Optimized protocol for the large scale production of HIV pseudovirions by transient transfection of HEK293T cells with linear fully deacylated polyethylenimine.
HIV vaccine strategies which employ pseudovirions (PVs) as the source of antigen require large amounts of particles. These are typically generated by transient transfection of mammalian cells and purification of the released PVs from the culture supernatant. Since efficiency and cost of transfection are key issues, in this report the transfection efficiencies, achieved by employing a panel of high-molecular-weight linear polyethylenimines (PEIs) and small cross-linked PEIs, were analyzed and compared to those obtained by transfections with calcium phosphate or the commercial reagent Polyfect. High efficiencies were obtained using several of the modified PEIs, and the transfections with these inexpensive reagents were very robust. The observed efficiencies (as quantitated by amounts of expressed gene product) were two to four fold superior to calcium phosphate transfection and approximately equal to that achieved using Polyfect which is, however, prohibitively expensive for large scale applications. An optimized and rapid protocol for the large scale production and purification of HIV-PVs from 293T cells growing in so-called cell stacks and transfected with the best reagent identified, PEI87, is described here. The generated PVs, obtained with a yield in the range of 0.4mg virion-associated HIV-CA/liter culture supernatant, exhibited only very minimal contamination with non-viral proteins and were thus suitable for vaccination applications.
Devitt
Gerard
G
Forschungsschwerpunkt Infektion und Krebs, F020, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany.
Thomas
Mini
M
Klibanov
Alexander M
AM
Pfeiffer
Tanya
T
Bosch
Valerie
V
D016915:AIDS Vaccines / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D005822:Genetic Vectors; D006678:HIV / Q000235:genetics* / Q000302:isolation & purification* / Q000502:physiology; D006801:Humans; D010957:Plasmids; D011094:Polyethyleneimine / Q000737:chemistry; D014162:Transfection / Q000379:methods*; D014771:Virion / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology
D016915:AIDS Vaccines; D011094:Polyethyleneimine