Methylated HBHA produced in M. smegmatis discriminates between active and non-active tuberculosis disease among RD1-responders

Abstract

Background: A 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 findings: 87 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.

Conclusions: In 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.

Figure 1. Concentration-dependent IFN-γ response after in vitro short-term whole blood stimulation with rHBHAms in subjects with LTBI.

Whole blood from 10 subjects with LTBI was stimulated with or without rHBHAms at different concentrations (between 25 and 1 µg/ml). IFN-γ response was evaluated after a short-term stimulation (1 day post-in vitro stimulation). A significant difference was found for the IFN-γ response obtained between 25 and 5 µg/ml and that obtained at a concentration of 1 µg/ml.

Figure 2. Response to rHBHAms is significantly impaired in patients with active TB.

Whole blood was stimulated with or without QFT-IT (A), rHBHAms (B) and CMV lysate (C). IFN-γ response was evaluated after a short-term stimulation (1 day post-in vitro stimulation). The data are shown after the subtraction of the results obtained in the unstimulated sample. A significant difference in terms of IFN-γ release to QFT-IT (A) and CMV lysate (C) was found between those with active TB and recent infection (p = 0.01 and p = 0.002 respectively). A significant lower response to rHBHAms (B) was found in patients with active TB compared to those with remote LTBI (p = 0.001) and past TB (p = 0.02).

Figure 3. Response to rHBHAms is significantly impaired in patients with active TB after short- or long- term stimulation.

A significant difference was found for the IFN-γ response to rHBHA between the subjects with and without active TB (p = 0.001) evaluated by short- or long- term stimulation.

Figure 4. IFN-γ response to rHBHAms in short- and long-term-“in vitro” stimulation and to QFT-IT: ROC analysis.

A ROC analysis for the response to rHBHAms obtained in whole blood by short-term stimulation (A), long- term stimulation (B) and QFT-IT (C) was performed using the active TB patients and the subjects without active TB as comparator groups.

Figure 5. Memory responses to rHBHAms.

Memory response (long-term stimulation) to rHBHAms was evaluated in the subjects who scored negative on the short- test. A significant difference was found between the IFN-γ value obtained in the short-term stimulation compared to the long-term stimulation among those without active TB (p = 0.0003). Differently, no significant difference was found between the short- and long- term stimulation among those with active TB (p = 0.4). The data are shown after the subtraction of the results obtained in the unstimulated samples. Dotted lines indicate the cut-off obtained for the short- and long-term tests.

Figure 6. CD4⁺ effector memory T lymphocytes produce IFN-γ in response to rHBHAms.

The phenotypic characteristics of the cells responding to the rHBHAms in the HBHA-responders were evaluated. As shown in a representative subject, a significant IFN-γ response to the rHBHAms was observed for CD4⁺ T-cells (D) over the negative control (B), whereas no response was detected for CD8⁺ T-cells (C) over the negative control (A). To characterize this immune response, naive and memory phenotypes were studied. Most of the CD4⁺ T-cells IFN-γ responding to the rHBHAms presented an effector memory phenotype (84%) defined as CD45R0⁺CD62L (E).