Study On Quantitation And Immunogenicity Of HPV16 L1 Protein

Cervical cancer is the fourth most common female malignancy worldwide.Persisting active high-risk human papillomavirus(HPV)infection is a prerequisite for development of cervical cancer in most cases.The HPV structure genes encode the major(L1)and minor(L2)capsid proteins that form the icosahedral capsid.The L1 protein self-assembles into virus-like particles(VLPs)with immunogenicity similar to infectious virions,which was the structural basis for the prophylactic vaccines.HPV parophylactic vaccine contains at least two types of Lls,and even more types of L1s,with HPV16 L1 being essential.Here we foused on the quantification and immunogenicity of HPV16 L1 antigen and obtained the following results through an in-depth research.1.Development of swandwich ELISA for the quantitative analysis of HPV16 L1 antigenCurrently three prophylactic vaccines are on the market in China,including Cervirax,Gardasil and Gardasil 9.Moreover,15 national HPV vaccines from 9 companies are at different stages of research and could hit the market in the near future.At this stage,each manufacturer uses its own sandwich ELISA to detect their HPV 16 LI antigen based on their monoclonal antibody,which makes the results uncomparable.So it is essential to screen a representative HPV16 type-specific monoclonal antibody to unify the ELISA quantification method.The neutralizing activities of 13 MAbs were assessed and HPV16.001 was found to have the highest neutralizing activity with an IC50 of 0.6ng/mL.Then four representative MAbs were chosen from the top six neutralizing MAbs for binding characterization analysis.HPV16.001 was found to have the highest binding affinity to HPV16 VLPs with an EC50 of 2.6ng/mL.And compared with other three MAbs,HPV16.001 had the same binding response curves with five antigens derived from three different expression systems.Through the competition studies,HPV16.001 was found to recognize the same epitope with most of the antibodies from the vaccinanted antisera,indicating it an immunodominant MAb.Then the quantitation method was beveloped based on HPV16.001 who recognized an immunodominant and neutralizing epitope of HPV16 L1 VLPs.Finally,parameters such as linear range,specificity,and precision were verified,which indicated that the sandwich ELISA established in this study was suitable for the quantitative detection of HPV16 L1 antigen,and could be used for the antigen identification and relative potency determination.2.Development of MS method for simultaneous quantification of major capsid L1 of HPV 16 and HPV18 in multivalent HPV vaccinesCurrently,HPV prophylactic vaccines are multivalent vaccines based on HPV L1 VLPs,from two-valent combination to 14-valent combination.Traditional protein quantification method such as the Smith Bicinchoninic Acid(BCA)method can only determine the total protein concentration without distinguishing the individual component.Although ELISA is routinely used for the identification and quantification of HPV type-specific VLPs,it has its limitations.Firstly,the ELISA assay relies on the availability of type-specific antibodies whose screening is laborious and time consuming.Secondly,HPV prophylactic vaccines contain adjuvants which could affect the quantification of ELISA assay.Thus,a MS method that can be used for simultaneous quantification of L1s of diffement genotypes in HPV prophylactic vaccines is urgently needed.Here,based on the strategy of stable isotope labeling method,considering the physical property and specificity of the tryptic peptide,AGAVGENVPDDLYIK and FSLDLDQYPLGR were respectively selected as the signature peptides of HPV16 L1 protein and HPV18 L1 protein.Heavy labeled isotopologs of the signature peptides were used as internal standards and simultaneous quantification of HP V16 L1 and HP V18 LI of the multivalent HPV vaccines was developed.After being treated with 0.1%RSF and trypsin,monovalent samples and multivalent vaccines were determined by the MS method.Notably,the internal standard method was used to calibrate the system for HPV L1 protein quantification.Their linear calibration curves were both obtained in the range of 20-500nmol/L(R2>0.990).The limits of detection(LOD)were as low as l.Onmol/L for HPV16 L1 and 0.5nmol/L for HPV18 L1,respectively.And the limits of quantitation(LOQ)were 2.8nmol/L for HPV 16 L1 and 1.7nmol/L for HPV18 L1,respectively.The intra-day accuracies were 83.96%?113.57%and 81.40%?100.43%,while the inter-day accuracies were 86.00%?100.20%and 87.10%?103.49%respectively.The intra-day precisions were 1.12%?4.65%and 0.79%?5.91%,while the inter-day precisions were 5.09%?10.59%and 4.17%?11.05%respectively.Specifically,using AGAVGENVPDDLYIK and FSLDLDQYPLGR as peptides for quantification of HPV16 and HPV18 L1 proteins in the six digest preparations,46.9±0.8?g and 17.2±0.28?g were determined for the Gardasil vaccines,while 51.2±2.0?g and 33.2±0.6?g were determined for the Gardasil 9 vaccines,both of which were generally in agreement with their label amounts.This method is superior to the current assays in terms of sensitivity,specificity,precision,accuracy and throughput;and could be used for simultaneous quantification of HPV16 and HPV 18 L1 proteins in multivalent vaccines.3.Characterization of broad-spectrum protective immunity of HPV16 vaccine strainWith the evolution of the HPV16,intra-genotype variants have accumulated over time.A collection of 1,204 naturally occurring HPV16 L1 protein sequences was chosen for study.Through analysis,270 of the 505 amino acid residues had natural mutations(0.08%-20.18%).Then whether the known MAbs or guinea pig sera immunized with HPV16 vaccine strain were able to neutralize these naturally occurring variants.To examine this question,constructs of 39 naturally occurring single amino acid substitutions in the L1 protein were generated by site-directed mutation.31 out of the 39 mutants of HPV16 L1 produced infectious PsVs that exhibited similar particle-to-infectivity ratios when compared to reference PsVs.Based on the paseudovirus based neutralization assay,we then assessed the ability of a set of known MAbs and guinea pig sera immunized with HPV16 vaccines to neutralize the infectivity of this series of PsVs.21 of the 31 PsVs-producing mutants showed different sensitivity to monoclonal antibodies(MAbs),with 20 mutants showed decreased sensitivity and 8 mutants displayed increased sensitivity.Notably,six mutants resulting in complete loss of reactivity to some of the tested MAbs.The vaccinated sera can neutralize all 31 variants,with the C428W and K430Q mutations displaying 9 and 11-fold lower susceptibilities to neutralization by the sera than the reference PsVs.The results suggest that the current HPV16 vaccine strain could induce broad-spectrum protective immunity against all the naturally occurring HPV16 variants discovered so far.However,the presence of serotypic divergence would suggest that individual genital HPV16 is undergoing antigenic drift that alters seroreactivity to the virions,which should be closely monitored.