로타바이러스 양입자 백신의 생산 및 면역원성 평가
Production of Rotavirus VLP vaccine and Evaluation of Immunogenecity
초록/요약
로타바이러스 VP2, VP6, 그리고 VP7을 배큘로바이러스 발현 시스템(Baculovirus Expression Vector System)에서 발현시켜 바이러스 양입자(virus-like particle)을 형성하였다. 배큘로바이러스 발현 시스템의 숙주세포로서 Sf9 세포주를 0.5L spinner flask부터 50L bioreactor에서 배양하여 최대 세포 농도 7.53 x 106 cells/㎖ ~ 1.12 x 107 cells/㎖까지 얻었으며, 이때 20.1~25.5 시간의 분열시간을 나타내었다. 바이러스 양입자를 생산하기 위하여 상용세포주와 상용바이러스주를 준비하였다. 각 재조합 배큘로바이러스로 부터 각 재조합 단백질의 발현을 확인하였고 삼중감염에 의한 바이러스 양입자 형성을 확인하였다. 최대의 로타바이러스 양입자를 생산할 수 있는 최적의 감염비율은 VP2, VP6, 그리고 VP7에 대하여 각각 1.0:2.0:0.2 이었으며 최적의 회수시기는 감염 후 6일차로 확인되었다. 로타바이러스 양입자를 정제하기 위해 여러 정제 공정을 평가한 결과, 초여과, DEAE 이온교환크로마토그래피, phenyl-sepharose 크로마토그래피, size exclusion 크로마토그래피로 구성되는 정제 공정이 그 구조를 유지하면서 효과적으로 바이러스 양입자를 정제할 수 있음을 보였다. 정제된 바이러스 양입자를 Freund’s incomplete adjuvant, aluminum hydroxide, QS-21 과 MF59와 함께 ICR 마우스에 근육 또는 피하 접종하였다. QS-21은 IgG 와 IgA를 함께 유도하여 로타바이러스 양입자의 면역보조제로서 가장 적합하였고, QS-21과 바이러스 양입자로 유도된 IgG는 면역 후 21주까지 유지되었다. 백신의 효력 연구를 위한 동물 공격 모델을 개발하기 위하여 로타바이러스 항원 검출 시약을 개발하였다. 개발된 검출 시약은 1.7 x 104 TCID50/㎖ 의 감도를 보였으며 상용화된 진단 시약과 비교 시 92.2%의 일치율을 보였다. 본 연구의 결과 배큘로바이러스 발현 시스템을 이용하여 50L 규모의 로타바이러스 양입자 백신을 생산할 수 있었고, QS-21을 면역보조제로 사용한 바이러스 양입자에서 최적의 면역 반응을 유도할 수 있음을 확인하였다. 이러한 결과는 로타바이러스 양입자 백신이 대량 생산 공정을 통해 상용화될 수 있는 백신 후보물질로서의 가능성을 보여 주었다
more초록/요약
Rotavirus VP2, VP6, and VP7 were expressed in Baculovirus Expression Vector System (BEVS) and Virus-Like Particle (VLP) was produced by tri-infection of recombinant baculoviruses for use as a vaccine candidate against rotavirus infection. Sf9 culture system, a host for BEVS, was established from 0.5L spinner flask to 50L bioreactor system where the maximal cell concentration was 7.53 x 106 cells/㎖ ~ 1.12 x 107 cells/㎖ with doubling time of 20.1 ~ 25.2 hours. Working cell bank and working virus seed bank were prepared for the production of VLP. The expression of recombinant protein from each recombinant baculovirus and VLP formation by tri-infection was confirmed. The optimal MOI ratio was determined as 1.0:2.0:0.2 for VP2, VP6, and VP7, respectively and the optimal harvest time also determined as 6 days of post infection. Several purification processes were evaluated and resulted that ultrafiltration, DEAE ion-exchange chromatography, phenyl-sepharose hydrophobic interaction chromatography, and size exclusion chromatography were effective for the purification of rotavirus VLP. ICR mice were immunized with purified VLP subcutaneously or intramuscularly with various adjuvants including IFA, aluminum hydroxide, QS-21 and MF59. QS-21 was showed to be the most appropriate adjuvant for rotavirus VLP vaccine to induce both rotavirus specific IgG and IgA. VLP formulated with QS-21 induced IgG that was maintained at least 21 weeks of post immunization. For the development of animal challenge model for vaccine efficacy study, a rotavirus antigen detection kit (RotaTek) was also developed and showed that the detection limit was 1.7 x 104 TCID50/㎖ and concordance rate was 92.2% by comparing with the commercialized kit. In this study, it was showed that the rotavirus VLP vaccine were produced in 50L scale using BEVS, and VLP formulated with QS-21 induced immune response. These results indicated that rotavirus VLP was a promising vaccine candidate produced in industrial scale.
more목차
Chapter Ⅰ. Introduction = 1
1. Epidemiology of Rotaviral Diseases = 1
2. Current Status of Rotaviral Vaccine = 7
3. Baculovirus Expression Vector System in Biotechnology = 11
4. Aims of Study = 14
Chapter Ⅱ. Large Scale cultivation of Sf9 cell = 15
1. Introduction = 15
2. Material and Method = 18
2.1 Cell Line = 18
2.2 Media = 18
2.3 Sf9 Cell Culture = 18
2.4 Glucose/Lactate Analysis = 21
2.5 Cell Count = 21
3. Results and Discussion = 23
3.1 Cell Bank Preparation = 23
3.2 Growth Characterization = 23
4. Summary = 30
Chapter Ⅲ. Optimization of VLP Production = 31
1. Introduction = 31
2. Materials and Methods = 33
2.1 Recombinant Baculoviruses = 33
2.2 Titration of Recombinant Baculovirus = 33
2.3 Semi Purification = 36
2.4 Monoclonal Antibody Preparation = 36
2.5 SDS-PAGE and Western Blot = 37
2.6 Electron Microscopy = 37
3. Results and Discussion = 38
3.1 Sf9 cell culture for Virus Seed Bank Preparation = 38
3.2 Intermediate Virus Seed Bank Preparation = 38
3.3 Working Virus Seed Bank Preparation = 39
3.4 Expression of Rotavirus Proteins = 41
3.5 Assembly of VLP = 46
3.6 Optimal MOI Evaluation = 46
3.7 Optimal Harvest Time Evaluation = 50
4. Summary = 53
Chapter Ⅳ. Purification of VLP and Efficacy Study = 54
1. Introduction = 54
2. Materials and Methods = 57
2.1 Protein Assay = 57
2.2 Antigen Assay = 57
2.3 Clarification = 58
2.4 Ultrafiltration = 58
2.5 Ultracentrifugation = 58
2.6 Ion-Exchange Chromatography = 59
2.7 Hydrophobic Interaction Chromatography = 60
2.8 Size Exclusion Chromatography = 62
2.9 Affinity Chromatography = 62
2.10 Immunization = 64
2.11 Antibody Titration = 65
3. Results and Discussion = 66
3.1 Purification = 66
3.2 Immunogenecity = 73
4. Summary = 79
Chapter Ⅴ. Development of Rotavirus Antigen Detection Kit = 80
1. Introduction = 80
2. Materials and Methods = 81
2.1 Antibodies = 81
2.2 HRP conjugation = 81
2.3 Antigen Detection Kit Preparation = 82
2.4 MA104 cell culture = 82
2.5 Rotavirus Production = 84
2.6 Immunofluorescence Assay (IFA) = 84
2.7 Titration of Rotavirus = 85
2.8 Clinical Sample = 85
2.9 Rotavirus Antigen Detection = 86
3. Results and Discussion = 87
3.1 Rotavirus Preparation = 87
3.2 Kit Evaluation = 87
3.3 Screening of Clinical Sample = 91
4. Summary = 93
Conclusion and Future Research = 94
Reference = 96
국문 요약 = 105
