Characterization And Application Of Anti-HPV16 Monoclonal Antibodies In The Development Of Immunochemical Assays For HPV16 Vaccine

Human papilloma virus(HPV)is responsible for a variety of cancers,persistent infection with high-risk HPVs may lead to cervical cancer,oropharyngeal cancer,rectal cancer and other cancers.The major capsid protein L1 of HPV16 could self-assemble into recombinant virus-like particles(VLPs).VLPs have great safety profiles and high efficacy because of no viral genetic materials and faithfully mimic the native virions.There are three HPV prophylactic vaccines,they have been approved for using against high-risk HPV infection in man countries.The HPV L1 VLPs are the key antigen components.The neutralizing antibodies are thought to be the primary immunogenicity biomarkers and correlate with the efficacy of prophylactic HPV vaccines.It is important to ensure safty and efficacy of HPV vaccines.In this study,a combination of immunochemical methods was developed by multiple monoclonal antibodies(mAbs)that recognized different epitopes.The assays could analyse the antigen quality attributes of HPV16 VLP-based vaccines and provide data-driven assurance to the life cycle management of HPV 16 VLP-based vaccines.Firstly,a total of 27 mAbs were exhibited.The characteristics of anti-HPV16 VLP mAbs of type-specificity,subtype,binding affinity,conformational sensitivity and neutralizating potency were characterized by a series of immunochemical analytical assays.Then,there are 10 representative mAbs were selected out for following studies.The competitive binding experiments of mAbs based on pairwise cross-competitions and competitions with human polyclonal antibodies were used for clustering mAbs into different epitope bins.There are at least five neutralizing epitopes recognized by these mAbs.According to the properties of mAbs and the results of clustering analysis,6 mAbs directed to different or proximal epitopes were selected for the development of immunochemical analysis methods for HPV16 VLP vaccines.The analytical system of HPV16 vacccines were consisted of three assays,including surface plasmon resonance(SPR)based on label-free binding analysis,competitive ELISA system(rICso)and sandwich ELISA system(rECso)based on two-sites.According to the testing of experimental conditions,a total of 6 mAbs were used on SPR,5 mAbs were used to rICso and 2 mAbs were used to rEC50.The results of three assays were all showed good reproducibility and robustness.Lastly,the multi-epitope immunochemical quality analysis system of HP V16 VLP vaccines was applied to confirm lot consistency in a pilot scale-up of 100 L and 500 L of a HPV16 vaccine candicate developed by our laboratory and INOVAX.The antigenicity tests of different antigen batches were measured by SPR and rICso.There was no significant difference between test samples and reference in antigenicity,confirmed the scalability reproducibility of vaccines in pilot scale-up.In summary,a panel of HPV16 L1 VLPs-specific mAbs was isolated.After characterization and clustering analysis,a tool box of representative mAbs that recognized different epitopes was obtained.The mAbs obtained here are good candidates for monitoring the vaccine production process to analyze potency in vitro and immune response induced in vivo.The multi-sites immunochemical analytical system for HPV16 vaccine was useful for guarantee the safety and efficacy.It also offered a reference model to establish the analytical system of other VLP-based vaccines.