Citation: Ávalos, I.; Lao, T.; Rodríguez, E.M.; Zamora, Y.; Rodríguez, A.; Ramón, A.; Lemos, G.; Cabrales, A.; Bequet-Romero, M.; Casillas, D.; et al. Chimeric Antigen by the Fusion of SARS-CoV-2 Receptor Binding Domain with the Extracellular Domain of Human CD154: A Promising Improved Vaccine Candidate. Vaccines 2022, 10, 897. https://doi.org/10.3390/ vaccines10060897 Academic Editor: François Meurens Received: 16 March 2022 Accepted: 27 May 2022 Published: 3 June 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 Chimeric Antigen by the Fusion of SARS-CoV-2 Receptor Binding Domain with the Extracellular Domain of Human CD154: A Promising Improved Vaccine Candidate Ileanet Ávalos, Thailin Lao , Elsa María Rodríguez, Yasser Zamora, Alianet Rodríguez, Ailyn Ramón, Gilda Lemos , Ania Cabrales, Monica Bequet-Romero , Dionne Casillas, Ivan Andújar , Luis Ariel Espinosa, Luis Javier González, Yanitza Alvarez, Yamila Carpio * and Mario Pablo Estrada * Center for Genetic Engineering and Biotechnology, CIGB, Ave. 31 E/158 y 190, Havana 10600, Cuba; ileanet.avalos@cigb.edu.cu (I.Á.); thailin.lao@cigb.edu.cu (T.L.); elsa.rodriguez@cigb.edu.cu (E.M.R.); yasser.zamora@cigb.edu.cu (Y.Z.); alianet.rodriguez@cigb.edu.cu (A.R.); ailyn.ramon@cigb.edu.cn (A.R.); gilda.lemos@cigb.edu.cu (G.L.); ania.cabrales@cigb.edu.cu (A.C.); monica.bequet@cigb.edu.cu (M.B.-R.); dionne.casillas@cigb.edu.cu (D.C.); ivan.andujar@cigb.edu.cu (I.A.); luis.espinosa@cigb.edu.cu (L.A.E.); luis.javier@cigb.edu.cu (L.J.G.); yanitza.alvarez@cigb.edu.cu (Y.A.) * Correspondence: yamila.carpio@cigb.edu.cu (Y.C.); mario.pablo@cigb.edu.cu(M.P.E.) Abstract: COVID-19 is a respiratory viral disease caused by a new coronavirus called SARS-CoV-2. This disease has spread rapidly worldwide with a high rate of morbidity and mortality. The receptor- binding domain (RBD) of protein spike (S) mediates the attachment of the virus to the host’s cellular receptor. The RBD domain constitutes a very attractive target for subunit vaccine development due to its ability to induce a neutralizing antibody response against the virus. With the aim of boosting the immunogenicity of RBD, it was fused to the extracellular domain of CD154, an immune system modulator molecule. To obtain the chimeric protein, stable transduction of HEK-293 was carried out with recombinant lentivirus and polyclonal populations and cell clones were obtained. RBD-CD was purified from culture supernatant and further characterized by several techniques. RBD-CD immuno- genicity evaluated in mice and non-human primates (NHP) indicated that recombinant protein was able to induce a specific and high IgG response after two doses. NHP sera also neutralize SARS-CoV-2 infection of Vero E6 cells. RBD-CD could improve the current vaccines against COVID-19, based in the enhancement of the host humoral and cellular response. Further experiments are necessary to confirm the utility of RBD-CD as a prophylactic vaccine and/or booster purpose. Keywords: SARS-CoV-2; RBD; HEK-293; lentivirus; vaccine 1. Introduction The coronavirus disease (COVID-19) is a respiratory viral disease caused by a new coronavirus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). This virus has become a severe threat to global health due to its rapid expansion around the world with a high rate of morbidity and mortality [1]. SARS-CoV-2 belongs to the beta- coronavirus genus of the family Coronaviridae. The Coronaviridae family comprises 4 genera: alpha-coronavirus, beta-coronavirus, gamma-coronavirus, and delta-coronavirus. Coronaviruses are enveloped viruses with a single-stranded, positive-sense RNA genome of 29–30 kb in size. These viruses infect several animal species including humans [2]. The SARS- CoV-2 genome encodes four major structural proteins: spike (S), membrane (M), envelope (E), and nucleocapsid (N). The S protein has become the main therapeutic target due to its critical role in viral attachment, entry, and fusion. This protein has two major subunits: the S1 and S2. The S1 subunit contains a receptor-binding domain (RBD) of 25 kDa approximately, which is responsible for the direct binding with the angiotensin- converting enzyme 2 (ACE2), a protein receptor on the surface of human cells that mediates Vaccines 2022, 10, 897. https://doi.org/10.3390/vaccines10060897 https://www.mdpi.com/journal/vaccines