高病原性禽流感H5N1可使人類被感染並對人類的生命造成威脅。血凝素 (hemagglutinin, HA) 是流感病毒上主要的膜蛋白也大量用於疫苗開發。HA是三具體蛋白，由兩個部分組成: HA球狀區域 (globular head) 跟HA軀幹部 (stem region)。在先前的研究已經證實，將HA球狀區域 g127+g138 (dm) 或g83+g127+g138 (tm)的醣遮蔽會誘導廣效性中和抗體去對抗不同亞型的H5N1；另外移除位於HA軀幹部 (stem region) 的醣基，可引發針對同型、異源型和異型病毒產生更高的中和抗體效價。在此篇研究中，我們把HA球狀區域醣遮蔽跟HA軀幹部去醣基化作結合(dm/st2和tm/st2)。利用禽流感H5N1 (A/Thailand/KAN-1/2004) 病毒的HA，用點突變的方式去生產重組H5HA蛋白及H5HA腺病毒載體，並使用不同免疫方式: 2劑重組蛋白和以腺病毒載體prime和重組蛋白boost的方式去免疫小鼠並採取血清做後續分析測試。經本篇研究證實，施打2劑重組蛋白或腺病毒載體prime和重組蛋白boost後，dm/st2 和 tm/st2血清組別對抗不同支系H5N1病毒的中和性抗體可被顯著提升。其中，重組蛋白dm/st2的組別可藉由增加具有軀幹部專一性的抗體來產生廣效性中和抗體對抗不同的異源型H5N1病毒也可以增強抑制病毒融合(anti-fusion)抗體。本篇論文研究結果可為發展廣效型H5N1禽流感病毒疫苗的開發，提供寶貴的資訊。

The Highly pathogenic avian influenza (HPAI) H5N1 viruses cause a serious threat to public health and severe mortality in humans. The hemagglutinin (HA) is a major envelope glycoprotein of influenza virus and also is the target for development influenza vaccines. HA is homo-trimeric complex structure protein, each monomer comprises two components, the globular head domain and stem region. The strategy of glycan-masking the HA globular head domain g127+g138 (dm) and g83+g127+g138 (tm) mutants was previously demonstrated to elicit more potent and broadly neutralizing antibodies against various H5N1 clades/subclades. Unmasking the HA2-stem region N484A (st2) was also discovered to induce more broadly neutralizing antibodies against the homologous and heterosubtypic influenza A viruses. In this study, we developed a new strategy that combines glycan masking and unmasking of hemagglutinin antigens by immunizations with recombinant HA protein, adenovirus vector, and the heterologous prime-boost regimen. Our results indicate that dm/st2 and tm/st2 elicited more neutralizing antibodies titer against heterologous virus of H5N1. Recombinant HA (rHA) dm/st2 induced significantly higher titers of stem-specific antibodies,and enhances H5HA stem-specific anti-fusion antibodies. These findings may provide information for developing more broadly protective HA stem-focused influenza vaccines.

中文摘要 II
Abstract III
致謝 IV
Content V
1. Introduction 1
1.1. Overview of Influenza A viruses 1
1.2. H5N1 avian influenza A viruses 2
1.3. Influenza A virus HA antigen 3
1.3.1. HA structure 3
1.3.2. HA globular head 4
1.3.3. HA Stem region 4
1.4. Influenza HA vaccine design 5
1.5. Glycan masking on the HA globular head for vaccine design 6
1.6. Glycan unmasking on the HA stem region for vaccine design 8
1.7. Study goals 9
2. Materials and Methods 11
2.1. Cell lines 11
2.2. Construction Design of the glycan masking and glycan unmasking recombination H5HA Proteins 11
2.3. Insect-baculovirus expression system for rH5HA proteins 13
2.3.1. Construction of recombinant plasmids 13
2.3.2. Production of recombinant HA proteins 14
2.3.3. Purification of recombinant HA proteins 15
2.3.4. Western blot 15
2.3.5. Commassie blue staining 16
2.3.6. Hemagglutination assay 16
2.4. Preparation of recombinant adenovirus 16
2.4.1. Plaque assay for recombinant adenovirus titration 17
2.4.2. Hemadsorption assay 18
2.5. Mouse Immunizations 18
2.5.1. Enzyme-linked immunosorbent assay (ELISA) 19
2.5.2. Preparation of H5N1 pseudotyped particle (H5pp) 20
2.5.3. H5pp neutralization assay 20
2.5.4. Protein absorption and antibody competition assay 21
2.5.5. Quantitative luciferase-based cell-cell fusion assay 21
2.5.6. Plaque assays and plaque reduction neutralization test (PRNT) 23
2.5.7. Influenza hemagglutination inhibition assay 23
2.6. Statistic analysis 24
3. Results 25
3.1. H5 antigen design by the combination of globular head glycan masking and HA2-stem unmasking strategies 25
3.2. Expression and characterization of soluble rHA -wt, rHA -dm/st2, or rHA -tm/st2 proteins 26
3.3. Binding of rHA proteins to RBS-specific mAbs and stem-specific mAbs 27
3.4. Construction and characterization of adenovirus vector expressing the full-length HA-wt, HA-dm/st2, and HA-tm/st2 27
3.5. Total IgG titers elicited by two-dose rHA immunization or the Ad-HA-prime rHA-boost immunization. 28
3.6. Neutralizing antibodies elicited by two-doses of rH5 protein or the Ad-HA-prime rHA-boost immunization. 29
3.7. Mapping the stem-specific antibodies elicited by two-dose rHA immunization or the Ad-HA-prime rHA-boost immunization. 32
3.8. Anti-fusion activity elicited by two-dose rHA immunization or the Ad-HA-prime rHA-boost immunization. 34
3.9. Hemagglutination inhibition and Neutralizing antibodies elicited by two-dose rHA immunization or the Ad-HA-prime rHA-boost immunization. 35
4. Discussion 37
5. References 41
6. Figures 47
Fig. 1 3D structure model of influenza of H5N1 viruses. 47
Fig. 2 Characterization of recombinant H5HA proteins 48
Fig. 3 Specific antibody binding analysis for rHA proteins 49
Fig. 4 Characterization of recombinant H5HA adenovirus vector. 50
Fig. 5 Total IgG titers elicited by two-doses of rH5 protein or the Ad-HA-prime rHA-boost immunization. 51
Fig. 6 Neutralizing antibodies elicited by two-doses of rHA protein or the Ad-HA-prime rHA-boost immunization. 54
Fig. 7 Neutralizing antibodies elicited by two-doses of rHA protein or the Ad-HA-prime rHA-boost immunization. 56
Fig. 8 Mapping of stem-specific antibodies elicited by two-doses of rHA protein or the Ad-HA-prime rHA-boost immunization. 58
Fig. 9 Competition assay of stem-specific antibodies elicited by two-doses of rHA protein or the Ad-HA-prime rHA-boost immunization 59
Fig. 10 Anti-fusion activity elicited by two-doses of rHA protein or the Ad-HA-prime rHA-boost immunization. 60
Fig. 11 Hemagglutination inhibition and neutralizing antibodies elicited by two-doses of rH5 protein or the Ad-HA-prime rHA-boost immunization. 61
7. Tables 62
Table. 1 Immunization groups by two-doses of rHA protein or the Ad-HA-prime rHA-boost immunization. 62

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