Mucosal administration of CpG oligodeoxynucleotide elicits strong CC and CXC chemokine responses in the vagina and serves as a potent Th1-tilting adjuvant for recombinant gD2 protein vaccination against genital herpes

Although sexually transmitted pathogens are capable of inducing pathogen-specific immune responses, vaginal administration of nonreplicating antigens elicits only weak, nondisseminating immune responses. The present study was undertaken to examine the potential of CpG-containing oligodeoxynucleotide (CpG ODN) for induction of chemokine responses in the genital tract mucosa and also as a vaginal adjuvant in combination with glycoprotein D of herpes simplex virus type 2 (HSV-2) for induction of antigen-specific immune responses. We found that a single intravaginal administration of CpG ODN in mice stimulates a rapid and potent response of CC chemokines macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES as well as of CXC chemokines MIP-2 and IP-10 in the vagina and/or the genital lymph nodes. Importantly, intravaginal vaccination with recombinant gD2 in combination with CpG ODN gave rise to a strong antigen-specific Th1-like immune response in the genital lymph nodes as well as the spleens of the vaccinated mice. Further, such an immunization scheme conferred both systemic and mucosal immunoglobulin G antibody responses as well as protection against an otherwise lethal vaginal challenge with HSV-2. These results illustrate the potential of CpG ODN for induction of potent chemokine responses in the genital tract and also as a vaginal adjuvant for generation of Th1-type mucosal and systemic immune responses towards a nonreplicating antigen derived from a sexually transmitted pathogen. These data have implications for the development of a mucosal vaccine against genital herpes and possibly other sexually transmitted diseases.     

Neutrophils Aid in Protection of the Vaginal Mucosae of Immune Mice against Challenge with Herpes Simplex Virus Type 2

Large numbers of polymorphonuclear leukocytes (PMNs) infiltrated the murine vaginal mucosa within 24 h after intravaginal inoculation with an attenuated strain of herpes simplex virus type 2 (HSV-2). The role of these cells in resolution of a primary genital infection and in protection of HSV-immune animals against challenge with a fully virulent HSV-2 strain was investigated. Depletion of greater than 95% of the PMNs at the vaginal mucosal surface prior to intravaginal inoculation with an attenuated HSV-2 strain resulted in significantly higher virus titers on days 3 to 7 but only slightly delayed resolution of the primary genital infection. These results suggest that neutrophils helped control the infection but that other immune mechanisms ultimately cleared the virus. Interestingly, depletion of PMNs from HSV-immune mice prior to challenge with a fully virulent HSV-2 strain resulted in a rise in virus titers to levels comparable to those of nonimmune mice and a more pronounced diminution of virus clearance from the vaginal mucosa despite the presence of HSV-specific B and T cells. Levels of gamma interferon (IFN-gamma) and HSV-specific antibody were comparable in neutrophil-depleted and control-treated immune mice following HSV-2 challenge, suggesting that RB6-8C5 treatment did not impair T- and B-cell function. Therefore, these results suggest that neutrophils play a role in limiting and clearing HSV-2 vaginal infections and that they are, in association with HSV-specific B and T cells, an important component in immune protection of the vaginal mucosa.     

Simian immunodeficiency virus rapidly penetrates the cervicovaginal mucosa after intravaginal inoculation and infects intraepithelial dendritic cells

Despite recent insights into mucosal human immunodeficiency virus (HIV) transmission, the route used by primate lentiviruses to traverse the stratified squamous epithelium of mucosal surfaces remains undefined. To determine if dendritic cells (DC) are used by primate lentiviruses to traverse the epithelial barrier of the genital tract, rhesus macaques were intravaginally exposed to cell-free simian immunodeficiency virus SIVmac251. We examined formalin-fixed tissues and HLA-DR(+)-enriched cell suspensions to identify the cells containing SIV RNA in the genital tract and draining lymph nodes within the first 24 h of infection. Using SIV-specific fluorescent in situ hybridization combined with immunofluorescent antibody labeling of lineage-specific cell markers, numerous SIV RNA(+) DC were documented in cell suspensions from the vaginal epithelium 18 h after vaginal inoculation. In addition, we determined the minimum time that the SIV inoculum must remain in contact with the genital mucosa for the virus to move from the vaginal lumen into the mucosa. We now show that SIV enters the vaginal mucosa within 60 min of intravaginal exposure, infecting primarily intraepithelial DC and that SIV-infected cells are located in draining lymph nodes within 18 h of intravaginal SIV exposure. The speed with which primate lentiviruses penetrate mucosal surfaces, infect DC, and disseminate to draining lymph nodes poses a serious challenge to HIV vaccine development.     

Vaccine-induced serum immunoglobin contributes to protection from herpes simplex virus type 2 genital infection in the presence of immune T cells

Herpes simplex type virus 2 (HSV-2) is a sexually transmitted pathogen that causes genital lesions and spreads to the nervous system to establish acute and latent infections. Systemic but not mucosal cellular and humoral immune responses are elicited by immunization of mice with a replication-defective mutant of HSV-2, yet the mice are protected against disease caused by subsequent challenge of the genital mucosa with virulent HSV-2. In this study, we investigated the role of immune serum antibody generated by immunization with a replication-defective HSV-2 vaccine prototype strain in protection of the genital mucosa and the nervous system from HSV-2 infection. Passive transfer of replication-defective virus-immune serum at physiologic concentrations to SCID or B-cell-deficient mice had no effect on replication of challenge virus in the genital mucosa but did significantly reduce the incidence and severity of genital and neurologic disease. In contrast, B-cell-deficient mice immunized with replication-defective HSV-2 were able to control replication of challenge virus in the genital mucosa, but not until 3 days postchallenge, and were not completely protected against genital and neurologic disease. Passive transfer of physiologic amounts of immune serum to immunized, B-cell-deficient mice completely restored their capacity to limit replication of challenge virus in the genital mucosa and prevented signs of genital and systemic disease. In addition, the numbers of viral genomes in the lumbosacral dorsal root ganglia of immunized, B-cell-deficient mice were dramatically reduced by transfer of immune serum prior to challenge. These results suggest that there is an apparent synergism between immune serum antibody and immune T cells in achieving protection and that serum antibody induced by vaccination with replication-defective virus aids in reducing establishment of latent infection after genital infection with HSV-2.     

Differential induction of adhesion molecule and chemokine expression by LTalpha3 and LTalphabeta in inflammation elucidates potential mechanisms of mesenteric and peripheral lymph node development

Lymphotoxin (LT) is a member of the proinflammatory TNF family of cytokines that plays a critical role in the development of lymphoid tissue. It has previously been reported that the presence of the LTalpha transgene under the control of the rat insulin promoter results in inflammation at the sites of transgene expression. LTalpha transgene expression results in expression of the adhesion molecules VCAM, ICAM, peripheral node addressin (a marker of peripheral lymph nodes), and mucosal addressin cellular adhesion molecule (a marker of mucosal lymphoid tissue, including mesenteric lymph nodes). In this study to determine the mechanisms by which LT promotes inflammation and lymphoid tissue organization, we analyzed the regulation of expression of adhesion molecules and chemokines in LT transgenic mice. The results demonstrate that LTalpha3 induces expression of the adhesion molecules VCAM, ICAM, and mucosal addressin cellular adhesion molecule as well as the chemokines RANTES, IFN-inducible protein-10, and monocyte chemotactic protein-1, while LTalphabeta is required for the induction of peripheral node addressin that may contribute to the recruitment of L-selectinhigh CD44low naive T cells. These data provide candidate mediators of LT-induced inflammation as well as potential mechanisms by which LTalpha and LTalphabeta may differentially promote the development of mesenteric and peripheral lymph nodes.     

Intranasal immunization with CpG oligodeoxynucleotides as an adjuvant dramatically increases IgA and protection against herpes simplex virus-2 in the genital tract

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.     

Efficient mucosal vaccination mediated by the neonatal Fc receptor

Almost all infectious diseases are initiated at mucosal surfaces, yet intramuscular or subcutaneous vaccination usually provides only minimal protection at sites of infection owing to suboptimal activation of the mucosal immune system. The neonatal Fc receptor (FcRn) mediates the transport of IgG across polarized epithelial cells lining mucosal surfaces. We mimicked this process by fusing a model antigen, herpes simplex virus type-2 (HSV-2) glycoprotein gD, to an IgG Fc fragment. Intranasal immunization, together with the adjuvant CpG, completely protected wild-type, but not FcRn knockout, mice after intravaginal challenge with virulent HSV-2 186. This immunization strategy induced efficient mucosal and systemic antibody, B- and T-cell immune responses, with stable protection for at least 6 months after vaccination in most of the immunized animals. The FcRn-IgG transcellular transport pathway may provide a general delivery route for subunit vaccines against many mucosal pathogens.     

Progesterone increases susceptibility and decreases immune responses to genital herpes infection

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.     

Injection of plasmid DNA into the gastric mucosa induces mucosal and systemic immunity

Nearly all mucosal surfaces participate in a common mucosal immune system, and application of an antigen to one mucosal surface elicits local as well as distant mucosal immune responses. However, whether the gastric mucosa is a part of this network has not been examined directly. We show here that the injection of plasmid DNA encoding beta-galactosidase into the gastric wall caused transfection of gastric mucosal epithelial cells, induced systemic and mucosal antibody responses at both local (digestive tract) and distant (genital and respiratory tracts) sites, and induced cytotoxic T lymphocyte responses in the spleen and the mesenteric and iliac lymph nodes.     

Mucosal and systemic immune responses to a human immunodeficiency virus type 1 epitope induced upon vaginal infection with a recombinant influenza A virus

The humoral and cellular immune responses in the genital mucosa likely play an important role in the prevention of sexually transmitted infections, including infection with human immunodeficiency virus type 1 (HIV-1). Here we show that vaginal infection of progesterone-treated BALB/c mice with a recombinant influenza virus bearing the immunodominant P18IIIB cytotoxic T-lymphocyte (CTL) epitope of the gp160 envelope protein from an HIV-1 IIIB isolate (P18IIIB; RIQRGPGRAFVTIGK) can induce a specific immune response in regional mucosal lymph nodes, as well as in a systemic site (the spleen). A single inoculation of mice with the recombinant influenza virus induced long-lasting (at least 5 months) antigen-specific CTL memory detectable as a rapid recall of effector CTLs upon vaginal infection with recombinant vaccinia virus expressing HIV-1 IIIB envelope gene products. Long-term antigen-specific CTL memory was also induced and maintained in distant mucosal tissues when mice were intranasally immunized with the recombinant influenza virus. These results indicate that mucosal immunization and, in particular, local vaginal immunization with recombinant influenza virus can provide strong, durable immune responses in the female genital tract of mice.     

In vivo role of nectin-1 in entry of herpes simplex virus type 1 (HSV-1) and HSV-2 through the vaginal mucosa

Herpes simplex virus type 2 (HSV-2) is transmitted through the genital mucosa during sexual encounters. In recent years, HSV-1 has also become commonly associated with primary genital herpes. The mechanism of viral entry of HSV-1 and HSV-2 in the female genital tract is unknown. In order to understand the molecular interactions required for HSV entry into the vaginal epithelium, we examined the expression of herpesvirus entry mediator nectin-1 in the vagina of human and mouse at different stages of their hormonal cycle. Nectin-1 was highly expressed in the epithelium of human vagina throughout the menstrual cycle, whereas the mouse vaginal epithelium expressed nectin-1 only during the stages of the estrous cycle in which mice are susceptible to vaginal HSV infection. Furthermore, the ability of nectin-1 to mediate viral entry following intravaginal inoculation was examined in a mouse model of genital herpes. Vaginal infection with either HSV-1 or HSV-2 was blocked by preincubation of the virus with soluble recombinant nectin-1. Viral entry through the vaginal mucosa was also inhibited by preincubation of HSV-2 with antibody against gD. Together, these results suggest the importance of nectin-1 in mediating viral entry for both HSV-1 and HSV-2 in the genital mucosa in female hosts.     

Intravaginal inoculation of rhesus macaques with cell-free simian immunodeficiency virus results in persistent or transient viremia.

The simian immunodeficiency virus (SIV)-rhesus macaque model of heterosexual human immunodeficiency virus transmission consists of atraumatic application of cell-free SIVmac onto the intact vaginal mucosa of mature female rhesus macaques. This procedure results in systemic infection, and eventually infected animals develop the clinical signs and pathologic changes of simian AIDS. To achieve 100% transmission with the virus stocks used to date, multiple intravaginal inoculations are required. The current titration study utilized two stocks of SIVmac and demonstrated that a single intravaginal dose of cell-free SIV can reliably produce infection in rhesus macaques. This study also demonstrated that some animals intravaginally inoculated with cell-free SIVmac develop transient viremia characterized by a limited ability to isolate virus from peripheral blood mononuclear cells and lymph node mononuclear cells and no seroconversion to SIV antigen. SIV could be isolated from the peripheral lymph nodes of transiently viremic animals only during periods of viremia and not at times when SIV was not detected in circulating mononuclear cells. Thus, peripheral lymphoid tissues were not reservoirs of infection in the transiently viremic animals. Taken together, these results suggest either that the SIV infection was cleared in the transiently viremic animals or that SIV infection is limited to a compartment of the genital mucosal immune system that cannot be assessed by monitoring SIV infection in peripheral blood mononuclear cells and peripheral lymphoid tissue.     

Susceptibility of CCR5-deficient mice to genital herpes simplex virus type 2 is linked to NK cell mobilization

Following genital herpes simplex virus type 2 (HSV-2) exposure, NK cells and T cells are mobilized to sites of infection to control viral replication and spread. The present investigation sought to determine the role of the chemokine receptor CCR5 in this process. Mice deficient in CCR5 (CCR5-/-) displayed a significant reduction in cumulative survival following infection in comparison to wild-type, HSV-2-infected controls. Associated with decreased resistance to viral infection, CCR5-/- mice yielded significantly more virus and expressed higher levels of tumor necrosis factor alpha, CXCL1, CCL2, CCL3, and CCL5 in the vagina, spinal cord, and/or brain stem than did wild-type mice. Whereas there was no difference in absolute number of leukocytes (CD45high), CD4 T cells, or CD8 T cells residing in the draining lymph nodes, spleen, spinal cord, or brain stem comparing HSV-2-infected wild-type to CCR5-/- mice prior to or after infection, there were significantly more NK cells (NK1.1+ CD3-) residing in the brain stem and spleen of infected wild-type mice. Functionally, NK activity from cells isolated from the brain stem of HSV-2-infected wild-type mice was greater than that from HSV-2-infected CCR5-/- mice. In addition, antibody-mediated depletion of NK cells resulted in an increase in HSV-2 levels in the vaginal, spinal cord, and brain stem tissue of wild-type but not CCR5-/- mice. Collectively, the absence of CCR5 expression significantly impacts the ability of the host to control genital HSV-2 infection, inflammation, and spread associated with a specific reduction in NK cell expansion, infiltration, and activity in the nervous system.     

CD8+ T-cell-mediated cross-clade protection in the genital tract following intranasal immunization with inactivated human immunodeficiency virus antigen plus CpG oligodeoxynucleotides

Human immunodeficiency virus (HIV) is a mucosally transmitted infection that rapidly targets and depletes CD4+ T cells in mucosal tissues and establishes a major reservoir for viral persistence in gut-associated lymphoid tissues. Therefore, vaccines designed to prevent HIV infections must induce potent and durable mucosal immune responses, especially in the genital tract. Here we investigated whether intranasal (i.n.) immunization with inactivated gp120-depleted HIV-1 antigen (Ag) plus CpG oligodeoxynucleotide (ODN) as an adjuvant induced local immune responses in the genital tract and cross-clade protection against intravaginal (IVAG) challenge. Lymphocytes isolated from the iliac lymph nodes (ILNs) and genital tracts of female mice i.n. immunized with HIV-1 Ag plus CpG showed significant HIV-specific proliferation and produced significantly higher levels of gamma interferon (IFN-gamma) and beta-chemokines than mice immunized with HIV-1 Ag alone or mixed with non-CpG ODN. CD8+ lymphocytes were dramatically increased in the genital tracts of mice immunized with HIV-1 Ag plus CpG, and protection following IVAG challenge with recombinant vaccinia viruses (rVVs) expressing HIV-1 gag was shown to be CD8 dependent. Finally, cross-clade protection was observed between clades A, C, and G but not B following IVAG challenge with rVVs expressing HIV-1 gag from different clades. These studies provide evidence that mucosal (i.n.) immunization induced strong local T-cell-mediated immune responses in the genital tract and cross-clade protection against IVAG challenge.     

Dendritic cell-mediated induction of mucosal cytotoxic responses following intravaginal immunization with the nontoxic B subunit of cholera toxin

The use of the nontoxic B subunit of cholera toxin (CTB) as mucosal adjuvant and carrier-delivery system for inducing secretory Ab responses has been documented previously with different soluble Ags. In this study, we have evaluated this approach for inducing CTL responses against a prototype Ag, OVA, in the female genital mucosa. We report here the ability of an immunogen comprised of CTB conjugated to OVA (CTB-OVA) given by intravaginal (ivag) route to induce genital OVA-specific CTLs in mice. Using adoptive transfer models, we demonstrate that ivag application of CTB-OVA activates OVA-specific IFN-gamma-producing CD4 and CD8 T cells in draining lymph nodes (DLN). Moreover, ivag CTB induces an expansion of IFN-gamma-secreting CD8+ T cells in DLN and genital mucosa and promotes Ab responses to OVA. In contrast, ivag administration of OVA alone or coadministered with CTB failed to induce such responses. Importantly, we demonstrate that ivag CTB-OVA generates OVA-specific CTLs in DLN and the genital mucosa. Furthermore, genital CD11b+ CD11c+ dendritic cells (DCs), but not CD8+ CD11c+ or CD11c- APCs, present MHC class I epitopes acquired after ivag CTB-OVA, suggesting a critical role of this DC subset in the priming of genital CTLs. Inhibition studies indicate that the presentation of OVA MHC class I epitopes by DCs conditioned with CTB-OVA involves a proteasome-dependent and chloroquine-sensitive mechanism. These results demonstrate that CTB is an efficient adjuvant-delivery system for DC-mediated induction of genital CTL responses and may have implications for the design of vaccines against sexually transmitted infections.     

Targeting the vaginal mucosa with human papillomavirus pseudovirion vaccines delivering simian immunodeficiency virus DNA

The majority of HIV infections occur via mucosal transmission. Vaccines that induce memory T and B cells in the female genital tract may prevent the establishment and systemic dissemination of HIV. We tested the immunogenicity of a vaccine that uses human papillomavirus (HPV)-based gene transfer vectors, also called pseudovirions (PsVs), to deliver SIV genes to the vaginal epithelium. Our findings demonstrate that this vaccine platform induces gene expression in the genital tract in both cynomolgus and rhesus macaques. Intravaginal vaccination with HPV16, HPV45, and HPV58 PsVs delivering SIV Gag DNA induced Gag-specific Abs in serum and the vaginal tract, and T cell responses in blood, vaginal mucosa, and draining lymph nodes that rapidly expanded following intravaginal exposure to SIV(mac251.) HPV PsV-based vehicles are immunogenic, which warrant further testing as vaccine candidates for HIV and may provide a useful model to evaluate the benefits and risks of inducing high levels of SIV-specific immune responses at mucosal sites prior to SIV infection.     

Mechanism of reduced T-cell effector functions and class-switched antibody responses to herpes simplex virus type 2 in the absence of B7 costimulation

T-cell costimulation molecules B7-1 and B7-2 play an important role in activation of T cells to cytolytic effector function and production of cytokines. Interaction with B7 also causes T cells to upregulate surface molecules, such as CD40L, that effectively stimulate antibody responses in conjunction with cytokines. We have shown that mice lacking both B7-1 and B7-2 (B7KO mice), when infected intravaginally with virulent herpes simplex virus type 2 (HSV-2), developed more severe disease and higher mortality than their wild-type counterparts. We have now investigated the effects of B7 costimulation deficiency on induction of immune responses to HSV-2 infection of the genital tract. Fewer gamma interferon (IFN-gamma)-producing T cells were present in the genital lymph nodes of B7KO mice compared to wild-type mice, either acutely after primary infection or in recall responses. Less IFN-gamma and especially interleukin-10 were produced by B7KO mice, and cytolytic T-lymphocyte activity was also attenuated. Reduced expression of CD25 on CD4(+) T cells after infection of B7KO mice was consistent with deficits in T-cell activation to effector functions. Although HSV-specific immunoglobulin M (IgM) titers were comparable for both B7KO mice and wild-type mice, B7KO mice had significant deficits in HSV-specific serum IgG responses, with markedly reduced levels of IgG2a and IgG1. In addition, significantly less IgG was detected in the vaginal secretions of B7KO mice than in those from wild-type mice. CD4(+) T-cell expression of CD40L was depressed in B7KO mice in vivo and in vitro. Together with reduced cytokine production, these results suggest a mechanism for decreased IgG class switching or production. Thus, in the absence of B7 costimulation, na?ve T cells fail to undergo proper activation in response to HSV-2, which limits T-cell cytokine production, cytotoxic T lymphocyte activity, and provision of help for class-switched antibody responses.     

Cyclooxygenase-2-mediated prostaglandin E2 production in mesenteric lymph nodes and in cultured macrophages and dendritic cells after infection with Salmonella

Although numerous studies have demonstrated the ability of intestinal epithelial cells to produce PGs after infection with wild-type strains of Salmonella, few studies have focused on Salmonella-induced prostanoids in mucosal lymphoid tissues. This is surprising in view of the profound effects PGs can have on the host response. To begin to address PG production at mucosal sites, mice were orally inoculated with Salmonella, and at varying times postinfection cyclooxygenase-2 (COX-2) mRNA expression and PGE(2) synthesis were investigated. COX-2 mRNA expression was highly inducible in the mesenteric lymph nodes, whereas COX-1 mRNA levels were constitutive. PGE(2) production also increased significantly in the mesenteric lymph nodes following exposure to viable Salmonella, but not after exposure to killed bacteria. This increased PGE(2) response could be blocked by treatment of mice with the selective COX-2 inhibitor, celecoxib. Treatment of mice with celecoxib during salmonellosis resulted in increased viable bacteria in the mesenteric lymph nodes by day 3 postinfection. However, celecoxib treatment prolonged the survival of lethally infected animals. In vitro studies demonstrated Salmonella-induced up-regulation of COX-2 mRNA expression and PGE(2) secretion by both macrophages and dendritic cells, which could also be blocked in the presence of celecoxib. Interestingly, exposure of these cultured APCs to viable Salmonella was a much greater stimulus for induction of PGE(2) synthesis than exposure to Salmonella-derived LPS. The present study demonstrates induction of PGE(2) synthesis in mesenteric lymph nodes, macrophages, and dendritic cells after infection with wild-type salmonella.     

Prolonged exposure to progesterone prevents induction of protective mucosal responses following intravaginal immunization with attenuated herpes simplex virus type 2

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-gamma) 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-gamma in the Depo 5 group coincided with clearance of the virus. In Depo 15 animals IFN-gamma 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.