Rv3615c is a highly immunodominant RD1 (Region of Difference 1)-dependent secreted antigen specific for Mycobacterium tuberculosis infection

Abstract

The 6-kDa early secretory antigenic target of Mycobacterium tuberculosis (ESAT-6) and the 10-kDa culture filtrate antigen (CFP-10), encoded in region of difference 1 (RD1) and secreted by the ESAT-6 system 1 (Esx-1) secretion system, are the most immunodominant and highly M. tuberculosis (MTB)-specific antigens. These attributes are responsible for their primary importance in tuberculosis (TB) immunodiagnosis and vaccine development. Rv3615c [Esx-1 substrate protein C (EspC)], encoded outside RD1, is similar in size and sequence homology to CFP-10 and ESAT-6, suggesting it might be a target of cellular immunity in TB. Using ex vivo enzyme-linked immunospot- and flow cytometry-based cytokine-secretion assay, we comprehensively assessed cellular immune responses to EspC in patients with active TB, latently infected persons, and uninfected bacillus Calmette-Guérin (BCG)-vaccinated controls. EspC was at least as immunodominant as ESAT-6 and CFP-10 in both active and latent TB infection. EspC contained broadly recognized CD4(+) and CD8(+) epitopes, inducing a predominantly CD4(+) T-cell response that comprised functional T-cell subsets secreting both IFN-γ and IL-2 as well as functional T-cell subsets secreting only IFN-γ. Surprisingly, T-cell responses to EspC were as highly specific (93%) for MTB infection as responses to ESAT-6 and CFP-10, with only 2 of 27 BCG-vaccinated controls responding to each antigen. Using quantitative proteomics and metabolically labeled mutant and genetically complemented MTB strains, we identified the mechanism of the specificity of anti-EspC immunity as the Esx-1 dependence of EspC secretion. The high immunodominance of EspC, equivalent to that of ESAT-6 and CFP-10, makes it a TB vaccine candidate, and its high specificity confers strong potential for T-cell-based immunodiagnosis.

Fig. 1.

(A and B) Frequency of peptide-specific IFN-γ SFC for EspC, ESAT-6, and CFP-10 in 47 TB cases (A) and 27 subjects with risk factors for LTBI (B), enumerated by ex vivo ELISpot. Horizontal lines indicate median responses. (C and D) T-cell epitope maps of EspC for 15 TB cases (C) and five persons with LTBI (D). IFN-γ production in response to EspC epitopes was primarily by CD4⁺ T cells as shown by cytokine-secretion assay and flow cytometry (E). Results are expressed as percentages of lymphocyte populations staining CD4⁺IFN-γ⁺ or CD8+IFN-γ⁺. Subjects T134, T137, T413, T266, and T116 had active TB (T266 and T116 with 2-mo treatment); subject T416 had LTBI.

Fig. 2.

The IFN-γ and IL-2 cytokine profiles of CD4⁺ T cells specific for EspC in active TB, during anti-TB therapy, and in LTBI. (A) Percentages plotted indicate the frequency of CD4⁺ T cells producing IFN-γ and/or IL-2 in response to EspC peptides. Background levels of nonspecific cytokine production have been subtracted. (B) Relative proportions of CD4⁺ T cells producing IFN-γ and/or IL-2 in the enriched fraction.

Fig. 3.

(A and B) Comparison of frequency of EspC-specific IFN-γ–secreting T cells in TB cases, LTBI, and controls (A) and IFN-γ–secreting T cells specific for EspC, ESAT-6, and CFP-10 in 27 BCG-vaccinated controls (B). Horizontal lines indicate median responses. EspC, identified by mass spectrometry, is present in H37Rv culture filtrates but not in the culture filtrate of the ΔRD1 (denoted as ΔEsx1) deletion mutant (C). EspC secretion is dependent on RD1; localization of EspC, EspA, and EsxA (ESAT-6) in MTB H37Rv strains carrying deletions of EspA, EspC, and EspD (denoted ΔEspACD) with or without additional deletion of RD1 (denoted ΔEsx1). Blots are shown for transposon of empty vector (pEmpty) and for transposon insertion of EspACD (pEspACD). Cell pellet (P) and culture filtrate (CF) proteins were resolved through SDS/PAGE, and the proteins were identified by Western blotting with specific antibodies (D).