Immunization with herpes simplex virus 2 (HSV-2) genes plus inactivated HSV-2 is highly protective against acute and recurrent HSV-2 disease

Abstract

To date, no vaccine that is safe and effective against herpes simplex virus 2 (HSV-2) disease has been licensed. In this study, we evaluated a DNA prime-formalin-inactivated-HSV-2 (FI-HSV2) boost vaccine approach in the guinea pig model of acute and recurrent HSV-2 genital disease. Five groups of guinea pigs were immunized and intravaginally challenged with HSV-2. Two groups were primed with plasmid DNAs encoding the secreted form of glycoprotein D2 (gD2t) together with two genes required for viral replication, either the helicase (UL5) and DNA polymerase (UL30) genes or the single-stranded DNA binding protein (UL29) and primase (UL52) genes. Both DNA-primed groups were boosted with FI-HSV2 formulated with monophosphoryl lipid A (MPL) and alum adjuvants. Two additional groups were primed with the empty backbone plasmid DNA (pVAX). These two groups were boosted with MPL and alum (MPL-alum) together with either formalin-inactivated mock HSV-2 (FI-Mock) or with FI-HSV2. The final group was immunized with gD2t protein in MPL-alum. After challenge, 0/9 animals in the group primed with UL5, UL30, and gD2t DNAs and all 10 animals in the mock-immunized control group (pVAX-FI-Mock) developed primary lesions. All mock controls developed recurrent lesions through day 100 postchallenge. Only 1 guinea pig in the group primed with pVAX DNA and boosted with FI-HSV2 (pVAX-FI-HSV2 group) and 2 guinea pigs in the group primed with UL5, UL30, and gD2t DNAs and boosted with FI-HSV2 (UL5, UL30, gD2t DNA-FI-HSV2 group) developed recurrent lesions. Strikingly, the UL5, UL30, gD2t DNA-FI-HSV2 group showed a 97% reduction in recurrent lesion days compared with the mock controls, had the highest reduction in days with recurrent disease, and contained the lowest mean HSV-2 DNA load in the dorsal root ganglia.

FIG. 1.

Immunization scheme and timeline. The vaccine components for each immunization group are shown in Table 1. Groups receiving a DNA prime and FI-HSV2 (or FI-Mock) boost (groups 1 to 4) were i.d. immunized between weeks 0 and 5 and then s.c. boosted on weeks 11 and 15. The gD2 subunit control group (gD2 su) received s.c. injections concurrently with groups receiving FI-HSV2 boosts. Each vaccine group consisted of 10 guinea pigs. The HSV-2 challenge (6 × 10⁵ PFU) was given intravaginally (i.vag.) at week 18. The acute disease phase was defined as up to 14 days postchallenge (weeks 18 to 20), and the recurrent phase was defined as days 15 to 100 postchallenge (to week 33). Guinea pigs were sacrificed, and the dorsal root ganglia (DRG) were harvested over the last 2 weeks of the experiment. The arrows show the weeks of test bleeds for antibody determination.

FIG. 2.

Virus-specific antibody levels in immunized guinea pigs. (A) Endpoint reciprocal IgG titers measured by an ELISA against either purified, dextran sulfate-released HSV-2 virion or purified, truncated gD2 protein. For reference, the weeks of immunization with FI-HSV2, FI-Mock, or gD2 subunit are shown by black arrows, and the week of i.vag. HSV-2 challenge is shown by a double-headed arrow. The symbols show the group means of the log₁₀ endpoint IgG titers as described in Materials and Methods, and the error bars depict the standard deviations (n = 10 for all groups.) Dashed lines represent the assay limit of sensitivity, and individual titers below the assay limit were assigned a value of one-half the assay limit for calculation and graphing purposes. (B) Virus neutralizing antibody titers were measured against HSV-2 that was purified from the serum-free medium of infected Vero cells. Symbols show the geometric mean titers (GMT) of the groups, and error bars represent the standard errors (SEM). The assay limit of sensitivity and treatment of below-limit values are as described for panel A.

FIG. 3.

Protection against acute HSV-2 disease. Guinea pigs were i.vag. challenged with HSV-2 and then scored daily by blinded observers over the 14-day acute disease period using the disease scoring scale as described in Materials and Methods. This scale represents the standard scale from 0 to 4, except that a score of 0.5 was given for slight erythema, swelling, or papules/patches and a score of 5 was given to animals that died of HSV-2 disease. (A) Mean disease scores for each vaccine group on each day postchallenge are shown by the symbols in the legend. (B) Disease scores from days 1 to 14 postchallenge were summed for each guinea pig (each symbol shows the value for one guinea pig), and the mean of each vaccine group is shown by the short horizontal line. The dotted line shows the acute disease-free baseline for clarity, and the numbers of guinea pigs in the groups with a cumulative score of 0 are 1, 6, 8, 8, and 8 for each group from left to right. Values that were significantly different from the value for the control group (pVAX-FI-Mock) by Kruskal-Wallis analysis plus Dunn's multiple-comparison test are indicated as follows: (*), P < 0.05; (**), P < 0.01; (***), P < 0.001).

FIG. 4.

Protection against acute-phase vaginal HSV-2 shedding. On the days shown postchallenge, each guinea pig was swabbed intravaginally, and the swabs were placed in 1 ml of DNG and frozen until standard plaque assay on Vero cells as described in Materials and Methods. (A) Levels of vaginal virus in the swabs are shown as group mean log₁₀ PFU, and error bars represent the standard deviations (n = 10 for all groups except for the UL5, UL30, gD2t-FI-HSV2 group where n = 9). The dotted line shows the limit of assay sensitivity. (B) Kinetics of viral clearance are represented as the percentage of guinea pigs in each group shedding virus on the indicated days postchallenge.

FIG. 5.

Protection against recurrent lesions and disease. HSV-2-challenged guinea pigs were scored for disease during the recurrent phase of infection, defined as days 15 to 100 postchallenge. (A) A lesion day was scored for a guinea pig that developed at least one lesion (disease score of 2 or rarely 3), and the cumulative number of lesion days for each vaccine group was averaged over the number of guinea pigs in each group. The pVAX-FI-HSV2 group, UL29, UL52, gD2t DNA-FI-HSV2 group, and gD2 subunit group each had 10 guinea pigs. The UL5, UL30, gD2t DNA-FI-HSV2 group had 9 guinea pigs, and as shown in Table 2 and as described in the text, the pVAX-FI-Mock group had 6 guinea pigs until day 34 and then 5 guinea pigs through day 100. (B) Each symbol represents the number of days in the recurrent phase that an individual (Indiv.) guinea pig had a score of 0.5 or above, and the vaccine group means are shown by the short horizontal lines. Note that the guinea pig in the pVAX-FI-Mock group that died on day 34 had resolved its primary disease and then accumulated 16 recurrent disease days before death. This was in contrast to an additional animal in this group that was omitted from the recurrent-phase study since it never resolved its primary disease. Values that were significantly different (P < 0.05) from the value for the control group (pVAX-FI-Mock) by Kruskal-Wallis analysis plus Dunn's multiple-comparison tests are indicated (*).

FIG. 6.

Protection against HSV-2 latent DNA load in the DRG. On weeks 46 to 48 of the study (28 to 30 weeks postchallenge), the lumbosacral DRG from each surviving guinea pig were removed, pooled, and frozen. The DRG DNA from each pool was extracted and quantified by spectrophotometry. HSV-2 DNA was quantified by TaqMan real-time quantitative PCR using primers and a probe specific for gG2. Each reaction mixture contained 250 ng of DRG DNA, and each standard curve reaction mixture contained 250 ng of DRG DNA from naïve guinea pigs and between 10 and 10⁴ copies of HSV-2 DNA isolated from purified HSV-2 virions. Each symbol represents the mean of 3 independent assays for an individual (Indiv.) guinea pig, and the horizontal lines represent vaccine group means. Two additional control groups consisted of the DRG from 4 naïve guinea pigs (Naïve) and from 3 guinea pigs that were immunized with FI-HSV2 but were not challenged with HSV-2 (FI-HSV2 Unchallenged). The broken line represents the detection limit of the assay of 10 copies of HSV-2 DNA per 250 ng of DRG DNA. Statistical significance levels for the values for each group compared with the pVAX-FI-Mock group are as described in the legend to Fig. 3.