Early to late VSV-G expression in AcMNPV BV enhances transduction in mammalian cells but does not affect virion yield in insect cells

Autores
Simonin, Jorge Alejandro; Cuccovia Warlet, Franco Uriel; Bauzá, María del Rosario; Plastine, María Del Pilar; Alfonso, Victoria; Olea, Fernanda Daniela; Cerrudo, Carolina Susana; Belaich, Mariano Nicolás
Año de publicación
2025
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Background/Objectives: 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. Methods: Three recombinant AcMNPV constructs were generated, each expressing VSV-G under distinct baculoviral promoters (ie1, gp64, and p10) 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. Results: All the pseudotyped viruses showed significantly enhanced transduction capacity versus controls, strongly correlating with VSV-G incorporation levels. The p10 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 (104 virions). Conclusions: Strategic VSV-G expression via very late promoters (particularly p10) 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.
Instituto de Biotecnología
Fil: Simonin, Jorge Alejandro. Universidad Nacional de Quilmes. Instituto de Microbiología Básica y Aplicada. Laboratorio de Ingeniería Genética y Biología Celular y Molecular (LIGBCM); Argentina
Fil: Simonin, Jorge Alejandro. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC); Argentina
Fil: Cuccovia Warlet, Franco Uriel. Universidad Nacional de Quilmes. Instituto de Microbiología Básica y Aplicada. Laboratorio de Ingeniería Genética y Biología Celular y Molecular (LIGBCM); Argentina
Fil: Cuccovia Warlet, Franco Uriel. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC); Argentina
Fil: Bauzá, María del Rosario. Universidad Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB). Laboratorio de Medicina Regenerativa Cardiovascular; Argentina
Fil: Bauzá, María del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Plastine, María Del Pilar. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina
Fil: Plastine, María Del Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Alfonso, Victoria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina
Fil: Alfonso, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Olea, Fernanda Daniela. Universidad Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB). Laboratorio de Medicina Regenerativa Cardiovascular; Argentina
Fil: Olea, Fernanda Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Cerrudo, Carolina Susana. Universidad Nacional de Quilmes. Instituto de Microbiología Básica y Aplicada. Laboratorio de Ingeniería Genética y Biología Celular y Molecular (LIGBCM); Argentina
Fil: Cerrudo, Carolina Susana. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC); Argentina
Fil: Belaich, Mariano Nicolás. Universidad Nacional de Quilmes. Instituto de Microbiología Básica y Aplicada. Laboratorio de Ingeniería Genética y Biología Celular y Molecular (LIGBCM); Argentina
Fil: Belaich, Mariano Nicolás. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC); Argentina
Fuente
Vaccines 13 (7) : 693 (July 2025)
Materia
Baculovirus
Virions
Cells
Vesicular Stomatitis Virus
Promoters
Vaccines
Virión
Células
Virus de la Estomatitis Vesicular
Autographa californica
Promotora
Vacuna
Nivel de accesibilidad
acceso abierto
Condiciones de uso
http://creativecommons.org/licenses/by-nc-sa/4.0/
Repositorio
INTA Digital (INTA)
Institución
Instituto Nacional de Tecnología Agropecuaria
OAI Identificador
oai:localhost:20.500.12123/23052
Acceder
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oai_identifier_str oai:localhost:20.500.12123/23052
network_acronym_str INTADig
repository_id_str l
network_name_str INTA Digital (INTA)
spelling Early to late VSV-G expression in AcMNPV BV enhances transduction in mammalian cells but does not affect virion yield in insect cellsSimonin, Jorge AlejandroCuccovia Warlet, Franco UrielBauzá, María del RosarioPlastine, María Del PilarAlfonso, VictoriaOlea, Fernanda DanielaCerrudo, Carolina SusanaBelaich, Mariano NicolásBaculovirusVirionsCellsVesicular Stomatitis VirusPromotersVaccinesViriónCélulasVirus de la Estomatitis VesicularAutographa californicaPromotoraVacunaBackground/Objectives: 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. Methods: Three recombinant AcMNPV constructs were generated, each expressing VSV-G under distinct baculoviral promoters (ie1, gp64, and p10) 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. Results: All the pseudotyped viruses showed significantly enhanced transduction capacity versus controls, strongly correlating with VSV-G incorporation levels. The p10 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 (104 virions). Conclusions: Strategic VSV-G expression via very late promoters (particularly p10) 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.Instituto de BiotecnologíaFil: Simonin, Jorge Alejandro. Universidad Nacional de Quilmes. Instituto de Microbiología Básica y Aplicada. Laboratorio de Ingeniería Genética y Biología Celular y Molecular (LIGBCM); ArgentinaFil: Simonin, Jorge Alejandro. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC); ArgentinaFil: Cuccovia Warlet, Franco Uriel. Universidad Nacional de Quilmes. Instituto de Microbiología Básica y Aplicada. Laboratorio de Ingeniería Genética y Biología Celular y Molecular (LIGBCM); ArgentinaFil: Cuccovia Warlet, Franco Uriel. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC); ArgentinaFil: Bauzá, María del Rosario. Universidad Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB). Laboratorio de Medicina Regenerativa Cardiovascular; ArgentinaFil: Bauzá, María del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Plastine, María Del Pilar. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Plastine, María Del Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alfonso, Victoria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Alfonso, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Olea, Fernanda Daniela. Universidad Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB). Laboratorio de Medicina Regenerativa Cardiovascular; ArgentinaFil: Olea, Fernanda Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cerrudo, Carolina Susana. Universidad Nacional de Quilmes. Instituto de Microbiología Básica y Aplicada. Laboratorio de Ingeniería Genética y Biología Celular y Molecular (LIGBCM); ArgentinaFil: Cerrudo, Carolina Susana. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC); ArgentinaFil: Belaich, Mariano Nicolás. Universidad Nacional de Quilmes. Instituto de Microbiología Básica y Aplicada. Laboratorio de Ingeniería Genética y Biología Celular y Molecular (LIGBCM); ArgentinaFil: Belaich, Mariano Nicolás. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC); ArgentinaMDPI2025-07-17T10:20:16Z2025-07-17T10:20:16Z2025-07info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfhttp://hdl.handle.net/20.500.12123/23052https://www.mdpi.com/2076-393X/13/7/6932076-393Xhttps://doi.org/10.3390/vaccines13070693Vaccines 13 (7) : 693 (July 2025)reponame:INTA Digital (INTA)instname:Instituto Nacional de Tecnología Agropecuariaenginfo:eu-repo/semantics/openAccesshttp://creativecommons.org/licenses/by-nc-sa/4.0/Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)2025-11-06T09:42:47Zoai:localhost:20.500.12123/23052instacron:INTAInstitucionalhttp://repositorio.inta.gob.ar/Organismo científico-tecnológicoNo correspondehttp://repositorio.inta.gob.ar/oai/requesttripaldi.nicolas@inta.gob.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:l2025-11-06 09:42:47.989INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuariafalse
dc.title.none.fl_str_mv Early to late VSV-G expression in AcMNPV BV enhances transduction in mammalian cells but does not affect virion yield in insect cells
title Early to late VSV-G expression in AcMNPV BV enhances transduction in mammalian cells but does not affect virion yield in insect cells
spellingShingle Early to late VSV-G expression in AcMNPV BV enhances transduction in mammalian cells but does not affect virion yield in insect cells
Simonin, Jorge Alejandro
Baculovirus
Virions
Cells
Vesicular Stomatitis Virus
Promoters
Vaccines
Virión
Células
Virus de la Estomatitis Vesicular
Autographa californica
Promotora
Vacuna
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_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_sort Early to late VSV-G expression in AcMNPV BV enhances transduction in mammalian cells but does not affect virion yield in insect cells
dc.creator.none.fl_str_mv Simonin, Jorge Alejandro
Cuccovia Warlet, Franco Uriel
Bauzá, María del Rosario
Plastine, María Del Pilar
Alfonso, Victoria
Olea, Fernanda Daniela
Cerrudo, Carolina Susana
Belaich, Mariano Nicolás
author Simonin, Jorge Alejandro
author_facet Simonin, Jorge Alejandro
Cuccovia Warlet, Franco Uriel
Bauzá, María del Rosario
Plastine, María Del Pilar
Alfonso, Victoria
Olea, Fernanda Daniela
Cerrudo, Carolina Susana
Belaich, Mariano Nicolás
author_role author
author2 Cuccovia Warlet, Franco Uriel
Bauzá, María del Rosario
Plastine, María Del Pilar
Alfonso, Victoria
Olea, Fernanda Daniela
Cerrudo, Carolina Susana
Belaich, Mariano Nicolás
author2_role author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Baculovirus
Virions
Cells
Vesicular Stomatitis Virus
Promoters
Vaccines
Virión
Células
Virus de la Estomatitis Vesicular
Autographa californica
Promotora
Vacuna
topic Baculovirus
Virions
Cells
Vesicular Stomatitis Virus
Promoters
Vaccines
Virión
Células
Virus de la Estomatitis Vesicular
Autographa californica
Promotora
Vacuna
dc.description.none.fl_txt_mv Background/Objectives: 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. Methods: Three recombinant AcMNPV constructs were generated, each expressing VSV-G under distinct baculoviral promoters (ie1, gp64, and p10) 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. Results: All the pseudotyped viruses showed significantly enhanced transduction capacity versus controls, strongly correlating with VSV-G incorporation levels. The p10 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 (104 virions). Conclusions: Strategic VSV-G expression via very late promoters (particularly p10) 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.
Instituto de Biotecnología
Fil: Simonin, Jorge Alejandro. Universidad Nacional de Quilmes. Instituto de Microbiología Básica y Aplicada. Laboratorio de Ingeniería Genética y Biología Celular y Molecular (LIGBCM); Argentina
Fil: Simonin, Jorge Alejandro. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC); Argentina
Fil: Cuccovia Warlet, Franco Uriel. Universidad Nacional de Quilmes. Instituto de Microbiología Básica y Aplicada. Laboratorio de Ingeniería Genética y Biología Celular y Molecular (LIGBCM); Argentina
Fil: Cuccovia Warlet, Franco Uriel. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC); Argentina
Fil: Bauzá, María del Rosario. Universidad Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB). Laboratorio de Medicina Regenerativa Cardiovascular; Argentina
Fil: Bauzá, María del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Plastine, María Del Pilar. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina
Fil: Plastine, María Del Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Alfonso, Victoria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina
Fil: Alfonso, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Olea, Fernanda Daniela. Universidad Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB). Laboratorio de Medicina Regenerativa Cardiovascular; Argentina
Fil: Olea, Fernanda Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Cerrudo, Carolina Susana. Universidad Nacional de Quilmes. Instituto de Microbiología Básica y Aplicada. Laboratorio de Ingeniería Genética y Biología Celular y Molecular (LIGBCM); Argentina
Fil: Cerrudo, Carolina Susana. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC); Argentina
Fil: Belaich, Mariano Nicolás. Universidad Nacional de Quilmes. Instituto de Microbiología Básica y Aplicada. Laboratorio de Ingeniería Genética y Biología Celular y Molecular (LIGBCM); Argentina
Fil: Belaich, Mariano Nicolás. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC); Argentina
description Background/Objectives: 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. Methods: Three recombinant AcMNPV constructs were generated, each expressing VSV-G under distinct baculoviral promoters (ie1, gp64, and p10) 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. Results: All the pseudotyped viruses showed significantly enhanced transduction capacity versus controls, strongly correlating with VSV-G incorporation levels. The p10 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 (104 virions). Conclusions: Strategic VSV-G expression via very late promoters (particularly p10) 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.
publishDate 2025
dc.date.none.fl_str_mv 2025-07-17T10:20:16Z
2025-07-17T10:20:16Z
2025-07
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/20.500.12123/23052
https://www.mdpi.com/2076-393X/13/7/693
2076-393X
https://doi.org/10.3390/vaccines13070693
url http://hdl.handle.net/20.500.12123/23052
https://www.mdpi.com/2076-393X/13/7/693
https://doi.org/10.3390/vaccines13070693
identifier_str_mv 2076-393X
dc.language.none.fl_str_mv eng
language eng
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
http://creativecommons.org/licenses/by-nc-sa/4.0/
Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)
eu_rights_str_mv openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by-nc-sa/4.0/
Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv MDPI
publisher.none.fl_str_mv MDPI
dc.source.none.fl_str_mv Vaccines 13 (7) : 693 (July 2025)
reponame:INTA Digital (INTA)
instname:Instituto Nacional de Tecnología Agropecuaria
reponame_str INTA Digital (INTA)
collection INTA Digital (INTA)
instname_str Instituto Nacional de Tecnología Agropecuaria
repository.name.fl_str_mv INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuaria
repository.mail.fl_str_mv tripaldi.nicolas@inta.gob.ar
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