Method of using HSV-2 single-cycle virus delta-gD and HSV-2 recombinant glycoprotein

Abstract

A method for vaccinating, immunizing, and / or treating a subject against herpes simplex virus infection or a disease caused by herpes simplex virus infection comprises administering to the subject an effective amount of HSV-2 short-cycle virus and an effective amount of recombinant HSV-2 glycoprotein D, wherein the HSV-2 short-cycle virus comprises HSV-2 having a deletion of the gene encoding glycoprotein D in its genome, and the HSV-2 is phenotypically complemented with HSV-1 glycoprotein D on the HSV-2 lipid bilayer.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Application No. 63 / 031,816, filed on 29 May 2020, which is incorporated herein by reference in its entirety. Federal Research Statement This invention was made with government support under grant numbers R01 AI17321-01 and AI057552, awarded by the National Institutes of Health (NIAID). The government has certain rights to this invention. [Background technology]

[0002] Herpes simplex virus 1 (HSV-1) and herpes simplex virus 2 (HSV-2) are common human pathogens. In developed countries, HSV-1 infects approximately 67% of the population by age 49 and is a leading cause of oral and eye diseases, a leading cause of infectious corneal blindness and fatal infectious encephalitis, and is emerging as a more common cause of genital diseases (Looker, KJ et al., PLoS ONE 2015, 10, e114989-23; Lafferty, WE et al, Journal of Infectious Diseases 2000, 181, 1454-1457; Roberts, CM et al, Sexually Transmitted Diseases 2003, 30, 797-800; Xu, F. et al., JAMA 2006, 296, 964-973). It is estimated that there are over 400 million people infected with HSV-2 worldwide, and it is a major cause of genital diseases in developing countries and a major risk factor for HIV acquisition and infection (Looker, KJ et al., PLoS ONE 2015, 10, e114989-23).

[0003] The enormous global health burden of these two related viruses has led to extensive vaccine development efforts, primarily focused on the production of neutralizing antibodies (nAbs) targeting viral envelope glycoprotein D (gD) as an immunoprotective correlate. One such vaccine was gD-2-AS04 (GlaxoSmithKline), a recombinant gD-2 protein vaccine formulated with aluminum hydroxide (alum) and monophosphoryl lipid A adjuvant. Despite promising preclinical studies and a Phase 3 clinical trial in seromismatched couples demonstrating protection in double seronegative women (not men) of HSV-1 and HSV-2, subsequent field trials did not find protection against HSV-2 infection or disease in double seronegative women (Stanberry, L. R, et al., N Engl J Med 2002, 347, 1652-1661; Belshe, RB, et al., N Engl J Med 2012, 366, 34-43). The vaccine was administered intramuscularly at 0, 1, and 6 months. Another vaccine that recently completed Phase I clinical trials is a replication-deficient HSV-2 strain lacking two genes (UL5 and UL29) involved in viral replication, and has been named dl5-29 (HSV529, Sanofi Pasteur) (Dropulic, LK, et al. Journal of Infectious Diseases 2019, 220, 990-1000).Preclinical studies have shown that the vaccine is safe, induces nAb and T-cell responses, and reduces the likelihood of latency in peripheral nerves (Da Costa, XJEA, et al., J. Virol. 2000, 74, 7963-7971; Da Costa, XJEA, et al., Virology 2001, 288, 256-263; Hoshino, Y., et al., Vaccine 2008, 26, 4034-4040; Hoshino, Y., et al., J. Virol. 2004, 79, 410-418; Hoshino, Y., et al. Journal of Infectious Diseases 2009, 200, 1088-1095; Bernard, M.-C., et al., PLoS ONE 2015, 10). (e0121518-21). In the Phase I trial, it was found that the vaccine was safe, and that the nAb response increased more than fourfold in HSV seronegative participants, but not sustainedly in seropositive participants. Furthermore, only a subset of participants induced a significant CD4 T cell response, and even fewer CD8 T cell responses (Dropulic, LK, et al., Journal of Infectious Diseases 2019, 220, 990-1000).

[0004] To obtain a single-cycle candidate HSV-2 vaccine strain named ΔgD-2, a single-cycle HSV-2 strain lacking glycoprotein D (ΔGD-2) was developed. In preclinical mouse studies, this vaccine strain ΔgD-2 induced high-titer non-neutralizing antibodies that activated Fc gamma receptors (FcγRs) and triggered antibody-dependent cell-mediated cytotoxicity (ADCC). Two subcutaneous doses provided complete protection for female and / or male mice from lethal vaginal or skin attacks by clinical isolates of HSV-1 and HSV-2, preventing the establishment of an incubation period (Petro, C., et al., eLife 2015; Petro, CD, et al., JCI Insight 2016, 1, 1-15; Burn, C., et al., Journal of Infectious Diseases 2017, 1-5; Kao, CM, et al., Journal of Infectious Diseases 2019, 42, 47-10). Furthermore, vaccination of female mice protected their offspring from subsequent HSV challenge within the first week of life (Kao, CM, et al., Journal of Infectious Diseases 2019, 42, 47-10). In contrast to adjuvant-added recombinant gD, ΔgD-2 enhanced the overall response and ADCC Ab response in HSV-1 seropositive mice and prevented subsequent fatal HSV-2 co-infection (Burn Aschner, C., et al., npj Vaccines 2020, 1-33).

[0005] Further strategies for preventing and treating infections and diseases caused by HSV-1 and / or HSV-2 would be beneficial. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Looker, KJ et al., PLoS ONE 2015, 10, e114989-23 [Non-licensed document 2] Lafferty, WE et al, Journal of Infectious Diseases 2000, 181, 1454-1457 [Non-licensed document 3] Roberts, CM et al, Sexually Transmitted Diseases 2003, 30, 797-800

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed Document 8

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

[0007] A method for vaccinating a subject against herpes simplex virus-2 (HSV-2) infection or a disease caused by HSV-2 infection, comprising administering an effective amount of HSV-2 single cycle virus and an effective amount of recombinant HSV-2 glycoprotein D to the subject to vaccinate the subject against herpes simplex virus-2 (HSV-2) infection or a disease caused by HSV-2 infection, wherein the HSV-2 single cycle virus has a deletion in the gene encoding glycoprotein D in its genome, and HSV-2 is phenotypically complemented by HSV-1 glycoprotein D on the lipid bilayer of HSV-2.

[0008] A method for immunizing a subject for herpes simplex virus-2 (HSV-2) infection or a disease caused by HSV-2 infection, comprising administering an effective amount of HSV-2 single-cycle virus and recombinant HSV-2 glycoprotein D to the subject to immunize the subject for herpes simplex virus-2 (HSV-2) infection or a disease caused by HSV-2 infection, wherein the HSV-2 single-cycle virus has a deletion in the gene encoding glycoprotein D in its genome, and the HSV-2 is phenotypically complemented by HSV-1 glycoprotein D on the lipid bilayer of HSV-2.

[0009] A method for treating or preventing HSV-2 infection in a subject, or treating or preventing a disease caused by HSV-2 infection in a subject, comprising administering an effective amount of herpes simplex virus-2 (HSV-2) single-cycle virus and recombinant HSV-2 glycoprotein D to a subject to treat or prevent HSV-2 infection or a disease caused by HSV-2 infection in the subject, wherein the HSV-2 single-cycle virus has a deletion in the gene encoding glycoprotein D in its genome, and the HSV-2 is phenotypically complemented by HSV-1 glycoprotein D on the lipid bilayer of HSV-2.

[0010] The above and other features are illustrated by the following drawings and detailed description. [Brief explanation of the drawing]

[0011] The following drawings are illustrative embodiments, and similar elements are numbered similarly. [Figure 1] The immunogenicity of the virus and adjuvant-added subunit HSV vaccine is regulated by the vaccination route. Female C57BL / 6 mice were given 5 x 10⁶ doses. 4 , 5x10 5 , or 5x10 6Mice were vaccinated subcutaneously (sc), intramuscularly (im), or intradermally (id) twice at 3-week intervals with pfu / mouse of dl5-29 or ΔgD-2, or 5 μg of gD-2-alum / MPL. One week after the second immunization, mice were bled retroorbitally, and sera were tested for total HSV-specific IgG (Figures 1A–C), neutralizing titers (Figures 1D–F), and FcγRIV activation by NFAT-luciferase reporter assay (Figures 1G–I) by ELISA. For gD-2-alum / MPL and dl5-29, 5 mice per group (N = 5) in one experiment, and for ΔgD-2, 5 mice per group (n = 5) in two independent experiments. Asterisks indicate significance by ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0012] [Figure 2] Differences in immunogenicity based on vaccine dose and route explain differences in protection. Female C57BL / 6 mice were vaccinated twice at 3-week intervals subcutaneously (sc), intramuscularly (im), or intradermally (id) with, 5x10 4 , 5x10 5 , or 5x10 6 pfu / mouse of dl5-29 or ΔgD-2, or 5 μg of gD-2-alum / MPL. Three weeks after the second vaccination, mice were inoculated with HSV-2 SD90 at 1mxLD90 on the skin. Disease scores over time for gD-2-alum / MPL (Figure 2A), dl5-29 (Figure 2B), and ΔgD-2 (Figure 2C) are shown. Survival rates are shown in Figures 2D–2J. For gD-2-alum / MPL and dl5-29, 5 mice per group (N = 5) in one experiment, and for ΔgD-2, 5 mice per group (n = 5) in two independent experiments. For survival curves, ** p < 0.01 by Gehan Breslow Wilcoxon test.

[0013] [Figure 3] HSV DNA detection in the sacral ganglia is parallel to survival data. Five μg of rgD-2-alum / MPL, or 5x105 Female C57BL / 6 mice, vaccinated with pfu / mouse dl5-29 or ΔgD-2 via the sc, im, or id routes, were challenged to the skin with 10xLD90 HSV-2 SD90. After the challenge, mice were monitored daily for 14 days. Sacral nerve tissue was collected at death for mice that succumbed to the challenge, or 14 days after the challenge for surviving animals. HSV DNA in the sacral ganglia was evaluated by qPCR, and the copy number of HSV-2 DNA per 10 ng of DNA is shown for rgD-2-aram / MPL (Figure 3B), dl5-29 (Figure 3A), and ΔgD-2 (Figure 3C). Mice that succumbed to the challenge are indicated by a cross symbol. There were no significant differences based on the vaccine administration route (ANOVA).

[0014] [Figure 4] Dynamics of T cell response after HSV vaccination. 5x10 5 Female C57BL / 6 mice were intramuscularly vaccinated twice at 3-week intervals with either pfu / mouse ΔgD-2 or 5 μg of gD-2-aram / MPL. Postorbital blood samples were collected from the mice before vaccination (-1 day) and at 1 and 2 weeks after the initial stimulation and booster immunization. CD11a + CD49d + Activated CD4 and CD8 T cells were evaluated (Figure 4C-D). A gating strategy for evaluating CD4 and CD8 T cell activation is shown (Figure 4A-B). Data were analyzed using Mixed Effects Analysis. * p<0.5, ** p<0.01, *** p<0.001, **** p<0.0001. Five mice per group (n=5).

[0015] [Figure 5] ΔgD-2 vaccination induces pluripotent CD4 and CD8 T cells that produce IFN-γ, TNF, and IL-2 in response to HSV-2 stimulation. Female C57BL / 6 mice were given 5 x 10⁶ doses. 5mice were intramuscularly vaccinated twice with pfu / ΔgD-2 and 5 μg of gD-2-arum / MPL at 3-week intervals. Splenocytes from vaccinated mice were collected 2 weeks after boost vaccination and stimulated for 18 hours with PHA or UV-inactivated HSV-2 SD90 with brefeldin A treatment before staining and flow cytometry analysis for IFN-γ, TNF, and IL-2 production. Gating strategies are shown (Figure 5A), and cytokine responses to CD4 (Figure 5B-D) and CD8 (Figure 5E-G) T cells are shown. Data were analyzed by Mixed Effects Analysis. * p<0.5, ** p<0.01, *** p<0.001. Five mice per group (n=5).

[0016] [Figure 6] Neutralizing antibody production enhances protection by low doses of ΔgD-2. Female C57BL / 6 mice were given 5 x 10⁶ doses. 4 Pfu / ΔgD-2, 5 μg gD-2-aram / MPL, or a combination of both vaccines were administered subcutaneously twice at 3-week intervals to the opposite side (opposite) or the same site (same). One week after the second vaccination, retroorbital blood was collected from the mice, and serum was evaluated for total HSV-specific IgG by ELISA (Figure 6A), neutralizing titer (Figure 6B), and FcγRIV activation (Figure 6C). Three weeks after the second vaccination, the mice were inoculated with 10xLD90 doses of HSV-2 (SD90) into the skin. Survival rates are shown in % (Figure 6D). Five mice per group in two independent experiments (N=5). (AC) * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, calculated using ANOVA. For survival curves, * p<0.05, ***p<0.001, Gehan-Breslow Wilcoxon test. [Modes for carrying out the invention]

[0017] The immunogenicity of a vaccine is influenced by how the viral antigen is presented by the vaccine (attenuated, replication-deficient, single-cycle, inactivated virus, or adjuvant-added subunit protein), as well as the dosage and route of administration. The route of vaccination is often based on practical considerations rather than immunological ones.

[0018] The immunogenicity and efficacy of genetically modified single-cycle herpes simplex virus-2 (HSV-2) with a deletion of glycoprotein D in the genome (ΔgD-2) were studied using different routes and vaccine doses, and compared with adjuvanted recombinant HSV-2 glycoprotein D (rgD-2) (similar to the gD-2-AS04 vaccine (GlaxoSmithKline)) with dl5-29 and aram and monophosphoryl lipid A. The immunogenicity and efficacy of co-administration of ΔgD-2 and rgD-2 were also evaluated. Surprisingly, it was found that co-administration of ΔgD-2 and recombinant HSV-2 glycoprotein D (rgD-2) did not interfere with the immunogenicity of either vaccine. In particular, simultaneous delivery of adjuvant-enhanced rgD-2 and relatively low doses of ΔgD-2 to the same or opposite flank did not interfere with the immunogenicity of either vaccine, and provided greater protection than adjuvant-enhanced rgD-2 alone. Furthermore, it was shown that the combination of low doses of ΔgD-2 and rgD-2 provided additive protection.

[0019] As used herein, “therapeutic dose,” “effective dose,” or “effective amount” refers to a quantity of a particular substance sufficient to achieve the desired effect in a subject.

[0020] "To treat" or "to treat" means administering the vaccine or product of the Disclosure to a subject or patient having or suspected to have one or more disease symptoms, in which case the vaccine or product has therapeutic or prophylactic activity. The vaccine or product may be administered in an amount effective to alleviate one or more disease symptoms of a subject to treat by inducing regression or inhibiting the progression of such symptoms to a clinically measurable extent. The term further includes delaying the onset of symptoms associated with the disorder and / or reducing the severity of symptoms of such disorder. These terms further include improving existing uncontrolled or undesirable symptoms, preventing additional symptoms, and improving or preventing the underlying causes of such symptoms.

[0021] "Preventing" means administering a sufficient amount of the vaccine or product of the disclosure to a subject who may be predisposed to the disease but does not have the disease, to significantly reduce the likelihood of the disease developing. In the context of viral infection, "preventing" includes the administration of an immune product resulting from the administration of a vaccine to a subject known to be at high risk of viral infection.

[0022] As used herein, the term “adjuvant” means any component added to a vaccine that enhances, enhances, and / or boosts the immune response to an antigen, but which, when administered alone, does not produce an immune response.

[0023] Disclosed herein are methods for vaccinating, immunizing, and / or treating subjects against herpes simplex virus (HSV) infection. Also disclosed herein are methods for vaccinating, immunizing, and / or treating subjects against diseases caused by HSV infection. HSV infection is herpes simplex virus 2 (HSV-2) infection, herpes simplex virus 1 (HSV-1) infection, or co-infection of HSV-1 and HSV-2. In one embodiment, HSV infection is herpes simplex virus-2 (HSV-2) infection. Diseases caused by HSV-1 infection, HSV-2 infection, or co-infection of HSV-1 and HSV-2 include herpes, oral herpes, tinea, genital herpes (genital ulcers), herpetic eczema, xiphenidal herpes, HSV keratitis, HSV retinitis, HSV encephalitis, or HSV meningitis. In one embodiment, the disease caused by HSV infection is genital ulcers.

[0024] The methods disclosed herein include administering to a subject an effective amount of HSV-2 single-cycle virus and an effective amount of recombinant HSV-2 glycoprotein D (rgD-2). The HSV-2 single-cycle virus is HSV-2 in which the gene encoding HSV-2 glycoprotein D is deleted in the genome, and HSV-2 is phenotypically complemented by HSV-1 glycoprotein D on the lipid bilayer. The gene encoding HSV-2 glycoprotein D is the Us6 gene of HSV-2, which is completely or partially deleted in the HSV-2 genome. In one embodiment, the gene encoding HSV-2 glycoprotein D is completely deleted in the HSV-2 genome.

[0025] HSV-1 glycoprotein D is not encoded by the HSV-2 genome. In particular, HSV-2 lacking HSV-2 glycoprotein D is phenotypically complemented by HSV-1 glycoprotein D by growing HSV-2 lacking the gene encoding HSV-2 glycoprotein in cells transfected to express HSV-1 gD. A complete description of the HSV-2 single-cycle virus can be found in WO2015 / 134368, which is incorporated herein by reference.

[0026] HSV-2 single-cycle viruses that have a deletion in the HSV-2 genome of the gene encoding HSV-2 glycoprotein D and are phenotypically complemented by HSV-1 glycoprotein D by growing HSV-2 in complementary cells expressing HSV-1 glycoprotein D are referred to herein interchangeably as "HSV-2ΔgD-2", "ΔgD-2", or "HSV-2 single-cycle virus".

[0027] Recombinant HSV-2 glycoprotein D is referred to herein interchangeably as "rgD-2," "recombinant HSV-2 gD," or "recombinant gD-2."

[0028] In multiple embodiments, recombinant HSV-2 gD is combined with an adjuvant, and the adjuvant-added recombinant HSV-2 gD is administered to the target.

[0029] The type of adjuvant is not limited and may be any adjuvant that can enhance, enhance, and / or promote the target immune response to recombinant HSV-2 gD compared to administration of recombinant HSV-2 without adjuvant (e.g., soluble recombinant HSV-2 gD). Non-limited examples of adjuvants include alum, potassium aluminum sulfate, aluminum hydroxide, aluminum sulfate hydroxyphosphate (AAHS), aluminum phosphate, calcium hydroxide phosphate, squalene, and plant saponins derived from Quillaja (e.g., Quill A). TMExamples include soybeans or Polygala senega. These include monophosphoryl lipid A (MPL), Freund's adjuvant (complete or incomplete), oil-in-water emulsions containing unmetabolized oils, paraffinic oil (e.g., EMULSIGEN™, MVP Laboratories, Ralston, Nebraska), mineral oil, vegetable oil or vegetable oil, squalane or squalene (e.g., MF59™), and / or animal oil, CpG oligodeoxynucleotide (ODN), QS-21, or combinations thereof. The adjuvant may be used with or without other specific immunostimulants such as 3-DMP, high molecular weight or monomeric amino acids such as polyglutamic acid or polylysine, or other immunoenhancing agents.

[0030] Disclosed herein are methods for vaccinating a target against herpes simplex virus 1 (HSV-1) infection, herpes simplex virus 2 (HSV-2) infection, or co-infection of HSV-1 and HSV-2. Also disclosed are methods for vaccinating a target against diseases caused by HSV-1 infection, HSV-2 infection, or co-infection of HSV-1 and HSV-2. A method for vaccinating a target against HSV-1 infection, HSV-2 infection, or co-infection of HSV-1 and HSV-2, or diseases caused by HSV-1 infection, HSV-2 infection, or co-infection of HSV-1 and HSV-2, includes administering an effective amount of recombinant HSV-2 single-cycle virus and an effective amount of recombinant HSV-2 glycoprotein D to the target to vaccinate against HSV-2 infection, HSV-1 infection, or co-infection of HSV-1 and HSV-2. HSV-2 single-cycle viruses contain HSV-2 with a deletion in the gene encoding glycoprotein D in its genome, and HSV-2 is phenotypically complemented by HSV-1 glycoprotein D on the lipid bilayer of HSV-2.

[0031] In one embodiment, a method of vaccinating a target against herpes simplex virus-2 (HSV-2) infection or a disease caused by HSV-2 infection includes administering an effective amount of HSV-2 single-cycle virus and an effective amount of recombinant HSV-2 gD to a target to vaccinate a target against HSV-2 infection or a disease caused by HSV-2 infection.

[0032] In one embodiment, a method of vaccinating a target against HSV-1 infection or a disease caused by HSV-1 infection includes administering an effective amount of HSV-2 single-cycle virus and an effective amount of recombinant HSV-2 gD to a target to vaccinate a target against HSV-1 infection or a disease caused by HSV-1 infection.

[0033] In one embodiment, a method of vaccinating a target for co-infection with HSV-1 and HSV-2 or for a disease caused by co-infection with HSV-1 and HSV-2 includes administering an effective amount of HSV-2 single-cycle virus and an effective amount of recombinant HSV-2 gD to a target, and vaccinating a target for co-infection with HSV-1 and HSV-2 or for a disease caused by co-infection with HSV-1 and HSV-2.

[0034] Methods for immunizing a subject against herpes simplex virus-1 (HSV-1) infection, herpes simplex virus-2 (HSV-2) infection, or co-infection with HSV-1 and HSV-2, as well as methods for immunizing a subject against diseases caused by HSV-1 infection, HSV-2 infection, or co-infection with HSV-1 and HSV-2, are also disclosed herein. A method for immunizing a subject against HSV-1 infection, HSV-2 infection, or co-infection with HSV-1 and HSV-2, or diseases caused by HSV-1 infection, HSV-2 infection, or co-infection with HSV-1 and HSV-2, includes administering an effective amount of recombinant HSV-2 single-cycle virus and an effective amount of recombinant HSV-2 gD to the subject to immunize the subject against HSV-1 infection, HSV-2 infection, or co-infection with HSV-1 and HSV-2, or diseases caused by HSV-1 infection, HSV-2 infection, or co-infection with HSV-1 and HSV-2. HSV-2 single-cycle viruses contain HSV-2 with a deletion in the gene encoding glycoprotein D in its genome, and HSV-2 is phenotypically complemented by HSV-1 glycoprotein D on the lipid bilayer of HSV-2.

[0035] In one embodiment, a method for immunizing a subject against HSV-2 infection or a disease caused by HSV-2 infection includes administering to the subject an effective amount of recombinant HSV-2 single-cycle virus and an effective amount of recombinant HSV-2 glycoprotein D to immunize the subject with respect to HSV-2 infection or a disease caused by HSV-2 infection.

[0036] Methods for preventing or treating HSV-2 infection, HSV-1 infection, or co-infection of HSV-2 and HSV-1 in subjects are also disclosed herein.

[0037] A method for preventing or treating HSV-2 infection, HSV-1 infection, or co-infection of HSV-2 and HSV-1 in a subject includes administering to the subject an effective amount of recombinant HSV-2 single-cycle virus and an effective amount of recombinant HSV-2 glycoprotein D to treat the subject with respect to HSV-2 infection, HSV-1 infection, or co-infection of HSV-2 and HSV-1. A method for preventing or treating disease caused by HSV-2 infection, HSV-1 infection, or co-infection of HSV-2 and HSV-1 in a subject includes administering to the subject an effective amount of recombinant HSV-2 single-cycle virus and an effective amount of recombinant HSV-2 glycoprotein D to treat the subject with respect to disease caused by HSV-2 infection, HSV-1 infection, or co-infection of HSV-2 and HSV-1. HSV-2 single-cycle viruses contain HSV-2 with a deletion in the gene encoding glycoprotein D in its genome, and HSV-2 is phenotypically complemented by HSV-1 glycoprotein D on the lipid bilayer of HSV-2.

[0038] In one embodiment, a method for treating or preventing herpes simplex virus-2 (HSV-2) infection in a subject, or a method for treating a disease caused by HSV-2 infection in a subject, includes treating the subject with respect to HSV-2 infection by administering an effective amount of recombinant HSV-2 single-cycle virus and an effective amount of recombinant HSV-2 glycoprotein D to the subject.

[0039] In some embodiments, a disclosed method for vaccinating, immunizing, and / or treating a subject includes administering an effective amount of a composition, pharmaceutical composition, or vaccine containing the HSV-2 single-cycle virus, and administering an effective amount of a composition, pharmaceutical composition, or vaccine containing recombinant HSV-2 gD.

[0040] In some embodiments, the administration of HSV-2 single-cycle virus and recombinant HSV-2 glycoprotein D is carried out substantially simultaneously. As used herein, “substantially simultaneously” means that the HSV-2 single-cycle virus and recombinant HSV-2 gD are administered within a short time interval from each other, for example, within 1 second to 24 hours, 1 second to 12 hours, 1 second to 8 hours, 1 second to 4 hours, 1 second to 2 hours (120 minutes), 1 second to 1 hour (60 minutes), or 1 second to 30 minutes.

[0041] In one embodiment, the HSV-2 single-cycle virus is administered 1 second to 60 minutes before the administration of recombinant glycoprotein D.

[0042] In one embodiment, the HSV-2 single-cycle virus is administered 1 second to 60 minutes after the administration of recombinant glycoprotein D.

[0043] In one embodiment, the administration of HSV-2 single-cycle virus and recombinant HSV-2 gD is performed simultaneously, for example, at the same time. As used herein, simultaneous administration or administration at the same time means that there is no discernible time between the administration of HSV-2 single-cycle virus and recombinant HSV-2 gD.

[0044] Compositions or pharmaceutical formulations or vaccines containing the HSV-2 single-cycle virus, and compositions or pharmaceutical formulations or vaccines containing recombinant HSV-2 gD, can be administered to subjects. Compositions or pharmaceutical formulations or vaccines containing the HSV-2 single-cycle virus and / or recombinant HSV-2 gD are prescribed for administration to subjects.

[0045] In one embodiment, HSV-2 single-cycle virus and recombinant HSV-2 gD are present in different compositions, pharmaceutical formulations, or vaccines for separate administration to a subject. In other words, HSV-2 single-cycle virus and recombinant HSV-2 gD are not present in the same composition, pharmaceutical formulation, or vaccine. However, the disclosure is not necessarily limited thereto, and compositions, pharmaceutical formulations, or vaccines containing both HSV-2 single-cycle virus and recombinant HSV-2 gD may be used, provided that the viability of the HSV-2 single-cycle virus is not impaired by the substances present therein.

[0046] In one aspect of this disclosure, the HSV-2 single-cycle virus and recombinant HSV-2 gD are formulated for separate administration to a subject and administered to the subject substantially simultaneously. In one aspect, the HSV-2 single-cycle virus is administered 1 second to 60 minutes before the administration of recombinant gD. In another aspect, the HSV-2 single-cycle virus is administered 1 second to 60 minutes after the administration of recombinant gD.

[0047] In one embodiment, the HSV-2 single-cycle virus and recombinant HSV-2 gD are formulated for separate administration to a subject and administered to the subject simultaneously. For example, a composition, pharmaceutical formulation, or vaccine containing the HSV-2 single-cycle virus and a composition, pharmaceutical formulation, or vaccine containing recombinant HSV-2 gD can be combined together before administration to the subject and administered simultaneously. Alternatively, for example, a composition, pharmaceutical formulation, or vaccine containing the HSV-2 single-cycle virus and a composition, pharmaceutical formulation, or vaccine containing recombinant HSV-2 gD can be administered to the subject simultaneously by separate means (e.g., different syringes).

[0048] In some embodiments, a composition, pharmaceutical formulation, or vaccine containing HSV-2 single-cycle virus or recombinant HSV-2 gD is formulated to be suitable for administration to a human subject. In particular, the composition, pharmaceutical formulation, or vaccine is formulated to be suitable for the intended route of administration to the subject. The intended route of administration of a composition, pharmaceutical formulation, or vaccine containing HSV-2 single-cycle virus may be the same as or different from the intended route of administration of a composition, pharmaceutical formulation, or vaccine containing recombinant HSV-2 gD. In one embodiment, HSV-2 single-cycle virus and recombinant HSV-2 gD are formulated independently for the same route of administration. In one embodiment, HSV-2 single-cycle virus and recombinant HSV-2 gD are formulated for different routes of administration.

[0049] In one embodiment, the composition, pharmaceutical formulation, or vaccine is formulated to be suitable for subcutaneous, intramuscular, intradermal, or vaginal administration to a subject. In the methods and compositions or pharmaceutical formulations or vaccines disclosed herein, administration is carried out in the auricle, oral cavity, conjunctiva, skin, subcutaneous, intracervical, sinus, trachea, enteral, epidural, hemodialysis, interstitium, intraperitoneal, intraamniotic, intraarterial, intraarticular, intrabiliary, intrabiliary, intrabronchial, intrasacral, intracardiac, intracartilage, intracavitary, intracavernosum, intracavitary, intracerebral, intracisional, intracerebral, intracorneal, intracoronary, intradermal, intradiscal, intratubular, intraepidermal, intraesophageal, intragastric, intravaginal, intravaginal, intraepidermal, intraesophageal, intragastric, intravaginal, intravaginal, intravaginal, intravaginal, intralymphatic Administration may be performed intratubularly, intramedullarily, intrameningally, intramuscularly, intraocularly, intraovarianly, intracardiacly, intraperitoneally, intrapleurally, intraprostatically, intrapulmonaryly, paranasal sinuses, intraspinal cord, intrasynovially, intratendinously, intratendinously, intratesticularly, intramedullarily, intratumorally, intratympanically, intrauterinely, intravascularly, intravenously, intraventricularly, intrabladderally, intravitreously, larynx, nose, transnasogastroctophoretically, orally, oropharynxically, parenterally, percutaneously, perijotarticularly, peridurally, rectally, by inhalation, retrobulbarly, subarachnoidally, subconjunctivally, sublingually, submucosally, topically, percutaneously, transmucosally, transplacentally, transtracheally, ureterally, urethraly, or vaginally. Combinations including at least one of the aforementioned routes of administration may also be used.

[0050] In one embodiment, a composition, pharmaceutical formulation, or vaccine comprising the HSV-2 single-cycle virus, and a composition, pharmaceutical formulation, or vaccine comprising recombinant HSV-2 gD are formulated for subcutaneous, intramuscular, or intradermal administration by injection. A composition, pharmaceutical formulation, or vaccine comprising the HSV-2 single-cycle virus can be administered to the same or a different area as a composition, pharmaceutical formulation, or vaccine comprising recombinant HSV-2 gD. For example, the HSV-2 single-cycle virus and recombinant HSV-2 gD can each be administered to the same or different limbs.

[0051] The compositions, pharmaceutical formulations, or vaccines disclosed herein may contain adjuvants. In one embodiment, a composition, pharmaceutical formulation, or vaccine comprising HSV-2 single-cycle virus or recombinant HSV-2 gD may contain an adjuvant. The compositions, pharmaceutical formulations, or vaccines disclosed herein may also contain pharmaceutically acceptable carriers.

[0052] As used herein, “pharmaceutically acceptable” means a substance approved by a federal or state regulatory agency, or a substance listed in the United States Pharmacopeia, other generally accepted pharmacopoeias, and other formulations safe for use in animals, more specifically, humans and / or non-human mammals. The term “pharmaceutically acceptable carrier” refers to excipients, diluents, preservatives, solubilizers, emulsifiers, adjuvants (also called immunological adjuvants), and / or vehicles administered with the antibody or fragment. Examples of pharmaceutically acceptable carriers include, but are not limited to, phosphate-buffered saline, sterile water (including USP Water for Injection), emulsions such as oil / water emulsions, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration include phosphate-buffered saline or standard (0.9%) saline, e.g., USP 0.9% sodium chloride solution. Compositions containing such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, edited by A. Gennaro, Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing, 2000 (the entire contents of each are incorporated herein)). In non-limiting examples, the carrier comprises one or more of the following: dibasic sodium phosphate, potassium chloride, monobasic potassium phosphate, polysorbate 80 (e.g., 2-[2-[3,5-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl(E)-octadeca-9-enoate), disodium edetate dihydrate, sucrose, monobasic sodium phosphate monohydrate, and dibasic sodium phosphate dihydrate. Pharmacochemically acceptable carriers are not limited as long as they are not incompatible with the viability of the HSV-2 single-cycle virus or the stability and / or conformation of recombinant HSV-2 gD.

[0053] In one embodiment, a composition, pharmaceutical formulation, or vaccine comprising recombinant HSV-2 gD may include stabilizers to prevent loss of protein activity or structural integrity due to denaturation, oxidation, or aggregation over a period of time during storage and transport prior to use. The composition, pharmaceutical formulation, or vaccine may further include salts, surfactants, pH and / or isotonic agents, or combinations thereof.

[0054] When the composition, pharmaceutical formulation, or vaccine is used as an injectable preparation, the pH value is within a nearly neutral pH range (pH 6.8–7.4). In one embodiment, the composition, pharmaceutical formulation, or vaccine is in liquid form. In one embodiment, the composition or pharmaceutical composition is isotonic.

[0055] In this disclosure, subjects are those who require treatment or prevention of HSV-2 infection, HSV-1 infection, or co-infection with HSV-2 and HSV-1. Subjects may also be those who require treatment or prevention of disease caused by HSV-2 infection, HSV-1 infection, or co-infection with HSV-2 and HSV-1. Subjects are mammalian subjects. For example, subjects are human subjects. HSV-2 single-cycle virus and recombinant HSV-2 gD can be formulated for administration to human subjects.

[0056] The methods disclosed herein induce an immune response in a subject, which triggers antibodies, cellular immune responses, and / or other immune factors (e.g., complement) that minimize and / or prevent viral dissemination and / or infection in the subject. In particular, the immune response includes the production of antibodies (FcR-activating antibodies) that activate Fc receptors and mediate an antibody-dependent cell-mediated cytotoxicity (ADCC) response. In one embodiment, administration of an effective dose of HSV-2 single-cycle virus induces the production of FcR-activating antibodies (also known as antibody-dependent cell-mediated cytotoxicity (ADCC) antibodies). An effective dose of HSV-2 single-cycle virus is a certain amount of plaque-forming units (pfus) of HSV-2 single-cycle virus to achieve the described objective.

[0057] This disclosure is further illustrated by the following non-limiting embodiments.

[0058] Examples material and method Female C57BL / 6 (BL / 6) mice of the same age were purchased from Jackson Laboratory (JAX, Bar Harbor, ME).

[0059] Vero cells (green monkey kidney cell line, ATCC), VD60 cells (Ligas, MW et al., J. Virol. 1988, 62, 1486-1494), and V5-29 cells (Da Costa, XJEA et al., Proc. Natl. Acad. Sci. USA 1999, 96, 6994-6998) were grown in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (Hyclone, Logan, UT) and 1% penicillin-streptomycin (Invitrogen). Clinical isolates used for viral attack included HSV-2 (SD90) (Dudek, TE, et al., Journal of Infectious Diseases 2011, 203, 1434-1441) and HSV-2 (4674). HSV-2 strain 4674 was obtained from Montefiore Clinical Virology Lab (Petro, CD, et al, JCI Insight 2016, 1, 1-15; Burn, C., et al, Journal of Infectious Diseases 2017, 1-5). The virus isolate was grown in Vero cells and its titer was measured (Petro, CD, et al., JCI Insight 2016, 1, 1-15).

[0060] ΔgD-2 was grown in complementary VD60 cells, and its titer was measured in both VD60 and Vero cells (Petro, C., et al., eLife 2015; Petro, CD, et al., JCI Insight 2016, 1, 1-15; Burn, C., et al., Journal of Infectious Diseases 2017, 1-5; Kao, CM, et al., Journal of Infectious Diseases 2019, 42, 47-10). Dl5-29 was grown in complementary V5-29 cells (Da Costa, XJEA, et al., J. Virol. 2000, 74, 7963-7971), and its titer was measured in complementary and non-complementary Vero cells. Recombinant gD-2 protein (5 μg) was supplied from the Einstein Macromolecular Therapeutics Development Facility and adjuvanted with 150 μg of alum (Imject Alum, Pierce Biotechnology, Rockland, Illinois) and 12.5 μg of MPL (Invivogen, San Diego, California) (rgD-2 / Alum-MPL) (Burn, C., et al., Journal of Infectious Diseases 2017, 1-5). The recombinant gD-2 is substantially the same as the recombinant gD-2 used in the AS04 vaccine (GlaxoSmithKline).

[0061] Vaccination and Challenge Protocol Female C57BL / 6 mice, 5 x 10 4 , 5×10 5 , or 5×10 6 pfu (based on viral titer by complementary cell lines) γgD-2 or dl5-29; 5 μg rgD-2 / arum MPL; or 5 x 10 4Pfu-based ΔgD-2 and 5 μg of rgD-2 / arum-MPL combinations were administered subcutaneously, intramuscularly, or intradermally (two doses at 3-week intervals). For intradermal vaccination, specialized intradermal microneedles designed for mice were used (Nanopass, Nes Ziona, ISR). Three weeks after the second vaccination, the skin of mice was attacked with HSV-2 SD90 at 10 times the lethal dose (LD90) for 90% of animals (Petro, C., et al., eLife 2015). Mice were monitored daily for epithelial and neurological diseases and scored as described. For skin diseases: 1) erythema at the injection site; 2) distant metastasis, herpes zoster, edema; 3) ulceration, epidermal spread, limb paralysis; 4) hind limb paralysis; 5) death. Mice were euthanized at score 4 and assigned a score of 5 the following day.

[0062] ELISA for HSV-specific antibodies Total HSV-binding IgG or isotype-specific HSV-binding IgG was measured by ELISA using serum collected one week after the second dose of recombinant monoclonal antibody or vaccine. ELISA plates were coated with lysates of Vero cells infected with HSV-2(G) at a MOI of 0.1 for 24 hours, or, as a control, with lysates of uninfected Vero cells. Serial dilutions of serum were incubated overnight at 4°C with the coated plates in two series, and bound IgG was quantified using a biotin-labeled secondary antibody (BD Pharmingen, CA). HSV-specific binding was quantified by subtracting background binding to uninfected Vero cell lysates from binding to HSV-infected Vero cell lysates.

[0063] FcγR activation assay Fc receptor activation, more specifically FcγRIV activation, was determined using a mouse FcγRIV ADCC reporter bioassay (Promega, Madison, WI) (Petro, CD et al., JCI Insight 2016, 1, 1-15; Burn, C., et al., Journal of Infectious Diseases 2017, 1-5). Target Vero cells were infected with HSV-2 (SD90) at a MOI of 0.1 for 12 hours. Infected or uninfected control cells were transferred to a white, flat-bottomed 96-well plate and incubated with thermoinactivated serum from vaccinated or control-immunized mice or human serum samples (see below) (1:5 dilution in DMEM) at room temperature for 15 minutes. Mouse FcγRIV was added at 37°C and 5% CO2 for 6 hours, and FcγRIV activation was detected by adding a luciferin substrate. SpectraMax M5 e The plates were read using molecular devices. The induction factor was calculated by comparing it to the luciferase activity in the absence of serum.

[0064] Neutralization assay Neutralizing titers were determined by a plaque reduction assay (Petro, C., et al., eLife 2015; Petro, CD, et al, JCI Insight 2016, 1, 1-15; Burn, C., et al., Journal of Infectious Diseases 2017, 1-5). Two-fold serial dilutions of thermoinactivated serum were incubated with virus (50 pfu / well) in two series at 37°C for 1 hour and applied to a Vero cell monolayer at 37°C for 1 hour. Cells were fixed with methanol and Giemsa stained after 48 hours of incubation. Plaques were counted, and the neutralizing titer was defined as the highest dilution ratio at which the number of plaques decreased by 50%.

[0065] Quantitative PCR for the determination of viral DNA in nerve tissue. Sacral nerve tissue was extracted at the time of euthanasia (when the mouse succumbed to the disease, or 14 days after HSV-2 challenge), and DNA was isolated using the Qiagen Blood and Tissue DNA Isolation Kit (Qiagen). 10 ng of DNA was loaded per sample, and HSV DNA was quantified using HSV-2 gB-specific primers and probes (HSV-2 forward primer (SEQ ID NO: 1) sequence 5'-TGCAGTTTACGTATAACCACATACAGC-3' (SEQ ID NO: 1); HSV-2 reverse primer sequence 5'-AGCTTGCGGGCCTCGTT-3' (SEQ ID NO: 2); HSV-2 probe sequence 5'-CGCCCCAGCATGTCGTTCACGT-3' (SEQ ID NO: 3) (Namvar, L., et al., Journal of Clinical Microbiology 2005, 43, 2058-2064). Mouse β-actin was used as a loading control (Applied Biosystems, Foster City, CA), and qPCR was performed using Applied Biosystems QuantStudio 7. The assay was performed using Flex. Based on the standard curve, this assay consistently detected copy numbers of 4 or greater. Samples with a detected copy number of less than 4 were considered negative (Petro, C., et al, eLife 2015; Petro, CD, et al, JCI Insight 2016, 1, 1-15; Burn, C., et al., Journal of Infectious Diseases 2017, 1-5).

[0066] Cell separation and flow cytometry Peripheral blood was collected by post-orbital sampling and pipetted into 5 ml of preheated ACK lysis buffer (Lonza BioWhittaker), then incubated at 37°C for 7 minutes. After lysis, the cells were washed twice with calcium and magnesium-free PBS. For splenocyte isolation, the spleen was isolated from vaccinated animals and mechanically digested by pressing it onto a 70 μm cell strainer. The cells were pelleted by centrifugation and resuspended in 2 ml of ACK lysis buffer. After 7 minutes at 37°C, RPMI was added, and the cells were pelleted by centrifugation. The cells were then washed and resuspended in RPMI for further processing.

[0067] For ex vivo stimulation, 2x10 per 200 μL of RPMI + 10% FBS 6 Individual splenocytes were plated in a U-bottom 96-well plate. Cells were treated with PHA (5 μg / mL) or 1 x 10⁶ cells. 6 Cells were treated with PFU UV-inactivated HSV-2 SD90 and incubated at 37°C for 18 hours. Brefelzin A (BioLegend, San Diego, California) was added during the last 5 hours of stimulation. For UV inactivation of the virus, HSV-2 SD90 was diluted in RPMI in a 24-well dish and exposed to a hand-held UV light placed on a 4-inch plate for 30 minutes. Cells were then treated for extracellular and intracellular staining for flow cytometry.

[0068] For flow cytometry analysis, 1 x 10⁶ per 100 μL 6 From 2 x 10 6Cells were incubated with Zombie Near-IR fixable viability dye and TruStain FcX (anti-mouse CD16 / CD32) antibody at room temperature for 10 minutes. For surface staining, cells were stained with anti-CD90.2-BV510, CD4-BV785, CD8-BV711, CD11a-APC, CD49d-APC / Fire750, KLRG1-BV605, and CD62L-BV570 (all BioLegend, San Diego, California) in a mixture of FACS Buffer and Brilliant Stain Buffer (BD Biosciences, Franklin Lakes, NJ) at room temperature for 30 minutes according to manufacturer's instructions. Cells were then washed and fixed by incubation in 200 ml of 2% PFA at room temperature for 20 minutes, followed by permeabilization by incubation in 0.3% Triton X-100 for 7 minutes. For intracellular staining, cells were incubated at 4°C for 30 minutes in 100 μL of a cocktail of anti-IFN-γ-PE, TNF-BV570, and IL-2-PerCP / Cy5.5 (BioLegend, San Diego, California). After staining, cells were washed, passed through a 40 μm cell strainer, and then analyzed using a 5-laser Cytek Aurora flow cytometer. 50,000 bio-CD90.2 cells were analyzed per sample. + Cells were collected, and data analysis was performed using FlowJo (BD Biosciences, Franklin Lakes, NJ).

[0069] statistical analysis The analysis was performed using GraphPad Prism version 8.2.1 software (GraphPad Software Inc., San Diego, CA). A p-value of 0.05 was considered statistically significant. Survival curves were compared using the Gehan-Breslow-Wilcoxon test. Other results were compared using ANOVA or mixed-effects analysis as described in the instructions.

[0070] result Dosage and delivery route affect the immunogenicity of HSV vaccines. To determine whether the dosage and / or delivery route affects immunogenicity, escalate doses of γgD-2 or dl5-29 (5 × 10) 4 , 5×10 5 , or 5×10 6 Mice were prime-boost immunized via the sc, im, or id pathway with 5 μg of gD protein adjuvant-added via pfu / dose (based on titer in complementary cell lines) or Aram and MPL. Total HSV-specific (ELISA), neutralization, and ADCC responses (measured using mouse FcγRIV activation as a surrogate) were quantified in serum obtained one week post-boost. Adjuvant-added gD protein vaccines induced significantly higher total HSV ELISA antibody responses when delivered via id compared to im or sc (p<0.001). Total HSV-specific antibody responses to dl5-29 and ΔgD-2 increased with increasing dose, but there was little difference when comparing the administration routes at each dose. For dl5-29, 5x10 4 At the pfu / mouse dose, the im pathway induced a significantly higher response compared to sc (p<0.05), and for ΔgD-2, 5x10 6 At pfu / mouse doses, the id pathway induced a higher Ab response compared to im (p<0.01, ANOVA) (Figure 1A-C).

[0071] Consistent with the increase in total HSV-specific Abs, a non-significant increase in neutralizing titer was observed after rgD-2 / arum-MPL id administration (Figure 1D-F). The neutralizing response to dl5-29 increased with dose but not when comparing administration routes. ΔgD-2 induced little to no neutralizing Ab response, regardless of dose or administration route, as expected from previous studies. In contrast, ΔgD-2 induced the most potent ADCC response compared to other vaccines, which increased with dose and 5x10 when comparing id or im administration to sc administration. 6The effect was significantly larger with dose. The adjuvant-added gD protein vaccine did not induce an ADCC response compared to control serum, regardless of the route of administration. The dl5-29 vaccine induced an intermediate ADCC response. This was 5x10 6 Compared to 30-fold FcγRIV activation induced by im or id administration of pfu ΔgD-2, 5x10 6 The highest levels were observed after administration of PFU (median 15-fold increase) (Figure 1G-I).

[0072] Differences in immunogenicity lead to differences in defense against lethal skin attacks. Mice vaccinated with Primeboost vaccine were attacked with a 10-fold dose of the LD90 (SD90) of the HSV-2 clinical isolate, which had previously been shown to be consistently lethal in a mouse model (Dudek, TE, et al., Journal of Infectious Diseases 2011, 203, 1434-1441), targeting the skin of the mice. Mice were monitored for signs of disease for two weeks, and euthanized if signs of severe skin or neurological disease were observed as previously described (Figure 2A-J) (Petro, C., et al, eLife 2015; Petro, CD, et al, JCI Insight 2016, 1, 1-15; Burn, C., et al., Journal of Infectious Diseases 2017, 1-5). Compared to im or sc administration, id administration resulted in a moderate but not statistically significant increase in protection provided by the adjuvant-added protein vaccine (Figures 2A-C), which corresponds to increased ELISA and nAb responses (Figures 1A, 1D). The administration route was dl5-29 5x10 4 It did not have a significant effect on post-administration survival (Figure 2E), and was not protective, but both the im and id pathways were 5x10 of dl5-29 5It provided superior protection over the sc route after administration (80% vs. 20%) (Figure 2F) and addressed the significant increase in ADCC (Figure 1H). All three routes were fully protective at the highest vaccine dose (Figure 2G). The only breakthrough in survival rate with ΔgD-2 was achieved with 5x10⁶ doses administered via the sc route. 4 This was observed at the dose (Figure 2H). Complete protection against lethality was observed at all other doses and routes (Figures 2I-J). When examining the association between ADCC and survival in the entire population regardless of dose, route of administration, or vaccine (n=145), 93 out of 96 mice showed a 4.5-fold increase in mFcγRIV activation compared to 14 out of 49 mice where mFcγRIV activation was less than 4.5-fold, and these mice survived (p<0.0001, chi-squared).

[0073] To assess whether the vaccination route affected the vaccine's ability to prevent the establishment of incubation, HSV viral DNA in the ganglia was quantified at death or 14 days post-attack. Despite increased Ab response after id vaccination with adjuvant-added gD protein, viral DNA recovered from the ganglia did not decrease with either immunization route. 5x10 5 The results for doses of dl5-29 and ΔgD-2 were parallel to disease scores and survival data. Compared to 4 out of 5 mice sc-immunized with dl5-29, which had ganglion HSV DNA, only 1 out of 5 mice immunized with im or id had ganglion HSV DNA. No viral DNA was recovered in mice vaccinated with the same dose of ΔgD-2 via any route (Figure 3A-C).

[0074] ΔgD-2 vaccination induces a strong CD4 and CD8 T cell memory response. To further phenotypologically determine the immune responses to γgD-2 and rgD-2 / arum-MPL, which induce functionally distinct Ab responses, mice were subjected to 5 × 10⁶ doses. 5pfu / mouse ΔgD-2 or 5 μg gD-2-alum / MPL were primed-boost vaccines administered via im at 3-week intervals and evaluated in peripheral blood pre-vaccination (-1 day) and at indicated times after both primed and booster vaccinations. ΔgD-2 induced activated CD4 and CD8 T cells after both primed and booster vaccinations, as measured by quantifying CD11a+CD49+ CD4 and CD8 T cells. In contrast, there was little detectable T cell response to adjuvant-added protein vaccines (Figure 4C-D). Splenocytes were collected from these mice at day 42 and stimulated with UV-inactivated SD90 or phytohemagglutinin (PHA) as viability controls to assess cytokine responses. When splenocytes isolated from mice vaccinated with ΔgD-2 (rather than those vaccinated with rgD-2 / Arum-MPL) were stimulated with an inactivated virus, significantly more IFN-γ, TNF, and IL-2-producing CD4+ T cells were observed compared to unstimulated cells (Figure 5B-D). The response was greater than that observed with PHA mitogen. There was also a non-significant increase in the cytokine-producing CD8 T cell response to the ΔgD-2 vaccine compared to unstimulated cells (Figure 5E-G).

[0075] The combination of low-dose ΔgD-2 and rgD-2 provides additional protection. To determine whether the combination of γgD-2 and rgD-2 / arum-MPL is beneficial or antagonistic, mice were given a dose of ΔgD-2 (5 × 10⁻¹) that did not provide complete protection. 4PFU (pfu / mouse) was vaccinated via sc. 5 μg of gD-2-arum / MPL, or a combination of both vaccines, was delivered to the contralateral or same flank. A less efficient vaccination route was used to highlight potential beneficial effects. Both combinations significantly increased the total HSV-specific antibody response compared to either vaccine alone (Figure 6A). These combinations did not show additive or antagonistic effects on the nAb response to rgD-2 / arum-MPL (Figure 6B) or the ADCC response to ΔgD-2 (Figure 6C), compared to the 20% and 60% protection observed with administration of rgD-2 / arum-MPL or ΔgD-2, respectively (Figure 6D), and resulted in 100% protection against skin attack at 10 times the LD90 (SD90) of HSV-2. There was no difference whether the combinations were administered to the contralateral or same flank.

[0076] Discussion Despite promising preclinical data from vaccines designed to induce neutralizing antibody responses primarily targeting gD, clinical studies using HSV vaccine candidates have proven disappointing. Preclinical studies using ΔgD-2 challenged the reliance on neutralizing antibodies, and ΔgD-2 10 5Subcutaneous vaccination with (or more) pfu has been demonstrated to provide reproducibly complete protection against lethal skin, vaginal, or eye attacks by clinical isolates of HSV-1 or HSV-2 (Petro, C., et al., eLife 2015; Petro, CD, et al., JCI Insight 2016, 1, 1-15; Burn, C., et al., Journal of Infectious Diseases 2017, 1-5; Ramsey, NLM, et al., J. Virol. 2020). As demonstrated in passive transmission studies, protection is mediated by ADCC rather than neutralizing Abs. Immunoserum from ΔgD-2-vaccinated mice, rather than mice vaccinated with rgD-2 / arum-MPL vaccine, completely protected naive wild-type mice but did not completely protect FcγRIV knockout mice from lethal attacks (Petro, C., et al, eLife 2015; Petro, CD, et al., JCI Insight 2016, 1, 1-15; Burn, C., et al., Journal of Infectious Diseases 2017, 1-5; Kao, CM, et al., Journal of Infectious Diseases 2019, 42, 47-10; Burn Aschner, C., et al, npj Vaccines 2020, 1-33).

[0077] This study provides further evidence that ADCC offers a more predictive correlation to immune protection compared to neutralization response in mice. The increased protection observed by increasing the dose and delivery route of dl5-29 was associated with a significant increase in ADCC, but not with the neutralization response. Furthermore, the only dose and route of vaccination with ΔgD-2 that did not provide 100% protection against the 10xLD90 challenge at SD90, namely 10 4PFU-mediated immunization via the sc pathway induced an average ADCC response of 2.8-fold (FcγRIV) activation. Regardless of vaccine dose or delivery route, only 3 out of 96 mice with FcγRIV doubling greater than 4.5 succumbed to the high-dose lethal attack.

[0078] Both the im and id routes of vaccination induced significantly higher overall and / or ADCC responses compared to the sc route. While the observation that im and id are more immunogenic than sc is consistent with studies using other vaccines, the relationship between the administration route and avian function (ADCC and neutralization) has not been explained to date. Immunogenicity improvements via the im or id pathways could reflect longer antigen retention, differential exposure to antigen-presenting cells in the dermis, and / or greater access to lymphatic drainage (Wahl, M., et al., Scand. J. Infect. Dis. 1987, 19, 617-621; Bryan, JP, et al., Clin. Infect. Dis. 1992, 14, 697-707; Rahman, F., et al., Hepatology 2000, 31, 521-527; Belshe, RB, et al., N Engl J Med 2004, 351, 2286-2294; Van Damme, P., et al., Vaccine 2009, 27, 454-459). For example, intramuscular administration of trivalent inactivated influenza vaccine resulted in a higher antibody response than subcutaneous vaccination in elderly patients (Gillet, Y., et al., BMC Med 2009, 7, 16). However, similar Ab and T cell responses have been reported with sc or im administration of attenuated measles, mumps, and rubella vaccines (Laurent, PE, et al, Vaccine 2010, 28, 5850-5856). Despite the technical difficulty of delivering consistent doses via the intradermal route, intradermal rabies vaccination has been the standard since the 1992 World Health Organization recommendation (Dubois, B., et al, Journal of Leukocyte Biology 1999, 66, 224-230). Intradermal vaccination is presumed to activate a stronger dendritic cell-mediated response, thus requiring a lower antigen dose (Peng, SL, et al., Proc. Natl. Acad. Sci. USA 2002, 99, 5545-5550).However, when comparing the id or im immune pathways at any dose of dl5-29 or ΔgD-2, we observed no significant difference in ADCC response or vaccine protection, suggesting that the id pathway does not offer a dose advantage for these vaccines. Nevertheless, we observed a statistically significant increase in the total HSV-specific Ab response to subunit vaccines with administration via the id pathway, which resulted in a non-significant increase in neutralizing titer and vaccine protection.

[0079] In addition to dose and delivery route, vaccine composition also influences immunogenicity, as evidenced by the exclusive neutralizing response to gD subunit vaccines, the non-neutralizing response to γgD-2 mediated by FcγR, and both the neutralizing and non-neutralizing responses induced by dl5-29. The absence of neutralizing antibodies after ΔgD-2 immunization may reflect the lack of a primary target for nAbs in mice. In other studies, we found that depleting gD-specific Abs from dl5-29 immunized serum significantly reduced neutralization rather than ADCC titer, indicating that gD is not a target of the ADCC response (Burn Aschner and Herold mspt. submitted). The switching of the IgG subclass to IgG2, which has the strongest affinity for mFcγRIV and is associated with ADCC in mice, requires intragerminal center interactions between antigen-presenting cells, T cells, and B cells (Sattentau, Q, et al, Nat Rev Micro 2008, 6, 815-826). Consistent with the T cell requirements for generating a robust ADCC response, we recorded potent activation of CD4 and CD8 T cells after prime and boost vaccination, although gD-2-aram / MPL induced little T cell activation. Stimulation of memory T cells isolated from ΔgD-2-vaccinated mice (not rgD-2 / aram-MPL-vaccinated mice) with inactivated virus produced IFN-γ, TNF, and IL-2, which were particularly potent with respect to CD4+ T cells. These differences support the idea that a vigorous T cell response contributes to the generation of the ADCC response.

[0080] Combinations of adjuvant-enhanced rgD-2 and low-dose γgD-2 delivered simultaneously to the same or contralateral flank did not interfere with the immunogenicity of either vaccine and were more protective than rgD-2-alum / MPL alone. This is consistent with co-infection mouse studies that have shown existing gD neutralizing agents do not interfere with the immunogenicity of ΔgD-2. Vaccination of HSV-1 seropositive mice with ΔgD-2 increased the ADCC (non-neutralizing) agent response, and subsequently, when the mice were attacked with a lethal dose of HSV-2, full protection was achieved (Burn Aschner, C., et al, npj Vaccines 2020, 1-33). Therefore, nAbs against gD alone are insufficient to protect mice (or humans so far), but a combination of both types of responses may be beneficial. While not bound by theory, one reason for the incomplete protection mediated by nAbs may be the ability of HSV to evade neutralization by spreading directly from cell to cell. However, it is important to note that delivering recombinant gD protein simultaneously with ΔgD-2 is different from having gD present in the viral envelope. In other studies, we have found that enveloped gD inhibits the production of IgG2 subclass switched Abs through interaction with herpesvirus entry mediator (HVEM), also known as tumor necrosis factor receptor superfamily member 14 (Burn Aschner and Herold, mspt under review). This likely contributes to the lower levels of ADCC produced by dl5-29, as shown in this study, and the lower levels produced in response to sublethal infection (Kao, CM, et al, Journal of Infectious Diseases 2019, 42, 47-10; Burn Aschner, C., et al, npj Vaccines 2020, 1-33).

[0081] In summary, this study provides further evidence that ADCC is a key component of immune defense. We initially hypothesized that the intradermal delivery pathway would be more immunogenic for all three vaccines, but this was observed only with the gD protein subunit vaccine. With ΔgD-2 and dl5-29, both the im and id pathways provided similar antibody responses and protection. Overall, ΔgD-2 induced the best ADCC response as well as the most robust protection against lethal challenge and latency.

[0082] Compositions, methods, and articles may, by alternative means, consist of, or essentially consist of, any suitable materials, processes, or components disclosed herein. Compositions, methods, and articles may be formulated additionally or by alternative means to avoid, or substantially avoid, any materials(s), processes, or components that are unnecessary for achieving the function or purpose of the composition, method, or article.

[0083] Terms such as “first,” “second,” etc., do not indicate order, quantity, or importance, but are used to distinguish one element from another. The terms “a,” “an,” and “the” do not imply a limitation of quantity and should be interpreted as covering both singular and plural forms unless otherwise indicated herein or unless the context clearly contradicts this. “Or” means “and / or” unless otherwise specified. Throughout this specification, references to “several embodiments,” “one embodiment,” etc., mean that certain elements described in relation to an embodiment may be included in at least one embodiment described herein, and may or may not be present in other embodiments. Furthermore, it should be understood that the elements described may be combined in any suitable way in various embodiments. “Those combinations” are free and include any combination that includes at least one of the enumerated components or characteristics together with similar or equivalent components or characteristics that may not be enumerated.

[0084] Unless otherwise specified herein, all test standards are the most recent standards in effect as of the filing date of this application, and, if priority is claimed, the filing date of the earliest priority application in which the test standards are described.

[0085] Unless otherwise defined, technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. All cited patents, patent applications, and other references are incorporated herein by reference in their entirety. However, in the event of any conflict or inconsistency between the terms of this application and the terms of an incorporated reference, the terms of this application shall prevail over the conflicting terms of the incorporated reference.

[0086] While specific embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents may arise for the applicant or those skilled in the art that are not currently foreseeable or may not be foreseeable. Therefore, the attached claims, which may be filed and amended, are intended to encompass all such alternatives, modifications, variations, improvements, and substantial equivalents.

Claims

1. (a) Herpes simplex virus-2 (HSV-2) single-cycle virus, and (b) Recombinant HSV-2 glycoprotein D (HSV-2 gD) A pharmaceutical composition for preventing HSV-2 infection or disease caused by HSV-2 infection in a subject, wherein the HSV-2 single-cycle virus comprises HSV-2 having a deletion in the gene encoding glycoprotein D in the HSV-2 genome, and the HSV-2 is phenotypically complemented by HSV-1 glycoprotein D on the lipid bilayer of HSV-2.

2. The pharmaceutical composition according to claim 1, wherein the administration of HSV-2 single-cycle virus and recombinant HSV-2 glycoprotein D is carried out substantially simultaneously.

3. The pharmaceutical composition according to claim 1 or 2, wherein the HSV-2 single-cycle virus and recombinant HSV-2 glycoprotein D are formulated for separate administration to a target.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein HSV-2 single-cycle virus and recombinant HSV-2 glycoprotein D are administered separately to the target.

5. A pharmaceutical composition according to any one of claims 1 to 4, wherein recombinant HSV-2 gD is added as an adjuvant.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the HSV-2 single-cycle virus is administered 1 second to 60 minutes after the administration of recombinant glycoprotein D.

7. A pharmaceutical composition according to any one of claims 1 to 6, wherein HSV-2 gD is formulated for subcutaneous, intramuscular, or intradermal administration.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the HSV-2 single-cycle virus and HSV-2 gD are administered by injection to the same limb of the subject.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein HSV-1 glycoprotein D is not encoded by the recombinant HSV-2 genome, and HSV-2 is phenotypically complemented by HSV-1 glycoprotein D by growing HSV-2 in complementary cells expressing HSV-1 glycoprotein D.

Images