Structural figures were illustrated by PyMOL40 and Chimera35. Statistical analyses Correlation between the inhibition ratio by mAbs and neutralization titer of the vaccinated guinea pig sera was determined using the calculated Pearson coefficient of correlation. H16.001 afforded unbiased potency characterization for various HPV16 vaccines and was potential for use in vaccine regulation practice. This study also showed a model process for selecting suitable mAbs for potency assays of other vaccines. expression system, and I1.16 from insect cell expression system. Interestingly, H16.001 and H16.8A9 reacted with all sorts of HPV16 VLPs comparably as manifested by almost overlapping binding response curves for five HPV16 VLP samples, indicating that H16.001 and H16.8A9 reactivities are tolerant and irrespective of the expression systems that generated the HPV16 VLPs, whereas H16.V5 reacted the best with expression system. I1.16 was derived from insect cell expression system. Immunodominance in anti-HPV16 sera of mAbs Immunodominance analysis for an mAb by competition ELISA (c/ELISA) or blocking ELISA (b/ELISA) usually reflect on the major neutralization epitope that generates corresponding antibodies in the anti-sera. We then measured the blocking ratio of the 4 mAbs against 14 guinea pig sera (Supplementary Table 1) from animals vaccinated with preparations of VLPs produced from 5 different manufacturing processes using yeast, expression system, the blue ones represent sera with E2.16 by expression system, and the purple ones represent sera with I1.16 by insect cell expression system. The comparison was assessed using the calculated Pearson coefficient of correlation, and the statistical analysis was performed using GraphPad Prism 7. Next, we explored the blocking ratio of the 4 mAbs against HPV16 reactive human sera (Supplementary Table 1, expression system, and I1.16 from insect cell expression system. HPV16 pentamers were kindly provided by Innovax Biotech Co., Ltd. Fifty post-vaccinated human serum samples were kindly provided by the vaccine manufacturer Shanghai Zerun Biotechnology (Shanghai, China), which were collected from a phase I clinical trial of a bivalent HPV16/18 vaccine, produced in Risedronate sodium Pichia Pastoris (ClinicalTrials.gov ID:2011L01085). Vaccines were administered according to their recommended three-dose vaccination schedules (months 0, 2, and 6). Serum samples were collected 7 months after the first immunization. Fab preparation To obtain H16.001 Fab, the mAb H16.001 was digested in the mixture solution containing 1 (w/w) papain, 20mM L-Cysteine, and 50?mM ethylenediamine tetraacetic acid at 37?C. Following 12-h incubation, 30?mM iodoacetamide was added to stop the digestion. The resulting Fab fragment was then purified by diethylethanolamine column (TOSOH, Japan) chromatography. Generation and purification of HPV16 PsVs 293FT cells were grown in complete Dulbeccos modified Eagles medium containing 10% fetal bovine serum, 2% HEPES, 1% nonessential amino acids, and 1% penicillin/streptomycin. The reporter plasmid pcDNA3.1-EGFP was constructed by inserting an EGFP reporter gene into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA). HPV16 L1L2 PsVs were generated and titrated as described previously4,23,24. The PsV titers were defined as 50% tissue culture infective dose, calculated with the ReedCMuench method25. For structure PLAT determination, PsV particles were purified by sucrose density gradient and subsequent CsCl density gradient ultracentrifugation26. Sera from immunized guinea pigs with HPV vaccines The animal study was approved by the Institutional Animal Care and Use Committee of National Institutes for Food and Drug Risedronate sodium Control. All animals were housed in accordance with relevant guidelines. Five samples of HPV16 VLPs, which were kindly provided Risedronate sodium by Ruike Biotechnology, Bowei Biotechnology, Health Guard Biotechnology, Innovax Biotech Co., Ltd., and Sino Biological Inc., were mixed with aluminum hydroxide adjuvant, respectively. Ten micrograms of the aforementioned adjuvant-adsorbed HPV16 VLPs were delivered intramuscularly for each guinea pig (200C220?g, female) and three guinea pigs were immunized with each of the vaccines. At the same time, the aluminum hydroxide.