無血清微載體細胞培養法製備病毒疫苗其繁殖動力與病毒抗原特性之研究

Propagation Kinetics and Antigenic Studies in Serum-Free Microcarrier Cell Culture for Viral Vaccine Development

劉家齊(Chia-Chyi Liu)

指導教授 ： 吳夙欽

國立清華大學/生命科學院/生命科學系/博士(2006年)

摘要

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。

關鍵字

無血清微載體細胞培養 ； 病毒疫苗 ； 腸病毒71型 ； 登革病毒 ； 生物反應器 ； Vero細胞 ； MRC-5細胞 ； 基因序列多樣性

並列摘要

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.

並列關鍵字

Serum-Free Microcarrier Cell Culture ； Viral Vaccine ； EV71 virus ； Dengue virus ； Bioreactor ； Vero cell ； MRC-5 cell ； Genetic quasispecises

參考文獻

Abubakar S, Chee HY, Al-Kobaisi MF, Xiaoshan J, Chua KB, Lam SK. (1999) Identification of enterovirus 71 isolates from an outbreak of hand, foot and mouth disease (HFMD) with fatal cases of encephalomyrlelitis in Malaysia. Virus Res. 61: 1-9.

Ada G. (1997) Overview of vaccines. Mol Biotechnol. 8(2): 123-34.

Assad S, Francis A. (1999) Over a decade of experience with a yeast recombinant hepatitis B vaccine. Vaccine. 18(1-2): 57-67.

Aviles G, Meissner J, Mantovani R, St Jeor S. (2003) Complete coding sequences of dengue-1 viruses from Paraguay and Argentina. Virus Res. 98(1): 75-82.

Barouch DH, Nabel GJ. (2005) Adenovirus vector-based vaccines for human immunodeficiency virus type 1. Hum Gene Ther. 16(2): 149-56.