Schistosomes are parasitic blood flukes that infect approximately 200 million people worldwide. The disease known as schistosomiasis is the second most significant tropical parasitic disease after malaria. Only one drug currently exists for schistosomiasis and there are concerns about resistance to the drug. There has been much effort to develop vaccines against schistosomiasis to produce long-term protection in endemic regions. Surface-associated proteins, and in particular, those expressed in the body wall, or tegument, have been proposed as potential vaccine targets. Annexins, also known as calcium-dependent phospholipid binding proteins, are among the abundant molecules associated with the apical membrane of the schistosome tegument. In this study, five annexins, identified from proteomic investigations of the tegument of S. mansoni, designated Anx(Sm)1, Anx(Sm)3, Anx(Sm)4, Anx(Sm)5 and Anx(Sm)12, were characterised.
The five S. mansoni annexin genes encoded proteins that varied in length from 996 to 1464 amino acids (aa). No signal peptide was predicted for any annexin. Bioinformatic analysis showed that the five S. mansoni annexins possess a primary structure consisting of a short but variable N-terminal region and a long C-terminal core containing four homologous annexin repeats (I-IV) composed of approximately 70 aa, which contain five alpha-helices. Comparison of the sequences of S. mansoni annexins with those of vertebrates showed that Anx(Sm)1, Anx(Sm)3 and Anx(Sm)5 have longer linker regions between repeats II and III. The life cycle expression profiles of the five annexins were assessed using quantitative PCR. This showed that mRNA levels of the annexins were low in the egg stage, but increased gradually after the transition of cercariae (the invasive schistosome larvae) to schistosomula (the post-invasive larvae). All five S. mansoni annexins were expressed as soluble full length recombinant proteins using a bacterial expression system. Circular dichroism (CD) demonstrated that recombinant annexins were folded, showing a secondary structure content rich in alpha-helices. The membrane binding affinity of each recombinant annexin was enhanced when the recombinant annexins are incubated in the presence of Ca2+. The five annexins were immunolocalised to the tegument, with immunoreactivity also occurring in cells and in muscle of adult parasites. Furthermore, Anx(Sm)4, Anx(Sm)5 and Anx(Sm)12 immunoreactivity was also confirmed for the gastrodermis, the syncytial lining of the digestive system in adult parasites. Recombinant annexins were recognised by the sera of mice infected with S. mansoni, indicating that the native proteins induce an antibody response in infection.
Attempts were made to functionally silence the annexins using interfering RNA interference technologies (RNAi). Each annexin-specific dsRNA brought about a reduction of mRNA transcripts by 80 – 96% in adults, and 87 – 95% in schistosomula. Knockdown of each annexin in adult parasites and schistosomula led to a 15 - 85% and 16 - 50% reduction of annexin-specific proteins levels. A motility assay of adult parasites showed that males were more susceptible to the annexin silencing than females. No other phenotypic changes were observed in adult parasites treated with annexin-specific dsRNA. By contrast, silencing of annexins in schistosomula appeared to inhibit schistosomula growth. Schistosomula, for which specific annexins had been silenced, were exposed to exogenous oxidative stress to test whether the annexins have a role in protecting the parasite against oxygen radicals, as proposed for annexins of other organisms. When Anx(Sm)4, Anx(Sm)5 and Anx(Sm)12 were silenced, the parasites displayed grossly swollen digestive regions, indicating that suppression of certain annexins can lead to the failure in the regulation of cellular signalling during stress.
A chimera peptide (chimeric-AT), containing the linker of Anx(Sm)3 and the second extracellular loop of S. mansoni tetraspanins 2 (Sm-TSP-2-EC2), was engineered as a vaccine construct. HLA-DR binding predictions showed that the Anx(Sm)3 linker and chimeric-AT were capable to bind with 11 alleles (21.6%) and 20 alleles (49%), respectively. Sera from mice infected with S. mansoni reacted strongly with the synthetic Anx(Sm)3 linker peptide and chimeric-AT, showing that they are immunogenic. Anx(Sm)3 and chimeric-AT adjuvated with alum induced high IgG1/IgG2a ratio, implying a Th2 type predominant immune response. Mice vaccinated with soluble recombinant Anx(Sm)3 showed low protective effect while chimeric-AT showed highest reductions in worm burden (20%), liver egg burden (17%) and intestinal egg burden (40.4%). The results showed that chimeric-AT triggered a high level of antibody production, suggesting this multi-antigen construct could be a potential vaccine candidate against schistosomiasis.
Collectively, studies conducted in this thesis have identified a number of biochemical characteristics of five annexins present in the host-interactive layer of S. mansoni. The biochemical and immunological characterisations of the annexins presented herein suggest that the molecules may be suitable for development for treatment of the disease. Targeting these molecules, particularly, the unique linker regions as vaccine candidates may provide attractive opportunity to explore anti-schistosomes intervention.