Synthetic peptides identify promiscuous human Th1 cell epitopes of the secreted mycobacterial antigen MPB70

Abstract

MPB70 is a secreted protein of Mycobacterium bovis and Mycobacterium tuberculosis which stimulates both cellular and humoral immune responses during infection with bovine and human tubercle bacilli. In addition, vaccination with MPB70 has been shown to induce Th1 cell responses and protection in animal models of tuberculosis. The present study was carried out to map the dominant human Th1 cell epitopes of MPB70 in relation to major histocompatibility complex (MHC) class II restriction in healthy subjects showing strong T-cell responses to complex mycobacterial antigens. Peripheral blood mononuclear cells (PBMC) from HLA-DR-typed donors were tested with complex mycobacterial antigens (whole-cell M. tuberculosis and M. tuberculosis culture filtrates), with MPB70 purified from the culture filtrate of M. bovis BCG Tokyo, and with 13 synthetic peptides (25-mers overlapping by 10 residues) covering the sequence of MPB70. The donors that responded to the complex antigens and MPB70 also responded to the cocktail of synthetic MPB70 peptides. Testing of PBMC with individual peptides showed that peptides p5 (amino acids [aa] 61 to 85), p6 (aa 76 to 100), p8 (aa 106 to 130), p12 (aa 166 to 190), and p13 (aa 181 to 193) were most frequently recognized in proliferation and gamma interferon (IFN-gamma) assays. Testing of antigen-specific CD4(+) T-cell lines with the individual peptides of MPB70 confirmed that peptides p8, p12, and p13 contain immunodominant Th1 cell epitopes of MPB70. MHC restriction analysis with HLA-typed donors showed that MPB70 and its immunodominant peptides were presented to T cells promiscuously. The T-cell lines responding to MPB70 and peptides p8, p12, and p13 in IFN-gamma assays mediated antigen-peptide-specific cytotoxic activity against monocytes/macrophages pulsed with the whole-protein antigen or the peptides. In conclusion, the promiscuous recognition of MPB70 and its immunodominant peptide defined epitopes (aa 106 to 130 and 166 to 193) by IFN-gamma-producing Th1 cells supports possible application of this secreted antigen to subunit vaccine design.

FIG. 1.

Thirteen 25-mer synthetic peptides covering the entire amino acid sequence of MPB70. The peptides overlap each other by 10 aa. The single-letter designations for amino acids are used.

FIG. 2.

Proliferation of PBMC of healthy donors in response to complex mycobacterial antigens, including ESAT-6, CFP-10, MPB70, and synthetic peptides of MPB70. Healthy donors (n = 14) were randomly chosen from the group of 34 positive responders to MPB70 shown in Table 1. Antigen- or peptide-induced proliferation of PBMC from each donor was determined as described in Materials and Methods. The results are expressed as the percentage of positive responders, which was defined as follows: [number of positive responders (SI, >3)/number of tested donors] × 100.

FIG. 3.

Inhibition of the proliferative responses of T-cell line SF1 in the presence of anti-HLA class II monoclonal antibodies. T-cell line SF1 was stimulated with MPB70, peptide p12, or peptide p13. Ab, antibody.