{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,20]],"date-time":"2026-06-20T07:03:07Z","timestamp":1781938987745,"version":"3.54.5"},"reference-count":20,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2025,12,15]],"date-time":"2025-12-15T00:00:00Z","timestamp":1765756800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Research, Development, and Innovation Authority (RDIA), Kingdom of Saudi Arabia","award":["12982-iau-2023-TAU-R-3-1-HW-"],"award-info":[{"award-number":["12982-iau-2023-TAU-R-3-1-HW-"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Background\/Objectives: SARS-CoV-2 continues to generate antigenically divergent variants that reduce the breadth of existing vaccine-induced antibody responses. Fc-fusion subunit vaccines offer advantages in stability, antigen display, and Fc-mediated immune engagement. This study aimed to design and evaluate a tetravalent RBD\u2013Fc fusion construct incorporating RBDs from Wuhan-Hu-1 and Omicron BA.4\/BA.5 and to determine whether this configuration can induce broad antibody recognition across SARS-CoV-2 variants. The objective was to assess its feasibility, biochemical properties, and initial immunogenicity. Methods: Immune responses to the construct were first assessed using the C-ImmSim simulation platform. The full-length fusion was synthesized, subcloned into pcDNA3.1(+), expressed in HEK293 cells, and purified by Protein G affinity chromatography. Protein integrity was evaluated by reducing SDS\u2013PAGE. BALB\/c mice (female, 8 weeks) were immunized with a prime\u2013boost\u2013boost schedule, and sera were analyzed by ELISA, considering binding to Wuhan-Hu-1, Omicron BA.4\/BA.5, and a panel of RBD variants. Results: In silico analysis predicted coordinated antigen clearance, class switching, memory B- and CD4+ T-cell formation, and transient cytokine induction. The recombinant protein was expressed efficiently, yielding a major ~56 kDa band and a ~23 kDa RBD fragment. Vaccinated mice generated strong IgG responses to Wuhan-Hu-1 and BA.4\/BA.5 RBDs and showed broad binding to major variant RBDs. Conclusions: The tetravalent RBD\u2013Fc fusion vaccine was successfully produced and elicited broad antibody binding across SARS-CoV-2 variants, supporting its potential as a versatile protein-based vaccine platform.<\/jats:p>","DOI":"10.3390\/vaccines13121244","type":"journal-article","created":{"date-parts":[[2025,12,15]],"date-time":"2025-12-15T09:36:39Z","timestamp":1765791399000},"page":"1244","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Integrated In Silico and In Vivo Evaluation of a Tetravalent SARS-CoV-2 RBD\u2013Fc Fusion Vaccine with Broad Cross-Variant Antibody Responses"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1515-2352","authenticated-orcid":false,"given":"Ahmad Bakur","family":"Mahmoud","sequence":"first","affiliation":[{"name":"Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Taibah University, Madinah 42353, Saudi Arabia"},{"name":"Health and Life Research Center, Taibah University, Madinah 42353, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-7331-0624","authenticated-orcid":false,"given":"Renad M.","family":"Alhamawi","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Taibah University, Madinah 42353, Saudi Arabia"},{"name":"Health and Life Research Center, Taibah University, Madinah 42353, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mustafa Yassin","family":"Taher","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Taibah University, Madinah 42353, Saudi Arabia"},{"name":"Health and Life Research Center, Taibah University, Madinah 42353, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Awadh S.","family":"Alsubhi","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Taibah University, Madinah 42353, Saudi Arabia"},{"name":"Health and Life Research Center, Taibah University, Madinah 42353, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5309-3552","authenticated-orcid":false,"given":"Mekky M.","family":"Abouzied","sequence":"additional","affiliation":[{"name":"Health and Life Research Center, Taibah University, Madinah 42353, Saudi Arabia"},{"name":"Department of Pharmacology and Toxicology, College of Pharmacy, Taibah University, Madinah 42353, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0131-8195","authenticated-orcid":false,"given":"Heba M.","family":"Zahid","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Taibah University, Madinah 42353, Saudi Arabia"},{"name":"Health and Life Research Center, Taibah University, Madinah 42353, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mohammed Abdullah","family":"Alotaibi","sequence":"additional","affiliation":[{"name":"Health and Life Research Center, Taibah University, Madinah 42353, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0570-1191","authenticated-orcid":false,"given":"Nada","family":"Almarghalani","sequence":"additional","affiliation":[{"name":"Health and Life Research Center, Taibah University, Madinah 42353, Saudi Arabia"},{"name":"King Abdullah International Medical Research Center, Jeddah 22384, Saudi Arabia"},{"name":"College of Science, King Abdulaziz University, Jeddah 22384, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Khulood","family":"Alotaibi","sequence":"additional","affiliation":[{"name":"Health and Life Research Center, Taibah University, Madinah 42353, Saudi Arabia"},{"name":"King Abdullah International Medical Research Center, Jeddah 22384, Saudi Arabia"},{"name":"College of Science, King Abdulaziz University, Jeddah 22384, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Abdulrahman","family":"Habash","sequence":"additional","affiliation":[{"name":"Pharmaceutical Care Division, King Faisal Specialist Hospital & Research Centre, Madinah 42353, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shaker Ahmed","family":"Alsharif","sequence":"additional","affiliation":[{"name":"Chest Department, King Fahad General Hospital, Madinah 42353, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7042-5985","authenticated-orcid":false,"given":"Almohanad","family":"Alkayyal","sequence":"additional","affiliation":[{"name":"Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, University of Tabuk, Tabuk 71491, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Wang, L., and Xiang, Y. 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