and Antigen Presentation and Diagnosis Development of Recombinant Overlapping Peptides Corresponding to ESAT-6/CFP-10.

Cellular immunity in () infection is important for the pathogenesis and final clearance of intracellular infection. In addition, it is valuable for the diagnosis of tuberculosis. In this pioneering work, we tested and antigen presentation and diagnostic application of a recombinant overlapping peptide-protein derived from two RD1 antigens ESAT-6 and CFP-10 (ROP-TB). The overlapping peptide sequence of ROP-TB is cleaved by the cathepsin S enzyme and covers the entire length of the two proteins. ROP-TB can be expressed and purified from . Once taken in by antigen-presenting cells, ROP-TB can be cleaved into a peptide pool by cathepsin S within the cells. We found that in dendritic cells, ROP-TB can be processed in 6 hours of co-culture, while the ESAT-6/CFP-10 fusion protein remained in the endosomal compartment. In -infected mice, ROP-TB stimulated stronger specific T cell responses than pooled synthetic peptides derived from ESAT-6 and CFP-10. With regard to the presentation of antigens, in a guinea pig model infected with , ROP-TB induced delayed type hypersensitivity (DTH) responses comparable to those of the tuberculin purified protein derivative (PPD) and ESAT-6/CFP-10 fusion protein. In -infected cattle, ROP-TB elicited DTH responses. Finally, in infected patients, ROP-TB stimulated cellular immune responses in majority of patients (16/18) of different HLA phenotypes while a single peptide derived from the same proteins did not elicit the immune responses in all patients. In summary, and data suggest that ROP-TB stimulates a strong cellular immune response irrespective of HLA phenotypes and is therefore suitable for use and diagnostics.