Chinese Science Bulletin, Volume 64, Issue 14: 1495-1505(2019) https://doi.org/10.1360/N972019-00132

Identification of H1N1 influenza virus-derived T-cell epitopes and the contribution in the cross-reactivities to avian influenza virus

More info
  • ReceivedFeb 20, 2019
  • AcceptedMar 21, 2019
  • PublishedApr 24, 2019


Influenza viruses, such as 2009 pandemic A(H1N1) influenza virus (2009 pH1N1) and avian influenza A (H7N9) virus (H7N9), which spread across the species, pose a great threat to human health and cause socioeconomic losses. Since 2009, circulation of 2009 pH1N1 has become a seasonal influenza virus. The sustaining epidemics have resulted in certain T-cell immune level among healthy populations.

T-cell epitopes are mainly derived from conserved internal proteins of influenza virus compared with B-cell epitopes, influenza virus-specific cross-reactive CD4+ and CD8+ T-cells broadly exist among the population, providing protection from subsequent infections by heterotypic viruses. Cytotoxic T lymphocytes (CTLs) specific for influenza A viruses mostly target internal, nonglycosylated proteins including M1, which are enriched with immunodominant CTL epitopes and markedly conserved among diverse strains compared to HA and NA. In comparison to M1 protein of 2009 pH1N1, the H7N9 M1 has 4 segments with clustering substitutions. In this study, we defined the cross-reactivity immunity between 2009 pH1N1 and H7N9 and identified four T-cell epitopes: H1-M42 (LMEWLKTR), H1-M102 (KLKREITFHGAK), H1-M202s (AMEVANQTR) and H1-M244 (MGVQMQRFK) in the mutant segments in 2009 pH1N1. T-cell responses were investigated using freshly isolated PBMCs from individuals (n = 16) through IFN-γ ELISPOT with these peptides derived from influenza virus M1 protein that are not conserved between 2009 pH1N1 and H7N9. We investigated the baseline of pre-existing immunity targeting M1 mutant segments of avian H7N9 influenza virus compared to 2009 pH1N1 among a healthy population. There was a certain level of T-cell immunity to H7N9 in the healthy population, but still weaker than that to 2009 pH1N1. Peptides derived from H7N9 which had mutations induced significant lower T-cell responses. The limited pre-existing T-cell immunity against H7N9 in healthy populations was partially contributed by H7N9 amino acid mutations in novel identified epitopes. Furthermore, we found that H1-M102 and H1-M244 could bind with HLA-A*1101 stably after renaturation in vitro, while the binding of H1-M42 and H1-M202s with HLA-A*3303 were relatively weak. Combined refolding and functional studies based on T-cell epitopes derived from influenza virus illustrated that minor mutation of an epitope can lead to a profound effect on the antigenicity of the peptide, which may also influence both HLA binding and TCR docking. Our study on T-cell immunity against influenza viruses provides an important reference for understanding the preexisting immune responses to avian influenza virus, and benefits the development of universal influenza vaccine. At the same time, considering that a few amino acid mutations will change the immunogenicity of the peptide completely, the study of antigenic variability of influenza virus major T cell immunogens such as M1, NP and PB1 are crucial in the development of universal vaccines.

Funded by




表S1 在2009 pH1N1和H7N9中M1蛋白的非保守肽库构建

本文以上补充材料见网络版csb.scichina.com. 补充材料为作者提供的原始数据, 作者对其学术质量和内容负责.


[1] Li J, Yu X F, Pu X Y, et al. Environmental connections of novel avian-origin H7N9 influenza virus infection and virus adaptation to the human. Sci China Life Sci, 2013, 56: 485-492 CrossRef PubMed Google Scholar

[2] Cui L, Liu D, Shi W, et al. Dynamic reassortments and genetic heterogeneity of the human-infecting influenza A (H7N9) virus. Nat Commun, 2014, 5: 3142 CrossRef PubMed ADS Google Scholar

[3] Liu D, Shi W, Shi Y, et al. Origin and diversity of novel avian influenza A H7N9 viruses causing human infection: Phylogenetic, structural, and coalescent analyses. Lancet, 2013, 381: 1926-1932 CrossRef Google Scholar

[4] Wang X, Wu P, Pei Y, et al. Assessment of human-to-human transmissibility of avian influenza A(H7N9) virus across 5 waves by analyzing clusters of case patients in mainland China, 2013–2017. Clin Infect Dis, 2013, 68: 623-631 CrossRef PubMed Google Scholar

[5] Chen H, Liu S, Liu J, et al. Nosocomial co-transmission of avian influenza A (H7N9) and A (H1N1) pdm09 viruses between 2 patients with hematologic disorders. Emerg Infect Dis, 2016, 22: 598-607 CrossRef PubMed Google Scholar

[6] Gaglani M, Pruszynski J, Murthy K, et al. Influenza vaccine effectiveness against 2009 pandemic influenza A (H1N1) virus differed by vaccine type during 2013–2014 in the United States. J Infect Dis, 2016, 213: 1546-1556 Google Scholar

[7] Lee L Y, Ha do L A, Simmons C, et al. Memory T cells established by seasonal human influenza A infection cross-react with avian influenza A (H5N1) in healthy individuals. J Clin Invest, 2008, 118: 3478–3490. Google Scholar

[8] Cheng V C, To K K, Tse H, et al. Two years after pandemic influenza A/2009/H1N1: What have we learned? Clin Microbiol Rev, 2012, 25: 223–263. Google Scholar

[9] Smith G J, Vijaykrishna D, Bahl J, et al. Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. Nature, 2009, 459: 1122–1125. Google Scholar

[10] Liu J, Wu B, Zhang S, et al. Conserved epitopes dominate cross-CD8+ T-cell responses against influenza A H1N1 virus among Asian populations. Eur J Immunol, 2013, 43: 2055-2069 CrossRef PubMed Google Scholar

[11] Jason A G, Maya F K, Yohan K, et al. Pre-existing immunity against swine-origin H1N1 influenza viruses in the general human population. Proc Natl Acad Sci USA, 2009, 106: 20365–20370. Google Scholar

[12] Zhao M, Liu K, Luo J, et al. Heterosubtypic protections against human-infecting avian influenza viruses correlate to biased cross-T-cell responses. MBio, 2018, 9: e01408–01418. Google Scholar

[13] Liu W J, Tan S, Zhao M, et al. Cross-immunity against avian influenza A (H7N9) virus in the healthy population is affected by antigenicity-dependent substitutions. J Infect Dis, 2016, 214: 1937-1946 CrossRef PubMed Google Scholar

[14] Liu J, Zhang S, Tan S, et al. Cross-allele cytotoxic T lymphocyte responses against 2009 pandemic H1N1 influenza A virus among HLA-A24 and HLA-A3 supertype-positive individuals. J Virol, 2012, 86: 13281–13294. Google Scholar

[15] Zhao M, Shu L M, Gao G F, et al. T-cell inmmunity against avian influenza A(H7N9) virus (in Chinese). Chin J Virol, 2018, 34: 911–919 [ 赵敏, 舒刘梅, 高福, 等. H7N9 亚型禽流感病毒的T细胞免疫研究. 病毒学报, 2018, 34: 911–919]. Google Scholar

[16] Sidney J, Peters B, Frahm N, et al. HLA class I supertypes: A revised and updated classification. BMC Immunol, 2008, 9: 1 CrossRef PubMed Google Scholar

[17] Garten R J, Davis C T, Russell C A, et al. Antigenic and genetic characteristics of swine-origin 2009 A (H1N1) influenza viruses circulating in humans. Science, 2009, 325: 197-201 CrossRef PubMed ADS Google Scholar

[18] Wrammert J, Koutsonanos D, Li G M, et al. Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection. J Exp Med, 2011, 208: 181–193. Google Scholar

[19] Rimmelzwaan G F, Katz J M. Immune responses to infection with H5N1 influenza virus. Virus Res, 2013, 178: 44-52 CrossRef PubMed Google Scholar

[20] Laidlaw B J, Decman V, Ali M A A, et al. Cooperativity between CD8+ T cells, non-neutralizing antibodies, and alveolar macrophages is important for heterosubtypic influenza virus immunity. PLoS Pathog, 2013, 9: e1003207 CrossRef PubMed Google Scholar

[21] Kreijtz J H C M, de Mutsert G, van Baalen C A, et al. Cross-recognition of avian H5N1 influenza virus by human cytotoxic T-lymphocyte populations directed to human influenza A virus. J Virol, 2008, 82: 5161-5166 CrossRef PubMed Google Scholar

[22] Ge X, Tan V, Bollyky P L, et al. Assessment of seasonal influenza A virus-specific CD4 T-cell responses to 2009 pandemic H1N1 swine-origin influenza A virus. J Virol, 2010, 84: 3312–3319. Google Scholar

[23] van de Sandt C E, Kreijtz J H C M, de Mutsert G, et al. Human cytotoxic T lymphocytes directed to seasonal influenza A viruses cross-react with the newly emerging H7N9 virus. J Virol, 2014, 88: 1684-1693 CrossRef PubMed Google Scholar

[24] Richards K A, Nayak J, Chaves F A, et al. Seasonal influenza can poise hosts for CD4+ T-cell immunity to H7N9 avian influenza. J Infect Dis, 2015, 212: 86-94 CrossRef PubMed Google Scholar

[25] Gao R, Cao B, Hu Y, et al. Human infection with a novel avian-origin influenza A (H7N9) virus. N Engl J Med, 2013, 368: 1888-1897 CrossRef PubMed Google Scholar

[26] Quiñones-Parra S, Grant E, Loh L, et al. Preexisting CD8+ T-cell immunity to the H7N9 influenza A virus varies across ethnicities. Proc Natl Acad Sci USA, 2014, 111: 1049-1054 CrossRef PubMed ADS Google Scholar

[27] Duan S, Meliopoulos V A, McClaren J L, et al. Diverse heterologous primary infections radically alter immunodominance hierarchies and clinical outcomes following H7N9 influenza challenge in mice. PLoS Pathog, 2015, 11: e1004642 CrossRef PubMed Google Scholar

[28] Gras S, Kedzierski L, Valkenburg S A, et al. Cross-reactive CD8+ T-cell immunity between the pandemic H1N1-2009 and H1N1-1918 influenza A viruses. Proc Natl Acad Sci USA, 2009, 107: 12599-12604 CrossRef PubMed ADS Google Scholar

[29] Valkenburg S A, Gras S, Guillonneau C, et al. Protective efficacy of cross-reactive CD8+ T cells recognising mutant viral epitopes depends on peptide-MHC-I structural interactions and T cell activation threshold. PLoS Pathog, 2010, 6: e1001039 CrossRef PubMed Google Scholar

  • Figure 1

    Comparison of H7N9 and 2009 pH1N1 M1 protein. The light blue boxes represent conserved sequences, dark blue boxes represent non-conserved sequences, black dots indicate specific amino acid mutation sites, and dotted boxes include non-conserved peptides. The dark green box represents the predicted short peptide from 2009 pH1N1 and the light green box represents the predicted short peptide from H7N9

  • Figure 2

    Determination of the individual peptide variants in H7N9 with decreased antigenicity. Recognition of M1 peptides derived from 2009 pH1N1 and H7N9 influenza virus were performed by ELISPOT using the in vitro expanded PBMC specific for 2009 pH1N1 M1. The individual peptides from the 2009 pH1N1 M1 peptides were used as stimulus. The peptides from H7N9 were mixed as peptide pool to stimulate the cells due to the limitation of the cell numbers. H7-M5 to H7-M9 derived from H7N9 were mixed into H7-Pool1; H7-M15 to H7-M20 derived from H7N9 were mixed into H7-Pool2; H7-M22 to H7-M27 of H7N9 into H7-Pool3; H7-M33 to H7-M39 of H7N9 into H7-Pool4. The PBMCs are from donors Z1, Z2, Z3, Z4, Z5, and Z6

  • Figure 3

    T-cell responses to the mutant overlapping peptides derived from 2009 pH1N1 M1. Data are shown as means ± SEM (standard errors of the means), the differences among mock and overlapping peptides (H1-M8, H1-M9, H1-M17, H1-M18, H1-M23, H1-M24, H1-M33, H1-M35 and H1-M39) were compared using ANOVA. *, P<0.05; **, P<0.01

  • Figure 4

    Identification of candidate peptides with the PBMCs of healthy donors by ELISPOT assay. The spots are a measure of IFN-γ secretion from PBMCs stimulated with various candidate peptides. The blue columns are peptides derived from 2009 pH1N1, while the green columns are peptides derived from H7N9. Bars represent the mean number of SFCs from independent donors, and all of them cut off the value of mock. Values are expressed as means ± SEM. T test was used for the statistical analyses. *, P<0.05

  • Figure 5

    The identification of T cell epitopes binding to HLA-A3. Binding of peptides H1-M42/H7-M42 and H1-M202s/H7-M202s to HLA-A*3303 was elucidated by in vitro refolding ((a), (b)). Peptides H1-M102/H7-M102 and H1-M244/H7-M244 could refold with the HLA-A*1101 chain and β2m ((c), (d)). After properly refolding, the high-absorbance peaks of the correctly refolded MHC I with the expected molecular mass of 45 kD eluted at the estimated volume of 16 mL on a SuperdexTM Increase 200 10/300 GL column. The profile is marked with the approximate positions of the molecular mass standards of 75.0, 44.0, and 13.7 kD. Peaks represent the aggregated heavy chain, the correctly refolded Ptal-N*01:01 complex (45 kD), and the extra β2m, respectively(d). Structure-based model of HLA-A*1101 (PDB Code:1q94) binding peptide H1-M244 (e) and H1-M244 compared with H7-M244 (f)

  • Table 1   Blood sample donor information









    A*1101, A*3101





    A*0201, A*0201

    B*0801, B*4001




    A*2402, A*2402

    B*1801, B*4001




    A*1101, A*0201

    B*1302, B*1501




    A*0206, A*0206




    A*2402, A*3303




    A*0210, A*3001

    B*1302, B*4006




    A*0201, A*1101

    B*1501, B*4601




    A*1101, A*2402





    A*1101, A*3101

    B*4001, B*5101




    A*1101, A*2402

    B*1501, B*1511




    A*1101, A*2402





    A*0201, A*3001

    B*4403, B*5401




    A*0207, A*6801

    B*3801, B*4601




    A*1101, A*1101

    B*1501, B*4001




    A*1101, A*3303

    B*5101, B*5801

    M, 男性; F, 女性. b) –, 志愿者的具体HLA分型未检测

  • Table 2   M1-derived peptides with low T-cell cross-reactivities due to the mutations in H7N9

    统计总结先前已报道的表位, 1代表来源于H1N1, 7代表来源于H7N9; b) 对应该确定多肽表位的HLA型; (c) Ⅱ类T细胞多肽表位在多肽H1-M8和H1-M9发生重叠; d) 与2009 pH1N1流感病毒相比, H7N9特异性肽序列中的突变位点以粗体和下划线标出

  • Table 3   Characteristics of the predicted peptides used in this study

    结合力通过http://www.cbs.dtu.dk/services/NetMHCpan/预测. b) 可以通过体外复性证明结合的用“+”表示, 其他用“–”表示

Copyright 2020 Science China Press Co., Ltd. 《中国科学》杂志社有限责任公司 版权所有