2a8cc7365042e641fa7e741ea2da03fb vaccines_a2022v10n2p250.pdf eacaa8557620ab141cfbf064419244d93e138060 vaccines_a2022v10n2p250.pdf 4b7d29055073ff00752f5316bf9d809c3a13b8163e39685fc8a052f278c59a67 vaccines_a2022v10n2p250.pdf Title: Optimization, Production, Purification and Characterization of HIV-1 GAG-Based Virus-like Particles Functionalized with SARS-CoV-2 Subject: Virus-like particles (VLPs) constitute a promising approach to recombinant vaccine development. They are robust, safe, versatile and highly immunogenic supra-molecular structures that closely mimic the native conformation of viruses without carrying their genetic material. HIV-1 Gag VLPs share similar characteristics with wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, making them a suitable platform for the expression of its spike membrane protein to generate a potential vaccine candidate for COVID-19. This work proposes a methodology for the generation of SARS-CoV-2 VLPs by their co-expression with HIV-1 Gag protein. We achieved VLP functionalization with coronavirus spike protein, optimized its expression using a design of experiments (DoE). We also performed the bioprocess at a bioreactor scale followed by a scalable downstream purification process consisting of two clarifications, an ion exchange and size-exclusion chromatography. The whole production process is conceived to enhance its transferability at current good manufacturing practice (cGMP) industrial scale manufacturing. Moreover, the approach proposed could be expanded to produce additional Gag-based VLPs against different diseases or COVID-19 variants. Keywords: VLP vaccines; HIV-1; SARS-CoV-2; COVID-19; transient transfection; HEK293; design of experiments; bioprocess; downstream process Author: Arnau Boix-Besora, Elianet Lorenzo, Jesús Lavado-García, Francesc Gòdia and Laura Cervera Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Feb 14 08:16:45 2022 CET ModDate: Mon Feb 14 08:22:44 2022 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 18 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3810724 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- KJSUBN+VnURWPalladioL Type 1 Custom yes yes no 10 0 VVORFN+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 XKBTOE+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 SBMECT+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 QQOOTE+CMSY10 Type 1 Builtin yes yes no 28 0 BZAVBL+VnURWPalladioL-Bold Type 1 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RepresentationInformation: vaccines_a2022v10n2p250.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-02-28 12:36:38 CET Size: 3810724 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 361 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 Line, Media and Culture Conditions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Plasmids and Transfection Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Children: Item: Title: Plasmid Expression Vectors Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Plasmid Amplification and Purification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: PEI-Mediated Transient Transfection Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Box Behnken Design Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Stirred Tank Reactor (STR) Bioprocess Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Sucrose Cushion Small-Scale Purification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Downstream Processing Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Children: Item: Title: Cell Harvest and Culture Supernatant Clarification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Ion-exchange chromatography (IEX) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Size-Exclusion Chromatography Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Immunocytochemistry Staining for Flow Citometry and Confocal Microscopy Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Children: Item: Title: Flow Cytometry Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Confocal Microscopy Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Transmission Electron Microscopy Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Children: Item: Title: Transmission Electron Microscopy: Negative Staining Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Transmission Electron Microscopy: Immunogold Labeling Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: Nanoparticle Tracking Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: Total Protein and dsDNA Quantification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Western Blot and SDS-PAGE Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Dot Blot Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Children: Item: Title: SARS-CoV-2 Spike Protein Co-Expression and Localization Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Item: Title: Characterization of the Produced S-VLPs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612679d6 Item: Title: Transient Transfection Optimization by a Box Behnken Design of Experiments Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@11758f2a Item: Title: Production in Stirred-Tank Bioreactor Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@e720b71 Item: Title: Downstream Process of the Produced S-VLPs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1b26f7b2 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@491cc5c9 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@74ad1f1f Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6a1aab78 Info: Title: Optimization, Production, Purification and Characterization of HIV-1 GAG-Based Virus-like Particles Functionalized with SARS-CoV-2 Author: Arnau Boix-Besora, Elianet Lorenzo, Jesús Lavado-García, Francesc Gòdia and Laura Cervera Subject: Virus-like particles (VLPs) constitute a promising approach to recombinant vaccine development. They are robust, safe, versatile and highly immunogenic supra-molecular structures that closely mimic the native conformation of viruses without carrying their genetic material. HIV-1 Gag VLPs share similar characteristics with wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, making them a suitable platform for the expression of its spike membrane protein to generate a potential vaccine candidate for COVID-19. This work proposes a methodology for the generation of SARS-CoV-2 VLPs by their co-expression with HIV-1 Gag protein. We achieved VLP functionalization with coronavirus spike protein, optimized its expression using a design of experiments (DoE). We also performed the bioprocess at a bioreactor scale followed by a scalable downstream purification process consisting of two clarifications, an ion exchange and size-exclusion chromatography. The whole production process is conceived to enhance its transferability at current good manufacturing practice (cGMP) industrial scale manufacturing. Moreover, the approach proposed could be expanded to produce additional Gag-based VLPs against different diseases or COVID-19 variants. 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