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Developmental Research Project DP012

Collaborating Institution: University of Florida, Gainsville, FL

Principal Investigator: Anthony Barbet, PhD

Title of the Project: Type 4 Secretion System for Vaccination against the Rickettsiales

Expected Product:  Development of a vaccine against the Type 4 Secretion System of Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis, that can be used against other bacteria in the order Rickettsiales.

Description: Immunization against organisms in the order Rickettsiales has been problematic because of strain or antigenic variation in immunodominant outer membrane proteins. It has been proposed that better vaccine targets would be subdominant, conserved proteins that have not been targets of immune selection. A possible target is the Type 4 Secretion System (T4SS) which is required to form membrane channels enabling protein secretion and for survival of intracellular bacteria. Although all Rickettsiales have a T4SS, this possibility has been most extensively investigated in the bovine pathogen Anaplasma marginale. In A. marginale, cattle vaccinated with outer membranes are protected against infection and respond strongly to membrane protein components. These responses are made against immunodominant, antigenically variable proteins as well as against VirB9 and VirB10 components of the T4SS. Unlike natural infections, outer membrane protein vaccination induces strong IgG2 responses in cattle against VirB9 and VirB10 as well as specific CD4+ T-cell proliferation and gamma interferon secretion. In the Rickettsiales there is evidence that VirB9 may be surface-exposed on outer membranes. Also, we have recently completed a comparative genomics study of seven strains of A. phagocytophilum that shows VirB9 and VirB10 are conserved between strains, unlike other T4SS components such as VirB2 and VirB6. There are two VirB9 paralogs, VirB9-1 and VirB9-2, both having strongly predicted signal peptides; of all the potentially exposed components of the T4SS theyare the least diverse among strains. In this project, we propose to test VirB9 and VirB10 as vaccines against A. phagocytophilum in a mouse model of infection. Recombinant VirB9 and VirB10 proteins will be prepared and used to immunize C3H mice. Antibody, T cell, and cytokine responses of mice will be determined, and then the mice will be challenged with A. phagocytophilum and the course of infection will be followed by real-time PCR. Protection against infection in terms of reduced bacteremia, length of bacterial persistence and pathology will be determined. A demonstration of successful vaccination against T4SS components would have significance for future vaccine development against all members of the Rickettsiales.