Production and intracellular trafficking of SARS CoV-2 spike protein in insect cells infected with recombinant baculovirus

Abstract

SARS CoV-2 belongs to the Coronaviridae family and is an enveloped virus with a positive polarity single stranded RNA genome. The virus's spike protein, embedded in the viral membrane, is the most important antigenic protein involved in binding the virus to the host cell receptor. This protein is the basic component of vaccines developed against the virus due to its antigenic character. Therefore, it is crucial to produce this protein heterologously. This study evaluated the potential of ExpiSf9 and Hi5 insect cells infected with recombinant baculoviruses carrying the spike gene to synthesize the spike protein. The synthesis of the spike protein in infected cells was analyzed using SDS-PAGE/silver staining, Western blotting, and immunofluorescence techniques. High levels of spike expression were detected in virus infected cultures at 72 hours post-infection compared to cellular proteins. The immunostaining results showed that spike proteins were present in the cell cytosol as aggregates, indicating that the proteins were transported via the endoplasmic reticulum-Golgi transport pathway. The Western blot analysis revealed that the spike proteins undergo post translational modifications, such as glycosylation and proteolytic cleavage, in both insect and mammalian cells. Based on this data, it has been concluded that the baculovirus expression system is a suitable and cost-effective method for producing the spike protein. This protein can be used as an antigenic component in the subunit vaccine against Covid 19.

Keywords

• Coronaviruses
• SARS CoV-2
• recombinant spike protein
• Covid-19
• baculovirus
• insect cells

References

1. [1] Su S, Wong G, Shi W, Liu J, Lai ACK, Zhou J, Liu W, Bi Y, Gao GF. Epidemiology, Genetic Recombination, and Pathogenesis of Coronaviruses. Trends Microbiol. 2016; 24(6): 490-502. https://doi.org/10.1016/j.tim.2016.03.003.
2. [2] Tam CW, Pang EP, Lam LC, Chiu HF. Severe acute respiratory syndrome (SARS) in Hong Kong in 2003: stress and psychological impact among frontline healthcare workers. Psychol Med. 2004; 34(7): 1197-1204. https://dx.doi.org/10.1017/s0033291704002247.
3. [3] Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret T, Emery S, Tong S, Urbani C, Comer JA, Lim W, Rollin PE, Dowell SF, Ling AE, Humphrey CD, Shieh WJ, Guarner J, Paddock CD, Rota P, Fields B, DeRisi J, Yang JY, Cox N, Hughes JM, LeDuc JW, Bellini WJ, Anderson LJ. A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med. 2003; 348(20): 1953-1966. https://doi.org/10.1056/nejmoa030781.
4. [4] Chan-Yeung M, Xu RH. SARS: epidemiology. Respirology. 2003; 8 Suppl(Suppl 1): 9-14. https://doi.org/10.1046/j.1440-1843.2003.00518.x.
5. [5] Azhar EI, Velavan TP, Rungsung I, Traore T, Hui DS, McCloskey B, El-Kafrawy SA, Zumla A. Middle East respiratory syndrome coronavirus-a 10-year (2012-2022) global analysis of human and camel infections, genomic sequences, lineages, and geographical origins. Int J Infect Dis. 2023; 131: 87-94. https://dx.doi.org/10.1016/j.ijid.2023.03.046.
6. [6] Stadler K, Masignani V, Eickmann M, Becker S, Abrignani S, Klenk HD, Rappuoli R. SARS--beginning to understand a new virus. Nat Rev Microbiol. 2003; 1(3): 209-218. https://dx.doi.org/10.1038/nrmicro775.
7. [7] Alyami MH, Alyami HS, Warraich A. Middle East Respiratory Syndrome (MERS) and novel coronavirus disease 2019 (COVID-19): From causes to preventions in Saudi Arabia. Saudi Pharm J. 2020; 28(11): 1481-1491. https://dx.doi.org/10.1016/j.jsps.2020.09.014.
8. [8] Zhang X, Tan Y, Ling Y, Lu G, Liu F, Yi Z, Jia X, Wu M, Shi B, Xu S, Chen J, Wang W, Chen B, Jiang L, Yu S, Lu J, Wang J, Xu M, Yuan Z, Zhang Q, Zhang X, Zhao G, Wang S, Chen S, Lu H. Viral and host factors related to the clinical outcome of COVID-19. Nature. 2020; 583(7816): 437-440. https://dx.doi.org/10.1038/s41586-020-2355-0.

9. [9] Tregoning JS, Flight KE, Higham SL, Wang Z, Pierce BF. Progress of the COVID-19 vaccine effort: viruses, vaccines and variants versus efficacy, effectiveness and escape. Nat Rev Immunol. 2021; 21(10): 626-636. https://dx.doi.org/10.1038/s41577-021-00592-1.
10. [10] He Q, Mao Q, An C, Zhang J, Gao F, Bian L, Li C, Liang Z, Xu M, Wang J. Heterologous prime-boost: breaking the protective immune response bottleneck of COVID-19 vaccine candidates. Emerg Microbes Infect. 2021; 10(1): 629 637. https://dx.doi.org/10.1080/22221751.2021.1902245.
11. [11] Cao L, Guo J, Li H, Ren H, Xiao K, Zhang Y, Xia B, Ji T, Yan D, Wang D, Yang Q, Zhou Y, Li X, Hou Z, Xu W. A beta strain-based spike glycoprotein vaccine candidate induces broad neutralization and protection against SARS CoV-2 variants of concern. Microbiol Spectr. 2023; 11(2): e0268722. https://dx.doi.org/10.1128/spectrum.02687 22.
12. [12] van Oers MM. Vaccines for viral and parasitic diseases produced with baculovirus vectors. Adv Virus Res. 2006; 68: 193-253. https://dx.doi.org/10.1016/S0065-3527(06)68006-8.
13. [13] Azali MA, Mohamed S, Harun A, Hussain FA, Shamsuddin S, Johan MF. Application of Baculovirus Expression Vector system (BEV) for COVID-19 diagnostics and therapeutics: a review. J Genet Eng Biotechnol. 2022; 20(1): 98. https://dx.doi.org/10.1186/s43141-022-00368-7.
14. [14] Hong Q, Liu J, Wei Y, Wei X. Application of Baculovirus Expression Vector System (BEVS) in Vaccine Development. Vaccines (Basel). 2023; 11(7): 1218. https://dx.doi.org/10.3390/vaccines11071218.
15. [15] Clem RJ, Passarelli AL. Baculoviruses: sophisticated pathogens of insects. PLoS Pathog. 2013; 9(11): e1003729. https://dx.doi.org/10.1371/journal.ppat.1003729.
16. [16] Martinez-Solis M, Gomez-Sebastian S, Escribano JM, Jakubowska AK, Herrero S. A novel baculovirus-derived promoter with high activity in the baculovirus expression system. PeerJ. 2016; 4: e2183. https://dx.doi.org/10.7717/peerj.2183.
17. [17] Kalyoncu S, Yilmaz S, Kuyucu AZ, Sayili D, Mert O, Soyturk H, Gullu S, Akinturk H, Citak E, Arslan M, Taskinarda MG, Tarman IO, Altun GY, Ozer C, Orkut R, Demirtas A, Tilmensagir I, Keles U, Ulker C, Aralan G, Mercan Y, Ozkan M, Caglar HO, Arik G, Ucar MC, Yildirim M, Yildirim TC, Karadag D, Bal E, Erdogan A, Senturk S, Uzar S, Enul H, Adiay C, Sarac F, Ekiz AT, Abaci I, Aksoy O, Polat HU, Tekin S, Dimitrov S, Ozkul A, Wingender G, Gursel I, Ozturk M, Inan M. Process development for an effective COVID-19 vaccine candidate harboring recombinant SARS-CoV-2 delta plus receptor binding domain produced by Pichia pastoris. Sci Rep. 2023; 13(1): 5224. https://dx.doi.org/10.1038/s41598-023-32021-9.
18. [18] Schaub JM, Chou CW, Kuo HC, Javanmardi K, Hsieh CL, Goldsmith J, DiVenere AM, Le KC, Wrapp D, Byrne PO, Hjorth CK, Johnson NV, Ludes-Meyers J, Nguyen AW, Wang N, Lavinder JJ, Ippolito GC, Maynard JA, McLellan JS, Finkelstein IJ. Expression and characterization of SARS-CoV-2 spike proteins. Nat Protoc. 2021; 16(11): 5339 5356. https://dx.doi.org/10.1038/s41596-021-00623-0.
19. [19] Chavda VP, Bezbaruah R, Valu D, Patel B, Kumar A, Prasad S, Kakoti BB, Kaushik A, Jesawadawala M. Adenoviral Vector-Based Vaccine Platform for COVID-19: Current Status. Vaccines (Basel). 2023; 11(2):432. https://dx.doi.org/10.3390/vaccines11020432.
20. [20 Uraki R, Imai M, Ito M, Yamayoshi S, Kiso M, Jounai N, Miyaji K, Iwatsuki-Horimoto K, Takeshita F, Kawaoka Y. An mRNA vaccine encoding the SARS-CoV-2 receptor-binding domain protects mice from various Omicron variants. NPJ Vaccines. 2024;9(1):4. https://doi.org/10.1038/s41541-023-00800-0.
21. [21] Kozak M, Hu J. DNA Vaccines: Their Formulations, Engineering and Delivery. Vaccines (Basel). 2024 Jan 11;12(1):71. https://doi.org/10.3390/vaccines12010071.
22. [22] Kost TA, Condreay JP, Jarvis DL. Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol. 2005; 23(5): 567-575. https://dx.doi.org/10.1038/nbt1095.
23. [23] Granados RR, Guoxun L, Derksen ACG, McKenna KA. A new insect cell line from Trichoplusia ni (BTI-Tn-5B1-4) susceptible to Trichoplusia ni single enveloped nuclear polyhedrosis virus. J Invertebr Pathol. 1994; 64(3): 260-266. https://dx.doi.org/10.1016/S0022-2011(94)90400-6.
24. [24] Gross DA, Silver DL. Purification of Integral Membrane Proteins and Lipid-Binding Assays In: Yang H, Li P, editors. Methods in Cell Biology. 116: Elsevier; 2013. pp. 191-211.
25. [25] Duan L, Zheng Q, Zhang H, Niu Y, Lou Y, Wang H. The SARS-CoV-2 spike glycoprotein biosynthesis, structure, function, and antigenicity: Implications for the design of spike-based vaccine immunogens. Front Immunol. 2020; 11: 576622. https://dx.doi.org/10.3389/fimmu.2020.576622.
26. [26] Çağlayan E, Turan K. Mutations in the SARS CoV2 spike gene and their reflections on the spike protein. Clin Exp Health Sci. 2022; 12: 472-478. https://dx.doi.org/10.33808/clinexphealthsci.981816.
27. [27] Carstens EB, Tjia ST, Doerfler W. Infection of Spodoptera frugiperda cells with Autographa californica nuclear polyhedrosis virus I. Synthesis of intracellular proteins after virus infection. Virology. 1979; 99(2): 386-398. https://dx.doi.org/10.1016/0042-6822(79)90017-5.
28. [28] Ikeda M, Kobayashi M. Cell-cycle perturbation in Sf9 cells infected with Autographa californica nucleopolyhedrovirus. Virology. 1999; 258(1): 176-188. https://dx.doi.org/10.1006/viro.1999.9706.
29. [29] Braunagel SC, Parr R, Belyavskyi M, Summers MD. Autographa californica nucleopolyhedrovirus infection results in Sf9 cell cycle arrest at G2/M phase. Virology. 1998; 244(1): 195-211. https://dx.doi.org/10.1006/viro.1998.9097.
30. [30] Abraham E, Bajusz C, Marton A, Borics A, Mdluli T, Pardi N, Lipinszki Z. Expression and purification of the receptor-binding domain of SARS-CoV-2 spike protein in mammalian cells for immunological assays. FEBS Open Bio. 2024; 14(3): 380-389. https://dx.doi.org/10.1002/2211-5463.13754.
31. [31] Chen J, Miao L, Li JM, Li YY, Zhu QY, Zhou CL, Fang HQ, Chen HP. Receptor-binding domain of SARS-Cov spike protein: soluble expression in E. coli, purification and functional characterization. World J Gastroenterol. 2005; 11(39): 6159-6164. https://dx.doi.org/10.3748/wjg.v11.i39.6159.
32. [32] Walls AC, Park YJ, Tortorici MA, Wall A, McGuire AT, Veesler D. Structure, function, and antigenicity of the SARS CoV-2 spike glycoprotein. Cell. 2020; 183(6): 1735. https://dx.doi.org/10.1016/j.cell.2020.11.032.
33. [33] Gong Y, Qin S, Dai L, Tian Z. The glycosylation in SARS-CoV-2 and its receptor ACE2. Signal Transduct Target Ther. 2021; 6(1): 396. https://dx.doi.org/10.1038/s41392-021-00809-8.
34. [34] 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. https://dx.doi.org/10.1016/j.antiviral.2020.104742.
35. [35] Hoffmann M, Kleine-Weber H, Pohlmann S. A Multibasic ccleavage site in the spike protein of SARS-CoV-2 is essential for infection of human in lung cells. Mol Cell. 2020; 78(4): 779-784. https://dx.doi.org/10.1016/j.antiviral.2020.104742.
36. [36] Kiefer AM, Niemeyer J, Probst A, Erkel G, Schroda M. Production and secretion of functional SARS-CoV-2 spike protein Chlamydomonas reinhardtii. https://dx.doi.org/10.3389/fpls.2022.988870.