A型流行性感冒病毒的兩種主要表面醣蛋白:血球凝集素(hemagglutinin, HA)及神經氨酸酶(neuraminidase, NA)不斷地進行抗原漂變(antigenic drift)及移型(antigenic shift)，因此A型流感病毒持續對全球健康造成威脅。每年季節性流感在人類導致嚴重的發病率及致死率。接種疫苗是當前控制A型流感病毒最有效的策略。相較於血球凝集素，神經氨酸酶抗原漂變率較低，且神經氨酸酶蛋白的酵素活化位序列具有高度的保守性。因此神經氨酸酶被廣泛地運用在藥物設計， 也被認為有潛力運用在流感疫苗的研發。近年來的文獻發現，神經氨酸酶抗體被認為具有對抗異型流感病毒感染的能力。本論文目的為利用桿狀病毒/昆蟲細胞表現系統，製備禽流感H5N1，pH1N1新興流感病毒及帶有 I149V, N344Y, I365T/ S366N及同時帶有四種(I149V, N344Y和I365T/ S366N)突變的pH1N1之神經氨酸酶重組蛋白，並以肌肉注射的方式，探討此重組蛋白於小鼠體內所引起的免疫原性(immunogenicity)。結果顯示，帶有I365T/S366N及四種(I149V, N344Y和I365T/ S366N) 突變的重組蛋白皆能於小鼠體內誘導產生對抗pH1N1, H5N1, H3N2及 H7N9病毒株的專一性神經氨酸酶抑制性抗體及提供免疫保護力。因此,我們認為帶有I365T/S366N及四種(I149V, N344Y and I365T/ S366N) 突變的重組蛋白是有潛力發展成為具交叉保護的流感疫苗候選抗原。

Influenza A virus remains a persistent threat to global health because of continuously antigen drift and shift of two major surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Every year, seasonal pandemics of influenza cause serious morbidity and mortality in human throughout the world. Vaccination is an important strategy to prevent from influenza virus infection. Compared to HA, NA is less antigenic drift and enzymatic active site of NA is highly conserved among different subtypes. Also, it is broadly used in drug design and it may be an ideal target for vaccine development. In recent study, NA is considered to provide heterosubtypic immunity. In this study, we produced H5N1-recombinant NA (rNA), pH1N1-rNA, pH1N1-rNA with I149V, N344Y, I365T/ S366N and tetra (I149V, N344Y and I365T/ S366N) mutants proteins using baculovirus-expression system. The immunogenicity of the pH1N1-rNA mutant proteins was investigated in BALB/c mice. Our results indicated that pH1N1-rNA with double mutants (I365T/S366N) and tetra mutants (I149V, N344Y and I365T/S366N) immunized group can induce the highest specific NA-inhibiting antibodies against homologous pH1N1 as well as heterosubtypic H5N1, H3N2 and H7N9 virus strains. Therefore, we suggested that double mutants (I365T/S366N) and tetra mutants (I149V, N344Y and I365T/S366N) of pH1N1-rNAs can be potential vaccine candidates for developing broadly-protective vaccines against influenza virus infection.

Content
中文摘要 I
Abstract II
致謝 III
Content IV
1. Introduction 1
1.1 Overview of Influenza viruses 1
1.2 Influenza A viruses Neuraminidase 2
1.3 NA-specific antibodies in influenza vaccine development 3
1.4 Study goals 4
2. Material and Methods 6
2.1 Cell lines 6
2.2 Bac-to-Bac® Baculovirus expression system 6
2.2.1 Construction of recombinant soluble NA proteins (rNA) 6
2.2.2 Site-directed mutagenesis PCR 7
2.2.3 Generating the recombinant Bacmids 9
2.2.4 Production of recombinant Baculovirus 10
2.3 Production and purification of soluble rNA proteins 10
2.3.1 Generation of the soluble rNA proteins 10
2.3.2 Purification of rNA proteins from supernatants 11
2.4 Characterize rNA proteins 12
2.4.1 Sodium dodecyl sulfate polyacrylamide (SDS-PAGE ) gel preparation and electrophoresis 12
2.4.2 Protein transfer and immuno-hybridization 12
2.5 Mouse immunization regimens 13
2.6 Fetuin-based rNA enzyme activity and Neuraminidase-inhibiting (NAi) assay 14
2.6.1 Detection of enzyme activity of rNA 14
2.6.2 Neuraminidase-inhibiting (NAi) antibodies assay 15
2.7 Enzyme-linked immunosorbent assay (ELISA) 16
2.8 Viral challenge 16
2.9 Statistic analysis 17
3. Results 18
3.1 Amino acid comparison between H5N1-NA and pH1N1-NA 18
3.2 Construction of soluble rNA protein of H5N1, pH1N1 and pH1N1 with the I149V, N344Y, I365T/S366N or tetra (I149V, N344Y, I365T/S366N) mutants 19
3.3 Characterization of the enzyme function of rNA proteins of H5N1, pH1N1 and pH1N1 with the I149V, N344Y, I365T/S366N or tetra (I149V, N344Y, I365T/S366N) mutants 20
3.4 NA-specific IgG antibodies induced by rNAs immunization 21
3.5 N1 Neuraminidase-inhibiting (NAi) antibodies elicited by wild-type or mutant rNA proteins immunizations against pH1N1 and H5N1 viruses 21
3.6 Heterosubtypic neuraminidase-inhibiting antibody elicited by wild-type or mutant rNA proteins immunizations against H3N2 and H7N9 viruses 23
3.7 Protective immunity against different subtypes of influenza viruses challenges 25
4. Discussion 27
5. References 32
6. Figures 36
Fig. 1 3D tetrameric structure of Neuraminidase and alignment of neuraminidase of pH1N1 and H5N1 viruses 36
Fig. 2 Construction, expression and characterization of wild-type or mutant recombinant Neuraminidase (rNA) proteins near enzymatic-sites 37
Fig. 3 The enzymatic activity of the of wild-type or mutant recombinant Neuraminidase (rNA) proteins 38
Fig. 5 NA-inhibiting antibodies elicited by wild-type or mutant rNA proteins immunization against homologous pH1N1 40
Fig. 6 NA-inhibiting antibodies elicited by wild-type or mutant rNA proteins immunization against heterosubtypic H5N1 VLPs 41
Fig. 7 NA-inhibiting antibodies elicited by wild-type or mutant rNA proteins immunization against heterosubtypic H3N2 viruses 42
Fig. 8 NA-inhibiting antibodies elicited by wild-type or mutant rNA proteins immunization against heterologous H7N9 VLPs 43
FIG. 9 Protective immunity of wild-type or mutant rNA proteins in mice by H1N1 viral challenges 44
FIG. 10 Protective immunity of wild-type or mutant rNA proteins in mice by H5N1 viral challenges 45
Fig. 11 Protective immunity of wild-type or mutant rNA proteins in mice by H7N9 viral challenges 46
Table 1. The enzyme kinetics of wild-type or mutant rNA proteins evaluted by Lineweaver-Burk plots 47

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