CCR7 Deficiency Allows Accelerated Clearance of Chlamydia from the Female Reproductive Tract.

Immune mechanisms responsible for pathogen clearance from the female reproductive tract (FRT) are incompletely defined; in particular, the contribution of lymphocyte trafficking to this process is unclear. CCR7-deficient mice have profoundly altered lymphocyte recirculation and display ectopic formation of lymphocyte aggregates within mucosal nonlymphoid tissues, including the FRT. In this study, we investigated how altered lymphocyte distribution in CCR7-deficient mice would affect host responses to Chlamydia muridarum within the reproductive tract. As expected, CCR7-deficient mice exhibited reduced lymphocyte trafficking to lymph nodes and a corresponding increase in T cell populations within the FRT. After intravaginal infection with Chlamydia, CCR7-deficient mice displayed markedly reduced Ag-specific CD4 T cell responses within the local draining iliac lymph nodes, yet robust Th1 and Th17 responses were prominent in the FRT. In addition, Chlamydia-specific Ab responses were dysregulated in CCR7-deficient mice, displaying an unexpected increase in the systemic IgA responses. Importantly, prominent mucosal immune responses in CCR7-deficient mice increased the efficiency of bacteria clearance from the FRT while reducing tissue-associated inflammation and pathology. Thus, increased numbers of lymphocytes within the FRT result in pathogen clearance with reduced immune-mediated pathology.

Comparison of Murine Cervicovaginal Infection by Chlamydial Strains: Identification of Extrusions Shed In vivo.

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections (STIs) and preventable blindness. Untreated, asymptomatic infection as well as frequent re-infection are common and may drive pelvic inflammatory disease, ectopic pregnancy, and infertility. In vivo models of chlamydial infection continue to be instrumental in progress toward a vaccine and further elucidating the pathogenesis of this intracellular bacterium, however significant gaps in our understanding remain. Chlamydial host cell exit occurs via two mechanisms, lysis and extrusion, although the latter has yet to be reported in vivo and its biological role is unclear. The objective of this study was to investigate whether chlamydial extrusions are shed in vivo following infection with multiple strains of Chlamydia. We utilized an established C3H/HeJ murine cervicovaginal infection model with C. trachomatis serovars D and L2 and the Chlamydia muridarum strain MoPn to monitor the (i) time course of infection and mode of host cell exit, (ii) mucosal and systemic immune response to infection, and (iii) gross and histopathology following clearance of active infection. The key finding herein is the first identification of chlamydial extrusions shed from host cells in an in vivo model. Extrusions, a recently appreciated mode of host cell exit and potential means of dissemination, had been previously observed solely in vitro. The results of this study demonstrate that chlamydial extrusions exist in vivo and thus warrant further investigation to determine their role in chlamydial pathogenesis.

Genomic variant representation in a Chlamydia population is dynamic and adaptive with dependence on in vitro and in vivo passage.

We have previously shown that Chlamydia muridarum has multiple genomic variants that concomitantly vary in their in vitro and in vivo phenotype. Herein, we used real-time polymerase chain reaction-based genotyping assays to query plaque-cloned isolates of C. muridarum for the frequency of eight selected polymorphisms. These strains had no history of passage in vivo since their original isolation from laboratory mice. There was significant variance in the frequency of two of the eight polymorphisms assessed with the remaining exhibiting a low rate of variance. To determine if any of these polymorphisms were more favorable for in vivo conditions, we blindly passaged non-clonal C. muridarum three times at 7-day intervals through the urogenital tract of mice. Seven of the eight polymorphisms varied in frequency following in vivo passage and four of these varied between C. muridarum strains. Selected isolates displayed variable growth rates and cytopathic effect in vitro. We conclude that multiple genotypic variants are present within the existing known C. muridarum strains and that the frequency of these variants changes upon introduction into the mouse host. These findings lend support to the concept that genotypic proportional representation in a chlamydial population is dynamic and adaptive.

Chlamydial infection of the gastrointestinal tract: a reservoir for persistent infection.

The mechanism by which chlamydiae persist in vivo remains undefined; however, chlamydiae in most animals persist in the gastrointestinal tract (GI) and are transmitted via the fecal-oral route. Oral infection of mice with Chlamydia muridarum was previously shown to establish a long-term persistent infection in the GI tract. In this study, BALB/c, DBA/2, and C57Bl/6 mice, infected orally with C. muridarum, were infected in the cecum for as long as 100 days in the absence of pathology. The primary target tissue was the cecum although the large intestine was also infected in most animals. A strong serum IgG and cecal IgA antibody response developed. Lymphocyte proliferation assays to chlamydial antigen on mesenteric lymph node cells were positive by day 10 and peaked on days 15-21, but the response returned to baseline levels by 50 days, despite the ongoing presence of the organism in the cecum. Because studies have shown that women and men become infected orally with chlamydiae, we propose that the GI tract is a site of persistent infection and that immune down-regulation in the gut allows chlamydiae to persist indefinitely. As a result, women may become reinfected via contamination of the genital tract from the lower GI tract.

Vaccination with the recombinant major outer membrane protein elicits antibodies to the constant domains and induces cross-serovar protection against intranasal challenge with Chlamydia trachomatis.

To determine the ability of the major outer membrane protein (MOMP) to elicit cross-serovar protection, groups of mice were immunized by the intramuscular (i.m.) and subcutaneous (s.c.) routes with recombinant MOMP (rMOMP) from Chlamydia trachomatis serovars D (UW-3/Cx), E (Bour), or F (IC-Cal-3) or Chlamydia muridarum strain Nigg II using CpG-1826 and Montanide ISA 720 VG as adjuvants. Negative-control groups were immunized i.m. and s.c. with Neisseria gonorrhoeae recombinant porin B (Ng-rPorB) or i.n. with Eagle's minimal essential medium (MEM-0). Following vaccination, the mice developed antibodies not only against the homologous serovar but also against heterologous serovars. The rMOMP-vaccinated animals also mounted cell-mediated immune responses as assessed by a lymphoproliferative assay. Four weeks after the last immunization, mice were challenged i.n. with 10(4) inclusion-forming units (IFU) of C. muridarum. The mice were weighed for 10 days and euthanized, and the number of IFU in their lungs was determined. At 10 days postinfection (p.i.), mice immunized with the rMOMP of C. muridarum or C. trachomatis D, E, or F had lost 4%, 6%, 8%, and 8% of their initial body weight, respectively, significantly different from the negative-control groups (Ng-rPorB, 13%; MEM-0, 19%; P < 0.05). The median number of IFU recovered from the lungs of mice immunized with C. muridarum rMOMP was 0.13 × 10(6). The median number of IFU recovered from mice immunized with rMOMP from serovars D, E, and F were 0.38 × 10(6), 7.56 × 10(6), and 11.94 × 10(6) IFU, respectively. All the rMOMP-immunized animals had significantly less IFU than the Ng-rPorB (40 × 10(6))- or MEM-0 (70 × 10(6))-immunized mice (P < 0.05). In conclusion, vaccination with rMOMP can elicit protection against homologous and heterologous Chlamydia serovars.

Usefulness of 11C-choline positron emission tomography for genital chlamydial infection assessment in a BALB/c murine model.

PURPOSE: The aim of this study is to explore the feasibility of 11C-Choline PET in the assessment of the degree of inflammation in the Chlamydia muridarum genital infection model. PROCEDURES: Forty female Balb/c mice received 2.5 mg of medroxyprogesterone acetate i.m. 9 and 2 days prior to the infection: 21 mice were infected by C. muridarum into the vaginal vault, 12 mice were treated with inactivated chlamydiae, and 7 mice were SPG buffer-treated as negative controls. Three healthy control mice were not treated with progesterone. Mice in each category were randomly subdivided in two groups: (1) sacrificed at 5, 10, 15, and 20 days for histological analysis and (2) undergoing 11C-Choline PET at days 5, 10, and 20 post-infection (20 MBq of 11C-Choline, uptake time of 10 min, acquisition through a small-animal PET tomograph for 15 min). RESULTS: Infected animals showed a significantly higher standardized uptake value than both controls and animals inoculated with heat-inactivated chlamydiae in each PET scan (P<0.05). All organs of the infected animals had scores of inflammation ranging between 2 and 3 at day 5, decreasing to 1-2 at day 20. CONCLUSIONS: This preliminary result demonstrated that 11C-Choline PET can highlight a specific proliferation mechanism of inflammatory cells induced by C. muridarum, thanks to a very high sensitivity in detecting very small amounts of tracer in inflammatory cells.

A comparison of the effects of a chlamydial vaccine administered during or after a C. muridarum urogenital infection of female mice.

Research into an efficacious Chlamydia trachomatis vaccine is ongoing, however, there has been no examination into the timing of vaccine administration to either asymptomatically or previously infected individuals. Using the female Chlamydia muridarum genital tract mouse model, we examined this aspect of vaccine development. Our results show timing of vaccination affected the production of systemic antibodies, but had minimal effects on mucosal antibody production. Vaccination during an active infection or after a resolved infection did not provide protection against re-exposure to Chlamydia, and did not exacerbate the development of pathological sequelae in infected mice. This demonstrates that vaccination may not be protective in individuals who are seropositive for an acute or previous chlamydial infection.

High-throughput multistrain polymerase chain reaction quantification of Chlamydia trachomatis from clinical and preclinical urogenital specimens.

Chlamydia trachomatis (CT) is the most prevalent sexually transmitted bacterial pathogen worldwide and causes severe reproductive tract infections. Currently, nucleic acid amplification tests (NAATs) are the gold standard for clinical diagnosis, but most NAATs are labor intensive and limited to specific CT serovars. We developed and validated a quantitative polymerase chain reaction (qPCR) assay that reproducibly detected CT serovars D, E, F, Ia, and Chlamydia muridarum over a linear range of 2 log(10) to 10 log(10) genomes with low coefficients of variation from both experimental and human urine samples. CT DNA loads from human vaginal, endocervical, and male urethral swabs correlated well with the BD ProbeTec ET assay (Becton Dickinson Diagnostic Systems, Franklin Lakes, NJ) run in parallel. In a preclinical microbicide evaluation, C. muridarum DNA loads in mouse swabs and tissues correlated well with an immunofluorescence assay. The optimized qPCR system provided enhanced sensitivity and facilitated the quantitative evaluation of clinical and experimental preclinical samples for anti-CT therapeutic and microbicide evaluation.

Intranasal vaccination with a secreted chlamydial protein enhances resolution of genital Chlamydia muridarum infection, protects against oviduct pathology, and is highly dependent upon endogenous gamma interferon production.

There is currently no licensed vaccine against Chlamydia trachomatis, the leading cause of sexually transmitted bacterial disease worldwide. Conventional vaccination attempts using surface-exposed chlamydial antigens have achieved only partial success. We have employed a novel vaccination strategy using a secreted protein, chlamydial protease-like activity factor (CPAF), which has been shown to degrade host major histocompatibility complex transcription factors and keratin-8 and therefore may allow immune evasion and establishment of a productive infection. Intranasal immunization using recombinant CPAF (rCPAF) plus interleukin-12 (IL-12) (rCPAF+IL-12 immunization) was used to assess the protective immunity against genital Chlamydia muridarum infection in BALB/c mice. rCPAF+IL-12 immunization induced robust gamma interferon (IFN-gamma) production and minimal IL-4 production by splenocytes upon in vitro recall with rCPAF. The total and immunoglobulin G2a (IgG2a) anti-rCPAF antibody levels in serum were significantly elevated after rCPAF+IL-12 vaccination, as were the total antibody, IgG2a, and IgA levels in bronchoalveolar lavage and vaginal fluids when the animals were compared to animals that received rCPAF alone. rCPAF+IL-12-vaccinated mice displayed significantly reduced bacterial shedding upon chlamydial challenge and accelerated resolution of infection compared to mock-immunized (phosphate-buffered saline) animals. Moreover, rCPAF+IL-12-immunized animals exhibited protection against pathological consequences of chlamydial infection, including the development of hydrosalpinx and oviduct dilatation. This vaccination regimen also reduced the development of fibrosis and the influx of neutrophils into the upper genital tract when the animals were compared to mock-immunized (phosphate-buffered saline) animals after bacterial challenge. rCPAF+IL-12-mediated resolution of the bacterial infection and protection against Chlamydia-induced inflammatory disease were highly dependent on endogenous IFN-gamma production. Together, these results demonstrate that secreted chlamydial antigens may be novel vaccine candidates to induce protective immunity.