Timothy P. Hickling

Identification of Conserved Residues in the E2 Envelope Glycoprotein of the Hepatitis C Virus That Are Critical for CD81 Binding

ABSTRACT Hepatitis C virus (HCV) cell entry involves interaction between the viral envelope glycoprotein E2 and the cell surface receptor CD81. Knowledge of conserved E2 determinants important for successful binding will facilitate development of entry inhibitors designed to block this interaction. Previous studies have assigned the CD81 binding function to a number of discontinuous regions of E2. To better define specific residues involved in receptor binding, a panel of mutants of HCV envelope proteins was generated, where conserved residues within putative CD81 binding regions were sequentially mutated to alanine. Mutant proteins were tested for binding to a panel of monoclonal antibodies and CD81 and for their ability to form noncovalent heterodimers and confer infectivity in the retroviral pseudoparticle (HCVpp) assay. Detection by conformation-sensitive monoclonal antibodies indicated that the mutant proteins were correctly folded. Mutant proteins fell into three groups: those that bound CD81 and conferred HCVpp infectivity, those that abrogated both CD81 binding and HCVpp infectivity, and a final group containing mutants that were able to bind CD81 but were noninfectious in the HCVpp assay. Specific amino acids conserved across all genotypes that were critical for CD81 binding were W420, Y527, W529, G530, and D535. These data significantly increase our understanding of the CD81 receptor-E2 binding process.

Characterization of the hepatitis C virus E2 epitope defined by the broadly neutralizing monoclonal antibody AP33

The mouse monoclonal antibody (MAb) AP33, recognizing a 12 amino acid linear epitope in the hepatitis C virus (HCV) E2 glycoprotein, potently neutralizes retroviral pseudoparticles (HCVpp) carrying genetically diverse HCV envelope glycoproteins. Consequently, this antibody and its epitope are highly relevant to vaccine design and immunotherapeutic development. The rational design of immunogens capable of inducing antibodies that target the AP33 epitope will benefit from a better understanding of this region. We have used complementary approaches, which include random peptide phage display mapping and alanine scanning mutagenesis, to identify residues in the HCV E2 protein critical for MAb AP33 binding. Four residues crucial for MAb binding were identified, which are highly conserved in HCV E2 sequences. Three residues within E2 were shown to be critical for binding to the rat MAb 3/11, which previously was shown to recognize the same 12 amino acid E2 epitope as MAb AP33 antibody, although only two of these were shared with MAb AP33. MAb AP33 bound to a panel of functional E2 proteins representative of genotypes 1‐6 with higher affinity than MAb 3/11. Similarly, MAb AP33 was consistently more efficient at neutralizing infectivity by diverse HCVpp than MAb 3/11. Importantly, MAb AP33 was also able to neutralize the cell culture infectious HCV clone JFH‐1. In conclusion, these data identify important protective determinants and will greatly assist the development of vaccine candidates based on the AP33 epitope. (HEPATOLOGY 2006;43:492–601.)

Specific interaction of hepatitis C virus glycoproteins with mannan binding lectin inhibits virus entry

Mannan-binding lectin (MBL) is a soluble innate immune protein that binds to glycosylated targets. MBL acts as an opsonin and activates complement, contributing to the destruction and clearance of infecting microorganisms. Hepatitis C virus (HCV) encodes two envelope glycoproteins E1 and E2, expressed as non-covalent E1/E2 heterodimers in the viral envelope. E1 and E2 are potential ligands for MBL. Here we describe an analysis of the interaction between HCV and MBL using recombinant soluble E2 ectodomain fragment, the full-length E1/E2 heterodimer, expressed in vitro, and assess the effect of this interaction on virus entry. A binding assay using antibody capture of full length E1/E2 heterodimers was used to demonstrate calcium dependent, saturating binding of MBL to HCV glycoproteins. Competition with various saccharides further confirmed that the interaction was via the lectin domain of MBL. MBL binds to E1/E2 representing a broad range of virus genotypes. MBL was shown to neutralize the entry into Huh-7 cells of HCV pseudoparticles (HCVpp) bearing E1/E2 from a wide range of genotypes. HCVpp were neutralized to varying degrees. MBL was also shown to neutralize an authentic cell culture infectious virus, strain JFH-1 (HCVcc). Furthermore, binding of MBL to E1/E2 was able to activate the complement system via MBL-associated serine protease 2. In conclusion, MBL interacts directly with HCV glycoproteins, which are present on the surface of the virion, resulting in neutralization of HCV particles.

Severe fibrosis in hepatitis C virus-infected patients is associated with increased activity of the mannan-binding lectin (MBL)/MBL-associated serine protease 1 (MASP-1) complex.

Mannan‐binding lectin (MBL) binds microorganisms via interactions with glycans on the target surface. Bound MBL subsequently activates MBL‐associated serine protease proenzymes (MASPs). A role for MBL in hepatitis C virus (HCV) infection had been indicated by previous studies examining MBL levels and polymorphisms in relation to disease progression and response to treatment. We undertook this study to investigate a possible relationship between disease progression and functional MBL/MASP‐1 complex activity. A functional assay for MBL/MASP‐1 complex activity was employed to examine serum samples from patients with chronic HCV infection, non‐HCV liver disease and healthy controls. Intrapatient consistency of MBL/MASP‐1 complex activity levels was assessed in sequential samples from a subgroup of patients. Median values of MBL/MASP‐1 complex activity were higher in sera from patients with liver disease compared with healthy controls. MBL/MASP‐1 complex activity levels correlate with severity of fibrosis after adjusting for confounding factors (P = 0·003). MBL/MASP‐1 complex activity was associated more significantly with fibrosis than was MBL concentration. The potential role of MBL/MASP‐1 complex activity in disease progression is worthy of further study to investigate possible mechanistic links.

Recombinant Human L-Ficolin Directly Neutralizes Hepatitis C Virus Entry

L-ficolin is a soluble pattern recognition molecule expressed by the liver that contributes to innate immune defense against microorganisms. It is well described that binding of L-ficolin to specific pathogen-associated molecular patterns activates the lectin complement pathway, resulting in opsonization and lysis of pathogens. In this study, we demonstrated that in addition to this indirect effect, L-ficolin has a direct neutralizing effect against hepatitis C virus (HCV) entry. Specific, dose-dependent binding of recombinant L-ficolin to HCV glycoproteins E1 and E2 was observed. This interaction was inhibited by soluble L-ficolin ligands. Interaction of L-ficolin with E1 and E2 potently inhibited entry of retroviral pseudoparticles bearing these glycoproteins. L-ficolin also inhibited entry of cell-cultured HCV in a calcium-dependent manner. Neutralizing concentrations of L-ficolin were found to be circulating in the serum of HCV-infected individuals. This is the first description of direct neutralization of HCV entry by a ficolin and highlights a novel role for L-ficolin as a virus entry inhibitor.