Quantitative Characterization Of Recombinant VLP Epitope Integrity And Its Application In Vaccine Quality Analysis

Recombinant virus-like particles are bionanoparticles that faithfully mimic the surface features of native virions.VLP is highly ordered assembly comprised of one or more capsid proteins,without nucleic acids.The analysis of the integrity of the key epitopes on VLPs is important for process development and improvement.It is of great significance to VLP vaccine research and development during preclinical and clinical stage of vaccine development.First of all,this research adopts the conformation sensitivity(rEC50)and quantitative determination of single(EC50)affinity efficacy,mAb classification methods to anti-HBsAg,anti-HPV16,anti-HPV18 properties identification,obtained representative mAbs(42B6,5F11;1D12,8A9;3C3,13H12)for further assay development.The representative mAbs were analyzed with respect to their biological functions through functional assays such as the pseudovirion neutralization assay,competition pattern with human sera from natural HPV infected individuals,HPV type specificity analysis assay,etc.Secondly,this study used representative mAbs to develop quantitative analysis method for epitope integrity:established HBsAg VLP sandwich ELISA("mass ELISA" 42B6:Ag:42B6-HRP,"antigenicity ELISA" 42B6:Ag:5B11-HRP)with good assay precision(RSD%<10%);developed HPV16 sandwich ELISA(1D12:Ag:8A9-HRP)and HPV18 sandwich ELISA(13H12:Ag:3 C3-HRP)with good assay precision(RSD%<10%).Solution based competitive ELISA analysis method for HPV 16 with mAbs 1D12,8A9 and PD1 and for HPV18 with mAbs 13H12,3C3 and 11A9 were developed to support product comparability studies for process upgrade.Lastly,the quantitative analysis method for epitope integrity was established.A pair of sandwich ELISA assays("Mass ELISA" 42B6:Ag:42B6-HRP,"Antigenicity ELISA" 42B6:Ag:5F11-HRP)were used to quantitatively analyze the antigenicity changes due to redox treatment of HBsAg particles.Turbidity scanning method of thermal stability for HBsAg VLP with and without 10 mM DTT treatment was carried out.Results from these assays confirmed that the presence of the disulfide bonds was the key factor of HBsAg VLP antigenicity and particle thermal stability.Quantitative analysis of HPV16 VLP,HPV18 VLP key epitope loss upon thimerosal treatment with sandwich ELISA and competitive ELISA methods was performed.Turbidity scan and DSC methods were used to assess thermal stability of HPV16 and HVP18 VLPs with and without thimerosal(0.01%)treatment.It was shown that thimerosal treatment,likely through specific interaction with free thiols,induced a decline in the thermal stability of VLPs.While epitope integrity is critical,antigen thermal stability is also required for an efficacious vaccine.In summary,a combination of different methods for VLP epitope were developed and used for quantitative analysis on recombinant VLPs-HBsAg,HPV16 and HPV 18 L1-based VLPs.In depth characterization of a panel of mouse mAbs for each of these three VLP antigens,including affinity ranking,degree of conformational sensitivity through intentional disruption of antigen conformation.More importantly,the functional assays such as viral neutralization assays were carried out,to confirm their validity as an antigenicity and immunogenicity marker,for the elite mAbs chosen for future quality analysis.Different immunochemical methods,developed based on these mAbs,offer valuable toolbox for process control and for product comparability when a process upgrade becomes necessary.