Identification of vaccine candidates in larval excretory/secretory products of Teladorsagia circumcincta

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The aim of this project is to define potential protective antigens in excretory/secretory (ES) products of larval Teladorsagia circumcincta. We will focus on antigens released by larvae harvested at 6 hours, day 3 and day 5 p.i., as host responses to these stages are important in preventing the establishment of parasites in immune sheep. Antigens will be identified by comparing local IgA and IgE responses of immune versus non-immune sheep. These responses will be studied in efferent gastric lymph and abomasal mucus, as local increases in parasite specific antibody have been linked to protective immunity. Antigens will be separated by 2-dimensional gel electrophoresis and electroblotted onto nitrocellulose membranes. The antigens will be probed with lymph and mucus material obtained from adult sheep that are immune to challenge infection and from young susceptible sheep. The antigen recognition profiles in the groups will be compared and only those spots reactive with antibody from the immune cohort will be subjected to further analysis, by excision of spots of interest from replicate gels and digestion with trypsin. The resultant peptide mixture will be pre-screened by MALDI-ToF mass spectrometry (Micromass) and subsequently, the tryptic peptides will be resolved by microcapillary (75 micrometers) C18 reversed phase nanochromatography (200nl/min, LC Packings system) and electrosprayed directly into the source of either our QToFmicro tandem mass spectrometer or our LTQ Linear Ion Trap instrument. Double charged peptides will be fragmented in the collision cell and the resultant product ion spectra will be used for de novo sequencing. If necessary, we will use 18O water in the tryptic digest to aid in differentiation of the b and y ion series. We will match the sequence tags to EST and cDNA sequences from T. circumcincta or other nematode species (including Caenorhabditis elegans). For such matches, we expect to ascertain putative function based on homology. For other proteins, there may be no homologues identified, as there is no full genome sequence database for T. circumcincta. These proteins will be subjected to more extensive LC-MS MS to derive peptide sequences that will allow us to design primers for PCR for gene cloning experiments. Subsequently the antigen spots will be listed in an order of merit based on the strength of immune response; broad disposition of immunoreactivity with material from immune (as opposed to non-immune) animals, predicted function, size of expressed product and presence of putative glycosylation sites. For each ES time point, two antigens will be selected for gene cloning. The peptide sequences and protein homologies identified will be used to design primers. The primers will be used in reverse transcriptase-polymerase chain reaction experiments to isolate the full length complimentary DNA¿s encoding these antigens. Joint with BB/C518422/1