Development, production and characterization of SARS-CoV-2 virus-like particles (Coronaviridae: Orthocoronavirinae: Betacoronavirus: Sarbecovirus)

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Introduction. The COVID-19 pandemic caused by SARS-CoV-2 has created serious health problems worldwide. The most effective way to prevent the occurrence of new epidemic outbreaks is vaccination. One of the modern and effective approaches to vaccine development is the use of virus-like particles (VLPs).

The aim of the study is to develop a technology for production of VLP based on recombinant SARS-CoV-2 proteins (E, M, N and S) in insect cells.

Materials and methods. Synthetic genes encoding coronavirus proteins E, M, N and S were used. VLP with various surface proteins of strains similar to the Wuhan virus, Delta, Alpha and Omicron were developed and cloned into the pFastBac plasmid. The proteins were synthesized in the baculovirus expression system and assembled into VLP in the portable Trichoplusia ni cell. The presence of insertion in the baculovirus genome was determined by PCR. ELISA and immunoblotting were used to study the antigenic activity of VLP. VLP purification was performed by ultracentrifugation using 20% sucrose. Morphology was assessed using electron microscopy and dynamic light scattering.

Results. VLPs consisting of recombinant SARS-CoV-2 proteins (S, M, E and N) were obtained and characterized. The specific binding of antigenic determinants in synthesized VLPs with antibodies to SARS-CoV-2 proteins has been demonstrated. The immunogenic properties of VLPs have been studied.

Conclusion. The production and purification of recombinant VLPs consisting of full-length SARS-CoV-2 proteins with a universal set of surface antigens have been developed and optimized. Self-assembling particles that mimic the coronavirus virion induce a specific immune response against SARS-CoV-2.

作者简介

Oleg Latyshev

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: oleglat80@mail.ru
ORCID iD: 0000-0002-5757-3809

PhD, Senior Scientist, Laboratory of molecular diagnostics

俄罗斯联邦, 123098, Moscow

Olga Zaykova

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: zaykova_o_n@mail.ru
ORCID iD: 0000-0003-4708-2069

Researcher at the Laboratory of Molecular Diagnostics

俄罗斯联邦, 123098, Moscow

Olesya Eliseeva

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: olesenka80@mail.ru
ORCID iD: 0000-0002-0723-9749

PhD, Senior Scientist, Laboratory of molecular diagnostics

俄罗斯联邦, 123098, Moscow

Tatyana Savochkina

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: tasavochkina@yandex.ru
ORCID iD: 0000-0003-4366-8476

Researcher, Laboratory of Molecular Diagnostics

俄罗斯联邦, 123098, Moscow

Yana Chernoryzh

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: revengeful_w@mail.ru
ORCID iD: 0000-0001-9848-8515

Candidate of Medical Sciences, Researcher, laboratory of molecular diagnostics

俄罗斯联邦, 123098, Moscow

Anton Syroeshkin

Peoples’ Friendship University of Russia

Email: syroeshkin-av@rudn.ru
ORCID iD: 0000-0003-3279-7520

Doctor of Biological Sciences, Professor, Head of the Department of Pharmaceutical and Toxicological chemistry

俄罗斯联邦, 117198, Moscow

Gleb Petrov

Peoples’ Friendship University of Russia

Email: petrov-gv@rudn.ru
ORCID iD: 0009-0004-1123-7393

3rd year PhD Student, Assistant at the Department of Pharmaceutical and Toxicological chemistry

俄罗斯联邦, 117198, Moscow

Galina Vorkunova

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: g.k.vorkunova@gmail.com
ORCID iD: 0000-0003-1346-3744

Doctor of Biological Sciences, Leading Researcher, Laboratory of Molecular Diagnostics National Research Center

俄罗斯联邦, 123098, Moscow

Victor Larichev

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: vlaritchev@mail.ru
ORCID iD: 0000-0001-8262-5650

Doctor of Med. Sci, Leading Researcher, laboratory of biology and indication of arbovirus infections

俄罗斯联邦, 123098, Moscow

Irina Fediakina

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: irfed2@mail.ru
ORCID iD: 0000-0001-6421-9632

PhD, Lead Researcher of laboratory of viral ecology

俄罗斯联邦, 123098, Moscow

Stanislav Cherepushkin

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: cherepushkin1@gmail.com
ORCID iD: 0000-0002-1734-5369

Researcher of laboratory of molecular diagnostics

俄罗斯联邦, 123098, Moscow

Valeriy Tsibezov

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: tsibezov@yandex.ru
ORCID iD: 0000-0003-2150-5764

PhD, Leading Researcher of laboratory of specific virus prevention products

俄罗斯联邦, 123098, Moscow

Ksenia Yuzhakova

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: chekh-ks@mail.ru
ORCID iD: 0000-0003-3130-5029

Researcher of laboratory of molecular diagnostics

俄罗斯联邦, 123098, Moscow

Nadezhda Kulikova

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: nad007@rambler.ru
ORCID iD: 0000-0002-3008-3383

Researcher of laboratory of molecular diagnostics

俄罗斯联邦, 123098, Moscow

Varvara Lebedeva

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: lebedevavv@yandex.ru
ORCID iD: 0000-0002-3088-0403

Researcher of laboratory of molecular diagnostics

俄罗斯联邦, 123098, Moscow

Dmitriy Yakunin

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: yd364@mail.ru
ORCID iD: 0009-0009-4531-5739

Graduate student of laboratory of molecular diagnostics

俄罗斯联邦, 123098, Moscow

Alina Kozlova

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: malinkakozlova88@gmail.com
ORCID iD: 0000-0003-2749-3258

PhD, researcher, laboratory of biology and indication of arbovirus infections

俄罗斯联邦, 123098, Moscow

Marina Baranets

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: shizotorex@mai.ru
ORCID iD: 0000-0002-3466-3588

PhD, researcher, laboratory of biology and indication of arbovirus infections

俄罗斯联邦, 123098, Moscow

Kirill Yurlov

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: kir34292@yandex.ru
ORCID iD: 0000-0002-4694-2445

Researcher, laboratory of cellular engineering

俄罗斯联邦, 123098, Moscow

Ekaterina Lesnova

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: wolf252006@yandex.ru
ORCID iD: 0000-0002-2801-6843

Researcher, laboratory of Molecular Diagnostics

俄罗斯联邦, 123098, Moscow

Tatyana Grebennikova

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

编辑信件的主要联系方式.
Email: t_grebennikova@mail.ru
ORCID iD: 0000-0002-6141-9361

Doctor of Biological Sciences, Professor, Corresponding Member RAS, deputy Director for Science of the Division of the Ivanovsky Virology Institute Head of the Control Center of National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya

俄罗斯联邦, 123098, Moscow

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2. Fig. 1. Gene inserts of the appropriate size in recombinant baculoviruses obtained as a result of PCR. 1 – diagnostic fragment of the S protein gene (597 bp); 2 – marker of molecular weights; 3 – insert containing the M protein gene (768 bp); 4 – insert containing the N protein gene (1359 bp); 5 – an insert containing the E protein gene (327 bp).

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3. Fig. 2. Western blot of recombinant VLP proteins after treatment with positive (a) and negative serum (b). Tracks: 1 – negative control (cells T.ni); 2 – N; 3 – M; 4 – E; 5 – S (Wuhan-like virus); 6 – S (Omicron); 7 – S (Delta); 8 – S (Alpha); 9 – positive control (SARS-CoV-2).

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4. Fig. 3. Morphology and physical dimensions of purified VLPs. a – electron microscopy of VLP; b – histogram of VLP size distribution.

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5. Fig. 4. Results of a study of the immune specificity of VLP in ELISA with human sera characterized by NT. The X-axis shows human sera characterized in NT; the Y-axis shows optical density at a wavelength of 450 nm, legend – VLP at concentrations of 8, 2, 0.5 and 0.125 micrograms/well: otp 1 – serum negative in NT; otp 2 – serum negative in NT collected more than 20 years ago; 1 : 320 – serum positive in NT, titer 1 : 320; 1 : 160 –serum positive in NT, titer 1 : 160; 1 : 80 – serum positive in NT, titer 1 : 80.

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版权所有 © Latyshev O.E., Zaykova O.N., Eliseeva O.V., Savochkina T.E., Chernoryzh Y.Y., Syroeshkin A.V., Petrov G.V., Vorkunova G.K., Larichev V.F., Fediakina I.T., Cherepushkin S.A., Tsibezov V.V., Yuzhakova K.A., Kulikova N.Y., Lebedeva V.V., Yakunin D.Y., Kozlova A.A., Baranets M.S., Yurlov K.I., Lesnova E.I., Grebennikova T.V., 2024

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