Patricia S. Coulson

Transcriptome analysis of the acoelomate human parasite Schistosoma mansoni

Schistosoma mansoni is the primary causative agent of schistosomiasis, which affects 200 million individuals in 74 countries. We generated 163,000 expressed-sequence tags (ESTs) from normalized cDNA libraries from six selected developmental stages of the parasite, resulting in 31,000 assembled sequences and 92% sampling of an estimated 14,000 gene complement. By analyzing automated Gene Ontology assignments, we provide a detailed view of important S. mansoni biological systems, including characterization of metazoa-specific and eukarya-conserved genes. Phylogenetic analysis suggests an early divergence from other metazoa. The data set provides insights into the molecular mechanisms of tissue organization, development, signaling, sexual dimorphism, host interactions and immune evasion and identifies novel proteins to be investigated as vaccine candidates and potential drug targets.

Chitinase and Fizz Family Members Are a Generalized Feature of Nematode Infection with Selective Upregulation of Ym1 and Fizz1 by Antigen-Presenting Cells

ABSTRACT Ym1 and Fizz1 are secreted proteins that have been identified in a variety of Th2-mediated inflammatory settings. We originally found Ym1 and Fizz1 as highly expressed macrophage genes in a Brugia malayi infection model. Here, we show that their expression is a generalized feature of nematode infection and that they are induced at the site of infection with both the tissue nematode Litomosoides sigmodontis and the gastrointestinal nematode Nippostrongylus brasiliensis. At the sites of infection with N. brasiliensis, we also observed induction of other chitinase and Fizz family members (ChaFFs): acidic mammalian chitinase (AMCase) and Fizz2. The high expression of both Ym1 and AMCase in the lungs of infected mice suggests that abundant chitinase production is an important feature of Th2 immune responses in the lung. In addition to expression of ChaFFs in the tissues, Ym1 and Fizz1 expression was observed in the lymph nodes. Expression both in vitro and in vivo was restricted to antigen-presenting cells, with the highest expression in B cells and macrophages. ChaFFs may therefore be important effector or wound-repair molecules at the site of nematode infection, with potential regulatory roles for Ym1 and Fizz1 in the draining lymph nodes.

The radiation-attenuated vaccine against schistosomes in animal models: paradigm for a human vaccine?

Publisher Summary Schistosomes can be established in the mammalian host; cercariae must penetrate the skin, transform into schistosomula, locate their favored site in the vasculature, and mature to adulthood. Although most schistosomes do not survive beyond six weeks after infection in the rat, and any that do are stunted, the course of migration and development before this time is very similar to that observed in hosts that are more permissive. There is abundant evidence to indicate that the resistance displayed by mice vaccinated with irradiated cercariae is mediated by specific immune mechanisms, unlike that observed in chronically infected animals. For example, it is schistosome species-specific and can be transferred across a parabiotic union between vaccinated and naive animals. Attenuation can be achieved by exposure of parasites to radiation from an ultraviolet, X-ray, or gamma source. The dose of radiation applied to parasites is an important factor influencing the eventual level of protection, although opinions have differed regarding the optimum amount. The chapter discusses why radiation-attenuated parasites should induce a protective response when an equal number of normal parasites do not. It describes two approaches feasible for developing a vaccine against schistosomiasis. One is to identify protective responses operating in the chronically infected human population, and try to recreate them. The other is to employ immune mechanisms not normally elicited by the parasite, and to which it has presumably not evolved an evasion strategy.

Migration of the schistosomula of Schistosoma mansoni in mice vaccinated with radiation-attenuated cercariae, and normal mice: an attempt to identify the timing and site of parasite death.

The migration of the schistosomula of Schistosoma mansoni labelled with [75Se]methionine, has been followed from the skin to the hepatic portal system. Parasites were detected in all mouse tissues by compressed organ autoradiography. Two separate experiments were performed to track parasites in normal mice, and in mice previously vaccinated with irradiated cercariae. In normal mice, the profile of numbers of autoradiographic foci detected in the skin, lungs, systemic and splanchnic organs was described with time post-infection. The distribution of parasites to systemic organs, following exit from the lungs, paralleled the fractional distribution of cardiac output. Accumulation of schistosomula in the hepatic portal system was complete by day 21 post-infection. Only 2-3 passes of parasites around the vascular system would be required to produce the hepatic portal population. No significant decline in total foci was detected in the first 12 days post-infection. The majority of parasite elimination appeared to occur in the lungs as late as day 21, with lesser proportions in the systemic organs and skin infection site. The pattern of migration in vaccinated mice was similar to that in normal animals. One difference observed was the longer duration of stay in the skin; however, the majority of parasites eventually reached the lungs. The systemic phase of migration occurred on a reduced scale, as did accumulation of parasites in the hepatic portal system. The decline in total foci in vaccinated mice commenced approximately 7 days earlier than in normal mice and proceeded to a lower end-point. Again the majority of parasite elimination appeared to occur in the lungs with lesser proportions in the systemic organs and skin infection site. It is suggested that resistance to reinfection in vaccinated mice has two additive components which combine to retard the migration of schistosomula within the vasculature, preventing them from reaching the hepatic protal system.

Antibodies to Glycans Dominate the Host Response to Schistosome Larvae and Eggs: Is Their Role Protective or Subversive?

Multiple exposures of chimpanzees to the radiation-attenuated schistosome vaccine provoked a strong parasite-specific cellular and humoral immune response. Specific IgM and IgG were directed mainly against glycans on antigens released by cercariae; these were also cross-reactive with soluble antigens from larvae, adult worms, and eggs. Egg deposition was the major antigenic stimulus after challenge of vaccinated and control chimpanzees with normal parasites, eliciting strong antiglycan responses to egg secretions. Glycan epitopes recognized included LacdiNAc, fucosylated LacdiNAc, Lewis(X) (weakly), and those on keyhole limpet hemocyanin. Antibodies to peptide epitopes became prominent only during the chronic phase of infection, as glycan-specific IgM and IgG decreased. Because of their intensity and cross-reactivity, the antiglycan responses resulting from infection could be a smoke screen to subvert the immune system away from more vulnerable larval peptide epitopes. Their occurrence in humans might explain the long time required for antischistosome immunity to build up after infection.

Impaired immunity and altered pulmonary responses in mice with a disrupted interferon-gamma receptor gene exposed to the irradiated Schistosoma mansoni vaccine.

A high level of protection against Schistosoma mansoni is elicited in mice by the irradiated cercaria vaccine and interferon‐γ (IFN‐γ) is a key cytokine in the pulmonary effector response. The role of this cytokine has been investigated in mice with a targeted disruption of the IFN‐γ receptor gene (IFN‐γR−/− mice). The level of protection was impaired relative to that elicited in C57BL/6 and 129 wild‐type (WT) animals. These two groups developed compact effector foci, of largely mononuclear cell composition, around individual challenge parasites migrating through the lungs. In contrast the IFN‐γR−/− mice showed a massive and generalized leucocytic infiltration of the airways and interstitium in which eosinophils were a prominent feature. Cultures of airway leucocytes from C57BL/6 mice produced abundant IFN‐γ whilst those from IFN‐γR−/− mice produced interleukin‐4 (IL‐4), IL‐5 and IL‐10, indicating default to the Th2 pathway; the WT animals showed an intermediate response. The pattern of cytokine gene transcripts in whole lung tissue agreed remarkably well with the level of cytokine protein detected in leucocyte cultures, with the exception of substantial IL‐4 mRNA but negligible protein in C57BL/6 mice. The loose but intense infiltrate of leucocytes in the lungs of IFN‐γR−/− mice was clearly ineffective in eliminating challenge parasites, whereas the level of IFN‐γ protein and mRNA in the lungs of C57BL/6 and WT mice correlated with the size and compactness of effector foci. On the basis of these and earlier observations, we suggest that a primary role for IFN‐γ is to promote intercelluar adhesion between the leucocytes in an effector focus, promoting its ability to block parasite migration.

Cellular and humoral immune responses and protection against schistosomes induced by a radiation-attenuated vaccine in chimpanzees.

ABSTRACT The radiation-attenuated Schistosoma mansoni vaccine is highly effective in rodents and primates but has never been tested in humans, primarily for safety reasons. To strengthen its status as a paradigm for a human recombinant antigen vaccine, we have undertaken a small-scale vaccination and challenge experiment in chimpanzees (Pan troglodytes). Immunological, clinical, and parasitological parameters were measured in three animals after multiple vaccinations, together with three controls, during the acute and chronic stages of challenge infection up to chemotherapeutic cure. Vaccination induced a strong in vitro proliferative response and early gamma interferon production, but type 2 cytokines were dominant by the time of challenge. The controls showed little response to challenge infection before the acute stage of the disease, initiated by egg deposition. In contrast, the responses of vaccinated animals were muted throughout the challenge period. Vaccination also induced parasite-specific immunoglobulin M (IgM) and IgG, which reached high levels at the time of challenge, while in control animals levels did not rise markedly before egg deposition. The protective effects of vaccination were manifested as an amelioration of acute disease and overall morbidity, revealed by differences in gamma-glutamyl transferase level, leukocytosis, eosinophilia, and hematocrit. Moreover, vaccinated chimpanzees had a 46% lower level of circulating cathodic antigen and a 38% reduction in fecal egg output, compared to controls, during the chronic phase of infection.

Immune effector mechanisms against schistosomiasis: looking for a chink in the parasite's armour

A recombinant antigen vaccine against Schistosoma mansoni remains elusive, in part because the parasite deploys complex defensive and offensive strategies to combat immune attack. Nevertheless, research on rodent and primate models has shown that schistosomes can be defeated when appropriate responses are elicited. Acquired protection appears to involve protracted inhibition of larval migration or key molecular processes at the adult surfaces, not rapid cytolytic killing mechanisms. A successful vaccine will likely require a cocktail of antigens rather than a single recombinant protein. In addition, ways need to be found of keeping the immune system on permanent alert, either to achieve adequate inhibition of protein function in adults, or because a trickle of incoming parasites does not amplify the secondary response.

From genomes to vaccines via the proteome

An effective vaccine against schistosomiasis mansoni would be a valuable control tool and the high levels of protection elicited in rodents and primates by radiation-attenuated cercariae provide proof of principle. A major obstacle to vaccine development is the difficulty of identifying the antigens that mediate protection, not least because of the size of the genome at 280Mb DNA encoding 14,000 to 20,000 genes. The technologies collectively called proteomics, including 2D electrophoresis, liquid chromatography and mass spectrometry, now permit any protein to be identified provided there is extensive DNA data, and preferably a genome sequence. Applied to soluble (cytosolic) proteins from schistosomes, proteomics reveals the great similarity in composition between life cycle stages, with several WHO vaccine candidates amongst the most abundant constituents. The proteomic approach has been successfully applied to identify the secretions used by cercaria to penetrate host skin, the gut secretions of adult worms and the proteins exposed on the tegument surface. Soluble proteins can also be separated by 2D electrophoresis before western blotting to identify the full range of antigenic targets present in a parasite preparation. The next step is to discover which target proteins represent the weak points in the worms defences.

IL-4 receptor signaling is required for mannose receptor expression by macrophages recruited to granulomata but not resident cells in mice infected with Schistosoma mansoni.

High levels of mannose receptor (MR) expression and pinocytic activity are a hallmark of Th2 cytokine-driven alternative MΦ activation in vitro. We examined expression of MR in situ and its regulation by Th1/Th2 cytokines in IL-4Rα −/− and wild-type (WT) mice challenged with Schistosoma mansoni. Parasite eggs induce a vigorous granulomatous response, driven by Th2 cytokines in WT mice, but by Th1 cytokines in IL-4Rα −/− mice. MR was expressed by granuloma MΦ in WT mice but not in IL-4Rα −/− mice, whose MΦ nevertheless exhibited a mature phenotype and morphology. By contrast expression of MR in resident tissue MΦ and endothelial cells appeared unaffected by Th1/Th2 cytokines. Our results revealed that Th1/Th2 cytokines differentially regulate MR according to cell type and play a critical role in regulating MR expression by MΦ recruited to granulomata. We also present evidence that components of schistosome eggs and a fraction of their secretions are ligands of MR, and suggest that MR activity may be of functional significance in the granulomatous response.