Design and immunogenicity assessment of HIV-1 virus-like particles as a candidate vaccine

Zhang, XiZhen; Wang, XiaoDan; Zhao, DongHai; Meng, XiangYu; Zhao, XingHong; Yu, XiangHui; Kong, Wei

Recombinant HEK293 cells expressing the HIV-1 structural proteins Gag and Env were obtained by cotransfecting plasmid D-GPEi and pcDNA3.1() into 293 cells. The plasmid D-GPEi was composed of the CMV promoter, a kanamycin resistance gene, prokaryotic cell high copy factors, and intron A. The main structural proteins Gag, pol and Env of a common China HIV-1 strain were cloned into this expression vector, with Gag fused to the N terminal of Pol as described [23] . All plasmid cloning was consistent with USA FDA clinical trial safety controls. Briefly, HEK293 cells were maintained in Dulbecco's modified minimal essential medium (DMEM) supplemented with 10% fetal calf serum (HyClone Laboratories, Logan, UT, USA). D-GPEi and pcDNA3.1() plasmids were cotransfected into 293 cells using Lipofectamine-2000. Adherent G418 resistant cells were trypsinized and serial diluted into medium containing G418 48 h after transfection. Stable 293 cell lines were maintained in standard growth medium with G418 (1 mg mL 1 ). Cells were harvested and assayed by Western blot after 2-3 weeks to assess clonality. The positive supernatants were collected by centrifugation at 4000 r min 1 for 30 min and passed through a 0.22 μm filter. VLPs were pelleted at 26000 r min 1 for 1.5 h at 4°C in a Beckman SW28 rotor through a 30% sucrose cushion twice, and their assembly was confirmed by standard electron microscopy analysis and Western blot analysis.