In vitro selection of a neutralization-resistant hepatitis C virus escape mutant

Effective immunization against hepatitis C virus (HCV) infections is likely to require the induction of both robust T and B cell immunity. Although neutralizing antibodies may play an important role in control of infection, there is little understanding of the structure of the HCV envelope glycoproteins and how they interact with such antibodies. An additional challenge for vaccine design is the genetic diversity of HCV and the rapid evolution of viral quasispecies that escape antibody-mediated neutralization. We used a cell culture-infectious, chimeric HCV with the structural proteins of genotype 1a virus to identify envelope residues contributing to the epitope recognized by a broadly neutralizing, murine monoclonal antibody, AP33. By repetitive rounds of neutralization followed by amplification, we selected a population of viral escape mutants that resist stringent neutralization with AP33 and no longer bind the antibody. Two amino acid substitutions, widely separated in the linear sequence of the E2 envelope protein (N415Y and E655G), were identified by sequencing of cloned cDNA and shown by reverse genetics analysis to contribute jointly to the AP33 resistance phenotype. The N415Y mutation substantially lowered virus fitness, most likely because of a defect in viral entry, but did not reduce binding of soluble CD81 to immobilized HCV-pseudotyped retrovirus particles. The in vitro selection of an HCV escape mutant recapitulates the ongoing evolution of antigenic variants that contributes to viral persistence in humans and reveals information concerning the conformational structure of the AP33 epitope, its role in viral replication, and constraints on its molecular evolution.     

Structural basis of hepatitis C virus neutralization by broadly neutralizing antibody HCV1

Hepatitis C virus (HCV) infects more than 2% of the global population and is a leading cause of liver cirrhosis, hepatocellular carcinoma, and end-stage liver diseases. Circulating HCV is genetically diverse, and therefore a broadly effective vaccine must target conserved T- and B-cell epitopes of the virus. Human mAb HCV1 has broad neutralizing activity against HCV isolates from at least four major genotypes and protects in the chimpanzee model from primary HCV challenge. The antibody targets a conserved antigenic site (residues 412–423) on the virus E2 envelope glycoprotein. Two crystal structures of HCV1 Fab in complex with an epitope peptide at 1.8-Å resolution reveal that the epitope is a β-hairpin displaying a hydrophilic face and a hydrophobic face on opposing sides of the hairpin. The antibody predominantly interacts with E2 residues Leu⁴¹³ and Trp⁴²⁰ on the hydrophobic face of the epitope, thus providing an explanation for how HCV isolates bearing mutations at Asn⁴¹⁵ on the same binding face escape neutralization by this antibody. The results provide structural information for a neutralizing epitope on the HCV E2 glycoprotein and should help guide rational design of HCV immunogens to elicit similar broadly neutralizing antibodies through vaccination.     

Depletion of interfering antibodies in chronic hepatitis C patients and vaccinated chimpanzees reveals broad cross-genotype neutralizing activity

Using human immune globulins made from antihepatitis C virus (HCV)-positive plasma, we recently identified two antibody epitopes in the E2 protein at residues 412–426 (epitope I) and 434–446 (epitope II). Whereas epitope I is highly conserved among genotypes, epitope II varies. We discovered that epitope I was implicated in HCV neutralization whereas the binding of non-neutralizing antibody to epitope II disrupted virus neutralization mediated by antibody binding at epitope I. These findings suggested that, if this interfering mechanism operates in vivo during HCV infection, a neutralizing antibody against epitope I can be restrained by an interfering antibody, which may account for the persistence of HCV even in the presence of an abundance of neutralizing antibodies. We tested this hypothesis by affinity depletion and peptide-blocking of epitope-II-specific antibodies in plasma of a chronically HCV-infected patient and recombinant E1E2 vaccinated chimpanzees. We demonstrate that, by removing the restraints imposed by the interfering antibodies to epitope-II, neutralizing activity can be revealed in plasma that previously failed to neutralize viral stock in cell culture. Further, cross-genotype neutralization could be generated from monospecific plasma. Our studies contribute to understanding the mechanisms of antibody-mediated neutralization and interference and provide a practical approach to the development of more potent and broadly reactive hepatitis C immune globulins.     

Virus‐neutralizing antibodies to hepatitis C virus

For a long time, the lack of an appropriate cell culture system has hampered the study of neutralizing antibody responses against hepatitis C virus (HCV). However, the last decade has seen the development of several model systems that have significantly advanced our understanding of viral entry and antibody neutralization. Studies of acutely infected patients suggest that a strong and early production of neutralizing antibodies may contribute to control the virus during the acute phase of HCV infection and facilitate viral elimination by cellular immune responses. It also emerges that the early antibody response mainly targets hypervariable region 1 (HVR1) of the envelope glycoprotein E2. This host response can lead to viral escape from neutralization by rapid amino acid changes in this hypervariable region. In contrast, cross‐reactive neutralizing antibodies seem to appear later during HCV infection, and several mechanisms contribute to reduce their accessibility to their cognate epitopes. These include the masking of major conserved neutralizing epitopes by HVR1, specific N‐linked glycans and the lipid moiety of the viral particle. Other potential mechanisms of evasion from the neutralizing antibody response include a modulation by high‐density lipoproteins and interfering antibodies as well as the capacity of the virus to be transferred by cell‐to‐cell contacts. Finally, the recent identification of several highly conserved neutralizing epitopes provides some opportunities for the design and development of vaccine candidates that elicit a protective humoral immune response.     

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.     

Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus

Hepatitis C virus (HCV) infects ～2% of the world's population. It is estimated that there are more than 500,000 new infections annually in Egypt, the country with the highest HCV prevalence. An effective vaccine would help control this expanding global health burden. HCV is highly variable, and an effective vaccine should target conserved T- and B-cell epitopes of the virus. Conserved B-cell epitopes overlapping the CD81 receptor-binding site (CD81bs) on the E2 viral envelope glycoprotein have been reported previously and provide promising vaccine targets. In this study, we isolated 73 human mAbs recognizing five distinct antigenic regions on the virus envelope glycoprotein complex E1E2 from an HCV-immune phage-display antibody library by using an exhaustive-panning strategy. Many of these mAbs were broadly neutralizing. In particular, the mAb AR4A, recognizing a discontinuous epitope outside the CD81bs on the E1E2 complex, has an exceptionally broad neutralizing activity toward diverse HCV genotypes and protects against heterologous HCV challenge in a small animal model. The mAb panel will be useful for the design and development of vaccine candidates to elicit broadly neutralizing antibodies to HCV.     

Neutralizing Antibodies and Pathogenesis of Hepatitis C Virus Infection

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. The interplay between the virus and host innate and adaptive immune responses determines the outcome of infection. There is increasing evidence that host neutralizing responses play a relevant role in the resulting pathogenesis. Furthermore, viral evasion from host neutralizing antibodies has been revealed to be an important contributor in leading both to viral persistence in acute liver graft infection following liver transplantation, and to chronic viral infection. The development of novel model systems to study HCV entry and neutralization has allowed a detailed understanding of the molecular mechanisms of virus-host interactions during antibody-mediated neutralization. The understanding of these mechanisms will ultimately contribute to the development of novel antiviral preventive strategies for liver graft infection and an urgently needed vaccine. This review summarizes recent concepts of the role of neutralizing antibodies in viral clearance and protection, and highlights consequences of viral escape from neutralizing antibodies in the pathogenesis of HCV infection.     

Perspectives for the utilization of neutralizing human monoclonal antibodies as anti-HCV drugs

Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge. Law M, Maruyama T, Lewis J, Giang E, Tarr AW, Stamataki Z, Gastaminza P, Chisari FV, Jones IM, Fox RI, Ball JK, McKeating JA, Kneteman NM, Burton DR. A major problem in hepatitis C virus (HCV) immunotherapy or vaccine design is the extreme variability of the virus. We identified human monoclonal antibodies (mAbs) that neutralize genetically diverse HCV isolates and protect against heterologous HCV quasispecies challenge in a human liver-chimeric mouse model. The results provide evidence that broadly neutralizing antibodies to HCV protect against heterologous viral infection and suggest that a prophylactic vaccine against HCV may be achievable. [Abstract reproduced by permission of Nat Med 2008;14:25-27].     

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.     

Hepatitis C virus entry and neutralization

The processes of hepatitis C virus (HCV) entry and antibody-mediated neutralization are intimately linked. The high frequency of neutralizing antibodies (nAbs) that inhibit E2-CD81 interaction(s) suggests that this is a major target for the humoral immune response. The observation that HCV can transmit to naive cells by means of CD81-dependent and -independent routes in vitro awaits further investigation to assess the significance in vivo but may offer new strategies for HCV to escape nAbs. The identification of claudins in the entry process highlights the importance of cell polarity in defining routes of HCV entry and release, with recent experiments suggesting a polarized route of viral entry into cells in vitro. In this review, the authors summarize the current understanding of the mechanism(s) defining HCV entry and the role of nAbs in controlling HCV replication.     

Hepatitis C virus entry into hepatocytes: molecular mechanisms and targets for antiviral therapies

Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. Preventive modalities are absent and the current antiviral treatment is limited by resistance, toxicity, and high costs. Viral entry is required for initiation, spread, and maintenance of infection, and thus is a promising target for antiviral therapy. HCV entry is a highly orchestrated process involving viral and host cell factors. These include the viral envelope glycoproteins E1 and E2, CD81, scavenger receptor BI, and tight junction proteins claudin-1 and occludin. Recent studies in preclinical models and HCV-infected patients have demonstrated that the virus has developed multiple strategies to escape host immune responses during viral entry. These include evasion from neutralizing antibodies and viral spread by cell-cell transmission. These challenges have to be taken into account for the design of efficient antiviral strategies. Thus, a detailed understanding of the mechanisms of viral entry and escape is a prerequisite to define viral and cellular targets and develop novel preventive and therapeutic antivirals. This review summarizes the current knowledge about the molecular mechanisms of HCV entry into hepatocytes, highlights novel targets and reviews the current preclinical and clinical development of compounds targeting entry. Proof-of-concept studies suggest that HCV entry inhibitors are a novel and promising class of antivirals widening the preventive and therapeutic arsenal against HCV infection.     

Neutralizing antibodies to hepatitis C virus (HCV) in immune globulins derived from anti-HCV-positive plasma

The role of humoral immunity in hepatitis C virus (HCV) infections is uncertain. Nevertheless, there is increasing evidence for neutralizing antibodies to HCV in the serum or plasma of chronically infected individuals. Immune globulins prepared by ethanol fractionation of plasma had long been considered safe until a commercial immune globulin product, Gammagard, prepared from plasma from which units containing anti-HCV had been excluded, transmitted HCV to recipients. Studies suggested that the exclusion might have removed neutralizing antibodies from the plasma and hence compromised the safety of the resulting immune globulins. In the present study, by using chimpanzees and a recently validated in vitro system based on neutralization of infectious HCV pseudoparticles, we found broadly reactive neutralizing and protective antibodies in experimental immune globulin preparations made from anti-HCV-positive donations. Neutralizing antibodies were also found in Gammagard lots made from unscreened plasma that did not transmit hepatitis C but not in Gammagard lots, which were prepared from anti-HCV-screened plasma, that did transmit hepatitis C. The results provide an explanation for the mechanism by which the safety of this product was compromised. Immune globulins made from anti-HCV-positive plasma and containing broadly reactive neutralizing antibodies may provide a method of preventing HCV infection.     

In vitro assay for neutralizing antibody to hepatitis C virus: evidence for broadly conserved neutralization epitopes

Our understanding of the humoral immune response to hepatitis C virus (HCV) is limited because the virus can be studied only in humans and chimpanzees and because previously described neutralization assays have not been robust or simple to perform. Nevertheless, epidemiologic and laboratory studies suggested that neutralizing Ab to HCV might be important in preventing infection. We have recently described a neutralization assay based on the neutralization of pseudotyped murine retrovirus constructs bearing HCV envelope glycoproteins on their surface. We have applied the assay to well characterized clinical samples from HCV-infected patients and chimpanzees, confirmed the existence of neutralizing Ab to HCV, and validated most previously reported neutralizations of the virus. We did not find neutralizing anti-HCV in resolving infections but did find relatively high titers (>1:320) of such Ab in chronic infections. Neutralizing Ab was directed not only to epitope(s) in the hypervariable region of the E2 envelope protein but also to one or more epitopes elsewhere in the envelope of the virus. Neutralizing Ab was broadly reactive and could neutralize pseudotype particles bearing the envelope glycoproteins of two different subgenotypes (1a and 1b). The ability to assay neutralizing anti-HCV should permit an assessment of the prospects for successful Ab-mediated passive and active immunoprophylaxis against hepatitis C.     

Immunopathogenesis of hepatitis C virus infection

HCV infection becomes persistent in many patients who are otherwise immune competent. There is increasing support for potential contribution of innate immune response and viral interference with its components to the subsequent outcome. As for the adaptive immune response, humoral immunity may be largely ineffective despite evidence for neutralizing antibody response directed to the E2 HVR region, perhaps due to rapid selection of antibody escape variants. Cellular immune response does seem to play a role in the virologic outcome during acute infection based on strong association of a sustained vigorous and multispecific antiviral CD4 and CD8 T cell response with HCV clearance during acute infection. Following clearance, vigorous CD4 T cell response to HCV is maintained for many years, whereas the memory CD8 T cell response may be maintained with variable efficiency. If unable to clear the virus quickly, the T cell response (particularly if focused) may also select for T cell escape variants that are poorly recognized by the circulating T cells or even actively inactivate them through T cell antagonism. In established chronic infection. HCV-specific T cell response is quantitatively weak, providing only minimal selection pressure for further escape mutation. Although earlier studies using conventional in vitro techniques suggest that this low-frequency T cell response may help control the virus and liver disease progression, the role and nature of these apparently defective T cells in the outcome of chronic HCV infection remains to be fully determined. In summary, much progress has been made in the field of HCV immune pathogenesis since the initial identification of HCV. Although more work is needed to define the mechanism of HCV persistence and liver cell injury, there is considerable hope as well as challenge for potential development of vaccine and immunotherapy for HCV infection (see article by Drs. Inchaupsé and Feinstone). A better understanding of the relevant host and viral factors for clinical and virologic outcome, and the mechanism of selective immune defect against HCV, will be invaluable in our ability to treat the many patients infected with HCV.     

Human combinatorial libraries yield rare antibodies that broadly neutralize hepatitis C virus

One way to dissect the antibody response to an invading microorganism is to clone the antibody repertoire from immune donors and subsequently characterize the specific antibodies. Recently, methodological advances have allowed investigations of neutralizing antibodies against hepatitis C virus (HCV) in vitro. We have investigated three human mAbs, previously isolated from an individual infected with HCV of genotype 2b, that are known to cross-react in a binding assay to the envelope E2 protein of genotypes 1a and 1b. We now report that two of them have a neutralizing activity with a breadth not previously observed. Indeed, mAbs 1:7 and A8 recognized E2 from all of the six major genotypes, and they neutralized retroviral pseudoparticles [HCV pseudoparticles (HCVpp)] carrying genetically equally diverse HCV envelope glycoproteins. Importantly, these antibodies were also able to neutralize the cell culture infectious HCV clone JFH-1 in vitro, with IC(50) values of 60 ng/ml and 560 ng/ml, respectively. The conformational epitopes of these two broadly reactive antibodies were overlapping yet distinct and involved amino acid residues in the 523-535 region of E2, known to be important for the E2-CD81 interaction. The third antibody clone, representing a dominant population in the initial screen for these antibodies, was less broadly reactive and was unable to neutralize the genotype 2a infectious clone JFH-1. Our results confirm at the clonal level that broadly neutralizing human anti-HCV antibodies can be elicited and that the region amino acids 523-535 of the HCV envelope glycoprotein E2 carries neutralizing epitopes conserved across all genotypes.     

Vaccine development for hepatitis C

Given the global disease burden and public health impact of hepatitis C, the development of an effective vaccine is of paramount importance. However, many challenging obstacles loom ahead of this goal. The hepatitis C virus (HCV), being an RNA virus, can mutate rapidly in adaptation to the environment, thus contributing to the high sequence divergence of multiple viral isolates in the world. The highest heterogeneity has been found in the hypervariable region of the envelope glycoprotein 2, which contains a principal neutralization epitope. HCV also causes persistent infection in a high percentage of immunocompetent hosts despite active immune response. The lack of an efficient tissue culture system for propagating HCV and testing neutralizing antibodies adds further complexity to the task of vaccine development. The immunologic correlates associated with disease progression or protection are yet to be defined, but recent studies suggest that a vigorous multispecific cellular immune response is important in the resolution of infection. Induction of high-titer, long-lasting, and cross-reactive antienvelope antibodies and a vigorous multispecific cellular immune response that includes both helper and cytotoxic T lymphocytes may be necessary for an effective vaccine. Several promising approaches have been used to develop an HCV vaccine. Novel vaccine candidates based on molecular technology such as recombinant proteins, peptides, viruslike particles, naked DNA, and recombinant viruses are being explored. The final vaccine product may require multiple components that target various aspects of protective immunity. Finally, sterilizing immunity may not be necessary if a vaccine can be developed to prevent chronic infection, which is the major cause of morbidity and mortality from this disease.     

Viral Safety of Blood Derivatives by Immune Neutralization

Despite careful donor selection and virus inactivation procedures, transmission of viruses by transfusion of blood and blood derivatives is still a threat. Outbreaks of hepatitis A among hemophiliacs having received highly purified, immune globulin depleted coagulation factor concentrates, put the importance of immune neutralization of viruses in blood derivatives in focus. Neutralizing antibodies may block several steps in the virus infection of a cell, from binding of virus to the cellular receptor to the uncoating of virus after uptake in the cell. The efficacy of antibody neutralizing activity depends on the availability and stability of the neutralizing epitopes. Hepatitis A and B viruses are very efficiently neutralized by antibodies and immune escape mutants rarely emerge. Anti-parvovirus B19 antibodies do not fully inactivate the virus, at least in low concentrations, but may prevent development of disease. The neutralizing epitopes on hepatitis C virus and human immunodeficiency virus are located on hypervariable regions of virus membrane proteins. The effects of neutralizing antibodies are thus marginal as immune escape mutants emerge at a relatively high frequency for both viruses. The neutralizing activity of anti-cytomegalovirus antibodies is also questionable as persons may become reinfected with cytomegolvirus despite high levels of antibodies. Plasma and plasma derivatives produced from large donor pools have the potential of being very efficient transmitters of viruses. Neutralizing antibodies are Nature's own, and very important barriers against the spread of many known and unknown viruses contaminating the plasma pools.     

A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis. The virus does not replicate efficiently in cell cultures, and it is therefore difficult to assess infection-neutralizing antibodies and to evaluate protective immunity in vitro. To study the binding of the HCV envelope to cell-surface receptors, we developed an assay to assess specific binding of recombinant envelope proteins to human cells and neutralization thereof. HCV recombinant envelope proteins expressed in various systems were incubated with human cells, and binding was assessed by flow cytometry using anti-envelope antibodies. Envelope glycoprotein 2 (E2) expressed in mammalian cells, but not in yeast or insect cells, binds human cells with high affinity (Kd approximately 10(-8) M). We then assessed antibodies able to neutralize E2 binding in the sera of both vaccinated and carrier chimpanzees, as well as in the sera of humans infected with various HCV genotypes. Vaccination with recombinant envelope proteins expressed in mammalian cells elicited high titers of neutralizing antibodies that correlated with protection from HCV challenge. HCV infection does not elicit neutralizing antibodies in most chimpanzees and humans, although low titers of neutralizing antibodies were detectable in a minority of infections. The ability to neutralize binding of E2 derived from the HCV-1 genotype was equally distributed among sera from patients infected with HCV genotypes 1, 2, and 3, demonstrating that binding of E2 is partly independent of E2 hypervariable regions. However, a mouse monoclonal antibody raised against the E2 hypervariable region 1 can partially neutralize binding of E2, indicating that at least two neutralizing epitopes, one of which is hypervariable, should exist on the E2 protein. The neutralization-of-binding assay described will be useful to study protective immunity to HCV infection and for vaccine development.     

Inhibition of HIV-1 entry by antibodies: potential viral and cellular targets

Vaccine-induced antibodies that interfere with viral entry are the protective correlate of most existing prophylactic vaccines. However, for highly variable viruses such as HIV-1, the ability to elicit broadly neutralizing antibody responses through vaccination has proven to be extremely difficult. The major targets for HIV-1 neutralizing antibodies are the viral envelope glycoprotein trimers on the surface of the virus that mediate receptor binding and entry. HIV-1 has evolved many mechanisms on the surface of envelope glycoproteins to evade antibody-mediated neutralization, including the masking of conserved regions by glycan, quaternary protein interactions and the presence of immunodominant variable elements. The primary challenge in the development of an HIV-1 vaccine that elicits broadly neutralizing antibodies therefore lies in the design of suitable envelope glycoprotein immunogens that circumvent these barriers. Here, we describe neutralizing determinants on the viral envelope glycoproteins that are defined by their function in receptor binding or by rare neutralizing antibodies isolated from HIV-infected individuals. We also describe the nonvariable cellular receptors involved in the HIV-1 entry process, or other cellular proteins, and ongoing studies to determine if antibodies against these proteins have efficacy as therapeutic reagents or, in some cases, as vaccine targets to interfere with HIV-1 entry.