Inhibition Of Hepatitis C Virus Hypervariable Region 1 On Immunogenicity Of E2 Protein

1. Inhibition of hepatitis C virus hypervariable region 1 on immunogenicity of E2 proteinHepatitis C virus (HCV) is an enveloped, positive-sense RNA virus, which belongs to the family of Flaviviridae. The 9.6 kb genome encodes one large polyprotein that is processed by viral and cellular proteases to produce the viral structural proteins (core and glycoproteins E1 and E2) as well as nonstructural (NS) proteins (p7 through NS5B). Chronic liver disease caused by HCV is an important global healthy problem that currently affects 170 million people worldwide. A major impediment in HCV research and drug development has been the lack of culture systems supporting virus production.The current therapy for HCV infection is PegIFN-a in combination with rivavirin. Although the sustained viral response rate for HCV genotype 2 and 3 is more tha 75%, it is no more than 50%for HCV genotype 1, the most widespread type in the world. Since HCV genome was cloned, some European and American developed countries have been always trying to develop its vaccine, but until now, there is no effective vaccine available.HCV E2 protein can induce neutralization antibody, that can block infection of HCV. HCV E2 protein is the key antigen that induces neutralizing antibody. Although E2 protein is of high variety, serial conserved epitopes of E2 protein have been identified in recent years. Named as it's feature, hypervariable region 1 is a 27aa peptides in the N-terminus of E2 protein. HVR1 is required for HCV infection, and also, is an important target for neutralizing antibody. HVR1 is considered as an independent factor of HCV chronic infection. In early HCV infection, the anti-E2 antibody is mainly directed against HVR1. As the infection turns to be chronic, cross neutralizing antibody which is against conserved epitopes can be detected. In this study, soluble E2 and HVR1-deleting E2 expressing plasmids pCI-la661 and pCI-1a661Δwere constructed, and used to immunize mice to study the effect of HVR1 on the immunogenicity of HCV envelope 2 proteins.Methods:1. Soluble E2 and soluble HVR1-deleting E2-expressing plasmids were constructed; 2. HEK 293 T was transfected with pCI-1a661 and pCI-1a661Δplasmids. Two kinds of E2 proteins were detected with Western blotting, ELISA and immunofluorescent assay using conformation-dependent antibody.3. Binding activity of HVR1-deleting E2 protein with SRB1 and CD81 were assayed with flow cytometry.4. Mice were immunized with two kinds of plasmids.5. E2 and HVR1 antibodies were utilized in detection in ELISA and Immunofluorescent assay; 6. Different genotypes of HCVpp were used to determine the cross reaction of the immunized mouse sera; 7. Different genotypes of HCVpp were produced and neutralizing activity of mouse sera was assayed.Results and conclusion:1. E2 and HVR1-deleting E2 were detectable in transfection cells and cell culture supernatant. Deleting HVR1 didn't affect the conformation of HCV E2 protein; 2. Deleting HVR1 made E2 protein lose the binding ability with SR-B1 but enhance the binding ability with CD81.3. The antibody induced by pCI-1a661 was against both E2 and HVR1, though was mainly against HVR1; detecting mouse sera showed that the anti-HVRl-deleting E2 antibody induced by pCI-la661Δwas significantly stronger than that of pCI-1a661.4. Assays using different genotypes of E1E2 protein as a test angigen showed that lack of HVR1 could significantly improve the E2 protein-induced cross-neutralizing antibody.5. HCVpp neutralizing test showed that the neutralizing ability of sera from pCI-1a661-immunized mice was mainly dependent on anti-HVR1 antibody; deleting HVR1 can significantly improve E2 protein-induced cross-neutralizing antibody. These results indicate that:the immunogenicity of other epitopes of E2 protein were significantly inhibited by HVR1. This might be the reason why acute HCV patients are unlikely to generate cross-neutralizing antibodies. Lack of HVR1 can significantly increase the E2 protein-induced cross-neutralizing antibody, which may be of great significance in the development of HCV vaccine.2. Cloning and sequencing of HBV full-length S gene and genome from an HBsAg-negative/anti-HBs-positive patientThe HBV is an enveloped double-stranded DNA virus that is the prototype member of the Hepadnavidae. HBV infection may lead to severe sequelae such as acute hepatitis, chronic hepatitis, cirrhosis and hepatocellular carcinoma. It has been estimated that there are currently 350 million patients chronically infected with hepatitis B virus worldwide. There may be half of people in China are infected or have been infected by HBV. The number of carrier may be 120 million, which is a severe public health problem.HBV polymerase is a reverse transcriptase and lack of proofreading function, so HBV genome mutants frequently. HBV surface antigen(HBsAg) is the key protein that mediate the infection and induce neutralized antibody, although the HBsAg gene is hypervariable in HBV genome. HBV mutations are considered to be related with immune escape and nucleoside drug resistance. In this study, one patient with HBsAg negative, anti-HBsAg positive but HBV DNA positive was recruited. The S gene and whole genome were cloned. A lot of different mutants were found in S gene. Its significance deserves further research.Methods:1. S gene,including PreS and HBsAg gene, was cloned using PCR; 2. HBV genome was cloned using PCR; 3.Different PreS and S gene clones were sequenced and aligned.Results and conclusion:1. Seventeen whole S gene clones and eleven S gene clones were acquired; 2. The results showed that complex mutations were existed in whole S gene: deletion mutations in preSl 57-117 amino acids(61 aa) were detected which was reported in China and other countries. We also found that there was at least one stop codon in these HBsAg variants. Even a N-terminal stop codon was discovered in some of these variants. These kinds of variants were predominant (11/17). Four amino acids, NTTT, were inserted into 115 to 116 sites of HBsAg. Because of these deletion mutations, functional envelope protein could not be produced. S gene and HBV DNA polymerase gene are completely overlaps, but these variants were still predominant with mutated polymerase. The mechanism and significance were worth in-depth study. We still found the insertion of NTTT in HBsAg which has never been reported (also found in several HBsAg and HBV DNA positive patients). Whether this mutation was involved in immune evasion remains to be further studied.3. Seven HBV whole genomic clones were obtained.