Comparative Molecular Biology Approaches for the Production of Poliovirus Virus-Like Particles Using <italic toggle="yes">Pichia pastoris</italic>

ABSTRACT For enteroviruses such as poliovirus (PV), empty capsids, which are antigenically indistinguishable from mature virions, are produced naturally during viral infection. The production of such capsids recombinantly, in heterologous systems such as yeast, have great potential as virus-like par...

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Autores principales:	Lee Sherry, Keith Grehan, Joseph S. Snowden, Michael L. Knight, Oluwapelumi O. Adeyemi, David J. Rowlands, Nicola J. Stonehouse
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	enterovirus poliovirus virus-like particle Pichia pastoris Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/488d04d86fa140579e59ec88d5d3ee47
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spelling	oai:doaj.org-article:488d04d86fa140579e59ec88d5d3ee472021-11-15T15:29:16ZComparative Molecular Biology Approaches for the Production of Poliovirus Virus-Like Particles Using <italic toggle="yes">Pichia pastoris</italic>10.1128/mSphere.00838-192379-5042https://doaj.org/article/488d04d86fa140579e59ec88d5d3ee472020-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00838-19https://doaj.org/toc/2379-5042ABSTRACT For enteroviruses such as poliovirus (PV), empty capsids, which are antigenically indistinguishable from mature virions, are produced naturally during viral infection. The production of such capsids recombinantly, in heterologous systems such as yeast, have great potential as virus-like particle (VLP) vaccine candidates. Here, using PV as an exemplar, we show the production of VLPs in Pichia pastoris by coexpression of the structural precursor protein P1 and the viral protease 3CD. The level of expression of the potentially cytotoxic protease relative to that of the P1 precursor was modulated by three different approaches: expression of the P1 precursor and protease from different transcription units, separation of the P1 and protease proteins using the Thosea asigna virus (TaV) 2A translation interruption sequence, or separation of the P1 and protease-coding sequences by an internal ribosome entry site sequence from Rhopalosiphum padi virus (RhPV). We also investigate the antigenicity of VLPs containing previously characterized mutations when produced in Pichia. Finally, using transmission electron microscopy and two-dimensional classification, we show that Pichia-derived VLPs exhibited the classical icosahedral capsid structure displayed by enteroviruses. IMPORTANCE Although the current poliovirus immunization program has been extremely successful in reducing the number of cases of paralytic polio worldwide, now more cases are caused by vaccine-derived polioviruses than by wild poliovirus. Switching to inactivated poliovirus vaccines will reduce this over time; however, their production requires the growth of large amounts of virus. This biosafety concern can be addressed by producing just the virus capsid. The capsid serves to protect the genetic material, which causes disease when introduced into a cell. Therefore, empty capsids (virus-like particles [VLPs]), which lack the viral RNA genome, are safe both to make and to use. We exploit yeast as a versatile model expression system to produce VLPs, and here we specifically highlight the potential of this system to supply next-generation poliovirus vaccines to secure a polio-free world for the future.Lee SherryKeith GrehanJoseph S. SnowdenMichael L. KnightOluwapelumi O. AdeyemiDavid J. RowlandsNicola J. StonehouseAmerican Society for Microbiologyarticleenteroviruspoliovirusvirus-like particlePichia pastorisMicrobiologyQR1-502ENmSphere, Vol 5, Iss 2 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	enterovirus poliovirus virus-like particle Pichia pastoris Microbiology QR1-502
spellingShingle	enterovirus poliovirus virus-like particle Pichia pastoris Microbiology QR1-502 Lee Sherry Keith Grehan Joseph S. Snowden Michael L. Knight Oluwapelumi O. Adeyemi David J. Rowlands Nicola J. Stonehouse Comparative Molecular Biology Approaches for the Production of Poliovirus Virus-Like Particles Using <italic toggle="yes">Pichia pastoris</italic>
description	ABSTRACT For enteroviruses such as poliovirus (PV), empty capsids, which are antigenically indistinguishable from mature virions, are produced naturally during viral infection. The production of such capsids recombinantly, in heterologous systems such as yeast, have great potential as virus-like particle (VLP) vaccine candidates. Here, using PV as an exemplar, we show the production of VLPs in Pichia pastoris by coexpression of the structural precursor protein P1 and the viral protease 3CD. The level of expression of the potentially cytotoxic protease relative to that of the P1 precursor was modulated by three different approaches: expression of the P1 precursor and protease from different transcription units, separation of the P1 and protease proteins using the Thosea asigna virus (TaV) 2A translation interruption sequence, or separation of the P1 and protease-coding sequences by an internal ribosome entry site sequence from Rhopalosiphum padi virus (RhPV). We also investigate the antigenicity of VLPs containing previously characterized mutations when produced in Pichia. Finally, using transmission electron microscopy and two-dimensional classification, we show that Pichia-derived VLPs exhibited the classical icosahedral capsid structure displayed by enteroviruses. IMPORTANCE Although the current poliovirus immunization program has been extremely successful in reducing the number of cases of paralytic polio worldwide, now more cases are caused by vaccine-derived polioviruses than by wild poliovirus. Switching to inactivated poliovirus vaccines will reduce this over time; however, their production requires the growth of large amounts of virus. This biosafety concern can be addressed by producing just the virus capsid. The capsid serves to protect the genetic material, which causes disease when introduced into a cell. Therefore, empty capsids (virus-like particles [VLPs]), which lack the viral RNA genome, are safe both to make and to use. We exploit yeast as a versatile model expression system to produce VLPs, and here we specifically highlight the potential of this system to supply next-generation poliovirus vaccines to secure a polio-free world for the future.
format	article
author	Lee Sherry Keith Grehan Joseph S. Snowden Michael L. Knight Oluwapelumi O. Adeyemi David J. Rowlands Nicola J. Stonehouse
author_facet	Lee Sherry Keith Grehan Joseph S. Snowden Michael L. Knight Oluwapelumi O. Adeyemi David J. Rowlands Nicola J. Stonehouse
author_sort	Lee Sherry
title	Comparative Molecular Biology Approaches for the Production of Poliovirus Virus-Like Particles Using <italic toggle="yes">Pichia pastoris</italic>
title_short	Comparative Molecular Biology Approaches for the Production of Poliovirus Virus-Like Particles Using <italic toggle="yes">Pichia pastoris</italic>
title_full	Comparative Molecular Biology Approaches for the Production of Poliovirus Virus-Like Particles Using <italic toggle="yes">Pichia pastoris</italic>
title_fullStr	Comparative Molecular Biology Approaches for the Production of Poliovirus Virus-Like Particles Using <italic toggle="yes">Pichia pastoris</italic>
title_full_unstemmed	Comparative Molecular Biology Approaches for the Production of Poliovirus Virus-Like Particles Using <italic toggle="yes">Pichia pastoris</italic>
title_sort	comparative molecular biology approaches for the production of poliovirus virus-like particles using <italic toggle="yes">pichia pastoris</italic>
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/488d04d86fa140579e59ec88d5d3ee47
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Comparative Molecular Biology Approaches for the Production of Poliovirus Virus-Like Particles Using <italic toggle="yes">Pichia pastoris</italic>

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