Avian Influenza virus M2e protein: Epitope mapping, competitive ELISA and phage displayed scFv for DIVA in H5N1 serosurveillance Noor Haliza Hasan A thesis submitted for the degree of Doctor of Philosophy School of Animal Veterinary Sciences The University of Adelaide Australia March 2017 1 Table of Contents Abstract………………………………………………………………………………………..v Thesis declaration …………………………………………………………………………...vii Acknowledgement ………………………………………………………………………… viii List of Tables ………………………………………………………………………………...xi List of Figures …………………………………………………………………………… xiii List of Abbreviations ………………………………………………………………………xvii List of Publications …………………………………………………………………………xix Introduction and Literature review ................................................ 1 Introduction ........................................................................................................... 3 Thesis outline ......................................................................................................... 5 Avian Influenza Virus (AIV): enzootic H5N1 and DIVA test in Indonesia ......... 6 AIV genes, M2 and M2e protein ........................................................................... 7 M2 protein ........................................................................................................... 11 M2e as potential universal vaccine ...................................................................... 12 M2e as DIVA marker .......................................................................................... 13 Antigenic mapping .............................................................................................. 14 Antibody and Antigenic determinants ................................................................. 15 M2e antigenic determinants ................................................................................ 17 Enzyme-linked immunosorbent assay (ELISA) for AIV surveillance and DIVA .............................................................................................................................................. 19 ELISA principles, components and types ........................................................... 19 AIV and ELISA ................................................................................................... 23 Phage display technology .................................................................................... 24 Naïve and immunized library as a source of phage library ................................. 26 Filamentous bacteriophage for phage display ..................................................... 27 Recombinant antibody display format ................................................................. 28 Phagemid as phage display vector ....................................................................... 30 Research Rationale and Aims .............................................................................. 32 References ........................................................................................................... 33 Avian influenza virus and DIVA strategies .................................. 52 Avian influenza virus (AIV) ................................................................................ 55 i2 Gene segments and proteins ................................................................................ 55 AIV transmissibility ............................................................................................ 56 AIV evolution ...................................................................................................... 56 AIV pathogenicity ............................................................................................... 57 LPAIV and HPAIV in poultry ............................................................................. 58 Virulence shift of LPAIV to HPAIV ................................................................... 58 Evolutionary pattern of H5N1 ............................................................................. 59 AIV and vaccination ............................................................................................ 61 Current understanding of DIVA strategies for AIV ............................................ 62 Sentinel birds ....................................................................................................... 63 Recombinant subunit vaccines ............................................................................ 63 Heterologous NA vaccine .................................................................................... 64 AIV nonstructural 1 (NS1) protein: Differential immune response .................... 65 Matrix 2 ectodomain (M2e) protein: Highly conserved protein.......................... 66 Haemagglutinin subunit 2 (HA2) glycoprotein (gp): Highly conserved epitope 66 DIVA strategies applicability and developments ................................................ 69 DIVA vaccine-based strategies: recombinant subunit and heterologous NA ..... 69 DIVA test-based strategies: NS1, M2e and HA2 proteins .................................. 71 Recommendations for DIVA programs ............................................................... 79 Acknowledgements ............................................................................................. 80 References ........................................................................................................... 80 Epitope mapping of avian influenza M2e protein: different species recognise various epitopes ................................................. 94 Introduction ......................................................................................................... 98 Material & Methods .......................................................................................... 100 Peptides for mouse and rabbit immunization and antigenic mapping ............... 100 Antibodies (sera) ............................................................................................... 101 Indirect M2e-ELISA and antigenic mapping .................................................... 104 Statistical and bioinformatics analyses .............................................................. 105 Ethics statement ................................................................................................. 105 Results ............................................................................................................... 106 Chicken, mouse and rabbit antibodies selection using indirect-M2e ELISA .... 106 Chicken sera recognized at least 2 different epitopes spanning M2e residue 5-18 and 10-17 ........................................................................................................... 106 Chicken sera reactivity pattern is highly influenced by its immunogen as well as individual chicken immune response ................................................................ 111 3 ii Mouse monoclonal antibodies recognized epitopes M2e2-18 and M2e11-18 while rabbit polyclonal antibodies recognized epitope M2e6-13 .................................. 111 Discussion .......................................................................................................... 114 Acknowledgment ............................................................................................... 120 References ......................................................................................................... 120 Characterization of monoclonal antibodies to the avian influenza virus H5N1 M2e protein and their potential use for diagnostic tool competitive ELISA development ......................................... 127 Introductions ...................................................................................................... 130 Material and Methods ........................................................................................ 133 M2e peptide as antigen for indirect and competitive ELISA ............................ 133 Monoclonal antibody (mAb) ............................................................................. 133 Sera .................................................................................................................... 134 Indirect M2e-ELISA .......................................................................................... 136 Development and Standardization of competitive ELISA ................................ 137 Data analysis and statistics ................................................................................ 138 Results ............................................................................................................... 138 Chicken polyclonal and mouse monoclonal antibodies anti-M2e titers ............ 138 Selection of mAb 3H4 as the competitor in M2e-based competitive ELISA ... 138 cELISA using mAb 3H4 distinguished infected from vaccinated chicken sera 139 Sensitivity and specificity of M2e-based cELISA ............................................ 143 Discussion .......................................................................................................... 144 Acknowledgement ............................................................................................. 148 References ......................................................................................................... 148 Isolation of reactive single-chain variable fragment antibodies against AIV-M2e protein (scFv anti-M2e) using phage display technology from H5N1-immunized chicken IgY ....................... 153 Introduction