【特邀约稿】-“以不变应万变',浅析基于M2e抗原的甲型流...

rojjer

"以不变应万变"，浅析基于M2e抗原的甲型流感通用疫苗研究

(Deng Lei，PhD )

   流感病毒容易通过呼吸道飞沫和接触传播并导致呼吸道疾病。流感病毒感染可通过接种疫苗进行有效预防。1917年至1918年的灾难性的甲型流感病毒H1N1大流行，又称为西班牙流感，导致全球大约五千万人死亡 [1, 2]。九十年后的H1N1大流行，又称墨西哥猪流感，在全球传播的头一年里导致了约20余万人死亡 [3]。2013年报道的人感染禽流感病毒H7N9带来了严重的经济损失。截止2014年秋季，关闭活禽市场带来的直接以及间接的经济损失高达18亿美元 [4]。

甲型流感病毒属于正黏液病毒科的负链RNA病毒。甲型流感病毒通过两个主要膜抗原的组合进行分型，血球凝集素(Hemagglutinin，HA)及神经氨酸酶（Neuraminidase，NA） [5]。只有甲型流感病毒能引起世界范围的大流行。该病毒拥有极其广泛的宿主：猪、禽类、狗、马、人类以及海鸟等。

    不同的甲型流感病毒毒株通过在同一感染宿主内发生基因重配（抗原转换 antigenic shift）进而产生潜在的大流行毒株 [6, 7]。传统流感大流行监测工作会特别关注季节性流感毒株HA被高致病性H5或者H7替换。

通过自然感染在人群中产生的特异获得性免疫反应会对流行的毒株制造免疫选择性压力。流感病毒毒株会通过一种细微的免疫逃脱机制又被称作抗原漂移(antigenic drift)进行逃脱免疫选择性压力，病毒株携带的突变主要集中在HA和NA。正因如此，人类流感疫苗毒株每年都会提前五到六个月开始预测更新，尽量与未来流行的毒株相匹配。但这种努力不是每次都能准确预测。例如2009年的墨西哥流感爆发就让流感病毒研究的科学家感到意外。自从上世纪70年代以来，人流感疫苗整合了H1亚型，但墨西哥流感病毒H1与1977年至2008年间的人类季节性流感病毒H1的抗原特征相去甚远，却与1918年西班牙流感毒株H1的抗原特征相似 [8]。

甲型流感病毒M2四聚体是三型膜蛋白，具有选择性离子通道的功能。结构上分为胞外区M2e（N端2-24氨基酸位点）、跨膜区（25-46氨基酸位点）以及胞内区（C端47-97氨基酸位点）。M2e序列在1918年到2008年间所发现的人类甲型流感病毒株中高度保守 [9]，且该区域包含人类B 细胞和T细胞抗原簇 [10, 11]。因此该位点的整合对于人类甲型流感广谱疫苗的研发变得至关重要。

1999年Neirynck首次报道的M2e被整合到病毒样颗粒乙肝病毒核心蛋白N端 [12]。研究表明，通过注射小鼠该疫苗能提供抵抗多种毒株感染的体液免疫保护作用。如今，已经有多种形式的M2e整合疫苗，如病毒样颗粒疫苗、DNA疫苗、多抗原肽疫苗以及其他载体蛋白疫苗等 (Reviewed in [9, 13])。

    M2e疫苗本质上通过产生M2e特异性体液免疫提供免疫保护作用。M2e特异性IgG抗体通过结合展示在被感染细胞膜上的M2以及表达在免疫细胞膜上的Fcγ受体介导清除感染细胞的作用 [14]。作用机理有抗体依赖的细胞介导的细胞毒性作用（ADCC）、抗体依赖的细胞介导的吞噬作用（ADCP）和补体依赖的细胞毒性作用（CDC）。

    M2e特异性单克隆IgG抗体在临床试验中用作免疫治疗剂。通过筛选从M2e血清阳性的健康人类PBMC分离的IgG+记忆B细胞而得到的M2e单克隆抗体TCN031 和TCN032能有效地结合PR8感染细胞细胞膜上的M2四聚体，但与M2e肽段结合的亲和力较低 [15]。Theraclone公司目前已经完成了TCN032单抗的第二阶段临床试验 [16]。与对照组相比，1至7天内接受M2e单抗试剂的实验组症状减弱。但在第三天出现最严重症状的时间点上没有区别，这是因为M2e抗体保护机制需要召集效应免疫细胞进行感染细胞的清除。

与中和性抗体相比，M2e抗体介导的保护作用特征是感染允许型（infection-permissive），即使在已经具M2e抗体的个体中，甲型流感病毒的第一轮感染复制很可能是无障碍的。因此，甲型流感病毒内部抗原的CD8 CTL免疫反应才会随之产生 [17]。虽然每年接种流感疫苗与具广谱保护作用的CD8 T细胞反应的下降的关系仍然不明确，但很有可能的是，从孩童时期开始接种传统流感疫苗会长期性减弱具广谱保护作用的CD8 T细胞反应 [18]。

    在M2e疫苗设计中，增加M2e抗原簇的拷贝数会提高M2e特异性免疫反应 [19]。笔者所在实验室研发的M2eHBc结构的疫苗，发现融合两拷贝和三拷贝M2e的结构比单拷贝M2e更有效地诱导更高滴度水平的M2e抗体 [19]。此外，研究表明接受了M2e单克隆抗体的SCID小鼠会产生逃脱的病毒颗粒，对应M2e（Pro10）抗原簇的抗体的免疫压力会导致逃脱的病毒颗粒的M2e序列的第10个氨基酸位点突变为Leu或者His [20]。所以在M2e多拷贝设计中加入包含Leu10的M2e可以增强M2e疫苗的保护作用。然而，接种了融合全长23个氨基酸序列的human consensus M2e疫苗的实验动物在PR8感染后未发现有逃脱的病毒颗粒。

    M2e是序列高度保守的，该位点对于研发广谱甲型流感疫苗有着重大的意义。实验证明多种形式的M2e疫苗能提供免疫保护抵抗多种甲型流感病毒的感染。M2e疫苗的保护作用机制不是中和病毒，所以第一轮病毒感染能有效地让病毒复制产生内部抗原，进而内部抗原被APC展示激活特异性T细胞杀伤。然而，M2e疫苗绝不是现有使用的传统疫苗的替代品。随着未来更深入的M2e疫苗的研究，终将会找到M2e在未来甲型流感疫苗中的地位。（Deng Lei）

参考文献:

1.Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65(1):4-21. PubMed PMID: 2021692.

2. Johnson NP, Mueller J. Updating the accounts: global mortality of the 1918-1920 "Spanish" influenza pandemic. Bull Hist Med. 2002;76(1):105-15. PubMed PMID: 11875246.

3. Dawood FS, Iuliano AD, Reed C, Meltzer MI, Shay DK, Cheng PY, et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis. 2012;12(9):687-95. doi: 10.1016/S1473-3099(12)70121-4. PubMed PMID: 22738893.

4. Qi X, Jiang D, Wang H, Zhuang D, Ma J, Fu J, et al. Calculating the burden of disease of avian-origin H7N9 infections in China. BMJ Open. 2014;4(1):e004189. Epub 2014/01/21. doi: 10.1136/bmjopen-2013-004189. PubMed PMID: 24441057; PubMed Central PMCID: PMC3902515.

5. Tong S, Zhu X, Li Y, Shi M, Zhang J, Bourgeois M, et al. New world bats harbor diverse influenza A viruses. PLoS pathogens. 2013;9(10):e1003657. Epub 2013/10/17. doi: 10.1371/journal.ppat.1003657. PubMed PMID: 24130481; PubMed Central PMCID: PMC3794996.

6. Ito T, Couceiro JN, Kelm S, Baum LG, Krauss S, Castrucci MR, et al. Molecular basis for the generation in pigs of influenza A viruses with pandemic potential. Journal of virology. 1998;72(9):7367-73. PubMed PMID: 9696833; PubMed Central PMCID: PMCPMC109961.

7. Smith GJ, Bahl J, Vijaykrishna D, Zhang J, Poon LL, Chen H, et al. Dating the emergence of pandemic influenza viruses. Proceedings of the National Academy of Sciences of the United States of America. 2009;106(28):11709-12. doi: 10.1073/pnas.0904991106. PubMed PMID: 19597152; PubMed Central PMCID: PMCPMC2709671

8. Xu R, Ekiert DC, Krause JC, Hai R, Crowe JE, Jr., Wilson IA. Structural basis of preexisting immunity to the 2009 H1N1 pandemic influenza virus. Science. 2010;328(5976):357-60. Epub 2010/03/27. doi: 10.1126/science.1186430. PubMed PMID: 20339031; PubMed Central PMCID: PMC2897825.

9. Deng L, Cho KJ, Fiers W, Saelens X. M2e-based universal influenza A vaccines (Review). Vaccines. 2015;3:105-36.

10. Jameson J, Cruz J, Terajima M, Ennis FA. Human CD8+ and CD4+ T lymphocyte memory to influenza A viruses of swine and avian species. J Immunol. 1999;162(12):7578-83. PubMed PMID: 10358215.

11. Jameson J, Cruz J, Ennis FA. Human cytotoxic T-lymphocyte repertoire to influenza A viruses. Journal of virology. 1998;72(11):8682-9. PubMed PMID: 9765409; PubMed Central PMCID: PMCPMC110281.

12. Neirynck S, Deroo T, Saelens X, Vanlandschoot P, Jou WM, Fiers W. A universal influenza A vaccine based on the extracellular domain of the M2 protein. Nat Med. 1999;5(10):1157-63. Epub 1999/09/30. doi: 10.1038/13484. PubMed PMID: 10502819.

13. Zhang H, Wang L, Compans WR, Wang BZ. Universal Influenza Vaccines, a Dream to Be Realized Soon. Viruses. 2014;6(5):1974-91.

14. El Bakkouri K, Descamps F, De Filette M, Smet A, Festjens E, Birkett A, et al. Universal vaccine based on ectodomain of matrix protein 2 of influenza A: Fc receptors and alveolar macrophages mediate protection. J Immunol. 2011;186(2):1022-31. doi: 10.4049/jimmunol.0902147. PubMed PMID: 21169548.

15. Grandea AG, 3rd, Olsen OA, Cox TC, Renshaw M, Hammond PW, Chan-Hui PY, et al. Human antibodies reveal a protective epitope that is highly conserved among human and nonhuman influenza A viruses. Proceedings of the National Academy of Sciences of the United States of America. 2010;107(28):12658-63. doi: 10.1073/pnas.0911806107. PubMed PMID: 20615945; PubMed Central PMCID: PMCPMC2906546.

16. Ramos EL, Mitcham JL, Koller TD, Bonavia A, Usner DW, Balaratnam G, et al. Efficacy and safety of treatment with an anti-m2e monoclonal antibody in experimental human influenza. J Infect Dis. 2015;211(7):1038-44. doi: 10.1093/infdis/jiu539. PubMed PMID: 25281755.

17. Schotsaert M, Ysenbaert T, Neyt K, Ibanez LI, Bogaert P, Schepens B, et al. Natural and long-lasting cellular immune responses against influenza in the M2e-immune host. Mucosal Immunol. 2013;6(2):276-87. doi: 10.1038/mi.2012.69. PubMed PMID: 22806098.

18. Bodewes R, Kreijtz JH, Rimmelzwaan GF. Yearly influenza vaccinations: a double-edged sword? Lancet Infect Dis. 2009;9(12):784-8. doi: 10.1016/S1473-3099(09)70263-4. PubMed PMID: 19879807.

19. De Filette M, Min Jou W, Birkett A, Lyons K, Schultz B, Tonkyro A, et al. Universal influenza A vaccine: optimization of M2-based constructs. Virology. 2005;337(1):149-61. doi: 10.1016/j.virol.2005.04.004. PubMed PMID: 15914228.

20.Zharikova D, Mozdzanowska K, Feng J, Zhang M, Gerhard W. Influenza type A virus escape mutants emerge in vivo in the presence of antibodies to the ectodomain of matrix protein 2. Journal of virology. 2005;79(11):6644-54. doi: 10.1128/JVI.79.11.6644-6654.2005. PubMed PMID: 15890902; PubMed Central PMCID: PMCPMC1112148.

本文由论坛会员Dr Deng Lei 友情撰写，Dr Deng目前为比利时UGent-VIB 研究院博士，从事流感通用疫苗及治疗性抗体的研究工作，近年在Journal of Virology，Vaccine，PLos one等国际杂志上发表论文7篇，参与著作一部，同时作为一项专利的执行人。

本文由中国病毒学论坛原创，欢迎转载，转载请注明出处和作者。

rojjer

更多资料详见Dr Deng代表性论文：
1，Deng L, Cho KJ, Fiers W, Saelens X.M2e-Based Universal Influenza A Vaccines.Vaccines (Basel). 2015 Feb 13;3(1):105-36

Abstract：窗体顶端

Abstract：The successful isolation of a human influenza virus in 1933 was soon followed by the first attempts to develop an influenza vaccine. Nowadays, vaccination is still the most effective method to prevent human influenza disease. However, licensed influenza vaccines offer protection against antigenically matching viruses, and the composition of these vaccines needs to be updated nearly every year. Vaccines that target conserved epitopes of influenza viruses would in principle not require such updating and would probably have a considerable positive impact on global human health in case of a pandemic outbreak. The extracellular domain of Matrix 2 (M2e) protein is an evolutionarily conserved region in influenza A viruses and a promising epitope for designing a universal influenza vaccine. Here we review the seminal and recent studies that focused on M2e as a vaccine antigen. We address the mechanism of action and the clinical development of M2e-vaccines. Finally, we try to foresee how M2e

based vaccines could be implemented clinically in the future.

2，Deng L, Ibañez LI, Van den Bossche V et al.Protection against Influenza A Virus Challenge with M2e-Displaying Filamentous Escherichia coli Phages.PLoS One. 2015 May 14;10(5):e0126650.

Abstract:Human influenza viruses are responsible for annual epidemics and occasional pandemics that cause severe illness and mortality in all age groups worldwide. Matrix protein 2 (M2) of influenza A virus is a tetrameric type III membrane protein that functions as a proton selective channel. The extracellular domain of M2 (M2e) is conserved in human and avian influenza A viruses and is being pursued as a component for a universal influenza A vaccine. To develop a M2e vaccine that is economical and easy to purify, we genetically fused M2e amino acids 2-16 to the N-terminus of pVIII, the major coat protein of filamentous bacteriophage f88. We show that the resulting recombinant f88-M2e2-16 phages are replication competent and display the introduced part of M2e on the phage surface. Immunization of mice with purified f88-M2e2-16 phages in the presence of incomplete Freund's adjuvant, induced robust M2e specific serum IgG and protected BALB/c mice against challenge with human and avian influenza A viruses. Thus, replication competent filamentous bacteriophages can be used as efficient and economical carriers to display conserved B cell epitopes of influenza A.

cao1976

学习了，很不错，可否写出成英文，发表在journal  of applied virology上。