RESUMO
Peptide sequences that bind to a wide range of ligands such as monoclonal antibodies, receptors and carbohydrates have been successfully identified after screening phage display peptide libraries. However, these procedures tend to select mainly medium to low affinity-binding clones. A modified screening procedure has been developed in order to improve the efficiency of this process such that high avidity/affinity binding clones are preferentially selected. Three different solid phase binding surfaces were evaluated for the attachment of antibody during the screening procedure and a stepwise decrease in the pH of the elution buffer introduced during the final round of biopanning. The monoclonal antibody MA 18/7 was used to screen a 15-mer peptide library. This antibody is well-characterised and its binding site has been mapped to residues 28-37 of the pre-S1 protein of the hepatitis B virus. The antibody was either biotinylated and attached to polystyrene plates via a streptavidin-biotin 'bridge', or bound directly to 1/4 in. polystyrene beads, or to 11 microm latex beads. A significant enrichment of binding clones was observed when the monoclonal antibody was attached directly to polystyrene or latex beads as compared to the biotinylated antibody. All mimotopes identified after biopanning with the antibody attached to the polystyrene beads possessed a central core motif, identical or similar to the sequence DPAF contained within the epitope binding site of MA 18/7 on the native pre-S molecule. However, this motif was only observed in 30% of clones isolated after biopanning using the 11 microm latex beads and in 2% of clones isolated after biopanning on the streptavidin-coated plates. Immunoblotting with the monoclonal antibody MA 18/7 confirmed binding to clones containing the DPAF sequence or a similar motif. A stepwise reduction in the pH of the elution buffer in the final round of biopanning resulted in the removal of clones that possessed low affinity binding motifs, thereby increasing the percentage of clones containing high affinity binding motifs in the final elution step at pH 2.0. Thus, the combined use of polystyrene beads and a stepwise decrease in the pH of the elution buffer in the final round of biopanning resulted in the elimination of non-binding clones and an increase in the efficiency in isolating high affinity binding clones.
Assuntos
Mapeamento de Epitopos/métodos , Epitopos de Linfócito B/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Hepatite B/imunologia , Peptídeos/imunologia , Precursores de Proteínas/imunologia , Sequência de Aminoácidos , Afinidade de Anticorpos , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Biblioteca de PeptídeosRESUMO
The amino acid sequence 139-147 from hepatitis B surface antigen (HBsAg) has previously been shown to represent a B-cell epitope with potential as a component of a synthetic peptide vaccine against hepatitis B. In this paper, two regions of HBsAg which act as T-cell epitopes in inbred mice have been identified (residues 23-34 and residues 160-171). The ability of synthetic peptides representing these epitopes to provide help for the production of antibody against the 139-147 epitope has been assessed following their co-linear synthesis with the B-cell epitope and following co-immunization of the peptides in an uncoupled form. Both these strategies result in the induction of anti-peptide antibodies which specifically react with recombinant HBsAg. The results presented give further support to the concept that synthetic peptides representing appropriately chosen B- and T-cell epitopes from HBsAg could form the basis of a synthetic vaccine against hepatitis B.
Assuntos
Epitopos/imunologia , Anticorpos Anti-Hepatite/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Imunização , Fragmentos de Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Formação de Anticorpos , Especificidade de Anticorpos , Linfócitos B/imunologia , Feminino , Antígenos de Superfície da Hepatite B/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Linfócitos T/imunologia , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/farmacologiaRESUMO
An extended (48 amino acid) synthetic peptide analogue of the hepatitus B virus (HBV) S protein (HBsAg) 'a' determinant has been produced by using 9-fluorenylmethoxycarbonyl (fmoc) chemistry and a low substitution polystyrene resin as the solid phase support. This peptide (S121/48) elicited a sustained anti-peptide antibody response in BALB/c (H-2(d)) mice when immunised with Freund's complete adjuvant (FCA). Cross-reactive, anti-HBs antibodies were induced, directed against a significant proportion of the conformationally restrained epitope repertoire on the native HBsAg particles. Similar responses were obtained by injection of guinea pigs, a species known both to be exquisitely sensitive to HBsAg and to produce a wide range of B cell responses to HBsAg antigens. Taken together, these data show for the first time, that a synthetic peptide mimicking conformational epitopes can be produced by chemical synthesis and can be used to induce significant titres of anti-HBs antibodies after a single injection. This immunogen has considerable potential for incorporation into novel delivery systems, e.g., microspheres, thus offering the potential of a controlled release, single dose hepatitis B vaccine.
Assuntos
Anticorpos Anti-Hepatite B/sangue , Antígenos de Superfície da Hepatite B/química , Vacinas contra Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Peptídeos/imunologia , Animais , Eletroforese em Gel de Poliacrilamida , Epitopos/imunologia , Cobaias , Hepatite B/prevenção & controle , Antígenos de Superfície da Hepatite B/imunologia , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Mimetismo Molecular , Peptídeos/administração & dosagem , Peptídeos/síntese químicaRESUMO
Direct DNA intramuscular or intradermal injection of plasmids containing viral genes under the control of viral promoters is an efficient means of stimulating both class I and class II-mediated antiviral responses. Viral hepatitis B and C are suitable candidates for this approach, particularly as therapeutic immunogens for chronically infected individuals. Several groups have shown that the S gene of HBV is expressed in murine muscle and stimulates a high titre and long-lasting anti-HBs response. Uniquely, CD8+ CTL responses are also induced to HBsAg. No vaccine exists for HCV. Therefore the structural genes (C + E1 + E2) have been cloned as a 2,831 bp fragment from a genotype la isolate into the vector pcDNA3. The resulting plasmid DNA was injected directly into the quadriceps muscle of three-week-old BALB/c mice. Intracellular-expressed E1 and E2 proteins thus represent the complete spectrum of native structural epitopes, including those dependent on glycosylation and protein folding. Mouse antisera were tested for reactivity against conserved sequences using overlapping 7-mer peptides. Two conserved, overlapping epitopes were identified in E2 spanning residues 581-591 and 590-603. This domain represents one of seven major E2 antigenic domains recognized by HCV human antibodies, one of three with antigenic homologies to related flavivirus proteins. Thus antigen is presented with high efficiency following DNA injection and offers the potential of high rates of seroconversion and virus clearance in those predisposed to virus-induced chronic liver disease.
Assuntos
Hepatite C/prevenção & controle , Vacinas de DNA/uso terapêutico , Vacinas contra Hepatite Viral/uso terapêutico , Animais , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Genes Virais/genética , Hepacivirus/genética , Hepacivirus/imunologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Regiões Promotoras Genéticas , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Proteínas Estruturais Virais/genéticaRESUMO
Recent developments in peptide technology enable the use of random peptide libraries for identifying linear amino acid sequences (mimotopes) which can mimic conformational epitopes without necessarily exhibiting amino acid sequence homology with the native linear sequence. In this study a 15-mer random peptide library displayed on the surface of a filamentous phage has been used to characterise the conformational epitopes recognised by a monoclonal antibody raised against the envelope protein gp120 of feline immunodeficiency virus (FIV). Three mimotopes were identified that reacted with the selecting antibody in an immunoblot assay. Sequence analysis revealed that, whereas the three mimotopes had several amino acids in common, there was no significant homology with the primary amino acid sequence of gp120 although some amino acids were shared between the variable region (V3) and the three mimotopes. Petide mimtopes of complex retroviral glycoproteins may have potential uses as novel vaccines and for the serological diagnosis of FIV.
Assuntos
Anticorpos Monoclonais , Anticorpos Antivirais , Bacteriófagos/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Vírus da Imunodeficiência Felina/imunologia , Biblioteca de Peptídeos , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Gatos , Immunoblotting , Dados de Sequência MolecularRESUMO
The pre-S-specific monoclonal antibody MA 18/7 has been shown to inhibit the binding of HBV to HepG2 cells and liver membranes. This antibody can thus be used to identify the critical residues of the pre-S region involved in the hepatocyte-binding domain. Using overlapping 7-mer peptides representing the pre-S region of HBV, the epitope recognized by MA 18/7 was shown to contain sequences from both the pre-S1 and pre-S2 regions, thus indicating that the hepatocyte-binding domain is conformationally dependent. To further characterize the primary structure of the hepatocyte-binding domain on the pre-S protein, a phage-displayed 15-mer peptide library and a 8-mer solid phase peptide library were used to analyze the fine specificity of the monoclonal antibody MA 18/7. Several mimotopes were identified with the phage-displayed peptide library, the majority of which possess a central motif with at least three identical residues present within the native pre-S1 sequence. No significant consensus sequences were found when these mimotopes were compared to the pre-S2 sequence. Mimotopes identified using the solid-phase peptide library also contained a similar motif. All phage mimotopes and a single mimotope from the solid-phase peptide library competed with recombinant HBsAg particles containing the pre-S1 region for binding to MA 18/7. Mouse antisera raised against four mimotopes from the phage display library reacted with HBsAg particles containing pre-S sequences. The data show that the structure of the pre-S molecule around the conserved DPAF motif in the pre-S region may have a functional role in binding HBV to cellular receptors, and that the central motif identified in mimotopes of this region may offer a novel strategy target for the improvement of existing hepatitis B vaccines which, at present, are mostly devoid of pre-S specificities.
Assuntos
Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Fígado/virologia , Precursores de Proteínas/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Biblioteca de Peptídeos , Precursores de Proteínas/química , Precursores de Proteínas/metabolismoRESUMO
An adult male farmer with chronic active hepatitis and cirrhosis despite previous circulating anti-HBs antibodies was studied. No markers of other hepatotropic viral infection were observed. HBV DNA was detected in serum by PCR and was characterized further by restriction fragment length polymorphism (RFLP) and sequencing of cloned PCR products derived from the S gene. The HBV DNA was ascribed to genotype F, and single-strand conformational polymorphism (SSCP) demonstrated the co-circulation of multiple quasispecies. Some of the variants exhibited changes located within the neutralizing "a" determinant, located between amino acids 124-147 of the S protein. Within this region, two clones showed either C124R or C124Y mutations. Other mutations were Q129R, C138R, C139R, and S140T (one clone each). Outside the "a" determinant several substitutions were documented. The high degree of the quasispecies variability was probably linked to the severity of the infection. Most members of the patient's family were infected with HBV, all with genotype F.