Maturation of West Nile virus modulates sensitivity to antibody-mediated neutralization

West Nile virions incorporate 180 envelope (E) proteins that orchestrate the process of virus entry and are the primary target of neutralizing antibodies. The E proteins of newly synthesized West Nile virus (WNV) are organized into trimeric spikes composed of pre-membrane (prM) and E protein heterodimers. During egress, immature virions undergo a protease-mediated cleavage of prM that results in a reorganization of E protein into the pseudo-icosahedral arrangement characteristic of mature virions. While cleavage of prM is a required step in the virus life cycle, complete maturation is not required for infectivity and infectious virions may be heterogeneous with respect to the extent of prM cleavage. In this study, we demonstrate that virion maturation impacts the sensitivity of WNV to antibody-mediated neutralization. Complete maturation results in a significant reduction in sensitivity to neutralization by antibodies specific for poorly accessible epitopes that comprise a major component of the human antibody response following WNV infection or vaccination. This reduction in neutralization sensitivity reflects a decrease in the accessibility of epitopes on virions to levels that fall below a threshold required for neutralization. Thus, in addition to a role in facilitating viral entry, changes in E protein arrangement associated with maturation modulate neutralization sensitivity and introduce an additional layer of complexity into humoral immunity against WNV.     

A dynamic landscape for antibody binding modulates antibody-mediated neutralization of West Nile virus

Neutralizing antibodies are a significant component of the host's protective response against flavivirus infection. Neutralization of flaviviruses occurs when individual virions are engaged by antibodies with a stoichiometry that exceeds a required threshold. From this "multiple-hit" perspective, the neutralizing activity of antibodies is governed by the affinity with which it binds its epitope and the number of times this determinant is displayed on the surface of the virion. In this study, we investigated time-dependent changes in the fate of West Nile virus (WNV) decorated with antibody in solution. Experiments with the well-characterized neutralizing monoclonal antibody (MAb) E16 revealed a significant increase in neutralization activity over time that could not be explained by the kinetics of antibody binding, virion aggregation, or the action of complement. Additional kinetic experiments using the fusion-loop specific MAb E53, which has limited neutralizing activity because it recognizes a relatively inaccessible epitope on mature virions, identified a role of virus "breathing" in regulating neutralization activity. Remarkably, MAb E53 neutralized mature WNV in a time- and temperature-dependent manner. This phenomenon was confirmed in studies with a large panel of MAbs specific for epitopes in each domain of the WNV envelope protein, with sera from recipients of a live attenuated WNV vaccine, and in experiments with dengue virus. Given enough time, significant inhibition of infection was observed even for antibodies with very limited, or no neutralizing activity in standard neutralization assays. Together, our data suggests that the structural dynamics of flaviviruses impacts antibody-mediated neutralization via exposure of otherwise inaccessible epitopes, allowing for antibodies to dock on the virion with a stoichiometry sufficient for neutralization.     

Potent Dengue Virus Neutralization by a Therapeutic Antibody with Low Monovalent Affinity Requires Bivalent Engagement

We recently described our most potently neutralizing monoclonal antibody, E106, which protected against lethal Dengue virus type 1 (DENV-1) infection in mice. To further understand its functional properties, we determined the crystal structure of E106 Fab in complex with domain III (DIII) of DENV-1 envelope (E) protein to 2.45 Å resolution. Analysis of the complex revealed a small antibody-antigen interface with the epitope on DIII composed of nine residues along the lateral ridge and A-strand regions. Despite strong virus neutralizing activity of E106 IgG at picomolar concentrations, E106 Fab exhibited a ∼20,000-fold decrease in virus neutralization and bound isolated DIII, E, or viral particles with only a micromolar monovalent affinity. In comparison, E106 IgG bound DENV-1 virions with nanomolar avidity. The E106 epitope appears readily accessible on virions, as neutralization was largely temperature-independent. Collectively, our data suggest that E106 neutralizes DENV-1 infection through bivalent engagement of adjacent DIII subunits on a single virion. The isolation of anti-flavivirus antibodies that require bivalent binding to inhibit infection efficiently may be a rare event due to the unique icosahedral arrangement of envelope proteins on the virion surface.     

Efficient neutralization of foot-and-mouth disease virus by monovalent antibody binding.

Neutralization of an aphthovirus by monovalent binding of an antibody is reported. Foot-and-mouth disease virus (FMDV) clone C-S8c1 was neutralized by monoclonal antibody (MAb) SD6, which was directed to a continuous epitope within a major antigenic site of the G-H loop of capsid protein VP1. On a molar basis, the Fab fragment was at most fivefold less active in neutralization than the intact antibody, and both blocked virus attachment to cells. Neither the antibody nor the Fab fragment caused aggregation of virions, as evidenced by sucrose gradient sedimentation studies of the antibody-virus complex formed at antibody to virion ratios of 1:50 to 1:10,000. The results of neutralization of infectivity and of ultracentrifugation are fully consistent with structural data based on X-ray crystallographic and cryoelectron microscopy studies, which showed monovalent interaction of the antibody with a critical receptor binding motif Arg-Gly-Asp. The conclusions of these neutralization studies are that (i) bivalent binding of antibody is not a requisite for strong neutralization of aphthoviruses and (ii) aggregation of viral particles, which has been proposed to be the dominant neutralization mechanism of antibodies that bind monovalently to virions, is not necessary for the neutralization of FMDV C-S8c1 by MAb SD6.     

Stoichiometry of Monoclonal Antibody Neutralization of T-Cell Line-Adapted Human Immunodeficiency Virus Type 1

In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes are coexpressed. By the coexpression of Env glycoproteins that either can or cannot bind a neutralizing MAb in an env transcomplementation assay, virions were generated in which the proportion of MAb binding sites could be regulated. As the proportion of MAb binding sites in Env chimeric virus increased, MAb neutralization gradually increased. Virus neutralization by virion aggregation was minimal, as MAb binding to HIV-1 Env did not interfere with an AMLV Env-mediated infection by HIV-1(AMLV/HIV-1) pseudotypes of CD4(-) HEK293 cells. MAb neutralization of chimeric virions could be described as a third-order function of the proportion of Env antigen refractory to MAb binding. This scenario is consistent with the Env oligomer constituting the minimal functional unit and neutralization occurring incrementally as each Env oligomer binds MAb. Alternatively, the data could be fit to a sigmoid function. Thus, these data could not exclude the existence of a threshold for neutralization. However, results from MAb neutralization of chimeric virus containing wild-type Env and Env defective in CD4 binding was readily explained by a model of incremental MAb neutralization. In summary, the data indicate that MAb neutralization of T-cell line-adapted HIV-1 is incremental rather than all or none and that each MAb binding an Env oligomer reduces the likelihood of infection.     

A fusion-loop antibody enhances the infectious properties of immature flavivirus particles

Flavivirus-infected cells secrete a mixture of mature, partially immature, and fully immature particles into the extracellular space. Although mature virions are highly infectious, prM-containing fully immature virions are noninfectious largely because the prM protein inhibits the cell attachment and fusogenic properties of the virus. If, however, cell attachment and entry are facilitated by anti-prM antibodies, immature flavivirus becomes infectious after efficient processing of the prM protein by the endosomal protease furin. A recent study demonstrated that E53, a cross-reactive monoclonal antibody (MAb) that engages the highly conserved fusion-loop peptide within the flavivirus envelope glycoprotein, preferentially binds to immature flavivirus particles. We investigated here the infectious potential of fully immature West Nile virus (WNV) and dengue virus (DENV) particles opsonized with E53 MAb and observed that, like anti-prM antibodies, this anti-E antibody also has the capacity to render fully immature flaviviruses infectious. E53-mediated enhancement of both immature WNV and DENV depended on efficient cell entry and the enzymatic activity of the endosomal furin. Furthermore, we also observed that E53-opsonized immature DENV particles but not WNV particles required a more acidic pH for efficient cleavage of prM by furin, adding greater complexity to the dynamics of antibody-mediated infection of immature flavivirus virions.     

Binding of anti-membrane-proximal gp41 monoclonal antibodies to CD4-liganded and -unliganded human immunodeficiency virus type 1 and simian immunodeficiency virus virions

The broadly neutralizing monoclonal antibodies (MAbs) 4E10, 2F5, and Z13e1 target membrane-proximal external region (MPER) epitopes of HIV-1 gp41 in a manner that remains controversial. The requirements for initial lipid bilayer binding and/or CD4 ligation have been proposed. To further investigate these issues, we probed for binding of these MAbs to human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) virions with protein A-conjugated gold (PAG) nanoparticles using negative-stain electron microscopy. We found moderate levels of PAG associated with unliganded HIV-1 and SIV virions incubated with the three MAbs. Significantly higher levels of PAG were associated with CD4-liganded HIV-1 (epitope-positive) but not SIV (epitope-negative) virions. A chimeric SIV virion displaying the HIV-1 4E10 epitope also showed significantly higher PAG association after CD4 ligation and incubation with 4E10. MAbs accumulated rapidly on CD4-liganded virions and slowly on unliganded virions, although both reached similar levels in time. Anti-MPER epitope-specific binding was stable to washout. Virions incubated with an irrelevant MAb or CD4-only (no MAb) showed negligible PAG association, as did a vesicle-rich fraction devoid of virions. Preincubation with Fab 4E10 inhibited both specific and nonspecific 4E10 IgG binding. Our data provide evidence for moderate association of anti-MPER MAbs to viral surfaces but not lipid vesicles, even in the absence of cognate epitopes. Significantly greater MAb interaction occurs in epitope-positive virions following long incubation or CD4 ligation. These findings are consistent with a two-stage binding model where these anti-MPER MAbs bind first to the viral lipid bilayer and then to the MPER epitopes following spontaneous or induced exposure.     

A conformational change in Sindbis virus glycoproteins E1 and E2 is detected at the plasma membrane as a consequence of early virus-cell interaction.

A conformational change in the structure of Sindbis (SB) virus was detected after virion attachment to baby hamster kidney cells but before internalization. The alteration was manifested as increased virion binding of certain glycoprotein E1 and E2 monoclonal antibodies (MAbs) that recognized transitional epitopes. These epitopes were inaccessible to MAb on native virions but became accessible to their cognate MAbs in the early stages of infection. Transit of virions through a low-pH compartment apparently was not required for the conformational change. Exposure of transitional epitopes was unaffected by treatment of BHK cells with NH4Cl and occurred normally in Chinese hamster ovary cells temperature sensitive for endosomal acidification. However, the rearrangement was correlated with both the time course and temperature dependence of SB virus penetration, and the rearrangement occurred earlier with an SB virus mutant having an accelerated penetration phenotype. In addition, MAb to a transitional epitope, a probe specific for rearranged particles, retarded penetration of infectious virions. These results suggested that the SB virus E1/E2 glycoprotein spike undergoes a structural rearrangement as a consequence of virion interaction with the cell surface and that this altered virion form may be an important early intermediate in an entry pathway leading to productive infection.     

Further studies on the mechanism of rabies virus neutralization by a viral glycoprotein-specific monoclonal antibody, #1-46-12

We previously reported that a conformational epitope-specific monoclonal antibody (mAb; #1-46-12) neutralized the rabies virus by binding only a small number (less than 20) of the antibody molecules per virion, while a linear epitope-specific mAb (#7-1-9) required more than 250 IgG molecules for the neutralization. We also isolated both the epitope-negative (R-31) and-positive (R-61) escape mutants that resisted mAb #1-46-12. Co-infection studies with wild type (wt) and R-61 mutant have shown that although the infectivity of R-61 mutant was not affected by the binding of about 300 IgG molecules per virion, incorporation of a small number of wt G protein into the R-61 virion resulted in dramatic loss of the resistance. In this study, we further investigated properties of the mutant G proteins. The R-61 G protein lost reactivity to the mAb when solubilized, even keeping a trimer form, suggesting that membrane-anchorage is essential for the maintenance of its epitope-positive conformation. On the other hand, incorporation of wt G proteins into the R-31 virions did not affect their resistance to the mAb very much. Although we have not so far found the presumed conformational changes induced by the mAb-binding, we think that these results are not inconsistent with our previously proposed novel model (referred to as a domino effect model) for the virus neutralization by mAb #1-46-12 other than a classical spike-blocking model, which implicates successive spreading of the postulated antibody-induced conformational changes of G protein to the neighboring spikes until abolishing the host cell-binding ability of the virion.     

Role of complement and the Fc portion of immunoglobulin G in immunity to Venezuelan equine encephalomyelitis virus infection with glycoprotein-specific monoclonal antibodies.

We have previously characterized with monoclonal antibodies (MAbs) seven unique epitopes on the two envelope glycoproteins of Venezuelan equine encephalomyelitis (VEE) virus vaccine strain TC-83. The epitopes important in protection from VEE virus infection were determined in passive antibody transfer studies, with virulent VEE (Trinidad donkey) virus as the challenge virus. Selected high-avidity MAbs to the three major protective epitopes (E2c, E1b, and E1d) were assayed for in vitro complement activity. All three fixed murine complement to high titer. Limited pepsin digestion of the anti-E2c in the presence of cysteine resulted in a rapid decrease and complete loss of complement-fixing ability by 2 h, but the majority of mice, except at the lowest dilution of MAb, were protected until the Fc termini were cleaved at 3 h. Anti-E2c F(ab')2 would neutralize VEE (Trinidad donkey) virus more efficiently than either Fab' or Fab; none of the fragments would fix complement or was effective in passive protection. C5-deficient mice and mice depleted of C3 with cobra venom factor were still protected from VEE (Trinidad donkey) virus challenge after passive transfer of either anti-E2c or anti-E1b MAb. The results show that the anti-E2c MAb mediates neutralization through bivalent binding at a critical site on the virion and that Fc effector functions, other than complement, are necessary for protection. Although the ability of the anti-E2c MAb to fix complement was associated with its ability to protect in vivo, no direct cause-and-effect relationship was found. Since the epitope defined by the anti-E1d antibody is found on the cell membrane, but is not expressed on the infectious virion, protection in mice was most likely mediated at the cellular level, possibly by inhibition of the final stages of virion maturation.     

The infectivity of prM-containing partially mature West Nile virus does not require the activity of cellular furin-like proteases

Cleavage of the flavivirus prM protein by a cellular furin-like protease is a hallmark of virion maturation. While this cleavage is a required step in the viral life cycle, it can be inefficient. Virions that retain uncleaved prM may be infectious. We investigated whether cleavage by furin of prM on partially mature West Nile virus (WNV) during virus entry contributes to infectivity. Using quantitative assays of WNV infection, we found that virions incorporating considerable amounts of uncleaved prM protein were insensitive to treatment of cells with a potent inhibitor of furin activity. Thus, partially mature WNV does not require furin-like proteases for infectivity.     

Monoclonal antibody AP33 defines a broadly neutralizing epitope on the hepatitis C virus E2 envelope glycoprotein

Hepatitis C virus (HCV) remains a significant threat to the general health of the world's population, and there is a pressing need for the development of new treatments and preventative vaccines. Here, we describe the generation of retrovirus-based pseudoparticles (HCVpp) incorporating a panel of full-length E1E2 clones representative of the major genotypes 1 through 6, and their application to assess the reactivity and neutralizing capability of antisera and monoclonal antibodies raised against portions of the HCV E2 envelope protein. Rabbit antisera raised against either the first hypervariable region or ectodomain of E2 showed limited and strain specific neutralization. By contrast, the monoclonal antibody (MAb) AP33 demonstrated potent neutralization of infectivity against HCVpp carrying E1E2 representative of all genotypes tested. The concentration of AP33 required to achieve 50% inhibition of infection by HCVpp of diverse genotypes ranged from 0.6 to 32 mug/ml. The epitope recognized by MAb AP33 is linear and highly conserved across different genotypes of HCV. Thus, identification of a broadly neutralizing antibody that recognizes a linear epitope is likely to be of significant benefit to future vaccine and therapeutic antibody development.     

Partial maturation: an immune-evasion strategy of dengue virus?

Cleavage of the precursor membrane (prM) protein is required for the activation of flavivirus infectivity. However, many studies have shown that, for dengue virus in particular, prM cleavage and maturation is inefficient. Heterogeneity of wild-type dengue virus preparations with regard to the presence of uncleaved prM in the virion is mirrored in the substantial levels of prM-specific antibodies that are produced following dengue infection. What might be the evolutionary advantage for the virus to produce so many prM-containing particles? In this review we summarize the latest achievements of dengue research that contribute to a better understanding of the role of prM-containing virions in the pathogenesis of dengue.     

Identification of a new quaternary neutralizing epitope on human immunodeficiency virus type 1 virus particles

The selection of human monoclonal antibodies (MAbs) specific for human immunodeficiency virus (HIV) type 1 by binding assays may fail to identify Abs to quaternary epitopes on the intact virions. The HIV neutralization assay was used for the selection of human MAb 2909, which potently neutralizes SF162 and recognizes an epitope on the virus surface but not on soluble proteins. Three regions of gp120, the V2 and V3 loops and the CD4 binding domain, contribute to the epitope recognized by MAb 2909. The existence of such a unique MAb, which defines a complex epitope formed by a quaternary structure, suggests that there may be other new neutralizing HIV epitopes to target with vaccines.     

Antibodies against Immature Virions Are Not a Discriminating Factor for Dengue Disease Severity

Humoral immunity plays an important role in controlling dengue virus (DENV) infection. Antibodies (Abs) developed during primary infection protect against subsequent infection with the same dengue serotype, but can enhance disease following secondary infection with a heterologous serotype. A DENV virion has two surface proteins, envelope protein E and (pre)-membrane protein (pr)M, and inefficient cleavage of the prM protein during maturation of progeny virions leads to the secretion of immature and partially immature particles. Interestingly, we and others found that historically regarded non-infectious prM-containing DENV particles can become highly infectious in the presence of E- and prM-Abs. Accordingly, we hypothesized that these virions contribute to the exacerbation of disease during secondary infection. Here, we tested this hypothesis and investigated the ability of acute sera of 30 DENV2-infected patients with different grades of disease severity, to bind, neutralize and/or enhance immature DENV2. We found that a significant fraction of serum Abs bind to the prM protein and to immature virions, but we observed no significant difference between the disease severity groups. Furthermore, functional analysis of the Abs did not underscore any specific correlation between the neutralizing/enhancing activity towards immature DENV2 and the development of more severe disease. Based on our analysis of acute sera, we conclude that Abs binding to immature virions are not a discriminating factor in dengue pathogenesis.     

Histidine at residue 99 and the transmembrane region of the precursor membrane prM protein are important for the prM-E heterodimeric complex formation of Japanese encephalitis virus

The formation of the flavivirus prM-E complex is an important step for the biogenesis of immature virions, which is followed by a subsequent cleavage of prM to M protein through cellular protease to result in the production and release of mature virions. In this study, the intracellular formation of the prM-E complex of Japanese encephalitis virus was investigated by baculovirus coexpression of prM and E in trans in Sf9 insect cells as analyzed by anti-E antibody immunoprecipitation and sucrose gradient sedimentation analysis. A series of carboxyl-terminally truncated prM mutant baculoviruses was constructed to demonstrate that the truncations of the transmembrane (TM) region resulted in a reduction of the formation of the stable prM-E complex by approximately 40% for the TM1 (at residues 130 to 147 [prM130-147]) truncation and 20% for TM2 (at prM153-167) truncation. Alanine-scanning site-directed mutagenesis on the prM99-103 region indicated that the His99 residue was the critical prM binding element for stable prM-E heterodimeric complex formation. The single amino acid mutation at the His99 residue of prM abolishing the prM-E interaction was not due to reduced expression or different subcellular location of the mutant prM protein involved in prM-E interactions as characterized by pulse-chase labeling and confocal scanning microscopic analysis. Recombinant subviral particles were detected in the Sf9 cell culture supernatants by baculovirus coexpression of prM and E proteins but not with the prM H99A mutant. Sequence alignment analysis was further conducted with different groups of flaviviruses to show that the prM H99 residues are generally conserved. Our findings are the first report to characterize the minimum binding elements of the prM protein that are involved in prM-E interactions of flaviviruses. This information, concerning a molecular framework for the prM protein, is considered to elucidate the structure/function relationship of the prM-E complex synthesis and provide the proper trajectory for flavivirus assembly and maturation.     

Structural Basis of Differential Neutralization of DENV-1 Genotypes by an Antibody that Recognizes a Cryptic Epitope

We previously developed a panel of neutralizing monoclonal antibodies against Dengue virus (DENV)-1, of which few exhibited inhibitory activity against all DENV-1 genotypes. This finding is consistent with reports observing variable neutralization of different DENV strains and genotypes using serum from individuals that experienced natural infection or immunization. Herein, we describe the crystal structures of DENV1-E111 bound to a novel CC′ loop epitope on domain III (DIII) of the E protein from two different DENV-1 genotypes. Docking of our structure onto the available cryo-electron microscopy models of DENV virions revealed that the DENV1-E111 epitope was inaccessible, suggesting that this antibody recognizes an uncharacterized virus conformation. While the affinity of binding between DENV1-E111 and DIII varied by genotype, we observed limited correlation with inhibitory activity. Instead, our results support the conclusion that potent neutralization depends on genotype-dependent exposure of the CC′ loop epitope. These findings establish new structural complexity of the DENV virion, which may be relevant for the choice of DENV strain for induction or analysis of neutralizing antibodies in the context of vaccine development.     

4E10-resistant variants in a human immunodeficiency virus type 1 subtype C-infected individual with an anti-membrane-proximal external region-neutralizing antibody response

The broadly neutralizing monoclonal antibody (MAb) 4E10 recognizes a linear epitope in the C terminus of the membrane-proximal external region (MPER) of gp41. This epitope is particularly attractive for vaccine design because it is highly conserved among human immunodeficiency virus type 1 (HIV-1) strains and neutralization escape in vivo has not been observed. Multiple env genes were cloned from an HIV-1 subtype C virus isolated from a 7-year-old perinatally infected child who had anti-MPER neutralizing antibodies. One clone (TM20.13) was resistant to 4E10 neutralization as a result of an F673L substitution in the MPER. Frequency analysis showed that F673L was present in 33% of the viral variants and in all cases was linked to the presence of an intact 2F5 epitope. Two other envelope clones were sensitive to 4E10 neutralization, but TM20.5 was 10-fold less sensitive than TM20.6. Substitutions at positions 674 and 677 within the MPER rendered TM20.5 more sensitive to 4E10 but had no effect on TM20.6. Using chimeric and mutant constructs of these two variants, we further demonstrated that the lentivirus lytic peptide-2 domain in the cytoplasmic tail affected the accessibility of the 4E10 epitope, as well as virus infectivity. Collectively, these genetic changes in the face of a neutralizing antibody response to the MPER strongly suggested immune escape from antibody responses targeting this region.     

The broadly neutralizing anti-human immunodeficiency virus type 1 4E10 monoclonal antibody is better adapted to membrane-bound epitope recognition and blocking than 2F5

The broadly neutralizing 2F5 and 4E10 monoclonal antibodies (MAbs) recognize epitopes within the membrane-proximal external region (MPER) that connects the human immunodeficiency virus type 1 (HIV-1) envelope gp41 ectodomain with the transmembrane anchor. By adopting different conformations that stably insert into the virion external membrane interface, such as helical structures, a conserved aromatic-rich sequence within the MPER is thought to participate in HIV-1-cell fusion. Recent experimental evidence suggests that the neutralizing activity of 2F5 and 4E10 might correlate with the MAbs' capacity to recognize epitopes inserted into the viral membrane, thereby impairing MPER fusogenic activity. To gain new insights into the molecular mechanism underlying viral neutralization by these antibodies, we have compared the capacities of 2F5 and 4E10 to block the membrane-disorganizing activity of MPER peptides inserted into the surface bilayer of solution-diffusing unilamellar vesicles. Both MAbs inhibited leakage of vesicular aqueous contents (membrane permeabilization) and intervesicular lipid mixing (membrane fusion) promoted by MPER-derived peptides. Thus, our data support the idea that antibody binding to a membrane-inserted epitope may interfere with the function of the MPER during gp41-induced fusion. Antibody insertion into a cholesterol-containing, uncharged virion-like membrane is mediated by specific epitope recognition, and moreover, partitioning-coupled folding into a helix reduces the efficiency of 2F5 MAb binding to its epitope in the membrane. We conclude that the capacity to interfere with the membrane activity of conserved MPER sequences is best correlated with the broad neutralization of the 4E10 MAb.     

Human monoclonal antibody to hepatitis C virus E1 glycoprotein that blocks virus attachment and viral infectivity

Human antibodies elicited in response to hepatitis C virus (HCV) infection are anticipated to react with the native conformation of the viral envelope structure. Isolation of these antibodies as human monoclonal antibodies that block virus binding and entry will be useful in providing potential therapeutic reagents and for vaccine development. H-111, an antibody to HCV envelope 1 protein (E1) that maps to the YEVRNVSGVYH sequence and is located near the N terminus of E1 and is able to immunoprecipitate E1E2 heterodimers, is described. Binding of H-111 to HCV E1 genotypes 1a, 1b, 2b, and 3a indicates that the H-111 epitope is highly conserved. Sequence analysis of antibody V regions showed evidence of somatic and affinity maturation of H-111. Finally, H-111 blocks HCV-like particle binding to and HCV virion infection of target cells, suggesting the involvement of this epitope in virus binding and entry.