Intermediate and definitive hosts of wild Schistosoma mansoni: ecological niche modeling of hosts in low endemicity areas

ABSTRACT The relationship between the environment and animal life began to be seen as an important tool to help control zoonoses. Climate variations lead to changes in the environment, which can influence the spatial distribution of species and, consequently, the spread of diseases to humans. Considered the main non-human definitive host species of Schistosoma mansoni in Brazil, the wild rodent Nectomys squamipes plays an important role as a reservoir in maintaining the schistosomiasis cycle in the absence of humans. This study demonstrates the results of ecological niche modeling of intermediate and definitive wild hosts of S. mansoni in the Regional Health Superintendence of Barbacena (Minas Gerais State), which has registered 31 municipalities, 80% of which are classified as endemic for parasitosis. Environmental variables associated with the distribution of each species were used based on information from the scientific collections of Global Biodiversity Information Facility (GBIF) and Species Link to project the ecological niche model in the geographic space. Abiotic variables such as the mean annual temperature, isothermality, and precipitation seasonality were obtained from World Clim. Ecological niche modeling of the wild host, N. squamipes, revealed the occurrence of the species in geographic overlap with the Biomphalaria species. Knowing the influence of bioclimatic variables and identifying favorable conditions for the establishment, occurrence, and distribution of species are important information for developing strategic actions for the surveillance and control of this endemic species. The presence of the definitive wild host needs to be considered by control programs of schistosomiasis.

Challenges to Schistosomiasis Control Program in Brazil: setbacks in the control program and critical analysis of the disease notification

ABSTRACT Background: In 1970, Brazil implemented the Schistosomiasis Control Program (PCE, Portuguese acronym for Programa de Controle da Esquistossomose) was implemented in Brazil, where, through successive treatment interventions, the epidemiology and transmission of schistosomiasis have changed significantly over time. This study aimed to evaluate the PCE's effectiveness by critically analyzing the disease notification system. Methods: An ecological study was conducted using data on reported schistosomiasis cases in Brazil between 2007 and 2020. Results: The highest number of municipalities actively participating in the PCE was 750, recorded in 2007. Conversely, participation reached its lowest point in 2020, with only 259 municipalities involved. Over the past decade, there has been a drastic decline in the number of municipalities with active schistosomiasis control programs. During the same period, there was an observed increase in the number of deaths caused by schistosomiasis, while the number of reported cases decreased. This suggests an inverse correlation. Conclusions: The present data suggest that schistosomiasis cases are not correctly diagnosed or reported, reflecting a twisted image of the magnitude of this public health problem in Brazil.

Descriptive study on risk of increased morbidity of schistosomiasis and graft loss after liver transplantation

ABSTRACT Solid-organ transplantation procedures have witnessed a surge in frequency. Consequently, increased attention to associated infections and their impact on graft success is warranted. The liver is the principal target for infection by the flatworm Schistosoma mansoni. Hence, rigorous screening protocols for this parasite should be implemented for liver transplantation donors and recipients. This study investigated the risks posed by schistosomiasis-infected liver tissues for successful liver transplantation (LT), considering donors and recipients, by analyzing reported cases. Among the 43 patients undergoing LT (donors = 19; recipients = 24), 32 were infected with S. mansoni, five were infected with other Schistosoma species, and no identification was made in four patients. Reported follow-up periods ranged from 1 to 132 months, and all patients achieved successful recovery. As these helminths do not replicate in their vertebrate hosts, immunosuppressive treatment is not expected to promote increased morbidity or reactivation. Moreover, suspected or confirmed schistosomiasis infections often have a benign course, and generally, should not prevent LT. The available literature was reviewed and a provisional screening protocol has been proposed.

Water, sanitation and schistosomiasis mansoni: a study based on the Brazilian National Prevalence Survey (2011-2015) / Água, saneamento e esquistossomose mansoni: um estudo baseado no Inquérito Nacional de Prevalência do Brasil (2011-2015)

Abstract This study aimed to explore the association between water, sanitation, and the prevalence of schistosomiasis mansoni in students aged 7 to 17 years from all 27 federative units in Brazil. It was a cross-sectional study conducted based on data on the prevalence of schistosomiasis mansoni referring to 197,567 students from 521 Brazilian municipalities, who participated in the National Survey on the Prevalence of Schistosomiasis Mansoni and Soil-transmitted Helminth Infections (2011-2015). Univariable and multivariable generalized linear models of the negative binomial type were adjusted using 25 and 5% significance levels, respectively, considering municipalities as the unit of analysis. While a protective association was found between access to filtered water in schools and schistosomiasis mansoni prevalence, sanitation in schools was indicated as a risk factor. The collection of wastewater through a network is not universal in Brazil, and even when present, it is not necessarily carried out by the treatment of collected effluents, thus often resulting in the direct discharge of raw sewage into water resources. Regarding septic tanks, only the presence of infrastructure alone does not guarantee its correct use by the population.

Resumo O presente trabalho teve como objetivo explorar a associação entre água, saneamento e a prevalência de esquistossomose mansoni em estudantes de 7 a 17 anos de todas as 27 unidades federativas do Brasil. Tratou-se de um estudo transversal, conduzido com base nos dados de prevalência de esquistossomose mansoni referentes a 197.567 estudantes de 521 municípios brasileiros que participaram do Inquérito Nacional da Prevalência de Esquistossomose Mansoni e Geo-helmintoses (2011-2015). Modelos lineares generalizados do tipo binomial negativo, univariável e multivariável foram construídos considerando níveis de significância de 25% e 5%, respectivamente, e os municípios como unidade de análise. Embora os resultados tenham indicado associação protetora entre o acesso à água filtrada nas escolas e a prevalência de esquistossomose mansoni, o acesso ao saneamento nas escolas foi apontado como um fator de risco. A coleta de águas residuais por rede não é universal no Brasil e, mesmo quando presente, não é necessariamente procedida pelo tratamento dos efluentes coletados, resultando, muitas vezes, no lançamento direto do esgoto bruto em matrizes aquosas. Com relação a soluções individuais como fossa sépticas, a presença da infraestrutura por si só não garante o seu uso correto pela população.

Discovery of new Schistosoma mansoni aspartyl protease inhibitors by structure-based virtual screening

BACKGROUND Schistosomiasis is a neglected tropical disease caused by trematodes of the genus Schistosoma, with a limited treatment, mainly based on the use of praziquantel (PZQ). Currently, several aspartic proteases genes have already been identified within the genome of Schistosoma species. At least one enzyme encoded from this gene family (SmAP), named SmCD1, has been validated for the development of schistosomicidal drugs, since it has a key role in haemoglobin digestion by worms. OBJECTIVE In this work, we integrated a structure-based virtual screening campaign, enzymatic assays and adult worms ex vivo experiments aiming to discover the first classes of SmCD1 inhibitors. METHODS Initially, the 3D-structures of SmCD1, SmCD2 and SmCD3 were generated using homology modelling approach. Using these models, we prioritised 50 compounds from 20,000 compounds from ChemBridge database for further testing in adult worm aqueous extract (AWAE) and recombinant SmCD1 using enzymatic assays. FINDINGS Seven compounds were confirmed as hits and among them, two compounds representing new chemical scaffolds, named 5 and 19, had IC50 values against SmCD1 close to 100 μM while presenting binding efficiency indexes comparable to or even higher than pepstatin, a classical tight-binding peptide inhibitor of aspartyl proteases. Upon activity comparison against mammalian enzymes, compound 50 was selective and the most potent against the AWAE aspartic protease activity (IC50 = 77.7 μM). Combination of computational and experimental results indicate that compound 50 is a selective inhibitor of SmCD2. Compounds 5, 19 and 50 tested at low concentrations (10 uM) were neither cytotoxic against WSS-1 cells (48 h) nor could kill adult worms ex-vivo, although compounds 5 and 50 presented a slight decrease on female worms motility on late incubations times (48 or 72 h). MAIN CONCLUSION Overall, the inhibitors identified in this work represent promising hits for further hit-to-lead optimisation.

Schistosomiasis of liver graft as a differential diagnosis of abnormal liver tests after transplantation: report of two cases

ABSTRACT Schistosomiasis is a major health problem that affects over 200 million people worldwide. There are few reports of Schistosoma mansoni found in liver transplants as well as scarce information about the course of the disease and the long-term effects on the graft. Herein, we report two cases of schistosomiasis in liver transplant recipients who presented abnormal serum liver enzymes, with evidence of gradual improvement after antiparasitic treatment. Furthermore, we discuss the possible role of screening the parasite infection in potential liver transplant recipients from endemic areas.

Association of Tumor Necrosis Factor-α (-G308A) Polymorphism with risk of Upper Gastrointestinal Bleeding from Schistosomiasis in Pernambuco

ABSTRACT Background: We evaluated the association between polymorphisms in the tumor necrosis factor alpha (TNF-α) (-G308A) gene and upper gastrointestinal bleeding (UGIB) in schistosomiasis. Methods: This was a transverse study involving 294 Brazilian patients infected with Schistosoma mansoni. Results: The homozygous A/A genotype in TNF-α (-G308A) showed a risk association (prevalence ratio = 1.90, p = 0.008) with UGIB. There was no statistically significant difference in serum TNF-α levels between the clinical groups. Conclusions: The polymorphic TNF-α (-G308A) can be a risk factor for UGIB, in addition to being a potentially predictive factor for the severity of UGIB in schistosomiasis.

Accuracy of the urine point-of-care circulating cathodic antigen assay for diagnosing Schistosomiasis mansoni infection in Brazil: A multicenter study

ABSTRACT Background: The World Health Organization recommends a market-ready, urine-based point-of-care diagnostic test for circulating cathodic antigens (CCA) to determine the prevalence of S. mansoni. This study evaluated the performance of the URINE CCA (SCHISTO) ECO TESTE® (POC-ECO), which is currently available in Brazil. Methods: Residents from eight sites with different prevalence estimates provided one urine sample for POC-ECO and one stool sample for Kato-Katz (KK) and Helmintex® (HTX) testing as an egg-detecting reference for infection status. Results: None of the study sites had significantly higher POC-ECO accuracy than KK. Conclusions: POC-ECO is not currently recommended in Brazilian schistosomiasis elimination programs.

Avaliação do impacto da temperatura e da concentração de IPTG na expressão da proteína Rzv-TSP2 derivada de Schistosoma mansoni

Schistosomiasis is endemic in tropical countries and affects 240 million people around the world, with the poorest countries and regions being the most affected because they have poor or no sanitation conditions. In Brazil, the parasite Schistosoma mansoni is the cause of the disease, whose records point 423,117 cases in the country between the years 2009 and 2019. A vaccine would be the best way to reduce the occurrence of schistosomiasis, however, due to its chronic nature of infections, it has not yet been possible to create a vaccine that produces consistent immunogenicity. Therefore, the aim of this work is to optimize the production of the vaccine candidate SmTSP-2 (protein located in the tegument of the parasite) fused to rizavidin, aiming its use in the construction of a Multiple Antigen Presenting System (MAPS). For this, two independent variables involved in the expression process were evaluated: temperature and concentration of the expression inducer IPTG. Using the Response Surface Methodology (RSM) and analyzing the results in Western Blot, it was possible to observe that temperature has a more significant role in protein expression compared to IPTG concentration: at 22oC the expression of Rzv-TSP2 occurred in soluble and insoluble fractions, in both IPTG concentrations tested (0.1 mM and 1 mM). At 37°C, the protein was only expressed in the insoluble fraction at 1 mM IPTG concentration.

A esquistossomose é endêmica em países tropicais e afeta 240 milhões de pessoas ao redor do mundo, sendo os países e regiões mais pobres os mais afetados por possuírem condições de saneamento precárias ou inexistentes. No Brasil, o parasito Schistosoma mansoni é o causador da doença, cujo os registros apontam 423.117 casos no país entre os anos de 2009 e 2019. Uma vacina seria a melhor maneira de reduzir a ocorrência da esquistossomose, entretanto, devido ao seu caráter crônico de infecções, ainda não foi possível criar uma vacina que produzisse uma imunogenicida de consistente. Dado a isso, o objetivo deste trabalho é otimizar a produção do candidato vacinal SmTSP-2 (proteína localizada no tegumento do parasito) fusionada a rizavidina, visando sua utilização na construção de um sistema de apresentação de múltiplos antígenos (MAPS). Para isso, foram avaliadas duas variáveis independentes envolvidas no processo de expressão: temperatura e concentração do indutor de expressão IPTG. Utilizando a metodologia RSM (Response Surface Methodology) e analisando os resultados por Western Blot, foi possível observar que a temperatura possui um papel mais significativo na expressão da proteína em comparação a concentração de IPTG. Enquanto a 22oC a expressão de Rzv-TSP2 se deu nas frações solúvel e insolúvel, em ambas as concentrações de IPTG testadas (0,1 mM e 1 mM), na temperatura de 37oC as proteínas só foram expressas na fração insolúvel, sob a concentração de 1 mM de IPTG.

Otimização do método extrativo por maceração para obtenção de fitoquímicos de Ochtodes secundiramea (Rhodophyta)

Schistosomiasis is a parasitic infectious disease that affects a vast area of the planet, reaching thousands of people in low-income regions, with little or no basic sanitation. In Brazil, the disease is endemic and caused by the worm Schistosoma mansoni. Currently, the only drug used for the treatment and control of the disease is praziquantel. However, there are studies demonstrating possible resistance to this drug. Another method recommended by the WHO (World Health Organization) is the molluscicide niclosamide, used to combat snails, however, affecting fish, amphibians and other organisms. Therefore, research is carried out looking for alternative treatments based on natural products. Previous studies have shown that crude extracts of the seaweed Ochtodes secundiramea have schistosomicidal activity. In order to obtain new extracts with higher yields of phytochemicals and cost-benefit relevance for possible prototypes of new more efficient drugs against Schistosoma mansoni, in this work we present the optimization of the extractive method from the maceration process, which demonstrated good yield using a larger volume of dichloromethane solvent and with chloroform solvent in a smaller volume.

A esquistossomose é uma doença infecto parasitária que afetando vasta área do planeta, atingindo milhares de pessoas em regiões de baixa renda, com pouca e até inexistência de saneamento básico. No Brasil, a doença é endêmica e causada pelo verme Schistosoma mansoni. Atualmente o único medicamento usado para o tratamento e controle da doença é o praziquantel. Contudo, existem estudos demonstrando uma possível resistência a este medicamento. Outro método recomendado pela OMS (Organização Mundial de Saúde) é o moluscicida niclosamida, usado no combate aos caramujos, porém, afetando peixes, anfíbios entre outros microorganismos. Portanto, são realizadas pesquisas a procura por tratamentos alternativos com base em produtos naturais. Estudos anteriores demonstraram que os extratos brutos da alga marinha Ochtodes secundiramea apresentam atividade esquistossomicida. Afim de obter novos extratos com maiores rendimentos de fitoquímicos e relevância custo-benefício para possíveis protótipos de novos fármacos mais eficientes frente ao combate do Schistosoma mansoni, neste trabalho apresentamos a otimização do método extrativo a partir do processo de maceração, o qual demonstrou bom rendimento utilizando maior volume de solvente diclorometano e com solvente clorofórmio em menor volume.

Praziquantel-encapsulated niosomes against Schistosoma mansoni with reduced sensitivity to praziquantel / Niosomas encapsulados en prazicuantel contra Schistosoma mansoni con sensibilidad reducida al prazicuantel

Introduction: Praziquantel (PZQ) is the only commercially available drug for schistosomiasis. The current shortage of alternative effective drugs and the lack of successful preventive measures enhance its value. The increase in the prevalence of PZQ resistance under sustained drug pressure is, therefore, an upcoming issue. Objective: To overcome the tolerance to PZQ using nanotechnology after laboratory induction of a Schistosoma mansoni isolate with reduced sensitivity to the drug during the intramolluscan phase. Materials and methods: Shedding snails were treated with PZQ doses of 200 mg/kg twice/ week followed by an interval of one week and then repeated twice in the same manner. The success of inducing reduced sensitivity was confirmed in vitro via the reduction of cercarial response to PZQ regarding their swimming activity and death percentage at different examination times. Results: Oral treatment with a single PZQ dose of 500 mg/kg in mice infected with cercariae with reduced sensitivity to PZQ revealed a non-significant reduction (35.1%) of total worm burden compared to non-treated control mice. Orally inoculated PZQ- encapsulated niosomes against S. mansoni with reduced sensitivity to PZQ successfully regained the pathogen's sensitivity to PZQ as evidenced by measuring different parameters in comparison to the non-treated infected animals with parasites with reduced sensitivity to PZQ. The mean total worm load was 1.33 ± 0.52 with a statistically significant reduction of 94.09% and complete eradication of male worms. We obtained a remarkable increase in the percentage reduction of tissue egg counts in the liver and intestine (97.68% and 98.56%, respectively) associated with a massive increase in dead eggs and the complete absence of immature stages. Conclusion: PZQ-encapsulated niosomes restored the drug sensitivity against laboratory- induced S. mansoni adult worms with reduced sensitivity to PZQ.

Introducción. El prazicuantel es el único fármaco disponible comercialmente para la esquistosomiasis. La escasez actual de medicamentos alternativos y la falta de medidas preventivas eficaces aumentan su valor. La creciente prevalencia de la resistencia al prazicuantel bajo una presión prolongada del fármaco es, por tanto, un tema emergente. Objetivos. Superar la tolerancia al prazicuantel mediante nanotecnología después de la inducción en laboratorio de un aislamiento de Schistosoma mansoni con sensibilidad reducida al fármaco durante la fase intramolusco. Materiales y métodos. Los caracoles que liberaban cercarias se trataron con prazicuantel en dosis de 200 mg/kg dos veces por semana, seguidas de un intervalo de una semana, y luego se repitieron dos veces de la misma manera. La inducción exitosa de la sensibilidad reducida se confirmó in vitro mediante la reducción de la reacción de las cercarias al prazicuantel con respecto a su actividad de natación y el porcentaje de muerte en diferentes momentos de examen. El éxito en inducir una menor sensibilidad se confirmó in vitro mediante la reducción de la reacción de las cercarias al prazicuantel. Resultados. El tratamiento oral con una dosis única de prazicuantel de 500 mg/kg en ratones infectados con cercarias con sensibilidad reducida al prazicuantel, reveló una reducción no significativa (35,1 %) de la carga total de gusanos en comparación con los ratones de control no tratados. Los niosomas encapsulados en prazicuantel inoculados por vía oral contra S. mansoni con sensibilidad reducida al prazicuantel, permitieron reestablecer con éxito la sensibilidad del patógeno al medicamento, como lo demostró la medición de diferentes parámetros en comparación con los animales infectados no tratados con parásitos con sensibilidad reducida a prazicuantel. La carga media total de gusanos fue de 1,33 ± 0,52, con una reducción estadísticamente significativa del 94,09 %, y la erradicación completa de los gusanos machos adultos. Se obtuvo un aumento notable en el porcentaje de reducción del recuento de huevos en el tejido del hígado y el intestino (97,68 % y 98,56 %, respectivamente), asociado con un aumento masivo de huevos muertos y ausencia total de estadios inmaduros. Conclusión. Los niosomas encapsulados en prazicuantel restauraron la sensibilidad al fármaco contra gusanos adultos de S. mansoni con sensibilidad reducida al prazicuantel inducida en el laboratorio.

Single-cell RNA-seq analyses show that long non-coding RNAs are conspicuously expressed in Schistosoma mansoni gamete and tegument progenitor cell populations

Schistosoma mansoni is a flatworm that causes schistosomiasis, a neglected tropical disease that affects over 200 million people worldwide. New therapeutic targets are needed with only one drug available for treatment and no vaccine. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides with low or no protein-coding potential. In other organisms, they have been shown as involved with reproduction, stem cell maintenance and drug resistance, and they tend to exhibit tissue-specific expression patterns. S. mansoni expresses thousands of lncRNA genes; however, the cell type expression patterns of lncRNAs in the parasite remain uncharacterized. Here, we have re-analyzed publicly available single-cell RNA-sequencing (scRNA-seq) data obtained from adult S. mansoni to identify the lncRNAs signature of adult schistosome cell types. A total of 8023 lncRNAs (79% of all lncRNAs) were detected. Analyses of the lncRNAs expression profiles in the cells using statistically stringent criteria were performed to identify 74 lncRNA gene markers of cell clusters. Male gamete and tegument progenitor lineages clusters contained most of the cluster-specific lncRNA markers. We also identified lncRNA markers of specific neural clusters. Whole-mount in situ hybridization (WISH) and double fluorescence in situ hybridization were used to validate the cluster-specific expression of 13 out of 16 selected lncRNA genes (81%) in the male and female adult parasite tissues; for one of these 16 gene loci, probes for two different lncRNA isoforms were used, which showed differential isoform expression in testis and ovary. An atlas of the expression profiles across the cell clusters of all lncRNAs detected in our analysis is available as a public website resource (http://verjolab.usp.br:8081). The results presented here give strong support to a tissue-specific expression and to a regulated expression program of lncRNAs in S. mansoni. This will be the basis for further exploration of lncRNA genes as potential therapeutic targets.

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.

Reliability of point-of-care circulating cathodic antigen assay for diagnosing schistosomiasis mansoni in urine samples from an endemic area of Brazil after one year of storage at -20 degrees Celsius

ABSTRACT Background The World Health Organization recommends reliable point-of-care (POC) diagnostic testing to eliminate schistosomiasis. Lateral flow immunoassay that detects schistosome circulating cathodic antigen (CCA) in urine to establish prevalence thresholds for intervention in endemic areas is recommended. Stored urine may be useful if surveying at-risk populations is delayed or interrupted by unforeseen circumstances, such as the current COVID-19 pandemic. This study evaluated the manufacturer's claim that Schistosoma mansoni infection can be reliably diagnosed in urine samples stored at -20°C for one year. Methods Two-hundred-forty-two subjects from an endemic site in Brazil provided one urine sample each for testing with URINE CCA (SCHISTO) ECO TESTE® (POC-ECO) and one stool sample each for testing with Kato-Katz (KK) and Helmintex® (HTX) as a robust reference standard for infection status. At least 2 ml of urine from each participant was stored at -20°C; after one year, 76 samples were randomly selected for POC-ECO retesting. Results: The POC-ECO agreement between freshly collected and stored urine was inadequate considering trace results as positive (Cohen's kappa coefficient κ = 0.08) and negative (κ = 0.36). POC-ECO accuracy was not significantly greater than that of routine KK (54%; 95% confidence interval: 42.1%-65.5%). Conclusions The precision and accuracy of POC-ECO have to be optimized in both freshly collected and stored urine before it can be recommended for use in control programs in Brazil.

Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR associated protein 9-mediated editing of Schistosoma mansoni genes: Identifying genes for immunologically potent drug and vaccine development

ABSTRACT Schistosomiasis is a neglected acute and chronic tropical disease caused by intestinal (Schistosoma mansoni and Schistosoma japonicum) and urogenital (Schistosoma haematobium) helminth parasites (blood flukes or digenetic trematodes). It afflicts over 250 million people worldwide, the majority of whom reside in impoverished tropical and subtropical regions in sub-Saharan Africa. Schistosomiasis is the second most common devastating parasitic disease in the world after malaria and causes over 200,000 deaths annually. Currently, there is no effective and approved vaccine available for human use, and treatment strongly relies on praziquantel drug therapy, which is ineffective in killing immature larval schistosomula stages and eggs already lodged in the tissues. The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9)-mediated gene editing tool is used to deactivate a gene of interest to scrutinize its role in health and disease, and to identify genes for vaccine and drug targeting. The present review aims to summarize the major findings from the current literature reporting the usage of CRISPR/Cas9-mediated gene editing to inactivate genes in S. mansoni (acetylcholinesterase (AChE), T2 ribonuclease omega-1 (ω1), sulfotransferase oxamniquine resistance protein (SULT-OR), and α-N-acetylgalactosaminidase (SmNAGAL)), and freshwater gastropod snails, Biomphalaria glabrata (allograft inflammatory factor (BgAIF)), an obligatory component of the life cycle of S. mansoni, to identify their roles in the pathogenesis of schistosomiasis, and to highlight the importance of such studies in identifying and developing drugs and vaccines with high therapeutic efficacy.

In vitro and in silico evaluation of the schistosomicidal activity of eugenol derivatives using biochemical, molecular, and morphological tools

Background Eugenol shows both antibacterial and antiparasitic activities, suggesting that it might be evaluated as an option for the treatment of praziquantel-resistant schistosome. Methods The in vitro activities of three eugenol derivatives (FB1, FB4 and FB9) on adult worms from Schistosoma mansoni were examined by fluorescence and scanning electron microscopy to analyze effects on the excretory system and integument damage, respectively. Biochemical tests with verapamil (a calcium channel antagonist) and ouabain (a Na+/K+-ATPase pump inhibitor) were used to characterize eugenol derivative interactions with calcium channels and the Na+/K+-ATPase, while in silico analysis identified potential Na+/K+-ATPase binding sites. Results The compounds showed effective doses (ED50) of 0.324 mM (FB1), 0.167 mM (FB4), and 0.340 mM (FB9). In addition, FB4 (0.322 mM), which showed the lowest ED50, ED90 and ED100 (p < 0.05), caused the most damage to the excretory system and integument, according to both fluorescence and scanning electron microscopy analysis. The death of adult worms was delayed by ouabain treatment plus FB1 (192 versus 72 hours) and FB9 (192 versus 168 hours), but the response to FB4 was the same in the presence or absence of ouabain. Besides, no changes were noted when all of the eugenol derivatives were combined with verapamil. Moreover, FB1 and FB9 inhibited Na+/K+-ATPase activity according to in silico analysis but FB4 did not show a time-dependent relationship and may act on targets other than the parasite Na+/K+-ATPase. Conclusion Eugenol derivatives, mainly FB4 when compared to FB1 and FB9, seem to act more effectively on the integument of adult S. mansoni worms.(AU)

Biochemical characterization of the recombinant schistosome tegumental protein SmALDH_312 produced in E. coli and baculovirus expression vector system

BACKGROUND The heterologous expression of parasitic proteins is challenging because the sequence composition often differs significantly from host preferences. However, the production of such proteins is important because they are potential drug targets and can be screened for interactions with new lead compounds. Here we compared two expression systems for the production of an active recombinant aldehyde dehydrogenase (SmALDH_312) from Schistosoma mansoni, which causes the neglected tropical disease schistosomiasis. RESULTS We produced SmALDH_312 successfully in the bacterium Escherichia coli and in the baculovirus expression vector system (BEVS). Both versions of the recombinant protein were found to be active in vitro, but the BEVS-derived enzyme showed 3.7-fold higher specific activity and was selected for further characterization. We investigated the influence of Mg2+, Ca2+ and Mn2+, and found out that the specific activity of the enzyme increased 1.5-fold in the presence of 0.5 mM Mg2+. Finally, we characterized the kinetic properties of the enzyme using a design-of-experiment approach, revealing optimal activity at pH 7.6 and 41C. CONCLUSIONS Although, E. coli has many advantages, such as rapid expression, high yields and low costs, this system was outperformed by BEVS for the production of a schistosome ALDH. BEVS therefore rovides an opportunity for the expression and subsequent evaluation of schistosome enzymes as drug targets

Robust immune response induced by Schistosoma mansoni TSP-2 antigen coupled to bacterial outer membrane vesicles

Purpose: The use of adjuvants can significantly strengthen a vaccine’s efficacy. We sought to explore the immunization efficacy of bacterial outer membrane vesicles (OMVs) displaying the Schistosoma mansoni antigen, SmTSP-2, through a biotin-rhizavidin coupling approach. The rationale is to exploit the nanoparticulate structure and the adjuvant properties of OMVs to induce a robust antigen-specific immune response, in light of developing new vaccines against S. mansoni. Materials and Methods: OMVs were obtained from Neisseria lactamica and conjugated with biotin. The recombinant SmTSP-2 in fusion with the biotin-binding protein rhizavidin (rRzvSmTSP-2) was produced in E. coli and coupled to biotinylated OMVs to generate an OMV complex displaying SmTSP-2 on the membrane surface (OMV:rSmTSP-2). Transmission electron microscopy (TEM) and dynamic light scattering analysis were used to determine particle charge and size. The immunogenicity of the vaccine complex was evaluated in C57BL/6 mice. Results: The rRzvSmTSP-2 protein was successfully coupled to biotinylated OMVs and purified by size-exclusion chromatography. The OMV:rSmTSP-2 nanoparticles showed an average size of 200 nm, with zeta potential around – 28 mV. Mouse Bone Marrow Dendritic Cells were activated by the nanoparticles as determined by increased expression of the co-stimulatory molecules CD40 and CD86, and the proinflammatory cytokines (TNF-α, IL-6 and IL-12) or IL-10. Splenocytes of mice immunized with OMV:rSmTSP-2 nanoparticles reacted to an in vitro challenge with SmTSP-2 with an increased production of IL-6, IL-10 and IL-17 and displayed a higher number of CD4+ and CD8+ T lymphocytes expressing IFN-γ, IL-4 and IL-2, compared to mice immunized with the antigen alone. Immunization of mice with OMV:rSmTSP-2 induced a 100-fold increase in specific anti-SmTSP-2 IgG antibody titers, as compared to the group receiving the recombinant rSmTSP-2 protein alone or even co-administered with unconjugated OMV. Conclusion: Our results demonstrate that the SmTSP-2 antigen coupled with OMVs is highly immunogenic in mice, supporting the potential effectiveness of this platform for improved antigen delivery in novel vaccine strategies.

Primary and memory response of human monocytes to vaccines: role of nanoparticulate antigens in inducing innate memory

Innate immune cells such as monocytes and macrophages are activated in response to microbial and other challenges and mount an inflammatory defensive response. Exposed cells develop the so-called innate memory, which allows them to react differently to a subsequent challenge, aiming at better protection. In this study, using human primary monocytes in vitro, we have assessed the memory-inducing capacity of two antigenic molecules of Schistosoma mansoni in soluble form compared to the same molecules coupled to outer membrane vesicles of Neisseria lactamica. The results show that particulate challenges are much more efficient than soluble molecules in inducing innate memory, which is measured as the production of inflammatory and anti-inflammatory cytokines (TNFα, IL-6, IL-10). Controls run with LPS from Klebsiella pneumoniae compared to the whole bacteria show that while LPS alone has strong memory-inducing capacity, the entire bacteria are more efficient. These data suggest that microbial antigens that are unable to induce innate immune activation can nevertheless participate in innate activation and memory when in a particulate form, which is a notion that supports the use of nanoparticulate antigens in vaccination strategies for achieving adjuvant-like effects of innate activation as well as priming for improved reactivity to future challenges.

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.