{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,21]],"date-time":"2026-05-21T17:51:18Z","timestamp":1779385878648,"version":"3.53.1"},"reference-count":34,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2016,4,25]],"date-time":"2016-04-25T00:00:00Z","timestamp":1461542400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"the National High Technology Research and Development Program of China","award":["2012AA02A402"],"award-info":[{"award-number":["2012AA02A402"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Pseudovirion-based neutralization assay is considered the gold standard method for evaluating the immune response to human papillomavirus (HPV) vaccines. In this study, we developed a multicolor neutralization assay to simultaneously detect the neutralizing antibodies against different HPV types. FluoroSpot was used to interpret the fluorescent protein expression instead of flow cytometry. The results of FluoroSpot and flow cytometry showed good consistency, with R2 &gt; 0.98 for the log-transformed IC50 values. Regardless of the reporter color, the single-, dual-, and triple-color neutralization assays reported identical results for the same samples. In low-titer samples from naturally HPV-infected individuals, there was strong agreement between the single- and triple-color assays, with kappa scores of 0.92, 0.89, and 0.96 for HPV16, HPV18, and HPV58, respectively. Good reproducibility was observed for the triple-color assay, with coefficients of variation of 2.0%\u201341.5% within the assays and 8.3%\u201336.2% between the assays. Three triple-color systems, HPV16-18-58, HPV6-33-45, and HPV11-31-52, were developed that could evaluate the immunogenicity of a nonavalent vaccine in three rounds of the assay. With the advantages of an easy-to-use procedure and less sample consumption, the multiple-color assay is more suitable than classical assays for large sero-epidemiological studies and clinical trials and is more amenable to automation.<\/jats:p>","DOI":"10.3390\/v8040107","type":"journal-article","created":{"date-parts":[[2016,4,25]],"date-time":"2016-04-25T10:02:36Z","timestamp":1461578556000},"page":"107","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":41,"title":["Development of a Triple-Color Pseudovirion-Based Assay to Detect Neutralizing Antibodies against Human Papillomavirus"],"prefix":"10.3390","volume":"8","author":[{"given":"Jianhui","family":"Nie","sequence":"first","affiliation":[{"name":"Division of HIV\/AIDS and Sexually Transmitted Virus Vaccines, National Institutes for Food and Drug Control (NIFDC), No. 2 Tiantanxili, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yangyang","family":"Liu","sequence":"additional","affiliation":[{"name":"Division of HIV\/AIDS and Sexually Transmitted Virus Vaccines, National Institutes for Food and Drug Control (NIFDC), No. 2 Tiantanxili, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weijin","family":"Huang","sequence":"additional","affiliation":[{"name":"Division of HIV\/AIDS and Sexually Transmitted Virus Vaccines, National Institutes for Food and Drug Control (NIFDC), No. 2 Tiantanxili, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9769-5141","authenticated-orcid":false,"given":"Youchun","family":"Wang","sequence":"additional","affiliation":[{"name":"Division of HIV\/AIDS and Sexually Transmitted Virus Vaccines, National Institutes for Food and Drug Control (NIFDC), No. 2 Tiantanxili, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2016,4,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"87","DOI":"10.3322\/caac.21262","article-title":"Global cancer statistics, 2012","volume":"65","author":"Torre","year":"2015","journal-title":"CA Cancer J. Clin."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1093\/emph\/eov003","article-title":"Papillomaviruses: Viral evolution, cancer and evolutionary medicine","volume":"2015","author":"Bravo","year":"2015","journal-title":"Evol. Med. Public Health"},{"key":"ref_3","first-page":"217","article-title":"Prophylaxis of cervical cancer and related cervical disease: A review of the cost-effectiveness of vaccination against oncogenic HPV types","volume":"16","author":"Armstrong","year":"2010","journal-title":"J. Manag. Care Pharm."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"740","DOI":"10.4161\/hv.27619","article-title":"Correlation between ELISA and pseudovirion-based neutralisation assay for detecting antibodies against human papillomavirus acquired by natural infection or by vaccination","volume":"10","author":"Zhao","year":"2014","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"92","DOI":"10.1016\/j.pedhc.2010.06.015","article-title":"Uptake of the human papillomavirus vaccine: A review of the literature and report of a quality assurance project","volume":"26","author":"Cassidy","year":"2012","journal-title":"J. Pediatr. Health Care"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"711","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":"ref_7","first-page":"108","article-title":"Simultaneous quantitation of antibodies to neutralizing epitopes on virus-like particles for human papillomavirus types 6, 11, 16, and 18 by a multiplexed luminex assay","volume":"10","author":"Opalka","year":"2003","journal-title":"Clin. Diagn. Lab. Immunol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"6686","DOI":"10.1016\/j.vaccine.2012.08.068","article-title":"Seroprevalence of seven high-risk HPV types in the Netherlands","volume":"30","author":"Scherpenisse","year":"2012","journal-title":"Vaccine"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1845","DOI":"10.1373\/clinchem.2005.052381","article-title":"Multiplex human papillomavirus serology based on in situ-purified glutathione s-transferase fusion proteins","volume":"51","author":"Waterboer","year":"2005","journal-title":"Clin. Chem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"F55","DOI":"10.1016\/j.vaccine.2012.06.083","article-title":"The biology and life-cycle of human papillomaviruses","volume":"30","author":"Doorbar","year":"2012","journal-title":"Vaccine"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"751","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":"ref_12","doi-asserted-by":"crossref","first-page":"205","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":"ref_13","doi-asserted-by":"crossref","first-page":"693","DOI":"10.1002\/jmv.21435","article-title":"Detection of HPV types and neutralizing antibodies in Gansu province, China","volume":"81","author":"Wu","year":"2009","journal-title":"J. Med. Virol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Sehr, P., Rubio, I., Seitz, H., Putzker, K., Ribeiro-Muller, L., Pawlita, M., and Muller, M. (2013). High-throughput pseudovirion-based neutralization assay for analysis of natural and vaccine-induced antibodies against human papillomaviruses. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0075677"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1542","DOI":"10.1002\/jmv.23995","article-title":"Optimization and validation of a high throughput method for detecting neutralizing antibodies against human papillomavirus (HPV) based on pseudovirons","volume":"86","author":"Nie","year":"2014","journal-title":"J. Med. Virol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1016\/j.jim.2014.12.013","article-title":"Development and evaluation of multiplexed immunoassay for detection of antibodies to HPV vaccine types","volume":"417","author":"Panicker","year":"2015","journal-title":"J. Immunol. Methods"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"3490","DOI":"10.4049\/jimmunol.1500918","article-title":"Single-cell analysis of B cell\/antibody cross-reactivity using a novel multicolor FluoroSpot assay","volume":"195","author":"Hadjilaou","year":"2015","journal-title":"J. Immunol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"9201","DOI":"10.1128\/JVI.75.19.9201-9209.2001","article-title":"Enhancement of capsid gene expression: Preparing the human papillomavirus type 16 major structural gene L1 for DNA vaccination purposes","volume":"75","author":"Leder","year":"2001","journal-title":"J. Virol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"365","DOI":"10.1016\/j.virol.2005.04.011","article-title":"Cross-neutralization of cutaneous and mucosal Papillomavirus types with anti-sera to the amino terminus of L2","volume":"337","author":"Pastrana","year":"2005","journal-title":"Virology"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1016\/j.virol.2004.04.050","article-title":"Codon optimization of the human papillomavirus 11 (HPV 11) L1 gene leads to increased gene expression and formation of virus-like particles in mammalian epithelial cells","volume":"326","author":"Mossadegh","year":"2004","journal-title":"Virology"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1016\/j.virol.2005.09.047","article-title":"Factors influencing subcellular localization of the human papillomavirus L2 minor structural protein","volume":"345","author":"Kieback","year":"2006","journal-title":"Virology"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"266","DOI":"10.1016\/j.virol.2006.08.037","article-title":"Neutralization of HPV16, 18, 31, and 58 pseudovirions with antisera induced by immunizing rabbits with synthetic peptides representing segments of the HPV16 minor capsid protein L2 surface region","volume":"358","author":"Kondo","year":"2007","journal-title":"Virology"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"857","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":"ref_24","doi-asserted-by":"crossref","first-page":"841","DOI":"10.1002\/jmv.21124","article-title":"Modification of human papillomavirus-like particle vaccine by insertion of the cross-reactive L2-epitopes","volume":"80","author":"Kondo","year":"2008","journal-title":"J. Med. Virol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2067","DOI":"10.1128\/JVI.02190-08","article-title":"Role of heparan sulfate in attachment to and infection of the murine female genital tract by human papillomavirus","volume":"83","author":"Johnson","year":"2009","journal-title":"J. Virol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1007\/s12250-010-3078-4","article-title":"Detection of HPV types and neutralizing antibodies in women with genital warts in Tianjin City, China","volume":"25","author":"Wu","year":"2010","journal-title":"Virol. Sin."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Buck, C.B., and Thompson, C.D. (2007). Production of papillomavirus-based gene transfer vectors. Curr. Protoc. Cell Biol.","DOI":"10.1002\/0471143030.cb2601s37"},{"key":"ref_28","first-page":"175","article-title":"A note on some points of calculation method of LD50 by Reed and Muench","volume":"20","author":"Matumoto","year":"1949","journal-title":"Jpn. J. Exp. Med."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"6520","DOI":"10.1016\/j.vaccine.2011.07.007","article-title":"The first international standard for antibodies to HPV 16","volume":"29","author":"Ferguson","year":"2011","journal-title":"Vaccine"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"F83","DOI":"10.1016\/j.vaccine.2012.04.106","article-title":"Human papillomavirus vaccines\u2014Immune responses","volume":"30","author":"Stanley","year":"2012","journal-title":"Vaccine"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1007\/978-1-61779-325-7_6","article-title":"Dual- and triple-color fluorospot","volume":"792","author":"Ahlborg","year":"2012","journal-title":"Methods Mol. Biol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"936","DOI":"10.1086\/597309","article-title":"The impact of quadrivalent human papillomavirus (HPV; types 6, 11, 16, and 18) L1 virus-like particle vaccine on infection and disease due to oncogenic nonvaccine HPV types in sexually active women aged 16\u201326 years","volume":"199","author":"Wheeler","year":"2009","journal-title":"J. Infect. Dis."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"926","DOI":"10.1086\/597307","article-title":"The impact of quadrivalent human papillomavirus (HPV; types 6, 11, 16, and 18) L1 virus-like particle vaccine on infection and disease due to oncogenic nonvaccine HPV types in generally HPV-naive women aged 16\u201326 years","volume":"199","author":"Brown","year":"2009","journal-title":"J. Infect. Dis."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2161","DOI":"10.1016\/S0140-6736(07)60946-5","article-title":"Efficacy of a prophylactic adjuvanted bivalent L1 virus-like-particle vaccine against infection with human papillomavirus types 16 and 18 in young women: An interim analysis of a phase III double-blind, randomised controlled trial","volume":"369","author":"Paavonen","year":"2007","journal-title":"Lancet"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/8\/4\/107\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T19:22:48Z","timestamp":1760210568000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/8\/4\/107"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2016,4,25]]},"references-count":34,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2016,4]]}},"alternative-id":["v8040107"],"URL":"https:\/\/doi.org\/10.3390\/v8040107","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2016,4,25]]}}}