A multivalent chimeric vaccine composed of Schistosoma mansoni SmTSP-2 and Sm29 was able to induce protection against infection in mice

Schistosoma mansoni is a blood fluke parasite responsible for schistosomiasis. The best long-term strategy to control schistosomiasis is through immunization combined with drug treatment. In this study, we cloned, expressed and purified SmTSP-2 fused to the N- and C-terminal halves of Sm29 and tested these chimeras as vaccine candidates using an adjuvant approved to be used in humans. The results demonstrated that vaccination with SmTSP-2 fused to N- or C-terminus of Sm29-induced reduction in worm burden and liver pathology when compared to control animals. Additionally, we detected high levels of mouse-specific IgG, IgG1 and IgG2a against both chimeras and significant amounts of IFN-γ and TNF-α and no IL-4. Finally, studies with sera from patients resistant to infection and living in schistosomiasis endemic areas revealed high levels of specific IgG to both chimeras when compared to healthy individuals. In conclusion, SmTSP-2/Sm29 chimeras tested here induced partial protection against infection and might be a potential vaccine candidate

Altered Patterns of Gene Expression Underlying the Enhanced Immunogenicity of Radiation-Attenuated Schistosomes

Schistosoma mansoni is a blood-dwelling parasitic worm that causes schistosomiasis in humans throughout Africa and parts of South America. A vaccine would enhance attempts to control and eradicate the disease that currently relies on treatment with a single drug. Although a manufactured vaccine has yet to generate high levels of protection, this can be achieved with infective parasite larvae that have been disabled by exposure to radiation. How these weakened parasites are able to induce protective immunity when normal parasites do not, is the question addressed by our experiments. We have used a technique of gene expression profiling to compare the patterns in normal and disabled parasites, over the period when they would trigger an immune response in the host. We found that only a handful of genes were differentially expressed, all of them diminished in the disabled parasite. However, a more sensitive technique to examine groups of genes revealed that those involved in nervous system and muscle function were depressed in the disabled parasites. We suggest that reduced mobility of these larvae permits them longer contact with the immune system, thus enabling a strong protective immune response to develop

Inconsistent Protective Efficacy and Marked Polymorphism Limits the Value of Schistosoma japonicum Tetraspanin-2 as a Vaccine Target

Schistosoma mansoni tetraspanin 2 (Sm-TSP-2) is considered a lead target for vaccine development against schistosomiasis mansoni because: (1) It is located in the schistosome tegument and is involved in tegument formation; (2) It is strongly recognized by IgG1 and IgG3 antibodies from individuals putatively resistant to schistosome infection, but not chronically infected people, and (3) It induces high levels of protection against challenge infection in the mouse model. We amplified 211 homologous TSP-2 sequences from male and female S. japonicum worms, which revealed 7 different cDNA subclasses. We expressed in E. coli a region of one of the clusters which exhibited a high frequency of transcription in female worms, and showed the purified recombinant protein (Sj-TSP-2e) was recognised by 43.1% of sera obtained from confirmed schistosomiasis japonica patients. Vaccination of mice with the recombinant protein induced high levels of IgG1 and IgG2 antibodies, but no consistent protective efficacy against challenge infection was elicited in three independent trials. The highly polymorphic nature of the Sj-TSP-2 gene at the transcriptional level may limit the value of Sj-TSP-2 as a target for future S. japonicum vaccine development. Further analysis of the distribution of the different subclasses/alleles of the Sj-TSP-2 gene in S. japonicum populations from different endemic areas would be informative

Suppression of mRNAs Encoding Tegument Tetraspanins from Schistosoma mansoni Results in Impaired Tegument Turnover

Schistosomes express a family of integral membrane proteins, called tetraspanins (TSPs), in the outer surface membranes of the tegument. Two of these tetraspanins, Sm-TSP-1 and Sm-TSP-2, confer protection as vaccines in mice, and individuals who are naturally resistant to S. mansoni infection mount a strong IgG response to Sm-TSP-2. To determine their functions in the tegument of S. mansoni we used RNA interference to silence expression of Sm-tsp-1 and Sm-tsp-2 mRNAs. Soaking of parasites in Sm-tsp dsRNAs resulted in 61% (p = 0.009) and 74% (p = 0.009) reductions in Sm-tsp-1 and Sm-tsp-2 transcription levels, respectively, in adult worms, and 67%–75% (p = 0.011) and 69%–89% (p = 0.004) reductions in Sm-tsp-1 and Sm-tsp-2 transcription levels, respectively, in schistosomula compared to worms treated with irrelevant control (luciferase) dsRNA. Ultrastructural morphology of adult worms treated in vitro with Sm-tsp-2 dsRNA displayed a distinctly vacuolated and thinner tegument compared with controls. Schistosomula exposed in vitro to Sm-tsp-2 dsRNA had a significantly thinner and more vacuolated tegument, and morphology consistent with a failure of tegumentary invaginations to close. Injection of mice with schistosomula that had been electroporated with Sm-tsp-1 and Sm-tsp-2 dsRNAs resulted in 61% (p = 0.005) and 83% (p = 0.002) reductions in the numbers of parasites recovered from the mesenteries four weeks later when compared to dsRNA-treated controls. These results imply that tetraspanins play important structural roles impacting tegument development, maturation or stability

Suppression of mRNAs Encoding Tegument Tetraspanins from Schistosoma mansoni Results in Impaired Tegument Turnover

Schistosomes express a family of integral membrane proteins, called tetraspanins (TSPs), in the outer surface membranes of the tegument. Two of these tetraspanins, Sm-TSP-1 and Sm-TSP-2, confer protection as vaccines in mice, and individuals who are naturally resistant to S. mansoni infection mount a strong IgG response to Sm-TSP-2. To determine their functions in the tegument of S. mansoni we used RNA interference to silence expression of Sm-tsp-1 and Sm-tsp-2 mRNAs. Soaking of parasites in Sm-tsp dsRNAs resulted in 61% (p = 0.009) and 74% (p = 0.009) reductions in Sm-tsp-1 and Sm-tsp-2 transcription levels, respectively, in adult worms, and 67%–75% (p = 0.011) and 69%–89% (p = 0.004) reductions in Sm-tsp-1 and Sm-tsp-2 transcription levels, respectively, in schistosomula compared to worms treated with irrelevant control (luciferase) dsRNA. Ultrastructural morphology of adult worms treated in vitro with Sm-tsp-2 dsRNA displayed a distinctly vacuolated and thinner tegument compared with controls. Schistosomula exposed in vitro to Sm-tsp-2 dsRNA had a significantly thinner and more vacuolated tegument, and morphology consistent with a failure of tegumentary invaginations to close. Injection of mice with schistosomula that had been electroporated with Sm-tsp-1 and Sm-tsp-2 dsRNAs resulted in 61% (p = 0.005) and 83% (p = 0.002) reductions in the numbers of parasites recovered from the mesenteries four weeks later when compared to dsRNA-treated controls. These results imply that tetraspanins play important structural roles impacting tegument development, maturation or stability

Schistosome antigens and immune response in specific and non-specific hosts

Department of ParasitologyKatedra parazitologieFaculty of SciencePřírodovědecká fakult

Isolation of cDNAs Encoding Secreted and Transmembrane Proteins from Schistosoma mansoni by a Signal Sequence Trap Method

Surface and secreted proteins of schistosomes orchestrate the basic physiologic requirements of a parasitic existence. These proteins are often exposed to host tissues during penetration, migration, feeding, and immune evasion, and they are obvious targets for control strategies. Signal sequence trap (SST) represents a novel approach that selects for cDNAs encoding secreted and surface proteins with N-terminal signal peptides, so we constructed a randomly primed adult Schistosoma mansoni cDNA library fused to a signalless reporter gene encoding placental alkaline phosphatase. The library was used to transfect COS-7 cells, which were then assayed for the presence of reporter at the cell surface. Eighteen S. mansoni cDNA fragments were isolated and sequenced. Expression profiles of the novel clones were determined for different developmental stages; some transcripts were restricted to single-sex adult worms, while others were ubiquitously distributed. Most clones contained signal peptides or signal anchors as determined by the SignalP algorithm. Open reading frames (ORFs) were categorized as follows: (i) previously identified S. mansoni cDNAs encoding proteins of known function; (ii) cDNAs encoding proteins of known function in other organisms but novel for Schistosoma; (iii) S. mansoni expressed sequence tags (ESTs) of unknown function; and (iv) completely novel ORFs without homologues (including ESTs) from any phylum. Clones of particular interest included tetraspanins similar to human cell surface antigens, a protein kinase, and ORFs transcribed in the antisense orientation to previously characterized S. mansoni cDNAs. This is the first report describing the use of SST as a tool for identifying secreted proteins from any pathogenic organism

Targeting schistosome cholinesterases for vaccine and drug development

The nervous system of schistosomes has been successfully targeted by anthelmintic drugs but the use of many of these has discontinued because of toxic side effects and so there is a need to better understand key neuronal processes at a molecular level to develop safer and more effective intervention strategies that target this vital system. Cholinesterases - acetylcholinesterases (AChE)s and butyrylcholinesterases (BChE)s - are key enzymes that play a pivotal role in the nervous system of schistosomes by regulating neurotransmission through acetylcholine hydrolysis and, accordingly, are an example of such an intervention target. The first results chapter (chapter two) of this thesis investigated the anti-schistosome efficacy of polypyridylruthenium (II) complexes and showed they were active against all intra-mammalian stages of S. mansoni. Two compounds, Rubb₁₂-tri and Rubb₇-tnl, which were among the most potent in their ability to kill schistosomula and adult worms and inhibit egg hatching in vitro, were assessed for their efficacy in a mouse model of schistosomiasis using 5 consecutive daily i.v. doses of 2 mg/kg (Rubb₁₂-tri) and 10 mg/kg (Rubb₇-tnl). Mice treated with Rubb₁₂-tri showed an average 42% reduction (P = 0.009), over two independent trials, in adult worm burden. Liver egg burdens were not significantly decreased in either drug-treated group but ova from both of these groups showed significant decreases in hatching ability (Rubb₁₂-tri - 68%, Rubb₇-tnl - 56%) and were significantly morphologically altered (Rubb₁₂-tri - 62% abnormal, Rubb₇-tnl - 35% abnormal). I hypothesize that the drugs exerted their activity, at least partially, through inhibition of both neuronal and tegumental acetylcholinesterases (AChEs), as worms treated in vitro showed significant decreases in activity of these enzymes. Further, treated parasites exhibited a significantly decreased ability to uptake glucose, significantly depleted glycogen stores and withered tubercules (a site of glycogen storage), implying drug-mediated interference in this nutrient acquisition pathway. Chapter three of this thesis provided the first comprehensive molecular characterization of three S. mansoni cholinesterases (SmChEs), designated as SmAChE1, SmBChE1 and SmAChE3, which were identified from the interrogation of the now wholly annotated S. mansoni genome. Anti-SmChE antibodies localized the proteins to the tegument and neuromusculature of adults and schistosomula and developmental expression profiling differed among the molecules, suggestive of functions extending beyond traditional cholinergic signaling for each of them. I also reported the presence of ChE activity in parasite ES products for the first time and proteomically identified the molecules responsible (SmAChE1 and SmBChE1). Functional recombinant versions of the three SmChEs were produced in Pichia pastoris and enzyme nomenclature (AChE or BChE) was verified based on substrate preference. Lastly, in the first characterization study of a BChE from helminths, evidence is provided that SmBChE1 may act as a bio-scavenger of AChE inhibitors as the addition of recombinant SmBChE1 to parasite cultures mitigated the effect of the anti-schistosomal AChE inhibitor dichlorvos whereas SmBChE1-silenced parasites displayed increased sensitivity to dichlorvos. SmChEs were further characterized by RNAi-based experiments in chapter four of this thesis. RNAi-mediated silencing of individual SmChEs, or simultaneous silencing of all three SmChEs, significantly suppressed transcript and protein expression levels and AChE activity in parasites. In a dissection of the hypothesis that tegumental AChE mediates exogenous glucose scavenging by the parasite, I showed that RNAi-mediated knockdown of SmAChE1 and SmAChE3, but not SmBChE1, significantly reduced glucose uptake by schistosomes. Parasite survivability in vitro and in vivo was significantly impaired with the silencing of SmChEs, either individually or in combination, attesting to the essentiality of these molecules. Chapter five of this thesis explored the vaccine potential of SmChEs. When treated in vitro with anti-SmChE IgG, parasites displayed significantly decreased ChE activity, which eventually resulted in death. Vaccination with individual SmChEs, or a combination of all three SmChEs, significantly reduced worm burdens (28% - 38%, averaged across two independent trials) compared to controls. Liver egg burdens were significantly decreased for all mice across both trials (13% - 46%) except those vaccinated with SmAChE1 in trial 1. Egg viability, as determined by egg hatching from liver homogenates, was significantly reduced in the groups vaccinated with the SmChE cocktail (40%) and SmAChE3 (46%). Surviving worms from each vaccinated group were significantly stunted and depleted of glycogen stores, compared to controls. In conclusion, this thesis has identified the burgeoning potential of a new class of antischistosome drugs that, at least in part, target the nervous system of the parasite and provided a comprehensive characterization of a family of ChEs from S. mansoni, giving compelling evidence for the essentiality of the proteins and their utility as intervention targets against schistosomiasis

Proteomic analysis of the Schistosoma mansoni surface membranes

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