Considerable evidence implicates Actinobacillus actinomycetemcomitans (Aa) as the etiologic agent of localized juvenile periodontitis (LJP). The goal of this application is to develop and test a fundamentally new and improved approach to identify Aa genes which are expressed during in vivo, but not in vitro, growth. Such genes are likely to be important to Aa's ability to cause LJP. The approach, called In Vivo Induced Antigen Technology (IVIAT), is superior to other related technologies because it does not rely on animal models to mimic the growth of the pathogen in humans. IVIAT uses antibodies present in pooled sera from LJP patients as probes to identify the genes of interest. This application has five specific aims. In the first specific aim, pooled antisera from 20 LJP patients will be exhaustively absorbed with in vitro-grown whole cells and cell extracts of Aa strain HK1651. The resulting serum will be used to probe two independent genomic expression libraries of HK1651 in Escherichia coli using colony blotting methods.
In Specific Aim 2, the cloned DNA inserts in reactive clones will be analyzed for IPTG inducibility and sequenced to determine open reading frames likely to be responsible for expression of the in vivo-induced antigens.
In Specific Aim 3, the open reading frames will be subcloned into an appropriate expression vector and at least 5 mg of the expressed protein will be purified by affinity chromatography. The purified proteins will be bound to a matrix support in Specific Aim 4 and used to affinity purify reactive antibodies from the absorbed LJP patient serum. These purified antibodies are used in Specific Aim 5 to definitively prove that the in vivo induced antigen is actually expressed by Aa during its growth in LJP patients. To accomplish this, plaque samples will be collected from first molars of 10 LJP patients. Aa cells in the plaque samples and their expression of the in vivo induced antigen will be demonstrated with differential fluorescence microscopy using a monoclonal antibody specific for Aa and the purified antibodies prepared in Specific Aim 4. The results of these studies are expected to improve our understanding of the pathogenic mechanisms employed by Aa in periodontal disease causation by identifying virulence-associated genes that would not be found by conventional microbiological or biochemical methods. The results should also validate IVIAT as a useful tool for a broader range of human pathogens. In vivo induced antigens discovered using this method are excellent candidates for consideration as potential diagnostic tools, new targets for antimicrobial therapy, or for vaccine design.
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