Production and characterization of novel Anti-HIV Fc-fusion proteins in plant-based systems: Nicotiana benthamiana & tobacco BY-2 cell suspension

Noemi Gutierrez-Valdes; Francesc Cunyat; Juliette Balieu; Marie-Laure Walet-Balieu; Matthew j. Paul; Jonas De groot; Amaya Blanco-Perera; Jorge Carrillo; Patrice Lerouge; Mariëlle Jansma-Van Seters; Jussi j. Joensuu; Muriel Bardor; Julian Ma; Julià Blanco; Anneli Ritala

doi:10.1016/j.nbt.2024.08.499

Production and characterization of novel Anti-HIV Fc-fusion proteins in plant-based systems: Nicotiana benthamiana & tobacco BY-2 cell suspension

Noemi Gutierrez-Valdes, Francesc Cunyat, Juliette Balieu, Marie-Laure Walet-Balieu, Matthew j. Paul, Jonas De groot, Amaya Blanco-Perera, Jorge Carrillo, Patrice Lerouge, Mariëlle Jansma-Van Seters, Jussi j. Joensuu, Muriel Bardor, Julian Ma, Julià Blanco, Anneli Ritala (Corresponding Author)

Research output: Contribution to journal › Article › Scientific › peer-review

1 Downloads (Pure)

Abstract

Multifunctional anti-HIV Fc-fusion proteins aim to tackle HIV efficiently through multiple modes of action. Although results have been promising, these recombinant proteins are hard to produce. This study explored the production and characterization of anti-HIV Fc-fusion proteins in plant-based systems, specifically Nicotiana benthamiana plants and tobacco BY-2 cell suspension. Fc-fusion protein expression in plants was optimized by incorporating codon optimization, ER retention signals, and hydrophobin fusion elements. Successful transient protein expression was achieved in N. benthamiana, with notable improvements in expression levels achieved through N-terminal hydrophobin fusion and ER retention signals. Stable expression in tobacco BY-2 resulted in varying accumulation levels being at highest 2.2.mg/g DW. The inclusion of hydrophobin significantly enhanced accumulation, providing potential benefits for downstream processing. Mass spectrometry analysis confirmed the presence of the ER retention signal and of N-glycans. Functional characterization revealed strong binding to CD64 and CD16a receptors, the latter being important for antibody-dependent cellular cytotoxicity (ADCC). Interaction with HIV antigens indicated potential neutralization capabilities. In conclusion, this research highlights the potential of plant-based systems for producing functional anti-HIV Fc-fusion proteins, offering a promising avenue for the development of these novel HIV therapies.

Original language	English
Pages (from-to)	142-154
Number of pages	13
Journal	New Biotechnology
Volume	83
DOIs	https://doi.org/10.1016/j.nbt.2024.08.499
Publication status	Published - 25 Nov 2024
MoE publication type	A1 Journal article-refereed

Funding

This research was funded by the European Union's Horizon 2020 Research and Innovation Program, specifically through the Pharma-Factory project under grant agreement No. 774078. The authors also wish to acknowledge the valuable technical assistance provided by Jaana Rikkinen, Kaisa Rinta-Harri, and Tuuli Teikari from VTT. Furthermore, the University of Rouen Normandie's support is gratefully acknowledged. The glycoanalysis work received financial backing from the French government through the ANR agency, both under the Grand Défi Bioproduction and Biologics (ANR-21-F2II-0005) and the ANR PRCE DAGENTA project (ANR-21-CE20-0038-001).

Keywords

BY-2 cell suspension
HIV
Hydrophobins
KDEL ER retention signal
Nicotiana benthamiana
Plant molecular farming
Plant-produced biologics glycosylation

Access to Document

10.1016/j.nbt.2024.08.499Licence: CC BY

j.nbt.2024.08.499Final published version, 6.49 MBLicence: CC BY

Cite this

Gutierrez-Valdes, N., Cunyat, F., Balieu, J., Walet-Balieu, M.-L., Paul, M. J., De groot, J., Blanco-Perera, A., Carrillo, J., Lerouge, P., Seters, M. J.-V., Joensuu, J. J., Bardor, M., Ma, J., Blanco, J., & Ritala, A. (2024). Production and characterization of novel Anti-HIV Fc-fusion proteins in plant-based systems: Nicotiana benthamiana & tobacco BY-2 cell suspension. New Biotechnology, 83, 142-154. https://doi.org/10.1016/j.nbt.2024.08.499

@article{2f0ada8286cd450cb62f9f20f446fcec,

title = "Production and characterization of novel Anti-HIV Fc-fusion proteins in plant-based systems: Nicotiana benthamiana & tobacco BY-2 cell suspension",

abstract = "Multifunctional anti-HIV Fc-fusion proteins aim to tackle HIV efficiently through multiple modes of action. Although results have been promising, these recombinant proteins are hard to produce. This study explored the production and characterization of anti-HIV Fc-fusion proteins in plant-based systems, specifically Nicotiana benthamiana plants and tobacco BY-2 cell suspension. Fc-fusion protein expression in plants was optimized by incorporating codon optimization, ER retention signals, and hydrophobin fusion elements. Successful transient protein expression was achieved in N. benthamiana, with notable improvements in expression levels achieved through N-terminal hydrophobin fusion and ER retention signals. Stable expression in tobacco BY-2 resulted in varying accumulation levels being at highest 2.2.mg/g DW. The inclusion of hydrophobin significantly enhanced accumulation, providing potential benefits for downstream processing. Mass spectrometry analysis confirmed the presence of the ER retention signal and of N-glycans. Functional characterization revealed strong binding to CD64 and CD16a receptors, the latter being important for antibody-dependent cellular cytotoxicity (ADCC). Interaction with HIV antigens indicated potential neutralization capabilities. In conclusion, this research highlights the potential of plant-based systems for producing functional anti-HIV Fc-fusion proteins, offering a promising avenue for the development of these novel HIV therapies.",

keywords = "BY-2 cell suspension, HIV, Hydrophobins, KDEL ER retention signal, Nicotiana benthamiana, Plant molecular farming, Plant-produced biologics glycosylation",

author = "Noemi Gutierrez-Valdes and Francesc Cunyat and Juliette Balieu and Marie-Laure Walet-Balieu and Paul, {Matthew j.} and {De groot}, Jonas and Amaya Blanco-Perera and Jorge Carrillo and Patrice Lerouge and Seters, {Mari{\"e}lle Jansma-Van} and Joensuu, {Jussi j.} and Muriel Bardor and Julian Ma and Juli{\`a} Blanco and Anneli Ritala",

year = "2024",

month = nov,

day = "25",

doi = "10.1016/j.nbt.2024.08.499",

language = "English",

volume = "83",

pages = "142--154",

journal = "New Biotechnology",

issn = "1871-6784",

publisher = "Elsevier",

}

Gutierrez-Valdes, N, Cunyat, F, Balieu, J, Walet-Balieu, M-L, Paul, MJ, De groot, J, Blanco-Perera, A, Carrillo, J, Lerouge, P, Seters, MJ-V, Joensuu, JJ, Bardor, M, Ma, J, Blanco, J & Ritala, A 2024, 'Production and characterization of novel Anti-HIV Fc-fusion proteins in plant-based systems: Nicotiana benthamiana & tobacco BY-2 cell suspension', New Biotechnology, vol. 83, pp. 142-154. https://doi.org/10.1016/j.nbt.2024.08.499

TY - JOUR

T1 - Production and characterization of novel Anti-HIV Fc-fusion proteins in plant-based systems: Nicotiana benthamiana & tobacco BY-2 cell suspension

AU - Gutierrez-Valdes, Noemi

AU - Cunyat, Francesc

AU - Balieu, Juliette

AU - Walet-Balieu, Marie-Laure

AU - Paul, Matthew j.

AU - De groot, Jonas

AU - Blanco-Perera, Amaya

AU - Carrillo, Jorge

AU - Lerouge, Patrice

AU - Seters, Mariëlle Jansma-Van

AU - Joensuu, Jussi j.

AU - Bardor, Muriel

AU - Ma, Julian

AU - Blanco, Julià

AU - Ritala, Anneli

PY - 2024/11/25

Y1 - 2024/11/25

N2 - Multifunctional anti-HIV Fc-fusion proteins aim to tackle HIV efficiently through multiple modes of action. Although results have been promising, these recombinant proteins are hard to produce. This study explored the production and characterization of anti-HIV Fc-fusion proteins in plant-based systems, specifically Nicotiana benthamiana plants and tobacco BY-2 cell suspension. Fc-fusion protein expression in plants was optimized by incorporating codon optimization, ER retention signals, and hydrophobin fusion elements. Successful transient protein expression was achieved in N. benthamiana, with notable improvements in expression levels achieved through N-terminal hydrophobin fusion and ER retention signals. Stable expression in tobacco BY-2 resulted in varying accumulation levels being at highest 2.2.mg/g DW. The inclusion of hydrophobin significantly enhanced accumulation, providing potential benefits for downstream processing. Mass spectrometry analysis confirmed the presence of the ER retention signal and of N-glycans. Functional characterization revealed strong binding to CD64 and CD16a receptors, the latter being important for antibody-dependent cellular cytotoxicity (ADCC). Interaction with HIV antigens indicated potential neutralization capabilities. In conclusion, this research highlights the potential of plant-based systems for producing functional anti-HIV Fc-fusion proteins, offering a promising avenue for the development of these novel HIV therapies.

AB - Multifunctional anti-HIV Fc-fusion proteins aim to tackle HIV efficiently through multiple modes of action. Although results have been promising, these recombinant proteins are hard to produce. This study explored the production and characterization of anti-HIV Fc-fusion proteins in plant-based systems, specifically Nicotiana benthamiana plants and tobacco BY-2 cell suspension. Fc-fusion protein expression in plants was optimized by incorporating codon optimization, ER retention signals, and hydrophobin fusion elements. Successful transient protein expression was achieved in N. benthamiana, with notable improvements in expression levels achieved through N-terminal hydrophobin fusion and ER retention signals. Stable expression in tobacco BY-2 resulted in varying accumulation levels being at highest 2.2.mg/g DW. The inclusion of hydrophobin significantly enhanced accumulation, providing potential benefits for downstream processing. Mass spectrometry analysis confirmed the presence of the ER retention signal and of N-glycans. Functional characterization revealed strong binding to CD64 and CD16a receptors, the latter being important for antibody-dependent cellular cytotoxicity (ADCC). Interaction with HIV antigens indicated potential neutralization capabilities. In conclusion, this research highlights the potential of plant-based systems for producing functional anti-HIV Fc-fusion proteins, offering a promising avenue for the development of these novel HIV therapies.

KW - BY-2 cell suspension

KW - HIV

KW - Hydrophobins

KW - KDEL ER retention signal

KW - Nicotiana benthamiana

KW - Plant molecular farming

KW - Plant-produced biologics glycosylation

UR - http://www.scopus.com/inward/record.url?scp=85201434482&partnerID=8YFLogxK

U2 - 10.1016/j.nbt.2024.08.499

DO - 10.1016/j.nbt.2024.08.499

M3 - Article

SN - 1871-6784

VL - 83

SP - 142

EP - 154

JO - New Biotechnology

JF - New Biotechnology

ER -

Production and characterization of novel Anti-HIV Fc-fusion proteins in plant-based systems: Nicotiana benthamiana & tobacco BY-2 cell suspension

Abstract

Funding

Keywords

Access to Document

Other files and links

Fingerprint

Cite this