The long term objective of this study is to use a lipooligosaccharide (LOS)-derived oligosaccharide (OS) conserved epitope of Neisseria gonorrhoeae as a vaccine target by creating its protein surrogate image in the form of a chimeric (mouse/human) anti-idiotope monoclonal antibody (MAb) suitable for clinical use. In accordance with Jerne's """"""""network"""""""" theory, the primary antigen is the LOS of N. gonorrhoeae; the primary antibody, AB1, is directed against a LOS-derived OS epitope that is conserved and widely expressed in vivo; and the secondary antibody, AB2, is directed against an idiotypic determinant of AB1. We have produced an AB2 murine MAb using an AB1 murine MAb as immunogen. Preliminary results indicate that this murine AB2 antibody can elicit an AB3 humoral immune response in mice and rabbits against LOS, which is bactericidal to gonococci in the presence of complement. The present goal is to convert the AB2 antibody into a chimeric mouse/human antibody, and test its ability to elicit in experimental animals an anti-LOS antibody response that would be potentially protective in humans, as determined by in vitro correlates of protective immunity. furthermore, the proposed studies will test the hypothesis that the effectiveness of the anti-idiotope antibody can be improved by site-directed mutagenesis. The cDNAs encoding the variable regions of the murine AB2 antibody will be cloned, sequenced, and linked to human constant region genes by recombinant DNA techniques, to create vectors encoding a mouse/human AB2 chimeric antibody. The chimeric antibody, which will be expressed following transfection of hybridoma cells, is expected to generate a minimal anti- species antibody response if used in humans. the affinity of the AB2 chimeric antibody for the LOS-binding site of AB1 will be increased by oligonucleotide-directed low frequency random mutagenesis of its complementarity determining regions (CDRs). The native and engineered AB2 antibodies will be tested for their ability to elicit anti-LOS antibodies (AB3) in mice and rabbits, and human anti-LOS antibodies in Hu-PBL-SCID mice (severe combined immunodeficiency mice reconstituted with human peripheral blood leukocytes). As no animal model for gonococcal infection exists, the potential of the engineered AB2 antibodies as vaccine candidates against N. gonorrhoeae will be evaluated by in vitro testing of the anti-LOS AB3 antibodies elicited in the experimental animals, for mediation of opsonophagocytosis and complement killing of gonococci.
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