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Here, we describe a self-assembling protein nanoparticle against MPXV using lumazine synthase to present viral surface proteins. Multivalent nanoparticles elicited broader and stronger immune responses against MPXV and provided superior heterologous protection in rodent models against lethal VACV challenges compared to monovalent formulations. The three antigens with the best protective efficacy (intracellular mature virus antigens M1 and E8, and extracellular enveloped virus antigen B6) were further combined as the trivalent cocktail or mosaic nanoparticle. The trivalent nanoparticles elicited higher humoral responses compared to the modified vaccinia virus Ankara, and were protective against lethal VACV challenge in mice, with the protection correlation revealed. These findings highlight the potential of multivalent nanoparticle as vaccines against MPXV and other orthopoxviruses.<\/jats:p>","DOI":"10.1371\/journal.ppat.1013389","type":"journal-article","created":{"date-parts":[[2025,8,21]],"date-time":"2025-08-21T17:23:02Z","timestamp":1755796982000},"page":"e1013389","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":6,"title":["Multivalent mpox protein nanoparticle vaccines confer cross-protection against orthopoxvirus infection"],"prefix":"10.1371","volume":"21","author":[{"given":"Lijun","family":"Zhang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chuanyu","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chenxi","family":"Yang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xueqi","family":"Xiao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Senyu","family":"Xu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shengfeng","family":"Wan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kun","family":"Xu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yan","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8352-1327","authenticated-orcid":true,"given":"Lianpan","family":"Dai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2025,8,21]]},"reference":[{"key":"ppat.1013389.ref001","doi-asserted-by":"crossref","first-page":"102402","DOI":"10.1016\/j.tmaid.2022.102402","article-title":"Phylogenomic analysis of the monkeypox virus (MPXV) 2022 outbreak: Emergence of a novel viral lineage?","volume":"49","author":"N Luna","year":"2022","journal-title":"Travel Med Infect Dis"},{"issue":"1","key":"ppat.1013389.ref002","doi-asserted-by":"crossref","first-page":"641","DOI":"10.1186\/s12879-024-09537-0","article-title":"Clinical features and viral load variations of Mpox: a retrospective study in Chongqing, China","volume":"24","author":"S Yang","year":"2024","journal-title":"BMC Infect Dis"},{"issue":"19","key":"ppat.1013389.ref003","doi-asserted-by":"crossref","first-page":"1783","DOI":"10.1056\/NEJMra2208860","article-title":"Monkeypox","volume":"387","author":"A Gessain","year":"2022","journal-title":"N Engl J Med"},{"issue":"8","key":"ppat.1013389.ref004","doi-asserted-by":"crossref","first-page":"679","DOI":"10.1056\/NEJMoa2207323","article-title":"Monkeypox Virus Infection in Humans across 16 Countries - April-June 2022","volume":"387","author":"JP Thornhill","year":"2022","journal-title":"N Engl J Med"},{"key":"ppat.1013389.ref005","unstructured":"WHO. Multi-country outbreak of mpox, External situation report#32- 30 April 2024. 2024. Available from: https:\/\/wwwwhoint\/."},{"issue":"7","key":"ppat.1013389.ref006","doi-asserted-by":"crossref","first-page":"615","DOI":"10.1001\/jama.2022.12513","article-title":"The WHO Declaration of Monkeypox as a Global Public Health Emergency","volume":"328","author":"JB Nuzzo","year":"2022","journal-title":"JAMA"},{"key":"ppat.1013389.ref007","unstructured":"Ghebreyesus TA. Fifth Meeting of the International Health Regulations (2005) (IHR) Emergency Committee on the Multi-Country Outbreak of Mpox (Monkeypox). 2023."},{"key":"ppat.1013389.ref008","unstructured":"WHO. Mpox (Monkeypox) in the Democratic Republic of the Congo. 2023."},{"key":"ppat.1013389.ref009","unstructured":"WHO. 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Patient\u2019s guide to mpox treatment with tecovirimat (TPOXX). 2023; [cited October 23, 2023]. 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