{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T01:40:23Z","timestamp":1760060423699,"version":"build-2065373602"},"reference-count":26,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2025,8,26]],"date-time":"2025-08-26T00:00:00Z","timestamp":1756166400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Key Research and Development Program of China","award":["2025YFC2311705"],"award-info":[{"award-number":["2025YFC2311705"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Vaccination is highly effective in preventing human papillomavirus (HPV) infection, but traditional pseudovirion-based neutralization assays (PBNA) are technically demanding, labor-intensive, and costly, limiting their use in multivalent vaccine studies. We developed and validated an automated, high-throughput PBNA in a 384-well format that quantifies neutralizing antibodies against 15 HPV types using triple-color pseudotyped viruses. Non-interfering type triplets were defined from cross-neutralization assays of serum against pseudotyped viruses, enabling simultaneous detection of three fluorescence signals per well. The workflow integrates a cap-decapper, semi-automatic sample addition and dilution, and a microplate stacker with automated imaging to reduce hands-on time. The 384-well method showed strong concordance with the conventional 96-well PBNA while increasing daily sample throughput by approximately 6.7-fold, reducing assay duration (including ~4-fold faster imaging), and lowering reaction volume by ~5-fold. Analytical validation demonstrated acceptable specificity, accuracy, repeatability, linearity and robustness for high-throughput use. Serostatus cutoff values were established in an age-appropriate female population to support classification of positive versus negative sera. This platform provides a scalable tool for evaluating neutralizing antibodies after natural infections or vaccination and is well suited for large clinical trials and the development of next-generation and multivalent HPV vaccines.<\/jats:p>","DOI":"10.3390\/v17091164","type":"journal-article","created":{"date-parts":[[2025,8,27]],"date-time":"2025-08-27T08:19:32Z","timestamp":1756282772000},"page":"1164","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Optimization of a High-Throughput Human Papillomavirus Neutralizing Antibody Assay Based on Pseudotyped Viruses for the 15-Valent Human Papillomavirus Vaccine Types"],"prefix":"10.3390","volume":"17","author":[{"given":"Huan","family":"Liu","sequence":"first","affiliation":[{"name":"Division of HIV\/AIDS and Sex-Transmitted Virus Vaccines, Institute for Biological Product Control, National Institutes for Food and Drug Control (NIFDC), Beijing 102629, China"},{"name":"Hebei Key Laboratory of Public Health Safety, School of Public Health, Hebei University, Baoding 071000, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Haiyang","family":"Qin","sequence":"additional","affiliation":[{"name":"Division of HIV\/AIDS and Sex-Transmitted Virus Vaccines, Institute for Biological Product Control, National Institutes for Food and Drug Control (NIFDC), Beijing 102629, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lingling","family":"Nie","sequence":"additional","affiliation":[{"name":"Division of HIV\/AIDS and Sex-Transmitted Virus Vaccines, Institute for Biological Product Control, National Institutes for Food and Drug Control (NIFDC), Beijing 102629, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yanru","family":"Shen","sequence":"additional","affiliation":[{"name":"Division of HIV\/AIDS and Sex-Transmitted Virus Vaccines, Institute for Biological Product Control, National Institutes for Food and Drug Control (NIFDC), Beijing 102629, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiayi","family":"Li","sequence":"additional","affiliation":[{"name":"Division of HIV\/AIDS and Sex-Transmitted Virus Vaccines, Institute for Biological Product Control, National Institutes for Food and Drug Control (NIFDC), Beijing 102629, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pengcheng","family":"Xiu","sequence":"additional","affiliation":[{"name":"Division of HIV\/AIDS and Sex-Transmitted Virus Vaccines, Institute for Biological Product Control, National Institutes for Food and Drug Control (NIFDC), Beijing 102629, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shasha","family":"Wang","sequence":"additional","affiliation":[{"name":"National Laboratory Animal Quality Testing Center, Institute of Laboratory Animal Resources, National Institutes for Food and Drug Control, Beijing 102629, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Meng","family":"Wang","sequence":"additional","affiliation":[{"name":"Division of HIV\/AIDS and Sex-Transmitted Virus Vaccines, Institute for Biological Product Control, National Institutes for Food and Drug Control (NIFDC), Beijing 102629, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Youchun","family":"Wang","sequence":"additional","affiliation":[{"name":"Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming 650118, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1474-0427","authenticated-orcid":false,"given":"Jianhui","family":"Nie","sequence":"additional","affiliation":[{"name":"Division of HIV\/AIDS and Sex-Transmitted Virus Vaccines, Institute for Biological Product Control, National Institutes for Food and Drug Control (NIFDC), Beijing 102629, 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2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries","volume":"74","author":"Bray","year":"2024","journal-title":"CA Cancer J. Clin."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"74","DOI":"10.1016\/j.virol.2018.04.003","article-title":"Towards quality and order in human papillomavirus research","volume":"519","author":"Eklund","year":"2018","journal-title":"Virology"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1016\/j.virol.2013.04.023","article-title":"Cross-roads in the classification of papillomaviruses","volume":"445","year":"2013","journal-title":"Virology"},{"unstructured":"International Human Papillomavirus Reference Center (2025, August 20). HPV Reference Clones. Available online: https:\/\/www.hpvcenter.se\/human_reference_clones\/.","key":"ref_4"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"001540","DOI":"10.1099\/jgv.0.001540","article-title":"The human papillomavirus oncoproteins: A review of the host pathways targeted on the road to transformation","volume":"102","author":"Scarth","year":"2021","journal-title":"J. Gen. Virol."},{"doi-asserted-by":"crossref","unstructured":"Williamson, A.L. (2023). Recent Developments in Human Papillomavirus (HPV) Vaccinology. Viruses, 15.","key":"ref_6","DOI":"10.3390\/v15071440"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"e29050","DOI":"10.1002\/jmv.29050","article-title":"Validation of Luminex immunological and competitive Luminex immunological assays for clinical immunogenicity assessment of a 14-valent recombinant human papillomavirus vaccine","volume":"95","author":"Zhang","year":"2023","journal-title":"J. Med. Virol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1016\/S1470-2045(05)70101-7","article-title":"Prophylactic quadrivalent human papillomavirus (types 6, 11, 16, and 18) L1 virus-like particle vaccine in young women: A randomised double-blind placebo-controlled multicentre phase II efficacy trial","volume":"6","author":"Villa","year":"2005","journal-title":"Lancet Oncol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1097\/01.AOG.0000192397.41191.fb","article-title":"Efficacy of human papillomavirus-16 vaccine to prevent cervical intraepithelial neoplasia: A randomized controlled trial","volume":"107","author":"Mao","year":"2006","journal-title":"Obstet. Gynecol."},{"unstructured":"WHO (2009). Human Papillomavirus Laboratory Manual, WHO. [1st ed.].","key":"ref_10"},{"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","first-page":"445","article-title":"Generation of HPV pseudovirions using transfection and their use in neutralization assays","volume":"119","author":"Buck","year":"2005","journal-title":"Methods Protoc."},{"key":"ref_13","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"},{"doi-asserted-by":"crossref","unstructured":"Sehr, P., Rubio, I., Seitz, H., Putzker, K., Ribeiro-M\u00fcller, L., Pawlita, M., and M\u00fcller, M. (2013). High-throughput pseudovirion-based neutralization assay for analysis of natural and vaccine-induced antibodies against human papillomaviruses. PLoS ONE, 8.","key":"ref_14","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."},{"doi-asserted-by":"crossref","unstructured":"Nie, J., Liu, Y., Huang, W., and Wang, Y. (2016). Development of a Triple-Color Pseudovirion-Based Assay to Detect Neutralizing Antibodies against Human Papillomavirus. Viruses, 8.","key":"ref_16","DOI":"10.3390\/v8040107"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"642","DOI":"10.1016\/j.ygyno.2013.05.033","article-title":"HPV vaccine cross-protection: Highlights on additional clinical benefit","volume":"130","author":"Ricci","year":"2013","journal-title":"Gynecol. Oncol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1007\/978-981-99-0113-5_5","article-title":"Pseudotyped Virus for Papillomavirus","volume":"1407","author":"Wu","year":"2023","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"563","DOI":"10.1080\/09537104.2018.1514106","article-title":"An optimized and validated 384-well plate assay to test platelet function in a high-throughput screening format","volume":"30","author":"Bragina","year":"2019","journal-title":"Platelets"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1177\/108705719900400206","article-title":"A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays","volume":"4","author":"Zhang","year":"1999","journal-title":"J. Biomol. Screen."},{"doi-asserted-by":"crossref","unstructured":"GlaxoSmithKline Vaccine HPV-007 Study Group, Romanowski, B., de Borba, P.C., Naud, P.S., Roteli-Martins, C.M., De Carvalho, N.S., Teixeira, J.C., Aoki, F., Ramjattan, B., and Shier, R.M. (2009). Sustained efficacy and immunogenicity of the human papillomavirus (HPV)-16\/18 AS04-adjuvanted vaccine: Analysis of a randomised placebo-controlled trial up to 6.4 years. Lancet, 374, 1975\u20131985.","key":"ref_21","DOI":"10.1016\/S0140-6736(09)61567-1"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1293","DOI":"10.1016\/S1473-3099(17)30468-1","article-title":"Changes in the prevalence of human papillomavirus following a national bivalent human papillomavirus vaccination programme in Scotland: A 7-year cross-sectional study","volume":"17","author":"Kavanagh","year":"2017","journal-title":"Lancet Infect. Dis."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1016\/j.vaccine.2022.11.057","article-title":"High vaccine effectiveness persists for ten years after HPV16\/18 vaccination among young Dutch women","volume":"41","author":"Hoes","year":"2023","journal-title":"Vaccine"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e29859","DOI":"10.1002\/jmv.29859","article-title":"Validation of a triple-color pseudovirion-based neutralization assay for immunogenicity assessment of a 14-valent recombinant human papillomavirus vaccine","volume":"96","author":"Gao","year":"2024","journal-title":"J. Med. Virol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"718","DOI":"10.1093\/ajcp\/aqab181","article-title":"Nationwide Prevalence and Genotype Distribution of High-Risk Human Papillomavirus Infection in China","volume":"157","author":"Zeng","year":"2022","journal-title":"Am. J. Clin. Pathol."},{"doi-asserted-by":"crossref","unstructured":"Wang, R., Guo, X.L., Wisman, G.B., Schuuring, E., Wang, W.F., Zeng, Z.Y., Zhu, H., and Wu, S.W. (2015). Nationwide prevalence of human papillomavirus infection and viral genotype distribution in 37 cities in China. BMC Infect. 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