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R Cortese

Publications and source records attributed to R Cortese.

At least 37 records · Page 2Linked to original sources
PubMed1999

A loop-mimetic inhibitor of the HCV-NS3 protease derived from a minibody.

We have been interested for some time in establishing a strategy for deriving lead compounds from macromolecule ligands such as minibody variants. A minibody is a minimized antibody variable domain whose two loops are amenable to combinatorial mutagenesis. This approach can be especially useful when dealing with 'difficult' targets. One such target is the NS3 protease of hepatitis C virus (HCV), a human pathogen that is believed to infect about 100 million individuals worldwide and for which an effective therapy is not yet available. Based on known inhibitor specificity (residues P6-P1) of NS3 protease, we screened a number of minibodies from our collection and we were able to identify a competitive inhibitor of this enzyme. We thus validated an aspect of recognition by HCV NS3 protease, namely that an acid anchor is necessary for inhibitor activity. In addition, the characterization of the minibody inhibitor led to the synthesis of a constrained hexapeptide mimicking the bioactive loop of the parent macromolecule. The cyclic peptide is a lead compound prone to rapid optimization through solid phase combinatorial chemistry. We therefore confirmed that the potential of turning a protein ligand into a low molecular weight active compound for lead discovery is achievable and can complement more traditional drug discovery approaches.

Binding Sites↗
PubMed1999

Development of animal models for adeno-associated virus site-specific integration.

The adeno-associated virus (AAV) is unique in its ability to target viral DNA integration to a defined region of human chromosome 19 (AAVS1). Since AAVS1 sequences are not conserved in a rodent's genome, no animal model is currently available to study AAV-mediated site-specific integration. We describe here the generation of transgenic rats and mice that carry the AAVS1 3.5-kb DNA fragment. To test the response of the transgenic animals to Rep-mediated targeting, primary cultures of mouse fibroblasts, rat hepatocytes, and fibroblasts were infected with wild-type wt AAV. PCR amplification of the inverted terminal repeat (ITR)-AAVS1 junction revealed that the AAV genome integrated into the AAVS1 site in fibroblasts and hepatocytes. Integration in rat fibroblasts was also observed upon transfection of a plasmid containing the rep gene under the control of the p5 and p19 promoters and a dicistronic cassette carrying the green fluorescent protein (GFP) and neomycin (neo) resistance gene between the ITRs of AAV. The localization of the GFP-Neo sequence in the AAVS1 region was determined by Southern blot and FISH analysis. Lastly, AAV genomic DNA integration into the AAVS1 site in vivo was assessed by virus injection into the quadriceps muscle of transgenic rats and mice. Rep-mediated targeting to the AAVS1 site was detected in several injected animals. These results indicate that the transgenic lines are proficient for Rep-mediated targeting. These animals should allow further characterization of the molecular aspects of site-specific integration and testing of the efficacy of targeted integration of AAV recombinant vectors designed for human gene therapy.

Animals↗
PubMed1998

Targeted integration of adeno-associated virus-derived plasmids in transfected human cells.

Adeno-associated virus (AAV) integrates its genomic DNA into a defined region of human chromosome 19 (AAVS1). The specificity of integration is dependent on the presence of the inverted terminal repeats (ITR) and on expression of the rep gene. To develop vectors capable of targeting the insertion of a selected DNA sequence into a specific location of human chromosome, we determined whether the rep gene can mediate site-specific integration when cloned outside of an ITR-flanked transgene cassette. HeLa and Huh-7 cells were transfected with a plasmid containing the rep gene, as well as the green fluorescent protein (GFP) and neomycin (neo) resistance gene inserted between the ITRs of AAV. Southern blot analysis of individual clones detected Rep-mediated site-specific integration of the ITR-flanked DNA in 25% and 12% of the HeLa and Huh-7 clones, respectively. The localization of the GFP-Neo sequence on chromosome 19 also was confirmed by fluorescent in situ hybridization analysis of the transfected HeLa clones. Sequence analysis of the ITR-AAVS1 junction of one of the transfected Huh-7 clones indicated that the insertion of the ITR DNA fragment had occurred at nucleotide 1003. These results have implications for the development of AAV-derived vectors capable of directing the site-specific integration of a gene of interest.

Base Sequence↗
PubMed1998

Efficient display of an HCV cDNA expression library as C-terminal fusion to the capsid protein D of bacteriophage lambda.

We describe the construction and characterization of a hepatitis C virus (HCV) cDNA expression library displayed as a fusion to the carboxy terminus of the capsid protein D of bacteriophage lambda. cDNA inserts were obtained by tagged random-priming of the HCV genome and cloned into a lambda vector from which chimeric phage bearing both wild-type D protein and D fusion products on the capsid surface were produced. The resulting library was affinity-selected with anti-HCV human monoclonal antibodies recognizing linear or conformational epitopes, and human sera from HCV-infected patients. Selection was monitored by immuno-screening experiments, ELISA, and sequence analysis of positive clones. The performance of this library was compared with two additional HCV cDNA display libraries generated as N-terminal fusions to the III and VIII capsid proteins of filamentous phage M13. The results obtained demonstrate the great potential of the lambda display system for constructing complex cDNA libraries for natural ligand discovery.

Antibodies, Monoclonal↗
PubMed1998

Towards a solution for hepatitis C virus hypervariability: mimotopes of the hypervariable region 1 can induce antibodies cross-reacting with a large number of viral variants.

The hypervariable region 1 (HVR1) of the putative envelope protein E2 of hepatitis C virus (HCV) is the most variable antigenic fragment in the whole viral genome and is mainly responsible for the large inter-and intra-individual heterogeneity of the infecting virus. It contains a principal neutralization epitope and has been proposed as the major player in the mechanism of escape from host immune response. Since anti-HVR1 antibodies are the only species shown to possess protective activity up to date, developing an effective prevention therapy is a very difficult task. We have approached the problem of HVR1 variability by deriving a consensus profile from >200 HVR1 sequences from different viral isolates and used it as a template to generate a vast repertoire of synthetic HVR1 surrogates displayed on M13 bacteriophage. This library was affinity selected using many different sera from infected patients. Phages were identified which react very frequently with patients' sera and bind serum antibodies that cross-react with a large panel of HVR1 peptides derived from natural HCV variants. When injected into experimental animals, the 'mimotopes' with the highest cross-reactivity induced antibodies which recognized the same panel of natural HVR1 variants. In these mimotopes we identified a sequence pattern responsible for the observed cross-reactivity. These data may hold the key for future development of a prophylactic vaccine against HCV.

Amino Acid Sequence↗
PubMed1998

Product inhibition of the hepatitis C virus NS3 protease.

The nonstructural protein NS3 of the hepatitis C virus (HCV) harbors a serine protease domain that is responsible for most of the processing events of the nonstructural region of the polyprotein. Its inhibition is presently regarded as a promising strategy for coping with the disease caused by HCV. In this work, we show that the NS3 protease undergoes inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B cleavage sites, whereas no inhibition is observed with a cleavage product of the intramolecular NS3-NS4A junction. The Ki values of the hexamer inhibitory products [Ki(NS4A) = 0.6 microM, Ki(NS5A) = 1.4 microM, and Ki(NS4B) = 180 microM] are lower than the Km values of the respective substrate peptides [Km(NS4A-NS4B) = 10 microM, Km(NS5A-NS5B) = 3.8 microM, and Km(NS4B-NS5A) > 1000 microM]. Mutagenesis experiments have identified Lys136 as an important determinant for product binding. The phenomenon of product inhibition can be exploited to optimize peptide inhibitors of NS3 protease activity that may be useful in drug development.

Amino Acid Sequence↗
PubMed1998

Potent peptide inhibitors of human hepatitis C virus NS3 protease are obtained by optimizing the cleavage products.

In the absence of a broadly effective cure for hepatitis caused by hepatitis C virus (HCV), much effort is currently devoted to the search for inhibitors of the virally encoded protease NS3. This chymotrypsin-like serine protease is required for the maturation of the viral polyprotein, cleaving it at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B sites. In the course of our studies on the substrate specificity of NS3, we found that the products of cleavage corresponding to the P6-P1 region of the substrates act as competitive inhibitors of the enzyme, with IC50s ranging from 360 to 1 microM. A detailed study of product inhibition by the natural NS3 substrates is described in the preceding paper [Steinkühler, C., et al. (1997) Biochemistry 37, 8899-8905]. Here we report the results of a study of the structure-activity relationship of the NS3 product inhibitors, which suggest that the mode of binding of the P region-derived products is similar to the ground-state binding of the corresponding substrates, with additional binding energy provided by the C-terminal carboxylate. Optimal binding requires a dual anchor: an "acid anchor" at the N terminus and a "P1 anchor" at the C-terminal part of the molecule. We have then optimized the sequence of the product inhibitors by using single mutations and combinatorial peptide libraries based on the most potent natural product, Ac-Asp-Glu-Met-Glu-Glu-Cys-OH (Ki = 0.6 microM), derived from cleavage at the NS4A-NS4B junction. By sequentially optimizing positions P2, P4, P3, and P5, we obtained several nanomolar inhibitors of the enzyme. These compounds are useful both as a starting point for the development of peptidomimetic drugs and as structural probes for investigating the substrate binding site of NS3 by modeling, NMR, and crystallography.

Amino Acid Sequence↗
PubMed1998

Codon-based mutagenesis using dimer-phosphoramidites.

A new approach for the synthesis of randomized DNA sequences containing the 20 codons corresponding to all natural amino acids is described. The strategy is based on the use of dinucleotide phosphoramidite building blocks within a resin-splitting procedure. Through this protocol, a minimal number of seven dimers is sufficient to encode all 20 natural amino acids. This synthesis procedure is extremely flexible and allows codon usage from different hosts to be accommodated.

Base Sequence↗
PubMed1998

DNA-based selection and screening of peptide ligands.

Phage display selection strategies rely on the physical link between the displayed heterologous protein ligand and the DNA encoding it. Thus, genes expressing a ligand with a specific binding affinity can be selected rapidly. To improve the specificity and sensitivity of this technology for potential use in identifying ligands to a specific antibody present in a complex mixture, we incorporated a DNA selection step along with the phage display technology. Ligands for hepatitis C virus (HCV) antibodies present in serum were identified by panning a phage-displayed random peptide library against pools of serum HCV antibodies. An additional DNA hybridization screening step using single-stranded DNA isolated from one of the pools increased the specificity and sensitivity, resulting in the selection of an HCV antibody ligand with diagnostic potential.

Amino Acid Sequence↗
PubMed1998

Engineering, characterization and phage display of hepatitis C virus NS3 protease and NS4A cofactor peptide as a single-chain protein.

The polyprotein encoded by hepatitis C virus (HCV) genomic RNA is processed into functional polypeptides by both host- and virus-encoded proteases. The HCV-encoded NS3 protease and its cofactor peptide NS4A form a non-covalent complex, which participates in processing the viral polyprotein. This proteolytic activity is believed to be essential for virus proliferation and thus the NS3 protease is a prime target for developing anti-HCV pharmacological agents. Recent X-ray crystallography structural studies have revealed the nature of this non-covalent complex between NS3 protease and the 'active' central segment of NS4A, providing the opportunity to design a single-chain polypeptide. To this end, the DNA sequence encoding for the NS4A peptide (residues 21-34) was genetically fused via a short linker, capable of making a beta-turn, to the N-terminus of the NS3 protease domain. This engineered single-chain NS3-protease (scNS3) is fully active with kinetic parameters virtually identical with those of the NS3/ NS4A non-covalent complex. Moreover, the scNS3 protease can be displayed on filamentous phage and affinity selected using an immobilized specific inhibitor. The scNS3 expressed as a soluble protein and in a phage-display format facilitates enzyme engineering for further structural studies and in vitro selection of potential drug-resistant mutants. These are important steps towards developing effective anti-protease compounds.

Amino Acid Sequence↗
PubMed1998

Site-specific integration in mammalian cells mediated by a new hybrid baculovirus-adeno-associated virus vector.

Baculovirus can transiently transduce primary human and rat hepatocytes, as well as a subset of stable cell lines. To prolong transgene expression, we have developed new hybrid vectors which associate key elements from adeno-associated virus (AAV) with the elevated transducing capacity of baculovirus. The hybrid vectors contain a transgene cassette composed of the beta-galactosidase (beta-Gal) reporter gene and the hygromycin resistance (Hygr) gene flanked by the AAV inverted terminal repeats (ITRs), which are necessary for AAV replication and integration in the host genome. Constructs were derived both with and without the AAV rep gene under the p5 and p19 promoters cloned in different positions with respect to the baculovirus polyheidrin promoter. A high-titer preparation of baculovirus-AAV (Bac-AAV) chimeric virus containing the ITR-Hygr-beta-Gal sequence was obtained with insect cells only when the rep gene was placed in an antisense orientation to the polyheidrin promoter. Infection of 293 cells with Bac-AAV virus expressing the rep gene results in a 10- to 50-fold increase in the number of Hygr stable cell clones. Additionally, rep expression determined the localization of the transgene cassette in the aavs1 site in approximately 41% of cases as detected by both Southern blotting and fluorescent in situ hybridization analysis. Moreover, site-specific integration of the ITR-flanked DNA was also detected by PCR amplification of the ITR-aavs1 junction in transduced human fibroblasts. These data indicate that Bac-AAV hybrid vectors can allow permanent, nontoxic gene delivery of DNA constructs for ex vivo treatment of primary human cells.

Animals↗
PubMed1998

Identification of peptides binding to IgG in the CSF of multiple sclerosis patients.

Phage displayed random peptide libraries were screened in order to identify phagotopes reacting with the IgG present in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients. Six families of phagotopes each composed of different isolates were identified. These phagotopes were used as reagents, to characterise IgG present in the CSF of MS patients. The following results were obtained: (a) CSF antibodies from different patients display different specificities; (b) anti-phagotope antibodies are also present in the serum of MS patients; (c) Anti-phagotope antibodies are equally frequent in the serum of MS patients and of healthy individuals; (d) some of the anti-phagotope antibodies are enriched in the CSF of MS patients. These data show that the natural antigen(s) recognised by CSF antibodies is rather common in the general population. By using the selected phagotopes as immunogens in rabbits, we have derived large quantities of anti-phagotope antisera which can provide for useful tools toward the identification of the natural antigen(s) recognised by MS CSF antibodies.

Adult↗
PubMed1998

CSF-enriched antibodies do not share specificities among MS patients.

The specificity of the oligoclonal immunoglobulins found in MS CSF is unknown. We have previously shown that random peptide libraries displayed on phage can be used to identify specific ligands for CSF antibodies. Here we describe the use of this tool in the attempt to identify MS-specific CSF-enriched antibody reactivities with potential pathogenic, diagnostic or prognostic value. Applying different experimental strategies, several ligands reacting with CSF-enriched antibodies were identified. When tested against a panel of 55 MS patients, none of the ligands found were recognized by antibodies shared by any two patients. We used the selected peptides to demonstrate the stability in time of CSF-enriched antibodies notwithstanding disease progression.

Adult↗
PubMed1997

Ciliary neurotrophic factor corrects obesity and diabetes associated with leptin deficiency and resistance.

Receptor subunits for the neurocytokine ciliary neurotrophic factor (CNTF) share sequence similarity with the receptor for leptin, an adipocyte-derived cytokine involved in body weight homeostasis. We report here that CNTF and leptin activate a similar pattern of STAT factors in neuronal cells, and that mRNAs for CNTF receptor subunits, similarly to the mRNA of leptin receptor, are localized in mouse hypothalamic nuclei involved in the regulation of energy balance. Systemic administration of CNTF or leptin led to rapid induction of the tis-11 primary response gene in the arcuate nucleus, suggesting that both cytokines can signal to hypothalamic satiety centers. Consistent with this idea, CNTF treatment of ob/ob mice, which lack functional leptin, was found to reduce the adiposity, hyperphagia, and hyperinsulinemia associated with leptin deficiency. Unlike leptin, CNTF also reduced obesity-related phenotypes in db/db mice, which lack functional leptin receptor, and in mice with diet-induced obesity, which are partially resistant to the actions of leptin. The identification of a cytokine-mediated anti-obesity mechanism that acts independently of the leptin system may help to develop strategies for the treatment of obesity associated with leptin resistance.

Animals↗
PubMed1997

Identification of a novel type 1 diabetes-specific epitope by screening phage libraries with sera from pre-diabetic patients.

We used random peptide libraries displayed on phage to search for ligands to insulin dependent diabetes mellitus-related antibodies and were able to identify several candidate disease-related peptides. One of them, clone 92, showed a significant difference in the frequency of reactivity with the sera of patients and normal controls. Human immunoglobulins immunopurified on phage 92 specifically stained the islets on human pancreatic sections. When injected into rabbits, the selected peptide elicited antibodies that also stained human and rat pancreatic sections, with a pattern similar to that observed with immunoglobulins purified from the sera of patients. No reactivity was observed in other tissues. Our results indicate that the peptide identified in this work mimics a novel, diabetes-related self-antigen.

Animals↗
PubMed1997

Induction of anti-carbohydrate antibodies by phage library-selected peptide mimics.

One of the prerequisites for the development of polysaccharide subunit vaccines is the induction of an efficient immune response to carbohydrate antigens like lipopolysaccharide (LPS) or capsular polysaccharide antigens of pathogens. In an attempt to overcome the problems that arise from the T-independent immune response induced by such antigens, selecting peptide sequences that mimic protective carbohydrate epitopes has been proposed. In this study, we investigate a new selection strategy for immunogenic peptide mimics using the phage-displayed peptide library technology. Two monoclonal antibodies (mAb) of the A isotype (mIgA), mIgA C5 and mIgA I3, specific for the O-antigen (O-Ag) part of the human pathogen Shigella flexneri serotype 5a LPS and protective against homologous infection were used to screen two phage-displayed nonapeptide libraries in pVIII. Using mIgA C5, 13 different specific clones were selected, and 6 using mIgA I3; 5 of the latter also interacted in enzyme-linked immunosorbent assay with the first mAb. All of the 19 clones selected were separately used to immunize mice, but only 2 of them, p100c (mIgA I3-specific) and p115 (interacting with both mIgA) were able to induce anti-O-Ag antibodies. The immune response was specific for the O-Ag of the S. flexneri serotype 5a, and also selectively recognized the corresponding bacterial strain. The amino acid sequences of p100c and p115 immunogenic peptide mimics were YKPLGALTH (flanked by two Cys residues) and KVPPWARTA, respectively. These results are the first example of immunogenic mimicry of carbohydrates by phage-displayed peptides, and indicate a new strategy of selection of immunogens for the development of anti-polysaccharide vaccines.

Animals↗
PubMed1997

Immunogenicity of filamentous phage displaying peptide mimotopes after oral administration.

Selected human sera can be used to identify disease-related peptide epitopes (mimotopes) displayed on bacteriophages. Parenteral administration of such recombinant phages is an effective route of immunization in different experimental animals, indicating that mimotopes could be an important source of leads for new vaccines. Here it is shown that intranasal or intragastric administration of phage in mice induces an immunological response both to the wild type proteins of the phage and to mimotopes displayed on them. Using mimotopes of human HBV surface antigen and of human HCV peptides, the authors show that the response induced by oral administration is specifically cross-reactive with the original antigen. These findings indicate that phage displaying selected mimotopes could be useful for the development of orally effective vaccines.

Adjuvants, Immunologic↗