Methylated HBHA Produced in M. Smegmatis Discriminates Between Active and Non-Active TB Disease among the QFT-IT-responders

Background: Challenge in tuberculosis (TB) research is to develop a test to distinguish, among the responders to an IFN-γ release assay (IGRA), those who control M. tuberculosis (Mtb) replication from those that cannot. IFN-γ response to the Heparin-binding-hemagglutinin (HBHA) of Mtb has been associated with latent TB infection (LTBI), but the cumbersome procedures to purify the methylated and immunological active form of the protein from Mtb or M. bovis BCG have prevented the implementation of a diagnostic test. Aim of this study was to evaluate the IFN-γ response to methylated HBHA of Mtb produced in M. smegmatis (rHBHAms) in subjects at different stages of TB who scored positive to QuantiFERON-TB Gold in-tube (QFT-IT). Methodology/Principal findings: 87 subjects at different stages of TB who scored positive to QFT-IT were selected. IFN-γ response to in vitro whole blood stimulation with rHBHAms was evaluated by short-term and long-term tests and detected by ELISA or flow cytometry. We demonstrated that the IFN-γ response to rHBHAms is mediated by CD4 + T-cells with an effector-memory phenotype. This response, evaluated also by short-term-tests is significantly lower in active TB than in remote LTBI (p=0.0010), past TB (p=0.0152) and recent infection (p=0.05). These results were confirmed by long-term tests and by qualitative analysis using ROC analysis. Conclusions: For the first time to our knowledge, we showed that the T-cell response to a recombinant and methylated HBHA of Mtb produced in M. smegmatis in a whole blood system is immunogenic and potentially useful to discriminate between active and non-active TB disease among those responsive to QFT-IT.