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。     

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.     

密码修饰可增强沙眼衣原体MOMP DNA质粒免疫小鼠的免疫应答

目的 探讨密码修饰对沙眼衣原体主要外膜蛋白（MOMP）基因表达和DNA质粒免疫的影响。方法 根据人类密码子使用偏好对鼠肺炎沙眼衣原体（Chlamydia trochomatis mouse pneumonitis）MOMP基因进行优化修饰，设计合成人源化MOMP基因（HuMOMP）。构建以pcDNA3为载体的含HuMOMP及含野生型MOMP基因（WtMOMP）的真核表达质粒。瞬时转染COS-1细胞，Western blot方法比较HuMOMP基因及WtMOMP基因在哺乳动物细胞中的蛋白表达水平。DNA质粒肌内免疫BALB／c小鼠，检测小鼠血清特异性抗体、DTH和淋巴细胞增殖反应，比较优化密码MOMP基因和野生型MOMP基因DNA质粒免疫的免疫效果。结果 人工合成HuMOMP基因，成功构建重组真核表达质粒pcDNA3-HuMOMP及pcDNA3-WtMOMP。转染细胞Western blot结果显示，HuMOMP基因的蛋白表达水平明显高于WtMOMP基因。ELISA结果表明，HuMOMP DNA质粒免疫组小鼠血清特异性IgG抗体有所升高；细胞免疫检测结果显示，HuMOMP DNA质粒免疫组小鼠足垫肿胀程度增强、淋巴细胞增殖实验刺激指数（SI）增高，两者与WtMOMP DNA质粒免疫组相比P〈0．05，差异均有统计学意义。结论 人源化密码修饰能够增强沙眼衣原体MOMP基因在哺乳动物细胞中的蛋白表达及DNA质粒免疫小鼠的免疫反应，这对于新型衣原体DNA疫苗的研究具有重要意义。     

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.     

Primary human T-cell responses to the major outer membrane protein of Chlamydia trachomatis.

The major outer membrane protein (MOMP) of Chlamydia trachomatis is the main candidate antigen for a synthetic vaccine against chlamydial infection. Antibodies to surface-exposed epitopes on MOMP neutralize chlamydial infectivity but little is known about T-cell recognition of the molecule. We have measured primary human T-cell responses to recombinant fragments of MOMP as well as to the whole organism and synthetic MOMP peptides. Using antigen-pulsed low density cells (LDC) we were able to stimulate proliferative responses with T cells from most naive individuals. This response was antigen dose dependent and displayed an absolute requirement for dendritic cells in the antigen-presenting cell (APC) population. Several T-cell epitopes were identified in MOMP and one which stimulated T cells from 80% of donors was resolved as a 12 amino acid synthetic peptide. Dual cell surface labelling and cell cycle analysis by FACS revealed that both CD4+ and CD8+ T cells were stimulated in these cultures. The fact that we were able to obtain proliferative responses and interferon-gamma (IFN-gamma) production to MOMP using cells from cord bloods confirmed that these are genuine primary responses. These experiments have identified a region on MOMP, to which T cells from most humans make a primary response, which may be useful in a chlamydial vaccine. The approach is useful for vaccine development in general.     

T-cell epitopes in variable segments of Chlamydia trachomatis major outer membrane protein elicit serovar-specific immune responses in infected humans

We previously identified 18 stimulatory Chlamydia trachomatis major outer membrane protein (MOMP) peptides containing at least 23 epitopes presented with various HLA class II allotypes. Only one peptide contained an epitope localized in a variable segment (VS2). Continued studies reported here identified a total of five VS peptides containing T-cell epitopes that are distributed among MOMPs VS1, VS2, and VS4. Only MOMP-primed T-cell cultures from subjects infected with serovar E responded to the serovar E VS peptides, while the response of such cultures to constant-segment peptides was independent of the infecting serovar. Furthermore, MOMP-primed T cells proliferated in response only to the VS peptides encoded in serovar E but not to the corresponding peptides derived from serovar F, I, or J, confirming that these responses were serovar specific.     

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.     

Neutralization of Chlamydia trachomatis infectivity with antibodies to the major outer membrane protein.

Rabbit immunoglobulin G (IgG) antibodies raised against the major outer membrane protein of the Chlamydia trachomatis lymphogranuloma venereum strain 434 neutralized the infectivity of the parasite for HeLa 229 cells. The mechanism by which anti-major outer membrane protein IgG prevented C. trachomatis from establishing infection was studied by using intrinsically 14C-radiolabeled elementary bodies. Neutralized elementary bodies were filterable through a polycarbonate filter (pore diameter, 600 nm), demonstrating that reduction in infectivity was not due to the aggregation of elementary bodies by cross-linking IgG. Antibody-neutralized elementary bodies attached to and penetrated HeLa cells at rats nearly identical to those for infectious organisms exposed to nonneutralizing control IgG. These results suggest that antibody interferes with the infectious process of the parasite after its internalization. Anti-major outer membrane protein Fab fragments could not be substituted for neutralizing IgG antibodies. The requirement for intact IgG implies that cross-linking of antibodies to the major outer membrane protein on the surfaces of the organisms may be instrumental in neutralization.     

Characterization of immune responses following intramuscular DNA immunization with the MOMP gene of Chlamydia trachomatis mouse pneumonitis strain

Studies were carried out to characterize the cellular and humoral immune responses evoked by intramuscular DNA vaccination with the major outer membrane protein (MOMP) gene of Chlamydia trachomatis mouse pneumonitis strain. The data demonstrate that DNA vaccinated mice develop antigen-specific delayed-type hypersensitivity, lymphocyte proliferation and interferon-gamma (IFN-gamma) production. Serum antibody responses (mainly immunoglobulin G2a; IgG2a) were evoked in two-thirds of the mice. We conclude that intramuscular DNA immunization with the MOMP gene evokes cellular and humoral immune responses suggestive of a T helper 1 (Th1) bias.     

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.     

沙眼衣原体主要外膜蛋白在减毒鼠伤寒杆菌中的表达及 …

目的 采用作为疫苗载体的减毒鼠伤寒杆菌致死性平衡系统,构建能异源表达沙眼衣原体（Ｃｔ）主要外膜蛋白（ＭＯＭＰ）的重组菌株。方法 以Ｄ型Ｃｔ的ＤＮＡ为模板,用所设计的特异性引物扩增编码Ｃｔ ＭＯＭＰ高变区（ＶＤＩ～ＶＤＩＶ）基因,并将扩增产物定向克隆至质粒ｐＵＣ１９中。序列分析后,再亚克隆至与减毒鼠伤寒杆菌Ｘ４５５０互补的质粒ｐＹＡ３３４１中,以重组质粒转化减毒鼠伤寒杆菌Ｘ４５５０。结果 对构建的重     

Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis.

Elementary bodies (EB) of Chlamydia trachomatis serotypes C, E, and L2 were extrinsically radioiodinated, and whole-cell lysates of these serotypes were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Autoradiography of the polypeptide profiles identified a major surface protein with an apparent subunit molecular weight of 39,500 that was common to each C. trachomatis serotype. The abilities of nonionic (Triton X-100), dipolar ionic (Zwittergent TM-314), mild (sodium deoxycholate and sodium N-lauroyl sarcosine), and strongly anionic (SDS) detergents to extract this protein from intact EB of the L2 serotype were investigated by SDS-PAGE analysis of the soluble and insoluble fractions obtained after each detergent treatment. Only SDS readily extracted this protein from intact EB. Sarkosyl treatment selectively solubilized the majority of other EB proteins, leaving the 39,500-dalton protein associated with the Sarkosyl-insoluble fraction. Ultrastructural studies of the Sarkosyl-insoluble EB pellet showed it to consist of empty EB particles possessing an apparently intact outer membrane. No structural evidence for a peptidoglycan-like cell wall was found. Morphologically these chlamydial outer membrane complexes (COMC) resembled intact chlamydial EB outer membranes. The 39,500-dalton outer membrane protein was quantitatively extracted from COMC by treating them with 2% SDS at 60 degrees C. This protein accounted for 61% of the total COMC-associated protein, and its extraction resulted in a concomitant loss of the COMC membrane structure and morphology. The soluble extract obtained from SDS-treated COMC was adsorbed to a hydroxylapatite column and eluted with a linear sodium phosphate gradient. The 39,500-dalton protein was eluted from the column as a single peak at a phosphate concentration of approximately 0.3 M. The eluted protein was nearly homogeneous by SDS-PAGE and appeared free of contaminating carbohydrate, glycolipid, and nucleic acid. Hyperimmune mouse antiserum prepared against the 39,500-dalton protein from serotype L2 reacted with C. trachomatis serotypes Ba, E, D, K, L1, L2, and L3 by indirect immunofluorescence with EB but failed to react with serotypes A, B, C, F, G, H, I, and J, with the C. trachomatis mouse pneumonitis strain, or with the C. psittaci feline pneumonitis, guinea pig inclusion conjunctivitis, or 6BC strains. Thus, the 39,500-dalton major outer membrane protein is a serogroup antigen of C. trachomatis organisms.     

Protective monoclonal antibodies to Chlamydia trachomatis serovar- and serogroup-specific major outer membrane protein determinants.

Monoclonal antibodies exhibiting Chlamydia trachomatis serovar specificity (serovar A, B-Ba, or C) and serogroup specificity (B, intermediate, or C serogroup) were produced and characterized. These antibodies reacted with the major outer membrane protein, recognized epitopes located at the chlamydial cell surface, and passively neutralized chlamydial toxicity for mice. The antibodies should be useful reagents for defining the molecular structure of these protective epitopes, a necessary step toward the development of a subunit or recombinant C. trachomatis vaccine.     

Mapping antigenic sites on the major outer membrane protein of Chlamydia trachomatis with synthetic peptides.

The antigenicity of the major outer membrane protein (MOMP) of Chlamydia trachomatis was comprehensively evaluated by using overlapping hexapeptide homologs of serovar B MOMP and polyclonal rabbit antisera in a peptide enzyme-linked immunosorbent assay. Of 367 hexapeptides, 152 showed reactivities with at least one antiserum. Seven hexapeptides located within variable domain (VD) IV (residues 288 to 316) were found to be most reactive in terms of their binding titer and frequency, suggesting that VD IV is the immunodominant region within the MOMP as detected by this assay. Peptide-reactive antibodies could also recognize corresponding epitopes on either viable or acetone-permeabilized organisms. The antigenic specificity and immunoaccessibility of epitopes located in VD IV were resolved by absorbing antisera with chlamydial elementary bodies. Six antigenic sites were found in this region and included a B-type-specific site (S1), four subserogroup-specific sites (S2 and S4 to 6), and one species-specific site (S3), each displaying varying degrees of surface exposures on elementary bodies from different C. trachomatis serovars.     

Antigenic analysis of the major outer membrane protein of Chlamydia trachomatis with murine monoclonal antibodies.

We prepared monoclonal antibodies against prototype strains of the 15 serovars of Chlamydia trachomatis and identified a subset of reagents that reacted with the major outer membrane protein(s) (MOMPs) of one or more serovars. We then determined the specificities of these anti-MOMP monoclonal antibodies by radioimmunoassay and immunoblot assays against the 15 serovars of C. trachomatis and a C. psittaci strain. We identified 14 different anti-MOMP antibody specificities, including serovar-, several orders of subspecies-, and species-specific determinants. In addition, one antibody reacted with all C. trachomatis serovars and a C. psittaci strain, indicating the presence of a genus-specific epitope on MOMP. Many of the cross-reactions of the subspecies-specific antibodies were similar to those previously reported by use of the microimmunofluorescence technique. We also observed a number of cross-reactions that were unexpected but consistent with data derived by the microimmunofluorescence test. All antibodies, except the genus-specific antibodies, reacted with whole elementary bodies in a radioimmunoassay, suggesting surface exposure of the epitopes. These data confirm and extend previous observations that MOMPs among C. trachomatis serovars are antigenically complex and diverse. In addition, these data indicate that the cross-reaction patterns of some monoclonal antibodies directed against MOMP are similar to those detected by the microimmunofluorescence test and are consistent with the hypothesis that such determinants are contained within MOMPs.     

Molecular cloning and expression of Chlamydia trachomatis major outer membrane protein antigens in Escherichia coli.

DNA obtained from Chlamydia trachomatis (serovar L2) was partially digested with DNase I and inserted into the beta-galactosidase gene of bacteriophage lambda gt11. Seven recombinants were selected that produced immunoreactive fusion proteins which were detected with anti-C. trachomatis rabbit serum. One recombinant, designated lambda gt11/L2/33, reacted with various monoclonal antibodies that recognize species-, subspecies-, and type-specific determinants on the chlamydial major outer membrane protein (MOMP). Immunoblot analysis of a lambda gt11/L2/33 lysogen revealed a fusion protein that expressed a approximately 15,000-dalton carboxyl-terminal peptide of the chlamydial MOMP. This moiety of the MOMP possesses epitopes responsible for each of the unique reactivities demonstrated by anti-MOMP monoclonal antibodies. The lambda gt11/L2/33 recombinant contained a 1.1-kilobase DNA insert which hybridized to DNA isolated from each of the 15 C. trachomatis serovars.     

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.     

A poliovirus hybrid expressing a neutralization epitope from the major outer membrane protein of Chlamydia trachomatis is highly immunogenic.

Trachoma and sexually transmitted diseases caused by Chlamydia trachomatis are major health problems worldwide. Epitopes on the major outer membrane protein (MOMP) of C. trachomatis have been identified as important targets for the development of vaccines. In order to examine the immunogenicity of a recombinant vector expressing a chlamydial epitope, a poliovirus hybrid was constructed in which part of neutralization antigenic site I of poliovirus type 1 Mahoney (PV1-M) was replaced by a sequence from variable domain I of the MOMP of C. trachomatis serovar A. The chlamydial sequence included the neutralization epitope VAGLEK. This hybrid was viable, grew very well compared with PV1-M, and expressed both poliovirus and chlamydial antigenic determinants. When inoculated into rabbits, this hybrid was highly immunogenic, inducing a strong response against both PV1-M and C. trachomatis serovar A. Antichlamydia titers were 10- to 100-fold higher than the titers induced by equimolar amounts of either purified MOMP or a synthetic peptide expressing the VAGLEK epitope. Furthermore, rabbit antisera raised against this hybrid neutralized chlamydial infectivity both in vitro, for hamster kidney cells, and passively in vivo, for conjunctival epithelia of cynomolgus monkeys. Because poliovirus infection induces a strong mucosal immune response in primates and humans, these results indicate that poliovirus-chlamydia hybrids could become powerful tools for the study of mucosal immunity to chlamydial infection and for the development of recombinant chlamydial vaccines.     

The frequency of Chlamydia trachomatis major outer membrane protein-specific CD8+ T lymphocytes in active trachoma is associated with current ocular infection

Chlamydia-specific cytotoxic T lymphocytes are able to control model infections but may be implicated in disease pathogenesis. HLA-A2 peptide tetramers to Chlamydia trachomatis major outer membrane protein 258-266 (MOMP258-266) and MOMP260-268 were used to characterize HLA class I-restricted CD8+ T cells in Gambian children aged 4 to 15 years with clinical signs of active trachoma and/or infection with C. trachomatis. The frequencies of circulating HLA-A2 tetramer binding cells (TBC) were determined in whole blood samples by flow cytometric analysis. Initial screening of subjects with an anti-HLA-A2 antibody confirmed the presence of either HLA-A2 or HLA-A28. These were subsequently further divided by molecular subtyping. The C. trachomatis-specific HLA-A2 peptide tetramers were able to bind T cells with receptors from subjects which were restricted by either the HLA-A2 or the HLA-A28 restriction element. In this population, the median value of C. trachomatis-specific CD8+ T cells was 0.02%, with frequencies of up to 3.71% of CD8+ T cells reactive with a single tetramer in a minority of subjects. TBC were detected more often in subjects who were infected at the ocular surface, and their presence was associated with infection episodes of longer duration. Detection of C. trachomatis-specific TBC was not associated with the presence of disease or with the estimated load of ocular C. trachomatis infection at the time of sample collection. High frequencies of C. trachomatis-specific cells did not predict subsequent appearance or resolution of the clinical disease signs of active trachoma.     

沙眼衣原体主要外膜蛋白T、B细胞多表位基因的表达及其鉴定

目的 对沙眼衣原体（Ct）主要外膜蛋白（MOMP）细胞毒性T淋巴细胞（CTL）表位进行预测和选择，并进行Ct MOMP多表位基因克隆和多表位蛋白的原核表达及其抗原性分析，为研制多表位Ct疫苗提供基础资料。方法利用SYFPEITHI法和多项式方案结合预测HLA-A2限制性的0MOMP的CTL表位，选取含多个CTL表位的基因片段作为目的基因，兼顾目的基因上下游存在的TH和B细胞表位的基因，共同组成含CTL、TH和B细胞表位的多表位基因。多表位基因经密码子优化后进行全序列合成，克隆入原核表达载体pET-32a（＋），经IPTG诱导在大肠杆菌BL21（DE3）表达并纯化，经SDS-PAGE和Western blot分析鉴定表达产物。结果 经预测筛选得到了多个MOMPHLA-A2限制性的CTL表位，成功克隆了含CTL、TH、B表位的MOMP多表位基因，并在大肠杆菌中获得了高效表达。表达产物的相对分子质量（Mr）约24×10^3，与预期肘，相符，并用Western blot方法初步证实多表位蛋白有抗原特异性。结论 成功设计了Ct MOMP的T、B细胞多表位基因，并证实在原核表达系统中获得正确表达的多表位蛋白具有良好的抗原性。