Neutralizing Monoclonal Antibodies against Hepatitis C Virus E2 Protein Bind Discontinuous ...

59 Pages·2011·2.5 MB·English

JVI Accepts, published online ahead of print on 4 May 2011
J. Virol. doi:10.1128/JVI.00586-11
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
1 Neutralizing Monoclonal Antibodies against Hepatitis C Virus E2 Protein Bind
2 Discontinuous Epitopes and Inhibit Infection at a Post-Attachment Step
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4 Michelle C. Sabo1, Vincent Luca2, Jannick Prentoe7, Sharon E. Hopcraft8, Keril J. Blight4,
5 MinKyung Yi5, Stanley M. Lemon6, Jonathan K. Ball9, Jens Bukh7, Matthew J. Evans8,
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6 Daved H. Fremont1,2, and Michael S. Diamond1,3,4 o
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8 Departments of Pathology & Immunology1, Biochemistry and Molecular Biophysics2 d
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9 Medicine3, Molecular Microbiology4, Washington University School of Medicine, St. f
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10 Louis, MO 63110.
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11 5Department of Microbiology and Immunology, University of Texas Medical Branch, p
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12 Galveston, Texas 77559-1073. jvi.
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13 6Division of Infectious Diseases, Department of Medicine, Inflammatory Diseases m
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14 Institute, and the Lineberger Comprehensive Cancer Center, University of North rg
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15 Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7292. n

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16 7Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and a
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17 Clinical Research Centre, Copenhagen University Hospital, Hvidovre, and Department of
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18 International Health, Immunology and Microbiology, Faculty of Health Sciences, ,
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19 University of Copenhagen, Copenhagen, Denmark. 1
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20 8Department of Microbiology, Mount Sinai School of Medicine, New York, New York y

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21 10029. e
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22 9School of Molecular Medical Sciences and the Nottingham Digestive Diseases
23 Biomedical Research Unit, The University of Nottingham, Queen’s Medical Centre,
24 Nottingham NG7 2UH, UK
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28 Address correspondence to:
29 Michael S. Diamond, M.D. PhD., Departments of Medicine, Molecular Microbiology, and
30 Pathology & Immunology, Washington University School of Medicine, 660 South Euclid
31 Ave. Box 8051, Saint Louis, MO 63110, USA, [email protected]. T: (314) 362-
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32 2842; F:[phone] o
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33 n
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34 Running Title: MAb neutralization of Hepatitis C virus d
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35 Manuscript : Figures: 8; Tables: 4; Supplementary Tables: 1 f
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36 Abstract: 242 words
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37 p
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38 ABSTRACT
39 The E2 glycoprotein of hepatitis C virus (HCV) mediates viral attachment and
40 entry into target hepatocytes and elicits neutralizing antibodies in infected patients. To
41 characterize the structural and functional basis of HCV neutralization, we generated a
42 novel panel of 78 monoclonal antibodies (MAbs) against E2 proteins from genotypes 1a
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43 and 2a HCV strains. Using high-throughput focus-forming reduction or luciferase-based o
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44 neutralization assays with chimeric infectious HCV containing structural proteins from
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45 both genotypes, we defined eight MAbs that significantly inhibited infection of the d
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46 homologous HCV strain in cell culture. Two of these bound E2 proteins from strains f
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47 representative of HCV genotypes 1-6, and one of these MAbs, H77.39, neutralized
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48 infection of strains from five of these genotypes. The three most potent neutralizing p
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49 MAbs in our panel, H77.16, H77.39 and J6.36, inhibited infection at an early post- jvi.
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50 attachment step. Receptor binding studies demonstrated that H77.39 inhibited binding of m
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51 soluble E2 protein to both CD81 and SR-B1, J6.36 blocked attachment to SR-B1 and rg
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52 modestly reduced binding to CD81, and H77.16 blocked attachment to SR-B1 only. n

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53 Using yeast surface display, we localized epitopes for the neutralizing MAbs on the E2 a
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54 protein. Two of the strongly inhibitory MAbs, H77.16 and J6.36, showed markedly
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55 reduced binding when amino acids within hypervariable region 1 (HVR1) and at sites ,
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56 ~100 to 200 residues away were changed, suggesting binding to a discontinuous 1
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57 epitope. Collectively, these studies help to define the structural and functional complexity y

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58 of antibodies against HCV E2 protein with neutralizing potential. e
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59
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60 INTRODUCTION
61 Hepatitis C virus (HCV) is a blood-borne, hepatotropic virus that infects ~170
62 million people worldwide. Approximately 70% of infected individuals progress to chronic
63 liver disease, which carries an increased risk of cirrhosis and hepatocellular carcinoma
64 (7). In general, treatment of chronic HCV is complicated by resistance due to extensive
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65 genetic diversity. HCV has been classified into seven major genotypes, which differ by o
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66 ~30% at the nucleotide level (4), and this positive-sense, single-stranded RNA virus has
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67 a capacity for rapid evolution of variant viruses during persistent infection. The current d
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68 treatment, pegylated IFN-α2a and ribavirin, has variable side effects and response rates fro
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69 depending on the virus and host genotype (16). No vaccine is currently available, and
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70 pre-clinical development has been hampered by a lack of understanding of which p
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71 conserved epitopes on the HCV structural proteins should be targeted. i.
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72 HCV contains a ~9.6kb RNA genome that is translated as a single polyprotein m
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73 and then cleaved by viral and host proteases into structural proteins (core, E1, E2), p7, rg
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74 and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (39). Viral n

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75 attachment and entry is mediated by the envelope glycoproteins, E1 and E2. Four a
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76 attachment or entry receptors that are required for infection of hepatocytes have been
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77 identified including CD81 (53), scavenger-receptor B1 (SR-B1) (56), and the tight ,
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78 junction proteins claudin-1 (CLDN1) (14), and occludin (OCLN) (54). The importance of 9

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79 E2 binding to the large extracellular loop of CD81 has been established in vitro (13, 18, y

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80 28, 50, 53), and interactions between E2 hypervariable region 1 (HVR1) and SR-B1 e
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81 have been reported (3, 5, 56). The structural basis of binding of E2 to its cognate cell
82 attachment factors, however, is poorly understood, in part because high-resolution
83 structures of the HCV glycoproteins or intact virion have not been solved.
84 The role of the humoral immune response in controlling HCV infection in patients
85 remains controversial, as patients with persistent infection develop high-titer antibodies
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86 that do not appear to clear infection (reviewed in (7)). Nonetheless, there are emerging
87 data that classes of monoclonal and polyclonal antibodies against HCV have protective
88 activity. Binding to CD81 by soluble forms of E2 (sE2, truncated proximal to the
89 transmembrane domain) is inhibited by antibodies that also neutralize infection of
90 pseudotyped HCV particles (HCVpp) derived from the structural proteins of multiple
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91 genotypes (1, 45). Perhaps more convincing, experiments in chimpanzees and chimeric o
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92 mice have shown that passive transfer of anti-E2 antibodies protects against infection
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93 (15, 37, 64), and immunization with E1-E2 virus-like particles and E2 glycoprotein in d
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94 chimpanzees induces protective antibodies (10, 29, 37). Moreover, in a comprehensive f
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95 study of neutralizing MAbs derived from infected patients, MAbs that bound regions
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96 comprised of amino acid residues 396–424, 436–447 and 523–540 on E2 neutralized p
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97 HCVpp derived from multiple genotypes (37). Thus, anti-E2 antibodies apparently can jvi.
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98 restrict HCV infection, although the exact steps (attachment, entry, or fusion) in the viral m
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99 entry process that are inhibited and the corresponding E2 binding epitopes have not rg
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100 been elucidated. n

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101 To gain more insight into the molecular and structural basis of anti-E2 antibody a
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102 neutralization of HCV infection, we generated a panel of 78 mouse monoclonal
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103 antibodies (MAbs) against soluble, recombinant E2 proteins derived from genotypes 1a ,
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104 (H77 strain) and 2a (J6 strain) HCV strains. These MAbs were analyzed for inhibitory 1
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105 activity against infectious HCV in cell culture and assessed for mechanism of action with y

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106 respect to inhibition of ligand binding on the cell surface. By combining this functional e
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107 analysis with a high-throughput yeast surface display mapping strategy, we identified
108 neutralizing MAbs that bound to distinct regions of E2, including MAbs that recognized
109 determinants with discontinuous epitopes with primary sequences greater than 100
110 amino acids apart. These experiments suggest that neutralizing MAbs blocking distinct
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111 stages of the HCV cell entry process recognize discontinuous epitopes on the E2
112 protein.
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114 MATERIALS AND METHODS
115 Cells and viruses. Huh-7.5 cells were cultured in Dulbecco’s Modified Eagle
116 Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Equitech), non-
117 essential amino acids (Gibco), and antibiotics (penicillin G and streptomycin) at 37°C in
118 a 5% CO incubator. SF9 cells were cultured in Grace’s Insect cell medium (Gibco)
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119 supplemented with 10% FBS at 28oC. HI-5 cells were cultured in Ex-cell media (Gibco) o
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120 at 27oC. CHO cells were grown in Ham’s F12 medium (Gibco) supplemented with 10% n
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121 FBS (HyClone) at 37°C. d
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122 The genotype 2 J6/JFH1/JC1 HCV chimera that expresses luciferase (58) was a f
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123 generous gift from Apath Inc. The HJ3-5 H77/JFH1 chimera, which expresses the core-
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124 NS2 segment of the genotype 1a polyprotein within a genotype 2a background has been p
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125 described (40, 67). The genotype 1a H77C/JFH1(57), genotype 2a J6/JFH1 (38), jvi.
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126 genotype 3a S52/JFH1 (23), genotype 4a ED43/JFH1 (57), genotype 5a SA13/JFH1 m
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127 (32), and genotype 6a HK6a/JFH1 (24) infectious HCV recombinants used in cross- rg
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128 neutralization studies also have been described. Cell culture compensatory and adaptive n

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129 mutations for the HJ3-5, S52/JFH1, ED43/JFH1, SA13/JFH1 and HK6a/JFH1 chimeras a
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130 have been described (24, 40, 67).
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131 To generate virus stocks from infectious cDNA clones, plasmids were linearized ,
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132 and RNA transcription was performed using the T7 DNA-dependent RNA polymerase 1
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133 (MEGAscript Kit, Ambion). Infectious HCV RNA (2 µg) was electroporated as described y

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134 (38), and virus was harvested at 48, 72, and 96 hours, sterile filtered (0.2 µm filter, e
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135 Corning Inc), and buffered with 10mM HEPES pH 7.2 (Mediatech, Inc.). Virus was
136 stored at 4°C for short-term usage or aliquots were prepared and stored at -80°C. Virus
137 titration on Huh-7.5 cells was performed by TCID assay as previously described (38).
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138 Generation of CHO cells stably expressing HCV cell entry factors. Human
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139 SR-BI and CD81 genes were expressed in CHO cells via lentivirus transduction in the
140 context of pTRIP, a self-inactivating lentiviral provirus that expresses no HIV proteins but
141 instead employs an internal cytomegalovirus (CMV) promoter to express cloned genes.
142 An intermediate plasmid, called TRIP-GFP-linker, was generated as a backbone into
143 which SR-BI and CD81 were cloned (all entry factor templates were kindly provided by
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144 C. Rice, Rockefeller University, NY). TRIP-GFP-linker was generated by amplifying the o
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145 GFP sequence with the forward oligonucleotide 5’-CGC AAA TGG GCG GTA GGC GTG
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146 and reverse oligonucleotide 5’-CTC GAG CTA GTC GAC TTC GAA ACT AGT GCT d
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147 AGC CCG CGG CTT GTA CAG CTC GTC CAT GCC. This PCR product was digested f
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148 with restriction enzymes BamHI and XhoI and ligated into the TRIP-GFP plasmid
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149 digested with the same enzymes. The human SR-BI sequence was amplified with p
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150 forward oligonucleotide 5’-CCG CGG ATG GGC TGC TCC GCC AAA GCG and reverse jvi.
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151 oligonucleotide 5’-GCT AGC CAG TTT TGC TTC CTG CAG CAC from the previously m
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152 described TRIP-hu-SR-BI plasmid (54) to generate TRIP-GFP-hu-SR-BI-linker. This rg
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153 PCR product was digested with SacII and NheI and ligated into similarly digested TRIP- n

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154 GFP-linker. The human CD81 sequence was amplified from an expression construct, a
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155 TRIP-GFP-hu-CD81(56), with forward oligonucleotide 5’-GCT AGC ATG GGA GTG
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156 GAG GGC TGC ACC and reverse oligonucleotide 5’-ACT AGT GTA CAC GGA GCT ,
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157 GTT CCG GAT. This PCR product was digested with NheI and SpeI and ligated into 1
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158 similarly digested TRIP-GFP-linker to generate TRIP-GFP-hu-CD81-linker. y

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159 Pseudoparticle production was performed as previously described (54) by co- e
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160 transfection of three plasmids encoding a TRIP provirus containing a transgene, HIV
161 Gag-Pol, and the VSV-G glycoprotein. 293-T cells were seeded at 1.8 x 106 cells/well
162 into a poly-L-lysine (Sigma)-coated six-well plate. Transfection was performed the next
163 day using a total of 1.5 µg of DNA plasmid, with 6 µl of TransIT-LT1 transfection reagent
164 (Mirus). Supernatants were collected 24, 48, and 72 h post-transfection, filtered (0.45-
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165 µm pore size), and mixed with 100 µl of 1 M HEPES buffer. All transductions were
166 performed in the presence of 4 µg/ml Polybrene (Sigma). Receptor expression was
167 verified by flow cytometry using the following protocol: cells were lifted using PBS
168 supplemented with 4 mM EDTA and 10% FBS, washed, and pelleted in a V-bottom
169 plate. Cells (105) were incubated with either 20 µg/ml of mouse anti-hu-CD81 (BD
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170 Biosciences) or rabbit anti-hu-SR-B1 (Ab-Cam) for 30 minutes on ice, washed, and then o
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171 incubated with goat anti-mouse IgG or goat anti-rabbit IgG secondary antibody lo
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172 conjugated to Alexa Fluor 647 (Molecular Probes). Cells were washed twice and e
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173 receptor expression was analyzed on a FACSArray flow cytometer (Becton-Dickinson) r
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174 using FloJo software (Tree Star). h
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175 Cloning, expression, and purification of recombinant HCV E2. The E2 :
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176 protein ectodomain of strains H77 (aa 384-661) (35) or J6 (aa 385-661) was cloned into i.
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177 a baculovirus expression vector (pFastBac derivative) from plasmids containing the
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178 structural proteins of H77 (gift of M. Gale, Jr., University of Washington) or the infectious g
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179 J6/JFH1/JC1 (58) viral genome (gift of Apath, Inc). The baculovirus expression vector n

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180 adds a honeybee melittin signal peptide at the NH terminus and a thrombin-cleavable a
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181 His6 tag and stop codon at the COOH-terminus. Recombinant baculoviruses expressing 2
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182 HCV E2 ectodomains were generated as described previously (31), amplified in SF9 2
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183 cells, and used for large scale infection of Hi-5 cells under serum-free conditions. 9

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184 Supernatant was concentrated and buffer exchanged into binding buffer (300 mM
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185 sodium citrate, 150 mM sodium chloride, 50 mM sodium phosphate pH 8.0) using a e
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186 Centramate tangential flow concentrator. E2 was purified by sequential nickel-affinity
187 and size-exclusion chromatography and monodispersed fractions of monomeric protein
188 were collected and used for subsequent studies.
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189 Generation, purification, and labeling of anti-HCV MAbs. MAbs were
190 generated by five independent splenocyte-myeloma fusions as described (43). Mice
191 were immunized via an intraperitoneal route with sE2 produced from either genotype 1a
192 (H77) or 2a (J6) HCV strains after complexing with RIBI Adjuvant System (Corixa Corp)
193 or complete Freund’s adjuvant (Sigma Chemical). Mice were boosted between two and
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194 five times with homologous HCV sE2 protein complexed with either incomplete Freund’s o
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195 adjuvant (Sigma), RIBI Adjuvant System (Corixa), or Sigma Adjuvant system (Sigma),
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196 depending on commercial availability, until adequate titers (>1:2500 by ELISA) were d
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197 achieved. Mice with the highest serum titers were boosted intravenously with purified f
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198 sE2 (50 µg) three days prior to fusion of splenocytes with P3X63Ag8.53 myeloma cells
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199 (12). Hybridomas producing anti-HCV E2 antibodies were identified after binding to p
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200 Saccharomyces cerevesiae yeast expressing sE2 on their surface by flow cytometry, i.
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201 subcloned by limiting dilution, and isotyped by ELISA. For large-scale production, MAbs m
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202 were generated from ascites or adapted to growth in Hybridoma Serum Free Media rg
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203 (Gibco) and purified using protein A or G affinity chromatography (Pierce). In some n

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204 experiments, MAbs were labeled with Alexa Fluor 647 (Molecular Probes) or NHS-FITC a
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205 (Pierce) MAb labeling kits according to the manufacturer’s instructions.
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206 Virus neutralization assays. Neutralization of HCV infection by viruses ,
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207 containing genotype 1a structural proteins (H77/JFH1) was assessed by a focus forming 9

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208 unit (FFU) assay. Serial dilutions of HCV-specific MAb, control MAb (WNV E16 (43)), y

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209 anti-human CD81 (clone JS81, BD Biosciences), or anti-human SR-B1 (clone 396, Ab- e
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210 Cam) were pre-incubated with 2.4 x 102 FFU of virus for one hour at 37°C. Virus-MAb
211 mixtures were added to Huh-7.5 cells (1.2 x 104 cells per well) in a 48-well tissue culture
212 plate pre-coated with poly-L lysine (Sigma). After 72 hours, cells were fixed with
213 methanol (0oC), and incubated sequentially with a mouse anti-NS5A (APA-1, 40 ng/ml)
214 (38) (a generous gift of Apath, Inc.) and secondary goat anti-mouse HRP diluted 1:3000
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