Immunization with a vaccine combining herpes simplex virus 2 (HSV-2) glycoprotein C (gC) and gD subunits improves the protection of dorsal root ganglia in mice and reduces the frequency of recurrent vaginal shedding of HSV-2 DNA in guinea pigs compared to immunization with gD alone

Abstract

Attempts to develop a vaccine to prevent genital herpes simplex virus 2 (HSV-2) disease have been only marginally successful, suggesting that novel strategies are needed. Immunization with HSV-2 glycoprotein C (gC-2) and gD-2 was evaluated in mice and guinea pigs to determine whether adding gC-2 to a gD-2 subunit vaccine would improve protection by producing antibodies that block gC-2 immune evasion from complement. Antibodies produced by gC-2 immunization blocked the interaction between gC-2 and complement C3b, and passive transfer of gC-2 antibody protected complement-intact mice but not C3 knockout mice against HSV-2 challenge, indicating that gC-2 antibody is effective, at least in part, because it prevents HSV-2 evasion from complement. Immunization with gC-2 also produced neutralizing antibodies that were active in the absence of complement; however, the neutralizing titers were higher when complement was present, with the highest titers in animals immunized with both antigens. Animals immunized with the gC-2-plus-gD-2 combination had robust CD4+ T-cell responses to each immunogen. Multiple disease parameters were evaluated in mice and guinea pigs immunized with gC-2 alone, gD-2 alone, or both antigens. In general, gD-2 outperformed gC-2; however, the gC-2-plus-gD-2 combination outperformed gD-2 alone, particularly in protecting dorsal root ganglia in mice and reducing recurrent vaginal shedding of HSV-2 DNA in guinea pigs. Therefore, the gC-2 subunit antigen enhances a gD-2 subunit vaccine by stimulating a CD4+ T-cell response, by producing neutralizing antibodies that are effective in the absence and presence of complement, and by blocking immune evasion domains that inhibit complement activation.

Fig. 1.

ELISA titers, neutralizing antibody titers, and C3b blocking antibody titers. Mice were mock immunized with CpG and alum or immunized with 5 μg of gC-2 antigen, 250 ng of gD-2 antigen, or 5 μg of gC-2 plus 250 ng of gD-2 antigen. Serum was collected 10 days after the third immunization and examined by ELISA for antibody to gC-2 (A) or gD-2 (C). Each symbol represents the serum from a single mouse tested in duplicate and is plotted as the mean log₁₀ titer. The error bars in panel C represent standard deviations. (B) IgG was purified from sera of mice immunized with gC-2 or gD-2 antigen and evaluated in a C3b blocking assay. The top panel is a cartoon illustrating anti-gC-2 IgG blocking gC-2 binding to C3b (left figure), while anti-gD-2 IgG has no effect (right figure). Increasing concentrations of anti-gC-2 IgG resulted in decreased binding of gC-2 to C3b, while anti-gD-2 IgG had no effect on gC-2 binding. An IgG level of 28 μg represents the amount of IgG in an approximately 1:160 dilution of mouse serum. Data plotted are representative of three separate experiments. (D) HSV-2 or HSV-2 gCnull was incubated with PBS or anti-gD-2 IgG in the presence or absence of 2.5% human complement. Results are averages for duplicate wells and are representative of three separate experiments. (E) Serial dilutions of sera from gC-2 (left)-, gD-2 (middle)-, and gC-2-plus-gD-2 (right)-immunized animals were evaluated for neutralizing antibody to HSV-2 strain 2.12, without complement (solid lines) or with 2.5% human serum as the source of complement (dashed lines). Sera from mock-immunized animals were used as a control, and their data are plotted in each panel. Each data point is the mean ± standard deviation for sera from 5 mice tested in duplicate wells.

Fig. 2.

Passive transfer of anti-gC-2 IgG protects complement-intact mice from inoculation site and zosteriform disease. Inoculation site (A and B) and zosteriform (C and D) disease scores are shown for C57BL/6 and C3 knockout (C3KO) mice passively immunized intraperitoneally with 200 μg murine anti-gC-2 IgG or 200 μg murine nonimmune IgG 24 h before epidermal HSV-2 challenge with 5 × 10⁵ PFU of HSV-2 strain 2.12. Results shown are the means ± standard deviations for 5 mice per group.

Fig. 3.

Dose escalation of gD-2 and gC-2 antigens in the mouse vaginal model. BALB/c mice were mock immunized with CpG and alum or immunized with 50, 100, or 250 ng of gD-2 antigen or with 1, 2, or 5 μg of gC-2 antigen, given with CpG and alum. Mice were challenged intravaginally with 2 × 10⁵ PFU of HSV-2 strain 2.12. Animals were observed for survival (A and B), vaginal disease (C and D), and vaginal titers (E and F). Results shown are means ± standard deviations for 5 mice per group.

Fig. 4.

Combined immunization with gC-2 and gD-2 antigens protects DRG after flank infection. BALB/c mice were mock immunized with CpG and alum or immunized with 5 μg of gC-2 antigen, 250 ng of gD-2 antigen, or 5 μg of gC-2 plus 250 ng of gD-2, with CpG and alum (n = 10 per group). Mice were challenged by flank inoculation with 4 × 10⁵ PFU of HSV-2 strain 2.12. Five animals in each group were observed for survival (A) and scored for severity of disease at the inoculation site (B) and zosteriform site (C). (D) The other 5 animals were euthanized on day 5 to measure DRG viral titers. The dotted line represents the limit of detection of the assay, which is <2 PFU per mouse. Results are plotted as means ± standard deviations.

Fig. 5.

Combined immunization with gC-2 and gD-2 antigens protects DRG better after vaginal challenge than immunization with gD-2 antigen alone. BALB/c mice were immunized as described in the legend to Fig. 3. Mice were challenged with 2 × 10⁵ PFU of HSV-2 strain 2.12 by vaginal inoculation. Five mice from each group were monitored for survival and disease, and the other five were evaluated for DRG infection. (A) Severity of vaginal disease. (B) Representative pictures of vaginal disease. The mock-immunized mouse had a score of 4 based on erythema, exudate, hair loss, and necrosis; the gC-2-immunized mouse had a score of 2 based on erythema and hair loss; the gD-2-immunized mouse had a score of 1 based on hair loss; and the gC-2-plus-gD-2-immunized mouse had no disease. (C) Vaginal swab titers. (D) DRG viral titers measured at 4 days postinfection. The dotted line represents the limit of detection of the assay, which is <2 PFU per mouse. (E) HSV-2 reactivation from sacral DRG. Data plotted are means ± standard deviations.

Fig. 6.

Combined immunization with gC-2 and gD-2 antigens protects DRG better than that with gD-2 alone at a higher gD-2 antigen dose. BALB/c mice were mock immunized with CpG and alum or immunized with 5 μg of gC-2 antigen, 2 μg of gD-2 antigen, or 5 μg of gC-2 plus 2 μg of gD-2 antigen, with CpG and alum (n = 10 per group), and were challenged with 5 × 10⁴ PFU of HSV-2 strain MS by vaginal inoculation. Five mice in each group were monitored for survival, disease, and vaginal titers, and the other five were monitored for DRG infection. (A) Vaginal disease; (B) vaginal titers; (C) DRG viral titers. Results plotted are means ± standard deviations. (D) Real-time qPCR was performed on DNAs isolated from sacral DRG at 4 dpi. LoQ represents the lower limit of accurate quantitation of the qPCR (5 DNA copies).

Fig. 7.

Persistence of antibody responses and protection. BALB/c mice were mock immunized with CpG and alum or immunized with the combination of 5 μg gC-2 plus 250 ng gD-2 antigen, given with CpG and alum. ELISA titers are given for antibodies to gD-2 (A) and gC-2 (B) 3, 15, 30, and 42 weeks after the third immunization. Results plotted are mean log₁₀ titers ± standard deviations. (C) Mice were challenged with 2 × 10⁵ PFU of HSV-2 strain 2.12 by vaginal inoculation 3, 15, 30, or 42 weeks after the third immunization. Challenge study results are means ± standard deviations for 5 mice per group.

Fig. 8.

IFN-γ⁺ and TNF-α⁺ CD4⁺ and CD8⁺ T cells in gC-2- and gD-2-immunized mice. Mice were immunized three times at 2-week intervals. Representative dot plots are shown for CD4⁺ T cells (A) and CD8⁺ T cells (B) from one mock- and one gC-2-plus-gD-2-immunized mouse. The means and standard errors for all animals evaluated for CD4⁺ T-cell (C) and CD8⁺ T-cell (D) responses are shown for the individual cytokines or both IFN-γ and TNF-α for mock- and gC-2-plus-gD-2-immunized mice (n = 4 for mock controls and n = 9 for CD4⁺ and CD8⁺ T-cell measurements).

Fig. 9.

ELISA and neutralizing antibody titers. Guinea pigs were mock immunized with CpG and alum (n = 5) or immunized with 10 μg of gC-2 antigen (n = 5), 5 μg of gD-2 antigen (n = 10), or 10 μg of gC-2 plus 5 μg of gD-2 antigen (n = 10). Sera were collected 14 days after the third immunization and examined by ELISA for antibody against gC-2 (A) or gD-2 (B). Each symbol represents serum from a single guinea pig tested in duplicate. Results are plotted as mean log₁₀ titers. (C) Serial dilutions of sera from gC-2 (left)-, gD-2 (middle)-, and gC-2-plus-gD-2 (right)-immunized animals were evaluated for neutralizing antibody to HSV-2 strain MS, without complement (solid lines) or with 2.5% human serum as the source of complement (dashed lines). Sera from mock-immunized animals were used as a control. Each data point represents the mean ± standard deviation for sera from 3 guinea pigs tested in duplicate wells.

Fig. 10.

Protection provided by gC-2 and gD-2 immunization of guinea pigs. Guinea pigs were immunized as described in the legend to Fig. 9 and were challenged intravaginally with 5 × 10⁵ PFU HSV-2 strain MS. (A) Survival; (B) vaginal disease; (C) vaginal titers. Results are means ± standard deviations for 5 animals per group for the mock- and gC-2-immunized animals and for 10 animals per group for the gD-2- and gC-2-plus-gD-2-immunized animals. (D) Average number of days with recurrent lesions per animal. All mock-immunized animals died during acute infection; therefore, recurrent disease is plotted only for the gC-2-, gD-2-, and gC-2-plus-gD-2-immunized animals.

Fig. 11.

Vaginal shedding of HSV-2 DNA as measured by qPCR. The heat charts show vaginal viral shedding for 10 guinea pigs in the gD-2-immunized group (top) and 10 animals in the gC-2-plus-gD-2-immunized group (bottom), measured at 28 to 48 dpi.