Background Schistosomiasis is a neglected disease affecting hundreds of millions worldwide. Of the three main species affecting humans, Schistosoma haematobium is the most common, and is the leading cause of urogenital schistosomiasis. S . haematobium infection can cause different urogenital clinical complications, particularly in the bladder, and furthermore, this parasite has been strongly linked with squamous cell carcinoma. A comprehensive analysis of the molecular composition of its different proteomes will contribute to developing new tools against this devastating disease. Methods and findings By combining a comprehensive protein fractionation approach consisting of OFFGEL electrophoresis with high-throughput mass spectrometry, we have performed the first in-depth characterisation of the different discrete proteomes of S . haematobium that are predicted to interact with human host tissues, including the secreted and tegumental proteomes of adult flukes and secreted and soluble egg proteomes. A total of 662, 239, 210 and 138 proteins were found in the adult tegument, adult secreted, soluble egg and secreted egg proteomes, respectively. In addition, we probed these distinct proteomes with urine to assess urinary antibody responses from naturally infected human subjects with different infection intensities, and identified adult fluke secreted and tegument extracts as being the best predictors of infection. Conclusion We provide a comprehensive dataset of proteins from the adult and egg stages of S . haematobium and highlight their utility as diagnostic markers of infection intensity. Protein composition was markedly different between the different extracts, highlighting the distinct subsets of proteins that different development stages present in their different niches. Furthermore, we have identified adult fluke ES and tegument extracts as best predictors of infection using urine antibodies of naturally infected people. This study provides the first steps towards the development of novel tools to control this important neglected tropical disease.

Helminth parasites release extracellular vesicles which interact with the surrounding host tissues, mediating host–parasite communication and other fundamental processes of parasitism. As such, vesicle proteins present attractive targets for the development of novel intervention strategies to control these parasites and the diseases they cause. Herein, we describe the first proteomic analysis by LC-MS/MS of two types of extracellular vesicles (exosome-like, 120 k pellet vesicles and microvesicle-like, 15 k pellet vesicles) from adult Schistosoma haematobium worms. A total of 57 and 330 proteins were identified in the 120 k pellet vesicles and larger 15 k pellet vesicles, respectively, and some of the most abundant molecules included homologues of known helminth vaccine and diagnostic candidates such as Sm-TSP2, Sm23, glutathione S-transferase, saponins and aminopeptidases. Tetraspanins were highly represented in the analysis and found in both vesicle types. Vaccination of mice with recombinant versions of three of these tetraspanins induced protection in a heterologous challenge (S. mansoni) model of infection, resulting in significant reductions (averaged across two independent trials) in liver (47%, 38% and 41%) and intestinal (47%, 45% and 41%) egg burdens. These findings offer insight into the mechanisms by which anti-tetraspanin antibodies confer protection and highlight the potential that extracellular vesicle surface proteins offer as anti-helminth vaccines.

Helminths are multicellular parasites affecting nearly three billion people worldwide. To orchestrate a parasitic existence, helminths secrete different molecules, either in soluble form or contained within extracellular vesicles (EVs). EVs are secreted by most cell types and organisms, and have varied roles in intercellular communication, including immune modulation and pathogenesis. Areas covered: In this review, we describe the nucleic acid and proteomic composition of EVs from helminths, with a focus on the protein vaccine candidates present on the EV surface membrane, and discuss the potential utility of helminth EVs and their constituent proteins in the fight against helminth infections. Expert commentary: A significant number of proteins present in helminth-secreted EVs are known vaccine candidates. The characterization of helminth EV proteomes will shed light on host-pathogen interactions, facilitate the discovery of new diagnostic biomarkers, and provide a novel approach for the development of new control measures against helminth infections.

Background Sensitive diagnostics are needed for effective management and surveillance of schistosomiasis so that current transmission interruption goals set by WHO can be achieved. We aimed to screen the Schistosoma haematobium secretome to find antibody biomarkers of schistosome infection, validate their diagnostic performance in samples from endemic populations, and evaluate their utility as point of care immunochromatographic tests (POC-ICTs) to diagnose urogenital schistosomiasis in the field. MethodsWe did a biomarker identification study, in which we constructed a proteome array containing 992 validated and predicted proteins from S haematobium and screened it with serum and urine antibodies from endemic populations in Gabon, Tanzania, and Zimbabwe. Arrayed antigens that were IgG-reactive and a select group of antigens from the worm extracellular vesicle proteome, predicted to be diagnostically informative, were then evaluated by ELISA using the same samples used to probe arrays, and samples from individuals residing in a lowendemicity setting (ie, Pemba and Unguja islands, Zanzibar, Tanzania). The two most sensitive and specific antigens were incorporated into POC-ICTs to assess their ability to diagnose S haematobium infection from serum in a fielddeployable format. FindingsFrom array probing, in individuals who were infected, 208 antigens were the targets of significantly elevated IgG responses in serum and 45 antigens were the targets of significantly elevated IgG responses in urine. Of the five proteins that were validated by ELISA, Sh-TSP-2 (area under the curve [AUC] serum =0⋅98 [95% CI 0⋅95-1⋅00]; AUC urine =0⋅96 [0⋅93-0⋅99]), and MS3_01370 (AUC serum =0⋅93 [0⋅89-0⋅97]; AUC urine =0⋅81 [0⋅72-0⋅89]) displayed the highest overall diagnostic performance in each biofluid and exceeded that of S haematobium-soluble egg antigen in urine (AUC=0⋅79 [0⋅69-0⋅90]). When incorporated into separate POC-ICTs, Sh-TSP-2 showed absolute specificity and a sensitivity of 75% and MS3_01370 showed absolute specificity and a sensitivity of 89%.Interpretation We identified numerous biomarkers of urogenital schistosomiasis that could form the basis of novel antibody diagnostics for this disease. Two of these antigens, Sh-TSP-2 and MS3_01370, could be used as sensitive, specific, and field-deployable diagnostics to support schistosomiasis control and elimination initiatives, with particular focus on post-elimination surveillance.Funding Australian Trade and Investment Commission and Merck Global Health Institute.

BackgroundSchistosomiasis is a neglected disease affecting hundreds of millions worldwide. Of the three main species affecting humans, Schistosoma haematobium is the most common, and is the leading cause of urogenital schistosomiasis. S. haematobium infection can cause different urogential clinical complications, particularly in the bladder, and furthermore, this parasite has been strongly linked with squamous cell carcinoma. A comprehensive analysis of the molecular composition of its different proteomes will contribute to developing new tools against this devastating disease.Methods and FindingsBy combining a comprehensive protein fractionation approach consisting of OFFGEL electrophoresis with high-throughput mass spectrometry, we have performed the first in-depth characterisation of the different discrete proteomes of S. haematobium that are predicted to interact with human host tissues, including the secreted and tegumental proteomes of adult flukes and secreted and soluble egg proteomes. A total of 662, 239, 210 and 138 proteins were found in the adult tegument, adult secreted, soluble egg and secreted egg proteomes, respectively. In addition, we probed these distinct proteomes with urine to assess urinary antibody responses from naturally infected human subjects with different infection intensities, and identified adult fluke secreted and tegument extracts as being the best predictors of infection.ConclusionWe provide a comprehensive dataset of proteins from the adult and egg stages of S. haematobium and highlight their utility as diagnostic markers of infection intensity for the development of novel tools to control this important neglected tropical disease.Author SummarySchistosomiasis is a neglected tropical disease affecting millions of people worldwide. Of the main three species affecting humans, Schistosoma haematobium is the most common, and is the leading cause of urogenital schistosomiasis. This parasite can cause a range of clinical complications associated with bladder pathogenesis, including squamous cell carcinoma as well as genital malignancy in women. Herein, we have performed the first comprehensive characterisation of the proteins implicated in host-parasite interactions (secreted and surface proteins from the adult flukes and secreted and soluble egg proteins) in order to advance our understanding of the parasite’s biology. Furthermore, we have characterised the different antibody responses in urine from infected human subjects from an endemic area presenting different infection intensities. The data obtained in this study can be used as a first step towards the development of novel tools for the control of urogenital schistosomiasis.

Schistosoma haematobium is the leading cause of urogenital schistosomiasis and it is recognised as a class 1 carcinogen due to the robust association of infection with bladder cancer. In schistosomes, tetraspanins (TSPs) are abundantly present in different parasite proteomes and could be potential diagnostic candidates due to their accessibility to the host immune system. The large extracellular loops of six TSPs from the secretome (including the soluble excretory/secretory products, tegument and extracellular vesicles) of S. haematobium (Sh-TSP-2, Sh-TSP-4, Sh-TSP-5, Sh-TSP-6, Sh-TSP-18 and Sh-TSP-23) were expressed in a bacterial expression system and polyclonal antibodies were raised to the recombinant proteins to confirm the anatomical sites of expression within the parasite. Sh-TSP-2, and Sh-TSP-18 were identified on the tegument, whereas Sh-TSP-4, Sh-TSP-5, Sh-TSP-6and Sh-TSP-23 were identified both on the tegument and internal tissues of adult parasites. The mRNAs encoding these TSPs were differentially expressed throughout all schistosome developmental stages tested. The potential diagnostic value of three of these Sh-TSPs was assessed using the urine of individuals (stratified by infection intensity) from an endemic area of Zimbabwe. The three Sh-TSPs were the targets of urine IgG responses in all cohorts, including individuals with very low levels of infection (those positive for circulating anodic antigen but negative for eggs by microscopy). This study provides new antigen candidates to immunologically diagnose S. haematobium infection, and the work presented here provides compelling evidence for the use of a biomarker signature to enhance the diagnostic capability of these tetraspanins.

29Helminth parasites release extracellular vesicles which interact with the surrounding host 30 tissues, mediating host-parasite communication and other fundamental processes of 31 parasitism. As such, vesicle proteins present attractive targets for the development of 32 novel intervention strategies to control these parasites and the diseases they cause. Herein, 33 we describe the first proteomic analysis by LC-MS/MS of two types of extracellular 34 vesicles (exosome-like, 120k pellet vesicles and microvesicle-like, 15k pellet vesicles) 35 from adult Schistosoma haematobium worms. A total of 57 and 330 proteins were 36 identified in the 120k pellet vesicles and larger 15k pellet vesicles, respectively, and some 37 of the most abundant molecules included homologues of known helminth vaccine and 38 diagnostic candidates such as Sm-TSP2, Sm23, glutathione S-tranferase, saposins and 39 aminopeptidases. Tetraspanins were highly represented in the analysis and found in both 40 vesicle types. Vaccination of mice with recombinant versions of three of these 41 tetraspanins induced protection in a heterologous challenge (S. mansoni) model of 42 infection, resulting in significant reductions (averaged across two independent trials) in 43 liver (47%, 38% and 41%) and intestinal (47%, 45% and 41%) egg burdens. These 44 findings offer insight into the mechanisms by which anti-tetraspanin antibodies confer 45 protection and highlight the potential that extracellular vesicle surface proteins offer as 46 anti-helminth vaccines.47 48 Introduction 52Schistosomiasis is the second most important parasitic disease, only after malaria, 53 in terms of social, economic and public health impact [1]. Schistosoma haematobium, the 54 causative agent of urogenital schistosomiasis, is highly prevalent in 53 Middle East and 55 African countries [1] and it is also sporadically seen in India [2] and France [3]. Urogenital 56 schistosomiasis affects more than 90 million people, mostly in sub-Saharan Africa where 57 180 million inhabitants are at risk, and is responsible for 150,000 deaths per year [4]. 58Furthermore, the most common complications associated with this disease include 59 schistosomal haematuria, bladder wall pathology, hydronephrosis, and dysuria [4]. 60The current control programs against schistosomiasis are aimed at reducing the 61 morbidity caused by the parasite by regularly treating infected populations with 62 praziquantel [5]. Despite the efforts made to control this devastating disease, 63 schistosomiasis is still spreading to new geographical areas [3,6]. Furthermore, 64 praziquantel has shown reduced efficacy in field studies [7] and is not effective against 65 the immature stages of the parasite [8,9]. Hence, a vaccine that reduces disease severity 66 and/or reduces transmission is needed to control and eliminate schistosomiasis [10,11]. 67Despite efforts over decades, there is no licensed vaccine [1,12]. Even though various 68 vaccine candidates have advanced into clinical trials targeting Schistosoma mansoni, ...

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