Modulation of immune responses upon progesterone administration and vaccine potential of a 5-fluorouracil resistant mutant in a murine respiratory infection of Chlamydia muridarum

Immune responses to chlamydial infections are poorly understood due to the complex intracellular lifestyle of the bacterium and the lack of genetic manipulation tools. A clear understanding of the role of immune response to infection is mandatory for generation of a successful chlamydial vaccine. My project focuses on two aspects (i) study the effect of progesterone administration on immune responses to chlamydial infection in mice and (ii) test the vaccine potential of a metabolic mutant of C. muridarum..;Female, age matched, C57Bl/6 mice were injected subcutaneously with either one or two doses of Depo-Provera, a long acting progestational formulation, prior to infection with C. muridarum and immune response was studied to compare the dose effect on host susceptibility to infection. Immunity to chlamydial infection is dominated by a protective Th1 response typically characterized by the production of the cytokine, IFN-gamma. Hence, cytokine production in culture supernatants of antigen stimulated spleen and lymph node cells and lung homogenates was determined by sandwich ELISA. We further investigated the bacterial burden in the lungs by in vitro infectivity assays. Serum levels of anti-chlamydial antibody was quantitated to study the effect of the hormone on humoral immunity. In addition, histopathological changes in the lungs were examined to determine the severity of the disease.;IFN-gamma expression was significantly decreased, IL-10 production was increased and the mice suffered greater bacterial loads in the lung together with severe pathological changes in the lung. Taken together, these results indicate that progesterone pretreatment at higher doses alters the immune response to chlamydial infection. Our data suggest that progesterone has marked immunosuppressive effect when administered in higher doses in comparison to a single dose pretreatment. These results have important implications for the development of a successful chlamydial vaccine which also utilize progesterone pretreatment regimens for vaccine efficacy studies in mice.;In the second part of the study, a metabolic mutant of C. muridarum was analyzed for its potential as a vaccine. Chlamydiae are obligate intracellular parasites and hence are auxotrophic for three of the four ribonucleoside triphosphates. Among the few differences that exist between C. muridarum , a mouse pathogen and C. trachomatis, a human pathogen is that the former synthesizes an enzyme called Uracil phosphoribosyltransferase (UPRT) encoded by the upp gene that is necessary for the mouse pathogen to maintain its UTP pools. UPRT catalyzes the synthesis of Uracilmonophoshpate (UMP) and also catalyzes the formation of the cytotoxic 5-Fluorouracil monophosphate (5-FUMP) from 5-Fluorouracil (5-FU). Mutations in the upp gene were generated by growing the bacterium in the presence of increasing concentrations of 5-FU, in a mammalian cell line (CHO UrdC-) that is resistant to the toxic effects of 5-FU. Only Chlamydiae that have an inactivating mutation in the upp gene can survive the toxic effects of 5-FU. One such particular mutant that has a point mutation in its upp gene was isolated and was studied in vivo in a mouse respiratory infection model.;Progesterone, a steroidal sex hormone produced by the ovaries and the placenta, has major roles in the modulation of host immune responses both locally and systemically. This hormone has a well documented role in enhancing susceptibility of the host to the development of a wide range of infections by means of either immunosuppression or by tipping the balance towards a non-protective Th2 response. Mouse genital tract infection models of C. muridarum routinely employ progesterone pretreatment regimens to facilitate the establishment of infection.;Body weight loss, bacterial load in lungs, humoral and cell mediated immune responses and histopathological changes in the lungs of mice were studied in the WT verus upp mutant infection. Interestingly, the upp mutant was found to be significantly attenuated in mice with minimal damage to the host. On the other hand, the mutant grew equally well in the mammalian HeLa229 cell line. These results prompted us to study the vaccination efficiency of this mutant in the mouse respiratory model. For this purpose we immunized young female, BALB/c mice with a small dose of either the mutant or WT chlamydia and all mice were challenged with a high dose of WT C. muridarum following complete recovery from primary infection. Impressively, all mice that were challenged developed sterilizing immunity and exhibited resistance to reinfection. Collectively, these results indicate that mutations in the metabolic pathways of chlamydial species can attenuate the bacterium and therefore a live attenuated vaccine is likely an achievable goal in human chlamydial vaccine development.