日本血吸虫23kDa膜蛋白（Sj23）和脂肪酸结合蛋白（Sj14）的双价DNA疫苗免疫保护性研究
|
摘要
日本血吸虫病是一种广泛流行的人兽共患寄生虫性疾病,导致严重的发病率和死亡率。尽管有一些抗血吸虫病的药物和公共卫生控制措施,血吸虫病并没有被控制,局部地区仍在扩散。在大多数病例中化疗能有效控制血吸虫病的病程发展,但流行区病人迅速的再感染需要反复的化疗。这不仅增加治疗成本,而且有引起耐药性的危险。因此,研制安全有效血吸虫病疫苗是长期综合防治血吸虫病的重要措施之一。
自1990年Wolff报道编码外源蛋白的质粒DNA注射到小鼠的骨骼肌,外源蛋白能在骨骼肌中持续稳定的表达以来,抗血吸虫病疫苗进入核酸疫苗的新阶段。核酸疫苗相对于传统的致弱疫苗和亚单位疫苗具有低成本,热稳定性和能诱导广泛的体液和细胞免疫反应等优点。人们陆续制备出许多单价DNA疫苗,尽管可以产生特异性抗体和细胞毒性T淋巴细胞反应,但与致弱尾蚴产生的高保护力相比,单价DNA疫苗的免疫保护效果并不令人满意,至今还没有一商品化的疫苗问世。究其原因血吸虫是多细胞生物,基因组比细菌病毒复杂得多,且在与宿主长期进化的过程中产生了多种免疫逃避机制,所以单一的疫苗难以诱导出足够有效的保护力抵御攻击感染。发展多价疫苗是提高保护力的新策略,它具有多个保护性表位分子,能针对血吸虫不同种株和生活史中不同阶段的特异性抗原产生较好的保护力。
本研究将不同的日本血吸虫抗原分子23kDa膜蛋白(Sj23)和脂肪酸结合蛋白(Sj14)引入到同一真核表达载体pVIVO2-mcs,构建双价共表达核酸疫苗,以期提高免疫保护效果。以BALB/c小鼠为动物模型观察了单价疫苗,共表达、融合表达双价疫苗、鸡尾酒混合双价核酸疫苗和四价疫苗的免疫保护效果,并初步以免疫剂量、免疫途径、免疫次数、末次免疫后攻击感染的时间四个因素探讨了双价疫苗的免疫优化方案。
研究工作分为以下四个部分:
一.日本血吸虫Sj23与Sj14的双价共表达DNA疫苗(pVIVO2-mcs-Sj14/Sj23和pVIVO2-mcs-Sj23/Sj14)的构建和鉴定
以pVIVO2-IL12-Sj23和pVIVO2-IL12-Sj14为模板,根据Sj23、Sj14基因序列各设计2对引物,用PCR技术得到Sj23和Sj14的DNA片段。将这两个片段分别插入到真核表达载体pVIVO2-mcs的一个多克隆位点(BamHI/EcoRI),构建两个单价DNA疫苗pVIVO2-mcs-Sj23和pVIVO2-mcs-Sj14。与此类似,用含有不同酶切位点的另一对引物通过PCR技术得到Sj23和Sj14的DNA片段,经酶切后插入单价疫苗的另一个位点(AvrII/BspHI)上,构建双价DNA疫苗pVIVO2-mcs-Sj14/Sj23和pVIVO2-mcs-Sj23/Sj14,酶切分析鉴定阳性克隆,并进行序列测定。结果:双酶切鉴定重组质粒pVIVO2-mcs-Sj23、pVIVO2-mcs-Sj14、pVIVO2-mcs-Sj23/Sj14和pVIVO2-mcs-Sj14/Sj23,所得小片段的大小分别是440bp和710bp。测序结果显示插入的cDNA片段与公布的Sj23或Sj14片段序列一致,提示质粒构建成功。
华中科技大学生命科学院利用重组PCR技术将日本血吸虫Sj14和Sj23的基因片段用(Gly4Ser) 3进行连接,得到融合基因Sj14·Sj23和Sj23·Sj14,再将该片段重组于pVIVO2-mcs载体多克隆位点(BamHI/EcoRI)中,成功构建两种融合双价DNA疫苗pVIVO2-mcs-Sj23·Sj14,pVIVO2-mcs-Sj14·Sj23。与此类似,用含有不同酶切位点的另一对引物通过PCR技术得到融合基因Sj14·Sj23和Sj23·Sj14,经酶切后插入双价疫苗的另一个位点(AvrII/BspHI)上,成功构建两种四价DNA疫苗pVIVO2-mcs-Sj23·Sj14/Sj14·Sj23,pVIVO2-mcs-Sj14·Sj23/Sj23·Sj14。将pVIVO2-mcs-Sj23/Sj14和pVIVO2-mcs-Sj23·Sj14以等量混合,获得鸡尾酒混合疫苗。
选用的载体pVIVO2-mcs是Invivogen公司新一代的具有两个翻译单位的双基因真核表达载体,由于真核表达载体具有人铁蛋白FerL和FerH复合启动子,可以消除两个转录单位的转录干扰。两个启动子的活性可进一步被SV40和CMV增强子增强。
二.日本血吸虫Sj23与Sj14的双价共表达DNA疫苗(pVIVO2-mcs-Sj14/Sj23)的真核表达
在进行动物免疫接种前,必须确定构建的DNA质粒能够在真核细胞合成表达相应的目的抗原分子。为此,选取双价共表达DNA疫苗pVIVO2-mcs-Sj14/Sj23,观察其在体内外的真核表达。
1.质粒pVIVO2-mcs-Sj14/Sj23的体外瞬时表达
采用脂质体转染技术将质粒pVIVO2-mcs-Sj14/Sj23和pVIVO2-mcs进行体外瞬时转染HEK-293细胞,转染后48小时通过RT-PCR检测Sj14和Sj23 mRNA的表达,以间接免疫荧光法检测相应蛋白的表达。
RT-PCR结果显示pVIVO2-mcs-Sj14/Sj23转染后获得了440bp和710bp两条目的条带,间接免疫荧光染色显示部分转染细胞的胞膜和胞浆中有目的蛋白的表达。表明上述重组质粒能够在哺乳动物细胞中表达。
2.质粒pVIVO2-mcs-Sj14/Sj23在BALB/c小鼠体内骨骼肌细胞的表达及持续时间18只BALB/c小鼠随机分为两组,分别以pVIVO2-mcs-Sj14/Sj23和pVIVO2-mcs质粒100μg/只经股四头肌免疫,免疫后4周、8周、12周、16周、20周、24周分别处死小鼠,取注射侧股四头肌做冰冻切片,间接免疫荧光定位显示免疫小鼠肌细胞的胞膜和胞浆均获得目的蛋白的表达,且表达持续6个月以上。三. DNA疫苗免疫保护力的观察
120只5周龄雄性BALB/c小鼠随机分为12组,每组小鼠分别肌肉免疫下述DNA各100μg : pVIVO2-mcs, pVIVO2-mcs-Sj23, pVIVO2-mcs-Sj14, pVIVO2-mcs-Sj23/Sj14, pVIVO2-mcs–Sj14/Sj23, pVIVO2-mcs-Sj23·Sj14, pVIVO2-mcs–Sj14·Sj23, pVIVO2-mcs-Sj23·Sj14/Sj14·Sj23, pVIVO2-mcs-Sj14·Sj23/Sj23·Sj14, pVIVO2-Sj23/Sj14+香菇多糖(100μg),鸡尾酒式混合疫苗(pVIVO2-mcs-Sj23/Sj14与pVIVO2-mcs-Sj23.Sj14各50μg),对照组各鼠免疫生理盐水100μl。免疫后4周,每只小鼠经腹部皮肤感染40±2条日本血吸虫尾蚴,感染后6周剖杀,收集成虫计算虫荷均数并计算每克肝组织的虫卵数(EPG)。用TY70图像分析系统,测量肝脏切片中单卵肉芽肿直径(μm),以减虫率、肝减卵率和单个虫卵肉芽肿直径减小率评价各组质粒免疫保护力。
实验结果:与空质粒相比,各实验组均有显著性免疫保护力(P<0.05)。双价疫苗组pVIVO2-mcs-Sj14/Sj23, pVIVO2-mcs-Sj23·Sj14, pVIVO2-mcs-Sj14·Sj23,鸡尾酒疫苗混合组和pVIVO2-mcs-Sj23/Sj14+香菇多糖组免疫保护力显著高于单价疫苗组pVIVO2-mcs-Sj23和四价疫苗组pVIVO2-mcs-Sj23·Sj14/Sj14·Sj23,pVIVO2-mcs-Sj14·Sj23/Sj23·Sj14(P<0.05),并且超过50%。pVIVO2-mcs-Sj23/Sj14+多糖佐剂组免疫保护力最佳,获得68.89%的减虫率和84.04%肝减卵率。但四价疫苗组与空质粒对照组肝脏虫卵数无显著性差异,单价疫苗组,双价疫苗组间肝脏虫卵数无显著性差异(P>0.05)。减虫率超过50%的五组双价DNA疫苗组单卵肉芽肿直径的减小率18-39%,其中以融合双价疫苗组pVIVO2-mcs-Sj23·Sj14效果最好。
实验结果表明:双价DNA疫苗免疫保护效果较单价疫苗和四价疫苗好;在共表达、融合表达和鸡尾酒混合三种形式的双价DNA疫苗中,以鸡尾酒混合疫苗免疫保护效果最优,共表达、融合表达的双价DNA疫苗保护力无显著性差异;香菇多糖是DNA疫苗有效的佐剂。本次构建的单价,双价和四价疫苗均可有效的降低虫荷数,但不具备抗生殖能力,五组双价DNA疫苗可一定程度的减小虫卵肉芽肿直径,有一定的抗病理作用。
四.日本血吸虫Sj23与Sj14的双价共表达DNA疫苗(pVIVO2-mcs-Sj14/Sj23)免疫优化方案的研究
为探讨最佳免疫条件,选取双价DNA疫苗pVIVO2-mcs-Sj14/Sj23进行第二次动物试验。应用SPSS12.0软件设计四因素三水平的免疫正交方案,通过统计学方法,以最少的实验组数获得与设置所有实验组相近的结果。四因素三水平分别是:免疫剂量(50μg、100μg、200μg)、免疫次数(1次、2次、3次,每次间隔2周)、免疫途经(肌内注射、背部皮下注射、尾根部皮内注射)、末次免疫后攻击感染的时间(4周、8周、12周)。110只5周龄雄性BALB/c小鼠随机分为11组,包括9个实验组和2个对照组(空质粒组、生理盐水组)。9个实验组用SPSS12.0软件按正交设计的原则,以四因素三水平表进行设计。两个对照组分别肌内注射100μg的pVIVO2-mcs和100μl的生理盐水,免疫后四周,每只小鼠经腹部皮肤感染40±2条日本血吸虫尾蚴,感染后6周剖杀,计算减虫率和每克肝脏减卵率。不同的实验组免疫时间不同,但11组小鼠攻击感染和剖杀时间一致。以减虫率和肝减卵率评价各组质粒免疫保护力。
结果是9个实验组的成虫数和肝虫卵数均显著低于空质粒组和生理盐水对照组(P<0.05)。根据均值的结果分析:在四个因素三个水平的范围内,以免疫剂量50μg,免疫2次,皮内注射,末次免疫后12周攻击感染是较优化的的免疫方案。然而根据SPSS12.0的统计学分析,F< F临界值,即P>0.05,说明四个因素三个水平在统计学上无显著性差异(P>0.05)。
本课题研究初步得出以下结论:
1.成功构建两种单价DNA疫苗pVIVO2-mcs-Sj23,pVIVO2-mcs-Sj14和两种共表达双价DNA疫苗pVIVO2-mcs-Sj23/Sj14和pVIVO2-mcs-Sj14/Sj23。
2.双价DNA疫苗可以在体内外表达,且体内表达持续时间至少在6个月以上。
3.适宜佐剂可增强抗原免疫力和维护免疫的回忆性效应,首次提出香菇多糖是DNA疫苗有效的佐剂,取得很好的结果,值得进一步研究。
4.为提高DNA疫苗保护力,我们首先采用多价多位点的技术路线,以共表达、融合表达和鸡尾酒混合三种形式构建双价DNA疫苗,均可诱导小鼠产生较显著的抗血吸虫攻击感染的免疫保护力(有五组减虫率超过50%),保护作用显著高于单价DNA疫苗,无疑在多价疫苗的设计上提供了新的思路和依据。
5.为提高DNA疫苗保护力,从免疫剂量、免疫次数、免疫途径、末次免疫后攻击感染时间四个因素个三个水平,探索和研究免疫优化方案。在统计学上,四个因素三个水平无显著性差异,但倾向于此范围内:50μg、免疫2次、皮内免疫和免疫12周后攻击感染为本次试验的最佳免疫方案。
Schistosomiasis japanica is a amphixenosis that could result in severe morbidity and mortality. Despite major advances in control with drugs against schistosoma and public health measures, schistosomiasis continues to spread to new geographic areas. In most cases schistosomiasis can be successfully treated by chemotherapy, but rapid reinfection rates for patients in endemic areas require repeated treatment. It will not only increase therapic cost but also provoke the dangers of potential resistance. Thus, safe and efficacious vaccine development is an one of important measures for long-term integrated control of schistosomiasis.
DNA vaccination was introduced in 1990 reported by Wollf. It was demonstrated that foreign protein could be persistent and steady expression upon direct intramuscular injection of plasmid DNA in myocytes. Since then, vaccine against schistosomiasis went into a new stage of nucleic acid vaccine. The advantages of DNA vaccines over the traditional attenuated cercarie or subunit vaccine are the low cost of production, thermal stability and their ability to induce a wide variety of cellular and humoral immune responses. In succession several DNA vaccines encoding single antigens have been studied. The protection levels obtained by univalent DNA vaccines were far from comparable to those achieved using the attenuated vaccines, although specific antibodies and CTL responses were generated. To this day, there is still not yet a commercial DNA vaccine to the wold. These results are perhaps explained by the fact that the schistosome parasite is such an antigenically complex organism. Its genome is more complicated than that of bacteria and virus. Further more many mechanisms of immunized escape generated during long-term evolutional process together with host. Thus, the immune responses induced by single antigen vaccination may not be strong enough to combat the challenging infection. The development of multivalent vaccines is a novel strategy. There are a greater amount of protective epitopes on multivalent vaccines to obtain protection against different parasite strains and stages of parasite life cycle.
In our research, Sj14 and Sj23 were cloned into plasmid pVIVO2 simultaneously to construct bivalent co-expression DNA vaccine to enhance the protection. In the present study, the protective abilities of immunization with univalent vaccine , the co-expression, gene fusion, cocktail bivalent vaccine and quadrivalent vaccine were evaluated in BALB/c mice. It was designed preliminary to explore the optimal conditions from four factors including immunized dosage, inoculated times, inoculated routes and the challenge time after last immunization.
The main contents of this thesis include four parts:
Part I Construction and confirmation of co-expression bivalent DNA vaccine pVIVO2-mcs-Sj14/Sj23 and pVIVO2-mcs-Sj23/Sj14 against schistosoma japonicum encoding Sj23 and Sj14
The complete Sj23 and Sj14 sequences were amplified by PCR from pVIVO2-IL12-Sj23 and pVIVO2-IL12-Sj14 using two pairs primers specific for their genes. The products were cloned into one cloning site (BamHI/EcoRI) of the vector pVIVO2-mcs, generating pVIVO2-mcs-Sj23 and pVIVO2-mcs-Sj14. Similarly, Sj23 and Sj14 were obtained by using different primer pairs with different enzyme cutting sequences. After being digested, they were cloned into the other site (AvrII/BspHI) of pVIVO2-mcs-Sj23 and pVIVO2-mcs-Sj14, enabling the co-expression of two genes (pVIVO2-mcs-Sj23/Sj14 and pVIVO2-mcs-Sj14/Sj23). The two bivalent vaccines were confirmed by restriction analysis and sequencing. Result: After digestion by restriction enzyme several recombinant plasmids of pVIVO2-mcs-Sj23、pVIVO2-mcs-Sj14、pVIVO2-mcs-Sj23/Sj14 and pVIVO2-mcs-Sj14/Sj23,molecular weight of small fragments were nearly 440bp and 710bp each. Complete sequencing of the cDNA inserts confirmed the homology to the published Sj23 and Sj14 sequences. The results indicated that the recombinant plasmids were successfully constructed.
At school of Life Science in Huazhong University of Science and Technology, fusion genes Sj23·Sj14 and Sj14·Sj23, which were connected by (Gly4Ser) 3, were amplified in successive PCR’s instead with the method of splicing by overlap extention (SOE). Fusion genes were cloned into one cloning site (BamHI/EcoRI) of the vector pVIVO2, generating the construction of a fusion gene for expression (pVIVO2-mcs-Sj23·Sj14 and pVIVO2-mcs-Sj14·Sj23). Similarly, fusion genes Sj23·Sj14 and Sj14·Sj23 were obtained by using different primer pairs with different enzyme cutting sequences. After being digested, they were cloned into the other site (AvrII/BspHI) of pVIVO2-mcs-Sj23·Sj14 and pVIVO2-mcs-Sj14·Sj23, enabling the co-expression of two genes (pVIVO2-mcs-Sj23·Sj14/Sj14·Sj23 and pVIVO2-mcs-Sj14·Sj23/Sj23·Sj14).The cocktail vaccine was produced by mixing pVIVO2-mcs- Sj23/Sj14 and pVIVO2-mcs-Sj23·Sj14 at a molar raio of 1:1.
The eukaryotic expression plasmid pVIVO2 (Invivogen, USA), a new generation multigenic vector with two transcription units, was used as a DNA vaccine vector. Since the eukaryotic expression plasmid pVIVO2 (Invivogen, USA) carries the human FerL and FerH composite promoters, the use of both promoters can eliminate the risk of transcriptional interference between the two expression cassettes. The activity of both promoters is further increased by the addition of the SV40 and CMV enhancers.
Part II Eukaryotic expression of bivalent DNA vaccine pVIVO2-mcs-Sj14/Sj23 co-expression Sj 23 and Sj14
It was initially important to determine that the DNA vaccine construct could direct synthesis of the immunogens in eukaryotic cells before immunization. Therefore pVIVO2-mcs-Sj14/Sj23 was selected to test the eukaryotic expression in vitro and in vivo.
1. Transient expression of DNA pVIVO2-mcs-Sj14/Sj23 in vitro
HEK-293 cells were transiently transfected by pVIVO2-mcs-Sj14/Sj23 and pVIVO2-mcs according to the manufacturer’s instructions using LipofectamineTM 2000 Reagent. The expression of Sj23 and Sj14 were determined by testing the presence of mRNA by RT-PCR and the presence of protein using the indirect immunofluorescence assay technique 48 hours after transfection.
The result of RT-PCR showed that products of the expected size (about 440bp and 710bp) were obtained using cDNA derived from cells transfected with pVIVO2–mcs- Sj14/Sj23. The presence of protein detected at the plasma and/or surface of transfected cells was confirmed by immunofluorescence microscopy. These results demonstrated that the plasmid pVIVO2-mcs-Sj14/Sj23 could be expressed in mammalian cells.
2. Expression of plasmid pVIVO2-mcs-Sj14/Sj23 in muscles of BALB/c mice and persistence time.
BALB/c mice (18 total) were divided into two groups. Each group was immunized by intramuscular injection with 100μg pVIVO2-mcs or pVIVO2-mcs-Sj14/Sj23. Mice were sacrificed 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, and 24 weeks separately. The quadriceps muscles of injection site were taken and made into frozen sections. The presence of proteins (Sj23 / Sj14) in the plasma and on the surface of skeletal muscle cells was confirmed by immunofluorescence microscopy. The expression of proteins was persistent at least 6 months.
Part III Protection immunity induced by DNA vaccines
120 BALB/c mice were divided randomly into twelve groups. Each group was immunized by intramuscular injection at a dosage of 100μg with one of the following DNA vaccine: pVIVO2-mcs, pVIVO2-mcs-Sj23, pVIVO2-mcs-Sj14, pVIVO2-mcs-Sj23/Sj14, pVIVO2-mcs-Sj14/Sj23, pVIVO2-mcs-Sj23·Sj14, pVIVO2-mcs-Sj14·Sj23, pVIVO2-mcs-Sj23·14/Sj14·23,pVIVO2-mcs-Sj14·23/Sj23·14, pVIVO2-mcs-Sj23/Sj14 (100μg)+ lentinan (100μg), the cocktail with pVIVO2-mcs-Sj23/Sj14(50μg) and pVIVO2-mcs-Sj23·Sj14(50μg), and the control group with 100μl normal saline. Four weeks after immunization, each mouse was challenged with 40±2 normal S. japonicum cercarie by abdominal skin penetration then perfused 6 weeks later. The number of adult worm recovered. The average of adult worm and eggs per gram (EPG) of liver were calculated. Granuloma of liver tissue was analyzed using a TY70 Imaging Analysis System. The diameters of all egg granulomas were measured. The protection was evaluated with worm reduction rate, egg reduction rate of the liver and granuloma diameter of single egg reduction rate.
Result showed that the worm and egg burden in test groups were significantly lower than control groups (including normal saline and pVIVO2-mcs groups) (P<0.05). The levels of worm reduction of pVIVO2-mcs-Sj14/Sj23, pVIVO2-mcs-Sj23·Sj14, pVIVO2-mcs-Sj14·Sj23, cocktail group and pVIVO2-mcs-Sj23/Sj14+ lentinan group were higher than that of univalent vaccine pVIVO2-mcs-Sj23 and quadrivalent vaccines pVIVO2-mcs-Sj23·Sj14/Sj14·Sj23,pVIVO2-mcs-Sj14·Sj23/Sj23·Sj14 (P<0.05), which were higher than 50%. Worm reduction rate in pVIVO2-mcs-Sj23/Sj14+lentinan group was the highest among the test groups , and the worm/EPG reduction rates were 68.89%/84.04%. Conversely, the egg reduction rates of univalent vaccine and quadrivalent vaccine showed no significant difference when compared with bivalent vaccine (P>0.05). Furthermore, granulomas diameters of the five groups whose worm reduction rates were higher than 50% were measured. Granuloma diameter reduction rates were 18-39%. Among these seven groups (including two control groups), the average diameter of granuloma in pVIVO2-mcs- Sj23·Sj14 group was shown to be the smallest.
Results showed that the protective immunity of bivalent vaccines is better than that of univalent and quadrivalent vaccine. At inducing protection of two genes co-expressed, fusion gene expression and cocktail vaccine, cocktail vaccine was shown to be the best. There appears to be no difference between two genes co-expressed and fusion gene expression in protection. Lentinan is effective adjuvant to increase potence of DNA vaccine. In this trail, univalent, bivalent and quadrivalent vaccine could decrease the worm burden, but had not anti-fecundity. The five groups whose worm reduction rates were higher than 50% could reduce granuloma diameter at some extent, and they had anti-pathology.
Part IV Study on the optimal schedule with co-expression bivalent DNA vaccine pVIVO2-mcs-Sj14/Sj23
In order to explore the optimal immunization conditions, bivalent vaccine pVIVO2-mcs-Sj14/Sj23 was selected to carry out second experiment (shown in Table 1). The orthogonal trial was designed by SPSS12.0 with four factors and three level of each factor. According to statistic approach, it could reduce the number of test groups chosen for incubation and help to obtain similar results with all test groups.
Four factors used were immunized dosage, inoculated times, inoculated routes and the challenge time after last immunization. There were 3 different concentrations of dosage (50μg, 100μg and 200μg) inoculated, respectively. Inoculated times were once, twice, and thrice(interval time was two weeks).The three immunized routes were intramuscular, subcutaneous and intradermal injection. Challenge time was 4, 8 and 12 weeks after the last immunization. 110 BALB/c mice were divided randomly into eleven groups, including two control groups and nine test groups. Nine groups were designed with four factors three levels in orthogonal trial by using SPSS12.0 software. Two control groups were immunized by intramuscular injection with 100μg of pVIVO2 and 100μl of normal saline initially then challenged 4 weeks later. Each mouse was challenged with 40±2 normal S. japonicum cercarie by abdominal skin penetration then perfused 6 weeks later. The worm reduction rate and the egg reduction rate of the liver were calculated. Different groups were immunized at different time points, but all groups were challenged and perfused at the same time. The protection was evaluated with worm and egg reduction rate of the liver.
Results showed that the worm and egg burden in test groups were significantly lower than control groups (including normal saline and pVIVO2 groups) (P<0.05). From the analysing of average value, it was concluded that the optimal schedule was 50μg, two times immunization, intradermal injection and challenged 12 weeks after the last immunization in three levels of four factors. However, the result of orthogonal design was obtained by SPSS12.0, in which F< F critical value, then P>0.05 (data not shown). There was no significant impact on immunity protection in three levels of each factor (P>0.05).
Together, these data demonstrated:
1. Two monovalent DNA vaccines pVIVO2-mcs-Sj23, pVIVO2-mcs-Sj14 and co-expression bivalent DNA vaccines pVIVO2-mcs-Sj14/Sj23, pVIVO2-mcs-Sj23/Sj14 were successfully constructed.
2. The bivalent vaccine such as pVIVO2-mcs-Sj14/Sj23 could be expressed in vitro and vivo. The expression in vivo could be persistent at least 6 months.
3. The eligible adjuvant could increase protective immunity and maintain memory effect. As one effective adjuvant to increase potence of DNA vaccine, lentinan was introduced at first time. It was worthy to reseach one step further.
4. In order to enhance protective immunity, multivalence technique line was applied. The bivalent DNA vaccines were constructed by the methods of co-expressing two genes, a fusion gene expression and the combination of two kinds of plasmids (cocktail vaccine). These bivalent vaccines could induce notable protective immunity against schistosoma. There were five groups whose worm reduction rates were higher than 50%. The level protections of bivalent DNA vaccines were higher than that of univalent vaccine significantly. Undoubtedly, it provided new idea on the contrivance of multivalent vaccine.
5. In order to enhance protective immunity, we explored optimal schedule from four factors of immunized dosage, inoculated times, inoculated routes and the challenge time after last immunization and three levels each. There was no significant difference in the level of protection with four factors in three levels. While optimal schedule trended to be 50μg, two times of immunization, intracutaneous injection and attack at 12th week after last immmunization.
自1990年Wolff报道编码外源蛋白的质粒DNA注射到小鼠的骨骼肌,外源蛋白能在骨骼肌中持续稳定的表达以来,抗血吸虫病疫苗进入核酸疫苗的新阶段。核酸疫苗相对于传统的致弱疫苗和亚单位疫苗具有低成本,热稳定性和能诱导广泛的体液和细胞免疫反应等优点。人们陆续制备出许多单价DNA疫苗,尽管可以产生特异性抗体和细胞毒性T淋巴细胞反应,但与致弱尾蚴产生的高保护力相比,单价DNA疫苗的免疫保护效果并不令人满意,至今还没有一商品化的疫苗问世。究其原因血吸虫是多细胞生物,基因组比细菌病毒复杂得多,且在与宿主长期进化的过程中产生了多种免疫逃避机制,所以单一的疫苗难以诱导出足够有效的保护力抵御攻击感染。发展多价疫苗是提高保护力的新策略,它具有多个保护性表位分子,能针对血吸虫不同种株和生活史中不同阶段的特异性抗原产生较好的保护力。
本研究将不同的日本血吸虫抗原分子23kDa膜蛋白(Sj23)和脂肪酸结合蛋白(Sj14)引入到同一真核表达载体pVIVO2-mcs,构建双价共表达核酸疫苗,以期提高免疫保护效果。以BALB/c小鼠为动物模型观察了单价疫苗,共表达、融合表达双价疫苗、鸡尾酒混合双价核酸疫苗和四价疫苗的免疫保护效果,并初步以免疫剂量、免疫途径、免疫次数、末次免疫后攻击感染的时间四个因素探讨了双价疫苗的免疫优化方案。
研究工作分为以下四个部分:
一.日本血吸虫Sj23与Sj14的双价共表达DNA疫苗(pVIVO2-mcs-Sj14/Sj23和pVIVO2-mcs-Sj23/Sj14)的构建和鉴定
以pVIVO2-IL12-Sj23和pVIVO2-IL12-Sj14为模板,根据Sj23、Sj14基因序列各设计2对引物,用PCR技术得到Sj23和Sj14的DNA片段。将这两个片段分别插入到真核表达载体pVIVO2-mcs的一个多克隆位点(BamHI/EcoRI),构建两个单价DNA疫苗pVIVO2-mcs-Sj23和pVIVO2-mcs-Sj14。与此类似,用含有不同酶切位点的另一对引物通过PCR技术得到Sj23和Sj14的DNA片段,经酶切后插入单价疫苗的另一个位点(AvrII/BspHI)上,构建双价DNA疫苗pVIVO2-mcs-Sj14/Sj23和pVIVO2-mcs-Sj23/Sj14,酶切分析鉴定阳性克隆,并进行序列测定。结果:双酶切鉴定重组质粒pVIVO2-mcs-Sj23、pVIVO2-mcs-Sj14、pVIVO2-mcs-Sj23/Sj14和pVIVO2-mcs-Sj14/Sj23,所得小片段的大小分别是440bp和710bp。测序结果显示插入的cDNA片段与公布的Sj23或Sj14片段序列一致,提示质粒构建成功。
华中科技大学生命科学院利用重组PCR技术将日本血吸虫Sj14和Sj23的基因片段用(Gly4Ser) 3进行连接,得到融合基因Sj14·Sj23和Sj23·Sj14,再将该片段重组于pVIVO2-mcs载体多克隆位点(BamHI/EcoRI)中,成功构建两种融合双价DNA疫苗pVIVO2-mcs-Sj23·Sj14,pVIVO2-mcs-Sj14·Sj23。与此类似,用含有不同酶切位点的另一对引物通过PCR技术得到融合基因Sj14·Sj23和Sj23·Sj14,经酶切后插入双价疫苗的另一个位点(AvrII/BspHI)上,成功构建两种四价DNA疫苗pVIVO2-mcs-Sj23·Sj14/Sj14·Sj23,pVIVO2-mcs-Sj14·Sj23/Sj23·Sj14。将pVIVO2-mcs-Sj23/Sj14和pVIVO2-mcs-Sj23·Sj14以等量混合,获得鸡尾酒混合疫苗。
选用的载体pVIVO2-mcs是Invivogen公司新一代的具有两个翻译单位的双基因真核表达载体,由于真核表达载体具有人铁蛋白FerL和FerH复合启动子,可以消除两个转录单位的转录干扰。两个启动子的活性可进一步被SV40和CMV增强子增强。
二.日本血吸虫Sj23与Sj14的双价共表达DNA疫苗(pVIVO2-mcs-Sj14/Sj23)的真核表达
在进行动物免疫接种前,必须确定构建的DNA质粒能够在真核细胞合成表达相应的目的抗原分子。为此,选取双价共表达DNA疫苗pVIVO2-mcs-Sj14/Sj23,观察其在体内外的真核表达。
1.质粒pVIVO2-mcs-Sj14/Sj23的体外瞬时表达
采用脂质体转染技术将质粒pVIVO2-mcs-Sj14/Sj23和pVIVO2-mcs进行体外瞬时转染HEK-293细胞,转染后48小时通过RT-PCR检测Sj14和Sj23 mRNA的表达,以间接免疫荧光法检测相应蛋白的表达。
RT-PCR结果显示pVIVO2-mcs-Sj14/Sj23转染后获得了440bp和710bp两条目的条带,间接免疫荧光染色显示部分转染细胞的胞膜和胞浆中有目的蛋白的表达。表明上述重组质粒能够在哺乳动物细胞中表达。
2.质粒pVIVO2-mcs-Sj14/Sj23在BALB/c小鼠体内骨骼肌细胞的表达及持续时间18只BALB/c小鼠随机分为两组,分别以pVIVO2-mcs-Sj14/Sj23和pVIVO2-mcs质粒100μg/只经股四头肌免疫,免疫后4周、8周、12周、16周、20周、24周分别处死小鼠,取注射侧股四头肌做冰冻切片,间接免疫荧光定位显示免疫小鼠肌细胞的胞膜和胞浆均获得目的蛋白的表达,且表达持续6个月以上。三. DNA疫苗免疫保护力的观察
120只5周龄雄性BALB/c小鼠随机分为12组,每组小鼠分别肌肉免疫下述DNA各100μg : pVIVO2-mcs, pVIVO2-mcs-Sj23, pVIVO2-mcs-Sj14, pVIVO2-mcs-Sj23/Sj14, pVIVO2-mcs–Sj14/Sj23, pVIVO2-mcs-Sj23·Sj14, pVIVO2-mcs–Sj14·Sj23, pVIVO2-mcs-Sj23·Sj14/Sj14·Sj23, pVIVO2-mcs-Sj14·Sj23/Sj23·Sj14, pVIVO2-Sj23/Sj14+香菇多糖(100μg),鸡尾酒式混合疫苗(pVIVO2-mcs-Sj23/Sj14与pVIVO2-mcs-Sj23.Sj14各50μg),对照组各鼠免疫生理盐水100μl。免疫后4周,每只小鼠经腹部皮肤感染40±2条日本血吸虫尾蚴,感染后6周剖杀,收集成虫计算虫荷均数并计算每克肝组织的虫卵数(EPG)。用TY70图像分析系统,测量肝脏切片中单卵肉芽肿直径(μm),以减虫率、肝减卵率和单个虫卵肉芽肿直径减小率评价各组质粒免疫保护力。
实验结果:与空质粒相比,各实验组均有显著性免疫保护力(P<0.05)。双价疫苗组pVIVO2-mcs-Sj14/Sj23, pVIVO2-mcs-Sj23·Sj14, pVIVO2-mcs-Sj14·Sj23,鸡尾酒疫苗混合组和pVIVO2-mcs-Sj23/Sj14+香菇多糖组免疫保护力显著高于单价疫苗组pVIVO2-mcs-Sj23和四价疫苗组pVIVO2-mcs-Sj23·Sj14/Sj14·Sj23,pVIVO2-mcs-Sj14·Sj23/Sj23·Sj14(P<0.05),并且超过50%。pVIVO2-mcs-Sj23/Sj14+多糖佐剂组免疫保护力最佳,获得68.89%的减虫率和84.04%肝减卵率。但四价疫苗组与空质粒对照组肝脏虫卵数无显著性差异,单价疫苗组,双价疫苗组间肝脏虫卵数无显著性差异(P>0.05)。减虫率超过50%的五组双价DNA疫苗组单卵肉芽肿直径的减小率18-39%,其中以融合双价疫苗组pVIVO2-mcs-Sj23·Sj14效果最好。
实验结果表明:双价DNA疫苗免疫保护效果较单价疫苗和四价疫苗好;在共表达、融合表达和鸡尾酒混合三种形式的双价DNA疫苗中,以鸡尾酒混合疫苗免疫保护效果最优,共表达、融合表达的双价DNA疫苗保护力无显著性差异;香菇多糖是DNA疫苗有效的佐剂。本次构建的单价,双价和四价疫苗均可有效的降低虫荷数,但不具备抗生殖能力,五组双价DNA疫苗可一定程度的减小虫卵肉芽肿直径,有一定的抗病理作用。
四.日本血吸虫Sj23与Sj14的双价共表达DNA疫苗(pVIVO2-mcs-Sj14/Sj23)免疫优化方案的研究
为探讨最佳免疫条件,选取双价DNA疫苗pVIVO2-mcs-Sj14/Sj23进行第二次动物试验。应用SPSS12.0软件设计四因素三水平的免疫正交方案,通过统计学方法,以最少的实验组数获得与设置所有实验组相近的结果。四因素三水平分别是:免疫剂量(50μg、100μg、200μg)、免疫次数(1次、2次、3次,每次间隔2周)、免疫途经(肌内注射、背部皮下注射、尾根部皮内注射)、末次免疫后攻击感染的时间(4周、8周、12周)。110只5周龄雄性BALB/c小鼠随机分为11组,包括9个实验组和2个对照组(空质粒组、生理盐水组)。9个实验组用SPSS12.0软件按正交设计的原则,以四因素三水平表进行设计。两个对照组分别肌内注射100μg的pVIVO2-mcs和100μl的生理盐水,免疫后四周,每只小鼠经腹部皮肤感染40±2条日本血吸虫尾蚴,感染后6周剖杀,计算减虫率和每克肝脏减卵率。不同的实验组免疫时间不同,但11组小鼠攻击感染和剖杀时间一致。以减虫率和肝减卵率评价各组质粒免疫保护力。
结果是9个实验组的成虫数和肝虫卵数均显著低于空质粒组和生理盐水对照组(P<0.05)。根据均值的结果分析:在四个因素三个水平的范围内,以免疫剂量50μg,免疫2次,皮内注射,末次免疫后12周攻击感染是较优化的的免疫方案。然而根据SPSS12.0的统计学分析,F< F临界值,即P>0.05,说明四个因素三个水平在统计学上无显著性差异(P>0.05)。
本课题研究初步得出以下结论:
1.成功构建两种单价DNA疫苗pVIVO2-mcs-Sj23,pVIVO2-mcs-Sj14和两种共表达双价DNA疫苗pVIVO2-mcs-Sj23/Sj14和pVIVO2-mcs-Sj14/Sj23。
2.双价DNA疫苗可以在体内外表达,且体内表达持续时间至少在6个月以上。
3.适宜佐剂可增强抗原免疫力和维护免疫的回忆性效应,首次提出香菇多糖是DNA疫苗有效的佐剂,取得很好的结果,值得进一步研究。
4.为提高DNA疫苗保护力,我们首先采用多价多位点的技术路线,以共表达、融合表达和鸡尾酒混合三种形式构建双价DNA疫苗,均可诱导小鼠产生较显著的抗血吸虫攻击感染的免疫保护力(有五组减虫率超过50%),保护作用显著高于单价DNA疫苗,无疑在多价疫苗的设计上提供了新的思路和依据。
5.为提高DNA疫苗保护力,从免疫剂量、免疫次数、免疫途径、末次免疫后攻击感染时间四个因素个三个水平,探索和研究免疫优化方案。在统计学上,四个因素三个水平无显著性差异,但倾向于此范围内:50μg、免疫2次、皮内免疫和免疫12周后攻击感染为本次试验的最佳免疫方案。
Schistosomiasis japanica is a amphixenosis that could result in severe morbidity and mortality. Despite major advances in control with drugs against schistosoma and public health measures, schistosomiasis continues to spread to new geographic areas. In most cases schistosomiasis can be successfully treated by chemotherapy, but rapid reinfection rates for patients in endemic areas require repeated treatment. It will not only increase therapic cost but also provoke the dangers of potential resistance. Thus, safe and efficacious vaccine development is an one of important measures for long-term integrated control of schistosomiasis.
DNA vaccination was introduced in 1990 reported by Wollf. It was demonstrated that foreign protein could be persistent and steady expression upon direct intramuscular injection of plasmid DNA in myocytes. Since then, vaccine against schistosomiasis went into a new stage of nucleic acid vaccine. The advantages of DNA vaccines over the traditional attenuated cercarie or subunit vaccine are the low cost of production, thermal stability and their ability to induce a wide variety of cellular and humoral immune responses. In succession several DNA vaccines encoding single antigens have been studied. The protection levels obtained by univalent DNA vaccines were far from comparable to those achieved using the attenuated vaccines, although specific antibodies and CTL responses were generated. To this day, there is still not yet a commercial DNA vaccine to the wold. These results are perhaps explained by the fact that the schistosome parasite is such an antigenically complex organism. Its genome is more complicated than that of bacteria and virus. Further more many mechanisms of immunized escape generated during long-term evolutional process together with host. Thus, the immune responses induced by single antigen vaccination may not be strong enough to combat the challenging infection. The development of multivalent vaccines is a novel strategy. There are a greater amount of protective epitopes on multivalent vaccines to obtain protection against different parasite strains and stages of parasite life cycle.
In our research, Sj14 and Sj23 were cloned into plasmid pVIVO2 simultaneously to construct bivalent co-expression DNA vaccine to enhance the protection. In the present study, the protective abilities of immunization with univalent vaccine , the co-expression, gene fusion, cocktail bivalent vaccine and quadrivalent vaccine were evaluated in BALB/c mice. It was designed preliminary to explore the optimal conditions from four factors including immunized dosage, inoculated times, inoculated routes and the challenge time after last immunization.
The main contents of this thesis include four parts:
Part I Construction and confirmation of co-expression bivalent DNA vaccine pVIVO2-mcs-Sj14/Sj23 and pVIVO2-mcs-Sj23/Sj14 against schistosoma japonicum encoding Sj23 and Sj14
The complete Sj23 and Sj14 sequences were amplified by PCR from pVIVO2-IL12-Sj23 and pVIVO2-IL12-Sj14 using two pairs primers specific for their genes. The products were cloned into one cloning site (BamHI/EcoRI) of the vector pVIVO2-mcs, generating pVIVO2-mcs-Sj23 and pVIVO2-mcs-Sj14. Similarly, Sj23 and Sj14 were obtained by using different primer pairs with different enzyme cutting sequences. After being digested, they were cloned into the other site (AvrII/BspHI) of pVIVO2-mcs-Sj23 and pVIVO2-mcs-Sj14, enabling the co-expression of two genes (pVIVO2-mcs-Sj23/Sj14 and pVIVO2-mcs-Sj14/Sj23). The two bivalent vaccines were confirmed by restriction analysis and sequencing. Result: After digestion by restriction enzyme several recombinant plasmids of pVIVO2-mcs-Sj23、pVIVO2-mcs-Sj14、pVIVO2-mcs-Sj23/Sj14 and pVIVO2-mcs-Sj14/Sj23,molecular weight of small fragments were nearly 440bp and 710bp each. Complete sequencing of the cDNA inserts confirmed the homology to the published Sj23 and Sj14 sequences. The results indicated that the recombinant plasmids were successfully constructed.
At school of Life Science in Huazhong University of Science and Technology, fusion genes Sj23·Sj14 and Sj14·Sj23, which were connected by (Gly4Ser) 3, were amplified in successive PCR’s instead with the method of splicing by overlap extention (SOE). Fusion genes were cloned into one cloning site (BamHI/EcoRI) of the vector pVIVO2, generating the construction of a fusion gene for expression (pVIVO2-mcs-Sj23·Sj14 and pVIVO2-mcs-Sj14·Sj23). Similarly, fusion genes Sj23·Sj14 and Sj14·Sj23 were obtained by using different primer pairs with different enzyme cutting sequences. After being digested, they were cloned into the other site (AvrII/BspHI) of pVIVO2-mcs-Sj23·Sj14 and pVIVO2-mcs-Sj14·Sj23, enabling the co-expression of two genes (pVIVO2-mcs-Sj23·Sj14/Sj14·Sj23 and pVIVO2-mcs-Sj14·Sj23/Sj23·Sj14).The cocktail vaccine was produced by mixing pVIVO2-mcs- Sj23/Sj14 and pVIVO2-mcs-Sj23·Sj14 at a molar raio of 1:1.
The eukaryotic expression plasmid pVIVO2 (Invivogen, USA), a new generation multigenic vector with two transcription units, was used as a DNA vaccine vector. Since the eukaryotic expression plasmid pVIVO2 (Invivogen, USA) carries the human FerL and FerH composite promoters, the use of both promoters can eliminate the risk of transcriptional interference between the two expression cassettes. The activity of both promoters is further increased by the addition of the SV40 and CMV enhancers.
Part II Eukaryotic expression of bivalent DNA vaccine pVIVO2-mcs-Sj14/Sj23 co-expression Sj 23 and Sj14
It was initially important to determine that the DNA vaccine construct could direct synthesis of the immunogens in eukaryotic cells before immunization. Therefore pVIVO2-mcs-Sj14/Sj23 was selected to test the eukaryotic expression in vitro and in vivo.
1. Transient expression of DNA pVIVO2-mcs-Sj14/Sj23 in vitro
HEK-293 cells were transiently transfected by pVIVO2-mcs-Sj14/Sj23 and pVIVO2-mcs according to the manufacturer’s instructions using LipofectamineTM 2000 Reagent. The expression of Sj23 and Sj14 were determined by testing the presence of mRNA by RT-PCR and the presence of protein using the indirect immunofluorescence assay technique 48 hours after transfection.
The result of RT-PCR showed that products of the expected size (about 440bp and 710bp) were obtained using cDNA derived from cells transfected with pVIVO2–mcs- Sj14/Sj23. The presence of protein detected at the plasma and/or surface of transfected cells was confirmed by immunofluorescence microscopy. These results demonstrated that the plasmid pVIVO2-mcs-Sj14/Sj23 could be expressed in mammalian cells.
2. Expression of plasmid pVIVO2-mcs-Sj14/Sj23 in muscles of BALB/c mice and persistence time.
BALB/c mice (18 total) were divided into two groups. Each group was immunized by intramuscular injection with 100μg pVIVO2-mcs or pVIVO2-mcs-Sj14/Sj23. Mice were sacrificed 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, and 24 weeks separately. The quadriceps muscles of injection site were taken and made into frozen sections. The presence of proteins (Sj23 / Sj14) in the plasma and on the surface of skeletal muscle cells was confirmed by immunofluorescence microscopy. The expression of proteins was persistent at least 6 months.
Part III Protection immunity induced by DNA vaccines
120 BALB/c mice were divided randomly into twelve groups. Each group was immunized by intramuscular injection at a dosage of 100μg with one of the following DNA vaccine: pVIVO2-mcs, pVIVO2-mcs-Sj23, pVIVO2-mcs-Sj14, pVIVO2-mcs-Sj23/Sj14, pVIVO2-mcs-Sj14/Sj23, pVIVO2-mcs-Sj23·Sj14, pVIVO2-mcs-Sj14·Sj23, pVIVO2-mcs-Sj23·14/Sj14·23,pVIVO2-mcs-Sj14·23/Sj23·14, pVIVO2-mcs-Sj23/Sj14 (100μg)+ lentinan (100μg), the cocktail with pVIVO2-mcs-Sj23/Sj14(50μg) and pVIVO2-mcs-Sj23·Sj14(50μg), and the control group with 100μl normal saline. Four weeks after immunization, each mouse was challenged with 40±2 normal S. japonicum cercarie by abdominal skin penetration then perfused 6 weeks later. The number of adult worm recovered. The average of adult worm and eggs per gram (EPG) of liver were calculated. Granuloma of liver tissue was analyzed using a TY70 Imaging Analysis System. The diameters of all egg granulomas were measured. The protection was evaluated with worm reduction rate, egg reduction rate of the liver and granuloma diameter of single egg reduction rate.
Result showed that the worm and egg burden in test groups were significantly lower than control groups (including normal saline and pVIVO2-mcs groups) (P<0.05). The levels of worm reduction of pVIVO2-mcs-Sj14/Sj23, pVIVO2-mcs-Sj23·Sj14, pVIVO2-mcs-Sj14·Sj23, cocktail group and pVIVO2-mcs-Sj23/Sj14+ lentinan group were higher than that of univalent vaccine pVIVO2-mcs-Sj23 and quadrivalent vaccines pVIVO2-mcs-Sj23·Sj14/Sj14·Sj23,pVIVO2-mcs-Sj14·Sj23/Sj23·Sj14 (P<0.05), which were higher than 50%. Worm reduction rate in pVIVO2-mcs-Sj23/Sj14+lentinan group was the highest among the test groups , and the worm/EPG reduction rates were 68.89%/84.04%. Conversely, the egg reduction rates of univalent vaccine and quadrivalent vaccine showed no significant difference when compared with bivalent vaccine (P>0.05). Furthermore, granulomas diameters of the five groups whose worm reduction rates were higher than 50% were measured. Granuloma diameter reduction rates were 18-39%. Among these seven groups (including two control groups), the average diameter of granuloma in pVIVO2-mcs- Sj23·Sj14 group was shown to be the smallest.
Results showed that the protective immunity of bivalent vaccines is better than that of univalent and quadrivalent vaccine. At inducing protection of two genes co-expressed, fusion gene expression and cocktail vaccine, cocktail vaccine was shown to be the best. There appears to be no difference between two genes co-expressed and fusion gene expression in protection. Lentinan is effective adjuvant to increase potence of DNA vaccine. In this trail, univalent, bivalent and quadrivalent vaccine could decrease the worm burden, but had not anti-fecundity. The five groups whose worm reduction rates were higher than 50% could reduce granuloma diameter at some extent, and they had anti-pathology.
Part IV Study on the optimal schedule with co-expression bivalent DNA vaccine pVIVO2-mcs-Sj14/Sj23
In order to explore the optimal immunization conditions, bivalent vaccine pVIVO2-mcs-Sj14/Sj23 was selected to carry out second experiment (shown in Table 1). The orthogonal trial was designed by SPSS12.0 with four factors and three level of each factor. According to statistic approach, it could reduce the number of test groups chosen for incubation and help to obtain similar results with all test groups.
Four factors used were immunized dosage, inoculated times, inoculated routes and the challenge time after last immunization. There were 3 different concentrations of dosage (50μg, 100μg and 200μg) inoculated, respectively. Inoculated times were once, twice, and thrice(interval time was two weeks).The three immunized routes were intramuscular, subcutaneous and intradermal injection. Challenge time was 4, 8 and 12 weeks after the last immunization. 110 BALB/c mice were divided randomly into eleven groups, including two control groups and nine test groups. Nine groups were designed with four factors three levels in orthogonal trial by using SPSS12.0 software. Two control groups were immunized by intramuscular injection with 100μg of pVIVO2 and 100μl of normal saline initially then challenged 4 weeks later. Each mouse was challenged with 40±2 normal S. japonicum cercarie by abdominal skin penetration then perfused 6 weeks later. The worm reduction rate and the egg reduction rate of the liver were calculated. Different groups were immunized at different time points, but all groups were challenged and perfused at the same time. The protection was evaluated with worm and egg reduction rate of the liver.
Results showed that the worm and egg burden in test groups were significantly lower than control groups (including normal saline and pVIVO2 groups) (P<0.05). From the analysing of average value, it was concluded that the optimal schedule was 50μg, two times immunization, intradermal injection and challenged 12 weeks after the last immunization in three levels of four factors. However, the result of orthogonal design was obtained by SPSS12.0, in which F< F critical value, then P>0.05 (data not shown). There was no significant impact on immunity protection in three levels of each factor (P>0.05).
Together, these data demonstrated:
1. Two monovalent DNA vaccines pVIVO2-mcs-Sj23, pVIVO2-mcs-Sj14 and co-expression bivalent DNA vaccines pVIVO2-mcs-Sj14/Sj23, pVIVO2-mcs-Sj23/Sj14 were successfully constructed.
2. The bivalent vaccine such as pVIVO2-mcs-Sj14/Sj23 could be expressed in vitro and vivo. The expression in vivo could be persistent at least 6 months.
3. The eligible adjuvant could increase protective immunity and maintain memory effect. As one effective adjuvant to increase potence of DNA vaccine, lentinan was introduced at first time. It was worthy to reseach one step further.
4. In order to enhance protective immunity, multivalence technique line was applied. The bivalent DNA vaccines were constructed by the methods of co-expressing two genes, a fusion gene expression and the combination of two kinds of plasmids (cocktail vaccine). These bivalent vaccines could induce notable protective immunity against schistosoma. There were five groups whose worm reduction rates were higher than 50%. The level protections of bivalent DNA vaccines were higher than that of univalent vaccine significantly. Undoubtedly, it provided new idea on the contrivance of multivalent vaccine.
5. In order to enhance protective immunity, we explored optimal schedule from four factors of immunized dosage, inoculated times, inoculated routes and the challenge time after last immunization and three levels each. There was no significant difference in the level of protection with four factors in three levels. While optimal schedule trended to be 50μg, two times of immunization, intracutaneous injection and attack at 12th week after last immmunization.
引文
1.Chitsulo L, Engels D, Montresor A, et al. The global status of schistosomiasis and its control.Acta Trop.2000,77(1):41-51.
2.林金祥,李莉莎.血吸虫病防治的回顾与现状.海峡预防医学杂志.2005,11(1):22-23.
3.郑江.我国血吸虫病防治应坚持以社会措施为主导的策略.国外医学·流行病学传染病学分册2005,32(1):4-7.
4.Fallon PG, Sturrock RF, Niang AC, et al. Short report: diminished susceptibility to praziquantel in a Senegal isolate of schistosoma mansoni. Am J Trop Med Hyg.1995,53(1):61-62.
5.石佑恩.血吸虫病疫苗的研究现状与展望.医药导报.2005, 24(4):265-266.
6.McManus DP, Bartley PB. A vaccine against Asian schistosomiasis. Parasitol Int. 2004 ,53(2):163-73.
7.Shi YE, Jiang CF, Han JJ, et al.Immunization of pigs against infection with schistosoma japonicum using ultraviolet-attenuated cercariae. Parasitology. 1993,106(Pt5):459-462.
8.李海山,刘勇,邵一鸣. DNA疫苗及其优化策略.国外医学病毒学分.2003,10(3):74-78.
9.Cherfas J. New hope for vaccine against schistosomiasis. Science. 1991,251(4994): 630-631.
10.Bergquist NR. Schistosomiasis vaccine development: progress and prospects. Mem Inst Oswaldo Cruz.1998,93(suppl 1):95-101.
11.Rainczuk A, Scorza T, Spithill TW, et al. A bicistronic DNA vaccine containing apical membrane antigen 1 and merozoite surface protein 4/5 can prime humoral and cellular immune responses and partially protect mice against virulent Plasmodium chabaudi adami DS malaria. Infect Immun. 2004,72(10): 5565-5573.
12.李柳哲,石佑恩,江侃,等.日本血吸虫多价DNA疫苗pBK-CMV-sj26(sj32)-sj23免疫效果的观察[J].同济医科大学学报.2001,30(5): 404-406.
13.Leachman SA, Shylankevich M, Slade MD. et al. Ubiquitin-fused and/or multiple early genes from cottontail rabbit papillomavirus as DNA vaccines. J Virol.2002,76(15): 7616-7624.
14.吴观陵.人体寄生虫学.第三版.人民卫生出版社. 2005,359.
15.Rogers MV, Davern KM, Smythe JA, et al. Immunoblotting analysis of the major integral membrane protein antigens of Schistosoma japonicum. Mol Biochem Parasitol.1988,29(1): 77-87.
16.Zhu Y, Ren J, Harn DA, et al. Protective immunity induced with 23 kDa membrane protein dna vaccine of Schistosoma japonicum Chinese strain in infected C57BL/6 mice. Southeast Asian J Trop Med Public Health.2003,34(4):697-701.
17.Shi F, Zhang Y, Ye P, et al. Laboratory and field evaluation of Schistosoma japonicum DNA vaccines in sheep and water buffalo in China. Vaccine. 2001,20(3-4): 462-467.
18.Zhu Y, Ren J, Da'dara A,et al. The protective effect of a Schistosoma japonicum Chinese strain 23 kDa plasmid DNA vaccine in pigs is enhanced with IL-12. Vaccine.2004, 23(1): 78-83.
19.Gobert GN, Stenzel DJ, Jones MK, et al. Immunolocalization of the fatty acid-binding protein Sj-FABPc within adult Schistosoma japonicum. Parasitology. 1997,115 (Pt1): 33-39.
20.谢来平,石佑恩.血吸虫脂肪酸结合蛋白的研究现状.国外医学寄生虫病分册. 1998,25(5): 193-196.
21.赵巍,吴海玮,苏川,等.日本血吸虫中国大陆株重组脂肪酸结合蛋白(Sj-FABPc)的特性和抗原表位的分析.中国人兽共患病杂志. 2000,16(6): 6-8.
22.余传信,朱荫昌,殷旭仁,等.日本血吸虫脂肪酸结合蛋白(SjC FABP)核酸疫苗诱导小鼠免疫保护作用研究.中国血吸虫病防治杂志.2002,14(3): 163-167.
23.朱晓华,石佑恩,胡萍,等.日本血吸虫脂肪酸结合蛋白DNA疫苗诱导小鼠保护性免疫力的研究.中国血吸虫病防治杂志. 2005, 17(1): 4-8.
24.董菁,成军,王勤环,等.乙型肝炎病毒囊膜中蛋白与白细胞介素18联合基因免疫.中华传染病杂志.2002,20(3): 148-151.
25.Singh M, Briones M, Ott G, et al. Cationic microparticles: a potentdelivery system for DNA vaccines. Proc Natl Acad Sci USA. 2000,97(2): 811-816.
26.Ertl HC, Xiang Z. Novel vaccine approaches. J Immunol.1996, 156 (10): 3579-3582.
27.许雪梅,宋国兴,司静懿. DNA疫苗机制研究进展.微生物学免疫学进展.2002,30(1): 65-70.
28.Forg P, von Hoegen P, Dalemans W, et al. Superiority of the ear pinna over muscle tissue as site for DNA vaccination. Gene Ther. 1998,5(6): 789–797.
29.谢来平,石佑恩.日本血吸虫DNA疫苗pCDSj32不同免疫次数及末次免疫后不同攻击时间对小鼠保护性免疫力的观察.中国人兽共患病杂志.2000,16(4):6-8。
30.Harn D, Chikunguwo S, Skelly P, et al. Synthetic peptides and DNA vaccines for schistosomiasis and Schistosoma japonicum[C]. Network Molecular Biology & Vaccine Meeting, 20-22, August, 1996. Manila, Philippines.
31.Armengol G, Ruiz LM, Orduz S. The injection of plasmid DNA in mouse muscle results in life long persistence of DNA, gene expression, and humoral response. Mol Biotechnol.2004, 27(2):109-118.
32.Lodmell DL, Ewalt LC, Parnell MJ, et al. One-time intradermal DNA vaccination in ear pinnae one year prior to infection protects dogs against rabies virus. Vaccine. 2006, 24(4): 412-416.
1. Capron A, Riveau GJ, Bartley PB, et al. Prospects for a schistosome vaccine. Curr Drug Targets Immune Endocr Metabol Disord.2002, 2(3): 281-290.
2. Waine GJ,McManus DP. Schistosomiasis vaccine development-the current picture. Bioessays.1997,19(5): 435-443.
3. Waine GJ, Yang W, Scott JC, et al. DNA-based vaccination using Schistosoma japonicum (Asian blood-fluke) genes. Vaccine. 1997, 15(8): 846-848.
4.李柳哲,石佑恩.日本血吸虫表膜蛋白23kDa的DNA疫苗的研制及其诱导小鼠的保护性免疫.中国血吸虫病防治杂志.1999,11:71-74.
5.余传信,朱荫昌,殷旭仁,等.日本血吸虫脂肪酸结合蛋白核酸疫苗诱导小鼠免疫保护作用研究.中国血吸虫病防治杂志.2002,14:163-167.
6. Davern KM, Wright MD, Herrmann VR, et al. Further characterisation of the Schistosoma japonicum protein Sj23, a target antigen of an immunodiagnostic monoclonal antibody. Mol Biochem Parasitol. 1991, 48(1): 67-75.
7. Becker MM, Kalinna BH, Waine GJ, et al. Gene cloning, overproduction and purification of a functionally active cytoplasmic fatty acid-binding protein (Sj-FABPC) from the human blood fluke Schistosoma japonicum. Gene.1994, 148(2):321-325.
8. Gan Y, Shi YE, Bu LG, et al. Immune responses against Schistosoma japonicum after vaccinating mice with a multivalent DNA vaccine encoding integrated membrane protein Sj23 and cytokine interleukin-12. Chin Med J (Engl). 2004,117(12):1842–1846.
9. Zhu XH, Shi YE, Gan Y. Construction and expression of the recombinant plasmid pVIVO2-IL12-Sj-14FABP for Schistosoma japonicum. Chin J of Zoon. 2005,21: 14–17.
10. Bergquist R, Al-Sherbiny M, Barakat R, et al. Blueprint for schistosomiasis vaccine development. Acta Trop. 2002, 82(2): 183-192.
11. Kumar A, Arora R, Kaur P,et al. "Universal" T helper cell determinantsenhance immunogenicity of a Plasmodium falciparum merozoite surface antigen peptide. J Immunol.1992,148(5):1499-1505.
12. Donnelly JJ, Wahren B, Liu MA. DNA Vaccines: Progress and Challenges. J Immunol. 2005,175(2):633 - 639.
13.李柳哲,石佑恩,江侃,等.日本血吸虫多价DNA疫苗pBK-CMV-sj26(sj32)-sj23免疫效果的观察.同济医科大学学报.2001,30(5): 404-406.
14. Ljungberg K, Rollman E, Eriksson L, et al. Enhanced immune responses after DNA vaccination with combined envelope genes from different HIV-1 subtypes. Virology. 2002,302(1): 44-57.
15. Kamath AT, CG Feng, M MacDonald, et al. Differential protective efficacy of DNA vaccines expressing secreted proteins of M. tuberculosis. Infect Immun. 1999, 67:1702-1707.
16. R.S.Phillips. Current Status of Malaria and Potential for Control.Clin. Microbiol. Rev. 2001,14:208 - 226.
17.刁薇,曹建平.日本血吸虫DNA疫苗的研究进展.国际医学寄生虫病杂志. 2006,33(3): 155-159.
18. Eisenstein RS, Munro HN. Translational regulation of ferritin synthesis by iron. Enzyme. 1990,44(1-4): 42-58.
1. Le TP, Coonan KM, Hedstrom RC, et al. Safety, tolerability and humoral immune responses after intramuscular administration of a malaria DNA vaccine to healthy adult volunteers. Vaccine. 2000, 18(18): 1893-1901.
2. Behr JP, Demeneix B, Loeffler JP, et al. Efficient gene transfer into mammalian primary endocrine cells with lipopolyamine-coated DNA. Proc Natl Acad Sci.1989, 86(18): 6982-6986.
3. J.萨姆布鲁克,D.W.拉塞尔.分子克隆试验指南[M].(第3版).黄培堂等译.北京:科学出版社, 2002.1272。
1. Ulmer JB, Donnelly JJ, Parker SE, et al. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science. 1993, 259(5102): 1745-1749.
2. Wolff JA, M alone RW, W illiama P, et al. Direct gene transfer mouse muscle in v ivo. Science. 1990,247(4949): 1465-1468.
3. Tang DC, De Vit M, Johnston SA. Genetic immunization is a simplemethod for eliciting an immune response. Nature.1992, 356 (6365):
152-154.
4. McMahon JM, Wells DJ. Electroporation for gene transfer to skeletal muscles: current status. BioDrugs.2004, 18(3): 155-165.
5. Tuteja R. DNA vaccines: a ray of hope. Crit Rev Biochem Mol Biol. 1999, 34(1): 1-24.
6. Wolff JA, Malone RW, Williams P, et al. Direct gene transfers into mouse muscle in vivo. Science.1990, 247:1465.
7. Loots K, Vleugels B, Ons E, et al. Evaluation of the persistence and gene expression of an anti-Chlamydophila psittaci DNA vaccine in turkey muscle. BMC Vet Res. 2006,2: 18.
8. Armengol G, Ruiz LM, Orduz S. The injection of plasmid DNA in mouse muscle results in lifelong persistence of DNA, gene expression, and humoral response. Mol Biotechnol. 2004, 27(2): 109-118.
9.吴观陵.人体寄生虫学[M].(第三版).人民卫生出版社,2005.363。
10. Ahmed R, D Gray D. Immunological memory and protective immunity: understanding their relation. Science.1996, 272: 54.
11. Chalberg TW, Genise HL, Vollrath D. phiC31 integrase confers genomic integration and long-term transgene expression in rat retina. Invest Ophthalmol Vis Sci. 2005,46(6): 2140-2146.
1. Hasan UA, Abai AM, Harper DR, et al. Nucleic acid immunization: concepts and techniques associated with third generation vaccines. J Immunol Methods. 1999, 229 (1-2): 1-22.
2. Kamath A T, Feng CG, Macdonald M, et al. Differential protective efficacy of DNA vaccines expression secreted proteins of Mycobacterium tuberculosis. Infect Immun. 1999, 67(4): 1702-1707.
3.李柳哲,石佑恩,江侃,等.日本血吸虫多价DNA疫苗的构建.中国寄生虫学与寄生虫病杂志.2001,19(4):205～208.
4.李柳哲,石佑恩,江侃,等.日本血吸虫多价DNA疫苗pBK-Sj26(Sj32)-Sj23免疫效果观察.同济医科大学学报.2001,30(5):404～406.
5.王晓婷,朱荫昌,管晓虹,等.日本血吸虫多价DNA疫苗免疫保护作用的研究.中国血吸虫病防治杂志.2006,18(1): 1-5.
6. Cherfas J. New hope for vaccine against schistosomiasis. Science. 1991, 251(4994): 630–631.
7.盛剑秋,杨淑英,段芳龄.香菇多糖的免疫调节作用研究进展.胃肠病学和肝病学杂志.1998,7(1): 92-96.
8.曹恒,熊娟,柳晶,等.偶联植物多糖对日本血吸虫疫苗PV1223增效作用及其机制的初步探讨.中国药学杂志. 2006,41(12): 899-901.
9. Hoffmann KF, Cheever AW, Wynn TA. IL-10 and the dangers of immune polarization: excessive type 1 and type 2 cytokine responses induce distinct forms of lethal immunopathology in murine schistosomiasis. J Immunol. 2000, 164 (12): 6406–6416.
10. Fallon PG. Immunopathology of schistosomiasis: A cautionary tale of mice and men. Immunology Today. 2000, 21(1): 29–35.
11. Johnson PA, Conway MA, Daly J, et al. Plasmid DNA encoding influenza virus haemagglutinin induces Th1 cells and protection against respiratory infection despite its limited ability to generate antibody responses. J Gen Virol. 2000, 81(Pt7):1737-1745.
12. Dupuis M, Denis-Mize K, Woo C, et al. Distribution of DNA vaccines determines their immunogenicity after intramuscular injection in mice. J Immunol. 2000, 165(5): 2850-2858.
1. McManus DP, Bartley PB. A vaccine against Asian schistosomiasis. Parasitol Int. 2004,53(2):163-73.
2. Tuteja R. DNA vaccines: a ray of hope. Crit Rev Biochem Mol Biol. 1999, 34(1): 1-24.
3. Giri M, Ugen KE, Weiner DB. DNA vaccines against human immunodeficiency virus type 1 in the past decade. Clin Microbiol Rev. 2004,17 (2): 370-389.
4. Da'dara AA, Skelly PJ, Fatakdawala M, et al. Comparative efficacy ofthe Schistosoma mansoni nucleic acid vaccine, Sm23, following microseeding or gene gun delivery. Parasite Immunol. 2002, 24(4): 179-187.
5. McCluskie, MJ, Brazolot Millan CL, Gramzinski RA, et al. Route and method of delivery of DNA vaccine influence immune responses in mice and non-human primates. Mol Med. 1999, 5(5): 287-300.
6. Tang DC, DeVit M, Johnston SA. Genetic immunization is a simple method for eliciting an immune response. Nature. 1992, 356(6365): 152-154.
7. Ertl HC, Xiang Z. Novel vaccine approaches. J Immunol.1996, 156 (10): 3579-3582.
8. Sin JI, Bagarazzi M, Pachuk C, et al. DNA priming-protein boosting enhances both antigen-specific antibody and Th1-type cellular immune responses in a murine herpes simplex virus-2 gD vaccine model. DNA Cell Biol. 1999,18(10): 771-779.
9. Pertmer TM, Eisenbraun MD, McCabe D, et al. Gene gun-based nucleic acid immunization: elicitation of humoral and cytotoxic T lymphocyte responses following epidermal delivery of nanogram quantities of DNA. Vaccine. 1995,13(15): 1427-1430.
10. Ayash-Rashkovsky M, Weisman Z, Zlotnikov S, et al. Induction of antigen-specific Th1-biased immune responses by plasmid DNA in Schistosoma-infected mice with a preexistent dominant Th2 immune profile. Biochem Biophys Res Commun. 2001, 282(5): 1169-1176.
11. Liu L, Zhou X, Liu H, et al. CpG motif acts as a 'danger signal' and provides a T helper type 1-biased microenvironment for DNA vaccination. Immunology. 2005, 115(2): 223-230.
12. Mora AL, Tam JP. Controlled lipidation and encapsulation of peptides as a useful approach to mucosal immunizations. J Immunol. 1998, 161(7): 3616 - 3623.
13. Fuller DH, Haynes JR. A qualitative progression in HIV type 1 glycoprotein 120-specific cytotoxic cellular and humoral immune responses in mice receiving a DNA-based glycoprotein 20 vaccine. AIDS Res Hum Retroviruses. 1994,10(11): 1433–1441.
14.谢来平,石佑恩.日本血吸虫DNA疫苗pCDSj32不同免疫次数及末次免疫后不同攻击时间对小鼠保护性免疫力的观察.中国人兽共患病杂志.2000,16(4):6-8。
15. Prayaga SK, Ford MJ, Haynes JR. Manipulation of HIV-1 gp120-specific immune responses elicited via gene gun-based DNA immunization. Vaccine. 1997, 15(12-13): 1349–1352.
16. Shiver JW, Davies ME, Perry HC, et al. Humoral and cellular immunities elicited by HIV-1vaccination. J Pharm Sci. 1996, 85(12):1317-1324.
17. Shiver JW, Davies ME, Yasutomi Y, et al. Anti-HIV env immunities elicited by nucleic acid vaccines. Vaccine. 1997,15(8):884-887.
18. Everett RS, Gerrard DE, Grant AL. Factors affecting production of luciferase and epitope-tagged IGF-I in porcine muscle after DNA injection. J Endocrinol. 2000, 166(2): 255-263.
19. MacGregor RR, Boyer JD, Ciccarelli RB, et al. Safety and immune responses to a DNA-based human immunodeficiency virus (HIV) type I env/rev vaccine in HIV-infected recipients: follow-up data. J Infect Dis. 2000, 181(1):406.
20. Le TP, Coonan KM, Hedstrom RC, et al. Safety, tolerability and humoral immune responses after intramuscular administration of a malaria DNA vaccine to healthy adult volunteers. Vaccine. 2000, 18(18): 1893-1901.
21. Harn D, Chikunguwo S, Skelly P, et al. Synthetic peptides and DNA vaccines for schistosomiasis. 2nd Schistosoma japonicum Network Molecular Biology & Vaccine Meeting, 20-22, August, 1996. Manila,Philippines.
22. Sedegah M, Hedstrom R, Hobart P, et al. Protection against malaria by immunization with plasmid DNA encoding circumsporozoite protein. Proc Natl Acad Sci USA. 1994, 91(21): 9866-9870.
23. Doolan DL, Hoffman SL. IL-12 and NK cells are required for antigen-specific adaptive immunity against malaria initiated by CD8+ T cells in the Plasmodium yoelii model. J Immunol. 1999,163(2): 884-892.
24. Johnson PA, Conway MA, Daly J, et al. Plasmid DNA encoding influenza virus haemagglutinin induces Th1 cells and protection against respiratory infection despite its limited ability to generate antibody responses. J Gen Virol. 2000, 81(Pt7):1737-1745.
25. Sedegah M, Brice GT, Rogers WO, et al. Persistence of protective immunity to malaria induced by DNA priming and poxvirus boosting: characterization of effector and memory CD8(+)-T-cell populations. Infect Immun. 2002,70(7): 3493-9.
26. Lodmell DL, Ewalt LC, Parnell MJ, et al. One-time intradermal DNA vaccination in ear pinnae one year prior to infection protects dogs against rabies virus. Vaccine. 2006, 24(4): 412-416.
27. Glenting J, Wessels S. Ensuring safety of DNA vaccines. Microb Cell Fact. 2005,4: 26.
28. Kudo-Saito C, Schlom J, Hodge JW. Intratumoral vaccination and diversified subcutaneous/intratumoral vaccination with recombinant poxviruses encoding a tumor antigen and multiple costimulatory molecules. Clin Cancer Res. 2004, 10(3): 1090–1099.
29. Casimiro DR, Chen L, Fu TM, et al. Comparative immunogenicity in rhesus monkeys of DNA plasmid, recombinant vaccinia virus, and replication-defective adenovirus vectors expressing a humanimmunodeficiency virus type 1 gag gene. J Virol. 2003, 77(11): 6305-6513.
1 van der Werf MJ, de Vlas SJ, Brooker S, et al. Acta Trop 2003;86:125–39.
2 Ross AG, Bartley PB, Sleigh AC, et al. N Engl J Med2002;346:1212–20.
3 Cristina T. Fonseca, Cristiana F. A. Brito, Jose B. Alves, et al. IL-12 enhances protective immunity in mice engendered by immunization with recombinant 14 kDa Schistosoma mansoni fatty acid-binding protein through an IFN-γand TNF-αdependent pathway . [J] . vaccine. 2004;22(3-4): 503-510.
4胡雪梅,张兆松,吴海伟,等.日本血吸虫rSj338及膜蛋白rSj22.6抗原表位联合免疫对小鼠的保护性研究[J].中国血吸虫病防治杂志,2002,14(2):88-91.
5 Zhu Y, Ren J, Da'dara A, et al.. The protective effect of a Schistosoma japonicum Chinese strain 23 kDa plasmid DNA vaccine in pigs is enhanced with IL-12. [J]. Vaccine, 2004, 23(1):78-83.
6 Dupre L, Kremer L, Wolowczuk I, et al. Immunostimulatory effect of IL-18-encoding plasmid in DNA vaccination against murine Schistosoma mansoni infection [J]. Vaccine. 2001;19(11-12):1373-80.
7 Da'dara AA, Skelly PJ, Wang MM, ET AL. Immunization with plasmid DNA encoding the integral membrane protein, Sm23, elicits a protective immune response against schistosome infection in mice [J]. Vaccine, 2001,20(3-4): 359-369.
8任建功,朱荫昌,D.A.Harn,等.日本血吸虫Sj23蛋白质疫苗对核酸疫苗增强作用的研究[J].中国血吸虫病防治杂志, 2002, 14(2):248-257.
9 Herve M, Dupre L, Ban E, et al. Features of the antibody response attributable to plasmid backbone adjuvanticity after DNA immunization [J]. Vaccine, 2001,19(3):4549-4556.
10 Tam JP. Recent advances in multiple antigen peptides[J]. J Immunol Met, 1996,196(1): 17-23.
11 Ferru I, Georges B, Bossus M, et al. Analysis of the immune response elicited by a Multiple Antigen Peptide ( MAP ), composed of two distinct protective antigens derived from the parasite Schistosoma mansoni[J]. Parasit Immunol, 1997,19(1): 1-11.
12 Argiro L, Henri S, Dessein H, et al. Induction of a protection agains S. mansoni with a MAP containing epitope of Sm37-GAPDH and Sm10-DLC. Effect of coadsorbtion with GM-CSF on alum[J]. Vaccine, 2000,18 ( 19 ): 2033-2038.
13 EI Ridi R, Montash M, Tallima H. Immunogenicity and vaccine potential of dipeptidic multiple antigen peptides from Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase[J]. Scand J Immunol, 2004,60(4): 392-402.
14李柳哲,石佑恩,江侃,等.日本血吸虫多价DNA疫苗pBK-CMV-sj26(sj32)-sj23免疫效果的观察[J].同济医科大学学报,2001,30(5): 404-406.
15 GAN Yan, SHI You-en, BU Ling-yi, et al. Immune responses against Schistosoma japonicum after vaccinating mice with a multivalent DNA vaccine encoding integrated membrane protein Sj23 and cytokine interleukin-12[J]. Chin Med J (Engl). 2004,117(12):1842-1846.
16朱晓华,石佑恩,甘燕.日本血吸虫Sj14FABP与细胞因子IL-12重组质粒的构建和表达[J].中国人兽共患病杂志, 2005,21(1): 14-17.
17 Waine GJ, Mazzer DR, Mc Manus DP. DNA immunization by intramuscular injection or gene gun induces specific IgG antibodies against a Schistosoma japonicum 22 kDa antigen, Sj22, when fusedto the murine Ig K-chain secretory leader sequence. [J]. Parasite Immunol, 1999 Jan;21(1):53-6.
18 Varaldo PB, Leite LC,Dias WO, et al . Recombinant Mycobacterium bovis BCG expressing the Sm14 antigen of Schistosoma mansoni protects mice from cercarial challenge. [J]. Infect Immun. 2004 Jun;72(6):3336-43.
19张仁利,吴少庭,高世同,等. CpG协同calpainDNA疫苗诱导BALB/c鼠TH1免疫应答和抗日本血吸虫感染[J].中华微生物学和免疫学杂志, 2003,23(10):782-786.
20 Chiaramonte MG, Hesse M, Cheever AW, et al. CpG oligonucleotides can prophylactically immunize against Th2-mediated schistosome egg-induced pathology by an IL-12-independent mechanism [J]. J Immunol. 2000 Jan 15;164(2):973-85.
2.林金祥,李莉莎.血吸虫病防治的回顾与现状.海峡预防医学杂志.2005,11(1):22-23.
3.郑江.我国血吸虫病防治应坚持以社会措施为主导的策略.国外医学·流行病学传染病学分册2005,32(1):4-7.
4.Fallon PG, Sturrock RF, Niang AC, et al. Short report: diminished susceptibility to praziquantel in a Senegal isolate of schistosoma mansoni. Am J Trop Med Hyg.1995,53(1):61-62.
5.石佑恩.血吸虫病疫苗的研究现状与展望.医药导报.2005, 24(4):265-266.
6.McManus DP, Bartley PB. A vaccine against Asian schistosomiasis. Parasitol Int. 2004 ,53(2):163-73.
7.Shi YE, Jiang CF, Han JJ, et al.Immunization of pigs against infection with schistosoma japonicum using ultraviolet-attenuated cercariae. Parasitology. 1993,106(Pt5):459-462.
8.李海山,刘勇,邵一鸣. DNA疫苗及其优化策略.国外医学病毒学分.2003,10(3):74-78.
9.Cherfas J. New hope for vaccine against schistosomiasis. Science. 1991,251(4994): 630-631.
10.Bergquist NR. Schistosomiasis vaccine development: progress and prospects. Mem Inst Oswaldo Cruz.1998,93(suppl 1):95-101.
11.Rainczuk A, Scorza T, Spithill TW, et al. A bicistronic DNA vaccine containing apical membrane antigen 1 and merozoite surface protein 4/5 can prime humoral and cellular immune responses and partially protect mice against virulent Plasmodium chabaudi adami DS malaria. Infect Immun. 2004,72(10): 5565-5573.
12.李柳哲,石佑恩,江侃,等.日本血吸虫多价DNA疫苗pBK-CMV-sj26(sj32)-sj23免疫效果的观察[J].同济医科大学学报.2001,30(5): 404-406.
13.Leachman SA, Shylankevich M, Slade MD. et al. Ubiquitin-fused and/or multiple early genes from cottontail rabbit papillomavirus as DNA vaccines. J Virol.2002,76(15): 7616-7624.
14.吴观陵.人体寄生虫学.第三版.人民卫生出版社. 2005,359.
15.Rogers MV, Davern KM, Smythe JA, et al. Immunoblotting analysis of the major integral membrane protein antigens of Schistosoma japonicum. Mol Biochem Parasitol.1988,29(1): 77-87.
16.Zhu Y, Ren J, Harn DA, et al. Protective immunity induced with 23 kDa membrane protein dna vaccine of Schistosoma japonicum Chinese strain in infected C57BL/6 mice. Southeast Asian J Trop Med Public Health.2003,34(4):697-701.
17.Shi F, Zhang Y, Ye P, et al. Laboratory and field evaluation of Schistosoma japonicum DNA vaccines in sheep and water buffalo in China. Vaccine. 2001,20(3-4): 462-467.
18.Zhu Y, Ren J, Da'dara A,et al. The protective effect of a Schistosoma japonicum Chinese strain 23 kDa plasmid DNA vaccine in pigs is enhanced with IL-12. Vaccine.2004, 23(1): 78-83.
19.Gobert GN, Stenzel DJ, Jones MK, et al. Immunolocalization of the fatty acid-binding protein Sj-FABPc within adult Schistosoma japonicum. Parasitology. 1997,115 (Pt1): 33-39.
20.谢来平,石佑恩.血吸虫脂肪酸结合蛋白的研究现状.国外医学寄生虫病分册. 1998,25(5): 193-196.
21.赵巍,吴海玮,苏川,等.日本血吸虫中国大陆株重组脂肪酸结合蛋白(Sj-FABPc)的特性和抗原表位的分析.中国人兽共患病杂志. 2000,16(6): 6-8.
22.余传信,朱荫昌,殷旭仁,等.日本血吸虫脂肪酸结合蛋白(SjC FABP)核酸疫苗诱导小鼠免疫保护作用研究.中国血吸虫病防治杂志.2002,14(3): 163-167.
23.朱晓华,石佑恩,胡萍,等.日本血吸虫脂肪酸结合蛋白DNA疫苗诱导小鼠保护性免疫力的研究.中国血吸虫病防治杂志. 2005, 17(1): 4-8.
24.董菁,成军,王勤环,等.乙型肝炎病毒囊膜中蛋白与白细胞介素18联合基因免疫.中华传染病杂志.2002,20(3): 148-151.
25.Singh M, Briones M, Ott G, et al. Cationic microparticles: a potentdelivery system for DNA vaccines. Proc Natl Acad Sci USA. 2000,97(2): 811-816.
26.Ertl HC, Xiang Z. Novel vaccine approaches. J Immunol.1996, 156 (10): 3579-3582.
27.许雪梅,宋国兴,司静懿. DNA疫苗机制研究进展.微生物学免疫学进展.2002,30(1): 65-70.
28.Forg P, von Hoegen P, Dalemans W, et al. Superiority of the ear pinna over muscle tissue as site for DNA vaccination. Gene Ther. 1998,5(6): 789–797.
29.谢来平,石佑恩.日本血吸虫DNA疫苗pCDSj32不同免疫次数及末次免疫后不同攻击时间对小鼠保护性免疫力的观察.中国人兽共患病杂志.2000,16(4):6-8。
30.Harn D, Chikunguwo S, Skelly P, et al. Synthetic peptides and DNA vaccines for schistosomiasis and Schistosoma japonicum[C]. Network Molecular Biology & Vaccine Meeting, 20-22, August, 1996. Manila, Philippines.
31.Armengol G, Ruiz LM, Orduz S. The injection of plasmid DNA in mouse muscle results in life long persistence of DNA, gene expression, and humoral response. Mol Biotechnol.2004, 27(2):109-118.
32.Lodmell DL, Ewalt LC, Parnell MJ, et al. One-time intradermal DNA vaccination in ear pinnae one year prior to infection protects dogs against rabies virus. Vaccine. 2006, 24(4): 412-416.
1. Capron A, Riveau GJ, Bartley PB, et al. Prospects for a schistosome vaccine. Curr Drug Targets Immune Endocr Metabol Disord.2002, 2(3): 281-290.
2. Waine GJ,McManus DP. Schistosomiasis vaccine development-the current picture. Bioessays.1997,19(5): 435-443.
3. Waine GJ, Yang W, Scott JC, et al. DNA-based vaccination using Schistosoma japonicum (Asian blood-fluke) genes. Vaccine. 1997, 15(8): 846-848.
4.李柳哲,石佑恩.日本血吸虫表膜蛋白23kDa的DNA疫苗的研制及其诱导小鼠的保护性免疫.中国血吸虫病防治杂志.1999,11:71-74.
5.余传信,朱荫昌,殷旭仁,等.日本血吸虫脂肪酸结合蛋白核酸疫苗诱导小鼠免疫保护作用研究.中国血吸虫病防治杂志.2002,14:163-167.
6. Davern KM, Wright MD, Herrmann VR, et al. Further characterisation of the Schistosoma japonicum protein Sj23, a target antigen of an immunodiagnostic monoclonal antibody. Mol Biochem Parasitol. 1991, 48(1): 67-75.
7. Becker MM, Kalinna BH, Waine GJ, et al. Gene cloning, overproduction and purification of a functionally active cytoplasmic fatty acid-binding protein (Sj-FABPC) from the human blood fluke Schistosoma japonicum. Gene.1994, 148(2):321-325.
8. Gan Y, Shi YE, Bu LG, et al. Immune responses against Schistosoma japonicum after vaccinating mice with a multivalent DNA vaccine encoding integrated membrane protein Sj23 and cytokine interleukin-12. Chin Med J (Engl). 2004,117(12):1842–1846.
9. Zhu XH, Shi YE, Gan Y. Construction and expression of the recombinant plasmid pVIVO2-IL12-Sj-14FABP for Schistosoma japonicum. Chin J of Zoon. 2005,21: 14–17.
10. Bergquist R, Al-Sherbiny M, Barakat R, et al. Blueprint for schistosomiasis vaccine development. Acta Trop. 2002, 82(2): 183-192.
11. Kumar A, Arora R, Kaur P,et al. "Universal" T helper cell determinantsenhance immunogenicity of a Plasmodium falciparum merozoite surface antigen peptide. J Immunol.1992,148(5):1499-1505.
12. Donnelly JJ, Wahren B, Liu MA. DNA Vaccines: Progress and Challenges. J Immunol. 2005,175(2):633 - 639.
13.李柳哲,石佑恩,江侃,等.日本血吸虫多价DNA疫苗pBK-CMV-sj26(sj32)-sj23免疫效果的观察.同济医科大学学报.2001,30(5): 404-406.
14. Ljungberg K, Rollman E, Eriksson L, et al. Enhanced immune responses after DNA vaccination with combined envelope genes from different HIV-1 subtypes. Virology. 2002,302(1): 44-57.
15. Kamath AT, CG Feng, M MacDonald, et al. Differential protective efficacy of DNA vaccines expressing secreted proteins of M. tuberculosis. Infect Immun. 1999, 67:1702-1707.
16. R.S.Phillips. Current Status of Malaria and Potential for Control.Clin. Microbiol. Rev. 2001,14:208 - 226.
17.刁薇,曹建平.日本血吸虫DNA疫苗的研究进展.国际医学寄生虫病杂志. 2006,33(3): 155-159.
18. Eisenstein RS, Munro HN. Translational regulation of ferritin synthesis by iron. Enzyme. 1990,44(1-4): 42-58.
1. Le TP, Coonan KM, Hedstrom RC, et al. Safety, tolerability and humoral immune responses after intramuscular administration of a malaria DNA vaccine to healthy adult volunteers. Vaccine. 2000, 18(18): 1893-1901.
2. Behr JP, Demeneix B, Loeffler JP, et al. Efficient gene transfer into mammalian primary endocrine cells with lipopolyamine-coated DNA. Proc Natl Acad Sci.1989, 86(18): 6982-6986.
3. J.萨姆布鲁克,D.W.拉塞尔.分子克隆试验指南[M].(第3版).黄培堂等译.北京:科学出版社, 2002.1272。
1. Ulmer JB, Donnelly JJ, Parker SE, et al. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science. 1993, 259(5102): 1745-1749.
2. Wolff JA, M alone RW, W illiama P, et al. Direct gene transfer mouse muscle in v ivo. Science. 1990,247(4949): 1465-1468.
3. Tang DC, De Vit M, Johnston SA. Genetic immunization is a simplemethod for eliciting an immune response. Nature.1992, 356 (6365):
152-154.
4. McMahon JM, Wells DJ. Electroporation for gene transfer to skeletal muscles: current status. BioDrugs.2004, 18(3): 155-165.
5. Tuteja R. DNA vaccines: a ray of hope. Crit Rev Biochem Mol Biol. 1999, 34(1): 1-24.
6. Wolff JA, Malone RW, Williams P, et al. Direct gene transfers into mouse muscle in vivo. Science.1990, 247:1465.
7. Loots K, Vleugels B, Ons E, et al. Evaluation of the persistence and gene expression of an anti-Chlamydophila psittaci DNA vaccine in turkey muscle. BMC Vet Res. 2006,2: 18.
8. Armengol G, Ruiz LM, Orduz S. The injection of plasmid DNA in mouse muscle results in lifelong persistence of DNA, gene expression, and humoral response. Mol Biotechnol. 2004, 27(2): 109-118.
9.吴观陵.人体寄生虫学[M].(第三版).人民卫生出版社,2005.363。
10. Ahmed R, D Gray D. Immunological memory and protective immunity: understanding their relation. Science.1996, 272: 54.
11. Chalberg TW, Genise HL, Vollrath D. phiC31 integrase confers genomic integration and long-term transgene expression in rat retina. Invest Ophthalmol Vis Sci. 2005,46(6): 2140-2146.
1. Hasan UA, Abai AM, Harper DR, et al. Nucleic acid immunization: concepts and techniques associated with third generation vaccines. J Immunol Methods. 1999, 229 (1-2): 1-22.
2. Kamath A T, Feng CG, Macdonald M, et al. Differential protective efficacy of DNA vaccines expression secreted proteins of Mycobacterium tuberculosis. Infect Immun. 1999, 67(4): 1702-1707.
3.李柳哲,石佑恩,江侃,等.日本血吸虫多价DNA疫苗的构建.中国寄生虫学与寄生虫病杂志.2001,19(4):205～208.
4.李柳哲,石佑恩,江侃,等.日本血吸虫多价DNA疫苗pBK-Sj26(Sj32)-Sj23免疫效果观察.同济医科大学学报.2001,30(5):404～406.
5.王晓婷,朱荫昌,管晓虹,等.日本血吸虫多价DNA疫苗免疫保护作用的研究.中国血吸虫病防治杂志.2006,18(1): 1-5.
6. Cherfas J. New hope for vaccine against schistosomiasis. Science. 1991, 251(4994): 630–631.
7.盛剑秋,杨淑英,段芳龄.香菇多糖的免疫调节作用研究进展.胃肠病学和肝病学杂志.1998,7(1): 92-96.
8.曹恒,熊娟,柳晶,等.偶联植物多糖对日本血吸虫疫苗PV1223增效作用及其机制的初步探讨.中国药学杂志. 2006,41(12): 899-901.
9. Hoffmann KF, Cheever AW, Wynn TA. IL-10 and the dangers of immune polarization: excessive type 1 and type 2 cytokine responses induce distinct forms of lethal immunopathology in murine schistosomiasis. J Immunol. 2000, 164 (12): 6406–6416.
10. Fallon PG. Immunopathology of schistosomiasis: A cautionary tale of mice and men. Immunology Today. 2000, 21(1): 29–35.
11. Johnson PA, Conway MA, Daly J, et al. Plasmid DNA encoding influenza virus haemagglutinin induces Th1 cells and protection against respiratory infection despite its limited ability to generate antibody responses. J Gen Virol. 2000, 81(Pt7):1737-1745.
12. Dupuis M, Denis-Mize K, Woo C, et al. Distribution of DNA vaccines determines their immunogenicity after intramuscular injection in mice. J Immunol. 2000, 165(5): 2850-2858.
1. McManus DP, Bartley PB. A vaccine against Asian schistosomiasis. Parasitol Int. 2004,53(2):163-73.
2. Tuteja R. DNA vaccines: a ray of hope. Crit Rev Biochem Mol Biol. 1999, 34(1): 1-24.
3. Giri M, Ugen KE, Weiner DB. DNA vaccines against human immunodeficiency virus type 1 in the past decade. Clin Microbiol Rev. 2004,17 (2): 370-389.
4. Da'dara AA, Skelly PJ, Fatakdawala M, et al. Comparative efficacy ofthe Schistosoma mansoni nucleic acid vaccine, Sm23, following microseeding or gene gun delivery. Parasite Immunol. 2002, 24(4): 179-187.
5. McCluskie, MJ, Brazolot Millan CL, Gramzinski RA, et al. Route and method of delivery of DNA vaccine influence immune responses in mice and non-human primates. Mol Med. 1999, 5(5): 287-300.
6. Tang DC, DeVit M, Johnston SA. Genetic immunization is a simple method for eliciting an immune response. Nature. 1992, 356(6365): 152-154.
7. Ertl HC, Xiang Z. Novel vaccine approaches. J Immunol.1996, 156 (10): 3579-3582.
8. Sin JI, Bagarazzi M, Pachuk C, et al. DNA priming-protein boosting enhances both antigen-specific antibody and Th1-type cellular immune responses in a murine herpes simplex virus-2 gD vaccine model. DNA Cell Biol. 1999,18(10): 771-779.
9. Pertmer TM, Eisenbraun MD, McCabe D, et al. Gene gun-based nucleic acid immunization: elicitation of humoral and cytotoxic T lymphocyte responses following epidermal delivery of nanogram quantities of DNA. Vaccine. 1995,13(15): 1427-1430.
10. Ayash-Rashkovsky M, Weisman Z, Zlotnikov S, et al. Induction of antigen-specific Th1-biased immune responses by plasmid DNA in Schistosoma-infected mice with a preexistent dominant Th2 immune profile. Biochem Biophys Res Commun. 2001, 282(5): 1169-1176.
11. Liu L, Zhou X, Liu H, et al. CpG motif acts as a 'danger signal' and provides a T helper type 1-biased microenvironment for DNA vaccination. Immunology. 2005, 115(2): 223-230.
12. Mora AL, Tam JP. Controlled lipidation and encapsulation of peptides as a useful approach to mucosal immunizations. J Immunol. 1998, 161(7): 3616 - 3623.
13. Fuller DH, Haynes JR. A qualitative progression in HIV type 1 glycoprotein 120-specific cytotoxic cellular and humoral immune responses in mice receiving a DNA-based glycoprotein 20 vaccine. AIDS Res Hum Retroviruses. 1994,10(11): 1433–1441.
14.谢来平,石佑恩.日本血吸虫DNA疫苗pCDSj32不同免疫次数及末次免疫后不同攻击时间对小鼠保护性免疫力的观察.中国人兽共患病杂志.2000,16(4):6-8。
15. Prayaga SK, Ford MJ, Haynes JR. Manipulation of HIV-1 gp120-specific immune responses elicited via gene gun-based DNA immunization. Vaccine. 1997, 15(12-13): 1349–1352.
16. Shiver JW, Davies ME, Perry HC, et al. Humoral and cellular immunities elicited by HIV-1vaccination. J Pharm Sci. 1996, 85(12):1317-1324.
17. Shiver JW, Davies ME, Yasutomi Y, et al. Anti-HIV env immunities elicited by nucleic acid vaccines. Vaccine. 1997,15(8):884-887.
18. Everett RS, Gerrard DE, Grant AL. Factors affecting production of luciferase and epitope-tagged IGF-I in porcine muscle after DNA injection. J Endocrinol. 2000, 166(2): 255-263.
19. MacGregor RR, Boyer JD, Ciccarelli RB, et al. Safety and immune responses to a DNA-based human immunodeficiency virus (HIV) type I env/rev vaccine in HIV-infected recipients: follow-up data. J Infect Dis. 2000, 181(1):406.
20. Le TP, Coonan KM, Hedstrom RC, et al. Safety, tolerability and humoral immune responses after intramuscular administration of a malaria DNA vaccine to healthy adult volunteers. Vaccine. 2000, 18(18): 1893-1901.
21. Harn D, Chikunguwo S, Skelly P, et al. Synthetic peptides and DNA vaccines for schistosomiasis. 2nd Schistosoma japonicum Network Molecular Biology & Vaccine Meeting, 20-22, August, 1996. Manila,Philippines.
22. Sedegah M, Hedstrom R, Hobart P, et al. Protection against malaria by immunization with plasmid DNA encoding circumsporozoite protein. Proc Natl Acad Sci USA. 1994, 91(21): 9866-9870.
23. Doolan DL, Hoffman SL. IL-12 and NK cells are required for antigen-specific adaptive immunity against malaria initiated by CD8+ T cells in the Plasmodium yoelii model. J Immunol. 1999,163(2): 884-892.
24. Johnson PA, Conway MA, Daly J, et al. Plasmid DNA encoding influenza virus haemagglutinin induces Th1 cells and protection against respiratory infection despite its limited ability to generate antibody responses. J Gen Virol. 2000, 81(Pt7):1737-1745.
25. Sedegah M, Brice GT, Rogers WO, et al. Persistence of protective immunity to malaria induced by DNA priming and poxvirus boosting: characterization of effector and memory CD8(+)-T-cell populations. Infect Immun. 2002,70(7): 3493-9.
26. Lodmell DL, Ewalt LC, Parnell MJ, et al. One-time intradermal DNA vaccination in ear pinnae one year prior to infection protects dogs against rabies virus. Vaccine. 2006, 24(4): 412-416.
27. Glenting J, Wessels S. Ensuring safety of DNA vaccines. Microb Cell Fact. 2005,4: 26.
28. Kudo-Saito C, Schlom J, Hodge JW. Intratumoral vaccination and diversified subcutaneous/intratumoral vaccination with recombinant poxviruses encoding a tumor antigen and multiple costimulatory molecules. Clin Cancer Res. 2004, 10(3): 1090–1099.
29. Casimiro DR, Chen L, Fu TM, et al. Comparative immunogenicity in rhesus monkeys of DNA plasmid, recombinant vaccinia virus, and replication-defective adenovirus vectors expressing a humanimmunodeficiency virus type 1 gag gene. J Virol. 2003, 77(11): 6305-6513.
1 van der Werf MJ, de Vlas SJ, Brooker S, et al. Acta Trop 2003;86:125–39.
2 Ross AG, Bartley PB, Sleigh AC, et al. N Engl J Med2002;346:1212–20.
3 Cristina T. Fonseca, Cristiana F. A. Brito, Jose B. Alves, et al. IL-12 enhances protective immunity in mice engendered by immunization with recombinant 14 kDa Schistosoma mansoni fatty acid-binding protein through an IFN-γand TNF-αdependent pathway . [J] . vaccine. 2004;22(3-4): 503-510.
4胡雪梅,张兆松,吴海伟,等.日本血吸虫rSj338及膜蛋白rSj22.6抗原表位联合免疫对小鼠的保护性研究[J].中国血吸虫病防治杂志,2002,14(2):88-91.
5 Zhu Y, Ren J, Da'dara A, et al.. The protective effect of a Schistosoma japonicum Chinese strain 23 kDa plasmid DNA vaccine in pigs is enhanced with IL-12. [J]. Vaccine, 2004, 23(1):78-83.
6 Dupre L, Kremer L, Wolowczuk I, et al. Immunostimulatory effect of IL-18-encoding plasmid in DNA vaccination against murine Schistosoma mansoni infection [J]. Vaccine. 2001;19(11-12):1373-80.
7 Da'dara AA, Skelly PJ, Wang MM, ET AL. Immunization with plasmid DNA encoding the integral membrane protein, Sm23, elicits a protective immune response against schistosome infection in mice [J]. Vaccine, 2001,20(3-4): 359-369.
8任建功,朱荫昌,D.A.Harn,等.日本血吸虫Sj23蛋白质疫苗对核酸疫苗增强作用的研究[J].中国血吸虫病防治杂志, 2002, 14(2):248-257.
9 Herve M, Dupre L, Ban E, et al. Features of the antibody response attributable to plasmid backbone adjuvanticity after DNA immunization [J]. Vaccine, 2001,19(3):4549-4556.
10 Tam JP. Recent advances in multiple antigen peptides[J]. J Immunol Met, 1996,196(1): 17-23.
11 Ferru I, Georges B, Bossus M, et al. Analysis of the immune response elicited by a Multiple Antigen Peptide ( MAP ), composed of two distinct protective antigens derived from the parasite Schistosoma mansoni[J]. Parasit Immunol, 1997,19(1): 1-11.
12 Argiro L, Henri S, Dessein H, et al. Induction of a protection agains S. mansoni with a MAP containing epitope of Sm37-GAPDH and Sm10-DLC. Effect of coadsorbtion with GM-CSF on alum[J]. Vaccine, 2000,18 ( 19 ): 2033-2038.
13 EI Ridi R, Montash M, Tallima H. Immunogenicity and vaccine potential of dipeptidic multiple antigen peptides from Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase[J]. Scand J Immunol, 2004,60(4): 392-402.
14李柳哲,石佑恩,江侃,等.日本血吸虫多价DNA疫苗pBK-CMV-sj26(sj32)-sj23免疫效果的观察[J].同济医科大学学报,2001,30(5): 404-406.
15 GAN Yan, SHI You-en, BU Ling-yi, et al. Immune responses against Schistosoma japonicum after vaccinating mice with a multivalent DNA vaccine encoding integrated membrane protein Sj23 and cytokine interleukin-12[J]. Chin Med J (Engl). 2004,117(12):1842-1846.
16朱晓华,石佑恩,甘燕.日本血吸虫Sj14FABP与细胞因子IL-12重组质粒的构建和表达[J].中国人兽共患病杂志, 2005,21(1): 14-17.
17 Waine GJ, Mazzer DR, Mc Manus DP. DNA immunization by intramuscular injection or gene gun induces specific IgG antibodies against a Schistosoma japonicum 22 kDa antigen, Sj22, when fusedto the murine Ig K-chain secretory leader sequence. [J]. Parasite Immunol, 1999 Jan;21(1):53-6.
18 Varaldo PB, Leite LC,Dias WO, et al . Recombinant Mycobacterium bovis BCG expressing the Sm14 antigen of Schistosoma mansoni protects mice from cercarial challenge. [J]. Infect Immun. 2004 Jun;72(6):3336-43.
19张仁利,吴少庭,高世同,等. CpG协同calpainDNA疫苗诱导BALB/c鼠TH1免疫应答和抗日本血吸虫感染[J].中华微生物学和免疫学杂志, 2003,23(10):782-786.
20 Chiaramonte MG, Hesse M, Cheever AW, et al. CpG oligonucleotides can prophylactically immunize against Th2-mediated schistosome egg-induced pathology by an IL-12-independent mechanism [J]. J Immunol. 2000 Jan 15;164(2):973-85.