We have focused on the periodontal pathogen, Actinobacillus actinomycetemcomitans (Aa). Individuals infected with this bacterium generate a specific humoral immune responses; some of the antigens recognized by serum antibodies have already been identified. On the other hand, the T cell epitopes on Aa have not yet been defined. Towards this end, we have undertaken an innovative, T cell hybridoma-based approach in order to dissect the T cell responses to Aa. In preliminary mice were orally inoculated with live bacteria and a panel of T cell hybridomas was generated. To our surprise, approximately 50% of the T cells reactive with Aa were specific for leukotoxin, a virulence factor produced by this oral pathogen. In order to characterize the immune response to Aa further, we now propose to:
Aim 1. Clone genes that encode other T cell epitopes by direct screening of an Aa genomic library. The identities of the Aa proteins recognized by the T cell hybridomas will be determined and recombinant peptides generated for use in Aims 2 and 3.
Aim 2 : Characterize the nature of the immune response in vivo to individual Aa antigens. Mice will be: a) immunized with purified recombinant peptides, b) immunized with bacteria, or c) orally inoculated with viable Aa. Immune activation to individual T cell epitopes will then be assessed by studies of antibody production, T cell activation and cytokine production (Th1 versus Th2), and protection in a murine inflammation model.
Aim 3. Determine whether the predominant T cell antigens in mice are similarly stimulatory in Aa-infected patients. Specifically, peripheral blood lymphocytes from EOP patients will be cultured in vitro with individual recombinant Aa peptides and T cell stimulation assessed by cytokine production and by spectrotyping.
These aims will: i) provide the first evidence regarding T cell antigenic epitopes on this pathogen and ii) assess the relationship between T cell epitopes that are immunodominant in mice and those seen in humans. The long term goal is to develop and validate a model for evaluating host-parasite interactions, vaccine potency, and immune protection for periodontal diseases.
