Rational Design And Characterization Of A Tri-type Human Papillomavirus Vaccine By Chimeric Construct Of High-risk Type 53 And Low-risk Types 11/6

Human papillomavirus(HPV),a non-enveloped double-stranded DNA virus,which was highly relevant with proliferative lesions of mucosal or epithelial tissue and a variety of benign and malignant tumors.Currently,more than 200 types of HPV have been identified,but only 9 of them can be prevented effectively,remaining 18 high-risk types related to cancer are not covered by the marketed vaccine.Due to the strict type specificity,it is difficult to elicit cross-type protection between different HPV types,so the marketed HPV vaccines all used a mixture of multiple HPV virus-like particles(VLPs)to prevent different types of infections,the protection spectrum of which is hard to expand.Moreover,most vaccines are derived from eukaryotic expression systems,which is not conducive to popularize in developing countries due to its high cost.Therefore,it is urgent to develop a broad-spectrum vaccine that can achieve "one defense multiple" cross-protection with fewer immunogens.Based on the understanding of the structure of HPV L1,our group innovatively developed HPV "7 defense 20" cross-type vaccine,aiming to construct 7 immunogens that can achieve "one defense multiple" cross-protection through homologous loop swap to cover 20 important HPV types,and prevents more than 99%of cervical cancers.However,we have found that the homologous loop swap was not effective in some genetically close types.In addition,it is not clear how to break through the barriers between the genetically distant types,high-risk and low-risk types.In response to these problems and challenges,combined with the development needs of the HPV "7 defense 20" cross-type vaccine,this study focuses on breaking through the type specificity between the high-risk type HPV53 and the high-risk-genetically close types HPV56/66 or the low-risk-genetically distant types HPV 11/6,rationally design a cross-type vaccine that can achieve "one defense multiple" protection and overcome the difficulties in the research of HPV "7 defense 20" cross-type vaccine.Firstly,this study verified the feasibility and effectiveness of the homologous loop swap to induce cross protection between the high-risk and genetically close types of HPV56/66/53 based on the E.coli expression system.Through N-terminal truncation,HPV56/66/53 wild-type VLPs with good soluble expression,uniformity,thermal stability and immunogenicity were constructed.And then,20 of HPV53/56,HPV53/66 and HPV56/66 double-type chimeric VLPs were constructed through homologous loop swap,and fully characterized by TEM,HPSEC,AUC,DSC,DLS,etc.The immunogenicity evaluation was further completed on mice,and based on the pseudovirus-based neutralization assays,a double-type chimeric vaccine candidate antigen H66-56HI that can ideally achieve the "one defense two" cross protection was obtained,which can elicit neutralizing antibodies against HPV56 and HPV66 comparable to the wild type VLPs.Furthermore,we used the double-type chimeric VLPs obtained before as the backbone to construct HPV56/66/53 triple-type chimeric VLPs through single loop swap,double loop swap and codon optimization,the particle properties and immunogenicity of which have been evaluated further.Moreover,obtained a chimera H66-56HI-53DE that can elicit a certain degree of triple type crossprotection at 5 μg and 1 μg doses,and provided a route reference to overcome the type specificity between genetically distant types or high-risk and low-risk types.Secondly,this study broke through the type-specific barrier between the genetically distant high-risk type HPV53 and the low-risk types HPV11/6.Combining with the above experience,the H11-6HI obtained in the previous study was used as the backbone,and 10 of HPV11/6/53 triple-type chimera with large expression,high purity,good particle uniformity and thermal stability were constructed by using single-loop or double-loop swapped.The immunogenicity was also evaluated on mice,and finally the triple-type chimeric vaccine candidate antigen HI 1-6HI-53DE-FG that can achieve the ideal "one defense three" cross protection was abtained,which can elicit neutralizing antibodies against HPV11/6/53 comparable to the wild type VLPs.Finally,the cryo-EM structures of HPV56,HPV66,HPV53,H66-56HI,H6656HI-53DE and HI 1-6HI-53DE-FG were analyzed,and the results showed that the candidate antigens all exhibited a T=7 icosahedral structure.In summary,this study based on the E.coli expression system successfully broke through the type specificity between genetically close high-risk types HPV56/66/53 and genetically distant high or low risk types HPV11/6/53 by homologous loop swap.And constructed a double-type chimeric vaccine candidate antigen H66-56HI that can achieve "one defense two" cross protection and a triple-type chimeric vaccine candidate antigen HI 1-6HI-53DE-FG that can achieve "one defense three" cross protection,completely supplemented the candidate molecular library of HPV "7 defense 20" crosstype vaccine.Moreover,summarized a multi-epitope transplantation strategy that was not limited to genetic relationships and barriers between high and low-risk types,which more comprehensively covered the current needs for HPV broad-spectrum vaccine study,and provided new ideas for the design of broad-spectrum vaccines for other multigenotype viruses.