Monoclonal Antibody AP33 Defines a Broadly Neutral
http://www.100md.com
病菌学杂志 2005年第17期
MRC Virology Unit, Institute of Virology, University of Glasgow, Church Street, Glasgow G11 5JR, United Kingdom
Institute of Infection, Immunity and Inflammation and Division of Microbiology, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom
Laboratoire de Vectorologie Rétrovirale et Thérapie Génique, INSERM U412, IFR128 BioSciences Lyon-Gerland, Ecole Normale Supérieure de Lyon, 69364 Lyon Cedex 07, France
ABSTRACT
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 μg/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.
INTRODUCTION
Hepatitis C virus (HCV), a positive-strand RNA virus belonging to the Flaviviridae family, is the major cause of non-A, non-B viral hepatitis. HCV has infected approximately 200 million people worldwide and current estimates suggest that as many as 3 million individuals are newly infected each year (4). Approximately 80% of those infected fail to clear the virus; a chronic infection ensues, frequently leading to severe chronic liver disease, cirrhosis, and hepatocellular carcinoma (2, 41). Current treatments for chronic infection are ineffective for approximately 50% of patients, and there is a pressing need to develop preventative and therapeutic vaccines.
Due to the error-prone nature of the RNA-dependent RNA polymerase and the high replicative rate in vivo (30, 46), HCV exhibits a high degree of genetic variability. Crucially, this propensity for genetic change allows the virus to respond to and overcome a variety of selective pressures, including host immunity and antiviral therapy (18, 26, 37, 44, 53). HCV can be classified into six genetically distinct genotypes and further subdivided into at least 70 subtypes, which differ by approximately 30% and 15% at the nucleotide level, respectively (59, 61). A significant challenge for the development of vaccines will lie in identifying protective epitopes that are conserved in the majority of viral genotypes and subtypes. This problem is compounded by the fact that the envelope proteins, the natural targets for the neutralizing response, are two of the most variable proteins (10).
The envelope proteins E1 and E2 are responsible for cell binding and entry (5, 8, 16, 51, 57). They are N-linked glycosylated (23, 31, 43, 62) transmembrane proteins with a N-terminal ectodomain and a C-terminal hydrophobic membrane anchor (12, 21, 22). In vitro expression experiments have shown that E1 and E2 proteins form a noncovalent heterodimer, which is proposed to be the functional complex on the virus surface (13, 14, 17, 22). Due to the lack of an efficient culture system, the exact mechanism of viral entry is unknown. That said, there is mounting evidence that entry into isolated primary liver cells and cell lines requires interaction with the cell surface receptors CD81 and scavenger receptor class B type 1 (SR-B1) (7, 8, 19, 58, 66), although these receptors individually are not sufficient to allow viral entry.
Current evidence suggests that cell-mediated immunity is pivotal in clearance and control of HCV replication in acute infection (32, 67). However, surrogate models of infection, such as animal infection and cell and receptor binding assays, have highlighted the potential role of antibodies in both acute and chronic infection (6, 24, 25, 36, 55, 57, 63, 68, 69). It is important to note that not all antibodies that inhibit binding of virus ligand to cell and/or receptors in in vitro assays necessarily neutralize infection. Unsurprisingly, antibodies able to inhibit binding to a cell receptor(s) and/or neutralize infection recognize both linear and conformational epitopes. The majority of antibodies that demonstrate broad neutralization of infection and/or inhibition of receptor binding are directed against conformational epitopes within E2 (1, 9, 33, 34, 36).
Induction of antibodies recognizing conserved conformational epitopes is extremely relevant to vaccine design, but this is likely to prove difficult, as the variable regions appear to be immunodominant (55). One such immunodominant linear epitope lies within the first hypervariable region (HVR-1) of E2 (68). The use of conserved HVR-1 mimotopes has been proposed to overcome problems of restricted specificity (11, 56, 70), but it is not yet known whether this approach will be successful. We and others have reported that a region immediately downstream of HVR-1 contains epitopes which elicit antibodies that potently inhibit CD81 binding (15, 27, 48, 64). One epitope, encompassing residues 412 to 423 and defined by the monoclonal antibody AP33, inhibits the interaction between CD81 and a range of presentations of E2, including soluble E2, E1E2, and virus-like particles (48). While AP33 is capable of blocking CD81 binding, it is unknown whether this will directly correlate with neutralization capacity and, if so, whether or not it will neutralize a diverse range of genetic variants of HCV; an essential property for any promising therapeutic antibody. In addition, it is unknown whether other linear epitopes downstream of HVR-1 could also be important in the development of an antibody-based vaccine.
We (7) and others (35) have recently developed a retroviral pseudoparticle assay whereby infectivity of the retroviral particles is conferred by HCV E1E2 envelope proteins. This assay can also be used to measure the neutralizing capacity of antibodies and sera (6, 40). In this report we describe the use of HCV pseudoparticles (HCVpp) reconstituted with E1E2 clones representative of genotypes 1 through 6 to determine the cross-neutralizing capacity of the AP33 antibody and of polyclonal antisera recognizing epitopes mapped to a region proximal to the AP33 epitope as well as HVR-1.
MATERIALS AND METHODS
Cell culture, plasmid expression constructs, generation of HCVpp, and infection assays. Human Huh-7 hepatoma cells (45) and human epithelial kidney (HEK) 293T cells (ATCC CRL-1573) were propagated as described (15). Plasmids expressing the HCV genotype 1a strain H77-derived full-length E1E2, murine leukemia virus (MLV) Gag-Pol, and the MLV transfer vector carrying green fluorescent protein (GFP) were described previously (7). cDNA sequences encoding the full-length E1E2, representing amino acid residues 170 to 746 of the HCV open reading frame referenced to strain H77 (65), were generated, cloned downstream from a human cytomegalovirus promoter in the expression vector pCR3.1 (Invitrogen) or phCMV-7a (7), and their nucleotide sequences were determined as described (39).
HCVpp were produced essentially as previously reported (7). Briefly, HEK 293T cells were cotransfected with the MLV Gag-Pol packaging vector, the MLV-GFP transfer construct, and a plasmid expressing HCV E1E2 using the calcium phosphate transfection method. In all experiments a no-envelope control was used in which the HCV glycoprotein-expressing construct was excluded from the cotransfections of HEK 293T cells. Two days following transfection, the medium containing HCVpp was collected, clarified, filtered through 0.45-μm pore-sized membranes, and used for infection of Huh-7 cells. Four days following infection, the cells were harvested and analyzed on a FACSCalibur (Becton Dickinson) using CellQuest software. The transduction efficiency was determined as the percentage of GFP-positive cells (following subtraction of the number of GFP-positive Huh-7 cells infected with the no-envelope control, which was typically 0.05%). The infectious titers, expressed as transducing units (TU) per ml, were calculated from the transduction efficiency [(initial number of target cells/volume of HCVpp) x (% GFP-positive cells/100)].
The soluble form of strain H77 E2 (E2660) was produced in COS-7 cells using a recombinant vaccinia virus described previously (49). The secreted form of soluble E2 was used to map antibody epitopes in an enzyme-linked immunosorbent assay (ELISA)-based peptide inhibition assay described below.
Neutralization of HCVpp infection of target cells. Medium containing HCVpp was mixed with appropriate amounts of antibody and incubated for 1 h at 37°C. The mixture was added to Huh-7 cells plated in a six-well tissue culture dish and the cells were incubated at 37°C for 3 h. Following removal of the inoculum, the cells were refed with fresh medium and incubated at 37°C for 4 days. The proportion of infected cells was determined by measurement of GFP by fluorescence-activated cell sorting as described above. The neutralizing activity was expressed as the IC50, defined as the concentration of antibody required to achieve 50% inhibition of infection.
Antibodies. The anti-E2 monoclonal antibodies (MAbs) AP33 and ALP98 and the rabbit polyclonal antisera R645 and R646 have been described previously (15, 48). MAb AP33 and R646 antiserum were purified on a protein G column according to the manufacturer's protocol (Amersham Biosciences). The antisera R1020 and R1021 were generated in New Zealand rabbits immunized with a branched peptide corresponding to the HVR-1 region (residues 384 to 411) of the genotype 1a H77. The immunization protocol used has been described previously (14).
Radiolabeling of proteins. HEK 293T cells were cotransfected with plasmids as described above. Eighteen hours following transfection, cells were washed with phosphate-buffered saline and incubated in methionine- and cysteine-free medium containing 25 μCi/ml of L-[35S]Redivue Pro-Mix (Amersham Biosciences) for 48 h. The medium of transfected cells was harvested and clarified by centrifugation. The cells were washed with phosphate-buffered saline, lysed in lysis buffer (20 mM Tris-HCl, pH 7.4, 20 mM iodoacetamide, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100), and the lysate was spun briefly to remove nuclei. The clarified cell lysates and the medium containing HCVpp were incubated with a mixture of anti-E2 MAbs AP33 and ALP98 for 2 h at 4°C and the immune complexes were precipitated using protein A-Sepharose. Following washes of the protein A-Sepharose beads, the immune complexes were analyzed by sodium dodecyl sulfate (SDS)-10% polyacrylamide gel electrophoresis (PAGE). The gels were dried and exposed overnight to a phosphor screen and the radiolabeled proteins visualized with a Bio-Rad Personal FX phosphorimager.
GNA capture ELISA and antibody adsorption. The ELISA to detect E2 glycoprotein was performed essentially as described previously (49). Briefly, E1E2 glycoproteins from clarified lysates of HEK 293T cells cotransfected with plasmids described above were captured onto Galanthus nivalis antigen (GNA) lectin-coated ELISA plates (Dynex Labsystems). The bound glycoproteins were detected using anti-E2 MAb AP33 or ALP98 or rabbit polyclonal serum R646, followed by an anti-species immunoglobulin G (IgG)-horseradish peroxidase (Sigma) and TMB (3,3', 5,5'-tetramethylbenzidine, Sigma) substrate. Absorbance values were determined at 450 nm.
For antibody adsorption experiments, cells expressing strain H77 E1E2 were lysed in phosphate-buffered saline by freeze-thawing and sonication. The lysates were clarified by brief centrifugation and were either untreated (native) or boiled (denatured) for 10 min after addition of SDS to 0.1% and dithiothreitol to 50 mM final concentration. The boiled lysates were allowed to cool and NP-40 was added to a final concentration of 1% to both denatured and native samples, which were then captured onto GNA-coated Nunc Maxisorp immuno test tubes (Gibco BRL, Life Technologies Ltd., Paisley, United Kingdom). Antiserum R646 was either left untreated or incubated with GNA alone or GNA-captured native or denatured E1E2 for 1 h at room temperature. This was repeated several times and unabsorbed antibodies were then analyzed for reactivity against native and denatured antigens in a GNA capture ELISA as described above.
Mapping of antibody epitopes by peptide competition ELISA. A series of overlapping peptides dissolved in distilled water were preincubated at concentrations stated in the text with antisera R1020, R645, and R646 at room temperature for 30 min. The peptide/antibody mix was then tested for reactivity against GNA-captured secreted E2 in an ELISA as described above. The epitopes recognized by antibodies in the antisera were identified from specific peptides that blocked secreted E2 recognition.
RESULTS
Generation of retroviral particles pseudotyped with HCV E1E2 representing genotypes 1 to 6. We recently reported the isolation of cDNA sequences encoding E1E2 glycoproteins from patients infected with different genotypes of HCV (39). To generate HCVpp enveloped with glycoproteins derived from different genotypes, HEK 293T cells were cotransfected with vectors expressing appropriate HCV E1E2 and MLV Gag-Pol along with an MLV transfer vector encoding the GFP reporter protein. The secreted particles were tested for their ability to infect Huh-7 target cells and the infectivity was determined by measurement by fluorescence-activated cell sorting of the transduced GFP reporter. The E1E2 sequences from the established infectious clone type 1a strain H77 (7) were used as control alongside the patient-derived clones throughout this series of experiments.
A total of 289 patient isolates were screened, and 39 of these were able to render pseudoparticles infectious. A representative selection of these functional clones, the properties of some of which were reported recently by us (39), are shown in Table 1. We identified at least one infectious clone representing each of the genotypes. HCVpp derived from some patient isolates were reproducibly more infectious in this assay than those from the control genotype 1a H77. For example, HCVpp derived from the construct UKN2B1.1 regularly transduced over 30% of the target cells, whereas those derived from 1aH77 only transduced 10 to 20%. Typically, the infectious titers, expressed as transducing units per milliliter (TU/ml), were about 105 TU/ml for UKN2B1.1 and 4 x 104 TU/ml for type 1a H77. In contrast, the genotype 3, 5, and 6 HCVpp gave very low titers (1 x 103 to 4 x 103 TU/ml) (Table 1).
To investigate why many of the isolates lacked infectivity, we checked whether HCV glycoproteins were expressed in the transfected HEK 293T cells. The relative level of the E2 glycoprotein in each cell lysate was determined by a GNA-capture ELISA, using polyclonal rabbit serum R646 and two monoclonal antibodies, AP33 and ALP98, all raised against type 1a E2 (15, 48). Lysates of HEK 293T cells cotransfected with each of the infectious clones together with the MLV Gag-Pol and the GFP reporter constructs contained levels of E2 that gave a strong, concentration-dependent signal with at least one of the two MAbs (Fig. 1). It is noteworthy that type 4 isolate (UKN4.21.16) and one of the two type 5 isolates tested (UKN5.14.4) were not recognized by the MAbs ALP98 and AP33, respectively. This is due to the presence of variant amino acids within the epitopes recognized by these antibodies (see below). The rabbit antiserum R646 almost exclusively recognized the genotype 1a H77 E2; it failed to recognize not only E2 from other genotypes, but also that from a different isolate of the same subtype 1a (Fig. 1A).
Lysates of HEK cells transfected with all the isolates that did not yield infectious HCVpp were also analyzed by GNA ELISA for the presence of E2. While some had no detectable or low levels of E2, others contained levels of E2 similar to those found in lysates of cells transfected with infectious clones (data not shown). We conclude that some isolates lack infectivity because they do not express E2, whereas others are noninfectious despite expressing high levels of E2, presumably because the E2 or the E1E2 complex that they encode is nonfunctional in some way, at least in the HCVpp system. It is possible that the HCVpp system is prone to introducing bias in terms of functionality of the viral glycoproteins.
The HCVpp infectivity is dependent on the incorporation of the full-length E1E2 complex into the envelope of the particles (7, 35). Although the ELISA data above confirmed the presence of E2 derived from different genotypes, presence of E1 could not be analyzed, because we lacked a broadly reactive anti-E1 antibody. Instead, we investigated E1E2 complex formation by immunoprecipitation. HEK 293T cells cotransfected with the HCV glycoprotein-expressing constructs and the MLV Gag-Pol and GFP transfer vector were radiolabeled with [35S]methionine/cysteine. The intracellular form of the radiolabeled proteins was immunoprecipitated with a mixture of anti-E2 MAbs AP33 and ALP98. As shown in Fig. 1B, E1 was coimmunoprecipitated along with E2 from the lysates of cells transfected with most of the glycoprotein-expressing constructs.
There was a degree of variation in the relative amounts of the proteins produced and interesting differences in the molecular weights of the precipitated proteins (particularly E1) were apparent. These may be due to differential glycosylation, as nucleotide sequence analysis show variations in the predicted glycosylation sites between different genotypes (39), although differences in amino acid residues may also be responsible. It is noteworthy that there was a significant variation in the relative stoichiometry between E1 and E2 of different genotypes. Similarly, E1E2 complexes secreted into the medium (a proportion of which were expected to be in the form of HCVpp) of the transfected cells were also detected by immunoprecipitation with the same MAbs (data not shown). Clearly, further investigation is warranted to determine the nature of the functional E1E2 complex formation in the different genotypes studied here.
Antibody-mediated neutralization of HCVpp infection of target cells. We have previously shown using in vitro assays that the MAb AP33 blocks the interaction between different E2 forms (derived from genotype 1a) and CD81 (48), a cell surface receptor which is essential for HCVpp entry into the target cells (8, 19, 35, 39). The latter studies have also implicated the HVR-1 of E2 in HCVpp infection, as antibodies to this region block entry. Here we tested the ability of MAb AP33 and rabbit antisera R645 and R646 (both raised against the soluble ectodomain of type 1a H77) and R1020 and R1021 (both specific to the HVR-1 region of strain H77 E2) to inhibit type 1a H77 HCVpp infection of cells. Of the rabbit antisera tested, R646 was able to completely abrogate infection, whereas R645 blocked infection to approximately 50%. Consistent with the previously established role of HVR-1 in infection, both the anti-HVR-1 antisera were able to neutralize infection by 65% (Fig. 2A). As expected, the corresponding preimmune rabbit sera had no effect on HCVpp infection. Similarly to R646, MAb AP33 completely blocked infection of Huh-7 cells by strain H77 HCVpp.
We next tested the ability of these antisera and the MAb AP33 to inhibit HCV genotypes other than 1a H77. We found that AP33 was broadly cross-neutralizing, whereas antisera R645, R646 and R1020 had very little effect on the infectivity of HCVpp incorporating the type 1b, 2a, or 2b glycoproteins (Fig. 2B). Having established that R646 does not significantly inhibit infection by HCVpp derived from genotypes other than 1a, we checked whether its specificity was limited to glycoproteins of type 1a H77, or whether it could inhibit other type 1a isolates. As shown in Fig. 2C, this antiserum was less effective on the two patient-derived isolates 1A14.8 and 1A14.36 than on 1a H77, against which it had an IC50 value of 200 ng IgG/ml.
The broad reactivity of MAb AP33 was examined further by testing it over a range of concentrations on a panel of HCVpp incorporating E1E2 derived from isolates representing the full complement of genotypes. As shown in Fig. 3, HCVpp harboring E1E2 of isolates representing all 6 genotypes were effectively neutralized, with the IC50 ranging from 0.6 μg/ml for type 5 to 32 μg/ml for genotype 3a. The infectivity of one of the genotype 5 HCVpp isolates, UKN5.14.4, was not affected by MAb AP33 (data not shown) since the E2 glycoprotein of this infectious isolate is not recognized by this antibody (Fig. 1A).
Epitope mapping of antibodies in the antiserum R646. To investigate the reason for the restricted specificity of R646, the linear epitope(s) recognized by this antiserum were determined using a series of 18-mer overlapping peptides spanning the entire E2 sequence (excluding insoluble regions) of strain H77. These peptides were tested initially at a concentration of 0.5 mg/ml for their ability to block recognition by R646 of the strain H77-derived soluble E2 captured by GNA in an ELISA. The only inhibition observed was by peptides representing the N-terminal portion of E2 (amino acids 384 to 451; data not shown). Peptides spanning this region were further tested at four concentrations, ranging from 31 to 830 μg/ml, in threefold dilutions (Fig. 4).
Antisera R645 and R1020 were tested in parallel. R1020 was raised against a peptide corresponding to the HVR-1 (amino acid residues 384 to 411) of the genotype 1a H77. Peptides 20 and 21 cover this region, and indeed peptide 21 competed effectively with E2 for the binding site of R1020 IgG, at all concentrations tested. Peptide 21 also inhibited R646 binding more strongly than any of the other peptides. In the presence of 830 μg/ml of peptide 21, R646 binding to E2 was reduced by 78%. Peptides 20, 23, 24, and 25 also appeared to inhibit in a concentration-dependent manner, albeit to a lesser extent. Peptide 22, which carries the MAb AP33 epitope (with the exception of its last two residues), failed to inhibit R646 recognition of soluble E2, indicating that R646 lacked antibodies specific to this epitope. This was confirmed by competition ELISA where the MAb AP33 failed to inhibit R646 recognition of soluble E2 (data not shown). Interestingly, the binding of antiserum R645 to E2 was not significantly affected by any of these peptides. These results suggest that antibodies in R646 serum recognize part of the HVR-1 of E2 and sequences downstream from it (amino acids 414 to 451).
It may be that R646 also recognizes one or more conformational epitopes that are not mimicked by any linear peptide. To test this hypothesis we preadsorbed purified R646 IgG with native and denatured E2, and then tested its capacity to recognize E2 in a GNA-capture ELISA and to inhibit infection by genotype 1a H77 HCVpp. As shown in Fig. 5, preadsorption with native but not with denatured E2 reduced both its recognition and its neutralization capacity by a factor of 10. This supports the idea that antibodies in the R646 serum recognize one or more conformational epitopes, present on native but not on denatured E2, which also contribute to neutralization of HCVpp infection.
Amino acid sequence alignment (Fig. 6a) of the N-terminal region of E2 of the different genotypes used in this study showed that the MAb AP33 epitope (residues 412 to 423) is relatively well conserved with only three variant amino acid sites (residues 412, 414, and 416). Other sites within this epitope are absolutely conserved. An exception to this was the corresponding amino acid sequence in the E2 encoded by the genotype 5 isolate UKN5.14.4, which has a 4 residue change in the sequence with a –1 shift in the location of the potential glycosylation site (NGS) relative to that of the other isolates (Fig. 6A). In contrast, significant variations were seen between different genotypes in sequences corresponding to the genotype 1a H77 linear epitopes recognized by R646 consistent with its restricted specificity. Finally, the epitope recognized by the ALP98 antibody (residues 644 to 651) was also conserved although an arginine to valine mutation at position 651 in the genotype 4 clones abolished recognition by this antibody (Fig. 6B).
DISCUSSION
Prior to the development of the retroviral pseudotype assay (7, 35) studies of the neutralizing potential of antibodies and sera were only possible using less direct surrogate assays, including receptor and cell binding assays (51, 57), together with animal models (25, 29). Previous studies have determined the neutralizing capacity of sera or antibodies against only a limited range of HCV genotypes (6, 28, 47). In this paper we have used the HCVpp assay to assess the capacity of a set of E2-specific antibodies and sera to neutralize HCVpp carrying E1E2 representative of all of the major genotypes 1 through 6. To facilitate this we have developed robust PCR methods capable of amplifying full-length E1E2 ex vivo.
In our previous work we noted that many E1E2 sequences recovered from patient-derived samples were not capable of conferring HCVpp infectivity (39), as has been reported recently (42). Here we extend our earlier findings and show that over 85% of recovered envelope genes fail to function in the HCVpp assay. There are a number of factors that may contribute to the high proportion of nonfunctional clones. First, although the PCR utilized proofreading enzymes to minimize the number of misincorporations during the amplification process, errors will still arise and there will inevitably be a proportion of clones that contain sporadic point mutations (60). Second, given the quasispecies nature of HCV (10, 54), some of the nonfunctional clones will contain naturally occurring deleterious mutations. Finally, a lack of infectivity might be attributable to inadequacies in the sensitivity of the HCVpp assay. Certainly, some but not all of the nonfunctional clones could be rendered infectious after transfer to a higher-expression vector (data not shown). Identifying the residues that affect infectivity in the HCVpp assay is of considerable interest, and the panel of functional and nonfunctional clones derived from the same source will enable us to address this issue.
Having established a panel of functional E1E2 clones representative of all the major genotypes, we went on to assess the capacity of the MAb AP33 and of rabbit antisera raised against the E2 protein of the H77 strain to neutralize HCVpp entry. Rabbit sera raised against HVR-1 of E2 were capable of neutralizing HCVpp incorporating H77 E1E2, but not glycoproteins derived from other genotypes. The high degree of HVR-1 genetic variability explains the poor cross-neutralization observed, in keeping with previous reports of the restricted neutralizing capacity of natural HVR-1-specific antibodies (70). Immunization of rabbits with the H77 E2 ectodomain also resulted in the induction of neutralizing antibodies, but again these responses were highly strain specific. Peptide mapping and competition assays showed that the most potently neutralizing serum (R646) recognized both conformational and linear determinants. Neutralization could be severely reduced by preadsorbing the serum with native but not denatured soluble E2, indicating that the neutralizing antibodies most likely recognize conformational epitopes.
Previous work has shown that antibodies elicited by immunization of chimpanzees with HCV envelope glycoproteins can partially protect against homologous challenge (29, 55). Similarly, a number of conformation sensitive human monoclonal antibodies capable of broadly inhibiting binding of E2 to CD81 have been described (1, 9, 33, 34, 36, 38). Some of these antibodies also neutralize HCVpp infection and as such they could have a future role in the treatment of HCV infection and they might also serve to define future vaccine candidates. However, studies with human immunodeficiency virus type 1 have shown that focusing the immune response on epitopes recognized by broadly neutralizing antibodies is a significant challenge. In this context, the finding that AP33 potently neutralizes the entry of HCVpp carrying highly divergent E1E2 is significant, particularly as its epitope is linear and highly conserved across different genotypes of HCV. The exact mechanism of neutralization by AP33 is unknown, although inhibition of CD81 binding is the most likely (48). The epitope recognized by AP33 has been mapped to residues 412 to 423 (QLINTNGSWHIN) and carries one potential N-linked glycosylation site (48).
It is interesting that HCVpp derived from one genotype 5 isolate (UKN5.14.4), although infectious, were not recognized (and therefore not neutralized) by the MAb AP33. This isolate has a 4-amino-acid change (QLIQNGSSWHIN) in the E2 region corresponding to the AP33 epitope, with a well-conserved N-linked glycosylation site shifted –1 relative to that in the other isolates. Subsequent analysis of over 5,500 sequences deposited into the GenBank database has shown the AP33 epitope to be highly conserved. The average diversity of sequences compared to the prototype AP33 epitope is 4.7%, and the majority of variable amino acids are located at the N terminus of the predicted epitope. Importantly, sequences similar to that present in UKN5.14.4 were not evident. Our preliminary data show that reversion of one of the four variant amino acids to its conserved counterpart (i.e., Q to N) renders the UKN5.14.4 HCVpp noninfectious (while also remaining nonreactive to MAb AP33) (not shown), highlighting the possible importance of this region of E2 in infection. This observation, together with the fact that the infectivity of UKN5.14.4 HCVpp is not affected by the N415Q and the –1 shift of the N-linked glycosylation sequence, further indicates that this particular isolate may represent a neutralization escape mutant. Further in-depth site-directed mutagenesis and structural studies will be needed to fully understand the AP33-E2 interaction and mechanisms of neutralization.
The IC50 of AP33 when neutralizing HCVpp of diverse genotypes ranged from approximately 0.6 up to 32 μg/ml. Immunization with peptides corresponding to HVR-1 can lead to the generation of sera capable of protection against homologous virus (25), and therefore induction of AP33-type antibodies, at titers capable of inducing neutralization, should be possible. That said, studies with the broadly neutralizing human immunodeficiency virus type 1 antibodies 2G12, b12, and 2F5 show that protection from in vivo challenge might require higher concentrations of antibody than their in vitro IC50 (52). However, many factors affect in vivo challenge and in vitro neutralization results. Currently it is unknown what antibody concentrations will offer protection against HCV infection or how predictive HCVpp neutralization is of natural protection.
One factor that will affect the neutralizing titers will be purity and homogeneity of the HCVpp inocula. We have used cell supernatants as the source of HCVpp without further purification; such preparations contain various forms of E2 (20). Normalization with respect to Gag-Pol protein or E2 achieves little, because neither protein is restricted to infectious HCVpp; the supernatant contains a heterogeneous mix of materials, including soluble E2, aggregates of E2, nonenveloped Gag-Pol particles, noninfectious enveloped particles, and infectious enveloped particles. The relative proportion of each of these almost certainly varies from one genotype to another. Purifying HCVpp through a sucrose cushion results in a significant loss of infectivity and only achieves the removal of the soluble E2 component. The theoretical ideal of isolating only infectious HCVpp is not realistically achievable.
In conclusion, we have shown that the epitope defined by the AP33 antibody is highly conserved across all the major genotypes and that this antibody is capable of broad neutralization, at least in the HCVpp assay. HCV particles in infected patients are associated with plasma lipoprotein (3), which may mask epitope availability, although antibodies to HVR-1 are clearly able to neutralize infection in chimpanzees (25). Furthermore, Petit et al. (50) more recently demonstrated that native virions have at least some E1E2 epitopes surface exposed. Nevertheless, it will be important to define whether or not MAb AP33 is indeed capable of neutralizing infection by patient-derived virions and, if so, whether passive transfer of this antibody can protect in animal model challenge experiments. Similarly, immunization and challenge studies using various immunogens containing the AP33 epitope will define its usefulness as a vaccine candidate. The identification of a conserved neutralizing linear epitope offers significant hope for the development of a successful HCV vaccine.
ACKNOWLEDGMENTS
This work was supported by the European Community (contract QLRT-2000-01120, ENHCV), the Medical Research Council United Kingdom, the University of Nottingham Research Committee, the Nottingham University Hospitals' Special Trustees, and the Agence Nationale pour la Recherche contre le SIDA et les Hépatites Virales.
A.O. and A.W.T. contributed equally to this work.
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Institute of Infection, Immunity and Inflammation and Division of Microbiology, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom
Laboratoire de Vectorologie Rétrovirale et Thérapie Génique, INSERM U412, IFR128 BioSciences Lyon-Gerland, Ecole Normale Supérieure de Lyon, 69364 Lyon Cedex 07, France
ABSTRACT
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 μg/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.
INTRODUCTION
Hepatitis C virus (HCV), a positive-strand RNA virus belonging to the Flaviviridae family, is the major cause of non-A, non-B viral hepatitis. HCV has infected approximately 200 million people worldwide and current estimates suggest that as many as 3 million individuals are newly infected each year (4). Approximately 80% of those infected fail to clear the virus; a chronic infection ensues, frequently leading to severe chronic liver disease, cirrhosis, and hepatocellular carcinoma (2, 41). Current treatments for chronic infection are ineffective for approximately 50% of patients, and there is a pressing need to develop preventative and therapeutic vaccines.
Due to the error-prone nature of the RNA-dependent RNA polymerase and the high replicative rate in vivo (30, 46), HCV exhibits a high degree of genetic variability. Crucially, this propensity for genetic change allows the virus to respond to and overcome a variety of selective pressures, including host immunity and antiviral therapy (18, 26, 37, 44, 53). HCV can be classified into six genetically distinct genotypes and further subdivided into at least 70 subtypes, which differ by approximately 30% and 15% at the nucleotide level, respectively (59, 61). A significant challenge for the development of vaccines will lie in identifying protective epitopes that are conserved in the majority of viral genotypes and subtypes. This problem is compounded by the fact that the envelope proteins, the natural targets for the neutralizing response, are two of the most variable proteins (10).
The envelope proteins E1 and E2 are responsible for cell binding and entry (5, 8, 16, 51, 57). They are N-linked glycosylated (23, 31, 43, 62) transmembrane proteins with a N-terminal ectodomain and a C-terminal hydrophobic membrane anchor (12, 21, 22). In vitro expression experiments have shown that E1 and E2 proteins form a noncovalent heterodimer, which is proposed to be the functional complex on the virus surface (13, 14, 17, 22). Due to the lack of an efficient culture system, the exact mechanism of viral entry is unknown. That said, there is mounting evidence that entry into isolated primary liver cells and cell lines requires interaction with the cell surface receptors CD81 and scavenger receptor class B type 1 (SR-B1) (7, 8, 19, 58, 66), although these receptors individually are not sufficient to allow viral entry.
Current evidence suggests that cell-mediated immunity is pivotal in clearance and control of HCV replication in acute infection (32, 67). However, surrogate models of infection, such as animal infection and cell and receptor binding assays, have highlighted the potential role of antibodies in both acute and chronic infection (6, 24, 25, 36, 55, 57, 63, 68, 69). It is important to note that not all antibodies that inhibit binding of virus ligand to cell and/or receptors in in vitro assays necessarily neutralize infection. Unsurprisingly, antibodies able to inhibit binding to a cell receptor(s) and/or neutralize infection recognize both linear and conformational epitopes. The majority of antibodies that demonstrate broad neutralization of infection and/or inhibition of receptor binding are directed against conformational epitopes within E2 (1, 9, 33, 34, 36).
Induction of antibodies recognizing conserved conformational epitopes is extremely relevant to vaccine design, but this is likely to prove difficult, as the variable regions appear to be immunodominant (55). One such immunodominant linear epitope lies within the first hypervariable region (HVR-1) of E2 (68). The use of conserved HVR-1 mimotopes has been proposed to overcome problems of restricted specificity (11, 56, 70), but it is not yet known whether this approach will be successful. We and others have reported that a region immediately downstream of HVR-1 contains epitopes which elicit antibodies that potently inhibit CD81 binding (15, 27, 48, 64). One epitope, encompassing residues 412 to 423 and defined by the monoclonal antibody AP33, inhibits the interaction between CD81 and a range of presentations of E2, including soluble E2, E1E2, and virus-like particles (48). While AP33 is capable of blocking CD81 binding, it is unknown whether this will directly correlate with neutralization capacity and, if so, whether or not it will neutralize a diverse range of genetic variants of HCV; an essential property for any promising therapeutic antibody. In addition, it is unknown whether other linear epitopes downstream of HVR-1 could also be important in the development of an antibody-based vaccine.
We (7) and others (35) have recently developed a retroviral pseudoparticle assay whereby infectivity of the retroviral particles is conferred by HCV E1E2 envelope proteins. This assay can also be used to measure the neutralizing capacity of antibodies and sera (6, 40). In this report we describe the use of HCV pseudoparticles (HCVpp) reconstituted with E1E2 clones representative of genotypes 1 through 6 to determine the cross-neutralizing capacity of the AP33 antibody and of polyclonal antisera recognizing epitopes mapped to a region proximal to the AP33 epitope as well as HVR-1.
MATERIALS AND METHODS
Cell culture, plasmid expression constructs, generation of HCVpp, and infection assays. Human Huh-7 hepatoma cells (45) and human epithelial kidney (HEK) 293T cells (ATCC CRL-1573) were propagated as described (15). Plasmids expressing the HCV genotype 1a strain H77-derived full-length E1E2, murine leukemia virus (MLV) Gag-Pol, and the MLV transfer vector carrying green fluorescent protein (GFP) were described previously (7). cDNA sequences encoding the full-length E1E2, representing amino acid residues 170 to 746 of the HCV open reading frame referenced to strain H77 (65), were generated, cloned downstream from a human cytomegalovirus promoter in the expression vector pCR3.1 (Invitrogen) or phCMV-7a (7), and their nucleotide sequences were determined as described (39).
HCVpp were produced essentially as previously reported (7). Briefly, HEK 293T cells were cotransfected with the MLV Gag-Pol packaging vector, the MLV-GFP transfer construct, and a plasmid expressing HCV E1E2 using the calcium phosphate transfection method. In all experiments a no-envelope control was used in which the HCV glycoprotein-expressing construct was excluded from the cotransfections of HEK 293T cells. Two days following transfection, the medium containing HCVpp was collected, clarified, filtered through 0.45-μm pore-sized membranes, and used for infection of Huh-7 cells. Four days following infection, the cells were harvested and analyzed on a FACSCalibur (Becton Dickinson) using CellQuest software. The transduction efficiency was determined as the percentage of GFP-positive cells (following subtraction of the number of GFP-positive Huh-7 cells infected with the no-envelope control, which was typically 0.05%). The infectious titers, expressed as transducing units (TU) per ml, were calculated from the transduction efficiency [(initial number of target cells/volume of HCVpp) x (% GFP-positive cells/100)].
The soluble form of strain H77 E2 (E2660) was produced in COS-7 cells using a recombinant vaccinia virus described previously (49). The secreted form of soluble E2 was used to map antibody epitopes in an enzyme-linked immunosorbent assay (ELISA)-based peptide inhibition assay described below.
Neutralization of HCVpp infection of target cells. Medium containing HCVpp was mixed with appropriate amounts of antibody and incubated for 1 h at 37°C. The mixture was added to Huh-7 cells plated in a six-well tissue culture dish and the cells were incubated at 37°C for 3 h. Following removal of the inoculum, the cells were refed with fresh medium and incubated at 37°C for 4 days. The proportion of infected cells was determined by measurement of GFP by fluorescence-activated cell sorting as described above. The neutralizing activity was expressed as the IC50, defined as the concentration of antibody required to achieve 50% inhibition of infection.
Antibodies. The anti-E2 monoclonal antibodies (MAbs) AP33 and ALP98 and the rabbit polyclonal antisera R645 and R646 have been described previously (15, 48). MAb AP33 and R646 antiserum were purified on a protein G column according to the manufacturer's protocol (Amersham Biosciences). The antisera R1020 and R1021 were generated in New Zealand rabbits immunized with a branched peptide corresponding to the HVR-1 region (residues 384 to 411) of the genotype 1a H77. The immunization protocol used has been described previously (14).
Radiolabeling of proteins. HEK 293T cells were cotransfected with plasmids as described above. Eighteen hours following transfection, cells were washed with phosphate-buffered saline and incubated in methionine- and cysteine-free medium containing 25 μCi/ml of L-[35S]Redivue Pro-Mix (Amersham Biosciences) for 48 h. The medium of transfected cells was harvested and clarified by centrifugation. The cells were washed with phosphate-buffered saline, lysed in lysis buffer (20 mM Tris-HCl, pH 7.4, 20 mM iodoacetamide, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100), and the lysate was spun briefly to remove nuclei. The clarified cell lysates and the medium containing HCVpp were incubated with a mixture of anti-E2 MAbs AP33 and ALP98 for 2 h at 4°C and the immune complexes were precipitated using protein A-Sepharose. Following washes of the protein A-Sepharose beads, the immune complexes were analyzed by sodium dodecyl sulfate (SDS)-10% polyacrylamide gel electrophoresis (PAGE). The gels were dried and exposed overnight to a phosphor screen and the radiolabeled proteins visualized with a Bio-Rad Personal FX phosphorimager.
GNA capture ELISA and antibody adsorption. The ELISA to detect E2 glycoprotein was performed essentially as described previously (49). Briefly, E1E2 glycoproteins from clarified lysates of HEK 293T cells cotransfected with plasmids described above were captured onto Galanthus nivalis antigen (GNA) lectin-coated ELISA plates (Dynex Labsystems). The bound glycoproteins were detected using anti-E2 MAb AP33 or ALP98 or rabbit polyclonal serum R646, followed by an anti-species immunoglobulin G (IgG)-horseradish peroxidase (Sigma) and TMB (3,3', 5,5'-tetramethylbenzidine, Sigma) substrate. Absorbance values were determined at 450 nm.
For antibody adsorption experiments, cells expressing strain H77 E1E2 were lysed in phosphate-buffered saline by freeze-thawing and sonication. The lysates were clarified by brief centrifugation and were either untreated (native) or boiled (denatured) for 10 min after addition of SDS to 0.1% and dithiothreitol to 50 mM final concentration. The boiled lysates were allowed to cool and NP-40 was added to a final concentration of 1% to both denatured and native samples, which were then captured onto GNA-coated Nunc Maxisorp immuno test tubes (Gibco BRL, Life Technologies Ltd., Paisley, United Kingdom). Antiserum R646 was either left untreated or incubated with GNA alone or GNA-captured native or denatured E1E2 for 1 h at room temperature. This was repeated several times and unabsorbed antibodies were then analyzed for reactivity against native and denatured antigens in a GNA capture ELISA as described above.
Mapping of antibody epitopes by peptide competition ELISA. A series of overlapping peptides dissolved in distilled water were preincubated at concentrations stated in the text with antisera R1020, R645, and R646 at room temperature for 30 min. The peptide/antibody mix was then tested for reactivity against GNA-captured secreted E2 in an ELISA as described above. The epitopes recognized by antibodies in the antisera were identified from specific peptides that blocked secreted E2 recognition.
RESULTS
Generation of retroviral particles pseudotyped with HCV E1E2 representing genotypes 1 to 6. We recently reported the isolation of cDNA sequences encoding E1E2 glycoproteins from patients infected with different genotypes of HCV (39). To generate HCVpp enveloped with glycoproteins derived from different genotypes, HEK 293T cells were cotransfected with vectors expressing appropriate HCV E1E2 and MLV Gag-Pol along with an MLV transfer vector encoding the GFP reporter protein. The secreted particles were tested for their ability to infect Huh-7 target cells and the infectivity was determined by measurement by fluorescence-activated cell sorting of the transduced GFP reporter. The E1E2 sequences from the established infectious clone type 1a strain H77 (7) were used as control alongside the patient-derived clones throughout this series of experiments.
A total of 289 patient isolates were screened, and 39 of these were able to render pseudoparticles infectious. A representative selection of these functional clones, the properties of some of which were reported recently by us (39), are shown in Table 1. We identified at least one infectious clone representing each of the genotypes. HCVpp derived from some patient isolates were reproducibly more infectious in this assay than those from the control genotype 1a H77. For example, HCVpp derived from the construct UKN2B1.1 regularly transduced over 30% of the target cells, whereas those derived from 1aH77 only transduced 10 to 20%. Typically, the infectious titers, expressed as transducing units per milliliter (TU/ml), were about 105 TU/ml for UKN2B1.1 and 4 x 104 TU/ml for type 1a H77. In contrast, the genotype 3, 5, and 6 HCVpp gave very low titers (1 x 103 to 4 x 103 TU/ml) (Table 1).
To investigate why many of the isolates lacked infectivity, we checked whether HCV glycoproteins were expressed in the transfected HEK 293T cells. The relative level of the E2 glycoprotein in each cell lysate was determined by a GNA-capture ELISA, using polyclonal rabbit serum R646 and two monoclonal antibodies, AP33 and ALP98, all raised against type 1a E2 (15, 48). Lysates of HEK 293T cells cotransfected with each of the infectious clones together with the MLV Gag-Pol and the GFP reporter constructs contained levels of E2 that gave a strong, concentration-dependent signal with at least one of the two MAbs (Fig. 1). It is noteworthy that type 4 isolate (UKN4.21.16) and one of the two type 5 isolates tested (UKN5.14.4) were not recognized by the MAbs ALP98 and AP33, respectively. This is due to the presence of variant amino acids within the epitopes recognized by these antibodies (see below). The rabbit antiserum R646 almost exclusively recognized the genotype 1a H77 E2; it failed to recognize not only E2 from other genotypes, but also that from a different isolate of the same subtype 1a (Fig. 1A).
Lysates of HEK cells transfected with all the isolates that did not yield infectious HCVpp were also analyzed by GNA ELISA for the presence of E2. While some had no detectable or low levels of E2, others contained levels of E2 similar to those found in lysates of cells transfected with infectious clones (data not shown). We conclude that some isolates lack infectivity because they do not express E2, whereas others are noninfectious despite expressing high levels of E2, presumably because the E2 or the E1E2 complex that they encode is nonfunctional in some way, at least in the HCVpp system. It is possible that the HCVpp system is prone to introducing bias in terms of functionality of the viral glycoproteins.
The HCVpp infectivity is dependent on the incorporation of the full-length E1E2 complex into the envelope of the particles (7, 35). Although the ELISA data above confirmed the presence of E2 derived from different genotypes, presence of E1 could not be analyzed, because we lacked a broadly reactive anti-E1 antibody. Instead, we investigated E1E2 complex formation by immunoprecipitation. HEK 293T cells cotransfected with the HCV glycoprotein-expressing constructs and the MLV Gag-Pol and GFP transfer vector were radiolabeled with [35S]methionine/cysteine. The intracellular form of the radiolabeled proteins was immunoprecipitated with a mixture of anti-E2 MAbs AP33 and ALP98. As shown in Fig. 1B, E1 was coimmunoprecipitated along with E2 from the lysates of cells transfected with most of the glycoprotein-expressing constructs.
There was a degree of variation in the relative amounts of the proteins produced and interesting differences in the molecular weights of the precipitated proteins (particularly E1) were apparent. These may be due to differential glycosylation, as nucleotide sequence analysis show variations in the predicted glycosylation sites between different genotypes (39), although differences in amino acid residues may also be responsible. It is noteworthy that there was a significant variation in the relative stoichiometry between E1 and E2 of different genotypes. Similarly, E1E2 complexes secreted into the medium (a proportion of which were expected to be in the form of HCVpp) of the transfected cells were also detected by immunoprecipitation with the same MAbs (data not shown). Clearly, further investigation is warranted to determine the nature of the functional E1E2 complex formation in the different genotypes studied here.
Antibody-mediated neutralization of HCVpp infection of target cells. We have previously shown using in vitro assays that the MAb AP33 blocks the interaction between different E2 forms (derived from genotype 1a) and CD81 (48), a cell surface receptor which is essential for HCVpp entry into the target cells (8, 19, 35, 39). The latter studies have also implicated the HVR-1 of E2 in HCVpp infection, as antibodies to this region block entry. Here we tested the ability of MAb AP33 and rabbit antisera R645 and R646 (both raised against the soluble ectodomain of type 1a H77) and R1020 and R1021 (both specific to the HVR-1 region of strain H77 E2) to inhibit type 1a H77 HCVpp infection of cells. Of the rabbit antisera tested, R646 was able to completely abrogate infection, whereas R645 blocked infection to approximately 50%. Consistent with the previously established role of HVR-1 in infection, both the anti-HVR-1 antisera were able to neutralize infection by 65% (Fig. 2A). As expected, the corresponding preimmune rabbit sera had no effect on HCVpp infection. Similarly to R646, MAb AP33 completely blocked infection of Huh-7 cells by strain H77 HCVpp.
We next tested the ability of these antisera and the MAb AP33 to inhibit HCV genotypes other than 1a H77. We found that AP33 was broadly cross-neutralizing, whereas antisera R645, R646 and R1020 had very little effect on the infectivity of HCVpp incorporating the type 1b, 2a, or 2b glycoproteins (Fig. 2B). Having established that R646 does not significantly inhibit infection by HCVpp derived from genotypes other than 1a, we checked whether its specificity was limited to glycoproteins of type 1a H77, or whether it could inhibit other type 1a isolates. As shown in Fig. 2C, this antiserum was less effective on the two patient-derived isolates 1A14.8 and 1A14.36 than on 1a H77, against which it had an IC50 value of 200 ng IgG/ml.
The broad reactivity of MAb AP33 was examined further by testing it over a range of concentrations on a panel of HCVpp incorporating E1E2 derived from isolates representing the full complement of genotypes. As shown in Fig. 3, HCVpp harboring E1E2 of isolates representing all 6 genotypes were effectively neutralized, with the IC50 ranging from 0.6 μg/ml for type 5 to 32 μg/ml for genotype 3a. The infectivity of one of the genotype 5 HCVpp isolates, UKN5.14.4, was not affected by MAb AP33 (data not shown) since the E2 glycoprotein of this infectious isolate is not recognized by this antibody (Fig. 1A).
Epitope mapping of antibodies in the antiserum R646. To investigate the reason for the restricted specificity of R646, the linear epitope(s) recognized by this antiserum were determined using a series of 18-mer overlapping peptides spanning the entire E2 sequence (excluding insoluble regions) of strain H77. These peptides were tested initially at a concentration of 0.5 mg/ml for their ability to block recognition by R646 of the strain H77-derived soluble E2 captured by GNA in an ELISA. The only inhibition observed was by peptides representing the N-terminal portion of E2 (amino acids 384 to 451; data not shown). Peptides spanning this region were further tested at four concentrations, ranging from 31 to 830 μg/ml, in threefold dilutions (Fig. 4).
Antisera R645 and R1020 were tested in parallel. R1020 was raised against a peptide corresponding to the HVR-1 (amino acid residues 384 to 411) of the genotype 1a H77. Peptides 20 and 21 cover this region, and indeed peptide 21 competed effectively with E2 for the binding site of R1020 IgG, at all concentrations tested. Peptide 21 also inhibited R646 binding more strongly than any of the other peptides. In the presence of 830 μg/ml of peptide 21, R646 binding to E2 was reduced by 78%. Peptides 20, 23, 24, and 25 also appeared to inhibit in a concentration-dependent manner, albeit to a lesser extent. Peptide 22, which carries the MAb AP33 epitope (with the exception of its last two residues), failed to inhibit R646 recognition of soluble E2, indicating that R646 lacked antibodies specific to this epitope. This was confirmed by competition ELISA where the MAb AP33 failed to inhibit R646 recognition of soluble E2 (data not shown). Interestingly, the binding of antiserum R645 to E2 was not significantly affected by any of these peptides. These results suggest that antibodies in R646 serum recognize part of the HVR-1 of E2 and sequences downstream from it (amino acids 414 to 451).
It may be that R646 also recognizes one or more conformational epitopes that are not mimicked by any linear peptide. To test this hypothesis we preadsorbed purified R646 IgG with native and denatured E2, and then tested its capacity to recognize E2 in a GNA-capture ELISA and to inhibit infection by genotype 1a H77 HCVpp. As shown in Fig. 5, preadsorption with native but not with denatured E2 reduced both its recognition and its neutralization capacity by a factor of 10. This supports the idea that antibodies in the R646 serum recognize one or more conformational epitopes, present on native but not on denatured E2, which also contribute to neutralization of HCVpp infection.
Amino acid sequence alignment (Fig. 6a) of the N-terminal region of E2 of the different genotypes used in this study showed that the MAb AP33 epitope (residues 412 to 423) is relatively well conserved with only three variant amino acid sites (residues 412, 414, and 416). Other sites within this epitope are absolutely conserved. An exception to this was the corresponding amino acid sequence in the E2 encoded by the genotype 5 isolate UKN5.14.4, which has a 4 residue change in the sequence with a –1 shift in the location of the potential glycosylation site (NGS) relative to that of the other isolates (Fig. 6A). In contrast, significant variations were seen between different genotypes in sequences corresponding to the genotype 1a H77 linear epitopes recognized by R646 consistent with its restricted specificity. Finally, the epitope recognized by the ALP98 antibody (residues 644 to 651) was also conserved although an arginine to valine mutation at position 651 in the genotype 4 clones abolished recognition by this antibody (Fig. 6B).
DISCUSSION
Prior to the development of the retroviral pseudotype assay (7, 35) studies of the neutralizing potential of antibodies and sera were only possible using less direct surrogate assays, including receptor and cell binding assays (51, 57), together with animal models (25, 29). Previous studies have determined the neutralizing capacity of sera or antibodies against only a limited range of HCV genotypes (6, 28, 47). In this paper we have used the HCVpp assay to assess the capacity of a set of E2-specific antibodies and sera to neutralize HCVpp carrying E1E2 representative of all of the major genotypes 1 through 6. To facilitate this we have developed robust PCR methods capable of amplifying full-length E1E2 ex vivo.
In our previous work we noted that many E1E2 sequences recovered from patient-derived samples were not capable of conferring HCVpp infectivity (39), as has been reported recently (42). Here we extend our earlier findings and show that over 85% of recovered envelope genes fail to function in the HCVpp assay. There are a number of factors that may contribute to the high proportion of nonfunctional clones. First, although the PCR utilized proofreading enzymes to minimize the number of misincorporations during the amplification process, errors will still arise and there will inevitably be a proportion of clones that contain sporadic point mutations (60). Second, given the quasispecies nature of HCV (10, 54), some of the nonfunctional clones will contain naturally occurring deleterious mutations. Finally, a lack of infectivity might be attributable to inadequacies in the sensitivity of the HCVpp assay. Certainly, some but not all of the nonfunctional clones could be rendered infectious after transfer to a higher-expression vector (data not shown). Identifying the residues that affect infectivity in the HCVpp assay is of considerable interest, and the panel of functional and nonfunctional clones derived from the same source will enable us to address this issue.
Having established a panel of functional E1E2 clones representative of all the major genotypes, we went on to assess the capacity of the MAb AP33 and of rabbit antisera raised against the E2 protein of the H77 strain to neutralize HCVpp entry. Rabbit sera raised against HVR-1 of E2 were capable of neutralizing HCVpp incorporating H77 E1E2, but not glycoproteins derived from other genotypes. The high degree of HVR-1 genetic variability explains the poor cross-neutralization observed, in keeping with previous reports of the restricted neutralizing capacity of natural HVR-1-specific antibodies (70). Immunization of rabbits with the H77 E2 ectodomain also resulted in the induction of neutralizing antibodies, but again these responses were highly strain specific. Peptide mapping and competition assays showed that the most potently neutralizing serum (R646) recognized both conformational and linear determinants. Neutralization could be severely reduced by preadsorbing the serum with native but not denatured soluble E2, indicating that the neutralizing antibodies most likely recognize conformational epitopes.
Previous work has shown that antibodies elicited by immunization of chimpanzees with HCV envelope glycoproteins can partially protect against homologous challenge (29, 55). Similarly, a number of conformation sensitive human monoclonal antibodies capable of broadly inhibiting binding of E2 to CD81 have been described (1, 9, 33, 34, 36, 38). Some of these antibodies also neutralize HCVpp infection and as such they could have a future role in the treatment of HCV infection and they might also serve to define future vaccine candidates. However, studies with human immunodeficiency virus type 1 have shown that focusing the immune response on epitopes recognized by broadly neutralizing antibodies is a significant challenge. In this context, the finding that AP33 potently neutralizes the entry of HCVpp carrying highly divergent E1E2 is significant, particularly as its epitope is linear and highly conserved across different genotypes of HCV. The exact mechanism of neutralization by AP33 is unknown, although inhibition of CD81 binding is the most likely (48). The epitope recognized by AP33 has been mapped to residues 412 to 423 (QLINTNGSWHIN) and carries one potential N-linked glycosylation site (48).
It is interesting that HCVpp derived from one genotype 5 isolate (UKN5.14.4), although infectious, were not recognized (and therefore not neutralized) by the MAb AP33. This isolate has a 4-amino-acid change (QLIQNGSSWHIN) in the E2 region corresponding to the AP33 epitope, with a well-conserved N-linked glycosylation site shifted –1 relative to that in the other isolates. Subsequent analysis of over 5,500 sequences deposited into the GenBank database has shown the AP33 epitope to be highly conserved. The average diversity of sequences compared to the prototype AP33 epitope is 4.7%, and the majority of variable amino acids are located at the N terminus of the predicted epitope. Importantly, sequences similar to that present in UKN5.14.4 were not evident. Our preliminary data show that reversion of one of the four variant amino acids to its conserved counterpart (i.e., Q to N) renders the UKN5.14.4 HCVpp noninfectious (while also remaining nonreactive to MAb AP33) (not shown), highlighting the possible importance of this region of E2 in infection. This observation, together with the fact that the infectivity of UKN5.14.4 HCVpp is not affected by the N415Q and the –1 shift of the N-linked glycosylation sequence, further indicates that this particular isolate may represent a neutralization escape mutant. Further in-depth site-directed mutagenesis and structural studies will be needed to fully understand the AP33-E2 interaction and mechanisms of neutralization.
The IC50 of AP33 when neutralizing HCVpp of diverse genotypes ranged from approximately 0.6 up to 32 μg/ml. Immunization with peptides corresponding to HVR-1 can lead to the generation of sera capable of protection against homologous virus (25), and therefore induction of AP33-type antibodies, at titers capable of inducing neutralization, should be possible. That said, studies with the broadly neutralizing human immunodeficiency virus type 1 antibodies 2G12, b12, and 2F5 show that protection from in vivo challenge might require higher concentrations of antibody than their in vitro IC50 (52). However, many factors affect in vivo challenge and in vitro neutralization results. Currently it is unknown what antibody concentrations will offer protection against HCV infection or how predictive HCVpp neutralization is of natural protection.
One factor that will affect the neutralizing titers will be purity and homogeneity of the HCVpp inocula. We have used cell supernatants as the source of HCVpp without further purification; such preparations contain various forms of E2 (20). Normalization with respect to Gag-Pol protein or E2 achieves little, because neither protein is restricted to infectious HCVpp; the supernatant contains a heterogeneous mix of materials, including soluble E2, aggregates of E2, nonenveloped Gag-Pol particles, noninfectious enveloped particles, and infectious enveloped particles. The relative proportion of each of these almost certainly varies from one genotype to another. Purifying HCVpp through a sucrose cushion results in a significant loss of infectivity and only achieves the removal of the soluble E2 component. The theoretical ideal of isolating only infectious HCVpp is not realistically achievable.
In conclusion, we have shown that the epitope defined by the AP33 antibody is highly conserved across all the major genotypes and that this antibody is capable of broad neutralization, at least in the HCVpp assay. HCV particles in infected patients are associated with plasma lipoprotein (3), which may mask epitope availability, although antibodies to HVR-1 are clearly able to neutralize infection in chimpanzees (25). Furthermore, Petit et al. (50) more recently demonstrated that native virions have at least some E1E2 epitopes surface exposed. Nevertheless, it will be important to define whether or not MAb AP33 is indeed capable of neutralizing infection by patient-derived virions and, if so, whether passive transfer of this antibody can protect in animal model challenge experiments. Similarly, immunization and challenge studies using various immunogens containing the AP33 epitope will define its usefulness as a vaccine candidate. The identification of a conserved neutralizing linear epitope offers significant hope for the development of a successful HCV vaccine.
ACKNOWLEDGMENTS
This work was supported by the European Community (contract QLRT-2000-01120, ENHCV), the Medical Research Council United Kingdom, the University of Nottingham Research Committee, the Nottingham University Hospitals' Special Trustees, and the Agence Nationale pour la Recherche contre le SIDA et les Hépatites Virales.
A.O. and A.W.T. contributed equally to this work.
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