During the course of this Program Project, we have demonstrated that roughly half of patients with early-onset periodontitis are seronegative for antigens of putative periodontal pathogens, functionality of antibodies that are produced is unexpectedly low, and periodontal treatment results in seroconversion and production of biologically more effective antibodies. These observations, combined with our demonstration that immunization of M. fascicularis with a vaccine containing P. gingivalis significantly suppresses alveolar bone loss, has led us to propose to continue characterization of major cell envelope antigens of selected periodontopathic bacteria both biochemically and immunologically, and to continue development of a vaccine for human periodontal infections. In the upcoming grant period we will focus our efforts on the cell envelope antigens of A. actinomycetemcomitans, P. gingivalis, and B. forsythus, and on antigens shared by these species. We will complete characterization of the carbohydrate antigen of A. actinomycetemcomitans, and assess its potential for use in a vaccine. We will complete our ongoing efforts to purify and characterize the 200 kDa cell envelope protein antigen from B. forsythus. This preparation and the 43 kDa, 53 kDa, and 67 kDa envelope proteins and cysteine protease from P. gingivalis will be evaluated for LPS contamination using a highly sensitive fluorescence probe method, and contaminants removed chromatographically using HPLC and by isoelectric focusing. LPS from P. gingivalis and B. forsythus will be purified and separated into high and low molecular weight fractions. Purity and homogeneity of protein and carbohydrate antigens will be documented. These antigens will be used for measurement of antibody levels in patients before and following therapy, and provided to investigators in Project IV. Monoclonal antibodies will be generated in mice for LPS epitopes shared by P. gingivalis and B. forsythus for development and testing of anti-idiotype vaccines. A similar approach will be taken for the oligosaccharide antigen of A. actinomycetemcomitans. Protein antigens will be used to immunize rabbits. The resulting antibodies, as well as antibodies in sera of monkeys immunized with the whole-cell P. gingivalis vaccine, will be extensively studied with the aim of identifying the antigen(s) with the most potential for inducing protective immunity in the monkey model. Measurements will include titer, avidity, enhancement of chemiluminescence and phagocytosis and killing, and blocking activity. Successful completion of this project will provide the fundamental information and purified antigens necessary for development of a vaccine against periodontal infections.