Study On Assembly Mechanism And Broad-spectrum Immunogenicity Of Human Papillomavirus Type-hybrid Virus Like Particles

Globally,Human papillomavirus(HPV)accounts for 5%of all cancers in humans,as they are also putatively involved in cancers of cervix,vagina,and penis.Approximately 560,000 new cancer cases worldwide and 100,000 in China are attributed to HPV infection annually.Vaccines protecting women from infection with high-risk HP Vs can reduce prevalence of cancers.It is known that there are at least 15 types of "high risk" HPV.There are two vaccines available in the market.They all contain HPV16 and 18 virus-like particles(VLPs),which were produced by self-assembly of the major capsid protein L1 of each HPV type,successfully induced type-specific neutralizing antibodies in the recipients of the large scale clinical trials and have been approved by several countries.Although HPV 16 and HPV 18 account for approximately 60%to 70%of cervical cancer,the remaining major issue to be addressed is how to prevent infection with other high-risk HPVs.Multivalent HPV vaccine which contains all high-risk HP Vs VLPs is a big chanellge to purification process.So a new type hybrid VLPs which has several type-common neutralization epitopes will make it ideal for protecting human from mutli high-risk HPV infection.In order to understand the mechanism of VLP assembly and to idnentify the corss-neutralizing epitope of L1,the assembly process were optimized.And the hybrids VLPs were well characterized.The possibility of the hybrid VLPs to be one of the next-generation vaccine candidates for a broad spectrum was evalutated.In this study,the different types of HPV L1 proteins with C175 and C428 mutants which couldn't assemble into VLPs were prepared.It was comfirmed to be pentamer by TEM,HPSEC and AUC.But HPV L1 with C175 and C428 mutants can co-assemble to hybrid VLPs.HPV L1 C175A and ?C428 can assemble into much more homogeneous hybrid VLPs,pH and molecular ratio of HPV L1 protein is key for hybrid VLP assemble.Under the condition of pH 6.5 and molecular ratio of 1:1(n:n)the VLPs assemble efficiency is highest.It had been comfirmed that several e types of mutant HPV L1s derived from eukaryotic expression system can assembled to homogeneous type hybrid VLPs.And as same as wild type HPV pseudovirus,the hybrid VLPs with L2 protein can pack exogenous DNA and infect 293FT cell.Further,L2 peptides were surface display on highly ordered,self-assembled type hybrid VLP.Then,the type hybrid VLPs were confirmed to be homogeneous T = 7 icosahedral by CryoEM 3D reconstruction.The ritio of C175 and ?C428 in hybrid VLPs was confirmed to be 1:1(n:n).In this study,the thermal stability of hybrid VLP show Tn of hybrid VLP was between the pentamer and native VLP.In this study,the antigenicity and immunogenicity of hybrid VLP were compared with corresponding native VLPs.The result showed,most of conformational antibodies derived from native VLP had decreasing reactivity to hybrid VLP.And the hybrid VLPs can induce the same of IgG mAbs titer but much higher neutraling mAbs titer against other types.Based on all these results,a homogenious type hybrid VLPs with fixed ratio can be prepared with mutliple type,expression systems.and show their unique antigenicity and immunogenicity of the broad spectrum.All these work will pave the way for further design of new generation of HPV vaccine.