PBMC were thawed and re-suspended in RPMI-1640 medium supplemented with 10% human AB serum (Biochrom, Merck, Germany), 2mM L-glutamine (PAA), 50 µM 2-mercaptoethanol (Sigma Aldrich, St. Louis, MO, USA) and 0.1 mg/ml gentamycin (Sigma Aldrich). For re-stimulation, PBMC (8 × 105/well) were incubated in a 96-well plate with the JEV antigen (12.5 µg/ml), TBE antigen (2.4 µg/ml), superantigen staphylococcal enterotoxin B (SEB; 1 µg/ml) or just medium in a final volume of 200 µl. The JEV antigen was kindly provided by Dr Klade, formerly of Intercell and now Valneva. The TBE antigen strain Neudörfl was provided by the company that was Baxter Innovation, Vienna, Austria and is now Pfizer. Plates were incubated at 37 °C and 5% of carbon dioxide for 48 hours. Thereafter, supernatants were harvested and stored at −20 °C until they were tested during the following days.
The quantification of IL-2, IFN-γ and IL-10 levels were performed by Luminex technology, according to the protocols, using the Fluorokine MAP Human Base Kit A (R&D Systems, Minneapolis, MN, USA) and a Luminex reader (AtheNA Multi-Lyte®, Zeus Scientific, Raritan, NJ, USA).
The quantification of IL-2, IFN-γ and IL-10 levels were performed by Luminex technology, according to the protocols, using the Fluorokine MAP Human Base Kit A (R&D Systems, Minneapolis, MN, USA) and a Luminex reader (AtheNA Multi-Lyte®, Zeus Scientific, Raritan, NJ, USA).