In addition, the frequency of IFN-γ-producing CD4 T lymphocytes from each donor in the absence of any stimulation was used to calculate the background for each stimulation. The resulting background levels were very low in every experiment, and no differences were observed between samples obtained before (t0 0.03% ± 0.04%) and after vaccination (t1 0.01% ± 0.03%). The frequency of antigen-specifi c CD4 T cells for each study participant was calculated by subtracting the relative background levels at t0 and t1.