Development and production of DNA-immunogen based SARS-CoV-2 genes
https://doi.org/10.20914/2310-1202-2023-4-96-101
Abstract
Due to the development of the pandemic and the need for mass vaccination, the development of next-generation vaccines against COVID 19 coronavirus infection is a major challenge for medicine and biotechnology. Due to the prevalence of COVID 19, it is still urgent to develop a safe and protective vaccine, especially one that stimulates T-cell immune response. This work presents the design and production of a DNA immunogen based on plasmid DNA encoding a hybrid protein containing immunogenic fragments of structural proteins of SARS-CoV 2 β-coronavirus. As a result, a vector construct based on plasmid DNA encoding a hybrid protein containing the most immunogenic parts of structural proteins M, S, N, E of SARS-CoV 2 β-coronavirus was created by genetic engineering methods for transient expression in mammalian cells; a strain of E. coli, a producer of DNA-coronavirus β-coronavirus, was created by electroporation. coli, a DNA-immunogen producer; a method of purification of recombinant plasmid DNA was developed on the basis of a sequential step-by-step process: this method allowed to obtain 100 mg of DNA-immunogen pCMV 3Tag 3a-CVVVV3 in a solution with a concentration of 1 mg/mL (100 ml), which corresponds to the concentration established in the State Pharmacopoeia of the Russian Federation (OFS.1.7.1.0013.0013.1). .7.1.0013.18 DNA-vaccines) quality parameters - the obtained result is stably reproduced in laboratory conditions; methods of plasmid DNA quality control were worked out and expression of hybrid protein was tested by Western blotting. The CVVV3 antigen was shown to bind specifically to IgG immunoglobulins from rabbit sera after immunization with pCMV 3Tag 3A-CVVVV3 DNA-immunogen. The proportion of superhelicalized plasmid DNA in the sample of DNA-immunogen pCMV 3Tag 3A-CVVVV3 was 85.64 %, the content of endotoxins was less than 25 U/mg; the concentration of residual proteins of the strain-producer in a solution of 1 mg/ml of DNA-immunogen pCMV 3Tag 3A-CVVVV3 was less than 100ng per 1 ml (mg of total recombinant DNA).
About the Authors
A. A. Riabchenkova
LLC ATG Service Gene
Russian Federation
Russian Federation
research scientist
E. R. Chirak
LLC ATG Service Gene
research scientist
E. L. Chirak
LLC ATG Service Gene
research scientist
N. N. Kolmakov
Institution Institute of experimental medicine
Cand. Sci. (Biol.)
V. V. Kopat
LLC ATG Service Gene
development director
I. V. Dukhovlinov
LLC ATG Service Gene
Cand. Sci. (Biol.), director of research
References
1. Dey A. Immunogenic potential of DNA vaccine candidate, ZyCoV-D against SARS-CoV 2 in animal models. Vaccine. 2021. vol. 39. no. 30. pp. 4108–4116.
2. Dhanda S.K. Prediction of HLA CD4 immunogenicity in human populations. Front. Immunol. 2018. vol. 9. pp. 1369.
3. Dukhovlinov I.V., Fedorova E.A., Kolmakov N.N., Chirak E.L., Alekseev A.V. Vaccine for the prevention or treatment of coronavirus infection based on a genetic design. Patent RF, no. 2747762, 2021.
4. He Q. Development of a Western Blot Assay for Detection of Antibodies against Coronavirus Causing Severe Acute Respiratory Syndrome. Clin. Diagn. Lab. Immunol. 2004. vol. 11. no. 2. pp. 417–422.
5. Lee J. Engineering DNA vaccines against infectious diseases. Acta Biomater. 2018. vol. 80. pp. 31–47.
6. Momin Т. Safety and Immunogenicity of a DNA SARS-CoV 2 vaccine (ZyCoV-D): Results of an open-label, non-randomized phase I part of phase I/II clinical study by intradermal route in healthy subjects in India. EClinicalMedicine. 2021. vol. 38.
7. Rodrigues João P.G.L.M. Insights on cross-species transmission of SARS-CoV 2 from structural modeling. PLoS Comput Biol. 2020. vol. 16. no. 12.
8. Vojdania A., Kharrazianb D. Potential antigenic cross-reactivity between SARS-CoV 2 and human tissue with a possible link to an increase in autoimmune diseases. Clinical Immunology. 2020. vol. 217. pp. 1–2.
9. Williams J.A. Generic plasmid DNA production platform incorporating low metabolic burden seed-stock and fed-batch fermentation processes. Biotechnol Bioeng. 2010. vol. 103. no. 6. pp. 1129–1143.
10. Zhao J. Antibody responses to SARS-CoV 2 in patients of novel Antibody responses to SARS-CoV 2 in patients of novel coronavirus disease 2019. Clin. Infect. Dis. 2020. vol. 71. no. 16. pp. 2027–2034.
11. Chashchin V.P., Gudkov A.B., Popova O.N., Odland Yu.O. et al. Characteristics of the main risk factors for health problems among the population living in areas of active environmental management in the Arctic. Human Ecology. 2014. no. 1. pp. 3-12. (in Russian).
12. Kontorovich A.E., Korzhubaev A.G., Eder L.V. Forecast of global energy supply: methodology, quantitative estimates, practical conclusions. Mineral Resources of Russia. Economics and Management. 2006. no. 5. pp. 18-27. (in Russian).
13. Raskina T.A., Pirogova O.A., Zobnina O.V., Pintova, G.A. Indicators of the osteoclastogenesis system in men with various clinical variants of ankylosing spondylitis. Modern rheumatology. 2015. vol. 9. no. 2. pp. 23-27. doi: 10.14412/1996-7012-2015-2-23-27 (in Russian).
14. Novikov A.A., Smolensky A.V., Mikhailova A.V. Approaches to assessing heart rate variability indicators (literature review). Bulletin of new medical technologies. Electronic edition. 2023. vol. 17. no. 3. pp. 85-94. (in Russian).
15. Abdurakhmanov G.M., Lopatin I.K., Ismailov Sh.I. Fundamentals of zoology and zoogeography. Moscow, Academy, 2001. (in Russian).
16. Kondratyev V.B. Global pharmaceutical industry. Industries and sectors of the global economy: features and development trends. 2015. pp. 226-249. Available at: http://perspektivy.info/rus/ekob/globalnaja_farmacevticheskaja_promyshlennost_2011-07-18.html (in Russian).
17. GOST 8.586.5-2005. State system for ensuring the uniformity of measurements. Measurement of flow and quantity of liquids and gases using standard restriction devices. Moscow, Standartinform, 2007. (in Russian).
18. Ura T., Yamashita A., Mizuki N., Okuda K. et al. New vaccine production platforms used in developing SARS-CoV-2 vaccine candidates. Vaccine. 2021. vol. 39. no. 2. pp. 197-201.
19. Chavda V.P., Pandya R., Apostolopoulos V. DNA vaccines for SARS-CoV-2: toward third-generation vaccination era. Expert Review of Vaccines. 2021. vol. 20. no. 12. pp. 1549-1560.
20. Borgoyakova M.B., Karpenko L.I., Rudometov A.P., Shanshin D.V. et al. Immunogenic properties of the DNA construct encoding the receptor-binding domain of the SARS-CoV-2 spike protein. Molecular Biology. 2021. vol. 55. pp. 889-898.
Review
For citations:
Riabchenkova A.A., Chirak E.R., Chirak E.L., Kolmakov N.N., Kopat V.V., Dukhovlinov I.V. Development and production of DNA-immunogen based SARS-CoV-2 genes. Proceedings of the Voronezh State University of Engineering Technologies. 2023;85(4):96-101. (In Russ.) https://doi.org/10.20914/2310-1202-2023-4-96-101
Views:
221
Email this article 
Post a Comment 
