In Vitro Immune Response To Vaccine Candidate Mycobacterium Tuberculosis Antigens In Healthy Adult Ethiopians

Introduction: The global burden of disease due to tuberculosis (TB) is huge and is one rnof the challenges in the fight against infectious diseases. The widespread use BCGrnvaccine was for many years believed to be one of the key control measures for TB, but itsrnefficacy has been questioned following the results of trials conducted in developingrncountries. Therefore, there is an urgent need particularly in developing countries, for arnvaccine with acceptable efficacy to combat the TB epidemic. However, protectivernimmunity in TB is incompletely understood. In murine models, cell-subset depletionrnexperiments in vivo showed that CD4 and CD8 άβ as well as γδ T cells all have a role inrnprotective immunity. Studies demonstrated that immunization of mice with live M.rntuberculosis induces protection and delayed type hypersensitivity (DTH), whereas heatkilledrnorganismsrninducernDTHrnonly.rnThisrnexperimentalrnobservationrnhasrnsparkedrninterestrninrnrnantigensrnrnreleased or secreted by mycobacteria in culture filtrates as vaccine candidatesrnand different studies on these antigens showed promising results. Ethiopia being one ofrnthe high TB burden countries, this project is designed with the objective to generaternbackground immunological data on immune response to selected candidate TB vaccinernantigens of healthy adult Ethiopians in preparation for a TB vaccine clinical trial. rnMethods: Tuberculin skin test (TST) and QuantiFERON®-TB-Gold (QFT-G) assayrnwere done. Peripheral blood monocytic cells (PBMCs) were isolated from healthy adultsrnand the immune response following PBMC stimulation with the different TB antigens,rnESAT-6, Ag85B and fused Ag85B-ESAT-6, was measured by the in vitro assays, IFN–γrnELISPOT assay and IFN–γ ELISA.rnResults: Our findings showed that 46.7 % and 43.9% of the study participants had rnpositive TST (TST10 mm) and QFT-G assay results respectively. The overall strengthrnof agreement between TST (10mm) and QFT-G assay was very good (Kappa = 0.83), rnwith concordant results in 98/107 (91.6%). Concordance between the two tests was goodrnin participants with BCG scar (85.4%, Kappa = 0.71), but it was found to be excellentrn(96.6%, Kappa =0.93) in those who had not BCG scar. By using IFN–γ ELISPOT assay,rnthe median (IQR) responses to Ag85B, ESAT-6 and fused Ag85B-ESAT-6 were found tornbe 256 (147-408), 305 (133-474) and 429 (267-601) SFC/Million of PBMC respectively.rnThe median (IQR) responses to Ag85B, ESAT-6 and fused Ag85B-ESAT-6 were foundrnto be 0(0.0-189.0), 328.0 (0.0- 2351.0) and 96.0(0.0- 1220.0) pg/ml of IFN-γ respectivelyrnas it was measured by IFN–γ ELISA.rnConclusion: An overall strong agreement between TST and QFT-G was observed at both rnTST10mm and TST>5 mm cutoff values in spite of the routine BCG vaccination atrnbirth. We also have demonstrated the possibility to have false TST positivity in adults asrnthe result of BCG vaccine administered in infancy, at cutoff of TST>5mm, but thernpossibility of true Latent TB infection (LTBI) is highly likely at larger indurations rn(TST10mm). QFT-G assay was not able to differentiate recent infection from remoternone. By using ELISPOT assay and ELISA, we were able to demonstrate the existence ofrneffector and central memory T-cells specific to the fused antigens, Ag85B-ESAT-6, andrnits components, ESAT-6 and Ag85B, in healthy adult populations. The presence ofrnsignificant ESAT-6 specific memory T-cells in the latent group makes the fused vaccinernvaluable to be used as post-exposure vaccine. The persistence of Ag85B specific T-cellsrnas the result of childhood BCG vaccination enables the fused vaccine to be used asrnbooster vaccine besides its potential use as post-exposure vaccine.

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