Recognition of mycobacterial epitopes by T cells across mammalian species and use of a program that predicts human HLA-DR binding peptides to predict bovine epitopes

Abstract

Bioinformatics tools have the potential to accelerate research into the design of vaccines and diagnostic tests by exploiting genome sequences. The aim of this study was to assess whether in silico analysis could be combined with in vitro screening methods to rapidly identify peptides that are immunogenic during Mycobacterium bovis infection of cattle. In the first instance the M. bovis-derived protein ESAT-6 was used as a model antigen to describe peptides containing T-cell epitopes that were frequently recognized across mammalian species, including natural hosts for tuberculosis (humans and cattle) and small-animal models of tuberculosis (mice and guinea pigs). Having demonstrated that some peptides could be recognized by T cells from a number of M. bovis-infected hosts, we tested whether a virtual-matrix-based human prediction program (ProPred) could identify peptides that were recognized by T cells from M. bovis-infected cattle. In this study, 73% of the experimentally defined peptides from 10 M. bovis antigens that were recognized by bovine T cells contained motifs predicted by ProPred. Finally, in validating this observation, we showed that three of five peptides from the mycobacterial antigen Rv3019c that were predicted to contain HLA-DR-restricted epitopes were recognized by T cells from M. bovis-infected cattle. The results obtained in this study support the approach of using bioinformatics to increase the efficiency of epitope screening and selection.

FIG. 1.

Identification of peptides from ESAT-6 recognized frequently by cattle and guinea pig T cells. (A) The ability of 11 ESAT-6-derived overlapping peptides to induce IFN-γ production by PBMC from 14 experimentally M. bovis-infected cattle was determined by ELISPOT assay. Results are expressed as a percentage (responder frequency) of animals producing IFN-γ (measured by ELISPOT assay) after peptide stimulation (tested at 20 μg/ml). Definition of responders: SFC with peptide − SFC without peptide ≥ 10, and SFC with peptide/SFC without peptide ≥ 2, where SFC is the number of spot-forming cells. The horizontal line indicates the cutoff for our definition of frequently (promiscuously) recognized peptides (50% responder frequency). (B) Proliferative responses of PBMC form three M. bovis-infected outbred guinea pigs to stimulation with ESAT-6-derived peptides (tested at 20 μg/ml). Positive response: SI (cpm with peptide/cpm without peptide) ≥ 2 (indicated by dashed horizontal line).

FIG. 2.

Hierarchy of ESAT-6 epitope recognition in humans, cattle, mice, and guinea pigs. The positions of the most frequently recognized immunodominant peptide determinants described are shown (2, 4, 20, 38, 44, 47). Presented are peptides recognized by PBMC from ≥50% of human TB patients and M. bovis-infected cattle tested, the majority of M. bovis-infected outbred guinea pigs tested, and of T cells from the mouse inbred strains with the H-2 haplotypes indicated that were infected with either M. bovis or M. tuberculosis.

FIG. 3.

Prediction of epitope recognized by bovine CD4⁺ T cells. (A) An ESAT-6 peptide 1 (residues 1 to 16) specific short-term T-cell line-derived from an M. bovis-infected calf was incubated with the overlapping peptides (tested at 20 μg/ml) indicated, and the production of IFN-γ after 4 days of culture was established. (B) The location of the experimentally defined epitope is shown in relation to the predicted sequence.

FIG. 4.

Identification of determinants from Rv3019c recognized by PBMC from M. bovis-infected cattle. The ability of five peptides (tested at 25 μg/ml) predicted to contain two to three ProPred motifs (underlined sequence regions) to induce IFN-γ production by PBMC from five experimentally M. bovis-infected cattle was determined by ELISPOT. Data are expressed as ΔSFC, calculated as SFC with peptide − SFC without peptide, where SFC is the number of spot-forming cells; each symbol represents responses of one animal to the indicated peptide. Cutoff for positive responses (mean ΔSFC plus 3 × standard deviation when noninfected animals were tested with peptides [shown as a vertical dotted line): ΔSFC > 15.