哺乳動物細胞培養在病毒疫苗生產的製程中是一項重要的技術，而微載體培養法使得依賴貼附生長的哺乳動物細胞能在懸浮培養的條件下大量化培養。微載體培養技術最新的發展趨勢是利用無血清培養基去克服含血清培養基的缺點，例如血清內含有大量未知蛋白質造成純化過程的妨礙，以及避免從牛隻血源遭到普恩蛋白(prions) 污染的潛在風險。腸病毒71型(EV71)與登革熱(DEN)在台灣是二個被極為重視的感染性疾病，其有效的疫苗目前仍然積極開發中。本篇論文在探討適當的無血清微載體細胞培養條件用於去活化腸病毒死毒疫苗與減毒登革熱活毒疫苗的製備。在腸病毒71型的研究中，分屬二種基因型的三株病毒株(EV71-075, EV71-117 and EV71-1207)以無血清微載體細胞培養法進行培養。Vero細胞株相較於WI-38與MRC-5細胞株能產出更高的病毒濃度。Vero細胞株的微載體培養建立在5 g/L Cytodex 1微載體並在感染病毒後置於32oC培養之條件下，能在細胞外得到大量的病毒顆粒，並且證實能於2 L生物反應器中以無血清培養基製備出大量的病毒。無血清微載體細胞培養法製備出的腸病毒以福馬林進行去活化的過程，相較於含血清培養基並未有顯著差異。就病毒引發的免疫性而言，在無血清培養的腸病毒經去活化後注射老鼠，相較於含血清培養能誘發略高的中和抗體反應，並且對三株腸病毒株皆具有交叉保護力。EV71-075與EV71-117病毒株的去活化病毒粒子高於EV71-1207病毒株約十倍的中和能力。在VP1蛋白質的胺基酸序列分析中，EV71-075與EV71-117為相同的序列而與EV71-1207有12個胺基酸的差異。在VP1蛋白質的核酸序列分析上，顯示EV71-075與EV71-117病毒株均屬於B4次基因型。在登革病毒的研究中，四種血清型病毒株與一種具感染性克隆株(DEN-1 strain HAWAII, DEN-2 strain NGC, DEN-3 strain H-87, DEN-4 strain H-241 and DEN-4 infectious clone strain 2A)以無血清微載體細胞培養法進行培養。Vero 與MRC-5細胞株的微載體培養建立在2 g/L Cytodex 1微載體，無血清微載體細胞培養法均能培養出大量的病毒。比較這些病毒株的病毒濃度，Vero細胞株均比MRC-5細胞株能產生更高的病毒量。從微載體培養法製備的克隆株病毒中分析出其具有基因序列多樣性，並觀察到以MRC-5細胞株培養比從Vero細胞株製備具有較低的基因序列的異質性。這些結果提供無血清微載體細胞培養法製備去活化腸病毒71型死毒疫苗與四價減毒登革熱活毒疫苗的重要資訊。

Mammalian cell culture is an important technology for the production of viral vaccines. Microcarrier culture introduces the possibility the practical high yield culture of anchorage-dependent mammalian cells in suspension. A new trend of microcarrier technology is to utilize serum-free culture to overcome the drawbacks of serum-containing media. These drawbacks include high serum-protein content for complicating downstream purification process and the risk for potential contamination of prions due to the bovine resource. Enterovirus 71 (EV71) and dengue virus (DEN) are two important infectious diseases in Taiwan. Effective vaccines for enterovirus 71 and dengue virus are still been developed. This research has proposed the large-scale preparation by a scalable cell culture system and established a serum-free microcarrier-based cell culture for development of inactivated enterovirus 71 vaccine and live-attenuated dengue virus vaccine. In EV71 virus research, three strains (EV71-1207, EV71-075 and EV71-117) that contain two genotypes were propagated in a serum-free microcarrier culture. Vero cells were found to produce higher titers of EV71 than WI-38 and MRC-5 cells. Microcarrier Vero cell cultures were established using 5g/L Cytodex 1 microcarriers and found to promote the extracellular release of EV71s from infected Vero cells at 32oC. The large-scale preparation of EV71s can be achieved using serum-free microcarrier Vero cell culture in a 2-liter bioreactor. No significant differences were observed for the formalin-inactivation kinetics of the three EV71 strains in serum-free and serum-containing cultures. The immunogenicity of the inactivated EV71 virions produced in serum-free cultures elicited slight higher levels of neutralizing antibody response in immunized mice and exhibited a cross-neutralization ability to resist three EV71 virus strains. The inactivated virions of EV71-075 and EV71-117 strains elicited approximately 1-log increase in ID50 values than EV71-1207 strain. EV71-075 and EV71-117 has identical amino acid sequences in the VP1 protein, while EV71-1207 revealed 12 amino acid sequences differences. Nucleotide sequence analysis indicated that the VP1 protein of EV71-075 and EV71-117 belonging to the B4 subgenotype. In dengue virus research, four serotypes dengue virus strains (DEN-1 strain HAWAII, DEN-2 strain NGC, DEN-3 strain H-87 and DEN-4 strain H-241) and a DEN-4 infectious clone (strain 2A) were propagated in serum-free microcarrier cultures. Vero and MRC-5 cells were grown on microcarriers using 2g/L Cytodex 1 and propagated these dengue viruses in serum-free cultures. The virus titers of dengue virus produced in microcarrier Vero cell cultures were higher than that produced in microcarrier MRC-5 cell cultures for each of these dengue strains. The genetic quasispecises of DEN-4 infectious clone virus were observed that MRC-5 cells produced the lower sequence heterogeneity than Vero cells in microcarrier cultures. These results constitute valuable information on the development of a serum-free microcarrier cell culture process for producing inactivated EV71 vaccine and tetravalent live-attenuated dengue vaccines.

CONTENT

中文摘要 I
ABSTRACT III
誌謝.........................…………………………………………………………………..V
CONTENT VI
FIGURES LIST IX
TABLES LIST XI

Chapter 1: Research Background, Incentive and Aims 1
1.1. Historic Aspects on Viral Vaccine Development 1
1.2. Cell Substrate for Viral Vaccine Production 2
1.3. Mammalian Cell Substrates for Viral Vaccine Production 4
1.4. Large Scale Culture for Anchorage Dependent Cells 7
1.5. A New Trend for Microcarrier Technology: Serum-free Microcarrier Cell Culture 11
1.6. Research Aims 13

Chapter 2: Establishment of Serum-free Microcarrier Cell Culture for Inactivated Enterovirus 71 Vaccine Development 14
2.1. Introduction 14
2.1.1. Enterovirus 71 virus and diseases 14
2.1.2. EV71 virus genome and structure 15
2.1.3. EV71 virus molecular epidemiology 16
2.1.4. EV71 virus vaccine development 18
2.1.5. Research aims for EV71 virus 20
2.2. Materials and Methods 21
2.2.1. Cells and media 21
2.2.2. EV71 virus strain 21
2.2.3. Stationary cultures with EV71-1207 virus 22
2.2.4. Determination of virus stability at different temperature 22
2.2.5. Preparation of microcarriers 23
2.2.6. Preparation of spinner flask cultures 23
2.2.7. Cultivation in bioreactor and fed-batch control 24
2.2.8. Measurements of cell density and virus titer 25
2.2.9. Preparation of inactivated EV71s and mouse immunization 26
2.2.10. Neutralizing assays for mouse antibodies 26
2.2.11. Cloning and sequence analysis of EV71 VP1 protein 27
2.2.12. Phylogenetic analysis and homology model for EV71 VP1 protein 28

2.3. Results 29
2.3.1. Selecting cell line for EV71 production 29
2.3.2. Effects of MOI on EV71-1207 virus production 29
2.3.3. EV71-1207 virus production in microcarrier culture 30
2.3.4. The stability of EV71-1207 virus at different temperature 30
2.3.5. Two temperature stages for EV71-1207 production in microcarrier culture 31
2.3.6. Microcarrier concentration evaluation 32
2.3.7. Vero cell growth curves in microcarrier culture with serum-free medium 33
2.3.8. Microcarrier cell culture for EV71-1207 virus strains in serum-free cultures 34
2.3.9. Microcarrier bioreactor culture 35
2.3.9.1. Serum-containing medium without feeding during infection 35
2.3.9.2. Serum-containing medium with feedings during infection 36
2.3.9.3. Serum-free medium with feedings during infection 36
2.3.10. Immunogenicity of formalin-inactivated EV71-1207 harvested from serum-containing and serum-free cultures 37
2.3.11. High immunogenic EV71 strains 38
2.3.12. Sequence analysis of the VP1 protein for high and low immunogenic EV71 strains 39
2.3.13. Serum-free microcarrier Vero cell culture for high immunogenic EV71 strains 39
2.3.14. Formalin-inactivation kinetics of highly immunogenic EV71 strains produced in serum-free microcarrier Vero cell cultures 40
2.3.15. Cross neutralization of high immunogenic EV71 strains produced in serum-free microcarrier Vero cell cultures 41
2.4. Discussion 42

Chapter 3: Establishment of Serum-free Microcarrier Cell Culture for Live-attenuated Dengue Virus Vaccine Development 53
3.1. Introduction 53
3.1.1. Dengue virus and disease 53
3.1.2. Dengue virus genome and structure 54
3.1.3. Dengue virus vaccine development 56
3.1.4. Dengue virus infectious clone and recombination vector 59
3.1.5. Dengue virus safety concern and RNA virus quasispecies 62
3.1.6. Research aims for dengue virus 64
3.2. Materials and Methods 65
3.2.1. Cells and Media 65
3.2.2. Four-serotype dengue viruses 65
3.2.3. Generation of DEN-4 infectious clone virus 66
3.2.4. Microcarrier preparation 67
3.2.5. Preparation of spinner flask cultures 67
3.2.6. Measurements of cell density and virus titer 68
3.2.7. Cloning and sequencing of DEN-4 infectious clone 69
3.2.8. Cloning and sequencing of DEN-4 infectious clone 70
3.3. Results 71
3.3.1. Vero and MRC-5 cells growth in serum-free microcarrier culture 71
3.3.2. Serum-free microcarrier culture for four serotypes dengue viruses 71
3.3.3. Generation of DEN-4 infectious clone virus in Vero and MRC-5 cells 74
3.3.4. Serum-free microcarrier cultures for DEN-4 infectious clone virus 74
3.3.5. The full-length genomic analysis of DEN-4 infectious clone virus in serum-containing and serum-free microcarrier cultures 75
3.3.6. Three genomic fragments sequence analysis of DEN-4 infectious clone virus produced in serum-containing and serum-free microcarrier cultures 76
3.4. Discussion 79

References 84
Figures 110
Tables 165



FIGURES LIST

Figure 1. The maximum virus titer of extracellular and intracellular EV71s produced in three mammalian cell lines. 110
Figure 2. Effects of MOI for EV71-1207 virus production. 111
Figure 3. The maximum virus titer of EV71-1207 virus strain in serum-containing microcarrier cultures. 112
Figure 4. The stability of EV71-1207 virus at different temperature. 113
Figure 5. Two temperature stages for EV71-1207 virus production in microcarrier culture. 114
Figure 6. Microcarrier concentration evaluation for EV71-1207 production in microcarrier culture. . 115
Figure 7. The specific virus titer of EV71-1207 virus produced in Vero-cell microcarrier cultures at different microcarrier concentrations. 116
Figure 8. Vero cell growth in serum-free microcarrier culture. 117
Figure 9. EV71-1207 virus production in serum-containing and serum-free cultures. 118
Figure 10. Microscopic observations of microcarrier-grown Vero cells in serum-containing and serum-free culture. 119
Figure 11. EV71-1207 virus production in serum-containing bioreactor culture. 120
Figure 12. EV71-1207 virus production in serum-free bioreactor culture. 121
Figure 13. Formalin-inactivation kinetics of EV71-1207 strains produced in microcarrier Vero cell cultures. 122
Figure 14. Immunogenicity of the formalin-inactivated EV71-1207 strain produced in microcarrier Vero cell cultures. 123
Figure 15. Immunogenicity of the formalin-inactivated three EV71 strains produced in microcarrier Vero cell cultures. 124
Figure 16. The VP1 amino acid sequences were aligned to compare the diversity of the three EV71 strains. 125
Figure 17. The cell growth curve of high immunogenic EV71 strains produced in microcarrier Vero cell cultures. 126
Figure 18. The extracellular virus titer of high immunogenic EV71 strains produced in microcarrier Vero cell cultures. 127
Figure 19. Formalin-inactivation kinetics of high immunogenic EV71 strains produced in microcarrier Vero cell cultures. 128
Figure 20. Cross neutralization of high immunogenic EV71 strains produced in microcarrier Vero cell cultures. 129
Figure 21. Phylogenetic analysis of EV71 based on the VP1 sequences. 130
Figure 22. The homology modeling of the VP1 protein. 131
Figure 23. Vero and MRC-5 cells grown on 2g/L Cytodex 1 microcarriers using serum-containing and serum-free medium. 132
Figure 24. DEN-1 virus (HAWAII strain) production in microcarrier cell cultures …………………………………………………………………..133
Figure 25. DEN-2 virus (NGC strain) production in microcarrier cell cultures. 134
Figure 26. DEN-3 virus (H-87 strain) production in microcarrier cell cultures. 135
Figure 27. DEN-4 virus (H-241 strain) production in microcarrier cell cultures. 136
Figure 28. Schematic diagram of the in vitro transcription of DEN-4 infectious clone virus (2A) and recovered by Vero or MRC-5 cells with serum-containing and serum-free cultures. 137
Figure 29. DEN-4 infectious clone virus (2A strain) production in microcarrier cell cultures. 138
Figure 30. The plaque morphology of DEN-4 infectious clone virus produced from Vero and MRC-5 cells in microcarrier cultures. 139
Figure 31. Schematic diagram of the dengue virus genome and three genomic fragments analyzed in this study. 140
Figure 32. Nucleotide sequence alignment of multiple clones of W13-W02R fragment (E-NS1) from microcarrier Vero cultures. 141
Figure 33. Nucleotide sequence alignment of multiple clones of W13-W02R fragment (E-NS1) from microcarrier MRC-5 cultures. 145
Figure 34. Nucleotide sequence alignment of multiple clones of W28-W29R fragment (NS3) from microcarrier Vero cultures. 149
Figure 35. Nucleotide sequence alignment of multiple clones of W28-W29R fragment (NS3) from microcarrier MRC-5 cultures. 153
Figure 35. Nucleotide sequence alignment of multiple clones of W21-W23R fragment (NS4B-NS5) from microcarrier Vero cultures. 157
Figure 35. Nucleotide sequence alignment of multiple clones of W21-W23R fragment (NS4B-NS5) from microcarrier MRC-5 cultures. 161


TABLES LIST

Table 1. The oligonucleotide primers used for full-length genomic sequence of DEN-4 virus. 165
Table 2. The maximum virus titers of four serotype dengue viruses for Vero and MRC-5 cells in microcarrier cultures. 166
Table 3. Differences of nucleotide and amino acid sequences of full-length genome between serum-containing and serum-free cultures for Vero and MRC-5 cells. 167
Table 4. Differences of nucleotide and amino acid sequences of W13-W02R fragment (E-NS1) between serum-containing and serum-free cultures for Vero and MRC-5 cells. 168
Table 5. Differences of nucleotide and amino acid sequences of W28-W29R fragment (NS3) between serum-containing and serum-free cultures for Vero and MRC-5 cells. 169
Table 6. Differences of nucleotide and amino acid sequences of W21-W23R fragment (NS4B-NS5) between serum-containing and serum-free cultures for Vero and MRC-5 cells. 170
Table 7. Nucleotide and amino acid diversity for three genomic fragments in microcarrier cultures. 171
Table 8. Two-sample t TEST for Vero and MRC-5 cells base on nucleotide and amino acid diversity. 172
Table 9. Two-sample t TEST for serum-containing and serum-free cultures base on nucleotide and amino acid diversity. 173

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