Recombinant epstein-barr virus proteins and anti-body-selected peptides: reagents for serodiagnosis and vaccination

The Epstein-Barr virus (EBV) is a ubiquitous human oncogemc herpesvirus which causes infectious mononucleosis (IM) and contributes to the development of many important cancers in man. The objectives of this thesis were twofold\ud (1)the development of recombinant EBV antigens as reagents for the serodiagnosis of EBV-related disease, \ud (2) the identification of peptides that bind EBV neutralising antibody using phage display technology.\ud \ud Serology testing is of paramount importance in the diagnosis of EBV-related diseases, with viral antigen for these tests usually being derived from cell culture. The production of the appropriate recombinant EBV antigens in a prokaryotic system would eliminate many of the disadvantages associated with cell culture-derived EBV antigens, le reproducibility, cross-reactivity and expense. The coding sequences for several targeted EBV proteins were cloned into an E coh expression vector (Thiofusion system). In this system recombinant proteins were fused to Ecoh thioredoxin thus offering the advantages of stability, solubility, mducibility and ease of purification. Three EBV antigens, pl8VCA, EA-D and EBNA1 were chosen for expression and purification. These recombinant antigens were assayed with sera that had been previously partially characterised for antibodies to EBV A cut-off point was determined by comparison of ELISA and Western blot results. The antibody profiles of each serotype, 1 e infectious mononucleosis-positive sera, sera from normal healthy individuals, VCA+/- sera and EBNA+/- sera, agreed with those determined using cell culture-derived antigen in other assay systems. Some potential cross reactivity with CMV was noted. These recombinant proteins may be of use as immunodiagnostic reagents for the diagnosis of EBV-related diseases.\ud \ud The EBV envelope glycoprotein gp350 mediates virus adsorption and penetration to it’s host cell and is the principal candidate subunit vaccine. The monoclonal antibody 72A1 is known to neutralise EBV through interaction with gp350. The aim was to identify peptides that bind to 72A1 using phage display technology. Using this antibody, peptide sequences were selected which showed specific similarities in ammo acid content and arrangement, however it was not possible to show specific interaction between the selected clones and 72A1 by other immunological methods. As only one group of clones from the set of 15mer sequences bears similarity to the native gp350 primary ammo acid sequence, it is most likely the epitope is conformational, or that all other selected peptides are mimotopes of a linear epitope