Induction Of Protective Immunity Against Chlamydia Muridarum Intravaginal Infection With A Chlamydial Glycogen Phosphorylase

Urogenital tract infection with Chlamydia trachomatis is a leading cause of sexually transmitted bacterial infections (2,17,21). Antibiotics that can cross mammalian cell membrane are effective in treating chlamydial infection. However, due to the lack of obvious symptoms, many infected individuals don't seek treatment, potentially leading to complications characterized with inflammatory pathologies in the upper genital tract, including pelvic inflammatory diseases, ectopic pregnancy and infertility (10,19,21). Obviously, a most effective way to prevent C. trachomatis-induced complications is vaccination. However, there is still no licensed C. trachomatis vaccine (18). Nevertheless, the failed human trachoma vaccine trials more than50years ago (7) and the extensive immunological studies since then (11,12,18) have led to a conclusion that a subunit vaccine approach is both necessary and feasible for preventing C. trachomatis infection and C. trachomatis-induced diseases in the upper genital tract.The precise pathogenic mechanisms of C. trachomatis-induced diseases remain unclear although both intracellular replication of C. trachomatis organisms and host responses to C. trachomatis antigens may significantly contribute to inflammatory pathologies during C. trachomatis infection (3,4,22,26). Importantly, C. muridarum organisms deficient in a cryptic plasmid failed to induce pathologies in the upper genital tract while the wild type organisms were fully capable of doing so (3,14). Although C. muridarum organisms cause no known human diseases, the organisms have been extensively used to study immunobiology and search for vaccine antigens of C. trachomatis (3,4,11-13,16,18). Extensive immunological studies, largely based on a C. muridarum intravaginal infection mouse model, have revealed that a Thl-dominant cell-mediated immunity is required for protection against Chlamydia urogenital tract infection (11,12,18). The plasmid-free C. trachomatis organisms also displayed a reduced pathogenicity in mice (15). The cryptic plasmid not only encodes8open reading frames (ORFs) of its own but also transcriptionally regulates22genomic ORFs (1). It is thus hypothesized that both the plasmid-encoded and-regulated ORFs may contribute to chlamydial pathogenesis.In the current study, we used a C. muridarum intravaginal infection mouse model to identify chlamydial antigens that can induce protective immunity against chlamydial intravaginal infection. Of the7plasmid-regulated ORFs evaluated, the chlamydial glycogen phosphorylase (GlgP), when delivered into mice intramuscularly, induced the most pronounced protective immunity. GlgP immunization not only significantly reduced vaginal shedding of live organisms on day14after infection but also decreased severity of hydropsalpinx. The GlgP-induced protection was accompanied with robust C. muridarumspecific antibody and Th1-dominant T cell responses. These observations have demonstrated that the intramuscular immunization of mice with purified protein approach can be used to identify vaccine candidate antigens for preventing chlamydial infection in the lower genital tract and chlamydial diseases in the upper genital tract. Part1Plasmid clone, Prokaryotic Expression and Protein Purification of Mice Chlamydia Regulatory ProteinsObjective:Chlamydia cryptic plasmids encoding22regulatory proteins, which regulate genome encoding proteins, so study of the proteins regulated by cryptic plasmid encoding proteins, can providing experimental basis for identifying CT subunit vaccine candidate antigens. This study using prokaryotic expression to obtain seven cryptic plasmid regulated proteins, and using affinity chromatography for purification. Obtain of purified protein lay a foundation for following-up experiments.Methods:This section of the experiment using molecular biology methods, obtain seven gene sequences from the Genebank, design primers, with Cm Nigg strain genomic DNA as template, using PCR to obtain seven protein gene sequences, and then cloned into the prokaryotic expression vector pGEX-6p. Which then transformed into BL21, induced by IPTG for the expression. After expression, using AKTA purifier system for purification(GE), and the protein was quantified by Bradford method, finally obtained adequate amounts of seven purified target protein.Results:After sequencing, the seven completely correct regulatory protein gene sequence, successfully cloned into prokaryotic expression vector pGEX-6p. IPTG induced expression, and purified by affinity chromatography, finally obtained seven electrophoresis grade purity target proteins.Conclusion:Obtained7cryptic plasmid regulated proteins successfully. Part2The vaccine protective mechanism study of Plasmid regulatory proteinObjective:In the first section, we use the prokaryotic expression and affinity chromatography obtained seven purified plasmid regulatory protein. So in the current study, we used a C. muridarum intravaginal infection mouse model to identify chlamydial antigens that can induce protective immunity against chlamydial intravaginal infection, and identify which antigen can induce the strongest protective immunity.Methods:This part of experiment using C. muridarum Nigg strain (also called MoPn), organisms were grown, purified and titrated as usual methods, and infected into Hela cells to obtain large quantities of C. muridarum. All mice were immunized intramuscularly (i.m.), then chlamydia and antigen was given to each mouse simultaneously, and used CpG-IFA as an immune adjuvant. To detect chlamydial content in vaginal shedding epithelial cells, vaginal swabs were collected weekly for4weeks after intravaginal infection. Mice were sacrificed60days after infection and the mouse urogenital tract tissues were isolated for gross pathology evaluation, and score hydrosalpinx in naked eye.Results:The GlgP-immunized group developed significantly reduced live organism shedding from the lower genital tract. Meanwhile, GlgP mice are less severe hydrosalpinx.Conclusion:The IFU shedding of the GlgP-immunized mice group is significant low and less hydrosalpinx. Part3The Study of GlgP protein vaccine protective mechanismObjective:In the first two sections, we have obtained seven purified cryptic plasmid regulatory proteins, and seven proteins were intramuscularly (i.m.) immunized mice, glycogen phosphorylase (GlgP) induced the strongest protective immunity. Therefore, in this section, we mainly explore the protective immune mechanism of glycogen phosphorylase (GlgP) in vivo.Methods:To identify antibody subtypes in vivo, the glycogen phosphorylase (GlgP) immunized serum were measured by standard cytokine ELISA kits. After stimulation, splenocytes were harvested from each immunized mice group, and the cultured supernatants were collected for cytokine measurements. At the same time, in order to further analysis of GlgP biological characteristics and protection mechanism, we use Western-Blot to analyze the distribution of GlgP in chlamydia.Results:ELISA, immunofluorescence, as well as the result of different cytokines stimulated spleen cells from immunized mice, were all confirmed that the antibody subtypes induced by Glgp immunization are mainly IgG2a, its protection mechanism is Th1cellular immune response can be induced. GlgP distribution consistant with the structural protein MoMP, EB and RB of Chlamydia both have distribution, while in the infected cell cytoplasm does not exist, GlgP high abundance in Chlamydia may be associated with the induction of protective immune responses to infection.Conclusion:The GlgP can induce Th1dominant cellular immune response and using the C. muridarum intravaginal infection model can screen other immunodominant antigens.