HK1229721B - Recombinant herpes simplex virus 2 (hsv-2) vaccine vectors - Google Patents

Description

Recombinant Herpes Simplex Virus 2 (HSV-2) Vaccine Vector

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/946,965, filed March 3, 2014, and U.S. Provisional Application No. 62/080,663, filed November 17, 2014, the contents of which are hereby incorporated by reference.

Government Funding Statement

This invention was made with U.S. Government support under Grant Nos. AI-061679 and AI-51519 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

Background of the Invention

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

Herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2) persist as major health problems worldwide, disproportionately affecting developing countries and impoverished communities and fueling the HIV epidemic. Because no effective vaccines currently exist for HSV-1, HSV-2, or HIV, vaccines against these infections are urgently needed. HSV-1 is the leading cause of infectious blindness, while HSV-2 is the leading cause of genital ulcers worldwide, although HSV-1 is now more commonly associated with genital tract disease in developed countries. Genital herpes is a recurring, lifelong disease that can be stigmatizing and psychologically impact those who suffer from it. HSV-2 infection significantly increases the likelihood of acquiring and transmitting HIV, and vertical transmission of either serotype often results in severe morbidity or mortality in infants. Recent clinical trials of HSV-2 vaccines based on subunit formulations of viral glycoprotein D, alone or in combination with glycoprotein B (gD and gB), have failed despite inducing systemic neutralizing antibodies. Surprisingly, the HSV-2 gD subunit (gD-2) vaccine provides partial protection against HSV-1, but does not provide protection against HSV-2. Several attenuated viruses have been evaluated preclinically, but clinical studies have so far been limited to therapeutic applications (reducing relapse frequency) and have not yet shown efficacy. Therefore, new vaccine strategies must be engineered and evaluated.

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

Summary of the Invention

An isolated recombinant herpes simplex virus-2 (HSV-2) is provided, wherein the herpes simplex virus-2 has a deletion in its genome of the gene ( _Us6 ) encoding HSV-2 glycoprotein D.

Also provided is an isolated recombinant HSV-2 virion having a deletion of the gene ( _Us6 ) encoding HSV-2 glycoprotein D in its genome.

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.

Also provided is a vaccine composition comprising the recombinant HSV-2 virus as described herein or the virion as described herein.

Also provided is a composition comprising a recombinant HSV-2 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 essential for HSV-2 viral replication or virulence.

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

Also provided is a method of stimulating an immune response in a subject, the method comprising administering to the subject (i) a 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 stimulate an immune response in the subject.

Also provided is a method for treating HSV-1, HSV-2, or HSV-1 and HSV-2 co-infection in a subject, or treating a disease caused by HSV-1, HSV-2, or co-infection in a subject, comprising administering to the subject (i) a recombinant HSV-2 virus as described herein; (ii) its 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 HSV-1, HSV-2, or co-infection in the subject, or treating a disease caused by HSV-1, HSV-2, or co-infection.

Also provided is a method of vaccinating a subject against HSV-1, HSV-2, or a co-infection, the method comprising administering to the subject (i) a 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 the subject against HSV-1, HSV-2, or a co-infection.

Also provided is a method of immunizing a subject against HSV-1, HSV-2, or a co-infection, the method comprising administering to the subject (i) a 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 the subject against HSV-1, HSV-2, or a co-infection.

In one embodiment of the vaccines, compositions and pharmaceutical compositions and methods of use thereof, the amount of recombinant HSV-2 is an amount of pfu of recombinant HSV-2 effective to achieve the stated purpose.

Also provided is a method for producing a recombinant herpes simplex virus-2 (HSV-2) virion having a gene deletion encoding HSV-2 glycoprotein D in its genome and comprising HSV-1 or HSV-2 glycoprotein D on its lipid bilayer, the method comprising infecting a cell containing a heterologous nucleic acid encoding HSV-1 or HSV-2 glycoprotein D with a recombinant herpes simplex virus-2 (HSV-2) having a gene deletion encoding HSV-2 glycoprotein D in its genome under conditions that allow the replication of the recombinant herpes simplex virus-2 (HSV-2), and recovering the recombinant HSV-2 virions produced by the cells.

Also provided is a recombinant nucleic acid having a sequence identical to that of a wild-type HSV-2 genome, except that the recombinant nucleic acid does not contain a sequence encoding HSV-2 glycoprotein D.

Also provided is an isolated recombinant herpes simplex virus-2 (HSV-2) having a deletion in its genome of a gene encoding HSV-2 glycoprotein D for use in treating or preventing HSV-1, HSV-2, or co-infection in a subject.

Also provided is an isolated recombinant HSV-2 virion having a deletion in its genome of a gene encoding HSV-2 glycoprotein D for use in treating or preventing HSV-1, HSV-2, or co-infection in a subject.

An isolated recombinant herpes simplex virus-2 (HSV-2) is provided, wherein the herpes simplex virus-2 has a deletion in its genome of the gene encoding HSV-2 glycoprotein D.

Also provided is an isolated recombinant HSV-2 virion having a deletion of the gene encoding HSV-2 glycoprotein D in its genome.

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 humans.

A vaccine composition comprising a virus as described herein or a virus particle as described herein.

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 essential for HSV-2 viral replication.

Also provided are pharmaceutical compositions comprising a virus as described herein or a virosome as described herein and a pharmaceutically acceptable carrier.

Also provided is a method of stimulating an immune response in a subject, the method comprising administering to the subject (i) a virus as described herein; (ii) a viral particle 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 stimulate an immune response in the subject.

Also provided is a method for treating HSV-2 infection in a subject or treating a disease caused by HSV-2 infection in a subject, the method comprising administering to the subject (i) a virus as described herein; (ii) a viral particle 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 HSV-2 infection or treat a disease caused by HSV-2 infection in the subject.

Also provided is a method of vaccinating a subject against HSV-2 infection, the method comprising administering to the subject (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 the subject against HSV-2.

Also provided is a method of immunizing a subject against HSV-2 infection, the method comprising administering to the subject (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 the subject against HSV-2.

Also provided is a method for producing recombinant herpes simplex virus-2 (HSV-2) virions having a gene deletion encoding HSV-2 glycoprotein D in its genome and comprising HSV-1 glycoprotein D on its lipid bilayer, the method comprising infecting cells comprising a heterologous nucleic acid encoding HSV-1 glycoprotein D with a recombinant herpes simplex virus-2 (HSV-2) having a gene deletion encoding HSV-2 glycoprotein D in its genome under conditions that allow the replication of the recombinant herpes simplex virus-2 (HSV-2), and recovering the recombinant HSV-2 virions comprising HSV-1 glycoprotein D on their lipid bilayer produced by the cells.

Also provided is a method for producing recombinant herpes simplex virus-2 (HSV-2) virions having a deletion of the gene encoding HSV-2 glycoprotein D in its genome and comprising a non-HSV-2 surface glycoprotein on its lipid bilayer, the method comprising infecting a cell comprising a heterologous nucleic acid encoding a non-HSV-2 surface glycoprotein with a recombinant herpes simplex virus-2 (HSV-2) having a deletion of the gene encoding HSV-2 glycoprotein D in its genome under conditions that allow the replication of the recombinant herpes simplex virus-2 (HSV-2), and recovering the recombinant HSV-2 virions comprising the non-HSV-2 surface glycoprotein on its lipid bilayer produced by the cell.

Also provided is a recombinant nucleic acid having a sequence identical to that of the HSV-2 genome, except that the sequence does not contain a sequence encoding HSV-2 glycoprotein D.

Also provided is an isolated recombinant herpes simplex virus-2 (HSV-2) having a deletion in its genome of a gene encoding HSV-2 glycoprotein D for use in treating or preventing HSV-2 infection in a subject.

Also provided is an isolated recombinant herpes simplex virus-2 (HSV-2) having a deletion in its genome of a gene encoding HSV-2 glycoprotein D for use in treating or preventing HSV-1 infection in a subject.

Also provided is an isolated recombinant HSV-2 virion having a deletion of the gene encoding HSV-2 glycoprotein D in its genome for use in treating or preventing HSV-2 infection in a subject.

Also provided is a method for treating HSV-1 infection or HSV-1 and HSV-2 co-infection in a subject, or treating a disease caused by HSV-2 infection or HSV-1 and HSV-2 co-infection in a subject, the method comprising administering to the subject (i) a virus as described herein; (ii) a viral particle 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 HSV-2 infection or a disease caused by HSV-2 infection in the subject, or an amount effective to treat HSV-1 and HSV-2 co-infection or a disease caused by HSV-1 and HSV-2 co-infection in the subject.

Also provided is a method of vaccinating a subject against HSV-1 infection or co-infection with HSV-1 and HSV-2, the method comprising administering to the subject (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 the subject against HSV-1 infection or co-infection with HSV-1 and HSV-2.

Also provided is a method of immunizing a subject against HSV-1 infection or co-infection with HSV-1 and HSV-2, the method comprising administering to the subject (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 the subject against HSV-1 infection or co-infection with HSV-1 and HSV-2.

Also provided is an isolated recombinant herpes simplex virus-2 (HSV-2) having a deletion in its genome of the gene encoding HSV-2 glycoprotein D and further comprising a heterologous antigen of a pathogen.

Also provided is a method of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) against an antigenic target in a subject, the method comprising administering to the subject an isolated recombinant herpes simplex virus-2 (HSV-2) having a deletion in its genome of the gene encoding HSV-2 glycoprotein D and further comprising a xenoantigen on its lipid bilayer in an amount effective to induce antibody-dependent cell-mediated cytotoxicity (ADCC) against the antigenic target.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: HSV-2ΔgD initiates abortive infection: HSV-2ΔgD-/+ successfully replicates only in cells that provide gD in trans (e.g., VD60 [40, 41]), but not in cells that do not encode _US6 , such as Vero cells (ATCC CCL-81, green monkey kidney) or CaSki (ATCC CRL-1550, Homo sapiens, cervix). Replication-incompetent HSV-2ΔgD (ΔgD-/- obtained from Vero cells) is unable to infect cells that do not encode _US6 , such as Vero and CaSki.

Figure 2A-C: A. Severe combined immunodeficient (SCID) mice inoculated with up to ¹⁰⁷ plaque-forming units (pfu) of HSV-2ΔgD-/+ virus do not show signs of disease following high-dose intravaginal or subcutaneous inoculation. In contrast, SCID mice inoculated with wild-type virus at a 1,000-fold lower viral dose ( ¹⁰⁴ pfu) succumbed to disease. Survival curves are shown in A, epithelial scores (scores of 0 to 5) for evidence of erythema, edema, or genital ulceration are shown in B, and neurological scores (scores of 0 to 5) for evidence of neuronal infection are shown in C.

Figure 3A-C: Immunization with HSV-2ΔgD-/+ virus stimulates anti-HSV-2 antibodies. Although subcutaneous-subcutaneous immunization stimulates significant levels of systemic and mucosal (vaginal wash) anti-HSV-2 antibodies, subcutaneous-intravaginal immunization with HSV-2ΔgD-/+ stimulates lower levels of systemic anti-HSV-2 antibodies and no increase in antibody levels in vaginal washes. Anti-HSV-2 antibody levels in serum are shown in A and anti-HSV-2 antibody levels in vaginal washes are shown in B. Mice immunized with ΔgD-/+ showed neutralization of anti-HSV-2 antibodies in serum after challenge with highly virulent HSV-2. The neutralizing capacity of antibodies stimulated by ΔgD-/+ immunization is shown in C (*p<0.05; **p<0.01; ***p<0.001).

Figure 4A-C: A: CD8+ gBT-I T cell counts in the spleens of C57Bl/6 mice primed and boosted with TgT cells and subsequently primed and boosted with HSV-2ΔgD-/+ or VD60 lysate (control). B: Percentage of gBT-I memory T cells in the spleens of vaccinated or control mice. C: 14 days after the booster immunization, splenocytes were isolated and restimulated in vitro with gB498-505 peptide and analyzed for cytokine production 6 hours later by intracellular cytokine staining and flow cytometry. (*p<0.05; **p<0.01; ***p<0.001).

Figure 5A-F: Immunization with HSV-2 ΔgD-/+ (10 ⁶ pfu/mouse) protected mice from lethal HSV-2 challenge. Mice were primed subcutaneously and boosted subcutaneously or intravaginally 3 weeks apart and then challenged with LD ₉₀ of virulent wild-type HSV-2 (4674) 3 weeks after intravaginal booster immunization. While control (immunized with VD60 cell lysate) mice succumbed to disease, as shown by significant weight loss (A) and death (B), ΔgD-/+-immunized mice showed significantly fewer lesions. In addition, ΔgD-/+-immunized mice showed less epithelial disease (C) and neurological lesions (D) after lethal challenge. In addition, compared with mice immunized with VD60 cell lysate as a control, after intravaginal challenge with a lethal dose of virulent HSV-2, ΔgD-/+-vaccinated mice showed significantly less viral load in vaginal washes (E), vaginal tissue, and dorsal root ganglia (DRG) (F). No infectious virus was recovered from ΔgD-/+-immunized mice in vaginal washes on day 4 or vaginal tissue and DRG on day 5 (*p<0.05;**p<0.01;***p<0.001).

Figure 6A-C: After challenge with virulent HSV-2, mice immunized with HSV-2ΔgD-/+ secreted fewer inflammatory cytokines in vaginal washes. Mice immunized with HSV-2ΔgD-/+ secreted less TNF-α, IL-6, and IL-1β in vaginal washes than mice immunized with VD60 lysate and challenged with virulent HSV-2. Differences in inflammatory cytokine expression were observed at different time points after challenge (*p<0.05; **p<0.01; ***p<0.001).

Figure 7A-D: Immunization with HSV-2ΔgD-/+ recruits T cells to the site of infection and associated lymph nodes (LN). After being challenged with virulent HSV-2, mice immunized subcutaneously with ΔgD-/+ showed an increase in the percentage of activated anti-HSV-2gBT-I CD8+T cells (A) and CD4+T cells (B) in the sacral lymph nodes (LN). LN were extracted and incubated with UV-inactivated ΔgD-/- for 6 hours and then stained with antibodies for flow cytometric analysis. After being challenged with virulent HSV-2, mice immunized subcutaneously with ΔgD-/+ in the vagina showed an increase in the number of anti-HSV-2gBT-I CD8+T cells (C) and CD4+T cells (D). Vaginal tissue was processed to extract T cells and stained with antibodies for flow cytometric analysis. Cell counts were performed using (CountBright ^™ , Lifetechnologies). (*p<0.05;**p<0.01).

Detailed Description of the Invention

An isolated recombinant herpes simplex virus-2 (HSV-2) is provided, wherein the herpes simplex virus-2 has a deletion in its genome of the gene encoding HSV-2 glycoprotein D.

In one embodiment, the HSV-2 glycoprotein D comprises the amino acid sequence shown in SEQ ID NO: 1:

MGRLTSGVGTAALLVVAVGLRVVCAKYALADPSLKMADPNRFRGKN LPVLDQLTDPPGVKRVYHIQPSLEDPFQPPSIPITVYYAVLERACRSVLLHA PSEAPQIVRGASDEARKHTYNLTIAWYRMGDNCAIPITVMEYTECPYNKS LGVCPIRTQPRWSYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDW TEITQFILEHRARASKYALPLRIPPAACLTSKAYQQGVTVDSIGMLPRFIPE NQRTVALYSLKIAGWHGPKPPYTSTLLPPELSDTTNATQPELVPEDPEDSAL LEDPAGTVSSQIPPNWHIPSIQDVAPHHAPAAPSNPGLIIGALAGSTLAVLVI GGIAFWVRRRAQMAPKRLRLPHIRDDDAPPSHQPLFY (HSV-2 reference strain HG52)

In one embodiment, the isolated recombinant HSV-2 further comprises herpes simplex virus-1 (HSV-1) glycoprotein D on its lipid bilayer.

In one embodiment, the HSV-1 glycoprotein D comprises the amino acid sequence shown in SEQ ID NO: 2:

MGGAAARLGAVILFVVIVGLHGVRGKYALADASLKMADPNRFRGK DLPVLDQLTDPPGVRRVYHIQAGLPDPFQPPSLPITVYYAVLERACRSVLL NAPSEAPQIVRGASEDVRKQPYNLTIAWFRMGGNCAIPITVMEYTECSYN KSLGACPIRTQPRWNYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKIND WTEITQFILEHRAKGSCKYALPLRIPPSACLSPQAYQQGVDSIGMLPRFI PENQRTVAVYSLKIAGWHGPKAPYTSTLLPPELSETPNATQPELAPEDPEDS ALLEDPVGTVAPQIPPNWHIPSIQDAATPYHPPATPNNMGLIAGAVGGSLLA ALVICGIVYWMRRRTQKAPKRIRLPHIREDDQPSSHQPLFY (HSV-1 reference strain F)

In one embodiment, the gene encoding HSV-2 glycoprotein D is the HSV-2 _US6 gene. (For example, see Dolan et al., J Virol. 1998 March; 72(3): 2010–2021. (PMCID: PMC109494) regarding the HSV-2 genome and the _US6 gene, "The Genome Sequence of Herpes Simplex Virus Type 2," which is hereby incorporated by reference in its entirety).

Also provided is an isolated recombinant HSV-2 virion having a deletion of the gene encoding HSV-2 glycoprotein D in its genome.

In one embodiment, the virion further comprises on its lipid bilayer HSV-1 or HSV-2 glycoprotein D. In one embodiment, the gene encoding HSV-2 glycoprotein D is the HSV-2 _US6 gene.

In one embodiment, the virus further comprises on its lipid bilayer HSV-1 or HSV-2 glycoprotein D. In one embodiment, the gene encoding HSV-2 glycoprotein D is the HSV-2 _US6 gene.

An isolated cell is provided, comprising a recombinant HSV-2 genome lacking the HSV- _2US6 gene.

In one embodiment, the cell is a complementing cell that provides expressed HSV 1 or 2 glycoproteins that are not encoded by the recombinant HSV-2 genome. In one embodiment, the complementing cell comprises a heterologous nucleic acid encoding HSV-1 or HSV-2 glycoprotein D. In one embodiment, the cell expresses HSV-1 glycoprotein D on its membrane. In one embodiment of the cell, HSV-1 glycoprotein D is encoded by a heterologous nucleic acid that is an HSV-1 or HSV-2 glycoprotein D gene, or a nucleic acid having a sequence identical to an HSV-1 or HSV-2 glycoprotein D gene.

Also provided is a vaccine composition comprising a recombinant HSV-2 virus as described herein or a virion as described herein. In one embodiment, the vaccine comprises an immunoadjuvant. In one embodiment, the vaccine does not comprise an immunoadjuvant. In one embodiment of the vaccine, composition, or pharmaceutical composition comprising recombinant HSV-2 described herein, the HSV-2 is live.

Also provided is a composition comprising a recombinant HSV-2 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 essential for HSV-2 viral replication or virulence.

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

In one embodiment, the composition or pharmaceutical composition or vaccine is formulated so that it is suitable for subcutaneous administration to a human subject. In one embodiment, the composition or pharmaceutical composition or vaccine is formulated so that it is suitable for intravaginal administration to a human subject. In one embodiment, the composition or pharmaceutical composition or vaccine is formulated so that it is suitable for intramuscular, intranasal or mucosal administration to a human subject.

Also provided is a method of stimulating an immune response in a subject, the method comprising administering to the subject (i) a 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 stimulate an immune response in the subject.

Also provided is a method for treating an HSV-2 infection in a subject or treating a disease caused by HSV-1, HSV-2, or a co-infection in a subject, comprising administering to the subject (i) a 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 co-infection in the subject or to treat a disease caused by an HSV-1, HSV-2, or co-infection. In one embodiment, the method comprises treating an HSV-1 or HSV-2 lesion caused by HSV-1, HSV-2, or a co-infection. In one embodiment of the method, the disease caused by HSV-1, HSV-2, or a co-infection is a genital ulcer. In one embodiment of this method, the disease caused by HSV-1, HSV-2, or a co-infection is herpes, oral herpes, herpes whitlow, genital herpes, eczema herpeticum, herpes gladiatorum, HSV keratitis, HSV retinitis, HSV encephalitis, or HSV meningitis.

In the embodiments herein relating to methods of treating or vaccinating against HSV-1, HSV-2, or co-infection (i.e., simultaneous infection with HSV-1 and HSV-2), separate, individual embodiments are provided for treating HSV-1 infection, treating HSV-2 infection, treating co-infection, vaccinating against HSV-1 infection, vaccinating against HSV-2 infection, and vaccinating against co-infection.

Also provided is a method of vaccinating a subject against HSV-1, HSV-2, or a co-infection, comprising administering to the subject (i) a 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 the subject against HSV-1, HSV-2, or a co-infection.

Also provided is a method of immunizing a subject against HSV-1, HSV-2, or a co-infection, the method comprising administering to the subject (i) a 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 the subject against HSV-1, HSV-2, or a co-infection.

In one embodiment of the method, a priming dose is administered subcutaneously or intravaginally to the subject and a second dose is administered subcutaneously or intravaginally. In an embodiment of the method, as many priming doses as necessary are administered subcutaneously or intravaginally to the subject to elicit anti-HSV antibodies and T cells.

Also provided is a method for producing recombinant herpes simplex virus-2 (HSV-2) virions, which have a gene deletion encoding HSV-2 glycoprotein D in their genome and contain HSV-1 or HSV-2 glycoprotein D on their lipid bilayer. The method comprises infecting cells containing heterologous nucleic acid encoding HSV-1 or HSV-2 glycoprotein D with a recombinant herpes simplex virus-2 (HSV-2) having a gene deletion encoding HSV-2 glycoprotein D in its genome under conditions that allow the replication of the recombinant herpes simplex virus-2 (HSV-2), and recovering the recombinant HSV-2 virions produced by the cells.

In one embodiment, the cell expresses HSV-1 or HSV-2 glycoprotein D on its membrane.

Also provided is a recombinant nucleic acid having the same sequence as the wild-type HSV-2 genome except that the recombinant nucleic acid does not contain a sequence encoding HSV-2 glycoprotein D. In one embodiment, the recombinant nucleic acid is DNA. In one embodiment, the recombinant nucleic acid is RNA.

Also provided is an isolated recombinant herpes simplex virus-2 (HSV-2) having a deletion in its genome of a gene encoding HSV-2 glycoprotein D for use in treating or preventing HSV-1, HSV-2, or a co-infection in a subject. In one embodiment, the isolated recombinant HSV-2 further comprises herpes simplex virus-1 (HSV-1) or herpes simplex virus-2 (HSV-2) glycoprotein D on its lipid bilayer. In one embodiment of the isolated recombinant HSV-2, the gene encoding HSV-2 glycoprotein D is the HSV-2 _US6 gene.

Also provided is an isolated recombinant HSV-2 virion having a deletion in its genome of a gene encoding HSV-2 glycoprotein D for use in treating or preventing HSV-1, HSV-2, or co-infection in a subject. In one embodiment, the virion further comprises HSV-1 or HSV-2 glycoprotein D on its lipid bilayer. In one embodiment, the gene encoding HSV-2 glycoprotein D is the HSV-2 U _S6 gene.

In one embodiment of the virus or virion as described, HSV-1, HSV-2 or a co-infection causes genital ulcers.

An isolated recombinant herpes simplex virus-2 (HSV-2) is provided, wherein the herpes simplex virus-2 has a deletion in its genome of the gene encoding HSV-2 glycoprotein D.

In one embodiment, the isolated recombinant HSV-2 further comprises a surface glycoprotein on its lipid bilayer that is herpes simplex virus-1 (HSV-1) glycoprotein D. In one embodiment, the isolated recombinant HSV-2 further comprises a non-HSV-2 viral surface glycoprotein on its lipid bilayer. In one embodiment, the isolated recombinant HSV-2 further comprises a bacterial surface glycoprotein on its lipid bilayer. In one embodiment, the isolated recombinant HSV-2 further comprises a parasite surface glycoprotein on its lipid bilayer, wherein the parasite is a mammalian parasite.

In one embodiment, the gene encoding HSV-2 glycoprotein D is the HSV-2 U _S6 gene. In one embodiment, the surface glycoprotein is encoded by a transgene that has been inserted into the genome of recombinant HSV-2. In one embodiment, the surface glycoprotein is present in the lipid bilayer of a cell by infecting it with a recombinant HSV-2 that has a gene deletion encoding HSV-2 glycoprotein D, wherein the cell is transfected or has been transfected to express the surface glycoprotein on its cell membrane, and wherein recombinant HSV-2 comprising the surface glycoprotein present on the lipid bilayer is produced from the cell. In one embodiment, the viral glycoprotein is from HIV, enterovirus, RSV, influenza virus, parainfluenza virus, porcine respiratory coronavirus virus, rabies virus, Lassa virus, bunyavirus, CMV, or a filovirus. In one embodiment, the glycoprotein is HIV gp120. In one embodiment, the filovirus is Ebola virus. In one embodiment, the virus is HIV, Mycobacterium tuberculosis, Chlamydia, Mycobacterium ulcerans, M. marinum, M. leprae, M. absenscens, Neisseria gonnorhea, or Treponeme. In one embodiment, the treponeme is Treponeme palidum.

Also provided is an isolated recombinant HSV-2 virion having a deletion of the gene encoding HSV-2 glycoprotein D in its genome.

In one embodiment, the isolated recombinant HSV-2 virion further comprises a surface glycoprotein on its lipid bilayer that is herpes simplex virus-1 (HSV-1) glycoprotein D. In one embodiment, the isolated recombinant HSV-2 virion further comprises a non-HSV-2 viral surface glycoprotein on its lipid bilayer. In one embodiment, the isolated recombinant HSV-2 virion further comprises a bacterial surface glycoprotein on its lipid bilayer. In one embodiment, the isolated recombinant HSV-2 virion further comprises a parasite surface glycoprotein on its lipid bilayer, wherein the parasite is a mammalian parasite. In one embodiment, the gene encoding HSV-2 glycoprotein D is the HSV-2 U _S6 gene. In one embodiment, the surface glycoprotein is encoded by a transgene that has been inserted into the recombinant HSV-2 genome of the virion. In one embodiment, the surface glycoprotein is present in the lipid bilayer of a cell by infecting a cell with a recombinant HSV-2 having a gene deletion encoding HSV-2 glycoprotein D, wherein the cell is transfected or has been transfected to express the surface glycoprotein on its cell membrane, and wherein recombinant HSV-2 comprising the surface glycoprotein present on the lipid bilayer is produced from the cell. In one embodiment, the virions have been recovered from this. In one embodiment, the viral glycoprotein is from HIV, enterovirus, RSV, influenza virus, parainfluenza virus, porcine respiratory coronavirus virus, rabies virus, Lassa virus, bunyavirus, CMV or a filovirus. In one embodiment, the glycoprotein is HIV gp120. In one embodiment, the filovirus is Ebola virus. In one embodiment, the virus is HIV, Mycobacterium tuberculosis, Chlamydia, Mycobacterium ulcerans, M. marinum, M. leprae, M. absenscens, Neisseria gonorrhea, or Treponeme. In one embodiment, the treponeme is Treponeme palidum.

Also provided is an isolated cell comprising a virus as described herein or a virion as described herein, wherein the cell is not present in humans. In one embodiment of the cell, the cell comprises a heterologous nucleic acid encoding HSV-1 glycoprotein D. In one embodiment of the cell, the cell expresses HSV-1 glycoprotein D on its membrane.

In one embodiment of the cell, the HSV-1 glycoprotein D is encoded by a heterologous nucleic acid, which is an HSV-1 glycoprotein D gene or a nucleic acid having the same sequence as an HSV-1 protein D gene.

A vaccine composition comprising a virus as described herein or a virion as described herein. In one embodiment of the vaccine composition, the vaccine composition comprises an immune adjuvant.

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 a deletion of at least a second gene, wherein the second gene is essential for HSV-2 viral replication. In one embodiment, the composition comprises serum from a mammal, or is derived from serum from a mammal, to which the virus or virion has been previously introduced to elicit an immune response.

Also provided are pharmaceutical compositions comprising a virus as described herein or a virosome as described herein and a pharmaceutically acceptable carrier.

Also provided is a method of stimulating an immune response in a subject, the method comprising administering to the subject (i) a virus as described herein; (ii) a viral particle 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 stimulate an immune response in the subject.

Also provided is a method for treating HSV-2 infection in a subject or treating a disease caused by HSV-2 infection in a subject, the method comprising administering to the subject (i) a virus as described herein; (ii) a viral particle 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 HSV-2 infection or treat a disease caused by HSV-2 infection in the subject.

Also provided is a method of vaccinating a subject against HSV-2 infection, the method comprising administering to the subject (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 the subject against HSV-2.

Also provided is a method of immunizing a subject against HSV-2 infection, the method comprising administering to the subject (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 the subject against HSV-2 infection.

HSV-2 and HSV-1 diseases are known in the art and are also described herein. Treatment and prevention of each of HSV-2 and HSV-1 diseases are independently encompassed. Treatment or prevention of HSV-2 and HSV-1 co-infection is also encompassed. Prevention is understood to mean an improvement in the progression of the disease or infection in a subject treated with a virus, virion, vaccine, or composition described herein, as compared to an untreated subject.

Also provided is a method for producing recombinant herpes simplex virus-2 (HSV-2) virions having a gene deletion encoding HSV-2 glycoprotein D in its genome and comprising HSV-1 glycoprotein D on its lipid bilayer, the method comprising infecting cells comprising heterologous nucleic acid encoding HSV-1 glycoprotein D with a recombinant herpes simplex virus-2 (HSV-2) having a gene deletion encoding HSV-2 glycoprotein D in its genome under conditions that allow the replication of the recombinant herpes simplex virus-2 (HSV-2), and recovering the recombinant HSV-2 virions comprising HSV-1 glycoprotein D on their lipid bilayer produced by the cells.

Also provided is a method for producing recombinant herpes simplex virus-2 (HSV-2) virions having a deletion of the gene encoding HSV-2 glycoprotein D in its genome and comprising a non-HSV-2 surface glycoprotein on its lipid bilayer, the method comprising infecting a cell comprising a heterologous nucleic acid encoding a non-HSV-2 surface glycoprotein with a recombinant herpes simplex virus-2 (HSV-2) having a deletion of the gene encoding HSV-2 glycoprotein D in its genome under conditions permissive for the replication of the recombinant herpes simplex virus-2 (HSV-2), and recovering the recombinant HSV-2 virions comprising the non-HSV-2 surface glycoprotein on its lipid bilayer produced by the cell.

Also provided is a recombinant nucleic acid having a sequence identical to that of the HSV-2 genome, except that the sequence does not contain a sequence encoding HSV-2 glycoprotein D.

Also provided is an isolated recombinant herpes simplex virus-2 (HSV-2) having a deletion in its genome of a gene encoding HSV-2 glycoprotein D for use in treating or preventing HSV-2 infection in a subject.

Also provided is an isolated recombinant herpes simplex virus-2 (HSV-2) having a deletion in its genome of a gene encoding HSV-2 glycoprotein D for use in treating or preventing HSV-1 infection in a subject.

Also provided is an isolated recombinant HSV-2 virion having a deletion of the gene encoding HSV-2 glycoprotein D in its genome for use in treating or preventing HSV-2 infection in a subject.

Also provided is a method for treating HSV-1 infection or HSV-1 and HSV-2 co-infection in a subject, or treating a disease caused by HSV-2 infection or HSV-1 and HSV-2 co-infection in a subject, the method comprising administering to the subject (i) a virus as described herein; (ii) a viral particle 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 HSV-2 infection in the subject or treating a disease caused by HSV-2 infection, or an amount effective to treat HSV-1 and HSV-2 co-infection in the subject or treating a disease caused by HSV-1 and HSV-2 co-infection.

Also provided is a method of vaccinating a subject against HSV-1 infection or co-infection with HSV-1 and HSV-2, the method comprising administering to the subject (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 the subject against HSV-1 infection or co-infection with HSV-1 and HSV-2.

Also provided is a method of immunizing a subject against HSV-1 infection or co-infection with HSV-1 and HSV-2, the method comprising administering to the subject (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 the subject against HSV-1 infection or co-infection with HSV-1 and HSV-2.

In one embodiment of the methods herein for immunizing, inoculating, or stimulating an immune response, passive transfer of virions or viruses, or antibodies or immune factors induced thereby, from one subject to another can be achieved. The product can be processed after being obtained from one subject and prior to administration to a second subject. In a preferred embodiment of the invention described herein, the subject is a mammalian subject. In one embodiment, the mammalian subject is a human subject.

Also provided is an isolated recombinant herpes simplex virus-2 (HSV-2) having a deletion in its genome of a gene encoding HSV-2 glycoprotein D and further comprising a heterologous antigen from a pathogen. In one embodiment, the heterologous antigen is a protein, peptide, polypeptide, or glycoprotein. In one embodiment, the heterologous antigen is a heterologous antigen relative to HSV-2, but is an antigen found on or in the relevant "pathogen." Viral and bacterial pathogens are described herein. In one embodiment, the pathogen is a bacterial pathogen of a mammal or a viral pathogen of a mammal. In one embodiment, the antigen or transgene encoding the pathogen is not actually taken or physically removed from the pathogen, but has a sequence identical to the pathogen antigen or encoding nucleic acid sequence. In one embodiment, the isolated recombinant HSV-2 comprises a heterologous antigen from the pathogen on its lipid bilayer. In one embodiment of the isolated recombinant HSV-2, the pathogen is bacterial or viral in nature. In one embodiment, the pathogen is a mammalian parasite. In one embodiment, the gene encoding HSV-2 glycoprotein D is the HSV-2 _US 6 gene. In one embodiment of 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.

Also provided is a method of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) against an antigenic target in a subject, the method comprising administering to the subject an isolated recombinant herpes simplex virus-2 (HSV-2) having a deletion in its genome of the gene encoding HSV-2 glycoprotein D and further comprising a xenoantigen on its lipid bilayer, in an amount effective to induce antibody-dependent cell-mediated cytotoxicity (ADCC) against the antigenic target.

Recombinant HSV-2ΔgD ^{−/+ gD −/+} expressing an appropriate transgene will selectively induce antibody and cellular immune responses that protect the skin or mucous membranes from pathogen infection.

In one embodiment, the xenoantigen is a surface antigen.

In one embodiment, the transgene encodes an antigen from HIV, Mycobacterium tuberculosis, Chlamydia, Mycobacterium ulcerans, Mycobacterium marinum, Mycobacterium leprae, M. absenscens, Neisseria gonorrhoeae, or a Treponema. In one embodiment, the Treponema is Treponema palidum. In one embodiment, the transgene encodes a gene for a Mycobacterium tuberculosis biofilm. In one embodiment, the transgene encodes a gene for HIV gp120.

In one embodiment, the xenoantigen is a surface antigen of the antigenic target. In one embodiment, the xenoantigen is a parasite antigen. In one embodiment, the xenoantigen is a bacterial antigen or a viral antigen.

In one embodiment, the antigenic target is a virus and is Lassa virus, human immunodeficiency virus, RSV, enterovirus, influenza virus, parainfluenza virus, porcine respiratory coronavirus, rabies virus, bunyavirus, or filovirus.

In one embodiment, the antigenic target is a bacterium and is Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycobacterium marinum, Mycobacterium leprae, M. absenscens, Chlamydia trachomatis, Neisseria gonorrhoeae, or Treponema pallidum.

In one embodiment, the isolated recombinant HSV-2 transgene is a gene encoding a Mycobacterium tuberculosis biofilm or wherein the transgene is a gene encoding HIV gp120.

In a preferred embodiment of the methods described herein, the subject is a human. In one embodiment of the methods described herein, the subject has not yet suffered from HSV-1, HSV-2 infection or co-infection. In one embodiment of the methods described herein, the subject has suffered from HSV-1, HSV-2 infection or co-infection.

As used herein, coinfection means co-infection with HSV-1 and HSV-2.

Unless otherwise indicated herein or otherwise clearly contradicted by context, all combinations of the various elements described herein are within the scope of the invention.

The present invention will be better understood from the following experimental details. However, those skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as more fully described in the appended claims.

Experimental details

Disclosed herein are genetically engineered deletion mutants of the gD (US6) gene of HSV-2 and their safety, immunogenicity, and vaccine efficacy evaluated against intravaginal challenge with HSV-2 in a mouse infection model. The gD gene was replaced with a DNA fragment encoding green fluorescent protein (gfp), and Vero cells expressing HSV-1 gD (VD60 cells) were transfected with this construct, and homologous recombinant viruses that formed green foci were screened. Molecular analysis revealed that precise recombinants had been engineered, which replicated to high titers in complementing VD60 cells but were non-infectious when propagated on non-complementing cells. Intravaginal challenge of wild-type or SCID mice with 10 ⁷ pfu/mouse of the complementing gD-null virus (designated herein as HSV-2ΔgD ^−/+ for viruses that are genotypically gD-deficient but phenotypically complemented by growth on VD60 cells) revealed no virulence, whereas doses as low as 10 ⁴ pfu/mouse of the parental wild-type virus were 100% lethal. Furthermore, immunization of mice with HSV-2ΔgD ^-/+ resulted in complete protection against intravaginal challenge with a clinical isolate of HSV-2. The robust humoral and cellular immunity elicited by HSV-2ΔgD ^-/+ was measured, leading to the conclusion that gD is required for productive infection in vivo and that an attenuated strain lacking this essential glycoprotein elicits protective immunity against HSV-2. Therefore, HSV-2ΔgD ^-/+ is a promising vaccine for the prevention or treatment of genital herpes.

Mechanisms and correlates of protection elicited by HSV-2ΔgD-/+. A gD-2 null virus was generated and demonstrated to be highly attenuated in immunocompetent and immunocompromised mice and, when tested as a vaccine candidate, induces a protective immune response against intravaginal challenge with HSV-2. Subcutaneous immunization with HSV-2ΔgD-/+ induces the humoral and cellular immune responses required for protection against intravaginal challenge with both HSV serotypes (HSV-2 and HSV-1).

HSV-2ΔgD-/+ initiates aborted infection: A strain of HSV-2 lacking _US6 was constructed to assess its contribution to the early signaling events that occur during cellular infection [41]. This virus is unable to infect host cells unless grown on a gD-complementing cell line encoding _US6 (e.g., VD60 cells encoding D-1 [40, 41]) under the control of its endogenous promoter (e.g., in one embodiment, the gD-1 promoter). Indeed, HSV-2ΔgD particles isolated from non-complementing cells do not infect epithelial cells (Figure 1) or neuronal cells (SK-N-SH, not shown). However, if propagated in VD60 cells, a phenotypically complemented virus (ΔgD-/+) is obtained that is fully capable of infecting cells that are common targets of wild-type HSV-2. However, following infection with ΔgD-/+, no infectious particles or viral plaques (pfu) were produced from these cells and the virus did not spread from infected to uninfected cells, reflecting the requirement of gD for these processes; thus, it was an abortive infection.

HSV-2ΔgD-/+ is safe in a murine infection model: The in vivo safety of ΔgD-/+ was evaluated in wild-type mice and severe combined immunodeficient (SCID) mice by subcutaneous or intravaginal inoculation of high doses. Throughout the experimental period, mice inoculated intravaginally with 10 ⁷ pfu of ΔgD-/+ (titrated on supplementary cells) did not show any signs of viral-induced pathology, while animals inoculated with 1,000-fold less wild-type virus (10 ⁴ pfu) succumbed to HSV-2 disease and began to die on day 8 after inoculation (Figure 2A). Throughout the experimental period, mice inoculated intravaginally with 10 ⁷ pfu of ΔgD-/+ did not show any signs of epithelial or neurological disease caused by the virus (Figures 2B and 2C). No infectious virus was recovered from reproductive tract tissues or DRGs as determined by plaque assay or co-culture of DRGs with Vero cells (not shown).

HSV-2ΔgD-/+ stimulates systemic and mucosal antibodies against HSV-2: Mice vaccinated subcutaneously and boosted (s.c.-s.c.) with ΔgD-/+ or subcutaneously with this candidate vaccine strain (10 ⁶ pfu/mouse) and boosted intravaginally (s.c.-s.c.) stimulated a humoral immune response against HSV-2, as evidenced by increased anti-HSV-2 antibodies in serum and vaginal washes (Figures 3A and 3B). Control animals were immunized with uninfected VD60 cell lysate (referred to as control). Antibodies were measured by ELISA using infected cell lysate as antigen (the response to uninfected cell lysate was used as background subtraction). Notably, the magnitude of the antibody response varied depending on the route of immunization. In fact, subcutaneous-subcutaneous immunization stimulated significantly more serum and vaginal wash antibodies against HSV-2 than subcutaneous-s.c.-s.c. immunization. The results of this study suggest that vaginal wash antibodies may represent IgG exudates from the blood and indicate that subcutaneous-subcutaneous administration is a more suitable route for eliciting high levels of systemic and local IgG antibodies against HSV-2. In addition, mice vaccinated and boosted subcutaneously (subcutaneous-subcutaneous) with ΔgD-/+ ( ¹⁰⁶ pfu/mouse) elicited neutralizing anti-HSV-2, as demonstrated by in vitro neutralization on Vero cell monolayers using virus and sera from these mice (Figure 3C).

HSV-2 Δ gD-/+ has stimulated HSV-2-specific T cell activation: before inoculation, gB498-505 specific transgenic CD8+T cells (gBT-I) are transferred into C57BL/6 mice. The mice of immunization are inoculated with 10 ⁶ pfu Δ gD-/+ or with VD60 cell lysates (control). Spleen is harvested on the 14th day after booster immunization and counting beads (CountBright ^™ , Lifetechnologies) are used, quantitatively (Fig. 4 A) by flow cytometry. On the same day, spleen is dyed to memory cell surface markers and analyzed by flow cytometry (Fig. 4 B). Finally, the splenocytes harvested on the same day are stimulated again in vitro for 6 hours with agonist gB498-505-peptide and intracellular cytokine staining method is used to measure the IFN-γ of these cells and produce. Compared with control mice, IFN-γ production (Fig. 4 C) is increased in the inoculation mice with Δ gD-/+ immunity. The responses in control mice are presumably a reflection of the persistence of gBT-1 T cells in naive mice after transfer. Similar results were obtained by multiplex cytokine analysis of supernatants from splenocytes restimulated in vitro with the gB498-505 peptide (not shown). These results demonstrate that the vaccine induces T cell responses.

Mice immunized with HSV-2ΔgD-/+ were protected against intravaginal lethal challenge with HSV-2ΔgD-/+: animals vaccinated subcutaneously-subcutaneously or subcutaneously-intravaginally gained less weight and survived challenge with an intravaginal lethal dose equivalent to _LD90 ( ^5x104 pfu/mouse), whereas mice immunized with VD60 control lysate succumbed to disease by day 10 (Figures 5A and 5B). The vaccine also provided complete protection against 10 times the _LD90 ( ^5x105 pfu/mouse, data not shown). This protection was associated with a significant reduction in epithelial disease scores (Figure 5C) and a complete absence of neurological signs (Figure 5D). Assessment was performed as previously described [44]. In addition, significantly less virus was recovered in the vaginal washes of ΔgD-/+-immunized mice compared to control mice on day 2 after vaginal challenge, suggesting rapid clearance (Figure 5E). In addition, no infectious virus was recovered in vaginal washes on day 4 ( FIG. 5E ) or in vaginal tissue or DRG isolated on day 5 post-challenge ( FIG. 5F ). The latter suggests that the vaccine prevents viral entry and/or replication in the DRG.

After challenge with virulent HSV-2, immunization with HSV-2ΔgD-/+ prevented inflammation at the site of infection: mice vaccinated with HSV-2ΔgD-/+ and challenged intravaginally with virulent HSV-2 showed significantly fewer inflammatory cytokines at the site of infection compared to animals vaccinated with VD60 lysate (control). In fact, vaccinated mice secreted significantly less TNF-α (Figure 6A), IL-6 (Figure 6B), and IL-1β (Figure 6C) in vaginal washes than control mice on days 2 and 7 after infection. Notably, increased levels of inflammatory cytokines are associated with increased HIV replication and unloading at the genitals when HSV-2 and HIV are co-infected [45, 46]. Similar phenomena were also observed in vitro [47].

T cells are recruited to the site of infection and relevant lymph nodes (LN) with HSV-2 Δ gD-/+ immunity. After attacking with virulent HSV-2, mice immunized with Δ gD-/+ subcutaneous-subcutaneous administration show that the percentage of anti-HSV-2gBT-1 CD8+T cells (Fig. 7 A) and CD4+T cells (Fig. 7 B) activated in the sacral lymph nodes (LN) increases. After attacking with virulent HSV-2, mice immunized with Δ gD-/+ subcutaneous-intravaginal administration show that the number of anti-HSV-2gBT-1 CD8+T cells (Fig. 7 C) and CD4+T cells (Fig. 7 D) in the vagina increases, which suggests that anti-HSV-2CD8+T cells and activated CD4+T cells (possibly anti-HSV-2) are recruited to the site of infection and relevant lymph nodes with Δ gD-/+ vaccination.

In other experiments, it was found that HSV-2-ΔgD ^-/+gD-1 immunization conferred protection against vaginal challenge with virulent HSV-2 in C57BL/6 and Balb/C. In addition, ΔgD ^-/+gD-1 immune mice challenged with HSV-2 intravaginally had no detectable HSV-2 in the vagina or nervous tissue 5 days after the attack. Unlike mice with morbid HSV-2 binding, it was found that HSV-2 ΔgD-/+gD-1 subcutaneous-subcutaneous antibodies recognized multiple HSV-2 proteins (both gD and gB). Serum antibodies from inoculated animals showed in vitro neutralization of HSV-1 and HSV-2. In addition, serum from ΔgD-/+gD-1 inoculated mice stimulated antibody-dependent cellular cytotoxicity (ADCC) of HSV-2 infected cells in vitro.

In summary, in wild-type (wt) mice and SCID mice, HSV-2ΔgD-/+gD-1 is weakened and completely safe. Recombinant HSV-2ΔgD-/+gD-1 protects against lethal HSV-2 intravaginal infection and HSV-2/HSV-1 skin infection. Protective effects were observed in two different mouse strains. There was no detectable infection and elimination immunity. Induction of HSV-2-specific CD8+T cells and systemic and mucosal HSV antibodies was also observed. IgG2a and IgG2b are the dominant anti-HSV isotypes. ADCC dependent on FcγRIII/II was also observed. Surprisingly, passive transfer of immune serum protected naive mice, while FcRn and FcγR knockout mice were not protected by immune serum.

discuss

The World Health Organization estimates that more than 500 million people worldwide are infected with herpes simplex virus type 2 (HSV-2), with approximately 20 million new cases each year [1]. The risk of infection increases with age and because the virus establishes a latent period under frequent subclinical or clinical reactivation conditions, the effects of infection are lifelong. Alarmingly, HSV-2 significantly increases the risk of acquiring and transmitting HIV [2-4]. The prevalence of HSV-2 varies across the globe, fluctuating from 8.4% in Japan to 70% in sub-Saharan Africa (where HIV prevalence is prevalent) [5,6]. In the United States, the prevalence of HSV-2 is approximately 16% and the prevalence of HSV-1 has dropped to approximately 54%. The decreasing prevalence of HSV-1 in the United States (and other European countries) is associated with an increase in genital HSV-1, as evidenced by the results of recent disappointing glycoprotein D (gD) subunit vaccine trials, in which the majority of genital herpes disease cases were caused by HSV-1 [7-9]. Although HSV-1 is associated with fewer recurrences and less genital viral shedding than HSV-2, both serotypes are transmitted perinatally and cause neonatal morbidity that is associated with significant morbidity and mortality, even with acyclovir treatment.[10-12] The morbidity associated with genital herpes, its synergy with the HIV epidemic, and its direct medical costs (over $500 million in the United States alone) highlight the need for a safe and effective vaccine.[13]

Subunit formulations consisting of viral envelope glycoproteins combined with adjuvants have dominated the HSV-2 vaccine field for almost 20 years and most clinical trials have centered on this strategy [8, 14-19]. Although subunit preparations are safe and elicit neutralizing antibodies, these preparations provide low efficacy against HSV-2 infection or disease in clinical trials [8, 14]. Surprisingly, an HSV-2 gD subunit vaccine provides protection against genital HSV-1 but not against HSV-2 [8, 20]. Subsequent studies found that serum HSV-2 gD antibody levels correlated with protection against HSV-1, suggesting that the antibody titer required for protection against HSV-2 may be higher than the antibody titer required for protection against HSV-1 [21]. In contrast, cell-mediated immunity (intracellular cytokine responses in response to overlapping gD peptides) was not associated with protection against either serotype [21]. The vaccine stimulated a CD4 ⁺ T cell response but not a CD8 ⁺ T cell response, although there was no difference in CD4 ⁺ T cell responses between vaccinated infected and uninfected women [21]. Genital or other mucosal antibody responses were not measured. A candidate HSV-2 vaccine in which gH was deleted from the genome did not reduce the frequency of viral recurrence in a clinical trial conducted in seropositive subjects, but the vaccine's efficacy against primary infection was not evaluated [29].

Clinical studies have shown an increased rate of HSV-2 reactivation in HIV-infected patients, and that the gD subunit vaccine, despite eliciting neutralizing serum antibodies, did not stimulate any CD8 ⁺ T cell responses. These clinical studies suggest that an effective vaccine must also stimulate a protective T cell response [28, 30-32]. Studies showing that HSV-1 reactive T cells are selectively retained in the human trigeminal ganglion further highlight the importance of T cells. CD4 ⁺ T cells and CD8 ⁺ T cells were identified as surrounding neurons, and there was heterogeneity in the viral proteins targeted, with viral protein 16 (VP16, intercalin) being recognized by multiple trigeminal ganglion T cells in the context of diverse HLA-A and B alleles; these results suggest that intercalin may be an important immunogen [33]. Similarly, cytotoxic T cells directed against intercalin were identified in humans with latent HSV-2 infection [34]. CD8 ⁺ T cells (including CD8 αα ⁺ T cells) persist at the skin-epidermal junction in the genital skin and mucous membranes after HSV reactivation, suggesting that they play a role in immune control [35].

Disclosed herein is an engineered HSV-2 virus genetically deficient in native HSV-2 gD. The HSV-2 gD gene encodes an envelope glycoprotein essential for viral entry and cell-to-cell spread. Glycoprotein D also binds to tumor necrosis factor receptor superfamily member 14 (TNFRSF14), an immune regulatory switch also known as herpesvirus entry mediator (HVEM). Because HVEM carries docking sites for more than one ligand and signal transduction differs depending on whether these molecules bind to HVEM in cis or trans, gD may have a regulatory effect on immune cells [36, 37]. In fact, recent studies suggest that gD competes with natural ligands for this receptor and modulates cytokine responses to the virus [38, 39]. The gD gene was replaced with a DNA fragment encoding green fluorescent protein (gfp), and homologous recombinant viruses that formed green foci were screened against complementing Vero cells (VD60 cells [40]) expressing HSV-1 gD (e.g., gD-1 under the gD-1 promoter) transformed with this construct. The mutant virus replicated to high titers in the complementing Vero cell line (designated HSV-2ΔgD ^−/+ when passaged on complementing cells) but was non-infectious in non-complementing cells (designated HSV-2ΔgD ^−/− when isolated from non-complementing cells). This virus was purified and characterized in vitro [41]. Intravaginal or subcutaneous inoculation of immunocompetent or immunocompromised (SCID) mice showed no virulence compared to lethal infection caused by the wild-type parental virus. Immunization (prime subcutaneously followed by a single booster administered subcutaneously or intravaginally) resulted in 100% protection against intravaginal challenge with virulent HSV-2. Robust humoral and cellular immunity was stimulated by HSV-2ΔgD ^-/+ , and it was concluded that _US6 (gD-2) is required for productive infection in vivo. This live attenuated strain will provide eliminating immunity against HSV. Passive serum or serum product transfer can also be used.

References

1.Looker, K.J., G.P.Garnett and G.P.Schmid, An estimate of the globalprevalence and incidence of herpes simplex virus type 2infection.Bull WorldHealth Organ, 2008.86(10):p.805-12,A.

2.Freeman, E.E et al., Herpes simplex virus 2infection increases HIVacquisition in men and women: systematic review and meta-analysis of longitudinal studies.AIDS, 2006.20(1):p.73-83.

3. Gray, R.H et al., Probability of HIV-1 transmission per coital act inmonogamous, heterosexual, HIV-1-discordant couples in Rakai, Uganda. Lancet, 2001.357(9263): p.1149-53.

4.Wald, A. and K. Link, Risk of human immunodeficiency virus infection inherpes simplex virus type 2-seropositive persons: a meta-analysis. J InfectDis, 2002.185(1):p.45-52.

5.Paz-Bailey, G et al., Herpes simplex virus type 2: epidemiology and management options in developing countries. Sex Transm Infect, 2007.83(1):p.16-22.

6.Doi, Y et al., Seroprevalence of herpes simplex virus 1and 2in apopulation-based cohort in Japan. J Epidemiol, 2009.19(2):p.56-62.

7. Bradley, H et al., Seroprevalence of herpes simplex virus types 1and 2--United States, 1999-2010. J Infect Dis, 2014.209(3):p.325-33.

8. Belshe, R.B et al., Efficacy results of a trial of a herpes simplexvaccine. N Engl J Med, 2012.366(1):p.34-43.

9.Bernstein, D.I et al., Epidemiology, clinical presentation, and antibody response to primary infection with herpes simplex virus type 1 and type 2inyoung women. Clin Infect Dis, 2013.56(3):p.344-51.

10. Kimberlin, D., Herpes simplex virus, meningitis and encephalitis inneonates. Herpes, 2004.11Suppl 2:p.65A-76A.

11. Ward, K.N et al., Herpes simplex serious neurological disease in young children: incidence and long-term outcome. Arch Dis Child, 2012.97(2):p. 162-5.

12. Lafferty, W.E et al., Recurrences after oral and genital herpes simplexvirus infection. Influence of site of infection and viral type. N Engl J Med, 1987. 316(23):p.1444-9.

13. Owusu-Edusei, K., Jr. et al., The estimated direct medical cost of selected sexually transmitted infections in the United States, 2008. Sex TransmDis, 2013.40(3):p.197-201.

14.Mertz, G.J et al., Double-blind, placebo-controlled trial of a herpessimplex virus type 2glycoprotein vaccine in persons at high risk for genitalherpes infection. J Infect Dis, 1990.161(4):p.653-60.

15. Group, H.S.V.S et al., Safety and immunogenicity of a glycoprotein Dgenital herpes vaccine in healthy girls 10-17years of age: results from arandomised, controlled, double-blind trial. Vaccine, 2013.31(51):p.6136-43.

16. Leroux-Roels, G et al., Immunogenicity and safety of different formulations of an adjuvanted glycoprotein D genital herpes vaccine in healthy adults: a double-blind randomized trial. Hum Vaccin Immunother, 2013.9(6):p. 1254-62.

17.Bernstein, D.I et al., Safety and immunogenicity of glycoprotein D-adjuvant genital herpes vaccine. Clin Infect Dis, 2005.40(9):p.1271-81.

18. Stanberry, L.R et al., Glycoprotein-D-adjuvant vaccine to prevent genital herpes. N Engl J Med, 2002.347(21):p.1652-61.

19. Corey, L et al., Recombinant glycoprotein vaccine for the prevention ofgenital HSV-2infection: two randomized controlled trials. Chiron HSV VaccineStudy Group. JAMA, 1999.282(4):p.331-40.

20.jh.richardus@rotterdam.nl, Safety and immunogenicity of aglycoprotein D genital herpes vaccine in healthy girls 10-17years of age: Results from a randomized, controlled, double-blind trial. Vaccine, 2013.31(51):p. 6136-43.

21. Belshe, R.B et al., Correlate of Immune Protection Against HSV-1Genital Disease in Vaccinated Women.J Infect Dis, 2013.

22.Gerber, S.I., B.J. Belval and B.C. Herold, Differences in the role of glycoprotein C of HSV-1and HSV-2 in viral binding may contribute to serotypedifferences in cell tropism. Virology, 1995.214(1):p.29-39.

23. Lubinski, J.M et al., The herpes simplex virus 1IgG fc receptor blocks antibody-mediated complement activation and antibody-dependent cellular cytotoxicity in vivo. J Virol, 2011.85(7):p.3239-49.

24.Para, M.F., L.Goldstein and P.G.Spear, Similarities and differences in the Fc-binding glycoprotein(gE) of herpes simplex virus types 1and 2andtentative mapping of the viral gene for this glycoprotein.J Virol, 1982.41(1):p. 137-44.

25.Hook, L.M et al., Herpes simplex virus type 1and 2glycoprotein Cprevents complement-mediated neutralization induced by natural immunoglobulinM antibody. J Virol, 2006.80(8):p.4038-46.

26.Lubinski, J.M et al., Herpes simplex virus type 1evades the effects ofantibody and complement in vivo.J Virol, 2002.76(18):p.9232-41.

27.Awasthi, S et al., Immunization with a vaccine combining herpes simplexvirus 2(HSV-2)glycoprotein C(gC)and gD subunits improves the protection ofdorsal root ganglia in mice and reduces the frequency of recurrent vaginal shedding of HSV-2DNA in guinea pigs compared to immunization with gD alone.JVirol,2011.85(20):p.10472-86.

28.Manservigi, R et al., Immunotherapeutic activity of a recombinantcombined gB-gD-gE vaccine against recurrent HSV-2infections in a guinea pigmodel. Vaccine, 2005.23(7):p.865-72.

29. de Bruyn, G et al., A randomized controlled trial of a replicationdefective(gH deletion) herpes simplex virus vaccine for the treatment of recurrent genital herpes among immunocompetent subjects. Vaccine, 2006.24(7):p.914-20.

30.Ouwendijk, W.J et al., T-cell immunity to human alphaherpesviruses. Curr Opin Virol, 2013.3(4):p.452-60.

31. Parr, M.B. and E.L. Parr, Mucosal immunity to herpes simplex virus type2infection in the mouse vagina is impaired by in vivo depletion of Tlymphocytes. J Virol, 1998.72(4):p.2677-85.

32.Noisakran, S. and D.J.Carr, Lymphocytes delay kinetics of HSV-1reactivation from in vitro explants of latent infected trigeminal ganglia. JNeuroimmunol, 1999.95(1-2):p.126-35.

33. van Velzen, M et al., Local CD4 and CD8T-cell reactivity to HSV-1 antigens documents broad viral protein expression and immune competence inlatently infected human trigeminal ganglia. PLoS Pathog, 2013.9(8):p. e1003547.

34. Muller, W.J et al., Herpes simplex virus type 2tegument proteins contain subdominant T-cell epitopes detectable in BALB/c mice after DNAimmunization and infection. J Gen Virol, 2009.90 (Pt 5): p.1153-63.

35. Zhu, J et al., Immune surveillance by CD8alphaalpha+skin-resident Tcells in human herpes virus infection. Nature, 2013.497(7450): p.494-7.

36.Steinberg, M.W et al., Regulating the mucosal immune system: the contrasting roles of LIGHT, HVEM, and their various partners. SeminImmunopathol, 2009.31(2):p.207-21.

37.Steinberg, M.W., T.C.Cheung and C.F.Ware, The signaling networks of the herpesvirus entry mediator (TNFRSF14) in immune regulation. Immunol Rev, 2011.244(1):p.169-87.

38. Kopp, S.J., C.S. Storti and W.J. Muller, Herpes simplex virus-2glycoprotein interaction with HVEM influences virus-specific recall cellular responses at the mucosa. Clin Dev Immunol, 2012.2012: p.284104.

39. Yoon, M et al., Functional interaction between herpes simplex virustype 2gD and HVEM transiently dampens local chemokine production after murinemucosal infection. PLoS One, 2011.6(1):p.e16122.

40. Ligas, M.W. and D.C. Johnson, A herpes simplex virus mutant in which glycoprotein D sequences are replaced by beta-galactosidase sequences bindsto but is unable to penetrate into cells. J Virol, 1988.62(5):p.1486-94.

41. Cheshenko, N et al., HSV activates Akt to trigger calcium release and promote viral entry: novel candidate target for treatment and suppression. FASEB J, 2013.27(7):p.2584-99.

42.Parr, E.L. and M.B. Parr, Immunoglobulin G is the mainantibody in mouse vaginal protective secretions after vaginal immunization with attenuated herpes simplex virus type 2.J Virol, 1997.71(11):p.8109-15.

43. Mbopi-Keou, F.

44.Hendrickson, B.A et al., Decreased vaginal disease in J-chain-deficientmice following herpes simplex type 2genital infection. Virology, 2000.271(1):p.155-62.

45.Nixon, B et al., Genital Herpes Simplex Virus Type 2Infection in Humanized HIV-Transgenic Mice Triggers HIV Shedding and Is Associated WithGreater Neurological Disease. J Infect Dis, 2013.

46. Carr, D.J. and L. Tomanek, Herpes simplex virus and the chemokines that mediate the inflammation. Curr Top Microbiol Immunol, 2006.303: p.47-65.

47. Stefanidou, M et al., Herpes simplex virus 2 (HSV-2) prevents dendritic cell maturation, induces apoptosis, and triggers release of proinflammatorycytokines: potential links to HSV-HIV synergy. J Virol, 2013. 87(3):p.1443-53.

48. Bourne, N et al., Herpes simplex virus (HSV) type 2glycoprotein D subunitvaccines and protection against genital HSV-1 or HSV-2disease in guinea pigs. JInfect Dis, 2003.187(4):p.542-9.

49. Bourne, N et al., Impact of immunization with glycoprotein D2/AS04 onherpes simplex virus type 2shedding into the genital tract in guinea pigs that become infected. J Infect Dis, 2005.192(12):p.2117-23.

50.Bernstein, D.I et al., The adjuvant CLDC increases protection of aherpes simplex type 2glycoprotein D vaccine in guinea pigs. Vaccine, 2010. 28(21):p.3748-53.

51.Bernstein, D.I et al., Potent adjuvant activity of cationic liposome-DNA complexes for genital herpes vaccines. Clin Vaccine Immunol, 2009.16(5):p.699-705.

52.Sweeney, K.A et al., A recombinant Mycobacterium smegmatis inducespotent bactericidal immunity against Mycobacterium tuberculosis. Nat Med, 2011.17(10):p.1261-8.

53.Kohl, S et al., Limited antibody-dependent cellular cytotoxicityantibody response induced by a herpes simplex virus type 2subunit vaccine. JInfect Dis, 2000.181(1):p.335-9.

54. John, M et al., Cervicovaginal secretions contribute to innateresistance to herpes simplex virus infection. J Infect Dis, 2005.192(10):p.1731-40.

55.Nugent, C.T et al., Analysis of the cytolytic T-lymphocyte response toherpes simplex virus type 1glycoprotein B during primary and secondaryinfection.J Virol, 1994.68(11):p.7644-8.

56.Mueller, S.N et al., Characterization of two TCR transgenic mouse lines specific for herpes simplex virus. Immunol Cell Biol, 2002.80(2):p.156-63.

57.Wallace, M.E et al., The cytotoxic T-cell response to herpes simplexvirus type 1infection of C57BL/6mice is almost entirely directed against asingle immunodominant determinant.J Virol, 1999.73(9):p.7619-26.

58.Milligan, G.N et al., T-cell-mediated mechanisms involved in resolution of genital herpes simplex virus type 2 (HSV-2) infection of mice. J ReprodImmunol, 2004.61(2):p.115-27.

59. Wang, K et al., A herpes simplex virus 2glycoprotein D mutant generated by bacterial artificial chromosome mutagenesis is severely impaired forinfecting neuronal cells and infects only Vero cells expressing exogenousHVEM.J Virol, 2012.86(23):p.12891-902.

60.Barletta, R.G et al., Identification of expression signals of themycobacteriophages Bxb1,L1and TM4using the Escherichia-Mycobacterium shuttleplasmids pYUB75 and pYUB76 designed to create translational fusions to thelacZ gene.J Gen Microbiol, 1992.138(1):p.23-30.

61.Yamaguchi, S et al., A method for producing transgenic cells using amulti-integrase system on a human artificial chromosome vector. PLoS One, 2011.6(2):p.e17267.

62. Xu, Z et al., Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome. BMC Biotechnol, 2013.13:p.87.

63.Hill, A et al., Herpes simplex virus turns off the TAP to evade hostimmunity. Nature, 1995.375(6530):p.411-5.

64.Shu, M et al., Selective degradation of mRNAs by the HSV host shutoffRNase is regulated by the UL47 tegument protein. Proc Natl Acad Sci U S A, 2013.110(18):p.E1669-75.

65. Umbach, J.L et al., MicroRNAs expressed by herpes simplex virus during latent infection regulate viral mRNAs. Nature, 2008.454(7205):p.780-3.

66. Cheshenko, N et al., Herpes simplex virus triggers activation ofcalcium-signaling pathways. J Cell Biol, 2003.163(2):p.283-93.

67.Cheshenko, N. and B.C. Herold, Glycoprotein B plays a predominant role in mediating herpes simplex virus type 2attachment and is required for entry and cell-to-cell spread. J Gen Virol, 2002.83(Pt 9):p.2247-55.

68.Cheshenko, N et al., Multiple receptor interactions trigger release ofmembrane and intracellular calcium stores critical for herpes simplex virus entry. Mol Biol Cell, 2007.18(8):p.3119-30.

69.Immergluck, L.C et al., Viral and cellular requirements for entry of herpes simplex virus type 1 into primary neuronal cells. J Gen Virol, 1998.79 (Pt 3): p.549-59.

70.Nixon, B et al., Genital Herpes Simplex Virus Type 2Infection inHumanized HIV-Transgenic Mice Triggers HIV Shedding and Is Associated WithGreater Neurological Disease.J Infect Dis, 2014.209(4):p.510-22.

71. Cheshenko, N et al., HSV usurps eukaryotic initiation factor 3subunit M for viral protein translation: novel prevention target. PLoS One, 2010.5(7):p.e11829.

72. Carbonetti, S et al., Soluble HIV-1 Envelope Immunogens Derived from anElite Neutralizer Elicit Cross-Reactive V1V2 Antibodies and Low PotencyNeutralizing Antibodies. PLoS One, 2014.9(1):p.e86905.

73.Janes, H et al., Vaccine-induced gag-specific T cells are associated with reduced viremia after HIV-1infection. J Infect Dis, 2013.208(8):p.1231-9.

74. Ferre, A.L et al., Immunodominant HIV-specific CD8+T-cell responses are common to blood and gastrointestinal mucosa, and Gag-specific responses dominate in rectal mucosa of HIV controllers. J Virol, 2010.84(19):p. 10354-65.

Claims (39)

1. A recombinant herpes simplex virus-2 (HSV-2) having a gene deletion encoding HSV-2 glycoprotein D in its genome and containing a herpes simplex virus-1 (HSV-1) glycoprotein on its lipid bilayer, wherein the HSV-1 glycoprotein is not encoded by the recombinant HSV-2 genome and wherein the HSV-1 glycoprotein is HSV-1 glycoprotein D. 2. The recombinant HSV-2 according to claim 1, wherein its lipid bilayer contains further non-HSV-2 viral surface glycoproteins. 3. The recombinant HSV-2 according to claim 1, wherein its lipid bilayer contains further bacterial surface glycoproteins. 4. The recombinant HSV-2 of claim 1, wherein the lipid bilayer contains further parasitic surface glycoproteins, wherein the parasite is a mammalian parasite. 5. The recombinant HSV-2 according to claim 1, wherein the gene encoding HSV-2 glycoprotein D is the HSV- _2US6 gene. 6. The recombinant HSV-2 according to claim 2, wherein the further viral surface glycoprotein is encoded by a transgene that has been inserted into the genome of the recombinant HSV-2. 7. The recombinant HSV-2 of claim 3, wherein the further bacterial surface glycoprotein is encoded by a transgene that has been inserted into the genome of the recombinant HSV-2. 8. The recombinant HSV-2 according to claim 4, wherein the further parasitic surface glycoprotein is encoded by a transgene that has been inserted into the genome of the recombinant HSV-2. 9. The recombinant HSV-2 according to any one of claims 2-8, wherein the HSV-1 glycoprotein D is present in its lipid bilayer by transfecting cells with a recombinant HSV-2 construct having a gene deletion encoding HSV-1 glycoprotein D, wherein the cells are transfected or have been transfected to express HSV-1 glycoprotein D on their cell membranes, and wherein recombinant HSV-2 containing the HSV-1 glycoprotein D present in the lipid bilayer is produced from the cells. 10. The recombinant HSV-2 viral particle according to any one of claims 1-9. 11. Isolated cells containing the virus according to any one of claims 1-9 or the viral particles according to claim 10, wherein the cells are not present in humans. 12. A vaccine composition comprising the virus according to any one of claims 1-9, or the viral particles according to claim 10. 13. A composition comprising the virus according to any one of claims 1-9, or the viral particle according to claim 10, wherein the genome of the virus or viral particle comprises at least a deletion of a second gene, wherein the second gene is essential for HSV-2 viral replication. 14. A pharmaceutical composition comprising the virus according to any one of claims 1-9, or the viral particle according to claim 10, and a pharmaceutically acceptable carrier. 15. Use of the virus of any one of claims 1-9 or the virion of claim 10 in the preparation of a medicament for eliciting an immune response in a subject. 16. Use of the virus of any one of claims 1-9 or the virion of claim 10 in the preparation of a medicament for treating HSV-1 or HSV-2 infection in a subject or for treating a disease caused by HSV-1 or HSV-2 infection in a subject. 17. The use according to claim 16, wherein diseases caused by HSV-2 infection include genital ulcers. 18. The use according to claim 16, wherein the disease caused by HSV-2 infection includes skin blisters or skin ulcers. 19. Use of the virus of any one of claims 1-9 or the virion of claim 10 in the preparation of a medicament for use in subjects inoculated against HSV-1 or HSV-2 infection. 20. Use of the virus of any one of claims 1-9 or the virion of claim 10 in the preparation of a medicament for immunizing subjects against HSV-1 or HSV-2 infection. 21. The use according to claim 16, 19 or 20, wherein the prepared medicine is used for administration of a pre-subcutaneous dose and for administration of a second dose subcutaneously or intravaginally. 22. A method for producing recombinant herpes simplex virus-2 (HSV-2) viral particles having a gene deletion encoding HSV-2 glycoprotein D in its genome and containing HSV-1 glycoprotein D on its lipid bilayer, the method comprising infecting a cell containing a heterologous nucleic acid encoding HSV-1 glycoprotein D with recombinant herpes simplex virus-2 (HSV-2) having a gene deletion encoding HSV-2 glycoprotein D in its genome, under conditions allowing the recombinant herpes simplex virus-2 (HSV-2) to replicate, and recovering the recombinant HSV-2 viral particles produced by the cells containing HSV-1 glycoprotein D on its lipid bilayer. 23. The method of claim 22, wherein the cell expresses HSV-1 glycoprotein D on its cell membrane. 24. A method for producing recombinant herpes simplex virus-2 (HSV-2) viral particles, said viral particles having a deletion in their genome of a gene encoding HSV-2 glycoprotein D and containing an HSV-1 surface glycoprotein on their lipid bilayer, wherein said HSV-1 surface glycoprotein is not encoded by said recombinant HSV-2 genome and wherein said HSV-1 surface glycoprotein is HSV-1 glycoprotein D, said method comprising, with recombinant herpes simplex virus-2 (HSV-2) having a deletion in its genome of a gene encoding HSV-2 glycoprotein D, infecting a cell containing a heterologous nucleic acid encoding the HSV-1 surface glycoprotein under conditions allowing said recombinant herpes simplex virus-2 (HSV-2) to replicate, and recovering the recombinant HSV-2 viral particles produced by said cells containing the HSV-1 surface glycoprotein on their lipid bilayer. 25. The method of claim 24, wherein the cell expresses HSV-1 surface glycoprotein on its cell membrane. 26. A recombinant herpes simplex virus-2 (HSV-2) having a gene deletion encoding HSV-2 glycoprotein D in its genome and containing herpes simplex virus-1 (HSV-1) glycoprotein on its lipid bilayer, wherein the HSV-1 glycoprotein is not encoded by the recombinant HSV-2 genome and wherein the HSV-1 glycoprotein is HSV-1 glycoprotein D, and further containing a heteroantigen of the pathogen. 27. The recombinant HSV-2 according to claim 26, wherein the lipid bilayer contains a heterologous antigen of the pathogen. 28. The recombinant HSV-2 of claim 26, wherein the pathogen is a bacterial or viral pathogen. 29. The recombinant HSV-2 of claim 26, wherein the pathogen is a mammalian parasite. 30. The recombinant HSV-2 according to any one of claims 26-29, wherein the gene encoding HSV-2 glycoprotein D is the HSV- _2US6 gene. 31. The recombinant HSV-2 according to any one of claims 26-29, wherein the xenoantigen is encoded by a transgene that has been inserted into the genome of the recombinant HSV-2. 32. The recombinant HSV-2 according to claim 31, wherein the transgene is a gene encoding a biofilm of Mycobacterium tuberculosis or the transgene is a gene encoding HIV gp120. 33. Use of isolated recombinant herpes simplex virus-2 (HSV-2) in the preparation of a medicament for inducing antibody-dependent cell-mediated cytotoxicity (ADCC) against an antigenic target in a subject, said herpes simplex virus-2 having a gene deletion encoding HSV-2 glycoprotein D in its genome and containing herpes simplex virus-1 (HSV-1) glycoprotein on its lipid bilayer, wherein said HSV-1 glycoprotein is not encoded by the recombinant HSV-2 genome, and wherein said HSV-1 glycoprotein is HSV-1 glycoprotein D, and also containing a xenoantigen on its lipid bilayer, said xenoantigen being present in an amount that effectively induces antibody-dependent cell-mediated cytotoxicity (ADCC) against an antigenic target. 34. The use according to claim 33, wherein the heteroantigen is a surface antigen of the antigenic target. 35. The use according to claim 33, wherein the heteroantigen is a parasitic antigen. 36. The use according to claim 33, wherein the heteroantigen is a bacterial antigen or a viral antigen. 37. The use according to claim 33 or 34, wherein the antigenic target is a virus and is Lassa virus, human immunodeficiency virus, RSV, enterovirus, influenza virus, parainfluenza virus, swine respiratory coronavirus, rabies virus, Bunyavirus or filovirus. 38. The use according to claim 33 or 34, wherein the antigenic target is a bacterium and is Mycobaterium tuberculosis, Mycobacterium ulcerans, Mycobacterium marinum, Mycobacterium leprae, Mycobacterium absenscens, Chlamydia trachomatis, Neisseria gonorrhoeae, or Treponema pallidum. 39. The use according to claim 36, wherein the isolated recombinant HSV-2 comprises a transgene, the transgene being a gene encoding a Mycobacterium tuberculosis biofilm or the transgene being a gene encoding HIV gp120.