Identification of genes coding for B cell antigens of Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC) by using phage display.
BACKGROUND
Contagious bovine pleuropneumonia (CBPP) is a mycoplasmal disease caused by Mycoplasma mycoides subsp. mycoides SC (MmmSC). Since the disease is a serious problem that can affect cattle production in parts of Africa, there is a need for an effective and economical vaccine. Identifying which of the causative agent's proteins trigger potentially protective immune responses is an important step towards developing a subunit vaccine. Accordingly, the purpose of this study was to determine whether phage display combined with bioinformatics could be used to narrow the search for genes that code for potentially immunogenic proteins of MmmSC. Since the production of IgG2 and IgA are associated with a Th1 cellular immune response which is implicated in protection against CBPP, antigens which elicit these immunoglobulin subclasses may be useful in developing a subunit vaccine.


RESULTS
A filamentous phage library displaying a repertoire of peptides expressed by fragments of the genome of MmmSC was constructed. It was subjected to selection using antibodies from naturally- and experimentally-infected cattle. Mycoplasmal genes were identified by matching the nucleotide sequences of DNA from immunoselected phage particles with the mycoplasmal genome. This allowed a catalogue of genes coding for the proteins that elicited an immune response to be compiled. Using this method together with computer algorithms designed to score parameters that influence surface accessibility and hence potential antigenicity, five genes (abc, gapN, glpO, lppB and ptsG) were chosen to be expressed in Escherichia coli. After appropriate site-directed mutagenesis, polypeptides representing portions of each of these proteins were tested for immunoreactivity. Of these five, polypeptides representing expression products of abc and lppB were recognised on immunoblots by sera obtained from cattle during a natural outbreak of the disease.


CONCLUSIONS
Since phage display physically couples phenotype with genotype, it was used to compile a list of sequences that code for MmmSC proteins bearing epitopes which were recognised by antibodies in the serum of infected animals. Together with the appropriate bioinformatic analyses, this approach provided several potentially useful vaccine or diagnostic leads. The phage display step empirically identified sequences by their interaction with antibodies which accordingly reduced the number of ORFs that had to be expressed for testing. This is a particular advantage when working with MmmSC since the mycoplasmal codon for tryptophan needs to be mutated to prevent it from being translated as a stop in E. coli.
Miltiadou
Dubravka R
DR
Immunology Section, Onderstepoort Veterinary Institute, Private Bag X5, Onderstepoort, Republic of South Africa. miltiadou@bigpond.com
Mather
Arshad
A
Vilei
Edy M
EM
Du Plessis
Dion H
DH
D000465:Algorithms; D000818:Animals; D000907:Antibodies, Bacterial / Q000032:analysis; D000942:Antigens, Bacterial / Q000235:genetics*; D002417:Cattle / Q000382:microbiology; D002418:Cattle Diseases / Q000382:microbiology; D019295:Computational Biology; D004269:DNA, Bacterial / Q000235:genetics; D018985:Epitopes, B-Lymphocyte / Q000235:genetics*; D005798:Genes, Bacterial; D015698:Genomic Library; D007070:Immunoglobulin A / Q000032:analysis; D007074:Immunoglobulin G / Q000032:analysis; D016297:Mutagenesis, Site-Directed; D009176:Mycoplasma mycoides / Q000235:genetics* / Q000276:immunology; D019151:Peptide Library; D011002:Pleuropneumonia, Contagious / Q000382:microbiology; D017422:Sequence Analysis, DNA
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D004269:DNA, Bacterial; D018985:Epitopes, B-Lymphocyte; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D019151:Peptide Library