The Research Of Human Papillomavirus Type 16/35/31 Cross-genotype Vaccine

5%of new cancer cases in the world each year caused by Human papillomavirus(HPV)(including cervical cancer,vaginal cancer in woman,and penile cancer in man),of which cervical cancer cases occupy the second highest rate of female malignancy.In China,nearly 100,000 women are diagnosed with cervical cancer every year,and the patients are getting younger and younger.Of these,99.7%were caused by HPV.Thus,research and development of HPV vaccine has been a hot issue in the world.Currently,cervical cancer vaccines on the market include Merck’s Gardasil(?),GSK’s Cervarix(?)and Merck’s Gardasil(?)9.The main components of all the three vaccines are virus-like particles(VLPs)of HPV.Studies have shown that VLPs can self-assemble in vitro under certain conditions.They have the similar conformations and nearly the same neutralizing epitopes as natural virions.After vaccination,they can stimulate the corresponding immune response in vivo and prevent the invasion of HPV.VLPs which do not package genetic material DNA and can’t replicate.It has been the mainstream form of HPV vaccines.However,the eukaryotic expression system brings high cost,complicated production process,and more adverse reactions with the increase of antigen types and dose.There is an urgent need for a kind of HPV vaccine with low cost,high safety factor,and a broad spectrum of protection.In this study,withthe E.coli expression system,we constructed chimeric VLPs by substitutingthe loops between genetically close HPV types.Then,identified characterizations of the recombinant protein and evaluated their immunogenicity,of which chimeric VLPs could elicit cross-protection were screened out.It will provide guidance for the development of broad-spectrum vaccines in future.Firstly,in this study,three types HPV 16,3 5 and 31 with close genetic relationship were used as research objects,The expression vectors of chimeric L1 proteins were successively constructed by the substitution of BC,DE,EF,FG and HI loops,between HPV16,35,and among HPV16,35,31.The expression was conducted with E.coli expression is established in our laboratory and two-step chromatography was used to obtain the mutant L1 protein,which was assembled into VLPs in the assembly solution.Secondly,we performed related property analysis on these recombinant VLPs.Observed under the negativestaining electron microscope,The VLPs were particles with diameter about 55 nm.The results of HPSEC and AUC analysis showed that the purity of most recombinant VLPs can reach more than 90%and the form were uniform.The results of DSC experiments also showed that the thermostability of mutant VLPs was not significantly different from wild-type.Finally,the antigenic and immunogenicity of mutant VLPs was analyzed.The indirect method of ELISA was used and we find that the binding abilityto antibodies of some mutant VLPs with certainloop substitutedchanged significantlyith the antibodies changed significantly.The immunogenicity of mutant VLPs was evaluated systematically.H16-35FG-31EF,H35-16FG-31BC with the best cross-protective effect were screened out as vaccine candidates.In summary,in this study,the recombinant E.coli expression system was used to construct the recombinant HPV16/35/31 loop-substituted VLPs.The morphology and various properties of wild-type HPV were similar with wild type.And the best cross-protective protein was screened by immunogenicity evaluation.It is expected that the cross-genotype vaccine will be launched in the near future and contribute to human health in the basis of our research.