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Feasibility of Probiotic Lactobacillus and Yeast As Oral Vaccine Carrier Against Coronaviruses Ho Phui San Department of Microbi

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Feasibility of Probiotic Lactobacillus and Yeast As Oral Vaccine Carrier Against Coronaviruses Ho Phui San Department of Microbi FEASIBILITY OF PROBIOTIC LACTOBACILLUS AND YEAST AS ORAL VACCINE CARRIER AGAINST CORONAVIRUSES HO PHUI SAN DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2005 FEASIBILITY OF PROBIOTIC LACTOBACILLUS AND YEAST AS ORAL VACCINE CARRIER AGAINST CORONAVIRUSES HO PHUI SAN (B. Sc. (Hons), NUS) A THESIS SUBMITTED FOR THE DEGREE OF PHILOSOPHY OF DOCTORATE DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2005 LIST OF PUBLICATIONS LIST OF PUBLICATIONS International peer review publications 1) Lee Y.K., Ho P.S., Low C.S., Arvilommi H. and Salminen S. (2004). Permanent colonization by Lactobacillus casei is hindered by the low rate of cell division in mouse gut. Appl Environ Microbiol. 70(2), 670-674. 2) Ho P.S., Kwang J. and Lee Y.K. (2005). Intragastric administration of Lactobacillus casei expressing transmissible gastroentritis coronavirus spike glycoprotein induced specific antibody production. Vaccine. 23(11), 1335-1342. 3) Lee Y.K., Hao W.L., Ho P.S., Nordling M.M., Low C.S., de Kok T.M. and Rafter J. (2005). Human fecal water modifies adhesion of intestinal bacteria to Caco-2 cells. Nutr Cancer. 52(1), 35-42. 4) Ho P.S. and Lee Y.K. Analysis of the ability of Saccharomyces boulardii, Saccharomyces cerevisiae and Pichia pastoris to adhere to intestinal cell line and murine gastrointestinal tract. (In preparation). 5) Ho P.S. and Lee Y.K. Development of a novel oral vaccine against severe acute respiratory syndrome coronavirus using yeast as the delivery vehicle. (In preparation). Conference publications 1) Ho P.S. and Lee Y.K. (2003). Daily consumption of Lactobacillus: Is it necessary? 7th NUS-NUH Annual Scientific Meeting, Singapore. 2) Ho P.S., Lee Y.K. and Kwang J. (2003). In vivo expression and immunogenicity of coronavirus spike protein by Lactobacillus in murine model. 2nd Asian Conference on Lactic Acid Bacteria (ACLAB), Taiwan. (Selected for Oral presentation). 3) Ho P.S., Lee Y.K. and Kwang J. (2003). Lactobacillus as oral vaccine carrier against Coronavirus. The 6th Asia Pacific Congress on Medical Virology (ASCMV), Malaysia. (Selected for Oral presentation). 4) Ho P.S., Lee Y.K. and Kwang J. (2004). Recombinant probiotic bacteria elicited systemic and local immune responses against coronavirus. 5th Combined Annual Scientific Meeting (CASM), Singapore. I LIST OF PUBLICATIONS 5) Ho P.S., Kwang J. and Lee Y.K. (2004). The Potential of Lactobacillus and yeast as an oral vaccine delivery vector against coronaviruses. 8th NUS-NUH Annual Scientific Meeting, Singapore. (Awarded Best Basic Science Poster Award). 6) Ho P.S., Kwang J. and Lee Y.K. (2005). Lactobacillus and yeast for vaccine delivery against coronaviruses. Joint meeting of the 3 Divisions of the International Union of Microbiological Societies (IUMS) 2005, Unites States of America. (Selected for Oral presentation). 7) Ho P.S., Lee Y.K. (2005). Feasibility of developing Saccharomyces spp. and Pichia spp. as vaccine delivery vehicle against coronavirus. Combined Scientific Meeting (CSM) 2005, Singapore. II ACKNOWLEDGEMENT ACKNOWLEDGEMENT I would like to express my utmost gratitude and appreciation to the following people who has made this project possible. Associate Professor Lee Yuan Kun for his continued patience and unfailing support throughout my course of study. It was a great pleasure for me to conduct this thesis under his supervision. His selfless help and invaluable advice and guidance will always be appreciated. Professor Jimmy Kwang for his guidance and for giving me the opportunity to work in his laboratory in Temasek Life Sciences Laboratory during the first year of my course. Mr Liu Wei who was formally with Temasek Life Sciences Laboratory, for imparting his invaluable knowledge and expertise on molecular techniques to me. Mr Low Chin Seng for all his technical help and advices, as well as for the laughters we have shared in the laboratory. Josephine Howe and the staff from Electron Microscopy Unit for their technical assistance. The staff from flow-cytometry laboratory from Clinical Research Center, especially Genie, who has already left, for their assistance. Mdm Chew Lai Meng for all the encouragements and the motherly advices. The postgraduates in our laboratory, Chow Wai Ling, Won Choong Yun, Lee Hui Cheng, Wang Shugui and not forgetting Janice Yong Jing Ying, who has already graduated, for their precious help and friendship along the way. Research life is definitely more meaningful with your companionships. The present and past honours students for their friendship and joy they have brought during the stay. My buddies outside NUS for their understanding, support and concern throughout this period. My family especially my husband for their patience and encouragement throughout these years. Many things have happened and your love and support have helped me to pull through this physically and emotionally draining period. III TABLE OF CONTENTS TABLE OF CONTENTS PAGE NO. CHAPTER 1 1 1.0 LITERATURE REVIEW 1 1.1 PROBIOTICS 1 1.1.1 Beneficial effects 2 1.1.2 Detrimental Effects of Probiotics 4 1.1.3 Methods to Analyze Adhesion of Intestinal Microorganism 5 1.2 LACTOBACILLUS 6 1.3 SACCHAROMYCES CEREVISIAE 8 1.3.1 Protein Expression in Saccharomyces Cerevisiae 9 1.4 SACCHAROMYCES BOULARDII 10 1.5 PICHIA PASTORIS 11 1.6 ADHESION 13 1.7 VACCINE 15 1.7.1 Types of Vaccines 15 1.7.1.1 Inactivated Vaccine 16 1.7.1.2 Live Attenuated Vaccine 16 1.7.1.3 Toxoid 18 1.7.1.4 Conjugated Vaccine 19 1.7.1.5 Subunit Vaccine 20 1.7.1.6 DNA Vaccine 21 1.7.2 Vaccine Delivery 22 1.7.2.1 Recombinant Vector Vaccines 22 1.7.2.1.1 Viral Vectors 23 1.7.2.1.2 Virus-like particles 24 1.7.2.1.3 Bacterial Vector 25 1.7.2.2 Transgenic Plant 26 1.7.2.3 Microencapsulation 27 IV TABLE OF CONTENTS 1.7.2.3.1 Liposome 27 1.7.2.3.2 Virosomes 28 1.7.2.3.3 Microspheres 29 1.7.3 Route of Vaccination 29 1.7.4 Immunity 30 1.7.4.1 Mucosal Immunity 32 1.7.4.1.1 Secretory IgA 34 1.7.4.2 Cytokines 35 1.7.4.2.1 Control of Virus Infections 37 1.8 CORONAVIRUSES 38 1.8.1 SEVERE ACUTE RESPIRATORY 41 SYNDROME CORONAVIRUS 1.8.1.1 SARS-CoV Spike Protein 43 1.8.1.2 Immunogenicity 44 1.8.2 TRANSMISSIBLE GASTROENTERITIS CORONAVIRUS 45 1.8.2.1 TGEV Spike Protein 47 1.8.2.2 Vaccines Developed Against TGEV 48 1.9 OBJECTIVES 49 CHAPTER 2 51 2.0 MATERIALS AND METHODS 51 2.1 ADHESION STUDIES 51 2.1.1 Bacteria and Yeast Strains 51 2.1.2 Adhesion Studies in Vivo 51 2.1.2.1 Preparation of Fluorogenic dye 51 2.1.2.2 Labeling of Lactobacillus spp. and Yeast With 52 Fluorescent Probe 2.1.2.3 Feeding of Mice With Labeled LcS or Yeast 53 2.1.2.4 Flow cytometric analysis 54 2.1.2.5 Mucus extraction from fecal material 54 V TABLE OF CONTENTS 2.1.3 Adhesion Studies In Vitro 55 2.2 MOLECULAR CLONING TECHNIQUES 55 2.2.1 Bacterial and Yeast Strains 55 2.2.2 Cloning and Expression Vectors 57 2.2.3 Restriction Digestion of DNA 59 2.2.4 Ligation and Transformation 59 2.2.5 Agarose Gel Electrophoresis 60 2.2.6 DNA Purification from Agarose Gel 61 2.2.7 Plasmid Isolation and Purification 61 2.3 GENERATION OF RECOMBINANT LCS EXPRESSING 62 TGEV SPIKE PROTEIN FRAGMENT 2.3.1 Complementary DNA (cDNA) Synthesis of TGEV gene 62 2.3.2 Amplification of TGEV Spike Gene 62 2.3.2.1 Primers Sequences and Related Information 62 2.3.2.2 Polymerase Chain Reaction (PCR) 63 2.3.3 Construction of Recombinant pLP500 Harboring TGEV 66 Spike Gene Fragment 2.3.3.1 Subcloning of rTGEV-S into pCR®-XL-TOPO® Vector 67 2.3.3.2 Cloning of rTGEV-S into pLP500 67 2.3.4 Preparation of Competent E.coli Cells for Chemical 67 Transformation 2.3.5 Generation of Recombinant L.casei Shirota Expressing rTGEV-S 68 2.3.5.1 Transformation of pLP500/rTGEV-S into E.coli 68 2.3.5.2 Confirmation of Transformants 68 2.3.5.3 Preparation of Competent Cells for Electroporation 69 2.3.5.4 Electroporation 70 2.3.5.5 Analysis of the Transformants 70 2.3.6 Expression of rTGEV-S Protein 71 VI TABLE OF CONTENTS 2.4 GENERATION OF RECOMBINANT P.PASTORIS 72 EXPRESSING TGEV SPIKE PROTEIN FRAGMENT 2.4.1 Amplification of TGEV Spike Gene 72 2.4.1.1 Primers Sequences and Related Information 72 2.4.1.2 Polymerase Chain Reaction (PCR) 72 2.4.2 Cloning of TGEV Spike Gene Fragment into pGAPZαC 72 2.4.3 Transformation of pGAPZαC/PrTGEV-S into E.coli 73 2.4.4 Confirmation of Transformants 73 2.4.5 Generation of Recombinant P.pastoris Expressing rTGEV-S 74 2.4.5.1 Linearization of Recombinant Vector 74 2.4.5.2 Transformation of pGAPZαC/PrTGEV-S into P.pastoris) 75 2.4.5.2.1 Preparation of Competent P.pastoris 75 2.4.5.2.2 Transformation of competent P.pastoris 75 2.4.5.3 Analysis of the Transformants 76 2.4.5.3.1 Total DNA isolation form P.pastoris 76 2.4.5.3.2 PCR of Total DNA 76 2.4.6 Expression of PrTGEV-S Protein 77 2.5 GENERATION OF RECOMBINANT P.PASTORIS EXPRESSING 77 SARS CoV SPIKE PROTEIN FRAGMENT 2.5.1 Amplification of SARS CoV Spike Gene 77 2.5.1.1 Primers Sequences and Related Information 78 2.5.1.2 Polymerase Chain Reaction (PCR) 78 2.6 SODIUM-DODECYL SULFATE POLYACRYLAMIDE GEL 79 ELECTROPHORESIS (SDS-PAGE) 2.7 IMMUNOBLOTTING 80 2.7.1 Antibodies Used For Immunoblotting 81 2.8 IMMUNIZATION OF MICE 81 2.8.1 Collection of Intestinal Secretions 82 2.9 LARGE SCALE EXPRESSION OF TGEV SPIKE PROTEIN 83 2.9.1 Polymerase Chain Reaction (PCR) 83 2.9.2 Cloning of TGEV Spike Gene Fragment into pGEX-4T-3 84 VII TABLE OF CONTENTS 2.9.3 Induction of TGEV-S-GST 84 2.9.4 Purification of TGEV-S-GST 85 2.10 ELISA 85 2.11 CYTOKINE PROFILING 86 2.11.1 Preparation of Peyer’s Patches and Cervical Lymph Node for 86 In Vitro Re-stimulation 2.11.2 Cytokine Assay 87 2.12 CELL CULTURE TECHNIQUES
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