Development of the Probiotic Yeast Saccharomyces boulardii as an Oral Vaccine Delivery System 公开

Hudson, Lauren Elizabeth (2016)

Permanent URL: https://etd.library.emory.edu/concern/etds/kw52j826q?locale=zh
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Abstract

Beneficial microorganisms can prevent and ameliorate the severity of numerous gastrointestinal disorders. Potential mechanisms of action of these so-called probiotics may include effects on the composition of the microbiota, reinforcement of the gastrointestinal epithelial barrier, modulation of mucosal immune responses, and direct anti-pathogen effects. Numerous microorganisms are currently studied and consumed typically as individual strains, but the ability to rationally design combination microorganism therapies tailored to particular diseases holds promise for further optimization of this adjuvant therapy. Recent studies also suggest the potential to use particular probiotics to synthesize and deliver oral vaccines and therapeutics. The yeast Saccharomyces boulardii may be particularly well suited to this purpose given its current consumption as a beneficial microbe, its ability to perform eukaryotic post translational modifications, and its lack of gastrointestinal colonization. In order to develop S. boulardii for this novel application of probiotic organisms, it will first be necessary to understand its interactions not with the inflamed or infected intestine, but with the healthy gut. The extent of S. boulardii uptake and interaction with mucosal immune cells may differ greatly in the healthy versus the inflamed intestine, potentially impacting responses to recombinant vaccine antigens. Furthermore, although genetic manipulation techniques for the closely related S. cerevisiae are well characterized, S. boulardii strains that can be manipulated without antibiotic selection and that can successfully express heterologous protein must be developed in order to serve as safe and efficient vaccine delivery vectors. Here we evaluate the interactions of S. boulardii with the healthy adult mouse intestine to provide insight into how this probiotic yeast may function as a vaccine delivery vector or prophylactic agent. We also present the generation of an auxotrophic mutant strain of S. boulardii that can be easily genetically manipulated without antibiotic selection markers and that can express heterologous protein. In vivo experiments also test the ability of recombinant auxotrophic mutant S. boulardii to induce immune responses specific to model vaccine antigens. These experiments thus provide a basis for further development and testing of S. boulardii as a vaccine delivery system to the mouse gastrointestinal tract.

Table of Contents

1) Table of Contents

2) List of Figures........................................................................................................ 11

3) List of Tables......................................................................................................... 13

4) Introduction........................................................................................................... 14

a) Therapeutic Uses of Probiotics............................................................................ 15

b) C. difficile Infection................................................................................................ 18

1) Risk factors for developing CDI.................................................................................................................. 19

2) Treatment of CDI and Disease Recurrence.......................................................................................... 21

3) Fecal Microbiota Transplantation and CDI......................................................................................... 22

4) Clinical Trials Evaluating Probiotic Efficacy Against CDI........................................................... 23

5) Microbial Taxa Associated with Colonization Resistance Against CDI............................. 25

6) Mechanisms of Colonization Resistance Against CDI................................................................... 26

1. Nutrient availability and competition for resources.............................................................................. 27

2. Bile salt metabolism and colonization resistance.................................................................................... 28

3. Production of anti-C. difficile compounds................................................................................................... 30

7) Other Mechanisms of Action of Beneficial Microbes and Probiotics Against CDI...... 31

1. Inactivation of C. difficile toxins....................................................................................................................... 31

2. Antibody-mediated control of C. difficile bacteria................................................................................... 32

3. Inhibition of mucus layer disruption............................................................................................................. 32

4. Maintenance of the intestinal epithelial cell barrier and tight junction expression............. 34

8) Summary of Potential Mechanisms of Action of FMT Against CDI and Implications for Probiotics 36

c) Ulcerative colitis.................................................................................................... 38

1) Risk factors for developing UC.................................................................................................................... 39

2) Ulcerative Colitis Pathophysiology.......................................................................................................... 40

1. Intestinal permeability......................................................................................................................................... 40

2. The microbiota and dysbiosis............................................................................................................................ 41

3. Aberrant immune responses.............................................................................................................................. 43

4. TNF-α............................................................................................................................................................................. 44

5. Th2 cells....................................................................................................................................................................... 44

6. Th17 cells.................................................................................................................................................................... 45

7. Neutrophils................................................................................................................................................................ 45

3) Treatment of UC.................................................................................................................................................... 47

4) Ulcerative Colitis and Fecal Microbiota Transplant...................................................................... 48

5) Clinical Trials of Probiotics and UC.......................................................................................................... 49

6) Protective Mechanisms of Probiotics against Ulcerative Colitis.......................................... 49

1. Maintenance of the microbiota.......................................................................................................................... 50

2. Maintenance of intestinal epithelial integrity and barrier function.............................................. 51

3. Dampening inflammation through modulation of the cytokine milieu....................................... 52

4. Effects on neutrophil infiltration and function........................................................................................ 54

7) Summary of Probiotic Mechanisms of Action in UC and Implications for Future Therapies 55

d) Discussion of Therapeutic Uses of Probiotics..................................................... 57

e) Novel Applications of Probiotics........................................................................... 59

f) Phylogenetic Classification of S. boulardii............................................................. 61

g) S. boulardii Stress Resistance and Kinetics of Gastrointestinal Transit.............. 62

h) Interactions of S. boulardii with Host Immune Cells........................................... 64

1) S. boulardii and the Cytokine Milieu......................................................................................................... 64

2) S. boulardii-induced Antibody Production......................................................................................... 65

i) Genetic manipulation and transformation of S. boulardii................................... 66

j) Saccharomyces recombinant antigen expression............................................... 67

k) Saccharomyces experimental vaccines............................................................... 68

l) Oral Tolerance....................................................................................................... 69

m) Summary............................................................................................................. 70

n) Figures and Tables............................................................................................... 72

5) General Materials and Methods....................................................................... 100

a) UV mutagenesis to generate auxotrophic yeast strains................................... 105

b) UV mutagenesis and screening for auxotrophic yeast strains........................ 108

c) Yeast transformation......................................................................................... 111

d) Oral gavage of mice with transformed yeast.................................................... 116

e) Harvest of murine Peyer's patches and isolation of viable yeast colonies...... 118

f) Discussion............................................................................................................ 120

g) Figures and Tables............................................................................................. 124

6) Characterization of the Probiotic Yeast Saccharomyces boulardii in the Healthy Mucosal Immune System 135

a) Introduction....................................................................................................... 135

b) Materials and Methods...................................................................................... 138

1) Yeast Strains......................................................................................................................................................... 138

2) Yeast Genomic Sequencing and Analysis............................................................................................ 138

3) Yeast Cell Wall Analyses................................................................................................................................ 139

4) Animal studies..................................................................................................................................................... 139

5) Immunohistochemistry............................................................................................................................... 140

6) ELISA......................................................................................................................................................................... 140

7) Flow Cytometry................................................................................................................................................. 141

8) ELISPOT................................................................................................................................................................... 142

9) RNA-sequencing................................................................................................................................................. 143

c) Results................................................................................................................. 144

1) S. boulardii MYA-797 is genomically distinct from S. cerevisiae......................................... 144

2) The S. boulardii cell wall is thicker relative to S. cerevisiae strains and mediates stress resistance 145

3) Association and uptake of S. boulardii into small intestinal Peyer's patches are low frequency events 146

4) S. boulardii induces marginal increases in total, but not antigen specific, antibody levels 147

5) S. boulardii induces limited changes in numbers of germinal center B cells and plasma cells 148

6) S. boulardii induces trends toward increased numbers of antibody secreting cells 149

7) S. boulardii induces minimal changes in MLN gene expression........................................... 149

d) Discussion and Conclusions............................................................................... 150

e) Acknowledgements........................................................................................... 154

f) Figures and Tables.............................................................................................. 155

7) Functional Heterologous Protein Expression by Genetically Engineered Probiotic Yeast Saccharomyces boulardii 169

a) Introduction....................................................................................................... 169

b) Materials and Methods...................................................................................... 172

1) Screening of UV Irradiated Cells.............................................................................................................. 172

2) Confirmation of URA3 mutations........................................................................................................... 173

3) pH and Bile Acid Testing............................................................................................................................... 173

4) Anaerobic Testing............................................................................................................................................. 173

5) Yeast Transformation.................................................................................................................................... 174

6) Analysis of GFP Fluorescence................................................................................................................... 174

7) Isolation of Viable Yeast from Murine Peyer's Patches............................................................ 175

c) Results................................................................................................................. 175

1) Diploid S. boulardii Require High Doses of UV Irradiation to Achieve 50% Cell Survival 176

2) Isolation of Three S. boulardii Mutants Unable to Grow Without Uracil..................... 177

3) S. boulardii Mutants are Resistant to Low pH and Bile Acid In Vitro.............................. 179

4) S. boulardii Mutants Show Increased Growth in Anaerobic Conditions...................... 180

5) S. boulardii Mutants Can Be Transformed and Express Functional GFP....................... 180

6) Viable Transformed S. boulardii Mutant 2 Expressing GFP can be Isolated from Murine Peyer's Patches 181

d) Discussion and Conclusions............................................................................... 183

e) Acknowledgements........................................................................................... 186

f) Figures and Tables.............................................................................................. 187

8) Vaccine Delivery to the Murine Gastrointestinal Tract Using an Auxotrophic Mutant Strain of the Probiotic Yeast Saccharomyces boulardii........................................................................................... 199

a) Introduction....................................................................................................... 199

b) Materials and Methods...................................................................................... 200

1) Constructs and Cloning................................................................................................................................. 200

2) Yeast Strain and Transformation............................................................................................................ 201

3) Immunoblotting................................................................................................................................................. 201

4) Animal studies..................................................................................................................................................... 202

5) ELISA and ELISPOT.......................................................................................................................................... 203

c) Results................................................................................................................. 204

1) Ovalbumin and Fc constructs can be expressed by Saccharomyces................................ 204

2) S. boulardii mutant admixed with purified ovalbumin does not induce significantly increased antibody responses 204

3) S. boulardii mutant expressing the ovalbumin vaccine construct does not induce significantly increased antibody responses 205

4) Addition of the mucosal adjuvant dmLT has a minimal effect on antibody responses in combination with transformed S. boulardii mutant 206

5) Vaccination with M2 expressing a nucleocapsid protein (NP) fragment fails to protect mice from lethal influenza challenge 207

d) Discussion........................................................................................................... 208

e) Acknowledgements........................................................................................... 209

f) Figures and Tables.............................................................................................. 210

9) Discussion............................................................................................................ 224

a) M cell targeting and antigen secretion may aid delivery of heterologous vaccine antigens to intestinal Peyer's patches 225

b) Potential tolerogenic factors must be evaluated for optimization of an S. boulardii vaccine delivery vector 226

c) S. boulardii itself does not serve as a sufficient adjuvant................................. 227

d) Use of the Fc fusion system as a mucosal adjuvant in probiotic yeast requires further optimization 229

e) Co-Administration of Alternative Heterologous Adjuvants May Promote Induction of Antigen Specific Responses and Modulate T Helper Phenotypes.................................................................................................... 231

f) Summary............................................................................................................. 232

10) Appendix A: Abbreviations............................................................................. 237

11) Appendix B: Permissions............................................................................... 238

12) References......................................................................................................... 239

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