Sj 26基因转染树突状细胞抗血吸虫感染作用

目的探讨日本血吸虫Sj26基因转染树突状细胞（DC）抗血吸虫感染保护性免疫作用。方法利用Sj26基因转染、空质粒pcDNA3转染和未处理DC分别免疫BALB／c小鼠3次，攻击感染后计数成虫和虫卵。结果Sj26基因转染DC免疫小鼠诱导34．5％的减虫率和48．5％的减卵率，明显高于空质粒pcDNA3转染DC组（16．8％和15.8％）和未处理DC组（14．6％和14．4％），空质粒pcDNA3转染DC和未处理DC组之间差异无统计学意义。结论Sj26基因转染DC可诱导抗血吸虫感染的保护性免疫作用。     

Vaccination of domestic pig with recombinant paramyosin. against Schistosoma japonicum in China

Paramyosin (PM), a myosin-like protein is a major antigen on Schistosoma japonicum (Sj). We reported that passive transfer of a monoclonal IgE SjE18varepsilon.1 which recognizes PM of Sj (SJPM), partially protected mice from challenge infection. In the present study, we developed an experimental model system of schistosomiasis japonica with domestic pigs in China and used it for the evaluation of vaccination with recombinant SJPM (rSJPM). Sixteen-week-old pigs were successfully infected by dermal penetration of 120 cercariae of a domestic strain of Sj (50-60% worm recovery 11 weeks after challenge). The pigs vaccinated with 400 UV attenuated cercariae showed a reduction of worm recovery (53%, p<0.001). The experimental groups were immunized intradermally with rSJPM and alum or TiterMax and were partially protected against the challenge infection (32-35% reduction).     

日本血吸虫微管蛋白基因的分离和cDNA序列分析

目的 分离和鉴定日本血吸虫(Schistosoma japonicum，Sj)新基因，为防治日本血吸虫病提供药物靶标或候选疫苗。方法构建日本血吸虫成虫cDNA文库，随机挑取重组阳性克隆进行测序，对部分序列进行引物步移法测序，获取其全长cDNA序列；采用生物信息学等技术对该cDNA序列进行开放阅读框(ORF)的寻找、编码氨基酸的推导、核苷酸和氨基酸同源性比较以及蛋白质二级结构的初步预测。结果获得了1个日本血吸虫新基因—微管蛋白基因，全长1439bp，编码443个氨基酸，与肝片形吸虫微管蛋白基因具有79％的同源性。编码蛋白的理论分子量为7．2198KDa，等电点为9．12；抗原表位可能位于cDNA序列373～396处。结论 获得了日本血吸虫微管蛋白基因的全长cDNA序列，为该基因功能的实验性鉴定工作奠定基础。     

Vaccination with recombinant paramyosin in Montanide ISA206 protects against Schistosoma japonicum infection in water buffalo

BackgroundSchistosomiasis japonica is a zoonosis and presents significant public health problems in China and the Philippines. Vaccines targeting domestic animals constitute attractive control measures.MethodsWe conducted three vaccine trials to evaluate the protective efficacy of recombinant full-length paramyosin (rSj97) in water buffalo. Animals were immunized with 3 doses of rSj97 adjuvanted with ISA206 at 250 μg/dose or 500 μg/dose at 4 wk intervals before challenge with 1000 Schistosoma japonicum cercariae. The primary outcome was worm burden assessed by portal perfusion 8–10 weeks post challenge. Safety measures included weight, temperature, body condition score, hemogram and routine assays for hepatic and renal function.ResultsThe three-dose regimen was well tolerated in all three trials. In the first trial, vaccinated buffalo had 51.5% lower worm burden post challenge compared to controls. In the second trial, buffalo immunized with 500 μg/dose of rSj97 had 57.8% lower worm burden compared to controls (p = 0.026). A similar but not significant reduction (60.9%) was observed with animals administered with 250ug rSj97/dose. In the third trial, buffalo immunized with a 500 μg/dose of rSj97 had 57.8% lower worm burden compared to controls (p = 0.014).ConclusionsThese findings indicated that rSj97 is a safe and promising vaccine candidate for schistosomiasis japonica in water buffalo.     

Immunogenicity of plasmid DNA encoding the 62 kDa fragment of Schistosoma japonicum myosin

The recombinant Schistosoma mansoni 62 kDa myosin fragment, rIrV-5, is highly protective in experimental animals, however, vaccination of mice and rats with the recombinant Schistosoma japonicum homologue, rSj62, did not induce significant resistance against S. japonicum infection. To explore alternative ways of presenting this antigen, we further constructed a plasmid (VRSj62) which encodes Sj62 using the VR1020 vector and tested it in vaccination experiments. Four immunisations with 10 microg VRSj62 DNA alone were sufficient to induce high and progressively increasing levels of IgG antibodies against rSj62 with increasing numbers of injections in CBA/Ca mice (IgG titre > or =1:25000), and three injections with 50 microg VRSj62 DNA alone induced significant IgG responses in C57Bl/6 mice (IgG titre, 1:1600). However, vaccination with plasmid DNA entrapped in cationic liposomes or together with pUC19 DNA as a source of CpG motifs, both of which have been reported to enhance immune responses, did not enhance specific antibody production. In spite of the stimulation of specific antibodies against rSj62 with the naked DNA construct no resistance to challenge was demonstrated.     

Protective immunity induced by rPb27 of Paracoccidioides brasiliensis

A cDNA coding for an antigenic protein (rPb27) from the pathogenic fungus Paracoccidioides brasiliensis was cloned and its protective activity was determined against paracoccidioidomycosis (PCM). The cDNA sequence contained an open reading frame (ORF) of 660 base pairs encoding a protein of 219 amino acids with a predicted molecular weight of 25kDa. The deduced amino acid sequence exhibited 100% identity to the 27kDa P. brasiliensis hypothetic protein (access number AA49615). The complete coding cDNA was cloned into a pGEX 4T-2 plasmid and expressed in Escherichia coli as a glutathione-S-transferase-tagged (GST) recombinant protein. Mice immunized with purified rPb27 were able to develop high levels of IgG2b, moderate levels of IgG1 and low levels of IgG2a. At the same time the levels of TGF-beta and IFN-gamma were high while a very low production of IL-10 was verified. Using confocal microscopy with anti-rPb27 mouse serum against P. brasiliensis yeast forms, surface and cytosolic staining pattern were observed. Moreover, immunization of mice with this antigen induced a significant degree of protection in the lungs (93%), liver (93%) and spleen (100%) at 60 days after challenge with infection. Thus, the granulomatous lesions revealed a greater degree of compaction and organization, with few lesions in the lungs and no dissemination of the fungus to other organs. These results showed that a recombinant protein of P. brasiliensis (rPb27) promoted acquired protection against infection with P. brasiliensis yeast forms, suggesting the use of this protein for future development as a prophylactic vaccine for PCM.     

日本血吸虫新基因精氨酸酶的扩增及序列分析

目的获取并真核克隆日本血吸虫新基因精氨酸酶全长cDNA.方法对本实验室曾获得精氨酸酶基因利用生物信息学进行分析,发现其5'端尚缺一段序列;根据已知序列设计引物,用5'端单巢式PCR从尾蚴文库中扩增,以获得精氨酸酶基因完整的阅读框(ORF);用生物信息学技术对获得的精氨酸酶编码基因进行结构与功能的分析;并将新基因的完整编码阅读框克隆入真核表达载体pCDNA3.1.结果用5'端单巢式PCR从尾蚴文库中获得了精氨酸酶5'端所缺序列,得到其完整的ORF,其ORF长1 095 bp,编码364个氨基酸.利用生物信息学技术鉴定其为日本血吸虫精氨酸酶的完整cDNA序列.重组质粒经双酶切DCR及测序鉴定证明日本血吸虫精氨酸酶真核表达质粒构建成功.结论成功获得、识别、扩增并克隆日本血吸虫精氨酸酶编码基因的全长cDNA.     

Protective immunity against Ixodes ricinus induced by a salivary serpin

Iris is a specific elastase inhibitor expressed in the salivary glands of the hard tick Ixodes ricinus. It belongs to the superfamily of serpins and interferes with both haemostasis and the immune response of the host. In this study, we first show that Iris is expressed in nymphs but not in the female midgut nor in males. We also show that Iris is present in the saliva. To examine its potency as anti-tick vaccine candidate, we set up three models of I. ricinus infestation on immunized animals: nymphs on mice, and adults and nymphs on rabbits. We report the rise of neutralizing antibodies following immunization of rabbits and mice. This comes with a significant protective immunity against ticks in rabbits only, resulting in a 30% mortality rate and a diminution of weight gain in both nymphs and adults and a prolongation of blood feeding time in adults. This is the first report on an anti-tick vaccine trial on I. ricinus using a protein able to interact with both host immunity and haemostasis, as a vaccinating antigen.     

Protective immunity induced by an inhaled SARS-CoV-2 subunit vaccine

Targeting the site of infection is a promising strategy for improving vaccine effectivity. To date, licensed COVID-19 vaccines have been administered intramuscularly despite the fact that SARS-CoV-2 is a respiratory virus. Here, we aim to induce local protective mucosal immune responses with an inhaled subunit vaccine candidate, ISR52, based on the SARS-CoV-2 Spike S1 protein. When tested in a lethal challenge hACE2 transgenic SARS-CoV-2 mouse model, intranasal and intratracheal administration of ISR52 provided superior protection against severe infection, compared to the subcutaneous injection of the vaccine. Interestingly for a protein-based vaccine, inhaled ISR52 elicited both CD4 and CD8 T-cell Spike-specific responses that were maintained for at least 6 months in wild-type mice. Induced IgG and IgA responses cross-reacting with several SARS- CoV-2 variants of concern were detected in the lung and in serum and protected animals displayed neutralizing antibodies. Based on our results, we are developing ISR52 as a dry powder formulation for inhalation, that does not require cold-chain distribution or the use of needle administration, for evaluation in a Phase I/II clinical trial.     

Recombinant paramyosin (rec-Sj-97) tested for immunogenicity and vaccine efficacy against Schistosoma japonicum in mice and water buffaloes.

A primary vaccine candidate antigen against schistosomiasis is paramyosin (pmy), a myofibrillar protein found exclusively in invertebrates. Here we report the results of vaccine trials against the Asian schistosome undertaken on inbred and outbred mice and water buffaloes using a bacterially expressed and purified form of Schistosoma japonicum pmy (rec-Sj-97). Vaccination of the mice resulted in high levels of specific anti-pmy IgG antibodies when compared with adjuvant controls and significant reduction in worm burdens and in liver eggs. Furthermore, a significant reduction in liver eggs was recorded in two of the three water buffalo vaccine trials undertaken and, in all three trials, high levels of specific anti-pmy IgG antibodies were generated. There was no evidence of any toxic effects and the vaccine preparations and Quil A adjuvant were clearly well tolerated. The development of a vaccine intended for livestock animals such as bovines would be beneficial in two ways; directly by blocking transmission of schistosomiasis to humans and economically by contributing to healthier livestock. We are encouraged by the consistent efficacy in the mouse and the buffalo vaccine trials that resulted in a significant decrease in liver eggs. Indeed, predictions from mathematical models indicate that an egg reduction effect of 42-45% in buffaloes would be sufficient when combined with human treatment to control schistosomiasis japonica in the marshes and lakes along the middle and upper reaches of the Yangtze River, the most highly endemic areas for the disease in China.     

Protective immunity against avian influenza induced by a fowlpox virus recombinant

Fowlpox virus, the prototypic virus of the genus Avipoxvirus has a natural host range limited to avian species. As such, fowlpox virus provides a suitable candidate for the development of a species-specific recombinant viral vector. This paper reports the development of a fowlpox virus recombinant expressing the haemagglutinin molecule from a highly virulent avian influenza virus. On immunization of chickens and turkeys with the recombinant, protection is afforded against a lethal challenge with either the homologous or a heterologous influenza virus strain.     

Protective immunity against foot-and-mouth disease virus induced by a recombinant vaccinia virus

We report the construction of a recombinant vaccinia virus expressing the precursor for the four structural proteins of FMD virus (FMDV) (P1) strain C3Arg85 using a procedure for isolation of recombinant vaccinia viruses based solely on plaque formation. Adult mice vaccinated with this recombinant vaccinia virus elicited high titers of neutralizing antibodies against both the homologous FMDV and vaccinia virus, measured by neutralization assays. Liquid phase blocking sandwich enzyme-linked immunosorbent assays (ELISAs) using whole virus as antigen showed high total antibody titers against homologous FMDV, similar to those induced by the conventional inactivated vaccine. When ELISAs were carried out with heterologous strains A79 or O1Caseros as antigens, sera from animals vaccinated with the recombinant virus cross-reacted. Mice boosted once with the recombinant vaccinia virus were protected against challenge with infectious homologous virus. These results indicate that recombinant vaccinia viruses are efficient immunogens against FMDV when used as a live vaccine in a mouse model.     

Protective immunity against pasteurellosis in cattle,induced by Pasteurella haemolytica ghosts

Pasteurella haemolytica is a cattle pathogen of significant economic impact. An effective vaccine against bovine pneumonic pasteurellosis is therefore of high importance. Apart from economic concerns, pasteurellosis caused by P. haemolytica is a serious disease leading to death in cattle if it remains untreated. In this study P. haemolytica-ghosts are presented as a promising vaccine candidate in cattle. To obtain sufficient vaccination material a fermentation protocol for P. haemolytica-ghost production was established. With the obtained experimental P. haemolytica-ghost vaccine, cattle immunization studies were performed based on a Pasteurella cattle challenge model developed specifically for vaccine validation. It was shown that protective immunization of cattle against homologous challenge was induced by adjuvanted P. haemolytica-ghosts. The level of protection was similar to a commercially available vaccine.     

Protective immunity spectrum induced by immunization with a vaccine from the TBEV strain Sofjin

Tick-borne encephalitis (TBE) circulates widely in the territory of Eurasia with up to 10,000 cases registered annually. The TBE virus (TBEV) includes three main subtypes: European, Siberian and Far-Eastern, and two new Asiatic variants, phylogenetically distant from the others. The inactivated antigen of European or Far-Eastern strains is used in commercial TBE vaccines. A set of 14 TBEV strains, isolated in 1937–2008, with different passage histories, representing all subtypes and variants, was used in this work. The chosen set covers almost all the TBE area.Sera of mice, immunized with the TBE vaccine Moscow, prepared from the TBEV strain Sofjin, were studied in a plaque neutralization test against the set of TBEV strains. The vaccine induced antibodies at a protective titer against all TBEV strains and Omsk hemorrhagic fever virus (OHFV) with Е protein amino acid distances of 0.008–0.069, but not against Powassan virus.We showed that after a course of two immunizations, factors such as the period between vaccinations (1–4 weeks), the challenging virus dose (30–1000 LD₅₀) and terms of challenge (1–4 weeks after the last immunization) did not significantly affect the assessment of protective efficacy of the vaccine in vivo. The protective effect of the TBE vaccine Moscow against the set of TBEV strains and the OHFV was demonstrated in in vivo experiments. TBE vaccine Moscow did not protect mice against 10 LD₅₀ of the Powassan virus.We showed that this range of Е protein amino acid distances between the vaccine strain and challenging virus do not have a decisive impact on the TBE vaccine protective effect in vitro and in vivo. Moreover, the TBE vaccine Moscow induces an immune response protective against a wide range of TBEV variants.     

日本血吸虫再感染危险因素的探讨

目的 探讨山丘型疫区日本血吸虫再感染的有关危险因素。方法 选取一山丘型疫区,随访观察居民吡喹酮普治后疫水接触及血吸虫再感染状况,对可能影响血吸虫再感染的有关因素进行非条件logistic回归分析。结果 多因素非条件logistic回归分析结果显示再感染与下列因素有关:化疗前每克粪便虫卵数(OR=2.066,95％CI:1.173～3.639),性别(OR=4.260,95％CI:1.275～14.235),4～10月份疫水接触平均指数B(OR=1.138,95％CI:1.045～1.240),性别与4～10月疫水接触平均指数B间的交互作用(OR=0.875,95％CI:0.817-0.982)。结论 性别、化疗前感染度、疫水接触的持续时间及暴露面积与再感染的发生有关,其中性别与疫水接触之间尚存在弱的拮抗作用。     

Cross protection between a laboratory passaged Chinese strain of Schistosoma japonicum and field isolates of S. japonicum from China

Our laboratory strain of Schistosoma japonicum has been isolated for 51 years, but is comparable to the indigenous Chinese parasite in terms of its infectivity to both the intermediate and definitive hosts. Vaccination with our strain protects mice against challenge with wild isolates of S. japonicum from China. Thus any defined antigen vaccine developed using our laboratory strain would be expected to protect against S. japonicum in the field in China.     

The strain specificity of vaccination with ultra violet attenuated cercariae of the Chinese strain of Schistosoma japonicum

Mice vaccinated with ultra violet (u.v.) attenuated cercariae of the Chinese mainland strain of S. japonicum were resistant to homologous challenge, but were not resistant to challenge with the Philippine strain of S. japonicum. Thus vaccination using u.v. attenuated S. japonicum cercariae is strain specific.     

Prevalence of renal involvement in Schistosoma japonicum infection

244 outpatients and 100 hospitalized patients with confirmed Schistosoma japonicum infection were prospectively surveyed for the presence of nephropathy. There was no association between schistosomiasis and renal disease in the outpatient group. Three hospitalized patients had evidence of significant nephropathy, but this number was not significantly higher than in a control group of 100 hospitalized age and sex-matched control patients without schistosomiasis. One schistosomiasis patient with severe nephrotic syndrome underwent percutaneous renal biopsy. Neither S. japonicum antigen nor antibody was found in the biopsy specimen. 64 of the 100 hospitalized patients had portal hypertension; in 28 patients there was hepatic decompensation. Only one of these hepatosplenic patients had evidence of renal disease. Thus renal involvement was uncommon in patients presenting various manifestations of chronic S. japonicum infection, including those with severe hepatosplenic disease. These results contrast markedly with S. mansoni infection, in which nephropathy associated with advanced liver disease is a distinct, well-recognized clinical entity.     

Effect of praziquantel on larval stages of Schistosoma japonicum

The effect of praziquantel on S. japonicum mother sporocysts, daughter sporocysts and cercariae was studied. At concentrations of 3 X 10(-7), 3 X 10(-6) and 3 X 10(-5) M and treatment times of 24 or 48 h, mother and daughter sporocysts and young cercarial embryos were not affected but nearly mature cercariae were killed and dissociated. The resistance of young cercariae could support the suggestion that the primitive cercarial epithelium arises from the sporocyst tegument. Treatment with praziquantel always stopped cercarial emission; this cessation lasted for a few days with the lowest concentration and for up to 25 d with the highest. The duration of treatment slightly affected the pattern of reappearance of cercariae but markedly affected the long-term reduction in numbers. Free cercariae treated with praziquantel lost their tails in 10 to 60 min, depending on the concentration.