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免疫佐剂对不同剂量丙肝核酸疫苗效果的影响
http://www.100md.com 2005年6月28日 《世界华人消化杂志》 2005年第12期
     金博,程留芳,中国解放军总医院消化内科 北京市 100853

    金博,Richard Y.-H. Wang, 裘奇, 史维国,国立卫生研究院,活伦麦哥纽荪临床中心,输血医学部传染病组, 白塞丝达,马里兰20892 美国

    金博,男, 1963-08-01生, 山东省淄博市人, 汉族.1985年第二军医大学本科毕业,1990年军医进修学院硕士研究生毕业, 2001级军医进修学院博士研究生,现为美国国家卫生研究院博士后,主要从事肝病研究.

    通讯作者:程留芳,100853, 北京复兴路28号, 中国解放军总医院消化内科. liufang_cheng@yahoo.com.cn

    电话:010-66937096 传真: 010-68154653

    收稿日期:2005-02-14 接受日期: 2005-03-16

    Effects of Distinct Adjuvant on HCV DNA Vaccine at Different Dosages

    Bo Jin, Richard Y.-H. Wang, Liu-Fang Cheng, Qi Qiu,James Wai-Kuo Shih

    Bo Jin, Liu-Fang Cheng, Department of Digestive Diseases, General Hospital of Chinese PLA, Beijing 100853, China

    Bo Jin, Richard Y.-H. Wang, Qi Qiu, James Wai-Kuo Shih, Department of Transfusion Medicine, Warren G, Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA

    Correspondence to: Dr. Liu-Fang Cheng, Department of Digestive Diseases, PLA General Hospital, 28 Fuxing Road, Beijing 100853, China. liufang_cheng@yahoo.com.cn

    Received: 2005-02-14 Accepted: 2005-03-16

    Abstract

    AIM: To explore the effects of different adjuvants on different dosages of hepatitis C virus (HCV) DNA vaccine.

    METHODS: Female Balb/c mice were primed by HCV DNA vaccine composed of HCV structure gene DNA, HCV nonstructure gene 3 (NS3) and NS5b at the dosages of either 100 mg/each or 50mg/each with liposome DDAB/EPC or aluminum hydroxide and boosted twice accordingly. The cytokine profiles induced by various HCV antigens on splenocytes from the immunized mice were assessed by ELISPOT assay using in vitro splenocyte culture stimulated with recombinant HCV core, E2, E1/E2, NS3 or NS5b protein.

    RESULTS: The frequency of IFN-g or IL-4 secreting cells found in splenocytes stimulated with HCV core, E2, or E1/E2 from the mice vaccinated with HCV recombinant DNA in DDAB/EPC adjuvant was significantly higher (P<0.05) than that from mice immunized with either naked DNA or DNA formulated in aluminum hydroxide. The frequency of IL-4 secreting cells from mice immunized with HCV DNA at a dosage of 100 mg/each mixed with aluminum hydroxide was significantly higher than that from naked DNA when the splenocytes were stimulated with all the antigens tested except E2 (P<0.05). At many cases, lymphocytes from mice received 100 mg/each DNA have more IFN-g or IL-4 productions compared with those from mice with 50 mg/each (P<0.05). The lymphocytes from mice primed and boosted with HCV DNA plus aluminum hydroxide can produce more IL-4 than IFN-g in contrast with the cytokine profile of mice immunized with naked DNA or DDAB/EPC adjuvant.

    CONCLUSION: Liposome DDAB/EPC has strong adjuvant effects on HCV DNA vaccine. Aluminum hydroxide is a Th2 adjuvant and can convert the Th1 nature of DNA vaccine to Th2-biased immunity. It seems that the dosage of 50 mg of HCV DNA vaccine is not adequate to elicit efficient immunity in mice.

    Key Words: Hepatitis B Virus; DNA vaccine;

    Jin B, Wang RYH, Cheng LF, Qiu Q, Shih JWK. Effects of Distinct Adjuvant on HCV DNA Vaccine at Different Dosages. Shijie Huaren Xiaohua Zazhi 2005;13(12):1429-1433

    摘要

    目的: 观察不同佐剂对不同剂量HCV DNA疫苗效果的影响.

    方法: 雌性Balb/c小鼠分别用脂质体DDAB/EPC和氢氧化铝为佐剂的HCV DNA疫苗(由HCV结构基因、NS3和NS5b各50 mg或100 mg等量混合)免疫3次, ELISPOT法观察脾淋巴细胞受HCV核心、E2、E1/E2、NS3和NS5b蛋白刺激后细胞因子的产生.

    结果: DDAB/EPC组产生IFN-g和IL-4较多, 用核心、E2或E1/E2刺激, 显著高于裸DNA和氢氧化铝组(P<0.05). 以氢氧化铝为佐剂用每种DNA100 mg剂量免疫的小鼠, 在受抗原刺激后, 除E2外, 其脾淋巴细胞产生IL-4的能力显著高于相应裸DNA组(P<0.05). 在多数情况下, 接受每种DNA10 mg剂量的小鼠产生IFN-g和IL-4的能力显著高于接种每种50 mg者(P<0.05). 与裸DNA组及DDAB/EPC组比较, 氢氧化铝组小鼠淋巴细胞产生的IL-4多于IFN-g.

    结论: DDAB/EPC对HCV DNA疫苗有很强的免疫佐剂效应,氢氧化铝是Th2佐剂,可将DNA疫苗Th1为主的免疫特性转换为以Th2为主, 50 mg的HCV DNA疫苗似不能诱导小鼠产生有效的免疫反应.

    关键词: 丙型肝炎病毒; 核酸疫苗; 免疫佐剂

    金博, Richard Y.-H. Wang, 程留芳, 裘奇, 史维国. 免疫佐剂对不同剂量丙肝核酸疫苗效果的影响. 世界华人消化杂志 2005;13(12):1429-14331 (PDF) 不同抗原刺激后,各疫苗组小鼠淋巴细胞IL-4/IFN-g的产生.

    3 讨论

    DNA分子im后,肌细胞可摄取DNA并在其细胞内表达该DNA分子所编码的蛋白质.肌细胞虽也可表达MHC-I,有时也可表达MHC-II,但不能表达启动(priming)和激活T细胞所需的共刺激分子,因此不能作为抗原提呈细胞(Antigen presenting cells,APC)而发挥作用.在注射部位局部有少量APC可被注射的DNA分子转染,并高水平表达MHC及辅助刺激分子,从而激活T细胞.另外,转染的肌细胞表达的抗原也可被APC摄取而交叉启动(cross-priming)T细胞.因此,APC是DNA免疫的关键细胞[6].而免疫佐剂对免疫反应的类型发挥重要作用.裸DNA疫苗免疫效果不理想的主要原因,一是由于DNA分子在进入细胞前被间质核糖核酸酶所降解,另外是注射的DNA分子主要转染肌细胞,只有少量转染APC.在脂质体制作过程中,经过脱水―再水合过程DNA分子可被脂质体包裹形成小单层囊泡,脂质体的外层脂质可以保护内部的DNA分子免受核糖核酸酶所降解.虽然理论上阳离子脂质体的正电性更易于与带负电荷的肌细胞膜接近,有利于DNA进入细胞,但细胞间质的体液会中和脂质体表面正电荷,从而影响其与肌细胞的结合.阳离子脂质体更可能是被包括树突细胞在内的APC所吞噬,诱导更强的免疫反应[7-8].本实验结果表明,小鼠接种阳离子脂质体DDAB/EPC包被的不同剂量HCV DNA疫苗后,脾淋巴细胞在受到HCV核心、E1/E2或E2抗原刺激后,产生的IFN-g显著多于裸DNA组及氢氧化铝组,提示此脂质体可显著提高HCV DNA疫苗的细胞免疫效能.NS3和NS5b刺激后DDAB/EPC组小鼠淋巴细胞IFN-g的产生与其他两组比较无显著性差异,可能是与不同的抗原反应特性有关,也可能与抗原在体外的刺激时间较短有关,或与HCV结构基因DNA对非结构基因DNA免疫效果可能有干扰作用有关.与IFN-g产生情况相似,DDAB/EPC组IL-4的产生也较其他两组显著升高,说明该脂质体对Th2介导的体液免疫也有增强作用.而通过对DDAB/EPC载体组数据的分析,可见其IL-4的产生在大多数情况下显著高于裸DNA载体组,提示该脂质体还可能有非特异性的体液免疫刺激作用.

    在幼稚T细胞分化为Th1细胞的过程中,IL-12发挥重要作用,而补体iC3b和C5a可以抑制IL-12的表达,铝佐剂本身可以激活补体系统,从而抑制IL-12的产生,进而抑制幼稚T细胞分化为Th1细胞;另一方面,铝佐剂可诱导IL-4的产生,而IL-4可抑制I型免疫反应,促进II型免疫反应.因此,激活补体和选择性地诱导细胞因子的产生可能是铝佐剂诱导II型免疫反应的主要机制.另外,肌肉注射铝佐剂可以导致组织的损伤和少量肌纤维的坏死,坏死细胞可释放一些未知的分子激活树突细胞[9-10].Ulmer et al[11]报道,铝佐剂可增强10 mg剂量的流感DNA疫苗的免疫反应,而抗体亚型检测提示各亚型效价均有提高,但代表Th1反应的IgG2a/IgG1比值与裸DNA组比较无明显变化,仍大于1.而应用磷酸铝为佐剂的HBV DNA疫苗,可增强小鼠的I型免疫反应[12].而我们应用铝佐剂混合HCV DNA疫苗免疫小鼠的结果与其不同,我们发现氢氧化铝佐剂可以显著提高被免疫小鼠脾淋巴细胞分泌IL-4的水平,而且其IL-4的水平超过IFN-g,提示铝佐剂在本实验中可以增强HCV DNA疫苗的Th2免疫反应,从而改变DNA疫苗以Th1免疫反应为主的特性.我们前期的研究表明,小鼠接种100 mg的HCV NS3 DNA疫苗,可以诱导较强的Th1免疫反应[13].我们用重组的HCV结构基因DNA和NS3 DNA、NS5b DNA各50 mg或100 mg等量混合,以裸DNA形式或与佐剂混合免疫balb/c小鼠,然后取小鼠脾淋巴细胞行ELISPOT检测,结果发现细胞因子IFN-g和IL-4的产生,300 mg组明显多于150 mg组,在相当多的情况下还有统计学差异,说明此种HCV DNA疫苗在50 mg的剂量时,诱导的小鼠免疫反应明显低于应用100 mg剂量者.类似的研究也表明,100 mg剂量的HCV E1E2裸DNA疫苗免疫小鼠后,可产生明显的免疫反应,而10 mg或50 mg剂量的同种疫苗免疫小鼠则不足以产生免疫反应[14-15].

    因此,根据我们的实验结果,DDAB/EPC对HCV-DNA疫苗的辅佐作用较好,可增强DNA疫苗诱导的Th1免疫反应,为Th1型佐剂;氢氧化铝可增强DNA疫苗诱导的Th2免疫反应,并可改变DNA疫苗的免疫反应特性,使其由Th1为主转为Th2为主,为Th2型佐剂;而小鼠的免疫反应,以每种DNA 100 mg的剂量为好.

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    编辑 潘伯荣 审读 张海宁, http://www.100md.com( 金 博, Richard Y.-H. Wang, 程留芳, 裘 奇, 史维国)
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