Method of obtaining purified SARS-CoV-2 antigen for structural and immunological studies in whole-virion vaccines against coronavirus infection

Abstract

BACKGROUND: Despite the large-scale development of vaccines against coronavirus infection, there is still no complete information about the antigenic structure of the SARS-CoV-2 virus particles. This article describes a method of obtaining a pure concentrated whole-virion sample that can be used in various studies.

AIM: The goal is to develop an optimal method of purification of the inactivated SARS-CoV-2 virus in order to obtain a standard with parameters of purity and antigen content sufficient for structural and immunological studies.

MATERIALS AND METHODS: To obtain a pure concentrated virus SARS-CoV-2 we used the sucrose density gradient ultracentrifugation. Fractions with the highest content of viral particles were evaluated based on the concentration of nucleocapsid N and glycoprotein S. The purity of the pooled fractions (the purified sample) was evaluated by the presence of impurity proteins, toxins and bovine serum albumin.

RESULTS: The optimal conditions for obtaining the inactivated purified SARS-CoV-2 whole virus were determined. A standard sample of the inactivated SARS-CoV-2 virus was isolated and characterized.

CONCLUSIONS: The obtained standard sample can be used in enzyme immunoassay to measure the amount of antigen in whole-virion vaccines against coronavirus infection, as well as in various structural studies of SARS-CoV-2 virus particle.

Keywords

whole-virion vaccines / coronavirus infection / SARS-CoV-2 / purified virus / sucrose gradient

References

[1] Fan S, Xiao K, Li D, et al. Preclinical immunological evaluation of an intradermal heterologous vaccine against SARS-CoV-2 variants. Emerg Microbes Infect. 2022;11(1):212–226. DOI: 10.1080/22221751.2021.2021807

[2] Fan S., Xiao K., Li D. et al. Preclinical immunological evaluation of an intradermal heterologous vaccine against SARS-CoV-2 variants // Emerg. Microbes Infect. 2022. Vol. 11, No. 1. P. 212–226. DOI: 10.1080/22221751.2021.2021807

[3] Che Y, Liu X, Pu Y, et al. Randomized, double-blinded, placebo-controlled phase 2 trial of an inactivated severe acute respiratory syndrome coronavirus 2 vaccine in healthy adults. Clin Infect Dis. 2021;73(11):e3949–e3955. DOI: 10.1093/cid/ciaa1703

[4] Che Y., Liu X., Pu Y. et al. Randomized, double-blinded, placebo-controlled phase 2 trial of an inactivated severe acute respiratory syndrome coronavirus 2 vaccine in healthy adults // Clin. Infect. Dis. 2021. Vol. 73, No. 11. P. e3949–e3955. DOI: 10.1093/cid/ciaa1703

[5] Saagar VK. Virus purification, detection and removal [dissertation]. Michigan Technological University; 2014. DOI: 10.37099/mtu.dc.etds/850

[6] Saagar V.K. Virus purification, detection and removal [dissertation]. Michigan Technological University, 2014. DOI: 10.37099/mtu.dc.etds/850

[7] Kozlovskaya LI, Piniaeva AN, Ignatyev GM, et al. Long-term humoral immunogenicity, safety and protective efficacy of inactivated vaccine against COVID-19 (CoviVac) in preclinical studies. Emerg Microbes Infect. 2021;10(1):1790–1806. DOI: 10.1080/22221751.2021.1971569

[8] Kozlovskaya L.I., Piniaeva A.N., Ignatyev G.M. et al. Long-term humoral immunogenicity, safety and protective efficacy of inactivated vaccine against COVID-19 (CoviVac) in preclinical studies // Emerg. Microbes Infect. 2021. Vol. 10, No. 1. P. 1790–1806. DOI: 10.1080/22221751.2021.1971569

[9] Limulus amebocyte lysate [Internet]. Endosafe. USA. License No. 1197. Available from: https://limulustest.ru/upload/iblock/625/LAL-Reagent %20Endosafe.PDF. Accessed: 08.06.2022.

[10] Limulus amebocyte lysate [Электронный ресурс] // Endosafe. USA. License No.1197. Режим доступа: https://limulustest.ru/upload/iblock/625/LAL-Reagent %20Endosafe.PDF. Дата обращения: 08.06.2022.

[11] Cubuk J, Alston JJ, Incicco JJ, et al. The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA. Nat Commun. 2021;12(1):1936. DOI: 10.1038/s41467-021-21953-3

[12] Cubuk J., Alston J.J., Incicco J.J. et al. The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA // Nat. Commun. 2021. Vol. 12, No. 1. P. 1936. DOI: 10.1038/s41467-021-21953-3

[13] OFS.1.2.3.0023.15. Ehlektroforez v poliakrilamidnom gele. Gosudarstvennaya farmakopeya Rossiiskoi Federatsii XIV. (In Russ.)

[14] ОФС.1.2.3.0023.15. Электрофорез в полиакриламидном геле // Государственная фармакопея Российской Федерации XIV.

[15] Bagrov DV, Glukhov GS, Moiseenko AV, et al. Structural characterization of β-propiolactone inactivated severe acute respiratory syndrome coronavirus (SARS-CoV-2) particles. Microsc Res Tech. 2022;85(2):562–569. DOI: 10.1002/jemt.23931

[16] Bagrov D.V., Glukhov G.S., Moiseenko A.V. et al. Structural characterization of β-propiolactone inactivated severe acute respiratory syndrome coronavirus (SARS-CoV-2) particles // Microsc. Res. Tech. 2022. Vol. 85, No. 2. P. 562–569. DOI: 10.1002/jemt.23931

[17] Bai Z, Cao Y, Liu W, Li J. The SARS-CoV-2 nucleocapsid protein and its role in viral structure, biological functions, and a potential target for drug or vaccine mitigation. Viruses. 2021;13(6):1115. DOI: 10.3390/v13061115

[18] Bai Z., Cao Y., Liu W., Li J. The SARS-CoV-2 nucleocapsid protein and its role in viral structure, biological functions, and a potential target for drug or vaccine mitigation // Viruses. 2021. Vol. 13, No. 6. P. 1115. DOI: 10.3390/v13061115

RIGHTS & PERMISSIONS

Eco-Vector