MURINE MODELS TO STUDY IMMUNITY AND IMMUNISATION AGAINST RESPIRATORY VIRAL PATHOGENS

MURINE MODELS TO STUDY IMMUNITY AND IMMUNISATION AGAINST RESPIRATORY VIRAL PATHOGENS POH WEE PENG (B.Sc.(Hons.)), Murdoch University, Western Australia A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENTS My journey through my post-graduate study years would not be possible without the presence of many individuals I would like to express my heartfelt gratitude, appreciation and thanks to: A/Prof Koh Dow Rhoon, for his supervision, guidance, patience and support throughout my time at the Immunobiology Laboratory, Dept of Physiology His insights into the world of Immunology have always fascinated me and encouraged me dwell in the field I hope to take away with me the meticulous planning of scientific research that he has shown me over the years A/Prof Vincent Chow T.K., for his co-supervision, constant support, encouragement, timely advice and allowing me the opportunity to undertake research projects in the Human Genome Laboratory (HGL), Dept of Microbiology I hope to take away with me the critical thinking and scientific reasoning skills that he has demonstrated His expert views in areas of virology have re-ignited my first love in research, ie Virology! A/Profs Hooi Shing Chuan and Soong Tuck Wah, past and present Heads of Department of Physiology, for their support and the opportunity they have given me to complete my post-graduate studies Prof Toshifumi Matsuyama and A/Prof Kiri Honma, Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Japan, for their collaboration, valuable feedback and opinions in the IRF-4 project A/Prof Paul A MacAry, for his advice and guidance in my research project, as well as mentoring me in the chromium release assay procedure A/Prof Lu Jinhua, for his collaboration and guidance in the SARS DNA vaccine project A/Prof Tan Kong Bing, Dr Wang Shi, and Mdm Connie Foo, Dept of Pathology, NUH, for their collaboration and valuable histopathology related-work Dr Teluguakula Narasaraju, Oklahoma State University, Stillwater, USA, for his guidance, encouragement and knowledge in the research project Mrs Phoon Meng Chee, Wu Yan and Xie Mei Lan, for their kind assistance and collaborative work in the viral neutralization assay Ms Asha Reka Das and Mdm Vasantha Nathan, Dept of Physiology Administrative Office, for their kind assistance, concern and encouragement Ms Ho Hwei Moon, Registrar’s Office, for her patience and kind assistance during the thesis submission Mdm Ho Chiu Han, Dept of Physiology and Ms Kelly Lau Suk Hiang, Dept of Microbiology, for their technical assistance i Dr Zia Rahman, Dr Tin Soe Kyaw and Dr Gong Yue, former PhD candidates from Immunobiology Laboratory, for their valuable insights and discussions Ooi Hui Ann, Fiona Setiawan and Cynthia Lee, former HGL Final Year Project students, whom I have mentored, for strengthening my knowledge in Immunology/Virology through their questions and enquiries “By your pupils you will be taught” Past and present students of HGL, namely, Audrey Ann Liew, for her kind assistance in intra-tracheal infections; Tan Kai Sen and Ng Wai Chii, for their technical assistance; Ng Huey Hian, Hsu Jung Pu, Edwin Yang and Ivan Budiman, for their friendship Principal Investigators, Research Fellows and laboratory staff at Translational Infectious Disease Laboratory, Dept of Microbiology, namely, Prof Naoki Yamamoto and Dr Yoichi Suzuki, for their guidance, patience and support during my thesis write-up; A/Prof Dr Chong Pei Pei, Visiting Scientist from Universiti Putra Malaysia, for her critical views on the thesis Emeritus Profs John William Penhale and Graham E Wilcox, School of Veterinary and Biomedical Sciences, Murdoch University, Western Australia, for introducing the world of Immunology and Virology to me so many years ago and their encouragement on my research project and post-graduate studies My aunty, Mdm Poh Siew Khiam, and my sister, Poh Wee Chin, for their unwavering support during my time as a PhD candidate My father, Poh Thuan Thak, for his undying love, support and constant encouragement throughout my life My wife, Christine Chen Yop Fong, and my children, Ethan and Edward; for their love, patience, support and understanding over my post-graduate study years This thesis is dedicated to My father, Poh Thuan Thak; My late mother, Annie Goh; My wife, Christine Chen, and my boys, Ethan and Edward Poh, for being the force behind the power in me ii Table of Contents Acknowledgements i Table of Contents iii Summary x List of Tables xii List of Figures xiii List of Publications, Abstracts and Poster Oral Presentations xvii Chapter 1 1.1 Prologue 1.2 Hypotheses Hypothesis I Hypothesis II 1.3 General Introduction 1.3.1 The respiratory system 1.3.2 Pulmonary Host Defense 1.3.3 Respiratory Viral Pathogens Chapter 2: A Novel DNA Vaccine Against SARS Coronavirus 11 2.1 Introduction 11 2.1.1 Coronaviruses 11 2.1.1.1 11 Structure 2.1.2 Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) 13 2.1.2.1 Innate immune responses to SARS-CoV infection 14 2.1.2.2 Adaptive cellular responses to SARS-CoV infection 15 2.1.2.3 Humoral immune response 16 iii to SARS- CoV infection 2.1.3 Vaccines 2.1.3.1 16 DNA vaccines against respiratory viruses and use of integrin to enhance its efficacy 18 2.1.4 Animal models in SARS-CoV infections 22 2.1.5 Spike glycoprotein in SARS-CoV vaccines 24 2.1.5 Novel SARS-like Coronavirus 28 2.2 Objectives 28 2.3 Hypothesis 29 2.4 Materials and Methods 29 2.4.1 Screening for H2-Kb-restricted peptides within the SARS spike glycoprotein 29 2.4.2 Peptides 30 2.4.3 Cell lines 30 2.4.4 MHC-peptide binding assay 30 2.4.5 Plasmid construction 31 2.4.6 Mice 36 2.4.7 Immunizations 37 2.4.8 Splenocyte re-stimulation in vitro 38 2.4.9 Cytotoxic T-lymphocyte assay 38 2.4.10 IFN-γ ELISPOT assay 40 2.4.11 Enzyme-linked immunosorbent assay (ELISA) 41 2.4.12 Amino acid sequence alignment 41 2.4.13 Statistical analysis 42 Results 42 2.5.1 Kyte-Doolittle Hydropathy and 42 2.5 iv Hopp-Woods Hydrophilic plots to determine antigenic regions 2.6 2.7 2.5.2 Screening and identification of H2-Kb-restricted epitopes within the SARS-CoV spike glycoprotein by MHC-peptide binding assay 47 2.5.3 Use of RMA/S cells as antigen-presenting cells in 51 Cr release assay 51 2.5.4 Cytotoxic T-lymphocytes are activated following DNA immunization 52 2.5.5 IFN-γ ELISPOT assay reveals T-cell epitopes of the SARS-CoV spike glycoprotein 58 2.5.6 Mice immunized with SARS-CoV S-His DNA vaccine induce significantly higher humoral immune responses compared to S-RGD/His immunized mice 61 2.5.7 Amino acid sequence comparison of SARS-CoV spike glycoprotein between Urbani, Tor2 and Beijing strains 61 Discussion 62 2.6.1 Efficacy enhancement of SARS Spike DNA vaccine fused to the integrin binding motif 65 Conclusion 72 Chapter 3: Understanding the role of Interferon Regulatory Factor-4 (IRF-4) transcription factor in severe influenza Pneumonitis 73 3.1 Introduction 73 3.1.1 Influenza Virus 73 3.1.1.1 Hemagglutinin (HA) 75 3.1.1.2 Neuraminidase (NA) 76 3.1.1.3 Influenza virus replication 76 3.1.1.4 Pathology and Pathogenesis 79 3.1.1.5 Innate immune responses in Influenza virus infections 80 v 3.1.1.6 Adaptive immune responses in Influenza virus infections 81 3.1.2 Cytokines in Virus Infections 83 3.1.3 Cytokines and Chemokines in the pathogenesis of Influenza Virus infections 84 3.1.4 Generation of transgenic and knock-out mice as models for studying respiratory virus infection 85 3.1.4.1 Transgenic mice 85 3.1.4.2 Knockout mice 86 3.1.4.3 Animal models in Influenza Virus Infection 87 3.1.5 Antiviral innate signaling pathwats activated by Interferons 91 3.1.6 Jak-Stat pathway in Type I IFN signaling 92 3.1.7 Interferon Regulatory Factors 94 3.1.6.1 Interferonic IRFs 95 3.1.6.2 Stress-responsive IRFs 96 3.1.6.3 Hematopoietic IRFs 96 3.1.6.4 Morphogenic IRFs 97 3.1.7 IRFs and TLR Signaling 99 3.1.8 Interferon Regulatory Factor-4 100 3.2 Objectives 106 3.3 Hypothesis 106 3.4 Materials and Methods 107 3.4.1 Experimental Methodology 107 3.4.2 Variables of IRF-4 PR8 H1N1 Influenza infection model 108 3.4.3 Animals 108 3.4.4 DNA extraction from Mouse Tail 108 vi 3.4.5 Genotyping 109 3.4.6 Virus 110 3.4.7 Viral plaque assay 111 3.4.8 TCID50 assay 112 3.4.9 Intra-tracheal infection 113 3.4.10 Intra-nasal infection 113 3.4.11 Mice euthanization, harvesting of organs and serum from blood 114 3.4.12 Lung homogenization 114 3.4.13 RNA extraction and purification 115 3.4.14 RNA quantification and RNA integrity 115 3.4.15 Reverse Transcription Reaction 116 3.4.16 Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) 117 3.4.17 Analyzing Quantitative Real-Time PCR data by the Comparative CT method 121 3.4.18 Histopathology 123 3.4.19 Lung Injury Score 123 3.4.20 Neutralization assay 124 3.4.21 Bioplex Cytokine assay 126 3.4.22 MouseRef-8 v2.0 expression BeadChip microarray 128 3.4.22.1 Illumina® TotalPrep™ RNA Amplification 128 3.4.22.2 Whole-genome gene expression direct hybridization assay 129 3.4.22.3 Microarray data analysis 130 3.4.22.4 GeneSpring GX 12.1 130 3.4.22.5 GeneSpring Fold Change Analysis 131 vii 3.4.23 Statistical Analysis 3.5 Results 132 133 3.5.1 Titration of Influenza A/Puerto Rico/8/1934 H1N1 virus in C57BL/6 IRF-4 and IRF-4 +/- mice by intra-tracheal route 133 3.5.2 Intra-tracheal infection of male and female IRF-4 +/+, +/- and -/- mice with 500pfu Influenza A/Puerto Rico/8/1934 H1N1 – Bodyweight and Survival Studies 137 3.5.3 Lung histopathology 143 3.5.4 Terminal Lung viral load determined by plaque assay and qRT-PCR 149 3.5.5 Neutralization assay 152 3.5.6 Lung homogenate cytokine level analysis 155 3.5.7 Quantitative Real-Time PCR to detect for gene expression 163 3.5.8 Microarray analysis 168 3.5.9 IRF-4 mice infection with mouse-adapted Influenza A/Aichi/2/1968 H3N2 173 3.6 Discussion 186 3.7 Conclusion 196 Chapter 4: Serendipitous observations and preliminary data on Spontaneous tumor formation in influenza virus-infected IRF-4 +/- mice and non-infected IRF-4 -/- mice 197 4.1 Introduction 197 4.2 Materials and methods 200 4.2.1 CD3 and CD20 immunohistochemistry 200 Results 200 4.3.1 Tumorigenesis model in recovered IRF-4 +/- infected with P12 mouse-adapted Influenza A/Aichi/2/68 H3N2 200 4.3.2 Spontaneous tumorigenesis IRF-4 -/- mice 205 4.3 viii 4.4 4.3.3 CD3 and CD20 staining 208 Discussion 210 Chapter 5: Conclusion / Epilogue 213 Chapter 6: Future Directions 216 Chapter 7: Reference List 219 Chapter 8: Appendix 280 8.1 Nucleotide and amino acid sequence of extracellular Domain of codon-optimized SARS-CoV spike glycoprotein (derived from TOR2 SARS-CoV strain) fused with SPD/myc in pcDNA3.1 (-) 8.2 Nucleotide and amino acid sequence of Spike glycoprotein of SARS-CoV TOR2 (GenBank: AY274119.3) 8.3 Nucleotide and amino acid sequence comparison of codonoptimized SARS-CoV Spike glycoprotein (Farzan) versus original SARS-CoV TOR2 strain 8.4 Amino acid sequence comparison of codon-optimized SARSCoV spike glycoprotein (Farzan) versus Beijing BJ302 SARSCoV spike protein used in ELIZA 8.5 Reagents for genotyping PCR 8.6 Reagents for plaque assay 8.7 Microarray procedure and data analysis 8.8 Statistical analysis of fold-changes in lung homogenate cytokine concentrations measured by multiplex cytokine assay 8.9 Absolute cytokine concentrations and fold-changes in lung homogenates 8.10 Statistical analysis of gene expression in lung homogenate Measured by Quantitative Real-Time PCR 8.11 Graphical data of individual gene expression analysis in lung homogenate measured by Quantitative Real-Time PCR ix IRF-5 Descriptivesa ACTB N -/+/+/+ Total 36 27 32 95 Mean 402 2.129 3.023 1.776 Std Dev iat ion 4024 1.3021 1.2832 1.5365 Std Error 0671 2506 2268 1576 95% Conf idence Interv al f or Mean Lower Bound Upper Bound 265 538 1.614 2.644 2.561 3.486 1.463 2.089 Minimum 1.6 Maximum 2.0 6.2 6.3 6.3 a gene = IRF5 ANOVAa ACTB Between Groups Within Groups Total Sum of Squares 121.135 100.794 221.929 df 92 94 Mean Square 60.568 1.096 F 55.283 Sig .000 a gene = IRF5 Post Hoc Tests Multi ple Comparisonsa Dependent Variable: ACTB Bonf erroni (I) genot y pe -/+/+/+ (J) genoty pe +/+/+ -/+/+ -/+/- Mean Dif f erence (I-J) St d Error -1.7271* 2665 -2.6216* 2543 1.7271* 2665 -.8945* 2735 2.6216* 2543 8945* 2735 Sig .000 000 000 005 000 005 95% Conf idence Interv al Lower Bound Upper Bound -2.377 -1.077 -3.242 -2.001 1.077 2.377 -1.562 -.228 2.001 3.242 228 1.562 * The mean dif f erence is signif icant at the 05 lev el a gene = IRF5 332 IRF-7 Descriptivesa ACTB N -/+/+/+ Total 36 30 33 99 Mean 1.718 8.563 18.420 9.359 Std Dev iat ion 2.1121 7.4828 14.6363 11.7216 Std Error 3520 1.3662 2.5478 1.1781 95% Conf idence Interv al f or Mean Lower Bound Upper Bound 1.004 2.433 5.768 11.357 13.230 23.610 7.022 11.697 Minimum 2.1 Maximum 10.2 24.2 59.4 59.4 a gene = IRF7 ANOVAa ACTB Between Groups Within Groups Total Sum of Squares 4829.874 8634.965 13464.839 df 96 98 Mean Square 2414.937 89.948 F 26.848 Sig .000 a gene = IRF7 Post Hoc Tests Multi ple Comparisonsa Dependent Variable: ACTB Bonf erroni (I) genot y pe -/+/+/+ (J) genoty pe +/+/+ -/+/+ -/+/- Mean Dif f erence (I-J) St d Error -6.8442* 2.3445 -16.7014* 2.2857 6.8442* 2.3445 -9.8571* 2.3925 16.7014* 2.2857 9.8571* 2.3925 Sig .013 000 013 000 000 000 95% Conf idence Interv al Lower Bound Upper Bound -12.557 -1.131 -22.271 -11.132 1.131 12.557 -15.687 -4.027 11.132 22.271 4.027 15.687 * The mean dif f erence is signif icant at the 05 lev el a gene = IRF7 333 IRF-8 Descriptivesa ACTB N -/+/+/+ Total 36 29 33 98 Mean 321 1.311 2.020 1.186 Std Dev iat ion 2287 9142 1.2841 1.1502 Std Error 0381 1698 2235 1162 95% Conf idence Interv al f or Mean Lower Bound Upper Bound 244 399 963 1.658 1.565 2.476 956 1.417 Minimum Maximum 1.0 3.6 4.6 4.6 a gene = IRF8 ANOVAa ACTB Between Groups Within Groups Total Sum of Squares 50.339 77.998 128.337 df 95 97 Mean Square 25.169 821 F 30.656 Sig .000 a gene = IRF8 Post Hoc Tests Multi ple Comparisonsa Dependent Variable: ACTB Bonf erroni (I) genot y pe -/+/+/+ (J) genoty pe +/+/+ -/+/+ -/+/- Mean Dif f erence (I-J) St d Error -.9892* 2261 -1.6990* 2184 9892* 2261 -.7098* 2306 1.6990* 2184 7098* 2306 Sig .000 000 000 008 000 008 95% Conf idence Interv al Lower Bound Upper Bound -1.540 -.438 -2.231 -1.167 438 1.540 -1.272 -.148 1.167 2.231 148 1.272 * The mean dif f erence is signif icant at the 05 lev el a gene = IRF8 334 MyD88 Descriptivesa ACTB N -/+/+/+ Total 36 29 33 98 Mean 344 1.619 2.348 1.396 Std Dev iat ion 3173 1.3673 1.7750 1.5327 Std Error 0529 2539 3090 1548 95% Conf idence Interv al f or Mean Lower Bound Upper Bound 237 452 1.099 2.139 1.719 2.978 1.089 1.704 Minimum Maximum 1.7 5.8 8.4 8.4 a gene = My D88 ANOVAa ACTB Between Groups Within Groups Total Sum of Squares 71.194 156.688 227.882 df 95 97 Mean Square 35.597 1.649 F 21.582 Sig .000 a gene = My D88 Post Hoc Tests Multi ple Comparisonsa Dependent Variable: ACTB Bonf erroni (I) genot y pe -/+/+/+ (J) genoty pe +/+/+ -/+/+ -/+/- Mean Dif f erence (I-J) St d Error -1.2744* 3205 -2.0042* 3095 1.2744* 3205 -.7298 3269 2.0042* 3095 7298 3269 Sig .000 000 000 084 000 084 95% Conf idence Interv al Lower Bound Upper Bound -2.055 -.493 -2.758 -1.250 493 2.055 -1.526 067 1.250 2.758 -.067 1.526 * The mean dif f erence is signif icant at the 05 lev el a gene = My D88 335 NFKB Descriptivesa ACTB N -/+/+/+ Total 36 29 33 98 Mean 262 763 1.410 797 Std Dev iat ion 2973 4849 1.5461 1.0596 Std Error 0495 0900 2691 1070 95% Conf idence Interv al f or Mean Lower Bound Upper Bound 162 363 578 947 862 1.958 584 1.009 Minimum 0 Maximum 1.6 2.0 8.1 8.1 a gene = NFKB ANOVAa ACTB Between Groups Within Groups Total Sum of Squares 22.728 86.173 108.901 df 95 97 Mean Square 11.364 907 F 12.528 Sig .000 a gene = NFKB Post Hoc Tests Multi ple Comparisonsa Dependent Variable: ACTB Bonf erroni (I) genot y pe -/+/+/+ (J) genoty pe +/+/+ -/+/+ -/+/- Mean Dif f erence (I-J) St d Error -.5004 2376 -1.1477* 2295 5004 2376 -.6474* 2424 1.1477* 2295 6474* 2424 Sig .114 000 114 027 000 027 95% Conf idence Interv al Lower Bound Upper Bound -1.080 079 -1.707 -.588 -.079 1.080 -1.238 -.057 588 1.707 057 1.238 * The mean dif f erence is signif icant at the 05 lev el a gene = NFKB 336 NS1 Descriptivesa ACTB N -/+/+/+ Total 33 22 29 84 Mean 1325841 3959.682 65040.694 544357.7 Std Dev iat ion 1838114.4622 3978.7929 100212.6060 1306331.5918 Std Error 319974.7 848.2815 18609.01 142532.5 95% Conf idence Interv al f or Mean Lower Bound Upper Bound 674073.865 1977607.961 2195.584 5723.780 26921.857 103159.531 260866.324 827848.992 Minimum 2720.6 146.1 700.1 146.1 Maximum 7893452.3 13111.6 447502.8 7893452.3 a gene = NS1 ANOVAa ACTB Between Groups Within Groups Total Sum of Squares 3E+013 1E+014 1E+014 df 81 83 Mean Square 1.662E+013 1.338E+012 F 12.419 Sig .000 a gene = NS1 Post Hoc Tests Multi ple Comparisonsa Dependent Variable: ACTB Bonf erroni (I) genot y pe -/+/+/+ (J) genoty pe +/+/+ -/+/+ -/+/- Mean Dif f erence (I-J) St d Error 1321881.2* 318407.1 1260800.2* 294448.7 -1321881.2* 318407.1 -61081.012 327072.8 -1260800.2* 294448.7 61081.0123 327072.8 Sig .000 000 000 1.000 000 1.000 95% Conf idence Interv al Lower Bound Upper Bound 543466.836 2100295.626 540957.361 1980643.077 -2100295.626 -543466.836 -860680.621 738518.596 -1980643.077 -540957.361 -738518.596 860680.621 * The mean dif f erence is signif icant at the 05 lev el a gene = NS1 337 TLR4 Descriptivesa ACTB N -/+/+/+ Total 36 29 33 98 Mean 280 558 1.008 607 Std Dev iat ion 2791 3325 8310 6189 Std Error 0465 0618 1447 0625 95% Conf idence Interv al f or Mean Lower Bound Upper Bound 185 374 432 685 713 1.303 483 731 Minimum 0 Maximum 1.6 1.7 4.6 4.6 a gene = TLR4 ANOVAa ACTB Between Groups Within Groups Total Sum of Squares 9.234 27.920 37.154 df 95 97 Mean Square 4.617 294 F 15.710 Sig .000 a gene = TLR4 Post Hoc Tests Multi ple Comparisonsa Dependent Variable: ACTB Bonf erroni (I) genot y pe -/+/+/+ (J) genoty pe +/+/+ -/+/+ -/+/- Mean Dif f erence (I-J) St d Error -.2787 1353 -.7284* 1307 2787 1353 -.4497* 1380 7284* 1307 4497* 1380 Sig .126 000 126 005 000 005 95% Conf idence Interv al Lower Bound Upper Bound -.608 051 -1.047 -.410 -.051 608 -.786 -.113 410 1.047 113 786 * The mean dif f erence is signif icant at the 05 lev el a gene = TLR4 338 TLR9 Descriptivesa ACTB N -/+/+/+ Total 35 27 33 95 Mean 541 1.508 1.181 1.038 Std Dev iat ion 5834 1.0388 8060 8975 Std Error 0986 1999 1403 0921 95% Conf idence Interv al f or Mean Lower Bound Upper Bound 340 741 1.097 1.919 896 1.467 855 1.221 Minimum Maximum 2.8 3.9 3.2 3.9 a gene = TLR9 ANOVAa ACTB Between Groups Within Groups Total Sum of Squares 15.298 60.416 75.714 df 92 94 Mean Square 7.649 657 F 11.648 Sig .000 a gene = TLR9 Post Hoc Tests Multi ple Comparisonsa Dependent Variable: ACTB Bonf erroni (I) genot y pe -/+/+/+ (J) genoty pe +/+/+ -/+/+ -/+/- Mean Dif f erence (I-J) St d Error -.9672* 2076 -.6409* 1966 9672* 2076 3263 2103 6409* 1966 -.3263 2103 Sig .000 005 000 372 005 372 95% Conf idence Interv al Lower Bound Upper Bound -1.473 -.461 -1.120 -.161 461 1.473 -.186 839 161 1.120 -.839 186 * The mean dif f erence is signif icant at the 05 lev el a gene = TLR9 339 TNF Alpha Descriptivesa ACTB N -/+/+/+ Total 36 30 32 98 Mean 1.409 3.584 21.467 8.624 Std Dev iat ion 1.8447 3.5722 17.1666 13.4468 Std Error 3075 6522 3.0347 1.3583 95% Conf idence Interv al f or Mean Lower Bound Upper Bound 784 2.033 2.250 4.918 15.278 27.656 5.928 11.320 Minimum 3.4 Maximum 8.6 15.0 68.1 68.1 a gene = TNF Alpha ANOVAa ACTB Between Groups Within Groups Total Sum of Squares 7914.451 9624.668 17539.119 df 95 97 Mean Square 3957.226 101.312 F 39.060 Sig .000 a gene = TNF Alpha Post Hoc Tests Multi ple Comparisonsa Dependent Variable: ACTB Bonf erroni (I) genot y pe -/+/+/+ (J) genoty pe +/+/+ -/+/+ -/+/- Mean Dif f erence (I-J) St d Error -2.1757 2.4882 -20.0585* 2.4455 2.1757 2.4882 -17.8828* 2.5579 20.0585* 2.4455 17.8828* 2.5579 Sig 1.000 000 1.000 000 000 000 95% Conf idence Interv al Lower Bound Upper Bound -8.240 3.888 -26.018 -14.099 -3.888 8.240 -24.117 -11.649 14.099 26.018 11.649 24.117 * The mean dif f erence is signif icant at the 05 lev el a gene = TNF Alpha 340 8.12 Graphical data of invidual gene expression analysis in lung homogenate Measured by Quantitative Real-Time PCR NS1 PR8 H1N1 SD WT +/+ / ACTB 65040.7 / ACTB 65040.7 / ACTB 100212.6 HZ +/- 3959.7 3959.7 3978.8 KO -/- 1325840.9 1325840.9 1838114.5 Mean Comparative Expression Level Mean Comparative Corrected Mean Comparative Expression Level, If MCE < 1, Genotype Expression Level Reduction Fold Change ((Fold Change) 21/MCE) ΔΔCp Terminal (Days 7-11 Post Infection) Mean Comparative Expression Level of NS1 PR8 H1N1 InfAV in IRF-4 mice 3.5E+06 lung homogenate 3.0E+06 *** 2.5E+06 *** 2.0E+06 1325840.9 1.5E+06 1.0E+06 5.0E+05 65040.7 3959.7 WT +/+ HZ +/- 0.0E+00 WT +/+ 1.1 1.1 1.3 HZ +/- 0.5 -1.9 -0.9 KO -/- 0.2 -4.4 -0.8 3.0 Mean Comparative Expression Level IRF-4 2.0 KO -/- Terminal (Days 7-11 Post Infection) IRF-4 Fold Change in IRF-4 mice lung homogenate ** 1.1 1.0 0.0 -1.0 -2.0 -1.9 -3.0 -4.0 -4.4 -5.0 -6.0 WT +/+ 0.3 -3.6 -0.82 HZ +/- 0.7 -1.5 -1.53 KO -/- IFN-Beta 0.2 -6.1 -0.43 WT +/+ 0.1 -14.9 -0.1 HZ +/- 0.1 -8.4 -0.2 KO -/- 0.0 -32.6 -0.2 KO -/- Terminal (Days 7-11 Post Infection) IFN-Alpha Fold Change in IRF-4 mice lung homogenate Mean Comprarative Expression Level WT +/+ 0.0 -1.0 -2.0 -1.5 -3.0 -4.0 -3.6 -5.0 -6.0 -6.1 -7.0 WT +/+ HZ +/- KO -/- Terminal (Days 7-11 Post-Infection) IFN-Beta Fold Change in IRF-4 mice lung homogenate Mean Comparative Expression Level IFN-Alpha HZ +/- 0.0 -5.0 -10.0 -8.4 -15.0 -14.9 -20.0 -25.0 -30.0 -35.0 WT +/+ HZ +/- -32.6 KO -/- Figure 8.7 mRNA expression analysis of Lung homogenate by qRT-PCR (1) 341 IFN-Gamma WT +/+ SD / ACTB / ACTB / ACTB 89.9 89.9 44.0 HZ +/- 4.3 4.3 3.7 KO -/- 2.4 2.4 2.7 Mean Comparative Expression Level Mean Comparative Corrected Mean Comparative Expression Level, If MCE < 1, Genotype Expression Level Reduction Fold Change ((Fold Change) 21/MCE) ΔΔCp 140.0 Terminal (Days 7-11 Post-Infection) IFN-Gamma Fold Change in IRF-4 mice lung homogenate *** 120.0 100.0 89.9 *** 80.0 60.0 40.0 20.0 WT +/+ WT +/+ HZ +/KO -/- 4.1 2.0 0.7 4.1 2.0 -1.4 4.0 2.0 -0.9 Mean Comparative Expression Level IRF-1 10.0 0.5 -1.9 -0.4 HZ +/- 0.5 -1.8 -0.4 KO -/- 0.1 -7.6 -0.1 3.0 3.0 1.3 HZ +/- 2.1 2.1 1.3 KO -/- 0.4 -2.5 -0.4 Mean Comparative Expression Level WT +/+ KO -/- *** 8.0 ** 6.0 4.1 4.0 2.0 2.0 0.0 -2.0 HZ +/- 1.0 0.0 -1.0 -2.0 -3.0 -4.0 -5.0 -6.0 -7.0 -8.0 -9.0 *** -1.4 KO -/- 5.0 4.0 *** -1.8 -1.9 -7.6 WT +/+ IRF-5 HZ +/- Terminal (Days 7-11 Post-Infection) IRF-3 Fold Change in IRF-4 mice lung homogenate Mean Comparative Expression Level WT +/+ 2.4 Terminal (Days 7-11 Post Infection) IRF-1 Fold Change in IRF-4 mice lung homogenate WT +/+ IRF-3 4.3 0.0 HZ +/- KO -/- Terminal (Days 7-11 Post Infection) IRF-5 Fold Change in IRF-4 mice lung homogenate *** 3.0 3.0 *** 2.1 2.0 1.0 0.0 -1.0 -2.0 -3.0 -2.5 -4.0 WT +/+ HZ +/- KO -/- Figure 8.8 mRNA expression analysis of Lung homogenate by qRT-PCR (2) 342 IRF-7 WT +/+ / ACTB 18.4 / ACTB 18.4 SD / ACTB 14.6 HZ +/- 8.6 8.6 7.5 KO -/- 1.7 1.7 2.1 Mean Comparative Expression Level Mean Comparative Corrected Mean Comparative Expression Level, If MCE < 1, Genotype Expression Level Reduction Fold Change ((Fold Change) 21/MCE) ΔΔCp 35.0 Terminal (Days 7-11 Post-Infection) IRF-7 Fold Change in IRF-4 mice lung homogenate 30.0 *** 25.0 *** 20.0 * 15.0 10.0 5.0 0.0 WT +/+ NFKb 2.0 1.3 HZ +/- 1.3 1.3 0.9 KO -/- 0.3 -3.1 -0.2 WT +/+ 2.3 2.3 1.8 HZ +/- 1.6 1.6 1.4 KO -/- 0.3 -2.9 -0.3 WT +/+ 1.4 1.4 1.5 HZ +/- 0.8 -1.3 -0.5 KO -/- 0.3 -3.8 -0.3 KO -/- Terminal (Days 7-11 Post Infection) IRF-8 Fold Change in IRF-4 mice lung homogenate Mean Comparative Expression Level 2.0 4.0 3.0 *** *** 2.0 2.0 1.3 1.0 0.0 -1.0 -2.0 -3.0 -3.1 -4.0 WT +/+ HZ +/- KO -/- Terminal (Days 7-11 Post Infection) MyD88 Fold Change in IRF-4 mice lung homogenate Mean Comparative Expression Level MyD88 WT +/+ Mean Comparative Expression Level IRF-8 HZ +/- 5.0 4.0 3.0 2.3 *** *** 1.6 2.0 1.0 0.0 -1.0 -2.0 -3.0 -2.9 -4.0 WT +/+ 4.0 HZ +/- Terminal (Days 7-11 Post Infection) NFKb Fold Change in IRF-4 mice lung homogenate *** 3.0 2.0 KO -/- 1.4 * 1.0 0.0 -1.0 -1.3 -2.0 -3.0 -4.0 WT +/+ HZ +/- -3.8 KO -/- Figure 8.9 mRNA expression analysis of Lung homogenate by qRT-PCR (3) 343 Table 8.1 Top 30 differentially expressed genes between IRF-4 +/+ WT vs +/- HZ vs -/- KO Comparison was made between the PR8 H1N1 InfAV-infected mice of three IRF-4 genotypes (IRF4 +/+ WT, IRF-4 +/- HZ and IRF-4 -/- KO) Significance analysis was performed with 1-way ANOVA with fold change analysis and the genelist is arranged by the p-value ProbeID 3130707 5810332 1580519 4570458 Symbol Pdgfb Mtap1b Anxa3 Mus musculus annexin A3 (Anxa3), mRNA Sn Slc7a8 2350440 610324 2760131 1580431 5820392 540138 240435 4850382 10411 3310274 20201 3310026 1690079 430670 1070307 670204 2230341 Tpbg Cfl1 1500012F01Rik Rnf30 Sfrs3 Sytl1 1700007G11Rik Akap7 Clec3b 6520725 5220725 2450239 4050609 630678 990438 2100196 Gpr43 Mus musculus mitogen activated protein kinase (Mapk9), transcript variant 1, mRNA Mus musculus free fatty acid receptor (Ffar2), mRNA Cotl1 Fnbp1 7.21E-05 1.14E-04 1.16E-04 1.24E-04 1.38E-04 1.47E-04 1.51E-04 1.86E-04 2.23E-04 2.76E-04 2.88E-04 Mus musculus metastasis associated (Mta3), mRNA Mus musculus coactosin-like (Dictyostelium) (Cotl1), mRNA Mus musculus formin binding protein (Fnbp1), transcript variant 1, mRNA FC ([-/- Test] vs [+/+ Test]) FC ([-/- Test] vs [+/- Test]) -1.2 -1.1 1.1 -1.1 -1.4 -1.3 -1.1 1.4 1.5 -1.3 1.5 1.9 -1.2 -1.3 -1.1 1.1 -2.3 -2.6 1.1 -2.9 -3.3 1.2 -2.4 -2.9 -1.1 1.2 1.3 1.0 1.4 1.4 1.1 -1.7 -1.8 1.1 1.5 1.4 -1.0 -1.6 -1.6 -1.0 1.5 1.6 1.1 -2.0 -2.1 -1.0 1.6 1.6 1.0 1.3 1.3 -1.1 -1.7 -1.6 1.1 -4.4 -5.0 -1.1 2.0 2.2 1.0 -1.5 -1.5 1.2 -1.7 -2.0 -1.1 -1.6 -1.5 -1.1 1.1 1.1 1.1 2.1 2.0 1.2 -1.6 -1.9 -1.1 -5.7 -5.0 1.1 -1.5 -1.6 1.1 -1.4 -1.5 -1.0 -1.8 -1.7 -1.1 -1.5 -1.4 2.05E-04 2.55E-04 Mus musculus inducible T-cell co-stimulator (Icos), mRNA Vti1a Mta3 6.26E-05 2.02E-04 A730042J05Rik Icos 6.04E-05 1.89E-04 A930008G19Rik Mapk9 5.82E-05 1.65E-04 Mus musculus selectin, platelet (p-selectin) ligand (Selplg), mRNA Ian3 2690292 5.12E-05 1.53E-04 Foxa1 FC ([+/- Test] vs [+/+ Test]) 4.59E-05 5.59E-05 Mus musculus RIKEN cDNA 2410042D21 gene (2410042D21Rik), mRNA Mus musculus trophoblast glycoprotein (Tpbg), mRNA Mus musculus cofilin 1, non-muscle (Cfl1), mRNA Mus musculus RIKEN cDNA 1500012F01 gene (1500012F01Rik), mRNA Mus musculus tripartite motif-containing 54 (Trim54), mRNA Mus musculus splicing factor, arginine/serinerich (SRp20) (Sfrs3), mRNA Mus musculus synaptotagmin-like (Sytl1), mRNA Mus musculus RIKEN cDNA 1700007G11 gene (1700007G11Rik), mRNA Mus musculus A kinase (PRKA) anchor protein (Akap7), mRNA Mus musculus C-type lectin domain family 3, member b (Clec3b), mRNA Cd8b Selpl 4.24E-05 4.79E-05 Mus musculus G protein-coupled receptor 68 (Gpr68), mRNA Cd3d 2410042D21Rik 4.17E-05 4.30E-05 Mus musculus solute carrier family (cationic amino acid transporter, y+ system), member (Slc7a8), mRNA BC021614 Gpr68 p 1.63E-05 Mus musculus microtubule-associated protein B (Mtap1b), mRNA 6550687 4290072 Definition 2.90E-04 3.02E-04 3.05E-04 344 Table 8.2 Fold Change of significant and non-signifcant differentially expressed genes between IRF-4 +/+ WT vs IRF-4 +/- HZ vs IRF-4 -/- KO, concurrently investigated for Cytokine Analysis by BioPlex and mRNA expression by qRT-PCR, arranged alphabetically by gene name p FC ([+/- Test] vs [+/+ Test]) FC ([-/- Test] vs [+/+ Test]) FC ([-/- Test] vs [+/Test]) 0.04464356 0.3512405 0.9349415 0.03324678 0.6306312 -1.1 -1.3 1.0 -1.4 -1.3 -1.3 1.5 1.2 -2.0 -1.5 -1.2 1.9 1.1 -1.4 -1.1 0.5489506 -1.1 1.0 1.2 0.61776745 1.2 -1.0 -1.2 0.01450484 -1.5 1.8 2.6 0.17187747 0.00694924 0.009921 -1.2 -1.2 1.0 2.8 1.6 1.7 3.3 1.9 1.7 -1.4 -1.2 -1.2 -1.2 -1.0 1.1 1.3 -1.2 -1.3 1.6 3.6 3.6 -1.1 1.0 3.6 -1.3 1.1 1.9 4.3 4.3 -1.0 -1.1 2.8 -1.0 1.7 1.9 1.2 1.0 -1.5 1.1 1.4 -1.1 -1.4 1.4 1.4 -1.5 Mus musculus interferon beta 1, fibroblast (Ifnb1), mRNA 0.00609144 Mus musculus interferon gamma (Ifng), mRNA 0.03848248 Mus musculus interferon gamma (Ifng), mRNA 0.22179013 0.00759154 Mus musculus interleukin 10 (Il10), mRNA 0.00768979 0.14338186 Mus musculus interleukin 13 (Il13), mRNA 0.2135997 0.00457027 Mus musculus interleukin induced (Il4i1), mRNA 0.36216855 -1.2 -1.2 -1.0 1.0 -1.1 1.2 -1.0 -1.3 1.2 2.1 -1.8 -1.7 1.4 -2.2 -1.1 -1.3 3.3 1.3 2.5 -1.5 -1.6 1.4 -2.0 -1.3 -1.3 4.3 1.2 Irf1 Mus musculus interferon regulatory factor (Irf1), mRNA 0.02809242 -1.5 -1.2 1.2 6660634 Irf1 Mus musculus interferon regulatory factor (Irf1), mRNA 0.14142697 -1.3 -1.4 -1.1 3360138 Irf1 Mus musculus interferon regulatory factor (Irf1), mRNA 0.3168505 -1.3 -1.1 1.2 1190088 4250291 4280193 Irf1 Irf3 Irf3 Mus musculus interferon regulatory factor (Irf1), mRNA 0.35852087 0.13625193 0.14759001 -1.1 1.1 1.0 1.0 1.2 1.1 1.1 1.2 1.1 1010397 Irf4 Mus musculus interferon regulatory factor (Irf4), mRNA 0.08393885 1.6 1.1 -1.5 3140646 6590653 Irf5 Irf7 -1.2 -1.4 -1.1 1.2 1.1 1.7 4810286 2070014 4050626 Myd88 Nfkb2 Tnf Mus musculus interferon regulatory factor (Irf5), mRNA 0.0900343 0.05245507 Mus musculus myeloid differentiation primary response gene 88 (Myd88), mRNA 0.21271646 0.9164811 Mus musculus tumor necrosis factor (Tnf), mRNA 0.13050207 -1.3 1.0 -1.4 -1.0 1.1 -1.0 1.2 1.0 1.4 ProbeID Symbol Definition 1190241 110112 2760047 6940184 7150500 Ccl1 Ccl2 Ccl3 Ccl4 Ccl4 1690768 Ccl5 2480296 Ccl8 3610082 Cxcl1 3140209 7550112 1030025 Cxcl10 Cxcl12 Cxcl12 20088 7550112 3060040 3060040 150746 3520349 520403 630274 Cxcl12 Cxcl12 Cxcl13 Cxcl13 Cxcl14 Cxcl14 Cxcl15 Cxcl16 5390639 Cxcl2 4040524 10598 4780072 Cxcl4 Cxcl9 Ifna1 Mus musculus chemokine (C-X-C motif) ligand 12 (Cxcl12), transcript variant 3, mRNA 0.11567923 0.00694924 0.00224982 0.00224982 0.7883063 0.8685516 0.13632338 0.0301476 Mus musculus chemokine (C-X-C motif) ligand (Cxcl2), mRNA 0.02922661 Mus musculus chemokine (C-X-C motif) ligand (Cxcl4), mRNA 0.1228403 0.3372854 Mus musculus interferon alpha (Ifna1), mRNA 0.02945433 6280592 6280477 2850092 3420139 1260632 2070468 5270719 3420754 6020224 Ifnb1 Ifng Ifng Il1 Il10 Il12a Il13 Il1b Il4i1 3400288 Mus musculus chemokine (C-C motif) ligand (Ccl1), mRNA Mus musculus chemokine (C-C motif) ligand (Ccl5), mRNA Mus musculus chemokine (C-C motif) ligand (Ccl8), mRNA Mus musculus chemokine (C-X-C motif) ligand (Cxcl1), mRNA Mus musculus chemokine (C-X-C motif) ligand 10 (Cxcl10), mRNA 345 Figure 8.10 This pathway is initiated by IFNG binding to its receptor and a subsequent phosphorylation cascade involving a number of the JAK and STAT family of proteins Several transcriptionally active complexes are formed (STAT1 homodimer, ISGF3 complex, STAT1:STAT1:IRF9 complex) and the pathway culminates with the transcriptional activation of target genes 346 [...]... Interferon Regulatory Factor-4 (IRF-4) is essential for the modulating or eliciting host adaptive immune responses against Influenza A pneumonitis 7 1.3 GENERAL INTRODUCTION 1.3.1 The respiratory system The respiratory system, which consists of the airways, lung and respiratory muscles functions to obtain oxygen from the external environment and to remove carbon dioxide from the body The respiratory tract... submucosa and regional lymph nodes 1.3.3 Respiratory Viral Pathogens Respiratory viruses contribute to significant morbidity and mortality in humans and cause large economic losses worldwide The infections that these pathogens cause are mostly self-limiting in healthy adults but are important factors in the illness and death of the very young, immunologically-compromised and elderly 1 It is estimated that respiratory. .. sporadic or epidemic respiratory infection in infants, children and adults (Mackie 2003) Of these viruses that replicate in the respiratory tract, coronaviruses, influenza virus, parainfluenza virus, respiratory syncytial virus and rhinoviruses produce infections that are primarily restricted to the respiratory mucosa and are not generally accompanied by systemic disease Serious respiratory diseases are... immunologically-compromised and elderly 1 It is estimated that respiratory viral pathogens account for about 5% of all deaths and for about 60% of deaths related to related to respiratory disease (Welliver and Ogra 1988) Species within the Adenoviridae, Coronaviridae, Herpesviridae, Orthomyxoviridae, Paramyxoviridae and Picoviridae are classified as causes of respiratory tract infection More than 200 antigenically distinct... of histograms of MHC-Peptide binding assay 2.9 51 2.10 51 2.11 51 2.12 Cytotoxicity of PBS control immunized mice 2.13 Cytotoxicity of splenocytes from mice immunized with DNA vector 2.14 Cytotoxicity of splenovytes from mice immunized with S-His DNA vaccine 2.15 Cytotoxicity of splenocytes from mice immunized with S-RGD/His DNA vaccine 2.16 ELISPOT of IFN-γ response against putative T-cell epitopes... immunology and medicine and contributes significantly to human and livestock health The goal of vaccination is to alter the adaptive immune system to obtain clinical benefits Attenuated vaccines made up of physiochemically-altered or genetically manipulated live pathogens, are most productive as they stimulate the most effective and lasting immunity to any natural infection These vaccines are able to elicit... Post Infection This allowed for its humoral immune response to be examined The cytokine analysis of lung homogenate was then analyzed using BioPlex multiplexing technology A cytokine profile in terms of pro-inflammatory cytokines, Th1 and Th2 cytokines and chemokines were generated In order to gain a better 5 understanding of mRNA expression of cytokines as well as other IRF family members in the lung... must therefore be able to defend against airborne particles and microorganisms present in the air The airway is made up of the upper and lower respiratory tract, where the former includes the nasal sinuses and nasopharynx and the latter begins at the larynx and continues to the trachea, terminating at the alveoli The lung, having the largest epithelial surface area in the body to facilitate efficient... A/Aichi/2/68 H3N2 virus than to A/PR/8/34 H1N1 Virus 9th Asia Pacific Congress for Medical Virology, Adelaide Convention Centre, South Australia, Australia Oral Presentation 6-8 June 2012 Young Investigator Conference Travel Scholarship Award Recipient xix CHAPTER 1 1.1 Prologue This thesis describes the study on the immunity and immunization against respiratory viral pathogens in the mouse animal... the club-shaped peplomers which project from the envelope and populate the virion surface (Holmes and Lai 1996) The S protein drives the entry of the virion into target cells via receptor-mediated endocytosis (Gillim-Ross and Subbarao 2006) It also functions as the major viral attachment protein that is critical to virus binding and fusion of the viral envelope (Holmes 2001) Source: (Holmes 2001) Figure ... as the bronchial submucosa and regional lymph nodes 1.3.3 Respiratory Viral Pathogens Respiratory viruses contribute to significant morbidity and mortality in humans and cause large economic losses... technology A cytokine profile in terms of pro-inflammatory cytokines, Th1 and Th2 cytokines and chemokines were generated In order to gain a better understanding of mRNA expression of cytokines as... respiratory system, which consists of the airways, lung and respiratory muscles functions to obtain oxygen from the external environment and to remove carbon dioxide from the body The respiratory