Construction of stable producer cells to make high‐titer lentiviral vectors for dendritic cell‐based vaccination

Abstract

Lentiviral vectors (LVs) enveloped with an engineered Sindbis virus glycoprotein can specifically bind to dendritic cells (DCs) through the surface receptor DC-SIGN and induce antigen expression, thus providing an efficient method for delivering DC-directed vaccines. In this study, we constructed a stable producer line (LV-MGFP) for synthesizing DC-SIGN-targeted HIV-1-based LVs (DC-LVs) encoding green fluorescent protein (GFP) by a concatemeric array transfection technique. We demonstrated that the established stable clones could routinely produce vector supernatants with titers above 107 transduction units per milliliter (TU/mL) during a continuous 3-month cell passage. The producer cells were also capable of generating similar titers of DC-LVs in serum-free medium. Moreover, the addition of 1-deoxymannojirimycin (DMJ) enabled the producer cells to manufacture DC-LVs with both improved titers and enhanced potency to evoke antigen-specific CD8+ T cell responses in mice. The stable lines could accommodate the replacement of the internal murine stem cell virus (MSCV) promoter with the human ubiquitin-c (Ubi) promoter in the lentiviral backbone. The resulting DC-LVs bearing Ubi exhibited the enhanced potency to elicit vaccine-specific immunity. Based on accumulated evidence, our studies support the application of this production method in manufacturing DC-LVs for preclinical and clinical testing of novel DC-based immunization.

Dendritic cell–targeted lentiviral vector immunization uses pseudotransduction and DNA-mediated STING and cGAS activation

“…Furthermore, LVs generated by either transient transfection using plasmids or plasmid-free packaging system similarly stimulated wild-type BMDCs (Fig 7I), suggesting that plasmid DNA within the vector preparations was not likely a dominant activator of DCs. LVs generated by plasmid DNA–free cell lines capably stimulate innate and adaptive immune responses in vivo (41, 42). These findings provide an explanation for the STING and cGAS dependence observed in the innate and adaptive immune responses generated by LVs and VLPs.…”

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

“…As shown in Figure 1B, isolation of individual clones yielded stable cell lines of varying quality 12 . Among 26 clones tested, 8 produced lentiviral particles at a titer of greater than 10 6 transduction units per ml (TU/ml), which is a typical benchmark for SVG-pseudotyped lentiviral vectors produced by transient transfection.…”

“…For example, co-transfection with large quantities of LV plasmids could theoretically increase the chance for the development of replication competent lentivirus 19 . Furthermore, transient transfections are more likely to produce variable results on a large scale than utilizing a stable producer line due to the large quantities of DNA inputs required 12 .…”

“…Lentiviral transfer plasmid TL20-GFP is a self-inactivating lentiviral transfer vector plasmid based on pCL20c-MSCV-GFP with a Dox-regulatable viral RNA genome expression system and replacement of the cytomegalovirus (CMV) enhancer with 7 tet operators( Figure 1C) 11,12,15 . Plasmid PGK-ble is a bleomycin resistant (ble) cassette driven by a weak PGK promoter 2 .…”

“…One of these lines, the GPR packaging line 11 , has the attractive advantage of being regulated by tetracycline. In this paper, we demonstrate how to adapt this system to produce self-inactivating lentiviral vectors that are specifically targeted toward dendritic cells (DC-LVs) 12 .…”