CA2942166C - Recombinant herpes simplex virus 2 (hsv-2) vaccine vectors - Google Patents

Abstract

Recombinant herpes simplex virus 2 (HSV-2) vaccine vectors, virions thereof, compositions and vaccines comprising such, and methods of use thereof are each provided.

Description

RECOMBINANT HERPES SIMPLEX VIRUS 2 (HSV-2) VACCINE VECTORS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S.

Provisional Application No. 61/946,965, filed March 3, 2014, and of U.S.

Provisional Application No. 62/080,663, filed November 17, 2014.

STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant numbers AI- 061679 and AI-51519 awarded by the National Institutes of Health.

The government has ce1iain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] 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, assist to more fully describe the art to which the subject invention pertains.

[0004] 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 HN.

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 impacts 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 glycoprotcins D 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 Date Reyue/Date Received 2021-06-15 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.

[0005] The present invention addresses this need for new and improved HSV-1 and HSV-2 vaccines.

SUMMARY OF THE INVENTION

[0006] An isolated, recombinant herpes simplex virus-2 (HSV-2) is provided having a deletion of an HSV-2 glycoprotein D-encoding gene ( Us 6) in the genome thereof.

[0007] Also provided is a virion of an isolated, recombinant HSV-2 having a deletion of an HSV-2 glycoprotein D-encoding gene (Us6) in the genome thereof.

[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 being.

[0009] Also provided 1s a vaccme 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] A pharmaceutical composition comprising the recombinant HSV-2 virus as described herein, or the virion as described herein, and a pharmaceutically acceptable earner.

[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) 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 coinfection or treat a disease caused by an HSV-1, HSV-2 or co-infection in a subject.

[0014] 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 co-infection.

[0015] 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) the 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 co-infection.

[0016] In an embodiment of the vaccmes, compositions and pharmaceutical compositions, and of the methods of use thereof, the amount of recombinant HSV-2 is an amount of pfu 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 deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof and comprising a HSV-1 or HSV-2 glycoprotein D on a lipid bilayer thereof, comprising infecting a cell comprising a heterologous nucleic acid encoding a HSV-1 or HSV-2 glycoprotein D with a recombinant herpes simplex virus-2 (HSV-2) having a deletion of an HSV-2 glycoprotcin D-cncoding gene 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.

[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 docs not comprise a sequence encoding an HSV-2 glycoprotein D.

[0019] Also provided is an isolated, recombinant herpes simplex virus-2 (HSV-2) having a deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof for treating or preventing an HSV-1, HSV-2 or co-infection in a subject.

[0020] Also provided is a virion of an isolated, recombinant HSV-2 having a deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof for treating or preventing an HSV-1, HSV-2 or co-infection in a subject.

[0021] An isolated, recombinant herpes simplex virus-2 (HSV-2) is provided having a deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof.

[0022] Also provided is a virion of an isolated, recombinant HSV-2 having a deletion of an HSV-2 glycoprotein O-encoding gene in the genome thereof.

[0023] 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.

[0024] A vaccine composition comprising a virus as described herein, or a virion as described herein.

[0025] 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.

[0026] Also provided is pharmaceutical composition comprising a virus as described herein, or a virion as described herein, and a pharmaceutically acceptable carrier.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] Also provided is a method of producing a vmon of a recombinant herpes simplex virus-2 (HSV-2), having a deletion of an HSV-2 glycoprotein D-encoding gene 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 virns-2 (HSV-2) having a deletion of an HSV-2 glycoprotein D-encoding gene 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.

[0032] Also provided is a method of producing a vmon of a recombinant herpes simplex virus-2 (HSV-2), having a deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof and comprising a non-HSV-2 surface glycoprotein on a lipid bilayer thereof, 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 a deletion of an HSV-2 glycoprotein D-encoding gene 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 surface glycoprotein on a lipid bilayer thereof produced by the cell.

[0033] Also provided is a recombinant nucleic acid is provided having the same sequence as a genome of a HSV-2 except that the sequence does not comprise a sequence encoding an HSV-2 glycoprotein D.

[0034] Also provided is an isolated, recombinant herpes simplex virus-2 (HSV-2) having a deletion of an HSV-2 glyeoprotein D-eneoding gene in the genome thereof for treating or preventing an HSV-2 infection in a subj eet.

[0035] Also provided is an isolated, recombinant herpes simplex virus-2 (HSV-2) having a deletion of an HSV-2 glyeoprotein D-eneoding gene in the genome thereof for treating or preventing an HSY -1 infection in a subj eet.

[0036] Also provided is a virion of an isolated, recombinant HSV-2 having a deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof for treating or preventing an HSV-2 infection in a subject.

[0037] Also provided is 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 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 co-infection or treat a disease caused by an HSV-1 and HSV-2 co-infection in a subject.

[0038] 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.

[0039] 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 HS V-1 infection, or HSV-1 and HSV-2 co-infection.

[0040] Also provided is an isolated, recombinant herpes simplex virus-2 (HSV-2) having a deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof and further comprising a heterogenous antigen of a pathogen.

[0041] 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 deletion of an HSV-2 glycoprotein O-encoding gene in the genome thereof and further comprising a heterogenous antigen on a lipid bilayer thereof in an amount effective to induce antibody dependent cell mediated cytotoxicity (ADCC) against an antigenic target.

BRJEF OESCRJPTION OF THE ORA WINGS

[0042] Fig. 1: HSV-2 L'.1gO initiates an abortive infection: HSV-2 L'.1gO-/+ only replicates successfully in cells that provide gO in trans (e.g. VO60 [40, 41]), but not in cells such as Vero cells (ATCC CCL-81, Green monkey kidney) or CaSki (ATCC CRL-1550, Homo sapiens, cervix) that do not encode U86.

Non-complemented HSV-2 L'.1gO (L1gO-/- obtained from Vero cells) cannot infect cells such as Vero and CaSki, which do not encode Us6-

[0043] Fig. 2A-C: A.

Severe combined immunodeficiency (SCIO) mice inoculated with up to 107 plaque-forming units (pfu) of HSV-2 L'.1gO-/+ virus do not manifest signs of disease after high dose intravaginal or subcutaneous inoculation.

In contrast SCIO mice inoculated with wild-type virus at a 1,000-fold lower viral dose (104 pfu) succumb to disease.

Survival curves are shown in A, epithelial scores (scale of O to 5) for evidence of erythema, edema, or genital ulcers in B and neurological scores (scale of O to 5) for evidence of neuronal infection in C.

[0044] Fig. 3A-C: Immunization with HSV-2 L'.1gO-/+ virus elicits anti-HSV-2 antibodies.

While sc.-sc. immunization elicits significant levels of both systemic and mucosal (vaginal washes) anti-HSV-2 antibodies, sc.-i.vag. immunization with HSV-2 L'.1gO-/+ elicits 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 antiHSV- 2 antibody levels in vaginal washes are shown in B.

Mice immunized with L'.1gD-/+ display neutralizing anti-HSV-2 antibodies in the serum after challenge with virulent HSV- 2.

The neutralizing capacity of the antibodies elicited by L'.1gO-/+ immunization is shown in C. (* p< 0.05; **p < 0.01; ***p < 0.001).

[0045] Fig. 4A-C: A: CO8+ gBT-I T cell counts in spleens of C57Bl/6 mice transferred with Tg T cells, then primed and boosted with HSV-2 11gO-/+ or VO60 lysate (Control). B: Percentage of gBT-I memory T cells in spleens of vaccinated or Control mice. C: 14 days after boost, splcnocytes were isolated and re-stimulated in vitro with gB498-505 peptide and analyzed 6 hr later for cytokine production by intracellular cytokine staining and flow cytometry. (*p<0.05; **p< 0.01; ***p<0.001).

[0046] Fig. 5A-F: Immunization with HSV-2 L'.1gO-/+ (106 pfu/mouse) protects mice from a lethal HSV-2 challenge.

Mice were primed subcutaneously and boosted 3-weeks apart either sc. or i.vag. and then challenged 3-weeks after boost intravaginally with an LO90 of virulent wild-type HSV-2(4674).

While Control (immunized with the VO60 cell lysate) mice succumbed to disease, as manifested by significant weight loss (A) and death (B), LlgD-/+-immunized mice displayed significantly less pathology.

Furthermore, LlgD-/+immunized mice showed less epithelial disease (C) and neurological pathology (D) after lethal challenge.

Additionally, LlgD-/+-vaccinated mice displayed significantly less viral loads in vaginal washes (E), vaginal tissue and dorsal root ganglia (DRG) (F) after intravaginal challenge with a lethal dose of virulent HSV-2 compared to mice immunized with VD60 cell lysate as a Control.

No infectious virus could be recovered from LlgD-/+immunized mice in Day 4 vaginal washes or Day 5 vaginal tissue and DRG. (*p<0.05; **p< 0.01; ***p<0.001).

[0047] Fig. 6A-C: Mice immunized with HSV-2 LlgD-/+ secrete less inflammatory cytokines in vaginal washes after challenge with virulent HSV-2.

Mice immunized with HSV-2 LlgD-/+ secrete less TNF-a, IL-6 and IL-I~ in vaginal washes than mice immunized with VD60 lysate and challenged with virulent HSV-2.

Differences in inflammatory cytokine expression are observed at different time-points after challenge. (*p<0.05; **p< 0.01; ***p<0.001).

[0048] Fig. 7A-D: Immunization with HSV-2 LlgD-/+ recruits T cells to the infection site and associated LNs.

Mice immunized sc.-sc. with LlgD-/+ displayed increased percentages of activated anti-HSV-2 gBT-I CD8+ (A) and CD4+ T cells (B) in sacral lymph nodes (LNs) after challenge with virulent HSV-2. LNs were extracted and incubated 6 h with UV-inactivated LlgD-/- and then stained with antibodies for flow cytometry analysis.

Mice immunized sc.-i.vag. with LlgD-/+ displayed increased numbers of anti-HSV-2 gBT-I CD8+ (C) and CD4+ T cells (D) in the vagina after challenge with virulent HSV-2.

Vaginal tissues were processed to extract T cells and stained with antibodies for flow cytometry analysis.

Cell counting was done with (CountBright™, Lifetechnologies). (*p<0.05; **p< 0.01).

DETAILED DESCRIPTION OF THE INVENTION

[0049] An isolated, recombinant herpes simplex virus-2 (HSV-2) is provided having a deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof.

[0050] In an embodiment, the HSV-2 glycoprotein D comprises the ammo acid sequence set forth in SEQ ID NO: 1: MGRLTSGVGTAALLVVAVGLRVVCAKYALADPSLKMADPNRFRGKNLPVLDQLT DPPGVKRVYHIQPSLEDPFQPPSIPITVYY A VLERACRSVLLHAPSEAPQIVRGASDE ARKHTYNL TIA WYRMGDNCAIPITVMEYTECPYNKSLGVCPIRTQPRWSYYDSFSA VSEDNLGFLMHAPAFETAGTYLRL VKINDWTEITQFILEHRARASCKY ALPLRIPPA ACLTSKA YQQGVTVDSIGMLPRFIPENQRTVALYSLKIAGWHGPKPPYTSTLLPPEL SDTTNATQPELVPEDPEDSALLEDPAGTVSSQIPPNWHIPSIQDV APHHAPAAPSNPG LIIGALAGSTLA VL VIGGIAFWVRRRAQMAPKRLRLPHIRDDDAPPSHQPLFY (HSV- 2 reference strain HG52)

[0051] In an embodiment, the isolated, recombinant HSV-2 further comprises a herpes simplex virus- I (HSV-1) glycoprotein D on a lipid bilayer thereof.

[0052] In an embodiment, the HSV-1 glycoprotein D comprises the amino acid sequence set forth in SEQ ID NO:2: MGGAAARLGA VILFVVNGLHGVRGKY ALADASLKMADPNRFRGKDLPVLDQLT DPPGVRRVYHIQAGLPDPFQPPSLPITVYY A VLERACRSVLLNAPSEAPQIVRGASE DVRKQPYNLTIA WFRMGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNYYDSFS A VSEDNLGFLMHAPAFETAGTYLRL VKINDWTEITQFILEHRAKGSCKY ALPLRIPP SACLSPQA YQQGVTVDSIGMLPRFIPENQRTV A VYSLKIAGWHGPKAPYTSTLLPPE LSETPNA TQPELAPEDPEDSALLEDPVGTV APQIPPNWHIPSIQDAA TPYHPP A TPNN MGLIAGA VGGSLLAALVICGNYWMRRRTQKAPKRIRLPHIREDDQPSSHQPLFY (HSV-1 reference strain F)

[0053] In an embodiment, the HSV-2 glycopro1ein D-encoding gene is an HSV-2 Us6 gene. (For example, see Dolan et al. J Viral. 1998 March; 72(3): 2010-2021. (PMCID: PMC109494) "The Genome Sequence of Herpes Simplex Virus Type 2" for HSV-2 genome and Us6 gene). [ 0054] Also provided is a virion of an isolated, recombinant HS V -2 having a deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof.

[0055] Tn an embodiment, the virion further comprises an HSV-1 or HSV-2 glycoprotein D on a lipid bilayer thereof.

In an embodiment, the HSV-2 glycoprotein Dencoding gene is an HSV-2 Us6 gene).

[0056] In an embodiment, the virus further comprises an HSV-1 or HSV-2 glycoprotein Don a lipid bilayer thereof.

In an embodiment, the HSV-2 glycoprotein D-encoding gene is an HSV-2 Us6 gene.

[0057] An isolated cell is provided comprising therein a recombinant HSV-2 genome which does not comprise an HSV-2 Us6 gene.

Date Reyue/Date Received 2021-06-15

[0058] In an embodiment, the cell is a complementing cell which provides expressed HSV 1 or 2 glycoprotein not encoded for by the recombinant HSV-2 genome.

In an embodiment, the complementing cell comprises a heterologous nucleic acid encoding a HSV-1 or HSV-2 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 the heterologous nucleic acid, which heterologous nucleic acid is a HSV-1 or HSV-2 glycoprotein D gene, or is a nucleic acid having a sequence identical to a HSV-1 or HSV-2 glycoprotein D gene.

[0059] Also provided is a vaccine composition comprising the recombinant HSV-2 virus as described herein, or the virion as described herein.

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.

[0060] 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.

[0061] A pharmaceutical composition compnsmg the recombinant HSV-2 virus as described herein, or the virion as described herein, and a pharmaceutically acceptable carrier.

[0062] 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.

[0063] Also provided is a method of eliciting an immune response m 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.

[0064] 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 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.

[0065] 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.

[0066] 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 co-infection.

[0067] 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) the 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 co-infection.

[0068] 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-HSY antibodies and T cells.

[0069] Also provided is a method of producing a virion of a recombinant herpes simplex virus-2 (HSV-2), having a deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof and comprising an HSV-1 or HSV-2 glycoprotein D on a lipid bilayer thereof, comprising infecting a cell comprising a heterologous nucleic acid encoding a HSV-1 or HSV-2 glycoprotein D with a recombinant herpes simplex virus-2 (HSV-2) having a deletion of an HSV-2 glycoprotein D-encoding gene 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 cell.

[0070] In an embodiment, the cell expresses HSV-1 or HSV-2 glycoprotein D on a membrane thereof.

[0071] 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.

In an embodiment, the recombinant nucleic acid is a DNA.

In an embodiment, the recombinant nucleic acid is an RNA.

[0072] Also provided is an isolated, recombinant herpes simplex virus-2 (HSV-2) having a deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof for treating or preventing an HSV-1, HSV-2 or co-infection in a subject.

In an embodiment, the isolated, recombinant HSV-2 further comprises a herpes simplex virus- I (HSV-1) or herpes simplex virus-2 (HSV-2) glycoprotein Don a lipid bilayer thereof.

In an embodiment of the isolated, recombinant HSV-2, the HSV-2 glycoprotein D-encoding gene is an HSV-2 U86 gene.

[0073] Also provided is a virion of an isolated, recombinant HSV-2 having a deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof for treating or preventing an HSV-1, HSV-2 or co-infection in a subject.

In an embodiment, the virion further comprises an HSY-I or HSV-2 glycoprotein Don a lipid bilayer thereof.

In an embodiment, the HSV- 2 glycoprotein D-encoding gene is an HSV-2 Us6 gene.

[0074] In an embodiment, of the virus or virion as described, the HSV-1, HSV-2 or coinfection causes a genital ulcer.

[0075] An isolated, recombinant herpes simplex virus-2 (HSV-2) is provided having a deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof.

[0076] In an embodiment, the isolated, recombinant HSV-2 further comprises a surface glycoprotein on a lipid bilayer thereof which is a herpes simplex virus- I (HSV-1) glycoprotein D.

In an embodiment, the isolated, recombinant HSV-2 further comprises a non-HSV-2 viral surface glycoprotein on a lipid bilayer thereof.

In an embodiment, the isolated, recombinant HSV-2 further comprises a bacterial surface glycoprotein on a lipid bilayer thereof.

In an embodiment, the isolated, recombinant HSV-2 further comprises a parasitic surface glycoprotein on a lipid bilayer thereof, wherein the parasite is a parasite of a mammal.

[0077] In an embodiment, the HSV-2 glycoprotein D-encoding gene is an HSV-2 US6 gene.

In an embodiment, the surface glycoprotein is encoded by a transgene that has been inserted into the genome of the recombinant HSV-2.

In an embodiment, 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, 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.

In an embodiment, the viral 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 gp120.

In an embodiment, the filovirus is an ebola virus.

In an embodiment, the virus is HIV, a M tuberculosis, a chlamydia, Mycobacterium ulcerans, Af. marinum, Af. leprae, A1. absenscens, Neisseria gonnorhea, or a Treponeme.

In an embodiment, the Treponeme is Treponeme palidum.

[0078] Also provided is a virion of an isolated, recombinant HSV-2 having a deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof.

[0079] 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-I (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 bacterial surface glycoprotein on a lipid bilayer thereof.

In an embodiment, the virion of the isolated, recombinant HSV-2 further comprises a parasitic surface glycoprotein on a lipid bilayer thereof, wherein the parasite is a parasite of a mammal.

In an embodiment, the HSV-2 glycoprotein D-encoding gene is an HSV-2 Us6 gene.

In an embodiment, the 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 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, 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.

In an embodiment, the virion has been recovered from such.

In an embodiment, the viral 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 gp120.

In an embodiment, the filovirus is an ebola virus.

In an embodiment, the virus is HIV, a M tuberculosis, a chlamydia, Mycobacterium ulcerans, M. marinum, M. leprae, M. absenscens, Neisseria gonnorhea, or a Treponeme.

In an embodiment, the Treponeme is Treponeme palidum.

[0080] 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 a HSV-1 glycoprotein D.

In an embodiment of the cell, the cell expresses HSV-1 glycoprotein D on a membrane thereof.

[0081] In an embodiment of the cell, the HSV-1 glycoprotein D is encoded by the heterologous nucleic acid, which heterologous nucleic acid is a HSV-1 glycoprotein D gene, or is a nucleic acid having a sequence identical to a HSV-1 glycoprotein D gene.

[0082] A vaccine composition comprising a virus as described herein, or a virion as described herein.

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

[0083] Also provided is a composition comprising a virus as described herein, or a virion as described herein, wherein the genome of the vims 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.

[0084] Also provided is pharmaceutical composition comprising a virus as described herein, or a virion as described herein, and a pharmaceutically acceptable carrier.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

Also treatment or prevention of a HSV-2 and HSV-1 co-infection are covered.

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.

[0090] Also provided is a method of producing a virion of a recombinant herpes simplex virus-2 (HSV-2), having a deletion of an HSV-2 glycoprotein D-encoding gene 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 deletion of an HSV-2 glycoprotein D-encoding gene 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.

[0091] Also provided is a method of producing a virion of a recombinant herpes simplex virus-2 (HSV-2), having a deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof and comprising a non-HSV-2 surface glycoprotein on a lipid bilayer thereof, 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 a deletion of an HSV-2 glycoprotein D-encoding gene 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 surface glycoprotein on a lipid bilayer thereof produced by the cell.

[0092] Also provided is a recombinant nucleic acid is provided having the same sequence as a genome of a HSV-2 except that the sequence does not comprise a sequence encoding an HSV-2 glycoprotein D.

[0093] Also provided is an isolated, recombinant herpes simplex virus-2 (HSV-2) having a deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof for treating or preventing an HSV-2 infection in a subject.

[0094] Also provided is an isolated, recombinant herpes simplex virus-2 (HSV-2) having a deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof for treating or preventing an HSY -1 infection in a subject.

[0095] Also provided is a virion of an isolated, recombinant HSV-2 having a deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof for treating or preventing an HSV-2 infection in a subject.

[0096] Also provided is 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 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 a11 HSV-1 and HSV-2 co-infection or treat a disease caused by an HSV-1 and HSV-2 co-infection in a subject.

[0097] Also provided is a method of vaccinating a subject for an HSV-1 infection, or HSV-1 and HSV-2 co-infection, comprising administe1ing 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.

[0098] 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.

[0099] 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.

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

In an embodiment, the heterogenous antigen is a protein, peptide, polypeptide or glycoprotein.

In an embodiment, the heterogenous antigen heterogenous antigen with respect to HSV-2, but is an antigen found on or in the relevant "pathogen." Pathogens, viral and bacterial, are described herein.

In an embodiment, the pathogen is a bacterial pathogen of a mammal or a viral 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 heterogenous antigen of a pathogen on a lipid bilayer thereof.

In a11 embodiment of the isolated, recombinant HSV-2, the pathogen is bacterial or viral. 1n an embodiment, the pathogen is a parasite of a mammal.

In an embodiment, the HSV-2 glycoprotein D-encoding gene is an HSV-2 Us6 gene.

In an embodiment, the isolated, recombinant HSV-2, the heterogenous antigen is encoded by a transgene that has been inserted into the genome of the recombinant HSV-2.

[00100] 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 deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof and further comprising a heterogenous antigen on a lipid bilayer thereof in an amount effective to induce antibody dependent cell mediated cytotoxicity (ADCC) against an antigenic target.

[00101] Recombinant HSV-2 J',,.gD-1 + gD-/+ expressing the appropriate transgenes will selectively induce antibodies and cellular immune responses that protect against skin or mucosal infections by pathogens.

[00102] In an embodiment, the heterogenous antigen is a surface antigen.

[00103] In an embodiment, the transgene encodes an antigen from an HN, a M tuberculosis, a chlamydia, A1ycobacterium ulcerans, M. marinum, M leprae, M absenscens, Neisseria gonnorhea, or a Treponeme.

In an embodiment, the Treponeme is Treponeme palidum.

In an embodiment, the trans gene is a M. tuberculosis biofilm-encoding gene.

In an embodiment, the transgene is an HIV gp120-encoding gene.

[00104] In an embodiment, the heterogenous antigen is a surface antigen of the antigenic target.

In an embodiment, the heterogenous antigen is a parasite antigen.

In an embodiment, the heterogenous antigen is a bacterial antigen or a viral antigen.

[00105] 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.

[00106] In an embodiment, the antigenic target is a bacteria and is Mycobaterium tuberculosis, Ni ulcerans, M marinum, il1. leprae, M absenscens, Chlamydia trachomatis, Neisseria gonorrhoeae or Treponema pallidum.

[00107] In an embodiment, the isolated, recombinant HSV-2 transgene is a M tuberculosis biofilm-cncoding gene or wherein the transgcnc is an HIV gp120-cncoding gene.

[00108] 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-infection.

In an embodiment of the methods described herein, the subject has been infected with HSV-1, HSV-2 or co-infection.

[00109] As described herein, a co-infection means a co-infection with HSV-1 and HSV- 2.

[00110] All combinations of the various clements described herein arc within the scope of the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[00111] 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.

EXPERIMENTAL DETAILS

[00112] Herein a genetically engineered deletion mutant of the gD (Us6) gene of HSV-2 is disclosed and its safety, immunogenicity, and vaccine efficacy evaluated against intravaginal HSV-2 challenge in the mouse infection model.

The gD gene was replaced with a DNA fragment encoding the green fluorescent protein (g/p) and Vero cells expressing HSV-1 gD (VD60 cells) were transfected with this construct and screened for homologous recombinant virus that formed green plaques.

Molecular analysis revealed that a precise recombination had been engineered, which replicates in the complementing VD60 cells to high titers but is noninfectious when propagated on non-complementing cells.

Intravaginal challenge of wild-type or SCID mice with 107 pfu/mouse of the complemented gD-null virus (designated herein as HSV-2 f:!.gD-I+ for the virus that is genotypically gD deleted, but phenotypically complemented by growth on VD60 cells) revealed no virulence, whereas doses as low as 104 pfu/mouse of parental wild-type virus were 100% lethal.

Moreover immunization of mice with HSV-2 f:!.gD-11 yielded complete protection against intravaginal challenge with a clinical isolate of HSV-2.

Robust humoral and cellular immunity elicited by HSV-2 f:!.gF1+ was measured and it is concluded that gD is required for productive infection in vivo and that an attenuated strain deleted in this essential glycoprotein elicits protective immunity against HSV-2.

Thus, HSV-2 f:!.gD-11 is a promising vaccine for prevention or treatment of genital herpes.

[00113] Mechanisms and correlates of protection elicited by HSV-2 f:!.gD-/+. A gD-2 null virus was generated, and it was demonstrated that it is highly attenuated in both immunocompetent and immunocompromised mice and when tested as a vaccine candidate, induced a protective immune response against intravaginal challenge with HSV-2.

Subcutaneous immunizations with HSV-2 f:!.gD-/+ will induce humoral and cellular immune responses that are required for protection against intravaginal challenge with both serotypes of HSY (HSV-2 and HSV-1).

[00114] HSV-2 f:!.gD-/+ initiates an abortive infection: An HSV-2 strain that is deleted for Us6 was constructed to assess its contribution in in early signaling events occurring during cell infection [ 41].

This virus is incapable of infecting host cells, unless it is grown on a gD-complementing cell line (e.g. VD60 cells encoding gD-1 [40, 41]) that encodes Us6 under the control of its endogenous promoter (for example, in an embodiment, the gD-1 promoter).

Indeed, HSV-2 t.gD particles isolated from non-complementing cells do not infect epithelial (Fig. 1) or neuronal cells (SK-N-SH, not shown).

However, if propagated in VD60 cells a phenotypically complemented virus (t.gD-/+) is obtained, which is fully capable of infecting cells that are common targets for wild-type HSV-2.

However, after infection with t.gD-/+ no infectious particles or viral plaques (pfu) are produced from these cells and the virus fails to spread from infected to uninfected cells, reflecting the requirement for gD in these processes; thus it is an abortive infection.

[00115] HSV-2 L1gD-/+ is safe in the murine infection model: L1gD-/+ was evaluated for safety in vivo in wild-type and severe combined immunodeficiency (SCID) mice by inoculating high doses subcutaneously or intravaginally.

Mice inoculated intravaginally with 107 pfu of L1gD-/+ (titered on complementing cells) did not manifest any signs ofvirusinduced pathology throughout the experiments, whereas animals inoculated with 1,000-fold less wild-type virus (104 pfu) succumbed to HSV-2 disease and died starting Day 8 after inoculation (Fig. 2A).

Mice inoculated intravaginally with 107 pfu of t.gD-/+ did not manifest any signs of virus-induced epithelial or neurological disease throughout the experiments (Fig. 2B and 2C).

No infectious virus was recovered from genital tract tissue or DRGs, as determined by plaque assay or co-cultivation of DRGs with Vero cells (not shown).

[00116] HSV-2 L1gD-/+ elicits systemic and mucosal antibodies to HSV-2: Mice inoculated and boosted subcutaneously (sc.-sc.) with t.gD-/+ or inoculated subcutaneously and boosted intravaginally (sc.-i.vag.) with this candidate vaccine strain (106 pfu/mouse) elicited a humoral immune response to HSV-2 as evidenced by an increase in serum and vaginal washes anti-HSV-2 antibodies (Fig. 3A and 3B).

The control animals were immunized with an uninfected VD60 cell lysatc (referred to as Control).

The antibodies were measured by ELISA using infected cell lysates as the antigen (response to uninfected cell lysates subtracted as background).

Noteworthy, the magnitude of the antibody response differs depending on the route of immunization.

Indeed, s.c.-s.c. immunization elicited significantly more scrum and vaginal wash antibodies to HSV-2 than s.c.-i.vag. immunization.

This finding suggests that the vaginal wash antibodies likely represent transudate of IgG from the blood and suggest that sc.-sc. is a more appropriate route for eliciting high levels of systemic and local IgG antibodies to HSV-2.

Additionally, Mice inoculated and boosted subcutaneously (sc.-sc.) with ilgD-/+ (106 pfu/mouse) elicited a neutralizing anti-HSV-2 as evidenced by in vitro neutralization of Vero cell monolayers with virus and sera from these mice (Fig. 3C).

[00117] HSV-2 ~gD-/+ elicits HSV-2-specific T cell activation: gB498-505-specific transgenic CD8+ T cells (gBT-I) were transferred into C57BL/6 mice prior to vaccination.

Vaccinated mice were inoculated with 106 pfu L'lgD-/+ or with VD60 cell lysates (Control).

Spleens were harvested on Day 14 after the boost and quantified by flow cytometry using counting beads (CountBright™, Lifetechnologies) (Fig. 4A).

At the same day, spleens were stained for memory surface markers and analyzed by flow cytomet1y (Fig. 4B).

Finally, splenocytes harvested the same day were re-stimulated in vitro for 6 hours with the agonist gB498-505-peptide and intracellular cytokine staining was performed to measure IFN-y production by these cells.

Immunization with ilgD-/+ increased the IFN-y production in the vaccinated compared to control mice (Fig. 4C).

The response in control mice presumably reflects the persistence of the gBT-I T cells in naive mice after transfer.

Similar results were obtained using multiplex cytokine analyses for supernatants of splenocytes re-stimulated in vitro with gB498-505-peptide (not shown).

These findings demonstrate that the vaccine induces T cell responses.

[00118] Mice immunized with HSV-2 ~gD-/+ are protected against intravaginal HSV-2 lethal challenge: Animals vaccinated with HSV-2 ilgD-/+ either sc.-sc. or sc.-i.vag. suffer less body weight after intravaginal lethal dose challenges equivalent to LD90 (5x 104 pfu/mouse) and survive challenges, whereas mice immunized with the VD60 control lysate succumbed to disease by Day 10 (Fig. 5A and 5B).

The vaccines also provided complete protection against 10 times the LD90 (5x105 pfu/mouse, data not shown).

This protection was associated with significantly reduced epithelial disease scores (Fig. 5C) and the complete absence of neurological signs (Fig. 5D).

Scoring was performed as previously described [ 44].

Furthermore, significantly less virus was recovered in vaginal washes in ilgD-/+-immunized mice, as compared to control mice at day 2 post-vaginal challenge suggesting rapid clearance (Fig. 5E).

Moreover no infectious virus was recovered in Day 4 vaginal washes (Fig. 5E) or in vaginal tissue or DRGs isolated on Day 5 after challenge (Fig. 5F).

The latter suggest that the vaccine prevents virus from reaching and/or replicating in the DRG.

[00119] Immunization with HSV-2 ~gD-/+ prevents inflammation at the infection site after challenge with virulent HSV-2: Mice vaccinated with HSV-2 ~gD-/+ and intravaginally challenged with virulent HSV-2 display significantly less inflammatory cytokines at the infection site as compared to animals inoculated with VD60 lysates (Control).

Indeed, vaccinated mice secreted significantly less TNF-a (Fig. 6A), IL-6 (Fig. 6B) and IL-1~ (Fig. 6C) in vaginal washes at Day 2 and 7 post-infection than Control mice.

Noteworthy, increased levels of inflammatory cytokines are associated with increased HIV replication and shedding at the genitalia in the co-infected with HSV-2 and HIV [45, 46]. A similar phenomenon is also observed in vitro [ 47].

[00120] Immunization with HSV-2 ~gD-/+ recruits T cells to the infection site and associated LNs.

Mice immunized sc.-sc. with ~gD-/+ displayed increased percentages of activated anti-HSV-2 gBT-I CD8+ (Fig. 7A) and CD4+ T cells (Fig. 7B) in sacral lymph nodes (LNs) after challenge with virulent HSV-2.

Mice immunized sc.-i.vag. with ~gD-/+ displayed increased numbers ofanti-HSV-2 gBT-I CD8+ (Fig. 7C) and CD4+ T cells (Fig. 7D) in the vagina after challenge with virulent HSV-2 suggesting that vaccination with ~gD-/+ recruits anti-HSV-2 CD8+ T cells and activated CD4+ T cells (likely anti-HSV-2) to the infection site and associated lymph nodes.

[00121] In further experiments, immunization with HSV-2-~g□-/+gD-l was found to confer protection in C57BU6 and Balb/C to vaginal challenge with virulent HSV-2.

In addition, intravaginal HSV-2 challenged ~g□-/+gD-l immunized mice had no detectable HSV-2 in vaginal or neural tissue at 5 days post-challenge. HSV-2 ~gD-/+gD-1 sc.sc. antibodies were found to recognize numerous HSV-2 proteins (both gD and gB) unlike HSV-2 morbid-bound mice.

Scrum antibodies from vaccinated animals showed neutralization of HSV-1 and HSV-2 in vitro.

Moreover, eerum from ~gD-/+gD-1 vaccinated mice elicited Antibody Dependent Cellular Cytotoxicity (ADCC) of HSV-2 infected cells in vitro.

[00122] In summary, HSV-2 ~gD-/+gD-1 is attenuated and completely safe in wi and SCID mice.

Recombinant HSV-2 ~gD-/+gD-1 protected against lethal HSV-2 intravaginal and HSV-2/HSV-l skin infection.

Protection was observed in two different mouse strains.

There was no detectable infection, and sterilizing immunity.

Also observed was induction of HSV-2 specific CD8+ T cells and systemic and mucosa! HSY Abs. lgG2a and lgG2b were the predominant anti-HSY isotype.

Also observed was FcyRIII/II-dependent ADCC.

Surprisingly, passive transfer of immune serum protects naive mice, and FcRn and FcyR knockout mice were not protected with immune sera.

Discussion

[00123] The World Health Organization estimated that over 500 million people were infected with herpes simplex virus type 2 (HSV-2) worldwide with approximately 20 million new cases annually [l].

Infection risk increases with age and because the virus establishes latency with frequent subclinical or clinical reactivation, the impact of infection is lifelong.

Alarmingly, HSV-2 significantly increases the risk of acquiring and transmitting HIV [2-4].

The prevalence of HSV-2 varies among global regions, fluctuating from 8.4% for Japan up to 70% for sub-Saharan Africa, a region where HIV prevalence is epidemic [5, 6].

In the US the prevalence ofHSV-2 is ~16% and that ofHSV-1 has declined to~ 54%.

The decreasing prevalence of HSV-1 in the US ( and other European nations) is linked to an increase in genital HSV-1 as evidenced by results in the recent disappointing glycoprotein D (gD) subunit vaccine trial in which the majority of cases of genital herpes disease were caused by HSV-1 [7-9].

While HSV-1 is associated with fewer recurrences and less genital tract viral shedding compared to HSV-2, both serotypes are transmitted perinataly and cause neonatal disease; neonatal disease is associated with high 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 cost, which surpasses 500 million dollars in the US alone, highlight the imperative to develop a safe and effective vaccine [13].

[00124] Subunit formulations consisting of viral envelope glycoproteins combined with adjuvants have predominated the HSV-2 vaccine field for nearly 20 years and the majority of clinical trials have focused on this strategy [8, 14-19].

Although subunit preparations are safe and elicit neutralizing antibodies, these formulations provided little efficacy against HSV-2 infection or disease in clinical trials [8, 14].

Surprisingly, an HSV-2 gD subunit vaccine provided protection against genital HSV-1, but not HSV-2 [8, 20].

Subsequent studies found that serum HSV-2 gD antibody levels correlated with protection against HSV-1, suggesting that the antibody titers required for HSV-2 protection may be higher than those needed to protect against HSV-1 [21].

In contrast, cell mediated immunity (intracellular cytokine responses to overlapping gD peptides) did not correlate with protection against either serotype [21].

The vaccine elicited CD4+, but not CD8+ T cell responses, but there were no differences in CD4- T cell responses between vaccinated infected and uninfected women [21].

Genital tract or other mucosal antibody responses were not measured.

An HSV-2 vaccine candidate with gH deleted from the genome failed to reduce the frequency of viral recurrences in a clinical trial conducted among seropositive subjects, although the vaccine was not evaluated for efficacy against primary infection [29].

[00125] Clinical studies showing increased rates of HSV-2 reactivation in HIV-infected patients combined with the failure of the gD subunit vaccine to elicit any CDS+ T cell response despite the induction of neutralizing serum antibodies suggest that an effective vaccine must also elicit protective T cell responses [28, 30-32].

The importance ofT cells is further highlighted by studies showing selective retention of HSV-1 reactive T -cells in human trigeminal ganglia. CD4+ and CDS+ T cells were identified surrounding neurons and, while there was heterogenity in the viral proteins targeted, the tegument protein, virion protein 16 (VP 16), was recognized by multiple trigeminal ganglia T cells in the context of diverse HLA-A and -B alleles; these findings suggest that tegument proteins may be important immunogens [33].

Similarly, cytotoxic T cells directed at tegument proteins were also identified in studies of humans latently infected with HSV-2 [34]. CD8 1 T cells (including CD8aa+ T cells) persist in genital skin and mucosa at the dermal-epidermal junction following HSY reactivation suggesting that they play a role in immune control [35].

[00126] Herein is disclosed an engineered an HSV-2 virus genetically deleted for 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-regulatmy switch also known as herpesvirus entry mediator (HVEM).

Because HVEM harbors docking sites for more than one ligand and signaling differs depending on whether these molecules bind to HVEM in cis or in trans, gD may have modulatory effects on immune cells [36, 37].

Indeed, recent studies suggest that gD competes with the natural ligands for this receptor and modulates the cytokine response to the virus [38, 39].

The gD gene was replaced with a DNA fragment encoding the green fluorescent protein (gfp) and transformed complementing Vero cells expressing HSV-1 gD (VD60 cells [40]) (e.g. gD-1 under gD-1 promoter)with this construct were screened for homologous recombinant virus that f01med green plaques.

The mutant virus replicates in the complementing Vero cell line to high titers (designated HSV-2 /1gD-11 when passaged on complementing cells), but is noninfectious in non-complementing cells (designated HSV-2 !1gU1 - when isolated from non-complementing cells).

This virus was purified and characterized in vitro [ 41].

Intravaginal or subcutaneous inoculation of immunocompetent or immunocompromised (SCIO) mice revealed no virulence compared to the lethal infection caused by parental wild-type virus.

Immunization (subcutaneous prime followed by a single boost administered either subcutaneously or intravaginally) was 100% protective against intravaginal challenge with virulent HSV-2.

Robust humoral and cellular immunity was elicited by HSV-2 1',,,gD-1 + and it was concluded that Us6 (gD-2) is required for productive infection in vivo.

This live attenuated viral strain will provide sterilizing immunity against HSV.

Also passive serum or serum product transfer can be employed.

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Claims (14)

1. What is claimed is: 1. A recombinant herpes simplex virus-2 (HSV-2) having a deletion ofHSV-2 Us6 gene in the genome thereof, and comprising a herpes simplex virus- I (HSV-1) glycoprotein D on a lipid bilayer thereof for use in the treatment or prevention of an HSV-1 infection or an HSV-2 infection in a subject.
2. 2. The recombinant HSV-2 for use according to claim 1, wherein the HSV-1 glycoprotein D is present on the lipid bilayer thereof by way of ex vivo infecting a cell with a recombinant HSV-2 having the deletion ofHSV-2 Us6 gene, wherein the cell is or has been transfected to express the HSV-1 glycoprotein D on a cell membrane thereof, and wherein the recombinant HSV-2 comprising the HSV-1 glycoprotein D present on the lipid bilayer thereof is produced from the cell.
3. 3. The recombinant HSV-2 for use according to claim 1 or 2, wherein said HSV-1 infection or said HSV-2 infection is an HSV-1 and HSV-2 coinfection in said subject.
4. 4. The recombinant HSV-2 for use according to claim 1 or 2, wherein the HSV-2 infection causes a genital ulcer.
5. 5. The recombinant HSV-2 for use according to any one of claims 1 to 4, wherein said recombinant HSV-2 is provided in a composition formulated for subcutaneous administration to a human subject.
6. 6. The recombinant HSV-2 for use according to any one of claim 1 to 4, wherein said recombinant HSV-2 is provided in a composition formulated for intravaginal administration to a human subject.
7. 7. The recombinant HSV-2 for use according to any one of claims 1 to 4, wherein said recombinant HSV-2 is provided in a composition formulated for intramuscular administration to a human subject.
8. 8. The recombinant HSV-2 for use according to any one of claims 1 to 4, wherein the subject is a human.
9. 9. A pharmaceutical composition comprising a recombinant HSV-2, and a pharmaceutically acceptable carrier for use in the treatment or prevention of an HSV-1 infection, Date Re9ue/Date Received 2023-11-20 an HSV-2 infection or an HSV-1 and HSV-2 coinfection in a subject, wherein the recombinant HSV-2 is a recombinant HSV-2 having a deletion ofHSV-2 Us6 gene in the genome thereof and comprising a herpes simplex virus-I (HSV-1) glycoprotein D on a lipid bilayer thereof.
10. 10. The pharmaceutical composition for use according to claim 9, wherein the HSV-1 glycoprotein Dis present on the lipid bilayer thereof by way of ex vivo infection a cell with the recombinant HSV-2 having the deletion of the HSV-2 Us6 gene, wherein the cell is or has been transfected to express the HSV-1 glycoprotein D on a cell membrane thereof, and wherein the recombinant HSV-2 comprising the HSV-1 glycoprotein D present on the lipid bilayer thereof is produced from the cell.
11. 11. The pharmaceutical composition for use according to claim 9 or 10, wherein said HSV-1 infection or said HSV-2 infection is an HSV-1 and HSV-2 coinfection in said subject.
12. 12. The pharmaceutical composition for use according to any one of claims 9 to 11, wherein the pharmaceutical composition is formulated for administration to a human subject.
13. 13. The pharmaceutical composition for use according to of any one of claims 9 to 12, wherein the pharmaceutical composition is formulated for subcutaneous administration to a human subject.
14. 14. The pharmaceutical composition for use according to any one of claims 9 to 12, wherein the pharmaceutical composition is formulated for intramuscular, intranasal or mucosal administration to a human subject. Date Re9ue/Date Received 2023-11-20