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Home SCIENCE NEWS Medicine & Health

Proteins for intranasal vaccines against COVID-19 were produced in tobacco leaves

February 2, 2022
in Medicine & Health
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The S-protein that makes up the “crown” of SARS-CoV-2 has a site from 319 to 541 amino acids (receptor-binding domain, or RBD). This site binds to the human angiotensin-converting enzyme and is important for the infection and “multiplication” of SARS-CoV-2 — without it, the virus could not attach to our cells. This means that there are likely no mutations in RBD, and a vaccine that trains the immune system to recognize this antigen will be relevant for a long time. If the coronavirus starts to “hide” from the immune system and modify this site, it will be harder for it to infect cells.

The plant-producer of the green fluorescent protein GFP

Credit: Courtesy of the Research Center of Biotechnology RAS

The S-protein that makes up the “crown” of SARS-CoV-2 has a site from 319 to 541 amino acids (receptor-binding domain, or RBD). This site binds to the human angiotensin-converting enzyme and is important for the infection and “multiplication” of SARS-CoV-2 — without it, the virus could not attach to our cells. This means that there are likely no mutations in RBD, and a vaccine that trains the immune system to recognize this antigen will be relevant for a long time. If the coronavirus starts to “hide” from the immune system and modify this site, it will be harder for it to infect cells.

RBD-based vaccines and other vaccines of the same type train the immune system to recognize the target precisely. Although they often need additives (adjuvants) to make the immune response to invasion stronger. One option is to attach an antigen to bacterial flagellin protein, in which the TLR5 innate immunity receptor immediately recognizes the enemy. This adjuvant has already been successfully used, for example, in vaccines against influenza A. Scientists from the Research Center of Biotechnology RAS learned how to produce a protein in which RBD is combined with flagellin in plant cells.

“Biotechnology allows synthesizing vaccine antigen proteins in animal cells, bacteria, yeast, plants, and other organisms. We put the sequence from the SARS-CoV-2 protein and the flagellin sequence of the bacterium Salmonella typhimurium into the pEff viral vector and infected the plant Nicotiana benthamiana, which belongs to the genus Tobacco. pEff causes plant cells to produce the proteins we need in large quantities. Using this vector, the authors of previous studies were able to obtain up to 1 milligram of a green fluorescent protein from each gram of fresh leaves in just a few days,” — says Eugenia Mardanova, one of the authors of the article, the senior researcher at the Laboratory of Molecular Cloning of the Research Center of Biotechnology RAS. This research is conducted as part of the activities of the world-class research center “Agrotechnologies for the Future”.

Protein production in plants does not require sophisticated equipment and can be easily scaled up. Unlike bacterial cells, which are also relatively easy to grow, plant cells can enable the protein posttranslational modifications. There is no chance that the product obtained in plants will be contaminated by pathogens since plant infections do not affect humans. Usually, transgenic plants are used as producers, but this technology has disadvantages: levels of production of the desired proteins are usually low, and it may take several months to obtain transgenic plants. Isolation of the final product from plant cells and purifying it from by-products is quite expensive. But if we use viral vectors that contain the gene for the desired protein and infect ordinary nontransgenic plants with them, we can get up to 5 mg of vector-encoded protein from each gram of leaf within a week.

Biotechnologists chose an RBD fragment with 319 to 524 amino acids and attached flagellin derived from Salmonella to it. This construct was inserted into the pEff vector, based on a potato mosaic virus harmless to humans. Then the scientists let the bacteria Agrobacterium tumefaciens to “absorb” the viral vector. These microorganisms transferred the viral vector into the plant cells. The scientists used only flagellin (without the RBD site) for a control. Four days later, when the required amount of protein was reached, the researchers harvested the leaves of the plants. The plant “biofactories” were able to produce a recombinant protein of 110-140 micrograms per gram.

“Our studies showed that using the pEff vector we can make plants producing recombinant protein we need — up to 100 micrograms per gram of biomass. This protein could be the basis of new vaccines against coronavirus infection that can be applied as nasal drops. Due to the adjuvant properties of flagellin, such vaccines should induce both a local and systemic immune response. We believe that this approach will make the production of drugs cheaper, and the vaccinations themselves will become more convenient, faster, and easier,” — says Nikolay V. Ravin, Head of the Laboratory of Molecular Cloning of the Institute of the Research Center of Biotechnology RAS.



Journal

Plants

DOI

10.3390/plants10122682

Article Title

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

Article Publication Date

6-Dec-2021

Tags: COVID19IntranasalleavesproducedproteinstobaccoVaccines
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