【作者】 李国新；

【导师】 童光志；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2008， 博士

【摘要】 猪流感(swine influenza,SI)是由猪流感病毒(SI virus,SIV)引起的一种急性高度传染性呼吸道疾病。目前,包括H1N1、H3N2和H1N2亚型在内的SI已成为危害许多国家猪群的疾病。而且,猪是禽、猪、人流感病毒的易感宿主,可最大限度地彼人等其他动物流感病毒和禽流感病毒所感染,成为流感病毒基因重组或重配的“混合器”,是流感病毒新流行毒株产生的“孵育器”。因此,猪流感的控制不仅有经济上的意义,更有其深远的公共卫生学意义。目前,广泛应用于临床的是单价或多价的猪流感病毒灭活疫苗。但猪流感病毒在鸡胚中连续传代有可能影响病毒的免疫原性。而且其免疫剂量大,同时还必须使用佐剂,从而增加了灭活疫苗的成本。因此,研制新一代疫苗以替代或作为灭活疫苗的有益补充就显得尤其必要。另外,猪流感疫苗的研究应该尽可能兼顾目前主要流行的几种亚型。DNA疫苗因为能够同时诱导体液免疫应答和细胞免疫应答而受到青睐。基于猪流感病毒血凝素HA的DNA疫苗的研究已经取得了一定的效果。但由于DNA疫苗蛋白表达量低,激发的免疫应答较弱,从而限制了它的应用。因此,提高DNA疫苗的免疫效果已成为基因免疫研究的热点。一系列的研究表明,多拷贝的C3d分子与靶抗原偶联极大地提高了特异抗原的免疫应答水平。另外,用DNA疫苗免疫后再用重组病毒加强免疫的prime-boost免疫策略也显示了很好的免疫效果。本研究探讨了上述两种策略在猪流感疫苗上的应用效果。为了探讨分子佐剂C3d对猪流感病毒HA-DNA疫苗免疫原性的影响,本研究构建了四种表达不同形式HA的质粒DNA,分别为:表达分泌型HA的质粒pCA-sHA,表达全长HA的质粒pCA-tmHA和融合表达三拷贝C3d(鼠源和猪源)与HA的质粒pCA-sHA/mC3d3和pCA-sHA/sC3d3。四种质粒与空载体pCAGGS分别免疫BALB/c小鼠后,分别用ELISA、HI试验和中和试验检测抗HA抗体水平,并用硫氰酸盐洗脱法检测抗体亲和力水平。结果显示,免疫后第8周,pCA-sHA组与pCA-tmHA组的抗体水平没有明显差别,而pCA-sHA/mC3d3和pCA-sHA/sC3d3免疫组的抗体水平显著高于这两组,并具有更强的抗体亲和力。淋巴细胞增殖试验及细胞因子检测实验结果显示,免疫后第8周,pCA-tmHA诱导了更强的淋巴细胞增殖反应和更高水平的IFN-γ,而pCA-sHA/mC3d3和pCA-sHA/sC3d3诱导了更高水平的IL-4,结果说明,HA表达形式的变化影响了免疫反应的类型,C3d与HA融合后通过诱导IL-4的产生而使免疫反应倾向于Th2型。在随后的同型或异型病毒攻击试验中,通过对攻击病毒的分离、攻击病毒在免疫小鼠体内的分布情况以及攻毒后的免疫小鼠的病理变化的检测,证实四种质粒DNA都在不同程度上加速了同型攻击病毒的清除,对免疫小鼠产生了一定的保护作用。而pCA-sHA/mC3d3和pCA-sHA/sC3d3免疫的小鼠对异型毒的攻击也产生了一定的保护作用。结果表明,三拷贝分子佐剂C3d与分泌型HA融合后显著提高了DNA疫苗诱导的抗体水平,加速了抗体亲和力成熟,并可以部分抵抗异型病毒的攻击。另外,结果也表明,猪源补体C3d可以用小鼠做模型来评价其分子佐剂的效力。为了评价用DNA疫苗和重组病毒联合免疫的效果,随后进行了第二次免疫攻毒试验。1-4组与5-8组分别免疫质粒pCA-sHA、pCA-tmHA、pCA-sHA/mC3d3和pCA-sHA/sC3d3,初次免疫后,间隔4周,1-4组再用同一种质粒加强免疫一次,5-8组用表达H3N2亚型猪流感病毒HA的重组伪狂犬病毒rPRV-HA加强免疫一次;第9组初次免疫用pCAGGS空载体,4周后用rPRV-HA加强免疫;第10组免疫PRV Bartha-K61疫苗株;第11组为不免疫不攻毒组对照。血清学分析结果显示,对于rPRV-HA加强免疫组,在有DNA疫苗作基础免疫的组,抗体水平高于rPRV-HA单独免疫组。用DNA疫苗加强免疫与用rPRV-HA加强免疫诱导的抗体水平没有明显差别。但质粒pCA-sHA/mC3d3和pCA-sHA/sC3d3做基础免疫组的抗体水平要显著高于质粒pCA-sHA、pCA-tmHA做基础免疫的组。这表明,与C3d共表达的融合形式HA不但增强了抗体应答,而且引发了良好的抗体免疫记忆,因此,在用rPRV-HA加强免疫后,抗体水平迅速上升,在加强免疫后第4周抗体水平显著高于其他组。细胞因子检测实验结果显示,DNA疫苗与rPRV-HA联合免疫组的IL-4和IFN-γ的水平显著高于rPRV-HA单独免疫组。并且,用rPRV-HA加强免疫诱导的IL-4和IFN-γ的水平高于用同种DNA疫苗加强免疫诱导的细胞因子水平。结果表明,用rPRV-HA加强免疫普遍增强了免疫应答。在随后的同型猪流感病毒的攻击试验中,各免疫组都在不同程度上加速了同型攻击病毒的清除,对免疫小鼠产生了一定的保护作用。其中,用质粒DNA免疫后再用rPRV-HA加强免疫产生了最好的保护作用。总之,本研究构建的融合表达3拷贝C3d与HA的质粒DNA可能作为猪流感的候选疫苗,为预防多种亚型猪流感疫苗的研究探索了新的思路。另外,用质粒DNA做基础免疫再用rPRV-HA加强免疫的免疫策略,也为猪流感疫苗的研究提供了新的模式。更多还原

【Abstract】 Swine influenza is a common respiratory disease in pigs, and it is an acute, feverish and infectious disease. Currently, three main subtypes of influenza virus are circulating in different swine populations throughout the world: H1N1, H3N2, and H1N2. Swine is the only animal which can be infected by either avian or human original influenza viruses, and can be considered as the intermediate host for the process of genetic reassortments between viruses of different hosts. This can lead to the generation of new strains of influenza, some of which may be transmitted to other species including humans. As such, the development of a highly effective influenza virus vaccine for pigs would be of benefit to both veterinary and human health.Currently, Bivalent, killed vaccines are now commercially available but antigenic variation in the hemagglutinin (HA) protein of influenza viruses passaged in eggs can reduce the efficacy of this vaccine in eliciting the desired protective immune responses. Furthermore, larger dose injection and addition of adjuvant increased cost. Therefore, it is important to development new vaccines that are capable of inducing virus-specific neutralizing antibody plus cell mediated immunity, which provides superior protection against the acute influenza diseases. Furthermore, the new vaccines should provide protection against several main subtypes of swine influenza virus.DNA vaccines can induce protective cellular and humoral immune responses and have therefore been used during the last decade to develop vaccines against a variety of different pathogens. Several studies indicated that DNA vaccines expressed HA can elicit an immune response and confers protection against an influenza virus challenge. However, developments of DNA vaccines were limited because of low protein expression level in vivo and weak immune response elicited especially in larger animals. Therefore, various tactics have been used to increase the potency of DNA vaccines. For example, several studies have convincingly demonstrated that conjugation of target antigens to multiple copies of C3d can dramatically enhance the antibody responses to the target antigens. Utilizing DNA as a priming vaccination and a different modality for boosting (e.g., live viral vectors) has induced remarkable levels of cellular immunity.This study evaluated the effects of C3d on the immunogenicity of the swine influenza HA expressed by a DNA vaccine. Plasmid encoding soluble HA, complete HA or soluble fused form of HA were constructed from H3N2 subtype of swine influenza virus. Mice of Four groups were respectively injected with plasmid pCA-sHA, pCA-tmHA, pCA-sHA/mC3d3, pCA-sHA/sC3d3 and empty vector pCAGGS. The immune response was monitored by an enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition (HI) assays, and virus neutralization tests. Analysis of antibody titers indicated that immunizations with pCA-sHA/mC3d3 or pCA-sHA/sC3d3 had higher titers of anti-HA antibodies and stronger antibody affinity compared to serum from mice vaccinated with pCA-sHA or pCA-tmHA. Furthermore, C3d fusion increased Th2-biased immune response by inducing IL-4 production. Splenocytes from mice vaccinated with pCA-sHA/mC3d3 or pCA-sHA/sC3d3 produced about three-fold more IL-4 than did splenocytes from mice vaccinated with pCA-sHA or pCA-tmHA. Seven days post-challenge with homologous virus (H3N2), no virus was isolated from the mice immunized with HA-expressing plasmids. However, 10 days post-challenge with heterologous virus (H1N1), only mice immunized with pCA-sHA/mC3d3 or pCA-sHA/sC3d3 had no virus or microscopic lesions in the kidneys and cerebrum. These results demonstrated that C3d gene fused to truncated HA gene significantly enhanced the humoral immunity of the soluble HA expressed by DNA vaccine. Furthermore, effects of molecule adjuvant wine C3d were evaluated in mice model.In order to investigate the immunogenicity and protective efficacy of a heterologous prime-boost immunization against swine influenza virus, mice were first primed with naked DNA (pCA-sHA, pCA-tmHA, pCA-sHA/mC3d3 and pCA-sHA/sC3d3, respectively) and then boosted with same plasmid or recombinant pseudorabies virus expressing HA (rPRV-HA). The mice immunized with the prime-boost regime developed a high level of specific antibody against HA and cytokine production in the splenocytes when compared with the mice immunized single rPRV-HA. After plasmid DNA immunization, mice boosted with rPRV-HA produced similar antibody level and higher cytokine production in the splenocytes than that boosted with same plasmid DNA. Four weeks after the booster immunization with rPRV-HA or plasmid DNA, the mice were challenged with homologous virus (H3N2). All groups immunized with DNA or rPRV-HA were protected to a great extent. Especially, prime-boost immunization with DNA followed by rPRV-HA improved protective immunity against homologous virus (H3N2).In conclusion, C3d enhanced antibody responses to hemagglutinin and protective immunity against SIV of different subtypes. The results implied the plasmid DNA expressing sHA/C3d3 fusion proteins may represent an effective candidate vaccine against swine influenza virus. Furthermore, this study also indicated that heterologous prime-boost immunization approach might be useful against SIV infection.更多还原