Induction of protective immunity against a Chlamydia trachomatis genital infection in three genetically distinct strains of mice

To establish the feasibility of inducing a protective immune response against a chlamydial genital infection in animals with different genetic backgrounds, groups of C3H/HeN (H-2k), BALB/c (H-2d) and C57BL/6 (H-2b) mice, were immunized intranasally with elementary bodies (EB) of the Chlamydia trachomatis mouse pneumonitis biovar. Following the intranasal immunization strong Chlamydia-specific humoral and cell-mediated immune (CMI) responses were detected in the three strains of mice. Eight weeks following immunization the animals were challenged with C. trachomatis in the genital tract. Vaginal cultures showed that the three strains of mice immunized with EB were significantly protected in comparison to the sham immunized animals. To determine the ability of this immunization protocol to protect against infertility six weeks after the genital challenge the animals were mated. Mice of the three strains immunized with EB showed significant protection as demonstrated by the number of animals that were fertile, and the number of embryos present in their uterine horns, in comparison to the sham immunized mice.     

沙眼衣原体主要外膜蛋白核酸疫苗诱导小鼠产生特异性细胞和体液免疫应答

目的构建沙眼衣原体主要外膜蛋白核酸疫苗,并观察其诱导小鼠产生的体液免疫和细胞免疫。方法将核酸疫苗(pcDNA3.1MOMP)或对照空质粒(pcDNA3.1)注射于4～6周龄小鼠后腿股四头肌,每次剂量为100mg。间隔2周加强免疫2次。末次免疫后,ELISA法测定脾淋巴细胞培养上清液中IFNγ及小鼠血清中抗MOMP水平;MTT法测定脾淋巴细胞特异性增殖反应。结果小鼠接种核酸疫苗后,能产生特异性抗体,第3次免疫后抗体最高滴度达1∶1024,培养上清液中IFNγ达(532.0±45.4)pg/mL;实验组小鼠脾淋巴细胞刺激指数为3.94±0.25,其抗原特异性反应明显高于对照组。结论沙眼衣原体主要外膜蛋白核酸疫苗能刺激机体产生特异的细胞免疫和体液免疫。     

Immunization with an acellular vaccine consisting of the outer membrane complex of Chlamydia trachomatis induces protection against a genital challenge.

The ability to induce protection against a genital challenge was studied in BALB/c female mice with three Chlamydia trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP) preparations as well as an acellular vaccine consisting of the chlamydial outer membrane complex (COMC). The MOMP preparations were extracted with three different types of detergents, sodium dodecyl sulfate (SDS), n-octyl-beta-D-glucopyranoside (OGP), and Zwittergent 3-14 (Z3-14). A positive immunization control consisted of mice inoculated intranasally with 10(4) C. trachomatis MoPn inclusion-forming units (IFU). Mice inoculated with ovalbumin served as a negative control. Furthermore, a sham-immunized, nonchallenged group was included as a fertility control. Two weeks after the last immunization, the mice were challenged in the left ovarian bursa with 10(5) C. trachomatis MoPn IFU. Vaginal swabs were collected for culture, vaginal and serum samples were assayed for chlamydial-specific antibodies, and splenocytes were collected to determine the lymphoproliferative response. At 42 days after the challenge, the mice were mated with proven male breeder mice. Animals that were considered to be pregnant (as determined by weight) were killed, and the embryos were counted. A significant humoral and cell-mediated immune response was observed in all the groups of mice inoculated with chlamydial antigens. Antibodies to variable domain (VD)1 of the MOMP were detected in serum samples from all the immunized groups. However, antibodies to VD3 and VD4 were detected only in the groups immunized with the Z3-14-MOMP and the COMC. Mice immunized with COMC developed significant immunoglobulin A chlamydia-specific antibodies in the vagina, while mice immunized with the detergent-extracted MOMPs had low antibody titers. Following the intrabursal challenge, a significant decrease in the intensity and duration of vaginal shedding was noted in the mice immunized with COMC and a moderate decrease was noted in the group immunized with OGP-MOMP. No protection against the infection was noted in the groups of animals immunized with SDS- and Z3-14-MOMP. Furthermore, of the mice immunized with the COMC preparation, only 25% (4 of 20) shed C. trachomatis, as determined by vaginal culture, while 83% (40 of 48) of the control mice inoculated with ovalbumin were culture positive (P < 0.05). In addition, after mating, the mice inoculated with COMC were found to have fertility rates comparable to those of the control sham-immunized, nonchallenged animals (70% [14 of 20] versus 81% [17 of 21], respectively [P > 0.05]), and there were no significant differences between the average number of embryos per mouse in the two groups (5.1 versus 5.9, respectively [P > 0.05]). In contrast, mice immunized with the purified MOMP preparations were not protected against infertility. In summary, a preparation of the COMC protected mice against infection and infertility, supporting the feasibility of the development of an acellular vaccine against C. trachomatis infections.     

Fc receptor regulation of protective immunity against Chlamydia trachomatis

The prevailing paradigm for designing potentially efficacious vaccines against the obligate intracellular bacterium, Chlamydia trachomatis, advocates regimens capable of inducing a mucosal antigen-specific T helper type 1 (Th1) response. However, recent reports indicate that rapid and efficient clearance of a secondary infection also requires certain B-cell functions. We investigated the hypothesis that Fc receptor (FcR)-mediated antibody effector mechanisms are important B-cell-related functions involved in controlling a chlamydial genital reinfection. Microbiological analysis of genital chlamydial infection in FcR knockout (FcRKO) mice lacking the activatory FcgammaRI (CD64) and FcRgammaIII (CD16), as well as the inhibitory FcgammaRIIB1 (CD32), revealed a greater intensity of secondary infection (i.e. bacterial shedding) in FcRminus sign/minus sign as compared to FcR+/+ mice; however, the course of the primary infection was indistinguishable in both animals. Pathologically, FcRKO mice suffered greater ascending infection than immunocompetent wild-type (WT) mice after a secondary infection. Immunological evaluation indicated that the presence of specific anti-chlamydial antibodies enhanced chlamydial antigen presentation for induction of a Th1 response by FcR+/+, but not FcRminus sign/minus sign, antigen-presenting cells. In addition, specific anti-chlamydial antibodies augmented both macrophage killing of infected epithelial cells by antibody-dependent cellular cytotoxicity (ADCC) and macrophage inhibition of productive growth of chlamydiae in co-cultures. These results indicate that B cells participate in anti-chlamydial immunity via FcR-mediated effector functions of antibodies, which are operative during reinfections. Such effector functions include ADCC, and possibly enhanced uptake, processing and presentation of chlamydial antigens for rapid induction of a Th1 response, all facilitating the early clearance of an infection. These findings suggest that a future anti-chlamydial vaccine should elicit both humoral and T-cell-mediated immune responses for optimal memory response and vaccine efficacy.     

Protection of sheep against Chlamydia psittaci infection with a subcellular vaccine containing the major outer membrane protein.

An outer membrane (OM) preparation from elementary bodies (EBs) of Chlamydia psittaci (ovine abortion strain) was used to vaccinate pregnant ewes in a single subcutaneous dose and was found to achieve protection after subcutaneous challenge with infectious organisms. Inactivated purified EBs used as a single-dose vaccine also gave protection. The ratio of live to dead lambs was significantly higher in the vaccinated groups (16:1 and 15:1, respectively) than in the placebo group (8:9). Polyacrylamide gel electrophoresis and immunoblotting showed that a 40-kilodalton protein was the main protein constituent of the OM preparation, and this was positively identified as the major outer membrane protein by protein microsequencing. Electron microscopy revealed that fine particulate structures on the outermost surface of the EB were also present in the OM preparation. The findings suggest that the major outer membrane protein is an important immunoprotective determinant in ovine abortion vaccines.     

Immunoassay for detecting Chlamydia trachomatis major outer membrane protein.

The development of a solid-phase immunoassay for the detection of the 39,500-dalton major outer membrane protein of the Chlamydia trachomatis lymphogranuloma venereum serotype L2 is described. The test uses immunoadsorbent-purified rabbit anti-L2 major outer membrane protein immunoglobulin G (IgG) passively adsorbed to microtiter plates as a capture antibody. This same IgG antibody was either conjugated to horseradish peroxidase or radioiodinated with 125I and used as a probe to detect major outer membrane protein bound to immobilized IgG. At its greatest sensitivity, the test was capable of detecting 0.5 to 1 ng of purified major outer membrane protein, 5 X 10(3) elementary body inclusion-forming units, and approximately 100 C. trachomatis intracytoplasmic inclusions per assay.     

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.     

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.     

Chlamydia trachomatis major outer membrane protein variants escape neutralization by both monoclonal antibodies and human immune sera.

We have identified two families of novel Chlamydia trachomatis isolates with amino acid changes within the major outer membrane protein (MOMP) variable domains: one family of Da, D*, and D- and one family of Ia and I-. In order to determine whether these MOMP variants can escape antibody neutralization of infectivity, we tested both the D and I prototype strains and the variants in a complement-independent in vitro neutralization assay. We found that variants can indeed escape neutralization by both monoclonal antibodies and polyclonal human immune sera that neutralize the prototype strain.     

Differential CD28 and inducible costimulatory molecule signaling requirements for protective CD4+ T-cell-mediated immunity against genital tract Chlamydia trachomatis infection

Th1 cells and gamma interferon (IFN-gamma) production play critical roles in protective immunity against genital tract infections by Chlamydia trachomatis. Here we show that inducible costimulatory molecule (ICOS)(-/-) mice develop greatly augmented host resistance against chlamydial infection. Protection following a primary infection was characterized by strong Th1 immunity with enhanced CD4(+) T-cell-mediated IFN-gamma production in the genital tract and high expression of T-bet in the draining para-aortic lymph node. This Th1 dominance was associated with low expression of interleukin 10 (IL-10) mRNA in the uteruses of protected ICOS(-/-) mice. By contrast, CD28(-/-) mice were severely impaired in their adaptive immune response, demonstrating a lack of CD4(+) T cells and IFN-gamma in the genital tract, with a substantial delay in bacterial elimination compared to that seen in wild-type (WT) mice. Upon reinfection, WT mice exhibited a transient local infection with evidence of regulatory T-cell (Treg)/Foxp3 mRNA and a more balanced Th1 and Th2 response in the genital tract than ICOS(-/-) mice, whereas 90% of the latter mice developed sterile immunity, poor expression of local Treg/Foxp3 mRNA, and macroscopic signs of enhanced local immunopathology. Therefore, different requirements for CD28 signaling and ICOS signaling clearly apply to host protection against a genital tract infection by C. trachomatis. Whereas, CD28 signaling is critical, ICOS appears to be dispensable and can have a dampening effect on Th1 development by driving Th2 immunity and anti-inflammation through IL-10 production and promotion of the Foxp3(+) Treg populations in the genital tract. Both the CD28-deficient and the ICOS-deficient mice demonstrated poor specific antibody production, supporting the fact that antibodies are not needed for protection against genital tract chlamydial infections.     

Antibody recognition of a neutralization epitope on the major outer membrane protein of Chlamydia trachomatis.

Two BALB/c mice were immunized with serovar C Chlamydia trachomatis elementary bodies, and 63 hybridomas producing monoclonal antibodies to C. trachomatis were recovered. Eight hybridomas which were specific for an identical peptide epitope (AGLQND) in serovar C major outer membrane protein variable domain I were identified. Detailed immunochemical study of the antigen-antibody interaction and genetic characterization of the antibody variable-region gene sequences showed that distinct B-cell clonal lineages were elicited by the epitope sequence. Since each antibody had a distinct pattern of fine specificity for recognition of the epitope and displayed different degrees of cross-reactivity with a related serovar (serovar A), we conclude that B-cell recognition of an immunodominant neutralization epitope can be pleiotropic. Differences in B-cell recognition of a neutralization epitope may delay the emergence by mutation of antigenic-drift variants of the C. trachomatis major outer membrane protein.     

Failure to detect Chlamydia trachomatis in cell culture by using a monoclonal antibody directed against the major outer membrane protein.

Two commercially available monoclonal antibodies for cell culture confirmation of Chlamydia trachomatis were compared in two prospective studies and one large retrospective study. In total, more than 33,000 genital specimens were cultured in parallel and stained with both antibodies, one of which was directed against the major outer membrane protein (MOMP) and one of which was directed against the lipopolysaccharide (LPS). We found the anti-LPS-based assay to be more sensitive and as specific as the anti-MOMP-based assay for C. trachomatis cell culture confirmation of genital specimens.     

E型沙眼衣原体重组主要外膜蛋白诱导恒河猴的交叉免疫效应

目的 探讨E型重组主要外膜蛋白(rMOMP)对恒河猴诱导产生的衣原体交叉免疫应答效应.方法 恒河猴分3组,每组2只,分别为佐剂蛋白组、佐剂组、对照组.于第0、2、4周双侧肱三头肌注射.末次免疫后两周,ELISA检测恒河猴血清中沙眼衣原体特异性IgG抗体和细胞因子IFN-γ,MTT法检测恒河猴淋巴细胞特异性增殖反应,观察恒河猴的迟发型超敏反应,以及恒河猴的血清抗体中和试验.结果 佐剂蛋白组产生了较强的抗rMOMP反应和高水平细胞因子.淋巴细胞特异性增殖反应、迟发型超敏反应明显强于对照组,抗体中和试验蛋白佐剂组血清能抑制D/E/H/L2型沙眼衣原体生长.结论 沙眼衣原体rMOMP能刺激恒河猴产生有效的交叉免疫.     

E型沙眼衣原体主外膜蛋白DNA疫苗和重组蛋白疫苗联合免疫诱导出的免疫效应

目的 研究沙眼衣原体主要外膜蛋白(MOMP)DNA疫苗和重组蛋白(rMOMP)疫苗联合免疫小鼠诱导出的免疫效应.方法 3～4周BALB/c雌鼠60只,分5组,每组12只,于第0、2、4周通过双侧股四头肌肌注免疫相应的疫苗.通过血清IgG抗体和IFN-γ含量、阴道冲洗液sIgA抗体含量、脾淋巴细胞增殖指数、迟发型超敏反应、阴道脱落细胞种植等指标进行小鼠免疫效应的测定.结果 DNA蛋白联合组小鼠血清IgG抗体水平A405值为0.629±0.052;sIgA,A450值为0.379±0.052;脾淋巴细胞增殖指数为5.682±0.484;淋巴细胞培养上清液中IFN-γ含量为(1265±128)ps/ml.DNA蛋白联合组小鼠的各项指标均好于EB阳性对照组除外的其他组(P<0.01).结论 沙眼衣原体DNA疫苗和蛋白疫苗联合免疫可以增强免疫保护效应.     

Evidence that the major outer membrane protein of Chlamydia trachomatis is glycosylated.

The major outer membrane protein (MOMP) of Chlamydia trachomatis was determined to be a glycoprotein on the basis of susceptibility to glycosidase digestion and the presence of carbohydrate by staining and radiolabeling. The MOMP of the serovar L2 organisms was isolated by electroelution from the protein band excised from the gel after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The incubation of MOMP with N-glycosidase F, an endoglycosidase that cleaves the N-glycan, and periodate resulted in two new molecular weight species. While MOMP treated with N-glycosidase F showed a lower-molecular-weight mobility, the periodate-treated MOMP increased in molecular weight. Both treatments abolished the ability of the MOMP to bind to HeLa cell components. In the immunoblot, the reactivity to the monoclonal antibody specific against the C. trachomatis species was preserved. The endoglycosidase specific to O-linked glycan, endo-alpha-N-acetylgalactosaminidase, had no visible effect on the isolated MOMP. Carbohydrate was detected in the MOMP by p-phenylenediamine staining of the protein band in the gel following SDS-PAGE. Autoradiograms of proteins of chlamydial organisms metabolically labeled with [3H]galactose or [3H]glucosamine and separated by SDS-PAGE revealed the MOMP band. The isolated MOMP was shown to bind specifically to concanavalin A, wheat germ agglutinin, and Dolichos biflorus agglutinin in the lectin binding assay. No binding was observed with Ulex europaeus agglutinin I, soybean agglutinin, or Ricinus communis agglutinin.     

Hypothesis: Chlamydia trachomatis infection of the female genital tract is controlled by Type 2 immunity

Chlamydia trachomatis is an obligate intracellular bacterium sexually transmitted to more than 90 million individuals each year. As this level of infectivity implies, C. trachomatis is a successful human parasite; a success facilitated by its ability to cause asymptomatic infection. Host defense against C. trachomatis in the female genital tract is not well defined, but current dogma suggests infection is controlled largely by T_H1 immunity. Conversely, it is well established that T_H2 immunity controls allergens, helminths, and other extracellular pathogens that cause repetitive or persistent T cell stimulation but do not induce the exuberant inflammation that drives T_H1 and T_H17 immunity. As C. trachomatis persists in female genital tract epithelial cells but does not elicit over tissue inflammation, we now posit that defense is maintained by Type 2 immune responses that control bacterial growth but minimize immunopathological damage to vital reproductive tract anatomy. Evaluation of this hypothesis may uncover novel mechanisms by which Type 2 immunity can control growth of C. trachomatis and other intracellular pathogens, while confirmation that T_H2 immunity was selected by evolution to control C. trachomatis infection in the female genital tract will transform current research, now focused on developing vaccines that elicit strong, and therefore potentially tissue destructive, Chlamydia-specific T_H1 immunity.     

Vaccination with the Chlamydia trachomatis major outer membrane protein can elicit an immune response as protective as that resulting from inoculation with live bacteria

BALB/c mice were vaccinated by the intramuscular (i.m.) and subcutaneous (s.c.) routes with a native preparation of the Chlamydia trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP), using Montanide ISA 720 and CpG-1826 as adjuvants. A negative control group was immunized with ovalbumin and the two adjuvants, and a positive control group was immunized intranasally (i.n.) with 10(4) inclusion-forming units (IFU) of C. trachomatis. Four weeks after the last i.m.-plus-s.c. immunization, mice were challenged in the ovarian bursa with 10(5) IFU of C. trachomatis MoPn. Six weeks after the genital challenge, animals were mated, and the pregnancies were monitored. After vaccination with MOMP, the mice developed strong Chlamydia-specific humoral and cellular immune responses. Following the genital challenge, of the mice vaccinated with the MOMP, only 15% (3/20) had positive vaginal cultures, while 85% (17/20) of the animals immunized with ovalbumin had positive cultures over the 6 weeks of observation (P < 0.05). Also, only 14% (3/21) of the animals inoculated i.n. with Chlamydia had positive vaginal cultures. After mating, 75% (15/20) of the mice vaccinated with MOMP carried embryos in both uterine horns. Of the animals vaccinated i.n. with the Chlamydia, 81% (17/21) had embryos in both uterine horns (P > 0.05). In contrast, only 10% (2/20) of the mice immunized with ovalbumin had embryos in both uterine horns (P < 0.05). In conclusion, immunization with a purified preparation of the MOMP is as effective as vaccination with viable C. trachomatis in eliciting a protective immune response against a genital challenge in mice.     

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.