Progesterone increases susceptibility and decreases immune responses to genital herpes infection

Abstract

Depo-provera, a long-acting progestational formulation, is widely used to facilitate infection of sexually transmitted diseases in animal models. We have previously reported that hormone treatments change susceptibility and immune responses to genital tract infections. In this study we compared the changes in susceptibility of mice to genital herpes simplex virus type 2 (HSV-2) after Depo-provera or a saline suspension of progesterone (P-sal). We found that following Depo-provera-treatment, mice had prolonged diestrus that lasted more than 4 weeks. This coincided with a 100-fold increase in susceptibility to genital HSV-2 compared to that of untreated mice. Mice given P-sal were in diestrous stage for 4 to 6 days before returning to irregular reproductive cycles. When these mice were infected at diestrus they showed a 10-fold increase in susceptibility compared to that of normal, untreated mice. P-sal-treated mice infected at estrus were susceptible to HSV-2, depending on the infectious dose. Normal, untreated mice in estrus were not susceptible to HSV-2, even at a high infectious dose of 10(7) PFU. In addition to alterations in susceptibility, Depo-provera treatment had inhibitory effects on immune responses to HSV-2. Mice immunized with HSV-2 protein (gB) and treated with Depo-provera showed significant lowering of local HSV-2-specific immunoglobulin G (IgG) and IgA in their vaginal washes. Mice immunized with an attenuated strain of HSV-2 2 weeks after Depo-provera treatment failed to develop protection when challenged intravaginally with wild-type HSV-2. In contrast, mice given progesterone and immunized at diestrus or estrus were completely protected from intravaginal challenge. These studies show that Depo-provera treatment changes susceptibility and local immune responses to genital HSV-2 infection. Animal models and vaccine strategies for sexually transmitted diseases need to consider the effect of hormone treatments on susceptibility and immune responses.

FIG. 1.

Vaginal smears from hormone-treated mice. Four groups of mice were treated with either Depo (2 mg/mouse) or progesterone (2, 1, or 0.5 mg/mouse) subcutaneously. Vaginal smears were taken daily, and the stage of the cycle was determined as described in Materials and Methods. Graphs show the cycles of each mouse followed for 20 (progesterone groups) or 30 days posttreatment (Depo group). Estrus is shown by peaks on the Y axis, whereas diestrus is denoted by the baseline.

FIG. 2.

Viral titers from mice following progesterone treatment. Three groups of mice (n = 6) were given subcutaneous progesterone doses of 2, 1, or 0.5 mg/mouse. Vaginal smears were taken daily as described in Materials and Methods. Mice were inoculated intravaginally with 10⁵ PFU of HSV-2 (strain 333) at estrus (▵) or diestrus (○). Vaginal washes were collected daily, and viral plaque assays were done as described in Materials and Methods. Plaques were counted and viral titers were expressed as PFU/milliliter. Geometric mean values are shown for each group. An asterisk indicates a value of >10⁸ PFU, the upper detection limit of the assay. The dashed lines show the lower detection limit of the assay. Results are representative of three independent experiments with similar results.

FIG. 3.

Viral titers of mice following Depo treatment. Three groups of mice (n = 5 to 6 mice in each group) were given Depo (2 mg/mouse) subcutaneously and were intravaginally inoculated 5 days later with different HSV-2 doses (10³ to 10⁵ PFU). Vaginal washes were collected daily, and viral plaque assays were done as described in Materials and Methods. Plaques were counted and viral titers were expressed as PFU/milliliter. Each symbol represents a single animal. Experiments were repeated twice with comparable results.

FIG. 4.

Viral titers of mice infected with different doses of HSV-2 (strain 333) following P-sal treatment. (A) Mice treated with P-sal (1 mg/mouse) and inoculated intravaginally at diestrus with different infectious doses. (B) Mice treated with P-sal and inoculated intravaginally at estrus with different infectious doses. Vaginal washes were collected daily, and viral plaque assays were done as described in Materials and Methods. Plaques were counted, and viral titers were expressed as PFU/milliliter. Each symbol represents a single animal (n = 5 to 6 mice in each group). Results shown are representative of two separate experiments.

FIG. 5.

Viral titers of normal, untreated mice following infection with HSV-2. Vaginal smears were taken daily as described in Materials and Methods. Mice were inoculated intravaginally with 10⁶ PFU of HSV-2 (strain 333) at estrus (○) or diestrus (▵). Vaginal washes were collected daily, and viral plaque assays were done as described in Materials and Methods. Plaques were counted, and viral titers were expressed as PFU/milliliter. Each symbol represents a single animal (n = 5 to 6 mice in each group). The dashed line shows the lower detection limit of the assay. Results are representative of two separate experiments.

FIG. 6.

Pathology of mice immunized with TK-HSV-2 and challenged with wild-type HSV-2. One group of mice was given Depo (2 mg/mouse) subcutaneously and was immunized intravaginally with 10⁵ PFU of TK-HSV-2 15 days later. A second group of mice was given progesterone (1 mg/mouse) subcutaneously. Vaginal smears were examined, and mice were sorted according to the stage of the cycle (diestrus or estrus) and were immunized intravaginally with 10⁵ PFU of TK-HSV-2. All mice in all three groups (n = 5 to 6 mice per group) were challenged intravaginally 15 days postimmunization with 10⁶ PFU of HSV-2 (strain 333) at diestrus. Pathology and survival were scored for all animals, as explained in Materials and Methods. The experiment was repeated two times with comparable results.

FIG. 7.

Mice were immunized intranasally with gB and CpG oligonucleotides, boosted 2 weeks later, and treated with Depo 3 weeks later. gB-specific IgG and IgA titers were measured prior to and 4 days after Depo treatment. Results were sorted out on the basis of the stage of the animal prior to Depo treatment. n = 6 to 10 mice per group. An asterisk indicates a P value of <0.05 compared to data for mice prior to Depo inoculation. Data shown are representative of two separate experiments.