Not for smoking
Proteins for intranasal COVID-19 vaccines were synthesized in tobacco leaves
Russian biotechnologists have connected a section of the coronavirus "spike" and a molecule from the salmonella flagellum to obtain an enhanced vaccine against SARS–CoV-2. It will work most effectively if it is buried in the nose. To produce such a protein "assembled from spare parts", the staff of the FITZ Biotechnology of the Russian Academy of Sciences learned in plant cells. An article about the study was published in the journal Plants (Mardanova et al., High-Yield Production of Receptor Binding Domain of SARS-CoV-2 Linked to Bacterial Flagellin in Plants Using Self-Replicating Viral Vector pEff).
In the structure of the S-protein, which makes up the "crown" of SARS–CoV-2, there is a site with 319 to 541 amino acids (receptor–binding domain, or RBD), which binds to the human angiotensin converting enzyme. This site is important for the infection and "reproduction" of SARS–CoV-2, because without it the virus could not attach to our cells. This means that there will most likely be 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 begins to "hide" from the immune system and change this area, it will be more difficult for it to infect cells. RBD vaccines and other vaccinations of the same type teach the immune system to accurately recognize the target, but they often need additives (adjuvants) to make the reaction to the invasion stronger. One of the options is to attach the antigen site to the bacterial flagellum protein (flagellin), in which the TLR5 innate immunity receptor immediately recognizes the enemy. This adjuvant has already been successfully used, for example, in vaccinations against influenza type A. Researchers of the FITZ Biotechnology of the Russian Academy of Sciences have learned how to produce a protein in which RBD is connected to flagellin in plant cells.
"Biotechnologies make it possible to synthesize proteins-antigens for vaccines in the cells of mammals, bacteria, yeast, plants and other organisms. We inserted a sequence from the SARS-CoV-2 protein and the flagellin sequence of the Salmonella typhimurium bacterium into the pEff viral vector, and infected the Nicotiana benthamiana plant, which belongs to the genus Tobacco, with it. By creating their copies, pEff forces plant cells to produce the proteins we need in large quantities. With the help of this vector, the authors of previous studies managed to obtain up to 1 milligram of immunogenic protein from each gram of fresh leaves in just a few days," comments one of the authors of the scientific article Evgenia Mardanova, senior researcher at the Laboratory of Molecular Cloning Systems of the FITZ Biotechnology of the Russian Academy of Sciences. This research is carried out within the framework of the world-class scientific center "Agrotechnologies of the Future".
Protein production in plants does not require complex equipment and can be easily scaled. Unlike bacterial cells, which are also relatively easy to grow, plant cells can impart posttranslational modifications to the protein. Then it will be folded into the final form, and it will not have to be further refined. You can also not be afraid that the drug obtained in plants will be contaminated with pathogens, since plant infections are not terrible for people. Usually transgenic plants are used as producers, but this technology has drawbacks: usually such plants synthesize little protein, and their production can take several months. It is quite expensive to isolate the final product from plant cells and purify it from by-products. But if you use viral vectors in which the gene of the desired protein is inserted and infect ordinary non-transgenic plants with them, then you can get up to 5 mg of protein encoded in the vector from each gram of leaves within a week.
Biotechnologists selected a fragment of RBD from 319 to 524 amino acids and attached flagellin obtained from salmonella to it. This construct was inserted into the pEff vector, a potato mosaic virus harmless to humans. Then the scientists allowed the bacterium Agrobacterium tumefaciens to "swallow" the viral vector. These microorganisms infected plant cells with the virus. For control, the scientists used only flagellin (without the RBD site). Four days later, when the level of the necessary protein reached its peak, the researchers collected the leaves of the plants. The producing plants were able to produce recombinant protein in the amount of 110-140 micrograms per gram.
"We have demonstrated that using the pEff vector, plants can produce up to 100 micrograms of the recombinant protein we need per gram of biomass. This protein can become the basis of new vaccines against coronavirus infection, which can be instilled into the nose. Due to the adjuvant action of flagellin, such vaccines should cause both a local immune response and an immune response of the whole organism. We hope that this approach will make the production of drugs cheaper, and the vaccinations themselves will become more convenient, faster and easier," Nikolay Ravin, head of the Laboratory of Molecular cloning Systems of the FITZ Biotechnology of the Russian Academy of Sciences, said about the scientific work.
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