ba9f869db4da3736ec149200ff8c78dd pmc_11209239.pdf 2403364e9dbcab41a6f2dfed217f7956790d86ed pmc_11209239.pdf cec7bbf31d34dc1d36ee17d278255d8298bd27554cd8888ff0b3e18584b3ea82 pmc_11209239.pdf Title: Production and Immunogenicity of FeLV Gag-Based VLPs Exposing a Stabilized FeLV Envelope Glycoprotein Subject: The envelope glycoprotein (Env) of retroviruses, such as the Feline leukemia virus (FeLV), is the main target of neutralizing humoral response, and therefore, a promising vaccine candidate, despite its reported poor immunogenicity. The incorporation of mutations that stabilize analogous proteins from other viruses in their prefusion conformation (e.g., HIV Env, SARS-CoV-2 S, or RSV F glycoproteins) has improved their capability to induce neutralizing protective immune responses. Therefore, we have stabilized the FeLV Env protein following a strategy based on the incorporation of a disulfide bond and an Ile/Pro mutation (SOSIP) previously used to generate soluble HIV Env trimers. We have characterized this SOSIP-FeLV Env in its soluble form and as a transmembrane protein present at high density on the surface of FeLV Gag-based VLPs. Furthermore, we have tested its immunogenicity in DNA-immunization assays in C57BL/6 mice. Low anti-FeLV Env responses were detected in SOSIP-FeLV soluble protein-immunized animals; however, unexpectedly no responses were detected in the animals immunized with SOSIP-FeLV Gag-based VLPs. In contrast, high humoral response against FeLV Gag was observed in the animals immunized with control Gag VLPs lacking SOSIP-FeLV Env, while this response was significantly impaired when the VLPs incorporated SOSIP-FeLV Env. Our data suggest that FeLV Env can be stabilized as a soluble protein and can be expressed in high-density VLPs. However, when formulated as a DNA vaccine, SOSIP-FeLV Env remains poorly immunogenic, a limitation that must be overcome to develop an effective FeLV vaccine. Keywords: Env; vaccine; virus-like particle (VLP); FeLV; SOSIP; veterinary science Author: Raquel Ortiz, Ana Barajas, Anna Pons-Grífols, Benjamin Trinité, Ferran Tarrés-Freixas, Carla Rovirosa, Víctor Urrea, Antonio Barreiro, Anna Gonzalez-Tendero, Maria Rovira-Rigau, Maria Cardona, Laura Ferrer, Bonaventura Clotet, Jorge Carrillo, Carmen Aguilar-Gurrieri and Julià Blanco Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Tue Jun 25 06:07:32 2024 CEST ModDate: Tue Jun 25 06:13:59 2024 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 14 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2021065 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- GGHKEL+VnURWPalladioL-Bold Type 1 Custom yes yes yes 10 0 MQZPFL+URWPalladioL-Bold Type 1 Custom yes yes yes 16 0 CQKXVD+URWPalladioL-Roma Type 1 Custom yes yes yes 22 0 OBRXIK+URWPalladioL-Ital Type 1 Custom yes yes yes 27 0 DDMOHK+VnURWPalladioL Type 1 Custom yes yes yes 32 0 HOWKDX+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 48 0 DKZMYW+CMSY10 Type 1 Builtin yes yes yes 65 0 OQIVQT+EURM10 Type 1 Builtin yes yes yes 70 0 HNDHMK+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 90 0 HNDHMM+PalatinoLinotype TrueType WinAnsi yes yes no 93 0 HNDHMO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 96 0 HNDIEM+PalatinoLinotype TrueType MacRoman yes yes no 99 0 MPUDFL+PazoMath Type 1 Builtin yes yes yes 107 0 HNDHMK+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 118 0 HNDHMM+PalatinoLinotype TrueType WinAnsi yes yes no 121 0 HNDHMO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 124 0 HNDIEM+PalatinoLinotype TrueType MacRoman yes yes no 127 0 HNDHMK+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 137 0 HNDHML+PalatinoLinotype CID TrueType Identity-H yes yes yes 140 0 HNDHMM+PalatinoLinotype TrueType WinAnsi yes yes no 146 0 HNDHMO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 149 0 HNDIEM+PalatinoLinotype TrueType MacRoman yes yes no 152 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-09-01 08:12:36 CEST RepresentationInformation: pmc_11209239.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-07-05 02:35:31 CEST Size: 2021065 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 374 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: Plasmids Destination: subsection.2.1 Item: Title: Cell Line, Culture Conditions, and Transfection Destination: subsection.2.2 Item: Title: Design, Production, and Purification of SOSIP-FeLV Soluble Protein Destination: subsection.2.3 Item: Title: Size-Exclusion High-Performance Liquid Chromatography (SE-HPLC) Destination: subsection.2.4 Item: Title: Analysis of VLP and Protein Production Destination: subsection.2.5 Children: Item: Title: Identification by Western Blot and Coomassie Blue Destination: subsubsection.2.5.1 Item: Title: Flow Cytometry Analysis of Recombinant Protein Expression Destination: subsubsection.2.5.2 Item: Title: Transmission (TEM) and Cryo-Transmission Electron Microscopy (Cryo-EM) Destination: subsubsection.2.5.3 Item: Title: Mice Immunization and Immunogenicity Analyses Destination: subsection.2.6 Children: Item: Title: Evaluation of Humoral Response by ELISA Destination: subsubsection.2.6.1 Item: Title: Design and Production of SU-huIgG Destination: subsubsection.2.6.2 Item: Title: Statistical Analysis Destination: subsubsection.2.6.3 Item: Title: Results Destination: section.3 Children: Item: Title: Generation of a Soluble Stable FeLV Env Trimer Destination: subsection.3.1 Item: Title: Generation of SOSIP-FeLV Gag-Based VLPs Destination: subsection.3.2 Item: Title: Immunogenicity of FeLV Gag-Based VLPs in C57BL/6 Mice Destination: subsection.3.3 Item: Title: Discussion Destination: section.4 Item: Title: References Destination: section.5 Info: Title: Production and Immunogenicity of FeLV Gag-Based VLPs Exposing a Stabilized FeLV Envelope Glycoprotein Author: Raquel Ortiz, Ana Barajas, Anna Pons-Grífols, Benjamin Trinité, Ferran Tarrés-Freixas, Carla Rovirosa, Víctor Urrea, Antonio Barreiro, Anna Gonzalez-Tendero, Maria Rovira-Rigau, Maria Cardona, Laura Ferrer, Bonaventura Clotet, Jorge Carrillo, Carmen Aguilar-Gurrieri and Julià Blanco Subject: The envelope glycoprotein (Env) of retroviruses, such as the Feline leukemia virus (FeLV), is the main target of neutralizing humoral response, and therefore, a promising vaccine candidate, despite its reported poor immunogenicity. The incorporation of mutations that stabilize analogous proteins from other viruses in their prefusion conformation (e.g., HIV Env, SARS-CoV-2 S, or RSV F glycoproteins) has improved their capability to induce neutralizing protective immune responses. Therefore, we have stabilized the FeLV Env protein following a strategy based on the incorporation of a disulfide bond and an Ile/Pro mutation (SOSIP) previously used to generate soluble HIV Env trimers. We have characterized this SOSIP-FeLV Env in its soluble form and as a transmembrane protein present at high density on the surface of FeLV Gag-based VLPs. Furthermore, we have tested its immunogenicity in DNA-immunization assays in C57BL/6 mice. Low anti-FeLV Env responses were detected in SOSIP-FeLV soluble protein-immunized animals; however, unexpectedly no responses were detected in the animals immunized with SOSIP-FeLV Gag-based VLPs. In contrast, high humoral response against FeLV Gag was observed in the animals immunized with control Gag VLPs lacking SOSIP-FeLV Env, while this response was significantly impaired when the VLPs incorporated SOSIP-FeLV Env. Our data suggest that FeLV Env can be stabilized as a soluble protein and can be expressed in high-density VLPs. However, when formulated as a DNA vaccine, SOSIP-FeLV Env remains poorly immunogenic, a limitation that must be overcome to develop an effective FeLV vaccine. 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