枸杞多糖对甲型H1N1流感裂解疫苗黏膜免疫的佐剂效力研究

发布时间：2019-06-04 14:27

【摘要】：流行性感冒病毒是威胁人类健康的主要病原体之一,2009年四月底,新甲型H1N1 (A/California/7/2009)流感在墨西哥爆发,随即波及至一百多个国家。世界卫生组织(WHO)于2009年6月11日将甲型H1N1流感大流行的预警提高至最高第六级,这意味着流感病毒在新千年的第一次全球大流行。这株新甲型H1N1流感病毒经重配形成,人群普遍缺乏抵抗力,感染者多为年轻人,一般两三天以内可传至他人。目前该病毒仍在人群中对流感的监控和防治是一项长期艰巨且不断具有新挑战的任务,对流感病毒及其疫苗的研究具有重大的公共卫生学意义。接种疫苗是预防流感病毒的最佳途径,传统疫苗采用肌肉注射的方式接种。但流感病毒主要经呼吸道感染,通过呼吸道黏膜进入动物和人体内,因而黏膜免疫在预防流感病毒感染的研究领域中显得尤为重要。通过鼻腔接种流感疫苗可诱导局部黏膜免疫反应,分泌特异性sIgA,不仅能及时阻止感染,而且能更有效地提供交叉保护。佐剂具有增强疫苗免疫原性、优化免疫应答、减少提供保护性免疫所需的抗原量等优点,因此安全有效的流感病毒灭活疫苗黏膜佐剂将有助于增强流感疫苗的免疫效果。枸杞多糖(LBP)是从植物枸杞的果实中提取的多糖,能诱导淋巴细胞增殖,调节细胞因子的分泌,促进抗体和补体形成,增强NK细胞活性,具有免疫增强作用。因此我们认为枸杞多糖可能对黏膜接种甲流疫苗具有佐剂效应。在本研究中,我们以不同剂量的甲型H1N1流感裂解疫苗(单价液)添加或不添加枸杞多糖佐剂分别滴鼻免疫BALB/c/小鼠,以仅免疫枸杞多糖和不接种疫苗的同龄健康小鼠作为对照,研究不同剂量甲流裂解疫苗的黏膜免疫效力以及枸杞多糖对其佐剂效力。用ELISA方法测定小鼠血清中IgG、IgG1、IgG2a抗体和小鼠鼻洗液中IgA抗体的滴度,用HI方法测定血清中抗体的滴度,以评价枸杞多糖对甲型H1N1流感病毒裂解疫苗黏膜免疫诱导小鼠体内抗体水平的影响。用Elispot方法检测脾脏细胞分泌细胞因子IFN-γ的情况,以评价小鼠体内细胞免疫水平。二次免疫两周后用致死量同源流感病毒攻击小鼠,通过测定肺部病毒滴度、观察小鼠体重丢失率及统计小鼠存活情况来评价添加或不添加佐剂的甲流裂解疫苗经黏膜免疫小鼠后抗致死量同源病毒的保护效果。实验结果显示,滴鼻接种高剂量甲流裂解疫苗组小鼠其血清中IgG水平高于低剂量组,对于相同剂量疫苗,枸杞多糖佐剂的添加可提高小鼠体内IgG水平,表明LBP可以增强甲流裂解疫苗在小鼠体内诱导的体液免疫。通过对分型抗体的测定,结果发现单独疫苗组和添加佐剂组均能在小鼠体内诱导产生IgG1和IgG2a,所有组别IgG1抗体水平均略高于IgG2a,表明裂解疫苗在体内诱导的体液免疫是Th1/Th2混合型的,且枸杞多糖能同时提高两种分型抗体的滴度,其对Th1、Tb2型免疫均有增强作用。在较高剂量疫苗佐剂添加组中,我们检测到—定量的IgA,说明滴鼻途径接种甲流疫苗诱导了局部粘膜免疫反应。HI试验结果表明枸杞多糖能在体内辅助甲流疫苗诱导产生更高水平的抗体。高剂量甲流裂解疫苗添加LBP佐剂组小鼠脾脏细胞分泌IFN-γ水平显著高于其他组别,表明其在小鼠体内诱导了较强烈的细胞免疫反应。小鼠抗致死量同源病毒攻击的保护能力与接种疫苗剂量呈正相关,且高剂量甲流裂解疫苗添加LBP佐剂组小鼠保护率达100%,枸杞多糖显示出良好的佐剂效应。因此,枸杞多糖可以作为黏膜佐剂增强甲流裂解疫苗的免疫效力。
[Abstract]:The influenza virus is one of the major pathogens that threaten human health. At the end of April 2009, the new H1N1 (A/ California/7/2009) flu broke out in Mexico and then spread to more than one hundred countries. The World Health Organization (WHO) raised the early warning of the influenza A (H1N1) pandemic to the highest level at the sixth level on 11 June 2009, which means the first global pandemic of the influenza virus in the new millennium. The new H1N1 influenza A virus has been reformulated, and the population is generally lacking in resistance, and most of the infected people are young people and can be transferred to others within two or three days. At present, the virus is still in the crowd, the monitoring and prevention of the influenza is a long-term and challenging task, and the research on the influenza virus and its vaccine has significant public health significance. The vaccine is the best way to prevent the influenza virus, and the traditional vaccine is administered by intramuscular injection. But the influenza virus is mainly infected by the respiratory tract and enters the animal and the human body through the respiratory tract mucosa, so that the mucosal immunity is particularly important in the research field of the prevention of influenza virus infection. Through the nasal administration of the influenza vaccine, the local mucosal immune response can be induced, the specific sIgA can be secreted, not only can the infection be prevented in time, but also the cross protection can be more effectively provided. The adjuvant has the advantages of enhancing the immunogenicity of the vaccine, optimizing the immune response, reducing the amount of the antigen required to provide the protective immunity, and the like, so the safe and effective influenza virus inactivated vaccine mucosal adjuvant can be used for enhancing the immune effect of the influenza vaccine. The lycium barbarum polysaccharide (LBP) is a polysaccharide extracted from the fruit of the plant of the plant, can induce the proliferation of lymphocytes, regulate the secretion of the cytokines, promote the formation of the antibody and the complement, enhance the activity of NK cells, and have the function of enhancing immunity. Therefore, we think that the lycium barbarum polysaccharide may have an adjuvant effect on the mucosal administration of the vaccine. In the present study, we add or not add Lycium barbarum polysaccharide adjuvant to BALB/ c/ mice respectively with different doses of the A/ H1N1 influenza lysis vaccine (monovalent liquid), to only immunize the Chinese wolfberry polysaccharide and the healthy mice of the same age who do not have the vaccine as the control, The mucosal immune efficacy and the effect of Lycium barbarum polysaccharide on the adjuvant of the vaccine were studied. The IgG, IgG1, IgG2a antibody and the titer of the IgA antibody in the mouse nasal wash were determined by the ELISA method, and the titer of the antibody in the serum was determined by the HI method. Cytokine IFN-1 was secreted by the spleen cells using the Eliot method in order to evaluate the level of cellular immunity in the mice. after two weeks of second immunization, the mice were attacked by a lethal amount of homologous influenza virus, and the titer of the lung virus was measured, To observe the loss of body weight and the survival of the mice, the protective effect of the anti-lethal dose of the vaccine after the administration of the mucosal immune mice was evaluated. The results showed that the level of IgG in the serum of the mice with high dose of high-dose methylprednisolone was higher than that of the low-dose group, and the addition of the adjuvant of the lycium barbarum polysaccharide could improve the level of IgG in the mice. It is shown that LBP can enhance the humoral immunity induced by the first-flow cracking vaccine in the mice. The results showed that the IgG1 and IgG2a could be induced in the mice and the level of IgG1 antibody in all groups was slightly higher than that of IgG2a, indicating that the humoral immunity induced by the split vaccine in the body was Th1/ Th2 hybrid. And the lycium barbarum polysaccharide can improve the titer of the two types of antibody at the same time, and has an enhanced effect on the Th1 and the Tb2 type of immunity. In the higher dose vaccine adjuvant addition group, we detected a quantitative IgA, indicating that the nasal route was inoculated with a vaccine to induce a local mucosal immune response. The results of HI test show that the lycium barbarum polysaccharide can induce a higher level of antibody in the body-assisted nail-flow vaccine. The level of IFN-1 in the spleen cells of mice with LBP adjuvant group was significantly higher than that of other groups, indicating that it induced a strong cellular immune response in the mice. The protective ability of the mouse anti-lethal homologous virus attack is positively correlated with the dose of the inoculated vaccine, and the protective rate of the mice with the high-dose alpha-flow cracking vaccine is up to 100%, and the lycium barbarum polysaccharide shows good adjuvant effect. Therefore, the lycium barbarum polysaccharide can be used as a mucosal adjuvant to enhance the immune efficacy of the first-flow cracking vaccine.
【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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