臺灣博碩士論文加值系統

全球性的流感病毒(Influenza virus)傳染，主要源於人體免疫系統無法產生
有效的抗體辨認流感病毒上的凝集素蛋白質(Hemagglutinin)，使得流感病毒在
人跟人之間可以有效率的傳染開來。凝集素，是病毒表面上主要的醣蛋白
(Glycoprotein)，它具有專一性結合唾液酸(Sialic acid)的能力。流感病毒藉
由凝集素結合人類呼吸道表皮細胞上的唾液酸，吸附住細胞並藉由細胞的吞噬作
用(Endocytosis)，進入宿主細胞。凝集素被認定是關鍵的抗原物質，若將凝集
素當成疫苗，可提供宿主細胞產生免疫反應，對抗病毒入侵人體。本研究結果指
出，與正常的凝集素比較，修飾凝集素醣化位置上的多醣結構(Cmplex type
N-linked glycans)成為單醣(Single N-linked N-acetylglucosamine ,GlcNAc)
後，製備成疫苗(Monoglycosyalted HA, HAmg)，可增加老鼠體內免疫反應並產生
較高濃度具有中和病毒能力的抗體。我們進一步測試凝集素上帶有不同程度醣類
物質時， 所提供交叉保護的免疫反應(Cross reactivity and protective
immunity)。結果顯示，老鼠經施打2007 A (H1N1)季節性流感(seasonal flu)
或2009 A (H1N1)猪流感病毒(Pandemic flu)所製備的單醣化凝集素疫苗後，都
可誘導出較高濃度並具有中和病毒能力的抗體，有效抑制病毒凝集紅血球
(Hemagglutination inhibition, HI) 及阻止病毒進入細胞的能力
(Microneutralization, MN)。除此之外，老鼠接種2007 A (H1N1)單醣凝集素
蛋白質疫苗後，在攻毒實驗(Challenge assay)測試下，可提供老鼠較高存活率
對抗100 倍致死劑量的2009 A (H1N1)猪流感病毒攻擊。本研究以新的策略製備
病毒表面凝集素為單醣化的蛋白質疫苗，提供交叉保護的免疫反應對抗流感病
毒，並在愛滋病(Human immunodeficiency virus, HIV)、C 型肝炎(Hepatitis
C virus,HCV)及等人類傳染性病毒疫苗設計提供新方法。

Influenza pandemics occur when influenza hemagglutinins (HA) are little recognized
by immunity and the viruses transmit efficiently from human to human. HA is the
major viral surface glycoprotein that binds to specific sialylated glycan receptors in
the respiratory tract and allows the virus to enter the cell. It has been recognized as the
key antigen in the host immune response to influenza virus in both natural infection
and vaccination. We have shown that HA with a single N-linked N-acetylglucosamine
(GlcNAc) at each glycosylation sites (monoglycosylated HA, HAmg) as a protein
vaccine increased the antibody response and neutralization activity as compared to the
fully glycosylated HA (HAfg). In this study, we examined the cross reactivity and
protective immunity of antibodies elicited by vaccination of HA, with various degrees
of glycosylations, toward seasonal and 2009 swine-origin influenza A (H1N1) viruses.
The HAmg vaccine elicited anti-HA antibodies with higher hemagglutination
inhibition, microneutralization activity and resulted in a better survival rate in virus
challenge experiments than HAfg. The protein vaccines of HAmg designed from 2009
A (H1N1) swine and 2007 A (H1N1) Brisbane are both sufficient to provide
cross-protection against infections with the highly pathogenic H1N1 viruses in mice.
This study provides new insights into the origin of cross reactivity and protective
immunity provided by monoglycosylated viral surface glycoproteins and reveals a
general strategy for vaccine design against human viruses.

Table of Contents
(A) Introduction ...........................................................................................................1
1. Influenza virus .........................................................................................................1
2. Components of the influenza A virus.......................................................................1
3. Basic structure of HA...............................................................................................2
4. Receptor binding of the HA.....................................................................................3
5. Importance of the glycosylation of influenza virus A hemagglutinin......................4
6. The current pandemic virus situation.......................................................................5
7. HA protein vaccines design .....................................................................................6
8. Significance..............................................................................................................7
9. Specific aims............................................................................................................8
10. Experimental design.................................................................................................9
(B) Material and Methods-1......................................................................................10
(Consensus H5N1 HA proteins for glycan array assay and vaccine design)
1. Protein expression and purification .........................................................................10
2. Glycan microarray fabrication .................................................................................11
3. Indirect binding assay ..............................................................................................11
4. Direct binding assay.................................................................................................12
5. Microneutralization assay ........................................................................................12
6. Mice, vaccination, and challenge.............................................................................12
(C) Materials and methods-2 ....................................................................................13
(H1N1 HA proteins for glycan array assay and vaccine design)
1. Gene constructs........................................................................................................13
2. Protein purification and endotoxin measurement ....................................................13
3. Viruses .....................................................................................................................14
4. Hemagglutination inhibition (HI) Assay .................................................................14
5. Microneutralization assay ........................................................................................15
6. Virus challenge experiments....................................................................................15
7. ELISA and isotyping of anti-HA antibodies............................................................16
(D) Results...................................................................................................................16
1. Expression, purification and characterization of the secreted HA proteins .............16
2. Mass spectrometric analysis of N-glycans from different HA proteins...................17
3. Creating defined HA glycoforms for quantitative glycan microarray profiling ......19
4. Vaccine design using monoglycosylated HA ..........................................................20
5. Characterization of the HA proteins from A/California/07/2009(H1N1) and
A/Brisbane/59/2007(H1N1) .....................................................................................21
6. The immune response elicited by HAfg, HAmg and HAug protein vaccines
in mice......................................................................................................................22
7. Cross-protection against Cal/09, WSN/33 and PR8/34 viruses with
Bris/07 HA protein vaccine......................................................................................23
8. Protection against Cal/09, WSN/33 and PR8/34 viruses with
Cal/09 HA protein vaccine.......................................................................................24
9. Structural integrity and oligomerization of monoglycosylated HA as a protein
vaccine .....................................................................................................................25
(E) Discussion .............................................................................................................26
(F) Reference ..............................................................................................................30
(G) Figures..................................................................................................................38
(H) Tables ...................................................................................................................60
List of Figures:
Figure 1. Schematic overviews and circular dichroism spectra of HAs with
different glycosylations............................................................................38
Figure 2. Glycan microarray analysis of HA with different glycosylations. ..........39
Figure 3. Comparison of HAfg and HAmg as vaccine..............................................40
Figure 4. Analysis of glycosylation sites on HAs H1, H3, and H5. .......................41
Figure 5. Construction of the H5 HA protein, purification, and gel-filtration
chromatography analysis. ......................................................................43
Figure 6. MS analysis of permethylated N-glycans from different HA proteins…44
Figure 7. MALDI-TOF analysis of HA variants. .................................................45
Figure 8. Langmuir isotherms. ..............................................................................46
Figure 9. Schematic overviews and characterization of HAs with different
glycosylations. .......................................................................................47
Figure 10. The immune response elicited by HAfg, HAmg and HAug protein
vaccines in mice.......................................................................................48
Figure 11. Cross-protection against Cal/09, WSN/33 and PR8/34 viruses with
Bris/07 HA protein vaccine. ....................................................................49
Figure 12. Protection against Cal/09, WSN/33 and PR8/34 viruses with
Cal/09 HA protein vaccine. ....................................................................50
Figure 13. Requirement of Structural integrity for monoglycosylated HA
to achieve immunogenicity and efficacy. ................................................51
Figure 14. Construction and purification of the Cal/09 and Bris/07 proteins...........52
Figure 15. Purification and characterization of the monomeric HAmg. ....................53
Figure 16. Construction and purification of Cal/09 full length HAmg. .....................54
Figure 17. Circular dichroism spectra of HA proteins with various degrees of
glycosylations. .........................................................................................55
Figure 18. Body weight of the mice vaccinated with Cal/09 and Bris/07
HA protein vaccines after challenge with lethal dose of H1N1
viruses. .....................................................................................................56
Figure 19. MALDI-TOF analysis of HA with various glycosylations. ....................57
Figure 20. The binding of rabbit antiserum from HAmg with different
HAs by ELISA.........................................................................................58
Figure 21. The binding of HA–glycan interactions in response to HAs
with different glycosylations....................................................................59
List of Tables:
Table 1. Assignments of major molecular ions observed in MALDI
spectra of permethylated N-glycans from HA trimers............................60
Table 2. Dissociation constants (KD,surf) and free energy changes (G)
of HA glycosylated variants when binding to α2,3
sialosides 1–15. .......................................................................................61

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