Use of the piggyBac transposon to create HIV-1 gag transgenic insect cell lines for continuous VLP production

Abstract

BackgroundInsect baculovirus-produced Human immunodeficiency virus type 1 (HIV-1) Gag virus-like-particles (VLPs) stimulate good humoral and cell-mediated immune responses in animals and are thought to be suitable as a vaccine candidate. Drawbacks to this production system include contamination of VLP preparations with baculovirus and the necessity for routine maintenance of infectious baculovirus stock. We used piggyBac transposition as a novel method to create transgenic insect cell lines for continuous VLP production as an alternative to the baculovirus system.ResultsTransgenic cell lines maintained stable gag transgene integration and expression up to 100 cell passages, and although the level of VLPs produced was low compared to baculovirus-produced VLPs, they appeared similar in size and morphology to baculovirus-expressed VLPs. In a murine immunogenicity study, whereas baculovirus-produced VLPs elicited good CD4 immune responses in mice when used to boost a prime with a DNA vaccine, no boost response was elicited by transgenically produced VLPs.ConclusionTransgenic insect cells are stable and can produce HIV Pr55 Gag VLPs for over 100 passages: this novel result may simplify strategies aimed at making protein subunit vaccines for HIV. Immunogenicity of the Gag VLPs in mice was less than that of baculovirus-produced VLPs, which may be due to lack of baculovirus glycoprotein incorporation in the transgenic cell VLPs. Improved yield and immunogenicity of transgenic cell-produced VLPs may be achieved with the addition of further genetic elements into the piggyBac integron.

A gene delivery system for insect cells mediated by arginine-rich cell-penetrating peptides

“…In addition to CPPs, transposon mutagenesis is another ideal chemical method of transfection techniques as it is based on a naturally occurring system in insect cells and has been widely used to transpose many insect species (Lynch et al, 2010). Especially, the piggyBac transposable element has been extensively studied as a useful tool in insect transgenesis recently due to its simplicity of movement and often high frequency of transformation (Ding et al, 2005; Lynch , 2010).…”

“…Drawbacks to use of the baculovirus expression system include the necessity for constant maintenance of baculovirus stocks, the need for fresh batch infections to be made each time and copurification of recombinant baculoviruses or baculoviral proteins with HIV-1 Gag virus-like-particles. Therefore, piggyBac transposition was applied as a novel method to replace the baculovirus expression system and to create transgenic insects for continuous production of HIV-1 Gag virus-like-particles (Lynch et al, 2010). In addition, the piggyBac transposon was surprisingly discovered to be used for cancer gene discovery in mice (Rad et al, 2010).…”

Abrogation of contaminating RNA activity in HIV-1 Gag VLPs

“…The purity of VLPs were assessed and Gag concentration of the GagCAT VLP preparation was measured by electrophoresis of purified VLPs through a 10% denaturing SDS polyacrylamide gel and western blotting [ 68 ]. To quantitate the Gag concentration a serial dilution of an HIV-1 p17/24 C clade protein standard (ARP695.2, FIT Biotech, Programme EVA, centre for AIDS reagents, NIBSC) was included on the gel.…”

HIV-1 Virus-Like Particles Produced by Stably Transfected Drosophila S2 Cells: a Desirable Vaccine Component

“…To quantify the amount of Gag p55 in supernatants produced by S2 clones in fed-batch culture, Western blot and densitometry analyses were performed as reported by Lynch et al (27). Briefly, culture supernatants were collected daily during 11 days of fed-batch culture (see above) and loaded onto 4 to 12% Bis-Tis gel (Invitrogen), along with a serially 2-folddiluted p24 standards (starting at 40 ng; Aalto BioReagents), and then transferred onto PVDF membranes.…”

“…Contamination of HIV-1 VLP preparations with live baculovirus is important with regard to immunogenicity and regulatory issues. Because of this, as an alternative to the baculovirus system, piggyback transposition that expressed HIV-1 Gag protein was developed to create transgenic insect cell lines for continuous HIV-1 Gag VLP production (27). Second, HIV-1 gp160 precursor envelope proteins are not cleaved in insect cells infected with RB (17), although the effect of uncleaved gp160 on immunogenicity has yet to be investigated.…”