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Depo-Provera (Depo) is a long-acting progestational formulation that is a popular form of contraception for women. In animal models of sexually transmitted diseases, it is used to facilitate infection. Here we report that treatment with Depo, in a mouse model of genital herpes simplex virus type 2 (HSV-2), altered immune responses depending on the length of time that animals were exposed to Depo prior to immunization. Mice immunized intravaginally (i.vag.) with an attenuated strain (TK−) of HSV-2 following longer (15 days) exposure to Depo (Depo 15 group) failed to show protection when challenged with wild-type HSV-2. In contrast, mice that were immunized shortly after Depo treatment (5 days; Depo 5 group) were fully protected and showed no genital pathology after HSV-2 challenge. High viral titers were detected in the vaginal washes of the Depo 15 group up to 6 days postchallenge. In contrast, no viral shedding was observed beyond day 3 postchallenge in the Depo 5 group. Following i.vag. TK− immunization, high levels of gamma interferon (IFN-γ) were detected locally in vaginal washes of the Depo 5 group but not the Depo 15 group. After HSV-2 challenge, an early peak of IFN-γ in the Depo 5 group coincided with clearance of the virus. In Depo 15 animals IFN-γ was present throughout the 6 days postinfection. HSV-2-specific T-cell cytokine responses measured in the lymph node cells of Depo 5 TK−-immunized mice indicated a significantly higher Th1 response than that of Depo 15 TK−-immunized mice. The protection after HSV-2 challenge in the Depo 5 group correlated with increased local HSV-2 glycoprotein B (gB)-specific immunoglobulin G (IgG) and IgA responses seen in the vaginal secretions. The Depo 15 group had poor gB-specific antibody responses in the genital tract after HSV-2 challenge. These results indicate that longer exposure to Depo leads to poor innate and adaptive immune responses to HSV-2 that fail to protect mice from subsequent genital challenges.
Sexually transmitted diseases (STDs), including human immunodeficiency virus (HIV) infection-AIDS, are a leading cause of morbidity and a huge economic burden on health systems in both developing and developed countries. Herpes simplex virus type 2 (HSV-2) is the causative agent of one of the most commonly transmitted viral STDs (1). Currently in North America, one out of every four adults is estimated to be seropositive for HSV-2 (6). Attempts at developing an effective vaccine have been unproductive over the last 4 decades. A successful prophylactic vaccine for sexually transmitted infections will have to induce a good local immune response in the genital tract where infection is initiated (13). In order to achieve this, factors that affect mucosal immune responses in the reproductive tract need to be taken into consideration. That these factors are important is demonstrated by a recent clinical trial of a glycoprotein D-based HSV-2 vaccine that reported limited success only in women and not in men (25). Gender-based factors that may affect the immune system may therefore play a critical role in the success of STD vaccines.
Various studies have shown that sex hormones have a profound effect on susceptibility to STDs and immune responses in females (8, 12, 15, 24, 27, 28). Hormonal contraception, such as oral contraceptives and progesterone-based methods (Depo-Provera [Depo]), has been shown in various studies to be a biologic factor linked to HIV type 1 acquisition (14). In our own studies we have seen that rats that are normally resistant to genital infection by Chlamydia trachomatis are rendered susceptible following progesterone treatment (11). Other studies have shown that simian immunodeficiency virus (SIV) genital infection and disease course are enhanced by progesterone implants (15).
Depo is a progesterone-based popular contraceptive. It is a long-acting (Depo) formulation of medroxyprogesterone acetate that was approved by the Food and Drug Administration for contraceptive use in women in 1992. It is effective for periods of 3 months, and patients receive injections at 3-month intervals (7). This form of contraception is currently being promoted as an attractive contraception option for adolescents due to its high efficacy and ease of compliance (26).
In mouse studies of HSV-2, Depo is commonly used to facilitate infection. Many studies have utilized Depo-treated mice to examine the infection kinetics and immune responses to HSV-2 (9, 16, 17, 21). In all of these studies mice are administered subcutaneous Depo and shortly after (within a week) immunized or infected. We recently reported that Depo changes the susceptibility of treated mice to HSV-2 infection. Depo-treated mice were found to be 100-fold more susceptible than untreated mice in diestrus (10). Additionally, we reported a significant lowering of antibody responses to HSV-2 in immunized mice following Depo treatment. Interestingly, in these studies when mice were exposed to Depo for a longer time (2 weeks) prior to immunization with TK− HSV-2, they failed to show any protection against subsequent challenge (10). Other studies using immunization protocols where mice were immunized 3 to 5 days after Depo treatment showed complete protection (16, 20, 21). The present study was undertaken to determine if indeed prolonged exposure to Depo prior to immunization results in failure to protect mice from subsequent challenge. Mice that were Depo treated 2 weeks prior to immunization were compared with mice that were exposed to Depo for 5 days before immunization. The effect of length of exposure to Depo on immune responses to HSV-2 was examined as well as its consequent effect on protection following challenge. Following immunization and challenge, viral shedding and pathology in the two groups of mice were compared. To examine the mechanism, the local immune responses in the genital tract and the draining lymph nodes (LN) were also measured. Finally mucosal antibody responses following challenge with wild-type HSV-2 were determined.
Inbred C57BL/6 mice, 8 to 10 weeks old, purchased from Charles River Canada (Constant, Quebec, Canada), were used in these studies. Mouse colonies were maintained on a 12-h-dark-12-h-light cycle. Mice were injected subcutaneously with 2 mg of Depo (Pharmacia-Upjohn, Don Mills, Ontario, Canada), based on previous studies (10).
Mice were anesthetized by injectable anesthetic (ketamine-xylazine, 0.75 and 0.25 ml, respectively) given intraperitoneally, placed on their backs, and inoculated intravaginally (i.vag.) with 10 μl of wild-type HSV-2 strain 333 or the attenuated TK− strain of HSV-2, each at a dose of 105 PFU per mouse. Mice were kept on their backs under the influence of anesthesia for 45 min to an hour to allow the inoculum to infect them.
Vaginal lavage for reproductive cycle staging and plaque assays was collected by pipetting 2× 30 μl of phosphate-buffered saline (PBS) in and out of the vagina several times. For vaginal smears, the fluid was smeared on glass slides and examined by light microscopy to determine the stage of the estrous cycle as described before (30). The following classification was used for identifying the stage of the cycle: estrus, >90% cornified epithelial cells; diestrus, >75% polymorphonuclear cells; diestrus-estrus, 50% epithelial cells-50% polymorphonuclear cells. For plaque assays the vaginal washes were frozen at −70°C. Mice treated with Depo remained in diestrus for 5 to 6 weeks, as described before (10). Vaginal smears of both Depo 5 and Depo 15 mice were examined to confirm that they were all in diestrus stage prior to immunization with TK− HSV-2 and challenge with wild-type HSV-2.
Genital pathology following infection with HSV-2 was monitored daily and scored on a five-point scale: 0, no infection; 1, slight redness of external vagina; 2, swelling and redness of external vagina; 3, severe swelling and redness of both vagina and surrounding tissue and hair loss in genital area; 4, genital ulceration with severe redness, swelling, and hair loss of genital and surrounding tissue; and 5, severe genital ulceration extending to surrounding tissue. Animals were sacrificed after they reached stage 4.
Vaginal washes or tissue homogenates were analyzed for viral titers by plaque assays. Vero cells were grown in alpha minimum essential medium (GIBCO Laboratories, Burlington, Canada) supplemented with 5% fetal calf serum (GIBCO), 1% penicillin-streptomycin, and l-glutamine (GIBCO). For plaque assays, Vero cells were grown to confluence in 12-well plates. Samples were diluted (10−2 to 10−7) and added to monolayers. Infected monolayers were incubated at 37°C for 2 h for viral absorption. Infected monolayers were overlaid with alpha minimum essential medium supplemented with 0.05% human immune serum globulin (Canadian Blood Services). Infection was allowed to occur for 48 h at 37°C. Monolayers were then fixed and stained with crystal violet, and viral plaques were counted under a light microscope. PFU per milliliter was calculated by taking a plaque count for every sample and taking into account the dilution factors.
HSV-2 glycoprotein B (gB)-specific antibody titers were determined by an enzyme-linked immunosorbent assay (ELISA) modified from a protocol described previously (8). Briefly Maxisorp 96 plates (Invitrogen, Burlington, Ontario, Canada) were coated overnight with 2.5 μg of recombinant gB protein (Chiron Inc., Emeryville, Calif.)/ml in PBS at 4°C. Plates were blocked with 2% bovine serum albumin for 2 h at room temperature and loaded with 100 μl of twofold serial dilutions of samples or controls. Incubation was carried out at 4°C overnight. Plates were washed and reacted for 1 h with one of the following biotinylated antibodies: goat anti-mouse IgG or goat anti-mouse IgA at a 1:1,000 dilution (PharMingen, Mississauga, Ontario, Canada). Plates were developed with extravidin-peroxidase (1:2,000 dilution) and 3,3′,5,5′-tetramethylbenzidine. Endpoint titers were determined and expressed as geometric mean titers ± standard errors (SEs) of the means. Background values were obtained by using vaginal lavage samples from nonimmunized mice. Two times the mean background optical density value was taken as the cutoff for determining positive values.
Iliac LN that drain the genital tract were dissected, and single-cell suspensions were prepared by teasing the LN. Debris was allowed to settle for 2 min, and supernatant containing single cells was recovered and spun down at 500 × g for 7 to 10 min. Cells were washed with RPMI 1640 medium containing 5% bovine serum albumin and plated at the density of 5 × 105 cells/well in a 96-well plate. Cells were tested for HSV-2-specific proliferation by addition of gB (10 μg/ml) (Chiron Inc.) in triplicate cultures. Total T-cell proliferation was measured by adding cells to plates coated with anti-CD3 antibody (20 μg/well; purified from clone 2C11, generously provided by D. P. Snider, McMaster University). Cultures were incubated for 48 h, and supernatants were collected and frozen for further testing. Proliferative responses were measured by uptake of 1 μCi of [3H]thymidine per well for the last 18 h of a 3-day culture. Results are reported as mean counts per minute ± SEs of the means of triplicate cultures.
Cytokine levels were measured in LN supernatants by using commercial ELISA kits from R&D Systems (Minneapolis, Minn.) according to the protocol recommended in the kits. Fifty-microliter aliquots of collected supernatants were run in duplicate. Absorbance was read at 450 nm. A standard curve was used to calculate the picograms-per-milliliter concentration in each sample. The ratios of gamma interferon (IFN-γ) to interleukin 10 (IL-10) and of IFN-γ to IL-4 were calculated for each animal, and mean ± SE was calculated for each experimental group.
Each experiment was repeated at least two times with 6 to 12 animals in each group. Data were analyzed by the unpaired two-tailed t test using SPSS 11.00 for Windows software. Significance was defined as a P value of <0.05.
Two groups of mice were treated with a single injection of Depo and immunized with 105 PFU of TK− HSV-2 i.vag. One group was vaccinated 15 days after Depo treatment (Depo 15 group), and the other group was vaccinated 5 days after administration of Depo (Depo 5 group). Following Depo treatment, all the mice were in diestrus between 5 and 6 weeks as reported before (10). Vaginal smears were examined prior to challenge to confirm that mice in both Depo 15 and Depo 5 groups were in diestrus. There was no difference in the pathology of animals in either group following immunization (data not shown). Animals in both groups showed signs of mild local infection with some redness and inflammation in the genital area for 3 to 4 days before resolving the infection.
Three weeks after immunization both groups were challenged i.vag. with 105 PFU of wild-type HSV-2 strain 333. Data from one of three separate experiments are shown in Fig. Fig.1.1. Gross daily examination following the challenge showed that animals in the Depo 5 group had no or minimal signs of vaginal pathology. In contrast, four out of six animals in the Depo 15 group started showing vaginal pathology within 72 h of HSV-2 challenge and by day 6 had severe pathology with scores of 3 to 4 on the scale and had to be euthanized. Similar results were seen in two other experiments, where 60 to 80% of Depo 15 animals succumbed to infection.
To examine if the differences in the protection seen in the two groups of mice were correlated with the ability of TK− virus to infect the genital tract, we examined viral titers after immunization as well as following challenge with wild-type HSV-2. Following TK− HSV-2 immunization viral titers in vaginal secretions were similar in both the Depo 15 and Depo 5 groups (Fig. (Fig.2A).2A). After HSV-2 challenge, viral titers measured in vaginal washes showed good correlation with pathology data (Fig. (Fig.2B).2B). All the animals in the Depo 5 group resolved the infection within 3 days, and subsequently no viral titers were detected in their vaginal washes. In contrast, four out of six animals in the Depo 15 group had high viral titers on all six days on which the shedding was measured. These animals were the same ones that exhibited severe pathology (Fig. (Fig.11).
The results from the previous experiments made it clear that 5-day Depo treatment allowed protection of mice from subsequent HSV-2 challenge but 15-day Depo treatment did not. Previous studies have shown that IFN-γ secreted into vaginal secretions is a good indicator of local antiviral responses (18). To examine if there were differences in local IFN-γ production in the infected animals, we examined IFN-γ levels in vaginal secretions. Following immunization with TK− HSV-2, the Depo 5 group showed an early peak of IFN-γ secreted into the vaginal secretions on day 2 postimmunization, as has been reported in other studies (Fig. (Fig.3B).3B). This early peak of IFN-γ was not present in the Depo 15 group. Very low levels of IFN-γ were detected in the vaginal washes of this group following immunization (Fig. (Fig.3A3A).
Following HSV-2 challenge, the Depo 5 animals showed an early peak of IFN-γ secretion starting on day 1 postchallenge. The levels decreased to below detectability by day 4, coinciding with the virus clearance pattern (Fig. (Fig.3D).3D). On the other hand, in Depo 15 animals, IFN-γ response peaked at day 2 postchallenge and showed the typical biphasic pattern seen in other studies (18), following primary exposure to virus, with high levels of IFN-γ still present on day 6 postchallenge (Fig. (Fig.3C3C).
Since we observed a decreased local IFN-γ response in vaginal secretions in the Depo 15 group following TK− HSV-2 immunization, we examined, 5 days after immunization in the two groups of mice, T-cell immune responses in the local LN draining the genital tract. The time kinetics of T-cell responses in the draining LN was determined prior to these experiments, and day 5 postimmunization was found to be the optimal time to measure HSV-2-specific LN T-cell responses (data not shown). To examine total T-cell responses following immunization, LN T cells were activated in vitro by anti-CD3 antibody. HSV-2-specific responses were measured by in vitro stimulation with gB, a highly immunogenic HSV-2 envelope glycoprotein. Following TK− immunization, LN cells from Depo 15 and Depo 5 groups had no significant differences in proliferative responses to in vitro gB challenge or stimulation by anti-CD3 antibody (Fig. (Fig.4A).4A). However, when cytokine profiles were examined in culture supernatants from in vitro gB-challenged LN cells, Depo 5 cultures had about threefold-higher levels of IFN-γ than did Depo 15 LN cultures (Fig. (Fig.4B).4B). IL-4 levels were lower and IL-10 levels were higher in the Depo 5 group than in the Depo 15 group, although these levels did not show statistical significance (Fig. (Fig.4C4C and D). However, when Th1/Th2 ratios were calculated, Depo 5 LN cultures had more-than-fourfold-higher IFN-γ/IL-4 ratios (P < 0.01) (Fig. (Fig.4E).4E). Similarly, a threefold increase in IFN-γ/IL-10 ratio was seen in supernatants of the Depo 5 group compared to the Depo 15 group (Fig. (Fig.4F4F).
T-cell responses were also examined 5 days following HSV-2 challenge (data not shown). In agreement with the observations made on viral shedding and IFN-γ response in the vaginal secretions, by day 5 postchallenge, T-cell responses in Depo 5 animals were past their peak and all the cytokine levels were comparable to those for uninfected mice. On the other hand, Depo 15 LN showed significantly higher levels of IFN-γ and Th1/Th2 ratios were significantly high, indicating a primary response to HSV-2.
Previous studies have demonstrated that protection against genital HSV-2 infection correlates with local antibody levels in the vaginal secretions (8, 20). Since differences were observed in protection against HSV-2 challenge between Depo 15 and Depo 5 mice, local antibodies to gB in vaginal secretions were measured to determine if Depo treatment had an effect on secondary antibody responses to HSV-2. Representative data from one of the three experiments are shown in Fig. Fig.5.5. All six mice in the Depo 15 group had poor gB-specific IgA responses, while three out of six mice in the Depo 5 group showed significant gB-specific IgA levels in the vaginal washes. Differences in gB-specific IgG were even more dramatic. All six mice in the Depo 5 group had gB-specific IgG responses, and four out of six had endpoint titers between 1,000 and 5,000. One out of six Depo 15 mice had a detectable level of gB-specific IgG in its vaginal secretions.
The results from this study clearly show that length of exposure to Depo had a significant influence on immune responses following immunization with an attenuated strain of HSV-2. The changes in immune responses resulted in mice being protected or not from subsequent challenge with wild-type virus. Previous studies have shown that immunization with TK− HSV-2 3 to 5 days following Depo treatment leads to complete protection against subsequent challenge (16, 21). In our study we found similar results, where shorter exposure to Depo (5 days) induced immune responses adequate to protect against subsequent i.vag. challenge. In contrast, longer exposure (2 weeks) to Depo inhibited induction of immune responses and failed to provide protection against subsequent genital challenge with wild-type HSV-2. Following immunization, there was a lack of induction of IFN-γ in vaginal secretions of Depo 15 mice, indicating that local innate responses were compromised. There was also a lowered Th1 response in the local draining LN of Depo 15 mice. Following challenge with wild-type HSV-2, inadequate protection in the Depo 15 group correlated with the absence of local antibody responses in the genital tract compared to the Depo 5 group.
While previous clinical and experimental studies have clearly shown that progesterone increases susceptibility to STDs, the present study starts to delineate the effects on immune responses. The most intriguing observation was the suppressive effect of progesterone on innate immune responses following immunization with TK− HSV-2. Previous studies have shown that in response to primary vaginal infection with HSV-2 there is a biphasic secretion of IFN-γ which plays an important role in clearance of virus (18). The early peak of IFN-γ seen in vaginal secretions is part of the innate response produced primarily by NK and NKT cells in response to the viral infection (18). Studies in other infection models have shown that IFN-γ produced by NK cells plays an important role in skewing the developing immune response to Th1 type (23). Local IFN-γ may also play an important role in activating the antigen-presenting cells and upregulating major histocompatibility complex class II on antigen-presenting cells, to enhance presentation of viral antigens (4). This early IFN-γ response is absent in animals with prolonged exposure to Depo, indicating a possible deficiency in the initiation of an innate response to HSV-2 immunization. In other studies from our group, mice that genetically lack the ability to mount this initial innate response (IFN-γ−/− and Rag-2−/−/γc−/− mice) also showed a significant increase in susceptibility to HSV-2 genital infection (2).
The progesterone effect was also evident on adaptive immune responses to HSV-2 immunization. The Th1/Th2 ratios were significantly lowered in Depo 15 mice following immunization, as were the local antiviral IgA and IgG antibody levels following challenge. One possibility is that the lack of the initial innate response affected subsequent induction of protective adaptive immune responses. The absence of IFN-γ in the early innate response could alter the Th1 response to more of a Th2 response and consequently affect mucosal antibody responses. On the other hand, both the innate and adaptive responses may be independently inhibited by Depo. It was recently reported that, when Depo was administered in previously immunized mice, the IgG and IgA antibody levels were lowered dramatically in the vaginal secretions, indicating that Depo may directly inhibit local antibody levels (10).
The cellular mechanism by which longer exposure to progesterone downregulates the protective immune response is not clear. Our data suggest that the effect requires more than 5 days of exposure to Depo. Results from other studies indicate that progesterone can exert a suppressive effect on neutrophils (19) and NK cell function (5). It is therefore possible that prolonged exposure to Depo may affect the ability of these cells to secrete appropriate chemokines and cytokines to initiate an antiviral innate immune response. Clinical studies in tumor research have also shown that continuous or intermittent scheduled administration of medroxyprogesterone acetate can lead to cell cycle arrest of human hematopoietic progenitors (22). If this mechanism is active in this HSV-2 model, then under the influence of long-term exposure to Depo progenitor immune cells may not be able to proliferate, resulting in a reduced antiviral response. We are currently investigating this possibility.
The finding that progesterone enhances susceptibility to sexually transmitted viruses has been demonstrated by a number of other studies (12, 14, 15). In the rhesus macaque model of SIV it has been shown that progesterone implants enhance SIV vaginal transmission and virus load (15). The increase in transmission in this and other studies has been attributed to the atrophy of vaginal epithelium under the influence of progesterone. The thin epithelium may allow the virus to easily penetrate the epithelial layer and/or establish infection in susceptible target cells under the epithelium. While the present study was done in mice, the results from this study may have important implications for women using Depo as a method of contraception. In addition to thinning the genital epithelium, if continuous use of Depo also affects immune responses in the genital tract of women, then it may further increase the risk of sexually transmitted infections, including HIV. This is especially important in adolescent girls who represent the group at highest risk of contracting STDs (3). Studies need to be done to further examine if women who are on Depo or other hormonal therapies have altered susceptibility to STDs and/or immune responses to vaccines. If so, additional precautions such as barrier methods of contraception are important for women who are currently using hormonal contraceptives and therapies, to protect themselves against increased risk of transmission.
The ultimate goal for a successful STD vaccine would be to induce sterile immunity in the genital tract. Previous studies have shown clearly that, in both animal models and women, reproductive tract immune responses are regulated by the sex hormones estradiol and progesterone (29). It was previously shown that estradiol and progesterone regulate susceptibility and immune responses in a rat model of C. trachomatis (12) and a mouse model of HSV-2 (10). The present study extends these findings by showing that hormonal treatments such as Depo may also affect the ability of the immune system to fight sexually transmitted viral infections. These studies emphasize the need to consider the hormonal status of women in order to develop effective STD vaccine strategies. It also has important implications for women who are currently on hormonal therapies.
This work was supported by research grants from the Canadian Institutes of Health Research (to C.K. and K.L.R.), the Ontario HIV Treatment Network (C.K.), and the Bickell Foundation (C.K.). C.K. is an Ontario HIV Treatment Network Scholar.
We acknowledge the technical help of Jen Newton and Amy Patrick. We also thank Denis Snider for critical reading of the manuscript.