| Hepatitis C virus (HCV) is an enveloped and single positive-stranded RNA virusbelonging to the Flaviviridae, and is a major cause of chronic liver disease. HCV infectionwill spontaneously clear in about15%of people, while80-85%of the infections transfer tochronic infection. People with HCV-related chronic hepatitis for more than20years havethe incidence of cirrhosis in10-15%; and1-7%of the patients with cirrhosis developedinto liver cancer. According to the last report of World Health Organization,130–150million people globally have chronic hepatitis C infection.350000to500000people dieeach year from hepatitis C-related liver diseases. The newly diagnosed patients infectedwith HCV in China are increasing annually:153,039people in2010,173,872people in2011,201,622people in2012, and223,094people in2013respectively. It is estimated thatthere are more than49million people with HCV infection in China.HCV RNA genome has been found for25years, which is in1989. During these25years, great progressed have been made in the molecular biology of HCV infection,immunity and pathogenesis, which lead to an outstanding achievement in somedirect-acting antiviral agents (DAAs). Oral anti-HCV drugs such as protease inhibitors (egBoceprevir and Telaprevir) and nucleotide NS5B polymerase inhibitors (eg Sofosbuvir)approved in US and Europe in recent years makes sustained virologic response rates (SVR)from40-50%to70-90%. And the treatment of a nucleotide polymerase inhibitor need notbe combined with interferon. However, these drugs in China and many other countrieshave not yet approved. In addition the effect of these drugs is also limited by HCVgenotypes. Moreover, the costs of these DAAs are too expensive. US FDA has approvedGilead’s$1,000-a-day hepatitis C pill, resulting in a total list price of$84,000for12weeks,according to Gilead spokeswoman Cara Miller. Moreover, resistance mutations against thevirus protease inhibitor drugs have been reported.The development of prophylactic vaccines for HCV infection has been of concern inEurope, Japan and other developed countries. There are more than ten kinds of vaccines,including prophylactic vaccines and therapeutic vaccines, approved for clinical trials, butthere is not vaccine available to use. The genome of HCV is of high variation. HCV isdivided into six or seven different genotypes and more subtypes. Like HIV, HCV geneticvariation is the main strategy to evade the host immune, which is also an important reasonfor the difficulties in developing a prophylactic vaccine. Although DAAs developed greatly in recent years, and there are always new DAAs in clinical trials showed good therapeuticeffect, the development of prophylactic vaccine for hepatitis C is still urgent, especially forthose with high-risks, such as practicing physicians, intravenous drug users, close contactsof patients with hepatitis C, gay, professional blood donors and regional clusters of HCVinfection and other populations.Envelope glycoproteins located at the surface of HCV particles, which mediated thecell entry of virus. Therefore HCV envelope proteins have become an important target forvaccine development. HCV envelope protein comprises an amino acid residue from aa192to aa746(amino acid sequence of genotype HCV-1a H77strain). Glycoprotein E1contains193amino acid residues from192to383, while glycoprotein E2comprises363amino acid residues from384to746. Studies found that glycoprotein E2contains threehypervariable regions, respectively HVR1(aa384-411), HVR2(aa460-485) and igVR (aa570-580). E2aa716-746is located at the C terminal as the transmembrane region.Receptor binding critical region of E2is located between aa384-661, called receptorbinding region (RBD). Disulfides (or cysteine residues) play a pivotal role in maintainingthe conformation of protein, the epitopes forming and in the membrane fusion. Theectodomain of HCV glycoprotein E2contains18cysteine residues, and highly conservedamong the seven genotypes, which suggested that disulfide bonds had an important role inthe structure and function of E2. In2011, Fraser et al studied HCV E1E2oxidized status,and found that free thiol is essential for HCV virus particle binding to the host cellreceptors. In2012, McCaffrey et al reported that cysteineresidue had an important role incell entry of HCVpp, CD81receptor binding and E1E2dimer formation. Fenouilletreported disulfide bonds obscures some epitopes of E2protein, and mice immunized withreduced E2protein can promote the induction of neutralizing antibodies. We can see thatdisulfide bonds played an important role in E2structure and function, however the role of18cysteine residues is still not clear, which is one of the aspects in this paper.It is clear now that neutralizing antibodies induced at the early stages of infectionpromote the virus removal process. However, most of the HCV infections developed tochronic infection, and the induced neutralizing antibody levels is only one factor in thisprocess. Current understanding of the characteristics of envelope protein neutralizingantibodies in chronic HCV patients is not clear. Concerning to the infection of HCV inChina, the study of envelope protein neutralizing antibodies in chronic HCV patients inChina will help to establish appropriate prevention measures and control strategies of HCV infection, and provided ideas for the development of preventive and therapeutic vaccineagainst HCV infection.1. Alanine scanning mutagenesis of hepatitis C virus E2cysteine residues: insightsinto E2biogenesis and antigenicityEnvelope glycoprotein2(E2) of hepatitis C virus plays an important role in mediatingviral cell entry and eliciting neutralizing antibodies. This protein contains18conservedcysteine (cys) residues in its ectodomain, which were predicted to form nine disulfidebonds. Although studies have shown that cysteines contributed to folding and function ofE2protein, significance of each cysteine regarding to function and antigenicity of de novosynthesized E2remains to be addressed. Here we performed a comprehensivecysteine-alanine (ala) mutagenesis scan of all18cysteine residues in full-length E1E2ofH77and Con-1strains. Results showed that all the individual cys-ala mutations did notaffect E2expression, but abrogated their corresponding pseudoparticles entry into Huh7cells. Six cysteine residues in H77E2(C494, C508, C552, C564, C607and C644) wereindispensable for recognition by two conformation-dependent anti-E2mAbs H53and H48,and two additional cysteine residues (C429and C503) were required for H48reactivity.Removal of any of these six cysteine residues notably decreased E1E2transmembranetransport and abolished E1E2incorporation into retroviral pseudotyped particles. Exceptfor C607, the cysteine residues required for H48reactivity were also required for E2binding to HCV receptor CD81. Any mutation of the eight cysteine residues required forH53or H48reactivity did not affect E2antigenicity and its competence of inducing HCVneutralizing antibodies in mice.Our research indicates that disulfide bonds play an important role in maintaining thenative conformation of E2. Six cysteine residues (cys494, cys508, cys552, cys564, cys607and cys644) are critical for E2conformation and functional maintenance. The findingsprovide new insight into HCV E2structure and have implications for the development ofHCV entry inhibitors and prophylactic vaccines.2. Characteristics of the envelope protein antibodies in chronic hepatitis C patients inChinaIn the present study, we firstly found that six individual mutation of cysteine in E2(C494A, C508A, C552A, C564A, C607A and C644A) abolished H77E1E2reactivity withsera from patients infected with genotype1a and1b. Further research found that Anti-E1 antibodies were absent in about85%of HCV patients’ sera, comparing that anti-E2antibodies were6.03%. It is reported that HCV-infected individuals can produce antibodiesthat recognize conserved conformational epitopes and inhibit the binding of HCV to CD81.To further clarify the E2antibodies profiles in chronic HCV patient, reactivity of HCVpatients’ sera with native and denatured E2protein were detected through ELISA. Resultsshowed that although E2antibodies were present in most of the HCV patients’ sera (94%),anti-E2linear antibodies were present in only about34%of the chronic HCV patients.Moreover, anti-E2linear antibodies titers were lower than that of conformation antibodies.The E2antigen we used in these assay is from H77strain of genotype1a. In order toconfirm the above results, E1E2genes were cloned from several chronic HCV patientsinfected with different genotype including1a,1b,3b and6n, and similar results wereobtained. C494A, a mutant affecting the global conformation of E2protein, abrogate E2binding to its autologous sera. Deletion of HVR1had a minimal effect on E2binding to itsautologous and heterologous sera. To further discover which regions of E2is critical for itsreactivity with patients’ sera, a serial of truncated E2plasmids (E2364-746,-716,-661,-644,-620,-607,-569,-564) were constructed. Those sera with anti-E2linear antibodieshad a relatively higher reactivity with truncated E2than sera absent in anti-E2linearantibodies, most of which were lost reactivity with truncated E2smaller than E2364-620.This indicates that E2conformational antibodies in patients’ sera strictly recognize a moreglobal conformational epitope of E2. Finally, the relationship between E2antibodies titersand liver injury were identified. Significant difference in GPT (p<0.001) and GOT (p=0.02)were discovered between group with higher titers of anti-E2antibodies and group withlower titers of anti-E2antibodies, while GGT (p=0.307), ALB (p=0.526), TBIL (p=0.134),HDL-c (p=0.664) were not. Significant difference in GPT (p<0.004) and GOT (p=0.021)were also discovered between group with anti-E2linear antibodies and group withoutanti-E2linear antibodies.There are two types of E2antigen in HCV patients: E2protein on the surface of thevirus particle and free E2proteins. Free E2proteins may release from the injured live cellsdue to liver cells hepatitis. The free E2proteins may expose more epitopes, and includedhigh levels of conformational epitopes and linear epitopes antibodies. 3. Cross-neutralizing activity of serum IgG from different genotypes of hepatitis CinfectionTo further explore whether these E2linear or conformational antibodies targeted toneutralize epitopes of E2, a G530A/D535A plasmid were constructed and its reactivitywith HCV patients’ sera were detected. Most of the sera with anti-E2linear antibodies hadsimilar level of reactivity with G530A/D535A mutant E2comparing to WT E2, while thesera absent in anti-E2linear antibodies had a remarkable decrease in reactivity withG530A/D535A mutant E2. Furthermore, the sera with anti-E2linear antibodies had arelatively higher reactivity with G530A/D535A mutant E2than sera absent in anti-E2linear antibodies.In order to detect whether these antibodies are capable of neutralizing HCV infection,purified IgG were prepared through affinity chromatograph from chronic hepatitis Cpatients’ sera. The envelope proteins antibodies in chronic HCV patients were individuallydifferent. Only40%of chronic HCV patients had anti-E2linear antibodies, and about60%of CHC patients showed no or very low titers of anti-E2linear antibodies. Moreoveranti-E1antibodies were rare in most of the CHC patients. However, IgG purified fromthese sera had a similar level of neutralizing activity. The neutralization assay showed thatserum IgG can be effectively neutralized HCV infection of genotype2a,4a and6a, whileshowed no or very low neutralizing activity to HCV infection in vitro of genotypes1a,3aand7a.To further monitor the cross neutralization activity of serum IgG from differentgenotypes of HCV patients, IgG were purified from1a,1b,2a,3a,3b and6n HCV patients,and neutralization captivity to seven genotypes HCVcc were performed using these IgG.The results showed that IgG from any patients were able to block the HCVcc infection ofgenotype2a,4a,6a, but had little effect on the infection of1a,3a, and7a HCVcc.The results of this paper deeps our understanding of hepatitis C envelope proteinstructure and immunogenicity, providing ideas and direction for the development of apreventive hepatitis C vaccine. Furthermore, the analysis of envelope protein antibodyneutralization activity in chronic HCV patients, promotes the establishment of a morerational HCV infection prevention and control strategies. |
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