The outer membrane of Gram-negative bacteria contains a """"""""heat- modifiable"""""""" protein called OmpA. OmpA (or its homolog) often plays a major role in the infection caused by Gram-negative pathogens. This proposal deals with the use of monoclonal antibodies (MAbs) to elucidate the antigenic determinants of OmpA from Salmonella typhimurium.
The specific aims of the project are to determine the immunochemical structure in OmpA, to evaluate the relative importance of epitopes localized to and C-terminal domains in immunoprotection, and to identify dominant epitopes that are the primary targets for immune recognition during Salmonella infections. We have already raised a large panel of anti-OmpA MAbs that appear to be directed against he majority, closed-channel conformer population of OmpA. We now propose to prepare new MAbs using the OmpA fraction enriched in the open-channel conformer population: The entire panel of MAbs will be used to analyze the immunochemical structure of OmpA by enzyme linked immunosorbent assays (ELISA), cytofluorimetry, and by Western immunoblot, and N-terminal sequence analysis of cyanogen bromide- generated peptides of OmpA. The relative importance of epitopes localized to and C-terminal domains will be evaluated by testing MAbs that recognize these two domains for their ability to passively protect mice against infection by S. typhimurium. Identification of dominant epitopes will involve the analysis of polyclonal immune sera by binding competition assays (ELISA and Western blots) with anti-MAbs. The effect of lipopolysaccharide structure on the specificity of anti-OmpA antibodies that arise during infection will be studied using smooth and rough variants of S. typhimurium. The long-term goals of this project is to determine the global structure of OmpA, and to elucidate the potential application of the immunodominant epitopes on this protein for insertion and expression of foreign genes.
