Genetics, immunity and DNA fingerprinting in the identification of protective antigens of Eimeria maxima
Damer P. Blake, Adrian L. Smith and Martin W. Shirley.
Institute for Animal Health, Compton, Berkshire, RG20 7NN, UK.
Email: damer.blake@bbsrc.ac.uk
Many protozoan genomes are the
subject of extensive sequencing projects, but the identification of genes that
encode antigens capable of stimulating a protective immune response remains a
demanding task. The rational identification of genuinely immunoprotective gene
products encoded by unsequenced genomes is even more daunting. The majority of
screens for vaccine candidates protective against protozoan infection have been
largely empirical, based upon the induction of an immune response rather than
protection. However, consideration of the genetics of immune escape by different
strains of Eimeria maxima has raised the possibility of the targeted
identification of a gene (or genes) under immune selection. Central to our
strategy is the concept that the inheritance of genetic markers is influenced by
their proximity within the genome to loci under deleterious selection. Initially
multiple independent hybrid populations were created through the mating of two
strains of E. maxima that each induce a lethal strain-specific protective immune
response in the host and show a differential response to the anticoccidial drug
robenidine. Escape by a sub-population within the progeny of this cross from a
double-barrier consisting of immune and chemotherapeutic selection lethal to
either parent proved that loci encoding molecules stimulating strain-specific
protective immunity or resistance to robenidine segregate independently.
Comparison of parasite replication in the presence of the selective
double-barrier, either individual component of the barrier or no barrier,
implicates a highly restricted number of key protective-antigen-encoding loci.
The selection of recombinant parasites through the double-barrier imposed
genetic-bottleneck was accompanied by the elimination of ~3% of the polymorphic
DNA markers that defined the parent strain used to immunise the host.
Hybridisation studies with Southern-blotted digested and undigested karyotypes
resolved by PFGE and a BAC library derived from the parent strain under immune
selection have revealed two clusters of these DNA markers correlated with
selection that cover independent regions of ~220 Kb (13 markers) and ~400 Kb (8
markers) within the genome. Our identification of polymorphic DNA markers that
associate with immune-mediated killing and are physically co-localised within
the genome support our strategy to identify loci under selection in Eimeria spp.
and should be applicable to other loci under selection in other Apicomplexa
including Plasmodium spp.