Dr. Michael J. Brennan, Ph.D. has been a Senior Investigator in the Laboratory of Mycobacterial Diseases and Cellular Immunology, Center for Biologics Evaluation and Research, Food and Drug Administration since 1992. The research activities in this laboratory are related to regulatory responsibilities which include the approval of vaccines, therapies and skin test reagents for mycobacterial diseases including tuberculosis (TB). A major research focus is on the discovery of mycobacterial cell surface proteins which mediate infection of host cells and the potential use of these proteins as vaccine candidates for the prevention of TB. The laboratory has a devoted BL-3 facility to test vaccines in animal models for TB following aerosol delivery. A major finding has been the identification and characterization of the Heparin-Binding Hemagglutinin (HBHA) a surface protein found on Mycobacterium tuberculosis (Mtb) which mediates infection of host cells and promotes the dissemination of Mtb from the lung to other tissues. HBHA is presently being studied as a novel subunit vaccine for tuberculosis. An additional focus is on the characterization of proteins expressed by a unique family of genes (PE family) found only in mycobacteria. Investigation of gene expression, protein characterization and immunological response to PE proteins has suggested that certain members of this family play an important role in Mtb survival within host cells. Targeting these unique antigens using vaccines or therapeutics may disrupt disease-causing infections including tuberculosis. Characterization of the HBHA mycobacterial adhesin. A novel protein (HBHA)has been identified on the surface of mycobacteria including M. tuberculosis, the causative agent of TB, which appears to promote the interaction of mycobacteria with host cells. Methods have been developed at CBER for the purification of this unique protein. Evidence suggests that this protein undergoes a post-translational modification that effects both function and immunogenicity. Important binding sites and immunological epitopes have been identified and characterized. Specific regions that interculate into the bacterial membrane, that form coiled-coil initiated interactions, and that bind to proteoglycans have been identified. The role of this protein in attachment of mycobacteria to host tissues and in other bacterial interactions continues to be investigated in detail using Mabs developed at CBER against HBHA. Importantly, HBHA appears to be crucial for promoting the dissemination of Mtb from the lung to other tissues, a process that is blocked by anti-HBHA antibodies. New evidence indidates that HBHA subunit vaccines are very effective at protecting against tuberculosis in the mouse model of TB. The current focus is on developing efficient methods for the purification of immunologically potent recombinant HBHA for use as a TB vaccine and on characterizing the nature of the protective immune response to the subunit HBHA vaccine. Characterization of the Novel PE Gene Family of Mycobacteria. One of the major outcomes of the recent sequencing of the Mycobacterium tuberculosis (Mtb) genome was the identification of the multigene families designated PE_PGRS and PE. Since little is known about the expression or function of these genes, we have used the gene Rv1818c (PE_PGRS) and its amino-terminal PE region, as prototypes of the PE_PGRS and PE families, to investigate their function and the immunological response to these proteins during experimental tuberculosis. Research studies have provided evidence that PEPGRS proteins are cell surface constituents that have a role in Mtb infection of macrophages and in bacterial-bacterial interactions. PE_PGRS and a PE fragment have been expressed in Mycobacterium smegmatis and these strains demonstrate differences in cell phenotype and colony formation suggesting that they insert into the cell surface. These strains are being used to further characterize the function of these proteins. An M. avium strain which lacks PE_PGRS genes but has PE genes is being used to determine if PE genes alone are expressed. PE_PGRS proteins elicit an antibody response following infection and immunization with DNA vaccine constructs. Recombinant PE and PE_PGRS proteins have been expressed and purified and used in immunoassays to show that the dominant B cell epitope resides in the PGRS domain. Recent studies suggest that the antibody response to PE_PGRS varies over time in vivo. No antibody response to the PE domain has been observed. The PGRS domain also appears to regulate protein stability as determined by the use of GFP fusion proteins and there is some evidence to suggest it may effect antigen presentation of the PE domain and of other mycobacterial proteins. FWe are also investigating the role of PE_PGRS proteins in regulating proteosome dependent antigen presentation and the possibility that PE_PGRS proteins aid Mtb in evading host immune recognition. This project incorporates FY2002 projects 1Z01BJ006009-09 and 1Z01BJ006017-05.
