Schistosoma mansoni: ultrastructural observations on the small intestine of murine host

Between 6 and 29 weeks, the small intestines of mice infected with Schistosoma mansoni were studied with the electron microscope. Granulomas were confined to the serosal-muscularis regions of the small intestine. Early granulomas were characterized by having several cell types with the most conspicuous type being the eosinophil. Older granulomas were more fibrotic. These were compared with hepatic granulomas of comparable age. In the vicinity of active eggs either in granulomas or in the lamina propria, mitochrondria from epithelial cells displayed intracristal granules. Intraperitoneal injections of soluble egg antigen isolated from viable S. mansoni eggs produced identical mitochondrial abberations. Cytochrome c-cytochrome oxidase activity was visualized in the mitochondria by the diaminobenzidine method.     

Schistosome Syntenin Partially Protects Vaccinated Mice against Schistosoma mansoni Infection

Background

Schistosomiasis is a neglected tropical disease caused by several species of trematode of the genus Schistosoma. The disease affects more than 200 million people in the world and causes up to 280,000 deaths per year, besides having high morbidity due to chronic illness that damages internal organs. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-term strategy to control disease is a combination of drug treatment and immunization with an anti-schistosome vaccine. Among the most promising molecules as vaccine candidates are the proteins present in the tegument and digestive tract of the parasite.

Methodology/Principal Findings

In this study, we describe for the first time Schistosoma mansoni syntenin (SmSynt) and we evaluate its potential as a recombinant vaccine. We demonstrate by real-time PCR that syntenin is mainly expressed in intravascular life stages (schistosomula and adult worms) of the parasite life cycle and, by confocal microscopy, we localize it in digestive epithelia in adult worms and schistosomula. Administration of siRNAs targeting SmSynt leads to the knock-down of syntenin gene and protein levels, but this has no demonstrable impact on parasite morphology or viability, suggesting that high SmSynt gene expression is not essential for the parasites in vitro. Mice immunization with rSmSynt, formulated with Freund''s adjuvant, induces a Th1-type response, as suggested by the production of IFN-γ and TNF-α by rSmSynt-stimulated cultured splenocytes. The protective effect conferred by vaccination with rSmSynt was demonstrated by 30–37% reduction of worm burden, 38–43% reduction in the number, and 35–37% reduction in the area, of liver granulomas.

Conclusions/Significance

Our report is the first characterization of syntenin in Schistosoma mansoni and our data suggest that this protein is a potential candidate for the development of a multi-antigen vaccine to control schistosomiasis.     

Solution Structure,Membrane Interactions,and Protein Binding Partners of the Tetraspanin Sm-TSP-2, a Vaccine Antigen from the Human Blood Fluke Schistosoma mansoni

The tetraspanins (TSPs) are a family of integral membrane proteins that are ubiquitously expressed at the surface of eukaryotic cells. TSPs mediate a range of processes at the surface of the plasma membrane by providing a scaffold for the assembly of protein complexes known as tetraspanin-enriched microdomains (TEMs). We report here the structure of the surface-exposed EC2 domain from Sm-TSP-2, a TSP from Schistosoma mansoni and one of the better prospects for the development of a vaccine against schistosomiasis. This is the first solution structure of this domain, and our investigations of its interactions with lipid micelles provide a general model for interactions between TSPs, membranes, and other proteins. Using chemical cross-linking, eight potential protein constituents of Sm-TSP-2-mediated TEMs were also identified. These include proteins important for membrane maintenance and repair, providing further evidence for the functional role of Sm-TSP-2- and Sm-TSP-2-mediated TEMs. The identification of calpain, Sm29, and fructose-bisphosphate aldolase, themselves potential vaccine antigens, suggests that the Sm-TSP-2-mediated TEMs could be disrupted via multiple targets. The identification of further Sm-TSP-2-mediated TEM proteins increases the available candidates for multiplex vaccines and/or novel drugs targeting TEMs in the schistosome tegument.     

Schistosoma mansoni: immediate hypersensitivity to adult antigens in the rat

Antigen fractions from adult S. mansoni, obtained from infected mice, were isolated by a variety of methods. A readily soluble fraction was obtained in good yield by freezing and thawing the schistosomes, while the less soluble residue was fractionated by the use of a number of the methods currently used for the extraction of tissue and cell surface antigens. The dialyzed, centrifuged products were characterized by acrylamide gel disc electrophoresis methods, agar gel precipitin reactions with antisera from rabbits immunized with whole schistosome homogenate, and by Prausnitz-Kustner (P-K) assay with sera from schistosome infected rats. The pattern of P-K reactivity suggested that there were a number of different antigen specificities involved in the reaginic antibody response to schistosome infection in rats. With repeated infection and increased duration of infection, more different antigens seemed to be involved in the reagin response. The schistosome antigen fraction obtained by freezing and thawing was especially reactive with both early infection rat sera and sera from multiply infected rats. Both the soluble fraction isolated by freezing and thawing and residue solubilized materials were found to be able to induce the formation of reagin antibodies on immunization with alum and B. pertussis vaccine.     

Eosinophils and immune mechanisms. IV. Culture conditions, antigen requirements, production kinetics and immunologic specificity of the lymphokine eosinophil stimulation promoter.

The culture conditions which contribute to the production of the lymphokine, eosinophil stimulation promoter (ESP), have been investigated. Spleen or lymph node cells from mice infected for 8–10 weeks with the helminth Schistosoma mansoni respond to challenge with a soluble egg antigenic preparation (SEA) from S. mansoni eggs by the elaboration of ESP into the culture medium. Exposure to as little as 0.1 μg of SEA elicits this response. Furthermore, 30 min of exposure to antigen is sufficient to stimulate subsequent ESP production. Production begins within 4 hr, is rapid for 12 hr, and continues for at least another 8 hr. The use of this information allows the standardization of ESP production to be such that the concentration of SEA is insufficient to interfere with the indirect assay of ESP upon eosinophil-rich peritoneal exudates from S. mansoni-infected mice. The specificity of the direct assay elicited by SEA was confirmed, and the ability of the lymphokine to stimulate eosinophil migration from eosinophil-rich peritoneal exudates from either S. mansoni-or Trichinella spiralis-infected mice was demonstrated.     

Vaccination Against Hydatidosis: Molecular Cloning and Optimal Expression of the EG95NC<Superscript>−</Superscript> Recombinant Antigen in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Cystic echinococcosis (CE) is a widely distributed zoonosis that is highly endemic in the Mediterranean basin. The disease represents a serious public health threat and causes economic losses. The parasite life-cycle involves dogs and ruminants as definitive and intermediate hosts; humans are accidently infected, causing serious clinical issues. Vaccination of ruminants and dog treatments represent the most efficient measures to prevent parasite transmission. The recombinant protein vaccine, EG95, has been used successfully in sheep vaccine trials against CE in several countries. In this study, we expressed the modified antigen, EG95NC-GST, in Escherichia coli for use as a vaccine against Echinococcus granulosus in ruminants. We tested three different media formulations for E. coli culture and established for each culture conditions for optimal levels of soluble EG95 expression. The results demonstrate that SOC and TB media provided high yields in cell density and EG95 protein expression. Purification of the recombinant protein with affinity chromatography (using FPLC) was also performed to increase the purity of the EG95NC^?-GST antigen.     

Sm10.3, a Member of the Micro-Exon Gene 4 (MEG-4) Family,Induces Erythrocyte Agglutination In Vitro and Partially Protects Vaccinated Mice against Schistosoma mansoni Infection

Background

The parasitic flatworm Schistosoma mansoni is a blood fluke that causes schistosomiasis. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-term strategy to control disease is a combination of drug treatment and immunization with an anti-schistosome vaccine. Numerous antigens that are expressed at the interface between the parasite and the mammalian host have been assessed. Among the most promising molecules are the proteins present in the tegument and digestive tract of the parasite.

Methodology/Principal Findings

In this study, we evaluated the potential of Sm10.3, a member of the micro-exon gene 4 (MEG-4) family, for use as part of a recombinant vaccine. We confirmed by real-time PCR that Sm10.3 was expressed at all stages of the parasite life cycle. The localization of Sm10.3 on the surface and lumen of the esophageal and intestinal tract in adult worms and lung-stage schistosomula was confirmed by confocal microscopy. We also show preliminary evidence that rSm10.3 induces erythrocyte agglutination in vitro. Immunization of mice with rSm10.3 induced a mixed Th1/Th2-type response, as IFN-γ, TNF-α, and low levels of IL-5 were detected in the supernatant of cultured splenocytes. The protective effect conferred by vaccination with rSm10.3 was demonstrated by 25.5–32% reduction in the worm burden, 32.9–43.6% reduction in the number of eggs per gram of hepatic tissue, a 23.8% reduction in the number of granulomas, an 11.8% reduction in the area of the granulomas and a 39.8% reduction in granuloma fibrosis.

Conclusions/Significance

Our data suggest that Sm10.3 is a potential candidate for use in developing a multi-antigen vaccine to control schistosomiasis and provide the first evidence for a possible role for Sm10.3 in the blood feeding process.     

FK506 Binding Protein from the Hyperthermophilic Archaeon Pyrococcus horikoshii Suppresses the Aggregation of Proteins in Escherichia coli

The 29-kDa FK506 binding protein (FKBP) gene is the only peptidyl-prolyl cis-trans isomerase (PPIase) gene in the genome of Pyrococcus horikoshii. We characterized the function of this FKBP (PhFKBP29) and used it to increase the production yield of soluble recombinant protein in Escherichia coli. The PPIase activity (k_cat/K_m) of PhFKBP29 was found to be much lower than that of other archaeal 16- to 18-kDa FKBPs by a chymotrypsin-coupled assay of the oligo-peptidyl substrate at 15°C. Besides this low PPIase activity, PhFKBP29 showed chaperone-like protein folding activity which enhanced the refolding yield of chemically unfolded rhodanese in vitro. In addition, it suppressed thermal protein aggregation in a temperature range of 45 to 100°C. When the PhFKBP29 gene was coexpressed with the recombinant Fab fragment gene of the anti-hen egg lysozyme antibody in the cytoplasm of E. coli, whose expressed product tended to form an inactive aggregate in E. coli, it improved the yield of the soluble Fab fragments with antibody specificity. PhFKBP29 exerted protein folding and aggregation suppression in E. coli cells.     

Increased immunogenicity and protection of recombinant Sm14 antigens by heat-killed Cutibacterium acnes in BALB/c mice infected with Schistosoma mansoni

After many years of the excessive use of praziquantel against Schistosoma mansoni (S. mansoni), it has already led to the development of drug resistance. While schistosomiasis is still affecting millions of people every year, vaccination may be one realistic alternative way to control the disease. Currently, S. mansoni 14-kDa fatty acid-binding protein (Sm14) has shown promising results as a vaccine antigen. Yet, the use of an adjuvant may be necessary to further increase the effectiveness of the vaccine. Herein, we investigated the potential of using heat-killed Cutibacterium acnes (C. acnes) as an adjuvant for recombinant Sm14 (rSm14). Immunization of mice with C. acnes-adjuvanted rSm14 showed increased humoral immune responses, compared with mice immunized with rSm14 alone. Additionally, C. acnes-adjuvanted rSm14 vaccination provided higher protection to mice against S. mansoni infection and liver injuries. These results suggest that C. acnes increases the immunogenicity of rSm14, which leads to better protection against S. mansoni infection. Therefore, heat-killed C. acnes may be a promising adjuvant to use with rSm14.     

Sm29, but Not Sm22.6 Retains its Ability to Induce a Protective Immune Response in Mice Previously Exposed to a Schistosoma mansoni Infection

Background

A vaccine against schistosomiasis would have a great impact in disease elimination. Sm29 and Sm22.6 are two parasite tegument proteins which represent promising antigens to compose a vaccine. These antigens have been associated with resistance to infection and reinfection in individuals living in endemic area for the disease and induced partial protection when evaluated in immunization trials using naïve mice.

Methodology/principals findings

In this study we evaluated rSm29 and rSm22.6 ability to induce protection in Balb/c mice that had been previously infected with S. mansoni and further treated with Praziquantel. Our results demonstrate that three doses of the vaccine containing rSm29 were necessary to elicit significant protection (26%–48%). Immunization of mice with rSm29 induced a significant production of IL-2, IFN-γ, IL-17, IL-4; significant production of specific antibodies; increased percentage of CD4+ central memory cells in comparison with infected and treated saline group and increased percentage of CD4+ effector memory cells in comparison with naïve Balb/c mice immunized with rSm29. On the other hand, although immunization with Sm22.6 induced a robust immune response, it failed to induce protection.

Conclusion/significance

Our results demonstrate that rSm29 retains its ability to induce protection in previously infected animals, reinforcing its potential as a vaccine candidate.     

An efficient in vitro refolding of recombinant bacterial laccase in Escherichia coli

Laccases (benzenediol oxygen oxidoreductases, EC 1.10.3.2) are important multicopper enzymes that are used in many biotechnological processes. A recombinant form of laccase from Bacillus sp. HR03 was overexpressed in Escherichia coli BL-21(DE3). Inclusion body (IB) formation happens quite often during recombinant protein production. Hence, developing a protocol for efficient refolding of proteins from inclusion bodies to provide large amounts of active protein could be advantageous for structural and functional studies. Here, we have tried to find an efficient method of refolding for this bacterial enzyme. Solubilization of inclusion bodies was carried out in phosphate buffer pH 7, containing 8 M urea and 4 mM β-mercaptoethanol and refolding was performed using the dilution method. The effect of different additives was investigated on the refolding procedure of denaturated laccase. Mix buffer (phosphate buffer and citrate buffer, 100 mM) containing 4 mM ZnSO₄ and 100 mM sorbitol was selected as an optimized refolding buffer. Also Kinetic parameters of soluble and refolded laccase were analyzed.     

Immunomodulatory effects of IL-12 released from poly-N-acetyl glucosamine gel matrix during schistosomiasis infection

We have reported recently that Interleukin-12 (IL-12) released from poly-N-acetyl glucosamine gel matrix (F2 gel/IL-12) is more effective than free IL-12 to enhance vaccination of mice with Schistosoma soluble worm antigen preparation. The aim of this study is to evaluate the effect of F2 gel/IL-12 on the inflammatory responses in mice undergoing schistosomiasis infection in absence of vaccination. To achieve this, mice undergoing Schistosoma mansoni infection or cured from this infection, after treatment with praziquantil (PZQ), were treated with subcutaneous injection of IL-12 for 3 consecutive days or once with F2 gel loaded with IL-12 (F2 gel/IL-12). The treatment was started on day 35 days after infection. For infection, mice were infected with 100 cercariae of S. mansoni using tail immersion method. We found that treatment with F2 gel/IL-12 induced significant decreases in the egg burden with a moderate reduction in the size of granuloma and decrease in the cellular granulomatous reaction in the lung as compared to infected mice treated with IL-12. These effects of F2 gel/IL-12 were more pronounced in infected mice previously treated with the anti-schistosomal drug PZQ. The total numbers of white blood cells in all treated mice showed similar profile. Treatment with IL-12 or F2 gel/IL-12, however, showed significant reduction in the number of mononuclear cells when compared with non-treated infected mice. In conclusion, this study showed the ability of IL-12 released from F2 gel to lower the inflammatory response to Schistosoma infection even in absence of vaccination.     

New Insights for Native Production of MSP119, the Disulfide-Rich C-Terminal Fragment from Plasmodium falciparum Merozoite Surface Protein 1

Malaria represents a major public health problem and an important cause of mortality and morbidity. The malaria parasites are becoming resistant to drugs used to treat the disease and still no efficient vaccine has been developed. One promising vaccine candidate is the merozoite surface protein 1 (MSP1), which has been extensively investigated as a vaccine target. The surface protein MSP1 plays an essential role in the erythrocyte invasion process and is an accessible target for the immune system. Antibodies to the carboxy-terminal region of the protein, named MSP1₁₉, can inhibit erythrocyte invasion and parasite growth. In order to develop an effective MSP1₁₉- based vaccine against malaria, production of an antigen that is recognized by protective antibodies is mandatory. To this aim, we propose a method to produce the disulfide-rich MSP1₁₉ in its native conformation based on its in vitro oxidative refolding. The native conformation of the renatured MSP1₁₉ is carefully established by immunochemical reactivity experiments, circular dichroism and NMR. MSP1₁₉ can successfully be refolded in vitro as an isolated protein or as a fusion with the maltose binding protein. The possibility to properly fold MSP1₁₉ in vitro paves the way to new approaches for high titer production of native MSP1₁₉ using Escherichia coli as a host.     

Modification in media composition to obtain secretory production of STxB-based vaccines using Escherichia coli

Shiga toxin B-subunit (STxB) from Shigella dysenteriae targets in vivo antigen to cancer cells, dendritic cells (DC) and B cells, which preferentially express the globotriaosylceramide (Gb3) receptor. This pivotal role has encouraged scientists to investigate fusing STxB with other clinical antigens. Due to the challenges of obtaining a functional soluble form of the recombinant STxB, such as formation of inclusion bodies during protein expression, scientists tend to combine STxB with vaccine candidates rather than using their genetically fused forms. In this work, we fused HPV16 E7 as a vaccine candidate to the recombinantly-produced STxB. To minimize the formation of inclusion bodies, we investigated a number of conditions during the expression procedure. Then various strategies were used in order to obtain high yield of soluble recombinant protein from E. coli which included the use of different host strains, reduction of cultivation temperature, as well as using different concentrations of IPTG and different additives (Glycin, Triton X-100, ZnCl₂). Our study demonstrated the importance of optimizing incubation parameters for recombinant protein expression in E. coli; also showed that the secretion production can be achieved over the course of a few hours when using additives such as glycine and Triton X-100. Interestingly, it was shown that when the culture mediums were supplemented by additives, there was an inverse ratio between time of induction (TOI) and the level of secreted protein at lower temperatures. This study determines the optimal conditions for high yield soluble E7-STxB expression and subsequently facilitates reaching a functionally soluble form of STxB-based vaccines, which can be considered as a potent vaccine candidate for cervical cancer.     

Serological Screening of the Schistosoma mansoni Adult Worm Proteome

Background

New interventions tools are a priority for schistosomiasis control and elimination, as the disease is still highly prevalent. The identification of proteins associated with active infection and protective immune response may constitute the basis for the development of a successful vaccine and could also indicate new diagnostic candidates. In this context, post-genomic technologies have been progressing, resulting in a more rational discovery of new biomarkers of resistance and antigens for diagnosis.

Methodology/Principal Findings

Two-dimensional electrophoresed Schistosoma mansoni adult worm protein extracts were probed with pooled sera of infected and non-infected (naturally resistant) individuals from a S. mansoni endemic area. A total of 47 different immunoreactive proteins were identified by mass spectrometry. Although the different pooled sera shared most of the immunoreactive protein spots, nine protein spots reacted exclusively with the serum pool of infected individuals, which correspond to annexin, major egg antigen, troponin T, filamin, disulphide-isomerase ER-60 precursor, actin and reticulocalbin. One protein spot, corresponding to eukaryotic translation elongation factor, reacted exclusively with the pooled sera of non-infected individuals living in the endemic area. Western blotting of two selected recombinant proteins, major egg antigen and hemoglobinase, showed a similar recognition pattern of that of the native protein.

Concluding/Significance

Using a serological proteome analysis, a group of antigens related to the different infection status of the endemic area residents was identified and may be related to susceptibility or resistance to infection.     

Recombinant Production of Human Interleukin 6 in Escherichia coli

In this study, we compared basic expression approaches for the efficient expression of bioactive recombinant human interleukin-6 (IL6), as an example for a difficult-to-express protein. We tested these approaches in a laboratory scale in order to pioneer the commercial production of this protein in Escherichia coli (E. coli). Among the various strategies, which were tested under Research and Development (R&D) conditions, aggregation-prone IL6 was solubilized most effectively by co-expressing cytoplasmic chaperones. Expression of a Glutathion-S-Transferase (GST) fusion protein was not efficient to increase IL6 solubility. Alteration of the cultivation temperature significantly increased the solubility in both cases, whereas reduced concentrations of IPTG to induce expression of the T7lac-promotor only had a positive effect on chaperone-assisted expression. The biological activity was comparable to that of commercial IL6. Targeting the expressed protein to an oxidizing environment was not effective in the generation of soluble IL6. Taken together, the presence of chaperones and a lowered cultivation temperature seem effective to isolate large quantities of soluble IL6. This approach led to in vivo soluble, functional protein fractions and reduces purification and refolding requirements caused by downstream purification procedures. The final yield of soluble recombinant protein averaged approximately 2.6 mg IL6/liter of cell culture. These findings might be beneficial for the development of the large-scale production of IL6 under the conditions of current good manufacturing practice (cGMP).     

Ribeiroia marini: pathogenesis and larval trematode antagonism in the snail, Biomphalaria glabrata

Larval trematode antagonism between Ribeiroia marini and Schistosoma mansoni was studied in the snail Biomphalaria glabrata. A laboratory-raised Puerto Rican strain of B. glabrata was exposed to single and double infections with given numbers of: (1) embryonated eggs of R. marini from laboratory rats, and (2) miracidia of S. mansoni from mice. Snails were maintained in outside environmental tanks in San Juan, Puerto Rico and larval trematode interactions were examined in a series of five experiments. Snails of all sizes were highly susceptible to single infections with R. marini. Rediae and cercariae caused extensive damage to the digestive gland and ovotestis resulting in premature death of snails. Heavily infected snails were castrated and stopped laying eggs. Snails infected first with S. mansoni were only partly susceptible to superinfection with R. marini given on Day 23. In a reverse experiment, snails infected first with R. marini were only partly susceptible to a second infection with S. mansoni given on Day 23. In simultaneous exposures, snails developed double infections (22%) with R. marini dominant and S. mansoni sporocyst and cercaria production reduced. While R. marini is not a strong direct antagonist against established S. mansoni infections, it has several attributes as a possible biological control agent: hardy eggs easily produced in rats; high infectivity to snails of all ages; and ability to castrate and prematurely kill B. glabrata. The R. marini-rat system described here provides a convenient laboratory and field model for the study of intrasnail trematode antagonism and biological control.     

Monoclonal Antibody-Based Dipstick Assay: A Reliable Field Applicable Technique for Diagnosis of Schistosoma mansoni Infection Using Human Serum and Urine Samples

A field applicable diagnostic technique, the dipstick assay, was evaluated for its sensitivity and specificity in diagnosing human Schistosoma mansoni infection. A monoclonal antibody (mAb) against S. mansoni adult worm tegumental antigen (AWTA) was employed in dipstick and sandwich ELISA for detection of circulating schistosome antigen (CSA) in both serum and urine samples. Based on clinical and parasitological examinations, 60 S. mansoni-infected patients, 30 patients infected with parasites other than schistosomiasis, and 30 uninfected healthy individuals were selected. The sensitivity and specificity of dipstick assay in urine samples were 86.7% and 90.0%, respectively, compared to 90.0% sensitivity and 91.7% specificity of sandwich ELISA. In serum samples, the sensitivity and specificity were 88.3% and 91.7% for dipstick assay vs. 91.7% and 95.0% for sandwich ELISA, respectively. The diagnostic efficacy of dipstick assay in urine and serum samples was 88.3% and 90.0%, while it was 90.8% and 93.3% for sandwich ELISA, respectively. The diagnostic indices of dipstick assay and ELISA either in serum or in urine were statistically comparable (P>0.05). In conclusion, the dipstick assay offers an alternative simple, rapid, non-invasive technique in detecting CSA or complement to stool examinations especially in field studies.