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Major Research Project RP008

Collaborating Institution: University of Texas Southwestern Medical Center, Dallas, TX

Principal Investigator: Michael Norgard, PhD

Title of the Project: Outer Membrane Proteins of Francisella tularensis as Acellular Vaccines

Expected Product: Development of new vaccines for tularemia.

Description: Outer membrane proteins (OMPs) and lipoproteins (LPs) of gram-negative bacteria are of enormous importance as virulence factors, stabilizers of outer membrane integrity, and protective immune targets (vaccines). To date, little is known regarding the OMPs of Francisella tularensis (Type A) strains that are pathogenic for humans (e.g., F. tularensis SCHU S4). However, selected OMPs could prove invaluable as subunit vaccines for tularemia. To this end, we have achieved two important milestones, allowing for the first time, the investigation of the OMPs as acellular vaccines for tularemia.

First, we developed a sucrose density gradient method for the physical separation of the outer and cytoplasmic membranes of F. tularensis (JF Huntley et al., 2007, J. Bacteriol. 189:561). This procedure has allowed us to investigate the OMP components of this important Category A select agent without confounding issues of protein contaminants from other cellular compartments (i.e., inner membrane and cytoplasm) (Aim 1). Second, we have demonstrated that OMP-immunized mice (using native OMPs contained within lysis-generated outer membrane vesicles [OMVs]) are 50% protected against intranasal challenge with SCHU S4 using Freund’s adjuvant (JF Huntley et al., 2008, Infect. Immun. 76:3664). Herein, we now report 80-100% efficacy using this same material in the human-relevant ISCOM-CpG adjuvant. Significant efficacy was also observed (i.e., 75-88%) following vaccination of mice with the OMV-ISCOM-CpG mixture by the intramuscular or subcutaneous routes, both of which are amenable to future human vaccine studies (Aim 2). We also are exploring methods to ensure sterility of the OMV preparations that will not compromise efficacy of the vaccine formulations. Lastly, we have begun to explore the humoral and cellular aspects of protective immunity induced by OMP vaccines (Aim 3). Vaccine-induced immunity correlated with potent humoral and cellular responses in mice, including the induction of triple-positive (IFN-γ, TNF-α, IL-2) CD4+ T-cells that are thought to both be optimal for effector function and exhibit long-term memory potential. The combined results have provided important new knowledge for understanding F. tularensis pathogenesis and for devising new acellular vaccines for tularemia.