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Biomedical subjects

P Monaci

Publications and source records attributed to P Monaci.

At least 19 recordsLinked to original sources
PubMed2000

"Affinity maturation" of ligands for HCV-specific serum antibodies.

We have previously screened a phage-displayed random peptide library using sera from patients and identified ligands binding to antibodies specifically associated with the hepatitis C virus infection. The ability of these peptides to detect HCV-specific antibodies was improved through an in vitro procedure which mimics the natural process of antibody affinity maturation operating in secondary immune response. Libraries were generated by mutating the sequence of the original peptide through a protocol that efficiently introduced substitution, insertion and deletion mutations on a single or population of clones. Screening these libraries isolated mutants that displayed increased specific reactivity with a broader range of sera from HCV-infected patients. Several variants of the original peptide were identified which discriminate between the various components of the specific polyclonal response. This methodology to select artificial ligands from RPL using sera and to enhance their diagnostic properties by affinity maturation makes the development of a diagnostic assay to detect disease-associated antibodies feasible, without requiring the natural antigen.

Amino Acid Sequence↗
PubMed2000

'In vitro evolution' of ligands for HCV-specific serum antibodies.

We developed a strategy to improve the properties of ligands selected from phage-displayed random peptide libraries. A site-directed mutagenesis protocol that introduces mutations and extends the size of a target sequence has been set up to generate diversity in a single or in a population of clones. The pool of mutants thus created is screened to identify variants with the desired properties. We refer to this strategy as in vitro evolution' of ligands. Here we report the application of this in vitro evolution protocol to the identification of improved ligands for HCV-specific serum antibodies. A single clone or population of clones were processed to generate a secondary library. Screening of these libraries with sera from HCV-infected patients identified peptides with an enhanced and broadened ability to detect HCV-specific serum antibodies.

Antibodies, Viral↗
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↗
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↗
PubMed1997

Identification of HCV core mimotopes: improved methods for the selection and use of disease-related phage-displayed peptides.

Disease-specific epitope discovery from random peptide libraries displayed on phage using sera from patients involves a number of screening steps with many immune and non-immune sera. To rapidly identify mimotopes of the human hepatitis C virus (HCV) core protein, we have used an anti-core human monoclonal antibody (mAb; B12.F8) as a probe in screening phage that were affinity-selected using a serum from an HCV infected patient. Three different positive phage were isolated displaying low or no homology with the natural antigen, but which still efficiently bound to the antigen binding site of the B12.F8 antibody. Testing the reactivity of these phage with forty-five sera from HCV infected patients showed that antibodies recognizing them are present in more than 80% of this population. These antibodies showed distinct fine specificity, as they bound the selected phage in a mutually exclusive fashion. Co-expression of two mimotopes in the same cells led to chimeric particles which were recognized by antibodies of different specificity. These data provide novel information on the potential use of the phage display technology for the characterization of antibody specificity as well as disease diagnosis and prevention.

Amino Acid Sequence↗
PubMed1996

Selection of antigenic and immunogenic mimics of hepatitis C virus using sera from patients.

Using sera from hepatitis C virus (HCV)-infected patients and noninfected subjects to screen random peptide libraries displayed on phage, we selected peptides specifically reacting with sera from infected patients. These phage- borne peptides were shown to mimic distinct HCV determinants. They detected in all cases the presence of anti-HCV Abs in a large panel of patients' sera, thus demonstrating the high sensitivity of the selected peptides as diagnostic markers. In addition, this diagnostic approach allowed a detailed characterization of the individual humoral response to viral infection. Phage-displayed HCV mimics were substitutes for the authentic HCV epitopes in inducing a strong specific response against HCV when used as immunogens in mice. These results support the search for HCV mimics with the potential to elicit a protective immune response as leads for the development of a mimotope-based vaccine against viral infection.

Amino Acid Sequence↗
PubMed1996

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.

Animals↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1995

A complex interplay of positive and negative elements is responsible for the different transcriptional activity of liver NF1 variants.

A full-length cDNA of the rat liver Nuclear Factor 1 (NF1L21) has been cloned and expressed in S. cerevisiae to analyse the architecture of its activation domain. NF1L21 displays a specific DNA-binding activity, as well as the ability to activate transcription from an artificial NF 1-responsive promoter in yeast. Interaction of two or more NF1L21 molecules with multiple sites on the same promoter activated transcription in a synergistic fashion. Functional analysis of the activation domain of NF1L21 reveals a tripartite structure. Two distinct positive elements are required for NF1L21 -mediated transcription activation. A proline-rich element sandwiched between these two positive domains attenuates their transactivation potential. A shorter NF1L variant (NFlL4) in which the distal positive element is replaced by a different sequence was also isolated. NF1L4 displays the same DNA-binding activity and dimerisation properties as NF1L21, but is unable to activate transcription in yeast.

Amino Acid Sequence↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1995

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.

Amino Acid Sequence↗