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Mechanisms of Inhibiting Immunogenicity of Hepatitis C Virus Envelope Protein 2 by Hypervariable Region 1

Author: LiuXiaoQing
Tutor: QiZhongTian
School: Second Military Medical University
Course: Microbiology
Keywords: Hepatitis C virus Hypervariable region 1 E2 protein DNA immunization Immunogenicity Cross - neutralizing antibodies
CLC: R392
Type: Master's thesis
Year: 2011
Downloads: 31
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Abstract


One of the major causative agent of hepatitis C virus (Hepatitis C Virus, HCV) is a single-stranded RNA virus, a member of the Flaviviridae is transmitted through blood and cause acute and chronic hepatitis. Currently there are around 170 million people infected with HCV, about three million cases of new infections each year. HCV chronic infection rates as high as more than 70% of HCV chronic infection in the developed countries is the main cause of end-stage liver disease such as cirrhosis, hepatocellular carcinoma. HCV infection with a variety of extrahepatic disease, such as lymphoma, Mixed cryoglobulinemia, glomerulonephritis, delayed cutaneous porphyria, and diabetes are closely related. Worldwide cases of hepatitis C have been infected by blood and blood products ways compared to previous nineties of the last century have dropped significantly, but blood and blood products in developing countries is still an important way to spread HCV. In addition, Sporadic unknown route of transmission of hepatitis C each year new cases worldwide remain high. The key measures to control HCV infection also depends on the development of an effective vaccine. Hepatitis C vaccine research in Europe and the United States and Japan and other developed countries since the HCV genome was cloned in 1989, has been a research focus, but so far no effective vaccine will be available. HCV envelope E2 protein is mediated by HCV cell invasion of key proteins, the amino terminal 27 amino acids of the protein is a region first identified containing neutralizing antibody epitope, and its hypervariable sequence called hypervariable District 1 (the hypervariable Region 1, HVR1), the height variation of HVRl was considered to curb the problem of hepatitis C vaccine development bottleneck. However, in recent years, based on the infection leave HCV particles (HCVpp) and HCV (HCVcc) in cell culture to produce and test showed that, HVRl not only neutralizing antibody epitopes Area, in the E2 protein of HCV envelope There is a the conservative spatial conformation and linear medium and antibody epitopes. Our previous study HVR1 immune properties: HVR1 has a strong immunogenicity, efficient induction of neutralizing antibodies, and simultaneously, HVR1 very significantly suppressed the E2 protein of the HCV envelope conserved epitope antibody response. In the present study, we further immunogenicity and protein structure analysis of the possible mechanism of HVR1 immune bait and identified one of the key amino acid sequence to provide new information for understanding HCV immune escape and HCV cell invasion mechanisms for HCV vaccine study new ideas. , HVR1 inhibition of E2 protein immunogenic peptides identified key methods: 1, the plasmid construct: genotype 1a H77 strain of HCV envelope protein gene as a prototype constructed HVR1 part of amino acid residues missing various secretory E2 protein expression plasmid, including the deletion of the entire HVR1 (77Δ), deletion HVR1 1-12 amino acid residues (77Δ1-12), missing the first 13-27 amino acid residues (77Δ13-27), deletion 16-24 The amino acid residues (77Δ16-24) containing the complete HVR1 (77) of the five kinds of plasmid. E2 protein detection: to detect the expression and secretion of various E2 protein by ELISA, Western blot and immunofluorescence. 3, DNA immunization: the plasmid intramuscular immunization of Balb / C mice. 4, antibody testing: detection by ELISA and immunofluorescence the mouse serum E2Δ antibody, HVR1 antibodies, as well as three known B cell epitopes (412,522,432) reaction. Results: missing HVR1 or which part of the residue of the E2 protein expression, secretion and conformation. Containing HVR1, or HVR1 first 16-24 bit amino plasmid can be immunized mice induced HVR1 antibodies, while suppressing the antibody response to other epitopes in the E2 protein, and removing the first 16-24 amino acids to eliminate this inhibition. Conclusion: 16-24 amino acids of the hypervariable region 1 for the inhibition of E2 protein immunogenicity critical peptides, the peptides are only B-cell epitope in our previous identified of H77 strain HVR1 in. HVR1 inhibiting HCV envelope E2 protein immunogenic mechanisms research methods: 1, to construct plasmid: HVR1 shift to the E2 protein carboxy terminal (77Δ-HC), increase in the E2 carboxy terminal the HVR1 peptide fragment (77-HC) ; the the HVR1 and HBsAg series (S-HN); HVR1 replace other epitope HA epitope (77HA). 2, the respective recombinant plasmid immunized mice. 3, detecting the generated mice HVR1 antibodies HA antibody, E2 antibody and HBsAg antibody, the correlation between the analysis of a variety of antibody levels. Results: 1 HVR1 positioning does not affect the inhibitory effect of E2 immunogenicity. 2 HVR1 inhibit HBsAg antibody response induced by. 3, HA epitope but also can inhibit the immunogenicity of the E2. Conclusion: HVR1 immunodominant epitope can inhibit the immunogenicity of HBsAg; E2 proteins of other regions the immunogenicity susceptible HVR1, and the impact of the HA epitope, resulting in reduced ability to induce antibodies. Serum virus in research methods: 1, some representative plasmid (77,77 Δ 77Δ13-27) with intramuscular the joint electrical pulses immune New Zealand white rabbits. 2, detection of rabbit serum antibody. 3, purified rabbit serum IgG, analysis of the neutralizing activity of IgG HCVpp and HCVcc. Results: 1, plasmid 77 can induce E2 antibody, but the dominant HVR1 antibodies; to E2Δ as a rabbit serum antigen detection, plasmid 77Δ induced 77Δ13-27 antibody was significantly stronger than the 77; 2, with different genotypes E1E2 antigen detection of serum antibodies in rabbits, indicating that deletion HVR1 can significantly improve the E2 protein induce cross neutralizing antibodies; HCVpp in and testing showed that 77 sera and the role is mainly dependent on the HVR1 antibodies, deletion of HVR1 or an HVR1 13 -27 amino acids can significantly promote the E2 induce cross neutralizing antibody. Conclusion: The rabbits immunized with the results obtained with mice immunized with the conclusion is the same, i.e. the HVR1 significantly inhibit the immunogenicity of other antibody epitopes in the E2 protein, deletion HVR1 or HVR1 13-27 amino acids of the E2 protein may Hepatitis C is an effective vaccine antigens, its development prospects worth the wait.

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