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研究生：	沈孜錡 Shen, Tzu-Chi
論文名稱：	血液透析腎臟疾病的病人接受COVID-19疫苗接種前後自然與後天免疫反應研究 Innate and adaptive immune response before and after receiving COVID-19 vaccines in renal disease patients with hemodialysis
指導教授：	陳柏齡 Chen, Po-Lin
學位類別：	碩士 Master
系所名稱：	醫學院 - 微生物及免疫學研究所 Department of Microbiology & Immunology
論文出版年：	2022
畢業學年度：	110
語文別：	英文
論文頁數：	53
中文關鍵詞：	終末期腎病、細胞因子、單核細胞、自然殺手細胞、B細胞、基因表達、咪喹莫特、抗體
外文關鍵詞：	End-stage renal disease (ESRD), cytokines, monocytes, natural killer cells, B cells, gene expression, imiquimod, antibody
相關次數：	點閱：83 下載：0
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背景: 終末期腎病 (ESRD) 是一種慢性疾病，是一種日益嚴重的疾病，給世界老齡人口的醫療保健帶來負擔。由於免疫系統受損，ESRD 病人可能會容易被微生物感染。由於近年來，SARS-CoV-2 正在全球蔓延，並造成大量人數的死亡。疾病研究表明，一旦感染 SARS-CoV-2，ESRD 病人的預後會比一般人更差。此外，研究發現 ESRD 病人對目前可用的 COVID-19 疫苗反應不佳。在我們的研究中，我們的研究旨在研究 ChAdOx1 nCoV-19 (AZ) 疫苗接種後的免疫反應，並研究導致 ESRD 病人和健康受試者之間免疫反應差異的潛在機制。
實驗和方法: 在 AZ 疫苗接種前後的不同時間點收集 ESRD 病人和健康人的血液樣本，分離 PBMC 並保留血漿以備將來使用。在用 Toll 受體激動劑刺激後測定免疫細胞分泌的細胞因子。測定AZ 疫苗接種後病人和健康受試者中針對 SARS-CoV-2 突變株的血清總抗體濃度和中和抗體。然後，為了研究抗體濃度與基因表現間的關連性，對從選定的抗體濃度較高或較低的病人中篩選出的 RNA 定序進行分析。使用GSEA分析基因表達圖。
結果: 我們觀察到咪喹莫特有效地激活 ESRD 病人的先天免疫細胞。在第二劑 AZ 後的三天，健康受試者的 NK 細胞中的 IFN-γ 水平顯著升高。在第一劑 AZ 後的三天，單核細胞數量減少並且檢測到更高的 CD86 和 IL-6 水平。兩劑 AZ 疫苗接種後，大多數病人的總中和抗體濃度增加，但大多數患者在加強接種前總抗體濃度下降。在接受兩劑 AZ 疫苗的病人中，針對 omicron 突變株的中和抗體濃度極低，但在第三劑 mRNA 疫苗施打後增強。在第一劑 AZ 疫苗接種後的一個月，不良反應者與炎症反應相關的基因表達增強，包括 IFN-γ、IFN-α、補體、IL6-JAK-STAT3 信號通路。相比之下，良好的反應者組在同一時間點具有缺氧、MYC 和氧化磷酸化途徑的基因表現增加。在第二劑疫苗接種後的三天，不良反應者組的蛋白質分泌、PI3K/Akt/mTOR 訊息傳遞、和血紅素代謝的基因表現有增加的現象。相比之下，良好的反應者組在同一時間點上增加脂肪生成、MYC和氧化磷酸化的基因表達。
結論: 與健康人相比，ESRD病人的單核細胞表現出發炎的特徵。在ESRD病人中注意到 AZ 疫苗接種後 NK 細胞的免疫反應受損。總體而言，AZ 疫苗通過代謝重編程觸發訓練有素的免疫力，從而改變免疫細胞的功能。

Background End-stage renal disease (ESRD) is one of the chronic diseases and presents an ever-growing disease which burdens the health care of aging population in the world. ESRD patients are likely to acquire infections due to impaired immune system. In recent years, SARS-CoV-2 virus is spreading globally and has caused a substantial mortality. Disease studies have shown that ESRD patients have a worse prognosis once they acquire SARS-CoV-2 infection. In addition, studies have discovered ESRD patients have a suboptimal response to currently available COVID-19 vaccines. In our research, our study aims to study the immune responses after ChAdOx1 nCoV-19 (AZ) vaccination and study the underneath mechanisms responsible for the differences of immune response between ESRD patients and healthy subjects.
Study subjects and methods We collected blood samples from ESRD patients and healthy subjects at different time points before and after AZ vaccinations and segregated PBMC and kept plasm for future use. Cytokines secreted from immune cells were determined after being stimulated with Toll-receptor agonists. Serum total antibody titers and neutralizing antibodies against SARS-CoV-2 variants in patients and healthy subjects after AZ vaccination were determined. And then, to investigate correlation between antibody titers and gene expression, RNA-sequencing isolated from the selected patients with higher or lower antibody titers was conducted. Gene expression profiles were analyzed using GSEA.
Results We observed that imiquimod activated innate immune cells effectively in ESRD patients. IFN-γ levels were significantly higher in NK cells from healthy subjects three days after the 2nd AZ. Monocyte numbers were decreased and higher CD86 and IL-6 levels were detected three days after 1st AZ. After two doses of AZ vaccination, most patients increased total antibody titers, but most patients declined total antibody titers before booster vaccination. The neutralizing titers against omicron variant in patients receiving two doses of AZ vaccines were extremely low but boosted after the 3rd dose with mRNA vaccine. At one month after the 1st dose of AZ vaccine, the poor responders had enhanced relevant gene expressions to inflammatory response, including IFN-γ, IFN-α, complement, IL6-JAK-STAT3 signaling pathways. In contrast, good responders tended to have enhanced gene expression for hypoxia, MYC and oxidative phosphorylation at the same time point. At three days after the 2nd vaccine, poor responders had an increase of gene expressions for protein secretion, PI3K/Akt/mTOR signaling, UV response and heme metabolism. In contrast, good responders had an increase of gene expressions for adipogenesis, MYC and oxidative phosphorylation at the same point.
Conclusion Monocytes in patients show inflammatory features when compared with their healthy subjects. Impaired immune responses in NK cells after AZ vaccination were noted in patients. Overall, AZ vaccines change the function of immune cells by triggering trained immunity through metabolic reprogramming.

Abstract I
中文摘要 III
致謝 V
Abbreviations VI
Chapter 1 Introduction 1
1.1 COVID-19 vaccine 2
 1.1.1 The ChAdOx1 nCoV-19 vaccine (AZD1222) 2
 1.1.2 mRNA vaccine 3
 1.1.3 MVC-COV1901 (Medigen Vaccine Biologics Corporation) 3
1.2 End-stage renal disease (ESRD) 4
1.3 Toll-like receptor (TLR) agonists 5
Chapter 2. Materials and methods 7
2.1 Volunteer vaccination study design 8
2.2 Blood collection and processing 8
2.3 Intracellular cytokines analysis with flow cytometry 9
2.4 SARS-CoV-2 neutralizing antibody assay 10
2.5 Anti-SARS-CoV-2 total antibody (Roche Elecsys) 10
2.6 RNA sequencing 11
2.7 Gene Set Enrichment Analysis (GSEA) and Cytoscape 11
2.8 Statistical analysis 11
Chapter 3. Results 13
3.1 Imiquimod promote regulation of CD56bright NK cells in ESRD patients 14
3.2 Unstimulated monocytes in ESRD patients have chronic inflammation characteristics when compared with healthy subjects 15
3.3 TLR-induced B cell subsets had lower cell number and highly active properties in ESRD patients 17
3.4 ESRD patients had lower IFN-γ levels in NK cells 3 days after the 2nd ChAdOx1 nCoV-19 (AZ1222) vaccine 18
3.5 Monocytes of ESRD patients had highly activated and pro-inflammatory properties 20
3.6 Number of B cells and IFN-α expression levels in ESRD patients were different with those in healthy subjects 21
3.7 Antibody titers in ESRD patients were increased after complete   vaccination 21
3.8 High and low antibody production after the 1st and 2nd doses AZ vaccines in ESRD patients are associated with different cellular metabolism	22
Chapter 4. Discussion 24
4.1 ChAdOx1 nCoV-19 (AZD1222) vaccine could not induce sufficient immune cell recruitment and cytokine secretion in ESRD patients 25
4.2 ESRD patients had lower seroconversion rate which showed lessened antibody titers and decreased inhibitive ability of variant of concern 26
4.3 The inflammatory response decreases vaccination-induced antibody responses 27
4.4 ESRD patients with higher or lower antibody titers had different metabolism	29
    Hypoxia 30
    mTOR 30
    MYC	31
Glycolysis and oxidative phosphorylation (OXPHOS) 31
Chapter 5. Reference 32
Chapter 6. Figures and Table 36
Figure 1. Experiment design of the study and samples collection of ESRD patients and healthy subjects. 37
Figure 2. TLR agonists altered NK cell subsets in healthy subjects and ESRD patients. 39
Figure 3. TLR agonists altered monocytes in healthy subjects and ESRD patients.	41
Figure 4. TLR agonists altered B cell subsets in healthy subjects and ESRD patients. 43
Figure 5. ChAdOx1 nCoV-19 vaccination altered NK cell subsets, in   healthy subjects and ESRD patients after 3 days of 1st and 2nd dose vaccination. 45
Figure 6. ChAdOx1 nCoV-19 vaccination altered monocytes in healthy      subjects and ESRD patients after 3 days of 1st and 2nd dose vaccination. 47
Figure 7. ChAdOx1 nCoV-19 vaccination altered B cell subsets in healthy subjects and ESRD patients after 3 days of 1st and 2nd dose vaccination. 49
Figure 8. Antibody titers production from healthy subjects and ESRD patients before and after booster vaccination 50
Figure 9. ESRD patients with high or low antibodies responses after 1 and 2 doses vaccination had different metabolism phenotypes. 52
Table 1. Demographic information and baseline hemogram in ESRD patients and healthy people of the study. 53
                                    

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校內：2027-09-15公開
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