在2013年中國爆發了新型禽流感H7N9的大流行，這個新型的病毒從2013到2017共造成了40%的死亡率。在台灣，有五個分別來自中國的第一波、第二波及第五波的境外感染。因此開發H7N9的疫苗來因應大流行是非常重要的事情。針對H7N9抗原所引起的低免疫反應，我們利用了趕狀病毒表現系統設計了兩個不同的表現載體，一個是分泌型的表現 (sH7)，一個是膜蛋白的表現 (mH7)。 mH7蛋白在純化過後形成具有8192的血球凝集力價的高度整齊排列的聚合體。相反的，sH7蛋白無法形成聚合體也對血球沒有凝集效果。儘管二級結構在sH7及mH7蛋白並沒有差別，在熱穩定實驗當中mH7在52度比sH7有著較好的穩定度。因此mH7可以在老鼠的實驗當中在注射第二劑後引發較好的免疫反應而sH7卻無法引起有效的免疫反應。因此mH7蛋白可以進一步的應用開發在H7N9流感病毒大爆發的疫苗開發上。
新型流感H5N2從2003年就開始在台灣流傳。在2012年，這個低致病性的H5N2病毒在雞隻當中演化成高致病性的病毒且造成了巨大的經濟損失。台灣疾病管制局在141個曾經接觸被感染的禽場的受測者當中發現了4.3%的血清陽轉率。HA蛋白的受體接受位的胺基酸突變是其中一個造成禽流感傳染給人的重要機制。我們利用了趕狀病毒表現系統表現H5N2 HA蛋白並且利用了結構學的方法發現在受體接受位的兩個胺基酸突變會造成受體專一性的改變。野生的HA蛋白與禽流感的sia-1醣有9個氫鍵的結合，顯示HA與禽流感有著較強的親和力 (0.48 μm)。與野生的HA比較，人類受器的sia醣在G228S突變體中往外位移了1.5 Å導致sia接近193號胺基酸。G228S變異體與人類的受器的親和力大於與禽類受器的親和力但Q226L變異體卻在與人類的受器結合方面有著較強的親和力。E190D、K193和N224K胺基酸的變異也導入Q226L變異體當中來試圖增加Q226L變異體與人類受器的結合能力。然而在E190D與K193T導入226L變異體後人類與禽類的結合能力確因此被消除。N224K胺基酸的突變與Q226L變異體結合後增強了Q226L變異體與人類受器結合的能力。這些發現就像在2013年爆發的新型H7N9流感上也存在著Q226L胺基酸的突變，對於禽類傳染人類的風險也就增加了，因此HA 受體結合位的胺基酸的變異對於禽類病毒傳變成為可以傳染人類的病毒有著非常重要的影響。

In 2013, avian influenza H7N9 widely caused human infections in China. This novel H7N9 virus caused about 40% case-fatality from 2013 to 2017. In Taiwan, five importeded human cases from china have been reported during the first, second and five waves. Therefore, development of influenza H7N9 vaccine is critical for pandemic preparedness. To overcome lower immunogenicity induced by recently recombinant H7N9 vaccine in mice study, recombinant full-length (mH7) and secreted ectodomain-based (sH7) protein were produced in our baculovirus expression system. After protein purification, mH7 was detected with highly order oligo-rosette in Small-angle X-ray scattering (SAXS) and had a high hemagglutinin (HA) titer 8192. In contrast to mH7, sH7 could not form an oligo-rosette structure and did not have HA titer. Although mH7 and sH7 did not have significant different in secondary structure, the mH7 still had better thermal stability (52 °C) than sH7 (43 °C). In a mice immunization study, the mH7 construct but not the sH7 construct could induce robust HI (GMT=101 vs. GMT=10) and neutralizing antibody titers (GMT=112 vs. GMT=25). In conclusion, further development of the mH7 vaccine candidate is desirable.
Novel avian influenza virus H5N2 has been circulating in Taiwan since 2003. In 2012, the low pathogenic H5N2 has evolved into high pathogenic resulting in huge economic lost. Taiwan Centers for Disease Control (CDC) found that 4.3% seroconversion in 141 subjects who had close contacts with infected poultry. The critical step of avian influenza to adapt into human to human transmission is amino acid substitution in HA receptor binding site (RBS). We employed the baculovirus platform to generate recombinant H5 HA for structural biology study and found that two amino acid mutations could change receptor binding specificity. In wild type HA and avian receptor analogue 3SLN (α2,3-Sialyl-acetyllactosamine) complex, sia-1 made 9 hydrogen bond with RBS and displayed stronger binding affinity to 3SLN (with 0.48 μm) in SPR experiment. Comparing to wild-type HA, the sia-1 of human receptor 6SLN (α2,6-Sialyl-acetyllactosamine) in G228S mutant was observed moving toward outside with 1.5 Å resulting in close to K193. The binding affinity of G228S with 6SLN (>5μm) is lower than 3SLN (3.3μm) whereas the Q226L mutant showed significantly human binding ability and abolished almost all binding capacity with avian glycans. K193T, N224K, and E190D were also introduced into Q226L mutant to enhance binding ability with human glycans, respectively. However, E190D and K193T mutations abolished wholly avian and human binding in glycan array and could not be detected in SPR experiment. N224K combined with Q266L enhanced 2-6 binding in glycan array and showed quantitatively switch from avian to human. In conclusion, one amino acid substitution could alter influenza virus receptor specific, like H7N9 pandemic-like virus in 2013, which is already have Q226L mutation. Therefore, the RBS variation increase the zoonotic transmission potential between avian and human.

Introduction 13
Part1: Improving immunogenicity of influenza virus H7N9 recombinant hemagglutinin for vaccine development 20
Materials and Methods 21
Recombinant HA construction 21
Baculovirus production 21
Expression and purification 22
Hemadsorption test 23
Recombinant protein stability assay 23
SDS-PAGE and Western blotting 24
Circular dichroism assay 24
Small-angle X-ray scattering (SAXS) 24
Animal vaccination 26
Serological test 27
Soluble hemagglutination assay 27
Results 28
Construction and identification of HA expressed in insect cells 28
Structural stability of H7 protein in protease and thermal treatment 29
Characterization of recombinant mH7 by small angle X-ray scattering 30
Antibody responses 30
Discussion 32
Figure legends 35
Fig 1. Diagram of two different influenza H7 expression constructs. (A) The sH7 ectodomain (18-513) was incorporated with N-terminal secretion signal (gp67), C-terminal hexa-His tag followed by trimerization foldon domain and thrombin cleavage site. (B) The mH7 construct was designed with a full-length (A/Taiwan/1/2013) sequence containing original signal peptide and TM domain under the polyhedron (PH) promoter. The protease cleavage site between HA1 and HA2 is pointed to with an arrow. After trypsin cleavage, the sH7 and mH7 were separated into HA1 and HA2. 35
Fig 2. Characterization of mH7 and sH7 using size exclusion chromatography. 36
Fig 3. Analysis of purified H7 protein after enzymatic digestion using SDS-PAGE and Western blot after purification and 30 days after purification. 37
Fig 4. Secondary structure analysis of sH7, sH7-m and mH7. 38
Fig 5. Thermal stability assay to evaluate the effect of foldon and TM domain with temperature escalation.. 39
Fig 6. HPLC/SAXS intensity and the evolution of the radius of gyration (Rg) and the corresponding rosette conformations (as shown) extracted along the sample elution time. 40
Fig 7. Hemagglutinin inhibition and neutralization titers in mice immunized with egg-derived influenza H7N9 whole virus antigen (NIBSC H7N9) and recombinant influenza H7N9 HA monomer (sH7-m). 41
Fig 8. Hemagglutinin inhibition and neutralization titers in mice immunized with egg-derived influenza H7N9 whole virus antigen (NIBSC H7N9) and recombinant influenza H7N9 membrane-based HA (mH7).. 42
Part2: Structural basis of receptor binding alternation of endemic Mexican-like chicken H5N2 viruses in Taiwan 43
Material and Method 44
Cloning, expression and protein purification 44
Crystallization, data collection and structural determination 45
Glycan microarray 46
Surface plamon resonance (SPR) analysis 46
Result 48
Sequence Evolution of Receptor Binding Site from H5N2 HA 48
Characterization of H5N2 HA and mutants receptor binding properties 49
Structural characterization of Mexican-like H5N2 HA and HA mutants 50
Enhancement of human receptor binding ability with several residue substitutions in Q226L mutant 51
Characterization the influence of N224K with Q226L mutant 52
Discussion 54
Figure legends 59
Fig 1. Structural comparison of HA receptor binding subdomain. 59
Fig 2. Receptor specificities of wild type H5N2 HA and mutants 60
Fig 3. 2Fo-Fc of electron density maps of human and avian receptor analogues in H5N2 HA crystal structure 61
Fig 4. Crystal structures of 0502 H5N2 HA and G228S mutant with avian (3SLN) and human (6SLN) receptor analogues. 62
Fig 5. Structural comparison of 3SLN and 6SLN in G228S mutant with WT HA 63
Fig 6. Biacore binding properties of H5N2 and mutants HA to avian receptor analogues 3SLN and human analogues 6SLN. 65
Fig 7. Receptor specificities of HA mutants in glycan array. 66
Fig 8. Receptor binding specific of N224KQ226L HA mutant. 68
Fig 9. Binding affinity of N224KQ226L HA mutant with Tetra-antennary. 69
Fig 10. Different orientation of Sia-1 of 3SLN and 6SLN in complex with Q226LG228S HA mutant. 71
Fig 11. Structural comparison of Q226LG228S HA mutant receptor binding subdomain (130-232). 72
Fig 12. Distribution of positive charge in HA1 head domain of H5N2. 73
Table 1. Sequence alignment of basic element in Receptor binding site for H5N2 from 2003 to 2015 in Taiwan. 74
Table 2. Comparison of evolution of receptor binding site among H5N2, H5N1 and H2N2 76
Table 4. list of glycan array 78
Reference 81

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