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 88 2.12.1 Cell Lines 88 2.12.2 Media for Cell Culture 89 2.12.3 Cultivation and Propagation of the Cell Lines 89 2.12.4 Cultivation of Cells in 6-Well, 24-Well and 96-Well Tissue 90 Culture Tray 2.12.5 Cultivation of Cells on Glass Coverslips 90 2.12.5.1 Pretreatment of Coverslips 90 2.12.5.2 Culture of Cells on Coverslips 90 2.12.6 Storage of Cells 91 2.13 INFECTION OF CELLS 92 2.13.1 Viruses 92 2.13.2 Infection of Cell Monolayers 92 2.13.4 Neutralization Assay 93 2.13.4.1 Plaque Neutralization Assay 93 2.13.4.2 Fluorescent Focus Assay 94 2.13.5 Extraction of Virus RNA Molecules 95 2.14 BIO-IMAGING VIA SCANNING ELECTRON MICROSCOPY (SEM) 95
VIII TABLE OF CONTENTS
CHAPTER 3 97 3.0 ADHESION STUDIES 97 3.1 INTRODUCTION 97 3.2 IN VITRO ADHESION STUDIES USING SCANNING 98 ELECTRON MICROSCOPY 3.3 IN VIVO ADHESION STUDIES 103 3.3.1 Murine Intestinal Surface Water and Mucus Content 103 3.3.2 Analysis of Lactobacillus casei Shirota Adhesion in Murine 103 Intestinal Tract 3.3.3 Analysis of Saccharomyces boulardii Adhesion in Murine 111 Intestinal Tract 3.3.4 Analysis of Saccharomyces cerevisiae Adhesion in Murine 118 Intestinal Tract 3.3.5 Analysis of Pichia pastoris Adhesion in Murine Intestinal Tract 125
CHAPTER 4 132 4.0 DEVELOPMENT OF ORAL VACCINE AGAINST 132 TRANSMISSIBLE GASRTOENTERITIS CORONAVIRUS 4.1 INTRODUCTION 132 4.2 GENERATION OF TGEV-S-GST 132 4.3 L.CASEI SHIROTA AS ORAL VACCINE CARRIER AGAINST 135 TGEV 4.3.1 Generation of Recombinant LcS Expressing rTGEV-S Protein 135 4.3.2 Immune responses induced by intragastric immunization of 141 LcS-rTGEV-S 4.3.3 Kinetics of cytokine production by Peyer’s patches from 149 mice orally immunized with LcS-rTGEV-S 4.4 P.PASTORIS AS ORAL VACCINE CARRIER AGAINST TGE 152 4.4.1 Generation of Recombinant P.pastoris (PP) Expressing 152 PrTGEV-S Protein
IX TABLE OF CONTENTS
4.4.2 Immune responses induced by intragastric immunization of 155 PP/PrTGEV-S 4.4.3 Kinetics of cytokine production by Peyer’s patches and cervical 162 lymph node from mice orally immunized with PP/PrTGEV-S
CHAPTER 5 165 5.0 DEVELOPMENT OF ORAL VACCINE AGAINST SEVERE ACUTE 165 RESPIRATORY SYNDROME CORONAVIRUS (SARS CoV) 5.1 INTRODUCTION 165 5.2 GENERATION OF RECOMBINANT P.PASTORIS EXPRESSING 165 SARS CoV SPIKE RECEPTOR-BINDING DOMAIN PROTEIN 5.3 IMMUNE RESPONSES INDUCED BY INTRAGASTRIC 171 IMMUNIZATION of PP/SARS-S-RBD 5.3.1 Kinetics of cytokine production by Peyer’s patches from mice 178 orally immunized with PP/SARS-S-RBD
CHAPTER 6 183 6.0 DISCUSSION 183 6.1 CONCLUSION 214 6.1.1 Adhesion Studies 214 6.1.2 Development of Oral Vaccine Against TGEV 215 6.1.3 Development of Oral Vaccine Against SARS CoV 216
REFERENCES 217
APPENDICES 287 APPENDIX 1 MATERIALS FOR BACTERIAL AND YEAST CULTURE 287
APPENDIX 2 MATERIALS FOR MOLECULAR CLONING 290
X TABLE OF CONTENTS
APPENDIX 3 MATERIALS FOR SODIUM DODECYL SULPHATE – 296 POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE)
APPENDIX 4 MATERIALS FOR IMMUNOBLOTTING 299 APPENDIX 5 MATERIALS FOR CELL CULTURE 300 APPENDIX 6 MATERIALS FOR VIRUS INFECTION, GROWTH OF 304 VIRUS AND PLAQUE ASSAY
APPENDIX 7 MATERIALS FOR BIO-IMAGING 306 APPENDIX 8 DATA FOR CHAPTER 3 309 APPENDIX 9 DATA FOR CHAPTER 4 329 APPENDIX 10 DATA FOR CHAPTER 5 345
XI LIST OF TABES
LIST OF TABLES
PAGE NO. Table 1.1 Comparison of current vaccination strategies with DNA 17 vaccination technology.
Table 1.2 Nature of immunologic reactivity after systemic or 31 mucosal immunization with conventional live or inactivated vaccines.
Table 1.3 Cytokines and biological activities. 35
Table 2.1 Bacterial and yeast strains used in adhesion study. 51
Table 2.2 Bacteria and yeast strains used in the study. 56
Table 2.3 Molecular vectors and related information. 57
Table 2.4 Primers used in this part of study. (Section 2.3.2.1) 63
Table 2.5 Primers used in this part of study. (Section 2.4.1.1) 72
Table 2.6 Primers used in this part of study. (Section 2.5.1.1) 78
Table 2.7 Primary and secondary antibodies used for 81 immunoblotting.
Table 2.8 Primers used in this part of study. (Section 2.9.1) 83
Table 2.9 Cell lines and related information. 92
Table 2.10 Viruses and related information. 92
Table 3.1 Fluorescence intensity profiles of lactobacilli harvested 109 from various sections of the intestinal mucosal surface on various days after orogastric intubation of LcS.
Table 3.2 Fluorescence intensity profiles of S.boulardii harvested 116 from various sections of the intestinal mucosal surface on various days post orogastric intubation.
XII LIST OF TABES
Table 3.3 Fluorescence intensity profiles of S.cerevisiae harvested 123 from various sections of the intestinal mucosal surface on various days post orogastric intubation.
Table 3.4 Fluorescence intensity profiles of P.pastoris harvested 130 from various sections of the intestinal mucosal surface on various days post orogastric intubation.
XIII LIST OF FIGURES
LIST OF FIGURES
PAGE NO. Figure 1.1 Current mucosal delivery systems, mucosal inductive sites 32 and the concept of Th1- and Th2-type immune responses.
Figure 1.2 Genome organization of SARS coronavirus. 39
Figure 1.3 Arrangement of the structural proteins and viral RNA 40 within an infectious virus particle of the coronavirus.
Figure 2.1 Porcine transmissible gastroenteritis virus spike protein S 66 mRNA sequences (GenBank accession number AF302263) downloaded from NCBI website (http//:www.ncbi.nlm.nih.gov).
Figure 2.2 Insertion of plasmid 5’ to the intact GAP promoter locus. 74
Figure 3.1 Observation by scanning electron microscopy of the 100 adherence of S.boulardii, S.cerevisiae and P.pastoris to human intestinal epithelial Caco-2 cells.
Figure 3.2 Adhesion of S.boulardii, S.cerevisiae and P.pastoris to 101 human mucus-secreting HT29 cells observed by scanning electron microscopy.
Figure 3.3 P.pastoris whole cells interacted with the mucus secreted 102 by the HT29 cells.
Figure 3.4 cFDA-SE labelling of LcS. 104
Figure 3.5 Plot of the residual median fluorescence intensity of LcS 105 adhering on the various sections of the intestinal tract against the generation number.
Figure 3.6 Plot of total LcS cell number adhered on various sections 106 of the intestinal tract against time after orogastric intubation.
Figure 3.7 Plot of the residual median fluorescence intensity of LcS 107 adhered on various sections of the intestinal tract against time after orogastric intubation.
XIV LIST OF FIGURES
Figure 3.8 Plots showing the division profiles of total population of 110 adhering LcS.
Figure 3.9 cFDA-SE labelling of S.boulardii. 111
Figure 3.10 Plot of the residual median fluorescent intensity of 112 S.boulardii adhered on the various sections of the intestinal tract against the generation number.
Figure 3.11 Plot of total S.boulardii cell number adhered on various 113 sections of the intestinal tract against time after orogastric intubation.
Figure 3.12 Plot of residual median fluorescence intensity of 114 S.boulardii adhered on various sections of the intestinal tract against time after orogastric intubation.
Figure 3.13 Plots showing the division profiles of total population of 117 adhering S.boulardii.
Figure 3.14 cFDA-SE labelling of S.cerevisiae. 118
Figure 3.15 Plot of the residual median fluorescent intensity of 119 S.cerevisiae adhered on the various sections of the intestinal tract against the generation number.
Figure 3.16 Plot of total S.cerevisiae cell number adhered on various 120 sections of the intestinal tract against time after orogastric intubation.
Figure 3.17 Plot of residual median fluorescence intensity of 121 S.cerevisiae adhered on various sections of the intestinal tract against time after orogastric intubation.
Figure 3.18 Plots showing the division profiles of total population of 124 adhering S.cerevisiae.
Figure 3.19 cFDA-SE labelling of P.pastoris. 125
Figure 3.20 Plot of the residual median fluorescent intensity of 126 P.pastoris adhered on the various sections of the intestinal tract against the generation number.
XV LIST OF FIGURES
Figure 3.21 Plot of total P.pastoris cell number adhered on various 127 sections of the intestinal tract against time after orogastric intubation.
Figure 3.22 Plot residual median fluorescence intensity of P.pastoris 128 adhered on various sections of the intestinal tract against time after orogastric intubation.
Figure 3.23 Plots showing the division profiles of total population of 131 adhering P.pastoris.
Figure 4.1 Agarose gel electrophoresis of PCR amplified TGEV 134 spike gene fragment to be ligated to the 3’ end of GST.
Figure 4.2 Restriction digested plasmids extracted from E.coli 134 transformants electrophorized on agarose gel.
Figure 4.3 Expression and purification of TGEV-S-GST protein by 135 recombinant E.coli after IPTG induction.
Figure 4.4 Agarose gel electrophoresis of PCR products of rTGEV- 136 S, amplified from cDNA synthesized from viral genomic RNA.
Figure 4.5 Schematic diagram of the construction of recombinant 137 Lactobacillus spp. expression vector harboring rTGEV-S gene fragment.
Figure 4.6 Electrophoretogram of BamHI and NheI digested pCR®- 138 XL-TOPO® vector in which rTGEV-S have been subcloned.
Figure 4.7 Electrophoretogram of BamHI and NheI digested 139 plasmids isolated from E.coli DH10β transformants.
Figure 4.8 PCR of recombinant pLP500/rTGEV-S extracted from 140 LcS transformants.
Figure 4.9 Expression of rTGEV-S protein from LcS-rTGEV-S. 141
Figure 4.10 Schematic diagram showing the oral vaccination regime 142 of recombinant LcS to BALB/c mice.
Figure 4.11 Production of rTGEV-S protein specific intestinal IgA and 143 serum IgG in mice fed with LcS-rTGEV-S.
XVI LIST OF FIGURES
Figure 4.12 rTGEV-S protein specific local IgA responses in murine 144 intestinal lavage after intragastric immunization.
Figure 4.13 Anti-rTGEV-S serum IgG titers induced after intragastric 148 immunization with recombinant LcS.
Figure 4.14 Inhibition of viral plaque formation by (A) intestine 148 lavages and (B) sera prepared from mice fed with recombinant LcS.
Figure 4.15 IL-2, IFN-γ, IL-4 and IL-5 production by Peyer’s patches 150 cells from mice immunized orally with LcS-rTGEV-S or LcS after re-stimulated with concanavalin A.
Figure 4.16 Kinetics of Th1 (IL-2 and IFN-γ) and Th2 (IL-4 and IL-5) 151 cytokines production by Peyer’s patches cells from mice immunized with LcS-rTGEV-S and LcS.
Figure 4.17 Agarose gel electrophoresis of PCR amplified PrTGEV-S 152 (2.3 kb).
Figure 4.18 PCR screening of plasmids isolated from transformants. 153 (Section 4.4.1)
Figure 4.19 Electrophoretogram of PCR products amplified from 154 genomic DNA isolated from P.pastoris transformants. (Section 4.4.1)
Figure 4.20 Expression of PrTGEV-S protein from P.pastoris 155 transformants.
Figure 4.21 Schematic diagram showing the oral vaccination regime 156 of PP/PrTGEV-S to BALB/c mice.
Figure 4.22 Immunogenicity of induced intestinal IgA and serum IgG 157 specific for PrTGEV-S protein.
Figure 4.23 PrTGEV-S protein specific local IgA response in murine 158 intestinal lavage after intragastric immunization.
Figure 4.24 Humoral immune responses after intragastric 160 immunization with recombinant P.pastoris (PP/PrTGEV- S).
XVII LIST OF FIGURES
Figure 4.25 Inhibition of viral plaque formation by (A) intestine 161 lavages and (B) sera prepared from mice orally fed with PP/PrTGEV-S and P.pastoris.
Figure 4.26 IL-2, IFN-γ, IL-4 and IL-5 production by Peyer’s patches 163 cells from mice immunized orally with PP/PrTGEV-S or P.pastoris after re-stimulated with concanavalin A.
Figure 4.27 Kinetics of Th1 (IL-2 and IFN-γ) and Th2 (IL-4 and IL-5) 164 cytokine production by Peyer’s patches cells from mice immunized with PP/PrTGEV-S and P.pastoris.
Figure 5.1 Agarose gel electrophoresis of PCR products of SARS-S- 167 RBD, amplified from pCR-XL-TOPO-S.
Figure 5.2 PCR screening of plasmids isolated from transformants. 167 (Section 5.2)
Figure 5.3 Electrophoretogram of PCR products amplified from 168 genomic DNA isolated from P.pastoris transformants. (Section 5.2)
Figure 5.4 Agarose gel electrophoresis for the comparison of the 169 amount of total DNA extracted from four P.pastoris transformants.
Figure 5.5 Expression of SARS-S-RBD protein from P.pastoris 170 transformants.
Figure 5.6 Schematic diagram showing the oral vaccination regime 171 of PP/SARS-S-RBD to BALB/c mice.
Figure 5.7 Immunogenicity of induced intestinal IgA and serum IgG 172 specific for SARS-S-RBD protein.
Figure 5.8 SARS-S-RBD protein specific local IgA response in 173 murine intestinal lavage after intragastric immunization.
Figure 5.9 Anti-SARS-S-RBD serum IgG titers induced after 176 intragastric immunization with PP/SARS-S-RBD.
Figure 5.10 Inhibition of MLV(SARS) infection by (A) intestine 177 lavages and (B) sera prepared from mice fed with PP/SARS-S-RBD.
XVIII LIST OF FIGURES
Figure 5.11 IL-2, IFN-γ, IL-4 and IL-5 production by cells from 180 Peyer’s patches (A) and CLN (B) of mice immunized orally with PP/SARS-S-RBD or P.pastoris after re- stimulated with concanavalin A.
Figure 5.12 Kinetics of Th1 (IL-2 and IFN-γ) and Th2 (IL-4 and IL-5) 181 cytokine production by Peyer’s patches cells from mice immunized with PP/SARS-S-RBD and P.pastoris.
Figure 5.13 Kinetics of Th1 (IL-2 and IFN-γ) and Th2 (IL-4 and IL-5) 182 cytokine production by CLN cells from mice immunized with PP/SARS-S-RBD and P.pastoris.
XIX LIST OF FIGURES
ABBREVIATIONS
Ω - ohm % - percentage °C - degree celsius μF - capacitance μg - microgram μl - microlitre Ampr - Ampicillin resistance BSA - bovine serum albumin bp - base pair cDNA - complementary deoxyribonucleic acid CD - Cluster determinant cm - centimetre cm2 - square centimetre Con A - concanavalin A CO2 - carbon dioxide dgd - double distilled water DNA - deoxyribonucleic acid dNTP - deoxynucleotide triphosphate E.coli - Escherichia coli EDTA - ethylenediamine tetraacetic acid ELISA - enzyme-linked immunosorbent assay Emr - Erythromycin resistance EtBr - ethidium bromide eGFP - Green fluorescence protein FCS - foetal calf serum FFU - fluorescence-forming unit g - gram(s) GST - glutathione-S-transferase hr - hour(s) Ig - immunoglobulin IFN-γ - interferon-γ IL-2 - interleukin-2 IL-4 - interleukin-4 IL-5 - interleukin-5 Kanr - Kanamycin resistance kb - kilobases kDa - kilodalton kV - kilovolts LcS - Lactobacillus casei Shirota M - molar M.O.I. - multipicity of infection mA - milliampere
XX LIST OF FIGURES
min - minute(s) mg - milligram ml - millilitres mm - millimetre mM - millimolar M - molar Mr - molecular weight ng - nanogram ORF - open reading frame pg - picogram PAGE - polyacrylamide gel electrophoresis PBS - phosphate buffer saline PCR - polymerase chain reaction PFU - plaque forming unit P.pastoris - Pichia pastoris RBD - receptor binding domain RNA - ribonucleic acid rpm - revolutions per minute s - second SARS CoV - severe acute respiratory syndrome coronavirus virus S.boulardii - Saccharomyces boulardii S.cerevisiae - Saccharomyces cerevisiae TGEV - transmissible gastroenteritis coronavirus UV - ultra violet v/v - volume by volume x g - centrifugal force Zeor - Zeocin resistance
XXI SUMMARY
SUMMARY
Increased awareness of the fact that most infectious agents use mucosal membranes as portals of entry has led to efforts to develop vaccines and antigen delivery systems that can efficiently induce mucosal immunity. Mucosal immunization offers many benefits, including reduced vaccine-associated side effects and the potential to overcome the known barriers of parenteral vaccination which includes preexisting systemic immunity from previous vaccination, or, in young animals, preexisting systemic immunity from maternal antibodies (Liljeqvist & Stahl, 1999). Live vaccine vehicles offer a powerful approach for inducing protective immunity against pathogenic microorganisms, where genetically engineered agents provide a method for delivering heterologous antigens derived from other pathogens. In this study, the potential of utilizing Lactobacillus spp. and yeast as oral vaccine delivery vehicle against coronaviruses was investigated
In the first part of the study, the adhesion and colonization capacities of L.casei
Shirota (LcS), S.boulardii, S.cerevisiae and P.pastoris on mucosal surfaces were determined. In vitro interactions between the yeasts and human intestinal cells, Caco-2 and HT29 were observed under scanning electron microscope, where P.pastoris demonstrated a stronger affinity to the intestinal cells than S.boulardii and S.cerevisiae.
The in vivo adhesion abilities of LcS and the three yeasts were then determined in various segments of the gastrointestinal tract of mice fed with fluorescently labeled LcS or yeast.
Adhesion of LcS and all three yeasts to murine intestinal tract, as determined by flow cytometry analysis of the intestinal samples, were observed. The half times for wash-out
XXII SUMMARY and the doubling times of the studied microorganisms were deduced from the data obtained. Among the three yeasts, S.boulardii was found to have a higher adhesiveness as the half time for wash-out was the longest. S.cerevisiae and P.pastoris detached from the upper segments of the intestine were capable of readhering to the lower segments of the intestinal tract. A large part of LcS, S.boulardii, S.cerevisiae and P.pastoris fed were able to replicate in the intestinal environment, though at a slower rate in comparison to the growth rates achievable in laboratory conditions. The results obtained in this part of the study were very encouraging especially when LcS was able to adhere and exist in the intestinal tract for a reasonable period of time, while P.pastoris was observed to possess higher capability to readhere and replicate in various segments of murine intestinal tract in comparison to the other two yeasts.
Since LcS and P.pastoris have proven their potential to be developed as vehicles for oral delivery of coronavirus antigens in the adhesion studies, recombinant LcS and
P.pastoris which constitutively express and secrete an N-terminal antigenic fragment of
Transmissible Gastroenteritis coronavirus (TGEV) spike protein were constructed.
Western blot analysis of the expressed protein, performed using convalescence swine serum against TGEV, demonstrated the immunogenicity of the expressed proteins.
However, the expression of the TGEV spike protein fragment by LcS was less efficient than expression by recombinant P.pastoris. Nevertheless, oral immunization of Balb/c mice with recombinant LcS or P.pastoris elicited specific local and systemic immunological responses, characterized by significant production of antigen specific intestinal IgA and serum IgG. Isotyping of the IgG subclass revealed that most of the
IgG responses induced by recombinant LcS and P.pastoris were of IgG2a isotype. In
XXIII SUMMARY
agreement to the higher levels of IgG2a in the sera of orally immunized mice, a T-helper
1 (Th1) biased cellular response was observed in the Peyer’s patches of mice fed with recombinant LcS or P.pastoris. However, in contrast to the potent neutralization activities of the antibodies generated by recombinant P.pastoris, antibodies induced by recombinant LcS demonstrated low neutralization abilities. The poor neutralization capacity of the antibodies elicited by the recombinant LcS was attributed to the lack of post-translational modifications of the delivered TGEV spike protein fragment in the prokaryotic expression system. Hence, results are in favor of P.pastoris as an oral vaccine carrier for the delivery of coronavirus antigen.
In the final part of the study, attempts were made to develop P.pastoris as an oral vaccine delivery vehicle against Severe Acute Respiratory Syndrome coronavirus (SARS
CoV). Recombinant P.pastoris capable of constitutive and extracellular expression of a fragment of SARS CoV spike glycoprotein, consisting of the receptor-binding domain
(RBD) of the virus, was engineered. Immunogenicity of the expressed SARS CoV spike protein fragment was confirmed with the aid of SARS-positive human sera in Western blotting. The recombinant SARS CoV vaccine induced in Balb/c mice high titers of systemic and mucosal neutralizing antibodies (intestinal IgA and serum IgG) as well as potent cell-mediated immune responses, by intragastric administration. In accordance to a higher level of IgG1 being produced, a T-helper 2 (Th2) dominated cellular response was observed in the Peyer’s patches and cervical lymph nodes of immunized animals.
Results obtained in this part of the study indicate the use of P.pastoris as vehicle for the oral delivery coronavirus antigens to immunize animals is a promising approach.
XXIV CHAPTER 1 LITERATURE REVIEW
1.0 LITERATURE REVIEW
1.1 PROBIOTICS
The term probiotic, as an antonym to the term antibiotic, was originally proposed by Lilley and Stillwell in 1965 to be used as substances that favor the growth of microorganisms (Lilley & Stillwell, 1965). More than two decades later, Fuller (1989) broadly defined probiotics as “live microbial feed supplement that beneficially affects the host animal by improving its intestinal microbial balance”. By the turn of the century, probiotics are commonly defined as viable microorganisms (bacteria or yeasts) that exhibit beneficial effects on the health of the host when ingested (Salminen et al., 1998a).
Microorganisms that are probiotic in humans include yeast (Guslandi et al., 2000), bacilli
(Pinchuk et al., 2001), Escherichia coli (Katz & Fiocchi, 2001), enterococci (Lund and
Edlund, 2001) and the more commonly used bifidobacteria and lactic acid bacteria such as lactobacilli, lactococci and streptococci (Isolauri et al., 2002; Madsen, 2001).
Probiotics have been used for many years in the animal feed industry, but they are now being increasingly made available in fermented dairy products, and can be purchased over the counter as freeze-dried preparations in health food stores. The US
Food and Drug Administration classifies some species of lactic acid bacteria that are found in both fermented food and in the gastrointestinal tract as GRAS (generally recognized as safe) organisms for human use (Teitelbaum & Walker, 2002). Today, probiotics are not only widely used in foods especially in the preparation of fermented dairy products, but also in pharmaceutical preparations.
1 CHAPTER 1 LITERATURE REVIEW
1.1.1 Beneficial effects
Several healths related effects associated with the intake of probiotics have been reported in human studies. Probiotics have been used therapeutically to modulate immunity where consumption of Lactobacillus acidophilus and Bifidobacterium bifidum significantly enhances the non-specific immune phagocytic activity of circulating blood granulocytes (Schiffrin et al., 1995). Several species of lactobacilli, killed by irradiation, were found to stimulate differentially dendritic cell activity with respect to interleukin-12 and tumour necrosis factor-α production (Christensen et al., 2002). Reports have also shown that infants suffering from rotavirus-induced diarrhea supplemented with a strain of Lactobacillus casei have enhanced concentration of circulating immunoglobulin A.
This correlates with a shortened duration of diarrhea, giving an implication that probiotics might be effective in alleviating the effects of the infection (Kaila, 1992).
Furthermore, it has been consistently reported that individual consuming probiotics fermented products had shortened episodes or reduced risk of the disease occurrence
(Salminen et al., 1996). Production of antimicrobials effective against intestinal pathogens and blocking the way from enteroinvasive microbes have also been suggested as possible causes of diarrhea prevention and cure (Salminen et al., 1998b). Besides infection-associated diarrhea, probiotics have also been demonstrated in humans to reduce the effects of non-infection associated diarrhea as a result of lactose intolerance by improving lactose digestion as well as by slowing orocecal transit (Sanders, 1993 &
Montes et al., 1995). A recent metaanalysis of nine double blind placebo controlled
2 CHAPTER 1 LITERATURE REVIEW studies of the use of probiotic yeast and lactobacilli to prevent antibiotic associated diarrhea showed consistent benefit (D’Souza et al., 2002).
Patients with chronic kidney failure usually have bacterial overgrowth in the small intestine, resulting in high levels dimethylamine and nitrosodimethylamine in the blood that can cause hepatotoxicity. Probiotic treatment of patients seemed to be effective as these toxic compounds were significantly lower in patients treated with
Lactobacillus acidophilus, resulting in better quality of life for these patients (Morishita et al., 1997).
The anti-tumor effects of probiotics have also been documented. The possible anti-carcinogenic effects in humans are very difficult to detect. However, positive effects on superficial bladder cancer by Lactobacillus casei Shirota have been reported (Aso et al., 1995). Reports have also indicated that consumption of probiotics could reduce levels of free amines and fecal microbial enzymes, such as β-glucoronidase, nitroreductase and urease, involved in the metabolic activation of miscellaneous mutagens and carcinogens, thereby reducing the risk of cancer development (Goldin &
Gorbach, 1984). In particular, the observations that Lactobacillus acidophilus consumption resulted in lower amounts of extractable fecal or urinary mutagens from human volunteers may also indicate a long-term anti-carcinogen effect (Lidbeck et al.,
1991).
Although the causes of inflammatory bowel disease including ulcerative colitis and Crohn's disease remain incompletely understood, increasing evidence implicates intestinal microflora in the pathogenesis of this disorder. Studies with a strain of
Lactobacillus plantarum showed the administration of the probiotic to specific pathogen
3 CHAPTER 1 LITERATURE REVIEW
free animals attenuated established inflammatory processed (Schultz et al., 2002).
Patients who were in remission of Crohn’s disease who were treated with probiotic yeast
Saccharomyces boulardii were found to have a significantly reduced chance of relapse
(Guslandi et al., 2000). There are increasing reports indicating that several probiotic strains are able to inhibit the attachment of pathogenic bacteria to intestinal epithelial cells through their ability to increase the production of intestinal mucins. Lactobacillus plantarum 299v and Lactobacillus rhamnosus GG were reported to inhibit the adherence of enteropathogenic Escherichia coli to HT29 and Caco-2 cells by elevating the expression of mucins (MUC2 and MUC3) mRNA, and subsequent production of mucins
(Mack et al., 1999; Mattar et al., 2002). When purified MUC2 and MUC3 mucins were added to cells, adherence of pathogenic Escherichia coli was inhibited. Therefore, modulation of microflora with probiotics may offer a plausible therapeutic approach
(Schultz et al., 2003).
1.1.2 Detrimental Effects of Probiotics
Probiotic agents are increasingly used for the treatment and prevention of a variety of infectious and inflammatory conditions. They are generally safe, but complications of probiotic use can occur. Though infections associated with probiotic strains of lactobacilli are extremely rare, invasive disease can be associated with probiotic lactobacilli. Cases of bacteremia and sepsis associated with ingestion of a Lactobacillus
spp. have been reported in 2 patients and a child with short gut syndrome (De Groote et
al., 2005).
4 CHAPTER 1 LITERATURE REVIEW
Saccharomyces cerevisiae is a well-known yeast used in the food industry and has
been documented to cause different forms of invasive infection (Cassone et al., 2003).
However, the most important clinical syndrome caused by Saccharomyces cerevisiae is
fungemia, because it is usually the most severe and well-proven clinical manifestation of
the disease. Saccharomyces cerevisiae fungemia has been described not only in
immunosuppressed patients and critically ill patients, but also in relatively healthy subjects (Herbrecht & Nivoix, 2005).
1.1.3 Methods to Analyze Adhesion of Intestinal Microorganism
Besides adhesion studies carried out in vitro, colonization studies in animals have also been carried out as in vitro studies of adhesive properties of probiotic strains might not be truly reflective of the interaction in vivo. In vitro adhering Lactobacillus casei
rhamnosus GG and Lactobacillus johnsonii La 1 were found to adhere to various
segments of the intestine when orally fed to C3H/He/Oujco gnotobiotic mice (Hudault et
al., 1997; Bernet-Camard et al., 1997). Some Bifidobacterium strains were also capable
of colonizing to the gastrointestinal tract in vivo (Crociani et al., 1995). Oral
administration of in vitro adhering Bifidobacterium infantis strain 1 and Bifidobacterium
spp. CA1 and F9 strains established high levels of bacteria in the mucosa and intestinal
contents of the gastrointestinal tract (Lievin et al., 2000).
Fecal samples have also been used in colonization studies with probiotic bacteria.
These, however, reflect only the bacteriologic situation in the fecal material and might
not give an accurate picture of the various portions of the gut. Colonic biopsies,
combined with molecular biological techniques offers a more accurate means of
5 CHAPTER 1 LITERATURE REVIEW
determining colonization (Johansson et al., 1993; Alander et al., 1997). In particular,
Alander and coworkers (1999) have shown that Lactobacillus rhamnosus GG can persist
in colonic mucosa for several days before subsequently being discharged in the fecal
samples.
1.2 LACTOBACILLUS
The genus Lactobacillus comprises a remarkably diverse and heterogenous group
of Gram-positive bacilli that are ubiquitous as normal indigenous flora of humans and
other animals. In addition to their role as members of the indigenous microbiota,
lactobacilli can also be found naturally in fermented food and have been commonly used
in the production of fermented products (Sharpe, 1981). Lactobacillus strains have a
number of properties that make them attractive candidates as delivery vehicles for the
presentation of compounds of pharmaceutical interest (vaccines and immunomodulators)
to the mucosal surfaces. Besides being considered GRAS organisms, certain strains of
Lactobacillus are able to colonize the gut and are believed to show health promoting
activities (Havenaar & Huis in’t Veld, 1993; Pouwels et al., 1998). However, it is
important to note that different strains of the same species are often different in their
abilities to colonize different habitats. For instances, Lactobacillus acidophilus strains
may be members of the human gastrointestinal microflora and colonize specific sites
within the human intestine, whereas Lactobacillus acidophilus strains used in food
production rarely interact with the intestinal mucosa (Clements et al., 1983; Marteau et al., 1993; Salminen et al., 1996).
6 CHAPTER 1 LITERATURE REVIEW
Interest in the expression of heterologous genes in Lactobacillus has increased in
the recent years, as techniques for genetic manipulation of strains have been developed.
Generally, heterologous proteins can be expressed in Lactobacillus strains either using expression vectors or via chromosomal integration of expression cassettes. Currently, most of the efforts have been focused on the development of expression system that utilizes expression vectors. Most vectors can be amplified in Escherichia coli and
Lactobacillus spp. and comprise a broad host range replicon from Lactobacillus pentosus.
An antibiotic resistance marker is also included to permit their replication in a wide variety of lactic acid in bacterial strains (Posno et al., 1991). In addition, the heterologous protein can be chosen to be expressed intracellularly and be transported over the membrane and anchored to the cell wall, or to be secreted into the culture medium, under the control of regulatable or constitutive promoter from Lactobacillus.
For instance, expression of cloned genes in the vectors can be driven by highly efficient, constitutive promoter of the Lactobacillus casei L-(+)-lactate dehydrogenase gene or regulatable promoter of the Lactobacillus amylovorous α-amylase gene (Boot et al.,
1996). Most of the Lactobacillus strains can now be transformed by electroporation,
including members of L.acidophilus A1 group which were previously refractory to
transformation techniques (Walker et al., 1996).
In the recent years, there has been great interest in the development of
Lactobacillus strains as vaccine vehicles. This has lead to the construction of
recombinant strains expressing vaccine antigens (Mercenier et al., 1996; Reveneau et al.
2002). The immunogencity of the expressed recombinant antigens have been examined.
Gerritse et al. demonstrated that mice orally fed with trinitrophenylated-Lactobacillus
7 CHAPTER 1 LITERATURE REVIEW
strains resulted in an induction of specific mucosal immunoglobulin A (Gerritse et al.,
1990). In addition, this study indicated that Lactobacillus spp. could also provide T-cell
help to the hapten expressed on the cell surface. Besides mucosal immune responses,
systemic immune responses were also elicited following intraperitoneal immunization of
strains expressing Escherichia coli β-galactosidase (Claassen et al., 1995).
1.3 SACCHAROMYCES CEREVISIAE
The most well-known and commercially significant yeasts are the related species
and strains of Saccharomyces cerevisiae. These organisms have long been utilized to
ferment the sugars of rice, wheat, barley, and corn to produce alcoholic beverages and in
the baking industry for the fermentation of bread dough. Saccharomyces cerevisiae is a
very attractive organism to work with since it is non-pathogenic. Due to its long history
of application in the production of consumable products such as ethanol and baker's yeast,
it has been classified as a GRAS organism (Generally Regarded As Safe). Also, the well-
established fermentation and process technology for large-scale production with
Saccharomyces cerevisiae make this organism attractive for several biotechnological
purposes. Not only being useful in daily brewers and bakers practice, yeast, as a simple,
unicellular eukaryote was developed as a unique powerful model system for biological
research. Its prominent useful features are the cheap and easy cultivation, short
generation times, the detailed genetic and biochemical knowledge accumulated in many
years of research and the ease of the application of molecular techniques for its genetic manipulation. Therefore, this organism provides a highly suitable system to study basic biological processes that are relevant for many other higher eukaryotes including man.
8 CHAPTER 1 LITERATURE REVIEW
1.3.1 Protein Expression in Saccharomyces Cerevisiae
Escherichia coli has been conventionally used for the expression of foreign proteins. While the simplicity of Escherichia coli makes it a desirable host for production of a foreign protein, it also has its disadvantage as a host cell. Being a prokaryote, proteins expressed in Escherichia coli are not post-translationally modified.
As such, the expressed protein might not be functional due to the lack of post- translational modifications. Because of the handicap encountered when using
Escherichia coli to produce eukaryotic proteins, other organisms like mammalian, insect and yeast cells have been studies as suitable replacements for Escherichia coli. Of the three, yeast cells are the most desirable as they combine the ease of genetic manipulation and rapid growth characteristics of a prokaryotic organism with the subcellular machinery for performing post-translational modification of eukaryotic cells (Cregg et al., 1993).
While many foreign proteins have been successfully expressed in
Saccharomyces cerevisiae, it has several limitations. Generally, the product yields are low, reaching a maximum of 1-5 percent of the total protein. In addition, proteins expressed in Saccharomyces cerevisiae seemed to be hyperglycosylated, which may result in differences in immunogenicity, diminished activity, or decreased serum retention of the foreign protein (Cregg et al., 1987). Problems may also be encountered during the purification of proteins expressed in Saccharomyces cerevisiae as many of the secreted proteins are not found secreted into the culture medium but trapped in the periplasmic space (Buckholz & Gleeson, 1991).
9 CHAPTER 1 LITERATURE REVIEW
1.4 SACCHAROMYCES BOULARDII
Saccharomyces boulardii which has been registered under the name
Saccharomyces cerevisiae Hansen CBS 5926, as a non-pathogenic, non-colonizing yeast
that is very closely related to the brewer's yeast, Saccharomyces cerevisiae. Recognized
to have probiotic effectiveness, Saccharomyces boulardii is sold in supplement form over
the counter in Europe for prevention and treatment of diarrhea of different aetiologies
(Czerucka & Rampal, 2002; Broussard & Surawicz, 2004; van der Aa Kuhle et al.,
2005). Saccharomyces boulardii is generally administered in lyophilized powder and the
application as a food additive has only been reported in a limited number of formulation
such as in the fermentation of vegetable raw materials and incorporation into commercial
yoghurts (Nguyen & Herve, 1997; Periti & Tonelli, 2001; Lourens-Hattingh & Viljoen,
2001; Sindhu & Khetarpaul, 2002).
Several possible mechanisms for the protective effect of Saccharomyces boulardii
against infections of the gastrointestinal tract have been proposed. Type I fimbrinated
Escherichia coli which binds to mannose as receptor, were found to be more strongly
bound to the surface of Saccharomyces boulardii than other probiotic strains of
Saccharomyces cerevisiae when observed under scanning electron microscopy (Sharon &
Ofck, 1986; Stefano et al., 1998). It could be reasoned that the outer membrane of
Saccharomyces boulardii, being richer in mannose than other yeast, enabled more type I
Escherichia coli to be bound to Saccharomyces boulardii. On the other hand, the
therapeutic effect of Saccharomyces boulardii in the prevention and recurrence of
Clostridium difficile associated diarrhea is likely attributable to the secretion of a 54 kDa
protease that can digest toxins A and B of the pathogen (Castagliuolo et al., 1999). This
10 CHAPTER 1 LITERATURE REVIEW
proteolytic activity of Sacchromyces boulardii may explain the protective effect against
Clostriduim difficile associated diarrhea.
In addition to diarrhea associated problems, Sarrchromyces boulardii has also
been reported to be effective in relieving constipation in the elderly without affecting the
mucosal barrier and beneficial in treating inflammatory bowel disease (Ouwehand et al.,
2002; Guslandi et al., 2000). In a recent study made by Lee et al. (2005), Saccharomyces
boulardii was demonstrated to be able to stimulate the expression peroxisome
proliferator-activated receptor-gamma (PPAR-gamma) expression, which plays a role in
the regulation of inflammation in intestinal epithelial cells. It was hypothesized that the anti-inflammatory effects of Saccharomyces boulardii are mediated through up regulation of PPAR-gamma expression, thus reducing the response of human colon cells to proinflammatory cytokines.
1.5 PICHIA PASTORIS
Pichia pastoris is established industrial methylotrophic yeast that uses methanol as its sole carbon source to produce energy and cellular materials at ultra-high levels.
Though not commonly used in the food industry, U.S. Food and Drug administration has permitted Pichia pastoris as a food additive in feed formulation for animals (U.S. Food and Drug Administration). In addition, Pichia pastoris has been developed to be a competent host for the production of foreign proteins (Romanos et al., 1992; Ilgen et al.,
2004).
11 CHAPTER 1 LITERATURE REVIEW
Being an excellent protein expression host, Pichia pastoris combines many of the benefits of E. coli expression with the advantages of expression in a eukaryotic system.
When compared to other eukaryotic expression systems, Pichia pastoris offers many advantages, neither does it have endotoxin problem associated with bacteria nor viral contamination of proteins produced in animal cell culture. Since the proteins produced in
Pichia pastoris are typically folded correctly and secreted into the medium, the fermentation of genetically engineered Pichia pastoris provides an excellent alternative to Escherchia coli expression systems. In addition, Pichia pastoris is capable of generating post-translational modifications that are more similar to human proteins modifications than Saccharomyces cerevisiae. For example, the expression of Hepatitis
B surface antigen (I-IBsAg) in Pichia pastoris leads to production of particles that are not only immunoreactive with anti-HBsAg antibodies but are also similar to Dane particles isolated from the sera of human carriers (Cregg et al., 1987). A comparison of protein secreted by Saccharomyces cerevisiae and Pichia pastoris has shown distinct differences between N-linked oligosaccharide structures. The length of the carbohydrates chains is much shorter in Pichia pastoris, ranging from 8 to 14 mannose residues, as compared to
50 to 150 mannose residues typically found in Saccharomyces cerevisiae glycoproteins.
In addition, glycans from Pichia pastoris do not have alpha 1,3-linked mannose residues, which are characteristic of Saccharomyces cerevisiae (Cregg et al., 1993). These may in turn explain why Pichia pastoris does not appear to hypermannosylate it glycoproteins to the same extend as Saccharomyces.cerevisiae (Bretthauer & Castellino, 1999).
Pichia pastoris has a strong inducible promoter that controls the expression of alcohol oxidase (AOX1), which is required for the metabolism of methanol (Ledeboer et
12 CHAPTER 1 LITERATURE REVIEW
al., 1985). The expression of this enzyme, coded for by AOX1 gene, is tightly regulated and induced by methanol to a level as high as thirty-five percent of the total cellular protein (Faber et al., 1995). As such, gene of interest under the control of the AOX1
promoter for inducible expression or the GAP promoter for constitutive expression will
result in high yield of functional proteins. A number of proteins have been produced
using this system, including tetanus toxin fragment, Bordatella pertussis pertactin, human
serum albumin and lysozyme (Tschopp et al., 1987; Digan et al., 1989; Clare et al., 1991;
Cregg et al., 1993; Chen et al., 1996). In addition, as Pichia pastoris grows on a simple mineral media and does not secrete high amounts of endogenous proteins, hence heterologous protein secreted into the culture is relatively pure and purification is easier to accomplish (Faber et al., 1995).
1.6 ADHESION
It is generally agreed that to have positive effects, a probiotic strain has to survive in the intestine in sufficient numbers to interact with the gut microflora and the host.
Adhesion also provides an interaction with the mucosal surface facilitating the contact with gut associated lymphoid tissue mediating local and systemic immune effects.
Hence, only adherent probiotics have been thought to effectively induce immune effects and further to stabilize the intestinal mucosal barrier (Salminen et al., 1996).
In order to permanently establish a bacterial strain in the host’s intestine, the organism must be able to attach to intestinal mucosal cells (O’Sullivan et al., 1992). The length of the lag phase of growth that an organism exhibits when encountering a new
13 CHAPTER 1 LITERATURE REVIEW environment is a decisive factor in determining whether the probiotic will successfully establish in the gastrointestinal tract.
Appropriate polarized and fully differentiated human intestinal cell models in culture that mimic the human situation have been extensively used to study specific human intestinal cell functions (Zweibaum et al., 1991; Louvard et al., 1992). Adhesion of organisms to the gut can be mimicked using cultured intestinal cell lines, which exhibits the specific characteristics of the several cell phenotypes that line the epithelium.
The parental human epithelial cell line Caco-2 has been shown to undergo morphological and functional enterocytic differentiation in vitro, while HT29 displays the functions of mucus secreting cells. In in vitro models, adhesion of probiotic strains to intestinal cells can be highly variable. Variation in adhesion can occur within the same strain and differences between strains can be significant.
Some strains of Lactobacillus, including Lactobacillus acidophilus strain
BG2FO4 and HN017, Lactobacillus johnsonii strain La 1, Lactobacillus rhamonsus strain DR20 and Lactobacillus casei subsp. rhamnosus Lcr38 and GG strains, adhered to the enterocyte-like Caco-2 (Coconnier et al., 1992; Bernet et al., 1994; Hudault et al.,
1997; Tuomola & Salminen, 1998; Forestier et al., 2001; Gopal et al., 2001). In particular, Lactobacillus acidophilus BG2FO4 and Lactobacillus johnsonii La 1 have also been reported to interact with the mucus secreted by HT29 cells. Like lactobacilli,
Bifidobacterium strains also display adhesiveness. Bifidobacterium breve 4, B.infantis 1 and Bifidobacterium lactis DR10 strains have been found adhering to both the brush border of Caco-2 cells and the mucus secreted by HT29 cells (Bernet et al., 1993; Gopal et al., 2001).
14 CHAPTER 1 LITERATURE REVIEW
1.7 VACCINE When a new disease emerges or an existing one becomes a more significant
health threat than it has been in the past, the need for a new way to prevent the disease
will be recognized. Vaccination has been one of the most important interventions
designed to prevent disease to be employed on a worldwide basis. The concept of
vaccinology originated in the 18th century when the English physician Edward Jenner used cowpox virus inoculations in an attempt to combat smallpox infections (Tan, 2004).
Since then, smallpox has been eradicated worldwide and other diseases such as poliomyelitis have virtually disappeared in countries of the Western Hemisphere.
Over the last quarter century, scientific advances have created new tools and technologies to improve the safety and efficacy of vaccines. These improvements are spurred by social and commercial needs to enhance existing vaccines or to create new ones against an expanding spectrum of diseases. Live, attenuated, pathogenic bacteria are traditional vaccines that are originally developed to prevent infection by homologous pathogens. More recently, strategies have been developed to use these bacterial vaccines
as vectors to deliver a variety of protective, vaccine antigens via the mucosal route to
protect not only against heterologous microbial infections, but also against non-
traditional threats such as biowarfare and cancer (Roland et al., 2005).
1.7.1 Types of Vaccines
The effectiveness of vaccines and immunization in the prevention of major
infectious diseases is recognized as one of the greatest successes in medicine. Advances
in recombinant DNA technology and other areas of bioengineering hold promise for
improvements in available vaccines and to accelerate the development of several novel
15 CHAPTER 1 LITERATURE REVIEW
and safe immunogenic vaccines. Table 1.1 sets out to illustrate the characteristics of
several forms of vaccines.
1.7.1.1 Inactivated Vaccine
Treating the pathogen with chemicals like formaldehyde or subjecting them to
heat treatment for inactivation produces inactivated vaccines. Such vaccines are stable
and safe due to the inability of inactivated microorganism to revert to the virulent form.
However, most inactivated vaccines stimulate a relatively weak immune response and
several doses are required to generate sufficient immune responses to confer protection
against invasion of pathogens. Nevertheless, inactivated tick-borne encephalitis vaccine has been successfully used in Europe for almost 30 years, and continues to be improved by additional purification, new stabilizers, an adjuvant, and better immunization schedules (Eckels & Putnak, 2003).
1.7.1.2 Live Attenuated Vaccine
Live attenuated vaccines are prepared by repeatedly culturing of viruses under
special laboratory conditions that causes it to lose its virulence. Measles vaccine, a live attenuated strain of measles virus, is one of the safest and most effective human vaccines
available. Since the 1960s, it has been given to billions of children (Despres et al., 2005).
Attenuated vaccines have an advantage over inactivated vaccines in that they produce
both antibody-mediated and cell mediated immunity, and generally require only one
booster dose to be effective. However, live vaccines have to be carefully tested and
16
Table 1.1 Comparison of current vaccination strategies with DNA vaccination technology. Adapted from Kowalczyk and Ertl (1999) and Hasan et al. (1999).
Types of vaccines Inactivated Attenuated Subunit Live recombinant DNA Vaccine vector Antibody response Y Y Y Y Y Antibody rise Fast Fast Fast Fast Slow Cytotoxic T lymphocyte induction N Y Variable Y Y T helper induction Y Y Y Y Y Complete antigen repertoire Y Y N N Possible Immune response(s) to vaccine carrier N N N Possibly N Duration of response Short Long Short Long Long No. of doses required Multiple One Multiple Multiple One or probably more Safety (for pregnant/ Y N Y N Probably immunocompromized individuals) Risk of reversion N Y N Y N Neonatalimmunization Y Y Y Y N Stability Stable Not very Stable Not very Stable Ease of production Variable Variable Difficult Difficult Easy Cost Variable Variable Expensive Expensive Inexpensive
17 CHAPTER 1 LITERATURE REVIEW monitor, as it is possible that the attenuated organism may revert back to a virulent form and cause disease when administered. On a safe side, people with compromised immune systems are generally not given live vaccines.
1.7.1.3 Toxoid
Some bacteria - for example those that cause tetanus and diphtheria - produce a toxin that is largely responsible for the disease. For this type of bacterial infection, the toxin can be used in a modified form as the vaccine. A toxoid is an inactivated toxin that is made by treating toxins with heat or chemical such as formalin, a solution of formaldehyde and sterile water, but still retains the ability to stimulate the immune system to make antitoxins. Because it is not a live vaccine, a person's immunity tends to wane with time, which is why booster doses are required. One of the most common vaccines is the tetanus vaccine. The first tetanus toxoid was produced in 1924 and was used successfully to prevent tetanus in the armed services during World War II.
Prompted by outbreaks after World War II, the World Health Organization (WHO) sponsored trials of diphtheria toxoid in Romania (Pappenheimer, 1984). In the mid-
1940s, tetanus vaccine was combined with diphtheria toxoid and inactivated pertussis bacteria to make the combination DTP vaccine for routine childhood immunization. In
1991, DTaP vaccine, a more purified version of the DTP was licensed in the United
States. (http://www.vaccineinformation.org/tetanus/qandavax.asp).
18 CHAPTER 1 LITERATURE REVIEW
1.7.1.4 Conjugated Vaccine
Conjugate vaccines are so-called because their production involves the conjugation of the polysaccharide antigen with a protein from a second type of organism that immature immune system can recognize. The bacteria for which conjugated vaccines are designed have an important structural feature in common. They are surrounded by a thick and slippery capsule, which determines the extent of their virulence. The capsule is made of carbohydrate and provides an antigenic target for recognition by the immune system. However, as carbohydrate antigens are T-cell independent, the antigen is not processed by antigen presenting cells, and B cells are able to make antibodies without help from T cells, protection wanes rapidly within two years from vaccination (Wald et al., 1981). This conjugation will in turn convert the T- cell independent carbohydrate antigen into a T-cell dependent antigen, with all the associated benefits in terms of immunological response (Overturf & American Academy of
Pediatrics, 2000). Various protein carriers have been used for conjugation, including diphtheria and tetanus toxoid.
The three most common causes of bacterial meningitis - Neisseria meningitidis
(the meningococcus), Streptococcus pneumoniae (the pneumococcus) and Haemophilus influenzae type B - are all capsulated. Past attempts to make vaccines against these pathogens using traditional methods have been less successful than for other diseases, and these diseases provide the best examples of the application of conjugation to vaccine technology.
19 CHAPTER 1 LITERATURE REVIEW
1.7.1.5 Subunit Vaccine
Subunit vaccines, also known as "component" vaccines, contain only the
antigenic fragment, rather than the whole microbe. Subunit vaccines can be made from
isolated proteins, bacteria, or a combination thereof, from wild type organism as well as
from those produced by genetic engineering. They are usually well tolerated with low reactogenicity, and are not able to multiply or revert to pathogenicity (Ellis, 2004). As such, subunit vaccines can be developed eliminating many of the safety issues
characteristic of attenuated vaccines while maintaining epitopes critical to inducing a
protective immune response (Byrd et al., 2005). Subunit vaccines are capable of
inducing systemic as well as mucosal immune responses, and function by eliciting
primarily an antibody and inducing immunological memory. Likewise, it has been
shown that certain subunit vaccines can induce cell-mediated immunity when
administered with certain adjuvant or via certain delivery systems (Ellis, 2004).
As typified by the Hepatitis B vaccine, in which a component of a virus is cloned,
over-expressed, and purified form a recombinant microorganism and delivered as a
subunit vaccine in combination with an adjuvant (Isaka et al., 2001; Vandepapeliere et
al., 2005). One of the highly efficient adjuvant is a bacterial DNA containing cytosine-
phosphate-guanosine (CpG) motifs (Krieg, 2002). The adjuvanticity of CpG DNA,
which result from the binding of the CpG-rich DNA to Toll-like receptors, is associated
with the induction of both pro-inflammatory and Th1-inducing cytokines and
chemokines, and the induction of Major Histocompatibility Complex (MHC) and co-
stimulatory molecules on antigen presenting cell (APC). The resulting immune responses
20 CHAPTER 1 LITERATURE REVIEW in mice are Th1 dominated with high levels of cytotoxic T lymphocytes, interferon-γ
(IFN-γ) production and IgG2a antibody production.
1.7.1.6 DNA Vaccine
DNA vaccines, also known as ‘genetic’, nucleic acid’ or ‘polynucleotide’ vaccines, deliver genes encoding protein antigens into host cell, enabling antigens production to occur in vivo (Davis, 1997). Generally, in DNA vaccination, direct parenteral injection of the plasmid DNA results in the in vivo synthesis of the encoded protein with post-translational modification similar to those that occur during natural infection and the most commonly studies routes for administration of DNA vaccines have been intramuscular (Donnelly et al., 2003). However, it is believed that majority of the
DNA injected intramuscularly is degraded by extracellular deoxyribinucleases (Kawabata et al., 1995). Carrier-mediated delivery may provide protection from degradation by introducing DNA directly into target cells. For instances, the gene gun delivery system allows direct injection of DNA into the cytosol of host cells, which often results in higher levels of antigen-specific stimulated immunity (Bennett et al., 1999; Belperron et al.,
1999; Yoshida et al., 2000).
The feasibility of DNA vaccines lies in the fact that they can be constructed easily within a short time frame and produced at large-scale. In addition, the ability to genetically manipulate DNA offers the advantage of vaccines designed to produce co- stimulatory molecules, or that are able to target protein production to specific cell compartment. DNA vaccines can also be manipulated to express multiple antigens
(Garmony et al., 2005). Even though studies have demonstrated that DNA vaccines are able to induce cellular immune responses, it has also been reported that DNA vaccines
21 CHAPTER 1 LITERATURE REVIEW
may induce relatively poor humoral immune responses and also lacks potency in humans following intramuscular administration (Donnelly et al., 2003). Nevertheless, DNA vaccine vectors have already been shown to have potential for immunization against
Yersinia pestis and Bacillus anthracis (Price et al., 2001; Williamson et al., 2002;
Grosfeld et al., 2003).
1.7.2 Vaccine Delivery
Many vaccine candidates are highly purified, sometimes monomeric antigens and as a result, not very immunogenic. As such, the application of new strategies to develop effective delivery vehicles is essential in modern vaccine design by facilitating the efficient delivery of target antigens. A variety of antigen delivery systems have been developed, including various inert systems as well as different live-attenuated bacterial or viral vector systems, to optimize the presentation of antigens.
1.7.2.1 Recombinant Vector Vaccines
Live recombinant vector vaccines are constructed by inserting antigenic genes into live, infectious, but non-disease-causing viruses or bacteria such as vaccinia virus or
Bacille Calmette-Guerin (Rezende et al., 2005; Drexler et al., 2004). The viruses or bacteria are used as vectors, or carriers, to deliver harmless genes from another disease causing organism into the cells of the body. The recombinant vector expresses proteins from the inserted genes within the human body and these proteins stimulate immune response against the pathogen.
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1.7.2.1.1 Viral Vectors
Recombinant viruses represent a particularly promising avenue of vaccine research, both for improving existing vaccines and for developing new ones. This process normally involves taking a proven safe and efficacious vaccine virus, such as vaccinia or adenovirus, and modifying its genome to include genes coding for immunogenic proteins from other pathogens. The recombinant viral vectors enter cells and allow the proteins from foreign pathogens to be generated inside the cells and be presented to the immune system in the same way that proteins from a virus-infected cell would be (Souza et al., 2005). Virus-derived vectors offer several potential advantages over traditional vaccine technologies. These include, most notably, high-level production of protein antigens directly within cells of the immunized host, potential adjuvant effects of the viral delivery system itself and the possibility of efficient delivery of antigen directly to components of the immune system, such as antigen-presenting cells. In order to be utilized as a vaccine carrier, the ideal viral vector should be safe and enable efficient presentation of required pathogen specific antigens to the immune system.
Vaccinia virus is now commonly used to make recombinant vector vaccines as vaccinia virus is relatively large and has ample room to accommodate additional genetic materials. Concerns about the safety of vaccinia virus have been addressed by the development of vectors based on attenuated viruses. One of them, modified vaccinia virus Ankara (MVA) can be considered as current vaccinia virus strain of choice for clinical investigation (Drexler et al., 2004). Currently, recombinant MVA-based constructs are developed as vaccines for a variety of diseases. Progress has been made towards the clinical assessment of MVA-based vectors in the treatment of HIV
23 CHAPTER 1 LITERATURE REVIEW
infections, malaria, measles, or cancer, either alone or in combination with DNA vaccines
(Allen et al., 2000; Drexler et al., 1999; Schneider et al., 1998; Weidinger et al., 2001).
The popularity of adenovirus (Ad) as a recombinant viral vector is largely due to
the successful and safe immunization of millions of US military recruits with live enteric-
coated capsules containing unattenuated Ad serotypes 4 and 7 in an attempt to prevent
outbreaks of respiratory diseases caused by Ad. The safety of these vaccinations has been
well documented (Top Jr et al., 1971). So far, no significant side effects or illnesses
related to the use of these vaccines have been reported.
1.7.2.1.2 Virus-like particles
Also known as pseudovirus, virus-like particles (VLP) are self-assembling, non-
replicating viral core structures, often from non-enveloped viruses, that are produced
through recombination in vitro. VLP are cheap and easily generated, as well as highly
immunogenic. In addition, VLP can also be used as carriers or adjuvants for foreign
antigens expressed recombinantly on the surface of the pseudovirus, and for DNA
vaccines carried within VLP (Garcea & Gissmann, 2004). VLP are especially useful
from a mucosal vaccine point of view as they offer opportunity to use the natural route of
transmission of the present virus for the delivery of vaccine. Successful use of VLP has
been reported from several mucosal viral pathogens including papilomavirus, calicivirus
and hepatitis E virus.
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1.7.2.1.3 Bacterial Vector
Among the many live bacterial vectors developed, there are two main categories,
those based on attenuated pathogens and those that use commensally bacteria. Antigen
delivery directly to mucosal sites via non-pathogenic commensally bacterial vectors could provide a significant advantage over some of the more traditional vaccines in that live bacteria are capable of stimulating sustained systemic as well as local immune responses (Liljeqvist & Stahl, 1999). In addition, commensally bacteria are also able to colonize the niche invaded by the pathogen and stimulate an immune response at their
portal of entry to prevent infection. This ability in combination with the facts that
bacteria are easily administered, are cost efficient and are well tolerated, makes live
commensally bacteria attractive vehicles for subunit vaccine delivery (Medina &
Guzman, 2001). In addition, the antigens can be tailored to be expressed intracellularly,
anchored on the cell surface or be secreted into the medium (Reveneau et al., 2002).
Generally, these expression systems use the conserved pathway that all Gram-positive
bacteria utilize to export and anchor protein on the cell surface (Medaglini et al., 2001;
Schneewind et al., 1992).
One such promising commensally bacterium is the Lactobacillus spp., a normal inhabitant of the gastrointestinal tract. Recombinant Lactobacillus spp. has been engineered to produce C fragment of tetanus toxin, Streptococcus pnuemoniae antigens and protective antigen of Bacillus anthracis (Shaw et al., 2000; Reveneau et al., 2002;
Oliveira et al., 2003; Zegers et al., 1999). Immunization studies using recombinant
Lactobacillus spp. so far have been rather encouraging.
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Similarly, studies have been conducted to test the feasibility of using attenuated
Salmonella spp. to carry antigenic domains of Hepatitis B virus and TGEV (transmissible gastroenteritis coronavirus) (Schodel et al., 1994; Smerdou et al., 1996).
1.7.2.2 Transgenic Plant
Transgenic plants have been increasingly utilized for the expression of immunogenically relevant antigens since the application was first reported by Mason et al. (1992). Since then, plant transgenics and biotechnology have been used to produce proteins with potential human and animal pharmaceutical value (Ma et al., 2003, Sala et al., 2003). This biotechnological strategy has the enormous advantage of not requiring fermentation control systems under strict conditions of biosafety and sterility, which makes this technology particularly attractive for its simplicity and low cost. More importantly, production of vaccine antigens in plants overcomes the risk of contamination with animal pathogens.
In addition to the utilization of plants as bioreactors for the production of vaccine antigens, plants also provide an adequate system for oral delivery of recombinant immunogens by including them in the diet. A number of viral and bacterial antigens
produced in plants have already demonstrated their immunogenicity when orally
administered, though the expression level was relatively low. Among them include
Escherichia coli heat-labile enterotoxin, cholera toxin B subunit, foot and mouth disease
virus and rotavirus enterotoxin (Haq et al., 1995; Arakawa et al., 1998; Wigdorovitz et
al., 1999; Yu & Langridge, 2001). This approach is particularly relevant for enteric
pathogens, because oral immunization may be able to elicit appropriate immune
26 CHAPTER 1 LITERATURE REVIEW
mechanisms for the induction of protective responses. Although this expression system constitutes an interesting alternative to conventional methodologies, the main disadvantage is the low concentration of the expressed antigen, requiring multiple immunization doses to evoke a protective immune response (Santoss & Wigdorovitz,
2005).
1.7.2.3 Microencapsulation
Microencapsulation is a technology by which solid or liquid materials are transferred onto spherical particles and protect an antigen from chemical and enzymatic
degradation in the body. This technology is especially useful for delivery of vaccines
like subunit vaccines as subunit antigens alone are usually weak immunogens and require
adjuvant or carrier system to induce protective immunity (Ellis, 2004). The use of
subunit vaccines like antigen-encapsulated microspheres can act to effectively deliver
specific antigens so as to optimize their immunological response.
1.7.2.3.1 Liposome
Liposomes are spherical, self-enclosed structures of varying size consisting of a
lipid bilayer and an aqueous inner compartment that are generated in vitro (Felnerova et
al., 2004). They have enormous versatility with regards to composition, size,
physicochemical, and immunological characteristics. The two key problems of
immunotherapy, biodistribution and targeting, can be addressed at least partially solved
by the use of liposome formulations. A liposome can protect the antigen from the
environment until controlled release occurs at the target site. The lipid membrane and its
27 CHAPTER 1 LITERATURE REVIEW
surface are versatile enough to allow the integration of specific targeting and release
device. Molecules such as monoclonal antibodies, ligands for cellular receptors or
specific peptide sequences can be attached to liposomal surface to provide specific
binding (Brignole et al., 2003). However, an unfavourable characteristic of liposomes is their potential for unspecific interaction with macromolecules and cellular surfaces,
resulting in short half-life. This interaction can be reduced by coating the surface of
liposome with inert molecules to form a spatial barrier (Allen et al., 2002).
1.7.2.3.2 Virosomes
Virosomes are liposomal formulations that involved viral envelope proteins
anchored in their lipid membrane and offer the same versatility with regard to lipid
composition as liposome. Virosomes were not only found suitable as vaccines against
parental virus, but were further exploited as carrier systems for unrelated vaccine
antigens. Additional antigens can be included in virosomal vaccine formulations through
direct incorporation into membrane, adsorption to the virosomal surface, chemical
coupling to a hydrophobic anchor (Felnerova et al., 2004). These virus-like particles
have proven to be effective immunogens with unique adjuvant properties (Moser et al.,
2003). The most advanced virosomal systems for targeted delivery and release are the
immunopotentiating reconstituted influenza virosomes (Moser et al., 2003; Gluck &
Metcalfe, 2003).
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1.7.2.3.3 Microspheres
Microspheres are spherical polymeric particles with diameters of 1 to 1000 μm,
prepared from various polymers such as poly (ortho-esters), poly (anhydrides), poly
(phosphazenes), with polyglycolic- (PLG) and polylactic acids and poly (lactic-co-
glycolic acid) (PLGA) being the most promising (Heller, 1985; Leong et al., 1986;
Lakshmi et al., 2003; Gupta et al., 1998). Besides acting as an adjuvant by increased
uptake by antigen presenting cells, they permit sustained release of antigen over time
(Morris et al., 1994). Smaller size microspheres (less than 10 μm) are often more
immunogenic than larger sized ones due to the preferable uptake of smaller microspheres
by the Membranous epithelial cells (M cells) in the Peyer’s patches of the
intestine. (Eldridge et al.,1991). Among biodegradable polymers, PLGA is well
documented for its safety, being biocompatible and approved for human usage (Frazza &
Schmitt, 1971).
1.7.3 Route of Vaccination
Vaccines can be administered either through injection or oral consumption. A
major advantage of mucosal administration of vaccine antigens is that both mucosal and
systemic immune responses are normally induced, while parenteral immunization is
ineffective for induction of mucosal immunity (Jackson et al., 1993). There are important
practical-logistic reasons to try to replace some of the existing injectable vaccines used to
prevent systemic infections with orally administered vaccines. Oral vaccines would be
easier to administer than parenteral vaccines. They would carry less risk of transmitting the type of infections like hepatitis B and HIV infections, which are associated with the
29 CHAPTER 1 LITERATURE REVIEW
use of injectable vaccines in several parts of the world. Oral vaccines can also be expected to have greater acceptability than injectable vaccines by causing no sore arms, etc. Oral administration could also lead to simplified manufacturing vaccine, there by increasing the potential for local vaccine production in developing countries.
1.7.4 Immunity
Immune responses to vaccines are influenced by the route of immunization
(injection or oral), the form of the antigen (live, killed, soluble, peptide subunit, particulate, etc.) and the presence in the vaccine of biologically active elements that mediate specific tissue tropisms. There is now substantial evidence supporting the
existence of at least two immune systems, a peripheral immune system and a mucosal
immune system (Ogra et al., 1994). These systems operate separately but simultaneously
in most species including humans. Protective immunity acquired during convalescence
is usually referred to as systemic immunity, which might be a combination of mucosal
and peripheral immunity or be dominated by an incomplete form of immunity dictated by
a specific pathogen (Salgame et al., 1991; Mosmann and Sad, 1996).
The way antigens are acquired by individual lymphatic tissues affects the
outcome of an immune response. For example, the same antigen may produce
qualitatively different immune responses in lymph nodes, spleen or Peyer's patches
(Anderson et al., 1990). Generally, the stimulation of the immune system systemically
either via injection or blood-borne routes result in production of protective antibody
especially antigen specific IgG and T cells only within the sterile, internal environment of
the body. On the other hand, stimulation of the mucosal immune response can result in
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production of protective B and T cells in both mucosal and systemic environments so that
infections are aborted before they get systemic into the body (McCluskie & Davis, 1999;
Ogra et al., 2001). The nature of serum and secretory immune responses induced after immunization by conventional live attenuated or killed vaccines is summarized in Table
1.2.
Table 1.2 Nature of immunologic reactivity after systemic or mucosal immunization with conventional live or inactivated vaccines. Adapted from Kiyono et al. (1996).
Response to immunization by indicated route and type of vaccine Systemic Mucosal Features of response Live Killed Live Killed Immunologic response similar to ± - + - natural infection Development of systemic immune + + + ± response Persistence of systemic immune ± ± + - response Development of secretory immune ± ± + ± response Persistence of secretory immune ± - + ± response Development of secretory immunity in other mucosal sites + - + ± and milk Protection against mucosal natural ± - + + reinfection Protection against systemic disease + + + ± after natural reinfection
+, Always; ±, occasional or inconsistent; -, absent.
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1.7.4.1 Mucosal Immunity
Mucous membranes line the gastrointestinal tract, the respiratory tract, and the urogenital tract and represent the most important portal of entry for human pathogens.
The mucosal surfaces are protected from pathogens by non-specific mechanisms such as the production of mucus forming a physical barrier, and substances such as lysozyme, lactoferri and lactoperoxidase, which kill microorganisms or inhibit their replication
(Brandtzaeg, 1995). Specific defense mechanisms at the mucosal site include cellular (T cell) and humoral (antibody) responses (Figure 1.1).
Figure 1.1 Current mucosal delivery systems, mucosal inductive sites and the concept of Th1- and Th2-type immune responses. Following uptake in the inductive site, the antigen can influence the nature of CD4+ Th cell responses, which in turn regulate cell mediated immunity and the isotype of mucosal vesus serum antibody responses. Adapted from Kiyono et al. (1996).
32 CHAPTER 1 LITERATURE REVIEW
In order to elicit a mucosal antibody response, viral antigens must reach the
organized mucosa-associated lymphoid tissue, which includes the gut-associated lymphoid tissue in the intestine or bronchus-associated lymphoid tissue in the lung.
Areas that contain clusters of follicles (30 or more) in the submucosal region of the intestines are called Peyer's patches, which are organized centers collecting antigen from macrophages that migrate from the mucosal area. The intraluminal surface of Peyer’s patches are covered with a follicle-associated epithelium, a one-cell-layer complex composed of columnar epithelial cells, globlet cells and M cells (Owen, 1977; Wolf et
al., 1981). M cells endocytose antigens from the surface of the mucosa and exocytose
them to the underlying dendritic and macrophage populations, which express MHC Class
II molecules. These immune cells process and present the antigen to CD4+ T cells and naive B cells destined to become cytokine producing cells and plasma cells, respectively, in mucosal immunity (Owen & Nemanic, 1978; Panja & Mayer, 1994).
The adhesion molecule, mucosal addressin cellular adhesion molecule-1
(MAdCAM-1), is located on the high endothelial venule of Peyer’s patches and regulates trafficking of naïve T and B cells into and through the mucosal immune system
(Brandtzaeg et al., 1999; Berlin et al., 1993). It is this interaction of T and B cells and the balance of cytokines, which controls IgA production for transport and mucosal protection. Transport of IgA through epithelial cells occurs by a receptor-mediated process via a polymeric immunoglobulin receptor, which is expressed on the basolateral membrane of the cell (Mostov, 1994). Upon binding to the receptor, the IgA-receptor complex is endocytosed and carried to the apical membrane of the cell. The receptor is
33 CHAPTER 1 LITERATURE REVIEW
then cleaved between its external and intramembranous domains and release the IgA into
the secretions of the lumen.
1.7.4.1.1 Secretory IgA
The principal antibody involved in mucosal immunity is secretory IgA (Russell et
al., 1997). IgA is the most abundant immunoglobulin with approximately 75% of the antibody-producing cells in the body manufacturing IgA, the majority of which is released into the gastrointestinal fluid, saliva, tears, urine and other secretions
(Brandtzaeg, 1994). Up to 40 mg/Kg body weight of IgA is manufactured and secreted daily in humans, which is many orders of magnitude greater than that of all other immunoglobulin isotypes (Brandtzaeg, 1994). In general, IgA does not mediate strong inflammatory responses but favors the principle of immune exclusion rather than destroying action (Stokes et al., 1975). Secretory IgA prevents absorption of pathogens or their toxic products at the mucosal epithelium, which allows them to be flushed away in the stream of secreted fluids and mucous washing over the epithelial membranes. In addition, IgA may also facilitate transport of pathogens and toxins out of the body by causing them to be conveyed into bile and other exocrine secretions (Mazanec et al.,
1993). Antigen-specific IgA has recently been shown to neutralize viral pathogens during transport across M cells of Peyers patches, where non-degradative endosomal transport might otherwise deliver a pathogen into the host (Owen and Jones, 1974; Neutra and Kraehenbuhl, 1994, Mazanec, 1992). Secretory IgM and IgG, mainly derived from serum, also appear at the mucosal sites, but to a lesser extend (Wagner et al., 1987; Ogra et al., 1974).
34 CHAPTER 1 LITERATURE REVIEW
1.7.4.2 Cytokines
Cytokines are small-to-medium-sized proteins, or glycoproteins, produced by cells in response to a variety of inducing stimuli. Majority of the cytokines are directly mitogenic for their target cells. They are secreted by their producer cells and are mostly directly mitogenic for their target cells (Clemens, 1991a). Cytokines that are produced by or act upon cells of the immune system are known as lymphokines. Factors commonly referred to lymphokines include interferon-γ and the interleukins. The generic name ‘interleukin’ was proposed in 1979 for factors produced and released by activated T lymphocytes that act on other lymphocytes to produce biological effects. In fact, the group of interleukins also includes species produced by cells of other hematopoietic lineages, such as monocytes. Interleukins act as intercellular signaling agents between lymphocytes. A brief detail of the biological activities associated with individual cytokines is illustrated in table 1.3.
Table 1.3 Cytokines and biological activities.
Cytokine Biological activities
Interferon-α & β All interferons induce antiviral activity in a wide variety of (IFN-α & β) cell types. They are multifunctional and pleiotropic regulators of immune functions. Stimulators of differentiation in many cells. Interferon-γ (IFN-γ) Antiviral activity. Factor involved in macrophage activation. Inducer of class II MHC antigens. Costimulator for proliferation of B lymphocytes. Enhances IgG2a secretion by B cells. Tumour necrosis factor Multifunctional and pleiotropic stimulators of cellular α & β (TNF α & β) responses. Inducers of certain cytokines and cell adhesion molecules. Regulators of proliferation/differentiation in lymphocytes and haemopoietic progenitors.
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Interleukin-1 (IL-1) Activates T cells. Induces growth factors and inflammatory mediators. Synergises with certain haemopoietic growth factors. Highly pleiotropic. Induces fever.
Interleukin-2 (IL-2) Stimulates proliferation of Th cells, proliferation/differentiation of CTL, and also acts on NK cells, B cells and macrophages. Induces other cytokines.
Interleukin-3 (IL-3) In combination with lineage-restricted cytokines, stimulates early growth of granulocyte, monocyte, erythroid and megakaryocyte precursor cells. Also supports mast cell growth. Interleukin-4 (IL-4) Stimulates or aids proliferation of B cells and mast cells. Promotes B-cells Ig class switching and induces IgE secretion. Induces class II MHC on B cells and macrophages. Generation Th2 cell subset from naïve Th0 cell population.
Interleukin-5 (IL-5) Induces proliferation and differentiation of eosinophil progenitors. Possible differentiation factor for B cells and Ig class switching (IgA).
Interleukin-6 (IL-6) Stimulates terminal differentiaion of B cells. Enhances Ig secretion by B cells. Cofactor for haemopoietic colony growth and thymocytes. Stimulates hepatocytes to produce acute phase proteins
Interleukin-7 (IL-7) Supports growth of immune T and B lymphocytes. Costimulatory activity on mature T cells
Interleukin-8 (IL-8) A member of the chemokine superfamily. Chemotactic for neutrophils and T cells. Activates neutrophils, but not lymphocytes and monocytes
Interleukin-9 (IL-9) Sustains antigen-independent growth of certain Th cells. Enhances mast cell proliferative response to IL-2
Interleukin-10 (IL-10) Inhibits cytokines synthesis (e.g. IFN-γ) by Th1 cells and cytotoxic T lymphocytes. Suppresses antigen-dependent proliferation of Th1 cells. Enhances B-cell proliferation and Ig synthesis.
36 CHAPTER 1 LITERATURE REVIEW
Interleukin-12 (IL-12) Stimulates cytokines synthesis (e.g. IFN-γ) by T cells and natural killer (NK) cells. Enhances proliferation of Th1 cells in vitro. Interleukin-14 (IL-14) Induces proliferation and of activated B cells (but not resting B cells). Inhibits Ig secretion by mitogen-stimulated B cells.
Interleukin-18 (IL-18) Induce production of IFN-γ by T-cells and enhaces NK cell cytotoxicity.
Adapted from Meager T. (1998).
Intestinal epithelial cells (IEC) are known to have an important role in the
transport of IgA produced by local lamina propria lymphocytes into the external secrestions (Brandtzaeg, 1985). IEC have been shown to produce several immunological important cytokines such as IL-6, TGF-β, IL-8 and TNF-α upon stimulation by bacteria,
bacterial products, or inflammatory cytokines (Panja et al., 1995; Koyama & Podolsky,
1989; Eckmann et al., 1993; McGee et al., 1995). This implies that IEC plays an
important role in the mucosal immune response, where IEC have been suggested to be
capable of regulating B and T cell response via secreted cytokines (McGee et al., 1995).
1.7.4.2.1 Control of Virus Infections
The body combats invading viruses in many ways, including immune and non-
immune pathways, and cytokines are essential to co-ordinate these effects. In particular,
sequences upstream of the IFN genes switch on transcription in response to viruses and
IFNs directly induce a state of resistance to viral replication in a wide variety of target
cells (Taylor & Grossberg, 1990). In contrast to IFN-γ which is induced by mitogenic or
antigenic stimulation of T lymphocytes, IFNα and β can be induced directly by viruses
37 CHAPTER 1 LITERATURE REVIEW
following infection of host cells. Viruses also induce other cytokines, particularly TNFs and IL-2, but these cytokines do not induce an anti-viral state in cells. Production of new
viral particles in an infected cell requires protein synthesis. Among the novel proteins
synthesized by IFN-treated cells are two enzymes that can regulate protein synthesis (De
Maeyer & De Maeyer-Guignard, 1988). One of these enzymes, 2’5’-oligoadenylate
synthetase, uses ATP to synthesize short oligomers of up to 12 adenylate residues, which
allosterically activate a latent ribonuclease, RNase L, which degrades viral and cellular
mRNA and ribosomal RNA. The other enzyme is a phosphokinase, which
phosphorylates initation factor eIF-2, a protein required for all polypeptide chain initation. Phosphorylated eIF-2 inhibits initiation of viral and cellular protein synthesis
(Clemens, 1991b).
1.8 CORONAVIRUSES
Coronaviruses are enveloped and spherical in shape, with a diameter of 120-160 nm and contain a single molecule of linear, positive sense, single-stranded sRNA. The viral genomic RNA is the largest viral RNA genome known, ranging from 27.6 to 31 kb, and functions as an mRNA that is infectious (Figure 1.2).
38 CHAPTER 1 LITERATURE REVIEW
Figure 1.2 Genome organization of SARS coronavirus. (Adapted from Lio and Goldman, 2004).
The virions contains approximately 7-10 functional genes, 4 or 5 of which encode
structural proteins. Coronavirus contains a large surface glycoprotein (spike), an integral
membrane protein (M), which is integrated in the virus envelope, a small membrane
protein (E) and a nucleocapsid protein (N). The spike protein is large, ranging from 1160 to 1452 amino acids, and in some coronaviruses, is cleaved into S1 and S2 subunits. The spike protein, a glycoprotein with a globular and stem region, is responsible for attachment to cells, hemagglutination, membrane fusion, and induction of neutralizing
antibodies. In some coronaviruses such as murine hepatitis virus (MHV), a second layer of peplomers formed by the hemagglutinin esterase (HE) has also been observed. The M
protein contains 225-260 amino acids and is the main inducer of α-interferon. Together
with the E protein (80-109 amino acids), the M protein plays an essential role in
coronavirus particle assembly. The N protein (377-455 amino acids), on the other hand,
is a highly basic phosphoprotein that modulates viral RNA synthesis, binds to the viral
RNA and forms a helical nucleocapsid (Figure 1.3).
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Figure 1.3 Arrangement of the structural proteins and viral RNA within an infectious virus particle of the coronavirus. (Adapted from Holmes, 2003.)
Coronaviruses, with a relatively restricted host ranges infects birds and a number
of mammals, including humans via respiratory, fecal-oral and mechanical transmission.
They exhibit a marked tropism for epithelial cells of the respiratory and enteric tracts, but
other organs including liver, kidney, heart and eyes can be affected too. These viruses can be divided into three antigenic groups according to serotypes. Group I and group II contain mammalian viruses. The porcine transmissible gastroenteritis virus, canine and feline coronavirus, and feline infectious peritonitis virus constitute Group I; porcine hemagglutinating encephalomyelitis virus and bovine coronavirus make up Group II that contain a hemagglutinin esterase gene homologous to that of Influenza C virus (Lai &
Holmes, 2001). Coronavirus of group I use the aminopeptidase N as receptor for cell entry while those of group II use receptors members of the biliary glycoprotein subfamily of the carcinoembryonic antigen family (Yokomori & Lai, 1992; Tresnan et al., 1996).
Group III contains only avian viruses, which includes infectious bronchitis virus and turkey coronavirus.
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1.8.1 SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS
Severe acute respiratory syndrome (SARS), also called atypical pneumonia, is a human severe respiratory infectious disease that emerges recently in Asia, North America and Europe. SARS is characterized by severe pulmonary infection with a high degree of transmissibility and mortality (Chan-Yeung and Yu, 2003; Lew et al., 2003; Riley et al.,
2003). First evident in Guangdong province of China around November 2002, SARS quickly became a global outbreak, affecting more 35 countries and regions in the world across five continents. By the end of June 2003, there were 8,450 cases and 810 deaths
(www.cdc.gov/mmwr/mguide_sars.html), with an estimated mortality rates of 13.2 % for patients younger than 60 years and 43.3 % for patients aged 60 years or older (Donnelly et al., 2003).
A novel virus, named as SARS coronavirus (SARS-CoV), was identified as the etiological agent and was subsequently confirmed by fulfillment of Koch’s postulates
(Peiris et al., 2003; Ksiazek et al., 2003; Drosten et al., 2003; Fouchier et al., 2003).
Like other coronaviruses, SARS-CoV is a single stranded, positive-sense RNA virus with a genome of approximately 29,727 nucleotides and is organized into 13 to 15 open reading frames (ORFs) (Groneberg et al., 2005; Rota et al., 2003; Thiel et al., 2003).
The virion is 80-140 nm in diameter, consisting of a nucleocapsid (N) core surrounded by
an envelope containing three membrane proteins, spike (S), membrane (M) and envelope
(E), with 20-40 nm complex surface projections surrounding the periphery (Rota et al.,
2003; Ksiazek et al., 2003). S protein is thought to be involved with receptor binding, E protein plays a role in viral assembly, M is important for budding, and N protein is associated with viral RNA packaging (Holmes, 2003). Phylogenetic analysis of the
41 CHAPTER 1 LITERATURE REVIEW
SARS-CoV has demonstrated that this new pathogen is distinct from the three known serological groups of coronaviruses (Drosten et al., 2003; Ksiazek et al., 2003).
However, in a recent study of the replicase gene, SARS-CoV was classified as an early split-off of group 2 coronaviruses, which includes human HCV-OC43, mouse hepatitis virus and bovine coronavirus (Snijder et al., 2003). This was further supported by the conserved cysteine distribution pattern of the major surface spike glycoprotein
(Eickmann et al., 2003). Despite the evolutionary overlap between SARS-CoV and
Group II coronavirus genome sequences, the SARS genome lacks a gene for hemagglutinin-esterase (HE) protein, which is common to a majority of Group II coronaviruses (Groneberg et al., 2005).
The putative natural reservoir of SARS-CoV has been reported to be the
Himalayan or masked palm civet, Paguma larvata (order Carnivora, family Viverridae).
However, additional virological and serological evidence suggests that raccoon dog and
Chinese ferret badger (order Carnivora, families Canidae and Mustelidae, respectively) may also be naturally infected with SARS-CoV (Guan et al., 2003). As the SARS-CoV polymerase gene has a recombination breakpoint, suggesting multiple genetic origins for this molecule, these evolutionary mechanisms may have facilitated the adaptation of the animal-borne SARS-CoV ancestor to the human host (Rest & Mindell, 2003). The modes of SARS-CoV transmission include shedding of the virus from respiratory tract via droplets, close contact, and formites. The incubation period of SARS ranges from 2 to 26 days, with a mean incubation of 6.4 days (Chan-Yeung & Xu, 2003; Yu, 2004).
Unlike the known human HCoV-299E and OC43, which infect the upper respiratory tract and cause common cold, the new SARS-CoV predominantly causes
42 CHAPTER 1 LITERATURE REVIEW
infection in the lower respiratory tract, causing lung lesions with high morbidity and
mortality (Lee et al., 2003; Tsang et al., 2003). Infection is usually characterized by
high-grade fever and chills, myalgia, headache, subsequently by a dry non-producive
cough and shortness of breath, loss of appetite and dyspnea, and may progress to
generalized interstitial infiltrates in the lung (Booth et al., 2003; Hui et al., 2003).
Gastrointestinal involvement is also common, with more than 20% of patients presenting
with watery diarrhea (Leung et al., 2003).
1.8.1.1 SARS-CoV Spike Protein
The S glycoprotein of SARS-CoV, a 1,255-amino-acid type I integral membrane
glycoprotein, is the prominent protein present in the viral membrane and present as the
typical spike structure found on the viral surface. The S glycoprotein consists of a leader
(amino acid 1 to 14), an ectodomain (amino acid 15 to 1190), a membrane-spanning
domain (amino acid 1191 to 1227), and a short intracellular tail (amino acid 1227 to
1255) (Rota et al., 2003). Sequences analysis of SARS-CoV revealed that the full-length
S protein has 23 potential N-linked glycosylation sites, and the glycoslation of this surface protein is a high-mannose structure (Rota et al., 2003; Krokhin et al., 2003).
Unlike many other coronaviruses, such as mouse hepatitis virus (MHV) in which the S protein is post-translationally cleaved into S1 and S2 subunit, no typical cleavage motif has been identified in the SARS-CoV S protein (Frana et al., 1985; Luytjes et al.,
1987; Rota et al., 2003). Nonetheless, its S1 and S2 domains were predicted by sequence alignment with other coronavirus S proteins (Spiga et al., 2003; Rota et al., 2003).
Because S protein is not cleaved, defining the exact location of the boundary between S1
43 CHAPTER 1 LITERATURE REVIEW
and S2 is difficult; presumably it is somewhere between residues around 672 and 758
(Xiao et al., 2003; Li et al., 2003). S1 forms the bulbous globular head and is responsible
for cell attachment. Its amino acid sequence is less conserved than S2, which forms the
membrane-anchored stalk region and is responsible for membrane fusion (Duquerroy et
al., 2005). The S1 domain (residues 12-672) of SARS-CoV S protein mediates virus
binding with angiotensin-converting enzyme 2 (ACE2), a metallopeptidase that has been
identified as a functional receptor for SARS-CoV (Li et al., 2003; Prabakaran et al.,
2004). The S2 domain (residues 681-1255), which contains a putative fusion peptide and
two heptad repeat (HR1 and HR2) regions, is responsible for fusion between viral and
target cellular membranes (Spiga et al., 2003; Rota et al., 2003).
Mapping of the receptor-binding domain have been ongoing. In particular,
Babcock et al. (2004) have determined that the first 510 amino acids of the SARS-CoV S glycoprotein contain the entire ligand-binding domain. Amino-terminal truncation of the first 510 residues of S protein further demonstrated that amino acids 270 to 510 contain the minimal receptor-binding domain of the SARS-CoV S glycoprotein. It was also found that independently folded fragments as short as 193 residues can specifically bind receptor molecules (Babcock et al., 2004).
1.8.1.2 Immunogenicity
Besides mediating the initial stages of viral entry, the S protein of SARS-CoV is also known to be responsible for inducing host immune responses and virus neutralization by antibodies (Buchholz et al., 2004). Recent studies have shown that S protein is one of the major antigens eliciting immune responses during infection where
44 CHAPTER 1 LITERATURE REVIEW sera from convalescent SARS patients also neutralized the virus (Hofmann et al., 2004;
Lu et al., 2004; He et al., 2004). The entry of SARS-CoV into cells can also be inhibited, when the antibodies specific for S protein compete with ACE2 for binding to the S glycoprotein, thus preventing virus-receptor interaction (Sui et al., 2004).
Several candidate vaccines such as DNA and vaccinia virus-based vaccines that express full-length S protein or soluble fragments including the whole ectodomain and fragments containing the receptor-binding domain induced SARS-CoV-specific neutralizing antibodies and protective immunity against SARS-CoV challenge (Gao et al., 2003; Yang et al., 2004; Bisht et al., 2004). These reports highlight the potential for developing peptide-based vaccine candidate if neutralizing epitopes of S could be identified.
1.8.2 TRANSMISSIBLE GASTROENTERITIS CORONAVIRUS
First isolated in 1946, Transmissible gastroenteritis coronavirus (TGEV), a member of the genus Coronavirus, family Coronaviridae, order Nidovirales, is an enteropathogenic coronavirus that causes a highly contagious enteric disease in swine of all ages (Doyle & Hutchings, 1946; Cavanagh, 1997). Within this family, TGEV forms an antigenic cluster with feline infectious peritonitis virus, feline enteric coronavirus, canine coronavirus and porcine respiratory coronavirus (Sanchez et al., 1990). The first clinical signs of TGEV in pigs are a roughening of the haircoat, shivering, vomiting and extreme thrist. This is followed by a severe, watery diarrhea that has a very putrid, characteristic odor, where the pigs will become dehydrated, weak and die within 2 to 5 days (NebGuide). While older animals generally recover, the mortality rate in piglets
45 CHAPTER 1 LITERATURE REVIEW
under the age of 2 weeks old approaches 100% (Siddell et al., 1983; Saif & Wesley,
1992). The enzootic form of the disease is becoming a major problem in swine nurseries
(Saif & Wesley, 1999). It has been estimated to cost US pig producers about 100 million
dollars annually due to death or poor growth of infected pigs (Wesley & Woods, 2001).
The viral genome consists of a single-stranded, positive-sense 28.5 kb RNA and is
constructed of three major structural proteins: a phosphorylated nucleoprotein (N protein)
and two glycoproteins, the membrane (M) and the spike (S) protein with 382, 262 and
1431 amino acids respectively (Saif & Wedley, 1982; Kapke & Brian, 1986; Laude et al.,
1987; Rasschaert & Laude, 1987). The S protein has a membrane-anchoring domain and is highly glycosylated. It is also thought to be the viral attachment protein which interacts with the cell receptor, porcine aminopeptidase N (APN) (Schultze et al., 1996;
Krempl et al., 1997). As the major inducer of TGEV-neutralizing antibodies, the S protein has been used mainly for the induction of protective immunity to TGEV (Delmas et al., 1986; Jim´enez et al., 1986; Laude et al., 1992).
Passive immunity is of primary importance in providing neonatal piglets with immediate protection against TGEV infection (Saif & Wesley, 1992). The protection of suckling piglets against TGEV infection is based on the uptake of specific lactogenic antibodies, mainly of the IgA class, in the milk of the TGEV immune sows. The induction of lactogenic immunity can be stimulated by presentation of selected antigens to the immune system in gut associated lymphoid tissues (Wesley et al., 1988;
Montgomery et al., 1974).
46 CHAPTER 1 LITERATURE REVIEW
1.8.2.1 TGEV Spike Protein
The S protein is 1431 residues long, highly glycosylated, with a membrane-
anchoring domain near its carboxy-terminus and a globular domain assumed to
correspond to its amino-terminal half (Rasschaert & Laude, 1987).
The surface S protein initiates the infection by binding to the cell surface and also mediates the subsequent fusion between the viral and cellular membranes. The S protein has two binding activities. Binding to aminopeptidase N has been reported to occur between residues 522 and 744, and is required for the initiation of infection (Godet et al,
1994; Delmas et al., 1992). Aminopeptidase N is an ectoenzyme abundantly expressed at
the apical membrane of the enterocytes and serves as a receptor for TGEV. In addition,
S protein has a salic acid binding activity which enables TGEV to recognize terminal
salic acid residues on glycoproteins and glycolipids, allowing TGEV to agglutinate erythrocytes (Schultze et al., 1996). Studies with mutants of TGEV indicated that residues with a short stretch of amino acid (145 to 209) are important for recognition of salic acids (Krempl et al., 1997; Krepml et al., 2000), where point mutations of the residues resulted in the loss of both the hemagglutinating activity and enteropathogenicity
(Krempl et al., 1997).
Neutralizing antibodies against the virus are directed mainly to the S glycoprotein,
and relevant epitopes in neutralization have been mapped into the N-terminal domain of
this protein (Garwes et al., 1978; Jim’enez et al., 1986; Correa et al., 1988). In this
protein, four antigenic sites (A, residues 538-591; B, residues 97-144; C, residues 49-52
and D, residues 378-395) have been defined by mutual competition of monoclonal
antibodies (Correa et al., 1988; Delmas et al., 1986; Gebauer et al., 1991). Epitopes
47 CHAPTER 1 LITERATURE REVIEW
critical for neutralization were found to cluster in the immunodominant A, and D sites,
and to a minor extend site B (Delmas et al., 1986; Jim’enez et al., 1986). Sites A, B and
D have been found to be highly conserved among TGEV strains, whereas antigenic
variation was observed on site C epitope (Enjuanes et al., 1992). Site A and B have been
demonstrated to be conformational and glycosylation dependent, while sites C and D are
continuous and glycosylation independent, although a small effect of glycosylation has
been observed in site D (Correa et al., 1988; Correa et al., 1990; Posthumus et al., 1990a;
Posthumus et al., 1990b; Gebauer et al., 1991). Site A being the major inducer of
neutralizing antibodies has been divided into three antigenic subsites: Aa, Ab and Ac,
with subset Ab being the most antigenic (Correa et al., 1988; De Diego M et al., 1992).
1.8.2.2 Vaccines Developed Against TGEV
To date, genetically engineered and attenuated agents have been utilized for the
development of vaccines against TGEV. The ability of Salmonella typhimurium to
colonize the intestinal tract has prompted the development of Salmonella typhimurium as a bivalent vector to induce an immune response against both Salmonella and TGEV
(Smerdou et al., 1996). When attenuated Salmonella vaccine constructs expressing
TGEV S protein were orally fed to mice, TGEV-neutralizing antibodies was detected in a
significant number of immunized animals (Chen & Schifferli, 2003).
In addition, transgenic plants expressing the S protein were also created. Extracts
from potato tubers or arabidopsis expressing the N-terminal domain of the S glycoprotein
that were inoculated intraperitoneally or orally to mice developed serum IgG specific for
TGEV (Gomez et al., 2000, Gomez et al., 1998). Antigens from transgenic tobacco
48 CHAPTER 1 LITERATURE REVIEW
plants also induced TGEV-specific immune responses in pigs as determined by virus
neutralization and ELISA (Tuboly et al., 2000).
Recombinant adenoviruses and baculoviruses expressing either fragments or the
full-length TGEV S protein were used to study the induction of antibodies in hamster and
swine. In particular, Torres et al. has constructed ten adenovirus recombinants
expressing either full length or truncated amino-terminal fragments of the S protein
(Torres et al., 1995). These recombinants induced immune responses in both hamsters
and swine, which neutralized infectivity, though higher titers of antibodies was induced
in swine than in hamsters.
1.9 OBJECTIVES
Development of less invasive and more readily administered vaccines has become a priority for public health agencies, and new vaccine formulation and delivery
technologies have emerged. There is great interest in the use of mucosal membranes for
noninvasive vaccine administration, especially when the mucosal surfaces of the gastrointestinal and respiratory tracts represent the principal portals of entry for most pathogens. However, most vaccines licensed for use in humans and animals are injected intramuscularly or subcutaneously. The mucosal immune responses induced by parenteral immunization are generally weaker, are more variable, and last for a briefer time than do mucosally induced responses (McGhee et al., 1992; Liu et al., 1998; Kaul et
al., 1998; McCluskie et al., 2002; Goonetilleke et al., 2003). As such, the focus of this study is to determine the feasibility of using bacteria and yeast as vectors for the delivery of coronavirus antigens via the intragastric route.
49 CHAPTER 1 LITERATURE REVIEW
Hence, the specific objectives of this study are as follow: a) To analyze the adhesion capabilities of Lactobacillus casei Shirota, Saccharomyces
boulardii, Saccharomyces cerevisiae and Pichia pastoris on mucosal surfaces. b) To select one of the yeasts for development into an oral vaccine carrier. c) To engineer L.casei Shirota and the selected yeast to express an antigenic fragment of
Transmissible Gastroenteritis coronavirus spike protein. d) To conduct immunization studies and compare the efficacy of using prokaryotic and
eukaryotic vectors for oral delivery of the coronavirus antigen. e) To generate oral vaccine for Severe Acute Respiratory Syndrome coronavirus using
the suitable vector (Lactobacillus spp. or yeast).
50 CHAPTER 2 MATERIALS & METHODS
2.0 MATERIALS AND METHODS
2.1 ADHESION STUDIES
2.1.1 Bacteria and Yeast Strains
The bacteria and yeast used in this study are given in Table 2.1. Lactobacillus
o casei Shirota was cultured in MRS medium (Merck, Germany) at 37 C with 5 % CO2.
Saccharomyces boulardii, Saccharomyces cerevisiae and Pichia Pastoris GS115 were
cultured in Yeast Extract Peptone Dextrose medium [(YPD) (Appendix 1)] at 30 oC with
shaking at 200 rpm.
Table 2.1 Bacterial and yeast strains used in adhesion study.
Strain Abbreviations Characteristics Source
Lactobacillus casei LcS Probiotic bacteria used in Yakult, Singapore Shirota fermented beverages. Saccharomyces S.boulardii Probiotic yeast consumed American Type boulardii commonly in the form of Culture Collection tablets. (ATCC), USA Saccharomyces S.cerevisiae Baker’s yeast used to Levure Sèche de cerevisiae ferment dough when Boularger, France baking bread. Pichia pastoris P.pastoris Yeast used for high Invitrogen, USA GS115 throughput protein expression.
2.1.2 Adhesion Studies in Vivo
2.1.2.1 Preparation of Fluorogenic dye
Five-(and 6-) carboxyfluorescein diacetate succinimidyl ester [(cFDA-SE)
(Molecular Probes, USA)] is a non-fluorescent membrane-permeable ester which non-
specific prokaryotic and eukaryotic intracellular esterases convert to a fluorescent
51 CHAPTER 2 MATERIALS & METHODS derivative, that is in turn covalently linked to intracellular proteins via the probe’s succinimidyl group (Weston & Parish, 1990). cFDA-SE was prepared as previously described by Logan and co-workers (1998), with slight modifications. In brief, a 3.6 mM stock solution of cFDA-SE was prepared by first dissolving in 20 μl of di-methyl- sulphoxide [(DMSO) (Merck, Germany)] and further diluting in 1ml of reagent grade ethanol. This was then filter-sterilized with 0.2 μm Acrodisc (Gelman, USA) before being aliquoted and stored at –20oC.
2.1.2.2 Labeling of Lactobacillus spp. and Yeast with Fluorescent Probe
Overnight culture of Lactobacillus spp. or yeast were inoculated into fresh broth
(Appendix 1) and incubated at the appropriate temperature (37 oC for Lactobacillus spp. and 30 oC yeast. The log-phase cultures were centrifuged at 3000 x g for 10 min, whereafter the pellet was washed twice in sterile PBS (Appendix 5). The pellet were then adjusted to give a cell concentration of 1010 CFU ml-1 prior to labeling with 50 μM cFDA-SE at 37 oC with shaking for 20 min. Pelleting the cells and washing twice in PBS to remove excess cFDA-SE terminated fluorescent labeling. Labeled LcS and yeast were resuspended in PBS (Appendix 5) to the desired concentration and viewed under conventional optical-fluorescence microscope (Olympus 1X81, Japan) with excitation wavelength of 480 nm and emission wavelength of 518 nm to check for labeling. The labeled organisms were also inoculated into fresh culture media and allowed to replicate for a few generations where the median fluorescence intensities were determined via flow cytometric analysis (Section 2.1.2.4) before proceeding further.
52 CHAPTER 2 MATERIALS & METHODS
2.1.2.3 Feeding of Mice with Labeled LcS or Yeast
Seven-week-old female BALB/c mice were obtained from the Laboratory
Animals Centre, National University of Singapore, and maintained at the University’s
Animal Holding Unit of the Department of Microbiology. The mice had free access to standard mouse diet and water. For each of the microorganisms investigated, a group of
15 mice had been orally dosed with approximately 109 cFDA-SE labeled LcS,
S.boulardii, S.cerevisiae or P.pastoris by orogastric intubation. Another group of 12
mice that have been orally fed with sterile PBS served as control. The food and water
intake of the experimental and control batches were measured daily. In addition, the
daily faeces production was measured and collected for mucin extraction. Groups of
three mice were sacrificed on days 1, 2, 3, 4 and 6 post-dosing by CO2 asphyxiation. The
duodenum, jejunum, ileum and colon were removed from each animal and individual
sections were cut longitudinally. Any visible residual food particles or faecal material
were removed. For determination of the intestinal water volume (IV), each intestinal part
was gently blotted dry (i.e. until no visible water mark remained on the filter) using pre-
weighed filter paper (90 nm Dia, Whatman®, UK), and the increase in the weight of the
paper being taken as the IV of the respective intestinal portion. The various intestinal
parts were then examined for the presence of adhering cFDA-SE labeled LcS. This was
accomplished by adding 150 μl of PBS to every 1.0 cm of tissue and dislodging microbes
from the mucosal surface of the tissues with the aid of a plunger from a syringe (1.0 ml,
Terumo®, Japan). Cell extracts were fixed with formaldehyde (final vol = 0.75 % v/v)
and filtered using SEFAR NITEX 60 μm polyester mesh filter (SEFAR, USA) to remove intestinal cells present in the intestinal extract prior to flow cytometric analysis.
53 CHAPTER 2 MATERIALS & METHODS
2.1.2.4 Flow cytometric analysis
Enumeration of cFDA-SE-stained LcS, S.boulardii, S.cerevisiae or P.pastoris
yeast from cell extracts were performed on a Coulter EPICS® ELITE flow sorter (Miami,
USA) at an excitation wavelength of 488 nm using a 15 mW Argon laser with a 75 mm sort sense flow cell at 82.7 kPa sheath pressure. Upon excitation at the wavelength of
488 nm in the flow cytometer, cFDA-SE gives a maximal emission signal in green at the wavelength of 518 nm. Data were recorded in the FCS2.0 file format using Coulter Epics
Elite (ver 4.01) software, then analyzed and converted into plots using WinMDI (ver 2.8)
software (J.Trotter, Scripps Research Inst., La Jolla, CA, USA). The sample volume was
estimated from the counts of the reference glass beads included in the sample.
2.1.2.5 Mucus extraction from fecal material
Mucus was isolated from murine feces by extraction and dual ethanol precipitation with modification from Miller and Hoskins (1981). In short, fecal samples collected daily were diluted 4 times in PBS containing protease inhibitors (1mM phenylmethysulfonyl fluoride, 2 mM iodoacetamide and 10 mM EDTA), and slowly stirred for 1 hr at 4 oC. The suspension was centrifuged at 15, 000 x g at 4 oC for 30 min
and the supernatant collected. Absolute ethanol was added to the supernatant to a final
concentration of 70 %. This was then centrifuged at 10, 000 x g at 4 oC for 30 min and
the precipitate were collected. The pellet was dissolved in milli-Q water and again
precipitated with ethanol (70 % v/v) whereafter the extracted mucus was lyophilised.
The dry weight of the mucus was then measured and the amount of mucus in the feces
was calculated.
54 CHAPTER 2 MATERIALS & METHODS
2.1.3 Adhesion Studies In Vitro
Fifty milliliters overnight culture of LcS, S.boulardii, S.cerevisiae or P.pastoris
were centrifuged at 5000 x g for 8 min and washed once with sterile PBS. The pellet was
resuspended in sterile PBS and 109 cfu of LcS, S.boulardii, S.cerevisiae or P.pastoris was
added to confluent HT-29 or Caco-2 cells grown on coverslips (Section 2.12.5). The
o bacteria or yeast and cell mixture was incubated at 37 C with 5 % CO2 for 1 hr to allow
adhesion to take place. The culture media was then removed and the coverslips were
gently rinsed twice with 1.5 ml PBS (Appendix 5), after which the coverslips were fixed
and processed for viewing by scanning electron microscopy (Section 2.14).
2.2 MOLECULAR CLONING TECHNIQUES
2.2.1 Bacterial and Yeast Strains
The bacteria and yeast used in this study are given in Table 2.2. Escherichia coli
TOP10, DH10β and JM105 were culture in Luria Bertani [(LB) (Appendix 1)] broth at 37
oC with shaking at 200 rpm, while LcS was cultured in MRS medium (Merck, Germany)
o at 37 C with 5 % CO2. P.pastoris was cultured in Yeast Extract Peptone Dextrose
medium [(YPD) (Appendix 1)] at 30 oC with shaking. Recombinant E.coli, LcS or
P.pastoris harboring plasmids with antibiotic resistant genes were grown in the respective
medium with the appropriate antibiotics.
55 CHAPTER 2 MATERIALS & METHODS
Table 2.2 Bacteria and yeast strains used in the study.
Strain Abbreviation Characteristics Source
One Shot® TOP10 TOP10 Ampicillin sensitive Invitrogen, Escherichia coli (Amps); bacterial host USA ® - for pCR F mcrA (mrr-hsdRMS- -XL- TOPO® mcrBC) 80lacZ M15 clones containing TGEV lacX74 recA1 ara 139 (ara- spike gene fragment leu)7697 galU galK rpsL (StrR) endA1 nupG DH10β Escherichia coli DH10β Ampicillin sensitive Promega, USA s - (Amp F mcrA (mrr-hsdRMS- ); bacterial host for pLP500 and mcrBC) 80lacZ M15 construction of clones lacX74 recA1 endA1 ara of TGEV spike gene 139 (ara-leu)7697 galU R fragment galK λ-rpsL (Str ) nupG JM105 Escherichia coli JM105 Ampicillin sensitive ATCC, USA s F' traD36 proA+ proB+ lacIq (Amp ); bacterial host delta(lacZ)M15 delta(pro-lac) for pGEX-4T-3 hsdR4 sbcB15 rpsL thi endA1 clones of TGEV lambda- spike gene fragment Lactobacillus casei strain LcS Erythromycin Yakult, sensitive (Erys); Singapore Shirota expression host and oral vaccine delivery vehicle of TGEV Spike protein fragment Pichia pastoris GS115 P.pastoris Zeocin sensitive Invitrogen, S - (Zeo USA his4 ); expression host and oral vaccine delivery vehicle of TGEV Spike protein fragment and SARS RBD protein
56 CHAPTER 2 MATERIALS & METHODS
2.2.2 Cloning and Expression Vectors
The following table (Table 2.3) shows the list of cloning and expression vectors that were used in this study.
Table 2.3 Molecular vectors and related information.
Plasmid Purpose Source
Shuttle vector to facilitate cloning DNA fragment into pCR®-XL-TOPO® Lactobacillus expression Invitrogen, USA. vector, Zeor, Kanr, 3.5 kb.
Lactobacillus expression vector for heterologous TNO-Nutrition protein expression under and Food control of constitutive pLP500 Research lactate dehygrogenase (ldh) Institute, promoter. Ampr, Eryr, 7.3 Netherlands. kb.
P.pastoris expression vector for recombinant protein expression under control of pGAPZαC Invitrogen, USA. constitutive GAP promoter. Zeor, 3.1 kb.
Glutathione S-transferase fusion vectors under the Amersham pGEX-4T-3 control of tac promoter Biosciences, UK Ampr, 4.9 kb.
Zeor : Zeocin resistance gene; Kanr : Kanamycin resistance gene; Ampr : Ampicillin resistance gene; Eryr : Erythromycin resistance gene.
pCR®-XL-TOPO® is a TA cloning vector that not only enable fast and efficient cloning of Taq-amplified PCR products, but also facilitates subsequent cloning of the
57 CHAPTER 2 MATERIALS & METHODS gene of interest into the desired expression vector. The restriction map and complete sequence is shown in Appendix 2.
pLP500 which mediates the expression and secretion of foreign protein under the control of the constitutive lactate dehygrogenase (ldh) promoter, was kindly given by
Prof. Pouwels PH of TNO-Nutrition and Food Research Institute, Netherlands. A very useful feature of pLP500 is that the plasmid is able to replicate not only in Lactobacillus spp., but also in some strains of E.coli. All elements needed for these specific functions, promoter, ribosome binding region, translation initiation sequences, secretion signal sequence and terminal sequence, are separated by unique restriction sites, facilitating the use of these plasmids in the directed expression of cloned genes. Immediately downstream from the translation initiation region, a multiple cloning sites sequence harboring a few unique restriction sites was introduced to allow cloning of heterologous gene sequences. The restriction map and complete sequence of pLP500 is shown in
Appendix 2.
pGAPZαC uses the GAP promoter of glyceraldehyde-3phosphate dehydrogenase
(GADPH) to constitutively express recombinant protein in P.pastoris. Recombinant proteins can be expressed as fusions to the N-terminal peptide encoding the
Saccharomyces cerevisiae α-factor secreting signal for constitutive and secretable expression of protein of interest. The restriction map and complete sequence of pLP500 is shown in Appendix 2.
pGEX-4T-3 is a Glutathione S-transferase (GST) fusion vector that allows protein of interest to be tagged with GST for easy purification. The restriction map and complete sequence is shown in Appendix 2.
58 CHAPTER 2 MATERIALS & METHODS
2.2.3 Restriction Digestion of DNA
Enzymes from New England Biolabx Inc. (USA) were used for the enzymatic digestion of inserts or plasmids in buffers and conditions as specified by the manufacturer. In general, an appropriate amount of DNA and the corresponding 10x restriction buffer were dispensed into microcentrifuge tubes and made up to the correct volume with double glass distilled water [(dgd) (Megapure, Corning, USA)]. A ratio of
2-4 units of restriction endonuclease per µg of substrate DNA was employed, thereafter the contents were mixed by flicking the reaction tube and incubated at 37 ºC for 2 hr.
The total enzyme added must not exceed one tenth of the total volume, as the enzyme preparations used in this study contained 50 % glycerol, which in excess affect the activities of the enzymes. The digestion results were interpreted by running the digestion mix on agarose gel.
2.2.4 Ligation and Transformation
One unit of T4 DNA ligase (New England Biolabs, USA) was used in a total reaction volume of 20 µl. The DNA concentration in the ligation mix was about 70-80
µg/ml and the vector:insert ratio was 1:2. Both vector and the insert were cleaved with the same set of restriction endonucleases to generate complementary sticky ends to facilitate ligation. The reaction was carried out at 16 ºC for at least 2 hr in 1x T4 DNA ligase reaction buffer. The ligation mixture was used directly to transform competent
E.coli cells. The ligated vector (10 µl) was added to 50 µl of E.coli competent cells and incubated on ice for 30 min. The cells were the heat-shock at 42 ºC for 1 min, after which the transformation mixture was returned immediately onto ice for 2 min. For the
59 CHAPTER 2 MATERIALS & METHODS
recovery of the transformed cells, 1 ml of LB broth (Appendix 1) was added to the cells
and incubated at 37 ºC with shaking (200 rpm) for 1 hr. The cells were the plated on pre-
warmed LB agar plate (Appendix 1) with the appropriate selective marker and incubated
overnight at 37 ºC for the selection of clones.
2.2.5 Agarose Gel Electrophoresis
To prepare a 1.0 % agarose gel, 1.0 g of agarose (Sigma, USA) was mixed with
100 ml of 1x TBE buffer (Appendix 2) in a 250 ml conical flask and heated in a
microwave to dissolve the agarose. After cooling to about 45 ºC, ethidium bromide
[(EtBr)(1μg/ml)(Sigma, USA)] was added and mixed, after which the agarose-EtBr
mixture was poured into a gel tray assembled within a gel box on a leveled platform and
the comb placed into position. The gel was allowed to set at room temperature for
30 min. The solidified gel was then submerged in the Bio-Rad Wide Mini-sub Cell GC
tank filled with 1x TBE buffer (Appendix 2). Ten to twenty µl of DNA samples were
mixed with gel loading buffer (Appendix 2) before loading into the wells. Ten μl of 1 kb
or 100 bp DNA ladder (New England Biolabs, USA; Promega, USA) was also loaded as
a relative molecular size reference. The sample was then electrophorized at 120 Volts for
30 min. The DNA bands were then visualized and photographed under UV trans- illuminator using ChemiGenius2 Bio imaging system (Syngene, UK).
2.2.6 DNA Purification from Agarose Gel
DNA electrophoretically separated on agarose gel was extracted using QIAquick
II Gel Extraction Kit (Qiagen, Germany) accordingly to the manufacturer’s protocol.
60 CHAPTER 2 MATERIALS & METHODS
Briefly, the DNA band of interest was excised from the gel using a clean scalpel and
placed into a sterile eppendorf tube where 10 μl of Capture Buffer was added for every
10 mg of gel slice. The tube was vortexed vigorously and incubated at 60 oC until the gel
was completely dissolved (~10 min). The dissolved sample was transferred to the
QIAquick spin column placed in a collection tube, which was incubated at room
temperature for 1 min. After incubation, the sample was centrifuged at 15, 000 x g for 30 sec. The flow-through was discarded and 500 μl of Wash Buffer was added. The column was then centrifuged at 15, 000 x g for 30 sec and the spin column was transferred to a sterile 1.5 ml eppendorf tube. For the elution of DNA, 50 μl of nuclease free water was added directly to the top of the glass fiber matrix in the spin column, and incubated at room temperature for 1 min. The purified DNA was recovered by centrifugation at 15, 000 x g for 1 min, and stored at -20 ºC.
2.2.7 Plasmid Isolation and Purification
Well-defined colonies were picked from the culture plate and inoculated into 5 ml of LB broth (Appendix 1) containing appropriate concentration of antibiotic (100 μg/ml of ampicillin or 50 μg/ml of kanamycin) in sterile 15 ml tubes. The tubes were incubated at 37 °C with shaking at 200 rpm overnight. The plasmids were then isolated following the procedures of the Wizard® Plus SV Miniprep DNA Purification System (Promega,
USA).
Briefly, the cells were harvested by centrifugation at 8000 x g for 5 min and the
pellet was resuspended in 250 μl of Cell Resuspension Solution and transferred to a
sterile eppendorf tube. The cells were first lysed with 250 μl of Cell Lysis Solution, and
61 CHAPTER 2 MATERIALS & METHODS
10 μl of Alkaline Protease Solution was added to inactivate endonucleases and other
proteins released by the lysed cells. The reaction mixture was then neutralized with 350
μl of Wizard® Plus SV Neutralization Solution and was centrifuged at 8000 x g. The
cleared lysate was transferred to a Spin Column, and washed twice with 750 μl and 250
μl of Column Wash Solution previously diluted with 95 % ethanol (Merck, Germany).
The spin column was then transferred to a sterile 1.5 ml eppendorf tube and the plasmid
DNA was eluted with 100 μl of nuclease free water.
2.3 GENERATION OF RECOMBINANT LCS EXPRESSING TGEV SPIKE
PROTEIN FRAGMENT
2.3.1 Complementary DNA (cDNA) Synthesis of TGEV gene
RNA sample extracted from TGEV (Section 2.13.5) was first reverse transcripted into cDNA. The reaction mixture consisted of 2 μl of 10 x Reaction Buffer, 4 μl of
25 mM MgCl2, 2 μl of Deoxynucleotide Mix, 1 μl of gene specific reverse primer, 1 μl
RNAse inhibitor, 0.8 μl Superscript Reverse Transcriptase (Invitrogen, USA), and 8.2 μl of RNA sample. The mixture was briefly vortexed and pulsed before being incubated at
25 oC for 10 min and then 42 oC for 60 min. The cDNA was then stored at –20 oC.
2.3.2 Amplification of TGEV Spike Gene
2.3.2.1 Primers Sequences and Related Information
The sequence, size and Tm of the primers used for the cloning of TGEV-S gene
into pLP500 are listed in Table 2.4.
62 CHAPTER 2 MATERIALS & METHODS
Table 2.4 Primers used in this part of study.
Primer Sequence Size Tm Polarity 5’ 3’ (nts) (oC) Tgeplp-FI CGGGATCCAATGCCATTGATTTATGGAGACAA 29 76 Forward
Tgeplp-RII CGGCTAGCTTAGACCTAACAGTTCACTG 28 70 Reverse pLP5005’ GCATGCACATAAGAAAGGATGAT 23 58 Forward
2.3.2.2 Polymerase Chain Reaction (PCR)
Two μl of the cDNA was used for the amplification of TGEV Spike gene via
PCR. The primers Tgeplp-FI and Tgeplp-RII were synthesized to specifically amplify a
2.3 kb N-terminal fragment (nucleotides 30-2293) of the TGEV spike gene (rTGEV-S)
that encompasses all the major antigenic domains of the spike protein (Figure 2.1,
GenBank accession number: AF302263). The 8-bp overhang contains a BamHI
restriction site in Tgeplp-FI, and a 8-bp overhang coding NheI in Tgeplp-RII for cloning
procedures. The reaction mixture consisted of 37.5 μl of dgd water, 5 μl of 10x reaction buffer, 4 μl of dNTPs (5mM), 0.5 μl of forward primer (50 pmol/μl), 0.5 μl of reverse
primer (50 pmol/μl), 0.5 μl of DNA Polymerase (Qiagen, Germany) and 2 μl DNA template. The reaction mixture was vortexed before it was amplified in a PCR machine
(Hybaid PCR ExpressTM, Hybraid Ltd). This mixture was heated to 94 oC for 5 min, and
then amplified for 30 cycles, each consisted of 94 oC for 30 sec, 55 oC for 30 sec and 72
oC for 2 min, which enabled repetitive DNA denaturation, primer hybridization and
primer extension, respectively. An extra step of 72 oC for 7 min allowed final extension
to occur and a holding step at 4 oC.
63 CHAPTER 2 MATERIALS & METHODS
DEFINITION - Porcine transmissible gastroenteritis virus spike protein S mRNA
ACCESSION - AF302263
translation="MKKLFVVLVVMPLIYGDNFPCSKLTNRTIGNHWNLIETFLLNYS SRLPPNSDVVLGDYFPTVQPWFNCIRNNSNDLYVTLENLKALYWDYATENITWNHKQR LNVVVNGYPYSITVTTTRNFNSAEGAIICICKGSPPTTTTESSLTCNWGSECRLNHKF PICPSNSEANCGNMLYGLQWFADAVVAYLHGASYRISFENQWSGTVTLGDMRATTLET AGTLVDLWWFNPVYDVSYYRVNNKNGITVVSNCTDQCASYVANVFTTKPGGFIPSDFS FNNWFLLTNSSTLVSGKLVTKQPLLVNCLWPVPSFEEAASTFCFEGAGFDQCNGAVLN NTVDVIRFNLNFTTNVQSGKGATVFSLNTTGGVTLEISCYNDTVSDSSFFSYGEIPFG VTDGPRYCYVLYNGTALKYLGTLPPSVKEIAISKWGHFYINGYNFFSTFPIDCISFNL TTGDSDVFWTIAYTSYTEALVQVENTAITKVTYCNSYVNNIKCSQLTANLNNGFYPVS SSEVGLVNKSVVLLPSFYTHTIVNITIGLGMKRSGYGQPIASTLSNITLPMQDKNTDV YCIRSDQFSVYVHSTCKSSLWDNIFKRNCTDVLDATAVIKTGTCPFSFDKLNNYLTFN KFCLSLSPVGANCKFDVAARTRTNDQVVRSLYVIYEEGDNIVGVPSDNSGLHDLSVLH LDSCTDYNIYGRTGVGIIRQTNRTLLSGLYYTSLSGDLLGFKNVSDGVIYSVTPCDVS AQAAVIDGTIVGAITSINSELLGLTHWTTTPNFYYYSIYNYTNDRIRGTAIDSNDVDC EPVITYSNIGVCKNGAWVFINVTHSDGDVQPISTGNVTIPTNFTISVQVEYIQVYTTP VSIDCSRYVCNGNPRCNKLLTQYVSACQTIEQSLAMGARLENMEVDSMLFVSENALKL ASVEAFNSSETLDPIYKEWPNIGGSWLEGLKYILPSDNSKRKYRSAIEDLLFDKVVTS GLGTVDEDYKRCTGGYDIADLVCAQYYNGIMVLPGVANADKMTMYTASLAGGITLGAL GGGAVAIPFAVAVQARLNYVALQTDVLNKNQQILASAFNQAIGNITQSFGKVNDAIHQ TSRGLATVAKALAKVQDVVNTQGQALSHLTVQLQNNFQAISSSISDIYNRLDELSADA QVDRLITGRLTALNAFVSQTLTRQAEVRASRQLAKDKVNECVRSQSQRFGFCGNGTHL FSLANAAPNGMIFFHTVLLPTAYETVTAWAGICALDGDRTFGLVVKDVQLTLFRNLDD KFYLTPRTMYQPRVATSSDFVQIEGCDVLFVNATVSDLPSIIPDYIDINQTVQDILEN FRPNWTVPELTFDIFNATYLNLTGEIDDLEFRSEKLHNTTVELAILIDNINNTLVNLE WLNRIETYVKWPWYVWLLIGLVVIFCIPLLLFCCCSTGCCGCIGCLGSCCHSICSRRQ FENYEPIEKVHVH"
1 atgaaaaaat tatttgtggt tttggtcgta atgccattga tttatggaga caattttcct 61 tgttctaaat tgactaatag aactataggc aaccattgga atctcattga aacctttctt 121 ctaaactata gtagtaggtt accacctaat tcagatgtgg tgttaggtga ttattttcct 181 actgtacaac cttggtttaa ttgcattcgc aataatagta atgaccttta tgttacattg 241 gaaaatctta aagcattgta ttgggattat gctacagaaa acatcacttg gaatcacaaa 301 caacggttaa acgtagtcgt taatggatac ccatactcca tcacagttac aacaacccgc 361 aattttaatt ctgctgaagg tgctattata tgcatttgta agggctcacc acctactacc 421 accacagaat ctagtttgac ttgcaattgg ggtagtgagt gcaggttaaa ccataagttc 481 cctatatgtc cttctaattc agaggcaaat tgtggtaata tgctgtatgg cctacaatgg 541 tttgcagatg cggttgttgc ttatttacat ggtgctagtt accgtattag ttttgaaaat 601 caatggtctg gcactgttac acttggtgat atgcgtgcga ctacattaga aaccgctggc 661 acgcttgtag acctttggtg gtttaatcct gtttatgatg tcagttatta tagagttaat
64 CHAPTER 2 MATERIALS & METHODS
721 aataaaaatg gtattaccgt agtttccaat tgcactgatc aatgtgctag ttatgtggct 781 aatgttttta ctacaaagcc aggaggtttt ataccatcag attttagttt taataattgg 841 ttccttctaa ctaatagctc cacgttggtt agtggtaaat tagttaccaa acagccgtta 901 ttagttaatt gcttatggcc agtccctagc tttgaagaag cagcttctac attttgtttt 961 gaaggtgctg gctttgatca atgtaatggt gctgttttaa ataacactgt agacgtcatt 1021 aggtttaacc ttaattttac tacaaatgta caatcaggta agggtgccac agtgttttca 1081 ttgaacacaa cgggtggtgt cactcttgaa atctcatgtt ataatgatac agtgagtgac 1141 tcgagctttt tcagttacgg tgaaattccg ttcggcgtaa ctgatggacc acggtactgt 1201 tacgtactct ataatggcac agctcttaag tatctaggaa cattaccacc tagtgtcaag 1261 gagattgcta ttagtaagtg gggccatttt tatattaatg gttacaattt ctttagcaca 1321 tttcctattg attgtatatc ttttaatttg accactggtg atagtgacgt tttctggaca 1381 atagcttaca catcgtacac tgaagcatta gtacaagttg aaaacacagc tattacaaag 1441 gtgacgtatt gtaatagtta cgttaataac attaaatgct ctcaacttac tgctaatttg 1501 aataatggat tttatcctgt ttcttcaagt gaagttggtc ttgtcaataa gagtgttgtg 1561 ttactaccta gcttttacac acataccatt gttaacataa ctattggtct tggtatgaag 1621 cgtagtggtt atggtcaacc catagcctca acattaagta acattacact accaatgcag 1681 gacaagaaca ccgatgtgta ctgtattcgt tctgaccaat tttcagttta tgttcattct 1741 acttgcaaaa gttctttatg ggacaatatt tttaagcgaa actgcacgga cgttttagat 1801 gccacagctg ttataaaaac tggtacttgt cctttctcat ttgataaatt gaacaattac 1861 ttaactttta acaagttctg tttgtcgttg agtcctgttg gtgctaattg taagtttgat 1921 gtagctgccc gtacaagaac caatgatcag gttgttagaa gtttgtatgt aatatatgaa 1981 gaaggagaca acatagtggg tgtaccgtct gataatagtg gtttgcacga tttgtcagtg 2041 ctacacctag attcctgcac agattacaat atatatggta gaactggtgt tggtattatt 2101 agacaaacta acaggacgct acttagtggc ttatattaca catcactatc aggtgatttg 2161 ttagggttta aaaatgttag tgatggtgtc atctactctg taacgccatg tgatgtaagc 2221 gcacaagcag ctgttattga tggcaccata gttggggcta tcacttccat taacagtgag 2281 ctgttaggtc taacacattg gacaacaaca cctaattttt attactactc tatatataat 2341 tacacaaatg ataggattcg tggcactgca attgacagta atgatgttga ttgtgaacct 2401 gtcataacat attctaacat aggtgtttgt aaaaatggtg cttgggtttt tattaacgtc 2461 acacattctg atggagacgt gcaaccaatt agcactggta atgtcacgat acctacaaac 2521 tttaccatat ccgtgcaagt cgaatacatt caggtttaca ctacacccgt gtcaatagac 2581 tgttcaagat atgtttgtaa tggtaaccct aggtgtaaca aattgttaac acaatacgtt 2641 tctgcatgtc aaactattga gcaatcactt gcaatgggtg ccagacttga aaacatggag 2701 gttgattcca tgttgtttgt ttctgaaaat gcccttaaat tggcttctgt cgaagcattc 2761 aatagttcag aaactttaga tcctatttac aaagaatggc ctaatatagg tggttcttgg 2821 ctagaaggtc taaaatatat acttccgtcc gataatagca aacgtaagta tcgttcagct 2881 atagaggact tgctttttga taaggttgta acatctggtt taggtacagt tgatgaagat 2941 tataaacgtt gtacaggtgg ttatgacata gctgacttag tatgtgctca atactataat 3001 ggcatcatgg tgctacctgg tgtggctaat gctgacaaaa tgactatgta cacagcatcc 3061 cttgcaggtg gtataacatt aggtgcactt ggtggaggcg ccgtggctat accttttgca 3121 gtagcagttc aggctagact taattatgtt gctctacaaa ctgatgtatt gaataaaaac 3181 cagcagatcc tggctagtgc tttcaatcaa gctattggta acattacaca gtcatttggt 3241 aaggttaatg atgctataca tcaaacatca cgaggtcttg ccactgttgc taaagcattg
65 CHAPTER 2 MATERIALS & METHODS
3301 gcaaaagtgc aagatgttgt caacacacaa gggcaagcat taagccacct aacagtacaa 3361 ttgcaaaata atttccaagc cattagtagt tctattagtg acatttataa taggcttgat 3421 gaattgagtg ctgatgcaca agttgacagg ctgatcacag gaagacttac agcacttaat 3481 gcatttgtgt ctcagactct aaccagacaa gcggaggtta gggctagtag acaacttgcc 3541 aaagacaagg ttaatgaatg cgttaggtct cagtctcaga gattcggatt ctgtggtaat 3601 ggtacacatt tgttttcact cgcaaatgca gcaccaaatg gcatgatctt ctttcacaca 3661 gtgctattac caacggctta tgaaactgtg actgcttggg caggtatttg tgctttagat 3721 ggtgatcgca cttttggact tgtcgttaaa gatgtccagt tgactttgtt tcgtaatcta 3781 gatgacaagt tctatttgac ccccagaact atgtatcagc ctagagtggc aactagttct 3841 gattttgttc aaattgaagg gtgcgatgtg ctgtttgtta atgcaactgt aagtgatttg 3901 cctagtatta tacctgatta tattgatatt aatcagactg ttcaagacat attagaaaat 3961 tttagaccaa attggactgt acctgagttg acatttgaca tttttaacgc aacctattta 4021 aacctgactg gtgaaattga tgacttagaa tttaggtcag aaaagctaca taacactact 4081 gtagaacttg ccattcttat tgacaacatt aacaatacat tagtcaatct tgaatggctc 4141 aatagaattg aaacctatgt aaaatggcct tggtatgtgt ggctactaat aggcttagta 4201 gtaatatttt gcataccatt actgctattt tgctgttgta gtacaggttg ctgtggatgc 4261 ataggttgtt taggaagttg ttgtcactct atatgcagta gaagacaatt tgaaaattac 4321 gaacctattg aaaaagtgca cgtccattaa
Figure 2.1 Porcine transmissible gastroenteritis virus spike protein S mRNA sequences (GenBank accession number AF302263) downloaded from NCBI website (http//:www.ncbi.nlm.nih.gov). The sequences included in the forward primer (Tgeplp-FI) with a start codon ATG (underlined) at the 5’ end, and the sequences included in the reverse primer (Tgeplp-RII) with stop codon TAA (underlined) at the 3’ end, were in bold, respectively.
2.3.3 Construction of Recombinant pLP500 Harboring TGEV Spike Gene
Fragment
The amplified rTGEV-S was first subcloned into pCR®-XL-TOPO® vector using
TOPO® XL PCR Cloning Kit (Invitrogen, USA) accordingly to manufacturer’s instructions, before being cloned into the expression Lactobacillus/E.coli shuttle vector, pLP500.
66 CHAPTER 2 MATERIALS & METHODS
2.3.3.1 Subcloning of rTGEV-S into pCR®-XL-TOPO® Vector
In brief, 4 μl of gel purified rTGEV-S and 1 μl of pCR®-XL-TOPO® vector were
incubated for 5 min at room temperature, after which 1 μl of 6X TOPO® Cloning Stop
Solution was added and mixed for several seconds. The mixture was pulse spun before
being transformed into One Shot® cell (Invitrogen, USA). Two μl was transferred into a vial of One Shot® cell, and incubated on ice for 30 min. Heat shock was carried out at 42
°C for 30 sec, after which the transformation mix was incubated on ice for 2 min before
250 μl of SOC medium (Appendix 1) was added. After 1 hr of incubation at 37 °C, 100
μl of the transformation mix was spread on a pre-warmed LB plate (Appendix 1)
containing 50 μg/ml kanamycin (Appendix 1) and incubated overnight at 37 °C for the selection of clones.
2.3.3.2 Cloning of rTGEV-S into pLP500
Five μl of the plasmids isolated from Section 2.3.3.1 was digested with BamHI and NheI (New Englands Biolabs, UK) as described in Section 2.2.3. The digested samples were separated on agarose-EtBr gel. The 2.3 kb rTGEV-S was excised from the gel and purified using QIAquick II Gel Extraction Kit (Qiagen, Germany) as in Section
2.2.6 and ligated to pLP500 (pre-digested with BamHI and NheI) to generate recombinant vector pLP500/rTGEV-S.
2.3.4 Preparation of Competent E.coli Cells for Chemical Transformation
A 100 ml DH10β E.coli culture was incubated at 37 °C until OD600 = 0.5. The
cells were placed on ice for 10 min before being centrifuged for 5 min at a speed of 4000
67 CHAPTER 2 MATERIALS & METHODS
rpm. The supernatant was removed and the pellet was resuspended with 50 ml of cold
0.1M CaCl2. The cells were incubated on ice for 45 min, spun and resuspended in 8 ml
of 0.1M CaCl2. Two ml of sterile glycerol was added before the cells were aliquoted and
kept at -80 °C for long term storage.
2.3.5 Generation of Recombinant L.casei Shirota Expressing rTGEV-S
2.3.5.1 Transformation of pLP500/rTGEV-S into E.coli
Ten μl of the ligation mixture (Section 2.3.3.2) was added to 50 μl of DH10β
E.coli competent cells and incubated on ice for 30 min. This was followed by heat shock
at 42 °C for 1 min, after which the cells were placed on ice for another 2 min. One
hundred μl of transformed cells was then plated out on pre-warmed LB plate containing
100 μg/ml of ampicillin and incubated overnight at 37 °C for the selection of clones.
Well-defined colonies were picked from the plate and grown in LB containing ampicillin
(Appendix 1). The plasmids were then isolated following the procedures of the Wizard®
Plus SV Miniprep DNA Purification System (Promega, USA) as mention in Section
2.2.7, and glycerol stocks (20 % v/v) of the bacterial cultures were also prepared and stored at –80 °C.
2.3.5.2 Confirmation of Transformants
Restriction digestion analysis of the vectors isolated from Section 2.3.5.1 was conducted to determine if the plasmids contain rTGEV-S insert. Five μl of the vector was digested with BamHI and NheI. The digestion mixtures were vortexed and incubated
68 CHAPTER 2 MATERIALS & METHODS
at 37 °C for 2 hr. The digested DNA was then electrophoresized in 1.0 % agarose-EtBr gel at 120 Volts for 30 min and DNA bands were then visualized and photographed.
Nucleotide sequencing was also performed to verify if the rTGEV-S was appropriately inserted in the vector. The sequencing reaction was performed using the specific forward primer, pLP5005’, that primed the pLP500 vector backbone sequence
(Table 2.4), and ABI PRISM BigDye™ Terminator Cycle Sequencing Ready Reaction
Kit (Applied Biosystem, USA). The composition of the sequencing reaction, sequencing profiles and purification of the extension product was performed as recommended by the manufacturer’s protocol. In brief, the precipitation reaction was carried out by adding 80
µl of freshly prepared precipitation solution (3.0 µl of 3 M sodium acetate (pH 4.6); 62.5
µl of non-denatured 95 % ethanol; 14.5 µl of dgd water) to each tube of vector to be sequenced. The precipitation mixture was mixed by vortexing briefly and left to stand at room temperature for 15 min. The DNA was then pelleted by centrifuging at 14, 000 rpm for 20 min at room temperature. The supernatant from each of the tubes were removed carefully, after which 250 µl of 70 % ethanol was added. The mixture was vortex briefly and centrifuge for 5 min at 14, 000 rpm. The supernatant was then carefully removed and left to air dry for 15 min at room temperature. The precipitated extension product was stored in -20 oC until they were sent for electrophoresis.
2.3.5.3 Preparation of Competent Cells for Electroporation
Hundred ml of MRS medium was inoculated with 10 ml of overnight culture of
o LcS and incubated at 37 C in a CO2-enriched atmosphere. The cells were harvested
during log phase (about 4-5 hr incubation) by centrifugation of the culture at 9500 rpm
69 CHAPTER 2 MATERIALS & METHODS
for 10 min at room temperature. The cell pellet was resuspended in chilled washing
buffer (Appendix 2) and spun down at 5000 rpm for 10 min. The washing step was
repeated once before resuspending the cells in electroporation buffer (Appendix 2) and
adjusted to a concentration of 1010-1011 cells/ml. The exact number of cells per ml was
determined by plating them on MRS agar. Aliquots (100 μl) of the electroporation
competent LcS were stored at -20 oC
2.3.5.4 Electroporation
Transformation of LcS with pLP500/rTGEV-S was carried out by electroporation.
0.5 µg of the recombinant plasmid was added into 100 µl of LcS competent cell (Section
2.3.5.3). The electroporation mixture was transferred to an electroporation cuvette with a
0.2 cm gap width (Bio-Rad, USA). One single pulse was applied with the Bio-Rad Gene
Pulser (Bio-Rad, USA) and pulse controller under the following conditions: potential 2.5
kV; capacity, 25 µF; resistance, 200 Ω. One ml of MRS broth (Merck, Germany) was
added to the cells immediately after the pulse. The cell suspension was then transferred
to a sterile eppendorf tube and incubated at 37 °C for 3 hr. The cells were pelleted and
resuspended in 100 µl of MRS medium, before being plated on MRS agar (Merck,
Germany) with erythromycin (5 µg/ml, Appendix 1). The MRS plates were incubated at
37 °C with 5 % CO2, for 48-72 hr until colonies were formed.
2.3.5.5 Analysis of the Transformants
To confirm that the LcS colonies with Eryr phenotype were true transformants,
plasmids were extracted from the transformants using method described by Posno (1991)
70 CHAPTER 2 MATERIALS & METHODS with slight modifications. In brief, 3 ml overnight culture of the transformants were centrifuged and resuspended in 400 µl of lysis buffer (Appendix 2). After 2 hr of incubation at 37 °C, the cells were pelleted and resuspended in 400 µl of 20 mM Tris-Cl
(pH 8.0), and lysed with 1 % (v/v) sodium dodecyl sulfate (SDS). High molecular weight
DNA was denatured by the addition of 35 µl of 1.0 N NaOH and incubating the mixture at room temperature for 10 min. The lysate was then neutralized by addition of 70 µl of 2
M Tris-Cl (pH 7.0). A final concentration of 0.5 M NaCl was added, before the lysate was extracted once with phenol and once with phenol-chloroform (1:1). Nucleic acid was precipitated with 1 volume of isopropanol and leaving the mixture at -20 °C for at least 1 hr. The nucleic acid pellet was washed once with 70 % ethanol before being dissolved in 30 µl of TE buffer with 50 µg of RNaseA (Qiagen, Germany) per ml and incubated at 37 °C for 15 min. The extracted plasmids were analyzed for the presence of rTGEV-S insert via PCR (Section 2.3.2.2) using the cloning primers.
2.3.6 Expression of rTGEV-S Protein
Overnight culture of recombinant LcS harboring the pLP500 encoding rTGEV-S
(LcS-rTGEV-S) was inoculated into 100 ml of MRS broth and incubated at 37 °C with shaking at 200 rpm. As the vector pLP500 used in this study is a constitutive vector that expresses heterologous protein in a secretable form, supernatant from LcS-rTGEV-S was harvested at 12 hr and concentrated ten times using Ultrafree-CL PBGC Biomax-10
(Millipore, USA). The concentrated supernatant was analyzed with 10 % Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblotting as described in Section 2.6 and 2.7.
71 CHAPTER 2 MATERIALS & METHODS
2.4 GENERATION OF RECOMBINANT P.PASTORIS EXPRESSING TGEV
SPIKE PROTEIN FRAGMENT
2.4.1 Amplification of TGEV Spike Gene
2.4.1.1 Primers Sequences and Related Information.
The sequence, size and Tm of the primers used for the cloning of TGEV spike gene fragment into pGAPZαC are listed in Table 2.5.
Table 2.5 Primers used in this part of study.
Primer Sequence Size Tm Polarity 5’ 3’ (nts) (oC) pGAPtge-FI GGAATTCGCCATTGATTTATGGAGACAA 28 73 Forward pGAPtge-RII GCTCTAGAGTTAGACCTAACAGTTCACTG 29 78 Reverse
α-factor TACTATTGCCAGCATTGCTGC 21 53 Forward
2.4.1.2 Polymerase Chain Reaction (PCR)
A 2.3 kb TGEV spike gene fragment (PrTGEV-S, nucleotides 33-2288) was amplified from 2 μl of TGEV cDNA, synthesized in Section 2.3.1, with the aid of the primers pGAPtge-FI and pGAPtge-RII. The 7-bp overhang contains an EcoRI restriction site in pGAPtge-FI, and a 8-bp overhang coding XbaI in pGAPtge-RII for cloning procedures and PCR was carried out as described in Section 2.3.2.2.
2.4.2 Cloning of TGEV Spike Gene Fragment into pGAPZαC
Twenty μl of PrTGEV-S generated in Section 2.4.1.2 was cleaved with EcoRI and
XbaI (New Englands Biolabs, UK) and ligated with EcoRI and XbaI digested pGAPZαC to generate recombinant vector pGAPZαC/PrTGEV-S.
72 CHAPTER 2 MATERIALS & METHODS
2.4.3 Transformation of pGAPZαC/PrTGEV-S into E.coli
Ten μl of the vector mixture was transformed into 50 μl of DH10β E.coli
competent cells as described in Section 2.2.4. One hundred μl of transformed cells was
then plated out on pre-warmed Low Salt LB plate (Appendix 1) with 25 μg/ml of zeocin
and incubated overnight at 37 °C for the selection of clones. Well-defined colonies were
picked from the plate and grown in Low Salt LB medium containing zeocin. The
plasmids were then isolated following the procedures of the Wizard® Plus SV Miniprep
DNA Purification System (Promega, USA) as mention in Section 2.2.7, and glycerol stocks (20 % v/v) of the bacterial cultures were also prepared and stored at –80 °C.
2.4.4 Confirmation of Transformants
To verify if the transformants harbour the recombinant pGAPZαC/PrTGEV-S vector, PCR was performed on the isolated vectors using the primers pGAPtge-FI and pGAPtge-RII.
Nucleotide sequencing was also performed to check if the insert was in-frame with the α-factor secretion signal. The sequencing reaction was performed using the α- factor sequencing primer (Table 2.5) and ABI PRISM BigDye™ Terminator Cycle
Sequencing Ready Reaction Kit (Applied Biosystem, USA). The composition of the sequencing reaction, sequencing profiles and purification of the extension product was performed as recommended by the manufacturer’s protocol. Precipitation of the extension product was performed as described in Section 2.3.5.2. The precipitated extension product was stored in -20 oC until they were sent for electrophoresis.
73 CHAPTER 2 MATERIALS & METHODS
2.4.5 Generation of Recombinant P.pastoris Expressing rTGEV-S
2.4.5.1 Linearization of Recombinant Vector
The recombinant vector was linearized to permit homologous recombination
between transforming DNA and regions of homology within the genome (Cregg et al.,
1985, 1989) to generate stable transformants of P.pastoris. Gene insertion events occurs
at the GAP promoter locus as a result of a single crossover event between the locus and
the PGAP region on pGAPZαC, leading to the insertion of one or more copies of the vector
up-steam or downstream of the GAP locus (Figure 2.2).
Figure 2.2 Insertion of plasmid 5’ to the intact GAP promoter locus.
Five µg of pGAPZαC/PrTGEV-S was linearized with AvrII, which cuts once in
the GAP promoter region to linearize the vector. The digest was electrophoresized to
ensure complete linearization of vector and purified using QIAquick II Gel Extraction Kit
(Qiagen, Germany) as described in Section 2.2.6.
74 CHAPTER 2 MATERIALS & METHODS
2.4.5.2 Transformation of pGAPZαC/PrTGEV-S into P.pastoris
Transformation of the linearized vector into P.pastoris was carried out using
Pichia EasyComptm Kit (Invitrogen, USA), following manufacturer’s instructions. The
following provides the protocol in brief.
2.4.5.2.1 Preparation of Competent P.pastoris
Ten ml of overnight culture of P.pastoris was diluted to an OD600 of 0.2 in 10 ml
of YPD and grown at 30 °C with shaking until the OD600 reached 0.6-1.0. The cells were
centrifuged at 500 x g for 5 min at room temperature, where the pellet was resuspended
in 10 ml of Solution I from Pichia EasyComptm Kit (Invitrogen, USA). The cells were
pelleted again and resuspended in 1 ml of Solution I before being aliquoted into microcentrifuge tubes and stored at -80 °C.
2.4.5.2.2 Transformation of competent P.pastoris
Three µg of linearized pGAPZαC/PrTGEV-S was added to 50 µl of competent
P.pastoris GS115 cells, after which 1 ml of Solution II from Pichia EasyComptm Kit
(Invitrogen, USA) was added to the transformation mixture and mixed by flicking the tube. The transformation reaction was incubated at 30 °C for 1 hr, where the transformation mixture was mixed by flicking the tube every 15 min. The cells were then heat shocked at 42 °C for 10 min and split into 2 microcentrifuge tubes, where 1 ml of
YPD medium (Appendix 2) was added to each tube. The cells were incubated at 30 °C for 1 hr to allow expression of zeocin resistance. The cells were then centrifuged at 3000 x g for 5 min at room temperature and resuspended in 1 ml of Solution III from Pichia
75 CHAPTER 2 MATERIALS & METHODS
EasyComptm Kit (Invitrogen, USA). The cells were pelleted again before being
resuspended in 100 µl of Solution III and plated onto YPD plates with 100 µg/ml zeocin
(Appendix 1). The plates were incubated at 30 °C for 2 to 4 days until colonies appeared.
2.4.5.3 Analysis of the Transformants
2.4.5.3.1 Total DNA isolation form P.pastoris
One ml of overnight culture of transformant was centrifuged at 7000 rpm for 1
min, after which the pellet was resuspended in 200 µl of Breaking buffer (Appendix 2).
A single scoop of 400 μ glass beads (BioSpec Products Inc., USA) was added to the cells,
followed by 200 µl of Phenol/Chloroform (1:1 v/v). The lysis mixture was vortexed
continuously for 5 min and centrifuged at 14000 rpm for 5 min, after which the aqueous
DNA phase was transferred to a new microcentrifuge tube. This step was repeated before
500 µl of absolute ethanol was added and the mixture was centrifuged at 14, 000 rpm for
10 min. The DNA pellet was washed with 500 µl of 70 % ethanol before being resuspended in 200 µl of 0.3 M NaAc. The DNA was precipitated again with 500 µl of absolute ethanol, washed with 500 µl 70 % ethanol and air dried before resuspended in
50 ul of dgd water. The samples were stored at -20 °C until ready for usage.
2.4.5.3.2 PCR of Total DNA
In order to determine if the insert PrTGEV-S has integrated into the transformant
genome, PCR of the DNA isolated from the P.pastoris transformants was conducted
using the primers pGAPtge-FI and pGAPtge-RII. The generated products were analyzed
on a 1.0 % agarose-EtBr gel.
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2.4.6 Expression of PrTGEV-S Protein
Overnight culture of the recombinant P.pastoris (PP/PrTGEV-S) was grown from which 0.1 ml was used to inoculate 50 ml of YPD medium in a 250 ml flask. The culture was incubated at 30 °C in a shaking incubator. One ml of the expression culture was transferred to a 1.5 ml microcentrifuge tube on Day 1, 2, 3 and 4 post-inoculation. The culture was centrifuged and the supernatant was transferred to a separate tube. The supernatants were snap frozen in liquid nitrogen before being stored at -80 °C, until ready for the analysis of protein expression through Western blotting (Section 2.6 & 2.7).
2.5 GENERATION OF RECOMBINANT P.PASTORIS EXPRESSING SARS CoV
SPIKE PROTEIN FRAGMENT
2.5.1 Amplification of SARS CoV Spike Gene
Babcock and coworkers (2004) reported that amino acids 270 to 510 were the receptor-binding region of Severe Acute Respiratory Syndrome Coronavirus (SARS
CoV) spike protein. More importantly, antibodies raised against recombinant protein containing amino acid residues 318-510 of the SARS spike protein were able to induce highly potent neutralizing antibody responses against SARS CoV. As such, gene encoding these 241 amino acids were amplified and cloned into P.pastoris.
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2.5.1.1 Primers Sequences and Related Information
The sequence, size and Tm of the primers used for the cloning of the receptor- binding region of SARS CoVspike gene into pGAPZαC are listed in Table 2.6.
Table 2.6 Primers used in this part of study.
Primer Sequence Size Tm Polarity 5’ 3’ (nts) (oC) RBD-SSF1 GGGGTACCTACAATCACAGATGCT 25 58 Forward
RBD-SSR1 GCTCTAGATCAGTGGATAATTTTGGT 27 70 Reverse
α-factor TACTATTGCCAGCATTGCTGC 21 53 Forward
2.5.1.2 Polymerase Chain Reaction (PCR)
With the aid of the primers, RBD-SSF1 and RBD-SSR1, a 720 bp fragment of
SARS CoV receptor-binding domain (nucleotides 810-1530) of spike gene (SARS-S-
RBD) was amplified from pCR-XL-TOPO-S, a vector encoding the SARS CoV spike gene, kindly provided by Associate Professor Vincent Chow of Department of
Microbiology, National University of Singapore. Both primers were designed to contain an 8-bp overhang that encodes a KpnI restriction site in RBD-SSF1, and XbaI in RBD-
SSR1 for cloning procedures. PCR was carried out as described in Section 2.3.2.2.
The same approach of cloning and generation of recombinant PrTGEV-S encoding P.pastoris (Section 2.4.3 to 2.4.6) was employed for the generation of recombinant P.pastoris expressing SARS-S-RBD (PP/SARS-S-RBD). Expression of the
SARS-S-RBD protein was determined as in Section 2.4.7.
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2.6 SODIUM-DODECYL SULFATE POLYACRYLAMIDE GEL
ELECTROPHORESIS (SDS-PAGE)
The discontinuous Laemmli gel system (Laemmli, 1970) was used for
electrophoresis of proteins in this study. The electrophoresis apparatus used was from
Mini-Protean®II Electrophoresis Cell (Bio-Rad, USA).
A 10 % separation gel (Appendix 3) was cast between two glass plates. The gel mixture was poured to about 1.5 cm below the comb bottom. Seventy percentage ethanol was layered over the gel mixture to ensure a straight meniscus and to exclude oxygen which inhibits gel polymerization reaction. The gel is allowed to polymerize for approximately 45 min before the ethanol was decanted and washed with dgd water to flush out the residual polyacrylamide. Stacking gel (Appendix 3) was then layered over the separation gel with comb in position and allowed to polymerize for 30 min. The comb was removed and the gel assembly was immersed into the electrophoresis chamber filled with running buffer (Appendix 3).
Prior to loading, 25 μl of each protein sample was boiled for 1 min with 5 μl of loading dye (Appendix 3). Six μl of the prestained protein marker (New England Biolabs,
UK) was treated as mentioned before. Electrophoresis was carried out using constant current of 20 mA for approximately 1.5 hr.
After electrophoresis, the gel was removed from the glass plates and soaked in staining solution (Appendix 3) for 1 hr. The gel was then rinsed briefly to remove the excess stain and immersed in destaining solution (Appendix 3), until the protein bands could be clearly visualized.
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2.7 IMMUNOBLOTTING
For the immunoblotting of proteins, the gel was transferred into a blotting cell.
The 0.45 μm nitrocellulose membrane (Bio-Rad, USA), filter paper and sponge for
transblot sandwich were presoaked in transfer buffer (Appendix 4). The separation gel
was placed between a wet nitrocellulose membrane and a piece of pre-wet filter paper.
The set-up was sandwiched between two pieces of sponge in the transblot cell. The cell
was immersed into the transfer chamber filled with transfer buffer with the nitrocellulose
membrane side adjacent to the positive electrode. The transfer was carried out at 90
Volts for 2 hr in a cold room.
After the transfer, the blot was blocked in PBS containing 5 % skim milk for 1 hr,
after which the blot was washed thrice with wash buffer (Appendix 4) for 5 min each.
Primary antibodies (Table 2.7), diluted in PBS, was added to the blot and incubated
overnight at 4 °C on an orbital shaker (Bellco, USA).
The immunoblot was washed with three times with wash buffer, before incubated
with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies
(Table 2.7). The secondary antibody was diluted in PBS and incubation was carried out at room temperature for 1 hr.
After the incubation period, the blot was washed thrice for 10 min each time.
Visualization of the immunolabeled bands was then carried out using SuperSignal West
Pico Chemiluminescent Substrate chemiluminescence reagent (Pierce Biotechnology,
USA), accordingly to manufacturer’s instruction.
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2.7.1 Antibodies Used For Immunoblotting
Table 2.7 Primary and secondary antibodies used for immunoblotting.
Supernant Primary antibodies Source Secondary Source to analyze & dilution antibodies & dilution LcS- TGEV convalescence Prof. Jimmy HRP-conjugated ZYMED rTGEV-S swine serum (1:1000) Kwang, anti-swine IgG Laboratories TLL (1:10 000) Inc., USA
PP/PrTGE TGEV convalescence Prof. Jimmy HRP-conjugated ZYMED V-S swine serum (1:1000) Kwang, anti-swine IgG Laboratories TLL (1:10 000) Inc., USA
PP/SARS- SARS CoV Singapore HRP-conjugated Bethyl S-RBD convalescent human SARS anti human IgG Laboratories sera (1:500) Patient (1:10 000) Inc., USA
HRP: Horseradish peroxidase
The convalescence swine serum against TGEV was obtained from pigs that were inoculated by oral route with 105 TCID50 of TGEV Miller Strain and challenged intramuscularly 45 days post-inoculation. For the detection of specific antibody binding, horseradish peroxidase (HRP)-conjugated anti-swine IgG (ZYMED Laboratories Inc.,
USA) diluted at 1:10,000 were used.
2.8 IMMUNIZATION OF MICE
Groups of three female BALB/c mice (8 weeks old) were immunized orally either with recombinant rTGEV-S expressor LcS (LcS-rTGEV-S), PrTGEV-S or SARS-S-RBD expressing P.pastoris (PP/PrTGEV-S and PP/SARS-S-RBD). Control non-expressor strain (harboring empty vector) or sterile PBS (Appendix 5) was also administrated as controls. Freshly cultured bacteria were harvested and washed twice with sterile PBS
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(pH 7.2). Oral doses of 2 x 109 cells (0.1 ml of the suspension) were administered on
three consecutive days at days 0, 1 and 2. A booster immunization was given at days 14,
15 and 16 and a second booster was given at days 28, 29 and 30. Tail bleeding and
lavaging was performed at the appropriate timing to collect sera and intestinal fluid from the mice. Mice were then sacrificed, where the Peyer’s patches and/or the cervical lymph node (CLN) were aseptically removed.
2.8.1 Collection of Intestinal Secretions
Lavaging of mice was performed as following the method used by Elson et al.
(1984) with slight modifications. In brief, four doses of 0.5 ml of lavage solution [25mM
NaCl, 40 mM Na2SO4, 10 mM KCl, 20 mM NaHCO3, and 48.5 mM polyethylene glycol
(average MW = 3350)] were given intragastrically at 15 min intervals. Thirty minutes after the last dose of lavage solution, the mice were given 0.1 mg of pilocarpine (Alcon-
Couvreur, Belgium) intraperitoneally. A discharge of intestinal contents occurred
regularly over the next 10-20 min. The collected intestinal discharges were transferred to
15 ml centrifuge tubes and the volume brought up to 6 ml with PBS. The samples were
vortexed vigorously before being centrifuged at 6000 x g for 10 min for the clarification
of the intestinal content, of which 3 ml of the supernatant was transferred to a round-
bottomed polycarbonate centrifuge tube. The secretions were further clarified by
centrifugation at 14, 000 x g at 4 °C for 20 min after the addition of 30 µl of 100 mM
phenlymethylsulfonyl fluoride [(PMSF) (Sigma, USA)] in 95 % ethanol. Two microliters
of the clarified secretions were removed, to which 20 µl of 100 mM PMSF in 95 %
ethanol and 20 µl of 1 % sodium azide (Sigma, USA) were added. The samples were
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then left to stand for 15 min, after which 100 µl of fetal calf serum serum was added to
provide an alternate substrate for any remaining proteases. The intestinal lavages were
stored at -20 °C until assay.
2.9 LARGE SCALE EXPRESSION OF TGEV SPIKE PROTEIN
A 1.3 kb N-terminal fragment of TGEV spike protein, expressed as a GST fusion
protein (TGEV-S-GST), was used for the analytical studies of the recombinants
LcS/rTGEV-S and PP/PrTGEV-S generated.
2.9.1. Polymerase Chain Reaction (PCR)
The sequence, size and Tm of the primers used for the cloning of N-terminal fragment of TGEV spike gene into pGEX-4T-3 are listed in Table 2.8.
Table 2.8 Primers used in this part of study.
Primer Sequence Size Tm Polarity 5’ 3’ (nts) (oC) pGEXF1 CCGGAATTCTGTTTATGATGTCAGTTATTAT 31 77 Forward pGEXR1 ACGCGTCGACATTATCAGACGGTACACCCAC 31 84 Reverse
Amplification of an N-terminal fragment (nucleotides 690-2010) of the TGEV spike gene was carried out using the primers, pGEXF1 and pGEXR1. Both primers were designed to contain a 9-bp overhang, which encodes an EcoRI restriction site in pGEXF1, and a 10-bp overhang that encodes SalI in pGEXR1 for cloning procedures.
PCR was carried out as described in Section 2.3.2.2.
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2.9.2 Cloning of TGEV Spike Gene Fragment into pGEX-4T-3
Twenty μl of TGEV gene fragment generated in Section 2.9.1 was cleaved with
EcoRI and SalI (New Englands Biolabs, UK) and ligated with EcoRI and SalI digested
pGEX-4T-3, downstream of GST gene.
Transformation of the vector mixture into JM105 E.coli competent cell was carried out as in Section 2.2.4. One hundred μl of transformed cells was then plated out on pre-warmed LB plate (Appendix 1) containing 100 μg/ml of ampicillin and incubated overnight at 37 °C for the selection of clones. Well-defined colonies were picked from the plate and grown in LB medium (Appendix 1) containing 100 μg/ml of ampicillin. The plasmids were then isolated following the procedures of the Wizard® Plus SV Miniprep
DNA Purification System (Promega, USA) as mention in Section 2.2.7, and glycerol stocks (20 % v/v) of the bacterial cultures were also prepared and stored at –80 °C.
2.9.3 Induction of TGEV-S-GST
Overnight culture of transformant was diluted 1:10 in 50 ml of fresh LB medium and incubated at 37 °C until OD600 reached 0.6-0.8. Protein expression was induced with
1 mM of Isopropyl-ß-D-thiogalactopyranosid [(IPTG) (Sigma, USA)] and incubating the transformants at 37 °C for 3 hr. The cells were then pelleted by centrifuging at 6000 rpm for 5 min at 4 °C. The supernatant was removed and kept for analysis, while the insoluble protein fraction was extracted via sonication of the pellet on ice. The cell extract was then centrifuged at 13, 000 rpm for 20 min at 4 °C, where the supernatant was kept and the insoluble fraction was resuspended with 1.5 ml of 1 M urea before being sonicated again.
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The insoluble protein fractions were then further extracted consecutively with 1 M, 6 M, 8
M urea and 0.01 % SDS (Appendix 2).
2.9.4 Purification of TGEV-S-GST
Two hundred microliters of Glutathione Sepharose 4B (Amersham Biosciences,
England) was washed thrice with 3 ml of chilled PBS. Cell lysate containing the fusion
protein was then added to the beads and incubated at 4 °C for 1 hr with gentle agitation.
The matrix was then transferred to a disposable column. Unbound proteins were
removed by washing the column twice with chilled PBS. Finally, the purified proteins
were eluted by adding reduced glutathione (Amersham Biosciences, England) to the
column and the eluates were collected. All samples were kept at -80 °C until ready to be
analyzed by SDS-PAGE (Section 2.6).
2.10 ELISA
Polystyrene microtitre plates were coated overnight at 4 oC with 1 μg of SARS-
Associated Coronavirus Spike mosaic recombinant protein S(M) [(SARS-S(M)(Virogen,
USA)] or affinity purified TGEV-S-GST. The coating antigen, TGEV-S-GST, was
extracted from cell lysate of recombinant E.coli harboring and expressing a 1.3 kb amino
terminal fragment of S protein in the form of GST-fusion protein (TGEV-S-GST), and
purified by passing the cell lysate through Glutathione Sepharose 4B affinity column
(Amersham Biosciences, England). Serum or intestinal lavage samples were diluted two
fold and used as primary antibodies. Bound antibodies were detected using horseradish
peroxidase conjugated goat anti-mouse IgA or IgG (ZYMED Laboratories Inc., USA),
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followed by colour development using O-phenylene diamine dihydrochloride (Sigma, St.
Louis, USA) as substrate. The plate was read using an ELISA TouchScreen plate reader
(Tecan, Austria), running in Magellan2 software, at the wavelength of 490 nm. For the
determination of relative levels of IgG subclasses, anti-murine IgG1 and IgG2a
conjugated with horseradish peroxidase (ZYMED Laboratories, USA) were substituted for anti-mouse IgG horseradish peroxidase.
2.11 CYTOKINE PROFILING
2.11.1 Preparation of Peyer’s Patches and Cervical Lymph Node for In Vitro Re-
stimulation
Single-cell suspensions of the Peyer’s patches (PP) and/or cervical lymph node
(CLN) were prepared by mechanical dissociation. The cells were resuspended and
adjusted to 4 x 106 cells/ml in RPMI with 10 % heat-inactivated FCS (Appendix 5). The
PP and CLN cells from mice immunized orally with LcS-rTGEV-S or PP/PrTGEV-S
were cultured in the presence of 5 μg of TGEV-S-GST, while cells isolated from mice
fed with PP/SARS-S-RBD were re-stimulated with 5 μg of SARS-S(M). The T-cell
mitogen concanavalin A [(5 μg/ml)(Vector Laboratories, USA)] was added to positive
control wells while an equal volume of RPMI medium was added to the negative control
wells. The culture supernatants were harvested on days 2, 3, 4 and 5 after restimulation
to determine the kinetics of production of different cytokines. All supernatants were
frozen at -80 oC until cytokine analysis.
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2.11.2 Cytokine Assay
The level of IL-2, IL-4, IL-5 and IFN-γ in the culture supernatants was determined using Bio-PlexTM Cytokine Assays (Bio-Rad, USA), following
manufacturer’s instruction. In brief, required wells of a 96-well filter plate were pre-wet
with 150 μl of Bio-Plex assay buffer. The buffer was then removed by vacuum filtration using MultiScreen Vacuum Manifolds (Millipore, USA) and the bottom of the filter plate was blotted to dry with a clean paper towel. Fifty microliters of multiplex beads (anti- cytokine conjugated) was added per well and the buffer was removed by vacuum filtration. The beads in each well were washed twice with 100 μl of Bio-Plex wash buffer before 50 μl of the culture supernatant (Section 2.11.1) was added to each well. The filter
plate was sealed with plate sealing tape, wrapped in aluminum foil to protect the beads
from light and incubated at room temperature on a shaker at 1100 rpm for the first 30 sec,
then at 300 rpm for 30 min. The buffer was then removed and the beads were washed
thrice with 100 μl of Bio-Plex wash buffer before 25 μl of 50 x Bio-Plex detection antibody was then added to each well. The plate was sealed and covered with aluminum foil, and incubated at room temperature on a shaker at a speed of 1100 rpm for the first
30 sec, then 300 rpm for 30 min. The buffer was first removed, after which the beads were washed thrice with 100 μl of Bio-Plex wash buffer. The beads were then resuspended with 125 μl of Bio-Plex assay buffer and the plate was shook at 1100 rpm for 30 sec immediately before reading the plate on Bio-Plex System (Bio-Rad, USA).
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2.12 CELL CULTURE TECHNIQUES
All solutions and media for cell culture were made using chemicals of ultrapure
grades and dgd. All cell cultures and media preparation works were performed under
aseptic conditions in a laminar flow cupboard (Gelman Sciences, Australia) or biohazard
hood (Nuaire, USA). Prepared media were stored in sterile glass bottles with screwed
capped with non-toxic silicone rubber washers.
Cells used in this study were grown in sterile plastic tissue culture flasks (25 cm2,
75 cm2 or 175 cm2), 6-well, 24-well or 96-well tissue culture plates from Falcon (USA).
Cells were either cultured in humidified incubator (Nuaire, USA) with carbon dioxide (5
%) or dry incubator at 37 °C.
2.12.1 Cell Lines
Cell lines that were used in this study were listed in the Table 2.9. The culture media, passage level and origin of the cell lines are indicated.
Table 2.9 Cell lines and related information.
Cell Line Type of cell Culture media Passage Level Origin line used for experiments Minimum American ST cell, Swine essential Type Culture Adherent 20-35 testicular cells medium Collection (Eagle) Center, USA Caco-2 cell, Minimum American colorectal essential Type Culture Adherent 25-40 adenocarcino medium Collection ma (Eagle) Center, USA HT-29 cell, American colorectal McCoy's 5a Type Culture Adherent 20-30 adenocarcino medium Collection ma Center, USA
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Vero cell, American African Green Type Culture Adherent Medium 199 35-50 Monkey Collection Kidney cells Center, USA
2.12.2 Media for Cell Culture
Cell culture media supplemented with 10 % fetal calf serum was used as the
growth medium (Appendix 5) for the respective cell lines (Section 2.12.1). The pH of the
medium was adjusted to 7.2 - 7.4 with sodium bicarbonate (Appendix 5).
2.12.3 Cultivation and Propagation of the Cell Lines
New flasks of cells were sub-cultured from confluent monolayer at a ratio of 1:8
(for 25 cm2 flask) and 1:4 (for 75 cm2 and 175 cm2 flask). The growth medium
(Appendix 5) was first discarded, after which the monolayer was then rinsed twice with about 5 ml of phosphate-buffered saline (PBS - Appendix 5). The cell was rinsed with trypsin-versene (ATV - Appendix 5) and incubated with fresh ATV for 3 min at 37 °C for the detachment of the cell monolayer from the flask. Appropriate amounts of the growth media (depending on the size of the flask), were added to the cell suspension to inactivate the enzymatic activity of the ATV. Cell aggregates were dispersed by pipetting to give a single cell suspension. The suspended cells were then distributed into new tissue culture flasks. The cells were then incubated at 37 °C. The monolayer would reach confluence in 3 to 4 days and was used for all the experiments.
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2.12.4 Cultivation of Cells in 6-Well, 24-Well and 96-Well Tissue Culture Tray
A confluent cell monolayer in a 25 cm2 tissue culture flask was used to seed a 6-,
24-, or 96-well tray (Falcon, USA). The cell monolayer was treated as previously
described (Section 2.11.3) and the cell suspension was made up to a final volume of 30
ml using cell culture medium (Appendix 5). Five ml, 1 ml and 300 µl aliquot of this
suspension was transferred into each well of the 6-, 24- and 96-well tray, respectively.
The trays were then incubated at 37 °C in the humidified incubator (Nuaire, USA) with
carbon dioxide (5 %). The monolayers were confluent within 3 days and ready for use.
2.12.5 Cultivation of Cells on Glass Coverslips
Cells were grown on coverslip for scanning electron microscopy (Section 2.14).
2.12.5.1 Pretreatment of Coverslips
Glass coverslips of 13 mm in diameter (ARH, UK) were washed in 95 % ethanol
for 30 min and then boiled in dgd water for about 15 min. The coverslips were then dried
overnight at 37 °C and dry heat sterilized in a hot air oven at 160 °C for 2 hr.
2.12.5.2 Culture of Cells on Coverslips
The individual coverslip was aseptically placed in a 24-well tissue culture tray. A
confluent cell monolayer in a 25 cm2 flask was used to propagate the cells as described in
Section 2.12.4. The cells were subcultured into the wells at a ratio of 1:25 using cell culture medium (Appendix 5). The trays were incubated at 37 °C in a humidified incubator supplemented with 5 % carbon dioxide until they were about 50 % confluent.
90 CHAPTER 2 MATERIALS & METHODS
2.12.6 Storage of Cells
A confluent cell monolayer in 25 cm2 tissue culture flask was was treated as for
propagation of cell line in Section 2.12.4. The cell suspension was spun in a sterile 15 ml
centrifuge tube at 800 x g for 10 min, after which the pellet was resuspended in 2 ml of
preserving medium (Appendix 5). One ml aliquot of this suspension (about 1 x 107 viable cells) was dispensed into cryovials with screw caps (Iwaki, Canada). These vials were placed in Cryo-freezing container [(1 °C/min) (Nalgene, USA)] in the -80 °C freezer overnight. The vials were then stored in liquid nitrogen.
The frozen cell lines were regenerated for experiments by fast thawing at 37 °C in a water bath. The cell suspension was then transferred into a sterile 10 ml centrifuge tube that contained 4 ml of cell culture medium (Appendix 5) for the specific cell line to dilute the dimethyl sulphoxide (DMSO) in the preserving medium that would be toxic to the cells. The cell suspension was then pelleted by centrifuging at 800 x g for 10 min. The supernatant (preserving medium) was discarded and the cell pellet was resuspended in 5 ml of fresh growth medium (Appendix 5) of the specific cell line. This suspension was transferred into a 25 cm2 tissue culture flask and was incubated at 37 °C overnight.
Growth medium was changed the following day and the cells were allowed to grow to
confluency.
2.13 INFECTION OF CELLS
2.13.1 Viruses
91 CHAPTER 2 MATERIALS & METHODS
The viruses used in this study are given in Table 2.10. The viruses were propagated in either ST cells or Vero cells throughout this study and would be herein referred by the abbreviations given below.
Table 2.10 Viruses and related information.
Source Timing p.i. of Abbreviatio Virus Family Virus Strain obvious n CPE/ Cell type Kind gift from Professor Transmissible Jimmy Kwang, 18 to 24 Coronaviridae Gastroenteritis Miller TGEV Temasek Life hrs Coronavirus Sciences (ST cells) Laboratory, Singapore Kind gift from Professor SARS CoV S Tsanan protein- Giroglou, pseudotyped 72 hrs Retroviridae - MLV(SARS) Georg-Speyer- murine Vero cells Haus, Institute leukemia virus for Biomedical vector particles Research, Germany
2.13.2 Infection of Cell Monolayers
Cell culture medium of the 3 days old confluent cell monolayer was discarded and the monolayer was washed with 5 ml of virus diluent (Appendix 6). Different volumes of virus suspension [Multiplicity Of Infection (M.O.I.) of 10] used were: a) 0.2 ml for a 25 cm2 flask, b) 0.6 ml for a 75 cm2 flask and c) 1.5 ml for a 175 cm2 flask. The flask was incubated at 37 °C for 1 hr and rocked every 15 min to ensure even infection. After 1 hr, unabsorbed virus was washed off with 5 ml of maintenance medium [(MEM) (Appendix
92 CHAPTER 2 MATERIALS & METHODS
6)] and an appropriate volume of MEM was added to the flask. The infected ST cells
were then incubated at 37 °C until the appropriate harvest time (Table 2.10).
Mock-infected controls used in this study were prepared as abovementioned
except that an appropriate amount of virus diluent (Appendix 9) was used instead of the
virus.
2.13.4 Neutralization Assay
2.13.4.1 Plaque Neutralization Assay
Intestinal fluids and serum samples from mice were evaluated using plaque
neutralization assay. Fifty µl of the samples in 2-fold serial dilutions (from 1:2 to 1:256
or 1:512) were prepared in microcentrifuge tubes. Virus, adjusted to 5 x 104 PFU in 50
μl of virus diluent (Appendix 6) was added to the tube containing serially diluted serum or intestinal lavage. The antibody-virus mixture was mixed by vortexing for 30 sec, pulsed centrifuged and incubated at 37 oC for an hour. A 24-well plate with confluent
monolayers of cells (prepared as described in Section 2.12.4) was used for virus
infection. The cell monolayer was rinse once with virus diluent (Appendix 6), after
which 100 μl of the antibody-virus mixture was added to the appropriate wells. The plates were left at 37 oC for another hour, and rocked at 15 min intervals. After incubation, the inoculum was removed and the cell monolayer was rinsed once with virus diluent. Overlay medium (Appendix 6) was added to the wells and the plate was incubated at 37 °C for 4-6 days. The overlay medium was then discarded, after which the wells were washed thrice with sterile PBS and virus plaques were stained with 0.5 % crystal violet/25 % formaldehyde solution (Appendix 6) for 30 min at room temperature.
93 CHAPTER 2 MATERIALS & METHODS
Differences in the number of plaques formed between treatments were examined for the level of significance by Student’s t-test after analysis of variance.
2.13.4.2 Fluorescent Focus Assay
MLV(SARS), adjusted to 5 x 104 PFU in 50 μl of virus diluent (Appendix 6), was added to 50 µl of lavage and serum samples from mice in 2-fold serial dilutions (from 1:2 to 1:256 or 1:512). The antibody-virus mixture was mixed by vortexing for 30 sec, pulsed centrifuged and incubated at 37 oC for 1 hr to enhance virus absorption. Confluent
Vero cells in 24-well microtiter plate (prepared as described in Section 2.12.4) was rinse once with virus diluent (Appendix 6), after which 100 μl of the antibody-virus mixture was added to the appropriate wells. In contrast to infection with authentic virus, transduction with SARS CoV S protein-pseudotyped murine leukemia virus
[MLV(SARS)] into Vero cells was carried out by spinning the plate at 800 x g at 31 °C for 1 hr. The transduced cells were then incubated at 37 °C in 5 % CO2 for 24 hr. The supernatant was replaced with fresh culture medium and further incubated for another 48 hr. The plaque neutralization efficiency of the pseudovirus was analyzed using fluorescence focus assay. The titers of individual serotypes (FFU/ml) were determined by counting the number of fluorescent foci in the well with an inverted fluorescence microscope with suitable filters for fluorescein isothiocyanate at 1000× magnification.
2.13.5 Extraction of Virus RNA Molecules
Viral RNA extraction procedure was carried out using QIAmp® Viral RNA extraction kit from Qiagen, USA, following manufacturer’s instruction. In brief, 140 μl
94 CHAPTER 2 MATERIALS & METHODS
of virus supernatant (approximately 5 x 108 PFU/ml) were added to 560 µl of Buffer
AVL containing carrier RNA in a 1.5 ml microcentrifuge tube. The mixture was
incubated at 25 ºC for 10 min, followed by the addition of 560 µl of absolute ethanol.
The solution was then transferred to the QIAmp spin column and spun at 6000 x g for 1
min. The filtrate was discarded and the spin column was spun again at 6000 x g for 1
min after adding 500 µl of Buffer AW1. The above step was repeated with the addition
of Buffer AW2. Viral RNA was eluted from the spin column by adding 60 µl of Buffer
AVE and spun at 6000 x g for 1 min. The viral RNA was stored at -80 ºC until use.
2.14 BIO-IMAGING VIA SCANNING ELECTRON MICROSCOPY (SEM)
The cells on the coverslip were fixed with a primary fixative (2 % glutaraldehyde
– Appendix 7) at room temperature for 1 hr and washed twice with 0.1 M sodium
cacodylate buffer (Appendix 7) where the coverslip was soaked in the buffer for 5 min during each wash. The cells were then post-fixed with secondary fixative (1 % osmium tetroxide - Appendix 7) for 1 hr at 4 °C. Excess fixatives were removed by rinsing and soaking the samples twice for 5 min in 0.1 M sodium cacodylate buffer before performing the dehydration process through a series of concentrations of ethanol as follows:
Ethanol Concentrations Dehydration Time
50 % 15 min
75 % 15 min
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95 % 15 min
100 % 20 min (3x)
The dehydrated cells on the coverslips were then critical point dried with CO2 and mounted onto SEM stubs before being sputter-coated with 70 nm gold films. Coated samples were then viewed under the scanning electron microscope (JEOL 5660, Japan).
96 CHAPTER 3 RESULTS
3.0 ADHESION STUDIES
3.1 INTRODUCTION
In order for probiotic microorganisms, like lactobacilli and yeast, to function as
oral vaccine delivery vehicle, the ability of the microorganisms to adhere to the intestinal
tract is crucial. To date, limited studies have been performed to investigate the
colonization capability of lactobacilli and yeast. As such, the present work aims to
determine the adhesion and colonization capacity of Lactobacillus casei Shirota (LcS),
and three other yeasts, S.boulardii, S.cerevisiae and P.pastoris. In vitro adhesion of
lactobacilli was not performed in this study as the ability of lactobacilli to adhere to
intestinal cells in vitro has been previously elucidated (Morita et al., 2002; Coconnier et
al., 1992; Lee & Puong, 2002). In vitro interactions between the yeasts and human
intestinal cell lines were first studied using scanning electron microscope, while the
ability of exogenously administered lactobacilli and yeasts to adhere to the mucosal cells
and to multiply in murine intestinal tract were determined in vivo. L.casei Shirota and
yeasts fluorescently labeled with five (and 6)-carboxyfluorescein diacetate, succinimidyl
ester (cFDA-SE), were individually fed to mice and the intestinal segments were
extracted from mice sacrificed various days post feeding. The adhesiveness of each
organism on the mucosa surface was then determined via flow cytometry analysis of the intestinal samples.
97 CHAPTER 3 RESULTS
3.2 IN VITRO ADHESION STUDIES USING SCANNING ELECTRON
MICROSCOPY
To date, few studies have been performed to determine the adhesion capability of
yeast to intestinal cells. As a preliminary study, S.boulardii, S.cerevisiae and P.pastoris
were assessed for their abilities to adhere to human enterocyte-like Caco-2 cells and to
bind to mucus produced by the human mucus-secreting HT-29 cell in culture. In vitro
adhesion of Lactobacillus spp. was not carried out as previous studies have already
confirmed that interactions do exist between lactobacilli and cultured human intestinal
cells (Morita et al., 2002; Coconnier et al., 1992; Lee & Puong, 2002).
Attachment of yeasts to the intestinal cell surfaces was characterized by scanning
electron microscopic examination. Scanning electron micrographs clearly illustrated that
binding of the yeasts to Caco-2 occurred at the mucosal surface of the cultured intestinal
epithelial cells (Figure 3.1). In particular, P.pastoris was seen to interact with the brush
border of the differentiated Caco-2 cells (Figure 3.1C). Adhesiveness of each of the
yeasts was assessed by enumerating the number yeast cell attached to 20 randomly
chosen Caco-2 cells (Appendix 8d). Similar attachment were observed with S.boulardii
and S.cerevisiae (4.1 x 101 and 4.8 x 101 yeasts per cell) while P.pastoris demonstrated
stronger adhesiveness (7.14 x 102 yeast per cell) to Caco-2 cells when compared to
Saccharomyces spp.
S.boulardii, S.cerevisiae and P.pastoris were also observed to adhere to the mucus-secreting HT-29 cell line (Figure 3.2). The number of yeasts adhering to 20 randomly chosen HT-29 cell were also enumerated (Appendix 8d). Corresponding to the results obtained in the adhesion study performed using Caco-2 cell, a higher level of
98 CHAPTER 3 RESULTS adhesion to HT-29 cell was observed for P.pastoris (1.12 x 103 yeast per cell), while
S.boulardii and S.cerevisiae (3.4 x 101 and 4.3 x 101 yeast per cell) demonstrated adhesiveness comparable to that in Caco-2 cell. It is noteworthy that cells of P.pastoris conspicuously interacted with the secreted mucus of HT-29 cells (Figure 3.3). The high level of adhesion by P.pastoris obscured the brush border, which was present under the biofilm of P.pastoris of HT-29 cells.
It is apparent from the scanning electron micrographs in Figures 3.1 and 3.2, and the enumeration results that P.pastoris exhibited the strongest affinity amongst the three yeasts for human intestinal cells.
99 CHAPTER 3 RESULTS
A B
C D
Figure 3.1 Observation by scanning electron microscopy of the adherence of S.boulardii, S.cerevisiae and P.pastoris to human intestinal epithelial Caco-2 cells. Low magnification showing adhesion of (A) S.boulardii (magnification, x2500) and (B) S.cerevisiae (magnification, x3300) to Caco-2 cells. Adhesion of (C) P.pastoris onto the brush border of Caco-2 cells (magnification, x2500). (D) Scanning electron micrograph showing Caco-2 cells with sparse microvilli (magnification, x2500). 100 CHAPTER 3 RESULTS
A B
C D
Figure 3.2 Adhesion of (A) S.boulardii (magnification, x3500), (B) S.cerevisiae (magnification, x2500) and (C) P.pastoris (magnification, x3500) to human mucus-secreting HT29 cells observed by scanning electron microscopy. (D) Scanning electron micrograph of HT29 cells with sparse microvilli and secreted mucus droplets on the cell surface (magnification, x2500).
101 CHAPTER 3 RESULTS
A B
C
Figure 3.3 P.pastoris whole cells interacted with the mucus secreted by the HT29 cells (magnification, x2000 [A] and x3700 [B]). (C) High level of adhering P.pastoris formed a biofilm, which obscured the brush border (magnification, x2000).
102 CHAPTER 3 RESULTS
3.3 IN VIVO ADHESION STUDIES
3.3.1 Murine Intestinal Surface Water and Mucus Content
Many of the intestinal microorganisms were suspended in a layer of intestinal
water, coating the intestinal surfaces. Any ingestion of food and water could potentially exert a dilution effect on the intestinal water. Hence, it is likely that microorganisms that remained suspended in the intestinal water and did not adhere to the mucosal surfaces would be diluted and washed out from the intestinal surfaces together with the mucus as part of the excreta eventually. As such, the dynamics of the mucosal layer determines the resident time of the intestinal microbiota.
For determination of the intestinal water volume, mice were sacrificed where the intestinal tracts were extracted and cut longitudinally. Each intestinal part was gently blotted dry using pre-weighed filter paper and the increase in the weight of the paper being taken as the volume of intestinal water of the respective intestinal portion. The
amounts of intestinal water overlaying the duodenum, jejunum, ileum and colon was
found to be 34.4 + 2.9, 58.8 + 6.8, 21.6 + 2.2 and 8.0 + 1.0 mg, respectively. The mice
consumed 6.6 + 0.7 g of feed and water per animal per day and excreted an average of 1.2
+ 0.3 g of feces per animal per day. Since the mucus content of the feces was 0.9 + 0.1 mg/g-faeces, it could be derived that 1.1 + 0.2 mg mucus was dislodged from the intestine surface of a mouse per day.
3.3.2 Analysis of Lactobacillus casei Shirota Adhesion in Murine Intestinal Tract
The protein dye cFDA-SE has been used to track cells and to analyze cell division
(Logan et al., 2000; Ueckert et al., 1997). An in vitro culture of cFDA-SE stained LcS
103 CHAPTER 3 RESULTS
showed that the bacteria could be successfully stained by the protein dye (Figure 3.4). In
addition, division of cell was characterized by sequential two-fold reduction in cFDA-SE
fluorescence (Figure 3.5). For instance, one cell doubling in number (generation number
1) resulted in a 1.6-fold reduction in the cellular fluorescence intensity of the dividing
cells. A further doubling in cell number (generation number 2) led to a 4.0-fold reduction
in the initial cellular fluorescence intensity. There is no indication of leakage of the dye
and re-staining of cells, which would lead to disproportional changes in cellular
fluorescence intensity with generation number.
a b
c d
Figure 3.4 cFDA-SE labelling of LcS. (a) LcS visualized under transmitted light source. (b) Green fluorescence of LcS stained with cFDA-SE. (c) Superimposed image of (a) and (b). (d) Unlabelled bacteria do not autofluorescence when the image was captured under cFDA-SE excitation wavelength. (Magnification x1000)
104 CHAPTER 3 RESULTS
4.00E+02
3.50E+02
y = -129.48x + 343.2 3.00E+02 R2 = 0.9958
2.50E+02
2.00E+02
1.50E+02
1.00E+02
5.00E+01
fluorescenceMedian (units/cell) intensity 0.00E+00 0.00.51.01.52.02.5 Generation No.
Figure 3.5 Plot of the residual median fluorescence intensity of an in vitro culture of labeled LcS against the generation number. cFDA-SE labeled LcS was allowed to replicate in vitro where the median fluorescence intensities were determined at various generation number of LcS. Each value represents the average of 3 samples and the vertical bars represent the standard deviations.
The rate of wash-out of the lactobacilli from the intestinal mucosal surface was estimated from the slope of the plots of the residual concentration of lactobacilli labeled with cFDA-SE (total fluorescent cells irrespective of the intensity) extracted from the mucosal surface versus the time after orogastric intubation (Figure 3.6). The half times for the wash-out of lactobacilli were determined to be 3.98, 1.55, 1.34, and 2.48 days in the duodenum, jejunum, ileum, and colon, respectively.
105 CHAPTER 3 RESULTS
9.00E+04 Duodenum 8.00E+04 R2 = 0.9872 Jejunum 7.00E+04 2 R = 0.7934 Ileum 6.00E+04 2 R = 0.7647 Colon 5.00E+04 2 R = 0.9921
4.00E+04
3.00E+04
2.00E+04 Cell no. per intestine part 1.00E+04
0.00E+00 012345 Days post orogastric intubation
Figure 3.6 Plot of total LcS cell number adhered on various sections of the intestinal tract against time after orogastric intubation. Each value represents the average for three samples, and the vertical bars indicate standard deviations.
As shown in Figure 3.5, division of LcS resulted in the dilution of the cFDA
labeled protein by half. Thus, the doubling times of the lactobacilli adhered on the mucosal surfaces were estimated from the residual fluorescence intensities (median) of
cells extracted at different times after orogastric intubation (Figure 3.7). The doubling
times of LcS were 4.10, 4.78, 4.56, and 5.59 days in the duodenum, jejunum, ileum, and
colon, respectively. This suggests that LcS is able to divide at comparable rates in all
parts of the small intestine and colon.
106 CHAPTER 3 RESULTS
3.00E+01 Duodenum 2 R = 0.9367 2.50E+01 Jejunum R2 = 0.9826 Ileum R2 = 0.9985 2.00E+01 Colon 2 R = 0.7347 1.50E+01
(units/cell) 1.00E+01
Median fluorescence intensity 5.00E+00
0.00E+00 1246
Days post orogastric intubation
Figure 3.7 Plot of the residual median fluorescence intensity of LcS adhered on various sections of the intestinal tract against time after orogastric intubation. The median fluorescence intensity of LcS adhering in the ileum on Day 6 could not be determined due to some technical faults with the flow sorter during flow cytometric analysis of the sample. Each value represents the average for three samples, and the vertical bars indicate standard deviations.
The fluorescence intensity profiles of the adhered lactobacilli determined on the
various days after orogastric intubation are summarized in Table 3.1. The percentage of total population of LcS with fluorescence intensity greater than the median on Day 1 was
found to differ amongst various portion of the intestinal tract. This gave an indication
that the bacterial cells were not uniformly labeled by cFDA-SE, and could probably be due to the physiological status of the cells at the time of incubation with cFDA-SE.
Taking the median fluorescence intensities measured on day 1 as the reference, the fluorescence intensities are categorized into those that are higher than the median measured on day 1, those from the median intensity down to half of the median intensity
107 CHAPTER 3 RESULTS measured on day 1 (expected fluorescence intensity after one cell division), those from half of the median intensity down to one-fourth of the median intensity measured on day
1 (expected fluorescence intensity after two cell divisions), and those lower than one- fourth of the median intensity measured on day 1 (expected fluorescence intensity after three cell divisions). For example, in the duodenum on day 1, 35.92% of the lactobacilli showed fluorescence intensity higher than the median intensity (1.40 × 101 fluorescence units/cell), and 36.49% showed an intensity lower than 3.5 × 100 fluorescence units/cell
(expected fluorescence level after three cell divisions).
The cell division profiles of the adherent lactobacilli post oral administration were also illustrated in Figure 3.8. It was observed that the percentage of cells that did not undergo any cell division decreased over the days post feeding, while the percentage of cells that have undergone two or three cell divisions was found to increase. This gives an indication that LcS adhering to various portion of the gastrointestinal tract was able to undergo replication. Using the data from Table 3.1, 44.4% (15.96/35.92) of the labeled lactobacilli retained the original fluorescence intensity (no cell division), whereas 13.1% of the total labeled lactobacilli had divided at least three times (49.54 − 36.49) on day 6 in the duodenum. It was calculated that on day 6 after orogastric intubation, the percentages of the lactobacilli that remained non-divided and adhered on the intestinal mucosal surfaces were 44.4, 60.5, 37.2, and 56.3% in the duodenum, jejunum, ileum, and colon, respectively. On day 6 after orogastric intubation, 13.1, 3.9, 21.6, and 21.4% of the lactobacilli fed had divided at least three times.
108 CHAPTER 3 RESULTS
Table 3.1 Fluorescence intensity profiles of lactobacilli harvested from various sections of the intestinal mucosal surface on various days after orogastric intubation of LcS.
% of the total population with the following fluorescence intensity Section and (mean + SD)a Day <1/4 median 1/4-1/2 median 1/2-median >Median Duodenum 1 36.49 ± 3.51 10.13 ± 1.02 17.46 ± 1.98 35.92 ± 2.47 2 33.04 ± 2.87 15.16 ± 1.48 19.80 ± 1.21 32.00 ± 2.41 4 37.76 ± 2.69 18.64 ± 1.78 20.02 ± 2.04 23.58 ± 2.84 6 49.54 ± 3.98 18.31 ± 1.20 16.19 ± 1.18 15.96 ± 1.07
Jejunum 1 24.23 ± 1.79 12.64 ± 0.84 18.95 ± 1.51 44.18 ± 4.25 2 28.54 ± 2.07 16.53 ± 1.20 20.97 ± 1.99 33.96 ± 2.89 4 28.15 ± 2.10 18.06 ± 1.74 21.71 ± 2.10 32.08 ± 3.84 6 28.14 ± 3.07 24.51 ± 2.86 20.60 ± 2.51 26.75 ± 2.76
Ileum 1 30.06 ± 2.87 14.73 ± 1.17 15.78 ± 1.72 39.43 ± 3.20 2 43.57 ± 3.51 24.85 ± 2.71 15.93 ± 1.43 15.65 ± 1.33 4 47.87 ± 3.82 24.18 ± 2.14 12.57 ± 0.94 15.38 ± 1.14 6 51.61 ± 4.01 22.90 ± 1.88 10.83 ± 0.76 14.66 ± 1.21
Colon 1 40.70 ± 3.14 14.38 ± 1.13 21.05 ± 1.45 23.87 ± 1.71 2 37.74 ± 2.41 24.33 ± 2.03 25.00 ± 2.17 12.93 ± 0.72 4 65.00 ± 4.95 14.36 ± 1.24 7.12 ± 0.49 13.52 ± 1.00 6 62.14 ± 3.45 16.57 ± 1.52 7.86 ± 0.84 13.43 ± 1.17
a The medians of the fluorescence intensities (n = 3) measured on day 1 were 1.40 x 101, 9.33 x 100, 1.87 x 101, and 2.23 x 101 U/cell in the duodenum, jejunum, ileum and colon respectively. <1/4 median, lower than one-fourth of the median fluorescence intensity measured on day 1; 1/4-1/2 median, one-fourth to one-half of the median intensity; 1/2- median, half of median to median intensity; >median, higher then median intensity.
109 CHAPTER 3 RESULTS
No cell division 1 cell division Duodenum 2 100 R = 0.9797 100 Duodenum Jejunum R2 = 0.0623 2 R = 0.919 Jejunum Ileum R2 = 0.3964 2 R = 0.6324 Ileum Colon R2 = 0.8824 2 10 R = 0.5614 10 Colon R2 = 0.6617
% of total population % of total population 1 1 1246 1246 Days post orogastric intubation Days post orogastric intubation
2 cell divisions 3 cell divisions
Duodenum 100 Duodenum 100 R2 = 0.8407 R2 = 0.6237 Jejunum Jejunum R2 = 0.9415 R2 = 0.5191 Ileum Ileum R2 = 0.43 R2 = 0.896 Colon 10 Colon R2 = 0.0086 R2 = 0.6973 10
% of total population % of total population 1 1 1246 1246 Days post orogastric intubation Days post orogastric intubation
Duodenum Jejunum Ileum Colon
Figure 3.8 Plots showing the division profiles of total population of adhering LcS. Each value represents the average for three samples, and the vertical bars indicate standard deviations.
110 CHAPTER 3 RESULTS
3.3.3 Analysis of Saccharomyces boulardii Adhesion in Murine Intestinal Tract
Similarly, cFDA-SE was also utilized to analyze the ability of S.boulardii to adhere to murine intestinal cells. S.boulardii labeled with cFDA-SE was cultured in vitro to determine if the replication of the yeast will result in halving of the fluorescence intensity. Results had shown that the yeast could be successfully stained by the protein dye (Figure 3.9) and the division of cell was characterized by sequential two-fold reduction in cFDA-SE fluorescence (Figure 3.10). One cell doubling in number
(generation number 1) resulted in a 1.6-fold reduction in the cellular fluorescent intensity of the dividing cells. A further doubling in cell number (generation number 2) led to a
4.1-fold reduction in the initial cellular fluorescent intensity. There is no indication of
leakage of the dye and re-staining of cells, which would lead to disproportional changes in cellular fluorescent intensity with generation number.
a b
c d
Figure 3.9 cFDA-SE labelling of S.boulardii. (a) S.boulardii visualized under transmitted light source. (b) Green fluorescence of S.boulardii stained with cFDA-SE. (c) Superimposed image of (a) and (b). (d) Unlabelled yeast does not autofluorescence when the image was captured under cFDA-SE excitation wavelength. (Magnification x1000).
111 CHAPTER 3 RESULTS
4.50E+02
4.00E+02 y = -135.54x + 357.44 3.50E+02 R2 = 0.9679 3.00E+02
2.50E+02
2.00E+02
1.50E+02
1.00E+02
5.00E+01 Median fluorescence intensity (units/cell) 0.00E+00 0.0 0.5 1.0 1.5 2.0 2.5 Generation No.
Figure 3.10 Plot of the residual median fluorescent intensity of an in vitro culture of labeled S.boulardii against the generation number. cFDA-SE labeled S.boulardii was allowed to replicate in vitro where the median fluorescence intensities were determined at various generation number. Each value represents the average of 3 samples and the vertical bars represent standard deviations.
As shown in Figure 3.11, the adherence of yeast cells after oral feeding took place
mainly in the duodenum (81.5 % of total adhering S.boulardii on day 1). The rate of
wash-out of S.boulardii from the intestinal mucosal surface was estimated from the slope
of the plots of the residual concentration of the yeast labeled with cFDA-SE (total
fluorescent cells irrespective of the intensity) extracted from the mucosal surface versus
the time after orogastric intubation (Figure 3.11). The half times for the wash-out of
S.boulardii were determined to be 2.64, 3.02, 5.35, and 66.02 days in the duodenum,
jejunum, ileum, and colon, respectively.
112 CHAPTER 3 RESULTS
8.00E+05 Duodenum R2 = 0.9279 7.00E+05 Jejunum R2 = 0.9887 6.00E+05 Ileum R2 = 0.6247 Colon 5.00E+05 R2 = 0.0869
4.00E+05
3.00E+05
Cell no. per intestine part 2.00E+05
1.00E+05
0.00E+00 1234 Days post orogastric intubation
Figure 3.11 Plot of total S.boulardii cell number adhered on various sections of the intestinal tract against time after orogastric intubation. Each value represents the average for three samples, and the vertical bars indicate standard deviations.
The time taken by adhering S.boulardii to replicate in murine intestinal tract were estimated from the residual fluorescence intensities (median) of cells extracted at different times after orogastric intubation (Figure 3.12). The doubling times of
S.boulardii were found to be 1.33, 2.61, 10.91, and 20.83 days in the duodenum, jejunum, ileum, and colon, respectively.
113 CHAPTER 3 RESULTS
1.40E+01 Duodenum R2 = 0.9938 Jejunum 1.20E+01 R2 = 0.9815 Ileum R2 = 0.4144 1.00E+01 Colon R2 = 0.4271 8.00E+00 tensity (units/cell)
6.00E+00
4.00E+00
2.00E+00
in fluorescence Median 0.00E+00 1246 Days post orogastric intubation
Figure 3.12 Plot of residual median fluorescence intensity of S.boulardii adhered on various
sections of the intestinal tract against time after orogastric intubation. Each value represents the
average for three samples, and the vertical bars indicate standard deviations.
Table 3.2 summarizes the fluorescence intensity profiles of the adhered
S.boulardii determined on the various days after orogastric intubation. On day 1, 38.66% of the yeast showed a fluorescence intensity higher than the median intensity (1.22 × 101 fluorescence units/cell) in the duodenum, and 33.15% showed an intensity lower than
3.05 × 100 fluorescence units/cell (expected fluorescence level after three cell divisions).
The cell division profiles of the adherent yeast post oral administration were
illustrated in Figure 3.13. Interestingly, the percentages of S.boulardii that did not under
go cell division remained constantly high in the ileum and colon. This gives an
indication that the yeast adhering to ileum and colon could not replicate. As for the cells
attached to duodenum and jejunum, there is a gradual reduction in the percentages of cell
114 CHAPTER 3 RESULTS
that did not undergo division, which was complimented by a general increase in the
percentages of cells that have replicated at least once. Based on the data obtained from
Table 3.2, 35.5% (13.72/38.66) of the labeled S.boulardii retained the original fluorescence intensity (no cell division), whereas 16.3% of the total labeled yeast had divided at least three times (49.48 − 33.15) on day 6 in the duodenum. It was calculated that on day 6 after orogastric intubation, the percentages of S.boulardii that remained non-dividing and adhering on the intestinal mucosal surfaces were 35.59, 53.4, 91.5, and
96.6% in the duodenum, jejunum, ileum, and colon, respectively. On day 6 after orogastric intubation, 16.3, 14.0, 8.0, and 6.1% of the yeast fed had divided at least three times.
115 CHAPTER 3 RESULTS
Table 3.2 Fluorescence intensity profiles of S.boulardii harvested from various sections of the intestinal mucosal surface on various days post orogastric intubation.
% of the total population with the following fluorescence intensity Section and (mean + SD)a Day <1/4 median 1/4-1/2 median 1/2-median >Median Duodenum 1 33.15 ± 3.19 11.58 ± 1.01 16.61 ± 1.88 38.66 ± 2.90 2 35.49 ± 3.75 13.34 ± 1.08 19.38 ± 1.13 31.79 ± 3.40 4 40.03 ± 3.63 16.52 ± 1.16 20.87 ± 1.42 22.58 ± 2.17 6 49.48 ± 4.35 17.28 ± 1.48 19.52 ± 1.60 13.72 ± 1.65
Jejunum 1 24.87 ± 2.41 13.80 ± 1.75 17.44 ± 1.79 43.89 ± 3.74 2 27.22 ± 1.19 14.80 ± 1.84 19.45 ± 1.77 38.53 ± 3.32 4 27.91 ± 3.05 17.37 ± 2.42 22.81 ± 1.84 31.91 ± 3.44 6 38.85 ± 2.21 19.31 ± 1.17 18.41 ± 2.08 23.43 ± 2.56
Ileum 1 9.12 ± 0.99 11.82 ± 1.02 10.78 ± 1.75 68.28 ± 4.66 2 9.83 ± 1.09 12.95 ± 1.91 11.54 ± 1.38 65.68 ± 2.92 4 12.55 ± 1.10 13.62 ± 1.24 10.74 ± 1.21 63.09 ± 3.53 6 17.10 ± 0.84 11.99 ± 1.20 8.44 ± 1.19 62.47 ± 2.95
Colon 1 8.23 ± 1.23 10.08 ± 1.05 9.53 ± 1.29 72.16 ± 4.73 2 12.19 ± 1.19 9.26 ± 0.25 8.11 ± 1.17 70.44 ± 4.16 4 11.24 ± 1.23 9.65 ± 1.36 8.01 ± 1.41 71.10 ± 3.96 6 14.33 ± 1.68 8.28 ± 1.37 7.70 ± 1.20 69.69 ± 5.13
a The medians of the fluorescence intensities (n = 3) measured on day 1 were 1.22 x 101, 4.72 x 100, 2.6 x 100, and 2.21 x 100 U/cell in the duodenum, jejunum, ileum and colon respectively. <1/4 median, lower than one-fourth of the median fluorescence intensity measured on day 1; 1/4-1/2 median, one-fourth to one-half of the median intensity; 1/2- median, half of median to median intensity; >median, higher then median intensity.
116 CHAPTER 3 RESULTS
No Cell Division 1 Cell Division 100 100 Duodenum Duodenum R2 = 0.996 R2 = 0.5447 Jejunum Jejunum 2 2 R = 0.9895 R = 0.1199 10 Ileum 10 Ileum R2 = 0.9447 R2 = 0.5664 Colon Colon R2 = 0.692 R2 = 0.7861
% of total population % of total population 1 1 1246 1246 Days post orogastric intubation Days post orogastric intubation
2 Cell Divisions 3 Cell Divisions
100 100 Duodenum Duodenum R2 = 0.9542 R2 = 0.8335 Jejunum Jejunum R2 = 0.9253 R2 = 0.7806 Ileum Ileum 10 R2 = 0.0325 10 R2 = 0.906 Colon Colon R2 = 0.7084 R2 = 0.7821
% of total population of total % % of total population 1 1 1246 1246 Days post orogastric intubation Days post orogastric intubation
Duodenum Jejunum Ileum Colon
Figure 3.13 Plots showing the division profiles of total population of adhering S.boulardii. Each value represents the average for three samples, and the vertical bars indicate standard deviations.
117 CHAPTER 3 RESULTS
3.3.4 Analysis of Saccharomyces cerevisiae Adhesion in Murine Intestinal Tract
An in vitro culture of fluorescently labeled S.cerevisiae was analyzed to determine if the replication of the yeast would result in halving of the fluorescence intensity. The yeast was observed to be successfully tag with cFDA-SE when viewed under fluorescent microscope (Figure 3.14). Results had shown that division of cell was characterized by sequential two-fold reduction in cFDA-SE fluorescence (Figure 3.15).
There is no indication of leakage of the dye and re-staining of cells, which would lead to disproportional changes in cellular fluorescent intensity with generation number.
a b
c d
Figure 3.14 cFDA-SE labelling of S.cerevisiae. (a) S.cerevisiae visualized under transmitted light source. (b) Green fluorescence of S.cerevisiae stained with cFDA-SE. (c) Superimposed image of (a) and (b). (d) Unlabelled yeast does not autofluorescence when the image was captured under cFDA-SE excitation wavelength. (Magnification x1000).
118 CHAPTER 3 RESULTS
4.50E+02
4.00E+02
3.50E+02 y = -141.38x + 378.29 R2 = 0.9951 3.00E+02
2.50E+02
2.00E+02
1.50E+02
1.00E+02
5.00E+01
Median fluorescence intensity (units/cell) 0.00E+00 0.0 0.5 1.0 1.5 2.0 2.5
Generation No.
Figure 3.15 Plot of the residual median fluorescent intensity of an in vitro culture of labeled S.cerevisiae against the generation number. cFDA-SE labeled S.cerevisiae was allowed to replicate in vitro where the median fluorescence intensities were determined at various generation number. Each value represents the average of 3 samples and the vertical bars represent standard deviations.
The rate of wash-out of S.cerevisiae from the intestinal mucosal surface was estimated from the slope of the plots of the residual concentration of yeast labeled with
cFDA-SE (total fluorescent cells irrespective of the intensity) extracted from the mucosal surface versus the time after orogastric intubation (Figure 3.16). The half times for the wash-out of S.cerevisiae were determined to be 1.25, -4.46, 1.70, and 1.84 days in the duodenum, jejunum, ileum, and colon, respectively. The negative wash-out half time obtained for S.cerevisiae in the jejunum was attributable to the increase in cell number various days post feeding. The increase in the number of S.cerevisiae in the jejunum
could either be due to the replication of the adhering yeast or contributed by the re-
adherence of yeast detached from the duodenum. It could be observed from Figure 3.17
that the median fluorescence intensity of S.cerevisiae adhering to the jejunum remained
119 CHAPTER 3 RESULTS
relatively constant from Day 1 to 6 post administrations. This implied that the adhering
S.cerevisiae has not undergone replication, as division of the cells would result in a
decrease in the fluorescence intensity. Hence, the increase in number of S.cerevisiae in
the jejunum is more likely due to the reattachment of the detached cells from the
duodenum.
3.00E+06 Duodenum R2 = 0.7475 Jejunum 2.50E+06 R2 = 0.6836 Ileum R2 = 0.7931 2.00E+06 Colon R2 = 0.6973
1.50E+06
1.00E+06
5.00E+05 Cell no. per intestine part 0.00E+00
1234 -5.00E+05 Days post orogastric intubation
Figure 3.16 Plot of total S.cerevisiae cell number adhered on various sections of the intestinal tract against time after orogastric intubation. Each value represents the average for three samples, and the vertical bars indicate standard deviations.
The doubling times of S.cerevisiae adhering to different portions of murine intestinal tract were estimated from the residual fluorescence intensities (median) of cells extracted at different times after orogastric intubation (Figure 3.17). The doubling times of S.cerevisiae were 1.67, 9.10, 0.45, and 0.85 days in the duodenum, jejunum, ileum, and colon, respectively.
120 CHAPTER 3 RESULTS
4.00E+01 Duodenum R2 = 0.7947 3.50E+01 jeunum R2 = 0.4732 3.00E+01 Ileum R2 = 0.7704 Colon 2.50E+01 R2 = 0.5643 2.00E+01
1.50E+01
1.00E+01
5.00E+00
0.00E+00
Median fluorescence intensity (units/cell) intensity fluorescence Median 1246 -5.00E+00 Days post orogastric intubation
Figure 3.17 Plot of residual median fluorescence intensity of S.cerevisiae adhered on various sections of the intestinal tract against time after orogastric intubation. Each value represents the average for three samples, and the vertical bars indicate standard deviations.
Analysis of the fluorescence intensities of adherent S.cerevisiae various days after
orogastric intubation was conducted. The fluorescence profiles were summarized and
represented in Table 3.3. For example, on day 1, 42.15% of the yeast showed a
fluorescence intensity higher than the median intensity (3.17 × 101 fluorescence
units/cell) in the duodenum, and 25.05% showed an intensity lower than 7.93 × 100 fluorescence units/cell (expected fluorescence level after three cell divisions).
As illustrated by the cell division profiles of the adhered S.cerevisiae (Figure
3.18), the percentage of S.cerevisiae that did not under go cell division was constantly high in the jejunum. More than half of the cells that were attached to the jejunum did not replicate and only a small fraction of cell has undergone cell division. Replication of
S.cerevisiae was observed in the duodenum, ileum and colon, as indicated by a gradual
121 CHAPTER 3 RESULTS
reduction in the percentages of cell that did not undergo division, and an increase in the
percentages of cells that have replicated at least once. Based on the data obtained from
Table 3.3, 44.5% (18.76/42.15) of the labeled S.cerevisiae retained the original
fluorescence intensity (no cell division), whereas 16.6% of the total labeled yeast had
divided at least three times (41.57 − 25.02) on day 6 in the duodenum. On day 6 after
orogastric intubation, the percentages of S.cerevisiae that remained non-divided and adhered on the intestinal mucosal surfaces were 44.5, 93.3, 24.0, and 30.6% in the duodenum, jejunum, ileum, and colon, respectively. On day 6 after orogastric intubation,
16.6, 0.27, 34.23, and 26.09% of the yeast fed had divided at least three times.
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Table 3.3 Fluorescence intensity profiles of S.cerevisiae harvested from various sections
of the intestinal mucosal surface on various days post orogastric intubation.
% of the total population with the following fluorescence intensity Section and (mean + SD)a Day <1/4 median 1/4-1/2 median 1/2-median >Median Duodenum 1 25.02 ± 3.45 14.15 ± 1.45 18.68 ± 2.69 42.15 ± 3.86 2 28.62 ± 2.48 16.35 ± 1.11 19.74 ± 1.21 35.29 ± 3.05 4 34.85 ± 3.71 18.41 ± 1.98 20.67 ± 3.00 26.07 ± 2.69 6 41.57 ± 2.52 19.71 ± 2.01 20.96 ± 2.34 18.76 ± 1.85
Jejunum 1 11.32 ± 1.34 11.74 ± 0.82 11.33 ± 1.91 65.61 ± 2.79 2 10.40 ± 1.14 10.75 ± 1.29 12.08 ± 1.67 66.77 ± 2.97 4 10.49 ± 0.98 11.68 ± 1.49 15.42 ± 0.76 63.95 ± 2.56 6 11.59 ± 1.35 11.19 ± 2.09 16.00 ± 1.31 61.22 ± 2.84
Ileum 1 28.99 ± 3.07 18.36 ± 2.12 19.48 ± 1.56 33.17 ± 2.63 2 43.62 ± 4.58 16.42 ± 2.71 18.32 ± 2.27 21.64 ± 3.16 4 56.84 ± 3.78 16.14 ± 1.98 14.96 ± 1.96 12.06 ± 1.66 6 63.22 ± 4.52 15.56 ± 1.48 13.51 ± 1.42 7.97 ± 1.49
Colon 1 30.45 ± 3.01 16.47 ± 1.42 17.47 ± 1.93 35.61 ± 3.47 2 36.03 ± 3.50 18.23 ± 1.64 18.83 ± 1.66 26.97 ± 2.91 4 48.46 ± 4.14 17.44 ± 1.65 17.37 ± 1.99 16.73 ± 1.46 6 56.54 ± 4.22 16.40 ± 1.92 16.17 ± 1.33 10.89 ± 1.44
a The medians of the fluorescence intensities (n = 3) measured on day 1 were 3.17 x 101, 1.87 x 101, 4.06 x 100, and 6.05 x 100 U/cell in the duodenum, jejunum, ileum and colon respectively. <1/4 median, lower than one-fourth of the median fluorescence intensity measured on day 1; 1/4-1/2 median, one-fourth to one-half of the median intensity; 1/2- median, half of median to median intensity; >median, higher then median intensity.
123 CHAPTER 3 RESULTS
No Cell Division 1 Cell Division
100 100 Duodenum Duodenum R2 = 0.997 R2 = 0.9487 Jejunum Jejunum R2 = 0.7346 R2 = 0.9134 Ileum Ileum 10 R2 = 0.9617 10 R2 = 0.963 Colon Colon R2 = 0.989 R2 = 0.4067 % of total population % of total population 1 1 1246 1246 Days post orogastric intubation Days post orogastric intubation
2 Cell Divisions 3 Cell Divisions 100 100 Duodenum 2 Duodenum R = 0.9833 2 R = 0.9875 Jejunum Jejunum R2 = 0.0391 2 R = 0.0402 Ileum Ileum 10 R2 = 0.9732 2 10 R = 0.8508 Colon Colon R2 = 0.9813 2 R = 0.0231 % of total population
% of total population 1 1 1246 1246 Days post orogastric intubation Days post orogastric intubation
Duodenum Jejunum Ileum Colon
Figure 3.18 Plots showing the division profiles of total population of adhering S.cerevisiae. Each value represents the average for three samples, and the vertical bars indicate standard deviations.
124 CHAPTER 3 RESULTS
3.3.5 Analysis of Pichia pastoris Adhesion in Murine Intestinal Tract
An in vitro culture of cFDA-SE stained P.pastoris had shown that the yeast could be successfully labeled by the protein dye (Figure 3.19) and the division of cell was characterized by sequential two-fold reduction in cFDA-SE fluorescence (Figure 3.20). There is no indication of leakage of the dye and re- staining of cells, which would lead to disproportional changes in cellular fluorescent intensity with generation number.
a b
c d
Figure 3.19 cFDA-SE labelling of P.pastoris. (a) P.pastoris visualized under transmitted optical view. (b) Green fluorescence of P.pastoris stained with cFDA-SE. (c) Superimposed image of (a) and (b). (d) Unlabelled yeast does not autofluorescence when the image was captured under cFDA-SE excitation wavelength. (Manification x1000).
125 CHAPTER 3 RESULTS
450
400
350 y = -144.64x + 378.63 R2 = 0.9862 300
250
200
150 100
50
Median fluorescence intensity (units/cell) 0 0.00.51.01.52.02.5 Ge ne ra tion No.
Figure 3.20 Plot of the residual median fluorescent intensity of an in vitro culture of labeled P.pastoris against the generation number. cFDA-SE labeled P.pastoris was allowed to replicate in vitro where the median fluorescence intensities were determined at various generation number. Each value represents the average of 3 samples and the vertical bars represent the standard deviations.
The wash-out rate of P.pastoris adhered to intestinal mucosal surface was
estimated from the slope of the plots of the residual concentration of yeast labeled with
cFDA-SE (total fluorescent cells irrespective of the intensity) extracted from the mucosal
surface versus the time after orogastric intubation (Figure 3.21). The half times for the
wash-out of P.pastoris were determined to be 2.85, 2.21, -28.80, and –5.97 days in the
duodenum, jejunum, ileum, and colon, respectively. The negative half times for the
wash-out of P.pastoris in the ileum and colon were due to the increase in cell number
various days post feeding. As mentioned in the previous section, an increase in cell
number might not be due to the replication of the adhering yeast. The re-adherence o
cells detached from the upper portion of the intestine could also result in an increase in
cell number further down the intestinal tract. As observed in Figure 3.22, the median
126 CHAPTER 3 RESULTS
fluorescence intensity of P.pastoris adhering to the ileum increased over the days, while
the median fluorescence intensity of those attached to the colon decreased over the days.
An increase in cell number, accompanied by a decrease in median fluorescence intensity,
implied the occurrence of cell division. Hence, P.pastoris that adhered to the colon had
undergone replication. As for the increase in number of yeast in the ileum, there was a
corresponding increase in the median fluorescence intensity of the adhering P.pastoris.
This gave an indication that yeast detached from the upper portion of the intestinal tract
had reattached to the colon.
1.20E+06 Duodenum R2 = 0.805 Jejunum 1.00E+06 R2 = 0.9262
t Ileum R2 = 0.3905 8.00E+05 Colon R2 = 0.1959
6.00E+05
4.00E+05 Cell no. per intestine par 2.00E+05
0.00E+00 1234 Days post orogastric intubation
Figure 3.21 Plot of total P.pastoris cell number adhered on various sections of the intestinal tract against time after orogastric intubation. Each value represents the average for three samples, and the vertical bars indicate standard deviations.
127 CHAPTER 3 RESULTS
As each division of the yeast resulted in the halving of fluorescence intensity, the doubling times of P.pastoris adhered on the mucosal surfaces were estimated from the residual fluorescence intensities (median) of cells extracted at different times after orogastric intubation (Figure 3.22). The doubling times of P.pastoris were 1.54, 1.97, -
77.32, and 2.76 days in the duodenum, jejunum, ileum, and colon, respectively. The negative doubling time of P.pastoris in the ileum was due to the increase in the residual median fluorescence intensity of adhering yeast over the days in the ileum. As explained earlier, this increase in fluorescence intensity was resulted from the reattachment of
P.pastoris detached from the duodenum and jejunum.
2.50E+01 Duodenum R2 = 0.9483 l Jejunum R2 = 0.9844 2.00E+01 Ileum R2 = 0.14 Colon R2 = 0.7378 1.50E+01
1.00E+01
5.00E+00
Median fluorescence intensity (units/ce 0.00E+00 1246 Days post orogastric intubation
Figure 3.22 Plot residual median fluorescence intensity of P.pastoris adhered on various sections of the intestinal tract against time after orogastric intubation. Each value represents the average for three samples, and the vertical bars indicate standard deviations.
The fluorescence intensity profiles of the adhered P.pastoris determined on the various days after orogastric intubation have been illustrated in Table 3.4. On day 1,
128 CHAPTER 3 RESULTS
40.50% of the yeast showed a fluorescence intensity higher than the median intensity
(1.77 × 101 fluorescence units/cell) in the duodenum, and 26.69% showed an intensity
lower than 4.43 × 100 fluorescence units/cell (expected fluorescence level after three cell
divisions).
The cell division profiles of the adherent P.pastoris post oral administration were
also illustrated in Figure 3.23. The percentage of P.pastoris that adhered to the ileum but
did not replicate was constantly high in comparison to those that were attached to other
portions of the intestinal tract. This is in agreement to the observations that the increase
in number of P.pastoris in the ileum was contributed by the re-adhesion of detached yeast
rather than the replication of the adhering P.pastoris in the ileum. The percentages of cells that have undergone 1 or 2 cell divisions were uniformly low throughout the days, while those that have undergone 3 cell divisions were increasing, giving an indication that the adhering yeast were constantly dividing. Based on the data obtained from Table
3.2, 44.9% (18.22/40.50) of the labeled P.pastoris retained the original fluorescence intensity (no cell division), whereas 20.2% of the total labeled yeast had divided at least three times (46.89 − 26.69) on day 6 in the duodenum. It was calculated that on day 6 after orogastric intubation, the percentages of P.pastoris that remained non-divided and adhered on the intestinal mucosal surfaces were 44.9, 45.2, 94.9, and 49.4% in the duodenum, jejunum, ileum, and colon, respectively. On day 6 after orogastric intubation,
20.2, 23.7, 3.7, and 13.7% of the yeast fed had divided at least three times.
129 CHAPTER 3 RESULTS
Table 3.4 Fluorescence intensity profiles of P.pastoris harvested from various sections of the intestinal mucosal surface on various days post orogastric intubation.
% of the total population with the following fluorescence intensity Section and (mean + SD)a Day <1/4 median 1/4-1/2 median 1/2-median >Median Duodenum 1 26.69 ± 2.90 15.45 ± 1.20 17.36 ± 1.50 40.50 ± 3.73 2 27.04 ± 2.65 17.18 ± 1.92 19.52 ± 1.82 36.26 ± 2.47 4 38.47 ± 3.86 19.51 ± 1.94 17.35 ± 1.88 24.67 ± 2.51 6 46.89 ± 4.10 18.36 ± 1.30 16.52 ± 2.45 18.22 ± 1.75
Jejunum 1 23.09 ± 1.63 13.54 ± 1.62 18.62 ± 1.51 44.75 ± 3.77 2 29.97 ± 3.27 15.21 ± 1.39 19.98 ± 1.29 34.84 ± 2.07 4 39.05 ± 2.47 16.86 ± 1.86 20.76 ± 1.87 23.33 ± 2.35 6 46.75 ± 3.21 14.59 ± 1.54 18.44 ± 1.49 20.22 ± 2.06
Ileum 1 20.34 ± 1.44 13.13 ± 1.42 15.45 ± 0.73 51.08 ± 3.66 2 20.04 ± 2.68 16.08 ± 1.77 18.36 ± 1.66 45.52 ± 3.45 4 21.12 ± 2.28 14.58 ± 1.66 12.62 ± 1.59 51.68 ± 4.35 6 24.03 ± 2.59 14.00 ± 2.30 13.51 ± 1.96 48.46 ± 3.79
Colon 1 25.22 ± 2.83 12.40 ± 2.10 13.92 ± 1.71 48.46 ± 4.30 2 26.39 ± 2.63 15.65 ± 1.58 16.40 ± 2.01 41.56 ± 2.87 4 31.52 ± 3.39 16.61 ± 1.57 18.19 ± 1.91 33.68 ± 2.78 6 38.91 ± 2.48 17.68 ± 2.12 19.48 ± 2.44 23.93 ± 1.99
a The medians of the fluorescence intensities (n = 3) measured on day 1 were 1.77 x 101, 1.27 x 101, 1.17 x 101, and 6.96 x 100 U/cell in the duodenum, jejunum, ileum and colon respectively. <1/4 median, lower than one-fourth of the median fluorescence intensity measured on day 1; 1/4-1/2 median, one-fourth to one-half of the median intensity; 1/2- median, half of median to median intensity; >median, higher then median intensity.
130 CHAPTER 3 RESULTS
No Cell Division 1 Cell Division
100 100 Duodenum Duodenum R2 = 0.9715 R2 = 0.2229 Jejunum Jejunum R2 = 0.9562 R2 = 0.0008 Ileum Ileum 10 R2 = 0.0062 10 R2 = 0.3454 Colon Colon R2 = 0.9938 R2 = 0.9797
% of total population % of totlap population 1 1 1246 1246 Days post orogastric intubation Days post orogastric intubation
2 Cell Divisions 3 Cell Divisions 100 100
Duodenum Duodenum R2 = 0.6789 R2 = 0.9083 Jejunum Jejunum R2 = 0.1981 R2 = 0.9975 Ileum Ileum 2 10 2 10 R = 0.0133 R = 0.7408 Colon Colon R2 = 0.905 R2 = 0.9159
% of total population % of total population 1 1 1246 1246 Days post orogastric intubation Days post orogastric intubation
Duodenum Jejunum Ileum Colon
Figure 3.23 Plots showing the division profiles of total population of adhering P.pastoris. Each value represents the average for three samples, and the vertical bars indicate standard deviations.
131 CHAPTER 4 RESULTS
4.0 DEVELOPMENT OF ORAL VACCINE AGAINST
TRANSMISSIBLE GASRTOENTERITIS CORONAVIRUS
(TGEV)
4.1 INTRODUCTION
TGEV, an enteropathogenic coronavirus, causes acute diarrhea in newborn pigs
with a mortality rate that is close to 100%. Oral vaccination, which allows the neutralization of pathogens at the point of infection, would be useful in the development of protective measures against TGEV infection. In this part of the study, the feasibility of
using prokaryotic lactobacilli and eukaryotic yeasts as a mode of delivery of TGEV antigen was investigated. In the previous chapter, we have showed that LcS and the three strains of yeast were able to adhere to the intestinal cells. In particular, P.pastoris
demonstrated the highest adhesiveness both in vitro and in vivo when compared to
S.boulardii and S.cerevisiae. Hence, LcS and P.pastoris were genetically engineered to constitutively express and secrete an N-terminal fragment of TGEV spike protein.
Immunization studies of the recombinant LcS and P.pastoris were carried out using
BALB/c mice. The neutralization ability of the induced antibodies against TGEV were determined, while the cytokine production profiles of cells of the Peyer’s patches extracted from the immunized mice were also characterized.
4.2 GENERATION OF TGEV-S-GST
A 1.3 kb gene fragment encoding the N-terminal portion of TGEV spike protein was generated from first strand cDNA synthesized from the TGEV genomic RNA
(Figure 4.1) and cloned into pGEX-4T-3, downstream of GST gene. The selected gene
132 CHAPTER 4 RESULTS
fragment consisted of the two immunodominant A and D sites, where epitopes critical for
neutralization were found to be clustered within (Delmas et al., 1986; Jim’enez et al.,
1986). Plasmids isolated from positively selected transformants were analyzed for the presence of TGEV spike insert via restriction digestion. As shown in Figure 4.2, the presence of a band that is approximately 1.3 kb in size indicated that recombinant E.coli
harboring TGEV spike gene fragment have been successfully constructed. Nucleotide
sequencing of the recombinant vectors showed that the TGEV insert were cloned in- frame to the sequence encoding GST in the vector.
Induction of TGEV-S-GST protein by the recombinant E.coli was carried out for
3 hr using 1 mM IPTG, for the optimal expression of the recombinant protein. The recombinant protein consisting of the N-terminal fragment of TGEV spike protein (44 kDa) fused to the C-terminal of GST protein (26 kDa), would have a molecular weight of
70 kDa. The supernatant and different fractions of the insoluble proteins extracted using various concentrations of urea and 0.01% SDS were separated on 10% SDS-PAGE and visualized by Coomassie Blue staining (Figure 4.3). It could be observed from Figure
4.3A that the highest amount of TGEV-S-GST protein was extracted using 6M urea. The protein fraction solubilized using 6M urea was then re-folded by extensive buffer exchange and further purified by running the extract through a column containing glutathione sepharose 4B. As apparent from Figure 4.3B, TGEV-S-GST protein was relatively pure after the affinity purification step. The purified form of TGEV-S-GST protein was then included as positive control in subsequent Western blots or used as coating antigen in ELISA for the analytical studies of the recombinant LcS or P.pastoris that were developed as oral vaccine carriers against TGEV.
133 CHAPTER 4 RESULTS
M
1.5 kb 1.2 kb 1.3 kb 1.0 kb
Figure 4.1 Agarose gel electrophoresis of PCR amplified TGEV spike gene fragment to be ligated to the 3’ end of GST. Lane M denotes 100 bp DNA ladder.
M1 M2 1 2
6.0 kb
5.0 kb 5.0 kb
1.5 kb 1.2 kb 1.3 kb 1.0 kb
Figure 4.2 Restriction digested plasmids extracted from E.coli transformants electrophorized on agarose gel. Lane: 1 and 2, a 1.3 kb band corresponded to the TGEV spike gene insert was cleaved from the vector using EcoRI and Sal1, which corresponded to the 5.0 kb band. Lane M1 denotes 1 kb DNA ladder while lane M2 denotes 100 bp DNA ladder.
134 CHAPTER 4 RESULTS
A B
175 kDa 175 kDa 83 kDa 83 kDa 70 kDa 70 kDa 62 kDa 62 kDa
47.5 kDa 47.5 kDa
1 2 3 4 5
Figure 4.3 Expression and purification of TGEV-S-GST protein by recombinant E.coli after IPTG induction. (A) Over-production of TGEV-S-GST protein analyzed on 10% SDS-PAGE and visualized by Coomassie Blue staining. Lanes: 1-4, cell extract solubilized with 1M, 6M, 8M urea and 0.01% SDS respectively; 5, cell-free extract. (B) Re-folded and column purified TGEV- S-GST was observed to be relatively pure.
4.3 L.CASEI SHIROTA AS ORAL VACCINE CARRIER AGAINST TGEV
4.3.1 Generation of Recombinant LcS Expressing rTGEV-S Protein
In order to generate recombinant LcS expression vector harboring TGEV spike
gene, the genes encoding an N-terminal fragment of the TGEV spike protein was
amplified from first strand cDNA synthesized from the viral genomic RNA by RT-PCR
reverse transcription.
To obtain the rTGEV-S insert, primers Tgeplp-FI and Tgeplp-RI were used to
amplify the region between nucleotides 33-2286 of TGEV spike gene (Figure 4.4). This
insert encompasses all the four major antigenic domains of the spike gene. The design of
oligonucleotides for PCR was based on the sequences of the TGEV spike gene at the start
135 CHAPTER 4 RESULTS
and end of nucleotides 33-2286. The restriction sites of BamHI and NheI, which were not present in the TGEV spike gene sequence to be amplified, were included onto the primers for cloning of rTGEV-S into the expression vector (Table 2.4).
M
3.0 kb 2.0 kb 2.3 kb 1.5 kb
Figure 4.4 Agarose gel electrophoresis of PCR products of rTGEV-S, amplified from cDNA synthesized from viral genomic RNA. Lane M denotes 1 kb DNA ladder.
Numerous attempts to clone rTGEV-S directly into Lactobacillus spp. expression
vector had failed. The insert was therefore subcloned into pCR®-XL-TOPO® vector via
TA cloning (Figure 4.5). Taq polymerase-amplified rTGEV-S was directly inserted into
the linearized vector with single 3´-thymidine (T) overhangs and transformed into One
Shot® cell. The 2.3 kb insert was then cleaved from plasmid isolated from transformant
positively selected on LB plates containing 50 μg/ml kanamycin (Figure 4.6).
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BamH1 Nhe1 A rTGEV-S A
TA Cloning
Nhe1 BamH1 rTGEV-S Nhe1 BamH1 T T
pCR-XL- pCR- pLP500 XL- TOPO
Digestion with BamH1 + Nhe1
rTGEV-S
pLP500
Ligation
rTGEV-S
pLP500
Figure 4.5 Schematic diagram of the construction of recombinant Lactobacillus spp. expression vector harboring rTGEV-S gene fragment. Taq polymerase amplified rTGEV-S gene fragment was subcloned into pCR®-XL-TOPO® via TA cloning. The insert rTGEV-S was cleaved from pCR®-XL-TOPO® using BamH1 and Nhe1 and religated to pLP500 to generate the recombinant pLP500/rTGEV-S.
137 CHAPTER 4 RESULTS
M 1
3.0 kb 3.5 kb 2.0 kb 2.3 kb 1.5 kb
Figure 4.6 Electrophoretogram of BamHI and NheI digested pCR®-XL-TOPO® vector in which rTGEV-S have been subcloned. Lane: 1, a 2.3 kb band corresponded to the insert was cleaved from the vector, which corresponded to the 3.5 kb band. Lane M denotes 1 kb DNA ladder.
The cleaved insert was purified prior to cloning into Lactobacillus spp. expression vector pLP500 and transformed into E.coli DH10β competent cells. pLP500 is a plasmid with endogenous regulation elements from Lactobacillus, downstream of the secretion signal of prt P gene of L. casei for secretable protein expression. Transformants selected on LB agar plate containing 100 μg/ml ampicillin were checked for the presence of insert rTGEV-S via restriction digestion of the isolated plasmids (Figure 4.7).
A 2.3 kb band was obtained for two of the plasmids extracted from the transformants, indicating that recombinant pLP500 vector harboring the insert rTGEV-S have been successfully constructed. One of the resulting plasmid was verified by sequence analysis, indicating that rTGEV-S has been cloned in frame with the secretion signal sequences of the vector. The plasmid was then denoted as pLP500/rTGEV-S.
138 CHAPTER 4 RESULTS
M 1 2 3
7.0 kb 3.0 kb 2.3 kb 2.0 kb 1.5 kb
Figure 4.7 Electrophoretogram of BamHI and NheI digested plasmids isolated from E.coli DH10β transformants. Lanes: 1 and 2, a 2.3 kb band corresponding to the expected size of rTGEV-S was generated, indicating that both plasmids were recombinant clones of rTGEV-S; 3, only a single band (7.2 kb) was obtained giving an indication that recirculation of pLP500 could have taken place. Lane M denotes 1 kb DNA ladder.
After the initial construction, screening and confirmation of the presence of the
TGEV spike gene fragment in the recombinant plasmid in E.coli, pLP500/rTGEV-S was then electroporated into LcS. Transformants appeared after 48 hr of incubation on MRS agar plates containing erythromycin (5 µg/ml). The gene coding for the resistance to erythromycin was encoded by the pE194 portion of the pLP500 plasmid (Appendix 2).
Ampicillin resistance could not be used for the selection of LcS transformants as proteolytic degradation of secreted β-lactamases occur in lactobacilli (Sibakov et al.,
1991; Pouwels & Leer et al., 1993). PCR analysis of the isolated vectors using the primers Tgeplp-FI and Tgeplp-RI identified five recombinant LcS harboring pLP500/rTGEV-S (Figure 4.8) of which one of the recombinant LcS was denoted as LcS- rTGEV-S
139 CHAPTER 4 RESULTS
M 1 2 3 4 5 6
3.0 kb 2.0 kb 2.3 kb 1.5 kb
Figure 4.8 PCR of recombinant pLP500/rTGEV-S extracted from LcS transformants. Lanes: 1, pLP500 (negative control); 2-6, plasmids isolated from transformants. Lane M denotes 1 kb DNA ladder.
The expression of rTGEV-S protein by LcS-rTGEV-S was analyzed by immunoblot assay using recombinant LcS harboring either pLP500/rTGEV-S or the empty vector and wild type LcS as the control (Figure 4.9). Western blot analysis of concentrated supernatant from overnight culture of LcS-rTGEV-S revealed a protein band (75 kDa) corresponding to the expected molecular weight of the rTGEV-S protein
(lane 3).
As the supernatant had to be concentrated ten times before a distinct protein band could be seen, the expression of rTGEV-S protein by the recombinant LcS was apparently inefficient. Nevertheless, the reactivity of the 75 kDa band with a polyclonal
TGEV-specific convalescence swine serum gave an indication of the immunogenicity of the expressed protein. In contrast, no signal was detected in culture medium and culture supernatants of control strain harboring pLP500 or wild type LcS (lanes 2, 4 and 5).
140 CHAPTER 4 RESULTS
Samples of purified recombinant TGEV spike protein (TGEV-S-GST, 70 kDa) was also included as positive control (lane 1).
175 kDa
83 kDa 62 kDa 47.5 kDa
32.5 kDa
1 2 3 4 5
Figure 4.9 Expression of rTGEV-S protein from LcS-rTGEV-S. Concentrated supernatants from overnight cultures of LcS-rTGEV-S (lane 3), LcS-pLP500 (lane 4) or LcS (lane 5) were separated by SDS-PAGE 10% and transferred electrophoretically to nitrocellulose membrane before being incubated with convalescent swine anti-TGEV serum at a dilution of 1:1000. Antibodies bound to the 75 kDa rTGEV-S protein were detected with HRP-conjugated anti-swine IgG antibodies. Lanes; 1, purified recombinant TGEV-S-GST protein (positive control); 2, MRS broth (negative control).
4.3.2 Immune responses induced by intragastric immunization of LcS-rTGEV-S
To investigate the levels of in vivo immune responses generated by the
Lactobacillus spp. vaccine (LcS-rTGEV-S), three 8 weeks old female BALB/c mice were orally immunized with LcS-rTGEV-S. As shown in Figure 4.10, mice receiving three doses of LcS expressing rTGEV-S protein on days 0-2 were primed for a secondary response to rTGEV-S protein following booster oral administrations on days 14-16 and
28-30. BALB/c mice were chosen as the model as it was utilized by Liu et al. to evaluate the immunogenicity of TGEV DNA vaccine (Liu et al., 2001).
141 CHAPTER 4 RESULTS
1st Boosters 2nd Boosters
Day 0 1 2 14 15 16 28 29 30
18 32 48
Figure 4.10 Schematic diagram showing the oral vaccination regime of recombinant LcS to BALB/c mice. indicates oral administration of 2 x 109 cfu LcS-rTGEV-S to each mice on the specific days indicated; indicates the specific days in which lavaging and bleeding of the vaccinated mice were performed.
Immunological responses of the oral fed mice to rTGEV-S protein were
determined by probing blots of ten-fold concentrated LcS-rTGEV-S supernatant with
intestinal lavages and sera obtained from these mice. IgA is the most abundant
immunoglobulin that is released into the gastrointestinal fluid, saliva, tears, urine and
other secretions (Brandtzaeg, 1994), while IgG constitute the major type of
immunoglobulin in the serum. For this reason, the presence of antibodies against rTGEV-S protein in the lavages and sera were determined using goat anti-mouse IgA or
IgG conjugated with HRP respectively. As shown in figure 4.11, intestinal IgA in intestinal lavage and serum IgG bound to the 75kDa rTGEV-S protein where as no band was observed on membranes probed with preimmune lavage and serum. The detection of a 75 kDa rTGEV-S band on the respective membranes blotted with intestinal lavages and sera, indicated that both mucosal and systemic immune responses against TGEV were elicited.
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175 kDa
83 kDa 75 kDa 62 kDa 47.5 kDa
32.5 kDa
1 2 3 4
Figure 4.11 Production of rTGEV-S protein specific intestinal IgA and serum IgG in mice fed with LcS-rTGEV-S. Western blot was performed using intestinal lavages (lane 2) and sera (lane 4) from mice orally fed with LcS-rTGEV-S. Controls of preimmunized samples of intestinal lavage (lane 1) and serum (lane 3) were also included.
The local immunological response was further studied by measuring the anti-
rTGEV-S IgA response in murine intestinal lavages post intragastric immunization with
LcS-rTGEV-S. The concentration of mucosal IgA antibodies against rTGEV-S protein in the intestinal lavages was determined via ELISA, using purified TGEV-S-GST protein as coating antigen. Results were expressed as titers that were determined by expression of the test samples to a standard curve generated by serial dilution of commercially purchased IgA of known titer. As shown in figure 4.12, there was no substantial difference in mucosal IgA levels between experimental group and control groups prior to intervention while oral immunization of rTGEV-S protein expressing LcS elicited an antigen-specific mucosal IgA response. The IgA titer of the rTGEV-S protein specific response in BALB/c mice following immunization with recombinant LcS was detectable
143 CHAPTER 4 RESULTS as rapidly as day 18 (1.29 ± 0.24 ng/ml of lavage), raising to titers of 3.98 ± 0.18 ng/ml of lavage and 3.63 ± 0.19 ng/ml of lavage by day 32 and day 48 respectively. Since an approximate volume of 2 ml of intestinal lavage was extracted from each mouse, the maximum amount of IgA in the entire intestinal tract, based on the highest titer detected on day 32, would be 7.96 ± 0.36 ng. From the estimation of a previous study (Lee et al.,
2004), it was derived that the intestinal water covering the surface of the entire gastrointestinal tract of mice adds up to 122.8 ± 3.2 mg. As such, the maximum titer of
IgA induced by oral administration of recombinant LcS amounted to 64.82 ± 2.93 ng/ml of intestinal water.
4.5 Preimmune lavage 4.0 Day 18 ) 3.5 Day 32 Day 48 3.0
2.5
2.0
1.5
1.0 Mean IgA concentration (ng/ml 0.5
0.0 PBS LcS-rTGEV-S LcS-pLP500
Figure 4.12 rTGEV-S protein specific local IgA responses in murine intestinal lavage after intragastric immunization. Groups of three mice received three consecutive doses of 109 cfu of LcS-rTGEV-S, three times at two weeks interval. Control mice received 109 CFU of LcS containing pLP500 while a naïve group received buffer (PBS) dose. Intestinal lavages that were collected 2 days after the first boost, 2 and 18 days after the second boost, were analyzed via ELISA, using TGEV-S-GST as the coating antigen. Bars represent the mean IgA titer ± S.E.M in each group.
144 CHAPTER 4 RESULTS
Likewise, serum concentration of rTGEV-S protein specific IgG from immunized mice were also determined. All animals that were orally fed with rTGEV-S protein expressing LcS sero-converted after the first boost (Figure 4.13A). Elicitation of TGEV specific serum IgG was found to be prompter and stronger on comparison to the induction of mucosal IgA in the intestine as the concentration of serum IgG was 4.7 time higher that of intestinal IgA on day 18. A titer of 6.03 ± 0.32 ng/ml of serum of rTGEV-
S protein specific IgG was attained after the first boost which continued to increase to a level of 8.38 ± 0.18 ng/ml after the second boost. The subclasses of rTGEV-S protein specific IgG induced by the LcS-rTGEV-S vaccine were also determined. As shown in
Figure 4.13B, most of the IgG responses generated from oral immunization with LcS- rTGEV-S was of IgG2a isotype (ratio of IgG2a to IgG1, 2.27). As a study by Finkelman and coworkers (1990) indicated that the production of IgG1 type is induced by Th2-type cytokines and the IgG2a isotype is regulated by Th1-type cytokines. The result of the serum isotype analysis suggested that LcS-rTGEV-S induces stronger T-helper-cell-type
1 (Th1) immune responses. No significant induction of anti-rTGEV-S antibodies was observed in the control groups of mice that received PBS or LcS harboring the empty vector (Figures 4.12 & 4.13).
Taken together these results, the recombinant LcS generated in this study was able to elicit both TGEV specific systemic and mucosal antibody responses upon oral administration.
Plaque reduction assays were performed to further determine the neutralizing ability of the antibody against TGEV. The percentage of inhibition was expressed as a
145 CHAPTER 4 RESULTS
percentage of plaques obtained in reduction assay with anti-rTGEV-S intestinal lavages
or sera with respect to intestinal lavages or sera obtained from control mice fed with PBS.
Results demonstrated that anti-rTGEV-S IgA or IgG conferred statistically significant neutralizing effects (p<0.05) on TGEV infection (Figure 4.14). A near 15% ± 0.77%
reduction in the number of plaques was consistently observed when plaque reduction
assays were carried out using two to eight folds diluted intestinal lavages or sera from
LcS-rTGEV-S fed mice. The inhibitory effect decreased gradually on further dilutions
and reached a level similar to that of the control non-expressor strain at dilutions 1:128
and 1:64 of intestinal lavages and sera, respectively.
146 CHAPTER 4 RESULTS
A. Total Serum IgG 10.0 Preimmune serum 9.0 Day 18 8.0 Day 32
Day 48 7.0
6.0 5.0 4.0 3.0 2.0 Mean IgG concentration (ng/ml) 1.0 0.0 PBS LcS-rTGEV-S LcS-pLP500
B. IgG Isotype
6.00 IgG1 5.00 IgG2a
4.00
3.00
2.00
1.00
Mean IgG Isotype Concentration (ng/ml) Concentration Isotype Mean IgG 0.00 PBS LcS-rTGEV-S LcS-pLP500
Figure 4.13 Anti-rTGEV-S serum IgG titers induced after intragastric immunization with recombinant LcS. (A) Total sera from groups of three mice immunized orally with 109 cfu of LcS (rTGEV-S expressor or control non-expressor strains) were tested for the presence of rTGEV-S specific IgG by ELISA, using TGEV-S-GST as the coating antigen. Negative control sera from mice fed with PBS were also assayed. (B) Serum IgG1 and IgG2a antibodies specific to the rTGEV-S protein were shown in samples collected 2 days after the second boost. Bars represent the mean IgG titer ± S.E.M in each group.
147 CHAPTER 4 RESULTS
A. Intestinal Lavage
16.00%
Lcs-rTgev-S 14.00% Lcs 12.00% 10.00% 8.00% 6.00%
4.00% % Inhibition
2.00%
0.00% 2 4 8 16 32 64 128 256 -2.00%
-4.00% Sample Dilution
B. Serum
18.00% Lcs-rTgev-S 16.00% Lcs 14.00% 12.00%
10.00% 8.00% 6.00%
% Inhibition 4.00% 2.00%
0.00% -2.00% 2 4 8 16 32 64 128 256 -4.00% Sample Dilution
Figure 4.14 Inhibition of viral plaque formation by (A) intestine lavages (Day 48) and (B) sera (Day 48) prepared from mice fed with recombinant LcS. Maximum reduction in number of plaques, expressed as a percentage of plaques obtained for the negative control samples, using (A) intestinal lavages or (B) sera collected from mice fed with rTGEV-S-expressing LcS was 13.35 ± 0.77 % and 15.70 ± 0.77 % respectively. Results are mean values and standard errors of triplicates.
148 CHAPTER 4 RESULTS
4.3.3 Kinetics of cytokine production by Peyer’s patches from mice orally
immunized with LcS-rTGEV-S
Cellular immune responses can be determined through the pattern of cytokine production from cells of the Peyer’s patches of mice immunized with LcS-rTGEV-S.
Cells from the Peyer’s patches, that represent the intestine inductive sites from where cells migrate to the mesenteric lymph node then to the lamina propria, were isolated two weeks after the second booster doses. The level of IL-2, IFN-γ, and IL-4, IL-5, as being representative of Th1 and Th2 responses, in the supernatant of Peyer’s patches cell culture were examined at different times after in vitro re-stimulation with TGEV-S-GST protein. The viability and ability of the cells from the Peyer’s patches were assessed. As shown in Figure 4.15, the cells were capable of being stimulated in vitro and to produce cytokines upon re-stimulation with concanavalin A.
When re-stimulated with TGEV-S-GST protein, cells of the Peyer’s patches isolated from mice immunized with LcS-rTGEV-S produced a mixed Th1/Th2-like cytokine profile including IL-2, IFN-γ, IL-4 and IL-5 (Figure 4.16). With respect to the level of cytokine production, highest amount of IFN-γ (1320.61 ± 55.19 pg/ml) was produced by the sensitized cells from the Peyer’s patches. However, IL-2 production peaked earlier than IFN-γ, IL-4 and IL-5 production. Maximum production of IL-2
(375.55 ± 22.85 pg/ml) was attained on day 2, while highest concentration of IFN-γ, IL-4 and IL-5 (1320.61 ± 55.19 pg/ml; 539.55 ± 37.54 pg/ml; 405.35 ± 27.62 pg/ml) were detected on day 4 or day 5. As for the cells from control mice that received the native
LcS, no significant cytokine titers could be detected after in vitro re-stimulation with
TGEV-S-GST protein. Residual production from in vivo priming was also evaluated in
149 CHAPTER 4 RESULTS wells incubated without antigen in the presence of RPMI only. The results presented here indicated that oral administration of mice with recombinant LcS expressing rTGEV-
S protein was able to elicit specific Th1 and Th2-type cellular immune responses.
14
12 )
10
8
6
4 Cytokine Concentration (ng/ml 2
0 IL-2 IFN-γ IL-4 IL-5 IL-2 IFN-γ IL-4 IL-5
LcS-rTGEV-S LcS
Figure 4.15 IL-2, IFN-γ, IL-4 and IL-5 production by Peyer’s patches cells from mice immunized orally with LcS-rTGEV-S or LcS after re-stimulated with concanavalin A. Readings are the actual increment in cytokine concentrations after off setting the background cytokine production from the cells. Results are mean values and standard errors of triplicate means.
150 CHAPTER 4 RESULTS
1,600
1,400 Day 2 Day 3 1,200 Day 4 Day 5 1,000
800
600
400 Cytokine Concentration (pg/ml) Cytokine Concentration
200
0 RPMI RPMI RPMI RPMI RPMI RPMI RPMI RPMI rTGEV-S-GST rTGEV-S-GST rTGEV-S-GST rTGEV-S-GST rTGEV-S-GST rTGEV-S-GST rTGEV-S-GST rTGEV_S_GST IL-2 IFN-γ IL-4 IL-5 IL-2 IFN-γ IL-4 IL-5
LcS-rTGEV-S LcS
Figure 4.16 Kinetics of Th1 (IL-2 and IFN-γ) and Th2 (IL-4 and IL-5) cytokines production by Peyer’s patches cells from mice immunized with LcS-rTGEV-S and LcS. Cells were removed 2 weeks after the second boost and re-stimulated in vitro with TGEV-S- GST or cultured in the presence of RPMI medium only. Results shown are the mean values and standard errors of triplicates means.
151 CHAPTER 4 RESULTS
4.4 P.PASTORIS AS ORAL VACCINE CARRIER AGAINST TGEV
4.4.1 Generation of Recombinant P.pastoris (PP) Expressing PrTGEV-S Protein
The insert PrTGEV-S was obtained by amplifying the region between nucleotides
33-2286 of TGEV first strand cDNA using primers pGAPtge-FI and pGAPtge-RII
(Figure 4.17). This insert encompasses all the four major antigenic domains of the spike gene. The design of oligonucleotides for PCR was based on the sequences of the TGEV spike gene at the start and end of nucleotides 33-2286. The restriction sites of EcoRI and
XbaI, which were not present in the TGEV spike gene sequence to be amplified, were tagged onto the primers for cloning of the rTGEV-S into the expression vector (Table
2.5).
M
3.0 kb 2.0 kb 2.3 kb 1.5 kb
Figure 4.17 Agarose gel electrophoresis of PCR amplified PrTGEV-S (2.3 kb). Lane M denotes 1 kb DNA ladder.
PrTGEV-S was cloned into P.pastoris expression vector pGAPZαC and transformed into E.coli DH10β competent cells. pGAPZαC uses the GAP promoter to constitutively express recombinant proteins that are fused to the N-terminal peptide encoding the S.cerevisiae α-factor secretion signal in P.pastoris. Positively selected
152 CHAPTER 4 RESULTS
transformants on LB agar plate containing 25 μg/ml zeocin were then screened for the presence of PrTGEV-S by performing PCR on the isolated plasmids using the cloning set
of primers (Figure 4.18). A 2.3 kb band which corresponded to the expected size of
PrTGEV-S was generated from the PCR of all the five plasmids isolated from the
transformants, hence indicating that recombinant pGAPZαC vector harboring the insert
PrTGEV-S have been successfully constructed. Nucleotide sequencing conducted on one
of the recombinant vector verified that PrTGEV-S has been cloned in frame with the
secretion signal sequences of the vector. The plasmid was then denoted as
pGAPZαC/PrTGEV-S.
M 1 2 3 4 5
3.0 kb 2.0 kb 2.3 kb 1.5 kb
Figure 4.18 PCR screening of plasmids isolated from transformants. Lanes: 1-5, a 2.3 kb band corresponding to the expected size of PrTGEV-S was amplified from each plasmid. Lane M denotes 1 kb DNA ladder.
pGAPZαC/PrTGEV-S was linearized with AvrII prior to transformation into
P.pastoris to allow homologous recombination between the insert and regions of
homology within the genome (Cregg et al., 1985; Cregg et al., 1989). Such integrants show extreme stability even in the absence of selective pressure. PCR analysis of the
153 CHAPTER 4 RESULTS
total DNA extracted from the yeast transformants showed that PrTGEV-S has been
inserted into the genome of two P.pastoris transformants (Figure 4.19). Non-specific
amplification was not observed in the genome of P.pastoris transformed with pGAPZαC.
M 1 2 3
3.0 kb 2.0 kb 2.3 kb 1.5 kb
Figure 4.19 Electrophoretogram of PCR products amplified from genomic DNA isolated from P.pastoris transformants. Lanes: 1 & 2, a 2.3 kb band corresponding to the expected size of PrTGEV-S was amplified from total DNA extracted from yeast transformed with pGAPZαC/PrTGEV-S; 3, PCR of genomic DNA isolated from yeast transformed with empty vector. Lane M denotes 1 kb DNA ladder.
Immunoblot assay was then performed to check for the expression and
immunogenicity of PrTGEV-S protein (Figure 4.20). Supernatant from overnight culture
of recombinant P.pastoris (PP/PrTGEV-S) subjected to Western blotting revealed a
protein band (90 kDa) significantly larger than the expected molecular weight of
PrTGEV-S protein (lane 4). The increase in molecular weight (15 kDa) of the expressed
PrTGEV-S protein could be attributable to glycosylation of the protein in P.pastoris
expression system, where the N-glycosylation pathway of the yeast mirrors the pathway
in typical mammalian cells (Bretthauer & Castellino, 1999). Nevertheless, the ability of
polyclonal TGEV-specific convalescence swine serum to probe to the expressed
PrTGEV-S protein gave an indication that the glycosylated protein has retained its
154 CHAPTER 4 RESULTS immunogenicity. As observed in the immunoblot, no signal was detected in the culture supernatants of the control PP/pGAPZαC or wild type P.pastoris, as well as in the culture medium (lanes 2, 3 and 5 respectively). Purified TGEV-S-GST (70 kDa) was also included as positive control (lane 1).
175 kDa
83 kDa 62 kDa
47.5 kDa
32.5 kDa
1 2 3 4 5
Figure 4.20 Expression of PrTGEV-S protein from P.pastoris transformants. Supernatants from overnight cultures of P.pastoris (lane 2), PP/pGAPZαC (lane 3) or PP/PrTGEV-S (lane 4) were separated by 10% SDS-PAGE and transferred electrophoretically to nitrocellulose membrane before being incubated with convalescent swine anti-TGEV serum at a dilution of 1:1000. Bound antibodies were detected with HRP-conjugated anti-swine IgG antibodies. Lanes; 1, purified recombinant TGEV-S-GST protein (positive control); 5, MRS broth (negative control).
4.4.2 Immune responses induced by intragastric immunization of PP/PrTGEV-S
BALB/c mice orally dosed with 2 x 109 cfu of recombinant PP/PrTGEV-S on days 0-2 were primed for a secondary response to PrTGEV-S protein following booster oral administrations on days 14-16 and 28-30 (Figure 4.21).
155 CHAPTER 4 RESULTS
1st Boosters 2nd Boosters
Day 0 1 2 14 15 16 28 29 30
18 32 48
Figure 4.21 Schematic diagram showing the oral vaccination regime of PP/PrTGEV-S to BALB/c mice. indicates oral administration of the recombinant P.pastoris to each mice on the specific days indicated; indicates the specific days in which lavaging and bleeding of the vaccinated mice were performed.
Elicitation of immune responses to PrTGEV-S protein in the mice was determined by probing blots of PP/PrTGEV-S supernatant with intestinal lavages and sera extracted from these mice. Antibody binding was determined using goat anti-mouse IgA and IgG conjugated with HRP for the detection of IgA in the lavages and IgG in the sera respectively. The detection of a 90 kDa band, corresponding to the molecular weight of the expressed PrTGEV-S protein, on the respective membranes blotted with intestinal lavages and sera, indicated that both PrTGEV-S protein specific mucosal and systemic immune responses were elicited (Figure 4.22).
Further studies on the local and systemic immune responses against PrTGEV-S protein were also performed. The appropriate concentration of anti-PrTGEV-S IgA and
IgG in intestinal lavages and sera was determined via ELISA, using purified TGEV-S-
GST protein as capturing antigen. Results were expressed as titers that were determined by expression of the test samples to a standard curve generated by serial dilution of commercially purchased IgA or IgG of known titer.
156 CHAPTER 4 RESULTS
175 kDa 90 kDa 83 kDa 62 kDa
47.5 kDa
32.5 kDa
1 2 3 4
Figure 4.22 Immunogenicity of induced intestinal IgA and serum IgG specific for PrTGEV-S protein. Western blot was performed using intestinal lavages (lane 1) and sera (lane 3) from mice orally fed with PP/PrTGEV-S. Controls of preimmunized samples of intestinal lavage (lane 2) and serum (lane 4) were also included. The PrTGEV-S protein (90 kDa) in the supernatant of PP/PrTGEV-S was detected by intestinal IgA (lane 1) and serum IgG (lane 3), respectively. However, no band was observed on membranes probed with preimmune lavage and serum.
As shown in Figure 4.23, mice immunized with pGAPZαC and PBS control did not show significant PrTGEV-S protein specific antibody production, while oral administration of PP/PrTGEV-S elicited a rapid and strong antigen-specific mucosal IgA response in the immunized mice. The antibody titer against the protein PrTGEV-S in
BALB/c mice following immunization with PrTGEV-S protein expressing P.pastoris reached a high level of 4.51 ± 0.17 ng/ml of lavage on day 18. Since an approximate volume of 2 ml of intestinal lavage was extracted from each mouse, the maximum amount of IgA in the entire intestinal tract would be 9.02 ± 0.34 ng. From an estimation of a previous study (Lee et al., 2004), the intestinal water covering the surface of the entire gastrointestinal tract of mice adds up to 122.8 ± 3.2 mg. It was then derived that
157 CHAPTER 4 RESULTS the maximum titer of IgA induced by oral administration of PP/PrTGEV-S would be
73.45 ± 2.77 ng/ml of intestinal water.
5.00 Pre-immune lavage 4.50 Day 18 4.00 Day 32 Day 48 3.50 3.00 2.50 2.00
1.50
Mean IgA concentration (ng/ml) 1.00
0.50 0.00 PBS PP/PrTGEV-S PP/pGAPZ α C
Figure 4.23 PrTGEV-S protein specific local IgA response in murine intestinal lavage after intragastric immunization. Groups of three mice received three consecutive doses of 109 PP/PrTGEV-S, three times at two weeks interval. Control mice received 109 CFU of PP/pGAPZαC while a naïve group received buffer dose. Intestinal lavages that were collected 2 days after the first boost, 2 and 16 days after the second boost, were analyzed via ELISA, using TGEV-S-GST as the coating antigen. Bars represent the mean IgA titer ± S.E.M in each group.
Results from ELISA demonstrated that all mice that were orally fed with
PP/PrTGEV-S sero-converted by day 18 (Figure 4.24A). The level of PrTGEV-S protein specific IgG remained relatively constant from day 18 (9.41 ± 0.44 ng/ml of serum) to day 48 (9.51 ± 0.32 ng/ml of serum). The serum IgG isotypes of PrTGEV-S protein specific antibodies induced by PP/PrTGEV-S were shown in Figure 4.24B. The recombinant P.pastoris induced more IgG2a than IgG1 (ratio of IgG2a to IgG1, 1.94).
158 CHAPTER 4 RESULTS
The result of the serum isotype analysis suggested that PP/PrTGEV-S induced more T- helper-cell-type 1 (Th1) immune responses. No significant induction of anti-PrTGEV-S antibodies was observed in the control groups of mice that received PBS or
PP/pGAPZαC (Figures 4.23 & 4.24).
Results obtained in this part of the study have shown that the recombinant
P.pastoris harbouring PrTGEV-S was able to induce TGEV specific systemic and mucosal immune responses when orally fed to mice. Even though a stronger serological response (IgG) was elicited, prompt induction of mucosal IgA was observed as the concentration of PrTGEV-S protein specific IgA after the first boost reached a level similar to those after the second boost.
The ability of the induced antibodies to confer protection against TGEV infection was then determined via plaque reduction assay. The percentage of inhibition was expressed as a percentage of plaques obtained in reduction assay with anti-rTGEV-S intestinal lavages or sera with respect to intestinal lavages or sera obtained from control mice fed with PBS. As observed in Figure 4.25, incubation of anti-PrTGEV-S IgA or
IgG with TGEV conferred statistically significant and potent neutralizing effects
(p<0.05). TGEV infection was strongly inhibited by both anti-PrTGEV-S IgA and IgG up to the dilution of 1:32 of lavages or sera from PP/PrTGEV-S fed mice respectively. It is commendable that anti-PrTGEV-S IgA and IgG conferred a near 100% inhibition of
TGEV infection, up to dilutions of 1:4 and 1:8 respectively. The inhibitory effect decreased gradually on further dilutions and reached a level similar to that of the control non-expressor strain at dilutions 1:512 of intestinal lavages.
159 CHAPTER 4 RESULTS
A. Total Serum IgG
12.00 Pr e- immune s er um Day 18 10.00 Day 32 Day 48 8.00
6.00
4.00
2.00 Mean IgG concentration (ng/ml)
0.00 PBS PP/Pr TGEV - S PP/pGA PZ α C
B. IgG Isotype
7.00 IgG1
6.00 IgG2a
5.00
4.00
3.00
2.00
1.00 Mean IgG Isotype Concentration (ng/ml) 0.00 PBS PP/Pr TGEV - S PP/pGA PZ α C
Figure 4.24 Humoral immune responses after intragastric immunization with recombinant P.pastoris (PP/PrTGEV-S). (A) Total sera from groups of three mice immunized orally with 109 cfu of P.pastoris (PrTGEV-S protein expressor or control non-expressor strains) were tested for the presence of PrTGEV-S protein specific IgG by ELISA, using TGEV-S-GST as the coating antigen. (B) Serum IgG1 and IgG2a antibodies specific to PrTGEV-S protein were shown in sera collected 2 days after the second boost. Negative control sera from mice fed with PBS were also assayed. Bars represent the mean IgG titer ± S.E.M in each group.
160 CHAPTER 4 RESULTS
A. Intestinal Lavage
100.00% PP-rTGEV-S
80.00% PP
n 60.00%
40.00% % Inhibitio
20.00%
0.00% 2 4 8 16 32 64 128 256 512 Sample Dilution
B. Serum
100.00% PP/rTGEV-S
PP 80.00%
n 60.00%
40.00% % Inhibitio 20.00%
0.00%
2 4 8 16 32 64 128 256 512 Sample Dilution
Figure 4.25 Inhibition of viral plaque formation by (A) intestine lavages (Day 48) and (B) sera (Day 48) prepared from mice orally fed with PP/PrTGEV-S and P.pastoris. Maximum reduction in number of plaques, expressed as a percentage of plaques obtained for the negative control samples, using (A) intestinal lavages or (B) sera collected from mice fed with PrTGEV-S- expressing P.pastoris was 66.79 ± 2.11 % and 72.20 ± 2.37 % respectively. Results are mean values and standard errors of triplicates.
161 CHAPTER 4 RESULTS
4.4.3 Kinetics of cytokine production by Peyer’s patches from mice orally
immunized with PP/PrTGEV-S
The level of cytokines released by the Peyer’s patches cell reflects the direction and magnitude of the immune responses. Therefore, supernatant from Peyer’s patches cell cultures stimulated with TGEV-S-GST in vitro were harvested and the release of IL-
2, IFN-γ, IL-4 and IL-5 were assessed at different times after re-stimulation. The capacity of Peyer’s patch cells to be stimulated in vitro and to produce cytokines was confirmed by production of cytokines in the presence of concanavalin A (Figure 4.26).
Residual production from in vivo priming was evaluated in wells incubated with
RPMI only. Cell from control mice that received the native P.pastoris did not produce any significant cytokine titers after in vitro re-stimulation with TGEV-S-GST. Cells of the Peyer’s patches from mice immunized with recombinant P.pastoris produced a mixed
Th1/Th2-like cytokine profile including IL-2, IFN-γ, IL-4 and IL-5 (Figure 4.27).
However, the results obtained indicated that the oral vaccine (PP/PrTGEV-S) resulted in the induction of stronger Th1-type cellular immune responses and the levels of Th1 (IL-2 and IFN-γ)-type cytokines were significantly higher than that of Th2 (IL-4 and IL-5)-type cytokines. IFN-γ production was the highest among the four cytokines being studied (IL-
2, 455.01 ± 34.65 pg/ml; IL-4, 375.21 ± 19.62 pg/ml; IL-5, 273.35 ± 22.41 pg/ml), reaching a concentration of 1402.67 ± 24.68 pg/ml on day 4 after re-stimulation, while
IL-2 production was found to peak earlier than IFN-γ, IL-4 and IL-5 production. Highest concentration of IL-2 was produced on day 3 as compared to IFN-γ, IL-4 and IL-5, where the maximum concentrations of the cytokines were only produced on day 4 or day 5.
Nevertheless, the cytokine profiling study have shown that oral administration of mice
162 CHAPTER 4 RESULTS with recombinant P.pastoris expressing PrTGEV-S protein resulted in the induction of specific and potent Th1 and Th2-type cellular immune responses in the Peyer’s patches of immunized mice.
14
) 12
10
8
6
4
Cytokine Concentration (pg/ml Concentration Cytokine 2
0 IL-2 IFN-γ IL-4 IL-5 IL-2 IFN-γ IL-4 IL-5 PP/PrTGEV-S PP
Figure 4.26 IL-2, IFN-γ, IL-4 and IL-5 production by Peyer’s patches cells from mice immunized orally with PP/PrTGEV-S or P.pastoris after re-stimulated with concanavalin A. Readings are the actual increment in cytokine concentrations after off setting the background cytokine production from the cells. Results are mean values and standard errors of triplicate means.
163
1,600
Day 2 1,400 Day 3 Day 4 1,200 Day 5
1,000
800
600
400 (pg/ml) Cytokine Concentration
200
0
RPMI RPMI RPMI RPMI RPMI RPMI RPMI RPMI
rTGEV-S-GST rTGEV-S-GST rTGEV-S-GST rTGEV-S-GST rTGEV-S-GST rTGEV-S-GST rTGEV-S-GST
rTGEV_S_GST IL-2 IFN-γ IL-4 IL-5 IL-2 IFN-γ IL-4 IL-5
PP/PrTGEV-S PP
Figure 4.27 Kinetics of Th1 (IL-2 and IFN-γ) and Th2 (IL-4 and IL-5) cytokine production by Peyer’s patches cells from mice immunized with PP/PrTGEV-S and P.pastoris. Cells were removed 2 weeks after the second boost and re-stimulated in vitro with TGEV-S-GST or cultured in the presence of RPMI medium only. Results shown are the mean values and standard errors of triplicates means. 164 CHAPTER 5 RESULTS
5.0 DEVELOPMENT OF ORAL VACCINE AGAINST SEVERE
ACUTE RESPIRATORY SYNDROME CORONAVIRUS
(SARS CoV)
5.1 INTRODUCTION
SARS CoV is a highly contagious respiratory illness that first infected people in
parts of Asia, North America, and Europe, affecting 8,450 people by the end of June
2003. During the SARS outbreak, the spread of the disease was primarily controlled by strict quarantine protocols and patient-isolation measures, as well as by broad-spectrum antibiotics and antiviral regimens, where a combination of corticosteroid and ribavirin therapy was given (So et al., 2003; Tsang & Zhong, 2003; Tsang et al., 2003; Centre for
Disease Control and Prevention, 2003). There is, however, considerable skepticism over the use of these drugs in SARS and there is currently no consistently effective anti-SARS
CoV therapy (Cyranoski, 2003).
Comparison studies from the previous chapter have demonstrated that P.pastoris
as an oral vaccine delivery vehicle, allowed eukaryotic modification of the expressed
antigen. As a result, the antigen specific mucosal IgA and serum IgG induced confer
strong protection against viral infection. As such, in this final part of the study,
P.pastoris was chosen over LcS for the development of oral vaccine against SARS CoV.
5.2 GENERATION OF RECOMBINANT P.PASTORIS (PP) EXPRESSING
SARS CoV SPIKE RECEPTOR-BINDING DOMAIN PROTEIN
A 720 bp fragment of SARS CoV receptor-binding domain (SARS-S-RBD),
spanning within the region of nucleotides 810-1530 of SARS CoV spike gene, was
165 CHAPTER 5 RESULTS
amplified from pCR-XL-TOPO-S, a vector encoding the full length SARS CoV spike
gene, with the aid of the primers RBD-SSF1 and RBD-SSR1 (Figure 5.1). The
restriction sites of KpnI and XbaI, which were not present in the SARS spike gene
sequence to be amplified, were tagged onto the primers for cloning of the SARS-S-RBD
into the expression vector (Table 2.6).
The insert, SARS-S-RBD, was cloned into P.pastoris expression vector
pGAPZαC and transformed into E.coli DH10β competent cells. Transformants that were
positively selected on LB agar plate containing 25 μg/ml zeocin were assessed for the presence of SARS-S-RBD by conducting PCR on the isolated plasmids using the cloning set of primers (Figure 5.2). Recombinant pGAPZαC vector harboring the insert SARS-S-
RBD have been successfully constructed since a 720 bp band that corresponded to the expected size of SARS-S-RBD was amplified from all isolated plasmids. Results from nucleotide sequencing performed on one of the vectors showed that SARS-S-RBD was inserted in frame with the secretion signal sequences of the vector. The plasmid was then denoted as pGAPZαC/SARS-S-RBD.
In order to generate stable recombinants, pGAPZαC/SARS-S-RBD was linearized and transformed into P.pastoris so as to allow homologous recombination between the insert and regions of homology within the genome (Cregg et al., 1985; Cregg et al., 1989). PCR analysis of the total DNA was extracted from the P.pastoris transformants showed that SARS-S-RBD has been inserted into the genome of the four
P.pastoris transformants (Figure 5.3). Non-specific amplification was not observed in the genome of P.pastoris transformed with pGAPZαC [(PP/pGAPZαC) (lane 1)].
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M
1000 bp 750 bp 720 bp 500 bp 250,253 bp
Figure 5.1 Agarose gel electrophoresis of PCR products of SARS-S-RBD, amplified from pCR- XL-TOPO-S. Lane M denotes BenchTop 1 kb DNA ladder.
M 1 2 3
1000 bp 750 bp 720 bp 500 bp 250,253 bp
Figure 5.2 PCR screening of plasmids isolated from transformants. Lanes: 1-3, a 720 bp band corresponding to the expected size of SARS-S-RBD was amplified from each plasmid. The faint bands at the lower end of lanes 1-3 were likely to be primer-dimers. Lane M denotes BenchTop 1 kb DNA ladder.
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M 1 2 3 4 5
1000 bp 750 bp 720 bp 500 bp
Figure 5.3 Electrophoretogram of PCR products amplified from genomic DNA isolated from P.pastoris transformants. Lanes: 2-5, a 720 bp band corresponding to the expected size of SARS- S-RBD was amplified from total DNA extracted from yeast transformed with pGAPZαC/SARS- S-RBD; 1, PCR of genomic DNA isolated from yeast transformed with empty vector. Lane M denotes BenchTop1 kb DNA ladder.
It could be observed that SARS-S-RBD could only be weakly amplified from the
total DNA isolated from one of the recombinant P.pastoris (lane 3). On comparison of
the total DNA extracted from all the transformants using gel electrophoresis, it was noted
that equivalent amount of DNA were extracted from each recombinant (Figure 5.4). As such, the variation in the amount of SARS-S-RBD amplified could not be attributed to the difference in concentration of total DNA utilized during PCR. More likely, the insert might have been integrated into other regions of the genome, resulting in instability of the integrant and subsequent loss of the insert. In view of this, subsequent studies were performed using the recombinant P.pastoris from which SARS-S-RBD could be strongly amplified from the total DNA extracted [(PP/SARS-S-RBD) (Figure 5.3, lane 5)].
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M 1 2 3 4
10,000 bp
8,000 bp
Figure 5.4 Agarose gel electrophoresis for the comparison of the amount of total DNA extracted from four P.pastoris transformants. Lanes 1-4, genomic DNA from which PCR analysis was performed to check for the insertion of SARS-S-RBD in lane 2-5 of Figure 5.3 respectively. Lane M denotes BenchTop 1 kb DNA ladder.
To visualize the synthesis of SARS-S-RBD protein by recombinant P.pastoris, culture medium was harvested from overnight culture of PP/SARS-S-RBD. Total secreted proteins were separated on 10 % SDS-PAGE and analyzed by Western blotting using SARS CoV convalescent human serum (Figure 5.5). Two distinct bands of 24 kDa and 62 kDa reacted strongly with the convalescence serum (lane 2). Previous studies showed that proteins expressed under P.pastoris expression system generally produce two forms of the same protein (Calabozo et al., 2003; Farn´os et al., 2005). One of which corresponds to the expected size of the actual protein while the other with a higher molecular-mass, corresponded to glycosylated product. Hence, the 62 kDa band probably correspond to glycosylated SARS-S-RBD protein while the 24 kDa band is probably the unglycosylated SARS-S-RBD protein since the unglycosylated SARS-S-
RBD protein has a similar molecular weight.
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175 kDa
83 kDa 62 kDa 47.5 kDa
32.5 kDa 25 kDa
1 2 3 4 5
Figure 5.5 Expression of SARS-S-RBD protein from P.pastoris transformants. Supernatants from overnight cultures of PP/SARS-S-RBD (lane 2), PP/pGAPZαC (lane 3) or P.pastoris (lane 4) were separated by 10 % SDS-PAGE and transferred electrophoretically to nitrocellulose membrane before being incubated with SARS CoV human convalescent serum at a dilution of 1:500. Bound antibodies were detected with HRP-conjugated anti human IgG antibodies. Lanes; 1, SARS-S(M) (positive control); 5, MRS broth (negative control).
The ability of polyclonal SARS-specific convalescence human serum to probe to the expressed SARS-S-RBD protein gave an indication that both the glycosylated and nonglycosylated protein retained antigenic authenticity. As observed in the immunoblot, the convalescence serum did not react with proteins present in the culture supernatants of the control PP/pGAPZαC or wild type P.pastoris, as well as in the culture medium (lanes
3, 4 and 5, respectively). The positive control included in the analysis of the expression of SARS-S-RBD protein by the recombinant P.pastoris (PP/SARS-S-RBD) was different from the positive control used in the previous chapter (TGEV-S-GST). Commercially available SARS-Associated Coronavirus Spike mosaic recombinant protein S(M)
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[(SARS-S(M), 45 kDa (Virogen, USA)], was also utilized as the positive control in this part of the study (lane 1).
5.3 IMMUNE RESPONSES INDUCED BY INTRAGASTRIC
IMMUNIZATION OF PP/SARS-S-RBD
Immunological responses of mice inoculated with three doses of 2 x 109 cfu of
PP/SARS-S-RBD on days 0-2 and primed for a secondary response with booster doses on days 14-16 and 28-30 were examined (Figure 5.6).
1st Boosters 2nd Boosters
Day 0 1 2 14 15 16 28 29 30
18 32 48
Figure 5.6 Schematic diagram showing the oral vaccination regime of PP/SARS-S-RBD to BALB/c mice. indicates oral administration of 2 x 109 cfu PP/SARS-S-RBD to each mice on the specific days indicated; indicates the specific days in which lavaging and bleeding of the vaccinated mice were performed.
Intestinal lavages and sera extracted from these mice were checked for reactivity to SARS-S-RBD protein in the supernatant harvested from overnight culture of
PP/SARS-S-RBD. As shown in figure 5.7, both intestinal IgA in intestinal lavage and serum IgG were able detect the glycosylated (62 kDa) and nonglycosylated (24 kDa) forms of SARS-S-RBD protein. The detection of the SARS-S-RBD protein bands on the respective membranes blotted with intestinal lavages and sera indicated that both mucosal and systemic immune responses against SARS CoV were elicited. Non-specific binding
171 CHAPTER 5 RESULTS
was not observed on membranes probed with preimmune lavage (lane 2) and serum (lane
4). 83 kDa
62 kDa 47.5 kDa
32.5 kDa 25 kDa
1 2 3 4
Figure 5.7 Immunogenicity of induced intestinal IgA and serum IgG specific for SARS-S-RBD protein. Western blot was performed using intestinal lavages (lane 1) and sera (lane 3) from mice orally fed with PP/SARS-S-RBD. Controls of preimmunized samples of intestinal lavage (lane 2) and serum (lane 4) were also included. Intestinal IgA in intestinal lavage and serum IgG bound to the SARS-S-RBD protein where as no band was observed on membranes probed with preimmune lavage and serum.
The serum and intestinal antibody responses in the mice were further examined by
performing ELISA, using SARS-S(M) as the target antigen. The level of anti-SARS-S-
RBD IgG and IgA in sera and intestinal lavages were expressed as titers that were
determined by expression of the test samples to a standard curve generated by serial
dilution of commercially purchased IgA or IgG of known titer.
Eighteen days after the first vaccination with recombinant PP/SARS-S-RBD, the
immunized mice had produced a rapid and strong antigen-specific mucosal IgA response
(Figure 5.8). The anti-SARS-S-RBD IgA titer was 4.48 ± 0.36 ng/ml of lavage on day 18
and remained relatively constant after the second boost from day 32 to 48. No substantial
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difference in mucosal IgA levels between experimental group and control groups was
observed prior to intervention. It could also be derived that the maximum amount of IgA
in the entire intestinal tract was 8.96 ± 0.72 ng, since an approximate volume of 2 ml of
intestinal lavage was extracted from each mouse. On the basis that the intestinal water
covering the surface of the entire gastrointestinal tract of mice is 122.8 ± 3.2 mg (Lee et
al., 2004), the maximum titer of IgA induced by oral administration of PP/SARS-S-RBD
would be 72.96 ± 5.86 ng/ ml of intestinal water.
6.00 Preimmune lavage Day 18 l 5.00 Day 32 Day 48 4.00
3.00
2.00
Mean IgA concentration (ng/m 1.00
0.00 PBS PP/SARS-S-RBD PP/pGAPZ a C
Figure 5.8 SARS-S-RBD protein specific local IgA response in murine intestinal lavage after intragastric immunization. Groups of three mice received three consecutive doses of 109 PP/SARS-S-RBD, three times at two weeks interval. Control mice received 109 CFU of PP/pGAPZαC while a naïve group received buffer dose. Intestinal lavages that were collected 2 days after the first boost, 2 and 16 days after the second boost, were analyzed via ELISA, using SARS-S(M) as the coating antigen. Bars represent the mean IgA titer ± S.E.M in each group.
Mice immunized orally with PP/SARS-S-RBD developed high IgG serological
responses (Figure 5.9A). All animals receiving three consecutive vaccine doses and the
first booster doses developed antibody titer of 9.42 ± 0.77 ng/ml of serum on day 18,
173 CHAPTER 5 RESULTS
which was significantly greater than the antibody titers generated following inoculation
with PBS or PP/pGAPZαC. With the administration of the second booster doses, the IgG
antibody level remained at a constant level of 9.49 ± 1.01 to 9.45 ± 0.86 ng/ml of serum
from day 32 to day 48. Even though the application of a second boost did not further
stimulate stronger serological responses, the high level of SARS-S-RBD protein specific
IgG induced by day 18 indicated that the recombinant P.pastoris was able to elicit strong
and rapid responses in mice when being orally vaccinated. The subclasses of SARS-S-
RBD specific IgG induced by the P.pastoris vaccine were also determined. As shown in
Figure 5.9B, most of the IgG responses generated from oral immunization with
PP/SARS-S-RBD were of IgG1 isotype (ratio of IgG2a to IgG1, 0.50), indicating that
PP/SARS-S-RBD induced a stronger type 2 (Th2)-like immune responses.
The capacity of PP/SARS-S-RBD to induce neutralizing antibodies, which would be effective to confer protection against SARS CoV infection, was determined. Since
SARS CoV-related work with the involvement of live virus can only be conducted in biosafety level 3 or higher containment facilities, it was not possible to use SARS CoV as the target virus as such containment facilities was not available in the department. SARS
CoV S protein-pseudotyped murine leukemia virus [MLV(SARS)] (Giroglou et al.,
2004), a relatively safe surrogate for native SARS CoV with a tropism restricted to the same cell types and host species as SARS CoV was instead utilized in the plaque reduction assay. The MLV(SARS) displayed properties of MLV with infectivity of cell
and tissue tropism mediated solely by SARS CoV spike protein, as the spike protein, with
a partially deleted cytoplasmic tail, was incorporated into the envelop of MLV particles.
MLV(SARS) was produced by co-transfection of 293T cells with plasmids encoding C-
174 CHAPTER 5 RESULTS
terminally truncated SARS CoV spike protein, MLV proteins and eGFP (enhanced green
fluorescence protein). The pseudotype viruses released into the supernatant were then
collected where infection of MLV(SARS) can be carried out by transduction of
MLV(SARS) into Vero cells. Neutralization assays were performed by incubating
concentrated pseudotypes (5 x 104 PFU) with serial dilutions of serum or intestinal lavage
at 37 oC for 1 hr, after which the antibody-virus mixture was transduced into Vero cells.
The percentage of eGFP-positive cells, as an indicator of MLV(SARS) infected cells, was enumerated under fluorescence microscope at 72 hr post-transduction. The percentage of inhibition of fluorescence- forming unit (FFU) was expressed as a percentage of fluorescence cell obtained in the neutralization assay with anti-SARS-S-RBD intestinal lavages or sera with respect to intestinal lavages or sera obtained from control mice fed with PBS.
As presented in Figure 5.10, relatively high titers of neutralizing antibodies
(p<0.05) were observed in the serum of mice orally administered with PP/SARS-S-RBD.
To determine the neutralization activities in secretions, intestinal lavages were analyzed for neutralization activities. Strong neutralizing activities (p<0.05) were also detected in
the intestinal lavages extracted from mice immunized with PP/SARS-S-RBD. Inhibition of MLV(SARS) infection was close to 100 % by both anti-SARS-S-RBD IgA and IgG up to the dilution of 1:8 of lavages or sera respectively. It is noteworthy that even at the dilution of 1:32, anti-SARS-S-RBD IgA and IgG in the intestinal lavage and serum could block more than 50 % of the viral infection. The inhibitory effect decreased gradually on further dilutions and reached a level similar to that of the control non-expressor strain at dilution 1:512 of intestinal lavages and sera.
175 CHAPTER 5 RESULTS
A. Total Serum IgG
12.00 Pre-immune serum Day 18 10.00 Day 32 Day 48 8.00
6.00
4.00
2.00 Mean IgG concentration (ng/ml) concentration Mean IgG
0.00 PBS PP/SA RS- S- RBD PP/pGA PZ α C
B. IgG Isotype
7.00 IgG1
6.00 IgG2a
5.00
4.00
3.00
2.00
1.00
Mean IgG Isotype Concentration (ng/ml) 0.00 PBS PP/SA RS- S- RBD PP/pGA PZ α C
Figure 5.9 Anti-SARS-S-RBD serum IgG titers induced after intragastric immunization with PP/SARS-S-RBD. (A) Total sera from groups of three mice immunized orally with 109 P.pastoris (SARS-S-RBD protein expressor or control non-expressor strains) were tested for the presence of SARS-S-RBD protein specific IgG by ELISA, using SARS-S(M) as the coating antigen. (B) Serum IgG1 and IgG2a antibodies specific to the SARS-S-RBD protein were shown in samples collected 2 days after the second boost. Negative control sera from mice fed with PBS were also assayed. Bars represent the mean IgG titer ± S.E.M in each group.
176 CHAPTER 5 RESULTS
A. Intestinal Lavage
100.00% PP/SARS-S-RBD 90.00% PP 80.00% U 70.00% 60.00% 50.00%
40.00%
30.00% % Inhibition of FF Inhibition % 20.00% 10.00% 0.00% 2 4 8 16 32 64 128 256 512
Sample Dilution
B. Serum
100.00% 90.00% PP/SARS-S-RBD 80.00% PP U 70.00%
60.00%
50.00% 40.00% 30.00% % Inhibition of FF Inhibition % 20.00% 10.00%
0.00% 2 4 8 16 32 64 128 256 512 Sam ple Dilution
Figure 5.10 Inhibition of MLV(SARS) infection by (A) intestine lavages and (B) sera prepared from mice fed with PP/SARS-S-RBD. Maximum reduction in FFU, expressed as a percentage of the FFU obtained for the negative control samples, using (A) intestinal lavages or (B) sera collected from mice fed with SARS-S-RBD-expressing P.pastoris was 71.99 ± 2.44 % and 78.56 ± 2.07 % respectively. Results are mean values and standard errors of triplicates.
177 CHAPTER 5 RESULTS
5.3.1 Kinetics of cytokine production by Peyer’s patches and cervical lymph node
from mice orally immunized with PP/SARS-S-RBD
Since SARS CoV causes an acute respiratory illness, virus-specific memory T cells may need to be present in the airway to undergo rapid activation and differentiation upon acute infection. As such, the presence of antigen-specific T cells in cervical lymph nodes (CLN) was also determined. The SARS-S-RBD protein specific cytokine production by cells from both the Peyer’s patches and CLN of mice orally inoculated with PP/SARS-S-RBD was examined. The level of IL-2, IFN-γ, IL-4 and IL-5 produced by cells of the lymphoid tissue cultures were assessed at different times after in vitro re- stimulation with SARS-S(M). The capacity of lymphoid cells to be stimulated in vitro and to produce cytokines was confirmed by production of cytokines in the presence of concanavalin A (Figure 5.11).
As seen in Figure 5.12 and 5.13, cells of the Peyer’s patches and CLN of mice immunized with PP/SARS-S-RBD produced a mixed Th1/Th2-like cytokine profile, whereby both Th1 (IFN-γ and IL-2) and Th2 (IL-4 and IL-5)-type cytokines were produced by the re-stimulated cells. In general, it could be observed that much larger amounts of IL-4 and IL-5 as compared to IL-2 and IFN-γ were produced when the cells of the Peyer’s patches and CLN were re-stimulated in vitro with SARS-S(M). The highest concentration of cytokines produced by cells of the Peyer’s patches were 250.12
± 23.45 pg/ml for IL-2 on day 4, 135.62 ± 24.06 pg/ml for IFN-γ on day 4, 1188.67 ±
60.25 pg/ml for IL-4 on day 5 and 1449.88 ± 64.70 pg/ml for IL-5 on day 4. As for the cells of the CLN, maximum amount of cytokines detected were 134.99 ± 11.68 pg/ml for
IL-2 on day 4, 82.02 ± 5.57 pg/ml for IFN- γ on day 4, 462.20 ± 30.65 pg/ml for IL-4 on
178 CHAPTER 5 RESULTS
day 5 and 535.94 ± 16.69 pg/ml for IL-5 on day 4. These results indicated that oral
vaccination of recombinant P.pastoris expressing SARS-S-RBD protein resulted in the
induction of Th2-biased immune responses in the immunized mice. However, the
concentrations of all four cytokines in the CLN culture were generally lower than those in
the culture of the Peyer’s patches. The level of cytokines produced by cells from the
Peyer’s patches was 1.2 to 4.1 times higher than that from the cells of CLN.
Nevertheless, the ability of cell from the CLN to produce both Th1 and Th2 associated
cytokines gave an indication that oral vaccination of mice with PP/SARS-S-RBD was
able to induce sensitized T cell not only within the intestine milieu, but also in a distal
lymphoid tissue which is more closely associated with the immune protection of the nasal
organs.
Cervical lymph node and Peyer’s patches cells from control mice inoculated with
native P.pastoris did not produce any significant cytokine titers after in vitro re- stimulation with SARS-S(M). However, it was noticed that a higher basal level of all the four cytokines were produced by cells from the cervical lymph node.
179 CHAPTER 5 RESULTS
A. Peyer’s Patches 14
12
10
8
6 4
2 Cytokine Concentration (ng/ml) 0 IL-2 IFN-γ IL-4 IL-5 IL-2 IFN-γ IL-4 IL-5
PP/SARS-S-RBD PP
B. Cervical Lymph Node
18
16
14 12 10
8 6 4
2 Cytokine Concentration (ng/ml) 0 IL-2 IFN-γ IL-4 IL-5 IL-2 IFN-γ IL-4 IL-5
PP/SA RS- S- RBD PP
Figure 5.11 IL-2, IFN-γ, IL-4 and IL-5 production by cells from Peyer’s patches (A) and CLN (B) of mice immunized orally with PP/SARS-S-RBD or P.pastoris after re-stimulated with concanavalin A. Readings are the actual increment in cytokine concentrations after offsetting the background cytokine production from the cells. Results are mean values and standard errors of triplicate means.
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1,600
1,400 Day 2 Day 3
1,200 Day 4 Day 5 1,000
800
600
400
Cytokine Concentration (pg/ml) Cytokine Concentration 200
0
RPMI RPMI RPMI RPMI RPMI RPMI RPMI RPMI
SARS-S(M) SARS-S(M) SARS-S(M) SARS-S(M) SARS-S(M) SARS-S(M) SARS-S(M) SARS_S(M) IL-2 IFN-γ IL-4 IL-5 IL-2 IFN-γ IL-4 IL-5 PP/SARS-S-RBD PP
Figure 5.12 Kinetics of Th1 (IL-2 and IFN-γ) and Th2 (IL-4 and IL-5) cytokine production by Peyer’s patches cells from mice immunized with
PP/SARS-S-RBD and P.pastoris. Cells were removed 2 weeks after the second boost and re-stimulated in vitro with SARS-S(M) or cultured in the presence of RPMI medium only. Results shown are the mean values and standard errors of triplicates means.
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600
Day 2 500 Day 3
) Day 4 Day 5 400
300
200
Cytokine Concentration (pg/ml Cytokine Concentration 100
0
RPMI RPMI RPMI RPMI RPMI RPMI RPMI RPMI
SARS-S(M) SARS-S(M) SARS-S(M) SARS-S(M) SARS-S(M) SARS-S(M) SARS-S(M) SARS_S(M) IL-2 IFN-γ IL-4 IL-5 IL-2 IFN-γ IL-4 IL-5 PP/SARS-S-RBD PP
Figure 5.13 Kinetics of Th1 (IL-2 and IFN-γ) and Th2 (IL-4 and IL-5) cytokine production by CLN cells from mice immunized with PP/SARS-S-RBD and P.pastoris. Cells were removed 2 weeks after the second boost and re-stimulated in vitro with SARS-S(M) or cultured in the presence of RPMI medium only. Results shown are the mean values and standard errors of triplicates means. 182 CHAPTER 6 DISCUSSION & CONCLUSION
6.0 DISCUSSION
Mucosal surfaces in the gastrointestinal, urogenital, and respiratory tracts provide portals of entry for pathogens. It is therefore important to develop vaccines that induce protective mucosal immune responses where initial infection and replication of the pathogen occurred. Although parental vaccination is usually highly effective in eliciting a protective systemic immune response, it is not necessarily effective against mucosal infections (Mesteck, 1987; Mc Ghee et al., 1992).
With the widespread threat of many emerging pathogenic viruses (such as SARS
CoV, Hantan and influenza viruses) to human life, challenges have been spawned to develop safe anti-viral vaccine for preventive use. Ideally, the new generation of vaccines to be developed to combat mucosal pathogens should meet a number of prerequisites. They should be safe, cheap, stable, and easy to administer, preferably given orally in a single dose. The oral route of administration is a most convenient route of vaccination as compared with the more frequently used parenteral routes mainly because of a less stringent criteria for application and education of trained healthcare workers. More importantly, oral administration of antigens might stimulate the natural route of infection and be a more effective method of immunization (Staats et al., 1994).
The scientific consensus at this point of time is that live vaccines are potential candidates for single oral immunization.
For vaccines to be able to exert long-term protective effects, it is important that the vector is able to continuously express the antigen of interest within the mucosal surfaces. This can be achieved using live vaccine delivery vehicles, capable of adhering to the mucosal surfaces, hence permitting continuous expression of antigen. The use of
183 CHAPTER 6 DISCUSSION & CONCLUSION
attenuated pathogen is particularly interesting, being powerful tool to present antigens, since they can induce specific immune responses against heterologous antigen and simultaneous protection against the pathogen. Various attenuated mutants have been produced and tested, such as Listeria monocytogenes, Salmonella typhimurium, Shigella flexneri and Mycobacterium bovis (Shata et al., 2000; Dietrich et al., 2001; Medina &
Guzman, 2001). Although these attenuated strains have been extensively engineered to
minimize their pathogenicity, they maintian certain invasive and virulence properities
(Fischetti et al., 1996). To circumvent some of the safety and environmental issues
inherent to the wide-scale dissemination of engineered pathogens, it will be more feasible
to develop non-pathogenic vectors as vaccine vectors. In this study, the focus was on the
development of a mucosal delivery system for the gastrointestinal tract. The potential of
using Lactobacillus spp. (LcS) and yeast (S.boulardii, S.cerevisiae and P.pastoris), as the
respective candidates of prokaryotic and eukaryotic carriers, were investigated.
The rationale behind the selection of Lactobacillus spp. and yeast as the
investigative candidate in this study, is that both organisms have been used from time
immemorial in the production of food and have been consumed widely by humans and
animals (Sharpe, 1981; van der Aa Kuhle et al., 2005). In addition, many lactobacilli and yeasts have been shown to exert beneficial probiotic effects (Havenaar & Huis in’t
Veld, 1993; Broussard & Surawicz, 2004). The first protective immune response for lactobacilli expressing C fragment of tetanus toxin was obtained when total cell-free extracts of L.casei expressing the toxin fragment at the cell surface were injected subcutaneously (Maassen et al., 1999). Subsequently, L.casei and L.plantarum strains have been developed as delivery vectors for tetanus toxin fragment C, Helicobacter
184 CHAPTER 6 DISCUSSION & CONCLUSION
pylori urease B and the protective antigen of Bacillus anthracis to induce high
immunological response, equivalent to those using Specol as an adjuvant (Pouwels et al.,
1996; Zegers et al., 1999; Shaw et al., 2000; Corthesy et al., 2005). As such, amongst
the numerous species and strains of lactobacilli, LcS was selected in this study for
development of an oral vaccine carrier against coronaviruses.
At the moment, S.boulardii is the only yeast commercialized as probiotic in
human medicine. As several taxonomy studies have indicated that S.boulardii is closely
related to S.cerevisiae, this leads to the question whether S.cerevisiae possess probiotic
properties as well (Mitterdorfer et al., 2002a,b; van der Aa Ku¨hle & Jespersen, 2003).
As for the P.pastoris, though not commonly used in the food industry, it has been permitted by U.S. Food and Drug Administration as a food additive in feed formulation for animals (U.S. Food and Drug Administration). More importantly, Pichia expression system is an excellent host for the production of heterologous proteins and have been proven capable of generating post-translational modifications that are more similar to human proteins modifications than Saccharomyces cerevisiae (Cregg et al., 1987;
Romanos et al., 1992; Ilgen et al., 2004). A number of human glycoproteins expressed in
P.pastoris have been shown to have retained the native structure and biological properties
(Caputo et al., 1999; Gupta & Dighe, 1999; Lerner et al., 1999). Proteins synthesized by
P.pastoris have also been applied in various industries, such as for the production of caseinomacropeptide, a peptide with growth-inhibitory activity against oral opportunistic
pathogens and puroindolines, a functional component of wheat endosperm used for
bread-making (Dubreil et al., 1998; Kim et al., 2005).
185 CHAPTER 6 DISCUSSION & CONCLUSION
Very few yeasts have been studied as possible biotherapeutics. Despite the fact that S.boulardii is one of the first and currently the only yeast commercialized in human medicine, the adhesive properties of S.boulardii on intestinal surface have only been examined in few cases (Tasteyre et al., 2002; Ouwehand et al., 1999 & 2000; van der Aa
Kühle et al., 2005). The adhesive capacity of a strain can be examined in vitro by the use of intestinal cell lines. For this purpose, different human intestinal epithelial cell lines have been employed such as Caco-2 (Tuomola & Salminen, 1998; Kimoto et al., 1999;
Greene & Klaenhammer, 1994), T84 (Dahan et al., 2003) and HT29 (Gopal et al., 2001), which are all derived from colon adenocarcinoma (Adlerberth et al., 1996). In this study, the two well-characterized cultured colon carcinoma cell lines, Caco-2 and HT29, were selected to study the in vitro adhesion capability of S.boulardii, S.cerevisiae and
P.pastoris. The advantages of these two cellular models are that they expresses morphological and functional differentiation in vitro and show characteristics of mature enterocytes, including polarization, a functional brush border, and apical intestinal hydrolases (Pinto et al., 1982 & 1983). Caco-2 and HT29 cells forms two clearly distinguishable domains, an apical membrane and a basolateral membrane separated by tight junctions (Simon & Fuller, 1985). The apical surface (brush border) contains peptidases and disaccharidases while the basolateral domain contains several peptide receptors involved in controlling intestinal hydroelectrolytic secretion (Hauri, 1988;
Hauri et al., 1985; Pinto et al., 1982 & 1983; Laburthe & Amiranoff, 1990). Since the binding of microorganisms to mucosal cells involves the interaction of bacterial ligands with specific eukaryotic receptors, human intestinal Caco-2 and HT29 cells are probably the best available models to study intestinal attachment of yeasts.
186 CHAPTER 6 DISCUSSION & CONCLUSION
Scanning electron microscopic examination revealed the ability of S.boulardii,
S.cerevisiae and P.pastoris to adhere to Caco-2 and HT29 cells (Figures 3.1 & 3.2).
Congruent to the present study, Kuhle et al. (2005) and Ouwehand et al. (1999) observed that S.boulardii isolated from pharmaceutical product were able to adhere to human intestinal mucus and a primary neonatal piglet jejunum cell line, IPEC-J2. However, only a few of the food-borne S.cerevisiae possessed noteworthy adhesiveness with strains from blue veined cheeses, as the most adhesive (Kuhle et al., 2005). In this study,
P.pastoris demonstrated highest adhesiveness (7.14 x 102 yeast per cell) when compared
to the other two yeasts, which showed similar adhesiveness to Caco-2 (4.1 x 101 and 4.8
x 101 yeast per cell) and HT29 (3.4 x 101 and 4.3 x 101 yeast per cell) cells. The amount
of P.pastoris attached to the cell surface of Caco-2 was 15 to 17 times higher than those of S.boulardii and S.cerevisiae, while the number of P.pastoris adhering to HT29 cells was 26 to 33 times the number of adhering S.boulardii and S.cerevisiae. Interestingly,
P.pastoris seemed to have a stronger affinity to HT29 cells as the number of P.pastoris adhering to HT29 cells was 1.6 times higher than that adhering to Caco-2 cells. Besides direct adherence to epithelial cells to colonize a mucosal surface, microorganisms may adhere to mucous (Coconnier et al., 1992; Bernet et al., 1994). Scanning electron micrographs have revealed that P.pastoris interacts not only with the brush border of the cell, the yeast cells were also observed to interact conspicuously with the secreted mucus
(Figure 3.3). It is therefore likely that the increased adhesiveness to HT29 cells was attributable to the binding of P.pastoris to the mucous, forming an additional platform for the adherence of P.pastoris to the intestinal cells.
187 CHAPTER 6 DISCUSSION & CONCLUSION
Even though numerous adhesion studies performed using food-derived lactobacilli and human isolates indicated the ability of lactobacilli to adhere to human
intestinal cell lines (Jacobsen et al., 1999; Baccigalupi et al., 2005). Chauviere et al.
(1992) observed that not all strains of Lactobacillus spp. developed this adhesiveness to
human intestinal cells in vitro. Strains like L.acidophilus BG2FO4 and HN017,
L.johnsonii La1, L.rhamnosus DR20, L.casei subsp. rhamnosus and LcS were found to be
able to adhered to enterocyte-like Caco-2 cells (Coconnier et al., 1992; Bernet et al.,
1994; Hudault et al., 1997; Tuomola & Salminen, 1998; Forestier et al., 2001; Gopal et
al., 2001; Kankaanpaa et al., 2001; Lee & Puong, 2002; Morita et al., 2002; Lee et al.,
2003). Since the ability of LcS to adhere to intestinal cell lines have already been
demonstrated, in vitro adhesion assay of LcS was not included in the current study. In
view of the fact that the adhesion of Lactobacillus spp. to human intestinal cells was a
discriminative parameter, showing pronounced variation among the strains, including
variations depending on the cell culture used (Chauviere et al., 1992; Sarem et al., 1996;
Tuomola & Salminen, 1998; Jacobsen et al., 1999), determination of the adhesiveness of
LcS in vivo will better correlate to the ability of LcS to colonize the intestinal tract.
As Hautefort et al. (2000) have pointed out that in vitro adhesion to intestinal
cells is not sufficient to ensure the persistence of lactic acid strains in the digestive tract
of mice, it is not very clear if results obtained in the in vitro adhesion assays for
S.boulardii, S.cerevisiae and P.pastoris can be extrapolated to in vivo environments. It is
therefore necessary to determine if the in vitro adhesiveness of LcS, S.boulardii,
S.cerevisiae and P.pastoris is applicable in vivo, so as to better elucidate which of the
188 CHAPTER 6 DISCUSSION & CONCLUSION
investigated microorganisms will be more suitable for oral vaccine delivery purposes
with regard to their persistence in the intestinal tract.
As the fluids present at the mucosal are constantly motile, it is likely that
microorganisms which remained suspended in the intestinal water and did not adhere to
the mucosal surfaces would be diluted and washed out from the intestinal surfaces
quickly. The layer of intestinal water in mice where intestinal microorganisms were
suspended amounted to 0.33 to 0.89% (wt/wt) of the daily feed and water consumption
(in small intestine) and 0.67% (wt/wt) of daily fecal excretion (in colon). That is, there were more than 100 volume changes in the intestinal water content per day. One volume change in intestinal water would dilute the organisms suspended in it by half. Hence, intestinal microorganisms that did not adhere on mucosal surfaces would be diluted to an undetectable level soon after a single feeding. The probability of an orally administered microorganism interacting with antigen sampling inductive cells may be increased if the carrier is able to persist in the environment. This persistence may be achieved by direct adherence to the epithelial cells or to the mucous.
The observation that the average half time for the wash-out of LcS, S.boulardii,
S.cerevisiae and P.pastoris fed ranged from -28.80 to 66.02 days in the various parts of the animal intestine suggests that the lactobacilli and yeasts were able to compete with the indigenous microorganisms for adhesion on the mucosal surface along the full stretch of the intestinal tract. A negative value in the half time for wash-out indicates an increment in the number of adhering cell. The wash-out half times of LcS and the yeasts varied within the different segments of the intestinal tract, indicating that the affinity for adhesion of LcS, S.boulardii, S.cerevisiae and P.pastoris differed. The affinity for
189 CHAPTER 6 DISCUSSION & CONCLUSION
adhesion were in the order of duodenum > colon > jejunum > ileum for LcS (half times
for wash-out were 3.98, 2.48, 1.55, and 1.34 days respectively), colon > ileum > jejunum
> duodenum for S.boulardii (half times for wash-out were 66.02, 5.35, 3.02 and 2.64 days respectively), jejunum > colon > ileum > duodenum for S.cerevisiae (half times for wash-out were -4.46, 1.84, 1.70 and 1.25 days respectively), and ileum > colon > duodenum > jejunum for P.pastoris (half times for wash-out were -28.80, -5.97, 2.85 and
2.21 days respectively).
A study using human fecal samples demonstrated that the half time for the wash- out of Lactobacillus rhamnosus GG from the intestinal tract was about 1 day (Goldin et
al., 1992). The current study showed that the wash-out half times for LcS in the animal
model ranged from 1.34 to 3.98 days, indicating that LcS have a stronger affinity to
murine intestinal tract than Lactobacillus rhamnosus GG. Martins et al. (2005) observed
that several strains of S.cerevisiae were not capable of colonizing the digestive tract of
germ-free mice, and only one particular strain, S.cerevisiae 905, was detected in the feces
with a level never fell below 106 cfu/g. Since the intestinal microorganisms in a fecal
sample are diluted by fecal material, their counts could reach the limits of detection when
the organism could still be detected on intestinal mucosal surfaces (Alander et al., 1999;
Cesena et al., 2001). Moreover, the organism count of a fecal sample does not allow the
differentiation of growth and colonization in the various sections of the intestine. Hence,
the study of fecal samples has limitations in evaluating growth and colonization by
lactobacilli and yeasts.
Disparate to the results obtained from the in vitro adhesion assay where P.pastoris
was seen to be the most adhesive strain, S.boulardii appeared to have a higher affinity to
190 CHAPTER 6 DISCUSSION & CONCLUSION
the different segments of murine intestinal tract, as the half times for wash-out were
generally longer than those for S.cerevisiae and P.pastoris. The higher adhesiveness displayed by S.boulardii was expected as S.boulardii has been widely documented as probiotic yeast. Interestingly, S.cerevisiae and P.pastoris that failed to remain adhered to the upper segments of the gastrointestinal tract were able to reattach to the lower regions of the intestine. As observed by a concurrent increment in the number of adhering yeast cells and the median fluorescence intensity of S.cerevisiae and P.pastoris in the jejunum and ileum respectively, 25.8 % of the S.cerevisiae detached from the duodenum readhered to the jejunum, while 9.5 % of P.pastoris detached from the duodenum and jejunum were able to reattach to the ileum. About 1.1 mg of mucus per day was dislodged from intestinal surfaces and could be recovered in the feces of the mice.
Microorganisms adhered on mucosal surface would have dislodged together with mucus, and a fraction of the dislodged bacteria might have been released and readhered onto the newly exposed mucosal surface. The higher readhesion percentage of S.cerevisiae could be due to the binding of the detached S.cerevisiae onto those attaching to the jejunum.
Yeast flocculation, commonly seen in S.cerevisiae, is a spontaneous aggregation of cells and is mediated by mannose chains present on the yeast cell surface which react with
neighboring cells via N-terminal domains of the proteins encoded by dominant
flocculation genes (Miki et al., 1982; Kobayashi et al., 1998). The increase in number of
P.pastoris in the ileum was likely a result of the reattachment of detached yeast cells onto
the enterocytes as there has been no clear evidence in the literature, showing flocculation
of P.pastoris.
191 CHAPTER 6 DISCUSSION & CONCLUSION
As there has been no previous study on the adhesion of P.pastoris, the mechanism by which P.pastoris adhered to various portion of the intestinal tract was uncertain. It is possible that the adhesion of P.pastoris to the intestinal surfaces occurred by hydrophobic interactions since studies have suggested that S.cerevisiae CBS 7764 isolated from the intestine of rainbow trout adhere to fish intestinal mucus via hydrophobic interactions
(Andlid et al., 1998). However, adhesion of the yeasts can also be mediated by glycoprotein structures since flocculation of yeast cells have been known to be enhanced by carbohydrate binding domain on the cell surface (Javadekar et al., 2000; Jin et al.,
2001). There has been diverse speculations on the adhesion mechanism of Lactobacillus spp.. It has been suggested that besides a polysaccharide located at the bacterial surface, adherence of Lactobacillus acidophilus was also mediated by a second factor, a proteinaceous compound that was secreted into the culture medium (Hood & Zottola,
1989; Coconnier et al., 1992). For some Lactobacillus spp., adhesion to human cells has been ascribed to an adhesion promoting protein with a molecular mass of 29 kDa highly homologous to a collagen-binding protein from Lactobacillus reuteri NCBI 11951 and to a basic surface protein from Lactobacillus fermentum BR11 (Heinemann et al., 2000). A recent study by Baccigalupi et al. (2005) indicated that at least two small (less than 3 kDa) factors, that were loosely associated with the cell wall, were involved in mediating the in vitro adhesion of Lactobacillus fermentum to Caco-2 cells. On the other hand, LcS was shown to possess multiple surface adhesions and up to four adhesins could bind to the mucosal surface at any time (Lee et al., 2000). Despite the extensive research that has been undertaken, the molecular basis and the exact mechanism of lactobacilli adhesion is still not completely understood.
192 CHAPTER 6 DISCUSSION & CONCLUSION
It is interesting that the medians of the fluorescence intensities of LcS and yeasts
measured on day 1 in various sections of the intestine were not the same (Figure 3.7,
3.12, 3.17 and 3.22). The differences in the fluorescence intensity of the original LcS and yeasts population were probably due to the physiological status of the cells at the time of incubation with cFDA-SE. Various quantities of cFDA-SE might have been taken up by the cells, converted to the fluorescent derivative, and covalently linked to intracellular proteins with different efficiencies. This observation suggests that lactobacilli and yeasts
in different physiological states preferentially adhere to the various sections of the intestinal tract. This may have implications for the preparation of vaccine carriers for selective adhesion on specific sections of the intestinal tract, so as to be more efficient in delivering the antigen of interest to the site of infection. As such, the medians of the fluorescence intensity profiles of LcS, S.boulardii, S.cerevisiae and P.pastoris
determined on day 1 were taken as the first generation (original population). In the
various sections of the intestine, 37.2 to 60.5 % of the original population of LcS that had
adhered onto the intestinal surface remained attached and undivided on day 6 after
orogastric intubation (Table 3.1). However, more than 90 % of the yeasts that were
attached to the jejunum, ileum or colon remained undivided on day 6. In particular, 91.5
and 96.6 % of S.boulardii did not undergo cell replication 6 days after attachment to the
ileum and colon respectively. The lower percentage of non-dividing LcS in the intestinal
tract could be due to the presence of a larger proportion of viable lactobacilli, as report
has suggested that ingested LcS were able to survive intestinal transit (Donnet-Hughes et
al., 1999; Yuki et al., 1999). The cells that had remained undivided on day 6 could have
lost their viability after passing through the stomach and intestinal tract but still retained
193 CHAPTER 6 DISCUSSION & CONCLUSION
their adhesion capability. Studies have shown that non-viable probiotic organisms
retained their ability to adhere to intestinal mucus, even though the adhesiveness has been
compromised to varying degree (Ouwehand et al., 2000). Among the dividing cells, 3.9
to 21.6 % of the total adhered LcS have undergone three cell divisions on day 6 after
orogastric intubation, while 6.1 to 16.3 % of S.boulardii, 0.27 to 34.23 % of S.cerevisiae
and 3.7 to 23.7 % of P.pastoris were found to be in their fourth generation. This study has clearly demonstrated that a large part of the LcS, S.boulardii, S.cerevisiae and
P.pastoris fed were able to grow and divide in the intestinal environment; however, the rate of cell division were low.
Doubling time is the time required for a cell population to double either the number of cells or the active cell mass. Doubling time varies with organism and
environment and can range from 20 minutes for a fast growing organism under ideal
conditions, to hours and days for less than ideal conditions or for slow growing
organisms. The average doubling times of the lactobacilli measured in the duodenum,
jejunum, ileum, and colon were 4.10, 4.78, 4.56, and 5.59 days, respectively. These
values are low compared with doubling times of the bacterium cultured in MRS medium
under aerobic (1.7 h) and anaerobic (1.2 h) conditions. The average doubling times for
for S.boulardii, S.cerevisiae and P.pastoris were 1.33, 1.67 and 1.54 days in the
duodenum, 2.61, 9.10 and 1.97 days in jejunum, 10.91, 0.45 and –77.32 days in the
ileum, and 20.83, 0.85 and 2.76 days in the colon. The negative doubling time of
P.pastoris in the ileum was due to the reattachment of P.pastoris detached from the duodenum and jejunum, resulting in an increase in the median fluorescence intensity of the adhering yeast. Similar to LcS, the doubling time of S.boulardii, S.cerevisiae and
194 CHAPTER 6 DISCUSSION & CONCLUSION
P.pastoris were slow compared to a doubling time of 3.4 h and 3.5 h under aerobic and anaerobic conditions (Porthur et al., 2000). Since the nutritional composition of the feed
for mice is well balanced and rich, it is the intestinal content and conditions that
suppressed the growth of the lactobacilli and yeasts.
The process of washing out of a microorganism from the intestinal mucosal
surface can be described by the following relationship: wash-out rate = specific growth
rate − dilution rate. The dilution factor involves the rate at which the intestinal content
passes through and the ability of the organism to adhere to the mucosal surface. The effective dilution rate could be estimated from the values of the wash-out rate and the
specific growth rate, which were determined experimentally. For example, in the
duodenum, the average wash-out rates of LcS and S.boulardii were ln2/3.98 or 0.17 day−1 and ln2/2.64 or 0.26 day-1 respectively, and the specific growth rates were ln2/4.10 or
0.17 day−1 and ln2/1.33 or 0.52 day−1 respectively. Thus, the calculated effective dilution rates for LcS and S.boulardii in the duodenum were 0.34 day−1 and 0.78 day−1. In order
to persist on a mucosal surface (i.e., wash-out rate = 0), the lactobacilli and yeast would
need to grow and divide at an average specific growth rate that equals to the effective
dilution rate. This specific growth rate represents a doubling time of 2.04 and 0.88 days
for LcS and S.boulardii respectively in the environment of the duodenum. In order to
permanently colonize the jejunum, ileum, and colon, LcS would need to divide at
doubling times of 1.17, 1.03 and 1.72 days respectively, while S.boulardii would have to
divide at doubling times of 1.40, 3.59 and 15.83 days respectively. These doubling time
are 2.0 to 4.4 times faster than the actual doubling time for the lactobacilli in the
intestine. This indicated that for LcS to persist in the intestinal tract, the lactobacilli
195 CHAPTER 6 DISCUSSION & CONCLUSION
would have to double its population at a time that is 2.0 to 4.4 times faster than the
current doubling times. In comparison, S.boulardii would only have to achieve doubling
times that is 1.3 to 3.0 times shorter than the original doubling time to be able to colonize
the intestinal tract for a significant period of time.
For S.cerevisiae and P.pastoris to persist in the intestinal tract, the yeasts must
have doubling times shorter than 0.71, 0.36 and 0.58 days in the duodenum, ileum and
colon for S.cerevisiae, and 1.00, 1.04 and 5.13 days in the duodenum, jejunum and colon
for P.pastoris. These represented doubling times which are 1.3 to 2.3 times and 0.5 to
1.9 times the actual doubling time for S.cerevisiae and P.pastoris respectively. The
doubling times required by S.cerevisiae and P.pastoris to permanently colonize the
jejunum and ileum respectively were not calculated as the actual doubling time of
S.cerevisiae and P.pastoris in the jejunum and ileum could not be accurately determined.
As mentioned earlier, the increment in the number of S.cerevisiae and P.pastoris in the jejunum and ileum due to the readherence of S.cerevisiae detached from the duodenum, and reattachment of P.pastoris detached from the duodenum and jejunum, resulted in the derivation of negative wash-out times of S.cerevisiae and P.pastoris in the jejunum and ileum respectively.
The adhesion study suggests that in addition to direct competition between intestinal microorganism for adhesion, the rate of growth and division, or generation time, determine the ability of these organisms to colonize and persist on mucosal surfaces of the intestinal tract. In this study, we have demonstrated the ability of LcS, S.boulardii,
S.cerevisiae and P.pastoris to adhere to various portions of murine intestinal tract.
Permanent adhesion of vaccine carrier is not a crucial criteria as prolonged stimulation of
196 CHAPTER 6 DISCUSSION & CONCLUSION
the immune system might in turn result in the development of tolerance to the antigen of
interest (Mowat & Weiner, 1999). Since LcS was shown to be able to adhere to the
intestinal tract for a reasonable period of time, recombinant LcS expressing coronavirus antigen was generated to determine its feasibility as a vaccine delivery vehicle. Among the three yeast strains, P.pastoris was chosen to be further analyzed as this yeast has shown more potential to be developed as an oral vaccine carrier. In particular, P.pastoris
was capable of readhering and replicating in certain segments of the gastrointestinal tract.
These can be beneficial as the readhesion and replication of P.pastoris will allow the maintenance of a significant number of vaccine carriers in the intestinal tract, long enough for the effective elicitation of immunological responses to the delivered antigen.
On top of these, P.pastoris also offers an additional advantage over S.cerevisiae in that it is capable of generating post-translational modifications that are more similar to human proteins modifications than S.cerevisiae (Cregg et al., 1987).
Interest in the expression of heterologous genes in Lactobacillus spp. has been
increased in the recent years, as techniques for the genetic manipulation of strains, many
of which were previously believed to be refractory to genetic transformation, have been
developed. To date, only a few systems have been described using Lactobacillus spp. as
a carrier for expressing heterologous vaccine antigens in a form that can be presented to
and processed by the immune system of the mammalian host (Zegers et al., 1999; Shaw
et al., 2000; Reveneau et al., 2002). Key features of cloning vectors for the delivery of
antigens involve promoter sequences that allow either constitutive or inducible
expression. Well-known inducible expression systems are the nisin-inducible expression
system (Kleerebezem et al., 1997; Pavan et al., 2000), and systems based on sugar
197 CHAPTER 6 DISCUSSION & CONCLUSION
metabolism with promoters that are regulated by catabolite repression such as xylose
promoter (Lokman et al., 1997). On the other hand, there has been limited development
of constitutive expression system of Lactobacillus spp.. To our best knowledge, the only constitutive expression system was developed by Pouwels et al. (1996), where recombinant protein have been constitutively expressed under the constitutive promoter of L.casei L-(+)-lactate dehydrogenase gene. In addition, these vectors can contain secretion and anchor signals that allow expression of proteins in different cellular compartments (Reveneau et al., 2002, Pouwels et al., 2001). Mostly, plasmid-based expression systems are used for their ease of manipulation. However, integrated systems offer the benefit of greater genetic stability of the immunization strain, but this could compromise the expression levels of the antigen to be delivered. In contrast to the great interest of the scientific community in developing Lactobacillus strains as vaccine vehicles, the potential of using yeast, which is also commonly utilized in food processes, remained unexplored. Since the idea of developing live bacterial or yeast as antigen delivery vector is on the basis of the ability of the vaccine carrier to produce the antigen incessantly, hence conferring long-term protection, constitutive and secretable expression systems have been selected for the expression of TGEV spike protein fragment by both
LcS and P.pastoris. Ideally, the secreted antigen will be sampled by cells of the mucosal immune system to induce a specific mucosal immune response, and will also be transported from the lumen across the epithelial barrier to stimulate a specific systemic immune response as well.
In this study, we engineered for the first time, a Lactobacillus spp. to express a coronaviral protein, rTGEV-S, into the external mileu. This N-terminal fragment of the
198 CHAPTER 6 DISCUSSION & CONCLUSION
TGEV spike protein encompasses all the four major antigenic sites (A, B, C and D) of the virus, of which epitopes critical for neutralization were found to be clustered within
(Delmas et al., 1986; Jimenez et al., 1986; Correa et al., 1988; Gebauer et al., 1991). In swine testicular cells, the receptor binding sites of TGEV are mapped within site A and
D, since monoclonal antibodies specific for these sites inhibit the binding of the virus to the cells (Sune et al., 1990). At the same time, P.pastoris that constitutively expresses and secrete the same recombinant coronaviral protein (PrTGEV-S) was also generated.
The use of recombinant Lactobacillus spp. and yeast as a coronavirus vaccine candidate also offer major advantages over the existing immunization strategies. Vaccine delivery using bacterial and yeast vectors does not involve the usage of attenuated or chimeric viruses, hence excluding the possibility of potential risk of the virus undergoing recombination, changing of tropism and resulting in increased pathogenicity.
A 75 kDa band corresponded to the expected size of rTGEV-S protein was detected in the supernatant of overnight recombinant LcS culture, when probed with convalescent swine serum (Figure 4.9). The expressed rTGEV-S protein could only be detected when the supernatant was concentrated ten-fold. Likewise, a study by Oliveira et al. (2003) indicated that secretion of recombinant protein by L.casei was only detected when the supernatant was concentrated. As suggested Oliveira and coworkers, the low level of rTGEV-S protein being secreted by recombinant LcS could be due to the inefficient processing of the expressed protein. The partially processed protein might have remained attached to the cell surface, thus resulting in low level of protein being secreted into the supernatant. On the other hand, a protein band with a molecular weight significantly higher (90 kDa) than then expected size of PrTGEV-S protein (75 kDa) was
199 CHAPTER 6 DISCUSSION & CONCLUSION
detected in the supernatant of overnight culture of recombinant P.pastoris using
convalescent swine serum (Figure 4.20). Since P.pastoris is capable of generating post-
translational modifications, resulting in proteins which are correctly folded and secreted
into the medium, the increase in molecular weight of PrTGEV-S protein is likely
contributed by glycosylation of the expressed protein (Romanos et al., 1992; Cregg et al.,
1993; Ilgen et al., 2004). Nevertheless, the ability of the polyclonal anti-TGEV
antibodies in the convalescent swine serum to bind to rTGEV-S or PrTGEV-S protein
bands indicated that both recombinant LcS and P.pastoris were able to express and secrete TGEV spike protein fragment without compromising its antigenic properties.
The hallmark of the mucosal immune system is the production of IgA as IgA is
the predominant antibody at the mucosal surface that is locally produced at a level that
exceeds that of all of other immunoglobulins. Therefore, it is likely that to be effective, an oral vaccine will have to induce a specific intestinal IgA response. Both recombinant
LcS and P.pastoris were shown to be able to elicit mucosal and systemic humoral responses after oral immunization in mice. The mucosal and systemic immune responses elicited by the recombinant P.pastoris was significantly prompter and stronger than the immune responses induced by the recombinant LcS (Figures 4.12, 4.13, 4.23 & 4.24).
High level of PrTGEV-S specific IgA and IgG were already induce on day 18 by recombinant P.pastoris, reaching concentrations that were relatively close to the maximum level of IgA and IgG observed in the study (Figure 4.23 & 4.24A). In particular, the concentration of rTGEV-S specific IgA elicited by recombinant LcS was only 16 % of the highest level IgA attained (Figure 4.12). The discrepancies in the immunological responses of mice to the LcS and P.pastoris oral vaccines could be
200 CHAPTER 6 DISCUSSION & CONCLUSION
attributable to the differential expression level of TGEV spike protein fragment. As observed in in vitro culture of recombinant LcS and P.pastoris, the recombinant TGEV spike protein fragment might be more efficiently expressed and secreted by P.pastoris
adhering to the intestinal tract, resulting in a more rapid dissemination of PrTGEV-S
protein to sensitize the mucosal inductive sites in the intestinal tract. Inevitably, a
prompter immune response specific for PrTGEV-S protein could be elicited.
The oral immunization regime used, which consisted of three sets of three
successive daily doses of the experimental vaccine, was adapted from the procedure of
Challacombe (1983), who found that this pattern of immunization was consistently
effective when particulate oral vaccines were used to immunize mice. Three successive
daily doses of recombinant strains were required in order to ensure that systemic antibody
response to rTGEV-S or PrTGEV-S proteins can be elicited in all mice receiving
recombinant LcS or P.pastoris intra-gastrically. In additional to mucosal rTGEV-S and
PrTGEV-S specific IgA responses, the presence of rTGEV-S and PrTGEV-S specific IgG
antibodies in the serum of orally immunized mice indicated that this regime also induced
rTGEV-S and PrTGEV-S specific immune responses in the systemic compartment.
Although Lactobacillus spp. have been known to possess adjuvant properties (Meydani &
Ha, 2000; Marteau, 2002), recombinant LcS being used as a vaccine carrier in this study
did not induce a stronger systemic response in comparison to that elicited by recombinant
P.pastoris. Mice administered with wild type LcS showed antibody levels similar to the
control groups that received PBS only. As suggested by Maassen and coworkers (2000)
who found that many of the Lactobacillus strains did not affect the systemic humoral
immune responses, LcS may act very similarly to natural gut commensals of the mice and
201 CHAPTER 6 DISCUSSION & CONCLUSION
hence, do not disturb the existing immune status. Nevertheless, the subclasses of
rTGEV-S or PrTGEV-S specific IgG induced by the oral vaccines were also determined.
Most of the IgG responses generated by oral immunization with LcS-rTGEV-S or
PP/PrTGEV-S were of IgG2a isotype (Figures 4.13B & 4.24B).
Although mucosal IgA has been known to be an important effector molecule to
protect mucosal surfaces, the contribution to mucosal protection by the cellular immune
system should not be underestimated. Cell-mediated immunity occurs before the
induction of antibodies and forms an early line of defense against viral infections. Since
previous study has indicated that the production of IgG1 isotype is induced by Th2-type
cytokines, and that the IgG2a isotype is regulated by Th1-type cytokines (Finkelmann et
al., 1990; Gemmell et al., 2004), it could be presumed from a higher IgG2a level that
both recombinant LcS and P.pastoris induced a more prominent type 1 response. In
order to have a better understanding of the cellular immune responses induced by the
recombinant LcS and P.pastoris, the specific cytokine responses were assessed in the
Peyer’s patches of mice after oral immunization. As the Peyer’s patches represent the
inductive sites after oral administration, from which sensitized lymphocytes migrate to
the mesenteric lymph nodes, then to the lamina propria, it is of particular importance to
assess the immune responses elicited in the Peyer’s patches to determine the effectiveness
of the oral vaccines to induce cell-mediated immunity.
It was observed that the immunized animals displayed strong cell-mediated
immune responses. A specific Th1/Th2-like cytokine response was observed after in vitro re-stimulation of Peyer’s patches cells from mice orally immunized with recombinant LcS or P.pastoris (Figure 4.16 & 4.27). In accordance with the fact that
202 CHAPTER 6 DISCUSSION & CONCLUSION higher levels of IgG2a (Figures 4.13B & 4.24B) were induced by both LcS-rTGEV-S and
PP/PrTGEV-S, a stronger Th1-like cytokines response, as measured by the Th1-type cytokines, IFN-γ and IL-2, were elicited (Figures 4.16 & 4.27). In particular, high levels of IFN-γ were induced by the sensitized cells of Peyer’s patches of mice orally immunized with recombinant LcS or P.pastoris. The strong interferogenic activity exhibited by cells of the Peyer’s patches could be attributed to the nature of the delivered antigen, where TGEV has been reported to be a potent inducer of interferon (La
Bonnardiere & Laude, 1981; Riffault et al., 1997). Interferon gamma, a prototypical
Th1-type cytokine, is produced predominantly by CD4 Th1 cells and CD8 T cells. The level of IFN-γ expressed by cells of the Peyer’s patches reflects the magnitude of the T cell response. Since after in vitro re-stimulation, cytokines are most likely to be produced by T cells, it can be concluded that TGEV-specific lymphocytes were present in the intestine after oral immunization of mice with recombinant LcS or P.pastoris, capable of constitutive expression of TGEV spike protein fragment.
Interestingly, even though IFN-γ is known to down regulate Th2-type responses
(McGhee et al., 1989), the findings in this study indicated that the production of Th2-type cytokines (IL-4 and IL-5) were elicited and maintained in the mice orally dosed with recombinant LcS or P.pastoris (Figures 4.16 & 4.27). Since IL-4, a prototypical Th2- type cytokine, have been shown to play a dominant role in B cell-mediated immune responses (Agadjanyan et al., 1999), the induction of Th2 cytokine producing cells may contribute to the induction of TGEV-specific IgA and IgG responses in both the mucosal and systemic compartment of immunized mice.
203 CHAPTER 6 DISCUSSION & CONCLUSION
When developing vaccines, it is crucial that the vaccines are capable of conferring
protection against viral infection. As such, the evaluation of their protective immunity
are warranted. In order to confirm the efficacy of the induced antibodies in inhibiting
TGEV infection, intestinal lavages and sera from mice orally immunized with rTGEV-S
protein expressing LcS or PrTGEV-S protein expressing P.pastoris were tested for their ability to inhibit the infection of ST cells in plaque reduction neutralization assay. Sera and intestinal lavages collected from mice administered with recombinant LcS or
P.pastoris demonstrated statistically significant inhibition of plaque formation by TGEV
(Figures 4.14 & 4.25). However, the protection conferred by lavages and sera from mice fed with recombinant LcS were very much less efficient than those conferred by lavages and sera from mice fed with recombinant P.pastoris. The maximum reduction in the number of plaques using intestinal lavages and sera collected from mice orally immunized with recombinant LcS were lower that 15 %. These inhibition efficiencies were only 19 to 22 % of the maximum plaque inhibition by the lavages and sera of mice fed with the recombinant P.pastoris, which showed a reduction close to 100 % at lower dilutions. The poor protective immunity conferred by recombinant LcS could be due to the lack of post-translational modifications in the prokaryotic expression system. The native TGEV S glycoprotein is usually highly glycosylated (Rasschaert & Laude, 1987).
In particular, antigenic sites A and B, which have been included in the recombinant rTGEV-S protein, have been demonstrated to be conformational and glycosylation dependent (Correa et al., 1988; Correa et al., 1990; Posthumus et al., 1990a; Posthumus et al., 1990b; Gebauer et al., 1991). As such, the lack of correlation between antibody levels measured by ELISA and neutralizing antibody titers is not surprising as
204 CHAPTER 6 DISCUSSION & CONCLUSION
neutralizing antibodies probably represent a subset of the total antibody, and the levels of
neutralizing antibodies may be related to the availability of neutralizing epitopes on the
expressed antigen. The expressed rTGEV-S protein might not be functional due to the lack of post-translational modifications, especially when glycosylation is crucial for the immunogenicity of site A, the major inducer of neutralizing antibodies (Correa et al.,
1988; De Diego M et al., 1992). Interestingly, despite the lack of post-translational modifications in prokaryotic expression system, the 75 kDa fragment of rTGEV-S protein expressed by LcS was recognized by convalescent swine serum, indicating that the expressed protein possessed certain degree of immunogenicity, albeit not strong enough to elicit significant immune protection.
Since P.pastoris has been demonstrated to be an extremely efficient expression system that generate recombinant proteins which are post-translationally modified and typically correctly folded (Cregg et al., 1987; Romanos et al., 1992; Ilgen et al., 2004), it is likely that the high inhibitory percentage of TGEV plaque formation was due to the proper processing of the expressed product. In agreement, Gomez and coworkers (1998) have developed arabidopsis transgenic plants expressing N-terminal domain of TGEV spike protein that elicited neutralizing antibodies when used to immunize mice. On the other hand, inoculation of animals with recombinant E.coli expressing glycoprotein S did not induce neutralizing antibodies or confer protection in vivo (Hu et al., 1996). In another study, the use of plant as an oral vaccine delivery vehicle would only result in short-term induction of the immune system against the expressed antigen and hence, requires consistent and frequent feeding of mice with the transgenic plant if long-term effect is desired. In contrast, the development of a delivery system like P.pastoris that is
205 CHAPTER 6 DISCUSSION & CONCLUSION
able to adhere and colonize the intestinal tract permits continuous expression of antigen
in the intestine, resulting in extended protection of the vaccinated subject from viral
infection.
To this point, the results have provided evidence, favoring the choice of
P.pastoris over LcS as vaccine delivery vehicle for coronaviruses. Oral immunization of
mice with recombinant P.pastoris expressing PrTGEV-S protein induced strong humoral
and cellular immune responses. These responses were characterized by high levels of
specific mucosal IgA and systemic IgG, neutralizing antibodies, as well as high levels of
IL-2, IFN-γ, IL-4 and IL-5 producing cells in the Peyer’s patches of immunized mice, which were good indicators of effective immunization strategy.
The emergence of SARS that resulted in a pandemic in 2003 spurred a flurry of
interest in the development of antiviral strategies against SARS CoV. Because of the
threat of a reemerging epidemic, much effort has been placed on the development of a
prophylatic vaccine against the pathogenic SARS CoV (Bisht et al., 2004; Bukreyev et
al., 2004; Chen et al., 2005; Johnston, 2004; Weingartl et al., 2004; Yang et al., 2004).
The fact that high titers of virus neutralizing antibody to SARS CoV are found in sera of
patients recovering from infection and those infected with the virus show improvement
after passive antibody administration suggests a SARS CoV vaccine is feasible (Pearson
et al., 2003; Sui et al., 2004; Oxford et al., 2005). Currently, the development of SARS
vaccine are focussed essentially on the utilization of two different types of SARS CoV
derived immunogens, inactivated whole virus and recombinant SARS CoV structural
proteins. Although Takasaka et al. (2004) have reported that subcutaneous
administration of UV-inactivated SARS CoV viron elicits high level of humoral
206 CHAPTER 6 DISCUSSION & CONCLUSION immunity, there remains the threat of introducing live virus into the environment from partially inactivated vaccine, as there are no validated and effective inactivation measures developed yet.
Current efforts on SARS CoV vaccine have very much been focussed on the development of recombinant vectored or recombinant subunit vaccines, utilizing recombinant nucleocapsid or S proteins as the antigen. Of the structural proteins that compose SARS CoV, the S glycoprotein is probably the most significant target for neutralization (Buchholz et al., 2004). Li and coworkers (2003) identified angiotensin- converting enzyme 2 (ACE2) as a functional receptor for SARS CoV. This finding was further confirmed by several other research groups (Xiao et al., 2003; Wang et al., 2004;
Mossel et al., 2005). The interaction between ACE2 and SARS CoV S glycoprotein was further explored to elucidate the structure and function of ACE2 (Prabakaran et al.,
2004). ACE2 was found to interact with an independently folded receptor binding domain (RBD), which is a 193 residue fragment [S(318-510)] of the SARS CoV S protein (Wong et al., 2004). Since the RBD of SARS CoV alone exhibited potent antiviral activity by blocking S protein mediated infection, the ACE2-binding site of the
S glycoprotein has become an attractive target for vaccine design. Chen et al. (2005) have introduced the S protein into the deletion III region of the live attenuated modified vaccinia virus Ankara vector and the recombinant virus generated elicited potent neutralization antibodies in mice, rabbits and monkeys. On the other hand, mice immunized with a plasmid containing the S protein developed neutralizing antibodies and cell-mediated responses resulting in a six-fold reduction in viral titer (Yang et al., 2004).
The demonstration that S proteins of several CoVs (TGEV, porcine epidemic diarrhea
207 CHAPTER 6 DISCUSSION & CONCLUSION
virus, and infectious bronchitis virus) are immunogenic when administered orally in
animals (Zhou et al., 2003; Bae et al., 2003; Lamphear et al., 2004; Tuboly et al., 2000)
have led us to test yeast derived SARS antigen by this route.
In the present study, a 720 bp fragment of SARS CoV S glycoprotein, encoding
the receptor-binding domain of SARS CoV was introduced into the genome of
P.pastoris. As suggested by the results presented earlier, P.pastoris being a more appropriate vaccine carrier for coronaviruses was selected for the delivery of SARS-S-
RBD protein. P.pastoris is an eukaryotic expression system with high efficiency and proteins expressed by this system can get appropriate post-translational modifications,
including glycosylation (Kukuruzinska et al., 1987; Grinner & Tschopp, 1989; Romanos
et al., 1992). Recombinant SARS-S-RBD protein was successfully expressed by
P.pastoris, and the expressed SARS-S-RBD protein was similar to the native protein in its immune response. This can be demonstrated by the reaction of recombinant SARS-S-
RBD protein with SARS-positive sera in Western blotting, which revealed two distinct protein bands in the supernatant of overnight culture of recombinant P.pastoris (Figure
5.5). The smaller protein band corresponding to the expected size of SARS-S-RBD protein represents the unglycosylated protein while the larger protein protein band is likely to be the glycosylated SARS-S-RBD protein. The ability of recombinant
P.pastoris to generate glycosylated SARS-S-RBD protein is crucial as recent evidence
has determined that virus neutralization is sensitive to deglycosylation of the S protein
(Song et al., 2004). Interestingly, the increment in molecular weight of SARS-S-RBD protein due to glycosylation of the expressed protein was much higher than that of the glycosylated PrTGEV-S protein. One could reason that the variation in the increment of
208 CHAPTER 6 DISCUSSION & CONCLUSION
size of the glycoprotein could possibly be due to the presence of different number of
glycosylation sites, resulting in the different extend of glycosylation. In a study where
glycosylation variants of β-lactoglobulin with different number of glycosylation sites
were produced by P.pastoris, the extent of glycosylation were found to vary among the
mutants (Kalidas et al., 2001). It has been predicted that the S protein of SARS CoV
contains 23 potential N-glycosylation sites (Krokhin et al., 2003). However, mass
spectrometric analysis of the S protein confirmed the existence of 12 glycosylation sites in this protein, while another study by Ying et al. (2004) revealed that only 4
glycosylation sites were present. On the other hand, no literature specifying the number of glycosylation sites in the S protein of TGEV is available. As such, no comparison could be made between the number of glycosylation sites within the PrTGEV-S and
SARS-S-RBD protein in order to elucidate the reason for the discrepancies in the increment of size of the recombinant proteins.
In this study, oral immunization of PP/SARS-S-RBD induced SARS-S-RBD specific humoral responses, characterized by strong and persistent SARS-S-RBD specific
IgA and IgG levels in the intestinal lavages and sera from immunized mice (Figures 5.8
& 5.9). The level of antigen specific IgA and IgG induced by P.pastoris, in the delivery of PrTGEV-S or SARS-S-RBD proteins were highly similar. These, together with the strong mucosal and systemic immunological responses elicited against PrTGEV-S and
SARS-S-RBD proteins demonstrated the stability and suitability of P.pastoris as an oral vaccine delivery vehicle for coronaviruses. Interestingly, in contrast to a higher level of
IgG2a isotype induced by PP/PrTGEV-S, the SARS-S-RBD specific isotype was predominantly IgG1 (Figure 5.9B). The decreased ratio of IgG2a/IgG1 in mice orally fed
209 CHAPTER 6 DISCUSSION & CONCLUSION
with P.pastoris expressing SARS-S-RBD protein suggests a shift toward a Th2-type
response in immunized animals. The type of immune response is known to be important
in achieving maximum protection against various pathogens, such as respiratory syncytial virus (Kim et al., 1969; Srikiatkhachorn & Braciale, 1997). The level of IgG1 antibody is often associated with Th2 cells (Finkelmann et al., 1990; Gemmell et al., 2004). The
Th2 cells produce more IL-4, expanding Th2 cells which support the associated immune responses. The production of IL-4 support IgG1 subclass and antibodies such as IgE and
IgA are also produced during a Th2 dominated response (McGhee et al., 1999). In accordance to a more dominant IgG1 isotype induced by oral immunization of mice with
PP/SAR-S-RBD, a stronger Th2-like cytokine response was elicited. The Th2 biased response was quite unexpected as IFN-γ, a typical Th1-type cytokine, was reported to be elevated in the acute phase sera of Taiwan SARS patients (Huang et al., 2005). In addition, when a combination of whole killed SARS CoV and DNA vaccine containing a plasmid encoding the full-length SARS CoV S protein were used to immunize mice, a
Th1 dominated immune response was induced (Zakhartchouk et al., 2005). However, by database analysis, Wang et al. (2004) identified two T cell epitopes, S978 and S1203, in
SARS CoV S protein which are localized in the S2 protein of SARS CoV. The SARS
CoV specific peptides S978 and S1203 were found to induce a high frequency of IFN-γ secreting T cell response, indicating that the IFN-γ-related cytokine storm in SARS patients and the Th1 dominated responses induced by the SARS CoV vaccines (DNA and whole killed virus) might be contributed significantly by the T cell epitopes in the S2 fragment of SARS CoV S glycoprotein. This might provide an explanation for the less dominant Th1-like cytokines responses in the Peyer’s patches and CLN of mice orally
210 CHAPTER 6 DISCUSSION & CONCLUSION
immunized with P.pastoris expressing SARS-S-RBD protein, since the delivered antigen encompasses only the RBD within the S1 fragment of SARS CoV S glycoprotein. In
accordance to our findings, injection of plant-derived S1 fragment of the SARS-CoV S
protein resulted in a shift toward Th2-type response in mice (Pogrebnyak et al., 2005).
The specific cytokine responses were assessed both in the Peyer’s patches and
CLN of mice after oral ingestion of P.pastoris expressing SARS-S-RBD protein. It is of
particular importance to assess the cell mediated immune responses in the CLN after immunization by the oral route since SARS CoV is a respiratory pathogen and the immune responses are expected to be stronger at the sites of induction than at distant sites. Indeed, the amount of IL-4 and IL-5 produced by the re-stimulated cells of the
Peyer’s patches were generally higher than the concentrations of these cytokines
produced by the re-stimulated cells of the CLN of mice immunized with PP/SARS-S-
RBD. Several studies reported that gut associated lymphoid tissue can stimulate the
common mucosal immune system, in which immune cell stimulated at one site migrate to
other sites through the systemic (blood and lymph) circulation (Tilney, 1971; Rudzik et
al., 1975; McCluckie et al., 2001). The ability of cells of the CLN to generate a mixed
Th1/Th2-like cytokine response when re-stimulated with SARS-S(M) indicated that oral
immunization with PP/SARS-S-RBD resulted in dissemination of cytokine producing
cells from the mucosal tissue to the CLN. This further substantiates the potential use of
P.pastoris for the oral delivery of antigens to elicit specific immunological responses at sites close to or distant from the immunization site. However, at present, the available
data are insufficient to allow conclusions about effectiveness of PP/SARS-S-RBD as
SARS CoV vaccine to be made.
211 CHAPTER 6 DISCUSSION & CONCLUSION
Spike specific monoclonal and polyclonal antibodies that neutralize the virus have been developed (Berry et al., 2004; Zhang et al., 2004) and passive transfer of immune serum into naive mice protected them from infection with SARS CoV (Subbarao et al.,
2004). These suggest that neutralizing antibody alone can prevent viral infection. As such, it is crucial to determine the neutralizing activity of the antibodies elicited by
SARS-S-RBD protein. In contrast to plaque reduction neutralization assay utilized to analyze the inhibitory effects of antibodies elicited by PP/PrTGEV-S against TGEV infection, fluorescent focus assay was performed using intestinal lavages and sera from mice orally immunized with PP/SARS-S-RBD to determine the protective effects conferred by SARS-S-RBD specific intestinal IgA and serum IgG to inhibit infection of
Vero cells. As SARS CoV is a highly hazardous pathogen in which only trained and experienced personnel is suited to work with, the next best alternative is to utilize murine leukemia virus particle pseudotyped with SARS CoV S protein (Giroglou et al., 2004).
MLV(SARS) offers a retroviral pseudotype-based assay that facilitates the accurate determination of neutralizing antibody responses to SARS CoV without the use of replication competent virus. Cells infected with MLV(SARS) will fluoresce under fluorescence microscope as the pseudotyped virus was co-transfected with eGFP as a marker during its construction. The number of FFU can then be enumerated and expressed as a percentage of fluorescence cell obtained in the neutralization assay with anti-SARS-S-RBD intestinal lavages or sera with respect to intestinal lavages or sera from control mice. The recombinant P.pastoris expressing SARS-S-RBD elicited potent neutralizing antibodies when orally administered to mice (Figure 5.10). The anti-SARS-
S-RBD IgA and IgG (up to dilution of 1:8) were capable of almost completely inhibiting
212 CHAPTER 6 DISCUSSION & CONCLUSION
infection of Vero cell by MLV(SARS). Of note, more that 50 % of infection by
MLV(SARS) were blocked by anti-SAR-S-RBD IgA and IgG in the intestinal lavage
and serum at a dilution of 1:32. In agreement, rabbits and mice immunized with RBD
produced high titer of neutralizing antibodies against SARS CoV (He et al., 2004). It is
likely that the S protein-based oral vaccine induced potent neutralizing antibodies that
prevented the virus from entering the Vero cells by binding to the virions to block their interaction with ACE2. More importantly, van den Brink et al. (2005) showed that one human monoclonal antibody (CR3014) specific for RBD of one of the SAR CoV strains can effectively bind to most RBDs of the early and late SARS CoV strains. These data suggest that antibodies directed against RBD of a SARS CoV isolate may neutralize infection by a broad spectrum of SARS CoV strains.
The data obtained in our attempt to generate an oral vaccine against SARS CoV have demonstrated the feasibility of utilizing P.pastoris as an oral delivery vehicle for the coronavirus. Oral immunization of mice with PP/SAR-S-RBD induced strong humoral and cellular immune responses. These responses were characterized by high levels of
SARS-S-RBD specific serum IgG and secretory IgA, neutralizing antibodies, and IFN-γ
producing cells both in the Peyer’s patches and CLN. Thus, PP/SARS-S-RBD can act as
a potent oral vaccine to induce both humoral and systemic responses, as well as both
local and distant immunity.
213 CHAPTER 6 DISCUSSION & CONCLUSION
6.1 CONCLUSION
6.1.1 Adhesion Studies
• All the three yeasts (S.boulardii, S.cerevisiae and P.pastoris) demonstrated in vitro
adhesiveness to human intestinal Caco-2 and HT29 cells, where P.pastoris appeared
to have a stronger affinity to the intestinal cells in comparison to S.boulardii and
S.cerevisiae.
• In agreement, in vivo adhesion studies conducted on the Balb/c mice revealed that
S.boulardii, S.cerevisiae and P.pastoris were able to adhere and replicate in various
segments of murine gastrointestinal tract.
• P.pastoris was selected over the other two yeasts for further development into oral
vaccine carrier against coronavirus as P.pastoris was found to be better equipped with
the ability to re-adhere and replicate in certain segments of the gastrointestinal tract.
At the same time, in vivo adhesion analysis of LcS showed that the lactobacilli was
able to compete with the indigenous microorganisms for adhesion on the mucosal
surfaces of the intestinal tract, thus making it a suitable prokaryotic candidate for oral
vaccination purposes.
214 CHAPTER 6 DISCUSSION & CONCLUSION
6.1.2 Development of Oral Vaccine Against TGEV
• Recombinant LcS and P.pastoris capable of constitutively expressing and
secreting an N-terminal fragment that encompassed the major antigenic domains
of TGEV S protein were generated. The recombinant proteins (rTGEV-S and
PrTGEV-S protein) expressed by LcS-rTGEV-S and PP/PrTGEV-S were showed
to have retained their authentic antigenicities.
• More importantly, oral immunization of mice with LcS-rTGEV-S or PP/PrTGEV-
S elicited antigen specific mucosal and systemic humoral responses against the
delivered antigens. However, the antibodies induced by the recombinant LcS
conferred modest protection against TGEV infection in the plaque reduction
assays conducted. This is in opposed to the strong inhibitory effects exerted by
the antibodies induced by PP/PrTGEV-S.
• The low protective ability of the antibodies elicited by LcS-rTGEV-S was
attributed to the lack of post-translational modifications of the delivered rTGEV-S
protein in prokaryotic expression system, where some of the antigenic sites within
the rTGEV-S protein were reported to be dependent on glycosylation.
• Nevertheless, mice immunized with either LcS-rTGEV-S or PP/PrTGEV-S
displayed strong cell-mediated immune responses, where Th1-type dominated
responses were elicited. Our findings indicated that P.pastoris served as a better
oral vaccine carrier against coronaviruses, while Lactobacillus spp. is more
suitable for the delivery of antigens in which post-translational modifications is
not crucial for the immunogenicity of the antigens.
215 CHAPTER 6 DISCUSSION & CONCLUSION
6.1.3 Development of Oral Vaccine Against SARS CoV
• Earlier results indicated that P.pastoris is a highly potential candidate for the
delivery of coronavirus antigen via the oral route. In view of this, P.pastoris was
developed into an oral vaccine carrier for the delivery of SARS CoV antigen in
the final part of the study.
• Recombinant P.pastoris was engineered to express and secrete a fragment of the
S glycoprotein of SARS CoV (SARS-S-RBD). The recombinant protein
contained the RBD of SARS CoV, found to be essential for the generation of
protective immunological responses against the coronavirus. PP/SARS-S-RBD
was demonstrated to be able to express the recombinant protein without
compromising its antigenic properties.
• Mice orally immunized with PP/SARS-S-RBD developed strong mucosal and
systemic immune responses against SARS-S-RBD. The antibodies elicited
demonstrated potent neutralizing activity against a SARS CoV pseudotyped virus
particle, MLV(SARS).
• A Th2 dominated cell-mediated response was elicited both in the Peyer’s patches
and CLN of immunized animals, indicating that oral immunization of PP/SARS-
S-RBD is able to induce cellular responses at sites close to and distant from the
site of immunization.
• Findings obtained in this part of study further substantiate the potential of
utilizing P.pastoris or even other yeast strains as oral vaccine delivery vehicles
against viral antigens.
216 REFERENCES
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286 APPENDIX
APPENDIX 1
MATERIALS FOR BACTERIAL AND YEAST CULTURE
Materials Required for Bacterial Culture
(a) Luria-Bertani (LB) Broth, pH 7.0
Items Amount Source Bacto-tryptone 10.0 g BD Biosciences, USA Bacto-yeast extract 5.0 g BD Biosciences, USA NaCl 10.0 g Merck, Germany
The above components were dissolved in 1.0 liter of dgd. The solution was autoclaved at 121 ºC for 15 min (Hirayama, Japan). Storage was at 4 °C.
(b) Luria-Bertani (LB) Agar
Items Amount Source Bacto-tryptone 10.0 g BD Biosciences, USA Bacto-yeast extract 5.0 g BD Biosciences, USA NaCl 10.0 g Merck, Germany Agarose 10.0 g Oxoid, UK
The above components were dissolved in 1.0 liter of deionised water. The solution was autoclaved at 121 ºC for 15 min (Hirayama, Japan) and cooled to 60 °C before pouring plates. Storage was at 4 °C.
287 APPENDIX
(c) Low Salt (LB) Agar
Items Amount Source Bacto-tryptone 10.0 g BD Biosciences, USA Bacto-yeast extract 5.0 g BD Biosciences, USA NaCl 5.0 g Merck, Germany Agarose 10.0 g Oxoid, UK
The above components were dissolved in 1.0 liter of deionised water. The solution was autoclaved at 121 ºC for 15 min (Hirayama, Japan) and cooled to 60 °C before pouring plates. Storage was at 4 °C.
(d) SOC media
Items Amount Source Bacto-Tryptone 2.0 g BD Biosciences, USA Bacto -Yeast extract 0.5 g BD Biosciences, USA NaCl 0.58 g Merck, Germany KCl 0.25 g Merck, Germany MgCl2 2.03 g Merck, Germany MgSO4 2.46 g Merck, Germany Glucose 1.8 g Merck, Germany
The above components were dissolved in 1.0 liter of dgd. The solution was autoclaved at 121 ºC for 15 min (Hirayama, Japan). Storage was at 4 °C.
(e) Antibiotics
Ampicillin was prepared as stock solution of 100 mg/ml in dgd and diluted to100 μg/ml as the working concentration. Kanamycin was prepared as stock solution of 50 mg/ml in dgd and diluted to 50 μg/ml as the working concentration. Zeocin was prepared as stock solution of 10 mg/ml in dgd and diluted to 100 μg/ml as the working concentration.
Erythromycin was prepared as stock solution of 25 mg/ml in ethanol and diluted to 5
288 APPENDIX
μg/ml as the working concentration. All the antibiotic solutions were filtered through a
0.22 µm filter and stored at -20 °C prior to use.
Materials Required for Yeast Culture
(f) YPD Media
Items Amount Source Bacto-Peptone 20.0 g BD Biosciences, USA Bacto-yeast extract 10.0 g BD Biosciences, USA Dextrose 20.0 g Merck, Germany
The above components were dissolved in 1.0 liter of dgd. The solution was autoclaved
at 121 ºC for 15 min (Hirayama, Japan). Storage was at 4 °C.
(g) YPD Agar
Items Amount Source Bacto-Peptone 20.0 g BD Biosciences, USA Bacto-yeast extract 10.0 g BD Biosciences, USA Dextrose 20.0 g Merck, Germany Agarose 10.0 g Oxoid, UK Agar 10.0 g Oxoid, UK
The above components were dissolved in 1.0 liter of deionised water. The solution was
autoclaved at 121 ºC for 15 min (Hirayama, Japan) and cooled to 60 °C before pouring plates. Storage was at 4 °C.
289 APPENDIX
APPENDIX 2
MATERIALS FOR MOLECULAR CLONING
Materials Required for DNA Agarose Gel Electrophoresis
(a) 1 x Tris-Borate-EDTA Buffer (TBE)
The stock solution of 10x TBE buffer was purchased from the National University
Medical Institute (NUMI) Store. Ten ml of the 10x TBE buffer was added to 90.0 ml of deionized water to obtain the 1 x TBE buffer.
(b) 1.5 % Agarose Gel
Items Amount Source Agarose 20.0 g Seakem, USA 10 x TBE 25.0 ml Prepared by NUMI Store Ethidium Bromide (10 12.5 μl Sigma, USA mg/ml)
The above mixture was topped up to 250.0 ml (excluding ethidium bromide) with dgd and the mixture was boiled for 3 min in a microwave oven. The solution was allowed to cool to 60 °C followed by the addition of ethidium bromide. The solution was mixed well before pouring into the agarose gel apparatus in the fume cardboard.
(c) DNA Loading Buffer
Items Amount Source Bromophenol Blue 25.0 mg Sigma, USA Sucrose 4.0 g Merck, Germany Dgd 10.0 ml Corning, USA
290 APPENDIX
Materials for Recombinant Fusion Protein Extraction
(d) GST- Lysis buffer
Items Amount Source Tris-HCl 7.88 g Sigma, USA Magnesium chloride 0.1 g Sigma, USA Triton X-100 (v/v) 0.1 ml Sigma, USA PMSF 5.0 ml Appendix 5 (e) DTT 0.78 g Sigma, USA
The above materials were dissolved in 1.0 litre of dgd water. The pH was adjusted to 7.2
using either 1 M HCl or 1 M NaOH. Storage was at room temperature.
(e) Urea + 0.01% SDS
Items Amount Source Urea 30 g Sigma, USA SDS 0.1 g Sigma, USA Triton X-100 (v/v) 0.1 ml Sigma, USA PMSF 5.0 ml Appendix 5 (e) DTT 0.78 g Sigma, USA
Materials for Electroporation of LcS
(f) Washing Buffer
Items Amount Source KH2PO4-K2HPO4 50 mM Sigma, USA Sucrose 0.3 M Sigma, USA MgCl2 1 mM Sigma, USA
The solution was then sterilized by filtration through 0.22 μm filter unit (Sterivex G-S, Millipore, USA).
291 APPENDIX
(g) Electroporation Buffer
Items Amount Source Sucrose 952 mM Sigma, USA MgCl2 3.5 mM Sigma, USA
The solution was then sterilized by filtration through 0.22 μm filter unit (Sterivex G-S, Millipore, USA).
Materials for DNA Isolation from Yeast Cells
(h) Breaking Buffer
Items Amount Source Triton X-100 (v/v) 20 ml Sigma, USA SDS 10 ml Sigma, USA Na Cl 100 mM Sigma, USA Tris-Cl 10 mM Sigma, USA EDTA 1 mM Sigma, USA
292 APPENDIX
Map of plasmids
(i) pCR-XL-TOPO
293 APPENDIX
(j) pLP 500
294 APPENDIX
(k) pGEX-4T-3
295 APPENDIX
APPENDIX 3
MATERIAL FOR SODIUM DODECYL SULPHATE - POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE)
(a) Stacking Gel
Items Amount Source 30 % Acrylamide & 0.8 % 1.67 ml Merck, Germany bis-acrylamide 0.5 M Tris buffer pH 6.8 2.5 ml Merck, Germany 10 % SDS 0.1 ml Merck, Germany Deionised water 5.7 ml Elga, UK TEMED (N,N,N'- 20.0 μl Merck, Germany tetramethyl ethylene diamine) 10 % Ammonium 50.0 μl Merck, Germany persulphate
The chemicals were added in the above order.
(b) Separation Gel (12% SDS-PAGE)
Items Amount Source 30 % Acrylamide & 0.8 % 12.0 ml Merck, Germany bis acrylamide 1 M Tris buffer pH 8.8 11.25 ml Merck, Germany 10 % SDS 0.3 ml Merck, Germany Deionised water 6.35 ml Elga, UK TEMED (N,N,N'- 40.0 μl Merck, Germany tetramethyl ethylene diamine) 10 % Ammonium 80.0 μl Merck, Germany persulphate
The chemicals were added in the above order.
(c) Dye Mixture (Stock Solution)
Items Amount Source Bromophenol blue 0.1g Sigma, USA Glycerol 1.0 ml Analar, UK 0.5 M Tris buffer, pH 6.8 10.0 ml Merck, Germany
296 APPENDIX
(d) SDS-Sample Buffer
A 1ml cocktail of dye mixture [see Appendix 5 (c)] stock solution were mixed with 100
mg of dithioerythritol (Biorad, USA). To every 25.0 μl of sample, 5.0 μl of these cocktail were added. After mixing the sample with cocktail, the mixture was boiled for 1 min in a water bath before loading onto the gel.
(e) Running Buffer (pH 8.9)
Items Amount Source Tris 6.0 g Merck, Germany Glycine 28.7 g Merck, Germany SDS 2.0 g BioRad, USA
The above was dissolved in 1 litre of dgd to make a 2x concentrated solution. From this
concentrated solution, 250.0 ml were diluted to 500.0 ml and used as the top chamber
buffer. The remaining stock solution were diluted to 4 litres and used as the bottom
chamber buffer.
(f) Gel Fixing Solution
Items Amount (ml) Source Glycerol 25.0 Merck, Germany Glacial acetic acid 25.0 Merck, Germany Deionised water 450.0 Elga, UK
This solution was prepared fresh before usage.
(g) X-Ray Film Developer
Items Amount (ml) Source X-ray Film Developer 100.0 Kodak, USA Deionised water 900.0 Elga, UK
297 APPENDIX
(h) X-Ray Film Fixer
Items Amount (ml) Source X-ray Film Fixer 100.0 Kodak, USA Deionised water 900.0 Elga, UK
298 APPENDIX
APPENDIX 4
MATERIAL FOR IMMUNOBLOTTING
(a) Transfer Buffer
Items Amount Source Tris (hydroxymethyl)- 3.0 g Merck, Germany aminoethane Glycine 14.40 g Merck, Germany Methanol 200.0 ml Merck, Germany Dgd 800.0 ml Elga, UK
(b) 5% Skim Milk Blocking Solution
Items Amount Source Skim milk powder 5.0 g Diploma, Singapore PBS 100.0 ml Appendix 1 (d)
(c) 0.1 % Bovine Serum Albumin (BSA)/PBS
0.1 ml of BSA (CSL, Australia) was added into 100.0 ml of PBS [see Appendix 1 (d)].
The solution was prepared fresh.
(d) 10% Bovine Serum Albumin (BSA)/PBS
1.0 ml of BSA (CSL, Australia) was added into 100.0 ml of PBS [see Appendix 1 (d)].
The solution was prepared fresh.
(e) 20 mM Phenylmethylsulfonyl Fluoride (PMSF)
Items Amount Source Phenylmethylsulfonyl 0.35 g Sigma, USA fluoride
The PMSF is dissolved in dgd to a final volume of 100.0 ml by stirring overnight.
299 APPENDIX
APPENDIX 5
MATERIALS FOR CELL CULTURE
All materials prepared for cell culture were either sterile or sterilised. Double glass
distilled water [(dgd) (Corning, USA)] was used for all dilutions.
(a) Growth Medium, M199 for Vero cells (pH 7.2 - pH 7.4), filter sterilised.
Items Amount Source Medium M199 1 packet (commerical) CSL, Australia NaHCO3 2.2 g Merck, Germany Foetal calf serum 100.0 ml GIBCO BRL, New Zealand Dgd 900.0 ml Corning, USA
(b) Growth Medium, RPMI for single cell suspension of Peyer’s patches and cervical lymph nodes, (pH 7.2 - pH 7.4), filter sterilised.
Items Amount Source RPMI 1 packet (commerical) CSL, Australia NaHCO3 2.0 g Merck, Germany Foetal calf serum 100.0 ml GIBCO BRL, New Zealand Dgd 900.0 ml Corning, USA
The solution was then sterilized by filtration through 0.22 μm filter unit (Sterivex G-S, Millipore, USA).
All cell culture media were screened for microbial contamination before the addition of serum.
(c) Growth Medium, MEM for ST cells (pH 7.2 - pH 7.4), filter sterilised.
Items Amount Source MEM 900 ml of the 1 liter GIBCO BRL, New Zealand solution Foetal calf serum 100.0 ml GIBCO BRL, New Zealand
300 APPENDIX
(d) Growth Medium, EMEM for Caco-2 cells (pH 7.2 - pH 7.4), filter sterilised.
Items Amount Source Medium M199 900 ml of the 1 liter CSL, Australia solution Foetal calf serum 100.0 ml GIBCO BRL, New Zealand
(e) Growth Medium, McCoy’s 5a for HT-29 cells (pH 7.2 - pH 7.4), filter sterilised.
Items Amount Source McCoy’s 5a 900 ml of the 1 liter CSL, Australia solution Foetal calf serum 100.0 ml GIBCO BRL, New Zealand
The sera supplemented media were kept at 4 °C. The cell culture media were warmed
up in water bath to the temperature suitable for cell culture before use.
(f) Phosphate Buffered Saline (PBS)
Items Amount (g) Source NaCl 8.0 Merck, Germany KCl 0.2 Merck, Germany Na2HPO4 1.15 Merck, Germany KH2PO4 0.2 Merck, Germany
The above materials were dissolved in 1 litre of dgd water. The pH was adjusted to 7.2
using either 1 M HCl or 1 M NaOH. The solution was aliquoted and sterilised at 15
ib/in2 (1 kg/cm2) pressure for 20 min. Storage was at room temperature.
301 APPENDIX
(g) Trypsin-Versene (ATV) Solution (10x concentrate)
Items Amount Source NaCl 80.0 g Merck, Germany KCl 4.0 g Merck, Germany D-glucose 10.0 g Analar, UK NaHCO3 5.8 g Merck, Germany Trypsin 5.0 g Difco, New Zealand Versene (EDTA) 2.0 g Sigma, USA dgd 1000.0 ml Corning, USA
The mixture was heated to 37 °C with occasional shaking for 3 to 4 hr to dissolve the trypsin. The solution was then sterilised by filtration through 0.22 μm filter unit
(Sterivex G-S, Millipore, USA). To use, 10.0 ml of this solution was added to 90.0 ml of sterile dgd to give 1x ATV solution.
(h) Preservation Medium
Items Amount (ml) Source Dimethylsulphoxide 1.0 Sigma, USA (DMSO) Foetal calf serum 1.0 GIBCO BRL, New Zealand Growth medium 7.0 Appendix 5 (a-d)
(i) 0.4% Phenol Red Solution
0.8 g of phenol red powder (Merck, Germany) was dissolved in a mortar using 1 M
NaOH. The NaOH was also used to adjust the pH of the solution. The solution was made up to 200.0 ml with dgd. Working concentration of 0.1 % was made by diluting in dgd and sterilising at 15 Ib/in2 pressure for 15 min.
302 APPENDIX
(j) 0.04 % L-Glutamine Solution
Items Amount Source L-glutamine 5.85 g Merck, Germany NaCl 1.70 g Merck, Germany Dgd 200.0 ml Corning, USA
(k) 3 % Sodium Bicarbonate - 0.01 % Phenol Red Solution
Items Amount Source NaHCO3 30.0 g Merck, Germany 0.4 % phenol red solution 250.0 ml Appendix 5 (i) Dgd 975.0 ml Corning, USA
(l) 0.5 % Trypan Blue Solution
Items Amount Source Trypan blue 0.5 g Merck, Germany PBS 100.0 ml Appendix 5 (f)
The solution was autoclaved at 121 ºC for 15 min (Hirayama, Japan). The solution was filtered before use.
303 APPENDIX
APPENDIX 6
MATERIALS FOR VIRUS INFECTION, GROWTH OF VIRUS AND PLAQUE ASSAY
(a) Maintenance Medium (MEM) - pH 7.2-7.4
9.57 g of MEM (Minimal Essential Medium) powder was dissolved in 995.0 ml of dgd
with 5.0 ml of 0.4 % Phenol Red solution [Appendix 5 (i)]. Ninety mls of the solution
was aliquoted into 100.0 ml glass bottles and sterilised at 15 Ib/in2 pressure for 20 min.
Storage was at 4 °C. MEM was warmed to the required temperature before adding to
cells.
The complete MEM was prepared as follow:
Items Amount (ml) Source MEM 90.0 From above 10% BSA 1.0 CSL, Australia L-glutamine 1.0 Appendix 5 (j) 3% Sodium bicarbonate 2.0 Appendix 5 (k)
(b) Virus Diluent (pH 7.2 - 7.4)
Items Amount (ml) Source Hank's BSS (10x 10.0 CSL, Australia concentrate) 10% Bovine serum albumin 1.0 CSL, Australia Dgd 87.0 Corning, USA 3% Sodium bicarbonate 1.0 Appendix 5 (k)
The solution was then sterilised by filtration through 0.22 μm filter unit (Sterivex G-S,
Millipore, USA). Storage was at 4 °C. Virus diluent was warmed to room temperature before use.
304 APPENDIX
(c) Overlay Medium (pH 7.2 - 7.4)
Items Amount Source Medium M199 1 packet (commerical) CSL, Australia NaHCO3 2.2 g Merck, Germany Foetal calf serum 20.0 ml GIBCO, New Zealand Dgd 500.0 ml Corning, USA 2 % w/v 500.0 ml Behring Diagnostic, USA Carboxymethylcellulose (CMC)
The solution was then sterilised by filtration through 0.22 μm filter unit (Sterivex G-S,
Millipore, USA) before mixing with CMC (sterilising at 15 Ib/in2 pressure for 15 min).
Storage was at 4 °C. Overlay medium is warmed to the required temperature before
adding to cells.
(d) 0.5 % Crystal Violet / 25 % Formaldehyde Solution
Items Amount Source Crystal violet 5.0 g Sigma, USA 37 % Formaldehyde 300.0 ml Merck, Germany solution PBS 200.0 ml See Appendix 5 (f)
(e) 0.02 M Tris (hydroxymethyl)-aminoethane buffer, pH 7.4
Items Amount Source Tris-base 0.24 g Merck, Germany NaCl 0.87 g Merck, Germany
The solution was then sterilised by filtration through 0.22 μm filter unit (Sterivex G-S,
Millipore, USA).
305 APPENDIX
APPENDIX 7
MATERIALS FOR BIO-IMAGING
Materials for Indirect Immunofluorescence Microscopy
(a) Ammonium Chloride - PBS Solution
Ammonium chloride (Merck, Germany) was prepared fresh by dissolving in PBS [see
Appendix 5 (f)] to give a final concentration of 0.265 g/100.0 ml.
(b) 0.1 % Bovine Serum Albumin (BSA)
0.1 ml of BSA (CSL, Australia) was added into 100.0 ml of PBS [Appendix 5 (f)]. The solution was prepared fresh.
(c) DABCO Mountant
4.5 g of DABCO (Sigma, USA) was dissolved in 180.0 ml of glycerol (Merck, Germany) and stirred gently while heating. This was made up to 200.0 ml with PBS [see Appendix
5 (f)].
Materials for Processing of Samples for Tranmission Electron Microscopy
(d) 0.4 M Sodium Cacodylate Stock
Items Amount Source Sodim cacodylate 8.56 g Merck, Germany Dgd 100.0 ml Elga, UK
306 APPENDIX
(e) 0.2 M Sodium Cacodylate Stock (pH 7.4)
Items Amount (ml) Source 0.4 M Sodim cacodylate 50.0 Appendix 7 (d) 0.1 M HCl 25.2 Merck, Germany 0.1 M CaCl2 (1.1 g in 100 2.0 Merck, Germnay of dgd
(f) 10 % Paraformaldehyde in 0.1 M Cacodylate buffer (pH 7.4)
Items Amount Source Paraformaldehyde powder 10.0 Merck, Germany 0.2 M Sodium cacodylate 50.0 ml Appendix 7 (e) buffer (pH 7.4)
Paraformaldehyde powder was first dissolved in the buffer by heating to 65 ºC. A few
drops of 1 M NaOH solution were added to clear the solution. The solution was cooled
before making up to 100.0 ml with the buffer.
(g) 2.5 % Glutaradehyde, 2.0 % Paraformaldehyde in 0.1 M Cacodylate Buffer,
pH 7.4
Items Amount Source 0.2 M Cacodylate buffer 50.0 ml Appendix 7 (e) 10 % Paraformaldehyde 20.0 ml Appendix 7 (f) solution 25 % Glutaraldehyde 10.0 ml Merck, Germany CaCl2 0.01 g Merck, Germany Dgd 20.0 ml Elga, UK
(h) 1 % Osmium Tetroxide in 0.1 M Cacodylate Buffer, pH 7.4
Items Amount Source Osmium tetroxide (packed 1.0g Taab, UK in ampoule) 0.2 M Sodium cacodylate 100.0 ml Appendix 7 (e) buffer (pH 7.4)
307 APPENDIX
To prepare the solution, the ampoule was opened in a brown glass bottle with the buffer inside. The mixture was swirled to ensure that the osmium tetroxide dissolved totally.
The solution was aliquoted into 5.0 ml bottle and stored at -20 °C.
(i) Ryter Kellenburger Buffer Stock Solution
Items Amount Source Sodium veronal 2.9 g Analar, UK Sodium acetate (hydrated) 1.9 g Analar, UK NaCl 3.4 g Merck, Germany Dgd 100 ml Elge, UK
308 APPENDIX
APPENDIX 8
DATA FOR CHAPTER 3
(a) Average food and water intake per mouse per day
Food intake per mouse per day (g) 3.74 1.93 5.27 4.58 5.63 3.43 3.40 4.43 3.90 3.40 4.80 4.43 4.23 5.30 3.97 4.12 4.67 4.60 3.93 3.50 4.40 3.77 5.10 4.07 4.53 4.33 4.02 3.90 4.60 3.33 Avg Food Std dev Avg H2O Avg food & water intake (g) intake (g) intake (g) 4.18 0.73 2.42 6.60
(b) Fecal volume excreted per mouse per day
Fecal vol per mouse per day (g) 1.13 0.93 1.04 1.32 1.64 1.02 1.08 1.88 0.63 1.24 1.50 1.00 0.77 1.44 1.30 0.83 1.13 1.28 0.90 1.40 1.34 0.76 1.23 1.29 2.02 1.01 1.18 Avg fecal vol (g) Std dev 1.20 0.32
309 APPENDIX
(c) Amount of intestinal water overlaying different segments of murine intestinal tract
Duodenum Total length(cm) Length used(cm) IV (g) IV/ tissue (g) Day 1 Mouse 1 7.0 5.0 0.0440 0.0616 Mouse 2 6.0 6.0 0.0459 0.0459 Mouse 3 6.5 6.5 0.0370 0.0370
Day 2 Mouse 1 7.5 7.5 0.0380 0.0380 Mouse 2 8.0 6.5 0.0334 0.0411 Mouse 3 5.5 5.5 0.0456 0.0456
Day 4 Mouse 1 5.5 5.5 0.0288 0.0288 Mouse 2 6.5 5.0 0.0154 0.0200 Mouse 3 6.0 6.0 0.0351 0.0351
Day 6 Mouse 1 6.5 6.5 0.0395 0.0395 Mouse 2 8.5 6.0 0.0230 0.0326 Mouse 3 7.5 6.0 0.0244 0.0305
Day 7 Mouse 1 7.5 6.0 0.0249 0.0311 Mouse 2 6.0 6.0 0.0134 0.0134 Mouse 3 8.0 6.0 0.0161 0.0215
Control Mouse 1 7.5 6.8 0.0242 0.0267 Mouse 2 8.5 6.5 0.0294 0.0384 Avg 0.0344 Std dev 0.0029
Jejunum Total length(cm) Length used(cm) IV (g) IV/ tissue (g) Day 1 Mouse 1 9.3 5.0 0.0141 0.0262 Mouse 2 15.0 8.5 0.0481 0.0849 Mouse 3 11.0 11.0 0.0360 0.0360
310 APPENDIX
Day 2 Mouse 1 13.0 6.0 0.0037 0.0080 Mouse 2 11.0 6.5 0.0438 0.0741 Mouse 3 12.0 7.0 0.0259 0.0444
Day 4 Mouse 1 6.5 6.0 0.0315 0.0341 Mouse 2 13.5 6.0 0.0338 0.0761 Mouse 3 10.3 6.0 0.0387 0.0664
Day 6 Mouse 1 11.5 6.0 0.0604 0.1158 Mouse 2 13.0 6.0 0.0678 0.1469 Mouse 3 12.0 6.5 0.0304 0.0561
Day 7 Mouse 1 9.0 6.0 0.0319 0.0479 Mouse 2 13.0 6.0 0.0191 0.0414 Mouse 3 10.0 6.0 0.0186 0.0310
Control Mouse 1 8.0 7.5 0.0117 0.0125 Mouse 2 13.0 6.0 0.0467 0.1012 Avg 0.0588 Std dev 0.0068
Ileum Total length(cm) Length used(cm) IV (g) IV/ tissue (g) Day 1 Mouse 1 10.5 8.5 0.0269 0.0332 Mouse 2 13.5 11.0 0.0065 0.0080 Mouse 3 10.0 10.0 0.0096 0.0096
Day 2 Mouse 1 8.5 6.0 0.0560 0.0793 Mouse 2 9.0 6.5 0.0097 0.0134 Mouse 3 6.0 6.0 0.0006 0.0006
Day 4 Mouse 1 8.0 6.0 0.0035 0.0047 Mouse 2 9.5 6.0 0.0014 0.0022 Mouse 3 13.5 6.0 0.0052 0.0117
311 APPENDIX
Day 6 Mouse 1 9.0 6.0 0.0170 0.0255 Mouse 2 13.0 6.5 0.0045 0.0090 Mouse 3 15.0 6.5 0.0100 0.0231
Day 7 Mouse 1 9.5 6.0 0.0009 0.0014 Mouse 2 15.0 6.0 0.0120 0.0300 Mouse 3 13.0 6.0 0.0059 0.0128
Control Mouse 1 8.5 5.5 0.0242 0.0374 Mouse 2 10.0 6.5 0.0294 0.0452 Avg 0.0216 Std dev 0.0080
Colon Total length(cm) Length used(cm) IV (g) IV/ tissue (g) Day 1 Mouse 1 6.5 5.0 0.0055 0.0072 Mouse 2 7.0 6.0 0.0048 0.0056 Mouse 3 5.6 5.6 0.0039 0.0039
Day 2 Mouse 1 6.0 6.0 0.0029 0.0029 Mouse 2 6.5 6.5 0.0048 0.0048 Mouse 3 6.0 6.0 0.0067 0.0067
Day 4 Mouse 1 6.0 6.0 0.0050 0.0050 Mouse 2 5.5 6.0 0.0061 0.0056 Mouse 3 6.8 6.0 0.0047 0.0053
Day 6 Mouse 1 6.0 6.0 0.0033 0.0033 Mouse 2 7.5 6.0 0.0026 0.0033 Mouse 3 7.0 6.0 0.0057 0.0067
Day 7 Mouse 1 6.5 6.0 0.0249 0.0270 Mouse 2 6.0 6.0 0.0134 0.0134 Mouse 3 6.5 6.0 0.0161 0.0174
312 APPENDIX
Control Mouse 1 7.5 6.0 0.0242 0.0303 Mouse 2 6.5 6.0 0.0294 0.0319 Avg 0.0106 Std dev 0.0099
(d) Average number of yeast cell adhering to human intestinal cells
Average no. of yeast attached per cell Caco-2 HT29 S.boulardii 4.10E+01 3.40E+01 S.cerevisiae 4.80E+01 4.30E+01 P.pastoris 7.14E+02 1.12E+03
(e) Median fluorescence intensities and the corresponding generation number of in vitro culture of cFDA-SE labeled LcS, S.boulardii, S.cerevisiae and P.pastoris
L.casei Shirota Hr Median fluorescence intensity (units/cell) Average Std dev 0 3.50E+02 3.30E+02 3.70E+02 3.50E+02 2.00E+01 2 2.60E+02 2.80E+02 2.90E+02 2.77E+02 1.53E+01 4 1.40E+02 1.60E+02 1.80E+02 1.60E+02 2.00E+01 6 6.80E+01 6.30E+01 7.10E+01 6.73E+01 4.04E+00
CFU/ml Average Generation No. 0 2.36E+05 2.29E+05 2.40E+05 2.35E+05 0.00 2 3.39E+05 3.27E+05 3.19E+05 3.28E+05 0.48 4 5.85E+05 6.06E+05 5.96E+05 5.96E+05 1.34 6 1.04E+06 9.60E+05 1.20E+06 1.07E+06 2.18
S.boulardii Median fluorescence intensity Hr (units/cell) Average Std dev 0 3.70E+02 4.00E+02 3.60E+02 3.77E+02 2.08E+01 2 2.00E+02 2.20E+02 2.30E+02 2.17E+02 1.53E+01 4 1.40E+02 1.60E+02 1.30E+02 1.43E+02 1.53E+01 6 8.70E+01 9.20E+01 8.60E+01 8.83E+01 3.21E+00
313 APPENDIX
CFU/ml Average Generation No. 0 3.24E+05 3.39E+05 3.48E+05 3.37E+05 0.00 2 5.92E+05 5.74E+05 6.02E+05 5.89E+05 0.81 4 1.05E+06 9.76E+05 9.98E+05 1.01E+06 1.58 6 1.36E+06 1.51E+06 1.39E+06 1.42E+06 2.08
S.cerevisiae Median fluorescence intensity Hr (units/cell) Average Std dev 0 3.70E+02 4.00E+02 3.80E+02 3.83E+02 1.53E+01 2 2.60E+02 2.20E+02 2.90E+02 2.57E+02 3.51E+01 4 1.40E+02 1.50E+02 1.70E+02 1.53E+02 1.53E+01 6 9.20E+01 9.60E+01 8.90E+01 9.70E+01 3.51E+00
CFU/ml Average Generation No. 0 3.24E+05 3.10E+05 3.19E+05 3.18E+05 0.00 2 5.57E+05 5.77E+05 5.63E+05 5.66E+05 0.83 4 9.07E+05 9.19E+05 9.03E+05 9.10E+05 1.52 6 1.27E+06 1.33E+06 1.36E+06 1.32E+06 2.05
P.pastoris Median fluorescence intensity Hr (units/cell) Average Std dev 0 4.00E+02 3.90E+02 3.80E+02 3.90E+02 1.00E+01 2 2.60E+02 2.50E+02 2.80E+02 2.63E+02 1.53E+01 4 1.30E+02 1.50E+02 1.00E+02 1.27E+02 2.52E+01 6 8.30E+01 9.60E+01 8.90E+01 8.93E+01 6.51E+00
CFU/ml Average Generation No. 0 2.89E+05 2.96E+05 3.01E+05 2.95E+05 0.00 2 4.87E+05 4.95E+05 4.73E+05 4.85E+05 0.72 4 9.29E+05 9.16E+05 9.03E+05 9.16E+05 1.63 6 1.24E+06 1.33E+06 1.26E+06 1.28E+06 2.11
314 APPENDIX
(f) Number of LcS, S.boulardii, S.cerevisiae and P.pastoris adhered to different segments of murine gastrointestinal tract
L.casei Shirota Day Duo 1 Duo 2 Duo 3 Avg Duo Std dev 1 4.98E+03 7.01E+03 6.12E+03 6.04E+03 1.02E+03 2 4.12E+03 3.71E+03 5.88E+03 4.57E+03 1.15E+03 4 3.06E+03 2.98E+03 4.71E+03 3.58E+03 9.77E+02
Jej 1 Jej 2 Jej 3 Avg Jej Std dev 1 1.52E+04 1.64E+04 2.01E+04 1.72E+04 2.59E+03 2 5.98E+03 7.09E+03 4.69E+03 5.92E+03 1.20E+03 4 6.13E+03 3.90E+03 5.63E+03 5.22E+03 1.17E+03
Ileum 1 Ileum 2 Ileum 3 Avg Ileum Std dev 1 5.71E+04 7.99E+04 6.97E+04 6.89E+04 1.14E+04 2 1.80E+04 1.68E+04 1.21E+04 1.56E+04 3.09E+03 4 1.75E+04 1.08E+04 1.55E+04 1.46E+04 3.42E+03
Colon 1 Colon 2 Colon 3 Avg Colon Std dev 1 3.58E+04 2.84E+04 2.58E+04 3.00E+04 5.17E+03 2 2.44E+04 1.54E+04 2.07E+04 2.02E+04 4.53E+03 4 1.34E+04 8.80E+03 1.68E+04 1.30E+04 4.01E+03
S.boulardii Day Duo 1 Duo 2 Duo 3 Avg Duo Std dev 1 6.27E+05 7.25E+05 6.54E+05 6.69E+05 5.06E+04 2 5.09E+05 4.61E+05 4.79E+05 4.83E+05 2.42E+04 3 3.48E+05 3.67E+05 2.99E+05 3.38E+05 3.51E+04 4 2.48E+05 3.61E+05 3.02E+05 3.04E+05 5.65E+04
Jej 1 Jej 2 Jej 3 Avg Jej Std dev 1 9.19E+04 1.09E+05 7.04E+04 9.05E+04 1.94E+04 2 8.59E+04 6.91E+04 7.97E+04 7.82E+04 8.50E+03 3 4.78E+04 7.31E+04 5.22E+04 5.77E+04 1.35E+04 4 4.44E+04 3.88E+04 5.32E+04 4.55E+04 7.26E+03
315 APPENDIX
Ileum 1 Ileum 2 Ileum 3 Avg Ileum Std dev 1 4.02E+04 2.76E+04 3.93E+04 3.57E+04 7.03E+03 2 3.82E+04 4.05E+04 4.31E+04 4.06E+04 2.45E+03 3 1.97E+04 3.18E+04 2.09E+04 2.41E+04 6.67E+03 4 1.89E+04 2.67E+04 2.71E+04 2.42E+04 4.62E+03
Colon 1 Colon 2 Colon 3 Avg Colon Std dev 1 2.07E+04 2.54E+04 3.14E+04 2.58E+04 5.36E+03 2 3.28E+04 4.37E+04 5.27E+04 4.31E+04 9.97E+03 3 3.31E+04 2.69E+04 1.59E+04 2.53E+04 8.71E+03 4 1.87E+04 3.18E+04 2.46E+04 2.50E+04 6.54E+03
S.cerevisiae Day Duo 1 Duo 2 Duo 3 Avg Duo Std dev 1 2.74E+06 1.93E+06 2.27E+06 2.31E+06 4.10E+05 2 6.55E+05 7.65E+05 9.13E+05 7.78E+05 1.29E+05 3 9.06E+05 7.08E+05 7.44E+05 7.86E+05 1.06E+05 4 5.42E+05 4.13E+05 3.59E+05 4.38E+05 9.42E+04
Jej 1 Jej 2 Jej 3 Avg Jej Std dev 1 5.55E+05 7.18E+05 1.19E+06 8.22E+05 3.32E+05 2 1.15E+06 9.96E+05 1.30E+06 1.15E+06 1.51E+05 3 9.83E+05 1.17E+06 8.84E+05 1.01E+06 1.45E+05 4 1.60E+06 1.10E+06 1.22E+06 1.31E+06 2.63E+05
Ileum 1 Ileum 2 Ileum 3 Avg Ileum Std dev 1 5.16E+05 4.07E+05 6.52E+05 5.25E+05 1.23E+05 2 4.91E+05 5.84E+05 7.21E+05 5.99E+05 1.15E+05 3 2.43E+05 2.82E+05 3.58E+05 2.94E+05 5.84E+04 4 1.94E+05 1.05E+05 1.63E+05 1.54E+05 4.51E+04
Colon 1 Colon 2 Colon 3 Avg Colon Std dev 1 7.94E+05 7.79E+05 6.79E+05 7.51E+05 6.23E+04 2 3.80E+05 3.85E+05 2.33E+05 3.33E+05 8.66E+04 3 2.31E+05 1.04E+05 1.40E+05 1.58E+05 6.55E+04 4 6.96E+04 6.05E+05 5.00E+04 2.42E+05 3.15E+05
316 APPENDIX
Pichia pastoris Day Duo 1 Duo 2 Duo 3 Avg Duo Std dev 1 1.04E+06 1.09E+06 9.92E+05 1.04E+06 5.12E+04 2 1.07E+06 9.89E+05 1.05E+06 1.04E+06 4.19E+04 3 5.15E+05 5.37E+05 4.59E+05 5.04E+05 4.02E+04 4 5.31E+05 4.75E+05 4.99E+05 5.02E+05 2.81E+04
Jej 1 Jej 2 Jej 3 Avg Jej Std dev 1 7.56E+05 6.86E+05 7.32E+05 7.25E+05 3.58E+04 2 6.51E+05 7.43E+05 7.03E+05 6.99E+05 4.61E+04 3 4.62E+05 4.13E+05 4.39E+05 4.38E+05 2.45E+04 4 3.91E+05 3.47E+05 1.10E+05 2.83E+05 1.51E+05
Ileum 1 Ileum 2 Ileum 3 Avg Ileum Std dev 1 6.45E+05 6.02E+05 6.76E+05 6.41E+05 3.72E+04 2 6.46E+05 5.91E+05 5.75E+05 6.04E+05 3.72E+04 3 7.15E+05 7.36E+05 7.51E+05 7.34E+05 1.81E+04 4 7.11E+05 6.57E+05 6.99E+05 6.89E+05 2.84E+04
Colon 1 Colon 2 Colon 3 Avg Colon Std dev 1 3.81E+05 4.92E+05 4.18E+05 4.30E+05 5.65E+04 2 7.52E+05 7.10E+05 7.33E+05 7.32E+05 2.10E+04 3 6.89E+05 5.40E+05 6.42E+05 6.24E+05 7.62E+04 4 6.49E+05 5.79E+05 5.99E+05 6.09E+05 3.61E+04
(g) Residual median fluorescence intensities of cFDA-SE labeled LcS, S.boulardii, S.cerevisiae and P.pastoris adhering to different segments of murine intestinal tract
L.casei Shirota Control = 2.10E+00 Day Duo 1 Duo 2 Duo 3 Avg Std dev 1 1.49E+01 1.21E+01 1.51E+01 1.40E+01 1.68E+00 2 1.20E+01 1.53E+01 9.80E+00 1.24E+01 2.77E+00 4 7.10E+00 6.50E+00 8.10E+00 7.23E+00 8.08E-01 6 5.20E+00 6.00E+00 7.20E+00 6.13E+00 1.01E+00
317 APPENDIX
Control = 1.80E+00 Jej 1 Jej 2 Jej 3 Avg Std dev 1 9.95E+00 7.80E+00 1.02E+01 9.33E+00 1.33E+00 2 6.30E+00 7.20E+00 8.20E+00 7.23E+00 9.50E-01 4 6.50E+00 7.30E+00 4.90E+00 6.23E+00 1.22E+00 6 3.50E+00 5.00E+00 4.20E+00 4.23E+00 7.51E-01
Control = 2.00E+00 Ileum 1 Ileum 2 leum 3 Avg Std dev 1 7.20E+00 5.90E+00 5.00E+00 6.03E+00 1.11E+00 2 5.80E+00 4.50E+00 3.70E+00 4.67E+00 1.06E+00 4 4.20E+00 3.50E+00 3.10E+00 3.60E+00 5.57E-01
Control = 2.30E+00 Colon 1 Colon 2 Colon 3 Avg Std dev 1 1.98E+01 2.50E+01 2.21E+01 2.23E+01 2.61E+00 2 1.47E+01 1.58E+01 1.20E+01 1.42E+01 1.96E+00 4 1.51E+01 1.47E+01 1.53E+01 1.50E+01 3.06E-01 6 1.27E+01 1.46E+01 8.70E+00 1.20E+01 3.01E+00
S.boulardii Control = 2.26E+00 Day Duo 1 Duo 2 Duo 3 Avg Std dev 1 1.29E+01 1.17E+01 1.19E+01 1.22E+01 6.54E-01 2 9.61E+00 7.72E+00 7.56E+00 8.30E+00 1.14E+00 4 6.16E+00 4.87E+00 5.03E+00 5.35E+00 7.03E-01 6 3.65E+00 2.06E+00 1.98E+00 2.56E+00 9.42E-01
Control = 2.16E+00 Jej 1 Jej 2 Jej 3 Avg Std dev 1 5.76E+00 4.63E+00 3.78E+00 4.72E+00 9.93E-01 2 3.63E+00 4.76E+00 2.43E+00 3.61E+00 1.17E+00 4 3.50E+00 3.31E+00 2.51E+00 3.11E+00 5.25E-01 6 1.51E+00 2.83E+00 2.05E+00 2.13E+00 6.64E-01
318 APPENDIX
Control = 1.85E+00 Ileum 1 Ileum 2 leum 3 Avg Std dev 1 2.38E+00 2.31E+00 3.12E+00 2.60E+00 4.49E-01 2 2.27E+00 2.20E+00 3.11E+00 2.53E+00 5.06E-01 4 2.11E+00 1.80E+00 2.82E+00 2.24E+00 5.23E-01 6 3.12E+00 2.34E+00 1.86E+00 2.44E+00 6.36E-01
Control = 1.95E+00 Colon 1 Colon 2 Colon 3 Avg Std dev 1 2.96E+00 2.14E+00 1.52E+00 2.21E+00 7.22E-01 2 2.22E+00 2.12E+00 1.87E+00 2.07E+00 1.80E-01 4 2.90E+00 1.66E+00 2.01E+00 2.19E+00 6.39E-01 5 1.87E+00 3.22E+00 9.10E-01 2.00E+00 1.16E+00
S.cerevisiae Control = 9.54E+00 Day Duo 1 Duo 2 Duo 3 Avg Duo Std dev 1 3.43E+01 2.93E+01 3.17E+01 3.17E+01 2.49E+00 2 1.63E+01 1.46E+01 1.36E+01 1.48E+01 1.33E+00 4 8.32E+00 1.04E+01 1.09E+01 9.88E+00 1.38E+00 6 8.57E+00 8.48E+00 1.04E+01 9.15E+00 1.08E+00
Control = 3.43E+00 Jej 1 Jej 2 Jej 3 Avg Jej Std dev 1 1.94E+01 1.90E+01 1.76E+01 1.87E+01 9.66E-01 2 1.68E+01 1.22E+01 1.37E+01 1.42E+01 2.36E+00 4 1.39E+01 1.63E+01 1.24E+01 1.42E+01 1.97E+00 6 1.32E+01 1.67E+01 1.48E+01 1.49E+01 1.75E+00
Control = 1.34E+00 Ileum 1 Ileum 2 Ileum 3 Avg Ileum Std dev 1 4.22E+00 5.20E+00 2.75E+00 4.06E+00 1.23E+00 2 2.60E-01 9.10E-01 1.84E+00 1.00E+00 7.94E-01 4 -7.70E-01 -1.50E-01 6.10E-01 -1.03E-01 6.91E-01 6 5.80E-01 -4.20E-01 -4.00E-02 4.00E-02 5.05E-01
Control = 1.41E+00 Colon 1 Colon 2 Colon 3 Avg Colon Std dev
319 APPENDIX
1 6.65E+00 6.14E+00 5.37E+00 6.05E+00 6.44E-01 2 -1.20E-01 4.60E-01 4.80E-01 2.73E-01 3.41E-01 4 -3.00E-02 1.50E-01 1.20E+00 4.40E-01 6.64E-01 6 1.28E+00 8.00E-02 2.10E-01 5.23E-01 6.59E-01
P.pastoris Control = 2.79E+00 Day Duo 1 Duo 2 Duo 3 Avg Std dev 1 1.60E+01 1.79E+01 1.94E+01 1.77E+01 1.72E+00 2 1.36E+01 1.62E+01 1.40E+01 1.46E+01 1.40E+00 4 6.37E+00 7.48E+00 6.00E+00 6.62E+00 7.70E-01 6 5.03E+00 3.37E+00 5.44E+00 4.61E+00 1.10E+00
Control = 2.98E+00 Jej 1 Jej 2 Jej 3 Avg Std dev 1 1.33E+01 1.26E+01 1.22E+01 1.27E+01 5.67E-01 2 1.12E+01 9.63E+00 1.05E+01 1.04E+01 7.95E-01 4 7.38E+00 6.70E+00 5.38E+00 6.49E+00 1.02E+00 6 3.94E+00 4.28E+00 5.01E+00 4.41E+00 5.47E-01
Control = 2.88E+00 Ileum 1 Ileum 2 leum 3 Avg Std dev 1 1.28E+01 1.09E+01 1.15E+01 1.17E+01 9.51E-01 2 1.04E+01 9.68E+00 9.16E+00 9.75E+00 6.23E-01 4 1.54E+01 1.47E+01 1.41E+01 1.47E+01 6.77E-01 6 16.68- 1.29E+01 1.12E+01 1.21E+01 1.17E+00
Control = 2.62E+00 Colon 1 Colon 2 Colon 3 Avg Std dev 1 5.59E+00 7.94E+00 7.36E+00 6.96E+00 1.22E+00 2 4.67E+00 3.94E+00 3.06E+00 3.89E+00 8.06E-01 4 4.24E+00 3.30E+00 3.55E+00 3.70E+00 4.87E-01 6 3.65E+00 3.20E+00 2.99E+00 3.28E+00 3.37E-01
320 APPENDIX
(h) Half times for wash-out of LcS, S.boulardii, S.cerevisiae and P.pastoris from different segments of murine intestinal tract
In2/Tw = (In F2 - In F1)/( t2 - t1) Duodenum Jejunum Ileum Colon L.casei Shirota 1.90 3.70 3.50 3.80 S.boulardii 2.64 3.02 5.35 66.02 S.cerevisiae 1.25 -4.46 1.70 1.84 P.pastoris 2.85 2.21 -28.80 -5.97
(i) Fluorescence intensity profiles of cFDA-SE labeled LcS, S.boulardii, S.cerevisiae and P.pastoris in various segments of murine intestinal tract
L.casei Shirota Section and Day <1/4 median 1/4-1/2 median Std Std Duodenum Average Dev Average Dev 1 33.08 36.30 40.10 36.49 3.51 9.21 9.94 11.23 10.13 1.02 2 32.84 36.01 30.28 33.04 2.87 14.95 16.74 13.78 15.16 1.48 4 40.45 37.76 35.07 37.76 2.69 16.98 20.50 18.44 18.64 1.78 6 49.12 53.71 45.78 49.54 3.98 17.11 18.31 19.51 18.31 1.20
1/2-median >Median Std Std Duodenum Average Dev Average Dev 1 15.58 19.53 17.26 17.46 1.98 35.72 38.48 33.55 35.92 2.47 2 21.11 19.59 18.69 19.80 1.21 34.50 31.80 29.70 32.00 2.41 4 22.15 18.08 19.82 20.02 2.04 23.38 26.51 20.84 23.58 2.84 6 15.01 16.19 17.37 16.19 1.18 15.75 17.13 14.99 15.96 1.07
<1/4 median 1/4-1/2 median Std Std Jejunum Average Dev Average Dev 1 24.04 26.10 22.54 24.23 1.79 12.44 13.58 11.90 12.64 0.84 2 30.70 26.57 28.35 28.54 2.07 16.32 15.43 17.83 16.53 1.20 4 26.19 30.36 27.90 28.15 2.10 17.86 19.90 16.42 18.06 1.74 6 28.14 31.21 25.07 28.14 3.07 21.76 27.47 24.31 24.51 2.86
1/2-median >Median Std Std Jejunum Average Dev Average Dev 1 18.75 20.55 17.54 18.95 1.51 48.53 43.98 40.03 44.18 4.25
321 APPENDIX
2 20.77 19.08 23.05 20.97 1.99 33.76 36.95 31.17 33.96 2.90 4 23.90 19.71 21.51 21.71 2.10 31.88 36.02 28.34 32.08 3.84 6 20.40 18.19 23.20 20.60 2.51 26.55 29.60 24.09 26.75 2.76
<1/4 median 1/4-1/2 median Std Std Ileum Average Dev Average Dev 1 27.89 29.28 32.98 30.05 2.87 14.53 16.01 13.66 14.73 1.19 2 40.16 47.18 43.37 43.57 3.51 27.65 24.65 22.24 24.85 2.71 4 47.75 44.07 51.79 47.87 3.82 22.14 26.41 23.98 24.18 2.14 6 47.70 51.40 55.72 51.61 4.01 24.87 22.70 21.12 22.90 1.88
1/2-median >Median Std Std Ileum Average Dev Average Dev 1 15.58 17.59 14.16 15.78 1.72 39.23 42.73 36.33 39.43 3.20 2 17.45 15.73 14.60 15.93 1.43 14.47 15.39 17.08 15.65 1.33 4 12.57 13.51 11.63 12.57 0.94 16.52 15.38 14.24 15.38 1.14 6 11.69 10.63 10.17 10.83 0.76 14.66 15.87 13.45 14.66 1.21
<1/4 median 1/4-1/2 median Std Std Colon Average Dev Average Dev 1 43.94 37.66 40.90 40.83 3.14 14.18 13.35 15.60 14.38 1.13 2 37.58 35.41 40.23 37.74 2.41 26.45 24.13 22.40 24.33 2.03 4 60.15 64.80 70.05 65.00 4.95 13.12 15.60 14.36 14.36 1.24 6 61.94 65.69 58.79 62.14 3.45 16.37 15.15 18.18 16.57 1.52
1/2-median >Median Std Std Colon Average Dev Average Dev 1 19.71 22.58 20.85 21.05 1.45 22.26 25.68 23.67 23.87 1.72 2 24.79 22.93 27.27 25.00 2.17 13.75 12.73 12.31 12.93 0.72 4 7.66 7.02 6.68 7.12 0.49 13.32 14.61 12.64 13.52 1.00 6 7.76 8.75 7.07 7.86 0.84 14.69 13.23 12.37 13.43 1.17
S.boulardii Section and Day <1/4 median 1/4-1/2 median Std Std Duodenum Average Dev Average Dev 1 33.67 29.73 36.05 33.15 3.19 10.54 11.63 12.56 11.58 1.01 2 31.70 39.19 35.57 35.49 3.75 12.19 13.51 14.32 13.34 1.08 4 43.59 36.34 40.17 40.03 3.63 16.09 15.64 17.83 16.52 1.16 6 49.82 53.64 44.97 49.48 4.35 17.44 15.72 18.67 17.28 1.48
322 APPENDIX
1/2-median >Median Std Std Duodenum Average Dev Average Dev 1 14.63 18.37 16.82 16.61 1.88 35.87 41.65 38.46 38.66 2.90 2 19.33 20.53 18.27 19.38 1.13 28.69 35.42 31.25 31.79 3.40 4 22.43 20.55 19.64 20.87 1.42 22.72 20.35 24.68 22.58 2.17 6 17.85 19.68 21.04 19.52 1.60 11.99 15.27 13.89 13.72 1.65
<1/4 median 1/4-1/2 median Std Std Jejunum Average Dev Average Dev 1 27.18 22.37 25.06 24.87 2.41 13.65 12.13 15.62 13.80 1.75 2 27.22 26.03 28.41 27.22 1.19 12.97 16.65 14.79 14.80 1.84 4 24.49 28.88 30.35 27.91 3.05 14.97 17.32 19.81 17.37 2.42 6 41.07 36.65 38.83 38.85 2.21 17.99 20.21 19.73 19.31 1.17
1/2-median >Median Std Std Jejunum Average Dev Average Dev 1 15.48 17.85 18.99 17.44 1.79 40.26 43.67 47.73 43.89 3.74 2 19.45 17.68 21.22 19.45 1.77 41.89 38.45 35.26 38.53 3.32 4 21.04 22.68 24.72 22.81 1.84 28.56 35.44 31.73 31.91 3.44 6 20.38 18.62 16.24 18.41 2.08 23.14 26.13 21.03 23.43 2.56
<1/4 median 1/4-1/2 median Std Std Ileum Average Dev Average Dev 1 10.11 9.12 8.14 9.12 0.99 11.91 12.79 10.76 11.82 1.02 2 10.74 8.62 10.13 9.83 1.09 11.11 14.92 12.83 12.95 1.91 4 12.61 11.42 13.61 12.55 1.10 12.66 13.18 15.02 13.62 1.24 6 16.82 16.43 18.04 17.10 0.84 10.73 12.13 13.11 11.99 1.20
1/2-median >Median Std Std Ileum Average Dev Average Dev 1 12.44 8.96 10.93 10.78 1.75 73.02 63.70 68.11 68.28 4.66 2 10.39 11.15 13.07 11.54 1.38 64.25 69.04 63.75 65.68 2.92 4 10.23 12.12 9.88 10.74 1.21 65.87 64.28 59.12 63.09 3.53 6 7.22 8.51 9.60 8.44 1.19 59.66 65.54 62.22 62.47 2.95
323 APPENDIX
<1/4 median 1/4-1/2 median Std Std Colon Average Dev Average Dev 1 8.29 6.97 9.43 8.23 1.23 8.96 10.23 11.05 10.08 1.05 2 10.96 13.33 12.28 12.19 1.19 10.33 7.89 9.55 9.26 1.25 4 9.88 11.57 12.27 11.24 1.23 9.87 10.88 8.19 9.65 1.36 6 12.66 16.02 14.32 14.33 1.68 9.62 6.89 8.33 8.28 1.37
1/2-median >Median Std Std Colon Average Dev Average Dev 1 10.88 9.39 8.32 9.53 1.29 76.88 72.16 67.43 72.16 4.73 2 8.21 6.89 9.22 8.11 1.17 69.98 66.53 74.82 70.44 4.16 4 6.44 8.41 9.18 8.01 1.41 71.12 75.05 67.14 71.10 3.96 6 7.26 6.78 9.05 7.70 1.20 74.68 69.96 64.43 69.69 5.13
S.cerevisiae Section and Day <1/4 median 1/4-1/2 median Std Std Duodenum Average Dev Average Dev 1 21.52 25.12 28.41 25.02 3.45 12.71 14.12 15.61 14.15 1.45 2 25.98 30.89 28.99 28.62 2.48 17.43 16.41 15.22 16.35 1.11 4 35.17 30.99 38.38 34.85 3.71 18.39 16.44 20.39 18.41 1.98 6 39.64 40.64 44.42 41.57 2.52 19.63 21.76 17.74 19.71 2.01
1/2-median >Median Std Std Duodenum Average Dev Average Dev 1 16.15 21.51 18.37 18.68 2.69 42.12 46.02 38.31 42.15 3.86 2 19.87 20.88 18.48 19.74 1.21 32.19 38.28 35.40 35.29 3.05 4 23.63 20.74 17.63 20.67 3.00 25.96 23.44 28.81 26.07 2.69 6 18.81 20.61 23.45 20.96 2.34 20.82 17.24 18.23 18.76 1.85
<1/4 median 1/4-1/2 median Std Std Jejunum Average Dev Average Dev 1 9.84 11.64 12.47 11.32 1.34 11.93 10.84 12.44 11.74 0.82 2 9.17 11.42 10.61 10.40 1.14 11.99 9.42 10.84 10.75 1.29 4 9.44 10.66 11.37 10.49 0.98 13.37 11.13 10.54 11.68 1.49
324 APPENDIX
6 10.03 12.42 12.32 11.59 1.35 11.86 8.84 12.86 11.19 2.09
1/2-median >Median Std Std Jejunum Average Dev Average Dev 1 9.41 13.22 11.35 11.33 1.91 64.63 63.44 68.76 65.61 2.79 2 10.32 13.65 12.27 12.08 1.67 63.61 67.22 69.49 66.77 2.97 4 15.32 14.71 16.22 15.42 0.76 63.44 61.68 66.73 63.95 2.56 6 17.28 16.07 14.66 16.00 1.31 63.83 61.63 58.19 61.22 2.84
<1/4 median 1/4-1/2 median Std Std Ileum Average Dev Average Dev 1 26.46 32.41 28.11 28.99 3.07 20.44 16.20 18.45 18.36 2.12 2 48.43 43.11 39.32 43.62 4.58 16.92 13.49 18.84 16.42 2.71 4 52.79 60.28 57.46 56.84 3.78 13.96 16.62 17.84 16.14 1.98 6 59.22 62.32 68.12 63.22 4.52 15.83 16.89 13.96 15.56 1.48
1/2-median >Median Std Std Ileum Average Dev Average Dev 1 21.15 19.22 18.06 19.48 1.56 33.23 30.52 35.77 33.17 2.63 2 20.73 16.21 18.02 18.32 2.27 21.49 18.56 24.88 21.64 3.16 4 15.07 12.95 16.86 14.96 1.96 12.26 10.31 13.62 12.06 1.66 6 12.33 15.08 13.12 13.51 1.42 9.03 8.61 6.27 7.97 1.49
<1/4 median 1/4-1/2 median Std Std Colon Average Dev Average Dev 1 27.42 30.51 33.43 30.45 3.01 16.11 15.27 18.04 16.47 1.42 2 36.22 39.43 32.44 36.03 3.50 19.89 16.62 18.19 18.23 1.64 4 44.38 48.34 52.66 48.46 4.14 15.69 17.64 18.98 17.44 1.65 6 56.67 60.69 52.26 56.54 4.22 18.13 14.34 16.72 16.40 1.92
1/2-median >Median Std Std Colon Average Dev Average Dev 1 17.81 15.40 19.21 17.47 1.93 32.32 39.24 35.26 35.61 3.47
325 APPENDIX
2 20.54 18.74 17.22 18.83 1.66 26.74 29.99 24.19 26.97 2.91 4 15.27 19.22 17.61 17.37 1.99 18.14 16.83 15.22 16.73 1.46 6 14.71 17.33 16.46 16.17 1.33 10.87 12.33 9.46 10.89 1.44
P.pastoris Section and Day <1/4 median 1/4-1/2 median Std Std Duodenum Average Dev Average Dev 1 26.53 29.67 23.87 26.69 2.90 15.76 14.12 16.46 15.45 1.20 2 24.31 27.21 29.61 27.04 2.65 15.22 17.26 19.06 17.18 1.92 4 34.56 42.27 38.58 38.47 3.86 17.61 21.49 19.43 19.51 1.94 6 42.81 46.84 51.01 46.89 4.10 18.42 19.63 17.04 18.36 1.30
1/2-median >Median Std Std Duodenum Average Dev Average Dev 1 18.71 17.62 15.75 17.36 1.50 36.81 44.27 40.43 40.50 3.73 2 19.23 21.47 17.86 19.52 1.82 38.67 36.38 33.74 36.26 2.47 4 15.47 17.37 19.22 17.35 1.88 24.75 27.14 22.13 24.67 2.51 6 16.74 18.85 13.96 16.52 2.45 16.49 18.21 19.98 18.23 1.75
<1/4 median 1/4-1/2 median Std Std Jejunum Average Dev Average Dev 1 21.30 23.48 24.49 23.09 1.63 13.63 15.11 11.88 13.54 1.62 2 26.81 33.34 29.76 29.97 3.27 16.38 13.68 15.57 15.21 1.39 4 37.18 38.11 41.85 39.05 2.47 15.28 18.91 16.38 16.86 1.86 6 43.68 50.08 46.49 46.75 3.21 16.22 14.37 13.17 14.59 1.54
1/2-median >Median Std Std Jejunum Average Dev Average Dev 1 20.12 18.64 17.11 18.62 1.51 44.56 48.61 41.08 44.75 3.77 2 20.15 21.17 18.61 19.98 1.29 34.95 36.85 32.71 34.84 2.07 4 22.67 18.94 20.67 20.76 1.87 25.32 23.92 20.74 23.33 2.35 6 18.92 16.77 19.64 18.44 1.49 18.16 20.24 22.27 20.22 2.06
326 APPENDIX
<1/4 median 1/4-1/2 median Std Std Ileum Average Dev Average Dev 1 18.84 20.47 21.71 20.34 1.44 11.86 14.67 12.87 13.13 1.42 2 18.12 18.91 23.10 20.04 2.68 14.30 16.11 17.83 16.08 1.77 4 18.53 22.01 22.82 21.12 2.28 13.12 16.39 14.22 14.58 1.66 6 26.14 21.14 24.81 24.03 2.59 11.70 16.29 14.02 14.00 2.30
1/2-median >Median Std Std Ileum Average Dev Average Dev 1 15.35 16.22 14.78 15.45 0.73 54.67 47.36 51.22 51.08 3.66 2 16.91 18.00 20.17 18.36 1.66 41.78 46.22 48.57 45.52 3.45 4 14.10 12.83 10.94 12.62 1.59 46.92 55.44 52.68 51.68 4.35 6 15.39 13.66 11.47 13.51 1.96 52.29 44.71 48.37 48.46 3.79
<1/4 median 1/4-1/2 median Std Std Colon Average Dev Average Dev 1 22.26 25.48 27.91 25.22 2.83 10.32 14.52 12.36 12.40 2.10 2 25.57 29.33 24.27 26.39 2.63 14.36 15.18 17.42 15.65 1.58 4 28.21 34.98 31.37 31.52 3.39 14.89 17.96 16.97 16.61 1.57 6 38.56 41.54 36.62 38.91 2.48 19.86 17.55 15.63 17.68 2.12
1/2-median >Median Std Std Colon Average Dev Average Dev 1 12.35 15.74 13.68 13.92 1.71 53.24 47.22 44.92 48.46 4.30 2 14.28 18.29 16.62 16.40 2.01 41.65 44.38 38.64 41.56 2.87 4 18.21 16.27 20.09 18.19 1.91 30.98 33.53 36.54 33.68 2.78 6 19.14 17.22 22.07 19.48 2.44 21.98 25.96 23.84 23.93 1.99
327 APPENDIX
(j) Doubling times of LcS, S.boulardii, S.cerevisiae and P.pastoris in different segments of murine intestinal tract
In2/Td = (In X2 - In X1)/( t2 - t1)
Duodenum Jejunum Ileum Colon L.casei Shirota 4.10 4.80 4.60 5.60 S.boulardii 1.33 2.61 10.91 20.83 S.cerevisiae 1.67 9.10 0.45 0.85 P.pastoris 1.54 1.97 -77.32 2.76
328 APPENDIX
APPENDIX 9
DATA FOR CHAPTER 4
(a) Concentrations of rTGEV-S specific IgA in the intestinal lavages of mice orally immunized with LcS-rTGEV-S
Intestinal IgA (ng/ml) Pre-immune Average SD Day 18 Average SD serum PBS 0.20 0.24 0.05 0.16 0.22 0.06 0.24 0.23 0.29 0.28 LcS-rTGEV-S 0.17 0.23 0.06 1.52 1.29 0.24 0.24 1.29 0.28 1.05 LcS-pLP500 0.31 0.26 0.06 0.37 0.32 0.04 0.20 0.29 0.27 0.31
PBS Day 32 Average SD Day 48 Average SD 0.16 0.18 0.03 0.35 0.27 0.08 0.17 0.26 LcS-rTGEV-S 0.21 0.19 4.15 3.98 0.18 3.81 3.63 0.19 3.98 3.65 LcS-pLP500 3.80 3.44 0.21 0.24 0.04 0.29 0.24 0.06 0.28 0.18 0.24 0.25
329 APPENDIX
(b) Concentrations of rTGEV-S specific IgG in the sera of mice orally immunized with LcS-rTGEV-S
Serum IgG (ng/ml)
Pre-immune Average SD Day 18 Average SD serum PBS 0.23 0.28 0.06 0.37 0.31 0.06
0.27 0.30
0.34 0.25
LcS-rTGEV-S 0.24 0.20 0.04 5.71 6.03 0.32
0.19 6.04
0.17 6.34
LcS-pLP500 0.2 0.25 0.05 0.32 0.26 0.06
0.29 0.21
0.26 0.25
Day 32 Average SD Day 48 Average SD
PBS 0.18 0.22 0.04 0.34 0.28 0.06
0.21 0.27
0.26 0.23
LcS-rTGEV-S 8.56 8.38 0.18 8.29 8.05 0.25
8.37 8.06
8.21 7.80
LcS-pLP500 0.25 0.33 0.08 0.14 0.22 0.08
0.41 0.29
0.34 0.24
330 APPENDIX
(c) Concentrations of rTGEV-S specific IgG isotypes in the sera of mice orally immunized with LcS-rTGEV-S
Serum IgG Isotype IgG1 (ng/ml) IgG2a (ng/ml) Day 32 Average SD Day 32 Average SD PBS 0.14 0.14 0.04 0.13 0.15 0.03 0.18 0.18 0.11 0.14 LcS-rTGEV-S 2.24 1.98 0.22 4.77 4.49 0.28 1.83 4.48 1.88 4.22 LcS-pLP500 0.15 0.17 0.02 0.16 0.16 0.03 0.17 0.19 0.19 0.14
(d) Concentrations of PrTGEV-S specific IgA in the intestinal lavages of mice orally immunized with PP/PrTGEV-S
Intestinal IgA (ng/ml) Pre-immune Average SD Day 18 Average SD serum PBS 0.49 0.50 0.09 0.54 0.47 0.08 0.42 0.39 0.59 0.47 PP/PrTGEV-S 0.43 0.52 0.08 4.67 4.51 0.17 0.58 4.33 0.55 4.54 PP/pGAPZ α C 0.61 0.55 0.06 0.51 0.52 0.08 0.49 0.45 0.56 0.60
Day 32 Average SD Day 48 Average SD
331 APPENDIX
PBS 0.61 0.56 0.04 0.52 0.48 0.08 0.53 0.39 0.54 0.54 PP/PrTGEV-S 4.47 4.55 0.12 4.31 4.48 0.15 4.69 4.54 4.49 4.58 PP/pGAPZ α C 0.43 0.48 0.07 0.52 0.53 0.05 0.44 0.49 0.56 0.58
(e) Concentrations of PrTGEV-S specific IgG in the sera of mice orally immunized with PP/PrTGEV-S
Intestinal IgG (ng/ml) Pre-immune Average SD Day 18 Average SD serum PBS 0.53 0.58 0.06 0.62 0.61 0.06 0.65 0.55 0.56 0.67 PP/PrTGEV-S 0.46 0.57 0.10 8.95 9.41 0.44 0.59 9.46 0.66 9.82 PP/pGAPZ α C 0.52 0.50 0.08 0.57 0.51 0.06 0.57 0.46 0.41 0.51
Day 32 Average SD Day 48 Average SD PBS 0.59 0.58 0.09 0.64 0.54 0.09 0.48 0.51 0.66 0.48 PP/PrTGEV-S 10.07 9.99 0.49 9.53 9.51 0.32 10.43 9.18
332 APPENDIX
9.46 9.82 PP/pGAPZ α C 0.49 0.56 0.07 0.55 0.53 0.08 0.63 0.45 0.55 0.60
(f) Concentrations of PrTGEV-S specific IgG isotypes in the sera of mice orally immunized with PP/PrTGEV-S
Serum IgG Isotype IgG1 (ng/ml) IgG2a (ng/ml) Day 32 Average SD Day 32 Average SD PBS 0.34 0.29 0.05 0.22 0.27 0.04 0.28 0.28 0.24 0.30 PP/PrTGEV-S 2.95 2.81 0.18 5.81 5.46 0.31 2.61 5.32 2.88 5.24 PP/pGAPZ α C 0.29 0.26 0.04 0.31 0.27 0.05 0.28 0.22 0.21 0.29
(g) Inhibition of TGEV plaque formation by intestinal lavages from mice orally immunized with LcS/rTGEV-S
Number of plaques formed % Inhibition with respect to negative ctrl (PBS)
Sample LcS- LCS PBS Avg PBSPBS-LCS- Avg Std PBS- Avg Std diln rTGEV-S rTGEV-S Dev LCS dev 2 2.83E+04 3.21E+04 3.24E+04 3.27E+04 13.46% 13.35% 0.77% 1.83% 1.12% 0.77%
2.81E+04 3.23E+04 3.29E+04 3.27E+04 14.07% 1.22%
2.86E+04 3.26E+04 3.28E+04 3.27E+04 12.54% 0.31%
4 2.84E+04 3.24E+04 3.21E+04 3.25E+04 12.62% 12.41% 0.64% 0.31% 0.31% 0.92%
2.87E+04 3.21E+04 3.28E+04 3.25E+04 11.69% 1.23%
2.83E+04 3.27E+04 3.26E+04 3.25E+04 12.92% -0.62%
8 2.88E+04 3.25E+04 3.32E+04 3.27E+04 11.93% 12.33% 0.47% 0.58% 0.19% 0.45%
333 APPENDIX
2.85E+04 3.28E+04 3.27E+04 3.27E+04 12.84% -0.31%
2.87E+04 3.26E+04 3.22E+04 3.27E+04 12.23% 0.31%
16 2.88E+04 3.23E+04 3.21E+04 3.24E+04 11.11% 10.49% 0.82% 0.28% 0.40% 0.48%
2.93E+04 3.24E+04 3.25E+04 3.24E+04 9.57% 0.00%
2.89E+04 3.21E+04 3.26E+04 3.24E+04 10.80% 0.93%
32 3.07E+04 3.26E+04 3.24E+04 3.22E+04 4.56% 5.60% 1.09% -1.35% -0.83% 0.47%
3.00E+04 3.24E+04 3.21E+04 3.22E+04 6.74% -0.73%
3.04E+04 3.23E+04 3.20E+04 3.22E+04 5.49% -0.41%
64 3.17E+04 3.26E+04 3.27E+04 3.25E+04 2.56% 2.46% 0.77% -0.20% 0.10% 0.81%
3.15E+04 3.22E+04 3.26E+04 3.25E+04 3.18% 1.02%
3.20E+04 3.27E+04 3.23E+04 3.25E+04 1.64% -0.51%
128 3.25E+04 3.28E+04 3.21E+04 3.24E+04 -0.41% 0.00% 0.47% -1.34% -0.51% 0.78%
3.22E+04 3.25E+04 3.26E+04 3.24E+04 0.51% -0.41%
3.24E+04 3.23E+04 3.24E+04 3.24E+04 -0.10% 0.21%
256 3.29E+04 3.25E+04 3.24E+04 3.27E+04 -0.61% -0.31% 0.53% 0.61% 0.71% 0.47%
3.26E+04 3.23E+04 3.30E+04 3.27E+04 0.31% 1.22%
3.29E+04 3.26E+04 3.27E+04 3.27E+04 -0.61% 0.31%
(h) Inhibition of TGEV plaque formation by sera from mice orally immunized with LcS/rTGEV-S
Number of plaques formed % Inhibition with respect to negative ctrl (PBS)
Sample LCS- LCS PBS Avg PBS PBS- Avg Std PBS- Avg Std diln rTGEV-S LCS/rTGEV-S Dev LCS dev 2 2.73E+04 3.23E+04 3.24E+04 3.27E+04 16.51% 15.70% 0.77% 1.22% 0.51% 0.98%
2.78E+04 3.24E+04 3.29E+04 3.27E+04 14.98% 0.92%
2.76E+04 3.29E+04 3.28E+04 3.27E+04 15.60% -0.61%
4 2.79E+04 3.26E+04 3.21E+04 3.25E+04 14.15% 13.74% 0.47% -0.31% 0.31% 0.62%
2.82E+04 3.22E+04 3.28E+04 3.25E+04 13.23% 0.92%
2.80E+04 3.24E+04 3.26E+04 3.25E+04 13.85% 0.31%
334 APPENDIX
8 2.89E+04 3.23E+04 3.32E+04 3.27E+04 11.62% 11.42% 0.64% 1.19% 0.40% 0.75%
2.92E+04 3.28E+04 3.27E+04 3.27E+04 10.70% -0.31%
2.88E+04 3.26E+04 3.22E+04 3.27E+04 11.93% 0.31%
16 2.98E+04 3.22E+04 3.21E+04 3.24E+04 8.02% 7.20% 0.78% 0.59% -0.42% 0.93%
3.01E+04 3.26E+04 3.25E+04 3.24E+04 7.10% -0.62%
3.03E+04 3.28E+04 3.26E+04 3.24E+04 6.48% -1.23%
32 3.17E+04 3.23E+04 3.24E+04 3.22E+04 1.45% 1.66% 0.65% -0.41% -0.21% 0.65%
3.14E+04 3.24E+04 3.21E+04 3.22E+04 2.38% -0.73%
3.18E+04 3.20E+04 3.20E+04 3.22E+04 1.14% 0.52%
64 3.27E+04 3.23E+04 3.27E+04 3.25E+04 -0.51% 0.31% 0.77% 0.72% 0.82% 1.08%
3.24E+04 3.19E+04 3.26E+04 3.25E+04 0.41% 1.95%
3.22E+04 3.26E+04 3.23E+04 3.25E+04 1.02% -0.20%
128 3.27E+04 3.22E+04 3.21E+04 3.24E+04 -1.03% -0.41% 0.62% 0.51% 0.10% 0.71%
3.23E+04 3.26E+04 3.26E+04 3.24E+04 0.21% -0.72%
3.25E+04 3.22E+04 3.24E+04 3.24E+04 -0.41% 0.51%
256 3.31E+04 3.27E+04 3.24E+04 3.27E+04 -1.22% -0.61% 0.61% 0.00% 0.41% 0.47%
3.27E+04 3.24E+04 3.30E+04 3.27E+04 0.00% 0.92%
3.29E+04 3.26E+04 3.27E+04 3.27E+04 -0.61% 0.31%
(i) Inhibition of TGEV plaque formation by intestinal lavages from mice orally immunized with PP/PrTGEV-S
Number of plaques formed % Inhibition with respect to negative ctrl (PBS)
Sample PP/ PP PBS Avg PBSPBS- Avg Std PBS- Avg Std diln TGEV-S PP/rTGEV-S Dev PP dev 2 2.02E+03 3.17E+04 3.30E+04 3.30E+04 93.88% 96.08% 2.13% 3.94% 3.23% 0.63% 1.24E+03 3.21E+04 3.32E+04 96.24% 2.73% 6.20E+02 3.20E+04 3.28E+04 98.12% 3.03%
4 1.58E+03 3.23E+04 3.31E+04 3.31E+04 95.22% 95.18% 1.51% 2.32% 2.42% 0.46% 1.10E+03 3.21E+04 3.32E+04 96.67% 2.92%
335 APPENDIX
2.10E+03 3.24E+04 3.29E+04 93.65% 2.02%
8 4.12E+03 3.23E+04 3.30E+04 3.27E+04 87.42% 83.66% 3.25% 1.19% 0.70% 0.62% 5.90E+03 3.27E+04 3.25E+04 81.96% 0.00%
6.01E+03 3.24E+04 3.26E+04 81.62% 0.92%
16 7.81E+03 3.26E+04 3.38E+04 3.29E+04 76.29% 72.23% 3.98% 0.98% 1.61% 1.61%
9.20E+03 3.18E+04 3.21E+04 72.06% 3.44% 1.04E+04 3.28E+04 3.29E+04 68.33% 0.40%
32 1.32E+04 3.19E+04 3.30E+04 3.29E+04 59.92% 60.00% 2.49% 3.14% 2.23% 0.91% 1.23E+04 3.22E+04 3.32E+04 62.53% 2.23%
1.40E+04 3.25E+04 3.26E+04 57.55% 1.32%
64 1.89E+04 3.27E+04 3.29E+04 3.31E+04 42.96% 39.03% 3.48% 1.31% 1.51% 0.92% 2.06E+04 3.29E+04 3.34E+04 37.83% 0.70%
2.11E+04 3.23E+04 3.31E+04 36.32% 2.52%
128 2.55E+04 3.26E+04 3.25E+04 3.27E+04 22.16% 20.79% 4.00% 0.41% 1.32% 0.81% 2.74E+04 3.21E+04 3.29E+04 16.29% 1.93%
2.49E+04 3.22E+04 3.28E+04 23.93% 1.63%
256 2.97E+04 3.29E+04 3.34E+04 3.28E+04 9.54% 9.14% 1.86% -0.20% 0.81% 0.93% 3.05E+04 3.23E+04 3.27E+04 7.11% 1.62%
2.93E+04 3.25E+04 3.24E+04 10.76% 1.02%
512 3.16E+04 3.32E+04 3.31E+04 3.31E+04 4.38% 2.26% 1.92% -0.24% 0.93% 1.10%
3.25E+04 3.27E+04 3.28E+04 1.72% 1.09% 3.29E+04 3.24E+04 3.33E+04 0.67% 1.94%
336 APPENDIX
(j) Inhibition of TGEV plaque formation by sera from mice orally immunized with PP/PrTGEV-S
Number of plaques formed % Inhibition with respect to negative ctrl (PBS) Sample PP/ PP PBS Avg PBSPBS- Avg Std PBS-PP Avg Std diln rTGEV-S PP/rTGEV-S Dev dev 2 2.86E+02 3.10E+04 3.10E+04 3.16E+04 99.09% 98.56% 0.79% 1.90% 2.43% 1.80% 7.40E+02 3.02E+04 3.22E+04 97.66% 4.43% 3.35E+02 3.13E+04 3.16E+04 98.94% 0.95% 4 1.09E+03 3.13E+04 3.21E+04 3.19E+04 96.59% 98.34% 1.59% 1.78% 1.26% 0.65% 4.04E+02 3.17E+04 3.16E+04 98.73% 0.52% 9.70E+01 3.14E+04 3.19E+04 99.70% 1.46% 8 3.67E+02 3.17E+04 3.20E+04 3.17E+04 98.84% 98.39% 1.29% -0.03% 0.83% 0.81% 1.89E+02 3.12E+04 3.15E+04 99.40% 1.58% 9.71E+02 3.14E+04 3.16E+04 96.94% 0.95% 16 5.69E+03 3.11E+04 3.11E+04 3.12E+04 81.74% 85.01% 3.06% 0.18% 0.95% 0.69% 3.80E+03 3.08E+04 3.08E+04 87.81% 1.18% 4.53E+03 3.07E+04 3.16E+04 85.47% 1.50% 32 1.22E+04 3.12E+04 3.20E+04 3.19E+04 61.67% 65.70% 4.75% 2.30% 1.15% 1.27% 1.13E+04 3.20E+04 3.22E+04 64.49% -0.21% 9.28E+03 3.15E+04 3.16E+04 70.94% 1.36% 64 1.63E+04 3.19E+04 3.13E+04 3.13E+04 47.90% 42.23% 5.47% -2.03% -0.32% 1.61% 1.97E+04 3.09E+04 3.17E+04 36.99% 1.17% 1.82E+04 3.13E+04 3.08E+04 41.79% -0.11% 128 2.47E+04 3.12E+04 3.15E+04 3.14E+04 21.32% 26.91% 4.86% 0.53% 1.17% 0.84% 2.22E+04 3.11E+04 3.18E+04 29.22% 0.85% 2.19E+04 3.07E+04 3.08E+04 30.18% 2.13% 256 2.67E+04 3.14E+04 3.14E+04 3.15E+04 15.15% 11.86% 4.38% 0.21% 2.01% 1.57% 2.93E+04 3.06E+04 3.18E+04 6.89% 2.75% 2.72E+04 3.05E+04 3.12E+04 13.56% 3.07% 512 3.09E+04 3.17E+04 3.21E+04 3.20E+04 3.22% 1.69% 1.42% 0.95% 1.19% 2.12% 3.18E+04 3.22E+04 3.16E+04 0.42% -0.80% 3.15E+04 3.09E+04 3.22E+04 1.42% 3.42%
337 APPENDIX
(k) Cytokine production by cells of the Peyer’s patches isolate from mice orally immunized with LcS-rTGEV-S
IL-2 (pg/ml) LcS/rTGEV-S rTGEV-S-GST Avg Std Dev RPMI Avg Std Dev Day 2 351.23 375.55 22.85 16.86 16.16 3.04 396.57 18.79 378.84 12.83 Day 3 381.94 347.56 31.62 17.27 14.31 3.08 319.73 14.54 341.02 11.13 Day 4 376.12 335.51 37.86 18.46 16.34 2.95 301.19 12.97 329.22 17.59 Day 5 365.18 366.15 27.92 16.17 15.12 3.21 394.54 11.52 338.72 17.68
LcS rTGEV-S-GST Avg Std Dev RPMI Avg Std Dev Day 2 16.02 13.45 2.24 18.67 16.06 3.69 12.37 11.84 11.95 17.68 Day 3 13.59 13.00 1.89 15.34 15.16 1.12 10.89 13.96 14.52 16.17 Day 4 16.38 13.62 2.60 12.78 15.38 3.49 11.23 14.01 13.24 19.34 Day 5 15.76 13.94 1.79 16.84 14.48 2.43 12.19 11.98 13.86 14.63
IFN-g (pg/ml) LcS/TGEV
338 APPENDIX
rTGEV-S-GST Avg Std Dev RPMI Avg Std Dev Day 2 762.37 773.90 32.29 12.68 13.09 2.68 810.37 15.96 748.96 10.64 Day 3 982.65 1063.97 73.23 11.37 13.40 1.85 1124.69 13.85 1084.56 14.99 Day 4 1361.33 1320.61 55.19 16.89 12.94 3.49 1342.71 10.27 1257.79 11.67 Day 5 1314.21 1305.28 46.77 14.00 13.98 1.96 1254.69 15.93 1346.95 12.01
LcS rTGEV-S-GST Avg Std Dev RPMI Avg Std Dev Day 2 16.38 13.67 3.11 11.15 12.89 3.17 14.36 10.96 10.27 16.55 Day 3 12.31 14.17 1.63 10.87 12.69 1.85 15.33 14.56 14.86 12.63 Day 4 17.01 14.81 2.09 12.54 13.21 2.84 14.57 16.33 12.85 10.77 Day 5 12.37 13.26 2.17 11.23 13.31 1.89 15.73 13.78 11.68 14.91
IL-4 (pg/ml) LcS/TGEV rTGEV-S-GST Avg Std Dev RPMI Avg Std Dev Day 2 406.34 443.61 41.63 10.84 12.77 1.91 488.54 14.66 435.94 12.81 Day 3 498.74 525.52 27.76 17.98 15.23 2.79
339 APPENDIX
523.64 15.30 554.17 12.41 Day 4 507.97 539.55 37.54 10.37 13.12 2.64 529.62 15.64 581.05 13.34 Day 5 497.21 527.89 41.62 14.77 13.97 2.70 575.27 16.18 511.20 10.96
LcS rTGEV-S-GST Avg Std Dev RPMI Avg Std Dev Day 2 13.41 16.41 2.81 11.51 13.11 1.89 18.99 12.63 16.83 15.20 Day 3 19.94 17.71 1.93 16.47 14.81 1.44 16.53 14.08 16.67 13.89 Day 4 16.42 17.35 1.75 14.54 13.17 1.92 19.37 10.98 16.26 13.99 Day 5 15.53 15.74 2.58 12.78 14.35 1.72 18.42 16.19 13.27 14.07
IL-5 (pg/ml) LcS/TGEV rTGEV-S-GST Avg Std Dev RPMI Avg Std Dev Day 2 246.65 217.35 26.31 17.51 14.90 2.46 209.67 12.63 195.74 14.55 Day 3 296.48 328.43 33.54 11.38 13.78 2.42 325.44 13.75 363.36 16.21 Day 4 358.76 400.93 38.34 14.67 13.76 1.34 410.34 12.22 433.68 14.38
340 APPENDIX
Day 5 376.12 405.35 27.62 13.11 13.34 2.50 431.02 10.96 408.91 15.94
LcS rTGEV-S-GST Avg Std Dev RPMI Avg Std Dev Day 2 15.88 14.63 2.60 13.64 12.24 1.75 16.37 12.79 11.64 10.28 Day 3 14.08 13.51 0.74 16.37 14.24 1.94 12.67 12.58 13.79 13.76 Day 4 12.51 14.01 2.28 12.69 15.00 2.14 16.63 16.93 12.89 15.37 Day 5 14.21 14.07 2.06 11.74 13.68 1.86 16.05 13.84 11.94 15.45
(l) Cytokine production by cells of the Peyer’s patches isolate from mice orally immunized with PP/PrTGEV-S
IL-2 (pg/ml) PP/PrTGEV-S rTGEV-S- Avg Std Dev RPMI Avg Std Dev Con A Avg Std Dev GST Day 2 409.13 403.11 38.89 15.59 15.23 2.41 10673.62 10220.58 392.42 361.57 17.43 9986.27 438.64 12.66 10001.85 Day 3 456.73 455.01 34.65 16.74 14.44 2.02 - - - 419.53 12.96 - 488.76 13.63 - Day 4 471.17 454.26 20.20 15.46 15.48 2.34 - - - 431.89 17.83 - 459.71 13.15 - Day 5 375.54 400.40 27.28 15.62 15.07 2.47 - - - 396.07 12.38 - 429.58 17.22 -
341 APPENDIX
PP rTGEV-S- Avg Std Dev RPMI Avg Std Dev Con A Avg Std Dev GST Day 2 11.37 14.04 2.76 17.54 15.25 2.15 10854.27 10536.69 275.16 13.85 13.28 10386.32 16.89 14.92 10369.47 Day 3 11.79 13.89 1.84 13.76 13.76 1.90 - - - 14.67 15.66 - 15.22 11.87 - Day 4 10.87 13.29 2.93 14.52 14.70 1.74 - - - 16.55 13.06 - 12.44 16.53 - Day 5 15.76 13.94 1.79 11.98 13.19 1.43 - - - 12.19 14.77 - 13.86 12.82 -
IFN-γ (pg/ml) PP/PrTGEV-S rTGEV-S- Avg Std Dev RPMI Avg Std Dev Con A Avg Std Dev GST Day 2 774.71 799.70 22.82 11.74 13.63 2.03 6894.26 7049.30 160.13 819.44 15.77 7039.58 804.96 13.37 7214.07 Day 3 1061.27 1100.69 35.79 12.86 12.74 2.42 - - - 1131.14 10.26 - 1109.67 15.09 - Day 4 1401.43 1402.67 24.68 13.22 13.32 2.21 - - - 1378.63 15.57 - 1427.95 11.16 - Day 5 1333.78 1299.77 37.95 13.37 14.92 1.76 - - - 1258.84 14.55 - 1306.70 16.84 -
PP rTGEV-S- Avg Std Dev RPMI Avg Std Dev Con A Avg Std Dev GST Day 2 10.84 12.20 1.41 11.54 10.70 0.94 7341.27 7173.65 160.64 13.66 10.88 7158.62 12.11 9.68 7021.05 Day 3 13.83 13.96 1.28 15.47 13.81 1.53 - - - 15.30 13.53 - 12.75 12.44 - Day 4 11.56 12.88 1.75 11.70 10.88 0.83 - - - 14.86 10.91 - 12.21 10.04 - Day 5 13.66 15.36 1.98 12.88 14.15 1.72 - - - 17.54 13.46 - 14.88 16.11 -
342 APPENDIX
IL-4 (pg/ml) PP/PrTGEV-S rTGEV-S- Avg Std Dev RPMI Avg Std Dev Con A Avg Std Dev GST Day 2 29.68 30.92 5.14 11.81 11.83 1.80 9756.27 9937.24 167.63 26.51 13.64 10087.21 36.56 10.05 9968.24 Day 3 117.32 101.79 13.73 15.74 14.16 1.43 - - - 96.78 13.81 - 91.26 12.94 - Day 4 310.14 296.86 17.85 14.22 12.11 2.00 - - - 276.57 10.24 - 303.88 11.86 - Day 5 354.81 375.21 19.62 16.79 14.87 1.89 - - - 393.94 14.81 - 376.89 13.01 -
PP rTGEV-S- Avg Std Dev RPMI Avg Std Dev Con A Avg Std Dev GST Day 2 13.22 15.74 2.25 10.74 12.04 1.62 10244.31 9986.81 245.72 16.47 13.86 9961.25 17.54 11.52 9754.87 Day 3 17.66 16.30 1.19 15.41 13.68 1.67 - - - 15.81 13.56 - 15.44 12.07 - Day 4 19.79 17.84 1.71 15.06 13.96 1.11 - - - 17.18 12.84 - 16.56 13.97 - Day 5 15.38 15.59 1.71 15.81 13.50 2.42 - - - 17.40 13.71 - 13.99 10.99 -
IL-5 (pg/ml) PP/PrTGEV-S rTGEV-S- Avg Std Dev RPMI Avg Std Dev Con A Avg Std Dev GST Day 2 28.87 25.67 3.18 15.87 14.46 1.45 11365.82 11116.10 466.18 22.51 14.55 10578.26 25.63 12.97 11404.21 Day 3 92.62 80.45 11.28 13.76 13.78 1.93 - - - 70.33 15.71 - 78.41 11.86 - Day 4 133.14 150.11 19.45 12.94 14.88 1.89 - - - 145.87 15.00 - 171.33 16.71 - Day 5 293.91 273.35 22.41 11.76 11.36 0.99 - - - 249.46 12.09 - 276.67 10.24 -
343 APPENDIX
PP rTGEV-S- Avg Std Dev RPMI Avg Std Dev Con A Avg Std Dev GST Day 2 18.94 16.04 2.75 12.84 12.16 1.66 10748.96 10815.92 239.30 15.71 10.26 10617.24 13.47 13.37 11081.57 Day 3 16.86 14.91 1.91 15.44 13.29 1.95 - - - 14.83 11.63 - 13.04 12.80 - Day 4 11.23 13.41 2.22 17.02 14.53 2.24 - - - 13.34 12.69 - 15.67 13.87 - Day 5 12.87 14.13 1.63 14.54 13.82 1.50 - - - 15.97 12.10 - 13.55 14.83 -
344 APPENDIX
APPENDIX 10
DATA FOR CHAPTER 5
(a) Concentrations of SARS-S-RBD specific IgA in the intestinal lavages of mice orally immunized with PP/SARS-S-RBD
Intestinal IgA (ng/ml) Pre-immune Average SD Day 18 Average SD serum PBS 0.52 0.55 0.11 0.53 0.51 0.08 0.46 0.58 0.68 0.43 PP/SARS-S-RBD 0.59 0.51 0.09 4.89 4.48 0.36 0.42 4.24 0.52 4.31 PP/pGAPZ α C 0.43 0.52 0.10 0.61 0.55 0.07 0.62 0.56 0.52 0.48
Day 32 Average SD Day 48 Average SD PBS 0.61 0.56 0.04 0.52 0.48 0.08 0.53 0.39 0.54 0.54 PP/SARS-S-RBD 4.56 4.52 0.37 4.46 4.51 0.26 4.87 4.28 4.14 4.79 PP/pGAPZ α C 0.57 0.50 0.08 0.43 0.53 0.08 0.53 0.57 0.41 0.58
345 APPENDIX
(b) Concentrations of SARS-S-RBD specific IgG in the sera of mice orally immunized with PP/SARS-S-RBD
Intestinal IgG (ng/ml) Pre-immune Average SD Day 18 Average SD serum PBS 0.64 0.56 0.09 0.45 0.57 0.12 0.47 0.69 0.58 0.58 PP/SARS-S-RBD 0.49 0.55 0.05 9.67 9.42 0.77 0.56 8.55 0.59 10.03 PP/pGAPZ α C 0.68 0.61 0.09 0.43 0.53 0.11 0.63 0.51 0.51 0.65
Day 32 Average SD Day 48 Average SD PBS 0.67 0.56 0.09 0.58 0.53 0.10 0.53 0.41 0.49 0.60 PP/SARS-S-RBD 9.61 9.49 1.01 8.71 9.45 0.86 10.43 9.26 8.42 10.39 PP/pGAPZ α C 0.58 0.56 0.08 0.64 0.59 0.06 0.48 0.61 0.63 0.52
346 APPENDIX
(c) Concentrations of SARS-S-RBD specific IgG isotypes in the sera of mice orally immunized with PP/SARS-S-RBD
Serum IgG Isotype IgG1 (ng/ml) IgG2a (ng/ml) Day 32 Average SD Day 32 Average SD PBS 0.28 0.26 0.04 0.24 0.24 0.05 0.21 0.19 0.29 0.29 PP/SARS-S-RBD 4.87 5.26 0.48 2.35 2.59 0.32 5.12 2.96 5.79 2.47 PP/pGAPZ α C 0.23 0.26 0.04 0.27 0.27 0.05 0.26 0.22 0.30 0.31
(d) Inhibition of MLV(SARS) infection by intestinal lavages from mice orally immunized with PP/SARS-S-RBD
Number of fluorescence cell (FFU) % Inhibition with respect of negative ctrl (PBS) Sample PP/SARS- PP PBS Avg PBSPBS- Avg Std PBS- Avg Std diln S-RBD PP/SARS-S- Dev PP dev RBD 2 4.70E+02 3.76E+04 3.84E+04 3.93E+04 98.80% 98.45% 0.80% 4.24% 5.00% 0.92% 9.67E+02 3.69E+04 3.95E+04 97.54% 6.02% 3.89E+02 3.74E+04 3.99E+04 99.01% 4.75% 4 7.54E+02 3.79E+04 4.12E+04 4.04E+04 98.13% 98.28% 0.74% 6.19% 5.28% 0.87% 3.69E+02 3.86E+04 3.97E+04 99.09% 4.46% 9.57E+02 3.83E+04 4.03E+04 97.63% 5.20% 8 1.45E+03 3.74E+04 3.89E+04 3.99E+04 96.36% 94.90% 1.29% 6.20% 5.12% 1.01% 2.43E+03 3.79E+04 4.01E+04 93.91% 4.95% 2.23E+03 3.82E+04 4.06E+04 94.41% 4.20% 16 6.68E+03 3.85E+04 4.11E+04 4.03E+04 83.42% 83.90% 1.46% 4.47% 4.96% 0.66% 6.95E+03 3.84E+04 4.06E+04 82.75% 4.71%
347 APPENDIX
5.83E+03 3.80E+04 3.92E+04 85.54% 5.71% 32 8.96E+03 3.84E+04 4.06E+04 4.04E+04 77.83% 72.25% 4.83% 5.03% 4.78% 0.89% 1.23E+04 3.89E+04 3.96E+04 69.68% 3.79% 1.24E+04 3.82E+04 4.11E+04 69.26% 5.52% 64 2.00E+04 3.78E+04 3.94E+04 3.98E+04 49.83% 48.96% 4.20% 5.10% 4.77% 0.81% 1.89E+04 3.83E+04 3.89E+04 52.67% 3.85% 2.21E+04 3.77E+04 4.12E+04 44.40% 5.36% 128 2.27E+04 3.78E+04 3.83E+04 3.95E+04 42.36% 38.23% 3.91% 4.22% 5.32% 1.14% 2.46E+04 3.74E+04 3.98E+04 37.75% 5.24% 2.58E+04 3.69E+04 4.03E+04 34.59% 6.50% 256 3.07E+04 3.82E+04 4.09E+04 4.02E+04 23.74% 20.37% 3.76% 5.05% 5.30% 0.90% 3.18E+04 3.84E+04 4.13E+04 21.07% 4.56% 3.37E+04 3.77E+04 3.85E+04 16.31% 6.30% 512 3.52E+04 3.75E+04 4.11E+04 4.00E+04 12.47% 11.59% 2.49% 6.79% 5.80% 1.14% 3.48E+04 3.84E+04 4.02E+04 13.51% 4.56% 3.67E+04 3.78E+04 3.87E+04 8.78% 6.05%
(e) Inhibition of MLV(SARS) infection by sera from mice orally immunized with PP/SARS-S-RBD
Number of fluorescence cell (FFU) % Inhibition with respect to negative ctrl Sample PP/SARS PP PBS Avg PBSPBS - Avg Std PBS- Avg Std diln -S-RBD PP/SAR-S- Dev PP dev RBD 2 4.36E+02 3.73E+04 4.14E+04 3.98E+04 98.91% 99.40% 0.47% 6.36% 5.86% 0.87% 6.00E+01 3.73E+04 3.93E+04 99.85% 6.36% 2.20E+02 3.79E+04 3.88E+04 99.45% 4.85% 4 8.50E+02 3.72E+04 3.84E+04 3.99E+04 97.87% 99.08% 1.05% 6.78% 5.36% 1.29% 1.21E+02 3.79E+04 4.03E+04 99.70% 5.03% 1.30E+02 3.82E+04 4.10E+04 99.67% 4.28% 8 7.36E+02 3.78E+04 4.02E+04 4.01E+04 98.16% 96.80% 1.42% 5.74% 4.57% 1.42% 1.87E+03 3.89E+04 3.89E+04 95.34% 2.99% 1.25E+03 3.81E+04 4.12E+04 96.89% 4.99%
348 APPENDIX
16 5.90E+03 3.84E+04 4.16E+04 4.06E+04 85.46% 81.88% 3.48% 5.36% 4.37% 0.99% 7.44E+03 3.92E+04 4.08E+04 81.66% 3.38% 8.72E+03 3.88E+04 3.93E+04 78.51% 4.37% 32 1.28E+04 3.79E+04 3.98E+04 3.99E+04 67.87% 66.71% 4.87% 4.93% 4.52% 0.38% 1.54E+04 3.82E+04 3.86E+04 61.37% 4.18% 1.16E+04 3.81E+04 4.12E+04 70.90% 4.43% 64 2.19E+04 3.86E+04 4.11E+04 4.05E+04 45.93% 42.75% 5.08% 4.70% 5.03% 1.51% 2.56E+04 3.78E+04 4.00E+04 36.89% 6.67% 2.21E+04 3.90E+04 4.04E+04 45.44% 3.71% 128 2.76E+04 3.83E+04 3.94E+04 4.03E+04 31.46% 27.81% 4.88% 4.88% 3.97% 1.00% 2.83E+04 3.91E+04 4.13E+04 29.72% 2.90% 3.13E+04 3.86E+04 4.01E+04 22.27% 4.14% 256 3.31E+04 3.76E+04 3.98E+04 3.98E+04 16.76% 14.94% 3.43% 5.45% 4.27% 1.13% 3.54E+04 3.85E+04 3.88E+04 10.98% 3.19% 3.30E+04 3.81E+04 4.07E+04 17.07% 4.19% 512 3.48E+04 3.79E+04 4.11E+04 4.03E+04 12.49% 8.55% 3.52% 4.69% 6.29% 1.43% 3.75E+04 3.68E+04 3.93E+04 5.70% 7.46% 3.68E+04 3.71E+04 4.06E+04 7.46% 6.71%
(f) Cytokine production by cells of the Peyer’s patches isolate from mice orally immunized with PP/SARS-RBD
IL-2 (pg/ml) PP/SARS-S-RBD SARS Avg Std Dev RPMI Avg Std Dev Con A Avg Std Dev S(M) Day 2 221.74 231.58 29.11 11.26 12.21 1.10 11445.34 11238.75 193.78 208.66 13.41 11209.87 264.33 11.96 11061.03 Day 3 217.96 227.39 23.71 9.87 11.10 1.29 - - - 254.36 10.98 - 209.84 12.44 - Day 4 246.80 250.12 23.45 9.18 11.62 2.35 - - - 275.06 13.86 - 228.51 11.82 - Day 5 214.93 234.23 17.28 10.63 11.97 1.23 - - - 239.47 13.04 - 248.28 12.24 -
349 APPENDIX
PP SARS Avg Std Dev RPMI Avg Std Dev Con A Avg Std Dev S(M) Day 2 12.23 13.45 1.08 10.97 11.14 1.19 11495.62 11269.10 230.66 13.84 12.41 11277.18 14.27 10.05 11034.51 Day 3 12.74 11.76 0.93 13.58 11.81 1.67 - - - 11.67 11.61 - 10.88 10.25 - Day 4 11.97 11.79 1.86 13.41 11.31 2.13 - - - 13.56 11.37 - 9.85 9.16 - Day 5 12.23 12.30 1.60 12.60 12.18 1.32 - - - 10.74 10.71 - 13.93 13.24 -
IFN-γ (pg/ml) PP/SARS-S-RBD SARS Avg Std Dev RPMI Avg Std Dev Con A Avg Std Dev S(M) Day 2 121.54 126.61 20.85 12.84 11.19 1.62 6991.20 7179.07 167.35 149.53 9.61 7233.85 108.77 11.11 7312.17 Day 3 135.67 134.33 17.82 8.79 11.59 2.49 - - - 151.44 12.44 - 115.87 13.54 - Day 4 127.69 135.62 24.06 10.96 12.50 1.47 - - - 162.65 12.67 - 116.53 13.88 - Day 5 154.64 126.09 24.80 12.61 10.73 1.77 - - - 109.88 10.48 - 113.74 9.10 -
PP SARS Avg Std Dev RPMI Avg Std Dev Con A Avg Std Dev S(M) Day 2 12.24 12.20 1.21 8.89 11.08 1.97 7438.91 7214.67 211.55 10.97 11.63 7186.44 13.38 12.71 7018.65 Day 3 12.40 11.72 1.61 13.57 12.12 1.75 - - - 9.89 12.62 - 12.88 10.17 - Day 4 10.64 12.18 1.52 10.84 11.78 1.46 - - - 12.24 13.46 - 13.67 11.05 - Day 5 10.37 11.88 1.68 13.23 12.01 1.13 - - - 13.69 11.81 - 11.57 10.99 -
350 APPENDIX
IL-4 (pg/ml) PP/SARS-S-RBD SARS Avg Std Dev RPMI Avg Std Dev Con A Avg Std Dev S(M) Day 2 27.64 36.00 7.73 12.12 12.03 1.88 9807.92 10128.73 330.01 37.49 13.86 10467.22 42.88 10.11 10111.05 Day 3 287.65 282.44 26.32 13.21 11.66 1.81 - - - 253.91 9.67 - 305.77 12.10 - Day 4 668.34 625.69 39.25 10.86 11.51 0.93 - - - 591.08 12.57 - 617.64 11.10 - Day 5 1214.67 1188.67 60.25 10.14 11.45 1.26 - - - 1119.78 12.66 - 1231.55 11.54 -
PP SARS Avg Std Dev RPMI Avg Std Dev Con A Avg Std Dev S(M) Day 2 12.41 12.30 1.41 9.75 11.35 1.42 10396.78 10772.10 565.78 10.84 12.46 11422.86 13.66 11.83 10496.67 Day 3 14.20 12.48 1.54 11.51 11.73 1.65 - - - 11.22 13.48 - 12.01 10.21 - Day 4 13.84 12.03 1.59 13.52 12.07 1.83 - - - 11.36 10.01 - 10.88 12.69 - Day 5 12.98 12.09 1.68 10.15 11.94 1.60 - - - 10.15 12.44 - 13.13 13.23 -
IL-5 (pg/ml) PP/SARS-S-RBD SARS Avg Std Dev RPMI Avg Std Dev Con A Avg Std Dev S(M) Day 2 329.27 376.57 41.22 10.86 11.37 0.63 11784.39 11406.45 368.26 395.66 12.08 11386.27 404.78 11.17 11048.69 Day 3 587.66 616.12 29.29 13.64 11.76 1.63 - - - 614.51 10.75 - 646.18 10.88 - Day 4 1441.87 1449.88 64.70 12.52 11.11 1.36 - - - 1389.55 10.99 - 1518.21 9.81 - Day 5 786.51 732.16 47.99 13.37 11.70 1.51 - - - 714.33 11.28 - 695.64 10.44 -
351 APPENDIX
PP SARS Avg Std Dev RPMI Avg Std Dev Con A Avg Std Dev S(M) Day 2 14.21 12.81 1.89 11.67 11.18 1.88 11812.14 11355.82 436.96 10.66 12.77 10941.20 13.57 9.11 11314.13 Day 3 11.17 11.91 1.59 13.28 12.15 1.09 - - - 13.74 12.07 - 10.82 11.11 - Day 4 10.99 12.35 1.23 10.88 11.54 0.95 - - - 12.66 12.63 - 13.40 11.10 - Day 5 13.64 11.90 1.52 10.23 10.78 1.85 - - - 10.86 9.27 - 11.20 12.84 -
(g) Cytokine production by cells of the CLN isolate from mice orally immunized with PP/SARS-RBD
IL-2 (pg/ml) PP/SARS-S-RBD SARS S(M) Avg Std Dev RPMI Avg Std Dev Con A Avg Std Dev Day 2 62.38 56.09 7.19 21.36 19.26 1.90 15367.18 15114.36 251.97 57.65 18.75 14863.25 48.25 17.66 15112.66 Day 3 101.36 112.13 9.39 19.64 20.05 2.07 - - - 116.47 22.30 - 118.56 18.22 - Day 4 138.96 134.99 11.68 17.28 19.36 1.85 - - - 144.17 19.97 - 121.84 20.83 - Day 5 135.55 125.65 9.33 16.75 19.08 2.22 - - - 117.02 19.33 - 124.39 21.17 -
PP SARS S(M) Avg Std Dev RPMI Avg Std Dev Con A Avg Std Dev Day 2 17.65 19.25 1.56 19.88 19.60 1.93 14386.61 14301.84 277.51 19.32 21.37 13991.84 20.77 17.54 14527.08 Day 3 18.63 19.71 2.26 22.40 20.00 2.16 - - - 22.31 19.39 - 18.20 18.21 - Day 4 21.45 19.34 1.87 19.61 18.98 1.46 - - - 18.69 20.02 - 17.88 17.32 -
352 APPENDIX
Day 5 20.68 19.34 1.62 22.65 20.54 1.86 - - - 19.81 19.87 - 17.54 19.11 -
IFN-γ (pg/ml) PP/SARS-S-RBD SARS S(M) Avg Std Dev RPMI Avg Std Dev Con A Avg Std Dev Day 2 37.65 33.27 3.85 24.08 22.52 2.11 9865.37 10015.75 198.96 30.44 23.35 10241.36 31.72 20.12 9940.52 Day 3 64.51 70.96 5.86 23.84 21.84 2.11 - - - 72.43 19.63 - 75.94 22.04 - Day 4 84.29 82.02 5.57 21.10 23.08 2.32 - - - 75.67 22.51 - 86.10 25.63 - Day 5 85.64 79.85 6.96 25.84 23.88 1.76 - - - 81.78 23.38 - 72.12 22.43 -
PP SARS S(M) Avg Std Dev RPMI Avg Std Dev Con A Avg Std Dev Day 2 19.19 19.14 1.47 17.74 19.54 1.91 9697.52 9940.44 231.85 17.65 19.33 10159.36 20.58 21.54 9964.43 Day 3 18.61 20.00 1.52 22.15 20.29 1.74 - - - 21.63 20.03 - 19.77 18.69 - Day 4 20.45 18.86 1.39 17.66 19.40 2.00 - - - 17.88 18.97 - 18.24 21.58 - Day 5 16.79 18.31 1.39 19.50 19.33 1.60 - - - 19.52 17.65 - 18.61 20.84 -
IL-4 (pg/ml) PP/SARS-S-RBD SARS S(M) Avg Std Dev RPMI Avg Std Dev Con A Avg Std Dev Day 2 29.98 29.79 3.96 18.08 18.28 1.40 13620.37 13842.30 207.18 33.66 16.99 14030.61 25.74 19.76 13875.91 Day 3 76.67 70.06 5.80 21.34 19.57 1.59 - - - 67.68 18.26 - 65.84 19.11 - Day 4 297.61 318.15 18.10 16.87 18.28 1.39 - - - 325.08 19.65 - 331.77 18.33 - Day 5 437.87 462.20 30.65 21.04 19.59 2.06 - - -
353 APPENDIX
496.62 17.23 - 452.12 20.51 -
PP SARS S(M) Avg Std Dev RPMI Avg Std Dev Con A Avg Std Dev Day 2 20.64 20.10 1.42 19.75 19.62 1.32 14008.67 13829.97 156.73 21.17 18.24 13765.43 18.48 20.87 13715.82 Day 3 17.58 18.84 1.10 17.68 19.42 1.93 - - - 19.62 19.08 - 19.31 21.49 - Day 4 20.52 19.66 1.81 16.69 18.65 1.72 - - - 20.88 19.37 - 17.58 19.89 - Day 5 18.69 20.11 1.29 21.56 19.95 1.67 - - - 20.41 20.06 - 21.22 18.22 -
IL-5 (pg/ml) PP/SARS-S-RBD SARS S(M) Avg Std Dev RPMI Avg Std Dev Con A Avg Std Dev Day 2 124.35 126.66 9.37 18.63 19.69 1.12 16127.87 15866.10 277.96 136.97 19.57 15896.05 118.66 20.86 15574.38 Day 3 357.91 366.87 13.72 19.59 19.66 1.72 - - - 382.67 21.41 - 360.03 17.98 - Day 4 536.99 535.94 16.69 22.12 20.06 1.89 - - - 518.74 18.41 - 552.08 19.65 - Day 5 495.61 523.05 24.75 17.57 19.36 1.90 - - - 529.88 19.16 - 543.67 21.35 -
PP SARS S(M) Avg Std Dev RPMI Avg Std Dev Con A Avg Std Dev Day 2 17.58 19.38 1.67 21.55 19.14 2.11 16007.02 15724.84 325.14 20.89 17.64 15798.21 19.66 18.24 15369.28 Day 3 18.94 19.07 1.31 18.29 19.51 1.30 - - - 17.83 19.37 - 20.44 20.87 - Day 4 21.45 19.65 1.66 22.17 20.12 1.85 - - - 19.33 19.63 - 18.17 18.57 - Day 5 20.85 19.49 1.46 18.19 18.47 1.62 - - - 17.95 20.21 - 19.66 17.00 -
354 APPENDIX
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