US20210236625A1 - HSV-2-DELTA-gD VACCINES AND METHODS FOR THEIR PRODUCTION AND USE - Google Patents

HSV-2-DELTA-gD VACCINES AND METHODS FOR THEIR PRODUCTION AND USE Download PDF

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US20210236625A1
US20210236625A1 US17/051,992 US201917051992A US2021236625A1 US 20210236625 A1 US20210236625 A1 US 20210236625A1 US 201917051992 A US201917051992 A US 201917051992A US 2021236625 A1 US2021236625 A1 US 2021236625A1
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hsv
subject
δgd
antigen
cells
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William Jacobs, JR.
Betsy Herold
Joseph Dardick
Kayla A. Weiss
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Albert Einstein College of Medicine
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Definitions

  • Pathogen infections including influenza and HIV viral infections
  • Vaccines have been developed for many pathogens, including both influenza and HIV viral infections, however they have failings and limitations. Thus, a novel vaccine strategy must be engineered and evaluated.
  • HSV-2-based vaccines for various antigenic targets, including influenza and HIV.
  • a process for producing a vaccine vector directed against a heterologous antigen comprising:
  • nucleic acid comprising a promoter-FP construct, wherein FP is a nucleic acid encoding a fluorescent protein
  • HSV-2 herpes simplex virus-2
  • HSV-2 having a genome encoding a heterologous antigen made by the process described herein.
  • HSV-2 herpes simplex virus-2
  • AgD-2 herpes simplex virus-2
  • AgD-2 herpes simplex virus-2
  • Also provided is a vaccine composition comprising the recombinant HSV-2 virus as described herein.
  • composition comprising a recombinant HSV-2 virus as described herein, and a pharmaceutically acceptable carrier.
  • Also provided is a method of eliciting 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 as described herein; or (iii) a pharmaceutical composition as described herein, in an amount effective to elicit and/or enhance an immune response in a subject.
  • Also provided is a method of treating or reducing the likelihood of an influenza 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 an influenza infection in a subject.
  • Also provided is a method of treating or reducing the likelihood of an 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 an HIV infection in a subject.
  • Also provided is a method of vaccinating a subject for 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 a subject for influenza infection.
  • Also provided is a method of vaccinating a subject for 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 a subject for HIV infection.
  • Also provided is a method of quantitating a rate or amount of antibody-dependent cell-mediated killing (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 that comprises a genome deleted for the gene encoding HSV-2 gD, under conditions permitting 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 quantitating at one or more time points the amount of cells exhibiting fluorescent protein fluorescence and, optionally, one or more markers, so as to quantitate over time the amount of live infected cells, so as to thereby quantitating the rate or amount of ADCK in the population of cells.
  • ADCK antibody-dependent cell-mediated killing
  • FIGS. 1A-1B HSV-2 ⁇ gD::HA and HSV-2 ⁇ gD::HAstalk were created by co-transfection of plasmid DNA and ⁇ gD-2::P EF1 ⁇ -RFP (B3 ⁇ 2.8, also referred to as ⁇ gD-2::RFP) genomic DNA and verified with PCR.
  • FIG. 1A Purified pYUB2169 containing the desired gD::HA gene was cut with Pad and co-transfected into VD60 cells alongside HSV-2 ⁇ gD::PEF1 ⁇ -RFP genomic DNA. Electroporation was used. Resultant RFP-plaques were purified three times and assessed for the presence of HA.
  • FIG. 1A Purified pYUB2169 containing the desired gD::HA gene was cut with Pad and co-transfected into VD60 cells alongside HSV-2 ⁇ gD::PEF1 ⁇ -RFP genomic DNA. Electroporation was used. Resultant RFP-plaques were
  • Recombinant viruses were verified by PCR amplification of the extracellular HA domain from plasmid and recombinant viral DNA. Primers used were located 100 base pairs (bp) upstream and downstream of the extracellular HA domain. Recombinant viruses both showed the desired band, as visualized by comparison to plasmid DNA. No band at that size was present in wells which contained DNA from ⁇ gD*2 (B 3 ⁇ 2.9)(Lanes 1 and 4).
  • Lane 2 shows DNA from pYUB2169 containing full-length HA construct.
  • Lane 3 shows DNA from recombinant HSV-2 ⁇ gD-2::HA (B 3 ⁇ 2.10) containing the non-codon optimized full-length extracellular domain of HA.
  • Lane 5 shows DNA from recombinant HSV-2 ⁇ gD-2::HAstalk containing the codon-optimized stalk domain of HA.
  • Lane 6 shows DNA from B3 ⁇ 2.11.
  • FIG. 2 shows antibody response to vaccination with ⁇ gD-2::GFP (B 3 ⁇ 2.7) or ⁇ gD-2 (B 3 ⁇ 2.9).
  • FIG. 3 shows results of antibody-dependent cellular cytotoxicity of cells infected with ⁇ gD-2::GFP virus vs. unmarked ⁇ gD-2 virus.
  • FIG. 4 T-cell response to ⁇ gD-2::GFP virus vs. unmarked virus.
  • FIG. 5 ⁇ gD-2 vaccination protects against challenge with wild-type HSV-2 strain 4674, similar to protection with ⁇ gD-2::GFP.
  • FIG. 6 Construction of chimeric ⁇ gD-2::HA viruses and immune response in mice.
  • the regions corresponding to the HA1 Stalk region, the HA1 Head region, the HA2 Stalk region, the HA2 Transmembrane region, and the HA2 Cytosolic region are indicated in the amino acid sequence and functional domains of A/Puerto Rico/1934/8 (PR8) HA (SEQ ID NO: 2).
  • PR8 HA A/Puerto Rico/1934/8
  • FIG. 7 Presence of chimeric ⁇ gD-2::HA in recombinant viruses verified by PCR.
  • the lanes as follows: Lane 1: ⁇ gD-2genomic DNA, Lane 2: ⁇ gD-2::FL HA nOP genomic DNA, Lane 3: pBJJ1 plasmid DNA, Lane 4: ⁇ gD-2genomic DNA, Lane 5: ⁇ gD-2::HL HA nOP genomic DNA, Lane 6: pBJJ2 plasmid DNA.
  • Primers used for PCR amplification were located just upstream and downstream of the HA extracellular domains. No exogenous promoter was inserted into the expression cassette. As a result, chimeric gene expression was regulated by the endogenous promoter.
  • FIG. 8 Mice vaccinated with ⁇ gD-2::FL HA nOP ( ⁇ 3 ⁇ 1) and gD-2::HL HA OPT ( ⁇ 3 ⁇ 3) are fully protected against HSV-2 challenge but do not form anti-HA IgGs. Mice were prime-boost vaccinated on days 0 and 21 with a control VD60 cell lysate or 1 ⁇ 10 6 PFU of gD-2::FL HA nOP, gD-2::HL HA OPT, or ⁇ gD-2::RFP.
  • FIG. 8A At day 42, mice were challenged with a 10 ⁇ LD 90 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.
  • FIG. 8B At day 40 post-prime vaccination, mice were bled to look at serum antibodies. ELISAs performed against soluble PR8 HA show the absence of HA-specific IgGs in mice.
  • FIG. 9 is a map of pEGFP-N1 expression plasmid.
  • the PCMV::HA::SV40 polyA cassettes were restriction cloned into pBRL812, a plasmid containing >5.5 kB of the HSV-2 (G) genome upstream and downstream of US6.
  • pBRL812 was cut with AsiSI and PacI and co-transfected with ⁇ gD-2::RFP genomic DNA into VD60 cells. Allelic exchange was identified by lack of RFP expression and RFP negative plaques were picked and purified three times before verification of recombination by PCR.
  • FIG. 10 Map of the pBRL812 HSV recombination plasmid prepared as described in the above description of FIG. 9 .
  • FIG. 11A Verification of ⁇ gD-2::P CMV -FL HA nOP. PCR using primers just upstream and downstream of the P CMV ::HA::SV40 PolyA cassette.
  • unlabeled lane DNA ladder
  • Lane 1 pJHA1
  • Lane 2 1.2.1 potential recombinant isolate
  • Lane 3 negative control ⁇ gD-2 genomic DNA.
  • FIG. 11B Verification of ⁇ gD-2::P CMV -HL HA nOP using PCR using primers just upstream and downstream of the P CMV ::HA::SV40 PolyA cassette.
  • Unlabeled lane DNA ladder
  • Lane 1 pJHA2
  • Lane 2 2.2.2 potential recombinant isolate
  • Lane 3 2.3.1 potential recombinant isolate
  • Lane 4 negative control ⁇ gD-2 genomic DNA.
  • FIG. 11C Verification of ⁇ gD-2::P CMV -HL HA OPT. PCR using primers just upstream and downstream of the P CMV ::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 make the original ⁇ gD-2::P CMV -HL HA OPT stock.
  • FIG. 12 ⁇ gD-2::P CMV -HA recombinant viruses all express HA.
  • VD60 cells were infected with 3 MOI of ⁇ gD-2::P CMV -FL HA nOP, ⁇ gD-2::P CMV -HL HA nOP, or ⁇ gD-2::P CMV -HL HA OPT.
  • cells were harvested and stained for HSV-protein and HA expression.
  • HSV protein expression was measured using serum from mice vaccinated with ⁇ gD-2::RFP.
  • HA expression was measured using monoclonal anti-HA stalk IgG C179. Cells were stained with either cell permeable or cell impermeable methods.
  • FIG. 13 shows that ⁇ gD-2::P CMV -FL HA nOP does not spread from cell to cell.
  • FIGS. 14A-14E Kinetics of fluorescent protein expression and infection with HSV-2 ⁇ gD-2::RFP ( ⁇ gD-2::RFP) compared to the parental HSV-2 ⁇ gD-2::GFP ( ⁇ gD-2) strain.
  • FIG. 14A HSV-2 ⁇ gD-2::RFP ( ⁇ gD-2::RFP) was created by transfecting VD60 cells simultaneously with HSV-2 ⁇ gD-2::GFP ( ⁇ gD-2) genomic DNA and a plasmid containing pEF1 ⁇ ::RFP flanked on either side by regions homologous to the HSV-2 (G) genome.
  • FIGS. 14B and 14C Vero cells were infected with 1 MOI of HSV-2 ⁇ gD-2 (B) or ⁇ gD-2::RFP. Kinetics of infection, RFP expression, and GFP expression were monitored over time. Both viruses show similar kinetics of infection. ⁇ gD-2 induced very little GFP expression even at 24 hours post-infection while ⁇ gD-2::RFP induced high levels of RFP expression beginning at 4.5 hours post-infection.
  • FIGS. 14D and 14E show similar kinetics of infection. ⁇ gD-2 induced very little GFP expression even at 24 hours post-infection while ⁇ gD-2::RFP induced high levels of RFP expression beginning at 4.5 hours post-infection.
  • VD60 VD60 cells at 12 hours post-infection with 1 MOI of ⁇ gD-2::RFP. Infected VD60 cells are forming syncytia, indicative of productive infection. Vero cells have not formed any syncytia. Images taken with deconvolution at 10 ⁇ magnification.
  • FIGS. 15A-15C Validation of novel rapid fluorometric antibody-dependent cell-mediated killing (RFADCK) assay in both Raw 264.7 and J774.1 cells.
  • FIG. 15A Highly expressing target cells were isolated by flow analysis using dual expression of membrane and live/dead markers. The proportion of cells expressing high levels of HSV proteins was then gated by determining the mean RFP intensity for populations of infected target cells. The percent difference in the proportion of this population between treated and untreated groups was then calculated as ADCK.
  • FIG. 15B Infected target cells were incubated in the presence or absence of serum from ⁇ gD-2::RFP prior to co-culture with J774.1 macrophages.
  • FIG. 15C ADCK assays were performed in parallel using Raw 264.7 and J774.1 macrophages as effector cells.
  • mice vaccinated with ⁇ gD-2:RFP or VD60 cell lysate were compared to that of co-cultures without serum.
  • Data represents three independent experiments done in triplicate with J774.1 and Raw 264.7 cells at effector to target cell ratios of 10:1.
  • Target cells used in all experiments were HEK 293.
  • Statistics in B were done using student's t tests.
  • Statistics in C were done using one-way ANOVA. Error bars reflect SEM. *p ⁇ 0.05; ***p ⁇ 0.001.
  • FIGS. 17A-17B The ADCK assay can be adapted to knockout mouse strains and other model organisms.
  • FIG. 17A ADCK assay was carried out using bone marrow-derived macrophages (BMDMs) from both Fc ⁇ R ⁇ / ⁇ and WT mice. Baseline killing of infected target cells was determined in co-cultures containing serum from VD60 lysate vaccinated mice. Data represents the percent difference between co-cultures containing serum from ⁇ gD-2::RFP vaccinated mice and the baseline.
  • BMDMs bone marrow-derived macrophages
  • WT BMDMs carried out significantly more ADCK in the presence of HSV-2 ⁇ gD-2::RFP vaccinated serum than Fc ⁇ R ⁇ / ⁇ BMDMs (p ⁇ 0.05).
  • Killing of infected cells by Fc ⁇ R′ BMDMs in co-cultures containing serum from ⁇ gD-2::RFP vaccinated mice was indistinguishable from that of co-cultures containing serum from VD60 lysate mock-vaccinated mice.
  • FIG. 17B ADCK assay was carried out using BMDMs derived from na ⁇ ve guinea pig marrow. Baseline ADCK was determined by co-cultures containing serum from na ⁇ ve animals.
  • FIG. 18 Anti-HA antibodies elicited by vaccination with various PR8 influenza A virus (IAV) hemagglutinin (HA) HSV-2 recombinants (see Legend in figure).
  • IAV PR8 influenza A virus
  • HA hemagglutinin
  • FIG. 19 Anti-HA antibody isotype elicited by vaccination with the HSV-2 recombinant ⁇ gD-2::FL HA nOP. **p ⁇ 0.01.
  • FIG. 20 Anti-PR8 IgG Isotype ELISA.
  • FIGS. 21A-21C Mice vaccinated with ⁇ gD-2::FL HA PR8 ( ⁇ gD-2::HA PR8 ) are fully protected from challenge with PR8. Mice were prime-boost vaccinated subcutaneously 3 weeks apart with 5 ⁇ 10 6 PFU of ⁇ gD-2::RFP or ⁇ gD-2::HA PR8 or mock vaccinated with VD60 cell lysate.
  • FIGS. 21B and 21C Three weeks post-boost, mice were challenged intranasally with a 6 ⁇ LD 50 of PR8. Mice were sacrificed when they reached 75% of their initial weight. Mice immunized with ⁇ gD-2::HA PR8 were fully protected from PR8 challenge while mice that received control vaccinations all succumbed to infection before day 9. Statistics for neutralization titer were calculated by ANOVA. Survival statistics were calculated by Mantel-Cox log-rank test *p ⁇ 0.05; “p ⁇ 0.01; ***p ⁇ 0.001.
  • FIGS. 22A-22C show that recombinant gD-2::HA PR8 expresses high levels of PR8 protein.
  • ⁇ gD-2::RFP DNA was co-transfected into VD60 cells alongside an HA expression cassette containing the hemagglutinin (HA) gene from IAV H1N1 strain A/Puerto Rico/1934/8 (PR8) downstream of P CMV and upstream of a poly-adenylation signal.
  • HA hemagglutinin
  • extracellular and intracellular HA expression was measured by flow cytometry in Vero and VD60 cells infected with 3 MOI of ⁇ gD-2, ⁇ gD-2::HA PR8 , or ⁇ gD-2 containing a truncated version of the PR8 HA expression cassette ( ⁇ gD-2::HL HA PR8 ).
  • FIGS. 23A-23F show that mice immunized with ⁇ gD-2::HA PR8 develop high titers of functional and isotype switched anti-PR8 antibodies.
  • Mice were prime-boost vaccinated 21 days apart with either VD60 cell lysate, ⁇ gD-2::RFP vector, or ⁇ gD-2::HA PR8 . At day 28 post-prime, serum was collected for analysis.
  • Anti-PR8 antibodies were measured by ELISA against purified HA PR8 protein. Mice immunized with ⁇ gD::HA PR8 developed isotype switch anti-PR8 HA antibodies that were predominantly IgG2c and IgG2b.
  • FIGS. 24A-24L show that mice immunized with ⁇ gD-2::HA PR8 develop protection against IAV challenge. Mice were prime-boost immunized 21 days apart with VD60 cell lysate, ⁇ gD-2, or ⁇ gD-2::HA PR8 , bled at day 28 post-prime, and challenged intranasally 14 days later with a 6 ⁇ LD50 of IAV.
  • Neutralization titers were measured using microneutralization assays against the respective strains. Mice were sacrificed after reaching 70% of starting weight.
  • Statistics for neutralization assays done using 3-way ANOVA tests. Statistics for survival done using Mantel-Cox log-rank tests. *p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001.
  • FIGS. 25A-25F Mice immunized with ⁇ gD-2::HA PR8 develop fully protective ADCC immunity against HSV-2. Mice were prime-boost vaccinated 21 days apart with either VD60 cell lysate, ⁇ gD-2, or ⁇ gD-2::HA PR8 . In FIGS. 25A and 25B , the mice were bled 1-week post-boost and the serum was analyzed by ELISA. Mice that received ⁇ gD-2 or ⁇ gD-2::HA PR8 generated similarly high levels of HSV-specific IgG ( FIG. 25A ). Additionally, these IgGs were predominantly IgG2c. In FIG.
  • FIG. 26 shows the results of mFc ⁇ RIV ADCC reporter bioassay response to serially diluted serum samples collected from mice vaccinated with HSV-2 ⁇ gD::HA, HSV-2 ⁇ gD::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 mFc ⁇ RIV expressing Jurkat T cells were used as effector cells.
  • the target cells were incubated with serially diluted serum samples and effector cells. Bio-GloTM Reagent was added, and luminescence was measured.
  • mice showed significantly higher activation of mFc ⁇ RIV receptors in comparison to mice vaccinated with HSV-2 ⁇ gD-2::RFP and inactivated PR8 virus.
  • the p value for significance is ⁇ 0.05. Symbols: **P ⁇ 0.01, ***P ⁇ 0.001, **** P ⁇ 0.0001.
  • FIG. 27 is a map of the plasmid ZM109F.PB4, which was used to amplify full length HIV-1 Env (Glade C) and Rev, together with partial Nef of HIV-1.
  • FIG. 28 is a map of the pBkk412 plasmid.
  • FIG. 29A Screen of transformed E. coli colonies for the presence of the cloned insert.
  • FIG. 29B Analysis by PCR to identify clones having the correct nucleic acid size.
  • a process for producing a vaccine vector directed against a heterologous antigen comprising:
  • nucleic acid comprising a promoter-FP construct, wherein FP is a nucleic acid encoding a fluorescent protein
  • the promoter of the promoter-FP construct is a heterologous promoter.
  • a process for producing a vaccine vector directed against an antigen, the process comprising:
  • a nucleic acid comprising an P EF1 ⁇ -RFP construct, where P EF1 ⁇ is a promoter of Elongation Factor 1 ⁇ gene and RFP is a nucleic acid encoding red fluorescent protein, wherein P EF1 ⁇ and RFP are fused together (P EF1 ⁇ -RFP);
  • promoters can be employed. Heterologous promoters are preferred including those with high efficiency. Such promoters include the CMV promoter, including the cytomegalovirus major immediate-early promoter.
  • 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 alpha may be used.
  • the human EF1 ⁇ gene sequence is known in the art, for example see NCBI accession No. J04617.
  • heterologous promoters known in the art and usable in the invention include, but are not limited to, CMV enhancer fused to the chicken beta-actin promoter (CAG), mouse cytomegalovirus (mouse CMV), Chinese hamster elongation factor-1a (CHEF-1a), and phosphoglycerate kinase (PGK).
  • CAG chicken beta-actin promoter
  • mouse CMV mouse cytomegalovirus
  • CHEF-1a Chinese hamster elongation factor-1a
  • PGK phosphoglycerate kinase
  • the host cell is a complementing cell, for example a cell that phenotypically complements for HSV-1 glycoprotein D.
  • the host cell is a VD60 cell, which phenotypically complements HSV-1 gD.
  • the vaccine vector produced is genotypically deleted for HSV-2 gD and is phenotypically complemented for HSV-1 gD on a lipid bilayer thereof. In embodiments, the vaccine vector produced does not genotypically encode any HSV gD.
  • the host cell is co-transfected with (i) the HSV-2 genome of a) and (ii) a linear DNA fragment encoding, in order, (i) HSV-2 gD signal sequence, the heterologous antigen, HSV-2 gD transmembrane domain, HSV-2 gD cytosolic domain, but not encoding a HSV-2 gD extracellular domain, or (ii) HSV-2 gD signal sequence, the heterologous antigen, and transmembrane cytoplasmic tail of HSV-2 gD.
  • the host cell is co-transfected with (i) the HSV-2 genome of a) and (ii) linear DNA fragments encoding, in order, (i) a promoter, the heterologous antigen, and optionally a poly-A signal.
  • the co-transfecting is effected by electroporation.
  • nucleic acid-encodable fluorescent proteins for use in the invention include red, far red, yellow, green, orange, cyan or photo switchable fluorescent protein. Examples of such proteins are well known in the art. Supplies include Molecular Probes (ThermoFisher USA) and Takara (USA).
  • the fluorescent protein is 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 emission of 584 nm. Alternatively, firefly luciferase or nano-luciferase can be used.
  • the antigen is not an HSV-2 antigen, i.e. it is a heterologous antigen.
  • an antigen is heterologous when it is heterologous relative to HSV-2, i.e. is not naturally found on or in a wildtype HSV-2.
  • the heterologous antigen can be derived from a living organism, comprising for example, a virus, a bacteria, 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.
  • the virus can be a pathogenic virus, examples of which include cytomegalovirus (CMV), coxsackie virus, Crimean-Congo hemorrhagic fever virus, chikungunya virus, dengue virus, Dhori virus, Eastern equine encephalitis (EEE) virus, ebola virus, Epstein Barr virus (EBV), hepatitis virus, herpesvirus, human immunodeficiency (HIV) virus, human papilloma virus, human SARS corona virus, human T lymphotropic virus (HTLV), influenza virus, measles virus, mumps virus, Norwalk virus, rabies virus, rotavirus, rubella virus, severe fever with thrombocytopenia 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.
  • CMV cytomegalovirus
  • the bacteria can be a pathogenic bacteria, examples of which include Bacillus sp., Baronella sp., Bordatella sp., Borelli asp., Brucella sp., Campylobacter sp., Chlamydia sp., Clostridium sp., Corynebacterium sp., Enterococcus sp., Escherichia sp., Haemophilis sp., Helicobacter sp., Legionella sp., Leptospira sp., Listeria sp., Mycobacterium sp., Mycoplasma sp., Neisseria sp., Rickettsia sp., Pseudomonas sp., Salmonella sp., Shigella sp., Staphylococcus sp., Streptococcus sp., Treponema sp.
  • the parasite can be a pathogenic parasite, examples of which include Acanthamoeba spp., Balamuthia spp., Babesia sp., Balantidium coli, Blastocystic sp., Cryptospiridium sp., Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia, Isospora bello, Leishmania sp., Naegleria foweri, Plasmodium sp., Rhinosporidium seeberi, Sarcocystis sp., Toxoplasma gondii, Trichomonas sp., Trypanosoma sp., or a combination thereof.
  • Acanthamoeba spp. Balamuthia spp.
  • Babesia sp. Balantidium coli
  • Blastocystic sp. Cryptospir
  • the human cell or animal cell can be, for example, a cancer cell.
  • the heterologous antigen is an influenza antigen. In embodiments, the heterologous antigen is an influenza hemagglutinin (HA) antigen. In embodiments, the HA antigen is a full-length HA extracellular domain or is a HA stalk domain.
  • HA hemagglutinin
  • the heterologous antigen is an HIV antigen.
  • the HIV antigen is an Env gp145.
  • the heterologous antigen is under control of an upstream CMV promoter and has a downstream SV40 poly-A signal.
  • the SV40 poly-A signal is known in the art. It promotes polyadenylation and transcription termination.
  • the promoter is a promoter of Elongation Factor 1a gene (P EF1 ⁇ ) and wherein P EF1 ⁇ and FP are fused together (P EF1 ⁇ -FP).
  • the nucleic acid is codon-optimized for expression. See, for example, Table 4 in the examples hereinbelow.
  • HSV-2 herpes simplex virus-2
  • HSV-2 having a genome encoding a heterologous antigen made by the process described herein.
  • HSV-2 herpes simplex virus-2
  • HSV-2 herpes simplex virus-2
  • a recombinant herpes simplex virus-2 having (i) a complete deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof and (ii) (a) encoding: a promoter, a heterologous antigen signal sequence, and a heterologous antigen or (b) encoding: a promoter and a heterologous antigen.
  • HSV-2 herpes simplex virus-2
  • HSV-2 having (i) a partial deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof and (ii) (a) encoding, in order, an HSV-2 gD signal sequence, a heterologous antigen, an HSV-2 gD transmembrane domain, optionally an HSV-2 gD cytosolic domain, but not encoding an HSV-2 gD extracellular domain, or (b) encoding, in order, an HSV-2 gD signal sequence, a heterologous antigen, cytosolic domain of HSV-2 gD.
  • HSV-2 gD herpes simplex virus-2
  • HSV-2 herpes simplex virus-2
  • HSV-2 having (i) a partial deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof and (ii) (a) encoding, in order, an HSV-2 gD signal sequence, a heterologous antigen, an HSV-2 gD transmembrane domain, an HSV-2 gD cytosolic domain, but not encoding an HSV-2 gD extracellular domain, or (b) encoding, in order, an HSV-2 gD signal sequence, a heterologous antigen, and transmembrane cytoplasmic tail of HSV-2 gD.
  • HSV-2 gD herpes simplex virus-2
  • the recombinant HSV-2 further comprises a parasitic surface glycoprotein on a lipid bilayer thereof, wherein the parasite is a parasite of a mammal.
  • the HSV-2 glycoprotein D-encoding gene is an HSV-2 U S6 gene.
  • HSV-2 U S6 gene For example, see Dolan et al. J Virol. 1998 March; 72(3): 2010-2021. (PMCID: PMC109494) “The Genome Sequence of Herpes Simplex Virus Type 2” for HSV-2 genome and U S6 gene, hereby incorporated by reference in its entirety).
  • the HSV-2 glycoprotein D-encoding gene is equivalent of an HSV-2 U S 6 gene. Such equivalents are easily identifiable by those of skill in the art using readily available sequencing and alignment tools.
  • the heterologous antigen is an influenza antigen. In embodiments, the heterologous antigen is an influenza hemagglutinin (HA) antigen.
  • HA hemagglutinin
  • the HA antigen is a full-length HA extracellular domain or is a HA stalk.
  • the full-length HA includes a HA signal sequence.
  • the HA is an HA of a human influenza A or human influenza B.
  • examples of the hemagglutinin gene (“HA”) include GenBank V01098.1; NCBI Reference Sequence: NP_040980.1.
  • HA gene sequences, and the mature hemagglutinin peptide sequences are well known in the art, and multiple HA sequences are available to those skilled in the art at the NCBI database.
  • one skilled in the art can readily identify the commonly discussed stalk, extracellular domain and other regions of hemagglutinin.
  • seasonal influenza virus strain sequences including the HA sequence, are routinely sequenced and identified in the art.
  • influenza genes as heterologous antigens, that can be added to ⁇ gD-2 by allelic exchange with B3 ⁇ 2.8 ( ⁇ gD-2::P EF1 ⁇ -RFP) include neuraminidase (NA), matrix protein 1 (M1), influenza A virus (IAV) matrix protein 2 (M2), influenza B virus (IBV) matrix protein 2 (M2), nucleoprotein (NP), and influenza B virus NB.
  • NA neuraminidase
  • M1 matrix protein 1
  • IAV influenza A virus
  • IBV influenza B virus
  • NP nucleoprotein
  • NP nucleoprotein
  • influenza B virus NB nucleoprotein
  • the corresponding modified headless HA gene for each strain can be added as well as a version of each headless antigen where a trimerization domain has been added to increase stability.
  • Table 1 provides non-limiting influenza examples of other such antigen genes.
  • HA gene from A/Vietnam/1203/04
  • the HA, headless HA, NA, M1, M2, NP, and NB genes from, e.g., B/Yamagata/16/1988 and B/Victoria/2/1987 strains.
  • a cosmid pYUB2169 can be engineered to express a gene for a heterologous antigen (e.g., HIV envelope protein antigen Env gp145 or Influenza virus HA stalk antigen) of choice that is flanked by sequences of the HSV-2 signal sequence and the transmembrane-cytoplasmic domain sequence on either respective side.
  • a heterologous antigen e.g., HIV envelope protein antigen Env gp145 or Influenza virus HA stalk antigen
  • homologous recombination occurs between the HSV-2 partially deleted glycoprotein D gene (RFP gene inserted) of (1) and the pYUB2169-HIV Env gp145 or Influenza A virus HA stalk antigen expressing gene of (2).
  • RFP gene inserted the HSV-2 partially deleted glycoprotein D gene
  • a successful recombination is expected to lead to loss of RFP gene and generation of a HSV-2 gD ⁇ / ⁇ virus expressing a heterologous HIV or Influenza virus antigen (see FIG. 1A-1B ).
  • VD60 cell culture plaques that are negative for RFP expression contain HSV-2 gD ⁇ / ⁇ the virus particles are expected to express HIV Env gp145 or I influenza virus HA stalk antigen.
  • the HSV-2 gD ⁇ / ⁇ virus expressing a heterologous HIV Env gp145 or Influenza A virus HA stalk antigen thus generated can be used to vaccinate a person and elicit at strong antibody mediated response and protective immunity against HIV or Influenza Virus.
  • the heterologous antigen is an HIV antigen.
  • the HIV antigen is an HIV-1 or HIV-2 antigen.
  • the heterologous antigen is an HIV-1 antigen.
  • the HIV is a C-subtype.
  • the HIV antigen is an Env, Pol, Gag, or Nef.
  • the HIV antigen is an Env antigen.
  • the HIV antigen is a C-subtype Env antigen.
  • the antigen is an Env gp145.
  • the heterologous antigen is a fully intact membrane-proximal external region (MPER).
  • the heterologous antigen is extended by a polylysine tail.
  • the heterologous antigen is not extended by a polylysine tail.
  • HIV gp145 Env protein sequences are readily identifiable by alignment tools, and are routine to sequence.
  • the ectodomain of HIV Env gp145 is fused with the signal peptide and transmembrane cytoplasmic tail of HSV gD.
  • a vaccine composition comprising a recombinant virus as described herein.
  • the vaccine comprises an adjuvant which is not derived from the 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, and aluminum potassium sulfate, and monophosphoryl lipid A.
  • the vaccine does not comprise an adjuvant.
  • composition comprising a recombinant HSV-2 virus as described herein, and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are well-known in the art.
  • Also provided is a method of eliciting and/or enhancing an immune response 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 elicit and/or enhance immune response in a subject.
  • Also provided is a method of eliciting and/or enhancing an immune response in a subject against a pathogen expressing a heterologous antigen 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 elicit and/or enhance immune response in a subject, wherein the recombinant virus of (i), (ii) or (iii) expresses the heterologous antigen.
  • Also provided is a method of treating or reducing the likelihood of an influenza 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 an influenza infection in a subject.
  • the heterologous antigen is an influenza HA antigen.
  • Also provided is a method of vaccinating a subject for influenza 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 a subject for influenza infection.
  • Also provided is a method of treating or reducing the likelihood of an 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 an HIV infection in a subject.
  • the heterologous antigen is an HIV antigen.
  • Also provided is a method of vaccinating a subject for 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 a subject for HIV infection.
  • Also provided is a method of eliciting and/or enhancing an immune response 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 an HSV-2 glycoprotein D-encoding gene in the genome thereof and (ii) encoding a promoter, a influenza hemagglutinin (HA) antigen signal sequence, and an HA antigen in an amount effective to elicit and/or enhance an immune response in a subject.
  • HSV-2 herpes simplex virus-2
  • HA influenza hemagglutinin
  • Also provided is a method of treating or reducing the likelihood of an 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 an HSV-2 glycoprotein D-encoding gene in the genome thereof and (ii) encoding a promoter, a influenza hemagglutinin (HA) antigen signal sequence, and an HA antigen in an amount effective to treat or reduce the likelihood of an influenza infection in a subject.
  • HSV-2 herpes simplex virus-2
  • HA influenza hemagglutinin
  • Also provided is a method of vaccinating a subject for influenza infection comprising administering to the subject an amount of a recombinant herpes simplex virus-2 made by a process described herein and comprising (i) a complete deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof and (ii) encoding a promoter, a influenza hemagglutinin (HA) antigen signal sequence, and an HA antigen in an amount effective to vaccinate a subject for influenza infection.
  • a recombinant herpes simplex virus-2 made by a process described herein and comprising (i) a complete deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof and (ii) encoding a promoter, a influenza hemagglutinin (HA) antigen signal sequence, and an HA antigen in an amount effective to vaccinate a subject for influenza infection.
  • the HA antigen is a full-length HA extracellular domain.
  • the methods further comprise, subsequent to an initial administration of the recombinant herpes simplex virus-2 encoding a full-length HA extracellular domain, administering one or more amounts of a recombinant herpes simplex virus-2 having (i) a complete deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof and (ii) encoding a promoter, a HA antigen signal sequence, and an HA stalk, but not encoding a full-length HA.
  • the HSV-2 glycoprotein D comprises the amino acid sequence set forth in SEQ ID NO:1:
  • the HSV-2 in which the HSV-2 glycoprotein D-encoding gene is deleted is an HSV-2 having a genome (prior to the deletion) as set forth in one of the following Genbank listed sequences: HSV-2(G) (KU310668), HSV-2(4674) (KU310667), B3 ⁇ 1.1 (KU310657), B3 ⁇ 1.2 (KU310658), B3 ⁇ 1.3 (KU310659), B3 ⁇ 1.4 (KU310660), B3 ⁇ 1.5 (KU310661), B3 ⁇ 2.1 (KU310662), B3 ⁇ 2.2 (KU310663), B3 ⁇ 2.3 (KU310664), B3 ⁇ 2.4 (KU310665), B3 ⁇ 2.5 (KU310666).
  • Genbank listed sequences HSV-2(G) (KU310668), HSV-2(4674) (KU310667), B3 ⁇ 1.1 (KU310657), B3 ⁇ 1.2 (KU310658), B3 ⁇ 1.3 (KU310659), B3 ⁇ 1.4 (KU310660), B3 ⁇ 1.5 (KU310661),
  • a cell comprising therein a recombinant HSV-2 genome as described herein.
  • compositions or pharmaceutical compositions described herein comprising a recombinant HSV-2, the HSV-2 is live-attenuated.
  • composition comprising the recombinant HSV-2 virus as described herein, wherein the genome of the virus or virion comprises at least a deletion of a second gene, wherein the second gene is necessary for HSV-2 viral replication or virulence.
  • a pharmaceutical composition comprising the recombinant HSV-2 virus as described herein, and a pharmaceutically acceptable carrier.
  • the composition or pharmaceutical composition or vaccine is formulated so that it is suitable for subcutaneous administration to a human subject. In an embodiment, the composition or pharmaceutical composition or vaccine is formulated so that it is suitable for oral administration to a human subject. In an embodiment, the composition or pharmaceutical composition or vaccine is formulated so that it is suitable for intravaginal administration to a human subject. In an embodiment, the composition or pharmaceutical composition or vaccine is formulated so that it is suitable for intra-muscular, intra-nasal, or mucosal administration to a human subject.
  • administration can be auricular, buccal, conjunctival, cutaneous, subcutaneous, endocervical, endosinusial, endotracheal, enteral, epidural, via hemodialysis, interstitial, intrabdominal, intraamniotic, intra-arterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intracisternal, intracorneal, intracoronary, intradermal, intradiscal, intraductal, intraepidermal, intraesophagus, intragastric, intravaginal, intragingival, intraileal, intraluminal, intralesional, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrae
  • Also provided is a method of eliciting an immune response in a subject comprising administering to the subject an amount of (i) the recombinant HSV-2 virus as described herein in an amount effective to elicit an immune response in a subject.
  • the HSV-2 glycoprotein D-encoding gene is an HSV-2 US6 gene.
  • the HSV-2 recombinant virus encodes a heterologous surface glycoprotein.
  • the heterologous surface glycoprotein is an HSV-1 gD.
  • the HSV-2 recombinant virus comprises a non-genotypically encoded HSV-1 gD also encodes a heterologous surface glycoprotein that is not a herpesvirus glycoprotein and/or is and not involved in herpesviridae infection, and which is encoded by a transgene that has been inserted into the genome of the recombinant HSV-2.
  • the genome of the recombinant HSV-2 does not encode any herpes virus gD.
  • the surface glycoprotein is present on a lipid bilayer of the virus by way of infecting a cell with a recombinant HSV-2 having a deletion of an HSV-2 glycoprotein D-encoding gene, wherein the cell is or has been transfected to express the surface glycoprotein on a cell membrane thereof, and wherein the recombinant HSV-2 comprising the surface glycoprotein present on a lipid bilayer is produced from the cell.
  • the host cell is a HSV-1 gD complementing cell.
  • the host cell encodes an HSV-1 gD under the endogenous gene promoter.
  • the host cell is a HSV-1 gD complementing VD60 cell.
  • a vaccine composition comprising a recombinant virus as described herein.
  • the vaccine composition comprises an immunological adjuvant.
  • composition comprising a recombinant virus as described herein, wherein the genome of the virus comprises at least a deletion of a second gene, wherein the second gene is necessary for HSV-2 viral replication.
  • the recombinant virus as described herein does not comprise a deletion of a second gene.
  • Reducing the likelihood of a viral infection is understood to mean amelioration of the extent of development of the relevant disease or chances of infection in a subject treated with the virus, vaccine or compositions described herein, as compared to an untreated subject.
  • the subject is a mammalian subject.
  • the mammalian subject is a human subject.
  • vaccinating a subject with an antigen elicits a humoral immune response to that antigen in the subject.
  • a vaccinated individual is usually able to mount a more efficacious immune response to a subsequent challenge from a pathogen comprising that antigen than they would be able to prior to vaccination.
  • the subject has not yet been infected with influenza virus. In an embodiment of the methods described herein, the subject has not yet been infected with HIV. In an embodiment of the methods described herein, the subject has been infected with influenza virus. In an embodiment of the methods described herein, the subject has been infected with HIV.
  • influenza infection is a human influenza A infection. In embodiments, the influenza infection is a human influenza B infection. In embodiments, the HIV infection is an HIV-1 infection. In embodiments, the HIV infection is an HIV-2 infection.
  • Codon optimization is defined as modifying a nucleic acid sequence for enhanced expression in the cells of interest, e.g. human, by replacing at least one, more than one, or a significant number, of codons of the native sequence with codons that are more frequently or most frequently used in the genes of that vertebrate.
  • Various species exhibit particular bias for certain codons of a particular amino acid.
  • the present invention relates to codon optimized inserts, nucleic acids or vectors, or host cells comprising such.
  • Also provided is a method of quantitating a rate or amount of antibody-dependent cell-mediated killing (ADCK) in a population of cells comprising infecting a plurality of cells of the population of cell with a fluorescent protein-expressing recombinant HSV-2 that comprises a genome deleted for the gene encoding HSV-2 gD, under conditions permitting 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 quantitating at one or more time points the amount of cells exhibiting fluorescent protein fluorescence and, optionally, one or more markers, so as to quantitate over time the amount of live infected cells, so as to thereby quantitating the rate or amount of ADCK in the population of immune cells.
  • ADCK antibody-dependent cell-mediated killing
  • Also provided is a method of quantitating a rate or amount of antibody-dependent cell-mediated killing (ADCK) in a population of cells comprising infecting a plurality of cells of the population of cells with a fluorescent protein-expression expressing recombinant HSV-2 that comprises a genome deleted for the gene encoding HSV-2 gD, under conditions permitting expression of the fluorescent protein in the cells, contacting the plurality of infected cells with an antibody-containing solution, and quantitating at one or more time points the amount of cells exhibiting fluorescent protein fluorescence and, optionally, one or more markers, so as to quantitate over time the amount of live infected cells, so as to thereby quantitating the rate or amount of ADCK in the population of cells.
  • ADCK antibody-dependent cell-mediated killing
  • the recombinant HSV-2 is made by a process as described herein.
  • the method is performed in vitro.
  • the population of immune cells comprises a population of macrophages.
  • the macrophages are human.
  • the antibody-containing solution comprises serum.
  • the fluorescent protein is as described hereinabove.
  • the fluorescent protein is Red Fluorescent Protein.
  • only the cells quantitated as RFP high are considered live.
  • the plurality of cells are RFP high if they express RFP above the mean intensity of an RFp-expressing cell of the infected population of cells.
  • the method is performed with the population of immune cells present at effector:target ratio of 5:1 or greater.
  • the method is performed with the population of immune cells present at effector:target ratio of 10:1 or greater.
  • 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 fluorescence spectrometer, fluorescence microplate reader, and fluorescence microscopy or fluorescence plate reader. In embodiments, the one or more markers comprise a cell membrane marker and/or a live/dead marker.
  • the method further comprises quantitating at one or more time points the amount of cells exhibiting fluorescent protein fluorescence and, optionally, one or more markers, in a control population of infected cells otherwise identical, but not contacted with an antibody-containing solution and comparing the amount or rate quantitated to that quantitated for the population of cells contacted with the antibody-containing solution.
  • the marker in the recombinant HSV-2 ⁇ gD-2 can be a beta galactosidase or an alkaline phosphatase.
  • the methods, processes and compositions disclosed herein can comprise, mutatis mutandis, recombinant HSV-2 ⁇ gD-2 comprising a nucleic acid in the genome thereof encoding beta galactosidase or an alkaline phosphatase.
  • An engineered HSV-2 virus that replaces the gD gene ( ⁇ gD-2) with a gene that strongly expresses the red fluorescent protein (RFP) was constructed. Once achieved, this provided an improved screen for identifying and obtaining new recombinants.
  • the ⁇ gD-2::RFP recombinant was made from a ⁇ gD-2 that showed protection.
  • the red fluorescent ⁇ gD-2:RFP referred to herein also as Bax 2.8 or ⁇ gD-2::P EF1 ⁇ -RFP, retains the capacity of the original ⁇ gD-2 to elicit protective immunity against HSV infection but also appears to have unaltered in vitro replication kinetics.
  • ⁇ gD-2::RFP possesses significant advantages over HSV-2 ⁇ gD::GFP virus for making recombinants. Since the RFP gene is fused to a highly efficient promoter, for example promoter of the EF1 ⁇ (Elongation Factor 1 a) gene, its expression is easily detected using a fluorescent microscope. In addition, visualization of red fluorescence in cells infected with ⁇ gD-2::RFP does not have the background found with the green fluorescent ⁇ gD-2. This property enables unambiguous identification of recombinant viruses in which the ⁇ gD-2::P EF1 ⁇ -RFP allele is replaced by other allelic exchange substrates.
  • EF1 ⁇ Elongation Factor 1 a
  • HSV-2 ⁇ gD ⁇ /+gD-1 virus hereafter called “ ⁇ gD-2”
  • HSV-2(G) as the backbone was accomplished as detailed below.
  • an expression construct was generated by Gibson cloning into an E. coli plasmid, cloning the expression construct in between the SpeI and BclI sites of a shuttle (for example, cosmid pYUB2169) that contains 12 kb of the HSV-2(G) virus spanning from US1 To US9, electroporated the Pad restriction fragment from the pYUB2169 recombinant alongside ⁇ gD-2::RFP DNA into VD60 cells, and screening the resulting plaques for loss of red fluorescence. (See FIG. 1 ).
  • This system was used to generate two sets of recombinant ⁇ gD-2 strains containing hemagglutinin (HA) antigens derived from the influenza A virus (IAV) strain A/Puerto Rico/8/1934 (PR8).
  • the first set of recombinants contain chimeric gD::HA genes recombined into the US6 region of ⁇ gD-2 through the use of, for example, pYUB2169. These recombinant genes were made up of the extracellular domain of PR8 HA fused to the transmembrane, cytosolic, and signal sequence domains of HSV-2 (G) gD.
  • the second set of recombinants contain modified or unmodified PR8 HA genes fused to an upstream CMV promoter and downstream SV40 polyadenylation signal. These cassettes were inserted into the US6 region of ⁇ gD-2 using a modified pYUB2169 identified as pBRL812. Each set of recombinants contained three type of HA genes:
  • FL HA nOP Fluorescence-activated 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.
  • ⁇ gD-2::RFP ( ⁇ gD-2::PEF1 ⁇ -RFP) was constructed by (1) introducing a kanamycin marker in RFP plasmid ptwB, (2) amplifying the kan marker and RFP (dTomato) with oligos having ⁇ 50 bp homologous sequence upstream and downstream of US6, (3) recombining in DY331 cells, then adding the PCR product followed by transformation with cosmid pYUB2156. (4) The kan marker was removed by restriction digest and ligation. (5) HSV2(G) genomic DNA and pYUB2167 were co-transfected in VD60 cells to generate ⁇ gD-2::RFP by allelic exchange.
  • ⁇ gD-2 was obtained by co-transfection of cosmid pYUB2163 with the ⁇ gD-2::RFP genome in VD60 cells to generate the ⁇ gD-2 genome by allelic exchange. This genome is free of all markers and antibiotic resistance genes. The resultant unmarked virus was sorted based on lack of RFP expression. RFP-negative plaques were purified three times and the lack of the RFP gene was verified by PCR and sequencing.
  • ⁇ gD-2::FL HA nOP was constructed by (1) Gibson cloning the cytosolic, transmembrane, and signal sequence domains of HSV-2 (G) gD to the extracellular domain of custom synthesized PR8 HA (Genscript, Piscataway, N.J.) in the pYUB2169 plasmid and (2) co-transfecting the resultant plasmid, pBJJ1, with the ⁇ gD-2:RFP genome in VD60 cells to generate the desired recombinant by allelic exchange.
  • G HSV-2
  • PR8 HA Genescript, Piscataway, N.J.
  • ⁇ gD-2::HL HA nOP was constructed similarly to ⁇ gD-2::FL HA nOP, but using a custom synthesized PR8 HA gene where the HA1 head domain was replaced with 4 glycine residues (Genscript).
  • the plasmid used for transfection was called pBJJ2.
  • ⁇ gD-2::HL HA OPT was constructed similarly to ⁇ gD-2::HL HA nOP, but using a custom synthesized PR8 HA gene where the HA1 head domain was replaced with 4 glycine residues and every codon which had less than 9.5% representation in HSV-2 (G) gD for its cognate amino acid was replaced with the codon most abundant in HSV-2 (G) gD (Genscript).
  • the plasmid used for transfection was called pBJJ4.
  • ⁇ gD-2::P CMV -FL HA nOP was constructed by (1) restriction cloning the full length PR8 HA gene (Genscript) in between the xbaI and HindIII restriction sites of pEGFP-N1 (Addgene, Cambridge, Mass.) (2) restriction cloning the P CMV -FL HA nOP::SV40 polyA cassette from the resultant plasmid into the SpeI and BclI sites of pBRL812. The resulting plasmid, pJHA1, was cut with Pad and AsiSI and co-transfected with the ⁇ gD-2::RFP genome in VD60 cells to generate the desired recombinant by allelic exchange.
  • ⁇ gD-2::P CMV -HL HA nOP was constructed similarly to ⁇ gD-2::P CMV -FL HA nOP but using a custom synthesized PR8 HA gene where the HA1 head domain was replaced with 4 glycine residues (Genscript).
  • the plasmid used for transfection was called pJHA2.
  • ⁇ gD-2::P CMV -HL HA OPT was constructed similarly to ⁇ gD-2::P CMV -FL HA nOP but using a custom synthesized PR8 HA gene where the HA1 head domain was replaced with 4 glycine residues and every codon which had less than 9.5% representation in HSV-2 (G) gD for its cognate amino acid was replaced with the codon most abundant in HSV-2 (G) gD (Genscript).
  • the plasmid used for transfection was called pJHA4.
  • mice were vaccinated on Day 0 (d0) and d21, then sera was taken on d40.
  • ⁇ gD-2 elicited anti-HSV antibodies that are similar to those obtained with ⁇ gD-2::GFP (See FIG. 2 ).
  • ⁇ gD-2 vaccination protects mice against challenge with a wild-type HSV-2 4674, which is a clinical isolate obtained from the virology laboratory at Montefiore Hospital, Bronx, N.Y., as well as the GFP-marked construct.
  • Mice were vaccinated with either the wild-type HSV-2 or the GFP-marked construct virus on d0 and d21. Twenty-one days after boost, mice were challenged subcutaneously with 10 ⁇ LD90 HSV-2 4674 and followed for skin lesion changes, presence of virus in the dorsal root ganglia, body weight, and survival. The unmarked virus behaved nearly identically by all measures ( FIG. 5 ).
  • 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), a recombinant virus expressing the 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 ⁇ gD-2 ( ⁇ gD ⁇ /+gD-1) virus were propagated on complementing VD60 cells and titered on VD60 and Vero cells. Concentrated viral stocks were stored at ⁇ 80° C. and diluted in PBS to the desired concentration when needed. Construction of ⁇ gD-2::GFP. Plasmid pcDNA3-eGFP (13031; Addgene, Cambridge, Mass., USA) was used as a template to PCR amplify the pCMV-eGFP-Neon and OriE-Ampr regions flanked by Van91I restriction enzyme sites.
  • the pCMV-eGFP-Neon 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.
  • genomic regions flanking the left and right of the US6 gene (gD) in HSV-2 were PCR amplified using purified viral DNA (HSV-2 strain 4674) as a 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).
  • the resulting plasmid (eKO2-US6) was sequence verified and extracted from E. coli using an endotoxin-free miniprep kit (MO-BIO Laboratories, Carlsbad, Calif., USA). HSV-2 DNA (1 ⁇ g) was co-transfected with 100 ng of eKO2-US6 into VD60 cells using Effectene (Qiagen, Valencia, Calif., USA), according to the manufacturer recommendations.
  • Genotypic confirmation of the gD deletion in ⁇ gD-2::GFP was performed by PCR.
  • a primer set was used to confirm the presence of wild-type (WT) and ⁇ gD-2 virus DNA in the samples (primers RL-V91I-US6 and RL-V91IUS6), while another set of primers (Neo-Out and US8-Out) was used to amplify a DNA region comprising eKO2-US6 and the genomic target region.
  • Vero or VD60 cells were infected with parental WT or ⁇ gD-2 virus (grown on VD60 cells and thus competent for entry) at a multiplicity of infection (MOI) of 10 plaque-forming units (PFU)/cell (based on VD60 titer). After 1-h incubation, cells were washed twice with PBS, incubated in Optimem for 48 h at 37° C., and harvested and evaluated for gD expression by Western blot.
  • MOI multiplicity of infection
  • PFU plaque-forming units
  • Prevalence Prevalence Replace- Original in in Altered in ments Codon HA gD Codon gD Made ATA 38.89% 4.35% ATC 86.96% 14 ATT 30.56% 8.70% ATC 86.96% 11 GTA 28.53% 4.00% GTC 40.00% 8 CTT 9.62% 5.13% CTG 43.59% 5 TTA 11.54% 7.69% CTG 43.59% 6 TCT 28.13% 7.69% TCG 69.23% 9 TCA 53.13% 0.00% TCG 69.23% 17 CCT 5.00% 0.00% CCC 65.12% 1 CCA 50.00% 6.98% CCC 65.12% 10 ACT 23.33% 4.35% ACG 39.13% 7 ACA 46.67% 4.35% ACC 52.17% 14 GCA 45.45% 7.32% GCC 60.98% 15 CAT 53.85% 9.09% CAC 90.91% 7 TGT 62.50% 0.00% TGC 100.0% 10 AGT 53.33% 8.33% AGC 91.67% 8 AGA 5
  • ⁇ gD-2genomic DNA Lane 2; ⁇ gD-2::FL HA nOP genomic DNA, Lane 3; pBJJ1 plasmid DNA, Lane 4; ⁇ gD-2genomic DNA, Lane 5; ⁇ gD-2::HL HA nOP genomic DNA, and Lane 6; pBJJ2 plasmid DNA.
  • Primers used for PCR amplification were located just upstream and downstream of the HA extracellular domains. No exogenous promoter was inserted into the expression cassette.
  • chimeric gene expression was regulated by the endogenous promoter.
  • Sanger sequencing was used to verify the proper construction of chimeric gD::HA genes in recombinant viruses.
  • 6-8 week old C57BL/6 mice were prime-boost vaccinated at days 0 and 21 with ⁇ gD-2::FL HA nOP or ⁇ gD-2::HL HA OPT in parallel with the parental ⁇ gD-2::RFP strain and a control group mock-vaccinated with VD60 cell lysates.
  • 21 days post-boost the mice were skin challenged with a 10 ⁇ LD90 of HSV-2 4674 to determine whether anti-HSV immunity had been compromised.
  • mice that were vaccinated with ⁇ gD-2::FL HA nOP and ⁇ gD-2::HL HA OPT are fully protected against HSV-2 challenge but do not form anti-HA IgGs (see FIG. 8 ).
  • Mice were prime-boost vaccinated on days 0 and 21 with a control VD60 cell lysate or 1 ⁇ 10 6 PFU of ⁇ gD-2::FL HA nOP, ⁇ gD-2::HL HA OPT, or ⁇ gD-2::RFP.
  • mice vaccinated with ⁇ gD-2::FL HA nOP and ⁇ gD-2::HL HA OPT were fully protected from challenge.
  • FIG. 8A At day 40 post-prime, mice were bled to look at serum antibodies.
  • ELISAs performed against soluble PR8 HA show the absence of HA-specific IgGs in mice.
  • FIG. 8B See FIG. 8B .
  • ⁇ gD-2::P CMV -HA viruses and HA expression It was investigated if it would be more immunogenic to insert non-chimeric HA genes into the ⁇ gD genome in cassettes containing a constitutive promoter and poly-adenylation sequence. To accomplish this, the synthesized PR8 HA genes were restriction cloned into expression plasmid pEGFP-N1 (Addgene). HA genes were inserted in between xbaI and HindIII sites and replaced EGFP in the plasmid.
  • mice were vaccinated with ⁇ gD-2::P CMV -FL HA nOP.
  • the mice were prime vaccinated with 5 ⁇ 10 6 PFU of each virus (See FIG. 18 legend for the different viruses used).
  • n 5 mice/group, except 10 mice for ⁇ gD-2::FL HA nOP.
  • Five (5) of the ⁇ gD-2::FL HA nOPmice were boosted with a stalk HA expressing ⁇ gD-2, 5 ⁇ g/mL of purified PR8 HA was used as the source of antigen.
  • a goat anti-mouse Ig Ab was used for the total Ab ELISA.
  • ⁇ gD-2::P CMV -HA recombinant viruses all express HA.
  • VD60 cells were infected with 3 MOI of ⁇ gD-2::P CMV -FL HA nOP, ⁇ gD-2::P CMV -HL HA nOP, or ⁇ gD-2::P CMV -HL HA OPT.
  • cells were harvested and stained for HSV-protein and HA expression.
  • HSV protein expression was measured using serum from mice vaccinated with ⁇ gD-2::RFP.
  • HA expression was measured using monoclonal anti-HA stalk IgG C179. Cells were stained with either cell permeable or cell impermeable methods.
  • Vero cells were infected with 0.01 MOI of ⁇ gD-2::P CMV -FL HA nOP or ⁇ gD-2::P EF1 ⁇ -RFP. At 120 hours post-infection, there were no signs of productive infection. Additionally, 5 C57BL/6 mice were given subcutaneous injections with 5 ⁇ 10 6 PFU of ⁇ gD-2::P CMV -FL HA nOP and monitored daily for one week. They showed no signs of disease or distress.
  • the transmitted/founder clone of Env gp145 was chosen, which lacks the cytoplasmic tail, from donor CH505 in the CHAVI001 acute HIV-1 infection cohort, as this well-characterized HIV-1 Glade C glycoprotein is thought to be representative of those that pass the bottleneck of infection in a region of high HIV prevalence (Liao, Lynch et al. 2013). HIV Env is not expressed particularly well in the context of natural infection and often not expressed well exogenously, so steps were investigated to enhance antigen expression in our construct.
  • Env gp145 was found to be incorporated more efficiently into virus like particles (VLPs) than full length, and replacing the Env signal peptide and transmembrane domains with corresponding domains from host proteins or other viral glycoproteins increased incorporation into VLPs further (Wang, Liu et al. 2007).
  • VLPs virus like particles
  • a chimera of the ectodomain of HIV Env with the signal peptide and transmembrane cytoplasmic tail of HSV-2 gD was constructed.
  • the signal peptide of HSV2 gD is 25 residues in length and the ectodomain is 306 residues total (Eisenberg, Long et al.
  • allelic exchange construct was generated by Gibson assembly (Gibson, Young et al. 2009). Briefly, oligonucleotide primers were synthesized to amplify from HSV2 strain G genomic DNA arms of homologous sequence ⁇ 800 bp 5′ to the 25th codon of HSV2 US6 and ⁇ 800 bp 3′ to the 306th codon of HSV2 US6. The insert was amplified from the CH505 TF gp145 expression plasmid HV1300631 (gift of Huaxin Liao, Duke University) with primers that encompassed the 30th codon to codon 680. The fragments were cloned into pUC19 between EcoRI and BamHI restriction sites.
  • ADCK Functional in vitro macrophage antibody-dependent cell-mediated killing
  • Fc ⁇ R-binding antibodies mediate killing of HSV-infected cells by binding antigens on infected cell and then binding and activating Fc ⁇ Rs on innate leukocytes. This precipitates antibody-dependent cell-mediated cytotoxicity and phagocytosis (ADCC and ADCP), here referred to as antibody-dependent cell-mediated killing (ADCK).
  • ADCC and ADCP antibody-dependent cell-mediated cytotoxicity and phagocytosis
  • ADCK antibody-dependent cell-mediated killing
  • Current assays face many limitations including, but not limited to, inflexible target and effector cell lines, artificial antigen presentation systems, indirect or separate outputs for ADCC and ADCP, and the use of cumbersome radioactive isotopes.
  • a quantitative in vitro assay was constructed to study ADCK in response to anti-HSV antibodies.
  • a ⁇ gD-2 variant 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 decreasing proportion of which was determined to be the result of cell killing.
  • FACS analysis was used to quantify the decreasing proportion of live-infected target cells after co-culture with macrophages.
  • ADCC rapid fluorometric ADCC
  • target cells are stained with a persistent membrane die and a live-dead marker that dissipates upon the initiation of apoptosis.
  • ADCC activity is then measured as the decrease in proportion of membrane dye + cells that are also live-dead + .
  • ADCP is measured by the proportion of macrophages that are marked by the phagocytosis of fluorescent cells.
  • ADCC and ADCP cannot be cleanly separated by these methods, so they are referred to collectively in this text as antibody-dependent cell-mediated killing (ADCK).
  • the prior art assays have some additional drawbacks, as they are usually restricted in their choice of antibody or effector cell.
  • a similar assay was used in Petro et al. 2015, but the ability of HSV to infect, replicate in, and kill both target and effector cells limited the utility of the assay for studying immune cells and their Fc ⁇ Rs (Petro et al., 2015).
  • ⁇ gD-2 is a single-cycle virus in non-complementing cells and was recently shown not to induce dendritic cell death in vitro. It was investigated whether a brightly fluorescent ⁇ gD-2 strain would allow for precise investigation of the cellular mechanisms of ADCK.
  • An RFP expressing ⁇ gD-2 ( ⁇ gD-2::RFP) strain was constructed and developed with an RFADCK assay that precisely measures the effector activity of macrophages on HSV-2 ⁇ gD-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 infectious environment, measures both ADCC and ADCP, and is profoundly flexible, allowing for the use of polyclonal animal sera and different cell lines and mouse strains.
  • BMDMs bone marrow-derived macrophages
  • Vero CCL-81; ATCC, Manassas, Va.
  • VD60 cells Vero cells containing multiple copies of gD-1 under the endogenous gene promoter
  • 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 the supernatant from L929 cell cultures (ATCC).
  • Guinea pig BMDMs were differentiated using recombinant human M-CSF (BioLegend, San Diego, Calif.).
  • Raw 264.7, J774.1, and HEK 293 cells (ATCC) were passaged in DMEM supplemented with 10% FBS and 1% Pen-strep.
  • VD60 cells were co-transfected with HSV-2 ⁇ gD:GFP ( ⁇ gD-2) genomic DNA and cosmid DNA containing 40 kB of the HSV-2 genome in which the US6 gene was replaced with tdtomato downstream of an EF1 ⁇ promoter.
  • Resultant virus was plaque purified three times using RFP expression as a marker of homologous recombination. The purified ⁇ gD-2::RFP virus was verified by PCR and sequencing.
  • ⁇ gD-2 and ⁇ gD-2::RFP were propagated on VD60 cell, which complement the gD deletion and allow for multiple rounds of replication. All viral strains were titered by serial dilution and propagation on their respective cell types.
  • mice Female C57BL/6 mice, aged 4-6 weeks, obtained from Jackson Laboratory (JAX, Bar Harbor, Me.) were used to obtain serum. Male C57BL/6 mice, aged 4-6 weeks, were obtained from JAX and bone marrow cell suspensions were isolated by flushing their femurs with DMEM supplemented with 10% PBS and 1% Pen-strep. Female Hartley guinea pigs, aged 5-6 weeks, were purchased from Charles River laboratories (Wilmington, Mass.). 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 Albert Einstein College of Medicine Institutional Animal Care and Use Committee
  • mice or guinea pigs were prime vaccinated subcutaneously with 5 ⁇ 10 6 plaque forming units (PFU) of HSV-2 ⁇ gD::RFP or an equal volume of VD60 cell lysate in phosphate-buffered saline (PBS) to a total volume of 200 ⁇ L. Animals were boosted with the same dose 21 days later.
  • PFU plaque forming units
  • PBS phosphate-buffered saline
  • BMDMs Bone Marrow Derived Macrophages
  • J774.1 macrophages J774.1 macrophages
  • Raw 264.7 macrophages were incubated in LPS (Sigma-Aldrich) for 12 hours prior to co-culture with HEK 293 cells (ATCC).
  • HEK 293 cells were double stained with PKH67 membrane (Sigma-Aldrich) and Tag-it VioletTM (Biolegend) dyes according to manufacturer's instructions.
  • HEK cells were infected with HSV-2 ⁇ gD::RFP at a MOI of 3 in serum free DMEM 4 hours before co-culture.
  • the infection media was removed after 3.5 hours and was replaced with a 1:5 dilution of heat-inactivated mouse or guinea pig serum collected at day 40 from mice prime-boost injected as described previously with either HSV-2 ⁇ gD::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, N.Y.). The co-cultures were incubated for 12 hours, then fixed and analyzed by flow cytometry on a LSRII (BD Biosciences, Franklin Lakes, N.J.).
  • Raw 264.7 macrophages and HEK 293 cells were co-cultured at a ratio of 10:1 on glass-bottom 96-well plates (Matrical Bioscience, Spokane Wash.) and imaged with an inverted NIKON Eclipse TiE microscope using NIS Elements software with deconvolution.
  • FIG. 14E but not Vero cells ( FIG. 14D ) at 12 hours post-infection, indicating that the virus maintained its single-cycle replication phenotype.
  • the introduction of pEF1 ⁇ ::RFP into the gD locus of ⁇ gD-2::GFP elicited robust RFP expression while maintaining the viral kinetics and single-cycle phenotype of the parental ⁇ gD-2 strain.
  • Serum from ⁇ gD-2::RFP vaccinated mice induces significant ADCK in the RFADCK assay.
  • an in vitro protocol was developed based on previous RFADCC assays.
  • Target HEK 293T cells were stained with a membrane dye and a live-dead marker and infected with ⁇ gD-2::RFP at an MOI of 3. Infected target cells were incubated for 3.5 hours before infection media was removed and media or mouse serum was added for an additional 30-minute incubation.
  • the infected HEK 293T cells were co-cultured for 12 hours with J774.1 murine macrophage cells which had been stimulated with LPS 12 hours prior. Based on previously reported assays, we used an effector to target cell ratio of 10:1. At this ratio, serum from ⁇ gD-2::RFP vaccinated mice induced killing of approximately 70.5% of highly infected (RFP high ) target cells ( FIG. 15B ). This was significantly more than the 49.3% killed in the absence of serum (p ⁇ 0.05). It was investigated whether only RFP high cells would significantly decrease, since RFP in the model is a surrogate for viral protein expression and cells expressing higher levels these proteins will bind more antibodies cross-link Fc ⁇ Rs, initiating ADCK.
  • Infected target cells were defined as double positive HEK293T cells that expressed RFP in cultures lacking effector cells.
  • RFP high cells were defined as cells expressing RFP above the mean intensity of infected target cells.
  • RFP mid cells were defined as all infected target cells expressing RFP below the mean intensity ( FIG. 15A ).
  • ADCK is significantly increased in the presence of serum from mice vaccinated with ⁇ gD-2::RFP compared to serum from mice vaccinated with a control cell lysate.
  • Infected HEK 293 cells were incubated with serum from mice vaccinated with either ⁇ gD-2::RFP or a control VD60 cell lysate to determine whether killing mediated by different immortalized macrophage cells lines was caused by antigen-specific ADCK.
  • the proportion of RFP high cells after 16 hours of co-culture was compared to that of parallel assays lacking serum.
  • ADCK assay was carried out as described above with the exceptions that glass-bottom 96-well plates were used and the effector cells were Raw 264.7 murine macrophage cells which have been shown to carry out similar amounts of ADCC and ADCP. The assays were imaged on a deconvolution microscope at 60 ⁇ magnification approximately every 15 minutes for 24 hours. Infected cells are RFP + . Uninfected HEK293 cells are highly Mem + and Live/Dead + . Macrophages are unstrained. FIG.
  • 15A indicates that different types of cell killing of infected cells (white arrows) by macrophages are observed at the indicated time points: Macrophages (*) cluster around the left infected cell over time, and the right infected cell undergoes apoptotic blebbing observed at 4.5 hours. These resulting blebs are quickly phagocytosed by macrophages, which fluoresce with RFP observed at 7.5-hours. The controlled apoptosis of this infected cell indicates that ADCC has occurred, but phagocytosis by, and subsequent fluorescence of, macrophages would be measured by flow analysis as ADCP.
  • ADCK activity by bone marrow-derived macrophages is Fc ⁇ R-dependent.
  • RFADCK assays were performed with bone marrow-derived macrophages (BMDMs) from wild-type and Fc ⁇ R ⁇ / ⁇ mice to determine whether observed ADCK was Fc ⁇ R dependent.
  • ADCK activity was defined as the decrease in RFP high cells compared to parallel cultures with serum from VD60 lysate mock-vaccinated mice.
  • wild-type BMDMs killed significantly more infected target cells than Fc ⁇ R ⁇ / ⁇ BMDMs in the presence of serum from HSV-2 ⁇ gD-2::RFP vaccinated mice (p ⁇ 0.05; FIG. 17A ).
  • RFADCK using guinea pig bone marrow-derived macrophages recapitulates murine results.
  • guinea pig BMDMs were co-cultured with ⁇ gD-2::RFP infected HEK 293 cells in the presence of serum from ⁇ gD-2 vaccinated, VD60 lysate mock vaccinated, or na ⁇ ve guinea pigs.
  • the killing of RFP high cells in parallel co-cultures containing serum from na ⁇ ve animals was used as the baseline.
  • guinea pig BMDMs carried out significant ADCK (p ⁇ 0.001; FIG. 17B ).
  • mice were primed with FL nOP and boosted with HL nOP. In each case, 5 ⁇ 10 6 PFU of virus were given with each injection.
  • mice were bled and serum was collected. Enzyme-linked immunosorbent assays (ELISAs) were then run to check for antibodies against purified PR8 HA or Vero cells infected with HSV-2 4674.
  • ELISAs Enzyme-linked immunosorbent assays
  • Mice prime-boosted with FL nOP or primed with FL nOP and boosted with HL nOP developed IgG1 and IgG2c antibodies against PR8 HA. No other group formed anti-PR8 IgG antibodies. All groups except the VD60 lysate group developed similar and high titers of anti-HSV IgGs. Anti-HSV IgGs anti-HSV IgGs were elicited with all the vaccine strains.
  • Recombinant gD-2::HA PR8 expresses high levels of PR8 protein.
  • ⁇ gD-2::RFP gDNA was co-transfected into VD60 cells alongside an HA expression cassette containing the hemagglutinin (HA) gene from IAV H1N1 strain A/Puerto Rico/1934/8 (PR8) downstream of P CMV and upstream of a poly-adenylation signal as illustrated in FIG. 22A .
  • Extracellular and intracellular HA expression was measured by flow cytometry in Vero and VD60 cells infected with 3 MOI of ⁇ gD-2, ⁇ gD-2::HA PR8 , or ⁇ gD-2 containing a truncated version of the PR8 HA expression cassette ( ⁇ gD-2::HL HA PR8 ), and HA expression is shown in FIG. 22B .
  • mice immunized with gD-2::HA PR8 develop high titers of functional and isotype switched anti-PR8 antibodies.
  • Mice were prime-boost vaccinated 21 days apart with either VD60 cell lysate, ⁇ gD-2::RFP vector, or ⁇ gD-2::HA PR8 (5 mice per group).
  • serum was collected for analysis.
  • Anti-PR8 antibodies were measured by ELISA against purified HA PR8 protein.
  • Mice immunized with ⁇ gD-2::HA PR8 developed isotype switch anti-PR8 HA antibodies that were predominantly IgG2c and IgG2b. (See FIGS.
  • mice immunized with gD-2::HA PR8 develop protection against IAV challenge.
  • Mice were prime-boost immunized 21 days apart with VD60 cell lysate, ⁇ gD-2, or ⁇ gD-2::HA PR8 , bled at day 28 post-prime, and challenged intranasally 14 days later with a 6 ⁇ LD 50 of IAV.
  • Mice were sacrificed after reaching 70% of starting weight and neutralization titers were measured using microneutralization assays against the respective strains. As shown in FIGS.
  • the PCR fragment product which also contained bGH polyA signal and T7 promoter, was cloned into the multiple cloning site (MCS) of the pBkk412 plasmid under control of CMV promoter, using RE sbf I and blp I. (See FIG. 28 )
  • FIG. 29A Stable, competent E. coli cells were transformed and plated on Agar+ carbenicillin. Colonies were screened for the presence of the insert using PCR ( FIG. 29A ), and were used to prepare DNA for analysis. The DNA was amplified by PCR and clones (CL 15-6 and CL 26-11) having the correct size were selected for transient transfection to assess HIV-1 protein expression. ( FIG. 29B )
  • cells will be co-transfected with the linear recombinant plasmid and the HSV ⁇ gD-2 negative virus (e.g., ⁇ gD-2::RFP, ⁇ gD-2::GFP) and selected for recombinants that express the HIV protein and do not express the GFP or RFP marker.
  • HSV ⁇ gD-2 negative virus e.g., ⁇ gD-2::RFP, ⁇ gD-2::GFP
  • Expression of the HIV protein(s) by the new recombinant virus will be assessed by Western blot or flow cytometry (FACS). Mice will be immunized intramuscularly (prime and boost) and serum collected and screen for the presence of HIV-specific antibodies. The functionality (e.g., neutralizing and non-neutralizing functions) of the Abs will also be measured.
  • a humanized mouse model which renders mice susceptible to infection with HIV, will also be used to assess whether the vaccine protects the mice from HIV infection.
  • 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 fully or partially deleted in a gene encoding HSV-2 glycoprotein D, and (ii) a nucleic acid comprising a promoter-FP construct, wherein FP is a nucleic acid encoding a fluorescent protein; b) co-transfecting a host cell with (i) the HSV-2 genome of a) and (ii) a linear DNA fragment encoding the heterologous antigen under conditions whereby allelic recombination occurs between the HSV-2 genome and the DNA fragment; c) screening plaques resulting from b) to identify plaques not showing fluorescence under excitation light which elicits fluorescent protein fluorescence; d) recovering from those plaques not showing fluorescence in c) recombinant HSV-2 viruses or virions so as to obtain a vaccine vector directed against the heterologous antigen.
  • Embodiment 2 The process of embodiment 1, wherein the host cell is a HSV-1 glycoprotein D complementing cell.
  • Embodiment 3 The process of embodiment 1 or embodiment 2, wherein the promoter of the promoter-FP construct is a heterologous promoter.
  • Embodiment 4 The process of any of Embodiments 1-3, wherein the co-transfecting is effected by electroporation.
  • Embodiment 5 The process of any of Embodiments 1-4, wherein the fluorescent protein is Red Fluorescent Protein (RFP).
  • RFP Red Fluorescent Protein
  • Embodiment 6 The process of any 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) HSV-2 gD signal sequence, the heterologous antigen, HSV-2 gD transmembrane domain, HSV-2 gD cytosolic domain, but not encoding a HSV-2 gD extracellular domain, or (ii) HSV-2 gD signal sequence, the heterologous antigen, and cytosolic domain of HSV-2 gD.
  • Embodiment 7 The process of any 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 poly-A signal.
  • Embodiment 8 The process of any of Embodiments 1-7, wherein the heterologous antigen is an influenza antigen.
  • Embodiment 9 The process of any of Embodiments 1-8, wherein the heterologous antigen is an influenza hemagglutinin (HA) antigen.
  • HA hemagglutinin
  • Embodiment 10 The process of Embodiment 9, wherein the HA antigen is a full-length HA extracellular domain or is a HA stalk.
  • Embodiment 12 The process of Embodiment 11, wherein the HIV antigen is an Env gp145.
  • Embodiment 14 The process of any of Embodiments 1-13, wherein the promoter is a promoter of Elongation Factor 1a gene (P EF1 ⁇ ) and wherein P EF1 ⁇ and FP are fused together (P EF1 ⁇ -FP).
  • the promoter is a promoter of Elongation Factor 1a gene (P EF1 ⁇ ) and wherein P EF1 ⁇ and FP are fused together (P EF1 ⁇ -FP).
  • Embodiment 15 A vaccine vector, or a recombinant herpes simplex virus-2 (HSV-2) comprising a genome encoding a heterologous antigen, made by the process of any of Embodiments 1-14.
  • HSV-2 herpes simplex virus-2
  • Embodiment 16 A recombinant herpes simplex virus-2 (HSV-2) comprising: (i) a complete deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof; and (ii) (a) a linear DNA fragment, encoding a promoter, a heterologous antigen signal sequence, and a heterologous antigen or (b) encoding a promoter, and a heterologous antigen.
  • HSV-2 herpes simplex virus-2
  • Embodiment 17 A recombinant herpes simplex virus-2 (HSV-2) comprising: (i) a partial deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof; and (ii) (a) a linear DNA fragment encoding, in order, an HSV-2 gD signal sequence, a heterologous antigen, an HSV-2 gD transmembrane domain, optionally an HSV-2 gD cytosolic domain, but not encoding an HSV-2 gD extracellular domain, or (b) a linear DNA fragment encoding, in order, an HSV-2 gD signal sequence, a heterologous antigen, and transmembrane cytoplasmic tail of HSV-2 gD.
  • HSV-2 herpes simplex virus-2
  • Embodiment 18 The recombinant HSV-2 of Embodiment 16 or 17, further comprising a parasitic surface glycoprotein on a lipid bilayer thereof, wherein the parasite is a parasite of a mammal.
  • Embodiment 19 The recombinant HSV-2 of any of Embodiments 16-18, wherein the HSV-2 glycoprotein D-encoding gene is an HSV-2 U S 6 gene.
  • Embodiment 20 The recombinant HSV-2 of any of Embodiments 16-19, wherein the heterologous antigen is an influenza antigen.
  • Embodiment 21 The recombinant HSV-2 of any of Embodiments 16-20, wherein the heterologous antigen is hemagglutinin (HA) antigen.
  • HA hemagglutinin
  • Embodiment 22 The recombinant HSV-2 of Embodiment 21, wherein the HA antigen is a full-length HA extracellular domain or is a HA stalk.
  • Embodiment 23 The recombinant HSV-2 of any of Embodiments 16-19, wherein the heterologous antigen is an HIV antigen.
  • Embodiment 24 The recombinant HSV-2 of Embodiment 23, wherein the HIV antigen is an Env gp145.
  • Embodiment 25 A cell comprising therein a recombinant virus of any of Embodiments 16-24, wherein the cell is not present in a human being.
  • Embodiment 26 A vaccine composition comprising the recombinant virus of any of Embodiments 16-24.
  • Embodiment 27 A pharmaceutical composition comprising the virus of any of Embodiments 16-24, and a pharmaceutically acceptable carrier.
  • Embodiment 28 A method of eliciting and/or enhancing an immune response in a subject, the method comprising administering to the subject an amount of the recombinant virus of any of Embodiments 16-24, in an amount effective to elicit and/or enhance an immune response in a subject.
  • Embodiment 29 A method of eliciting 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 elicit and/or enhance an immune response in a subject.
  • Embodiment 30 A method of eliciting 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 elicit and/or enhance an immune response in a subject.
  • Embodiment 31 A method of treating or reducing the likelihood of an influenza infection in a subject, the method comprising administering to the subject an amount of the recombinant virus of any of Embodiments 16-22, in an amount effective to treat or reduce the likelihood of an influenza infection in a subject.
  • Embodiment 32 A method of treating or reducing the likelihood of an 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 an influenza infection in a subject.
  • Embodiment 33 A method of treating or reducing the likelihood of an 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 an influenza infection in a subject.
  • Embodiment 34 A method of treating or reducing the likelihood of an HIV infection in a subject, the method comprising administering to the subject an amount of the recombinant virus of any of Embodiments 16-19, 23 or 24, in an amount effective to treat or reduce the likelihood of an HIV infection in a subject.
  • Embodiment 35 A method of treating or reducing the likelihood of an 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 an HIV infection in a subject.
  • Embodiment 36 A method of treating or reducing the likelihood of an 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 an HIV infection in a subject.
  • Embodiment 37 A method of vaccinating a subject for influenza infection, the method comprising administering to the subject an amount of the recombinant virus of any of Embodiments 16-22, in an amount effective to vaccinate a subject for influenza infection.
  • Embodiment 38 A method of vaccinating a subject for influenza infection, the method comprising administering to the subject an amount of the vaccine of Embodiment 26, in an amount effective to vaccinate a subject for influenza infection.
  • Embodiment 39 A method of vaccinating a subject for influenza infection, the method comprising administering to the subject an amount of the pharmaceutical composition of Embodiment 27, in an amount effective to vaccinate a subject for influenza infection.
  • Embodiment 40 A method of vaccinating a subject for HIV infection, the method comprising administering to the subject an amount of the recombinant virus of any of Embodiments 16-19, 23 or 24, in an amount effective to vaccinate a subject for HIV infection.
  • Embodiment 41 A method of vaccinating a subject for HIV infection, the method comprising administering to the subject an amount of the vaccine of Embodiment 26, in an amount effective to vaccinate a subject for HIV infection.
  • Embodiment 42 A method of vaccinating a subject for HIV infection, the method comprising administering to the subject an amount of the pharmaceutical composition of Embodiment 27, in an amount effective to vaccinate a subject for HIV infection.
  • Embodiment 43 A method of eliciting 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 of Embodiments 1-14 and comprising (i) a complete deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof and (ii) encoding a promoter, an influenza hemagglutinin (HA) antigen signal sequence, and an HA antigen in an amount effective to elicit and/or enhance an immune response in a subject.
  • HSV-2 herpes simplex virus-2
  • Embodiment 44 A method of treating or reducing the likelihood of an 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 of Embodiments 1-13 and comprising (i) a complete deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof 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 an influenza infection in a subject.
  • HSV-2 herpes simplex virus-2
  • Embodiment 45 A method of vaccinating a subject for influenza infection, the method comprising administering to the subject an amount of a recombinant herpes simplex virus-2 made by the process of any of Embodiments 1-14 and comprising (i) a complete deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof and (ii) encoding a promoter, an influenza hemagglutinin (HA) antigen signal sequence, and an HA antigen in an amount effective to vaccinate a subject for influenza infection.
  • a recombinant herpes simplex virus-2 made by the process of any of Embodiments 1-14 and comprising (i) a complete deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof and (ii) encoding a promoter, an influenza hemagglutinin (HA) antigen signal sequence, and an HA antigen in an amount effective to vaccinate a subject for influenza infection.
  • Embodiment 46 The method of any of Embodiments 43-45, wherein the HA antigen is a full-length HA extracellular domain.
  • Embodiment 47 The method of Embodiment 46, further comprising, subsequent to an initial administration of the recombinant herpes simplex virus-2 encoding a full-length HA extracellular domain, administering one or more amounts of a recombinant herpes simplex virus-2 comprising (i) a complete deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof and (ii) encoding a promoter, a HA antigen signal sequence, and an HA stalk, but not encoding a full-length HA.
  • Embodiment 48 The method of any of Embodiments 28-47, wherein the subject is a human being.
  • Embodiment 49 A method of quantitating a rate or amount of antibody-dependent cell-mediated killing (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 that comprises a genome deleted for the gene encoding HSV-2 gD, under conditions permitting 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 quantitating at one or more time points the amount of the plurality of infected cells exhibiting fluorescent protein fluorescence and, optionally, one or more markers, so as to quantitate over time the amount of live infected cells, so as to thereby quantitating the rate or amount of ADCK in the population of cells.
  • ADCK antibody-dependent cell-mediated killing
  • Embodiment 50 The method of Embodiment 49, wherein the method is performed in vitro.
  • Embodiment 51 The method of Embodiment 49 or 50, wherein the population of immune cells comprises macrophages.
  • Embodiment 52 The method of any of Embodiments 49-51, wherein the antibody-containing solution comprises serum.
  • Embodiment 53 The method of any of Embodiments 49-51, wherein the fluorescent protein is Red Fluorescent Protein.
  • Embodiment 54 The method of any of Embodiments 49-53, wherein the amount of the plurality of infected cells exhibiting fluorescent protein fluorescence and, optionally, one or more markers, is measured by fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • Embodiment 55 The method of any of Embodiments 49-55, wherein the at least one marker comprises a cell membrane marker, a live/dead marker, or a combination thereof.
  • Embodiment 56 The method of any of Embodiments 49-55, further comprising quantitating at one or more time points the amount of cells exhibiting fluorescent protein fluorescence and, optionally, one or more markers, in a control population of infected cells otherwise identical but not contacted with an antibody-containing solution and comparing the amount or rate quantitated to the amount or rate quantitated for the population of cells contacted with the antibody-containing solution.
  • Embodiment 57 The method of any of Embodiments 49-56, wherein the population of immune cells is present at effector:target ratio of 5:1 or greater.
  • Embodiment 58 The method of any of Embodiments 49-57, wherein the recombinant HSV-2 is made by the process of any of Embodiments 1-14.

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