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.

Interleukin-15 (IL-15), natural killer (NK) cells, and NK T (NKT) cells, components of the innate immune system, are known to contribute to defense against pathogens, including viruses. Here we report that IL-15−/− (NK− and NKT−/+) mice and RAG-2−/−/γc−/− (NK− and NKT−) mice that lack all lymphoid cells were very susceptible to vaginal infection with a low dose of herpes simplex virus type 2 (HSV-2). IL-15−/− and RAG-2−/−/γc−/− mice were 100-fold more susceptible and RAG-2−/−, CD-1−/− (NKT−), and gamma interferon (IFN-γ)−/− mice were 10-fold more susceptible to vaginal HSV-2 infection than control C57BL/6 mice. NK and/or NKT cells were the early source of IFN-γ in vaginal secretions following genital HSV-2 infection. This study demonstrates that IL-15 and NK-NKT cells are critical for innate protection against genital HSV-2

SummaryThe induction and maintenance of long-term CTL memory at mucosal surfaces may be a critical component of protection against mucosal pathogens and is one goal towards development of effective mucosal vaccines. In these studies we have functionally evaluated short and longterm CTL memory in systemic and respiratory or genital-associated lymphoid tissues following mucosal or systemic routes of immunization. Our results indicate that shortly after immunizing mice with a recombinant adenovirus vector expressing glycoprotein ~ (gB) of herpes simplex virus (AdgB8), gB-specific CTL memory responses were observed in systemic and mucosal immune compartments regardless of the route of inoculation. In contrast, several months after immunization, anamnestic CTL responses compartmentalized exclusively to mucosal or systemic lymphoid tissues after mucosal or systemic immunization, respectively. Furthermore, the compartmentalized CTL memory responses in mucosal tissues were functionally observed for longer than 1.5 yr after intranasal immunization, and CTL precursor frequencies one year after immunization were comparable to those seen shortly after immunization. Therefore, to our knowledge, this is the first functional demonstration that the maintenance of anti-viral memory CTL in mucosal tissues is dependent on the route of immunization and the time of assessment. These results have important implications for our understanding of the development, maintenance, and compartmentalization of functional T cell memory and the development and evaluation of vaccines for mucosal pathogens, such as HSV and HIV.

Together, these data indicate that chronic viraemic HIV-1 is associated with increased TLR expression and responsiveness, which may perpetuate innate immune dysfunction and activation that underlies HIV pathogenesis, and thus reveal potential new targets for therapy.

Development of vaccines capable of preventing the transmission or limiting the severity of sexually transmitted viruses, such as HSV and HIV, will likely be dependent on the induction of potent long-lasting mucosal immune responses in the genital tract. Recently, synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs were shown to serve as potent adjuvants for the induction of mucosal immune responses. Here, we show that intranasal immunization with CpG ODN, plus recombinant glycoprotein B (rgB) of HSV-1, results in significantly elevated levels of specific anti-gB IgA Abs in vaginal washes that remained high throughout the estrous cycle. Additionally, dramatically elevated numbers of specific IgA Ab-secreting cells were present and persisted in the genital tract in response to intravaginal (IVAG) HSV-2 challenge. HSV-2-specific CTL were observed at moderate levels in the spleens of CpG or non-CpG ODN-immunized mice. In contrast, strong CTL responses were observed locally in the genital tissues of both groups following IVAG HSV-2 challenge. Interestingly, mice immunized intranasally with rgB plus CpG ODN, but not non-CpG ODN, were significantly protected following IVAG HSV-2 challenge. Measurement of virus in protected CpG-immunized mice revealed a log lower level of replication within the first few days after infection. In conclusion, these results indicate that intranasal immunization with CpG ODN plus protein mediates immunity in the female genital tract capable of protecting against a sexually transmitted pathogen.

We previously demonstrated that delivery of CpG oligodeoxynucleotide (ODN) to vaginal mucosa induced an innate mucosal antiviral state that protected against intravaginal challenge with herpes simplex virus (HSV)-2. We report that mucosal, but not systemic, delivery of ligands for Toll-like receptor (TLR)-3, but not TLR4, induced protection against genital HSV-2 challenge that was not accompanied by the local inflammation and splenomegaly seen after treatment with CpG ODN. Surprisingly, TLR4 messenger (m) RNA expression was shown to be higher than that of TLR3 or TLR9 in murine genital mucosa. Similarly, murine RAW264.7 cells were shown to express more mRNA for TLR4 than TLR3 or TLR9, yet treatment of these cells with double-stranded RNA provided greater protection than lipopolysaccharide or CpG ODN. These results indicate that TLR3 ligand induces a more potent antiviral response than TLR4 and TLR9 ligands and may be a safer means of protecting against sexually transmitted viral infections.

Mucosal surfaces are the entry sites for the vast majority of infectious pathogens and provide the first line of defense against infection. In addition to the epithelial barrier, the innate immune system plays a key role in recognizing and rapidly responding to invading pathogens via innate receptors, such as Toll-like receptors (TLR). Bacterial CpG DNA, a potent activator of innate immunity, is recognized by TLR9. Here, we confirm that local mucosal, but not systemic, delivery of CpG oligodeoxynucleotides (ODN) to the genital tract protects mice from a subsequent lethal vaginal herpes simplex virus type 2 (HSV-2) challenge. Since these effects were so local in action, we examined the genital mucosa. Local delivery of CpG ODN induced rapid proliferation and thickening of the genital epithelium and caused significant recruitment of inflammatory cells to the submucosa. Local CpG ODN treatment also resulted in inhibition of HSV-2 replication but had no effect on HSV-2 entry into the genital mucosa. CpG ODN-induced protection against HSV-2 was not associated with early increases in gamma interferon (IFN-␥) secretion in the genital tract, and CpG ODN-treated IFN-␥ ؊/؊ mice were protected from subsequent challenge with a lethal dose of HSV-2. Treatment of human HEK-293 cells transfected with murine TLR9 showed that the antiviral activity of CpG ODN was mediated through TLR9. These studies suggest that local induction of mucosal innate immunity can provide protection against sexually transmitted infections, such as HSV-2 or possibly human immunodeficiency virus, at the mucosal surfaces.

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.