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研究生： 沈靜芬
Shen, Ching-Fen
論文名稱： 健康及第一型糖尿病青少年施打mRNA新冠疫苗的系統疫苗學
Systems vaccinology of the mRNA vaccine in healthy and type 1 diabetes mellitus adolescents
指導教授： 謝奇璋
Shieh, Chi-Chang
柯文謙
Ko, Wen-Chien
學位類別： 博士
Doctor
系所名稱： 醫學院 - 臨床醫學研究所
Institute of Clinical Medicine
論文出版年： 2025
畢業學年度： 113
語文別： 英文
論文頁數： 98
中文關鍵詞： BNT162b2 mRNA 疫苗 、第一型糖尿病 、先天性免疫反應 、抗體反應 、細胞免疫 、免疫原性
外文關鍵詞： BNT162b2 mRNA vaccine, type 1 diabetes mellitus, innate immune response, antibody response, cellular immunity, immunogenicity
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    • 糖尿病與許多慢性疾病一樣，是2019冠狀病毒疾病（COVID-19）重症與死亡的重要風險因子。然而，目前對第一型糖尿病（T1D）青少年的免疫反應了解仍十分有限，這也限制了疫苗策略的制定與調整。本研究深入探討T1D青少年與健康對照組（HC）在接種輝瑞-BNT 162b2 mRNA疫苗後的免疫反應差異，本研究共招募24位年齡介於12至17歲的青少年，其中包含16位健康對照者與8位T1D患者，並於多個時間點進行全面性的免疫反應評估，包括接種前、第一劑疫苗後第3天、第二劑後第3天、1個月、5個月與1年，以及第三劑後1個月。這些評估涵蓋免疫細胞活化、全血RNA定序（轉錄體分析）、細胞激素反應、針對武漢原始株及其他變異株（VOCs）的結合與中和抗體生成、抗原特異性T細胞反應（AIM+ T細胞、IFN-γ釋放與增殖分析）、以及B細胞受體（BCR）庫分析。研究發現，在接種疫苗前，T1D青少年的B細胞亞群於接受PRR促進劑刺激後，透過TLR9所誘導的IFN-α產生能力較差，且其轉錄體分析顯示免疫相關基因表現較高。接種疫苗後，T1D青少年的單核細胞亞群中的細胞激素表現較低，且全血轉錄體顯示其疫苗誘導的先天免疫活化程度較低。儘管T1D青少年產生了與健康對照組相當的抗體反應，但其疫苗接種前的HbA1c水準與對變異株抗體交叉反應呈負相關。雖然T1D組在疫苗接種後展現與健康組相似的細胞免疫力（QuantiFERON分析所示），但對Omicron變異株的CD8+ T細胞交叉保護力較弱。此減弱的細胞交叉反應與其初始B細胞對TLR9反應差與第一劑疫苗誘導的經典單核細胞反應減弱有關。接種疫苗一年後，兩組在對變異株的抗體交叉反應及BCR多樣性方面表現相當，但健康組的體細胞突變（SHM）頻率提升較明顯。總結來說，儘管T1D青少年在疫苗接種後的抗體水平與健康對照組相當，但其先天與細胞免疫反應較為減弱，這可能與TLR9活性降低與HbA1c較高有關。然而，兩組的長期抗體反應與B細胞多樣性相似。本研究結果顯示，免疫功能與血糖控制是影響T1D青少年的疫苗效力的因素。

    Diabetes, along with other chronic medical conditions, is a significant risk factor for severe coronavirus disease 2019 (COVID-19) and subsequent mortality. However, information on the immune response of adolescents with type 1 diabetes (T1D) has been limited, which hinders the development and adjustment of future vaccination strategies. This study provides a comprehensive investigation into the immunological differences between adolescents with T1D and healthy control (HC) after receiving the Pfizer-BNT 162b2 mRNA vaccine. A total of 24 adolescents aged 12 to 17 years participated in the study, including 16 HCs and 8 individuals with T1D. Extensive immunological assessments were conducted at multiple time points: before vaccination, 3 days after the first dose, 3 days, 1 month, 5 months, and 1 year after the second dose, and 1 month following the third dose. These assessments included immune cell activation, whole blood RNA sequencing, cytokine responses, binding and neutralizing antibodies against both the Wuhan strain and variants of concern (VOCs), antigen-specific T cell responses (AIM+ T cells, IFN-γ release assays, proliferation assays), and B cell receptor (BCR) repertoire analysis. At baseline, adolescents with T1D demonstrated impaired TLR9-mediated IFN-α production in B cell subsets following ex vivo stimulation with pattern recognition receptor (PRR) agonists. Meanwhile, they exhibited a higher expression of immune-related gene sets in transcriptome analyses before vaccination. After vaccination, T1D adolescents showed reduced cytokine expression in monocyte subsets and lower levels of vaccine-induced innate immune activation based on gene expression analysis. Although T1D adolescents had robust antibody responses after vaccination—comparable to those of healthy controls—their HbA1c levels prior to vaccination negatively correlated with antibody cross-reactivity to VOCs. Furthermore, the T1D group demonstrated similar cellular immunity to HCs after vaccination (as measured by QuantiFERON), but they exhibited weakened CD8+ T cell cross-protection against the Omicron variant. This diminished cellular cross-reactivity was associated with poor TLR9 responses in naive B cells and reduced initial vaccine-induced classical monocyte responses. One year after vaccination, both groups showed comparable antibody cross-reactivity against VOCs and BCR repertoire diversity, except that healthy controls exhibited a more obvious increase in somatic hypermutation (SHM) frequencies over time. In summary, adolescents with T1D displayed reduced innate and cellular immune responses to the BNT162b2 mRNA vaccine compared to healthy controls, despite having similar levels of antibodies. Weaker responses were linked to diminished TLR9 activity and higher HbA1c levels. However, long-term antibody levels and B cell diversity were comparable between both groups. These results suggest that immune function and glycemic control may influence vaccine effectiveness in adolescents with T1D.

    中文摘要 i Abstract ii 致謝 iv 1. Introduction 1 1.1. COVID-19 pandemic and changing epidemiology 1 1.2. Vaccination as the way out of pandemic 2 1.3. Modification of mRNA and its effect on antigen presentation and immunogenicity 2 1.4. COVID-19 and vaccination in children 3 1.5. Diabetes as a risk factor for severe COVID-19 and associated system dysfunction 4 1.6. Vaccination recommendations for diabetic patients 4 1.7. Type 1 diabetes mellitus in adolescents and associated infectious risks 5 1.8. Immuno-pathogenesis and immune dysfunction in type 1 diabetes mellitus 5 1.9. Systems vaccinology and its implications in understanding the COVID-19 vaccination in type 1 diabetes mellitus 6 1.10. COVID-19 community outbreak and vaccine campaign rollout in Taiwan 6 1.11. Hypothesis and study aim 7 2. Materials and methods 9 2.1. Study design and participant recruitment 9 2.2. Isolation and preparation of PBMCs 10 2.3. Analysis of innate immune cell responses to PRR agonists 10 2.4. Analysis of early innate immune cell activation after vaccination 12 2.5. RNA sequencing and data analysis 12 2.6. Cytokine array analysis 13 2.7. Detection of SARS-CoV-2 binding antibodies 14 2.8. Detection of SARS-CoV-2 neutralizing antibodies 15 2.9. SARS-CoV-2 spike-specific T cell recall response 15 2.10. BrdU-based cell proliferation assay 16 2.11. QuantiFERON SARS-CoV-2 assay 17 2.12. B cell receptor (BCR) repertoire analysis 17 2.13. B cell receptor somatic hypermutation analysis (SHM) 18 2.14. Statistical analysis 18 3. Results 20 3.1. Overview of study population and experimental design 20 3.2. Immune cell responses before vaccination in healthy and T1D adolescents (TP1) 20 3.3. Early innate immune cell responses following the first and second doses of BNT162b2 mRNA vaccination (TP2, TP3) 21 3.4. Gene expression and immune gene set responses following BNT162b2 mRNA vaccination (TP1, TP2, TP3) 22 3.5. Serum cytokine levels in healthy controls and T1D adolescents following vaccination (TP1, TP2, TP3) 23 3.6. Antibody responses following BNT162b2 mRNA vaccination (TP3, TP4, TP5, TP7) 24 3.7. T cellular responses following BNT162b2 mRNA vaccination (TP5, TP6, TP7) 25 3.8. BCR repertoire and SHM frequencies in healthy and T1D adolescents (TP3, TP7) 27 4. Discussion 27 4.1. Impaired TLR9 agonist-induced B cell response and increased immune- related gene expression in T1D adolescents at baseline 28 4.2. COVID-19 mRNA vaccines-induced monocyte response and its association with overall immunogenicity 29 4.3. Unimpaired antibody but weakened CD8+ T cell reactivity to vaccination in T1D patients 29 4.4. Chronic auto-inflammation and immune cell exhaustion undermine vaccine-induced responses in T1D 30 4.5. Reduced S-specific T cell immunity in type 1 diabetes adolescents despite assay variability 30 4.6. Strengths and limitations 31 4.7. Future directions 32 5. Conclusion 34 6. References 80 7. Appendix 84

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