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The reduced prevalence of insulin resistance and type 2 diabetes in countries with endemic parasitic worm infections suggests a protective role for worms against metabolic disorders, however clinical evidence has been non-existent. This 2-year randomised, double-blinded clinical trial in Australia of hookworm infection in 40 male and female adults at risk of type 2 diabetes assessed the safety and potential metabolic benefits of treatment with either 20 (n = 14) or 40 (n = 13) Necator americanus larvae (L3) or Placebo (n = 13) (Registration ACTRN12617000818336). Primary outcome was safety defined by adverse events and completion rate. Homoeostatic model assessment of insulin resistance, fasting blood glucose and body mass were key secondary outcomes. Adverse events were more frequent in hookworm-treated participants, where 44% experienced expected gastrointestinal symptoms, but completion rates were comparable to Placebo. Fasting glucose and insulin resistance were lowered in both hookworm-treated groups at 1 year, and body mass was reduced after L3-20 treatment at 2 years. This study suggests hookworm infection is safe in people at risk of type 2 diabetes and associated with improved insulin resistance, warranting further exploration of the benefits of hookworms on metabolic health., (© 2023. The Author(s).)

Background: Although there is unprecedented interest in experimental human hookworm infection, details of hookworm manufacture and characterisation have been sparsely reported. In this report, we detail the production and characterisation of Necator americanus larvae for use in a recently published clinical trial., Methods: Faeces was obtained from an experimentally infected donor. Faecal hookworm DNA was determined by quantitative PCR. Paired samples were incubated in either sterile water or sterile water mixed with antimicrobials (amphotericin and gentamicin). Coproculture was performed by modified Harada-Mori method. The harvested larvae were then processed in either sterile water or antiseptic solution. Larval yield was then calculated (larvae per gram), larval viability was determined by thermally induced motility assay and microbial burden was determined at the day of harvest, at 48 h and at 7 days., Results: Twenty-eight faecal cultures were performed over 16 months. The faecal hookworm DNA content was variable over this time. There was no association of larval yield with faecal hookworm DNA content. Pre-treatment of faeces with antimicrobials did not influence larval yield. Larval motility was 85.3% (95% CI 79.3-91.3%). Incubation of larvae in antiseptics did not reduce viability at 14 days with a marginal mean of 68.6% (95% CI 59.1-78.1%) washed in water vs. 63.3% (95% CI 53.8 - 72.9%) when incubated in betadine (p = 0.38). Larvae washed in sterile water did not meet microbial bioburden criteria. Incubation in antiseptic resulted in acceptable microbial bioburden at 48 h but not at 7 days. Although the addition of gentamicin did reduce the microbial bio-burden acceptable levels, it was found to significantly lower larval motility at 7 days compared to incubation in sterile water and motility at 7 days 37.8% (95% CI 4.7-70.9%) vs. 67.3% (95% CI 35.2-99.3%, p < 0.001), respectively., Conclusions: Despite standardised culture methodologies and the use of a single donor, larval yield varied considerably between batches and had no association with faecal hookworm DNA. Larval viability decreases over time and the age of larvae at time of use are likely to be important. Microbial bioburden maybe temporarily reduced by incubation in antiseptics and has little effect on viability. Incubation of larvae in gentamicin is effective at reducing microbial bioburden but is deleterious to larval viability., (© 2022. The Author(s).)

Background: Approximately 400 million individuals are infected with hookworms globally. Protective vaccines are needed to prevent reinfections, which often occur after drug treatment in endemic areas. Ideal vaccines are highly efficacious and well tolerated, and do not present risks to patient safety. Peptide vaccines can generate specific, highly protective responses because they focus on minimal antigenic target(s) with a specific immunoprotective mechanism. Necator americanus aspartyl protease 1 (Na-APR-1) is one of the most promising hookworm vaccine antigens. The neutralizing epitope p3 (TSLIAGPKAQVEAIQKYIGAEL), together with universal the T H epitope P25 (KLIPNASLIENCTKAEL), has been used previously to produce peptide vaccines and was found to protect BALB/c mice against rodent hookworm infections, resulting in worm burden reductions of up to 98%. However, because of extensive digestion in the gastrointestinal tract, large oral vaccination doses were necessary to achieve this level of efficacy., Objective: We sought to overcome the limitations of oral vaccine delivery and to investigate protective efficacy and immune correlates of protection. Herein, we examined 5 different peptide vaccines following intraperitoneal injection, to compare their efficacy with that of the clinical protein antigen APR-1., Methods: BALB/c mice were immunized with p3-P25-based antigen that was adjuvanted with (1) lipid core peptide, (2) polymethyl methacrylate, (3) linear polyleucine, and (4) branched polyleucine (BL 10 ), or with (5) CpG/aluminum hydroxide adjuvant (alum)-adjuvanted control and protein-based (6) CpG/alum-adjuvanted Na-APR-1. The mice sera, saliva, and feces were sampled for immune response evaluation. The immunized mice were further challenged via hookworm larvae infection, and protection was evaluated by conducting intestinal hookworm counts., Results: BL 10 and lipid core peptide generated the highest serum anti-Na-APR-1 IgG and fecal anti-APR-1 IgG titers, but only BL 10 generated significant fecal anti-Na-APR-1 IgA titers. Upon challenge, immunization with CpG/alum-adjuvanted p3-P25, BL 10 , and lipid core peptide provided the highest worm burden reductions of 75%, 77%, and 59%, respectively, whereas the group immunized with Na-APR-1 had only modest worm reduction of 26%. The relationships between serum anti-Na-APR-1 IgG, fecal anti-Na-APR-1 IgA and IgG, and worm burden reduction were established with R 2 values greater than or equal to 0.9, and the crucial role of both anti-Na-APR-1 IgG and IgA responses was identified., Conclusions: We demonstrated for the first time that p3-based vaccine candidates are safer and can deliver higher protection against hookworm infection compared with the clinical vaccine candidate, Na-APR-1., (Copyright © 2022 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

The elimination of lymphatic filariasis (LF) is achieved through repeated mass drug administration (MDA) of anti-filarial medications, which interrupts transmission and prevents new infections. Accurate transmission assessments are critical to deciding when to stop MDA. Current methods for evaluating transmission may be insufficiently sensitive, resulting in post-MDA resurgence. We, therefore, evaluated potential diagnostic testing scenarios for post-MDA surveillance. Data were used from two surveys (a household cluster and a cohort) conducted in an area of Mandalay Region, Myanmar, with ongoing transmission following several rounds of MDA. First, age- and sex-adjusted seroprevalence were estimated for the area using the household survey. Next, three Bayesian networks were built from the combined datasets to compare antigens by immunochromatic testing (ICT) and/or Og4C3 enzyme-linked immunosorbent assay (ELISA) and antibody (Ab) detection methods (Wb123 or Bm14 Ab ELISA). The networks were checked for validity and then used to compare diagnostic testing scenarios. The adjusted prevalence from the household survey for antigen, Wb123 Ab and Bm14 Ab were 4.4% (95% CI 2.6-7.3%), 8.7% (5.96-12.5%) and 20.8% (16.0-26.6%), respectively. For the three networks, the True Skill Statistic and Area Under the Receiver Operating Characteristic Curve for antigen, Wb123 and Bm14 Ab were 0.79, 0.68 and 0.55; and 0.97, 0.92 and 0.80, respectively. In the Bayesian network analysis, a positive case was defined as testing positive to one or more infection markers. A missed result was therefore the probability of a positive case having a negative test result to an alternate marker. The probability of a positive case prior to any testing scenario was 17.4%, 16.8% and 26.6% for antigen, Wb123 Ab and Bm14 Ab, respectively. In the antigen-only testing scenario, the probability of a missed positive LF result was 5.2% for Wb123 and 15.6% for Bm14 Ab. The combination of antigen plus Bm14 Ab testing reduced the probability of missing a positive LF case as measured by Wb123 Ab to 0.88%. The combination of antigen plus Wb123 Ab was less successful and yielded an 11.5% probability of a missed positive result by Bm14 Ab testing. Across scenarios, there was a greater discordance between Bm14 and both antigen and Wb123 Ab in the 1-10 age group compared to older ages. These findings suggest that the addition of Bm14 Ab improves the sensitivity of LF testing for current or past infection. The combination of antigen plus Bm14 Ab should therefore be considered for inclusion in post-MDA surveillance to improve the sensitivity of transmission surveys and prevent the premature cessation of MDA.

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., Competing Interests: The authors have declared that no competing interests exist.

Background: Control of human hookworm infection would be greatly aided by the development of an effective vaccine. We aimed to develop a live attenuated human hookworm vaccine., Methods: This was a two-part clinical trial done at Q-Pharm in Brisbane (QLD, Australia) using a live ultraviolet C (UVC)-attenuated Necator americanus larvae vaccine. Part one was an open-label, dose-finding study using 50 L3 larvae suspended in water to a volume of 200 μL, attenuated with UVC exposure of 700 μJ (L3-700) or 1000 μJ (L3-1000). Part two was a randomised, double-blind, placebo-controlled, challenge study, in which participants were randomly assigned 2:1 to the vaccine group or placebo group. Healthy hookworm-naive adults aged 18-65 years with body-mass index 18-35 kg/m 2 received two doses of either placebo (Tabasco sauce) or vaccine (50 L3-700) on day 1 and day 42, followed by challenge with 30 unattenuated L3 larvae to both groups. All participants received a single oral dose of 400 mg albendazole 4 weeks after each inoculation and a 3-day course (400 mg orally daily) initiated on day 161 after the challenge phase, to eliminate any remaining infection. The primary outcome of part 1 was the level of larval attenuation the resulted in a grade 2 or 3 dermal adverse event. The primary outcome of part 2 was safety and tolerability, assessed by frequency and severity of adverse events in all randomly assigned participants. Prespecified exploratory outcomes in the challenge study were faecal N americanus DNA concentration, the number of N americanus larvae recovered per g of faeces cultured, hookworm antigen-specific serum IgG antibody responses, and hookworm antigen-specific peripheral blood cytokine responses. The trial is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12617001007325)., Findings: Between Sept 19, 2017, and Oct 24, 2018, seven participants were enrolled into three cohorts in part one (two participants in cohort 1, who received L3-700; two participants in cohort 2, who received L3-700; and three participants in cohort 3, who received L3-1000) and a further 15 were enrolled into part two. There were no serious adverse events in part one or part two. In part one, a greater number of skin penetration sites were observed after administration of L3-700 than L3-1000 (mean 15·75 [95% CI 11·18 to 20·32] with L3-700 vs 4·33 [-1·40 to 10·07] with L3-1000). Similarly, greater erythema (median 225 mm 2 [IQR 150 to 325] vs 25 mm 2 [12·5 to 80]) and a longer duration of the dermal reaction (median 8·0 days [IQR 3·5 to 11·5] vs 2·0 days [2·0 to 4·5]) were observed after L3-700 than L3-1000. The mean number of adverse events per participant did not differ between the groups (3·25 [95% CI 1·48 to 5·02] vs 3·00 [1·04 to 4·96]). Thus, L3-700 was used for vaccination in part two. In part two, ten participants were randomly assigned to receive L3-700 and five to placebo. Significantly more adverse events occurred after vaccination with attenuated larvae than with placebo (incident rate ratio [IRR] 2·13 [95% CI 2·09 to 5·51]; p=0·0030). There was no difference between groups in the frequency of adverse events after challenge (IRR 1·25 [0·78 to 2·01]; p=0·36). Most adverse events were mild in severity, with only one severe adverse event reported (erythematous and indurated pruritic rash >100 mm in a vaccine group participant after challenge). The eosinophil count increased in all participants after challenge, with a significantly greater increase among vaccinated participants than placebo participants (1·55 × 10 9 cells per L [IQR 0·92 to 1·81] in the vaccine group vs 0·49 × 10 9 cells per L [0·43 to 0·63] in the placebo group; p=0·014). Vaccinated participants had an IgG response to larval extract after challenge that was higher than that in placebo participants (increase in IgG titre 0·22 [IQR 0·10 to 0·41] vs 0·03 [-0·40 to 0·06]; p=0·020). Significantly fewer larvae per g of faeces were recovered in the vaccine group than in the placebo group after challenge (median larvae per g 0·8 [IQR 0·00 to 3·91] vs 10·2 [5·1 to 18·1]; p=0·014). The concentration of N americanus DNA in faeces was not significantly different between the vaccinated group and the placebo group (log 10 DNA intensity 4·28 [95% CI 3·92 to 4·63] vs 4·88 [4·31 to 5·46]; p=0·14). Peripheral blood mononuclear cells from vaccinated participants exhibited significantly greater cytokine production at day 112 than placebo participants for IFNγ, TNFα, IL-2, IL-4, and IL-5 (p<0·05), but not IL-10., Interpretation: Vaccination with UVC-attenuated N americanus larvae is well tolerated, induces humoral and cellular responses to hookworm antigens, and reduces larval output after challenge with unattenuated larvae. Larger studies are required to confirm protective efficacy., Funding: National Health and Medical Research Council of Australia., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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]; AUC urine =0·96 [0·93-0·99]), and MS3&#95;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&#95;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&#95;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., Competing Interests: MSP, AL, and JS are inventors on a provisional patent filed by the funding body that captures the data presented in this study. All other authors declare no competing interests., (© 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license.)

Approximately 0.4 billion individuals worldwide are infected with hookworm. An effective vaccine is needed to not only improve the health of those affected and at high risk, but also to improve economic growth in disease-endemic areas. An ideal anti-hookworm therapeutic strategy for mass administration is a stable and orally administered vaccine. Oral vaccines are advantageous as they negate the need for trained medical staff for administration and do not require strict sterility conditions. Vaccination, therefore, can be carried out at a significantly reduced cost. One of the most promising current antigenic targets for hookworm vaccine development is the aspartic protease digestive enzyme (APR-1). Antibody-mediated neutralization of APR-1 deprives the worm of nourishment, leading to reduced worm burdens in vaccinated hosts. Previously, we demonstrated that, when incorporated into vaccine delivery systems, the APR-1-derived p3 epitope (TSLIAGPKAQVEAIQKYIGAEL) was able to greatly reduce worm burdens (≥90%) in BALB/c mice; however, multiple, large doses of the vaccine were required. Here, we investigated a variety of p3-antigen conjugates to optimize antigen delivery and establish immune response/protective efficacy relationships. We synthesized, purified, and characterized four p3 peptide-based vaccine candidates with: (a) lipidic (lipid core peptide (LCP)); (b) classical polymeric (polymethylacrylate (PMA)); and (c) novel polymeric (polyleucine in a branched or linear arrangement, BL 10 or LL 10 , respectively) groups as self-adjuvanting moieties. BL 10 and LL 10 induced the highest serum anti-p3 and anti-APR-1 IgG titers. Upon challenge with rodent hookworms, the highest significant reduction in worm burden was observed in mice immunized with LL 10 . APR-1-specific serum IgG titers correlated with worm burden reduction. Thus, we provide the first vaccine-triggered immune response-protection relationship for hookworm infection.

Despite the enormous morbidity attributed to schistosomiasis, there is still no vaccine to combat the disease for the hundreds of millions of infected people. The anthelmintic drug, praziquantel, is the mainstay treatment option, although its molecular mechanism of action remains poorly defined. Praziquantel treatment damages the outermost surface of the parasite, the tegument, liberating surface antigens from dying worms that invoke a robust immune response which in some subjects results in immunologic resistance to reinfection. Herein we term this phenomenon Drug-Induced Vaccination (DIV). To identify the antigenic targets of DIV antibodies in urogenital schistosomiasis, we constructed a recombinant proteome array consisting of approximately 1,000 proteins informed by various secretome datasets including validated proteomes and bioinformatic predictions. Arrays were screened with sera from human subjects treated with praziquantel and shown 18 months later to be either reinfected (chronically infected subjects, CI) or resistant to reinfection (DIV). IgG responses to numerous antigens were significantly elevated in DIV compared to CI subjects, and indeed IgG responses to some antigens were completely undetectable in CI subjects but robustly recognized by DIV subjects. One antigen in particular, a cystatin cysteine protease inhibitor stood out as a unique target of DIV IgG, so recombinant cystatin was produced, and its vaccine efficacy assessed in a heterologous Schistosoma mansoni mouse challenge model. While there was no significant impact of vaccination with adjuvanted cystatin on adult worm numbers, highly significant reductions in liver egg burdens (45-55%, P <0.0001) and intestinal egg burdens (50-54%, P <0.0003) were achieved in mice vaccinated with cystatin in two independent trials. This study has revealed numerous antigens that are targets of DIV antibodies in urogenital schistosomiasis and offer promise as subunit vaccine targets for a drug-linked vaccination approach to controlling schistosomiasis., Competing Interests: BG was employed by Ares Trading, S.A. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pearson, Tedla, Becker, Nakajima, Jasinskas, Mduluza, Mutapi, Oeuvray, Greco, Sotillo, Felgner and Loukas.)

Introduction: Celiac disease is an autoimmune disorder where intestinal immunopathology arises after gluten consumption. Previous studies suggested that hookworm infection restores gluten tolerance; however, these studies were small (n = 12) and not placebo controlled., Methods: We undertook a randomized, placebo-controlled trial of hookworm infection in 54 people with celiac disease. The 94-week study involved treatment with either 20 or 40 Necator americanus third-stage larvae (L3-20 or L3-40) or placebo, followed by escalating gluten consumption (50 mg/d for 12 weeks, 1 g intermittent twice weekly for 12 weeks, 2 g/d sustained for 6 weeks, liberal diet for 1 year)., Results: Successful study completion rates at week 42 (primary outcome) were similar in each group (placebo: 57%, L3-20: 37%, and L3-40: 44%; P = 0.61), however gluten-related adverse events were significantly reduced in hookworm-treated participants: Median (range) adverse events/participant were as follows: placebo, 4 (1-9); L3-20, 1 (0-9); and L3-40, 0 (0-3) (P = 0.019). Duodenal villous height:crypt depth deteriorated similarly compared with their enrolment values in each group (mean change [95% confidence interval]: placebo, -0.6 [-1.3 to 0.2]; L3-20, -0.5 [-0.8 to 0.2]; and L3-40, -1.1 [-1.8 to 0.4]; P = 0.12). A retrospective analysis revealed that 9 of the 40 L3-treated participants failed to establish hookworm infections. Although week 42 completion rates were similar in hookworm-positive vs hookworm-negative participants (48% vs 44%, P = 0.43), quality of life symptom scores were lower in hookworm-positive participants after intermittent gluten challenge (mean [95% confidence interval]: 38.9 [33.9-44] vs 45.9 [39.2-52.6])., Discussion: Hookworm infection does not restore tolerance to sustained moderate consumption of gluten (2 g/d) but was associated with improved symptom scores after intermittent consumption of lower, intermittent gluten doses.

Soil-transmitted helminths, including hookworms and whipworms, infect billions of people worldwide. Their capacity to penetrate and migrate through their hosts' tissues is influenced by the suite of molecules produced by the infective developmental stages. To facilitate a better understanding of the immunobiology and pathogenicity of human hookworms and whipworms, we investigated the metabolomes of the infective stage of Nippostrongylus brasiliensis third-stage larvae (L3) which penetrate the skin and Trichuris muris eggs which are orally ingested, using untargeted liquid chromatography-mass spectrometry (LC-MS). We identified 55 polar metabolites through Metabolomics Standard Initiative level-1 (MSI-I) identification from N. brasiliensis and T. muris infective stages, out of which seven were unique to excretory/secretory products (ESPs) of N. brasiliensis L3. Amino acids were a principal constituent (33 amino acids). Additionally, we identified 350 putative lipids, out of which 28 (all known lipids) were unique to N. brasiliensis L3 somatic extract and four to T. muris embryonated egg somatic extract. Glycerophospholipids and glycerolipids were the major lipid groups. The catalogue of metabolites identified in this study shed light on the biology, and possible therapeutic and diagnostic targets for the treatment of these critical infectious pathogens. Moreover, with the growing body of literature on the therapeutic utility of helminth ESPs for treating inflammatory diseases, a role for metabolites is likely but has received little attention thus far.

The ability of the parasitic blood fluke Schistosoma mansoni and other parasitic helminths to manipulate host biology is well recognised, but the mechanisms that underpin these phenomena are not well understood. An emerging paradigm is that helminths transfer their biological cargo to host cells by secretion of extracellular vesicles (EVs). Herein, we show that two populations of S. mansoni secreted EVs - exosome-like vesicles (ELVs) and microvesicles (MVs) - are actively internalised in two distinct human cell lines that reflect the resident cell types encountered by the parasite in vivo: human umbilical vein endothelial cells (HUVECs) and THP-1 monocytes. RNA-sequencing of HUVECs co-cultured with S. mansoni ELVs compared with untreated HUVECs revealed differential expression of genes associated with intravascular parasitism, including vascular endothelial contraction, coagulation, arachidonic acid metabolism and immune cell trafficking and signalling. Finally, we show that antibodies raised against recombinant tetraspanin (TSP) proteins from the surface of S. mansoni EVs significantly blocked EV uptake by both HUVECs and THP-1 monocytes whereas pre-immunisation antibodies did not. To our knowledge, this is the first evidence demonstrating the internalisation of secreted EVs from any helminth into vascular endothelial cells, providing novel insight into the potential mechanisms underlying host-schistosome interactions. The ability of anti-TSP antibodies to block vesicle uptake by host target cells further supports the potential of TSPs as promising antigens for an anti-fluke vaccine. It also suggests a potential mechanism whereby the current candidate human schistosomiasis vaccine, Sm-TSP-2, exerts its protective effect in animal models., (Copyright © 2020 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

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, Sm 23, 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.

Schistosomiasis is a neglected tropical disease caused by parasitic blood flukes of the genus Schistosoma , which kills 300,000 people every year in developing countries, and there is no vaccine. Recently, we have shown that cholinesterases (ChEs)-enzymes that regulate neurotransmission-from Schistosoma mansoni are expressed on the outer tegument surface and present in the excretory/secretory products of larval schistosomula and adult worms, and are essential for parasite survival in the definitive host, highlighting their utility as potential schistosomiasis vaccine targets. When treated in vitro with anti-schistosome cholinesterase ( Sm ChE) IgG, both schistosomula and adult worms displayed significantly decreased ChE activity, which eventually resulted in parasite death. Vaccination with individual Sm ChEs, or a combination of all three Sm ChEs, significantly reduced worm burdens in two independent trials compared to controls. Average adult worm numbers and liver egg burdens were significantly decreased for all vaccinated mice across both trials, with values of 29-39% and 13-46%, respectively, except for those vaccinated with Sm AChE1 in trial 1. Egg viability, as determined by egg hatching from liver homogenates, was significantly reduced in the groups vaccinated with the Sm ChE cocktail (40%) and Sm AChE2 (46%). Furthermore, surviving worms from each vaccinated group were significantly stunted and depleted of glycogen stores, compared to controls. These results suggest that Sm ChEs could be incorporated into a vaccine against schistosomiasis to reduce the pathology and transmission of this debilitating disease.

Helminth parasites secrete extracellular vesicles (EVs) into their environment that have potential roles in host-parasite communication, and thus represent potentially useful targets for novel control strategies. Here, we carried out a comprehensive proteomic analysis of two different populations of EVs - 15k pellet and 120k pellet EVs - from Schistosoma mansoni adult worms. We characterised the proteins present in the membranes of the EVs (including external trypsin-liberated peptides, integral membrane proteins (IMPs) and peripheral membrane proteins (PMPs)), as well as cargo proteins, using LC-MS/MS. A total of 286 and 716 proteins were identified in 15k and 120k pellets, respectively. Some of the most abundant proteins identified from both 15k and 120k pellets include known vaccine candidates such as Sm-TSP-2, saponin B domain-containing proteins, calpain glutathione-S-transferase, Sm29 and cathepsin domain-containing proteins. Other abundant proteins that have not been tested as vaccines include DM9 domain-containing protein, 13 kDa tegumental antigen and histone H4-like protein. Sm23, a member of the tetraspanin family with known vaccine efficacy, was identified in the cargo and IMP compartments of only 15k pellet vesicles. Moreover, a collection of proteins with known or potential relevance in host-parasite communication including proteases, antioxidants and EV biogenesis/trafficking of both vesicle types were identified. Our results provide the first report of a comprehensive compartmental proteomic analysis of adult S. mansoni-derived EVs. Future research should investigate recombinant forms of these proteins as vaccine and serodiagnostic antigens as well as the roles of EV proteins in host-parasite communication., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Hookworms cause a major neglected tropical disease, occurring after larvae penetrate the host skin. Neutrophils are phagocytes that kill large pathogens by releasing neutrophil extracellular traps (NETs), but whether they target hookworms during skin infection is unknown. Using a murine hookworm, Nippostrongylus brasiliensis, we observed neutrophils being rapidly recruited and deploying NETs around skin-penetrating larvae. Neutrophils depletion or NET inhibition altered larvae behavior and enhanced the number of adult worms following murine infection. Nevertheless, larvae were able to mitigate the effect of NETs by secreting a deoxyribonuclease (Nb-DNase II) to degrade the DNA backbone. Critically, neutrophils were able to kill larvae in vitro, which was enhanced by neutralizing Nb-DNase II. Homologs of Nb-DNase II are present in other nematodes, including the human hookworm, Necator americanus, which also evaded NETs in vitro. These findings highlight the importance of neutrophils in hookworm infection and a potential conserved mechanism of immune evasion., Competing Interests: Declaration of Interests A.L. and P.G. are shareholders in Paragen Bio Pty Ltd, a biotechnology company focusing on the use of hookworm proteins to treat inflammation., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

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&#95;154600 and Smache2 -smp&#95;136690) and a butyrylcholinesterase (BChE) (Smbche1 -smp&#95;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., Competing Interests: The authors have declared that no competing interests exist

Four healthy volunteers were infected with 50 Necator americanus infective larvae (L3) in a controlled human hookworm infection trial and followed for 52 weeks. The kinetics of fecal egg counts in volunteers was assessed with Bayesian multilevel analysis, which revealed an increase between weeks 7 and 13, followed by an egg density plateau of about 1000 eggs/g of feces. Variation in egg counts was minimal between same-day measurements but varied considerably between days, particularly during the plateau phase. These analyses pave the way for the controlled human hookworm model to accelerate drug and vaccine efficacy studies., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

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., Competing Interests: The authors have declared that no competing interests exist.

Iatrogenic hookworm therapy shows promise for treating disorders that result from a dysregulated immune system, including inflammatory bowel disease (IBD). Using a murine model of trinitrobenzenesulfonic acid-induced colitis and human peripheral blood mononuclear cells, we demonstrated that low-molecular-weight metabolites derived from both somatic extracts (LMWM-SE) and excretory-secretory products (LMWM-ESP) of the hookworm, Ancylostoma caninum , display anti-inflammatory properties. Administration to mice of LMWM-ESP as well as sequentially extracted fractions of LMWM-SE using both methanol (SE-MeOH) and hexane-dichloromethane-acetonitrile (SE-HDA) resulted in significant protection against T cell-mediated immunopathology, clinical signs of colitis, and impaired histological colon architecture. To assess bioactivity in human cells, we stimulated primary human leukocytes with lipopolysaccharide in the presence of hookworm extracts and showed that SE-HDA suppressed ex vivo production of inflammatory cytokines. Gas chromatography-mass spectrometry (MS) and liquid chromatography-MS analyses revealed the presence of 46 polar metabolites, 22 fatty acids, and five short-chain fatty acids (SCFAs) in the LMWM-SE fraction and 29 polar metabolites, 13 fatty acids, and six SCFAs in the LMWM-ESP fraction. Several of these small metabolites, notably the SCFAs, have been previously reported to have anti-inflammatory properties in various disease settings, including IBD. This is the first report showing that hookworms secrete small molecules with both ex vivo and in vivo anti-inflammatory bioactivity, and this warrants further exploration as a novel approach to the development of anti-inflammatory drugs inspired by coevolution of gut-dwelling hookworms with their vertebrate hosts., (Copyright © 2019 American Society for Microbiology.)

Background: Schistosomiasis affects over 200 million people and there are concerns whether the current chemotherapeutic control strategy (periodic mass drug administration with praziquantel (PZQ)-the only licenced anti-schistosome compound) is sustainable, necessitating the development of new drugs., Methodology/principal Findings: We 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). We 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., Conclusions/significance: Our data provide compelling evidence that ruthenium complexes are effective against all intra-mammalian stages of schistosomes, including schistosomula (refractory to PZQ) and eggs (agents of disease transmissibility). Further, the results of this study suggest that schistosome AChE is a target of ruthenium drugs, a finding that can inform modification of current compounds to identify analogues which are even more effective and selective against schistosomes.

When normal lymphatic function is hampered, imperceptible subcutaneous edema can develop and progress to overt lymphedema. Low-cost reliable devices for objective assessment of lymphedema are well accepted in clinical practice and research on breast-cancer related lymphedema but are untested in populations with lymphatic filariasis (LF). This is a cross-sectional analysis of baseline data in a longitudinal study on asymptomatic, LF antigen-positive and -negative young people in Myanmar. Rapid field screening was used to identify antigen-positive cases and a group of antigen-negative controls of similar age and gender were invited to continue in the study. Tissue compressibility was assessed with three tissue tonometers, and free fluids were assessed using bio-impedance spectroscopy (BIS). Infection status was confirmed by Og4C3 antigen assay. At baseline ( n = 98), antigen-positive cases had clinically relevant increases in tissue compressibility at the calf using a digital Indurometer (11.1%, p = 0.021), and in whole-leg free fluid using BIS (9.2%, p = 0.053). Regression analysis for moderating factors (age, gender, hydration) reinforced the between-infection group differences. Results demonstrate that sub-clinical changes associated with infection can be detected in asymptomatic cases. Further exploration of these low-cost devices in clinical and research settings on filariasis-related lymphedema are warranted., Competing Interests: The authors declare no conflict of interest.

Background: Whipworms and blood flukes combined infect almost one billion people in developing countries. Only a handful of anthelmintic drugs are currently available to treat these infections effectively; there is therefore an urgent need for new generations of anthelmintic compounds. Medicinal plants have presented as a viable source of new parasiticides. Ajania nubigena, the Bhutanese daisy, has been used in Bhutanese traditional medicine for treating various diseases and our previous studies revealed that small molecules from this plant have antimalarial properties. Encouraged by these findings, we screened four major compounds isolated from A. nubigena for their anthelmintic properties., Methodology/principal Findings: Here we studied four major compounds derived from A. nubigena for their anthelmintic properties against the nematode whipworm Trichuris muris and the platyhelminth blood fluke Schistosoma mansoni using the xWORM assay technique. Of four compounds tested, two compounds-luteolin (3) and (3R,6R)-linalool oxide acetate (1)-showed dual anthelmintic activity against S. mansoni (IC50 range = 5.8-36.9 μg/mL) and T. muris (IC50 range = 9.7-20.4 μg/mL). Using scanning electron microscopy, we determined luteolin as the most efficacious compound against both parasites and additionally was found effective against the schistosomula, the infective stage of S. mansoni (IC50 = 13.3 μg/mL). Luteolin induced tegumental damage to S. mansoni and affected the cuticle, bacillary bands and bacillary glands of T. muris. Our in vivo assessment of luteolin (3) against T. muris infection at a single oral dosing of 100 mg/kg, despite being significantly (27.6%) better than the untreated control group, was markedly weaker than mebendazole (93.1%) in reducing the worm burden in mice., Conclusions/significance: Among the four compounds tested, luteolin demonstrated the best broad-spectrum activity against two different helminths-T. muris and S. mansoni-and was effective against juvenile schistosomes, the stage that is refractory to the current gold standard drug, praziquantel. Medicinal chemistry optimisation including cytotoxicity analysis, analogue development and structure-activity relationship studies are warranted and could lead to the identification of more potent chemical entities for the control of parasitic helminths of humans and animals.

Herein we show for the first time that Schistosoma mansoni adult worms secrete exosome-like extracellular vesicles ranging from 50 to 130nm in size. Extracellular vesicles were collected from the excretory/secretory products of cultured adult flukes and purified by Optiprep density gradient, resulting in highly pure extracellular vesicle preparations as confirmed by transmission electron microscopy and Nanosight tracking analysis. Extracellular vesicle proteomic analysis showed numerous known vaccine candidates, potential virulence factors and molecules implicated in feeding. These findings provide new avenues for the exploration of host-schistosome interactions and offer a potential mechanism by which some vaccine antigens exert their protective efficacy., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Necator americanus (hookworm) infects over half a billion people worldwide. Anthelminthic drugs are commonly used to treat the infection; however, vaccination is a more favorable strategy to combat this parasite. We designed new B-cell peptide epitopes based on the aspartic protease of N. americanus (Na-APR-1). The peptides were conjugated to self-adjuvanting lipid core peptide (LCP) systems via stepwise solid-phase peptide synthesis (SPPS) and copper catalyst azide-alkyne cycloaddition (CuAAC) reactions. The LCP vaccine candidates were able to self-assemble into nanoparticles, were administered to mice without the use of additional adjuvant, and generated antibodies that recognized the parent epitope. However, only one LCP derivative was able to produce a high titer of antibodies specific to Na-APR-1; circular dichroism analyses of this compound showed a β-sheet conformation for the incorporated epitope. This study provides important insight in epitope and delivery system design for the development of a vaccine against hookworm infections., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The tegument of Schistosoma mansoni plays an integral role in host-parasite interactions, particularly during the transition from the free-living cercariae to the intra-mammalian schistosomula stages. This developmental period is characterised by the transition from a trilaminate surface to a heptalaminate tegument that plays key roles in immune evasion, nutrition and excretion. Proteins exposed at the surface membranes of newly transformed schistosomula are therefore thought to be prime targets for the development of new vaccines and drugs for schistosomiasis. Using a combination of tegumental labelling and high-throughput quantitative proteomics, more than 450 proteins were identified on the apical membrane of S. mansoni schistosomula, of which 200 had significantly regulated expression profiles at different stages of schistosomula development in vitro, including glucose transporters, sterols, heat shock proteins, antioxidant enzymes and peptidases. Current vaccine antigens were identified on the apical membrane (Sm-TSP-1, calpain) or sub-tegumental (Sm-TSP-2, Sm29) fractions of the schistosomula, displaying localisation patterns that, in some cases, differ from that in the adult stage fluke. This work provides the first known in-depth proteomic analysis of the surface-exposed proteins in the schistosomula tegument, and some of the proteins identified are clear targets for the generation of new vaccines and drugs against schistosomiasis., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Schistosoma haematobium affects more than 100 million people throughout Africa and is the causative agent of urogenital schistosomiasis. The parasite is strongly associated with urothelial cancer in infected individuals and as such is designated a group I carcinogen by the International Agency for Research on Cancer. Using a protein microarray containing schistosome proteins, we sought to identify antigens that were the targets of protective IgG1 immune responses in S. haematobium-exposed individuals that acquire drug-induced resistance (DIR) to schistosomiasis after praziquantel treatment. Numerous antigens with known vaccine potential were identified, including calpain (Smp80), tetraspanins, glutathione-S-transferases, and glucose transporters (SGTP1), as well as previously uncharacterized proteins. Reactive IgG1 responses were not elevated in exposed individuals who did not acquire DIR. To complement our human subjects study, we screened for antigen targets of rhesus macaques rendered resistant to S. japonicum by experimental infection followed by self-cure, and discovered a number of new and known vaccine targets, including major targets recognized by our human subjects. This study has further validated the immunomics-based approach to schistosomiasis vaccine antigen discovery and identified numerous novel potential vaccine antigens.

Background: As part of the Global Programme to Eliminate Lymphatic Filariasis (LF), American Samoa conducted mass drug administration (MDA) from 2000-2006, and passed transmission assessment surveys in 2011-2012. We examined the seroprevalence and spatial epidemiology of LF post-MDA to inform strategies for ongoing surveillance and to reduce resurgence risk., Methods: ELISA for LF antigen (Og4C3) and antibodies (Wb123, Bm14) were performed on a geo-referenced serum bank of 807 adults collected in 2010. Risk factors assessed for association with sero-positivity included age, sex, years lived in American Samoa, and occupation. Geographic clustering of serological indicators was investigated to identify spatial dependence and household-level clustering., Results: Og4C3 antigen of >128 units (positive) were found in 0.75% (95% CI 0.3-1.6%) of participants, and >32 units (equivocal plus positive) in 3.2% (95% CI 0.6-4.7%). Seroprevalence of Wb123 and Bm14 antibodies were 8.1% (95% CI 6.3-10.2%) and 17.9% (95% CI 15.3-20.7%) respectively. Antigen-positive individuals were identified in all ages, and antibody prevalence higher in older ages. Prevalence was higher in males, and inversely associated with years lived in American Samoa. Spatial distribution of individuals varied significantly with positive and equivocal levels of Og4C3 antigen, but not with antibodies. Using Og4C3 cutoff points of >128 units and >32 units, average cluster sizes were 1,242 m and 1,498 m, and geographical proximity of households explained 85% and 62% of the spatial variation respectively., Conclusions: High-risk populations for LF in American Samoa include adult males and recent migrants. We identified locations and estimated the size of possible residual foci of antigen-positive adults, demonstrating the value of spatial analysis in post-MDA surveillance. Strategies to monitor cluster residents and high-risk groups are needed to reduce resurgence risk. Further research is required to quantify factors contributing to LF transmission at the last stages of elimination to ensure that programme achievements are sustained.

Onchocerca lupi is a neglected filarioid causing nodular lesions associated with acute or chronic ocular disease in dogs. Despite the recent appraisal of its zoonotic potential, human cases are increasingly reported in the Old and New Worlds. Therefore, the development of accurate tools for the rapid diagnosis of O. lupi infections in dogs is becoming a priority. In this study, we conducted a preliminary investigation aimed at evaluating the usefulness of a commercially available ELISA test for the detection of O. lupi antigens in canine sera. The potential use of this tool for larger epidemiological studies of canine onchocerciasis is discussed.