The prospective use of Brazilian marine macroalgae in Schistosomiasis control

Schistosomiasis is a parasitic disease that affects more than 250 million people. The treatment is limited to praziquantel and the control of the intermediate host with the highly toxic molluscicidal niclosamide. Marine algae are a poorly explored and promising alternative that can provide lead compounds, and the use of multivariate analysis could contribute to quicker discovery. As part of our search for new natural compounds with which to control schistosomiasis, we screened 45 crude extracts obtained from 37 Brazilian seaweed species for their molluscicidal activity against Biomphalaria glabrata embryos and schistosomicidal activities against Schistosoma mansoni. Two sets of extracts were taxonomically grouped for metabolomic analysis. The extracts were analyzed by GC–MS, and the data were subjected to Pattern Hunter and Pearson correlation tests. Overall, 22 species (60%) showed activity in at least one of the two models. Multivariate analysis pointed towards 3 hits against B. glabrata veliger embryos in the Laurencia/Laurenciella set, 5 hits against B. glabrata blastula embryos, and 31 against S. mansoni in the Ochrophyta set. Preliminary annotations suggested some compounds such as triquinane alcohols, prenylated guaianes, dichotomanes, and xenianes. Despite the putative identification, this work presents potential candidates and can guide future isolation and identification

Schistosoma mansoni venom allergen-like proteins: phylogenetic relationships, stage-specific transcription and tissue localization as predictors of immunological cross-reactivity

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

Schistosoma mansoni venom allergen-like proteins: phylogenetic relationships, stage-specific transcription and tissue localization as predictors of immunological cross-reactivity

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

Recombinant BCG expressing LTAK63 adjuvant induces superior protection against mycobacterium tuberculosis

In order to develop an improved BCG vaccine against tuberculosis we have taken advantage of the adjuvant properties of a non-toxic derivative of Escherichia coli heat labile enterotoxin (LT), LTAK63. We have constructed rBCG strains expressing LTAK63 at different expression levels. Mice immunized with BCG expressing low levels of LTAK63 (rBCG-LTAK63(lo)) showed higher Th1 cytokines and IL-17 in the lungs, and when challenged intratracheally with Mycobacterium tuberculosis displayed a 2.0-3.0 log reduction in CFU as compared to wild type BCG. Histopathological analysis of lung tissues from protected mice revealed a reduced inflammatory response. Immunization with rBCG-LTAK63(lo) also protected against a 100-fold higher challenge dose. Mice immunized with rBCG-LTAK63(lo) produced an increase in TGF-beta as compared with BCG after challenge, with a corresponding reduction in Th1 and Th17 cytokines, as determined by Real Time RT-PCR. Furthermore, rBCG-LTAK63(lo) also displays protection against challenge with a highly virulent Beijing isolate. Our findings suggest that BCG with low-level expression of the LTAK63 adjuvant induces a stronger immune response in the lungs conferring higher levels of protection, and a novel mechanism subsequently triggers a regulatory immune response, which then limits the pathology. The rBCG-LTAK63(lo) strain can be the basis of an improved vaccine against tuberculosis.

The antiviral effect of mollusk mucus on measles virus.

Measles is a viral disease highly contagious spread by respiratory transmission. Although infection can be controlled by vaccination, numerous cases of measles have been registered in many areas of the world, highlighting the need for additional interventions. Terrestrial gastropods exude mucus on their body surface when traveling, to protect the body from mechanical injury, desiccation or contact with harmful substances. The mucus of mollusks has been studied as a source of new natural compounds with diverse biological activities. In this study, the antiviral activity of the mucus of the land slug P. boraceiensis was demonstrated in vitro using Vero cells infected with measles virus. The crude sample and four fractions were tested in cultures infected with measles virus and the antiviral activity was assessed by the cytopathic effect in infected cell cultures as well as by immunofluorescence and qPCR. Fractions 39 and 50 of the mucus from P. boraceiensis were analyzed by HPLC-DAD-ESI-MS/MS and infrared spectroscopy. A mixture of polyunsaturated fatty acids was found in the two fractions. A reduction in the growth of the measles virus was observed, measured by qPCR, with a protection index of 80% in Vero cells infected with measles and treated with fraction 39. Fraction 39 exhibited the best antiviral action in vitro and high contents of hydroxy-tritriacontapentaenoic acid and hydroxy-pentatriacontapentaenoic acid were found in this fraction.

The antiviral effect of mollusk mucus on measles virus

Measles is a viral disease highly contagious spread by respiratory transmission. Although infection can be controlled by vaccination, numerous cases of measles have been registered in many areas of the world, highlighting the need for additional interventions. Terrestrial gastropods exude mucus on their body surface when traveling, to protect the body from mechanical injury, desiccation or contact with harmful substances. The mucus of mollusks has been studied as a source of new natural compounds with diverse biological activities. In this study, the antiviral activity of the mucus of the land slug P. boraceiensis was demonstrated in vitro using Vero cells infected with measles virus. The crude sample and four fractions were tested in cultures infected with measles virus and the antiviral activity was assessed by the cytopathic effect in infected cell cultures as well as by immunofluorescence and qPCR. Fractions 39 and 50 of the mucus from P. boraceiensis were analyzed by HPLC-DAD-ESI-MS/MS and infrared spectroscopy. A mixture of polyunsaturated fatty acids was found in the two fractions. A reduction in the growth of the measles virus was observed, measured by qPCR, with a protection index of 80% in Vero cells infected with measles and treated with fraction 39. Fraction 39 exhibited the best antiviral action in vitro and high contents of hydroxy-tritriacontapentaenoic acid and hydroxy-pentatriacontapentaenoic acid were found in this fraction. (C) 2016 Elsevier B.V. All rights reserved.

New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters

The expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such as Mycobacterium bovis BCG or M. smegmatis was made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong P-L5 promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinant M. smegmatis bacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in both M. smegmatis and M. bovis BCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of the Schistosoma mansoni antigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response

Schistosoma mansoni venom allergen like proteins present differential allergic responses in a murine model of airway inflammation.

BACKGROUND: The Schistosoma mansoni Venom-Allergen-Like proteins (SmVALs) are members of the SCP/TAPS (Sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7) protein superfamily, which may be important in the host-pathogen interaction. Some of these molecules were suggested by us and others as potential immunomodulators and vaccine candidates, due to their functional classification, expression profile and predicted localization. From a vaccine perspective, one of the concerns is the potential allergic effect of these molecules. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we characterized the putative secreted proteins SmVAL4 and SmVAL26 and explored the mouse model of airway inflammation to investigate their potential allergenic properties. The respective recombinant proteins were obtained in the Pichia pastoris system and the purified proteins used to produce specific antibodies. SmVAL4 protein was revealed to be present only in the cercarial stage, increasing from 0-6 h in the secretions of newly transformed schistosomulum. SmVAL26 was identified only in the egg stage, mainly in the hatched eggs' fluid and also in the secretions of cultured eggs. Concerning the investigation of the allergic properties of these proteins in the mouse model of airway inflammation, SmVAL4 induced a significant increase in total cells in the bronchoalveolar lavage fluid, mostly due to an increase in eosinophils and macrophages, which correlated with increases in IgG1, IgE and IL-5, characterizing a typical allergic airway inflammation response. High titers of anaphylactic IgG1 were revealed by the Passive Cutaneous Anaphylactic (PCA) hypersensitivity assay. Additionally, in a more conventional protocol of immunization for vaccine trials, rSmVAL4 still induced high levels of IgG1 and IgE. CONCLUSIONS: Our results suggest that members of the SmVAL family do present allergic properties; however, this varies significantly and therefore should be considered in the design of a schistosomiasis vaccine. Additionally, the murine model of airway inflammation proved to be useful in the investigation of allergic properties of potential vaccine candidates.