T-CELL-EPITOPE MAPPING OF THE MAJOR SECRETED MYCOBACTERIAL ANTIGEN AG85A IN TUBERCULOSIS AND LEPROSY

Lymphoproliferation and gamma interferon (IFN-gamma) secretion in resp onse to 28 overlapping 20-mer synthetic peptides covering the complete sequence of the mature (295-amino-acid) 85A component of the major se creted, fibronectin-binding antigen 85 complex from Mycobacterium tube rculosis and Mycobacterium bovis BCG (MTAg85A) was examined by using p eripheral blood mononuclear cell (PBMC) cultures from healthy tubercul in- and lepromin-positive volunteers and from patients with tuberculos is and leprosy. Peptide recognition was largely promiscuous, with a va riety of human leukocyte antigen haplotypes reacting to the same pepti des. PBMC from all tuberculin-positive subjects reacted to Ag85, and t he majority proliferated in response to peptide 6 (amino acids 51 to 7 0), peptides 13, 14, and 15 (amino acids 121 to 160), or peptides 20 a nd 21 (amino acids 191 to 220). PBMC from tuberculosis patients demons trated a variable reactivity to Ag85 and its peptides, and the stronge st 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 pr omiscuous manner), but despite a 90% homology between the 85A proteins of M. leprae and M. tuberculosis, the peptides recognized were differ ent. PBMC from lepromin-positive healthy contacts reacted against pept ide 2 (amino acids 11 to 30), peptide 5 (amino acids 41 to 60), and pe ptides 25 and 26 (amino acids 241 to 270). PBMC from paucibacillary pa tients reacted preferentially against peptide 1 (amino acids 1 to 20) and peptide 5. Multibacillary patients were not reactive to Ag85 or th e MT85A peptides. IFN-gamma production was generally detected simultan eously with positive lymphoproliferative responses, although peptide 1 mostly stimulated proliferation and peptides 27 and 28 mostly elicite d anIFN-y response. In conclusion, regions 41 to 80 and 241 to 295 dem onstrated powerful and promiscuous T-cell-stimulatory properties, resu lting in proliferative responses and IFN-gamma secretion, respectively , in the majority of reactive subjects tested in this study. These res ults could be of value in the development of a subunit vaccine for tub erculosis and leprosy.