{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,22]],"date-time":"2026-07-22T21:40:33Z","timestamp":1784756433837,"version":"3.55.0"},"reference-count":37,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2026,1,4]],"date-time":"2026-01-04T00:00:00Z","timestamp":1767484800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"EXIST Forschungstransfer","award":["\"ImpfKraft\""],"award-info":[{"award-number":["\"ImpfKraft\""]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Background: Emerging immune-evasive viral variants threaten the efficacy of current vaccines, underscoring the need for strategies that elicit broad and durable protection. Heterologous prime\u2013boost regimens combining distinct vaccine platforms can enhance humoral and cellular immunity through complementary mechanisms. Methods: Using an intramuscular immunization scheme aligned with clinical vaccination practice, CD-1 mice received homologous or heterologous prime\u2013boost regimens combining a replication-deficient Orf virus (Parapoxvirus orf, ORFV)-based spike vaccine (ORFV-S) with the licensed adjuvanted recombinant protein vaccine VidPrevtyn Beta. Spike-specific humoral and cellular immune responses were assessed. Results: ORFV-S alone induced potent and broad spike-specific IgG responses and achieved the strongest ACE2-binding inhibition across variants of concern. ORFV-S priming followed by VidPrevtyn Beta boosting markedly enhanced the magnitude and cross-variant breadth of antibody responses compared with homologous protein vaccination. Both homologous ORFV-S and heterologous regimens incorporating ORFV-S elicited strong CD4+ and CD8+ T-cell responses, whereas VidPrevtyn Beta alone induced only modest T-cell activity, demonstrating that ORFV-S effectively complements protein-based vaccines. Conclusions: The ORFV-S vector represents a potent vaccine platform capable of inducing broad humoral and cellular immunity. Its use in heterologous prime\u2013boost combinations enhances both antibody magnitude and breadth beyond homologous protein vaccination, supporting its application in vaccination strategies against evolving viral pathogens.<\/jats:p>","DOI":"10.3390\/vaccines14010064","type":"journal-article","created":{"date-parts":[[2026,1,5]],"date-time":"2026-01-05T13:35:47Z","timestamp":1767620147000},"page":"64","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Broadening SARS-CoV-2 Immunity by Combining ORFV and Protein-Based Vaccines"],"prefix":"10.3390","volume":"14","author":[{"given":"Alena","family":"Reguzova","sequence":"first","affiliation":[{"name":"Institute of Immunology, University Hospital T\u00fcbingen, Auf der Morgenstelle 15, 72076 T\u00fcbingen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Melanie","family":"M\u00fcller","sequence":"additional","affiliation":[{"name":"Institute of Immunology, University Hospital T\u00fcbingen, Auf der Morgenstelle 15, 72076 T\u00fcbingen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-5750-7274","authenticated-orcid":false,"given":"Madeleine","family":"Fandrich","sequence":"additional","affiliation":[{"name":"NMI Natural and Medical Sciences Institute at the University of T\u00fcbingen, 72770 Reutlingen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alex","family":"Dulovic","sequence":"additional","affiliation":[{"name":"NMI Natural and Medical Sciences Institute at the University of T\u00fcbingen, 72770 Reutlingen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-8482-2240","authenticated-orcid":false,"given":"Ralf","family":"Amann","sequence":"additional","affiliation":[{"name":"Institute of Immunology, University Hospital T\u00fcbingen, Auf der Morgenstelle 15, 72076 T\u00fcbingen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1038\/s41392-024-01917-x","article-title":"Emerging and reemerging infectious diseases: Global trends and new strategies for their prevention and control","volume":"9","author":"Wang","year":"2024","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1038\/s41541-023-00756-1","article-title":"Systematic review and meta-analysis of the factors affecting waning of post-vaccination neutralizing antibody responses against SARS-CoV-2","volume":"8","author":"Jacobsen","year":"2023","journal-title":"npj Vaccines"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"e13276","DOI":"10.1111\/irv.13276","article-title":"Recent Advances, Approaches and Challenges in the Development of Universal Influenza Vaccines","volume":"18","author":"Lim","year":"2024","journal-title":"Influenza Other Respir. Viruses"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"6829446","DOI":"10.1155\/arat\/6829446","article-title":"Advances in HIV Treatment and Vaccine Development: Emerging Therapies and Breakthrough Strategies for Long-Term Control","volume":"2025","author":"Okesanya","year":"2025","journal-title":"AIDS Res. Treat."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Hsiung, K.C., Chiang, H.J., Reinig, S., and Shih, S.R. (2024). Vaccine Strategies Against RNA Viruses: Current Advances and Future Directions. Vaccines, 12.","DOI":"10.3390\/vaccines12121345"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"738","DOI":"10.1016\/j.immuni.2022.04.010","article-title":"Inducing broad-based immunity against viruses with pandemic potential","volume":"55","author":"Sette","year":"2022","journal-title":"Immunity"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Livieratos, A., Gogos, C., Thomas, I., and Akinosoglou, K. (2025). Vaccination Strategies: Mixing Paths Versus Matching Tracks. Vaccines, 13.","DOI":"10.3390\/vaccines13030308"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Tang, J., Amin, M.A., and Campian, J.L. (2025). Past, Present, and Future of Viral Vector Vaccine Platforms: A Comprehensive Review. Vaccines, 13.","DOI":"10.3390\/vaccines13050524"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1510","DOI":"10.1038\/s41591-021-01463-x","article-title":"A \u2018mix and match\u2019 approach to SARS-CoV-2 vaccination","volume":"27","author":"Deming","year":"2021","journal-title":"Nat. Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"537","DOI":"10.3390\/idr14040057","article-title":"Mix-and-Match COVID-19 Vaccinations (Heterologous Boost): A Review","volume":"14","author":"Garg","year":"2022","journal-title":"Infect. Dis. Rep."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Qian, G., Gao, C., Zhang, M., Chen, Y., and Xie, L. (2024). A Review of Protein-Based COVID-19 Vaccines: From Monovalent to Multivalent Formulations. Vaccines, 12.","DOI":"10.3390\/vaccines12060579"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1597","DOI":"10.1038\/nm.3409","article-title":"Key roles of adjuvants in modern vaccines","volume":"19","author":"Reed","year":"2013","journal-title":"Nat. Med."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"102587","DOI":"10.1016\/j.eclinm.2024.102587","article-title":"Effectiveness of the Sanofi\/GSK (VidPrevtyn Beta) and Pfizer-BioNTech (Comirnaty Original\/Omicron BA.4-5) bivalent vaccines against hospitalisation in England","volume":"71","author":"Kirsebom","year":"2024","journal-title":"eClinicalMedicine"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Zhuang, Z., Zhuo, J., Yuan, Y., Chen, Z., Zhang, S., Zhu, A., Zhao, J., and Zhao, J. (2024). Harnessing T-Cells for Enhanced Vaccine Development against Viral Infections. Vaccines, 12.","DOI":"10.3390\/vaccines12050478"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Ura, T., Takeuchi, M., Kawagoe, T., Mizuki, N., Okuda, K., and Shimada, M. (2022). Current Vaccine Platforms in Enhancing T-Cell Response. Vaccines, 10.","DOI":"10.3390\/vaccines10081367"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"e70038","DOI":"10.1002\/rmv.70038","article-title":"Advancing ORFV-Based Therapeutics to the Clinical Stage","volume":"35","author":"Helmold","year":"2025","journal-title":"Rev. Med. Virol."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"AlDaif, B.A., and Fleming, S.B. (2025). Innate Immune Sensing of Parapoxvirus Orf Virus and Viral Immune Evasion. Viruses, 17.","DOI":"10.3390\/v17040587"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Muller, M., Reguzova, A., Loffler, M.W., and Amann, R. (2022). Orf Virus-Based Vectors Preferentially Target Professional Antigen-Presenting Cells, Activate the STING Pathway and Induce Strong Antigen-Specific T Cell Responses. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.873351"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1038\/s41541-024-00981-2","article-title":"A multiantigenic Orf virus-based vaccine efficiently protects hamsters and nonhuman primates against SARS-CoV-2","volume":"9","author":"Reguzova","year":"2024","journal-title":"npj Vaccines"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Metz, C., Haug, V., Muller, M., and Amann, R. (2024). Pharmacokinetic and Environmental Risk Assessment of Prime-2-CoV, a Non-Replicating Orf Virus-Based Vaccine against SARS-CoV-2. Vaccines, 12.","DOI":"10.3390\/vaccines12050492"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Esen, M., Fischer-Herr, J., Gabor, J.J., Gaile, J.M., Fleischmann, W.A., Smeenk, G.W., de Moraes, R.A., Belard, S., Calle, C.L., and Woldearegai, T.G. (2024). First-in-Human Phase I Trial to Assess the Safety and Immunogenicity of an Orf Virus-Based COVID-19 Vaccine Booster. Vaccines, 12.","DOI":"10.3390\/vaccines12111288"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2410574","DOI":"10.1080\/21645515.2024.2410574","article-title":"A novel orf virus vector-based COVID-19 booster vaccine shows cross-neutralizing activity in the absence of anti-vector neutralizing immunity","volume":"20","author":"Klinkardt","year":"2024","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Reguzova, A., Haug, V., Metz, C., M\u00fcller, M., Fandrich, M., Dulovic, A., and Amann, R. (2025). Heterologous prime-boost vaccination with VLA2001 and an ORFV-based vector enhances spike- and nucleocapsid-specific immunity in mice. Front. Immunol., 16.","DOI":"10.3389\/fimmu.2025.1675859"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Reguzova, A., Haug, V., M\u00fcller, M., Fandrich, M., Dulovic, A., and Amann, R. (2025). Heterologous ORFV\u2013Ad26 vaccination broadens antibody breadth and amplifies cellular immunity against SARS-CoV-2 spike. Front. Immunol., 16.","DOI":"10.3389\/fimmu.2025.1715442"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e240","DOI":"10.1093\/cid\/ciac498","article-title":"Antibody Binding and Angiotensin-Converting Enzyme 2 Binding Inhibition Is Significantly Reduced for Both the BA.1 and BA.2 Omicron Variants","volume":"76","author":"Junker","year":"2023","journal-title":"Clin. Infect. Dis."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"488","DOI":"10.1038\/s41586-022-04460-3","article-title":"T cell responses to SARS-CoV-2 spike cross-recognize Omicron","volume":"603","author":"Keeton","year":"2022","journal-title":"Nature"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"162","DOI":"10.1016\/j.chom.2023.12.003","article-title":"Post-pandemic memory T cell response to SARS-CoV-2 is durable, broadly targeted, and cross-reactive to the hypermutated BA.2.86 variant","volume":"32","author":"Nesamari","year":"2024","journal-title":"Cell Host Microbe"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1505","DOI":"10.3390\/v7031505","article-title":"Molecular genetic analysis of orf virus: A poxvirus that has adapted to skin","volume":"7","author":"Fleming","year":"2015","journal-title":"Viruses"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2209","DOI":"10.1056\/NEJMoa0908492","article-title":"Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand","volume":"361","author":"Pitisuttithum","year":"2009","journal-title":"N. Engl. J. Med."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"701","DOI":"10.1016\/j.medj.2021.03.014","article-title":"Reduced blood-stage malaria growth and immune correlates in humans following RH5 vaccination","volume":"2","author":"Minassian","year":"2021","journal-title":"Med"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"609","DOI":"10.1056\/NEJMoa2207566","article-title":"Efficacy and Safety of an Ad26.RSV.preF-RSV preF Protein Vaccine in Older Adults","volume":"388","author":"Falsey","year":"2023","journal-title":"N. Engl. J. Med."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2258","DOI":"10.1016\/S0140-6736(21)02717-3","article-title":"Safety and immunogenicity of seven COVID-19 vaccines as a third dose (booster) following two doses of ChAdOx1 nCov-19 or BNT162b2 in the UK (COV-BOOST): A blinded, multicentre, randomised, controlled, phase 2 trial","volume":"398","author":"Munro","year":"2021","journal-title":"Lancet"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"vlaf027","DOI":"10.1093\/immhor\/vlaf027","article-title":"A viral vector prime and protein boost vaccine regimen elicits higher binding antibody titers than a homologous viral vector prime-boost regimen","volume":"9","author":"Awakoaiye","year":"2025","journal-title":"ImmunoHorizons"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"101495","DOI":"10.1016\/j.trim.2021.101495","article-title":"T helper type (Th1\/Th2) responses to SARS-CoV-2 and influenza A (H1N1) virus: From cytokines produced to immune responses","volume":"70","author":"Molavi","year":"2022","journal-title":"Transpl. Immunol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1038\/s41541-025-01216-8","article-title":"A novel nanoparticle vaccine displaying multistage tuberculosis antigens confers protection in mice infected with H37Rv","volume":"10","author":"Ding","year":"2025","journal-title":"npj Vaccines"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"134","DOI":"10.1038\/s41541-023-00731-w","article-title":"Polyfunctional CD4 T-cells correlating with neutralising antibody is a hallmark of COVISHIELD(TM) and COVAXIN\u00ae induced immunity in COVID-19 exposed Indians","volume":"8","author":"Rakshit","year":"2023","journal-title":"npj Vaccines"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"110441","DOI":"10.1016\/j.isci.2024.110441","article-title":"Cytokine profile of anti-spike CD4+T cells predicts humoral and CD8+T cell responses after anti-SARS-CoV-2 mRNA vaccination","volume":"27","author":"Benhamouda","year":"2024","journal-title":"iScience"}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/14\/1\/64\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,7]],"date-time":"2026-01-07T05:27:11Z","timestamp":1767763631000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/14\/1\/64"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,4]]},"references-count":37,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,1]]}},"alternative-id":["vaccines14010064"],"URL":"https:\/\/doi.org\/10.3390\/vaccines14010064","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,4]]}}}