Study On HCV Pentavalent Recombinant Protein Vaccine And Construction Of Humanized Mouse Model

Liver diseases caused by hepatitis C virus(HCV)infection seriously threaten public health.Although the existing direct-acting antivirals have more than 95%response to chronic HCV infection,effective vaccines are irreplaceable means to completely eliminate HCV infection and reduce the burden of HCV related diseases.The high variation of HCV genome leads to the emergence of nearly 100 virus strains with different gene subtypes and serious immune escape.So far,no effective HCV vaccine has been successfully developed.Aiming at the difficulties in vaccine development and improving the antigen coverage of the vaccine,we designed and developed a pentavalent HCV E1E2 subunit vaccine simulating the natural conformation of HCV E1E2 to provide a new perspective.In addition,a humanized receptor mouse model of HCV infection was constructed to provide a model basis for the evaluation of HCV vaccine at the animal level.In this study,the full-length E1E2 fusion proteins of five HCV genotypes(H77-la,Con1-1b,J6-2a,S52-3a and HK6a-6a)were constructed and expressed by optimizing the synthesis.Immunofluorescence confocal and Western blotting analysis showed that E1 E2 fusion protein existed in the form of noncovalently linked heterodimer,with a predominantly a helical conformation by the CD spectra.At the same time,surface plasmon resonance(SPR),ELISA,flow cytometry(FACS),HCVcc(JFH1)blocking test and antibody binding test showed that E1E2 proteins had definite biological activity and antigenicity,indicating that the spatial conformation of E1E2 protein is folded correctly.The five genotypes of E1E2 proteins were equivalently mixed to form HCV E1E2 pentavalent protein vaccine.After immunization against BALB/c mice,the titer of antiserum can reach 106 and maintain for a long time.At the same time,the antiserum obtained mainly recognized the conformational epitope on E1E2 protein,and had broadspectrum cross-neutralizing activity against seven different genotypes of HCV viruses.In addition,ELISPOT results showed that the T cell activation signal of IL-4 and IFN-γsecretion were detected in splenocytes of E1 E2 protein immunized groups,indicating that E1E2 can cause antigen-specific T cell response.Because HCV infection is highly species-specific and its natural hosts are only humans and chimpanzees,experimental animals have become an important factor limiting research in the development and evaluation of new vaccines.In this study,HCV four receptor(CD81,SR-BI,CLDN1,OCLN)transgenic ICR mice(4hEFTg ICR mice)and STATI knockout ICR mice(STAT1-/-ICR mice)were prepared by microinjection with transgenic technology and CRISPR/Cas9 technology,respectively.The mouse model of HCV four receptor transgene combined with STAT1 knockout(4hEFTg STAT1-/-ICR mice)was obtained by reproductive methods such as hybridized and self-breed.HCV RNA can be detected in the blood of mice 3 months after HCV infection by tail vein injection,indicating that HCV viremia can be maintained for at least 3 months.In conclusion,we purified five genotypes of E1 E2 protein.Compared with monovalent E1E2 protein,pentavalent EIE2 protein has more immune advantages in cross-neutralizing antibody reaction and T cell response induction.It can be further explored as a candidate for HCV vaccine and provide experimental data reference for HCV vaccine research and development.In addition,the successfully constructed 4hEFTg STAT1-/-ICR mouse model provides a model basis for the study of HCV pathogenesis and the evaluation of HCV vaccine effect.