{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T07:24:29Z","timestamp":1790666669296,"version":"4.1.0"},"reference-count":70,"publisher":"Public Library of Science (PLoS)","issue":"2","license":[{"start":{"date-parts":[[2025,2,4]],"date-time":"2025-02-04T00:00:00Z","timestamp":1738627200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Intravacc B.V."}],"content-domain":{"domain":["www.plospathogens.org"],"crossmark-restriction":false},"short-container-title":["PLoS Pathog"],"abstract":"<jats:p>To counteract hand, foot, and mouth disease-causing viruses such as enterovirus A71 and coxsackievirus A6, virus-like particles (VLPs) have emerged as a leading contender for the development of a multivalent vaccine. However, VLPs have shown rapid conversion from a highly immunogenic state to a less immunogenic state and low particle integrity lifetimes compared to inactivated virus vaccines, thus raising concerns about their overall stability. Here, we produce VLPs to investigate capsid stability using cryogenic electron microscopy (cryo-EM), mass spectrometry (MS), biochemical assays, and atomic force microscopy (AFM). In contrast to prior studies and prevailing hypotheses, we show that insect-cell produced enterovirus VLPs include encapsidated RNA fragments with viral protein coding sequences. Our integrated approach reveals that CVA6 VLPs do not undergo viral maturation, in contrast to virions; that they can encapsidate RNA fragments, similarly to virions; and that despite the latter, they are more brittle than virions. Interestingly, this indicates that CVA6 VLP stability is more affected by lack of viral maturation than the presence of RNA. Our study highlights how the development of VLPs as vaccine candidates should encompass probing for unwanted (viral) RNA content and establishing control of their maturation to enhance stability.<\/jats:p>","DOI":"10.1371\/journal.ppat.1012873","type":"journal-article","created":{"date-parts":[[2025,2,4]],"date-time":"2025-02-04T18:33:05Z","timestamp":1738693985000},"page":"e1012873","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":6,"title":["Enterovirus-like particles encapsidate RNA and exhibit decreased stability due to lack of maturation"],"prefix":"10.1371","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7245-3031","authenticated-orcid":true,"given":"Louis","family":"Kuijpers","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Evdokia-Anastasia","family":"Giannopoulou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuzhen","family":"Feng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wouter","family":"van den Braak","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Abbas","family":"Freydoonian","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ramon","family":"Ramlal","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hugo","family":"Meiring","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bel\u00e9n","family":"Solano","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wouter H.","family":"Roos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Arjen J.","family":"Jakobi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leo A.","family":"van der Pol","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4029-0973","authenticated-orcid":true,"given":"Nynke H.","family":"Dekker","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2025,2,4]]},"reference":[{"key":"ppat.1012873.ref001","doi-asserted-by":"crossref","first-page":"500","DOI":"10.1136\/jech-2014-203836","article-title":"Enterovirus 71 related severe hand, foot and mouth disease outbreaks in South-East Asia: Current situation and ongoing challenge","volume":"68","author":"S Sabanathan","year":"2014","journal-title":"J Epidemiol Community Health"},{"key":"ppat.1012873.ref002","doi-asserted-by":"crossref","first-page":"1708","DOI":"10.1016\/S0140-6736(22)00313-0","article-title":"Efficacy, safety, and immunogenicity of an inactivated, adjuvanted enterovirus 71 vaccine in infants and children: a multiregion, double-blind, randomised, placebo-controlled, phase 3 trial","volume":"399","author":"TT Nguyen","year":"2022","journal-title":"Lancet"},{"key":"ppat.1012873.ref003","article-title":"Explanations for the high potency of HPV prophylactic vaccines","author":"J Schiller","year":"2018","journal-title":"Vaccine"},{"key":"ppat.1012873.ref004","doi-asserted-by":"crossref","first-page":"10021","DOI":"10.1128\/JVI.05107-11","article-title":"Crystal Structures of Enterovirus 71 3C Protease Complexed with Rupintrivir Reveal the Roles of Catalytically Important Residues","volume":"85","author":"J Wang","year":"2011","journal-title":"J Virol"},{"key":"ppat.1012873.ref005","doi-asserted-by":"crossref","first-page":"320","DOI":"10.1038\/334320a0","article-title":"Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA","volume":"334","author":"J Pelletier","year":"1988","journal-title":"Nature"},{"key":"ppat.1012873.ref006","article-title":"Picornavirus Morphogenesis","author":"P Jiang","year":"2014","journal-title":"Microbiol Mol Biol Rev"},{"key":"ppat.1012873.ref007","doi-asserted-by":"crossref","DOI":"10.1371\/journal.ppat.1006117","article-title":"Genetically Thermo-Stabilised, Immunogenic Poliovirus Empty Capsids; a Strategy for Non-replicating Vaccines","author":"H Fox","year":"2017"},{"key":"ppat.1012873.ref008","doi-asserted-by":"crossref","DOI":"10.1038\/s41541-020-00267-3","article-title":"Mammalian expression of virus-like particles as a proof of principle for next generation polio vaccines","author":"MW Bahar","year":"2021"},{"key":"ppat.1012873.ref009","article-title":"Virus-like particle vaccines for poliovirus types 1, 2, and 3 with enhanced thermostability expressed in insect cells","author":"Y Xu","year":"2019","journal-title":"Vaccine"},{"key":"ppat.1012873.ref010","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1099\/jgv.0.001771","article-title":"Thermal stabilization of enterovirus A 71 and production of antigenically stabilized empty capsids","volume":"103","author":"NJ Kingston","year":"2022","journal-title":"J Gen Virol"},{"key":"ppat.1012873.ref011","first-page":"1166","article-title":"Production of antigenically stable enterovirus A71 virus-like particles","volume":"57","author":"N Kingston","year":"2023","journal-title":"Pichia pastoris as vaccine candidate"},{"key":"ppat.1012873.ref012","first-page":"8","article-title":"Structural factors that control conformational transitions and serotype specificity in type 3 poliovirus","author":"DJ Filman","year":"1989","journal-title":"EMBO J"},{"key":"ppat.1012873.ref013","article-title":"Crystal structure of human enterovirus 71","author":"P Plevka","year":"2012","journal-title":"Science"},{"key":"ppat.1012873.ref014","first-page":"5","article-title":"Cryo-electron microscopy and image classification reveal the existence and structure of the coxsackievirus A6 virion","author":"CR B\u00fcttner","year":"2022"},{"key":"ppat.1012873.ref015","first-page":"96","article-title":"Structure of Human Enterovirus 70 and Its Inhibition by Capsid-Binding Compounds","author":"T F\u00fczik","year":"2022","journal-title":"J Virol"},{"key":"ppat.1012873.ref016","article-title":"Yeast-produced recombinant virus-like particles of coxsackievirus A6 elicited protective antibodies in mice","author":"Y Zhou","year":"2016","journal-title":"Antiviral Res"},{"key":"ppat.1012873.ref017","article-title":"Virus-like particle-based vaccine against coxsackievirus A6 protects mice against lethal infections","author":"C Shen","year":"2016","journal-title":"Vaccine"},{"key":"ppat.1012873.ref018","first-page":"14","article-title":"Efficient expression of enterovirus 71 based on virus-like particles vaccine","author":"HJ Kim","year":"2019","journal-title":"PLoS One"},{"key":"ppat.1012873.ref019","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.jtice.2017.10.020","article-title":"Production and purification of virus-like particles of different enterovirus subtypes as vaccines","volume":"82","author":"SY Lin","year":"2017","journal-title":"J Taiwan Inst Chem Eng"},{"key":"ppat.1012873.ref020","article-title":"Production of virus-like particles for vaccines","author":"J Fuenmayor","year":"2017","journal-title":"New Biotechnology"},{"key":"ppat.1012873.ref021","doi-asserted-by":"crossref","first-page":"653","DOI":"10.1016\/j.vaccine.2017.12.057","article-title":"A virus-like particle vaccine confers protection against enterovirus D68 lethal challenge in mice","volume":"36","author":"W Dai","year":"2018","journal-title":"Vaccine"},{"key":"ppat.1012873.ref022","doi-asserted-by":"crossref","first-page":"10099","DOI":"10.1073\/pnas.92.22.10099","article-title":"Efficient gene transfer into human hepatocytes by baculovirus vectors","volume":"92","author":"C Hofmann","year":"1995","journal-title":"Proc Natl Acad Sci U S A"},{"key":"ppat.1012873.ref023","first-page":"109","article-title":"Host RNAs, including transposons, are encapsidated by a eukaryotic single-stranded RNA virus","author":"A Routh","year":"2012","journal-title":"Proc Natl Acad Sci U S A"},{"key":"ppat.1012873.ref024","doi-asserted-by":"crossref","first-page":"92","DOI":"10.1007\/s12033-012-9598-4","article-title":"Construction and characterization of virus-like particles: A review","volume":"53","author":"A. 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Marchetti","year":"2016","journal-title":"Curr Opin Virol"},{"key":"ppat.1012873.ref050","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1103\/PhysRevE.75.031901","article-title":"Nonlinear finite-element analysis of nanoindentation of viral capsids","volume":"75","author":"MM Gibbons","year":"2007","journal-title":"Phys Rev E\u2014Stat Nonlinear, Soft Matter Phys."},{"key":"ppat.1012873.ref051","first-page":"49","article-title":"VIPERdb v3.0: A structure-based data analytics platform for viral capsids","author":"D Montiel-Garcia","year":"2021","journal-title":"Nucleic Acids Res"},{"key":"ppat.1012873.ref052","doi-asserted-by":"crossref","first-page":"733","DOI":"10.1038\/nphys1797","article-title":"Physical virology.","volume":"6","author":"WH Roos","year":"2010","journal-title":"Nat Phys"},{"key":"ppat.1012873.ref053","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s42003-021-01779-x","article-title":"Identification of a conserved virion-stabilizing network inside the interprotomer pocket of enteroviruses.","volume":"4","author":"JW Flatt","year":"2021","journal-title":"Commun Biol."},{"key":"ppat.1012873.ref054","article-title":"Activity of pleconaril against enteroviruses","author":"DC Pevear","year":"1999","journal-title":"Antimicrob Agents Chemother"},{"key":"ppat.1012873.ref055","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1073\/pnas.2022464118","article-title":"A pocket-factor\u2013triggered conformational switch in the hepatitis B virus capsid","volume":"118","author":"L Lecoq","year":"2021","journal-title":"Proc Natl Acad Sci U S A"},{"key":"ppat.1012873.ref056","first-page":"5","article-title":"The refined structure of human rhinovirus 16 at 2.15 \u00c5 resolution: Implications for the viral life cycle","author":"AT Hadfield","year":"1997","journal-title":"Structure"},{"key":"ppat.1012873.ref057","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1371\/journal.ppat.1009146","article-title":"Assembly of infectious 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