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通用流感mRNA候选疫苗的构建及免疫效果评价
Construction and evaluation of a universal influenza mRNA vaccine
- 陆军军医大学学报 2024年46卷第7期 页码：725-731
- 作者机构：
1.010110 呼和浩特, 内蒙古医科大学基础医学院
2.100853 北京, 中国人民解放军总医院第一医学中心肝胆胰外科医学部研究所
- 作者简介：
[杨鹏辉, E-mail: ypenghuiamms@hotmail.comYANG Penghui, E-mail: ypenghuiamms@hotmail.com
- 基金信息：
DOI：10.16016/j.2097-0927.202312014
中图分类号： R392.2;R392.7;R511.7收稿：2023-12-04，
修回：2024-01-16，
纸质出版：2024-04-15
- 稿件说明：
移动端阅览
田昱莹, 邓卓雅, 李聪, 等. 通用流感mRNA候选疫苗的构建及免疫效果评价[J]. 陆军军医大学学报, 2024,46(7):725-731.
Yuying TIAN, Zhuoya DENG, Cong LI, et al. Construction and evaluation of a universal influenza mRNA vaccine[J]. Journal of Army Medical University, 2024, 46(7): 725-731.
田昱莹, 邓卓雅, 李聪, 等. 通用流感mRNA候选疫苗的构建及免疫效果评价[J]. 陆军军医大学学报, 2024,46(7):725-731. DOI： 10.16016/j.2097-0927.202312014.
Yuying TIAN, Zhuoya DENG, Cong LI, et al. Construction and evaluation of a universal influenza mRNA vaccine[J]. Journal of Army Medical University, 2024, 46(7): 725-731. DOI： 10.16016/j.2097-0927.202312014.
摘要
目的
2
构建通用流感mRNA疫苗并全面评价其免疫保护效果。
方法
2
优化流感病毒株A/California/04/2009的血凝素(hemagglutinin
HA)、核蛋白(nucleoprotein
NP)和基质蛋白2胞外区(matrix protein 2 ectodomain
M2e)抗原序列
并将HA、NP和3个串联的M2e (3M2e)分别克隆至pcDNA3.1载体
通
过线性化、体外转录、酶学法加帽、酶促加尾合成mRNA
命名为mRNA-HA、mRNA-NP和mRNA-3M2e。3种mRNA分别转染293T细胞后
通过免疫荧光实验鉴定蛋白的表达。利用脂质纳米颗粒分别包裹mRNA-HA、mRNA-NP和mRNA-3M2e并测量其粒径和电位。再将3种mRNA等体积混合制备成Comb-mRNA疫苗。将28只6周雌性Balb/c小鼠(体质量为18~22 g)按简单随机分组法分为2组: LNP组(
n
=14)和Comb-mRNA组(
n
=14)。通过血凝抑制(hemagglutination inhibition
HI)、微量中和(microneutralization
MN)实验评价流感mRNA疫苗诱导小鼠产生的血清抗体滴度; 通过流式细胞术评价Comb-mRNA疫苗诱导的细胞免疫反应。采用5LD
50
野生型H1N1流感病毒株感染小鼠评价Comb-mRNA疫苗的免疫保护效果。
结果
2
成功构建mRNA-HA、mRNA-NP和mRNA-3M2e
且3种mRNA均能在293T细胞表达。脂质纳米颗粒包裹mRNA平均粒径为(119.53±6.5) nm
平均电位为(-8.23±1.3) mV。与LNP组比较
Comb-mRNA组疫苗的HI几何平均滴度(geometric mean titer
GMT)达179.6
MN的GMT达201.6
同时诱导IFNγ
+
CD4
+
/CD8
+
T细胞比例升高。Comb-mRNA组在加强免疫后2周能够提供对5LD
50
野生流感H1N1亚型病毒的保护。
结论
2
通用流感疫苗候选疫苗Comb-mRNA能够诱导小鼠免疫反应并保护小鼠免受病毒感染。
Abstract
Objective
2
To construct a universal influenza mRNA vaccine and evaluate its immunogenicity.
Methods
2
The antigen sequence of hemagglutinin (HA)
nucleoprotein (NP) and matrix protein 2 ectodomain (M2e) in influenza A/California/04/2009 was optimized.HA
NP and 3 tandem M2e (3M2e) were cloned into pcDNA3.1 vector
respectively.Then the mRNAs were synthesized by linearization
in vitro
transcription
enzymatic capping and enzymatic tailing
and named as mRNA-HA
mRNA-NP and mRNA-3M2e
respectively.The protein expression of the 3 kinds of mRNAs in 293T cells was detected by immunofluorescence assay.Comb-mRNA vaccine was prepared by enveloped mRNA-HA
mRNA-NP and mRNA-3M2e with lipid nanoparticles
respectively
and the particle size and potential were identified.Twenty-eight 6-week-old female BALB/c mice (18~22 g) were randomly divided into LNP group (
n
=14) and Comb-mRNA group (
n
=14).Hemagglutination inhibition (HI) method and microneutralization (MN) test were used to evaluate the s
erum antibody titer induced by Comb-mRNA vaccines.The mice were infected by 5LD
50
wild-type H1N1 influenza virus to evaluate the protective efficacy.
Results
2
The mRNA-HA
mRNA-NP and mRNA-3M2e were successfully constructed
and the 3 mRNAs could be expressed in 293T cells.The average size of mRNA encapsulated by lipid nanoparticles was 119.53±6.5 nm
and the average potential was-8.23±1.3 mV.The geometric mean titer (GMT) of HI and MN in the Comb-mRNA group were 179.6 and 201.6
compared with the LNP group.The ratio of IFN-γ
+
CD4
+
/CD8
+
T cells was increased.The Comb-mRNA group could provide protection against 5LD
50
wild type influenza H1N1 virus after 2 weeks of booster immunization.
Conclusion
2
Comb-mRNA
an influenza vaccine candidate
can induce immune responses and protect mice from influenza virus challenge.
关键词
Keywords
references
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