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安全、有效和稳定的抗原和佐剂是疫苗研究的重点和难点。本课题面向灭活口蹄疫病毒（Foot and mouth desease virus，FMDV）抗原易发生裂解导致免疫活性降低的问题，对影响灭活病毒颗粒结构稳定性和免疫活性的因素和规律进行了深入分析，提出利用过渡金属离子和离子液体（Ionic liquids，ILs）增强病毒衣壳蛋白五聚体界面相互作用、避免颗粒解聚提高其稳定性的新策略；并将抗原的稳定策略和佐剂设计相结合，设计和制备了基于离子液体的新型O/IL纳米乳液，提升了FMDV疫苗的稳定性和免疫应答，并将研究结果拓展应用于流感裂解疫苗佐剂的设计。主要研究结果和创新点如下： （1）发现过渡金属离子Ni2+和Cu2+与146S （即结构完整的FMDV）能发生特异性结合，且结合规律不同于非过渡金属离子Ca2+。通过等温滴定量热、微量热泳动分析、以及ICP-MS分析，发现Ni2+、Cu2+和Ca2+三种离子均能与FMDV发生焓驱动的自发性结合，即ΔG<0，?H<0、?S<0。其中Cu2+与146S结合亲和力最大；Ni2+和Cu2+在146S上的结合数量为Ca2+的10倍以上。（2）利用过渡金属离子的结合提高146S的稳定性和免疫活性，并对其机理进行分析。发现Ni2+、Cu2+、Ca2+与146S的结合均能提高抗原的热稳定性，其中Cu2+稳定效果最显著；此外，Ni2+和Cu2+的结合还能提高146S的耐酸稳定性，而Ca2+的结合则不利于颗粒的耐酸稳定性。提出了过渡金属离子与146S五聚体界面相邻组氨酸间通过配位作用形成“过渡金属离子桥”而稳定146S结构的机理。采用结合了Ni2+或Cu2+的146S对小鼠进行免疫，发现比单一的146S能产生更高的抗体滴度。通过146S与细胞表面受体整合素β6和硫酸乙酰肝素的亲和力实验，表明Cu2+的结合使抗原与两种受体的亲和力分别提高了3倍和10倍。（3）采用胆碱型离子液体调节146S微环境的质子强度，提高抗原的稳定性。发现胆碱型离子液体[Cho][Cl]和[Cho][SO4]可将146S的颗粒热变性温度Tm1值提高4℃；而[Cho][H2PO4]则降低了146S的热稳定性。通过分析不同离子液体对146S颗粒微环境pH值的影响，提出了[Cho][Cl]和[Cho][SO4]通过降低146S周围微环境的质子强度，而抑制146S颗粒五聚体交界处组氨酸的质子化从而稳定146S的机理。小鼠免疫实验表明添加[Cho][Cl]和[Cho][SO4]对146S的体内免疫活性无显著影响。（4）构建了基于离子液体的O/IL纳米乳液，并用于FMDV疫苗注射免疫佐剂，提高了机体的体液免疫应答。将胆碱型离子液体的阴离子替换为具有生物学活性的烟酸，构建了离子液体[Cho][nicotinic]（CANI），并以该离子液体替代水相、以角鲨烯为油相、Tween 80为表面活性剂制备出粒径约为160 nm的均一、稳定的O/IL纳米乳液。与市售乳液佐剂ISA-206相比，O/IL乳液能显著提高146S的稳定性，用于FMDV 疫苗注射免疫佐剂能显著增强机体的体液免疫应答，产生与使用ISA-206佐剂相当的IgG滴度。（5）将O/IL纳米乳液用于H1N1型流感裂解疫苗的注射和鼻粘膜免疫佐剂，均展现出很好的佐剂效应。将O/IL纳米乳液用于H1N1的注射免疫佐剂，产生的IgG滴度显著高于MF59佐剂；用于H1N1的鼻黏膜免疫佐剂，在增强的粘膜免疫应同时，还能提高体液和细胞免疫应答，产生的sIgA、IgG滴度和IFN-γ水平均显著高于使用MF59佐剂。O/IL乳液提高鼻黏膜免疫应答效果的作用机制包括：延长HA在鼻腔的停留时间，促进鼻黏膜相关淋巴组织中DC细胞和CD4+ T细胞的增殖。;With the development of vaccine industry, much research efforts have been devoted to impoving stability of vaccine antigens and design of safe and effective vaccine adjuvants. Inactivated foot and mouth disease virus (FMDV), which is the most effective vaccine antigen against highly contagious foot and mouth disease, is extremely unstable. The intact FMDV particle, also known as 146S, is easily dissociated during production, storage and application, leading to severve decrease in its immuniginicity. This work proposed new strategies for the stabilization and developed a new adjuvant design based on in depth analyses of the factors and mechanism affecting the structural integrity and the immunogenicity of the 146S particles. Transition metal ions and ionic liquids (ILs) were selected and examined for possible enhancement of the interaction between the inter-pentamers of virus capsids so that the particle disassociation of particle is prevented and its stability is improved. Furthermore, an ILs-based O/IL nanoemulsion was constructed and tested as a novel adjuvant for the FMDV vacccine. This O/IL nanoemulsion was also applied as a new adjuvant for an influenza virus vaccine. The results and novelty are as follows.(1) Transition metal ions Ni2+ and Cu2+ were found to bind specifically to 146S through a mechanism different from bingding of nontransition metal ions Ca2+. Combining with isothermal titration calorimetry, microscale thermophoresis and ICP-MS analyses, the thermodynamics and capacities of Ni2+, Cu2+ and Ca2+ binding to 146S were studied and discussed. All three metal ions could bind spontaneously to 146S through an enthalpy driving proces (ΔG<0, ?H<0, ?S<0), among which the Cu2+ showed the highest binding affinity. The binding capacities of Ni2+ and Cu2+ on 146S capsids were about 10 times higher than Ca2+. These results suggested that the binding modes and amino acid types involved in binding of these two different types of metal ions on 146S might be different.(2) The binding of transition metal ions resulted in improvment in stability and immunogenicity of the 146S. The binding of Ni2+, Cu2+ or Ca2+ could all improve the thermostability of 146S, and binding of Cu2+ was the most effective. The binding of Ni2+ and Cu2+ also improved the acid-resistant stability of 146S, while binding of Ca2+ was not conducive. Aminal experiments indicated that the immunization of 146S bound with Ni2+ or Cu2+ induced higher antibody titers in mice. The the affinities between 146S with two important cell surface receptors, integrin β6 and heparin sulfate, was increased by 3 and 10 times, respectively, after binding of Cu2+. Based on the above results and combined with the characteristics of numerous histidine residues at inter-pentameric interface of FMDV, we speculate that“transition metal ion bridges”were formed to stabilize 146S by coordination inertactions between transition metal ions and adjacent histidines at the inter-pentameric interface of 146S.(3) Using choline-based ILs to stabilize the 146S by adjusting the microenvironmental proton intensity surrounding the particle. Choline-based ILs [Cho][Cl] and [Cho][SO4] were found to improve the transition temperature (Tm) related to 146S dissociation by 4 ℃, while [Cho] [H2PO4] decreased the thermostability of 146S. By analyzing the effects of different choline-based ILs on the microenvironmental pH value of 146S particle, [Cho][Cl] and [Cho][SO4] were found to stabilize the FMDVD 146S mainly by reducing microenvironmental proton intensity surrounding the particle, so that the protonation of histidines at the inter-pentameric interface of 146S was supressed. (4) An O/IL nanoemulsion was constructed and tested as adjuvant for FMDV vaccine to enhance the humoral immune response. By substituting the anion of choline-based ILs with nicotinic acid with biological activity, a [Cho] [nicotinic] (CANI) IL was constructed. With CANI sulution as aqueous phase, squalene as the oil phase, and the Tween 80 as the surfactant, a stable O/IL nanoemulsion with uniform size about 160 nm was successfully constructed. Compared with commercial emulsion adjuvant ISA-206, the O/IL emulsion could significantly improve the stability of 146S. Using the O/IL nanoemulsion as adjuvant of 146S vaccine, significantly enhanced humoral immune response was induced by subcutaneous immunization. The level of IgG titer was comparable to that using ISA-206 adjuvant. (5) O/IL nanoemulsion exhibited excellent adjuvant effects for H1N1 split influenza vaccine both by subcutaneous and nasal mucosal vaccinations. Using the O/IL nanoemulsion as adjuvant of H1N1, IgG titers significantly higher than that by using MF59 adjuvant were induced by by subcutaneous vaccination. When was used as nasal mucosa adjuvant, the O/IL nanoemulsion not only enhanced the mucosal immune response, but also the humoral and cellular immune responses. Compared with MF59 adjuvant, the O/IL nanoemulsion induced significantly higher level of sIgA and IgG titers, as well as higher IFN-γ secretion. The excellent adjuvant effects of the O/IL nanoemulsion for mucosal immune was mainly benefited from prolonging the retention time of antigen in nasal cavity, promoting the proliferation of DC cells and CD4+ T cells in nasal mucosa associated lymphoid tissue (NALT).
|林旋. 口蹄疫疫苗抗原的稳定策略及新佐剂的设计[D]. 中国科学院大学,2020.|
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