect, the recombinant HSV-2 is AgD2P propagated in a VerB::gDl complementing cell, e.g. VerB::gDl.l, VerB::gD1.4 or VerB::gD1.6C.

[0094] In an embodiment, the isolated, recombinant HSV-2 further comprises a surface glycoprotein on a lipid bilayer thereof which is a herpes simplex virus- 1 (HSV-1) glycoprotein D. In an embodiment, the isolated, recombinant HSV-2 further comprises a heterologous, non-HSV-2, antigen or surface glycoprotein on a lipid bilayer thereof. In an embodiment, the isolated, recombinant HSV-2 further comprises a bacterial antigen or surface glycoprotein on a lipid bilayer thereof. In an embodiment, the isolated, recombinant HSV-2 further comprises a parasitic antigen or surface glycoprotein on a lipid bilayer thereof, wherein the parasite is a parasite of a mammal.

[0095] In an embodiment, the heterologous antigen or surface glycoprotein is encoded by a transgene that has been inserted into the genome of the recombinant HSV-2. In an embodiment, the heterologous antigen or surface glycoprotein is present on a lipid bilayer thereof by way of infecting a cell with a recombinant HSV-2 having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter, wherein the cell is or has been transfected to express the heterologous antigen or surface glycoprotein on a cell membrane thereof, and wherein the recombinant HSV-2 comprising the heterologous antigen or surface glycoprotein present on a lipid bilayer is produced from the cell. In an embodiment, the viral antigen or glycoprotein is from a HIV, an enterovirus, a RSV, an influenza virus, a parainfluenza virus, pig corona respiratory virus, a rabies virus, a Lassa virus, a bunyavirus, a CMV, or a filovirus. In an embodiment, the glycoprotein is an HIV gpl20. In an embodiment, the filovirus is an ebola virus. In an embodiment, the virus is HIV, a M. tuberculosis, a chlamydia, Mycobacterium ulcerous, M. marinum, M. leprae, M. absenscens, Neisseria gonnorhea, or a Treponeme. In an embodiment, the Treponeme is Treponeme palidum.

[0096] Also provided is a virion of an isolated, recombinant HSV-2 having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof. In one aspect, the HSV-2 virion is AgD2P propagated in a VerB::gDl complementing cell, e.g. VerB::gDl.l, VerB::gD1.4 or VerB::gD1.6C.

[0097] In an embodiment, the virion of the isolated, recombinant HSV-2 further comprises a surface glycoprotein on a lipid bilayer thereof which is a herpes simplex virus- 1 (HSV-1) glycoprotein D. In an embodiment, the virion of the isolated, recombinant HSV-2 further comprises a non-HSV-2 viral surface glycoprotein on a lipid bilayer thereof. In an embodiment, the virion of the isolated, recombinant HSV-2 further comprises a heterologous bacterial antigen or bacterial surface glycoprotein on a lipid bilayer thereof. In an embodiment, the virion of the isolated, recombinant HSV-2 further comprises a heterologous parasitic antigen or parasitic surface glycoprotein on a lipid bilayer thereof, wherein the parasite is a parasite of a mammal. In an embodiment, the virion of the isolated, recombinant HSV-2 further comprises a heterologous viral antigen or viral surface glycoprotein on a lipid bilayer thereof. In an embodiment, the HSV-2 glycoprotein D-encoding gene is an HSV-2 Us6 gene. In an embodiment, the heterologous antigen or surface glycoprotein is encoded by a transgene that has been inserted into the genome of the recombinant HSV-2 of the virion. In an embodiment, the heterologous antigen or surface glycoprotein is present on a lipid bilayer thereof by way of infecting a cell with a recombinant HSV-2 having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter, wherein the cell is or has been transfected to express the heterologous antigen or surface glycoprotein on a cell membrane thereof, and wherein the recombinant HSV-2 comprising the heterologous antigen and/or surface glycoprotein present on a lipid bilayer is produced from the cell. In an embodiment, the virion has been recovered from such. In an embodiment, the heterologous antigen is a viral glycoprotein from a HIV, an enterovirus, a RSV, an influenza virus, a parainfluenza virus, Pig corona respiratory virus, a rabies virus, a Lassa virus, a bunyavirus, a CMV, a filovirus, or a combination thereof. In an embodiment, the glycoprotein is an HIV gpl20. In an embodiment, the filovirus is an ebola virus. In an embodiment, the virus is HIV, a M. tuberculosis, a chlamydia, Mycobacterium ulcerous, M. marinum, M. leprae, M. absenscens, Neisseria gonnorhea, or a Treponeme. In an embodiment, the Treponeme is Treponeme palidum.

[0098] Also provided is an isolated cell comprising therein a virus as described herein or a virion as described herein, wherein the cell is not present in a human being. In an embodiment of the cell, the cell comprises a heterologous nucleic acid encoding an HSV-1 glycoprotein D. In an embodiment of the cell, the cell expresses HSV-1 glycoprotein D on a membrane thereof. In another embodiment, the cell comprises nucleic acid sequences encoding HSV-1 glycoprotein D which, though produce a complementing glycoprotein D, are devoid of sequences homologous to the HSV-2 virus or virion as described herein. In one aspect the cell is VerB::gDl, VerB::gDl.l, VerB::gD1.4. In another aspect, the cell is VerB::gD1.6C.

[0099] Also provided is a method of producing a virion of a recombinant herpes simplex virus-2 (HSV-2), having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof and comprising a non-HSV-2 antigen or surface glycoprotein on a lipid bilayer thereof, comprising infecting a cell comprising a heterologous nucleic acid encoding the non-HSV-2 antigen or surface glycoprotein with a recombinant herpes simplex virus-2 (HSV-2) having a complete deletion of an HSV-2 glycoprotein D- encoding gene and its promoter in the genome thereof under conditions permitting replication of the recombinant herpes simplex virus-2 (HSV-2) and recovering a recombinant HSV-2 virion comprising a non-HSV-2 antigen or surface glycoprotein on a lipid bilayer thereof produced by the cell. [0100] In an embodiment of the cell, the HSV-1 glycoprotein D is encoded by a heterologous nucleic acid, which heterologous nucleic acid is an HSV-1 glycoprotein D gene, or is a nucleic acid having a sequence identical to an HSV-1 glycoprotein D gene. In another embodiment of the cell, the heterologous nucleic acid encoding HSV-1 glycoprotein D gene is devoid of sequences that can undergo homologous recombination with the HSV-2 virus or virion with a deletion of the glycoprotein gene and its promoter as described herein. In one aspect, the cell is VerB::gDl, e.g. VerB::gDl.l, or VerB::gD1.4. In another aspect, the cell is VerB::gD1.6C.

[0101] A vaccine composition comprising a virus as described herein, or a virion as described herein. In one aspect, the vaccine composition comprises recombinant HSV-2 virus AgD2P propagated in a VerB::gDl complementing cell, e.g. VerB::gDl.l, VerB::gD1.4 or VerB::gD1.6C, or a combination thereof.

[0102] In an embodiment of the vaccine composition, the vaccine composition comprises an immunological adjuvant.

[0103] Also provided is a composition comprising a virus as described herein, or a virion as described herein, wherein the genome of the virus or virion comprises at least a deletion of a second gene, wherein the second gene is necessary for HSV-2 viral replication. In an embodiment, the composition comprises serum from, or is derived from serum from, a mammal into which the virus or virion has been previously introduced so as to elicit an immune response.

[0104] Also provided is pharmaceutical composition comprising a virus as described herein, or a virion as described herein, and a pharmaceutically acceptable carrier.

[0105] Also provided is a method of eliciting an immune response in a subject comprising administering to the subject an amount of (i) a virus as described herein; (ii) a virion as described herein, (iii) a vaccine as described herein; (iv) a composition as described herein; or (v) a pharmaceutical composition as described herein, in an amount effective to elicit an immune response in a subject.

[0106] Also provided is a method of treating 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 (i) a virus as described herein; (ii) a virion as described herein, (iii) a vaccine as described herein; (iv) a composition as described herein; or (v) a pharmaceutical composition as described herein, in an amount effective to treat an HSV-2 infection or treat a disease caused by an HSV-2 infection in a subject. [0107] Also provided is a method of vaccinating a subject for HSV-2 infection comprising administering to the subject an amount of (i) a virus as described herein; (ii) a virion as described herein, (iii) a vaccine as described herein; (iv) a composition as described herein; or (v) a pharmaceutical composition as described herein, in an amount effective to vaccinate a subject for HSV-2.

[0108] Also provided is a method of immunizing a subject against HSV-2 infection comprising administering to the subject an amount of (i) a virus as described herein; (ii) a virion as described herein, (iii) a vaccine as described herein; (iv) a composition as described herein; or (v) a pharmaceutical composition as described herein, in an amount effective to immunize a subject against HSV-2.

[0109] HSV-2 and HSV-1 diseases are known in the art and are also described herein. Both treatment and prevention of HSV-2 and HSV-1 diseases are each separately encompassed as well as treatment or prevention of a HSV-2 and HSV-1 co-infection. Prevention is understood to mean amelioration of the extent of development of the relevant disease or infection in a subject treated with the virus, virion, vaccine or compositions described herein, as compared to an untreated subject.

[0110] Also provided is a method of producing a virion of a recombinant herpes simplex virus-2 (HSV-2), having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof and comprising an HSV-1 glycoprotein D on a lipid bilayer thereof, comprising infecting a cell comprising a heterologous nucleic acid encoding a HSV-1 glycoprotein D with a recombinant herpes simplex virus-2 (HSV-2) having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof under conditions permitting replication of the recombinant herpes simplex virus-2 (HSV-2) and recovering a recombinant HSV-2 virion comprising an HSV-1 glycoprotein D on a lipid bilayer thereof produced by the cell.

[0111] Also provided is an isolated, recombinant herpes simplex virus-2 (HSV-2) having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof for treating or preventing an HSV-2 infection in a subject.

[0112] Also provided is an isolated, recombinant herpes simplex virus-2 (HSV-2) having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof for treating or preventing an HSV-1 infection in a subject.

[0113] Also provided is a virion of an isolated, recombinant HSV-2 having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof for treating or preventing an HSV-2 infection in a subject. [0114] Also provided is a method of treating an HSV-1 infection, or HSV-1 and HSV-2 co-inf ection, in a subject, or treating a disease caused by an HSV-2 infection or HSV- 1 and HSV-2 co-infection in a subject comprising administering to the subject an amount of (i) a virus as described herein; (ii) a virion as described herein, (iii) a vaccine as described herein; (iv) a composition as described herein; or (v) a pharmaceutical composition as described herein, in an amount effective to treat an HSV-2 infection or treat a disease caused by an HSV-2 infection in a subject or an amount effective to treat an HSV-1 and HSV-2 coinfection or treat a disease caused by an HSV-1 and HSV-2 co-infection in a subject.

[0115] Also provided is a method of vaccinating a subject for an HSV-1 infection, or HSV-1 and HSV-2 co-infection, comprising administering to the subject an amount of (i) a virus as described herein; (ii) a virion as described herein, (iii) a vaccine as described herein; (iv) a composition as described herein; or (v) a pharmaceutical composition as described herein, in an amount effective to vaccinate a subject for an HSV-1 infection, or HSV-1 and HSV-2 co-infection.

[0116] Also provided is a method of immunizing a subject against an HSV-1 infection, or HSV-1 and HSV-2 co-infection, comprising administering to the subject an amount of (i) a virus as described herein; (ii) a virion as described herein, (iii) a vaccine as described herein; (iv) a composition as described herein; or (v) a pharmaceutical composition as described herein, in an amount effective to immunize a subject against an HSV-1 infection, or HSV-1 and HSV-2 co-infection.

[0117] A method is provided of eliciting an immune response in a first subject against an HSV-2 and/or HSV-1 infection, comprising effectuating passive transfer to the first subject of an amount of a product from a pregnant female immunized with HSV-2 having a complete deletion of the entire HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof an wherein said HSV-2 is phenotypically complemented with a herpes simplex virus- 1 (HSV-1) glycoprotein D by propagating said HSV-2 in a complementing cell expressing said HSV-1 glycoprotein D, wherein the product comprises antibodies induced thereby, effective to elicit an immune response against an HSV-2 and/or HSV-1 infection in the first subject, wherein the first subject is a fetus or neonate. In one aspect, the pregnant female receives a HSV-2 composition comprising recombinant HSV-2 virus AgD2P propagated in a VerB::gDl complementing cell, e.g. VerB::gDl.l, VerB::gD1.4 or VerB::gD1.6C.

[0118] In embodiments, the product comprises serum of the pregnant female. [0119] In embodiments, the product comprises breast milk of the pregnant female.

[0120] In embodiments, the first subject is a fetus.

[0121] In embodiments, the first subject is a neonate.

[0122] In embodiments, the first subject is bom from a second subject.

[0123] In embodiments, the pregnant female is pregnant with the fetus.

[0124] In embodiments, the first subject and the pregnant female are human.

[0125] In embodiments, the product comprises antibodies elicited by immunization of the pregnant female with the HSV-2 having the deletion. In embodiments, the product comprises FcyRIV-activating antibodies. In embodiments, the product comprises IgGl and IgG2 antibodies. In embodiments, the product comprises anti-HSV-1 IgG or anti-HSV-2 IgG. In one aspect, the HSV-2 having the deletion comprises recombinant HSV-2 virus AgD2P propagated in a VerB::gDl complementing cell, e.g. VerB::gDl.l, VerB::gD1.4 or VerB::gD1.6C.

[0126] In embodiments, the method elicits an immune response in the first subject against an HSV-2.

[0127] In embodiments, the method elicits an immune response in the first subject against an HSV-1.

[0128] In embodiments, the product further comprises an immunological adjuvant.

[0129] In embodiments, the product further comprises an HSV-2 having a complete deletion of the HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof and wherein said HSV-2 is phenotypically complemented with a herpes simplex virus- 1 (HSV-1) glycoprotein D by propagating said HSV-2 in a complementing cell expressing said HSV-1 glycoprotein D. In one aspect, the HSV-2 having a deletion of the entire HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof comprises recombinant HSV-2 virus AgD2P and the complementing cell is a VerB::gDl complementing cell, e.g.

VerB::gDl.l, VerB::gD1.4 or VerB::gD1.6C.

[0130] A method is provided of inhibiting a perinatal HSV-1 and/or HSV-2 infection in a neonate comprising administering to a female pregnant with a fetus which will become the neonate an amount of an HSV-2 having a complete deletion of the HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof and wherein said HSV-2 is phenotypically complemented with a herpes simplex virus- 1 (HSV-1) glycoprotein D by propagating said HSV-2 in a complementing cell expressing said HSV-1 glycoprotein D, effective to inhibit a perinatal HSV-1 and/or HSV-2 infection in a neonate. In one aspect, the HSV-2 having a complete deletion of the HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof comprises recombinant HSV-2 virus AgD2P and the complementing cell is a VerB::gDl complementing cell, e.g. VerB::gDl.l, VerB::gD1.4 or VerB::gD1.6C.

[0131] A method is provided of inhibiting HSV-1 and/or HSV-2 viral dissemination from a mother to her neonate comprising administering to the mother an amount of an HSV-2 having a complete deletion of the HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof and wherein said HSV-2 is phenotypically complemented with a herpes simplex virus- 1 (HSV-1) glycoprotein D, effective to inhibit HSV-1 and/or HSV-2 viral dissemination from a mother to her neonate. In one aspect, the HSV-2 having a complete deletion of the HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof comprises recombinant HSV-2 virus AgD2P and the complementing cell is a VerB::gDl complementing cell, e.g. VerB::gDl.l, VerB::gD1.4 or VerB::gD1.6C.

[0132] In embodiments, the mother is pregnant with a fetus which will become the neonate.

[0133] In embodiments, the HSV-1 infection and/or HSV-1 viral dissemination is inhibited.

[0134] In embodiments, the HSV-2 infection and/or HSV-2 viral dissemination is inhibited.

[0135] In embodiments, an immune response is elicited against HSV-1.

[0136] In embodiments, an immune response is elicited against HSV-2.

[0137] In embodiments, the complementing cell is a VerB::gDl cell.

[0138] In embodiments, the complementing cell is a VerB::gD1.6C cell.

[0139] In embodiments, the pregnant female, the mother or the second subject is seronegative for HSV-1, seronegative for HSV-2, or seronegative for both HSV-1 and HSV- 2.

[0140] In embodiments, the pregnant female or the mother is subcutaneously administered the HSV-2 having a complete deletion of the HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof. In one aspect, the HSV-2 having a complete deletion of the HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof comprises recombinant HSV-2 virus AgD2P and the complementing cell is a VerB::gDl complementing cell, e.g. VerB::gDl.l, VerB::gD1.4 or VerB::gD1.6C. [0141] In embodiments, the pregnant female or the mother is administered a subcutaneous boost does of the HSV-2 having a complete deletion of the HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof after an initial subcutaneous administration of the HSV-2 having a complete deletion of the HSV-2 glycoprotein D- encoding gene and its promoter. In one aspect, the HSV-2 having a complete deletion of the HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof comprises recombinant HSV-2 virus AgD2P and the complementing cell is a VerB::gDl complementing cell, e.g. VerB::gDl.l, VerB::gD1.4 or VerB::gD1.6C.

[0142] In an embodiment of the methods herein for immunizing, vaccinating or eliciting an immune response, passive transfer of the virion or virus or the antibodies or immune factors induced thereby may be effected from one subject to another. The relevant product may be treated after obtention from one subject before administration to a second subject. In a preferred embodiment of the inventions described herein, the subject is a mammalian subject. In an embodiment, the mammalian subject is a human subject.

[0143] A method is provided of eliciting an immune response in a first subject against an HSV-2 and/or HSV-1 infection, comprising passive transfer to the subject of an amount of a product from a second subject immunized with HSV-2 having a complete deletion of the HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof an wherein said HSV-2 is phenotypically complemented with a herpes simplex virus- 1 (HSV-1) glycoprotein D by propagating said HSV-2 in a complementing cell expressing said HSV-1 glycoprotein D, wherein the product comprises the HSV-2 or the antibodies or immune factors induced thereby, effective to elicit an immune response against the HSV-2 and/or HSV-1 infection. In one aspect, the HSV-2 having a complete deletion of the HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof comprises recombinant HSV-2 virus AgD2P and the complementing cell is a VerB::gDl complementing cell, e.g. VerB::gDl.l, VerB::gD1.4 or VerB::gD1.6C.

[0144] In embodiments, the product comprises serum of the second subject.

[0145] In embodiments, the second subject is a pregnant female.

[0146] In embodiments, the first subject is a fetus or neonate. In embodiments, the second subject is pregnant with the first subject.

[0147] In embodiments, the product comprises antibodies elicited by immunization of the second subject with the HSV-2. [0148] Also provided is an isolated, recombinant herpes simplex virus-2 (HSV-2) having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof and further comprising a heterologous antigen of a pathogen. In an embodiment, the heterologous antigen is a protein, peptide, polypeptide or glycoprotein. In an embodiment, the heterologous antigen is a heterologous antigen with respect to HSV-2, but is an antigen found on or in the relevant “pathogen.” Pathogens, viral, bacterial or parasitic, are described herein. In an embodiment, the pathogen is a bacterial pathogen of a mammal, a viral pathogen of a mammal, or a parasitic pathogen of a mammal. In an embodiment, the antigen or the transgene encoding the pathogen is not actually taken or physically removed from the pathogen, but nevertheless has the same sequence as the pathogen antigen or encoding nucleic acid sequence. In an embodiment, the isolated, recombinant HSV-2 comprises a heterologous antigen of a pathogen on a lipid bilayer thereof. In an embodiment of the isolated, recombinant HSV-2, the pathogen is bacterial, parasitic, or viral. In an embodiment, the HSV-2 glycoprotein D-encoding gene is an HSV-2 US6 gene. In one aspect, the HSV-2 having a complete deletion of the HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof comprises recombinant HSV-2 virus AgD2P and the complementing cell is a VerB::gDl complementing cell, e.g. VerB::gDl.l, VerB::gD1.4 or VerB::gD1.6C. In an embodiment, the isolated, recombinant HSV-2, the heterologous antigen is encoded by a transgene that has been inserted into the genome of the recombinant HSV-2.

[0149] Also provided is a method of inducing antibody dependent cell mediated cytotoxicity (ADCC) against HSV-2 or HSV-1 in a subject comprising administering to the subject an isolated, recombinant herpes simplex virus-2 (HSV-2) having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof and further comprising a heterologous HSV-1 antigen or surface glycoproteins on a lipid bilayer thereof in an amount effective to induce antibody dependent cell mediated cytotoxicity (ADCC) against an antigenic target. In one aspect, the heterologous HSV-1 antigen is HSV-1 gD. In one aspect, the recombinant HSV-2 having a deletion of the entire HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof comprises recombinant HSV-2 virus AgD2P and the complementing cell is a VerB::gDl complementing cell, e.g. VerB::gD1.4 or VerB::gD1.6C.

[0150] Also provided is a method of inducing antibody dependent cell mediated cytotoxicity (ADCC) against a heterologous antigenic target in a subject comprising administering to the subject an isolated, recombinant herpes simplex virus-2 (HSV-2) having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof and further comprising the heterologous antigen on a lipid bilayer thereof in an amount effective to induce antibody dependent cell mediated cytotoxicity (ADCC) against the antigenic target. In one aspect, the recombinant HSV-2 having a deletion of the entire HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof comprises recombinant HSV-2 virus AgD2P and the complementing cell is a VerB::gDl complementing cell, e.g. VerB::gD1.4 or VerB::gD1.6C.

[0151] In one aspect, recombinant HSV-2 AgD2P (HSV-2 AgD2P ^{/+gD 1} is HSV-2 with a complete deletion of the nucleic acid encoding glycoprotein D gene and its promoter, produced in a complementing cell expressing HSV-1 glycoprotein D, and HSV-2 AgD2P-/- is HSV-2 with a complete deletion of the nucleic acid encoding glycoprotein D gene and its promoter when isolated from non-complementing cells) expressing the appropriate transgenes will selectively induce antibodies and cellular immune responses that protect against skin or mucosal infections by pathogens.

[0152] In an embodiment, the heterologous antigen is a surface antigen.

[0153] In an embodiment, the transgene encodes an antigen from an HIV, a M. tuberculosis, a chlamydia, Mycobacterium ulcerous, M. marinum, M. leprae, M. absenscens, Neisseria gonnorhea, a Treponeme, or a combination thereof. In an embodiment, the Treponeme is Treponeme palidum. In an embodiment, the transgene is a M. tuberculosis biofilm-encoding gene. In an embodiment, the transgene is an HIV gpl20-encoding gene.

[0154] In an embodiment, the heterologous antigen is a surface antigen of the antigenic target. In an embodiment, the heterologous antigen is a parasite antigen. In an embodiment, the heterologous antigen is a bacterial antigen or a viral antigen.

[0155] In an embodiment, the antigenic target is a virus and is a Lassa virus, a human immunodeficiency virus, an RSV, an enterovirus, an influenza virus, a parainfluenza virus, pig corona respiratory virus, a lyssavirus, a bunyavirus, or a filovirus.

[0156] In an embodiment, the antigenic target is a bacteria and is Mycobaterium tuberculosis, M. ulcerous, M. marinum, M. leprae, M. absenscens, Chlamydia trachomatis, Neisseria gonorrhoeae or Treponema pallidum.

[0157] In an embodiment, the isolated, recombinant HSV-2 transgene is a M. tuberculosis biofilm-encoding gene or wherein the transgene is an HIV gp!20-encoding gene. [0158] In a preferred embodiment of the methods described herein, the subject is a human. In an embodiment of the methods described herein, the subject has not yet been infected with HSV-1, HSV-2 or co-inf ection. In an embodiment of the methods described herein, the subject has been infected with HSV-1, HSV-2 or co-inf ection.

[0159] As described herein, a co-inf ection means a co-inf ection with HSV-1 and HSV-2.

[0160] All combinations of the various elements described herein are within the scope of the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0161] This invention will be better understood from the Experimental Details, which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.

EXAMPLES

EXAMPLE 1- Generation of HSV-2 AgD2P.

[0162] Herein a genetically engineered complete deletion mutant of the gD (Us6) gene of HSV-2 and its promoter is disclosed. The entire open reading frame (ORF) of the gD gene and its promoter in a 40 kb region of HSV-2 G DNA that contains entire Usi to Usi2 ORFs was first deleted by partially digesting HSV-2 G DNA with Sau3A, selecting for fragments around 40 kb, and cloning these fragments into the unique Bglll site of cosmid pYUB328 (Balasubramanian et al., J. Bacterial. 178,273-9, 1996). pYUB328 was designed to contain two Pad restriction enzyme sites flanking the Bglll site. Pad recognizes the AT -rich sequence TTAAT/TAA which is absent in the GC-rich sequences of herpesviruses. Digestion of cosmids made from these DNAs release the vector from 40 kb insert fragments providing recombinogenic fragments for genetic manipulation of HSV genome. The HSV-2 G DNA library was screened and pYUB2156 was identified as a fragment containing the genes Usi to Usi2 (FIG. 1A) as the recombinogenic substrate.

[0163] To delete the entire ORF of gD-2 and its promoter, the recombineering system first developed by Don Court (Yu et al., PNAS USA 97, 5978-83) was used. This system allows the precise insertion of a double- stranded PCR product into a plasmid or cosmid in E. coli. pYUB2156 was first transformed into the recombineering strain DY331 [Genotype AlacU169 gal490 pglA8 [/. cI857 (cro-bioA)] (srlA-recA)301::Tnl0')]. The PCR product that allowed the removal of gD-2 and its promoter and the insertion of a Swal-flanked kanamycin-resistance gene (kan) was generated by performing PCR on pMV306 (Addgene #26155). The primer pMV306K_1683-gD2 (see Table 1 below) links the 3’end of Uss to the 5’ end of kanamycin gene and the primer pMV306K_2950c-gD2 links the 3’end of kanamycin gene to the 5 ’end of Us7- The resulting DNA fragment was electroporated into DY331 containing pYUB2156 using the recombineering protocol. Recombinant plasmids with the deletion gD-2 deletion were selected for as kanamycin-resistant transformants. One representative plasmid pBRLl 170 was sequence verified to have the Swal-flanked kananycin gene inserted where gD (Use) had previously been in pYUB2156. Swal is another restriction enzyme that recognizes the AT-rich sequence AAT^ATTA that is not found in HSV-2 DNA or pYUB2156. Digestion of pBRLl 170 with Swal and recircularization yielded pBRLl 171 (FIG. IB) which was sequenced verified to contain the deletion of gD-2 and its promoter with the insertion of a Swal site. The pBRLl 171 was digested with PacI and these linear DNAs were electroprated with AgD-2::RFP DNA (Kaugars et al., PNAS USA 118:e2110714118). The AgD-2::RFP DNA had been made from the AgD-2::GFP construct (Petro et al., Elife 4, 2015; Petro et al., JCI Insight 1, 2016; Cheshenko et al., FASEB J. 27, 2584-99, 2013).

EXAMPLE 2 - VerB::gDl complementing cell generation.

[0164] The complementation component in this VerB::gDl cell line shared no sequence homology with the AgD2P virus but still expressed gDl at high levels using a minimal gD- 1 promoter and a polyadenylation signal sequence.

TABLE 1: PRIMER SEQUENCES

[0165] Vero-attB Parental Cell Line(s): As described further below, the parental cell lines, Vero cell lines VerB-al and VerB-o2, assessed for construction of VerB::gDl were recombinantly engineered. These Vero cell lines, derived from Vero cells (ATCC® CCL- 81™), were recombinantly engineered to contain a site-specific recombination sequence, attB (attachment site) which is a 38 bp sequence: 5’ GGCTTGTCGACGACGGCGGTCTCCGTCGTCAGGATCAT-3’ (SEQ ID NO:7) derived from Mycobacterium smegmatis. The attachment site was originally found to allow sitespecific integration into the genomic DNA of bacteriophage DNA that contains the 48 bp attachment sequence (attP) mediated by the Bxbl integrase. The utility of this system has been exploited to include integration of any exogenous DNA that contains an attP site (Figure 3, Step 1).

[0166] The VerB-al is a Vero (CCL-81™) cell line which contains the 38 bp attB sequence inserted into the Vero genome AAVS1 site 1529 (Described in US2022-0090146). The VCI'B-G2 is a Vero (CCL-81™) cell line which contains the attB sequence inserted into Vero genome AAVS1 intron 1 of PPP1R12C (Protein Phosphatase 1 Regulatory Subunit 12C) (Described in US2022-0090146).

[0167] Plasmid Vector Constructions: The attP-shuttle plasmid (pMAEl, FIG. 4B) was designed and synthesized (BioBasic™, Ontario, Canada) as a minimal plasmid to contain the Bxbl attP sequence

5 ’ -GGTTTGTCTGGTC AACC ACCGCGGTCTCAGTGGTGTACGGTAC AAACC-3 ’ (SEQ ID NO: 8), an upstream CMV promoter (pMAE7, FIG. 4C), multiple cloning site (MCS), and ampicillin selection marker.

[0168] A shuttle plasmid (pMAECL20, FIG. 4D, SEQ ID NO: 15) which contains the gD-1 expression cassette comprising the gD-1 minimal promoter, the gD-1 coding sequence and the attP attachment site was constructed. Specifically, the Us6 ORF sequence, encoding the native HSV-1 glycoprotein D (gD-1) was PCR amplified from the HSV-1 strain 17+ viral genome (see primers in Table 2) and inserted into pMAE7 downstream to the CMV promoter and in frame with the bGHpA element by NEBuilder®HiFi DNA Assembly (New England Biolabs®, Ipswitch, MA), generating pMAECLl (Figure 4B). Then, the minimal promoter sequence of gD-1 was PCR amplified from the HSV-1 strain 17+ viral genome (GenBank accession number JN555585) and cloned by NEBuilder®HiFi DNA Assembly (New England Biolabs®, Ipswitch, MA) into pMAECLl, replacing the CMV promoter and generating pMAECL17 (Figure 4C). The minimal promoter sequence (5’ non-transcribed gD region) contains: cap sites specifying the gD-1 mRNA termini (mRNA translation) putative CCAAT and TATA boxes (role in determining the level and initiation points of transcription). The sequences necessary for Early promoter activation lie within 83 bp of the RNA cap-sites. gDl minimal promoter sequence:

5’ATCCCCTAAGGGGGAGGGGCCATTTTACGAGGAGGAGGGGTATAACAAAGTCT GTCTTTAAAAAGCAGGGGTTAGGGAGTTGTTCGGTCATAAGCTTCAGCGCGAAC GACCAACTACCCCGATCATCAGTTATCCTTAAGGTCTCTTTTGTGTGGTGCGTTCC

GGT3’ (SEQ ID NO:1) (Watson, 1983 Gene, 26:307-312; Everett, 1983 Nucleic Acid Research 11 (19):6647-6666; Jogger et al., 2004 Virology 318

(doi: 10.1016/j.virol.2003.10.004); Fan et al., 2017 (DOI: 10.1038/srep43712). To avoid the presence of antibiotic resistance gene in the attP-shuttle vector which will be used to integrate the gD-1 expression cassette into the cellular genome, the ampicillin gene was substituted with the histidinol dehydrogenase (hisD) cassette from Salmonella typhimurium. The hisD (histidinol dehydrogenase) gene sequence is derived from Salmonella enterica subsp. enterica serovar Typhimurium str. LT2. Gene ID: 125593, Genomic Sequence: NC_003197.2; McClelland M, et al. Nature, 2001 Oct 25. PMID 11677609. hisD GENE SEQUENCE (SEQ ID NO: 16)

5’ATGAGCTTCAATACCCTGATTGACTGGAACAGCTGTAGCCCTGAACAGCAGCG TGCGCTGCTGACGCGTCCGGCGATTTCCGCCTCTGACAGTATTACCCGGACGGTC AGCGATATTCTGGATAATGTAAAAACGCGCGGTGACGATGCCCTGCGTGAATAC AGCGCTAAATTTGATAAAACAGAAGTGACAGCGCTACGCATTACCCCTGAAGAG ATCGCCGCCGCCGGCGCGCGTCTGAGCGACGAATTAAAACAGGCGATGGCCGCT GCCGTCAAAAATATTGAAACGTTCCATTCCGCGCAGACGCTACCGCCTGTAGATG TGGAAACCCAGCCAGGCGTGCGTTGCCAGCAGGTTACGCGTCCCGTCGCGTCTGT CGGTCTGTATATTCCCGGCGGCTCGGCTCCGCTCTTCTCAACGGTGCTGATGCTG GCGACGCCGGCGCGTATTGCGGGATGTCAGAACGTGGTTCTGTGCTCGCCGCCGC CCATCGCTGATGAAATCCTCTATGCGGCGCAACTGTGTGGCGTGCAGGAAATCTT TAACGTCGGCGGCGCGCAGGCGATTGCCGCTCTGGCCTTCGGCAGCGAGTCCGT ACCGAAAGTGGATAAAATTTTTGGCCCCGGCAACGCCTTTGTAACCGAAGCCAA GCGTCAGGTCAGCCAGCGCCTCGACGGCGCGGCTATCGATATGCCAGCCGGGCC GTCTGAAGTACTGGTGATAGCCGACAGCGGCGCAACACCGGATTTCGTCGCGTCT GACCTGCTCTCCCAGGCTGAGCACGGTCCGGATTCCCAGGTGATTCTGCTGACGC CTGATGCTGACATTGCCCGCAAGGTGGCGGAGGCGGTAGAACGTCAACTGGCGG AGCTGCCGCGCGCGGACACCGCCCGGCAGGCCCTGAGCGCCAGTCGTCTGATTG TGACCAAAGATTTAGCGCAGTGCGTCGCCATCTCTAATCAGTATGGGCCGGAAC ACTTAATCATCCAGACGCGCAATGCGCGCGATTTGGTGGATGCGATTACCAGCGC AGGCTCGGTATTTCTCGGCGACTGGTCGCCGGAATCCGCCGGTGATTACGCTTCC GGAACCAACCATGTTTTACCGACCTATGGCTATACTGCTACCTGTTCCAGCCTTG GGTTAGCGGATTTCCAGAAACGGATGACCGTTCAGGAACTGTCGAAAGCGGGCT TTTCCGCTCTGGCATCAACCATTGAAACATTGGCGGCGGCAGAACGTCTGACCGC CC ATAAAAATGCCGTGACCCTGCGCGTAAACGCCCTCAAGGAGC AAGCATGA 3 ’

[0169] The hisD cassette contains the histidinol dehydrogenase gene under the control of both the eukaryotic EF-la and the prokaryotic EM7 promoters. The hisD cassette was PCR amplified from the pBRLl 153 plasmid and cloned by NEBuilderOHiFi DNA Assembly (New England Biolabs®, Ipswitch, MA) into pMAECL17, replacing the ampicillin cassette and generating pMAECL20 (Figure 4D). The pMAECL20 plasmid sequence was verified by Sanger sequence analysis and is identified in SEQ ID NO: 15.

TABLE 2. LIST OF PRIMERS USED TO CONSTRUCT THE ATTP-SHUTTLE

PLASMID PMAECL20

[0170] Expression of the gD-1 protein was verified by immunofluorescence staining. Vero cells were seeded in 24- well plate (IxlO⁵ cells/well) and transfected with the pMAECL20 plasmid (1 pg/well) using Lipofectamine™ 3000 (Invitrogen™, Waltham, MA, manufacturer’s protocol). At 24 h post-transfection, cells were infected with AgD2::RFP at multiplicity of infection (MOI) 0.1 PFU or mock infected. Infected cells were incubated for Ih at 37°C, and virus inoculum removed and DMEM 2% FBS added to the cells. At 48 h post-infection, cells were fixed with 4% paraformaldehyde for 5 min at room temperature (RT), permeabilized with 0.2% Triton™ -X 100 in PBS for 10 min at RT, and incubated with a primary mouse antibody (dilution 1:500) against HSV-gD (sc69802, Santa Cruz Biotechnology®, Dallas, TX) in 3% BSA-PBS for 16 h at 4°C. Then, cells were washed with PBS and incubated with the secondary antibody AlexaFluor® 488 (AF488) goat anti-mouse IgG (dilution 1: 1000) (cat#A32723, Invitrogen™, Waltham, MA) for 1 h at RT. Images were captured using the EVOS™ 5000 Imaging System (Invitrogen™, Waltham, MA). The gD-1 promoter is transactivated only upon HSV infection in the presence of the immediate early ICP4 HSV protein (FIG. 5) (Xiao et al., 1997, Journal of Virology 71 (3): 1757-1765).

[0171] Transfection and Integration of gD-1 shuttle plasmid into VerB-attB cells: VerB-al and VcrB-c2 cell lines were seeded in 6-well plates (2xl0⁶ cells/well) in growth media (DMEM 5% FBS). Cells were transfected with the shuttle plasmid pMAECL20 (2 pg/well), containing the gD-1 expression cassette, using Lipofectamine™ 2000 (Invitrogen™, Waltham, MA, manufacturer’s protocol) and followed by co-transfection with the Bxbl integrase mRNA (1 pg/well), using RNAfection™ (System Biosciences®, Palo Alto, CA, manufacturer’s protocol). The Bxbl integrase mRNA was synthesized using the mRNA Express kit (SBI, catalog# MR-KIT-1, System Biosciences®, Palo Alto, CA), following manufacturer’s protocol.

[0172] Selection of VerB::gDl cells: At 3 days after transfection, cells were passaged at a 1: 10 split ratio into several 6-well plates in selection media (DMEM without histidine, 5% FBS, 0.5 mM I-histidinol). Selection of resistant cells, VerB::gDl (Vero cells that have integrated the gD-1 expression cassette into the attB site present in the cellular genome), occurred during 3 weeks with selection media exchanged every 3 days. Single cell colonies were isolated using cloning rings and subsequently expanded in selection media into separated dishes until reaching confluence. The VerB::gDl cell clones were selected for complementation and production yields of AgD2P virus.

[0173] Screening of VerB::gDl cell clones: VerB::gDl cell clones were seeded in 24- well plates (IxlO⁵ cells/well) in growth media (DMEM 5% FBS). On the next day, cells were infected with AgD2::RFP (HSV-2 virus with a deletion of the gD gene and insertion of the Red Fluorescence Protein gene) virus at an MOI of 0.01 plaque forming unit (PFU) for Ih at 37°C. Virus inoculum was removed and DMEM 2% FBS was added to the cells. Infected cells were imaged daily and cytopathic effect (CPE) characteristic of HSV infection was recorded. The VerB::gDl cell clones were selected for complementation and production yields of AgD2P virus.

[0174] Four VerB::gDl cell clones (Al, A2, A3, A4) derived from the VerB-o2 cell line were selected based on more robust complementation of AgD2::RFP virus production (FIG. 6). These cell clones were subsequently adapted to serum-free medium (VP-SFM Thermo Fisher Scientific®, Waltham, MA) supplemented with 2mM Glutamax™ (Thermo Fisher Scientific®, Waltham, MA).

[0175] VerB::gDl Characterization: The growth properties of AgD2P virus in VerB::gDl cells is described below for virus growth curve, productivity, and phenotype.

[0176] AgD2P virus Growth Curve: VerB::gDl cells and VD60 cells were seeded in 12-well plate (2xl0⁵ cells/well). On the next day, cells were infected with AgD2P virus at MOI 0.1 PFU. Infected cells were harvested at different time points after infection (0, 8, 16, 24, 48, 72 hpi). Infected cells were lysed by 3 cycles of freeze and thaw and cell-associated virus collected. AgD2P virus titers were determined by plaque assay in VD60 cells (Figure 7A, B).

[0177] AgD2P virus produced in VerB::gDl cells grows at a slower rate than in VD60 cells, and the viral plaques formed in VerB::gDl cells are smaller than plaques observed in VD60 cells (FIG. 7A). However, AgD2P virus produced in VerB::gDl cells did reach 90% CPE 2 days later than in VD60 cells (FIG. 7B).

[0178] AgD2P virus Productivity: VerB::gDl and VD60 cells were seeded in 10 cm dishes (3xl0⁶ cells/dish). On the next day, cells were infected with AgD2P virus at different MOIs (0.01; 0.05; 0.1; 0.5 PFU) and incubated at temperatures (34, 35, 37°C). Additionally, media of infected cells was removed at 3 days post infection (dpi) and fresh media added. Infected cells were harvested when CPE reached 90%; cells were lysed by 3 cycles of freeze and thaw, and cell associated virus collected. The AgD2P virus produced under the different conditions were titrated by plaque assay in VD60 cells (Table 3, FIG. 8).

TABLE 3. TITRATION OF AgD2P IN COMPLEMENTING CELL LINES

[0179] Of the four VerB::gD clones, VerB::gDl.l and VerB::gD1.4 cell clones were able to complement and produce AgD2P virus similar to the ones obtained in VD60 cells. Specifically, VerB::gDl.l and VerB::gD1.4 infected with AgD2P virus at MOI 0.01 PFU at 34°C produced AgD2P virus yields 1 log lower than the yields in VD60 cells (Table 3).

[0180] Phenotype Assay: to test the inability of AgD2P virus to induce CPE on Vero cells or test the absence of homologous recombination between AgD2P virus and gD sequences in complementing cell lines (VerB::gDl compared to VD60), VerB::gDl cells were seeded in 10 cm²-dish (3xl0⁶ cells/dish). On the next day, cells were infected with AgD2P virus MOI 0.01 PFU, and cells was harvested at 4-8 dpi. Infected cells were lysed by 3 cycles of freeze and thaw and cell associated virus were collected.

[0181] AgD2P virus produced in VerB::gDl cells was used to infect Vero cells in serial passages. For each passage, Vero cells were seeded in 10 cm²-dish (3xl0⁶ cells/dish), and infected with 50 ul of cell lysate from the previous passage. Cells were imaged and harvested at 8 dpi. Infected cells were lysed by 3 cycles of freeze and thaw and cell associated virus were collected. AgD2P virus produced in VerB::gDl cells is not able to spread and produce infectious virions when serially passaged in non-complementing Vero cells. This experiment shows that no replication competent virus was detectable when AgD2P virus produced in VerB::gDl cells are passaged in Vero cells (FIG. 9). EXAMPLE 3 - PRODUCTION of VerB::gD1.6C

MATERIAL AND METHODS

[0182] Cells and viruses'. Vero (African green monkey kidney cell line; CCL-81; American Type Culture Collection (ATCC), Manassas, VA, USA) cells, VD60 cells [Vero cells encoding gD-1 under endogenous promoter (Ligas et al., 1988)], were passaged in DMEM supplemented with 10% fetal bovine serum (FBS, Gemini Bio-Products, West Sacramento, CA). VerB::gDl cells (Vero cells with minimal gD-1 expression cassette), or VerB::gD1.6C (Vero cells with modified minimal gD-1 promoter expression cassette) were passaged in VP-SFM (a serum-free, ultra low protein (5 ug/ml) medium containing no proteins, peptides, or other components of animal or human origin) supplemented with 2 mM GlutaMax™ (Thermo Fisher Scientific®, Waltham, MA).

[0183] HSV-2 strain MS (ATCC) was propagated and titrated on Vero cells. HSV- 2(G) AgD2P was propagated on complementing gD-1 cells VerB::gDl, and viral titers determined by plaque assays on VerB::gD1.6C, VerB::gDl, VD60 and Vero cells in parallel; no plaques are detected on the non-complementing Vero control cells.

[0184] Generation of VerB::gD1.6C. A shuttle plasmid that contained a modified version of the gDl small promoter was constructed. The specific sequence of the gD promoter is not well determined. After different minimal promoters were assayed, the minimal promoter able to produce titers of AgD2P virus similar to titers obtained in VD60 cells was selected. Specifically, this promoter had three extra nucleotides (CGA) inserted upstream the 5’ region of the previously described gD-1 small promoter sequence (5’CGAATCCCCTAAGGGGGAGGGGCCATTTTACGAGGAGGAGGGGTATAACAAA GTCTGTCTTTAAAAAGCAGGGGTTAGGGAGTTGTTCGGTCATAAGCTTCAGCGCG AACGACCAACTACCCCGATCATCAGTTATCCTTAAGGTCTCTTTTGTGTGGTGCG TTCCGGT 3’, identified in SEQ ID NO:32, based on the HSV-1 17+ strain, following Q5 Site Directed Mutagenesis kit (NEB) manufacturer’s protocol. The previously constructed pMAECL20 plasmid was used as a template and primers (# 444: 5’CGAATCCCCTAAGGGGGAGGG3’ SEQ ID NO:33, # 445: 5’CCCAAGCTTGGGTTACTAGTG3’ SEQ ID NO:34) were designed for the insertion of 3 nucleotides, generating the pMAECL41 plasmid (FIG. 10). VerB::gD1.6C cellular genomic DNA was extracted using Monarch® High Molecular weight DNA extraction kit (NEB) following manufacturer’s protocol. Cellular genomic DNA was prepared for NGS following Nanopore sequencing protocol.

[0185] Transfection and Integration of gD-1 shutle plasmid pMAECL41 into VerB- attB cells'. VerB-al cell line was seeded in 6-well plates (2xl0⁶ cells/well) in growth media (DMEM 5% FBS). Cells were transfected with the shuttle plasmid pMAECL41 (2 pg/well), containing the gD-1 expression cassette as described above, using Lipofectamine™ 2000 (Invitrogen™, Waltham, MA, manufacturer’s protocol) and followed by co-transfection with the Bxbl integrase mRNA (1 pg/well), using RNAfection™ (System Biosciences®, Palo Alto, CA, manufacturer’s protocol). The Bxbl integrase mRNA was synthesized using the mRNA Express kit (SBI, catalog# MR-KIT-1, System Biosciences®, Palo Alto, CA), following manufacturer’s protocol.

[0186] Selection of VerB::gD1.6C cells'. At 3 days after transfection, cells were passaged at a 1: 10 split ratio into several 6-well plates in selection media (DMEM without histidine, 5% FBS, 0.5 mM I-histidinol). Selection of resistant cells, VerB::gD1.6C (Vero cells that have integrated the gD-1 expression cassette with minimal promoter having additional 3 nucleotide modification into the attB site present in the cellular genome), occurred during 3 weeks with selection media exchanged every 3 days. Single cell colonies were isolated using Namocell (Biotechne®) single cell dispenser and subsequently expanded in selection media into separated dishes until reaching confluence. The VerB::gD1.6C cell clones were selected for complementation and production yields of AgD2P virus.

[0187] Screening of VerB::gD1.6C cell clones'. VerB::gD1.6C cell clones were seeded in 24-well plates (IxlO⁵ cells/well) in growth media (DMEM 5% FBS). On the next day, cells were infected with AgD2::RFP (HSV-2 virus with a deletion of the gD gene and insertion of the Red Fluorescence Protein gene) virus at an MOI of 0.01 plaque forming unit (PFU) for Ih at 37°C. Virus inoculum was removed and DMEM 2% FBS was added to the cells. Infected cells were imaged daily and cytopathic effect (CPE) characteristic of HSV infection was recorded.

[0188] In vitro growth curves: Multi-step growth curves were performed as previously described. For multi-step growth of each virus, Vero cells were infected at a MOI 0.01 PFU/cell and cell-associated virus were harvested every 12 up to 72 h post-infection (pi) and stored at -80°C. Infectious virus was measured by performing plaque assays with supernatants and lysed cells.

[0189] AgD2P virus Productivity. VerB::gD1.6C and VD60 cells were seeded in 10 cm² dishes (3xl0⁶ cells/dish). On the next day, cells were infected with AgD2P virus at MOI 0.01 PFU and incubated at 37°C. Infected cells were harvested when CPE reached 90%; cells were lysed by 3 cycles of freeze and thaw, and cell-associated virus collected. The AgD2P virus produced under the different conditions were titrated by plaque assay in VD60 cells.

[0190] Immunofluorescence assay: VerB::gD1.6C, VD60 and Vero cells were seeded on 24-well plate (2xl0⁴ cells per well), and on the following day infected with AgD2P or HSV-2 strain MS virus at MOI 0.1 PFU or mock infected. Infected cells were incubated for Ih at 37°C, and virus inoculum removed and DMEM 2% FBS added to the cells. At 48 h post-infection, cells were fixed with 4% paraformaldehyde for 5 min at room temperature (RT), permeabilized with 0.2% Triton-X 100™ in PBS for 10 min at RT, and incubated with a primary mouse antibody (dilution 1:500) against HSV-gD (sc69802, Santa Cruz Biotechnology®, Dallas, TX) in 3% BSA-PBS for 16 h at 4°C. Then, cells were washed with PBS and incubated with the secondary antibody AlexaFluor® 488 (AF488) goat anti-mouse IgG (dilution 1: 1000) (cat#A32723, Invitrogen™, Waltham, MA) for 1 h at RT. Images were captured using the EVOS™ 5000 Imaging System (Invitrogen™, Waltham, MA).

[0191] Western blot assay .•VerB::gD1.6C, VD60 and Vero cells were seeded on 12- well plates (2xl0⁵ cells per well), and on the following day infected with AgD2P, AgD2 or HSV-2 strain MS virus at MOI 1 PFU or mock infected. Infected cells were incubated for Ih at 37°C, and virus inoculum removed and DMEM 2% FBS added to the cells. At different time points after infection (6, 8, 12, 20 hpi), cells were washed and lysed with RIPA buffer (RIPA Eysis and Extraction Buffer, ThermoScientific™) supplemented with protease inhibitor cocktail (cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail, Roche Diagnostics). After incubation at 4°C for 30 minutes, cell extract was obtained after centrifugation. Samples were mixed with SDS-loading buffer (4x Laemmli Sample Buffer, Bio-Rad) and boiled for 5 minutes. Proteins were separated in a 7% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Immuno-blot, Bio-Rad). The membranes were blocked for 1 hour with 5% skim milk in PBS. Membranes were then incubated for 1 h with the HSV-gD (sc69802, Santa Cruz Biotechnology®, Dallas, TX) primary antibody at a dilution of 1: 1,000. After washing, the membranes were incubated for 1 h with goat anti-mouse IgG conjugated with horseradish peroxidase (Sigma- Aldrich®) at a dilution of 1: 10,000. Target proteins were visualized by enhanced chemiluminescence (ECE Western blotting analysis system; GE Healthcare) and imaged on the iBright™ 1500 Western Blot Imaging System (Invitrogen™).

[0192] Murine immunization and viral challenge studies: Experiments were performed with approval from Albert Einstein College of Medicine Institutional Animal Care and Use Committee, Protocol #20130913 and #20150805. C57BL/6 mice are purchased from Jackson Laboratory (Bar Harbor, ME).

[0193] Female C57BL/6 and BALB/c mice were purchased from Jackson Laboratory (Bar Harbor, ME) at 4-6 weeks of age. Mice were primed and boosted 3 weeks later with 5xl0⁵ or 5xl0⁶ PFU of AgD2P (propagated in VerB::gD1.6C cells), AgD2 (propagated in VD60 cells) or equal amount of VerB::gD1.6C cell lysates (Control), intramuscularly (i.m., medial to the hind limb and pelvis) at 100 pl/mouse. The titer was determined by a plaque assay on complementing cells (VD60 and VerB::gD1.6C). The skin scarification model was used to challenge mice two weeks after boost with lxLD90 (10⁵ pfu/mouse) of HSV-2 strain MS. For HSV skin infections, mice are depilated on the right flank with Nair™ and allowed to rest for 24 h. Depilated mice are anesthetized with isoflurane (Isothesia, Henry-Schein), then abraded on the exposed skin with a disposable emory board for 20-25 strokes and subsequently challenged with HSV-2 strains for in vivo virulence studies or for vaccine efficacy studies. Mice were monitored for 14 days and scored as follows: 1: primary lesion or erythema, 2: distant site zosteriform lesions, mild edema/erythema, 3: severe ulceration and edema, increased epidermal spread, 4: hind-limb paresis/paralysis. Mice that are euthanized at a score of 4 were assigned a value of 5 on all subsequent days for statistical analyses.

[0194] FcyRIV activation assay: To measure a proxy of ADCC activity, the FcyRIV activation core assay (Promega, Cat# M1215) was used, following the manufacturer's protocol. The target Vero cells were seeded at 2 xlO⁴ cells/well on white, flat-bottomed 96- well plates in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific, Cat# 12634010) containing 5% FBS and incubated at 37°C, 5% CO2. After 6-8 hours, the medium was removed, and cells were infected with the HSV-2 strain MS virus at an MOI 1 PFU/cell in DMEM with 2% FBS and incubated overnight at 37°C, 5% CO2. The next day, serum from vaccinated mice was 2-fold serialy diluted and heat-inactivated at 56°C for 30 minutes. Media was aspirated from the infected cells, the serum was added in duplicates and incubated for Ih at 37°C. The proprietary effector cells, Jurkat T-cells expressing mouse FcyRIV, were added and incubated at 37°C, 5% CO2 for 6 hours. The Promega Bio-Gio™ Luciferase Assay substrate was added, and luminescence (RLU) was measured using a Biotek Synergy™ Hl Hybrid Multi-Mode Reader. Fold FcyRIV induction was calculated as: (RLU of individual sample with effector and target cells - RLU of background)/(RLU of activation of effector cells by target cells without antibody - RLU of background). N=10. [0195] Statistical analysis'. Analyses were conducted using GraphPad Prism version 9.1.0 software (GraphPad Software Inc., San Diego, CA). A p value of 0.05 was deemed statistically significant. Two-way analysis of variance (ANOVA) with main column effect was used to analyze weight loss curves, one-way ANOVA was used for FcyRIV activation assay analysis, and Gehan-Breslow-Wilcoxon test was used for survival curves. All data are shown as mean ± SD, n=10 mice.

RESULTS

[0196] In an effort to increase the yields of AgD2P virus produced in the VerB::gDl cells, an alternative cell line was constructed (VerB::gD1.6C) which contains a modified version of the minimal gD-1 promoter that was present on the VerB::gDl cell lines. The modification on the gD minimal promoter comprised a site directed mutagenesis step to insert 3 nucleotides (CGA) upstream the gDl minimal promoter sequence (FIG. 10). The shuttle plasmid containing the gDl expression cassette (pMAECL41) was demonstrated to express gD in transfected cells in the presence of AgD2P virus co-infection (FIG. 11). The selected VerB::gD1.6C cells can complement AgD2P virus infection and induce the expression of gD- 1 protein at similar levels as the ones induced by HSV-2 strain MS in Vero cells, although VD60 cells still induce a higher level of expression (FIG. 12 and 13).

[0197] The new cell line (VerB::gD1.6C) was able to complement and produce AgD2P at similar titers as VD60, and a one log increase over titer yield when grown on VerB::gDl cells containing a nonmodified gD-1 minimal promoter. Specifically, VerB::gD1.6C infected with AgD2P virus at MOI 0.01 PFU at 37°C produced AgD2P virus yields similar to the yields in VD60 cells (FIG. 14 A, B).

[0198] Immunization with low doses of HSV-2 AgD2P is protective against skin challenges. A dose de-escalation study was conducted to determine protection at lower doses of vaccination. C57BL/6 mice (10 mice/group) were intramuscularly primed and boosted 21 days later with 5xl0⁵ PFU and 5xl0⁶ PFU of AgD2P. Two weeks after the boost the mice were challenged by skin scarification with an 1 x LD90 (10⁵ PFU) of HSV-2 strain MS, a previously described clinical isolate. Mice were evaluated daily for signs of epithelial and neurological disease after challenge. Mice immunized with either dose of AgD2P (referred to as AgD2P/6C in the figures) or AgD2 (referred to as AgD2/VD60 in the figures) candidate vaccines showed significantly reduced epithelial disease scores, only mild signs of epithelial disease, mostly due to the skin scarification model of infection that was resolved after 5 days and the complete absence of neurological signs. Control non-immunized mice showed severe skin disease that evolved to paralysis and succumbed to disease by Day 7. Scoring was performed as previously described. It was demonstrated that both AgD2P and AgD2 candidate vaccines are highly attenuated in immunocompetent mice and induced a protective immune response against challenge with HSV-2 (FIG. 15 A, B).

[0199] HSV antibodies measured by FcyRVI activation assay were detected in the serum of all vaccinated mice two weeks after boost, and the increase in antibody titer with administration of higher vaccine doses was demonstrated for both AgD2P and AgD2 candidate vaccines. No significant difference was detected between the two candidate vaccines (FIG. 16 A, B). The HSV-2 AgD2P vaccine induced systemic immune responses as FcyRVLdependent ADCC.

[0200] This study confirms that HSV-2 AgD2P is attenuated and completely safe in wild type mice. Vaccination with HSV-2 AgD2P affords complete protection against HSV-2 skin infection and potentially against lethal HSV-2 intravaginal and HSV-1 skin infection. Vaccine efficacy was confirmed in an optimized skin model, which is reflective of human primary disease. The protective effect of HSV-2 AgD2P against HSV-2 will differentiate it from other candidate vaccines such as HSV-2 AUL5/AUL29, which failed to fully protect against the clinical isolate SD90, or gD subunit vaccine which provided partial protection against HSV-1, but no protection against HSV-2.

EXAMPLE 4 - AgD2P vaccine induces T cell responses.

[0201] HSV-2 AgD2P elicits HSV-2-specific T cell activation: gB498-505-specific transgenic CD8+ T cells (gBT-I) will be transferred into C57BL/6 mice prior to vaccination. Vaccinated mice will be inoculated with 5xl0⁶ pfu AgD2P or with VerB::gDl cell lysates (Control). Spleens will be harvested on Day 14 after the boost and quantified by flow cytometry using counting beads (CountBright™, Lifetechnologies). At the same day, spleens will be stained for memory surface markers and analyzed by flow cytometry. Finally, splenocytes harvested the same day will be re-stimulated in vitro for 6 hours with the agonist gB 498 -505 -peptide and intracellular cytokine staining was performed to measure IFN-y production by these cells. Multiplex cytokine analyses for supernatants of splenocytes restimulated in vitro with gB498-505-peptide will be performed. These findings will demonstrate that the vaccine induces T cell responses. [0202] Immunization with HSV-2 AgD2P recruits T cells to the infection site and associated lymph nodes (LNs). Mice will be immunized with AgD2P and the increased percentages of activated anti-HSV-2 gBT-I CD8+ and CD4+ T cells in sacral lymph nodes (LNs) after challenge with virulent HSV-2 will be measured. Increased numbers of anti- HSV-2 gBT-I CD8+ and CD4+ T cells in the vagina after challenge with virulent HSV-2 suggests that vaccination with AgD2P recruits anti-HSV-2 CD8+ T cells and activated CD4+ T cells (likely anti-HSV-2) to the infection site and associated lymph nodes.

[0203] EXAMPLE 5 - MICE IMMUNIZED WITH HSV-2 AgD2P ARE PROTECTED AGAINST INTRA VAGINAL HSV-2 LETHAL CHALLENGE. Animals will be vaccinated with HSV-2 AgD2P sc.-i.vag. will be evaluated by loss of body weight after intravaginal lethal dose challenges equivalent to LD90 (5xl0⁴ pfu/mouse) and survival challenges, and compared with mice immunized with the VerB::gDl control lysate which normally succumb to disease by Day 10. The vaccines will be assayed for complete protection against 10 times the LD90 (5xl0⁵ pfu/mouse and protection will be assessed by significantly reduced epithelial disease scores and the complete absence of neurological signs. Scoring is performed as described in the art. Furthermore, virus recovered in vaginal washes in HSV-2 AgD2P-immunized mice, as compared to control mice at day 2 post- vaginal challenge, will be measured as a correlate of rapid clearance. Moreover, measurement of infectious virus recovered in Day 4 vaginal washes or in vaginal tissue or DRGs isolated on Day 5 after challenge will suggest if the vaccine prevents virus from reaching and/or replicating in the DRG.

[0204] In further experiments, immunization with HSV-2- AgD2P will be used to measure conferred protection in C57BL/6, SCID, and Balb/C to vaginal challenge with virulent HSV-2. In addition, intravaginal HSV-2 challenged AgD2P immunized mice will be assayed for HSV-2 in vaginal or neural tissue at 5 days post-challenge. HSV-2 AgD22 antibodies and serum antibodies will be tested for recognition of numerous HSV-2 proteins (both gD and gB) and neutralization of HSV-1 and HSV-2 in vitro. Moreover, serum from AgD2P vaccinated mice will be tested for Antibody Dependent Cellular Cytotoxicity (ADCC) of HSV-2 infected cells in vitro.

[0205] In summary, these experiments will show that HSV-2 AgD2P is attenuated and completely safe in wt and SCID mice. Recombinant HSV-2 AgD2P provides a composition that can potentially protect against lethal HSV-2 intravaginal and HSV-2/HSV-1 skin infection. Protection conferring sterilizing immunity with no detectable infection, will be observed in two different mouse strains. In addition, the HSV-2 AgD2P vaccine will likely induce immune responses including HSV-2 specific CD8+ T cells and systemic and mucosal HSV Abs, with IgG2a and IgG2b as the predominant anti-HSV isotype, as well as FcyRIIFIV-dependent ADCC. Passive transfer of immune serum will protect naive mice, whereas FcRn and FcyR knockout mice will not be protected with immune sera.

EXAMPLE 5 - VACCINE ASSAYS.

[0206] The ability of the vaccine to protect against clinical HSV-1 and HSV-2 isolates will be further confirmed, as well as the local immune response at the site of infection. Classically, HSV primarily infects genital or oral nucleated epidermal cells due to breaches in the skin or mucocutaneous layers in humans. To more closely model human HSV infection, a skin scarification model was used for these studies, which displays viral kinetics and histopathology similar to humans.

[0207] Immunization with low doses of HSV-2 AgD2P are protective against lethal intravaginal and skin challenges.

[0208] Mice immunized with HSV-2 AgD2P are protected from high viral challenges of virulent HSV-2 clinical isolates. To evaluate if the HSV-2 AgD2P vaccine protects against diverse HSV-1 and HSV-2 strains, five HSV-1 will be obtained (denoted B 3x1.1 - B 3x1.5) and five HSV-2 (denoted B3x2.1 - B3x2.5) clinical isolates from the Clinical Virology Lab at Montefiore located in the Bronx, NY as well as a South African HSV-2 clinical isolate (SD90). The isolates are grown on Vero cells and passaged no more than three times before sequencing and phenotyping. Illumina® sequencing showed that the strains exhibit substantial genetic diversity with pairwise distances as high as 6.3% between B3xl.5 and the other B3xl isolates and 5.0% between B3x2.2 and the other B3x2 isolates. In vivo virulence of each clinical strain was compared to laboratory strains by challenging Balb/C mice using the skin scarification model with IxlO⁵ PFU of the HSV-1 strains or 5xl0⁴ PFU of HSV-2 strains. The clinical isolates demonstrated a range of virulence with B3xl.l, B3xl.3, B3x2.3, and SD90 inducing more rapid disease with the highest morbidity in naive mice. Similar results were observed in the vaginal challenge model with the same 4 isolates exhibiting the most virulent disease (not shown). Interestingly, no differences between the isolates were observed by in vitro single and multistep growth curves on Vero cells.

[0209] To assess if HSV-2 AgD2P protects against the different isolates, C57BL/6 or Balb/C mice will be primed and boosted with 5xl0⁶ PFU/mouse of HSV-2 AgD2P (or VerB::gDl lysate as the control immunogen) and then challenged with an LD90 dose of the 4 more virulent clinical isolates (Table 1) using the skin scarification model. Epithelial disease, neurological disease, days for recovery post challenge, and percent survival will be measured.

TABLE 4: HSV STRAINS USED IN VACCINE EFFICACY STUDIES

Viral Strain Origin of Isolate HSV Serotype Lethal

B³xl.l United States Type 1 5xl

B³xl.3 United States Type 1 lxl

SD90 South Africa Type 2 5xl

B³X2.3 United States Type 2 lxl

4674 United States Type 2 5xl

Note: *Plaque forming units that cause 90% morbidity in Balb/C mice skin challenge model

[0210] To further evaluate the robustness of the immune response, the challenge dose will be increased in the C57BL/6 mice to lOx and lOOx the LD90 doses of SD90 and lOx the LD90 of B3xl.l. Signs of neurological disease will be monitored, and days of survival of the HSV-2 AgD2P vaccinated mice will be measured. Virus in skin biopsies by post-challenge will be measured in the HSV-2 AgD2P vaccinated mice and mock vaccinated mice (unvaccinated, uninfected). Moreover, replicating or latent virus will be detected by plaque assay or qPCR , respectively, in DRGs isolated on Day 14 post-challenge in the HSV-2 AgD2P vaccinated mice (n=10 mice per each challenge dose and strain). Similarly, reactivating virus will be measured from DRGs isolated from HSV-2 AgD2P vaccinated mice (isolated Day 5 post-challenge with LD90 of SD90) a reco-cultured for 3 weeks with Vero cells.

[0211] Skin biopsies from HSV-2 AgD2P-immunized and control vaccinated mice obtained on Day 5 post-challenge or unimmunized, uninfected mice (mock) will be evaluated by immunohistochemistry and/or immunofluorescence for immune cell responses, such as an increase in CD3+ T cells and B220+ B cells compared to control immunized mice. The T- cells will be further characterized by staining for CD4 or CD8; in HSV-2 AgD2P compared to control-immunized mice, as well as Ibal+ monocyte/macrophage cells and Ly6G+ neutrophils in HSV-2 AgD2P compared to control immunized mice. [0212] Inflammatory cytokines/chemokines detected in skin homogenates in the HSV-2 AgD2P compared to control immunized mice will be measured 5 days post-challenge. Levels of TNFa, IL-ip, IL-6, and chemokines CXCL9 and CXCL10 will be compared.

[0213] This study will confirm that vaccination with HSV-2 AgD2P affords complete protection against a panel of genetically diverse HSV-1 and HSV-2 clinical isolates and prevents the establishment of latency. Vaccine efficacy will be confirmed in an optimized skin model, which is reflective of human primary disease. The protective effect of HSV-2 AgD2P against a broad array of HSV-1 and HSV-2 clinical isolates will differentiate it from other candidate vaccines such as HSV-2 AUL5/AUL29, which failed to fully protect against the clinical isolate SD90, or gD subunit vaccines and others that have only been tested against one or two laboratory viral strains.

EXAMPLE 6 - IMMUNIZATION OF FEMALE ADULT MICE (PRIOR TO CONCEPTION) WITH HSV-2 AgD2P ELICITS ANTIBODY RESPONSES THAT PROTECT NEONATAL MICE (7-14 DAYS OF LIFE) FROM INTRANASAL VIRAL CHALLENGE WITH CLINICAL ISOLATES OF HSV-1 OR HSV-2 AND PROTECTION CORRELATES WITH THE ABILITY OF THE ANTIBODIES TO ACTIVATE THE MURINE FcyRIV TO ELICIT ADCK.

MATERIAL AND METHODS

[0214] Cells and viruses: Vero (African green monkey kidney cell line; CCL-81, CCL-82; American Type Culture Collection (ATCC), Manassas, VA, USA) cells, VD60 cells (Vero cells containing multiple copies of gD-1 gene under its endogenous promoter], or VerB::gD1.6C (Vero cells with modified minimal gD-1 promoter expression cassette), and CaSki (human cervical epithelial cell line; CRL-1550; ATCC) will be passaged in DMEM supplemented with 10% fetal bovine serum (FBS, Gemini Bio-Products, West Sacramento, CA). THP-1 (human monocyte cell line; TIB-202; ATCC) cells will be passaged in RPML 1640 (Life Technologies) supplemented with 10% FBS and sub-cultured according to ATCC guidelines. HSV-2(4674) will be propagated on CaSki cells. Laboratory strains HSV-2(G), the clinical isolates, HSV-2 (4674) and HSV-1 (B3xl.l), and the laboratory strain, HSV-2 (333) ZAG, which expresses green fluorescence protein (GFP) under control of the CMV promoter inserted at an intergenic site within the virus, HSV-1(17), and HSV-l(F) will be propagated on Vero cells. HSV-2(G) AgD2P was propagated on complementing gD-1 cells VerB::gDl and viral titers determined by plaque assays on VerB::gD1.6C, VerB::gDl, VD60 and Vero cells in parallel; no plaques are detected on the non-complementing Vero control cells. South African isolate HSV-2(SD90) is provided by David Knipe and will be propagated on Vero cells. Five HSV-1 (B3xl.l through B3xl.5) and five HSV-2 (B3x2.1 through B3x2.5) de-identified clinical isolates are provided by the Clinical Virology Lab at Montefiore and will be passaged three times on Vero cells for a low-passage working stock.

[0215] In vitro growth curves: Single-step and multi-step growth curves are performed as previously described. For single-step growth of each virus, Vero cells are infected with virus at a multiplicity of infection (moi) of 5 PFU/cell and supernatants and cells are collected every 4, 8, 16 and 24 hours (h) post-infection (pi) and stored at -80°C. For multi-step growth of each virus, Vero cells are infected at a moi of 0.01 PFU/cell and supernatants and cells are harvested every 12 h pi up to 72 hours. Infectious virus is measured by performing plaque assays with supernatants and lysed cells.

[0216] Viral DNA isolation and sequencing of clinical isolates: HSV DNA is prepared by infecting confluent Vero cells in a T150 flask with each of the B3x clinical isolates at an MOI of 10. Cells are harvested 16 hpi and washed twice with PBS. DNA is extracted using DNeasy® Blood and Tissue (Qiagen) following the manufacturer’s recommendations. DNA is quantitated by Qubit™ dsDNA hs assay (Life Technologies). Paired-end libraries are prepared by the Nextera® XT DNA library preparation kit (Illumina®) following the manufacturer’s instructions. Libraries were sequenced on an Illumina® MiSeq™ Desktop Sequencer. Viral genome sequences are assembled with the VirAmp pipeline following removal of host sequence by alignment to the Macaca mulatta genome as a substitute for the incomplete Chlorocebus sabaeus (source of Vero cells) genome. HSV-1 and HSV-2 genomes are annotated with Genome Annotation Transfer Utility on ViPR by comparison to HSV-1 (GenBank accession no. JN555585.1) & HSV-2(HG52) (JN561323) prior to submission to GenBank. Whole genome alignments including the previously sequenced HSV-2 (SD90e) (KF781518), HSV-2(333) (KP192856), ChHV 105640 (NC_023677.1), & HSV-l(F) (GU734771.1) are performed using ClustalW and phylogenetic trees were constructed using the UPGMA method with 1000 bootstrap replicates in MEGA6. All positions containing gaps or missing data are eliminated. GenBank numbers for the genome sequences are as follows: HSV-2(G) (KU310668), HSV-2(4674) (KU310667), B3xl.l (KU310657), B3xl.2 (KU310658), B3xl.3 (KU310659), B3xl.4 (KU310660), B3xl.5 (KU310661), B3x2.1 (KU310662), B3x2.2 (KU310663), B3x2.3 (KU310664), B3x2.4 (KU310665), B3x2.5 (KU310666). [0217] Murine immunization and viral challenge studies: Experiments are performed with approval from Albert Einstein College of Medicine Institutional Animal Care and Use Committee, Protocol #20130913 and #20150805. Female C57BL/6 and BALB/c mice are purchased from Jackson Laboratory (J AX, Bar Harbor, ME) at 4-6 weeks of age. Mice are primed and boosted 3 weeks later with 5xl0⁴ - 5xl0⁶ PFU of AgD-2P or equal amount of VerB::gDl cell lysates (Control) intramuscularly (im, medial to the hind limb and pelvis) at lOOpl/mouse. The titer is determined by a plaque assay on complementing cells (VD60 or VerB::gDl, or VerB::gD1.6C).

[0218] For intravaginal HSV infections, mice are treated with 2.5 mg of medoxyprogesterone acetate (MPA; Sicor Pharmaceuticals, Irvine, CA) sc five days prior to challenge. Mice are then inoculated intravaginally with an LD90 (5x105 pfu/mouse) of HSV- 2 (4674) at 30 pl/mouse and scored for disease and monitored for survival for 14 days as described in the art. For HSV skin infections, mice are depilated on the right flank with Nair™ and allowed to rest for 24 hr. Depilated mice are anesthetized with isoflurane (Isothesia, Henry-Schein), then abraded on the exposed skin with a disposable emory board for 20-25 strokes and subsequently challenged with IxlO⁵ PFU HSV-1 or 5xl0⁴ PFU HSV-2 strains for in vivo virulence studies or challenged with an LD90, lOxLDgo, or 1 OOxLDgo of select HSV strains (see Table 4) for vaccine efficacy studies. Mice are monitored for 14 days and scored as follows: 1: primary lesion or erythema, 2: distant site zosteriform lesions, mild edema/erythema, 3: severe ulceration and edema, increased epidermal spread, 4: hind-limp paresis/paralysis and 5: death. Mice that are euthanized at a score of 4 were assigned a value of 5 on all subsequent days for statistical analyses.

[0219] HSV RT-qPCR: DNA is extracted from weighed tissue samples using DNeasy® Blood and Tissue (Qiagen) following the manufacturer’s recommendations. Extracted DNA is then normalized to 10 ng of DNA per reaction and viral DNA quantified using real-time quantitative PCR (RT-qPCR, qPCR) using AB solute™ qPCR ROX Mix (Thermo Scientific). Primers for HSV polymerase (UL30) are purchased from Integrated DNA Technologies (Cat#: 1179200494) and used to detect viral genomic DNA. Isolated HSV-2 viral DNA is calibrated for absolute copy amounts using QuantStudio® 3D Digital PCR (dPCR, ThermoFisher Scientific) and subsequently used as a standard curve to determine HSV viral genome copies. Samples that read 4 or less copy numbers are considered negative. Data are presented as log 10 HSV genomes per gram of DRG (dorsal root ganglia) tissue. [0220] Detection of antibodies and cytokines in skin biopsies: Skin biopsies are obtained from HSV-2 AgD2P or VerB::gDl or VerB::gD1.6C lysates (control) immunized mice (-5-10 mm in diameter by mechanical excision) day 21 post-boost or day 2 and 5 post viral skin challenge. The tissue is weighed and homogenized in RNase/DNase free Lysing Matrix A tubes (MP Biomedicals, Santa Ana, CA) with serum-free DMEM at 6.0m/sec for three 30sec cycles in the FastPrep-24™ 5G (MP Biomedicals). Samples are spun at 5000 rpm for 10 min at 4°C and the resulting supernatant is evaluated for anti-HSV antibodies, cytokines and chemokines. Anti-HSV antibodies are detected by ELISA as previously described using uninfected, HSV-1, or HSV-2(4674)-infected Vero cell lysates as the coating antigen. Biotin anti-mouse Ig K or biotin anti-mouse IgA, IgM, IgGl, IgG2a, IgG2b, or IgG3 at 1 pg/ml (Becton Dickenson, San Diego) are used as secondary detection antibodies. Wells are read on a SpectraMax® (M5 series) ELISA plate reader at an absorbance of 450 nm. The resulting absorbance is determined by subtracting values obtained for uninfected cell lysates to values obtained with infected cell lysates. Total anti-HSV Ig is reported as the optical density (OD) at 450 nm normalized to relative tissue weight at a 1: 1000 dilution of tissue homogenate. Anti-HSV IgG, IgA, IgM, or IgGl -3 are reported as the optical density (OD) at 450 nm at all dilutions except IgGl-3 which is reported only at a 1: 100 dilution of skin homogenate.

[0221] Skin homogenate supernatants are assayed for interleukin-6 (IL-6), IL-1 beta (IL-ip), IL-33, tumor necrosis factor alpha (TNFa), monokine induced by interferon-gamma (MIG, CXCL9), interferon-inducible cytokine (IP- 10, CXCL10) using a Milliplex® mouse cytokine/chemokine immunoassay (Millipore, Danvers, MA) and a Luminex Magpix® system and analyzed with Milliplex Analyst (Version 3.5.5.0; VigeneTech Inc.).

[0222] Histopathology, immunohistochemistry and immunofluorescence of skin tissue: Mice are euthanized on Day 5 post-challenge and the skin at the viral (or mock) infection site is excised and formalin fixed for 48hrs at RT. Samples are processed routinely to be paraffin-embedded and sectioned. Slides for histopathology are stained with hematoxylin and eosin (H&E). Samples are evaluated histologically by a board certified veterinary pathologist which are blinded of samples identity. For immunohistochemistry (IHC), the samples are sectioned to 5 pm, deparaffinized in xylene followed by graded alcohols. Antigen retrieval is performed in 10 mM sodium citrate buffer at pH 6.0, heated to 96°C, for 30 minutes. Endogenous peroxidase activity is blocked using 3% hydrogen peroxide in water. The sections are stained by routine IHC methods, using SuperPicTure™ (ThermoFisher Scientific, Cat:87-9673) against rabbit primary antibodies to anti-CD3 (Ready to use format, ThermoFisher Scientific, Cat: RM-9107-R7), anti-B220 (BD Biosciences Cat: 550286), or anti-Ibal (1:3000 dilution Wako Pure Chemical Industries, Richmond, VA) and then stained with diaminobenzidine as the final chromogen. All immunostained sections are lightly counterstained with hematoxylin. Stained cross- sections are photomicrographed with a Zeiss Axio Observer inverted light microscope at 20x magnification from apical layer (epidermal) to basal layer (striated muscle) at 3 different locations per sample. Stainedpositive cells are enumerated as described in the art. Data is represented as the average of % positive cells= (positive nucleated cells/total nucleated cells) of three photomicrographed sections per sample.

[0223] For Immunofluorescent studies, skin tissue is excised 5 days post-HSV or mock skin challenge and then frozen in OCT media. Samples are cut into 5pm sections and stored at -80°C. Frozen slides are then fixed in -20°C acetone for 15 mins, washed with wash buffer (WB, 0.05% Tween 20 in PBS), then blocked for 2hrs with blocking buffer (2% BSA, 5% heat inactivated goat serum in PBS) at RT. Slides are washed twice and incubated with anti-CD4 (GK1.5, 1:200), anti-CD8 (YTS169.4, 1:250), anti-Ly6G (1A8, 1:500) in blocking buffer for 1 hr at RT. Slides are thoroughly washed and incubated with an goat anti-rat secondary antibody conjugated with either Alexa Fluor® 555 or Alexa Fluor® 488 (1:500 or 1:200, respectively) for 30 min at RT. Slides are washed and mounted with media containing DAPI (ProLong® Diamond Antifade Mountant with DAPI, ThermoFisher Scientific). Slides are imaged using a Nikon Eclipse Ti-U inverted light microscope at 20x magnification from apical layer (epidermal) to basal layer (striated muscle) at two different locations per sample. For %CD4+ and %CD8+ quantification, total nucleated cells are calculated by DAPI positive objects > 5pm via a software algorithm from Velocity (version 6.3, Perkin Elmer). CD4 or CD8 positive cells are counted manually for fluorescence and incorporation of a DAPI+ nuclei to exclude non-specific staining of hair follicles and cellular debris in the skin sections. Data is represented as the average of %positive cells= (positive cells/total DAPI cells) of two images per sample.

[0224] Antibody dependent cellular phagocytosis (ADCP) assay. To determine HSV specific ADCP, a protocol modified from the art. Briefly, 2xl0⁸ 1pm Neutravadin®-red fluorescent beads (Invitrogen, F-8775) are coated with 0.3mg of biotinylated HSV-2 infected or uninfected (control) Vero cells overnight at 4°C in 500pl of BlockAid™ (ThermoFisher Scientific, B-10710). Beads are washed twice with 1% BSA in PBS and then IxlO⁶ beads/well are added in a 96 round bottom plate. Serum from immunized mice at 1 week post boost is heat-inactivated at 56°C for 30 min and diluted 1:5 in serum-free RPMI. 50pl of diluted serum is added to wells that contained the HSV lysates or control cell lysates coated beads and incubated for 2 h at 37°C. 2xl0⁴ cells/well THP-1 cells are added to each at a final volume of 200|al/well and incubated for 8 hr at 37°C at 5% CO2. Subsequently, lOOpl of supernatant is removed and stored at -20°C then resuspended with lOOpl 4% paraformaldehyde. Samples are then read on 5-laser LSRII flow cytometer (Becton Dickenson, San Diego) at the Einstein Flow Cytometry Core Facility. Phagocytic score is reported by gating on events representing THP-1 cells then applying the following equation: [(% of cells bead positive X MFI of cells positive for beads)/10⁶] using FlowJo software (version 10, Tree Star Inc.). IFN-y secretion from activated THP-1 cells via antibody phagocytosis is determined by analyzing stored cultured supernatants using a Milliplex human custom immunoassay (Millipore, Danvers, MA) and a Euminex Magpix® system as previously described.

[0225] Virus detection in tissue. Skin and dorsal root ganglia (DRG) are weighed and homogenized as described above. Supernatants of homogenized tissue are then overlaid on confluent Vero cell monolayers (2xl0⁵ cells/well in a 48-well plate) for 1 h. Wells are washed with PBS and then with 199 medium (Gibco®) containing 1% heat-inactivated FBS, overlaid with 0.5% methylcellulose and incubated at 37°C for 48 h. Cells are fixed with 2% paraformaldehyde, stained with a crystal violet solution and the number of PFU quantified. Neuronal ex-vivo co-culture assays are performed as previously described in the art.

[0226] Immunizations and viral challenge studies: Female mice will be vaccinated with 5 x 10⁶ pfu of AgD2P (grown on VerB::gDl cells or VerB::gD1.6C), uninfected control VerB::gDl cell lysates, or 5pg of recombinant gD-2 protein combined with 150 pg of aluminum (Alum) (Imject Alum; Pierce Biotechnology, Rockland, IE) and 12.5 pg of monophosphoryl lipid A (MPL) (Invivogen, San Diego, CA) (rgD-2/Alum-MPL). Vaccines will be administered subcutaneously at 4 weeks of age (prime) and at 7 weeks (boost) in a final volume of 100 pl/mouse. One week after boost, immunized females will be housed with males (2: 1) and monitored for a vaginal plug. Male mice will subsequently be removed and the pregnant females monitored daily until delivery. Pups will be inoculated intranasally on days 1, 7 or 14 of life with 5pl of virus administered by micropipette to both nares at a dose that resulted in 90% lethality (LD90) in unvaccinated 7 day-old neonatal mice (LD90) (typically' 10⁵ pfu/mouse of HSV-1 (B3xl.l) or 10³-10⁴ pfu/mouse HSV-2 (4674)). Pups will be monitored daily for signs of disease and will immediately be euthanized if they develop signs of encephalitis (e.g., paralysis, tremors, gai instability, hunched posture). [0227] Detection of HSV DNA by quantitative polymerase chain reaction (qPCR): At time of euthanasia (when mice succumbed to disease or day 14 post-challenge), trigeminal ganglia will be collected, DNA will be extracted using DNeasy® Blood and Tissue (Qiagen) and 10 ng DNA per sample will be assayed for presence of HSV by real-time qPCR (Absolute™ qPCR ROX Mix (Thermo Scientific) using primers that target HSV polymerase (UL30) (forward primer sequence, 5’-GGCCAGGCGCTTGTTGGTGTA-3’ (SEQ ID NO:29); reverse primer sequence, 5’-ATCACCGACCCGGAGAGGGA-3’ (SEQ ID NO:30); probe sequence, 5’-CCGCCGAACTGAGCAGACACCCGC-3’ (SEQ ID NO:31)) (Integrated DNA Technologies). Mouse P-actin will be used as a loading control (Applied Biosystems, Foster City, CA) and qPCR will be run in an Applied Biosystems QuantStudio 7 Flex. Samples with fewer than four copies will be considered negative.

[0228] Quantification of antibody responses, neutralizing titers and FcyR activation: HSV-specific total or isotype specific IgG will be determined by a previously described enzyme-linked immunosorbent assay (ELISA) in serum or in breastmilk. Breastmilk will be collected from pregnant female mice on days 8-12 post-parturition by separating them from their offspring for at least 2 hours and administration of a single-dose of 2 lU/kg of oxytocin via intraperitoneal injection 5 minutes before milking. Droplets of milk will be manually expressed and then pipetted into sterile Eppendorf tubes and frozen at -20°C until use. Briefly, ELISA plates will be coated with Vero cell lysates harvested 24 h after infection with HSV-l(B3xl.l) or HSV-2(4674) at a multiplicity of Infection (MOI) of 0.1 pfu/cell or uninfected control lysates. Serial dilutions of serum will be incubated with the coated plates overnight at 4°C and bound IgG, IgGl, IgG2, or IgG3 will be quantified using specific biotin-labeled secondary Abs (BD Biosciences, San Jose, CA). The anti-HSV IgG level will be determined after subtracting optical densitometry (OD) values obtained for uninfected cell lysates. The neutralization titer will be defined as the highest heat-inactivated serum dilution to result in 50% reduction in the number of plaques relative to plaques formed in the absence of immune serum (-100 plaques per well). FcyRIV activation will be determined using the mFcylV ADCC Reporter Bioassay (Promega, Madison, WI) with HSV-2(4674) or HSV- 1(B 3x1.1) -infected Vero cells as the targets.

[0229] Antibody-Dependent Cell-Mediated Killing (ADCK) assay: Effector immune cells will be isolated from spleens and livers and pooled from 5-10 naive neonatal or 3-5 naive adult mice. Red blood cells will be removed by lysis with ammonium-chloride- potassium (ACK) lysis buffer (Gibco, Grand Island, NY). Pooled cells will be counted and resuspended in complete RPMI media. Target cells will be HaCaT cells infected for 4 h at 37°C with a MOI of 1 pfu/cell of HSV-2 (333) ZAG, or as a control, uninfected HaCaT cells. The targets will be dissociated with CellStripper™ (Coming®), resuspended at 10⁷ cells/ml in DMEM and 2 x 10⁵ cells (in lOOpl) will be added to each well of a 96-well U bottom plate. The targets will be incubated with pooled heat- inactivated serum (1:5 dilution in DMEM) isolated from HSV-2 AgD2P or VerB::gDl or VerB::gD1.6C control vaccinated adult mice for 15 minutes at room temperature. The pooled effector cells will be then added at an effector to target cell ratio of 10: 1 for 1 hr. The cultures will be stained with Live/Dead Red fixable dye (Invitrogen) for 30 min, fixed with 5% paraformaldehyde in phosphate buffered saline (PBS) and resuspended in FACs buffer (2% heat-inactivated FBS in PBS). The cells will be analyzed by flow cytometry on an LSRII (Becton Dickinson) and the percentage of GFP-positive (HSV-2 infected) dead cells determined using FlowJo analysis software.

[0230] FcyR expression: Immune cells isolated as for the ADCK assay from spleens and livers of adult or neonatal mice will be adjusted to IxlO⁷ cells/mL in FACS Buffer and 100 pl of cell suspension will be added to a 96-well round-bottom microtiter plate. Cells will be stained for 30 minutes at 4°C in the dark with CD1 lb-APC-Cy7, Ey6G-AlexaFluor® 700, Ey6C-PE, F4/80-APC, CD3-PE-Cy7, Eive/dead Red fixable dye (BD Biosciences, Invitrogen) and relevant anti-mFcy-FITC (I, II, III, IV; BioEegend) and then fixed with 4% paraformaldehyde in PBS for 15 min. For analysis, doublets will be excluded based on forward and side light scatter area (FSC-A, SSC-A) versus width (FSC-W, SSC-W). Cell populations will be defined as follows: macrophages, CDl lbintF4/80hi; neutrophils, CDl lbhiF4/801oLy6GhiLy6Cint; monocytes, CDl lbhiF4/801oLy6GintLy6Chi. Samples will be read on LSRII flow cytometer (BD Biosciences) and data will be analyzed using FlowJo analysis software. The percent of FcyR positive cells will be calculated based on %FM0 FITC positive-%FITC positive.

[0231] Statistical Analysis: Statistical analyses will be performed using GraphPad Prism version 6 or 7 software (GraphPad Software Inc., San Diego, CA). A p value less than or equal to 0.05 will be considered statistically significant. Survival curves will be compared using Mantel-Cox test; other results will be compared using analysis of variance with multiple testing as indicated.

RESULTS

[0232] To determine if maternal vaccination could protect pups from HSV postnatally, adult female mice will be vaccinated with two doses of HSV-2 AgD2P, rgD- 2/Alum-MPL, or as a control, an uninfected VD60, and an uninfected Verb::gDl cell lysate prior to mating, and their pups challenged intranasally with clinical isolates of HSV-1 or HSV-2 at an -LD90 dose. Pups will be initially challenged on day of life 7, which is presumed to correspond immunologically to a term human infant. Protection against lethal HSV-1 and HSV-2 disease will be observed in pups bom to HSV-2 AgD2P-immunized dams compared to pups born to control-vaccinated dams (p< 0.001) . Protection from challenge in different aged pups will be assessed.

[0233] To determine whether the protection against lethal disease observed in 7 or 14 day old pups is associated with protection against the establishment of a latent reservoir, trigeminal ganglia will be assayed for HSV DNA by qPCR either at time of demise or, in the surviving mice, on day 14 post-viral challenge to measure whether a significant reduction in viral DNA is recovered from pups bom to dams vaccinated with HSV-2 AgD2P compared to mice vaccinated with rgD2-Alum/MPL or VerB::gDl or VerB::gD1.6C control lysates, which will indicate level of viral clearance by maternally derived Abs.

[0234] Protection correlates with FcyRIV-activating antibodies: HSV binding (ELISA), neutralizing and FcyRIV-activating Ab levels will be quantified in blood obtained from pups who were infected on Day 7 of life with HSV-1 or HSV-2 at time of euthanasia for disease (mean Day 6 post-challenge) or at the end of the experiment for pups who survived challenge (Day 14 post-challenge) to determine if vaccination with gD-2/Alum-MPL and HSV-2 AgD2P elicited similar titers of HSV-1 or HSV-2 binding IgG Abs or little or no HSV-specific IgG, The functionality of the Abs in semm of pups bom to dams immunized with gD-2/Alum-MPL will be tested for neutralizing activity and activation of the murine FcyRIV indicative of ADCC activity

[0235] Optimal protection requires antibodies acquired from breastmilk: A difference in protection observed when pups were challenge on Day 1 of life compared to Day 7 or 14 suggests that transplacentally acquired antibodies alone are not sufficient and/or there are age-dependent differences in FcyR effector cell function(s). To explore these possibilities, pups bom to HSV-2 AgD2P-immunized mothers will be switched at birth and nursed by mothers who had been immunized with VerB::gDl or VerB::gD1.6C cell lysates (only transplacentally-acquired Abs) and, conversely, mice bom to VerB::gDl or VerB::gD1.6C control-immunized mothers were nursed by HSV-2 AgD2P-immunized mothers (only breastmilk-acquired Abs). Positive controls will be mice that were both born to and nursed by HSV-2 AgD2P-immunized mothers and negative controls will be mice that were born to and nursed by VerB::gDl or VerB::gD1.6C control lysate-immunized mothers. The pups will be challenged intranasally on Days 7 or 14 of life and monitored for two weeks for protection from an LD90 of HSV-1 and HSV-2 Protection against latency will also be assessed in neonates which have received both transplacental and breastmilk Abs.

[0236] Results will be correlated with the amount of neonatal Ab levels present in the pup’s serum at time of challenge. For these studies, blood will be obtained on Days 3, 7 or 14 in “switched” mice who were not challenged with virus. Neonatal levels of HSV-2 specific IgG will be measure in mice bom to, but not nursed by a HSV-2 AgD2P-immunized mother and in mice that were only nursed by a HSV-2 AgD2P-immunized mother. The presence of HSV-specific antibodies in breastmilk will be confirmed in samples collected on Days 8-12.

[0237] Decreased protection in mice challenged on day 1 is linked to antibodydependent cell killing activity: Expression of any of the FcyRs will be observed in immune cell sub-populations isolated from the livers and spleens of 1-3 day old compared to adult mice. The ADCK effector function of the immune cells isolated from 1-3 day old pups will be compared to Day 7 or adult cells. For these studies, pooled single cell suspensions of immune cells isolated from different aged mice (effector cells) will be incubated with HSV- 2(ZAG) (which expresses GFP)-infected HaCAT target cells in the presence of serum harvested from HSV-2 AgD2P or VerB::gDl or VerB::gD1.6C immunized adult mice and the ability of the effector cells to kill the target cells will be quantified.

[0238] Comparing convalescent serum from mice infected with a sublethal dose of HSV-2 for protecting pups from subsequent viral challenge. Adult female mice (C567BE/6) will be infected intranasally with a dose of 10⁵ pfu HSV-2 (4674) or with PBS (control). Two weeks after infection, serum will be collected from the mice and tested for HSV-2 specific antibodies and the type of antibodies and their neutralizing activity assessed, compared to serum from HSV-2 AgD2P-vaccinated mice. The seropositive or control mice will be subsequently mated and the resulting pups will be challenged intranasally on Day 7 with HSV-2, and protection will be observed.

[0239] Results of the murine studies will demonstrate whether immunization of female adult mice (prior to conception) with HSV-2 AgD2P elicits Ab responses that protect neonatal mice (7-14 days of life) from intranasal viral challenge with clinical isolates of HSV-1 or HSV-2 and if protection correlates with the ability of the Abs to activate the murine FcyRIV to elicit ADCK. Per clinical observation that neutralizing Abs, which are the dominant response to natural infection, provide partial protection against neonatal disease. REFERENCES

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Claims

1. An isolated, recombinant herpes simplex virus-2 (HSV-2) having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof.

2. The isolated, recombinant HSV-2 of Claim 1, further comprising a surface glycoprotein on a lipid bilayer thereof which is a herpes simplex virus- 1 (HSV-1) glycoprotein D.

3. The isolated, recombinant HSV-2 of Claim 1, further comprising a non-HSV-2 viral surface glycoprotein on a lipid bilayer thereof.

4. The isolated, recombinant HSV-2 of Claim 1, further comprising a bacterial surface glycoprotein on a lipid bilayer thereof.

5. The isolated, recombinant HSV-2 of Claim 1, further comprising a parasitic surface glycoprotein on a lipid bilayer thereof, wherein the parasite is a parasite of a mammal.

6. The isolated, recombinant HSV-2 of any of Claims 1-5, wherein the HSV-2 glycoprotein D-encoding gene is an HSV-2 Us6 gene.

7. The isolated, recombinant HSV-2 of any of Claims 2-6, wherein the surface glycoprotein is encoded by a transgene that has been inserted into the genome of the recombinant HSV-2.

8. The isolated, recombinant HSV-2 of any of Claims 2-6, wherein the surface glycoprotein is present on a lipid bilayer thereof by way of infecting a cell with a recombinant HSV-2 having a deletion of an HSV-2 glycoprotein D-encoding gene and its promoter, wherein the cell is or has been transfected to express the surface glycoprotein on a cell membrane thereof, and wherein the recombinant HSV-2 comprising the surface glycoprotein present on a lipid bilayer is produced from the cell.

9. A virion of an isolated, recombinant HSV-2 having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof.

10. The virion of Claim 9, further comprising a surface glycoprotein on a lipid bilayer thereof which is a herpes simplex virus- 1 (HSV-1) glycoprotein D.

11. The virion of Claim 9, further comprising a non-HSV-2 viral surface glycoprotein on a lipid bilayer thereof.

12. The virion of Claim 9, further comprising a bacterial surface glycoprotein on a lipid bilayer thereof.

13. The virion of Claim 9, further comprising a parasitic surface glycoprotein on a lipid bilayer thereof, wherein the parasite is a parasite of a mammal.

14. The virion of any of Claims 9-13, wherein the HSV-2 glycoprotein D- encoding gene is an HSV-2 Us6 gene.

15. The virion of any of Claims 10-14, wherein the surface glycoprotein is encoded by a transgene that has been inserted into the genome of the recombinant HSV-2.

16. The virion of any of Claims 9-14, wherein the surface glycoprotein is present on a lipid bilayer thereof by way of infecting a cell with a recombinant HSV-2 having a deletion of an HSV-2 glycoprotein D-encoding gene and its promoter, wherein the cell is or has been transfected to express the surface glycoprotein on a cell membrane thereof, and wherein the virion comprising the surface glycoprotein present on a lipid bilayer thereof is produced from the cell.

17. An isolated cell comprising therein a virus of any of Claims 1-8 or a virion of any of Claims 9-16, wherein the cell is not present in a human being.

18. The cell of Claim 17, comprising a heterologous nucleic acid encoding a HSV-1 glycoprotein D.

19. The cell of any of Claims 17 or 18, expressing an HSV-1 glycoprotein D on a membrane thereof.

20. The cell of any of Claims 18 or 19, wherein the heterologous nucleic acid encoding HSV-1 glycoprotein D comprises a minimal promoter or a modified minimal promoter, a nucleic acid encoding a HSV-1 glycoprotein D, and a polyadenylation signal sequence.

21. The cell of any of Claim 18-20, wherein the heterologous nucleic acid encoding a HSV-1 glycoprotein D comprises the expression cassette comprising HSV-1 gD minimal promoter sequence or a modified minimal promoter sequence, HSV-1 encoding nucleic acid, and a polyadenylation sequence.

22. The cell of any of Claims 18-21, wherein the heterologous nucleic acid is inserted in the cell’s genome.

23. The cell of any of Claims 17-22, wherein the cell is a Vero cell.

24. The cell of Claim 23, wherein the cell is VerB::gDl or VerB::gD1.6C.

25. A vaccine composition comprising the virus of any of Claims 1-8, or the virion of any of Claims 9-16 and a pharmaceutically acceptable carrier.

26. A HSV-2 virus or virion produced in the cell of Claim 24, wherein the HSV-2 virus or virion is a recombinant herpes simplex virus-2 (HSV-2), has a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof, and comprises an HSV-1 glycoprotein D on a lipid bilayer thereof.

27. A vaccine composition comprising the virus of Claim 26 and a pharmaceutically acceptable carrier.

28. The vaccine composition of any of Claims 25 or 27, further comprising an adjuvant.

29. A composition comprising the virus of any of Claims 1-8, or the virion of any of Claims 9-16, and a pharmaceutically acceptable carrier, wherein the genome of the virus or virion comprises at least a deletion of a second gene, wherein the second gene is necessary for HSV-2 viral replication.

30. A pharmaceutical composition comprising the virus of any of Claims 1-8, or the virion of any of Claims 9-16, and a pharmaceutically acceptable carrier.

31. A pharmaceutical composition comprising the virus of Claim 26, and a pharmaceutically acceptable carrier.

32. A method of eliciting an immune response in a subject comprising administering to the subject an amount of (i) the virus of any of Claims 1, 2, 6-8; (ii) the virion of any of Claims 9, 10 14-16, or 26 (iii) the vaccine of Claim 25, 27, or 28; (iv) the composition of Claim 28; or (v) the pharmaceutical composition of Claim 29 or 30.

33. A method of inducing antibody dependent cellular cytotoxicity (ADCC) against HSV-1 and/or HSV-2 in a subject comprising administering to the subject an amount of (i) the virus of any of Claims 1, 2, 6-8; (ii) the virion of any of Claims 9, 10 14-16, or 26 (iii) the vaccine of Claim 25, 27, or 28; (iv) the composition of Claim 28; or (v) the pharmaceutical composition of Claim 29 or 30.

34. A method of treating 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 (i) the virus of any of Claims 1, 2, 6-8; (ii) the virion of any of Claims 9, 10 14-16, or 26 (iii) the vaccine of Claim 25, 27, or 28; (iv) the composition of Claim 28; or (v) the pharmaceutical composition of Claim 29 or 3.

35. The method of Claim 34, wherein the disease caused by an HSV-2 infection comprises a genital ulcer.

36. The method of Claim 34, wherein the disease caused by an HSV-2 infection comprises a skin vesicle or skin ulcer.

37. A method of vaccinating a subject for HSV-2 infection comprising administering to the subject an amount of (i) the virus of any of Claims 1, 2, 6-8; (ii) the virion of any of Claims 9, 10 14-16, or 26 (iii) the vaccine of Claim 25, 27, or 28; (iv) the composition of Claim 28; or (v) the pharmaceutical composition of Claim 29 or 30.

38. A method of immunizing a subject against HSV-2 infection comprising administering to the subject an amount of (i) the virus of any of Claims 1, 2, 6-8; (ii) the virion of any of Claims 9, 10 14-16, or 26 (iii) the vaccine of Claim 25, 27, or 28; (iv) the composition of Claim 28; or (v) the pharmaceutical composition of Claim 29 or 30.

39. The method of Claim 37 or 38, wherein the subject is administered a subcutaneous priming dose and is administered a second dose subcutaneously or intravaginally.

40. A method of producing a virion of a recombinant herpes simplex virus-2 (HSV-2) having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof and comprising an HSV-1 glycoprotein D on a lipid bilayer thereof, the method comprising infecting a cell comprising a heterologous nucleic acid encoding the HSV-1 glycoprotein D with a recombinant herpes simplex virus-2 (HSV-2) having the complete deletion of the HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof under conditions permitting replication of the recombinant herpes simplex virus-2 (HSV-2), and recovering the recombinant HSV-2 virion comprising the HSV-1 glycoprotein D on the lipid bilayer thereof produced by the cell.

41. The method of Claim 40, wherein the cell expresses the HSV-1 glycoprotein D on a cell membrane thereof.

42. The method of Claim 40 or 41, wherein the cell is VerB::gDl or VerB::gD1.6C.

43. A method of producing a virion of a recombinant herpes simplex virus-2 (HSV-2) having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof and comprising a non-HSV-2 surface glycoprotein on a lipid bilayer thereof, the method comprising infecting a cell comprising a heterologous nucleic acid encoding the non-HSV-2 surface glycoprotein with a recombinant herpes simplex virus- 2 (HSV-2) having the complete deletion of the HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof under conditions permitting replication of the recombinant herpes simplex virus-2 (HSV-2), and recovering a recombinant HSV-2 virion comprising the non-HSV-2 surface glycoprotein on the lipid bilayer thereof produced by the cell.

44. The method of Claim 43, wherein the cell expresses the non-HSV-2 surface glycoprotein on a cell membrane thereof.

45. The method of Claim 43 or 44, wherein the non-HSV-2 surface glycoprotein is a viral non-HSV-2 surface glycoprotein.

46. The method of Claim 43 or 44, wherein the non-HSV-2 surface glycoprotein is a bacterial non-HSV-2 surface glycoprotein or is a parasite non-HSV-2 surface glycoprotein.

47. A recombinant nucleic acid having the same sequence as a genome of an HSV-2 except that the nucleic acid sequence does not comprise a sequence encoding a complete HSV-2 glycoprotein D and its promoter.

48. An isolated, recombinant herpes simplex virus-2 (HSV-2) having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof for treating or preventing an HSV-2 infection in a subject.

49. An isolated, recombinant herpes simplex virus-2 (HSV-2) having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof for treating or preventing an HSV-1 infection in a subject.

50. The isolated, recombinant HSV-2 of Claim 48 or 49, further comprising a herpes simplex virus- 1 (HSV-1) glycoprotein D on a lipid bilayer thereof.

51. The isolated, recombinant HSV-2 of any of Claims 48, 49 or 50, wherein the HSV-2 glycoprotein D-encoding gene is an HSV-2 Us6 gene.

52. A virion of an isolated, recombinant HSV-2 having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof for treating or preventing an HSV-2 infection in a subject.

53. The virion of Claim 52, further comprising an HSV-1 glycoprotein D on a lipid bilayer thereof.

54. The virion of Claim 52 or 53, wherein the HSV-2 glycoprotein D-encoding gene is an HSV-2 Us6 gene.

55. The virus of any of Claims 48-51, or the virion of any of Claims 52-54, wherein the HSV-2 infection causes a genital ulcer.

56. A method of treating an HSV-1 infection, or HSV-1 and HSV-2 co-infection, in a subject, or treating a disease caused by an HSV-2 infection or HSV-1 and HSV-2 coinfection in a subject comprising administering to the subject an amount of (i) the virus of any of Claims 1, 2, 6-8; (ii) the virion of any of Claims 9, 10 14-16, or 26 (iii) the vaccine of Claim 25, 27, or 28; (iv) the composition of Claim 28; or (v) the pharmaceutical composition of Claim 29 or 30.

57. A method of vaccinating a subject for an HSV-1 infection, or HSV-1 and HSV-2 co-infection, comprising administering to the subject an amount of (i) the virus of any of Claims 1, 2, 6-8; (ii) the virion of any of Claims 9, 10 14-16, or 26 (iii) the vaccine of Claim 25, 27, or 28; (iv) the composition of Claim 28; or (v) the pharmaceutical composition of Claim 29 or 30.

58. A method of immunizing a subject against an HSV-1 infection, or HSV-1 and HSV-2 co-infection, comprising administering to the subject an amount of (i) the virus of any of Claims 1, 2, 6-8; (ii) the virion of any of Claims 9, 10 14-16, or 26 (iii) the vaccine of Claim 25, 27, or 28; (iv) the composition of Claim 28; or (v) the pharmaceutical composition of Claim 29 or 30.

59. An isolated, recombinant herpes simplex virus-2 (HSV-2) having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof and further comprising a heterologous antigen of a pathogen.

60. The isolated, recombinant HSV-2 of Claim 59, comprising the heterologous antigen of a pathogen on a lipid bilayer thereof.

61. The isolated, recombinant HSV-2 of Claim 59 or 60, wherein the pathogen is bacterial or viral.

62. The isolated, recombinant HSV-2 of Claim 59 or 60, wherein the pathogen is a parasite of a mammal.

63. The isolated, recombinant HSV-2 of any of Claims 59-62, wherein the HSV-2 glycoprotein D-encoding gene is an HSV-2 Us6 gene.

64. The isolated, recombinant HSV-2 of any of Claims 59-63, wherein the heterologous antigen is encoded by a transgene that has been inserted into the genome of the recombinant HSV-2.

65. The isolated, recombinant HSV-2 of Claim 64, wherein the transgene is a M. tuberculosis biofilm-encoding gene or wherein the transgene is an HIV gpl20-encoding gene.

66. A method of inducing antibody dependent cell mediated cytotoxicity (ADCC) against an antigenic target in a subject comprising administering to the subject an amount of an isolated, recombinant herpes simplex virus-2 (HSV-2), the HSV-2 having a complete deletion of an HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof and further comprising a heterologous antigen on a lipid bilayer thereof.

67. The method of Claim 66, wherein the heterologous antigen is a surface antigen of the antigenic target.

68. The method of Claim 66 or 67, wherein the heterologous antigen is a parasite antigen.

69. The method of any of claim 66-68, wherein the heterologous antigen is a bacterial antigen or a viral antigen.

70. The method of any of Claims 66-69, wherein the antigenic target is a virus and is a HSV-1, a HSV-2, a Lassa virus, a human immunodeficiency virus, an RSV, an enterovirus, an influenza virus, a parainfluenza virus, pig corona respiratory virus, a lyssavirus, a bunyavirus, or a filovirus.

71. The method of any of Claims 66-69, wherein the antigenic target is a bacteria and is Mycobaterium tuberculosis, M. ulcerous, M. marinum, M. leprae, M. absenscens, Chlamydia trachomatis, Neisseria gonorrhoeae or Treponema pallidum.

72. The method of Claim 66, wherein the isolated, recombinant HSV-2 surface antigen is a M. tuberculosis biofilm-encoding gene or wherein the transgene is an HIV gpl20-encoding gene.

73. A method of eliciting an immune response in a first subject against an HSV-2 and/or HSV-1 infection, comprising effectuating passive transfer to the first subject of an amount of a product from a second subject immunized with HSV-2 having a complete deletion of the HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof and wherein said HSV-2 is phenotypically complemented with a herpes simplex virus- 1 (HSV-1) glycoprotein D by propagating said HSV-2 in a complementing cell expressing said HSV-1 glycoprotein D, wherein the complementing cell is VerB::gDl or VerB::gD1.6C, and wherein the product comprises antibodies induced thereby.

74. The method of Claim 73, wherein the product comprises serum.

75. The method of Claim 73, wherein the first subject and second subject are human.

76. The method of Claim 74, wherein the first subject and second subject are human.

77. A method of eliciting an immune response in a first subject against an HSV-2 and/or HSV-1 infection, comprising effectuating passive transfer to the first subject of an amount of a product from a pregnant female immunized with HSV-2 having a complete deletion of the HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof and wherein said HSV-2 is phenotypically complemented with a herpes simplex virus- 1 (HSV-1) glycoprotein D by propagating said HSV-2 in a complementing cell expressing said HSV-1 glycoprotein D, wherein the complementing cell is VerB::gDl or VerB::gD1.6C, and wherein the product comprises antibodies induced thereby, wherein the first subject is a fetus or neonate.

78. The method of Claim 77, wherein the product comprises serum of the pregnant female.

79. The method of Claim 77 or Claim 78, wherein the product comprises breast milk of the pregnant female.

80. The method of any of Claims 77-79, wherein the first subject is a fetus.

81. The method of any of Claims 77-79, wherein the first subject is a neonate.

82. The method of Claim 81, wherein the first subject is bom from a second subject.

83. The method of Claim 77, wherein the pregnant female is pregnant with the fetus.

84. The method of any of Claims 77-83, wherein the first subject and the pregnant female are human.

85. The method of any of Claims 77-84, wherein the product comprises antibodies elicited by immunization of the pregnant female with the HSV-2 having the deletion.

86. The method of any of Claims 75-83, wherein the method elicits an immune response in the first subject against an HSV-2.

87. The method of Claims 86, wherein the immune response is an antibody dependent cellular cytotoxicity against HSV-2.

88. The method of any of Claims 77-86, wherein the method elicits an immune response in the first subject against an HSV-1.

89. The method of Claims 88, wherein the immune response is an antibody dependent cellular cytotoxicity against HSV-1.

90. The method of any of Claims 77-89, wherein the product further comprises an immunological adjuvant.

91. The method of Claim 90, wherein the product further comprises an HSV-2 having a complete deletion of the HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof and wherein said HSV-2 is phenotypically complemented with a herpes simplex virus- 1 (HSV-1) glycoprotein D by propagating said HSV-2 in a complementing cell expressing said HSV-1 glycoprotein D, wherein said complementing cell is VerB::gDl or VerB::gD1.6C.

92. A method of inhibiting a perinatal HSV-1 and/or HSV-2 infection in a neonate comprising administering to a female pregnant with a fetus which will become the neonate an amount of an HSV-2 having a complete deletion of the HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof and wherein said HSV-2 is phenotypically complemented with a herpes simplex virus- 1 (HSV-1) glycoprotein D by propagating said HSV-2 in a complementing cell expressing said HSV-1 glycoprotein D, wherein said complementing cell is VerB::gDl or VerB::gD1.6C, effective to inhibit a perinatal HSV-1 and/or HSV-2 infection in a neonate.

93. A method of inhibiting HSV-1 and/or HSV-2 viral dissemination from a mother to her neonate comprising administering to the mother an amount of an HSV-2 having a complete deletion of the HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof and wherein said HSV-2 is phenotypic ally complemented with a herpes simplex virus- 1 (HSV-1) glycoprotein D by propagating said HSV-2 in a complementing cell expressing said HSV-1 glycoprotein D, wherein said complementing cell is VerB::gDl or VerB::gD1.6C, effective to inhibit HSV-1 and/or HSV-2 viral dissemination from a mother to her neonate.

94. The method of Claim 93, wherein the mother is pregnant with a fetus which will become the neonate.

95. The method of Claim 93 or Claim 94, wherein the HSV-1 infection or HSV-1 viral dissemination is inhibited.

96. The method of Claim 93 or Claim 94, wherein the HSV-2 infection or HSV-2 viral dissemination is inhibited.

97. The method of Claim 78, wherein an immune response is elicited against HSV-1.

98. The method of Claim 97, wherein the immune response is induction of antibody dependent cellular cytotoxicity against HSV-1.

99. The method of Claim 78, wherein an immune response is elicited against HSV-2.

100. The method of Claim 78, wherein the immune response is induction of antibody dependent cellular cytotoxicity against HSV-2.

101. The method of Claim 78, Claim 93 or Claim 95, wherein the complementing cell is a VerB::gDl cell.

102. The method of any of Claims 78 to 101, wherein the pregnant female, the mother, or the second subject is seronegative for HSV-1, seronegative for HSV-2, or seronegative for both HSV-1 and HSV-2.

103. The method of any of Claims 78 to 102, wherein the pregnant female or the mother is subcutaneously administered the HSV-2 having a deletion of the entire HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof.

104. The method of Claim 103, wherein the pregnant female or the mother is administered a subcutaneous boost dose of the HSV-2 having a deletion of the entire HSV-2 glycoprotein D-encoding gene and its promoter in the genome thereof after an initial subcutaneous administration of the HSV-2 having a deletion of the entire HSV-2 glycoprotein D-encoding gene and its promoter.

105. A transgenic Vero cell comprising a HSV-1 glycoprotein D (gDl) expression cassette integrated in a target locus on a chromosome of the Vero cell genome, wherein the target locus is a recombination sequence comprising a Bxbl attB sequence from Mycobacterium smegmatis, and wherein the expression cassette comprises a minimal native promoter, a gD-1 open reading frame, and a polyadenylation signal sequence.

106. The transgenic Vero cell of Claim 105, wherein the target locus comprises adeno-associated virus integration site 1 (AAVS1) on chromosome 6 of the Vero cell genome.

107. The transgenic Vero cell of Claim 106, wherein the attB site is between positions 1529 and 1530 of the AAVS1 locus, between positions 2155 and 2156 of the AAVS1 locus, between positions 2408 and2409 of the AAVS1 locus, or a combination thereof.

108. The transgenic Vero cell of any of claims 105-106, wherein the attB sequence has the sequence of GGCTTGTCGACGACGGCGGTCTCCGTCGTCAGGATCAT (SEQ ID NO: 7).

109. The transgenic Vero cell of any of Claims 105-108, wherein the gD-1 expression cassette comprising HSV-1 gD minimal promoter sequence, HSV-1 encoding nucleic acid, and a polyadenylation sequence is inserted between positions 1529 and 1530 of the AAVS1 locus.

110. The transgenic Vero cell of any of Claims 105-109, wherein the transgenic Vero cell comprises an attL sequence at a first end of the integrated gD-1 expression cassette and an attR sequence at a second end of the integrated gD-1 expression cassette.

111. The transgenic Vero cell of any of claims 105-110, wherein the expression cassette does not comprise a gene encoding an antibiotic resistance marker, a CMV sequence or a combination thereof.

112. A cell line comprising the Vero cell of any of Claims 105-111.

113. The cell line of Claim 112, wherein the cell line is VerB::gDl.

114. A transgenic Vero cell comprising a HSV-1 glycoprotein D (gDl) expression cassette integrated in a target locus on a chromosome of the Vero cell genome, wherein the target locus is a recombination sequence comprising a Bxbl attB sequence from Mycobacterium smegmatis, and wherein the expression cassette comprises a modified minimal native promoter in SEQ ID NO:32, a gD-1 open reading frame, and a polyadenylation signal sequence.

115. The transgenic Vero cell of Claim 114, wherein the target locus comprises adeno-associated virus integration site 1 (AAVS1) on chromosome 6 of the Vero cell genome.

116. The transgenic Vero cell of Claim 115, wherein the attB site is between positions 1529 and 1530 of the AAVS1 locus, between positions 2155 and 2156 of the AAVS1 locus, between positions 2408 and2409 of the AAVS1 locus, or a combination thereof.

117. The transgenic Vero cell of any of Claims 114-115, wherein the attB sequence has the sequence of GGCTTGTCGACGACGGCGGTCTCCGTCGTCAGGATCAT (SEQ ID NO: 7).

118. The transgenic Vero cell of any of Claims 113-117, wherein the gD-1 expression cassette comprising HSV-1 gD modified minimal promoter sequence of SEQ ID NO:32, HSV-1 encoding nucleic acid, and a polyadenylation sequence is inserted between positions 1529 and 1530 of the AAVS1 locus.

119. The transgenic Vero cell of any of Claims 113-118, wherein the transgenic Vero cell comprises an attL sequence at a first end of the integrated gD-1 expression cassette and an attR sequence at a second end of the integrated gD-1 expression cassette.

120. The transgenic Vero cell of any of Claims 114-119, wherein the expression cassette does not comprise a gene encoding an antibiotic resistance marker, a CMV sequence or a combination thereof.

121. A cell line comprising the Vero cell of any of Claims 113-120.

122. The cell line of Claim 121, wherein the cell line is VerB::gD1.6C.