IFN-γ ex-vivo ELISpot was performed on freshly isolated Peripheral Blood Mononuclear Cells (PBMC) from volunteers in the Starter Group and Group A at: D0, D14, D28, D56, D84 and D168, Group B at: D0, D14, D28, D56, D63, D84, D140 and D224 and Group C at: D0, D14, D28, D42, D56, D119, D126, D147, D203 and D287, as previously described [14] (link) using the Human IFN-γ ELISpot (ALP) kit (Mabtech).
Ag85A-specific responses were measured using a single pool of Ag85A peptides (66 15mer peptides, overlapping by 10 amino acids). Anti-vector responses were measured using ChAdOx1 (2 IU:1 PBMC) (Vector Core Facility, The Jenner Institute, Oxford, UK) and responses to MVA were measured using separate pools of CD4 (27 14-21mer peptides) and CD8 (36 9mer peptides) epitopes from Vaccinia and MVA (Peptide Protein research, UK) (final concentration 2 μg/ml) and Purified Protein Derivative (PPD) (Statens Serum Institute, Denmark) (20 μg/ml). Staphylococcal enterotoxin B (SEB) (Sigma) (10 μg/ml) was used as a positive control and unstimulated PBMC as a negative control.
Ag85A-specific responses were measured using a single pool of Ag85A peptides (66 15mer peptides, overlapping by 10 amino acids). Anti-vector responses were measured using ChAdOx1 (2 IU:1 PBMC) (Vector Core Facility, The Jenner Institute, Oxford, UK) and responses to MVA were measured using separate pools of CD4 (27 14-21mer peptides) and CD8 (36 9mer peptides) epitopes from Vaccinia and MVA (Peptide Protein research, UK) (final concentration 2 μg/ml) and Purified Protein Derivative (PPD) (Statens Serum Institute, Denmark) (20 μg/ml). Staphylococcal enterotoxin B (SEB) (Sigma) (10 μg/ml) was used as a positive control and unstimulated PBMC as a negative control.
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