Cross Reactivity And Reactivity Frequency Of Mimotopes Of Hepatitis C Virus Hypervariable Region 1

The hepatitis C virus (HCV) is a positive-strand RNA virus that belongs to the genus Hepacivirus in the family Flaviviridae. HCV is the major etiologic agent of transfusion-associated hepatitis and is associated with a chronic infection that leads to liver cirrhosis and hepatocellular carcinoma. It is estimated that there are over 170 million HCV infected individuals worldwide and about 40 million in China. The combination of pegylated interferon with ribavirin is a normal therapeutic strategy which is costly, carries the risk of significant side effects, and shows variable therapeutic effect for diversity of HCV genetype. The research of HCV vaccine is still in progress, and the screening of effective epitopes is becoming more and more important for developing HCV prevential and therapeutic HCV vaccine.The HCV E2 protein contains some principal neutralizing epitopes, which can induce protective antibodies. The hypervariable region 1(HVR1) of HCV , a 27 amino acid segment located at the N- terminus of the E2 protein. It has been reported that the antibodies induced by HVR1 can block the infection of HCV in vivo and in chimpanzee, which imply that HVR1 could be a suitable target as antigen. Although the extreme variability of HVR1 makes the antibody fail to generally protect previously, the recent researchs show that some HVR1 epitopes have high cross reactivity, which brings hope for HCV vaccines development.Research shows that, mice which were immunized by amino acid sequence different from national can induce antibady reacting with heterologous strains. The epitopes could analogue different HVR1 are worth studying.1. Expression and purification of the HVR1 mimotope proteins and detection of the cross reactivity of the proteins and HCV positive sera.12 antigenic mimotope of HCV HVR1 region were selected based on literitures. The 12 mimotope DNA fragments fused with the Trx gene were cloned into a prokaryotic expression vector pET32a respectively. The 12 recombianted vectors (pET32a-Trx-p)were proved to be coincident with expection by restriction enzyme analysis and DNA sequencing. The constructs directed high levels of expression of these fusion proteins in E. coli BL21(DE3).By SDS-PAGE, the molecular weight of these proteins was around 14-16KD. After purification by the metal-affinity chromatography on nickel-nitrilotriacetic acid (Ni-NTA)agarose, the 12 fused Trx-P mimotopes were used for immunization of BALB/c mice. At the same time, the thioredoxin expressed by empty pET32a vector was purified and immuned the BALB/c mice as a control.The cross-reaction of these momitope proteins were detected with human HCV positive sera by ELISA. The momitope proteins could react with most of the sera. The reaction rates were 46.7%(P1),43.3%(P2),66.7%(P3),56.7%(P4),70%(P5),70%(P6),53.3%(P7),56.7%(P8),70%(P9),60%(P10),60%(P11),53.3%(P12). The results showed that the combination of the 12 momitope proteins have positive reaction with 29 samples of 30 sera and the same result was observed when combining the mimotope pipetides P5,P6,P7 and P9. The fusion proteins P5,P6 and P9 were able to react with 21 samples of 30 HCV positive sera, achieving 70% reaction rate,which is much higher than another 9 proteins.2. Immunnization by fused momitope proteins, analysis of reactivity frequence of the immuned mouse sera and synthesized HVR1 momitopes proteins, or fused momitope proteins.BALB/c mice were injected with the fused mimotope pipetides and thioredoxin emulsified in Freund,s complete adjuvan(tCFA)or incomplete Freund,s adjuvan(tIFA) with a routine immunization , which contains 1 time multi-point subcutaneous injection on abdo minal part with 2 weeks interval, 2 times totally. The immuned mouses were taken blood from bulbus oculi at the week 8 and the sera were preserved at 4℃.The antibody of the immuned mice elicited by the 12 pipetides respectively were evaluated by Western blotting. The collected mice sera were able to react with coresponding pipetides, which showed that the sera antibodies response of these pipetides was effectively induced in BALB/c mice. The ELISA results confirmed that the P5 pipetide has the highest positive reaction rate,and then the reaction of the synthesized P5 momitope protein with the sera antibodies of the immuned mouse by other momitope perteins was analyzed . The cross-reaction of P5 was detected with the antibodies of other 11 momitope proteins, but not in control.The mouse sera immuned by the 12 momitope proteins could react with the synthesized J4 and H77 HVR1 pipetides, which indicated that the 12 pipetides could efficiently elicit antibodies which can neutralize different HCV strains.3. Neutralizing experiment of HCV pseudotype virusThe mixture of pcDNA-HCVE1E2,pCMV-gag-pol and pCMV-GFP was transfected into the HEK293T cells to package HCV pseudotype virus(HCVpp), then the supernatant was taken to infect the human hepatoma cells Huh7.5. 52 h after infection, the GFP fluorescence was observed under a fluorescence microscope.The Huh 7.5 coinfected supernatant of the HEK293T cells transfected with three vectors could be viewed the generation of green fluorescence, which indicated the exact package of HCVpp was generated in the supernatant of HEK293T. When the Huh7.5 cell was treated with the supernatant contained HCVpp and the mice sera immuned by P5 and P6 mimotope proteins, the fluorescence signal decreased dramaticaly. The fluorescence of the Huh7.5 treated by supernatant contained HCVpp and the Trx immune mouse sera was not impacted. This result showed that the sera of the BALB/c mouse immuned by these mimotope proteins contain antibodies to the HCV E1E2 protein which were able to inhibit the infection of HCVpp to Huh7.5.Conclusion: 12 HCV HVR1 mimotope proteins were used in this study. They could be specially recoganized by majority HCV positive sera. The mice immuned by the mimotope proteins can generate the antibodies which were able to cross-react to different HVR1 regions, which present good neutralizing effect to HCVpp. The study indicates that these mimotopes may be of potential use in the development of HCV vaccines.