上海交通大学学报（医学版）

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2023 , Vol. 43 >Issue 11: 1374 - 1383

DOI: https://doi.org/10.3969/j.issn.1674-8115.2023.11.005

论著 · 基础研究

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 ,
  • 俞康莹 ,
  • 陈颖盈 ,
  • 季萍 ,
  • 王颖
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  • 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
沈海浅（1985—），男，硕士生；电子信箱：1802670873@qq.com。
王 颖，电子信箱：ywangssmu@shsmu.edu.cn。

收稿日期: 2023-08-21

  录用日期: 2023-10-19

  网络出版日期: 2023-11-28

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)

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

  • Haiqian SHEN ,
  • Kangying YU ,
  • Yingying CHEN ,
  • Ping JI ,
  • Ying WANG
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  • 1.Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
    2.Shanghai Institute of Virology, Shanghai 200025, China
WANG Ying, E-mail: ywangssmu@shsmu.edu.cn.

Received date: 2023-08-21

  Accepted date: 2023-10-19

  Online published: 2023-11-28

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)

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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

本文引用格式

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 . DOI: 10.3969/j.issn.1674-8115.2023.11.005

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

参考文献

1 PAGET J, TAYLOR R J, et al. Estimates of mortality associated with seasonal influenza for the European Union from the GLaMOR project[J]. Vaccine, 2022, 40(9): 1361-1369.
2 PAULES C I, SULLIVAN S G, SUBBARAO K, et al. Chasing seasonal influenza: the need for a universal influenza vaccine[J]. N Engl J Med, 2018, 378(1): 7-9.
3 MATHIEU E, RITCHIE H, ORTIZ-OSPINA E, et al. A global database of COVID-19 vaccinations[J]. Nat Hum Behav, 2021, 5(7): 947-953.
4 POLACK F P, THOMAS S J, KITCHIN N, et al. Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine[J]. N Engl J Med, 2020, 383(27): 2603-2615.
5 LAU J J, CHENG S M S, LEUNG K, et al. Real-world COVID-19 vaccine effectiveness against the Omicron BA.2 variant in a SARS-CoV-2 infection-naive population[J]. Nat Med, 2023, 29(2): 348-357.
6 NORDSTR?M P, BALLIN M, NORDSTR?M A. Effectiveness of heterologous ChAdOx1 nCoV-19 and mRNA prime-boost vaccination against symptomatic Covid-19 infection in Sweden: a nationwide cohort study[J]. Lancet Reg Health Eur, 2021, 11: 100249.
7 AI J W, ZHANG H C, ZHANG Q R, et al. Recombinant protein subunit vaccine booster following two-dose inactivated vaccines dramatically enhanced anti-RBD responses and neutralizing titers against SARS-CoV-2 and Variants of Concern[J]. Cell Res, 2022, 32(1): 103-106.
8 HEKELE A, BERTHOLET S, ARCHER J, et al. Rapidly produced SAM? vaccine against H7N9 influenza is immunogenic in mice[J]. Emerg Microbes Infect, 2013, 2(8): e52.
9 VOGEL A B, LAMBERT L, KINNEAR E, et al. Self-amplifying RNA vaccines give equivalent protection against influenza to mRNA vaccines but at much lower doses[J]. Mol Ther, 2018, 26(2): 446-455.
10 AREVALO C P, BOLTON M J, LE SAGE V, et al. A multivalent nucleoside-modified mRNA vaccine against all known influenza virus subtypes[J]. Science, 2022, 378(6622): 899-904.
11 LEUNG A K K, HAFEZ I M, BAOUKINA S, et al. Lipid nanoparticles containing siRNA synthesized by microfluidic mixing exhibit an electron-dense nanostructured core[J]. J Phys Chem C Nanomater Interfaces, 2012, 116(34): 18440-18450.
12 SCHOENMAKER L, WITZIGMANN D, KULKARNI J A, et al. mRNA-lipid nanoparticle COVID-19 vaccines: structure and stability[J]. Int J Pharm, 2021, 601: 120586.
13 BADEN L R, EL SAHLY H M, ESSINK B, et al. Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine[J]. N Engl J Med, 2021, 384(5): 403-416.
14 LIU X Q, LI Y H, WANG Z F, et al. Safety and superior immunogenicity of heterologous boosting with an RBD-based SARS-CoV-2 mRNA vaccine in Chinese adults[J]. Cell Res, 2022, 32(8): 777-780.
15 GUI Y Z, CAO Y, HE J J, et al. Safety and immunogenicity of a modified COVID-19 mRNA vaccine, SYS6006, as a fourth-dose booster following three doses of inactivated vaccines in healthy adults: an open-labeled Phase 1 trial[J]. Life Metab, 2023, 2(3): load019.
16 Moderna announces interim phase 3 safety and immunogenicity results for mRNA-1010, a seasonal influenza vaccine candidate[EB/OL]. (2023-02-16)[2023-10-10]. https://news.modernatx.com/news/news-details/2023/Moderna-Announces-Interim-Phase-3-Safety-and-Immunogenicity-Results-for-mRNA-1010-a-Seasonal-Influenza-Vaccine-Candidate/default.aspx.
17 BURGER L. Sanofi says it's back to the drawing board on mRNA flu vaccines[EB/OL]. (2023-06-29)[2023-10-10]. https://www.nasdaq.com/articles/sanofi-says-its-back-to-the-drawing-board-on-mrna-flu-vaccines.
18 CureVac advances seasonal flu study to phase 2 in collaboration with GSK following selection of promising mRNA vaccine candidate with broad coverage[EB/OL]. (2023-09-12)[2023-10-10]. https://www.curevac.com/en/curevac-advances-seasonal-flu-study-to-phase-2-in-collaboration-with-gsk-following-selection-of-promising-mrna-vaccine-candidate-with-broad-coverage/.
19 Clinical trial of mRNA universal influenza vaccine candidate begins[EB/OL]. (2023-09-12)[2023-10-10]. https://www.nih.gov/news-events/news-releases/clinical-trial-mrna-universal-influenza-vaccine-candidate-begins.
20 TAKANO T, SATO T, KOTAKI R, et al. Heterologous SARS-CoV-2 spike protein booster elicits durable and broad antibody responses against the receptor-binding domain[J]. Nat Commun, 2023, 14(1): 1451.
21 CLEMENS S A C, WECKX L, CLEMENS R, et al. Heterologous versus homologous COVID-19 booster vaccination in previous recipients of two doses of CoronaVac COVID-19 vaccine in Brazil (RHH-001): a phase 4, non-inferiority, single blind, randomised study[J]. Lancet, 2022, 399(10324): 521-529.
22 ATMAR R L, LYKE K E, DEMING M E, et al. Homologous and heterologous covid-19 booster vaccinations[J]. N Engl J Med, 2022, 386(11): 1046-1057.
23 PARK H J, BANG Y J, KWON S P, et al. Analyzing immune responses to varied mRNA and protein vaccine sequences[J]. NPJ Vaccines, 2023, 8(1): 84.
24 AL KHAMES AGA Q A, ALKHAFFAF W H, HATEM T H, et al. Safety of COVID-19 vaccines[J]. J Med Virol, 2021, 93(12): 6588-6594.
25 HOSSEINI R, ASKARI N. A review of neurological side effects of COVID-19 vaccination[J]. Eur J Med Res, 2023, 28(1): 102.
26 FRAIMAN J, ERVITI J, JONES M, et al. Serious adverse events of special interest following mRNA COVID-19 vaccination in randomized trials in adults[J]. Vaccine, 2022, 40(40): 5798-5805.
27 SHINKAI M, SONOYAMA T, KAMITANI A, et al. Immunogenicity and safety of booster dose of S-268019-b or BNT162b2 in Japanese participants: an interim report of phase 2/3, randomized, observer-blinded, noninferiority study[J]. Vaccine, 2022, 40(32): 4328-4333.
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