Nelly Villalobos

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.

Limitations of current diagnostic procedures for the diagnosis of Taenia solium cysticercosis in rural pigs

The aim of the present study was to evaluate diagnostic procedures for porcine cysticercosis. Sera were obtained from 32 pigs reared in commercial farms, 47 pigs before and after experimental infection, 42 carefully necropsied rural pigs and 191 slaughtered pigs from rural communities in which the presence of the Taenia solium metacestode was assessed by tongue dissection. Sera were analyzed by ELISA to detect antibodies against T. solium antigens and to detect parasite antigens. Most sera from the necropsied rural pigs were also evaluated by the Western blot method. Antigen and antibody ELISA detection assays showed high sensitivity and specificity when applied to sera from pigs reared in commercial farms. In contrast, all methods (Ag-ELISA, Ab-ELISA assays, EITB and tongue inspection) showed lower sensitivity and specificity when applied to the generally lightly infected rurally reared pigs. The probability distribution of cysts in carcasses were also determined. These results emphasize the difficulties in detecting cysticercosis in rural pigs with low levels of cyst burdens.

CYSTICERCOSIS: IDENTIFICATION AND CLONING OF PROTECTIVE RECOMBINANT ANTIGENS

We describe the cloning and the evaluation of the protective capacity of 5 recombinant antigens expressed during the cysticercus stage of both Taenia crassiceps and Taenia solium. A cDNA library was constructed in bacteriophage lambda ZAP using mRNA isolated from larvae of T. crassiceps of the ORF strain. The recombinant phage library was screened with polyclonal antibodies against 56- and 74-kDa protective antigen fractions. This screening identified 13 recombinant clones, 5 of which were also strongly recognized by pooled sera from pigs experimentally infected with T. solium. The native antigens are proteins of 56 (clones KETcl, 4, 7) and 74 and 78 kDa (clones KETc11, 12) of T. crassiceps cysticerci. Vaccination experiments using these 5 recombinant clones against murine cysticercosis point to the relevance of KETcl, 4, 7, and 12 in host protection, whereas immunization with the clone KETc11 does not modify the parasite load in females and facilitates the parasitosis in males. We report here the DNA and the deduced amino acid sequence (100 amino acids) of the first protective antigen (KETc7) of potential interest for T. solium pig cysticercosis prevention.

Castration and pregnancy of rural pigs significantly increase the prevalence of naturally acquired Taenia solium cysticercosis

Cuentepec is a rural village of central Mexico, where 1300 pigs were bred at the time of the study in conditions that favor Taenia solium transmission. The tongues of 1087 (84%) of these pigs were visually examined and 33% were found to be cysticercotic. Castration of male pigs increased prevalence from 23 to 50% (P < 0.001) and pregnancy in sows also increased their prevalence from 28 to 59% (P < 0.001). Thus, endocrinological conditions characterized by low levels of androgens or high levels of female hormones probably influence the susceptibility of pigs to T. solium cysticercosis as observed in mice infected with Taenia crassiceps. Delaying castration of male pigs and confinement of sows during pregnancy might significantly decrease the prevalence of pig-cysticercosis and help curb transmission without much cost or difficulty.

Spatial distribution of Taenia solium porcine cysticercosis within a rural area of Mexico.

Cysticercosis is caused by Taenia solium, a parasitic disease that affects humans and rurally bred pigs in developing countries. The cysticercus may localize in the central nervous system of the human, causing neurocysticercosis, the most severe and frequent form of the disease. There appears to be an association between the prevalence of porcine cysticercosis and domestic pigs that wander freely and have access to human feces. In order to assess whether the risk of cysticercosis infection is clustered or widely dispersed in a limited rural area, a spatial analysis of rural porcine cysticercosis was applied to 13 villages of the Sierra de Huautla in Central Mexico. Clustering of cases in specific households would indicate tapeworm carriers in the vicinity, whereas their dispersal would suggest that the ambulatory habits of both humans and pigs contribute to the spread of cysticercosis. A total of 562 pigs were included in this study (August–December 2003). A global positioning system was employed in order to plot the geographic distribution of both cysticercotic pigs and risk factors for infection within the villages. Prevalence of pig tongue cysticercosis varied significantly in sampled villages (p = 0.003), ranging from 0% to 33.3% and averaging 13.3%. Pigs were clustered in households, but no differences in the clustering of cysticercotic and healthy pigs were found. In contrast, the presence of pigs roaming freely and drinking stagnant water correlated significantly with porcine cysticercosis (p = 0.07), as did the absence of latrines (p = 0.0008). High prevalence of porcine cysticercosis proves that transmission is still quite common in rural Mexico. The lack of significant differentiation in the geographical clustering of healthy and cysticercotic pigs weakens the argument that focal factors (e.g., household location of putative tapeworm carriers) play an important role in increasing the risk of cysticercosis transmission in pigs. Instead, it would appear that other wide-ranging biological, physical, and cultural factors determine the geographic spread of the disease. Extensive geographic dispersal of the risk of cysticercosis makes it imperative that control measures be applied indiscriminately to all pigs and humans living in this endemic area.

Th1 and Th2 indices of the immune response in pigs vaccinated against Taenia solium cysticercosis suggest various host immune strategies against the parasite

Kinetics of the production of serum antibody levels and Th1 (IL-2, IFN-gamma) and Th2 (IL-4, IL-10) cytokines was studied in five pigs vaccinated with a synthetic tri-peptide vaccine (S3Pvac) against Taenia solium, a vaccine that has been shown protects pigs against naturally acquired infection. Healthy pigs of mixed genetic background, similar to those bred in rural villages of Mexico, were vaccinated with S3Pvac or with adjuvant alone, kept in sanitary conditions and bled at different times after vaccination to study the development of their specific immune response. Peripheral blood mononuclear cells (PBMCs) of vaccinated pigs showed a significant increment in the production of Th1 cytokines (IL-2 and IFN-gamma) but not of Th2 cytokines (IL-4 and IL-10) after specific PBLs stimulation with all the individual peptides. A Th1-inclined cytokine profile leading to an exacerbated local inflammation at the early installation stage of the cysticercus may possibly interfere with their successful establishment in the serum antibodies against total cysticercus antigens and against each of the three different peptides comprising S3Pvac were detected 7-51 days after vaccination. Antibodies against GK-1 interfered with the cysticerci development into intestinal tapeworms in prednisolone-treated hamsters. The sub-lethal crippling effect of anti-GK-1 antibodies upon cysticerci indicates to a therapeutic application of S3Pvac in infected pigs having potential epidemiological consequences, as it could aid in decreasing the number of tapeworms expected to develop from the few cysticerci that survive in the vaccinated pigs.

INHIBITORY ROLE OF ANTIBODIES IN THE DEVELOPMENT OF TAENIA SOLIUM AND TAENIA CRASSICEPS TOWARD REPRODUCTIVE AND PATHOGENIC STAGES

Untreated Taenia solium cysticerci obtained from different naturally infected pigs vary notably in their capacity to develop into intestinal tapeworms in prednisolone-treated hamsters, whereas cells derived from Taenia crassiceps cysticerci after 2 mo of infection almost always develop to cysticerci in the peritoneal cavity of susceptible BALB/cAnN mice. Preincubation of whole cysticerci or parasite cells with mice immunoglobulins raised against an 18-mer peptide epitope (GK-1) common to both parasites significantly interferes with both transformations. These crippling effects of antiparasite antibodies suggest new forms of immunological interference with parasite biology other than simple killing. Antibodies that cripple biological functions of the parasite, e.g., their development to reproductive or pathogenic stages, make them important protagonists in taeniasis/cysticercosis disease as classic parasitocidal antibodies. Different serum levels of crippling antibodies in the infected pigs could be responsible for the varied ability of cysticerci to convert to tapeworms. Antigens capable of inducing crippling antibodies, e.g., GK-1, could be useful as a therapeutic vaccine for pigs in order to reduce parasite transmission.

Vaccination against Taenia solium cysticercosis in underfed rustic pigs of México: roles of age, genetic background and antibody response.

Vaccination of pigs of mixed genetic make-up, raised as rustically as done in rural Mexico, resulted in effective protection to experimental challenge against Taenia solium cysticercosis. Maximum protection was achieved if pigs were immunized at 70 days of age. There was large variation of viable parasite load within vaccinated pigs and controls, which is suggestive of significant genetic factors influencing susceptibility, besides immunization. Our results strengthen the advisability of pig vaccination for control of T. solium cysticercosis, since it lowers the number of viable cysticerci capable of transforming into tapeworms.

Recombinant S3Pvac-phage anticysticercosis vaccine: Simultaneous protection against cysticercosis and hydatid disease in rural pigs

This paper provides macroscopic and histological evidence on the statistically significant protective effects of S3Pvac-phage vaccination against porcine cysticercosis and hydatidosis. The study included 391 rustically bred pigs (187 vaccinated and 204 controls). Vaccination significantly reduced the prevalence of cysticercosis by 61.7%. Vaccination also significantly reduced by 56.1% the prevalence of hydatidosis caused by Echinococcus granulosus in pigs. The presence of the vaccine epitopes in both cestodes is probably involved in the cross-protection observed. Increased inflammation was found in 5% of cysticerci recovered from controls, versus 24% from vaccinated pigs (P<0.01). Hydatid cysts were non-inflammatory in either group. Vaccination was effective to prevent one single disease, but it failed to prevent the simultaneous infections with both parasites in a same pig. The widening of the S3Pvac-phage vaccine protective repertoire to include hydatidosis is a convenient feature that should reduce the prevalence of two frequent zoonoses that affect rustic porcine breading with a single action. Thus, the costs of two different vaccination programs would be reduced to a single one with significant reduction in both zoonoses.

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.