At day 6, infected cell culture (200 ml) was clarified by centrifugation at 30009g for 30 min at 4°C. VLPs in the supernatant were concentrated by ultracentrifugation (L8-60M ultracentrifuge, Beckman SW-32.1 Ti rotor) at 100,0009g for 2 h at 4°C, and pellets were resuspended in 0.2 M Tris-HCl, pH 7.3. VLPs were loaded onto a 10-60% discontinuous sucrose gradient and ultracentrifugated at 100,0009g for 1 h at 4°C as described before [17] . Fractions were collected by bottom puncture. The fractions containing VLPs were pooled. An additional discontinuous sucrose gradient (35-60%) ultracentrifugation was performed. Sucrose was removed by overnight dialysis against 1 liter of PBS. VLPs were concentrated by dialysis against polyethylene glycol (PEG; 50%) [6] or by ultrafiltration [19] . VLPs were concentrated using an Amicon Ultra 30 kDa centrifuge filter device (Millipore Corporation, Billerica, Germany). VLPs were stored at 4°C in PBS.