The interaction of complement and antibody in the in vitro neutralization of infectivity of Chlamydia trachomatis serovar L2

• Main Author: Hall, Raymond Thomas
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/5340

Chiamydia trachomatis is an obligate intracellular human pathogen responsible for important diseases, including trachoma, the leading cause of preventable blindness, infecting more than 500 million people worldwide. It is also the etiologic agent of a sexually transmitted disease causing serious sequelae such as salpingitis, leading to ectopic pregnancy or infertility. Chiamydial infectivity of tissue culture cells can be neutralized by anti-chiamydial antibody, however, the presence of complement with antibody results in maximal neutralization. This thesis investigated the interaction of complement and anti chiamydial antibody, and sought to provide a mechanism for the enhancement effect in complement-dependent neutralization of chiamydial infectivity. Three approaches were used to study this problem: 1) flow cytometry was optimized for detection of purified chiamydial elementary bodies, and in situ binding experiments were performed; 2) outer membrane protein-complement C3b complexes were extracted and immunoblotted in order to study in situ binding qualitatively; and 3) in vitro neutralization assays were used to determine the step in the complement cascade at which antibody interacts with complement in mediating neutralization of infectivity. The flow cytometry experiments tested the hypothesis that antibody affects the quantity of C3b bound to the surface of whole elementary bodies in situ. The results indicated that antibody did not augment either the rate or magnitude of C3b binding, and that complement was fixed predominantly via the alternative pathway. Immunoblotting of outer membrane protein-C3b complexes tested the hypothesis that antibody was determining the specific C3 target proteins on the chiamydial cell surface. However, the results showed that the major outer membrane protein was the primary C3b target protein in either the presence or absence of antibody. Immunoblotting experiments were repeated with outer membrane protein-C3b complexes treated with hydroxylamine. C3b appeared to be bound by hydroxyl esters, not amino esters, to the outer membrane proteins, and this was unchanged by antibody. The final set of experiments utilized in vitro neutralization assays. The results demonstrated that antibody must be present before the formation of C5 convertase, and that if antibody was added at later stages of activation of the complement cascade, neutralization did not occur. The data also showed that neutralization occurred via activation of the alternative complement pathway, and infectivity was not neutralized when the classical pathway alone was isolated. Interestingly, neutralization occurred, although to a lesser degree, when the terminal complement components C7 and C8 were missing, suggesting that terminal components are not essential for neutralization. Flow cytometry binding experiments were repeated to measure the effect of anti-chlamydial antibody on in situ binding of terminal components C9 and C5b-9 neoantigen. The results indicated that the presence of antibody significantly increased C9 and, to a lesser extent, C5b-9 neoantigen binding. In conclusion, the experiments in this thesis demonstrated that antibody mediated complement-dependent neutralization of C. trachomatis serovar L2 at the stage of alternative pathway C5 convertase formation. Anti-chlamydial antibody probably configures the C3b molecules in specific locations on the major outer membrane protein; however, the binding experiments were unable to detect any augmentation of C3b binding by antibody. Neutralization may occur as a result of the covalently bound complement complex on the major outer membrane protein inhibiting reorganization of the EB into an RB. The role of terminal complement components in neutralization is unclear. These data showed an increase in terminal components bound in the presence of antibody, yet neutralization occurred when C7 and C8 were excluded. It is likely that there are several mechanisms of complement-dependent neutralization, some requiring the terminal components, and others not.