Early to Late VSV-G Expression in AcMNPV BV Enhances Transduction in Mammalian Cells but Does Not Affect Virion Yield in Insect Cells
<b>Background/Objectives:</b> Baculoviruses represent promising gene delivery vectors for mammalian systems, combining high safety profiles with substantial cargo capacity. While pseudotyping with vesicular stomatitis virus G-protein (VSV-G) enhances transduction efficiency, optimal expr...
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MDPI AG
2025-06-01
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| Series: | Vaccines |
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| Online Access: | https://www.mdpi.com/2076-393X/13/7/693 |
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| author | Jorge Alejandro Simonin Franco Uriel Cuccovia Warlet María del Rosario Bauzá María del Pilar Plastine Victoria Alfonso Fernanda Daniela Olea Carolina Susana Cerrudo Mariano Nicolás Belaich |
| author_facet | Jorge Alejandro Simonin Franco Uriel Cuccovia Warlet María del Rosario Bauzá María del Pilar Plastine Victoria Alfonso Fernanda Daniela Olea Carolina Susana Cerrudo Mariano Nicolás Belaich |
| author_sort | Jorge Alejandro Simonin |
| collection | DOAJ |
| description | <b>Background/Objectives:</b> Baculoviruses represent promising gene delivery vectors for mammalian systems, combining high safety profiles with substantial cargo capacity. While pseudotyping with vesicular stomatitis virus G-protein (VSV-G) enhances transduction efficiency, optimal expression strategies during the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection cycle remain unexplored. This study investigates how VSV-G expression timing affects pseudotype incorporation into budded virions (BVs) and subsequent transduction efficacy. <b>Methods:</b> Three recombinant AcMNPV constructs were generated, each expressing VSV-G under distinct baculoviral promoters (<i>ie1</i>, <i>gp64</i>, and <i>p10</i>) and GFP via a CMV promoter. VSV-G incorporation was verified by Western blot, while transduction efficiency was quantified in mammalian cell lines (fluorescence microscopy/flow cytometry) and rat hind limbs. Viral productivity was assessed through production kinetics and plaque assays. <b>Results:</b> All the pseudotyped viruses showed significantly enhanced transduction capacity versus controls, strongly correlating with VSV-G incorporation levels. The <i>p10</i> promoter drove the highest VSV-G expression and transduction efficiency. Crucially, BV production yields and infectivity remained unaffected by VSV-G expression timing. The in vivo results mirrored the cell culture findings, with p10-driven constructs showing greater GFP expression at low doses (10<sup>4</sup> virions). <b>Conclusions:</b> Strategic VSV-G expression via very late promoters (particularly <i>p10</i>) maximizes baculoviral transduction without compromising production yields. This study establishes a framework for optimizing pseudotyped BV systems, demonstrating that late-phase glycoprotein expression balances high mammalian transduction with preserved insect-cell productivity—a critical advancement for vaccine vector development. |
| format | Article |
| id | doaj-art-a946c33b3ffe4f43897072ce40cfa7eb |
| institution | Kabale University |
| issn | 2076-393X |
| language | English |
| publishDate | 2025-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Vaccines |
| spelling | doaj-art-a946c33b3ffe4f43897072ce40cfa7eb2025-08-20T03:32:35ZengMDPI AGVaccines2076-393X2025-06-0113769310.3390/vaccines13070693Early to Late VSV-G Expression in AcMNPV BV Enhances Transduction in Mammalian Cells but Does Not Affect Virion Yield in Insect CellsJorge Alejandro Simonin0Franco Uriel Cuccovia Warlet1María del Rosario Bauzá2María del Pilar Plastine3Victoria Alfonso4Fernanda Daniela Olea5Carolina Susana Cerrudo6Mariano Nicolás Belaich7Laboratorio de Ingeniería Genética y Biología Celular y Molecular (LIGBCM), Instituto de Microbiología Básica y Aplicada, Comisión de Investigaciones Científicas de la Provincia de Buenos Aires, Universidad Nacional de Quilmes, Buenos Aires B1876BXD, ArgentinaLaboratorio de Ingeniería Genética y Biología Celular y Molecular (LIGBCM), Instituto de Microbiología Básica y Aplicada, Comisión de Investigaciones Científicas de la Provincia de Buenos Aires, Universidad Nacional de Quilmes, Buenos Aires B1876BXD, ArgentinaLaboratorio de Medicina Regenerativa Cardiovascular, Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Favaloro, Buenos Aires C1078AAI, ArgentinaInstituto de Agrobiotecnología y Biología Molecular (IABIMO), Instituto Nacional de Tecnología Agropecuaria (INTA), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Hurlingham, Buenos Aires B1686WAA, ArgentinaInstituto de Agrobiotecnología y Biología Molecular (IABIMO), Instituto Nacional de Tecnología Agropecuaria (INTA), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Hurlingham, Buenos Aires B1686WAA, ArgentinaLaboratorio de Medicina Regenerativa Cardiovascular, Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Favaloro, Buenos Aires C1078AAI, ArgentinaLaboratorio de Ingeniería Genética y Biología Celular y Molecular (LIGBCM), Instituto de Microbiología Básica y Aplicada, Comisión de Investigaciones Científicas de la Provincia de Buenos Aires, Universidad Nacional de Quilmes, Buenos Aires B1876BXD, ArgentinaLaboratorio de Ingeniería Genética y Biología Celular y Molecular (LIGBCM), Instituto de Microbiología Básica y Aplicada, Comisión de Investigaciones Científicas de la Provincia de Buenos Aires, Universidad Nacional de Quilmes, Buenos Aires B1876BXD, Argentina<b>Background/Objectives:</b> Baculoviruses represent promising gene delivery vectors for mammalian systems, combining high safety profiles with substantial cargo capacity. While pseudotyping with vesicular stomatitis virus G-protein (VSV-G) enhances transduction efficiency, optimal expression strategies during the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection cycle remain unexplored. This study investigates how VSV-G expression timing affects pseudotype incorporation into budded virions (BVs) and subsequent transduction efficacy. <b>Methods:</b> Three recombinant AcMNPV constructs were generated, each expressing VSV-G under distinct baculoviral promoters (<i>ie1</i>, <i>gp64</i>, and <i>p10</i>) and GFP via a CMV promoter. VSV-G incorporation was verified by Western blot, while transduction efficiency was quantified in mammalian cell lines (fluorescence microscopy/flow cytometry) and rat hind limbs. Viral productivity was assessed through production kinetics and plaque assays. <b>Results:</b> All the pseudotyped viruses showed significantly enhanced transduction capacity versus controls, strongly correlating with VSV-G incorporation levels. The <i>p10</i> promoter drove the highest VSV-G expression and transduction efficiency. Crucially, BV production yields and infectivity remained unaffected by VSV-G expression timing. The in vivo results mirrored the cell culture findings, with p10-driven constructs showing greater GFP expression at low doses (10<sup>4</sup> virions). <b>Conclusions:</b> Strategic VSV-G expression via very late promoters (particularly <i>p10</i>) maximizes baculoviral transduction without compromising production yields. This study establishes a framework for optimizing pseudotyped BV systems, demonstrating that late-phase glycoprotein expression balances high mammalian transduction with preserved insect-cell productivity—a critical advancement for vaccine vector development.https://www.mdpi.com/2076-393X/13/7/693baculovirusAcMNPVVSV-Gviral-based gene delivery |
| spellingShingle | Jorge Alejandro Simonin Franco Uriel Cuccovia Warlet María del Rosario Bauzá María del Pilar Plastine Victoria Alfonso Fernanda Daniela Olea Carolina Susana Cerrudo Mariano Nicolás Belaich Early to Late VSV-G Expression in AcMNPV BV Enhances Transduction in Mammalian Cells but Does Not Affect Virion Yield in Insect Cells Vaccines baculovirus AcMNPV VSV-G viral-based gene delivery |
| title | Early to Late VSV-G Expression in AcMNPV BV Enhances Transduction in Mammalian Cells but Does Not Affect Virion Yield in Insect Cells |
| title_full | Early to Late VSV-G Expression in AcMNPV BV Enhances Transduction in Mammalian Cells but Does Not Affect Virion Yield in Insect Cells |
| title_fullStr | Early to Late VSV-G Expression in AcMNPV BV Enhances Transduction in Mammalian Cells but Does Not Affect Virion Yield in Insect Cells |
| title_full_unstemmed | Early to Late VSV-G Expression in AcMNPV BV Enhances Transduction in Mammalian Cells but Does Not Affect Virion Yield in Insect Cells |
| title_short | Early to Late VSV-G Expression in AcMNPV BV Enhances Transduction in Mammalian Cells but Does Not Affect Virion Yield in Insect Cells |
| title_sort | early to late vsv g expression in acmnpv bv enhances transduction in mammalian cells but does not affect virion yield in insect cells |
| topic | baculovirus AcMNPV VSV-G viral-based gene delivery |
| url | https://www.mdpi.com/2076-393X/13/7/693 |
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