Evidence for differences in immunologic and pathogenesis properties of herpes simplex virus 2 strains from the United States and South Africa

Abstract

Background: Genital infection with herpes simplex virus 2 (HSV-2) is linked to an increased risk of infection with human immunodeficiency virus (HIV) in areas such as Sub-Saharan Africa. Thus, an effective genital herpes vaccine would be an important weapon in the fight against HIV/AIDS.

Methods: To test whether a current vaccine candidate can protect against HSV-2 from Sub-Saharan Africa, we examined the ability of an HSV-2 vaccine strain, dl5-29, and other HSV-2 replication-defective mutant strains to protect against genital challenge with US or South African strains in a murine model.

Results: Immunization with dl5-29 reduces infection by both viruses but is significantly more efficacious against the US virus than against the African virus. Furthermore, another US vaccine strain was more efficacious against US than against African viruses, and the converse was observed for the parallel African vaccine strain. Nevertheless, protection against the African viruses was significantly less with all vaccines used in this study.

Conclusions: We conclude that there may be differences in protective epitopes and pathogenesis between the US and African strains that raise the need for increased doses of the existing vaccine candidate or an HSV-2 vaccine strain based on viruses from that region.

Figure 1.

The dl5-29 vaccine strain protects mice against intravaginal infection with herpes simplex virus 2 ( HSV-2). Groups of mice (n = 14) were either mock-immunized or immunized with dl5-29 at a dose of 10⁴, 10⁵, or 10⁶ plaque-forming units (PFU) at weeks 0 and 4 and then challenged at week 8 with either HSV-2 SD90-3P or HSV-2 G virus. A, Clinical disease score, as described in Materials and Methods. B, Viral shedding. Results are shown as means ± standard errors of the mean. Dotted line indicates limit of detection of 2 PFU. C, Protection from paralysis.

Figure 2.

Protein expression by the 2 vaccine strains. Vero cells were either mock-infected (lane 1) or infected with wild-type herpes simplex virus 2 (HSV-2) 186syn⁺-1 (lane 2), its replication-defective derivative, 5BlacZ (lane 3), wild-type SD90-3P (lane 4), or its replication-defective derivative, SD90-8LacZ (lane 5), at a multiplicity of infection of 10 for 6 h. At 30 min before harvesting, cells were labeled with sulfur 35–labeled methionine. Proteins were resolved by SDS-PAGE, and the gel was subjected to autoradiography; the resulting autoradiograph is shown. Infected cell proteins 8 (ICPs) and the ICP8–β-galactosidase fusion protein are indicated.

Figure 3.

Primary herpes simplex virus (HSV)–specific CD8⁺ T cell responses as measured by major histocompatibility complex class I (MHC-I) pentamer staining. C57Bl/6 mice were immunized with 5BlacZ virus, SD90-8LacZ virus, or uninfected V5-29 cell lysate (n = 8). At days 6, 7, and 8 after immunization, whole blood was collected via the tail vein, and peripheral blood mononuclear cells were prepared and stained with MHC-I pentamers specific for the H2K^b restricted HSV-2 gB epitope SSIEFARL and antibody against murine CD8a. The percentage of live lymphocytes that stained positive for both CD8a and the MHC-I pentamer are shown over time. Lower panels show dot plot graphs of representative cellular responses.

Figure 4.

Secondary herpes simplex virus (HSV)–specific T cell responses as measured by intracellular cytokine staining. C57Bl/6 mice were immunized on days 0 and 28 with 5BlacZ virus, SD90-8LacZ virus, or uninfected V5-29 cell lysate (n = 8). On day 34, splenocytes were collected and stimulated with either the gB-SSIEFARL peptide to determine CD8⁺ responses or HSV-2–infected splenocytes to measure CD4⁺ responses. Results are shown as the mean percentage ± SEM of CD8⁺ or CD4⁺ T lymphocytes that specifically express interferon (IFN) γ after HSV-specific stimulation. A, CD8⁺ T cell responses. B, CD4⁺ T cell responses. Splenocytes from immunized mice were stimulated by splenocytes infected overnight with wild-type HSV-2 186syn⁺-1, SD90-3P or G strain. Dot plots below graphs are representative of cellular responses.

Figure 5.

Protective efficacy of 5BlacZ and SD90-8LacZ against wild-type herpes simplex virus 2 (HSV-2) viral challenges. Groups of female Balb/c mice (n = 16–18) were immunized at 0 and 4 weeks with 10⁵ plaque-forming units (PFU) of 5BlacZ, SD90-8LacZ or mock-immunized with uninfected V5-29 cell lysate (n = 8). At 8 weeks the mice were challenged intravaginally with 50 times the 50% lethal dose of wild-type HSV-2 strains from the United States, 186syn⁺-1, G, or 89-390, or from South Africa (SA), SD90-3P, SD15, or SD66. A, Average disease score during first 10 days of infection. B, Average disease score analyzed by geographic group of vaccine strain and challenge viruses. C, Average viral shedding levels during first 5 days of infection. D, Average viral shedding analyzed by geographic group of vaccine strain challenge viruses. E, Protection from paralysis at day 21 after infection. F, Protection from paralysis analyzed by geographic group of vaccine strain and challenge viruses. *P ≤ .05 (Mann-Whitney-Wilcoxon 2-sample test for A–D, Fisher exact test for E and F).

Figure 6.

Reduction of disease, virus shedding or lethality for groups of US or South African (SA) challenge viruses. Results from the studies in Figure 5 have been reanalyzed to show the reduction in disease in mice immunized with US or South African vaccines and then challenged with the US or South African challenge viruses.