Abstract
Studies on Mycobacterium tuberculosis (MTB) antigens are of interest in order to improve vaccine efficacy and to define biomarkers for diagnosis and treatment monitoring. The methodologies used for these investigations differ greatly between laboratories and discordant results are common. The IFN-gamma response to two well characterized MTB antigens ESAT-6 and CFP-10, in the form of recombinant proteins and synthetic peptides, was evaluated in HIV-1 uninfected persons in both long-term (7 day) and 24 hour, commercially available QuantiFERON TB Gold in Tube (QFT-GIT), whole blood assays. Our findings showed differences in the IFN-gamma response between 24 hour and 7 day cultures, with recombinant proteins inducing a significantly higher response than the peptide pools in 7 day whole blood assays. The activity of peptides and recombinant proteins did not differ in 24 hour whole blood or peripheral blood mononuclear cell (PBMC) based assays, nor in the ELISpot assay. Further analysis by SELDI-TOF mass spectrometry showed that the peptides are degraded over the course of 7 days of incubation in whole blood whilst the recombinant proteins remain intact. This study therefore demonstrates that screening antigenic candidates as synthetic peptides in long-term whole blood assays may underestimate immunogenicity.
Highlights
Assay of the T cell response to antigens of Mycobacterium tuberculosis (MTB) is a research priority and critical in the evaluation of new antigenic candidates to improve immunodiagnosis and vaccines
When recombinant proteins were used as stimulants, 27/29 (93%) donors responded to either recombinant ESAT-6 or CFP-10, and this difference was significant when compared to the peptides (p,0.0001 Wilcoxon matched pairs test, Figure 1, Panel B)
In this study we investigated the secretion of IFN-c in the commercially available QuantiFERON TB Gold in Tube (QFTGIT) and long-term (7 day) whole blood assays, as well as the ELISpot assay in response to ESAT-6 and CFP-10
Summary
Assay of the T cell response to antigens of Mycobacterium tuberculosis (MTB) is a research priority and critical in the evaluation of new antigenic candidates to improve immunodiagnosis and vaccines. Newer IFN-gamma release assays use MTB specific ESAT-6 and CFP-10, two co-expressed immunodominant antigens encoded by the region of difference (RD)-1 of MTB These antigens were first evaluated in a 6-day lymphocyte stimulation test (LST) and found to be highly specific to infer tuberculosis infection [4,5]. IFN-gamma release assays were subsequently developed that differed from the classical LST in terms of cell type to be stimulated (whole blood or peripheral blood mononuclear cells), the formulation of the antigenic candidate (overlapping synthetic peptides or recombinant proteins), duration of incubation (from ,24 hours to 7 days), or the methods used to read out the results (proliferation, ELISA, ELISpot, flow cytometry) [1,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30]. Discordance between reported results have been attributed mainly to the variation in the duration of incubation and have been suggested to be a result of the measurement of various components of the T cell response where short-term assays detect circulating effector cells while long-term assays detect the secondary effector cells that differentiate from central memory cells during culture [28,31,32,33,34]
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