small molecules

2026-07-08 | Unmasking the Hidden Culprit: Capsule Endoscopy Reveals Diffuse Hookworm Infestation as a Cause of Obscure Gastrointestinal Bleeding and Refractory Anemia-A Case Report.

A 70-year-old woman with transfusion-dependent anemia and ongoing gastrointestinal bleeding had inconclusive findings on imaging, upper endoscopy, and colonoscopy. Capsule endoscopy revealed the diagnosis: diffuse hookworm infestation causing active bleeding throughout the small bowel and colon. Targeted anthelmintic therapy resolved her anemia completely.

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2026-06-29 | Profound Iron Deficiency Anemia Associated With Endoscopically Visualized Nematode Infestation Mimicking Gastric Malignancy: A Case Report From Trinidad and Tobago.

Severe iron deficiency anemia (IDA) secondary to parasitic infestation is uncommon in modern clinical practice but remains an important diagnostic consideration in endemic regions and vulnerable populations. We present the case of a 61-year-old male patient who presented to the emergency department following a syncopal episode and fall and was found to have profound anemia with an initial hemoglobin level of 2.5 g/dL and a serum ferritin of 1.96 ng/ml. Initial diagnostic workup with contrast-enhanced computed tomography of the abdomen and pelvis excluded acute intra-abdominal hemorrhage but demonstrated marked thickening of the gastric fundus and body, raising concern for an underlying gastric malignancy. Laboratory investigations additionally revealed raised eosinophil percentage on complete blood count analysis. Following hemodynamic stabilization and packed red blood cell transfusion, the patient underwent esophagogastroduodenoscopy. Endoscopic evaluation revealed short-segment Barrett's esophagus (Prague classification C0M2) and multiple motile nematodes attached to the duodenal mucosa, consistent with an endoscopically visualized nematode infestation involving the proximal small bowel. In the context of endemic exposure, raised eosinophil percentage, profound microcytic anemia, and low ferritin, this was considered morphologically suggestive of hookworm infestation. Further dietary and social history revealed habitual barefoot outdoor exposure and regular consumption of wild meat, supporting environmental risk factors for parasitic infection. Stool microscopy was negative, and histopathology did not identify parasitic organisms or malignancy. The patient was treated with albendazole and proton pump inhibitor therapy, with subsequent clinical improvement, recovery of hemoglobin levels following transfusion and anti-helminthic treatment, and sustained hematologic stability on follow-up. This case highlights the importance of considering parasitic infection in the differential diagnosis of severe microcytic anemia and gastric wall thickening, particularly in endemic settings. It also demonstrates how infectious gastrointestinal pathology may mimic advanced malignancy on cross-sectional imaging.

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2026-06-07 | Soil-transmitted helminth infections among migrants and travelers: a 10-year retrospective study.

Soil-transmitted helminth (STH) infections, caused by Ascaris lumbricoides, Trichuris trichiura, and hookworms, are widely distributed in tropical regions and significantly contribute to morbidity and mortality. These parasites share similar life cycles, and treatment is primarily based on benzimidazoles. This study aims to describe the clinical and epidemiological characteristics of STH cases among migrants and travelers who attended the Vall d'Hebron-Drassanes International Health Unit between 2014 and 2024, the treatments administered, and their efficacy. This retrospective descriptive study reviewed STH cases associated with migration or travel during the period 2014-2024. Diagnosis was established either through identification of eggs or larvae in stool samples or through the detection of adult Ascaris lumbricoides worms in digestive samples, always associated with a migratory process or international travel. Clinical and epidemiological data were collected and analyzed using SPSS. A total of 361 STH cases were identified, with a mean age of 32 years. Of these, 77% were migrants, 15.7% were travelers visiting friends and relatives (VFR), and 7.2% were travelers. Hookworm infection was diagnosed in 126 cases, Trichuris trichiura in 113 cases, Ascaris lumbricoides in 87 cases, and 35 patients presented with multiple STH infections. Forty-six (12.7%) were symptomatic, predominantly with gastrointestinal symptoms, and 38% presented with eosinophilia. Ninety-five percent of patients received benzimidazole-based treatment, and therapeutic failure was observed in 7.8% of cases. Cure rates were highest for A. lumbricoides (nearly 100%) and remained above 90% for T. trichiura, whereas hookworm infections showed lower rates, ranging from 83% with albendazole to 92% with mebendazole. Multiple STH infections showed, proportionally, higher rates of therapeutic failure. Most STH cases were diagnosed in asymptomatic migrants and VFRs, highlighting the importance of screening for imported diseases in migrant populations. The observed increase in therapeutic failure with commonly used regimens highlights the need to standardize treatment protocols and consider the incorporation of new drugs and combination therapies.

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2026-04-30 | Maternal anemia in helminth-infected pregnant women: Dose-response with hookworm intensity and other risk factors in a Ugandan cross-sectional study.

Anemia remains a significant global public health challenge, affecting approximately 25% of the world's population and disproportionately impacting pregnant women, with an estimated 40% prevalence worldwide. Intestinal helminth infections exacerbate this burden, with anemia prevalence reported as high as 55.6% among infected pregnant women compared to 16.4% in those uninfected. This study aimed to determine the burden of anemia and its determinants among pregnant women with helminthiasis attending antenatal care at tertiary Hospitals in western Uganda. Methods: A hospital-based cross-sectional study was conducted from 1st November 2022-1st March 2023, enrolling 420 pregnant women diagnosed with helminthiasis attending antenatal clinics. Data were collected via interviewer-administered questionnaires and laboratory records. Descriptive statistics and multivariable logistic regression analyses were performed using IBM SPSS version 23 to identify factors independently associated with anemia. Results: The prevalence of anemia was 26.2% (95% CI: 22.2-30.6), with 39.1% (95% CI: 30.5-48.4) mild, 35.5% (95% CI: 27.1-44.7) moderate, and 25.5% (95% CI: 18.2-34.3) severe cases. Multivariate analysis showed that moderate and heavy hookworm intensities increased anemia risk nearly two-fold (aOR = 1.90, 95% CI: 1.16-3.54; p = 0.028) and 2.4-fold (aOR = 2.40, 95% CI: 1.06-5.41; p = 0.041), respectively. Protective factors included being a student (aOR = 0.17, 95% CI: 0.05-0.63; p = 0.008), deworming ≥3 months prior (aOR = 0.11, 95% CI: 0.01-0.88; p = 0.038), using borehole water (aOR = 0.60, 95% CI: 0.36-1.00; p = 0.048), and gestational age 14-27 weeks (aOR = 0.57, 95% CI: 0.35-0.92; p = 0.020). Conclusions: Maternal anemia showed a clear dose-response with hookworm intensity, with risk increasing from moderate to heavy infection. Delayed deworming and unsafe water increased odds, whereas being a student and second-trimester gestation were protective. Timely anthelminthic treatment, improved water access, and strengthened antenatal care are critical in endemic settings.

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2026-04-18 | Correlation of School-based Albendazole Mass Drug Administration on Malaria and Soil Transmitted Helminths Co-infection in School-aged Children

Background: Malaria and Soil-Transmitted Helminth (STH) infections present a significant public health challenge in endemic regions, often occurring together and worsening health outcomes. This cross-sectional study examined the prevalence of the co-infection among school-aged children in the Unwana Community, Ebonyi State, and showed the effects of school-based Albendazole Mass Drug Administration (MDA). Methods: A total of 249 pupils from three constituencies in the community were sampled between June and August 2025. Malaria was diagnosed using Rapid Diagnostic Tests (RDTs), while STH infections were identified in stool samples using standard techniques. Data on MDA receipt, socio-economic factors, and preventive measures were gathered through a questionnaire. Additionally, health center records on community MDA were collected. Results: The overall prevalence of infections was 38.6% (n=96) for malaria, 36.5% (n=91) for STH, and 7.2% (n=18) for co-infection. The STHs identified included Ascaris lumbricoides, Trichuris trichiura, and hookworm. A. lumbricoides had the highest prevalence at 16.5% (n=41). Double STH infections included A. lumbricoides-hookworm (2.4%, n=6), A. lumbricoides-T. trichiura (2.8%, n=7), and T. trichiura-hookworm (1.2%, n=3). Significant associations were found between malaria, STH infections, and the demographics of the schoolchildren (P<0.05); however, there was no association between co-infection and the demographics of the children (P>0.05). Not receiving Albendazole at school was identified as the only significant factor influencing malaria-STH co-infection (Adjusted Odds Ratio = 3.760, P = 0.042). Analysis of health center records revealed inconsistencies in community-level Albendazole MDA, with only the U.C.P.S 2 constituency receiving doses in 2021, 2022, and 2023. Conclusion: The prevalence of co-infection indicates an ongoing public health challenge within the community. Additionally, the significant risk factor identified for malaria and STH co-infection suggests that implementing school-based mass drug administration (MDA) could be a more effective strategy for controlling STH among school-aged children in Unwana.

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vaccines

2026-06-10 | A novel hookworm vaccine candidate identified through immunomic profiling of human protective immunity.

Hookworms infect nearly half a billion people, causing approximately 2.1 million disability-adjusted life-years lost. While anthelmintic drugs are available, they do not prevent reinfection. A vaccine could provide long-term immunity, but none currently exists. We recently demonstrated that human subjects could be safely vaccinated with irradiated hookworm larvae, eliciting humoral and cellular immune responses that induce partial protection against challenge infection with non-irradiated parasites. Using immunomic approaches, we identified IgG targets in vaccinated individuals by screening a proteome microarray containing recombinant proteins sourced from the secretome of Necator americanus. We characterized the most immunoreactive N. americanus proteins and identified their orthologs in the rodent hookworm Nippostrongylus brasiliensis. These N. brasiliensis proteins were expressed recombinantly and tested for efficacy in a mouse challenge model. Our lead vaccine candidate NBR17057 - a homolog of Na_11335 provided robust protection against infection, evidenced by >90% reductions in intestinal worm burden and fecal egg counts, and elevated antigen-specific IgG levels. Vaccination also stimulated IgA+ B-cells, IgG1+/IgA+ plasma cells, and IgG1+/IgA+ germinal centre B-cells in inguinal lymph nodes, indicating gut immunity. Additionally, high levels of IgG1+/IgA+ B-cells, germinal centre B-cells, and plasma cells in the spleen suggest protection against systemic larval migration. The presence of IgG1+/IgA+ memory B-cells in both the lymph nodes and spleen indicates potential long-term immunity against reinfection. Our findings suggest that a N. brasiliensis L3 larval-stage protein NBR17057 and its homolog in N. americanus Na_11335 could serve as a promising subunit vaccine candidate targeting the infective larval stage of human hookworms.

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2025-11-24 | A framework for guiding integrated disease control measures through multipathogen surveillance.

Global health programs have traditionally focused on single diseases. There is potential for synergy through integrated intervention delivery, particularly in areas with overlapping geographic disease burden, but there is limited methodology developed for assessing potential efficiency gains through integration. Here, we applied a measure of diversity, Rao's quadratic index, to quantify multipathogen burden across two large-scale surveys: Bangladesh (90 clusters, 2,396 children) and Cambodia (100 clusters, 2,150 women). In both settings, we observed geographic clustering of multiple pathogens, indicating potential for more efficient, integrated disease control strategies. We assessed the efficiency of a multipathogen-targeted strategy compared to traditional single-pathogen approaches by calculating the percent reduction in the number of spatial clusters needed to reach 75% of the disease burden (infections or unvaccinated individuals) in a hypothetical intervention. In Bangladesh, integrating deworming with measles vaccination guided by Rao's quadratic index improved efficiency by 15% for Ascaris lumbricoides, 31% for hookworm, and 38% for Trichuris trichiura, compared to a measles-focused approach. In Cambodia, a Rao-guided strategy performed similarly to the best single-pathogen strategy for Strongyloides stercoralis, and reduced the number of spatial clusters that would need to be targeted by 57% (lymphatic filariasis), 83% (Plasmodium falciparum), and 59% (Plasmodium vivax). We also found that higher multipathogen burden was significantly associated with lower household wealth, suggesting that Rao-guided strategies may be more effective in reaching under-resourced populations. These findings support the use of multipathogen burden metrics to guide integrated program delivery, offering potential for greater efficiency in disease control.

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2025-06-03 | Reverse vaccinology for hookworms: a rational selection of vaccinable antigens against parasitic nematodes.

Reverse vaccinology is a time- and cost-effective approach to identify potential vaccinable antigens for further in vivo experimental validation. Despite its wide application to multiple organisms, the use of in silico vaccine development tools to parasitic nematodes has been limited. Herein, we have used the rodent hookworm Nippostrongylus brasiliensis as a mouse model for the human hookworm Necator americanus to identify potential vaccine candidates against the latter. Our strategy combined advanced bioinformatic evaluations with knowledge-based criteria. A cumulative rating of antigenic properties was performed resulting in a global prioritization scoring for an updated N. brasiliensis proteome of 22,796 proteins assigned. Evaluation criteria included homology to the human counterpart N. americanus, absence of mammalian homologs, cellular location by computational predictors, as well as mass spectrometry data, proteolytic activity of the evaluated protein within the parasite, presence of conserved domains, predicted humoral epitopes, and MHC class II epitope population coverage. To assign one global score representing these characteristics, cumulative scoring was performed. This analysis provided a group of 56 potential candidates, including 11 proteins associated with parasite survival and establishment. Remarkably, the second highest score was assigned to an aspartic protease homologous of the N. americanus vaccine-candidate Na-APR-1, which supports the relevance of this approach. Allergenicity and toxicity of the selected molecules were also predicted to anticipate side effects of future candidates. This comprehensive approach provides valuable insights for the rational design of new vaccines against N. americanus, the results of which, however, must be validated in vivo.

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2025-04-18 | Manufacture of Necator americanus as an infectious challenge agent: Accelerating human hookworm vaccine development.

Hookworms infect 450 million people globally and account for the loss of 5 million disability-adjusted life years annually. Over the last decade, the Human Hookworm Vaccine (HHV) candidate N. americanus Glutathione-S-Transferase-1 (Na-GST-1) has advanced to efficacy testing. This manuscript describes the manufacture of third-stage N. americanus larvae (NaL3) as an infectious challenge agent to provide "proof-of-concept" for the efficacy of Na-GST-1 prior to more extensive and more resource-intensive vaccine field trials in hookworm endemic areas. NaL3 were produced from fecal samples of three hookworm-infected human donors by a modified Harada and Mori method that complied with current Good Manufacturing Practices (cGMP). A series of lot release tests assessed the purity (bioburden), viability (potency), and identity (speciation) of NaL3 before administration to participants in a hookworm vaccine challenge model (HVCM) in Washington, DC. Twenty-four production runs yielded an average of 947 NaL3 per lot, which were approved for clinical to inoculate of 39 participants in a Hookworm Vaccine Challenge Model. This manuscript describes the unique manufacture and testing for NaL3 in compliance with cGMP.

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2025-01-20 | Proteomic characterization and comparison of the infective and adult life stage secretomes from Necator americanus and Ancylostoma ceylanicum.

More than 470 million people globally are infected with the hookworms Ancylostoma ceylanicum and Necator americanus, resulting in an annual loss of 2.1 to 4 million disability-adjusted-life-years. Current infection management approaches are limited by modest drug efficacy, the costs associated with frequent mass drug administration campaigns, and the risk of reinfection and burgeoning drug resistance. Subunit vaccines based on proteins excreted and secreted (ES) by hookworms that reduce worm numbers and associated disease burden are a promising management strategy to overcome these limitations. However, studies on the ES proteomes of hookworms have mainly described proteins from the adult life stage which may preclude the opportunity to target the infective larva. Here, we employed high resolution mass spectrometry to identify 103 and 57 ES proteins from the infective third larvae stage (L3) as well as 106 and 512 ES proteins from the adult N. americanus and A. ceylanicum respectively. Comparisons between these developmental stages identified 91 and 41 proteins uniquely expressed in the L3 ES products of N. americanus and A. ceylanicum, respectively. We characterized these proteins based on functional annotation, KEGG pathway analysis, InterProScan signature and gene ontology. We also performed reciprocal BLAST analysis to identify orthologs across species for both the L3 and adult stages and identified five orthologous proteins in both life stages and 15 proteins that could be detected only in the L3 stage of both species. Last, we performed a three-way reciprocal BLAST on the L3 proteomes from both hookworm species together with a previously reported L3 proteome from the rodent hookworm Nippostrongylus brasiliensis, and identified eight L3 proteins that could be readily deployed for testing using well established rodent models. This novel characterization of L3 proteins and taxonomic conservation across hookworm species provides a raft of potential candidates for vaccine discovery for prevention of hookworm infection and disease.

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cell therapies

2026-01-19 | Exploratory study characterizing gastrointestinal physiological changes during controlled human hookworm infection.

Hookworm infects over 400 million people globally and causes gastrointestinal morbidity, yet its physiological effects remain poorly defined. Controlled human hookworm infection is also being explored as a therapy for gut diseases. We performed an exploratory study to evaluate the impact of experimental Necator americanus infection on gastrointestinal transit, motility, and luminal pH in 10 healthy adults (mean age 41 yr, 60% females) infected with 30 larvae via skin application. Assessments using the SmartPill Wireless Motility Capsule were performed at baseline, week 6 (acute infection), and week 24 or 48 (chronic infection). Parameters included gastric emptying time, small bowel and colonic transit, whole gut transit, intraluminal pressures, contraction frequency, motility index, and segmental pH, analyzed with paired t tests or ANOVA. All participants developed patent infections. No significant differences were observed in gastric emptying, small bowel, colonic, or whole gut transit times, nor in motility indices or contraction frequencies. However, during acute infection, duodenal (6.14 ± 0.19 vs. 5.80 ± 0.24, P < 0.05) and small bowel pH (6.96 ± 0.37 vs. 6.50 ± 0.37, P < 0.05) were significantly reduced compared with baseline, normalizing by the chronic phase. No other significant pH alterations were detected. Thus, these data suggest that controlled N. americanus infection in healthy adults induces a transient reduction in duodenal and small-intestinal pH without affecting gastrointestinal transit or motility. This acidification may contribute to acute-phase symptoms and nutrient malabsorption in endemic settings, whereas the absence of sustained motility disturbance supports the safety of controlled hookworm infection for therapeutic investigation.NEW & NOTEWORTHY This exploratory study using SmartPill technology found that controlled hookworm infection in healthy adults caused a transient drop in duodenal and small-intestinal pH during the acute phase, but no lasting changes in gut motility or transit. The findings, the first of their kind in humans, suggest that the physiological effects of controlled doses of hookworm are subtle and short-lived, offering reassurance for therapeutic trials while highlighting a potential mechanism for symptoms and malabsorption in endemic regions.

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2023-07-26 | Effect of experimental hookworm infection on insulin resistance in people at risk of type 2 diabetes

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.

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2023-03-20 | Effect of experimental hookworm infection on insulin resistance in people at risk of Type 2 Diabetes: a randomized, placebo-controlled trial

ABSTRACT The reduced prevalence of insulin resistance and type 2 diabetes (T2D) 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 randomized controlled trial of hookworm infection in 40 people at risk of T2D involved treatment with either 20 or 40 Necator americanus third-stage larvae (L3) or Placebo. Primary outcome was safety, with homeostatic model assessment of insulin resistance (HOMA-IR), fasting blood glucose (FBG), and body mass being the key metabolic outcomes. Adverse events were more frequent in hookworm-treated participants, where 44% experienced transient gastrointestinal symptoms. FBG and HOMA-IR were significantly lowered in both hookworm-treated groups at 1-year, and body mass was significantly lowered after L3-20 treatment at 2-years. Overall, hookworm infection was well tolerated in people at risk of T2D, and were associated with improved insulin resistance, warranting further exploration of the benefits of hookworms on metabolic health.

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2021-03-01 | Immune System Investigation Using Parasitic Helminths

Coevolutionary adaptation between humans and helminths has developed a finely tuned balance between host immunity and chronic parasitism due to immunoregulation. Given that these reciprocal forces drive selection, experimental models of helminth infection are ideally suited for discovering how host protective immune responses adapt to the unique tissue niches inhabited by these large metazoan parasites. This review highlights the key discoveries in the immunology of helminth infection made over the last decade, from innate lymphoid cells to the emerging importance of neuroimmune connections. A particular emphasis is placed on the emerging areas within helminth immunology where the most growth is possible, including the advent of genetic manipulation of parasites to study immunology and the use of engineered T cells for therapeutic options. Lastly,we cover the status of human challenge trials with helminths as treatment for autoimmune disease, which taken together, stand to keep the study of parasitic worms at the forefront of immunology for years to come.

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2019-12-01 | Safety and tolerability of experimental hookworm infection in humans with metabolic disease: study protocol for a phase 1b randomised controlled clinical trial

Abstract 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.

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proteins

2026-06-04 | Transgenic hookworm secretes anti-tetrodotoxin human single chain antibody.

Biologics, protein- and peptide-based drugs derived from living organisms or cell lines have emerged as effective therapies across a broad range of indications. However, parenteral administration and the need for frequent dosing increase costs and limit compliance, creating an urgent need for innovative platforms capable of continuously, safely, and efficiently delivering sustained biologics in situ within the host. Here, we describe a bioengineered hookworm platform to manufacture and deliver biologic therapeutics in vivo. As proof of concept, we engineer the Ancylostoma ceylanicum secretome by inserting a human single-chain variable fragment antibody (s16-HuScFv) into its genome. Transgene expression does not perturb surrounding gene expression, and heritable transgenesis is confirmed. The s16-HuScFv transgene product is secreted into host circulation and partially neutralizes tetrodotoxin. Given the availability of controlled human infections, our disease-agnostic bioengineered hookworm platform offers a next-generation approach to address a suite of chronic human diseases, and with a single-dose administration, could potentially produce and deliver biologic medicines within the human host for years.

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2026-05-04 | Chromosomal markerless integration of anthelmintic Cry proteins into the Bacillus thuringiensis genome.

Bacillus thuringiensis (Bt) is a Gram-positive bacterium that during sporulation produces insecticidal Crystal (Cry) proteins, which play a major role in insect control today. Some Bt Cry proteins, e.g., Cry5Ba, target nematodes and, when given orally, can cure animals of gastrointestinal nematode (GIN) parasites. To eliminate concerns about treating humans and animals with spores and live bacteria, we developed an asporogenous system for scalable and safe Cry protein delivery called IBaCC (Inactivated Bacteria with Cytosolic Crystal(s)), which results in production of a bioactive crystal and a dead bacterium. However, to date, IBaCC involves expression of Cry proteins from antibiotic-selectable plasmids to ensure maintenance. Here, we develop and validate tools for markerless and stable integration and expression of Cry proteins in Bt. We markerlessly integrate an expression construct for Cry5Ba into either the spo0A or the sigK locus and demonstrate robust Cry5Ba expression. We also integrate our Cry5Ba expression construct into both loci simultaneously, increasing expression further. We demonstrate that an expression construct for a second anthelmintic Cry protein, Cry21Aa, can be integrated either alone or in combination with Cry5Ba in a single Bt strain. We furthermore show that these markerless integrants are stable in the absence of a selectable marker. These integrated strains, processed to IBaCC, demonstrate excellent ex vivo nematicidal bioactivity toward the larval stages of the sheep GIN parasite Haemonchus contortus and adult stages of the human hookworm GIN parasite Ancylostoma ceylanicum . This study demonstrates the successful markerless integration of 1-2 identical or dissimilar Cry proteins into Bt. These Cry integrants, in which genes essential to sporulation are deleted or replaced, provide robust Cry expression, stability, and bioactivity. These studies represent an important advance in Bt genetics and toward a safe, deployable, and cost-effective anthelmintic therapy to treat GIN parasitic infections in humans and animals.

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2025-06-27 | Differential Emodepside Efficacy in Drug-Resistant and Drug-Susceptible Ancylostoma caninum Highlights Variability in Potassium Channel Activity

Multi-anthelmintic resistance in hookworms poses a significant challenge to both human and veterinary health, underscoring the need for novel treatment strategies. In this study, we evaluated the in vitro efficacy of three anthelmintics—pyrantel, ivermectin, and emodepside—against L3 larvae of drug-susceptible (WMD) and triple-anthelmintic-resistant (BCR) isolates of Ancylostoma caninum. While pyrantel was largely ineffective and ivermectin induced high mortality in both isolates, emodepside displayed a surprising trend: the drug-resistant BCR isolate was more susceptible than the drug-susceptible WMD isolate. To explore the underlying mechanism, we performed survival assays in the presence of penitrem A, a BK channel (SLO-1) inhibitor. The addition of penitrem A reversed the enhanced emodepside sensitivity in BCR, implicating elevated basal expression of SLO-1 channels as a potential factor. These findings suggest that emodepside, via its action on SLO-1, may offer a promising therapeutic avenue to combat multidrug-resistant hookworm infections.

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2024-10-25 | Bacillus thuringiensis Cry14A family proteins as novel anthelmintics against gastrointestinal nematode parasites.

Bacillus thuringiensis crystal (Cry) proteins have been expressed in commercial transgenic crops for nearly 30 years, providing safe and effective control of insect pests and significantly reducing the application of hazardous chemical pesticides. B. thuringiensis crystal proteins have also been shown to target parasitic nematodes, including plant parasitic nematodes. Recently, transgenic soybean crops expressing Cry14Ab have been shown to provide control against the soybean cyst nematode Heterodera glycines, marking the first time a crystal protein is being commercialized in transgenic crops for control of a nematode pest. However, apart from H. glycines and the free-living nematode, Caenorhabditis elegans, the breadth of nematode activity of Cry14Ab, e.g., against gastrointestinal parasitic nematodes (GINs), has not been reported. Here we study the efficacy of Cry14Ab against a wide range of gastrointestinal nematode parasites (GINs) in vitro and in vivo. We find that Cry14Ab is effective in vitro against the barber's pole worm Haemonchus contortus larvae, small strongyles cyathostomin larvae, the hookworm Ancylostoma ceylanicum adults, the roundworm Ascaris suum L4 larvae, and the whipworm Trichuris muris adults. In rodents infected with GIN parasites, Cry14Ab is effective as an in vivo anthelmintic against the hookworms A. ceylanicum and N. americanus, against the mouse parasite Heligmosomoides polygyrus bakeri, and against the roundworm A. suum. Cry14Ab also variably reduces the reproduction of the whipworm T. muris in vivo. Using optimized profile Markov Models, we looked for other putative anthelmintic Cry proteins and, within this list, identified a Bt crystal protein, GenBank accession no. MF893203, that we produced and demonstrated intoxicated GINs. This protein, with 90% amino acid identity to Cry14Ab, is active against C. elegans, A. ceylanicum adults, and A. suum L4 larvae in vitro. MF893203 was given the official designation of Cry14Ac. Cry14Ac is also an effective in vivo anthelmintic against A. ceylanicum hookworms in hamsters and intestinal A. suum in mice. Taken together, our results demonstrate that Cry14Ab and Cry14Ac have wide therapeutic utility against GINs.

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2022-09-13 | A newly identified secreted larval antigen elicits basophil-dependent protective immunity against N. brasiliensis infection.

Hookworms infect more that 400 million people and cause significant socio-economic burden on endemic countries. The lack of efficient vaccines and the emergence of anthelminthic drug resistance are of major concern. Free-living hookworm larvae infect their hosts via the skin and live as adult worms in the small intestine where they feed on host tissue and blood. Excretory/secretory (E/S) products, released by helminths as they migrate through their host, are thought to play a key role in facilitating infection and successful establishment of parasitism. However, E/S products can also elicit protective immune responses that might be harnessed for vaccine development. By performing Western blots with serum of Nippostrongylus brasiliensis (Nb) infected mice as a model for human hookworm infection, we identified a largely overlapping set of IgG1- and IgE-reactive antigens in E/S from infective L3 stage larvae. Mass spectrometry analysis led to the identification of a new protein family with 6 paralogues in the Nb genome which we termed Nb-LSA1 for "Nippostrongylus brasiliensis larval secreted protein 1". The recombinantly expressed 17 kDa family member Nb-LSA1a was recognized by antibodies in the serum of Nb immune mice. Immunization of mice with Nb-LSA1a in alum elicited a strong IgG1 response but no detectable antigen-specific IgE. Most importantly, immunized mice were largely protected against a challenge Nb infection. This effect was dependent on the presence of basophils and occurred before the parasites reached the intestine. Therefore, basophils appear to play a critical role for rapid control of infection with L3 stage larvae in mice immunized with a single secreted larval protein. A better understanding of basophil-mediated protective immunity and identification of potent larval antigens of human hookworms could help to develop promising vaccination strategies.

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other

2025-01-10 | Altering the intracellular trafficking of Necator americanus GST-1 antigen yields novel hookworm mRNA vaccine candidates.

The antigen Na-GST-1, expressed by the hookworm Necator americanus, plays crucial biochemical roles in parasite survival. This study explores the development of mRNA vaccine candidates based on Na-GST-1, building on the success of recombinant Na-GST-1 (rNa-GST-1) protein, currently assessed as a subunit vaccine candidate, which has shown promise in preclinical and clinical studies. By leveraging the flexible design of RNA vaccines and protein intracellular trafficking signal sequences, we developed three variants of Na-GST-1 as native (cytosolic), secretory, and plasma membrane-anchored (PM) antigens. After one immunization in mice, mRNA vaccines induced an earlier onset of antigen-specific antibodies compared to rNa-GST-1. Following two immunizations, mRNA vaccines induced similar or superior levels of antigen-specific antibodies compared to rNa-GST-1. Secretory Na-GST-1 was comparable to rNa-GST1 in producing neutralizing antibodies against Na-GST-1's thiol transferase activity, while native Na-GST-1 induced a more robust CD8+ T cell response due to its intracellular accumulation. Although PM Na-GST-1 elicited one of highest titers of antigen-specific antibody and a diverse set of memory T-cell populations, it resulted in a lower ratio of neutralizing antibodies after IgG purification compared to the other vaccine candidates. These findings emphasize the importance of antigen localization in tailoring immune responses and suggest that extracellular antigens are more effective for inducing humoral responses, whereas cytosolic antigen accumulation enhances MHC-1 peptide presentation. Future studies will determine if these in vitro and immunogenicity findings translate to in vivo efficacy. Altogether, mRNA vaccines offer numerous possibilities in the development of multivalent vaccines with single or multiple antigens.

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2025-01-08 | Randomized, observer-blind, controlled Phase 1 study of the safety and immunogenicity of the Na-GST-1/Alhydrogel hookworm vaccine with or without a CpG ODN adjuvant in hookworm-naïve adults.

Recombinant Necator americanus Glutathione-S-Transferase-1 (Na-GST-1) formulated on Alhydrogel (Na-GST-1/Alhydrogel) is being developed to prevent anemia and other complications of N. americanus infection. Antibodies induced by vaccination with recombinant Na-GST-1 are hypothesized to interfere with the blood digestion pathway of adult hookworms in the host. Phase 1 trials have demonstrated the safety of Na-GST-1 formulated on Alhydrogel, but further optimization of the vaccine adjuvant formulation may improve humoral immune responses, thereby increasing the likelihood of vaccine efficacy. A randomized, observer-blind, dose escalation Phase 1 trial was conducted in 24 healthy, hookworm-naïve adults. In each cohort of 12 participants, 4 were randomized to receive 100 µg of Na-GST-1/Alhydrogel and 8 to receive 30 µg or 100 µg of Na-GST-1/Alhydrogel plus the Cytosine-phospho-Guanine (CpG) oligodeoxynucleotide Toll-like receptor-9 agonist, CpG 10104, in the first and second cohorts, respectively. Progression to the second cohort was dependent upon evaluation of 7-day safety data after all participants in the first cohort had received the first dose of vaccine. Three intramuscular injections of study product were administered on days 0, 56, and 112, after which participants were followed for 6 months. IgG and IgG subclass antibody responses to Na-GST-1 were measured by qualified indirect ELISAs at pre- and post-vaccination time points. Na-GST-1/Alhydrogel administered with or without CpG 10104 was well-tolerated. The most common solicited adverse events were mild injection site tenderness and pain, and mild headache. There were no vaccine-related serious adverse events or adverse events of special interest. Both dose concentrations of Na-GST-1/Alhydrogel plus CpG 10104 had significantly higher post-vaccination levels of antigen-specific IgG antibody compared to Na-GST-1/Alhydrogel without CpG, starting after the second injection. Peak anti-Na-GST-1 IgG levels were observed between 2 and 4 weeks following the third dose, regardless of Na-GST-1 formulation. IgG levels decreased but remained significantly above baseline in all groups by day 290, at which point all participants (20 of 20 evaluable participants) still had detectable IgG. Longitudinal antigen-specific IgG1 and IgG3 subclass responses mirrored those of total IgG, whereas IgG4 responses were lower in the groups that received the vaccine with the CpG adjuvant compared to the non-CpG group. Vaccination of hookworm-naïve adults with Na-GST-1/Alhydrogel plus CpG 10104 was safe and minimally reactogenic. Addition of CpG 10104 to Na-GST-1/Alhydrogel resulted in significant improvement in IgG responses against the vaccine antigen. These promising results have led to inclusion of the CpG 10104 formulation of Na-GST-1/Alhydrogel in a Phase 2 proof-of-concept controlled human infection trial.

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2024-12-04 | Disruption of the C5a/C5aR1 axis confers protection against hookworm infection in lung.

Hookworms are soil-transmitted parasitic nematodes that penetrate the host skin before migrating to the lungs. With an estimated 500-700 million people infected worldwide, hookworm infections are a neglected tropical disease and a significant cause of morbidity, particularly in children, pregnant women, and immunocompromised individuals. Although there is ample evidence that complement activation is pivotal to elicit a protective host immune response against invasive pathogens, its role in hookworm infection remains insufficiently explored. Here, we investigated the complement anaphylatoxin, C5a, during the early lung stage of infection with Nippostrongylus brasiliensis in C57BL/6J wild type and C5aR1-/- mice. Despite the previously reported ability of lung larvae to evade complement activation, C5a was detectable locally in lung tissue and bronchoalveolar lavages. Surprisingly, C5aR1 presence directly contributed to the pathogenicity of hookworm infection. The burden of viable parasites in the lungs was mitigated in C5aR1-/- mice, compared to C57BL/6J mice 48 hours post-infection. Additionally, C5aR1-/- mice showed significantly reduced lung injury, lower cytokine release, attenuated alveolar hemorrhage, and limited alveolar-capillary barrier disruption. Neutrophils were the most abundant and highest C5aR1-expressing cell type in the alveolar space after infection. Deficiency of C5aR1 reduced the influx of neutrophils, monocytes, and eosinophils to the pulmonary airways. RNA sequencing of alveolar neutrophils revealed C5aR1-dependent regulation of the novel nuclear protein, DEDD2. In conclusion, our findings highlight the impact of C5aR1 signaling in neutrophils during hookworm infection uncovering an unexpected downside of complement activation in parasitic infection.

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2024-11-18 | Isolation of anti-Ancylostoma-secreted protein 5 (ASP5) antibody from a naïve antibody phage library.

Ancylostoma species are parasitic nematodes that release a multitude of proteins to manipulate host immune responses to facilitate their survival. Among the released proteins, Ancylostoma-secreted protein 5 (ASP5) plays a pivotal role in mediating host-parasite interactions, making it a promising target for interventions against canine hookworm infections caused by Ancylostoma species. Antibody phage display, a widely used method for generating human monoclonal antibodies was employed in this study. A bacterial expression system was used to produce ASP5 for biopanning. A single-chain fragment variable (scFv) monoclonal antibody against ASP5 was generated from the naïve Human AntibodY LibrarY (HAYLY). The resulting scFv antibody was characterized to elucidate its antigen-binding properties. The identified monoclonal antibody showed good specificity and binding characteristics which highlights its potential for diagnostic applications for hookworm infections.

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2021-07-05 | Establishing a controlled hookworm human infection (CHHI) model for Africa: A report from the stakeholders meeting held in Lambaréné, Gabon, November 10–11, 2019

Abstract Background Hookworm is a major contributor to worldwide disease burden with over 230 million people infected. It has been identified as one of the Neglected Tropical Diseases that can be controlled and even eliminated through mass drug administration and other effective interventions. Mathematical models have shown that hookworm can only be eliminated via a vaccine. Controlled Hookworm Human Infection (CHHI) models can facilitate rapid development of vaccines and drugs. Methods As a first step towards the establishment of CHHI in Africa, we held a stakeholders meeting in Lamberene, Gabon from 10 to 11 November 2019. Results Discussions revolved around the roles of the different regulatory institutions concerned; the need to strengthen existing regulatory capacity and the role of legislation; creating Gabon-specific ethical guidelines to govern Controlled Human Infection (CHI) studies; development of a study protocol; consideration of cultural and social peculiarities; the need for regular joint review meetings between interested parties throughout the process of protocol implementation; and participant compensation. Moreover, operational considerations concerning the introduction of CHHI in Gabon include the use of the local strain of hookworm for the challenge infections, capacity building for the local production of challenge material, and the establishment of adequate quality assurance procedures. Conclusion The workshop addressed several of the anticipated hurdles to the successful implementation of CHHI in Gabon. It is our aim that this report will stimulate interest in the implementation of this model in the sub-Saharan African setting.

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