Mouse strain-dependent variation in the course and outcome of chlamydial genital tract infection is associated with differences in host response.

Whether there is a pathogenic or protective outcome to chlamydial infection may be defined by the host response. We infected C57BL/6 (C57) and C3H/HeN (C3H) mice with the human biovar of Chlamydia trachomatis, serovar E, and, in select experiments, with the mouse pneumonitis agent of C. trachomatis (MoPn). We compared the courses of infection, histopathology, and host responses that resulted from these infections. The duration of infection with either chlamydial biovar was significantly increased in the C3H strain of mice. The intensity of infection was examined in mice infected with serovar E, and it was significantly increased in the C3H strain. Histopathology revealed the incidence of severe hydrosalpinx to be significantly greater in C3H mice than in C57 mice. In contrast, severe distention of the uterine horns was observed in all infected C57 mice compared to none of the C3H mice infected with serovar E and only 25% of those infected with MoPn. Acute inflammation was significantly increased in the uterine horns of C57 mice compared to that of C3H mice. Examination of antigen-specific responses revealed qualitatively similar responses in the two strains. Determination of gamma interferon- versus interleukin 4- producing cells revealed the predominance of a Th1 response in both strains. Serum enzyme-linked immunosorbent assays for immunoglobulin G1 (IgG1) and IgG2a revealed a predominance of IgG2a antibody in both strains, although the levels of antibody were significantly greater in C3H mice. Lymphocyte proliferation studies revealed increased proliferation in the iliac nodes of both strains at 1 to 3 weeks after infection. Because of the early eradication of infection observed in the C57 strain, we explored the relative production of tumor necrosis factor alpha (TNF-alpha) in the two strains. TNF-alpha levels were significantly increased in the genital tract secretions of C57 mice compared to that of C3H mice during the first week of infection. Increased TNF-alpha may be beneficial to the host by leading to earlier eradication of infection, thereby preventing infection of the oviduct and thus the major disease sequelae associated with chlamydial infection of the genital tract.     

Immunization with the Chlamydia trachomatis mouse pneumonitis major outer membrane protein by use of CpG oligodeoxynucleotides as an adjuvant induces a protective immune response against an intranasal chlamydial challenge

Recently, we have shown that a vaccine consisting of a purified preparation of the Chlamydia trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP) and Freund's adjuvant can protect mice against a genital challenge. Here, we wanted to determine if CpG motifs could be used as an immune modulator to the MOMP to induce protection in mice against an intranasal (i.n.) challenge. One-week-old BALB/c mice were immunized intramuscularly and subcutaneously either once or three times at 2-week intervals with MOMP and CpG suspended in aluminum hydroxide (alum). Negative controls received ovalbumin, CpG, and alum. Positive controls were immunized i.n. with C. trachomatis MoPn elementary bodies (EB). Six weeks after the last immunization, mice were challenged i.n. with 10(4) inclusion-forming units (IFU) of the C. trachomatis MoPn serovar. Mice that received MOMP, CpG, and alum had a strong immune response, as shown by a high titer of serum antibodies to Chlamydia and significant lymphoproliferation of T-cells following stimulation with C. trachomatis EB. After the i.n. challenge mice immunized with MOMP, CpG, and alum showed significantly less body weight loss than the corresponding control mice immunized with ovalbumin, CpG, and alum. Ten days after the challenge the animals were euthanized, their lungs were weighed, and the numbers of IFU in the lungs were determined. The average weight of the lungs of the mice immunized with MOMP, CpG, and alum was significantly less than average weight of the lungs of the mice immunized with ovalbumin, CpG, and alum. Also, the average number of IFU recovered per mouse immunized with MOMP, CpG, and alum was significantly less than the average number of IFU per mouse detected in the mice inoculated with ovalbumin, CpG, and alum. In conclusion, our data show that CpG sequences can be used as an effective adjuvant with the C. trachomatis MoPn MOMP to elicit a protective immune response in mice against a chlamydial respiratory challenge.     

Role for CD8+ T cells in antichlamydial immunity defined by Chlamydia-specific T-lymphocyte clones.

The role of CD8+ T cells in antichlamydial immunity was investigated in a murine model of chlamydial genital infection by using T-cell clones generated against the Chlamydia trachomatis agent of mouse pneumonitis (MoPn). Two CD8+ T-cell clones tested (2.1F and 2.14-9) were chlamydia antigen specific and MHC restricted and reacted against MoPn as well as the Chlamydia psittaci agent of guinea pig inclusion conjunctivitis and C. trachomatis serovar E, suggesting the recognition of a genus-specific antigen. Upon adoptive transfer into persistently MoPn-infected nu/nu mice, 55.6% of the recipients of clone 2.1F (15 of 27) resolved the infection but recipients of clone 2.14-9 did not. The ability to resolve the MoPn infection correlated with the capacity of clone 2.1F to elaborate a combination of gamma interferon and tumor necrosis factor alpha. The results suggested that in addition to CD4+ T cells, CD8+ T cells may also contribute to antichlamydial T-cell immunity in vivo.     

Protective efficacy of major outer membrane protein-specific immunoglobulin A (IgA) and IgG monoclonal antibodies in a murine model of Chlamydia trachomatis genital tract infection.

The protective efficacy of immunoglobulin A (IgA) and IgG monoclonal antibodies (MAbs) specific for the major outer membrane protein of Chlamydia trachomatis MoPn was evaluated in a murine genital tract infection model. MAbs were delivered into serum and vaginal secretions of naive mice by using the backpack hybridoma tumor system, and protective efficacy was assessed over the first 8 days following challenge by quantitative determination of chlamydial recovery from cervicovaginal swabs, histopathological evaluation of genital tract tissue, and immunohistochemical detection of chlamydial inclusions. IgA and IgG significantly reduced the incidence of infection following vaginal challenge with 5 50% infectious doses, but such protection was overwhelmed by 10- and 100-fold higher challenge doses. Both MAbs also consistently reduced vaginal shedding from infected animals with all three challenge doses compared with the negative control MAb, although the magnitude of this effect was marginal. Blinded pathological evaluation of genital tract tissues at 8 days postinfection showed a significant reduction in the severity of the inflammatory infiltrate in oviduct tissue of infected IgA- and IgG-treated animals. Immunohistochemical detection of chlamydial inclusions revealed a marked reduction in the chlamydial burden of the oviduct epithelium; this finding is consistent with the reduced pathological changes observed in this tissue. These studies indicate that the presence of IgA or IgG MAbs specific to major outer membrane proteins has a marginal effect in preventing chlamydial colonization and shedding from the genital tract but has a more pronounced effect on ascending chlamydial infection and accompanying upper genital tract pathology.     

Chlamydial protease-like activity factor induces protective immunity against genital chlamydial infection in transgenic mice that express the human HLA-DR4 allele

There is no licensed vaccine available against Chlamydia trachomatis, the leading cause of bacterial sexually transmitted disease. We have found that intranasal immunization with recombinant chlamydial protease-like activity factor (CPAF) induces CD4(+) T-cell- and gamma interferon (IFN-gamma)-dependent protective immunity against murine genital chlamydial infection, thus making CPAF a viable vaccine candidate for further characterization. HLA-DR4 is the predominant allele involved in chlamydial antigen presentation to CD4(+) T cells in humans. We used engineered mice that lack endogenous major histocompatibility complex class II (MHC-II) alleles but express a human HLA allele (HLA-DR4 transgenic [tg] mice) to examine primary immune and CPAF-mediated responses against genital Chlamydia muridarum challenge. Upon primary bacterial exposure, HLA-DR4 tg mice developed Chlamydia-specific IFN-gamma and antibody production and resolved the infection within 30 days, similar to challenged conventional C57BL/6 animals. Moreover, C. muridarum-challenged HLA-DR4 tg mice exhibited CPAF-specific antibody and IFN-gamma production. Upon CPAF-plus-interleukin-12 (IL-12) vaccination, HLA-DR4 tg animals exhibited robust CPAF-specific IFN-gamma production and elevated titers of anti-CPAF total antibody and immunoglobulin G2a (IgG2a) and lower titers of IgG2b and IgG1 antibodies. HLA-DR4 tg and C57BL/6 mice vaccinated with CPAF plus IL-12 resolved the primary genital chlamydial infection significantly earlier than mock-immunized animals, whereas similarly vaccinated MHC class II-deficient mice displayed minimal antigen-specific immune responses and failed to resolve the infection even at 30 days postchallenge. Together, these results demonstrate the importance of human HLA-DR4 molecules in the recognition and presentation of CPAF epitopes, leading to the generation of protective antichlamydial immunity and making these mice a valuable model for mapping HLA-DR4-restricted chlamydial epitopes.     

Local Th1-like responses are induced by intravaginal infection of mice with the mouse pneumonitis biovar of Chlamydia trachomatis.

A critical role for cell-mediated immunity (CMI) has been demonstrated for effecting the resolution of genital infections of mice infected intravaginally with the mouse pneumonitis biovar of Chlamydia trachomatis (MoPn). However, little is known about expression of CMI in the murine genital tract. The mouse MoPn model was used to examine CMI responses in the genital tract and associated lymph nodes during the course of infection. MoPn-specific lymphocytes were present in the genital mucosa, with the maximum level of proliferation in response to MoPn at 3 weeks postinfection. MoPn-stimulated cells secreting gamma interferon were also detected in the cells from the genital mucosa, but few interleukin-4-secreting cells were seen at any time postinfection, indicating the induction of a Th1-like response in the cells of the genital mucosa. The iliac node draining the genital tract was the major node stimulated as a result of a genital infection and exhibited a predominant Th1-like pattern of cytokine secretion as well. Mesenteric lymph node cells demonstrated poor proliferative responses to MoPn and few antigen-stimulated cytokine-secreting cells after the primary infection. However, 7 days after a second infection administered 50 days following the primary infection, there was a marked increase in both proliferative responses and the frequencies of MoPn-stimulated gamma interferon- and interleukin-4-secreting cells. These studies provided information regarding the local CMI response to MoPn in mice which may prove valuable in the development of vaccination strategies for the prevention of chlamydial genital infections.     

Induction of protective immunity against Chlamydia trachomatis genital infection by a vaccine based on major outer membrane protein-lipophilic immune response-stimulating complexes

The significance of delivery systems in modern vaccine design strategies is underscored by the fact that a promising vaccine formulation may fail in vivo due to an inappropriate delivery method. We evaluated the immunogenicity and efficacy of a candidate vaccine comprising the major outer membrane protein (MOMP) of Chlamydia trachomatis delivered with the lipophilic immune response-stimulating complexes (ISCOMs) as a vehicle with adjuvant properties, in a murine model of chlamydial genital infection. Immunocompetent BALB/c mice were immunized intranasally (IN) or intramuscularly (IM) with MOMP, MOMP-ISCOMs, and live or heat-inactivated C. trachomatis serovar D. The level of local genital mucosal Th1 response was measured by assaying for antigen-specific Th1 cell induction and recruitment into the genital mucosa at different times after immunization. Immunization with MOMP-ISCOMs by the IM route induced the greatest and fastest local genital mucosal Th1 response, first detectable 2 weeks after exposure. Among the other routes and regimens tested, only IN immunization with MOMP-ISCOMs induced detectable and statistically significant levels of local genital mucosal Th1 response during the 8-week test period (P < 0.001). In addition, when T cells from immunized mice were adoptively transferred into syngeneic naive animals and challenged intravaginally with Chlamydia, recipients of IM immunization of MOMP-ISCOMs cleared their infection within 1 week and were resistant to reinfection. Animals that received IN immunization of MOMP-ISCOMs were partially protected, shedding fewer chlamydiae than did control mice. Altogether, the results suggested that IM delivery of MOMP-ISCOMs may be a suitable vaccine regimen potentially capable of inducing protective mucosal immunity against C. trachomatis infection.     

Vaccination against Chlamydia genital infection utilizing the murine C. muridarum model

Chlamydia trachomatis genital infection is a worldwide public health problem, and considerable effort has been expended on developing an efficacious vaccine. The murine model of C. muridarum genital infection has been extremely useful for identification of protective immune responses and in vaccine development. Although a number of immunogenic antigens have been assessed for their ability to induce protection, the majority of studies have utilized the whole organism, the major outer membrane protein (MOMP), or the chlamydial protease-like activity factor (CPAF). These antigens, alone and in combination with a variety of immunostimulatory adjuvants, have induced various levels of protection against infectious challenge, ranging from minimal to nearly sterilizing immunity. Understanding of the mechanisms of natural infection-based immunity and advances in adjuvant biology have resulted in studies that are increasingly successful, but a vaccine licensed for use in humans has not yet been brought to fruition. Here we review immunity to chlamydial genital infection and vaccine development using the C. muridarum model.     

Priming with Chlamydia trachomatis major outer membrane protein (MOMP) DNA followed by MOMP ISCOM boosting enhances protection and is associated with increased immunoglobulin A and Th1 cellular immune responses

We previously reported that DNA vaccination was able to elicit cellular immune responses and partial protection against Chlamydia trachomatis infection. However, DNA immunization alone did not generate immune responses or protection as great as that induced by using live organisms. In this study, we evaluated the immunologic effects of a combinational vaccination approach using C. trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP) DNA priming followed by boosting with immune-stimulating complexes (ISCOM) of MOMP protein (MOMP ISCOM) for protection of BALB/c mice against MoPn lung infection. Substantially better protection to challenge infection was observed in mice given combinational vaccination compared with mice given MOMP ISCOM immunization alone, and the protection approximated that induced by live organisms. Enhanced protection was correlated with stronger delayed-type hypersensitivity, higher levels of gamma interferon production, and increased immunoglobulin A antibody responses in lung homogenates. The results indicate that DNA priming followed by ISCOM protein boosting may be useful in designing a fully protective chlamydial vaccine.     

Analysis of the humoral immune response to chlamydial genital infection in guinea pigs.

Studies using the guinea pig model of chlamydial genital infection with the Chlamydia psittaci agent of guinea pig inclusion conjunctivitis (GPIC) have shown that serum and local antibodies play a role both in the resolution of infection and in protection against reinfection. Thus, this model is suited for further exploration of immune mechanisms and for vaccine studies with chlamydial macromolecules. We have further characterized the model by assessing the antigen-specific antibody response to experimental genital infection by using immunoblotting to assay both genital secretions and serum. The GPIC agent was characterized by analysis of outer membrane proteins, which indicated that the GPIC agent possessed a major outer membrane protein (MOMP), with a molecular mass of 39 kilodaltons (kDa), and a 61-kDa protein, analogous to cysteine-rich 60-kDa proteins or doublets of Chlamydia trachomatis strains. As indicated by immunoblotting, most infected animals produced serum immunoglobulin G antibodies to MOMP, the 61-kDa proteins, an 84-kDa outer membrane protein, and lipopolysaccharide. Such serum antibodies persisted for at least 813 days after primary genital infection. Immunoglobulin A antibodies against the 61-kDa proteins, lipopolysaccharide, and MOMP, but not the 84-kDa protein, were detected in secretions. Animals challenged with GPIC 825 days after primary infection became infected again despite the presence of serum antibodies, but the period of chlamydial shedding was significantly shorter and less intense than in primary infections. Although the specific mechanism is not known, these data suggest that a long-lasting immune effect is capable of altering the course of infection late after primary infection. Correlation of the antigen-specific antibody response and other immune parameters with the duration and degree of protective immunity induced by infection or vaccination may be helpful in further understanding the nature of such protective immunity.     

Reactivation of chlamydial genital tract infection in mice.

A model was developed to study chlamydial quiescence in C3H/HeN (C3H) and C57BL/6N (C57) mice following genital tract infection by Chlamydia trachomatis MoPn. Reactivation of chlamydial shedding following immunosuppression indicated that viable MoPn remained in the genital tract for up to 4 or 5 weeks after the apparent clearance of a primary infection. Either cyclophosphamide or cortisone acetate treatment could cause reactivation, but cyclophosphamide was more effective. However, the frequency of reactivation by either drug diminished with time in both mouse strains. Progesterone treatment prior to infection of C57 mice greatly reduced the frequency of reactivation by cyclophosphamide and also correlated with the development of marked fluid accumulation and distension of the uterine horns in the vast majority of those animals. This pathology was apparent by 5 to 7 weeks postinfection and was consistently seen through 110 days postinfection. Neither of these phenomena was observed in C57 mice that had not been treated with progesterone or in C3H mice under any conditions tested. The infecting dose of MoPn did not clearly influence the frequency of reactivation in either inbred strain as defined by this model.     

Subclinical chlamydial infection of the female mouse genital tract generates a potent protective immune response: implications for development of live attenuated chlamydial vaccine strains

Chlamydia trachomatis is a major cause of sexually transmitted disease (STD) for which a vaccine is needed. CD4(+) T-helper type 1 (Th1) cell-mediated immunity is an important component of protective immunity against murine chlamydial genital infection. Conventional vaccine approaches have not proven effective in eliciting chlamydial-specific CD4 Th1 immunity at the genital mucosa. Thus, it is possible that the development of a highly efficacious vaccine against genital infection will depend on the generation of a live attenuated C. trachomatis vaccine. Attenuated strains of C. trachomatis do not exist, so their potential utility as vaccines cannot be tested in animal models of infection. We have developed a surrogate model to study the effect of chlamydial attenuation on infection and immunity of the female genital tract by treating mice with a subchlamydiacidal concentration of oxytetracycline following vaginal infection. Compared to untreated control mice, antibiotic-treated mice shed significantly fewer infectious organisms (3 log(10)) from the cervico-vagina, produced a minimal inflammatory response in urogenital tissue, and did not experience infection-related sequelae. Antibiotic-treated mice generated levels of chlamydia-specific antibody and cell-mediated immunity equivalent to those of control mice. Importantly, antibiotic-treated mice were found to be as immune as control untreated mice when rechallenged vaginally. These findings demonstrate that subclinical chlamydial infection of the murine female genital tract is sufficient to stimulate a potent protective immune response. They also present indirect evidence supporting the possible use of live attenuated chlamydial organisms in the development of vaccines against chlamydial STDs.     

Clearance of Chlamydia trachomatis from the murine genital mucosa does not require perforin-mediated cytolysis or Fas-mediated apoptosis

The molecular mechanisms of resistance to genital infection with the mouse pneumonitis (MoPn) strain of Chlamydia trachomatis are unknown. A role for major histocompatibility complex class II-restricted, interleukin-12-dependent CD4(+) T cells has been established, but the functional activity of these cells does not depend on secretion of gamma interferon. Here we examined the potential contribution of T-cell-mediated cytotoxicity and apoptosis to mucosal clearance of MoPn by using mice deficient in the molecular mediators of target cell lysis. Animals lacking perforin, Fas, Fas ligand, or both perforin and Fas ligand were infected genitally with C. trachomatis MoPn and monitored for expression of immunity to chlamydial antigens and clearance of MoPn from the genital mucosa. In each case, the profile of spleen cytokine production, the magnitude of the host antibody response, and the kinetics of chlamydial clearance were similar to those of genetically intact controls. Compensatory overproduction of tumor necrosis factor alpha, an alternate mediator of apoptosis in certain cell types, did not appear to account for the ability of mutant mice to resolve Chlamydia infections. These results fail to support CD4(+) T-cell-mediated apoptosis or CD8(+) T-cell-mediated cytotoxicity as being critical to the clearance of C. trachomatis MoPn urogenital infections.     

Chlamydia trachomatis serovar E isolates from patients with different clinical manifestations have similar courses of infection in a murine model: host factors as major determinants of C trachomatis mediated pathogenesis

BACKGROUND: Some investigators have proposed an association between certain Chlamydia trachomatis serovars and the clinical course of infection in humans. A recent study of over 1100 patients with culture confirmed and serotyped C trachomatis urogenital infection detected no such association. AIMS: To corroborate these results using a murine model of female genital tract infection. METHODS: Various parameters of infection were assessed in mice intravaginally infected with human genital isolates of C trachomatis serovar E from four cases with either a clear symptomatic or asymptomatic clinical course in both the patient and their partner. RESULTS: No differences were seen among the strains in the incidence or duration of infection, polymorphonuclear granulocyte responses, or upper genital tract progression. CONCLUSIONS: An investigation to determine the correlation between the clinical manifestations of different isolates of C trachomatis serovar E in humans and certain parameters of microbial pathogenesis in a mouse model failed to reveal an association between the measured parameters and the tendency of serovar E to produce symptomatic versus asymptomatic infections in humans. These findings suggest that differences in the clinical course of infection in humans seen with these strains may be more related to host factors than to genetic variation among strains.     

Effect of immunoglobulin G isotype on the infectivity of Chlamydia trachomatis in a mouse model of intravaginal infection.

It has been previously shown with an in vitro neutralization system that monoclonal antibodies (MAbs) to the major outer membrane protein (MOMP) of Chlamydia trachomatis, depending on the isotype of the MAb and the host cell used, can either neutralize or enhance the infectivity of this organism. MAbs to variable domain 4 (VD 4) of MOMP have been described that neutralize the infectivity of C. trachomatis when tested in a system in which either the host cell does not have detectable Fc gammaRIII receptors or complement is added to block the interaction of the MAb with the receptor. However, if Fc gammaRIII receptors are available, immunoglobulin G2b (IgG2b) MAbs to the VD 4 are able to enhance the infectivity of this pathogen. Two MAbs that recognize the sequence TLNPTIA in VD 4 of the MOMP but differ in isotype, E4 (IgG2b) and E21 (IgG1), were used to test whether in vivo the isotype of the MAb modulates the outcome of a vaginal infection in a murine model. A third MAb, CP33 (IgG2b), that recognizes the chlamydial lipopolysaccharide but does not neutralize infectivity of C. trachomatis, was also tested. Elementary bodies (EBs) of C. trachomatis, serovar E (BOUR), were pretreated with the three MAbs and were used to inoculate the vaginas of C3H/HeJ mice which had been pretreated with progesterone. Subsequently mice were monitored over a 5-week period with vaginal cultures. In the groups that were inoculated with EBs pretreated with MAbs directed to VD 4 of MOMP, there was a significant decrease (P < 0.05) in the number of mice infected. Only 30% of the mice were infected in the MAb E4-treated group, and 10% were infected in the MAb E21 group. This was in contrast to the groups inoculated with EBs pretreated with MAb CP33 and control untreated EBs, which resulted in 100 and 79% of the mice infected, respectively. Therefore, in this setting in which EBs were introduced in vivo coated with MAb, there was no enhancement of infection by IgG2b MAbs; rather, the results paralled the in vitro neutralization results, in which cells lacking Fc gammaRIII receptors were employed. Mice were also given the MAbs, as well as purified IgG as a control, by intraperitoneal injection before and after intravaginal inoculation with C. trachomatis. Despite relatively high levels of MAbs in serum and detectable levels of MAbs in the vagina at the time of infection, there was only modest protection in animals receiving MAb E21, with 60% of the mice infected in contrast to 90% of the mice receiving MAb E4, MAb CP33, and IgG. However, by the second week of infection compared to controls, there was a significant increase (P < 0.05) in the amount of chlamydiae recovered from the vaginas of mice that had received the two IgG2b MAbs, E4 and CP33. In summary, the presence of IgG2b MAbs directed to surface components of C. trachomatis at certain times during the course of infection may play a role in enhancing the infectivity of this pathogen.     

Specific-pathogen-free pigs as an animal model for studying Chlamydia trachomatis genital infection

The purpose of the present study was to evaluate pigs as a large-animal model for female genital infection with two Chlamydia trachomatis human serovar E strains. Sixteen-week-old specific-pathogen-free female pigs (gilts) were intravaginally infected with the trachoma type E reference strain Bour or the urogenital serovar E strain 468. Several conclusions can be drawn from our findings on the pathogenicity of a primary C. trachomatis genital infection in gilts. First of all, we demonstrated that the serovar E strains Bour and 468 could ascend in the genital tract of gilts. The serovar E strains could replicate in the superficial columnar cervical epithelium and in the superficial epithelial layer of the uterus, which are known to be the specific target sites for a C. trachomatis genital infection in women. Second, inflammation and pathology occurred at the replication sites. Third, the organisms could trigger a humoral immune response, as demonstrated by the presence of immunoglobulin M (IgM), IgG, and IgA in both serum and genital secretion samples. Our findings imply that the pig model might be useful for studying the pathology, pathogenesis, and immune response to a C. trachomatis infection of the genital system.     

Resolution of chlamydial genital infection in B-cell-deficient mice and immunity to reinfection.

The purpose of this investigation was to determine the relative roles of the humoral and cell-mediated immune responses in the resolution of chlamydial genital infection of mice and resistance to reinfection. To this end, female BALB/c mice were rendered B cell deficient by treatment with heterologous anti-immunoglobulin M (IgM) serum from birth. Controls were similarly treated with either normal serum or phosphate-buffered saline. Before inclusion in each experiment, anti-IgM-treated mice were screened for the absence of IgM in serum and for the presence of cell-mediated immune responses. In addition, spleen cells from anti-IgM-treated mice responded to concanavalin A and phytohemagglutinin but not to lipopolysaccharide. By these criteria, mice were designated B cell deficient. B-cell-deficient mice and controls were inoculated intravaginally with a suspension of mouse pneumonitis agent (MoPn), a Chlamydia trachomatis biovar. All B-cell-deficient mice resolved the infection. Additionally, no significant difference was seen in the course of the infection in B-cell-deficient mice when compared with controls. In contrast to control mice, B-cell-deficient mice displayed no detectable antibody responses to MoPn in serum or in genital secretions. However, both B-cell-deficient mice and controls developed delayed-type hypersensitivity and T-cell proliferative responses to MoPn. When challenged 53 days after primary infection, no significant difference was seen in the resistance of B-cell-deficient mice to reinfection when compared with that of the controls. These data indicate that cell-mediated immune mechanisms play an important role in the resolution of and resistance to chlamydial genital infection in this model.     

B-cell-deficient mice develop complete immune protection against genital tract infection with Chlamydia trachomatis.

We evaluated the ability of mice made genetically deficient for B cells to resolve a primary infection and to develop protective immunity against vaginal challenge with a human isolate of Chlamydia trachomatis bacteria. The B-cell-deficient microMT mice cleared a primary ascending infection with similar or faster kinetics compared with wild-type mice. The presence of chlamydial inclusion bodies and the degree of inflammation in the upper genital tract was comparable and showed similar kinetics in microMT as in wild-type mice. Following resolution of the primary infection the mice were challenged by 100 ID50 of live bacteria and the level of protection and the extent of local inflammation was assessed. Strikingly, all microMT mice, as well as most of the wild-type mice, demonstrated complete immune protection with no bacterial shedding. While high titres of chlamydia-specific antibodies were stimulated locally and systemically in wild-type mice, no antibodies were detected in microMT mice. However, in both strains, immunohistochemical analysis of the upper genital tract demonstrated the presence of large numbers of CD4+ T cells and increased levels of interferon-gamma (IFN-gamma)-producing cells. The results unequivocally demonstrate that antibodies are not required for full protection to develop against ascending infection with a high dose of C. trachomatis in the female genital tract. Our study confirms the notion that cell-mediated immunity, in particular that owing to CD4+ T helper I (Th1)-type cells, is critical for host resistance against C. trachomatis in mice.     

CD4+ T cells play a significant role in adoptive immunity to Chlamydia trachomatis infection of the mouse genital tract.

The ability of CD4+ and CD8+ T cells to adoptively immunize mice against Chlamydia trachomatis infection of the mouse genital tract was studied. Adoptive transfer experiments were performed with splenic CD4+ or CD8+ T cells obtained from mice following resolution of a primary genital tract infection and after a secondary chlamydial challenge. The results show that donor CD4+ T cells, but not CD8+ T cells, obtained from mice following resolution of a primary infection or after secondary challenge were effective in transferring significant antichlamydial immunity to the genital tracts of naive animals. The lymphokine profiles in the culture supernatants of proliferating Chlamydia-specific CD4+ T cells obtained from mice following resolution of a primary infection and after secondary challenge were assayed by an enzyme-linked immunoadsorbent assay. Protective CD4+ T cells restimulated in vitro secreted interleukin 2, gamma interferon, and interleukin 6, lymphokine profiles characteristic of both Th1- and Th2-like responses. Resting CD4+ T cells obtained from mice 4 months following resolution of a primary infection were also capable of conferring significant levels of adoptive protective immunity to naive mice. These findings support an important role for CD4+ T cells in acquired immunity to chlamydial infection of the genital tract and indicate that protective CD4+ immune responses in this model are relatively long lived.     

Immunization with the Chlamydia trachomatis Mouse Pneumonitis Major Outer Membrane Protein Can Elicit a Protective Immune Response against a Genital Challenge

Infertility, ectopic pregnancy, and chronic abdominal pain are frequent complications of genital infections with Chlamydia trachomatis. In an attempt to produce a vaccine to protect against this pathogen we purified and refolded the C. trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP). This preparation, mixed with Freund's adjuvant using vortexing or sonication, was used to immunize BALB/c mice that were subsequently challenged in the upper genital tract. Vaginal cultures were taken on a weekly basis, and mice were mated 6 weeks after the challenge. Gels of the vortexed MOMP showed a predominant band with a molecular size of 62 kDa and weaker bands at 42 and 132 kDa, while the sonicated MOMP had a single band with a molecular size of 42 kDa. Following immunization with these two preparations, strong humoral and cell-mediated immune responses were detected in the mice inoculated with the vortexed MOMP. On the other hand, mice immunized with the sonicated MOMP had a strong humoral immune response but a relatively weak cell-mediated immune response, as determined by a T-cell lymphoproliferative assay and level of cytokine production by splenocytes. Vaginal cultures showed that the mice immunized with the vortexed MOMP were significantly protected, as determined by a decrease in the number of animals with positive cultures, the length of time the mice shed viable organisms, and the number of inclusion-forming units recovered per mouse. Animals immunized with the sonicated MOMP, on the other hand, showed a weaker level of protection based on the same three parameters. After mating, the number of fertile animals and number of embryos per mouse were significantly higher for the mice immunized with vortexed MOMP, but not for the mice immunized with sonicated MOMP, compared to those of the control groups. In conclusion, immunization with a purified and refolded preparation of the C. trachomatis MoPn MOMP confers a significant level of protection in mice against a genital challenge.