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研究生：	林鈺珊 Lin, Yu-Shan
論文名稱：	發展可行性的口服疫苗預防困難梭狀芽孢桿菌的感染 Development of an orally available vaccine for the prevention of Clostridium difficile infections
指導教授：	黃一修 Huang, I-Hsiu
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：	2018
畢業學年度：	106
語文別：	英文
論文頁數：	108
中文關鍵詞：	困難梭狀芽孢桿菌、毒素B 、多肽聚合物、疫苗
外文關鍵詞：	Clostridium difficile, toxin B, polypeptide-based polymer, vaccine
相關次數：	點閱：41 下載：0
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困難梭狀芽孢桿菌是一個革蘭氏陽性會形成孢子的厭氧菌，並在全世界造成抗生素相關腹瀉。一旦感染困難梭狀芽孢桿菌其症狀包括腹瀉、偽膜性腸炎和毒性巨腸症。感染的風險包括抗生素的使用和長期的住院治療。最常透過口服萬古黴素、硝基甲嘧唑乙醇和非達黴素這些抗生素來治療困難梭狀芽孢桿菌的感染。然而，如此的治療會破壞腸道正常菌叢的定殖。再者，大約25-30%的病患會有復發現象的產生。因此發展一個有效的疫苗來預防困難梭狀芽孢桿菌的感染是需要的。困難梭狀芽孢桿菌主要的毒力因子為兩個糖基化毒素，毒素A和毒素B，因此這兩者常作為理想的目標來設計疫苗。在本篇研究中，我們建構了重組蛋白毒素B（rTcdB）包含毒素B的受體結合區域1852-2363殘基位置作為抗原。這個重組蛋白抗原會分別被由聚賴氨酸接枝吲哚和聚賴胺酸嵌段聚蘇胺酸所形成的具有生物降解性和pH值敏感性的多肽聚合物包裹進去。我們假設多肽聚合物可以作為一個佐劑和抗原傳遞系統來誘發粘膜免疫反應。透過使用老鼠模式，我們將會評估這些多肽聚合物疫苗在口服給予後所誘發的專一性抗體反應如何，還有是否能夠有效對抗困難梭狀芽孢桿菌的感染。從本篇結果指出口服含有重組毒素B的多肽聚合物疫苗，能夠誘發抗毒素B的專一性免疫球蛋白G和免疫球蛋白A抗體並能夠有效對抗困難梭狀芽孢桿菌的感染。

Clostridium difficile is a gram-positive, spore-forming anaerobic bacterium and is the leading cause of antibiotic associated diarrhea worldwide (AAD). Once infected with C. difficile, symptoms can include diarrhea, pseudomembranous colitis and toxic megacolon. Risk factors of infection include antibiotic usage, frequent hospitalization, aging, and proton pump inhibitor usage. The most common treatment for CDI involves oral antibiotic therapy using vancomycin, metronidazole, or fidaxomicin with success rate range from 64 to 82%. However, it is expensive and may disrupt intestinal microbiota. Approximately 25–30% of CDI patients will experience recurrence. Development of an effective vaccine for CDI is therefore desirable and necessary to prevent infection. The major virulence factor of C. difficile are the two glucosylating toxins, toxin A and B and are therefore ideal targets for vaccine development. In this study, we constructed a recombinant protein, rTcdB, consisted of residues 1852-2363 of the toxin B receptor binding domain as an antigen. The rTcdB antigen was encased in polypeptide-based polymer made up of poly(L-lysine)20-graft-Indo0.3 and poly(L-lysine)20-block-poly(L-threonine)40, which are biodegradable and pH-sensitive. We hypothesized that the polypeptide-based polymer could serve as an adjuvant and antigen delivery system to induce mucosal immunity. Using a mouse model, we evaluated the potential of the polymer- based vaccine in inducing antigen specific antibody responses after oral injection and protection against C. difficile infection. Our results indicated that oral vaccination with rTcdB polymer could induce specific anti-rTcdB IgG and IgA responses and conferred protection against C. difficile infection.

Contents
中文摘要	I
Abstract	II
誌謝	IV
Abbreviations	XII
Chapter 1 Introduction	1
1.1 Characteristics of Clostridium difficile	1
1.2 Epidemiology of Clostridium difficile infection	1
1.3 Clinical manifestations	2
1.3.1 Asymptomatic carriers	2
1.3.2 Watery diarrhea	3
1.3.3 Pseudomembranous colitis	3
1.3.4 Fulminant colitis	3
1.4 C. difficile toxins	4
1.5 C. difficile vaccine development	5
1.6 Mucosal immunity in vaccine development	6
1.7 Major challenge on mucosal vaccine development	7
1.8 Natural polypeptide-based polymers in vaccine delivery	8
1.9 Specific aim	9
Chapter 2 Materials and Methods	10
2.1 Materials	10
2.1.1 Bacterial strains	10
DH5α	10
ClearColi BL21 (DE3) electrocompetent cell	10
R20291	11
VPI10463	11
BI-1	11
NCKUH K14	11
NCKUH A6	12
Netherlands	12
2.1.2 Plasmid	12
pET-21b (+)	12
2.1.3 Animal	12
C57BL/6 JNarl mice	12
2.1.4 Cell line	13
Vero cell	13
2.1.5 Chemicals and other material	13
Chemicals and enzymes	13
2.2 Methods	16
2.2.1 Bacterial strains, plasmid and growth conditions	16
2.2.2 Electroporation of pET21b-tcdB plasmid into ClearColi	16
2.2.3 Purification of recombinant TcdB protein in ClearColi	17
2.2.4 Size-exclusion chromatography	18
2.2.5 Synthesis of poly(L-lysine)-block-poly(L-threonine) polymers	18
2.2.6 Preparation of genipin cross-linked gel particles	19
2.2.7 Encapsulation of antigen in the gel particles	19
2.2.8 Characterization of genipin cross-linked gel particles	20
2.2.9 Stability of PLL20-b-PLT40 polymer at distinct pH environments	20
2.2.10 Mice immunization and sample collection	21
2.2.11 Detection of vaccine-induced specific IgG and IgA by enzyme linked immunosorbent assays (ELISA)	21
2.2.12 Preparation of C. difficile spores	22
2.2.13 C. difficile challenge and sample collection	23
2.2.14 Cytokine and chemokine measurement	24
2.2.15 RNA extraction for mice colon	25
2.2.16 Real-time quantitative reverse transcription-polymerase chain reaction (q-RT-PCR) for mice colon analysis	26
2.2.17 Fecal cytotoxicity and GAL cytotoxicity in infected mice	27
2.2.18 Cell culture	28
2.2.19 Cytotoxicity of different kinds of polymers	28
2.2.20 Cytotoxicity of polymers to mice	29
2.2.21 Toxin B neutralizing antibody assay	29
2.2.22 Toxin B neutralizing assay in different C. difficile strains	30
2.2.23 Cytokine ELISPOT	30
2.2.24 Statistics	31
Chapter 3 Results	32
3.1. Recombinant protein purification and preparation of rTcdB-encapsulated polymers	32
3.2 Induction of antigen-specific IgG and IgA antibodies in poly(L-lysine)20-graft-Indo0.3 and poly(L-lysine)20-block-poly(L-threonine)40 polymers	33
3.3 Induction of antigen-specific IgG and IgA antibodies in modified poly(L-lysine)20-block-poly(L-threonine)40 polymer	34
3.4 Cytotoxicity of different kinds of polymers in vitro and in vivo	34
3.5 Characterization of PLL20-b-PLT40 polymer at distinct pH environments	35
3.6 Vaccine-derived neutralizing antibodies against toxin B	36
3.7 Protective efficacy of 25μg rTcdB-encapsulated PLL20-b-PLT40 polymer in mice model	36
3.8 The long-term immune response provided by 25μg rTcdB-encapsulated PLL20-b-PLT40 polymer	37
3.9 Protective efficacy of 25μg rTcdB-encapsulated PLL20-b-PLT40 polymer in long-term mice model	38
3.10 Comparation of different rTcdB concentration (25μg and 50μg) of PLL20-b-PLT40 polymer induced rTcdB-specific IgG and IgA titers	39
3.11 Protective efficacy of 50 μg rTcdB-encapsulated PLL20-b-PLT40 polymer in mice model	39
3.12 Induction of local antibodies responses in the infected mice	41
3.11 Expression level of inflammatory cytokines and chemokines in the infected mice	42
3.14 Fecal cytotoxicity and GAL cytotoxicity from the infected mice	43
3.15 Cellular immune effects of the PLL20-b-PLT40 (50 μg rTcdB) polymer in mice	43
3.16 The long-term immune response provided by 50μg rTcdB-encapsulated PLL20-b-PLT40 polymer	45
3.17 Protective efficacy of 50μg rTcdB-encapsulated PLL20-b-PLT40 polymer in long-term mice model	45
3.18 Toxin B neutralization activity in different C. difficile strains	46
Chapter 4 Discussion	48
Chapter 5 Conclusion	55
References	56
Tables	62
Figures	65
Supplementary data	103
Supplementary Material	104


                                    

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