Hepatitis C epitopes from phage-displayed cDNA libraries and improved diagnosis with a chimeric antigen

Abstract

A novel method for cloning DNase I fragments into bacteriophage display vector fUSE2 was used to create libraries expressing hepatitis C virus (HCV) protein fragments on the phage surface. Selection by panning with a mixture of sera from five HCV-seropositive individuals enabled identification of antigenic determinants in NS3 (amino acids 1,383-1,415), NS4 (amino acids 1, 930-1,938), and NS5 (amino acids 2,088-2,104). The NS3 result is the most accurate location to date of a major conformational determinant that cannot be mimicked by short peptides. Any expressed sequence from the phage library can be excised with Bgl II and cloned directly into the Bgl II site of an appropriate plasmid for bacterial expression. This enables production of chimeric proteins containing multiple antigenic determinants, illustrated by co-expression of the NS4P (amino acids 1,930-1,938) epitope with an NS4N fragment (amino acids 1,644-1,812) containing at least three linear HCV epitopes. When used to screen 35 individual HCV-positive sera by enzyme-linked immunosorbent assay (ELISA), the chimeric antigen detected eight more positives than NS4N alone and gave increased immunoreactivity with others. This approach of identifying antigenic regions by phage display and then co-expressing them as chimeric proteins may be generally applicable to the production of improved diagnostic antigens and recombinant vaccines.

Epitope Mapping of Neutralizing Botulinum Neurotoxin A Antibodies by Phage Display

“…The BOT1-selected S25 and 3D12 clones are larger (500 to 600 bp) than those in the library (150 to 300 bp). In contrast, other gene fragment selections (50 to 400 bp) from multivalent rather than monovalent display libraries yield small epitopes (i.e., 50 to 200 bp) (3,4,8,9,11,20,27) that are perhaps related to multivalent, smaller fragments with higher functional affinity.…”

Vaccinology in the genome era

“…The use of phage display methods and protein microarray technologies can overcome this limitation and lead to the identification of the complete immunoproteome, where immunogenic polypeptides and proteins can be identified independently of their level of expression in vitro. Bacterial and phage-based expression libraries of either large polypeptides or small peptides encoded by random synthetic oligonucleotides of identical length have frequently been used (82). A novel genomebased approach that combines serological antigen identification and the use of a comprehensive genomic peptide library, potentially expressing all genome-encoded amino acid sequences, has been recently developed and termed antigenomics (83).…”

Synthetic peptide vaccines

“…Помимо выявления линейных В-эпитопов, предпринимаются также попытки картировать и моделировать конформационные В-эпитопы с помощью синтетических или получаемых методом фагового дисплея комбинаторных пептидных библиотек, охватывающих огромное разнообразие аминокислотных последовательностей длиной обычно от 6 до 12-15 а.о. [47]. Кроме того, определить В-эпитопы можно путём масс-спектрометрического анализа комплексов антиген-антитело, выявляя участок молекулы антигена, защищенный от протеолиза или модификации паратопом антитела [43,48].…”