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Viruses
Volume 17
Issue 9
10.3390/v17091164
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This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
Article

Optimization of a High-Throughput Human Papillomavirus Neutralizing Antibody Assay Based on Pseudotyped Viruses for the 15-Valent Human Papillomavirus Vaccine Types

by
Huan Liu
1,2,†,
Haiyang Qin
1,†,
Lingling Nie
1,
Yanru Shen
1,
Jiayi Li
1,
Pengcheng Xiu
1,
Shasha Wang
3,
Meng Wang
1,
Youchun Wang
4,*,
Jianhui Nie
1,*,
Weijin Huang
1,* and
Li Zhang
1,*
1
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
2
Hebei Key Laboratory of Public Health Safety, School of Public Health, Hebei University, Baoding 071000, China
3
National Laboratory Animal Quality Testing Center, Institute of Laboratory Animal Resources, National Institutes for Food and Drug Control, Beijing 102629, China
4
Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming 650118, China
*
Authors to whom correspondence should be addressed.
†
These authors equally contributed to this study.
Viruses 2025, 17(9), 1164; https://doi.org/10.3390/v17091164 (registering DOI)
Submission received: 3 August 2025 / Revised: 23 August 2025 / Accepted: 23 August 2025 / Published: 26 August 2025
(This article belongs to the Section Viral Immunology, Vaccines, and Antivirals)
Versions Notes

Abstract

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.
Keywords: human papillomavirus; pseudovirion-based neutralization assay (PBNA); immunogenicity; high-throughput assay human papillomavirus; pseudovirion-based neutralization assay (PBNA); immunogenicity; high-throughput assay

Share and Cite

MDPI and ACS Style

Liu, H.; Qin, H.; Nie, L.; Shen, Y.; Li, J.; Xiu, P.; Wang, S.; Wang, M.; Wang, Y.; Nie, J.; et al. Optimization of a High-Throughput Human Papillomavirus Neutralizing Antibody Assay Based on Pseudotyped Viruses for the 15-Valent Human Papillomavirus Vaccine Types. Viruses 2025, 17, 1164. https://doi.org/10.3390/v17091164

AMA Style

Liu H, Qin H, Nie L, Shen Y, Li J, Xiu P, Wang S, Wang M, Wang Y, Nie J, et al. Optimization of a High-Throughput Human Papillomavirus Neutralizing Antibody Assay Based on Pseudotyped Viruses for the 15-Valent Human Papillomavirus Vaccine Types. Viruses. 2025; 17(9):1164. https://doi.org/10.3390/v17091164

Chicago/Turabian Style

Liu, Huan, Haiyang Qin, Lingling Nie, Yanru Shen, Jiayi Li, Pengcheng Xiu, Shasha Wang, Meng Wang, Youchun Wang, Jianhui Nie, and et al. 2025. "Optimization of a High-Throughput Human Papillomavirus Neutralizing Antibody Assay Based on Pseudotyped Viruses for the 15-Valent Human Papillomavirus Vaccine Types" Viruses 17, no. 9: 1164. https://doi.org/10.3390/v17091164

APA Style

Liu, H., Qin, H., Nie, L., Shen, Y., Li, J., Xiu, P., Wang, S., Wang, M., Wang, Y., Nie, J., Huang, W., & Zhang, L. (2025). Optimization of a High-Throughput Human Papillomavirus Neutralizing Antibody Assay Based on Pseudotyped Viruses for the 15-Valent Human Papillomavirus Vaccine Types. Viruses, 17(9), 1164. https://doi.org/10.3390/v17091164

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