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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 (T regs ) frequencies in both settings, markers of T regs function, including inducible T-cell costimulatory (ICOS), tumor necrosis factor receptor 2 (TNFR2), and latency-associated peptide (LAP), as well as in vitro T regs 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., (© 2024. The Author(s).)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ribeiro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2024 The Authors. Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of the Australian and New Zealand Society for Immunology, Inc.)

In this chapter, we describe the scientific, technical, clinical and regulatory aspects of establishing a controlled human hookworm infection (CHHI) model in non-endemic and endemic geographical regions, to facilitate a pathway towards accelerated vaccine development. The success achieved in establishing the CHHI platform specifically allows the Human Hookworm Vaccine Initiative (HHVI) to accelerate its progress by establishing a human hookworm vaccination/challenge model (HVCM) in a hookworm endemic area of Brazil. The HVCM will permit the rapid and robust determination of clinical efficacy in adults, allowing for early selection of the most efficacious human hookworm vaccine (HHV) candidate(s) to advance into later-stage pivotal paediatric clinical trials and reduce the overall number of participants required to assess efficacy (Diemert et al. 2018)., (© 2021. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

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., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

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

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.

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.

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.

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., (© 2016 John Wiley & Sons Ltd.)

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., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.)

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.

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.

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., (Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.

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.

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.

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.

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.

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 alpha- and beta-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.

Background: Hookworms survive for several years (5 to 7 years) in the host lumen, inducing a robust but largely ineffective immune response. Among the most striking aspects of the immune response to hookworm (as with many other helminths) is the ablation of parasite-specific T cell proliferative response (hyporesponsiveness). While the role of the adaptive immune response in human helminth infection has been well investigated, the role of the innate immune responses (e.g., dendritic cells and eosinophils) has received less attention and remains to be clearly elucidated., Methodology/principal Findings: We report on the differentiation/maturation of host dendritic cells in vitro and the eosinophil activation/function associated with human hookworm infection. Mature DCs (mDCs) from Necator americanus (Necator)-infected individuals showed an impaired differentiation process compared to the mDCs of non-infected individuals, as evidenced by the differential expression of CD11c and CD14. These same hookworm-infected individuals also presented significantly down-regulated expression of CD86, CD1a, HLA-ABC, and HLA-DR. The lower expression of co-stimulatory and antigen presentation molecules by hookworm-infected-derived mDCs was further evidenced by their reduced ability to induce cell proliferation. We also showed that this alternative DC differentiation is partially induced by excreted-secreted hookworm products. Conversely, eosinophils from the same individuals showed a highly activated status, with an upregulation of major cell surface markers. Antigen-pulsed eosinophils from N. americanus-infected individuals induced significant cell proliferation of autologous PBMCs, when compared to non-infected individuals., Conclusion: Chronic N. americanus infection alters the host's innate immune response, resulting in a possible modulation of the maturation process of DCs, a functional change that may diminish their ability for antigen presentation and thus contribute to the ablation of the parasite-specific T cell proliferative response. Interestingly, a concomitant upregulation of the major cell surface markers of eosinophils was observed in hookworm-infected individuals, indicative of antigen-specific immune responses, especially antigen presentation. We showed that in addition to the postulated role of the eosinophils as effector cells against helminth infection, activated cells may also be recruited to sites of inflammation and contribute to the immune response acting as antigen presenting cells.

The impact of the interaction between excreted and/or secreted (ES) Necator americanus products and NK cells from Necator-infected individuals was analyzed. We investigated the binding of ES products to NK cells, the expression of NK cell receptors (CD56, CD159a/NKG2A, CD314/NKG2D, CD335/NKp46, and KLRF1/NKp80), the frequency of gamma interferon (IFN-gamma)-producing NK cells after whole-blood in vitro stimulation, and the capacity of N. americanus ES products to induce NK cell chemotaxis. In contrast to those from noninfected individuals, NK cells from Necator-infected individuals demonstrated no binding with N. americanus ES products. This phenomenon was not due to alterations in NK cell receptor expression in infected subjects and could not be reproduced with NK cells from uninfected individuals by incubation with immunoregulatory cytokines (interleukin-10/transforming growth factor beta). Further, we found that a significantly greater percentage of NK cells from infected subjects than NK cells from uninfected individuals spontaneously produced IFN-gamma upon ex vivo culture. Our findings support a model whereby NK cells from Necator-infected individuals may interact with ES products, making these cells refractory to binding with exogenous ES products. During N. americanus infection, human NK cells are attracted to the site of infection by chemotactic ES products produced by adult Necator worms in the gut mucosa. Binding of ES products causes the NK cells to become activated and secrete IFN-gamma locally, thereby contributing to the adult hookworm's ability to evade host immune responses.

To determine the cellular immune response during different stages of hookworm infection, we infected two human volunteers with Necator americanus and followed their immune responses against stage-specific, crude antigen extracts through larval migration, pre-patency, and early patency. After chemotherapy, the volunteers were followed for an additional 6 months. Low-dose N. americanus infection resulted in mild clinical signs and peripheral blood eosinophilia occurred during the estimated time of arrival of juvenile worms in the intestine. After an initial rise in proliferative responses against larval and adult worm antigens, the cellular reactivity decreased until the end of pre-patency, rose again during patency, and dropped after chemotherapy. Before infection and during the course of infection, elevated concentrations of TNF-alpha were detected in supernatants of peripheral blood mononuclear cells (PBMC) stimulated in vitro with third-stage (L3) or adult worm excretory-secretory (ES) antigens, which dropped off after chemotherapy. After stimulation with L3 antigen, elevated concentrations of CCL17 were detected in supernatants during pre-patency and patency. Interestingly, a prominent rise in IL-10 secretion was detected in ES antigen-stimulated PBMC cultures during late pre-patency. During reinfection studies in endemic areas, such distinct cytokine and chemokine profiles might be additional markers to better classify egg-negative patients.

The L3-secreted Ancylostoma Secreted Protein-2 from the human hookworm Necator americanus (Na-ASP-2) has been selected as a candidate vaccine antigen in anticipation of clinical trials. Its crystal structure revealed that Na-ASP-2 has structural and charge similarities to CC-chemokines, suggesting that it might act as a chemokine mimic when released by the infective larvae during tissue migration. Using the air pouch model of acute inflammation, we found that Na-ASP-2 induced a significant leukocyte influx to the skin pouch, mostly comprised of neutrophils (60%) and monocytes (30%) that was transient and resolved in 24h. Other hookworm larval proteins did not cause any inflammatory leukocytes to migrate into air pouches. In vitro chemotaxis assays confirmed our results and demonstrated that leukocyte migration was a direct effect of Na-ASP-2 exposure and not caused by other molecules released by host cells in the inflammatory microenvironment or by the expression vector.

A popular hypothesis to explain parasite survival in the presence of a pronounced T helper 2 phenotype in helminth-parasitized populations has been Fc epsilonRI blockade by parasite-induced polyclonal IgE. To begin to test the hypothesis that Fc epsilonRI-bearing cells would be refractory to activation in parasitized populations, we investigated basophil function in 43 individuals from a hookworm endemic area. Study individuals had high levels of total IgE and eosinophilia and a mean hookworm burden of 2,257 epg. Basophils from all members of this parasitized population were shown to release histamine to a number of agonists, including anti IgE and a hookworm allergen, calreticulin. These data would indicate that Fc epsilonRI blockade at the level of the basophil did not occur in this parasitized population despite the presence of possible immunologic blocking agents. This would suggest that this effector arm of the T helper 2 phenotype remains operative in infected populations.

We describe how hookworms interact with their human hosts by comparing lymphocyte phenotyping, proliferative responses, and cytokine and chemokine secretion patterns in adults who are either mono-infected with Necator americanus or egg-negative controls resident in an area of high transmission in Brazil. Cellular immune responses against crude hookworm antigen extracts from different developmental stages were evaluated simultaneously. Principal component analysis (PCA) was used to reduce the standardized immune responses. Random effects multivariate regression was then used to investigate whether principal components (PC) differ between the two groups once potential confounders and effect modifiers have been accounted for. Although hookworm patients had reduced percentages of T and B cells, they had higher levels of activated CD4(+) T and CD19(+) B cells. This state of 'immune activation' coincided with lower proliferative responses, especially to third-stage larval antigen. Cytokine levels in mono-infected adults were also lower and characterized by a mixed Th1/Th2-type profile. Excretory/secretory antigen from adult worms was a potent modulator of the immune response, resulting in diminished TNF-alpha and IL-10 secretion in peripheral blood mononuclear cells (PBMC) from hookworm infected patients. We propose that the longevity of hookworms in their human hosts results from a stage-specific, down-modulation of the immune response.

The recommended control option against onchocerciasis is repeated ivermectin treatment, which will need to be implemented for decades, and it remains unknown how repeated ivermectin therapy might affect immunity against Onchocerca volvulus in the long term. O. volvulus-specific antibody reactivity and cellular cytokine production were investigated in onchocerciasis patients receiving ivermectin (150 microg/kg) annually for 16 years. In treated patients, the T helper type 2 (Th2) cytokine interleukin (IL)-5 and T regulatory IL-10 in response to O. volvulus antigen (OvAg) and bacteria-derived Streptolysin O (SL-O) diminished to levels found in infection-free endemic controls; also, cellular release of Th1-type interferon (IFN)-gamma at 16 years post initial ivermectin treatment (p.i.t.) approached control levels. In ivermectin-treated onchocerciasis patients, IL-5 production in responses to the mitogen phytohaemagglutinin (PHA) decreased, but IL-10 in response PHA increased, and neither attained the cytokine production levels of endemic controls. At 16 years p.i.t., O. volvulus-specific IgG1 and IgG4 subclass reactivity still persisted at higher levels in onchocerciasis patients than in O. volvulus exposed but microfilariae-free endemic controls. In addition, cytokine responses remained depressed in onchocerciasis patients infected concurrently with Mansonella perstans and Necator americanus or Entamoeba histolytica/dispar. Thus, long-term ivermectin therapy of onchocerciasis may not suffice to re-establish fully a balanced Th1 and Th2 immune responsiveness in O. volvulus microfilariae-negative individuals. Such deficient reconstitution of immune competence may be due to an as yet continuing and uncontrolled reinfection with O. volvulus, but parasite co-infections can also bias and may prevent the development of such immunity.

It is unclear how immunity limits hookworm infection. Australian researchers, using capsule and conventional gastrointestinal endoscopy in volunteers inoculated with Necator americanus, have reported that virtually all larvae reach the intestine within six weeks. Unlike the neutral response surrounding resident hookworms, newly arrived adults provoke an eosinophilic enteropathy. This allergic reaction curtails the attachment of hookworms and accompanies the passage of additional worms as they are expelled from the proximal small intestine.

The protective immunity elicited by ultraviolet-irradiated third-stage infective larvae of Necator americanus (UV-NaL3) and Ancylostoma caninum (UV-AcL3) was evaluated in laboratory mice (a non-permissive model) and hamsters (a permissive model). After optimizing the time of exposure to UV-irradiation, both oral and subcutaneous vaccination routes with UV-AcL3 in mice were explored. Oral vaccination was more effective at reducing the number of challenge AcL3 entering the lungs, whereas subcutaneous vaccination was more effective at blocking muscle entry. When UV-irradiated NaL3 and non-irradiated AcL3 were used as vaccines in hamsters, both of them were effective at reducing adult hookworm burdens. However, the length of protection afforded by UV-irradiated L3 was substantially greater than that resulting from immunization with non-irradiated L3. A single dose was less effective than multiple doses. The protective immunity elicited by UV-irradiated NaL3 given once every other week for a total of three immunizations was similar to that elicited by non-irradiated AcL3 given during the same schedule. Protection was not significantly affected by administering the L3 on a weekly basis for a total of three immunizations, even though the antibody titers were reduced using this schedule. These studies will facilitate the elucidation of the mechanisms underlying larval protection.

To characterize the immune response following primary human hookworm infection, an adult volunteer was infected with 50 L3 larvae of Necator americanus, reinfected 27 months later and followed for a further 6 months. Clinical signs, blood picture, ex-vivo peripheral blood cytokine production (IFN-gamma, IL-5, IL-13, IL-10 to mitogen and hookworm antigen), acute phase proteins (APP) (C-reactive protein, CRP and alpha1-antitrypsin, alpha1-AT) and antibody levels were determined. Dermatitis, oedema, mild nausea and abdominal discomfort followed the primary infection. Eosinophil counts peaked early during both infections but remained elevated ( approximately 18%) throughout. Transient production of IL-5, IL-13 and APP also followed infection but there were negligible levels of IFN-gamma or IL-10. The onset of nausea, oedema and the initial rise in CRP, alpha1-AT, eosinophilia and IL-5 coincided (days 13-27) with the late larval migration and early establishment of the preadult worms in the intestine. Apart from the eosinophilia these responses declined to baseline levels within 4 months and were less pronounced on re-infection.

A vaccine against the human hookworm Necator americanus is urgently required to reduce hookworm-induced morbidity in endemic areas. In the present study, recombinant hookworm calreticulin, a nominated vaccine candidate, has been tested in mice. Mice given calreticulin had 43-49% fewer worms in their lungs, compared to non-vaccinated controls, following challenge infection with infective hookworm larvae. These levels of protection were achieved in the absence of adjuvant following intraperitoneal administration of three doses of 15 microg antigen. Antigen was also encapsulated in PLG microparticles. Encapsulated calreticulin elicited higher levels of anti-calreticulin IgG1 than free antigen but failed to induce protective immunity. The protection induced by free calreticulin was associated with low levels of serum IgE and moderate lung eosinophilia whilst administration of calreticulin-loaded microparticles was associated with high levels of serum IgE and higher lung eosinophil activity, suggesting that the classical Th2 phenotype may not always be associated with protective immunity, albeit in experimental necatoriasis.

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 1.68 A 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 alphabetaalpha 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.

Hookworms infect almost one billion people and are a major cause of iron-deficiency anaemia in developing countries of the tropics. Despite their prevalence and the morbidity they cause, little is known about the immune response to this complex eukaryotic parasite. Recent publications have shed light on the human cellular immune responses to hookworms, as well as mechanisms that hookworms utilize to skew the immune response in its favour. Unlike most other human helminth infections, neither age- nor exposure-related immunity develops in the majority of infected people. A vaccine is therefore a highly desirable goal. To this end, gene sequencing efforts have resulted in the deposition of more than 10,000 hookworm cDNA sequences in the public domain, providing a molecular snapshot of this intriguing parasite and providing novel tools for the development of new control strategies. Significant progress has been made in the development of anti-hookworm recombinant vaccines, and clinical trials are expected to begin in the near future.

Advances in hookworm immunoepidemiology are reviewed. Recent studies demonstrate a mixed Th1/Th2 response in human hookworm infection, with immunosuppression of specific and nonspecific IFN-gamma responses. There is increasing evidence for protective immunity in human hookworm infection, including anti-larval IL-5- and IgE-dependent mechanisms, and for immunological interactions between hookworm infection and other diseases.

Cytokine and proliferative responses to Necator americanus infection were measured in a treatment-reinfection study of infected subjects from an area of Papua New Guinea where N. americanus is highly endemic. Before treatment, most subjects produced detectable interleukin (IL)-4 (97%), IL-5 (86%), and interferon (IFN)- gamma (64%) in response to adult N. americanus antigen. Pretreatment IFN- gamma responses were negatively associated with hookworm burden, decreasing by 18 pg/mL for each increase of 1000 eggs/gram (epg) (n=75; P<.01). Mean IFN- gamma responses increased significantly after anthelmintic treatment, from 166 to 322 pg/mL (n=42; P<.01). The intensity of reinfection was significantly negatively correlated with pretreatment IL-5 responses, decreasing by 551 epg for each 100 pg/mL increase in production of IL-5 (n=51; P<.01). These data indicate that there is a mixed cytokine response in necatoriasis, with worm burden-associated suppression of IFN- gamma responses to adult N. americanus antigen. Resistance to reinfection is associated with the parasite-specific IL-5 response.

Human hookworm infections are distributed widely in tropical areas and have a significant impact on host morbidity and human health. In the present study, we investigated the cellular responsiveness and cytokine production in peripheral blood mononuclear cells (PBMC) from Necator americanus-infected schoolchildren who had recently received chemotherapy, and compared them with non-infected endemic controls. Hookworm patients and treated, egg-negative individuals showed a lower cellular reactivity against phytohaemagglutinin (PHA) and hookworm antigen when compared with egg-negative endemic controls. The baseline production of proinflammatory tumour necrosis factor-alpha (TNF-alpha) in PBMC from infected patients and treated, egg-negative individuals was elevated. On the other hand, PHA- or hookworm antigen-induced interleukin (IL)-12 and interferon (IFN)-gamma secretion was higher in endemic controls than in hookworm patients, who either continued egg-positive or were egg-negative after treatment. Also, PBMC from endemic controls secreted more IL-5 and IL-13 than the other patient groups. Opposite to that, the spontaneous as well as the antigen-driven IL-10 secretion was lower in endemic controls when compared with the other groups. In summary, patently hookworm-infected as well as egg-negative treated patients disclosed an elevated spontaneous cellular secretion of proinflammatory TNF-alpha, a prominent secretion of regulatory Th2-type IL-10 and an impaired production of IL-12, IFN-gamma, IL-5 and IL-13.

Helminth parasites are well known to induce an immune response in their hosts characterised by elevated IgE, peripheral blood or local tissue eosinophilia, and in some cases, intestinal mastocytosis. This immunological response has a strong T-helper 2 (Th2) cytokine bias and is reminiscent of the immunological constellation found in allergic diseases. However, the molecular forces driving the Th2 response to helminth parasites are still not understood. By using the human hookworm parasite Necator americanus as an example, the authors of the current article propose that in the course of its life cycle, this parasite becomes innately allergenic through the secretion of a molecular array designed to promote tissue migration and homing, feeding and survival against immunological attack. This complex array comprises proteases, lectins and other classes of molecules. Subsequent immunological and physiological events seemingly protect the host from both the allergic sequelae of exposure to environmental allergens and, moreover, from the parasite itself.

Hookworm infection is one of the most important parasitic infections of humans, possibly outranked only by malaria as a cause of misery and suffering. An estimated 1.2 billion people are infected with hookworm in areas of rural poverty in the tropics and subtropics. Epidemiological data collected in China, Southeast Asia and Brazil indicate that, unlike other soil-transmitted helminth infections, the highest hookworm burdens typically occur in adult populations, including the elderly. Emerging data on the host cellular immune responses of chronically infected populations suggest that hookworms induce a state of host anergy and immune hyporesponsiveness. These features account for the high rates of hookworm reinfection following treatment with anthelminthic drugs and therefore, the failure of anthelminthics to control hookworm. Despite the inability of the human host to develop naturally acquired immune responses to hookworm, there is evidence for the feasibility of developing a vaccine based on the successes of immunising laboratory animals with either attenuated larval vaccines or antigens extracted from the alimentary canal of adult blood-feeding stages. The major antigens associated with each of these larval and adult hookworm vaccines have been cloned and expressed in prokaryotic and eukaryotic systems. However, only eukaryotic expression systems (e.g., yeast, baculovirus, and insect cells) produce recombinant proteins that immunologically resemble the corresponding native antigens. A challenge for vaccinologists is to formulate selected eukaryotic antigens with appropriate adjuvants in order to elicit high antibody titres. In some cases, antigen-specific IgE responses are required to mediate protection. Another challenge will be to produce anti-hookworm vaccine antigens at high yield low cost suitable for immunising large impoverished populations living in the developing nations of the tropics.

In this murine (BALB/c) model of necatoriasis, high levels of protection against challenge infection by Necator americanus larvae (n=300) were afforded by successive vaccinations at 14-day intervals, either subcutaneously or percutaneously, with gamma-irradiated N. americanus larvae (n=300). Percutaneous vaccination was significantly more effective than the subcutaneous route, with pulmonary larval burdens at 3 days post-infection being reduced by 97.8 vs. 89.3%, respectively, after three immunisations (P<0.05). No worms were recovered from the intestines of thrice vaccinated mice. Two percutaneous vaccinations also reduced worm burdens, by 57% in the lungs and 98% in the intestines; P<0.05. In vaccinated animals, lung pathology (mainly haemorrhage) following infection was greatly reduced compared with non-vaccinated animals. In vaccinated mice (but not in non-vaccinated mice) mast cells accumulated in the skin and were degranulated. RT-PCR analyses of mRNAs in the skin of vaccinated animals indicated increased expression of interleukin (IL)-4 relative to gamma-interferon (gamma-IFN). Lymphocytes from the axillary (skin-draining) lymph nodes of vaccinated mice, stimulated in vitro with concanavalin A, exhibited enhanced secretion of IL-4 protein and a higher IL-4/gamma-IFN protein ratio than lymphocytes from non-vaccinated animals. In vaccinated mice, levels of IgG1 and IgG3 (directed against larval excretory/secretory products) were elevated for the most part compared with those in non-vaccinated animals. These data demonstrate the successful vaccination of BALB/c mice against human hookworm infection and suggest that a localised Th2 response may be important for conferring protection against necatoriasis.

Passage of helminth larvae through the lungs can cause pulmonary eosinophilia that may have evolved as a means of parasite attrition. If allergic responses represent a misdirected activation of this arm of the immune system, then mechanisms governing eosinophil recruitment during infection would be expected to be closely related to those seen in allergy. We studied primary Necator americanus infection and compared this to multiply-infected or vaccinated mice. The arrival of larvae in the lungs triggered rapid eosinophil recruitment, which was greatly enhanced in previously sensitized mice. Interestingly, the presence of larvae in the lung was sufficient to trigger eosinophil chemoattractant production, including the chemokines eotaxin and MIP-1alpha, and was not enhanced by prior exposure to the parasites. Infection stimulated IL-5 production in all groups; however, this and IgE production were greatly enhanced in sensitized animals. Elevated IL-5 increased bone marrow production of eosinophils, and eosinophilia was abrogated by treatment with anti-IL-5 antibody. Therefore, trapping of larvae in the pulmonary vasculature is sufficient to trigger eosinophil recruitment, by induction of chemokines and IL-5. Primed cognate Th2 immunity does not increase local chemokine production, but does increase IL-5 production, which greatly enhances the availability of eosinophils for recruitment to the lung.

Peripheral lymphocyte subsets CD3, CD4, CD8 were studied using monoclonal antibodies to determine the mechanism of immunosuppression observed in an earlier study with total T-cells using the sheep erythrocyte rosetting technique. The study was carried out in 37 Nigerian patients with hookworm infection (Necator americanus and Ancylostoma duodenale and 17 healthy Nigerians as controls. The leucocyte migration inhibition (LMI) test was also carried out to assess the functional integrity of the lymphocytes, while the hookworm status of the patients was assessed by the Stoll technique for measuring egg counts. The results of the T-cell studied showed that CD3 and CD4 cell percentages were significantly depressed in hookworm patients compared to controls (P<0.05). The difference between mean CD8 percentages and absolutes in hookworms patients and controls were not significant. Absolute CD3 and CD4 numbers were not significantly lowered compared to controls in the study and CD4/CD8 ratios were insignificantly reduced. Patients with polyparasitism including hookworm infection showed significant reduction in CD3 and CD4 cells when compared with controls and with patients with hookworm infection alone. The leucocyte migration inhibition response to purified protein derivative of M. tuberculosis (PPD) was significantly decreased in hookworm patients compared to controls, confirming that functionally cell mediated immunity is depressed in hookworm infection.

In this study parasite-specific antibody, cellular reactivity and Thl-type or Th2-type cytokine responses were investigated in humans concurrently infected with Necator americanus and Oesophagostomum bifurcum. The prospects for O. bifurcum-specific serodiagnosis based on IgG4 and IgE were evaluated. IgG4 showed low specificity for O. bifurcum due to antigen cross-reactivity with N. americanus, while IgE specifically distinguished between hookworm and O. bifurcum, and, in doubly infected patients, levels of O. bifurcum-specific as well as N. americanus-specific IgE were significantly elevated compared to those with N. americanus mono-infections. Cellular immunity was not strictly dominated by a Thl- or Th2- type reactivity. In co-infected patients cellular unresponsiveness to parasite antigens was observed, while cellular production of tumour necrosis factor alpha (TNF-alpha) and gamma-interferon (IFN-gamma) was greater in those doubly infected. Th2-type cytokines (interleukin-5 and interleukin-10) were produced in equal amounts by peripheral blood mononuclear cells from individuals with mono- and coinfections. Such mixed Thl-type and Th2-type immune responsiveness associated with persisting gastrointestinal parasitic nematodes may reflect a state of infection at which parasite-induced inflammatory and enteropathogenic responses co-exist, and furthermore, helminth coinfection will not only suppress parasite-specific cellular responsiveness but may also direct cytokine production towards a "permissive Th1-type cytokine profile" that favours parasite persistence.

The hookworm Necator americanus establishes infections of impressive longevity in the immunologically hostile environment of its human host. In the process, it promotes pronounced T-helper 2 (Th2) cell activity, which in turn seemingly affords the host at least a degree of protection. Given the relatively asymptomatic nature of infection, we argue here that Necator americanus might be approaching a mutualistic symbiotic relationship with humans. In our view, infection is controlled by the immune system while being supported by a subtle immune-evasion strategy that is tolerated and possibly beneficial to the host in certain immunological circumstances, such as in counterbalancing potentially damaging Th1 responses.

Immmoglobulin E-rich plasma from patients from Papua New Guinea infected with Necator americanus has been used to probe an adult N. americanus cDNA library for the presence of hookworm allergens. Using this approach, one hookworm allergen has been identified as calreticulin, which was subsequently expressed in Escherichia coli. Little serological cross reactivity was seen between the recombinant calreticulins of this hookworm and its host. Prospective roles for hookworm calreticulin in the host-parasite relationship are discussed in depth.

The blood and broncho-alveolar leucocyte (BAL) responses, to a primary Necator americanus infection, were studied in male BALB/c and NIH mice. Following percutaneous infection, a significant blood leucocytosis occurred in both the BALB/c and NIH strains. The peak response occurred, in both strains, on day 10 p.i. and this was reflected in the eosinophil response which peaked at the same time-point. The change in blood eosinophil numbers, as a result of infection, was found to be the greatest recorded for any cell type. In BALB/c mice, however, infection elicited a significantly more intense total leucocyte, lymphocyte and eosinophil response than in NIH mice. In contrast, the BAL response was more intense in the NIH strain. Peak BAL responses were observed between days 12 and 16 p.i., in both strains, and the change in broncho-alveolar eosinophil numbers, as a result of infection, was found to be the greatest recorded for any cell type. The relationship between the observed leucocyte responses and resistance to the migrating larvae of a primary infection is discussed.

Described is an epidemiological investigation of hookworm infections in a rural community in Zimbabwe, where Necator americanus is the only human helminth species present. Among a cohort of 120 individuals the overall prevalence of infection was 78%. Intensity of infection was quantified both as egg counts (range: 0-2563 eggs per g of stool) and worm burden (range: 0-100 worms). Although both these measures provide useful quantitative data, they are tedious to determine in large-scale epidemiological studies and may present social and logistic difficulties. As an alternative screening method, we therefore investigated isotype-specific responses to adult worm antigens of N. americanus. The results show that specific IgG4 responses correlate positively and significantly with both measures of intensity and may be a useful marker of hookworm infection.