Proteins from the wall-forming bodies of Eimeria maxima: function and immunogenicity
Smith, N.C.1, Belli, S.I.1, Katrib, M.1, Witcombe, D.M.1, Mai, K., Skene, C.1, Gleeson, M.T.1, Wallach, M.G.1, Ferguson, D.J.P.2, Luxford, C.3, Davies, M.3 and Finger, A.4
1Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, Australia; 2Nuffield Department of Pathology, University of Oxford, UK; 3The Heart Research Institute, Sydney, Australia; 4Abic Veterinary Products Ltd, Netanya, Israel.
Wall-forming bodies type 1 of Eimeria maxima are large, electron-dense vacuoles that stain positively with antibodies in an enriched preparation of native forms of 56, 82 and 230 kDa gametocyte proteins (APGA). Wall-forming bodies type 2 also react positively with these antibodies, but are less electron-dense than wall-forming bodies type 1, have whorled or donut-shaped profiles and remain enclosed in the rough endoplasmic reticulum. At the initiation of oocyst wall formation, wall-forming bodies type 1 first align at the periphery of the macrogamete, then release their contents to form an electron-dense outer oocyst wall that reacts with anti-APGA antibodies. Wall-forming bodies type 2 subsequently give rise to an electron-lucent inner wall. Proteomic and Western blot analysis has revealed that the 56 and 82 kDa proteins are processed into smaller, tyrosine-rich proteins that can cross-link and harden to form the oocyst wall of Eimeria maxima. This observation helps to explain the molecular basis of action of the subunit vaccine, CoxAbic®, which includes native 56 and 82 kDa proteins and stimulates strong antibody responses to protect the hatchlings of vaccinated hens from coccidiosis. Genes encoding these two immunodominat components of CoxAbic® were cloned and the recombinant proteins expressed in E. coli and purified. Both recombinant proteins are immunogenic, eliciting dose-dependent antibody responses comparable to that provoked by CoxAbic® and able to recognise the native proteins in that vaccine. Furthermore, the recombinant proteins are able to inhibit the binding of anti-APGA antibodies to APGA, indicating that the recombinant versions have some promise as components of a second-generation subunit vaccine against poultry coccidiosis.