Selection of mycobacterium tuberculosis - Specific T-cell epitopes for immune diagnosis

M. tuberculosis (MTB) infection evokes a Type-1 immune response with Interferon-γ (IFN-γ) production, such that the detection of MTB-specific IFN-γ producing T-cells is a useful diagnostic strategy to measure cellular immune responses. However, a strong limitation in evaluating the efficacy of T-cell response-based assays is represented by the restriction of the host HLA haplotypes. In the present study we describe the details of the "reverse immune-genetics" approach to the identification of relevant peptide T-cell epitopes and we applied this technology to all gene products of the RD1 genomic region of MTB. The 11 RD1 proteins were analysed by the quantitative implemented HLA peptide-binding motifs analysis and 15 peptides belonging to 6 RD1 proteins were designed as being multiepitopic, HLA-class II promiscuous and MTB-specific. These peptides were tested by ELISpot technique. All RD1 selected peptides showed greater intensity of T-cell responses in active tuberculosis subjects [334.1 ±271.7 mean spots/million PBMCs] and PPD-positive MTB-exposed subjects [204.3 ±143.2] compared to PPD-negative, non-MTB-exposed and non-BCG-vaccinated [17.7 ±16.1; p < 0.05 all comparisons]. Our data suggest that the quantitative implemented peptide binding motifs analysis is able to select MTB-specific peptide epitopes and this approach reduces the screening of peptides of about 95%.