本論文主要研究利用免疫聚焦性 (immunofocusing) 策略，即醣遮蔽與去醣基化的方式，將抗原辨識的位置轉到保守的區域，進一步引發廣效性中和抗體。針對禽流感H5N1病毒的HA進行點突變，以產生不同免疫聚焦性抗原的類病毒顆粒和H5HA腺病毒載體，此外利用在M2融合FliC和Fc分子佐劑來增強免疫效果。本篇研究證實，小鼠在接種兩劑免疫聚焦性(dm: g127+g138和tm: g83+g127+g138)類病毒顆粒後，血清對抗不同支系H5N1病毒的中和性抗體可被顯著提升，尤其添加了FliC和Fc分子佐劑的效果會比wild type來的更有差異。然而(st2: N484A)免疫聚焦性類病毒顆粒在加上FliC和Fc分子佐劑即失去引起中和性抗體的功能。本論文進一步以腺病毒載體prime和類病毒顆粒boost的免疫方式，但是並不能產生比兩劑免疫聚焦性類病毒顆粒免疫更高的中和性抗體，且免疫血清並不能跨型中和pH1N1。因此更進一步利用同時添加不同類型的HA搭配GM-CSF和FliC來免疫小鼠，結果發現免疫小鼠血清可以同時保護H5H7或H1H5H7來達到廣效性中和抗體的成效。此研究成果可作為未來發展廣效型H5N1禽流感病毒疫苗和廣效型多重流感病毒疫苗的參考。

The study was aimed at developing the influenza virus-like particles (VLPs) containing the immunofocusing HA antigens that can “refocus” antibody responses from the immunodominant antigenic sites to the conserved cross-protective antigenic sites. The immunofocusing VLPs can thus elicit more broadly neutralizing antibodies against the heterologous clades of HPAI H5N1 viruses. The immunofocusing VLPs were constructed to the glycan masking on the HA globular head by double mutations (dm: g127+g138) or triple mutations (tm: g83+g127+g138) or the glycan unmasking on the HA2-stem (st2) region. Immunofocusing VLPs by M2 fusion with FliC (TLR5 agonist) or IgG-2a Fc fragment were obtained to enhance the VLP immunogenicity. Immunization results showed that the dm VLPs and the tm VLPs, but not the st2 VLPs, induced more potent broadly neutralizing antibodies against three heterologous HPAI H5N1 clades (Indonesia, clade 2.1.3.2; Qinghai, clade 2.2; Anhui, clade2.3.4). Furthermore, followed by using adenovirus vector priming VLPs boosting of the immunofocusing dm or tm HAs also resulted in increasing potent broadly neutralizing antibodies against three heterologous HPAI H5N1 clades (Indonesia, clade 2.1.3.2; Qinghai, clade 2.2; Anhui, clade2.3.4) However, the sera of Ad-Has priming VLPs boosting cannot cross protect hetersubtypic influenza A virus. Therefore, the dual-adjuvanted GM-CSF/FliC H5N1 VLP were further contructed to include H7 or H1H7 as multi-subtype influenza VLP vaccines. Results show that H5H7-GM-CSF/FliC VLPs can simultaneously against H5pp and H7pp. For H1H5H7-GM-CSF/FliC VLPs can simultaneously against pH1N1, H5pp, and H7pp It is our hope that these findings provide useful information for developing universal influenza vaccines.

Content
Development of immunofocusing virus-like particles as broadly protective influenza vaccines I
中文摘要 II
Abstract III
Acknowledgements IV
Abbreviations V
Content VI
1. Introduction 1
1.1 Overview of Influenza A viruses 1
1.2 Epidemiology of H1N1, H5N1, and H7N9 influenza viruses 3
1.3 Influenza virus-like particle vaccine technologies 7
1.4 Development of multi-subtype influenza VLP vaccine 10
1.5 N-linked glycosylation of influenza HA envelope proteins 12
1.6 Immunofocusing HA by N-linked glycan masking on Globular head 14
1.7 Immunofocusing HA by N-linked glycan unmasking on HA2 stem 15
1.8 Study goals for research 16
2. Materials and methods 17
2.1 Cell lines 17
2.2 Immunofocusing VLP construction design 17
2.3 Bac-to-Bac baculovirus expression system 18
2.3.1 Construction of recombinant plasmids 18
2.3.2 Generation of recombinant baculovirus 19
2.4 Production and purification of immunofocusing H5N1 and multi-subtype VLPs 20
2.5 SDS-PAGE and Western blot analyses 20
2.6 Hemagglutination (HA) assay and hemagglutination inhibition (HI) assay 21
2.7 Production and purification of KAN-1 H5HA proteins 21
2.8 Preparation of recombinant adenovirus 22
2.9 Plaque assay for recombinant adenovirus titration 23
2.10 Hemadsorption assay 23
2.11 Mouse Immunizations 24
2.12 Preparation of H5N1 pseudotyped particle (H5pp) 25
2.13 H5N1 pseudotyped particle (H5N1pp) neutralization assay 25
2.14 ELISA 26
2.15 Transmission electron microscopy (TEM) 27
2.16 pH1N1 and H3N2 virus plaque neutralization assay 27
2.17 Statistic analysis 28
3. Results 29
3.1 Construction and characterization of wt-VLPs, FliC-wt-VLPs, and Fc-wt-VLPs. 29
3.2 Construction and characterization of immunofocusing (dm, tm, st2) FliC-VLPs and Fc-VLPs 30
3.3. Mouse immunizations with immunofocusing (dm, tm, st2) FliC-VLPs and Fc-VLPs 31
3.4 Neutralizing antibodies elicited by two-dose immunofocusing FliC-VLP+FliC-VLP and Fc-VLP+Fc-VLP immunizations 32
3.5 Mouse immunizations with immunofocusing (dm, tm, st2) adenovirus vector priming and immunofocusing (dm, tm, st2) FliC-VLP and Fc-VLP boosting. 33
3.6 Neutralizing antibodies elicited by Ad-HA+FliC-VLP or Ad-HA+Fc-VLP prime-boost immunization 35
3.7 IC50 values ratio comparison of 2 doses immunofocusing VLPs and adenovirus prime VLPs boost immunization 36
3.8 pH1N1 hemagglutination inhibition and plaque neutralization assay 37
3.9 Bi- and tri-subtype GM-CSF/FliC-fused influenza VLPs expression and characterization 37
3.10 H5HA-specific total IgG titers elicited by multi-subtype VLPs immunization 38
3.11 Neutraling antibodies elicited by bi-subtype (H5H7) and tri-subtype (H1H5H7) GM-CSF/FliC VLPs 39
4. Discussion 40
5. References 42
6. Figure Legends 56
Fig. 1 Construction and characterization of wt-VLPs, FliC-wt-VLPs, and Fc-wt-VLPs 56
Fig. 2 Construction and characterization of immunofocusing (dm, tm, st2) FliC-VLPs and Fc-VLPs 56
Fig. 3 Mouse immunizations with immunofocusing (dm, tm, st2) FliC-VLPs and Fc-VLPs 56
Fig. 4 Neutralizing antibodies elicited by two-dose immunofocusing FliC-VLP+FliC-VLP and Fc-VLP+Fc-VLP immunizations 56
Fig. 5 Neutralizing antibodies elicited by two-dose FliC-VLP + FliC-VLP or Fc-VLP + Fc-VLP immunizations 57
Fig. 6 Hemadsorption assay for Ad-HA vector-infected 293A cells 57
Fig. 7 Mouse immunizations with adenovirus vector priming, followed by a booster of FliC-VLPs or Fc-VLPs 57
Fig. 8 Neutraling antibodies elicited by two-dose immunofocusing Ad-HA+FliC-VLP and Ad-HA+Fc-VLP immunizations 57
Fig. 9 Neutraling antibodies elicited by two-dose immunofocusing Ad-HA+FliC-VLP and Ad-HA+Fc-VLP immunizations 57
Fig. 10 IC50 values ratio comparison of two-dose immunofocusing FliC-VLP+FliC-VLP, Fc-VLP+Fc-VLP, Ad+FliC-VLP, and Ad+Fc-VLP immunizations 58
Fig. 11 pH1N1 hemagglutination inhibition and plaque neutralization assay 58
Fig. 12 Bi- and tri-subtype GM-CSF/FliC-fused influenza VLP expression and characterization 58
Fig. 13 Total IgG titers and cross-reactive HI and Neutralizing antibodies elicited by bi- and tri-subtype GM-CSF/FliC VLPs 58
Fig. S1 Construction, characterization, and hemagglutination assay of C3d-wt-VLPs and immunofocusing (dm, tm, st2) C3d-VLPs 59
Fig. S2 Mouse immunizations with immunofocusing (dm, tm, st2) C3d-VLPs 59
Fig. S3 Neutralizing antibodies elicited by two doses immunofocusing C3d-VLP+C3d-VLP immunizations 59
Fig. S4 Mouse immunizations with immunofocusing (dm, tm, st2) adenovirus vector priming and immunofocusing (dm, tm ,st2) C3d-VLP boosting 60
Fig. S5 Neutralizing antibodies elicited two-dose immunofocusing Ad-HA+C3d-VLP immunizations. 60
Fig. S6 pH1N1 hemagglutination inhibition and plaque neutralization assay 60
7. Figures 61
Fig. 1 Construction and characterization of wt-VLPs, FliC-wt-VLPs, and Fc-wt-VLPs 61
Fig. 2 Construction and characterization of immunofocusing (dm, tm, st2) FliC-VLPs and Fc-VLPs 62
Fig. 3 Mouse immunizations with immunofocusing (dm, tm, st2) FliC-VLPs and Fc-VLPs 63
Fig. 4 Neutralizing antibodies elicited by two-dose immunofocusing FliC-VLP+FliC-VLP and Fc-VLP+Fc-VLP immunizations 64
Fig. 5 Neutralizing antibodies elicited by two-dose immunofocusing FliC-VLP+FliC-VLP or Fc-VLP+Fc-VLP immunizations 65
Fig. 6 Hemadsorption assay for Ad-HA vector-infected 293A cell 66
Fig. 7 Mouse immunizations with adenovirus vector priming, followed by a booster of FliC-VLPs or Fc-VLPs 67
Fig. 8 Neutraling antibodies elicited by two-dose immunofocusing Ad+FliC-VLP and Ad+Fc-VLP immunizations 68
Fig. 9 Neutraling antibodies elicited by two-dose immunofocusing Ad+FliC-VLP and Ad+Fc-VLP immunizations 69
Fig. 10 IC50 values ratio comparison of two-dose immunofocusing FliC-VLP+FliC-VLP, Fc-VLP+ Fc-VLP, Ad+FliC-VLP, and Ad+Fc-VLP immunizations 70
Fig. 11 pH1N1 hemagglutination inhibition and plaque neutralization assay. 71
Fig. 12 Bi- and tri-subtype GM-CSF/FliC-fused influenza VLP expression and characterization. 72
Fig. 13 Total IgG titers and cross-reactive HI and Neutralizing antibodies elicited by bi- and tri-subtype GM-CSF/FliC VLPs. 73
7. Supplementary Tables and Figures 74
Fig. S1 Construction, characterization, and hemagglutination assay of C3d-wt-VLPs and immunofocusing (dm, tm, st2) C3d-VLPs 77
Fig. S2 Mouse immunizations with immunofocusing (dm, tm, st2) C3d-VLPs 78
Fig. S3 Neutralizing antibodies elicited by two doses immunofocusing C3d-VLP+C3d-VLP immunizations 79
Fig. S4 Mouse immunizations with immunofocusing (dm, tm, st2) adenovirus vector priming and immunofocusing (dm, tm ,st2) C3d-VLP boosting 80
Fig. S5 Neutralizing antibodies elicited two-dose immunofocusing ad+C3d-VLP immunizations 81
Fig. S6 pH1N1 hemagglutination inhibition and plaque neutralization assay 82

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