C3d对分泌型柯萨奇病毒B组3型VP1 DNA疫苗的免疫增强作用
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摘要
目的:柯萨奇病毒属小RNA病毒科肠道病毒属,其中B组3型(Coxsackievirus group B type 3, CVB3)是引起柯萨奇病毒爆发性流行的最常见的血清型之一,其感染是导致急、慢性心肌炎和扩张性心肌病重要原因,因此对其防治手段的研究日益受到人们的重视。由于目前尚无特别有效的防治方法,所以研制CVB3疫苗成为预防该病毒感染的重要策略。DNA疫苗自90年代发展以来,在抗病毒免疫中具有诱导机体产生特异性体液免疫和细胞免疫应答的优势。国内外研究发现,编码CVB3主要衣壳蛋白VP1的DNA疫苗能够诱导小鼠产生体液和细胞免疫应答,并对感染小鼠具有一定的保护作用,但存在免疫原性差、免疫保护作用不能令人满意的问题。因此,增强VP1基因的免疫原性,提高CVB3特异性体液和细胞免疫应答,提高其免疫保护作用是本研究的主要设计思路。
补体是天然免疫系统的重要组成部分,近年来的研究表明,补体裂解片段还可以触发和调节获得性免疫应答。补体片段C3d由补体蛋白C3活化裂解而来,是不能被蛋白酶裂解的最小片段。C3d的受体是II型补体受体(complement receptor type II, CR2),主要分布在成熟的B淋巴细胞和滤泡树突状细胞(follicular dendritic cells, FDCs)。C3d与抗原偶联后可通过特异性受体CR2将抗原信号提供给CR2+细胞,从而诱导特异性免疫应答。有研究表明,C3d与CR2结合可以为抗原与B细胞受体(B cell Ag-receptor,BCR)的交联提供共刺激信号,降低B细胞的活化阈值,促进抗体的亲和力成熟,调节B细胞与T细胞之间的相互作用,从而提高疫苗的免疫原性。鉴于C3d的分子佐剂作用,本研究将编码带有白细胞介素2(interleukin-2,IL-2)信号肽的分泌型CVB3 VP1(secreted form of CVB3 VP1, sVP1)和三拷贝C3d基因拼接,将连接产物插入真核表达质粒pcDNA3,构建重组质粒pcDNA3/sVP1-C3d3,通过研究该疫苗免疫BALB/c小鼠后诱生的特异性免疫应答和病毒攻击后的免疫保护作用来探讨这种疫苗的应用效果。
方法:(1)用合适的限制性内切酶对含有C3d3编码基因的质粒pSG5.C3d3.YL进行双酶切,经酶切鉴定确认C3d3片段正确。(2)以编码sVP1蛋白的DNA为模板,用自行设计的sVP1的特异性引物进行PCR扩增,将扩增产物与pGEM-T载体连接,转化DH5α大肠杆菌,提取质粒进行酶切鉴定和测序筛选重组子。(3)经酶切鉴定和测序确认后,取双酶切的sVP1片段与经过同样两种酶酶切的pcDNA3载体连接,构建真核表达质粒pcDNA3/sVP1。(4)将C3d3从pSG5.C3d3.YL的BglII和XbalI位点之间切出,同时以BamHI和XbalI双酶切pcDNA3/sVP1,将C3d3插入pcDNA3/sVP1,构建真核表达重组质粒pcDNA3/sVP1-C3d3。(5)用碱裂解法从转化的DH5α大肠杆菌中大量提取质粒pcDNA3/sVP1、pcDNA3/sVP1-C3d3和pcDNA3,用聚乙二醇(polyethylene glycol, PEG)沉淀法进行纯化。(6)动物实验:将6～8周龄雄性BALB/c小鼠随机分为3组,分别为pcDNA3对照组、pcDNA3/sVP1对照组和pcDNA3/sVP1-C3d3组,每组14只,纯化后的质粒以生理盐水稀释后,股四头肌注射免疫小鼠,每次每只接种100μg,每4周免疫1次,共免疫3次;每次免疫后第14天,眼眶静脉取血,分离血清,用微量中和试验(固定病毒-稀释血清法)检测血清中CVB3特异性中和抗体的水平;第3次免疫后3周,每组3只小鼠取脾脏制备淋巴细胞悬液,采用细胞计数试剂盒(cell counting kit-8, CCK-8)法进行特异性淋巴细胞增殖活性和特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte, CTL)杀伤活性的检测;另每组8只小鼠用致死量的CVB3(5LD50)进行腹腔注射,观察并记录小鼠死亡情况至感染后第21天;每组剩余的3只小鼠用3LD50CVB3攻击,注射病毒后第7天处死,用于血中病毒滴度的测定,同时取不同处理组的心脏制备石蜡切片,HE染色,观察各组小鼠心肌病理学改变。
结果:(1)以限制性内切酶双酶切从质粒中获得C3d3片段,PCR扩增出sVP1基因片段,采用DNA测序和双酶切证实这两段基因片段的长度均与报道的基因序列相一致。(2)成功构建真核表达质粒pcDNA3/sVP1-C3d3,双酶切结果证实插入和连接均正确。(3)三次免疫接种小鼠后,各组血清中和抗体的平均效价分别为:pcDNA3/sVP1组1:8.91,1:15.87,1:28.28;pcDNA3/sVP1-C3d3组1:8.41,1:25.19,1:33.65;pcDNA3组各次平均效价均低于1:5.00。单因素方差分析表明,三次免疫后,pcDNA3/sVP1组和pcDNA3/sVP1-C3d3组中和抗体滴度逐次增加( P<0.05 );第三次免疫后pcDNA3/sVP1-C3d3组中和抗体滴度高于pcDNA3/sVP1组(P<0.05)。(4)小鼠脾脏淋巴细胞增殖试验分别以CVB3和刀豆蛋白A(concanavalin A, ConA)体外刺激淋巴细胞,检测细胞增殖活性。单因素方差分析表明,以CVB3刺激时,与pcDNA3组和pcDNA3/sVP1组相比,pcDNA3/sVP1-C3d3组的增殖活性显著升高(P<0.05);以ConA刺激时, pcDNA3/sVP1-C3d3组和pcDNA3/sVP1组均显著高于pcDNA3组(P<0.05),但这两组之间相比无统计学差异。(5)小鼠脾脏特异性CTL杀伤活性经单因素方差分析表明,pcDNA3/sVP1-C3d3免疫组在所有实验组中杀伤活性最高,而pcDNA3免疫组则最低,两两比较P<0.05。(6)用5LD50攻击小鼠,各组21天生存率分别为:pcDNA3/sVP1-C3d3组50%,pcDNA3/SVP1组25%,pcDNA3组无生存。但经χ2检验,两两比较差别无统计学意义。用Kaplan-Meier法进行生存分析表明,各组生存率曲线分布有差别(P<0.05),进一步两两比较说明pcDNA3/sVP1-C3d3组小鼠的生存状况好于pcDNA3组( P<0.05 ),但pcDNA3/SVP1-C3d3组与pcDNA3/sVP1组比较差异无统计学意义。(7)与两个对照组相比,pcDNA3/sVP1-C3d3组小鼠血中病毒滴度显著降低(P<0.05)。(8)心肌病理学检查发现pcDNA3/sVP1-C3d3组较对照组心肌病理学变化均有所减轻,但各组之间并无明显差别。
结论:(1)通过限制性内切酶双酶切成功得到C3d3基因片段。(2)成功构建真核表达质粒pcDNA3/sVP1-C3d3。(3)用致死量CVB3攻击小鼠后,经pcDNA3/sVP1-C3d3免疫后的小鼠生存率有所提高,Kaplan-Meier生存分析表明pcDNA3/ sVP1-C3d3组的生存状况优于pcDNA3组。(4)小鼠血中中和抗体滴度随免疫次数的增加而提高,三次免疫后C3d能够显著提高sVP1基因免疫所诱导的中和抗体水平。(5) pcDNA3/ sVP1-C3d3能够增强小鼠淋巴细胞增殖活性和特异性CTL杀伤水平。(6)中和抗体滴度、特异性淋巴细胞增殖指数、CTL杀伤率、血中病毒滴度和心肌切片病理学分析结果和小鼠生存率一致,说明C3d可以显著增强sVP1基因免疫诱导的特异性免疫应答,但其对于小鼠受到致死量病毒攻击时的免疫保护作用尚不能达到满意效果。
Object: Coxsackievirus group B(CVB), a member of the picornavirus group, is an important human pathogen. Among the six serotypes of CVB, CVB3 is increasingly recognized to be closely associated with acute/chronic myocardial disease and dilated cardiomyopathy cases. DNA vaccine is known for its ability to induce both humoral and cellular immune responses when delivered into animals. Its potential to generate specific CTL affords it the opportunity to establish new preventive procedures against viral infection. VP1, the major capsid protein of CVB3, can induce the production of effective neutralizing antibody. Although the vaccine constructed with VP1 alone could induce the production of antibody, the titers of antibody were usually too low to protect the host from lethal CVB3 challenge. Therefore, it appears to be an important strategy in our study to improve immunological potency and induce CVB3-specific immune responses, as well as enhance the protective efficacy.
The complement system plays an important role in innate immunity. More recently, there has been an accumulating body of evidence indicating that the fragment of complement also participates in the adaptive immune response. C3d is one of the final degradation products of the third complement protein 3, which can’t be hydrolysed any more by enzyme. C3d receptor, complement receptor type II (CR2, CD21), is primarily presented on the membranes of mature B cells and follicular dendritic cells (FDCs). When C3d is bond to foreign antigens (Ags), it can simultaneously bind to CR2+ cells. The attachment of foreign Ag to CR2+ cells through C3d facilitates the cross-linking of the Ag to the B cell membrane-bound receptor, which can lower the threshold of activating B cells, promote the affinity maturation of antibody and regulate the interaction of B cells and T cells. In this study, a fusion gene vaccine conjugating C3d3 to a secreted form of CVB3 VP1 was constructed. The immune effect was then evaluated by specific immune response and protection of mice with DNA vaccinations.
Methods: (1) C3d3 cDNA was obtained by cutting the presented plasmid pSG5.C3d3.YL with proper restriction endonucleases. (2) The secreted form of CVB3 VP1 (sVP1) cDNA was amplified by PCR and cloned into pGEM-T vector. pGEM-T/sVP1 was then transformed E.coli DH5αand cultured in medium containing Ampicilin (100μg/ml) 2YT. The plasmids were extracted and identified by restriction endonuleases digestion as well as sequencing. (3) sVP1 was obtained from pGEM-T/sVP1 by proper endonucleases digestion and then inserted into pcDNA3 vector which was cut by the same endonucleases. As a result, a eukaryotic expressing plasmid of pcDNA3/sVP1 was formed. (4) Three copies of C3d were introduced into pcDNA3/sVP1 to construct pcDNA3/sVP1-C3d3 and restriction endonuleases digestion was used to identify the fused insertion. (5) Large preparation were performed to obtain the plasmids of pcDNA3/sVP1 pcDNA3/sVP1-C3d3 and pcDNA3 from transformed E.coli DH5α, and purified by PEG. (6) BALB/c mice aged 6-8 weeks were inoculated intramuscularly (i.m.) three times at four weeks’interval with 100μg of pcDNA3/sVP1-C3d3, pcDNA3/sVP1 and pcDNA3 plasmids respectively. Fourteen days after every injection, sera of each group were collected and the titers for CVB3-specific neutralizing antibodies were measured. Three weeks after the third immunization, splenocytes from three immunized mice of each group were stimulated by inactivated CVB3 and harvested to analyze the lymphocytic proliferative activity and specific CTL cytotoxic activity by CCK-8 assay. At the same time, another eight mice were challenged with 5LD50 CVB3 and the number of surviving animals was monitored up to three weeks post infection. Furthermore, the rest mice of each group were challenged with 3LD50 CVB3 and sacrificed after seven days in order to evaluate the titers of blood viruses. Meanwhile, hearts of mice was fixed with 10% formalin, dehydrated with grading ethanol, embedded in paraffin, and cut into 5μm sections. Some sections were stained with hemotoxylin and eosin to observe the changes of the myocardium.
Results: (1) Gene fragment of C3d3 was obtained by restriction endonucleases digestion and sVP1 fragment was amplified by PCR. After restriction endonucleases digestion, both of the length was the same as expected. DNA sequencing showed that sVP1 was identical to the sequence recorded in Genbank. (2) Three copies of C3d were introduced into pcDNA3/sVP1 to form pcDNA3/sVP1-C3d3. The result of enzyme digestion was identical as expected and suggested that the eukaryotic expressing plasmid pcDNA3/sVP1-C3d3 had been constructed successfully. (3) When mice were immunized with the plasmids, the antibody titers increased with the time of inoculation. The mean titers of neutralizing antibody after every immunization in pcDNA3/sVP1 group were 1:8.91,1:15.87,1:28.28, respectively, and that in pcDNA3/sVP1-C3d3 group were 1:8.41,1:25.19,1:33.65, while it is always lower than 1:5 in mice immunized with pcDNA3. More specifically, mice with pcDNA3/sVP1-C3d3 elicited the strongest neutralizing antibody after the third immunization. When compared with that of pcDNA3/sVP1, the difference was significant (P<0.05). (4) Splenocytes from vaccinated mice showed different proliferative activity due to different stimulants of CVB3 or ConA. When stimulated by CVB3, the splenocytes from mice with pcDNA3/sVP1-C3d3 showed significantly stronger proliferative activity than that with pcDNA3/sVP1 (P<0.05). While when stimulated by ConA, the proliferative activity of mice with pcDNA3/sVP1-C3d3 and pcDNA3/sVP1 were both stronger than that with pcDNA3, but the difference between the two stronger groups was not significant. (5) DNA expressing sVP1 fused to three copies of C3d efficiently enhanced the specific lymphocytic CTL cytotoxic activity, which was remarkably stronger than that in the mice immunized with pcDNA3/sVP1. (6) Protection of mice from death after lethal CVB3 (5LD50) challenge was augmented to 50% with fused DNA, while mice with pcDNA3/sVP1 was only 25% and no one survived in pcDNA3 group. Chi-square test indicated that differences between any two groups were not significant by the multiple comparisons. Kaplan-Meier test was used to compare the differences of survival curves, which indicated that the survive state of pcDNA3/sVP1-C3d3 group is better than that of pcDNA3 group (P<0.05), but difference between pcDNA3/sVP1-C3d3 and pcDNA3/sVP1 was not significant (P>0.05). (7) After 3LD50 CVB3 challenge, the virus titers of blood in pcDNA3/sVP1-C3d3 group were only 2.58±0.66, significantly lower than that of pcDNA3/sVP1 group (3.61±0.41). (8) The histopathology changes in mice immunized with fused DNA were less serious compared with that in mice of other groups, but significant difference couldn’t be found between them.
Conclusion: (1) The gene fragment of C3d3 was obtained by restriction endonucleases digestion successfully. (2) The eukaryotic expressing plasmid pcDNA3/sVP1-C3d3 was constructed successfully. (3) The survival rate of mice with pcDNA3/sVP1-C3d3 peaked to 50%, and pcDNA3/sVP1 was 25%, while no mice survived in pcDNA3 group. The differences were not significant by Chi-square test. While, the result of Kaplan-Meier test showed that the survival state of pcDNA3/sVP1-C3d3 group was better than that of pcDNA3 group (P<0.05). (4) After administration of pcDNA3/sVP1-C3d3 and pcDNA3/sVP1 to BALB/c mice, the neutralizing antibody increased along with the inoculation times. More meaningfully, C3d3 could induce much stronger antibody from mice. (5) Specific lymphocytic proliferative activity and CTL cytotoxic activity in immunized mice with pcDNA3/sVP1-C3d3 were markedly stronger than those in mice immunized with pcDNA3/sVP1 and pcDNA3. (6) Overall, the results of neutralizing antibody titers, specific lymphocytic proliferative activity, specific CTL cytotoxic activity, viruses titers and histopathology all indicate that C3d can strongly enhance the specific immune response induced by sVP1 gene vaccination, but the protection of mice from death after lethal CVB3 (5LD50) challenge are still not significant now.
补体是天然免疫系统的重要组成部分,近年来的研究表明,补体裂解片段还可以触发和调节获得性免疫应答。补体片段C3d由补体蛋白C3活化裂解而来,是不能被蛋白酶裂解的最小片段。C3d的受体是II型补体受体(complement receptor type II, CR2),主要分布在成熟的B淋巴细胞和滤泡树突状细胞(follicular dendritic cells, FDCs)。C3d与抗原偶联后可通过特异性受体CR2将抗原信号提供给CR2+细胞,从而诱导特异性免疫应答。有研究表明,C3d与CR2结合可以为抗原与B细胞受体(B cell Ag-receptor,BCR)的交联提供共刺激信号,降低B细胞的活化阈值,促进抗体的亲和力成熟,调节B细胞与T细胞之间的相互作用,从而提高疫苗的免疫原性。鉴于C3d的分子佐剂作用,本研究将编码带有白细胞介素2(interleukin-2,IL-2)信号肽的分泌型CVB3 VP1(secreted form of CVB3 VP1, sVP1)和三拷贝C3d基因拼接,将连接产物插入真核表达质粒pcDNA3,构建重组质粒pcDNA3/sVP1-C3d3,通过研究该疫苗免疫BALB/c小鼠后诱生的特异性免疫应答和病毒攻击后的免疫保护作用来探讨这种疫苗的应用效果。
方法:(1)用合适的限制性内切酶对含有C3d3编码基因的质粒pSG5.C3d3.YL进行双酶切,经酶切鉴定确认C3d3片段正确。(2)以编码sVP1蛋白的DNA为模板,用自行设计的sVP1的特异性引物进行PCR扩增,将扩增产物与pGEM-T载体连接,转化DH5α大肠杆菌,提取质粒进行酶切鉴定和测序筛选重组子。(3)经酶切鉴定和测序确认后,取双酶切的sVP1片段与经过同样两种酶酶切的pcDNA3载体连接,构建真核表达质粒pcDNA3/sVP1。(4)将C3d3从pSG5.C3d3.YL的BglII和XbalI位点之间切出,同时以BamHI和XbalI双酶切pcDNA3/sVP1,将C3d3插入pcDNA3/sVP1,构建真核表达重组质粒pcDNA3/sVP1-C3d3。(5)用碱裂解法从转化的DH5α大肠杆菌中大量提取质粒pcDNA3/sVP1、pcDNA3/sVP1-C3d3和pcDNA3,用聚乙二醇(polyethylene glycol, PEG)沉淀法进行纯化。(6)动物实验:将6～8周龄雄性BALB/c小鼠随机分为3组,分别为pcDNA3对照组、pcDNA3/sVP1对照组和pcDNA3/sVP1-C3d3组,每组14只,纯化后的质粒以生理盐水稀释后,股四头肌注射免疫小鼠,每次每只接种100μg,每4周免疫1次,共免疫3次;每次免疫后第14天,眼眶静脉取血,分离血清,用微量中和试验(固定病毒-稀释血清法)检测血清中CVB3特异性中和抗体的水平;第3次免疫后3周,每组3只小鼠取脾脏制备淋巴细胞悬液,采用细胞计数试剂盒(cell counting kit-8, CCK-8)法进行特异性淋巴细胞增殖活性和特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte, CTL)杀伤活性的检测;另每组8只小鼠用致死量的CVB3(5LD50)进行腹腔注射,观察并记录小鼠死亡情况至感染后第21天;每组剩余的3只小鼠用3LD50CVB3攻击,注射病毒后第7天处死,用于血中病毒滴度的测定,同时取不同处理组的心脏制备石蜡切片,HE染色,观察各组小鼠心肌病理学改变。
结果:(1)以限制性内切酶双酶切从质粒中获得C3d3片段,PCR扩增出sVP1基因片段,采用DNA测序和双酶切证实这两段基因片段的长度均与报道的基因序列相一致。(2)成功构建真核表达质粒pcDNA3/sVP1-C3d3,双酶切结果证实插入和连接均正确。(3)三次免疫接种小鼠后,各组血清中和抗体的平均效价分别为:pcDNA3/sVP1组1:8.91,1:15.87,1:28.28;pcDNA3/sVP1-C3d3组1:8.41,1:25.19,1:33.65;pcDNA3组各次平均效价均低于1:5.00。单因素方差分析表明,三次免疫后,pcDNA3/sVP1组和pcDNA3/sVP1-C3d3组中和抗体滴度逐次增加( P<0.05 );第三次免疫后pcDNA3/sVP1-C3d3组中和抗体滴度高于pcDNA3/sVP1组(P<0.05)。(4)小鼠脾脏淋巴细胞增殖试验分别以CVB3和刀豆蛋白A(concanavalin A, ConA)体外刺激淋巴细胞,检测细胞增殖活性。单因素方差分析表明,以CVB3刺激时,与pcDNA3组和pcDNA3/sVP1组相比,pcDNA3/sVP1-C3d3组的增殖活性显著升高(P<0.05);以ConA刺激时, pcDNA3/sVP1-C3d3组和pcDNA3/sVP1组均显著高于pcDNA3组(P<0.05),但这两组之间相比无统计学差异。(5)小鼠脾脏特异性CTL杀伤活性经单因素方差分析表明,pcDNA3/sVP1-C3d3免疫组在所有实验组中杀伤活性最高,而pcDNA3免疫组则最低,两两比较P<0.05。(6)用5LD50攻击小鼠,各组21天生存率分别为:pcDNA3/sVP1-C3d3组50%,pcDNA3/SVP1组25%,pcDNA3组无生存。但经χ2检验,两两比较差别无统计学意义。用Kaplan-Meier法进行生存分析表明,各组生存率曲线分布有差别(P<0.05),进一步两两比较说明pcDNA3/sVP1-C3d3组小鼠的生存状况好于pcDNA3组( P<0.05 ),但pcDNA3/SVP1-C3d3组与pcDNA3/sVP1组比较差异无统计学意义。(7)与两个对照组相比,pcDNA3/sVP1-C3d3组小鼠血中病毒滴度显著降低(P<0.05)。(8)心肌病理学检查发现pcDNA3/sVP1-C3d3组较对照组心肌病理学变化均有所减轻,但各组之间并无明显差别。
结论:(1)通过限制性内切酶双酶切成功得到C3d3基因片段。(2)成功构建真核表达质粒pcDNA3/sVP1-C3d3。(3)用致死量CVB3攻击小鼠后,经pcDNA3/sVP1-C3d3免疫后的小鼠生存率有所提高,Kaplan-Meier生存分析表明pcDNA3/ sVP1-C3d3组的生存状况优于pcDNA3组。(4)小鼠血中中和抗体滴度随免疫次数的增加而提高,三次免疫后C3d能够显著提高sVP1基因免疫所诱导的中和抗体水平。(5) pcDNA3/ sVP1-C3d3能够增强小鼠淋巴细胞增殖活性和特异性CTL杀伤水平。(6)中和抗体滴度、特异性淋巴细胞增殖指数、CTL杀伤率、血中病毒滴度和心肌切片病理学分析结果和小鼠生存率一致,说明C3d可以显著增强sVP1基因免疫诱导的特异性免疫应答,但其对于小鼠受到致死量病毒攻击时的免疫保护作用尚不能达到满意效果。
Object: Coxsackievirus group B(CVB), a member of the picornavirus group, is an important human pathogen. Among the six serotypes of CVB, CVB3 is increasingly recognized to be closely associated with acute/chronic myocardial disease and dilated cardiomyopathy cases. DNA vaccine is known for its ability to induce both humoral and cellular immune responses when delivered into animals. Its potential to generate specific CTL affords it the opportunity to establish new preventive procedures against viral infection. VP1, the major capsid protein of CVB3, can induce the production of effective neutralizing antibody. Although the vaccine constructed with VP1 alone could induce the production of antibody, the titers of antibody were usually too low to protect the host from lethal CVB3 challenge. Therefore, it appears to be an important strategy in our study to improve immunological potency and induce CVB3-specific immune responses, as well as enhance the protective efficacy.
The complement system plays an important role in innate immunity. More recently, there has been an accumulating body of evidence indicating that the fragment of complement also participates in the adaptive immune response. C3d is one of the final degradation products of the third complement protein 3, which can’t be hydrolysed any more by enzyme. C3d receptor, complement receptor type II (CR2, CD21), is primarily presented on the membranes of mature B cells and follicular dendritic cells (FDCs). When C3d is bond to foreign antigens (Ags), it can simultaneously bind to CR2+ cells. The attachment of foreign Ag to CR2+ cells through C3d facilitates the cross-linking of the Ag to the B cell membrane-bound receptor, which can lower the threshold of activating B cells, promote the affinity maturation of antibody and regulate the interaction of B cells and T cells. In this study, a fusion gene vaccine conjugating C3d3 to a secreted form of CVB3 VP1 was constructed. The immune effect was then evaluated by specific immune response and protection of mice with DNA vaccinations.
Methods: (1) C3d3 cDNA was obtained by cutting the presented plasmid pSG5.C3d3.YL with proper restriction endonucleases. (2) The secreted form of CVB3 VP1 (sVP1) cDNA was amplified by PCR and cloned into pGEM-T vector. pGEM-T/sVP1 was then transformed E.coli DH5αand cultured in medium containing Ampicilin (100μg/ml) 2YT. The plasmids were extracted and identified by restriction endonuleases digestion as well as sequencing. (3) sVP1 was obtained from pGEM-T/sVP1 by proper endonucleases digestion and then inserted into pcDNA3 vector which was cut by the same endonucleases. As a result, a eukaryotic expressing plasmid of pcDNA3/sVP1 was formed. (4) Three copies of C3d were introduced into pcDNA3/sVP1 to construct pcDNA3/sVP1-C3d3 and restriction endonuleases digestion was used to identify the fused insertion. (5) Large preparation were performed to obtain the plasmids of pcDNA3/sVP1 pcDNA3/sVP1-C3d3 and pcDNA3 from transformed E.coli DH5α, and purified by PEG. (6) BALB/c mice aged 6-8 weeks were inoculated intramuscularly (i.m.) three times at four weeks’interval with 100μg of pcDNA3/sVP1-C3d3, pcDNA3/sVP1 and pcDNA3 plasmids respectively. Fourteen days after every injection, sera of each group were collected and the titers for CVB3-specific neutralizing antibodies were measured. Three weeks after the third immunization, splenocytes from three immunized mice of each group were stimulated by inactivated CVB3 and harvested to analyze the lymphocytic proliferative activity and specific CTL cytotoxic activity by CCK-8 assay. At the same time, another eight mice were challenged with 5LD50 CVB3 and the number of surviving animals was monitored up to three weeks post infection. Furthermore, the rest mice of each group were challenged with 3LD50 CVB3 and sacrificed after seven days in order to evaluate the titers of blood viruses. Meanwhile, hearts of mice was fixed with 10% formalin, dehydrated with grading ethanol, embedded in paraffin, and cut into 5μm sections. Some sections were stained with hemotoxylin and eosin to observe the changes of the myocardium.
Results: (1) Gene fragment of C3d3 was obtained by restriction endonucleases digestion and sVP1 fragment was amplified by PCR. After restriction endonucleases digestion, both of the length was the same as expected. DNA sequencing showed that sVP1 was identical to the sequence recorded in Genbank. (2) Three copies of C3d were introduced into pcDNA3/sVP1 to form pcDNA3/sVP1-C3d3. The result of enzyme digestion was identical as expected and suggested that the eukaryotic expressing plasmid pcDNA3/sVP1-C3d3 had been constructed successfully. (3) When mice were immunized with the plasmids, the antibody titers increased with the time of inoculation. The mean titers of neutralizing antibody after every immunization in pcDNA3/sVP1 group were 1:8.91,1:15.87,1:28.28, respectively, and that in pcDNA3/sVP1-C3d3 group were 1:8.41,1:25.19,1:33.65, while it is always lower than 1:5 in mice immunized with pcDNA3. More specifically, mice with pcDNA3/sVP1-C3d3 elicited the strongest neutralizing antibody after the third immunization. When compared with that of pcDNA3/sVP1, the difference was significant (P<0.05). (4) Splenocytes from vaccinated mice showed different proliferative activity due to different stimulants of CVB3 or ConA. When stimulated by CVB3, the splenocytes from mice with pcDNA3/sVP1-C3d3 showed significantly stronger proliferative activity than that with pcDNA3/sVP1 (P<0.05). While when stimulated by ConA, the proliferative activity of mice with pcDNA3/sVP1-C3d3 and pcDNA3/sVP1 were both stronger than that with pcDNA3, but the difference between the two stronger groups was not significant. (5) DNA expressing sVP1 fused to three copies of C3d efficiently enhanced the specific lymphocytic CTL cytotoxic activity, which was remarkably stronger than that in the mice immunized with pcDNA3/sVP1. (6) Protection of mice from death after lethal CVB3 (5LD50) challenge was augmented to 50% with fused DNA, while mice with pcDNA3/sVP1 was only 25% and no one survived in pcDNA3 group. Chi-square test indicated that differences between any two groups were not significant by the multiple comparisons. Kaplan-Meier test was used to compare the differences of survival curves, which indicated that the survive state of pcDNA3/sVP1-C3d3 group is better than that of pcDNA3 group (P<0.05), but difference between pcDNA3/sVP1-C3d3 and pcDNA3/sVP1 was not significant (P>0.05). (7) After 3LD50 CVB3 challenge, the virus titers of blood in pcDNA3/sVP1-C3d3 group were only 2.58±0.66, significantly lower than that of pcDNA3/sVP1 group (3.61±0.41). (8) The histopathology changes in mice immunized with fused DNA were less serious compared with that in mice of other groups, but significant difference couldn’t be found between them.
Conclusion: (1) The gene fragment of C3d3 was obtained by restriction endonucleases digestion successfully. (2) The eukaryotic expressing plasmid pcDNA3/sVP1-C3d3 was constructed successfully. (3) The survival rate of mice with pcDNA3/sVP1-C3d3 peaked to 50%, and pcDNA3/sVP1 was 25%, while no mice survived in pcDNA3 group. The differences were not significant by Chi-square test. While, the result of Kaplan-Meier test showed that the survival state of pcDNA3/sVP1-C3d3 group was better than that of pcDNA3 group (P<0.05). (4) After administration of pcDNA3/sVP1-C3d3 and pcDNA3/sVP1 to BALB/c mice, the neutralizing antibody increased along with the inoculation times. More meaningfully, C3d3 could induce much stronger antibody from mice. (5) Specific lymphocytic proliferative activity and CTL cytotoxic activity in immunized mice with pcDNA3/sVP1-C3d3 were markedly stronger than those in mice immunized with pcDNA3/sVP1 and pcDNA3. (6) Overall, the results of neutralizing antibody titers, specific lymphocytic proliferative activity, specific CTL cytotoxic activity, viruses titers and histopathology all indicate that C3d can strongly enhance the specific immune response induced by sVP1 gene vaccination, but the protection of mice from death after lethal CVB3 (5LD50) challenge are still not significant now.
引文
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15 Green DT, Newton BR, Rota RA, et al. C3d enhancement of neutralizing antibodies to measle hemagglutinin. Vaccine, 2002, 20: 242~248
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23 赵欣荣, 李大金, 袁敏, 等. 分子佐剂 C3d 增强人绒毛膜促性腺激素 β 基因避孕疫苗的免疫效应及转变免疫应答模式的研究. 中华医学杂志, 2003, 83(21): 1906~1909
24 Toapanta FR, Ross TM. Mouse strain-dependent differences in enhancement of immune responses by C3d. Vaccine, 2004, 22(13~14): 1773~1781
25 Hess MW, Schwendinger MG, Eskelinen EL, et al. Tracing up take of C3dg-conjugated antigen into B cells via complement receptor type 2(CR2, CD21). Immunobiology, 2000, 95(8): 2617~2623
26 Han S, Hathcock K, Zheng B, et al. Cellular interaction in germinal centers: roles of CD40 ligand and B7-2 in established germinal centers. Immunology, 1995, 155(2): 556~557
27 Borriello F, Sethna MP, Boye SD, et al. B7-1 and B7-2 have overlapping critical roles in immunoglobulin class swithing and germinal center formation. Immunity, 1997, 6(3): 303~313
28 余敏, 李大金, 王秀丽等. 分子佐剂 C3d 上调 Raji 细胞协同刺激分子 B7-1 和 B7-2 的表达. 分子细胞生物学报, 2006, 39(1): 79~81
29 Kozono Y, Abe R, Kozono H, et al. Cross-linking CD21/CD35 or CD19 increases both B7-1 and B7-2 expression on murine splenic B cells. Immunology. 1998,160(4): 1565~1572
30 Mongini PK, Tolani S, Fattah RJ, et al. Antigen receptor triggered up regulation of CD80 and CD86 in human B cells: augmenting role of the CD19/CD21 costimulatory complex and IL-4. Cell Immunol, 2002, 216(1~2): 50~64
31 Wu X, Jiang N, Fang YF, et al. Impaired affinity maturation in CR2-/- mice is rescued by adjuvants without improvement in germinal center development. Immunology, 2000, 165(6): 3119~3127
32 Surahat S, Braun RP, Lewis PJ, et al. Fusion of C3d molecule with bovine rotavirus vp7 or bovine herpesvirus type 1 glycoprotein D inhibits immune response following DNA immunization. Vet Immunol Immunopathol, 2001, 83(1~2): 79~92
33 Bergmann-Leitner ES, Scheiblhofer S, Weiss R, et al. C3d binding to the circumsporozoite protein carboxy-terminus deviates immunity against malaria. Int Immunol, 2005, 17(3): 245~255
34 Bergmann-Leitner ES, Leitner WW, Tsokos GC. Complement C3d: from molecular adjuvant to target of immune escape mechanisms. Clin Immunol, 2006, 121(2): 177~185
35 Haas KM, Toapanta FR, Oliver JA, et al. Cutting edge: C3d functions as a molecular adjuvant in the absence of CD21/CD35 expression. Immunology, 2004, 172(10): 5833~5837
36 Mitsuyoshi JK, Hu Y, Test SY. Role of complement receptor type 2 and endogenous complement in the humoral immune response to conjugates of complement C3d and pneumococcal serotype 14 capsular polysaccharide. Inf Immun, 2005, 73(11): 7311~7316