New Genome-Wide Algorithm Identifies Novel In-Vivo Expressed Mycobacterium Tuberculosis Antigens Inducing Human T-Cell Responses with Classical and Unconventional Cytokine Profiles

Abstract

New strategies are needed to develop better tools to control TB, including identification of novel antigens for vaccination. Such Mtb antigens must be expressed during Mtb infection in the major target organ, the lung, and must be capable of eliciting human immune responses. Using genome-wide transcriptomics of Mtb infected lungs we developed data sets and methods to identify IVE-TB (in-vivo expressed Mtb) antigens expressed in the lung. Quantitative expression analysis of 2,068 Mtb genes from the predicted first operons identified the most upregulated IVE-TB genes during in-vivo pulmonary infection. By further analysing high-level conservation among whole-genome sequenced Mtb-complex strains (n = 219) and algorithms predicting HLA-class-Ia and II presented epitopes, we selected the most promising IVE-TB candidate antigens. Several of these were recognized by T-cells from in-vitro Mtb-PPD and ESAT6/CFP10-positive donors by proliferation and multi-cytokine production. This was validated in an independent cohort of latently Mtb-infected individuals. Significant T-cell responses were observed in the absence of IFN-γ-production. Collectively, the results underscore the power of our novel antigen discovery approach in identifying Mtb antigens, including those that induce unconventional T-cell responses, which may provide important novel tools for TB vaccination and biomarker profiling. Our generic approach is applicable to other infectious diseases.

Proteomic analysis of protein purified derivative of Mycobacterium bovis

“…Altogether, ninety-eight mycobacterial antigens which play a central role in DTH have been identified in all examined bPPDs. Furthermore, from a comparative point of view, a very large number of T cell antigens was found to be shared between bPPDs of this study and MtbPPDs [26, 28–30, 32, 39–41]. …”

“…Fourteen of them belong to a novel series of in vivo expressed M. tuberculosis (IVE-TB) T cell antigens [31, 32]. Two reactivation associated antigens, namely Mb0391c (gene name: clpB) and Mb2492c (gene name: rplB) and three lipoprotein T cell antigens, namely Mb0959 (gene name: pstS1), Mb0956c (gene name: pstS2) and Mb0951 (gene name: pstS3), known to generate very high levels of cytokine secretion [33], were also detected.…”

Introducing the ESAT-6 free IGRA, a companion diagnostic for TB vaccines based on ESAT-6

“…The immunodiagnostic potential of EspF was first demonstrated in cattle using the M . bovis equivalent Mb389528, and human recognition was very recently shown as part of a larger screening study29. The third new antigen Rv2348c is a hypothetical protein with unknown function.…”

“…One of these Ags, ESX-1 substrate protein C, (EspC, Rv3615c), has previously been identified as a highly recognized and Mtb specific antigen1319202122. Other potential candidates include the ESX-1 associated EspJ (Rv3878)2324, EspF (Rv3865)2526272829, the RD1 encoded PPE68 (Rv3873)23, PE35 (Rv3872)30, EccD1 (Rv3877)31 and the RD7 encoded Rv234832 (Table 1). By screening fragments in pools of three to seven peptides, we aimed to cover several antigens in the cocktail while retaining a low total number of peptides.…”

Cell-Mediated Immune Responses to in vivo-Expressed and Stage-Specific Mycobacterium tuberculosis Antigens in Latent and Active Tuberculosis Across Different Age Groups

“…Ideally, Mtb proteins selected as targets for new TB vaccines should be expressed during active Mtb lung infection and efficiently trigger immune effector cells capable of controlling or clearing the infection without inflicting major tissue damage. Recently, we identified a new class of Mtb antigens, named IVE-TB, encoded by Mtb genes that were highly and consistently expressed in the lung of susceptible (C3HeB/FeJ) as well as resistant (C57BL/6J) mice following aerosol Mtb (Erdman) challenge (10,11). Besides their high expression in Mtb infected lungs, these IVE-TB proteins constitute an attractive group of novel candidate antigens for multiple other reasons (11): (i) they are conserved among 219 Mtb clinical isolates and thus cover a wide array of Mtb strains; (ii) they have high homology with BCG and thus have potential as booster vaccines; (iii) they have high homology with pathogenic mycobacteria, including M. leprae and NTM and thus have potential as broader vaccines; (iv) they contain a large number of epitopes predicted to bind to HLA-Ia and HLA-II alleles (coverage of 85% of the human population);…”