title: 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 10(7) 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.
authors.affiliation: Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, 3710 McClintock Avenue, RTH-509, Los Angeles, California 90089, USA.
authors.forename: Chi-Lin
authors.initials: CL
authors.lastname: Lee
authors.forename: Michael
authors.initials: M
authors.lastname: Chou
authors.forename: Bingbing
authors.initials: B
authors.lastname: Dai
authors.forename: Liang
authors.initials: L
authors.lastname: Xiao
authors.forename: Pin
authors.initials: P
authors.lastname: Wang
mesh_terms: D000818:Animals; D001709:Biotechnology / Q000379:methods*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002460:Cell Line; D016895:Culture Media, Serum-Free; D003713:Dendritic Cells / Q000276:immunology*; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics*; D051379:Mice; D012845:Sindbis Virus / Q000235:genetics; D013194:Staining and Labeling; D013678:Technology, Pharmaceutical / Q000379:methods*; D046509:Tissue Culture Techniques / Q000379:methods; D014161:Transduction, Genetic; D014611:Vaccination / Q000379:methods*; D014759:Viral Envelope Proteins / Q000235:genetics; D019562:Viral Load
chemical_list: D016895:Culture Media, Serum-Free; D014759:Viral Envelope Proteins; D049452:Green Fluorescent Proteins