The search for appropriate antigens for sensitive and specific serodiagnosis of human cystic echinococcosis (CE) applicable in clinical practice has been underway for decades. To date, there is no available recombinant protein clinically useful for unambiguous CE diagnosis (overviewed in Chapter 1). Accordingly, the majority of my study aimed to isolate genes of Echinococcus granulosus encoding
proteins applicable for the serodiagnosis ofhydatid disease.
To achieve this goal, a pool of sera from mice infected with oncospheres (eggs) of E. granulosus was used to screen a cDNA library prepared from RNA isolated from protoscolex larvae from sheep hydatid cysts (Chapter 3). Three sequences were isolated after screening 1.6 x 104 recombinants. One clone termed EpCl showed a high performance for recognition of mouse infection sera using phage plaque expressed protein, indicating that EpCl can be a candidate for CE serodiagnosis. EpCI comprised of 420 bp nucleotide sequence with an open reading frame (ORP) of 231 bp encoding 76 amino acids. The complementary DNA (cDNA) fragment was
subcloned into an expression vector, pET-41b(+), and the resulting recombinant EpCl glutathione Stransferase (GST) fusion protein (rEpCI-GST) was expressed in Escherichia coli and affinity purified against the GST tag (Chapter 4). The purified recombinant protein was subjected to serological reactions and showed that immunoglubulin G (IgG) was the dominant antibody isotypes generated against rEpCI-GST as determined by ELISA. A total of 896 human sera were used to evaluate the diagnostic sensitivity and specificity of the fusion protein by IgG-immunoblotting. The sera from 324 CE, 172 neurocysticercosis (NCC), 89 alveolar echinococcosis (AE), 241 other infections/clinical presentations and 70 confirmed negative controls yielded an overall sensitivity of 92.2% and specificity of 95.6% with rEpCI-GST. The combined levels of sensitivity and specificity achieved with the
rEpCI-GST fusion protein for diagnosis of CE are unprecedented taking into account the large panel of sera that were tested (Chapter 5).
BLAST showed that the predicted protein sequence of EpCl displayed high sequence identity to a Taenia solium (the cause of neurocysticercosis (NCC)) protein sequence, indicating that EpC1 may cross-react with antibodies in sera from NCC patients. To determine diagnostic antibody-binding regions on EpCl recognised by CE sera, ten truncated regions (Pl-lO) of the immunogenic antigen were expressed in E. coli as GST fusion proteins and subjected to Western immunoblotting using hyper-immune sera from mice and rabbits prepared against recombinant
EpCl, sera from confirmed CE and NCC patients, and sera from mice experimentally infected with oncospheres of E. granulosus. The immunoblotting analysis indicated that one peptide, designated peptide 5 (P5) (fused with GST) was positively recognised by the four sources of CE sera, while sera from NCC patients did not react with any of the peptides used. There are 4 amino acid substitutions in P5 compared with the T. solum sequence and these may form part of epitope inducing CE-specific antibody. 424 human sera collected from subjects in communities in Urumqi, Xinjiang, a hyperendemic area in China for CE showed 4.5% and 3.3% positivity to EpCl and P5, respectively, whereas, 19.8% of these sera reacted positively against hydatid cyst fluid antigen B in Western blots. EpCl or its contained P5 peptide may prove more accurate for seroepidemiological surveys of
CE (Chapter 6).
Motif analysis showed that the deduced amino acid sequence of EpCl has two significant EF-hand motif structures that resemble the calcium-binding loop of EF-hands. A calcium binding assay showed that the recombinant EpCl fusion protein strongly bound calcium. Immunolocalization assays showed that EpCl was extensively expressed and accumulated at the surface of cells in the germinal membrane, brood capsules and immature protoscoleces, whereas, cells of the mature protoscoleces were weakly or not stained, suggesting that the molecule has function associated with cell growth or cell differentiation (Chapter 4). Further observation showed that EpCl anti-serum recognised antigens on the surface of the germinal
cells including inner face of cells where contact with hydatid cyst fluid (HCF), indicating that the protein is a secreted membrane protein, playing a role of transforming calcium.
Another clone encoded thioredoxin peroxidase (EgTPx) of E. granulosus (Chapter 7). A full-length cDNA sequence coding for E. granulosus thioredoxin peroxidase (EgTPx) was expressed as a fusion protein with GST exhibited significant thioldependent peroxidase activity that protected plasmid DNA from damage by metal catalysed oxidation (MCO) in vitro. Furthermore, the suggested anti-oxidant role for EgTPx was reinforced in an in vivo assay whereby its expression in BL21 bacterial
cells markedly increased the tolerance and survival of the cells to high concentrations ofH20 2 compared with controls. Immunolocalization studies revealed that EgTPx was specifically expressed in all tissues of the protoscolex and brood capsules. Higher intensity of labelling was detected in many, but not all, calcareous corpuscle cells in protoscoleces. The purified recombinant EgTPx protein was used to screen sera from heavily infected mice and patients with confirmed hydatid infection. Only a portion ofthe sera reacted positively with the EgTPx-GST fusion protein in Western blots, suggesting that EgTPx may form antibody-antigen complexes or that responses to the EgTPx antigen may be immunologically regulated. Recombinant EgTPx may prove useful for the screening of specific inhibitors that could serve as new drugs for treatment of hydatid disease. Moreover, given
that TPx from different parasitic phyla were phylogenetically distant from host TPx molecules, the development of anti-parasite TPx inhibitors that do not react with host TPx might be feasible.
The third clone, EgG5, is described in Chapter 8. EgG5 is a full-length cDNA sequence of 1893 bp with an open reading frame of 1680 bp, corresponding to 559 amino acids. The protein has 99.9% nucleotide identity to the major protoscolex surface antigens of E. multilocularis (EMlO) and E. granulosus (EG10), which was found in germinal cluster cells that are forming protoscoleces. EgG5 was weakly expressed in mature protoscoleces. The protein fused with
GST was expressed in E. coli and the purified fusion EgG5-GST was used for diagnostic evaluation with 100 serum samples from CE patients and 243 serum samples from patients with other diseases including AE and NCe. The assay achieved 71% of sensitivity and 58.8% of specificity, which limits the usefulness ofEgG5 in serodiagnosis ofhuman cystic echinococcosis (CE).