High-Yield Production of Receptor Binding Domain of SARS-CoV-2 Linked to Bacterial Flagellin in Plants Using Self-Replicating Viral Vector pEff

Mardanova, Eugenia S.; Kotlyarov, Roman Y.; Ravin, Nikolai V.

doi:10.3390/plants10122682

Cited by 12 publications

(16 citation statements)

References 47 publications

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“…Such a modification may also negate the cost-effectiveness of the procedure proposed in our study, however, and so further analysis is required. The expression system used to drive recombinant protein production also has a dramatic effect on purification yield, as demonstrated by the use of the potato virus X-based pEff vector [ 34 ]. Incorporating this vector into our protocol in the future could easily fast-track the scaling-up process.…”

Section: Discussionmentioning

confidence: 99%

Scalable agroinfiltration-based production of SARS-CoV-2 antigens for use in diagnostic assays and subunit vaccines

et al. 2022

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Agroinfiltration is a method used in biopharming to support plant-based biosynthesis of therapeutic proteins such as antibodies and viral antigens involved in vaccines. Major advantages of generating proteins in plants is the low cost, massive scalability and the rapid yield of the technology. Herein, we report the agroinfiltration-based production of glycosylated SARS-CoV-2 Spike receptor-binding domain (RBD) protein. We show that it exhibits high-affinity binding to the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) and displays folding similar to antigen produced in mammalian expression systems. Moreover, our plant-expressed RBD was readily detected by IgM, IgA, and IgG antibodies from the serum of SARS-CoV-2 infected and vaccinated individuals. We further demonstrate that binding of plant-expressed RBD to ACE2 is efficiently neutralized by these antibodies. Collectively, these findings demonstrate that recombinant RBD produced via agroinfiltration exhibits suitable biochemical and antigenic features for use in serological and neutralization assays, and in subunit vaccine platforms.

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Section: Discussionmentioning

confidence: 99%

Scalable agroinfiltration-based production of SARS-CoV-2 antigens for use in diagnostic assays and subunit vaccines

et al. 2022

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“…Receptor Binding Domain (RBD) Ceballo et al, 2022Diego-Martin et al, 2020Khorattanakulchai et al, 2022König-Beihammer et al, 2022Makatsa et al, 2021Maharjan et al, 2021Mamedov et al, 2021Mardanova et al, 2021Rattanapisit et al, 2020Rattanapisit et al, 2021Royal et al, 2021Schwestka et al, 2021Siriwattananon et al, 2021 Spike protein Rattanapisit et al, 2020Shanmugaraj et al, 2020 stability (Wrapp et al, 2020). The transmembrane and cytoplasmic domains of the S protein were replaced by a thrombin cleavage site and a T4 foldon sequence for trimerization (Amanat et al, 2020).…”

Section: Transient Expression (Nicotiana Benthamiana)mentioning

confidence: 99%

Production of the SARS-CoV-2 Spike protein and its Receptor Binding Domain in plant cell suspension cultures

Rebelo¹,

Folgado²,

Ferreira³

et al. 2022

Front. Plant Sci.

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The COVID-19 pandemic, caused by the worldwide spread of SARS-CoV-2, has prompted the scientific community to rapidly develop efficient and specific diagnostics and therapeutics. A number of avenues have been explored, including the manufacture of COVID-related proteins to be used as reagents for diagnostics or treatment. The production of RBD and Spike proteins was previously achieved in eukaryotic cells, mainly mammalian cell cultures, while the production in microbial systems has been unsuccessful until now. Here we report the effective production of SARS-CoV-2 proteins in two plant model systems. We established transgenic tobacco BY-2 and Medicago truncatula A17 cell suspension cultures stably producing the full-length Spike and RBD recombinant proteins. For both proteins, various glycoforms were obtained, with higher yields in Medicago cultures than BY-2. This work highlights that RBD and Spike can be secreted into the culture medium, which will impact subsequent purification and downstream processing costs. Analysis of the culture media indicated the presence of the high molecular weight Spike protein of SARS-CoV-2. Although the production yields still need improvement to compete with mammalian systems, this is the first report showing that plant cell suspension cultures are able to produce the high molecular weight Spike protein. This finding strengthens the potential of plant cell cultures as production platforms for large complex proteins.

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“…RBD was expressed in plants alone [ 41 , 42 , 43 ] with a maximal purification yield of about 20 μg per gram of fresh leaf tissue or as a fusion protein [ 44 , 45 , 46 ]. Plant-produced RBD antigens elicited high titers of antibodies with a potent virus-neutralizing activity [ 43 ].…”

Section: Introductionmentioning

confidence: 99%

“…Plant-produced RBD antigens elicited high titers of antibodies with a potent virus-neutralizing activity [ 43 ]. RBD fused to flagellin of Salmonella typhimurium was transiently expressed in plants at the purification level of about 100 μg/g fresh weight and such fusion could be used for the development of intranasal vaccine [ 45 ]. Plant-produced RBD fused with the Fc fragment of human IgG1 was immunogenic in mice and non-human primates [ 44 ].…”

Section: Introductionmentioning

confidence: 99%

High-Yield Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope and Receptor Binding Domain of SARS-CoV-2 in Plants Using Viral Vectors

Mardanova

Kotlyarov

Stuchinskaya

et al. 2022

IJMS

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Capsid protein of Hepatitis E virus (HEV) is capable of self-assembly into virus-like particles (VLPs) when expressed in Nicotiana benthamiana plants. Such VLPs could be used as carriers of antigens for vaccine development. In this study, we obtained VLPs based on truncated coat protein of HEV bearing the M2e peptide of Influenza A virus or receptor-binding domain of SARS-CoV-2 spike glycoprotein (RBD). We optimized the immunogenic epitopes’ presentation by inserting them into the protruding domain of HEV ORF2 at position Tyr485. The fusion proteins were expressed in Nicotiana benthamiana plants using self-replicating potato virus X (PVX)-based vector. The fusion protein HEV/M2, targeted to the cytosol, was expressed at the level of about 300–400 μg per gram of fresh leaf tissue and appeared to be soluble. The fusion protein was purified using metal affinity chromatography under native conditions with the final yield about 200 μg per gram of fresh leaf tissue. The fusion protein HEV/RBD, targeted to the endoplasmic reticulum, was expressed at about 80–100 μg per gram of fresh leaf tissue; the yield after purification was up to 20 μg per gram of fresh leaf tissue. The recombinant proteins HEV/M2 and HEV/RBD formed nanosized virus-like particles that could be recognized by antibodies against inserted epitopes. The ELISA assay showed that antibodies of COVID-19 patients can bind plant-produced HEV/RBD virus-like particles. This study shows that HEV capsid protein is a promising carrier for presentation of foreign antigen.

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High-Yield Production of Receptor Binding Domain of SARS-CoV-2 Linked to Bacterial Flagellin in Plants Using Self-Replicating Viral Vector pEff

Cited by 12 publications

References 47 publications

Scalable agroinfiltration-based production of SARS-CoV-2 antigens for use in diagnostic assays and subunit vaccines

Scalable agroinfiltration-based production of SARS-CoV-2 antigens for use in diagnostic assays and subunit vaccines

Production of the SARS-CoV-2 Spike protein and its Receptor Binding Domain in plant cell suspension cultures

High-Yield Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope and Receptor Binding Domain of SARS-CoV-2 in Plants Using Viral Vectors

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