Knowledge Management System Of Institute of process engineering,CAS
|Thesis Advisor||张松平, 苏志国|
接种安全、有效的疫苗是预防和控制传染性疾病最有效的手段。然而，病毒的抗原多样性和高度变异性为传统疫苗制备技术带来挑战。本论文以典型流感病毒疫苗为研究对象，从疫苗合成生物学的概念出发，以去铁铁蛋白（Apo-ferritin，AFt）和乙肝病毒核心抗原（Hepatitis B core antigen，HBc）病毒样颗粒（Virus-like particles，VLPs）为“底盘”，发挥其抗原提呈和免疫刺激的佐剂效应；以流感病毒的HA、M2e和NP抗原为“插件”，借助化学修饰偶联、基因编辑、及创新的物理加热包埋技术，模拟各抗原“插件”在天然病毒上的空间分布特点，在VLPs“底盘”上仿生构建可自由组合的新型流感疫苗。论文研究结果和创新点如下：（1）利用化学偶联技术，将流感病毒的外部抗原M2e或HA共价结合至AFt颗粒外表面制备出M2e-AFt和HA-AFt结合疫苗，免疫小鼠所激发的特异性抗体水平和攻毒保护率均显著高于游离抗原，且HA-AFt优于M2e-AFt。进一步将两种抗原共同偶联于AFt表面，制备出 (M2e+HA)-AFt外部组装双抗原疫苗，可同时激发高水平的M2e和HA特异性抗体，与单抗原疫苗相比，为免疫小鼠提供相对更强的广谱免疫保护力，对同源毒株的保护率达到100%，异源毒株保护率70%。（2）从颗粒变化角度对AFt纳米颗粒的热稳定性和亚基间孔道随温度变化规律进行研究，发现AFt在25-50℃间加热30 min颗粒结构保持完整，但粒径从9.3 nm增大至12.2 nm，表明亚基间孔道膨胀使颗粒尺寸增大；进一步升高加热温度，导致AFt二级结构发生显著变化，甚至发生颗粒降解或聚集。根据AFt在高温下颗粒亚基间孔道增大的发现，提出了通过加热在AFt颗粒内部装载流感病毒内部抗原NP的新思路。将AFt和NP溶液50℃加热处理45 min，可成功将约20个NP装载入单个AFt颗粒内部，显著高于传统酸解法的装载效率（每个颗粒仅包埋约4.5个NP），且更易于操作和过程控制。（3）结合上述建立的AFt表面偶联HA抗原、内部热包埋NP抗原策略，成功构建了以AFt颗粒为“底盘”，表面偶联有HA、内部包埋NP的HA-AFt+NP内-外组装仿生双抗原流感疫苗。该疫苗能同时激发强的HA和NP特异性IgG抗体反应，且内部包埋NP抗原协同增强HA抗原的 IgG2a和Th1型免疫应答。在无佐剂条件下，仿生疫苗针对同源和异源性H1N1毒株免疫保护率均达到100%。且对于H5N1、H3N2等多种异源毒株的血凝抑制滴度显著高于HA和HA-AFt单抗原疫苗，显示针对多种流感病毒毒株具有潜在的广谱保护效率。（4）基于HBc VLPs具有类似于AFt的热稳定性及孔道热膨胀特性，进一步以HBc VLPs为底盘，结合基因编辑和创新的加热装载技术，构建基于HBc VLPs的内-外组装仿生双抗原流感疫苗。通过基因编辑，将M2e或NP抗原融合表达于HBc VLPs颗粒外表面，通过60℃加热30 min，将NP或M2e装载在颗粒的内部，分别构建同时展示M2e与NP抗原的M2eext-HBc+NPint（仿生疫苗）和NPext-HBc+M2eint（非仿生疫苗）内-外组装双抗原流感疫苗。两种疫苗均能同时激发较强的M2e和NP特异性IgG抗体水平，且两种抗原间存在抗体水平协同增强效应。更为重要的是，以异源的H1N1病毒感染免疫后的小鼠，仿生疫苗的保护率为100%，且小鼠体重几乎无变化；而非仿生疫苗的保护率仅有62.5%，且伴随12.5%的体重减轻。免疫机制分析显示，M2eext-HBc+NPint仿生双抗原疫苗具有显著增强的生发中心（GC）B cells生成和更高水平的总CD8+效应记忆T cells反应。综上结果表明，以VLPs颗粒为“底盘”，以仿生策略灵活组装和组合抗原“插件”，为快速构建更加有效、广谱的流感疫苗，以及研制针对其他新发、突发传染性疾病的疫苗提供新策略。;The vaccination of safe and effective vaccines is the most effective way to prevent and control infectious diseases. However, the high antigenic diversity and high mutation rates of viruses impose significant limitations on traditional vaccine manufacturing processes. Influenza caused by influenza virus is one of such infectious diseases that threaten human health seriously thus demands universal vaccines urgently. This work from vaccine synthetic biology point of view proposed a series of nanovaccines against influenza virus by taking apo-ferritin (AFt) and Hepatitis B core antigen (HBc) virus-like particles as “chassis”, employing influenza virus hemagglutinin (HA), matrix protein 2 ectodomain (M2e) and nucleoprotein (NP) antigen as “plugins”. The presentation of antigens on the VLPs were realized by chemical modification and conjugation, genetic engineering, and a novel temperature-shift based physical encapsulation technologies to simulate their spatial distribution in the natural virus. The spatially programmed VLPs influenza vaccines allowed the presentation of structurally diverse antigens and enhanced their antigen immunogenicity. The results and novelties are as follows: (1) The M2e-AFt and HA-AFt conjugate vaccines were prepared by covalently conjugating the external M2e antigen or full-length HA antigen of influenza virus on the outer surface of AFt nanoparticles through chemical coupling technology. Upon intraperitoneal immunization in mice, the two conjugate vaccines stimulated significantly higher antigen-specific antibody and better protective immune responses than the free antigen, and HA-AFt was superior to M2e-AFt. The (M2e+HA)-AFt externally assembled dual-antigen influenza vaccine was further developed by covalently coupling the external full-length HA and M2e antigens on the AFt surface simultaneously, which could elicit both high levels of M2e and HA antigen-specific antibodies in mice. Compared with the single-antigen vaccine, the dual-antigen vaccine provided stronger broadly protective immune responses for immunized mice, with a 100% protection rate against homologous strain and a 70% protection rate against heterologous strain.(2) From the perspective of particulate structural change, the thermal stability and the variation of pore channel between subunits of AFt nanoparticles with the temperature was investigated. It was found that the intact structures of AFt nanoparticles were retained after heating at 25 to 50℃ for at least 30 min. However, its particle size increased from 9.3 to 12.2 nm, indicating a thermally induced expansion in the nanopores thus resulted in an increase of particle size of AFt nanoparticles. Further increasing the heating temperature led to significant changes in the secondary structure of AFt and even degradation or aggregation of the AFt nanoparticle. Benefited from the discovery of the expansion in the nanopores of AFt nanoparticles at high temperatures, a novel temperature-shift based encapsulation process was proposed for encapsulation of influenza virus internal nucleoprotein (NP) antigens inside AFt nanoparticles. By mixing NP with AFt solution at 50℃ for 45 min, about 20 NP peptides could be encapsulated into each AFt nanoparticle, which was significantly higher than that obtained by traditional pH-dependent disassembly-reassembly encapsulation process (Only 4.5 NP peptides per AFt nanoparticle). Moreover, the new process was easier to operate and control than the pH-dependent strategy.(3) By combining the abovementioned temperature-shift based encapsulation strategy with the chemical conjugation of HA antigens on the AFt surface, an internally-externally assembled biomimetic dual-antigen influenza vaccine HA-AFt+NP with HA chemical conjugated on the surface and NP encapsulated inside the cage was constructed based on the AFt “chassis”. Both HA and NP antigen-specific humoral immune responses were elicited by this HA-AFt+NP vaccine, and the IgG2a and Th1 immune responses of the surface-coupled HA antigen were synergistically enhanced by the embedded NP antigen. Compared with free HA antigen and the HA-AFt single-antigen vaccine, the biomimetic HA-AFt+NP dual-antigen vaccine conferred 100% protection against a lethal infection of both homologous and heterologous H1N1 strains in the absence of adjuvants, and higher hemagglutination inhibition (HAI) titers against heterologous H5N1 and H3N2 virus strains, which indicated a more potential cross-protective effect of the HA-AFt+NP vaccine against a variety of influenza virus strains.(4) Based on the thermal stability and thermally associated pore-expansion characteristics of HBc VLP nanocages similar to AFt, an internally-externally assembled biomimetic dual-antigen influenza vaccine was further constructed with HBc VLPs as “chassis”, by combining genetic engineering with a novel temperature-shift based encapsulation technology. By displaying NP or M2e antigens on the exterior of HBc VLPs through genetic fusion, and further encapsulating M2e or NP antigens inside the VLPs at 60℃ for 30 min, two internally-externally assembled dual-antigen influenza vaccines M2eext-HBc+NPint (A biomimetic vaccine) or NPext-HBc+NPint (A non-biomimetic vaccine) simultaneously targeting M2e and NP antigens was constructed. Both of the dual-antigen vaccines could elicit M2e and NP antigen-specific antibodies simultaneously in mice and synergistically enhanced effects in antibody levels were observed between the loaded two antigens in the dual-antigen vaccines. Most importantly, after a lethal challenge of the heterologous H1N1 virus, the biomimetic vaccine M2eext-HBc+NPint conferred the mice 100% protection without noticeable body weight loss in the absence of any adjuvant. While the protective efficacy conferred by the non-biomimetic vaccine NPext-HBc+M2eint was only 62.5%, accompanying 12.5% body weight loss in the immunized mice. Analysis of the immune mechanisms showed that the biomimetic M2eext-HBc+NPint dual-antigen vaccine was able to stimulate the significantly more efficient formation of germinal center (GC) B cells and a higher level of effector memory CD8+ T cell population.All above results demonstrated that with VLPs as the “chassis” for biomimetic assembly of a flexible combination of different antigens as “plugins”, the present work not only provides a new strategy for the rapid construction of more effective and universal vaccines against influenza virus but also promising for the production of effective vaccines against other emerging or emergent infectious pathogens.
|魏江雪. 以病毒样颗粒为“底盘”的流感疫苗仿生合成[D]. 中国科学院大学,2020.|
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