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Adjuvants play a relevant role in enhancing vaccine-induced immune responses. One such molecule is interferon-stimulated gene 15 (ISG15), a key modulator of antiviral immunity that acts both through ISGylation-dependent mechanisms and as a cytokine-like molecule. Methods: In this study, we assessed the immunostimulatory potential of ISG15 as an adjuvant in Modified Vaccinia virus Ankara (MVA)-based vaccine candidates targeting Zika virus (ZIKV) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Early innate responses and immune cell infiltration were analyzed in immunized mice by flow cytometry and cytokine profiling. To elucidate the underlying mechanism of action of ISG15, in vitro co-infection studies were performed in macrophages. Finally, we evaluated the magnitude and functional quality of the elicited antigen-specific cellular immune responses in vivo. Results: Analysis of early innate responses revealed both platform- and variant-specific effects. ISG15AA preferentially promoted natural killer (NK) cell recruitment at the injection site, whereas ISG15GG enhanced myeloid cell infiltration in draining lymph nodes (DLNs), particularly when delivered via MVA. Moreover, in vitro co-infection of macrophages with MVA-based vaccine vectors and the ISG15AA mutant led to a marked increase in proinflammatory cytokine production, highlighting a dominant role for the extracellular, ISGylation-independent functions of ISG15 in shaping vaccine-induced immunity. Notably, co-infection of ISG15 with MVA-ZIKV and MVA-SARS-CoV-2 vaccine candidates enhanced the magnitude of antigen-specific immune responses in both vaccine models. Conclusions: ISG15, particularly in its ISGylation-deficient form, acts as a promising immunomodulatory adjuvant for viral vaccines, enhancing both innate and adaptive immune responses. Consistent with previous findings in the context of Human Immunodeficiency virus type 1 (HIV-1) vaccines, this study further supports the potential of ISG15 as an effective adjuvant for vaccines targeting viral infections such as ZIKV and SARS-CoV-2.<\/jats:p>","DOI":"10.3390\/vaccines13070696","type":"journal-article","created":{"date-parts":[[2025,7,1]],"date-time":"2025-07-01T07:42:06Z","timestamp":1751355726000},"page":"696","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["ISG15 as a Potent Immune Adjuvant in MVA-Based Vaccines Against Zika Virus and SARS-CoV-2"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5167-5724","authenticated-orcid":false,"given":"Juan","family":"Garc\u00eda-Arriaza","sequence":"first","affiliation":[{"name":"Department of Molecular and Cellular Biology, Centro Nacional de Biotecnolog\u00eda (CNB), Consejo Superior de Investigaciones Cient\u00edficas (CSIC), 28049 Madrid, Spain"},{"name":"Centro de Investigaci\u00f3n Biom\u00e9dica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III (ISCIII), 28029 Madrid, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michela","family":"Falqui","sequence":"additional","affiliation":[{"name":"Department of Preventive Medicine, Public Health and Microbiology, Faculty of Medicine, Universidad Aut\u00f3noma de Madrid, 28029 Madrid, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8983-6784","authenticated-orcid":false,"given":"Patricia","family":"P\u00e9rez","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Biology, Centro Nacional de Biotecnolog\u00eda (CNB), Consejo Superior de Investigaciones Cient\u00edficas (CSIC), 28049 Madrid, Spain"},{"name":"Centro de Investigaci\u00f3n Biom\u00e9dica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III (ISCIII), 28029 Madrid, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0365-1565","authenticated-orcid":false,"given":"Roc\u00edo","family":"Coloma","sequence":"additional","affiliation":[{"name":"Department of Preventive Medicine, Public Health and Microbiology, Faculty of Medicine, Universidad Aut\u00f3noma de Madrid, 28029 Madrid, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6276-7766","authenticated-orcid":false,"given":"Beatriz","family":"Perdiguero","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Biology, Centro Nacional de Biotecnolog\u00eda (CNB), Consejo Superior de Investigaciones Cient\u00edficas (CSIC), 28049 Madrid, Spain"},{"name":"Centro de Investigaci\u00f3n Biom\u00e9dica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III (ISCIII), 28029 Madrid, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8308-7524","authenticated-orcid":false,"given":"Enrique","family":"\u00c1lvarez","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Biology, Centro Nacional de Biotecnolog\u00eda (CNB), Consejo Superior de Investigaciones Cient\u00edficas (CSIC), 28049 Madrid, Spain"},{"name":"Centro de Investigaci\u00f3n Biom\u00e9dica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III (ISCIII), 28029 Madrid, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Laura","family":"Marcos-Villar","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Biology, Centro Nacional de Biotecnolog\u00eda (CNB), Consejo Superior de Investigaciones Cient\u00edficas (CSIC), 28049 Madrid, Spain"},{"name":"Centro de Investigaci\u00f3n Biom\u00e9dica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III (ISCIII), 28029 Madrid, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2969-1840","authenticated-orcid":false,"given":"David","family":"Astorgano","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Biology, Centro Nacional de Biotecnolog\u00eda (CNB), Consejo Superior de Investigaciones Cient\u00edficas (CSIC), 28049 Madrid, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8073-8114","authenticated-orcid":false,"given":"Irene","family":"Campa\u00f1a-G\u00f3mez","sequence":"additional","affiliation":[{"name":"Department of Preventive Medicine, Public Health and Microbiology, Faculty of Medicine, Universidad Aut\u00f3noma de Madrid, 28029 Madrid, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9473-283X","authenticated-orcid":false,"given":"Carlos \u00d3scar S.","family":"Sorzano","sequence":"additional","affiliation":[{"name":"Biocomputing Unit and Computational Genomics, CNB, CSIC, 28049 Madrid, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0846-2827","authenticated-orcid":false,"given":"Mariano","family":"Esteban","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Biology, Centro Nacional de Biotecnolog\u00eda (CNB), Consejo Superior de Investigaciones Cient\u00edficas (CSIC), 28049 Madrid, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5414-7935","authenticated-orcid":false,"given":"Carmen Elena","family":"G\u00f3mez","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Biology, Centro Nacional de Biotecnolog\u00eda (CNB), Consejo Superior de Investigaciones Cient\u00edficas (CSIC), 28049 Madrid, Spain"},{"name":"Centro de Investigaci\u00f3n Biom\u00e9dica en 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