Inhibition of the binding of HCV serum particles to human hepatocytes by E1E2‐specific D32.10 monoclonal antibody

Abstract

The aim of this study was to determine the inhibition of binding activity of the monoclonal antibody (mAb) D32.10 which recognizes a highly conserved discontinuous antigenic determinant (E1:297-306, E2:480-494, and E2:613-621) expressed on the surface of serum-derived HCV particles (HCVsp) of genotypes 1a, 1b, 2a, and 3a. To this end, an in vitro direct cell-binding assay based on the attachment of radiolabeled HCVsp was developed, and Scatchard plots were used to analyze ligand-receptor binding data. HCV adsorption was also assessed by quantitating cell-associated viral RNA by a real-time RT-PCR method. Saturable concentration-dependent specific binding of HCVsp to Huh-7 or HepaRG cells was demonstrated. The Scatchard transformed data showed two-site interaction for Huh-7 and proliferative HepaRG cells: the high-affinity binding sites (K(d1) = 0.1-0.5 microg/ml) and the low-affinity binding sites (K(d1) = 5-10 microg/ml), and one-site high-affinity binding model between E1E2/D32.10-positive HCVsp and hepatocyte-like differentiated HepaRG cells. The E1E2-specific mAb D32.10 inhibited efficiently (>60%) and selectively the binding with an IC(50) <or=0.5 microg/ml in all the experimental approaches using serum HCV of genotype either 3 or 1b. This supports the involvement of the E1E2/D32.10 discontinuous antigenic determinant in the interactions between human hepatocytes and HCVsp, and suggests that D32.10-like antibodies present in sera from patients infected with HCV could play a protective role.

Non-neutralizing epitopes induce robust hepatitis C virus (HCV)-specific antibody-dependent CD56+ natural killer cell responses in chronic HCV-infected patients

“…A total of 17 linear peptides were reported to be recognized by defined anti-hepatitis C virus (HCV) E1/E2 monoclonal antibodies. Underlined epitopes 1 [11], 5 [12,13], 7 [14], 8 [15,16], 10 [17] and 12 [18,19] are overlapped with six putative linear neutralizing epitopes, and the other 11 epitopes (2, 3, 4, 6, 9, 11, 13, 14, 15, 16 and 17) are targets recognized by well-characterized anti-HCV E1/E2 monoclonal antibodies [20][21][22][23][24][25]. *The first and final positions are numbered with reference to the consensus sequence of the polyprotein of HCV strain H77 (Genbank accession number AB009606).…”

Structural and functional characterization of the single‐chain Fv fragment from a unique HCV E1E2‐specific monoclonal antibody

“…Unlike well‐characterized anti‐HCV E2 human mAbs with potent cross‐neutralizing activity, D32.10‐like antibodies have been shown to be associated with spontaneous virus clearance and predictive for complete viral response in chronically‐infected patients under SOC antiviral therapy [9]. The E1E2 epitope targeted by the D32.10 mAb has been shown to be preferentially involved in interactions between the virus and polarized hepatocytes [7]. In the recently proposed 3D‐model of HCV E2 ectodomain (E2e) [19], the E2 segment, aa 613–621, recognized by D32.10 encompassed residues 613–618 important for CD81 binding.…”

“…4A and B, lanes 0). As the native antibody, the scFv showed a higher reactivity with the HCVsp isolate 1 (HCV-Lat, GT3) than with the HCV isolate 2 (HCV-Fan, GT1a/1b), independently of the genotype but dependent on surface pattern of HCV particles [6,7]. It is worth noticing that the scFv significantly inhibited in a concentration-dependent manner the binding of IgG D32.10 to both HCVsp isolates (>50% for the isolate 1 and >70% for the isolate 2 at a concentration of 50 lg/ml corresponding to a molecular ratio IgG/scFv = 2) ( Fig.…”

“…This epitope is highly conserved between the different genotypes of HCV (1a, 1b, 2a, 2b, 3a, 4, 5 and 6). Furthermore, the mAb D32.10 is so far the only antibody able to efficiently inhibit the interactions between HCVsp and hepatocytes [7] as well as in vitro HCV infection [8]. The relevance of this mAb in vivo was proven by using 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.…”

Determination of the Human Antibody Response to the Neutralization Epitopes Encompassing Amino Acids 313–327 and 432–443 of Hepatitis C Virus E1E2 Glycoproteins

“…Studies have suggested that viral clearance is associated with a rapid induction of neutralizing antibodies in the early phase of infection [19], [20], and a large collection of antibodies has been reported to prevent HCV pseudoparticles (HCVpp) or Cell culture-produced HCV (HCVcc) infection[9], [21]–[29]. One other antibody, named D32.10, plays a protective role by inhibiting the interaction between serum-derived envelope HCV particles and hepatocytes [30], [31].…”