Contributed Papers: Oral Presentations Immunology |
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..