Thailin Lao, Ileanet Avalos, Elsa María Rodríguez, Yasser Zamora, Alianet Rodriguez, Ailyn Ramón, Yanitza Alvarez, Ania Cabrales, Ivan Andújar, Luis Javier González, Pedro Puente, Cristina García, Leonardo Gómez, Rodolfo Valdés, Mario Pablo Estrada , Yamila Carpio
Despite that more than one hundred vaccines against SARS-CoV-2 have been developed and that some of them were evaluated in clinical trials, the latest results revealed that these vaccines still face great challenges. Among the components of the virus, the N-protein constitutes an attractive target for a subunit vaccine because it is the most abundant, highly conserved and immunogenic protein. In the present work, a chimeric protein (N-CD protein) was constructed by the fusion of the N-protein to the extracellular domain of human CD154 as the molecular adjuvant. HEK-293 cells were transduced with lentiviral vector bearing the N-CD gene and polyclonal cell populations were obtained. The N-CD protein was purified from cell culture supernatant and further characterized by several techniques. Immunogenicity studies in mice and non-human primates showed the N-CD protein induced high IgG titers in both models after two doses. Moreover, overall health monitoring of non-human primates demonstrated that animals were healthy during 228 days after first immunization. Data obtained support further investigation in order to develop this chimeric protein as vaccine candidate against COVID-19 and other coronavirus diseases.
Coronavirus disease 2019 (COVID-19), a disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has become a serious threat to human health causing millions of deaths around the world. SARS-CoV-2 has a ribonucleic acid (RNA) genome, single stranded and positive orientation. This genome is 29.88 Kb and contains approximately 12 open reading frames. Like in all coronaviruses (CoVs), these genes encode four major structural proteins: the nucleocapsid (N) that is associated with the RNA genome and three membrane proteins that are the spike glycoproteins (S), the integral membrane glycoprotein (M) and the envelope protein (E). Protein S contains a receptor-binding domain (RBD) and this RBD domain has an approximate molecular mass of 25 kDa. This domain is involved in viral binding and fusion to the cell membrane, and induces neutralizing antibodies that block binding to the angiotensin II receptor (ACE2) present in host cells. Until now, the RBD domain constitutes the fundamental target for the development of subunit vaccines against SARS-CoV2 [1–3]. The appearance of new circulating variants of the virus due to mutations in the RBD domain has raised important concerns about the geographic and temporal efficacy of vaccines based on this protein domain .
Materials and methods
The plasmid pDisplay-CMV-N-CD was constructed by steps. First, the plasmid pDisplay-CMV-linker-CD was generated through the cloning of a linker sequence followed by the gene of the extracellular domain of Homo sapiens CD154 (NM_000074.2), as described by Ávalos et al.  (see also S1 Table for more details). This linker sequence corresponds to a 6 His tail and a linker (Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser). Second, a sequence encoding for amino acids (aa) 2–419 of the SARS-CoV-2 N protein was amplified by RT-PCR using mRNA samples extracted from convalescent COVID-19 patients. N protein-F and N protein-R oligonucleotide primers (S1 Table) were designed taking into account the sequence reported for SARS-CoV-2 isolate Wuhan-Hu-1 (GenBank: MN908947.3). The PCR product was cloned into a pGEM-Teasy vector (Promega, Madison, USA) and sequenced. Then, this N gene was amplified by PCR using specific oligonucleotide primers N-Display-F and N-Display-R (S1 Table), adding a BamHI restriction site to the 5´ end.
Functionality of the N-CD expression cassette in the pDisplay-CMV-N-CD and pl6twblast-CMV-N-CD plasmids
Recombinant N-CD protein comprises the SARS-CoV-2 N protein fused by a linker sequence to the extracellular domain of human CD154. Sequencing results showed a different N protein variant compared with Wuhan1 strain. This variant bears an amino acid replacement of RG (AGGGGA) by KR (AAACGA) according a previous report .
The Western Blotting analysis of cell culture supernatant of HEK-293 cells transfected with the plasmid pDisplay-CMV-N-CD, using an anti-SARS-CoV-2 N protein monoclonal antibody (S1C Fig) and sera from convalescent COVID-19 patients (S1D Fig), showed a band between 75 and 100 kDa bands of the molecular weight marker, under reducing conditions. The expected molecular weight for N-CD protein is 73,1 kDa. High molecular aggregates were also detected when the electrophoresis was performed in non-reducing conditions. A band around 50 kDa corresponding to SARS-CoV-2 N protein expressed in E. coli, used as positive control, was also detected.
Since the emergence of SARS-CoV-2 variants with enhanced transmissibility, such as alpha, beta, delta and omicron (https://www.who.int/en/activities/tracking-SARS-CoV-2-variants/); there is a growing concern that these new variants could impair the efficacy of current vaccines . Indeed, the variants present mutations that are found in the antigenic supersite in the N-terminal domain of the spike protein or in the ACE2-binding site of the RBD, which is a major target of potent virus-neutralizing antibodies elicit by vaccines [22–24]. Taking into account this evidence, many scientists have proposed to use the N protein of SARS-CoV-2 as an important target for vaccine development [8, 9, 25–27]. New vaccines candidates based on the combination of Spike protein or RBD and N protein, could control SARS-CoV-2 in a more efficient manner. The combination of both antigens may elicit T cell and neutralizing antibody responses that cross-react with different SARS-CoV-2 variants [28–32].
We acknowledge the valuable technical support of Regla Margarita Somoza (Quality Control Department, CIGB) in SDS-PAGE and Western Blotting analysis. We are grateful to Gilda Lemos Perez (Biomedical Research Department, CIGB) for providing reagents and the knowledge to perform ELISA experiments. We thank to Gabriela Calderin Hernandez (Biomedical Research Department, CIGB) for help during the purification of the N-CD protein. We are grateful to CIGB Sancti Spiritus for the N protein and antibodies supply, especially Yeosvany Cabrera for his unconditional support. Special thanks to Randall Morse for his valuable comments and review of this manuscript.
Citation: Lao T, Avalos I, Rodríguez EM, Zamora Y, Rodriguez A, Ramón A, et al. (2023) Production and characterization of a chimeric antigen, based on nucleocapsid of SARS-CoV-2 fused to the extracellular domain of human CD154 in HEK-293 cells as a vaccine candidate against COVID-19. PLoS ONE 18(9): e0288006. https://doi.org/10.1371/journal.pone.0288006
Editor: Engin Berber, Lerner Research Institute - Cleveland Clinic, UNITED STATES
Received: January 27, 2023; Accepted: June 16, 2023; Published: September 26, 2023
Copyright: © 2023 Lao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: The authors received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.