Synthetic peptide antigens induce antibodies to Taenia ovis oncospheres
Introduction
Taenia ovis is a cestode parasite of sheep that is responsible for economic losses due to the condemnation of infected meat. In 1989, Johnson et al. described a recombinant antigen, termed 45W, which protected sheep against challenge infection with the T. ovis parasite[1].
In vaccination trials, various forms of 45W have been shown to elicit differing immune responses. These antigens provide an opportunity to investigate the specificity of the immune response toward individual epitopes and further, offer the possibility of defining protective epitopes. The cDNA which codes for 45W (Fig. 1) represents an incomplete copy of a gene belonging to a small gene family[2]. Another member of this family, also isolated as an incomplete cDNA, codes for a protein termed 45S. Immunisation with this protein, however, did not provide protection against challenge infection[1], even though it is highly homologous to 45W[2](98.4% amino acid homology). In contrast, the full length 45S protein, encoded by the complete gene, induced a high level of protection against challenge[3]; this protein possesses an additional 67 amino acid residues, at the N- and C-termini compared with 45S2, 3. Therefore, the lack of protection afforded by the 45S antigen may be due to its incomplete sequence rather than the small number of amino acid differences between 45S and 45W. The protective activity of the full length 45S antigen could be attributed to sequences within one or both of the N- and C-terminal regions.
A derivative of 45W, termed 45WB/X, has been found to induce a high level of protection in sheep, despite the truncation of 17 N-terminal amino acids and 19 C-terminal amino acids compared with the full length 45S antigen[3]. Hence, the main difference between a protective antigen, 45WB/X, and a nonprotective antigen, 45S, appears to reside in the 23 N-terminal amino acids and/or the nine C-terminal amino acids of 45WB/X. This hypothesis is partly supported by B-cell epitope mapping which has identified a nine amino acid sequence within the N-terminal region of 45WB/X which distinguishes between protected and nonprotected animals vaccinated with 45WB/X[3].
The importance of specific antibody in providing protection against the parasite has been demonstrated by the transfer of protective immunity from 45W vaccinated ewes to lambs via colostrum[4](also GBL Harrison, unpublished observation). In ruminants, antibodies transferred via colostrum are predominantly of the IgG1 isotype[5]implicating this immunoglobulin isotype in protection afforded by the 45W vaccine. Additionally, significant 45W-specific IgG1 titres and negligible IgG2 titres have been found in the sera of lambs passively protected via maternal antibody from 45W immunised ewes[6]. This finding is consistent with the proposed mechanism of parasite killing, which is thought to be by antibody dependent complement mediated lysis of oncospheres, a mechanism demonstrated in vitro for a number of taeniid species7, 8, 9, 10, 11.
The protective efficacy of both the recombinant 45W antigen[12]and its native equivalent[13]was not diminished by treatment with a range of denaturing agents, indicating that the host-protective epitopes present in 45W are resistant to denaturation. This finding, together with the inference that protective epitopes appear to be located in short sequences at the N- and/or C-termini, encouraged us to synthesise peptides corresponding to the N- and C-terminal regions of 45WB/X and examine their immunogenic properties.
Section snippets
Recombinant proteins
Recombinant 45W and the related proteins used in this study were all in the form of glutathione-S-transferase (GST) fusion proteins, except where 45WB/X was used as an antigen in enzyme linked immunosorbent assays (ELISAs) and in the lymphocyte proliferation assay. In these instances a polyhistidine fusion protein (45WB/X-PH)[6]was used.
Synthetic peptides
The 30 amino acid N-terminal peptide was designed to incorporate the 23 amino acids identified as including potentially protective epitopes, five amino acids
Characterisation of synthetic peptides
The synthetic peptides indicated in Fig. 1, each eluted as a single main species when examined by analytical reverse-phase HPLC (Fig. 2). The purity of each peptide was further examined by amino acid analysis which resulted in experimental amino acid ratios in accordance with expected ratios (data not shown). In addition, the main species detected by MALDI mass spectrometry displayed molecular weights in close accordance with theoretical molecular weights (C-terminal peptide: expected mass
Discussion
The 45WB/X derived N-terminal peptide used in this study was found to be a potent immunogen, whereas the C-terminal peptide was not a strong immunogen unless it was conjugated to a carrier protein. Furthermore, approximately 40% of the antibodies elicited by immunisation with 45WB/X are directed toward the N-terminal peptide sequence, suggesting that this region of the protein displays significant immunological activity. Although the crystal and solution phase structures of these peptides are
Acknowledgements
This work was supported by the Meat Research Corporation, Australia and the National Health and Medical Research Council. The authors would like to thank Mary Macris for performing the mass spectrometry, Charles Gauci for assistance with sheep trials, John Bryant for care of the sheep and Joseph Cox for image scanning.
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