Improved vaccine production by engineering the agroinfiltration platform
Lead Research Organisation:
John Innes Centre
Department Name: Biochemistry and Metabolism
Abstract
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Technical Summary
This proposal aims to significantly enhance vaccine production through transient expression in plants using agroinfiltration of Nicotiana benthamiana. This method is effective for producing virus-like particles (VLPs) used in vaccines, but faces low yields due to three key challenges that we aim to address in the following objectives:
The first objective is to prevent crosslinking in agroinfiltrated leaf extracts, which hinder VLP purification. Our preliminary data indicate that polyphenol oxidases (PPOs) are responsible. The objective is to confirm this, improve VLP purification across different envelope viruses, and create PPO knockout plants for broader adoption.
The second objective is to prevent undesired cleavage of the Spike protein of SARS-CoV-2. Our current data suggests that cleavage occurs in the secretory pathway by a membrane-localised protease before the Spike protein trimerises. We will determine the subcellular location of cleavage and identify and confirm the cleavage site. We will deplete 15 groups of candidate proteases by Virus-Induced Gene Silencing (VIGS) to identify conditions with reduced Spike protein cleavage. Selected proteases will either be permanently deleted using genome editing or their activity will be suppressed using inducible RNAi or protease inhibitors.
The third objective aims to improve protein folding by co-expressing three types of chaperones. While human calreticulin has shown promise, the goal is to test additional lectin-based chaperones and expand this approach by assessing protein prolyl isomerases (PPIs) and protein disulfide isomerases (PDIs). This will enhance the production of the Spike protein, known for its folding complexity due to many N-glycans, proline residues, and disulfide bonds.
The first objective is to prevent crosslinking in agroinfiltrated leaf extracts, which hinder VLP purification. Our preliminary data indicate that polyphenol oxidases (PPOs) are responsible. The objective is to confirm this, improve VLP purification across different envelope viruses, and create PPO knockout plants for broader adoption.
The second objective is to prevent undesired cleavage of the Spike protein of SARS-CoV-2. Our current data suggests that cleavage occurs in the secretory pathway by a membrane-localised protease before the Spike protein trimerises. We will determine the subcellular location of cleavage and identify and confirm the cleavage site. We will deplete 15 groups of candidate proteases by Virus-Induced Gene Silencing (VIGS) to identify conditions with reduced Spike protein cleavage. Selected proteases will either be permanently deleted using genome editing or their activity will be suppressed using inducible RNAi or protease inhibitors.
The third objective aims to improve protein folding by co-expressing three types of chaperones. While human calreticulin has shown promise, the goal is to test additional lectin-based chaperones and expand this approach by assessing protein prolyl isomerases (PPIs) and protein disulfide isomerases (PDIs). This will enhance the production of the Spike protein, known for its folding complexity due to many N-glycans, proline residues, and disulfide bonds.
Organisations
People |
ORCID iD |
| George Lomonossoff (Principal Investigator) |