Immunization against genital herpes with a vaccine virus that has defects in productive and latent infection

Abstract

An effective vaccine for genital herpes has been difficult to achieve because of the limited efficacy of subunit vaccines and the safety concerns about live viruses. As an alternative approach, mutant herpes simplex virus strains that are replication-defective can induce protective immunity. To increase the level of safety and to prove that replication was not needed for immunization, we constructed a mutant herpes simplex virus 2 strain containing two deletion mutations, each of which eliminated viral replication. The double-mutant virus induces protective immunity that can reduce acute viral shedding and latent infection in a mouse genital model, but importantly, the double-mutant virus shows a phenotypic defect in latent infection. This herpes vaccine strain, which is immunogenic but has defects in both productive and latent infection, provides a paradigm for the design of vaccines and vaccine vectors for other sexually transmitted diseases, such as AIDS.

Figures

Figure 1

Lack of dl5-29 mutant virus growth in mouse tissues. BALB/c mice (Taconic Farms; aged 6 weeks; n = 3 per time point per virus) were inoculated intranasally with 1 × 106 pfu of HSV-2 strain 186 wt virus (wt), HSV-2 strain 186ΔKpn TK− virus (TK−), or HSV-2 strain 186 dl5-29 virus (dl5-29). At the times shown, the nasal septa were removed and homogenized in sterile, low endotoxin PBS, and an aliquot of each homogenate was assayed for infectious virus by standard plaque assay on V5-29 cells, which complement the growth of dl5-29 mutant virus. Shown are the geometric mean values (log10) of the titers ± SEM.

Figure 2

Levels of HSV-2 DNA in trigeminal ganglia (TG) at various times after intranasal inoculation. Mice were inoculated with 1 × 106 pfu of the HSV-2 replication-defective double mutant dl5-29 (♦) or the replication-competent TK− mutant 186ΔKpn (○; n = 8 trigeminal ganglia per time point per virus) or given saline as a negative control (n = 4 trigeminal ganglia per time point; not shown). On days 1, 3, 7, 14, 21, or 28 after inoculation, the trigeminal ganglia were harvested and assayed for viral DNA by quantitative PCR. The lower limit of detection was 10 molecules per trigeminal ganglia. No viral DNA was detected in the mock-infected animals. Individual values of each trigeminal ganglia are shown along with the geometric mean for each series.

Figure 3

Reduction of the shedding of the challenge virus from the genital tract in animals immunized with recombinant mutant HSV strains. Female 6-week-old BALB/c mice were placed randomly into four groups of six mice each. All animals were injected twice, 4 weeks apart, by the subcutaneous route in the rear flank with 2 × 106 pfu of dl5 (○), dl29 (▵), dl5-29 (□), or as a control, uninfected cell lysate (⋄). The mice were challenged 4 weeks after the boost injection by intravaginal challenge with 5 × 105 pfu of HSV-2 strain G (≈50 times LD50). Virus shed in the genital tract was collected by vaginal swabs daily for 1 week after the challenge and quantified by titration in Vero cell monolayer cultures. Shown are the geometric means of the virus titers ± SEM. The lower limit of detection was 1 pfu/ml.