Antibodies elicited by the secretions from schistosome cercariae and eggs are predominantly against glycan epitopes.

The glycoproteins secreted by Schistosoma mansoni cercariae and eggs play a key role in parasite transmission to and from the mammalian host. We used secreted preparations from these two life cycle stages to characterize the reactivity of sera from baboons exposed to normal and/or attenuated cercariae, in comparison with somatic antigen preparations and defined glycan epitopes. Periodate treatment of native antigens revealed that responses to the two secreted preparations were overwhelmingly directed against glycans rather than peptides. Considerable immunological cross-reactivity between glycans in the two preparations was inferred from a comparison of sera from infected-only and vaccinated-only animals, predominantly exposed to egg and cercarial secretions, respectively. In contrast, when somatic antigen preparations derived from adult worms or eggs were used to probe sera, a stronger antipeptide response was seen that accounted for up to 66% of maximum reactivity. Probing of sera with defined glycan structures confirmed the time course of responses and the presence of cross-reactive epitopes. In spite of the intense antiglycan response elicited in mice by administration of live eggs, no protection against a cercarial challenge was observed. Our data further support the hypothesis that antiglycan responses are a smokescreen with negligible protective potential.

Resistance to re-infection after exposure to normal and attenuated schistosome parasites in the baboon model.

The baboon model of schistosomiasis has been used extensively to study parasite biology, immune responses and pathological manifestations after natural and experimental infections. The body of knowledge accumulated so far has placed this animal model at the pinnacle in the continuing search for new interventions and might hold the key to the development of new anti-schistosome vaccines. In this review paper, we highlight previous and recent studies that have elevated the baboon to be the model of choice for schistosomiasis research. In particular, the long-term studies of re-infection after chemotherapy as well as the interaction between vaccination, chemotherapy and infection are highlighted.

An overview of animal models in experimental schistosomiasis and refinements in the use of non-human primates.

The complex nature of the schistosome parasite and its interaction with the mammalian host necessitates the continued use of live intact animal models in schistosomiasis research. This review acknowledges this necessity and highlights some of the important insights into the pathogenesis of the disease that have been gained from using various animal models. The use of non-human primates as more relevant models of human schistosomiasis is stated. In addition, the importance of animal welfare consideration when using primates for research is emphasized. Finally, some guidelines for the refined capture, handling and early humane endpoints for non-human primates to be used in experimental schistosomiasis are suggested.

Repeated exposure induces periportal fibrosis in Schistosoma mansoni-infected baboons: role of TGF-beta and IL-4.

Recently, we observed that repeated Schistosoma mansoni infection and treatment boost Th2-associated cytokines and TGF-beta production in baboons. Other studies have shown that some chronically infected baboons develop hepatic fibrosis. Because TGF-beta, IL-2, and IL-4 have been shown to participate in development of fibrosis in murine schistosomiasis, the present study examined whether repeated exposure stimulates hepatic fibrosis in olive baboons. To test this hypothesis, animals were exposed to similar numbers of S. mansoni cercariae given once or repeatedly. After 19 wk of infection, animals were cured with praziquantel and reinfected once or multiple times. Hepatic granulomatous inflammation and fibrosis were assessed from serial liver biopsies taken at weeks 6, 9, and 16 after reinfection and egg Ag (schistosome egg Ag)-specific cytokine production by PBMC were measured simultaneously. Periportal fibroblast infiltration and extracellular matrix deposition (fibrosis), angiogenesis, and biliary duct hyperplasia developed in some animals. The presence and amount of fibrosis directly correlated with the frequency of exposure. Fibrosis was not associated with adult worm or tissue egg burden. The amount of fibrosis correlated with increased schistosome egg Ag-driven TGF-beta at 6, 9, and 16 wk postinfection (rs = 0.9, 0.8, and 0.54, respectively, all p < 0.01) and IL-4 production (p = 0.02) at 16 wk postinfection and not IFN-gamma, IL-2, IL-5, or IL-10. These data suggest that repeated exposure is a risk factor for periportal fibrosis by a mechanism that primes lymphocytes to produce increased levels of profibrotic molecules that include TGF-beta and IL-4.

Cytokine control of the granulomatous response in Schistosoma mansoni-infected baboons: role of exposure and treatment.

Variations in exposure and treatment may contribute to heterogeneity in immunity and granuloma-induced pathology in human schistosomiasis. To examine this hypothesis, olive baboons were either repeatedly infected with Schistosoma mansoni cercariae or received an equivalent dose in a single infection. They were then cured with praziquantel and reinfected with a single exposure. Serial liver biopsies were obtained throughout the course of the experiment, and cytokine responses by peripheral blood mononuclear cells were measured every 2 to 3 weeks. Reinfection after treatment resulted in a twofold-smaller granuloma size at 6 and 9 weeks after infection compared to the size for the same period after primary infection (P < 0.001) but had no effect at 16 or 19 weeks postinfection. The pattern of exposure did not influence granuloma size. During primary infection schistosome-soluble egg antigen (SEA)-induced cytokine production correlated with granulomatous inflammation. Cytokine levels peaked during the acute infection, declined with chronic infection, and became undetectable after treatment. Reinfection after treatment stimulated a two- to three-fold increase in SEA-specific interleukin-4 (IL-4), IL-5, IL-10, IL-2, and transforming growth factor beta (TGF-beta) production and a marked rise in SEA-specific immunoglobulin E (IgE) and IgG regardless of the type of exposure. Cytokine production was significantly greater in repeatedly exposed animals (P < 0.001). SEA-induced gamma interferon production, however, did not increase with reinfection after treatment. SEA-induced TGF-beta was the only cytokine that remained elevated as the infection become chronic and correlated with diminished hepatic granuloma size, implying its participation in down-modulation. These studies demonstrate that baboons partially retain their ability to down-modulate the granulomatous response after treatment.

Role of adult worm antigen-specific immunoglobulin E in acquired immunity to Schistosoma mansoni infection in baboons.

Allergic-type immune responses, particularly immunoglobulin E (IgE), correlate with protective immunity in human schistosomiasis. To better understand the mechanisms of parasite elimination we examined the immune correlates of protection in baboons (Papio cynocephalus anubis), which are natural hosts for Schistosoma mansoni and also develop allergic-type immunity with infection. In one experiment, animals were exposed to a single infection (1,000 cercariae) or were exposed multiple times (100 cercariae per week for 10 weeks) and subsequently were cured with praziquantel prior to challenge with 1, 000 cercariae. Singly and multiply infected animals mounted 59 and 80% reductions in worm burden, respectively (P < 0.01). In a second experiment, animals were inoculated with S. mansoni ova and recombinant human interleukin 12 (IL-12). This produced a 37 to 39% reduction in adult worm burden after challenge (P < 0.05). Parasite-specific IgG, IgE, IgM, and peripheral blood cytokine production were evaluated. The only immune correlate of protection in both experiments was levels of soluble adult worm antigen (SWAP)-specific IgE in serum at the time of challenge infection and/or 6 weeks later. Baboons repeatedly infected with cercariae or immunized with ova and IL-12 developed two- to sixfold-greater levels of SWAP-specific IgE in serum than did controls, and this correlated with reductions in worm burden (r2, -0.40 to -0.64; P, <0. 01). Thus, in baboons and unlike mice, adult worm-specific IgE is uniquely associated with acquired immunity to S. mansoni infection. This similar association of parasite-specific IgE and protection among primates infected with schistosomiasis, along with similar pathology, anatomy, and genetic make-up, indicates that baboons provide an excellent permissive experimental model for better understanding the mechanisms of innate and acquired immunity to schistosomiasis in humans.

Schistosoma mansoni: development and modulation of the granuloma after or multiple exposures in the baboon (Papio cynocephalus anubis).

The ability of the host to modulate the granulomatous response around ova trapped in tissues determines the severity of disease to schistosome infections. Multiple factors may affect this modulation such as age, prior sensitization, history of treatment, and exposure. The present study examines the effect of different patterns of exposure on the sequential development and modulation of granuloma in juvenile Kenyan baboons (Papio cynocephalus anubis) after receiving either a single infection (SI) of 1500 Schistosoma mansoni cercariae or multiple infections (MI) of 150 cercariae, once a week for 10 weeks. Prior to sacrifice at 17 weeks postinfection (p.i.), liver biopsies were obtained at Weeks 0, 6, 9, and 13. SI animals experienced more prolonged dysentery and greater weight loss compared to MI animals. Peak hepatic granuloma size (mean 355 +/- 65.5 microns diameter), the maximum percentage of eosinophils in the granuloma (61%), and severity of disease occurred at 6 weeks in SI animals. Peak granuloma size and pathology did not appear until Week 9 in the MI animals (mean 317.7 +/- 67.3 microns diameter). Granuloma size, tissue eosinophilia, and gross pathology diminished by Week 13 p.i. and were virtually absent in both groups by Week 17. The decrease in granuloma size, pathology, and clinical illness resolved more rapidly in the MI baboons. Singly infected baboons were more susceptible to infection (83 +/- 12% of cercariae developed into adult worms) compared to MI baboons (67 +/- 7%, P < 0.01). Eggs recovered from tissues at necropsy were primarily confined to the large intestine (85% of total egg recovered), followed by liver (10%) and small intestine (5%). Significantly more eggs were recovered from MI compared to SI animals, indicating a higher fecundity of female worms in the MI baboons. These date demonstrate that granulomatous responses develop more slowly and modulate more rapidly with repeated infection compared to a single heavy infection and suggest the type of exposure may affect the pathologic response to infection.

Vaccination of vervet monkeys against cutaneous leishmaniosis using recombinant Leishmania 'major surface glycoprotein' (gp63).

Vervet monkeys (Cercopithicus aethiops) were shown to give a positive delayed-type hypersensitivity (DTH) reaction to gp63, a major surface glycoprotein of Leishmania parasites, and also produce antibodies to the molecule following a triple vaccination with a total dose of 150 micrograms of recombinant gp63 mixed with Bacille Calmette Guerin (BCG). However, peripheral blood leucocytes (PBL) from these animals neither proliferated nor produced any interferon-gamma (IFN-gamma) following in vitro stimulation with the antigen. Analysis of lymphocyte subsets following vaccination did not reveal any striking phenotypic alteration of cellular sub-populations in PBL. When vaccinated animals were rechallenged, via the needle, with virulent Leishmania major promastigotes containing salivary gland extracts from vector sandflies, only partial protection was achieved. We concluded from these studies that rgp63 produced in Escherichia coli is a safe vaccine molecule which gives only partial protection following vaccination in the vervet monkey host. The molecule requires further improvement for vaccine and/or immunodiagnosis application.

Visceral leishmaniasis in vervet monkeys: immunological responses during asymptomatic infections.

Nine vervet monkeys (Cercopithecus aethiops) were infected intradermally with 8 x 10(7) virulent L. donovani promastigotes. Four animals developed clinical visceral leishmaniasis and died over a period of 18 months. The remaining five animals have remained asymptomatic for a period of 3 years now. Attempts to isolate parasites from spleen and liver through biopsies were fruitless. Immunological responses of these subclinically infected animals were examined. Enzyme-linked immunosorbent assay (ELISA) and western blot analyses demonstrated Leishmania specific antibodies in these animals, but the antibody titres were low. When proliferation of peripheral blood monocytes (PBMC) to Concanavalin A (Con A) of these animals was compared with control 'disease free animals' there were no significant differences in response. However, L. donovani antigen (fixed promastigotes) specific proliferation was demonstrated in the five subclinically infected animals. High and varying levels of interferon gamma (IFN-gamma) were secreted in PBMC cultures from the five vervet monkeys when stimulated with either Con A or L. donovani antigens. In control animals, IFN-gamma was only detected when PBMC were stimulated with Con A. Marked delayed-type hypersensitivity (DTH) responses were demonstrated in the five subclinically infected animals 48 h after injection with formalin fixed promastgotes. It was concluded that the visceral Leishmania disease spectrum due to L. donovani observed in humans could be induced in vervet monkeys and that L. donovani asymptomatic/cryptic infected animals have competent humoral and cellular responses to homologous parasites.

Immunization with Theileria parva parasites from buffaloes results in generation of cytotoxic T cells which recognize antigens common among cells infected with stocks of T. parva parva, T. parva bovis, and T. parva lawrencei.

Immunity to infection by the protozoan parasite Theileria parva in cattle is partially attributable to cytotoxic T cells, which kill lymphocytes infected with the schizont stage of the parasite. Here we evaluated five stocks of buffalo-derived T. parva lawrencei parasites and two stocks of cattle-derived T. parva parva parasites for their ability to induce in vivo cytotoxic T cells which can kill lymphocytes infected with a wide variety of strains of T. parva parasites. A group of seven full-sibling cattle, produced by embryo transfer and matched for at least one major histocompatibility complex class I haplotype, were immunized by infection and treatment with the parasite stocks. Target cells used in in vitro cytotoxicity assays were infected with five buffalo-derived parasite stocks and five cattle-derived parasite stocks, including T. parva parva and T. parva bovis. Immunization with any of the seven parasite stocks resulted in the generation of cytotoxic T cells which recognized parasite antigens on most if not all of the target cell lines tested, although the T. parva bovis stock was the least effective at doing so. Further in-depth analyses performed with peripheral blood mononuclear cells from one of the cattle immunized with T. parva lawrencei parasites showed that the pattern of killing of the panel of target cells was altered when either cells infected with different parasite stocks or clones of infected cells were used as stimulator cells in vitro, suggesting the presence of more than one population of parasite-specific cytotoxic effector cells in the peripheral blood mononuclear cells. However, clones of these cytotoxic effector cells recognized common or cross-reactive antigen epitopes expressed by the entire panel of infected target cells. These T-cell clones will be useful for identifying common T-cell antigen epitopes of T. parva and the parasite genes encoding them.