基于甲型流感病毒H1N1亚型血凝素的mRNA疫苗制备和加强免疫策略研究

doi:10.3969/j.issn.1674-8115.2023.11.005

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (11): 1374-1383.doi: 10.3969/j.issn.1674-8115.2023.11.005

• 论著 · 基础研究 • 上一篇

基于甲型流感病毒H1N1亚型血凝素的mRNA疫苗制备和加强免疫策略研究

沈海浅¹(), 俞康莹¹, 陈颖盈¹, 季萍¹, 王颖¹^,²()

^1.上海交通大学医学院上海市免疫学研究所，上海 200025
^2.上海市病毒研究院，上海 200025

收稿日期:2023-08-21 接受日期:2023-10-19 出版日期:2023-11-28 发布日期:2023-11-28
通讯作者: 王颖 E-mail:1802670873@qq.com;ywangssmu@shsmu.edu.cn
作者简介:沈海浅（1985—），男，硕士生；电子信箱：1802670873@qq.com。
基金资助:
国家重点研发计划(2021YFC2301502);上海交通大学“交大之星”计划医工交叉研究基金(YG2023ZD02)

Construction of an mRNA vaccine encoding hemagglutinin of influenza A H1N1 virus and investigation on booster immunization strategy

SHEN Haiqian¹(), YU Kangying¹, CHEN Yingying¹, JI Ping¹, WANG Ying¹^,²()

^1.Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
^2.Shanghai Institute of Virology, Shanghai 200025, China

Received:2023-08-21 Accepted:2023-10-19 Online:2023-11-28 Published:2023-11-28
Contact: WANG Ying E-mail:1802670873@qq.com;ywangssmu@shsmu.edu.cn
Supported by:
National Key Research and Development Program of China(2021YFC2301502);Key Project of Medical Engineering Cross Research Fund for "Star of Jiao Tong University" of Shanghai Jiao Tong University(YG2023ZD02)

摘要/Abstract

摘要：

目的·制备甲型流感病毒H1N1亚型血凝素（hemagglutinin，HA）mRNA疫苗，并探讨不同加强免疫策略的免疫保护作用。方法·以荧光素酶（firefly luciferase，Fluc）为报告基因，构建Fluc mRNA-脂质纳米颗粒（lipid nanoparticle，LNP）疫苗，通过小鼠活体成像实验鉴定Fluc mRNA-LNP疫苗肌内注射后在体内的表达情况。进一步构建H1N1亚型（A/Michigan/45/2015）HA（M15-HA）的mRNA-LNP疫苗，将20、10、5和1 μg M15-HA mRNA-LNP疫苗通过肌内注射分别免疫不同剂量组小鼠2次（间隔3周），酶联免疫吸附试验（enzyme-linked immunosorbent assay，ELISA）测定小鼠第2次免疫2周和4周后血清抗体滴度，血凝抑制试验检测功能性抗体水平。第2次免疫后40 d，采用1 μg mRNA疫苗或10 μg HA蛋白亚单位疫苗对1 μg剂量免疫组小鼠进行加强免疫，接种2周和4周后用ELISA和血凝抑制试验分别检测特异性抗体及功能性抗体水平。结果·活体成像实验结果显示，小鼠接种Fluc mRNA-LNP疫苗1 d后即能在小鼠体内检测到荧光素酶活性。制备获得的M15-HA mRNA-LNP疫苗2次免疫小鼠2周和4周后，所有剂量组小鼠的特异性抗体水平均较免疫前显著上升（均P=0.000）；血凝抑制试验结果显示，20 μg和10 μg剂量组的功能性抗体水平均较PBS对照组显著升高（均P<0.05）。对1 μg低剂量组小鼠进行HA蛋白或M15-HA mRNA-LNP的加强免疫后，均诱导产生了更高水平的特异性抗体和功能性抗体，并能维持较长时间；2种不同加强免疫策略之间差异无统计学意义。结论·成功制备M15-HA mRNA-LNP疫苗，显示出良好的免疫原性和抗体中和活性；低剂量mRNA疫苗免疫2次后，同源mRNA疫苗和异源蛋白疫苗加强免疫均可以诱导更强的免疫反应。

关键词: 流感疫苗, 甲型流感病毒, 血凝素, mRNA疫苗, 加强免疫

Abstract:

Objective ·To construct an mRNA vaccine encoding hemagglutinin (HA) of influenza A H1N1 virus, and explore the protective effects of different booster vaccination strategies. Methods ·Firefly luciferase (Fluc) was used as the reporter gene to construct Fluc mRNA vaccine enveloped in lipid nanoparticles (LNP). The in vivo expression of Fluc mRNA-LNP after intramuscular injection was determined by live imaging assay in mice. Furthermore, M15-HA mRNA-LNP derived from H1N1 subtype (A/Michigan/45/2015) was constructed. Mice were immunized with 20, 10, 5, or 1 μg doses of M15-HA mRNA-LNP twice (with an interval of 3 weeks) through intramuscular injection. Serum antibody titers were measured by enzyme-linked immunosorbent assay (ELISA) at 2 weeks and 4 weeks after the second immunization, and functional antibody levels were detected by hemagglutination inhibition test. The third booster vaccination was performed 40 d after the second immunization in 1 μg dose group with 1 μg M15-HA mRNA-LNP or 10 μg HA subunit vaccine. The levels of specific antibody and functional antibody were detected by ELISA and hemagglutination inhibition test, respectively 2 weeks and 4 weeks later. Results ·Live imaging assay showed that luciferase activity could be detected in mice 1 d after injection of Fluc mRNA-LNP. At 2 weeks and 4 weeks after the second immunization of M15-HA mRNA-LNP, HA-specific antibodies were significantly higher than those before the immunization in all vaccination groups at different doses (P=0.000). The hemagglutination inhibition test showed that the levels of functional antibodies in the 20 μg dose and 10 μg dose groups were significantly higher than those in the PBS control group (P<0.05). After 1 μg dose group mice were immunized with HA protein or M15-HA mRNA-LNP, higher levels of HA-specific antibody and functional antibody were induced and maintained for a long time. There was no significant difference between the two different booster immunization strategies. Conclusion ·M15-HA mRNA-LNP vaccine is constructed with immunogenicity and antibody neutralization activity. Low-dose mRNA priming vaccination followed by both homologous mRNA vaccine and heterologous protein subunit vaccine booster vaccination can induce stronger immune recall responses.

Key words: influenza vaccine, influenza A virus, hemagglutinin, mRNA vaccine, booster immunization

中图分类号:

R392.12

沈海浅, 俞康莹, 陈颖盈, 季萍, 王颖. 基于甲型流感病毒H1N1亚型血凝素的mRNA疫苗制备和加强免疫策略研究[J]. 上海交通大学学报（医学版）, 2023, 43(11): 1374-1383.

SHEN Haiqian, YU Kangying, CHEN Yingying, JI Ping, WANG Ying. Construction of an mRNA vaccine encoding hemagglutinin of influenza A H1N1 virus and investigation on booster immunization strategy[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(11): 1374-1383.

图/表 7

图1 FLuc mRNA-LNP的制备及鉴定Note： A. Full length of FLuc mRNA (2 000 nt) determined by gel electrophoresis. M—RNA ladder; lane 1—FLuc mRNA after IVT; lane 2—FLuc mRNA after 5′ capping. B. Luciferase activity detection. C. The particle size of FLuc mRNA-LNP measured by dynamic light scattering method.

Fig 1 Preparation and identification of FLuc mRNA-LNP

图2 小鼠经肌内注射FLuc mRNA-LNP不同天数后体内荧光素酶活性检测

Fig 2 Detection of luciferase activity in mice at different days after intramuscular injection of FLuc mRNA-LNP

图3 M15-HA mRNA-LNP制备及鉴定Note： A. Full length of M15-HA mRNA (2 100 nt) determined by gel electrophoresis. M—RNA ladder; lane 1—M15-HA mRNA after IVT; lane 2—M15-HA mRNA after 5′ capping. B. Detection of M15-HA protein in HEK293T cells transfected with M15-HA mRNA by Western blotting. M—protein marker; lane 1—the control without transfection; lane 2—the cells transfected with M15-HA mRNA. C. The particle size of M15-HA mRNA-LNP measured by dynamic light scattering method.

Fig 3 Preparation and identification of M15-HA mRNA-LNP

图4 M15-HA mRNA-LNP免疫小鼠后血清抗原特异性IgG抗体滴度变化Note： A. Schematic diagram of the experimental design. B. M15-HA-specific IgG antibody titers determined by ELISA. ①P=0.000, compared with D0 in the same dose group; ②P=0.000, ③P=0.034, ④P=0.015, compared with D14 in the 1 μg group.

Fig 4 Determination of serum antigen-specific IgG antibody titers in M15-HA mRNA-LNP-vaccinated mice

图5 血凝抑制试验检测M15-HA mRNA-LNP免疫小鼠功能性抗体滴度Note： HI—hemagglutination inhibition test. ①P=0.000, ③P=0.038, ④P=0.033, ⑤P=0.028, ⑥P=0.005, compared with the corresponding time point in the PBS group; ②P=0.000, compared with D35 in the 20 μg group.

Fig 5 Determination of functional antibody titers in M15-HA mRNA-LNP-vaccinated mice by hemagglutination inhibition test

图6 小鼠经过同源或异源加强免疫后外周血清抗原特异性IgG抗体滴度变化Note： A. Schematic diagram of the experimental design. B. M15-HA-specific IgG antibody titers were determined by ELISA. ①P=0.025, compared with D49 in the same group.

Fig 6 Determination of serum antigen-specific IgG antibody titers in mice after homologous or heterologous immunization

图7 血凝抑制试验检测小鼠经同源或异源加强免疫后功能性抗体滴度的变化Note： ①P=0.008, compared with D49 in the same group.

Fig 7 Determination of functional antibody titers in the mice after homologous or heterologous immunization by hemagglutination inhibition test

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基于甲型流感病毒H1N1亚型血凝素的mRNA疫苗制备和加强免疫策略研究

Construction of an mRNA vaccine encoding hemagglutinin of influenza A H1N1 virus and investigation on booster immunization strategy

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