Dose-ranging study for trials of therapeutic infection with Necator americanus in humans.

Epidemiological studies suggest that a hookworm infection producing 50 eggs/gram of feces may protect against asthma. We conducted a dose-ranging study to identify the dose of hookworm larvae necessary to achieve 50 eggs/gram of feces for therapeutic trials of asthma. Ten healthy subjects without asthma or airway hyperresponsiveness to inhaled methacholine received 10, 25, 50, or 100 Necator americanus larvae administered double blind to an area of skin on the arm. Subjects were seen weekly for 12 weeks and were then treated with mebendazole. Skin itching at the entry site and gastrointestinal symptoms were common at higher doses. Lung function did not change. Levels of blood eosinophils and IgE increased transiently, and levels of IgG increased progressively. All doses resulted in at least 50 eggs/gram of feces in the eight subjects who completed the study. Infection with 10 N. americanus larvae is well tolerated, elicits a modest host eosinophil response, and is potentially suitable for use in preliminary clinical therapeutic trials.

Antigenic cross-reactivity between Necator americanus and Ascaris lumbricoides in a community in Papua New Guinea infected predominantly with hookworm.

Sero-epidemiological data are presented in which antigenic cross-reactivity between Necator americanus and Ascaris lumbricoides has been investigated in a community in Papua New Guinea infected predominantly with N. americanus. It is our contention that the antigenic cross-reactivity which undoubtedly exists between these species accounted for (i) a peak in antibody levels against N. americanus in 10-13 years old children (driven by infection with A. lumbricoides), and (ii) the maintenance of apparent antibody levels against A. lumbricoides in older age groups (driven by infection with N. americanus in the absence of overt infection with A. lumbricoides). Cross-reactivity was analysed further, and apparently N. americanus-specific epitopes identified, by immunoblotting. These observations could have considerable bearing on the interpretation of data from sero-epidemiological studies which failed to take account of concurrent infection with these parasites.

The identification of a species-specific antigen from Necator americanus.

A 17 kD protein of Necator americanus was isolated by SDS-PAGE and used to raise monospecific antisera in rabbits. ELISA and Western blotting against a range of parasite extracts demonstrated the species specificity of this protein. It is expressed at all stages of the life-cycle, appears to be accumulated through the larval stages to adulthood, and can be localized in the oesophageal glands and cuticle of the adult parasite. The possible nature and diagnostic potential of this protein is discussed.

Epidemiology and immunology of Necator americanus infection in a community in Papua New Guinea: humoral responses to excretory-secretory and cuticular collagen antigens.

Baseline data from an immuno-epidemiological study of hookworm infection in a rural village in Madang Province, Papua New Guinea are reported. Necator americanus was found to be the commonest helminth infection, with a prevalence of near 100% and intensity of 40 worms per host in adults. Enterobius vermicularis, Ascaris lumbricoides and Trichuris trichiura were also present, at prevalences of 53, 10 and 3% respectively; Ancylostoma duodenale was absent. The frequency distribution of N. americanus was highly over-dispersed, and was well described by a negative binomial distribution with aggregation parameter, k, of 0.370. Intensity of infection was significantly related to host age, but did not differ between the sexes. Haemoglobin levels and haematocrit values were indicative of anaemia in the community, but were unrelated to hookworm infection. Levels of antibodies (IgG, IgA and IgM combined) against adult Necator cuticular collagen and excretory-secretory (ES) products were determined. Serum concentrations of the two types of antibody were significantly correlated with each other. Significant positive correlations were found between anti-ES antibody levels and hookworm egg production, and between anti-collagen antibody levels and host age. It is suggested that the level of anti-collagen antibodies may reflect cumulative exposure to infection, whereas levels of anti-ES antibodies may be more dependent on current worm burden. No evidence was found to suggest that either antibody response is important in regulating parasite population growth. Similarly, the presence of a positive correlation between eosinophil concentration and infection intensity in adults indicates that eosinophilia reflects, rather than determines, the host's worm burden.

The action of a mast cell protease on the cuticular collagens of Necator americanus.

Collagen-like proteins, thought to be responsible for maintaining the structural integrity of the nematode cuticle, were isolated from adult Necator americanus and shown to be susceptible to digestion by purified mast cell proteases. Although these collagens would appear normally to be masked by superficially expressed (surface) antigens, it is suggested that a sufficiently avid and specific immune response could remove this potentially protective coat, rendering the structurally important underlying layers open to immune attack.

Acquired resistance to the human hookworm Necator americanus in mice.

BALB/c mice were exposed to primary or secondary infection with the hamster-adapted strain of Necator americanus, and the course of infection was monitored through worm recovery and immunological assays. Significantly fewer viable larvae were recovered from the skin site of reinfected mice on day 2 post-infection, and fewer larvae resided in the lungs of challenged mice 3-5 days after infection, suggesting that the skin was involved in resistance to secondary infection. The serum antibody response to L3 antigen was enhanced during secondary infection, peaking on day 9, and the bronchoalveolar leucocyte (BAL) response was more intense at this stage. Thus the secondary BAL response was initiated more promptly than the primary response, peaking on day 13 at twice the intensity of the primary response and five times above the resting level. Differential counts revealed that by far the most significant changes in cell populations were those observed for eosinophils in lavage fluid. At the peak of the response a 925-fold increase over control levels was detected in mice undergoing a challenge infection. Some cellular and serological components of the secondary response were defined in the present work and it was concluded that reinfected mice have the capacity to trap parasites during their passage through the skin and development in the lungs.

Cuticle preparations from Necator americanus and their immunogenicity in the infected host.

Cuticle samples have been prepared from the human parasitic nematode Necator americanus using detergent solubilisation with sodium dodecyl sulphate and subsequent reduction of disulphide bonds using 2-beta-mercaptoethanol. It would appear that N. americanus, like many other nematodes, relies on relatively small collagenous proteins, linked by disulphide bonds, to maintain the integrity of its cuticle. These molecules are normally hidden from the immune system during the course of infection but are potentially immunogenic. It is suggested that waves of antibodies, firstly with specificity against superficial cuticular epitopes followed by antibodies against deeper lying, structural elements, should be induced during vaccination to effectively destroy the nematode at the cuticular level.

The clinical and immunologic responses of normal human volunteers to low dose hookworm (Necator americanus) infection.

Five normal human volunteers were exposed to approximately 50 infective larvae of Necator americanus and were observed for the development of clinical signs or symptoms and for changes in blood eosinophil levels, IgG antibody titers, total and parasite-specific IgE, and lymphocyte blastogenic responses for 6-10 weeks. Bronchoalveolar lavage was performed on four subjects prior to infection and at times when larval migration through the pulmonary tree was likely. Eggs were demonstrated in the stools of four volunteers who remained untreated for more than 6 weeks; one volunteer had to be treated at day 40 because of severe gastrointestinal symptoms. All others also complained of abdominal pain and flatulence between days 35-40. All volunteers developed marked blood eosinophilia which peaked between days 38-64 and ranged from 1,350-3,828 eosinophils/mm3. Small increases in total and parasite-specific IgE and IgG were noted in some volunteers. One volunteer showed a significant lymphocyte blastogenic response. With the exception of mucosal erythema, bronchoalveolar lavage results were unremarkable. Our data indicate that a single small inoculum of hookworm larvae is capable of producing significant transient gastrointestinal morbidity and marked blood eosinophilia but does not induce other prominent T cell- and B cell-dependent immune responses.

Adherence of human eosinophils to infective filariform larvae of Necator americanus in vitro.

Eosinophilia is common in hookworm infection but the interaction between eosinophils and the larval stage of the parasite is poorly understood. The present study was conducted to test the ability of the eosinophils to adhere to infective filariform larvae of Necator americanus in vitro. Adherence of eosinophils to the larvae was found to be serum dependent. Antibody facilitated eosinophil adherence but this was maximal in the presence of complement. The adherence was greatly diminished by EGTA treated normal human serum (NHS) and was completely abolished when NHS was treated with either EDTA or heat-inactivation, suggesting that the process can be facilitated through complement activation via the alternative pathway. As with other nematodes, the surface of hookworm larvae appeared to be both antigenic and complement-activating. Although it is not known whether eosinophil adherence has any larvicidal effect, the present study demonstrated for the first time a definite interaction between human eosinophils and hookworm filariform larvae.

Antigen expression during development of the human hookworm, Necator americanus (Nematoda).

The accumulated and de novo synthesized antigens expressed by L3, L4 and adult Necator americanus, recognized by both the natural host, man, and the experimental host, the hamster, were identified by immunoblotting and immunoprecipitation analysis. Following infection of neonatal hamsters serum samples were taken on days 17, 35 and 117. Only serum taken 117 days after infection showed significant reactivity in immunoblotting experiments, recognizing adult epitopes of 30,000, 33,000, 48,000 and 69,000 mol. wt thereby suggesting that few accumulated antigens are shared between developmental stages. By contrast, immunoprecipitation analysis of metabolically labelled proteins suggested that L3 and in particular L4 larvae synthesize some antigens which comigrate with those synthesized and accumulated by adult worms. In addition, L4 larvae synthesize a 41,000 mol. wt excretory/secretory (ES) stage specific antigen. Parallel experiments using serum samples from infected humans, demonstrated that hamsters and man recognize many antigens of identical molecular weight. Notable in this respect are accumulated adult antigens of 30,000, 33,000, 48,000 and 69,000 and de novo synthesized antigens of 30,000, 33,000, 44,000, 46,000 and 69,000 mol. wt. Some individual human sera mainly recognized L3 antigens of 47,000-69,000 mol. wt in immunoblotting experiments whilst others simultaneously recognized adult epitopes. This differential recognition of developmental stages by individual human sera suggests that genetic or epidemiological factors are operative and warrants further study. Overall, these studies confirm the pronounced immunogenicity of Necator americanus in both man and an animal model and pave the way for analysis of the relevance of these antigens to field situations.

Necator americanus in neonatally infected hamsters. The time-course of infection and antibody response to the surface antigens of L4 and adult worms.

Neonatal hamsters were infected with a hamster-adapted strain of Necator americanus, and the time-course of infection was followed by worm and faecal egg counts. Parasite eggs were first recorded during the 6th week of infection, increasing rapidly thereafter to peak in weeks 7-10. Male hamsters excreted more eggs than females, but both sexes were equally susceptible to infection and harboured comparable worm burdens. Faecal egg counts declined from week 10 onwards and this was associated with a loss of worms from animals with heavy infections. Low level infections were stable over the first 114 d of infection but worm fecundity nevertheless still declined over this period. Both hamster sexes responded similarly to surface antigens on adult worms, the antibody levels rising from week 5 onwards to reach a plateau in weeks 6-7, which persisted until the experiments were terminated. The major antigens recognised on the surface of adult worms had molecular masses corresponding to 25 kDa, 32 kDa, a doublet with the heaviest polypeptide resolving at 46 kDa, and a triplet with the heaviest at 67 and 93kDa. In contrast L4 had only 2 major cuticular antigens resolving at 41 and 93kDa. The 93kDa molecule on L4 and adult worms may be antigenically related.

The recognition of antigens on the surface of adult and L4 Necator americanus by human and hamster post-infection sera.

The surface antigens of adult Necator americanus were recognized by post-infection hamster sera and resolved at molecular weight 93,000, 67,000, 46,000, 43,000, 32,000 and 25,000. L4 larvae in contrast had one major surface antigen, resolving at 93,000. These antigens were also recognized by a range of human sera, although on a differential basis. This suggests that the human sera tion. However, the results do indicate that the hamster model might be of immunological relevance to the human disease state, in that infected hamster recognized the full cuticular antigen spectrum of adult Necator. This, at least, gives the experimenter a convenient reference point from which to conduct further experiments incorporating parameters such as re-infection, anthelmintic treatment and genetic variability to study the effect of these modifications on the serological response.

Identification of hookworm (Necator americanus) antigens and their translation in vitro.

During in vitro culture adult (day 35) Necator americanus synthesise a wide range of protein species many of which are excreted or secreted into the culture medium. Both post infection (day 117) hamster sera and sera from infected humans precipitate antigens of 15, 30, 33, 44, 46 and 69 kDa although individual human sera exhibit some variability in absolute specificity. In immunoblotting experiments antigens of 33 kDa are routinely recognised by human sera although two-dimensional gel analysis suggests that more than one polypeptide is involved. RNA isolated from adult worms direct the in vitro synthesis of numerous polypeptides possessing antigenic determinants recognised by sera from infected hamsters and humans. Post-translational modification of N. americanus encoded polypeptides is not, therefore, a prerequisite for antigenicity.

Site of resistance to Necator americanus in hamsters.

Resistance to the development of human hookworm, Necator americanus was examined in 3- to 6-week-old young adult hamsters. Only 3% of N. americanus infective third stage larvae (NaL3) reached maturity in the intestines of young adults as opposed to as many as 60% in 2-day-old baby hamsters. This seemingly effective resistance prevailing in young adults was investigated in some detail. The skin, the first site of contact for the invading NaL3, was bypassed during the infection process. Completely in vitro exsheathed NaL3 (ExNaL3) were used, and young adult hamsters were infected parenterally, by-passing the skin. Even after exsheathing the larvae artificially before infection and by-passing the skin, no improvement was seen in the development of N. americanus in the intestines of young adults. Higher infection doses also did not increase the worm burden. Some of the factors limiting the development of parasites in young adults were examined. N. americanus were monitored in lungs and intestines during various intervals after infection. Similar parasite burdens were apparent in lungs of baby as well as young adult hamsters. In the intestines, a significantly lower burden of N. americanus was seen during various intervals in young adults compared to the baby hamsters. Moreover, N. americanus were expelled soon after reaching the intestine. This comparative monitoring revealed the intestine as the seat of resistance against the establishment of N. americanus in young adult hamsters.

Antigen stripping from the nematode epicuticle using the cationic detergent cetyltrimethylammonium bromide (CTAB).

The cuticular antigens of adult Nematospiroides dubius were selectively removed using the cationic detergent cetyltrimethylammonium bromide (CTAB). Nonionic, zwitterionic or anionic detergents were ineffective in comparison. The biochemical profile of the antigens removed by detergent was identical to that of surface antigens removed by homogenization, with the added advantage that detergent-stripped antigens lacked many of the background antigens (excretory/secretory--ES and somatic) seen in homogenates. In addition, the detergent was shown to act in a non-invasive manner as electron micrographs failed to reveal any gross damage to the nematode outer cuticle. The observed selective release of significant quantities of relatively clean nematode surface antigen by CTAB in a non-invasive or destructive manner provides the impetus for definitive studies on the relevance of surface antigens (in the absence of ES or somatic antigens) to the overall immunogenicity of this and other parasites.

Necator americanus in infant rabbits: complete development, humoral antibody, leukocyte response and serum protein changes following infection.

Necator americanus, originally isolated from man, developed completely in infant rabbits (RSG-1). This infection was serially passed in infant rabbits up to the 6th generation without using any immunosuppressant. Two day old infant rabbits demonstrated a high degree of susceptibility to N. americanus. Studies revealed the retention of worms in the intestine for more than 150 days during which egg production was high. In this experimental model the leukocyte, antibody and serum protein responses due to N. americanus infection were monitored. Results showed that eosinophil numbers rose significantly from day 28 to 170. A maximum was observed on day 80 (41%) in rabbits of the fourth generation. Antibodies to N. americanus were detectable in infected rabbits by counterimmunoelectrophoresis using third stage larval antigen (Rabbit strain). Generally, total globulins increased with decrease in albumin. Serum protein increase was associated with alpha-2, beta and gamma globulin components.

Homology between roundworm (Ascaris) and hookworm (N. americanus) antigens detected by human IgE antibodies.

Utilizing the techniques of radioallergosorbent test (RAST), inhibition of RAST and isoelectric focusing on polyacrylamide gels, antigenic cross-reactivity has been shown between extracts of Ascaris lumbricoides. Ascaris suum and Necator americanus (hookworm). Antigenic homology is extensive between the two Ascaris preparations. The antigens common to hookworm and Ascaris, while representing a minor component of Ascaris body fluid, are nonetheless potent stimulators of IgE antibody production in subjects infected only with hookworm larvae. These shared antigens appear to have a pI of less than 5.1. The hookworm specific antigens also have a pI of less than 5.1, whereas those specific to Ascaris have pI values in the ranges of 5.0 to 6.0 and 7.0 to 8.6. These latter antigens, i.e. those with pI between 7.0 and 8.6, apparently elicit an immune response only during an active infection with Ascaris.