A novel blood-feeding detoxification pathway in Nippostrongylus brasiliensis L3 reveals a potential checkpoint for arresting hookworm development.

As part of on-going efforts to control hookworm infection, the "human hookworm vaccine initiative" has recognised blood feeding as a feasible therapeutic target for inducing immunity against hookworm infection. To this end, molecular approaches have been used to identify candidate targets, such as Necator americanus (Na) haemoglobinase aspartic protease-1 (APR-1), with immunogenicity profiled in canine and hamster models. We sought to accelerate the immune analysis of these identified therapeutic targets by developing an appropriate mouse model. Here we demonstrate that Nippostrongylus brasiliensis (Nb), a phylogenetically distant strongylid nematode of rodents, begins blood feeding early in its development and that immunisation with Na-APR-1 can block its growth and completion of its life cycle. Furthermore, we identify a new haem detoxification pathway in Nb required for blood feeding that can be blocked by drugs of the quinolone family, reducing both infection burden and the associated anaemia in rodents. Collectively, our findings show that haem metabolism has potential as a checkpoint for interrupting hookworm development in early stages of the hookworm life cycle and that the Nippostrongylus brasiliensis rodent model is relevant for identifying novel therapeutic targets against human hookworm.

Rhesus macaques self-curing from a schistosome infection can display complete immunity to challenge.

The rhesus macaque provides a unique model of acquired immunity against schistosomes, which afflict >200 million people worldwide. By monitoring bloodstream levels of parasite-gut-derived antigen, we show that from week 10 onwards an established infection with Schistosoma mansoni is cleared in an exponential manner, eliciting resistance to reinfection. Secondary challenge at week 42 demonstrates that protection is strong in all animals and complete in some. Antibody profiles suggest that antigens mediating protection are the released products of developing schistosomula. In culture they are killed by addition of rhesus plasma, collected from week 8 post-infection onwards, and even more efficiently with post-challenge plasma. Furthermore, cultured schistosomula lose chromatin activating marks at the transcription start site of genes related to worm development and show decreased expression of genes related to lysosomes and lytic vacuoles involved with autophagy. Overall, our results indicate that enhanced antibody responses against the challenge migrating larvae mediate the naturally acquired protective immunity and will inform the route to an effective vaccine.

Fifteen Years of Sm-p80-Based Vaccine Trials in Nonhuman Primates: Antibodies From Vaccinated Baboons Confer Protection in vivo and in vitro From Schistosoma mansoni and Identification of Putative Correlative Markers of Protection.

Recent advances in systems biology have shifted vaccine development from a largely trial-and-error approach to an approach that promote rational design through the search for immune signatures and predictive correlates of protection. These advances will doubtlessly accelerate the development of a vaccine for schistosomiasis, a neglected tropical disease that currently affects over 250 million people. For over 15 years and with contributions of over 120 people, we have endeavored to test and optimize Sm-p80-based vaccines in the non-human primate model of schistosomiasis. Using RNA-sequencing on eight different Sm-p80-based vaccine strategies, we sought to elucidate immune signatures correlated with experimental protective efficacy. Furthermore, we aimed to explore the role of antibodies through in vivo passive transfer of IgG obtained from immunized baboons and in vitro killing of schistosomula using Sm-p80-specific antibodies. We report that passive transfer of IgG from Sm-p80-immunized baboons led to significant worm burden reduction, egg reduction in liver, and reduced egg hatching percentages from tissues in mice compared to controls. In addition, we observed that sera from Sm-p80-immunized baboons were able to kill a significant percent of schistosomula and that this effect was complement-dependent. While we did not find a universal signature of immunity, the large datasets generated by this study will serve as a substantial resource for further efforts to develop vaccine or therapeutics for schistosomiasis.

Quantification of total sheep IgE concentration using anti-ovine IgE monoclonal antibodies in an enzyme immunoassay.

Monoclonal antibodies (mAbs) which recognize separate epitopes on ovine immunoglobulin E (IgE) have been used to develop a non-competitive antibody sandwich enzyme immunoassay (EIA) for quantitating ovine IgE. Purified anti-IgE mAb (YD3) coated onto polystyrene microtitre plates was used to capture IgE in serum samples. Biotinylated anti-IgE mAb (XB6) followed by streptavidin conjugated with horseradish peroxidase were used to detect captured IgE. Tetramethylbenzidine and H2O2 were used as enzyme substrate. A reference serum was prepared by pooling sheep sera containing elevated IgE levels. This reference serum was assigned a value of 100 units ml-1 and used to prepare standard curves for the EIA. The linear region of log-log transformed standard curve data covered a range of 0.05-0.8 units ml-1. The equation of a linear regression line fitted to this curve was used to determine sample concentrations. Using purified IgE, 1 unit of reference serum was equivalent to 0.86 micrograms ml-1 IgE. Maximum intra- and inter-assay coefficients of variation for the EIA were 4.6% and 9.7%, respectively. Subjecting serum samples to 15 freeze/thaw cycles, storage at room temperature for 16 days or incubation at 37 degrees C for 8 h resulted in minimal loss of IgE detection. Incubation of serum at 56 degrees C resulted in rapid reduction in detection of IgE by the EIA. The assay was used to determine IgE levels in adult sheep monospecifically infected with weekly doses of the nematode Trichostrongylus axei. Serum IgE levels increased from 9 to 16 days following first infection and reached maximum levels by days 35-58. Serum IgE responses closely followed IgE positive cell responses in the abomasal mucosa.

Proteolytic enzymes secreted by larval stage of the parasitic nematode Trichinella spiralis.

Excretory/secretory products (ES), collected from in vitro cultures of muscle larvae (L1) of Trichinella spiralis (Owen, 1835) were examined for the presence of proteolytic enzymes. Several discrete proteinases in the size range of 25-55 kDa were identified by substrate gel electrophoresis and were characterised according to pH optima, substrate specificity and inhibitor sensitivity using azocasein assay. Serine, cysteine and metalloproteinases active at pH 5-7 were identified. The serine proteinases were found to predominate and some of them were found to be specific for the larval stage of the parasite. The results from the substrate analysis indicated the presence of collagenolytic and elastolytic activities. The proteinase activity was inhibited by IgG isolated from T. spiralis-infected mice, an observation of relevance to understanding host/parasite interactions and, ultimately, the development of anti-Trichinella vaccine.

Antibody response to a protease secreted by Trichinella spiralis muscle larvae.

In the present study we analyzed the humoral response of Trichinella spiralis-infected mice to a 35-kDa protease (purified from the excretory-secretory products of T. spiralis muscle larvae) by a Western-blot procedure and an enzyme-linked immunosorbent assay (ELISA) technique using a panel of postinfection mouse anti-Trichinella sera. The results demonstrated that this response was time-dependent and that infected mice could be distinguished from controls. In addition, inhibition assays demonstrated that these antisera were capable of abolishing the proteinase activity of the 35-kDa protease in vitro. The occurrence of proteases seems to be a very common feature in parasite crude extracts and excretory-secretory products (McKerrow 1989). It is also known that these enzymes are implicated in important host-parasite interactions, and for this reason, recent reports have proposed the use of parasite proteases both as alternative targets for an induced immune response and as a rich source of antigenic material for diagnostic testing (Hotez et al. 1985; Yamasaki et al. 1989; Song et al. 1990; Frank and Grieve 1991; Britton et al. 1992; Song and Chappell 1993). We have recently purified a protease (mol. wt., 35 kDa) from the excretory-secretory (ES) products of Trichinella spiralis (GM-1 strain) muscle larvae and established some of the biochemical properties of this protease (Armas-Serra et al. 1994).(ABSTRACT TRUNCATED AT 250 WORDS)

Eosinophil-mediated killing of Haemonchus contortus larvae: effect of eosinophil activation and role of antibody, complement and interleukin-5.

Eosinophils have previously been shown to accumulate around the tissue invasive (L3) stage of sheep gastrointestinal parasites in vivo. In this study, eosinophils obtained from mammary washes of sheep, were shown to immobilize and kill H. contortus larvae in vitro in the presence of antibody specific against a defined L3 surface antigen. Eosinophils obtained from sheep primed by repeated infusion of H. contortus larvae were more effective than eosinophils obtained after a single infusion of parasite extract in Fasciola hepatica infected ewes suggesting the former were activated in vivo. The level of larval immobilization in the presence of antibody was significantly increased when complement was added to cultures containing activated eosinophils. The addition of interleukin-5 to larval cultures containing antibody and complement resulted in a significant increase in larval immobilization with unactivated eosinophils suggesting that eosinophil effector function is enhanced following priming with this cytokine. Ultrastructural analysis of the eosinophil/larvae interaction at 6 h of incubation revealed degranulation of adhering eosinophils onto the surface of larvae. By 24 h of incubation, many larvae showed signs of damage and most eosinophils had degenerated. These results suggest that eosinophil-mediated killing may be an effector mechanism for the elimination of L3 H. contortus larvae in immune sheep.

Macrophage modifying factor secreted by the tetrathyridia of Mesocestoides corti (Cestoda): monoclonal antibody to the modifying factor antagonizes its immunological activity.

Immunomodulation of macrophage activity by in vitro secretions of Mesocestoides corti has been previously demonstrated. The modifying activity secreted by M. corti had the effect of reducing the normal accessory function of macrophages in a Con-A-activated lymphocyte proliferation assay. This paper describes the purification of the modifying activity by FPLC techniques and the generation of a monoclonal antibody (MoAb) to this molecule in mice. The MoAb bound immunomodulatory FPLC fractions of M. corti in an ELISA. When MoAb was applied in conjunction with immunomodulatory parasite secretions to macrophages in vivo or in vitro, the modifying effect of the secretions was abolished. This profound effect of the MoAb should help to elucidate the mechanisms by which metacestode parasites avoid host immune responses and may enable therapeutic intervention.

Characterization of proteinases in extracts of adult Haemonchus contortus, the ovine abomasal nematode.

The degradation of several protein substrates, including the blood proteins haemoglobin, albumin and fibrinogen, by proteinases present in extracts of adult Haemonchus contortus was examined over a broad pH range. These proteinases were further characterized on the basis of substrate specificity, inhibitor sensitivity and molecular size by spectrophotometric and substrate gel analysis. The majority of the proteinases capable of degrading the blood proteins tested were active at acidic pH and could be ascribed to the cysteine proteinase class. In addition, evidence is presented that these proteinases are differentially recognized and inhibited by immune sera and that parasites capable of withstanding protective host immune responses exhibit modified expression of proteinases.

Mice with a targeted deletion of the IgE gene have increased worm burdens and reduced granulomatous inflammation following primary infection with Schistosoma mansoni.

Although IgE has been considered to play an essential role in host defense against parasitic helminth infections such as Schistosoma mansoni, in vivo evidence of a protective function of IgE in infected mice is lacking. In the present study, mice with a null mutation of the C epsilon gene, and thus incapable of making IgE (IgE deficient), were infected by S. mansoni cercariae percutaneously. In two independent experiments, IgE-deficient mice were significantly more susceptible to primary infection, developing worm burdens twofold greater than those of wild-type mice (p < 0.001). In contrast, resistance to challenge infection following three immunizations with irradiated cercariae was similar in the two groups. The percentage of reduction in worm burdens in immunized IgE-deficient animals compared with unimmunized mice was 50%; immunized wild-type mice had a reduction of 55% compared with the baseline parasite count. Levels of parasite-specific IgG1 were more than twofold lower in IgE-deficient mice after primary infection (p = 0.005), whereas no significant difference was observed in the IgG1 response of animals previously immunized with irradiated cercariae. IgE-deficient animals also developed significantly smaller granulomas (by 37-40%) around schistosome eggs deposited in their livers compared with wild-type animals (p < 0.001). The spleens of IgE-deficient mice contained significantly more Ag-specific IL-4-secreting cells following primary infection. These data show that IgE participates in parasite elimination in primary infection with S. mansoni and in the generation of humoral immunity and cytokine responses to the parasite.

Praziquantel-induced exposure of Schistosoma mansoni alkaline phosphatase: drug-antibody synergy which acts preferentially against female worms.

The efficacy of praziquantel-treatment of murine Schistosoma mansoni-infections can be enhanced by concurrent administration of rabbit anti-sera with specificity for parasite antigens. Monospecific rabbit serum raised against S. mansoni worm alkaline phosphatase, that was reactive with the enzyme on the drug-treated female surface, was found to significantly and preferentially increase the mortality of female worms by PZQ. Immunoglobulins purified from the anti-alkaline phosphatase antiserum inhibited 54% of schistosome alkaline phosphatase enzymatic activity on the surface of praziquantel-treated worms. We propose that synergistic antibody-mediated death of drug-damaged worms is a consequence of the inhibition of drug-exposed alkaline phosphatase on the female worm surface by passively transferred antibody.

Role of IgE in primary murine Schistosomiasis mansoni.

Schistosoma mansoni infection proceeds in normal mice in the absence of detectable levels of polyclonal or specific immunoglobulin (Ig)E until worms mature and deposit eggs. Hence, the course of a primary S. mansoni infection is not expected to vary appreciably in mice with defects in the IgE production. Experimental increase of IgE production early after infection may, however, influence worm development. In the first approach towards this goal, BALB/c mice were injected with interleukin(IL)4 to raise the level of endogenously synthesized IgE. A significant increase in serum polyclonal IgE and antischistosome IgG1 during the prepatent period was not associated with significant changes in worm and egg burden or liver pathology. During the second approach, mice were injected with IgE which was affinity purified from serum of BALB/c mice infected for 16 weeks with S. mansoni. The purified IgE bound to carbohydrate-independent epitopes of soluble antigens from 3 h larvae, adult worms and eggs and recognized the schistosomular surface membrane. No differences in worm and egg load or granuloma number and size were noted between untreated and exogenous IgE-injected mice. Together, the data demonstrate that by itself IgE does not influence the outcome of infection in primary murine S. mansoni.

Characterization of Meloidogyne javanica surface coat with antibodies and their effect on nematode behaviour.

The surface coat of the 2nd-stage juveniles (J2) of plant-parasitic nematodes is considered to be involved in interactions with microorganisms in the soil and rhizosphere, as well as with the host plant. Characterization of surface antigens might be important in the development of new nematode control strategies. In this study, polyclonal and monoclonal antibodies raised against Meloidogyne javanica, M. incognita and other plant-parasitic nematodes were tested for their binding to the surface coat and secreted-excreted products of M. javanica. Some of the monoclonal and polyclonal antibodies raised against M. incognita showed cross-reactivity with the surface coat of M. javanica. Western blot analysis of M. javanica surface coat extracts revealed labelling of several polypeptides with a 48 kDa main band for the polyclonal antibody IACR-PC Mi 373, and a 55 kDa main band for PC Mj E2. Further characterization of the antigens recognized by the polyclonal antibody PC Mj E2, in planta, showed that they were present in the parasitic stages J2 and J3 and that the surface coat was shed during root penetration. The hypodermis of the infective juveniles was labelled by PC Mj E2 and the monoclonal antibody IACR-Misec 3F.4, suggesting that these surface antigens are produced in the hypodermis. Nematode behaviour was affected by all the antibodies that bound to the surface coat of the pre-parasitic J2, and we demonstrated that the movement pattern of the M. javanica J2 was affected by these antibodies. Continuous binding of the antibodies to the M. javanica surface inhibited the infection of Arabidopsis thaliana roots on agar plates.

Stem cell factor (c-kit ligand) influences eosinophil recruitment and histamine levels in allergic airway inflammation.

The increased reactivity of mast cells during allergic airway inflammation has been linked to several aspects of pulmonary disease. A primary inducer of mast cell differentiation, proliferation, and activation has been identified as c-kit ligand or stem cell factor (SCF). In the present study, we used an established murine model of allergic eosinophilic airway inflammation to examine the role of SCF during an Ag-specific airway response. Initial data demonstrates increased SCF protein production at 8 h postchallenge in both lungs and serum of allergen-challenged, but not vehicle-challenged, mice. The immunolocalization of SCF in Ag-challenged lungs suggested that macrophage populations were the primary source of SCF, while epithelial cell regions also stained positive. Intense immunohistochemical staining of macrophages in bronchoalveolar lavage samples recovered from Ag-sensitized mice indicate that these cells may be a significant source of SCF in the lungs. Alveolar macrophages from the airways of normal mice stimulated with either TNF (0.1-10 ng/ml) or IL-4 (10 ng/ml) produced significant levels of SCF. Furthermore, neutralization studies demonstrated that the inhibition of airway SCF during allergen challenge significantly decreased eosinophil, but not neutrophil, infiltration throughout the response. Furthermore, when mice were treated with anti-SCF Ab, histamine levels were significantly reduced at 8 h postchallenge, the time of significant SCF production. Together, these data indicate that the production of SCF during Ag-induced lung inflammation by alveolar macrophages can play a significant role in the subsequent recruitment of eosinophils, possibly via mast cell activation and degranulation.