Preparation Of Chimeric Virus-like Particle Expressing HCV Neutralizing Epitopes And Preliminary Analysis Of The Neutralizing Antibody Induced In Immunized BLAB/c Mice

HCV (hepatitis C virus, HCV) infection is a major social and public health problemworldwide. Chronic HCV infection is associated with a high risk to end-stage liverdisease, cirrhosis and hepatocellular carcinoma. The treatment to HCV infection is limitedto using interferon (interferon, IFN) along, or combining with the broad-spectrum antiviraldrug ribavirin. However there are some side effect, and the responsiveness to IFN variedin different types of HCV infection. So far, there have not yet developed an effective HCVvaccine, and the most important reason is their high degree of genetic variability of HCV.Most of the variability came from the hypervariable region of the envelope protein genesand HVR1E2gene region. Series of antibodies can be induced after HCV infection. Butonly neutralizing antibody induced by the envelope protein can protect us from infection.Therefore screening of the E1/E2antigen epitopes, particularly the ones that can inducebroad-spectrum cross-neutralization of the antigen epitope maybe could effective solvethe problem. With the progress of research in recent years, the applications of HCV pseudo-virus particles (HCV pseudo-particle, HCVpp), HCV in vitro culture system(HCVcc) and the establishment of evaluation methods of neutralizing antibody,researchers have recognized on neutralizing antibodies in the body’s adaptive immunityeffect, especially in cross-protection functions. The research of HCV vaccine to inducecross-protective effect of broad-spectrum neutralizing antibodies will become the newstrategy.1. Construction of eukaryotic vector expressing chimeric HCV neutralizing epitopeswith HBV S gene and analysis of the expression in293T cellsObjective Construction of eukaryotic vector expressing chimeric HCV neutralizingepitopes with HBV S gene and analysis of the expression in293T cells. Methods Threeconservative HCV linear neutralizing epitopes in E1and E2region and a mimotopeagainst HVR1according to the literatures were selected. The amino acid sequence weredetermined as ITGHRMAWDMMMNWS，QLINTNGSWHIN，GVPTYSWGENET，ETYVSGGSAARNAYGLTSLFTVGPAQK. Age I was inserted between127and128amino acid sequence of HBV S gene amplified by PCR. By producing four downstreamforward primer of S gene contained different epitope sequences of the HCV epitopes, therecombinants of HBV S were constructed. Recombinant HBV S genes were subclonedinto pCI-neo and obtained recombinant eukaryotic expressing vector pCI-HBSEm（pCI-HBSE1-4）. Restrictive enzyme digestion analysis of recombinant vector confirmedthere had expected size of each band by agarose gel electrophoresis.293T cells were thentransfected with pCI-HBSEm for48hours. Results strong green fluorescence weredetected in the cytoplasm of293T cells transfected with four kinds of recombinantplasmids by IFA (indirect immunofluorescence assay), while the293T cells transfectedempty vector had no green fluorescence; The results of western blot showed lysates of293T cell transfected with Four kinds of recombinant plasmid could detected proteinbands at about27KD and the one with empty vector had not. Conclusion we successfullyconstruct eukaryotic expression vectors of HCV linear neutralizing epitope and HBV Schimeric gene, and the pCI-HBSEm can be expressed in293T cells. 2. Preparation and identification of chimeric HBV S antigen virus-like particleexpressing of HCV neutralizing antibody epitopesObjective Preparation and identification of chimeric HBV S antigen virus-like particleexpressing of HCV neutralizing antibody epitopes. Methods293T cells cultured inDMEM untill90%density were transfected with recombinant eukaryotic expressingvector pCI-HBSEm using lipofectamin transfection. VLPs (Viral-like particles) wereharvested from the supernatant after48hours. For the purification of VLPs, we prepared9layers gradient of density of20%-60%sucrose solutions for gradient centrifugation.Enriched VLPs will detected in some layer by measuring HBsAg by Electrochemicalluminescence assay. Then the collected fragments were dialyzed, concentrated bycentrifugal concentrator tubes, and identified by electron microscopy. The VLPs werequantified by measuring HBsAg, then compared with the commercialized hepatitis Bvaccine. Results The purified chimeric VLPs were confirmed by EM. Spherical particlesof about22nm coincide with sub-viral particles formed with HBsAg-S proteins. Thequantitative of HBsAg reached5.51×103ng/ml for greatest value, as well as theconcentration of hepatitis B vaccine was20×103ng/ml. Conclusion we successfullyprepared chimerized VLPs with HBV S and HCV linear neutralizing epitope for a highconcentration after purification and it was the basement for studying the neutralizingantibody induced by chimerized VLPs.3. Immunization of BLAB/C Mice by chimeric VLPs expressing HCV neutralizingepitopes and analysis of the induced neutralizing antibodyObjective Immunizing BLAB/C Mice by chimeric VLPs expressing HCV neutralizingepitopes and analyzing the induced neutralizing antibody. Methods42male BLAB/Cmice were randomly divided into seven groups for six per group. Injecting thecorresponding antigen/adjuvant mixture which were named VLPs-HBsE1、VLPs-HBsE2、VLPs-HBsE3、VLPs-HBsE4、VLPs-pools、VLPs-S and Adj. The first fourgroups of mice were injected with corresponding0.3ml500ng VLPs-SEm and0.3mladjuvant which mixed to0.6ml; The VLPs-pools group of mice injected0.3ml mixture offour kinds of VLPs of equal quantity and0.3ml adjuvant which mixed to0.6ml; The VLPs-HBs group were injected with0.3ml500ng VLPs-HBs and adjuvant which mixedto0.6ml; The Adj group injected0.6ml adjuvant to each one. We planed three timesimmunization and each interval was14days, with complete Freund’s adjuvant as theimmune adjuvant for the first time, and the last two times with incomplete Freund’sadjuvant. Collected100μl blood from tail vein of mice at the date of first immunizationand repeated every14days for six times. The serum were collected and stored at-20℃forneutralizing antibody detecting. Analysising antisera of mice by coating E1-E4peptideas antigens, four kinds of mixed antigens peptide and positive serum of anti-HBs byELISA.Measuring the OD values to reflect the production of corresponding neutralizingantibody. Results Each coated antigen reacted with corresponding antisera in immunizedmice. Conclusion It showed that chimerized virus-like particles can induce neutralizingantibodies in BALB/c mice, while assessment of the antibodies still needs in-depth study.