A conserved epitope III on hepatitis C virus E2 protein has alternate conformations facilitating cell binding or virus neutralization.

Epitope III, a highly conserved amino acid motif of 524APTYSW529 on the hepatitis C virus (HCV) E2 glycoprotein, resides in the critical loop that binds to the host receptor CD81, thus making it one of the most important antibody targets for blocking HCV infections. Here, we have determined the X-ray crystal structure of epitope III at a 2.0-Å resolution when it was captured by a site-specific neutralizing antibody, monoclonal antibody 1H8 (mAb1H8). The snapshot of this complex revealed that epitope III has a relatively rigid structure when confined in the binding grooves of mAb1H8, which confers the residue specificity at both ends of the epitope. Such a high shape complementarity is reminiscent of the "lock and key" mode of action, which is reinforced by the incompatibility of an antibody binding with an epitope bearing specific mutations. By subtly positioning the side chains on the three residues of Tyr527, Ser528, and Trp529 while preserving the spatial rigidity of the rest, epitope III in this cocrystal complex adopts a unique conformation that is different from previously described E2 structures. With further analyses of molecular docking and phage display-based peptide interactions, we recognized that it is the arrangements of two separate sets of residues within epitope III that create these discrete conformations for the epitope to interact selectively with either mAb1H8 or CD81. These observations thus raise the possibility that local epitope III conformational dynamics, in conjunction with sequence variations, may act as a regulatory mechanism to coordinate "mAb1H8-like" antibody-mediated immune defenses with CD81-initiated HCV infections.

Multiple epitopes of hepatitis B virus surface antigen targeted by human plasma-derived immunoglobulins coincide with clinically observed escape mutations.

Hepatitis B immune globulin (HBIG) is a human plasma-derived immunoglobulin G concentrate that contains a high titer of neutralizing antibodies (anti-HBs) to the hepatitis B virus (HBV) surface antigen (HBsAg). HBIG is known to be highly effective in treating HBV infections, however, a more systematic characterization of the antibody binding sites on HBsAg and their correlation with emerging "escape" mutations in HBsAg was lacking. By using anti-HBs antibodies from HBIG lots to screen random peptide phage display libraries, we identified five clusters of peptides that corresponded to five distinct anti-HBs binding sites on the HBsAg. Three sites, Site II (C121-C124), Site III (M133-P135), and Site IV (T140-G145), were mapped within the "a" determinant, while the two other sites, Site I (Q101-M103) and Site V (I152-S154), were outside the "a" determinant. We then tested in binding assays HBsAg peptides containing clinically relevant mutations previously reported within these sites, such as Y134S, P142S, and G145R, and observed a significant reduction in anti-HBs binding activity to the mutated sites, suggesting a mechanism the virus may use to avoid HBIG-mediated neutralization. The current HBIG treatment could be improved by supplementing it with site-specific neutralizing monoclonal antibodies that target these mutations for control of HBV infections.

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

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.

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.

In vitro enhancement of Zika virus infection by preexisting West Nile virus antibodies in human plasma-derived immunoglobulins revealed after P2 binding site-specific enrichment.

Human immunoglobulin preparations contain a diverse range of polyclonal antibodies that reflect past immune responses against pathogens encountered by the blood donor population. In this study, we examined a panel of intravenous immunoglobulins (IGIVs) manufactured over the past two decades (1998-2020) for their capacity to neutralize or enhance Zika virus (ZIKV) infection in vitro. These IGIVs were selected specifically based on their production dates in relation to the occurrences of two flavivirus outbreaks in the U.S.: the West Nile virus (WNV) outbreak in 1999 and the ZIKV outbreak in 2015. As demonstrated by enzyme-linked immunosorbent assay (ELISA) experiments, IGIVs made before the ZIKV outbreak already harbored antibodies that bind to various peptides across the envelope protein of ZIKV because of the WNV outbreak. Using phage display, the most dominant binding site was mapped precisely to the P2 peptide between residues 211 and 230 within domain II, where BF1176-56, an anti-ZIKV monoclonal antibody, also binds. When tested in permissive Vero E6 cells for ZIKV neutralization, the IGIVs, even after undergoing rigorous enrichment for P2 binding specificity, failed, as did BF1176-56. Meanwhile, BF1176-56 enhanced ZIKV infection in both FcγRII-expressing K562 cells and human peripheral blood mononuclear cells. However, for enhancement by the IGIVs to be detected in these cells, a substantial increase in their P2 binding specificity was required, thus linking the P2 site with ZIKV enhancement in vitro. Our findings warrant further study of the significance of elevated levels of anti-WNV antibodies in IGIVs, considering that various mechanisms operating in vivo may modulate ZIKV infection outcomes.IMPORTANCEWe investigated the capacity of intravenous immunoglobulins manufactured previously over two decades (1998-2020) to neutralize or enhance Zika virus infection in vitro. West Nile virus antibodies in IGIVs could not neutralize Zika virus initially; however, once the IGIVs were concentrated further, they enhanced its infection. These findings lay the groundwork for exploring how preexisting WNV antibodies in IGIVs could impact Zika infection, both in vitro and in vivo. Our observations are historically significant, since we tested a panel of IGIV lots that were carefully selected based on their production dates which covered two major flavivirus outbreaks in the U.S.: the WNV outbreak in 1999 and the ZIKV outbreak in 2015. These findings will facilitate our understanding of the interplay among closely related viral pathogens, particularly from a historical perspective regarding large blood donor populations. They should remain relevant for future outbreaks of emerging flaviviruses that may potentially affect vulnerable populations.

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.

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.