| AbstractChlamydia trachomatis is an obligate intracellular prokaryotic parasite which is the leading cause of preventable blindness and the most prevalent bacterial pathogen causing sexually transmitted disease in clinic. Although chlamydial infection is susceptible to antibiotics treatment, many urogenitally infected individuals don't seek treatment due to lack of obvious clinic symptoms, thus, becoming vulnerable to developing persistent infection and long-term sequelae including ectopic pregnancy and infertility. Vaccination is considered the most effective means for preventing chlamydial infection and diseases. However, the inactivated whole organism-based vaccines failed in human trachoma vaccine trials. Thus, extensive efforts were made to develop effective and safe subunit vaccines in past decades. However, there is still no licensed C. trachomatis vaccine probably due to insufficient knowledge on protective and pathogenic determinants of chlamydial organisms. A genome-wide profiling of the immune response to Chlamydia trachomatis infection and screening immundominant antigens will contribute to identify protective and pathogenic determinants of chlamydial organisms, to clarify chlamydial pathogenesis, and to develop a serum diagnosis kit or a chlamydial vaccine. Part?A Genome-Wide Profiling of the Humoral Immune Response to Chlamydia trachomatis Infection in Humans Objectives1. Cloning and expression of GST fusion proteins encoded by Chlamydia trachomatis serovar D genome-wide ORFs, to lay the foundation for study on Chlamydial proteomics.2. To establish the humoral immune response profile to Chlamydia trachomatis infection in humans and screen immundominant antigens; to identify infection-dependent antigens by comparing antigen profiles recognized by live chlamydial organism-infected versus dead organism-immunized hosts. To provide the experimental evidence for clarifying chlamydial pathogenesis, and developing a serum diagnosis kit or a chlamydial vaccine.Methods1. The informations of all Chlamydia trachomatis serovar D ORFs were analyzed according to the publised papers and sequences on website http://stdgen.northwestern.edu/. Specific primers of 918 chlamydial genes full length or fragments were synthesized according to the gene sequences. PCR was used to amply the target genes from Chlamydia trachomatis serovar D genomic DNA. The pGEX-6p-CTs expression plasmids were constructed and been transformated into E.coli XL-1 blue. The expression of recombinant Ct proteins were induced by IPTG. The proteome of Chlamydia trachomatis serovar D was established.2. All the bacterial lysates containing the chlamydial fusion proteins were added to the 96 well microplates in ORF order for setting up the whole genome scale protein array. Serum samples were collected from patients which were urogenitally infected with Chlamydia. The ELISA was carried out to detect the reactivity of STI antisera with the whole genome scale protein array. The humoral immune response profile to Chlamydia trachomatis infection in humans was established and the chlamydial immunodominant antigens were identified.3. To identify the specificity of the reactivity of the antisera with chlamydial proteins:?Sera from healthy female individuals (without C. trachomatis infection) were harvest and used as negative controls and the reactivities of these sera with the whole genome scale protein array by ELISA. All positively detected antigens were re-measured with the same antibody samples with or without further absorption with lysates made from either HeLa cells alone or C. trachomatis serovar D-infected HeLa cells;?The reactivity of all positively detected antigens were re-measured by Western blot;?The immunoprecipitation and Western blot assays were carried out to detect whether the same antisera can precipitate endogenous chlamydial antigens from chlamydia-infected HeLa cell lysates under a non-denaturing condition.4. To produce antibodies to the whole chlamydial organisms, rabbits or mice were immunized with UV-inactivated C. trachomatis serovar D organisms; Rabbits were injected intramuscularly while Balb/c mice intraperitoneally. Balb/c mice were infected with live chlamydia EB intranasally. Balb/c and C57BL mice were infected with live chlamydia EB intravaginally. All the antisera were harvested and the reactivity with the whole genome scale protein array by ELISA.?We compared the human-recognized C. trachomatis antigens with those detected by antisera from rabbits and different strains of mice to identify whether these hosts are appropriate for using to produce antibodies to C. trachomatis antigens and/or identify/evaluate chlamydial vaccine candidate antigens;?We compared the antigen profiles recognized by live organism-infected humans versus dead organism-immunized animals to identify infection-dependent antigens and infection-independent antigens.5. The expressiong of infection-dependent antigens and infection-independent antigens on whole cell lysates samples with or without human antibody precipitation along with the purified chlamydial RB and EB organisms were detected by an Western blot.Results1. We found that there were 918 predicted ORFs encoded by C. trachomatis serovar D genome and plasmid.In the current study,908 genes were cloned,788 ORFs were expressed in full length and 120 in one or more fragments. Totally,933 pGEX-6p-CTs were constructed and all of them were expressed as GST fusion proteins in E.coli XL1-Blue. The remaining 10 ORFs were not expressed good quality fusion proteins despite our best efforts. These difficult ORFs include CT081, CT219, CT267, CT786, CT039.1, CT221.1, CT480.1, CT814.1, DEG02 and DEG16.2. All the Chlamydia GST fusion proteins were arrayed onto ten 96 well microplates in ORF order. To minimize the plate to plate variations, each plate was included with a GST-alone negative control well and CPAF positive control well. Thus,the chlamydial whole genome scale protein array was set up.3. Antisera from 99 women urogenitally infected with C. trachomatis were collected. The reactivity of these antisera with the chlamydial whole genome scale protein array was detected by ELISA. The humoral immune response profile to Chlamydia trachomatis infection in humans was set up and 27 chlamydial immunodominant antigens were identified. 719 out of the 908 ORF-encoded chlamydial proteins were recognized by at least one of the 99 human antisera. Among the 719 antigens,124 were recognized by 10%,75 by 20%,50 by 30% and 38 by 40% or more antisera respectively. The 27 proteins recognized by 50% or more antisera were designated as immunodominant antigens. These 27 immunodominant antigens cover a wide range of proteins including those localized in the organism membrane such as CT681, CT443, CT812 and the ABC transporters CT067 and CT381, proteins in the inclusion membrane such as CT119, CT147, CT442, CT529 and CT813, and proteins secreted into host cell cytosol such as CT858 and pCT03. In addition, some type?secretion system- related proteins such as CT089 and CT456, metabolic enzymes and proteases such as CT240, CT798, CT806, CT828 and CT841as well as various hypothetical proteins are also among the immunodominant antigens. Although 11 of the 27 antigens were previously reported to be immunodominant, the remaining 16 proteins represent the newly discovered immundominant antigens in the current study, including CT022, CT067, CT101, CT142, CT143, CT240, CT381, CT442, CT443, CT456, CT695, CT798, CT806, CT828, CT841 and CT875.4. The reactivity of positively reacted antigens were re-measured with the same antibody samples with or without further absorption with lysates made from either HeLa cells alone or C. trachomatis serovar D-infected HeLa cells. The results showed that the absorption with chlamydia-infected HeLa lysates blocked antibody binding to all fusion proteins while a similar absorption with HeLa alone lysates failed to do so. No significant reactivity of the serum samples from healthy individuals was found. These observations have demonstrated that human antibody binding to the whole genome scale protein array is specific to chlamydial antigens.5. The reactivity of all positively detected antigens were re-measured by Western blot and the results showed that Most of the chlamydial GST fusion proteins identified by this antiserum in the proteome array ELISA were also detected by the antiserum in Western blot. However, some antigens were not detected by the serum sample in the Western blot or only minimally detected and the intensity of their reactivity with the human antiserum on the Western blot was incompatible with the OD values. The immunoprecipitation and Western blot assays showed that the antigens which positively reacted in ELISA but not in Western blot were successfully precipitated by the same human antiserum and the amounts of antigens precipitated relative to the amounts of antigens available in the infected cell lysates were largely consistent with the corresponding OD values detected in the proteome array ELISA. Thus, these chlamydial proteins were conformation-dependent antigens. The results demonstrated that our fusion protein-based whole genome scale proteome array ELISA approach could detected both linear and conformation-dependent antigens, so that our method was advantage over the Western blot approaches. Among the 27 immunodominant antigens identified using our proteome array with human sera,10 were not or only minimally detected in Western blot,they were GST-CT022, GST-CT067, GST-CT101, GST-CT142, GST-CT240, GST-CT695C, GST-CT798C, GST-CT806, GST-CT828 and GST-pCT03 (Pgp3).6.?As for the types of antigens recognized by the 3 host species, they were human,rabbit and mouth,we found that many antigens dominantly recognized by humans were also strongly recognized by the two rodents species,suggesting that rodents can be used to study chlamydial Immunology and evaluate the protection efficacy of chlamydial vaccine candidate antigens.?A more careful analysis of antigens recognized by the live Chlamydia infected Balb/c and C57BL mice revealed that Intranasal (in) or intravaginal (iv) infection of Balb/c mice both induced antibodies to 54 chlamydial antigens respectively while intravaginal infection of C57BL mice induced antibodies to 27 antigens only, suggestion that a correlation between the number of antigens recognized by the host and the severity of infection.?There were especially significant differences in the types of antigens recognized by antisera from live organism infection and dead organism immunization individuals. When the 719 antigens recognized by human antibodies were resorted based on their recognition by the immunized rabbits and mice, we found that 563 of the 719 human-recognized antigens were not detected by either rabbits or mice, thus classified as infection-dependent antigens, while the remaining 156 as infection-independent antigens since they were recognized by the dead organism-immunized animals.7. The expressiong of infection-dependent antigens and infection-independent antigens on whole cell lysates samples with or without human antibody precipitation along with the purified chlamydial RB and EB organisms were detected by an Western blot. The results showed that many infection-dependent antigens were detected abundantly in infected whole cell lysates but only minimally in purified EBs, suggesting that all infection-dependent antigens are produced during live infection and many may not be incorporated into the infectious particles. Conclusions1. There were 918 predicted ORFs of all Chlamydia trachomatis serovar D genome.2.788 Chlamydia trachomatis gene full length and 120 gene fragments were successfully cloned into pGEX-6p vector,933 pGEX-6p-CTs plasmids were constructed.3.908 chlamydial ORFs full length or fragments were successfully expressed and 933 recombinant GST-CTs fusion proteins,Thus Chlamydia trachomatis serovar D proteome. A whole genome scale proteome array of Chlamydia trachomatis serovar D was set up.4. The B cell ANTIGENome of women urogenitally infected with C. trachomatis were mapped and the humoral immune response profile to Chlamydia trachomatis infection in humans was established.27 chlamydial immunodominant antigens were identified, including 16 proteins were the newly discovered immundominant antigens in the current study, including CT022, CT067, CT101, CT142, CT143, CT240, CT381, CT442, CT443, CT695, CT798, CT806, CT828, CT841 and CT875.5. Our fusion protein-based whole genome scale proteome array ELISA approach could detected both linear and conformation-dependent antigens. Among the 27 immunodominant antigens identified using our proteome array with human sera,10 were conformation-dependent antigens, they were GST-CT022, GST-CT067, GST-CT101, GST-CT142, GST-CT240, GST-CT695C, GST-CT798C, GST-CT806, GST-CT828 and GST-pCT03 (Pgp3).6. Newly systematically identified 563 Chlamydia trachomatis infection-dependent antigens.14 of the 38 antigens which were recognized by(?)40% patients sera were infection- dependent antigens,they are CT089, CT116, CT142, CT153, CT228, CT442, CT529, CT694, CT798, CT806, CT813, CT828, CT858, CT866.Objectives1. The reactivity of Type?Secretion System associated proteins with tients rogenitally infected with C. trachomatis (sexually transmitted infection, TI) and trachoma patients were further detected.2. The mAbs against Type?Secretion System associated protein Tarp (CT456),which was an immnuodominant antigen,were prepared.3. The expression of Tarp protein in Chlamydia infected HeLa cells were detected.4. Protection efficacy of Tarp protein recombinant vaccine was estimated.5. The immunodominant regions of Tarp protein were identified.Methods1. Searching for the reported Chlamydia trachomatis type?secretion system associated proteins in the internet.2. Antisera from 24 women urogenitally infected with C. trachomatis and 8 trachoma patients were collected. The reactivity of these antisera with the chlamydial type?secretion system associated proteins was further detected by ELISA. To identify the specificity of the reactivity of the antisera with chlamydial proteins, sera from healthy female individuals (without C. trachomatis infection) were harvest and the patient sera were preabsorpted with lysates made from either HeLa cells alone or C. trachomatis serovar D-infected HeLa cells, the reactivities of these sera with the t3ssassociated proteins by ELISA.3. Hybridoma technique was carried out to prepare mAbs against Tarp protein. The specificities to these monoclonal antibodies were determined by ELISA. The isotype and chlamydial species specificity of these monoclonal antibodies were determined by an indirect immunofluores-cence assay.4. The expression of Tarp protein in Chlamydia infected HeLa cells were detected by an indirect immunofluorescence assay. The expression of Tarp protein in purified EB, RB and Chlamydia infected HeLa cells lysate were detected by Western blot.5. Evaluation of the immune protection efficacy of Tarp protein recombinant vaccme:?GST fusion protein of Chlamydia muridarum Tarp(ORF TC0471)was cloned and expressed.The GST-TC0741 protein were cleavaged with a precision protease and freed TC0741 recombinant protein was made.?Mopn urogenitally infected Balb/c mouse model was set up. The endogenous Tarp-specific antibody and T cell response were measured by ELISA after the antisera and spleen cells of infected mice were collected respectively.?Mice were immunized with recombinant TC0741 protein,mouse antisera and spleen cells were collected respectively. The exogenous Tarp-specific antibody production and isotypes were detected by ELISA. The specific of anti-MoPn antibody were detected by an indirect immunofluorescence assay. The levels of IFN-??IL-4 and IL-5 were detected from the spleen cell culture supernants after the mouse spleen cells were re-stimulated with MoPn antigens by ELISA.?Balb/c mice were immunized with recombinant TC0741 protein emulsified in the CpF-IFA agjuvant via intramuscular injection, The intact MoPn organisms similarly emulsified in the same adjuvant were used as a positive control, adjuvant alone as a negative control. Thirty days after the immunization, the mice were intravaginally challenged with MoPn. To test the clearance of MoPn infection, the IFU values of MoPn organism shedding were monitored by an indirect immunofluorescence assay.The mouse urogenital tract tissues were isolated and the Histopathological changes were assessed by an H&E(hematoxylin and eosin) dyeing method.6. To map the immunodominant regions of Tarp protein and provide important information for further understanding the biological function and the protective immunity of Tarp,11 fragments of Tarp were cloned and expressed as GST fusion proteins and the reactivity of these fusion proteins with antisera from patients infected with C. trachomatis in the urogenital tract or in the ocular tissue and from rabbits immunized with C. trachomatis organisms and with mAbs against Tarp protein were detected by ELISA.Results1. We found that chlamydial type?secretion system associated proteins was predicted to consist of Inc proteins and other 49 chlamydial proteins or so by searching in the internet.The reactivities were detected by ELISA and the results showed that CT456 (Tarp)?CT089 and CT858 recombinant proteins were immunodominantly recognized by both STI and trachoma antisera,the reactivity ot Tarp even stronger than that of CPAF with 100% recognition frequency and the highest average OD value. The trachoma antisera can't predominantly recognize the three Inc proteins CT119,529 and 813despite the fact that they are immunodominant during human urogenital infection with C. trachomatis. MOMP from C. trachomatis serovar D was lack of recognition by trachoma antisera.2. Nine hybridoma cell lines stable in secreting specific against Tarp protein monoclonal antibodies were successfully obtained, they were R4D5, N5B11, R5B6.2, R2H1.2, R12B12, R2H7, R5G8.1, R8G7.2 and M4F4. All the 9 mAbs were IgG,2 mAbs (R5B6.2 and R8G7.2) were belong to IgG2a isotype and the other 7 mAbs (R4D5, R2H1.2, R12B12, R2H7, N5B11, M4F4 and R5G8.1) were belong to IgGl isotype. Results of indirect immunofluorescence assay showed that all the 9 mAbs can react strongly with chlamydia serovar A,D,and L2, but not MoPn,6BC,and AR39.3. The expression of Tarp protein in Chlamydia infected HeLa cells were detected by an indirect immunofluorescence assay. The results showed that:Tarp was detected in the first 8 hours and no longer detectable by 13 hours after infection. Although chlamydial inclusions became very obvious at 24 hours after infection, Tarp remained undetectable. However, by 28 hours after infection, Tarp started to reappear and progressively increased as the incubation continued. Tarp protein was only detected in Chlamydia trachomatis serovar D infected HeLa cell lysates and purified EBs but not RBs on a Western blot. Our results suggesting that Tarp protein was an EB associated protein.4. TC0471 full length sequence was amplified by PCR from Chlamydia muridarum genomic DNA and pGEX-6p-TC0741 plasmid was constructed; GST-TC0741 fusion protein was successfully expressed; freed TC0741 recombinant protein was made by cleavaged the GST-TC0741 protein with a precision protease.5. The MoPn infected mice developed high titers of antibodies that recognized both MoPn-derived antigens and Tarp fusion proteins but not the fusion tag GST alone. High levels of IFN-y were induced after the spleen cells from MoPn infected mice were re-srimulated with either Tarp protein or MoPn. The levels of IFN-ywere increased with the amount of Tarp protein. No IL-4 and only minimum levels of IL-5 were detected from the spleen cell culture supernatants of the MoPn infected mice. These observations together have demonstrated that mice infected with MoPn developed Tarp-specific humoral and cellular immune responses.6. The immune response induced by exogenous Tarp protein was evaluated after mice were immunized with recombinant TC0741 protein emulsified with adjuvant. The antisera induced by MoPn or Tarp but not adjuvant alone visualized antigens in MoPn inclusions in animmunofluorescence assay. the anti-Tarp antibody labeling was blocked by either the immunogen GST-Tarp or MoPn-infected cell lysates while the anti-MoPn antibody labeling was effectively blocked by MoPn-infected cell lysates. We further quantitated the titers of the MoPn antigen-reactive antibodies and isotyped the mouse IgG antibodies in an ELISA Immunization with either MoPn or Tarp with adjuvant but not adjuvant alone induced high titers of antibodies that specifically recognized MoPn endogenous antigens. Immunization with either Tarp or MoPn induced more IgG2a than IgG1,that is IgG2a/IgG1>1. The spleen cells from mice immunized with either MoPn or Tarp produced significantly higher levels of IFN than those from adjuvant alone immunization. No IL-4 and only minimum levels of IL-5 were detected from the spleen cell culture supernatants of the immunized mice. These results further strengthened the conclusion that immunization with Tarp induced a MoPn-specific cellular and humoral immune responses, it was a Thl-dominant immune response.7. To test the clearance of MoPn infection, the IFU values of MoPn organism shedding were monitored by an indirect immunofluorescence assay. Live MoPn organisms were detected in the vaginal swabs collected from the adjuvant alone group of mice up to 27 days after infection. Immunization with the intact MoPn organisms shortened the infection time course to 15 days with a significant reduction in the infectious titers from the vaginal swabs within 6 days after infection. Importantly, immunization with Tarp resulted in a significant reduction in the vaginal shedding of live organisms on day 21 and most of the Tarp-immunized mice cleared infection by day 24 post infection. Thus, Tarp-induced immunity enhanced the resolution of MoPn infection from the mouse lower genital tracts although not as potently as the intact organism-induced immunity.8. Gross appearance assessment showed that the incidence of bilateral hydrosalpinx in Tarp-immunized (33%) or MoPn-immunized (10%) mice is significantly lower than that in the adjuvant alone-immunized mice(70%). After, we further evaluated the severity of inflammation microscopically using histology sections. Both the inflammatory cell infiltration and luminal dilatation were blindly semi-quantitated. There was no significant difference in either inflammation or lumenal dilatation scores in the uterine horn tissues between the three groups of mice. However, mice immunized with either Tarp or MoPn displayed significantly lower scores in both inflammatory cell infiltration and lumenal dilatation than the control group of mice, suggesting that the immunity induced by either Tarp or MoPn can reduce the inflammatory damages in the mouse oviducts.9.11 fragments of Tarp were successfully expressed as GST fusion proteins and the reactivity of these fusion proteins with antisera from patients infected with C. trachomatis in the urogenital tract or in the ocular tissue and from rabbits immunized with C. trachomatis organisms and with mAbs against Tarp protein were detected by ELISA. A major immunodominant region was strongly recognized by all antibodies. This region covers amino acids 152 to 302, which consist of three repeats (152-201,202-251 and 252-302). Several other minor immunodominant regions were also identified, including 1-156,310-431 and 582-682, recognized by antisera from both human and rabbit; 425-581, only recognized by human antisera; and 683-847, preferentially recognized by rabbit antisera. This immunodominance was also confirmed by the observation that six out of the nine mAbs bound to the major immunodominant region and that the other three each bound to one of the minor fragments,1-119,120-151 and 310-431. Conclusions1. Tarp protein was an immunodominant antigen not only in STI patient but also in trachoma patient.2. Nine hybridoma cell lines stable in secreting specific against Tarp protein monoclonal antibodies were successfully set up.3. Tarp was a chlamydial EB associated protein.4. Immunization of mice with Tarp induced Thl-dominant immunity that significantly reduced the shedding of live organisms from the lower genitaltract and attenuated inflammatory pathologies in the fallopian tube tissues. These observations have demonstrated that Tarp can induce protective immunity against chlamydial infection and pathology in mice,it cab be used as a novel promising chlamydia vaccine candidate antigen.5. The region covers amino acids 152 to 302 was the most immunodominant region of Tarp protein. |
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