Production of Virus-Like Particles

To generate VLPs, suspension cultures of Sf9 (2 × 10⁶ cells/mL) were infected with the recombinant baculoviruses Bac-EV71-P1/3CD, Bac-CVA16-P1/3CD, Bac-CVA10-P1/3CD, or Bac-CVA6-P1/3CD at a multiplicity of infection of 1 followed by culturing at 27 °C for 3 days. Sf9 cells from each culture were then collected by centrifugation and lysed with 0.15 M PBS containing 1% NP-40. Cell lysates were centrifuged at 12,000 rpm for 15 min to remove cellular debris, and the resultant supernatants were precipitated overnight at 4 °C with 8% (w/v) polyethylene glycol 8000 and 200 mM NaCl. After centrifugation at 12,000 rpm for 15 min, the resulting pellets were collected and resuspended in 0.15 M PBS buffer, followed by clarification by centrifugation. Next, 20% sucrose cushion and 10–50% sucrose-gradient ultracentrifugation steps were carried out as previously described^{37 (link)}. Finally, VLP-rich fractions were pooled and buffer-exchanged into 0.15 M PBS buffer using Amicon Ultra 100 K centrifugal filters (Millipore, USA). For comparison, the control antigen was generated from uninfected Sf9 cells following the same protocol. Purified VLPs and control antigen were quantified using the Bradford protein assay kit (Bio-Rad, USA) according to the manufacturer’s instructions.