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Biomedical subjects

R Burioni

Publications and source records attributed to R Burioni.

At least 19 recordsLinked to original sources
PubMed2001

Nonneutralizing human antibody fragments against hepatitis C virus E2 glycoprotein modulate neutralization of binding activity of human recombinant Fabs.

Evidence from clinical and experimental studies indicates that hepatitis C virus E2 (HCV/E2) glycoprotein is the major target of a putatively protective immune response. However, even in the presence of a vigorous production of anti-HCV/E2 antibodies, reinfection can occur. Dissection of the human immune response against HCV/E2 indicated that blocking of binding of HCV/E2 to target cells [neutralization of binding (NOB) activity] varies widely among antibody clones. Moreover, in vivo, simultaneous binding of antibodies to distinct epitopes can induce conformational changes and synergies that may be relevant to understanding the anti-HCV immune response. In this study, human recombinant Fabs were generated by affinity-selecting a phage display repertoire library with antibody-coated HCV/E2. These Fabs, which share the same complementarity-determining region DNA sequences, had higher affinity than other anti-HCV/E2 Fabs but showed no NOB activity even at the highest concentrations. Binding of Fabs to HCV/E2 caused conformational changes modifying Fab-binding patterns and reducing, with a negative synergistic effect, Fab-mediated NOB activity. These data suggest that some antibody clones have the potential to modify HCV/E2 conformation and that, in this state, binding of this glycoprotein to its cellular target is less prone to inhibition by some antibody clones. This can explain why high anti-HCV/E2 antibody titers do not directly correlate with protection from infection. Information on the interactions among different antibody clones can contribute to understanding virus-host interplay and developing more effective vaccines.

Amino Acid Sequence↗
PubMed2001

Molecular profile of a human monoclonal antibody Fab fragment specific for Epstein-Barr virus gp350/220 antigen.

Experimental evidence indicates Epstein Barr virus (EBV) envelope glycoprotein gp350/220 elicits a potent virus neutralizing response in the infected human host that may play an important role in restricting viral pathogenesis. In this study, we report the molecular cloning in combinatorial phage display vectors, of the IgG1 repertoire of an individual naturally infected with EBV, and describe the recovery and characterization of a monoclonal antibody recognizing gp350/220. A detailed understanding of the human antibody response in EBV infection will identify antibodies of potential use in anti-viral prophylaxis and will advance the production of more effective vaccine candidates.

Amino Acid Sequence↗
PubMed2001

Mapping B-cell epitopes of hepatitis C virus E2 glycoprotein using human monoclonal antibodies from phage display libraries.

Clinical and experimental evidence indicates that the hepatitis C virus (HCV) E2 glycoprotein (HCV/E2) is the most promising candidate for the development of an effective anti-HCV vaccine. Identification of the human epitopes that are conserved among isolates and are able to elicit protective antibodies would constitute a significant step forward. This work describes the mapping of the B-cell epitopes present on the surface of HCV/E2, as recognized by the immune system during infection, by the analysis of the reciprocal interactions of a panel of human recombinant Fabs derived from an HCV-infected patient. Three unrelated epitopes recognized by antibodies with no neutralization-of-binding (NOB) activity were identified; a fourth, major epitope was defined as a clustering of minor epitopes recognized by Fabs endowed with strong NOB activity.

Antibodies, Monoclonal↗
PubMed2000

n--> infinity limit of O(n) ferromagnetic models on graphs

Thirty years ago, H. E. Stanley showed that an O(n) spin model on a lattice tends to a spherical model as n-->infinity. This means that at any temperature the corresponding free energies coincide. This fundamental result is no longer valid on more general discrete structures lacking in translation invariance, i.e., on graphs. However, only the singular parts of the free energies determine the critical behavior of the two statistical models. Here we show that for ferromagnetic models such singular parts still coincide even on graphs in the thermodynamic limit. This implies that the critical exponents of O(n) models on graphs for n-->infinity tend to the spherical ones and depend only on the graph spectral dimension.

Journal Article↗
PubMed2000

No evidence of Helicobacter pylori sequences in pancreatic juices of patients affected by chronic pancreatitis.

BACKGROUND: The course of chronic pancreatitis is often unpredictable and many factors are likely to be involved in the progression of the disease. In physiological condition, pancreatic juice exerts significant antibacterial activity, which is impaired in patients with chronic pancreatitis. AIM: Hypothesizing that Helicobacter pylori could, in these conditions, lead to an ascending infection, we aimed to assess the presence of H. pylori sequences in pancreatic juices of patients with chronic pancreatitis. METHODS: 40 patients (mean age 52+/-3 yr) with alcoholic chronic pancreatitis and H. pylori infection were examined. Pancreatic juices were collected during endoscopic retrograde cholangiopancreatography. Using polymerase chain reaction (PCR) with two primers homologous to a portion of urease-C gene, H. pylori DNA was detected. Gastric biopsies, microscopically positive to H. pylori were used as positive controls. RESULTS: All gastric biopsies produced H. pylori-specific DNA products. Conversely, no H. pylori urease-C gene sequences have been detected in any of the pancreatic juices. CONCLUSION: Our data suggest that the impaired antibacterial activity of pancreatic juices in patients affected by chronic pancreatitis does not have a permissive role for a superimposing H. pylori infection in the pancreas. The possibility that Helicobacter species other than pylori may be involved in a superimposing infection requires further investigation.

Chronic Disease↗
PubMed1998

A vector for the expression of recombinant monoclonal Fab fragments in bacteria.

The availability of genes coding for monoclonal Fab fragments of a desired specificity permits their expression in bacteria and provides a simple method for the generation of good quality reagents. In this paper we describe a new phagemid vector for the production of recombinant Fabs from genes obtained from phage display combinatorial libraries. The phagemid features an antibiotic resistance cassette which, once inserted between the heavy chain fragment and the light chain genes, avoids unwanted recombination and preserves useful restriction sites not affecting the Fab production rate.

Antibodies, Monoclonal↗
PubMed1998

Dissection of human humoral immune response against hepatitis C virus E2 glycoprotein by repertoire cloning and generation of recombinant Fab fragments.

Demonstration of antibodies inhibiting key viral functions is the basis for the design of an effective vaccine. Dissection of the human antibody response by repertoire cloning may be a powerful means to address this issue. In this study, a panel of human monoclonal recombinant Fab fragments specific for hepatitis C virus (HCV) E2 envelope protein was generated. The selection procedure was designed to select for cross-genotype reactive antibodies. Sequences coding five different human recombinant Fabs specific for the HCV/E2 protein were cloned and characterized. The ability of the cloned antibody fragments to inhibit adhesion of recombinant envelope E2 protein to target cells was assayed. While affinity of the different antibody fragments appeared similar, activity in inhibiting E2 binding to target cells varied considerably from one Fab fragment to another. Two Fabs were not able to inhibit E2 binding at high concentration (40 microg/mL), while three other Fab clones were active in neutralizing 50% of the E2 binding at concentrations ranging from 3 to 0.35 microg/mL.

Amino Acid Sequence↗
PubMed1997

Use of recombinant human antibody fragments for detection of cytomegalovirus antigenemia.

The determination and quantitation of peripheral blood leukocytes (PBLs) expressing human cytomegalovirus (HCMV) antigens is widely employed in clinical virology for rapid diagnosis of HCMV-related infections. We describe how CMV antigenemia may be accurately detected by means of human recombinant monoclonal Fab fragments rescued from a combinatorial phage display library prepared from an HCMV-infected donor. Fourteen recombinant Fabs were tested against HCMV-positive PBLs from a patient with ongoing HCMV infection. Three clones were found to react specifically with the nuclei of these cells. These three recombinant Fabs were subsequently tested, individually and pooled together, against 60 PBL samples taken from immunosuppressed patients. The reactivity observed was comparable to that obtained with mouse monoclonal antibodies commercially available for this purpose. The three recombinant Fabs were shown to react specifically with the 65-kDa viral tegument phosphoprotein encoded by UL83 (pUL83), which is the most abundant viral antigen in HCMV-infected PBLs.

Antibodies, Monoclonal↗
PubMed1995

Engineering human monoclonal antibody fragments: a recombinant enzyme-linked Fab.

A new plasmid vector, pCRP, allowing the expression of human recombinant monoclonal antibody Fab fragments fused with a bacterial acid phosphatase has been constructed. pCRP can accept heavy- and light-chain cDNAs cloned from combinatorial antibody libraries displayed on filamentous phages with the pCombIII system and is able to direct expression to soluble Fabs in which the carboxy-terminus of the heavy chain is fused to the amino-terminus of the mature PhoN nonspecific acid phosphatase of Providencia stuartii. Using the pCRP vector, we expressed two different human recombinant Fabs cloned from combinatorial libraries (one anti-tetenus toxoid and the other anti-HIV-1 gp120) fused with the acid phosphatase. In both cases chimeric antibodies were obtained which retained the antigen-binding ability and the enzymatic activity. Similar Fab-enzyme fusions can be successfully used, even unpurified, in enzyme immunoassays.

Acid Phosphatase↗
PubMed1995

Cloning and characterization of human recombinant antibody Fab fragments specific for types 1 and 2 herpes simplex virus.

Twenty-six bacterial clones producing human recombinant Fab fragments specific for Herpes Simplex virus (HSV) antigens were obtained from an IgG1k human antibody combinatorial library displayed on filamentous phage, following panning against an HSV lysate. All the Fabs reacted against the HSV lysate in enzyme-linked immunosorbent assay and were able to recognize both type 1 and type 2 HSV in an indirect immunofluorescence assay (IFA). DNA sequencing of the heavy chain variable regions showed that these Fabs were different from those already described. One of these Fabs (Fab19) was purified and subjected to further characterization. Purified Fab19 was able to specifically recognize several different HSV-1 and HSV-2 strains (including 2 reference strains and 12 clinical isolates) in IFA. It was also able to neutralize the infectivity of both HSV-1 and HSV-2 strains, although the neutralizing activity was somewhat lower against HSV-2. In fact, 100% neutralization of infectivity was observed at a Fab concentration of 2 micrograms/100 TCD50 for the majority of HSV-1 strains, while a concentration of 8 micrograms/100 TCD50 was needed for 100% neutralization of all the HSV-2 strains tested. Owing to the above properties, Fab19 appears to be useful for diagnostic purposes and might also prove useful for in vivo immunoprophylaxis and therapy of HSV infections.

Animals↗
PubMed1995

A new plasmid cloning vector for direct detection of recombinant clones, based on inactivation of a bacterial acid phosphatase-encoding gene.

A new plasmid cloning vector for Escherichia coli (pPhoR) suitable for direct detection of recombinant clones has been constructed. The plasmid is a multicopy of a vector which carries a pUC-derived origin of replication and beta-lactamase gene, and the phoN acid phosphatase-encoding gene from Providencia stuartii. Foreign DNA fragments can be cloned into unique restriction sites located within the phoN gene causing a loss of the acid phosphatase activity which is normally overproduced by E. coli strains carrying pPhoR. Since PhoN production can be easily detected by a plate histochemical assay, recombinant clones carrying foreign DNA fragments inserted in the phoN gene can be easily detected as PhoN-negative clolonies on the above medium. The efficiency of the pPhoR-based cloning system for direct cloning of PCR amplimers of a variable region of the HIV-1 genome was comparable to that of conventional cloning systems for direct detection of recombinant based on beta-galactosidase inactivation. Advantage of the pPho-R-based system include reduced costs for histochemical assays and the possibility of being used with any E. coli host.

Acid Phosphatase↗
PubMed1994

Recombinant human Fab to glycoprotein D neutralizes infectivity and prevents cell-to-cell transmission of herpes simplex viruses 1 and 2 in vitro.

Herpes simplex viruses 1 and 2 (HSV-1 and -2) are associated with a number of conditions of varying severity, which are only partially responsive to current therapies. Human antibodies to the viruses offer a potential alternative. We describe here the generation of panels of human monoclonal Fab fragments to HSV-1 and -2 by panning a phage display combinatorial antibody library against whole lysates from the two viruses. Each lysate selected a largely distinct set of Fabs, although all of the Fabs were cross-reactive with both viruses. In a plaque-reduction assay, one Fab neutralized HSV-1 at 0.25 microgram/ml (50% reduction) and HSV-2 at 0.05 microgram/ml. This Fab also inhibited plaque formation when applied to virus-infected monolayers, completely abolishing HSV-2 plaque development at 25 micrograms/ml 72 hr postinfection, indicating the ability of the Fab to prevent cell-to-cell spread of virus. The Fab was shown to recognize viral glycoprotein D and to neutralize virus primarily by a postattachment mechanism. Recombinant Fabs may be useful for topical administration, although whole antibody will probably be required for systemic use.

Amino Acid Sequence↗