Comparison of titer and neutralization of antibody, SARS-CoV-2 spike protein expressed in eukaryotic and prokaryotic cells

Karimi, Sahar; Nazarian, Shahram; Sotoodehnejadnematalahi, Fattah; Dorostkar, Roohollah; Amani, Jafar

Comparison of titer and neutralization of antibody, SARS-CoV-2 spike protein expressed in eukaryotic and prokaryotic cells

Document Type : Brief Communication

Authors

sahar karimi ¹

Shahram Nazarian ²

fattah Sotoodehnejadnematalahi ¹

Roohollah Dorostkar ³

Jafar Amani ⁴

¹ Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran

² Department of Biology, Imam Hossein University, Tehran, Iran

³ Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran

⁴ Applied Microbiology Research Center, System Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

Abstract

Given the global epidemic of COVID-19, it is important to design a vaccine for prevention. The virus belongs to the beta-coronavirus family and forms appendages on the surface of the glycoprotein spike virus membrane. Studies on SARS-CoV-1 and related MERS-CoV vaccines have shown that the spike protein on the virus surface is a suitable target for the vaccine. In this experimental study, we compare the recombinant fragment of spike protein (rfsp) expressed in the eukaryotic host CHO-K1 cell and prokaryotic E. coli in terms of immunogenicity, neutralizing activity, and epitopes recognition Similar to the virus strain and the ability to bind to the serum of improved patients, in two types of alpha and delta variants. The results showed that both rfSP proteins are a potential new antigen candidate for the development of the Covid 19 vaccine, but the CHO cell maintains the biological activity of the protein by performing post-translational modification processes such as glycolysis. This increases the present likelihood of developing virus-like epitopes and increases the titer of rfsp-specific antibodies in the serum of immunized mice. Therefore, priority is given to rfsp expressed in CHO cells to evaluate vaccine efficacy.

Keywords

Covid-19

vaccine

spike protein

glycolysis

CHO-K1 cell

20.1001.1.23222115.1401.14.1.7.3

Subjects

Pharmaceutical Biotechnology

1. Liu, A. and Y. Li, Antibody responses against SARS‐CoV‐2 in COVID‐19 patients. Journal of medical virology, 2020.
2. Liu, C., et al., Research and development on therapeutic agents and vaccines for COVID-19 and related human coronavirus diseases. 2020, ACS Publications.
3. Yuan, M., et al., Structural basis of a shared antibody response to SARS-CoV-2. Science, 2020. 369(6507): p. 1119-1123.
4. Kar, T., et al., A candidate multi-epitope vaccine against SARS-CoV-2. Scientific reports, 2020. 10(1): p. 1-24.
5. Amanat, F. and F. Krammer, SARS-CoV-2 vaccines: status report. Immunity, 2020. 52(4): p. 583-589.
6. Johari, Y.B., et al., Production of trimeric SARS‐CoV‐2 spike protein by CHO cells for serological COVID‐19 testing. Biotechnology and bioengineering, 2021. 118(2): p. 1013-1021.
7. Watanabe, Y., et al., Site-specific glycan analysis of the SARS-CoV-2 spike. Science, 2020. 369(6501): p. 330-333.
8. Walls, A.C., et al., Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell, 2020. 181(2): p. 281-292. e6.
9. Ansarin, K., et al., Effect of bromhexine on clinical outcomes and mortality in COVID-19 patients: a randomized clinical trial. BioImpacts: BI, 2020. 10(4): p. 209.
10. Coutard, B., et al., The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade. Antiviral research, 2020. 176: p. 104742.
11. Hoffmann, M., H. Kleine-Weber, and S. Pöhlmann, A multibasic cleavage site in the spike protein of SARS-CoV-2 is essential for infection of human lung cells. Molecular cell, 2020. 78(4): p. 779-784. e5.
12. Mehdipour, A.R. and G. Hummer, Dual nature of human ACE2 glycosylation in binding to SARS-CoV-2 spike. Proceedings of the National Academy of Sciences, 2021. 118(19).
13. Lopez Bernal, J., et al., Effectiveness of Covid-19 vaccines against the B. 1.617. 2 (Delta) variant. N Engl J Med, 2021: p. 585-594.
14. Liu, H., et al., The basis of a more contagious 501Y. V1 variant of SARS-COV-2. Cell research, 2021. 31(6): p. 720-722.
15. Mlcochova, P., et al., SARS-CoV-2 B. 1.617. 2 Delta variant emergence and vaccine breakthrough. 2021.
16. Shajahan, A., et al., Deducing the N-and O-glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2. Glycobiology, 2020. 30(12): p. 981-988.
17. Watanabe, Y., et al., Exploitation of glycosylation in enveloped virus pathobiology. Biochimica et Biophysica Acta (BBA)-General Subjects, 2019. 1863(10): p. 1480-1497.
18. Vakili, A., et al., Designing and Expression of Recombinant Chimeric Protein Containing CtxB and OmpW from Vibrio Cholerae and Evaluation of Its lmmunogenicity. Iranian Journal of Immunology, 2018. 15(3): p. 207-220.
19. Tahmoorespur, M., Evaluation of Different Expression Systems for the Production of Pharmaceutical Recombinant Proteins with Emphasis on Mammalian Cells. Journal of Biosafety, 2016. 9(1): p. 51-65.
20. Van Hove, J., et al., High-level production of recombinant human lysosomal acid alpha-glucosidase in Chinese hamster ovary cells which targets to heart muscle and corrects glycogen accumulation in fibroblasts from patients with Pompe disease. Proceedings of the National Academy of Sciences, 1996. 93(1): p. 65-70.
21. Du, L., et al., Recombinant receptor-binding domain of SARS-CoV spike protein expressed in mammalian, insect and E. coli cells elicits potent neutralizing antibody and protective immunity. Virology, 2009. 393(1): p. 144-150.
22. Takrim, S., et al., Expression, Purification and Immunogenicity Evaluation of Recombinant Fusion Protein (F) from Newcastle Virus in Animal Model. Modares Journal of Biotechnology, 2019. 10(1): p. 15-21.
23. Kamionka, M., Engineering of therapeutic proteins production in Escherichia coli. Current pharmaceutical biotechnology, 2011. 12(2): p. 268-274.
24. Sørensen, H.P. and K.K. Mortensen, Advanced genetic strategies for recombinant protein expression in Escherichia coli. Journal of biotechnology, 2005. 115(2): p. 113-128.
25. Fitzgerald, G.A., et al., Expression of SARS-CoV-2 surface glycoprotein fragment 319–640 in E. coli, and its refolding and purification. Protein expression and purification, 2021. 183: p. 105861.
26. Sahdev, S., S.K. Khattar, and K.S. Saini, Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Molecular and cellular biochemistry, 2008. 307(1): p. 249-264.
27. Rosser, M.P., et al., Transient transfection of CHO-K1-S using serum-free medium in suspension: a rapid mammalian protein expression system. Protein Expression and Purification, 2005. 40(2): p. 237-243.
28. Eifler, N., et al., Functional expression of mammalian receptors and membrane channels in different cells. Journal of structural biology, 2007. 159(2): p. 179-193.
29. Du, L., et al., Antigenicity and immunogenicity of SARS-CoV S protein receptor-binding domain stably expressed in CHO cells. Biochemical and biophysical research communications, 2009. 384(4): p. 486-490.
30. Tegel, H., et al., Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta (DE3). Protein expression and purification, 2010. 69(2): p. 159-167.
31. Reis, C.A., R. Tauber, and V. Blanchard, Glycosylation is a key in SARS-CoV-2 infection. Journal of Molecular Medicine, 2021: p. 1-9.
32. Djukic, T., et al., Expression, purification and immunological characterization of recombinant nucleocapsid protein fragment from SARS-CoV-2. Virology, 2021. 557: p. 15-22.
33. Graham, R.L., E.F. Donaldson, and R.S. Baric, A decade after SARS: strategies for controlling emerging coronaviruses. Nature Reviews Microbiology, 2013. 11(12): p. 836-848.