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Title: The development and application of sheep monoclonal antibody technology to epitope mapping of viral synthetic peptides
Author: Flynn, James Norman
ISNI:       0000 0001 3471 5512
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1989
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Modern Approaches to the development of novel vaccines require a thorough understanding of both how the immune system responds to antigenic challenge and the epitopes involved in the elicitation of protective immunity to the pathogen. In an attempt to define the fine specificities of the immune response in sheep to synthetic peptide antigens a system for the production of ovine monoclonal antibodies was developed. Due to the lack of suitable ovine myeloma cell lines, any attempt at the production of ovine monoclonal antibodies will require the use of heterohybridomas. A cloned sheep x mouse aminopterin sensitive heterohybridoma (cell line 1C6.3a 6T.1D7) developed and proved more efficacious than a mouse myeloma cell line or subsequently developed sheep x sheep x mouse heterohybridoma cell lines in the generation of stable heterohybridoma cell lines which secreted antibody for long periods of time in tissue culture. Following the development of 106.3a 6T.1D7 the lymphocytes in efferent lymph, lymph nodes and peripheral blood were evaluated as sources of immune B cells surtable for the generation of antibody secreting sheep x sheep x mouse heterohybridomas. Lymphocytes collected from cannulated efferent lymphatic vessels and prepared from peripheral lymph nodes were superior sources of immune B cells for monoclonal antibody production. Secondary antigenic stimulation of these tissues localised antigen specific B cells at these sites and geared the cellular machinery of B cells for immunoglobulin synthesis and release. This resulted in the generation of greater numbers of specific antibody secreting heterohybridomas when these tissues were fused on the fourth day following secondary antigenic stimulation. Having established optimal conditions for the generation of sheep monoclonal antibodies, this technology was applied to synthetic peptides of VP1 from the O^K strain of FMDV in an attempt to define the B cell epitopes recognised by the sheep immune system. Using these sheep monoclonal antibodies it was shown that by synthesising a 1+0-residue peptide of residues 114.1 - 158 and 200-213 of VP1 of FMLV in tandem separated by a di-proline spacer, unique conformational epitopes were generated which were absent from the constituent 21-residue and 19-residue peptides. These unique epitopes were not directly associated with the proline residues used as linking amino acids. These unique epitopes appear to be associated with the induction of higher serum neutralisation titres and protective immunity in sheep innoculated with the uncoupled 4O-residue peptide. The same synthetic peptides were examined for the presence of T cell epitopes and it appeared that residues 11+8-157 present in both the 1+0- and 21-residue peptides contained helper T cell epitope(s). No such helper T cell epitopes were identified within residues 200-213, and thus sheep were non-responsive to the 19-nesidue peptide when administered uncoupled. This unresponsiveness could be overcome by making a single amino acid substitution of a glycine residue for an alanine residue at position 208. These results demonstrate that by the use of sheep monoclonal antibodies and polyclonal T cells it was possible to identify epitopes important in the immunogenicity of viral synthetic peptides. The implications of these findings are discussed with regard to the generation of synthetic peptide vaccines.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Immune response in sheep