Evi Struble

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.

Structural evidence for a bifurcated mode of action in the antibody-mediated neutralization of hepatitis C virus

Hepatitis C virus (HCV) envelope glycoprotein E2 has been considered as a major target for vaccine design. Epitope II, mapped between residues 427–446 within the E2 protein, elicits antibodies that are either neutralizing or nonneutralizing. The fundamental mechanism of antibody-mediated neutralization at epitope II remains to be defined at the atomic level. Here we report the crystal structure of the epitope II peptide in complex with a monoclonal antibody (mAb#8) capable of neutralizing HCV. The complex structure revealed that this neutralizing antibody engages epitope II via interactions with both the C-terminal α-helix and the N-terminal loop using a bifurcated mode of action. Our structural insights into the key determinants for the antibody-mediated neutralization may contribute to the immune prophylaxis of HCV infection and the development of an effective HCV vaccine.

Discrete conformations of epitope II on the hepatitis C virus E2 protein for antibody-mediated neutralization and nonneutralization

Significance X-ray crystallographic analysis revealed that one of the critical antibody-binding sites on the hepatitis C virus exists in different shapes. The structural transition among these shapes is governed by a highly conserved glycine residue that serves as a flexible joint connecting the two essential parts of the binding site; that is, the C-terminal α-helix and the N-terminal loop. It is the particular spatial arrangement of these parts that determines the specificity of antibody recognition and, consequently, the outcome of either neutralization or nonneutralization of the virus. These structural insights may be beneficial for the immune prophylaxis and treatment of HCV infections. The X-ray crystal structure of epitope II on the E2 protein of hepatitis C virus, in complex with nonneutralizing antibody mAb#12, has been solved at 2.90-Å resolution. The spatial arrangement of the essential components of epitope II (ie, the C-terminal α-helix and the N-terminal loop) was found to deviate significantly from that observed in those corresponding complexes with neutralizing antibodies. The distinct conformations are mediated largely by the flexibility of a highly conserved glycine residue that connects these components. Thus, it is the particular tertiary structure of epitope II, which is presented in a spatial and temporal manner, that determines the specificity of antibody recognition and, consequently, the outcome of neutralization or nonneutralization.

Amino acid residue-specific neutralization and nonneutralization of hepatitis C virus by monoclonal antibodies to the E2 protein.

ABSTRACT Antibodies to epitopes in the E2 protein of hepatitis C virus (HCV) reduce the viral infectivity in vivo and in vitro. However, the virus can persist in patients in the presence of neutralizing antibodies. In this study, we generated a panel of monoclonal antibodies that bound specifically to the region between residues 427 and 446 of the E2 protein of HCV genotype 1a, and we examined their capacity to neutralize HCV in a cell culture system. Of the four monoclonal antibodies described here, two were able to neutralize the virus in a genotype 1a-specific manner. The other two failed to neutralize the virus. Moreover, one of the nonneutralizing antibodies could interfere with the neutralizing activity of a chimpanzee polyclonal antibody at E2 residues 412 to 426, as it did with an HCV-specific immune globulin preparation, which was derived from the pooled plasma of chronic hepatitis C patients. Mapping the epitope-paratope contact interfaces revealed that these functionally distinct antibodies shared binding specificity for key amino acid residues, including W437, L438, L441, and F442, within the same epitope of the E2 protein. These data suggest that the effectiveness of antibody-mediated neutralization of HCV could be deduced from the interplay between an antibody and a specific set of amino acid residues. Further understanding of the molecular mechanisms of antibody-mediated neutralization and nonneutralization should provide insights for designing a vaccine to control HCV infection in vivo.

Hepatitis C virus with a naturally occurring single amino-acid substitution in the E2 envelope protein escapes neutralization by naturally-induced and vaccine-induced antibodies

Mutations arising in neutralizing epitopes of hepatitis C virus may play a role in the ability of the virus to escape control by neutralizing antibodies and in the establishment of chronic infections. An amino-acid substitution, Q412H, within a major conserved neutralization epitope EP I (aa 412-426) in the E2 glycoprotein is observed in chronic HCV carriers. We found that naturally acquired polyclonal EP I-specific antibodies have an equivalent binding capacity toward either the wild type or the Q412H mutant peptide encompassing the EP I epitope. While EP I-specific antibodies neutralized J6/JFH1 virus in vitro, they did not neutralize J6/JFH1 virus containing the Q412H mutation. Furthermore, we found that plasma obtained from a chimpanzee that had anti-E1/E2 antibodies following experimental immunization, neutralized the wild type J6/JFH1 virus but failed to neutralize the mutant virus. Thus, mutation Q412H found in naturally occurring variants could represent an antibody escape mutation. These data may have important implications for vaccine design.

A neutralization epitope in the hepatitis C virus E2 glycoprotein interacts with host entry factor CD81.

The identification of a specific immunogenic candidate that will effectively activate the appropriate pathway for neutralizing antibody production is fundamental for vaccine design. By using a monoclonal antibody (1H8) that neutralizes HCV in vitro, we have demonstrated here that 1H8 recognized an epitope mapped between residues A524 and W529 of the E2 protein. We also found that the epitope residues A524, P525, Y527 and W529 were crucial for antibody binding, while the residues T526, Y527 and W529 within the same epitope engaged in the interaction with the host entry factor CD81. Furthermore, we detected “1H8-like” antibodies, defined as those with amino acid-specificity similar to 1H8, in the plasma of patients with chronic HCV infection. The time course study of plasma samples from Patient H, a well-characterized case of post-transfusion hepatitis C, showed that “1H8-like” antibodies could be detected in a sample collected almost two years after the initial infection, thus confirming the immunogenicity of this epitope in vivo. The characterization of this neutralization epitope with a function in host entry factor CD81 interaction should enhance our understanding of antibody-mediated neutralization of HCV infections.

Multiple in silico tools predict phenotypic manifestations in congenital thrombotic thrombocytopenic purpura

Congenital thrombotic thrombocytopenic purpura (cTTP) is a rare, recessively inherited genetic disorder with varying clinical presentation that is caused by ADAMTS13 mutations. Several studies have found limited associations between ADAMTS13 mutations and cTTP phenotype. The use of in silico tools that examine multiple mutation characteristics may better predict phenotype. We analysed 118 ADAMTS13 mutations found in 144 cTTP patients reported in the literature and examined associations of several mutation characteristics, including N‐terminal proximity, the evolutionary conservation of the affected amino acid position, as well as amino acid charge/phosphorylation and genetic codon usage to disease phenotype. Structure‐altering mutations were examined for their impact on ADAMTS13 function based on existing ADAMTS13 crystallographic data (AA 77‐685). Our in silico data indicate that: (i) The position of the mutation in the N‐ or C‐terminus, (ii) evolutionary conservation and (iii) codon usage of the affected mutation position are associated with disease parameters, such as age of onset, organ damage and fresh frozen plasma prophylaxis. In conclusion, the usage of multiple in silico tools presents a promising strategy in refining predictions for the diverse presentation of cTTP. Enhancing our utilization of in silico tools to find genotype‐phenotype associations will create better‐tailored approaches for individual patient treatment.

Antibody-mediated synergy and interference in the neutralization of SARS-CoV at an epitope cluster on the spike protein.

Abstract Incomplete neutralization of virus, especially when it occurs in the presence of excess neutralizing antibody, represents a biological phenomenon that impacts greatly on antibody-mediated immune prophylaxis of viral infection and on successful vaccine design. To understand the mechanism by which a virus escapes from antibody-mediated neutralization, we have investigated the interactions of non-neutralizing and neutralizing antibodies at an epitope cluster on the spike protein of severe acute respiratory syndrome coronavirus (SARS-CoV). The epitope cluster was mapped at the C-terminus of the spike protein; it consists of structurally intertwined epitopes recognized by two neutralizing monoclonal antibodies (mAbs), 341C and 540C, and a non-neutralizing mAb, 240C. While mAb 341C binds to a mostly linear epitope composed of residues 507PAT509 and V349, mAb 240C binds to an epitope that partially overlaps the former by at least two residues (P507 and A508). The epitope corresponding to mAb 540C is a conformational one, involving residues L504 and N505. In neutralization assays, non-neutralizing 240C disrupted virus neutralization by mAb 341C and/or mAb 540C, whereas a combination of mAbs 341C and 540C blocked virus infectivity synergistically. These findings indicate that the epitope cluster on the spike protein may serve as an evolutionarily conserved platform at which a dynamic interplay between neutralizing and non-neutralizing antibodies occurs, thereby determining the outcome of SARS-CoV infection.

A View of the E2-CD81 Interface at the Binding Site of a Neutralizing Antibody against Hepatitis C Virus

ABSTRACT Hepatitis C virus (HCV) glycoprotein E2 is considered a major target for generating neutralizing antibodies against HCV, primarily due to its role of engaging host entry factors, such as CD81, a key cell surface protein associated with HCV entry. Based on a series of biochemical analyses in combination with molecular docking, we present a description of a potential binding interface formed between the E2 protein and CD81. The virus side of this interface includes a hydrophobic helix motif comprised of residues W437LAGLF442, which encompasses the binding site of a neutralizing monoclonal antibody, mAb41. The helical conformation of this motif provides a structural framework for the positioning of residues F442 and Y443, serving as contact points for the interaction with CD81. The cell side of this interface likewise involves a surface-exposed hydrophobic helix, namely, the D-helix of CD81, which coincides with the binding site of 1D6, a monoclonal anti-CD81 antibody known to block HCV entry. Our illustration of this virus-host interface suggests an important role played by the W437LAGLF442 helix of the E2 protein in the hydrophobic interaction with the D-helix of CD81, thereby facilitating our understanding of the mechanism for antibody-mediated neutralization of HCV. IMPORTANCE Characterization of the interface established between a virus and host cells can provide important information that may be used for the control of virus infections. The interface that enables hepatitis C virus (HCV) to infect human liver cells has not been well understood because of the number of cell surface proteins, factors, and conditions found to be associated with the infection process. Based on a series of biochemical analyses in combination with molecular docking, we present such an interface, consisting of two hydrophobic helical structures, from the HCV E2 surface glycoprotein and the CD81 protein, a major host cell receptor recognized by all HCV strains. Our study reveals the critical role played by hydrophobic interactions in the formation of this virus-host interface, thereby contributing to our understanding of the mechanism for antibody-mediated neutralization of HCV.

Potentiation of C1-esterase inhibitor by heparin and interactions with C1s protease as assessed by surface plasmon resonance.

BACKGROUND Human C1-esterase inhibitor (C1-INH) is a multifunctional plasma protein with a wide range of inhibitory and non-inhibitory properties, mainly recognized as a key down-regulator of the complement and contact cascades. The potentiation of C1-INH by heparin and other glycosaminoglycans (GAGs) regulates a broad spectrum of C1-INH activities in vivo both in normal and disease states. SCOPE OF RESEARCH: We have studied the potentiation of human C1-INH by heparin using Surface Plasmon Resonance (SPR), circular dichroism (CD) and a functional assay. To advance a SPR for multiple-unit interaction studies of C1-INH we have developed a novel (consecutive double capture) approach exploring different immobilization and layout. MAJOR CONCLUSIONS Our SPR experiments conducted in three different design versions showed marked acceleration in C1-INH interactions with complement protease C1s as a result of potentiation of C1-INH by heparin (from 5- to 11-fold increase of the association rate). Far-UV CD studies suggested that heparin binding did not alter C1-INH secondary structure. Functional assay using chromogenic substrate confirmed that heparin does not affect the amidolytic activity of C1s, but does accelerate its consumption due to C1-INH potentiation. GENERAL SIGNIFICANCE This is the first report that directly demonstrates a significant acceleration of the C1-INH interactions with C1s due to heparin by using a consecutive double capture SPR approach. The results of this study may be useful for further C-INH therapeutic development, ultimately for the enhancement of current C1-INH replacement therapies.