负载GST抗原树突状细胞疫苗抗日本血吸虫感染的保护效应研究
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摘要
血吸虫病是一种严重危害人民健康,影响社会经济发展的重大传染病。主要分布于非洲、中东、南美和东南亚的76个国家和地区。在东南亚,目前血吸虫病主要流行于5个国家,即中国、菲律宾、印尼、柬埔寨和老挝。虽然经过几十年持续的防治,主要是大规模的人群化疗,各国流行区人群的感染率都比较低,但是流行的状况不稳定。我国为日本血吸虫病流行区。2008年全国调查资料显示,共有现症患者40余万,受威胁人口1亿。血吸虫病引起政府的高度重视,确定为重点防治的传染性疾病之一。
我国血吸虫病防治工作面临的主要问题,包括重复感染严重存在、唯一有效化疗药吡喹酮的长期使用可能产生抗药株、大量哺乳动物保虫宿主的存在。因而寻找长效防治措施成为主要目标。疫苗预防作用被作为血吸虫病综合防治重要的措施之一而倍受关注。世界卫生组织(WHO)专家认为,化疗手段(短效)必须与长效的措施(疫苗)相结合。在疫苗研制的依据上,多年的探索表明,至少在啮齿类和灵长类的某些动物模型,获得性免疫应答可杀灭血吸虫。但是,尽管历经半个世纪的努力,血吸虫病疫苗目前仍然没有取得突破性进展,部分原因在于血吸虫在长期与宿主的共进化过程中,建立起复杂的保护体系,抵御和防御宿主免疫系统的攻击。在日本血吸虫病疫苗领域,近年来最大的进展在于克隆了数以百计的候选抗原,但单一候选抗原分子诱导的保护力鲜有超过40%。而世界卫生组织曾经公布的被认为最有希望的6种曼氏血吸虫病的候选抗原已全部完成测试,无一能稳定诱导40%以上的保护力。WHO认为可能是抗原供应前后制剂稳定性差,以及抗原和佐剂质控不严所致。多年的研究提示抗原递呈作用的重要性,佐剂的选择和免疫接种方案优化也需进一步探讨。
树突状细胞是机体免疫系统岗哨工作的承担者,对自身或来自外界的抗原进行识别和加工。它们不仅识别加工抗原,还能活化初始T细胞,并且调节T细胞应答的性质,即诱导免疫耐受或是使免疫向Thl/Th2方向极化。DC这些重要的生物功能使通过对DC干涉调节免疫应答成为可能。目前已知,佐剂可通过激活DC而增强抗原应答,DC本身也被称为天然佐剂。经抗原体外刺激的DC免疫机体后可有效地诱导T细胞依赖的免疫力,还可诱导小鼠产生强的抗体应答。目前,基于DC的治疗和免疫预防广泛用于癌症和感染性疾病,取得显著进展。在血吸虫候选抗原中,谷胱甘肽S转移酶GST被认为是最有前途的候选抗原。有鉴于此,本文尝试用GST负载的树突状细胞联合CpG ODN免疫小鼠,研究其诱导的体液免疫和细胞免疫,并评价其免疫保护力。研究共分三部分：
一日本血吸虫抗原负载树突状细胞的实验研究
含日本血吸虫大陆株26kDa GST抗原基因的工程菌(Sj26GST/PBV220/DH5α)温控表达后,超声粉碎,应用亲和层析法纯化出重组GST抗原(简称GST),并经SDS-PAGE和Western blotting鉴定。可溶性虫卵抗原按常规制备。共采用两种树突状细胞进行血吸虫抗原负载的研究。小鼠骨髓来源树突状细胞(BMDC)制备培养方法参照文献,用IL-4和GM-CSF诱导培养。培养至第7天调整细胞浓度至2×105/ml接种于24孔培养板,每孔1ml。分别加入不同浓度的SEA、GST刺激细胞,并设LPS和PBS对照组。流式细胞仪检测树突状细胞表面分子CD40、 CD80、CD86的表达情况。结果表明,同本血吸虫抗原GST负载BMDC的最适浓度为1μg/ml.与LPS刺激不同,SEA、GST负载BMDC后,BMDC表面分子CD40、CD80、CD86表达并未上调,表现为不成熟表型。树突状细胞DC2.4按常规方法培养。分别加入不同浓度的GST进行抗原负载,比较树突状细胞表面分子CD40、CD80、CD86的表达水平。FITC标记的抗GST单克隆抗体检测GST抗原负载的情况。荧光显微镜下观察GST抗原负载的树突状细胞内可见特异的绿色荧光,表明抗原被细胞所摄取。流式细胞术分析结果表明,抗原刺激DC2.4的最适浓度为10μg/ml。日本血吸虫抗原GST、SEA负载DC2.4后,可检测到DC2.4表面CD40、CD86分子的平均荧光强度增强,而CD80增强不明显。
二负载GST抗原的树突状细胞疫苗联合CpG-ODN免疫小鼠对日本血吸虫感染的保护性效应研究
比较两种不同CpG ODN序列对树突状细胞DC2.4的刺激效果。流式分析结果表明,CpG ODN最佳刺激浓度为0.5μM, CpG0ODN1序列促DC成熟效果要优于CpG0DN2,且能进一步促进GST负载的树突状细胞的成熟。动物实验共分7组,每组小鼠10只,分别为：DC免疫组、BSA刺激DC免疫组、GST刺激DC免疫组、GST+CpG ODN刺激DC免疫组、CpG ODN刺激DC免疫组、GST蛋白免疫组、不免疫对照组(以下简称DC、BSA-DC、GST-DC、GST+CpG ODN-DC、CpG ODN-DC、GSTpr组)。1-5组每鼠皮下注射1×10。个细胞,共免疫三次,每次间隔两周。第6组第一次每鼠免疫50μg蛋白加完全弗氏佐剂,第二次免疫50μg蛋白加不完全弗氏佐剂,第三次免疫10μg蛋白加不完全弗氏佐剂。末次免疫两周后,每组半数小鼠留作细胞免疫相关检测分析。其余小鼠经皮肤感染30条尾蚴。每次免疫前及攻击感染前采血。攻击感染6周后门静脉灌注法收集成虫,计算减虫率。收集肝脏计数虫卵,计算每克肝组织的虫卵数(EPG)和减卵率。结果：与对照组比较,DC、BSA-DC、GST-DC、 GST+CpG ODN-DC、CpG ODN-DC和GSTpr组小鼠的减虫率分别为9.33%、2.7%、21.33%、53.33%、0和24%,减卵率分别为10.76%、19.37%、39.05%、64.24%、33.64%和34.90%,GST+CpG ODN-DC组小鼠有显著的减虫和减卵效果(P<0.05)。
三GST抗原负载树突状细胞疫苗联合CpG ODN对日本血吸虫感染的免疫保护机制研究
ELISA方法检测免疫血清特异性抗体及其亚类。结果表明,三次免疫后,GST蛋白免疫组产生的抗体滴度最高,并明显高于其他各组。GST-DC组和GST+CpG ODN-DC细胞免疫组的抗体滴度也明显提高,加强免疫后抗体亚类IgGl、IgG2a水平呈现上升趋势。末次免疫后每组小鼠各取5只,分别取小鼠脾脏和淋巴结,制备单细胞悬液。CCK-8法检测淋巴细胞增殖情况。结果表明,抗原刺激后,GST-DC组、GST+CpG ODN-DC组有较明显的增殖。淋巴细胞分离液分离脾脏淋巴细胞,同时以GST抗原(15μg/m1)体外刺激细胞,收集48h、72h和96h培养上清,ELISA法检测诱生的IL-4、IFN-γ和IL-10细胞因子水平。结果表明,与对照组相比,各组IL-4、IL-10水平并未明显提高,组间无明显差异；而相比对照组,GST+CpG ODN-DC组和GST-DC组IFN-γ水平均显著提高,与对照组相比上升了10倍(P<0.05)。上述结果表明,GST+CpG ODN-DC和GST-DC疫苗免疫C57BL/6小鼠后,诱导了体液免疫和细胞免疫应答,以Thl型细胞免疫为主导。
Schistosomiasis remains to be one of the major parasitic diseases of mankind, particularly in sub-Saharan Africa, Middle East, South America and Southeast Asia. And Schistosoma japonicum, one of the species of human schistosomiasis, is still endemic in China, Philippines, Indonesia, Cambodia and Laos. Owing to the availability of effective chemotherapeutic agents, large-scale population chemotheary, the prevalence in the endemic area is low. But epidemic status is unsteady. China is epidemic for Schistosoma japonicum, with400,000cases of schistosomiasis japonica and100million population at risk according to national survey in2008. And now it is regarded as one of the severe infectious diseases that should be focus on diseases control by China government.
Schistosomiasis control in China has obstacles and confronted problems such as the reinfection in particular; the potential drug resistance in repeated chemotherapy, with the only effective drug praziquantel; a wide range of mammalian reservoir host. To develop of a long-term effective control measure has become a major task, of which vaccine development brought widely attention. Experts of WHO suggest that chemotherapy (short-term) must be combined with vaccine (long-term) in schistosomiasis control measures. In the recent years, efforts have accumulated a plenty of evidences in some animal models, including rodent and primate, that schistosome can be defeated by acquired immune response. But a recombinant antigen vaccine against schistosome remains elusive, in part because the parasite deploys complex defensive and offensive strategies to combat immune attack. The most significant progress made in recent years in vaccine against Schistosoma japonicum is gene cloning encoding vaccine candidates. Hundreds of antigens were cloned and expressed and their protective effects were tested in animal but only few induced protection over40%. The6candidate antigens regarded as the most promising ones by WHO have been retested, and unfortunately no one induced protection over40%steadily. The probable reasons may be the inconsistency of antigen preparation and quality control of antigen and adjuvant. A lot of research progress highlights to be recognized the importance of antigen presenting, adjuvance choosing and optimization of immunization routes.
Dendritic cells (DC) are the crucial sentinel cells of the adaptive immune system, continuously sampling the antigenic environment. They not only process and present antigens to activate naive T lymphocytes but also regulate the nature of the T cell response obtained, determining whether it leads to tolerance or to Th1or Th2effector T cell responses. These central roles make manipulation of the DC system an attractive strategy for modulating immune responses. It is now clear that one of the roles of adjuvants which enhance response to antigens is to activate the DC system, while DC themselves have been termed "Nature's Adjuvants". Injection of in vitro pulsed DC may provide an efficient way for inducing T-dependent immunity without the use of external adjuvant and induce strong specific humoral responses in vivo. DC has been used in a very direct way in immunotherapy or prevention of cancer and infectious diseases and has made great success. Of all the vaccine candidate antigens for schistosomiasis, GST is regarded as the most promising one. On that account, we try to use GST pulsed DC to vaccinate mice and evaluate their humoral and cell-mediated immune response. The study included following three parts.
1. Loading antigen of Schistosoma japonicum to dendritic cell
The recombinant plasmid Sj26GST-PBV220containing GST gene was expressed in E.coli DH5a by temperature control. And then26kDa GST was purified by affinity chromatography and ananlyzed by SDS-PAGE and Western blotting. Soluble egg antigen was prepared as routine. We used two kinds of dendritic cells, bone marrow derived dendritic cell and DC2.4, to study the cell phenotype after antigen loaing. Bone marrow derived dendritic cells of mice were induced and cultured in the presence of GM-CSF and IL-4, as was described in reference. After culture for7days, cells were used to seed a flat-bottomed24-well culture microtitre plate, with2×105cells per well dispensed into1ml. Antigens, both GST and SEA were added in different concentration to load BMDC. LPS or PBS were also used to pulse BMDC and served as controls. The expression level of CD40, CD80, CD86were examined and analyzed by fluorescence-activated cell sorter (FACS). Results showed that the optimal concentration for antigen loading to BMDC was1μg/ml. The expression level of CD40, CD80, CD86were not raised in the surface of BMDC loaded with SEA or GST, which was significantly different from that loaded with LPS. So the phenotype of BMDC loaded with SEA or GST was an immature one. Dendritic cell2.4, a cell line of bone marrow derived dendritic cell of mouse, was cultured as routine. Antigen of GST in different concentration was added and the cells were pulsed for16h and then analyzed by FACS. Anti-GST monoclonal antibody conjugated with FITC was used to analyze the antigen loading. Specific green fluorescence was observed in GST pulsed DC under a fluorescence microscope, which indicated a successful antigen loading. According to the results of FACS analysis, the optimal loading concentration for GST antigen was10μg/ml. After loading, the mean fluorescence intensity of CD40and CD86, but not CD80on the surface of DC2.4, were significantly enhanced.
2. Research on protective efficacy induced by dendritic cells pulsed with GST against Schistosoma japonicum infection
The difference of immunostimulatory activities between two kinds of CpG ODN was compared. According to the results of FACS analysis, CpG ODN1was superior to the other in that it induced more mature dendritic cell. Seventy mice were divided into seven groups, vaccinated with GST protein, DC2.4alone or DC2.4pulsed with BSA, GST, GST+CpG ODN, CpG ODN respectively. For the cells vaccinated groups, each mouse was immunized with106cells subcutaneously for three times, at2weeks interval. and then challenged with30±1cercaria two weeks post immunization. For the GST protein vaccinated group, each mouse was vaccinated subcutaneously for50μg GST emulsified with complete Freund's adjuvant and then with incomplete Freund's adjuvant, and10μg GST with incomplete Freund's adjuvant for the last immunization. Two week post-immunization, half of mice in each group were sacrificed for cell-mediated immune response analysis and the rest were challenged with30±1cercaria. Six weeks later, all mice were sacrificed and worms were recovered. The average of adult worm and eggs per gram (EPG) of liver were calculated. Sera were collected before immunization and2week after each immunization. The protective efficacy was evaluated with worm reduction rate and egg reduction rate of the liver. Results showed that worm and egg burden in test groups were significantly lower than unvaccinated control group. The worm reduction rates of groups of DC, BSA-DC, GST-DC, GST+CpG ODN-DC, CpG ODN-DC and GST protein, were9.33%,2.67%,21.33%,53.33%,0,24%, respectively. And the EPG reduction rates were10.76%、19.37%、39.05%、64.24%、33.64%and34.90%respectively. GST+CpG ODN pulsed DC vaccine induced significant worm reduction rate and EPG reduction rate (P<0.05)
3. The study on immune mechanism induced by GST+CpG ODN pulsed DC vaccine
Titres of antigen-specific total IgG,1gG1,1gG2a,1gG2b antibodies in serum samples obtained from vaccinated mice were determined by a GST-based ELISA. Results showed that the antibody level was the highest in the group vaccinated with GST protein. Of the five groups vaccinated with DC, both GST-DC group and GST+CpG ODN-DC group exhibited higher antibody level, comparing to the unvaccinated control groups. And levels of1gG1and lgG2a were both raised as the times of vaccination increased. Two weeks after the last immnuzation, mice were sacrified and spleen and mesenteric lymph nodes were harvested under sterile conditions. These spleens and lymph nodes were pressed through a nylon mesh respectively to remove tissue debris and generate a single-cell suspension. Lymphocyte proliferation assay was performed by the method of CCK8. Results showed that lymphocytes from GST-DC group and GST+CpG ODN group challenged with antigen in vitro proliferated significantly. Spleen lymphocytes were separated using lymphocyte separating reagents. Cytokines released from spleen lymphocyte stimulated in vitro with15μg of GST per ml were determined in the culture supernatants collected at48h,72h and96h. The concentrations of IL-4, IL-10and IFN-y were determined by ELISA. Splenocytes from mice vaccinated with GST-DC and GST+CpG ODN-DC secreted larger amounts of IFN-y than that in other groups (P<0.05). And IL-4and IL-10level among varied groups were not significantly different. These results suggest a potential Th1/Th2immune response but Th1dominating.
我国血吸虫病防治工作面临的主要问题,包括重复感染严重存在、唯一有效化疗药吡喹酮的长期使用可能产生抗药株、大量哺乳动物保虫宿主的存在。因而寻找长效防治措施成为主要目标。疫苗预防作用被作为血吸虫病综合防治重要的措施之一而倍受关注。世界卫生组织(WHO)专家认为,化疗手段(短效)必须与长效的措施(疫苗)相结合。在疫苗研制的依据上,多年的探索表明,至少在啮齿类和灵长类的某些动物模型,获得性免疫应答可杀灭血吸虫。但是,尽管历经半个世纪的努力,血吸虫病疫苗目前仍然没有取得突破性进展,部分原因在于血吸虫在长期与宿主的共进化过程中,建立起复杂的保护体系,抵御和防御宿主免疫系统的攻击。在日本血吸虫病疫苗领域,近年来最大的进展在于克隆了数以百计的候选抗原,但单一候选抗原分子诱导的保护力鲜有超过40%。而世界卫生组织曾经公布的被认为最有希望的6种曼氏血吸虫病的候选抗原已全部完成测试,无一能稳定诱导40%以上的保护力。WHO认为可能是抗原供应前后制剂稳定性差,以及抗原和佐剂质控不严所致。多年的研究提示抗原递呈作用的重要性,佐剂的选择和免疫接种方案优化也需进一步探讨。
树突状细胞是机体免疫系统岗哨工作的承担者,对自身或来自外界的抗原进行识别和加工。它们不仅识别加工抗原,还能活化初始T细胞,并且调节T细胞应答的性质,即诱导免疫耐受或是使免疫向Thl/Th2方向极化。DC这些重要的生物功能使通过对DC干涉调节免疫应答成为可能。目前已知,佐剂可通过激活DC而增强抗原应答,DC本身也被称为天然佐剂。经抗原体外刺激的DC免疫机体后可有效地诱导T细胞依赖的免疫力,还可诱导小鼠产生强的抗体应答。目前,基于DC的治疗和免疫预防广泛用于癌症和感染性疾病,取得显著进展。在血吸虫候选抗原中,谷胱甘肽S转移酶GST被认为是最有前途的候选抗原。有鉴于此,本文尝试用GST负载的树突状细胞联合CpG ODN免疫小鼠,研究其诱导的体液免疫和细胞免疫,并评价其免疫保护力。研究共分三部分：
一日本血吸虫抗原负载树突状细胞的实验研究
含日本血吸虫大陆株26kDa GST抗原基因的工程菌(Sj26GST/PBV220/DH5α)温控表达后,超声粉碎,应用亲和层析法纯化出重组GST抗原(简称GST),并经SDS-PAGE和Western blotting鉴定。可溶性虫卵抗原按常规制备。共采用两种树突状细胞进行血吸虫抗原负载的研究。小鼠骨髓来源树突状细胞(BMDC)制备培养方法参照文献,用IL-4和GM-CSF诱导培养。培养至第7天调整细胞浓度至2×105/ml接种于24孔培养板,每孔1ml。分别加入不同浓度的SEA、GST刺激细胞,并设LPS和PBS对照组。流式细胞仪检测树突状细胞表面分子CD40、 CD80、CD86的表达情况。结果表明,同本血吸虫抗原GST负载BMDC的最适浓度为1μg/ml.与LPS刺激不同,SEA、GST负载BMDC后,BMDC表面分子CD40、CD80、CD86表达并未上调,表现为不成熟表型。树突状细胞DC2.4按常规方法培养。分别加入不同浓度的GST进行抗原负载,比较树突状细胞表面分子CD40、CD80、CD86的表达水平。FITC标记的抗GST单克隆抗体检测GST抗原负载的情况。荧光显微镜下观察GST抗原负载的树突状细胞内可见特异的绿色荧光,表明抗原被细胞所摄取。流式细胞术分析结果表明,抗原刺激DC2.4的最适浓度为10μg/ml。日本血吸虫抗原GST、SEA负载DC2.4后,可检测到DC2.4表面CD40、CD86分子的平均荧光强度增强,而CD80增强不明显。
二负载GST抗原的树突状细胞疫苗联合CpG-ODN免疫小鼠对日本血吸虫感染的保护性效应研究
比较两种不同CpG ODN序列对树突状细胞DC2.4的刺激效果。流式分析结果表明,CpG ODN最佳刺激浓度为0.5μM, CpG0ODN1序列促DC成熟效果要优于CpG0DN2,且能进一步促进GST负载的树突状细胞的成熟。动物实验共分7组,每组小鼠10只,分别为：DC免疫组、BSA刺激DC免疫组、GST刺激DC免疫组、GST+CpG ODN刺激DC免疫组、CpG ODN刺激DC免疫组、GST蛋白免疫组、不免疫对照组(以下简称DC、BSA-DC、GST-DC、GST+CpG ODN-DC、CpG ODN-DC、GSTpr组)。1-5组每鼠皮下注射1×10。个细胞,共免疫三次,每次间隔两周。第6组第一次每鼠免疫50μg蛋白加完全弗氏佐剂,第二次免疫50μg蛋白加不完全弗氏佐剂,第三次免疫10μg蛋白加不完全弗氏佐剂。末次免疫两周后,每组半数小鼠留作细胞免疫相关检测分析。其余小鼠经皮肤感染30条尾蚴。每次免疫前及攻击感染前采血。攻击感染6周后门静脉灌注法收集成虫,计算减虫率。收集肝脏计数虫卵,计算每克肝组织的虫卵数(EPG)和减卵率。结果：与对照组比较,DC、BSA-DC、GST-DC、 GST+CpG ODN-DC、CpG ODN-DC和GSTpr组小鼠的减虫率分别为9.33%、2.7%、21.33%、53.33%、0和24%,减卵率分别为10.76%、19.37%、39.05%、64.24%、33.64%和34.90%,GST+CpG ODN-DC组小鼠有显著的减虫和减卵效果(P<0.05)。
三GST抗原负载树突状细胞疫苗联合CpG ODN对日本血吸虫感染的免疫保护机制研究
ELISA方法检测免疫血清特异性抗体及其亚类。结果表明,三次免疫后,GST蛋白免疫组产生的抗体滴度最高,并明显高于其他各组。GST-DC组和GST+CpG ODN-DC细胞免疫组的抗体滴度也明显提高,加强免疫后抗体亚类IgGl、IgG2a水平呈现上升趋势。末次免疫后每组小鼠各取5只,分别取小鼠脾脏和淋巴结,制备单细胞悬液。CCK-8法检测淋巴细胞增殖情况。结果表明,抗原刺激后,GST-DC组、GST+CpG ODN-DC组有较明显的增殖。淋巴细胞分离液分离脾脏淋巴细胞,同时以GST抗原(15μg/m1)体外刺激细胞,收集48h、72h和96h培养上清,ELISA法检测诱生的IL-4、IFN-γ和IL-10细胞因子水平。结果表明,与对照组相比,各组IL-4、IL-10水平并未明显提高,组间无明显差异；而相比对照组,GST+CpG ODN-DC组和GST-DC组IFN-γ水平均显著提高,与对照组相比上升了10倍(P<0.05)。上述结果表明,GST+CpG ODN-DC和GST-DC疫苗免疫C57BL/6小鼠后,诱导了体液免疫和细胞免疫应答,以Thl型细胞免疫为主导。
Schistosomiasis remains to be one of the major parasitic diseases of mankind, particularly in sub-Saharan Africa, Middle East, South America and Southeast Asia. And Schistosoma japonicum, one of the species of human schistosomiasis, is still endemic in China, Philippines, Indonesia, Cambodia and Laos. Owing to the availability of effective chemotherapeutic agents, large-scale population chemotheary, the prevalence in the endemic area is low. But epidemic status is unsteady. China is epidemic for Schistosoma japonicum, with400,000cases of schistosomiasis japonica and100million population at risk according to national survey in2008. And now it is regarded as one of the severe infectious diseases that should be focus on diseases control by China government.
Schistosomiasis control in China has obstacles and confronted problems such as the reinfection in particular; the potential drug resistance in repeated chemotherapy, with the only effective drug praziquantel; a wide range of mammalian reservoir host. To develop of a long-term effective control measure has become a major task, of which vaccine development brought widely attention. Experts of WHO suggest that chemotherapy (short-term) must be combined with vaccine (long-term) in schistosomiasis control measures. In the recent years, efforts have accumulated a plenty of evidences in some animal models, including rodent and primate, that schistosome can be defeated by acquired immune response. But a recombinant antigen vaccine against schistosome remains elusive, in part because the parasite deploys complex defensive and offensive strategies to combat immune attack. The most significant progress made in recent years in vaccine against Schistosoma japonicum is gene cloning encoding vaccine candidates. Hundreds of antigens were cloned and expressed and their protective effects were tested in animal but only few induced protection over40%. The6candidate antigens regarded as the most promising ones by WHO have been retested, and unfortunately no one induced protection over40%steadily. The probable reasons may be the inconsistency of antigen preparation and quality control of antigen and adjuvant. A lot of research progress highlights to be recognized the importance of antigen presenting, adjuvance choosing and optimization of immunization routes.
Dendritic cells (DC) are the crucial sentinel cells of the adaptive immune system, continuously sampling the antigenic environment. They not only process and present antigens to activate naive T lymphocytes but also regulate the nature of the T cell response obtained, determining whether it leads to tolerance or to Th1or Th2effector T cell responses. These central roles make manipulation of the DC system an attractive strategy for modulating immune responses. It is now clear that one of the roles of adjuvants which enhance response to antigens is to activate the DC system, while DC themselves have been termed "Nature's Adjuvants". Injection of in vitro pulsed DC may provide an efficient way for inducing T-dependent immunity without the use of external adjuvant and induce strong specific humoral responses in vivo. DC has been used in a very direct way in immunotherapy or prevention of cancer and infectious diseases and has made great success. Of all the vaccine candidate antigens for schistosomiasis, GST is regarded as the most promising one. On that account, we try to use GST pulsed DC to vaccinate mice and evaluate their humoral and cell-mediated immune response. The study included following three parts.
1. Loading antigen of Schistosoma japonicum to dendritic cell
The recombinant plasmid Sj26GST-PBV220containing GST gene was expressed in E.coli DH5a by temperature control. And then26kDa GST was purified by affinity chromatography and ananlyzed by SDS-PAGE and Western blotting. Soluble egg antigen was prepared as routine. We used two kinds of dendritic cells, bone marrow derived dendritic cell and DC2.4, to study the cell phenotype after antigen loaing. Bone marrow derived dendritic cells of mice were induced and cultured in the presence of GM-CSF and IL-4, as was described in reference. After culture for7days, cells were used to seed a flat-bottomed24-well culture microtitre plate, with2×105cells per well dispensed into1ml. Antigens, both GST and SEA were added in different concentration to load BMDC. LPS or PBS were also used to pulse BMDC and served as controls. The expression level of CD40, CD80, CD86were examined and analyzed by fluorescence-activated cell sorter (FACS). Results showed that the optimal concentration for antigen loading to BMDC was1μg/ml. The expression level of CD40, CD80, CD86were not raised in the surface of BMDC loaded with SEA or GST, which was significantly different from that loaded with LPS. So the phenotype of BMDC loaded with SEA or GST was an immature one. Dendritic cell2.4, a cell line of bone marrow derived dendritic cell of mouse, was cultured as routine. Antigen of GST in different concentration was added and the cells were pulsed for16h and then analyzed by FACS. Anti-GST monoclonal antibody conjugated with FITC was used to analyze the antigen loading. Specific green fluorescence was observed in GST pulsed DC under a fluorescence microscope, which indicated a successful antigen loading. According to the results of FACS analysis, the optimal loading concentration for GST antigen was10μg/ml. After loading, the mean fluorescence intensity of CD40and CD86, but not CD80on the surface of DC2.4, were significantly enhanced.
2. Research on protective efficacy induced by dendritic cells pulsed with GST against Schistosoma japonicum infection
The difference of immunostimulatory activities between two kinds of CpG ODN was compared. According to the results of FACS analysis, CpG ODN1was superior to the other in that it induced more mature dendritic cell. Seventy mice were divided into seven groups, vaccinated with GST protein, DC2.4alone or DC2.4pulsed with BSA, GST, GST+CpG ODN, CpG ODN respectively. For the cells vaccinated groups, each mouse was immunized with106cells subcutaneously for three times, at2weeks interval. and then challenged with30±1cercaria two weeks post immunization. For the GST protein vaccinated group, each mouse was vaccinated subcutaneously for50μg GST emulsified with complete Freund's adjuvant and then with incomplete Freund's adjuvant, and10μg GST with incomplete Freund's adjuvant for the last immunization. Two week post-immunization, half of mice in each group were sacrificed for cell-mediated immune response analysis and the rest were challenged with30±1cercaria. Six weeks later, all mice were sacrificed and worms were recovered. The average of adult worm and eggs per gram (EPG) of liver were calculated. Sera were collected before immunization and2week after each immunization. The protective efficacy was evaluated with worm reduction rate and egg reduction rate of the liver. Results showed that worm and egg burden in test groups were significantly lower than unvaccinated control group. The worm reduction rates of groups of DC, BSA-DC, GST-DC, GST+CpG ODN-DC, CpG ODN-DC and GST protein, were9.33%,2.67%,21.33%,53.33%,0,24%, respectively. And the EPG reduction rates were10.76%、19.37%、39.05%、64.24%、33.64%and34.90%respectively. GST+CpG ODN pulsed DC vaccine induced significant worm reduction rate and EPG reduction rate (P<0.05)
3. The study on immune mechanism induced by GST+CpG ODN pulsed DC vaccine
Titres of antigen-specific total IgG,1gG1,1gG2a,1gG2b antibodies in serum samples obtained from vaccinated mice were determined by a GST-based ELISA. Results showed that the antibody level was the highest in the group vaccinated with GST protein. Of the five groups vaccinated with DC, both GST-DC group and GST+CpG ODN-DC group exhibited higher antibody level, comparing to the unvaccinated control groups. And levels of1gG1and lgG2a were both raised as the times of vaccination increased. Two weeks after the last immnuzation, mice were sacrified and spleen and mesenteric lymph nodes were harvested under sterile conditions. These spleens and lymph nodes were pressed through a nylon mesh respectively to remove tissue debris and generate a single-cell suspension. Lymphocyte proliferation assay was performed by the method of CCK8. Results showed that lymphocytes from GST-DC group and GST+CpG ODN group challenged with antigen in vitro proliferated significantly. Spleen lymphocytes were separated using lymphocyte separating reagents. Cytokines released from spleen lymphocyte stimulated in vitro with15μg of GST per ml were determined in the culture supernatants collected at48h,72h and96h. The concentrations of IL-4, IL-10and IFN-y were determined by ELISA. Splenocytes from mice vaccinated with GST-DC and GST+CpG ODN-DC secreted larger amounts of IFN-y than that in other groups (P<0.05). And IL-4and IL-10level among varied groups were not significantly different. These results suggest a potential Th1/Th2immune response but Th1dominating.
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2. Sher A, Hieny S, James SL, et al. Mechanisms of protective immunity against Schistosoma mansoni infection in mice vaccinated with irradiated cercariae. Ⅱ. Analysis of immunity in hosts deficient in T lymphocytes, B lymphocytes, or complement. J Immunol,1982; 128 (4):1880-1884
3. Dillon, G.P. et al. (2008) Altered patterns of gene expression underlying the enhanced immunogenicity of radiation-attenuated schistosomes. PLoS Negl Trop Dis 2, e240
4. Kelly EA, Colley DG. In vivo effects of monoclonal anti-L3T4 antibody on immune responsiveness of mice infected with Schistosoma mansoni. Reduction of irradiated cercariae-induced resistance. J Immunol,1988,140(8):2737-2745
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