摘要
日本血吸虫病是一种广泛流行并严重影响人畜健康的寄生虫病。我国目前对血吸虫病的防治重点在于灭螺及人畜同步化疗。但是由于吡喹酮仅对侵入皮肤3 h的童虫和成虫有效,且目前在很多国家已经出现对吡喹酮敏感性降低的报道。因此,安全有效的疫苗对血吸虫病的防治具有重要的作用。
日本血吸虫疫苗的研究经历了死疫苗、减毒活疫苗、基因工程疫苗和核酸疫苗等过程。其中核酸疫苗中最常用的是DNA疫苗,因其具有制备简单、保存及运输方便和能诱导广泛和持久的体液免疫和细胞免疫的优点而备受研究者青睐。目前血吸虫DNA疫苗在血吸虫研究中占据着主导地位。但是截至目前为止,单价DNA疫苗的免疫保护效果并不令人满意,WHO推荐的40%或以上的保护力仍然没有达到。究其原因在于,血吸虫作为一种多细胞生物,基因组非常复杂,且在与宿主长期进化的过程中产生了多种免疫逃避机制。
新近证实,调节性T细胞(Tregs)与寄生虫逃避宿主免疫攻击关系非常密切。在很多感染性疾病中,尤其是寄生虫感染时Tregs的水平显著升高,从而成为了寄生虫一个逃生的“窗口”帮助其逃避宿主的免疫攻击。在疟疾感染的小鼠模型中,消耗Tregs有利于机体清除病原体并降低小鼠出现致死性感染的可能。因此探究血吸虫感染中Tregs的变化,并针对该变化采取相应的治疗方案必将有助于血吸虫疫苗的研究。
西咪替丁(CIM)作为H2受体阻滞剂在临床上一直用于治疗胃酸引起的胃肠功能紊乱并有确定的疗效。除此之外,CIM在临床上还广泛的用于治疗疱疹病毒,HIV病毒等感染性疾病引起的免疫功能下降,以及阻抑肿瘤的发生及生长。研究表明,其作用机制主要在于抑制抑制性T细胞的功能从而发挥免疫增强作用。有报道称,直接将CIM和毗喹酮合用可显著提高吡喹酮的杀虫效果。同时也有很多将CIM作为一种免疫调节剂来增强疫苗的免疫原性的报道。
因此,本研究选用CIM和日本血吸虫26KD谷胱甘肽S-转移酶(Sj26)DNA疫苗合用,一方面观察CIM和日本血吸虫pEGFP-Sj26GST DNA疫苗联合使用的免疫保护作用；另一方面观察CIM和日本血吸虫pEGFP-Sj26GST DNA疫苗联合使用后宿主体内Tregs的变化,并探讨其免疫机制。
本课题分为以下三个部分：
一、西咪替丁对日本血吸虫感染小鼠免疫应答的影响研究
目的：探讨不同剂量的C1M对血吸虫感染小鼠免疫应答的影响。
方法：32只6-8周龄的BALB/c雌性小鼠随机分成3组,即50 mg/kg CIM组(CIM50)、25 mg/kg CIM组(CIM25)及感染对照组(Control)。CIM50组采用50mg/kg的CIM皮下注射三次,每次间隔两周；CIM25组采用25 mg/kg的CIM皮下注射三次,每次间隔两周,即第1、14、28天分别用药一次；Control为单纯感染组。第42天时各组小鼠攻击感染日本血吸虫尾蚴40条/鼠。感染后第6周杀鼠取脾淋巴细胞计数,检测脾淋巴细胞中CD4+CD25+FoxP3+Tregs百分比及IL-2、IFN-γ、IL-4和IL-5水平。
结果：和感染对照组相比,使用25mg/kg CIM组调节性T细胞的比例显著降低(P<0.01)且小鼠脾细胞培养上清中IL-2、IFN-γ的水平均较对照组升高(P<0.05),而IL-4、IL-5的水平虽较对照组升高,但无显著性差异(P>0.05)。而和感染对照组相比,使用50mg/kg CIM组在调节性T细胞的比例及小鼠脾细胞培养上清中细胞因子的水平上均与对照组无显著性差异(P>0.05)。
结论：CIM可作为免疫调节剂,增强小鼠的免疫功能,从而提高小鼠对血吸虫感染的免疫保护作用,但是该作用与剂量有关。
二、西咪替丁和日本血吸虫pEGFP-Sj26GST DNA疫苗联合使用的免疫保护作用研究
目的：观察CIM对pEGFP-Sj26GST DNA疫苗免疫保护作用的影响。
方法：从本室保存的DH5a大肠杆菌体内提取已构建好的pEGFP-Sj26GST重组质粒,进行鉴定。50只BALB/c小鼠随机分为5组,即感染对照组、CIM单用组、pEGFP-N3空载体对照组、pEGFP-Sj26GST DNA疫苗组及CIM和pEGFP-Sj26GST DNA疫苗合用组。其中疫苗组每只小鼠在第1、14及28天时分别经股四头肌注射100μgpEGFP-Sj26GST DNA疫苗或pEGFP-N3空载体,CIM组每只小鼠从首次免疫前两天开始每天皮下注射25 mg/kg的CIM直至感染前。末次免疫后2周,各组小鼠均经腹部皮肤感染日本血吸虫尾蚴40条/鼠。感染后第6周杀鼠冲虫,计数每只小鼠的虫荷及肝内虫卵数。计算减虫率和肝组织中每条雌虫的减卵率。取出小鼠肝组织用福尔马林固定,脱水、石蜡切片、HE染色,检测各组小鼠肝组织内虫卵肉芽肿的变化。
结果：将大量提取出的pEGFP-Sj26GST重组质粒及pEGFP-N3空载体质粒在核酸分析仪上检测发现浓度达到3mg/ml且纯度较高。动物保护性试验结果显示,CIM和pEGFP-Sj26GST DNA疫苗联合使用后减虫率高达79%,显著高于pEGFP-Sj26GST疫苗单独使用组(27%)及其他各组。肝脏内虫卵计数发现其肝减卵率与pEGFP-Sj26GSTDNA疫苗单用组相比,联合使用CIM和pEGFP-Sj26GST DNA疫苗也有显著的降低(22.48%vs 68.35%)。小鼠肝组织HE染色显示,和感染对照组相比,CIM和PEGFP-Sj26GST DNA疫苗合用组的虫卵肉芽肿数目(16.25±2.87 vs 4.5±1.76)显著减少。显微镜下可见,感染对照组的肝肉芽肿体积较大,而pEGFP-Sj26GST疫苗组及与CIM合用组的肝肉芽肿体积则明显缩小。
结论：CIM可作为佐剂增强血吸虫pEGFP-Sj26GST DNA疫苗的免疫保护效果。且CIM和疫苗合用可以显著减少血吸虫感染宿主肝内虫卵肉芽肿的数目。
三、西咪替丁增强日本血吸虫pEGFP-Sj26GST DNA疫苗的免疫保护作用的机制研究
目的：观察CIM对宿主体内CD4+CD25+Tregs及相关细胞因子的影响,探讨CIM增强pEGFP-Sj26GST DNA疫苗免疫保护作用的机制。
方法：80只雌性BALB/c小鼠随机分成五组,即感染对照组、CIM组、pEGFP-N3空载体对照组、pEGFP-Sj26GST DNA疫苗组及CIM和pEGFP-Sj26GST DNA疫苗合用组。其中疫苗组每只小鼠在第1、14及28天时分别经股四头肌注射100μgpEGFP-Sj26GST DNA疫苗或pEGFP-N3空载体,即每只小鼠免疫三次,每次间隔2周。CIM组每只小鼠从首次免疫前两天开始每天皮下注射25mg/kg的CIM直至感染前。各组取6只小鼠在末次免疫后一周剖杀,取眼眶血,静置后离心取上清,检测其血清中特异性GST抗体的含量。将剩余的50只小鼠在末次免疫后2周均经腹部皮肤感染日本血吸虫尾蚴40条/鼠。感染后第6周,剖杀小鼠,无菌取脾,制备单个脾细胞悬液,流式细胞仪检测脾淋巴细胞中CD4+CD25+Tregs百分比。将无菌取出的脾细胞悬液在体外用丝裂原ConA刺激后培养48小时,MTT法检测在体外各组脾细胞对丝裂原的增值比率。体外培养72小时离心收集各组脾细胞上清,夹心ELISA法分别检测脾细胞上清中Th1细胞因子γ-干扰素(IFN-γ)、白细胞介素-2(IL-2)和Th2细胞因子白细胞介素-4(IL-4)、白细胞介素-5(IL-5)和白细胞介素-10(IL-10)等细胞因子含量。
结果：
1)使用pEGFP-Sj26GST DNA疫苗后小鼠血清中特异性的抗Sj26GST抗体的水平明显高于空载体组及单用CIM组(p<0.05),且将pEGFP-Sj26GST DNA疫苗和CIM合用后小鼠血清中特异性的抗Sj26GST抗体的水平亦显著高于单独使用pEGFP-Sj26GST DNA疫苗组(p<0.05)。
2)和感染对照组相比,单用pEGFP-Sj26GST DNA疫苗可以降低其脾淋巴细胞中CD4+CD25+Foxp3+T细胞的比例(p<0.05),而将CIM和pEGFP-Sj26GST DNA疫苗合用则可以显著降低其脾淋巴细胞中CD4+CD25+Foxp3+ T细胞的比例(p<0.01)。
3)和感染对照组相比,单用CIM后脾淋巴细胞对ConA的增殖反应明显提高,而合用CIM及pEGFP-Sj26GST DNA疫苗后,脾淋巴细胞对ConA的增殖反应则较单独使用pEGFP-Sj26GST DNA疫苗组亦有显著提高(p<0.05)。
4)CIM和pEGFP-Sj26GST DNA疫苗合用组小鼠脾细胞培养上清中IL-12、IFN-γ的水平均较感染对照组高(P<0.05),IL-10的含量较对照组降低(P<0.05),而IL-4、IL-5的水平虽较对照组升高,但无显著性差异(P>0.05)。
结论：
1)提取出的pEGFP-Sj26GST DNA疫苗具有较强的免疫原性,将CIM与疫苗合用后能诱导机体产生更高水平的特异性抗体。
2)CIM和疫苗合用可以降低血吸虫感染宿主脾淋巴细胞中CD4+CD25+ Tregs的百分比。
3)CIM可以辅助DNA疫苗在体外增强T淋巴细胞对丝裂原的反应,增强T细胞的功能。
4)CIM和疫苗合用可以增强机体的Thl型免疫反应,提高疫苗的保护性免疫效果。
课题的特色和创新点：
1)证明了CIM可作为一种免疫调节剂增强机体的Thl型免疫应答。
2)首次将CIM与日本血吸虫的DNA疫苗合用,并证实能有效提高抗日本血吸虫感染的免疫保护作用。
3)证明CIM增强疫苗保护作用的机制与其下调宿主CD4+CD25+Tregs的水平有关。
Schistosoma japonicum (S. japonicum) is an amphixenosis that could result in severe morbidity and mortality. Current schistosomiasis control in Asia is based primarily on snail eradication and large scale chemotherapy using the effective drug praziquantel. Although active drug praziquantel is available, evidence is now accumulating that if it can reduce the overall incidence of severe forms of the disease. Moreover, the parasite resistance to the drug has appeared. Thus, safe and efficacious vaccine development is an important measure for long-term integrated control of schistosomiasis.
The vaccines against S. japonicum, including dead vaccine, attenuated live vaccine, genetically engineering and nucleic acid vaccine. The advantages of DNA vaccines over the traditional vaccine are the low cost of production, thermal stability and their ability to induce a wide variety of cellular and humoral immune response. Currently, DNA vaccine has become the highest priority of vaccine against schistosome. But to this day, there is still not yet a commercial DNA vaccine to the world. These results are perhaps explained by the fact that the schistosome parasite is such an antigenically complex organism with very complicated genome. Furthermore, they have achieved many mechanisms of immunized escape generated during long-term evolutional process together with host.
More recently, there is convincing evidence that CD4+CD25+ Tregs are a variety of T cell subsets with suppressive properties, which play an important role in the progress of parasites escaping from the host immune assault. Evidence suggests that regulatory T cells help to protect the host from the hepatocyte damage induced by S. mansoni eggs and to prevent death from the infection through immune-mediated pathology. Thus, there is a need to explore the level of regulatory T cells in the host infected of S. japonicum, and to adopt related method for immunization of S. japonicum vaccine.
Cimetidine (CIM) is a histamine-2-receptor antagonist that with highly successful use in gastric acid-mediated gastrointestinal disorders. Its use in clinical medicine is reported not only reduces or inhibits some infectious diseases induced impairment of the immune response such as chronic Herpes virus infection and HIV, but suppresses angiogenesis and tumor growth with few adverse biologic effects. Evidence suggests that CIM enhances a variety of immunologic functions because of its inhibitory effects on suppressor T cell function. There was convincing evidence that simultaneous administration of praziquantel and CIM could improve further the efficacy of the single therapy with praziquantel for cysticercosis and other arasitic diseases, such as schistosomiasis. Moreover, they had also achieved successful results to simultaneous administration cimetidine and variable vaccine.
So in the present study, we evaluated the feasibility of using CIM to co-immunization with a S. japonicum DNA vaccine, the gene encoding 26 kDa glutathione S-transferase of S. japonicum (Sj26), which is one of promising vaccine candidated selected by WHO. Explore the adjuvant effect and mechanism of CIM in the protective efficacy against S. japonicum generated by pEGFP-Sj26GST DNA vaccine.
The main contents and results of this thesis include three parts:
Part 1:Studies on the effect of CIM on the immune responds of mice infected with S. japonicum
Objective:Explore the effect of different schedule of CIM treatment on the S. japonicum infected mice.
Methods:BALB/c mice were randomly divided into three groups.50 mg/kg CIM group (CIM50)、25 mg/kg CIM group (CIM25) and infected control group (control).Animals in CIM50 treated with 50 mg/kg CIM subcutaneously (s.c) injection three times; Animals in CIM25 treated with 25 mg/kg CIM subcutaneously (s.c) injection three times; Six weeks of the treatment, mice in each group were challenged percutaneously with cercariae for 20 min. After 6 weeks post infection, all mice were succumbed. The percent of CD4+CD25+ Tregs in spleen was measured with flow cytometer. The expression of gamma interferon (IFN-γ), interleukin-2 (IL-2), interleukin-4 (IL-4) and interleukin-5 (IL-5) in cultural suspension of splenic cells was detected by sandwich-ELISA after stimulation with ConA.
Results:Compare with the Control group, the percentage of CD4+CD25+ regulatory T cells was significiantly decreased in the spleens of CIM25 Group mice (P<0.01), the cytokine production of splenocytes showed that animals in CIM25 group promoted an elevated level of IFN-γand IL-2 (P<0.05). But compare with the Control group, neither the percentage of CD4+CD25+ regulatory T cells nor the cytokine production of splenocytes in the spleens of CIM50 Group mice showed significiantly different (P>0.05).
Conclusion:CIM can be used as an immunomodulator drug and could be useful in protective immunity against schistosome infection of mice in a dose-response manner.
Part 2:Studies on the effcet of co-immunization with CIM and pEGFP-Sj26GST DNA vaccine on the protective efficacy in mice
Objective:To study the protective efficacy of co-immunization with pEGFP-Sj26GST DNA vaccine and CIM against S.japonicum in mice.
Methods:The plasmid pEGFP-Sj26GST and pEGFP vector were amplified as DNA vaccine to immunize BALB/c mice.50 female BALB/c mice were randomly divided into five groups, each with 10 animals. CIM with pEGFP-Sj26GST DNA vaccine immunization was used as co-treated group. The pEGFP or pEGFP-Sj26GST alone was used as the immune challenge group. CIM alone was used as adjuvant groups. Sixteen non-treated mice were used as infected control group. Animals treated with CIM received a daily subcutaneously (s.c) injection of 25 mg/kg from day-2 through the entire 6-week. After 1, 14 and 28 days of treatment mice were immunized intramuscularly with 100μg pEGFP-Sj26GST DNA vaccine or pEGFP vector. Control animals received an equivalent volume of Sodium Chloride via the same route. After two weeks of the final boost, mice were challenged percutaneously with 40 of cercariae for 20 min by the cover glass method. On day 42 following the challenge, adult worms were perfused from hepatic portal system and also manually removed from mesenteric veins after mice were sacrificed. Percent of worm reduction and percent reduction in liver eggs in co-immunization group were compared with that in pEGFP-Sj26GST alone group and infected control group. Liver tissue sections were stained by histopathological examination to detect S. japonicum egg granuloma. The histopathological examination showed a significantly (P<0.01) greater number of egg granulomas in the control group (16.25±2.87) than in the CIM plus pEGFP-Sj26GST group (4.5±1.76). Sections of groups of mice immunized with pEGFP-Sj26GST DNA vaccine plus CIM showed fewer and smaller egg granuloma. In contrast, sections of the control groups showed a greater number of portal egg granulomas.
Results:The recombinant plasmid pEGFP-Sj26GST was successfully abstracted, and could be used as DNA vaccine. Compared with the infected control group, mice inoculated intramuscularly with pEGFP-Sj26GST showed worm reduction of 27%(P<0.05), and co-injection of CIM and pEGFP-Sj26GST dramatically enhanced worm reduction to 79% (P<0.01), there was significant difference between the two groups. In addition, there was substantial reduction in the number of eggs present in the livers and feces of the pEGFP-Sj26GST alone and plus CIM immunized groups (P<0.05) when compared to the control groups.
Conclusion:Compared with the pEGFP-Sj26GST DNA vaccine alone group, the mice co-immunization with pEGFP-Sj26GST and CIM could significiant enhance the protective efficacy against S. japonicum. The immunization with pEGFP-Sj26GST plus CIM could impair the development of schistosome egg granulomas.
Part 3:Studies on the mechanism of CIM to enhance the protective efficacy induced by pEGFP-Sj26GST DNA vaccine
Objective:To explore the effect of pEGFP-Sj26GST DNA vaccine and cimetidie on the percentages of CD4+CD25+ Tregs; To explore the mechanism of CIM in the protective efficacy against S. japonicum generated by pEGFP-Sj26GST DNA vaccine.
Methods:80 female BALB/c mice were randomly divided into five groups, each with 16 animals. CIM with pEGFP-Sj26GST DNA vaccine immunization was used as co-treated group. The pEGFP or pEGFP-Sj26GST alone was used as the immune challenge group. CIM alone was used as adjuvant groups. Sixteen non-treated mice were used as infected control group. Animals treated with CIM received a daily subcutaneously (s.c) injection of 25 mg/kg from day-2 through the entire 6-week. After 1,14 and 28 days of treatment mice were immunized intramuscularly with 100μg pEGFP-Sj26GST DNA vaccine or pEGFP vector. That means the animals were inoculated three times with two weeks interval. Control animals received an equivalent volume of Sodium Chloride via the same route. One week after last immunization,6 mice from each group were sacrificed to determine the anti-Sj26GST antibodies in the individual serum samples (diluted in PBS) by GST-ELISA. Two weeks after the last immunization, mice were challenged percutaneously with 40 of cercariae for 20 min by the cover glass method. On day 42 following the challenge, spleens were obtained sterile, preparation single cell suspension. Flow cytometry was performed to detect the percentage of CD4+CD25+ Tregs. Single suspensions of splenocyte were harvested as describe previously and 2×105 cells/well were cultured in 96-well plates, in triplicate, in RPMI-1640 medium containing 5% FCS at 37℃in a 5% CO2 incubator for 48 h. Then we used MTT to express the splenocyte proliferation responses to ConA in vitro. The concentrations of IFN-γ, IL-12, IL-4, IL-5 and IL-10 in cell culture supernatants were determined as described by the manufacturer of the ELISA Kits.
Results:
1) The mean optical density values (mean±standard deviation) of anti-Sj26GST antibody in the mice immunized with pEGFP-Sj26GST was higher than that in the control group injected with CIM or saline alone (P<0.05). And at the same time, co-injection of CIM and pEGFP-Sj26GST did produce a significantly higher level of anti-Sj26GST antibody (P<0.05), compared with vaccination with pEGFP-Sj26GST alone.
2) The percentage of CD4+CD25+Foxp3+ T cells in spleens of mice treated with pEGFP-Sj26GST was significantly decreased from infected control mice (P<0.05). Meanwhile the percentage of CD4+CD25+Foxp3+ T cells in groups with pEGFP-Sj26GST plus CIM was dramatically dropped down and was significantly lower than those in the infected control groups (P<0.01).
3) Mice immunized with CIM and pEGFP-Sj26GST was seen to produce vigorous lymphocyte responses, compared with the control group (P<0.05).
4) Co-injection of CIM and pEGFP-Sj26GST significantly increased the production of IFN-y and IL-12 compared with pEGFP-Sj26GST alone (P<0.05). However, spleen cells showed no difference in IL-4 and IL-5 production among all five of the groups tested and the expression of the anti-inflammatory cytokine (IL-10) were significantly decreased (P<0.05).
Conclusion:
1) The recombinant plasmid pEGFP-Sj26GST can be used as DNA vaccine. The co-immunization CIM with pEGFP-Sj26GST DNA vaccine could produce a significantly higher level of anti-Sj26GST antibody in BALB/c mice.
2) The percentage of CD4+CD25+Foxp3- T cells in groups with pEGFP-Sj26GST plus CIM was dramatically dropped down and was significantly lower than those in the pEGFP-Sj26GST alone group.
3) Mice immunized with CIM and pEGFP-Sj26GST could produce an enhanced cell-mediated response in vivo.
4) CIM could alter the Th1-Th2 balance in murine hosts even during a Th2-promoting S. japonicum infection. The immunization with pEGFP-Sj26GST plus CIM could significantly enhance the protection of DNA vaccine through enhancing Th1 type immune responses. hi conclusion, these data demonstrated:
1. In our study we confirmed the finding that CIM could exerts immunomodulating properties through enhancing Thl type immune responses during S. japonicum infection.
2. It was firstly reported that the co-immunization CIM with pEGFP-Sj26GST DNA vaccine could significantly enhanced the protection of DNA vaccine.
3. Our data showed for the first time that vaccination of mice with pEGFP-Sj26GST DNA vaccine plus CIM could significantly impair the suppressive function of CD4+CD25+ Treg cells.
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