Analysis of Specific Neutralizing Antibody Titers Against Cervical Cancer Vaccine Using the Pseudovirion-Based Neutralization Assay

Abstract

        Currently, the cervical cancer vaccines have been included in Thailand Expanded Program on Immunization. Therefore, measuring immune responses in vaccinated individuals, as well as assessing vaccine potency for quality control, is important. This study aimed to develop and validate a pseudovirion-based neutralization assay (PBNA) for quantifying antibodies against human papillomavirus (HPV). This study aimed to develop and validate a PBNA for quantifying antibodies against HPV. The ability of human standard sera specific to HPV types 16 and 18 to inhibit the entry of the corresponding HPV pseudovirions into cultured 293FT cells was examined and expressed as IC₅₀/100μL. The developed method was subsequently verified. The produced HPV pseudovirions were found to be specific to the corresponding antibodies of each HPV type and were not inhibited by negative serum. The accuracy showed a %recovery in the range of 70-130%. A linear relationship was observed between log fluorospot and the dilution level of the standard serum for both types, with a correlation coefficient, > 0.98. The resulting graph was linear over the range of 20–80% inhibitory rate. The limits of quantitation for antibodies against types 16 and 18 were 1.49 IC₅₀/100μL and 1.56 IC₅₀/100μL, respectively. The assay also showed good precision, with the % coefficient of variance of no more than 25%. In the ruggedness test, using different cell concentrations and cell passage, the IC₅₀/100μL values were not significantly different at 95% confidence level (p-value > 0.05). In conclusion, the developed PBNA was validated for HPV16 and HPV18 in both standard human sera and type-specific mouse sera. This assay can serve as a platform for the development of animal-derived reference standards
for other HPV-VLP types used in vaccine production.