A replication-competent, neuronal spread-defective, live attenuated herpes simplex virus type 1 vaccine

Abstract

Herpes simplex virus type 1 (HSV-1) produces oral lesions, encephalitis, keratitis, and severe infections in the immunocompromised host. HSV-1 is almost as common as HSV-2 in causing first episodes of genital herpes, a disease that is associated with an increased risk of human immunodeficiency virus acquisition and transmission. No approved vaccines are currently available to protect against HSV-1 or HSV-2 infection. We developed a novel HSV vaccine strategy that uses a replication-competent strain of HSV-1, NS-gEnull, which has a defect in anterograde and retrograde directional spread and cell-to-cell spread. Following scratch inoculation on the mouse flank, NS-gEnull replicated at the site of inoculation without causing disease. Importantly, the vaccine strain was not isolated from dorsal root ganglia (DRG). We used the flank model to challenge vaccinated mice and demonstrated that NS-gEnull was highly protective against wild-type HSV-1. The challenge virus replicated to low titers at the site of inoculation; therefore, the vaccine strain did not provide sterilizing immunity. Nevertheless, challenge by HSV-1 or HSV-2 resulted in less-severe disease at the inoculation site, and vaccinated mice were totally protected against zosteriform disease and death. After HSV-1 challenge, latent virus was recovered by DRG explant cocultures from <10% of vaccinated mice compared with 100% of mock-vaccinated mice. The vaccine provided protection against disease and death after intravaginal challenge and markedly lowered the titers of the challenge virus in the vagina. Therefore, the HSV-1 gEnull strain is an excellent candidate for further vaccine development.

FIG. 1.

Safety studies of NS-gEnull as a vaccine candidate. (A to C) Mice were monitored for survival, inoculation site disease, and zosteriform disease (P = 0.01 in panel A for comparison of NS-gEnull or mock vaccination with rNS-gEnull; P < 0.01 in panels B and C for comparison of these groups). There were five mice in each group. (D) Skin samples were harvested for viral titers at the inoculation site (P < 0.01 on days 1 and 3; P > 0.05 on day 6). There were three mice in each group. (E) Skin samples were evaluated by immunohistochemistry to detect HSV-1 antigens (arrows). (F) DRG were evaluated for viral titers (P < 0.01 on day 3; P = 0.4 on day 6). There were three mice in each group. (G) Single-step growth curves of NS, NS-gEnull, and rNS-gEnull in Vero cells demonstrated comparable replication of the viruses. Results in panels B, C, D, and F are means ± SEMs. LOD refers to the limit of detection for the assay, which was 20 PFU.

FIG. 2.

NS-gEnull vaccination protects against flank challenge with 10⁵ PFU HSV-1(NS). (A to C) Mice were monitored for survival, inoculation site disease, and zosteriform disease (P = 0.003 in panel A for comparison of each vaccinated group with mock-vaccinated mice; P < 0.001 in panels B and C for comparison of these groups). There were five mice in each group. (D) Skin titers after challenge of mice vaccinated with 5 × 10⁵ PFU of NS-gEnull or mock vaccinated (P > 0.05 on day 1; P = 0.02 on day 3; P > 0.05 on day 6). Three mice were included for each data point. (E) Immunohistochemistry of skin for HSV-1 antigens (arrows). (F) Viral titers in DRG of mice vaccinated with 5 × 10⁵ PFU or mock vaccinated (P = 0.02 on days 3 and 6; P = 0.4 on day 8). There were three mice in each group. (G) HSV-1 viral DNA copies per 5 × 10⁵ copies of adipsin within the DRG of challenged mice (P = 0.03 on day 3; P = 0.02 on day 6; P = 0.4 on day 8). The asterisk indicates the average log₁₀ copy number including an outlier of 10⁴ PFU, possibly due to contamination, on day 1 (P = 0.48 with the day 1 outlier; P = 0.42 without the outlier). There were three mice in each group. Data points in panels B, C, D, F, and G are means ± SEMs. LOD refers to the limit of detection for the assay, which was 20 PFU.

FIG. 3.

NS-gEnull vaccination protects against disease and reduces the recovery of latent virus from DRG after flank challenge with HSV-1(KOS) at 10⁵ PFU. (A and B) Mice were monitored for inoculation site disease (P = 0.03) and zosteriform disease (P = 0.04 on day 5; P < 0.001 on days 6 and 7). The data points are means ± SEMs. (C) DRG explant cocultures were performed 28 days after challenge (P < 0.001). There were 10 mice in each group.

FIG. 4.

NS-gEnull vaccination is protective when administered by scarification, i.m., or SubQ. (A) Mice were monitored for survival (P = 0.02 for comparison of mock vaccination with each immunization route). (B) Inoculation site disease (P < 0.001 for comparison of disease in mock-vaccinated mice with that for each immunization route). (C) Zosteriform disease (P = 0.03 for comparison of mock vaccination with each immunization group). (D) Twenty-eight days after challenge, DRG explant cultures were performed. (E) HSV-1 neutralizing antibody titers (P = 0.03 for comparison of scarification or the i.m. route with the SubQ route; P > 0.05 for comparison of the i.m. route and scarification). There were five mice in each group, except for the epidermal scarification group, which had four mice. Results in panels B, C, and E are means ± SEMs.

FIG. 5.

NS-gEnull vaccination protects against flank challenge (chall.) with HSV-1(NS), HSV-1(F), and HSV-1(17) at 10⁵ PFU. (A) Mice were monitored for survival (P = 0.08 for strain NS, P = 0.08 for strain F, and P = 0.3 for strain 17 for comparison of the survival of immunized and mock-immunized mice). vac., vaccination. (B) Inoculation site disease (P < 0.001 for comparison of the same groups as in panel A). (C) Zosteriform disease (P < 0.001 for comparison of these groups). Three mice were challenged with strain F or 17, and four mice were challenged with strain NS. The results in panels B and C are means ± SEMs.

FIG. 6.

NS-gEnull vaccination cross-protects against HSV-2(2.12) challenge at 10⁵ PFU. (A) Mice were monitored for survival (P < 0.05). (B) Inoculation site disease (P > 0.05 on day 1; P = 0.01, P = 0.03, and P = 0.001 on days 5 to 7, respectively). (C) Zosteriform disease (P > 0.05 on days 3 and 4; P = 0.01, P = 0.02, and P = 0.005 on days 5 to 7, respectively). There were five mice in each group. Data points in panels B and C are means ± SEMs. (D) One year after challenge, explant cultures of DRG were monitored for virus reactivation.

FIG. 7.

NS-gEnull vaccination protects against vaginal challenge with HSV-1(NS) at 10⁵ PFU. (A) Mice were monitored for survival (P = 0.01). (B) Vaginal titers (P < 0.01 on day 1; P < 0.001 on days 2 to 5; P > 0.05 on days 6 and 7). Data points are means ± SEMs. LOD refers to the limit of detection for the assay, which was 2 PFU. (C) Vaginal disease in each of five mice on day 8 after challenge. There were five mice in each group, except that vaginal swabs on days 1 to 4 were from nine mice. Arrows indicate lesions.