4618bd74bbc3f06d37670198ac38da66 pmc_8954388.pdf 4d59ec3939aa064f6bbe096a84f1c6436eca8eb0 pmc_8954388.pdf e65f61287b4456504952537d25474b33fdef66e909b68e6aa00d38dc8968d9d4 pmc_8954388.pdf Title: Transduction of HEK293 Cells with BacMam Baculovirus Is an Efficient System for the Production of HIV-1 Virus-like Particles Subject: Gag virus-like particles (VLPs) are promising vaccine candidates against infectious diseases. VLPs are generally produced using the insect cell/baculovirus expression vector system (BEVS), or in mammalian cells by plasmid DNA transient gene expression (TGE). However, VLPs produced with the insect cell/BEVS are difficult to purify and might not display the appropriate post-translational modifications, whereas plasmid DNA TGE approaches are expensive and have a limited scale-up capability. In this study, the production of Gag VLPs with the BacMam expression system in a suspension culture of HEK293 cells is addressed. The optimal conditions of multiplicity of infection (MOI), viable cell density (VCD) at infection, and butyric acid (BA) concentration that maximize cell transduction and VLP production are determined. In these conditions, a maximum cell transduction efficiency of 91.5 1.1%, and a VLP titer of 2.8 0.1 109 VLPs/mL are achieved. Successful VLP generation in transduced HEK293 cells is validated using super-resolution fluorescence microscopy, with VLPs produced resembling immature HIV-1 virions and with an average size comprised in the 100–200 nm range. Additionally, evidence that BacMam transduction occurs via different pathways including dynamin-mediated endocytosis and macropinocytosis is provided. This work puts the basis for future studies aiming at scaling up the BacMam baculovirus system as an alternative strategy for VLP production. Keywords: BacMam baculovirus; HIV-1; virus-like particle; HEK293 cells; super-resolution fluorescence microscopy; endocytosis Author: Eduard Puente-Massaguer, Byron Cajamarca-Berrezueta, Aleix Volart, Irene González-Domínguez and Francesc Gòdia Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Mar 18 13:28:47 2022 CET ModDate: Fri Mar 18 15:51:50 2022 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 16 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2693717 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- LPJXBF+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 KORLVE+VnURWPalladioL Type 1 Custom yes yes no 15 0 RWSBEW+URWPalladioL-Bold Type 1 Custom yes yes no 19 0 SBHUWU+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 MNPOMR+URWPalladioL-Ital Type 1 Custom yes yes no 28 0 RGUFCP+CMSY10 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Linotype TrueType WinAnsi yes yes no 172 0 ABCDEE+Palatino Linotype,Italic TrueType WinAnsi yes yes no 176 0 ABCDEE+Palatino Linotype CID TrueType Identity-H yes yes yes 180 0 ABCDEE+Palatino Linotype,Bold TrueType WinAnsi yes yes no 197 0 ABCDEE+Palatino Linotype TrueType WinAnsi yes yes no 201 0 ABCDEE+Palatino Linotype,Italic TrueType WinAnsi yes yes no 205 0 ABCDEE+Palatino Linotype,Bold TrueType WinAnsi yes yes no 221 0 ABCDEE+Palatino Linotype TrueType WinAnsi yes yes no 225 0 ABCDEE+Palatino Linotype,Italic TrueType WinAnsi yes yes no 229 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-04-27 02:32:39 CEST RepresentationInformation: pmc_8954388.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-04-26 17:59:43 CEST Size: 2693717 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 428 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Cell Lines, Media, and Culture Conditions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: BacMam Transfer Plasmid Construction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Generation of BacMam Working Stock and Titration Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Butyric Acid Toxicity Assays Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: VLP Production in HEK293 Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: D-Optimal Design Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Flow Cytometry Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Spectrofluorometry Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Nanoparticle Tracking Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Super-Resolution Confocal Microscopy Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Electron Microscopy Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Inhibition of BacMam Baculovirus Uptake Pathways Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Children: Item: Title: Screening of Transduction Conditions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Optimization of HEK293 Cell Transduction by Design of Experiments (DoE) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Characterization of VLP Production with the BacMam Expression System Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Info: Title: Transduction of HEK293 Cells with BacMam Baculovirus Is an Efficient System for the Production of HIV-1 Virus-like Particles Author: Eduard Puente-Massaguer, Byron Cajamarca-Berrezueta, Aleix Volart, Irene González-Domínguez and Francesc Gòdia Subject: Gag virus-like particles (VLPs) are promising vaccine candidates against infectious diseases. VLPs are generally produced using the insect cell/baculovirus expression vector system (BEVS), or in mammalian cells by plasmid DNA transient gene expression (TGE). However, VLPs produced with the insect cell/BEVS are difficult to purify and might not display the appropriate post-translational modifications, whereas plasmid DNA TGE approaches are expensive and have a limited scale-up capability. In this study, the production of Gag VLPs with the BacMam expression system in a suspension culture of HEK293 cells is addressed. The optimal conditions of multiplicity of infection (MOI), viable cell density (VCD) at infection, and butyric acid (BA) concentration that maximize cell transduction and VLP production are determined. In these conditions, a maximum cell transduction efficiency of 91.5 1.1%, and a VLP titer of 2.8 0.1 109 VLPs/mL are achieved. Successful VLP generation in transduced HEK293 cells is validated using super-resolution fluorescence microscopy, with VLPs produced resembling immature HIV-1 virions and with an average size comprised in the 100 200 nm range. Additionally, evidence that BacMam transduction occurs via different pathways including dynamin-mediated endocytosis and macropinocytosis is provided. This work puts the basis for future studies aiming at scaling up the BacMam baculovirus system as an alternative strategy for VLP production. 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