Andrea Toledo

Two Epitopes Shared by Taenia crassiceps and Taenia solium Confer Protection against Murine T. crassiceps Cysticercosis along with a Prominent T1 Response

ABSTRACT Taenia crassiceps recombinant antigens KETc1 and KETc12 have been shown to induce high level of protection against experimental murine T. crassiceps cysticercosis, an experimental model successfully used to test candidate antigens for use in vaccination against porcine Taenia solium cysticercosis. Based on the deduced amino acid sequence, KETc1 and KETc12 were chemically synthesized in linear form. Immunization with KETc1 induced 66.7 to 100% protection against murine cysticercosis, and immunization with KETc12 induced 52.7 to 88.1% protection. The elicited immune response indicated that both peptides contain at least one B-cell epitope (as demonstrated by their ability to induce specific antibodies) and one T-cell epitope that strongly stimulated the proliferation of T cells primed with either the free peptide or total cysticercal T. crassiceps antigens. The high percentage of spleen cells expressing inflammatory cytokines points to the likelihood of a T1 response being involved in protection. The protective capacity of the peptides and their presence in all developmental stages of T. solium point to these two epitopes as strong candidates for inclusion in a polyepitopic synthetic vaccine against T. solium pig cysticercosis.

In vitro culture of Taenia crassiceps larval cells and cyst regeneration after injection into mice.

Taenia crassiceps cysticerci were disrupted through trypsinization to isolate cells which can be maintained in culture for up to 15 days. When injected intraperitoneally into susceptible BALB/cAnN mice, complete cysticerci were recovered in a number that is proportional to the quantity of injected cells. Thus, cysticerci contain cells which can reconstitute complete cysts, suggesting that individual cells play a role, independent to budding, during asexual multiplication of T. crassiceps cysticerci in the peritoneal cavity of mice. In contrast, injection of the cells into resistant C57BL/6J mice does not result in the recovery of complete cysts. These findings provide a new experimental model to identify resistance factors in the hosts, for the in vitro screening of anti-cysticerci drugs and for the genetic manipulation of cysticerci through recombinant DNA techniques.

TAENIA CRASSICEPS CYSTICERCOSIS: PROTECTIVE EFFECT AND IMMUNE RESPONSE ELICITED BY DNA IMMUNIZATION

The nucleotide sequence of a protective recombinant antigen of Taenia crassiceps cysticerci present in all stages of Taenia solium (KETc7), cloned into pcDNA3 plasmid with the signal peptide sequence of the β-glycan receptor (pTc-sp7), has been shown to be effective in protecting mice against experimental infection of T. crassiceps. To explore further the possibilities of this form of immunization and the immune response induced, mice were injected intramuscularly (i.m.) or intradermally (i.d.) with 3 doses of pTc-sp7. Similar levels of resistance were found using either i.m. or i.d. immunization. Spleen cells from i.d. and i.m. DNA immunized mice induced a specific T-cell response to T. crassiceps antigens and to a synthetic peptide from the immunogen itself (GK-1). Proliferated cells were especially enriched in CD8+ CD4− T-lymphocytes. A clear increase in the percentage of CD3+ cells that produce γ-interferon and interleukin-2 was detected when measuring the intracellular cytokine production, an indication of the pTc-sp7 capacity to induce an effective cellular response. These results provide encouraging information on the use of KETc7 in the prevention of cysticercosis as well as a first insight into the characterization of the immune response induced by pTc-sp7 that hints to the relevance of cellular immunity in protection.

Further evaluation of the synthetic peptide vaccine S3Pvac against Taenia solium cysticercosis in pigs in an endemic town of Mexico

Taenia solium cysticercosis is a parasitic disease frequently affecting human health and the pig industry in many developing countries. A synthetic peptide vaccine (designated S3Pvac) against porcine cysticercosis has been developed previously as an aid to interrupt transmission and has been shown to be effective. The results of the present study support the effectiveness of the vaccine under endemic field conditions. However, given the time-frame of the vaccination trial, no changes in the local levels of transmission were detectable before and after vaccination using sentinel pigs. Thus, this investigation shows the limited usefulness of single vaccination as the sole means of interrupting Taenia solium transmission in an endemic region.

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.

Prevalence of virulence genes in Escherichia coli strains isolated from piglets in the suckling and weaning period in Mexico

Faecal Escherichia coli isolates from suckling (n = 503) and weaning (n = 450) piglets with and without diarrhoea from 10 farms in Mexico were examined for identification and prevalence of virulence genes. E. coli isolates were tested further for enterotoxin (LT, STa, STb, Stx1, Stx2 and EAST1), fimbrial (F4, F5, F6, F17, F18 and F41) and eae adhesin genes by multiplex PCR. Of the 953 isolates of E. coli examined by multiplex PCR, 650 (68.2 %) isolates were positive for at least one adhesin gene. Among the isolates from diarrhoeic piglets, F41 (72 %) was the most prevalent adhesin followed by eae adhesin (27 %), F6 (12 %), F17 (9 %), F18 (9 %), F5 (8 %) and F4 (3 %). Enterotoxin genes were detected in 424 (44.5 %) of the isolates, of which EAST1 (38 %) and STa (30 %) were the most common, followed by STb (17 %), Stx2 (6 %) and LT (5 %). Twenty-three per cent of isolates from suckling piglets and 43 % of isolates from weaned piglets carried both enterotoxin and adhesin genes, the most common virotypes being F41 : STa, F41 : EAST1, EAST1 : eae, F41 : F6, F41 : STa : STb, F41 : eae and F17 : eae. The present study examined for the first time, to our knowledge, the prevalence of 13 virulence genes among E. coli strains isolated from piglets with and without diarrhoea in Mexico. The results suggested that there are a wide variety of virulence genes associated with diarrhoea in piglets. This study provides baseline information on the significance of specific virotypes associated with suckling and weaning periods in piglets in Mexico.

Effective Protection Against Experimental Taenia solium Tapeworm Infection in Hamsters by Primo-Infection and by Vaccination with Recombinant or Synthetic Heterologous Antigens

The disease caused by Taenia solium is progressively being recognized as a growing global threat for public human health and pig husbandry that requires the development of effective control measures. A central participant in the taeniasis/cysticercosis transmission network is the human carrier of the adult tapeworm because of its great potential in spreading the infection. Herein, evidence is presented that a primary infection of golden hamsters with orally administered T. solium cysticerci improved the hosts resistance against a secondary infection. Likewise, previous vaccination increased the hamsters resistance. Similar high levels of protection (>78%) were induced by systemic or oral vaccination with the S3Pvac anticysticercosis synthetic peptide vaccine or the highly immunogenic recombinant chimera based on the protective peptide KETc1 bound to Brucella spp. lumazine synthase (BLS-KETc1). Increased resistance after primo-infection and vaccination possibly results from changes in the immune conditions prevailing in the hosts intestine. The contribution to protection from the KETc1 and BLS epitopes in a chimeric vaccine is under study. Preventive vaccination of definitive hosts of T. solium against the tapeworm, the most relevant step in the taeniasis/cysticercosis transmission, may greatly impact the dynamics of endemic disease and has not been studied or tried previously.

RENEWED HOPE FOR A VACCINE AGAINST THE INTESTINAL ADULT TAENIA SOLIUM

Review of experimental and observational evidence about various cestode infections of mammalian hosts revives hope for the development of an effective vaccine against adult intestinal tapeworms, the central protagonists in their transmission dynamics. As for Taenia solium, there are abundant immunological data regarding cysticercosis in humans and pigs, but information about human taeniasis is scarce. A single publication reporting protection against T. solium taeniasis by experimental primo infection and by vaccination of an experimental foster host, the immunocompetent female hamster, kindles the hope of a vaccine against the tapeworm to be used in humans, its only natural definitive host.

Intrahepatic DNA Vaccination: Unexpected Increased Resistance Against Murine Cysticercosis Induced by Non-specific Enhanced Immunity

Experimental murine cysticercosis caused by Taenia crassiceps has proved to be a useful model with which to test the efficacy of new vaccine candidates and delivery systems against pig cysticercosis. A high level of protection against murine cysticercosis was previously observed by intramuscular or intradermal DNA immunization with the use of the sequence of the recombinant KETc7 antigen cloned in pcDNA3 (pTc-sp7). To determine the effect of KETc7 differential expression in DNA vaccination, KETc7 was cloned in pGEM 11Zf(+) under the control of the tissue-specific regulatory promoter phosphoenolpyruvate carboxykinase (pPc-sp7). A high level of protection was induced by intrahepatic immunization with pPc-sp7, pTc-sp7 and the empty vector in the absence of any specific immunity. The empty vector pGEM 11Zf(+), the plasmid with the highest content of CpG sequences, provided to the most efficient protection. This protection was related to an increased number of splenocytes, enhanced nonspecific splenocyte proliferation, and intensified intrahepatic INF-γ production. Overall, intrahepatic plasmid CpG-DNA immunization provokes an exacerbated nonspecific immune response that can effectively control Taenia crassiceps cysticercosis.

A lateral flow assay (LFA) for the rapid detection of extraparenchymal neurocysticercosis using cerebrospinal fluid

A lateral flow assay (LFA) for the diagnosis and monitoring of extraparenchymal neurocysticercosis, has been developed. The assay is based on the use of the monoclonal antibody HP10, and when applied to cerebrospinal fluid, correctly identified 34 cases of active extraparenchymal neurocysticercosis, but was negative with 26 samples from treated and cured neurocysticercosis patients and with 20 samples from unrelated neurological diseases. There was complete agreement between the HP10 Ag-ELISA results and the HP10-LFA. The HP10-LFA thus has utility for diagnosis and treatment of extraparenchymal neurocysticercosis, frequently a more dangerous form of the infection.