Immunogenicity and protective immunity induced by attenuated recombinant L. monocytogenes vaccines. A. Immunogenicity. Nine groups of 3 BALB/c mice were sham-vaccinated, or vaccinated twice at weeks 0 and 5 with 1 × 106 CFU of the parental recombinant L. monocytogenes vector (rLmΔactA) or the rLm vaccines expressing various F. tularensis antigens as indicated on the horizontal scale of the graph. Mice vaccinated twice with 5 × 104 CFU LVS served as positive controls. At week 9, mice were euthanized, a single cell suspension of splenocytes was prepared and incubated with the F. tularensis antigen (10 μg/ml) indicated on the horizontal scale of the graphs, and the culture then pulsed with 0.25 μCi of [3H]thymidine/ml for 2 h. The mean CPM varied from group to group, ranging from 90 to 380 in the absence of the F. tularensis protein and from 350 to 1500 in the presence of F. tularensis protein. The Stimulation Index (SI) was determined by dividing the mean CPM in the presence of the F. tularensis protein by the mean CPM in the absence of the F. tularensis protein. Values are the mean ± SE of the SI. The asterisks indicate that the difference in SI between the mice in a control group and the mice in the rLm/iglC-vaccinated group was significant. *, P < 0.05 and **, P < 0.01 by student t-test. B. Protective Immunity. Ten groups of 8 mice were immunized as described in A. At week 9, mice were anesthetized with Isoflurane, challenged i.n. with 1 × 106 CFU LVS (30 × LD50s), and monitored for illness and survival for three weeks. The differences in survival between the mice in the vaccinated groups and mice in the sham-vaccinated group were evaluated using a logrank analysis. ns, not significant (P > 0.05). Mean survival time (MST) was calculated by dividing the sum of the survival times of all mice in a group by the total number of mice challenged, with surviving animals given a survival time of 21 days, when the experiment was terminated.