L. plantarum expressing the target antigen was cultured in LM medium supplemented with 10 mg/ml Cm, and grown at 30uC to an OD 600 of 1.0. That is the equivalent of 1610 9 cells/ml corresponding to approximately 125 mg of total protein. The cells were harvested by centrifugation at 3000 g for 10 min at 4uC and resuspended in 20% glycerol/phosphate buffered salt solution (Gibco, Grand Island, NY) in 1% of the initial volume. Cell suspensions in aliquots of 2 ml were frozen quickly in a dry ice bath and stored at 280uC. Aliquots were thawed at 4uC and 400 ml (4610 10 cells) were placed in a ball-tipped syringe for oral gavage inoculation. Groups of four female C3H-HeJ mice (6-8 weeks old, Charles River, Boston, MA) were immunized by intragastric inoculation of 4610 10 Lactobacillus expressing OspA recombinant antigens. L. plantarum (Lp) was used as control. Mice received the first immunization, twice daily, for 8 days (days 1-4 and 8-11). After resting for two weeks the mice were bled (day 28). On days 29-32 they received the 1 st oral boost and rested for an additional 2 weeks. On day 49, the mice were bled. On days 50-53 they received the 2 nd oral boost and rested for an additional 2 weeks. On day 68 mice were terminated, and blood, stool and bronchoalveolar lavage fluids (BAL) were collected. The animals were housed in a pathogen free colony on hardwood chip bedding in microisolator cages at the University of Tennessee Health Science Center, Memphis, TN. They were maintained on a 12 h light-dark cycle, in a room kept al 23uC with 50-60% relative humidity; they were given tap water and sterile irradiated rodent chow ad libitum.