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MPXV may establish new reservoirs in non-endemic countries and can be considered a possible biological weapon. Human-to-human MPXV transmission is increasing with a growing susceptibility, coincident with the declining herd immunity against smallpox. The emerging threat of MPXV highlights the urgent need for protection from new zoonotic infections, as mankind is completely unprepared for encounters with new viruses. Preventive vaccination remains the most effective control against orthopoxviruses (OPXVs) such as MPXV and <jats:italic>prime-boost<\/jats:italic> vaccination strategies can significantly influence vaccine efficacy and enhance immune responses. Our study aimed at characterizing potential vaccine candidates against OPXV infections in a murine model using DNA, viral and protein recombinant vaccines using different <jats:italic>prime-boost<\/jats:italic> regimens. The experiments employed Vaccinia virus (VACV) A33, B5, L1, and A27 envelope proteins as immunogens for both <jats:italic>priming<\/jats:italic> and <jats:italic>boosting<\/jats:italic>. <jats:italic>Priming<\/jats:italic> was carried out using a mixture of four plasmids (4pVAXmix), and <jats:italic>boosts<\/jats:italic> employed fowlpox (FWPV) recombinants (4FPmix) and\/or the purified recombinant proteins (4protmix), all of them expressing the same antigens. One or two doses of the same immunogens were tested and identical protocols were also compared for intranasal (i.n.) or intramuscular (i.m.) viral administration, before challenge with the highly pathogenic VACV VV<jats:sub>IHD\u2212J<\/jats:sub> strain. Our results show that a single dose of any combined immunogen elicited a very low antibody response. Protein mixtures administered twice boosted the humoral response of DNA immunizations by electroporation (e. p.), but did not protect from viral challenge. The antibody neutralizing titer was inversely correlated with animals\u2019 weight loss, which was initially similar in all of the groups after the challenge, but was then reversed in mice that had been primed twice with the DNA recombinants and boosted twice with the FWPV recombinants.<\/jats:p>","DOI":"10.1186\/s12985-024-02534-4","type":"journal-article","created":{"date-parts":[[2024,11,7]],"date-time":"2024-11-07T09:10:04Z","timestamp":1730970604000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Different immunogens and prime-boost vaccination strategies affect the efficacy of recombinant candidate vaccines against pathogenic orthopoxviruses"],"prefix":"10.1186","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5683-6216","authenticated-orcid":false,"given":"Antonia","family":"Radaelli","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0222-6102","authenticated-orcid":false,"given":"Carlo","family":"Zanotto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chiara","family":"Brambilla","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tommaso","family":"Adami","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francesca","family":"Paolini","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aldo","family":"Venuti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Adriana","family":"Manuka","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Irsida","family":"Mehmeti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carlo","family":"De Giuli Morghen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,11,7]]},"reference":[{"key":"2534_CR1","doi-asserted-by":"publisher","first-page":"620","DOI":"10.1089\/hs.2017.0061","volume":"15","author":"GD Koblentz","year":"2017","unstructured":"Koblentz GD. 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