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. 2001 Dec;75(24):12412-20.
doi: 10.1128/JVI.75.24.12412-12420.2001.

Production and characterization of monoclonal antibodies specific for a conserved epitope within hepatitis C virus hypervariable region 1

Affiliations

Production and characterization of monoclonal antibodies specific for a conserved epitope within hepatitis C virus hypervariable region 1

C Li et al. J Virol. 2001 Dec.

Abstract

Frequent mutations in hypervariable region 1 (HVR1) of the main envelope protein of hepatitis C virus (HCV) is a major mechanism of persistence by escaping the host immune recognition. HVR1 contains an epitope eliciting neutralizing antibodies. This study was aimed to prepare broadly cross-reacting, high-affinity, monoclonal antibodies (MAb) to the HVR1 C terminus of HCV with potential therapeutic neutralizing capacity. A conserved amino residue group of glycine (G) at position 23 and glutamic acid (Q) at position 26 in HVR1 was confirmed as a key epitope against which two MAbs were selected and characterized. MAbs 2P24 and 15H4 were immunoglobulin G1 kappa chain [IgG1(kappa)], cross-reacted with 32 and 30 of 39 random C-terminal HVR1 peptides, respectively, and did not react with other HCV peptides. The V(H) of 2P24 and 15H4 heavy chains originated from Igh germ line v gene family 1 and 8, respectively. In contrast, the V(L) kappa sequences were highly homologous. The affinity (K(d)) of 2P24 and 15H4 (10(-9) or 10(-8) M with two immunizing peptides and 10(-8) M with two nonimmunizing HVR1 peptides) paralleled the reactivity obtained with peptide enzyme immunoassay. MAbs 2P24 and 15H4 captured 25 of 31 (81%) HCV in unselected patients' plasmas. These antibodies also blocked HCV binding to Molt-4 cells in a dose-dependent fashion. The data presented suggest that broadly cross-reactive MAbs to a conserved epitope within HCV HVR1 can be produced. Clinical application for passive immunization in HCV-related chronic liver disease and after liver transplantation is considered.

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Figures

FIG. 1
FIG. 1
Competitive binding of 15H4 and 2P24 to MH2. A total of 0.25 μg of biotinylated 2P24 or 15H4/ml was mixed with nonbiotinylated 15H4 or 2P24 at concentrations of 0, 1, 2.5, 5, 7.5, and 10 μg/ml. Then, 100 μl of the mixture was added to MH2 HVR1 peptide-coated well. Mouse myeloma IgG1 (mIgG1) and HCV core region peptide S5 (Control) were used as MAb and peptide controls, respectively. Bound biotinylated MAb was detected by avidin-peroxidase with ortho-phenylenediamine substrate.
FIG. 2
FIG. 2
Amino acid sequences of the HVR1 MAbs. Amino acid sequences are presented with a single-letter code. Dashes indicate identity with the lead sequence. Asterisks indicate gaps compared with the reference antibody sequence. FWR1, -2, -3, and -4 correspond to framework regions 1, 2, 3, and 4, resepctively. CDR1, -2 and -3 correspond to complementarity-determining regions 1, 2, and 3, respectively.
FIG. 3
FIG. 3
Capture of HCV from plasma samples of representative patients by HVR1 MAbs 2P24 and 15H4. Captured HCV was detected by RT-nested PCR. A mouse myeloma IgG1 was used as a negative control. Lanes: 1, PCR molecular weight markers; 2 to 4, EH with antibodies 2P24, 15H4, and IgG1, respectively; 5 to 7, UKS3 with antibodies 2P24, 15H4, and IgG1, respectively; 8 to 10, UK12700 with antibodies 2P24, 15H4, and IgG1, respectively; 11 to 13, G4720 with antibodies 2P24, 15H4, and IgG1, respectively.
FIG. 4
FIG. 4
Inhibition of MAb HCV capture by HVR1 peptides. G4720 and G1245 (reactive with 2P24 and 15H4 by EIA) and G878 and G1224 (reactive at a low level or not reactive with 2P24 and 15H4 by EIA) are patient-derived HVR1 peptides. SMH2-2 (reactive with 15H4 but not with 2P24 by EIA) is a mutated HVR1 peptide. S5 is an HCV core region peptide used as control. A mouse myeloma IgG1 was used as a negative antibody control. Peptides (50 μg/ml) were preincubated with 2P24 and 15H4 or control IgG1 (10 μg/ml) and then incubated with HCV UKS3 plasma (1:10 diluted). Captured HCV was detected by RT-nested PCR. Lanes: 1, PCR molecular weight markers; 2 and 3, peptide G4720 with MAbs 2P24 and 15H4, respectively; 4 and 5, peptide G878 with MAbs 2P24 and 15H4, respectively; 6 and 7, peptide G1245 with MAbs 2P24 and 15H4, respectively; 8 and 9, peptide G1224 with MAbs 2P24 and 15H4, respectively; 10 and 11, peptide SMH2-2 with MAbs 2P24 and 15H4, respectively; and 12 to 14, peptide S5 with MAbs 2P24, 15H4, and IgG1, respectively.
FIG. 5
FIG. 5
Blocking of HCV binding to Molt-4 with MAbs 2P24 and 15H4. (A) EH virus. A total of 1.7 × 104 IU of EH virus was preincubated with different concentrations of a 1:1 mixture of 2P24 and 15H4 and added to 2 × 105 Molt-4 cells. Lanes: 1, PCR molecular weight markers; 2, PC; 3 to 6, MAbs 2P24 and 15H4 at 50, 25, 2.5, and 0.25 μg/ml, respectively; 7 to 10, MAb control at 50, 25, 2.5, and 0.25 μg/ml, respectively; 11, NC. (B) UKS3 virus. A total of 100 μl of 1:10 diluted UKS3 viruses were separately preincubated with 2P24 or 15H4 and then added to Molt-4 cells. Lanes: 1, PCR molecular weight markers; 2, PC; 3 and 4, MAb 2P24 at 25 and 2.5 μg/ml, respectively; 5 and 6, MAb 15H4 at 25 and 2.5 mg/ml, respectively; 7 and 8, MAb control at 25 and 2.5 μg/ml, respectively; 9, NC. In both panels, bound HCV was detected by RT-nested PCR; PC indicates positive control (HCV binding without MAb preincubation) and NC indicates negative control (normal plasma). MAb negative control is a mouse myeloma IgG1.

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