Characterizing evolutionarily conserved influenza a virus sequences as vaccine targets

CHARACTERIZING EVOLUTIONARILY CONSERVED INFLUENZA A VIRUS SEQUENCES AS VACCINE TARGETS HEINY B.Sc (Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2008 Parts of this dissertation work have contributed to the following publications: Heiny AT, Miotto O, Srinivasan KN, Khan AM, Zhang GL, Brusic V, Tan TW, August JT (2007) Conserved protein sequences of all influenza A viruses as vaccine targets PLoS ONE 2(11): e1190 (Citation: 1) Contribution: Conceived, designed and performed the experiments; analyzed the data and drafted the manuscript Miotto O, Heiny AT, Tan TW, August JT, Brusic V (2008) Identification of human-to-human transmissibility factors in PB2 proteins of influenza A by largescale mutual information analysis BMC Bioinformatics Suppl 1: S18 (Citation: 6; Impact Factor: 3.49) Contribution: Collected and annotated the sequence data; analyzed the data Khan AM, Heiny AT, Lee KX, Srinivasan KN, Tan TW, August JT, Brusic V (2006) Large-scale analysis of antigenic diversity of T-cell epitopes in dengue virus BMC Bioinformatics Suppl 5: S4 (Citation: 7; Impact Factor: 3.49) Contribution: Participated in the design of the study Khan AM, Miotto O, Heiny AT, Salmon J, Srinivasan KN, Nascimento EJ, Marques ET Jr, Brusic V, Tan TW, August JT (2007) A systematic bioinformatics approach for selection of epitope-based vaccine targets Cell Immunol 244(2): 141-7 (Citation: 6; Impact Factor: 1.81) Contribution: Participated in the development of the methodology platform Zhang GL, Khan AM, Srinivasan KN, Heiny AT, Lee KX, Kwoh CK, August JT and Brusic V (2008) Hotspot Hunter: a computational system for large-scale screening and selection of candidate immunological hotspots in pathogen proteomes BMC Bioinformatics Suppl 1: S19 (Citation: 1; Impact Factor: 3.49) Contribution: Participated in the design of the study and pilot testing of the system Khan AM, Miotto O, Nascimento EJM, Srinivasan KN, Heiny AT, Zhang GL, Marques ET, Tan TW, Brusic V, Salmon J, August JT (2008) Conservation and Variability of Dengue virus Proteins: Implications for Vaccine Design PLoS Neglected Tropical Diseases 2(8): e272 Contribution: Participated in the design of experiments, data analysis, and methods development ii ACKNOWLEDGEMENT I would like to express my gratitude to my supervisors  Assoc Prof Tan Tin Wee (Department of Biochemistry, National University of Singapore), Dr Vladimir Brusic (Dana-Farber Cancer Institute, Harvard Medical School), and Prof J Thomas August (Johns Hopkins University)  for the opportunity given to me, for their guidance, advice, continuous support and encouragement I also would like to thank Mr Asif Khan (PhD candidate), Mr Olivo Miotto (PhD candidate), Ms Hu Yong Li (graduate student) and Dr KN Srinivasan (previously a postdoctoral fellow at Prof August’s laboratory) for their help and suggestions; Dr Paul August, Dr Lin Hong Huang, and Ms Zhang Guang Lan for their expert knowledge in programming and technical help; Dr Paul Tan Thiam Joo for his critical review of the thesis; Dr Songsak Tongchusak, Mr Mark de Silva and Mr Lim Kuan Siong for their support iii TABLE OF CONTENTS Introduction 1.1 The genome, structure and life cycle of influenza A virus .6 1.2 Influenza virus sequences and classification 12 1.3 Current vaccines against influenza and their limitations 15 1.4 Antigenic variation of influenza A viruses .18 1.5 Immune responses to influenza 22 1.6 The approach, aims and contribution of this work 28 Materials and Methods 30 2.1 General thesis overview 30 2.2 Sequence data collection and processing 32 2.3 Measuring the diversity of human and avian influenza A viruses 33 2.4 Selection of highly conserved human and avian influenza A virus sequences 35 2.5 Determining the immune-relevance of the highly conserved sequences 36 2.5.1 HLA supertype-restricted T-cell epitopes 36 2.5.2 Experimentally identified T-cell epitopes 39 2.6 Retrospective study on the stability of highly conserved sequences 39 2.7 Functional sites of influenza A virus proteins 40 Results and Discussion .41 3.1 Summary of results 41 3.2 Avian and human influenza A virus sequences 43 3.3 The diversity of human and avian influenza A virus proteins 45 3.4 Highly conserved sequences of influenza A virus were present mostly in the internal proteins 52 3.5 Highly conserved sequences contain numerous antigenic determinants 55 3.5.1 Prediction data 55 3.5.2 Experimental data 62 3.6 Stability of highly conserved sequences through the viral evolutionary history .64 3.7 Functional sites of influenza A virus proteins 68 Conclusions and Discussion 71 Future Work .76 REFERENCES 79 APPENDIX A WHO recommended seasonal vaccine composition 96 APPENDIX B List of identified highly conserved sequences 97 APPENDIX C Predicted epitopes in the highly conserved sequences 100 APPENDIX D Classification of HLA I and II molecules into supertypes 112 APPENDIX E Functional sites of influenza A virus proteins 113 APPENDIX F Permission to reproduce copyrighted materials 120 APPENDIX G Poster and oral presentations 122 APPENDIX H Publications .123 iv LIST OF FIGURES Figure Influenza A virus genome and structure .8 Figure Influenza A virus replication cycle .11 Figure Outline of thesis overview 31 Figure Nonamer entropy plot for avian influenza A viruses for the period 19771986, 1987-1996, and 1997-2006 47 Figure Nonamer entropy plot for avian and human H5N1 influenza A virus sequences 48 Figure Nonamer entropy plot of human H1N1, H3N2, and H1N2 influenza A virus sequences 50 Figure Entropy-sequence conservation relationship plot from data in this study 51 Figure Highly conserved sequences of influenza A viruses in human H1N1, H3N2, H1N2, H5N1, avian H5N1 and other avian subtypes circulating between 1997 and 2006 54 Figure Binding sequences present in the highly conserved sequences 63 Figure 10 Functional sites of the influenza A virus PB2, PB1, PA, NP and M1 proteins 69 Figure 11 Functional sites of the influenza A virus HA, NA, NS1, NS2, M2 and PB1F2 proteins 70 v LIST OF TABLES Table Influenza A virus proteins and their sequence lengths .7 Table Public influenza sequence databases 13 Table A summary of sialic acid linkages and binding site preferences 21 Table HLA I and II molecules that were included in this thesis and their corresponding supertypes 26 Table Number of amino acid sequences of influenza A virus proteins from the past decade (1997-2006) 44 Table Number of influenza A virus proteins sequences prior to 1997 .44 Table A summary of the identified highly conserved sequences in the avian and human influenza A virus sequences from 1997 to 2006 53 Table List of predicted epitopes within the highly conserved sequences 57 Table Summary of the number of predicted epitopes within the highly conserved sequences 59 Table 10 Summary of the number of highly conserved sequences with predicted epitopes per HLA supertype .61 Table 11 Highly conserved sequences that remain conserved in the retrospective study using data from prior to 1997 65 Table 12 List of highly conserved sequences that were less than 80% conserved in sequences prior to 1997 67 vi LIST OF ABBREVIATIONS ABK ANN APC ARB AVANA BIG CDC cRNA DC DMID ELISA ELISPOT ER FDA MHC HA or H HIV HLA HMM HPAI IC50 ICTVdb IEDB IFN M1 M2 NA or N NCBI NIAID nM NP NS1 NS2 PA PB1 PB2 RNA RNP SN ssRNA SP TAP TLR vRNA WHO Aggregator of Biological Knowledge Artifical Neural Network Antigen Presenting Cell Average Relative Binding Antigenic Variability Analyser tool Beijing Institute of Genomics Centers for Disease Control and Prevention complementary RiboNucleic Acid Dendritic Cell Division of Microbiology and Infectious Diseases Enzyme-Linked ImmunoSorbent Assay Enzyme-Linked Immunosorbent Spot Endoplasmic Reticulum Food and Drug Administration Major Histocompatibility Complex Hemagglutinin Human Immunodeficiency Virus Human Leukocyte Antigen Hidden Markov Model Highly Pathogenic Avian Influenza Median Inhibition Concentration The Universal Virus Database by International Committee on Taxonomy of Viruses Immune Epitope Database Interferon Matrix protein Matrix protein Neuraminidase National Center for Biotechnology Information National Institute of Allergy and Infectious Diseases Nanomolar Nucleoprotein Non-Structural protein Non-Structural protein Polymerase Acidic protein Polymerase Basic protein Polymerase Basic protein Ribonucleic Acid Ribonucleoprotein particle Sensitivity single-stranded Ribonucleic Acid Specificity Transporter of Antigen Processing Toll-Like Receptor viral Ribonucleic Acid World Health Organization vii SUMMARY Influenza A viruses generate an extreme genetic diversity through point mutation and gene segment exchange, resulting in many new strains and variants that emerge from the avian reservoirs, among which was the emergence of highly pathogenic H5N1 virus Given the looming threat of emergence of an influenza pandemic, a vaccine that will provide broad spectrum coverage to influenza A subtypes/strains is a critical need One feasible approach is a vaccine containing conserved immunogenic protein sequences that represent the genotypic diversity of the currently known and newly emerged avian and human influenza viruses as an alternative to current vaccines that address only the known circulating virus strains This thesis focuses on bioinformatics approaches in characterizing the proteomes of known influenza A viruses for identification of highly conserved sequences that are potentially immunogenic in a broad spectrum of the human population Tools and methodologies for automated aggregation and annotation of influenza A virus sequences from public databases and identification of highly conserved sequences were developed A total of 36,343 sequences of various influenza subtypes from avian and human hosts were collected and classified into six major subgroups (human H1N1, human H1N2, human H3N2, human H5N1, avian H5N1 and other avian subtypes) for analysis Fifty-five (55) highly conserved sequences that were present in at least 80% of all sequences from the past decade (1997-2006) in each virus subgroups were defined and were present mostly in the internal proteins PB2, PB1, PA, NP and M1 Forty-nine (49) of 55 conserved sequences contained clusters of potential and reported T-cell epitopes Forty-nine (49) conserved sequences remained unchanged in at least 80% of human and avian sequences prior to 1997 Many of the highly conserved sequences are located in the functionally important domain/site of influenza A proteins, suggesting their relative importance to virus survival The identified sequences that are both highly conserved and immune-relevant are suitable candidates for a T-cell epitopebased vaccine and can be designed to provide a continuum of immune responses to influenza A infection (Word Count: 330) viii Introduction One of the most important threats to human health is infection by influenza A viruses, which has its natural reservoir in aquatic birds (de Jong et al., 2000; Treanor, 2004; Killbourne, 2006) While global influenza pandemics have occurred only three times in the past century, the H1N1 pandemic of 1918-1919 caused an estimated 20-50 million deaths, making it one of the most serious disease outbreaks in recorded history The recent evolution of the highly pathogenic avian influenza (HPAI) virus of H5N1 subtype, while not human-to-human transmissible, has emphasized the continuous threat of influenza viruses on a global scale (Peiris et al., 2007) The initial outbreak of the HPAI virus in poultry (Hong Kong, 1997) was soon followed by reports of human infections by avian influenza H5N1 with high fatality rate Since then, the World Health Organization (WHO) has reported a total of 382 human cases in 14 countries with 241 deaths (as of 30 April 2008) Outbreaks of HPAI virus of H7 subtype in poultry have also been reported, with several confirmed cases of human infection (Belser et al., 2008) It is widely predicted that because of the increased human population density and increased travel, a new pandemic on the scale of the H1N1 infection would have a devastating effect globally Besides the threat of pandemic, influenza A virus also poses challenges in the form of recurrent flu epidemics Influenza epidemics occur periodically, affecting the majority of world populations, of all age groups In the United States, 5-20% of the population is affected, resulting in some 200,000 hospitalizations and 30,000 deaths each year (Centers for Disease Control and Prevention, http://www.cdc.gov/flu/) Singapore is also affected by annual influenza, 20% of population is estimated to have flu with clinical symptoms, with the mortality rate of approximately 14.8 per 100,000 person-years (Lee et al., 2007) The socioeconomic burden caused by influenza from hospitalization costs, lost of work productivity, and death is huge (Szucs, 1999) Molinari et al (2007) conducted a study to estimate the economic impact of influenza epidemics in the United States based on 2003 population and concluded that the total economic burden of seasonal influenza, including both direct and indirect costs, to be more than $87 billion per year Vaccination has been the main strategy for prevention and control of disease (Nichol, 2008) However, despite the availability of seasonal influenza vaccines, their efficacy varies, depending on the match between vaccine strains and the circulating virus strains (Carrat and Flahault, 2007) This is mainly caused by the ability of influenza A virus to undergo rapid genetic changes known as antigenic drift, and occasionally major changes in the form of antigenic shift As a result of such variability, vaccination can only target against a small number of selected circulating strains In addition, the situation is further complicated by the disproportionate correlation between genetic change and antigenicity (Smith et al., 2004), making it difficult to anticipate the effectiveness of a preemptive vaccine formulation The issue of matching the vaccine formulation to viruses in circulation is complicated by the lengthy process of vaccine production The manufacturing process of the seasonal flu vaccine, which is based on technology that is more than 60 years old, takes about six to eight months It includes growing the selected strains of viruses in eggs for distribution to vaccines manufacturers, expansion of virus seed pools in embryonated chicken eggs, harvesting of virions from the allantoic fluids and inactivation, purification of hemagglutinin (HA) and neuraminidase (NA) subunits, to the final blending and packaging (Treanor, 2004) Because of the lengthy vaccine production process, the manufacturing has to begin very much in advance of a flu Protein Highly conserved sequences Class I Class II A2, A26 A1, B39 CTHLEVCFM THLEVCFMY LEVCFMYSD EVCFMYSDF VCFMYSDFH CFMYSDFHF FMYSDFHFI 130-YYLEKANKIKSE-141 (3) YYLEKANKI YLEKANKIK LEKANKIKS 143-THIHIFSFTGEEMA-156 (2) HIFSFTGEE IFSFTGEEM 185-RGLWDSFRQSERGEETIEE-203 (5) GLWDSFRQS FRQSERGEE RQSERGEET QSERGEETI SERGEETIE 298-HEGEGIPLYDAIKC-311 (2) HEGEGIPLY IPLYDAIKC 560-SRPMFLYVRTNGTSK-574 (6) SRPMFLYVR RPMFLYVRT MFLYVRTNG FLYVRTNGT LYVRTNGTS YVRTNGTSK DR A26 A3 A24, B58 A2, A24, B8, B39, B62 A2, A24, B39 A1, A2, A3, A24 DR DR DR DR A3 DR A2 B62 A1 B44 A1, A26, B44, B62 B7 DR B27 B7 A2, B8 A3, B7 109 DR DR DR DR Protein NP M1 Highly conserved sequences Class I 1-MASQGTKRSYEQMET-15 (3) MASQGTKRS ASQGTKRSY GTKRSYEQM 35-GIGRFYIQMCTELKL-49 (5) IGRFYIQMC GRFYIQMCT FYIQMCTEL YIQMCTELK IQMCTELKL 66-MVLSAFDERRN-76 (2) MVLSAFDER VLSAFDERR 78-YLEEHPSAGKDPKKTGGPIY-97 (4) LEEHPSAGK HPSAGKDPK PSAGKDPKK DPKKTGGPI 110-LYDKEEIRRIWRQANNG-126 (4) KEEIRRIWR IRRIWRQAN RRIWRQANN RIWRQANNG 241-DQVRESRNPGNAEIEDL-257 (2) QVRESRNPG SRNPGNAEI 410-QPTFSVQRNLPF-421 (1) FSVQRNLPF 1-MSLLTEVETYVLSI-14 (4) SLLTEVETY Class II A1 A1, A26, B62 A26, B8 DR B27 A2, A24, B39, B44 A3 A2, A24, B27, B39, B44, B62 DR DR DR A3, A26 A3 A3 B7 A3 B7, B8 A3 DR B27 A2 A3 B27, B39 A1, A26, B7, B8, B39, B58, B62 A1, A2, A3, A26, B58, B62 110 DR Protein Highly conserved sequences Class I LLTEVETYV LTEVETYVL EVETYVLSI 122-GALASCMGLIYNRMG-136 (4) GALASCMGL ALASCMGLI LASCMGLIY SCMGLIYNR 175-HENRMVLASTTAKAMEQMAGSSEQAAEAME-204 (9) NRMVLASTT RMVLASTTA MVLASTTAK VLASTTAKA LASTTAKAM TTAKAMEQM EQMAGSSEQ QMAGSSEQA SSEQAAEAM 208-QARQMVQAMR-217 (2) QARQMVQAM ARQMVQAMR 111 Class II A2 A1, B8, B39 A1, A2, A26 A2 A2, A3 A1, A3, A26, B58 B62 A3 B27 A2, B62 A3 A2, A3 B7, B8, B62 A1, A26, B58 B62 A2, B62 A1 B7, B8, B62 A3, B27 DR DR DR DR APPENDIX D Classification of HLA I and II molecules into supertypes HLA Class A (HLA I) Supertypes* A1 A2 A3 B (HLA I) A24 A26 B7 B8 B27 DR (HLA II) B39 B44 B58 B62 DR1 DR3 DR4 DR7 DR8 DR11 DR13 DR15 DR51 Alleles A1, A*3001, A*3002, A*3003, A*3004 A*0201, A*0202, A*0204, A*0205, A*0206, A*0209, A*0214, A*0217, A*6802, A*6901 A3, A*0301, A*1101, A*3101, A*3303, A*6601, A*6801 A24, A*2402 A*2601, A*2602, A*2603 B*07, B*0702, B*3501, B*5102, B*5101, B*5103, B*51, B*5301, B*5401 B*08, B*0802 B*27, B*2701, B*2702, B*2703, B*2704, B*2705, B*2706 B*1509, B*1510, B*3801, B*3901, B*3909 B18, B40, B*4001, B*4006, B44, B*4402, B*4403 B*1513, B*5701, B*5702, B*5801, B*5802 B*1501, B*1502, B*1503, B*1508 DR1*0101, DR1*0102 DR1*0301, DR1*0305, DR1*0306, DR1*0307, DR1*0308, DR1*0309, DR1*0311, DR1*1107 DR1*0401, DR1*0402, DR1*0404, DR1*0405, DR1*0408, DR1*0410, DR1*0423, DR1*0426 DR1*0701, DR1*0703 DR1*0801, DR1*0802, DR1*0804, DR1*0806, DR1*0813, DR1*0817 DR1*1101, DR1*1104, DR1*1106, DR1*1128, DR1*1305, DR1*1307, DR1*1311, DR1*1321 DR1*1102, DR1*1114, DR1*1120, DR1*1121, DR1*1301, DR1*1302, DR1*1304, DR1*1322, DR1*1323, DR1*1327, DR1*1328 DR1*1501, DR1*1502, DR1*1506 DR5*0101, DR5*0105 * Definition of supertypes is based on clustering of specificity materices by Lund et al., 2004 112 APPENDIX E Functional sites of influenza A virus proteins Protein PB2 Position Functional significance 1-120 Contain mitochondrial targeting signal 1-131 PB1 binding region 1-159 Contain sequences important for RNA polymerizing activity and cap-Idependent RNAse activity, in association with PA 501-617 for RNA polymerization and PB2305-559, PB11-222, PA301-517, PA601-716 for cap-Idependeing RNAse activity 1-269 NP binding region 10 51-259 206-259 242-282 305-559 318-483 363-404 402-559 Leucine at this position was essential for mitochondrial localization of PB2 to preserve mitochondrial function (membrane potential in infected cells) during influenza virus infection Leucine at this position was essential for mitochondrial localization of PB2 to preserve mitochondrial function (membrane potential in infected cells) during influenza virus infection Binds to PB1, and position 206-259 may be the most important region for binding to PB1 because lost of this regions resulted in lost of binding activity Binding of monoclonal antibodies to this region inhibited in vitro transcription by the viral RNP complex with either globin mRNA or ApG as primers N-terminal RNA cap binding region Contain sequences important for cap-I-dependent RNAse activity, in association with PB21-159, PB11-222, PA301-517 and PA601-716 Minimal cap binding domain Middle RNA cap binding region Cap-binding domain 113 References Carr et al., 2006 Toyoda et al., 1996; Poole et al., 2004 Masunaga et al., 1999 Poole et al., 2004 Carr et al., 2006 Carr et al., 2006 Ohtsu et al., 2001 Hatta et al., 2000 Honda et al., 1999 Masunaga et al., 1999 Guilligay et al., 2008 Fechter et al., 2003 Masunaga et al., 1999 Protein PB2 Position 449-495 538-577 544-556 580-683 580-759 627 627 627 627 627 687-759 701 PB1 736-739 1-12 1-25 1-48 1-222 Functional significance Nuclear localization signal C-terminal RNA cap binding region Cap-binding sequences NP binding region PB1 binding region Host range determinant (EK) Determinant of cold sensitivity in RNA replication Determine H5N1 pathogenicity in mice (Lys, high; Glu, low) Affect virus replication in mice, probably also in human Data suggests that lysine at 627 confers advantage of efficient growth in upper and lower respiratory tract of mammals to avian H5N1 May affect assembly of polymerase, which could be crucial for transcriptional efficiency Lysine at position 627 was a major determinant of pathogenicity and tissue distribution of the Dutch highly pathogenic human influenza A H7N7 virus Bind to human importin α5 to mediate nuclear import Detemine the ability of H5N1 viruses of duck origin to replicate and causes lethal infection in mice (AspAsn) Nuclear localization signal Constitute the core of interaction interface with PA and important for polymerase activity and virus growth Position V3, N4, P5, L7, L8, F9, and L10 are important for binding to PA Binds to PA Domain alpha, contain residues required for binding to PA Contain sequences important for cap-I-dependent RNAse activity, in association with PB21-159, PB2305-559, PA301-517, PA601-716 114 References Mukaigawa and Nayak, 1991 Honda et al., 1999 Li et al., 2001 Poole et al., 2004 Poole et al., 2004 Subbarao et al., 1993; Massin et al., 2001; Labadie et al., 2007; Naffakh et al., 2000 Hatta et al., 2001 Hatta et al., 2007 Deng et al., 2005 Munster et al., 2007 Tarendeau et al., 2007 Li et al., 2005 Mukaigawa and Nayak, 1991 Perez and Donis, 2001 Ohtsu et al., 2001 Perez and Donis, 1995 Masunaga et al., 1999 Protein PB1 PA Position Functional significance 180-195 Nuclear localization signal 180-252 Region containing two discontinuous nuclear localization signals of PB1 in A/WSN/33 Both signal regions are required for nuclear localization 202-252 Nuclear localization signal 233-249 Proposed to contain a new 5' vRNA promoter-binding site 267-493 cRNA binding region 286-483 RNA-dependent RNA-polymerase catalytic domain 541-757 PB2-binding site 600-757 Binds to PB2, and position 718-732 may be the most important region for binding to PB2 because lost of this regions resulted in lost of binding activity 102 K102A mutation in A/WSN/33 virus caused a general decrease in transcription and replication in vivo, as a result of significant decrease in cap binding and viral RNA promoter-binding activities 108 D108A mutation in A/WSN/33 virus caused selective inhibition of transcription D108A mutations caused complete inhibition of endonuclease acitivity in vitro 109-117 Alanine-scanning mutations in this N-terminal region caused PA degradation mediated by UPS (affect protein stability) 124-139 Contain nuclear localization signal 134 K134A mutation in A/WSN/33 virus caused selective inhibition of transcription K134A mutation caused complete inhibition of endonuclease acitivity in vitro 154 A point mutation at position 154 (Glu to Gly) completely eliminated nuclear transport Position 154 is located between two NLS (region I 124-139 and region II 186-247), suggesting its importance in allowing a proper relative position of regions I and II 115 References Jones et al., 1986 Nath and Nayak, 1990 Jones et al., 1986 Jung and Brownlee, 2006 Gonzalez and Ortin, 1999 UniProtKB: Q910D6 Toyoda et al., 1996 Ohtsu et al., 2002 Hara et al., 2006 Hara et al., 2006 Hara et al., 2006 Nieto et al., 1994 Hara et al., 2006 Nieto et al., 1994 Protein PA NP Position Functional significance 157 Mutation at position 157 abrograted proteolysis induction completely and led to selective loss of the ability to synthesize cRNA from the viral RNA template but not to transcribe viral RNA 162 Mutation at position 162 produced a moderate decrease in proteolysis induction and resulted in intermediate selective loss of the ability to synthesize cRNA from the viral RNA template 186-247 Contain nuclear localization signal 226 Leu to Pro at position 226 caused temperature sensitivity in vRNA synthesis 301-517 Contain sequences important for cap-I-dependent RNAse activity, in association with PB21-159, PB2305-559, PB11-222 and PA601-716 501-617 Contain sequences important for RNA polymerizing activity, in association with PB21-159 510 H510A mutation in A/WSN/33 caused abolishment of transcriptional activity (vRNA to mRNA), however no change in replication (VRNA to cRNA to vRNA) ability; suggesting defect in endonuclease activity 601-716 Contain sequences important for cap-I-dependent RNAse activity, in association with PB21-159, PB2305-559, PB11-222 and PA301-517 624 Active site of serine protease activity of PA is required for maximal viral growth but not essential for viral growth and pathogenesis (Study using S624A virus vs A/WSN/33 virus) 638 R638A mutation in A/WSN/33 caused severe attenuation of viral growth in culture by promoting the synthesis of defective interfering RNAs; Mutation in this position possibly destabilized PA-RNA template interactions during elongation 668-692 Binds to PB1 1-13 Nonconventional nuclear localization signal (nNLS) 116 References Perales et al., 2000 Perales et al., 2000 Nieto et al., 1994 Kawaguchi et al., 2005 Masunaga et al., 1999 Masunaga et al., 1999 Fodor et al., 2002 Masunaga et al., 1999 Toyoda et al., 2003 Fodor et al., 2003 Toyoda et al., 1996; Ohtsu et al., 2001 Neumann et al., 1997; Wang et al., 1997; Weber et al., 1998 Protein NP Position Functional significance 1-20 Interact with RAF-2p48 RAF-2p48 (aliases: BAT1, UAP56) binding to free NP facilitates NP-RNA interaction, leading to enhanced viral RNA synthesis 1-80 Contain signal for NP nuclear accumulation 1-180 RNA-binding region Further subdivided into two smaller regions, 1-77 and 79-180, that also retained RNA-binding activity 3-13 Nuclear localization signal (unconventional NLS, NLS1), necessary for efficient viral mRNA synthesis NLS1 is not essential for virus replication 80-320 Contain signal for NP nuclear accumulation 91-188 RNA binding region Highly conserved among NPs from A-, B-, and C-type influenza viruses and similar to RNA binding domain of a plant virus movement protein 120 WA mutation resulted in partial loss of RNA-binding activity 139 WA mutation resulted in partial loss of RNA-binding activity 160-200 Contain signal for NP cytoplasmic accumulation 189-358 Binding to NP-1 (self-associate) 198-216 Nuclear localization signal (bipartite NLS, NLS2), essential for vRNA transcription and NP's nucleolar accumulation NLS2 is essential for virus replication 199 RA mutation resulted in 60% loss of homomultimerization affinity 240-400 Contain signal for NP cytoplasmic accumulation 267 RA mutation resulted in complete loss of RNA-binding activity 320-400 Contain signal for NP nuclear accumulation 330 WA mutation resulted in complete loss of RNA-binding activity 336-345 Highly conserved nuclear accumulation sequence (NAS) 340-498 Contains a PB2 binding site and a sequence regulating the NP-PB2 interaction in the last 33 aa of NP 371-498 Binding to NP-2 (self-associate) 117 References Momose et al., 2001 Bullido et al., 2000 Albo et al., 1995 Ozawa et al., 2007 Bullido et al., 2000 Kobayashi et al., 1994 UniProtKB: P03466 (A/PR/8/34) UniProtKB: P03466 (A/PR/8/34) Bullido et al., 2000 Elton et al., 1999 Ozawa et al., 2007; Weber et al., 1998 UniProtKB: P03466 (A/PR/8/34) Bullido et al., 2000 UniProtKB: P03466 (A/PR/8/34) Bullido et al., 2000 UniProtKB: P03466 (A/PR/8/34) Davey et al., 1985; Weber et al., 1998 Biswas et al., 1998 Biswas et al., 1998 Protein NP M1 HA Position Functional significance 412 FA mutation resulted in complete loss of RNA-binding activity 416 RA mutation resulted in complete loss of homomultimerization and complete loss of RNA-binding activity 1-164 Membrane-binding region 101-105 Nuclear localization signal 165-252 C-terminal domain of M1 protein that mediates vRNP binding 190 Determine preferential binding to α2,6 or α2,3 linkages 225 Determine preferential binding to α2,6 or α2,3 linkages 226 Sialic acid binding site, determine receptor specificity (i.e 3Gal- or 6Gal-) 226 228 324 NA NS1 343-344 11-33 144 275 431 1-73 34-38 38 92 137-146 Gln to Leu in H2 and H3 human viruses, together with Gly228Ser, changed the receptor binding specificity from human to avian Gly to Ser in H2 and H3 human viruses, together with Gln226Leu, changed the receptor binding specificity from human to avian Glutamic acid to Lysine substitution associated with high pathogenicity of H5N2 avian influenza viruses from Mexico Cleavage site Involved in apical transport and lipid raft association (by similarity) Glycosylation site, associated with substrate specificy of N8 Associated with substracte specificy of N8 Associated with substracte specificy of N8 RNA-binding and homodimerization region (by similarity) Nuclear localization signal (by similarity) Required for binding dsRNA (R38) Glutamic acid at this position of H5N1 virus confers virulence and resistance to interferons and TNF-alpha in pigs Nuclear export signal (by similarity) 118 References UniProtKB: P03466 (A/PR/8/34) UniProtKB: P03466 (A/PR/8/34) UniProtKB: P03485 (A/PR/8/34) UniProtKB: P03485 (A/PR/8/34) Baudin et al., 2001 Matrosovich et al., 2000 Matrosovich et al., 2000 Rogers et al., 1983; Matrosovich et al., 2000 Naeve et al., 1984; Connor et al., 1994 Naeve et al., 1984; Connor et al., 1994 García et al., 1996 UniProtKB: P03452 (A/PR/8/34) UniProtKB: P03468 (A/PR/8/34) Saito and Kawano, 1997 Kobasa et al., 1999 Kobasa et al., 1999 UniProtKB: P03496 (A/PR/8/34) UniProtKB: P03496 (A/PR/8/34) Wang et al., 1999 Seo et al., 2002 UniProtKB: P03496 (A/PR/8/34) Protein NS1 NS2 M2 PB1-F2 Position Functional significance 149 Determine the ability of avian H5N1 viruses to antagonize the induction of IFN-alpha and IFN-beta production in chickens (virulence; A at this position associated with high virulence) 149 Determine the ability of avian H5N1 viruses to replicate in chickens cells (host range; A at this position made the virus highly virulent while V at this position made the virus unable to replicate in chicken cells) 180-215 CPSF4-binding (by similarity) 216-221 Nuclear localization signal (by similarity) 223-230 PABPN1-binding (by similarity) 12-21 Nuclear export signal 59-116 M1-binding domain on NS2 85-94 Nuclear export signal (by similarity) 10-20 Contain sequences associated with host restriction specificities for human, avian, and swine 37 Essential for channel activity, possibly by being protonated during channel activation, and by forming the channel gate and the selective filter (by similarity) 46-75 Necessary and sufficient for mitochondrial targeting Subdomain 63-75 and both Lys73 and Arg75 are minimally required for mitochondrial localization 66 N66S mutation in H5N1 virus from HK 1997 outbreak was found to be associated with increased pathogenicity This mutation was also seen in 1918 pandemic virus A/Brevig/Mission/18 A study in mice with reversion of this mutation in the 1918 virus (SN) resulted in attenuation, reduced morbidity and mortality (vs wt) 65-87 Mitochondrial targeting sequence 119 References Li et al., 2006 Li et al., 2006 UniProtKB: P03496 (A/PR/8/34) UniProtKB: P03496 (A/PR/8/34) UniProtKB: P03496 (A/PR/8/34) UniProtKB: P03508 (A/PR/8/34) PDB: 1PD3 UniProtKB: P03508 (A/PR/8/34) Liu et al., 2005 UniProtKB: P06821 (A/PR/8/34) Yamada et al,, 2004 Conenello et al., 2007 UniProtKB: P0C0U1 (A/PR/8/34) APPENDIX F Permission to reproduce copyrighted materials Original Message -Subject:Re: Request for Permission to Reproduce Copyright Work in Thesis Date:Thu, 22 May 2008 11:10:59 +0200 From:Rebecca Cox To:heiny@nus.edu.sg References: Dear Heiny, You have my permission to reproduce the figure Yours sincerely Rebecca Cox On May 22, 2008, at 10:47 AM, Heiny wrote: 22 May 2008 Dr R.J Cox Influenza Research Centre The Gade Institute University of Bergen Dear Dr Cox, RE: Request for Permission to Reproduce Copyright Work I am a graduate student at the National University of Singapore (“NUS”), and am currently working on a thesis for submission to NUS as part of my academic program My thesis is likely to be published electronically by NUS on the internet and on NUS’ own intranet, and copies of my thesis will be made available to NUS and end users My thesis seeks to characterize the evolutionarily conserved influenza A virus sequences as vaccine targets, using bioinformatics approach I am writing to seek your kind permission to use and reproduce, for purposes of submission, publication and distribution of my thesis as described above, the following works for which I understand you to be the copyright owner: (1) The whole of Figure (A diagrammatic representation of influenza A virus showing protein and RNA composition) in the article "Cox RJ, Brokstad KA, Ogra P (2004) Influenza virus: immunity and vaccination strategies Comparison of the immune response to inactivated and live, attenuated influenza vaccines Scand J Immunol 59(1):1-15." Due acknowledgment of your permission and ownership of copyright in the work will be made in my thesis I would be much obliged if you could let me hear from you at your earliest convenience Yours sincerely, Heiny 120 Original Message -Subject:RE: Request for Permission to Reproduce Copyright Work Date:Tue, 27 May 2008 10:57:15 +0100 From:Murdock, Andrew To: References: Dear Heiny, I am happy to give permission for you to reproduce the requested image from McSwiggen JA and Seth S A potential treatment for pandemic influenza using siRNAs targeting conserved regions of influenza A Expert Opin Biol Ther 2008 Mar;8(3):299-313 This permission applies only to this figure and only for use in your dissertation Please let me know if you have any questions Best wishes, Andrew Andrew Murdock, Ph.D Commissioning Editor Expert Opinion, Informa Healthcare Telephone House 69-77 Paul Street London, EC2A 4LQ UK Tel: +44 (0)20 7017 4861 Fax:+44 (0)20 7017 7667 E-mail: andrew.murdock@informa.com Informa Healthcare is a trading name of Informa UK Limited Registered in England under no 1072954 Registered Office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH **************************************************************************************************** The Editors of the Expert Opinion publication series welcome unsolicited article proposals In the first instance, please direct a very brief summary, together with working title and author contact details, for the attention of the Editor **************************************************************************************************** The information contained in this email or its attachments is confidential and is intended solely for the use of the named addressee Disclosure, copying, use, or distribution of the information in it by any other person than the addressee is prohibited and may be unlawful If you are not an addressee, please immediately inform the sender and delete this message 121 APPENDIX G Poster and oral presentations Poster presentation  Conserved sequences of human and avian influenza A viruses are candidates for a pan-influenza, epitope-based vaccine 15th International Conference on Intelligent Systems for Molecular Biology and 6th European Conference on Computational Biology, Austria, 2007 Oral presentations  Highly conserved and immune-relevant sequences of human and avian influenza A viruses Bangkok International Conference on Avian Influenza 2008: Integration from Knowledge to Control, Thailand, 2008  Identifying vaccine targets from evolutionarily conserved regions in avian and human influenza A viruses ASEAN Workshop on Reverse Genetics-Based Vaccines for Avian Influenza, Thailand, 2008 122 APPENDIX H Publications Heiny AT, Miotto O, Srinivasan KN, Khan AM, Zhang GL, Brusic V, Tan TW, August JT (2007) Conserved protein sequences of all influenza A viruses as vaccine targets PLoS ONE 2(11): e1190 (Citation: 1) p 124 Contribution: Conceived, designed and performed the experiments; analyzed the data and drafted the manuscript Miotto O, Heiny AT, Tan TW, August JT, Brusic V (2008) Identification of human-to-human transmissibility factors in PB2 proteins of influenza A by large-scale mutual information analysis BMC Bioinformatics Suppl 1: S18 (Citation: 6; Impact Factor: 3.49) p 138 Contribution: Collected and annotated the sequence data; analyzed the data Khan AM, Heiny AT, Lee KX, Srinivasan KN, Tan TW, August JT, Brusic V (2006) Large-scale analysis of antigenic diversity of T-cell epitopes in dengue virus BMC Bioinformatics Suppl 5: S4 (Citation: 7; Impact Factor: 3.49) p 156 Contribution: Participated in the design of the study Khan AM, Miotto O, Heiny AT, Salmon J, Srinivasan KN, Nascimento EJ, Marques ET Jr, Brusic V, Tan TW, August JT (2007) A systematic bioinformatics approach for selection of epitope-based vaccine targets Cell Immunol 244(2): 141-7 (Citation: 6; Impact Factor: 1.81) p 168 Contribution: Participated in the development of the methodology platform Zhang GL, Khan AM, Srinivasan KN, Heiny AT, Lee KX, Kwoh CK, August JT and Brusic V (2008) Hotspot Hunter: a computational system for large-scale screening and selection of candidate immunological hotspots in pathogen proteomes BMC Bioinformatics Suppl 1: S19 (Citation: 1; Impact Factor: 3.49) p 175 Contribution: Participated in the design of the study and pilot testing of the system Khan AM, Miotto O, Nascimento EJM, Srinivasan KN, Heiny AT, Zhang GL, Marques ET, Tan TW, Brusic V, Salmon J, August JT (2008) Conservation and Variability of Dengue virus Proteins: Implications for Vaccine Design PLoS Neglected Tropical Diseases 2(8): e272 Contribution: Participated in the design of experiments, data analysis, and methods development 123 p 184 ... multiple strains of influenza A viruses With the availability and easy access to virus sequences in the public databases, as well as the advancement of bioinformatics tools for analysis of large amount... the availability of seasonal influenza vaccines, their efficacy varies, depending on the match between vaccine strains and the circulating virus strains (Carrat and Flahault, 2007) This is mainly... 13 Classification of influenza A viruses The conventional classification of influenza A viruses are based on the antigenic differences of the HA and NA surface glycoproteins There are 16 HA subtypes