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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. A beneficial effect of parasitic worms on metabolic health has been postulated based on epidemiological and animal studies. Here, the authors show in a phase I clinical trial that treatment of people at risk of type 2 diabetes with hookworms is safe and may improve key measures of metabolic health. [ABSTRACT FROM AUTHOR]

Background: The human hookworm, Necator americanus, is a parasite that infects almost half a billion people worldwide. Although treatment is available, vaccination is favorable to combat the spread of this parasite due to its wide distribution and continuous reinfection cycle in endemic communities.Methods: We have designed a lipopeptide oral delivery system using a B-cell epitope derived from the aspartic protease Na-APR-1 from N americanus, attached to a T-helper epitope. Lipopeptides were self-assembled into nanoparticles or entrapped in liposomes that were electrostatically coated with alginate and trimethyl chitosan polymer shields. The adjuvant-free vaccine candidates were orally administered to mice and generated a humoral immune response against both peptide antigen, and the parent protein in the hookworm gut.Results: The vaccine candidates were evaluated in a rodent hookworm challenge model, resulting in up to 98% and 99% decreases in mean intestinal worm and egg burdens in immunized mice, respectively.Conclusions: Lipopeptide survived the gastrointestinal conditions, induced humoral immune responses and drived protection against parasite challenge infection. [ABSTRACT FROM AUTHOR]

Background: Abdominal obesity and presence of the metabolic syndrome (MetS) significantly increase the risk of developing diseases such as Type 2 diabetes mellitus (T2DM) with escalating emergence of MetS and T2DM constituting a significant public health crisis worldwide. Lower prevalence of inflammatory and metabolic diseases such as T2DM in countries with higher incidences of helminth infections suggested a potential role for these parasites in the prevention and management of certain diseases. Recent studies confirmed the potential protective nature of helminth infection against MetS and T2DM via immunomodulation or, potentially, alteration of the intestinal microbiota. This Phase 1b safety and tolerability trial aims to assess the effect of inoculation with helminths on physical and metabolic parameters, immune responses, and the microbiome in otherwise healthy women and men. Methods: Participants eligible for inclusion are adults aged 18–50 with central obesity and a minimum of one additional feature of MetS recruited from the local community with a recruitment target of 54. In a randomised, double-blind, placebo-controlled design, three groups will receive either 20 or 40 stage three larvae of the human hookworm Necator americanus or a placebo. Eligible participants will provide blood and faecal samples at their baseline and 6-monthly assessment visits for a total of 24 months with an optional extension to 36 months. During each scheduled visit, participants will also undergo a full physical examination and complete diet (PREDIMED), physical activity, and patient health (PHQ-9) questionnaires. Outcome measurements include tolerability and safety of infection with Necator americanus, changes in metabolic and immunological parameters, and changes in the composition of the faecal microbiome. Discussion: Rising cost of healthcare associated with obesity-induced metabolic diseases urgently calls for new approaches in disease prevention. Findings from this trial will provide valuable information regarding the potential mechanisms by which hookworms, potentially via alterations in the microbiota, may positively influence metabolic health. Trial registration: The protocol was registered on ANZCTR.org.au on 05 June 2017 with identifier ACTRN12617000818336. Alternatively, a Google search using the above trial registration number will yield a direct link to the trial protocol within the ANZCTR website. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 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 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. [ABSTRACT FROM AUTHOR]

Gastrointestinal (GI) parasites, hookworms in particular, have evolved to cause minimal harm to their hosts, allowing them to establish chronic infections. This is mediated by creating an immunoregulatory environment. Indeed, hookworms are such potent suppressors of inflammation that they have been used in clinical trials to treat inflammatory bowel diseases (IBD) and celiac disease. Since the recent description of helminths (worms) secreting extracellular vesicles (EVs), exosome-like EVs from different helminths have been characterized and their salient roles in parasite--host interactions have been highlighted. Here, we analyze EVs from the rodent parasite Nippostrongylus brasiliensis, which has been used as a model for human hookworm infection. N. brasiliensis EVs (Nb-EVs) are actively internalized by mouse gut organoids, indicating a role in driving parasitism. We used proteomics and RNA-Seq to profile the molecular composition of Nb-EVs. We identified 81 proteins, including proteins frequently present in exosomes (like tetraspanin, enolase, 14-3-3 protein, and heat shock proteins), and 27 sperm-coating protein-like extracellular proteins. RNA-Seq analysis revealed 52 miRNA species, many of which putatively map to mouse genes involved in regulation of inflammation. To determine whether GI nematode EVs had immunomodulatory properties, we assessed their potential to suppress GI inflammation in a mouse model of inducible chemical colitis. EVs from N. brasiliensis but not those from the whipworm Trichuris muris or control vesicles from grapes protected against colitic inflammation in the gut of mice that received a single intraperitoneal injection of EVs. Key cytokines associated with colitic pathology (IL-6, IL-1β, IFNγ, and IL-17a) were significantly suppressed in colon tissues from EV-treated mice. By contrast, high levels of the anti-inflammatory cytokine IL-10 were detected in Nb-EV-treated mice. Proteins and miRNAs contained within helminth EVs hold great potential application in development of drugs to treat helminth infections as well as chronic non-infectious diseases resulting from a dysregulated immune system, such as IBD. [ABSTRACT FROM AUTHOR]

Hookworms produce a vast repertoire of structurally and functionally diverse molecules that mediate their long-term survival and pathogenesis within a human host. Many of these molecules are secreted by the parasite, after which they interact with critical components of host biology, including processes that are key to host survival. The most important of these interactions is the hookworm’s interruption of nutrient acquisition by the host through its ingestion and digestion of host blood. This results in iron deficiency and eventually the microcytic hypochromic anemia or iron deficiency anemia that is the clinical hallmark of hookworm infection. Other molecular mechanisms of hookworm infection cause a systematic suppression of the host immune response to both the parasite and to bystander antigens (eg, vaccines or allergens). This is achieved by a series of molecules that assist the parasite in the stealthy evasion of the host immune response. This review will summarize the current knowledge of the molecular mechanisms used by hookworms to survive for extended periods in the human host (up to 7 years or longer) and examine the pivotal contributions of these molecular mechanisms to chronic hookworm parasitism and host clinical outcomes. [ABSTRACT FROM AUTHOR]

The large extracellular loop of the Schistosoma mansoni tetraspanin, Sm-TSP-2, when fused to a thioredoxin partner and formulated with Freund's adjuvants, has been shown to be an efficacious vaccine against murine schistosomiasis. Moreover, Sm-TSP-2 is uniquely recognised by IgG1 and IgG3 from putatively resistant individuals resident in S. mansoni endemic areas in Brazil. In the present study, we expressed Sm-TSP-2 at high yield and in soluble form in E. coli without the need for a solubility enhancing fusion partner. We also expressed in E. coli a chimera called Sm-TSP-2/5B, which consisted of Sm-TSP-2 fused to the immunogenic 5B region of the hookworm aspartic protease and vaccine antigen, Na-APR-1. Sm-TSP-2 formulated with alum/CpG showed significant reductions in adult worm and liver egg burdens in two separate murine schistosomiasis challenge studies. Sm-TSP-2/5B afforded significantly greater protection than Sm-TSP-2 alone when both antigens were formulated with alum/CpG. The enhanced protection obtained with the chimeric fusion protein was associated with increased production of anti-Sm-TSP-2 antibodies and IL-4, IL-10 and IFN-γ from spleen cells of vaccinated animals. Sera from 666 individuals from Brazil who were infected with S. mansoni were screened for potentially deleterious IgE responses to Sm-TSP-2. Anti-Sm-TSP-2 IgE to this protein was not detected (also shown previously for Na-APR-1), suggesting that the chimeric antigen Sm-TSP-2/5B could be used to safely and effectively vaccinate people in areas where schistosomes and hookworms are endemic. Author Summary: There are currently no vaccines available to combat helminth (worm) infections in humans. The most devastating of the diseases caused by human helminths are schistosomiasis (or bilharzia) and hookworm disease. By fusing one of the lead schistosomiasis vaccine antigens, Sm-TSP-2, with a protective fragment from one of the lead hookworm vaccine antigens, Na-APR-1, we have produced a chimeric vaccine, termed Sm-TSP-2/5B that might provide protection against two debilitating and co-endemic neglected tropical diseases. Sm-TSP-2/5B provided increased protection compared to Sm-TSP-2 alone when formulated with human approved adjuvants and tested in a mouse model of schistosomiasis. Moreover, IgE against Sm-TSP-2 or Na-APR-1 has not been detected in the blood of residents from an area in Brazil that is endemic for schistosomes and hookworms, indicating that vaccines based on these molecules would be unlikely to generate allergic reactions in recipients from developing countries. [ABSTRACT FROM AUTHOR]

We present immunological data from two clinical trials where the effect of experimental human hookworm (Necator americanus) infection on the pathology of celiac disease was evaluated. We found that basal production of Interferon- (IFN-)&ggr; and Interleukin- (IL-)17A from duodenal biopsy culture was suppressed in hookworm-infected participants compared to uninfected controls. Increased levels of CD4+CD25+Foxp3+ cells in the circulation andmucosa are associated with active celiac disease. We show that this accumulation also occurs during a short-term (1 week) oral gluten challenge, and that hookworm infection suppressed the increase of circulating CD4+CD25+Foxp3+ cells during this challenge period. When duodenal biopsies from hookworm-infected participants were restimulated with the immunodominant gliadin peptide QE65, robust production of IL-2, IFN-c and IL-17A was detected, even prior to gluten challenge while participants were strictly adhering to a gluten-free diet. Intriguingly, IL-5 was produced only after hookworm infection in response to QE65. Thus we hypothesise that hookworm-induced TH2 and IL-10 cross-regulation of the TH1/TH17 inflammatory response may be responsible for the suppression of these responses during experimental hookworm infection. [ABSTRACT FROM AUTHOR]

Background: Helminth parasites cause untold morbidity and mortality to billions of people and livestock. Anthelmintic drugs are available but resistance is a problem in livestock parasites, and is a looming threat for human helminths. Testing the efficacy of available anthelmintic drugs and development of new drugs is hindered by the lack of objective high-throughput screening methods. Currently, drug effect is assessed by observing motility or development of parasites using laborious, subjective, low-throughput methods. Methodology/Principal Findings: Here we describe a novel application for a real-time cell monitoring device (xCELLigence) that can simply and objectively assess anthelmintic effects by measuring parasite motility in real time in a fully automated high-throughput fashion. We quantitatively assessed motility and determined real time IC50 values of different anthelmintic drugs against several developmental stages of major helminth pathogens of humans and livestock, including larval Haemonchus contortus and Strongyloides ratti, and adult hookworms and blood flukes. The assay enabled quantification of the onset of egg hatching in real time, and the impact of drugs on hatch rate, as well as discriminating between the effects of drugs on motility of drug-susceptible and –resistant isolates of H. contortus. Conclusions/Significance: Our findings indicate that this technique will be suitable for discovery and development of new anthelmintic drugs as well as for detection of phenotypic resistance to existing drugs for the majority of helminths and other pathogens where motility is a measure of pathogen viability. The method is also amenable to use for other purposes where motility is assessed, such as gene silencing or antibody-mediated killing. Author Summary: Parasitic worms cause untold morbidity and mortality on billions of people and livestock. Drugs are available but resistance is problematic in livestock parasites and is a looming threat for human helminths. Currently, new drug discovery and resistance monitoring is hindered as drug efficacy is assessed by observing motility or development of parasites using laborious, subjective, low-throughput methods evaluated by eye using microscopy. Here we describe a novel application for a cell monitoring device (xCELLigence) that can simply and objectively assess real time anti-parasite efficacy of drugs on eggs, larvae and adults in a fully automated, label-free, high-throughput fashion. This technique overcomes the current low-throughput bottleneck in anthelmintic drug development and resistance detection pipelines. The widespread use of this device to screen for new therapeutics or emerging drug resistance will be an invaluable asset in the fight against human, animal and plant parasitic helminths and other pathogens that plague our planet. [ABSTRACT FROM AUTHOR]

Hookworm infection and schistosomiasis rank among the most important health problems in developing countries. Both cause anaemia and malnutrition, and schistosomiasis also results in substantial intestinal, liver and genitourinary pathology. In sub-Saharan Africa and Brazil, co-infections with the hookworm, Necator americanus, and the intestinal schistosome, Schistosoma mansoni, are common. The development of vaccines for these infections could substantially reduce the global disability associated with these helminthiases. New genomic, proteomic, immunological and X-ray crystallographic data have led to the discovery of several promising candidate vaccine antigens. Here, we describe recent progress in this field and the rationale for vaccine development. [ABSTRACT FROM AUTHOR]

The aspartic protease of Necator americanus, Na-APR-1, is a vaccine antigen that induces antibodies that neutralize hemoglobin proteolysis in the gut of the worm. To define the epitopes recognized by these antibodies, monoclonal antibodies (mAbs) were raised and assessed for neutralizing activity. Three immunoglobulin (Ig) G1 mAbs bound to the intestine of N. americanus and inhibited Na-APR-1 enzymatic activity. Overlapping fragments of Na-APR-1 were expressed, and one (APR-1/5B) was recognized by all 3 mAbs; the epitope was further characterized as AGPKAQVEAIQKY (A291Y). This same peptide with a Phe/Tyr303 substitution was recognized by mAbs in APR-1 orthologues from Ancylostoma species hookworms. IgG from humans infected with hookworms did not recognize A291Y but, rather, recognized the S107L epitope. APR-1/5B was fused to other helminth vaccine antigens, including Schistosoma mansoni Sm-TSP-2 and N. americanus Na-GST-1; antibodies against both chimeras neutralized the enzymatic activity of Na-APR-1. These findings support the incorporation of Na-APR-1 into a multivalent vaccine against hookworm and/or schistosomiasis. [ABSTRACT FROM AUTHOR]

Hookworms digest hemoglobin from erythrocytes via a proteolytic cascade that begins with the aspartic protease, APR-1. Ac-APR-1 from the dog hookworm, Ancylostoma caninum, protects dogs against hookworm infection via antibodies that neutralize enzymatic activity and interrupt blood-feeding. Toward developing a human hookworm vaccine, we expressed both wild-type (Nα-APR-1wt) and mutant (Nα-APR-1mut--mutagenesis of the catalytic aspartic acids) forms of Nα-APR-1 from the human hookworm, Necator americanus Refolded Nα-APR-1wt was catalytically active, and Nα-APR-1mut was catalytically inactive but still bound substrates. Vaccination of canines with Nα-APR-1mut and heterologous challenge with A. caninum resulted in significantly reduced parasite egg burdens (P=0.034) and weight loss (P=0.022). Vaccinated dogs also had less gut pathology, fewer adult worms, and reduced blood loss compared to controls but these did not reach statistical significance. Vaccination with Nα-APR-1mut induced antibodies that bound the native enzyme in the parasite gut and neutralized enzymatic activity of Nα-APR-1wt and APR-1 orthologues from three other hookworm species that infect humans. IgG1 against Nα-APR-1mut was the most prominently detected antibody in sera from people resident in high-transmission areas for N. americanus, indicating that natural boosting may occur in exposed humans. Nα-APR-1mut is now a lead antigen for the development of an antihematophagy vaccine for human hookworm disease. [ABSTRACT FROM AUTHOR]

Blood-feeding parasites use mechanistically distinct proteases to digest hemoglobin (Hb), often as multienzyme cooperative cascades. We investigated the roles played by 3 distinct proteases from adults of the human hookworm Necator americanus. The aspartic protease Na-APR-1 and the cysteine protease Na-CP-3 were expressed in catalytically active form in yeast, and the metalloprotease Na-MEP-1 was expressed in catalytically active form in baculovirus. Antibodies to all 3 proteases were used to immunolocalize each native enzyme to the intestine of adult N. americanus. Recombinant Na-APR-1 cleaved intact human Hb. In contrast, Na-CP-3 and Na-MEP-1 could not cleave Hb but instead cleaved globin fragments that had been hydrolyzed by Na-APR-1, implying an ordered process of hemoglobinolysis. Seventy-four cleavage sites within Hb α- and β-chains were characterized after digestion with all 3 proteases. All of the proteases demonstrated promiscuous subsite specificities within Hb; noteworthy preferences included aromatic and hydrophobic P1 residues and hydrophobic P1' residues for Na- APR-1 and hydrophobic P1 residues for Na-MEP-1. We conclude that Hb digestion in N. americanus involves a network of distinct proteases, some of which act in an ordered fashion, providing a potential mechanism by which some of these hemoglobinases exert their efficacy as recombinant vaccines against hookworm infection. [ABSTRACT FROM AUTHOR]

Hookworm infection and schistosomiasis are two of the world's most important human parasitic infections, affecting hundreds of millions of people in developing countries. Measured together in disability-adjusted life years, hookworm infection and schistosomiasis rank closely behind malaria as the most prevalent human parasitic diseases. A major approach for the control of these two helminth infections relies on periodic, mass chemotherapy with anthelminthics. However, high rates of post-treatment reinfection, the declining efficacy with repeated treatment, rebound morbidity (in the case of schistosomiasis) and the potential for the emergence of anthelminthic drug resistance threaten the sustainability of mass drug administration as the only form of control. Hence, there is a strong rationale for developing a vaccine that simultaneously targets both hookworms and schistosomes because of similarities in the pathobiology of both parasites, the ability of both helminths to cause anemia and their coendemicity in sub-Saharan Africa, Brazil and East Asia. A multivalent anthelminthic vaccine for hookworm infection and schistosomiasis would represent an important new tool for combating disease and poverty. [ABSTRACT FROM AUTHOR]

Background: Hookworms infect 730 million people in developing countries where they are a leading cause of intestinal blood loss and iron-deficiency anemia. At the site of attachment to the host, adult hookworms ingest blood and lyse the erythrocytes to release hemoglobin. The parasites subsequently digest hemoglobin in their intestines using a cascade of proteolysis that begins with the Ancylostoma caninum aspartic protease 1, APR-1. Methods and Findings: We show that vaccination of dogs with recombinant Ac-APR-1 induced antibody and cellular responses and resulted in significantly reduced hookworm burdens (p = 0.056) and fecal egg counts (p = 0.018) in vaccinated dogs compared to control dogs after challenge with infective larvae of A. caninum. Most importantly, vaccinated dogs were protected against blood loss (p = 0.049) and most did not develop anemia, the major pathologic sequela of hookworm disease. IgG from vaccinated animals decreased the catalytic activity of the recombinant enzyme in vitro and the antibody bound in situ to the intestines of worms recovered from vaccinated dogs, implying that the vaccine interferes with the parasite's ability to digest blood. Conclusion: To the best of our knowledge, this is the first report of a recombinant vaccine from a hematophagous parasite that significantly reduces both parasite load and blood loss, and it supports the development of APR-1 as a human hookworm vaccine. [ABSTRACT FROM AUTHOR]

Objective: To describe a zoonotic ancylostomiasis (canine), acquired from domestic pets by patients living in developed, urban communities.Design: An 8-year, retrospective case study.Setting: A clinical gastroenterologic practice in Townsville and a university parasitology department in Brisbane, Australia.Patients: Nine patients, each with enteric hookworm infection diagnosed by finding a single organism in situ; five were treated by us, and the rest were referred to us for parasite identification.Measurements: Clinical and demographic data, complete blood examinations, total serum immunoglobulin E assay, and serologic testing with enzyme-linked immunosorbent assay and Western blot using excretory-secretory antigens of Ancylostoma caninum. Gut biopsy specimens were examined histologically, and hookworms were identified using morphologic criteria.Results: The infections in three of the patients were diagnosed during the initial 6 years and six in the last 2 years. All owned a dog and described activity potentially exposing them to infection with canine hookworm larvae. Three patients had a laparotomy for acute abdominal pain, and six had colonoscopies (five with pain and one without symptoms). Six of the nine had blood eosinophilia (mean, 0.97 x 10(9)/L), and five of eight had elevated immunoglobulin E levels (mean level, 756 micrograms/L); six of eight had eosinophilic inflammation of the gut. In six patients, the worm was identified as A. caninum, whereas in three, damage to the specimen did not allow specific identification; however, they were unlikely to be human parasite species. Although all parasites were in the adult stage, none were sexually mature. Positive serologic findings in seven of the eight patients tested confirmed presence of antibody to the parasite.Conclusions: Human enteric infections with A. caninum are being diagnosed more frequently in northeastern Australia. Although infection may be subclinical, the chief symptom is abdominal pain, sometimes sudden and severe. The pathologic finding is focal or diffuse eosinophilic inflammation caused by a type 1 hypersensitivity response to secreted antigens. Infection by sexually immature worms is scant and nonpatent, indicating poor adaptation to the human host. Serologic testing assists in identification of occult infection. Advanced hygiene and sanitation afford little protection because the parasite reservoir is a large and growing pool of infected domestic pets. [ABSTRACT FROM AUTHOR]

Highlights findings of a study by Jeffrey Bethony and colleagues which examined the antibody responses of individuals from hookworm endemic areas of Brazil and China against antigens secreted by third-stage infective hookwork larvae, and Ancylostoma Secreted Proteins (ASP) 1 and 2. Cloning and expression of ASPs; Association between the risk of hookworm infection and increased levels of IgG4 anti-ASP-2; In vitro and in vivo effects of anti-ASP-2 antibody in canine vaccination with ASP-2.

The intestinal microbiota plays a critical role in the development of the immune system. Recent investigations have highlighted the potential of helminth therapy for treating a range of inflammatory disorders, including celiac disease (CeD); however, the mechanisms by which helminths modulate the immune response of the human host and ameliorate CeD pathology are unknown. In this study, we investigated the potential role of alterations in the human gut microbiota in helminth-mediated suppression of an inflammatory disease. We assessed the qualitative and quantitative changes in the microbiota of human volunteers with CeD prior to and following infection with human hookworms, and following challenge with escalating doses of dietary gluten. Experimental hookworm infection of the trial subjects resulted in maintenance of the composition of the intestinal flora, even after a moderate gluten challenge. Notably, we observed a significant increase in microbial species richness over the course of the trial, which could represent a potential mechanism by which hookworms can regulate gluten-induced inflammation and maintain intestinal immune homeostasis. [ABSTRACT FROM AUTHOR]

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/m2 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 mm2 [IQR 150 to 325] vs 25 mm2 [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 × 109 cells per L [IQR 0·92 to 1·81] in the vaccine group vs 0·49 × 109 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 (log10 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. [ABSTRACT FROM AUTHOR]

The prevalence of autoimmune diseases is on the rise globally. Currently, autoimmunity presents in over 100 different forms and affects around 9% of the world's population. Current treatments available for autoimmune diseases are inadequate, expensive, and tend to focus on symptommanagement rather than cure. Clinical trials have shown that live helminthic therapy can decrease chronic inflammation associated with inflammatory bowel disease and other gastrointestinal autoimmune inflammatory conditions. As an alternative and better controlled approach to live infection, we have identified and characterized two peptides, Acan1 and Nak1, from the excretory/secretory component of parasitic hookworms for their therapeutic activity on experimental colitis. We synthesizedAcan1 and Nak1 peptides fromthe Ancylostoma caninum and Necator americanus hookworms and assessed their structures and protective properties in human cell-based assays and in a mouse model of acute colitis. Acan1 and Nak1 displayed anticolitic properties via significantly reducing weight loss and colon atrophy, edema, ulceration, and necrosis in 2,4,6-trinitrobenzene sulfonic acid-exposed mice. These hookworm peptides prevented mucosal loss of goblet cells and preserved intestinal architecture. Acan1 upregulated genes responsible for the repair and restitution of ulcerated epithelium, whereas Nak1 downregulated genes responsible for epithelial cell migration and apoptotic cell signaling within the colon. These peptides were nontoxic and displayed key immunomodulatory functions in human peripheral blood mononuclear cells by suppressing CD4+ T cell proliferation and inhibiting IL-2 and TNF production. We conclude that Acan1 and Nak1 warrant further development as therapeutics for the treatment of autoimmunity, particularly gastrointestinal inflammatory conditions. [ABSTRACT FROM AUTHOR]

Abstract: Hookworm activation-associated secreted proteins can be structurally classified into at least three different groups. The hallmark feature of Group 1 activation-associated secreted proteins is a prominent equatorial groove, which is inferred to form a ligand binding site. Furthermore, a conserved tandem histidine motif is located in the centre of the groove and believed to provide or support a yet to be determined catalytic activity. Here, we report three-dimensional crystal structures of Na-ASP-2, an L3-secreted activation-associated secreted protein from the human hookworm Necator americanus, which demonstrate transition metal binding ability of the conserved tandem histidine motif. We further identified moderate phosphohydrolase activity of recombinant Na-ASP-2, which relates to the tandem histidine motif. By panning a random 12-mer peptide phage library, we identified a peptide with high similarity to the human calcium-activated potassium channel SK3, and confirm binding of the synthetic peptide to recombinant Na-ASP-2 by differential scanning fluorimetry. Potential binding modes of the peptide to Na-ASP-2 were studied by molecular dynamics simulations which clearly identify a preferred topology of the Na-ASP-2:SK3 peptide complex. [Copyright &y& Elsevier]

Abstract: mRNAs encoding cathepsin B-like cysteine proteases (CatBs) are abundantly expressed in the genomes of blood-feeding nematodes. Recombinant CatBs have been partially efficacious in vaccine trials in animal models of hookworm infection, supporting further investigation of these enzymes as new control tools. We recently described a family of four distinct CatBs (Na-CP-2, -3, -4, -5) from the human hookworm, Necator americanus. Here we show that these N. americanus CatBs form a robust clade with other hookworm CatBs and are most similar to intestinal CatBs from Haemonchus contortus. All four mRNAs (Na-cp-2, -3, -4 and -5) are up-regulated during the transition from a free-living larva to a blood-feeding adult worm and are also expressed in gut tissue of adult N. americanus that was dissected using laser microdissection microscopy. Recombinant Na-CP-3 was expressed in soluble, secreted form in the yeast Pichia pastoris, while Na-CP-2, -4 and -5 were expressed in insoluble inclusion bodies in Escherichia coli. Recombinant Na-CP-3 was not catalytically active when secreted by yeast but underwent auto-activation to an active enzyme at low pH in the presence of dextran sulphate. Activated Na-CP-3 digested gelatin and cleaved the fluorogenic substrate Z-Phe-Arg-aminomethylcoumarin (AMC) but not Z-Arg-Arg-AMC. Recombinant Na-CP-3 did not digest intact hemoglobin but digested globin fragments generated by prior hydrolysis with N. americanus aspartic hemoglobinases. Antibodies raised in mice to all four recombinant proteins showed minimal cross-reactivity with each other, and each antiserum bound to the intestine of adult N. americanus, supporting the intestinal expression of their mRNAs. These data show that N. americanus expresses a family of intestinal CatBs, many of which are likely to be involved in nutrient acquisition and therefore are potential targets for chemotherapies and vaccines. [Copyright &y& Elsevier]

Abstract: We have previously reported the successful adaptation of human hookworm Necator americanus in the golden hamster, Mesocricetus auratus. This animal model was used to test a battery of hookworm (N. americanus and Ancylostoma caninum) recombinant antigens as potential vaccine antigens. Hamsters immunized a leading vaccine candidate N. americanus–Ancylostoma secreted protein 2 (Na-ASP-2) and challenged with N. americanus infective larvae (L3), resulted in 30–46.2% worm reduction over the course of three vaccine trials, relative to adjuvant controls. In addition, significant reduction of worm burdens was also observed in the hamsters immunized with adult hookworm antigens A. caninum aspartic protease 1 (Ac-APR-1); A. caninum-glutathione-S transferase 1 (Ac-GST-1) and Necator cysteine proteases 2 (Na-CP-2) (44.4%, 50.6%, and 29.3%, respectively). Our data on the worm burden reductions afforded by these hookworm antigens approximate the level of protection reported previously from dogs challenged with A. caninum L3, and provide additional evidence to support these hookworm antigens as vaccine candidates for human hookworm infection. The hamster model of N. americanus provides useful information for the selection of antigens to be tested in downstream vaccine development. [Copyright &y& Elsevier]

Summary: The three main soil-transmitted helminth infections, ascariasis, trichuriasis, and hookworm, are common clinical disorders in man. The gastrointestinal tract of a child living in poverty in a less developed country is likely to be parasitised with at least one, and in many cases all three soil-transmitted helminths, with resultant impairments in physical, intellectual, and cognitive development. The benzimidazole anthelmintics, mebendazole and albendazole, are commonly used to remove these infections. The use of these drugs is not limited to treatment of symptomatic soil-transmitted helminth infections, but also for large-scale prevention of morbidity in children living in endemic areas. As a result of data showing improvements in child health and education after deworming, and the burden of disease attributed to soil-transmitted helminths, the worldwide community is awakening to the importance of these infections. Concerns about the sustainability of periodic deworming with benzimidazole anthelmintics and the emergence of resistance have prompted efforts to develop and test new control tools. [ABSTRACT FROM AUTHOR]

Human hookworm infection is a major cause of anemia and malnutrition of adults and children in the developing world. As part of on-going efforts to control hookworm infection, The Human Hookworm Vaccine Initiative has identified candidate vaccine antigens from the infective L3 larval stages of the parasite, including a family of pathogenesis-related (PR) proteins known as the Ancylostoma-secreted proteins (ASPs). A novel crystal structure of Na-ASP-2, a PR-1 protein secreted by infective larvae of the human hookworm Necator americanus, has been solved to resolution limits of 1.68Å and to an R-factor of 17% using the recombinant protein expressed in and secreted by Pichia pastoris. The overall fold of Na-ASP-2 is a three-layer αβα sandwich flanked by an N-terminal loop and a short, cysteine-rich C terminus. Our structure reveals a large central cavity that is flanked by His129 and Glu106, two residues that are well conserved in all parasitic nematode L3 ASPs. Na-ASP-2 has structural and charge similarities to chemokines, which suggests that Na-ASP-2 may be an extra-cellular ligand of an unknown receptor. Na-ASP-2 is a useful homology model for NIF, a natural antagonistic ligand of CR3 receptor. From these modeling studies, possible binding modes were predicted. In addition, this first structure of a PR-1 protein from parasitic helminths may shed light on the molecular basis of host–parasite interactions. [Copyright &y& Elsevier]

Abstract: Hookworm infection is one of the world''s most common neglected tropical diseases and a leading cause of iron deficiency anemia in low- and middle-income countries. A Human Hookworm Vaccine is currently being developed by the Sabin Vaccine Institute and is in phase 1 clinical testing. The candidate vaccine is comprised of two recombinant antigens known as Na-GST-1 and Na-APR-1, each of which is an important parasite enzyme required for hookworms to successfully utilize host blood as a source of energy. The recombinant proteins are formulated on Alhydrogel® and are being tested in combination with a synthetic Toll-like receptor 4 agonist. The aim of the vaccine is to induce anti-enzyme antibodies that will reduce both host blood loss and the number of hookworms attached to the gut. Transfer of the manufacturing technology to the Oswaldo Cruz Foundation (FIOCRUZ)/Bio-Manguinhos (a Brazilian public sector developing country vaccine manufacturer) is planned, with a clinical development plan that could lead to registration of the vaccine in Brazil. The vaccine would also need to be introduced in the poorest regions of Africa and Asia, where hookworm infection is highly endemic. Ultimately, the vaccine could become an essential tool for achieving hookworm control and elimination, a key target in the 2012 London Declaration on Neglected Tropical Diseases. [Copyright &y& Elsevier]

Presents information on hookworm infections in humans, which is caused by an infection with the helminth nematode parasites Necator americanus and Ancylostoma duodenale and is transmitted through contact with contaminated soil. Description of hookworm infections and where they most often occur; Major sites of entry for the parasites, including the hands and feet; Symptoms of the infection; How males have greater exposure to infections; Treatment.