Molecular epidemiology, evolutionary history and patterns of antigenic variation in the parasitic trematode 'Schistosoma turkestanicum'

Schistosoma turkestanicum is a parasitic blood fluke widely distributed across Asia. Infection with S.turkestanicum in mammals, including domestic livestock, causes the debilitating disease schistosomiasis and although this parasite is not known to complete a full lifecycle in a human host it is a causative agent of cercarial dermatitis(CD) and a potential agent of neuroschistosomiasis. In 2010 a natural focus of this parasite was reported infecting Red deer in the Gemenc region of Hungary and prior molecular clock analyses places the population in this region around the time of the last ice age. So far only a few geographic isolates have been sequenced for this parasite limiting our understanding of the routes of transmission of this pathogen and there is no literature review collating known geographic localities, current and historic, which may shed light on the distribution of S.turkestanicum. There have also been few studies on the phylogeography of the intermediate host vector of this parasite, the fresh water snail Radix auricularia, where the presence of this species determines the location of transmission sites. Presently there is no vaccine to protect humans or livestock from infection with S.turkestanicum, or any other schistosome species of medical importance, and no vaccine candidates have been characterised or proposed for this species. Advances in computational methods of sequence annotation, structural prediction and antigenic characterisation enable rapid comparison of potential vaccine target antigens to predict vaccine efficacy before testing in vivo. Currently many schistosome genomes, including S.turkestanicum, are not annotated, hindering the screening of these genomes for potential vaccine candidates. Additionally, there have been few studies on the population dynamics of potential Schistosome vaccine candidate proteins where the diversity and accelerated evolution of antigenic proteins within a population is hypothesised to be responsible for the current lack in efficacy of Schistosome vaccine candidates. 6 In this thesis population and molecular clock analyses were carried out on S.turkestanicum and their intermediate hosts R.auricularia, from multiple geographic localities to track the historic spread of this Asian parasite to Europe. The results of this study identified the introduction of S.turkestanicum in Hungary likely followed the introduction of R.auricularia, around 316,000 YA. Furthermore transmission of S.turkestanicum occurred multiple times in the past between Hungary, the Middle-East and China however intermediate host population movement was much more conservative, with only one predicted colonisation dated at around 420,000 YA and a potential isolation event separating Asian and European localities. This ancient restriction of movement could be a result of geographic barriers preventing gene flow which divide Europe and Asia. An extensive literature review on S.turkestanicum revealed the presence of this parasite in 14 different mammalian species in 14 localities ranging from central Europe to the Russian Far East and highlighted a lack of study of infection prevalence in wild mammals and a lack of recent studies in Middle and Far-Eastern regions. Additionally, in the absence of whole genome annotations for multiple schistosome species, including S.turkestanicum, an annotation pipeline was developed to predict putative orthologous proteins and plot antigenicity to aid in the evaluation of these proteins as vaccine candidates. Potential vaccine candidates, such as Schistosome Tetraspanin Orphan Receptor (St-TOR), Tetraspanin-1 (Tsp1), Tetraspanin-2 (Tsp2), Tetraspanin-23 (Tsp23), as well as one candidate which has been less well studied, CD63-like protein, were successfully predicted and further subject to population analyses in the S.turkestanicum population. The large extracellular regions (LEL) used as vaccine candidates in clinical trials for schistosome vaccines, were found to be the most variable regions between schistosome species where gene divergence of Tsp23 LEL, CD63-like LEL and STOR ED-1 peptide corresponds to species divergence suggesting the possibility that host-parasite interaction proteins may drive speciation in schistosomes. The observation of a number of DNA artefacts in otherwise high scoring sequence regions across the vaccine candidate datasets lead to the development of the Basecap program to help prevent the incorporation of error as real SNP's into later population and functional 7 analyses using Sanger sequences. The use of Basecap led to a dramatic reduction in diversity for the schistosome vaccine candidate CD63-like LEL and highlights the importance of read merging methods based on score as well as manual inspection of SNPs within a population. Most artefacts in all 3 test datasets led to the interpretation of non-synonymous changes into the sequence data and notably Tajima's D estimates were found to be highly sensitive to these artefacts which could have large implications for functional studies. Inter-host sequencing of these potential vaccine candidates identified variation within the S.turkestanicum population in the functional LEL regions of Tsp23 and CD63-like protein. Reverse vaccinology methods predicted immune epitope regions at variable sites and these variable sites included both neutral variants and variants predicted to be under balancing selection. This suggests that parasite diveristy in the potential vaccine candidate regions Tsp23 and CD63-like protein LEL regions may be maintained by frequency dependant selection, thowing into question their suitability as vaccine candidates. Conversely, there was no diversity observed for ED1 peptide region of S.turkestanicum in Hungary and further sequencing identified a completed lack of diversity in geographic isolates, from different host species, suggesting that the function of the ED1 peptide may be highly conserved between individuals in multiple host species and in different geographic localities. The function of St-TOR as an immune-modulator may complicate its use as a candidate, however, the lack of variation observed in this study, in combination with the prediction of only a single expressed transcript suggests that the St-TOR ED1 peptide could be an ideal vaccine candidate for S.turkestanicum. The functions of host-parasite interaction proteins in schistosomes warrant further study, for example it would be interesting to explore the potential role of cholesterol in tetraspanin protein function and the CD63-like protein could represent an interesting therapeutic target for inflammatory diseases with a putative IL-10 regulatory function.