Screening Of Chlamydia Trachomatis Subunit Vaccine Candidate Antigens And Study On The Biological Characteristics Of GlgA Protein

Chlamydia trachomatis urogenital tract infection cause serious diseases but vaccine is not available. Subunit vaccine is the development direction but a single one can not simultaneously attack multiple stages of chlamydia infection processes. Discovery and identification of more novel vaccine candidate antigens, immunization with combinations of various antigens with each able to induce unique effector mechanisms, and optimization the immunization regimen are the preferred research strategy for C. trachomatis vaccine. Using mouse model evaluate the 5 immunodominant antigens identified by our previously experiment, study the protection efficiency of chlamydial plasmid encoded protein Pgp3, and screening of the infection dependent antigens will provide useful information for human C. trachomatis vaccine development.Chlamydial pathogenic mechanisms remain unclear which is a important factor to hinder the successful development of chlamydial vaccine. Chlamydiae are strictly intracellular pathogen, which can synthesis glycogen in the inclusion body. If glycogen storage disorder, C. trachomatis will lose pathogenic ability. Thus, localization analysis of Chlamydia trachomatis 6 glycogen metabolism-related enzymes and identifying the biologic characteristics may provide useful data for C. trachomates glycogen metabolism, pathogenesis and vaccine research. Part Ⅰ Evaluating the protection efficacy of Chlamydia trachomatis’ immunodominant antigens and screening the infection-phase dependent antigensObjective 1. Evaluate the protection efficacy of 5 immunodominant antigens(Art J,Omc B,MIP,Inc,HP) identified by both human and mouse antisera, to provide useful imformation for designing human vaccine against C. trachomatis infection.2. Evaluate the protection efficacy of chlamydial plasmid encoded and secreted protein Pgp3, Comparing the different protection efficacy between intranasal and intramuscular immunizations.3. Genome wide screen Chlamydia infection-phase dependent antigens, provide new targets for pathogenic mechanism research and vaccine development.Methods 1. The 5 ORFs were cloned into p GEX vectors and expressed as fusion proteins with glutathione-stransferase(GST) fused to the N-terminus. The purified proteins were used to intramuscularly immunize mice with Cp G-IFA as adjuvant. Indirect immunofluorescence assay was used to detect the lower genital tract bacterial shedding in a mouse intravaginal infection model; Assessment of severity of hydrosalpinx and inflammation in the upper genital tract by naked eyes or immunohistochemistry; ELISA were used to detection specific antibody titerd and cytokines level of splenocytes after different stimulations.2. The secreted protein Pgp3 were cloned into p GEX vectors and its prokaryotic recombinant GST fusion protein was purified to immunize Balb/c mice intranasally or intramuscularly with Cp G-IFA as adjuvant. The chlamydia shedding from the lower genital tract, the upper genital tract gross pathology and histopathological characteristics, as well as the immune response were compare between different route.3. Large scale purification of Chlamydia trachomatis serovar D EB organism. 50 IFU live EB were used to infect C3H/He J mice in the airway, 1×106 inactived EB plus adjuvant were used to immunize mice intranasally. Collecting sera to reacted with chlamydial whole genome encoded protein, ELISA screen the antigens which only be recognized by C. trachomatis infected mice sera. Using these sreened antigens stimulate the spleenocytes of chlamydia infected mice, ELISA detect IFN-γand IL-17 production level.Results 1. The five p GEX6p-TCs expression plasmid was successfully constructed, the purifed proteins all with high quality. We screened all of them in a mouse intravaginal infection model for induction of protective immunity. Only MIP immunization significantly reduced lower genital tract shedding and upper genital tract pathology. MIP immunization induced high levels of MIP-specific Ab responses and cytokine IFN-γ production.2. The mouse groups intranasal immunized with Pgp3 displayed statistically significant reduction in shedding of live organisms on day 15 post infection, but intramuscular immunized group delay to day 21. Pgp3 i.n. immunization significantly reduced both the hydrosalpinx and inflammation and lumen dilatation scores in the oviduct tissues, which shows significant difference against intramuscular immunization grou, p<0.01.3. Intranasal immunization induces significant high Ig G2a/Ig G1, P<0.01; The Ig A level in urogenital tract mucus in intranasal immunized group is significantly higher than intramuscular group. Splenic cells from each group after stimulated with Pgp3, IFN-γproduction level is much high in intranasal group than intramuscular immunization group, but IL-17 is no difference and IL-5 is reversed.4. Both live EB intranasal infected mice and UV-EB intramuscular immunized mice develop similar amount antibodies. Among the antigens encoded by Chlamydia genome 908 ORFs, only 60 of them can be recognized by at least one antisera. 30 antigens can only be recognized EB infected group mice sera, we call them infection-phase dependent antigens. 12 antigens which can be recognized by both chlamydial infected and immunized mice sera with recognition frequence in each group >=50%, CT875 can only be recognized by immunized mice sera with recognition frequence >=50%, 5 antigens which only recognized by EB infected mice sera with frequence >=50%.5. The 9 antigens which only recognized by EB infected mice sera with frequence equal or more than 40% were used to restimulate splenocytes of chlamydial urogenital tract infected mice. 7 antigens stimulation with high level Th1 cytokine IFN-γproduction. 8 antigens restimulation induced significant higher level of IL-17 production.Conclusions 1. Among the 5 immunodominant antigens evaluated in mouse intravaginal infection model, Only MIP immunized animals provide protection. These protection mechanisms are correlated well with a Th1-dominant immune response.2. Pgp3 protein intranasal immunization compare with intramuscular route immunization induce more powerful Th1-dominant immune response. Only Pgp3 protein intranasal immunization provide protection against mice urogenital tract infection.3. Both C. trachomatis EB lung infection and muscle immunization induce high level of specific antibodies against chlamydia organisms. 30 antigens are infection-phase dependent antigens which can only be recognized by chlamydia infected mice sera.6. Chlamydial antigens, p CT03(Pgp3), CT823, CT806, CT148, CT039 and CT315 restimulates splenocytes of chlamydial infected animals, induce Th1 dominant and lower Th17 cellular immune response.Part Ⅱ Evaluating the protection efficacy of Chlamydia trachomatis’ immunodominant antigens and screening the infection-phase dependent antigensObjective 1. Localization analysis of Chlamydia trachomatis 6 glycogen metabolism-related enzymes, to provide powerful experimental tools and data for Chlamydia trachomates glycogen metabolism study.2. Study on secretion characteristics of chlamydial glycogen synthase Glg A, analysis of Glg A overexpression on Chlamydia infection, and collecting C. trachomtis urogenital tract infected patients serum to analysis the immunogenicity of Glg A protein in case of human natural infection, which may lay foundation for the in-depth study of Glg A function and pathogenic mechanism.Methods 1. The 6 ORFs CT042(Glg X), CT087(Mal Q), CT248(Glg P), CT295(Mrs A), CT489(Glg C), CT798(Glg A) from C. trachomatis serovar D genome were cloned into p GEX vectors. The corresponding fusion protein were used to immunize mice for producing polyclonal antibodies. Using multiple experimental methods, such as succession antibody dilution, confocal immunofluorescence microscope observation, GST fusion protein adsorption and western blot to analysis the specificity of Glg A antibody, and provide convencing data to confirm the secretion phenomenon of Glg A protein.2. He La cells with or without C. trachomatis infection were fractionated and resolved in SDS-PAGE. The resolved proteins were used for Western blot detection of Glg A distrubution. Time course expression and the percent of cells secretion Glg A or CPAF during C. trachomatis L2 infection were detected by Immunofluorescence assay. This assay also used to identify whether Glg A protein secretion phenomenon is prevalent in different serovars. p FLAG-CMV4 was constructed to evaluate the effect of chlamydial Glg A expression on glycogen synthesis and subsequent chlamydial infection. Collecting sera from acute C. trachomatis infection or tubal factor infertility patients, as well as rabbits intramuscularly immunized with dead C. trachomatis organisms to analysis the immunogenicity of Glg A protein in human natural infection.Results 1. Chlamydia trachomatis 6 glycogen metabolism-related enzymes have good immunogenicity, after immunize mice induced high titer of specific antibody production. Only the anti-GST-CT798(Glg A) detected signals that were free of chlamydial organisms, dilution of antibody deduce the stain intensity but not change the staining pattern. Under a confocal microscope, the labeling with anti-Glg A polyclonal antibody was different form HSP60 antibody which was completely overlap with MOMP.2. The mouse anti-Glg A(p Ab), anti-CPAF(m Ab) and anti-Pgp3(m Ab) labelings were removed only by the corresponding but not irrelevant fusion proteins. He La cell lysates, Ct-infected He La cell lysates, GST-Glg A or GST-CPAF fusion proteins were resolved in SDS-polyacrylamide gel and transferred onto nitrocellulose membrane for Western blot detection with Glg A p Ab, CPAF m Ab and MOMP m Ab, these antibodies only detected their corresponding antigens without any significant cross-reactivity.3. The distrubition of Glg A in fractions of C. trachomatis-infected cells was different from CPAF. Glg A endogenous expression was first detected associated with the chlamydial inclusions at 12 h while secretion out of the inclusions was first detected at 24 h. The secreted Glg A and CPAF remained in the infected cells throughout the infection cycle. He La cells infected with L2 for 40 h, The % of cells positive for Glg A secretion into host cell cytoplasm has no difference with those positive for CPAF secretion. Glg A secretion into host cell cytosol was detected in all Chlamydia trachomatis biovars/serovars.4. Expression of chlamydial Glg A in HeLa cells resulted in elevated levels of glycogen but has no affect on the subsequent chlamydial infection. Glg A was recognized by antisera from women diagnosed with either acute C. trachomatis infection(STI patients) or tubal factor infertility(TFI patients) with a recognition frequency of 60% or 29% respectively. However, the secreted proteins CPAF, Pgp3 and CT795 did not display such a preference with a recognition frequency of 100%, 95% & 35% by STI antisera and 92%, 96% & 50% by TFI antisera respectively.Conclusions 1. First discovered and confirmed that Chlamydia trachomatis glycogen metabolism-related enzyme CT798(Glycogen synthase, Glg A) is a secreted protein which can be secreted into host cell cytosol during chlamydia infection and has no cross-reaction with the other two secreted proteins CPAF and Pgp3.2. The other 5 Chlamydia trachomatis glycogen metabolism-related enzymes CT042(Glg X),CT087(Mal Q),CT248(Glg P),CT295(Mrs A),CT489(Glg C) were all located in chlamydial organisms.3. Chlamydia trachomatis cytosolic secreted proteins Glg A and CPAF have similar secretion characteristics, but the distribution pattern is different.4. Among all the glycogen-related enzymes analyzed, Glg A has the highest immunogenicity. The recognition frequence of Glg A antigen by antisera from women diagnosed with acute C. trachomatis infection(STI patients) was significantly higher than tubal factor infertility(TFI patients). By combine with others chlamydial antigens, Glg A may be develop as a new diagnostic marker for chlamydial acute infection.