Citation: Vakhrusheva, A.V.; Kudriavtsev, A.V.; Kryuchkov, N.A.; Deev, R.V.; Frolova, M.E.; Blagodatskikh, K.A.; Djonovic, M.; Nedorubov, A.A.; Odintsova, E.; Ivanov,A.V.; et al. SARS-CoV-2 Subunit Virus-like Vaccine Demonstrates High Safety Profile and Protective Efficacy: Preclinical Study. Vaccines 2022, 10, 1290. https:// doi.org/10.3390/vaccines10081290 Academic Editors: Esther Blanco, Juan Bárcena and S. Louise Cosby Received: 12 June 2022 Accepted: 4 August 2022 Published: 10 August 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Article SARS-CoV-2 Subunit Virus-like Vaccine Demonstrates High Safety Profile and Protective Efficacy: Preclinical Study Anna V. Vakhrusheva 1,2, * , Aleksandr V. Kudriavtsev 1,2 , Nickolay A. Kryuchkov 3 , Roman V. Deev 4 , Maria E. Frolova 5 , Konstantin A. Blagodatskikh 6 , Milana Djonovic 1 , Andrey A. Nedorubov 7 , Elena Odintsova 7 , Aleksandr V. Ivanov 8 , Ekaterina A. Romanovskaya-Romanko 9 , Marina A. Stukova 9 , Artur A. Isaev 5,6 and Igor V. Krasilnikov 8,9 1 Betuvax LLC, 121096 Moscow, Russia 2 Department of Bioengineering, Biological Faculty, Lomonosov Moscow State University, 119991 Moscow, Russia 3 Clinical Excellence Group LLC, 127051 Moscow, Russia 4 Department of Pathological Anatomy, I.I. Mechnikov North-Western State Medical University, 195067 Saint Petersburg, Russia 5 PJSC Human Stem Cells Institute, 129110 Moscow, Russia 6 Center of Genetics and Reproductive Medicine “Genetico”, 119333 Moscow, Russia 7 Center for Preclinical Studies, Sechenov First Moscow State Medical University, 119435 Moscow, Russia 8 Biotechnology Developments LLC, 119285 Moscow, Russia 9 Department of Vaccinology, Smorodintsev Research Institute of Influenza, Ministry of Health of the Russian Federation, 197376 Saint Petersburg, Russia * Correspondence: vakhrusheva@betuvax.ru; Tel.: +7-985-6171544 Abstract: Public health threat coming from a rapidly developing COVID-19 pandemic calls for developing safe and effective vaccines with innovative designs. This paper presents preclinical trial results of “Betuvax-CoV-2”, a vaccine developed as a subunit vaccine containing a recombinant RBD- Fc fusion protein and betulin-based spherical virus-like nanoparticles as an adjuvant (“Betuspheres”). The study aimed to demonstrate vaccine safety in mice, rats, and Chinchilla rabbits through acute, subchronic, and reproductive toxicity studies. Along with safety, the vaccine demonstrated protective efficacy through SARS-CoV-2-neutralizing antibody production in mice, rats, hamsters, rabbits, and primates (rhesus macaque), and lung damage and infection protection in hamsters and rhesus macaque model. Eventually, “Betuvax-CoV-2” was proved to confer superior efficacy and protection against the SARS-CoV-2 in preclinical studies. Based on the above results, the vaccine was enabled to enter clinical trials that are currently underway. Keywords: SARS-CoV-2; RBD-based vaccine; nanoparticle vaccine; recombinant vaccine; betulin; COVID-19 1. Introduction The world population is currently struggling with an outbreak of COVID-19 caused by the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) [1]. The World Health Organization (WHO) reported over 400 million confirmed cases of COVID-19 and up to 6 million deaths by 8 February 2022 [2]. As a result, much effort was made to develop safe and efficient vaccines. By 7 February 2022, there were 183 vaccine candidates developed, and only 10 of them were approved for use by the WHO [3]. Nevertheless, by mid-February 2022, only 61.9% of the world population received at least one dose of a vaccine against COVID-19, that being approximately 10 billion administered doses, but mostly in high-income countries, whereas in low-income countries, only 10.6% [4]. Although, in the past, the only known coronavirus infections were associated with mild symptoms as in a “common cold”, the severe clinical conditions that SARS-CoV-2 causes and the magnitude of the coronavirus pandemic sparked the scientific interest in developing Vaccines 2022, 10, 1290. https://doi.org/10.3390/vaccines10081290 https://www.mdpi.com/journal/vaccines