T-cell-epitope mapping of the major secreted mycobacterial antigen Ag85A in tuberculosis and leprosy

Abstract

Lymphoproliferation and gamma interferon (IFN-y) secretion in response to 28 overlapping 20-mer synthetic peptides covering the complete sequence of the mature (295-amino-acid) 85A component of the major secreted, fibronectin-binding antigen 85 complex from Mycobacterium tuberculosis and Mycobacterium bovis BCG (MTAg85A) was examined by using peripheral blood mononuclear cell (PBMC) cultures from healthy tuberculinand lepromin-positive volunteers and from patients with tuberculosis and leprosy. Peptide recognition was largely promiscuous, with a variety of human leukocyte antigen haplotypes reacting to the same peptides. PBMC from all tuberculin-positive subjects reacted to Ag85, and the majority proliferated in response to peptide 6 (amino acids 51 to 70), peptides 13, 14, and 15 (amino acids 121 to 160), or peptides 20 and 21 (amino acids 191 to 220). PBMC from tuberculosis patients demonstrated a variable reactivity to Ag85 and its peptides, and the strongest proliferation was observed against peptide 7 (amino acids 61 to 80). MTAg85A peptides were also recognized by PBMC from healthy lepromin-positive volunteers and paucibacillary leprosy patients (again in a promiscuous manner), but despite a 90%o homology between the 85A proteins ofM. leprae and M. tuberculosis, the peptides recognized were different. PBMC from lepromin-positive healthy contacts reacted against peptide 2 (amino acids 11 to 30), peptide 5 (amino acids 41 to 60), and peptides 25 and 26 (amino acids 241 to 270). PBMC from paucibacillary patients reacted preferentially against peptide 1 (amino acids 1 to 20) and peptide 5. Multibacillary patients were not reactive to Ag85 or the MT85A peptides. IFN-y production was generally detected simultaneously with positive lymphoproliferative responses, although peptide 1 mostly stimulated proliferation and peptides 27 and 28 mostly elicited an IFN-y response. In conclusion, regions 41 to 80 and 241 to 295 demonstrated powerful and promiscuous T-cell-stimulatory properties, resulting in proliferative responses and IFN-y secretion, respectively, in the majority of reactive

Epitope focus, clonal composition and Th1 phenotype of the human CD4 response to the secretory mycobacterial antigen Ag85

“…4 in [7], SI . 5 in [13]. Finally, discrepancies may also be attributed to the different peptide concentrations used in the in vitro assays, that ranged from 1 mg/ml in this study, to 10 mg/ml in [7], to 20 mg/ml in [13] and up to 250 m g/ml in [9].…”

Cutinase‐like proteins of Mycobacterium tuberculosis : characterization of their variable enzymatic functions and active site identification

“…Secreted gene products of M. tuberculosis are important for virulence and survival of the bacillus and pathogenesis of tuberculosis. For example, the mycolyl transferases of the Antigen 85 complex are critical for cell wall development (5) and are major targets of the immune responses in animal and human mycobacterial infections (43,44). The first secreted CULPs of M. tuberculosis to be identified were Culp1 and Culp2, which were present in short-term culture filtrate (30).…”

Mycobacterium tuberculosis Antigen 85A Induces Th-1 Immune Responses in Systemic Sarcoidosis

“…A unique pattern of recognition for MTB Ag85A peptides between subjects with tuberculosis and leprosy has also been described. Tuberculosis subjects are reported to recognize MTB Ag85A peptides 6, 13, 14, 15, 20, and 21; lepromatous patients recognize MTB Ag85A peptides 1, 2, 5, 6, 25, and 28 (13). It is thought that the dual recognition of MTB Ag85A observed between tuberculosis and leprosy patients is due to the close genetic homology of M. tuberculosis and M. leprae; the unique pattern of recognition reflects that they are distinct.…”

“…This complex also has been shown to induce strong CD4+ T cell responses during infection with MTB. In patients infected with MTB or M. leprae, MTB Antigen 85A (Ag85A) has been shown to induce strong T cell proliferative responses as well as generate the production of interferon-γ (13). Disruption of the genes encoding the three Ag85 components of MTB suggests that Ag85A may be the most essential component for bacterial survival within macrophages (14).…”