eVLP production in insect Sf9 or mammalian 293T cells

The generation of Ebola VLPs in insect cells (eVLP) has been described previously [26]. Briefly, recombinant baculoviruses co-expressing Ebola VP40 and GP (rBV-GP-VP40) proteins or only expressing Ebola VP40 (rBVVP40) proteins infect Spodoptera frugiperda Sf9 insect cells at an MOI of 1. After 48h, the supernants were collected and VP40 and eVLPs proteins were purified in a discontinuous sucrose gradient (10–50%). A visible band between the 30% and 50% sucrose layers was harvested, concentrated by ultracentrifugation and then resuspended in PBS.

Ebola VP40 and GP genes were cloned into pIRES2-EGFP. Mammalian 293T cells were transfected with pIRES2-EGFP-VP40 alone or in combined with pIRES2-EGFP-GP expression vectors at equal DNA concentrations. 48h post-transfection, the supernatants (free of FBS) were collected and clarified with a cell spin. VLPs were purified by centrifugation through a sucrose cushion at 26000 rpm in a Beckman SW-28 rotor for 2 h at 4°C. eVLPs were resuspended in PBS. VP40 and eVLPs containing VP40 and GP proteins produced in mammalian 293T cells was designated VP40m and eVLPm respectively. The final concentration of eVLP protein was quantitated using the DC protein assay (Bio-Rad, Hercules, CA).