Osteopontin is an autoantigen of the somatostatin cells in human islets: identification by screening random peptide libraries with sera of patients with insulin-dependent diabetes mellitus.

Random peptide libraries (RPLs) screening with IDDM sera has identified 5 disease-specific 'mimotopes' displayed on phage (phagotopes). We characterised one phagotope (CH1p), by raising a rabbit antibody against the peptide insert on phage, which was employed in immunohistochemistry, Western blotting and cDNA libraries screening. The CH1p mimotope was detected in somatostatin cells of human islets and experimentally raised anti-osteopontin antibodies or human sera positive for the phagotope, detected a similar subpopulation of islet cells. The screening of cDNA library identified a clone corresponding to human osteopontin. In summary, RPLs proved to be successful in the identification of a novel islet-related autoantigen (osteopontin), whose significance in disease remains to be established.

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.

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.

Selection of biologically active peptides by phage display of random peptide libraries.

Random peptide libraries displayed on phage are used as a source of peptides for epitope mapping, for the identification of critical amino acids responsible for protein-protein interactions and as leads for the discovery of new therapeutics. Efficient and simple procedures have been devised to select peptides binding to purified proteins, to monoclonal and polyclonal antibodies and to cell surfaces in vivo and in vitro.

Selection of phage-displayed peptides mimicking type 1 diabetes-specific epitopes.

Phage display technology represents a powerful tool for the identification of peptides reacting with disease-related antibodies present in human sera. The application of this technology to type 1 diabetes could provide a set of novel reagents for diabetes prediction and could also lead to the identification of novel autoantigens or even of environmental factors possibly causing the disease. In the present study, sera of prediabetic and high risk individuals were used to select candidate peptides from phage-displayed random peptide libraries. Diabetes specific phage clones were then identified from these through screening and counter screening, using sera from diabetic and non-diabetic individuals. The results presented in this paper demonstrate the feasibility of this methodology to identify peptides reacting preferentially with antibodies present in the serum of diabetic patients.

Derivation of vaccines from mimotopes. Immunologic properties of human hepatitis B virus surface antigen mimotopes displayed on filamentous phage.

We have previously reported the identification, using human immune sera, of mimotopes of human hepatitis B virus surface Ag (HBsAg) displayed on filamentous phage. To test if these mimotopes could be useful in developing a vaccine against the human hepatitis B virus (HBV), we have compared the humoral immune response of animals immunized either with a recombinant HBsAg vaccine, or with mimotopes. Immunogens were prepared by fusing the mimotopes on different carrier molecules (phage coat protein pIII and pVIII, recombinant human H ferritin, HBV core peptide) and by synthesizing multiple antigenic peptides carrying the mimotopes' amino acid sequences. These immunogens were injected into mice and rabbits and sera were collected and tested for the presence of HBsAg-specific Abs. Our data confirm that mimotopes can induce a humoral immune response resembling that induced by the original Ag, and HBsAg mimotopes displayed on phage prove to be the best immunogens, inducing the most reproducible and potent immunization. Mimotopes that react as HBV subtype-specific Ags do not show this specificity as immunogen and induce a nonsubtype-restricted response. Furthermore, mimotopes displayed on phage elicit a strong response to HBsAg in a strain of mouse reported to show a low response to it. These results indicate that mimotopes identified from random peptide libraries through utilizing human immune sera could be important leads for the derivation of new vaccines.

Identification of biologically active peptides using random libraries displayed on phage.

The construction of new and increasingly diverse libraries, as well as the implementation of more powerful selection schemes, has led to the identification of linear peptides that mimic complex epitopes. Phage display techniques are allowing the selection of disease-related peptides, which reproduce the antigenic and immunogenic properties of natural antigens, using whole sera from patients. The range of applications of phage technology has been extended to include the search for peptides binding to molecules other than antibodies, such as cell receptors and enzymes.

Peptide and protein display on the surface of filamentous bacteriophage.

The isolation of ligands that bind biologically relevant molecules is fundamental to the understanding of biological processes and to the search for therapeutics. Filamentous phage can be used to display foreign peptides and proteins in physical association with their DNA coding sequences. Repertoires larger than 10(8) phage clones expressing different peptide sequences can be prepared using molecular genetic techniques. The strategies utilizing this technology promise to provide not only new binding and possibly catalytic activities, but also lead structures for the development of new drugs and vaccines.

Mimicking of discontinuous epitopes by phage-displayed peptides, II. Selection of clones recognized by a protective monoclonal antibody against the Bordetella pertussis toxin from phage peptide libraries.

We have screened phage peptide libraries to establish if clones binding to a monoclonal antibody (mAb), specific for a discontinuous epitope, could be isolated and if the selected phage particles would be able to elicit an in vivo immuno-response against the original antigen. Two phage peptide libraries, consisting of 9 random amino acids inserted in the major coat protein (pVIII), were independently screened with a mAb which is capable of neutralizing the Bordetella pertussis toxin (PTX) in in vitro and in vivo assays. The epitope of PTX recognized by this and other protective mAb has been shown to be discontinuous. Six different positive phage clones were selected; their binding to the mAb could be competed for by PTX, showing that these clones bind to the antigen-binding site of the mAb. Three of the clones were used (alone or as a mixture) to immunize BALB/c mice. The sera showed a good immunoresponse both against the phage bearing the epitopes and against synthetic multiple-antigen peptides of the same sequence. The immune sera, however, showed no detectable signal against PTX and no capacity to neutralize the CHO-cell-clustering activity of the toxin. The results show that the selected recombinant phage are capable of mimicking the discontinuous epitope as far as binding to the corresponding mAb, but they are unable to elicit a detectable production of antibodies specific for the original antigen.

Mimicking of discontinuous epitopes by phage-displayed peptides, I. Epitope mapping of human H ferritin using a phage library of constrained peptides.

We have constructed a random nonapeptide library in the N-terminal region of the major coat protein VIII of bacteriophage f1, with two cysteines flanking the insert, and preliminary data suggest that many of the clones display at least some of their peptides in cyclized form. This library was used to select oligopeptides binding to the monoclonal antibody (mAb) H107, recognising the assembled native conformation of recombinant human H-subunit ferritin (H Fer), whose three-dimensional structure is known. Comparison of the selected oligopeptides with one another allowed us to derive two consensus sequences characterized by conserved amino acid (aa) residues. Analysis of the distribution of the aa side chains exposed on the surface of H Fer reveals that most of the aa defining both consensus sequences are present either at the end of the big loop or at the end of the A helix. These two regions of the H Fer, though separated in the linear sequence, are very close in the folded molecule. Interestingly, each consensus sequence derived from the selected phage-displayed peptides is characterized by aa present both at the end of the big loop and at the end of the A helix. These two H Fer regions are good candidates for mimicry by the selected peptides and therefore for constituting part of the H107 epitope. To provide support to this hypothesis, we constructed several H Fer mutants carrying point mutations in different positions of these two regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of ferritin iron uptake: activity of the H-chain and deletion mapping of the ferro-oxidase site. A study of iron uptake and ferro-oxidase activity of human liver, recombinant H-chain ferritins, and of two H-chain deletion mutants.

To study the functional differences between human ferritin H- and L-chains and the role of the protein shell in the formation and growth of the ferritin iron core, we have compared the kinetics of iron oxidation and uptake of ferritin purified from human liver (90% L) and of the H-chain homopolymer overproduced in Escherichia coli (100% H). As a control for iron autocatalytic activity, we analyzed the effect of Fe(III) on the iron uptake reaction. The results show that the H-chain homopolymer has faster rates of iron uptake and iron oxidation than liver ferritin in all the conditions analyzed and that the difference is reduced in the conditions in which iron autocatalysis in high: i.e. at pH 7 and in presence of iron core. We have also analyzed the properties of two engineered H-chains, one lacking the last 22 amino acids at the carboxyl terminus and the other missing the first 13 residues at the amino terminus. These mutant proteins assemble in ferritin-like proteins and maintain the ability to catalyze iron oxidation. The deletion at the carboxyl terminus, however, prevents the formation of a stable iron core. It is concluded that the ferritin H-chain has an iron oxidation site which is separated from the sites of iron transfer and hydrolysis and that either the integrity of the molecule or the presence of the amino acid sequences forming the hydrophobic channel is necessary for iron core formation.