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Schistosomiasis causes significant morbidity and mortality. Vaccine efforts to date indicate the need to increase the immunogenicity of Schistosoma antigens. The multiple antigen-presenting system, whereby proteins are genetically fused to rhizavidin and affinity linked to biotinylated templates, enables the generation of robust immune responses. The objective of this work was to express and purify the S. mansoni antigens, SmTSP-2 and SmCD59.2, in fusion with rhizavidin. The fusion with rhizavidin greatly decreased the expression level of rSmTSP-2, but not rSmCD59.2, and both were expressed in the insoluble fraction, requiring optimization of culture conditions. Evaluation of different E. coli strains and media showed that BL21-DE3 cultured in Terrific Broth provided the highest expression levels of both proteins. Investigation of a range of time and temperature of induction showed that E. coli strains expressing rRzv:SmTSP-2 and rRzv:SmCD59.2 showed the highest protein production at 23 °C for 15 h. Recombinant proteins were purified by a single step of affinity chromatography allowing isolation of these proteins in high concentration and purity. The optimization process increased final soluble protein yield of rRzv:SmTSP-2 by fourfold and rRzv:SmCD59.2 by tenfold, providing ~ 20 mg/L of each protein. Optimized fusion protein production will allow antigen use in biotin-rhizavidin affinity platforms., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Determining Schistosoma mansoni infection rate and intensity is challenging due to the low sensitivity of the Kato-Katz (KK) test that underestimates the true disease prevalence. Circulating cathodic antigen (CCA) excreted in urine is constantly produced by adult worms and has been used as the basis of a simple, non-invasive point of care test (POC-CCA) for Schistosoma mansoni infections. Although the abundance of CCA in urine is proportional to worm burden, the POC-CCA test is marketed as a qualitative test, making it difficult to investigate the wide range of infection intensities. This study was designed to compare the prevalence and intensity of S. mansoni by KK and POC-CCA and quantify, on fresh and frozen (<-20°C) urine samples, CCA using the visual scores and the ESEquant LR3 reader., Methodology: Stool and urine samples were collected from 759 school-aged children. The prevalence and intensity of S. mansoni were determined using KK and POC-CCA. The degree of the positivity of POC-CCA was estimated by quantifying CCA on fresh and frozen urine samples using visual scores and strip reader. The prevalence, the infection intensity as well the relative amounts of CCA were compared., Results: The S. mansoni infection rates inferred from POC-CCA and KK were 40.7% and 9.4% respectively. Good correlations were observed between infection intensities recorded by; i) the reader and visual scoring system on fresh (Rho = 0.89) and frozen samples (Rho = 0.97), ii) the reader on fresh urine samples and KK (epg) (Rho = 0.44). Nevertheless, 238 POC-CCA positive children were negative for KK, and sixteen of them had high levels of CCA. The correlation between results from the reader on fresh and frozen samples was good (Rho = 0.85). On frozen samples, CCA was not detected in 55 samples that were positive in fresh urine samples., Conclusion: This study confirmed the low sensitivity of KK and the high capacity of POC-CCA to provide reliable data on the prevalence and intensity of S. mansoni infections. The lateral flow reader enabled accurate quantification of CCA under field conditions on fresh and frozen urine samples with less time and effort than KK., Competing Interests: The authors have declared that no competing interests exist.

Aims: Schistosomes infect approximately 250 million people worldwide. To date, there is no effective vaccine available for the prevention of schistosome infection in endemic regions. There remains a need to develop means to confer long-term protection of individuals against reinfection. In this study, an annexin, namely annexin B30, which is highly expressed in the tegument of Schistosoma mansoni was selected to evaluate its immunogenicity and protective efficacy in a mouse model., Methods and Results: Bioinformatics analysis showed that there were three potential linear B-cell epitopes and four conformational B-cell epitopes predicted from annexin B30, respectively. Full-length annexin B30 was cloned and expressed in Escherichia coli BL21(DE3). In the presence of adjuvants, the soluble recombinant protein was evaluated for its protective efficacy in two independent vaccine trials. Immunization of CBA mice with recombinant annexin B30 formulated either in alum only or alum/CpG induced a mixed Th1/Th2 cytokine profile but no significant protection against schistosome infection was detected., Conclusion: Recombinant annexin B30 did not confer significant protection against the parasite. The molecule may not be suitable for vaccine development. However, it could be an ideal biomarker recommended for immunodiagnostics development., (© 2019 John Wiley & Sons Ltd.)

Currently, three recombinant antigens based vaccines are under clinical trials against Schistosomiasis, but there is no vaccine available for prophylaxis or therapeutic. This study was conducted to construct a multi-epitope based vaccine against Schistosoma mansoni via utilizing Sm14, Sm21.7, Sm23, Sm29, Smp80, Sm-CB and SM-TSP-2 antigens. Helper T lymphocyte (HTL), cytotoxic T lymphocyte (CTL) and IFN-γ epitopes were predicted. Furthermore, Pan HLA DR-binding epitope was added to the vaccine. Moreover, 50S ribosomal protein L7/L12 of Mycobacterium tuberculosis as a novel TLR4 agonist was applied. The TAT peptide was added to the vaccine to augment intracellular delivery. The selected epitopes were linked together through appropriate linkers and chimeric vaccine was constructed with 617 amino acids with molecular weight of 65.43 kDa. Physico-chemical properties revealed a soluble protein with antigenic and non-allergic properties. Further analyses validated the stability of the construct that was able to interact with TLR4. Immunoinformatics analysis demonstrated the strong potential of constructed vaccine to stimulate T and B-cell mediated immune responses. In summary, obtained data indicated that the proposed vaccine can properly induce both T and B cells immune responses and could possibly be utilized for prophylactic or therapeutic aims in response to infection caused by S. mansoni., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Schistosoma mansoni venom allergen-like proteins (SmVALs) are part of a diverse protein superfamily partitioned into two groups (group 1 and group 2). Phylogenetic analyses of group 1 SmVALs revealed that members could be segregated into subclades (A-D); these subclades share similar gene expression patterns across the parasite lifecycle and immunological cross-reactivity. Furthermore, whole-mount in situ hybridization demonstrated that the phylogenetically, transcriptionally and immunologically-related SmVAL4, 10, 18 and 19 (subclade C) were all localized to the pre-acetabular glands of immature cercariae. Our results suggest that SmVAL group 1 phylogenetic relationships, stage-specific transcriptional profiles and tissue localization are predictive of immunological cross-reactivity., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Accurate and sensitive point-of-care diagnostic tools are critical for schistosomiasis control and elimination. The existing ultrasensitive lateral flow assay for the detection of Schistosoma circulating anodic antigen (CAA) has demonstrated excellent sensitivity but is time-consuming and requires significant laboratory infrastructure that limits its applicability at the point of care. To address this challenge, we sought to develop an alternative sample preparation method to concentrate CAA from large-volume urine samples requiring little-to-no laboratory equipment. The developed method relies on electrostatic interactions between the negatively-charged CAA biomarker and positively-charged poly(amidoamine) (PAMAM) dendrimers functionalized to the surface of magnetic particles. After CAA capture on the surface of the PAMAM-functionalized magnetic beads, the supernatant was removed, and CAA was eluted into a small-volume, high-salt elution buffer. This concentrated eluate was subsequently applied to the existing lateral flow assay. The PAMAM-functionalized magnetic bead-based CAA concentration method was extensively characterized for its robustness, evaluated on a set of endemic urine samples, and compared to spin filter-based concentration methods. The novel bead-based sample preparation method used only disposable laboratory materials, resulted in a 200-fold improvement in CAA limits of detection, and performed just as well as infrastructure-intensive and high-cost spin filter methods. Additionally, the functionalized beads were robust to variations in sample pH and storage conditions. The PAMAM-functionalized magnetic bead-based CAA concentration method represents a promising step toward ultrasensitive schistosomiasis diagnosis at the point of care.

Background: Schistosoma mansoni venom allergen-like protein (SmVAL) is a gene family composed of 29 members divided into group 1 encoding proteins potentially secreted, and group 2 encoding intracellular components. Some members were found to be upregulated in the transition of germ ball - cercariae - day 3 schistosomula, suggesting that group 1 SmVAL proteins are associated with the invasion of the human host, although their functions are not completely established. Recently, we have described the localization of SmVAL7 (group 1) and SmVAL6 (group 2) transcripts in the oesophageal gland and in the oral and ventral suckers of adult parasites, respectively. The expression patterns of the two genes suggest that SmVAL7 protein plays a role in the blood-feeding process while SmVAL6 is associated with the parasite attachment and movement in the vasculature. In this way, searching for additional secreted SmVAL proteins that could be involved in key processes from skin penetration to the beginning of blood-feeding, we investigated the tissue localization of SmVAL4, 13, 16 and 24 by whole-mount in situ hybridization (WISH)., Results: We report here the localization of group 1 SmVAL4 and 24 transcripts in the pre-acetabular glands of developing germ balls. Time course experiments of in vitro cultured schistosomula after cercariae transformation demonstrated that SmVAL4 protein is secreted during the first 3 h of in vitro culture, correlating with the emptying of acetabular glands as documented by confocal microscopy. In addition, the localization of SmVAL13 transcripts in adult male anterior oesophageal gland suggests that the respective protein may be involved in the first steps of the blood-feeding process. SmVAL16 was localized close to the neural ganglia and requires further investigation., Conclusions: Our findings demonstrate that SmVAL proteins have localizations that place them in strategic positions to be considered as potential vaccine candidates as some members are exposed to interaction with the immune system and may participate in key processes of mammalian invasion and parasitism establishment.

Background: The heptalaminate-covered, syncytial tegument is an important anatomical adaptation that enables schistosome parasites to maintain long-term, intravascular residence in definitive hosts. Investigation of the proteins present in this surface layer and the immune responses elicited by them during infection is crucial to our understanding of host/parasite interactions. Recent studies have revealed a number of novel tegumental surface proteins including three (SmCD59a, SmCD59b and Sm29) containing uPAR/Ly6 domains (renamed SmLy6A SmLy6B and SmLy6D in this study). While vaccination with SmLy6A (SmCD59a) and SmLy6D (Sm29) induces protective immunity in experimental models, human immunoglobulin responses to representative SmLy6 family members have yet to be thoroughly explored., Methodology/principal Findings: Using a PSI-BLAST-based search, we present a comprehensive reanalysis of the Schistosoma mansoni Ly6 family (SmLy6A-K). Our examination extends the number of members to eleven (including three novel proteins) and provides strong evidence that the previously identified vaccine candidate Sm29 (renamed SmLy6D) is a unique double uPAR/Ly6 domain-containing representative. Presence of canonical cysteine residues, signal peptides and GPI-anchor sites strongly suggest that all SmLy6 proteins are cell surface-bound. To provide evidence that SmLy6 members are immunogenic in human populations, we report IgG1 (as well as IgG4 and IgE) responses against two surface-bound representatives (SmLy6A and SmLy6B) within a cohort of S. mansoni-infected Ugandan males before and after praziquantel treatment. While pre-treatment IgG1 prevalence for SmLy6A and SmLy6B differs amongst the studied population (7.4% and 25.3% of the cohort, respectively), these values are both higher than IgG1 prevalence (2.7%) for a sub-surface tegumental antigen, SmTAL1. Further, post-treatment IgG1 levels against surface-associated SmLy6A and SmLy6B significantly drop (p = 0.020 and p < 0.001, respectively) when compared to rising IgG1 levels against sub-surface SmTAL1., Conclusions/significance: Collectively, these results expand the number of SmLy6 proteins found within S. mansoni and specifically demonstrate that surface-associated SmLy6A and SmLy6B elicit immunological responses during infection in endemic communities.

Background: The soluble antigen preparation of adult schistosomes (SWAP) has often been used to probe host responses against these blood-dwelling parasites. Despite its long-established use there is limited knowledge about its composition. The information we provide here on the molecular composition of SWAP may contribute as a guide for a rational selection of antigenic targets., Methods: Label-free quantitative shotgun proteomics was employed to determine the composition and abundance of SWAP constituents. Briefly, paired adult Schistosoma mansoni worms were sonicated in PBS pH 7.2 and ultracentrifuged for recovery of the soluble supernatant. An aliquot was subjected to trypsin digestion. Resulting peptides were separated under ultra-high performance liquid chromatography and analysed using an orbitrap mass spectrometer. Spectral data were interrogated using SequestHT against an in-house S. mansoni database. Proteins were quantified by recording the mean area under curve obtained for up to three most intense detected peptides. Proteins within the 90(th) percentile of the total SWAP mass were categorized according to their sub-cellular/tissue location., Results: In this work we expanded significantly the list of known SWAP constituents. Through application of stringent criteria, a total of 633 proteins were quantitatively identified. Only 18 proteins account to 50% of the total SWAP mass as revealed by their cumulative abundance. Among them, none is predicted as a secreted molecule reinforcing the point that SWAP is dominated by cytosolic and cytoskeletal proteins. In contrast, only 3% of the mass comprised proteins proposed to function at the host-parasite interfaces (tegument and gut), which could conceivably represent vulnerable targets of a protective immune response. Paradoxically, at least 11 SWAP proteins, comprising ~25% of its mass, have been tested as vaccine candidates., Conclusions: Our data suggest that use of SWAP to probe host responses has greatest value for diagnostic purposes or assessing intensity of infection. However, the preparation is of limited utility as an antigen source for investigating host responses to proteins expressed at or secreted from worm-host interfaces. The data also pose the question as to why vaccination with SWAP, containing so many proposed vaccine candidates, has no additive or even synergistic effects on the induction of protection.

Vaccines are an important public health measure for prevention and treatment of diseases. In addition to the vaccine immunogen, many vaccines incorporate adjuvants to stimulate the recipient's immune system and enhance vaccine-specific responses. While vaccine development has advanced from attenuated organism to recombinant protein or use of plasmid DNA, the development of new adjuvants that safely increase immune responses has not kept pace. Previous studies have shown that the complex mixture of molecules that comprise saline soluble egg antigens (SEA) from Schistosoma mansoni eggs functions to promote CD4(+) T helper 2 (Th2) responses. Therefore, we hypothesized that coadministration of SEA with a Listeria vector HIV-1 Gag (Lm-Gag) vaccine would suppress host cytotoxic T lymphocyte (CTL) and T helper 1 (Th1) responses to HIV-1 Gag epitopes. Surprisingly, instead of driving HIV-1 Gag-specific responses toward Th2 type, we found that coadministration of SEA with Lm-Gag vaccine significantly increased the frequency of gamma interferon (IFN-γ)-producing Gag-specific Th1 and CTL responses over that seen in mice administered Lm-Gag only. Analysis of the functionality and durability of vaccine responses suggested that SEA not only enlarged different memory T cell compartments but induced functional and long-lasting vaccine-specific responses as well. These results suggest there are components in SEA that can synergize with potent inducers of strong and durable Th1-type responses such as those to Listeria. We hypothesize that SEA contains moieties that, if defined, can be used to expand type 1 proinflammatory responses for use in vaccines., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Schistosomiasis is caused by several worm species of the genus Schistosoma and afflicts up to 600 million people in 74 tropical and sub-tropical countries in the developing world. Present disease control depends on treatment with the only available drug praziquantel. No vaccine exists despite the intense search for molecular candidates and adjuvant formulations over the last three decades. Cysteine peptidases such as papain and Der p 1 are well known environmental allergens that sensitize the immune system driving potent Th2-responses. Recently, we showed that the administration of active papain to mice induced significant protection (P<0.02, 50%) against an experimental challenge infection with Schistosoma mansoni. Since schistosomes express and secrete papain-like cysteine peptidases we reasoned that these could be employed as vaccines with inbuilt adjuvanticity to protect against these parasites. Here we demonstrate that sub-cutaneous injection of functionally active S. mansoni cathepsin B1 (SmCB1), or a cathepsin L from a related parasite Fasciola hepatica (FhCL1), elicits highly significant (P<0.0001) protection (up to 73%) against an experimental challenge worm infection. Protection and reduction in worm egg burden were further increased (up to 83%) when the cysteine peptidases were combined with other S. mansoni vaccine candidates, glyceraldehyde 3-phosphate dehydrogenase (SG3PDH) and peroxiredoxin (PRX-MAP), without the need to add chemical adjuvants. These studies demonstrate the capacity of helminth cysteine peptidases to behave simultaneously as immunogens and adjuvants, and offer an innovative approach towards developing schistosomiasis vaccines.

The Schistosoma mansoni Venom Allergen-Like proteins (SmVALs) are members of the SCP/TAPS (Sperm-Coating Protein/Tpx-1/Ag5/PR-1/Sc7) protein superfamily, which may be important in host-pathogen interactions. Whole mount in situ hybridisation demonstrated a distinct expression pattern in oral and ventral suckers of adult worms for SmVAL6 and in the oesophageal gland for SmVAL7 transcripts, respectively. Additionally, immunocytochemistry analysis corroborated SmVAL7 expression in the oesophageal gland. Analysis of protein expression across the parasite's life cycle revealed that the SmVAL6 protein is upregulated in cercariae and adult male worms. Furthermore, SmVAL6 protein was identified by mass spectrometry in tegument fractions of adult worms. Finally, we speculate on possible functions of these two SmVALs at the host-parasite interface., (Copyright © 2012 Australian Society for Parasitology Inc. All rights reserved.)

Soluble egg antigens (SEA) of the human parasite Schistosoma mansoni are among the strongest natural stimuli of Th2 responses. Omega-1, a major glycoprotein in SEA, initiates these characteristic Th2 responses through conditioning of dendritic cells (DCs). In view of the reported immunomodulatory potential of SEA glycans, we have investigated omega-1 glycosylation, using an approach combining mass spectrometric techniques and enzyme treatments at the glycopeptide level. We demonstrate that omega-1 has two fully occupied N-glycosylation sites, each mainly carrying core-difucosylated diantennary glycans with one or more Lewis X motifs in the antennae. Using a specific approach of nanoscale LC-MS(/MS) and MALDI-TOF(/TOF) MS in combination with exoglycosidase treatments of tryptic glycopeptides, we were able to provide a detailed, site-specific glycosylation analysis of a single, native S. mansoni glycoprotein. The obtained knowledge of the glycans present on omega-1 contributes to a full understanding of the mode of action of this immunomodulatory glycoprotein.

Background: This work demonstrates successful propagation of HCV in HepG2 and human blood cells as well as viral shedding into their culture media. The influence of Schistosoma mansoni crude soluble egg antigens (SEA) on the rate of viral propagation in both mammalian cells was also monitored., Methodology: HepG2 cells were inoculated with HCV viremic human sera and some wells were exposed to HCV infection in presence of SEA. Cells were harvested for RT-PCR and Western blotting analysis. HepG2 media was collected for HCV ELISA. Blood samples from HCV-infected humans were cultured in the presence and absence of SEA. Media were collected at different time points post culturing and subjected to HCV ELISA., Results: The ELISA concentration of HCV antigens were generally higher in media of infected HepG2 cells compared to media of control cells at all time intervals post infection. Western blots showed reactivity to immunogenic peptides of different molecular weights in lysate of infected HepG2 cells that were not evidenced in uninfected cells. In presence of SEA, RT-PCR results revealed earlier detection of viral RNA in infected HepG2 cells compared to in absence of such bilharzial antigen. Also, ELISA results revealed higher levels of detected HCV antigens in media of both infected HepG2 and blood cells cocultured with S. mansoni SEA compared to that of cultured infected cells in absence of the parasite antigens., Conclusion: HepG2 cells as well as whole blood cultures maintain HCV replication. Furthermore, SEA has the potential to enhance HCV propagation.

Background: Schistosomiasis affects more than 200 million individuals worldwide, with a further 650 million living at risk of infection, constituting a severe health problem in developing countries. Even though an effective treatment exists, it does not prevent re-infection, and the development of an effective vaccine still remains the most desirable means of control for this disease., Methodology/principal Findings: Herein, we report the cloning and characterization of a S. mansoni Stomatin-like protein 2 (SmStoLP-2). In silico analysis predicts three putative sites for palmitoylation (Cys11, Cys61 and Cys330), which could contribute to protein membrane association; and a putative mitochondrial targeting sequence, similar to that described for human Stomatin-like protein 2 (HuSLP-2). The protein was detected by Western blot with comparable levels in all stages across the parasite life cycle. Fractionation by differential centrifugation of schistosome tegument suggested that SmStoLP-2 displays a dual targeting to the tegument membranes and mitochondria; additionally, immunolocalization experiments confirm its localization in the tegument of the adult worms and, more importantly, in 7-day-old schistosomula. Analysis of the antibody isotype profile to rSmStoLP-2 in the sera of patients living in endemic areas for schistosomiasis revealed that IgG1, IgG2, IgG3 and IgA antibodies were predominant in sera of individuals resistant to reinfection as compared to those susceptible. Next, immunization of mice with rSmStoLP-2 engendered a 30%-32% reduction in adult worm burden. Protective immunity in mice was associated with specific anti-rSmStoLP-2 IgG1 and IgG2a antibodies and elevated production of IFN-gamma and TNF-alpha, while no IL-4 production was detected, suggesting a Th1-predominant immune response., Conclusions/significance: Data presented here demonstrate that SmStoLP-2 is a novel tegument protein located in the host-parasite interface. It is recognized by different subclasses of antibodies in patients resistant and susceptible to reinfection and, based on the data from murine studies, shows protective potential against schistosomiasis. These results indicate that SmStoLP-2 could be useful in a combination vaccine.

Infection of the mammalian host by the parasitic helminth Schistosoma mansoni is accompanied by the release of excretory/secretory molecules (ES) from cercariae which aid penetration of the skin. These ES molecules are potent stimulants of innate immune cells leading to activation of acquired immunity. At present however, it is not known which cells take up parasite antigen, nor its intracellular fate. Here, we develop a technique to label live infectious cercariae which permits the imaging of released antigens into macrophages (MPhi) and dendritic cells (DCs) both in vitro and in vivo. The amine reactive tracer CFDA-SE was used to efficiently label the acetabular gland contents of cercariae which are released upon skin penetration. These ES products, termed '0-3hRP', were phagocytosed by MHC-II(+) cells in a Ca(+) and actin-dependent manner. Imaging of a labelled cercaria as it penetrates the host skin over 2 hours reveals the progressive release of ES material. Recovery of cells from the skin shows that CFDA-SE labelled ES was initially (3 hrs) taken up by Gr1(+)MHC-II(-) neutrophils, followed (24 hrs) by skin-derived F4/80(+)MHC-II(lo) MPhi and CD11c(+) MHC-II(hi) DC. Subsequently (48 hrs), MPhi and DC positive for CFDA-SE were detected in the skin-draining lymph nodes reflecting the time taken for antigen-laden cells to reach sites of immune priming. Comparison of in vitro-derived MPhi and DC revealed that MPhi were slower to process 0-3hRP, released higher quantities of IL-10, and expressed a greater quantity of arginase-1 transcript. Combined, our observations on differential uptake of cercarial ES by MPhi and DC suggest the development of a dynamic but ultimately balanced response that can be potentially pushed towards immune priming (via DC) or immune regulation (via MPhi).

Schistosoma mansoni is a parasitic trematode of the portal-mesenteric veins with a closed-end intestine. Adult worms regurgitate their intestinal content after digestion, together with constituents of the lining gut. Some of these molecules circulate in the blood and are antigenic. We obtain a "vomit" preparation and preliminary evaluate its biochemical composition and antigenic capacity. The "vomit" preparation was obtained after changes in temperature and solutions of incubation of adult worms between 4 and 37 degrees C. Supernatant was assayed for protein, carbohydrate concentration and enzymatic activities associated to the intestine and to the worm tegument. The antigenicity of the product was evaluated using Western blot (WB) analysis against sera of experimentally infected mice, before and after drug cure, sera from people infected with S. mansoni and from individuals infected with other parasitoses. More carbohydrate than protein was detected in the preparations. Cysteine proteinase (CP), N-acetyl-beta-D: -glucosaminidase and alkaline phosphatase activities were detected. The latter enzyme activity is a marker of the tegument, suggesting that in spite of careful conditions used to avoid the presence of tegumental material, manipulation of the worms always resulted in the release of tegumental molecules. Cationic exchange chromatography was useful to separate various components of this "vomit" preparation, particularly enzymes responsible for CP activity. Two highly immunogenic and specific duplets were observed in the WB analysis, 31/32- and 38/40-kDa components, the former probably referring to the intestinal CPs Sm31/Sm32. None of the two duplets disappeared after successful chemotherapy during the time of evaluation in mice or humans.

The human parasitic trematode Schistosoma mansoni has a complex life cycle that includes the freshwater snail Biomphalaria glabrata as intermediate host. Within each stage, the parasite synthesizes a wide array of glycoconjugates, exhibiting, in part, unique carbohydrate structures. In addition, the parasite expresses definitive host-like sugar epitopes, such as Lewis X determinants, supporting the concept of carbohydrate-mediated molecular mimicry as an invasion and survival strategy. In the present study, we investigated whether common carbohydrate determinants occur also at the level of the intermediate host. To this end, a structural characterization of hemolymph glycoprotein-N-glycans of B. glabrata was performed. N-glycans were released from tryptic glycopeptides and labeled with 2-aminopyridine. Sugar chains serologically cross-reacting with S. mansoni glycoconjugates were isolated by immunoaffinity chromatography using a polyclonal antiserum directed against schistosomal egg antigens and fractionated by Aleuria aurantia lectin affinity chromatography and high-performance liquid chromatography. Obtained glycans were analyzed by different mass spectrometric techniques as well as by monosaccharide constituent and linkage analysis. The results revealed a highly heterogeneous oligosaccharide pattern. Cross-reacting species represented about 5% of the total glycans and exhibited a terminal Fuc(alpha1-3)GalNAc unit, a (1-2)-linked xylosyl residue, or both types of structural motifs. In conclusion, our study demonstrates the presence of common carbohydrate epitopes also at the level of S. mansoni and its intermediate host.

Schistosoma mansoni lung-stage larvae appear to not bind antibodies from radiation vaccine or infection sera in the membrane immunofluorescence test. However, treatment of ex vivo lung-stage schistosomula with methyl-beta-cyclodextrin, a hydrophobic oligosaccharide that specifically extracts cholesterol from plasma membranes, induced readily detectable binding of specific antibodies in a concentration- and time-dependent manner. Surface membrane antigen binding of specific antibodies was also conclusively demonstrated by quantitative absorption of anti-schistosome sera with intact ex vivo larvae. These data together suggest that confinement of lung-stage schistosomula surface membrane antigens in cholesterol-rich sites allows only monovalent antibody binding, which can be detected by absorption and not by direct serology.

The glycobiology of Schistosoma mansoni is dominated by developmentally regulated expression of various fucosylated structures, most notably the Lewis X epitope and a multifucosylated sequence, Fuc alpha1-->2Fuc alpha1-->, in its various forms. For the infective cercarial stage, Lewis X has been structurally identified on glycosphingolipids and N-glycans of total glycoprotein extracts, and a population of multifucosylated glycoproteins were found to carry a unique terminal sequence, +/-Fuc alpha1-->2Fuc alpha1-->[3GalNAc beta1-->4(Fuc alpha1-->2Fuc alpha1--> 2Fuc alpha1-->3) GlcNAc beta1-->3Gal alpha1-->](n), on their O-glycans. Using a mass spectrometry approach coupled with chromatographic separation, sequential exoglycosidase digestion, periodate oxidation, and other chemical derivatization, we demonstrate that Lewis X could also be carried on the cercarial O-glycans, but the two distinctive sets of fucosylated epitopes were conjugated to two different core structures. Lewis X, lacNAc, or single GlcNAc was found to attach directly to the -->3Gal beta1-->3GalNAc core and indirectly via another beta-Gal residue branching off from C6 of the reducing end GalNAc to give a biantennary-like structure. The -->3(+/-Gal beta1-->6)Gal beta1-->3(-->3Gal beta1-->6)GalNAc core thus characterized represents a novel core type for O-glycans. In contrast, the previously characterized multifucosylated terminal sequences were carried on conventional type 1 and 2 cores. The smallest structures of the reductively released O-glycans were defined as GalNAc beta1-->4GlcNAc beta1-->3Gal beta1-->3GalNAcitol with a total of two to four fucoses attached to the terminal lacdiNAc. alpha-Galactosylation of the nonreducing terminal beta-GalNAc instead of fucose capping leads to further elongation with another lacdiNAc unit that could also extend directly from C6 of the reducing end GalNAc and similarly elongated or terminated.

Sm13, a 13-kDa Schistosoma mansoni tegumental antigen, is one of the principal polypeptides recognized by antibodies from mice protectively vaccinated with adult-worm tegumental membranes. To obtain the complete gene encoding Sm13 we subcloned and sequenced a cDNA and a fragment of a genomic clone. The collated sequence contains 1,088 nucleotides and represents the full-length open reading frame of the gene, encoding a protein of 104 amino acids with a calculated molecular mass of 11,923 Da, compatible with the native protein identified in the tegumental membranes. The sequence derived from genomic DNA contains a 45-nucleotide intron. The analysis of the predicted protein suggests the presence of both N- and C-terminal hydrophobic membrane-spanning segments, and the coding region contains no homology in the currently available data bases. Additionally, the coding region is preceded by putative CCAAT and TATA boxes that may be involved in the control of expression. Western-blot analysis and indirect immunofluorescence resulted in the identification of a 13-kDa protein (Sm13) in the tegument of adult worms. The present study reveals that Sm13 behaves as an integral membrane protein upon partitioning in Triton X-1 14 and that it is present in worms of 3 weeks or older but not in schistosomula or miracidia. Moreover, it is also specifically recognized by sera from some schistosomiasis patients in enzyme-linked immunosorbent assay and Western-blot analysis, suggesting that it is immunogenic in human schistosomiasis.

Sm15 and Sm25 are two of the principal tegumental antigens recognized by antibodies from mice protectively vaccinated with adult worm tegumental membranes and may therefore be potential vaccine candidate antigens. Using antibodies affinity purified from anti-tegumental membrane anti-sera, and antibodies raised against the recombinant antigens, Sm15 and Sm25 were shown to be located specifically in the tegument of adult worms being distributed throughout the syncitium but not associated with the outer membrane.

Sodium dodecyl sulphate- poly acrylamide gel electronphoresis (SDS-PAGE) fractionation of tegumental surface antigens (STEG-Ags.) of 7-day cultured normal and irradiated schistosomula showed no obvious qualitative differences. The observed polypeptide bands of both normal irradiated STEG-Ags. were almost identical and have similar corresponding molecular weights. The immunoblotting assay, using different types of mouse sera, revealed similarity between the bands of both normal and irradiated STEG-Ags. recognized by each type of mouse serum. No qualitative rather than quantitative differences have been observed. The quantitative differences were reflected in intensively staining of some bands from normal STEG-Ag. rather than their corresponding bands of the same molecular weights from irradiated STEG-Ag.

The neutral glycolipid fraction from mouse-propagated, Schistosoma mansoni adult worms has been investigated as to its chromatographic and antigenic properties, and whether it fulfills the serodiagnostic antigen requirements of sensitivity and specificity in the detection of schistosomiasis. Serological analyses were performed by thin-layer chromatography immunostaining and ELISA. In the acute-phase form of mouse schistosomiasis, the kinetics of development of neutral glycolipid-specific antibody levels was correlated with the intensity of the initial infection and the response was dominated by IgG, as represented by the subclass IgG1. With the experimental animal helminthiases screened, glycolipid antigenicity fulfilled the fundamental traits for a serodiagnostic reagent. In the chronic-phase form of human schistosomiasis mansoni, neutral glycolipid-specific antibody levels were not correlated with the intensity of infection, as estimated from the faecal content of parasite eggs, whilst the isotypic response was dominated by IgM and IgG, the latter represented primarily by IgG1 and secondarily by IgG3. With other human helminthiases, glycolipid antigenicity was incomplete, in that, the specificity was only partially fulfilled. The reason for this incomplete specificity has been clarified, in part, by the detection of cryptic schistosomiasis infections in the cohorts of African patient sera examined.

Utilizing a Biomphalaria alexandrina-derived lectin (BaSII) of proven specificity to a Schistosoma mansoni-associated fucosyllactose [(Fuc alpha 1-2) Gal beta 1-4 Glc] determinant, two determinant-bearing glycoproteins of 40 and 37 kDa were found to be synthesized by the cercarial stage of the parasite. The two glycoproteins were isolated by BaSII affinity column chromatography from extracts of cercariae metabolically radiolabelled with 35S-methionine. Treatments with endoglycosidases, alkaline borohydride, as well as concanavalin A column chromatography and analysis by two-dimensional gels indicated that the two glycoproteins are synthesized as a single 33 kDa polypeptide backbone that is differentially glycosylated with one and/or two determinant-bearing N-linked complex-type glycan units of either the biantennary, or, to a lesser extent, the tri- or tetra-antennary types. The two glycoproteins lack other conventional high mannose-type or O-linked glycans, and the distinct structures of the complex-type oligosaccharides accounted solely for the expression of three isomorphs for each determinant-bearing glycoprotein. Based on the structural relatedness of the fucosyllactose determinant to the antigenic mammalian blood group H trisaccharide, our observations may have implications in mechanisms of host-parasite interactions as well as immunoprophylaxis.

Molecular cloning of components of protective antigenic preparations has suggested that related parasite fatty acid-binding proteins could form the basis of the protective immune crossreactivity between the parasitic trematode worms Fasciola hepatica and Schistosoma mansoni. Molecular models of the two parasite proteins showed that both molecules adopt the same basic three-dimensional structure, consisting of a barrel-shaped molecule formed by 10 antiparallel beta-pleated strands joined by short loops, and revealed the likely presence of crossreactive, discontinuous epitopes principally derived from amino acids in the C-terminal portions of the molecules. A recombinant form of the S. mansoni antigen, rSm14, protected outbred Swiss mice by up to 67% against challenge with S. mansoni cercariae in the absence of adjuvant and without provoking any observable autoimmune response. The same antigen also provided complete protection against challenge with F. hepatica metacercariae in the same animal model. The results suggest that it may be possible to produce a single vaccine that would be effective against at least two parasites, F. hepatica and S. mansoni, of veterinary and human importance, respectively.

A cDNA clone of Echinococcus granulosus has been isolated from an expression library screened with sera from cystic echinococcosis patients. The deduced amino acid sequence shows 56% homology to the heavy chain of human ferritin. E. granulosus ferritin contains 173 amino acid residues and has a calculated molecular weight of 19830 Da and a statistical isoelectric point of 7.6. Functionally important amino acid residues of the ferroxidase centre are conserved in comparison with other ferritins. In vitro-translated E. granulosus ferritin was tested for its diagnostic potential by immunoprecipitation. The antigenic reactivity exhibited a good potential for the further development of E. granulosus ferritin as an immunodiagnostic tool for human hydatidosis.

The gut-associated excretory antigen circulating cathodic antigen (CCA) was isolated by immunoaffinity chromatography from adult Schistosoma mansoni worms, which were collected from infected golden hamsters. This antigen is probably involved in protection of the schistosome gut and is increasingly used in highly sensitive and specific immunodiagnostic assays. Amino acid analysis before and after alkaline borohydride treatment of CCA and monosaccharide analysis indicated that CCA is O-glycosylated mostly via GalNAc-Thr. After reductive alkaline treatment, the O-linked carbohydrate chains were fractionated by gel-permeation chromatography, followed by normal-phase HPLC on LiChrosorb-NH2. Carbohydrate-positive fractions were investigated by one-dimensional and two-dimensional 1H-NMR spectroscopy, fast atom bombardment mass spectrometry and collision-induced-dissociation tandem mass spectrometry. The analyses showed that the low-molecular-mass O-linked oligosaccharide alditols (the minor fraction) consist of disaccharides to hexasaccharides having the Gal beta (1-3)GalNAc-OL core in common. The major carbohydrate fraction comprises a population of polysaccharides, containing Lewis x repeating units (-3)Gal beta (1-4)[Fuc alpha (1-3)]GlcNAc beta (1-). CCA-specific monoclonal antibodies and IgM antibodies in patient sera recognized the fucosylated O-linked carbohydrate antigenic structures. Since CCA evokes a strong IgM antibody response and carbohydrate structures containing repeating Lewis x units are found on circulating neutrophils, it is proposed that the antigenic poly-Lewis x polysaccharide of CCA is involved in the induction of auto-immunity against granulocytes, resulting in the mild to moderate neutropenia observed during schistosome infection.

Stage-specific expression of the mRNA encoding the cercarial-specific 8 kDa CaBP has been described previously. To gain information on possible function(s) of this protein we raised antibodies to the CaBP in rabbits immunized with a CaBP-TrpE fusion protein synthesized in bacteria. Western blots showed high levels of CaBP in cercariae and 3 h schistosomula, trace amounts in 24 h schistosomula, and none in miracidia sporocyst and adult worm, as found for the mRNA. The CaBP molecule has a short half-life (< or = 4 h) similar to that of the mRNA. Other experiments demonstrate that the CaBP may interact with a putative target molecule in a calcium-dependent manner to form a complex of 45 kDa. Immunogold electron microscopy showed CaBP in selected regions of cercariae and 3 h schistosomula: tegument, head gland, subtegumental cells, flame cells, intestinal wall and the body-tail junction. Other investigators have shown that the head gland and subtegumental cells synthesize and translocate granules to the tegument during transformation from cercariae (living freely in water) to schistosomula (residing in vertebrate host). These observations and the time-course of CaBP detection suggest that the CaBP synthesized in the head gland and subtegumental cells is translocated to the tegument where it plays a role in tegument modifications required for adaptation to parasite life in the host. CaBP was not found in muscles and mitochondria, suggesting that it is not involved in the rapid motility and aerobic metabolism characteristic of cercariae.

Sm23, a surface protein of the human parasite Schistosoma mansoni, belongs to the family of "cysteine-rich, hydrophobic proteins," which are expressed on mammalian hematopoietic cells or tumor cells. Sm23 shares the highly conserved hydrophobicity profile of these proteins, which predicts four transmembrane segments, but is in addition linked to the membrane by a glycosylphosphatidylinositol (GPI) anchor. Our results suggest that Sm23 uses both the potential transmembrane domains and the GPI anchor for membrane insertion: (a) Sm23 was not released from the surface after cleavage with phosphatidylinositol-specific phospholipase C (PIPLC). (b) In a Triton X-114 phase-separation system, native [3H]ethanolamine- or [35S]methionine-labeled Sm23 partitioned into the detergent phase. Upon removal of the GPI anchor by PIPLC, the majority of the molecules stayed in the detergent-phase as expected of a transmembrane protein. (c) When full-length recombinant Sm23 was transcribed and translated in vitro, the polypeptide chain was inserted into microsomal membranes: Sm23 stayed associated with the membranes when they were incubated with carbonate buffer at pH 11.5, and membrane bound Sm23 was protected from digestion with proteinase K. (d) Recombinant Sm23, when expressed in the baculovirus expression system, was transported to the surface of infected insect cells, and similarly to the native protein it was not released from these cells after cleavage with PIPLC.

The technique of high performance electrophoretic chromatography (HPEC) has been used to fractionate soluble antigens from adult Schistosoma mansoni worms on the basis of molecular weight (MW), prior to screening for their ability to stimulate T lymphocyte activity. Approximately 250 micrograms of protein were separated by continuous electrophoresis through an SDS polyacrylamide gel into 30-50 aqueous samples of minimal volume (80 microliters). Each consecutive sample contained a limited number of proteins of progressively greater MW, although the resolution of the fractionation was affected by a number of factors including acrylamide concentration, gel length, gel diameter and electrophoretic current. Following the extraction of SDS using Calbiosorb resin, the aqueous fractions were used directly to stimulate cultures of lymphocytes taken from the lymph nodes of infected or vaccinated mice. The most promising fractions were those containing proteins which induced the release of high levels of interferon-gamma relative to the extent of proliferation. This suggests that these proteins are good inducers of Th1 lymphocyte activity.

The complete sequence of the cDNA encoding a 20 kDa calcium-binding protein of Schistosoma mansoni (Sm20) has been determined. The predicted amino acid sequence contains 4 EF hand domains but examination of the predicted secondary structure of Sm20, together with the specific residues in each calcium-binding domain, suggests that only 1 EF hand (domain IV) is functional. Sm20 is most homologous to calmodulin, troponin C and the regulatory light-chain of myosin, particularly those of invertebrates. However, troponin C and the regulatory light-chain of myosin can be distinguished from Sm20 by size and by their differential levels of expression during the life-cycle. Sm20 also appears to be distinct from calmodulin but may be functionally equivalent to the soluble sarcoplasmic calcium-binding proteins of molluscs and crustacea which may act as a reservoir for calcium in muscle. Sm20 is encoded by a small multi-gene family whose members are clustered within a 15 kb region of the genome. A 20 kDa antigen, cross-reactive with Sm20, is expressed in Schistosoma haematobium, Fasciola hepatica and Paragonomus mexicanus.

The passive transfer of monoclonal antibodies, direct vaccination and in vitro assays have all shown that antigens associated with the tegumental membranes of Schistosoma mansoni are capable of mediating protective immune responses against the parasite in animal models. Furthermore, the principal antigens are highly antigenic during natural infection in man and stimulate strong humoral and cellular responses although, at present, their role in mediating protective immune responses in man remains equivocal. This presentation will review the current state of knowledge of the structure and expression of the major antigenic tegumental proteins of the schistosome and will attempt to relate the relevance of their structural features to possible function both in terms of protective immunity and the parasite's ability to survive within the definitive host. A focus will be the recent advances that have been made in the identification of means of anchoring of the antigenic proteins to the tegumental membrane. In addition, the implications of the structural complexity of the tegumental proteins in terms of their possible utility in vaccination and diagnosis will be considered.

T cell-deprived mice acutely infected with S. mansoni suffer microvesicular hepatocyte damage which is not seen in infected, immunological intact animals. A cationic fraction (CEF6) of the PBS-soluble portion of S. mansoni eggs (SEA) induces antibodies which, on passive transfer, prevent hepatocyte damage. CEF6 contains 2 antigens, omega 1 and alpha 1, and has also been shown to be a useful serodiagnostic reagent. This paper describes the purification and characterization of the 2 antigens present in CEF6. omega 1 is a monomeric glycoprotein with a pI greater than 9.0 and a molecular weight of 31 kDa. Alpha 1 consists of two immunologically cross-reactive dimers, 41 and 36 kDa in non-reducing conditions, each of which consists of one unique and one common glycoprotein subcomponent. In ELISA with mouse and human infection sera omega 1 is shown to be S. mansoni specific and is better able to distinguish S. mansoni infections from other schistosome infections than are unfractionated SEA, CEF6 or alpha 1. Passive transfer of monospecific anti-omega 1 sera into S. mansoni infected, T cell-deprived mice completely prevented the occurrence of microvesicular hepatocyte damage in these animals. Monospecific anti-alpha 1 serum had no hepatoprotective capacity.