Cross-reactive epitope mimics in a fragmented-genome phage display library derived from the rickettsia, Cowdria ruminantium.
BACKGROUND
Epitopes can be mapped by comparing immunoaffinity-selected peptides from fragmented-gene display libraries with the target gene. With larger libraries derived from unsequenced genomes, this is not possible. Spurious epitope mimics may be created by expressing DNA in a variety of meaningless reading frames and orientations.


OBJECTIVE
To determine empirically whether panning a large fragmented-genome phage display library with antibodies to MAP1, the major antigenic protein of the rickettsial parasite Cowdria ruminantium, would result in the selection of irrelevant, cross-reactive mimotopes.


METHODS
A gene III phage library displaying peptides derived from C. ruminantium was constructed using cloned DNA from a bacteriophage lambda genomic library. After in vivo excision, plasmids were cleaved with PvuII followed by PCR. Genes with a PvuII site, including MAP1 were therefore not amplified. DNA was sonicated, partially digested with DNase and cloned into the display vector fUSE2. Affinity-purified MAP1 antibodies were used for panning. Peptides expressed by panned phages were tested for recognition in Western blot and ELISA. Oligonucleotides representing antigenic sequences were used to locate their encoding DNA sequences in the original lambda library. The phage display library was also panned with two monoclonal antibodies (Mabs) against bluetongue virus (BTV).


RESULTS
Five different peptide sequences were selected from the MAP1-deficient phage display library. None was identical to MAP1, but four peptides had regions that were similar, both to each other, and to the parasite protein. They produced strong signals in ELISA and Western blot. None could be located to any C. ruminantium open reading frame. Two BTV Mabs recognised a sequence similar to their authentic epitope.


CONCLUSIONS
Large genome-targeted phage display libraries may be sufficiently diverse to allow the selection of peptides that mimic actual antigenic determinants. This diversity may be exploited in the search for useful epitopes.
Fehrsen
J
J
Immunology Division, Onderstepoort Veterinary Institute, South Africa.
du Plessis
D H
DH
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000276:immunology; D001435:Bacteriophages / Q000235:genetics; D015153:Blotting, Western; D003429:Cross Reactions; D016995:Ehrlichia ruminantium / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D016680:Genome, Bacterial; D016056:Immunodominant Epitopes / Q000276:immunology*; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D016056:Immunodominant Epitopes; D019151:Peptide Library; D010455:Peptides; C086402:major antigenic protein 1, Cowdria ruminantium