Immune profiling of leprosy and tuberculosis patients to 15-mer peptides of Mycobacterium leprae and M. tuberculosis GroES in a BCG vaccinated area: implications for development of vaccine and diagnostic reagents

Abstract

Mycobacterium leprae (ML) GroES has been shown to induce strong T cell responses in tuberculoid as well as in exposed healthy contacts of leprosy patients, and therefore this antigen has been the focus of study as a potential vaccine candidate. Paradoxically, we have shown that ML GroES also induces extremely high titres of IgG1 antibody in leprosy patients across the disease spectrum, a response associated with disease progression. IgG1 antibodies in leprosy also show a negative association with interferon-gamma, a critical T cell cytokine responsible for macrophage activation and intracellular killing of mycobacteria. We therefore queried if antibody and T cell responses were being evoked by different epitopes in ML GroES proteins. To address the issue of epitope recognition in mycobacterial diseases, we have analysed 16 peptides (15-mer peptides) spanning the entire ML and M. tuberculosis GroES protein in leprosy (n = 19) and tuberculosis (n = 9) patients and healthy endemic controls (n = 8). Our analysis demonstrates clearly that the dominant peptides evokingT cell and IgG subclass antibodies were different. The target of both T and B cell responses were cross-reactive epitopes in all groups. Differences in disease and healthy states related to the strength (mean intensity) of the T cell and antibody response. IgG1 and IgG3 antibodies were associated with disseminated disease and IgG 2 and IgG4 with disease limitation. Such comprehensive immune profiling of antigen-specific responses is critical to understanding the disease pathogenesis and also if these reagents are to be exploited for either diagnostic or vaccine purposes.

Figure 1

Amino acid sequence of MT and ML GroES proteins.

Figure 2

T cell (LTT, IFN-γ and IL-10) and B cell (IgG1–IgG4) antibody responses to the overlapping 15-mer peptides of ML GroES. Results are expressed as percentage giving a positive signal (determined as described in Materials and methods section). Solid lines indicate T cell responses and broken lines indicate B cell responses. Responses for leprosy patients [lepromatous (a) and tuberculoid leprosy (b)], healthy endemic controls (c) and pulmonary tuberculosis patients (d) are compared. The number in each group is shown in brackets next to the group. The symbol for each parameter is identical in the four panels. The x-axis shows the stimulating peptides. Prefix T/l before the peptides indicates that the peptide is shared with the M. tuberculosis (MT) GroES homologue. Prefix L indicates that this peptide has amino acid interchanges in the MT homologue.

Figure 3

Mean intensity of T cell responses (LTT, IFN-γ and IL-10) in donors giving a positive signal to the 15-mer peptides of ML GroES in the four study groups described in Figure 2. Results are expressed after deducting background cpm incorporation or spontaneous secretion of cytokines in the absence of stimulus. Different groups are shown with different symbols. All other details including the donors in each group are as described in Figure 2.

Figure 4

Mean intensity of IgG subclass antibody responses to the 15-mer peptides in each group. Results are given as mean of the four groups described after deducting background binding in the absence of antigen. Different groups are shown as boxes with different fillings, while the vertical line indicates 1 SE around the mean (1 SEM). All other details including the donors in each group are as described in Figure 2.