發展並評估適用於猴腎細胞平台之流感疫苗全能株 (master donor virus) 於流感大流行之應用

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研究生：	陳柏伶 Chen, Po-Ling.
論文名稱：	發展並評估適用於猴腎細胞平台之流感疫苗全能株 (master donor virus) 於流感大流行之應用 Development and evaluation of Vero cell-based master donor viruses (MDVs) for influenza pandemic preparedness
指導教授：	李敏西 Lee, Min-Shi 王慧菁 Wang, Lily Hui-Ching
口試委員:	胡勇誌 Hu, Alan Yung-Chih 殷献生 Yin, Hsien-Sheng 陳俊叡 Chen, Juine-Ruey
學位類別：	博士 Doctor
系所名稱：	生命科學暨醫學院 - 分子與細胞生物研究所 Institute of Molecular and Cellular Biology
論文出版年：	2020
畢業學年度：	108
語文別：	英文
論文頁數：	76
中文關鍵詞：	流感疫苗、禽流感、猴腎細胞
外文關鍵詞：	pandemic influenza
相關次數：	點閱：4 下載：0
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季節型流感每年造成數百萬起感染病例與數十萬死亡病例，此外H5與H7N9禽流感病毒更有引發流感大流行之潛在風險。為了防範流感病毒造成的危害與經濟損失，疫苗是不可或缺的工具。目前流感疫苗的生產包括：雞胚蛋、細胞培養與重組蛋白生產平台。雞胚蛋生產平台約提供90%的季節性流感疫苗，低成本高產率是其優點。然而此生產平台高度依賴雞胚蛋的供應，為大流行流感疫苗的供給埋下不確定因素。細胞培養與重組蛋白生產平台容易放大製程且可加快疫苗供給，因此適合用於大流行疫苗生產。但這兩個平台生產成本較高，目前在季節性流感疫苗的市場上難以與低成本的雞胚蛋平台競爭。不過近年來雞胚蛋平台所生產的H3N2季節性流感疫苗有免疫力不佳的問題，主要是因為在生產過程中出現改變HA抗原性的突變。這種突變發生率在細胞培養與重組蛋白生產平台中較低，因此以這兩種平台所生產的H3N2疫苗還能保有足夠的免疫力。綜合以上考量，美國總統在2019年所發布的行政命令中提到：流感疫苗的生產將逐步以細胞培養與重組蛋白生產平台取代雞胚蛋生產平台。細胞培養平台所生產的流感疫苗其免疫力優於重組蛋白疫苗，而重組蛋白疫苗則有快速與低成本等優勢。本研究著重於猴腎細胞平台，猴腎細胞與狗腎細胞為主要兩種生產流感疫苗的細胞株。流感病毒在狗腎細胞中有較佳的產率，但此細胞株的應用性較低，目前只用於流感疫苗生產。猴腎細胞有較廣泛的應用性，可用於小兒麻痺、日本腦炎與腸病毒疫苗的生產，但流感病毒在猴腎細胞的產能不佳。為提高流感病毒在猴腎細胞中的產能，需要開發高成長全能病毒株(master donor virus, MDV)。本研究開發兩株適用於猴腎細胞生產平台之高成長全能病毒株：Vero-15與vB5。Vero-15來自猴腎細胞馴化之H5N1疫苗株，vB5則是由馴化病毒之突變基因設計而得。進一步利用此兩株MDV(Vero-15與vB5)與兩株來自其他實驗室的MDV(PR8與PR8-HY)搭配五株H5與H7N9禽流感病毒來製備重組疫苗株共20株，並分析這些重組疫苗株在猴腎細胞的生長情形。結果發現難以使用單獨一株MDV為此五種禽流感病毒製備高產能重組疫苗株，但搭配兩株MDV(例如Vero-15與vB5)即可達成。此外，我們也進行動物試驗證實:猴腎細胞平台所生產之H5N1與H7N9流感疫苗在小鼠中能激起顯著的抗體反應。

Avian influenza H5 and H7N9 viruses pose a threat to public health because they frequently cause zoonotic transmission and may lead to pandemics. Vaccination is an important tool for pandemic preparedness. Currently, there are egg-based, cell-based and recombinant protein-based production platforms for influenza vaccines. The egg-based platform, which has the advantages such as low cost and high productivity, is the main platform for seasonal influenza vaccines. However, this platform highly relies on the supply of embryonated eggs which could be a great concern during pandemics. The cell-base and recombinant protein-based platforms are highly scalable and could shorten the preparation time, which are critical for pandemic preparedness. However, these two platforms have the main drawback of high cost which makes these two platforms less attractive than the egg-based platform for supplying seasonal influenza vaccines. Recently, egg-derived influenza H3N2 viruses could generate mutations in HA protein, which may be related to low vaccine effectiveness of egg-derived influenza vaccines observed in the 2017-2018 season. Therefore, US President announced Executive Order in Sep 2019 to establish “Influenza Vaccine Task Force” for introducing new technology, such as cell-based and recombinant vaccines to replace egg-based influenza vaccines. Recombinant protein-based platform, using baculovirus expression system, has higher productivity and shorter production time than cell-based platform, but cell-based platform has better immunogenicity than recombinant protein-based platform. In this study, we focus on the Vero cell-based platform. MDCK and Vero cells have been used for production of influenza vaccines. Influenza viruses grow efficiently in MDCK cells, but this cell line could be only used for production of influenza vaccines. Vero cells have a broader usage in human vaccine production, such as poliovirus, JEV and EV71, but influenza viruses sometimes did not grow efficiently in Vero cells. To enhance the growth of influenza viruses in Vero cells, Vero cell-derived high-growth master donor viruses (MDVs) are desirable. In this study, we developed two MDVs, Vero-15 and vB5. Vero-15 is a Vero cell-adapted high growth H5N1 vaccine virus, and vB5 is a designed MDV which was generated to carry Vero cell-adapted mutations using reverse genetics. We further used Vero-15, vB5 and two external MDVs (PR8 and PR8-HY) to generate reassortant viruses for five avian influenza viruses, and evaluate growth efficiency of these reassortant viruses in Vero cells. Based on our results, using one MDV could not generate high growth reassortants (HGRs, >107 TCID50/ml) for all avian influenza viruses. By combining two MDVs (Vero-15 and vB5), we could generate HGRs for these 5 avian influenza viruses with pandemic potentials. In addition, we further proved that the Vero cell-derived influenza H5N1 and H7N9 vaccine candidates could elicit robust antibody response in mice.

Contents
Introduction    1
Aims and strategy    6
Material and methods    7
Cells and Viruses    7
Plasmid construction    7
Electroporation    8
Virus titration    8
Plaque assay and virus selection    9
Growth curve    9
Viral RNA expression    10
A preparation of inactivated, whole-virus antigens    11
Single radial immunodiffusion (SRID)    11
Densitometry of SDS-PAGE    11
Mouse immunisation    12
Intravenous pathogenicity index (IVPI)    12
Serological assays    12
Ethics statement    13
Part I: Using Vero cell-adapted H5N1 HGR (Vero-15) as a master donor virus (MDV)    14
Result    14
Using MDV Vero-15 to generate Vero cell-derived HGRs for Eurasian-lineage H5N1 clade 2 viruses    14
Development of American-lineage H5N2 vaccine viruses (published on Viruses, 2019)    14
Discussion    16
MDV Vero-15 is suitable for Eurasian-lineage H5N1 viruses, but not American-lineage H5N2 viruses    16
The influence of adapted mutations on Vero-derived H5N2 vaccine viruses    17
Part II: development of novel MDVs for generating Vero cell-derived high-growth RG viruses    20
Result    20
Design of MDVs based on the Vero cell-adapted mutations    20
Generation of reassortant viruses with candidate master donor viruses    20
Growth efficiency in serum-containing and serum-free culture system    21
Growth efficiency of rRG6-PR8 and rRG6-vB5 in microcarrier culture system    21
Growth efficiency of reassortant viruses bearing a single mutation    22
Evaluation of four MDVs for generating HGRs    22
Mouse immunogenicity study    23
Discussion    25
Multifunctional platform for pandemic preparedness    25
H7N9 influenza vaccine development    25
The low immunogenicity of H5N1 antigens    27
Growth efficiency of serum-containing and serum-free culture systems    27
Safety of Vero cell-derived MDVs    28
The roles of PB2-S360Y mutation and truncated NS1 in Vero cells    28
Conclusion    29
Acknowledgement    30
Tables and Figures    31
Primer list    31
Table 1. Genetic analysis of Vero-adapted viruses compared with PR8: A/Puerto Rico/8/34 (H1N1), the current master donor strain.    34
Table 2. Genetic differences of six internal genes between PR8 from NIBSC, US CDC, St. Jude Children Hospital, and PR8-HY    35
Table 3. A comparison between reassortant viruses and their parental viruses.    36
Table 4. The profile of generated H5N2 reassortant viruses.    37
Table 5. A summary of quality tests of E7-V15 C11, an adapted H5N2 high-growth reassortant.    38
Table 6. Intravenous pathogenicity index test for E7-V15 C11.    39
Table 7. The HI titre of E7-V15 C11 virus-infected chickens.    40
Table 8. Virus titres (log (TCID50)) of organs from infected ferrets were tested with Vero cells.    41
Table 9. The antigenicity of E7-V15 C11 viruses analysed using HI assay.    41
Table 10. The receptor binding affinity of H5N2 RG virus and its parental virus.    43
Table 11. The genetic background of five potential MDVs carrying different internal genes.    44
Table 12. The HA and virus titre of reassortant virus stocks.    45
Table 13. A comparison of virus growth among PR8-NIBSC (PR8), vB5, Vero-15 (V15) and PR8-HY (HY) MDVs.    46
Table 14. The properties of purified antigens.    47
Figure 1. The life cycle of influenza A viruses    48
Figure 2. The methods to generate high-growth reassortants (HGRs) as vaccine seed viruses.    49
Figure 3. The strategies for developing the Vero cell-derived MDVs by using reverse genetics.    50
Figure 4. Growth curve of E7-V15 C11 virus.    51
Figure 5. The change of body weight and temperature in pathogenicity assay in ferrets.    52
Figure 6. Trypsin dependency assay of E7-V15 C11.    53
Figure 7. The adapted mutation on NS1 monomer.    54
Figure 8. The plaque formation of reassortant viruses in Vero cells.    55
Figure 9. The growth property of reassortant viruses in M199 medium in 6-well plates.    56
Figure 10. The growth property of reassortant viruses in SF medium in T75 flasks.    58
Figure 11. The growth property of rRG6-vB5 in SF medium in spinner flasks.    60
Figure 12. Comparing the growth of viruses with PB2 mutation and/or with truncated NS1 in VP-SFM medium.    61
Figure 13. RNA expression of reassortant viruses in Vero cells.    62
Figure 14. Purified virus morphology under transmission electron microscope (TEM).    63
Figure 15. Mouse immunisation with H5N1 inactivated whole virus antigens.    65
Figure 16. The immunogenicity of inactivated H7N9 whole virus antigen in mice.    67
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