【作者】 董金杰；

【导师】 张勇； 王永录；

【作者基本信息】 甘肃农业大学 ， 兽医博士（专业学位）， 2022， 博士

【摘要】 口蹄疫是当今国际上最重要的动物传染病之一,能够侵染所有的偶蹄动物。疫苗免疫仍是防控口蹄疫的首要选择。由于口蹄疫病毒血清型多且容易发生变异,致使流行毒株和疫苗株之间抗原匹配性不高,影响疫苗免疫效力和疫病预防效果。mRNA疫苗具有开发速度快、易制备、可以体外快速设计合成和大规模生产,且能够诱导高效保护性免疫应答和生物安全风险低等优点。因此,针对口蹄疫病毒变异造成的免疫逃逸问题,开发其mRNA应急储备疫苗意义重大。本研究开展了下述研究:1.O型口蹄疫mRNA疫苗体外转录系统的构建。根据结构蛋白VP1（viral structural protein 1）基因序列和功能,以猪口蹄疫病毒O型毒株（O/MYA98/BY/2010株）灭活抗原为模板,扩增VP1基因,并与p SFV1载体连接构建重组质粒p SFV1-VP1。将其转化至大肠杆菌DH5α中扩大培养,提取质粒,经Spe I单酶切制备线性化的DNA模板,体外转录成mVP1,转染BHK-21细胞进行表达。研究表明,mRNA转染BHK-21细胞24 h后,经免疫荧光法（immunofluorescence assay,IFA）检测,细胞可见特异性亮绿色胞浆荧光,而对照组细胞无荧光,同时用Western blot平行验证时,mRNA转染的细胞在35k D处出现目的条带。表明该重组mRNA可在体外细胞有效表达,充分证明了此转录系统的可行性。2.mRNA疫苗递送系统的构建及FMDV LNP/mVP1疫苗制备。由于无处不在的RNA酶,人们构建了大量的纳米级生物材料来递送mRNA。阳离子脂质纳米颗粒能够通过静电吸附作用结合mRNA,同时保护mRNA免受核酸酶的降解,并通过膜融合方式将mRNA递送到细胞内。本研宄研制了一种阳离子脂质纳米颗粒（Lipid nanoparticles,LNP）,对其形态、粒子直径、电位、安全性、细胞毒性和结合mRNA的能力进行了测定。通过体外细胞转染对其功能进行评价。体外细胞实验表明LNP能够保护绿色荧光蛋白（p CMV-N-EGFP）不被降解,顺利进入细胞翻译抗原蛋白。随后将前期获得的mVP1与LNP自组装成FMDV LNP/mVP1疫苗。通过免疫荧光方法检测,FMDV LNP/mVP1疫苗可在体外细胞BHK-21中表达。本研宄通过粒径、电位等对疫苗进行了探讨,FMDV LNP/mVP1疫苗平均直径为202.2 nm,电位为17.9 m V,为后续动物试验检测奠定基础。3.FMDV LNP/mVP1疫苗的免疫原性研究。以豚鼠为免疫动物模型,比较LNP、FMDV LNP/mVP1、FMDV CV、PBS的豚鼠血清抗原特异性Ig G抗体滴度、病毒中和抗体效价、豚鼠脾细胞分泌的Th1型细胞因子IFN-γ和Th2型细胞因子IL-4的表达情况以及各组豚鼠脾细胞T淋巴细胞亚群变化。在35 dpv时对免疫豚鼠攻毒,评价FMDV LNP/mVP1疫苗对攻毒豚鼠的保护效果。结果表明,相对LNP和PBS对照组,FMDV LNP/mVP1疫苗组豚鼠的抗体滴度升高,在35 dpv时达到检测周期中的峰值,细胞因子IFN-γ、IL-4的表达量均显著提高（P<0.01）。FMDV LNP/mVP1疫苗组豚鼠脾细胞中CD4⁺T细胞、CD8⁺T细胞所占比例相对LNP和PBS对照组均提高,其中CD4⁺T细胞提高显著（P<0.05）;经对攻毒豚鼠连续观察14天发现,PBS组和LNP组的所有豚鼠均出现明显的病变。与阴性对照组相比,FMDV LNP/mVP1疫苗免疫动物中以100GPID₅₀病毒攻击的1只豚鼠（1/4）出现轻微的临床症状,其余动物均未出现。该疫苗对50 GPID₅₀的病毒攻击提供了4/4的保护,对100 GPID₅₀的病毒攻击提供了3/4的保护。表明研制的mRNA疫苗不但能够刺激动物产生较好的体液免疫,而且能够激发动物产生细胞免疫,同时能使免疫豚鼠抵御强度攻击,具有比较理想的攻毒保护效果。综上所述,本研究以口蹄疫病毒VP1作为候选基因所制备的mRNA疫苗可以诱导较强的体液免疫与细胞免疫应答,免疫原性良好,为研制口蹄疫紧急预防接种疫苗提供了新途径,为其它mRNA疫苗的设计和免疫策略的制定提供了实验和理论依据。更多还原

【Abstract】 Foot and mouth disease（FMD）,one of the important infectious diseases,can affect all cloven-hoofed animals.Vaccination is still the first choice for the prevention and control of FMD.Due to the large number of serotypes and easy variation of foot and mouth disease virus（FMDV）,low antigen matching between candidate vaccine strains and epidemic strains will affect the immune efficacy of vaccine and the effect of disease control.The mRNA vaccine has the advantages of fast speed,easy preparation,rapid design,synthesis and large-scale production in vitro,inducing high-efficiency protective immune response with low biosafety risk.Therefore,it is of great significance to develop its mRNA vaccine emergency reserve in order to deal with immune escape caused by FMDV mutation.The following studies were carried out in this study:1.Construction of in vitro transcription system for mRNA vaccine against FMDV type O.According to the molecular and functional characteristics of structural protein VP1 gene,the VP1 gene was amplified and ligated into p SFV1 vector to construct the recombinant plasmid p SFV1-VP1 using the inactivated Porcine FMDV type O strain as the template.The plasmid was transformed into E.coli DH5α,and the plasmid was extracted.The linearized DNA template digested with Spe I was transcribed into mVP1 in vitro.Subsequently,the mRNA was transfected into BHK-21 cells and verified its expression properly.The results suggested that BHK-21 cells transfected with mRNA after 24 h showed specific bright green cytoplasmic fluorescence using immunofluorescence assay（IFA）,while the control group had no fluorescence.Simultaneously,the target band verified by western blot appeared at 35k D in the mRNA transfected cells.These results showed that the recombinant mRNA can be effectively expressed in vitro,with evidence of the feasibility of this transcription system.2.Construction of mRNA vaccine delivery system and preparation of FMDV LNP/mVP1 vaccine.A large number of nano biomaterials have been constructed to deliver mRNA as a result of the ubiquitous RNases.Cationic lipid nanoparticles can bind mRNA by electrostatic adsorption,protecting mRNA from nuclease degradation,delivering mRNA to cells by membrane fusion.A cationic lipid nanoparticles（LNP）was constructed in this study,and its morphology,particle diameter,zeta potential,cytotoxicity and binding ability to mRNA were determined.Further,its function was evaluated by cell transfection in vitro.Cell experiment in vitro showed that LNP could protect p CMV-N-EGFP from degradation and smoothly deliver it to cells to translate antigen proteins.Subsequently,LNP and mVP1 previously obtained were self-assembled into FMDV LNP/mVP1 vaccine.The result showed FMDV LNP/mVP1 vaccine can be expressed in BHK-21 cells in vitro using immunofluorescence assay.In this study,vaccines were investigated by particle size and potential.The particle size of FMDV LNP/mVP1 vaccine was about 202.2 nm,and potential was 17.9 m V,which laid the foundation for subsequent animal experiments.3.The study on immunogenicity of FMDV LNP/mVP1 vaccine.Guinea pigs were used as immune animal model to compare guinea pigs serum antigen-specific Ig G antibody titers,virus neutralizing antibody titer,Th1 cytokine IFN-γ,and Th2cytokine IL-4 and the changes in the T cell subsets of the spleen cells in LNP,FMDV LNP/mVP1,FMDV CV and PBS group.The guinea pigs immunized were challenged at 35 dpv to evaluate the protective effect of FMDV LNP/mVP1 vaccine.The results showed that the antibody titer of FMDV LNP/mVP1 vaccine group was increased and reached the peak in the detection cycle at 35 dpv,and the expression levels of cytokines IFN-γand IL-4 were significantly increased（P<0.01）compared with LNP and PBS control group.The proportion of CD4⁺T cells and CD8⁺T cells of the spleen cells in the FMDV LNP/mVP1 group were increased compared with the LNP and PBS control group,with CD4⁺T cells increased significantly（P<0.05）.Observation of guinea pigs after challenge for 14 days,the result showed that all guinea pigs in PBS group and LNP group had obvious lesions.Among the animals immunized with FMDV LNP/mVP1 vaccine,one guinea pig（1/4）against 100 GPID₅₀（50%Guinea pig infectious dose）virus challenge had mild clinical symptoms,and the other animals were protected.The mRNA vaccine could provide 4/4 protection against 50 GPID₅₀virus challenge in vaccinated guinea pigs and 3/4 protection against 100 GPID₅₀virus challenge.Those results suggested that the FMDV LNP/mVP1 vaccine constructed in this study induced robust humoral immunity and cellular immune responses.At the same time,guinea pigs immunized with mRNA vaccine can free from FMDV challenge with an ideal protective effect.In conclusion,the developed mRNA vaccine prepared with FMDV VP1 gene as the candidate gene in this study can elicit humoral and cellular immune response with sound immunogenicity,providing a new approach for the development of emergency vaccine against FMDV,and giving experimental and theoretical basis for the design of other mRNA vaccines and the formulation of immunization strategies.更多还原