NEUTRALIZING EPITOPES OF CHLAMYDIA TRACHOMATIS

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The goal of this proposal is to identify and characterize continuous neutralizing epitopes of the major outer membrane protein (MOMP) of Chlamydia trachomatis. This organism is the leading cause of preventable blindness and sexually transmitted diseases, therefore protection through an effective vaccine would be desirable. Vaccine trials in the past which have employed the whole organism for immunization have failed, partly due to hypersensitivity reactions. An alternate approach to immunization is to engineer a subunit vaccine where only epitopes that elicit a protective host response are included. To identify such key epitopes MOMP was chosen as the focus of this investigation since it is surface exposed, has been shown to elicit neutralizing antibodies and DNA sequence data is available and thus the derived amino add sequence. More specifically variable domain (VD) IV will be the main focus of this proposal since antibodies that neutralize the B-,B-related and C-related complexes have been mapped to this region. However, MAbs that have mapped to this region fail to neutralize members of the C-complex Therefore, in addition to VD IV, VD I and VD 11 will be examined with C-complex serovars in order to be more successful in identifying continuous neutralizing epitopes that are broadly reactive and neutralize these members of the species. Peptides representing VD IV from serovars L2 and E and VD I, II and IV from serovar C will be used to immunize mice. Both the polyclonal sera as well as MAbs resulting from the immunizations will be examined for their ability to neutralize C. trachomatis. This will be accomplished by performing in vitro neutralization assays. In addition, the Geysen technique will be used to map recognition sites by ELISA using overlapping hexameric peptides to the VD used in the immunizations. The functional, i.e., neutralizable, site will be determined by competition experiments in which the neutralizing antibodies will be competed for by viable C. trachomatis and peptides to the region of interest. The spectrum of serovars neutralized by the antibodies will be established as well as the ability of the antibodies to act in an additive or synergistic manner. Mice will be immunized with peptide conjugates or passively immunized with MAbs that are identified as broadly neutralizing in terms of serovars. These animals will then be challenged with C. trachomatis in both a systemic as well as a mucosa] model of infection. In addition, studies aimed at determining the mechanism(s) of inhibition of the broad neutralizing MAbs will be performed. The work proposed in this investigation will provide structure-function information about a key structural protein of this organism and will be useful in the future design of vaccines to control the spread and growth of this important human pathogen.