Intramuscular Immunization of Mice with a Live-Attenuated Triple Mutant of Yersinia pestis CO92 Induces Robust Humoral and Cell-Mediated Immunity To Completely Protect Animals against Pneumonic Plague

Abstract

ABSTRACTEarlier, we showed that the ΔlppΔmsbBΔailtriple mutant ofYersinia pestisCO92 with deleted genes encoding Braun lipoprotein (Lpp), an acyltransferase (MsbB), and the attachment invasion locus (Ail), respectively, was avirulent in a mouse model of pneumonic plague. In this study, we further evaluated the immunogenic potential of the ΔlppΔmsbBΔailtriple mutant and its derivative by different routes of vaccination. Mice were immunized via the subcutaneous (s.c.) or the intramuscular (i.m.) route with two doses (2 × 106CFU/dose) of the above-mentioned triple mutant with 100% survivability of the animals. Upon subsequent pneumonic challenge with 70 to 92 50% lethal doses (LD50) of wild-type (WT) strain CO92, all of the mice survived when immunization occurred by the i.m. route. Since Ail has virulence and immunogenic potential, a mutated version of Ail devoid of its virulence properties was created, and the genetically modifiedailreplaced the nativeailgene on the chromosome of the ΔlppΔmsbBdouble mutant, creating a ΔlppΔmsbB::ailL2vaccine strain. This newly generated mutant was attenuated similarly to the ΔlppΔmsbBΔailtriple mutant when administered by the i.m. route and provided 100% protection to animals against subsequent pneumonic challenge. Not only were the two above-mentioned mutants cleared rapidly from the initial i.m. site of injection in animals with no histopathological lesions, the immunized mice did not exhibit any disease symptoms during immunization or after subsequent exposure to WT CO92. These two mutants triggered balanced Th1- and Th2-based antibody responses and cell-mediated immunity. A substantial increase in interleukin-17 (IL-17) from the T cells of vaccinated mice, a cytokine of the Th17 cells, further augmented their vaccine potential. Thus, the ΔlppΔmsbBΔailand ΔlppΔmsbB::ailL2mutants represent excellent vaccine candidates for plague, with the latter mutant still retaining Ail immunogenicity but with a much diminished virulence potential.