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Analysis of Immune Responses Directed toward a Recombinant Early Secretory Antigenic Target Six-Kilodalton Protein-Culture Filtrate Protein 10 Fusion Protein in Mycobacterium bovis-Infected Cattle

Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, Missouri 65211,¹ U.S. Department of Agriculture, Agricultural Research Service, National Animal Disease Center, Bacterial Diseases of Livestock Research Unit, Ames, Iowa 50010,² Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164,³ College of Veterinary Medicine, Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, Iowa 50011,⁴ University of Texas Medical Branch, Department of Pediatrics and the Sealy Center for Vaccine Development, Galveston, Texas 77555⁵

Received 15 April 2005/ Returned for modification 13 June 2005/ Accepted 21 June 2005

Cell-mediated immune responses are critical for protective immunity to mycobacterial infections. Recent progress in defining mycobacterial antigens has determined that region of difference 1 (RD1) gene products induce strong T-cell responses, particularly the early secretory antigenic target 6-kDa (ESAT-6) protein and culture filtrate protein 10 (CFP10). However, comprehensive analysis of the immune response towards these antigens is incompletely characterized. To evaluate recall responses to ESAT-6 and CFP10, peripheral blood mononuclear cells from M. bovis-infected cattle were stimulated in vitro with a recombinant ESAT-6 (rESAT-6)-CFP10 fusion protein and compared to responses induced by M. bovis-derived purified protein derivative. Following antigenic stimulation, activation marker expression was evaluated. Significant proliferative responses (P < 0.05) were evident in CD4⁺, CD8⁺, immunoglobulin M-positive, and CD172a⁺ cell fractions after 6 days of culture. Expression of CD25 and CD26 was increased (P < 0.05) on CD4⁺, CD8⁺, and T-cell-receptor-positive cells. CD4⁺ and CD8⁺ cells also exhibited significant changes (P < 0.05) in expression of CD45 isoforms. Using a flow cytometry-based proliferation assay, it was determined that CD45R expression is downregulated (P < 0.05) and that CD45RO expression is upregulated (P < 0.05) on proliferating (i.e., activated) CD4⁺ cells. Collectively, data indicate that recall immune responses directed toward the rESAT-6-CFP10 fusion protein or purified protein derivative are comparable and that recall to mycobacterial antigens correlates with a CD45RO⁺ phenotype.

Editor: J. L. Flynn