Identification of Eimeria tenella genes encoding for secretory proteins and evaluation of candidates by DNA immunisation studies in chickens.
In order to identify secretory proteins as possible new vaccine candidates, a cDNA-library from E. tenella sporozoites was generated in yeast and was used to select secreted and surface proteins. Herein 191 clones were isolated and analysis of the nucleic acid sequences revealed 162 deduced open reading frames with a prediction for signal peptides. These sequences are characterized by high redundancy, comprising 25 unique protein fragments with a high degree of stage specificity. Only three sequences showed identical homology to already known E. tenella proteins. The majority, 16 fragments, revealed homology to known or hypothetical proteins, and six fragments had no sequence homologues in protein databases. In order to obtain optimised conditions for a DNA vaccination trial in chickens, with which our selected new sequences could be tested, we performed variant DNA immunisations with the well-characterized E. tenella antigen SO7. The cDNA of the SO7 antigen was subcloned into two different eucaryotic expression vectors, i.e. pcDNA3 and pVR1012. In addition, the SO7 sequence was fused to the stabilizing sequence of the enhanced green fluorescence protein (EGFP). All SO7 constructs induced a SO7 specific immune response after intramuscular application and no significant differences were found on using constructs with or without the EGFP fusion or with different vector systems. Full-length open reading frames from six selected Eimeria sequences were introduced into the eucaryotic expression vector pcDNA3. Subsequent immunisation trials revealed a decrease in parasite excretion for three constructs after challenge infection in comparison to the control animals. Our approach represents a rapid screening to identify and test putative new vaccine candidates from E. tenella sporozoites that could also be adopted to other apicomplexan parasites.
Department of Molecular Parasitology, Humboldt University Berlin, Philippstrasse 13, 10115 Berlin, Germany.
Christian
C
Klotz
Florian
F
Gehre
Richard
R
Lucius
Thomas
T
Pogonka
D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology; D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D002645:Chickens; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics / Q000276:immunology; D016786:Eimeria tenella / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D015723:Gene Library; D006801:Humans; D007114:Immunization / Q000379:methods; D007074:Immunoglobulin G / Q000276:immunology; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D016052:Protozoan Vaccines / Q000235:genetics / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D019444:Vaccines, DNA / Q000235:genetics / Q000276:immunology*
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D016054:DNA, Protozoan; D007074:Immunoglobulin G; D015800:Protozoan Proteins; D016052:Protozoan Vaccines; D019444:Vaccines, DNA