HSV-2-DELTA-gD Vaccines and Methods for Their Production and Use

Abstract

Recombinant herpes simplex virus-2 (HSV-2) vaccine vectors, compositions and vaccines comprising the same, and methods of use thereof are each provided. [Selected Figure] Figure 2

Description

[Technical field]

[0001] Statement of government support This invention was made with Government support under Grant Nos. AI117321 and AI007501 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0002] 2. Background of the Invention Pathogen infections, including influenza and HIV virus infections, have a large impact on global health. Vaccines have been developed for many pathogens, including both influenza and HIV virus infections, but they have failures and limitations. Therefore, new vaccine strategies must be engineered and evaluated. Summary of the Invention

[0003] Summary of the Invention Disclosed herein are new and improved HSV-2-based vaccines for a variety of antigenic targets, including influenza and HIV.

[0004] A process for producing a vaccine vector directed against a heterologous antigen is provided, the process comprising: a) Below: (i) a gene encoding HSV-2 glycoprotein D, which is either completely or partially deleted, and (ii) a nucleic acid comprising a promoter-FP construct, where FP is a nucleic acid encoding a fluorescent protein; providing an HSV-2 genome comprising: b) co-transfecting a host cell with (i) the HSV-2 genome and (ii) the linear DNA fragment encoding the heterologous antigen under conditions in which allelic recombination occurs between the HSV-2 genome of a) and the linear DNA fragment; c) screening the plaques resulting from b) to identify plaques that do not exhibit fluorescence under excitation light that induces fluorescent protein fluorescence; d) Recovering recombinant HSV-2 viruses or virions from those plaques that do not show fluorescence in c) to obtain a vaccine vector directed against the heterologous antigen.

[0005] Also provided is a recombinant herpes simplex virus-2 (HSV-2) having a genome encoding a heterologous antigen produced by the processes described herein.

[0006] Also provided is a recombinant herpes simplex virus-2 (HSV-2) having (i) a complete deletion in its genome of the gene encoding HSV-2 glycoprotein D (designated ΔgD-2), and (ii) (a) a promoter, a heterologous antigen signal sequence, encoding a heterologous antigen, or (b) a promoter, encoding a heterologous antigen.

[0007] Also provided is a recombinant herpes simplex virus-2 (HSV-2) having (i) a partial deletion in its genome of the HSV-2 glycoprotein D-encoding gene, and (ii) (a) encoding, in order, the HSV-2 gD signal sequence, a heterologous antigen, the HSV-2 gD transmembrane domain, optionally the HSV-2 gD cytoplasmic domain, but not the HSV-2 gD extracellular domain, or (b) encoding, in order, the HSV-2 gD signal sequence, a heterologous antigen, and the transmembrane cytoplasmic tail of HSV-2 gD.

[0008] Also provided is an isolated cell comprising a recombinant HSV-2 virus as described herein, wherein the cell is not present in a human.

[0009] Vaccine compositions comprising a recombinant HSV-2 virus as described herein are also provided.

[0010] Also provided is a pharmaceutical composition comprising a recombinant HSV-2 virus as described herein and a pharma- ceutically acceptable carrier.

[0011] Also provided is a method of inducing and / or enhancing an immune response in a subject, comprising administering to the subject an amount of (i) a recombinant HSV-2 virus as described herein; (ii) a vaccine described herein; or (iii) a pharmaceutical composition described herein, in an amount effective to induce and / or enhance an immune response in the subject.

[0012] Also provided is a method of treating or reducing the likelihood of influenza infection in a subject, comprising administering to a subject an amount of (i) a recombinant HSV-2 virus as described herein; (ii) a vaccine as described herein; or (iii) a pharmaceutical composition as described herein, in an amount effective to treat or reduce the likelihood of influenza infection in the subject.

[0013] Also provided is a method of treating or reducing the likelihood of HIV infection in a subject, comprising administering to the subject an amount of (i) a recombinant HSV-2 virus as described herein; (ii) a vaccine as described herein; or (iii) a pharmaceutical composition as described herein, in an amount effective to treat or reduce the likelihood of HIV infection in the subject.

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

[0015] Also provided is a method of vaccinating a subject against HIV infection, comprising administering to the subject an amount of (i) a recombinant HSV-2 virus as described herein; (ii) a vaccine as described herein; or (iii) a pharmaceutical composition as described herein, in an amount effective to vaccinate the subject against HIV infection.

[0016] Also provided is a method for quantifying the rate or amount of antibody-dependent cell-mediated cytotoxicity (ADCK) in a population of cells, comprising infecting a plurality of cells of a population of cells with a fluorescent protein-expressing recombinant HSV-2 comprising a genome deleted for the gene encoding HSV-2 gD under conditions allowing expression of the fluorescent protein in the cells, contacting the plurality of infected cells with an antibody-containing solution and a population of immune cells, and quantifying the amount of cells exhibiting the fluorescent protein fluorescence, and optionally one or more markers, at one or more time points, to quantify the amount of live infected cells over time, thereby quantifying the rate or amount of ADCK in the population of cells. [Brief description of the drawings]

[0017] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]Figure 1A-1B. HSV-2 ΔgD::HA and HSV-2 ΔgD::HA stalks were generated by co-transfection of plasmid DNA and ΔgD-2::PEF1α-RFP (also called B3x2.8, ΔgD-2::RFP) genomic DNA and verified by PCR. Figure 1A. Purified pYUB2169 containing the desired gD::HA gene was cut with PacI and co-transfected with HSV-2 ΔgD::PEF1α-RFP genomic DNA into VD60 cells. Electroporation was used. The resulting RFP-plaques were purified three times and evaluated for the presence of HA. Figure 1B. Recombinant viruses were verified by PCR amplification of the extracellular HA domain from the plasmid and recombinant viral DNA. The primers used were located 100 base pairs (bp) upstream and downstream of the extracellular HA domain. Both recombinant viruses showed the desired bands as visualized by comparison with the plasmid DNA. There was no band of that size in wells that contained DNA from ΔgD*2 (B3x2.9) (lanes 1 and 4). Lane 2 shows DNA from pYUB2169 containing the full-length HA construct. Lane 3 shows DNA from recombinant HSV-2 ΔgD-2::HA (B3x2.10) containing the non-codon-optimized full-length extracellular domain of HA. Lane 5 shows DNA from recombinant HSV-2 ΔgD-2::HA stalk containing the codon-optimized stalk domain of HA. Lane 6 shows DNA from B3x2.11.

[0018] [Diagram 2] FIG. 2 shows the antibody response to vaccination with ΔgD-2::GFP(B3x2.7) or ΔgD-2(B3x2.9).

[0019] [Diagram 3] FIG. 3 shows the results of antibody-dependent cellular cytotoxicity of cells infected with ΔgD-2::GFP virus versus unmarked ΔgD-2 virus.

[0020] [Figure 4]Figure 4. T cell responses to ΔgD-2::GFP virus versus unmarked virus.

[0021] [Diagram 5] Figure 5. ΔgD-2 vaccination protects against challenge with wild-type HSV-2 strain 4674 similar to protection with ΔgD-2::GFP.

[0022] [Figure 6] Figure 6. Construction of chimeric ΔgD-2::HA virus and immune response in mice. Regions corresponding to the HA1 stalk region, HA1 head region, HA2 stalk region, HA2 transmembrane region, and HA2 cytoplasmic region are shown in the amino acid sequence and functional domains of A / Puerto Rico / 1934 / 8 (PR8) HA (SEQ ID NO:2). For the chimeric ΔgD-2::HA virus, the HA1 stalk, HA1 head (or four glycine residues), and HA2 stalk were fused to the transmembrane, cytoplasmic, and signal sequence domains of HSV-2(G)gD. For the headless ΔgD-2::HA gene, the HA1 head domain was replaced with four glycine residues.

[0023] [Figure 7] Figure 7. Presence of chimeric ΔgD-2::HA in recombinant viruses verified by PCR. Lanes are as follows: lane 1: ΔgD-2 genomic DNA, lane 2: ΔgD-2::FL HA nOP genomic DNA, lane 3: pBJJ1 plasmid DNA, lane 4: ΔgD-2 genomic DNA, lane 5: ΔgD-2::HL HA nOP genomic DNA, lane 6: pBJJ2 plasmid DNA. Primers used for PCR amplification were located immediately upstream and downstream of the HA extracellular domain. No exogenous promoter was inserted into the expression cassette. As a result, expression of the chimeric gene was regulated by the endogenous promoter.

[0024] [Figure 8]Figure 8. Mice vaccinated with ΔgD-2::FL HA nOP (β3χ1) and gD-2::HL HA OPT (β3χ3) are fully protected from HSV-2 challenge but do not form anti-HA IgG. Mice were prime-boost vaccinated on days 0 and 21 with control VD60 cell lysate or 1x106 PFU of gD-2::FL HA nOP, gD-2::HL HA OPT, or ΔgD-2::RFP. Figure 8A. On day 42, mice were challenged with 10xLD90 of wild-type HSV-2 4674. Mice vaccinated with gD-2::FL HA nOP and gD-2::HL HA OPT were fully protected from challenge. Figure 8B. Mice were bled for serum antibodies 40 days after prime vaccination. ELISA performed on soluble PR8HA shows the absence of HA-specific IgG in mice.

[0025] [Figure 9] Figure 9 is a map of the pEGFP-N1 expression plasmid. The PCMV::HA::SV40 polyA cassette was restriction cloned into pBRL812, a plasmid containing over 5.5 kB of the HSV-2(G) genome upstream and downstream of US6. pBRL812 was cut with AsiSI and PacI and cotransfected with ΔgD-2::RFP genomic DNA into VD60 cells. Allelic exchange was identified by the lack of RFP expression, and RFP-negative plaques were selected and purified three times before verification of recombination by PCR.

[0026] [Figure 10] Figure 10. Map of the pBRL812 HSV recombinant plasmid prepared as described in the legend to Figure 9 above.

[0027] [Figure 11A]Figure 11A. Validation of ΔgD-2::PCMV-FL HA nOP. PCR using primers immediately upstream and downstream of the PCMV::HA::SV40 PolyA cassette. In Figure 11, unlabeled lane: DNA ladder, lane 1: pJHA1, lane 2: 1.2.1 potential recombinant isolate, and lane 3: negative control ΔgD-2 genomic DNA.

[0028] [Figure 11B] Figure 11B. Validation of ΔgD-2::PCMV-HL HA nOP using PCR with primers immediately upstream and downstream of the PCMV::HA::SV40 PolyA cassette. Unlabeled lanes: DNA ladder, lane 1: pJHA2, lane 2: 2.2.2 potential recombinant isolate, lane 3: 2.3.1 potential recombinant isolate, and lane 4: negative control ΔgD-2 genomic DNA.

[0029] [Figure 11C] Figure 11C. Validation of ΔgD-2::PCMV-HL HA OPT. PCR using primers immediately upstream and downstream of the PCMV::HA::SV40 PolyA cassette. Lane 1: pJHA4, lane 2: 1.2.2 potential recombinant isolate, lane 3: 1.2.3 potential recombinant isolate, unlabeled lane: DNA ladder. The latter (1.2.3 potential recombinant) isolate was expanded to create the original ΔgD-2::PCMV-HL HA OPT stock.

[0030] [Figure 12]Figure 12: ΔGD-2::PCMV-HA recombinant viruses all express HA. VD60 cells were infected with 3 MOI of ΔgD-2::PCMV-FL HA nOP, ΔgD-2::PCMV-HL HA nOP, or ΔgD-2::PCMV-HL HA OPT. 16 hours post-infection, cells were harvested and stained for HSV protein and HA expression. HSV protein expression was measured using serum from mice vaccinated with ΔgD-2::RFP. HA expression was measured using monoclonal anti-HA stalk IgG C179. Cells were stained with either a cell-permeable or cell-impermeable method. Statistics were calculated by Student's t-test of the geometric mean between control fluorescence and fluorescence in wells stained for HA expression. Although only the ΔgD-2::PCMV-FL HA nOP induced high levels of HA expression at the infected cell membrane, all three viruses induced expression as measured by cell permeability staining.

[0031] [Figure 13] FIG. 13 shows that ΔgD-2::PCMV-FL HA nOP does not spread between cells.

[0032] [Figure 14]Figures 14A-14E. Kinetics of fluorescent protein expression and infection in HSV-2 ΔgD-2::RFP (ΔgD-2::RFP) compared to the parental HSV-2 ΔgD-2::GFP (ΔgD-2) strain. Figure 14A. HSV-2 ΔgD-2::RFP (ΔgD-2::RFP) was generated by co-transfecting VD60 cells with HSV-2 ΔgD-2::GFP (ΔgD-2) genomic DNA and a plasmid containing pEF1α::RFP flanked on both sides by regions of homology to the HSV-2(G) genome. Homologous recombination between the genomic DNA and the plasmid DNA gave rise to ΔgD-2::RFP. Recombinant viruses were isolated by three rounds of plaque purification and verified by PCR and viral genome sequencing. Figures 14B and 14C. Vero cells were infected with HSV-2 ΔgD-2 (B) or ΔgD-2::RFP at 1 MOI. The kinetics of infection, RFP expression, and GFP expression were monitored over time. Both viruses show similar infection kinetics. ΔgD-2 induced little GFP expression even at 24 hours postinfection, whereas ΔgD-2::RFP induced high levels of RFP expression starting at 4.5 hours postinfection. Figures 14D and 14E. Images show Vero (D) and VD60 (E) cells 12 hours postinfection with ΔgD-2::RFP at 1 MOI. Infected VD60 cells formed syncytia, indicating productive infection. Vero cells did not form syncytia. Images taken at 10x magnification and deconvolved.

[0033] [Figure 15]Figures 15A-15C. Validation of the novel rapid fluorometric antibody-dependent cell-mediated cytotoxicity (RFADCK) assay in both Raw 264.7 and J774.1 cells. Figure 15A. High expressing target cells were isolated by flow analysis using dual expression of membrane and live / dead markers. The percentage of cells expressing high levels of HSV proteins was then gated by determining the mean RFP intensity for the population of infected target cells. The percentage difference in the proportion of this population between treated and untreated groups was then calculated as ADCK. Figure 15B. Infected target cells were incubated in the presence or absence of serum from ΔgD-2::RFP prior to co-culture with J774.1 macrophages. After 12 hours of co-culture, cells were fixed and the percentage of RFP high and RFP intermediate target cells was quantified. In assay wells containing serum from ΔGD-2:RFP vaccinated mice, there were significantly fewer RFP high cells rather than RFP intermediate or RFP high + intermediate cells at the end of coculture (P<0.05, mean=70.5, 49.3). Values ​​represent three independent experiments performed at least in duplicate. Figure 15C. ADCK assays were performed in parallel using Raw 264.7 and J774.1 macrophages as effector cells. The number of RFP high cells present in cocultures containing serum from mice vaccinated with ΔgD-2:RFP or VD60 cell lysate was compared to that of serum-free cocultures. Serum from ΔgD-2:RFP vaccinated mice induced more damage in cocultures containing both Raw 264.7 and J774.1 cells (p=0.09, p<0.001; mean=18.8%, 38.3%). Data represent three independent experiments performed in triplicate with J774.1 and Raw 264.7 cells at an effector to target cell ratio of 10:1. Target cells used in all experiments were HEK293. Statistics in B were performed using Student's t-test. Statistics in C were performed using one-way ANOVA. Error bars reflect SEM. *p<0.05; ***p<0.001.

[0034] [Figure 16] Figure 16. ADCC and ADCP, although not easily distinguishable, are mediated by Raw 264.7 macrophages in the presence of serum from HSV-2 ΔgD-2::RFP vaccinated mice. Quantification of the number of infected cells that died during the three movies suggests that the addition of serum from HSV-2 ΔgD-2::RFP vaccinated mice induces more damage than serum from mice given control VD60 cell lysates, when compared to movies taken with co-cultures lacking serum (p=0.14 for both groups, mean=18.5, 3.7, n=58 infected cells). Images represent partial frames of one movie taken at 60x magnification. Images were taken every 15 min for 24 h. HEK293 cells were used as target cells. Raw 264.7 cells were used as macrophages. All assays were performed at an effector to target cell ratio of 10:1. Statistics were performed using Student's t-test.

[0035] [Figure 17]Figures 17A-17B. The ADCK assay can be adapted to knockout mouse strains and other model organisms. Figure 17A. The ADCK assay was performed using bone marrow derived macrophages (BMDMs) from both FcγR− / − and WT mice. Baseline killing of infected target cells was determined in co-cultures containing serum from VD60 lysate vaccinated mice. Data represent the percent difference between baseline and co-cultures containing serum from ΔgD-2::RFP vaccinated mice. At an embodiment effector:target ratio of 10:1, WT BMDMs performed significantly more ADCK than FcγR− / − BMDMs in the presence of HSV-2 ΔgD-2::RFP vaccinated serum (p<0.05). Killing of infected cells by FcγR− / − BMDMs in co-cultures containing serum from ΔgD-2::RFP vaccinated mice was indistinguishable from that of co-cultures containing serum from VD60 lysate mock vaccinated mice. Figure 17B. ADCK assays were performed using BMDMs derived from naive guinea pig bone marrow. Baseline ADCK was determined with co-cultures containing serum from naive animals. Due to limited availability of bone marrow, an effector:target ratio of 5:1 was used. At this ratio, BMDMs in co-cultures containing pooled serum from ΔgD-2 vaccinated guinea pigs performed significantly more ADCK than BMDMs in co-cultures containing pooled serum from VD60 mock vaccinated guinea pigs (p<0.001). Each pooled sample contained serum from 6-9 animals. Data represent two independent assays performed in triplicate. HEK293 cells were used as target cells. Statistics were performed by Student's t-test. Error bars reflect SEM. *p<0.05; ***p<0.001.

[0036] [Figure 18] FIG. 18: Anti-HA antibodies induced by vaccination with various PR8 influenza A virus (IAV) hemagglutinin (HA) HSV-2 recombinants (see figure legend).

[0037] [Figure 19] Figure 19: Anti-HA antibody isotypes induced by vaccination with HSV-2 recombinant ΔgD-2::FL HA nOP. **p<0.01.

[0038] [Figure 20] Figure 20: Anti-PR8 IgG isotype ELISA.

[0039] [Figure 21] Figures 21A-21C: Mice vaccinated with ΔgD-2::FLHAPR8 (ΔgD-2::HAPR8) are fully protected from challenge with PR8. Mice were prime-boost vaccinated subcutaneously at 3-week intervals with 5x106 PFU of ΔgD-2::RFP or ΔgD-2::HAPR8 or mock vaccinated with VD60 cell lysate. Figure 21A. One week after the boost, mice were bled and serum neutralizing titers were measured against A / Puerto Rico / 1934 / 8 IAV (PR8). Mice immunized with ΔgD-2::HAPR8 developed significant neutralizing Ab titers against PR8 (mean=304). The dotted line represents the limit of detection for the assay. Figures 21B and 21C. Three weeks after the boost, mice were challenged intranasally with 6xLD50 of PR8. Mice were sacrificed when they reached 75% of their initial body weight. All control vaccinated mice succumbed to infection before day 9, whereas mice immunized with ΔGD-2::HAPR8 were completely protected from PR8 challenge. Statistics for neutralization titers were calculated by ANOVA. Survival statistics were calculated by Mantel-Cox log-rank test *p<0.05; **p<0.01; ***p<0.001.

[0040] [Figure 22]Figures 22A-22C show that recombinant gD-2::HAPR8 expresses high levels of PR8 protein. In Figure 22A, ΔGD-2::RFP DNA was cotransfected into VD60 cells with a HA expression cassette containing the hemagglutinin (HA) gene from IAV H1N1 strain A / Puerto Rico / 1934 / 8 (PR8) downstream of PCMV and upstream of the polyadenylation signal. In Figure 22B, extracellular and intracellular HA expression was measured by flow cytometry in Vero and VD60 cells infected with 3 MOI of ΔgD-2, ΔgD-2::HAPR8, or ΔgD-2 containing a truncated version of the PR8 HA expression cassette (ΔgD-2::HL HAPR8).

[0041] [Diagram 23] Figures 23A-23F show that mice immunized with ΔgD-2::HAPR8 express high titers of functional and isotype-switched anti-PR8 antibodies. Mice were prime-boost vaccinated at 21-day intervals with either VD60 cell lysate, ΔgD-2::RFP vector, or ΔgD-2::HAPR8. 28 days after prime, serum was collected for analysis. Figures AE. Anti-PR8 antibodies were measured by ELISA against purified HA PR8 protein. Mice immunized with ΔGD::HAPR8 developed isotype-switched anti-PR8 HA antibodies that were primarily IgG2c and IgG2b. In Figure 23F, serum from mice immunized with ΔgD-2::HAPR8 induced significant hemagglutination inhibition compared to serum from mice immunized with ΔgD-2 (p<0.001; mean HAI titer=80). Mice immunized with ΔgD-2::RFP did not develop any hemagglutination-inhibiting antibodies. All graphs show one representative experiment with n=5 mice / group. Statistics were calculated using Student's T-test. **p<0.01.

[0042] [Figure 24]Figures 24A-24L show that mice immunized with ΔgD-2::HAPR8 develop protection against IAV challenge. Mice were prime-boost immunized with VD60 cell lysate, ΔgD-2, or ΔgD-2::HAPR8 at 21-day intervals, bled 28 days after prime, and challenged intranasally with 6xLD50 of IAV 14 days later. In Figures 24A-24C, mice immunized with ΔgD-2::HAPR8 developed significant neutralizing titers against PR8 (mean titer PR8=1:304; pVD60<0.01; pΔgD-2<0.05; n=10 mice / group). Mice immunized with ΔgD-2::HAPR8 were also completely protected from weight loss and death following subsequent challenge with PR8 (p<0.001, n=5 mice / group). Figures 24D-24F. Mice immunized with ΔgD::HAPR8 developed significant neutralizing antibodies against A / California / 2009 H1N1 (A / Cal / 2009) IAV (mean titer A / Cal=26; pVD60<0.01 pΔgD-2=0.1; n=10 mice / group). Mice immunized with ΔGD::HAPR8 were partially protected from weight loss and death after challenge with A / Cal / 2009 (P<0.05, n=15 mice / group). As shown in Figures 24G-24I, mice immunized with ΔgD-2::HAPR8, ΔgD-2::RFP, or VD60 cell lysates did not develop neutralizing antibody titers against influenza strains A / Victoria / 3 / 75 H3N2 (A / Vic), and A / Aichi / 68 H3N2 (X-31), and were not protected from weight loss and death after challenge (n neutralization = 10 mice / group; n A / Vic challenge = 15 mice / group; n X-31 challenge = 5 mice / group). Neutralization titers were measured using a microneutralization assay against each strain. Mice were sacrificed after reaching 70% of their starting body weight. Statistics for neutralization assays performed using 3-way ANOVA test. Statistics for survival performed using Mantel-Cox log-rank test. *p<0.05; **p<0.01; ***p<0.001.

[0043] [Diagram 25]Figures 25A-25F. Mice immunized with ΔgD-2::HAPR8 develop fully protective ADCC immunity against HSV-2. Mice were prime-boost vaccinated at 21-day intervals with either VD60 cell lysate, ΔgD-2, or ΔgD-2::HAPR8. In Figures 25A and 25B, mice were bled 1 week after the boost and sera were analyzed by ELISA. Mice that received ΔgD-2 or ΔgD-2::HAPR8 produced similarly high levels of HSV-specific IgG (Figure 25A). Moreover, these IgGs were predominantly IgG2c. In Figure 25C, rapid fluorescent antibody-dependent cellular cytotoxicity (RFADCK) assays were performed using the same sera as in Figures P25A and 25B. Sera from mice vaccinated with ΔgD-2 or ΔgD-2::HAPR8 induced significant ADCK activity in the presence of J774.1 macrophages and ΔgD-2 infected cells compared to sera from mice given VD60 cell lysate (P<0.01). There was no difference between the ADCK activity induced by sera from mice vaccinated with ΔgD-2 and ΔgD-2::HAPR8. In Figures 25D to 25F, mice were challenged by skin scarification with 10xLD90 of HSV-2 4674 21 days after boost. Mice receiving VD60 cell lysate succumbed to HSV-2 by 10 and developed severe epithelial and neurological disease. Mice receiving ΔgD-2 and ΔgD-2::HAPR8 were completely protected from morbidity and mortality after challenge. n=5 mice / group from a typical experiment. Statistics for RFADCK assay performed using 3-way ANOVA test. Survival statistics performed using the Mantel-Cox log-rank test. **p<0.01;****p<0.0001.

[0044] [Figure 26]FIG. 26 shows the results of mFcγRIV ADCC reporter bioassay responses to serially diluted serum samples collected from mice vaccinated with HSV-2 ΔgD::HA, HSV-2 ΔgD::RFP and inactivated A / Puerto Rico / 8 / 1934 H1N1 virus (PR8). Madin-Darby Canine Kidney (MDCK) cells infected with PR8 virus were used as target cells and Jurkat T cells expressing mFcγRIV were used as effector cells. Target cells were incubated with serially diluted serum samples and effector cells. Bio-Glo™ reagent was added and luminescence was measured. Recombinant HSV-2 ΔgD-2::HA vaccinated mice showed significantly higher activation of mFcγRIV receptors compared to mice vaccinated with HSV-2 ΔgD-2::RFP and inactivated PR8 virus. The p-value for significance is <0.05. Symbols: **P≦0.01, ***P≦0.001, ****P≦0.0001.

[0045] [Figure 27] FIG. 27 is a map of plasmid ZM109F.PB4, which was used to amplify full-length HIV-1 Env (clade C) and Rev along with a partial Nef of HIV-1.

[0046] [Figure 28] Figure 28 is a map of the pBkk412 plasmid.

[0047] [Figure 29A] Figure 29A. Screening of transformed E. coli colonies for the presence of the cloned insert.

[0048] [Figure 29B] Figure 29B. Analysis by PCR to identify clones with the correct nucleic acid size.

[0049] Detailed Description A process for producing a vaccine vector directed against a heterologous antigen is provided, the process comprising: a) Below: (i) a gene encoding HSV-2 glycoprotein D, which is either completely or partially deleted, and (ii) a nucleic acid comprising a promoter-FP construct, where FP is a nucleic acid encoding a fluorescent protein; providing an HSV-2 genome comprising: b) co-transfecting a host cell with (i) the HSV-2 genome and (ii) the linear DNA fragment encoding the heterologous antigen under conditions in which allelic recombination occurs between the HSV-2 genome of a) and the linear DNA fragment; c) screening the plaques resulting from b) to identify plaques that do not exhibit fluorescence under excitation light that induces fluorescent protein fluorescence; d) recovering recombinant HSV-2 viruses or virions from those plaques that do not show fluorescence in c) to obtain a vaccine vector directed against the heterologous antigen.

[0050] In an embodiment, the promoter of the promoter-FP construct is a heterologous promoter.

[0051] Also provided is a process for producing a vaccine vector directed against an antigen, the process comprising: a) Below: (i) a deletion in the gene encoding HSV-2 glycoprotein D, and (ii) P EF1α - a nucleic acid comprising an RFP construct, where P EF1α is the promoter of the elongation factor 1α gene, and RFP is a nucleic acid encoding red fluorescent protein, where P EF1α and RFP are fused together (P EF1α -RFP) providing an HSV-2 genome comprising: b) co-transfecting a host cell under conditions allowing allelic recombination with (i) the HSV-2 genome of a) and (ii) a linear DNA fragment encoding, in order, (i) the HSV-2 gD signal sequence, the antigen, the HSV-2 gD transmembrane domain, the HSV-2 gD cytoplasmic domain, but not the HSV-2 gD extracellular domain, or (ii) the HSV-2 gD signal sequence, the antigen, and the transmembrane cytoplasmic tail of HSV-2 gD; c) screening the plaques resulting from b) to identify plaques that do not exhibit red fluorescence under an excitation light that induces red fluorescent protein fluorescence; d) Recovering recombinant HSV-2 viruses or virions from those plaques that do not show red fluorescence in c) to obtain a vaccine vector directed against the antigen.

[0052] A variety of promoters can be used. Heterologous promoters, including those with high efficiency, are preferred. Such promoters include CMV promoters, including the major immediate early promoter of cytomegalovirus. The human elongation factor-1 alpha (EF-1 alpha) constitutive promoter is of human origin and can be used to drive ectopic gene expression in vitro and in vivo. A variety of PEF-1 alphas may be used. Human EF1α gene sequences are known in the art, see, for example, NCBI Accession No. J04617. Other heterologous promoters known in the art and usable in the present invention include, but are not limited to, CMV enhancer fused to chicken beta actin promoter (CAG), mouse cytomegalovirus (mouse CMV), Chinese hamster elongation factor-1 alpha (CHEF-1α), and phosphoglycerate kinase (PGK).

[0053] In an embodiment of the process, the host cell is a complementing cell, e.g., a cell that phenotypically complements HSV-1 glycoprotein D. In an embodiment of the process, the host cell is a VD60 cell that phenotypically complements HSV-1 gD. In an embodiment, the vaccine vector produced is genotypically deleted for HSV-2 gD and phenotypically complemented for HSV-1 gD on its lipid bilayer. In an embodiment, the vaccine vector produced does not genotypically encode any HSV gD.

[0054] In an embodiment, a host cell is co-transfected with (i) the HSV-2 genome of a) and (ii) a linear DNA fragment encoding, in order, (i) the HSV-2 gD signal sequence, the heterologous antigen, the HSV-2 gD transmembrane domain, the HSV-2 gD cytoplasmic domain, but not the HSV-2 gD extracellular domain, or (ii) the HSV-2 gD signal sequence, the heterologous antigen, and the transmembrane cytoplasmic tail of HSV-2 gD.

[0055] In an embodiment, a host cell is co-transfected with (i) the HSV-2 genome of a) and (ii) a linear DNA fragment encoding, in order, (i) a promoter, a heterologous antigen, and optionally a polyA signal.

[0056] In an embodiment, the co-transfecting is accomplished by electroporation.

[0057] Examples of nucleic acid-encodable fluorescent proteins for use in the present invention include red, far-red, yellow, green, orange, cyan, or photoswitchable fluorescent proteins. Examples of such proteins are well known in the art. Suppliers include Molecular Probe (ThermoFisher USA) and Takara (USA). In certain embodiments, the fluorescent protein is a red fluorescent protein. Fluorescent proteins with an excitation range of 554-584 nanometers (nm) and an emission range of 562-610 nm are preferred. Examples include Red Fluorescent Protein, mCherry, mTomato, J-Red, and mOrange. RFP has an excitation of 556 nm and an emission of 584 nm. Alternatively, firefly luciferase or nanoluciferase can be used.

[0058] In an embodiment, the antigen is not an HSV-2 antigen, i.e., it is a heterologous antigen. As used herein, an antigen is heterologous if it is heterologous relative to HSV-2, i.e., if it is not naturally found on or in wild-type HSV-2.

[0059] The heterologous antigen can be derived from a living organism, including, for example, a virus, a bacterium, a parasite, a human cell, an animal cell, or a combination thereof. The heterologous antigen can be a surface protein or a non-surface protein.

[0060] The virus can be a pathogenic virus, examples of which include cytomegalovirus (CMV), coxsackievirus, Crimean-Congo hemorrhagic fever virus, chikungunya virus, dengue virus, Dorivirus, Eastern equine encephalitis (EEE) virus, Ebola virus, Epstein-Barr virus (EBV), hepatitis virus, herpes virus, human immunodeficiency (HIV) virus, human papillomavirus, human SARS coronavirus, human T-lymphotropic virus (HTLV), influenza virus, measles virus, mumps virus, Norwalk virus, rabies virus, rotavirus, rubella virus, severe fever with thrombocytopenic syndrome (SFTS) virus, respiratory syncytial virus (RSV), varicella-zoster virus, Western equine encephalitis virus, West Nile virus, yellow fever virus, Zika virus, or a combination thereof.

[0061] The bacteria can be pathogenic bacteria, examples of which include Bacillus species, Ballonella species, Bordatalla species, Borelli asp., Brucella species, Campylobacter species, Chlamydia species, Clostridium species, Corynebacterium species, Enterococcus species, Escherichia species, Haemophilis species, Helicobacter species, Legionella species, Leptospira species, Listeria species, Mycobacterium species, Mycoplasma species, Neisseria meningitidis, Rickettsia species, Pseudomonas species, Salmonella species, Shigella species, Staphylococcus species, Streptococcus species, Treponema species, Vibrio species, Yersinia species, or combinations thereof.

[0062] The parasite can be a pathogenic parasite, examples of which include Acanthamoeba subsp., Balamuthia subsp., Babesia spp., Balantidium coli, Blastocystic spp., Cryptospiridium spp., Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia, Isospora vero, Leishmania spp., Naegleria fowleri, Plasmodium spp., Rhinosporidium sayberi, Sarcocystis spp., Toxoplasma gondii, Trichomonas spp., Trypanosoma spp., or combinations thereof.

[0063] The human or animal cell can be, for example, a cancer cell.

[0064] In an embodiment, the heterologous antigen is an influenza antigen. In an embodiment, the heterologous antigen is an influenza hemagglutinin (HA) antigen. In an embodiment, the HA antigen is the full-length HA extracellular domain or the HA stalk domain.

[0065] In an embodiment, the heterologous antigen is an HIV antigen. In an embodiment, the HIV antigen is Env gp145.

[0066] In an embodiment, the heterologous antigen is under the control of an upstream CMV promoter and has a downstream SV40 polyA signal, which is known in the art and promotes polyadenylation and transcription termination.

[0067] In an embodiment, the promoter is selected from the group consisting of the elongation factor 1 alpha gene (P EF1α ), where P EF1α and FP are fused together (P EF1α -FP).

[0068] In embodiments, the nucleic acids are codon optimized for expression, see, e.g., Table 4 in the Examples below.

[0069] Vaccine vectors produced by the processes described herein are also provided.

[0070] Also provided is a recombinant herpes simplex virus-2 (HSV-2) having a genome encoding a heterologous antigen produced by the processes described herein.

[0071] Also provided is a recombinant herpes simplex virus-2 (HSV-2) having (i) a complete deletion of the HSV-2 glycoprotein D-encoding gene in its genome and (ii) (a) a promoter, a heterologous antigen signal sequence, and encoding a heterologous antigen, or (b) a promoter and encoding a heterologous antigen.

[0072] Also provided is a recombinant herpes simplex virus-2 (HSV-2) having (i) a partial deletion in its genome of the HSV-2 glycoprotein D-encoding gene and (ii) (a) encoding, in order, the HSV-2 gD signal sequence, a heterologous antigen, the HSV-2 gD transmembrane domain, optionally the HSV-2 gD cytoplasmic domain, but not the HSV-2 gD extracellular domain, or (b) encoding, in order, the HSV-2 gD signal sequence, a heterologous antigen, the cytoplasmic domain of HSV-2 gD.

[0073] Also provided is a recombinant herpes simplex virus-2 (HSV-2) having (i) a partial deletion in its genome of the HSV-2 glycoprotein D-encoding gene and (ii) (a) encoding, in order, the HSV-2 gD signal sequence, a heterologous antigen, the HSV-2 gD transmembrane domain, the HSV-2 gD cytoplasmic domain, but not the HSV-2 gD extracellular domain, or (b) encoding, in order, the HSV-2 gD signal sequence, a heterologous antigen, and the transmembrane cytoplasmic tail of HSV-2 gD.

[0074] In an embodiment, the recombinant HSV-2 further comprises a parasite surface glycoprotein on its lipid bilayer, wherein the parasite is a mammalian parasite.

[0075] In one embodiment, the gene encoding the HSV-2 glycoprotein D is S6 (See, e.g., Dolan et al. J Virol. 1998 March; 72(3): 2010-2021. (PMCID: PMC109494), which is incorporated by reference in its entirety.) The HSV-2 genome and S6 (See "The Genome Sequence of Herpes Simplex Virus Type 2" for the gene. In an embodiment, the gene encoding HSV-2 glycoprotein D is S6 genes. Such equivalents are readily identifiable by those skilled in the art using readily available sequencing and alignment tools.

[0076] In an embodiment, the heterologous antigen is an influenza antigen. In an embodiment, the heterologous antigen is an influenza hemagglutinin (HA) antigen.

[0077] In an embodiment, the HA antigen is a full-length HA extracellular domain or HA stalk. In an embodiment, the full-length HA includes an HA signal sequence. In an embodiment, the HA is a human influenza A or human influenza B HA. Examples of hemagglutinin genes ("HA") include GenBank V01098.1; NCBI Reference Sequence: NP_040980.1. HA gene sequences, and mature hemagglutinin peptide sequences well known in the art, and multiple HA sequences available to the skilled artisan in the NCBI database. In addition, the skilled artisan can easily identify the stalk, extracellular domain, and other regions of the hemagglutinin that are commonly discussed. In addition, seasonal influenza virus strain sequences, including HA sequences, are routinely sequenced and identified in the art.

[0078] B3x2.8(ΔgD-2::P EF1αOther influenza genes as heterologous antigens that can be added to ΔgD-2 by allelic exchange with -RFP include neuraminidase (NA), matrix protein 1 (M1), including influenza A virus (IAV) matrix protein 2 (M2), influenza B virus (IBV) matrix protein 2 (M2), nucleoprotein (NP), and influenza B virus NB. Combinations including at least one of the foregoing can also be used. Corresponding modified headless HA genes for each strain can be added, as well as versions of each headless antigen with a trimerization domain added to increase stability. Table 1 provides non-limiting influenza examples of other such antigen genes. Other examples include inserting the HA gene from A / Vietnam / 1203 / 04, HA, headless HA, NA, M1, M2, NP, and NB genes, for example, from B / Yamagata / 16 / 1988 and B / Victoria / 2 / 1987 strains. [Table 1]

[0079] One method for generating recombinant HSV-2 gD− / − viral vectors expressing HIV or influenza A viral antigens is as follows: 1. To generate an engineered HSV-2 genome by partially deleting the glycoprotein D encoding gene Us6 so that only the glycoprotein D signal sequence and transmembrane cytoplasmic domain sequence remain (no extracellular domain sequence). The deleted portion of the glycoprotein D gene is P EF1 -RFP gene, where red fluorescent protein (RFP) is P EF1 It is expressed under the control of a promoter. 2. Cosmid pYUB2169 can be engineered to express a gene for a heterologous antigen of choice (e.g., the HIV envelope protein antigen Env gp145 or the influenza virus HA stalk antigen) flanked by sequences of the HSV-2 signal sequence and transmembrane cytoplasmic domain sequence on each side. 3. Both the HSV-2 gD- / -RFP genome in (1) and the engineered cosmid pYUB2169 in (2) can be introduced into the VD60 cell line. 4. Homologous recombination occurs between the HSV-2 truncated glycoprotein D gene (with the inserted RFP gene) in (1) and the pYUB2169-HIV Env gp145 or influenza A virus HA stalk antigen expression gene in (2) in the VD60 cell line. Successful recombination is expected to result in the loss of the RFP gene and the generation of HSV-2 gD- / - viruses expressing heterologous HIV or influenza virus antigens (see Figure 1A-1B). 5. VD60 cell culture plaques that are negative for RFP expression contain HSV-2 gD- / - and the viral particles are expected to express HIV Env gp145 or influenza I virus HA stalk antigen. 6. HSV-2gD− / − viruses thus generated expressing heterologous HIV Env gp145 or influenza A virus HA stalk antigens can be used to vaccinate humans and induce potent antibody-mediated responses and protective immunity against HIV or influenza viruses.

[0080] In an embodiment, the heterologous antigen is an HIV antigen. In an embodiment, the HIV antigen is an HIV-1 or HIV-2 antigen. For example, the heterologous antigen is an HIV-1 antigen. In an embodiment, the HIV is a C subtype. In an embodiment, the HIV antigen is an Env, Pol, Gag, or Nef. In an embodiment, the HIV antigen is an Env antigen. In an embodiment, the HIV antigen is a C subtype Env antigen. In an embodiment, the antigen is Env gp145. In an embodiment, the heterologous antigen is a fully intact membrane proximal external region (MPER). In an embodiment, the heterologous antigen is extended by a polylysine tail. In an embodiment, the heterologous antigen is not extended by a polylysine tail. The HIV gp145 Env protein sequence is easily identifiable by alignment tools and is routine to sequence. In an embodiment, the extracellular domain of HIV Env gp145 is fused to the signal peptide and transmembrane cytoplasmic tail of HSV gD.

[0081] Also provided is a cell that contains a recombinant virus as described herein, wherein the cell is not present in a human.

[0082] Also provided is a vaccine composition comprising the recombinant virus as described herein.In an embodiment, the vaccine comprises an adjuvant that is not derived from HSV-2.Adjuvants are well known in the art and include alum, oil-in-water or water-in-oil emulsions, aluminum salts such as aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, and monophosphoryl lipid A.In an embodiment, the vaccine does not comprise an adjuvant.

[0083] Also provided is a pharmaceutical composition comprising a recombinant HSV-2 virus as described herein and a pharma- ceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art.

[0084] Also provided are methods of inducing and / or enhancing an immune response in a subject, the method comprising administering to the subject an amount of (i) a recombinant virus as described herein; (ii) a vaccine as described herein; or (iii) a pharmaceutical composition as described herein, in an amount effective to induce and / or enhance an immune response in the subject.

[0085] Also provided is a method of inducing and / or enhancing an immune response in a subject against a pathogen expressing a heterologous antigen, the method comprising administering to the subject an amount of (i) a recombinant virus as described herein; (ii) a vaccine as described herein; or (iii) a pharmaceutical composition as described herein, in an amount effective to induce and / or enhance an immune response in the subject, wherein the recombinant virus of (i), (ii) or (iii) expresses a heterologous antigen.

[0086] A method for treating or reducing the likelihood of influenza infection in a subject, comprising administering an amount of (i) a recombinant virus as described herein; (ii) a vaccine as described herein; or (iii) a pharmaceutical composition as described herein, in an amount effective to treat or reduce the likelihood of influenza infection in the subject. For treating or reducing the likelihood of influenza, the heterologous antigen is an influenza HA antigen. Also provided is a method for vaccinating a subject against influenza infection, comprising administering to the subject (i) a recombinant virus as described herein; (ii) a vaccine as described herein; or (iii) a pharmaceutical composition as described herein, in an amount effective to vaccinate the subject against influenza infection.

[0087] Also provided is a method of treating or reducing the likelihood of HIV infection in a subject, comprising administering to the subject an amount of (i) a recombinant virus as described herein; (ii) a vaccine as described herein; or (iii) a pharmaceutical composition as described herein, in an amount effective to treat or reduce the likelihood of HIV infection in the subject. For treating or reducing the likelihood of HIV, the heterologous antigen is an HIV antigen. Also provided is a method of vaccinating a subject against HIV infection, comprising administering to the subject an amount of (i) a recombinant virus as described herein; (ii) a vaccine as described herein; or (iii) a pharmaceutical composition as described herein, in an amount effective to vaccinate the subject against HIV infection.

[0088] Also provided is a method of inducing and / or enhancing an immune response in a subject, the method comprising administering to the subject an amount of a recombinant herpes simplex virus-2 (HSV-2) made by a process described herein and comprising (i) a complete deletion of the HSV-2 glycoprotein D-encoding gene in its genome and (ii) encoding a promoter, an influenza hemagglutinin (HA) antigen signal sequence, and an HA antigen, in an amount effective to induce and / or enhance an immune response in the subject.

[0089] Also provided is a method of treating or reducing the likelihood of influenza infection in a subject, comprising administering to the subject an amount of a recombinant herpes simplex virus-2 (HSV-2) made by a process described herein and comprising (i) a complete deletion of the HSV-2 glycoprotein D-encoding gene in its genome and (ii) encoding a promoter, an influenza hemagglutinin (HA) antigen signal sequence, and an HA antigen, in an amount effective to treat or reduce the likelihood of influenza infection in the subject.

[0090] Also provided is a method of vaccinating a subject against influenza infection, the method comprising administering to the subject an amount of a recombinant herpes simplex virus-2 produced by a process described herein and comprising (i) a complete deletion of the HSV-2 glycoprotein D-encoding gene in its genome and (ii) encoding a promoter, an influenza hemagglutinin (HA) antigen signal sequence, and an HA antigen, in an amount effective to vaccinate the subject against influenza infection.

[0091] In an embodiment, the HA antigen is the full-length HA extracellular domain.

[0092] In an embodiment, the method further comprises administering, following the initial administration of a recombinant herpes simplex virus-2 encoding a full-length HA extracellular domain, one or more amounts of a recombinant herpes simplex virus-2 having (i) a complete deletion of the HSV-2 glycoprotein D encoding gene in its genome and (ii) encoding a promoter, an HA antigen signal sequence, and an HA stalk, but not a full-length HA.

[0093] In an embodiment of the method or process, the HSV-2 glycoprotein D is represented by SEQ ID NO:1: MGRLTSGVGTAALLVVAVGLRVVCAKYALADPSLKMADPNRFRGKNLPVLDQLTDPPGVKRVYHIQPSLEDPFQPPSIPITVYYAVLERACRSVLLHAPSEAPQIVRGASDEARKHTYNLTIAWYRMGDNCAIIPITVMEYTECPYNKSLGVCPIRTQPRWSYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKIN DWTEITQFILEHRARASCKYALPLRIPPAACLTSKAYQQGVTVDSIGMLPRFIPENQRTVALYSLKIAGWHGPKPPYTSTLLPPELSDTTNATQPELVPEDPEDSALLEDPAGTVSSQIPPNWHIPSIQDVAPHHAPAAPSNPGLIIGALAGSTLAVLVIGGIAFWVRRRAQMAPKRLRLPHIRDDDAPPSHQPLFY (HSV-2 reference stock HG52) The amino acid sequence shown in

[0094] In one embodiment, the HSV-2 in which the gene encoding HSV-2 glycoprotein D has been deleted is an HSV-2 having a genome (before the deletion) set forth in one of the following Genbank listed sequences: HSV-2 (G)(KU310668), HSV-2(4674)(KU310667), B3x1.1(KU310657), B3x1.2(KU310658), B3x1.3(KU310659), B3x1.4(KU310660), B3x1.5(KU310661)), B3x2.1(KU310662), B3x2.2(KU310663), B3x2.3(KU310664), B3x2.4(KU310665), B3x2.5(KU310666). These sequences are incorporated herein by reference.

[0095] A cell containing a recombinant HSV-2 genome as described herein is provided.

[0096] Also provided is a vaccine composition comprising a recombinant HSV-2 virus as described herein. In one embodiment, the vaccine comprises an immunological adjuvant. In one embodiment, the vaccine does not comprise an immunological adjuvant. In an embodiment of the vaccine, composition or pharmaceutical composition described herein comprising recombinant HSV-2, the HSV-2 is attenuated.

[0097] Also provided are compositions comprising a recombinant HSV-2 virus as described herein, wherein the viral or virion genome comprises a deletion of at least a second gene, the second gene being required for HSV-2 viral replication or virulence.

[0098] A pharmaceutical composition comprising a recombinant HSV-2 virus as described herein and a pharma- ceutically acceptable carrier.

[0099] In one embodiment, the composition or pharmaceutical composition or vaccine is formulated such that it is suitable for subcutaneous administration to a human subject. In one embodiment, the composition or pharmaceutical composition or vaccine is formulated such that it is suitable for oral administration to a human subject. In one embodiment, the composition or pharmaceutical composition or vaccine is formulated such that it is suitable for vaginal administration to a human subject. In one embodiment, the composition or pharmaceutical composition or vaccine is formulated such that it is suitable for intramuscular, intranasal, or mucosal administration to a human subject. In embodiments of the methods herein and of the compositions or pharmaceutical compositions or vaccine formulations herein, administration can be via auricular, buccal, conjunctival, cutaneous, subcutaneous, intracervical, intrasinus, intratracheal, enteral, epidural, via hemodialysis, interstitial, intraperitoneal, intra-amniotic, intra-articular, intrabiliary, intra-bronchial, intravesical, intracardiac, intracartilage, intrasacral, intracavernous, intraluminal, intracerebral, intracisternal, intracorneal, intracoronary, intradermal, intradiscal, intraductal, intraepidermal, intraesophageal, intragastric, intravaginal, intragingival, intraintestinal, intraluminal, intralesional, intralymphatic, intramedullary, intrameningeal, intramuscular, ocular, The route of administration may be intravenous, intraovarian, intraepicardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intranasal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intrapleural, intratubular, intratumoral, intratympanic, intrauterine, intravascular, intravenous, intraventricular, intravesical, intravitreal, laryngeal, intranasal, nasogastric, ophthalmic, oral, oropharyngeal, parenteral, percutaneous, periarticular, epidural, rectal, by inhalation, retrobulbar, subarachnoid, subconjunctival, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, ureteral, urethral, ​​or vaginal. Combinations comprising at least one of the foregoing routes of administration may also be used.

[0100] Also provided is a method of inducing an immune response in a subject, the method comprising: (i) administering to the subject an amount of a recombinant HSV-2 virus as described herein, in an amount effective to induce an immune response in the subject.

[0101] In one embodiment, the gene encoding HSV-2 glycoprotein D is the HSV-2 US6 gene. In one embodiment, the HSV-2 recombinant virus encodes a heterologous surface glycoprotein. In one embodiment, the heterologous surface glycoprotein is HSV-1 gD. In one embodiment, the HSV-2 recombinant virus comprises a non-genomically encoded HSV-1 gD, which is not a herpesvirus glycoprotein and / or is not involved in Herpesviridae infection, and also encodes a heterologous surface glycoprotein encoded by a transgene inserted into the genome of the recombinant HSV-2. In one embodiment, the genome of the recombinant HSV-2 does not encode herpesvirus gD. In one embodiment, the surface glycoprotein is present on the lipid bilayer of the virus by infecting a cell with a recombinant HSV-2 having a deletion of the gene encoding HSV-2 glycoprotein D, where the cell is transfected or has been transfected to express the surface glycoprotein on its cell membrane, and recombinant HSV-2 comprising the surface glycoprotein present on the lipid bilayer is produced from the cell. In one embodiment, the host cell is an HSV-1 gD-complementing cell. In one embodiment, the host cell encodes HSV-1 gD under an endogenous gene promoter. In one embodiment, the host cell is an HSV-1 gD-complementing VD60 cell. (See, e.g., Ligas et al., J Virol. 1988 May; 62(5):1486-94, incorporated herein by reference).

[0102] Also provided is a cell that contains a recombinant virus as described herein, where the cell is not present in a human.

[0103] A vaccine composition comprising a recombinant virus as described herein.In one embodiment of the vaccine composition, the vaccine composition comprises an immunological adjuvant.

[0104] Also provided is a composition comprising a recombinant virus as described herein, wherein the genome of the virus comprises a deletion of at least a second gene, the second gene being required for HSV-2 viral replication.

[0105] In one embodiment, the recombinant virus as described herein does not comprise a deletion of a second gene.

[0106] Reducing the likelihood of viral infection is understood to mean improving the degree of onset of the associated disease or the chance of infection in a subject treated with a virus, vaccine or composition described herein compared to an untreated subject.

[0107] In the embodiment of the method of the present specification for immunizing, vaccinating or inducing an immune response, the passive transfer of the virus or the antibody or immune factor derived thereby may be effected from one subject to a second subject. The relevant product may be treated after obtaining from one subject before being administered to another subject. In the preferred embodiment of the present invention described herein, the subject is a mammalian subject. In one embodiment, the mammalian subject is a human subject.

[0108] In one embodiment, vaccinating a subject with an antigen induces a humoral immune response in the subject against that antigen, and the vaccinated individual is typically able to mount a more effective immune response to a subsequent challenge with a pathogen that contains that antigen than prior to vaccination.

[0109] In one embodiment of the methods described herein, the subject is not yet infected with influenza virus. In one embodiment of the methods described herein, the subject is not yet infected with HIV. In one embodiment of the methods described herein, the subject is infected with influenza virus. In one embodiment of the methods described herein, the subject is infected with HIV.

[0110] In an embodiment, the influenza infection is a human influenza A infection. In an embodiment, the influenza infection is a human influenza B infection. In an embodiment, the HIV infection is an HIV-1 infection. In an embodiment, the HIV infection is an HIV-2 infection.

[0111] "Codon optimization" is defined as modifying a nucleic acid sequence for enhanced expression in a target, for example, human cell, by replacing at least one, two or more, or a significant number of codons of a native sequence with codons that are more frequently or most frequently used in vertebrate genes. Different species show a particular bias for certain codons of specific amino acids. In one aspect, the present invention relates to codon-optimized inserts, nucleic acids or vectors, or host cells containing them.

[0112] Also provided is a method for quantifying the rate or amount of antibody-dependent cell-mediated cytotoxicity (ADCK) in a population of cells, comprising infecting a plurality of cells of a population of cells with a fluorescent protein-expressing recombinant HSV-2 comprising a genome deleted for the gene encoding HSV-2 gD under conditions allowing expression of the fluorescent protein in the cells, contacting the plurality of infected cells with an antibody-containing solution and a population of immune cells, and quantifying the amount of cells exhibiting the fluorescent protein fluorescence, and optionally one or more markers, at one or more time points, so as to quantify the amount of live infected cells over time, thereby quantifying the rate or amount of ADCK in the population of immune cells.

[0113] Also provided is a method for quantifying the rate or amount of antibody-dependent cell-mediated cytotoxicity (ADCK) in a population of cells, comprising infecting a plurality of cells of the population of cells with a fluorescent protein-expressing recombinant HSV-2 comprising a genome deleted for the gene encoding HSV-2 gD under conditions allowing expression of the fluorescent protein in the cells, contacting the plurality of infected cells with an antibody-containing solution, and quantifying the amount of cells exhibiting the fluorescent protein fluorescence, and optionally one or more markers, at one or more time points, to quantify the amount of live infected cells over time, thereby quantifying the rate or amount of ADCK in the population of cells.

[0114] In an embodiment, the recombinant HSV-2 is made by a process as described herein.

[0115] In an embodiment, the method is carried out in vitro. In an embodiment, the population of immune cells comprises a population of macrophages. In an embodiment, the macrophages are human. In an embodiment, the antibody-containing solution comprises serum. In an embodiment, the fluorescent protein is as described herein above. In one embodiment, the fluorescent protein is red fluorescent protein. In one embodiment, RFP 高い In one embodiment, a plurality of cells are considered viable if they express RFP above the average intensity of RFp-expressing cells in the infected population of cells. 高い In embodiments, the method is performed on a population of immune cells present at an effector:target ratio of 5:1 or greater. In embodiments, the method is performed on a population of immune cells present at an effector:target ratio of 10:1 or greater.

[0116] In embodiments, the amount of cells exhibiting fluorescent protein fluorescence and, optionally, one or more markers, is measured by fluorescence activated cell sorting (FACS). In embodiments, the amount of cells exhibiting fluorescent protein fluorescence and, optionally, one or more markers, is measured by a fluorescence spectrometer, a fluorescence microplate reader, and a fluorescence microscope or a fluorescence plate reader. In embodiments, the one or more markers include a cell membrane marker and / or a live / dead marker.

[0117] In an embodiment, the method further comprises quantifying the amount of cells exhibiting fluorescent protein fluorescence, and optionally one or more markers, in an identical control population of infected cells that has not been contacted with the antibody-containing solution at one or more time points, and comparing the quantified amount or percentage to that quantified for the population of cells contacted with the antibody-containing solution.

[0118] In other embodiments of the invention relating to assays for ADCK, the marker in the recombinant HSV-2 ΔgD-2 can be beta-galactosidase or alkaline phosphatase instead of a fluorescent protein. Thus, the methods, processes and compositions disclosed herein can include, mutatis mutandis, recombinant HSV-2 ΔgD-2 that includes a nucleic acid in its genome encoding beta-galactosidase or alkaline phosphatase.

[0119] As used herein, for example with Option A and / or Option B, "and / or" includes the separate embodiments of (i) Option A, (ii) Option B, and (iii) Option A plus Option B.

[0120] All combinations of the various elements described herein are within the scope of the invention unless otherwise indicated herein or clearly contradicted by context.

[0121] The present invention may be better understood from the following examples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0122] Working Example Example 1 An engineered HSV-2 virus was constructed in which the gD gene (ΔgD-2) was replaced with a gene that strongly expresses red fluorescent protein (RFP). Once accomplished, this provided an improved screen for identifying and obtaining new recombinants. The ΔgD-2::RFP recombinant was made from ΔgD-2, which showed protection. 3 x2.8 or ΔgD-2::P EF1α Red fluorescent ΔgD-2::RFP, also referred to as -RFP, retains the ability of the original ΔgD-2 to induce protective immunity against HSV infection, but also appears to have an unaltered in vitro replication kinetic. Furthermore, ΔgD-2::RFP has a significant advantage over the HSV-2 ΔgD::GFP virus for making recombinants. Because the RFP gene is fused to a highly efficient promoter, such as the promoter of the EF1α (elongation factor 1α) gene, its expression is easily detected using a fluorescent microscope. In addition, visualization of red fluorescence in cells infected with ΔgD-2::RFP is without the background found with green fluorescent ΔgD-2. This feature is consistent with the ΔgD-2::RFP-infected cells. EF1α This allows the unambiguous identification of recombinant viruses in which the -RFP allele has been replaced by another allelic exchange substrate.

[0123] Construction of HSV-2 ΔgD- / +gD-1 virus (hereafter referred to as "ΔgD-2") using HSV-2(G) as the backbone was accomplished as detailed below. To make the allelic exchange construct, an expression construct was generated by Gibson cloning into an E. coli plasmid, cloning the expression construct between the SpeI and BclI sites of a shuttle (e.g., cosmid pYUB2169) containing 12 kb of HSV-2(G) virus spanning US1 to US9, electroporating a PacI restriction fragment from the pYUB2169 recombinant into VD60 cells along with ΔgD-2::RFP DNA, and screening the resulting plaques for loss of red fluorescence (see FIG. 1).

[0124] Using this system, two sets of recombinant ΔgD-2 strains containing hemagglutinin (HA) antigens derived from influenza A virus (IAV) strain A / Puerto Rico / 8 / 1934 (PR8) were generated. The first set of recombinants contained chimeric gD::HA genes recombined into the US6 region of ΔgD-2, for example, via the use of pYUB2169. These recombinant genes were composed of the extracellular domain of PR8HA fused to the transmembrane, cytoplasmic and signal sequence domains of HSV-2(G)gD. The second set of recombinants contained modified or unmodified PR8HA genes fused to an upstream CMV promoter and a downstream SV40 polyadenylation signal. These cassettes were inserted into the US6 region of ΔgD-2 using a modified pYUB2169 identified as pBRL812. Each set of recombinants contained three types of HA genes: FL HA nOP-Full length extracellular domain hemagglutinin, non-codon optimized HL HA nOP-headless extracellular domain hemagglutinin, non-codon optimized HL HA OPT-headless extracellular domain hemagglutinin, codon optimized.

[0125] The following constructs were made:

[0126] ΔgD-2::RFP (ΔgD-2::PEF1α-RFP) was constructed by (1) introducing a kanamycin marker into the RFP plasmid ptwB, (2) amplifying the kan marker and RFP(dTomato) with oligos carrying ~50 bp of homologous sequences upstream and downstream of US6, (3) recombining into DY331 cells, adding the PCR product, and then transforming with cosmid pYUB2156. (4) The kan marker was removed by restriction digestion and ligation. (5) HSV2(G) genomic DNA and pYUB2167 were cotransfected into VD60 cells to generate ΔgD-2::RFP by allelic exchange.

[0127] ΔgD-2 was obtained by cotransfection of cosmid pYUB2163 with the ΔgD-2::RFP genome in VD60 cells and generating the ΔgD-2 genome by allelic exchange. This genome is free of all markers and antibiotic resistance genes. The resulting unmarked viruses were classified based on the lack of RFP expression. RFP-negative plaques were purified three times and the absence of the RFP gene was verified by PCR and sequencing.

[0128] The ΔgD-2::FL HA nOP was constructed by (1) Gibson cloning the cytoplasmic, transmembrane and signal sequence domains of HSV-2(G)gD into the extracellular domain of custom synthesized PR8 HA (Genscript, Piscataway, NJ) in the pYUB2169 plasmid and (2) cotransfecting the resulting plasmid, pBJJ1, with the ΔgD-2:RFP genome in VD60 cells to generate the desired recombinants by allelic exchange.

[0129] The ΔgD-2::HL HA nOP was constructed similarly to the ΔgD-2::FL HA nOP, but using a custom synthetic PR8 HA gene (Genscript) in which the HA1 head domain was replaced with four glycine residues. The plasmid used for transfection was designated pBJJ2.

[0130] ΔgD-2::HL HA OPT was constructed similarly to ΔgD-2::HL HA nOP, but with the HA1 head domain replaced with four glycine residues, and each codon that had less than 9.5% representation in HSV-2(G)gD for its cognate amino acid replaced with the most abundant codon in HSV-2(G)gD using a custom synthetic PR8 HA gene (Genscript). The plasmid used for transfection was designated pBJJ4.

[0131] ΔgD-2::P CMV The -FL HA nOP was generated by (1) restriction cloning the full-length PR8 HA gene (Genscript) between the xbaI and HindIII restriction sites of pEGFP-N1 (Addgene, Cambridge, MA) and (2) cloning P from the resulting plasmid into the SpeI and BclI sites of pBRL812. CMV A -FL HA nOP::SV40 polyA cassette was constructed by restriction cloning into pJHA1. The resulting plasmid, pJHA1, was cut with PacI and AsiSI and cotransfected with the ΔgD-2::RFP genome in VD60 cells to generate the desired recombinants by allelic exchange.

[0132] ΔgD-2::P CMV -HL HA nOP, ΔgD-2::P CMV -FL HA was constructed using a custom synthetic PR8 HA gene (Genscript) similar to nOP, but with the HA1 head domain replaced with four glycine residues. The plasmid used for transfection was designated pJHA2.

[0133] ΔgD-2::P CMV -HL HA OPT, ΔgD-2::P CMVA custom synthetic PR8 HA gene (Genscript) was constructed similar to the -FL HA nOP, but with the HA1 head domain replaced with four glycine residues, and each codon that had less than 9.5% representation in HSV-2(G)gD for its cognate amino acid was replaced with the most abundant codon in HSV-2(G)gD. The plasmid used for transfection was designated pJHA4. [Table 2]

[0134] Whole genome sequencing was used to verify the deletion of US6. To test immunogenicity, mice were vaccinated on day 0 (d0) and d21, and then serum was collected on d40. ΔgD-2 elicited anti-HSV antibodies that were similar to those obtained with ΔgD-2::GFP (see Figure 2).

[0135] Mechanistically, the most important correlates of protection appear to be ADCC and ADCP. Sera from ΔgD-2 vaccinated mice induced significantly more ADCC / ADCP immunity than sera from ΔgD-2::GFP vaccinated mice (Figure 3). CD8 and CD4 T cell responses were similar at 40 days using the same vaccination strategy (Figure 4).

[0136] ΔgD-2 vaccination protects mice against challenge with wild-type HSV-2 4674 and GFP-marked constructs, clinical isolates obtained from the Virology Laboratory at Montefiore Hospital, Bronx, NY. Mice were vaccinated on d0 and d21 with either wild-type HSV-2 or GFP-marked construct viruses. 21 days after the boost, mice were challenged subcutaneously with 10× LD90 HSV-2 4674 and followed for changes in skin lesions, presence of virus in dorsal root ganglia, weight, and survival. The unmarked virus performed nearly identically by all measures (Figure 5).

[0137] material and method HSV-1(17) (Brown et al., 1973), HSV-2(G) (Ejercito et al., 1968), HSV-1(F) (Ejercito et al., 1968), and HSV-2(333)ZAG (Nixon et al., 2013), recombinant viruses expressing green fluorescent protein (GFP), were propagated on Vero cells. HSV-2 4674 (Nixon et al., 2013) was propagated on HaCAT cells. VD60 cells, Vero cells encoding gD-1 under the endogenous gene promoter (Ligas and Johnson, 1988), were passaged in DMEM supplemented with 10% fetal bovine serum. HSV-2 ΔgD-2 (ΔgD- / +gD-1) virus stocks were propagated on complementing VD60 cells and titered on VD60 and Vero cells. Concentrated virus stocks were stored at -80°C and diluted to desired concentrations in PBS as needed.

[0138] Construction of ΔgD-2::GFP. Plasmid pcDNA3-eGFP (13031; Addgene, Cambridge, MA, USA) was used as a template to construct pCMV-eGFP-Neo flanked by Van91I restriction enzyme sites. r and OriE-Amp r The region was PCR amplified. pCMV-eGFP-Neo rThe region was PCR amplified using primers Fwd-pCMV and Rev-NeoR-Term (see Table 3 for a list of primers). The OriE-Ampr region was PCR amplified using primers Fwd-Origin and Rev-AmpR.

[0139] In parallel, the left and right flanking genomic regions of the US6 gene (gD) in HSV-2 were PCR amplified using purified viral DNA (HSV-2 strain 4674) as template and primers LL-V91I-US6 plus LR-V91I-US6 for the left homology arm and primers RL-V91I-US6 and RR-V91I-US6 for the right homology arm (see Table 3 for sequence alignment). All four PCR fragments were gel purified, digested with Van91I (Fermentas Molecular Biology Tools, Thermo Scientific, West Palm Beach, FL, USA), ligated with Quick-Ligase [New England Biolabs (NEB), Ipswich, MA, USA], and transformed into NEB5-α competent cells.

[0140] The resulting plasmid (eKO2-US6) was sequence verified and extracted from E. coli using an endotoxin-free miniprep kit (MO-BIO Laboratories, Carlsbad, CA, USA). HSV-2 DNA (1 μg) was cotransfected with 100 ng of eKO2-US6 into VD60 cells using Effectene (Qiagen, Valencia, CA, USA) according to the manufacturer's recommendations.

[0141] Four days after transfection, plates were screened for green plaques and the supernatants were collected and overlaid on fresh VD60 cells for 1 h, then washed and overlaid with 4% low melting point agarose prepared in Optimem (Invitrogen, Carlsbad, CA, USA). Single green fluorescent plaques were picked and purified three times using this method. Noncomplemented virus was generated by growing virus stocks on VD60 cells and harvesting infected cell lysates from Vero cells as described for HSV-1 gD deletion virus (Ligas, 1988).

[0142] Genotypic confirmation of the gD deletion in ΔgD-2::GFP was performed by PCR. A primer set was used to confirm the presence of wild-type (WT) and ΔgD-2 viral DNA in the samples (primers RL-V91I-US6 and RL-V91IUS6), while another primer set (Neo-Out and US8-Out) was used to amplify the DNA region containing eK02-US6 and the genomic target region. To confirm the deletion of gD expression, Vero or VD60 cells were infected with parental WT or ΔgD-2 viruses (which grow on VD60 cells and are therefore competent for entry) at a multiplicity of infection (MOI) of 10 plaque-forming units (PFU) / cell (based on VD60 titer). After 1 h of incubation, cells were washed twice with PBS, incubated in Optimem for 48 h at 37°C, harvested and gD expression was assessed by Western blot. [Table 3]

[0143] Sequence homology for the PCR fragment used to construct the eKO2-US6 plasmid. Searching the published Addgene pcDNA3-eGFP (id:13031, 6159bp) sequence for the pCMV-eGFP-neomycin resistance primer using Blast2 yields a 1819bp fragment: [ka]

[0144] Probing the pcDNA3-eGFP sequence with the pUC Origin-Ampicillin Resistance primer yields a 1754 bp fragment: [ka]

[0145] Searching the blast.ncbi.nlm.nih.gov / Blast.cgi database for the left US6 homology arm of herpes simplex virus (type 2) (taxid:10310) returned a sequence from human HSV-2 strain HG52, complete genome: BLAST for the left-US6 homology arm yields a 991 bp homologous region, a 1029 bp fragment: [ka]

[0146] BLAST on the right-US6 homology arm yields a 1072 bp homologous region, a 1110 bp fragment: [ka]

[0147] Table 4: Codons replaced in the custom synthesized codon-optimized HA gene. When synthesizing the HA gene, versions were designed in which codons in PR8 HA that had less than 9.5% representation in HSV-2(G)gD were replaced with the codons for each amino acid that had the highest representation in HSV-2(G)gD. [Table 4]

[0148] The presence of the chimeric gD::HA in the recombinant virus was verified by PCR (see FIG. 7). The lanes from left to right are: lane 1; ΔgD-2 genomic DNA, lane 2; ΔgD-2::FL HA nOP genomic DNA, lane 3; pBJJ1 plasmid DNA, lane 4; ΔgD-2 genomic DNA, lane 5; ΔgD-2::HL HA nOP genomic DNA, and lane 6; pBJJ2 plasmid DNA. The primers used for PCR amplification were located immediately upstream and downstream of the HA extracellular domain. No exogenous promoter was inserted into the expression cassette. As a result, the expression of the chimeric gene was controlled by the endogenous promoter. Sanger sequencing was used to confirm the proper construction of the chimeric gD::HA gene in the recombinant virus. For genetic validation, 6-8 week old C57BL / 6 mice were prime-boost vaccinated with ΔgD-2::FL HA nOP or ΔgD-2::HL HA OPT on days 0 and 21 in parallel with controls mock vaccinated with the parental ΔgD-2:RFP strain and VD60 cell lysate. Twenty-one days after the boost, mice were challenged intracutaneously with 10xLD90 of HSV-2 4674 to determine whether anti-HSV immunity was compromised.

[0149] Mice vaccinated with ΔgD-2::FL HA nOP and ΔgD-2::HL HA OPT are fully protected against HSV-2 challenge but do not form anti-HA IgG (see FIG. 8). Mice were vaccinated with either control VD60 cell lysate or 1×10 6Mice were prime-boost vaccinated on days 0 and 21 with PFU of ΔgD-2::FL HA nOP, ΔgD-2::HL HA OPT, or ΔgD-2::RFP. On day 42, mice were challenged with 10xLD90 of HSV-2 4674. Mice vaccinated with ΔgD-2::FL HA nOP and ΔgD-2::HL HA OPT were fully protected from challenge (see Figure 8A). 40 days after prime, mice were bled to check for serum antibodies. ELISA performed against soluble PR8 HA shows the absence of HA-specific IgG in mice. (See Figure 8B).

[0150] ΔgD-2::P CMV Construction of -HA virus and HA expression. It was investigated whether inserting a non-chimeric HA gene into the ΔgD genome in a cassette containing a constitutive promoter and polyadenylation sequence would be more immunogenic. To achieve this, the synthetic PR8 HA gene was restriction cloned into the expression plasmid pEGFP-N1 (Addgene). The HA gene was inserted between the xbaI and HindIII sites, replacing EGFP in the plasmid.

[0151] Mice were transfected with ΔgD-2::P CMV Mice were vaccinated with 5×10 6 Prime vaccinations were with PFU of each virus (see Figure 18 legend for different viruses used). n=5 mice / group, except for 10 mice for ΔgD-2::FL HA nOP. Five of the ΔgD-2::FL HA nOP mice were boosted with stalk HA expressing ΔgD-2, and 5 μg / mL purified PR8 HA was used as the source of antigen. Goat anti-mouse Ig Ab was used for total Ab ELISA. 14 days after prime, anti-PR8 HA ELISA showed that sera from mice vaccinated with stalk HA (ΔgD-2::FL HA nOP) showed overall Ab responses, but did not score above background in the isotype ELISA (see Figure 18). Importantly, ΔgD-2::PCMV Vaccination with -FL HA nOP(β3χ4) resulted in significantly more IgG2c than IgG1 (see FIG. 19). Vaccination with full-length HA induced the greatest anti-HA response, which was predominantly IgG2c. This is consistent with the same IgG isotype response that can be obtained with recombinant viral vaccines against exogenous antigens relative to endogenous HSV proteins.

[0152] ΔgD-2::P CMV As shown in Figure 12, VD60 cells were infected with 3 MOI of ΔgD-2::P CMV -FL HA nOP, ΔgD-2::P CMV -HL HA nOP, or ΔgD-2::P CMV ΔgD-2::RFP vaccinated mice were infected with ΔgD-2::RFP-HL HA OPT. At 16 hours post-infection, cells were harvested and stained for HSV protein and HA expression. HSV protein expression was measured using serum from mice vaccinated with ΔgD-2::RFP. HA expression was measured using monoclonal anti-HA stalk IgG C179. Cells were stained with either cell-permeable or cell-impermeable methods. Statistics were calculated by Student's t-test of geometric means between control fluorescence and 0 fluorescence of wells stained for HA expression. β 3 Although only χ4 induced high levels of HA expression in the infected cell membrane, all three viruses induced expression as measured by cell permeability staining.

[0153] To determine whether insertion of full-length PR8 HA into ΔgD-2 introduces a novel pathway of cell entry, Vero cells were infected with ΔgD-2::P- CMV -FL HA nOP or ΔgD-2::P EF1α At 120 hours postinfection, there were no signs of productive infection. In addition, five C57BL / 6 mice were infected with 5 × 10 6 PFU of ΔgD-2::P CMVThe mice were given subcutaneous injections of ΔgD-2::P-FL HA nOP and monitored daily for one week. They showed no signs of disease or distress. CMV In vitro assays were performed in which cells were infected with titrations of either -FL HA nOP and ΔgD-2::PEF1α-RFP. At 70 hours post-infection, cells were fixed and stained for HSV-2 glycoprotein B (gB). Staining was performed using 10% aliquots of each virus. -3 Dilution shows the presence of isolated infected cells but no evidence of cell-to-cell virus spread (see Figure 13). This is not surprising given that the influenza A and B proton pump genes, both named M2, require intermediate release of virions from endosomes. Because β3χ4-6 do not contain any of the proton pump genes, they lack a mechanism to exit cellular endosomes after entry.

[0154] Example 2 Engineering HIV constructs to be expressed in HSV-2 ΔgD virus: We investigated producing vaccine vectors in which glycoprotein antigens from HIV are expressed from an ADCC-inducing HSV-2 ΔgD vector. The rationale for using this vector is that it elicits non-neutralizing ADCC-inducing IgG antibodies to HSV, and this type of immune response has correlated with protection in the only HIV vaccine trial to show efficacy so far (Haynes, Gilbert et al. 2012). For the glycoprotein antigen, we selected a transfer / founder clone of Env gp145 lacking the cytoplasmic tail from donor CH505 in the CHAVI001 acute HIV-1 infection cohort because this well-characterized HIV-1 clade C glycoprotein is thought to be representative of those that pass the hurdle of infection in areas of high HIV prevalence (Liao, Lynch et al. 2013). Because HIV Env is not particularly well expressed in the context of natural infection and is often poorly expressed exogenously, steps were explored to enhance antigen expression in our constructs. Env gp145 has been found to be more efficiently incorporated into virus-like particles (VLPs) than the full length, and replacing the Env signal peptide and transmembrane domain with corresponding domains from host proteins or other viral glycoproteins further increased incorporation into VLPs (Wang, Liu et al. 2007). To provide efficient incorporation of HIV Env into HSV VLPs, chimeras were constructed of the ectodomain of HIV Env and the signal peptide and transmembrane cytoplasmic tail of HSV-2 gD. The signal peptide of HSV2 gD is 25 residues long and the ectodomain is a total of 306 residues (Eisenberg, Long et al. 1984, Nicola, Willis et al. 1996). Allelic exchange constructs were generated by Gibson assembly (Gibson, Young et al. 2009).Briefly, oligonucleotide primers were synthesized to amplify the HSV2 strain G genomic DNA arms of homologous sequences ~800 bp 5' to codon 25 of HSV2 US6 and ~800 bp 3' to codon 306 of HSV2 US6. The insert was amplified from CH505 TF gp145 expression plasmid HV1300631 (gift of Huaxin Liao, Duke University) with primers encompassing codon 30 to codon 680. The fragment was cloned into pUC19 between EcoRI and BamHI restriction enzyme recognition sites.

[0155] Example 3 introduction Functional in vitro macrophage antibody-dependent cell-mediated killing (ADCK) assay using RFP-expressing HSV-2 ΔgD strain (made in Example 1): Single-cycle herpes simplex virus type 2 (HSV-2) strain deleted in glycoprotein D (ΔgD-2) induces killing anti-HSV immunity by inducing antibodies that bind and activate Fcγ receptors (FcγR). Mouse FcγRIV was highly activated in the presence of serum from vaccinated mice and expressed on macrophages, monocytes, and neutrophils. The exact mechanism of FcγR-mediated cytotoxicity is not fully understood and more tools are needed. FcyR-binding antibodies mediate the killing of HSV-infected cells by binding to antigens on infected cells and then binding and activating FcγR on naive leukocytes. This promotes antibody-dependent cell-mediated cytotoxicity and phagocytosis (ADCC and ADCP), referred to herein as antibody-dependent cell-mediated cell killing (ADCK). Current assays face many limitations, including but not limited to, inflexible target and effector cell lines, artificial antigen-presenting systems, indirect or separate outputs for ADCC and ADCP, and the use of cumbersome radioisotopes.

[0156] To overcome these limitations, a quantitative in vitro assay was constructed to test ADCK in response to anti-HSV antibodies. A ΔgD-2 mutant that highly expresses the gene for red fluorescent protein (rfp) was used to mark infection. RFP and cell viability markers were then used to identify live infected target cells, the reduction in their percentage being determined to be the result of cytotoxicity. FACS analysis was used to quantitate the reduction in the percentage of live infected target cells after co-culture with macrophages.

[0157] Using the new assay, it was shown that serum from mice vaccinated with ΔgD-2 induced significant amounts of ADCK by both immortalized macrophage cell lines and bone marrow-derived macrophages (BMDMs) from both mice and guinea pigs. Live imaging of the assay using raw macrophages showed that the injury readout could not be readily attributed to cytotoxicity or phagocytosis, but that the processes could occur simultaneously, highlighting the importance of using an assay that measures both. Furthermore, FcγR - / - ADCK was eliminated when BMDM from mice were used, indicating that the primary readout is FcγR dependent and that the assay is suitable for studying ADCK in different knockout mouse strains. This assay allows for the precise study of ADCK and its associated genes and FcγR in different species and animal models.

[0158] Traditionally, ADCC has been a cumbersome 51 Previously, ADCC was measured by Cr release assay, but many FACS methods have been developed to measure ADCC in response to different antigens and antibodies. The collective term for these methods is rapid fluorescent ADCC (RFADCC). In these assays, target cells are stained with a persistent membrane dye (die) and a live / dead marker that dissipates upon the onset of apoptosis. ADCC activity is then measured as a function of live / dead ratios. + A membrane pigment +In a similar assay, ADCP is measured by the percentage of macrophages marked by phagocytosis of fluorescent cells. However, as the data herein show, ADCC and ADCP cannot be clearly separated by these methods, so in this text they are collectively referred to as antibody-dependent cell-mediated cytotoxicity (ADCK). Prior art assays have some additional drawbacks, as they are usually limited in the selection of antibodies or effector cells. A similar assay was used in Petro et al. 2015, but the ability of HSV to infect, replicate, and cytology both target and effector cells limited the usefulness of the assay for testing immune cells and their FcγRs (Petro et al., 2015).

[0159] ΔgD-2 is a single cycle virus in non-complementing cells and was recently shown not to induce dendritic cell death in vitro. We investigated whether a brightly fluorescent ΔgD-2 strain would allow precise study of the cellular mechanisms of ADCK. We constructed and developed an RFP expressing ΔgD-2 (ΔgD-2::RFP) strain, an RFADCK assay that precisely measures macrophage effector activity against HSV-2 ΔgD-2:RFP-infected cells in vitro. The assay was validated for both immortalized and primary cell lines using J774 cells, Raw 264.7 macrophages, and bone marrow-derived macrophages (BMDMs). Unlike current RFADCC assays, this method simulates an infection environment, measures both ADCC and ADCP, and is highly flexible, allowing the use of polyclonal animal sera and different cell lines and mouse strains.

[0160] Cell lines: Vero (CCL-81; ATCC, Manassas, VA) and VD60 cells (Vero cells containing multiple copies of gD-1 under the endogenous gene promoter) were passaged in DMEM (Thermo Fisher Scientific, Waltham, MA) supplemented with 5% fetal bovine serum (FBS, Gemini Bio-Products, West Sacramento, CA) and 10 U / ml penicillin and 10 μg / ml streptomycin sulfate (Thermo Fisher Scientific). Bone marrow derived macrophages (BMDMs) were obtained from C57Bl / 6 mice as previously described. Bone marrow precursors were stored in DMEM supplemented with 50% FBS and 10% DMSO (Sigma-Aldrich, St. Louis, MO) if not used immediately. Mouse BMDMs were differentiated using supernatants from L929 cell cultures (ATCC). Guinea pig BMDMs were differentiated using recombinant human M-CSF (BioLegend, San Diego, CA). Raw 264.7, J774.1, and HEK 293 cells (ATCC) were passaged in DMEM supplemented with 10% FBS and 1% Pen-strep.

[0161] Generation of ΔgD-2::RFP and virus propagation. VD60 cells were cotransfected with HSV-2 ΔgD:GFP (ΔgD-2) genomic DNA and cosmid DNA containing 40 kB of the HSV-2 genome in which the US6 gene was replaced by tdtomato downstream of the EF1α promoter. The resulting virus was plaque-purified three times using RFP expression as a marker for homologous recombination. The purified ΔgD-2::RFP virus was verified by PCR and sequencing.

[0162] ΔgD-2 and ΔgD-2::RFP were propagated in VD60 cells, which complement the gD deletion and allow multiple rounds of replication. All virus strains were titered by serial dilution and growth on each cell type.

[0163] Animals: 4-6 week old female C57BL / 6 mice obtained from Jackson Laboratory (JAX, Bar Harbor, ME) were used to obtain serum. 4-6 week old male C57BL / 6 mice were obtained from JAX and bone marrow cell suspensions were isolated by flushing femurs with DMEM supplemented with 10% FBS and 1% Pen-strep. 5-6 week old female Hartley guinea pigs were purchased from Charles River Laboratories (Wilmington, MA). Bone marrow cell suspensions were isolated by flushing femurs and tibias with DMEM supplemented with 10% FBS and 1% Pen-strep. Vaccinations were administered by subcutaneous injection. All procedures were approved by the Institutional Animal Care and Use Committee of the Albert Einstein College of Medicine.

[0164] Immunization: 6-8 week old mice or guinea pigs were immunized with 5 × 10 6 Animals were primed subcutaneously with plaque forming units (PFU) of HSV-2 ΔgD::RFP or an equal volume of VD60 cell lysate in phosphate buffered saline (PBS) in a total volume of 200 μL. Animals were boosted with the same dose 21 days later.

[0165] Antibody-dependent cellular cytotoxicity assay: Bone marrow-derived macrophages (BMDM), J774.1 macrophages, and Raw 264.7 macrophages were incubated with LPS (Sigma-Aldrich) for 12 h and then co-cultured with HEK 293 cells (ATCC). HEK 293 cells were coated with PKH67 membranes (Sigma-Aldrich) and Tag-it Violet membranes (Sigma-Aldrich) according to the manufacturer's instructions. TM(Biolegend) dye. HEK cells were infected with HSV-2 ΔgD::RFP at an MOI of 3 in serum-free DMEM 4 hours prior to co-culture. Infection medium was removed 3.5 hours later and replaced with a 1:5 dilution of heat-inactivated mouse or guinea pig serum collected on day 40 from mice prime-boost injected as described above with either HSV-2 ΔgD::RFP or VD60 cell lysate. HEK 293T cells were incubated in serum for 30 minutes at 37°C and then added to macrophage cultures in 96-well tissue culture plates (Corning Inc, New York City, NY). Co-cultures were incubated for 12 hours, then fixed and analyzed by flow cytometry on an LSRII (BD Biosciences, Franklin Lakes, NJ). For live imaging experiments, Raw 264.7 macrophages and HEK 293 cells were cocultured at a 10:1 ratio on glass-bottom 96-well plates (Matrical Bioscience, Spokane WA) and imaged on an inverted NIKON Eclipse TiE microscope using NIS Elements software with deconvolution.

[0166] Statistical analysis: Data were compiled in GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). Statistical analysis using the tests indicated in the figures was also performed using GraphPad Prism. Statistical significance was indicated in the figures by * , p < 0.5; *** , p<0.001.

[0167] result Insertion of rfp into US6 of HSV-2 ΔgD does not alter the growth phenotype of the virus. Vero cells were infected with HSV-2 ΔgD-2::GFP at a multiplicity of infection (MOI) of 1 to determine the kinetics of GFP expression. Fluorescence microscopy was used to visualize GFP expression, which was low even at 24 hours postinfection (Figure 14B). GFP at the US6 locus of ΔgD-2 was replaced with RFP under the EF1α promoter to create ΔgD-2::RFP (Figure 14A). To determine the kinetics of RFP expression, non-complementing Vero cells were infected with HSV-2 ΔgD-2::RFP or ΔgD-2 at 1 MOI. Fluorescence microscopy visualization showed that RFP expression began with ΔgD-2::RFP at 4.5 hours postinfection and continued to increase until 24 hours postinfection (Figure 14C). Phase contrast imaging showed that virus infection with ΔgD-2::RFP appeared similar to that of the parental ΔgD-2 strain in Vero cells (Figures 14B and 14C). Infection with ΔgD-2::RFP at an MOI of 3 resulted in syncytia formation in VD60 at 12 hours postinfection (Figure 14E), but not in Vero cells (Figure 14D), indicating that the virus maintained its single-cycle replication phenotype. Introduction of pEF1α::RFP into the gD locus of ΔgD-2::GFP induced strong RFP expression while maintaining the viral kinetics and single-cycle phenotype of the parental ΔgD-2 strain.

[0168] Serum from mice vaccinated with ΔgD-2::RFP induces significant ADCK in the RFADCK assay. To measure the level of ADCK activity induced by serum from mice vaccinated with ΔgD-2::RFP, we developed an in vitro protocol based on our previous RFADCC assay. Target HEK293T cells were stained with membrane dyes and live / dead markers and infected with ΔgD-2::RFP at an MOI of 3. After 3.5 h of incubation of infected target cells, the infection medium was removed and medium or mouse serum was added and incubated for an additional 30 min. Infected HEK293T cells were co-cultured for 12 h with J774.1 mouse macrophage cells that had been stimulated with LPS 12 h prior. Based on the previously reported assay, a 10:1 effector to target cell ratio was used. At this ratio, serum from mice vaccinated with ΔgD-2::RFP induced highly infected (RFP 高い ) induced killing of approximately 70.5% of target cells (Figure 15B), which was significantly more than the 49.3% killed in the absence of serum (p<0.05). RFP in the model is a surrogate for viral protein expression, and cells expressing higher levels of these proteins are more likely to be bound by antibodies, cross-link FcyR, and initiate ADCK. 高い We investigated whether there was a significant decrease in only RFP cells between the two treatment groups. 中間 or RFP 中間+高い No significant difference was observed in killing of (total infected cells) (Figure 15B). Infected target cells were defined as double-positive HEK293T cells expressing RFP in cultures lacking effector cells. RFP 高い Cells were defined as those expressing RFP above the average intensity of infected target cells. 中間 Cells were defined as all infected target cells expressing RFP at less than the average intensity (Figure 15A).

[0169] ADCK is significantly increased in the presence of serum from mice vaccinated with ΔgD-2::RFP compared to serum from mice vaccinated with control cell lysate. Infected HEK293 cells were incubated with serum from mice vaccinated with either ΔgD-2::RFP or control VD60 cell lysate to determine whether the damage mediated by the different immortalized macrophage cell lines was caused by antigen-specific ADCK. For each treatment group, RFP after 16 hours of co-culture was 高い The percentage of cells was compared to that in a parallel assay lacking serum. In cultures containing J774.1 macrophages, serum from mice vaccinated with ΔgD-2::RFP suppressed RFP expression more than serum from mice mock vaccinated with VD60. 高い induced significantly more cell killing (p<0.001; FIG. 15c). A similar trend was seen in cultures containing Raw 264.7 macrophages (p=0.09). Furthermore, for both J774.1 and Raw 264.7 macrophages, ADCK measured in cultures containing serum from mice mock vaccinated with VD60 was indistinguishable from cultures lacking serum (mean % difference=-5.9±7.6, -0.3±3.5), indicating that nonspecific antibodies do not induce killing in our assay. These results confirm that the ADCK measured in the RFADCK assay is the result of HSV-specific antibodies induced by vaccination with ΔgD-2::RFP.

[0170] Tracking of infected cells by live imaging of the ADCK assay indicates that cytotoxicity and phagocytosis are not mutually exclusive. ADCK assays were performed as described above, except that glass-bottom 96-well plates were used and effector cells were Raw 264.7 mouse macrophage cells, which have been shown to perform equivalent amounts of ADCC and ADCP. Assays were imaged approximately every 15 minutes for 24 hours at 60x magnification under a deconvolution microscope. Infected cells were imaged approximately every 15 minutes for 24 hours at 60x magnification. + Uninfected HEK293 cells are highly Mem +and Life / Death + The macrophages remain intact. Figure 15A shows that different types of cytotoxicity of infected cells by macrophages (white arrows) are observed at the indicated time points: macrophages ( * ) gather around the infected cell on the left over time, while the infected cell on the right undergoes apoptotic blebbing observed at 4.5 h. These resulting blebs are rapidly phagocytosed by macrophages, whose fluorescence in RFP was observed at 7.5 h. This controlled apoptosis of the infected cells indicates that ADCC has occurred, while phagocytosis by macrophages and subsequent macrophage fluorescence as ADCP would be measured by flow analysis. Although the total number of infected cells captured was low, resulting in high variability (n=58 infected cells / group), quantification of infected cell injury across movies from three experiments shows similar mean ADCK in the presence of Raw 264.7 macrophage cells as previously measured by the RFADCK assay (mean RFADCK =18.8%, average ムービー = 18.5% injury; Figures 15C, 16B).

[0171] ADCK activity by bone marrow-derived macrophages is FcγR dependent. RFADCK assays were performed on wild-type and FcγR - / - This was performed in bone marrow derived macrophages (BMDM) from mice to determine whether the ADCK observed was FcγR dependent. ADCK activity was compared to parallel cultures with serum from mice mock vaccinated with VD60 lysate. 高い At an effector to target cell ratio of 10:1, wild-type BMDMs expressed FcγR in the presence of serum from mice vaccinated with HSV-2 ΔgD-2::RFP. - / - ΔgD-2::RFP vaccinated mice killed significantly more infected target cells than BMDMs (p<0.05; Fig. 17A). Furthermore, FcγR - / - RFP by BMDM 高いCellular killing was indistinguishable from killing in the presence of serum from mice mock vaccinated with VD60 lysate (mean % difference = 3.6 ± 4.0), indicating that the ADCK activity measured in our assay is entirely FcγR dependent.

[0172] RFADCK using guinea pig bone marrow-derived macrophages recapitulates the mouse results. To determine the ability of effector cells from another species to perform ADCK in our assay, guinea pig BMDMs were co-cultured with ΔgD-2::RFP-infected HEK293 cells in the presence of serum from guinea pigs vaccinated with ΔgD-2, mock vaccinated with VD60 lysate, or naïve RFP in parallel co-cultures containing serum from naïve animals. 高い Cellular injury was used as a baseline. In the presence of serum from animals vaccinated with ΔgD-2, guinea pig BMDMs underwent significant ADCK (p<0.001; FIG. 17B).

[0173] Anti-PR8 IgG isotype ELISA. Mice were subcutaneously injected with ΔgD-2::RFP, ΔgD-2 expressing full-length PR8 hemagglutinin (FL nOP, β3χ4), ΔgD-2 expressing headless PR8 hemagglutinin (HL nOP, β3χ5), or ΔgD-2 expressing headless PR8 hemagglutinin codon-optimized for HSV-2(G) (HL OPT, β3χ6), or mock vaccinated with VD60 cell lysate. Five mice / group were prime-boost vaccinated separately for each treatment. (See FIG. 20). Five mice were primed with FL nOP and boosted with HL nOP. In each case, 5×10 6PFU of virus was given with each injection. On day 30 (9 days after boost), mice were bled and serum was collected. Enzyme-linked immunosorbent assay (ELISA) was then performed to check for antibodies against purified PR8HA or Vero cells infected with HSV-2 4674. Mice primed-boosted with FL nOP or primed with FL nOP and boosted with HL nOP developed IgG1 and IgG2c antibodies against PR8 HA. No other groups formed anti-PR8 IgG antibodies. All groups except the VD60 lysate group developed similar high titers of anti-HSV IgG. Anti-HSV IgG Anti-HSV IgG was induced with all vaccine strains.

[0174] Example 4 ΔgD-2::HA PR8 Mice vaccinated with 5 × 10 6 PFU of ΔgD-2::RFP or ΔgD-2::HA PR8 Mice were primed and boosted subcutaneously at 3-week intervals or mock vaccinated with VD60 cell lysate. Mice were bled one week after the boost and serum neutralizing titers were measured against A / Puerto Rico / 1934 / 8 IAV (PR8) (see FIG. 21A). PR8 Mice immunized with 6xLD2 developed significant neutralizing antibody titers against PR8 (mean=304). The dotted line represents the detection limit of the assay. Three weeks after the boost, mice were immunized with 6xLD2 50 Mice were challenged intranasally with the PR8 strain of ΔgD-2::HA. Mice were sacrificed when they reached 75% of their initial body weight. As shown in Figures 21B and 21C, ΔgD-2::HA PR8 Mice immunized with were fully protected from PR8 challenge, whereas all control-vaccinated mice succumbed to infection by day 9.

[0175] Recombinant gD-2::HA PR8 express high levels of PR8 protein. To assess expression, ΔgD-2::RFP gDNA was transfected into P CMVThe hemagglutinin (HA) expression cassette containing the hemagglutinin (HA) gene from IAV H1N1 strain A / Puerto Rico / 1934 / 8 (PR8) downstream of the ΔgD-2 gene and upstream of the polyadenylation signal was co-transfected into VD60 cells. Extracellular and intracellular HA expression was controlled by ΔgD-2, ΔgD-2::HA at an MOI of 3. PR8 , ΔgD-2 (ΔgD-2::HL HA), which contains a truncated version of the PR8 HA expression cassette PR8 ), HA expression was measured by flow cytometry in Vero and VD60 cells infected with VD60 and VD60 mAbs is shown in FIG. 22B.

[0176] gD-2::HA PR8 Mice immunized with ΔgD-2::RFP vector or ΔgD-2::HA develop high titer functional and isotype-switched anti-PR8 antibodies. PR8 Mice were prime-boost vaccinated at 21-day intervals with either ΔgD-2::HA or ΔgD-2::HA. Sera were collected for analysis 28 days after prime vaccination. Anti-PR8 antibodies were measured by ELISA against purified HA PR8 protein. PR8 Mice immunized with 、 They generated isotype-switched anti-PR8 HA antibodies that were predominantly IgG2c and IgG2b (see Figures 23A-23E). PR8 Sera from mice immunized with induced significant hemagglutination inhibition compared to sera from mice immunized with ΔgD-2 (p<0.001; mean HAI 力価 =80) (see FIG. 23F). Mice immunized with ΔgD-2::RFP did not develop hemagglutination-inhibiting antibodies.

[0177] gD-2::HA PR8 Mice immunized with VD60 cell lysate, ΔgD-2, or ΔgD-2::HA develop protection against IAV challenge. PR8 Prime-boost immunization was performed at 21-day intervals with 6xLD50 Mice were sacrificed after reaching 70% of their starting body weight and neutralization titers were measured using microneutralization assays for each strain. As shown in Figures 24A-24C, ΔgD-2::HA PR8 Mice immunized with developed significant neutralizing titers against PR8 (mean titers PR8 =1:304;p VD60 <0.01; p ΔgD-2 <0.05; n = 10 mice / group). ΔgD-2::HA PR8 Mice immunized with ΔgD-2::HA were also completely protected from weight loss and death after challenge with PR8 (p<0.001, n=5 mice / group). As shown in Figures 24D-24F, ΔgD-2::HA PR8 Mice immunized with IgG1 developed significant neutralizing antibodies against A / California / 2009 H1N1 (A / Cal / 2009) IAV (mean titers A / Cal =26;p VD60 p <0.01 ΔgD-2 = 0.1; n = 10 mice / group). PR8 Mice immunized with ΔgD-2::HA were partially protected from weight loss and death following challenge with A / Cal / 2009 (P<0.05, N=15 mice / group). As shown in Figures 24G-24I, mice immunized with ΔgD-2::HA were partially protected from weight loss and death following challenge with A / Cal / 2009 (P<0.05, N=15 mice / group). PR8 Mice immunized with ΔgD-2::RFP, or VD60 cell lysates did not develop neutralizing antibody titers against A / Victoria / 3 / 75 H3N2 (A / Vic) and A / Aichi / 68 H3N2 (X-31) and were not protected from weight loss and death after challenge (n 中和 n = 10 mice / group A / Vic Challenge = 15 mice / group; n X-31 challenge = 5 mice / group).

[0178] ΔgD-2::HA PR8 Mice immunized with VD60 cell lysate, ΔgD-2, or ΔgD-2::HA develop fully protective ADCC immunity against HSV-2. PR8Mice were prime-boost vaccinated at 21-day intervals (5 mice per group) with either ΔgD-2 or ΔgD-2::HA. In Figures 25A and 25B, mice were bled one week after the boost and sera were analyzed by ELISA. PR8 Mice receiving ΔgD-2 or ΔgD-2::HA similarly produced high levels of HSV-specific IgG (Figure 25A). Moreover, these IgGs were predominantly IgG2c. In Figure 25C, rapid fluorescent antibody-dependent cell-mediated cytotoxicity (RFADCK) assays were performed using the same sera as in Figures 25A and 25B. PR8 Sera from mice vaccinated with ΔgD-2 induced significant ADCK activity in the presence of J774.1 macrophages and ΔgD-2-infected cells compared with sera from mice given VD60 cell lysate (P<0.01). PR8 There was no difference between the ADCK activity induced by sera from mice vaccinated with 10xLD 90 Mice were challenged by skin scarification with 100 mg of HSV-2 4674. Mice receiving VD60 cell lysate succumbed to HSV-2 by day 10 and developed severe epithelial and neurological disease. PR8 Mice that received the antibody were completely protected from morbidity and mortality following challenge (see Figures 25D to 25F).

[0179] mFcγRIV ADCC reporter bioassay response. Serum samples collected from mice vaccinated with ΔgD-2::HA, ΔgD-2::RFP, and inactivated A / Puerto Rico / 8 / 1934 H1N1 virus (PR8) were serially diluted and subjected to mFcγRIV ADCC reporter bioassay. PR8 virus-infected Madin-Darby canine kidney (MDCK) cells were used as target cells and mFcγRIV-expressing Jurkat T cells were used as effector cells. Target cells were incubated with serially diluted serum samples and effector cells. Bio-Glo TMReagent was added and luminescence was measured. Mice vaccinated with HSV-2 ΔgD::HA showed significantly greater activation of the mFcγRIV receptor at each dilution tested compared to mice vaccinated with ΔgD-2::RFP and inactivated PR8 virus (see FIG. 26).

[0180] Example 5 Cloning of HIV-1 Env in the pBkk412 Plasmid Containing the HSV-2 Genes. The full-length HIV-1 Env (clade C) and Rev genes, along with a partial Nef gene, were PCR amplified from plasmid ZM109F.PB4 (Figure 27) obtained from the NIH AIDS Reagent Program.

[0181] The PCR fragment product, which also contained the bGH polyA signal and the T7 promoter, was cloned into the multiple cloning site (MCS) of the pBkk412 plasmid under the control of the CMV promoter using the REs sbfI and blpI (see FIG. 28).

[0182] Stable competent E. coli cells were transformed and plated on agar + carbenicillin. Colonies were screened for the presence of inserts using PCR (Figure 29A) and used to prepare DNA for analysis. DNA was amplified by PCR and clones with the correct size (CL 15-6 and CL 26-11) were selected for transient transfection to evaluate HIV-1 protein expression (Figure 29B).

[0183] Upon confirmation of HIV protein expression in the transfected cells, cells are co-transfected with the linear recombinant plasmid and HSV ΔgD-2 negative virus (e.g., ΔgD-2::RFP, ΔgD-2::GFP) and selected for recombinants that express HIV proteins and do not express the GFP or RFP markers. Expression of HIV proteins by the new recombinant viruses is assessed by Western blot or flow cytometry (FACS). Mice are immunized intramuscularly (prime and boost) and serum is collected and screened for the presence of HIV-specific antibodies. Ab function (e.g., neutralizing and non-neutralizing function) is also measured. Humanized mouse models that render mice susceptible to HIV infection are also used to assess whether the vaccine protects mice from HIV infection.

[0184] Several embodiments of the processes, recombinant viruses, and methods described herein are presented below.

[0185] Embodiment 1: A process for producing a vaccine vector directed against a heterologous antigen, the process comprising: a) providing an HSV-2 genome comprising: (i) a gene encoding a completely or partially deleted HSV-2 glycoprotein D, and (ii) a nucleic acid comprising a promoter-FP construct, where FP is a nucleic acid encoding a fluorescent protein; b) co-transfecting a host cell with (i) the HSV-2 genome and (ii) a linear DNA fragment encoding a heterologous antigen under conditions in which allelic recombination occurs between the HSV-2 genome of a) and the DNA fragment; c) screening the plaques resulting from b) to identify those that do not show fluorescence under an excitation light that induces fluorescent protein fluorescence; d) recovering recombinant HSV-2 viruses or virions from those plaques that do not show fluorescence in c) to obtain a vaccine vector directed against the heterologous antigen.

[0186] Embodiment 2: The process of embodiment 1, wherein the host cell is an HSV-1 glycoprotein D-complementing cell.

[0187] Embodiment 3: The process of embodiment 1 or embodiment 2, wherein the promoter of the promoter-FP construct is a heterologous promoter.

[0188] Embodiment 4: The process of any one of embodiments 1-3, wherein co-transfecting is accomplished by electroporation.

[0189] Embodiment 5: The process of any one of embodiments 1-4, wherein the fluorescent protein is a red fluorescent protein (RFP).

[0190] Embodiment 6: The process of any one of embodiments 1-5, wherein a host cell is co-transfected with (i) the HSV-2 genome of a) and (ii) a linear DNA fragment encoding, in order, the HSV-2 gD signal sequence, the heterologous antigen, the HSV-2 gD transmembrane domain, the HSV-2 gD cytoplasmic domain, but not the HSV-2 gD extracellular domain, or (ii) the HSV-2 gD signal sequence, the heterologous antigen, and the cytoplasmic domain of HSV-2 gD.

[0191] Embodiment 7: The process of any one of embodiments 1-5, wherein the host cell is co-transfected with (i) the HSV-2 genome of a) and (ii) a linear DNA fragment encoding, in order, (i) a promoter, the heterologous antigen, and optionally a polyA signal.

[0192] Embodiment 8: The process of any one of embodiments 1-7, wherein the heterologous antigen is an influenza antigen.

[0193] Embodiment 9: The process of any one of embodiments 1-8, wherein the heterologous antigen is an influenza hemagglutinin (HA) antigen.

[0194] Embodiment 10: The process of embodiment 9, wherein the HA antigen is a full-length HA extracellular domain or an HA stalk.

[0195] Embodiment 11: The process of any one of embodiments 1-7, wherein the heterologous antigen is an HIV antigen.

[0196] Embodiment 12: The process of embodiment 11, wherein the HIV antigen is Env gp145.

[0197] Embodiment 13: The process of any one of embodiments 1-12, wherein the heterologous antigen is under the control of an upstream CMV promoter and has a downstream SV40 polyA signal.

[0198] Embodiment 14: The process of any one of embodiments 1-13, wherein the promoter is an elongation factor 1 alpha gene (P EF1α ) promoter, P EF1α and FP are both fused (P EF1α -FP), process.

[0199] Embodiment 15: A vaccine vector or recombinant herpes simplex virus-2 (HSV-2) comprising a genome encoding a heterologous antigen produced by the process of any one of embodiments 1-14.

[0200] Embodiment 16: A recombinant herpes simplex virus-2 (HSV-2), comprising: (i) a complete deletion of the HSV-2 glycoprotein D-encoding gene in its genome; and (ii) (a) a linear DNA fragment encoding a promoter, a heterologous antigen signal sequence, and a heterologous antigen, or (b) a promoter and a heterologous antigen.

[0201] Embodiment 17: A recombinant herpes simplex virus-2 (HSV-2) comprising: (i) a partial deletion in its genome of the HSV-2 glycoprotein D-encoding gene; and (ii) (a) a linear DNA fragment encoding, in order, the HSV-2 gD signal sequence, a heterologous antigen, an HSV-2 gD transmembrane domain, optionally an HSV-2 gD cytoplasmic domain, but not the HSV-2 gD extracellular domain, or (b) a linear DNA fragment encoding, in order, the HSV-2 gD signal sequence, a heterologous antigen, and the transmembrane cytoplasmic tail of HSV-2 gD.

[0202] Embodiment 18: The recombinant HSV-2 of embodiment 16 or 17, further comprising a parasite surface glycoprotein on the lipid bilayer, wherein the parasite is a mammalian parasite.

[0203] Embodiment 19: The recombinant HSV-2 of any one of embodiments 16-18, wherein the HSV-2 glycoprotein D coding gene is HSV-2 U S 6 gene, recombinant HSV-2.

[0204] Embodiment 20: The recombinant HSV-2 of any one of embodiments 16-19, wherein the heterologous antigen is an influenza antigen.

[0205] Embodiment 21: The recombinant HSV-2 of any one of embodiments 16-20, wherein the heterologous antigen is a hemagglutinin (HA) antigen.

[0206] Embodiment 22: The recombinant HSV-2 of embodiment 21, wherein the HA antigen is the full-length HA extracellular domain or the HA stalk.

[0207] Embodiment 23: The recombinant HSV-2 of any one of embodiments 16-19, wherein the heterologous antigen is an HIV antigen.

[0208] Embodiment 24: The recombinant HSV-2 of embodiment 23, wherein the HIV antigen is Env gp145.

[0209] Embodiment 25: A cell comprising the recombinant virus of any one of embodiments 16-24, wherein the cell is not present in a human.

[0210] Embodiment 26: A vaccine composition comprising the recombinant virus of any one of embodiments 16-24.

[0211] Embodiment 27: A pharmaceutical composition comprising the virus of any one of embodiments 16-24 and a pharma- ceutical acceptable carrier.

[0212] Embodiment 28: A method for inducing and / or enhancing an immune response in a subject, the method comprising administering to the subject an amount of the recombinant virus of any one of embodiments 16-24 in an amount effective to induce and / or enhance an immune response in the subject.

[0213] Embodiment 29: A method of inducing and / or enhancing an immune response in a subject, the method comprising administering to the subject an amount of the vaccine of embodiment 26 in an amount effective to induce and / or enhance an immune response in the subject.

[0214] Embodiment 30: A method of inducing and / or enhancing an immune response in a subject, the method comprising administering to the subject an amount of the pharmaceutical composition of embodiment 27 in an amount effective to induce and / or enhance an immune response in the subject.

[0215] Embodiment 31: A method of treating or reducing the likelihood of influenza infection in a subject, the method comprising administering to the subject an amount of the recombinant virus of any one of embodiments 16-22 in an amount effective to treat or reduce the likelihood of influenza infection in the subject.

[0216] Embodiment 32: A method of treating or reducing the likelihood of influenza infection in a subject, the method comprising administering to the subject an amount of the vaccine of embodiment 26 in an amount effective to treat or reduce the likelihood of influenza infection in the subject.

[0217] Embodiment 33: A method of treating or reducing the likelihood of influenza infection in a subject, the method comprising administering to the subject an amount of the pharmaceutical composition of embodiment 27 in an amount effective to treat or reduce the likelihood of influenza infection in the subject.

[0218] Embodiment 34: A method of treating or reducing the likelihood of HIV infection in a subject, the method comprising administering to the subject an amount of the recombinant virus of any one of embodiments 16-19, 23 or 24 in an amount effective to treat or reduce the likelihood of HIV infection in the subject.

[0219] Embodiment 35: A method of treating or reducing the likelihood of HIV infection in a subject, the method comprising administering to the subject an amount of the vaccine of embodiment 26 in an amount effective to treat or reduce the likelihood of HIV infection in the subject.

[0220] Embodiment 36: A method of treating or reducing the likelihood of HIV infection in a subject, the method comprising administering to the subject an amount of the pharmaceutical composition of embodiment 27 in an amount effective to treat or reduce the likelihood of HIV infection in the subject.

[0221] Embodiment 37: A method of vaccinating a subject against influenza infection, the method comprising administering to the subject an amount of the recombinant virus of any one of embodiments 16-22 in an amount effective to vaccinate the subject against influenza infection.

[0222] Embodiment 38: A method of vaccinating a subject against influenza infection, the method comprising administering to the subject an amount of the vaccine of embodiment 26 in an amount effective to vaccinate the subject against influenza infection.

[0223] Embodiment 39: A method of vaccinating a subject against influenza infection, the method comprising administering to the subject an amount of the pharmaceutical composition of embodiment 27 in an amount effective to vaccinate the subject against influenza infection.

[0224] Embodiment 40: A method of vaccinating a subject against HIV infection, the method comprising administering to the subject an amount of the recombinant virus of any one of embodiments 16-19, 23 or 24 in an amount effective to vaccinate the subject against HIV infection.

[0225] Embodiment 41: A method of vaccinating a subject against HIV infection, the method comprising administering to the subject an amount of the vaccine of embodiment 26 in an amount effective to vaccinate the subject against HIV infection.

[0226] Embodiment 42: A method of vaccinating a subject against HIV infection, the method comprising administering to the subject an amount of the pharmaceutical composition of embodiment 27 in an amount effective to vaccinate the subject against HIV infection.

[0227] Embodiment 43: A method of inducing and / or enhancing an immune response in a subject, the method comprising administering to the subject an amount of a recombinant herpes simplex virus-2 (HSV-2) made by the process of any one of embodiments 1-14 and comprising (i) a complete deletion of the HSV-2 glycoprotein D-encoding gene in its genome and (ii) a promoter, an influenza hemagglutinin (HA) antigen signal sequence, and encoding the HA antigen, in an amount effective to induce and / or enhance an immune response in the subject.

[0228] Embodiment 44: A method of treating or reducing the likelihood of influenza infection in a subject, the method comprising administering to the subject an amount of a recombinant herpes simplex virus-2 (HSV-2) made by the process of any one of embodiments 1-13 and comprising (i) a complete deletion of the HSV-2 glycoprotein D-encoding gene in its genome and (ii) encoding a promoter, an influenza hemagglutinin (HA) antigen signal sequence, and an HA antigen, in an amount effective to treat or reduce the likelihood of influenza infection in the subject.

[0229] Embodiment 45: A method of vaccinating a subject against influenza infection, the method comprising administering to the subject an amount of a recombinant herpes simplex virus-2 produced by the process of any one of embodiments 1-14 and comprising (i) a complete deletion of the HSV-2 glycoprotein D-encoding gene in its genome, and (ii) encoding a promoter, an influenza hemagglutinin (HA) antigen signal sequence, and an HA antigen.

[0230] Embodiment 46: The method of any one of embodiments 43-45, wherein the HA antigen is a full-length HA extracellular domain.

[0231] Embodiment 47: The method of embodiment 46, further comprising administering, following the initial administration of a recombinant herpes simplex virus-2 encoding a full-length HA extracellular domain, one or more amounts of a recombinant herpes simplex virus-2 comprising (i) a complete deletion of the HSV-2 glycoprotein D encoding gene in the genome and (ii) an encoding sequence for a promoter, an HA antigen signal sequence, and an HA stalk, but not a full-length HA.

[0232] Embodiment 48: The method of any one of embodiments 28-47, wherein the subject is a human.

[0233] Embodiment 49: A method for quantifying the rate or amount of antibody-dependent cell-mediated cytotoxicity (ADCK) in a population of cells, the method comprising: infecting a plurality of cells of the population of cells with a fluorescent protein-expressing recombinant HSV-2 comprising a genome deleted for the gene encoding HSV-2 gD under conditions allowing expression of the fluorescent protein in the cells, contacting the plurality of infected cells with an antibody-containing solution and a population of immune cells, and quantifying the amount of a plurality of infected cells exhibiting fluorescent protein fluorescence and, optionally, one or more markers at one or more time points, so as to quantify the amount of live infected cells over time, thereby quantifying the rate or amount of ADCK in the population of cells.

[0234] Embodiment 50: The method of embodiment 49, wherein the method is performed in vitro.

[0235] Embodiment 51: The method of embodiment 49 or 50, wherein the population of immune cells comprises macrophages.

[0236] Embodiment 52: The method of any one of embodiments 49-51, wherein the antibody-containing solution comprises serum.

[0237] Embodiment 53: The method of any one of embodiments 49-51, wherein the fluorescent protein is a red fluorescent protein.

[0238] Embodiment 54: The method of any one of embodiments 49-53, wherein the amount of a plurality of infected cells exhibiting fluorescent protein fluorescence and, optionally, one or more markers, is measured by fluorescence-activated cell sorting (FACS).

[0239] Embodiment 55: The method of any one of embodiments 49-55, wherein at least one marker comprises a cell membrane marker, a live / dead marker, or a combination thereof.

[0240] Embodiment 56: The method of any one of embodiments 49-55, further comprising quantifying at one or more time points the amount of cells exhibiting fluorescent protein fluorescence, and optionally one or more markers, in an identical control population of infected cells not contacted with the antibody-containing solution, and comparing the quantified amount or percentage with the amount or percentage quantified for the population of cells contacted with the antibody-containing solution.

[0241] Embodiment 57: The method of any one of embodiments 49-56, wherein the population of immune cells is present at an effector:target ratio of 5:1 or greater.

[0242] Embodiment 58: The method of any one of embodiments 49-57, wherein the recombinant HSV-2 is produced by the process of any one of embodiments 1-14.

[0243] Throughout this application, various publications are referenced. Full citations for these references may be found at the end of the specification. The disclosures of these publications, as well as all patents, patent application publications and books referenced herein, are hereby incorporated by reference in their entireties into the subject application in order to more fully describe the state of the art to which the subject invention pertains.

[0244] Those skilled in the art will readily appreciate that the specific methods and results discussed above are merely illustrative of the invention, as more fully described in the claims that follow. reference [Table 1-1] [Table 1-2]

Claims

1. 1. A process for producing a herpes simplex virus type 2 (HSV-2) vaccine vector directed against a heterologous antigen, comprising: a) providing an HSV-2 genome having a deleted glycoprotein D (gD-2) gene, wherein the deleted gD-2 is replaced with a nucleic acid comprising a promoter-RFP construct, where RFP is a nucleic acid encoding a red fluorescent protein; b) HSV-1 glycoprotein D (gD-1) complementing eukaryotic host cells: (i) the HSV-2 genome of a) and (ii) co-transfecting with a linear DNA fragment encoding a heterologous antigen, wherein the linear DNA fragment consists of DNA encoding one of the following in the following order: CMV promoter-heterologous antigen-polyadenylation signal, wherein the heterologous antigen is codon-optimized for HSV-2 expression; c) growing the transfected cells of b) under conditions in which allelic recombination occurs between said HSV-2 genome and said DNA fragment; d) screening the plaques resulting from c) to identify those that do not exhibit red fluorescence; and e) recovering recombinant HSV-2 viruses or virions from those plaques not showing red fluorescence in d) to obtain HSV-2 vaccine vectors directed against the heterologous antigen.

2. 2. The process of claim 1, wherein the heterologous antigen is an influenza hemagglutinin (HA) antigen.

3. 3. The process according to claim 1 or 2, wherein the heterologous antigen is an HIV antigen, and the HIV antigen is Env gp145.

4. The process according to any one of claims 1 to 3, wherein the promoter of the promoter-FP construct is the elongation factor 1 alpha gene (P ＥＦ１α ), where P ＥＦ１α and FP are both fused (P ＥＦ１α -FP), process.

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