Screening of Highly Expressed Mycobacterial Genes Identifies Rv3615c as a Useful Differential Diagnostic Antigen for the Mycobacterium tuberculosis Complex

Rv3615c was identified as the antigen with the greatest responder frequency in M. bovis-infected cattle in our initial selection of highly expressed candidates. In this screen IFN-γ levels were measured using the Bovigam ELISA, and the level of IFN-γ production upon stimulation with Rv3615c (A) or PPD-B (B) for each animal in each group is shown. Symbols: •, uninfected cattle; ▴, M. bovis-infected cattle; ▪, BCG-vaccinated cattle; ▾, M. bovis-infected ESAT-6/CFP-10-negative (ESAT-6/CFP-10 -ve) cattle. The data are expressed as background-subtracted OD₄₅₀ (Δ OD450), and the dashed lines indicate the cutoff for positivity. Significance testing was performed using the nonparametric Wilcoxon rank sum test adjusted for multiple comparisons.

To confirm the presence and location of the T-cell epitopes in Rv3615c, the responses of constituent peptides from the Rv3615c pool were determined using an IFN-γ ELISPOT assay with PBMC isolated from M. bovis-infected cattle. The results are expressed in SFU per 10⁶ PBMC for each animal. The cattle used were animals 3420, 3450, and 3606.

FACS analysis with PBMC isolated from three M. bovis-infected cattle performed after stimulation with RPMI medium, each individual Rv3615c peptide (5 μg ml⁻¹), or the Rv3615c peptide pool (5 μg ml⁻¹). Secretion of IFN-γ was stopped by addition of brefeldin A (10 μg ml⁻¹). Lymphocytes were analyzed for intracellular IFN-γ production and the presence of CD4 and CD8 T-cell subset markers using anti-CD4-Alexa Fluor 647, anti-CD8-fluorescein isothiocyanate, and anti-IFN-γ-phycoerythrin conjugated antibodies. A live/dead differential stain analysis was performed using ViVid and a Dake Cyan ADP. The results are expressed as percentages of the lymphocyte population staining as CD4⁺ IFN-γ⁺ (bars above the x axis) or CD8⁺ IFN-γ⁺ (bars below the x axis) for each animal (animals 3420, 3450, and 3606).