基于杆状病毒表面展示技术的H5N1流感疫苗初步研究

发布时间：2018-04-16 15:12

本文选题：H5N1 + 杆状病毒载体疫苗　；参考：《中国疾病预防控制中心》2012年硕士论文

【摘要】：自1997年首次报道高致病性禽流感H5N1病毒能够直接感染人并导致死亡事件以来,在过去的十多年内,H5N1在全球范围内传播的不可预测性、对家禽和人类的高致病性以及病毒复杂的进化和重配等特点,充分说明了它可导致流感大流行的可能。毫无疑问,疫苗接种能够为潜在的H5N1流感大流行的爆发提供最有效的防控手段。而基于鸡胚生产的传统型流感疫苗由于生产能力有限等因素,无法满足流感大流行发生后的快速需求。因此,亟需开发出一种新型流感疫苗生产体系来及时生产流感大流行疫苗。为了获取免疫原性更强、保护效果更广以及生产系统更为快速的禽流感疫苗,本研究基于杆状病毒表面展示技术,开展了针对禽流感新型疫苗的初步研究。主要研究内容如下： (1)利用改造的pFast BacTM Dual载体质粒构建pFast-HA-NA表达质粒,通过Bac-to-Bac杆状病毒表达系统,在其表面共同展示禽流感病毒H5N1亚型(A/Hubei/1/2010)的主要免疫原性蛋白HA和NA,制备出新型禽流感疫苗Bac-HA-NA; (2)采用BALB/c小鼠模型评价该疫苗的免疫原性。通过两种策略进行免疫,即肌肉注射100μ1Bac-HA-NA (HA血凝滴度Log28)和滴鼻免疫25μ1Bac-HA-NA (HA血凝滴度Log210),二周后对每组小鼠进行加强免疫,免疫方式、免疫剂量与第一次免疫完全相同。利用间接ELISA实验检测血清中HA特异性IgG抗体滴度,ELISPOT实验检测小鼠淋巴细胞分泌IFN-y能力。 (3)利用A/PR/8/34H1N1病毒对小鼠进行攻击实验,评价该疫苗的交叉保护效率。研究结果表明： 1)载体疫苗Bac-HA-NA的杆状病毒表面能够成功的同时表达HA和NA蛋白,HA蛋白大小为72kDa, NA蛋白大小为55kDa,并且表达的HA蛋白血凝滴度达到log211,表达的NA蛋白与重配的湖北株H5N1有相似的NA酶活特性,对奥司他韦和扎那米韦有相似的药物敏感性。 2) Bac-HA-NA载体疫苗采用肌肉注射途径在小鼠体内诱导的HA特异性IgG抗体滴度达到1：40000,而滴鼻免疫途径在小鼠体内的HA特异性IgG抗体水平为阴性结果。 3)肌肉注射途径能够在小鼠体内诱导较强的细胞免疫水平,达到479SFCs/million splenocytes。滴鼻免疫途径也能够在小鼠体内产生一定的细胞免疫水平,达到325SFCs/million splenocytes。 4)肌注免疫对A/PR/8/34(H1N1)的交叉保护效果能达到25%,而滴鼻免疫则无交叉保护效果。本研究结果表明,基于杆状病毒表面展示系统构建的流感载体疫苗Bac-HA-NA,可以产生有效的体液免疫和细胞免疫,并具有部分交叉保护作用。由于杆状病毒生产系统的高安全性、易于分离纯化,生产周期短而显优于传统流感疫苗生产系统,可以用于将来的流感大流行疫苗的生产。
[Abstract]:Since 1997, when the highly pathogenic avian influenza H5N1 virus was first reported to be capable of directly infecting humans and causing deaths, the global spread of H5N1 has been unpredictable over the past decade or so.The high pathogenicity of poultry and humans and the complex evolution and reassortment of viruses fully illustrate the possibility that it can lead to influenza pandemic.There is no doubt that vaccination can provide the most effective means of prevention and control for a potential H5N1 pandemic.The traditional influenza vaccine based on chicken embryo can not meet the rapid demand of influenza pandemic due to limited production capacity and other factors.Therefore, it is urgent to develop a new influenza vaccine production system to produce influenza pandemic vaccine in time.In order to obtain avian influenza vaccine with stronger immunogenicity, wider protective effect and faster production system, a preliminary study on new avian influenza vaccine was carried out based on baculovirus surface display technology.The main contents of the study are as follows:1) the pFast-HA-NA expression plasmid was constructed by using the modified pFast BacTM Dual vector plasmid. By Bac-to-Bac baculovirus expression system, the main immunogenicity proteins HA and NAA of H5N1 subtype A / Hubei / 1 / 2010) were jointly displayed on its surface, and a new avian influenza vaccine, HBac-HA-NAA, was prepared.BALB/c mouse model was used to evaluate the immunogenicity of the vaccine.Two strategies were used to immunize the mice: intramuscular injection of 100 渭 1Bac-HA-NA HA hemagglutination titer Log28) and nasal immunization with 25 渭 1Bac-HA-NA HA hemagglutination titer Log210. After two weeks, the mice in each group were immunized more intensively, and the immunization mode was exactly the same as that in the first immunization.Indirect ELISA assay was used to detect the titer of HA specific IgG antibody in serum. Elispot assay was used to detect the ability of murine lymphocytes to secrete IFN-y.A/PR/8/34H1N1 virus was used to attack mice to evaluate the cross protection efficiency of the vaccine.The results show that:1) the baculovirus surface of the vector vaccine Bac-HA-NA can successfully express HA and na protein at the same time. The size of HA protein is 72 kDa. na protein size is 55 kDa. the HA protein hemagglutination titer of expressed HA protein reaches log211, and the expressed na protein is matched with H5N1 of Hubei strain.Have similar characteristics of na enzyme activity,There is a similar sensitivity to oseltamivir and zanamivir.2) the titer of HA specific IgG antibody induced by Bac-HA-NA vector vaccine in mice by intramuscular injection was 1: 40000, but the level of HA-specific IgG antibody induced by nasal drip was negative in mice.3) the intramuscular injection pathway could induce strong cellular immunity in mice and reach 479SFCs/million splenocytes.Nasal drip immune pathway can also produce a certain level of cellular immunity in mice, reaching 325SFCs/million splenocytes.4) the cross protective effect of intramuscular immunization on Ar / R / 8 / 34 H1 was 25%, but nasal drip had no cross protective effect.The results show that the influenza vector vaccine Bac-HA-NAbased on baculovirus surface display system can produce effective humoral and cellular immunity and has partial cross-protection.Because of the high safety of baculovirus production system, easy separation and purification, the production cycle is short and superior to the traditional influenza vaccine production system, so it can be used in the production of influenza pandemic vaccine in the future.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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