Assessment of in vitro immunity to Mycobacterium tuberculosis in a human peripheral blood infection model using a luciferase reporter construct of M. tuberculosis H37Rv.

Protective immune responses to tuberculosis in man are primarily cell-mediated and require the interaction of specific T cells, cytokines and activated macrophages. In the present study, Mycobacterium tuberculosis H37Rv labelled with luciferase reporter enzyme was used to analyse the anti-mycobacterial immunity in man using an in vitro whole blood infection model. Peripheral blood samples obtained from M. bovis bacille Calmette-Guérin (BCG)-vaccinated tuberculin-positive healthy volunteers (n = 23) were cultured with M. tuberculosis H37Rv reporter strain. The growth of bacteria in the whole blood cultures was monitored after 48 and 96 h of infection. The results showed that the growth of M. tuberculosis was significantly inhibited after 96 h (P < 0.029) of culture. Among the cytokines studied, interleukin (IL)-10 and IL-12 were not detected at all, whereas low levels of interferon (IFN)-gamma after 96 h (0.4 IU/ml) and tumour necrosis factor (TNF)-alpha after 48 (135 pg/ml) and 96 h (47 pg/ml) of culture were detected in the supernatants of whole blood infected with M. tuberculosis. The magnitude of bacterial growth correlated directly with the concentration of TNF-alpha detected after 48 h (r = 0.722) and 96 h (r = 0.747) of culture (P <or= 0.0001 and P <or= 0.0001, respectively). However, the addition of monoclonal antibodies specific to TNF-alpha and IFN-gamma to the blood cultures did not alter mycobacterial growth indicating the role of other mechanisms/factors in restricting the growth of M. tuberculosis in whole blood cultures.