| Hepatitis C virus (HCV), belonging to the family Flaviviridae, is a single-stranded RNA virus with enveloped virion. HCV is a major etiological agent of transfusion-associated hepatitis and chronic liver disease worldwide. HCV has infected an estimated 170 million people all over the world, and each year 3 million new patients infected with HCV are diagnosed. It is currently believed that more than 70% of HCV-infected individuals will become chronic carriers and faced with high risk of developing cirrhosis and hepatocellular carcinomas (HCC). HCV infection is also closely associated with lymphoma, mixed cryoglobulinemia, glomerulonephritis, porphyria cutanea tarda, diabetes mellitus and so on.Since the adoption of blood screen for HCV in 1990s, the incidence of HCV infection has dropped dramatically. However, blood and blood products are still important ways of HCV transmission. Moreover, there are still many new cases of HCV infection due to unknown transmission ways every year. The key measure to control HCV infection is to develop an effective vaccine. Since HCV was first molecularly cloned in 1989, HCV vaccine has been a research focus in developed countries such as Europe, America and Japan, but up until now, there is no vaccine available to prevent HCV infection.Hypervariable region 1 (HVR1), the peptide consisting of 27aa peptide in the N-terminus of E2 protein, play an important role in HCV entry. The high variability of HVRl is considered to be the bottleneck for HCV vaccine development. However, in recent years, virus neutralization assays with the infectious HCV pseudotyped particles (HCVpp) and cell culture produced HCV (HCVcc) demonstrated that HVRl is not the only region containing neutralizing epitope in HCV E2 proteins, and some conserved conformation and linear neutralizing epitopes have been identified in other regions of E2 protein .Our previous studies found that HVR1 could efficiently induce neutralizing antibodies. In addition, the antibody responses of the conserved epitopes in E2 were significantly inhibited by HVR1.In this study, we further analyze the possible mechanisms of how HVR1 functions as immune bait through the immunogenicity and protein structure, and identify the key peptide in HVR1 that inhibits the immune responses of other epitiopes in E2. These results may provide new insight for understanding the mechanism of HCV immune evasion and propose a new idea for HCV vaccine research. 1. Identification of key peptide in HVR1 which suppresses the immunogenicity of conserved epitopes in E2 protein.Methods: 1. Construction of plasmids: based on H77 isolate, soluble HVR1-completely deleted or partially deleted E2-expressing plasmids were constructed, containing 77 (HVR1 not deleted), 77Δ(HVR1-comletely deleted), 77Δ1-12 (HVR1 1-12aa deleted), 77Δ13-27 (HVR1 13-27aa deleted), 77Δ16-24 (16-24aa deleted); 2. Expression of these recombinant E2 proteins were detected by Western blotting, ELISA and immunofluorescent assays; 3. Mice immunization: the above plasmids were injected into tibialis anterior muscles; 4. Detection antibodies level: anti-E2 and HVR1 antibodies and the responses of immune sera with known B cell epitopes (412, 522, and 432) were detected by ELISA and immunofluorescent assay.Results: HVR1 complete deletion or partial deletion did not affect expression, secretion, conformation of E2 proteins. HVR1 antibodies can be induced by immunization with containing HVR1 or 16-24aa HVR1 plasmids, meanwhile the immune responses of other epitopes in E2 were inhibited. Removal of HVR1 or just removal of HVR1 16-24aa not only aborted the inhibition function, but also enhanced responses with a variety of known epitopes.Conclusions: HVR1 16-24aa is the key peptide in HVR1 which suppressesthe immunogenicity of conserved epitopes in E2 protein. This region is also the only B cell epitope identified in our previous study.2.Influence of HCV hypervariable region 1 on immunogenicity of E2 proteinMethods: 1. Construction of plasmids: HVR1 located at N-terminal of E2 (77), HVR1 located at carboxyl-terminal of E2 (77Δ-HC), HVR1 located at both ends of E2 (77-HC); Connection HVR1 with HBsAg (S-HN); Replacement HVR1 epitope with HA epitope (77HA); 2. Mice were immunized with kinds of plasmids; 3. Anti-HVR1/HA antibodies and anti-E2Δ/HBsAg antibodies in mice immune sera were detected, meanwhile, the correlation between the various antibodies level were analyzed.Results: 1. Positions of HVR1 didn't affect the functions that HVR1 suppressed the immune responses of conserved epitopes in E2. 2. HVR1 inhibited the immune responses of HBsAg. 3. HA epitope inhibited immunogenicity of other epitopes in E2.Conclusions: HVR1 is a strong immune epitope, while the immunogenicity of other epitopes of E2 protein are easily subjected to high immunogenic epitopes, causing that HVR1 inhibits the immune responses of conserved epitopes, which may explain why it is difficult to induce cross-protective antibodies in acute HCV infection.3. Neutralization assays of immune sera.Methods: 1. Immunization New Zealand white rabbit with several representative plasmids (77, 77Δ, 77Δ13-27) by electroporation; 2. Purification of IgG antibodies; 3. Neutralization assays (HCVpp and HCVcc.)Results: 1. The antibodies induced by 77 were against both E2 and HVR1, mainly against HVR1; the E2Δantibodies induced by 77Δ,77Δ13-27 was significantly stronger than that of 77. 2. Lack of HVR1 or HVR1 13-27aa could significantly improve the E2 protein-induced cross-neutralizing antibodies. 3. Neutralizing tests showed that the neutralizing ability of 77-immunized sera was mainly dependent on anti-HVR1 antibody; deleting HVR1 or HVR1 13-27aa could significantly improve cross-neutralizing antibodies.Conclusions: Results of immunization New Zealand white rabbit were consistent with of immunization mice: the immunogenicity of other epitopes in E2 protein was significantly inhibited by HVR1. Lack of HVR1 or HVR1Δ13-27 can significantly increase the E2 protein-induced cross-neutralizing antibodies, which may be of great significance in the development of HCV vaccine.
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