Efficacy of an HSV-1 Neuro-Attenuated Vaccine in Mice Is Reduced by Preventing Viral DNA Replication

Abstract

We previously isolated an HSV-1 mutant, KOS-NA, that contains two non-synonymous mutations in UL39. One of the mutations, resulting in an R950H amino acid substitution in ICP6, renders KOS-NA severely neuro-attenuated and significantly reduces HSV-1 latency. Vaccination of mice with KOS-NA prior to corneal challenge provides significant protection against HSV-1-mediated eye diseases even at a very low immunizing dose, indicating its utility as a vaccine scaffold. Because KOS-NA contains a neuro-attenuating mutation in a single gene, we sought to improve its safety by deleting a portion of the UL29 gene whose protein product, ICP8, is essential for viral DNA replication. Whereas KOS-NA reduced replication of HSV-1 challenge virus in the corneal epithelium and protected mice against blepharitis and keratitis induced by the challenge virus, KOS-NA/8- and an ICP8- virus were significantly less efficacious except at higher doses. Our results suggest that the capacity to replicate, even at significantly reduced levels compared with wild-type HSV-1, may be an important feature of an effective vaccine. Means to improve safety of attenuated viruses as vaccines without compromising efficacy should be sought.

Keywords: corneal infection; herpes simplex virus 1; replication-defective; vaccine.

Figure 1

Eye titers of challenge virus from immunized mice. Groups of 10 BALB/c mice were vaccinated with medium (control) or (A) low (2 × 10⁴ pfu), (B) medium (1 × 10⁵ pfu), or (C) high (5 × 10⁵ pfu) doses of KOS-NA, ICP8-, and KOS-NA/8-. The control group is the same for all three graphs. All groups were challenged with HSV-1 strain mP (4 × 10⁵ pfu/eye), 30 days post-immunization. Eye swabs were taken at the times indicated (0–5 days post-infection), and titers were determined by standard plaque assays. Data represent the geometric means ± SEM from 3 independent experiments combined for n = 30 mice per group. *** p < 0.0001; ** p = 0.0002–0.0008; and * p = 0.0011–0.0064 for KOS-NA compared with ICP8- and KOS-NA/8- viruses.

Figure 2

Protection of mice from blepharitis after corneal challenge. Groups of 10 mice immunized with (A) low (2 × 10⁴ pfu), (B) medium (1 × 10⁵ pfu), or (C) high (5 × 10⁵ pfu) doses of the indicated viruses and 1 group of mice immunized with supernatant (control) were challenged by corneal infection with HSV-1 strain mP (4 × 10⁵ pfu/eye) and scored daily for signs of eyelid disease. Values are the mean ± SEM score of eyelids from 3 independent experiments combined for n = 30 mice. *** p < 0.0001; ** p = 0.0002-0.0003; * p = 0.0011–0.0092 note significant differences comparing KOS-NA to ICP8- and/or KOS-NA/8-.

Figure 3

Protection of mice from severe keratitis after corneal challenge. The corneas of the same groups of mice described in Figure 2 were evaluated at 9 days post-challenge for signs of keratitis at (A) low (2 × 10⁴ pfu), (B) medium (1 × 10⁵ pfu), or (C) high (5 × 10⁵ pfu) vaccine doses. Values represent the mean disease scores ± SEM compiled from 3 independent experiments; n = 11 to 30 mice examined per group. p-values shown were determined by one way ANOVA with Dunnett’s correction for multiple groups compared to KOS-NA.

Figure 4

Body weight change upon virus challenge. Groups of 10 BALB/c mice were vaccinated with supernatant (control) or (A) low (2 × 10⁴ pfu), (B) medium (1 × 10⁵ pfu), or (C) high (5 × 10⁵ pfu) doses of KOS-NA, ICP8-, and KOS-NA/8-. At 30 days post-vaccination, mice were weighed and challenged with HSV-1 strain mP (4 × 10⁵ pfu/eye) by corneal infection. Mice were then weighed daily post-challenge. Data shown are the mean weight change per vaccination group ± SEM from 3 independent experiments combined for n = 30 mice per group. *** p < 0.0004; ** p = 0.0032–0.0089; * p = 0.012–0.030 represent significant differences between KOS-NA relative to ICP8- and/or KOS-NA/8- as determined by one way ANOVA with Dunnett’s correction for multiple groups.

Figure 5

Survival of vaccinated mice after virus challenge. Mice were vaccinated with supernatant (control) or a low (2 × 10⁴ pfu/eye) dose of KOS-NA, ICP8-, and KOS-NA/8-. At 30 days post-vaccination, mice were challenged with HSV-1 strain mP (4 × 10⁵ pfu/eye) by corneal infection, and their survival was observed up to 14 days post-challenge. Data shown are survival ± SEM from 3 independent experiments. Survival of KOS-NA-vaccinated mice was statistically different from ICP8- by the log-rank test.

Figure 6

Titers of HSV-specific antibody in immunized mice. Groups of 10 BALB/c mice were immunized with low (2 × 10⁴ pfu), medium (1 × 10⁵ pfu), or high (5 × 10⁵ pfu) doses of the indicated viruses, and 1 group of mice was immunized with supernatant (control) as a negative control. Blood was collected 21 days post-immunization, and HSV-specific serum IgG was quantified by ELISA. Data represent the geometric mean titer for each group ± SEM and are the combined results of 3 independent experiments for n = 30 mice per group. *** p < 0.0004, ** p = 0.0011–0.0055, and * p = 0.011 indicate significant differences for KOS-NA compared to ICP8- and KOS-NA/8- viruses using one way ANOVA.