Immunization with a dominant-negative recombinant Herpes Simplex Virus (HSV) type 1 protects against HSV-2 genital disease in guinea pigs

Abstract

Background: CJ9-gD is a novel dominant-negative recombinant herpes simplex virus type 1 (HSV-1) that is completely replication-defective, cannot establish detectable latent infection in vivo, and expresses high levels of the major HSV-1 antigen glycoprotein D immediately following infection. In the present study, CJ9-gD was evaluated as a vaccine against HSV-2 genital infection in guinea pigs.

Results: Animals immunized with CJ9-gD developed at least 700-fold higher titers of HSV-2-specific neutralization antibodies than mock-immunized controls. After challenge with wild-type HSV-2, all 10 control guinea pigs developed multiple genital lesions with an average of 21 lesions per animal. In contrast, only 2 minor lesions were found in 2 of 8 CJ9-gD-immunized animals, representing a 40-fold reduction on the incidence of primary genital lesions in immunized animals (p < 0.0001). Immunization significantly reduced the amount and duration of viral shedding and provided complete protection against neurological symptoms, while 90% of mock-immunized animals succumbed due to the severity of disease. Importantly, immunized animals showed no signs of recurrent disease or viral shedding during a 60-days observation period after recovery from primary infection, and carried 50-fold less latent viral DNA load in their dorsal root ganglia than the surviving mock-vaccinated controls (p < 0.0001).

Conclusions: Collectively, we demonstrate that vaccination with the HSV-1 recombinant CJ9-gD elicits strong and protective immune responses against primary and recurrent HSV-2 genital disease and significantly reduces the extent of latent infection.

Figure 1

Induction of HSV-2-specific neutralizing antibodies in immunized guinea pigs. Two sets of guinea pigs (n = 8; n = 10) were injected s.c. with 5 × 10⁶ PFU/animal of CJ9-gD or with DMEM and boosted after 3 weeks. Blood was taken 3 weeks after each immunization and 5 weeks after challenge. After heat inactivation, serum from each animal was assayed separately for HSV-2-specific neutralizing antibody titers on Vero cell monolayers. The results represent average titers ± SEM. P-value was assessed by Student's t-test (* p < 0.005).

Figure 2

Reduction of challenge HSV-2 vaginal replication in guinea pigs immunized with CJ9-gD. One set of 8 and one set of 10 guinea pigs were inoculated s.c. with either 5 × 10⁶ PFU/animal of CJ9-gD or DMEM and boosted after 3 weeks. At 6 weeks guinea pigs were challenged intravaginally with 5 × 10⁵ PFU of HSV-2 strain MS. Vaginal swabs were taken on days 1, 2, 3, 5, 7, and 9 post-challenge. Infectious virus in swab materials was assessed by standard plaque assay on Vero cell monolayers. Viral titers are expressed as the mean ± SEM in individual vaginal swabs (A). The duration of viral shedding is represented as the mean number of days during which infectious virus was detected in swab materials following challenge ± SEM (B). P-values were assessed by Student's t-test (* p < 0.05, ** p < 0.005, *** p < 0.0005)

Figure 3

Prevention of primary HSV-2 genital lesions in guinea pigs immunized with CJ9-gD. Mock-immunized and CJ9-gD-immunized guinea pigs described in Fig. 2 were monitored daily for clinical symptoms following challenge with wild-type HSV-2. The average number of lesions per immunized animals was compared with that found in mock-immunized guinea pigs. The indicated values represent the mean number of lesions ± SD on day 6 post-challenge. P-value was assessed by Student's t-test (* p < 0.0001).

Figure 4

Prevention of primary HSV-2 disease in guinea pigs immunized with CJ9-gD. After challenge with wild-type HSV-2, individual guinea pigs described in legend of Fig. 3 were observed during a 60-day follow-up period for the incidence of genital and disseminated HSV-2 disease using the following score: 0 = no disease; 1 = redness or swelling; 2 = a few small vesicles; 3 = several large vesicles; 4 = several large ulcers with maceration; 5 = paralysis; and 6 = death. Presented is the disease score for the first 15 days after challenge (A.) and the percentage of survival until day 60 after challenge (B.).

Figure 5

Protection from latent viral infection in guinea pigs immunized with CJ9-gD. Sixty days after challenge, 12 lower lumbar and sacral dorsal root ganglia (DRG) per guinea pig were harvested from all 8 immunized guinea pigs and the 2 surviving mock-immunized controls. The whole DNA was extracted and quantified for the presence of latent viral DNA using quantitative real-time PCR. The amount of viral DNA per guinea pig (A) was determined. The results are indicated as mean values ± SEM. P-value was assessed by Student's t-test (* p < 0.0001).