Oncospheral Penetration Glands and Secretory Blebs Are the Sources of Taenia ovis Vaccine Antigens

Taenia ovis is a cestode parasite infecting primarily sheep as intermediate hosts and dogs as definitive hosts. The first highly effective, recombinant vaccine against a parasitic organism was developed against T. ovis infection in sheep. Three separate host-protective antigens (To16, To18, and To45W) have been cloned from the oncosphere of the parasite. We localize these antigens in the oncosphere by using quantitative immunogold labeling and transmission electron microscopy. The three antigens were uniquely associated with penetration gland cells. The cytoplasm and secretory granules of both penetration gland type 1 and type 2 cells exhibited statistically significant levels of staining for each of the three antigens. The intensity of labeling of the penetration gland type 1 cell was approximately three to five times greater (P < 0.01) compared to the level of staining intensity seen in the penetration gland type 2 cell. In activated oncospheres, secretory blebs were found to contain granules with a structure similar to those observed in the penetration gland cells. The granules within the secretory blebs were shown to stain specifically for the presence of each of the three host-protective antigens. The absence of surface location of the T. ovis antigens suggests that the parasite may not be susceptible to vaccine-induced antibody- and complement-mediated attack until some postoncospheral development has occurred after infection of the intermediate host.Taenia ovis (phylum Platyhelminthes, class Cestoda, family Taeniidae) is a parasite primarily infecting dogs as definitive hosts and sheep or goats as intermediate hosts. T. ovis is an economically important species, causing cysticercosis in intermediate hosts, leading to economic losses in the sheep meat industry, particularly in Australia and New Zealand (1, 12, 17). T. ovis was the first parasite against which a highly effective recombinant vaccine (To45W) was developed (9). After discovery of the host-protective To45W antigen, two further host-protective recombinant antigens (To16 and To18) were also identified (4). T. ovis has provided a model for the development of vaccines against other taeniid species, and collectively these are the most-effective defined-antigen vaccines against any parasitic infection (15, 16). All of the host-protective antigens for the taeniid cestodes have been isolated from the oncosphere. These vaccines appear to target the parasites either at the time they infect the intermediate host, or shortly thereafter, because vaccinated animals show little, if any, evidence of the presence of parasites following a challenge infection. The principal immune mechanism is believed to be antibody and complement-mediated lysis of the oncosphere or early developing parasite (26).There has been much speculation about the association of the host-protective immune response and the role of oncospheral penetration gland secretions as secretory blebs; however, this has never been confirmed. Miyazato et al. (20) identified secretory blebs arising from oncospheres of Hymenolepis nana in vivo while they were penetrating the intestinal mucosa, indicating that the blebs are not simply in vitro artifacts. Silverman and Hansen (30) proposed that resistance to reinfection in taeniids was most likely to be due to exogenous antigens released during cysticercus development. Rickard and Bell (23-25) showed that some host-protective antigens from oncospheres were excretory/secretory products. Silverman and Maneely (29), Heath (5), and Rajasekariah et al. (21, 22) proposed that the secretions contained in secretory blebs produced from the penetration glands of activated oncospheres may be a source of host-protective antigens. However, there has been little direct evidence to support the hypothesis. Benitez et al. (2) identified an antigen, referred to as HP6, as being associated with the penetration glands of Taenia saginata oncospheres. Rajasekariah et al. (21, 22) reported that host-protective antigens were present in oncospheres prior to their activation and were not simply produced de novo by metabolically active parasites.Recently, three host-protective antigens in T. ovis oncospheres were localized by using immunohistochemical and light microscopic methods (7). These studies revealed that the antigens were present uniquely in two pairs of oncospheral cells, arranged bilaterally. The techniques used in the studies described by Jabbar et al. (7) did not, however, provide details about the type and characteristics of the cells which contained the antigens, or whether the T. ovis host-protective antigens were present in the secretions from activated oncospheres. Alternative methods for examination of the specimens that can provide much greater detail, while still allowing immunological localization of specific antigens, are immunogold labeling and transmission electron microscopy. A recent comprehensive reconstruction of the cellular structure of T. ovis oncosphere from serial sections provides a clear basis for an accurate localization of antigens in T. ovis oncospheres (8). We present quantitative data from immunogold labeling which localizes To16, To18, and To45W antigens in both nonactivated and activated oncospheres and secretory blebs.     

Characterisation of antibody responses in pigs induced by recombinant oncosphere antigens from Taenia solium

Recombinant antigens cloned from the oncosphere life cycle stage of the cestode parasite Taenia solium (T. solium) have been proven to be effective as vaccines for protecting pigs against infections with T. solium. Previous studies have defined three different host protective oncosphere antigens, TSOL18, TSOL16 and TSOL45. In this study, we evaluated the potential for combining the antigens TSOL16 and TSOL18 as a practical vaccine. Firstly, in a laboratory trial, we compared the immunogenicity of the combined antigens (TSOL16/18) versus the immunogenicity of the antigens separately. Secondly, in a field trial, we tested the ability of the TSOL16/18 vaccine to induce detectable antibody responses in animals living under environmental stress and traditionally reared in areas where T. solium cysticercosis is endemic; and finally, we characterised the immune response of the study population. Pigs of 8–16 weeks of age were vaccinated with 200 μg each of TSOL16 and TSOL18, plus 5 mg of Quil-A. Specific total IgG, IgG₁ and IgG₂ antibody responses induced by TSOL16 and TSOL18 were determined with ELISA. The immunogenicity of both antigens was retained in the combined TSOL16/18 vaccine. The combined vaccine TSOL16/18 induced detectable specific anti-TSOL18 antibody responses in 100% (113/113) and specific anti-TSOL16 in 99% (112/113) of the vaccinated animals measured at 2 weeks following the booster vaccination. From the two IgG antibody subtypes analysed we found there was stronger response to IgG₂.     

Peptide nucleic acid delivery to human mitochondria

Peptide nucleic acids (PNAs) are synthetic polynucleobase molecules, which bind to DNA and RNA with high affinity and specificity. Although PNAs have enormous potential as anti-sense agents, the success of PNA-mediated gene therapy will require efficient cellular uptake and sub-cellular trafficking. At present these mechanisms are poorly understood. To address this, we have studied the uptake of biotinylated PNAs into cultured cell lines using fluorescence confocal microscopy. In human myoblasts, initial punctate staining was followed by the release of PNAs into the cytosol and subsequent localisation and concentration in the nucleus. To determine whether PNAs could also be used as therapeutic agents for mtDNA disease, we attempted to localise PNAs to the mitochondrial matrix. When attached to the presequence peptide of the nuclear-encoded human cytochrome c oxidase (COX) subunit VIII, the biotinylated PNA was successfully imported into isolated organelles in vitro. Furthermore, delivery of the biotinylated peptide-PNA to mitochondria in intact cells was confirmed by confocal microscopy. These studies demonstrate that biotinylated PNAs can be directed across cell membranes and to a specific sub-cellular compartment within human cells - highlighting the importance of these novel molecules for human gene therapy.     

Vaccines for prevention of cysticercosis

Neurocysticercosis due to Taenia solium infection is an important cause of human morbidity and mortality. Despite the availability of effective anthelmintics, the disease remains prevalent in many parts of the world and there is a need for new and improved measures for control of the infection. An effective vaccine to prevent infection in pigs, the parasite's natural intermediate host, would be a valuable new option to assist with T. solium control. Several approaches are being used currently towards the development of a T. solium vaccine and these approaches are reviewed briefly, with emphasis on the use of recombinant oncosphere antigens. Highly effective vaccines have been developed against cysticercosis in sheep and cattle caused by Taenia ovis and Taenia saginata, respectively. This success has encouraged the adoption of a similar strategy for T. solium. The recent finding that one oncosphere antigen, TSOL18, can induce complete protection against T. solium infection in pigs, highlights the potential for development of a practical vaccine. A vision is proposed for the development of a safe, effective, inexpensive vaccine for pigs, which can be administered in an edible form. Through an international collaborative effort, research is progressing towards the realisation of such a vaccine and its use to reduce the global burden of neurocysticercosis.     

Differential serodiagnosis for cystic and alveolar echinococcosis using fractions of Echinococcus granulosus cyst fluid (antigen B) and E. multilocularis protoscolex (EM18)

Echinococcus granulosus cyst fluid and E. multilocularis protoscolex extract were fractionated by a single step of preparative isoelectric focusing, resulting in an antigen B-rich fraction (8-kD) and an Em18-rich fraction, respectively. The usefulness of both fractions for differential serodiagnosis of cystic (CE) and alveolar (AE) echinococcosis was evaluated by a large-scale immunoblot analysis on a battery of 354 serum samples. These included 66 from AE patients originating from four different endemic areas, 173 from CE patients originating from seven different endemic areas, 71 from patients with other parasitic diseases, 15 from patients with hepatomas, and 29 from healthy individuals. In an immunoblot with the antigen B-rich fraction, 92% (158 of 173) of the CE sera as well as 79% (52 of 66) of the AE sera reacted with the 8-kD subunit. No cross-reactivity occurred with any sera from patients with cysticercosis, other parasitic diseases, or with hepatomas, or from healthy controls. In an immunoblot with the Em18-rich fraction, all but two sera from AE patients (64 of 66, 97%) recognized Em18, and only nine of 34 CE sera from China reacted with it. All other (139) CE sera from six other countries were negative as were all (115) other non-echinococcosis sera. These findings indicate that antigen B (8-kD) is not species-specific for E. granulosus but is genus-specific for Echinococcus, and that the Em18 antigen is a reliable serologic marker for species-specific differentiation of AE from CE.     

Neurocysticercosis: regional status,epidemiology, impact and control measures in the Americas

The analysis of epidemiological data concerning human cysticercosis point to important advances in understanding the magnitude and distribution of this parasitic disease in Latin America, as well as the relationship of the elements that conform the life cycle of Taenia solium. The data indicate that the main risk factor for acquiring human neurocysticercosis and swine cysticercosis is the presence of the tapeworm carrier in the household. Therefore, several intervention measures for the control of cysticercosis have been evaluated: mass treatment in order to cure tapeworm carriers, health education towards understanding the risk factors, pig control by restraining them, experimental vaccination of pigs and treatment of swine cysticercosis. In this paper, we review the information obtained in these areas. We hope it will be useful in other endemic countries that wish to elaborate an action plan for the control and ultimate eradication of T. solium.     

A novel mitochondrial tRNA phenylalanine mutation presenting with acute rhabdomyolysis

We describe a patient who presented with acute rhabdomyolysis and had 68% cytochrome c oxidase (COX)-deficient fibers in skeletal muscle. Further investigations confirmed a respiratory chain defect that was associated with a novel heteroplasmic point mutation in the phenylalanine tRNA gene of the mitochondrial genome (mtDNA). Analysis of single muscle fibers revealed a significantly greater level of mutant mtDNA in COX-negative fibers. This is the first case of a mitochondrial tRNA gene point mutation presenting with acute rhabdomyolysis and recurrent myoglobinuria.     

DNA Delivery to Mitochondria: Sequence Specificity and Energy Enhancement

Purpose  

Mitochondria are competent for DNA uptake in vitro, a mechanism which may support delivery of therapeutic DNA to complement organelle DNA mutations. We document here key aspects of the DNA import process, so as to further lay the ground for mitochondrial transfection in intact cells.     

Sporadic mitochondrial myopathy due to a new mutation in the mitochondrial tRNASer(UCN) gene

We describe a young woman with a progressive mitochondrial myopathy that started with muscle weakness and went on to include deafness, dementia and ataxia. Skeletal muscle showed the histological and biochemical features of mitochondrial respiratory chain dysfunction. Genetic analysis identified a novel, heteroplasmic, A to G transition in tRNA(Ser(UCN)) at position 7480 affecting a highly conserved base in the anticodon loop. Single-fibre PCR showed highest levels of mutation in cytochrome c-oxidase-deficient fibres and quantification in two biopsies taken 5 years apart showed no change in percentage heteroplasmy. The mutation was present at lower levels in the patient's blood, but was not found in either her mother's or sister's blood and skeletal muscle, suggesting a sporadic occurrence. This is the eighth disease-causing mutation in this tRNA gene and confirms serine (UCN) as one of the most common sites for mtDNA mutation.