Elispot plate

LNMCs were stimulated or not for 4 days with 0.1 ug/ml of gp140 and 1 ug/ml of R848 (In vivoGen), 10 ng/ml of IL-2 (Miltenyi Biotec) and 100 ng/ml of IL-21 (Miltenyi Biotec). In the stimulated conditions cells were treated or not with 100 ng/ml of IL-4 (Miltenyi Biotec) or IFN-γ (Miltenyi Biotec). ELISPOT plates (BD) were coated with 15 ug/ml of anti-Ig antibodies (Mabtech) at 4°C overnight. Next, plates were washed and cells were added for 24 hours at 37°C followed by addition of biotinylated antibody against IgG or biotinylated proteins gp140 or the control protein keyhole limpet hemocyanin (KLH), and finally addition of a streptavidin-HRP (Mabtech). Frequencies of gp140-specific antibody secreting cells (ASC) were calculated from triplicate wells plated with 100.000 LNMCs per well. Specificity was verified with PBMCs of HIV-uninfected individuals.

PBMCs were stimulated or not for 5 days with 1 ug/mL of R848 (InvivoGen, San Diego, CA, USA) and 10 ng/mL of IL-2 (Miltenyi Biotec, Bergisch Gladbach, Germany). ELISPOT plates (BD) were coated with 15 μg/mL of anti-IgG (Mabtech, Stockholm, Sweden) and stored at 4 °C overnight. Next, the plates were washed, and cells were added for 24 h at 37 °C, followed by the addition of the biotinylated antibody against IgG or biotinylated proteins, and finally, the addition of a streptavidin-HRP (Mabtech, Stockholm, Sweden). Frequencies of the SARS-CoV-2-specific antibody-secreting cells (ASC) were calculated from triplicate or duplicate wells plated with 300.000 PBMCs per well. PBMCs from the pre-pandemic samples were used as controls.

ELISPOT assays were performed as previously described [42 (link)]. Briefly, ELISPOT plates (BD Biosciences, San Jose, CA, USA) were coated overnight at 4 °C with murine interferon gamma (IFN)-γ or interleukin (IL)-5 specific monoclonal antibodies. Splenocytes (2 × 10⁵ cells), collected after 7 days of the last immunization, were added each of triplicate wells and stimulated with 10 μg/mL RSV-F protein. After incubation at 37 °C for 24 h, the cells were then lysed with deionized (DI) water, and the plates were incubated at RT with biotinylated IFN-γ or IL-5 antibody for 2 h and peroxidase-labeled streptavidin for another 1 h. After washing with PBS, 100 μL of the final substrate solution was added to each well, and spot development was monitored. The plates were washed with DI water to stop the reaction. IFN-γ and IL-5 spot-forming cells (SFC) were counted automatically using an ELISPOT reader (BD Biosciences) and analyzed using ImmunoSpot image analyzer software v4.0 (BD Biosciences).

Cellular immune responses were evaluated by ELISPOT assay, as described previously, with a few modifications [32 (link)]. Briefly, ELISPOT plates (BD Bioscience, USA) were coated with monoclonal anti-mouse interferon-gamma (IFN-γ) and interleukin-4 (IL-4) capture antibodies diluted in sterile PBS (5 μg/mL) and incubated at 4 °C overnight. Then, the coating antibody was discarded and blocked with complete RPMI 1640 medium (PAN Biotech, Bayern, Germany) with 10% fetal bovine serum (PAN Biotech, Bayern, Germany) and 1% Antibiotic-Antimycotic (Glibco, Burlington, MA, USA) for 2 h at RT. Freshly isolated splenocytes were seeded at a density of 1 × 10⁶ cells/well. Cells were stimulated with purified proteins, epitope-specific peptides (10 μg/well), or phytohemagglutinin as a positive control (Gibco, Burlington, MA, USA), or only medium maintain as a negative control. Next, plates were incubated for 48 h at 37 °C with 5% CO₂. Then, cells were discarded from the plates and sequentially treated with biotinylated anti-mouse IFN-γ and IL-4 antibodies, stereptavidin HRP, and substrate solution. The substrate reaction was terminated by washing with deionized water and dried in the dark at RT. Finally, Spots were enumerated by CTL-Immunospot S5 UV analyzer (Cellular technologies, Shaker Heights, OH, USA).