Immune Protect Responses Of Dendritic Cells Transfected With Recombinant Adenovirus Containing MOMP Gene Of Chlamydia Trachomatis Serovar E

Background:Chlamydia trachomatis (Ct) is the most prevalent sexually transmitted bacterial pathogen in the world, with an estimated 90 million clinically diagnosed cases occurring annually. Although antibiotic therapy appears to eliminate Ct infection effectively, asymptomatic infections are common (>50%) and when left untreated, can progress to or or result in chronic disease, including pelvic inflammatory disease, ectopic pregnancy, and infertility. More recently, genital Ct infection has been found to be highly associated with the increased risk for acquiring human immunodeficiency virus infection and the persistence of high-risk types of human papillomavirus infection. It is likely that the most effective form of intervention for diseases caused by Ct will be a vaccine.Early vaccine studies showed that whole inactivated bacterial cells administered intramuscularly were partially protective in human and primate trials, the protection was of short duration and was serovar specific, in some of the vaccinated individuals, a severe disease developed, this hypersensitivity reaction was thought to be due to a component present in the organism. Based on these observations, recent efforts have focused on developing a vaccine using the major outer membrane protein (MOMP) of Ct as the antigen. Ct is grouped to 19 human serovars, while serovar E is most prevalent one. For difficulty of the Ct culture and its less susceptible to mice, the research of serovar E Ct vaccine is limited. Purpose:1. Obtain the high titer and activity Ct; construct the mice model of Ct infected genital track; choose the proper Ct titer to estimate the vaccine.2. Estimate the effection of the dendritic cells transfected with recombinant adenovirus containing momp gene of Ct serovar E.3. Research the DC function after infected by Ct, so we can find the new approach to research the pathogenic mechanism of Ct.Research contents:Basing on our lab's research, we mainly do research the protect effection of this vaccine. Summarizing all the experiments, we do three part researches for estimate the competence vaccine.1. Culture of Ct serovar E and construction of the chlamydial genital tract infection mice modelCt serovar E was cultured in Hep-2 cells. First we used DEAE-dextran treat the Hep-2 cells, made it susceptible to Ct serovar E infection, then added the infectious sample(containing living Ct serovar E), and cultured for 2-3h; discarded the infectious sample, added cell culture liquid and cultured for 42-48h, and the chlamydial inclusion will was found in the cells. We collected the infected cells and sonicated to make the Ct elementary bodies free. The Ct elementary bodies were centrifuge and purified using discontinuous renograffin gradient.Next we constructed a mouse chlamydial genital tract infection model by vaginal inoculation of Ct serovar E, Our results show that, the vaginal inoculation of Ct serovar E results in an infection that naturally ascends from the lower genital tract (vagina and cervix) to upper genital tract tissues (uterine horns and oviducts). In our study, we subgroup inoculated with 10~7,10~6, 10~5, 10~4IFU Ct infect the mice ,10~6 IFU is the suitable to the mouse model of chlamydial genital tract infection, Pathologic findings include: The initial inflammatory response elicited by infection is characterized by a marked mucosal edema, genital tract hyperemia, infiltration of neutrophils. While the suitable titer to estimate the vaccine is the 10~5 IFU which is the smallest titer to infect the mice. 2. The immunity protection of dendritic cells transfected with recombinant adenovirus containing momp gene of Ct serovar EIn this part, we mainly do estimate the dendritic cell vaccine transfected with recombinant adenovirus containing momp gene of Ct serovar E. we did two part experiments:1) Our results demonstrate that DC transfected with Ad-MOMP secrete IL-12 and stimulate na(?)ve T cells to proliferate and secrete gamma interferon ex vivo, and elicit a Chlamydia-specific Th1-biased immune response and immunoglobulin A response.2) When the Ct serovar E infected the mice again in vivo, we found the Ad-MOMP-DC immunity mice could resist the Ct infect comparing with the Ad -DC immunity mice. The body weight loss of the Ad-MOMP-DC immunity mice was less than that of the Ad -DC group; Detecting the IFU of vaginal swab and the number of the Ct, we found the Ad-MOMP-DC group was obviously less than Ad -DC group; Observing the pathogical slide, we found the inflammation of the Ad-MOMP-DC group was less than Ad -DC group. Analyzing the results, we can safely conclude that the Ad-MOMP-DC group have the protect effection resisting the Ct infect again,3,C. muridarum infect Dendritic Cells and affect the function of Dendritic CellsApart finding the competence vaccine, we also work on finding the pathogenicmechanism of the serovar E. Thinking of the virulence of the serovar E Ct, we chose theChlamydia muridarum to do the follow two part experiments:1) We used the C. muridarum infect the DC induced from the mouse bone.C.muridarum can live in the DC slowly, Comparing to the inclusion forming in the Hep-2,the inclusion in the DC was atypical, It did not form the infection elementary body. Inthis part, we succeeded in constructing the cell model.2) Detecting the supernatant of the infected DC culture with ELISA, we found thatinfected DC can secrete IL-12 highly and promote proliferation of T cell, the resultssuggest that DC antigen present function isn't being obviously affected. So may be thereare others pathogenic mechanism or the function of infected DC is different in vivo. Weshould try to do a further research on this in the future. Conclusion:1. We have obtained the high activity serovar E Ct; we also have constructed the Ct infected genital track mice model successfully and we found the proper titer of the Ct to estimate the vaccine is 10~5IFU2. We can see the Ad-MOMP-DC vaccine have the protection immunity, this vaccine can resist the serovar E Ct infection again.3. In the thirth part, we can conclude that the DC infected by Ct can differentiate to DC1...