Identification by mass spectrometry of CD8(+)-T-cell Mycobacterium tuberculosis epitopes within the Rv0341 gene product

Abstract

Identification of Mycobacterium tuberculosis proteins that can provide immunological protection against tuberculosis is essential for the development of a more effective vaccine. To identify new vaccine targets, we have used immunoaffinity chromatography to isolate class I HLA-A*0201-peptide complexes from M. tuberculosis-infected cells and sequenced the isolated peptides by mass spectrometry. From this material, we have identified three peptides derived from a single M. tuberculosis protein that is encoded by the M. tuberculosis Rv0341 gene. Although no known protein encoded by the Rv0341 gene has been described, it is predicted to give rise to a 479-amino-acid protein with a molecular mass of 43.9 kDa. The three peptides identified are all nested and were found to be antigenic, in that they were capable of inducing peptide-specific, CD8(+) T cells from healthy blood donors in vitro and capable of recognizing and lysing M. tuberculosis-infected dendritic cells. This methodology provides a powerful tool for the identification of M. tuberculosis proteins that can be evaluated as potential vaccine candidates.

FIG. 1.

LC/MS/MS analysis of unfractionated MHC class I peptides isolated from M. tuberculosis-infected human macrophage cell line U937/A2. Shown are the total ion chromatogram of HLA-A*0201 purified peptides (top) and MS/MS spectra of Rv0341_33-45 (middle) and Rv0341_33-44 (bottom).

FIG. 2.

Comparative LC/MS analysis of unfractionated MHC class I peptides from uninfected and M. tuberculosis-infected U937/A2 macrophage cells. The similarity of the two samples is evidenced in the selected-ion chromatograms, as each peak in the infected-cell sample is present in the uninfected-cell sample, with the exception of Rv0341_33-45 (∗).

FIG. 3.

Peptide recognition by M. tuberculosis peptide-specific CTL. PBMC from healthy donors were stimulated in vitro with peptide-pulsed autologous DC. CTL lines were generated with peptides Rv0341_33-42 (a and b), Rv0341_33-44 (c and d), and Rv0341_33-45, (e and f). The cytolytic activity of the CD8⁺ T-cell lines was measured in a ⁵¹Cr release assay with either T2 cell (a, c, and e) or DC (b, d, and f) targets pulsed with either no peptide (○) or the peptide used for in vitro stimulation (•) or with cells infected with M. tuberculosis (▾).

FIG. 4.

Patterns of peptide recognition exhibited by M. tuberculosis peptide-generated CTL. The patterns of recognition by the CD8⁺ T-cell lines raised against M. tuberculosis peptides Rv0341_33-42 (a), Rv0341_33-44 (b), and Rv0341_33-45 (c) were measured in a ⁵¹Cr release assay. T2 target cells were either unpulsed (○) or pulsed with peptide Rv0341_33-42 (•), peptide Rv0341_33-44 (▾), or peptide Rv0341_33-45 (▪).