【作者】 陈丹丹；

【导师】 都小姣； 金迪华；

【作者基本信息】 华南理工大学 ， 药学（专业学位）， 2023， 硕士

【摘要】 2019年由新型冠状病毒引起的新型冠状病毒肺炎大流行给全世界带来了巨大的灾难,导致了过亿例感染和数百万例患者死亡。疫苗被认为是阻断传播和控制疾病的希望之一,其中信使RNA（messenger RNA,mRNA）疫苗因其优异的保护效果、安全性以及易于生产在传染性疾病领域研究中脱颖而出。但裸露的mRNA存在不稳定、难入细胞等问题。使用递送载体能够有效改善mRNA应用面临的难题。目前上市的3款mRNA疫苗均采用脂质纳米粒（LNP）递送体系,但其易在肝脏积累,并存在专利限制等问题,亟需发展新型高效mRNA递送体系。我们利用团队合作开发的高效递送体系(iCLAN,由单甲氧基聚乙二醇-聚乳酸-羟基乙酸共聚物(PEG_2k-b-PLGA_2k)、可电离脂质（N,N-二甲基氨基）丁酸（二亚油基）甲酯（Dlin-MC3-DMA）和（1,2-二油氧基丙基）三甲基氯化铵（DOTAP）自组装而成)递送表达SARS-CoV-2奥密克戎突变株S蛋白的mRNA（iCLAN@S＿omicron）,评估其诱导免疫应答的功效。研究显示该纳米载体粒径约为80 nm,表面电势为-6 mV,在4℃保存一个月粒径不发生明显改变,并能被不同细胞（包括免疫细胞、体细胞）摄取和表达相应蛋白质。通过荷载奥密克戎突变株S蛋白的mRNA,在体内外证实了iCLAN@S＿omicron肌肉注射后能够被树突状细胞（DC）及和巨噬细胞等靶细胞摄取,有效表达S蛋白,并诱导小鼠淋巴结生发中心（GC）反应,引起体液免疫应答,产生抗原特异性IgG抗体,其中,加强免疫后7天,小鼠血清中针对Omicron病毒的IgG抗体提高了9.45倍;而加强免疫后28天,小鼠血清中抗Omicron的Ig G抗体提高了15.41倍。此外,疫苗接种后还诱导小鼠产生较强的中和抗体,30μg/只剂量组比10μg/只剂量组中和抗体活性高3.37倍。进一步地,本文验证了该疫苗能引起机体固有免疫激活及适应性免疫的反应。最后,通过毒性评估证实了该体系在给药后一周对机体不产生明显毒性。综上,我们证实了iCLAN递送mSARS-CoV-2具有诱导体液、固有免疫应答的潜力,可以为mRNA疫苗递送载体的设计提供参考。更多还原

【Abstract】 The COVID-19 pandemic caused by SARS-CoV-2 in 2019 has brought great disaster to the world,resulting in more than 100 million infections and millions of deaths.Vaccines are considered to be one of the hopes for blocking transmission and controlling diseases,among which messenger RNA（mRNA）vaccines stand out in the field of infectious diseases because of their excellent protective effect,safety,and easy production.However,the exposed mRNA is unstable and difficult to enter cells.The use of delivery vectors can effectively improve the challenges faced by mRNA applications.At present,the three mRNA vaccines on the market and clinically mainly use lipid nanoparticles（LNP）delivery system,but it is easy to accumulate in the liver,and there are some problems such as patent restrictions.Therefore,there is an urgent need to develop new efficient mRNA delivery systemsWe use a team developed efficient delivery system(iCLAN,Delivery of mRNA expressing the S protein of SARS-CoV-2 Omicron mutant strain（iCLAN@S＿omicron）,which is self-assembled from a monomethoxy polyethylene glycol-polylactic acid-glycolic acid copolymer(PEG_2k-b-PLGA_2k),an ionable lipid（N,N-dimethylamino）butyric acid（dilinyl）methyl ester Dlin-MC3-DMA,and（1,2-dioleopropyl）trimethylammonium chloride（DOTAP）To evaluate its efficacy in inducing immune responses.The study showed that the nanocarrior had a particle size of about 80 nm and a surface potential of-6 mV.The particle size did not change significantly after storage at 4℃for one month.By loading Omicron mutant S protein mRNA,it was confirmed that after intramuscular injection of iCLAN@S＿omicron,it could be taken up by target cells such as dendritic cells（DC）and macrophages,effectively express S protein,and induce lymph node GC reaction and humoral immune response in mice.The specific IgG antibody against Omicron virus was increased by 9.45 times 7 days after booster immunization.The IgG antibody against Omicron increased by 15.41 times 28 days after booster immunization.In addition,strong neutralizing antibodies were induced in the mice after vaccination,and the neutralizing antibody activity in the 30μg/mouse dose group was 3.37 times higher than that in the 10μg/mouse dose group.Furthermore,we demonstrated that the vaccine could induce both innate and adaptive immune responses.Finally,the toxicity evaluation confirmed that the system did not produce obvious toxicity to the body one week after administration.In summary,we have demonstrated the potential of iCLAN to deliver m SARS-CoV-2 to induce humoral and innate immune responses,which can provide reference for the design of mRNA vaccine delivery vectors.更多还原