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

Abstract

ABSTRACT 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 γδ 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.