HLA-Promiscuous Th1-Cell Reactivity of MPT64 (Rv1980c), a Major Secreted Antigen of <i>Mycobacterium tuberculosis</i>, in Healthy Subjects

Abstract

Objective: To determine HLA-promiscuous Th1-cell reactivity of MPT64 (Rv1980c) in healthy humans. Materials and Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from healthy subjects (n = 61) and HLA typed genomically. PBMCs were tested for Th1-cell reactivity (antigen-induced proliferation and interferon-γ secretion) with complex antigens (whole cells, culture filtrate and cell walls) and single secreted antigens (Ag85B, MPT64 and MPB70) of Mycobacterium tuberculosis. In addition, culture-filtrate-induced T-cell lines were established from PBMCs of 14 donors and tested with the above antigens in Th1-cell assays. Furthermore, 3 T-cell lines were tested for cytotoxic activity against MPT64-pulsed monocytes/macrophages, and a T-cell line was analysed for HLA restriction in antigen presentation using anti-HLA class I and class II monoclonal antibodies and HLA-DR-typed antigen-presenting cells. Results: PBMCs showed strong Th1-cell reactivity with all of the complex mycobacterial antigens, whereas MPT64 induced moderate Th1-cell reactivity, which was comparable to the reactivity induced by other previously characterized secreted antigens of M. tuberculosis, i.e. Ag85B and MBP70. Furthermore, HLA heterogeneity of the responding donors suggested that MPT64 was presented to Th1 cells promiscuously. Testing of the T-cell lines confirmed that Th1 cells contributed to the promiscuous antigen-specific reactivity observed with PBMCs and exhibited cytotoxic activity against MPT64-pulsed monocytes/macrophages. In addition, a T-cell line investigated for HLA restriction analysis showed that MPT64 was presented to T cells in association with HLA-DR molecules in a promiscuous manner. Conclusion: MPT64 is promiscuously recognized by human Th1 cells with cytotoxic activity and therefore deserves consideration as a candidate vaccine against tuberculosis in humans.

Polymorphism of Antigen MPT64 in Mycobacterium tuberculosis Strains

“…Biochemical, immunological, and molecular biological characterization of M. tuberculosis has led to the identification of several antigens that may be useful in the development of improved diagnostic methods and vaccines (2). MPT64 (Rv1980c), a 24-kDa protein of M. tuberculosis, is an important protein secreted by this pathogen (3,4). It is hypothesized that actively secreted proteins of M. tuberculosis are the first to interact with the host immune system, and therefore that such proteins are important for activating the immune response in individuals infected with M. tuberculosis.…”

Comparative Evaluation of MPT83 (Rv2873) for T Helper-1 Cell Reactivity and Identification of HLA-Promiscuous Peptides in Mycobacterium bovis BCG-Vaccinated Healthy Subjects

“…Although several computational methods are available for HLA-DR binding and the prediction of T cell epitopes, including ProPred, NetMHCII 2.2, Petrochemitric, and IEDB Consensus, etc. (24,56,62,67), ProPred was evaluated first because comparative evaluations have previously been shown the usefulness of this method in predicting mycobacterial T-cell epitopes (5,10,44,53). The overall results of computational analysis suggest that MPT83 was highly promiscuous for binding to HLA-DR molecules, as all of the HLA-DR specificities included in the computational methods were predicted to bind the MPT83 sequence (see Fig.…”

“…However, none of the single peptides qualified to replace MPT83 as the best Th1 cell-stimulating peptide, i.e., P14 (aa 196 to 220), although a promiscuous HLA-DR binder with strong Th1 cell responses showed negative responses for 22% of MPT83-responding donors in Th1 cell assays. Thus, this protein behaves like other major antigenic proteins of M. tuberculosis, e.g., Ag85B, PPE68, MPT70, ESAT-6, and CFP10, etc., in which no single peptide could replace the complete protein sequences for Th1 cell reactivity (5,10,44,47,53). However, being a major Th1 cell antigen capable of stimulating Th1 cells from HLA-DR heterogeneous subjects, this protein may be considered in formulations to develop a new vaccine candidate against TB.…”

“…At the time of blood collection, all the donors were PPD skin test positive (Ͼ10 mm, as determined with tuberculin PPD RT 23 from the Statens Serum Institute, Copenhagen, Denmark). PBMCs were isolated from the buffy coats of healthy donors by using standard procedures (9,44). In brief, each buffy coat was diluted with warm tissue culture medium (RPMI 1640) at a ratio of 1:2 and gently mixed.…”

“…Cellular proliferation results are presented below by using the stimulation index (SI), which is defined as follows: SI ϭ cpm in antigen-stimulated cultures/cpm in cultures without antigen. An SI of Ն2 was considered a positive proliferative response in response to a given antigen (6,44).…”