초록 ▼

○ 연구결과
-제1세부과제를 통해 Pilotscale의 Fc분자 표면발현 PED 바이러스를 3lot생산하였으며,cellbank를 확립하였다.생산된 시험백신을 이용한 임상시험을 국가검정기준에 준하는 기준으로 임상시험을 실시하였으며,목적동물에서의 전임상 시험을 실시하였다. 또한 상용화된 백신과의 비교시험을 통해 효능의 우수성을 확인하였다.
-IFA 및 FACS 분석을 통해 바이러스 표면의 Fc발현을 확인하고 이를 통해 백신의 항원으로 충분히 작용할 수 있는 가능성을 확인하였고,Fc 발현 세포주(Vero/Fc 및 MARC145

○ 연구결과
-제1세부과제를 통해 Pilotscale의 Fc분자 표면발현 PED 바이러스를 3lot생산하였으며,cellbank를 확립하였다.생산된 시험백신을 이용한 임상시험을 국가검정기준에 준하는 기준으로 임상시험을 실시하였으며,목적동물에서의 전임상 시험을 실시하였다. 또한 상용화된 백신과의 비교시험을 통해 효능의 우수성을 확인하였다.
-IFA 및 FACS 분석을 통해 바이러스 표면의 Fc발현을 확인하고 이를 통해 백신의 항원으로 충분히 작용할 수 있는 가능성을 확인하였고,Fc 발현 세포주(Vero/Fc 및 MARC145)증식 PEDV 및 PRRSV 정상 세포주 (Vero및 MARC 145)에 재감염 시킨 후 각 세포막에 남아 있는 uncoatedFc 단백질을 IFA 확인하여 바이러스에 Fc분자 함유 여부를 다시 한 번 확인하였다.
-다양한 Oiladjuvant,carbopol,aviridin등에 대한 백신과의 물리적 적합 여부 검토 및 Adjuvant 기본 면역 유도능을 확인하였으며 oiladjuvant를 이용한 경우 상대적으로 뛰어난 면역 유도능을 타냄을 확인하였다.
- PEDV-Fcharboring 검증하기 위해 western blotting과 virus survivalrate를 측정하법으 이용한 평가법을 확립하였으며,백신의 면역 유도능을 평가하는 방법 또한 확립하였다.

Abstract ▼

Opsonization of foreign materials with IgG enhances their removal from the host by macrophages through antibody-dependent cell-mediated cytotoxicity. Currently various Fc receptorsdiscovered on the diverse immune cells and they has own immunological roles. Fc part of the antigen binding IgG interact

Opsonization of foreign materials with IgG enhances their removal from the host by macrophages through antibody-dependent cell-mediated cytotoxicity. Currently various Fc receptorsdiscovered on the diverse immune cells and they has own immunological roles. Fc part of the antigen binding IgG interacts with Fc receptor(FcRs) on the diverse immune cells influence immune reactions. For example, cytokine secretion, antigen re-presentation and phagocytosis are activated. This is due to an increased uptake of antigen-antibody complex into antigen presenting cells (APC) through the APC cell surface FcRs resulting in increased processing and presentation. Therefore if we make Fc molecules expressed on the viral surface it can be used for more efficacious vaccine antigen. Most enveloped virus use host cell membrane for making their own envelop. So if we make swine IgG Fc molecules expressed on the surface of the mammalian cell and then enveloped virus propagate on this transformed cell, virus has reverse oriented Fc molecules on the virus envelop. In this study, We make Fc molecules expressed Vero cell and Marc145 cell using swine IgG Fc gene expression vector and porcine epidemic disease virus(PEDV) and Porcine reproductive and respiratory syndrome virus(PRRSV) are grown in genetically modified Vero cells and PED virus and PRRS virus may acquire Fc on the viral envelop. We testthe immunological enhancement of the Fc molecules acquired PED virus as an inactivated antigen. Swine IgG Fc molecule expression vector (pBudCE4.1 GFP-Fc) was constructed using Fc and transferrin receptor gene amplified from swine spleen. Vero cells were transformed with Fc expression vector and transformed cells were selected in the Zeocine containing media. PED virus propagated on the transformed Vero cells. Fc molecules on the Vero cell and PED virus were tested by ELISA, Western blot analysis and immunofluorescent analysis. Immunological enhancement test of the Fc harboring PED virus(PED-Fc) was performed using thetitration of the serum antibody of the mouse immunized with PED-Fc virus. Immune cell proliferation, cytokine secretion, Serum neutralization antibody titer were measured in the serum of the pig immunized with PED-Fc virus. Immunofluorescent analysis showed that transformed Vero cells successfully express swine IgG Fc molecules on the cell membrane. PED virus harboring Fc molecules on the virus envelop was identified by ELISA assay. Vero cells transiently transformed by the plasmid vector and PED-Fc virus, the expression of approximately 50 kDa molecules containing Fc was detected by Western blotting assay in reduced condition. The size of detected band was same to the expected molecular weight of the chimera Fc monomer. PED-Fc virus showed more strong immune stimulations in the mouse immunization test. IgG antibody titer increase 2 times, virus neutralization titer increase 16 times compare with normal PED virus. At the T cell proliferation assay also show PED-Fc virus increase 3 times compare with normal PED virus. Immunization with PED-Fc virus showed higher IgG2a/IgGl ratio comparing with normal PED virus. The result indicates that PED-Fc induces Th1 biased immune responses. In the swine model, when the animals are immunized with PED-Fc virus(10⁶ TCID₅₀/ml), serum neutralization antibody titer was 64 through 128. This SN titer 4 times higher than that of the same does normal PED immunization pigs. In this study, we found that PED-Fc virus elect more strong immune responses compare with normal PED virus. PED-Fc virus immunized mouse and pig show more strong humoralas wellas cellular immune responses. The PED- Fc has a possibility as an inactivated vaccine, which have comparable effect to live attenuate vaccines to prevent PED disease. In addition, the newly established vaccines strategy might be able to apply for other viruses, which contain host cell derived envelope, using Vero cells or other host cells expressing Fc molecules

목차 Contents

• 표지 ... 1
• 제 출 문 ... 2
• 요 약 문 ... 3
• SUMMARY (영문 용약문) ... 17
• CONTENTS(영문 목차) ... 19
• 목 차 ... 20
• 제 1 장 연구개발 과제의 개요 ... 21
• 1. 경제 사회적 측면 ... 21
• 2. 기술적 측면 ... 22
• 제 2 장 국내외 기술개발 현황 ... 32
• 1. Fc 관련 연구 현황 ... 32
• 2. PED 백신 개발 현황 ... 32
• 3. PRRS 백신 개발현황 ... 34
• 4. 연구배경에 따른 연구의 범위 ... 36
• 제 3 장 연구 개발 수행 내용 및 결과 ... 38
• 1절 PED-Fc 백신의 개발 ... 38
• 2절 PRRS-Fc 백신의 개발 ... 102
• 3절 Fc 분자발현 양돈용 백신의 경제성 분석 ... 132
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 142
• 제 5 장 연구개발의 성과 및 성과활용 계획 ... 145
• 1. 연구 성과 ... 145
• 2. 연구 성과 활용 계획 ... 145
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 146
• 1. 해외 PED발생 현황 ... 146
• 2. PRRSV 불활화 백신 연구 ... 146
• 제 7 장 참고문헌 ... 147
• 끝페이지 ... 148