RD1 and RD9 Proteins and Peptides of Mycobacterium tuberculosis and Antibody Reactivity RD1 and RD9 Proteins and Peptides of Mycobacterium tuberculosis and Antibody Reactivity Hanif SNM1, Mustafa AS2 1Department of Basic Sciences, University of Pikeville, Kentucky, USA.; 2Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait Abstract Introduction: The major antigens encoded by Mycobacterium tuberculosis-specific genomic regions of differences (RDs) could be useful in the development of new vaccines and/or diagnostic reagents using T-cell and/or antibody assays. In particular, RD1 proteins PE35, PPE68, ESXA, and ESXB and RD9 protein ESXV and their peptides have been identified as major T-cell antigens. However, little is known about their antibody reactivities in different mammalian species. Aims: To determine the antigen-specific antibody reactivities to the above antigens and their peptides in three different mammalian species, i.e. rabbits, mice, and humans. Methods: Sera were obtained from i. rabbits immunized with purified recombinant proteins PE35, PPE68, ESXA, ESXB, and ESXV, ii. mice immunized with recombinant DNA vaccine constructs of pUMVC6 and pUMVC7 containing RD1 and RD9 genes, and iii. tuberculosis patients and healthy humans. Enzyme-linked immunosorbent assays (ELISAs) were performed with the sera to determine the antibody reactivity to purified recombinant proteins, peptide pools, and individual peptides of RD1 and RD9 proteins. Results: The ELISA results with sera from rabbits immunized with pure recombinant proteins showed positive antibody reactivity with all of the immunizing proteins and their synthetic peptide pools. Testing of the sera with individual peptides showed positive antibody reactivity with PE35 peptides P1 (aa 1-25), P2 (aa 16-40), P5 (aa 61-85) and P6 (aa 76-99); PPE68 peptides P9 (aa 121-145), P11 (aa 151-175), P14 (aa 196-220), P22 (aa 316-340), P23 (aa 331-355) and P24 (aa 346-371); all peptides (P1 to P6) of ESXA and ESXB, and ESXV peptides P1 (aa 1-25), P2 (aa 16-40), P3 (aa 31-55), P5 (aa 61-85) and P6 (aa 76-94). The sera from mice immunized with DNA vaccine constructs showed antibody reactivity to all proteins and the peptide P6 (aa 76-99) of PE35 and peptides P19 (aa 271-295) and P24 (aa 346-371) of PPE68. In humans, the peptides P11 (aa 151-175), P14 (aa 196-220), P22 (aa 316-340), P23 (aa 331-355) and P24 (aa 346-371) of PPE68 and the peptides P4 (aa 46-70) P5 (aa 61-85) and P6 (aa 76-94) of ESXV showed positive reactivity with sera from tuberculosis patients and healthy subjects. Conclusions: The results demonstrate the presence of several antibody epitopes in each protein, but variations in the epitopes recognized were observed among mice, rabbits, and humans, which could be due to mammalian species differences and/or mode of antigen delivery.