• Characterization of Schu S4 delta aroD as a putative vaccine candidate against pulmonary tularemia

      Cunningham, Aimee L.; Santiago, Araceli E.; Mann, Barbara J., Ph.D.; Qin, Aiping; Grassel, Christen; Vogel, Stefanie N.; Barry, Eileen M. (2015-03-09)
      Francisella tularensis is the etiological agent of the human disease tularemia and a Tier 1 Select Agent. There is a need for an efficacious vaccine against this pathogen due to its low infectious dose, high mortality rate, and ability to be spread by aerosol. To this end, we have engineered a defined live attenuated strain derived from the highly human virulent F. tularensis WT strain Schu S4, designated Schu S4ΔaroD; this strain lacks aroD, the 3rd enzyme in the chorismate biosynthesis pathway used for synthesis of aromatic amino acids. Schu S4ΔaroD is attenuated for growth in broth cultures and in both J774 and primary murine peritoneal macrophages, with a pulmonary LD50 > 105 CFU in C57BL/6 mice (compared to WT Schu S4 LD50 < 10 CFU). Intranasal immunization with Schu S4ΔaroD protects against high-dose WT pulmonary challenge in C57BL/6 mice. A single 50 CFU dose of Schu S4ΔaroD generated 80% protection against 100 CFU challenge, and addition of a 105 CFU booster to the initial 50 CFU vaccination is 80% and 40% protective against 500 or 1000 CFU challenge, respectively. Further studies revealed that a 103 or 105 CFU priming dose followed by a 108 CFU boost is 100% protective against 1000 CFU WT Schu S4 pulmonary challenge. This level of protection has not previously been demonstrated by any tularemia vaccine candidate. Bacterial burden following i.n. vaccination found that regardless of initial dose, immunized mice show identical bacterial loads (107 CFU/g tissue in the lung and 105 CFU/g tissue in the liver) at the peak of infection. However, time to peak burden was dose-dependent, as animals receiving a higher initial inoculum (at least 5600 CFU) reach this peak at day 3, as opposed to day 7 in lower initial inoculum groups. Peak bacterial burden correlates with maximal associated histopathology in the liver and peak pro-inflammatory cytokine and chemokine production in lungs, liver, and spleen (TNF-α, IL-1β, Cox-2, KC, iNOS, MCP-1). Our study shows Schu S4ΔaroD serves as an efficacious defined live attenuated vaccine against pulmonary tularemia. We have revealed a dose-dependent response to Schu S4ΔaroD, which may help determine the protective capacity of this vaccine and assist in development of vaccine-associated correlates of protection in the mouse model.
    • In vivo characterization of the murine intranasal model for immunologic assessment of Salmonella typhi vaccine and live vector strains and use of this model to assess an S. typhi live vector strain expressing a Plasmodium falciparum antigen

      Pickett, Thames Emily; Levine, Myron M. (Myron Max), 1944- (1999)
      Attenuated Salmonella typhi live vector vaccine strains are highly immunogenic in mice following intranasal, but not orogastric inoculation. To elucidate the relationship between organs within which vaccine organisms are found and the induction of specific serum IgG antibodies, we examined the in vivo distribution of S. typhi vaccine strain CVD 908-htrA following intranasal administration. Vaccine organisms were cultured from the nasal-associated lymphoid tissue (NALT), lungs, and Peyer's patches two minutes after intranasal inoculation. Vaccine organisms persisted longer in NALT than in other organs. By decreasing the volume of intranasal inoculum containing 109 CFU (from a single 30 mul or 10 mul dose to four 2.5 mul doses given over the course of one hour), we were able to significantly reduce the number of vaccine organisms isolated from the lungs (p < 0.05), without reducing the number of vaccine organisms in NALT. Reducing the number of vaccine organisms in the lungs resulted in a significant decrease in the serum tetanus antitoxin response elicited by CVD 908-htrA expressing tetanus toxin fragment C under the control of the redox-responsive nir15 promoter. In contrast, a similar construct expressing tetanus toxin fragment C under control of the constitutive lpp promoter stimulated a strong serum IgG tetanus antitoxin response with both inoculation regimens. The data suggest that following intranasal inoculation, the NALT is a sufficient inductive site for elicitation of an immune response against both the live vector and heterologous antigen and, as occurs following oral inoculation of humans, attenuated S. typhi vaccine organisms elicit serum IgG responses. The murine intranasal model was used to assess the immunogenicity of CVD 908-htrA expressing an antigen from Plasmodium falciparum, the etiological agent of human malaria. A truncated version of the circumsporozoite protein (tCSP) was expressed as a translational fusion to tetanus toxin fragment C, under the control of either the nir15 or the ompC promoter. Mice inoculated with both constructs mounted appreciable serum IgG responses against S. typhi LPS; serum IgG responses against the fusion protein were weak or non-existent. However mice inoculated with both constructs exhibited specific lymphoproliferative responses against the live vector, tetanus toxoid and CSP.