{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,29]],"date-time":"2026-05-29T07:02:27Z","timestamp":1780038147509,"version":"3.53.1"},"reference-count":16,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,5,29]],"date-time":"2026-05-29T00:00:00Z","timestamp":1780012800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003569","name":"Ministry of Food and Drug Safety","doi-asserted-by":"publisher","award":["23202MFDS151, RS-2024-00331833"],"award-info":[{"award-number":["23202MFDS151, RS-2024-00331833"]}],"id":[{"id":"10.13039\/501100003569","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003725","name":"National Research Foundation of Korea","doi-asserted-by":"publisher","award":["2021R1C1C1008626"],"award-info":[{"award-number":["2021R1C1C1008626"]}],"id":[{"id":"10.13039\/501100003725","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003710","name":"Korea Health Industry Development Institute","doi-asserted-by":"publisher","award":["RS-2025-02215878, RS-2021-KH118940"],"award-info":[{"award-number":["RS-2025-02215878, RS-2021-KH118940"]}],"id":[{"id":"10.13039\/501100003710","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:sec>\n                    <jats:title>Introduction<\/jats:title>\n                    <jats:p>Evaluating next-generation prophylactic human papillomavirus (HPV) vaccines requires high-throughput and scalable neutralization assays spanning a wide array of genotypes. Standard secreted alkaline phosphatase (SEAP)-based platforms, however, are limited by tedious protocols, such as mandatory serum heat inactivation, and restricted sensitivity. To address these bottlenecks, we developed an advanced pseudovirus (PsV) platform using Cypridina luciferase (cLuc), a versatile dual-reporter system that facilitates both extracellular secretion and intracellular monitoring.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>A comprehensive library of cLuc-engineered PsVs was constructed, covering 28 distinct mucosal and cutaneous HPV genotypes. We verified their structural integrity and antigenic presentation via neutralization profiling with L2 N-terminus-specific monoclonal antibodies (JWW-1\/JWW-2).<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>The cLuc-PsVs demonstrated robust infectivity along with excellent analytical linearity. Critically, this system eliminated the need for thermal inactivation of sera while maintaining an exceptional correlation (R\u00b2 &amp;gt; 0.99) with traditional SEAP assays during evaluations of hyperimmune mouse sera. Beyond in vitro screens, the platform's translational value was confirmed in a murine cervicovaginal challenge model, where real-time bioluminescence imaging enabled non-invasive tracking of mucosal infection across all 28 genotypes.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Discussion<\/jats:title>\n                    <jats:p>This dual-purpose cLuc-PsV platform provides a highly efficient, sensitive alternative to conventional neutralization assays. By unifying high-throughput in vitro quantification with longitudinal in vivo imaging, this system offers a robust tool to accelerate the preclinical screening of multivalent HPV vaccine candidates.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fimmu.2026.1863344","type":"journal-article","created":{"date-parts":[[2026,5,29]],"date-time":"2026-05-29T06:11:16Z","timestamp":1780035076000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["An advanced, highly sensitive and versatile Cypridina luciferase-based HPV pseudovirus system for broad-spectrum neutralization and non-invasive In vivo monitoring"],"prefix":"10.3389","volume":"17","author":[{"given":"Hyemi","family":"Kim","sequence":"first","affiliation":[{"name":"Department of Microbiology and Immunology, Yonsei University College of Medicine","place":["Seoul, Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chai Won","family":"Kim","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Yonsei University College of Medicine","place":["Seoul, Republic of Korea"]},{"name":"Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine","place":["Seoul, Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kihyuck","family":"Kwak","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Yonsei University College of Medicine","place":["Seoul, Republic of Korea"]},{"name":"Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine","place":["Seoul, Republic of Korea"]},{"name":"Institute for Immunology and Immunological Diseases, Yonsei University College of Medicine","place":["Seoul, Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,5,29]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1016\/S1473-3099(07)70158-5","article-title":"Worldwide prevalence and genotype distribution of cervical human papillomavirus DNA in women with normal cytology: a meta-analysis","volume":"7","author":"de Sanjos\u00e9","year":"2007","journal-title":"Lancet Infect Dis"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1016\/S1470-2045(14)71179-9","article-title":"Next generation prophylactic human papillomavirus vaccines","volume":"16","author":"Schiller","year":"2015","journal-title":"Lancet Oncol"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa1405044","article-title":"A 9-valent HPV vaccine against infection and intraepithelial neoplasia in women","volume":"372","author":"Joura","year":"2015","journal-title":"N Engl J Med"},{"key":"B4","doi-asserted-by":"publisher","DOI":"10.1016\/j.virol.2003.12.027","article-title":"Reactivity of human sera in a sensitive, high-throughput pseudovirus-based papillomavirus neutralization assay for HPV16 and HPV18","volume":"321","author":"Pastrana","year":"2004","journal-title":"Virology"},{"key":"B5","doi-asserted-by":"publisher","DOI":"10.4161\/hv.4.6.6912","article-title":"Correlation between direct ELISA, single epitope-based inhibition ELISA and pseudovirion-based neutralization assay for measuring anti-HPV-16 and anti-HPV-18 antibody response","volume":"4","author":"Dessy","year":"2008","journal-title":"Hum Vaccin"},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.78.2.751-757.2004","article-title":"Efficient intracellular assembly of papillomaviral vectors","volume":"78","author":"Buck","year":"2004","journal-title":"J Virol"},{"key":"B7","doi-asserted-by":"publisher","first-page":"24","DOI":"10.1016\/j.coviro.2013.11.002","article-title":"The cellular pathways of infectious human papillomavirus entry","volume":"4","author":"Day","year":"2014","journal-title":"Curr Opin Virol"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.1089\/adt.2006.053","article-title":"Bioluminescent assays for high-throughput screening","volume":"5","author":"Fan","year":"2007","journal-title":"ASSAY Drug Dev Technol"},{"key":"B9","doi-asserted-by":"publisher","DOI":"10.1271\/bbb.68.565","article-title":"cDNA cloning and characterization of a secreted luciferase from the luminous Japanese ostracod, Cypridina hilgendorfii","volume":"68","author":"Nakajima","year":"2004","journal-title":"Biosci Biotechnol Biochem"},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymthe.2004.10.016","article-title":"Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo","volume":"11","author":"Tannous","year":"2005","journal-title":"Mol Ther"},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.02639-06","article-title":"A protective and broadly cross-neutralizing epitope of human papillomavirus L2","volume":"81","author":"Gambhira","year":"2007","journal-title":"J Virol"},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.79.5.2839-2846.2005","article-title":"Maturation of papillomavirus capsids","volume":"79","author":"Buck","year":"2005","journal-title":"J Virol"},{"key":"B13","doi-asserted-by":"publisher","DOI":"10.1038\/nri2510","article-title":"Towards an understanding of the adjuvant action of aluminium","volume":"9","author":"Marrack","year":"2009","journal-title":"Nat Rev Immunol"},{"key":"B14","doi-asserted-by":"publisher","DOI":"10.1038\/nm1598","article-title":"Genital transmission of HPV in a mouse model is potentiated by nonoxynol-9 and inhibited by carrageenan","volume":"13","author":"Roberts","year":"2007","journal-title":"Nat Med"},{"key":"B15","doi-asserted-by":"publisher","DOI":"10.1016\/j.virol.2013.04.017","article-title":"L2, the minor capsid protein of papillomavirus","volume":"445","author":"Wang","year":"2013","journal-title":"Virology"},{"key":"B16","doi-asserted-by":"publisher","DOI":"10.1093\/infdis\/jis554","article-title":"Cellular immune responses to human papillomavirus (HPV)-16 L1 in healthy volunteers immunized with recombinant HPV-16 L1 virus-like particles","volume":"206","author":"Pinto","year":"2012","journal-title":"J Infect Dis"}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2026.1863344\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,29]],"date-time":"2026-05-29T06:11:17Z","timestamp":1780035077000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2026.1863344\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,29]]},"references-count":16,"alternative-id":["10.3389\/fimmu.2026.1863344"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2026.1863344","relation":{},"ISSN":["1664-3224"],"issn-type":[{"value":"1664-3224","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,29]]},"article-number":"1863344"}}