A Q fever vaccine remains an important public health and national security goal. The objective of this application is to clone the O antigen epitopes from C. burnetii lipopolysaccharide (LPS) expressed on a heterologous LPS lipid A and core or N-linked glycoprotein, and combine this with protective T cellstimulating recombinant proteins in Recombinant Attenuated Salmonella Vaccines (RASV). The vaccine candidates will be characterized for the protective capacity of recombinant antigens that confer strong T-cell responses in vaccine-induced and infection-derived immunity in BL6 and MHC ll-humanized (DR1 or DR4) mice and outbred guinea pigs. We will accomplish the objective by pursuing the following specific aims: (1) Identify and characterize genes required forsubunit LPS O-antigen expression. Protective carbohydrate epitopes on the O-side chain of C. burnetii LPS can be expressed on a heterologous lipid A-core acceptor in Salmonella by cloning essential genes encoding the biosynthetic pathways for O antigen synthesis. Selection for recombinant O antigen carbohydrate synthesis will include phage resistance, LPS-specific antibody reactivity, and direct cloning of predicted genes for O antigen synthesis. (2) Heterologously express O polysaccharide as an N-linked glycoprotein. Protective carbohydrate epitopes on the O-side chain of C. burnetii LPS will be expressed as an N-linked glycoprotein using the transferase (PgIB) and acceptor (AcrA) proteins from Campylobacterjejuni. The coupled O side chain carbohydrates will be purified, and structurally and antigenically characterized. A C. burnetii antigenic protein (Com-1) will be modified with an acceptor motif to replace AcrA and evaluated for the ability to act as an antigenic N-linked carrier protein. (3) Identify and characterize mimetope peptides that antigenically model O-polysaccharide. Combinatorial peptide libraries will be screened for targets that cross react with antibody specific for C. burnetii O polysaccharide. These peptides will be tested for protective antibody responses upon vaccination by recognizing C. burnetii LPS. Candidate peptides will be conjugated to KLH or TT carrier protein and used to stimulate antibody responses in mice. (4) Evaluate the protective capacity of heterologously expressed C. burnetii Oside chain carbohydrates and proteins in mouse and guinea pig challenge models. Live RASV, microvesicles prepared from RASV, and purified recombinant proteins conjugated to heterologous LPS will be compared.
Vaccine development is a primary focus of the RCE system and the WRCE. C. burnetii is an understudied Select Agent in the RCE system, and the WRCE provides important coverage of the agent in meeting the goals outlined by NIH. Support for the development of a relevant animal model (guinea pig aerosol) in the first funding cycle translated into a tool used in this renewal application for vaccine development. Ongoing, separately funded studies to identify T-cell protein epitopes, both CD4 (NIH, RO1 Samuel, PI) and CD8 (PSRCE, Peters PI, Samuel subcontract), will be used to support the ultimate protein partner choices for combination with LPS epitopes.
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