Characterization of S3Pvac Anti-Cysticercosis Vaccine Components: Implications for the Development of an Anti-Cestodiasis Vaccine

Background: Cysticercosis and hydatidosis seriously affect human health and are responsible for considerable economic loss in animal husbandry in non-developed and developed countries. S3Pvac and EG95 are the only field trial-tested vaccine candidates against cysticercosis and hydatidosis, respectively. S3Pvac is composed of three peptides (KETc1, GK1 and KETc12), originally identified in a Taenia crassiceps cDNA library. S3Pvac synthetically and recombinantly expressed is effective against experimentally and naturally acquired cysticercosis.Methodology/ Principal Findings: In this study, the homologous sequences of two of the S3Pvac peptides, GK1 and KETc1, were identified and further characterized in Taenia crassiceps WFU, Taenia solium, Taenia saginata, Echinococcus granulosus and Echinococcus multilocularis. Comparisons of the nucleotide and amino acid sequences coding for KETc1 and GK1 revealed significant homologies in these species. The predicted secondary structure of GK1 is almost identical between the species, while some differences were observed in the C terminal region of KETc1 according to 3D modeling. A KETc1 variant with a deletion of three C-terminal amino acids protected to the same extent against experimental murine cysticercosis as the entire peptide. on the contrary, immunization with the truncated GK1 failed to induce protection. Immunolocalization studies revealed the non stage-specificity of the two S3Pvac epitopes and their persistence in the larval tegument of all species and in Taenia adult tapeworms.Conclusions/ Significance: These results indicate that GK1 and KETc1 may be considered candidates to be included in the formulation of a multivalent and multistage vaccine against these cestodiases because of their enhancing effects on other available vaccine candidates

Transfection of Platyhelminthes

Flatworms are one of the most diverse groups within Lophotrochozoa with more than 20,000 known species, distributed worldwide in different ecosystems, from the free-living organisms in the seas and lakes to highly specialized parasites living in a variety of hosts, including humans. Several infections caused by flatworms are considered major neglected diseases affecting countries in the Americas, Asia, and Africa. For several decades, a particular interest on free-living flatworms was due to their ability to regenerate considerable portions of the body, implying the presence of germ cells that could be important for medicine. The relevance of reverse genetics for this group is clear; understanding the phenotypic characteristics of specific genes will shed light on developmental traits of free-living and parasite worms. The genetic manipulation of flatworms will allow learning more about the mechanisms for tissue regeneration, designing new and more effective anthelmintic drugs, and explaining the host-parasite molecular crosstalk so far partially inaccessible for experimentation. In this review, availability of transfection techniques is analyzed across flatworms, from the initial transient achievements to the stable manipulations now developed for free-living and parasite species

Hormones and Parasites, Their Role in <em>Taenia solium</em> and <em>Taenia crassiceps</em> Physiology and Development

The host’s hormonal environment determines the susceptibility, the course, and severity of several parasite infections. In most cases the infection disturbs the host environment, and activates immune responses that end up affecting the endocrine system. In the other hand, a number of reports indicate that parasites have reproductive systems, and some others have shown that these organisms synthetize sex steroid hormones. We have shown that cysticerci, the larval stage of Taenia solium and Taenia crassiceps ORF and WFU, synthesize steroid hormones. This capacity was modified by drugs that act inhibiting the steroid synthesizing enzymes, or blocking the parasite’s hormone receptors. We have also shown that the cysticerci of T. crassiceps WFU and T. solium have the capacity to synthesize corticosteroids as deoxicorticosterone and corticosterone. We also reviewed the effects of insulin on these parasites, and the receptors found for this hormone. A deep knowledge of the parasite’s endocrine properties will contribute to understand their reproduction and the reciprocal interactions with the host. Likewise, may also help designing tools to combat the infection in clinical situations

Proteomic Study of Entamoeba histolytica Trophozoites, Cysts, and Cyst-Like Structures.

The cyst stage of Entamoeba histolytica is a promising therapeutic target against human amoebiasis. Our research team previously reported the production in vitro of Cyst-Like Structures (CLS) sharing structural features with cysts, including rounded shape, size reduction, multinucleation, and the formation of a chitin wall coupled to the overexpression of glucosamine 6-phosphate isomerase, the rate-limiting enzyme of the chitin synthesis pathway. A proteomic study of E. histolytica trophozoites, cysts, and in vitro-produced CLS is reported herein to determine the nature of CLS, widen our knowledge on the cyst stage, and identify possible proteins and pathways involved in the encystment process. Total protein extracts were obtained from E. histolytica trophozoites, CLS, and partially purified cysts recovered from the feces of amoebic human patients; extracts were trypsin-digested and analyzed by LC-MS/MS. In total, 1029 proteins were identified in trophozoites, 550 in CLS, and 411 in cysts, with 539, 299, and 84 proteins unique to each sample, respectively, and only 74 proteins shared by all three stages. About 70% of CLS proteins were shared with trophozoites, even though differences were observed in the relative protein abundance. While trophozoites showed a greater abundance of proteins associated to a metabolically active cell, CLS showed higher expression of proteins related to proteolysis, redox homeostasis, and stress response. In addition, the expression of genes encoding for the cyst wall proteins Jessie and Jacob was detected by RT-PCR and the Jacob protein identified by Western blotting and immunofluorescence in CLS. However, the proteomic profile of cysts as determined by LC-MS/MS was very dissimilar to that of trophozoites and CLS, with almost 40% of hypothetical proteins. Our global results suggest that CLS are more alike to trophozoites than to cysts, and they could be generated as a rapid survival response of trophozoites to a stressful condition, which allows the parasite to survive temporarily inside a chitin-like resistant cover containing Jacob protein. Our findings lead us to suggest that encystment and CLS formation could be distinct stress responses. In addition, we show that cysts express a high number of genes with unknown function, including four new, highly antigenic, possibly membrane-located proteins that could be targets of therapeutic and diagnostic usefulness

Validation of proteins identified by RT-PCR.

<p>Five proteins were selected for RT-PCR validation. Left above: malic enzyme. Left middle: F1-6BA, fructose 1,6-bisphosphate aldolase. Left below: GAPDH: glyceraldehyde 3-phosphate dehydrogenase. Right above: Gal/GalNAc lectin LC3 fragment. Right middle: peroxiredoxin. Right below: ARF, ADP-ribosylation factor, used as loading control. T: trophozoite, CLS: cyst-like structure.</p

Proteins shared among trophozoites, cysts, and CLS as identified by LC-MS/MS.

<p>Proteins shared among trophozoites, cysts, and CLS as identified by LC-MS/MS.</p

Jacob protein expression in CLS and cyst.

<p>A)15 μg of sample protein were resolved on 12% acrylamide gel (Coomassie blue-stained) and transferred to a PVDF membrane. Anti-Jacob 1:1000 and a goat HRP-conjugated anti-rabbit 1:50,000 antibodies were used. Unique bands of around 30-kDa and 62-kDa were observed in CLS and cyst samples, respectively, but not in a trophozoites sample. T: Trophozoite, CLS: Cyst-like structure, C: Cyst. B) Immunofluorescence on fixed and permeabilized cyst and CLS using anti-Jacob 1:200 and a goat FITC-conjugated anti-rabbit 1:200 antibodies.</p

Proteins identified in cysts in both studies.

<p>Proteins identified in cysts in both studies.</p

Comparison of proteomic data obtained from <i>E</i>. <i>histolytica</i> trophozoites, cysts and CLS.

<p>A) Proteome comparison using the BioVenn software. B) Percentage of annotated and hypothetical proteins identified in each sample. C) Global protein association between samples, labeled as total, annotated, or hypothetical proteins as determined by correlation test. R-values closer to 1 indicate a closer association. T: Trophozoite; CLS: cyst-like structure; C: cyst.</p