pCD5 expression vectors containing the HA ectodomainencoding sequences were transfected into HEK293S GnTI(2)cells [22] using polyethyleneimine I (PEI) in a 1:5 ratio (mg DNA: mg PEI). At 6 h post transfection, the transfection mixture was replaced by 293 SFM II expression medium (Invitrogen), supplemented with sodium bicarbonate (3.7 g/liter), glucose (2.0 g/liter), Primatone RL-UF (3.0 g/liter), penicillin (100 units/ml), Streptomycin (100 mg/ml), glutaMAX (Gibco), and 1,5% dimethyl sulfoxide. Tissue culture supernatants were harvested 5-6 days post transfection. HA protein expression and secretion was confirmed by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) followed by western blotting using a mouse anti-Strep-tag antibody (IBA, Germany). HA proteins were purified using Strep-tactin Sepharose beads according to the manufacturer's instructions (IBA, Germany). The concentration of purified protein was determined by using a Nanodrop 1000 spectrophotometer (Isogen Life Sciences) according to the manufacturer's instructions.