Antigenicity and immunogenicity of SARS-CoV-2 surface glycoprotein fragment in CHO cells
-
Sahar Karimi
,
Shahram Nazarian
,
Fattah Sotoodehnejadnematalahi
,
Roohollah Dorostkar
,
Jafar Amani
Abstract
Background and Objectives: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein that projects from the virus surface is highly immunogenic. It is considered to be the target of many neutralizing antibodies as well as a target in vaccine design efforts. Evaluation the immunogenicity of a recombinant fragment of the spike protein (rfsp) that is comprised of Receptor Binding Domain (RBD), S1/S2 cleavage site, and fusion peptide (FP) as immunogenic proteins of SARS-COV-2, in BALB/c mice and evaluation of the efficacy of epitopes rfsp as a multi-subunit chimeric vaccine.
Materials and Methods: The present study made use of CHO-K1 (Chinese hamster ovary K1) cells to create a cell line for constant expression rfsp. The rfsp was purified with Ni-NTA chromatography and confirmed by Western blotting. The immunogenicity and neutralizing antibody efficacy of rfsp were evaluated in BALB/c mice. ELISA was employed to test rfsp via sera of COVID-19 convalescent patients infected with SARS-CoV-2 alpha and delta variants.
Results: Our results showed significant differences in antibody titers in immunized mice compared to the control groups and neutralizing antibodies were positive, sera from mice immunized are capable of bound SARS-CoV-2 virus, chimer peptide is capable bound antibodies patients infected with SARS-CoV-2 and patients infected with delta variant SARS-CoV-2.
Conclusion: Overall, these results indicate that rfsp protein would be a novel potential antigen candidate for the development of a subunit SARS CoV-2 vaccine and rfsp has the potential to be a useful option for the development of the assays for serodiagnosis of SARS-CoV-2 infection.
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15. Shajahan A, Supekar NT, Gleinich AS, Azadi P. Deducing the N-and O-glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2. Glycobiology 2020; 30: 981-988.
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21. Rosser MP, Xia W, Hartsell S, McCaman M, Zhu Y, Wang S, et al. Transient transfection of CHO-K1-S using serum-free medium in suspension: a rapid mammalian protein expression system. Protein Expr Purif 2005; 40: 237-243.
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25. Amanat F, Krammer F. SARS-CoV-2 vaccines: status report. Immunity 2020; 52: 583-589.
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27. Sahdev S, Khattar SK, Saini KS. Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Mol Cell Biochem 2008; 307: 249-264.
28. Eifler N, Duckely M, Sumanovski LT, Egan TM, Oksche A, Konopka JB, et al. Functional expression of mammalian receptors and membrane channels in different cells. J Struct Biol 2007; 159: 179-193.
29. Jiang S, Hillyer C, Du L. Neutralizing antibodies against SARS-CoV-2 and other human coronaviruses. Trends Immunol 2020; 41: 355-359.
30. Tegel H, Tourle S, Ottosson J, Persson A. Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta (DE3). Protein Expr Purif 2010; 69: 159-167.
31. Reis CA, Tauber R, Blanchard V. Glycosylation is a key in SARS-CoV-2 infection. J Mol Med (Berl) 2021; 99: 1023-1031.
32. Du L, Zhao G, He Y, Guo Y, Zheng B-J, Jiang S, et al. Receptor-binding domain of SARS-CoV spike protein induces long-term protective immunity in an animal model. Vaccine 2007; 25: 2832-2838.
33. Djukic T, Mladenovic M, Stanic-Vucinic D, Radosavljevic J, Smiljanic K, Sabljic L, et al. Expression, purification and immunological characterization of recombinant nucleocapsid protein fragment from SARS-CoV-2. Virology 2021; 557: 15-22.
34. Graham RL, Donaldson EF, Baric RS. A decade after SARS: strategies for controlling emerging coronaviruses. Nat Rev Microbiol 2013; 11: 836-848.
35. He Q, Mao Q, Peng X, He Z, Lu S, Zhang J, et al. Immunogenicity and protective efficacy of a recombinant protein subunit vaccine and an inactivated vaccine against SARS-CoV-2 variants in non-human primates. Signal Transduct Target Ther 2022; 7: 69.
36. Siriwattananon K, Manopwisedjaroen S, Shanmugaraj B, Prompetchara E, Ketloy C, Buranapraditkun S, et al. Immunogenicity studies of plant-produced SARS-CoV-2 receptor binding domain-based subunit vaccine candidate with different adjuvant formulations. Vaccines (Basel) 2021; 9: 744.
2. Liu A, Li Y, Peng J, Huang Y, Xu D. Antibody responses against SARS‐CoV‐2 in COVID‐19 patients. J Med Virol 2021; 93: 144-148.
3. Johari YB, Jaffé SRP, Scarrott JM, Johnson AO, Mozzanino T, Pohle TH, et al. Production of trimeric SARS‐CoV‐2 spike protein by CHO cells for serological COVID‐19 testing. Biotechnol Bioeng 2021; 118: 1013-1021.
4. Yuan M, Liu H, Wu NC, Lee CD, Zhu X, Zhao F, et al. Structural basis of a shared antibody response to SARS-CoV-2. Science 2020; 369: 1119-1123.
5. Kar T, Narsaria U, Basak S, Deb D, Castiglione F, Mueller DM, et al. A candidate multi-epitope vaccine against SARS-CoV-2. Sci Rep 2020; 10: 10895.
6. Watanabe Y, Allen JD, Wrapp D, McLellan JS, Crispin M. Site-specific glycan analysis of the SARS-CoV-2 spike. Science 2020; 369: 330-333.
7. Walls AC, Park Y-J, Tortorici MA, Wall A, McGuire AT, Veesler D. Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell 2020; 181: 281-292. e6.
8. Ansarin K, Tolouian R, Ardalan M, Taghizadieh A, Varshochi M, Teimouri S, et al. Effect of bromhexine on clinical outcomes and mortality in COVID-19 patients: A randomized clinical trial. Bioimpacts 2020; 10: 209-215.
9. Coutard B, Valle C, de Lamballerie X, Canard B, Seidah NG, Decroly E. The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade. Antiviral Res 2020; 176: 104742.
10. Hoffmann M, Kleine-Weber H, Pöhlmann S. A multibasic cleavage site in the spike protein of SARS-CoV-2 is essential for infection of human lung cells. Mol Cell 2020; 78: 779-784. e5.
11. Mehdipour AR, Hummer G. Dual nature of human ACE2 glycosylation in binding to SARS-CoV-2 spike. Proc Natl Acad Sci U S A 2021; 118(19): e2100425118.
12. Liu H, Zhang Q, Wei P, Chen Z, Aviszus K, Yang J, et al. The basis of a more contagious 501Y. V1 variant of SARS-CoV-2. Cell Res 2021; 31: 720-722.
13. Lopez Bernal J, Andrews N, Gower C, Gallagher E, Simmons R, Thelwall S, et al. Effectiveness of Covid-19 vaccines against the B. 1.617. 2 (Delta) variant. N Engl J Med 2021; 385: 585-594.
14. Mlcochova P, Kemp SA, Dhar MS, Papa G, Meng B, Ferreira IATM, et al. SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion. Nature 2021; 599: 114-119.
15. Shajahan A, Supekar NT, Gleinich AS, Azadi P. Deducing the N-and O-glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2. Glycobiology 2020; 30: 981-988.
16. Watanabe Y, Bowden TA, Wilson IA, Crispin M. Exploitation of glycosylation in enveloped virus pathobiology. Biochim Biophys Acta Gen Subj 2019; 1863: 1480-1497.
17. Du L, Zhao G, Li L, He Y, Zhou Y, Zheng B-J, et al. Antigenicity and immunogenicity of SARS-CoV S protein receptor-binding domain stably expressed in CHO cells. Biochem Biophys Res Commun 2009; 384: 486-490.
18. Steger K, Brady J, Wang W, Duskin M, Donato K, Peshwa M. CHO-S antibody titers> 1 gram/liter using flow electroporation-mediated transient gene expression followed by rapid migration to high-yield stable cell lines. J Biomol Screen 2015; 20: 545-551.
19. Omasa T, Onitsuka M, Kim W-D. Cell engineering and cultivation of Chinese hamster ovary (CHO) cells. Curr Pharm Biotechnol 2010; 11: 233-240.
20. Guo L, Wang L, Yang R, Feng R, Li Z, Zhou X, et al. Optimizing conditions for calcium phosphate mediated transient transfection. Saudi J Biol Sci 2017; 24: 622-629.
21. Rosser MP, Xia W, Hartsell S, McCaman M, Zhu Y, Wang S, et al. Transient transfection of CHO-K1-S using serum-free medium in suspension: a rapid mammalian protein expression system. Protein Expr Purif 2005; 40: 237-243.
22. Stuible M, Gervais C, Lord-Dufour S, Perret S, L’Abbé D, Schrag J, et al. Rapid, high-yield production of full-length SARS-CoV-2 spike ectodomain by transient gene expression in CHO cells. J Biotechnol 2021; 326: 21-27.
23. Jahanshahlu L, Rezaei N. Monoclonal antibody as a potential anti-COVID-19. Biomed Pharmacother 2020; 129: 110337.
24. Márquez-Ipiña AR, González-González E, Rodríguez-Sánchez IP, Lara-Mayorga IM, Mejía-Manzano LA, Sánchez-Salazar MG, et al. Serological test to determine exposure to SARS-CoV-2: ELISA based on the receptor-binding domain of the spike protein (S-RBDN318-V510) expressed in Escherichia coli. Diagnostics (Basel) 2021; 11: 271.
25. Amanat F, Krammer F. SARS-CoV-2 vaccines: status report. Immunity 2020; 52: 583-589.
26. Wang N, Shang J, Jiang S, Du L. Subunit vaccines against emerging pathogenic human coronaviruses. Front Microbiol 2020; 11: 298.
27. Sahdev S, Khattar SK, Saini KS. Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Mol Cell Biochem 2008; 307: 249-264.
28. Eifler N, Duckely M, Sumanovski LT, Egan TM, Oksche A, Konopka JB, et al. Functional expression of mammalian receptors and membrane channels in different cells. J Struct Biol 2007; 159: 179-193.
29. Jiang S, Hillyer C, Du L. Neutralizing antibodies against SARS-CoV-2 and other human coronaviruses. Trends Immunol 2020; 41: 355-359.
30. Tegel H, Tourle S, Ottosson J, Persson A. Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta (DE3). Protein Expr Purif 2010; 69: 159-167.
31. Reis CA, Tauber R, Blanchard V. Glycosylation is a key in SARS-CoV-2 infection. J Mol Med (Berl) 2021; 99: 1023-1031.
32. Du L, Zhao G, He Y, Guo Y, Zheng B-J, Jiang S, et al. Receptor-binding domain of SARS-CoV spike protein induces long-term protective immunity in an animal model. Vaccine 2007; 25: 2832-2838.
33. Djukic T, Mladenovic M, Stanic-Vucinic D, Radosavljevic J, Smiljanic K, Sabljic L, et al. Expression, purification and immunological characterization of recombinant nucleocapsid protein fragment from SARS-CoV-2. Virology 2021; 557: 15-22.
34. Graham RL, Donaldson EF, Baric RS. A decade after SARS: strategies for controlling emerging coronaviruses. Nat Rev Microbiol 2013; 11: 836-848.
35. He Q, Mao Q, Peng X, He Z, Lu S, Zhang J, et al. Immunogenicity and protective efficacy of a recombinant protein subunit vaccine and an inactivated vaccine against SARS-CoV-2 variants in non-human primates. Signal Transduct Target Ther 2022; 7: 69.
36. Siriwattananon K, Manopwisedjaroen S, Shanmugaraj B, Prompetchara E, Ketloy C, Buranapraditkun S, et al. Immunogenicity studies of plant-produced SARS-CoV-2 receptor binding domain-based subunit vaccine candidate with different adjuvant formulations. Vaccines (Basel) 2021; 9: 744.
| Files | ||
| Issue | Vol 15 No 1 (2023) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v15i1.11929 | |
| Keywords | ||
| Spike; Vaccine; CHO-K1; SARS-CoV-2; Delta variant | ||
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This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Karimi S, Nazarian S, Sotoodehnejadnematalahi F, Dorostkar R, Amani J. Antigenicity and immunogenicity of SARS-CoV-2 surface glycoprotein fragment in CHO cells. Iran J Microbiol. 2023;15(1):128-137.
