Neutralizing activity and safety of human monoclonal antibodies against hepatitis C virus

Abstract

AIM:

Assessment of the neutralizing activity of human monoclonal antibodies against HCV and also study their safety in experimental small animals (Swiss mice).

MATERIALS AND METHODS:

Assessment of neutralizing activity of human monoclonal antibodies against HCV envelope regions (E1, E2) by two methods: by HCV cc infectious system 1) and by using positive HCV positive serum as source of HCV particles genotype 4a (neutralizing assay 2). Dot ELISA was used to study the activity of the generated antibodies. Safety and toxicity of the generated human antibodies were tested by assessing the changes in the biochemistry of liver function and kidney function tests, Complete blood counts (CBC) and studying the pathological changes with different concentrations of purified human antibodies were carried out..

RESULTS:

Human Abs # 5 & 11 showed neutralizing activity by (neutralizing assay 2) but were not neutralizing by HCV cc assay. Human Abs # 12 & 15 showed neutralizing activity by the two methods i.e our generated human antibodies Abs# 5 &11 & 12 & 15 were neutralizing for HCV genotype 4a and Abs # 12 & 15 were neutralizing for HCV genotypes 4a and 2a. Liver and kidney functions and CBC results indicated that doses of 10 $\mu$ g, 100 $\mu$ g were safe. The histopathological results indicated that the dose of 10 $\mu$ g of purified human monoclonal antibodies per mouse body weight was safe.

CONCLUSION:

The generated human monoclonal antibodies can be used to develop potent immunotherapy agents that can be administrated for the post-transplantation patients to prevent the recurrence of HCV infection. Also, the monoclonal antibodies can be used to develop a candidate vaccine against HCV.

Keywords

HCV monoclonal antibodies liver transplantation vaccine development

1. Introduction

Hepatitis C virus represents a big health problem worldwide with about 3 million infections every year and a total number of 170 million case in the world. HCV infection leads to chronic infection in 50% to 80% of infected cases, this chronic infection leads to damage of the liver that may cause cirrhosis or liver cancer (hepatocellular carcinoma (HCC)). The HCV genome comprises of a single stranded positive-sense RNA of roughly 9.6 kb, which contains 3000 base pairs flanked by untranslated regions (UTRs) at both closures [1, 2]. Pegylated IFN (PEG-IFN) in combination with ribavirin (RBV) or Non-pegylated interferon (IFN) has been the basic components for HCV treatment. 70% to 90% of individuals infected with HCV genotype 2 and 3 were able to achieve the sustained virological response (SVR), while only 30% to 40% of infected patients with genotype 1 reached the SVR [3, 4, 5, 6, 7, 8]. The HCV treatment has been improved during the last years. Many molecules with an antiviral activity have been synthesized such as HCV synthetic peptides, antisense oligodeoxynucleotides, monoclonal antibodies, and the direct-acting antivirals [9, 10, 11]. In December 2013, the Food and Drug Administration (FDA) approved the Sofosbuvir (SOF) as a new drug against HCV. SOF is one of the second-generation direct acting antivirals DAAs which are characterized by increased SVR rates, comfortable ways of administration and good safety profiles [12]. The DAAs can be taken with or without ribavirin according to the patient case. Liver transplant occurs when HCV patients reach the end stage of liver disease. Unfortunately, HCV reinfection occurs for nearly half of liver transplanted patients in addition to the high risk of death one year after liver transplantation. Monoclonal antibodies (mAb) are monospecific antibodies that can attach to the same epitope [13]. Synthesis of monoclonal antibodies is made through homogenous hybrid cells (B cells). Hybrid cells are cell lines formed from the fusion between a myeloma cell (B cancer cell) and an antibody producing B cell. In 1975, the first mAb produced from a cell line was established [14], Human monoclonal antibodies can be produced through several ways such as transgenic mice, phage display, immortalization with Epstein Barr Virus (EBV), and humanized mouse monoclonal antibodies. The best way for production of fully human monoclonal antibodies is the immortalization of B lymphocyte cells [15]. In vitro, Epstein Barr Virus has the ability to immortalize all human B lymphocyte cells which enable the secretion of specific human monoclonal antibodies from the isolated monoclonal cell lines [16]. EBV immortalization leads to the production of human monoclonal antibodies of different types such as IgG, IgM, IgE, and IgA. Steinitz mentioned that human monoclonal antibodies are promising products of passive immunization for several problems like bacterial infections, viral diseases, and cancer [16]. In the present study, we established Human cell line producing human monoclonal antibodies, explored their viral neutralization, and studied their safety in small experimental animals (Swiss mice). In the hope to establish a potent immunotherapy from monoclonal antibodies targeting HCV. Immunotherapy may represent an important tool for HCV patients especially who undergo liver transplantation surgeries to prevent recurrence of HCV infection. Also these antibodies can be used to study candidate vaccine development.

2. Materials and methods

2.1 Isolation of B-cells (CD22+) from peripheral blood lymphocytes (PBMCs)

10 ml peripheral blood was collected from HCV positive patient (Informed consent was obtained). The blood specimen was layered on Ficoll-Hypaque solutions (Amersham Pharmacia, Milan, Italy). After centrifugation at 3000 rpm for 20 min, mononuclear cells were collected from the interface. Magnetic selection (Miltenyi Biotec, Bologna, Italy) was used to select and purify the CD22+ Lymphocytes.

2.2 Epstein-barr virus (EBV) production

B95.8 cell line (kindly obtained from Dr. James robinson, Tulan University, USA.) was used to produce free EBV particles. B95.8 was cultured in complete Dulbecco’s Modified Eagle’s Medium (DMEM) and was incubated in a CO2 incubator at 37C and 5% CO2. After culturing the cells, they were exposed to freezing and thawing. After that, centrifugation was occurred to obtain the media that contain the EBV particles.

2.3 Isolation of B-cells (CD22+) from human PBMCs positive for HCV antibodies negative for HCV RNA

Immortalization of B lymphocyte cells (CD22+) was occurred with some modifications. In brief, Mini-Macs cell separator (MACS) was used to isolate CD22 B cells, then 50 B cells/well were cultured in 96 well plate (Costar, Corning, USA) and immortalized using 30% v/v EBV in addition to B cell stimulators, 1 $\mu$ g/ml CpG2006 (Invitrogen, USA). After 1 week, cells were re-stimulated with 50 U/ ml IL-2 (Roche Diagnostics, Germany) and 1 $\mu$ g/ml CpG2006. The experiment was continued for 3 weeks with renewing the culture medium.

2.4 Dot blot ELISA analysis for antibody production

Culture supernatant (5 $\mu$ l) of potentially immortalized B cells were spotted on a nitrocellulose blotting membrane (Protran BA-85, VWR International). After drying, blots were blocked with 5% (w/v) FCS in PBS (blocking buffer) for 30 min on a shaker. Membranes were then incubated for 1 h with HRP-labelled rabbit anti-human IgG (Dako) 1/2500 in blocking buffer and extensively washed in PBS supplemented with 0.05% (v/v) Triton X-100. Antibody production was detected using diaminobenzidine (DAB) substrate (Sigma Aldrich). Human sera (in dilution 1:100) were included as positive control. Culture medium was used as negative control. Also supernatant from control cells was included as another negative control.

2.5 Viral neutralization

Hepatoma Huh7.5 cells were cultured and transfected with HCVcc (J6/JFH-1) infectious system as described [17]. The J6/JFH-1 infectious system was a gift from Charles Rice (The Rockefeller University, USA, MTA# 642 to Dr. Mostafa K. El-Awady 2007). This experiment was done as mentioned early by [9, 18] with some modifications. In brief, Huh7.5 cells were cultured in in complete DMEM media and were incubated in CO2 incubator at 37 ${}^{\circ}$ C and 5% CO ${}_{2}$ overnight. HCV infected serum and the media obtained from infected Huh7.5 cells with HCVcc (J6/JFH-1) infectious system were used as a source of virus. For the neutralization assays, produced monoclonal antibodies mixed with equal volume of HCVcc (J6/JFH-1) or HCV infected serum in 10% FBS/DMEM media and incubated at 37 ${}^{\circ}$ C for one hour. The two mixtures were used to infect Huh7.5 cells seeded in 24 well plates and then incubated at 37 ${}^{\circ}$ C for three hours. The virus-containing media was removed, washed with sterile phosphate buffer saline (PBS) and replaced with 15% FBS DMEM media and incubated at 37 ${}^{\circ}$ C. After 48 hours, media were removed and cells were washed twice with sterile PBS. RNA was extracted from cells of each well using a method described by [19]. The RNA was amplified to detect the presence of HCV RNA as described in our previous studies [20].

2.6 Study the safety/toxicity of the human monoclonal antibodies in small experimental animals (Swiss mice)

To explore the dose range for future pharmacological studies, three doses namely 10 $\mu$ g, 100 $\mu$ g, 250 $\mu$ g/25 gm mouse body weight were tested for safety assays including liver functions [ALT, AST, Albumin), kidney function (S. Creatinin), and Complete blood counts (Platelets, Leucocytes counts and RBCs counts) were assessed for all mice groups injected with human antibodies compared with their values before injection. For Histopathological changes, We demonstrated the histopathological changes observed after injection of 10 $\mu$ g, 100 $\mu$ g, 250 $\mu$ g/25 gm of purified whole molecule human monoclonal antibodies per 25 gm body weight of mice (4 animals per group). Animals were sacrificed after 6-weeks (chronic effect) from immunization. Tissues examined were: liver, spleen, kidney, and lungs.

3. Results

3.1 Isolation of B-cells (CD22+) from human PBMCs positive for HCV antibodies negative for HCV RNA

Immortalization and propagation of separated CD22+ cells by EBV (30%), CPG2006 and IL2 were followed up and checked microscopically using inverted microscope and the growth of cell clones started to appear after 3 days. Figure 1 showed the immortalization of isolated CD22+ positive cells by EBV (30%) as procedures described in materials and methods.

Figure 1.

Immortalization of isolated CD22+ positive cells by EBV (30%) in addition of CPG2006 and IL2. (A) Control cells; (B) Immortalized cells with EBV after 1 week; (C) Immortalized cells with EBV after 2 weeks; (D) Immortalized cells with EBV after 3 weeks.

3.2 Dot blot analysis for antibody production

Using dot ELISA, as shown in Fig. 2, Positive human sera showed dark brown spot as positive control while culture medium or media supernatant of negative control cells showed no spot. Supernatant from different wells of immortalized cells showed good dark brown spot indicated that immortalized cells secreted human antibodies.

Figure 2.

Dot blot analysis for antibody production after two weeks of cell immortalization by EBV.

3.3 Neutralizing activity of the generated human monoclonal antibodies using both HCV cc and HCV positive sera samples as a source of viral HCV particles

We assessed the neutralizing activity of the generated human monoclonal antibodies using HCV cc and patient’s HCV positive serum ample as a source of HCV as shown in Fig. 3. The neutralizing activity of the generated human antibodies were tested, one based on HCV cc system, and the other protocol based on using serum sample positive for HCV RNA as a source of the viral particle. Human antibody # 12, 15 showed neutralizing activity based on HCV cc and serum as a source of HCV viral particles. Ab # 11 and 5 showed neutralization activity only for serum samples but no neutralization using HCV cc system. i.e all tested human antibodies # 12, 15, 11, 5 showed neutralization based on serum sample as a source of viral particles (no band 174 bp) and only antibodies # 12, 15 have capability to neutralizing HCV serum and HCV cc systems. I.e our generated human antibodies Abs Abs # 5 & 11 & 12 & 15 were neutralizing for HCV genotype 4a and Abs # 12 & 15 were neutralizing for HCV genotypes 4a and 2a.

3.4 Study the safety/toxicity of the human monoclonal antibodies in small experimental animals (Swiss mice)

3.4.1 Hematology

RBCs count, total leucocytes and platelets counts were measured in all groups of mice immunized with 10 $\mu$ g, 100 $\mu$ g, 250 $\mu$ g/25 gm of human monoclonal antibodies per 25 gm body weight of mice (4 animals per group), as shown in Fig. 4.

3.4.2 Biochemical changes in liver and kidney function tests

Biochemical parameters of immunized Swiss mice with different doses of human monoclonal antibodies were assessed. The data presented in Fig. 5 show that the dose 10 $\mu$ g/25 gm body weight of our human monoclonal antibodies did not cause any increase of serum ALT level, AST, Albumin or creatinine above the mean levels of non-immunized mice after 6 weeks. However slight but non significant increase (at $P>$ 0.05), were observed in mice receiving 100 $\mu$ g/25 of our human monoclonal antibodies with regards to albumin and creatinine. On the other hand both serum AST, ALT, significantly increase in sera of mice immunized with doses 10 $\mu$ g/25 gm body weight ( $P<$ 0.001). Biochemical changes in liver and kidney function tests were measured in interval times

3.4.3 Histopathological changes

The data shown in Table 1 indicated that the doses of 10 $\mu$ g of purified human monoclonal antibodies per 25 gm mouse body weight were safe for all examined tissues observed after 6-weeks post injection, while a dose of 100 $\mu$ g/25 gm mouse body weight of human monoclonal antibodies induced slight histopathological changes after 6-weeks in liver, spleen, lung, and kidneys. Dose of 250 $\mu$ g/25 gm mouse body weight of purified human monoclonal antibodies induced strong and dangerous histopathological changes after 6-weeks in liver, spleen, lung and kidneys. So, we suggest that it is better to assess doses less than 10 $\mu$ g 25 gm mouse body weight and also it is better to use Fab fragment. So the pathological changes Group A 10 $\mu$ g (good) is better than group B 100 $\mu$ g (moderate) than group C, 250 $\mu$ g (Very bad) (Table 1).

Table 1
Histopathological changes of the human monoclonal antibodies in small Experimental animals (Swiss mice)

Histopathological changes
Control groups
Liver	Spleen	Kidney	Lung
Normal	Normal	Normal	Normal (slight emphysema)
Group A ( 10 $\mu$ g per mouse)
Liver	Spleen	Kidney	Lung
Nearly normal structure	Somewhat increase of mega karyocytes	Nearly normal structure	Nearly normal structure
Group B ( 100 $\mu$ g per mouse)
Liver	Spleen	Kidney	Lung
Hepatocytomegaly, acinus formation	Increase the numbers of megalokaryocytes per field.	Glomerulonephrosis	Slight bronchitis
Group C ( 250 $\mu$ g per mouse)
Liver	Spleen	Kidney	Lung
Modular state of tumor (not capsulated)	Increase the number of megakaryocytes per field	Degeneration of kidney tubules	Emphysema

Figure 3.

(A, B, C, D, E, F) neutralizing activity of generated human antibodies # 5, 11, 12, 15. Tested generated human monoclonal antibodies # 12, 15, 11, 5 showed neutralization based on serum sample as a source of viral particles (no band at 174 bp) and only antibodies # 12, 15 have capability to neutralizing HCV serum and HCV cc systems.

Figure 4.

Changes in counts of red blood cells in blood of mice immunized with different concentrations (10 $\mu$ g, 100 $\mu$ g, 250 $\mu$ g/25 gm mouse body weight) of human antibodies.

Figure 5.

Changes in liver function ALT, AST, Albumin and kidney function (creatinin) in serum of mice immunized with different concentrations (10 $\mu$ g, 100 $\mu$ g, 250 $\mu$ g/25 gm mouse body weight) of human antibodies.

4. Discussion

Human monoclonal antibodies can be produced through several ways such as transgenic mice, phage display, immortalization with Epstein Barr Virus (EBV), and humanized mouse monoclonal antibodies.

There are several studies aimed to produce monoclonal antibodies through immortalization of B lymphocyte cells [10, 21, 22, 23, 24]. In the present study, we aimed to establish a cell line that produce human monoclonal antibodies targeting HCV, these human monoclonal antibodies may be an important tool for HCV patients especially who are undergoing liver transplantation surgeries to prevent recurrence of HCV infection. First of all, we isolated B cells from peripheral blood mononuclear cells via microbeads conjugated with monoclonal antibodies target human CD22+. After that, the separated B cells (CD 22+) were immortalized using CPG2006, IL2, and 30% EBV, this method was reported by [21, 22], this method of immortalization gave higher reactive antibodies. Dot ELISA confirmed that the generated antibodies were human antibodies as we used conjugate antihuman peroxidase (Fig. 2). So, we optimized a reproducible immortalization method for B cell from patients negative for HCV RNA and positive for HCV antibodies for rapid human monoclonal IgG antibodies production from CD22 ${}^{+}$ IgG peripheral blood B cells by B cell infection with EBV followed plus an extra stimulation of IL-2 and CpG2006.

Neutralizing antibodies have an important function to control the HCV infection. Neutralizing antibodies are highly secreted in the early stage of HCV infection by patients who have the ability to resolve the infection [25, 26]. The HCV envelope proteins E1 and E2 are the main targets for the host immune defense (humoral immunity). So, neutralizing monoclonal antibodies targeting these HCV envelope proteins are promising tools to develop good therapeutic and prophylactic approaches in addition to accurate studying of the host-virus interactions.

The results of the neutralization assay using the HCV cc infectious system and HCV infected serum showed that two antibodies have neutralizing activity (Fig. 3). Sabo et al. reported that neutralizing antibodies are basic for inhibition of viral entry and post binding entry of the virus to the target cells [27]. Our generated human antibodies Abs# 5 & 11 & 12 & 15 were neutralizing for HCV genotype 4a and Abs # 12 & 15 were neutralizing for HCV genotypes 4a and 2a. This means that epitopes for these neutralizing human monoclonal antibodies can be used to design new HCV candidate vaccines for patients with HCV genotypes 4a and 2a. Also in this study we tested the safety and toxicity of the generated human antibodies by assessment of the changes in biochemistry of liver function tests (ALT, AST, Albumin) and changes in kidney function test (serum creatinin) with different concentration of purified human antibodies (10 $\mu$ g, 100 $\mu$ g, 250 $\mu$ g/25 gm mouse body weight). Results indicated that doses of 10 $\mu$ g, 100 $\mu$ g were safe and almost no changes in biochemical changes in liver and kidney function tests (ALT, AST, Albumin and Creatinin) were observed, but dose of 250 $\mu$ g/25 gm mouse body weight causes changes in liver function tests. For pathological changes, the histopathological changes after injection of 10 $\mu$ g, 100 $\mu$ g, 250 $\mu$ g/25 gm of whole molecule human monoclonal antibodies per 250 $\mu$ g/25 gm mouse body weight of mice (4 animals per group) were investigated. Animals were sacrificed after 6-weeks (chronic effect) from immunization. Tissues examined were: liver, spleen, kidney, and lungs. The histopathological data indicated that the dose of 10 $\mu$ g of purified human monoclonal antibodies per mouse body weight was safe for all examined tissues after 6-weeks of 10 $\mu$ g of human monoclonal antibodies injection, while a dose of 100 $\mu$ g/mouse body weight of human monoclonal antibodies induced slight histopathological changes after 6-weeks in liver, spleen, lung, and kidneys. Dose of 250 $\mu$ g/mouse body weight of human monoclonal antibodies induced strong and dangerous histopathological changes after 6-weeks in liver, spleen, lung, and kidneys. So, we suggest that it is better to use doses less than 10 $\mu$ g/mouse body weight and also it is better to use Fab fragment. In conclusion, the produced monoclonal can be used to develop a potent immunotherapy that can be administrated for the post-transplantation patients to prevent the recurrence of HCV infection and also these neutralizing antibodies can be used to design new candidate HCV vaccine particularly for patients with HCV genotypes 4a and 2a.

Footnotes

Acknowledgments

This study was supported in part by projects STDF 894 and STDF5041 to Dr. Ashraf Tabll.

References

Choo

Q.L.

Kuo

et al., Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome, Science 244(4902) (1989), 359–362.

Scheel

T.K.

and Rice

C.M.

, Understanding the hepatitis C virus life cycle paves the way for highly effective therapies, Nat Med 19(7) (2013), 837–849.

Bacon

B.R.

Gordon

S.C.

et al., Boceprevir for previously treated chronic HCV genotype 1 infection, N Engl J Med 364(13) (2011), 1207–1217.

Jacobson

I.M.

McHutchison

J.G.

et al., Telaprevir for previously untreated chronic hepatitis C virus infection, N Engl J Med 364(25) (2011), 2405–2416.

Sherman

K.E.

Flamm

S.L.

et al., Response-guided telaprevir combination treatment for hepatitis C virus infection, N Engl J Med 365(11) (2011), 1014–1024.

Manns

M.P.

McHutchison

J.G.

et al., Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial, Lancet 358(9286) (2001), 958–965.

Fried

M.W.

Shiffman

M.L.

et al., Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection, N Engl J Med 347(13) (2002), 975–982.

Hadziyannis

S.J.

Sette

et al., Peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis C: a randomized study of treatment duration and ribavirin dose, Ann Intern Med 140(5) (2004), 346–355..

Abdelhafez

T.H.

Bader El Din

N.G.

et al., Mice Antibody Response to Conserved Nonadjuvanted Multiple Antigenic Peptides Derived from E1/E2 Regions of Hepatitis C Virus, Viral Immunol 30(5) (2017), 359–365.

10.

Tabll

El Abd

et al., Establishment of human clones producing neutralizing human monoclonal antibodies to the envelope E1/E2 protein of hepatitis C virus by EBV immortalization of immune CD22(+) B cells, Hum Antibodies 22(3-4) (2013), 55–65.

11.

Touni

Tabll

A.A.

et al., Diagnosis of Hepatitis C Virus Infection Using a Novel Mouse Monoclonal Antibody to Detect Serum HCV E1/E2 Antigens, Clin Lab 63(4) (2017), 669–678.

12.

Ponziani

F.R.

Mangiola

et al., Future of liver disease in the era of direct acting antivirals for the treatment of hepatitis C, World J Hepatol 9(7) (2017), 352–367.

13.

Tansey

E.M.

and Catterall

P.P.

, Monoclonal antibodies: a witness seminar in contemporary medical history, Med Hist 38(3) (1994), 322–327.

14.

Kohler

and Milstein

, Continuous cultures of fused cells secreting antibody of predefined specificity, Nature 256(5517) (1975), 495–497.

15.

Carter

P.J.

, Potent antibody therapeutics by design, Nat Rev Immunol 6(5) (2006), 343–357.

16.

Steinitz

, Production of human monoclonal antibodies by the epstein-barr virus method, Methods Mol Biol 1060(2014), 111–122.

17.

Scheel

T.K.

Gottwein

J.M.

et al., Development of JFH1-based cell culture systems for hepatitis C virus genotype 4a and evidence for cross-genotype neutralization, Proc Natl Acad Sci U S A 22;105(3) (2008), 997–1002.

18.

Stamataki

Coates

et al., Hepatitis C virus envelope glycoprotein immunization of rodents elicits crossreactive neutralizing antibodies, Vaccine 25 (2007), 7773–7784.

19.

Chomczynski

and Sacchi

, Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal Biochem 162 (1987), 156–159.

20.

El-Awady

M.K.

Ismail

S.M.

et al., Assay for hepatitis C virus in peripheral blood mononuclear cells enhances sensitivity of diagnosis and monitoring of HCVassociated hepatitis, Clin Chim Acta 283 (1999), 1–14.

21.

Fraussen

Vrolix

et al., A novel method for making human monoclonal antibodies, J Autoimmun 35(2) (2010), 130–134.

22.

Schieffelin

J.S.

Costin

J.M.

et al., Neutralizing and non-neutralizing monoclonal antibodies against dengue virus E protein derived from a naturally infected patient, Virol J 4 (2010), 7:28.

23.

Amoli

M.M.

Carthy

et al., EBV Immortalization of human B lymphocytes separated from small volumes of cryo-preserved whole blood, Int J Epidemiol 37 (2008), i41–i45.

24.

Funaro

Gribaudo

et al., Generation of potent neutralizing human monoclonal antibodies against cytomegalovirus infection from immune B cells, BMC Biotechnol 12 (2008), 8:85.

25.

Sautto

Tarr

A.W.

et al., Structural and antigenic definition of hepatitis C virus E2 glycoprotein epitopes targeted by monoclonal antibodies, Clin Dev Immunol 2013 (2013), 450963.

26.

Lavillette

Morice

et al., Human serum facilitates hepatitis C virus infection, and neutralizing responses inversely correlate with viral replication kinetics at the acute phase of hepatitis C virus infection, J Virol 79(10) (2005), 6023–6034.

27.

Sabo

M.C.

Luca

V.C.

et al., Neutralizing monoclonal antibodies against hepatitis C virus E2 protein bind discontinuous epitopes and inhibit infection at a postattachment step, J Virol 85(14) (2011), 7005–7019.