A purified 28,000 dalton protein from Schistosoma mansoni adult worms protects rats and mice against experimental schistosomiasis

We have purified a 28,000 dalton (P28) protein from Schistosoma mansoni adult worms and used it to immunize Fischer rats. Immunofluorescence assays demonstrated that the P28 antigen was mainly located in the parenchyma of the schistosomulum and of the adult worm, including the dorsal spines of the parasite. Western blot analysis revealed that this antigen was present in three species of schistosomes: S. mansoni, S. japonicum, and S. bovis. The antibody response raised against this protein was able to kill S. mansoni schistosomula in in vitro cytotoxicity assays in the presence of rat eosinophils. The inhibition of this cytotoxic activity by an aggregated myeloma IgG2a indicated that one of the major isotypes involved in this in vitro model is IgG2a. The passive transfer of P28 antisera induced a significant level of protection against experimental infection. Moreover, we have immunized Fischer rats and BALB/c mice with the purified 28,000 dalton protein and observed a marked decrease (up to 70%) in the parasite burden in both experimental infection models.     

Apoptosis governs the elimination of Schistosoma japonicum from the non-permissive host Microtus fortis

The reed vole, Microtus fortis, is the only known mammalian host in which schistosomes of Schistosoma japonicum are unable to mature and cause significant pathogenesis. However, little is known about how Schistosoma japonicum maturation (and, therefore, the development of schistosomiasis) is prevented in M. fortis. In the present study, the ultrastructure of 10 days post infection schistosomula from BALB/c mice and M. fortis were first compared using scanning electron microscopy and transmission electron microscopy. Electron microscopic investigations showed growth retardation and ultrastructural differences in the tegument and sub-tegumental tissues as well as in the parenchymal cells of schistosomula from M. fortis compared with those in BALB/c mice. Then, microarray analysis revealed significant differential expression between the schistosomula from the two rodents, with 3,293 down-regulated (by ≥ 2-fold) and 71 up-regulated (≥ 2 fold) genes in schistosomula from the former. The up-regulated genes included a proliferation-related gene encoding granulin (Grn) and tropomyosin. Genes that were down-regulated in schistosomula from M. fortis included apoptosis-inhibited genes encoding a baculoviral IAP repeat-containing protein (SjIAP) and cytokine-induced apoptosis inhibitor (SjCIAP), genes encoding molecules involved in insulin metabolism, long-chain fatty acid metabolism, signal transduction, the transforming growth factor (TGF) pathway, the Wnt pathway and in development. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) and PI/Annexin V-FITC assays, caspase 3/7 activity analysis, and flow cytometry revealed that the percentages of early apoptotic and late apoptotic and/or necrotic cells, as well as the level of caspase activity, in schistosomula from M. fortis were all significantly higher than in those from BALB/c mice.     

日本血吸虫尾蚴经人工方法转变的童虫体外培养的研究

本文介绍了日本血吸虫体外培养系统。建立了较适于童虫生长发育的B41培养基。尾蚴经人工方法转变的童虫在体外可发育至雌雄合抱,雌雄生殖器官形成。雌虫可达产卵阶段,但未具备正常的产卵机能。培养的血吸虫在体外至少可存活110天。     

Schistosoma japonicum: a method for transformation by electroporation

Despite of our knowledge of genetic make up of schistosomes, a number of genes have not been characterized largely due to lack of effective transformation protocols. Here we present electroporation as a strategy for effective introduction of plasmids DNA into schistosomula and adults. Using plasmids of pEGFP-C1 as an expression vector, we first verified that the CMV promoter could direct EGFP to express in primary culture cells from Schistosoma japonicum. Subsequently, the plasmids were introduced into schistosomula and adults by electroporation and EGFP expression was demonstrated using molecular and microscopical methods. Our findings indicate that electroporation is an effective method for transformation of S. japonicum.     

日本血吸虫Sj Cyclophilin B基因的克隆、表达及免疫保护效果分析

本研究克隆和表达了日本血吸虫Cyclophilin B(Sj CyPB)编码基因的cDNA,分析其在日本血吸虫不同发育阶段虫体的表达情况,评估该重组抗原在小鼠体内诱导的抗血吸虫免疫保护效果。本研究以日本血吸虫童虫cDNA为模板,RT-PCR扩增其基因全长cDNA,提交序列到NCBI,登录号为GQ403665。荧光实时定量PCR分析该基因在日本血吸虫不同发育阶段虫体的表达情况,构建重组表达质粒,表达纯化重组蛋白。利用Western blotting检测重组蛋白的抗原性。以重组抗原免疫小鼠,评估其对小鼠诱导的免疫保护效果。结果表明,RT-PCR获得了Sj CyPB编码基因的全长cDNA,其开放阅读框为672bp。经分析确定其为CyPs家族中的CyPB基因,命名为Sj CyPB。荧光实时定量PCR分析表明,该基因在18d童虫期表达量最高,32d次之。构建了重组表达质粒pGEX-6P-1-SjCyPB,并在大肠杆菌中成功表达,表达产物分子量为49.5kDa。Western blotting试验显示该重组蛋白具有良好的抗原性,在小鼠免疫试验中,与空白对照组比较,免疫组小鼠获得31.5%的减虫率和41.01%的肝脏减卵率。本研究获得了日本血吸虫童虫期高表达的Sj CyPB基因的全长cDNA,成功构建了Sj CyPB原核重组表达质粒,并在大肠杆菌中成功表达,证实该重组抗原在小鼠体内诱导产生了部分免疫保护效果。     

Production and properties of a mouse monoclonal IgE antibody to Schistosoma japonicum

A monoclonal IgE antibody was prepared by fusion of NS-1 myeloma cells with spleen cells of C3H/He mice immunized with an extract of adult worms of Schistosoma japonicum (Sj). The antibody was able to elicit passive cutaneous anaphylaxis in the rat skin against Sj with the highest titer of 1/256,000 in an ascitic form but did not cross-react with any of antigens extracted from S. mansoni, Fasciola hepatica, Paragoniumus westermani, or Trichinella spiralis. Western blot analysis indicated that the monoclonal IgE antibody recognized epitopes on molecules of 82 kDa, 97 kDa, 160 kDa, and 200 kDa, at least some of which were recognized by IgG antibodies of patients with chronic schistosomiasis japonica. The IgE antibody also recognized a 97-kDa antigen expressed on the surface of mechanically transformed schistosomula. Passive transfer of the antibody into mice in an early stage of challenge infection resulted in a partial but significant reduction of recovery of adult worms. However, similar treatment was not effective for the protection if the antibody was given in the postlung stage of the infection. Moreover, eosinophil-mediated damage to schistosomula was observed in vitro in the presence of the monoclonal anti-Sj IgE antibody, whereas the damage was not observed in the presence of another monoclonal IgE antibody with dinitrophenyl specificity.     

Existence of host DNA sequences in schistosomes--horizontal and vertical transmission

The localization of repetitive DNA sequences in the mouse genome such as mouse type 2 Alu sequence (B2) and mouse retrovirus-related sequences was shown in the body of adult Schistosoma japonicum and Schistosoma mansoni by applying an in situ PCR and hybridization technique. Using the same method, mouse major histocompatibility complex (MHC) class I sequence was also found in schistosomes. Furthermore, mouse MHC class I sequence and type A retroviral sequence were detected in S. japonicum and S. mansoni cercarial DNA by blot hybridization. These findings indicated that horizontal and vertical transmission of host DNA sequences occurred in schistosomes. The incorporation and propagation of host sequences in schistosomes and the roles played by such host sequences form the focus of this brief review.     

Schistosoma japonicum: protective immunity induced by schistosomulum-derived cells in a mouse model

We previously reported that immunization with intact live cells from schistosomula of Schistosoma japonicum (S.j) partially protected the Kunming strain of mice from challenge infection. In the present work, 2 immune protective experiments were designed to further validate the protective effect induced by this type of vaccine and to optimize the immunization protocol, including the number of inoculations and parasite stages from which immunogenic cells were derived. Three antigens derived from 18-day-old postinfection live (LLC) and dead (DLC) larval worm cells and from dead 42-day-old postinfection adult worm cells (DAC) were used as immunogens. Our results demonstrate that live cells from 18-day-old worms are capable of inducing significant protection in mice using a murine-Sj challenge model as shown by reduction rates of worm recoveries and egg burdens. The development of adult worms was stunted. A Th1-biased immune response was reflected in the protected groups as evidenced by the ratio of IgG2a/IgG1. A 38-kDa polypeptide was recognized by sera from LLC immunized animals. We demonstrate that live parasite cells are a source of novel protective antigens that can be exploited for vaccine development.     

日本血吸虫SjOST48基因的克隆、表达及其重组蛋白免疫保护效果分析

为了鉴定日本血吸虫SJCHGC01743基因并评估其重组蛋白作为新的血吸虫病候选疫苗抗原的潜力,利用PCR技术扩增日本血吸虫SJCHGC01743基因,应用荧光实时定量PCR分析该基因在日本血吸虫不同发育阶段虫体的转录水平,以p ET-28a(+)为载体构建重组表达质粒并诱导其在大肠杆菌中表达。将纯化的重组蛋白免疫BALB/c小鼠制备免疫血清,利用Western blotting检测重组蛋白的免疫原性,应用间接免疫荧光技术对SJCHGC01743进行蛋白组织定位,利用间接ELISA方法检测小鼠血清中特异性抗体水平。将重组抗原免疫小鼠,评估其免疫保护效果。PCR扩增得到1 248 bp不含信号肽的c DNA序列,同源性分析结果显示,该基因为日本血吸虫寡糖转移酶OST48亚基,命名为Sj OST48。实时定量PCR分析显示Sj OST48在检测的童虫和成虫各个期别虫体中均有转录,其中在28 d虫体中的转录水平最高,在42 d雌虫中的转录量显著高于雄虫。构建的重组表达质粒p ET-28a(+)-Sj OST48成功在大肠杆菌中表达,重组蛋白r Sj OST48分子量约50 k Da。Western blotting分析表明r Sj OST48能被小鼠免疫血清识别,具有良好的免疫原性,间接免疫荧光实验表明Sj OST48蛋白主要分布于虫体体被,少量分布于实质。ELISA检测结果表明r Sj OST48能诱导产生较高的特异性Ig G、Ig G1和Ig G2a抗体。动物免疫保护实验结果表明Sj OST48能诱导小鼠产生32.62%(P0.05)的减虫率及57.61%(P0.01)的肝脏减卵率。本研究为深入探讨日本血吸虫Sj OST48基因的生物学功能及筛选新的血吸虫疫苗候选分子奠定了基础。     

日本血吸虫新抗原基因Sj—Ts4的克隆、表达及免疫保护性

为探讨旋毛虫感染小鼠后抗血吸虫感染的分子机制 ,并为血吸虫病疫苗的研究提供新的抗原分子 ,用旋毛虫感染鼠血清筛选日本血吸虫成虫cDNA文库。经过 3轮筛选 ,获得 9个阳性克隆 ,其中新基因Sj Ts4有一完整的编码框 ,编码2 10个氨基酸。该蛋白质的理论分子量为 2 3kD ,等电点为 7.72。将Sj Ts4亚克隆于原核表达载体 pGEX 5X 3,以GST融合蛋白的形式在E .coliDH5α中表达。Western印迹分析显示 ,重组Sj Ts4蛋白 (rSj Ts4)能被慢性感染鼠血清识别。rSj Ts4或与弗氏完全佐剂混合后免疫小鼠 ,均可以诱导产生特异性的IgG抗体 ,抗体效价可高达 1∶2 5 6 0 0。两免疫组的减虫率分别为31.36 %和 36 .80 % ,与对照组相比都具有统计学意义     

ERK1-/- mice exhibit Th1 cell polarization and increased susceptibility to experimental autoimmune encephalomyelitis

Activation of MAPK ERK1/2 has been shown to play an important role in Th1/Th2 polarization and in regulating cytokine production from APCs. The ERK family consists of two members ERK1 and ERK2, which share approximately 84% identity at the amino acid level and can compensate for each other for most functions. Despite these features, ERK1 and ERK2 do serve different functions, but there is very little information on the contribution of individual forms of ERK on innate and adaptive immune responses. In this study, we describe that ERK1(-/-) mice display a bias toward Th1 type immune response. Consistent with this observation, dendritic cells from ERK1(-/-) mice show enhanced IL-12p70 and reduced IL-10 secretion in response to TLR stimulation. Furthermore, serum from ERK1(-/-) mice had 100-fold higher total IgG2b and 10-fold higher total IgG2a and IgG1 Ab isotype titers, and enhanced levels of Ag-specific IgG2b Ab titers, compared with wild-type mice. Consistent with this enhanced Th1 bias, ERK1(-/-) mice showed enhanced susceptibility to myelin oligodendrocyte glycoprotein (MOG)35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE) and developed EAE earlier, and with increased severity, compared with wild-type mice. Importantly, there was a profound skewing toward Th1 responses in ERK1(-/-) mice, with higher IFN-gamma production and lower IL-5 production in MOG35-55-primed T cells, as well as an augmentation in the MOG-specific IgG2a and IgG2b Th1 Ab isotypes. Finally, increased infiltrating cells and myelin destruction was observed in the spinal cord of ERK1(-/-) mice. Taken together, our data suggest that deficiency of ERK1 biases the immune response toward Th1 resulting in increased susceptibility to EAE.     

<Emphasis Type="Italic">Trichobilharzia regenti</Emphasis>: Antigenic structures of intravertebrate stages

Like several other bird schistosomes, neurotropic schistosome of Trichobilharzia regenti can invade also mammals, including humans. Repeated infections cause cercarial dermatitis, a skin inflammatory reaction leading to parasite elimination in non-specific mammalian hosts. However, in experimentally primo-infected mice, the worms escape from the skin and migrate to the central nervous system. In order to evade host immune reactions, schistosomes undergo cercaria/schistosomulum transformation accompanied with changes of surface antigens. The present study is focused on localization of the main antigens of T. regenti; cercariae, schistosomula developed under different conditions and adults were compared. Antigens were localized by immunofluorescence and ultrastructural immunocytochemistry using sera of mice repeatedly infected with T. regenti. Detected antibody targets were located in glycocalyx and penetration glands of cercariae and in tegument of cercariae, schistosomula and adults. Shedding of cercarial glycocalyx significantly reduced surface reactivity; further decrease was reported during ongoing development of schistosomula. Spherical bodies, probably transported from subtegumental cell bodies to worm surface, were identified as the most reactive tegumental structures. Based on similar results for schistosomula developed in specific, non-specific hosts and in vitro, it seems that the ability of T. regenti to decrease the surface immunoreactivity during ontogenesis is independent on the host type.     

日本血吸虫SjRAD23基因的克隆表达及基因重组抗原的免疫保护效果

辐射敏感蛋白23具有核苷酸切除修复功能,在泛素蛋白酶体途径中起到重要作用。本研究利用PCR技术克隆了日本血吸虫辐射敏感蛋白23(Sj RAD23)编码的c DNA序列,成功获得Sj RAD23的基因序列,其ORF为1 053 bp。构建Sj RAD23基因重组表达质粒p ET28a(+)-Sj RAD23,并在大肠杆菌BL21中成功诱导表达,重组蛋白在上清和沉淀中都有存在。利用免疫组化技术检测该蛋白在虫体的分布情况,该蛋白广泛分布在日本血吸虫虫体被膜。用重组蛋白免疫BALB/c小鼠后,免疫小鼠血清中检测到较高水平的特异性Ig G、Ig G1和Ig G2a。Western blotting分析显示重组蛋白能够被日本血吸虫成虫可溶性抗原免疫小鼠血清所识别。用重组蛋白r Sj RAD23免疫结果与206佐剂对照组比较,r Sj RAD23在BALB/c小鼠中诱导了35.94%减虫率,40.59%肝脏减卵率。结果表明Sj RAD23具有作为疫苗候选分子的潜力。     

Protective monoclonal antibodies from mice vaccinated or chronically infected with Schistosoma mansoni that recognize the same antigens

An IgM monoclonal antibody, designated mAb 1.G1, has been generated from spleen cells of mice immunized with irradiated Schistosoma mansoni cercariae. As determined by indirect immunofluorescence, mAb 1.G1 binds to the surface membrane of schistosomula and to the ciliated plates of miracidia. mAb 1.G1 also binds to the protonephridial systems of live adult worms and denuded, acetone-fixed schistosomula. Western blot analysis shows that the target epitope of this mAb is found on Nonidet P-40-solubilized schistosomular antigens ranging in molecular size from 85 to 130 kDa and ciliated plate antigens of miracidia at 92, 95, and 102 kDa. The recognized epitope in an 8 M urea adult worm extract is found on a 97-kDa molecule. In addition, mAb 1.G1 mediates a high level of complement-dependent cytotoxic activity against schistosomula when used in an in vitro assay. In passive immunization experiments, approximately 40% protection was provided mice when mAb 1.G1 was administered either at the time of challenge or when given 8 days postchallenge. However, when administered 15 days postchallenge, mAb 1.G1 failed to mediate passive protection. The ability of mAb 1.G1 to mediate protection in vivo correlates with its recognition of epitopes on the surfaces of live schistosomula up to 8 days but not at 15 days. Western blot analysis showed that the antigens were contained within Nonidet P-40 extracts of schistosomula during the same time period. Furthermore, a second monoclonal antibody (mAb 4.4B) derived from mice chronically infected with S. mansoni exhibits the identical properties as described for mAb 1.G1.     

Expression of Lex antigen in Schistosoma japonicum and S.haematobium and immune responses to Lex in infected animals: lack of Lex expression in other trematodes and nematodes

Adults of the human parasitic trematode Schistosoma mansoni, which causes hepatosplenic/intestinal complications in humans, synthesize glycoconjugates containing the Lewis x (Lex) Galbeta1-->4(Fucalpha1-- >3)GlcNAcbeta1-->R, but not sialyl Lewis x (sLex), antigen. We now report on our analyses of Lexand sLexexpression in S.haematobium and S.japonicum, which are two other major species of human schistosomes that cause disease, and the possible autoimmunity to these antigens in infected individuals. Antigen expression was evaluated by both ELISA and Western blot analyses of detergent extracts of parasites using monoclonal antibodies. Several high molecular weight glycoproteins in both S. haematobium and S. japonicum contain the Lexantigen, but no sialyl Lexantigen was detected. In addition, sera from humans and rodents infected with S.haematobium and S.japonicum contain antibodies reactive with Lex. These results led us to investigate whether Lexantigens are expressed in other helminths, including the parasitic trematode Fasciola hepatica , the parasitic nematode Dirofilaria immitis (dog heartworm), the ruminant nematode Haemonchus contortus , and the free-living nematode Caenorhabditis elegans . Neither Lexnor sialyl-Lexis detectable in these other helminths. Furthermore, none of the helminths, including schistosomes, express Lea, Leb, Ley, or the H- type 1 antigen. However, several glycoproteins from all helminths analyzed are bound by Lotus tetragonolobus agglutinin , which binds Fucalpha1-->3GlcNAc, and Wisteria floribunda agglutinin, which binds GalNAcbeta1-->4GlcNAc (lacdiNAc or LDN). Thus, schistosomes may be unique among helminths in expressing the Lexantigen, whereas many different helminths may express alpha1,3-fucosylated glycans and the LDN motif.      

Schistosoma mansoni tegument protein Sm29 is able to induce a Th1-type of immune response and protection against parasite infection

Background

Schistosomiasis continues to be a significant public health problem. This disease affects 200 million people worldwide and almost 800 million people are at risk of acquiring the infection. Although vaccine development against this disease has experienced more failures than successes, encouraging results have recently been obtained using membrane-spanning protein antigens from the tegument of Schistosoma mansoni. Our group recently identified Sm29, another antigen that is present at the adult worm tegument surface. In this study, we investigated murine cellular immune responses to recombinant (r) Sm29 and tested this protein as a vaccine candidate.

Methods and Findings

We first show that Sm29 is located on the surface of adult worms and lung-stage schistosomula through confocal microscopy. Next, immunization of mice with rSm29 engendered 51%, 60% and 50% reduction in adult worm burdens, in intestinal eggs and in liver granuloma counts, respectively (p<0.05). Protective immunity in mice was associated with high titers of specific anti-Sm29 IgG1 and IgG2a and elevated production of IFN-γ, TNF-α and IL-12, a typical Th1 response. Gene expression analysis of worms recovered from rSm29 vaccinated mice relative to worms from control mice revealed a significant (q<0.01) down-regulation of 495 genes and up-regulation of only 22 genes. Among down-regulated genes, many of them encode surface antigens and proteins associated with immune signals, suggesting that under immune attack schistosomes reduce the expression of critical surface proteins.

Conclusion

This study demonstrates that Sm29 surface protein is a new vaccine candidate against schistosomiasis and suggests that Sm29 vaccination associated with other protective critical surface antigens is the next logical strategy for improving protection.