高病原性禽流感可造成大量禽類死亡及在人類中造成高死亡率，而2009年爆發之流行性感冒更造成人類嚴重的威脅。由於血凝素(hemagglutinin, HA)在流感病毒演化過程中的高度變異性，對於發展可以對抗多種類流感病毒的廣效型疫苗而言仍是一大挑戰。相較於高變異性的HA球狀區域(globular head)，位於軀幹部(stem region)的N-linked 醣基在不同A型流感分離株中具有較佳的保留性。此篇研究針對禽流感H5N1 (A/Thailand/ KAN-1/2004) 和2009年爆發之流行性感冒pH1N1(A/Texas/05/2009)的HA軀幹部進行高保留性N-linked醣基預測與移除，並利用桿狀病毒表現系統(baculovirus expression system)製備軀幹去醣基化(stem-deglycosylated)的重組血凝素蛋白─H5 N19, 20, 34A、H5 N484A、H1 N32, 33, 45A和H1 N503A。在老鼠接種軀幹去醣基化免疫原後，進一步分析血清中的IgG、中和性抗體以及軀幹部專一性抗體反應。本篇研究證實，藉由高保留性N-linked醣基的移除可以誘發中和性抗體以對抗同源性的A型流感病毒。尤其是移除pH1N1 HA第503個胺基酸上的N-linked醣基，推判在免疫老鼠對抗異源性H5N1病毒的交叉保護與中和性免疫反應有重要影響。此研究成果可以作為未來發展廣效型疫苗的參考。

Due to rapid variation of hemagglutinin (HA), the major virion surface glycoprotein of influenza A virus, it remains challenge to develop broader and more effective universal vaccines. Compared to the highly variable HA globular head, N-linked glycan on the stem region is more conserved among different strains and most subtypes. In this study, we predicted and removed the conserved N-linked glycans in the H5N1 (A/Thailand/ KAN-1/2004) and pH1N1(A/Texas/05/2009) stem region. The stem-deglycosylated H5 N19, 20, 34A, H5 N484A, H1 N32, 33, 45A, and H1 N503A recombinant HA proteins (rHAs) were purified by baculovirus expression system. Further, after vaccination, we analyzed anti-HA total IgG and neutralizing antibodies (nAbs) titer against different subtypes of influenza viruses, and the stem-specific antibody responses were also identified. We successfully produced stem-deglycosylated rH5HAs and rH1HAs that could induce neutralizing antibodies against homologous serotype of influenza A virus. In addition, the removal of N-linked glycan on pH1N1 stem region 503 residue may be important to elicit neutralizing antibodies and cross-reactive protection against heterosubtypic H5N1, suggesting that H1 N503A protein might be used as a cross-reactive vaccine candidate.

中文摘要 I
Abstract II
致謝 III
Content IV
1. Introduction 1
1.1 Overview of influenza A virus 1
1.2 Hemmaglutinin of influenza A virus 4
1.2.1 Structure of HA 4
1.2.1.1 Globular head of HA 5
1.2.1.2 Stem region of HA 6
1.2.2 Glycosylation of HA 6
1.2.2.1 Glycosylation on HA globular head 7
1.2.2.2 Glycosylation on HA stem region 8
1.3 Influenza A vaccine development based on HA 8
1.4 Study goals 11
2. Materials and methods 13
2.1 Cell lines 13
2.2 Construction of recombinant HA proteins 13
2.2.1 Construction design 13
2.2.2 Plasmid preparation 14
2.3 Bac-to-Bac baculovirus expression system 14
2.3.1 Generating the recombinant bacmid 14
2.3.2 Recombinant bacmid clones amplification 15
2.3.3 Transfection by Turbofect 15
2.4 Production and purification of recombinant HA proteins 16
2.4.1 Production of recombinant HA proteins 16
2.4.2 Purification of recombinant HA proteins 16
2.5 HA proteins characterization 17
2.5.1 Western blotting 17
2.5.2 Protein transfer and immuno-hybridization 17
2.5.3 Coomassie blue staining 18
2.5.4 HA glycosylation pattern test by peptide-N-glycosidase F (PNGaseF) treatment 18
2.6 Mouse immunization 18
2.7 Enzyme-linked immunosorbent assay (ELISA) 19
2.8 H5N1 pseudotyped particle (H5N1pp) neutralization assays 19
2.9 Plague assay 20
2.10 Plague reduction neutralization test (PRNT) 20
2.11 Absorption assay 21
2.13 Statistic analysis 21
3. Results 22
3.1 N-linked glycosylation sites located in the stem region of H5N1 and pH1N1 HA proteins 22
3.2 Bindings of stem-deglycosylated rH5HA and rH1HA to stem-specific mAbs C179, CR6261, and FI6v3 23
3.3 HA-specific IgG titers elicited by stem-deglycosylated rH5HA and rH1HA immunization 24
3.4 Neutralizing antibody titers elicited by stem-deglycosylated rH5HA and rH1HA immunization 25
3.5 Stem-specific antibodies elicited by stem-deglycosylated rH5HA and rH1HA immunization 27
4. Discussion 28
5. References 34
6. Figures 41
Fig. 1 Sequence alignment and N-linked glycans prediction by NetNGlyc Server 41
Fig.2 3D structure model of influenza HA containing potential N-linked glycans on stem region 42
Fig. 3 Characterization of recombinant influenza HA glycoproteins 43
Fig. 4 Specific antibody binding analysis for WT and stem-deglycosylated HA proteins 44
Fig. 5 Total IgG titers elicited by stem-deglycosylated H5HAs and H1HAs 45
Fig. 6 Neutralizing antibodies elicited by stem-deglycosylated H5HAs against H5N1 (KAN-1) pseudotyped virus particle and H1N1 (A/California/04/2009) virus 46
Fig. 7 Neutralizing antibodies elicited by stem-deglycosylated H1HA proteins against H1N1 (A/California/04/2009) virus and H5N1 (KAN-1) pseudotyped virus particle 47
Fig. 8 Mapping the stem-specific antibodies elicited by stem-deglycosylated rH5HAs and rH1HAs 48
7. Supplements 49
Fig.S1 Characterization of stem-deglycosylated rH5HA and rH1HA proteins 49
Fig.S2 Characterization of Δstem-H5HA andΔstem-H1HA 50
Fig.S3 TNF-α production of DCs by stem-deglycosylated rH5HA or rH1HA treatment 51
Fig.S4 IL-12 p40 activity of DCs by stem-deglycosylated rH5HA or rH1HA stimulus 52
Fig.S5 CD40, CD86, and IAb surface markers on DCs analysis due to stem-deglycosylated rH5HA or rH1HA stimulus 53

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