Recombinant Receptor Binding Domain Protein Induces Partial Protective Immunity in Rhesus Macaques Against Middle East Respiratory Syndrome Coronavirus Challenge()

Lan, Jiaming; Yao, Yanfeng; Deng, Yao; Chen, Hong; Lu, Guangwen; Wang, Wen; Bao, Linlin; Deng, Wei; Wei, Qiang; Gao, George F.; Qin, Chuan; Tan, Wenjie

To evaluate the antigen-specific T-cell response induced by rRBD protein in immunised monkeys, an IFN-γ ELISpot assay was performed as described previously (Wen et al., 2013) . After PBMCs were isolated from EDTA-treated blood, IFN-γ ELISpot assay was carried out according to the manufacturer's instructions. Briefly, 2.5 × 10 5 cells per well were stimulated with pools of 15-mer peptides overlapping by 11 amino acids spanning the RBD protein of MERS-CoV at a final concentration of 4 μg/mL into a PVDF membrane-bottomed plate pre-coated with anti-monkey IFN-γ capture antibody. A set of negative control wells comprising media only were included on each plate. After stimulation for 20-24 h, IFN-γ-spot-forming cells were detected by staining membranes with the detection antibody (7-B6-1-biotin) followed by streptavidin-horseradish peroxidase and development with TMB (3, 3′, 5, 5′-tetramethylbenzidine) substrate solution. Results are presented as spot-forming cells per 10 6 PBMCs. Values were reported as the mean of the test wells minus the mean of all negative-control wells.