Targeting schistosome cholinesterases for vaccine and drug development

The nervous system of schistosomes has been successfully targeted by anthelmintic drugs but the use of many of these has discontinued because of toxic side effects and so there is a need to better understand key neuronal processes at a molecular level to develop safer and more effective intervention strategies that target this vital system. Cholinesterases - acetylcholinesterases (AChE)s and butyrylcholinesterases (BChE)s - are key enzymes that play a pivotal role in the nervous system of schistosomes by regulating neurotransmission through acetylcholine hydrolysis and, accordingly, are an example of such an intervention target. The first results chapter (chapter two) of this thesis investigated the anti-schistosome efficacy of polypyridylruthenium (II) complexes and showed they were active against all intra-mammalian stages of S. mansoni. Two compounds, Rubb₁₂-tri and Rubb₇-tnl, which were among the most potent in their ability to kill schistosomula and adult worms and inhibit egg hatching in vitro, were assessed for their efficacy in a mouse model of schistosomiasis using 5 consecutive daily i.v. doses of 2 mg/kg (Rubb₁₂-tri) and 10 mg/kg (Rubb₇-tnl). Mice treated with Rubb₁₂-tri showed an average 42% reduction (P = 0.009), over two independent trials, in adult worm burden. Liver egg burdens were not significantly decreased in either drug-treated group but ova from both of these groups showed significant decreases in hatching ability (Rubb₁₂-tri - 68%, Rubb₇-tnl - 56%) and were significantly morphologically altered (Rubb₁₂-tri - 62% abnormal, Rubb₇-tnl - 35% abnormal). I hypothesize that the drugs exerted their activity, at least partially, through inhibition of both neuronal and tegumental acetylcholinesterases (AChEs), as worms treated in vitro showed significant decreases in activity of these enzymes. Further, treated parasites exhibited a significantly decreased ability to uptake glucose, significantly depleted glycogen stores and withered tubercules (a site of glycogen storage), implying drug-mediated interference in this nutrient acquisition pathway. Chapter three of this thesis provided the first comprehensive molecular characterization of three S. mansoni cholinesterases (SmChEs), designated as SmAChE1, SmBChE1 and SmAChE3, which were identified from the interrogation of the now wholly annotated S. mansoni genome. Anti-SmChE antibodies localized the proteins to the tegument and neuromusculature of adults and schistosomula and developmental expression profiling differed among the molecules, suggestive of functions extending beyond traditional cholinergic signaling for each of them. I also reported the presence of ChE activity in parasite ES products for the first time and proteomically identified the molecules responsible (SmAChE1 and SmBChE1). Functional recombinant versions of the three SmChEs were produced in Pichia pastoris and enzyme nomenclature (AChE or BChE) was verified based on substrate preference. Lastly, in the first characterization study of a BChE from helminths, evidence is provided that SmBChE1 may act as a bio-scavenger of AChE inhibitors as the addition of recombinant SmBChE1 to parasite cultures mitigated the effect of the anti-schistosomal AChE inhibitor dichlorvos whereas SmBChE1-silenced parasites displayed increased sensitivity to dichlorvos. SmChEs were further characterized by RNAi-based experiments in chapter four of this thesis. RNAi-mediated silencing of individual SmChEs, or simultaneous silencing of all three SmChEs, significantly suppressed transcript and protein expression levels and AChE activity in parasites. In a dissection of the hypothesis that tegumental AChE mediates exogenous glucose scavenging by the parasite, I showed that RNAi-mediated knockdown of SmAChE1 and SmAChE3, but not SmBChE1, significantly reduced glucose uptake by schistosomes. Parasite survivability in vitro and in vivo was significantly impaired with the silencing of SmChEs, either individually or in combination, attesting to the essentiality of these molecules. Chapter five of this thesis explored the vaccine potential of SmChEs. When treated in vitro with anti-SmChE IgG, parasites displayed significantly decreased ChE activity, which eventually resulted in death. Vaccination with individual SmChEs, or a combination of all three SmChEs, significantly reduced worm burdens (28% - 38%, averaged across two independent trials) compared to controls. Liver egg burdens were significantly decreased for all mice across both trials (13% - 46%) except those vaccinated with SmAChE1 in trial 1. Egg viability, as determined by egg hatching from liver homogenates, was significantly reduced in the groups vaccinated with the SmChE cocktail (40%) and SmAChE3 (46%). Surviving worms from each vaccinated group were significantly stunted and depleted of glycogen stores, compared to controls. In conclusion, this thesis has identified the burgeoning potential of a new class of antischistosome drugs that, at least in part, target the nervous system of the parasite and provided a comprehensive characterization of a family of ChEs from S. mansoni, giving compelling evidence for the essentiality of the proteins and their utility as intervention targets against schistosomiasis

Novel cholinesterase paralogs of Schistosoma mansoni have perceived roles in cholinergic signaling and drug detoxification and are essential for parasite survival

Cholinesterase (ChE) function in schistosomes is essential for orchestration of parasite neurotransmission but has been poorly defined with respect to the molecules responsible. Interrogation of the S. mansoni genome has revealed the presence of three ChE domain-containing genes (Smche)s, which we have shown to encode two functional acetylcholinesterases (AChE)s (Smache1 -smp_154600 and Smache2 -smp_136690) and a butyrylcholinesterase (BChE) (Smbche1 -smp_125350). Antibodies to recombinant forms of each SmChE localized the proteins to the tegument of adults and schistosomula and developmental expression profiling differed among the three molecules, suggestive of functions extending beyond traditional cholinergic signaling. For the first time in schistosomes, we identified ChE enzymatic activity in fluke excretory/secretory (ES) products and, using proteomic approaches, attributed this activity to the presence of SmAChE1 and SmBChE1. Parasite survival in vitro and in vivo was significantly impaired by silencing of each smche, either individually or in combination, attesting to the essential roles of these molecules. Lastly, in the first characterization study of a BChE from helminths, evidence is provided that SmBChE1 may act as a bio-scavenger of AChE inhibitors as the addition of recombinant SmBChE1 to parasite cultures mitigated the effect of the anti-schistosome AChE inhibitor 2,2- dichlorovinyl dimethyl phosphate-dichlorvos (DDVP), whereas smbche1-silenced parasites displayed increased sensitivity to DDVP.Funding: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was funded by NHMRC program grant APP1037304, an NHMRC Senior Principal Research Fellowship (APP1117504) to A.L. and a James Cook University Postgraduate Scholarship to B.T

Characterisation of tetraspanins from Schistosoma haematobium and evaluation of their potential as novel diagnostic markers

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-6 and 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

Immunomics-guided discovery of serum and urine antibodies for diagnosing urogenital schistosomiasis:A biomarker identification study

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. Methods: We 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 low-endemicity 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 field-deployable format. Findings: From 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]; AUCurine=0·96 [0·93-0·99]), and MS3_01370 (AUCserum=0·93 [0·89-0·97]; AUCurine=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

Global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990-2015: a systematic analysis for the Global Burden of Disease Study 2015

Forouzanfar MH, Afshin A, Alexander LT, et al. Global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990-2015: a systematic analysis for the Global Burden of Disease Study 2015. LANCET. 2016;388(10053):1659-1724.Background The Global Burden of Diseases, Injuries, and Risk Factors Study 2015 provides an up-to-date synthesis of the evidence for risk factor exposure and the attributable burden of disease. By providing national and subnational assessments spanning the past 25 years, this study can inform debates on the importance of addressing risks in context. Methods We used the comparative risk assessment framework developed for previous iterations of the Global Burden of Disease Study to estimate attributable deaths, disability-adjusted life-years (DALYs), and trends in exposure by age group, sex, year, and geography for 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks from 1990 to 2015. This study included 388 risk-outcome pairs that met World Cancer Research Fund-defined criteria for convincing or probable evidence. We extracted relative risk and exposure estimates from randomised controlled trials, cohorts, pooled cohorts, household surveys, census data, satellite data, and other sources. We used statistical models to pool data, adjust for bias, and incorporate covariates. We developed a metric that allows comparisons of exposure across risk factors-the summary exposure value. Using the counterfactual scenario of theoretical minimum risk level, we estimated the portion of deaths and DALYs that could be attributed to a given risk. We decomposed trends in attributable burden into contributions from population growth, population age structure, risk exposure, and risk-deleted cause-specific DALY rates. We characterised risk exposure in relation to a Socio-demographic Index (SDI). Findings Between 1990 and 2015, global exposure to unsafe sanitation, household air pollution, childhood underweight, childhood stunting, and smoking each decreased by more than 25%. Global exposure for several occupational risks, high body-mass index (BMI), and drug use increased by more than 25% over the same period. All risks jointly evaluated in 2015 accounted for 57.8% (95% CI 56.6-58.8) of global deaths and 41.2% (39.8-42.8) of DALYs. In 2015, the ten largest contributors to global DALYs among Level 3 risks were high systolic blood pressure (211.8 million [192.7 million to 231.1 million] global DALYs), smoking (148.6 million [134.2 million to 163.1 million]), high fasting plasma glucose (143.1 million [125.1 million to 163.5 million]), high BMI (120.1 million [83.8 million to 158.4 million]), childhood undernutrition (113.3 million [103.9 million to 123.4 million]), ambient particulate matter (103.1 million [90.8 million to 115.1 million]), high total cholesterol (88.7 million [74.6 million to 105.7 million]), household air pollution (85.6 million [66.7 million to 106.1 million]), alcohol use (85.0 million [77.2 million to 93.0 million]), and diets high in sodium (83.0 million [49.3 million to 127.5 million]). From 1990 to 2015, attributable DALYs declined for micronutrient deficiencies, childhood undernutrition, unsafe sanitation and water, and household air pollution; reductions in risk-deleted DALY rates rather than reductions in exposure drove these declines. Rising exposure contributed to notable increases in attributable DALYs from high BMI, high fasting plasma glucose, occupational carcinogens, and drug use. Environmental risks and childhood undernutrition declined steadily with SDI; low physical activity, high BMI, and high fasting plasma glucose increased with SDI. In 119 countries, metabolic risks, such as high BMI and fasting plasma glucose, contributed the most attributable DALYs in 2015. Regionally, smoking still ranked among the leading five risk factors for attributable DALYs in 109 countries; childhood underweight and unsafe sex remained primary drivers of early death and disability in much of sub-Saharan Africa. Interpretation Declines in some key environmental risks have contributed to declines in critical infectious diseases. Some risks appear to be invariant to SDI. Increasing risks, including high BMI, high fasting plasma glucose, drug use, and some occupational exposures, contribute to rising burden from some conditions, but also provide opportunities for intervention. Some highly preventable risks, such as smoking, remain major causes of attributable DALYs, even as exposure is declining. Public policy makers need to pay attention to the risks that are increasingly major contributors to global burden. Copyright (C) The Author(s). Published by Elsevier Ltd

Global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for 249 causes of death, 1980-2015 : a systematic analysis for the Global Burden of Disease Study 2015

Background Improving survival and extending the longevity of life for all populations requires timely, robust evidence on local mortality levels and trends. The Global Burden of Disease 2015 Study (GBD 2015) provides a comprehensive assessment of all-cause and cause-specific mortality for 249 causes in 195 countries and territories from 1980 to 2015. These results informed an in-depth investigation of observed and expected mortality patterns based on sociodemographic measures. Methods We estimated all-cause mortality by age, sex, geography, and year using an improved analytical approach originally developed for GBD 2013 and GBD 2010. Improvements included refinements to the estimation of child and adult mortality and corresponding uncertainty, parameter selection for under-5 mortality synthesis by spatiotemporal Gaussian process regression, and sibling history data processing. We also expanded the database of vital registration, survey, and census data to 14 294 geography-year datapoints. For GBD 2015, eight causes, including Ebola virus disease, were added to the previous GBD cause list for mortality. We used six modelling approaches to assess cause-specific mortality, with the Cause of Death Ensemble Model (CODEm) generating estimates for most causes. We used a series of novel analyses to systematically quantify the drivers of trends in mortality across geographies. First, we assessed observed and expected levels and trends of cause-specific mortality as they relate to the Socio-demographic Index (SDI), a summary indicator derived from measures of income per capita, educational attainment, and fertility. Second, we examined factors affecting total mortality patterns through a series of counterfactual scenarios, testing the magnitude by which population growth, population age structures, and epidemiological changes contributed to shifts in mortality. Finally, we attributed changes in life expectancy to changes in cause of death. We documented each step of the GBD 2015 estimation processes, as well as data sources, in accordance with Guidelines for Accurate and Transparent Health Estimates Reporting (GATHER). Findings Globally, life expectancy from birth increased from 61.7 years (95% uncertainty interval 61.4-61.9) in 1980 to 71.8 years (71.5-72.2) in 2015. Several countries in sub-Saharan Africa had very large gains in life expectancy from 2005 to 2015, rebounding from an era of exceedingly high loss of life due to HIV/AIDS. At the same time, many geographies saw life expectancy stagnate or decline, particularly for men and in countries with rising mortality from war or interpersonal violence. From 2005 to 2015, male life expectancy in Syria dropped by 11.3 years (3.7-17.4), to 62.6 years (56.5-70.2). Total deaths increased by 4.1% (2.6-5.6) from 2005 to 2015, rising to 55.8 million (54.9 million to 56.6 million) in 2015, but age-standardised death rates fell by 17.0% (15.8-18.1) during this time, underscoring changes in population growth and shifts in global age structures. The result was similar for non-communicable diseases (NCDs), with total deaths from these causes increasing by 14.1% (12.6-16.0) to 39.8 million (39.2 million to 40.5 million) in 2015, whereas age-standardised rates decreased by 13.1% (11.9-14.3). Globally, this mortality pattern emerged for several NCDs, including several types of cancer, ischaemic heart disease, cirrhosis, and Alzheimer's disease and other dementias. By contrast, both total deaths and age-standardised death rates due to communicable, maternal, neonatal, and nutritional conditions significantly declined from 2005 to 2015, gains largely attributable to decreases in mortality rates due to HIV/AIDS (42.1%, 39.1-44.6), malaria (43.1%, 34.7-51.8), neonatal preterm birth complications (29.8%, 24.8-34.9), and maternal disorders (29.1%, 19.3-37.1). Progress was slower for several causes, such as lower respiratory infections and nutritional deficiencies, whereas deaths increased for others, including dengue and drug use disorders. Age-standardised death rates due to injuries significantly declined from 2005 to 2015, yet interpersonal violence and war claimed increasingly more lives in some regions, particularly in the Middle East. In 2015, rotaviral enteritis (rotavirus) was the leading cause of under-5 deaths due to diarrhoea (146 000 deaths, 118 000-183 000) and pneumococcal pneumonia was the leading cause of under-5 deaths due to lower respiratory infections (393 000 deaths, 228 000-532 000), although pathogen-specific mortality varied by region. Globally, the effects of population growth, ageing, and changes in age-standardised death rates substantially differed by cause. Our analyses on the expected associations between cause-specific mortality and SDI show the regular shifts in cause of death composition and population age structure with rising SDI. Country patterns of premature mortality (measured as years of life lost [YLLs]) and how they differ from the level expected on the basis of SDI alone revealed distinct but highly heterogeneous patterns by region and country or territory. Ischaemic heart disease, stroke, and diabetes were among the leading causes of YLLs in most regions, but in many cases, intraregional results sharply diverged for ratios of observed and expected YLLs based on SDI. Communicable, maternal, neonatal, and nutritional diseases caused the most YLLs throughout sub-Saharan Africa, with observed YLLs far exceeding expected YLLs for countries in which malaria or HIV/AIDS remained the leading causes of early death. Interpretation At the global scale, age-specific mortality has steadily improved over the past 35 years; this pattern of general progress continued in the past decade. Progress has been faster in most countries than expected on the basis of development measured by the SDI. Against this background of progress, some countries have seen falls in life expectancy, and age-standardised death rates for some causes are increasing. Despite progress in reducing age-standardised death rates, population growth and ageing mean that the number of deaths from most non-communicable causes are increasing in most countries, putting increased demands on health systems. Copyright (C) The Author(s). Published by Elsevier Ltd.Peer reviewe

Measuring the health-related Sustainable Development Goals in 188 countries : a baseline analysis from the Global Burden of Disease Study 2015

Background In September, 2015, the UN General Assembly established the Sustainable Development Goals (SDGs). The SDGs specify 17 universal goals, 169 targets, and 230 indicators leading up to 2030. We provide an analysis of 33 health-related SDG indicators based on the Global Burden of Diseases, Injuries, and Risk Factors Study 2015 (GBD 2015). Methods We applied statistical methods to systematically compiled data to estimate the performance of 33 health-related SDG indicators for 188 countries from 1990 to 2015. We rescaled each indicator on a scale from 0 (worst observed value between 1990 and 2015) to 100 (best observed). Indices representing all 33 health-related SDG indicators (health-related SDG index), health-related SDG indicators included in the Millennium Development Goals (MDG index), and health-related indicators not included in the MDGs (non-MDG index) were computed as the geometric mean of the rescaled indicators by SDG target. We used spline regressions to examine the relations between the Socio-demographic Index (SDI, a summary measure based on average income per person, educational attainment, and total fertility rate) and each of the health-related SDG indicators and indices. Findings In 2015, the median health-related SDG index was 59.3 (95% uncertainty interval 56.8-61.8) and varied widely by country, ranging from 85.5 (84.2-86.5) in Iceland to 20.4 (15.4-24.9) in Central African Republic. SDI was a good predictor of the health-related SDG index (r(2) = 0.88) and the MDG index (r(2) = 0.2), whereas the non-MDG index had a weaker relation with SDI (r(2) = 0.79). Between 2000 and 2015, the health-related SDG index improved by a median of 7.9 (IQR 5.0-10.4), and gains on the MDG index (a median change of 10.0 [6.7-13.1]) exceeded that of the non-MDG index (a median change of 5.5 [2.1-8.9]). Since 2000, pronounced progress occurred for indicators such as met need with modern contraception, under-5 mortality, and neonatal mortality, as well as the indicator for universal health coverage tracer interventions. Moderate improvements were found for indicators such as HIV and tuberculosis incidence, minimal changes for hepatitis B incidence took place, and childhood overweight considerably worsened. Interpretation GBD provides an independent, comparable avenue for monitoring progress towards the health-related SDGs. Our analysis not only highlights the importance of income, education, and fertility as drivers of health improvement but also emphasises that investments in these areas alone will not be sufficient. Although considerable progress on the health-related MDG indicators has been made, these gains will need to be sustained and, in many cases, accelerated to achieve the ambitious SDG targets. The minimal improvement in or worsening of health-related indicators beyond the MDGs highlight the need for additional resources to effectively address the expanded scope of the health-related SDGs.Peer reviewe

Cancer Incidence, Mortality, Years of Life Lost, Years Lived With Disability, and Disability-Adjusted Life Years for 29 Cancer Groups From 2010 to 2019: A Systematic Analysis for the Global Burden of Disease Study 2019.

The Global Burden of Diseases, Injuries, and Risk Factors Study 2019 (GBD 2019) provided systematic estimates of incidence, morbidity, and mortality to inform local and international efforts toward reducing cancer burden. To estimate cancer burden and trends globally for 204 countries and territories and by Sociodemographic Index (SDI) quintiles from 2010 to 2019. The GBD 2019 estimation methods were used to describe cancer incidence, mortality, years lived with disability, years of life lost, and disability-adjusted life years (DALYs) in 2019 and over the past decade. Estimates are also provided by quintiles of the SDI, a composite measure of educational attainment, income per capita, and total fertility rate for those younger than 25 years. Estimates include 95% uncertainty intervals (UIs). In 2019, there were an estimated 23.6 million (95% UI, 22.2-24.9 million) new cancer cases (17.2 million when excluding nonmelanoma skin cancer) and 10.0 million (95% UI, 9.36-10.6 million) cancer deaths globally, with an estimated 250 million (235-264 million) DALYs due to cancer. Since 2010, these represented a 26.3% (95% UI, 20.3%-32.3%) increase in new cases, a 20.9% (95% UI, 14.2%-27.6%) increase in deaths, and a 16.0% (95% UI, 9.3%-22.8%) increase in DALYs. Among 22 groups of diseases and injuries in the GBD 2019 study, cancer was second only to cardiovascular diseases for the number of deaths, years of life lost, and DALYs globally in 2019. Cancer burden differed across SDI quintiles. The proportion of years lived with disability that contributed to DALYs increased with SDI, ranging from 1.4% (1.1%-1.8%) in the low SDI quintile to 5.7% (4.2%-7.1%) in the high SDI quintile. While the high SDI quintile had the highest number of new cases in 2019, the middle SDI quintile had the highest number of cancer deaths and DALYs. From 2010 to 2019, the largest percentage increase in the numbers of cases and deaths occurred in the low and low-middle SDI quintiles. The results of this systematic analysis suggest that the global burden of cancer is substantial and growing, with burden differing by SDI. These results provide comprehensive and comparable estimates that can potentially inform efforts toward equitable cancer control around the world.Funding/Support: The Institute for Health Metrics and Evaluation received funding from the Bill & Melinda Gates Foundation and the American Lebanese Syrian Associated Charities. Dr Aljunid acknowledges the Department of Health Policy and Management of Kuwait University and the International Centre for Casemix and Clinical Coding, National University of Malaysia for the approval and support to participate in this research project. Dr Bhaskar acknowledges institutional support from the NSW Ministry of Health and NSW Health Pathology. Dr Bärnighausen was supported by the Alexander von Humboldt Foundation through the Alexander von Humboldt Professor award, which is funded by the German Federal Ministry of Education and Research. Dr Braithwaite acknowledges funding from the National Institutes of Health/ National Cancer Institute. Dr Conde acknowledges financial support from the European Research Council ERC Starting Grant agreement No 848325. Dr Costa acknowledges her grant (SFRH/BHD/110001/2015), received by Portuguese national funds through Fundação para a Ciência e Tecnologia, IP under the Norma Transitória grant DL57/2016/CP1334/CT0006. Dr Ghith acknowledges support from a grant from Novo Nordisk Foundation (NNF16OC0021856). Dr Glasbey is supported by a National Institute of Health Research Doctoral Research Fellowship. Dr Vivek Kumar Gupta acknowledges funding support from National Health and Medical Research Council Australia. Dr Haque thanks Jazan University, Saudi Arabia for providing access to the Saudi Digital Library for this research study. Drs Herteliu, Pana, and Ausloos are partially supported by a grant of the Romanian National Authority for Scientific Research and Innovation, CNDS-UEFISCDI, project number PN-III-P4-ID-PCCF-2016-0084. Dr Hugo received support from the Higher Education Improvement Coordination of the Brazilian Ministry of Education for a sabbatical period at the Institute for Health Metrics and Evaluation, between September 2019 and August 2020. Dr Sheikh Mohammed Shariful Islam acknowledges funding by a National Heart Foundation of Australia Fellowship and National Health and Medical Research Council Emerging Leadership Fellowship. Dr Jakovljevic acknowledges support through grant OI 175014 of the Ministry of Education Science and Technological Development of the Republic of Serbia. Dr Katikireddi acknowledges funding from a NHS Research Scotland Senior Clinical Fellowship (SCAF/15/02), the Medical Research Council (MC_UU_00022/2), and the Scottish Government Chief Scientist Office (SPHSU17). Dr Md Nuruzzaman Khan acknowledges the support of Jatiya Kabi Kazi Nazrul Islam University, Bangladesh. Dr Yun Jin Kim was supported by the Research Management Centre, Xiamen University Malaysia (XMUMRF/2020-C6/ITCM/0004). Dr Koulmane Laxminarayana acknowledges institutional support from Manipal Academy of Higher Education. Dr Landires is a member of the Sistema Nacional de Investigación, which is supported by Panama’s Secretaría Nacional de Ciencia, Tecnología e Innovación. Dr Loureiro was supported by national funds through Fundação para a Ciência e Tecnologia under the Scientific Employment Stimulus–Institutional Call (CEECINST/00049/2018). Dr Molokhia is supported by the National Institute for Health Research Biomedical Research Center at Guy’s and St Thomas’ National Health Service Foundation Trust and King’s College London. Dr Moosavi appreciates NIGEB's support. Dr Pati acknowledges support from the SIAN Institute, Association for Biodiversity Conservation & Research. Dr Rakovac acknowledges a grant from the government of the Russian Federation in the context of World Health Organization Noncommunicable Diseases Office. Dr Samy was supported by a fellowship from the Egyptian Fulbright Mission Program. Dr Sheikh acknowledges support from Health Data Research UK. Drs Adithi Shetty and Unnikrishnan acknowledge support given by Kasturba Medical College, Mangalore, Manipal Academy of Higher Education. Dr Pavanchand H. Shetty acknowledges Manipal Academy of Higher Education for their research support. Dr Diego Augusto Santos Silva was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil Finance Code 001 and is supported in part by CNPq (302028/2018-8). Dr Zhu acknowledges the Cancer Prevention and Research Institute of Texas grant RP210042

Schistosoma haematobium Extracellular Vesicle Proteins Confer Protection in a Heterologous Model of Schistosomiasis.

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.This work was funded by the NHMRC program grant APP1037304. A.L. was funded by an NHMRC Senior Principal Research Fellowship (APP1117504). B.A.T. was funded by a James Cook University Postgraduate Scholarship. G.G.M. was funded by an AITHM Postgraduate Scholarship.S