Protection from genital herpes disease, seroconversion and latent infection in a non-lethal murine genital infection model by immunization with an HSV-2 replication-defective mutant virus

Abstract

Viral vaccines have traditionally protected against disease, but for viruses that establish latent infection, it is desirable for the vaccine to reduce infection to reduce latent infection and reactivation. While seroconversion has been used in clinical trials of herpes simplex virus (HSV) vaccines to measure protection from infection, this has not been modeled in animal infection systems. To measure the ability of a genital herpes vaccine candidate to protect against various aspects of infection, we established a non-lethal murine model of genital HSV-2 infection, an ELISA assay to measure antibodies specific for infected cell protein 8 (ICP8), and a very sensitive qPCR assay. Using these assays, we observed that immunization with HSV-2 dl5-29 virus reduced disease, viral shedding, seroconversion, and latent infection by the HSV-2 challenge virus. Therefore, it may be feasible to obtain protection against genital disease, seroconversion and latent infection by immunization, even if sterilizing immunity is not achieved.

Keywords: animal infection model; genital disease; herpes simplex virus; latent infection; seroconversion; vaccines.

Figure 1. Disease and viral shedding in mice infected intravaginally with HSV-2 186ΔKpn

Mice were infected intravaginally with varying doses of HSV-2 186ΔKpn virus, and disease and viral shedding were monitored. Five animals per group were evaluated. A) Disease scores in BALB/c mice infected intravaginally with HSV-2 186ΔKpn. B) Viral shedding in the vaginal cavity of mice infected intravaginally with HSV-2 186ΔKpn.

Figure 2. Development of a sensitive PCR assay to detect latent HSV-2 DNA

A) A representative qPCR standard curve generated using NaI gradient double-banded HSV-2 DNA and ICP8 primers and probes showing 6 logs of detection with a lower limit of 1 copy/reaction and an efficiency of 96%. B) An independent experiment showing detection of latent HSV-2 DNA in mouse DRGs infected with HSV-2 186ΔKpn. Green circles correspond to standard points ranging from 2 to 2×10⁶ copies. Black crosses correspond to values for experimental samples determined to contain from 3.8 to 12 copies per reaction.

Figure 3. Development of an ELISA to detect HSV ICP8-specific antibody

A) Recombinant ICP8-FLAG protein used for coating ELISA plates. Two independent preparations of HSV-1 ICP8 protein were produced in insect cells and purified with an anti-FLAG antibody. Shown is a gel stained with Simply Blue Coomassie blue staining. The positive control lane is a preparation used in a previous biochemical study (Bryant et al., 2012). B) ELISA titers for anti-ICP8 antibodies from mice inoculated with HSV-2 186ΔKpn virus delivered intravaginally. n = 5 mice/group, plot shows mean titer ± SEM.

Figure 4. Protection against disease, virus shedding, and seroconversion

Mice were immunized intramuscularly (IM) or subcutaneously (SQ) with HSV-2 dl5-29 or mock-immunized and challenged intravaginally with HSV-2 186ΔKpn virus. A) Disease Scores. B) Viral shedding into the vaginal cavity. C) ELISA titers. n = 10 (Mock-vaccinated) or 9 (vaccinated groups) mice/group, plots show mean ± SEM. p values: Mock = 0.001, SQ = 0.0052, IM = 0.2234.

Figure 5. Protection against latent infection

At 30 days after challenge with HSV-2 186ΔKpn, DNA was collected from the dorsal root ganglia of BALB/c mice. In two independent experiments, we measured latent viral DNA in all mock-vaccinated animals. Results are expressed as copies of viral DNA per microgram of total DNA. ICP8 was used to measure viral DNA copies. Murine adipsin was used as an internal control. Dotted line shows the lower limit of detection of 2 copies/reaction. n = 10 (Mock-vaccinated) or 9 (vaccinated groups) mice/group, plot shows mean ± SEM. Mock-vaccinated group was significantly higher that both SQ and IM vaccinated groups (p = 0.0028 and 0.0026 respectively).

Figure 6. Correlation of protection from acute and latent infection

Pairwise comparisons of the three protection parameters were evaluated for all animals in this study. A) Viral shedding versus anti-ICP8 titers. B) Copies of latent viral DNA in DRGs versus anti-ICP8 titers. C) Viral shedding versus copies of latent viral DNA in DRGs. Pearson coefficients (r) and p-values (p) were calculated as described in Materials and Methods.