Fig 1.

Immunization and challenge protocols. (a) Immunogenicity study. BALB/c mice were primed i.d. with LVS ΔcapB at week 0 and boosted i.d. with rLm/iglC or with L. monocytogenes ΔactA (vector control) at week 4. Mice sham immunized with PBS or immunized once with LVS or LVS ΔcapB at week 0 and not subsequently boosted served as controls. At week 6, all mice were euthanized, their sera were assayed for antibody titer, and their splenic lymphocytes were assayed for antigen-specific production of IFN-γ and IL-17A or for cytokine-producing CD4⁺ or CD8⁺ T cells by multiparameter intracellular cytokine staining and flow cytometry. (b) Efficacy study. Mice were primed with LVS ΔcapB or rLVS ΔcapB/IglC at week 0 and boosted with rLm/iglC at week 4. Mice not immunized (not shown) or immunized once with LVS ΔcapB, rLVS ΔcapB/IglC, rLVS ΔcapB/IglA, or LVS at week 4 served as controls. At week 10, 6 weeks after the last or only immunization, such that the immunization-challenge interval was held constant, all mice were challenged with the F. tularensis subsp. tularensis Schu S4 strain by aerosol, after which the mice were monitored for weight loss, signs of illness, and survival for 3 weeks.