Immunisation of two rodent species with new live-attenuated mutants of CO92 induces protective long-term humoral- and cell-mediated immunity against pneumonic plague.

We showed recently that the live-attenuated Δ Δ Δ and Δ Δ mutants of CO92 provided short-term protection to mice against developing subsequent lethal pneumonic plague. These mutants were either deleted for genes encoding Braun lipoprotein (Lpp), an acetyltransferase (MsbB) and the attachment invasion locus (Ail) (Δ Δ Δ) or contained a modified version of the gene with diminished virulence (Δ Δ). Here, long-term immune responses were first examined after intramuscular immunisation of mice with the above-mentioned mutants, as well as the newly constructed Δ Δ Δ mutant, deleted for the plasminogen-activator protease () gene instead of . specific IgG levels peaked between day 35 and 56 in the mutantimmunised mice and were sustained until the last tested day 112. Splenic memory B cells peaked earlier (day 42) before declining in the Δ Δ mutant-immunised mice while being sustained for 63 days in the Δ Δ Δ and Δ Δ Δ mutant-immunised mice. Splenic CD4 T cells increased in all immunised mice by day 42 with differential cytokine production among the immunised groups. On day 120, immunised mice were exposed intranasally to wild-type (WT) CO92, and 80-100% survived pneumonic challenge. Mice immunised with the above-mentioned three mutants had increased innate as well as CD4 responses immediately after WT CO92 exposure, and coupled with sustained antibody production, indicated the role of both arms of the immune response in protection. Likewise, rats vaccinated with either Δ Δ Δ or the Δ Δ Δ mutant also developed long-term humoral and cell-mediated immune responses to provide 100% protection against developing pneumonic plague. On the basis of the attenuated phenotype, the Δ Δ Δ mutant was recently excluded from the Centers for Disease Control and Prevention select agent list.