Algal-Derived Halogenated Sesquiterpenes from Laurencia dendroidea as Lead Compounds in Schistosomiasis Environmental Control.

Schistosomiasis has been controlled for more than 40 years with a single drug, praziquantel, and only one molluscicide, niclosamide, raising concern of the possibility of the emergence of resistant strains. However, the molecular targets for both agents are thus far unknown. Consequently, the search for lead compounds from natural sources has been encouraged due to their diverse structure and function. Our search for natural compounds with potential use in schistosomiasis control led to the identification of an algal species, Laurencia dendroidea, whose extracts demonstrated significant activity toward both Schistosoma mansoni parasites and their intermediate host snails Biomphalaria glabrata. In the present study, three seaweed-derived halogenated sesquiterpenes, (-)-elatol, rogiolol, and obtusol are proposed as potential lead compounds for the development of anthelminthic drugs for the treatment of and pesticides for the environmental control of schistosomiasis. The three compounds were screened for their antischistosomal and molluscicidal activities. The screening revealed that rogiolol exhibits significant activity toward the survival of adult worms, and that all three compounds showed activity against S. mansoni cercariae and B. glabrata embryos. Biomonitored fractioning of L. dendroidea extracts indicated elatol as the most active compound toward cercariae larvae and snail embryos.

Systems Biology Analysis of the Radiation-Attenuated Schistosome Vaccine Reveals a Role for Growth Factors in Protection and Hemostasis Inhibition in Parasite Survival.

In spite of several decades of research, an effective vaccine against schistosomiasis remains elusive. The radiation-attenuated (RA) cercarial vaccine is still the best model eliciting high protection levels, although the immune mechanisms have not yet been fully characterized. In order to identify genes and pathways underlying protection we investigated patterns of gene expression in PBMC and skin draining Lymph Nodes (LN) from mice using two exposure comparisons: vaccination with 500 attenuated cercariae versus infection with 500 normal cercariae; one versus three doses. Vaccinated mice were challenged with 120 normal parasites. Integration of PBMC and LN data from the infected group revealed early up-regulation of pathways associated with Th2 skewing and polarization of IgG antibody profiles. Additionally, hemostasis pathways were downregulated in infected mice, correlating with platelet reduction, potentially a mechanism to assist parasite migration through capillary beds. Conversely, up regulation of such mechanisms after vaccination may explain parasite blockade in the lungs. In contrast, a single exposure to attenuated parasites revealed early establishment of a Th1 bias (signaling of IL-1, IFN-γ; and Leishmania infection). Genes encoding chemokines and their receptors were more prominent in vaccinated mice, indicating an enhanced capacity for inflammation, potentially augmenting the inhibition of intravascular migration. Increasing the vaccinations from one to three did not dramatically elevate protection, but there was a clear shift towards antibody-mediated effectors. However, elements of the Th1 bias were still evident. Notable features after three vaccinations were markers of cytotoxicity (including IL-6 and NK cells) together with growth factors and their receptors (FGFR/VEGF/EGF) and the apoptosis pathway. Indeed, there is evidence for the development of anergy after three vaccinations, borne out by the limited responses detected in samples after challenge. We infer that persistence of a Th1 response puts a limit on expression of antibody-mediated mechanisms. This feature may explain the failure of multiple doses to drive protection towards sterile immunity. We suggest that the secretions of lung stage parasites would make a novel cohort of antigens for testing in protection experiments.

Rhesus macaques self-curing from a schistosome infection can display complete immunity to challenge.

The rhesus macaque provides a unique model of acquired immunity against schistosomes, which afflict >200 million people worldwide. By monitoring bloodstream levels of parasite-gut-derived antigen, we show that from week 10 onwards an established infection with Schistosoma mansoni is cleared in an exponential manner, eliciting resistance to reinfection. Secondary challenge at week 42 demonstrates that protection is strong in all animals and complete in some. Antibody profiles suggest that antigens mediating protection are the released products of developing schistosomula. In culture they are killed by addition of rhesus plasma, collected from week 8 post-infection onwards, and even more efficiently with post-challenge plasma. Furthermore, cultured schistosomula lose chromatin activating marks at the transcription start site of genes related to worm development and show decreased expression of genes related to lysosomes and lytic vacuoles involved with autophagy. Overall, our results indicate that enhanced antibody responses against the challenge migrating larvae mediate the naturally acquired protective immunity and will inform the route to an effective vaccine.

Stage and tissue expression patterns of Schistosoma mansoni venom allergen-like proteins SmVAL 4, 13, 16 and 24.

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.

Two SmDLC antigens as potential vaccines against schistosomiasis.

The Schistosoma mansoni transcriptome revealed new members of the dynein light chain family (DLC/LC8). The antigenicity and immunogenicity of these proteins, and their potential as vaccine candidates were investigated. Two DLC genes (DLC12_JI392413.1 and DLC13_JI387686.1) were cloned and the recombinant proteins produced in E. coli. The immunization of mice with the rDLCs, using alhydrogel as adjuvant, resulted in high titers of antibodies, indicated that these proteins are highly immunogenic. The anti-DLCs antibodies presented cross reactivity with both recombinant antigens and also recognized proteins from S. mansoni adult worm extracts. The DLC12 and DLC13 immunized animals were challenged by infection with cercariae and a protective profile was observed in three different assays, with a significant decreased in worm burden, of 43% and 51% respectively, when compared to the non-vaccinated group. The granulomas formation due to egg retention in the hepatic tissues was evaluated 45 days after infection. Smaller granulomas were observed in the liver of DLC immunized animals, up to 70% reduction in comparison to the granulomas size in the non-vaccinated animals. Fifty-five days after infection, the average size of the hepatic granulomas was still 25-35% smaller in the DLCs vaccinated groups. The interference of DLC immunization on the hepatic granuloma formation may reflect the lower worm burden and consequent decrease on the number of eggs retained in the liver, resulting in lower pro-inflammatory level in the tissue. The protective effect of DLCs immunization, decreasing the worm burden and delaying the rate of granuloma formation, suggests that these antigens should be further studied as potential vaccine candidates.

Avaliação da imunogenicidade da catepsina L1 de Schistosoma mansoni

As esquistossomoses constituem um dos principais problemas de saúde pública nos países em desenvolvimento. Estima-se que 200 milhões de pessoas estejam infectadas e 600 milhões vivam em áreas de risco. Os esquistossomos jovens e adultos vivem no interior dos vasos sanguíneos de seus hospedeiros, alimentando-se dos eritrócitos, sendo a hemoglobina essencial para o crescimento, desenvolvimento e reprodução dos vermes. Muitos parasitos utilizam as proteases para realizar algumas funções do seu ciclo de vida como: penetração através da pele, digestão das células do hospedeiro para a sua alimentação e evasão da resposta imune. A Catepsina L é uma cisteína protease da superfamília Papaína, detectada no intestino, indicando que essa enzima contribui para a proteólise da hemoglobina ingerida. Por desempenharem esses papéis na biologia dos esquistossomos, as enzimas proteolíticas são consideradas alvos potenciais para desenvolver e direcionar terapias anti-esquistossomóticas. Neste trabalho, foi desenvolvido um plasmídeo que permite a expressão da Catepsina L1 de Schistosoma mansoni em Escherichia coli. Os cDNAs que codificam a Catepsina L1 foram subclonados no vetor pAE, que proporciona alto nível de expressão de proteínas heterólogas sob o controle do promotor T7. Esses clones foram utilizados para transformar a cepa BL21-SI de E. coli, que é induzível por NaCl. A proteína recombinante foi expressa na forma de corpúsculos de inclusão, purificada sob condições desnaturantes por meio de cromatografia de afinidade ao níquel e utilizada em ensaios de proteção e desafio

Avaliação da imunogenicidade da catepsina L1 de Schistosoma mansoni / Evaluation of imunogenicity from Schistosoma mansoni cathepsin L1

As esquistossomoses constituem um dos principais problemas de saúde pública nos países em desenvolvimento. Estima-se que 200 milhões de pessoas estejam infectadas e 600 milhões vivam em áreas de risco. Os esquistossomos jovens e adultos vivem no interior dos vasos sanguíneos de seus hospedeiros, alimentando-se dos eritrócitos, sendo a hemoglobina essencial para o crescimento, desenvolvimento e reprodução dos vermes. Muitos parasitos utilizam as proteases para realizar algumas funções do seu ciclo de vida como: penetração através da pele, digestão das células do hospedeiro para a sua alimentação e evasão da resposta imune. A Catepsina L é uma cisteína protease da superfamília Papaína, detectada no intestino, indicando que essa enzima contribui para a proteólise da hemoglobina ingerida. Por desempenharem esses papéis na biologia dos esquistossomos, as enzimas proteolíticas são consideradas alvos potenciais para desenvolver e direcionar terapias anti-esquistossomóticas. Neste trabalho, foi desenvolvido um plasmídeo que permite a expressão da Catepsina L1 de Schistosoma mansoni em Escherichia coli. Os cDNAs que codificam a Catepsina L1 foram subclonados no vetor pAE, que proporciona alto nível de expressão de proteínas heterólogas sob o controle do promotor T7. Esses clones foram utilizados para transformar a cepa BL21-SI de E. coli, que é induzível por NaCl. A proteína recombinante foi expressa na forma de corpúsculos de inclusão, purificada sob condições desnaturantes por meio de cromatografia de afinidade ao níquel e utilizada em ensaios de proteção e desafio