猪带绦虫六钩蚴TSO45W基因工程疫苗研究进展

囊虫病是一种危害严重的人兽共患寄生虫病。研制有保护性作用的疫苗将是控制和消灭该病的有效方法,猪带绦虫保护性抗原的研究是猪囊虫病免疫的基础。45W蛋白是猪带绦虫六钩蚴时期的重要抗原,但天然45W抗原来源有限,限制了其应用,而基因工程重组抗原的应用可解决质量控制和抗原来源的问题。本文就近年来国内外对猪带绦虫六钩蚴TSO45W基因工程疫苗的研究进展作一综述。     

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.     

Evaluation of Taenia solium and Taenia crassiceps cysticercal antigens for the serodiagnosis of neurocysticercosis

We evaluated the usefulness of seven cysticercal antigen extracts, four from Taenia solium cysticerci (whole parasite-TsoW, membrane-TsoMe, vesicular fluid-TsoVF and scolex-TsoSc) and three from T. crassiceps cysticerci (whole parasite-TcraW, membrane-TcraMe and vesicular fluid-TcraVF), for serodiagnosis of neurocysticercosis with an enzyme-linked immunosorbent assay (ELISA). Cysticercus-specific IgG were screened in serum samples from 23 patients with neurocysticercosis, 32 patients with other infections and 48 healthy persons. The best results were obtained with the TsoVF-ELISA (91.3% sensitivity; 96.2% specificity) and TcraVF-ELISA (91.3% sensitivity; 95% specificity). The ELISA done with whole parasite and membrane extracts from cysts of T. solium and T. crassiceps and the scolex extract from T. solium cysts showed a low performance in terms of sensitivity, ranging from 47.8% to 73.9%. None of the antigen preparations from T. solium and T. crassiceps cysticerci used in this study showed outstanding performance for the serodiagnosis of neurocysticercosis. However, considering the results obtained with the seven antigen preparations, vesicular fluid from T. solium and T. crassiceps cysticerci may be useful for detecting specific antibodies in sera from patients with neurocysticercosis.     

Helminth antigens (Taenia solium, Taenia crassiceps, Toxocara canis, Schistosoma mansoni and Echinococcus granulosus) and cross-reactivities in human infections and immunized animals

Helminth antigens were investigated in the search for accessible heterologous antigens capable to discriminate different helminthiases, by the enzyme linked immunosorbent assay (ELISA) and the immunoblot assay (IB). Antigens used were: Taenia solium cysticercus total saline (Tso); Taenia crassiceps cysticercus vesicular fluid (Tcra-VF); T. crassiceps cysticercus glycoproteins (Tcra-GP and Tcra-(18-14)-GP); Toxocara canis larva excretory-secretory (TES); Schistosoma mansoni adult total saline (Sm) and Echinococcus granulosus hydatid fluid (Eg). The assayed sera were from patients with: cysticercosis (n = 18); toxocariasis (n = 40); schistosomiasis (n = 19) and hydatidosis (n = 50) with proven clinical and laboratory diagnosis, and sera from rabbits immunized with Tso, Tcra-VF, TES and Eg. Cross-reactivity occurred mostly between infections caused by Taenia and Echinococcus or in immunized rabbits, by ELISA. Moreover, the cross-reactivity among helminthiases was found with the use of antigens belonging to phylogenetically related parasite species, Eg, Tso and Tcra-VF, by sharing same antigenic components. Lower cross-reactivities were obtained by IB technique, when only peptides were considered as antigens, and the use of T. crassiceps purified glycoproteins demonstrated high sensitivity and specificity in the diagnosis of human cysticercosis, similarly to that using homologous antigen (Tso) by the same technique.     

Antibody responses and epitope specificities to the Taenia solium cysticercosis vaccines TSOL18 and TSOL45-1A

Taenia solium is a cestode parasite that causes cysticercosis in humans and pigs. This study examined the antibody responses in pigs immunized with the TSOL18 and TSOL45-1A recombinant vaccines against T. solium cysticercosis. Immunization with these proteins induced specific, complement-fixing antibodies against the recombinant antigens that are believed to be associated with vaccine-induced protection against T. solium infection. Sera from immunized pigs were used to define the linear B-cell epitopes of TSOL18 and TSOL45-1A. Prominent reactivity was revealed to one linear epitope on TSOL18 and two linear epitopes on TSOL45-1A. These, and oncosphere antigens from other taeniid cestodes, contain a protein sequence motif suggesting that they may show a tertiary structure similar to the fibronectin type III domain (FnIII). Comparison of the location of linear antigenic epitopes in TSOL18 and TSOL45-1A within the proposed FnIII structure to those within related cestode vaccine antigens reveals conservation in the positioning of the epitopes between oncosphere antigens from different taeniid species.     

Cysticercosis vaccine: cross protecting immunity with T. solium antigens against experimental murine T. crassiceps cysticercosis

Vaccination of mice with an antigen extract from Taenia solium cysticerci induced protection against challenge with T. crassiceps cysticerci as successfully as did antigen extracts from T. crassiceps. Vaccination was more effective in male than in female mice and in the resistant strain (BALB/B) more so than in the susceptible strain (BALB/c). While only the resistant strain was completely protected by vaccination, the parasite load of the susceptible strain was significantly reduced by vaccination. Cross immunity between the human and murine parasites establishes murine T. crassiceps cysticercosis as a convenient laboratory model in which to test promising T. solium antigens aimed at vaccine development against T. solium cysticercosis. Further, results point to strong interactions of the immune system with sexual and histocompatibility factors in the host's dealing with cysticercosis.     

Vaccination of pigs to control human neurocysticercosis

Taenia solium taeniasis/cysticercosis is a zoonotic disease complex in which the pig is an obligate intermediate host. The infection is widespread, particularly in the developing world, and neurocysticercosis is a major cause of human neurologic disease where the parasite is endemic. Despite easy availability, effective anti-parasitic drugs have not been deployed effectively to control disease transmission. We have investigated a vaccine strategy to prevent parasite infection of the pig intermediate host. Such a strategy would interrupt the parasite's life cycle and eliminate the source of infection for humans. Two recombinant antigens selected from the parasite oncosphere life cycle stage were tested in vaccination trials in pigs that were challenged orally with Taenia solium eggs. Both antigens were highly effective in protecting the pigs against infection with the parasite (98.6% and 99.9% protection, respectively). No viable cysts were found in eight pigs vaccinated with one of the antigens. A recombinant subunit vaccine based on oncosphere antigens has the potential to improve the available control measures for T. solium and thereby reduce or eliminate neurocysticercosis.     

Deciphering western blots of tapeworm antigens (Taenia solium, Echinococcus granulosus, and Taenia crassiceps) reacting with sera from neurocysticercosis and hydatid disease patients

Complex antigen mixtures displayed in Western blots may be immediately and quantitatively categorized with respect to specificity and immunogenicity by immunoplotting. This involves plotting the frequency with which each antigen band reacts with a set of immune sera against the frequency of the same band when reacted with another set of immune sera. Immunoplotting has proven to be a powerful method of analyzing Western blots of reactions between vesicular fluids from the metacestodes of Taenia solium, E. granulosus, and T. crassiceps, and sera from human cases of neurocysticercosis and hydatid disease. Immunoplotting readily sorts out those antigens useful for discriminative immunodiagnosis from the multitude of bands in the sera of sick and healthy people. It aids in assessing the antigenic similarity between the human parasites and the murine parasite T. crassiceps, validating the latter as an alternative source of antigens for immunodiagnosis of cysticercosis and hydatid disease.     

猪带绦虫六钩蚴TSO18基因研究进展

猪囊尾蚴病是一种严重危害人类健康的人兽共患寄生虫病,疫苗是预防控制猪囊尾蚴病的重要手段.TSO18基因是猪带绦虫六钩蚴阶段重要的免疫原基因,被认为是最具有前途的疫苗候选基因.该文综述了猪带绦虫六钩蚴TSO18基因的分子生物学及其疫苗开发的研究进展.     

Excretory/secretory antigens (ES) from in-vitro cultures of Taenia crassiceps cysticerci,and use of an anti-ES monoclonal antibody for antigen detection in samples of cerebrospinal fluid from patients with neurocysticercosis

Antigens were obtained from cysticerci of the ORF strain of Taenia crassiceps, by culture of cysts in protein-free hybridoma medium (PFHM). Budding of new vesicles was observed after 24-48 h. Excretory/secretory (ES) antigens (peptides of <20 kDa) were recovered in the medium after culture for 48 h. SDS-PAGE analysis of vesicular-fluid (VF) antigens (obtained by rupturing T. crassiceps cysticerci in PFHM) and the ES antigens indicated partial homology between the two preparations. ES peptides of 18- and 14-kDa were recognized by polyclonal antibodies produced in rabbits immunized either with the VF antigens or with a total-antigen preparation of T. solium cysticerci. Antibodies present in samples of serum or cerebrospinal fluid (CSF) from patients with neurocysticercosis also reacted with ES peptides. An anti-ES monoclonal antibody detected antigens in the CSF from 10 patients with neurocysticercosis, showing the antigenic homology of the ES antigens with those of T. solium cysticerci in human infections.     

ELISA test for the diagnosis of cysticercosis in pigs using antigens of Taenia solium and Taenia crassiceps cysticerci

In the present study ELISA was standardized for the diagnosis of swine cysticercosis based on necropsy parameters and confirmed positive and negative control sera. Serum samples from pigs with other infections were also assayed to determine possible cross-reactions. Four antigens were assayed: from Taenia crassiceps vesicular fluid (VF-Tcra) and crude larvae extract (T-Tcra), and from Taenia solium extracts of scolex (S-Ts) and of larvae (T-Ts). A checkerboard evaluation of antigen, serum and conjugate dilutions, as well as the use of Tween-20 and skim cow milk in wash and blocking solution had a marked effect on improving ELISA performance. All the antigens showed a good performance, but VF-Tcra was the best, with 96.0% and 80.0% sensitivities for cut-offs respectively at 2sd and 3sd, and corresponding specificities of 97.5% and 100.0%. Cross-reactivity was observed only with hydatidosis and ascaridiosis. In view of the high performance observed, the ELISA test should be recommended for the diagnosis of cysticercosis in suspected swine in slaughterhouses and for the screening of cysticercosis in swine production. These results will support integrated measures of cysticercosis control throughout the chain of swine production, effectively contributing to public health.     

Multiple genotypes of Taenia solium--ramifications for diagnosis,treatment and control

Mitochondrial DNA sequences of Taenia solium have fully been analyzed. Analysis of the full length of cytochrome c oxidase subunit 1 (1620 bp) and cytochrome b (1068 bp) genes of T. solium, isolated from Asia (China, Thailand, Indonesia and India), from Latin America (Mexico, Ecuador, Bolivia, Peru and Brazil) and from Africa (Tanzania, Mozambique and Cameroon), has revealed that the two phylogenies obtained were similar to each other regardless of the genes examined. The isolates from Asia formed a single cluster, whereas those from Latin America combined with those from Africa to form an additional cluster. It was estimated that these two genotypes emerged approximately 4-8 x 10(5) years ago. These results together with recent study of the ancient of human taeniid cestodes emerged several MYA in Africa, historical data on swine domestication, distribution of pigs and colonization patterns suggest that T. solium was introduced recently into Latin America and Africa from different regions of Europe during the colonial age, which started 500 years ago, and that T. solium of another origin independently spread in Asian countries, perhaps from China. Why did not T. solium of European origin invade or spread into Asia during the colonial age? Analysis of T. solium distribution must include other Taenia species, especially T. saginata and T. asiatica, which can not be differentiated from each other morphologically. BESS T-base analysis for differentiation of all human Taenia species including the two genotypes of T. solium, and T. saginata and T. asiatica has also been characterized. BESS T-base analysis differentiates African isolates from Latin American isolates as well but more samples should be analyzed for obtaining conclusive evidence for the latter. Serological analysis of cyst fluid of T. solium cysticerci obtained in China and Indonesia and from Mozambique and Ecuador indicates geographical differences in their banding patterns. These differences are discussed in the light of possible differences in pathology of T. solium worldwide. As it has been speculated that the ancient T. solium emerged several million years ago in Africa, it is necessary to analyze more isolates from Africa. Such working hypothesis may be evaluated combined with symptomatology and serology when we get additional DNA data from such areas, since there are some varieties of manifestation of neurocysticercosis with or without subcutaneous cysticercosis and of antigens of cyst fluid of T. solium from Asia and from Africa and/or America. Transfer of techniques of molecular identification and sero- and immuno-diagnoses between researchers and technicians from endemic countries using their own materials should be promoted with the aim of better international cooperation for the control of cysticercosis.     

Vaccine effect of intact metacestodes of Taenia crassiceps against T. taeniaeformis infection in rats

Wistar rats inoculated intraperitoneally with 10 viable metacestodes of Taenia crassiceps without adjuvant once on day 0 showed strong resistance to challenge with 200 eggs of T. taeniaeformis on day 30. When rats were killed one month after challenge, there were 80.4% and 46.1% reductions in the number of cystic and total metacestodes of T. taeniaeformis in the liver, respectively. When five rats were killed 16 months after challenge, they showed almost complete immunity against the challenge, with 99.4% and 91.1% reductions in the number of cystic and total metacestodes, respectively. There were only a few degenerated, pin-point metacestodes of T. taeniaeformis in the liver of all five rats; one harbored one cystic metacestode as well. However, there were no such reductions in rats injected initially with cyst fluid antigens of T. crassiceps with Freund's complete adjuvant. An additional experiment was carried out using 500 eggs of T. taeniaeformis in order to confirm the vaccine effect against higher egg dose. There were 96.6%, 87.9%, 83.9%, and 79.3% reductions in the number of cystic metacestodes in rats initially inoculated with 10 viable, 10 formalized, and 10 frozen metacestodes, and injected with sodium deoxycholate-solubilized metacestode antigens, respectively. It is strongly suggested that rats singly dosed with 10 viable or non-viable, intact metacestodes of T. crassiceps without adjuvant became highly resistant to challenge infection with eggs of T. taeniaeformis, which resulted in almost no cystic metacestode establishment.     

Epidemiologic study and control of Taenia solium infections with praziquantel in a rural village of Mexico

This study reports the results of an epidemiologic survey for the detection of Taenia solium in a rural village of 559 inhabitants in Sinaloa, Mexico, as well as a large scale treatment of the population with praziquantel. The study was carried out in two stages. In stage 1, serial stool analysis of 392 persons detected a cluster of three T. solium tapeworms. A fourth T. solium tapeworm was detected through a household census, giving a 1.32% prevalence rate for this helminth. Over 70% of the population over five years of age was treated with a 10 mg/kg dose of praziquantel, and no additional tapeworms were found. Environmental studies for the detection of Taenia sp. eggs in soil, water, and and objects from the houses of tapeworm-infected individuals showed only one soil sample containing eggs compatible with Taenia sp. A total of 72 domestic pigs were examined for the presence of cysticerci under the tongue. One animal had cysts, and belonged to a household that had two T. solium tapeworm infections. Stage 2 of the study was carried out one year after large scale antihelminthic treatment (LSAT), and no infections with Taenia sp. eggs were found. No cysticercus-infected pigs were detected. Intestinal parasitosis decreased from 69.2% to 37.5%. It is concluded that LSAT with praziquantel is efficient in decreasing endemic foci of T. solium. Seropositivity to T. solium bladder fluid antigens was tested by enzyme-linked immunosorbent assay and found to be 11% before LSAT and 7% one year later. In family members living with T. solium tapeworm carriers, the number of seropositive individuals was 28%. The relative risk ratio of seropositivity for persons living in the same household with a T. solium tapeworm carrier was 2.95. Positive response was significantly higher in the 30-39-year-old age group, in which 30% were seropositive in stage 1, compared with 7% one year after LSAT. High seropositivity rates were significantly associated with tapeworm clusters as well as with individuals with a clinical history of seizures.     

Taenia crassiceps WFU cysticerci synthesize corticosteroids in vitro: metyrapone regulates the production

Taenia solium and Taenia crassiceps WFU cysticerci and tapeworms have the ability to synthesize sex steroid hormones and have a functional 3β-hydroxisteroid dehydrogenase. Corticosteroids (CS) like corticosterone and dexamethasone have been shown to stimulate in vitro estrogen production by Taenia crassiceps WFU cysticerci. The aim of this work was to study the ability of T. crassiceps WFU cysticerci to synthesize corticosteroids, and the effect of the inhibitor metyrapone on the CS synthesis. For this purpose T. crassiceps WFU cysticerci were obtained from the abdominal cavity of mice, thoroughly washed and pre-incubated in multiwells for 24 h in DMEM plus antibiotics/antimycotics. The tritiated CS precursor progesterone ((3)H-P4) was added to the culture media and parasites cultured for different periods. Blanks containing the culture media plus the (3)H-P4 were simultaneously incubated. Blanks and parasite culture media were ether extracted and analyzed by thin layer chromatography (TLC) in two different solvent systems. Corticosterone production was measured in the culture media by RIA. In some experiments metyrapone (0.1-0.5 mM) was added for 24, 48 or 72 h. Results showed that cysticerci mainly synthesized tritiated 11-deoxy corticosterone (DOC) and small amounts of corticosterone that was also detected by RIA. Small amounts of (3)H-11-deoxy cortisol were also found. Corticosteroid synthesis was time dependent. The addition of metyrapone significantly inhibited tritiated DOC, deoxycortisol and corticosterone synthesis. These results show for the first time that parasites have the capacity to synthesize CS that is modulated by metyrapone. Data suggest that DOC is the main corticosteroid in the parasites.     

猪带绦虫重组BCG-TSOL18疫苗构建及其表达

目的 构建猪带绦虫重组BCG-TSOL18疫苗,研究TSOL18基因在BCG中的表达情况。方法 通过酶切的方法 从重组质粒pGEX-TSOL18获取猪带绦虫TSOL18基因,将其定向克隆到大肠杆菌-分枝杆菌穿梭表达质粒pMV261中,构建猪带绦虫重组质粒pMV261-TSOL18,进行酶切、PCR和测序鉴定;再将其电穿孔转化入BCG,构建猪带绦虫重组BCG-TSOL18疫苗,进行PCR鉴定;SDS-PAGE和Western blot分析TSOL18基因在BCG中的表达情况。结果 通过酶切成功获得了393 bp的TSOL18基因片段;酶切、PCR和测序鉴定证明成功构建了猪带绦虫重组质粒pMV261-TSOL18;PCR鉴定证实猪带绦虫重组质粒pMV261-TSOL18成功转入BCG中,提示猪带绦虫重组BCG-TSOL18疫苗构建成功;SDS-PAGE分析发现在相对分子质量(Mr)约为14.7 kD处有明显的TSOL18目的蛋白条带,Western blot证实表达的TSOL18目的蛋白能被兔抗血清和囊虫病猪血清所识别。结论 成功构建了猪带绦虫重组BCG-TSOL18疫苗,TSOL18基因能够在BCG中成功表达,表达的TSOL18重组蛋白具有特异的抗原性,为该疫苗的进一步研究奠定了基础。     

猪带绦虫囊尾蚴与亚洲带绦虫囊尾蚴蛋白双向电泳图谱分析

将2头20日龄三元杂交乳猪分别感染猪带绦虫虫卵和亚洲带绦虫虫卵,8×104个/头。感染后40 d分别收集寄生在肝脏的未成熟猪带绦虫囊尾蚴(简称猪囊尾蚴)和亚洲带绦虫囊尾蚴,制备囊尾蚴蛋白,并进行蛋白双向电泳分析,用ImageMaster 2D Plantinum 6.0软件分析差异表达蛋白。结果显示,感染后40 d肝脏寄生的未成熟猪囊尾蚴和亚洲带绦虫囊尾蚴蛋白的双向电泳凝胶上分别有(236±12)和(231±14)个蛋白斑点,差异表达2倍以上的蛋白共10个,其中猪囊尾蚴表达上调的蛋白3个,下调的蛋白7个。     

Epidemiological study of Taenia solium taeniasis/cysticercosis in a rural village in Yucatan state, Mexico.

A survey to detect human taeniasis and cysticercosis was conducted in a community in Yucatan state, Mexico, an area endemic for Taenia solium. Information on the environmental, demographic and risk factors associated with transmission of T. solium within the community was recorded on questionnaires. Although no Taenia eggs or proglottides were found in the initial faecal samples collected from each of the 475 subjects, the results of a capture-ELISA for T. solium coproantigen were positive for 10 of the subjects (of both genders and various ages). After treatment with niclosamide, proglottides were detected in purge samples from seven of these 10 subjects. The prevalence of parasitologically confirmed taeniasis was therefore 1.5% (seven in 475). The other three ELISA-positive cases delayed supplying faecal material post-treatment, and it is nuclear whether they had expelled proglottides before providing the samples. All 10 ELISA-positive subjects became ELISA-negative after treatment. Seroprevalence of human cysticercosis, based on the detection in immunoblots of antibodies to antigens of 8- and 26-kDa from a crude saline extract of T. solium metacestodes, was 3.7% (i.e. five positives out of 134 subjects). None of the seropositive cases demonstrated clinical symptoms of infection. Again, the positive cases were of both genders and various ages. Although tongue palpation indicated that 17 (23%) of 75 pigs kept within the community had T. solium cysticercosis, the results of immunoblotting demonstrated antibodies to the 8- and/or 26-kDa antigens of T. solium in 26 (35%). The pigs allowed to roam throughout the community were far more likely to have cysticercosis than those kept in pens (odds ratio = 42, with a 95% confidence interval of 5.05-920.2; P < 0.00001). Not surprisingly, the risk factors associated with human taeniasis and cysticercosis included the eating of infected pork and close proximity to a carrier of T. solium. The main risk factor identified for porcine cysticercosis was free-range husbandry, permitting access to human faeces. This is the first comprehensive report of taeniasis and cysticercosis in a rural population from the Yucatan peninsula of Mexico.     

Development of a serologic assay to detect Taenia solium taeniasis

We developed a serologic assay to identify adult Taenia solium tapeworm carriers using excretory/secretory (TSES) antigens collected from in vitro cultured T. solium tapeworms. To identify taeniasis-specific antigens we used an immunoblot assay with serum samples from T. solium tapeworm carriers and cysticercosis patients. Antigens were identified that reacted with antibodies present in serum samples from taeniasis cases and not with those from cysticercosis patients. Using serum samples collected from persons with confirmed T. solium tapeworm infections, the test was determined to be 95% (69 of 73) sensitive. Serum samples (n = 193) from persons with other parasitic infections, including T. saginata tapeworm infections, do not contain cross-reacting antibodies to TSES, indicating that the assay is 100% specific. These data suggest that the immunoblot assay using TSES antigens can be used to identify persons with current or recent T. solium tapeworm infections and provides a new, important tool for epidemiologic purposes, including control and prevention strategies.     

Human T and B cell epitope mapping of Taenia solium paramyosin

Taenia solium paramyosin is an immunodominant antigen in human and porcine cysticercosis that has shown promise as a vaccine candidate against schistosomiasis and some filariasis. There are few studies to identify the immunologically relevant regions of paramyosin. In this work, we characterize the humoral and cellular response of neurocysticercotic patients against T. solium paramyosin. Western blots using different recombinant fragments of T. solium paramyosin, showed that the sera from neurocysticercotic patients were strongly reactive against the carboxyl end region, with poor recognition of the central and amino regions. In contrast, the cellular immune response of patients did not show preferential recognition of any region of paramyosin.