백일해 주요 항원의 재조합 단백질 설계 및 초기 면역원성 평가 = Design of recombinant proteins based on major pertussis antigens and preliminary evaluation of their immunogenicity

다국어 초록 (Multilingual Abstract)

Design of recombinant proteins based on major pertussis antigens and preliminary evaluation of their immunogenicity Min Jeong Kim Adviser: Prof. Kim Dong-Min., M.D., Ph.D. Department of Biomedical Science Graduate School of Chosun University Background: Pertussis remains a significant public health concern despite widespread vaccination with whole-cell and acellular pertussis vaccines. The recent resurgence of pertussis has been associated with waning immunity following acellular pertussis vaccination and the limited ability of current vaccines to prevent bacterial transmission. These limitations highlight the need for improved vaccine strategies and the identification of novel antigens capable of inducing effective and durable immune responses. Recombinant protein-based vaccine approaches offer the advantage of incorporating selected protective epitopes while allowing flexible antigen design and optimization. Therefore, this study aimed to generate recombinant proteins based on major Bordetella pertussis antigens and evaluate their antigenicity, preliminary immunogenicity, and pertussis toxin (PT)-neutralizing potential. Methods: Three recombinant proteins, Pertussis FsmS1, Pertussis rFeSOD, and CRM197EK-Pertussis FsmS1, were designed based on major Bordetella pertussis antigens, expressed in an Escherichia coli system, and purified by affinity chromatography. Their antigenic reactivity was screened by indirect ELISA using anti-pertussis serum and sera from DTP-vaccinated individuals. Based on the screening results, Pertussis FsmS1 was selected for further immunogenicity evaluation in BALB/c mice using ECLSQ or Alum as adjuvants. Antigen-specific antibody responses were assessed by ELISA against recombinant FsmS1 and purified FHA. Functional PT-neutralizing activity was evaluated using a CHO cell clustering assay, and preliminary B. pertussis culture conditions and challenge model conditions were examined to support future protection studies. Results: All three recombinant proteins were successfully expressed in soluble form and purified with the expected molecular weights. Among the tested antigens, Pertussis FsmS1 exhibited stronger IgG reactivity than Pertussis rFeSOD against both anti-pertussis serum and sera from DTP-vaccinated individuals, supporting its selection as the lead antigen candidate. Immunization with Pertussis FsmS1 formulated with either ECLSQ or Alum induced robust antigen-specific IgG responses in mice. Antibody responses against both purified FHA and recombinant Pertussis FsmS1 remained detectable at high serum dilutions (10⁴–10⁵), indicating substantial humoral immunogenicity. Furthermore, sera from immunized mice showed reactivity against B. pertussis lysate antigens. However, despite the strong binding antibody responses, PT-induced CHO cell clustering was not clearly inhibited by sera from Pertussis FsmS1-immunized mice, suggesting no clear PT-neutralizing activity under the experimental conditions tested. Preliminary bacterial growth analyses and OD600-CFU correlations supported the feasibility of establishing a future B. pertussis challenge model. Conclusion: Pertussis FsmS1 demonstrated favorable antigenicity and stron g humoral immunogenicity in both human sera and immunized mice, supporti ng its potential as an early-stage recombinant pertussis vaccine antigen candi date. Nevertheless, the absence of detectable PT-neutralizing activity indicates that induction of binding antibodies alone may be insufficient for functional pr otection. These findings suggest that further antigen optimization focusing on PT-neutralizing epitopes and validation in appropriate challenge models will b e necessary for the development of next-generation pertussis vaccines. Keywords: Bordetella pertussis; pertussis toxin; recombinant antigen; immu nogenicity; toxin-neutralizing activity; vaccine candidate

목차 (Table of Contents)

• Ⅰ. 서론 1
• A. 연구 배경 1
• B. 백일해 백신과 주요 항원 4
• ⅰ. Pertussis FsmS1 5
• ⅱ. Pertussis rFeSOD 5
• C. CHO cell assay를 이용한 가능성 평가의 중요성 6
• D. 연구 목적 7
• Ⅱ. 연구 방법 8
• A. 연구 윤리 및 실험 설계 8
• B. 백일해 주요 항원 기반 재조합 단백질 설계 8
• C. 유전자 클로닝 및 발현 벡터 구축 9
• D. 재조합 단백질 발현 및 정제 9
• E. 항원 선별을 위한 indirect ELISA 11
• F. 마우스 면역화 및 항혈청 확보 11
• G. CHO cell assay를 이용한 pertussis toxin 중화능 평가 14
• H. Bordetella pertussis 배양 및 challenge 조건 검토 15
• I. 통계 분석 및 결과 해석 기준 17
• Ⅲ. 연구 결과 18
• A. 재조합 단백질 발현 및 정제 결과 18
• B. 항원 선별을 위한 indirect ELISA 결과 26
• C. Pertussis FsmS1 면역화 마우스 혈청의 항체 반응 33
• D. CHO cell assay를 통한 Pertussis toxin 중화 활성 평가 39
• E. Bordetella pertussis challenge model 적용 가능성 검토 42
• Ⅳ. 고찰 49
• ⅰ. 재조합 단백질 발현 및 정제 성공 49
• ⅱ. 사람 혈청 반응성의 의미 49
• ⅲ. 마우스 면역원성의 의미 50
• ⅳ. CHO cell assay 기반 기능성 평가 51
• ⅴ. 현재 백일해 백신 한계와 본 연구의 의의 53
• ⅵ. 연구의 제한점 54
• ⅶ. 향후 연구 방향 및 결론 55
• 참고문헌 57