# Patent

## Title
HSV-1 and HSV-2 vaccines and methods of use thereof

## Abstract

This invention provides methods of treating, suppressing, inhibiting, reducing an incidence, reducing the pathogenesis of, ameliorating the symptoms of, or ameliorating the secondary symptoms of a primary or recurring Herpes Simplex Virus (HSV) infection, or prolonging the latency to a relapse of an HSV infection, and disorders and symptoms associated with same and inducing an anti-HSV immune response in a subject comprising the step of contacting the subject with a composition comprising a mutant HSV strain comprising an inactivating mutation in a Us8 gene, followed by a second contacting with the composition.

## Background
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
The invention described herein was supported in whole or in part by grants from the National Institutes of Health (Grant No. R01AI33063). The United States government has certain rights in the invention.
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a National Phase Application of PCT International Application No. PCT/US10/29493, International Filing Date Mar. 31, 2010 which is a continuation-in-part of U.S. patent application Ser. No. 12/415,152, filed on Mar. 5, 2009, which are incorporated by reference herein in their entirety.


FIELD OF INVENTION
This invention provides methods of treating, suppressing, inhibiting, reducing an incidence, reducing the pathogenesis of, ameliorating the symptoms of, or ameliorating the secondary symptoms of a primary or recurring Herpes Simplex Virus (HSV) infection, or prolonging the latency to a relapse of an HSV infection, and disorders and symptoms associated with same and inducing an anti-HSV immune response in a subject comprising the step of contacting the subject with a composition comprising a mutant HSV strain comprising an inactivating mutation in a Us8 gene, followed by a second contacting with the composition
BACKGROUND OF THE INVENTION
Human infection with herpes simplex virus (HSV) type 1 or 2 is typically acquired through intimate contact and causes oral and genital lesions. HSV-1 usually causes oral ulcers and HSV-2 normally causes genital ulcers, but the reverse can also occur. A person infected with HSV-1 or HSV-2 will always be a carrier of the virus. After initial infection, lesions heal and HSV exists in a dormant, latent state in sensory neurons. Periodically, HSV reactivates from latently infected neurons and causes new ulcers to form at the skin surface. Newborn infants and immunosuppressed individuals are particularly vulnerable to HSV infection, often having a disseminated infection with fatal results. Ocular HSV infection, a leading cause of blindness, is another serious consequence of the virus. Furthermore, genital HSV infection results in a two-fold increase in HIV transmission rate. Therefore, a vaccine to prevent infection with and transmission of HSV is urgently needed.
SUMMARY OF THE INVENTION
This invention provides methods of treating, suppressing, inhibiting, reducing an incidence, reducing the pathogenesis of, ameliorating the symptoms of, or ameliorating the secondary symptoms of a primary or recurring Herpes Simplex Virus (HSV) infection, or prolonging the latency to a relapse of an HSV infection, and disorders and symptoms associated with same and inducing an anti-HSV immune response in a subject comprising the step of contacting the subject with a composition comprising a mutant HSV strain comprising an inactivating mutation in a Us8 gene, followed by a second contacting with the composition.
In one embodiment, the present invention provides a method of inducing an anti-Herpes Simplex Virus (HSV) immune response in a subject comprising the step of contacting a subject with a composition comprising a mutant HSV strain, wherein said mutant HSV strain comprises an inactivating mutation in a Us8 gene, followed by a second contacting of said composition comprising said mutant HSV strain.
In another embodiment, the present invention provides a method of treating a Herpes Simplex Virus (HSV) infection in a subject comprising the step of administering to said subject a composition comprising a mutant HSV strain, wherein said mutant HSV strain comprises an inactivating mutation in a Us8 gene, followed by a second administration of said composition comprising said mutant HSV strain.
In another embodiment, the present invention provides a method of suppressing, inhibiting, or reducing an incidence of a Herpes Simplex Virus (HSV) infection in a subject comprising the step of administering to said subject a composition comprising a mutant HSV strain, wherein said mutant HSV strain comprises an inactivating mutation in a Us8 gene, followed by a second administration of said composition comprising said mutant HSV strain.
In another embodiment, the present invention provides a method of reducing the pathogenesis of, ameliorating the symptoms of, ameliorating the secondary symptoms of, or prolonging the latency to a relapse of a Herpes Simplex Virus (HSV) infection in a subject, comprising the step of administering to said subject a composition comprising a mutant HSV strain, wherein said mutant HSV strain comprises an inactivating mutation in a Us8 gene, followed by a second administration of said composition comprising said mutant HSV strain.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. HSV spread in neurons.
FIG. 2. Typical HSV-1 infection of a mouse flank.
FIG. 3. Spectrum of skin disease in the mouse flank/vaccination model.
FIG. 4. Survival of mice following infection with HSV-1(gE null) vaccine or virulent HSV-1(Rescue gE null).
FIG. 5. Primary skin disease after infection with HSV-1(gE null) vaccine or virulent HSV-1(Rescue gE null).
FIG. 6. Secondary (zosteriform) skin disease after infection with HSV-1(gE null) vaccine or virulent HSV-1(Rescue gE null).
FIG. 7. Viral titers in skin after infection with vaccine or virulent HSV-1.
FIG. 8. HSV detection in skin after infection with vaccine or wild-type HSV-1.
FIG. 9. Viral titers in dorsal root ganglia (DRG) after infection with vaccine or virulent HSV-1.
FIG. 10. Survival of vaccinated or mock-vaccinated mice following challenge with 105 pfu of WT HSV-1 (NS).
FIG. 11. Primary skin disease scores in vaccinated mice challenged with 105 pfu of WT HSV-1 (NS).
FIG. 12. Viral titers in skin in vaccinated mice challenged with 105 pfu of WT HSV-1 (NS).
FIG. 13. HSV detection in skin of mock-vaccinated or vaccinated mice, challenged with 105 pfu of WT HSV-1 (NS).
FIG. 14. Secondary skin disease in vaccinated mice challenged with 105 pfu of WT HSV-1 (NS). N=3.
FIG. 15. Viral titers of ganglia from mock-vaccinated or vaccinated mice, challenged with 105 pfu of WT HSV-1 (NS). N=3.
FIG. 16. Vaccine protects ganglia from latent infection.
FIG. 17. Cross protection of mice vaccinated with 5×105 pfu HSV-1ΔgE against flank challenge with 105 pfu HSV-2(2.12). “Mock Vac” denotes mock vaccination; “DgE Vac” denotes vaccination with 5×105 pfu HSV-1ΔgE. Error bars represent the Standard Error of the Mean (“SEM”).
FIG. 18. Protection of mice vaccinated with 5×105 pfu HSV-1ΔgE against latency following flank challenge with 5×105 pfu HSV-1(KOS). Error bars represent the SEM.
FIG. 19. Protection of mice vaccinated with 5×105 pfu HSV-1ΔgE against death, visible Disease and Extensive Viral Replication Following Vaginal Challenge with 105 pfu HSV-1(NS). A. Top panel—survival curves; bottom panel—viral titer as assessed by vaginal swabs. B. Photographs of mice on day 8 post-challenge. Error bars: SEM.
FIG. 20. Protection of mice vaccinated with 5×105 pfu HSV-1ΔgE by different routes (“Ep. Scar.”: epidermal scarification; “SubQ”: subcutaneously; IM: intramuscular) against flank challenge with 105 pfu HSV-1(NS). “DgE Vac” denotes HSV-1ΔgE; Error bars represent the SEM.
FIG. 21. Induction of neutralizing antibody response in mice vaccinated with 5×105 pfu HSV-1ΔgE by different routes. Percentages depicted are compared to serum from mock-vaccinated mice. n=3 (Ep. Scar & Sub Q), n=2 (IM), assays were done in duplicate. Error bars represent the SEM.
FIG. 22. Protection of mice vaccinated with 5×105 pfu HSV-1ΔgE against flank challenge with 105 pfu HSV-1(NS, F or 17). Error bars represent the SEM.
FIG. 23. Protection of mice vaccinated with 5×105 pfu HSV-1ΔgE against flank challenge with 105-7 pfu HSV-1(NS). Error bars represent the SEM.
FIG. 24. A. Western blot to detect gD (Us6) and gI (Us7) in infected cell extracts. B. Stability of the KOS-gDA3C virus in vitro. An Ssp1 digest of a PCR-amplified gD gene fragment of KOS or KOS-gDA3C. C. Stability of the KOS-gDA3C mutant virus in vivo. A PCR-amplified gD fragment obtained from the DRG of KOS-gDA3C-infected mice were cut with Ssp1 or left uncut.
FIG. 25. Entry of KOS, rKOS-gDA3C and KOS-gDA3C virus into (A) Vero, (B) B78-H1, (C) A10 or (D) C10 cells. Results are the mean±SE of three separate infections each done in triplicate.
FIG. 26. Single-step (A, B) and multi-step (C, D) growth curves of KOS, rKOS-gDA3C and KOS-gDA3C performed in A10 (A, C) or C10 (B, D) cells. Results are the mean±SE of three separate infections.
FIG. 27. Disease in the murine flank model. Inoculation (A) and zosteriform (B) site disease scores in mice inoculated with 5×105 PFU of KOS, rKOS-gDA3C or KOS-gDA3C. Error bars represent SE. C. Photographs of mice flanks taken 10 days post-infection with KOS, rKOS-gDA3C, or KOS-gDA3C.
FIG. 28. Virus titers and genome copy numbers in DRG. DRG were dissected from mice infected with KOS, rKOS-gDA3C or KOS-gDA3C and assayed for virus titers (A) or viral genome copy number (B). Results represent the mean±SE.
FIG. 29. Prior infection with KOS-gDA3C protects against WT HSV-1 challenge. Results represent mean disease scores+SE at the inoculation (A) and zosteriform (B) sites from days 3-7 post-infection. DRG viral titers (C) and genome copy number (D) were measured 5 days post-challenge with NS. Results represent the mean±SE.
FIG. 30. Model for KOS-gDA3C infection in mice. KOS infects epithelial cells (E) and produces disease at the inoculation site. The virus spreads to neurons (N) in the DRG, replicates and spreads to adjacent neurons and then travels back to epithelial cells in the skin to cause zosteriform disease. KOS-gDA3C is impaired in entry and infects fewer epithelial cells, which results in fewer neurons becoming infected in the DRG. The defect in entry also reduces infection of adjacent neurons in the DRG and results in reduced zosteriform disease.
FIG. 31. A. Alignment of HSV-1(NS) gE (SEQ ID NO: 2) with HSV-2(HG52) gE (SEQ ID NO: 18). B. Alignment of HSV-2(2.12) gE (SEQ ID NO: 6) with HSV-2 (HG52) gE (SEQ ID NO: 18). C. Strategy for generation of gE-2 deletion.
FIG. 32. A schematic diagram of HSV-2ΔgE(gfp) deletion and insertion of gfp2 cassette under the control of a CMV promoter. The gfp2 cassette, which allows for screening of recombinant viruses by fluorescence, is inserted in the US8 reading frame just after the bases encoding amino acid 123. The portion of the US8 gene encoding the 156 c-terminal amino acids of gE remain but are not expressed. The flanking regions used for the recombination are indicated. The mutation was made in wild-type strain HSV-2(2.12).
FIG. 33. Characterization of HSV-2ΔgE(gfp) protein expression. Western blot of protein isolated from Vero cells infected with HSV-2(2.12)(WT) or HSV-2ΔgE(gfp) mutants and stained with HSV-2 antibodies raised against VP5, gE, US9 and gD.
FIG. 34. Growth of HSV-2ΔgE(gfp) in vitro. In vitro single-step growth kinetics of HSV-2ΔgE(gfp) and WT HSV-2 in vitro in both epithelial (Vero cells) (A) and primary neuronal (superior cervical ganglia from rat embryos) (B) cell lines. For growth curves, cells were infected at an MOI of 3.0 and the zero hour time-point collected immediately. After a 1 hour incubation, cells were acid washed with a pH 3.0 citrate buffer and the 1 hour time-point was collected. Samples collected were titered on Vero cells by plaque assay. (C) Plaque sizes in Vero cells were measured at 96 hpi. 25 plaques were averaged for each virus.










Statistics Table














Groups




FIG.
P value
different
Statistical Test
















34A-Vero single-
=0.2938
No
2-way ANOVA



step growth



34B-SCG single
=0.8907
No
t-test



step growth



34C-Plaque size
<0.0001
Yes
2-way ANOVA










FIG. 35. Mouse retinal infection with HSV-2ΔgE(gfp). Thin cryosections were made of retina (A) and optic nerve (B) following mouse retina infection with 4×105 PFU of either HSV-2(2.12) or HSV-2ΔgE(gfp). Representative immunofluoresence images from three mice per data point on days 3 and 5 are shown and use an anti-HSV-2 polyclonal (DAKO) and a Dapi nuclear stain.
FIG. 36. Anterograde and retrograde retinorecipient areas of the brain following HSV-2ΔgE(gfp) retina infection of the mouse. Mouse retinas were infected with 4×105 PFU HSV-2ΔgE(gfp) or HSV-2(2.12). Brains were cryosectioned and stained with anti-HSV-2 rabbit polyclonal primary antibody and goat anti-rabbit HRP conjugated secondary antibody. Representative sections are shown from infection of 3 mice per virus strain.
FIG. 37. Safety of HSV-2ΔgE(gfp) in the mouse flank model. One day prior to infection, hair was removed from the right flank of mice using clippers and depilatory cream (NairTM). The following day, mice were infected by scarification on denuded flank skin with 5×105 pfu HSV-2(2.12) or HSV-2ΔgE(gfp) by making 60 gentle scratches in several different directions on a 1 cm square area of the skin. Mice were monitored daily for (A) survival, (B) inoculation site disease and (C) zosteriform disease. Disease was scored on a scale of 0 (no disease) to 4 (most severe). There were 5 mice per group. (D) A representative photo from each group taken on day 7 post-inoculation is shown. The boxed area of each picture is the site of inoculation and the arrows indicate zosteriform disease. Statistics Table-2-Way ANOVA (comparisons with p<0.05 are considered significantly different)















FIG.
2.12 vs. ΔgE
ΔgE vs. Mock









37B-inoculation
P < 0.0001
p = 0.0046



site disease



37C-zosteriform
p < 0.0001
N/A



disease










FIG. 38. Virus yield of HSV-2ΔgE(gfp) in skin and dorsal root ganglion (DRG) following mouse flank scarification. One day prior to infection, hair was removed from the right flanks of mice using clippers and depilated with depilatory cream (NairTM). The following day, mice were infected by scarification on denuded flank skin with 5×105 pfu HSV-2(2.12) or HSV-2ΔgE(gfp) by making 60 gentle scratches in several different directions on a 1 cm square area of the skin. Mice were sacrificed at intervals to evaluate viral titers in the skin at the site of inoculation. Skin at the site of inoculation (A) and DRG (B) were removed from groups of mice (n=3) on days 1, 3, 6 and 8. Tissues were pulverized and the viral content was quantified by plaque assay. p<0.0001 for skin titers and p=0.0006 for DRG titers. There were 3 mice for each data point. The limit of detection for the titering assay was 5 pfu.
FIG. 39. Safety and virus yield of HSV-1 and HSV-2 WT and vaccine strains in the mouse vaginal model. HSV. Pathogenicity (A), safety (B), and replication (C) of HSV-1 and HSV-2 wild-type and vaccine strains were assessed in the mouse vaginal model. Mice were treated with Depo Provera and infected 5 days later with 5×105 pfu of each virus. Mice were monitored daily for survival and scored for disease. The limit of detection for the titering assay was 20 pfu. There were 5 mice per group. Statistics Table-2-Way ANOVA (comparisons where p<0.05 are considered significantly different).















FIG.
NS vs. gEnull
2.12 vs. ΔgE-2









39B-disease scores
P < 0.0001
P < 0.0001



39C-swab titers
p = 0.0053
p = 0.0004










FIG. 40. Intramuscular (IM) and subcutaneous (subQ) HSV-2ΔgE(gfp) vaccine efficacy following challenge with HSV-2(MS) in the mouse flank model; protection against death and disease. Mice were vaccinated with 5×105 pfu HSV-2ΔgE(gfp) or mock vaccinated either intra-muscularly (IM) in the right rear thigh or subcutaneously (SubQ) in the neck scruff. One day prior to challenge, hair was removed from the right flanks of mice using clippers and depilated with depilatory cream (NairTM). The following day, 28 days after vaccination, mice were challenged by flank scarification on denuded flank skin with 5×105 pfu (1,736 LD50s) HSV-2(MS) by making 60 gentle scratches in several different directions on a 1 cm square area of the skin. Mice were monitored daily for survival (A), inoculation site disease (B) and zosteriform disease (C). Disease was scored on a scale of 0 (no disease) to 4 (most severe). There were 5 mice per group. Statistics Table-2-Way ANOVA (comparisons where p<0.05 are considered significantly different).

















IM vs.

IM vs.
SubQ vs



IM vs.
mock
SubQ vs.
mock
mock


FIG.
SubQ
vac
mock vac
chall
chall







40B-
p =
p =
P < 0.0001
p =
p =


inoculation site
0.8732
0.0335

0.9925
0.0789


disease


40C-
p =
p =
p = 0.0112
p =
p =


zosteriform
0.9999
0.0012

0.99993
0.9456


disease









FIG. 41. HSV-2ΔgE(gfp) vaccine efficacy (IM) following HSV-2(MS) challenge in the mouse flank model; protection of tissues. Mice were vaccinated IM with 5×105 pfu HSV-2ΔgE(gfp) or mock vaccinated. 28 days later, mice were challenged by flank scarification on denuded flank skin with 5×105 pfu (1,736 LD50s) HSV-2(MS). Skin at the site of inoculation and DRG were removed from groups of mice (n=3) on days 1, 3, 6 and 8. Tissues were pulverized and the viral content was quantified by plaque assay. Mice were sacrificed at intervals to evaluate viral titers in the skin at the site of inoculation (A) and the DRG (B). There were 3 mice for each data point. The limit of detection for the titering assay was 5 pfu. Statistics Table-2-Way ANOVA (comparisons where p<0.05 are considered significantly different).














FIG.
p =









41A-skin titers
P < 0.0001



41B-DRG titers
p = 0.0011










FIG. 42. Vaccine efficacy (SubQ/IM) following challenge with 50 LD50s of HSV-2(MS) in the mouse vaginal model. Mice were vaccinated IM or SubQ with 5×105 pfu HSV-2ΔgE(gfp) or mock vaccinated. Five days prior to challenge, mice were treated with Depo Provera. Twenty-eight days following vaccination, mice were challenged by vaginal instillation of 250 pfu (50 LD50s) HSV-2(MS). Mice were monitored daily for disease and survival. Disease was scored on a scale of 0 (no disease) to 4 (most severe disease). Vaginal swab samples were collected daily and assayed by plaque assay to quantify virus. Mice were monitored daily for survival (A) and scored for disease (B). Mice were swabbed intra-vaginally on days 1-7 (C). The limit of detection for the titering assay was 20 pfu. There were 5 mice per group. Statistics Table-2-Way ANOVA (comparisons where p<0.05 are considered significantly different).













FIG.
IM vs. SubQ
IM vs. mock
SubQ vs. mock







42B-Disease Scores
p = 0.0099
P < 0.0001
P < 0.0001


42C-Swab Titers
p = 0.0005
P < 0.0001
P < 0.0001









FIG. 43. Protection against vaginal challenge with a 104 LD50 dose of HSV-2(MS) after one or two doses of HSV-2ΔgE(gfp). Mice were vaccinated IM in the right hind leg gastrocnemius muscle with either one or two doses (three weeks apart) of 5×105 pfu HSV-2ΔgE(gfp) or mock vaccinated. Five days prior to challenge, mice were treated with Depo Provera. Twenty-eight days following the date of the second vaccination, mice were challenged by vaginal instillation of 5×104 pfu (104 LD50s) HSV-2(MS) Mice were monitored daily for survival (A) and scored for disease (B). Disease was scored on a scale of 0 (no disease) to 4 (most severe disease). Vaginal swab samples were collected daily and assayed by plaque assay to quantify virus (C). n=5 per group. The limit of detection for the titering assay was 20 pfu. There were 5 mice per group. (D) Photos from each mouse taken on day 7 post-inoculation are shown. Scores given for each mouse on day 7 are indicated below each photo. Statistics Table-2-Way ANOVA (comparisons where p<0.05 are considered significantly different).













FIG.
1X vs. 2X
1X vs. mock
2X vs. mock







43B-Disease Scores
p = 0.1024
P < 0.0001
P < 0.0001


43C-Swab Titers
P < 0.0001
P < 0.0001
P < 0.0001









FIG. 44. Shows safety evaluation of HSV-2ΔgE(gfp) in BALB/c and SCID mice following intramuscular (IM), intravenous (IV), intracranial, and intravaginal inoculation.
FIG. 45. Shows antibody response to HSV-2 gD measured by ELISA after one or two immunizations with HSV-2ΔgE(gfp).
FIG. 46. Shows antibody response to HSV-2 gC measured by ELISA after one or two immunizations with HSV-2ΔgE(gfp).
FIG. 47. Shows neutralizing antibody response after one or two immunizations with HSV-2ΔgE(gfp).
FIG. 48. Shows HSV-2ΔgE(gfp) administered as a prophylactic vaccine at varying immunizing doses in female BALB/c mice. A. shows survival of animals following administration of varying doses of the gE2-null strains and mock control. B. Animals were scored for vaginal disease on a scale of 0-4, where 0 is no disease, and one point was assigned for each of the following: erythema/swelling, exudate, hair loss in the perineal area, and ulcers or necrosis in the vaginal area. C. Animals were evaluated for vaginal titers. D. Animals were evaluated for viral titers or viral DNA in dorsal root ganglia (DRG) 4 days post-infection or 35 days post-infection (labeled as latent viral load).
FIG. 49. Assessment of DRG at day 35 for wild-type or vaccine strain DNA.
FIG. 50. Evaluation of HSV-2ΔgE(gfp) as a prophylactic vaccine in Hartley Strain guinea pigs. A. mock immunized guinea pigs challenged with 5×103 or 5×105 PFU. B. Vaginal disease scores in mock immunized animals and in animals immunized with HSV-2ΔgE(gfp). C. Vaginal titers in mock immunized animals and in animals immunized with HSV-2ΔgE(gfp). D. Shows the number of recurrences and the number of animals having a recurrence between days 15-49 post-infection. E. real-time qPCR for HSV-2 DNA.
FIG. 51. Immunization with HSV-2ΔgE(gfp) as a therapeutic vaccine to treat recurrent infections in guinea pigs. A. ELISA of anti-gC-2 antibodies. B. ELISA of anti-gD-2 antibodies. C. guinea pigs immunized with HSV-2ΔgE(gfp) or mock immunized. D. real-time qPCR for HSV-2 DNA.



DETAILED DESCRIPTION OF THE INVENTION
This invention provides methods of vaccinating a subject against Herpes Simplex Virus (HSV) infection and disorders and symptoms associated with same, and impeding, inhibiting, reducing the incidence of, and suppressing HSV infection, neuronal viral spread, formation of zosteriform lesions, herpetic ocular disease, herpes-mediated encephalitis, and genital ulcer disease in a subject, comprising the step of contacting the subject with a mutant strain of the HSV, containing an inactivating mutation in a gene encoding a gE, gI, Us9, other protein, or combinations thereof. In another embodiment, the mutant strain of the HSV, comprises an inactivating mutation in a gene encoding gE, which in one embodiment, is a gE null mutation. In another embodiment, the present invention provides pharmaceutical compositions comprising a mutant strain of HSV which comprises an inactivating mutation in a gene encoding gE, which in one embodiment, is a gE null mutation. In another embodiment, this invention provides pharmaceutical compositions comprising a mutant strain of HSV which comprises an inactivating mutation in a gene encoding a gE, gI, Us9, other protein, or combinations thereof.
In one embodiment, the present invention provides a method of inducing an anti-Herpes Simplex Virus (HSV) immune response in a subject comprising the step of contacting a subject with a composition comprising a mutant HSV strain, wherein said mutant HSV strain comprises an inactivating mutation in a Us8 gene, followed by a second administration of said composition comprising said mutant HSV strain.
In another embodiment, the present invention provides a method of treating a Herpes Simplex Virus (HSV) infection in a subject comprising the step of administering to said subject a composition comprising a mutant HSV strain, wherein said mutant HSV strain comprises an inactivating mutation in a Us8 gene, followed by a second administration of said composition comprising said mutant HSV strain.
In another embodiment, the present invention provides a method of suppressing, inhibiting, or reducing an incidence of a Herpes Simplex Virus (HSV) infection in a subject comprising the step of administering to said subject a composition comprising a mutant HSV strain, wherein said mutant HSV strain comprises an inactivating mutation in a Us8 gene, followed by a second administration of said composition comprising said mutant HSV strain.
In one embodiment, the present invention provides a method of reducing the pathogenesis of, ameliorating the symptoms of, ameliorating the secondary symptoms of, or prolonging the latency to a relapse of a Herpes Simplex Virus (HSV) infection in a subject, comprising the step of administering to said subject a composition comprising a mutant HSV strain, wherein said mutant HSV strain comprises an inactivating mutation in a Us8 gene, followed by a second administration of said composition comprising said mutant HSV strain.
In another embodiment, the present invention provides a method of inducing an anti-Herpes Simplex Virus (HSV) immune response in a subject comprising the step of contacting a subject with a composition comprising a mutant HSV strain, wherein said mutant HSV strain comprises an inactivating mutation in a Us8 gene and wherein said composition is administered in a first inoculation or “priming inoculation” and in a second inoculation or “boosting inoculation”.
In another embodiment, the present invention provides a method of treating a Herpes Simplex Virus (HSV) infection in a subject comprising the step of administering to said subject a composition comprising a mutant HSV strain, wherein said mutant HSV strain comprises an inactivating mutation in a Us8 gene and wherein said composition is administered in a first inoculation or “priming inoculation” and in a second inoculation or “boosting inoculation”.
In another embodiment, the present invention provides a method of suppressing, inhibiting, or reducing an incidence of a Herpes Simplex Virus (HSV) infection in a subject comprising the step of administering to said subject a composition comprising a mutant HSV strain, wherein said mutant HSV strain comprises an inactivating mutation in a Us8 gene and wherein said composition is administered in a first inoculation or “priming inoculation” and in a second inoculation or “boosting inoculation”.
In another embodiment, the present invention provides a method of reducing the pathogenesis of, ameliorating the symptoms of, ameliorating the secondary symptoms of, or prolonging the latency to a relapse of a Herpes Simplex Virus (HSV) infection in a subject, comprising the step of administering to said subject a composition comprising a mutant HSV strain, wherein said mutant HSV strain comprises an inactivating mutation in a Us8 gene and wherein said composition is administered in a first inoculation or “priming inoculation” and in a second inoculation or “boosting inoculation”.
In one embodiment, the present invention provides a method of vaccinating a subject against an HSV infection, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein.
In another embodiment, the present invention provides a method of impeding HSV-1 infection in a subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein.
In another embodiment, the present invention provides a method of impeding HSV-2 infection in a subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein.
“HSV-1” refers, in one embodiment, to a Herpes Simplex Virus 1. In another embodiment, the term refers to a KOS strain. In another embodiment, the term refers to an F strain. In another embodiment, the term refers to an NS strain. In another embodiment, the term refers to a CL101 strain. In another embodiment, the term refers to a “17” strain. In another embodiment, the term refers to a “17+syn” strain. In another embodiment, the term refers to a MacIntyre strain. In another embodiment, the term refers to an MP strain. In another embodiment, the term refers to an HF strain. In another embodiment, the term refers to any other HSV-1 strain known in the art.
“HSV-2” refers, in one embodiment to a Herpes Simplex Virus 2. In another embodiment, the term refers to an HSV-2 333 strain. In another embodiment, the term refers to a 2.12 strain. In another embodiment, the term refers to an HG52 strain. In another embodiment, the term refers to an MS strain. In another embodiment, the term refers to an 186 strain. In another embodiment, the term refers to a G strain. In another embodiment, the term refers to any other HSV-2 strain known in the art.
In another embodiment, the present invention provides a method of impeding primary HSV infection in a subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In another embodiment, the present invention provides a method of impeding primary HSV-1 infection in a subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein In another embodiment, the present invention provides a method of impeding primary HSV-2 infection in a subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein
The terms “impeding HSV infection” and “impeding primary HSV infection” refer, in one embodiment, to decreasing the titer of infectious virus by 90%. In another embodiment, the titer is decreased by 50%. In another embodiment, the titer is decreased by 55%. In another embodiment, the titer is decreased by 60%. In another embodiment, the titer is decreased by 65%. In another embodiment, the titer is decreased by 70%. In another embodiment, the titer is decreased by 75%. In another embodiment, the titer is decreased by 80%. In another embodiment, the titer is decreased by 85%. In another embodiment, the titer is decreased by 92%. In another embodiment, the titer is decreased by 95%. In another embodiment, the titer is decreased by 96%. In another embodiment, the titer is decreased by 97%. In another embodiment, the titer is decreased by 98%. In another embodiment, the titer is decreased by 99%. In another embodiment, the titer is decreased by over 99%.
In another embodiment, the terms refer to decreasing the extent of viral replication by 90%. In another embodiment, replication is reduced by 50%. In another embodiment, replication is reduced by 55%. In another embodiment, replication is reduced by 60%. In another embodiment, replication is reduced by 65%. In another embodiment, replication is reduced by 70%. In another embodiment, replication is reduced by 75%. In another embodiment, replication is reduced by 80%. In another embodiment, replication is reduced by 85%. In another embodiment, replication is reduced by 92%. In another embodiment, replication is reduced by 95%. In another embodiment, replication is reduced by 96%. In another embodiment, replication is reduced by 97%. In another embodiment, replication is reduced by 98%. In another embodiment, replication is reduced by 99%. In another embodiment, replication is reduced by over 99%.
Methods for measuring HSV infection are well known in the art, and include, in one embodiment, determination of appearance and severity of skin lesions and viral-mediated illness (Examples 1 and 4). Other embodiments of methods for measuring viral infection are described, for example, in Burgos J S et al. (Herpes simplex virus type 1 infection via the bloodstream with apolipoprotein E dependence in the gonads is influenced by gender. J Virol. 2005 February; 79(3):1605-12) and Parr M B et al. (Intravaginal administration of herpes simplex virus type 2 to mice leads to infection of several neural and extraneural sites. J Neurovirol. 2003 December; 9(6):594-602). Other methods of determining the extent of HSV replication and HSV infection are well are described, for example, in Lambiase A et al. (Topical treatment with nerve growth factor in an animal model of herpetic keratitis. Graefes Arch Clin Exp Ophthalmol. 2007 May 4), Ramaswamy M et al. (Interactions and management issues in HSV and HIV coinfection. Expert Rev Anti Infect Ther. 2007 April; 5(2):231-43), and Jiang C et al. (Mutations that decrease DNA binding of the processivity factor of the herpes simplex virus DNA polymerase reduce viral yield, alter the kinetics of viral DNA replication, and decrease the fidelity of DNA replication. J Virol. 2007 April; 81(7):3495-502).
In one embodiment, vaccination with gE-null HSV strains of the present invention protects against subsequent infection with virulent HSV. In another embodiment, the vaccination protects against disease caused by virulent HSV. In another embodiment, the vaccine strain does not itself cause significant disease, which in one embodiment is herpes (Examples 1, 4, and 19), and, in another embodiment, the vaccine strain does not itself result in significant symptoms.
“Inactivating mutation” in gE refers, in one embodiment, to a mutation that abrogates HSV neuronal spread. In another embodiment, the terms refer to a mutation that abrogates cell-to-cell spread of HSV. In another embodiment, the terms refer to abrogation of spread along axons. In another embodiment, the spread is retrograde (defined herein below). In another embodiment, the spread is anterograde (defined herein below). In another embodiment, spread in both anterograde and retrograde directions is abrogated.
“Anterograde” refers, in one embodiment, to spread from ganglia to skin. In another embodiment, the term refers to spread from the cell body towards the axon. In another embodiment, the term refers to any other definition accepted in the art.
“Retrograde” refers, in one embodiment, to spread from the site of infection to ganglia. In another embodiment, the term refers to spread from the axon towards the cell body. In another embodiment, the term refers to any other definition accepted in the art.
In one embodiment, a “defect” or “deficiency” describes an impairment, which in one embodiment, refers to a 10%, 25%, 40%, 50%, 60%, 75%, or 90% decrease in a particular function.
In one embodiment, neuronal spread is decreased by 90%. In another embodiment, neuronal spread is decreased by 60%. In another embodiment, the reduction is 65%. In another embodiment, the reduction is 70%. In another embodiment, the reduction is 75%. In another embodiment, the reduction is 80%. In another embodiment, the reduction is 85%. In another embodiment, the reduction is 95%. In another embodiment, the reduction is 96%. In another embodiment, the reduction is 97%. In another embodiment, the reduction is 98%. In another embodiment, the reduction is 99%. In another embodiment, the reduction is over 99%.
In one embodiment, the term refers to abrogating the ability of gE protein to sequester host anti-HSV antibodies. In another embodiment, sequestration of anti-HSV antibodies by gE is reduced by 90%. In another embodiment, sequestration is reduced by 50%. In another embodiment, the reduction is 65%. In another embodiment, the reduction is 70%. In another embodiment, the reduction is 75%. In another embodiment, the reduction is 80%. In another embodiment, the reduction is 85%. In another embodiment, the reduction is 95%. In another embodiment, the reduction is 96%. In another embodiment, the reduction is 97%. In another embodiment, the reduction is 98%. In another embodiment, the reduction is 99%. In another embodiment, the reduction is over 99%.
In one embodiment, the term refers to abrogating the ability of gE protein to bind IgG monomers. In another embodiment, binding of IgG monomers by gE is reduced by 90%. In another embodiment, binding is reduced by 50%. In another embodiment, the reduction is 65%. In another embodiment, the reduction is 70%. In another embodiment, the reduction is 75%. In another embodiment, the reduction is 80%. In another embodiment, the reduction is 85%. In another embodiment, the reduction is 95%. In another embodiment, the reduction is 96%. In another embodiment, the reduction is 97%. In another embodiment, the reduction is 98%. In another embodiment, the reduction is 99%. In another embodiment, the reduction is over 99%.
In one embodiment, the term refers to abrogating the ability of gE protein to bind IgG complexes. In another embodiment, binding of IgG complexes by gE is reduced by 90%. In another embodiment, binding is reduced by 50%. In another embodiment, the reduction is 65%. In another embodiment, the reduction is 70%. In another embodiment, the reduction is 75%. In another embodiment, the reduction is 80%. In another embodiment, the reduction is 85%. In another embodiment, the reduction is 95%. In another embodiment, the reduction is 96%. In another embodiment, the reduction is 97%. In another embodiment, the reduction is 98%. In another embodiment, the reduction is 99%. In another embodiment, the reduction is over 99%.
In one embodiment, an inactivating mutation in gE comprises a deletion of amino acids 124-508. In another embodiment, an inactivating mutation in gE comprises a deletion of amino acids 110-500. In another embodiment, an inactivating mutation in gE comprises a deletion of amino acids 1-552. In another embodiment, an inactivating mutation in gE comprises a deletion of amino acids 1-50. In another embodiment, an inactivating mutation in gE comprises a deletion of amino acids 1-100. In another embodiment, an inactivating mutation in gE comprises a deletion of amino acids 1-250. In another embodiment, an inactivating mutation in gE comprises a deletion of amino acids 100-300. In another embodiment, an inactivating mutation in gE comprises a deletion of amino acids 1-400. In another embodiment, an inactivating mutation in gE comprises a deletion of amino acids 200-500. In another embodiment, an inactivating mutation in gE comprises a deletion of amino acids 24-71. In another embodiment, an inactivating mutation in gE comprises a deletion of amino acids 30-508. In another embodiment, an inactivating mutation in gE comprises a deletion of approximately amino acids 40-70. In another embodiment, an inactivating mutation in gE comprises insertion of a non-native sequence into a portion of the gene encoding gE, wherein said gE is inactivated as a result. In another embodiment, an inactivating mutation in gE comprises substitution of amino acid residues, such as a substitution of polar for non-polar residues, non-polar for polar residues, charged for uncharged residues, positively charged for negatively charged residues, or vice versa, or a combination thereof, as is known to one of skill in the art. In another embodiment, an inactivating mutation in gE consists essentially of any of the mutations listed hereinabove. In another embodiment, an inactivating mutation in gE consists of any of the mutations listed hereinabove. In another embodiment, an inactivating mutation in gE or other protein as described in the instant invention for a first HSV strain may also be mutated in an equivalent location of the corresponding protein in a second HSV strain, wherein the equivalent location of the insertion, deletion or substitution may be inferred by sequence alignment, as is well known in the art, wherein the region that aligns with the sequence of the mutation in the first strain would be mutated in the second strain.
“Inactivating mutation” in gD refers, in one embodiment, to a mutation that inhibits protein/receptor interactions, which in one embodiment is an interaction with a HVEM cell receptor, a nectin-1 cell receptor, or both, and in another embodiment, to a mutation that inhibits viral entry into a cell, inhibits downstream activation of gB, gH, and gL, inhibits fusion of the viral envelope with cell membrane, or a combination thereof. In one embodiment, an inactivating mutation in gD is in the amino terminus of the gD peptide, which in one embodiment is residue 1-15, and in another embodiment, the mutation inhibits formation of a hairpin loop structure when gD is bound to HVEM. In another embodiment, the mutation is at amino acids 3, 38, or both, and in one embodiment, alanine and tyrosine residues at those locations are replaced with cysteine residues (A3C/Y38C) to create a 3-38 disulfide bond and/or a fixed hairpin loop at the amino terminus.
In one embodiment, inactivating mutations of the present invention are accomplished using tools known in the art. In one embodiment, the nucleic acids used in this invention and those encoding proteins of and for use in the methods of the present invention can be produced by any synthetic or recombinant process such as is well known in the art. Nucleic acids can further be modified to alter biophysical or biological properties by means of techniques known in the art. For example, the nucleic acid can be modified to increase its stability against nucleases (e.g., “end-capping”), or to modify its lipophilicity, solubility, or binding affinity to complementary sequences. In another embodiment, transposons may be used to create inactivating mutations of a gene, where in one embodiment, the transposon may be Tn551, Minos, Hermes or piggyback. In another embodiment, the transposon may be AT-2 (tyl based transposon, Perkin Elmer; Devine et al. (1997) Genome Res. 7:551-563), GPS-1 (New England Biolabs), GPS-2 (New England Biolabs), EZ::tn (Tn5 based transposon, Epicenter Technologies), SIF (Tn7 based transposon, Biery et al. (2000) Nucl Acid Res 28:1067-1077), or Mu (Finnzymes, Haapa et al. (1999) Nucl Acid Res 13:2777-2784). In one embodiment, Southern blot analysis of digested DNA from individual transposon mutants may be used to verify transposon insertion. In another embodiment, sequence analysis, PCR and/or hybridization may be utilized to determine transposon insertion. Mutations may also be elicited using ethylmethanesulfonate (EMS) or radiation. In another embodiment, mutagenesis with chemical agents may be used. Such chemical mutagens may comprise, in other embodiments, chemicals that affect nonreplicating DNA such as HNO2 and NH2OH, as well as agents that affect replicating DNA such as acridine dyes, which have been shown to cause frameshift mutations. Methods for creating mutants using radiation or chemical agents are well known in the art, and any method may be utilized for the methods of this invention (see, for example, Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, Mukund V., Appl. Biochem. Biotechnol. 36, 227 (1992).
In one embodiment, DNA is synthesized chemically from the four nucleotides in whole or in part by methods known in the art. Such methods include those described in Caruthers (1985; Science 230:281-285). DNA can also be synthesized by preparing overlapping double-stranded oligonucleotides, filling in the gaps, and ligating the ends together (see, generally, Sambrook et al. (1989; Molecular Cloning—A Laboratory Manual, 2nd Edition. Cold Spring Habour Laboratory Press, New York)). In another embodiment, inactivating mutations are prepared from wild-type DNA by site-directed mutagenesis (see, for example, Zoller et al. (1982; DNA. 1984 December; 3(6):479-88); Zoller (1983); and Zoller (1984; DNA. 1984 December; 3(6):479-88); McPherson (1991; Directed Mutagenesis: A Practical Approach. Oxford University Press, NY)). The DNA obtained can be amplified by methods known in the art. One suitable method is the polymerase chain reaction (PCR) method described in Saiki et al. (1988; Science. 1988 Jan. 29; 239(4839):487-491), Mullis et al., U.S. Pat. No. 4,683,195, and Sambrook et al. (1989).
In one embodiment, the present invention provides a method of impeding the establishment of a latent HSV infection in a subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In another embodiment, the latent HSV infection that is prevented follows primary HSV infection. In another embodiment, the subject has been infected with HSV before vaccination. In another embodiment, the subject is at risk for HSV infection. In another embodiment, whether or not the subject has been infected with HSV at the time of vaccination, vaccination by a method of the present invention is efficacious in protecting a subject against latent HSV infection, following primary HSV infection.
In one embodiment, the present invention provides a method of inhibiting an HSV flare in a subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In another embodiment, the flare that is prevented follows exposure of the subject to HSV. In another embodiment, the subject has been infected with HSV before vaccination. In another embodiment, the subject is at risk for HSV infection. In another embodiment, whether or not the subject has been infected with HSV at the time of vaccination, vaccination by a method of the present invention is efficacious in protecting a subject against a formation of a flare, following an exposure of the subject to HSV.
In one embodiment, the present invention provides a method of protecting a subject against an HSV flare, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In another embodiment, the flare that is prevented follows exposure of the subject to HSV. In another embodiment, the subject has been infected with HSV before vaccination. In another embodiment, the subject is at risk for HSV infection. In another embodiment, whether or not the subject has been infected with HSV at the time of vaccination, vaccination by a method of the present invention is efficacious in protecting a subject against a formation of a flare, following exposure of the subject to HSV.
In one embodiment, the present invention provides a method of reducing the incidence of an HSV flare, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In another embodiment, the flare that is prevented follows exposure of the subject to HSV. In another embodiment, the subject has been infected with HSV before vaccination. In another embodiment, the subject is at risk for HSV infection. In another embodiment, whether or not the subject has been infected with HSV at the time of vaccination, vaccination by a method of the present invention is efficacious in reducing the incidence of a flare, following exposure of the subject to HSV.
In one embodiment, the present invention provides a method of inhibiting HSV recurrence in a subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In another embodiment, the recurrence that is prevented follows exposure of the subject to HSV. In another embodiment, the subject has been infected with HSV before vaccination. In another embodiment, the subject is at risk for HSV infection. In another embodiment, whether or not the subject has been infected with HSV at the time of vaccination, vaccination by a method of the present invention is efficacious in protecting a subject against a recurrence, following an exposure of the subject to an HSV.
In one embodiment, the present invention provides a method of reducing the incidence of HSV recurrence, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In another embodiment, the recurrence that is prevented follows exposure of the subject to HSV. In another embodiment, the subject has been infected with HSV before vaccination. In another embodiment, the subject is at risk for HSV infection. In another embodiment, whether or not the subject has been infected with HSV at the time of vaccination, vaccination by a method of the present invention is efficacious in reducing the incidence of a recurrence, following exposure of the subject to HSV.
“Flare” or “recurrence” refers, in one embodiment, to reinfection (in one embodiment, of skin tissue) following latent neuronal HSV infection. In another embodiment, the terms refer to reactivation of HSV after a latency period. In another embodiment, the terms refer to symptomatic HSV lesions following a non-symptomatic latency period.
In another embodiment, the present invention provides a method of suppressing HSV-1 infection in a subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant HSV strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In another embodiment, the mutant HSV strain is a mutant HSV-1 strain. In another embodiment, the mutant HSV strain is a mutant HSV-2 strain.
In another embodiment, the present invention provides a method of suppressing HSV-2 infection in a subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant HSV strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In another embodiment, the mutant HSV strain is a mutant HSV-1 strain. In another embodiment, the mutant HSV strain is a mutant HSV-2 strain.
According to any of the methods of the present invention and in one embodiment, the subject is human. In another embodiment, the subject is murine, which in one embodiment is a mouse, and, in another embodiment is a rat. In another embodiment, the subject is canine, feline, bovine, or porcine. In another embodiment, the subject is mammalian. In another embodiment, the subject is any organism susceptible to infection by HSV.
In one embodiment, the subject is infected by HSV, while in another embodiment, the subject is at risk for infection by HSV. In one embodiment, a subject at risk for HSV infection is a neonate. In another embodiment, a subject at risk for HSV infection is immunocompromised. In another embodiment, a subject at risk for HSV infection is elderly. In another embodiment, a subject at risk for HSV infection is an immunocompromised neonate or an immunocompromised elderly subject.
In another embodiment, the present invention provides a method of protecting a subject against formation of a zosteriform lesion or an analogous outbreak in a human subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein.
In another embodiment, the present invention provides a method of impeding formation of an HSV zosteriform lesion or an analogous outbreak in a human subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein.
In another embodiment, the zosteriform lesion or analogous outbreak that is impeded follows exposure of the subject to HSV. In another embodiment, the subject has been infected with HSV before vaccination. In another embodiment, the subject is at risk for HSV infection. In another embodiment, whether or not the subject has been infected with HSV at the time of vaccination, vaccination by a method of the present invention is efficacious in impeding formation of a zosteriform lesion or analogous outbreak, following an exposure of the subject to an HSV.
In another embodiment, the present invention provides a method of impeding HSV zosteriform spread or an analogous condition in a human subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In another embodiment, the zosteriform spread or analogous condition that is impeded follows exposure of the subject to HSV. In another embodiment, the subject has been infected with HSV before vaccination. In another embodiment, the subject is at risk for HSV infection. In another embodiment, whether or not the subject has been infected with HSV at the time of vaccination, vaccination by a method of the present invention is efficacious in impeding formation of a zosteriform spread or analogous condition, following exposure of the subject to HSV.
“Zosteriform” refers, in one embodiment, to skin lesions characteristic of an HSV infection, particularly during reactivation infection, which, in one embodiment, begin as a rash and follow a distribution near dermatomes, commonly occurring in a strip or belt-like pattern. In one embodiment, the rash evolves into vesicles or small blisters filled with serous fluid. In one embodiment, zosteriform lesions form in mice as a result of contact with HSV. In another embodiment, zosteriform lesions form in humans as a result of contact with HSV.
“Zosteriform spread” refers, in one embodiment, to an HSV infection that spreads from the ganglia to secondary skin sites within the dermatome. In another embodiment, the term refers to spread within the same dermatome as the initial site of infection. In another embodiment, the term refers to any other definition of “zosteriform spread” known in the art. “Outbreak”, in another embodiment, refers to a sudden increase in symptoms of a disease or in the spread or prevalence of a disease, and in one embodiment, refers to a sudden increase in zosteriform lesions, while in another embodiment, “outbreak” refers to a sudden eruption of zosteriform lesions.
In one embodiment, the present invention provides a method of impeding the formation of a dermatome lesion or an analogous condition in a human subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In another embodiment, the dermatome lesion or analogous condition that is impeded follows exposure of the subject to HSV. In one embodiment, dermatome lesions form in humans as a result of contact with HSV. In another embodiment, dermatome lesions in humans most often develop when the virus reactivates from latency in the ganglia and in one embodiment, spreads down nerves, in one embodiment, causing a recurrent infection. In another embodiment, dermatome lesions form in mice as a result of contact with HSV. In another embodiment, the subject has been infected with HSV before vaccination. In another embodiment, the subject is at risk for HSV infection. In another embodiment, whether or not the subject has been infected with HSV at the time of vaccination, vaccination by a method of the present invention is efficacious in impeding the formation of a dermatome lesion or analogous condition, following exposure of the subject to HSV.
In one embodiment, vaccination with gE-null HSV strains of the present invention protects against latent HSV infection (Example 5) and formation of zosteriform and dermatome lesions (Examples 4 and 20) after subsequent infection with virulent HSV. In another embodiment, the vaccination protects against disease caused by or associated with latent HSV infection. In another embodiment, the vaccination does not itself cause significant disease (Examples 2 and 19). In another embodiment, the vaccination protects against death, vaginal disease and recurrent infection (Example 30 and 31). In another
“Virulent HSV” refers, in one embodiment, to a naturally occurring HSV strain. In another embodiment, the term refers to an HSV strain capable of causing infection. In another embodiment, the term refers to an HSV strain capable of establishing latent infection.
In another embodiment, the present invention provides a method of impeding neuronal spread of an HSV in a subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In another embodiment, the neuronal spread that is impeded follows exposure of the subject to HSV. In another embodiment, the subject has been infected with HSV before vaccination. In another embodiment, the subject is at risk for HSV infection. In another embodiment, whether or not the subject has been infected with HSV at the time of vaccination, vaccination by a method of the present invention is efficacious in impeding neuronal viral spread, following an exposure of the subject to HSV.
Methods of measuring neuronal HSV spread are well known in the art, and include, in one embodiment, determination of the presence and extent of secondary dermatome lesion (Example 2). Other embodiments of methods for measuring viral spread are described, for example, in Labetoulle M et al. (Neuronal propagation of HSV1 from the oral mucosa to the eye. Invest Ophthalmol V is Sci. 2000 August; 41(9):2600-6) and Thompson K A et al. (Herpes simplex replication and dissemination is not increased by corticosteroid treatment in a rat model of focal Herpes encephalitis. J Neurovirol. 2000 February; 6(1):25-32).
In one embodiment, the present invention provides a method of reducing the incidence of herpetic ocular disease in a subject, comprising the step of contacting the subject with a mutant strain of HSV, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In another embodiment, the subject is infected by HSV. In another embodiment, the subject is at risk of infection by the HSV.
In one embodiment, the present invention provides a method of reducing the severity of herpetic ocular disease in a subject, comprising the step of contacting the subject with a mutant strain of HSV, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In another embodiment, the subject is infected by HSV. In another embodiment, the subject is at risk of infection by HSV. In another embodiment, the vaccine strain is from a different species from the challenge strain. In another embodiment, the vaccine strain is of the same species as the challenge strain.
In another embodiment, the present invention provides a method of reducing the incidence of an HSV-1 corneal infection, herpes keratitis or any other herpetic ocular disease in a subject, the method comprising the step of administering to said subject a mutant strain of HSV of the present invention, thereby reducing an incidence of an HSV-1 corneal infection or herpes keratitis in a subject. In another embodiment, administering to said subject a mutant strain of HSV of the present invention elicits an immune response against the HSV-1.
Methods for determining the presence and extent of herpetic ocular disease, corneal infection, and herpes keratitis are well known in the art, and are described, for example, in Labetoulle M et al. (Neuronal propagation of HSV1 from the oral mucosa to the eye. Invest Ophthalmol V is Sci. 2000 August; 41(9):2600-6) and Majumdar S i (Dipeptide monoester ganciclovir prodrugs for treating HSV-1-induced corneal epithelial and stromal keratitis: in vitro and in vivo evaluations. J Ocul Pharmacol Ther. 2005 December; 21(6):463-74).
In one embodiment, the present invention provides a method of reducing the incidence of a genital ulcer disease in a subject, comprising the step of contacting the subject with a mutant strain of HSV, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In another embodiment, the subject is infected by HSV. In another embodiment, the subject is at risk of infection by HSV
In one embodiment, the present invention provides a method of reducing the severity of genital ulcer disease in a subject, comprising the step of contacting the subject with a mutant strain of HSV, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In another embodiment, the subject is infected by HSV. In another embodiment, the subject is at risk of infection by HSV.
In one embodiment, genital ulcer disease is characterized by ulcerative lesions on the genitals. Methods for determining the presence and extent of genital ulcer disease are well known in the art.
In one embodiment, the present invention provides a method of reducing the incidence of HSV-1-mediated encephalitis in a subject, comprising the step of contacting the subject with a mutant strain of HSV, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein. “HSV-1 encephalitis” refers, in one embodiment, to encephalitis caused by HSV-1. In another embodiment, the term refers to encephalitis associated with HSV-1. In another embodiment, the term refers to any other type of HSV-1-mediated encephalitis known in the art. In another embodiment, the subject is infected by HSV. In another embodiment, the subject is at risk of infection by HSV. In another embodiment, the vaccine strain is from a different species from the challenge strain. In another embodiment, the vaccine strain is of the same species as the challenge strain.
In another embodiment, the present invention provides a method of reducing the incidence of HSV-2-mediated encephalitis in a subject, comprising the step of contacting the subject with a mutant strain of HSV, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein. “HSV-2 encephalitis” refers, in one embodiment, to encephalitis caused by HSV-2. In another embodiment, the term refers to encephalitis associated with HSV-2. In another embodiment, the term refers to any other type of HSV-2-mediated encephalitis known in the art. In another embodiment, the subject is infected by HSV. In another embodiment, the subject is at risk of infection by HSV
In one embodiment, the present invention provides a method of reducing the severity of herpes-mediated encephalitis in a subject, comprising the step of contacting the subject with a mutant strain of HSV, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In one embodiment, the subject is infected by HSV. In another embodiment, the subject is at risk of infection by HSV.
In one embodiment, the herpes-mediated encephalitis treated or prevented by a method of the present invention is a focal herpes encephalitis. In another embodiment, the herpes-mediated encephalitis is a neonatal herpes encephalitis. In another embodiment, the herpes-mediated encephalitis is any other type of herpes-mediated encephalitis known in the art.
In one embodiment, the present invention provides a method of reducing the incidence of disseminated HSV infection in a subject, comprising the step of contacting the subject with a mutant strain of HSV, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In one embodiment, the subject is infected by HSV. In another embodiment, the subject is at risk of infection by the HSV.
In one embodiment, the present invention provides a method of reducing the severity of disseminated HSV infection in a subject, comprising the step of contacting the subject with a mutant strain of HSV, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In another embodiment, the subject is infected by HSV. In another embodiment, the subject is at risk of infection by HSV.
In one embodiment, the present invention provides a method of reducing the incidence of a neonatal HSV-1 infection in an offspring of a subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant HSV strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In one embodiment, the offspring is contacted the subject with the mutant HSV strain. In another embodiment, the subject is infected by HSV. In another embodiment, the subject is at risk of infection by HSV.
In one embodiment, the present invention provides a method of reducing the incidence of a neonatal HSV-2 infection in an offspring of a subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant HSV strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In one embodiment, the subject is infected by HSV. In another embodiment, the subject is at risk of infection by HSV.
In one embodiment, the present invention provides a method of reducing the transmission of an HSV-1 infection from a subject to an offspring thereof, the method comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant HSV strain contains an inactivating mutation in a Us8 gene encoding a gE protein.
In one embodiment, the present invention provides a method of reducing the transmission of an HSV-2 infection from a subject to an offspring thereof, the method comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant HSV strain contains an inactivating mutation in a Us8 gene encoding a gE protein.
In one embodiment, the present invention provides a method of reducing HIV-1 transmission to an offspring, the method comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant HSV strain contains an inactivating mutation in a Us8 gene encoding a gE protein. As is known in the art, HSV-2 infection increases HIV-1 viral shedding in genital secretions (Nagot N et al. Reduction of HIV-1 RNA levels with therapy to suppress herpes simplex virus. N Engl J Med. 2007 Feb. 22; 356(8):790-9). Thus, methods of the present invention of inhibiting HSV-2 infection are also believed to be efficacious for reducing HIV-1 transmission to an offspring. In another embodiment, the mutant HSV strain is an HSV-1 strain. In another embodiment, the mutant HSV strain is an HSV-2 strain.
In one embodiment, the present invention provides a method of reducing HIV-1 transmission to a sexual partner, the method comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant HSV strain contains an inactivating mutation in a Us8 gene encoding a gE protein. As is known in the art, HSV-2 infection increases HIV-1 viral shedding in genital secretions. Thus, methods of the present invention of inhibiting HSV-2 infection are also believed to be efficacious for reducing HIV-1 transmission to a sexual partner. In another embodiment, the mutant HSV strain is an HSV-1 strain. In another embodiment, the mutant HSV strain is an HSV-2 strain.
In one embodiment, the present invention provides a method of reducing susceptibility to HIV-1, the method comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant HSV strain contains an inactivating mutation in a Us8 gene encoding a gE protein. As is known in the art, HSV-2 infection increases HIV-1 replication (Ouedraogo A et al Impact of suppressive herpes therapy on genital HIV-1 RNA among women taking antiretroviral therapy: a randomized controlled trial. AIDS. 2006 Nov. 28; 20(18):2305-13). Thus, methods of the present invention of inhibiting HSV-2 infection are also believed to be efficacious for reducing susceptibility to HIV-1. In another embodiment, the mutant HSV strain is an HSV-1 strain. In another embodiment, the mutant HSV strain is an HSV-2 strain.
In one embodiment, the present invention provides a method of reducing the severity of a neonatal HSV infection in an offspring of a subject, comprising the step of contacting the subject with a mutant strain of HSV, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In another embodiment, the subject is infected by HSV. In another embodiment, the subject is at risk of infection by HSV.
In one embodiment, the present invention provides a method of reducing the incidence of a disease, disorder, or symptom associated with or secondary to a herpes-mediated encephalitis in a subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein.
In one embodiment, the present invention provides a method of treating a disease, disorder, or symptom associated with or secondary to a herpes-mediated encephalitis in a subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein, thereby treating a disease, disorder, or symptom associated with or secondary to a herpes-mediated encephalitis in a subject.
In one embodiment, the disease, disorder, or symptom is fever. In another embodiment, the disease, disorder, or symptom is headache. In another embodiment, the disease, disorder, or symptom is stiff neck. In another embodiment, the disease, disorder, or symptom is seizures. In another embodiment, the disease, disorder, or symptom is partial paralysis. In another embodiment, the disease, disorder, or symptom is stupor. In another embodiment, the disease, disorder, or symptom is coma. In another embodiment, the disease, disorder, or symptom is any other disease, disorder, or symptom known in the art that is associated with or secondary to herpes-mediated encephalitis.
In another embodiment, “symptoms” may be any manifestation of a HSV infection, including, but not limited to blisters, ulcerations, or lesions on the urethra, cervix, upper thigh, and/or anus in women and on the penis, urethra, scrotum, upper thigh, and anus in men, inflammation, swelling, fever, flu-like symptoms, sore mouth, sore throat, pharyngitis, pain, blisters on tongue, mouth or lips, ulcers, cold sores, neck pain, enlarged lymph nodes, reddening, bleeding, itching, dysuria, headache, muscle pain, etc., or a combination thereof.
In another embodiment, the disease, disorder, or symptom is fever. In another embodiment, the disease, disorder, or symptom is headache. In another embodiment, the disease, disorder, or symptom is stiff neck. In another embodiment, the disease, disorder, or symptom is seizures. In another embodiment, the disease, disorder, or symptom is partial paralysis. In another embodiment, the disease, disorder, or symptom is stupor. In another embodiment, the disease, disorder, or symptom is coma. In another embodiment, the disease, disorder, or symptom is any other disease, disorder, or symptom known in the art that is associated with or secondary to a herpes-mediated encephalitis.
In one embodiment, a mutant HSV-1 strain of the present invention protects a subject against infection and disorders and symptoms associated with infection with wild-type HSV-1. In another embodiment, the disorders and symptoms include herpes labialis (cold sores or fever blisters). In another embodiment, the disorders and symptoms include HSV-mediated cornea disease. In another embodiment, the disorders and symptoms include herpes-mediated retinitis. In another embodiment, the disorders and symptoms include herpes-mediated encephalitis. In another embodiment, the disorders and symptoms include HSV-1-mediated genital ulcer disease. In another embodiment, a mutant HSV-1 strain of the present invention provides substantial protection against HSV-1 infection and partial protection against one or more symptoms associated with HSV-2 infection. In another embodiment, these HSV-2 symptoms include the symptoms described hereinabove.
Methods of determining the presence and severity of herpes-mediated encephalitis are well known in the art, and are described, for example, in Bonkowsky J L et al. (Herpes simplex virus central nervous system relapse during treatment of infantile spasms with corticotropin. Pediatrics. 2006 May; 117(5):e1045-8) and Khan O A et al. (Herpes encephalitis presenting as mild aphasia: case report. BMC Fam Pract. 2006 Mar. 24; 7:22).
In one embodiment, the present invention provides a method of treating a disease, disorder, or symptom associated with an HSV infection in a subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein.
In one embodiment, the present invention provides a method of reducing the incidence of a disease, disorder, or symptom associated with an HSV infection in a subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein.
In one embodiment, the present invention provides a method of treating a disease, disorder, or symptom secondary to an HSV infection in a subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein.
In one embodiment, the present invention provides a method of reducing the incidence of a disease, disorder, or symptom secondary to an HSV infection in a subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein.
In one embodiment, the disease, disorder, or symptom secondary to an HSV infection is oral lesions. In another embodiment, the disease, disorder, or symptom is genital lesions. In another embodiment, the disease, disorder, or symptom is oral ulcers. In another embodiment, the disease, disorder, or symptom is genital ulcers. In another embodiment, the disease, disorder, or symptom is fever. In another embodiment, the disease, disorder, or symptom is headache. In another embodiment, the disease, disorder, or symptom is muscle ache. In another embodiment, the disease, disorder, or symptom is swollen glands in the groin area. In another embodiment, the disease, disorder, or symptom is painful urination. In another embodiment, the disease, disorder, or symptom is vaginal discharge. In another embodiment, the disease, disorder, or symptom is blistering. In another embodiment, the disease, disorder, or symptom is flu-like malaise. In another embodiment, the disease, disorder, or symptom is keratitis. In another embodiment, the disease, disorder, or symptom is herpetic whitlow. In another embodiment, the disease, disorder, or symptom is Bell's palsy. In another embodiment, the disease, disorder, or symptom is herpetic erythema multiforme. In another embodiment, the disease, disorder, or symptom is a lower back symptom (e.g. numbness, tingling of the buttocks or the area around the anus, urinary retention, constipation, and impotence). In another embodiment, the disease, disorder, or symptom is a localized eczema herpeticum. In another embodiment, the disease, disorder, or symptom is a disseminated eczema herpeticum. In another embodiment, the disease, disorder, or symptom is a herpes gladiatorum. In another embodiment, the disease, disorder, or symptom is a herpetic sycosis. In another embodiment, the disease, disorder, or symptom is an esophageal symptom (e.g. difficulty swallowing or burning, squeezing throat pain while swallowing, weight loss, pain in or behind the upper chest while swallowing). In another embodiment, the disease, disorder, or symptom is any other disease, disorder, or symptom is known in the art.
The HSV infection treated or ameliorated by methods and compositions of the present invention is, in one embodiment, a genital HSV infection. In another embodiment, the HSV infection is an oral HSV infection. In another embodiment, the HSV infection is an ocular HSV infection. In another embodiment, the HSV infection is a dermatologic HSV infection.
In one embodiment, the HSV infection is an HSV-2 infection. In another embodiment, the HSV is an HSV-1 infection. In another embodiment, the HSV infection is any other type of HSV infection known in the art.
In one embodiment, the present invention provides a method of inducing rapid clearance of an HSV-1 infection in a subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In another embodiment, the mutant HSV strain is a mutant HSV-1 strain. In another embodiment, the mutant HSV strain is a mutant HSV-2 strain.
In one embodiment, the present invention provides a method of inducing rapid clearance of an HSV-2 infection in a subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In another embodiment, the mutant HSV strain is a mutant HSV-1 strain. In another embodiment, the mutant HSV strain is a mutant HSV-2 strain.
In one embodiment, the present invention provides a method of inducing an anti-HSV immune response in a subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein, thereby inducing an anti-HSV immune response in a subject. In another embodiment, the mutant HSV strain is a mutant HSV-1 strain. In another embodiment, the mutant HSV strain is a mutant HSV-2 strain.
In one embodiment, the present invention provides a method of inducing an anti-HSV neutralizing antibody response in a subject, comprising the step of contacting the subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein. In another embodiment, the mutant HSV strain is a mutant HSV-1 strain. In another embodiment, the mutant HSV strain is a mutant HSV-2 strain. In another embodiment, no antibody response to gD-2 is detected after the first immunization with HSV-2ΔgE(gfp) as shown in Example 25 herein.
In one embodiment, a first immunization with the live virus vaccine HSV-2ΔgE(gfp) results in an antibody response. In another embodiment, a second administration or immunization with the live virus vaccine provided herein results in a high titer and more pronounced antibody response as compared to the first immunization (see Example 26, herein).
In another embodiment, a second administration or immunization with the live virus vaccine provided herein produces high titers of anti-gC-2 and anti-gD-2 antibodies than one immunization alone (see Example 31, herein).
In one embodiment, the present invention provides a method of inhibiting HSV labialis in a subject, comprising the step of vaccinating the subject against an HSV by a method of the present invention.
In one embodiment, the examples of the present invention provide experimental support for a method of vaccinating against HSV infection by contacting the subject with a mutant strain of HSV, containing one or more inactivating mutations.
In another embodiment, the present invention provides a method of inhibiting HSV labialis in a subject, comprising the step of impeding an HSV infection in the subject by a method of the present invention.
In one embodiment, the immune response induced by methods and compositions of the present invention is a cellular immune response. In another embodiment, the immune response comprises a CD8+ cytotoxic T lymphocyte (CTL) response. In another embodiment, the immune response comprises a CD4+ helper T cell response. In another embodiment, the immune response comprises a humoral immune response. In one embodiment, an immune response refers to an in vivo or in vitro reaction in response to a challenge by an immunogen. In one embodiment, an immune response is expressed by antibody production, cell-mediated immunity, immunologic tolerance, or a combination thereof.
The route of administration of the mutant strains in the methods of the present invention is, in one embodiment, epidermal. In another embodiment, the mutant strain is administered by epidermal scarification or scratching. In another embodiment, the mutant strain is administered intramuscularly. In another embodiment, the mutant strain is administered subcutaneously. In another embodiment, the mutant strain is administered intranasally. In another embodiment, the mutant strain is administered transdermally. In another embodiment, the mutant strain is administered intravaginally. In another embodiment, the mutant strain is administered transmucosally, which in one embodiment, is intra-respiratory mucosally. In another embodiment, the mutant strain is administered intranasally. In another embodiment, the mutant strain is administered in an aerosol. In another embodiment, the mutant strain is administered via any other route known in the art.
In one embodiment, the inactivating mutation in the gE-encoding gene of HSV strains as described in the methods and compositions of the present invention is a deletion mutation. In another embodiment, the inactivating mutation is an insertion mutation. In another embodiment, the inactivating mutation is a substitution mutation. In another embodiment, the inactivating mutation is a gE-null mutation. In another embodiment, the inactivating mutation is any other type of mutation known in the art.
In one embodiment, the inactivating mutation in the glycoprotein-encoding gene of HSV strains as described in the methods and compositions of the present invention is a deletion mutation. In another embodiment, the inactivating mutation is an insertion mutation. In another embodiment, the inactivating mutation is a substitution mutation. In another embodiment, the inactivating mutation is a null mutation. In another embodiment, the inactivating mutation is any other type of mutation known in the art. In one embodiment, the insertion, deletion or substitution mutation comprises an insertion, deletion or substitution of a single amino acid, while in another embodiment, it comprises an insertion, deletion or substitution of 1-5 amino acids, 1-10 amino acids, 5-20 amino acids, 10-50 amino acids, 25-100 amino acids, 100-500 amino acids, 300-400 amino acids, 200-1000 amino acids, or 1000 or more amino acids.
In one embodiment, the present invention provides an isolated mutant HSV-1 strain comprising a first inactivating mutation in a gene encoding a gE protein and a second inactivating mutation. In another embodiment, the gene encoding a gE protein is a Us8 gene. In another embodiment, the mutation is a gE-null mutation. In one embodiment, an isolated mutant HSV-1 strain as described in the methods and compositions of the present invention further comprises one or more additional mutations, which in one embodiment are inactivating mutations. In another embodiment, the second or additional inactivating mutation is in a Us7 gene. In another embodiment, the second or additional inactivating mutation is in a Us9 gene. In another embodiment, the second inactivating mutation is in any gene which confers neurovirulence. In another embodiment, the second inactivating mutation is in any gene required for virus entry into a host cell. In another embodiment, the second inactivating mutation is in a host shut-off gene. In another embodiment, the second inactivating mutation is in the thymidine kinase gene. In another embodiment, the second inactivating mutation is in any other HSV-1 gene known in the art. In another embodiment, the isolated mutant HSV-1 strain contains inactivating mutations in a gene encoding a gE protein, a Us7 gene, and a Us9 gene. In another embodiment, an isolated mutant HSV-1 strain as described in the methods and compositions of the present invention further comprises an additional mutation in a gene encoding a gD protein.
In one embodiment, the present invention provides an isolated mutant HSV-2 strain comprising a first inactivating mutation in a gene encoding a gE protein and a second inactivating mutation. In another embodiment, the gene encoding a gE protein is a Us8 gene. In another embodiment, the mutation is a gE-null mutation. In one embodiment, an isolated mutant HSV-2 strain as described in the methods and compositions of the present invention further comprises one or more additional mutations, which in one embodiment are inactivating mutations. In another embodiment, the second or additional inactivating mutation is in a Us7 gene. In another embodiment, the second or additional inactivating mutation is in a Us9 gene. In another embodiment, the second inactivating mutation is in any gene which confers neurovirulence. In another embodiment, the second inactivating mutation is in any gene required for virus entry into a host cell. In another embodiment, the second inactivating mutation is in a host shut-off gene. In another embodiment, the second inactivating mutation is in the thymidine kinase gene. In another embodiment, the second inactivating mutation is in any other HSV-2 gene known in the art. In another embodiment, the isolated mutant HSV-2 strain contains inactivating mutations in a gene encoding a gE protein, a Us7 gene, and a Us9 gene. In another embodiment, an isolated mutant HSV-2 strain as described in the methods and compositions of the present invention further comprises an additional mutation in a gene encoding a gD protein.
In one embodiment, the Us7 gene that is mutated is highly conserved amongst alpha-herpesviruses. In another embodiment, the Us7 gene that is mutated is required for anterograde spread of the virus. In another embodiment, the Us7 gene that is mutated is required for retrograde spread of the virus.
In one embodiment, the Us9 gene that is mutated is highly conserved amongst alpha-herpesviruses. In another embodiment, the Us9 gene that is mutated is required for anterograde spread of the virus. In another embodiment, the Us9 gene that is mutated is required for retrograde spread of the virus.
In one embodiment, the mutation in Us7 and/or Us9 is an inactivating mutation. In another embodiment, the mutation is a deletion mutation. In another embodiment, the mutation is an insertion mutation. In another embodiment, the mutation is a substitution mutation. In another embodiment, the mutation is any other type of mutation known in the art.
In one embodiment, a mutant strain of the present invention further comprises an additional inactivating mutation in a gene encoding a membrane protein not required for virus entry. In one embodiment, a gene encoding a membrane protein not required for virus entry is Us7 gene or Us9 gene. In another embodiment, a gene encoding a membrane protein not required for virus entry is Us5, Us4, UL53, or UL10. In another embodiment, a mutant strain of the present invention further comprises an additional inactivating mutation in a gene encoding a membrane protein required for virus entry, which in one embodiment, is Us6.
In one embodiment, the additional gene that is mutated is highly conserved amongst alpha-herpesviruses. In another embodiment, the additional gene that is mutated is required for anterograde spread of the virus. In another embodiment, the additional gene that is mutated is required for retrograde spread of the virus.
In one embodiment, the additional gene that is mutated is a virion membrane protein. In one embodiment, the additional gene is a virion membrane protein not required, or non-essential, for virus entry. In another embodiment, the membrane protein is a glycoprotein. In another embodiment, the additional gene is glycoprotein J. In another embodiment, the additional gene is glycoprotein G. In another embodiment, the additional gene is glycoprotein K. In another embodiment, the additional gene is glycoprotein M. In another embodiment, the additional gene is selected from glycoproteins J, G, K, and M.
In one embodiment, the additional mutation is introduced to enhance inhibition of anterograde spread of the mutant HSV-1 strain. In another embodiment, the additional mutation is required, in combination with a gE mutation, to confer inhibition of anterograde spread of the mutant HSV-1 strain. In another embodiment, the gE mutation is insufficient to confer inhibition of anterograde spread of the mutant HSV-1 strain. In another embodiment, the additional mutation is sufficient, in the absence of a gE mutation, to confer inhibition of anterograde spread of the mutant HSV-1 strain.
In one embodiment, the additional mutation is introduced to enhance inhibition of anterograde spread of the mutant HSV-2 strain. In another embodiment, the additional mutation is required, in combination with a gE mutation, to confer inhibition of anterograde spread of the mutant HSV-2 strain. In another embodiment, the gE mutation is insufficient to confer inhibition of anterograde spread of the mutant HSV-2 strain. In another embodiment, the additional mutation is sufficient, in the absence of a gE mutation, to confer inhibition of anterograde spread of the mutant HSV-2 strain.
In one embodiment, the additional mutation is introduced to enhance attenuation of virulence in the HSV-1 or HSV-2 strain or both. In another embodiment, the additional mutation is required, in combination with a gE mutation, to attenuate virulence.
In one embodiment, the additional gene that is mutated is a virion membrane protein. In another embodiment, the additional gene is a virion membrane protein required for virus entry. In another embodiment, the additional gene is glycoprotein B. In another embodiment, the additional gene is glycoprotein D. In another embodiment, the additional gene is glycoprotein H. In another embodiment, the additional gene is glycoprotein L.
In one embodiment, the mutant HSV strain as described in the methods and compositions of the present invention is replication-competent. In another embodiment, the mutant HSV strain as described in the methods and compositions of the present invention is replication-competent in skin tissue of the subject. In another embodiment, the mutant strain is replication-competent in skin cell of the subject. In another embodiment, the mutant strain is replication-competent in skin tissue of the species to which the subject belongs. In another embodiment, the mutant strain is replication-competent in a cell line derived from skin tissue of the subject's species. In another embodiment, the mutant strain is replication-competent in a culture of skin cells of the subject's species. In another embodiment, the mutant strain is replication-competent in a cell line derived from a skin cell of the subject's species.
“Replication competent” refers, in one embodiment, to an ability to replicate. In another embodiment, the term includes strains that exhibit impaired but still detectable levels of replication. In another embodiment, the term refers to a strain that exhibits measurable replication.
In one embodiment, the mutant HSV strain as described in the methods and compositions of the present invention is defective in its ability to spread from the site of inoculation to the dorsal root ganglia (DRG). In one embodiment, the dorsal root ganglia contain the neuron cell bodies of nerve fibres. In another embodiment, the mutant HSV strain is defective in retrograde spread. In another embodiment, the mutant HSV strain is impaired in retrograde spread. In another embodiment, the mutant HSV strain is significantly impaired in retrograde spread. In another embodiment, the mutant HSV strain is impaired in retrograde spread but is replication-competent in skin.
In one embodiment, the mutant HSV strain as described in the methods and compositions of the present invention is defective in spread from DRG to the skin. In another embodiment, the mutant HSV strain is defective in anterograde spread. In another embodiment, the mutant HSV strain is impaired in anterograde spread. In another embodiment, the mutant HSV strain is significantly impaired in anterograde spread. In another embodiment, the mutant HSV strain is impaired in anterograde spread but is replication-competent in skin. In another embodiment, the mutant HSV strain is impaired in anterograde spread but is replication-competent at the site of inoculation.
In one embodiment, the mutant HSV strain as described in the methods and compositions of the present invention is defective in spread from DRG to the skin. In another embodiment, the mutant HSV strain is defective in retrograde spread. In another embodiment, the mutant HSV strain is impaired in retrograde spread. In another embodiment, the mutant HSV strain is significantly impaired in retrograde spread. In another embodiment, the mutant HSV strain is impaired in retrograde spread but is replication-competent in skin. In another embodiment, the mutant HSV strain is impaired in anterograde spread but is replication-competent at the site of inoculation.
“DRG” refers, in one embodiment, to a neuronal cell body. In another embodiment, the term refers to any other definition of “DRG” used in the art.
In one embodiment, a mutant HSV strain of the present invention is replication-defective, either in a particular tissue (e.g. in neural tissue) or in general. Methods for measuring viral replication are well known in the art and include, in one embodiment, titering assays of tissue samples near a site of inoculation (Examples herein). In another embodiment, recovery of infectious virus from tissues near a site of inoculation is utilized (Examples herein). Other embodiments as described in the methods for measuring viral replication are described, for example, in Thi T N et al. (Rapid determination of antiviral drug susceptibility of herpes simplex virus types 1 and 2 by real-time PCR. Antiviral Res. 2006 March; 69(3):152-7); Schang L M et al. (Roscovitine, a specific inhibitor of cellular cyclin-dependent kinases, inhibits herpes simplex virus DNA synthesis in the presence of viral early proteins. J Virol. 2000 March; 74(5):2107-20); and Kennedy P G et al. (Replication of the herpes simplex virus type 1 RL1 mutant 1716 in primary neuronal cell cultures—possible relevance to use as a viral vector. J Neurol Sci. 2000 Oct. 1; 179(S 1-2):108-14).
In one embodiment, a mutant strain as described in the methods and compositions of the present invention is impaired in its spread in neural tissue of the subject. In another embodiment, the mutant strain is impaired in its spread in a culture of neural cells of the subject. In another embodiment, the mutant strain is impaired in its spread in neural tissue of the species to which the subject belongs. In another embodiment, the mutant strain is impaired in its spread in a cell line derived from neural tissue of the subject's species. In another embodiment, the mutant strain is impaired in its spread in a culture of neural cells of the subject's species. In another embodiment, the mutant strain is impaired in its spread in a cell line derived from a neural cell of the subject's species.
In one embodiment, a mutant strain as described in the methods and compositions of the present invention is impaired in its ability to enter neural tissue of the subject. In another embodiment, the mutant strain is impaired in its ability to enter a culture of neural cells of the subject. In another embodiment, the mutant strain is impaired in its ability to enter neural tissue of the species to which the subject belongs. In another embodiment, the mutant strain is impaired in its ability to enter a cell line derived from neural tissue of the subject's species. In another embodiment, the mutant strain is impaired in its ability to enter a culture of neural cells of the subject's species. In another embodiment, the mutant strain is impaired in its ability to enter a cell line derived from a neural cell of the subject's species.
In one embodiment, the mutant HSV strain as described in the methods and compositions of the present invention induces an anti-HSV immune response. In another embodiment, the immune response impedes replication of the HSV in the subject. In another embodiment, the immune response impedes neuronal spread of the HSV in the subject. In another embodiment, the immune response results in killing of HSV-infected cells in the subject.
In one embodiment, the mutant HSV strain as described in the methods and compositions of the present invention is a mutant HSV-1 strain. In another embodiment, the mutant HSV-1 strain confers protection against an HSV-1 infection, spread, or a consequence thereof (e.g. zosteriform lesions or herpetic ocular disease). In another embodiment, the mutant HSV strain is a mutant HSV-2 strain. In another embodiment, the mutant HSV strain is HSV-2ΔgE or HSV-2ΔgE(gfp). In another embodiment, the mutant HSV-2 strain confers protection against an HSV-2 infection, spread, or a consequence thereof (e.g. zosteriform lesions or herpetic ocular disease). In another embodiment, the mutant HSV strain is any other mutant HSV strain known in the art. In another embodiment, the mutant HSV strain provided herein confers protection in a dose dependent manner. In another embodiment, the mutant HSV strain provided herein confers protection to the DRG against high dose challenge with an HSV-2 strain (see Example 28, herein).
In another embodiment, the disorders and symptoms include HSV infections in an immunocompromised subject, including subjects with HIV. In another embodiment, a mutant HSV-1 strain of the present invention prevents or inhibits transmission of genital HSV-1 from a vaccinated mother to her newborn infant. In another embodiment, a mutant HSV strain of the present invention treats, suppresses, prevents or inhibits HSV in elderly subjects.
In one embodiment, a mutant HSV-2 strain of the present invention protects a subject against infection and disorders and symptoms associated with infection with wild-type HSV-2. In another embodiment, the mutant HSV-2 strain prevents or inhibits transmission of genital HSV-2 from the vaccinated mother to her newborn infant. In another embodiment, the mutant HSV-2 strain prevents or inhibits genital ulcer disease. In another embodiment, the mutant HSV-2 strain provides substantial protection against both HSV-2 and HSV-1 infection.
According to any of the methods of the invention, and in one embodiment, the infection is an HSV-1 infection. In another embodiment, the infection is an HSV-2 infection.
According to any of the methods of the invention, and in one embodiment, the vaccine strain is from a different species from the strain against which protection is conferred (“challenge strain”). In another embodiment, the vaccine strain is of the same species as the challenge strain.
In one embodiment, a vaccine as described in the methods and compositions of the present invention protects a subject against a challenge with heterologous HSV. In another embodiment, the heterologous challenge is a different strain of the same species. In another embodiment, in the case of a mutant HSV-1 vaccine strain, the vaccine confers protection against a heterologous HSV-1 strain. In another embodiment, in the case of a mutant HSV-2 vaccine strain, the vaccine confers protection against a heterologous HSV-2 strain. In another embodiment, the heterologous strain has an antigenic protein that is significantly heterologous relative to the vaccine strain. In another embodiment, the antigenic protein is gD. In another embodiment, the antigenic protein is gB. In another embodiment, the antigenic protein is any other antigenic protein known in the art.
In one embodiment, the heterologous strain has a gD protein that is significantly heterologous relative to the vaccine strain. In another embodiment, the gD protein of the heterologous strain shares 50% homology with the vaccine strain. In another embodiment, the homology shared between the gD protein of the heterologous strain and the vaccine strain is 55%. In another embodiment, the homology shared is 60%. In another embodiment, the homology shared is 65%. In another embodiment, the homology shared is 70%. In another embodiment, the homology shared is 75%. In another embodiment, the homology shared is 80%. In another embodiment, the homology shared is 85%. In another embodiment, the homology shared is 90%. In another embodiment, the homology shared is 95%. In another embodiment, the homology shared is 98%. In another embodiment, the homology shared is greater than 98%.
In one embodiment, the heterologous strain has a gB protein that is significantly heterologous relative to the vaccine strain. In another embodiment, the gB protein of the heterologous strain shares 50% homology with the vaccine strain. In another embodiment, the homology shared between the gB protein of the heterologous strain and the vaccine strain is 55%. In another embodiment, the homology shared is 60%. In another embodiment, the homology shared is 65%. In another embodiment, the homology shared is 70%. In another embodiment, the homology shared is 75%. In another embodiment, the homology shared is 80%. In another embodiment, the homology shared is 85%. In another embodiment, the homology shared is 90%. In another embodiment, the homology shared is 95%. In another embodiment, the homology shared is 98%. In another embodiment, the homology shared is greater than 98%.
In one embodiment, the heterologous challenge strain is HSV-1 NS. In another embodiment, the heterologous challenge strain is HSV-1(F). In another embodiment, the heterologous challenge strain is HSV-1(17). In another embodiment, the heterologous challenge strain is any other HSV-1 strain known in the art.
In one embodiment, the heterologous challenge strain is HSV-2(2.12). In another embodiment, the heterologous challenge strain is any other HSV-2 strain known in the art.
In one embodiment, the heterologous challenge strain is a different HSV species. In another embodiment, in the case of a mutant HSV-1 vaccine strain, the vaccine confers protection against HSV-2 challenge. In another embodiment, in the case of a mutant HSV-2 vaccine strain, the vaccine confers protection against HSV-1 challenge.
In one embodiment, a vaccine as described in the methods and compositions of the present invention protects a subject against a challenge with a large inoculum of HSV. In another embodiment, the large inoculum is 106 plaque-forming units (pfu). In another embodiment, the inoculum is 1.5×106 pfu. In another embodiment, the inoculum is 2×106 pfu. In another embodiment, the inoculum is 3×106 pfu. In another embodiment, the inoculum is 4×106 pfu. In another embodiment, the inoculum is 5×106 pfu. In another embodiment, the inoculum is 7×106 pfu. In another embodiment, the inoculum is 1×107 pfu. In another embodiment, the inoculum is 1.5×107 pfu. In another embodiment, the inoculum is 2×107 pfu. In another embodiment, the inoculum is 3×107 pfu. In another embodiment, the inoculum is 4×107 pfu. In another embodiment, the inoculum is 5×107 pfu. In another embodiment, the inoculum is 7×107 pfu. In another embodiment, the inoculum is 108 pfu. In another embodiment, the inoculum is 103-106 pfu. In another embodiment, the inoculum is 103-105 pfu. In another embodiment, the inoculum is 104-106 pfu. In another embodiment, the inoculum is 3×104-3×106 pfu. In another embodiment, the inoculum is 104-107 pfu. In another embodiment, the inoculum is 3×104-3×107 pfu. In another embodiment, the inoculum is 105-108 pfu. In another embodiment, the inoculum is 3×105-3×108 pfu. In another embodiment, the inoculum is more than 108 pfu.
In one embodiment, a vaccine as described in the methods and compositions of the present invention exhibits enhanced safety relative to gE-containing HSV vaccine strains, due to its inability to infect the ganglia. In another embodiment, a method of the present invention exhibits enhanced safety relative to gE-containing HSV vaccine strains, due to its inability to spread in neurons. In another embodiment, the vaccine provided herein is safe when administered through multiple routes as exemplified herein (see Example 24).
Various embodiments of dosage ranges of mutant HSV particles can be used, in another embodiment, in methods of the present invention. In another embodiment, the dosage is 103 pfu. In another embodiment, the dosage is 2×103 pfu. In another embodiment, the dosage is 3×103 pfu. In another embodiment, the dosage is 5×103 pfu. In another embodiment, the dosage is 104 pfu. In another embodiment, the dosage is 1.5×104 pfu. In another embodiment, the dosage is 104 pfu. In another embodiment, the dosage is 2×104 pfu. In another embodiment, the dosage is 3×104 pfu. In another embodiment, the dosage is 5×104 pfu. In another embodiment, the dosage is 7×104 pfu. In another embodiment, the dosage is 105 pfu. In another embodiment, the dosage is 2×105 pfu. In another embodiment, the dosage is 3×105 pfu. In another embodiment, the dosage is 5×105 pfu. In another embodiment, the dosage is 7×105 pfu. In another embodiment, the dosage is 106 pfu. In another embodiment, the dosage is 2×106 pfu. In another embodiment, the dosage is 3×106 pfu. In another embodiment, the dosage is 5×106 pfu. In another embodiment, the dosage is 7×106 pfu. In another embodiment, the dosage is 107 pfu. In another embodiment, the dosage is 2×107 pfu. In another embodiment, the dosage is 3×107 pfu. In another embodiment, the dosage is 5×107 pfu. In another embodiment, the dosage is 7×107 pfu. In another embodiment, the dosage is 108 pfu. In another embodiment, the dosage is 2×108 pfu. In another embodiment, the dosage is 3×108 pfu. In another embodiment, the dosage is 5×108 pfu. In another embodiment, the dosage is 7×108 pfu.
In another embodiment, the dosage is 103 pfu/dose. In another embodiment, the dosage is 2×103 pfu dose. In another embodiment, the dosage is 3×103 pfu/dose. In another embodiment, the dosage is 5×103 pfu/dose. In another embodiment, the dosage is 104 pfu/dose. In another embodiment, the dosage is 1.5×104 pfu/dose. In another embodiment, the dosage is 104 pfu/dose. In another embodiment, the dosage is 2×104 pfu/dose. In another embodiment, the dosage is 3×104 pfu/dose. In another embodiment, the dosage is 5×104 pfu/dose. In another embodiment, the dosage is 7×104 pfu/dose. In another embodiment, the dosage is 105 pfu/dose. In another embodiment, the dosage is 2×105 pfu/dose. In another embodiment, the dosage is 3×105 pfu/dose. In another embodiment, the dosage is 5×105 pfu/dose. In another embodiment, the dosage is 7×105 pfu/dose. In another embodiment, the dosage is 106 pfu/dose. In another embodiment, the dosage is 2×106 pfu/dose. In another embodiment, the dosage is 3×106 pfu/dose. In another embodiment, the dosage is 5×106 pfu/dose. In another embodiment, the dosage is 7×106 pfu/dose. In another embodiment, the dosage is 107 pfu/dose. In another embodiment, the dosage is 2×107 pfu/dose. In another embodiment, the dosage is 3×107 pfu/dose. In another embodiment, the dosage is 5×107 pfu/dose. In another embodiment, the dosage is 7×107 pfu/dose. In another embodiment, the dosage is 108 pfu/dose. In another embodiment, the dosage is 2×108 pfu/dose. In another embodiment, the dosage is 3×108 pfu/dose. In another embodiment, the dosage is 5×108 pfu/dose. In another embodiment, the dosage is 7×108 pfu/dose. In another embodiment, the dose is more than 108 pfu. In another embodiment, the dose is 103-106 pfu. In another embodiment, the dose is 103-105 pfu. In another embodiment, the dose is 104-106 pfu. In another embodiment, the dose is 3×104-3×106 pfu. In another embodiment, the dose is 104-107 pfu. In another embodiment, the dose is 3×104-3×107 pfu. In another embodiment, the dose is 105-108 pfu. In another embodiment, the dose is 3×105-3×108 pfu.
In another embodiment, the dose of mutant HSV particles administered to a subject is the above-described dose per gram body weight. In one embodiment, the dose of mutant HSV particles administered to a subject is 2.5×104 pfu/gram body weight.
In one embodiment, the methods of the present invention comprise administering to a subject or contacting a subject with a mutant HSV of the present invention and with a herpes simplex virus subunit vaccine, which in one embodiment, is described in WO 2008/085486, published 17 Jul. 2008, which is incorporated by reference herein in its entirety. In one embodiment, a mutant HSV of the present invention and a subunit vaccine are administered at one time, while in another embodiment, a mutant HSV is administered and then, at a later time point, a subunit vaccine is administered, while in another embodiment, a subunit vaccine is administered and then, at a later time point, a mutant HSV is administered.
In one embodiment, the time period separating a first and second administration of a mutant HSV, or of a mutant HSV and another vaccine composition is 3-6 weeks. In another embodiment, the first and second administration (or contacting) are 1 week apart. In another embodiment, the first and second administration (or contacting) are 2 weeks apart. In another embodiment, the first and second administration (or contacting) are 3 weeks apart. In another embodiment, the first and second administration (or contacting) are 4 weeks apart. In another embodiment, the first and second administration (or contacting) are 5 weeks apart. In another embodiment, the first and second administration (or contacting) are 6 weeks apart. In another embodiment, the first and second administration (or contacting) are 7 weeks apart. In another embodiment, the first and second administration (or contacting) are 8 weeks apart. In another embodiment, the first and second administration (or contacting) are 1 month apart. In another embodiment, the first and second administration (or contacting) are 2 months apart.
It is to be understood that in one embodiment, methods of the present invention described hereinabove as comprising the step of contacting a subject with a mutant HSV strain, wherein the mutant strain contains an inactivating mutation in a Us8 gene encoding a gE protein may further comprise the step of a second contacting (or administration) of said subject with the same or another mutant HSV strain.
In one embodiment, “treating” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or lessen the targeted pathologic condition or disorder as described hereinabove. Therefore, in one embodiment, compositions for use in the methods of the present invention are administered to/contacted with a subject before exposure to HSV. In another embodiment, compositions for use in the methods of the present invention are administered to/contacted with a subject after exposure to HSV.
Thus, in one embodiment, treating may include directly affecting or curing, suppressing, inhibiting, preventing, reducing an incidence, reducing the severity of, delaying the onset of, reducing symptoms associated with the disease, disorder or condition, or a combination thereof. Thus, in one embodiment, “treating” refers inter alia to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative therapeutics, or a combination thereof. In another embodiment, treating refers to reducing the pathogenesis of, ameliorating the symptoms of, ameliorating the secondary symptoms of, or prolonging the latency to a relapse of a Herpes Simplex Virus (HSV) infection in a subject. In one embodiment, “preventing” refers, inter alia, to delaying the onset of symptoms, preventing relapse to a disease, decreasing the number or frequency of relapse episodes, increasing latency between symptomatic episodes, or a combination thereof. In one embodiment, “suppressing” or “inhibiting”, refers inter alia to reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, or a combination thereof.
In one embodiment, symptoms are primary, while in another embodiment, symptoms are secondary. In one embodiment, “primary” refers to a symptom that is a direct result of the subject viral infection, while in one embodiment, “secondary” refers to a symptom that is derived from or consequent to a primary cause. In one embodiment, the compositions and strains for use in the present invention treat primary or secondary symptoms or secondary complications related to HSV infection.
In another embodiment, “symptoms” may be any manifestation of a HSV infection, comprising blisters, ulcerations, or lesions on the urethra, cervix, upper thigh, and/or anus in women and on the penis, urethra, scrotum, upper thigh, and anus in men, inflammation, swelling, fever, flu-like symptoms, sore mouth, sore throat, pharyngitis, pain, blisters on tongue, mouth or lips, ulcers, cold sores, neck pain, enlarged lymph nodes, reddening, bleeding, itching, dysuria, headache, muscle pain, etc., or a combination thereof.
The gE protein as described in the methods and compositions of the present invention has, in one embodiment, the sequence:







(SEQ ID No: 2)


MDRGAVVGFLLGVCVVSCLAGTPKTSWRRVSVGEDVSLLPAPGPTGRGPT


 


QKLLWAVEPLDGCGPLHPSWVSLMPPKQVPETVVDAACMRAPVPLAMAYA


 


PPAPSATGGLRTDFVWQERAAVVNRSLVIHGVRETDSGLYTLSVGDIKDP


 


ARQVASVVLVVQPAPVPTPPPTPADYDEDDNDEGEDESLAGTPASGTPRL


 


PPPPAPPRSWPSAPEVSHVRGVTVRMETPEAILFSPGETFSTNVSIHAIA


 


HDDQTYSMDVVWLRFDVPTSCAEMRIYESCLYHPQLPECLSPADAPCAAS


 


ATWTSRLAVRSYAGCSRTNPPPRCSEAHMEPVPGLAWQAASVNLEFRDAS


 


PQHSGLYLCVVYVNDHIHAWGHITISTAAQYRNAVVEQPLPQRGADLAEP


 


ATHPHVGAPPHAPPTHGALRLGAVMGAALLLSALGLSVWACMTCWRRRAW


 


RVKSRASGKGPTYIRVADSELYADWSSDSEGERDQVPWLAPPERPDSPST


 


NGSGFEILSPTAPSVYPRSDGHQSRRQLTTFGSGRPDRRYSQASDSSVF 


 


W.






In another embodiment, the gE protein is a homologue of SEQ ID No: 2. In another embodiment, the gE protein is a variant of SEQ ID No: 2. In another embodiment, the gE protein is an isomer of SEQ ID No: 2. In another embodiment, the gE protein is a fragment of SEQ ID No: 2. In another embodiment, the gE protein comprises SEQ ID No: 2.

In another embodiment, the gE protein is encoded by a nucleotide sequence having the sequence:







(SEQ ID No: 3)


atggatcgcggggcggtggtggggtttcttctcggtgtttgtgttgtatc


 


gtgcttggcgggaacgcccaaaacgtcctggagacgggtgagtgtcggcg


 


aggacgtttcgttgcttccagctccggggcctacggggcgcggcccgacc


 


cagaaactactatgggccgtggaacccctggatgggtgcggccccttaca


 


cccgtcgtgggtctcgctgatgccccccaagcaggtgcccgagacggtcg


 


tggatgcggcgtgcatgcgcgctccggtcccgctggcgatggcgtacgcc


 


cccccggccccatctgcgaccgggggtctacgaacggacttcgtgtggca


 


ggagcgcgcggccgtggttaaccggagtctggttattcacggggtccgag


 


agacggacagcggcctgtataccctgtccgtgggcgacataaaggacccg


 


gctcgccaagtggcctcggtggtcctggtggtgcaaccggccccagaccg


 


accccacccccgaccccagccgattacgacgaggatgacaatgacgaggg


 


cgaggacgaaagtctcgccggcactcccgccagcgggaccccccggctcc


 


cgcctccccccgcccccccgaggtcaggcccagcgcccccgaagtctcac


 


atgtgcgtggggtgaccgtgcgtatggagactccggaagctatcctgatt


 


cccccggggagacgttcagcacgaacgtctccatccatgccatcgcccac


 


gacgaccagacctactccatggacgtcgtctggagaggacgacgtgccga


 


cctcgtgtgccgagatgcgaatatacgaatcgtgtctgtatcacccgcag


 


ctcccagaatgtctgtccccggccgacgcgccgtgcgccgcgagtacgtg


 


gacgtctcgcctggccgtccgcagctacgcggggtgttccagaacaaacc


 


ccccaccgcgctgttcggccgaggctcacatggagcccgtcccggggctg


 


gcgtggcaggcggcctccgtcaatctggagttccgggacgcgtccccaca


 


acactccggcctgtatctgtgtgtggtgtacgtcaacgaccatattcacg


 


cctggggccacattaccatcagcaccgcggcgcagtaccggaacgcggtg


 


gtggaacagcccctcccacagcgcggcgcggataggccgagcccacccac


 


ccgcacgtcggggcccctccccacgcgcccccaacccacggcgccctgcg


 


gttaggggcggtgatgggggccgccctgctgctgtctgcactggggagtc


 


ggtgtgggcgtgtatgacctgaggcgcaggcgtgcctggcgggcggttaa


 


aagcagggcctcgggtaaggggcccacgtacattcgcgtggccgacagcg


 


agctgtacgcggactggagctcggacagcgagggagaacgcgaccaggtc


 


ccgtggctggcccccccggagagacccgactctccctccaccaatggatc


 


cggctttgagatcttatcaccaacggctccgtctgtatacccccgtagcg


 


atgggcatcaatctcgccgccagctcacaacctaggatccggaaggcccg


 


atcgccgttactcccaggcctccgattcgtccgtcttctggtaa.






In another embodiment, the gE protein is encoded by a nucleotide molecule that a homologue of SEQ ID No: 3. In another embodiment, the nucleotide molecule is a variant of SEQ ID No: 3. In another embodiment, the nucleotide molecule is an isomer of SEQ ID No: 3. In another embodiment, the nucleotide molecule is a fragment of SEQ ID No: 3. In another embodiment, the nucleotide molecule comprises SEQ ID No: 3.

In another embodiment, the gE protein as described in the methods and compositions of the present invention has the sequence:







(SEQ ID No: 4)


MDRGAVVGFLLGVCVVSCLAGTPKTSWRRVSVGEDVSLLPAPGPTGRGPT


 


QKLLWAVEPLDGCGPLHPSWVSLMPPKQVPETVVDAACMRAPVPLAMAYA


 


PPAPSATGGLRTDFVWQERAAVVNRSLVIYGVRETDSGLYTLSVGDIKDP


 


ARQVASVVLVVQPAPVPTPPPTPADYDEDDNDEGEGEDESLAGTPASGTP


 


RLPPSPAPPRSWPSAPEVSHVRGVTVRMETPEAILFSPGEAFSTNVSIHA


 


IAHDDQTYTMDVVWLRFDVPTSCAEMRIYESCLYHPQLPECLSPADAPCA


 


ASTWTSRLAVRSYAGCSRTNPPPRCSAEAHMEPFPGLAWQAASVNLEFRD


 


ASPQHSGLYLCVVYVNDHIHAWGHITINTAAQYRNAVVEQPLPQRGADLA


 


EPTHPHVGAPPHAPPTHGALRLGAVMGAALLLSALGLSVWACMTCWRRRA


 


WRAVKSRASGKGPTYIRVADSELYADWSSDSEGERDQVPWLAPPERPDSP


 


STNGSGFEILSPTAPSVYPRSDGHQSRRQLTTFGSGRPDRRYSQASDSSV


 


FW.






In another embodiment, the gE protein is a homologue of SEQ ID No: 4. In another embodiment, the gE protein is a variant of SEQ ID No: 4. In another embodiment, the gE protein is an isomer of SEQ ID No: 4. In another embodiment, the gE protein is a fragment of SEQ ID No: 4. In another embodiment, the gE protein comprises SEQ ID No: 4.

In another embodiment, the gE protein is encoded by a nucleotide sequence having the sequence:







(SEQ ID No: 5)


atggatcgcggggcggtggtggggtttcttctcggtgtttgtgttgtatc


 


gtgcttggcgggaacgcccaaaacgtcctggagacgggtgagtgtcggcg


 


aggacgtttcgttgctaccagctccggggcctacggggcgcggcccgacc


 


cagaaactactatgggccgtggaacccctggatgggtgcggccccttaca


 


cccgtcgtgggtctcgctgatgccccccaagcaggtacccgagacggtcg


 


tggatgcggcgtgcatgcgcgctccggtcccgctggcgatggcatacgcc


 


cccccggccccatctgcgaccgggggtctacggacggacttcgtgtggca


 


ggagcgcgcggccgtggttaaccggagtctggttatttacggggtccgag


 


agacggacagcggcctgtataccctgtctgtgggcgacataaaggacccg


 


gctcgccaagtggcctcggtggtcctggtggtgcaaccggccccagttcc


 


gactccacccccgaccccagccgattacgacgaggatgacaatgacgagg


 


gcgagggcgaggacgaaagtctagccggcactcccgccagcgggaccccc


 


cggctcccgccacccccgcccccccgaggtcaggcccagcgcccccgaag


 


tctcacacgtgcgtggggtgaccgtgcgtatggagactccggaagctatc


 


ctgattcccccggggaggcgtttagcacgaacgtctccatccatgccatc


 


gcccacgacgaccagacctacaccatggacgtcgtctggttgaggttcga


 


cgtgccgacctcgtgtgccgagatgcgaatatacgaatcgtgtctgtatc


 


atccgcagctcccagagtgtctgtccccggccgacgctccgtgcgccgcg


 


agtacgtggacgtctcgcctggccgtccgcagctacgcggggtgaccaga


 


acaaaccccccgccgcgctgacggccgaggctcacatggagcccacccgg


 


ggctggcgtggcaggcggcctcagtcaatctggagaccgggacgcgtccc


 


cacaacactccgggctgtatctgtgcgtggtgtacgtcaacgaccatatt


 


cacgcatggggccacattaccatcaacaccgcggcgcagtaccggaacgc


 


ggtggtggaacagcccctcccacagcgcggcgcggataggccgagcccac


 


ccacccgcacgtcggggcccctccccacgcgcccccaacccacggcgccc


 


tgcggttaggggcggtgatgggggccgccctgctgctgtctgcgctgggg


 


ttgtcggtgtgggcgtgtatgacctgttggcgcaggcgtgcctggcgggc


 


ggttaaaagcagggcctcgggtaaggggcccacgtacattcgcgtggccg


 


acagcgagctgtacgcggactggagctcggacagcgagggagaacgcgac


 


caggtcccgtggctggcccccccggagagacccgactctccctccaccaa


 


tggatccggattgagatcttatcaccaacggctccgtctgtatacccccg


 


tagcgatgggcatcaatctcgccgccagctcacaacctttggatccggaa


 


ggcccgatcgccgttactcccaggcctccgattcgtccgtcttctggtaa.






In another embodiment, the gE protein is encoded by a nucleotide molecule that a homologue of SEQ ID No: 5. In another embodiment, the nucleotide molecule is a variant of SEQ ID No: 5. In another embodiment, the nucleotide molecule is an isomer of SEQ ID No: 5. In another embodiment, the nucleotide molecule is a fragment of SEQ ID No: 5. In another embodiment, the nucleotide molecule comprises SEQ ID No: 5.

In another embodiment, the gE protein as described in the methods and compositions of the present invention has the sequence:







MARGAGLVFFVGVWVVSCLAAAPRTSWKRVTSGEDVVLLPAPAERTRAHK


 


LLWAAEPLDACGPLRPSWVALWPPRRVLETVVDAACMRAPEPLAIAYSPP


 


FPAGDEGLYSELAWRDRVAVVNESLVIYGALETDSGLYTLSVVGLSDEAR


 


QVASVVLVVEPAPVPTPTPDDYDEEDDAGVTNARRSAFPPQPPPRRPPVA


 


PPTHPRVIPEVSHVRGVTVHMETLEAILFAPGETFGTNVSIHAIAHDDGP


 


YAMDVVWMRFDVPSSCADMRIYEACLYHPQLPECLSPADAPCAVSSWAYR


 


LAVRSYAGCSRTTPPPRCFAEARMEPVPGLAWLASTVNLEFQHASPQHAG


 


LYLCVVYVDDHIHAWGHMTISTAAQYRNAVVEQHLPQRQPEPVEPTRPHV


 


RAPHPAPSARGPLRLGAVLGAALLLAALGLSAWACMTCWRRRSWRAVKSR


 


ASATGPTYIRVADSELYADWSSDSEGERDGSLWQDPPERPDSPSTNGSGF


 


EILSPTAPSVYPHSEGRKSRRPLTTFGSGSPGRRHSQASYPSVLW


(SEQ ID No: 6; this protein was mutated in 


Examples 1-5 herein).






In another embodiment, the gE protein is a homologue of SEQ ID No: 6. In another embodiment, the gE protein is a variant of SEQ ID No: 6. In another embodiment, the gE protein is an isomer of SEQ ID No: 6. In another embodiment, the gE protein is a fragment of SEQ ID No: 6. In another embodiment, the gE protein comprises SEQ ID No: 6.

In another embodiment, the gE protein is encoded by a nucleotide sequence having the sequence:







atggctcgcggggccgggttggtgttttttgttggagtttgggtcgtatc


 


gtgcctggcggcagcacccagaacgtcctggaaacgggtaacctcgggcg


 


aggacgtggtgttgcttccggcgcccgcggaacgcacccgggcccacaaa


 


ctactgtgggccgcggaacccctggatgcctgcggtcccctgcgcccgtc


 


gtgggtggcgctgtggcccccccgacgggtgctcgagacggtcgtggatg


 


cggcgtgcatgcgcgccccggaaccgctcgccatagcatacagtcccccg


 


ttccccgcgggcgacgagggactgtattcggagttggcgtggcgcgatcg


 


cgtagccgtggtcaacgagagtctggtcatctacggggccctggagacgg


 


acagcggtctgtacaccctgtccgtggtcggcctaagcgacgaggcgcgc


 


caagtggcgtcggtggttctggtcgtggagcccgcccctgtgccgacccc


 


gacccccgacgactacgacgaagaagacgacgcgggcgtgacgaacgcac


 


gccggtcagcgttccccccccaaccccccccccgtcgtccccccgtcgcc


 


cccccgacgcaccctcgtgttatccccgaggtgtcccacgtgcgcggggt


 


aacggtccatatggagaccctggaggccattctgtttgcccccggggaga


 


cgtttgggacgaacgtctccatccacgccattgcccacgacgacggtccg


 


tacgccatggacgtcgtctggatgcggtttgacgtgccgtcctcgtgcgc


 


cgatatgcggatctacgaagcttgtctgtatcacccgcagcttccagagt


 


gtctatctccggccgacgcgccgtgcgccgtaagttcctgggcgtaccgc


 


ctggcggtccgcagctacgccggctgttccaggactacgcccccgccgcg


 


atgttttgccgaggctcgcatggaaccggtcccggggttggcgtggctgg


 


cctccaccgtcaatctggaattccagcacgcctccccccagcacgccggc


 


ctctacctgtgcgtggtgtacgtggacgatcatatccacgcctggggcca


 


catgaccatcagcaccgcggcgcagtaccggaacgcggtggtggaacagc


 


acctcccccagcgccagcccgagcccgtcgagcccacccgcccgcacgtg


 


agagccccccatcccgcgccctccgcgcgcggcccgctgcgcctcggggc


 


ggtgctgggggcggccctgttgctggccgccctcgggctgtccgcgtggg


 


cgtgcatgacctgctggcgcaggcgctcctggcgggcggttaaaagccgg


 


gcctcggcgacgggccccacttacattcgcgtggcggacagcgagctgta


 


cgcggactggagttcggacagcgagggggagcgcgacgggtccctgtggc


 


aggaccctccggagagacccgactctccctccacaaatggatccggcttt


 


gagatcttatcaccaacggctccgtctgtatacccccatagcgaggggcg


 


taaatctcgccgcccgctcaccacctttggttcgggaagcccgggccgtc


 


gtcactcccaggcctcctatccgtccgtcctctggtaa


(SEQ ID No: 7; this gene was mutated in Examples 


1-5 herein.






In another embodiment, the gE protein is encoded by a nucleotide molecule that a homologue of SEQ ID No: 7. In another embodiment, the nucleotide molecule is a variant of SEQ ID No: 7. In another embodiment, the nucleotide molecule is an isomer of SEQ ID No: 7. In another embodiment, the nucleotide molecule is a fragment of SEQ ID No: 7. In another embodiment, the nucleotide molecule comprises SEQ ID No: 7.

In one embodiment, the gE protein is encoded by one of the following GenBank Accession Numbers: DQ889502, NC—001806, NC—001798, Z86099, X14112, L00036, X02138, and X04798, and any of AJ626469-AJ626498. In another embodiment, the gE protein is homologous to a sequence disclosed in one of the above GenBank Accession Numbers. In another embodiment, the gE protein is a variant of a sequence disclosed in one of the above GenBank Accession Numbers. In another embodiment, the gE protein is a fragment of a sequence disclosed in one of the above GenBank Accession Numbers.
In one embodiment, a gE protein HSV-1 glycoprotein E (gE) is a virion surface protein which is necessary for spread in neurons, and in one embodiment, is necessary for spread along axons in either direction, both to (“retrograde”), and from (“anterograde”), the neuronal cell body. In another embodiment, gE also facilitates evasion of the host immune system by sequestering host antibodies against HSV-1, rendering them inactive. In one embodiment, a gE-deleted HSV-1 replicates in the skin, but cannot spread along neurons to establish latency or escape the host's antibody response. Thus, in one embodiment, infection with the live attenuated gE-deleted HSV-1 or HSV-2 will elicit a robust immune response in the skin and protect the host from future encounters with the wild-type virus.
In one embodiment, the gE protein is a HSV-1 gE protein. In another embodiment, the gE protein is a HSV-1(NS) gE protein. In another embodiment, the gE protein is a HSV-1(17) gE protein. In another embodiment, the gE protein is a HSV-1(F) gE protein. In another embodiment, the gE protein is a HSV-1(KOS) gE protein. In another embodiment, the gE protein is a HSV-1(CL101) gE protein. In another embodiment, the gE protein is a HSV-1(MacIntyre) gE protein. In another embodiment, the gE protein is a HSV-1(MP) gE protein. In another embodiment, the gE protein is a HSV-1(17+syn) gE protein. In another embodiment, the gE protein is a HSV-1(HF) gE protein. In another embodiment, the gE protein is any other HSV-1 gE protein known in the art.
In one embodiment, the gE protein is a HSV-2 gE protein. In another embodiment, the gE protein is a HSV-2(HG52) gE protein. In another embodiment, the gE protein is a HSV-2(2.12) gE protein. In another embodiment, the gE protein is a HSV-2(MS) gE protein. In another embodiment, the gE protein is a HSV-2(186) gE protein. In another embodiment, the gE protein is a HSV-2(G) gE protein. In another embodiment, the gE protein is any other HSV-2 gE protein known in the art.
In another embodiment, the gE protein is any other HSV-1 or HSV-2 gE protein which in one embodiment has greater than 80% homology, in another embodiment, greater than 85% homology, in another embodiment greater than 95% homology, and in another embodiment greater than 98% homology to one of the gE proteins or nucleic acid sequences listed hereinabove. In another embodiment, the gE protein has 98.6% homology to HSV(NS) or HSV(17), or both.
In one embodiment, the gE protein is any other gE protein known in the art. In another embodiment, the gE protein is encoded by any Us8 nucleotide known in the art. In one embodiment, the Us8 gene has a nucleic acid sequence that corresponds to that set forth in Genbank Accession Nos: GeneID:2703448 or GeneID:1487360, or encodes a protein sequence of glycoprotein E, which in one embodiment, corresponds to that set forth in Genbank Accession Nos: NP—044670.1 or NP—044538.1.
In one embodiment, the HSV strain of and for use in the methods of the present invention comprise an additional inactivating mutation, which in one embodiment, is an inactivation of the Us6 gene. In one embodiment, the Us6 gene has a nucleic acid sequence that corresponds to that set forth in Genbank Accession Nos: GeneID:2703444, GeneID:1487358, NC—001806, NC—001798, EU029158, EF177451, EF177450, EF157322, EF157321, EF157320, EF157319, Z86099, AJ004801, X14112, AF147806, AY779754, AY779753, AY779752, AY779751, AY779750, AY517492, AY155225, AB016432, AF021342, U12183, U12182, U12181, U12180, or InterPro:IPRO02896, or encodes a protein sequence of glycoprotein D, which in one embodiment, corresponds to that set forth in Genbank Accession Nos: NP—044668.1, NP—044536.1, CAA38245, AAB59754, AAA19629, AAA19631, AAA19630, AAK93950, ABS84899, ABM66848, ABM66847, AAW23134, AAW23133, AAW23132, AAW23131, AAW23130, AAS01730, ABM52981, ABM52980, ABM52979, ABM52978, AAN74642, AAO26211, AAL90884, AAL90883, AAK19597, AAA45785, BAA00020, AAB60555, AAB60554, AAB60553, AAB60552, AAA98962, AAA98963, AAA45842, AAA45786, VGBEDZ, CAB06713, CAA32283, AAB72102, or CAB06713.1.
In another embodiment, the gD protein is any other HSV-1 or HSV-2 gD protein which in one embodiment has greater than 80% homology, in another embodiment, greater than 85% homology, in another embodiment greater than 95% homology, and in another embodiment greater than 98% homology to one of the gD proteins or nucleic acid sequences listed hereinabove.
In one embodiment, the HSV strain of and for use in the methods of the present invention comprise an additional inactivating mutation, which in one embodiment, is an inactivation of the Us9 gene. In one embodiment, the Us9 gene has a nucleic acid sequence that corresponds to that set forth in Genbank Accession Nos: GeneID:2703452 or GeneID:1487362, or encodes a protein sequence of Us9 membrane protein, which in one embodiment, corresponds to that set forth in Genbank Accession Nos: NP—044672.1 or NP—044540.1.
In one embodiment, the HSV strain of and for use in the methods of the present invention comprise an additional inactivating mutation, which in one embodiment, is an inactivation of the Us5 gene. In one embodiment, the Us5 gene has a nucleic acid sequence that corresponds to that set forth in Genbank Accession Nos: GeneID:2703406 or GeneID:1487357, or encodes a protein sequence of glycoprotein J, which in one embodiment, corresponds to that set forth in Genbank Accession Nos: NP—044667.1 or NP—044535.1.
In one embodiment, the HSV strain of and for use in the methods of the present invention comprise an additional inactivating mutation, which in one embodiment, is an inactivation of the Us4 gene. In one embodiment, the Us4 gene has a nucleic acid sequence that corresponds to that set forth in Genbank Accession Nos: GeneID:2703404 or GeneID:1487356, or encodes a protein sequence of glycoprotein G, which in one embodiment, corresponds to that set forth in Genbank Accession Nos: NP—044666.1 or NP—044534.1.
In one embodiment, the HSV strain of and for use in the methods of the present invention comprise an additional inactivating mutation, which in one embodiment, is an inactivation of the UL53 gene. In one embodiment, the UL53 gene has a nucleic acid sequence that corresponds to that set forth in Genbank Accession Nos: GeneID:2703425 or GeneID:1487342, or encodes a protein sequence of glycoprotein K, which in one embodiment, corresponds to that set forth in Genbank Accession Nos: NP—044656.1 or NP—044524.1.
In one embodiment, the HSV strain of and for use in the methods of the present invention comprise an additional inactivating mutation, which in one embodiment, is an inactivation of the UL10 gene. In one embodiment, the UL10 gene has a nucleic acid sequence that corresponds to that set forth in Genbank Accession Nos: GeneID:2703379 or GeneID:1487293, or encodes a protein sequence of glycoprotein M, which in one embodiment, corresponds to that set forth in Genbank Accession Nos: NP—044611.1 or NP—044479.1.
In one embodiment, the HSV strain of and for use in the methods of the present invention comprise an additional inactivating mutation, which in one embodiment, is an inactivation of the UL1 gene. In one embodiment, the UL1 gene has a nucleic acid sequence that corresponds to that set forth in Genbank Accession Nos: GeneID:2703393 or GeneID:1487292, or encodes a protein sequence of glycoprotein L, which in one embodiment, corresponds to that set forth in Genbank Accession Nos: NP—044602.1 or NP—044470.1.
In one embodiment, the HSV strain of and for use in the methods of the present invention comprise an additional inactivating mutation, which in one embodiment, is an inactivation of the UL22 gene. In one embodiment, the UL22 gene has a nucleic acid sequence that corresponds to that set forth in Genbank Accession Nos: GeneID:2703373 or GeneID:1487306, or encodes a protein sequence of glycoprotein H, which in one embodiment, corresponds to that set forth in Genbank Accession Nos: NP—044623.1 or NP—044491.1.
In one embodiment, the HSV strain of and for use in the methods of the present invention comprise an additional inactivating mutation, which in one embodiment, is an inactivation of the UL27 gene. In one embodiment, the UL27 gene has a nucleic acid sequence that corresponds to that set forth in Genbank Accession Nos: GeneID:2703455 or GeneID:1487312, or encodes a protein sequence of glycoprotein B, which in one embodiment, corresponds to that set forth in Genbank Accession Nos: NP—044629.1 or NP—044497.1.
In one embodiment, the HSV strain of and for use in the methods of the present invention comprise an additional inactivating mutation, which in one embodiment, is an inactivation of the UL44 gene. In one embodiment, the UL44 gene has a nucleic acid sequence that corresponds to that set forth in Genbank Accession Nos: GeneID:2703410 or GeneID:1487331, or encodes a protein sequence of glycoprotein C, which in one embodiment, corresponds to that set forth in Genbank Accession Nos: NP—044646.1 or NP—044514.1.
In one embodiment, the HSV strain of and for use in the methods of the present invention comprise an additional inactivating mutation, which in one embodiment, is an inactivation of the UL49a gene. In one embodiment, the UL49a gene has a nucleic acid sequence that corresponds to that set forth in Genbank Accession Nos: GeneID:2703419 or GeneID:1487337, or encodes a protein sequence of glycoprotein N, which in one embodiment, corresponds to that set forth in Genbank Accession Nos: NP—044652.1 or NP—044520.1.
In another embodiment, the additional mutation is in an HSV-1 or HSV-2 glycoprotein that, in one embodiment, has greater than 80% homology, in another embodiment, greater than 85% homology, in another embodiment greater than 95% homology, and in another embodiment greater than 98% homology to one or more of the glycoproteins listed hereinabove.
In one embodiment, HSV strains of and for use in the instant invention may comprise an inactivating mutation in a gene encoding gD, which in one embodiment is Us6. In another embodiment, HSV strains of and for use in the instant invention may comprise an inactivating mutation in a gene encoding gE, which in one embodiment is Us8. In another embodiment, HSV strains of and for use in the instant invention may comprise an inactivating mutation in a gene encoding gE and in a gene encoding gD. In one embodiment, the Us6 mutation is introduced to attenuate an HSV strain comprising a Us8 mutation that is highly virulent. In one embodiment, the Us6 mutation reduces virus entry. Us6 mutations, as well as any of the mutations of the present invention may be in either HSV-1 or HSV-2 or both. In one embodiment, HSV-1 gD and HSV-2 gD have a large degree of homology. In one embodiment, the amino acid sequences of HSV-1 gD and HSV-2 gD have 81% homology, or in another embodiment, greater than 80% homology, or in another embodiment, greater than 85% homology, or in another embodiment, greater than 90% homology, or in another embodiment, greater than 95% homology. In one embodiment, the nucleic acid sequences of HSV-1 gD and HSV-2 gD have 85% homology, or in another embodiment, greater than 80% homology, or in another embodiment, greater than 85% homology, or in another embodiment, greater than 90% homology, or in another embodiment, greater than 95% homology.
In one embodiment, the gD protein derived of the methods and compositions of the present invention has the sequence:







(SEQ ID No: 14)


MGGTAARLGAVILFVVIVGLHGVRGKYALADASLKMADPNRFRGKDLPVL


 


DQLTDPPGVRRVYHIQAGLPDPFQPPSLPITVYYAVLERACRSVLLNAPS


 


EAPQIVRGASEDVRKQPYNLTIAWFRMGGNCAIPITVMEYTECSYNKSLG


 


ACPIRTQPRWNYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTE


 


ITQFILEHRAKGSCKYALPLRIPPSACLSPQAYQQGVTVDSIGMLPRFIP


 


ENQRTVAVYSLKIAGWHGPKAPYTSTLLPPELSETPNATQPELAPEDPED


 


SALLEDPVGTVAPQIPPNWHIPSIQDAATPYHPPATPNNMGLIAGAVGGS


 


LLAALVICGIVYWMHRRTRKAPKRIRLPHIREDDQPSSHQPL.






In another embodiment, the gD protein is a homologue of SEQ ID No: 14. In another embodiment, the gD protein is a variant of SEQ ID No: 14. In another embodiment, the gD protein is an isomer of SEQ ID No: 14. In another embodiment, the gD protein is a fragment of SEQ ID No: 14. In another embodiment, the gD protein comprises SEQ ID No: 14. In one embodiment, the gD amino acid sequence is an HSV-1 amino acid sequence.

In another embodiment, the gD protein is encoded by a nucleotide sequence having the sequence:







(SEQ ID No: 15)


gtggccccggcccccaacaaaaatcacggtagcccggccgtgtgacacta


 


tcgtccataccgaccacaccgacgaacccctaagggggaggggccatttt


 


acgaggaggaggggtataacaaagtctgtctttaaaaagcaggggttagg


 


gagttgttcggtcataagcttcagcgcgaacgaccaactaccccgatcat


 


cagttatccttaaggtctcttttgtgtggtgcgttccggtatggggggga


 


ctgccgccaggttgggggccgtgattttgtttgtcgtcatagtgggcctc


 


catggggtccgcggcaaatatgccttggcggatgcctctctcaagatggc


 


cgaccccaatcgctttcgcggcaaagaccttccggtcctggaccagctga


 


ccgaccctccgggggtccggcgcgtgtaccacatccaggcgggcctaccg


 


gacccgaccagccccccagcctcccgatcacggatactacgccgtgagga


 


gcgcgcctgccgcagcgtgctcctaaacgcaccgtcggaggccccccaga


 


ttgtccgcggggcctccgaagacgtccggaaacaaccctacaacctgacc


 


atcgcaggatcggatgggaggcaactgtgctatccccatcacggtcatgg


 


agtacaccgaatgctcctacaacaagtctctgggggcctgtcccatccga


 


acgcagccccgctggaactactatgacagcttcagcgccgtcagcgagga


 


taacctggggacctgatgcacgcccccgcgatgagaccgccggcacgtac


 


ctgcggctcgtgaagataaacgactggacggagattacacagatatcctg


 


gagcaccgagccaagggctcctgtaagtacgccctcccgctgcgcatccc


 


cccgtcagcctgcctctccccccaggcctaccagcagggggtgacggtgg


 


acagcatcgggatgctgccccgcttcatccccgagaaccagcgcaccgtc


 


gccgtatacagcttgaagatcgccgggtggcacgggcccaaggccccata


 


cacgagcaccctgctgcccccggagctgtccgagacccccaacgccacgc


 


agccagaactcgccccggaagaccccgaggattcggccctcaggaggacc


 


ccgtggggacggtggcgccgcaaatcccaccaaactggcacatcccgtcg


 


atccaggacgccgcgacgccttaccatcccccggccaccccgaacaacat


 


gggcctgatcgccggcgcggtgggcggcagtctcctggcagccctggtca


 


tttgcggaattgtgtactggatgcaccgccgcactcggaaagccccaaag


 


cgcatacgcctcccccacatccgggaagacgaccagccgtcctcgcacca


 


gcccttgattactagatacccccccttaatgggtgcgggggggtcaggtc


 


tgcggggttgggatgggaccttaactccatataaagcgagtctggaaggg


 


gggaaaggcggacagtcgataagtcggtagcgggggacgcgcacctgacc


 


gcctgtcgcacccacagctattcgcgaaccgtcccgattcgggat.






In another embodiment, the gD protein is encoded by a nucleotide molecule that a homologue of SEQ ID No: 15. In another embodiment, the nucleotide molecule is a variant of SEQ ID No: 15. In another embodiment, the nucleotide molecule is an isomer of SEQ ID No: 15. In another embodiment, the nucleotide molecule is a fragment of SEQ ID No: 15. In another embodiment, the nucleotide molecule comprises SEQ ID No: 15. In one embodiment, the gD nucleotide sequence is an HSV-1 nucleotide sequence.

In one embodiment, the gD protein as described in the methods and compositions of the present invention has the sequence:







(SEQ ID No: 16)


MGRLTSGVGTAALLVVAVGLRVVCAKYALADPSLKMADPNRFRGKNLPVL


 


DQLTDPPGVKRVYHIQPSLEDPFQPPSIPITVYYAVLERACRSVLLHAPS


 


EAPQIVRGASDEARKHTYNLTIAWYRMGDNCAIPITVMEYTECPYNKSLG


 


VCPIRTQPRWSYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTE


 


ITQFILEHRARASCKYALPLRIPPAACLTSKAYQQGVTVDSIGMLPRFIP


 


ENQRTVALYSLKIAGWHGPKPPYTSTLLPPELSDTTNATQPELVPEDPED


 


SALLEDPAGTVSSQIPPNWHIPSIQDVAPHHAPAAPSNPGLIIGALAGST


 


LAVLVIGGIAFWVRRRAQMAPKRLRLPHIRDDDAPPSHQPLFY.






In another embodiment, the gD protein is a homologue of SEQ ID No: 16. In another embodiment, the gD protein is a variant of SEQ ID No: 16. In another embodiment, the gD protein is an isomer of SEQ ID No: 16. In another embodiment, the gD protein is a fragment of SEQ ID No: 16. In another embodiment, the gD protein comprises SEQ ID No: 16. In one embodiment, the gD amino acid sequence is an HSV-2 amino acid sequence.

In another embodiment, the gD protein is encoded by a nucleotide sequence having the sequence:









(SEQ ID No: 17)



atggggcgtt tgacctccgg cgtcgggacg gcggccctgc



 



tagttgtcgc ggtgggactc cgcgtcgtct gcgccaaata 



 



cgccttagca gacccctcgc ttaagatggc cgatcccaat



 



cgatttcgcg ggaagaacct tccggttttg gaccagctga



 



ccgacccccc cggggtgaag cgtgtttacc acattcagcc 



 



gagcctggag gacccgttcc agccccccag catcccgatc



 



actgtgtact acgcagtgct ggaacgtgcc tgccgcagcg



 



tgctcctaca tgccccatcg gaggcccccc agatcgtgcg



 



cggggcttcg gacgaggccc gaaagcacac gtacaacctg



 



accatcgcct ggtatcgcat gggagacaat tgcgctatcc



 



ccatcacggt tatggaatac accgagtgcc cctacaacaa



 



gtcgttgggg gtctgcccca tccgaacgca gccccgctgg



 



agctactatg acagctttag cgccgtcagc gaggataacc



 



tgggattcct gatgcacgcc cccgccttcg agaccgcggg 



 



tacgtacctg cggctagtga agataaacga ctggacggag



 



atcacacaat ttatcctgga gcaccgggcc cgcgcctcct 



 



gcaagtacgc tctccccctg cgcatccccc cggcagcgtg



 



cctcacctcg aaggcctacc aacagggcgt gacggtcgac



 



agcatcggga tgctaccccg ctttatcccc gaaaaccagc



 



gcaccgtcgc cctatacagc ttaaaaatcg ccgggtggca



 



cggccccaag cccccgtaca ccagcaccct gctgccgccg



 



gagctgtccg acaccaccaa cgccacgcaa cccgaactcg



 



ttccggaaga ccccgaggac tcggccctct tagaggatcc 



 



cgccgggacg gtgtcttcgc agatcccccc aaactggcac



 



atcccgtcga tccaggacgt cgcgccgcac cacgcccccg



 



ccgcccccag caacccgggc ctgatcatcg gcgcgctggc



 



cggcagtacc ctggcggtgc tggtcatcgg cggtattgcg



 



ttttgggtac gccgccgcgc tcagatggcc cccaagcgcc



 



tacgtctccc ccacatccgg gatgacgacg cgcccccctc



 



gcaccagcca ttgttttact ag.






In another embodiment, the gD protein is encoded by a nucleotide molecule that a homologue of SEQ ID No: 17. In another embodiment, the nucleotide molecule is a variant of SEQ ID No: 17. In another embodiment, the nucleotide molecule is an isomer of SEQ ID No: 17. In another embodiment, the nucleotide molecule is a fragment of SEQ ID No: 17. In another embodiment, the nucleotide molecule comprises SEQ ID No: 17. In one embodiment, the gD nucleic acid sequence is an HSV-2 nucleic acid sequence.

In one embodiment, an inactivating mutation in a gene encoding gD comprises a mutation in which an alanine at amino acid 3 of HSV-1 gD or HSV-2 gD is mutated to a cysteine (A3C). In another embodiment, an inactivating mutation in a gene encoding gD comprises a mutation in which an alanine at residue 3 of HSV-1 gD or HSV-2 gD is mutated to a cysteine (A3C), a tyrosine at residue 2 to alanine (Y2A), a leucine at residue 4 to alanine (L4A), or a combination thereof. In another embodiment, an inactivating mutation in a gene encoding gD comprises a deletion of the alanine at residue 3 of HSV-1 gD or HSV-2 gD, a deletion of the tyrosine at residue 2, a deletion of leucine at residue 4, or a combination thereof. In another embodiment, an inactivating mutation in a gene encoding gD comprises a mutation at amino acid positions 38, 222, 223, 215, or a combination thereof. In another embodiment, an inactivating mutation in a gene encoding gD comprises a Y38C mutation, while in another embodiment, it comprises a R222N, F223I, D215G mutation, or combination thereof.
In another embodiment, an inactivating mutation in a gene encoding gD comprises mutations in amino acids 2 and 3, 3 and 4, 2-4, 1-5, 1-7, or 1-10. In another embodiment, an inactivating mutation in a gene encoding gD consists essentially of a mutation in which an alanine at amino acid 3 of HSV-1 gD or HSV-2 gD is mutated to a cysteine (A3C). In another embodiment, an inactivating mutation in a gene encoding gD consists of a mutation in which an alanine at amino acid 3 of HSV-1 gD or HSV-2 gD is mutated to a cysteine (A3C). In one embodiment, the numbering used to describe the location of the mutation refers to amino acid numbering of the mature peptide after cleaving of the signal sequence, which in one embodiment, is the first 25 amino acids for HSV-1 or HSV-2 gD, as is known in the art.
As provided herein and in one embodiment, a mutant HSV strain of the present invention comprising a mutation in gD has reduced virulence (Example 16) and ability to reach DRG (Example 17). In another embodiment, vaccination with a mutant HSV strain comprising a mutation in gD of the present invention protects against latent HSV infection (Example 18) after subsequent infection with virulent HSV. In another embodiment, the vaccination protects against disease caused by or associated with latent HSV infection. In another embodiment, the vaccination does not itself cause significant disease.
In one embodiment, the composition comprising a mutant HSV strain of the present invention further comprises an adjuvant. In one embodiment, the adjuvant comprises a CpG oligonucleotide. In another embodiment, the adjuvant comprises an aluminum salt. In another embodiment, the adjuvant comprises both a CpG oligonucleotide and an aluminum salt. In another embodiment, the adjuvant comprises any other adjuvant disclosed hereinabove. In another embodiment, the adjuvant comprises any combination of adjuvants disclosed hereinabove. An appropriate dose of adjuvant may readily be titrated by a skilled artisan and is routine in the art.
In another embodiment, the booster vaccination follows a single priming vaccination. “Priming vaccination” refers, in another embodiment, to a vaccination that comprises a mutant HSV of the present invention. In another embodiment, the term refers to a vaccine initially administered.
In another embodiment, a single booster vaccination is administered after the priming vaccination. In another embodiment, two booster vaccinations are administered after the priming vaccination. In another embodiment, three booster vaccinations are administered after the priming vaccination.
In one embodiment, the priming and booster vaccinations of the present invention are administered at a single site, while in another embodiment, they are administered at separate sites.
In some embodiments, any of the mutant HSV strains of and for use in the methods of this invention will comprise an inactivating mutation of the present invention, in any form or embodiment as described herein. In some embodiments, any of the mutant HSV strains of this invention will consist of an inactivating mutation of the present invention, in any form or embodiment as described herein. In some embodiments, the mutant HSV strains of this invention will consist essentially of an inactivating mutation of the present invention, in any form or embodiment as described herein. In some embodiments, the term “comprise” refers to the inclusion of the inactivating mutation, such as a mutation in gE or in gD, as well as inclusion of other mutations that may be known in the art. In some embodiments, the term “consisting essentially” of refers to a strain, whose only functional mutation is the indicated functional mutation, however, other mutations may be included that are not involved directly in the utility of the strain. In some embodiments, the term “consisting” refers to a strain, which contains mutation of a particular gene or a particular mutation.
In one embodiment, plasmid complementation may be used to complement the inactivating mutation, which in one embodiment, allows at least one round of infection with a mutant HSV of the invention.
In one embodiment, the present invention provides a composition for impeding formation of zosteriform lesions in a subject, the composition comprising a mutant HSV strain of the present invention.
In one embodiment, the present invention provides a composition for impeding herpetic ocular disease in a subject, the composition comprising a mutant HSV strain of the present invention.
In one embodiment, the present invention provides a composition for vaccinating a subject against an HSV infection, the composition comprising a mutant HSV strain of the present invention.
In one embodiment, the present invention provides a composition for impeding HSV infection in a subject, the composition comprising a mutant HSV strain of the present invention.
In one embodiment, the present invention provides a composition for impeding herpes-mediated encephalitis in a subject, the composition comprising a mutant HSV strain of the present invention.
In one embodiment, a gE protein of the present invention is homologous to a peptide disclosed or enumerated herein. The terms “homology,” “homologous,” etc., when in reference to any protein or peptide, refer, in one embodiment, to a percentage of amino acid (AA) residues in the candidate sequence that are identical with the residues of a corresponding native polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Methods and computer programs for the alignment are well known in the art.
Homology is, in one embodiment, determined by computer algorithm for sequence alignment, by methods well described in the art. For example, computer algorithm analysis of nucleic acid sequence homology can include the utilization of any number of software packages available, such as, for example, the BLAST, DOMAIN, BEAUTY (BLAST Enhanced Alignment Utility), GENPEPT and TREMBL packages.
In one embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 2-7 of greater than 70%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 2-7 of greater than 72%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-7 of greater than 75%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 2-7 of greater than 78%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-7 of greater than 80%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-7 of greater than 82%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 2-7 of greater than 83%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-7 of greater than 85%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-7 of greater than 87%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 2-7 of greater than 88%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-7 of greater than 90%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-7 of greater than 92%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 2-7 of greater than 93%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-7 of greater than 95%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 2-7 of greater than 96%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-7 of greater than 97%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-7 of greater than 98%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-7 of greater than 99%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-7 of 100%.
In one embodiment, homology is determined via determination of candidate sequence hybridization, methods of which are well described in the art (See, for example, “Nucleic Acid Hybridization” Hames B D and Higgins S J, Eds. (1985); Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y). In other embodiments, methods of hybridization are carried out under moderate to stringent conditions, to the complement of a DNA encoding a native caspase peptide. Hybridization conditions being, for example, overnight incubation at 42° C. in a solution comprising: 10-20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA.
Protein and/or peptide homology for any AA sequence listed herein is determined, in one embodiment, by methods well described in the art, including immunoblot analysis, or via computer algorithm analysis of AA sequences, utilizing any of a number of software packages available, via established methods. Some of these packages include the FASTA, BLAST, MPsrch or Scanps packages, and, in another embodiment, employ the use of the Smith and Waterman algorithms, and/or global/local or BLOCKS alignments for analysis, for example.
In one embodiment, “variant” refers to an amino acid or nucleic acid sequence (or in other embodiments, an organism or tissue) that is different from the majority of the population but is still sufficiently similar to the common mode to be considered to be one of them, for example splice variants.
In one embodiment, “isomer” refers to one of any of two or more substances that are composed of the same elements in the same proportions but differ in chemical and/or biological properties because of differences in the arrangement of atoms, which in one embodiment are stereoisomers, in another embodiment, constitutional isomers or tautomers. In one embodiment, an isomer is an optical isomer or entantiomer, a geometric isomer, a D- and L-isomer, positional isomer, or a cis-trans isomer.
In one embodiment of the present invention, “nucleic acids” or “nucleotide” refers to a string of at least two base-sugar-phosphate combinations. The term includes, in one embodiment, DNA and RNA. “Nucleotides” refers, in one embodiment, to the monomeric units of nucleic acid polymers. RNA is, in one embodiment, in the form of a tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, small inhibitory RNA (siRNA), micro RNA (miRNA) and ribozymes. The use of siRNA and miRNA has been described (Caudy A A et al., Genes & Devel 16: 2491-96 and references cited therein). DNA can be, in other embodiments, in form of plasmid DNA, viral DNA, linear DNA, or chromosomal DNA or derivatives of these groups. In addition, these forms of DNA and RNA can be single-, double-, triple-, or quadruple-stranded. The term also includes, in another embodiment, artificial nucleic acids that contain other types of backbones but the same bases. In one embodiment, the artificial nucleic acid is a PNA (peptide nucleic acid). PNA contain peptide backbones and nucleotide bases and are able to bind, in one embodiment, to both DNA and RNA molecules. In another embodiment, the nucleotide is oxetane-modified. In another embodiment, the nucleotide is modified by replacement of one or more phosphodiester bonds with a phosphorothioate bond. In another embodiment, the artificial nucleic acid contains any other variant of the phosphate backbone of native nucleic acids known in the art. The use of phosphothiorate nucleic acids and PNA are known to those skilled in the art, and are described in, for example, Neilsen P E, Curr Opin Struct Biol 9:353-57; and Raz N K et al. Biochem Biophys Res Commun. 297:1075-84. The production and use of nucleic acids is known to those skilled in art and is described, for example, in Molecular Cloning, (2001), Sambrook and Russell, Eds., and Methods in Enzymology: Methods for molecular cloning in eukaryotic cells (2003) Purchio and G. C. Fareed.
In one embodiment, the present invention provides a kit comprising a compound or composition utilized in performing a method of the present invention. In another embodiment, the present invention provides a kit comprising a composition, tool, or instrument of the present invention.
“Contacting,” in one embodiment, refers to directly contacting the target cell with a mutant HSV strain of the present invention. In another embodiment, “contacting” refers to indirectly contacting the target cell with a mutant HSV strain of the present invention. Thus, in one embodiment, methods of the present invention include methods in which the subject is contacted with a mutant HSV strain which is brought in contact with the target cell by diffusion or any other active transport or passive transport process known in the art by which compounds circulate within the body.
In one embodiment of the methods of the present invention, the mutant HSV strain is carried in the subjects' bloodstream to the target cell. In another embodiment, the mutant HSV strain is carried by diffusion to the target cell. In another embodiment, the mutant HSV strain is carried by active transport to the target cell. In another embodiment, the mutant HSV strain is administered to the subject in such a way that it directly contacts the target cell.
Pharmaceutical Compositions and Methods of Administration
In one embodiment, the methods of the present invention comprise administering a pharmaceutical composition comprising the mutant HSV strain and a pharmaceutically acceptable carrier.
“Pharmaceutical composition” refers, in one embodiment, to a therapeutically effective amount of the active ingredient, i.e. the mutant HSV strain, together with a pharmaceutically acceptable carrier or diluent. A “therapeutically effective amount” refers, in one embodiment, to that amount which provides a therapeutic effect for a given condition and administration regimen.
The pharmaceutical compositions containing the mutant HSV strain can be, in one embodiment, administered to a subject by any method known to a person skilled in the art, such as parenterally, transmucosally, transdermally, intramuscularly, intravenously, intra-dermally, subcutaneously, intra-peritonealy, intra-ventricularly, intra-cranially, intra-vaginally or intra-tumorally.
In another embodiment as described in the methods and compositions of the present invention, the pharmaceutical compositions are administered orally, and are thus formulated in a form suitable for oral administration, i.e. as a solid or a liquid preparation. Suitable solid oral formulations include tablets, capsules, pills, granules, pellets and the like. Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In another embodiment of the present invention, the active ingredient is formulated in a capsule. In accordance with this embodiment, the compositions of the present invention comprise, in addition to the active compound and the inert carrier or diluent, a hard gelating capsule.
In another embodiment, the pharmaceutical compositions are administered by intravenous, intra-arterial, or intra-muscular injection of a liquid preparation. Suitable liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In another embodiment, the pharmaceutical compositions are administered intravenously and are thus formulated in a form suitable for intravenous administration. In another embodiment, the pharmaceutical compositions are administered intra-arterially and are thus formulated in a form suitable for intra-arterial administration. In another embodiment, the pharmaceutical compositions are administered intra-muscularly and are thus formulated in a form suitable for intra-muscular administration.
In another embodiment, the pharmaceutical compositions are administered topically to body surfaces and are thus formulated in a form suitable for topical administration. Suitable topical formulations include gels, ointments, creams, lotions, drops and the like. For topical administration, the mutant HSV strain is prepared and applied as a solution, suspension, or emulsion in a physiologically acceptable diluent with or without a pharmaceutical carrier.
In another embodiment, the pharmaceutical compositions provided herein are controlled-release compositions, i.e. compositions in which the mutant HSV strain is released over a period of time after administration. Controlled- or sustained-release compositions include formulation in lipophilic depots (e.g. fatty acids, waxes, oils). In another embodiment, the composition is an immediate-release composition, i.e. a composition in which all the mutant HSV strain is released immediately after administration.
Each of the above additives, excipients, formulations and methods of administration represents a separate embodiment of the present invention.
The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.
EXPERIMENTAL DETAILS SECTION
Example 1
HSV(gE NULL) does not Cause Disease
Materials and Experimental Methods
Examples 1-4
Virus Strains
Wild-type HSV-1 strain NS, a low-passage-number clinical isolate, was used for generation of mutant viruses. To construct HSV-1(gE null), the entire gE coding sequence was excised from pCMV3gE-1 with XbaI and cloned into pSPT18. pSPT18 has the sequence:








(SEQ ID No: 1)



gaatacaagcttgcatgcctgcaggtcgactctagaggatccccgggtaccgagctcgaattccggtctccctatagtgagtcgtattaatttcgata



 


agccagctgggcctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgg


 


gagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgatagcggagtgtatatactgg


 


cttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcg


 


ctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacag


 


aatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctc


 


cgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctc


 


cctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgta


 


ggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgt


 


cttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagtt


 


cttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagct


 


cttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttg


 


atcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatcctttt


 


aaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcga


 


tctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgata


 


ccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctc


 


catccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagttttgcgcaacgttgttgccattgctacaggcatcgtggtg


 


tcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctc


 


cttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccg


 


taagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggat


 


aataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccag


 


ttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaa


 


aaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcgga


 


tacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcat


 


gacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggaga


 


cggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcg


 


gcatcagagcagattgtactgagagtgcaccatatcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttg


 


agcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacgggcctgccaccatacccacgccgaaacaagcgctc


 


atgagcccgaagtggcgagcccgatcaccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatg


 


cgtccggcgtagaggatctggctagcgatgaccctgctgattggttcgctgaccatttccgggtgcgggacggcgttaccagaaactcagaaggttcg


 


tccaaccaaaccgactctgacggcagtttacgagagagatgatagggtctgcttcagtaagccagatgctacacaattaggcttgtacatattgtcgt


 


tagaacgcggctacaattaatacataaccttatgtatcatacacatacgatttaggtgacactata.






A 1.1-kb HpaI-BglII fragment from amino acids (AA) 124-508 was excised, and the HpaI site was changed to a BglII site. A 4.3-kb fragment derived from pD6P containing the Escherichia coli β-galactosidase gene (β-gal) under the control of the HSV ICP6 promoter was cloned into the BglII site. The resultant vector contains 374 bp of NS DNA sequences 5′ and 225 bp 3′ of the ICP6::lacZ cassette and was used to construct the gE null virus. The XbaI fragment containing the flanking sequence vector was isolated, and 750 ng was cotransfected into Vero cells with 1.0 μg of NS DNA by calcium phosphate transfection. The DNA-calcium phosphate mixture was removed, and cells were shocked with 15% glycerol. Cells were harvested when cytopathic effects were noted in 30-40% of cells and were sonicated to prepare a virus pool. Recombinant gE null virus expressing β-gal was selected by infecting Vero cells and overlaying with 0.5% agarose, 5.0% fetal bovine serum (FBS), and 300 μg of 5-bromo-D-galactopyranoside (X-Gal). Blue plaques were picked and purified twice in X-Gal agarose overlay and once by limiting dilution. Virus was purified from supernatant fluids of infected Vero cells on a 5-70% sucrose gradient.
HSV-1(Rescue gE null) was prepared by co-transfection of Vero cells with 1.0 μg of NS-gEnull DNA and 1.5 μg of wild-type gE fragment purified from pCMV3gE-1, which was obtained by digesting HSV-1 (NS) DNA with NruI. Progeny viruses were examined by immunoperoxidase staining using anti-gE MAb 1BA10 to confirm expression of gE on the surface of infected cells. Plaques were purified by limiting dilution, and virus pools were prepared.
Virus stocks were grown on confluent Vero cells (an African green monkey kidney epithelial cell line) at an MOI of 2.0. 24 hours post-infection, cells were scraped in the media and centrifuged at 3,000×g. All but 1 mL of supernatant was removed, and cells were resuspended, sonicated for 3 seconds and distributed into 50 mcL (microliter) aliquots. For mock infections, similar aliquots were made using uninfected cells.
Mouse Flank Infection Protocol
All experimental protocols were approved by the University of Pennsylvania animal and laboratory resources IACUC committee. Five- to six-week-old BalbC mice (National Cancer Institute) were allowed to acclimate to the biosafety level 2 animal facility with constant temperature and photoperiod (12 hours of light, 12 hours of darkness) for 1 week. Mice were shaved and depilated with depilatory cream (NairTM) along the right flank (for vaccination) or the left flank (for challenge), then washed with warm water. The next day, mice were anesthetized via intraperitoneal injection of 75 mcL of 14.3 mg/ml ketamine and 1.8 mg/ml xylazine in PBS, then infected by making 60 superficial scratches in a 1 cm2 area of the flank, 1 cm dorsal to the spine, with a 263⁄8-gauge needle through a 10 mcL droplet containing 5×103-5×105 pfu HSV (FIG. 1). In HSV flank-infected mice, secondary spread back to the skin from the neurons of one or more spinal ganglia results in a belt-like lesion (FIG. 2). Mice were sacrificed by asphyxiation with CO2, followed by cervical dislocation. Mice were observed at 24-hour intervals starting at day 3 post-inoculation to record the appearance and severity of skin lesions and illness. A standardized scoring system to describe lesion severity was used to provide consistency in observations (FIG. 3).
Vaccination and Challenge
For vaccination, mice were infected by making scratches through the inoculum, containing either HSV-1(gE null) or virus-free cell lysate (described above), on the right flank. Mice were challenged 28 days later on the opposite (left) flank by scratching through a droplet containing 1×105 pfu HSV-1(NS).
Dissections of Dorsal Root Ganglia and Skin
Dorsal root ganglia (DRG) along either the right (for analysis of HSV-1(gE null) vaccination or HSV-1(Rescue gE null) infection), or left (for analysis of HSV-1(NS) challenge) sides of the spinal column were removed, pooled, and placed in 110 mcL DMEM (2.5% FBS) and frozen at −80° C. until analysis.
A 1-cm2 area of skin at the site of inoculation was removed. Half of the sample was placed in a tube containing 110 mcL DMEM (2.5% FBS) and frozen at −80° C. until analysis. The other half was placed on an index card with like-samples and immersed in 4% paraformaldehyde in 1×PBS for 24 hours at 4° C., then the solution was replaced with 1×PBS. Samples remained at 4° C. until processing for histological analysis.
Sectioning, Histology and Immunohistochemistry
Paraffin embedding, sectioning and staining of skin samples was performed by the Pathology Core Facility at Children's Hospital of Philadelphia. Skin sections were stained for HSV-1 antigen using anti-HSV-1 rabbit polyclonal antibody (DAKO) and counter-stained with hematoxylin.
Results
Groups of five mice were flank-infected by scarification of 103.5, 104.5, or 105.5 plaque-forming units (pfu) of the vaccine strain, HSV-1(gE null). For comparison to the virulent form of HSV-1, another group of five mice was flank-infected with 105.5 pfu of HSV-1(Rescue gE null), the vaccine strain backbone with the gene encoding gE restored. HSV-1(Rescue gE null) infection resulted in illness and 60% mortality (FIG. 4). However, no clinical signs of disease, illness or death occurred following HSV-1(gE null) vaccination.
In addition, the HSV-1(Rescue gE null) infection caused severe primary lesions that appeared ulcerative and necrotic (FIG. 5). In contrast, HSV-1(gE null) infection caused a mild skin pathology at the site of inoculation indistinguishable from that of mock vaccination. Thus, all or essentially all of the skin pathology following the administration of HSV-1(gE null) resulted from the process of scratch inoculation itself.
These findings show that infection with gE null herpes viruses does not cause disease.
Example 2
HSV(gE NULL) does not Spread within Sensory Neurons
HSV-1(Rescue gE null) infection caused severe secondary (zosteriform) ulcerative and necrotic lesions that first appeared at 4-5 (days post-infection) dpi (FIG. 6). In contrast, no secondary lesions were seen following HSV-1(gE null) infection. Since secondary lesion formation along the dermatome depends on the ability of the virus to spread along neurons from the skin to the ganglia, and then back again, these results show that the vaccine is not able to spread within sensory neurons. Therefore, HSV(gE null) is unable to cause recurrent infection.
Example 3
HSV(gE NULL) Replicates within Skin Cells
To determine the extent of replication of HSV-1(gE null) in skin cells, mice (n=3) were infected with HSV-1(gE null) or virulent HSV-1(Rescue gE null). On days 1, 3, 6, 8 and 13 post-inoculation, mice were sacrificed, skin at the site of inoculation was removed, and DRG from the right side of each mouse were pooled. Titering to determine viral content of the homogenized tissue revealed that HSV-1(gE null) replicated in skin following vaccination, but less extensively than HSV-1(Rescue gE-null) (FIG. 7). Further, HSV-1(gE null)-vaccinated skin was infiltrated by immune cells, and the virus was cleared by day 6 (FIG. 8).
These results show that HSV(gE null) replicates in the skin, and thus is expected to elicit an inflammatory response by the host.
Levels of HSV-1(Rescue gE null) exceeded that of HSV-1(gE null) by 4 orders of magnitude (FIG. 9). In addition, no infectious virus was detected in DRG of mice infected with HSV-1(gE null). The absence of the characteristic belt-like pattern of lesions and of detectable virus in the ganglia confirmed that no neuronal viral spread occurred after HSV-1(gE null) vaccination.
Thus, HSV infection in the absence of gE is limited to the skin of the host, and the immune system is able to detect and clear the virus rapidly.
Example 4
HSV-1(gE NULL) Vaccination is Protective Against Wild-Type HSV-1 Infection
Mock-vaccinated or HSV-1(gE null) vaccinated mice were challenged with a lethal dose of 105 pfu WT HSV-1 (NS strain) 28 days after vaccination. Whereas 100% of mock-vaccinated mice died following challenge, 100% of HSV-1(gE null)-vaccinated mice survived the lethal challenge (FIG. 10). All tested doses of HSV-1(gE null) (103.5, 104.5, and 105.5 pfu) were protective against challenge. Additionally, very little primary (inoculation site) disease was observed after challenge of the HSV-1(gE null)-vaccinated mice (FIG. 11). Vaccinated mice had undetectable levels of challenge virus in skin, at least 5 orders of magnitude less than mock-vaccinated mice (FIG. 12).
In contrast to the severely diseased mock-vaccinated mice, primary disease healed rapidly in vaccinated mice. Confirming this observation, immunohistochemistry of equivalent skin samples demonstrated the presence of very little antigen in vaccinated mice on day 3, and clearance by day 6 (FIG. 13). Histological analyses also revealed that vaccinated mice had significant infiltration of immune cells, showing that the vaccine successfully primed the host immune system.
Unlike the severe dermatome lesions resulting from zosteriform spread of the virus in mock-vaccinated mice, no zosteriform disease was seen in vaccinated mice (FIG. 14). In addition, viral titers in pulverized DRG were undetectable in vaccinated mice and thus, at least 4 orders of magnitude less than in mock-vaccinated mice (FIG. 15).
Example 5
HSV(gE NULL) Vaccination Prevents Establishment of Latent Wild-Type HSV-1 Infection after Subsequent Challenge
Materials and Experimental Methods
Recovery of Infectious Virus from Tissues
DRG and skin samples were removed from mice immediately after sacrifice (at 1, 3, 6, 8 or 13 dpi). Three mice were infected for each data point, but like tissues from these mice were analyzed individually. Tissues were placed in 110 mcL DMEM containing 2.5% FBS and stored at −80° C. until analysis. To determine viral titer, tissue was thawed and pulverized with a disposable pestle. Infectious virus in 50 mcL of the supernatant, serially diluted (1:10, 1:100 and 1:1000), was quantified by plaque assay on confluent Vero cells in 12-well dishes.
Explant of DRG
To recover infectious virus from latently infected mice, DRG were removed from the same (right) side of infection with HSV-1(gE null). All DRG from an individual mouse were placed in one well of a 12-well dish of confluent Vero cells bathed in DMEM (2.5% FBS). Medium was changed every 2 days and cells were monitored for signs of CPE, an indication that DRG contained infectious virus.
Results
DRG were removed from vaccinated mice, 28 days post-challenge, and explanted in order to reactivate latent challenge virus. Reactivation from associated neurons of only 1/15 of the vaccinated mice occurred upon removal (FIG. 16).
Results of this and the previous Example show that HSV(gE null) protects mice from neuronal infection upon challenge and from developing latent infection typically associated with WT HSV infections.
Example 6
Vaccination of Mice with HSV-1ΔgE Cross Protects Against HSV-2 Challenge
Materials and Experimental Methods
Female Balb/C mice, 6-8 weeks old, were acclimated to the animal facility for 10 days. Mice were anesthetized and shaved and hair was removed by Nair treatment of the right flank. The following day, anesthetized mice were mock-vaccinated or vaccinated with 5×105 pfu HSV-1ΔgE (which is referred to, in one embodiment, as gE null) by scratching 60 times through a 10 mcl (microliter) droplet of inoculum with a 26 (5⁄8)-gauge needle. The opposite flank (left side) of each mouse was shaved and denuded as before, twenty-seven days later. Mice were challenged the following day (day 28) by scratch inoculation of 105 pfu HSV-2 (strain 2.12). Mice were observed and scored daily for inoculation site disease, zosteriform disease and survival. (Scoring: 0=no disease 4=severe necrotic disease). Error bars represent the standard error of the mean (SEM).
Results
To test the ability of HSV-1ΔgE vaccination to protect against HSV-2 disease, HSV-1ΔgE-vaccinated mice were epidermally challenged with wild-type HSV-2. None of the HSV-1ΔgE vaccinated mice died, while 80% of mock vaccinated mice died (FIG. 17, top panel). Vaccinated mice exhibited inoculation site disease that healed rapidly compared to the unvaccinated mice (FIG. 17, middle panel). Additionally, vaccinated mice were completely protected from the zosteriform disease and death observed in the mock-vaccinated mice (FIG. 17, bottom panel).
Thus, ΔgE HSV vaccination is capable of protecting subjects against heterologous HSV disease, even of a different species of herpes simplex.
Example 7
Vaccination with HSV-1ΔgE Protects Against HSV-1(KOS) and Inhibits Establishment of HSV-1 Latency
Materials and Experimental Methods
Vaccination and assessment of disease were performed as described for the previous Example, except that 5×105 pfu HSV-1, strain KOS, was used for the challenge. For measurement of latent infection, mice were sacrificed 41 d post-challenge, and DRG from both right and left sides were removed, placed in DMEM/10% FBS, minced with scissors, and explanted onto sub-confluent Vero cell monolayers. Cultures were monitored daily for 20 d for plaque formation, indicative of reactivation from latency.
Results
This experiment tested the ability of HSV-1ΔgE vaccination to protect mice from the disease associated with a heterologous HSV-1 wild-type strain. HSV-1ΔgE-vaccinated mice challenged with wild-type HSV-1, strain KOS, exhibited inoculation site disease that healed rapidly compared to unvaccinated mice (FIG. 18, top panel). Additionally, HSV-1ΔgE vaccination protected HSV-1 KOS-challenged mice completely against zosteriform disease (FIG. 18, middle panel). Since HSV-1 KOS infection of mice is not lethal, this strain was utilized to compare viral reactivation from latency in mock-versus HSV-1ΔgE-vaccinated mice at 4 weeks post-challenge. In mock-vaccinated mice, HSV-1(KOS) virus reactivated from explanted DRG in 100% of mice (n=10), whereas only 1/10 HSV-1ΔgE vaccinated mice (10%) exhibited reactivation (FIG. 18, table in bottom panel), which could have been latent infection by either the vaccine or the challenge virus. Therefore, HSV-1ΔgE vaccination is effective at protecting mice from both disease and establishment of latency by heterologous HSV viruses.
Example 8
Vaccination with HSV-1ΔgE Protects Against HSV-1 Vaginal Challenge
Vaccination was performed as described for Example 6. Medroxyprogesterone acetate (2 mg) (Sicor Pharmaceuticals, Inc., Irvine Calif.), diluted to 100 mcl total volume in a 0.9% NaCl/10 mM HEPES buffer, was injected subcutaneously 33 days later into the neck area of each mouse. Five days later (day 38), mice were anesthetized, intra-vaginally swabbed with a calcium alginate swab dipped in PBS, and challenged by intra-vaginal instillation of 5×105 pfu HSV-1 (strain NS). Mice were allowed to recover in their cages, resting in a prone position. Challenged mice were observed daily for vaginal disease and survival. Daily intra-vaginal swabs were taken for analysis by viral titering on Vero cells.
Results
The ability of HSV-1ΔgE vaccination to protect against vaginal challenge with wild-type HSV-1 was tested. All mice vaccinated with HSV-1ΔgE survived the challenge, whereas 60% of mock-vaccinated mice succumbed (FIG. 19A, top panel). All mock-vaccinated mice showed some sign of visible disease in the vaginal area; however HSV-1ΔgE-vaccinated mice showed no obvious visible signs of disease (FIG. 19B). By day 1 post-vaginal challenge, HSV-1ΔgE-vaccinated mice had 100-fold less infectious virus than mock-vaccinated animals, as detected in vaginal swab samples. By day three post-challenge, no infectious virus was detected in swabs from HSV-1ΔgE-vaccinated mice, a value that is at least 30,000-fold less than mock-vaccinated mice on the same day. Additionally, infection of HSV-1ΔgE-vaccinated mice cleared nearly three times faster than mock-vaccinated mice (FIG. 19A, bottom panel). Thus, vaccination with HSV-1ΔgE protects from death and disease associated with HSV-1 vaginal challenge and confers the ability to rapidly clear HSV infection.
Example 9
Vaccination with HSV-1ΔgE by Epidermal, Subcutaneous, and Intramuscular Routes Protects Against HSV-1 Challenge
Materials and Experimental Methods
Vaccination was performed with 5×105 pfu HSV-1ΔgE by scratching 60 times through a 10 mcl droplet of inoculum with a 26 (5⁄8)-gauge needle, injection of 100 mcl inoculum into the scruff of the neck subcutaneously, or by intramuscular injection of 100 mcl inoculum into the right rear thigh muscle.
For measurement of latent infection, mice were sacrificed 32 days post-challenge, and DRG from both right and left sides were removed, placed in DMEM/10% FBS, minced with scissors and explanted onto sub-confluent Vero cell monolayers. Cultures were monitored daily (for 15 days) for plaque formation, indicating reactivation from latency.
Results
The efficacy of intramuscular and subcutaneous routes of administration of the HSV-1ΔgE vaccine was compared to epidermal scarification used in previous Examples. All routes of vaccination were effective at protecting mice against death upon epidermal challenge with HSV-1(NS) (FIG. 20, top panel). Mice vaccinated by each of the three routes exhibited inoculation site disease upon HSV-1(NS) challenge that was only slightly greater than mock-challenged mice (FIG. 20, bottom left panel). HSV-1ΔgE vaccination by epidermal scarification or intramuscular injection protected mice completely against zosteriform disease (FIG. 20, bottom right panel). Following challenge, 1⁄5 mice vaccinated by the subcutaneous route had several discrete zosteriform lesions, which were not severe and resolved quickly (FIG. 20, bottom right panel). In addition, the ability of the vaccine to prevent latent infection was measured. HSV-1ΔgE vaccination by all routes protected against the establishment of latency (Table 1). Mock-vaccinated mice showed 100% reactivation (Table 1; also see FIG. 18).







TABLE 1







HSV-1 vaccination by different routes protects against HSV latency.










Vaccination Route
Reactivation from Latency







Mock
1 of 1



Epidermal Scarification
0 of 4



Intra-Muscular
0 of 5



Subcutaneous
1 of 5










Thus, HSV-1ΔgE administered by epidermal, intramuscular or subcutaneous routes each protects against acute disease, flares and latent disease by wild-type HSV challenge.
Example 10
Vaccination with HSV-1ΔgE by Epidermal Scarification, Subcutaneous Injection and Intramuscular Injection Induces Neutralizing Antibodies
Materials and Experimental Methods
Mice were vaccinated as described in the previous Example. On day 21, mice were bled through jugular veins. On day 28, the opposite flank (left side) of each mouse was shaved and denuded as before. Neutralization assays on serum samples were done by incubating 50 mcl serum dilution (1:10 to 1:320) with 102 pfu HSV-1(NS) in 5 mcl for 1 hour at 37° C., and then inoculating Vero cell monolayers.
Results
The efficacy of HSV-1ΔgE vaccination by the epidermal scarification, intramuscular, and subcutaneous routes of administration for induction of neutralizing antibodies were measured. HSV-1ΔgE vaccination of mice by all three routes induced neutralizing antibody formation; the epidermal scarification and intramuscular routes yielded significantly higher levels than subcutaneous vaccination (FIG. 21).
Example 11
Vaccination with HSV-1ΔgE Protects Against Flank Challenge with Various Heterologous HSV-1 Strains
Ability of HSV-1ΔgE vaccination to protect against heterologous, higher virulence wild-type HSV-1 strains (F and 17) was measured; with vaccination and challenged performed as described for Example 7, except that challenge utilized various strains. HSV-1ΔgE vaccination protected mice completely from death upon epidermal challenge with HSV-1(NS), HSV-1(F) and HSV-1(17) (FIG. 22, top panel). Vaccination also reduced inoculation site disease, although challenge with HSV-1(F) caused slightly more disease at the inoculation site than HSV-1(NS) or HSV-1(17) (FIG. 22, middle panel). Moreover, HSV-1ΔgE vaccination completely protected all mice challenged with HSV-1(NS) and HSV-1(17) from zosteriform disease and 2⁄3 mice challenged with HSV-1(F); the other mouse challenged with HSV-1(F) had two small zosteriform lesions (FIG. 22, bottom panel). Thus, HSV-1ΔgE vaccination protects mice against various heterologous strains of HSV-1.
Example 12
Vaccination with HSV-1ΔgE Protects Against Flank Challenge with Doses Up to 1×107 PFU of HSV-1(NS)
The ability of HSV-1ΔgE vaccination to protect against higher doses of wild-type HSV-1 was measured; with vaccination and challenged performed as described for Example 7, except that challenge utilized higher doses of 105, 106 or 107 pfu of HSV-1(NS). Vaccinated mice were all completely protected from death and zosteriform disease (FIG. 23, top and bottom panels). Vaccinated mice challenged with 105 pfu HSV-1(NS) exhibited inoculation site disease that was slightly more severe than mock-challenged mice, indicating that most of the disease was caused by the scarification (needle scratch). Challenge of HSV-1ΔgE vaccinated mice with 106 or 107 pfu was associated with significantly reduced disease at the inoculation site, which healed rapidly compared with mock-vaccinated mice (middle panel).
Example 13
Characterization and Stability of HSV gD Mutant
Materials and Experimental Methods
Examples 13-18
Virus Strains
Wild-type HSV-1 strain KOS was used to prepare gD mutants. To construct HSV-1(gD null), plasmid pSC594 was constructed by inserting A3C (alanine to cysteine) and Y38C (tyrosine to cysteine) mutations into plasmid pRM416 which contains the KOS gD open reading frame flanked by 474 base pairs 5′ and 985 base pairs 3′ of the open reading frame. HSV-1 gD-mull DNA and pSC594 DNA were co-transfected into VD60 cells. Recombinant virus was screened by replication in Vero cells and then plaque-purified. After each plaque purification, 600 base pairs were amplified by PCR at the 5′ end of the gD gene that included the sites of the mutations. The amplified gD fragments were screened by restriction enzyme mapping. Introduction of a new SspI site confirmed the presence of the A3C mutation and the loss of an RsaI site confirmed the presence of the Y38C mutation. Following further plaque purification, DNA sequencing was used to confirm the presence of the mutations. The clones were grown to high titer on Vero cells, purified on a 10% to 60% sucrose gradient, and subjected to a final DNA sequence analysis and restriction mapping, which revealed that only the A3C mutation remained. The KOS-gDA3C was further purified on a sucrose gradient and the entire gD gene was sequenced to confirm the presence of the A3C mutation and the absence of additional unintended mutations.
Rescued KOS-gDA3C virus, referred to as rKOS-gDA3C, was generated by co-transfection of Vero cells with KOS-gDA3C and pRM416 DNA.
Virus stocks were grown in Dulbecco's minimum essential medium (DMEM), supplemented with 10% fetal calf serum (FCS). B78-H1 cells, mouse melanoma cells that are non-permissive for HSV-1 entry, were grown in DMEM with 5% FCS. B78-H1-A10 cells (A10) and B78-H1-C10 cells (C10) stably express HVEM and nectin-1, respectively, and were grown in DMEM containing 5% FCS and 500 μg/ml of G418. The gD-null virus was propagated in Vero cells stably transfected with gD DNA (VD60 cells). HSV-1 strain NS, a low-passage clinical isolate, was used for challenge studies in mice. Viruses were grown in Vero cells, unless otherwise noted, purified on sucrose gradients and stored at −80° C.
Mouse Flank Infection Protocol
All experimental protocols were approved by the University of Pennsylvania animal and laboratory resources IACUC committee. Five-six-week-old Balb/c mice (Charles River) were allowed to acclimate to the biosafety level 2 animal facility with constant temperature and photoperiod (12 hours of light, 12 hours of darkness) for 1 week. Mice were shaved and depilated with depilatory cream (NairTM) along the right flank (for vaccination) or the left flank (for challenge), then washed with warm water. The next day, mice were anesthetized via intraperitoneal injection of 75 mcL of 14.3 mg/ml ketamine and 1.8 mg/ml xylazine in PBS, then infected by making 60 superficial scratches in a 1 cm2 area of the flank, 1 cm dorsal to the spine, with a 30-gauge needle through a 10 mcL droplet containing 5×105 pfu HSV. Mice were observed at 24-hour intervals starting at day 3 post-inoculation to record the appearance and severity of skin lesions and illness. Scores at the inoculation site ranged from 0 to 5 and at the zosteriform site from 0 to 10. One point was assigned per vesicle or if lesions were confluent multiple points were assigned based on the size of the confluent lesions.
Entry Assay
KOS-gDA3C, rKOS-gDA3C or KOS (400 pfu) was incubated for one hour at 4° C. with B78-H1, A10, C10 or Vero cells. Cells were warmed to 37° C. for 0, 10, 30, 60 or 120 minutes followed by washing to remove unbound virus and exposed to a citrate buffer pH 3.0 wash for 1 minute to inactivate virus that had bound but had not entered cells. After an additional wash, cells were overlaid with 0.6% low-melt agar in DMEM, and plaques were visualized and counted after 68 hours.
Single-Step and Multi-Step Growth Curves
Single-step growth curves were performed on B78-H1, A10 and C10 cells inoculated with KOS, KOS-gDA3C or rKOS-gDA3C virus at an MOI of 3. After one hour at 37° C., cells were treated with citrate buffer pH 3.0 for one minute, and cells and supernatant fluids were collected immediately (time 0) or at 2, 4, 8, 10, 12, 20 and 24 hours. Samples were freeze-thawed once, sonicated three times each for 10 seconds and titered on Vero cells. Multi-step growth curves were performed in a similar fashion, except infection was performed at an MOI of 0.01 and titers measured at 24, 48 and 72 hours.
Real-Time Quantitative PCR for Viral DNA in Dorsal Root Ganglia (DRG)
DRG nearest the site of inoculation were harvested and DNA was isolated using the Qia Amp-mini DNA kit (Qiagen). The Us9 gene was amplified to quantify viral genome copy number in DRG. The PCR reaction was performed in a 50 mcl volume with a minimum of 200 ng of DNA from DRG. Fifty pmol of forward 5′ cgacgccttaataccgactgtt (SEQ ID NO: 8) and reverse 5′ acagcgcgatccgacatgtc (SEQ ID NO: 9) primers and 15 pmol of Taqman probe 5′ tcgttggccgcctcgtcttcgct (SEQ ID NO: 10) were added. One unit of Ampli Taq Gold (Applied Bioscience) per 50 mcl reaction was added. Real time PCR amplification was performed on an ABI Prism7700 Sequence Detector (Applied Biosystems). A standard curve was generated from purified HSV-1 (NS) DNA. Mouse adipsin, a cellular housekeeping gene was also amplified from DRG DNA under identical conditions. The primers used for amplification were forward 5′ gatgcagtcgaaggtgtggtta (SEQ ID NO: 11) and reverse 5′ cggtaggatgacactcgggtat (SEQ ID NO: 12), while Taqman probe 5′ tctcgcgtctgtggcaatggc (SEQ ID NO: 13) was used for detection. The viral DNA copies were then normalized based on the murine adipsin copy number.
Results
Since the gD transcript is co-terminal 3′ with gI and gJ, the molecular mass of gD and gI was evaluated by western blots of cells infected with WT, rKOS-gDA3C, or KOS-gDA3C virus. The size of the proteins was similar for the three viruses (FIG. 24A), while DNA sequencing confirmed the integrity of the gJ gene in KOS-gDA3C (result not shown). The stability of the gDA3C mutation was confirmed by restriction digestion using SspI of PCR-amplified DNA fragments to confirm the presence of the cysteine residue at position 3. The Ssp1 site was maintained through 30 passages, suggesting that the change of alanine to cysteine at residue 3 was stable (FIG. 24B). This was confirmed by DNA sequence analysis after every five passages.
Mice were scratch-inoculated on the flank with KOS-gDA3C, and DRG harvested five days post-infection to confirm the stability of the gDA3C mutation in vivo. Virus was isolated from three individual plaques. All three isolates retained the Ssp1 site (FIG. 24C), suggesting that the cysteine residue at amino acid 3 was maintained, which was confirmed by DNA sequencing.
Example 14
HSV gD Mutant as an Entry-Impaired Live Virus Vaccine
The entry of KOS, rKOS-gDA3C, and KOS-gDA3C into cells that express HVEM (A10), nectin-1 (C10), both (Vero), or neither receptor (B78-H1) was evaluated. Entry of the three viruses into Vero cells was comparable (FIG. 25A), while each virus failed to enter B78-H1 cells (FIG. 25B). Entry of KOS-gDA3C into A10 cells was reduced by approximately 50% compared with KOS or rKOS-gDA3C (FIG. 25C), and entry into C10 cells was reduced by approximately 70% (FIG. 25D).
These findings show that the gDA3C mutation reduces entry mediated by both HVEM and nectin-1 receptors.
Example 15
Growth Curves of HSV gD Mutant Virus
Virus replication was examined by performing single-step growth curves at an MOI of 3. KOS, rKOS-gDA3C and KOS-gDA3C failed to infect B78-H1 cells (results not shown). Replication of the three viruses was comparable in A10 cells (FIG. 26A) and C10 cells (FIG. 26B), except that the titers of KOS-gDA3C were reduced at time 0 (at the end of the one-hour adsorption period), which reflects the entry defect seen in Example 14.
Multi-step growth curves were performed by infecting the cells at an MOI of 0.01 to allow multiple cycles of virus replication. Compared with KOS and rKOS-gDA3C, peak titers of KOS-gDA3C were reduced at 72 hours by approximately 1.5 log 10 in A10 cells (FIG. 26C) and 2 log 10 in C10 cells (FIG. 26D).
Example 16
HSV gD Mutant has Reduced Virulence
The virulence of the KOS-gDA3C mutant was evaluated in the mouse flank model. Mice were infected with 5×105 PFU of KOS, rKOS-gDA3C, or KOS-gDA3C and animals scored for disease at the inoculation and zosteriform sites. Mice infected with KOS-gDA3C had less severe disease at the inoculation site (FIG. 27A) and almost no zosteriform disease with only one of 30 mice developing 3 lesions on day 5 (FIG. 27B). Photographs of the zosteriform site disease are shown on day 10 (FIG. 27C).
These findings show that infection with the gD mutant herpes virus causes minimal disease.
Example 17
HSV gD has Reduced Ability to Reach DRG
Mice were inoculated with 5×105 PFU of KOS, rKOS-gDA3C, or KOS-gDA3C and at 5 days post-infection, the DRG were harvested to measure viral titers (FIG. 28A) and viral genome copy number (FIG. 28B), which were reduced for KOS-gDA3C compared with KOS or rKOS-gDA3C.
These findings show that the gD mutant herpes virus is defective in reaching the DRG.
Example 18
HSV gD Mutant as an Attenuated Live Virus Vaccine
Mice were mock-infected or infected with rKOS-gDA3C or KOS-gDA3C and allowed to recover. Although rKOS-gDA3C produced extensive disease, all animals survived, as did all mice infected with KOS. Thirty days later, mice previously infected with KOS-gDA3C or rKOS-gDA3C were challenged on the opposite flank with HSV-1 strain NS at 106 PFU (approximately 20 LD50). The challenge virus caused extensive disease at the inoculation (FIG. 29A) and zosteriform (FIG. 29B) sites in the mock group. KOS-gDA3C and rKOS-gDA3C protected against disease at the inoculation site and both viruses totally prevented zosteriform disease. None of the rKOS-gDA3C or KOS-gDA3C infected mice died after the NS strain challenge, while 100% of the mock-infected mice died (result not shown).
These findings show that KOS-gDA3C provided protection against challenge that was comparable to protection provided by the more virulent rKOS-gDA3C.
The ability of a prior infection with KOS-gDA3C to prevent the WT virus from reaching the DRG was evaluated. Mice were mock-infected or infected in the flank with 5×105 pfu of rKOS-gDA3C or KOS-gDA3C. Thirty days later, mice were challenged with 106 pfu of NS on the opposite flank. DRG that innervate the challenge site were harvested five days post-challenge. NS viral titers were approximately 6 log 10 in DRG of mice that were previously mock infected, while no virus was recovered from DRG of mice previously infected with rKOS-gDA3C or KOS-gDA3C (FIG. 29C).
Quantitative PCR was performed on the DRG at five days post-challenge. Approximately 5.8 log 10 HSV-1 genome copies were detected in DRG of previously mock-infected mice compared with 3.4 or 3.2 log 10 DNA copies in mice previously infected with rKOS-gDA3C or KOS-gDA3C, respectively (FIG. 29D).
These findings show that KOS-gDA3C is attenuated in causing skin lesions at the inoculation and zosteriform sites and in infecting DRG, yet it is as effective as rKOS-gDA3C in protecting mice against WT HSV-1 challenge.
These examples suggest that an HSV strain with a mutation in gD may be used as an attenuated live HSV vaccine. FIG. 30 shows a model in which at each step of the virus life cycle, less KOS-gDA3C is produced because of the defect in virus entry. These steps include the amount of virus produced in epidermal cells (labeled E), in DRG nuclei (labeled N), and that return to the skin at the zosteriform site.
Example 19
HSV-2(gE NULL) does not Cause Disease
Materials and Experimental Methods
Cells and Viruses
Vero cells (ATCC CCL81) are cultured in Dulbecco's modified Eagle's medium containing heat-inactivated 10% newborn calf serum (Life Technologies, Gaithersburg, Md.) plus 50 micrograms (mcg) of penicillin/ml, 50 mcg/ml of streptomycin/ml, and 0.15 mcg/ml of Fungizone® (Life Technologies) at 37° C. and 5% CO2. Clarified stocks of HSV-2 strains are prepared from infected Vero monolayers and stored at −80° C. until used. Titers of virus are determined by standard plaque assays.
FIG. 31A demonstrates sequence alignment between gE of HSV-2(HG52) and HSV-1(NS). FIG. 31B demonstrates sequence alignment between gE of HSV-2(2.12) and HSV-2(HG52). A deletion in base pairs (bp) 369-1479 of the 1635 bp HSV-2 (2.12) Us8 gene, encoding HSV-2 gE, was introduced as follows. Two PCR fragments from HSV-2(2.12), namely a 658 bp fragment corresponding to the region 5′ of the intended deletion and a 536 bp fragment 3′ of the intended deletion, were subcloned into the pBluescript SK+ multiple cloning site (MCS). The 5′ flanking region was subcloned into the KpnI and HindIII sites of the pBluescript SK+ MCS, and the 3′ flanking region was subcloned into the PstI and SacI sites of the MCS. This left a short stretch of the MCS between the 5′ and 3′ flanking regions that includes the EcoRI and EcoRV restriction sites and causes a frameshift such that only the first 123 amino acids of gE were expressed (FIG. 31C). The vector was co-transfected into Vero cells with HSV-2(2.12) genomic DNA to allow for homologous recombination. The virion DNA purified from resulting plaques was screened by PCR to detect incorporation of the deletion.
Mouse Vaginal Model of HSV-2 Infection
Mice are treated with 2.0 mg of Depo-Provera (Upjohn, Kalamazoo, Mich.) subcutaneously in the scruff of the neck 7 and 1 day prior to viral inoculation to synchronize their estrus cycles and to increase their susceptibility to HSV-2 vaginal infection. HSV-2 virus (104 pfu) is instilled in the vaginal cavity following wet and then dry vaginal swabbing with a calcium alginate swab (Fisher Scientific, Pittsburgh, Pa.). Animals are assessed daily for symptomatic disease (as indicated by hair loss and erythema near the vagina) through 14 days post-inoculation (p.i.). Survival is followed through 21 days p.i. As an additional indicator of infection, vaginal swabs are collected and tested for viral content on Vero cells.
Results
We previously showed that HSV-1 NS-gEnull is a safe and effective vaccine in mice owing to the defect in anterograde neuronal transport and deficient cell to cell spread of this strain. In our most recent work, we have constructed an HSV-2 gE deletion mutant, HSV-2ΔgE(gfp), that deletes a region of gE-2 similar to the region deleted in NS-gEnull. We have shown that, similar to HSV-1 NS-gEnull, HSV-2ΔgE(gfp) is defective in anterograde spread and deficient in cell-to-cell spread. We also demonstrate that this strain causes no disease in mice and serves as an effective vaccine against HSV-2 epidermal and mucosal challenge of mice, particularly when given in mice as two doses separated by approximately three weeks.
gE null HSV-2 virus was constructed from strain HSV-2(2.12), using a similar strategy as that used for HSV-1 (Example 1). The HSV-2 mutant, HSV-2ΔgE(gfp), was produced by deleting and inserting the gfp2 cassette under the control of a CMV promoter. The gfp2 cassette, which allowed for screening of recombinant viruses by fluorescence, was inserted in the US8 reading frame just after the bases encoding amino acid 123. The portion of the US8 gene encoding the 156 C-terminal amino acids of gE remained but was not expressed. The mutation was made in wild-type strain HSV-2(2.12) (FIG. 32).
The HSV-2ΔgE(gfp) mutant did not express gE but had normal expression of gD, VP5 and US9 (FIG. 33). In vitro single-step growth kinetics of HSV-2ΔgE(gfp) in vitro were similar to WT in both epithelial (Vero cells) (FIG. 34A) and primary neuronal (superior cervical ganglia from rat embryos) (FIG. 34B) cell lines. In Vero cells, HSV-2ΔgE(gfp) produced significantly smaller plaques than HSV-2(2.12) (FIG. 34C), indicating that HSV-2ΔgE(gfp) had impaired cell-to-cell spread. Thus, replication of HSV-2ΔgE(gfp) was normal, but cell to cell spread was impaired.
Mouse retina were infected with 4×105 PFU HSV-2(2.12) or HSV-2ΔgE(gfp), and immunofluoresence in the retina and optic nerve of infected mice were observed on days 3 and 5 post-infection. Viral antigen was seen in retinas infected with both viruses (FIG. 35A), suggesting that a productive infection occurred. However, antigen was not seen in the optic nerve sections following HSV-2ΔgE(gfp) retina infection (FIG. 35B), indicating that the virus was defective in anterograde axonal transport. Therefore, HSV-2ΔgE(gfp) was defective in anterograde spread in vivo.
Anterograde and retrograde retinorecipient areas of the brain following HSV-2ΔgE(gfp) retina infection of the mouse were examined. Mouse retinas were infected with 4×105 PFU HSV-2ΔgE(gfp) or HSV-2(2.12). HSV-2 antigen appeared in the dorsal lateral geniculate nucleus (LGN) (arrowhead) and the optic tract (arrow) in HSV-2(2.12) infected mice by 5 dpi but not in the brains of mice infected with HSV-2ΔgE(gfp) at 5 or 8 days post infection (dpi; FIG. 36A). HSV-2 antigen was present in the dorsal LGN (arrowhead), the ventral LGN (open arrowhead) and the intergeniculate leaflet (IGL) of the LGN (arrow) in the brains of HSV-2(2.12) infected mice but not in the brains of mice infected with HSV-2ΔgE(gfp) (FIG. 36B). HSV-2 antigen was detected in the superior colliculus (SC) (arrowhead) and the oculomotor and Edinger-Westphal nuclei (arrow) of mice infected with HSV-2(2.12) infected mice but not in the brains of mice infected with HSV-2ΔgE(gfp) (FIG. 36C). In mouse retinas infected with HSV-2(2.12), virus traveled through both anterograde and retrograde optic circuits. However, no spread was observed in either direction in the brains of mice infected with HSV-2ΔgE(gfp), indicating that the vaccine is defective in both anterograde and retrograde directional spread in vivo.
Next, the safety of HSV-2ΔgE(gfp) in the mouse flank model was determined Mice were infected by scarification on denuded flank skin with 5×105 pfu HSV-2(2.12) or HSV-2ΔgE(gfp) and monitored daily for survival, inoculation site disease and zosteriform disease. In contrast to HSV-2(2.12) flank infection, HSV-2ΔgE(gfp) did not cause any death (FIG. 37A), produced inoculation site disease similar to mock infection (FIG. 37B), and caused no zosteriform disease (FIG. 37C). Therefore, HSV-2ΔgE(gfp) causes no disease or death following mouse flank scarification and does not reactivate from DRG, indicating that this vaccine is safe in mice.
Explants:
DRG explanted >28 dpi from 5 mice infected with HSV-2ΔgE did not reactivate virus when cultured on Vero cells for 16 days. There was no positive control, since all mice infected with HSV-2(2.12) died. These results suggest that HSV-2ΔgE(gfp) is impaired in epithelial cell to cell spread and/or epithelial to neuronal cell spread within the host.
HSV-2ΔgE(Gfp) is not Detected in the Skin or the DRG Following Mouse Flank Scarification, Further Demonstrating the Safety of this Virus as a Vaccine.
Mice were infected by scarification on denuded flank skin with 5×105 pfu HSV-2(2.12) or HSV-2ΔgE(gfp). HSV-2(2.12) flank infection yielded virus in skin samples (FIG. 38A) on all days tested and in DRG on days 3, 6 and 8 (FIG. 38B). In contrast, tissues titered from mice infected with HSV-2ΔgE(gfp) had no detectable virus.
HSV-2ΔgE(Gfp) and HSV-1(NS)-gEnull Infections Cause No Disease or Death Following Vaginal Inoculation and Yield Lower Swab Titers than WT HSV Strains.
Mice were infected intravaginally with 5×105 pfu of HSV-2(2.12) or HSV-2ΔgE(gfp). All mice infected with the wild-type HSV-2 strain (2.12) died by day 8, whereas only 40% of the mice infected with the wild-type HSV-1 strain (NS) died (FIG. 39A). 100% of mice infected with HSV-2(2.12) develop severe disease, while mice infected with HSV-1(NS) develop more moderate disease (FIG. 39B). All mice infected with either HSV-1 NS-gEnull or HSV-2ΔgE(gfp) vaccine strains survived and showed no signs of disease indicating that these vaccines are non-pathogenic in mice (FIGS. 39A and B). Mice were swabbed intra-vaginally on days 0-3. Titers of all strains increased from days 1 to 2, indicating that both WT and vaccine strains are likely replicating (FIG. 39C).
Example 20
HSV-2(gE NULL) Vaccination is Protective Against Wild-Type HSV-2 Infection
HSV-2ΔgE(Gfp) Immunization by IM Route Protects Mice Better than SubQ Injection from Epidermal Challenge by Flank Scarification.
Mice were vaccinated with HSV-2ΔgE(gfp) or mock-vaccinated, infected IM or subQ with 5×105 pfu (1,736 LD50s) HSV-2(MS), and survival, inoculation site disease, and zosteriform disease were evaluated. While 100% of mock vaccinated mice died following flank scarification on denuded flank skin with 5×105 pfu (1,736 LD50s) HSV-2(MS), all mice given HSV-2ΔgE(gfp) by the IM route survived (FIG. 40A). However, only 4 of 5 vaccinated by the SubQ route survived. The IM route was also more effective at preventing both inoculation site (FIG. 40B) and zosteriform disease (FIG. 40C) than the SubQ route. One of 5 mice vaccinated IM developed severe zosteriform disease but recovered, but the remaining 4 vaccinated mice had no zosteriform lesions.
HSV-2ΔgE(Gfp) Given IM to Mice Results in Reduced Viral Loads in Skin and DRG Following Challenge by Epidermal Scarification.
Mice were vaccinated IM with 5×105 pfu HSV-2ΔgE(gfp) or mock-vaccinated, infected with 5×105 pfu (1,736 LD50s) HSV-2(MS), and viral titers were evaluated in skin (FIG. 41A) and DRG (FIG. 41B). Virus could be detected in both skin and DRG samples from mock vaccinated mice but not from mice vaccinated with HSV-2ΔgE(gfp), indicating that vaccination led to reduced viral loads following challenge.
HSV-2ΔgE(Gfp) Immunization by IM Route Protects Mice Better than SubQ Injection from Mucosal Challenge with 250 Pfu (50 LD50s) of HSV-2(MS) in the Mouse Vaginal Model.
Mice were vaccinated IM or subQ with HSV-2ΔgE(gfp) or mock-vaccinated, infected intravaginally with 250 pfu (50 LD50s) HSV-2(MS), and survival, inoculation site disease, and virus titer were evaluated. 100% of mock vaccinated mice died whereas all of the mice vaccinated with HSV-2ΔgE(gfp) by the IM route survived (FIG. 42A). In contrast, only 60% of the mice vaccinated by the SubQ route survived the vaginal challenge, indicating that the IM route is more effective at protecting from mucosal challenge. All mice that were mock vaccinated developed severe disease and some of the mice vaccinated by the SubQ route developed disease (FIG. 42B). However, mice that were vaccinated with HSV-2ΔgE(gfp) by the IM route, developed no visible signs of disease. SubQ vaccination with HSV-2ΔgE(gfp) caused reduced intravaginal viral loads relative to the mock vaccinated group (FIG. 42C). However, IM vaccination was even more effective at reducing the amount of virus in the vagina following challenge.
Explants:
Sacral DRG explanted >28 dpi from 4 of 5 mice vaccinated IM with HSV-2ΔgE and 5 of 5 SubQ vaccinated mice reactivated virus when cultured on Vero cells for 16 days. This indicates that one dose of the vaccine given by the IM route protected ganglia from challenge infection in a minority of cases.
Two Immunizations with HSV-2ΔgE(Gfp) are Significantly Better than One in Protecting Mice from Disease Following Challenge with 5×104 Pfu (104 LD50s) of HSV-2(MS) in the Mouse Vaginal Model.
Mice were vaccinated with either one or two doses (three weeks apart) of 5×105 pfu HSV-2ΔgE(gfp) or mock-vaccinated, infected intravaginally with 5×105 pfu (1,736 LD50s) HSV-2(MS), and survival, inoculation site disease, and vaginal virus titer were evaluated. While 100% of the mice from the mock vaccinated group died, groups vaccinated with either one or two doses of HSV-2ΔgE(gfp) were completely protected from death (FIG. 43A). One vaccination with HSV-2ΔgE(gfp) did not completely protect mice from disease; however, when given in two doses, mice showed no outward signs of disease (FIG. 43B). While one HSV-2ΔgE(gfp) vaccination reduced vaginal titers following challenge relative to wild-type, two vaccinations were significantly better (FIG. 43C). Both vaccinated groups had no detectable virus in swabs taken on day 5, whereas mock-vaccinated animals had approximately 104 pfu challenge virus on day 5, persisting at high levels (greater than 104 pfu) until day 7, just prior to death. Photos from each mouse taken on day 7 post-inoculation demonstrate the difference between vaginal disease in each group (FIG. 43D).
Example 21
Efficacy of HSV-2(gE NULL) Vaccination Against Existing HSV-2 Genital Infection in a Guinea Pig Model
Materials and Experimental Methods
Guinea Pig Model of Genital Herpes
On the day of inoculation, vaginal closure membranes are ruptured with a pre-moistened calcium alginate swab. Vaginal vault is swabbed with a dry calcium alginate swab, and 5×103 pfu of HSV-2 (strain MS) is instilled into the vaginal vault with a syringe and a 20-gauge plastic catheter. This dose is generally sublethal, while providing infection of nearly every inoculated animal. During acute genital infection, animals are evaluated daily through day 14 p.i. for genital skin disease and urinary retention. Disease is quantified by a skin lesion scoring system ranging from 0 (no disease) to 4 (severe disease characterized by large ulcers with maceration). Daily scoring of each animal proceeds from day 15-60 p.i. to establish frequency of external recurrent herpetic lesions.
Viral Shedding Detection
Guinea pigs spontaneously shed HSV-2 from the vaginal cavity even in the absence of signs of disease. Viral DNA can be detected in 10 to 20% of the vaginal swabs from latently infected guinea pigs, allowing for the study of viral shedding frequencies and comparisons of the magnitudes. Vaginal cavities are swabbed daily with a calcium alginate-tipped swab from days 15-60 p.i. DNA is extracted from each swab sample using the QIAmp® DNA extraction system (Qiagen, Inc, Chatsworth, Calif.), including mock swab blanks as monitors for sample contamination, and subjected to quantitative PCR for HSV-2 DNA, using primers targeting the DNA polymerase gene. A separate reaction is performed for each of the specimens to address template quality and quantity, using a second set of primers to amplify the single-copy guinea pig albumin gene. The resulting 498-bp amplimer is utilized for normalization of DNA concentration and a more quantitative estimate of the HSV-2 burden in each specimen. Positive specimens are compared to amplification of a series of 10-fold serial dilutions of established genomic equivalents using MS HSV-2 stocks. Reactions are run in a GeneAmp® PCR System 9600 (Perkin-Elmer Corp, Norwalk, Conn.) beginning with a “hot start” at 95° C. for 2 min; then 35 cycles of denaturation at 95° C. for 1 min, annealing for 1 min at 65° C., and 72° C. extension for 1 min 30 s; and a final 7-min extension at 72° C. Amplification products of each sample, positive and negative controls, and the series of known standards are detected by Southern blotting. HSV-2 burdens are extrapolated from the linear relationship established from band density of a dilution series of known genomic equivalents amplified in parallel to the samples.
Determination of HSV-2 DNA Copy Numbers in Guinea Pig Dorsal Root Ganglia.
Sacral dorsal root ganglia (6-8 per animal) are dissected on day 60 p.i. and weighed, viral DNA is extracted by using a QIAamp® DNA minikit (QIAGEN), and real-time PCR is performed. A standard curve is constructed for each experiment, using purified plasmid containing HSV-2 gD gene sequences. Data are normalized to probes specific for guinea pig lactalbumin DNA.
Immunization of guinea pigs: 60 days p.i., guinea pigs are immunized or mock immunized once or twice separated by approximately 3 weeks and animals followed for recurrent lesions by visual inspection of the vaginal orifice and surrounding skin and by daily swabs for HSV-2 DNA detected by PCR.
Results
The guinea pig model is utilized to evaluate the efficacy of herpes simplex vaccine against recurrent herpetic disease. This model provides a naturally occurring recurrent disease similar to that seen in human HSV-2 infections, and latently infected guinea pigs shed virus vaginally at a frequency similar to that observed in humans.
Guinea pigs previously infected by HSV-2 and then vaccinated with HSV-2(gE null) are expected to have significantly reduced frequency of genital lesion compared to mock-vaccinated animals and reduce the number of animals that experience any recurrences. In addition, HSV-2(gE null) vaccination is expected to significantly reduce the magnitude of viral shedding.
When HSV-2(gE null) vaccination is given prior to challenge to evaluate the effect of the vaccination on the establishment of latent HSV-2 infection, accumulation of wt HSV-2 viral genomes in guinea pig DRG is evaluated on day 60 post challenge. HSV-2(gE null) vaccination is expected to significantly reduce the number of viral genomes in the DRG.
This and the previous Example are expected to provide additional evidence that HSV-2(gE null) vaccines are efficacious in protecting subjects against HSV-2 infection and subsequent genital reactivation.
Example 22
Introduction of Additional Deletions to the Us Region in Order to Further Impair the Anterograde Spread of the ΔgE-2 Vaccine Strain
In order to further attenuate the ΔgE-2 vaccine strain, additional deletions are introduced into Us7 and Us9, encoding the gI and Us9 proteins, using a similar approach to that used to construct the HSV-2 Us8 deletion (FIG. 32). A cloning vector that contains two 500-1000 base pair flanking regions, each homologous to either the DNA sequence 5′ or 3′ of the intended deletion, is constructed. The DNA for these two regions is obtained by PCR of HSV-2(2.12) genomic DNA. The cloning vector is co-transfected with HSV-2 genomic DNA, so that the deletions are incorporated into the viral DNA by homologous recombination. The resulting plaques are screened for the correct Us deletion by PCR.
Example 23
Identification of Additional Mutations that Impair Anterograde Spread of the ΔgE-2 Vaccine Strain
RNAi gene silencing methodology is utilized to identify genes other than gE, Us7 and Us9 that are involved in virus spread. RNAi technology uses approximately 20-22 base-pair double-stranded RNA fragments with sequences identical to the viral gene targeted for silencing. To target sequences on viral genes of HSV-1 or HSV-2, small RNA double-stranded fragments identical in sequence to the viral RNA are synthesized using standard techniques known in the art, and are introduced by transfection technology into cells that are then infected with HSV-1 or HSV-2 wild-type or mutant virus. Spread of defective virus is detected by screening for small plaques in human epidermal keratinocytes (HaCaT) cells (Collins W J et al. Herpes simplex virus gE/gI expressed in epithelial cells interferes with cell-to-cell spread. J Virol. 2003 February; 77(4):2686-95). The genes targeted by the RNAi fragments that induce small plaques are used in gene deletion studies. Inactivating mutations are then introduced into the gene or genes identified by the above RNAi screening method to create mutant viruses. Spread properties of mutant viruses are evaluated in vitro using rat superior cervical ganglion cell neuron cultures (Wang F, Tang W, McGraw H M, Bennett J, Enquist L W, and Friedman H M. J. Virol 79:13362-72, 2005) and the mouse retina eye infection model (Wang F, Tang W, McGraw H M, Bennett J, Enquist L W, Friedman H M. J. Virol 79:13362-72, 2005). The viral mutant strains identified that modify spread in vitro or in vivo are introduced into strains containing deletions of gE, Us7 or Us9 to develop strains containing deletions in multiple genes to identify the optimum combination of mutations that causes little or no disease when inoculated into laboratory animals, that results in low levels or no viral DNA in DRG, and that provides maximum protection against disease and establishment of viral latency when challenged by infection with wild type HSV-1 or HSV-2.
In other experiments, efforts are focused on virion membrane proteins, e.g. glycoproteins J, G, K, and M. Membrane glycoproteins required for virus entry, e.g. glycoproteins B, D, H and L, are excluded. These virion membrane proteins are analyzed as described in the previous paragraph.
Example 24
HSV-2ΔgE(Gfp) Vaccines are Safe
The LD50 of the HSV-2ΔgE(gfp) were assessed (labeled as gE2-del virus in the table) in BALB/c and SCID mice. The LD50 was calculated by the Reed-Muench method. After infection, disease and death were monitored for 4 weeks. At least 5 mice were evaluated at each inoculation dose.
IM Safety
In BALB/c mice, wild-type virus was evaluated at 5×103 to 5×105 PFU, and the vaccine strain at 5×105 to 5×106 PFU. In SCID mice, wild type virus was used at 5×101 to 5×105 PFU, while the vaccine strain was evaluated at 5×104 to 5×106 PFU.
Intravaginal Infection
In BALB/c mice, wild-type virus was used at 1-50 PFU, while the vaccine strain was evaluated at 5×104 to 5×105 PFU. In SCID mice, wild-type virus was used at 5×101 to 5×103 PFU, while the vaccine strain was used at 5×104 to 5×106 PFU.
Intravenous Infection:
In BALB/c mice wild-type virus was used at 5×103 to 5×105 PFU, while the vaccine strain was used at 5×105 to 5×106 PFU. In SCID mice, wild-type virus was evaluated at 5×103 to 5×105 PFU, while the vaccine strain was evaluated at 5×104 to 5×106 PFU.
Intracranial Infection
BALB/c mice were inoculated with wild-type virus at 5 to 5×103 PFU, while the vaccine strain was inoculated at 5×104 to 5×106 PFU.
Results
No mice died or showed signs of illness at any dose of HSV-2ΔgE(gfp) (gE2-del virus) inoculated by these routes (highest inoculum was 5×106 PFU for all routes, except vaginal route in BALB/c mice that was at 5×105 PFU) (FIG. 44). In contrast, mice inoculated with HSV-2 strain 2.12 (the parental virus for vaccine strain) died at doses that were 102 to 105 PFU lower than the highest dose of vaccine virus that caused no disease) (FIG. 44). The LD50 of the vaccine virus were also evaluated after intracranial inoculation of 3-4 week old BALB/c mice injected with 25 μl of virus. 9/10 mice inoculated with 5×106 PFU of the vaccine strain died, and 1⁄5 mice died at 5×105 PFU (LD50=1.4×106 PFU). In contrast, the LD50 of WT virus was <5 PFU, which was the lowest dose tested (FIG. 44). Therefore, there is >2.8×105 PFU difference in the LD50 comparing HSV-2ΔgE(gfp) with wild-type virus following intracranial inoculation.
Example 25
Antibody Response to HSV-2 gD-2 after Immunization with HSV-2ΔgE(gfp)
Mice were bled prior to immunization (prebleed) or immunized with HSV-2 glycoprotein gD as a positive control. The gD-2 protein extends from amino acid 26-331 (amino acid 26 as the first amino acid after the signal sequence). The gD-2 construct has 306 amino acids and is referred to as bac-gD-2(306t) High-level expression and purification of secreted forms of herpes simplex virus type 1 glycoprotein gD synthesized by baculovirus-infected insect cells. HSV-2 gD was used at 2 μg/mouse mixed with CpG and alum as adjuvants, where CpG: TCC ATG ACG TTC CTG ACG TT (SEQ ID NO: 19) 50 μg per mouse was mixed with alum 25 μg/μg protein in a 50 μl volume. Mice were immunized IM in the calf three times separated by 2 week intervals (labeled gD imm+ct). For comparison, mice were immunized IM in the calf muscle with HSV-2ΔgE(gfp) using 5×105 PFU, given either once or twice separated by 4 weeks. Mice were bled 4 weeks after the first immunization (1×imm), 4 weeks after the second immunization (2×imm), and 3 and 5 months after the second immunization (3 mo, or 5 mo). n=5 mice per group, except gD imm (+ct), which involves a single mouse.
Results
No antibodies to gD-2 were detected in the pre-immune sera. Antibody titers after gD-2 immunization (positive control) were higher than antibodies produced to the live virus vaccine HSV-2ΔgE(gfp) when administered once or twice (FIG. 45) Importantly, no antibody response to gD-2 was detected after the first immunization; however, antibodies were detected after the second immunization that persisted for 3 and 5 months (FIG. 45).
Example 26
Antibody Response to HSV-2 gC-2 after Immunization with HSV-2ΔgE(gfp)
A similar experiment was performed as described above for gD-2; however, the mice were immunized with HSV-2 gC (gC-2) as the positive control and antibody was measured to gC-2 protein. The gC-2 immunogen extends from amino acid 27-426 (amino acid 1 is the methionine at the ATG site, and amino acid 27 is the first amino acid after the signal sequence). Based on the cloning method, an aspartic acid and proline were added at the amino terminus just prior to amino acid 27. The gC-2 protein is referred to as bac-gC-2(426t). Mice were immunized with gC-2 5 μg mixed with CpG and alum given 3 times separated by 2 weeks. Other mice were immunized with HSV-2ΔgE(gfp) at 5×105 PFU in the calf muscle given either once or twice, separated by 4 weeks. n=5 mice per group, except gC imm (+ct), which involves a single mouse.
Results
No gC-2 antibody was detected in pre-immune serum (labeled Prebleed). High titers of gC-2 antibody were detected following immunization with gC-2 mixed with CpG and alum (labeled as gC imm+ct) (FIG. 46). Low titers of gC-2 antibody were detected after one immunization with the live virus vaccine HSV-2ΔgE(gfp) (labeled as 1×imm). In contrast, higher antibody titers were detected after the second immunization (labeled as 2×imm) that persisted for 3 and 5 months (FIG. 46).
Example 27
Neutralizing Antibody Response after One or Two Immunizations with HSV-2ΔgE(gfp)
Female BALB/c mice at 5-6 weeks age were immunized IM in the calf muscle with HSV-2ΔgE(gfp) at 5×103, 5×104, or 5×105 PFU. Immunizations were given once or twice separated by 4 weeks. Serum was collected 4 weeks after the first or 4 weeks after the second immunization and tested for neutralizing antibodies by incubating serial dilutions of serum with 200 PFU of HSV-2 strain 2.12 for 1 h at 37° C. and measuring the virus titer by plaque assay on Vero cells. The end point titer was that dilution of serum that reduced the virus titer by ≧50%. Legend: I immu, immunized once; II immu, immunized twice. n=5-10 animals per group.
Results
At each immunization dose, neutralizing antibody titers were higher after the second immunization than the first. Titers were highest in mice immunized with 5×105 PFU (FIG. 47).
Example 28
Protection of Mice by HSV-2ΔgE(gfp) is Dose Dependent
Mice were immunized with 5×103, 5×104, or 5×105 PFU of HSV-2ΔgE(gfp) (labeled as gE-2null) given twice in the calf muscle separated by 4 weeks. Four weeks after the second immunization, mice were challenged intravaginally with 5×104 PFU of HSV-2 strain MS (approximately 10,000 LD50). Animals were followed for survival and were scored for vaginal disease on a scale of 0-4, where 0 is no disease, and one point was assigned for each of the following: erythema/swelling, exudate, hair loss in the perineal area, and ulcers or necrosis in the vaginal area (maximum score of 4 per animal per day). Moreover, animals were evaluated for vaginal titers, which were determined by swabbing the vagina and titering virus by plaque assay on Vero cells Animals were also evaluated for viral titers or viral DNA in dorsal root ganglia (DRG) 4 days post-infection or 35 days post-infection (labeled as latent viral load). DRG were harvested at either 4 days or 35 days post-challenge. For viral titers, the DRG samples were minced with small scissors and pulverized using a pestle and half the sample was titered on Vero cells. The remainder of the DRG sample was evaluated by real-time quantitative PCR(RT qPCR) by amplifying the HSV-2 US9 DNA. Mouse adipsin DNA was amplified in each well as a DNA control. PCR was performed in 96-well qPCR plates using 2×FAST Taqman master mix (Applied Biosystems). Standard curves were prepared using purified HSV-2 DNA (Advanced Biotechnologies) and mouse lung genomic DNA as a source of the adipsin gene (BioChain Institute). The standard curves were run in triplicate wells at 5, 50, 500, 5,000 and 50,000 copies of DNA. DRG DNA (Qiagen DNeasy) samples were run in duplicate and results are reported as the number of HSV-2 DNA copies per 105 adipsin genes. Primers for adipsin were: forward 5′-GCAGTCGAAGGTGTGGTTACG-3′-(SEQ ID NO: 20), and reverse 5′-GGTATAGACGCCCGGCTTTT-3′ (SEQ ID NO: 21). Reporter dye and probe for adipsin were: 5′-VIC-CTGTGGCAATGGC-3′ MGBNFQ (SEQ ID NO: 22) (minor grove binder non-fluorescent quencher). Primers for HSV-2 Us9 were: forward 5′-GCAGAAGCCTACTACTCGGAAA-3′ (SEQ ID NO: 23), reverse 5′-CCATGCGCACGAGGAAGT-3′ (SEQ ID NO: 24). Reporter dye and probe for Us9 were: 5′-6FAM-CGAGGCCAAC-3′-MGBNFQ (SEQ ID NO: 25). Primers for gpGAPDH (for studies described in FIG. 50 in guinea pigs) were: forward 5′-CATGACAACTTCGGCATTGTG-3′ (SEQ ID NO: 26), reverse 5′-TCTTCTGGGTGGCAGTGATG-3′ (SEQ ID NO: 27). Primers for HSV-2 gE were: forward 5′-CGTCTGGATGCGGTTTGAC-3′(SEQ ID NO: 28), and reverse 5′-CTGGAAGCTGCGGGTGATAC-3′ (SEQ ID NO: 29). Reporter dye and probe for gE were: 6FAM-5′-ATGCGGATCTACGAAGC-3′-MGBNFQ (SEQ ID NO: 30). Primers for GFP were: forward 5′-AGCAAAGACCCCAACGAGAA-3′ (SEQ ID NO: 31), and reverse 5′-GGCGGCGGTCACGAA-3′ (SEQ ID NO: 32). Reporter dye and probe for GFP were: 6FAM-5′-ATCACATGGTCCTGCTGG-3′-MGBNFQ (SEQ ID NO: 33). Reactions were performed using 5 μl of sample DNA in 25 μl volume using the TaqMan Gene Expression Master Mix (Applied Biosystems) and the ABI 7500 Fast machine.
Results
No mock immunized mouse survived, while survival was 60% in mice immunized with 5×103 PFU, and 100% in mice immunized with 5×104 or 5×105 PFU of the vaccine strain (labeled as gE-2null) (FIG. 48A). Vaginal disease scores were highest in mock immunized mice, and declined proportionally in mice immunized with 5×103, 5×104, or 5×105 PFU of the vaccine strain. The number of animals surviving until days 6, 7 and 8 in the mock immunized group is noted on the graph (FIG. 48B). Vaginal titers were highest in mock immunized mice, and declined proportionally in mice immunized with 5×103, 5×104, and 5×105 PFU of the vaccine strain.
D. Virus titers or viral load in the DRG measured by real-time qPCR. The graph on the left shows viral titers 4 days post-challenge in mock immunized mice or HSV-2ΔgE(gfp) immunized mice (labeled as gE2-del). Titers were  ̃4 log10 in mock immunized compared with  ̃log10 in HSV-2ΔgE(gfp) immunized mice (P<0.001). The middle graph shows viral load in DRG at day 4. Mock immunized mice had between 6-7 log10 copies of HSV-2 DNA in DRG compared with  ̃3 log10 copies in HSV-2ΔgE(gfp) immunized mice (P<0.001). The right graph evaluates DRG 35 days post-challenge. No mock immunized mouse survived to day 35. The DRG of HSV-2ΔgE(gfp) immunized mice (labeled as gE2-del) showed 2-3 log10 copies of HSV-2 DNA.
From the experiments shown in FIG. 48 it is concluded that the protection of mice by HSV-2ΔgE(gfp) is dose dependent, with greater protection at 5×105 PFU than at the lower immunizing doses, and that the vaccine strain provides substantial protection to the DRG against high dose challenge with HSV-2 strain MS.
Example 29
Assessment of DRG for Wild-Type or Vaccine Strain DNA
The day 35 DRG described in FIG. 48 were assessed for wild-type or vaccine strain DNA by amplifying the HSV-2 gE gene (present in wild-type virus, but not the vaccine strain) or GFP (present in the vaccine strain, but not wild-type virus (FIG. 49). The primers and probes are described in Example 28.
Results
Wild-type DNA (gE) was detected in all 9 mice, while GFP DNA (vaccine strain) was detected in one mouse only. Therefore, the DNA detected in the right graph of FIG. 48D is predominantly from the challenge strain HSV-2 MS and not the vaccine strain.
Example 30
Intramuscular Immunization with HSV-2ΔgE(gfp) Given Twice Provides Protection Against Death, Vaginal Disease and Recurrent Infection
Immunization studies were performed in female Hartley strain guinea pigs (175 to 225 grams at the time of first immunization) that were injected in the calf muscle of the left hind leg two times at 4-week intervals with either HSV-2ΔgE(gfp) (labeled as gE2-del) at 5×105 PFU or with Vero cell lysate. As a control for some experiments, 5 ug gC-2 subunit immunogen was injected intramuscularly three times at 2-week intervals with CpG and alum as adjuvants. Adjuvants for guinea pigs were CpG: TCG TCG TTG TCG TTT TGT CGT T (SEQ ID NO: 34) 100 μg per guinea pig was mixed with alum 20 μg/μg protein in a 50 μl volume. For challenge studies, guinea pigs were infected intravaginally with HSV-2 MS strain at 5×103 or 5×105 PFU in 50 ul using a soft catheter to inject the virus. Vaginal titers were measured 1 to 7 days post-infection by inserting a moistened swab into the vagina, and then placing the swab in 1 ml of DMEM-10% FBS. Samples were stored at −70° C. until titers were determined by plaque assay. Animals were observed daily, and disease severity was scored as follows: 1 point for erythema, 2 points for discrete lesions, 3 points for coalesced lesions and 4 for ulcerative lesions. Disease that occurred 1 to 14 days post-infection was considered as acute infection, while disease that developed 15 to 60 days post-infection was considered as recurrent infection. At day 60, sacral DRG were harvested to evaluate for viral DNA copy number by real-time qPCR, which was performed as described in FIG. 48 (Example 28). Blood was collected from the lateral saphenous vein of the hind limb at various times for antibody assays.
Results
All mock immunized guinea pigs challenged with 5×103 or 5×105 PFU died, while all HSV-2ΔgE(gfp) or gC-2 immunized guinea pigs survived (FIG. 50A). Vaginal disease scores were highest in mock immunized animals challenged with 5×105 PFU, and lowest in animals immunized with HSV-2ΔgE(gfp) (FIG. 50B). Vaginal titers were highest in the mock immunized animals and lowest in animals immunized with HSV-2ΔgE(gfp) or gC-2. No significant differences were detected comparing HSV-2ΔgE(gfp) with gC-2; however, both these groups were significantly different from mock immunized animals (P<0.001) (FIG. 50C). The table shows the number of recurrences and the number of animals having a recurrence between days 15-49 post-infection. No mock immunized animal survived long enough to assess recurrences, even at the lower challenge dose of 5×103 PFU of HSV-2 MS. Three of the 5 animals immunized with gC-2 had recurrences, and these 3 animals had a total of 10 recurrences that lasted a total of 12 days. In contrast, only 1 of 10 animals immunized with HSV-2ΔgE(gfp) had a recurrent infection (P<0.001), which lasted for 2 days (FIG. 50D). DRG were harvested at the end of the experiment and assessed for HSV-2 DNA by real-time qPCR. HSV-2 DNA at low levels was detected in 2 of 5 guinea pigs immunized with gC-2, compared with 0 of 5 guinea pigs immunized with HSV-2ΔgE(gfp) (FIG. 50E).
It is concluded that immunization with HSV-2ΔgE(gfp) given twice IM in the calf separated by 4 weeks at a dose of 5×105 PFU provides protection against death, vaginal disease and recurrent infection.
Example 31
Two Immunizations with HSV-2ΔgE(Gfp) after Recovery from a Primary HSV-2 Genital Infection Produce Higher Titers to gC-2 and gD-2 than One Immunization and is Effective in Reducing the Frequency or Recurrent Lesions
The therapeutic potential of the live virus vaccine was examined in female Hartley strain guinea pigs (175 to 225 grams). 30 guinea pigs were infected intravaginally with HSV-2 MS strain at 104 PFU in 50 ul using a soft catheter. 17 animals survived the infection. Antibody titers to gC-2 and gD-2 were determined 28 days post-infection in these 17 animals to confirm infection. Only 11 animals had antibodies to either gC-2 or gD-2, indicating infection in these 11 guinea pigs. 50 days after the initial infection, the 11 guinea pigs were immunized IM in the calf muscle of the left hind leg twice separated by 28 days. 6 animals were randomly assigned to receive two immunizations with HSV-2ΔgE(gfp) at 5×105 PFU, while 5 animals were mock immunized twice with Vero cell lysates. Animals were bled for antibodies 4 weeks after each immunization and antibodies measured to gC-2 (A) or gD-2 (B). Animals were scored for recurrent infections from day 16 after the first immunization until day 58 after the first immunization. One point was assigned for each lesion observed (C). DRG were harvested for HSV-2 DNA by real-time qPCR at the end of the experiment.
Results
After recovery from acute infection, only 1 of 6 guinea pigs had antibodies to gC-2 after the first immunization, compared to 5 of 6 after the second immunization, indicating that two immunizations induced higher ELISA titers to gC-2 than one immunization (FIG. 51A). After recovery from acute infection, 4 of 6 guinea pigs had gD-2 antibodies after both the first immunization and the second immunization; however, antibody titers were higher after the second immunization (P<0.01) (FIG. 51B). Recurrent lesions developed significantly more often in mock immunized than HSV-2ΔgE(gfp) immunized guinea pigs, indicating that the live virus vaccine is effective in reducing the frequency of recurrent infection (P<0.0001) (FIG. 51C). 3 of 5 DRG from mock immunized animals were positive for HSV-2 DNA at the end of the experiment, compared with 1 of 5 from HSV-2ΔgE(gfp) immunized animals (FIG. 51D).
It is concluded that when HSV-2ΔgE(gfp) is used as a therapeutic vaccine, two immunizations produce higher titers to gC-2 and gD-2 than one immunization, and that the vaccine is effective in reducing the frequency of recurrent lesions.
Having described the embodiments of the invention with reference to the accompanying drawings, it is to be understood that the invention is not limited to the precise embodiments, and that various changes and modifications may be effected therein by those skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

## Claims

1. A method of suppressing a recurrent Herpes Simplex Virus (HSV) infection in a subject comprising the steps of:
a) administering a composition comprising a mutant HSV-2 strain to said subject, wherein said mutant HSV-2 strain consists of a single inactivating mutation, wherein said inactivating mutation consists of the deletion of base pairs (bp) 369-1479 of SEQ ID NO: 7, and
b) following step a) a second administration of said composition comprising said mutant HSV-2 strain is performed.

2. The method of claim 1, wherein said composition is first administered before exposure to HSV.

3. The method of claim 1, wherein said composition is first administered after exposure to HSV.

4. The method of claim 1, wherein said mutant strain has a defect in anterograde neuronal transport, retrograde spread, and is deficient in cell-to-cell spread.

5. The method of claim 1, wherein said mutant strain is replication-competent.

6. The method of claim 1, wherein said mutant HSV strain induces an anti-HSV immune response in said subject.

7. The method of claim 6, wherein said anti-HSV immune response is an anti-HSV neutralizing antibody response.

8. The method of claim 1, wherein said HSV infection is an HSV-1 infection.

9. The method of claim 1, wherein said HSV infection is an HSV-2 infection.

10. The method of claim 1, wherein said HSV infection is a genital infection.

11. The method of claim 1, wherein said HSV infection is an oral infection.

12. The method of claim 1, wherein said HSV infection is an ocular HSV infection.

13. The method of claim 1, wherein said recurrent HSV infection is HSV labialis following a primary HSV infection.

14. The method of claim 1, wherein said composition is administered at least one additional time after said second administration.

15. The method of claim 1, wherein said composition is administered intramuscularly, epidermally, subcutaneously, intravaginally, or via intra-respiratory mucosal injection.