Cinzia Traboni

Binding of Hepatitis C Virus E2 Glycoprotein to CD81 Does Not Correlate with Species Permissiveness to Infection

ABSTRACT Hepatitis C virus (HCV) glycoprotein E2 binds to human cells by interacting with the CD81 molecule, which has been proposed to be the viral receptor. A correlation between binding to CD81 and species permissiveness to HCV infection has also been reported. We have determined the sequence of CD81 from the tamarin, a primate species known to be refractory to HCV infection. Tamarin CD81 (t-CD81) differs from the human molecule at 5 amino acid positions (155, 163, 169, 180, and 196) within the large extracellular loop (LEL), where the binding site for E2 has been located. Soluble recombinant forms of human CD81 (h-CD81), t-CD81, and African green monkey CD81 (agm-CD81) LEL molecules were analyzed by enzyme-linked immunosorbent assay for binding to E2 glycoprotein. Both h-CD81 and t-CD81 molecules were able to bind E2. Competition experiments showed that the two receptors cross-compete and that the t-CD81 binds with stronger affinity than the human molecule. Recently, h-CD81 residue 186 has been characterized as the critical residue involved in the interaction with E2. Recombinant CD81 mutant proteins were expressed to test whether human and tamarin receptors interacted with E2 in a comparable manner. Mutation of residue 186 (F186L) dramatically reduced the binding capability of t-CD81, a result that has already been demonstrated for the human receptor, whereas the opposite mutation (L186F) in agm-CD81 resulted in a neat gain of binding activity. Finally, the in vitro data were confirmed by detection of E2 binding to cotton-top tamarin (Saguinus oedipus) cell line B95-8 expressing endogenous CD81. These results indicate that the binding of E2 to CD81 is not predictive of an infection-producing interaction between HCV and host cells.

Generation of infectious and transmissible virions from a GB virus B full-length consensus clone in tamarins

The strong similarity between GB virus B (GBV-B) and hepatitis C virus (HCV) makes tamarins infected by GBV-B an acceptable surrogate animal model for HCV infection. Even more attractive, for drug discovery purposes, is the idea of constructing chimeric viruses by inserting HCV genes of interest into a GBV-B genome frame. To accomplish this, infectious cDNA clones of both viruses must be available. The characterization of several HCV molecular clones capable of infecting chimpanzees has been published, whereas only one infectious GBV-B clone inducing hepatitis in tamarins has been reported so far. Here we describe the infection of tamarins by intrahepatic injection of RNA transcribed from a genomic GBV-B clone (FL-3) and transmission of the disease from infected to naive tamarins via serum inoculation. The disease resulting from both direct and secondary infection was characterized for viral RNA titre and hepatitis parameters as well as for viral RNA distribution in the hepatic tissue. Host humoral immune response to GBV-B antigens was also monitored. The progression of the disease was compared to that induced by intravenous injection of different amounts of the non-recombinant virus.

Receptor phage. Display of functional domains of the human high affinity IgE receptor on the M13 phage surface.

In this paper we demonstrate that phage display technology is a suitable system for studying the interaction between the high‐affinity receptor for IgE (FcϵRI) and IgE. The α subunit extracellular domains of the human receptor were expressed on the surface of filamentous phage M 13 fused to the carboxyl‐terminal part of the gene III protein (pIII). Two constructs were made, the first with both the Ig‐like domains of the receptor α chain and the second with only the C‐terminal domain. The fusion genes were cloned in a phagemid vector to display monovalently the receptor on the phage surface. Our results indicate that the α receptor expressed on the phage is able to interact with IgE as demonstrated by an ELISA assay. In addition, by using the same system, we show that a single domain of the α receptor is sufficient for the interaction with IgE although with a binding affinity lower than that of the two‐domain receptor.

Processing of GB virus B non-structural proteins in cultured cells requires both NS3 protease and NS4A cofactor.

The identification of antivirals and vaccines against hepatitis C virus (HCV) infection is hampered by the lack of convenient animal models. The need to develop surrogate models has recently drawn attention to GB virus B (GBV-B), which produces hepatitis in small primates. In a previous study in vitro, it was shown that GBV-B NS3 protease shares substrate specificity with the HCV enzyme, known to be crucial for virus replication. In this report, GBV-B NS3 activity on GBV-B precursor proteins has been analysed in a cell-based system. It is shown that mature protein products are obtained that are compatible with the cleavage sites proposed on the basis of sequence homology with HCV and that GBV-B NS4A protein is required as a cofactor for optimal enzymatic activity. Experiments in vitro supported by a structural model mapped the region of NS4A that interacts with NS3 and showed that the GBV-B cofactor cannot be substituted for by its HCV analogue.

Phage display of a human antibody against Clostridium tetani toxin

9F12, a human antibody capable of neutralising tetanus toxin in an animal model, has been expressed as a Fab fragment in a phagemid system. The nucleotide sequence for the variable domain of both chains has been determined and compared to germline sequences.

A human antibody specific for hepatitis C virus core protein: synthesis in a bacterial system and characterization

The cDNA coding for the Fab fragment of the human B12.F8 antibody (Ab), directed against the putative nucleocapsid component (core protein) of hepatitis C virus (HCV), was cloned in the prokaryotic phagemid vector, pHEN-1, to obtain its expression in Escherichia coli. The functionality and specificity of the recombinant Ab, called B12Fab, were examined by Western blot and ELISA using recombinant HCV core protein as antigen. The specificity of B12Fab was further confirmed by ELISA with the 33-mer peptide epitope recognized by the original whole B12.F8 Ab. By immunofluorescence, the recombinant B12Fab was shown to recognize HCV core protein produced in cells transfected with HCV cDNA, indicating that the recombinant B12Fab is suitable as a diagnostic tool for tissue localization of the virus. The B12Fab also functioned when displayed on phage particles, providing the basis for future experiments of in vitro affinity maturation and selection of mutants. The variable chain coding regions of the recombinant B12Fab clone were sequenced and the V-gene usage was determined by comparison with the V kappa and VH germline sequences. The B12Fab V kappa chain belongs to the subgroup II and shows the highest degree of homology with the A3 germline gene, whereas the sequence of the VH chain is strictly related to that of the Humhv3019b18 gene of the VH3 family. These results are, to our knowledge, the first report of molecular cloning and characterization of a functional human Ab specific for an HCV antigen.

Tamarin alpha-interferon is active in mouse liver upon intramuscular gene delivery

The hepatitis C virus (HCV) is responsible for a severe and widespread form of hepatitis for which a durable and effective therapy has not yet been established. The only approved therapy against hepatitis C, alpha‐interferon protein intramuscular administration, presents numerous drawbacks that might be overcome by adopting a gene therapy approach. HCV exclusively infects humans and chimpanzees, hence an acceptable animal model for hepatitis C pharmacological studies is not available. Recently, tamarins infected by GB virus B (GBV‐B) have been proposed as a surrogate animal model for HCV infection. The aim of the present study was the production of tamarin interferon (tIFN) through delivery of tIFN‐coding DNA to evaluate the feasibility of a gene therapy approach based on IFN electro‐gene transfer (EGT) in future studies with primates.