84556fdc2778fdd51187ef17156aa152 pmc_33809060.pdf 21b7aadd8878612dae3bf93e7e1e42f9311ada9c pmc_33809060.pdf 74250454639923cd5a181c732ef0013789d3034dfc02522e0c4ed64726596171 pmc_33809060.pdf Title: Chimeric VLPs Based on HIV-1 Gag and a Fusion Rabies Glycoprotein Induce Specific Antibodies against Rabies and Foot-and-Mouth Disease Virus Subject: Foot and mouth disease is a livestock acute disease, causing economic losses in affected areas. Currently, control of this disease is performed by mandatory vaccination campaigns using inactivated viral vaccines. In this work, we describe the development of a chimeric VLP-based vaccine candidate for foot-and-mouth disease virus (FMDV), based on the co-expression of the HIV-1 Gag protein and a novel fusion rabies glycoprotein (RVG), which carries in its N-term the FMDV main antigen: the G-H loop. It is demonstrated by confocal microscopy that both Gag-GFP polyprotein and the G-H loop colocalize at the cell membrane and, that the Gag polyprotein of the HIV virus acts as a scaffold for enveloped VLPs that during the budding process acquires the proteins that are being expressed in the cell membrane. The obtained VLPs were spherical particles of 130 40 nm in diameter (analyzed by TEM, Cryo-TEM and NTA) carrying an envelope membrane that efficiently display the GH-RVG on its surface (analyzed by gold immunolabeling). Immunostainings with a FMDV hyperimmune serum showed that the heterologous antigenic site, genetically fused to RVG, is recognized by specific G-H loop antibodies. Additionally, the cVLPs produced expose the G-H loop to the liquid surrounding (analyzed by specific ELISA). Finally, we confirmed that these FMD cVLPs are able to induce a specific humoral immune response, based on antibodies directed to the G-H loop in experimental animals. Keywords: virus-like particles; rabies; foot-and-mouth disease; HIV-1; chimeric; vaccine Author: Diego Fontana, Ernesto Garay, Laura Cevera, Ricardo Kratje, Claudio Prieto and Francesc Gòdia Creator: LaTeX with hyperref Producer: MiKTeX pdfTeX-1.40.21 CreationDate: Thu Mar 25 10:06:57 2021 CET ModDate: Thu Mar 25 11:01:18 2021 CET Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 17 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 4797283 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- YNZXYR+VnURWPalladioL Type 1 Custom yes yes no 10 0 ISMLRP+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 GTPCVS+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 GZKUEI+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 QMDDXF+CMSY10 Type 1 Builtin yes yes no 28 0 WLFTRW+VnURWPalladioL-Bold Type 1 Custom yes yes no 32 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 59 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 63 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 67 0 DVTUXA+EURM10 Type 1 Builtin yes yes no 85 0 NNJLMH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 107 0 NNJLMI+PalatinoLinotype CID TrueType Identity-H yes yes yes 110 0 NNJLMJ+PalatinoLinotype TrueType WinAnsi yes yes no 116 0 NNJLNL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 119 0 NNJLMH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 133 0 NNJLMJ+PalatinoLinotype TrueType WinAnsi yes yes no 136 0 NNJLNL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 139 0 NNJLMH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 153 0 NNJLMI+PalatinoLinotype CID TrueType Identity-H yes yes yes 156 0 NNJLMJ+PalatinoLinotype TrueType WinAnsi yes yes no 162 0 NNJLNL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 165 0 NNJLMH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 180 0 NNJLMJ+PalatinoLinotype TrueType WinAnsi yes yes no 183 0 NNJLNL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 186 0 NNJLMH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 197 0 NNJLMI+PalatinoLinotype CID TrueType Identity-H yes yes yes 200 0 NNJLMJ+PalatinoLinotype TrueType WinAnsi yes yes no 206 0 NNJLNL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 209 0 NNJLMH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 228 0 NNJLMJ+PalatinoLinotype TrueType WinAnsi yes yes no 231 0 NNJLNL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 234 0 NNJLMH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 245 0 NNJLMJ+PalatinoLinotype TrueType WinAnsi yes yes no 248 0 NNJLNL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 251 0 CRUZTT+VnURWPalladioL-Italic Type 1 Custom yes yes no 267 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-10-02 03:24:09 CEST RepresentationInformation: pmc_33809060.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-03-31 12:09:34 CEST Size: 4797283 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 498 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Fusion Protein Construction. Plasmids Destination: subsection.2.1 Item: Title: Cell Line, Media, and Culture Conditions Destination: subsection.2.2 Item: Title: Transient Transfection Destination: subsection.2.3 Item: Title: VLP Purification Destination: subsection.2.4 Item: Title: Confocal Microscopy Destination: subsection.2.5 Children: Item: Title: Standard Confocal Laser Scanning Microscopy (CLSM) Destination: subsubsection.2.5.1 Item: Title: Super-Resolution Fluorescence Microscopy (SRFM) Destination: subsubsection.2.5.2 Item: Title: Flow Cytometry Destination: subsection.2.6 Item: Title: VLPs Analysis and Quantitation Destination: subsection.2.7 Children: Item: Title: Bi-specific” Sandwich ELISA Destination: subsubsection.2.7.1 Item: Title: Fluorescence-Based Quantification of VLP Production Destination: subsubsection.2.7.2 Item: Title: Nanoparticle Tracking Analysis (NTA) Destination: subsubsection.2.7.3 Item: Title: Electron Microscopy Destination: subsection.2.8 Children: Item: Title: Transmission Electron Microscopy (TEM) Destination: subsubsection.2.8.1 Item: Title: Cryogenic Electron Microscopy (CryoTEM) Destination: subsubsection.2.8.2 Item: Title: Mice Immunization and Serology Destination: subsection.2.9 Children: Item: Title: Immunization Plan Destination: subsubsection.2.9.1 Item: Title: Quantification of RV Specific Immunoglobulins Destination: subsubsection.2.9.2 Item: Title: Quantification of FMD Disease Epitope Specific Immunoglobulins Destination: subsubsection.2.9.3 Item: Title: Statistical Analysis Destination: subsection.2.10 Item: Title: Results Destination: section.3 Children: Item: Title: Design, Construction and Analysis of the Expression of GH-RVG Fusion Protein Destination: subsection.3.1 Item: Title: cVLP Expression and Characterization Destination: subsection.3.2 Item: Title: cVLPs Production Optimization Destination: subsection.3.3 Item: Title: Evaluation of cVLPs Immunogenicity in Mice Destination: subsection.3.4 Item: Title: Discussion Destination: section.4 Item: Title: References Destination: section.4 Info: Title: Chimeric VLPs Based on HIV-1 Gag and a Fusion Rabies Glycoprotein Induce Specific Antibodies against Rabies and Foot-and-Mouth Disease Virus Author: Diego Fontana, Ernesto Garay, Laura Cevera, Ricardo Kratje, Claudio Prieto and Francesc Gòdia Subject: Foot and mouth disease is a livestock acute disease, causing economic losses in affected areas. Currently, control of this disease is performed by mandatory vaccination campaigns using inactivated viral vaccines. In this work, we describe the development of a chimeric VLP-based vaccine candidate for foot-and-mouth disease virus (FMDV), based on the co-expression of the HIV-1 Gag protein and a novel fusion rabies glycoprotein (RVG), which carries in its N-term the FMDV main antigen: the G-H loop. It is demonstrated by confocal microscopy that both Gag-GFP polyprotein and the G-H loop colocalize at the cell membrane and, that the Gag polyprotein of the HIV virus acts as a scaffold for enveloped VLPs that during the budding process acquires the proteins that are being expressed in the cell membrane. The obtained VLPs were spherical particles of 130 40 nm in diameter (analyzed by TEM, Cryo-TEM and NTA) carrying an envelope membrane that efficiently display the GH-RVG on its surface (analyzed by gold immunolabeling). Immunostainings with a FMDV hyperimmune serum showed that the heterologous antigenic site, genetically fused to RVG, is recognized by specific G-H loop antibodies. Additionally, the cVLPs produced expose the G-H loop to the liquid surrounding (analyzed by specific ELISA). Finally, we confirmed that these FMD cVLPs are able to induce a specific humoral immune response, based on antibodies directed to the G-H loop in experimental animals. 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