A Flagellin-IL-15 Fusion Adjuvant for Enhanced Mucosal Immunity Against Influenza = 인플루엔자 백신의 점막 면역 증진을 위한 플라젤린-IL-15 융합 보조제

다국어 초록 (Multilingual Abstract)

A Flagellin-IL-15 Fusion Adjuvant for Enhanced Mucosal Immunity Against Influenza

Department of Biomedical Science
Graduate School, Chonnam National University

Bang, Yong Jun

Supervised by Professor Lee, Shee Eun

Abstract
Flagellin, the structural component of bacterial flagella with inherent structural stability, functions as a pathogen-associated molecular pattern (PAMP) that cognately activates innate immunity via cell surface Toll-like receptor 5 (TLR5) and cytosolic NLRC4 inflammasome pathways. It has unique adjuvant activity across diverse vaccine platforms. Interleukin-15 (IL-15) is a pleiotropic cytokine essential for the development, maintenance, and activation of natural killer (NK) cells and memory CD8⁺ T cells, which could be utilized in developing immunomodulatory strategies for vaccines and immunotherapeutics. However, its clinical application is limited because of poor pharmacokinetic characteristics and intrinsic instability, which would severely compromise in vivo efficacy. Although fusion with the high-affinity IL-15 receptor α extracellular Sushi domain has been employed to extend half-life and improve in vivo activity, IL-15-based formulations still face limitations in inducing stable immunomodulation. To address these challenges, we developed a structurally optimized fusion adjuvant, Sushi-IL15-FlaB (SIB), by fusing flagellin B (FlaB) with Sushi domain-fused IL-15. The recombinant SIB fusion protein was produced using the Expi293F expression system with high purity and stability. TLR5 agonist activity was confirmed using the HEK-Blue TLR5 reporter assay. Intranasal co-administration of SIB with formalin-inactivated influenza A/PR/8/34 (iPR8) significantly potentiated antibody responses in both mucosal and systemic immune compartments. Animals immunized with SIB-adjuvanted iPR8 vaccine manifested significantly enhanced protection against the lethal challenge with live A/PR/8/34 (H1N1) viruses. Co-immunization with iPR8 and SIB elicited strong antigen-specific IgG1, IgG2a, and IgG2c responses in both BALB/c and C57BL/6 mice. The protection in C57BL/6 was significantly stronger than BALB/c mice, suggesting a more balanced induction of cellular immunity by SIB adjuvant, given the cellular immunity predilection in the former mouse strain. Of note, SIB also markedly enhanced antigen-specific secretory IgA production, highlighting its much more potentiated mucosal adjuvant properties. Beyond antibody quantity, SIB augmented qualitative B cell immunity by expanding germinal center responses in cervical lymph nodes, increasing the frequency of antigen-specific memory B cells and supporting the emergence of plasma cells, changes consistent with improved high avidity binding. In parallel, SIB promoted polyfunctional CD8⁺ T cell responses characterized by IFN-γ and IL-2 double-positive, while CD4⁺ T cells predominantly exhibited an IL-2 single-positive profile, consistent with enhanced T-cell responsiveness and cellular immune capacity. In summary, SIB serves as a novel multifunctional fusion mucosal adjuvant that combines TLR5 activation with IL-15-mediated immune modulation. It significantly enhances both systemic and mucosal immune responses with balanced activation of both humoral and cellular immunity.

국문 초록 (Abstract)

인플루엔자 백신의 점막 면역 증진을 위한 플라젤린-IL-15 융합 보조제

방 용 준

의과학과(의생명학전공)
전남대학교 의과대학 의과학과
(지도교수: 이시은)

국문 초록
Flagellin은 고유의 구조적 안정성을 지닌 세균 편모의 구조 단백질로서 병원체연관분자패턴 (PAMP)으로 작동하며 세포 표면의 TLR5와 세포질 NLRC4 인플라마솜 경로를 통해 선천면역을 정합적으로 활성화한다. 이러한 특성은 다양한 백신 플랫폼에서 독특한 면역증강제 활성을 부여한다. Interleukin-15 (IL-15)는 자연살해세포 (NK cells)와 기억 CD8+ T 세포의 발생 유지 활성에 필수적인 다기능성 사이토카인으로 백신 및 면역치료에서의 면역조절 전략으로 유용하나 불리한 약동학적 특성과 고유 불안정성으로 인해 임상 적용이 제한된다. 고친화도 IL-15 수용체 α의 세포 외 Sushi 도메인 융합을 통해 반감기 연장과 체내 활성을 개선하려는 접근이 사용되어 왔지만 IL-15 기반 제형은 여전히 안정적인 면역조절 유도에 제약이 존재한다. 본 연구에서는 Sushi 도메인과 IL-15를 융합하고 여기에 flagellin B (FlaB)를 결합한 구조 최적화 융합 보조제 Sushi-IL15-FlaB (SIB)를 설계하여 SIB라 명명하였고 Expi293F 발현계를 이용해 고순도 및 고안정성으로 재조합 단백질을 생산하였다. HEK-Blue TLR5 리포터 분석으로 TLR5 활성을 확인하였으며 SIB를 포르말린 불활화 인플루엔자 A/PR/8/34 (iPR8)와 비강 병용 투여했을 때 점막 및 전신 면역 구획 모두에서 항체 반응이 유의하게 증폭되었다. SIB로 보조한 iPR8 백신으로 면역한 동물은 생체 내 A/PR/8/34 (H1N1) 치사 도전에서 현저히 향상된 보호를 보였다. BALB/c와 C57BL/6 두 계통 모두에서 항원특이적 IgG1, IgG2a, IgG2c가 강하게 유도되었고 C57BL/6에서의 보호가 더 강력하여 해당 계통의 세포성 면역 성향을 고려할 때 SIB가 보다 균형 잡힌 세포성 면역을 유도함을 시사하였다. 특히 SIB는 항원특이적 분비형 IgA를 크게 증가시켜 점막 보조제로서의 강화된 효능을 부각시켰다. 정량적 향상을 넘어 경부 림프절에서의 발달중심 반응 확대 항원특이적 기억 B 세포 빈도 증가 형질세포 출현을 통해 항체 결합 친화도의 향상을 시사하는 질적 B 세포 면역의 증대를 동반하였다. 병행하여 SIB는 IFN-γ와 IL-2 이중 양성을 특징으로 하는 다기능성 CD8⁺ T 세포를 촉진하였고 CD4⁺ T 세포는 주로 IL-2 단일 양성 양상을 보여 T 세포 반응성과 세포성 면역 역량의 증대를 입증하였다. 요약하면 SIB는 TLR5 자극과 IL-15 매개 면역조절을 하나의 분자로 통합한 새로운 다기능성 비강 점막 보조제로서 체액성과 세포성 면역을 균형 있게 강화하여 전신 및 점막 면역 반응과 보호 효능을 유의하게 향상시킨다.

목차 (Table of Contents)

• 1. Abstract
• 2. Introduction
• 2.1. The burden of influenza infections
• 2.2. The advantages of mucosal vaccination
• 2.3. Flagellin-based mucosal adjuvant platform
• 2.4. The limitation of IL15 as a vaccine adjuvant
• 2.5. The expected synergistic adjuvant effect of IL-15 and FlaB
• 2.6. Experimental design and hypothesis
• 3. Materials and Methods
• 3.1. Plasmid constructions, expression, and purification of recombinant proteins
• 3.2. Determination of TLR5-dependent NF-κB activation
• 3.3. CTLL-2 cell proliferation assay
• 3.4. The carboxyfluorescein succinimidyl ester (CFSE) labeling and analysis
• 3.5. Ethics statement
• 3.6. Intranasal immunization
• 3.7. Determination of antigen-specific antibody response by enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis 17
• 3.8. Hemagglutination inhibition (HAI) assay
• 3.9. Virus plaque reduction neutralization test (PRNT)
• 3.10. Preparation of formalin-inactivated A/PR/8/34 (iPR8)
• 3.11. Lethal challenge with live influenza virus
• 3.12. ELISPOT assay
• 3.13. Determination of antibody avidity by ELISA
• 3.14. Determination of antigen-specific cytokine production by ELISA
• 3.15. Germinal center observation by confocal microscopy
• 3.16. Flow cytometry
• 3.17. Statistical analysis
• 4. Results
• 4.1. Engineering and characterization of protein-based adjuvant Sushi- IL-15-FlaB (SIB)
• 4.2. The SIB-adjuvant induced significantly enhanced protection compared with B and SI in a H1N1 influenza lethal challenge BALB/c model
• 4.3. Enhanced generation of antigen-specific memory B and plasma cells by IN immunization with the SIB-adjuvanted vaccine in BALB/c mice
• 4.4. The SIB-adjuvant induced significantly enhanced protection compared with B and SI in a H1N1 influenza lethal challenge C57BL/6 model
• 4.5. Enhanced generation of antigen-specific memory B and plasma cells by IN immunization with the SIB-adjuvanted vaccine in C57BL/6 mice
• 4.6. Intranasal SIB-adjuvanted vaccination elicits enhanced germinal center formation in cervical lymph nodes
• 4.7. SIB-adjuvanted IN vaccine induces IFN-γ and IL-2 producing multifunctional T cell responses in cervical lymph nodes
• 5. Discussion
• 6. Conclusion
• 7. References
• 8. 국문초록
• 9. Acknowledgment