Methylated HBHA Produced in M. smegmatis Discriminates between Active and Non-Active Tuberculosis Disease among RD1-Responders

Abstract

BackgroundA challenge in tuberculosis (TB) research is to develop a new immunological test that can help distinguish, among subjects responsive to QuantiFERON TB Gold In tube (QFT-IT), those who are able to control Mtb replication (remote LTBI, recent infection and past TB) from those who cannot (active TB disease). IFN-γ response to the Heparin-binding-hemagglutinin (HBHA) of Mtb has been associated with LTBI, but the cumbersome procedures of purifying the methylated and immunological active form of the protein from Mtb or M. bovis Bacillus Calmette et Guerin (BCG) have prevented its implementation in a diagnostic test. Therefore, the aim of the present study was to evaluate the IFN-γ response to methylated HBHA of Mtb produced in M. smegmatis (rHBHAms) in individuals at different stages of TB who scored positive to QFT-IT.Methodology/Principal Findings87 individuals 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 by short-term-tests, is significantly lower in active TB than in remote LTBI (p = 0.0010) and past TB (p = 0.0152). These results were confirmed by long-term tests. The qualitative data confirmed that IFN-γ responses higher than the cut-off point identified by ROC analysis are associated with the status of non-active disease.ConclusionsIn this study we show that the T-cell response to a recombinant and methylated HBHA of Mtb produced in M. smegmatis is useful to discriminate between active and non-active TB disease among those responsive to QFT-IT in a whole blood system. Further studies are needed to improve the accuracy of the assay.

Exploiting the mycobacterial cell wall to design improved vaccines against tuberculosis

“…These results imply that during M. tuberculosis infection the host-specific immune response directed against HBHA differs depending on the clinical status. These findings are relevant from a diagnostic point of view, since it would be possible to discriminate patients with active TB from those infected but with no overt sign of the disease [56][57][58][59][60][61]. At the same time, these differential immune responses in TB patients versus healthy TBinfected subjects may suggest that an effective T cell response against HBHA could help to control M. tuberculosis replication and prevent disease development [54,62].…”

“…Lastly, the AERAS-422 vaccine, which basically combines concepts underlying VPM1002 and rBCG30 [25], was shown to induce effective protection in preclinical animal models; however, though the phase I clinical trial was stopped because of an adverse effect observed in two participants (www.clinicaltrials.gov ID# NCT01340820). [36] MΦ apoptosis [37] Expression in saprophytic mycobacteria [40] Deletion in BCG [41] Deletion in H37Rv [42] HBHA Bacterial agglutination [45; 46] Dissemination from the primary site of infection [45] Protection in the mouse model of TB [55] Diagnostic marker in QFT-test [59] ESX secretion systems ESX-1: Secretion of ESAT-6 and CFP-10 which are employed in IGRAs to diagnose TB infection [71] Encoded by the RD1 region which loss is the main molecular mechanism of attenuation of BCG [69] …”

The risk of tuberculosis in transplant candidates and recipients: a TBNET consensus statement

“…New diagnostic approaches are needed that allow for a more targeted identification of patients at risk to develop TB. This may involve modifications of in vitro immunodiagnostic assays such as the use of novel stimulatory antigens [15,164,165], alternative biomarkers other than IFN-c [166][167][168][169][170][171][172], variations in incubation time [173,174], the readout system [9,12,13,175,176] or the clinical specimen instead of blood [177][178][179]. In addition, both for the detection of LTBI with a risk of reactivation and for suspected active TB, a novel approach based on a whole blood transcriptional signature could provide a biomarker system with high discriminative potential [180].…”

Assessment of CD27 expression as a tool for active and latent tuberculosis diagnosis

“…3,4 They represent a breakthrough, however, they do not discriminate between active TB disease and LTBI. 1,3e7 IGRA accuracy for LTBI diagnosis may be enhanced using other Mtb-specific antigens 8,9 or peptides selected from ESAT-6/CFP-10, 10e12 evaluating the response at the site of TB disease 13,14 or investigating host biomarkers other than IFN-g in whole blood or peripheral blood mononuclear cells (PBMC). 10,14e21 Cytometry has been proposed as a potential tool to improve TB diagnosis by phenotypical and functional characterization of antigen-specific T-cells.…”