Design and immunological evaluations of the DNA vaccine specific for hepatitis B virus = B형 간염 DNA백신 제제의 설계 및 면역반응평가

국문 초록 (Abstract)

B형 간염은 전 세계적으로 발생빈도가 높을 뿐만 아니라 간경변이나 암과 같은 만성 간질환으로 진행될 가능성이 높아 중요한 보건문제가 되고 있다. 지구상에는 3억 5천만명의 만성 B형 간염 감염자가 있다고 알려져 있으며 B형 간염의 유병률은 아프리카와 아시아 지역에서 특히 높다. 현재 인터페론 등이 만성 B형 간염의 치료제로 사용되고 있으나 그 효과가 20-40%로 낮고 약물과 관련된 이상반응의 발생빈도가 높게 나타나 치료방법이 매우 제한적인 상황이다. 이러한 점을 감안할 때 B형 간염의 발생을 사전에 예방할 수 있는 백신의 중요성은 매우 크다고 할 수 있다. 현재까지 15년 이상 사용되어 온 혈장유래 및 재조합 B형 간염 백신은 면역원성이 높고 효과적인 것으로 알려져 왔다. 그러나 충분한 면역효과를 얻기 위해 3회의 백신 접종을 실시해야 하며, 백신에 대한 비반응자들의 비율이 높아 면역능을 증가시킨 새로운 B형간염 백신의 개발이 요구되고 있다.
최근 전통적인 백신과는 달리 체액성 면역반응 뿐만 아니라 세포성 면역반응을 유도하고 백신 자체의 안정성이 높으며 대량 생산이 용이하다는 DNA ?

다국어 초록 (Multilingual Abstract)

Hepatitis B virus (HBV) causes many problems in the all parts of the world. The prospects for controlling the infection caused by the HBV depend on the availability of safe, effective, and affordable vaccines. In particular, such dependency is almost true in most of the Asian countries. For the prevention of HBV infections, the plasma-derived and recombinant DNA vaccines have been used for more than 15 years, but the vaccine formulae are expensive and are not much effective for treatment of HBV carriers. Thus, improved vaccine formulae or new approaches of vaccination method are needed to enhance the efficacy and reduce the cost.
In recent, the development of molecular biology and immunology has led to a concept of DNA vaccination, which, in fact, is under replacement of the protein vaccine. The DNA vaccination induces both humoral and cellular immunities. Furthermore, the DNA vaccine appears to provoke more efficient immune response.
In the present study, pCDHBS plasmid DNA vaccine encoding the preS2 + S in the HBV was constructed and its immunological efficacy was characterized. Results showed that the plasmid-transfected cells expressed the protein specific for the HBV. A similar expression was determined in the intramuscular injection site of BALB/c mice. Cytokine-profile determination by ELISA and ELISPOT revealed that the DNA vaccine-stimulated T cells produced the Th1 type of IL-2 & IFN-γ and the Th2 type of IL-4 & IL-6, respectively. Especially, expressions of IL-2, IFN-γ, RANTES, and MIP-1β were significant, whereas IL-4 and IL-6 expressions were as much the same as the negative controls. These results were confirmed by the flow cytometry analysis. These results indicate that the HBV DNA vaccine formula induces the Th1 immune response more predominantly than Th2 response.
In conclusion, these data would be informative in development of HBV DNA vaccines, which can be helpful for preventing and/or treating infection due to the hepatitis vi

목차 (Table of Contents)

• LIST OF TABLES AND FIGURES i
• ABBREVIATIONS AND TERMINOLOGY iii
• ABSTRACT v
• INTRODUCTION 1
• MATERIALS AND METHODS 15
• 1. Plasmid Construction 15
• 2. Examination of Gene Expression 16
• 2.1 Cell Culture 17
• 2.2 In Vitro Expression of PCDHBS DNA Vaccine 17
• 2.3 In Vivo Expression of PCDHBS DNA Vaccine 18
• 2.4. Immunofluorescence Assay 19
• 3. Immunization Strategy 20
• 3.1 Mice 20
• 3.2 Immunization of Mice 20
• 4. Immunoassay 21
• 4.1 Cytokine and Chemokine Expression Analysis 21
• 4.2. ELISPOT Assay 22
• 4.3. Intracellular Cytokine Assay 24
• 5. Safety Test 25
• 6. Statistical analysis 25
• RESULTS 26
• 1. Plasmid Construction 26
• 2. Examinations of Gene Expression 29
• 2.1 In Vitro Expression of pCDHBS DNA VAccine 29
• 2.2 In Vivo Expression of pCDHBS DNA VAccine 32
• 2.3 Immunofluorescence Assay 34
• 3. Examinations of Mode of Action 36
• 3.1. Immunization with pCDHBS Induces Potent Antigen
• Specific Th1 Type Cellular Immune Response 36
• 3.2. Immunization with pCDHBS Induces Potent Antigen
• Specific Production of Chemokines MIP-1β and RANTES 42
• 3.3. pCDHBS Specific IFN-γ Secreting T Cells are Increased 45
• 3.4. The Increase of Th1 Type T cell Responses are Examined by Intracellular Cytokine Analyis 47
• 4. Safety Test 50
• DISCUSSION 52
• REFERENCES 55
• ABSTRACT IN KOREAN 66
• ACKNOWLEDGEMENT IN KOREAN