Structural basis for penetration of the glycan shield of hepatitis C virus E2 glycoprotein by a broadly neutralizing human antibody.

Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. A challenge for HCV vaccine development is to identify conserved epitopes able to elicit protective antibodies against this highly diverse virus. Glycan shielding is a mechanism by which HCV masks such epitopes on its E2 envelope glycoprotein. Antibodies to the E2 region comprising residues 412-423 (E2(412-423)) have broadly neutralizing activities. However, an adaptive mutation in this linear epitope, N417S, is associated with a glycosylation shift from Asn-417 to Asn-415 that enables HCV to escape neutralization by mAbs such as HCV1 and AP33. By contrast, the human mAb HC33.1 can neutralize virus bearing the N417S mutation. To understand how HC33.1 penetrates the glycan shield created by the glycosylation shift to Asn-415, we determined the structure of this broadly neutralizing mAb in complex with its E2(412-423) epitope to 2.0 Å resolution. The conformation of E2(412-423) bound to HC33.1 is distinct from the ß-hairpin conformation of this peptide bound to HCV1 or AP33, because of disruption of the ß-hairpin through interactions with the unusually long complementarity-determining region 3 of the HC33.1 heavy chain. Whereas Asn-415 is buried by HCV1 and AP33, it is solvent-exposed in the HC33.1-E2(412-423) complex, such that glycosylation of Asn-415 would not prevent antibody binding. Furthermore, our results highlight the structural flexibility of the E2(412-423) epitope, which may serve as an immune evasion strategy to impede induction of antibodies targeting this site by reducing its antigenicity.

Coupling to the surface of liposomes alters the immunogenicity of hepatitis C virus-derived peptides and confers sterile immunity.

We have previously demonstrated that antigens chemically coupled to the surface of liposomes consisting of unsaturated fatty acids were cross-presented by antigen presenting cells to cytotoxic T lymphocytes (CTLs). Liposomal form of immunodominant CTL epitope peptides derived from lymphocytic choriomeningitis virus exhibited highly efficient antiviral CTL responses in immunized mice. In this study, we coupled 15 highly conserved immunodominant CTL epitope peptides derived from hepatitis C virus (HCV) to the surface of liposomes. We also emulsified the peptides in incomplete Freund's adjuvant, and compared the immune responses of the two methods of presenting the peptides by cytotoxicity induction and interferon-gamma (IFN-γ) production by CD8(+) T cells of the immunized mice. We noticed significant variations of the immunogenicity of each peptide between the two antigen delivery systems. In addition, the immunogenicity profiles of the peptides were also different from those observed in the mice infected with recombinant adenoviruses expressing HCV proteins as previously reported. Induction of anti-viral immunity by liposomal peptides was tested by the challenge experiments using recombinant vaccinia viruses expressing corresponding HCV epitopes. One D(b)-restricted and three HLA-A(*)0201-restricted HCV CTL epitope peptides on the surface of liposomes were found to confer complete protection to immunized mice with establishment of long-term memory. Interestingly, their protective efficacy seemed to correlate with the induction of IFN-γ producing cells rather than the cytotoxicity induction suggesting that the immunized mice were protected through non-cytolytic mechanisms. Thus, these liposomal peptides might be useful as HCV vaccines not only for prevention but also for therapeutic use.

From Structural Studies to HCV Vaccine Design.

Hepatitis C virus (HCV) is a serious and growing public health problem despite recent developments of antiviral therapeutics. To achieve global elimination of HCV, an effective cross-genotype vaccine is needed. The failure of previous vaccination trials to elicit an effective cross-reactive immune response demands better vaccine antigens to induce a potent cross-neutralizing response to improve vaccine efficacy. HCV E1 and E2 envelope (Env) glycoproteins are the main targets for neutralizing antibodies (nAbs), which aid in HCV clearance and protection. Therefore, a molecular-level understanding of the nAb responses against HCV is imperative for the rational design of cross-genotype vaccine antigens. Here we summarize the recent advances in structural studies of HCV Env and Env-nAb complexes and how they improve our understanding of immune recognition of HCV. We review the structural data defining HCV neutralization epitopes and conformational plasticity of the Env proteins, and the knowledge applicable to rational vaccine design.

Structure-Based and Rational Design of a Hepatitis C Virus Vaccine.

A hepatitis C virus (HCV) vaccine is a critical yet unfulfilled step in addressing the global disease burden of HCV. While decades of research have led to numerous clinical and pre-clinical vaccine candidates, these efforts have been hindered by factors including HCV antigenic variability and immune evasion. Structure-based and rational vaccine design approaches have capitalized on insights regarding the immune response to HCV and the structures of antibody-bound envelope glycoproteins. Despite successes with other viruses, designing an immunogen based on HCV glycoproteins that can elicit broadly protective immunity against HCV infection is an ongoing challenge. Here, we describe HCV vaccine design approaches where immunogens were selected and optimized through analysis of available structures, identification of conserved epitopes targeted by neutralizing antibodies, or both. Several designs have elicited immune responses against HCV in vivo, revealing correlates of HCV antigen immunogenicity and breadth of induced responses. Recent studies have elucidated the functional, dynamic and immunological features of key regions of the viral envelope glycoproteins, which can inform next-generation immunogen design efforts. These insights and design strategies represent promising pathways to HCV vaccine development, which can be further informed by successful immunogen designs generated for other viruses.

Evaluation of hepatitis C virus core antigen assays in detecting recombinant viral antigens of various genotypes.

A single substitution within the hepatitis C virus core antigen sequence, A48T, which is observed in approximately 30% of individuals infected with genotype 2a virus, reduces the sensitivity of a commonly used chemiluminescence enzyme immunoassay. Quantitation of the antigen is improved by using a distinct anticore antibody with a different epitope.

Immunoreactivity of HCV/HBV epitopes displayed in an epitope-presenting system.

It has been demonstrated that the immunodominant region of the HCV core protein and the hepatitis B surface antigen (HBsAg) have high degree of reactivity. In order to construct a chimeric protein that carries HCV and HBV epitopes and possesses immunogenicity to both HCV and HBV, four epitopes derived from residues aa2-21 (epitope C1), aa22-40 (epitope C2) of the core protein, residues aa315-328 (epitope E) of E1 protein of HCV, and residues aa124-147 (epitope S) of HBsAg were chosen to be displayed in a conformation-specific manner on the outer surface of the Flock House virus capsid protein and expressed in E. coli cells. The reactivity of these epitopes with antisera from hepatitis C and hepatitis B patients and induction of immune response in guinea pigs were determined. The results showed that when displayed in this system, the chimeric protein carrying only epitope S could react with anti-HBsAg positive human sera, elicit an anti-HBsAg response in guinea pigs. The chimeric protein carrying epitopes C1, C2 and E could react with antibodies to different HCV genotypes, elicit an anti-HCV response in guinea pigs. The chimeric protein carrying epitopes C1, C2, E, and S could react with antibodies against HCV and HBV, elicit anti-HCV and anti-HBsAg responses in guinea pigs. The results suggested that these epitopes displayed in this form could be considered for development of epitope-based vaccines against HCV/HBV infections.

Mass spectrometric detection of the amino acid sequence polymorphism of the hepatitis C virus antigen.

A method for detection and identification of the hepatitis C virus antigen (HCVcoreAg) in human serum with consideration for possible amino acid substitutions is proposed. The method is based on a combination of biospecific capturing and concentrating of the target protein on the surface of the chip for atomic force microscope (AFM chip) with subsequent protein identification by tandem mass spectrometric (MS/MS) analysis. Biospecific AFM-capturing of viral particles containing HCVcoreAg from serum samples was performed by use of AFM chips with monoclonal antibodies (anti-HCVcore) covalently immobilized on the surface. Biospecific complexes were registered and counted by AFM. Further MS/MS analysis allowed to reliably identify the HCVcoreAg in the complexes formed on the AFM chip surface. Analysis of MS/MS spectra, with the account taken of the possible polymorphisms in the amino acid sequence of the HCVcoreAg, enabled us to increase the number of identified peptides.

Biophysical characterization of hepatitis C virus core protein: implications for interactions within the virus and host.

A primary function of the hepatitis C virus (HCV) core protein is to package the viral genome within a nucleocapsid. In addition, core protein has been shown to interact with more than a dozen cellular proteins, and these interactions have been suggested to play critical roles in HCV pathogenesis. A more complete knowledge of the biophysical properties of the core protein may help to clarify its role in HCV pathogenesis and nucleocapsid assembly and provide a basis for the development of novel anti-HCV therapies. Here we report that recombinant mature core protein exists as a large multimer in solution under physiological conditions. Far-UV circular dichroism (CD) experiments showed that the mature core protein contains stable secondary structure. Studies with truncated core protein demonstrated that the C-terminal region of the core protein is critical for its folding and oligomerization. Intrinsic fluorescence spectroscopy and near-UV CD analysis indicated that the tryptophan-rich region (residues 76-113) is largely solvent-exposed and not likely responsible for multimerization of the mature core protein in vitro.

Bacterial cell surface display for epitope mapping of hepatitis C virus core antigen.

Cell surface expression of protein has been widely used to display enzymes and antigens. Here we show that Pseudomonas syringae ice nucleation protein with a deletion of internal repeating domain (INC) can be used in Escherichia coli to display peptide in a conformationally active form on the outside of the folded protein by fusing to the C-terminus of INC. Diagnostic potential of this technology was demonstrated by effective mapping of antigenic epitopes derived from hepatitis C virus (HCV) core protein. Amino acids 1-38 and 26-53 of HCV core protein were found to react more sensitively in a native conformation with the HCV patient sera than commercial diagnostic antigen, c22p (amino acids 10-53) by display-ELISA. These results demonstrate that the bacterial cell surface display using INC is useful for peptide presentation and thus epitope mapping of antigen.

Chemical synthesis of NS-1 region of hepatitis C-viral polyprotein fragments: a comparison of PS-BDODMA resin and merrifield resin.

Three peptide fragments selected from the NS-1 region of hepatitis C-viral polyprotein (Leu-Ile-Asn-Thr-Asn-Ala-Ser-Trp-His-Ala-Asn-Arg-Thr-Ala-Leu-Ser Asn-Asp Ser-Lys Leu Asn Thr-Gly Ala NH(2), Leu-lle Asn Thr Asn Ala Ser-Trp-His-Ala-Asn-Arg-Thr Ala NH(2) and Leu-Asn-Cys(Acm)-Asn-Asp-Ser-Leu-Asn-Thr-Ala-NH(2)) have been synthesized on PS-BDODMA resin. The synthetic capability of the resin PS-BDODMA resin was compared with Merrifield resin. The peptides were synthesized by the stepwise fluoren-9-yl methoxycarbonyl (Fmoc) solid-phase method. The synthesized peptides were purified by HPLC and the identity of the peptides was established by mass spectrum and amino acid analysis. The synthesis of these peptides illustrates the application of the PS-BDODMA resin for the synthesis of long chain peptides in high yield and homogeneity compared to the Merrifield resin.

Purification of recombinant proteins with high isoelectric points.

The use of recombinant antigens is essential for the construction of robust and sensitive diagnostic assays. A critical step in the preparation of recombinant antigens is protein purification. Purification problems may be very different for related structural proteins expressed in the same host or for the same protein expressed in different hosts, because the biochemical characteristics of a recombinant protein, expressed in a heterologous system, are unique. In this chapter we make a brief introduction to protein purification procedures and we present a quick purification process suitable for the isolation of recombinant protein having high isoelectric points encoding non-conformational epitopes.

[Analysis of hepatitis C virus proteins based on amino acid sequence data and literature data]. / Analiz belkov virusa gepatita C na osnove aminokislotnykh posledovatel'nostei i literaturnykh dannykh.

To analyze the interrelationships between the amino acid sequences of the proteins of hepatitis C virus and the functional characteristics of different variants of this virus, a database of protein functional mapping of hepatitis C virus was developed. The database contains amino acid sequences (both full-size and fragmentary) retrieved from accessible databases and experimental data published in literature. The database also contains the results of comparison and treatment of primary data, including alignments and functional regions. On the basis of these data, variable and conservative regions of envelope proteins of hepatitis C virus were revealed. Antigenic and functional maps of structural and nonstructural proteins of the virus were constructed. The most variable region of the envelope protein E2 (HVR1) was analysed. It is assumed that the conservatism of some amino acid positions of HVR1 is related to the functions of this region.

Construction, expression, purification and biotin labeling of a single recombinant multi-epitope antigen for double-antigen sandwich ELISA to detect hepatitis C virus antibody.

Based on B cell epitope predictions, a recombinant antigen with multiple epitopes from four Hepatitis C Virus fragments (C, NS3, NS4 and NS5) were engineered. The recombinant gene was then highly expressed in E. coli. The non-modified and C-terminal-modified recombinant proteins were used for coating and biotin labeling, respectively, to establish the double-antigen sandwich ELISA. Ten positive reference samples confirmed by the CHIRON RIBA HCV 3.0 SIA kit were detected positive, Forty one plasma samples were positive among samples from 441 volunteers, which indicated that the recombinant antigen could readily react well with plasma HCV antibody. As critical reagents of double-antigen sandwich ELISA, the recombinant multi-epitope antigen and the C-terminal-modified and biotin-conjugated antigen show good antigenicity. In this study, we provide a simple approach to produce multiple epitopes within one recombinant protein in order to avoid the costly expression of less-effective pools of multiple proteins, which is the conventional strategy of diagnostic antigen production for HCV antibody detection.

Evaluation of core and NS4B synthetic peptide-based immunoassays for the detection of hepatitis C virus antibodies in clinical samples from Cameroon, Central Africa.

BACKGROUND: According to previous data, the antibodies produced during natural hepatitis C virus (HCV) infection frequently recognize amino acids 10-43 in the core protein and 1689-1740 or 1921-1940 in the non-structural 4B (NS4B) protein. The reactivity of these peptides with the corresponding antibodies has mainly been evaluated using serum samples from Western countries where HCV genotype 1 (HCV-1) is predominant, and no information is available concerning samples from sub-Saharan countries where high HCV variability has been reported. OBJECTIVE OF THIS STUDY: To evaluate the performance of HCV core and NS4B peptide-based immunoassays in the serodiagnosis of HCV infection in Cameroon subjects. STUDY DESIGN: Three core and four NS4B-based synthetic peptides derived from HCV genotypes 1b and 2a were designed and tested against a panel of 151 serum samples from Cameroon (40 positive for HCV-1, 32 for HCV-2, 39 HCV-4, and 40 HCV-negative). RESULTS: The three core peptides all demonstrated strong immunoreactivity, regardless of the HCV genotype from which they were derived, with greater than 90% and 92% sensitivity and specificity. In contrast, the NS4B-derived peptides exhibited lower sensitivities (24.3-65.8% depending on the HCV genotype) but higher specificities (100% for all four peptides tested). CONCLUSIONS: Our findings indicate that an HCV core peptide could be used for the diagnosis of chronic HCV infection. Among the NS4B peptides tested, a chimeric NS4B peptide encompassing both N- and C-terminal portions of the NS4B protein gave a much better performance than the two component N- and C-terminal peptides used individually.

Swerilactones A and B, anti-HBV new lactones from a tradtional Chinese herb: Swertia mileensis as a treatment for viral hepatitis.

Swerilactones A (1) and B (2), two novel lactones with an unprecedented 6/6/6/6/6 pentacyclic ring system, were isolated from the traditional Chinese herb of Swertia mileensis with activity against the hepatitis virus. Their structures and relative stereochemistry were elucidated based on spectroscopic methods and further confirmed by X-ray single crystal diffraction analysis. In vitro antihepatitis B virus (HBV) assay on Hep G 2.2.15 cell line showed that compound 1 inhibited HBsAg and HBeAg secretion with IC(50) values of 3.66 and 3.58 mM, respectively.

Antigen-specific proteolysis by hybrid antibodies containing promiscuous proteolytic light chains paired with an antigen-binding heavy chain.

The antigen recognition site of antibodies consists of the heavy and light chain variable domains (V(L) and V(H) domains). V(L) domains catalyze peptide bond hydrolysis independent of V(H) domains (Mei, S., Mody, B., Eklund, S. H., and Paul, S. (1991) J. Biol. Chem. 266, 15571-15574). V(H) domains bind antigens noncovalently independent of V(L) domains (Ward, E. S., Güssow, D., Griffiths, A. D., Jones, P. T., and Winter, G. (1989) Nature 341, 544-546). We describe specific hydrolysis of fusion proteins of the hepatitis C virus E2 protein with glutathione S-transferase (GST-E2) or FLAG peptide (FLAG-E2) by antibodies containing the V(H) domain of an anti-E2 IgG paired with promiscuously catalytic V(L) domains. The hybrid IgG hydrolyzed the E2 fusion proteins more rapidly than the unpaired light chain. An active site-directed inhibitor of serine proteases inhibited the proteolytic activity of the hybrid IgG, indicating a serine protease mechanism. The hybrid IgG displayed noncovalent E2 binding in enzyme-linked immunosorbent assay tests. Immunoblotting studies suggested hydrolysis of FLAG-E2 at a bond within E2 located approximately 11 kDa from the N terminus. GST-E2 was hydrolyzed by the hybrid IgG at bonds in the GST tag. The differing cleavage pattern of FLAG-E2 and GST-E2 can be explained by the split-site model of catalysis, in which conformational differences in the E2 fusion protein substrates position alternate peptide bonds in register with the antibody catalytic subsite despite a common noncovalent binding mechanism. These studies provide proof-of-principle that the catalytic activity of a light chain can be rendered antigen-specific by pairing with a noncovalently binding heavy chain subunit.

Establishment of hybrid cell lines producing monoclonal antibodies to a synthetic peptide from the E1 region of the hepatitis C virus.

We aimed at establishing hybridoma cells secreting monoclonal antibodies (mAbs) against E1 synthetic peptide of HCV. BALB/c mice were immunized with HCV E1-synthetic peptide (GHRMAWDMM) and its spleenocytes were fused with the P3NS1 myeloma cell line. Two highly reactive and specific mAbs (10C7 IgG2b mAb, and 10B2 IgG1 mAb) were generated. The target HCV E1 antigen was identified at approximately 38 kDa in serum of infected individuals. A newly developed ELISA detected the target antigen in 90% of sera from HCV RNA infected individuals with a specificity of 84%. So, the generated mAbs may provide promising probes for serodiagnosis of HCV infection.

Contribution of redox status to hepatitis C virus E2 envelope protein function and antigenicity.

Disulfide bonding contributes to the function and antigenicity of many viral envelope glycoproteins. We assessed here its significance for the hepatitis C virus E2 envelope protein and a counterpart deleted for hypervariable region-1 (HVR1). All 18 cysteine residues of the antigens were involved in disulfides. Chemical reduction of up to half of these disulfides was compatible with anti-E2 monoclonal antibody reaction, CD81 receptor binding, and viral entry, whereas complete reduction abrogated these properties. The addition of 5,5'-dithiobis-2-nitrobenzoic acid had no effect on viral entry. Thus, E2 function is only weakly dependent on its redox status, and cell entry does not require redox catalysts, in contrast to a number of enveloped viruses. Because E2 is a major neutralizing antibody target, we examined the effect of disulfide bonding on E2 antigenicity. We show that reduction of three disulfides, as well as deletion of HVR1, improved antibody binding for half of the patient sera tested, whereas it had no effect on the remainder. Small scale immunization of mice with reduced E2 antigens greatly improved serum reactivity with reduced forms of E2 when compared with immunization using native E2, whereas deletion of HVR1 only marginally affected the ability of the serum to bind the redox intermediates. Immunization with reduced E2 also showed an improved neutralizing antibody response, suggesting that potential epitopes are masked on the disulfide-bonded antigen and that mild reduction may increase the breadth of the antibody response. Although E2 function is surprisingly independent of its redox status, its disulfide bonds mask antigenic domains. E2 redox manipulation may contribute to improved vaccine design.

Hepatitis C virus epitope-specific neutralizing antibodies in Igs prepared from human plasma.

Neutralizing antibodies directed against hepatitis C virus (HCV) are present in Igs made from anti-HCV-positive plasma. However, these HCV-specific Igs are largely ineffective in vivo. The mechanism for the poor effectiveness is currently unknown. We hypothesize that the presence of nonneutralizing antibodies in HCV-specific Igs interferes with the function of neutralizing antibodies, resulting in the reduction or blockage of their effect. In the present study, we identified at least two epitopes at amino acid residues 412-419 (epitope I) and 434-446 (epitope II), located downstream of the hypervariable region I within the HCV E2 protein. We demonstrated that epitope I, but not epitope II, was implicated in HCV neutralization and that binding of a nonneutralizing antibody to epitope II completely disrupted virus neutralization mediated by antibody binding at epitope I. The dynamic interaction between nonneutralizing and neutralizing antibodies may thus play a key role in determining the outcomes of HCV infection. Further exploration of this interplay should lead to a better understanding of the mechanisms of neutralization and immune escape and may indicate pathways for the manufacture of an effective HCV-specific Ig product for immune prophylaxis of HCV infection.