The results from this study show that not only did Depo induce a prolonged diestrus stage in mice, allowing them to be easily infected with HSV-2, but there was also a change in the susceptibility of the treated mice. Susceptibility of Depo-treated mice increased by 100-fold compared to that of normal untreated mice in diestrus, and susceptibility of Depo-treated mice increased by 10-fold compared to that of mice treated with P-sal at a similar dose. Treatment with P-sal, which does not have the slow-release effect of Depo, also induced diestrus for 4 to 6 days in the treated animals. Compared to that of normal, untreated mice in diestrus, the P-sal-treated mice had 10-fold-increased susceptibility. The results also show that though some of the mice started cycling 4 to 6 days following progesterone treatment and showed estrus stage by vaginal cytology, these mice had increased susceptibility as well and were susceptible to genital HSV-2 infection at high inoculum doses (106 and 107 PFU). Normal, untreated mice in estrus were not susceptible to HSV-2 infection, even at very high infectious doses (107 PFU).
That hormone treatment enhances genital tract infection by agents such as HSV-2 has been known for a while (15
). Most studies, in fact, use Depo to induce the diestrous state in mice to get consistent infection. However, the possibility of changed susceptibility due to the hormone treatment has not been considered by most investigators. To the best of our knowledge, this is the first time that susceptibility changes following different progesterone treatments have been documented and compared. The results clearly show that, depending on the hormone treatment, susceptibility of mice to genital herpes infection can vary significantly. Compared to normal, untreated mice, treatment with progesterone increases susceptibility to HSV-2 significantly both at diestrus and estrus. Depo-treated mice are the most susceptible and remain so for prolonged periods of time. Studies with Depo-treated mice have to take into account the effect on their results of altered susceptibility due to hormone treatment.
The mechanism by which progesterone increases susceptibility in mice is not clear. Animals in estrus, when the epithelial lining in the vagina is several layers thick, normally have been shown to be resistant to genital HSV-2 infection (2
). In this study, untreated animals in estrus also were resistant to inoculation dose as high as 107
PFU. Under the influence of progesterone the epithelial lining in the vagina is thinned out, making it possible for microbial organisms to cross the protective barrier and establish infection. The high titers of viral shedding observed in Depo- and progesterone-treated mice support this possibility. What is unclear is why the mice administered the long-lasting formulation of progesterone, Depo, were more susceptible than those administered similar doses of P-sal at the same time point (days 4 to 6). On the other hand, it is interesting that the thickness of the epithelial lining may not be the only factor in modulating susceptibility. This is indicated by results where progesterone-treated animals in estrus were also susceptible at high infectious doses of HSV-2. It is possible that other factors, such as receptors for HSV-2 which would play a critical role in regulating susceptibility, may be differentially expressed under various hormone treatments. This may contribute to changes in susceptibility. Previous studies have also shown that local IgA levels in the genital tract are affected profoundly by progesterone (22
). IgA plays a critical role in defending the genital tract against viral infections (10
). Downregulation of IgA levels by Depo could also enhance infection by HSV-2. We are presently examining these possibilities.
The results from TK-HSV-2 immunization experiments underline the importance of taking into consideration the hormonal state in designing vaccines against STDs. Vaccination was successful or unsuccessful in our studies depending on the hormonal state at the time of initial exposure to the attenuated virus vaccine. Mice immunized 15 days post-Depo treatment showed the least protection following subsequent challenge with wild-type HSV-2. Mice immunized at diestrus or estrus following progesterone treatment showed complete protection against subsequent challenges. These results indicate that adequate immune responses were induced when mice were immunized at estrus or diestrus. The length of exposure to Depo treatment appears to be quite critical in determining whether the mice are protected from subsequent challenges with wild-type virus. In other studies we have seen that mice that were immunized within 4 to 5 days following Depo treatment did not show lack of subsequent protection compared to mice that were immunized 15 days after Depo treatment (A. Gillgrass, A. A. Ashkar, K. L. Rosenthal, and C. Kaushic, submitted for publication). This explains why other studies, where mice were immunized 3 to 5 days following Depo treatment, found that TK-HSV-2 immunization protects mice from subsequent challenges (13
Our data from the present study indicate that alterations in immune responses under different hormonal conditions may affect the ability to raise an adequate immune response that protects the genital tract from viral infections. In the intranasal gB immunization experiments, it was clear that local antibody levels to gB in the genital tract were significantly lowered following Depo treatment. Local antibody responses following mucosal immunization may be critical to subsequent protection against HSV-2 exposure in the genital tract (1
). In related studies we have observed that long-term exposure to Depo leads to a decreased antibody response in the genital tract following immunization with TK-HSV-2, and this correlates with a lack of protection against subsequent challenges (A. Gillgrass et al., submitted).
We and others have also demonstrated that immune responses in the genital tract are regulated by sex hormones (20
). Studies show that following progesterone treatment and at diestrus (when progesterone is the predominant hormone), immune responses, such as IgA and IgG levels in the uterine fluid, IgA transport, and immune cell trafficking in the uterus, are all suppressed (20
). Similar results have recently been reported for the reproductive tract of women (18
). Antigen-independent CD3+
T-lymphocyte cytolytic activity was found to be high in the proliferative phase under the influence of estradiol and was absent in the secretory phase of menstrual cycle when progesterone levels are high.
The results from this study also raise questions regarding the change in susceptibility of women to STDs. Studies have already indicated that intake of oral contraceptives influences susceptibility to candidiasis, HSV-2, human immunodeficiency virus type 1, and chlamydial infections in women (11
). A recent study also shows that estrogen may protect against vaginal transmission of simian immunodeficiency virus in a rhesus macaque model, while earlier studies have shown that subcutaneous progesterone implants made monkeys more susceptible to simian immunodeficiency virus vaginal transmission (12
). Depo treatment is a popular form of contraception. The results from these studies and clinical data on oral contraceptives make it very likely that there are changes in susceptibility to STDs in women who use Depo as a method of birth control (11
). This is of concern, since many women on hormonal birth control methods, such as Depo, do not use barrier methods, which would provide some degree of protection against transmission of STDs. Further studies are needed to completely understand the implication of the present study for women, since our experiments were done with inbred mice and the conclusions may not be directly applicable in humans.
In summary, the results of this study document for the first time that pretreatment with different formulations of progesterone not only induces diestrous state in mice but also significantly increases their susceptibility to genital HSV-2 infection. In addition, the study also indicates that Depo may suppress immune responses following immunization. These studies emphasize the need to take into consideration hormonal influences in designing vaccination strategies to preventing STDs.