Controlled human hookworm infection remodels plasmacytoid dendritic cells and regulatory T cells towards profiles seen in natural infections in endemic areas.

Hookworm infection remains a significant public health concern, particularly in low- and middle-income countries, where mass drug administration has not stopped reinfection. Developing a vaccine is crucial to complement current control measures, which necessitates a thorough understanding of host immune responses. By leveraging controlled human infection models and high-dimensional immunophenotyping, here we investigated the immune remodeling following infection with 50 Necator americanus L3 hookworm larvae in four naïve volunteers over two years of follow-up and compared the profiles with naturally infected populations in endemic areas. Increased plasmacytoid dendritic cell frequency and diminished responsiveness to Toll-like receptor 7/8 ligand were observed in both controlled and natural infection settings. Despite the increased CD45RA+ regulatory T cell (Tregs) frequencies in both settings, markers of Tregs function, including inducible T-cell costimulatory (ICOS), tumor necrosis factor receptor 2 (TNFR2), and latency-associated peptide (LAP), as well as in vitro Tregs suppressive capacity were higher in natural infections. Taken together, this study provides unique insights into the immunological trajectories following a first-in-life hookworm infection compared to natural infections.

Public engagement for the conduct of a controlled human infection study testing vaccines against Necator americanus (hookworm) in areas of active hookworm transmission in Brazil.

Controlled Human Infection Models (CHIS) involve administering human pathogens to healthy participants in controlled medical settings, which can elicit complex bioethical issues. Understanding how the community perceives such studies can significantly increase the participant's sense of cooperation and increases the researcher's and the participant's transparency. The current study describes the development of an educational intervention to achieve these ends as it aims to (1) analyze perceptions of the Controlled Human Infection Studies (CHIS), and (2) evaluate the participants' comprehension of the CHIS. METHODS: This is a qualitative action research that includes the development of an educational intervention with residents of a rural area in Minas Gerais, Brazil, where there is continuous natural transmission of the human pathogen Necator americanus ("hookworm"). In this area, it is intended to carry out a proposed phase 3 vaccine clinical trial in the future to test the efficacy of hookworm vaccines using controlled human infection. Two data collection strategies were used: an educational intervention and a focus group. RESULTS: The participants' perceptions showed distinct perspectives on CHIS. On one side, they recognized that the investigation is essential for the community, but on the other side, they thought that there would be resistance to its conduct by fear of infection. The idea that the study would generate a benefit for the greater good, contributing to the prevention of hookworm infection, was clearly stated. The participants perceived that the study offered concrete risks that could be reduced by constant monitoring by the researchers. They also mentioned the importance of access to information and the positive influence those who express interest in participating in the study can exert in the community. In relation to comprehension the participants memorized the information, mobilized it to explain everyday situations and created strategies to disseminate the study and engage the community in its development. By repeating and making sense of the information, the participant not only assimilates the knowledge transmitted, but also creates new knowledge. CONCLUSION: We concluded that an educational process of discussion and dialogue around participants' perceptions about the CHIS, promotes understanding and allows ways to disseminate information about the research to be collectively created.

Modulation of intestinal epithelial permeability by chronic small intestinal helminth infections.

Increased permeability of the intestinal epithelial layer is linked to the pathogenesis and perpetuation of a wide range of intestinal and extra-intestinal diseases. Infecting humans with controlled doses of helminths, such as human hookworm (termed hookworm therapy), is proposed as a treatment for many of the same diseases. Helminths induce immunoregulatory changes in their host which could decrease epithelial permeability, which is highlighted as a potential mechanism through which helminths treat disease. Despite this, the influence of a chronic helminth infection on epithelial permeability remains unclear. This study uses the chronically infecting intestinal helminth Heligmosomoides polygyrus to reveal alterations in the expression of intestinal tight junction proteins and epithelial permeability during the infection course. In the acute infection phase (1 week postinfection), an increase in intestinal epithelial permeability is observed. Consistent with this finding, jejunal claudin-2 is upregulated and tricellulin is downregulated. By contrast, in the chronic infection phase (6 weeks postinfection), colonic claudin-1 is upregulated and epithelial permeability decreases. Importantly, this study also investigates changes in epithelial permeability in a small human cohort experimentally challenged with the human hookworm, Necator americanus. It demonstrates a trend toward small intestinal permeability increasing in the acute infection phase (8 weeks postinfection), and colonic and whole gut permeability decreasing in the chronic infection phase (24 weeks postinfection), suggesting a conserved epithelial response between humans and mice. In summary, our findings demonstrate dynamic changes in epithelial permeability during a chronic helminth infection and provide another plausible mechanism by which chronic helminth infections could be utilized to treat disease.

Hookworm infection: Toward development of safe and effective peptide vaccines.

Hookworms are hematophagous nematode parasites that have infected a billion people worldwide. Anthelmintic drugs have limited efficacy and do not prevent reinfection. Therefore, prophylactic vaccines are in high demand. Whole parasite vaccines are allergic and unsafe; thus, research into subunit vaccines has been warranted. A comprehensive overview of protein or peptide subunit vaccines' safety, protective efficacy, and associated immune responses is provided herein. The differences between the immune responses against hookworm infection by patients from epidemic versus nonepidemic areas are discussed in detail. Moreover, the different immunologic mechanisms of protection are discussed, including those that rely on allergic and nonallergic humoral and antibody-dependent cellular responses. The allergic and autoimmune potential of hookworm antigens is also explored, as are the immunoregulatory responses induced by the hookworm secretome. The potential of oral mucosal immunizations has been overlooked. Oral immunity against hookworms is a long-lived and safer immune response that is associated with elimination of infection and protective against reinfections. However, the harsh conditions of the gastrointestinal environment necessitates special oral delivery systems to unlock vaccines' protective potential. The potential for development of safer and more effective peptide- and protein-based anthelmintic vaccines is explored herein.

Vaccination of human participants with attenuated Necator americanus hookworm larvae and human challenge in Australia: a dose-finding study and randomised, placebo-controlled, phase 1 trial.

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.

Dynamics of the bacterial gut microbiota during controlled human infection with Necator americanus larvae.

Hookworms are soil-transmitted helminths that use immune-evasive strategies to persist in the human duodenum where they are responsible for anemia and protein loss. Given their location and immune regulatory effects, hookworms likely impact the bacterial microbiota. However, microbiota studies struggle to deconvolute the effect of hookworms from confounders such as coinfections and malnutrition. We thus used an experimental human hookworm infection model to explore temporal changes in the gut microbiota before and during hookworm infection. Volunteers were dermally exposed to cumulative dosages of 50, 100 or 150 L3 Necator americanus larvae. Fecal samples were collected for microbiota profiling through 16S rRNA gene amplicon sequencing at weeks zero, four, eight, fourteen and twenty. During the acute infection phase (trial week zero to eight) no changes in bacterial diversity were detected. During the established infection phase (trial week eight to twenty), bacterial richness (Chao1, p = .0174) increased significantly over all volunteers. No relation was found between larval dosage and diversity, stability or relative abundance of individual bacterial taxa. GI symptoms were associated with an unstable microbiota during the first eight weeks and rapid recovery at week twenty. Barnesiella, amongst other taxa, was more abundant in volunteers with more GI symptoms throughout the study. In conclusion, this study showed that clinical GI symptoms following N. americanus infection are associated with temporary microbiota instability and relative abundance of specific bacterial taxa. These results suggest a possible role of hookworm-induced enteritis on microbiota stability.

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

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.

Human dendritic cell sequestration onto the Necator americanus larval sheath during ex-sheathing: a possible mechanism for immune privilege.

Despite the profound health implications of Necator americanus infection in humans, many aspects of its interaction with the host immune system are poorly understood. Here we investigated the early events at the interface of N. americanus larvae (L3) and human dendritic cells (DCs). Our data show that co-culturing DCs and the larvae trigger ex-sheathing of hookworms rapidly where a majority of DCs are sequestered onto the larval sheath allowing the ex-sheathed larvae to migrate away unchallenged. Intriguingly, DCs show negligible interaction with the ex-sheathed larvae, alluding to differences between the surface chemistry of the larva and its sheath. Furthermore, blocking of two key C-type lectin receptors on DC surface (i.e. DC-SIGN and mannose receptor) resulted in inhibition of ex-sheathing process and DC sequestration, highlighting the importance of C-type lectins on DCs in the induction of the ex-sheathing. Analyses of DC phenotype and cytokine profile after co-culture with the N. americanus larvae showed an immature phenotype as evidenced by the low expression of the maturation markers and cytokines. These data provide new insights into early events at the interface of human DCs and N. americanus larvae and could explain how L3 evade immune recognition upon initial interaction with DCs.

Immune polarization by hookworms: taking cues from T helper type 2, type 2 innate lymphoid cells and alternatively activated macrophages.

Cellular and molecular investigation of parasitic helminth infections has greatly accelerated the understanding of type 2 immune responses. However, there remains considerable debate regarding the specific leucocytes that kill parasites and whether these mechanisms are distinct from those responsible for tissue repair. Herein, we chronicle discoveries over the past decade highlighting current paradigms in type 2 immunity with a particular emphasis upon how CD4(+) T helper type 2 cells, type 2 innate lymphoid cells and alternatively activated macrophages coordinately control helminth-induced parasitism. Primarily, this review will draw from studies of the murine nematode parasite Nippostrongylus brasiliensis, which bears important similarities to the human hookworms Ancylostoma duodenale and Necator americanus. Given that one or more hookworm species currently infect millions of individuals across the globe, we propose that vaccine and/or pharmaceutical-based cure strategies targeting these affected human populations should incorporate the conceptual advances outlined herein.

Impact of experimental hookworm infection on the human gut microbiota.

The interactions between gastrointestinal parasitic helminths and commensal bacteria are likely to play a pivotal role in the establishment of host-parasite cross-talk, ultimately shaping the development of the intestinal immune system. However, little information is available on the impact of infections by gastrointestinal helminths on the bacterial communities inhabiting the human gut. We used 16S rRNA gene amplification and pyrosequencing to characterize, for the first time to our knowledge, the differences in composition and relative abundance of fecal microbial communities in human subjects prior to and following experimental infection with the blood-feeding intestinal hookworm, Necator americanus. Our data show that, although hookworm infection leads to a minor increase in microbial species richness, no detectable effect is observed on community structure, diversity or relative abundance of individual bacterial species.

Integrated school-based surveillance for soil-transmitted helminth infections and lymphatic filariasis in Gampaha district, Sri Lanka.

We explored the practicality of integrating surveillance for soil-transmitted helminthiasis (STH, assessed by Kato-Katz) with transmission assessment surveys for lymphatic filariasis (LF) in two evaluation units (EUs) in Gampaha district, Sri Lanka (population 2.3 million). The surveys were performed 6 years after five annual rounds of mass drug administration with diethylcarbamazine and albendazole. Each transmission assessment survey tested children (N = 1,462 inland EU; 1,642 coastal EU) sampled from 30 primary schools. Low filarial antigenemia rates (0% and 0.1% for the inland and coastal EUs) suggest that LF transmission is very low in this district. The STH rates and stool sample participation rates were 0.8% and 61% (inland) and 2.8% and 58% (coastal). Most STH detected were low or moderate intensity Trichuris trichiura infections. The added cost of including STH testing was ∼$5,000 per EU. These results suggest that it is feasible to integrate school-based surveillance for STH and LF.

Genome of the human hookworm Necator americanus.

The hookworm Necator americanus is the predominant soil-transmitted human parasite. Adult worms feed on blood in the small intestine, causing iron-deficiency anemia, malnutrition, growth and development stunting in children, and severe morbidity and mortality during pregnancy in women. We report sequencing and assembly of the N. americanus genome (244 Mb, 19,151 genes). Characterization of this first hookworm genome sequence identified genes orchestrating the hookworm's invasion of the human host, genes involved in blood feeding and development, and genes encoding proteins that represent new potential drug targets against hookworms. N. americanus has undergone a considerable and unique expansion of immunomodulator proteins, some of which we highlight as potential treatments against inflammatory diseases. We also used a protein microarray to demonstrate a postgenomic application of the hookworm genome sequence. This genome provides an invaluable resource to boost ongoing efforts toward fundamental and applied postgenomic research, including the development of new methods to control hookworm and human immunological diseases.

Generalized urticaria induced by the Na-ASP-2 hookworm vaccine: implications for the development of vaccines against helminths.

BACKGROUND: Necator americanus Ancylostoma-secreted protein 2 (Na-ASP-2) is secreted by infective hookworm larvae on entry into human hosts. Vaccination of laboratory animals with recombinant Na-ASP-2 provides significant protection against challenge infections. In endemic areas antibodies to Na-ASP-2 are associated with reduced risk of heavy N americanus infections. OBJECTIVE: To assess the safety and immunogenicity of recombinant Na-ASP-2 adjuvanted with Alhydrogel in healthy Brazilian adults previously infected with N americanus. METHODS: Participants were randomized to receive Na-ASP-2 or hepatitis B vaccine. Major IgG and IgE epitopes of the Na-ASP-2 molecule were mapped by using sera from these same subjects. Seroepidemiologic studies in adults and children residing in hookworm-endemic areas were conducted to assess the prevalence of IgE responses to Na-ASP-2. RESULTS: Vaccination with a single dose of Na-ASP-2 resulted in generalized urticarial reactions in several volunteers. These reactions were associated with pre-existing Na-ASP-2-specific IgE likely induced by previous hookworm infection. Surveys revealed that a significant proportion of the population in hookworm-endemic areas had increased levels of IgE to Na-ASP-2. Epitope mapping demonstrated sites on the Na-ASP-2 molecule that are uniquely or jointly recognized by IgG and IgE antibodies. CONCLUSION: Infection with N americanus induces increased levels of total and specific IgE to Na-ASP-2 that result in generalized urticaria on vaccination with recombinant Na-ASP-2. These data advance knowledge of vaccine development for helminths given their propensity to induce strong T(H)2 responses. Study data highlight the important differences between the immune responses to natural helminth infection and to vaccination with a recombinant helminth antigen.

Acquired hookworm immunity in the golden hamster (Mesocricetus auratus) elicited by living Necator americanus third-stage infective larvae.

The aim of the study is to demonstrate and understand the acquired immunity in golden hamsters (Mesocricetus auratus) elicited by primary Necator americanus infective third-stage larvae (L3) infection. Hamsters infected with 150 L3 for 1, 2, 3, 6 and 10 weeks, were challenged with the same number of L3 and sacrificed 25 days post challenge. The primarily infected hamsters exhibited 99-100% protection against subsequent L3 challenge compared to un-infected naive hamsters. The acquired immunity was developed as early as 1 week post L3 infection and lasted up to 10 weeks. Similar protective immunity was obtained in hamsters infected with N. americanus L3 and then treated orally with a single of 100mg/kg albendazole, followed by challenge with N. americanus L3 4 and 8 weeks post-treatment. The infected hamsters exhibited a rise in IgG antibodies against L3 and juvenile adult worm antigens. Histological examination showed that challenging L3 were trapped in the skin of primarily infected hamsters and surrounded or infiltrated by different inflammatory cells. The trapped L3 were damaged and dead followed by the formation of granulomas encasing dead worms. The results demonstrate that hamsters primarily infected with N. americanus L3 develop acquired immunity against re-infection.

A history of hookworm vaccine development.

The human hookworms Necator americanus and Ancylostoma duodenale remain among the most common infections of humans in areas of rural poverty in the developing regions of the world, with an estimated 1 billion people infected with one or more of these parasites. Herein, we review the nearly 100 years of research, development, animal testing, and fieldwork that have led to our current progress in recombinant hookworm vaccines. We begin with the identification of hookworm at the start of the 20th century in Southern US, then discuss the progress in developed countries to eliminate human hookworm infection, and then the industrial development and field use in the 1970s a canine hookworm vaccine(Ancylostoma caninum), and finally our progress to date in the development and clinical testing of an array of recombinant antigens to prevent human hookworm disease from N. americanus infection. Special attention is given to the challenges faced in the development of a vaccine against a blood-feeding nematode, including the epidemiology of infection (high prevalence of infection), pathogenesis (chronic infection that increases with the age of the host), and a robust immune response that fails to confer the protection in the host and a concomitant absence of correlates of protection by a successful vaccine could be developed and tested. Finally, we provide the optimal and acceptable profiles of a human hookworm vaccine, including the proposed indication, target population, and route of administration, as developed by the Human Hookworm Vaccine Initiative, the only group currently working on vaccines targeting this parasite.

Necator americanus and helminth co-infections: further down-modulation of hookworm-specific type 1 immune responses.

BACKGROUND: Helminth co-infection in humans is common in tropical regions of the world where transmission of soil-transmitted helminths such as Ascaris lumbricoides, Trichuris trichiura, and the hookworms Necator americanus and Ancylostoma duodenale as well as other helminths such as Schistosoma mansoni often occur simultaneously. METHODOLOGY: We investigated whether co-infection with another helminth(s) altered the human immune response to crude antigen extracts from either different stages of N. americanus infection (infective third stage or adult) or different crude antigen extract preparations (adult somatic and adult excretory/secretory). Using these antigens, we compared the cellular and humoral immune responses of individuals mono-infected with hookworm (N. americanus) and individuals co-infected with hookworm and other helminth infections, namely co-infection with either A. lumbricoides, Schistosoma mansoni, or both. Immunological variables were compared between hookworm infection group (mono- versus co-infected) by bootstrap, and principal component analysis (PCA) was used as a data reduction method. CONCLUSIONS: Contrary to several animal studies of helminth co-infection, we found that co-infected individuals had a further downmodulated Th1 cytokine response (e.g., reduced INF-γ), accompanied by a significant increase in the hookworm-specific humoral immune response (e.g. higher levels of IgE or IgG4 to crude antigen extracts) compared with mono- infected individuals. Neither of these changes was associated with a reduction of hookworm infection intensity in helminth co-infected individuals. From the standpoint of hookworm vaccine development, these results are relevant; i.e., the specific immune response to hookworm vaccine antigens might be altered by infection with another helminth.

Suppression of inflammatory immune responses in celiac disease by experimental hookworm infection.

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-)γ 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 and mucosa 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-γ 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.

Molecular cloning, biochemical characterization, and partial protective immunity of the heme-binding glutathione S-transferases from the human hookworm Necator americanus.

Hookworm glutathione S-transferases (GSTs) are critical for parasite blood feeding and survival and represent potential targets for vaccination. Three cDNAs, each encoding a full-length GST protein from the human hookworm Necator americanus (and designated Na-GST-1, Na-GST-2, and Na-GST-3, respectively) were isolated from cDNA based on their sequence similarity to Ac-GST-1, a GST from the dog hookworm Ancylostoma caninum. The open reading frames of the three N. americanus GSTs each contain 206 amino acids with 51% to 69% sequence identity between each other and Ac-GST-1. Sequence alignment with GSTs from other organisms shows that the three Na-GSTs belong to a nematode-specific nu-class GST family. All three Na-GSTs, when expressed in Pichia pastoris, exhibited low lipid peroxidase and glutathione-conjugating enzymatic activities but high heme-binding capacities, and they may be involved in the detoxification and/or transport of heme. In two separate vaccine trials, recombinant Na-GST-1 formulated with Alhydrogel elicited 32 and 39% reductions in adult hookworm burdens (P < 0.05) following N. americanus larval challenge relative to the results for a group immunized with Alhydrogel alone. In contrast, no protection was observed in vaccine trials with Na-GST-2 or Na-GST-3. On the basis of these and other preclinical data, Na-GST-1 is under possible consideration for further vaccine development.

Antigen-driven basophil activation is indicative of early Necator americanus infection in IgE-seronegative patients.

BACKGROUND: Parasitic worms induce a strong, polarized T(H)2-type immune response. The kinetics of gastrointestinal nematode-induced T(H)2-type responses, especially in the context of primary infection, have been extensively studied in experimental infection models but not in human subjects. OBJECTIVE: We sought to determine the kinetics of basophil sensitization in subjects infected with Necator americanus during the first 12 weeks after infection. METHODS: Thirty nonasthmatic subjects with allergic rhinoconjunctivitis were randomized in a double-blind manner to cutaneous administration of either 10 hookworm infective larvae or histamine placebo. Blood samples were taken at regular intervals for 12 weeks, and basophil activation was determined in whole blood by measuring CD63 and CD203c levels on stimulation with N americanus excretions/secretions. Parasite-specific immunoglobulin responses were assessed by means of ELISA and Western blotting. RESULTS: Median values reflecting basophil activation (CD203c/CD63 double-positive cells) in the excretion/secretion-stimulated infected group steadily increased after week 4, consistently achieving statistical significance compared with the placebo group between 6 and 12 weeks after infection. Only parasite-specific IgM levels increased significantly during this period, whereas total and parasite-specific IgE levels did not differ between groups. CONCLUSION: Basophils are sensitized early in the context of a low-dose primary infection with N americanus in the absence of measurable total and specific IgE serum level increase.

[Relevance of helminths in the prevention and healing of immune diseases]. / Relevancia de los helmintos en la prevención y curación de las enfermedades inmunes.

The incidence of immune diseases has had a steady significant increase in the industrialized countries since the end of the last century. Epidemiological data suggest that prevention of exposure to helminths may be one of the factors promoting the rise in these diseases. Animal models of immune diseases support this concept by showing that these parasites can prevent and reverse developed diseases. Helminths strongly modulate the host's immune system inducing regulatory immune cells and pathways that afford protection from these illnesses. Helminthic therapy has emerged as a promising treatment for several immune diseases; the potential benefits are startling and new therapeutic strategies for the control of these conditions may result.