新冠肺炎的的大流行與快速傳播在世界各地造成大量生命的損失。為了有效解決，多種疫苗快速地被研發並施打，而隨著疫苗的涵蓋率逐漸普及，仍持續出現突破性感染，因此疫苗接種者是否真的具備足夠保護力就成了更重要的課題。本研究中首先專注於抗體生成相關的分析。在第一部分裡，我們收集施打兩劑AZ疫苗的血液樣本，透過酵素結合免疫吸附分析法 (ELISA) 來檢測抗體含量，另外使用側向流體免疫分析法 (LFIA) 來提供更快速簡單檢測抗體含量的方法。在另一方面，我們也檢測了孕婦施打疫苗後的中和抗體是否會經由胎盤轉移給胎兒，還有其中和抗體是否能用以對抗Delta變異株，以及在孕期同時施打其它疫苗是否會影響中和抗體的生成效率，以此做為孕婦施打COVID-19疫苗相關方針的參考依據。而除了抗體檢測以外，本研究也希望能找到特定生物標記，用以代表或預測疫苗接種者的免疫反應。在這裡以血液中IFN-γ做為檢測標的，將其與抗體生成進行相關性分析。在後半部分的研究中，更加入了細胞免疫的分析，使用QuantiFERON SARS-CoV-2套組，檢測以抗原刺激T細胞後所釋放之IFN-γ含量，作為判斷細胞免疫反應高低的依據。綜合上述，本研究的目的便是透過對疫苗接種者後天免疫反應完整的分析來評估疫苗效力，促進疫苗的改良及優化，最終達到有效控制疫情的效果。

COVID-19 has caused significant loss of life around the world. To solve this problem, a variety of vaccines have been rapidly developed and administered. As the coverage of vaccines gradually popularized, breakthrough infections continue to occur. Therefore, whether the vaccine recipients obtain sufficient protection has become an even more important issue. In this study, we first focus on antibody production related analysis. In the first part, blood samples were collected after two doses of AZ vaccines. The antibody level was mainly detected by enzyme-linked immunosorbent assay (ELISA), and the lateral flow immunoassay (LFIA) was used to provide a more rapid and simple method. On the other hand, we tested whether the neutralizing antibody of pregnant women would be transferred to their fetus. Also, we would like to know if the neutralizing antibodies they produced can fight against SARS-CoV-2 Delta variant, and whether having other vaccines during pregnancy would influence the effectiveness of neutralizing antibody production. These results can be utilized as the reference of pregnant woman having COVID-19 vaccines. In addition to antibody detection, we also tried to find specific biomarkers that can represent or predict the immune response of vaccine recipients. Here, IFN-γ in blood is used as the detection target, and the correlation between it and antibody production is analyzed. In the subsequent experiments, cellular immunity was analyzed through QuantiFERON SARS-CoV-2 by measuring the antigen-induced-IFN-γ released. Based on the above, the purpose of this study is to evaluate vaccine efficacy through a complete analysis of the adaptive immune response of vaccine recipients, further promote vaccine improvement and optimization, and ultimately achieve the effect of effectively controlling the pandemic.

摘要--------------------I
ABSTRACT--------------------II
謝辭--------------------III
目錄--------------------IV
圖目錄--------------------VI
表目錄--------------------VIII
第一章 緒論--------------------.1
1-1研究背景--------------------1
1-1-1新型冠狀病毒肺炎 (COVID-19) --------------------1
1-1-2 新冠病毒疫苗--------------------3
1-1-3 Heterologous prime-boost疫苗策略--------------------4
1-1-4 體液免疫--------------------4
1-1-5 細胞免疫--------------------10
1-2 研究動機與目的--------------------11
第二章 研究方法--------------------14
2-1 實驗試劑及器材--------------------14
2-2 實驗方法--------------------15
2-2-1 酵素結合免疫吸附分析法 (Enzyme-linked immunosorbent assay,　ELISA) --------------------15
2-2-2 無病毒中和性抗體檢測--------------------16
2-2-3 QuantiFERON SARS-CoV-2--------------------18
2-2-4 統計方法--------------------21
第三章 中和抗體檢測之實驗結果與討論--------------------22
3-1 側向流體免疫分析法搭配以光譜為基礎的測讀儀用於新冠病毒中和抗體快速檢測--------------------22
3-1-1 前言--------------------22
3-1-2 受試者檢體--------------------24
3-1-3 結果--------------------24
3-1-4 結論--------------------34
3-2 孕婦接種新冠疫苗後抗體經胎盤轉移之評估--------------------35
3-2-1 前言--------------------35
3-2-2 受試者檢體--------------------36
3-2-3 結果--------------------36
3-2-4 結論--------------------40
3-3 疫苗接種者細胞激素 (IFN-γ) 與抗體生成之相關性--------------------42
3-3-1 前言--------------------42
3-3-2 受試者檢體--------------------42
3-3-3 結果--------------------43
3-3-4 結論--------------------47
第四章 細胞免疫之實驗結果與討論--------------------48
4-1 前言--------------------48
4-2 受試者檢體--------------------49
4-3 結果--------------------49
4-4 結論--------------------56
第五章 結論與未來展望--------------------58
5-1 結論--------------------58
5-2 未來展望--------------------61
參考文獻--------------------63
著作目錄--------------------68

1 Zhu, N. et al. A novel coronavirus from patients with pneumonia in China, 2019. New England journal of medicine (2020).
2 The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nature microbiology 5, 536-544 (2020).
3 Sun, P., Lu, X., Xu, C., Sun, W. & Pan, B. Understanding of COVID‐19 based on current evidence. Journal of medical virology 92, 548-551 (2020).
4 Jiang, S., Hillyer, C. & Du, L. Neutralizing antibodies against SARS-CoV-2 and other human coronaviruses. Trends in immunology 41, 355-359 (2020).
5 Cuffari, B. What are Spike Proteins?, (2021).
6 Worldometer. COVID Live - Coronavirus Statistics (2022).
7 Organization, W. H. Tracking SARS-CoV-2 variants, (2022).
8 ECDC. SARS-CoV-2 variants of concern as of 21 December 2022, (2022).
9 Li, Y. et al. A comprehensive review of the global efforts on COVID-19 vaccine development. ACS Central Science 7, 512-533 (2021).
10 Wu, S. C. Progress and concept for COVID‐19 vaccine development. Biotechnology journal (2020).
11 Kaur, S. P. & Gupta, V. COVID-19 Vaccine: A comprehensive status report. Virus research 288, 198114 (2020).
12 Strizova, Z., Smetanova, J., Bartunkova, J. & Milota, T. Principles and challenges in anti-COVID-19 vaccine development. International Archives of Allergy and Immunology 182, 339-349 (2021).
13 Krammer, F. SARS-CoV-2 vaccines in development. Nature 586, 516-527 (2020).
14 Amanat, F. & Krammer, F. SARS-CoV-2 vaccines: status report. Immunity 52, 583-589 (2020).
15 Sui, Y., Bekele, Y. & Berzofsky, J. A. Potential SARS-CoV-2 immune correlates of protection in infection and vaccine immunization. Pathogens 10, 138 (2021).
16 Doria-Rose, N. et al. Antibody persistence through 6 months after the second dose of mRNA-1273 vaccine for Covid-19. New England Journal of Medicine 384, 2259-2261 (2021).
17 Levin, E. G. et al. Waning immune humoral response to BNT162b2 Covid-19 vaccine over 6 months. New England Journal of Medicine 385, e84 (2021).
18 Andrews, N. et al. Duration of protection against mild and severe disease by Covid-19 vaccines. New England Journal of Medicine 386, 340-350 (2022).
19 Lu, S. Heterologous prime–boost vaccination. Current opinion in immunology 21, 346-351 (2009).
20 Sapkota, B. et al. Heterologous prime–boost strategies for COVID-19 vaccines. Journal of Travel Medicine 29, taab191 (2022).
21 Suntronwong, N. et al. Effects of boosted mRNA and adenoviral‐vectored vaccines on immune responses to omicron BA. 1 and BA. 2 following the heterologous CoronaVac/AZD1222 vaccination. Journal of Medical Virology 94, 5713-5722 (2022).
22 Seidel, A. et al. BNT162b2 booster after heterologous prime-boost vaccination induces potent neutralizing antibodies and T cell reactivity against SARS-CoV-2 Omicron BA. 1 in young adults. Frontiers in immunology 13 (2022).
23 Tenbusch, M. et al. Heterologous prime–boost vaccination with ChAdOx1 nCoV-19 and BNT162b2. The Lancet Infectious Diseases 21, 1212-1213 (2021).
24 Li, J. et al. Heterologous AD5-nCOV plus CoronaVac versus homologous CoronaVac vaccination: a randomized phase 4 trial. Nature medicine 28, 401-409 (2022).
25 Assawakosri, S. et al. Persistence of immunity against Omicron BA. 1 and BA. 2 variants following homologous and heterologous COVID-19 booster vaccines in healthy adults after a two-dose AZD1222 vaccination. International Journal of Infectious Diseases 122, 793-801 (2022).
26 Britannica, T. E. o. E. antibody, (2021).
27 Montesinos, I. et al. Evaluation of two automated and three rapid lateral flow immunoassays for the detection of anti-SARS-CoV-2 antibodies. Journal of Clinical Virology 128, 104413 (2020).
28 Furman, D. et al. Apoptosis and other immune biomarkers predict influenza vaccine responsiveness. Molecular systems biology 9, 659 (2013).
29 Agha, M., Blake, M., Chilleo, C., Wells, A. & Haidar, G. Suboptimal response to COVID-19 mRNA vaccines in hematologic malignancies patients. MedRxiv (2021).
30 Morawska, M. Reasons and consequences of COVID‐19 vaccine failure in patients with chronic lymphocytic leukemia. European Journal of Haematology 108, 91-98 (2022).
31 Lipsitch, M., Krammer, F., Regev-Yochay, G., Lustig, Y. & Balicer, R. D. SARS-CoV-2 breakthrough infections in vaccinated individuals: measurement, causes and impact. Nature Reviews Immunology 22, 57-65 (2022).
32 Tamariz-Amador, L.-E. et al. Immune biomarkers to predict SARS-CoV-2 vaccine effectiveness in patients with hematological malignancies. Blood cancer journal 11, 1-13 (2021).
33 Ewer, K. J. et al. T cell and antibody responses induced by a single dose of ChAdOx1 nCoV-19 (AZD1222) vaccine in a phase 1/2 clinical trial. Nature medicine 27, 270-278 (2021).
34 Altmann, D. M. & Boyton, R. J. SARS-CoV-2 T cell immunity: Specificity, function, durability, and role in protection. Science immunology 5, eabd6160 (2020).
35 Sahin, U. et al. COVID-19 vaccine BNT162b1 elicits human antibody and TH1 T cell responses. Nature 586, 594-599 (2020).
36 Kalimuddin, S. et al. Early T cell and binding antibody responses are associated with COVID-19 RNA vaccine efficacy onset. Med 2, 682-688. e684 (2021).
37 Le Bert, N. et al. SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls. Nature 584, 457-462 (2020).
38 Immunotec, O. T-SPOT.COVID Package Insert.
39 Jaganathan, S. et al. Preliminary evaluation of QuantiFERON SARS-CoV-2 and QIAreach anti-SARS-CoV-2 total test in recently vaccinated individuals. Infectious diseases and therapy 10, 2765-2776 (2021).
40 Krüttgen, A. et al. Evaluation of the QuantiFERON SARS-CoV-2 interferon-ɣ release assay in mRNA-1273 vaccinated health care workers. Journal of virological methods 298, 114295 (2021).
41 科技部之科技發展觀測平台 - 焦點主題：COVID-19預防、診斷及治療產品現況與對體外診斷市場之影響. (2020).
42 Genscript. cPass SARS-CoV-2 Neutralization Antibody Detection Kit Instructions for use. (2022).
43 QIAGEN. QuantiFERON® SARS-CoV-2 Extended set blood collection tubes instructions for use (Handbook). (2021).
44 QIAGEN. QuantiFERON® Control set blood collection tubes instructions for use (Handbook). (2021).
45 QIAGEN. QuantiFERON® SARS-CoV-2 Starter set blood collection tubes instructions for use (Handbook). (2021).
46 QIAGEN. QuantiFERON® ELISA instructions for use (Handbook). (2021).
47 Jung, B. K. et al. Performance Evaluation of the BZ COVID-19 Neutralizing Antibody Test for the Culture-Free and Rapid Detection of SARS-CoV-2 Neutralizing Antibodies. Diagnostics 11, 2193 (2021).
48 Huang, R.-L. et al. A Lateral Flow Immunoassay Coupled with a Spectrum-Based Reader for SARS-CoV-2 Neutralizing Antibody Detection. Vaccines 10, 271 (2022).
49 Ketas, T. J. et al. Antibody responses to SARS-CoV-2 mRNA vaccines are detectable in saliva. Pathogens and Immunity 6, 116 (2021).
50 Munoz, F. M. & Englund, J. A. A step ahead: infant protection through maternal immunization. Pediatric Clinics of North America 47, 449-463 (2000).
51 Kachikis, A. & Englund, J. A. Maternal immunization: Optimizing protection for the mother and infant. Journal of Infection 72, S83-S90 (2016).
52 Saso, A. & Kampmann, B. in Seminars in immunopathology. 627-642 (Springer).
53 Swamy, G. K. & Garcia-Putnam, R. Maternal immunization to benefit the mother, fetus, and infant. Obstetrics and Gynecology Clinics 41, 521-534 (2014).
54 Fouda, G. G., Martinez, D. R., Swamy, G. K. & Permar, S. R. The Impact of IgG transplacental transfer on early life immunity. Immunohorizons 2, 14-25 (2018).
55 Faucette, A. N., Unger, B. L., Gonik, B. & Chen, K. Maternal vaccination: moving the science forward. Human reproduction update 21, 119-135 (2015).
56 Zeng, H. et al. Antibodies in infants born to mothers with COVID-19 pneumonia. Jama 323, 1848-1849 (2020).
57 Flannery, D. D. et al. Assessment of maternal and neonatal cord blood SARS-CoV-2 antibodies and placental transfer ratios. JAMA pediatrics 175, 594-600 (2021).
58 Juncker, H. G. et al. Antibodies against SARS-CoV-2 in human milk: Milk conversion rates in the Netherlands. Journal of Human Lactation 37, 469-476 (2021).
59 Zdanowski, W. & Waśniewski, T. Evaluation of SARS-CoV-2 spike protein antibody titers in cord blood after COVID-19 vaccination during pregnancy in Polish healthcare workers: preliminary results. Vaccines 9, 675 (2021).
60 Gray, K. J. et al. Coronavirus disease 2019 vaccine response in pregnant and lactating women: a cohort study. American journal of obstetrics and gynecology 225, 303. e301-303. e317 (2021).
61 Shen, C.-J. et al. Evaluation of Transplacental Antibody Transfer in SARS-CoV-2-Immunized Pregnant Women. Vaccines (Basel) 10, 101 (2022).
62 AdipoGen. Manual of SARS-CoV-2 Neutralizing Antibodies Detection Kit (B.1.617.2 Variant, Delta). (2021).
63 Planas, D. et al. Reduced sensitivity of SARS-CoV-2 variant Delta to antibody neutralization. Nature 596, 276-280 (2021).
64 De Rosa, S. C. et al. Whole‐blood cytokine secretion assay as a high‐throughput alternative for assessing the cell‐mediated immunity profile after two doses of an adjuvanted SARS‐CoV‐2 recombinant protein vaccine candidate. Clinical & translational immunology 11, e1360 (2022).
65 Van Praet, J. T., Vandecasteele, S., De Roo, A., De Vriese, A. S. & Reynders, M. Humoral and cellular immunogenicity of the BNT162b2 messenger RNA coronavirus disease 2019 vaccine in nursing home residents. Clinical Infectious Diseases 73, 2145-2147 (2021).
66 Chalkias, S. et al. A bivalent omicron-containing booster vaccine against Covid-19. New England Journal of Medicine 387, 1279-1291 (2022).
67 Fu, Y.-C., Su, Y.-S., Shen, C.-F. & Cheng, C.-M. Vol. 12 1401 (Multidisciplinary Digital Publishing Institute, 2022).