Testing a DNA vaccine for Aggregatibacter actinomycetemcomitans, an oral pathogen

Objective: A. actinomycetemcomitans (Aa), a gram-negative capnophilic bacterium, has been identified as a primary cause of Localized Aggressive Periodontitis (LAP). Infected individuals generate antibody and T-cell responses, but typically cannot clear the infection. Thus, enhancing adaptive immunity to Aa and other periodontal pathogens via a vaccine will likely require both humoral and cellular immunity. Thus, a DNA prime – protein boost approach is being tested. Background: We had identified Aa proteins that elicit T-cell responses in mice and are recognized by infected human peripheral blood lymphocytes; these include Omp34, CagE, GroEL and leukotoxin. Methods: CagE and Omp34 were amplified by PCR from Aa JP-2 DNA and cloned into a prokaryotic expression vector, pRSET, to produce protein vaccines. A two-step PCR was employed to add a Kozak sequence to the 5' end of each construct; they were subsequently cloned into pVAX-1, a eukaryotic expression vector, for use as DNA vaccines. C57Bl/6 mice were immunized with the DNA vaccines using a Helios gene gun (BioRad). Additional mice were inoculated with corresponding protein antigens in alum adjuvant. Mice were immunized and then bled at intervals thereafter; serum antibody levels were determined using ELISAs. In addition, T-cell proliferation assays were performed using lymph node T-cells harvested after either 1 or 4 exposures to antigen. Results: As expected, there was only minimal serum antibody to the antigens one week after primary immunization. After 3 immunizations with either the DNA or protein CagE vaccine, the antibody titers remained surprisingly low. ELISAs for the other antigens are currently underway. However, T-cells were activated by cagE DNA, or by either protein antigen, but not by the omp DNA construct. Conclusions: These preliminary results suggest that a prime-boost approach might be needed to elicit protective responses to Aa in C57Bl/6 mice. Supported by COSTAR training grant NIDCR T-32 DE14318.