Induction of immunity in mice to Fasciola hepatica with a Fasciola/Schistosoma cross-reactive defined immunity antigen

The paradox of schistosomiasis is that infection confers immunity to its host, yet immunization with subcellular antigens of the parasite does not, in general, induce protective immunity. Infection or immunization with subcellular antigens of Fasciola hepatica confers high levels of immunity to a challenge infection with another trematode, Schistosoma mansoni. We have isolated by antibody affinity chromatography a Fasciola hepatica/Schistoma mansoni cross-reactive antigen, designated FhSmIII(M), and also have shown that this antigen confers immunity in mice to a challenge infection with S. mansoni. This antigen was compared with a crude F. hepatica worm extract (FhWWE) as to its ability to induce an IgG antibody response in mice, and to determine whether it had a protective effect in mice to a challenge infection with F. hepatica metacercariae. Mice immunized with FhSmIII(M) or FhWWE, and subsequently infected with F. hepatica, developed higher IgG antibody levels to FhSmIII(M), as measured by ELISA, than F. hepatica-infected controls. Mice immunized with FhWWE did not develop significant levels of resistance to challenge with F. hepatica metacercariae. Mice immunized with FhSmIII(M) and infected with F. hepatica metacercariae developed 69%-78% less worms than controls. An F. hepatica/S. mansoni cross-reactive, cross-protective defined immunity antigen confers in mice significant levels of protection to a challenge infection with F. hepatica.     

Factors associated with resistance to Schistosoma mansoni infection in an endemic area of Bahia, Brazil

Detailed knowledge of factors associated with resistance to Schistosoma mansoni infection in endemic areas might facilitate more effective schistosomiasis control. We conducted a cross-sectional study of persons resistant to schistosomiasis and found no association between socioeconomic status and resistance to infection. Mononuclear cells of resistant subjects produced higher levels of interleukin-5 (IL-5), IL-13 and interferon-γ upon stimulation with soluble egg antigen (SEA) compared with infected persons. When stimulated with Sm21.6 or Sm22.6, levels of IL-10 were higher in cell culture of resistant persons. Levels of IgE against soluble adult worm antigen (SWAP) and against interleukin-4-inducing principle from S. mansoni eggs (IPSE) and levels of IgG4 against SWAP, SEA, and Sm22.6 were lower in the resistant group compared with the susceptible group. Our data suggest that socioeconomic status could not fully explain resistance to S. mansoni infection observed in the studied area. However, a mixture of Th1 and Th2 immune responses and low levels of specific IgG4 against parasite antigens could be mediating resistance to infection.     

Antibody responses in schistosomiasis haematobium in Somalia. Relation to age and infection intensity

Antibody responses in schistosomiasis haematobium were studied in relation to age and infection intensity in Somalia. The area is highly endemic for Schistosoma haematobium but free of S. mansoni. Antibodies of the IgG class against particulate antigens of S. mansoni adult worms were investigated by immunofluorescence (gut and somatic associated antigens) and against soluble egg and adult worm antigens by ELISA. Total IgE levels were examined by Pharmacia IgE RIA, and specific IgE against soluble adult worm antigen by enzyme immunoassay. The IgG antibody response showed a characteristic pattern with highest reactivity against both gut associated and soluble egg antigens in the age group 10-14 years, when both prevalence and intensity of the infection were highest. Reactivity against somatic associated antigen was also high in this age group, but it increased slightly and remained at high level in the older ages. It is thought that such antigen is exposed mainly after the death of the parasite and that the antigenic stimulation may remain throughout most of the life of infected individuals. On the other hand, the IgG antibody reactivity against soluble adult worm antigen was low during childhood, but it increased significantly with age. It is suggested that repeated booster effects are needed for more potent response against these antigenic components. The finding of high levels of total IgE already in the youngest age groups, together with low specific IgE response, indicates that mainly other antigens are involved in the IgE production. The specific IgE response against soluble adult worm antigen was low but increased significantly with age.     

Antibody response to a purified parasite proteinase (SMw32) in Schistosoma mansoni infected mice

Indirect immunofluorescence of Schistosoma mansoni adult worm sections has revealed that the early immunoglobulin response is directed toward the parasite digestive tract. One of the components of the worm gut is a cysteine proteinase which degrades host hemoglobin ingested by the parasite. In this report the purified proteinase (SMw32) was used in ELISA and immunoblot analyses to study the specific antibody response during the course of an acute infection. We have found high titer IgG antibody in S. mansoni infected, but not uninfected, mice. The anti-proteinase response involves IgM, IgG1, IgG2a, and IgE isotypes. Total IgM and IgG levels increased by week 3 post-infection and remained elevated throughout the study (7 weeks). Increased titers (IgM, IgG) of specific anti-proteinase were also apparent by week 3 post-infection, long before fecal eggs were detectable. Mean anti-proteinase IgG stabilized at high titer by week 5 post-infection, while IgM titers decreased to near background levels. Anti-proteinase IgE was first detectable at week 4 and reached peak titers by weeks 6 and 7. The strong antibody response to the purified SMw32 proteinase is consistent with the early reactivity of S. mansoni infected mice and humans to a 31 kDa component of the worm gut described by others.     

Antibody response to Schistosoma mansoni adult worm cysteine proteinases in infected individuals

Antigens of the Schistosoma mansoni digestive tract are recognized early in the infective process. Two immunogenic components of the excretory/secretory products are proteolytic enzymes that degrade host hemoglobin in the lumen of the parasite gut. These enzymes, CP1 and CP2, belong to the class of cysteine proteinases. In this study, a preparation containing both proteinases has been used to detect proteinase antibodies in the sera of individuals living in Burundi. Of 133 individuals tested, 92% were excreting schistosome eggs. All patients with documented infections had positive anti-proteinase IgG titers (mean = 1:614), while 82% had positive IgM titers (mean = 1:267). Six weeks following praziquantel treatment, patients were assessed for egg excretion and antibody titer. Anti-proteinase IgG titers were significantly lower (mean = 1:259) than pre-treatment titers. Patients who were infected with S. japonicum or S. haematobium typically showed a cross-reactive IgG response. Patients from non-endemic regions yielded negative titers, and those with non-trematode parasites were negative (79%) or weakly positive. S. mansoni cysteine proteinases may be used for the detection of schistosome infections.     

Immunochemical characterization and diagnostic potential of a 63-kilodalton Schistosoma antigen.

Schistosoma circulating antigens were used for the detection of active infection. Anti-S. mansoni IgG2a monoclonal antibody (MAb) designated C5C4 was generated. The target epitope of this MAb was detected in adult worms, eggs, and cercariae antigenic extracts of S. mansoni and S. haematobium, had a molecular size of 63 kD, and was not detected in Fasciola hepatica and Ascaris. In addition, a 50-kD degradation product was identified only in the urine of infected individuals. Analysis by high-performance liquid chromatography of the purified antigen demonstrated only one peak. The 63-kD antigen was characterized as a protein containing 40.4% hydrophobic, 7.5% acidic, and 8.8% basic amino acids. The C5C4 MAb was used in a Fast Dot-ELISA for rapid and simple diagnosis of human schistosomiasis. The 63-kD circulating antigen was detected in 92% of urine samples from 330 S. mansoni-infected individuals, with 16% false-positive results among 130 noninfected individuals.     

Isolation and characterization of Schistosoma haematobium egg antigens

Schistosoma haematobium soluble egg antigen (ShSEA) was prepared from eggs isolated from the livers of hamsters or mice infected for at least 3 months. Immunoaffinity purified S. haematobium egg antigens (ShSh) were isolated by first passing ShSEA through a column containing anti-S. mansoni hamster IgG coupled to CNBr-activated Sepharose 4B, and recycling the unbound fraction until no more bound material could be eluted with an acid wash. The unbound fraction was then filtered through a second antibody affinity column containing anti-S. haematobium hamster IgG, and in the acid eluate the ShSh antigens were obtained. This antigenic preparation was shown by PAGE to contain at least 6 distinct bands ranging in molecular weight (Mr) from 116 to less than 31 Kd. A 40 Kd polypeptide was identified by both silver staining and EITB as specific for S. haematobium eggs. In addition, a 55 Kd worm-egg shared antigen was identified as a prominent band in EITB expressed during a primary S. haematobium hamster infection. The sera from hamsters harboring patent S. haematobium or S. mansoni infections were reacted by ELISA with ShSh antigens. The anti-Sh sera showed significantly higher absorbance values than the anti-Sm sera, demonstrating that only a minor population of S. mansoni cross-reactive egg antigens is still present in the ShSh antigens. Sera collected weekly for 13 weeks from hamsters with a primary infection of S. haematobium were then tested by ELISA against ShSh, ShSEA and SmSEA antigens. Antibody levels against both ShSEA and SmSEA were shown to increase early in infection (2 weeks). Moreover, antibody levels to ShSh did not increase until week 5 post-infection. These findings suggest that the purification procedure utilized results in the elimination of most of the S. mansoni worm antigens cross-reactive with S. haematobium eggs. The ShSh antigens had shown a high degree of sensitivity and stage-species specificity also suggesting their potential as antigens for the immunodiagnosis of schistosomiasis haematobia.     

Infection induces antibodies against the cercarial secretions, but not against the cercarial elastases of Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum and Trichobilharzia ocellata

Cercarial secretions from different species of the parasite Schistosoma and from Trichobilharzia ocellata contain a proteolytic activity, cercarial elastase, which was demonstrated by a 30 kDa band in gelatin gels. Sera of patients infected with Schistosoma mansoni, Schistosoma haematobium or Schistosoma japonicum contain immunoglobulin G which react in ELISA with cercarial secretions from all schistosomes and cross-react among the different parasite species. In Western blots, however, infection sera from patients, as well as heavily infected mice or rabbits, did not react with a 30-kDa protein. Moreover, when sections from infected snails (Biomphalaria, Bulinus and Lymnaea) were analysed by immunofluorescence using the same infection sera, only the tegument of the developing cercariae was recognized, but not the acetabular glands. In contrast, when antisera against purified cercarial elastase from either S. mansoni or S. haematobium were tested with sections of infected Biomphalaria or Bulinus, fluorescence was strong in the preacetabular glands of the cercariae of either species, but undetectable with the tegument. Cross-reactivity of both antisera extended to T. ocellata-infected Lymnaea, but not to S. japonicum-infected Oncomelania. In conclusion, although immunization with purified cercarial elastase results in antibody production, the enzyme does not induce an apparent antibody response following natural infection.     

Schistosoma mansoni cationic egg antigens (CEF6): immunoserology with oxamniquine-treated patients and involvement of CEF6 in the circumoval precipitin reaction

The serologic activity of a cationic fraction (denoted CEF6) of Schistosoma mansoni soluble egg antigen was compared in an ELISA with that of the unpurified soluble egg antigen for the ability to detect human infections and for the prediction of chemotherapeutic success in patients followed up to 5 years post-treatment with oxamniquine. The cationic fraction correctly identified 100% of 20 patients as infected with S. mansoni; moreover, 50% (10 of 20) seroconverted to negative by 2 years post-treatment and 100% of 15 patients tested were negative 5 years post-treatment. In general, the cationic fraction was superior to the unpurified soluble egg antigen for the detection of infection and for the prediction of chemotherapeutic success. The cationic fraction also exhibited greater immunologic specificity over the unpurified soluble egg antigen in that the latter exhibited higher titers than the former to antibodies against heterologous parasite antigens (Fasciola hepatica, Clonorchis sinensis, Paragonimus westermani adult worms; Trichinella spiralis larvae). Moreover, rabbit antisera raised against egg antigens isolated from the precipitation formed when fresh S. mansoni eggs are incubated with S. mansoni infection of immunization sera (known as circumoval precipitin reactions or COP) reacted strongly with the cationic fraction in ELISA. In addition, antiserum to the cationic fraction as well as antisera against either of the two antigenic components of this fraction, known as antigens alpha 1 and omega 1, all give positive COP reactions when incubated with fresh S. mansoni eggs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of immune responses of Schistosoma mansoni-infected mice with distinct chronic forms of the disease

BACKGROUND: Schistosoma mansoni-infected mice tend to present with either one of two different hepatic pathological patterns during chronic infection: periportal fibrosis (PF) with portal concentration of periovular granulomas and fibrosis or isolated granulomas (IG), with scattered periovular granulomas within the liver. These are models for the two clinical presentations of schistosomiasis, the severe hepatosplenic and the mild intestinal forms. In the present work, we examined the relationship between the development of these histopathological aspects and immunological markers in S. mansoni-infected mice. Although BALB/c mice with PF and IG had similar egg numbers in the liver, PF mice had higher liver collagen contents than mice with IG. Cultured spleen cells from mice with PF and IG had similar proliferation 20 and 40 weeks after S. mansoni infection upon stimulation with parasite egg antigen (SEA) or mitogen (Con A). Production of IL-4 upon SEA stimulation was higher in cell cultures from mice with PF, whereas IL-5 and IFN-gamma levels were not statistically different between PF and IG groups. Mice with IG had similar serum concentrations of total IgE and anti-SEA IgG1, IgG2a, IgG2b and IgG3 compared to sera from PF mice. Levels of IgG1 and IgG2a antibodies were the highest and the lowest detected, respectively. In conclusion, isogenic BALB/c mice infected with S. mansoni that develop periportal fibrosis or isolated granulomas have similar immunological patterns despite the two pathologic forms of schistosomal liver fibrosis.     

Effect of praziquantel on Schistosoma mansoni eggs: leucine aminopeptidase (LAP) activity and anti-LAP antibodies

High levels of leucine aminopeptidase (LAP) have been detected in Schistosoma mansoni eggs. In this report we demonstrate that this enzyme is immunogenic in mice infected with either S. mansoni or S. japonicum. The anti-schistosomal agent, praziquantel, causes rapid release of this enzyme from schistosome eggs in vitro. Praziquantel treatment of S. japonicum infected mice is associated with a significant decrease in anti-LAP antibodies. Anti-LAP IgM antibody levels decreased more than did anti-LAP IgG antibodies.     

Juvenile rhesus monkeys have lower type 2 cytokine responses than adults after primary infection with Schistosoma mansoni

Adults and children have differences in their susceptibility to schistosomiasis. The relative influences of age-dependent innate resistance and acquired immunity in the differences between susceptibility to schistosomiasis are difficult to assess in humans. Therefore, we exposed juvenile and adult female rhesus monkeys to primary infection with Schistosoma mansoni. In contrast to the adult animals, the juvenile rhesus monkeys had low levels of interleukin (IL)-4 and IL-5 production by peripheral blood mononuclear cells after schistosome infection, as well as lower levels of parasite-antigen-specific antibody (IgG, IgM, and IgA) responses, and produced limited antigen-specific or total IgE. Juvenile animals had statistically nonsignificant increased worm burdens and tissue or fecal egg counts, compared with that of adults, whereas circulating schistosome antigens were significantly higher in infected juvenile monkeys. These results suggest that juvenile rhesus monkeys have reduced type 2 cytokine responses after primary schistosome infections and perhaps are more susceptible to parasite infection.     

Schistosoma bovis as an immunological analogue of S. haematobium

The host-parasite relationships of Schistosoma bovis and S. haematobium have been compared in normal and T-cell-deprived mice, and have been found to contrast with that of S. mansoni. Deprived mice infected with either of the former two schistosome species survived as long as, or longer than, comparably infected immunologically intact controls, and hepatocytes of infected deprived mice were not damaged in the absence of granuloma formation. S. mansoni-infected deprived mice, however, die earlier than intact controls, and suffer extensive hepatocellular abnormalities. A high degree of cross-reactivity between S. bovis, S. haematobium and S. mansoni antibodies and antigens was noted in immunoprecipitation but a greater degree of homology between S. haematobium and S. bovis egg antigens was demonstrated by enzyme immunoassay (ELISA). S. haematobium and S. bovis thus resemble each other more closely than either resembles S. mansoni, and in view of the apparent antigenic similarities between S. haematobium and S. bovis and the relatively greater ease with which the S. bovis life-cycle can be maintained in the laboratory, the animal parasite may be useful in providing material for further immunological studies of the human infection.     

Vaccination of mice with gamma-irradiated Schistosoma japonicum cercariae

gamma-irradiated cercarial vaccines induce high levels of protection in mice against Schistosoma mansoni infection, however, the same has not been well established for S. japonicum. Here we describe vaccination studies in mice with gamma-irradiated S. japonicum cercariae testing the effectiveness of different irradiation doses, number of vaccinations, and mouse strains. In CBA/Ca mice, a single percutaneous exposure to 500 S. japonicum cercariae previously attenuated by 10, 20, 30, 40 or 50 krad gamma-irradiation induced significant, but comparable levels of protection (34-46%) against challenge infection. In a repeat experiment in C57Bl/6 mice, only groups vaccinated with 10 or 20 krad gamma-irradiated cercariae showed statistically significant, but lower levels of resistance (20-24%). Multiple vaccination of CBA/Ca mice with 500 20 krad gamma-irradiated cercariae did not improve the resistance level (40%). Analysis of IgG responses showed no clear correlation between antibody levels and levels of protection. Western blot analysis suggested that recognition of a 200-kDa antigen might be correlated with protection, that antigens of 42 and 50 kDa may be involved in the protection induced by single vaccination, but that different antigens might be protective in single vs multiple vaccinations. Sera from mice vaccinated with gamma-irradiated cercariae recognized many fewer antigens than more protective sera from mice vaccinated with UV-attenuated cercariae. These results suggest that the mouse may not be a suitable host for studies involving gamma-irradiated S. japonicum vaccines.     

Serodiagnosis of Schistosoma mansoni with microsomal adult worm antigen in an enzyme-linked immunosorbent assay using a standard curve developed with a reference serum pool

A standardized microtest plate enzyme-linked immunosorbent assay was developed using the microsomal fraction of adult worms of Schistosoma mansoni (MAMA) as antigen. The standard reference serum pool was prepared from acutely and chronically infected rhesus monkeys and was shown to be appropriate as a standard for measuring the levels of reactivity of the unknowns. The standard serum pool was arbitrarily designated as having 100 activity units per microliter. The levels of reactivity of the unknowns were expressed as activity units per microliter. Serum specimens were obtained from 190 patients infected with S. mansoni in the Caribbean, South America, and Africa. Serum was obtained from small numbers of patients infected with S. haematobium, S. japonicum, or S. mekongi. Controls were 136 patients with other helminthic infections, 142 patients with protozoal or other diseases with liver involvement, and 81 healthy serum donors. The J index (or predictability) of the assay was calculated to determine the significant level of reactivity. The assay has a predictability of 95% for both patients with S. mansoni infections and those with other infections. The sensitivity of the assay for S. mansoni infections was 96%, and the specificity (in terms of cross-reactions with infections with other parasite genera or with other liver diseases) was 99%. The heterologous Schistosoma species showed a markedly lower level of reactivity, with an overall sensitivity of 55%. This is in accord with the species-specificity previously recognized in MAMA, and emphasizes the need for standard reference pools of human sera prepared from patients infected with single species of each of the Schistosoma. Use of these pools in assays with antigens of the respective schistosome species would allow optimum serologic evaluation.     

Familial resemblance in humoral immune response to defined and crude Schistosoma mansoni antigens in an endemic area in Brazil.

This study addressed whether the humoral immune response to crude and defined Schistosoma mansoni antigens aggregates within families. The sample included 155 siblings from 42 nuclear families in Brazil. Sera examined by ELISA for antibody isotypes reactive to defined schistosome antigens and crude schistosome antigens (soluble adult worm antigen preparation and soluble egg antigen) demonstrated that there was a difference in sibling-pair correlations between defined and crude S. mansoni antigens. In contrast to the finding with crude antigens, egg-positive sibling pairs showed significant familial resemblance for all IgG subclasses and IgE to adult-stage antigens Smp20.8 and Smp50. Only the IgE and IgG4 isotypes showed familial resemblance to the egg-stage antigen, Smp40. Egg-negative sibling pairs showed significant familial resemblance only for IgE and IgG4 to Smp40. That both the IgE and IgG4 response to defined S. mansoni antigens showed familial resemblance is interesting in light of the converging evidence for the role of IgE and IgG4 in human susceptibility and resistance to reinfection.     

Reactivity of Schistosoma japonicum and S. mansoni antigen preparations in indirect haemagglutination (IHA) with sera of patients with homologous and heterologous schistosomiasis.

Sera of patients infected with Schistosoma japonicum, S. mansoni or S. haematobium were tested in an indirect haemagglutination assay (IHA) using soluble S. japonicum egg antigen (SjSEA) and soluble S. mansoni adult antigen prepared either from a Puerto Rican strain (SmAWA) or an Egyptian strain (SmBW; Cellognost-Schistosomiasis Kit). Reactions were best, in terms of titres and sensitivity, in homologous systems. Heterologous systems were less reliable, particularly those using sera from urinary schistosomiasis patients. It is suggested that IHA is a suitable test to detect Schistosoma infections, especially when homologous systems are used.     

Identification of a genus-specific Schistosoma mansoni soluble egg antigen reactive with the serum of infected patients

The ability of serum samples obtained from humans with schistosomiasis mansoni to recognize Schistosoma mansoni soluble egg antigens (SmSEA) was assessed by the enzyme-linked immunotransfer blot (EITB) method. The sera from 15 infected patients before and 2 years after treatment with oxamniquine (n = 30) recognized bands ranging in molecular weight from 9 to 200 Kd. A 31 Kd component of SmSEA was recognized by all infection sera, but not by normal human serum. The sera from 2 humans infected with S. haematobium and 2 with S. japonicum also recognized the 31 Kd band present in SmSEA, whereas those from 2 humans infected with Fasciola hepatica did not. The 31 Kd antigen was isolated by electrophoresing SmSEA through a 15% SDS-acrylamide gel, followed by excision and electroelution of the 31 Kd band. The purified 31 Kd was used in conjunction with ELISA at a concentration of 1 microgram/ml to confirm the apparent genus specificity of this protein. Thus, the sera of patients infected with S. mansoni, S. haematobium, or S. japonicum were clearly reactive in ELISA, whereas normal human serum or sera from patients with F. hepatica were negative. In conclusion, the 31 Kd protein appears to be a good candidate for developing a screening assay for the immunodiagnosis of schistosomiasis.     

Intradermal immunization of rats with plasmid DNA encoding Schistosoma mansoni 28 kDa glutathione S-transferase

Direct administration of plasmid DNA encoding an antigen represents an attractive approach to vaccination against infectious diseases, particularly in developing countries where easy-to-handle and cost-effective vaccines are needed. We have investigated the potential of DNA immunization to induce a specific antibody response against Schistosoma mansoni , using plasmid-DNA encoding the protective antigen, S. mansoni 28 kDa glutathione S-transferase (Sm28GST). Since S. mansoni parasite penetrates into its host through the skin, this tissue was chosen for plasmid DNA delivery. Following plasmid DNA administration into the skin of rats, the parasite antigen was detected in skin cells by immunohistochemistry. Three administrations of 200 μg plasmid at 14 day intervals led to the induction of a long-lasting specific IgG antibody response in the sera of immunized rats, with a predominance of IgG2a and IgG2b subclasses. Sera of immunized animals were able to mediate antibody-dependent cellular cytotoxicity in vitro , leading to the specific killing of parasite larvae. A parasite challenge performed on plasmid DNA-immunized animals induced a strong and rapid boosting effect on the specific IgG antibody response. These results demonstrate the potential of genetic immunization via the skin with plasmid DNA encoding Sm28GST for inducing immune responses with protective patterns against an S. mansoni infection .     

Real-time PCR for detection of low intensity Schistosoma japonicum infections in a pig model

Decades of successful Schistosoma japonicum control have increased the interest in how to diagnose low intensity infections. A real-time PCR assay targeting the mitochondrial NADH dehydrogenase I gene in S. japonicum was evaluated in infected pigs with very low egg output. Six out of 12 S. japonicum infected pigs were treated with praziquantel 8 weeks after infection and all pigs were followed for 16 weeks post-infection. One commercial and one non-commercial extraction method were evaluated in combination with PCR on faecal samples. PCR with either extraction method were equally sensitive as the DBL-filtration/sedimentation technique in the acute, productive stage. PCR recovered slightly more positive samples in the chronic stage, but most faecal samples were negative for both PCR and microscopy from week 9 post-infection irrespective of treatment. IgG antibody titers against soluble egg antigen IgG remained high throughout the study in both the treated and non-treated group. PCR was consistently negative in serum and urine samples and negative in most of the caecal biopsies. We conclude that the S. japonicum faecal PCR is a highly sensitive test. However, in clinical samples when faecal egg output almost reaches nil in the chronic stage despite persistent worm burdens, both the faecal PCR and microscopy results were negative. Real-time PCR is less labour intensive than most microscopy methods, but has a higher material cost per sample.