Gamma interferon responses induced by a panel of recombinant and purified mycobacterial antigens in healthy, non-mycobacterium bovis BCG-vaccinated Malawian young adults

Abstract

We have previously shown that young adults living in a rural area of northern Malawi showed greater gamma interferon (IFN-gamma) responses to purified protein derivatives (PPD) prepared from environmental mycobacteria than to PPD from Mycobacterium tuberculosis. In order to define the mycobacterial species to which individuals living in a rural African population have been exposed and sensitized, we tested T-cell recognition of recombinant and purified antigens from M. tuberculosis (38 kDa, MPT64, and ESAT-6), M. bovis (MPB70), M. bovis BCG (Ag85), and M. leprae (65 kDa, 35 kDa, and 18 kDa) in >600 non-M. bovis BCG-vaccinated young adults in the Karonga District of northern Malawi. IFN-gamma was measured by enzyme-linked immunosorbent assay (ELISA) in day 6 supernatants of diluted whole-blood cultures. The recombinant M. leprae 35-kDa and 18-kDa and purified native M. bovis BCG Ag85 antigens induced the highest percentages of responders, though both leprosy and bovine tuberculosis are now rare in this population. The M. tuberculosis antigens ESAT-6 and MPT64 and the M. bovis antigen MPB70 induced the lowest percentages of responders. One of the subjects subsequently developed extrapulmonary tuberculosis; this individual had a 15-mm-diameter reaction to the Mantoux test and responded to M. tuberculosis PPD, Ag85, MPT64, and ESAT-6 but not to any of the leprosy antigens. We conclude that in this rural African population, exposure to M. tuberculosis or M. bovis is much less frequent than exposure to environmental mycobacteria such as M. avium, which have antigens homologous to the M. leprae 35-kDa and 18-kDa antigens. M. tuberculosis ESAT-6 showed the strongest association with the size of the Mantoux skin test induration, suggesting that among the three M. tuberculosis antigens tested it provided the best indication of exposure to, or infection with, M. tuberculosis.

FIG. 1.

Frequency of IFN-γ responders to the panel of mycobacterial antigens. Whole-blood assays were stimulated with the recombinant and purified antigens at final concentrations of 10 μg/ml, and IFN-γ was measured in day 6 supernatants by ELISA. A responder was defined as a subject making >62 pg of IFN-γ/ml. The numbers of subjects tested with each antigen was 601 for the M. leprae (ML) 65-kDa antigen, 604 for the M. leprae 35-kDa antigen, 604 for the M. leprae 18-kDa antigen, 607 for the M. tuberculosis (MT) 38-kDa antigen, 458 for M. tuberculosis MPT64, 376 for M. tuberculosis ESAT-6, 607 for M. bovis MPB70, and 535 for M. bovis BCG Ag85.

FIG. 2.

Frequency distributions of IFN-γ responses to the panel of mycobacterial antigens. Whole-blood assays were stimulated with the recombinant and purified antigens at final concentrations of 10 μg/ml, and IFN-γ was measured in day 6 supernatants by ELISA. The numbers of subjects tested with each antigen was 601 for the M. leprae 65-kDa antigen, 604 for the M. leprae 35-kDa antigen, 604 for the M. leprae 18-kDa antigen, 607 for the M. tuberculosis 38-kDa antigen, 458 for M. tuberculosis MPT64, and 376 for M. tuberculosis ESAT-6 (a) and 607 for M. bovis MPB70 and 535 for M. bovis BCG Ag85 (b).

FIG. 3.

Association between responses to M. leprae or M. tuberculosis recombinant antigens within individuals. The percentage of individuals with IFN-γ responses >62 pg/ml to none, one, two, or all three of the leprosy recombinant antigens tested (65-, 35-, and 18-kDa antigens) and to none, one, two, or three of the tuberculosis recombinant antigens tested (38 kDa, MPT64, and ESAT-6) is shown. A total of 599 individuals were tested with the three leprosy antigens, and 284 were tested with the three tuberculosis antigens.

FIG. 4.

Association of IFN-γ responses to recombinant antigens with Mantoux skin test induration to M. tuberculosis PPD. Subjects were tested for IFN-γ production in response to the M. tuberculosis ESAT-6, MPT64, and 38-kDa antigens (A) and to the M. leprae (ML) 65-, 35-, and 18-kDa antigens; MPB70; and M. bovis BCG Ag85 (B), and for Mantoux skin test induration in response to M. tuberculosis PPD. The proportion of subjects making >62 pg of IFN-γ/ml in response to each antigen in each DTH category is indicated. A total of 339 subjects who had data on DTH response were tested for responses to ESAT-6, 349 were tested for responses to MPT64, 549 were tested for responses to the 38-kDa antigen, 491 were tested for responses to the 65-kDa antigen, 546 were tested for responses to the 35- and 18-kDa antigens, 549 were tested for responses to MPB70, and 482 were tested for responses to Ag85.