Multiple-epitope Immunochemical Assays For Quality And Stability Assessment Of Hepatitis E Vaccine

Hepatitis E(HE)is a world-wide serious infectious disease due to Hepatitis E Virus(HEV)infection.The mortality among pregnant women once infected with HEV can be up to 20%.It is estimated that a third of global population has been infected with HEV,sporadic cases of hepatitis E in developed countries are also increasing in recent years.China belongs to high incidence of hepatitis E endemic areas,sporadic hepatitis E is widespread everywhere.Hecoline which is currently the first and only licensed HEV prophylactic vaccine was jointly developped by our lab and Xiamen Innovax Biotech Co.,Ltd.The antigen in this vaccine,p239,is a truncated HEV ORF2 protein from a.a.368 to a.a.606.p239 can form VLPs as a stable structure and elicit a protective immune response against HEV.Although there are many study supports and strict,thorough production technics for Hecolin(?),the quality analysis system and stability assessment need to be developped further.Comparing to the biophysical and biochemical analytical methods,immunological analytical methods for vaccines offer potency markers for a vaccine.Mouse potency assay as an immunological analytical method is the gold standard for vaccine potency analysis.But in vitro immmochemical analysis for vaccine quality had gradually replaced part of animal assays in recent years due to advantages such as low cost,better precision and quick turn around time.The correlation between IVRP and animal based assay/ED50 had been verified on several VLP based vaccines such as HPV vaccine Gardasil(?).Analysis of critical immunodominant epitopes and neutralizing epitopes is essential as the in vitro assessment of the intended function(immunogenicity)of recombinant VLP based vaccines.Thus,with correlation established,in vitro relative potency assay(IVRP)based on in vitro immunochemical analysis can be used for process control,vaccine batch release and vaccine stability assessment.The objective of this study was to establish the in vitro immunochemical quality analysis system for HEV vaccine and to apply these assays in stability assessment of the vaccine or vaccine antigen.The utilization of multiple antibodies,recognizing different epitopes,in in vitro immunochemical quality analysis system can comprehensively monitor the conformation and activity of recombinant VLP based HEV vaccines from multiple facets.Firstly,we clustered the 27 mAbs anti-p239 mAbs based on their cross blocking profiles.The mAbs were clustered into six categories A1-A6 according to the different epitope regions.The properties of mAbs,i.e.,binding affinity to p239,degree of sensitivity to conformation were quantitated.Then 9 representative mAbs from five categories were selected for immunochemical analysis for HEV vaccine quality according to their property including serum blocking capability,binding affinity to p239,sensitivity to p239 conformation,etc.Secondly,immunochemical system for HEV vaccine quality was established with these 9 mouse mAbs.The system consisted of three assays:Surface plasmon resonance(SPR)based on label-free binding analysis,competitve ELISA system(rIC50)based on "in solution" antigen comformation detection,and a sandwich ELISA system(rEC50)based on the mAb 8C11’s critical epitope detection.In IVRP or rEC50 method,we applied data processing model of "Parallel Line Analysis"which included the procedures of raw data quality control and effectively fitting.It ensured the reproducibility of tests and the reliability of results.These three immunochemical assays involved six mAbs,five mAbs,and two mAbs,respectively.These methods were run with six lots of p239.The reproducibility of these three methods were assessed and the batch consistency were demonstrated.These methods can also be applied in "comparability exercise" during process scale up or in development of QbD(Quanlity by Design)system for HEV vaccine.Finally,in vitro immunochemical assays for HEV vaccine quality with multiple epitope detection was applied to the stability studies of Hecolin(?)and its antigen p239.Stability of p239 under the conditions of different tempreture,pH,salt concentration and additives were assessed.The immunochemical assays was also compared with traditional assays such as SDS-PAGE,SEC-HPLC,DSC,etc.Reasonable agreement was observed the results from these different methods.Heat stress study of HEV vaccine provided information of the vaccine stability under various conditions.Specifically,improved vaccine stability was shown when adsorbed onto aluminum based adjuvants.Systematic immunoassays and the stability imformation of HEV vaccine provided a strong technical assurance and data support for HEV future development such as replacing mouse potency assay with IVRP,process scale up to support market demand,and development of combination vaccines.In conclution,in vitro immunochemical analytical system for HEV vaccine quality was developped in this study and it was preliminary applied to the stability assessments of Hecolin(?)and its antigen p239.The system laid the foundation of IVRP for HEV vaccine.Comparing to animal based potency assays or the clinical trials in the late product stages of HEV vaccine,IVRP based on this system could contribute to reduce testing costs and analyzing time.It also offered a reference model to the relative potency assessments of other recombinant VLP based vaccines.