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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

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

Abstract

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

国家自然科学基金(81822040,81373141)


Supplement

补充材料

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.


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  • 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

    ID

    性别a)

    年龄(岁)

    HLA-A分型

    HLA-B分型

    Z1

    M

    33

    A*1101, A*3101

    b)

    Z2

    F

    30

    A*0201, A*0201

    B*0801, B*4001

    Z3

    M

    24

    A*2402, A*2402

    B*1801, B*4001

    Z4

    M

    28

    A*1101, A*0201

    B*1302, B*1501

    Z5

    M

    39

    A*0206, A*0206

    Z6

    F

    25

    A*2402, A*3303

    C1

    F

    29

    A*0210, A*3001

    B*1302, B*4006

    C2

    M

    28

    A*0201, A*1101

    B*1501, B*4601

    C3

    F

    24

    A*1101, A*2402

    B*1501

    C4

    M

    25

    A*1101, A*3101

    B*4001, B*5101

    C5

    F

    26

    A*1101, A*2402

    B*1501, B*1511

    C6

    M

    25

    A*1101, A*2402

    B*1302

    C7

    F

    25

    A*0201, A*3001

    B*4403, B*5401

    C8

    F

    23

    A*0207, A*6801

    B*3801, B*4601

    C9

    M

    37

    A*1101, A*1101

    B*1501, B*4001

    C10

    M

    29

    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

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