Potential conflict of interest: Nothing to report.
The monoclonal antibody (mAb) D32.10 recognizes a discontinuous epitope encompassing three regions E1 (amino acids 297-306), E2A (amino acids 480-494), and E2B (amino acids 613-621) juxtaposed on the surface of serum-derived hepatitis C virus (HCV) particles (HCVsp). The mAb D32.10 inhibits efficiently and specifically the binding of HCVsp to human hepatocytes. Therefore, we investigated the clinical relevance of anti-E1E2A,B response in the serum of patients infected with HCV. To this end, an enzyme-linked immunosorbent assay (ELISA) using synthetic E1-, E2A-, and E2B-derived peptides was used. The ELISA was validated in terms of sensitivity, specificity, and test efficiency. The detection of the anti-E1E2 D32.10 epitope-binding antibodies during natural HCV infection in more than 300 HCV-positive sera demonstrated significantly (P < 0.001) higher prevalence of these antibodies: (1) in patients who spontaneously cured HCV infection (46 of 52, 88.5%) showing high titers (70% ≥ 1/1000) compared to never-treated patients with chronic hepatitis C (7 of 50, 14%) who actively replicated the virus, and (2) in complete responders (20 of 52, 38.5%) who cleared virus following treatment and achieved a sustained viral response compared to nonresponders (4 of 40, 10%). Serum anti-E1E2 antibodies were monitored before, during, and after the current standard-of-care therapy (pegylated interferon plus ribavirin) in responder and nonresponder patients. Optimal cutoff values were assessed by receiver operating characteristic curve analysis. One month prior to therapy initiation, the threshold of 1131 (optical density × 1000) gave 100% and 86% positive and negative predictive values, respectively, for achieving or not achieving a sustained viral response. Conclusion: The anti-E1E2 D32.10 epitope-binding antibodies are associated with control of HCV infection and may represent a new relevant prognostic marker in serum. This unique D32.10 mAb may also have immunotherapeutic potential. (HEPATOLOGY 2010)
Hepatitis C virus (HCV) is the major etiological agent of liver disease worldwide, with approximately 180 million virus carriers. The majority (80%) of infected individuals progress to chronic hepatitis that increases their risk for developing cirrhosis and hepatocellular carcinoma.1 Spontaneous clearance, however, during the acute phase may occur in a minority of subjects (20%) without medical treatment.2 Therefore, identification of protective determinants is essential for understanding the role of neutralizing responses in disease pathogenesis, and for developing vaccines and antibody-based therapies. New tools were developed in recent years to study virus-host interactions. They include HCV-like particles (HCV-LP), HCV pseudotyped particle (HCVpp), and infectious cell culture HCV particles (HCVcc) produced by transfection of Huh-7 cells and derivatives with a particular genotype 2a clone called Japanese fulminant hepatitis 1 (JFH-1).3 These systems were used to evaluate the neutralizing activity of monoclonal antibodies (mAbs) and antibodies from patients.4 Thus, there was increasing evidence for a role of neutralizing antibodies in controlling HCV during all stages of infection,5, 6 but the presence of these antibodies were not associated with viral clearance in vivo7 or with response to antiviral therapy.8 The human neutralizing antibodies that were identified targeted the hypervariable region 1 (HVR1) at the E2 N-terminal part. Because of the extreme variability of the virus, escape variants emerged and poor cross-neutralization was observed.5, 6 Furthermore, high-density lipoprotein (HDL) was shown to attenuate the neutralization of HCVpp by antibodies from HCV-infected patients.7, 9 By contrast, the mouse mAb AP33, which recognizes a highly conserved linear epitope in E2 spanning amino acid (aa) residues 413 to 420, demonstrated potent neutralization of infectivity against both HCVpp and HCVcc.10 However, the prevalence of human serum AP33-like antibodies was low (<2.5%), suggesting that these antibodies do not play a major role in natural clearance of HCV infection.11
Previously, we have shown that the mouse mAb D32.10 recognized a unique discontinuous epitope formed by one sequence between aa 297-306 in the E1 protein, and two sequences between aa 480-494 and aa 613-621 in the E2 protein,12 all expressed close to each other on the surface of serum-derived envelope HCV particles.13 Furthermore, the mAb D32.10 is so far the only antibody able to efficiently inhibit the interactions between serum-derived envelope HCV particles and hepatocytes.14
In order to validate the relevance of the mAb D32.10 in vivo, we used the D32.10 epitope as a probe to look for the presence of anti-E1E2A,B D32.10 epitope-binding antibodies in the serum of HCV-infected patients. The prevalence of anti-E1E2 antibodies in serum was high in patients who either resolved the infection spontaneously, or who achieved a sustained viral response (SVR) after antiviral therapy. Thus, the E1E2A,B D32.10 epitope-binding antibody response appears as associated with control of HCV infection in vivo and may be predictive of the response to HCV treatment.
Human serum samples positive for HCV antibody were obtained from 194 individuals, tested by third-generation enzyme-linked immunosorbent assay (ELISA; Ortho Diagnostics), and classified according to four groups. Group 1: Fifty-two samples negative for HCV RNA were from 22 patients who had spontaneously resolved symptomatic or asymptomatic acute HCV infection in the past (≥ 10 years), and from 30 patients whose date of acute infection was unknown. Only 50% (26 of 52) of samples were analyzed for genotyping, and 25 of 26 were of genotype 1 (Table 1). Group 2: Fifty serum samples were from never-treated (NT) HCV chronic carriers. Fifty-eight percent (28 of 48) were of genotype 1 (Table 1). Their median HCV viral load was 5.8 log10 IU/mL (range: 3.4-7.8 log10 IU/mL) for 44 of 47 cases (Table 1). A total of 54% (26 of 48) showed elevated aminotransferases (median = 1.4 × upper limit of the normal range [ULN], range = 1.06-4.90 × ULN) whereas 46% (22 of 48) had normal levels (median = 0.75 × ULN, range = 0.3-1 × ULN). A total of 77% (27 of 35) exhibited no or low Metavir activity score (A0-A1) and 63% (27 of 43) had a Metavir fibrosis score of F0-F1 (Table 1). Group 3: Forty serum samples were from chronically infected patients who did not respond to multiple successive antiviral therapies with standard or pegylated interferon (PEG-IFN) in association with ribavirin in the majority of cases (77%, 30 of 39; Table 1). HCV RNA levels showed a median of 5.7 log10 IU/mL (range: 4.7-6.9 log10 IU/mL) for 27 of 35 cases (Table 1). Seventy-six percent (28 of 37) were of genotype 1, and had elevated aminotransferase levels (median = 2 × ULN, range = 1-9 × ULN). The majority (88%, 28 of 32) had minimal or moderate activity (A1-A2), but 75% (24 of 32) showed fibrosis stage ≥ F2, of whom five had cirrhosis (Table 1). Group 4: Fifty-two samples were obtained 6 months to 5 years after stopping antiviral treatment from patients who achieved an SVR and were thus considered as complete responders (CR, Table 1). Fifty-three percent (24 of 45) were of genotype 1. Their serum HCV RNA was negative and their aminotransferase levels normal (Table 1). These CR patients exhibited similar degrees of liver disease (Metavir score) than the nonresponder (NR) patients (Table 1). The control group was composed of 17 normal human serum (NHS) samples from blood donors (negative for HCV, hepatitis B virus [HBV], and human immunodeficiency virus [HIV]).
Table 1. Characteristics of Four Selected Groups of HCV-Antibody–Positive Patients
Where mentioned, sera were inactivated by heating to 56°C for 30 minutes before use. All serum samples had been stored at −80°C upon collection (INSERM Unit 871 and/or Hepatogastroenterology Unit of Hôtel-Dieu Hospital, Lyon, France).
Determination of Viral Load and Genotyping.
HCV viral loads were quantified at the virology laboratory of the Hôtel-Dieu Hospital on the patient samples, using either the Versant HCV RNA 3.0 branched DNA assay from Bayer HealthCare (Tarrytown, NY) or the Quantiplex HCV RNA branched DNA assay from Chiron Corp. (Emeryville, CA), as specified by the manufacturer. All the results were converted and expressed in international units per milliliter of serum (1 MEq = 159,000 IU). HCV genotype was determined locally by the Line Probe Assay INNO-LiPA HCV II (Innogenetics, Ghent, Belgium).
Three biotinylated peptides were synthesized: Peptide E1 (Bio-TFSPRRHWTTQGCNC-amide) covers aa residues 292-306 of the HCV E1 glycoprotein. Peptide E2A (Bio-PDQRPYCWHYPPKPC-amide) covers aa residues 480-494 and peptide E2B (Bio-LVDYPYRLWHYPCTI-amide) covers aa residues 608-622 of the HCV E2 glycoprotein.12 Peptides were dissolved in dimethyl sulfoxide (2.5% final), diluted with phosphate-buffered saline (PBS) to 1 mg/mL, and stored at −20°C. Streptavidin (Promega) was coated onto 96-well Maxisorp microtiter plates (Immulon, Dynex) by incubating 100 μL (stock solution: 1 mg/mL diluted 1/100 in 0.1 M carbonate buffer [pH 9.6], i.e., 10 μg/mL in final) in each well (1 μg/well) overnight at 4°C. The wells were blocked with 200 μL of PBS 1× (Cambrex) containing 10% goat serum (Eurobio, CAECHV00) for 1 hour at 37°C. Plates were washed three times with PBS, and 100 μL of the biotinylated peptide solution (10 μg/mL) was added. For each sample, triplicate wells were coated with either peptide E1, E2A, or E2B for 2 hours at 37°C. After another wash with PBS, 100 μL of human serum diluted 1/250 or 1/500 in PBSTG (PBS containing 0.05% Tween 20 and 10% goat serum) was added to the wells and incubated for 2 hours at 37°C. The plates were washed four times with PBST, and conjugate was added. Goat anti-human immunoglobulin G (IgG) peroxidase (Beckman Coulter France) was diluted 1/5000 in PBSTG, added to the wells, and incubated at 37°C. After 1 hour, the wells were washed four times with PBST, and the substrate (o-phenylenediamine dihydrochloride and H2O2) was added. The reaction was stopped after 30 minutes by adding 50 μL 2 N HCl. Color development was measured in an ELISA plate reader at 490 nm. Mean optical densities (OD) from triplicate wells containing either peptide E1, E2A, or E2B, and the mean OD of the control wells (NHS) were calculated. Serum samples from each patient were all processed on the same day, and each plate contained a positive control serum sample. The recognition cutoff for each peptide was calculated as the mean value obtained with at least three NHS + 3 standard deviations (SD).
Sensitivity and Specificity Validation of the Anti-E1E2A,B Test.
To this end, the anti-E1E2A,B ELISA test was carried out either in the absence of peptide (no peptide) or with the three biotinylated peptides E1, E2A, and E2B added together at the final concentration of 5 μg/mL each on the same well. The test was also performed by direct coating of nonbiotinylated peptides on the solid phase. For testing the specificity of detection, two irrelevant biotinylated peptides, peptide-1× (Bio-RSFKSWTGQTPGEFRESRRRDNP LG-amide; 25 aa) and peptide-2× (Bio-SCARRGCIR RRPGHAG-amide; 16 aa) were used in comparison with E1, E2A, and E2B peptides. The peptides 1× and 2× showed from 7%-20% of sequence homology with the D32.10 epitope sequences E1, E2A, and E2B. Peptide-1× did not contain any cysteine residue whereas peptide-2× contained two cysteine residues. Indeed, the critical residues for the D32.10 mAb recognition were identified as Cys306 (E1), Cys494 (E2A), and Cys620 (E2B).12
Serum Immunoglobulin Purification.
Serum IgG fraction was purified using protein G–sepharose 4-fast flow (Pharmacia France, Montigny-le-Bretonneux, France). Two hundred μL of heat-inactivated serum diluted by one-half in PBS was mixed with 200 μL of protein G–sepharose immunobeads for 30 minutes at 25°C and then centrifuged for 90 seconds at 3800g. The supernatant was discarded and the immunobeads were then washed three times with Immunopure IgG binding buffer (Pierce Protein, Perbio Science France SAS) by centrifugation for 90 seconds at 5000g each time. Immunopure IgG elution buffer (400 μL; Pierce Protein, Perbio Science France SAS) was added to the immunobeads, which were mixed thoroughly and then centrifuged for 90 seconds at 5000g. The supernatant was neutralized with 35 μL 1 M Tris-HCl (pH 8.0). The IgG concentration was determined using a micro BCA assay (Bio-Rad Laboratories, Marnes-la-Coquette, France). Purified IgGs were stored at −80°C. The protein mean concentration for four serums from NHS was 1.29 ± 0.13 mg/mL, for 12 serums from cured (C) patients was 2.09 ± 0.46 mg/mL, and for 14 serums from NT patients was 3.19 ± 0.27 mg/mL.
Statistical comparison between two groups of patients was performed with a t test, and P values were calculated using the GraphPad Prism 4 software. P value < 0.001 (represented as *** symbol) corresponds to “extremely significant” results, 0.001 < P < 0.01 (denoted by **) corresponds to very significant, 0.01 < P < 0.05 (denoted by *) corresponds to significant, and P > 0.05 is not significant (ns). The overall sensitivity of anti-E1E2 antibodies for the prediction of treatment response was calculated using receiver operating characteristic (ROC) curves in one-point studies and at different time points before and during treatment in follow-up studies. Optimal cutoff values were defined using the highest sum of sensitivity and specificity. For each optimal cutoff value, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated.
Determination of the Cutoff Value and Evaluation of Precision of Anti-E1E2A,B D32.10 Epitope-Binding Antibodies Detection Test.
Five negative controls (NHS) were initially tested at different dilutions: 1/50, 1/100, 1/250, 1/500, and 1/1000 (Fig. 1A). Standard dilutions selected were 1/250 and 1/500. The cutoff values for both dilutions were determined with 17 NHS (negative for HCV, HBV, and HIV; Fig. 1B). The mean OD values for E1, E2A, and E2B were 0.609 ± 0.033 for 1/250 dilution and 0.374 ± 0.036 for 1/500 dilution. The cutoff was calculated as the mean value + 3 SD, and corresponded to 0.708 for the 1/250 dilution and 0.482 for the 1/500 dilution. Each serum sample was tested in triplicate for the E1, E2A, and E2B peptides. For an easier representation of results, they were expressed as the average of OD obtained for E1, E2A, and E2B, which are very similar (Fig. 1B). The interpretation for the presence or absence of anti-E1E2A,B antibodies took place according to these mean OD values. If the mean value was greater than or equal to the cutoff for a fixed dilution, the sample was defined as positive or the limit for this dilution. If it was under the cutoff, the sample was considered negative. All the sera positive for anti-E1E2A,B contain antibodies that bind to the D32.10 epitope, i.e., to the three peptides, E1, E2A, and E2B. Inversely, the negative sera do not contain any of them. Therefore, these antibodies are called “D32.10 epitope-binding antibodies” throughout the text. At least five NHS were systematically included in each assay, and the cutoff was recalculated for each type of experiments.
The intra-assay variability was evaluated by testing a same positive sample 10 times in an intra-assay run, and showed a coefficient of variation < 4%. The inter-assay variability was evaluated by testing a same positive sample in triplicate in seven independent runs at different days by the same technician, and showed a coefficient of variation < 5%.
Seroprevalence of Anti-E1E2A,B D32.10 Epitope-Binding Antibodies in Natural Human HCV Infection.
Figure 2A shows the results obtained with samples from 52 patients cured of HCV infection (Group 1: C). Twenty-two patients who had spontaneously cleared a past infection (≥10 years) corresponded to the series 1, whereas the 30 other patients whose date of acute infection was unknown corresponded to the series 2. Among the total of 52 C patients, 46 were found positive for anti-E1E2A,B antibodies (88.5%) with a higher prevalence (21 of 22, 95.5%) in the series 1. Globally, relatively high titers of anti-E1E2A,B were detected in serum of these patients (Fig. 2B). The majority (80%) exhibited titers ≥ 1/1000 with 35% of cases ≥ 1/2000. If we compare these results with those of sera obtained from 50 never-treated HCV chronic carriers (Group 2: NT), only 7 of 50 (14%) were found positive. A significant difference (P < 0.001) in the anti-E1E2A,B prevalence between C patients (Group 1) and NT patients (Group 2) was observed (Fig. 3A). When we purified IgGs from a subset of HCV-negative (4 NHS) and HCV-positive serum samples belonging to each group of patients (12 C and 14 NT) prior to determination of the anti-E1E2A,B reactivity by ELISA (Fig. 3B), similar results were observed. Only the IgGs purified from C patients were found significantly positive (P < 0.001) for the anti-E1E2A,B reactivity up to the 1/2000 dilution as the corresponding serums. However, a much lower assay cutoff corresponding to OD = 0.433 instead of 0.883 was obtained.
The prevalence of anti-E1E2A,B D32.10 epitope-binding antibodies was also determined in sera obtained from 40 nonresponders (Group 3: NR) and 52 complete responders (Group 4: CR) who eradicated the virus after antiviral therapies and so achieved an SVR (Table 1). Only 4 of 40 NR patients were found positive (10%) at 1/250 dilution (Fig. 4A). In contrast to NR patients, 20 of 52 CR patients were positive (38.5%) for D32.10 epitope-binding antibodies (Fig. 4B). One patient (CR80) still under antiviral therapy showed a titer > 1/1000 dilution, 11 of 19 (58%) exhibited a titer ≥ 1/500 dilution, and 8 of 19 (42%) showed a titer = 1/250 dilution (Fig. 4B). We noticed that the CR patients who were positive (20 of 52) were tested between 6 months and 1 year after stopping treatment, whereas the CR patients who were negative (32 of 52, results not shown) were tested from 1-5 years after recovery with complete biochemical and virological responses. A significant difference in anti-E1E2A,B seroprevalence was observed with a P = 0.002 (chi-square test) between the Group 3 (NR) and the Group 4 (CR) patients (Fig. 4C). If we compare the NR patients (40) with the anti-E1E2A,B-positive CR patients (20 of 52) by ROC curve (Fig. 4D), the area under the curve (AUC) is estimated to 0.886 (P < 0.001) with an optimal cutoff of 0.845. Thus, patients with OD < 0.845 exhibit a predictive value for nonresponse (NPV) of 97.0%, whereas those with OD ≥ 0.845 exhibit a predictive value for SVR (PPV) of 70.4%.
Longitudinal Follow-Up Study of NR and CR Patients to Standard-of-Care Antiviral Therapy.
Because of the observed variability of anti-E1E2A,B response in the group of CR patients according to the time after stopping treatment, follow-up studies were performed among nine CR patients with SVR (seven of genotype 1 and two of genotype 2; five males and four females; mean age: 41.7 ± 0.6 years) and seven NR patients (all of genotype 1; five males and two females; mean age: 39.7 ± 3.0 years) to current standard-of-care therapy by a combination of PEG-IFN plus ribavirin. The first sample (M-1) was taken before starting treatment (Trt). The other serum samples were taken at time 0 of Trt (M0), then 1 (M+1), 2 (M+2), 3 (M+3), 6 (M+6) and 12 (M+12) months after the start of Trt, and 6 months after termination of Trt (6M stop Trt). The mean OD values for both groups of patients (NR and CR) were represented on the Fig. 5A for the samples M-1, M0, M+1, M+2, M+3 and M+6 from at least five patients in each group. Indeed, the antiviral therapy was often stopped after 6 months of Trt in the NR group. No significant positive results were observed in the NR group. In contrast, the anti-E1E2A,B response was found significantly (P < 0.05) positive for all serum samples in the CR group compared to the NR group. Notably, before the start (M-1) and 3 months after the start of Trt (M+3), the difference was highly significant (***P < 0.001). We observed that the anti-E1E2A,B response fluctuated over time with a peak at 1 month (M1) after starting treatment. Afterwards, the antibody response decreased (M2), but remained positive (CR3) or even rebounded (CR1, CR2) at 3-6 months (M3, M6) after the start of Trt (Fig. 5B). ROC curve analysis was conducted to assess the cutoffs of anti-E1E2 antibodies at M-1, M+1, M+3 and M+6 which best distinguished responder from NR patients (Fig. 5A,B). Table 2 indicates that at 1 month prior therapy initiation, a threshold of 1131 (OD × 1000) best distinguished responders from nonresponders with a 100% and 86% PPV and NPV, respectively, meaning that all patients above this threshold subsequently responded to therapy whereas 86% of those below this cutoff failed to achieve SVR. Similar cutoffs were obtained at the other time points with similar predictive values (Table 2). Although a unique standard breakpoint could not be determined, we did observe by ROC curve analysis that a significant difference always remained between NR patients and patients achieving a SVR.
Table 2. Predictive Values for SVR to Standard-of-Care Antiviral Therapy (ROC Curves)
Patients' Groups (n)
Follow-Up CR/NR (16)
M-1 = 1 month before starting treatment Trt; M+1 = at 1 month after starting Trt; M+3 = at 3 months; M+6 = at 6 months.
Cutoff (OD × 1000)
AUC (area under the ROC curve → 1)
Sensitivity and Specificity of Anti-E1E2A,B D32.10 Epitope-Binding Antibodies Detection Tests.
When the three biotinylated peptides E1, E2A, and E2B were added together on the same solid phase as peptide combination (E1-E2A-E2B, Fig. 6A), similar results were obtained compared to the format using separate peptides on three separate solid phase (E1+E2A+E2B, Fig. 6A). The samples positive for anti-E1E2A,B (CR+ or C) were always found significantly positive compared to samples negative for anti-E1E2A,B (NR and CR-). On the other hand, when the test was performed by coating directly the peptides on the solid phase without involving the streptavidin-biotin system (Fig. 6B), the serum samples from C group were again positive whereas those from NR group negative. However, in both cases a lower significance was observed : 0.001 < P < 0.01 (**, Fig. 6A) and 0.01 < P < 0.05 (*, Fig. 6B), respectively, instead of P < 0.001 (***). This likely results from steric hindrance in the first case (Fig. 6A) or improper position of peptides in the second case (Fig. 6B) leading to a decreased accessibility of human antibodies to their corresponding composite E1E2A,B D32.10 epitope. The format described and used all along this study thus appeared as the best fit in terms of sensitivity.
For strengthening the specificity of test, a subset of serum samples was analyzed in the absence of peptide (no peptide) or in the presence of two irrelevant peptides (1× and 2×). As shown in Fig. 6C, 10 samples from C group showing OD > 1.500 at 1/250 dilution were negative for both peptide-1× and peptide-2×. Only one anti-E1E2A,B–positive sample (C9) was found positive for the peptide-2× whereas the sample C12 showed a very low reactivity with both peptide-1× and peptide-2× (not shown). In conclusion, the anti-E1E2A,B reactivity was highly specific (80%-90%). Furthermore, in the absence of peptide (no peptide), all the positive samples for E1E2A,B were negative (specificity = 100%). Under these conditions, the mean OD values at 1/500 dilution for NHS were 0.058 ± 0.022 (7), and the cutoff value = 0.124. The NR patients were indeed negative with OD = 0.105 ± 0.045(5) and the C patients highly positive with OD = 1.196 ± 0.236(12) (positive/negative ratio ∼ 10).
Although the role of CD4 and CD8 T cells in controlling HCV infection is widely accepted, the role that antibodies may play in HCV clearance is still a matter of debate.15 Antibodies directed against the E1 and E2 viral envelope proteins may prevent or control viral infection if they are directed against epitopes implicated in virus entry. Therefore, because of the relevant properties of the unique mAb D32.10,12-14 the seroprevalence of E1E2A,B-specific D32.10 epitope-binding antibodies was investigated here at different phases of HCV infection. In sera from patients who had spontaneously resolved HCV infection, the prevalence of these antibodies was close to 90% with high titers > 1/1000 in 80% of cases. In contrast, their prevalence in sera from never treated chronic carrier patients was significantly much lower (<15%, P < 0.001). To ensure that high prevalence was well-specific, a subset of samples was tested for reactivity to two irrelevant immunogenic peptides 1× and 2× showing sequence homology from 7%-20% with the D32.10 epitope sequences E1, E2A and E2B. No reactivity was observed in 80%-90% of cases. Ten serum samples from patients who had resolved HCV infection and were highly positive for E1, E2A, and E2B were found unequivocally negative for both irrelevant peptides. Furthermore, in the absence of any peptide, the mean OD values for negative controls were much lower leading to cutoff = 0.124 for positive OD values > 1. The sensitivity of the test was also investigated by evaluating the proficiency of different formats: either not involving the streptavidin-biotin system for the capture of peptide-antibody complexes, or by coating the three biotinylated peptides together on the same solid phase. In both cases, the positivity was lower but remained significant (0.001 < P < 0.01). The principle of the test described and used here was demonstrated to be the best for the detection of D32.10 epitope-binding antibodies in human serum.
To further investigate the potential clinical significance of D32.10 epitope-binding antibodies, we explored whether they could be predictive for SVR after antiviral therapy in chronic carriers. A transversal study in nonresponder patients and in responder patients achieving an SVR revealed that the prevalence of antibodies was close to 40% in complete responders and only of 10% in nonresponders with significant difference (chi-square test, P = 0.002). ROC curve analysis allowed to discriminate non responders from complete responders achieving a SVR. However, for such patients undergoing antiviral therapy, it appeared better to perform longitudinal follow-up studies in order to determine the timing of antibody appearance and their long-term clearance kinetics. Indeed, we demonstrated in complete responders that the anti-E1E2A,B antibodies were present even before initiation of therapy (M-1), showed fluctuating profiles with a maximum 1 month and 3 months after beginning of treatment followed thereafter by progressive decrease over time but remained positive at 1-year after stopping treatment. In contrast, the nonresponder patients were negative all along the follow-up. ROC curve analysis indicated that 1 month prior therapy and at the time points: M+1 and M+3 a cutoff of approximately 1100-1200 (OD × 1000) was associated with SVR with a 100% and 86%-87% PPV and NPV, respectively. Thus, pretreatment anti-E1E2A,B antibodies may be predictive of the response to HCV treatment.
Overall, our data show that D32.10 epitope-binding antibodies and their corresponding epitope E1E2A,B play a major role in natural clearance of HCV infection, in contrast to other type of antibodies such as AP33-like antibodies.10, 11 Further supporting this, similar results were obtained by using purified IgG fractions, minimizing the possible effect of serum components. In addition, the reactivity was positive up to 1/2000 dilution of serum samples or purified IgGs, and independent of genotype 1 or non–genotype 1 of HCV strains. Identifying potentially neutralizing antibodies with epitopes conserved across all isolates of HCV is essential for the development of a successful vaccine capable of eliciting protective humoral immune response. Up to now, no parameters such as human leukocyte antigen, HCV genotype, coinfection with HIV, sex, race, or advanced age, even if they seem to influence the clinical course of disease, could accurately predict spontaneous resolution.2 Only a strong and multispecific cellular immune response was now considered to be an important host factor for spontaneous viral eradication.16 Low baseline IFN-γ–inducible protein-10 levels were shown to be significantly associated with SVR and predictive of the response during treatment in patients infected with HCV genotypes 1 and 4.17 Recently, genetic variation in IL-28B, which encodes the type II IFN-λ3, was shown to be associated with spontaneous18 or treatment-induced clearance of HCV.19 At present, clinical practice serum HCV RNA levels below 2 million copies/mL (≈800,000 IU/mL) and a rapid decrease (≥ 3 log10) of viral load after the onset of treatment are the only viral factors associated with favorable treatment results. Thus, monitoring of anti-E1E2A,B D32.10 epitope-binding antibodies in HCV-infected patients may prove helpful for clinicians to predict rapid viral response.
For the first time, our observations indicate that induction of these likely neutralizing antibodies appears to closely correlate with therapeutic outcome and complete elimination of HCV in contrast with several previous reports.5-9 In these last studies, viral escape from antibody-mediated neutralization occurred because the neutralizing antibodies identified were directed against the HVR1 of the E2 envelope protein. It resulted in interplay of the viral E2/HVR1 with HDL and scavenger receptor class B type I, an HDL receptor, which mediates protection from cross-neutralizing antibodies present in sera of both acute and chronic HCV-infected patients.5, 7 Here, the detected anti-E1E2A,B antibodies target unique highly conserved distinct regions outside the HVR1.
In conclusion, we demonstrated the clinical relevance of anti-E1E2A,B D32.10 epitope-binding humoral immune response in patients infected with HCV. We identified that the E1(aa297-306)-E2A(aa480-494)-E2B(aa613-621) D32.10 epitope was recognized by human antibodies only present in patients who resolved HCV infection spontaneously or under antiviral therapy. This suggests that E1E2A,B-specific antibodies are neutralizing, predictive for complete virus elimination and may represent a new relevant prognostic marker in serum. These findings have implications not only for the HCV diagnosis but also for the design of novel immunotherapeutic and preventive strategies against HCV.