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Asexual Blood-Stage Malaria Vaccine Candidate PfRipr5: Enhanced Production in Insect Cells

The malaria asexual blood-stage antigen PfRipr and its most immunogenic fragment PfRipr5 have recently risen as promising vaccine candidates against this infectious disease. Continued development of high-yielding, scalable production platforms is essential to advance the malaria vaccine research. In...

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Autores principales: Correia, Ricardo, Fernandes, Bárbara, Castro, Rute, Nagaoka, Hikaru, Takashima, Eizo, Tsuboi, Takafumi, Fukushima, Akihisa, Viebig, Nicola K., Depraetere, Hilde, Alves, Paula M., Roldão, António
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9280424/
https://www.ncbi.nlm.nih.gov/pubmed/35845392
http://dx.doi.org/10.3389/fbioe.2022.908509
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author Correia, Ricardo
Fernandes, Bárbara
Castro, Rute
Nagaoka, Hikaru
Takashima, Eizo
Tsuboi, Takafumi
Fukushima, Akihisa
Viebig, Nicola K.
Depraetere, Hilde
Alves, Paula M.
Roldão, António
author_facet Correia, Ricardo
Fernandes, Bárbara
Castro, Rute
Nagaoka, Hikaru
Takashima, Eizo
Tsuboi, Takafumi
Fukushima, Akihisa
Viebig, Nicola K.
Depraetere, Hilde
Alves, Paula M.
Roldão, António
author_sort Correia, Ricardo
collection PubMed
description The malaria asexual blood-stage antigen PfRipr and its most immunogenic fragment PfRipr5 have recently risen as promising vaccine candidates against this infectious disease. Continued development of high-yielding, scalable production platforms is essential to advance the malaria vaccine research. Insect cells have supplied the production of numerous vaccine antigens in a fast and cost-effective manner; improving this platform further could prove key to its wider use. In this study, insect (Sf9 and High Five) and human (HEK293) cell hosts as well as process-optimizing strategies (new baculovirus construct designs and a culture temperature shift to hypothermic conditions) were employed to improve the production of the malaria asexual blood-stage vaccine candidate PfRipr5. Protein expression was maximized using High Five cells at CCI of 2 × 10(6) cell/mL and MOI of 0.1 pfu/cell (production yield = 0.49 mg/ml), with high-purity PfRipr5 binding to a conformational anti-PfRipr monoclonal antibody known to hold GIA activity and parasite PfRipr staining capacity. Further improvements in the PfRipr5 expression were achieved by designing novel expression vector sequences and performing a culture temperature shift to hypothermic culture conditions. Addition of one alanine (A) amino acid residue adjacent to the signal peptide cleavage site and a glycine-serine linker (GGSGG) between the PfRipr5 sequence and the purification tag (His(6)) induced a 2.2-fold increase in the expression of secreted PfRipr5 over using the expression vector with none of these additions. Performing a culture temperature shift from the standard 27–22°C at the time of infection improved the PfRipr5 expression by up to 1.7 fold. Notably, a synergistic effect was attained when combining both strategies, enabling to increase production yield post-purification by 5.2 fold, with similar protein quality (i.e., purity and binding to anti-PfRipr monoclonal antibody). This work highlights the potential of insect cells to produce the PfRipr5 malaria vaccine candidate and the importance of optimizing the expression vector and culture conditions to boost the expression of secreted proteins.
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spelling pubmed-92804242022-07-15 Asexual Blood-Stage Malaria Vaccine Candidate PfRipr5: Enhanced Production in Insect Cells Correia, Ricardo Fernandes, Bárbara Castro, Rute Nagaoka, Hikaru Takashima, Eizo Tsuboi, Takafumi Fukushima, Akihisa Viebig, Nicola K. Depraetere, Hilde Alves, Paula M. Roldão, António Front Bioeng Biotechnol Bioengineering and Biotechnology The malaria asexual blood-stage antigen PfRipr and its most immunogenic fragment PfRipr5 have recently risen as promising vaccine candidates against this infectious disease. Continued development of high-yielding, scalable production platforms is essential to advance the malaria vaccine research. Insect cells have supplied the production of numerous vaccine antigens in a fast and cost-effective manner; improving this platform further could prove key to its wider use. In this study, insect (Sf9 and High Five) and human (HEK293) cell hosts as well as process-optimizing strategies (new baculovirus construct designs and a culture temperature shift to hypothermic conditions) were employed to improve the production of the malaria asexual blood-stage vaccine candidate PfRipr5. Protein expression was maximized using High Five cells at CCI of 2 × 10(6) cell/mL and MOI of 0.1 pfu/cell (production yield = 0.49 mg/ml), with high-purity PfRipr5 binding to a conformational anti-PfRipr monoclonal antibody known to hold GIA activity and parasite PfRipr staining capacity. Further improvements in the PfRipr5 expression were achieved by designing novel expression vector sequences and performing a culture temperature shift to hypothermic culture conditions. Addition of one alanine (A) amino acid residue adjacent to the signal peptide cleavage site and a glycine-serine linker (GGSGG) between the PfRipr5 sequence and the purification tag (His(6)) induced a 2.2-fold increase in the expression of secreted PfRipr5 over using the expression vector with none of these additions. Performing a culture temperature shift from the standard 27–22°C at the time of infection improved the PfRipr5 expression by up to 1.7 fold. Notably, a synergistic effect was attained when combining both strategies, enabling to increase production yield post-purification by 5.2 fold, with similar protein quality (i.e., purity and binding to anti-PfRipr monoclonal antibody). This work highlights the potential of insect cells to produce the PfRipr5 malaria vaccine candidate and the importance of optimizing the expression vector and culture conditions to boost the expression of secreted proteins. Frontiers Media S.A. 2022-06-30 /pmc/articles/PMC9280424/ /pubmed/35845392 http://dx.doi.org/10.3389/fbioe.2022.908509 Text en Copyright © 2022 Correia, Fernandes, Castro, Nagaoka, Takashima, Tsuboi, Fukushima, Viebig, Depraetere, Alves and Roldão. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Correia, Ricardo
Fernandes, Bárbara
Castro, Rute
Nagaoka, Hikaru
Takashima, Eizo
Tsuboi, Takafumi
Fukushima, Akihisa
Viebig, Nicola K.
Depraetere, Hilde
Alves, Paula M.
Roldão, António
Asexual Blood-Stage Malaria Vaccine Candidate PfRipr5: Enhanced Production in Insect Cells
title Asexual Blood-Stage Malaria Vaccine Candidate PfRipr5: Enhanced Production in Insect Cells
title_full Asexual Blood-Stage Malaria Vaccine Candidate PfRipr5: Enhanced Production in Insect Cells
title_fullStr Asexual Blood-Stage Malaria Vaccine Candidate PfRipr5: Enhanced Production in Insect Cells
title_full_unstemmed Asexual Blood-Stage Malaria Vaccine Candidate PfRipr5: Enhanced Production in Insect Cells
title_short Asexual Blood-Stage Malaria Vaccine Candidate PfRipr5: Enhanced Production in Insect Cells
title_sort asexual blood-stage malaria vaccine candidate pfripr5: enhanced production in insect cells
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9280424/
https://www.ncbi.nlm.nih.gov/pubmed/35845392
http://dx.doi.org/10.3389/fbioe.2022.908509
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