{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,30]],"date-time":"2026-03-30T11:09:10Z","timestamp":1774868950095,"version":"3.50.1"},"reference-count":50,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2022,1,2]],"date-time":"2022-01-02T00:00:00Z","timestamp":1641081600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>The COVID-19 pandemic is ongoing, and the need for safe and effective vaccines to prevent infection and to control spread of the virus remains urgent. Here, we report the development of a SARS-CoV-2 subunit vaccine candidate (Betuvax-CoV-2) based on RBD and SD1 domains of the spike (S) protein fused to a human IgG1 Fc fragment. The antigen is adsorbed on betulin adjuvant, forming spherical particles with a size of 100\u2013180 nm, mimicking the size of viral particles. Here we confirm the potent immunostimulatory activity of betulin adjuvant, and demonstrate that two immunizations of mice with Betuvax-CoV-2 elicited high titers of RBD-specific antibodies. The candidate vaccine was also effective in stimulating a neutralizing antibody response and T cell immunity. The results indicate that Betuvax-CoV-2 has good potential for further development as an effective vaccine against SARS-CoV-2.<\/jats:p>","DOI":"10.3390\/vaccines10010069","type":"journal-article","created":{"date-parts":[[2022,1,9]],"date-time":"2022-01-09T23:35:09Z","timestamp":1641771309000},"page":"69","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["Design and Immunological Properties of the Novel Subunit Virus-like Vaccine against SARS-CoV-2"],"prefix":"10.3390","volume":"10","author":[{"given":"Igor V.","family":"Krasilnikov","sequence":"first","affiliation":[{"name":"Department of Vaccinology, Smorodintsev Research Institute of Influenza of the Ministry of Health of the Russian Federation, 197376 Saint Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aleksandr V.","family":"Kudriavtsev","sequence":"additional","affiliation":[{"name":"Betuvax, 121096 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7948-1254","authenticated-orcid":false,"given":"Anna V.","family":"Vakhrusheva","sequence":"additional","affiliation":[{"name":"Betuvax, 121096 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maria E.","family":"Frolova","sequence":"additional","affiliation":[{"name":"PJSC Human Stem Cells Institute, 129110 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7608-1914","authenticated-orcid":false,"given":"Aleksandr V.","family":"Ivanov","sequence":"additional","affiliation":[{"name":"Razvitie BioTechnologiy, 119136 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marina A.","family":"Stukova","sequence":"additional","affiliation":[{"name":"Department of Vaccinology, Smorodintsev Research Institute of Influenza of the Ministry of Health of the Russian Federation, 197376 Saint Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ekaterina A.","family":"Romanovskaya-Romanko","sequence":"additional","affiliation":[{"name":"Department of Vaccinology, Smorodintsev Research Institute of Influenza of the Ministry of Health of the Russian Federation, 197376 Saint Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7750-9652","authenticated-orcid":false,"given":"Kirill A.","family":"Vasilyev","sequence":"additional","affiliation":[{"name":"Department of Vaccinology, Smorodintsev Research Institute of Influenza of the Ministry of Health of the Russian Federation, 197376 Saint Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nataliya V.","family":"Mushenkova","sequence":"additional","affiliation":[{"name":"Betuvax, 121096 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Artur A.","family":"Isaev","sequence":"additional","affiliation":[{"name":"PJSC Human Stem Cells Institute, 129110 Moscow, Russia"},{"name":"Center of Genetics and Reproductive Medicine \u201cGenetico\u201d, 119333 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,1,2]]},"reference":[{"key":"ref_1","unstructured":"(2021, November 17). WHO. Available online: https:\/\/www.who.int\/publications\/m\/item\/weekly-operational-update-on-covid-19---3-november-2021."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"237","DOI":"10.1146\/annurev-virology-110615-042301","article-title":"Structure, Function, and Evolution of Coronavirus Spike Proteins","volume":"3","author":"Li","year":"2016","journal-title":"Annu. Rev. Virol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"104742","DOI":"10.1016\/j.antiviral.2020.104742","article-title":"The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade","volume":"176","author":"Coutard","year":"2020","journal-title":"Antivir. Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"867","DOI":"10.1007\/s00408-020-00408-4","article-title":"ACE2: The Major Cell Entry Receptor for SARS-CoV-2","volume":"198","author":"Scialo","year":"2020","journal-title":"Lung"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1080\/14760584.2021.1875824","article-title":"COVID-19 vaccine: Where are we now and where should we go?","volume":"20","author":"Soleimanpour","year":"2021","journal-title":"Expert Rev. Vaccines"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"882","DOI":"10.1016\/j.micinf.2005.02.006","article-title":"Antibody responses to individual proteins of SARS coronavirus and their neutralization activities","volume":"7","author":"Qiu","year":"2005","journal-title":"Microbes Infect."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"198508","DOI":"10.1016\/j.virusres.2021.198508","article-title":"SARS-CoV-2 specific memory T cell epitopes identified in COVID-19-recovered subjects","volume":"304","author":"Zhao","year":"2021","journal-title":"Virus Res."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1038\/s41591-021-01230-y","article-title":"Looking beyond COVID-19 vaccine phase 3 trials","volume":"27","author":"Kim","year":"2021","journal-title":"Nat. Med."},{"key":"ref_9","unstructured":"(2021, November 17). Reuters. Available online: https:\/\/www.reuters.com\/article\/health-coronavirus-china-vaccine-int-idUSKBN2940CA."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2603","DOI":"10.1056\/NEJMoa2034577","article-title":"Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine","volume":"383","author":"Polack","year":"2020","journal-title":"N. Engl. J. Med."},{"key":"ref_11","unstructured":"(2021, November 17). WHO. Available online: https:\/\/www.who.int\/publications\/m\/item\/who-target-product-profiles-for-covid-19-vaccines."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2092","DOI":"10.1056\/NEJMoa2104840","article-title":"Thrombotic Thrombocytopenia after ChAdOx1 nCov-19 Vaccination","volume":"384","author":"Greinacher","year":"2021","journal-title":"N. Engl. J. Med."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2293","DOI":"10.1038\/s41423-021-00752-2","article-title":"Neutralizing antibodies for the prevention and treatment of COVID-19","volume":"18","author":"Du","year":"2021","journal-title":"Cell Mol. Immunol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Peters, M.H., Bastidas, O., Kokron, D.S., and Henze, C.E. (2020). Static all-atom energetic mappings of the SARS-CoV-2 spike protein and dynamic stability analysis of \u201cUp\u201d versus \u201cDown\u201d protomer states. PLoS ONE., 15.","DOI":"10.1101\/2020.05.12.091090"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"263","DOI":"10.1080\/07391102.2020.1811774","article-title":"Insights into the structural and dynamical changes of spike glycoprotein mutations associated with SARS-CoV-2 host receptor binding","volume":"40","author":"Ahamad","year":"2020","journal-title":"J. Biomol. Struct. Dyn."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"645","DOI":"10.2174\/1871520617666171012124820","article-title":"Immunopharmacological Activity of Betulin in Inflammation-associated Carcinogenesis","volume":"18","author":"Schwiebs","year":"2018","journal-title":"Anticancer Agents Med. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1021\/mp060096p","article-title":"Type 1 and 2 immunity following vaccination is influenced by nanoparticle size: Formulation of a model vaccine for respiratory syncytial virus","volume":"4","author":"Mottram","year":"2007","journal-title":"Mol. Pharm."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"e57309","DOI":"10.7554\/eLife.57309","article-title":"SARS-CoV-2 (COVID-19) by the numbers","volume":"9","author":"Flamholz","year":"2020","journal-title":"eLife"},{"key":"ref_19","unstructured":"(2021, January 30). RBC. Available online: https:\/\/amp.rbc.ru\/rbcnews\/rbcfreenews\/5e735ff09a7947be392f2bec."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1489","DOI":"10.1016\/j.cell.2020.05.015","article-title":"Targets of T cell responses to SARS-CoV-2 coronavirus in humans with COVID-19 disease and unexposed individuals","volume":"181","author":"Grifoni","year":"2020","journal-title":"Cell"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"100355","DOI":"10.1016\/j.xcrm.2021.100355","article-title":"Impact of SARS-CoV-2 variants on the total CD4+ and CD8+ T cell reactivity in infected or vaccinated individuals","volume":"2","author":"Tarke","year":"2021","journal-title":"Cell Rep. Med."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1016\/j.jconrel.2015.09.069","article-title":"Shape and size-dependent immune response to antigen-carrying nanoparticles","volume":"220","author":"Kumar","year":"2015","journal-title":"Control. Release"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"6533","DOI":"10.4049\/jimmunol.172.11.6533","article-title":"Cutting Edge: Effector Memory CD8+ T Cells Play a Prominent Role in Recall Responses to Secondary Viral Infection in the Lung","volume":"172","author":"Roberts","year":"2004","journal-title":"J. Immunol."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Tseng, C.T., Sbrana, E., Iwata-Yoshikawa, N., Newman, P.C., Garron, T., and Atmar, R.L. (2012). Immunization with SARS coronavirus vaccines leads to pulmonary immunopathology on challenge with the SARS virus. PLoS ONE., 7.","DOI":"10.1371\/annotation\/2965cfae-b77d-4014-8b7b-236e01a35492"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"347","DOI":"10.1038\/s41577-020-0323-4","article-title":"COVID-19 vaccine design: The Janus face of immune enhancement","volume":"20","author":"Hotez","year":"2020","journal-title":"Nat. Rev. Immunol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1038\/s41421-020-00199-1","article-title":"Immunization with the receptor-binding domain of SARS-CoV-2 elicits antibodies cross-neutralizing SARS-CoV-2 and SARS-CoV without antibody-dependent enhancement","volume":"6","author":"Zang","year":"2020","journal-title":"Cell Discov."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"572","DOI":"10.1038\/s41586-020-2599-8","article-title":"A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity","volume":"586","author":"Yang","year":"2020","journal-title":"Nature"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Jiang, H., and Mei, Y. (2021). SARS-CoV-2 Spike Impairs DNA Damage Repair and Inhibits V(D)J Recombination In Vitro. Viruses, 13.","DOI":"10.3390\/v13102056"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"BSR20210611","DOI":"10.1042\/BSR20210611","article-title":"SARS-CoV-2 spike protein S1 induces fibrin (ogen) resistant to fibrinolysis: Implications for microclot formation in COVID-19","volume":"41","author":"Grobbelaar","year":"2021","journal-title":"Biosci. Rep."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"136","DOI":"10.1016\/j.vaccine.2006.06.084","article-title":"Immunogenicity of a receptor-binding domain of SARS coronavirus spike protein in mice: Implications for a subunit vaccine","volume":"25","author":"Zakhartchouk","year":"2007","journal-title":"Vaccine"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"eabc3539","DOI":"10.1126\/scitranslmed.abc3539","article-title":"Antibody signature induced by SARS-CoV-2 spike protein immunogens in rabbits","volume":"12","author":"Ravichandran","year":"2020","journal-title":"Sci. Transl. Med."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"773","DOI":"10.1016\/j.bbrc.2004.09.106","article-title":"Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: Implication for developing subunit vaccine","volume":"324","author":"He","year":"2004","journal-title":"Biochem. Biophys Res. Commun."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1016","DOI":"10.3201\/eid1107.050219","article-title":"SARS vaccine development","volume":"11","author":"Jiang","year":"2005","journal-title":"Emerg. Infect Dis."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1038\/nature12328","article-title":"Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26","volume":"500","author":"Lu","year":"2013","journal-title":"Nature"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"9379","DOI":"10.1128\/JVI.01277-13","article-title":"The receptor binding domain of the new Middle East respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies","volume":"87","author":"Mou","year":"2013","journal-title":"J. Virol."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Du, L., Kou, Z., Ma, C., Tao, X., Wang, L., Zhao, G., Chen, Y., Yu, F., Tseng, C.K., and Zhou, Y. (2013). A truncated receptor-binding domain of MERS-CoV spike protein potently inhibits MERS-CoV infection and induces strong neutralizing antibody responses: Implication for developing therapeutics and vaccines. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0081587"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"10777","DOI":"10.1128\/JVI.01756-13","article-title":"Crystal structure of the receptor-binding domain from newly emerged Middle East respiratory syndrome coronavirus","volume":"87","author":"Chen","year":"2013","journal-title":"J. Virol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"986","DOI":"10.1038\/cr.2013.92","article-title":"Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4","volume":"23","author":"Wang","year":"2013","journal-title":"Cell Res."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"5071","DOI":"10.1016\/j.vaccine.2020.04.070","article-title":"The biological characteristics of SARS-CoV-2 spike protein Pro330-Leu650","volume":"38","author":"Su","year":"2020","journal-title":"Vaccine"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"282","DOI":"10.1038\/s41392-020-00402-5","article-title":"RBD-Fc-based COVID-19 vaccine candidate induces highly potent SARS-CoV-2 neutralizing antibody response","volume":"5","author":"Liu","year":"2020","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_41","first-page":"205","article-title":"Triterpenes from Euphorbia spinidens with immunomodulatory activity","volume":"8","author":"Ghannadian","year":"2013","journal-title":"Res. Pharm. Sci."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Pfarr, K., Danciu, C., Arlt, O., Neske, C., Dehelean, C., Pfeilschifter, J.M., and Radeke, H.H. (2015). Simultaneous and dose dependent melanoma cytotoxic and immune stimulatory activity of betulin. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0118802"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"772","DOI":"10.1016\/j.parint.2014.07.008","article-title":"Carbon nanotube based betulin formulation shows better efficacy against Leishmania parasite","volume":"63","author":"Saudagar","year":"2014","journal-title":"Parasitol. Int."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"14992","DOI":"10.3390\/ijms131114992","article-title":"Betulin complex in \u03b3-cyclodextrin derivatives: Properties and antineoplasic activities in in vitro and in vivo tumor models","volume":"13","author":"Soica","year":"2012","journal-title":"Int. J. Mol. Sci."},{"key":"ref_45","first-page":"289","article-title":"A comparison investigation on the solubilization of betulin and betulinic acid in cyclodextrin derivatives","volume":"7","author":"Wang","year":"2012","journal-title":"Nat. Prod. Commun."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"327","DOI":"10.1007\/s13318-016-0340-7","article-title":"Pharmacokinetics and tissue disposition of nonosystem entrapped betulin after endotracheal administration to rats","volume":"42","author":"Pozhacitskaya","year":"2017","journal-title":"Eur. J. Drug Metab. Pharmacokinet."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1016\/j.biopha.2015.07.033","article-title":"Self-assembled betulinic acid augments immunomodulatory activity associates with IgG response","volume":"75","author":"Dash","year":"2015","journal-title":"Biomed. Pharmacother."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"82","DOI":"10.1038\/s41421-021-00320-y","article-title":"RBD-homodimer, a COVID-19 subunit vaccine candidate, elicits immunogenicity and protection in rodents and nonhuman primates","volume":"7","author":"Pan","year":"2021","journal-title":"Cell Discov."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"eabd6160","DOI":"10.1126\/sciimmunol.abd6160","article-title":"SARS-CoV-2 T cell immunity: Specificity, function, durability, and role in protection","volume":"5","author":"Altmann","year":"2020","journal-title":"Sci. Immunol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"1998","DOI":"10.1002\/eji.202048908","article-title":"Divergent SARS-CoV-2-specific T- and B-cell responses in severe but not mild COVID-19 patients","volume":"50","author":"Oja","year":"2020","journal-title":"Eur. J. Immunol."}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/10\/1\/69\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,13]],"date-time":"2025-10-13T14:12:32Z","timestamp":1760364752000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/10\/1\/69"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,1,2]]},"references-count":50,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2022,1]]}},"alternative-id":["vaccines10010069"],"URL":"https:\/\/doi.org\/10.3390\/vaccines10010069","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,1,2]]}}}