{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T19:56:55Z","timestamp":1791230215570,"version":"4.1.0"},"reference-count":58,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2022,9,8]],"date-time":"2022-09-08T00:00:00Z","timestamp":1662595200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:p>\n                    SARS-CoV-2 has caused a global pandemic, infecting millions of people. An effective preventive\u00a0vaccine\u00a0against this virus is urgently needed. Here, we designed and developed a novel formulated recombinant receptor-binding domain (RBD) nucleocapsid (N) recombinant\u00a0vaccine\u00a0candidates. The RBD and N were separately expressed in\n                    <jats:italic>E. coli<\/jats:italic>\n                    and purified using column chromatography. The female Balb\/c mice were immunized subcutaneously with the combination of purified RBD and N alone or formulated with saponin adjuvant in a two-week interval in three doses. Neutralization antibody (Nabs) titers against the SARS-CoV-2 were detected by a Surrogate Virus Neutralization (sVNT) Test. Also, total IgG and IgG1, and IgG2a isotypes and the balance of cytokines in the spleen (IFN-\u03b3, Granzyme B, IL-4, and IL-12) were measured by ELISA. The percentages of CD4+ and CD8+ T cells were quantified by flow cytometry. The lymphoproliferative activity of restimulated spleen cells was also determined. The findings showed that the combination of RBD and N proteins formulated with saponin significantly promoted specific total IgG and neutralization antibodies, elicited robust specific lymphoproliferative and T cell response responses. Moreover, marked\u00a0increase in CD4+ and CD8+ T cells were observed in the adjuvanted RBD and N vaccine\u00a0group compared with other groups. The results suggest that the formulations are able to elicit a specific long-lasting mixed Th1\/Th2 balanced immune response. Our data indicate the significance of the saponin-adjuvanted RBD\/N vaccine in the design of SARS-CoV-2 vaccines and provide a rationale for the development of a protective long-lasting and strong vaccine.\n                  <\/jats:p>","DOI":"10.3389\/fimmu.2022.974364","type":"journal-article","created":{"date-parts":[[2022,9,8]],"date-time":"2022-09-08T07:31:11Z","timestamp":1662622271000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":26,"title":["Recombinant COVID-19 vaccine based on recombinant RBD\/Nucleoprotein and saponin adjuvant induces long-lasting neutralizing antibodies and cellular immunity"],"prefix":"10.3389","volume":"13","author":[{"given":"Amir","family":"Ghaemi","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Parisa","family":"Roshani Asl","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hedieh","family":"Zargaran","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Delaram","family":"Ahmadi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Asim Ali","family":"Hashimi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elahe","family":"Abdolalipour","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sahar","family":"Bathaeian","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Seyed Mohammad","family":"Miri","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2022,9,8]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1186\/s12985-020-01369-z","article-title":"SARS-CoV-2: characteristics and current advances in research","volume":"17","author":"Yang","year":"2020","journal-title":"Virol J"},{"key":"B2","doi-asserted-by":"publisher","first-page":"3","DOI":"10.1007\/978-1-0716-1884-4_1","article-title":"Challenges for vaccinologists in the first half of the twenty-first century","author":"Thomas","year":"2022","journal-title":"Vaccine Des. Springer"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1097\/PR9.0000000000000891","article-title":"The impact of the COVID-19 pandemic on research","volume":"8","author":"Alam","year":"2021","journal-title":"Transl Perioper Pain Med"},{"key":"B4","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.00635-20","article-title":"Design of potent membrane fusion inhibitors against SARS-CoV-2, an emerging coronavirus with high fusogenic activity","volume":"94","author":"Zhu","year":"2020","journal-title":"J Virol"},{"key":"B5","doi-asserted-by":"publisher","first-page":"1196","DOI":"10.3390\/vaccines9101196","article-title":"COVID-19 vaccine platforms: Challenges and safety contemplations","volume":"9","author":"Al-Jighefee","year":"2021","journal-title":"Vaccines"},{"key":"B6","doi-asserted-by":"publisher","first-page":"2949","DOI":"10.3390\/cells10112949","article-title":"Insights into COVID-19 vaccine development based on immunogenic structural proteins of SARS-CoV-2, host immune responses, and herd immunity","volume":"10","author":"Chaudhary","year":"2021","journal-title":"Cells"},{"key":"B7","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1038\/s43856-021-00012-4","article-title":"Neutralising SARS-CoV-2 RBD-specific antibodies persist for at least six months independently of symptoms in adults","volume":"1","author":"Wagner","year":"2021","journal-title":"Commun Med"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.1101\/2022.02.14.480394","article-title":"Increased potency and breadth of SARS-CoV-2 neutralizing antibodies after a third mRNA vaccine dose","author":"Muecksch","year":"2022","journal-title":"bioRxiv"},{"key":"B9","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1038\/s41392-021-00809-8","article-title":"The glycosylation in SARS-CoV-2 and its receptor ACE2","volume":"6","author":"Gong","year":"2021","journal-title":"Signal transduct. target. Ther"},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2022.02.029","article-title":"A randomized, double-blind phase I clinical trial of two recombinant dimeric RBD COVID-19 vaccine candidates: safety, reactogenicity and immunogenicity","volume":"18","author":"P\u00e9rez-Rodr\u00edguez","year":"2022","journal-title":"Vaccine"},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jmedchem.1c00881","article-title":"Potent neutralizing antibodies elicited by RBD-Fc-Based COVID-19 vaccine candidate adjuvanted by the Th2-skewing iNKT cell agonist","volume":"64","author":"Wang","year":"2021","journal-title":"J med. Chem"},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2021.637654","article-title":"A novel DNA vaccine against SARS-CoV-2 encoding a chimeric protein of its receptor-binding domain (RBD) fused to the amino-terminal region of hepatitis b virus preS1 with a W4P mutation","volume":"12","author":"Jeong","year":"2021","journal-title":"Front Immunol"},{"key":"B13","doi-asserted-by":"publisher","DOI":"10.1016\/S1473-3099(21)00127-4","article-title":"Safety and immunogenicity of a recombinant tandem-repeat dimeric RBD-based protein subunit vaccine (ZF2001) against COVID-19 in adults: two randomised, double-blind, placebo-controlled, phase 1 and 2 trials","volume":"21","author":"Yang","year":"2021","journal-title":"Lancet Infect Dis"},{"key":"B14","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1038\/s41392-021-00750-w","article-title":"The self-assembled nanoparticle-based trimeric RBD mRNA vaccine elicits robust and durable protective immunity against SARS-CoV-2 in mice","volume":"6","author":"Sun","year":"2021","journal-title":"Signal transduct. target. Ther"},{"key":"B15","doi-asserted-by":"publisher","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":"B16","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1038\/s41467-021-23942-y","article-title":"SARS-CoV-2 RBD trimer protein adjuvanted with alum-3M-052 protects from SARS-CoV-2 infection and immune pathology in the lung","volume":"12","author":"Routhu","year":"2021","journal-title":"Nat Commun"},{"key":"B17","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jmedchem.1c02025","article-title":"MPLA-Adjuvanted liposomes encapsulating s-trimer or RBD or S1, but not s-ECD, elicit robust neutralization against SARS-CoV-2 and variants of concern","volume":"65","author":"Wang","year":"2022","journal-title":"J med. Chem"},{"key":"B18","doi-asserted-by":"publisher","first-page":"109664","DOI":"10.1016\/j.celrep.2021.109664","article-title":"Combining spike-and nucleocapsid-based vaccines improves distal control of SARS-CoV-2","volume":"36","author":"Dangi","year":"2021","journal-title":"Cell Rep"},{"key":"B19","doi-asserted-by":"publisher","first-page":"1028","DOI":"10.3390\/vaccines9091028","article-title":"Immunological analysis of people in northeast China after SARS-CoV-2 inactivated vaccine injection","volume":"9","author":"Fu","year":"2021","journal-title":"Vaccines"},{"key":"B20","doi-asserted-by":"publisher","DOI":"10.1080\/21645515.2020.1833579","article-title":"Exploring the possible use of saponin adjuvants in COVID-19 vaccine","volume":"16","author":"Sharma","year":"2020","journal-title":"Hum Vaccines Immunother."},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.1002\/elsc.202000106","article-title":"Purification and characterization of the receptor-binding domain of SARS-CoV-2 spike protein from escherichia coli","volume":"21","author":"He","year":"2021","journal-title":"Eng Life Sci"},{"key":"B22","doi-asserted-by":"publisher","first-page":"201","DOI":"10.1186\/s12985-021-01672-3","article-title":"A formulated poly (I:C)\/CCL21 as an effective mucosal adjuvant for gamma-irradiated influenza vaccine","volume":"18","author":"Sabbaghi","year":"2021","journal-title":"Virol J"},{"key":"B23","doi-asserted-by":"publisher","DOI":"10.1007\/s00705-020-04900-3","article-title":"Protective cellular and mucosal immune responses following nasal administration of a whole gamma-irradiated influenza a (subtype H1N1) vaccine adjuvanted with interleukin-28B in a mouse model","volume":"166","author":"Sabbaghi","year":"2021","journal-title":"Arch Virol"},{"key":"B24","doi-asserted-by":"publisher","first-page":"103610","DOI":"10.1016\/j.ebiom.2021.103610","article-title":"Immunogenic T cell epitopes of SARS-CoV-2 are recognized by circulating memory and na\u00efve CD8 T cells of unexposed individuals","volume":"72","author":"Quiros-Fernandez","year":"2021","journal-title":"EBioMedicine"},{"key":"B25","doi-asserted-by":"publisher","first-page":"520","DOI":"10.3390\/vaccines9050520","article-title":"A synthetic peptide CTL vaccine targeting nucleocapsid confers protection from SARS-CoV-2 challenge in rhesus macaques","volume":"9","author":"Harris","year":"2021","journal-title":"Vaccines"},{"key":"B26","doi-asserted-by":"publisher","first-page":"428","DOI":"10.3390\/vaccines9050428","article-title":"Designing a sars-cov-2 t-cell-inducing vaccine for high-risk patient groups","volume":"9","author":"Rammensee","year":"2021","journal-title":"Vaccines"},{"key":"B27","doi-asserted-by":"publisher","first-page":"152091","DOI":"10.1016\/j.imbio.2021.152091","article-title":"Antigenic sites in SARS-CoV-2 spike RBD show molecular similarity with pathogenic antigenic determinants and harbors peptides for vaccine development","volume":"226","author":"Dakal","year":"2021","journal-title":"Immunobiology"},{"key":"B28","doi-asserted-by":"publisher","DOI":"10.1007\/s00705-017-3647-z","article-title":"Enhancement of therapeutic DNA vaccine potency by melatonin through inhibiting VEGF expression and induction of antitumor immunity mediated by CD8+ T cells","volume":"163","author":"Baghban Rahimi","year":"2018","journal-title":"Arch Virol"},{"key":"B29","doi-asserted-by":"publisher","first-page":"199","DOI":"10.1016\/S0952-7915(96)80058-6","article-title":"Immunoglobulin class switching","volume":"8","author":"Stavnezer","year":"1996","journal-title":"Curr Opin Immunol"},{"key":"B30","doi-asserted-by":"publisher","first-page":"18","DOI":"10.7774\/cevr.2012.1.1.18","article-title":"A review of vaccine development and research for industry animals in Korea","volume":"1","author":"Lee","year":"2012","journal-title":"Clin Exp Vaccine Res"},{"key":"B31","doi-asserted-by":"publisher","DOI":"10.1080\/21655979.2016.1191707","article-title":"Recent advances in the production of recombinant subunit vaccines in pichia pastoris","volume":"7","author":"Wang","year":"2016","journal-title":"Bioengineered"},{"key":"B32","doi-asserted-by":"publisher","first-page":"2673","DOI":"10.3390\/cells9122673","article-title":"Recent advances in the development of protein-and peptide-based subunit vaccines against tuberculosis","volume":"9","author":"Bellini","year":"2020","journal-title":"Cells"},{"key":"B33","doi-asserted-by":"publisher","first-page":"103720","DOI":"10.1016\/j.isci.2021.103720","article-title":"ACE2 can act as the secondary receptor in the Fc\u03b3R-dependent ADE of SARS-CoV-2 infection","volume":"25","author":"Wang","year":"2022","journal-title":"Iscience"},{"key":"B34","doi-asserted-by":"publisher","DOI":"10.1111\/imr.12367","article-title":"Fc receptors in antibody-dependent enhancement of viral infections","volume":"268","author":"Taylor","year":"2015","journal-title":"Immunol Rev"},{"key":"B35","doi-asserted-by":"publisher","first-page":"100228","DOI":"10.1016\/j.xcrm.2021.100228","article-title":"Long-term persistence of RBD+ memory b cells encoding neutralizing antibodies in SARS-CoV-2 infection","volume":"2","author":"Abayasingam","year":"2021","journal-title":"Cell Rep Med"},{"key":"B36","doi-asserted-by":"publisher","first-page":"108915","DOI":"10.1016\/j.celrep.2021.108915","article-title":"Linear epitope landscape of the SARS-CoV-2 spike protein constructed from 1,051 COVID-19 patients","volume":"34","author":"Li","year":"2021","journal-title":"Cell Rep"},{"key":"B37","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.2100272","article-title":"SARS-CoV-2 neutralizing antibody responses towards full-length spike protein and the receptor-binding domain","volume":"207","author":"Bayarri-Olmos","year":"2021","journal-title":"J Immunol"},{"key":"B38","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-020-00789-5","article-title":"Antibody-dependent enhancement and SARS-CoV-2 vaccines and therapies","volume":"5","author":"Lee","year":"2020","journal-title":"Nat Microbiol"},{"key":"B39","doi-asserted-by":"publisher","DOI":"10.1039\/C5SC03892H","article-title":"Peptide-based synthetic vaccines","volume":"7","author":"Skwarczynski","year":"2016","journal-title":"Chem Sci"},{"key":"B40","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2020.09.058","article-title":"Enhanced elicitation of potent neutralizing antibodies by the SARS-CoV-2 spike receptor binding domain fc fusion protein in mice","volume":"38","author":"Liu","year":"2020","journal-title":"Vaccine"},{"key":"B41","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jmedchem.1c02000","article-title":"Self-adjuvanting lipoprotein conjugate \u03b1GalCer-RBD induces potent immunity against SARS-CoV-2 and its variants of concern","volume":"65","author":"Wang","year":"2022","journal-title":"J med. Chem"},{"key":"B42","doi-asserted-by":"publisher","first-page":"1589","DOI":"10.1080\/22221751.2021.1951126","article-title":"Safety and immunogenicity of a recombinant interferon-armed RBD dimer vaccine (V-01) for COVID-19 in healthy adults: a randomized, double-blind, placebo-controlled, phase I trial","volume":"10","author":"Zhang","year":"2021","journal-title":"Emerg. Microbes infect."},{"key":"B43","doi-asserted-by":"publisher","first-page":"60","DOI":"10.1016\/j.trsl.2021.02.006","article-title":"Immunogenicity and crossreactivity of antibodies to the nucleocapsid protein of SARS-CoV-2: utility and limitations in seroprevalence and immunity studies","volume":"232","author":"Doba\u00f1o","year":"2021","journal-title":"Trans Res"},{"key":"B44","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.01279-20","article-title":"The SARS-CoV-2 n protein is a good component in a vaccine","volume":"94","author":"Ahl\u00e9n","year":"2020","journal-title":"J Virol"},{"key":"B45","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2020.587615","article-title":"Immunoinformatic analysis of SARS-CoV-2 nucleocapsid protein and identification of COVID-19 vaccine targets","volume":"2758","author":"Oliveira","year":"2020","journal-title":"Front Immunol"},{"key":"B46","doi-asserted-by":"publisher","first-page":"671","DOI":"10.1016\/j.chom.2020.03.002","article-title":"A sequence homology and bioinformatic approach can predict candidate targets for immune responses to SARS-CoV-2","volume":"27","author":"Grifoni","year":"2020","journal-title":"Cell Host Microbe"},{"key":"B47","doi-asserted-by":"publisher","first-page":"1095","DOI":"10.1016\/j.immuni.2020.10.006","article-title":"Unbiased screens show CD8+ T cells of COVID-19 patients recognize shared epitopes in SARS-CoV-2 that largely reside outside the spike protein","volume":"53","author":"Ferretti","year":"2020","journal-title":"Immunity"},{"key":"B48","doi-asserted-by":"publisher","first-page":"971","DOI":"10.1016\/j.immuni.2020.04.023","article-title":"Detection of SARS-CoV-2-specific humoral and cellular immunity in COVID-19 convalescent individuals","volume":"52","author":"Ni","year":"2020","journal-title":"Immunity"},{"key":"B49","doi-asserted-by":"publisher","DOI":"10.1002\/jmv.26769","article-title":"SARS-CoV-2 nucleocapsid protein intranasal inoculation induces local and systemic T cell responses in mice","volume":"93","author":"He","year":"2021","journal-title":"J Med Virol"},{"key":"B50","doi-asserted-by":"publisher","first-page":"1337","DOI":"10.3390\/vaccines9111337","article-title":"Production and characterization of nucleocapsid and RBD cocktail antigens of SARS-CoV-2 in nicotiana benthamiana plant as a vaccine candidate against COVID-19","volume":"9","author":"Mamedov","year":"2021","journal-title":"Vaccines"},{"key":"B51","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.174.8.4908","article-title":"Receptor-binding domain of severe acute respiratory syndrome coronavirus spike protein contains multiple conformation-dependent epitopes that induce highly potent neutralizing antibodies","volume":"174","author":"He","year":"2005","journal-title":"J Immunol"},{"key":"B52","doi-asserted-by":"publisher","first-page":"1984","DOI":"10.1016\/j.ymthe.2021.02.007","article-title":"Nanoluciferase complementation-based bioreporter reveals the importance of n-linked glycosylation of SARS-CoV-2 s for viral entry","volume":"29","author":"Azad","year":"2021","journal-title":"Mol Ther"},{"key":"B53","doi-asserted-by":"publisher","first-page":"1812","DOI":"10.3390\/biom11121812","article-title":"The nuts and bolts of SARS-CoV-2 spike receptor-binding domain heterologous expression","volume":"11","author":"Maffei","year":"2021","journal-title":"Biomolecules"},{"key":"B54","doi-asserted-by":"publisher","DOI":"10.3389\/fmolb.2021.629873","article-title":"Glycans of SARS-CoV-2 spike protein in virus infection and antibody production","volume":"8","author":"Zhao","year":"2021","journal-title":"Front Mol Biosci"},{"key":"B55","doi-asserted-by":"publisher","DOI":"10.1021\/acscentsci.0c01056","article-title":"Beyond shielding: the roles of glycans in the SARS-CoV-2 spike protein","volume":"6","author":"Casalino","year":"2020","journal-title":"ACS Cent Sci"},{"key":"B56","doi-asserted-by":"publisher","first-page":"100038","DOI":"10.1016\/j.yjsbx.2020.100038","article-title":"Structures and dynamics of the novel S1\/S2 protease cleavage site loop of the SARS-CoV-2 spike glycoprotein","volume":"4","author":"Lemmin","year":"2020","journal-title":"J Struct biol.: X"},{"key":"B57","doi-asserted-by":"publisher","first-page":"55","DOI":"10.1016\/j.vaccine.2017.11.030","article-title":"Leaf saponins of quillaja brasiliensis enhance long-term specific immune responses and promote dose-sparing effect in BVDV experimental vaccines","volume":"36","author":"Cibulski","year":"2017","journal-title":"Vaccine [Internet]"},{"key":"B58","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2016.01.029","article-title":"Novel ISCOMs from quillaja brasiliensis saponins induce mucosal and systemic antibody production, T-cell responses and improved antigen uptake","volume":"34","author":"Cibulski","year":"2016","journal-title":"Vaccine"}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2022.974364\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,9,8]],"date-time":"2022-09-08T07:31:16Z","timestamp":1662622276000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2022.974364\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,9,8]]},"references-count":58,"alternative-id":["10.3389\/fimmu.2022.974364"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2022.974364","relation":{},"ISSN":["1664-3224"],"issn-type":[{"value":"1664-3224","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,9,8]]},"article-number":"974364"}}