title: 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.
  
  
  RESULTS
  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.
authors.affiliation: Istituto di Microbiologia, Università Cattolica del Sacro Cuore, Roma, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Delogu
authors.forename: Teresa
authors.initials: T
authors.lastname: Chiacchio
authors.forename: Valentina
authors.initials: V
authors.lastname: Vanini
authors.forename: Ornella
authors.initials: O
authors.lastname: Butera
authors.forename: Gilda
authors.initials: G
authors.lastname: Cuzzi
authors.forename: Alessandra
authors.initials: A
authors.lastname: Bua
authors.forename: Paola
authors.initials: P
authors.lastname: Molicotti
authors.forename: Stefania
authors.initials: S
authors.lastname: Zanetti
authors.forename: Francesco Nicola
authors.initials: FN
authors.lastname: Lauria
authors.forename: Susanna
authors.initials: S
authors.lastname: Grisetti
authors.forename: Nicola
authors.initials: N
authors.lastname: Magnavita
authors.forename: Giovanni
authors.initials: G
authors.lastname: Fadda
authors.forename: Enrico
authors.initials: E
authors.lastname: Girardi
authors.forename: Delia
authors.initials: D
authors.lastname: Goletti
mesh_terms: D000328:Adult; D000942:Antigens, Bacterial / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D003710:Demography; D005260:Female; D006801:Humans; D007156:Immunologic Memory; D007371:Interferon-gamma / Q000276:immunology; D037102:Lectins / Q000096:biosynthesis* / Q000276:immunology; D008297:Male; D008745:Methylation; D008875:Middle Aged; D020102:Mycobacterium smegmatis / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D012372:ROC Curve; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins / Q000276:immunology; D013997:Time Factors; D014376:Tuberculosis / Q000175:diagnosis* / Q000276:immunology*
chemical_list: D000942:Antigens, Bacterial; D037102:Lectins; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins; C104578:heparin-binding hemagglutinin; D007371:Interferon-gamma