【作者】 龚伟；

【导师】 梅兴国；

【作者基本信息】 中国人民解放军军事医学科学院 ， 药剂学， 2009， 博士

【摘要】 完善的呼吸道合胞病毒(respiratory syncytial virus, RSV)疫苗,需同时具备激发细胞免疫和体液免疫的能力。CTL介导的细胞免疫在清除病原物方面作用显著,对RSV的预防尤为重要。因为,虽然RSV感染后体液免疫是主要的保护机制,但这种保护很不完全,患者在感染后较短时间内乃至终生易发生多次重复感染。同时,安全的RSV疫苗需要诱导平衡的免疫应答反应。单独的重组G蛋白或G蛋白片段仅诱导IgG1型抗体和Th2型细胞因子优势应答,不产生CD8+ T细胞免疫。前期我们将含有B细胞表位的G蛋白片段G1和CD8+ T细胞表位的片段F1/M2:81-95的融合蛋白G1F1/M2免疫BALB/c小鼠同时诱导了高滴度的特异性抗体和CTL应答,而且CD8+ T的诱导下调了IgG1与IgG2a的比例。但融合蛋白G1F1/M2比含载体蛋白的融合蛋白DsbA-G1F1/M2所诱导的中和抗体弱;而且尽管IgG1/IgG2a的比例有所降低,但仍未达到自然感染时的水平。因此为进一步增强G1F1/M2诱导体液免疫和细胞免疫的能力,尤其是进一步增强MHCⅠ分子限制的CD8+ T细胞的激活,提高CTL活性,进一步调整Th1/Th2的比例,使免疫反应更加平衡,我们在G1F/M2融合蛋白中引入可以促进抗原交叉提呈的蛋白转导结构域HIV TAT或HSP70构建新的重组蛋白抗原,并对其抗原性和保护性等进行了研究。在重组表达载体pET-G1F/M2的基础上,构建了两种新的重组融合蛋白表达载体,在原核表达系统中实现了重组蛋白的诱导表达,通过亲和层析分离纯化制备了用于动物免疫试验的重组抗原。通过设计引物进行PCR扩增的方式直接将蛋白转导结构域HIV TAT基因连接至G1F/M2基因的上游,构建新的重组融合蛋白TAT-G1F/M2。由于在原核表达系统中不能实现HSP70和G1F/M2的融合表达,因此构建了一个可以与HSP70高亲和力结合的融合蛋白Javelin-G1F/M2,在G1F/M2 N端引入的Javelin序列可以与热休克蛋白HSP70高亲和力结合。通过构建引物进行PCR扩增的方式直接将Javelin短肽基因引入至G1F/M2基因的上游。上述表达载体导入原核表达系统成功实现了重组融合蛋白的诱导表达,并采用Western-blot鉴定了重组蛋白的RSV抗原特异性。重组蛋白经Ni2+螯合亲和层析柱分离纯化,梯度透析复性后得到了纯度较高的重组蛋白。在体外先将Javelin-G1F/M2和HSP70孵育制备HSP70: Javelin-G1F/M2复合物进行动物免疫,对复合物诱导IgG抗体及亚型、中和抗体、CTL应答以及保护性等进行了考察,并与Javelin-G1F/M2皮下免疫组和Javelin-G1F/M2铝佐剂腹腔免疫组进行比较研究。在不添加其他佐剂,皮下免疫的条件下HSP70: Javelin- G1F/M2复合物可诱导高滴度的IgG抗体和强的CTL应答,其抗体滴度和CTL应答强度均优于Javelin- G1F/M2加铝佐剂腹腔免疫和单独Javelin-G1F/M2皮下免疫。HSP70: Javelin- G1F/M2复合物在诱导中和抗体方面与Javelin-G1F/M2加铝佐剂腹腔免疫基本相当。HSP70: Javelin-G1F/M2复合物可以诱导更为平衡的Th1/Th2应答,IgG的亚型IgG1/IgG2a的比值在攻毒前后均低于Javelin-G1F/M2加铝佐剂腹腔免疫组和单独Javelin-G1F/M2皮下免疫组;而且攻毒后肺组织嗜酸性粒细胞浸润的程度较Javelin-G1F/M2加铝佐剂腹腔免疫组轻。病毒滴定结果表明HSP70: Javelin- G1F/M2复合物可为小鼠提供完全的保护作用。对重组融合蛋白TAT- G1F/M2诱导IgG抗体及亚型、中和抗体、CTL应答以及保护性等进行了考察,并与G1F/M2进行了比较研究。TAT-G1F/M2诱导的IgG抗体滴度和中和抗体滴度与G1F/M2免疫组基本相当,但其诱导的CTL应答强度要高于G1F/M2免疫组。而且TAT- G1F/M2诱导的IgG的亚型IgG1/IgG2a的比值在攻毒前后均低于G1F/M2免疫组。保护性试验表明TAT-G1F/M2可为小鼠提供完全的保护作用。因此可见在G1F/M2融合蛋白的基础上引入蛋白转导结构域HIV TAT,或者将G1F/M2与热休克蛋白HSP70构建HSP70:G1F/M2复合物均可通过促进RSV抗原的交叉提呈,增强抗原G1F/M2的CTL应答,使Th1/Th2应答朝平衡的方向偏移,改善Th2优势应答导致的毒副作用。更多还原

【Abstract】 An ideal respiratory syncytial virus(RSV) vaccine should be capable of inducing humoral immune (antibody) , as well as cellular immune (CTLs). Cellular immune mediated by CTL plays an important role in elimination of pathogen, which is particularly important for prevention of RSV. Following RSV infection, humoral immunity is major protective mechanism, but this protection is not fully enough. After infection patients will be repeated infected during relatively short period of time or lifetime. Meanwhile, a safe RSV vaccine should be capable of inducing balanced immune response. However, previous studies in mice indicated that recombinant G protein or G protein fragment immunization resulted in IgG1 antibody and Th2-type response and failed to induce IgG2a, Th1 and MHC I-restricted CD8+T cell response.We engineered a fusion protein G1F/M2, consisting of an antibody epitope G: 125-225(G1) fragment from RSV-A G protein and a CD8+ T cell epitope (M2:81-95) from RSV-M2 protein. High titer of specific antibody and CTL response were induced in BALB/c mice intraperitoneal vaccinated with G1F/M2 formulated in Al(OH)3. And the induction of CD8+ T cells reduced the ratio of IgG1/IgG2a. But the titer of neutralization antibody induced by recombinant protein G1F/M2 was lower than DsbA-G1F1/M2, containing carrier protein DsbA. And despite the lower ratio of IgG1/IgG2a induced by G1F/M2, it was higher than that IgG1/IgG2a induced by RSV virus. The main aim of this study is to further enhance the G1F/M2’s ability of inducing humoral immunity and cellular immune, especially enhance MHC I-restricted CD8+T cell response and CTL activity. And then more balanced immune response will be achieved. So on the basis of recombinant protein G1F/M2, new recombinant proteins containing protein transduction domain HIV TAT or heat shock protein HSP70 were constructed to facilitate antigen cross-presentation. And studies of immunogenicity, protective efficacy and safety of recombinant proteins were investigated.Two new recombinant fusion proteins expression vectors were constructed. The inductions of recombinant proteins expression were achieved in prokaryotic expression system. And recombinant antigens were separated and purified by affinity chromatography. By primers design and PCR amplification, the gene of protein transduction domain HIV TAT was connected to the upstream of gene G1F/M2. A new recombinant protein expression vector, termed pET-TAT-G1F/M2, was constructed. Because recombinant fusion protein containing HSP70 and G1F/M2 can not be expressed in prokaryotic expression system, a new recombinant protein which can high-affinity bind to HSP70. By primers design and PCR amplification, peptide Javelin was introduced to the N terminal of G1F/M2. Through the high-affinity interaction between Javelin and HSP70, new recombinant fusion protein Javelin-G1F/M2 can form complex with HSP70. These expression vectors were transformed into prokaryotic expression system to achieve successful fusion protein expression. RSV-specific antigenicity of recombinant fusion protein were characterized by Western-blot. Recombinant fusion proteins were separated and purified by affinity chromatography on Ni2+ chelating Sepharose and refolded by gradient dialysis.HSP70: Javelin-G1F/M2 complex was prepared by incubation of Javelin-G1F/M2 and HSP70 in vitro. The titer of IgG antibody, IgG antibody subtype and neutralization antibody, CTL activities and protective efficacy induced by complex, Javelin-G1F/M2 and Javelin-G1F/M2 formulated in Al(OH)3 were investigated. High titer of IgG antibody and strong CTL responses were induced in BALB/c mice subcutaneous vaccinated with HSP70: Javelin-G1F/M2 complex without additional adjuvant. The titer of IgG antibody and the activity of CTL induced by HSP70: Javelin-G1F/M2 complex were markedly higher than those in mice subcutaneous vaccinated with Javelin-G1F/M2 or intraperitoneal vaccinated with Javelin-G1F/M2 formulated in Al(OH)3. The titer of neutralization antibody induced by HSP70: Javelin-G1F/M2 was equivalent with that induced by Javelin-G1F/M2 formulated in Al(OH)3. Compared with Javelin-G1F/M2 formulated in Al(OH)3, HSP70: Javelin-G1F/M2 complex can induce more balanced Th1/Th2 response. BALB/c mice were challenged with 106 pfu RSV-A at 14 days post immunization. before and after challenge the ratios of IgG1/IgG2a in mice subcutaneous vaccinated with HSP70: Javelin-G1F/M2 complex were lower than that of IgG1/IgG2a in mice subcutaneous vaccinated with Javelin-G1F/M2 or intraperitoneal vaccinated with Javelin-G1F/M2 formulated in Al(OH)3. After challenge, eosinophil infiltration in lungs of mice subcutaneous vaccinated with HSP70: Javelin-G1F/M2 complex was lighter. Virus titration results indicate that HSP70: Javelin-G1F/M2 complex protected the lungs against RSV-A challenge.The titer of IgG antibody, IgG antibody subtype and neutralization antibody, CTL activities and protective efficacy induced by TAT-G1F/M2 and G1F/M2 formulated in Al(OH)3 were investigated. The titer of IgG antibody and neutralization antibody induced by TAT-G1F/M2 was equivalent with that induced by G1F/M2. But the CTL activity of TAT-G1F/M2 group was higher than that of G1F/M2 group. Furthermore, TAT-G1F/M2 could induce lower ratio of IgG1/IgG2a in mice before and post challenge. Similarly, TAT-G1F/M2 could protect mice from RSV infection as well as G1F/M2.Therefore the cross presentation of antigen G1F/M2 can be enhanced by introduction of protein transduction domains HIV TAT or chaperoned by HSP70 in forms of HSP70:G1F/M2 complex. Ehanced cross presentation results in enhanced MHC I-restricted CD8+T cell response. So more balanced Th1/Th2 responses were induced and side effects caused by Th2 response were improved.更多还原