Raúl J. Bobes

The genomes of four tapeworm species reveal adaptations to parasitism.

Tapeworms (Cestoda) cause neglected diseases that can be fatal and are difficult to treat, owing to inefficient drugs. Here we present an analysis of tapeworm genome sequences using the human-infective species Echinococcus multilocularis, E. granulosus, Taenia solium and the laboratory model Hymenolepis microstoma as examples. The 115- to 141-megabase genomes offer insights into the evolution of parasitism. Synteny is maintained with distantly related blood flukes but we find extreme losses of genes and pathways that are ubiquitous in other animals, including 34 homeobox families and several determinants of stem cell fate. Tapeworms have specialized detoxification pathways, metabolism that is finely tuned to rely on nutrients scavenged from their hosts, and species-specific expansions of non-canonical heat shock proteins and families of known antigens. We identify new potential drug targets, including some on which existing pharmaceuticals may act. The genomes provide a rich resource to underpin the development of urgently needed treatments and control.

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.

Subarachnoidal and intraventricular human neurocysticercosis: application of an antigen detection assay for the diagnosis and follow-up.

Background  Neurocysticercosis (NC) is a parasitic disease of the central nervous system caused by the larval stage of Taenia solium. Although imaging studies are recommended for diagnosis and follow‐up of patients, their high cost and restricted availability limit their use. Among various immunological tests, the detection of HP10 antigen in cerebral spinal fluid (CSF) has proved to be a useful tool for the diagnosis of NC in the case of viable but not dead parasites.

Effective protection induced by three different versions of the porcine S3Pvac anticysticercosis vaccine against rabbit experimental Taenia pisiformis cysticercosis.

In an effort to develop an effective and affordable oral vaccine against porcine Taenia solium cysticercosis, the S3Pvac anti-cysticercosis vaccine was expressed in papaya calli. Taenia pisiformis experimental rabbit cysticercosis was used as a model to compare the efficacy of the oral vaccine vs. the injectable S3Pvac-synthetic and S3Pvac-phage versions. Oral S3Pvac-papaya significantly reduced the expected number of hepatic lesions and peritoneal cysticerci to a similar extent than the injectable vaccines. This study reports for the first time an effective oral vaccine against T. pisiformis cysticercosis, possibly useful against porcine T. solium cysticercosis.

Genome analysis of Excretory/Secretory proteins in Taenia solium reveals their Abundance of Antigenic Regions (AAR)

Excretory/Secretory (ES) proteins play an important role in the host-parasite interactions. Experimental identification of ES proteins is time-consuming and expensive. Alternative bioinformatics approaches are cost-effective and can be used to prioritize the experimental analysis of therapeutic targets for parasitic diseases. Here we predicted and functionally annotated the ES proteins in T. solium genome using an integration of bioinformatics tools. Additionally, we developed a novel measurement to evaluate the potential antigenicity of T. solium secretome using sequence length and number of antigenic regions of ES proteins. This measurement was formalized as the Abundance of Antigenic Regions (AAR) value. AAR value for secretome showed a similar value to that obtained for a set of experimentally determined antigenic proteins and was different to the calculated value for the non-ES proteins of T. solium genome. Furthermore, we calculated the AAR values for known helminth secretomes and they were similar to that obtained for T. solium. The results reveal the utility of AAR value as a novel genomic measurement to evaluate the potential antigenicity of secretomes. This comprehensive analysis of T. solium secretome provides functional information for future experimental studies, including the identification of novel ES proteins of therapeutic, diagnosis and immunological interest.

Genetic diversity of Taenia solium cysticerci from naturally infected pigs of central Mexico.

This study was designed to explore if each individual case of naturally acquired porcine cysticercosis, living in different geographic rural areas of central Mexico, is caused by one or more different specimens of Taenia solium tapeworm. The genetic variability among cysticerci from the same pig and that from different pigs was assessed by random amplified polymorphic DNA markers (RAPDs), through the percentage of polymorphic loci, the number of effective alleles, the expected heterozygosity and the Shannon index. The parasite populations reproductive structure was estimated through the association index (I(A)), and the degree of genetic differentiation and variation was determined using AMOVA. Using six different random primers, and a total of 181 cysticerci from 14 pigs, 88 different loci were amplified: 85% were polymorphic between pigs and 24% within pigs. The phenogram grouped the cysticerci into eight major clusters, with differences in the genetic distances among all cysticerci from 14 pigs ranging from 0.78 to 1. Most of the cysticerci grouped in accord with their different geographical origin and with their pig of origin. The similarity matrix produced from the phenogram (obtained by UPGMA) and the original similarity matrix yielded a good cophenetic correlation (r=0.82317, P=0.0004), which suggests that the phenogram accurately represents the original genetic similarities between isolates. The combination of I(A) (0.0-0.089) with the genetic diversity index (0.009-0.073) supports the idea that DNA diversity in T. solium cysticerci of naturally infected pigs is within the range expected from a recombination process occurring during sexual reproduction. The small genetic diversity found within the cysticerci of each pig (33.81%), when compared with that between pigs (66.19%), indicates that pigs are rarely infected by different tapeworms. It would then appear that porcine cysticercosis courses with effective concomitant immunity, as occurs in ovine cysticercosis.

Heterologous Prime-Boost Oral Immunization with GK-1 Peptide from Taenia crassiceps Cysticerci Induces Protective Immunity

ABSTRACT Oral immunization is a goal in vaccine development, particularly for pathogens that enter the host through the mucosal system. This study was designed to explore the immunogenic properties of the Taenia crassiceps protective peptide GK-1 administered orally. Mice were orally immunized with the synthetic GK-1 peptide in its linear form with or without the Brucella lumazine synthase (BLS) protein adjuvant or as a chimera recombinantly bound to BLS (BLS-GK-1). Mice were boosted twice with GK-1 only at 15-day intervals. A significant rate of protection of 64.7% was achieved in GK-1-immunized mice, and that rate significantly increased to 91.8 and 96% when mice were primed with GK-1 coadministered with BLS as an adjuvant and BLS as a carrier, respectively. Specific antibodies and T cell activation and proliferation accompanied the protection induced, revealing the potent immunogenicity of GK-1. Through immunohistochemical studies, GK-1 was detected in T and B cell zones of the Peyers patches (PP) and mesenteric lymph nodes. In the latter, abundant proliferating cells were detected by 5′-bromo-2′-deoxyuridine incorporation. No proliferation was detected in PP. Altogether, these results portray the potent immunogenic properties of GK-1 administered orally and reinforce the usefulness of BLS as an adjuvant and adequate vaccine delivery system for oral vaccines.

Taenia solium: Development of an Experimental Model of Porcine Neurocysticercosis

Human neurocysticercosis (NC) is caused by the establishment of Taenia solium larvae in the central nervous system. NC is a severe disease still affecting the population in developing countries of Latin America, Asia, and Africa. While great improvements have been made on NC diagnosis, treatment, and prevention, the management of patients affected by extraparenchymal parasites remains a challenge. The development of a T. solium NC experimental model in pigs that will allow the evaluation of new therapeutic alternatives is herein presented. Activated oncospheres (either 500 or 1000) were surgically implanted in the cerebral subarachnoid space of piglets. The clinical status and the level of serum antibodies in the animals were evaluated for a 4-month period after implantation. The animals were sacrificed, cysticerci were counted during necropsy, and both the macroscopic and microscopic characteristics of cysts were described. Based on the number of established cysticerci, infection efficiency ranged from 3.6% (1000 oncospheres) to 5.4% (500 oncospheres). Most parasites were caseous or calcified (38/63, 60.3%) and were surrounded by an exacerbated inflammatory response with lymphocyte infiltration and increased inflammatory markers. The infection elicited specific antibodies but no neurological signs. This novel experimental model of NC provides a useful tool to evaluate new cysticidal and anti-inflammatory approaches and it should improve the management of severe NC patients, refractory to the current treatments.

Protection against murine intestinal amoebiasis induced by oral immunization with the 29 kDa antigen of Entamoeba histolytica and cholera toxin

Entamoeba histolytica antigens recognized by salivary IgA from infected patients include the 29 kDa antigen (Eh29), an alkyl hydroperoxide reductase. Here, we investigate the potential of recombinant Eh29 and an Eh29-cholera toxin subunit B (CTxB) fusion protein to confer protection against intestinal amoebiasis after oral immunization. The purified Eh29-CTxB fusion retained the critical ability to bind ganglioside GM(1), as determined by ELISA. Oral immunization of C3H/HeJ mice with Eh29 administered in combination with a subclinical dose of whole cholera toxin, but not as an Eh29-CTxB fusion, induced elevated levels of intestinal IgA and serum IgG anti-Eh29 antibodies that inhibited trophozoites adherence to MDCK cell monolayers. The 80% of immunized mice seen to develop IgA and IgG immune responses showed no evidence of infection in tissue sections harvested following intracecal challenge with virulent E. histolytica trophozoites. These results suggest that Eh29 is capable of inducing protective anti-amoebic immune responses in mice following oral immunization and could be used in the development of oral vaccines against amoebiasis.

Identification and quantification of host proteins in the vesicular fluid of porcine Taenia solium cysticerci.

The host-parasite relationship in cestode infections is complex. One feature of this bidirectional molecular communication is the uptake of host proteins by the parasite. Here we describe the presence of several host proteins in the vesicular fluid of Taenia solium cysticerci dissected from the central nervous system and the skeletal muscle of naturally infected pigs. Using two-dimensional electrophoresis we compared the protein patterns of vesicular fluids of cysticerci vs. the sera of cysticercotic pigs. We found that the vesicular fluids of both groups of cysts showed 17 protein spots matching with the pigs sera spots. After mass spectrometry sequencing of these spots, five host proteins were identified: hemoglobin, albumin, serpin A3-8, haptoglobin, rho GTPase-activating protein 36-like. Three of the 17 spots corresponded to host protein fragments: hemoglobin, albumin and serpin A3-8. IgG heavy and light chains were also identified by Western blot using a specific antibody. Quantitative estimations indicated that the host proteins represented 11-13% of the protein content in the vesicular fluids. We also calculated the relative abundance of these host proteins in the vesicular fluids; all were represented in similar relative abundances as in host sera. This suggests that uptake of host proteins by cysticerci proceeds through an unspecific mechanism such as non-specific fluid pinocytosis.