HSV-2-delta-gD vaccines and methods for their production and use

1. A process for producing a recombinant Herpes simplex virus-2 (HSV-2) vaccine vector directed against a heterologous antigen, the process comprising:

a) providing a HSV-2 delta-gD2 genome with a deleted glycoprotein D gene (delta gD-2), wherein the deleted gD-2 gene is replaced with a nucleic acid comprising a promoter-red fluorescent protein (RFP) construct, wherein RFP is a nucleic acid encoding a red fluorescent protein;

b) co-transfecting a eukaryotic host cell which phenotypically complements HSV-2 delta-gD-2 by expressing glycoprotein D from HSV-1 (gD-1) on its lipid bilayer with the following:

(i) the HSV-2 delta-gD2 genome of a) and

(ii) a linear DNA fragment encoding the heterologous antigen

wherein the linear DNA fragment consists of an expression cassette comprising one of the following in the following order: CMV promoter—the heterologous antigen—a polyadenylation signal, wherein the heterologous antigen is codon-optimized for HSV-2 expression;

wherein the linear DNA fragment is flanked on either side by regions homologous to the genomic HSV-2 regions which flank the promoter-RFP construct such that homologous recombination between genomic DNA and the DNA fragment results in the insertion of the expression cassette into the HSV-2 genome between the homologous regions and results in deletion of the promoter-RFP from the HSV-2 genome;

c) propagating the transfected cell of b) under conditions whereby homologous recombination occurs between the HSV-2 delta-gD2genome and the DNA fragment;

d) screening plaques resulting from c) to identify plaques not showing red fluorescence; and

e) recovering from those plaques not showing red fluorescence in d) a recombinant HSV-2 virus or virion to obtain the HSV-2 recombinant vaccine vector directed against the heterologous antigen.

2. The process of claim 1 , wherein the promoter of the promoter-FP construct is a promoter heterologous to the HSV-2.

3. The process of claim 1 , wherein the heterologous antigen is an influenza antigen, an HIV antigen, or a combination thereof, and wherein the influenza antigen is full-length extracellular domain of the hemagglutinin (HA) antigen or is the HA antigen stalk, and the HIV antigen is Env gp145 antigen.

4. The process of claim 1 , wherein the heterologous antigen is under control of an upstream CMV promoter and has a downstream SV40 poly-A signal.

5. The process of claim 1 , wherein the promoter of the promoter-RFP construct is a promoter of Elongation Factor 1a gene (PEF1α) and wherein PEF1α and FP are fused together (PEF1α-FP).

6. A recombinant herpes simplex virus-2 (HSV-2) vaccine vector comprising a genome encoding a heterologous antigen, made by the process of claim 1 .

7. The recombinant HSV-2 of claim 6 , wherein the heterologous antigen is an influenza antigen, an HIV antigen, or a combination thereof, wherein the influenza antigen is full-length extracellular domain of the hemagglutinin (HA) antigen or is the HA antigen stalk, and the HIV antigen is Env gp145 antigen.

8. A vaccine composition comprising the recombinant HSV-2 of claim 7 .

9. A pharmaceutical composition comprising the recombinant HSV-2 of claim 7 , and a pharmaceutically acceptable carrier.

10. A method of eliciting antibody-dependent cell-mediated cytotoxicity (ADCC) immunity against HSV1 and or HSV-2 and/or a heterologous antigen in a subject, the method comprising administering to the subject an amount of the recombinant HSV-2 of claim 7 effective to elicit the immune response in the subject.

11. A method of treating an influenza infection in a subject, the method comprising administering to the subject an amount of the recombinant HSV-2 of claim 7 effective to treat an influenza infection in the subject, wherein the heterologous antigen is an influenza antigen.

12. A method of treating an HIV infection in a subject, the method comprising administering to the subject an amount of the recombinant HSV-2 of claim 7 effective to treat an HIV infection in a subject, wherein the heterologous antigen is an HIV Env gp145 antigen.

13. A method of vaccinating a subject for influenza infection, the method comprising administering to the subject an amount of the recombinant HSV-2 of claim 7 effective to vaccinate the subject for influenza infection, wherein the heterologous antigen is an influenza HA antigen.

14. A method of vaccinating a subject for HIV infection, the method comprising administering to the subject an amount of the recombinant HSV-2 of claim 7 effective to vaccinate the subject for the HIV infection and, wherein the heterologous antigen is HIV Env gp145 antigen.

15. 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 red 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 phagocytic immune cells, and

quantitating at multiple time points the amount of infected cells among the plurality of infected cells, which exhibit fluorescent protein fluorescence that is greater than the mean intensity of fluorescent protein fluorescence of the plurality of infected cells, and, optionally, quantitating one or more cell viability markers, so as to quantitate over time the amount of live infected cells and thereby quantitate the rate or amount of ADCK in the population of cells.

16. The method of claim 15 , wherein the population of immune cells comprises macrophages.

17. The method of claim 15 , wherein the antibody-containing solution comprises serum.

18. The method of claim 15 , wherein the one or more markers comprises a cell membrane marker, a live/dead marker, or a combination thereof.

19. The method of claim 15 , 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.