Comprehensive study of inactivated viral hemorrhagic septicemia virus (VHSV) vaccine-induced immunity and IL-10 quantification assay development in olive flounder (Paralichthys olivaceus) = 넙치(Paralichthys olivaceus)에서 불활화 바이러스성 출혈성 패혈증 바이러스(VHSV) 백신에 의해 유도되는 면역반응의 종합적 연구 및 IL-10 정량 분석법 개발

다국어 초록 (Multilingual Abstract)

Viral hemorrhagic septicemia virus (VHSV) is a highly pathogenic virus that causes fatal infections in various freshwater and saltwater fish. In Korea, VHSV infections have been recurrent in the major farmed species, olive flounder (Paralichthys olivaceus), causing persistent economic losses to the aquaculture industry. Vaccination is a key response strategy for effectively controlling VHSV infection, and inactivated vaccines are currently commercially available and widely used in the field. However, the immune protection mechanisms induced by inactivated vaccines in fish remain insufficiently understood, limiting our understanding of the immunological mechanisms that determine vaccine efficacy. This is because previous studies have primarily focused on limited immune markers, such as immune gene mRNA expression characteristics and serum antibody titer measurements. To overcome these limitations, this doctoral dissertation aimed to comprehensively elucidate the immune response induced by an inactivated VHSV vaccine in olive flounder by investigating immunogenicity analysis based on the preservation of the vaccine's antigenic structure, analysis of antibody-dependent immune responses after vaccination, and development of an analytical tool for detecting cytokines at the protein level.

In Chapter I, the immunogenicity of VHSV vaccines inactivated by formalin, β-propiolactone (BPL), binary ethylenimine (BEI), and heat treatment was comparatively analyzed in relation to changes in antigenicity. Viral challenge experiments revealed that the formalin- and BPL-inactivated VHSV vaccine groups exhibited higher survival rates and lower viral replication numbers, and significantly increased expression of antiviral, cytokine, and adaptive immune-related genes. Antigenicity analysis of the inactivated VHSV vaccines revealed that the formalin- and BPL-inactivated VHSV vaccines, which exhibited high protective efficacy, most effectively preserved the disulfide bond-dependent structure of the glycoprotein. This suggests that preservation of antigen structure during inactivated vaccine production significantly influences the immunogenicity and protective efficacy of the vaccine.

In Chapter II, we analyzed the antibody-dependent immune response induced in olive flounder after inactivated VHSV vaccination. After vaccination, B cell differentiation and the production of VHSV-specific antibody-secreting cells in the spleen were observed, which led to increased serum VHSV and VHSV G-specific IgM titers. Subsequent virus challenge experiments revealed a rapid decrease in VHSV G-specific IgM and a dramatic decrease in viral replication in the vaccinated group, suggesting that the induced antibodies contribute to virus clearance in the early stages of infection. Furthermore, we demonstrated that vaccination-induced IgM antibodies can directly bind to glycoproteins on the surface of VHSV-infected cells during the early stages of VHSV infection. Subsequently, antibody-dependent cytotoxicity against VHSV-infected cells was significantly increased in the vaccinated group. These results suggest that VHSV-specific IgM antibodies induced by the inactivated VHSV vaccine in the early stages of infection contribute not only to simple virus neutralization but also directly to the clearance of infected cells.

In Chapter III, a sandwich ELISA was developed and applied for the quantitative analysis of interleukin-10 (IL-10) in olive flounder. The established IL-10 sandwich ELISA demonstrated high sensitivity and specificity and enabled quantitative analysis of IL-10 protein expression in VHSV-infected olive flounder. High levels of IL-10 were consistently elevated in most individuals after VHSV infection, suggesting the potential involvement of IL-10 in modulating antiviral and inflammatory immune responses in olive flounder. Furthermore, a strong correlation was observed between IL-10 mRNA and protein expression levels, demonstrating that mRNA expression closely mirrors actual protein production. The development of this sandwich ELISA provides a practical analytical foundation for quantitatively assessing cytokine-based immune responses in fish immunity research and demonstrates its potential for use in future vaccine efficacy assessments. In conclusion, this study systematically elucidated the immune response induced by an inactivated VHSV vaccine in olive flounder, focusing on immunogenicity and antibody-dependent immune responses based on the preservation of antigenic structure.

In conclusion, this study systematically elucidates immune responses induced by an inactivated VHSV vaccine in olive flounder, with particular emphasis on antigen structure preservation–dependent immunogenicity and antibody-dependent immune mechanisms. Furthermore, the development and application of cytokine protein–level immune assays complement the limitations of conventional mRNA-based analyses. Collectively, the findings and analytical approaches presented in this dissertation provide an important experimental and methodological foundation for future studies on fish vaccine efficacy and immune mechanisms.

국문 초록 (Abstract)

바이러스성 출혈성 패혈증 바이러스(VHSV)는 다양한 담수 및 해수어에서 치명적인 감염을 유발하는 고병원성 바이러스이다. 한국에서는 주요 양식 어종인 넙치(Paralichthys olivaceus)에 VHSV 감염이 반복적으로 발생하여 양식 산업에 지속적인 경제적 손실을 초래하고 있다. 백신 예방은 VHSV 감염을 효과적으로 제어하기 위한 주요 대응 전략으로 활용되고 있으며, 현재 현장에서는 불활화 백신이 상용화되어 널리 사용되고 있다. 그러나 어류에서 불활화 백신에 의해 유도되는 면역 보호 기전은 아직 충분히 규명되지 않아,백신 효능을 결정하는 면역학적 기전에 대한 이해에는 여전히 한계가 존재한다.이는 기존 연구들이 주로 면역 유전자 mRNA 발현 특성 및 혈청 항체 역가측정과 같은 제한적인 면역 지표에 초점을 맞추었기 때문이다. 이러한 한계를 극복하기 위해 본 박사 학위 논문은 백신의 항원 구조 보존에 따른 면역원성
분석, 백신 접종 후 항체 의존성 면역 반응 분석, 단백질 수준에서의 사이토카인 검출을 위한 분석법 개발을 통해 불활화 VHSV 백신이 가자미에서
유도하는 면역 반응을 다면적으로 규명하는 것을 목표로 하였다. 제 1장에서는 formalin, β-propiolactone (BPL), binary ethylenimine (BEI), 열처리 불활화 VHSV 백신의 항원성 변화에 따른 면역원성을 비교 분석하였다. 바이러스 공격 실험 결과, formalin 및 BPL 불활화 VHSV 백신군은 더 높은 생존율과 더 낮은 바이러스 복제 수를 나타냈으며, 항바이러스, 사이토카인 및 적응 면역 관련 유전자의 발현이 유의하게 증가하였다. 불활화 VHSV 백신의 항원성 분석 결과, 높은 보호 효능을 나타낸 formalin 및 BPL 불활화 VHSV는 당단백질의 이황화 결합 의존성 구조를 가장 효과적으로 보존하는 것으로나타났다. 이는 불활화 백신 제조 시 항원 구조 보존이 백신의 면역원성과 보호효능에 중요한 영향을 미친다는 것을 시사한다.
제 2장에서는 불활화 VHSV 백신 접종 후 넙치에서 유도되는 항체 의존적 면역 반응을 분석하였다. 백신 접종 후 비장에서 B 세포의 분화와 VHSV특이적 항체 분비 세포의 생성이 확인되었으며, 이에 따라 혈청에서 VHSV 및 VHSV G 특이적 IgM의 역가가 증가하였다. 이후 바이러스 공격 실험에서 백신접종군은 VHSV G 특이적 IgM의 빠른 감소와 동시에 바이러스 복제 수가급격히 감소하는 양상을 보여, 유도된 항체가 감염 초기 단계에서 바이러스제거에 관여함을 시사하였다. 또한, VHSV 감염 초기 단계에서 백신 종으로 유도된 IgM 항체가 VHSV 감염 세포 표면의 당단백질에 직접 결합할 수 있음을 입증하였다. 이어서 백신 접종군에서 VHSV 감염 세포에 대한 항체의존적 세포독성이 유의하게 증가하였다. 이러한 결과는 감염 초기 단계에서불활화 VHSV 백신에 의해 유도된 VHSV 특이적 IgM 항체가 단순한 바이러스중화 반응뿐만 아니라 감염 세포 제거에도 직접적으로 기여함을 시사한다.제 3장에서는 넙치의 interleukin-10 (IL-10)에 대한 정량 분석을 위해sandwich ELISA를 개발하고 적용하였다. 확립된 IL-10 sandwich ELISA는높은 민감도와 특이도를 보여주었으며, VHSV에 감염된 넙치에서 IL-10 단백질발현을 정량적으로 분석할 수 있었다. VHSV 감염 후 대부분의 개체에서 높은수준의 IL-10이 지속적으로 증가하였으며, 이는 IL-10이 넙치의 항바이러스 및 염증성 면역 반응 조절에 관여할 가능성을 시사한다. 또한, IL-10의 mRNA발현 수준과 단백질 발현 수준 사이에 강한 상관관계가 관찰되어 mRNA 발현수준이 실제 단백질 생성 수준을 유사하게 반영함을 입증했다. 본 sandwichELISA의 개발은 어류 면역 연구에서 사이토카인 기반 면역 반응을 정량적으로평가하는 실용적인 분석 기반을 제공하며, 향후 백신 효능 평가에 활용될 가능성을 보여준다.결론적으로, 본 논문은 넙치에서 불활화 VHSV 백신에 의해 유도되는 면역반응을 항원 구조 보존에 따른 면역원성과 항체 의존적 면역 반응 측면에서 체계적으로 규명하였다. 또한 사이토카인 단백질 수준의 면역 분석법을 개발하고 적용함으로써 기존 mRNA 발현 중심의 분석이 갖는 한계를보완하였다. 본 연구에서 제시된 결과와 분석 방법은 향후 어류 백신의 효능평가 및 면역 기전 연구를 위한 중요한 실험적·방법론적 기반을 제공할 것으로 기대된다.

목차 (Table of Contents)

• Contents ·············································································································i
• List of Tables ····································································································· vi
• List of Figures ···································································································vii
• Abstract ············································································································ xi
• General introduction ······························································································ 1
• Chapter I: Comparative analysis of immunogenicity for inactivated viral hemorrhagic septicemia virus
• (VHSV) vaccines by different methods ········································································ 6
• 1. Introduction ································································································· 6
• 2. Materials and methods ·······················································································8
• 2.1 Virus ·······································································································8
• 2.2. Virus inactivation ·······················································································8
• 2.3. Experimental animals···················································································9
• 2.4. Immunization, challenge, and sampling····························································10
• 2.5. Measurement of virus-specific antibody titers in fish serum ····································10
• 2.5.1. Indirect enzyme-linked immunosorbent assay (ELISA) ····································11
• 2.5.2. Competitive ELISA··············································································11
• 2.6. RNA isolation and cDNA synthesis ································································12
• 2.7. Quantitative expression of immune genes ·························································12
• 2.8. Measurement of viral copy numbers································································13
• 2.9. Immunoreactivity of inactivated virus with anti-VHSV G mAbs·······························15
• 2.9.1. Indirect ELISA ···················································································15
• 2.9.2. Western blot ······················································································15
• 2.10. Statistical analysis ···················································································16
• 3. Results ·······································································································17
• 3.1 Protective efficacy of inactivated VHSV vaccine ·················································17
• 3.2. Virus copy numbers···················································································18
• 3.3. VHSV-specific antibody titers·······································································19
• 3.4. Expression kinetics of immune-related genes ·····················································21
• 3.5. Immunoreactivity of inactivated virus with anti-VHSV G mAbs·······························27
• 4. Discussion ···································································································29
• Chapter II: Analysis of antibody-dependent immune response of olive flounder (Paralichthys olivaceus)
• induced by inactivated viral hemorrhagic septicemia virus (VHSV) vaccine ····························38
• 1. Introduction ································································································38
• 2. Materials and methods ·····················································································41
• 2.1 Experimental fish ······················································································41
• 2.2. Virus and vaccine preparation ·······································································41
• 2.3. Immunization, challenge, and sampling····························································42
• 2.4. Leukocyte isolation ···················································································43
• 2.5. Characterization of B cell differentiation in spleen ···············································43
• 2.5.1. Flow cytometry···················································································43
• 2.5.2. Enzyme-Linked Immuno Spot (ELISpot)·····················································44
• 2.6. Measurement of antibody titers in serum ··························································45
• 2.6.1. Sandwich ELISA·················································································45
• 2.6.2. Indirect ELISA ···················································································45
• 2.6.3. Competitive ELISA··············································································46
• 2.7. Antibody-dependent cellular cytotoxicity (ADCC) assay········································47
• 2.7.1. Expression of VHSV G on the membrane of VHSV-infected cells ·······················47
• 2.7.2. Binding of olive flounder IgM to VHSV G on VHSV-infected cells ·····················47
• 2.7.3. Lactate dehydrogenase (LDH)-based cytotoxicity assay ···································48
• 2.8. Measurement of viral copy numbers································································49
• 2.9. Statistical analysis·····················································································49
• 3. Results ·······································································································50
• 3.1 Protective efficacy of the inactivated VHSV vaccine post-challenge ···························50
• 3.2. Kinetics of B-cell differentiation in spleen after vaccination····································52
• 3.3. Kinetics of antibody titer in serum post-vaccination and challenge ····························56
• 3.4. ADCC activity ·························································································58
• 4. Discussion ···································································································61
• Chapter III: Development of sandwich ELISA for IL-10 quantification in olive flounder (Paralichthys
• olivaceus) and its application to protein-level analysis during viral hemorrhagic septicemia virus
• (VHSV) infection·································································································66
• 1. Introduction ································································································66
• 2. Materials and methods ·····················································································69
• 2.1 RNA isolation and cDNA synthesis ·································································69
• 2.2. Plasmid construction··················································································69
• 2.3. Expression and purification of recombinant IL-10 protein ······································69
• 2.3.1. Production of recombinant IL-10 in prokaryotic cells ······································69
• 3.3.2. Production of recombinant IL-10 in eukaryotic cells········································70
• 2.4. Deglycosylation assay ················································································70
• 2.5. Production of anti-IL-10 monoclonal antibody (mAb) ···········································71
• 2.5.1 Mouse immunization and hybridoma cell production········································71
• 2.5.2. Screening and cloning ···········································································71
• 2.5.3. Antibody purification············································································72
• 2.5.4. Antibody biotinylation ··········································································72
• 2.5.5. Antibody isotyping···············································································72
• 2.6. Production of anti-IL-10 polyclonal antibody (pAb) ·············································73
• 2.6.1. Rabbit Immunization ············································································73
• 2.6.2. Antibody purification············································································73
• 2.7. Experimental fish······················································································73
• 2.8. Virus ····································································································74
• 2.9. VHSV challenge and sampling ······································································74
• 2.10. Real-time PCR ·······················································································75
• 2.11. Preparation of analytical protein samples·························································76
• 2.12. Characterization and validation of anti-IL-10 antibodies·······································76
• 2.12.1. SDS-PAGE and western blot ·································································76
• 2.12.2. Immunoprecipitation ···········································································77
• 2.13. Sandwich ELISA ····················································································77
• 2.13.1. Sensitivity and specificity assessment ·······················································78
• 2.13.2. Spike and recovery ·············································································78
• 2.13.3. Linearity and parallelism ······································································79
• 2.13.4. Positive signal verification analysis ··························································79
• 2.14. Statistical analysis ···················································································79
• 3. Results ·······································································································80
• 3.1 Antibody production and characterization··························································80
• 3.2. Characterization of IL-10 antibodies reactivity and molecular forms of IL-10 ···············83
• 3.3. Establishment and validation of IL-10 sandwich ELISA ········································85
• 3.3.1. Standard curve of sandwich ELISA ···························································85
• 3.3.2. Sensitivity and specificity·······································································86
• 3.3.3. Spike and recovery···············································································87
• 3.3.4. Linearity and parallelism········································································89
• 3.3.5. Positive signal verification analysis ···························································91
• 3.4. Mortality and virus copy number post-challenge with VHSV ··································93
• 3.5. Expression characteristics of IL-10 in olive flounder from challenge with VHSV ···········95
• 4. Discussion ···································································································99
• General discussion ·····························································································107
• References······································································································· 116
• Abstract (Korean) ······························································································148