Mapping targets of immune responses in complete dengue viral genomes

MAPPING TARGETS OF IMMUNE RESPONSES IN COMPLETE DENGUE VIRAL GENOMES MOHAMMAD ASIF KHAN (B Appl Sc (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF MASTERS OF SCIENCE DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2005 ACKNOWLEDGEMENTS Firstly, I thank Almighty God for His graces and guidance, and for giving the endurance to go through this strenuous exercise I would like to express my heartfelt gratitude to my two supervisors, Professor Vladimir Brusic of the Institute for Infocomm Research, Singapore (I2R), and A./Prof Tan Tin Wee of the Department of Biochemistry, NUS, for their advice, guidance, continuous support and encouragement that they have given me through the course of this project Special thanks to Professor Vladimir Brusic, an extraordinary teacher, mentor, and friend, who inspired this study and taught me a lot about life I owe my sincere thanks to the programmers, Seah Seng Hong, Vu Xuan Linh, Zhang Guang Lan, Matthew Longuet, Gregory Fitte, Anitha Veeramani, Kavitha Gopalakrishnan, and Judice Koh, all from I2R for their support in the development of the computational tools I am also grateful to Mr Kenneth Lee Xunjian, Ms Heiny Tan Anling, Dr K.N Srinivasan and Mr Paul T.J Tan from I2R for the invaluable support rendered when I was carrying out my project work I am deeply indebted to Dr Alex Sette and co-workers, from the La Jolla Institute for Allergy and Immunology in San Diego (California, USA), for conducting the experimental work Last but not least, I am thankful to my parents, my wife and siblings, and all my friends for their continuous support, help and company I would like to dedicate this thesis to my father Mohammad Shahid Khan, who worked hard and went the extra mile just to make sure I receive the best education possible i TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS .ii SUMMARY v LIST OF TABLES .vii LIST OF FIGURES ix LIST OF SYMBOLS xi CHAPTER I INTRODUCTION 1.1 Research issues investigated in this thesis 1.2 Contribution CHAPTER II LITERATURE REVIEW 11 2.1 Dengue virus 11 2.1.1 Dengue virus infection in humans 13 2.1.2 Immune responses to dengue virus infection 13 2.1.3 Targets of immune responses in dengue viral genome 15 2.2 Antigenic diversity of targets of immune responses in dengue virus 15 2.2.1 Mutation and recombination 16 2.2.2 Challenge for vaccine design: finding needles in a haystack 16 2.2.3 Overcoming antigenic diversity 18 2.3 Mapping targets of immune responses in dengue viral genomes 19 2.3.1 Immunological hot-spots: best targets to map 19 2.3.2 T-cell epitope mapping 23 2.3.3 Current status of mapping immune targets in dengue virus 25 2.4 Application of bioinformatics 29 2.5 Chapter summary 30 CHAPTER III MATERIALS AND METHODS 32 3.1 Project overview 32 3.2 Data collection 34 3.3 Data processing: cleaning and grouping 34 3.4 Data analysis 37 3.4.1 Analysis of antigenic diversity using exhaustive search method 38 3.4.2 Antigenic diversity in whole protein sequences 42 3.4.3 Antigenic diversity of immunological hot-spots 43 3.5 A novel method for selecting peptide targets for vaccine design 46 3.6 Experimental measurements to validate predictions 54 3.7 VAGAT: Viral Antigenome Analysis Tool 56 CHAPTER IV 4.1 RESULTS 57 Derived sequence datasets 57 ii 4.2 Factors that affect antigenic diversity 60 4.3 HLA specific targets for dengue vaccine formulation 66 4.4 Experimental validation 80 4.5 Mapping vaccine targets from other viral genomes 85 4.6 Chapter summary 87 CHAPTER V DISCUSSION 89 5.1 Antigenic diversity and implications for vaccine design 89 5.2 Strategies for dengue vaccine development 97 5.3 Future vaccines 99 CHAPTER VI CONCLUSIONS AND DIRECTIONS FOR FUTURE 102 6.1 Conclusions 102 6.2 Future work 105 REFERENCES 110 AUTHOR’S PUBLICATIONS 120 LIST OF SUPPLEMENTARY MATERIALS 123 APPENDIX 1: A list of candidate vaccine targets specific for HLA-A2, -A3, and -DR supertypes for each dengue serotype protein iii Chance favors the prepared mind By Albert Einstein Viruses are only obstacle to the domination of the Earth by mankind By Joshua Lederberg, Nobel Laureate The wonders of deadlines, they force you to complete your work By Asif Khan (2005) Do your homework before you take the plunge into the deep sea, But if you have to jump anyway, don’t take too long By Asif Khan (2005) Is not disease the rule of existence? There is not a lily pad floating on the river but has been riddled by insects Almost every shrub and tree has its gall, often times esteemed its chief ornament and hardly to be distinguished from the fruit If misery loves company, misery has company enough Now, at midsummer, find me a perfect leaf or fruit By Henry David Thoreau (1817-1862) It is within the power of man to eradicate infection from the earth By Louis Pasteur (1822-1895) iv SUMMARY Mapping and analysing targets of immune responses in viral variants presents us with an opportunity to improve our understanding of the immune responses to viruses and aid in identification of targets for vaccine formulation This thesis presents a systematic bioinformatics approach towards mapping and analysing potential immune targets in dengue viral sequences A special feature of dengue virus is that it exists in nature as four distinct serotypes − all studied in this thesis The potential immune targets mapped for each serotype were analysed to explore for ways to overcome the increasing diversity of the targets (antigenic diversity) Overcoming antigenic diversity is important for the study of vaccine formulation The bioinformatics approach developed in this project enables the systematic screening of dengue sequence data, which would otherwise be impossible to carry out experimentally The limits on experimentation are due to high viral diversity and the variations in immune responses among human individuals The work presented in this thesis has several novel aspects This thesis provides the first complete map of potential targets of cellular immune responses (Tcell epitope hot-spots), specific to three human leukocyte antigen (HLA) supertypes (A2, A3, and DR), for each dengue serotype Experimental validation of the predictions shows that we can successfully identify majority of the real hot-spots The most important contribution of this work is the discovery of previously unknown relationship between dengue virus variability and the immune system, which have important implications for vaccine design An interesting and important finding made in this study was the conserved localization of majority of the HLA-A2, -A3, and -DR supertype specific immune targets at the same region, though their sequences vary, across multiple intra-species v and inter-species sequence variants of Flaviviruses This finding is supported by experimental measurements of peptide binding affinity to HLA molecules Structural analysis showed that major portions of such regions are exposed on the threedimensional (3-D) structure of the protein This suggests influence of protein 3-D structure on antigen processing and consequent epitope selection by the immune system Immune targets in these regions merit further investigations as they could have important implications for vaccine design The most important finding in this study is that by focusing on specific targets of immune responses in the context of individual HLA supertypes, the large antigenic diversity (~3700 unique sequences of length ~114-900 amino acids) can be represented by small dataset (~60-260 peptides of average length 24-53 amino acids), which can be experimentally validated for vaccine formulation Further, the addition of a single new unique variant sequence is less likely to increase the diversity and the number of each HLA supertype relevant peptides Based on the insights gained, novel tools for screening and selection of peptides for vaccine formulation were designed The tools developed represent the starting point for selection of experiments that will validate targets relevant for vaccine design against multiple variants of dengue viruses and effective for human population at large The bioinformatics approach employed in this study is novel, generic and applicable to other viruses (499 Words) vi LIST OF TABLES Table 2.1 DENV proteins that have been demonstrated to date to be immunogenic during infection in humans 15 Table 2.2 DENV proteins that are immunogenic in humans 25 Table 2.3 Human T-cell epitopes experimentally mapped in dengue 27 virus genomes Table 3.1 Sample output from the computational method that uses 42 exhaustive search to perform antigenic diversity analysis Table 3.2 The four measures to rank consensus hot-spots 53 Table 3.3 The peptide regions in AAK74146 sequence that corresponded to the predicted DENV-3 envelope protein HLA-DR consensus hot-spots were selected for experimental validation of their hot-spot status 54 Table 3.4 The peptide regions in AAK62756 sequence that corresponded to the predicted WNV envelope protein HLA-DR consensus hot-spots were selected for experimental validation of their hot-spot status 55 Table 4.1 The number of collected, extracted and non-redundant protein sequences for each DENV serotype 58 Table 4.2 Comparison of the number of extracted and non-redundant 59 DENV protein sequences Table 4.3 Number of available complete and incomplete DENV-2 protein sequences Table 4.4 Minimum and maximum percentage sequence identity 60 range for each dengue protein (between any two complete non-redundant sequences only) - between and within serotypes Table 4.5 Minimal number of sequences required to represent entire 62 antigenic diversity of each protein from the individual serotypes Table 4.6 Effect of sequence length to antigenic diversity 65 Table 4.7 Effect of sequence region to antigenic diversity 65 Table 4.8 Potential consensus hot-spots for HLA-A2, -A3, and -DR 68 59 vii Table 4.9 Six potential inter-species consensus hot-spots (ISCH) in envelope protein with conserved localization in at least four and at most six Flaviviruses 73 Table 4.10 Minimal number of peptides required to represent antigenic diversity of HLA-A2, -A3, and -DR consensus hot-spots, for all the dengue serotypes 79 Table 4.11 Peptide binding analysis of the peptides selected with eight most prevalent molecules of HLA-DR supertype in the human population 84 viii LIST OF FIGURES Figure 2.1 Organization of the DENV genome 11 Figure 2.2 Representative polyprotein sequences for dengue virus serotypes and their protein products 12 Figure 2.3 Analysis of the activities of various mutated peptides in eliciting T-cell response 17 Figure 2.4 A schema depicting the concept of supertypes and promiscuous peptides 22 Figure 2.5 Clustering of T-cell epitopes in antigens 23 Figure 2.6 Experimental methods for mapping T-cell epitopes 24 Figure 3.1 A flowchart of the general overview of the project 33 Figure 3.2 The process of extracting the portion corresponding to the sample sequences from the blastp matches 36 Figure 3.3 Generation of overlapping 9-mers 38 Figure 3.4 Definition of antigenically redundant sequence 39 Figure 3.5 A schematic representation of the method used to remove 41 antigenically redundant sequences and analyse antigenic diversity Figure 3.6 A sample consensus hot-spot region in DENV-3 capsid protein Figure 3.7 A flowchart summarizing the screening and selection 47 processes for predicted peptides of each consensus hot-spot Figure 3.8 A flowchart describing the specification of the screening method 49 Figure 3.9 The importance of duplicates in predicted peptide sequences of consensus hot-spots 50 Figure 3.10 A flowchart describing the selection method 51 Figure 4.1 Antigenic diversity curves for DENV-2 structural and nonstructural proteins 63 Figure 4.2 Effect of sequence number on antigenic diversity 64 44 ix Transgenic animals expressing HLA alleles Immunise with synthetic peptide constructs of each individual serotype Challenge with peptides from the consensus hot-spots of individual serotype Eliminate functionally redundant constructs Identification of functionally active constructs Test the functionality of identified constructs in humans, using the sera from people that have clearly defined previous dengue virus infection of individual serotypes Identification of vaccine candidate constructs for each serotype 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cytotoxic CD4+ T-cell clones J Virol 70, 3108-3117 Zhang, G.L., Khan, A.M., Srinivasan, K.N., August, J.T., Brusic,V (2005) MULTIPRED: a computational system for prediction of promiscuous HLA binding peptides Nucleic Acids Res 33, W172-W179 Zhang, W., Chipman, P.R., Corver, J., Johnson, P.R., Zhang, Y., Mukhopadhyay, S., Baker, T.S., Strauss, J.H., Rossmann, M.G., Kuhn, R.J (2003) Visualization of membrane protein domains by cryo-electron microscopy of dengue virus Nat Struct Biol 10, 907-912 Zhao, B., Mathura, V.S., Rajaseger, G., Moochhala, S., Sakharkar, M.K., Kangueane, P (2003) A novel MHCp binding prediction model Hum Immunol 64, 1123-1143 Zivna, I., Green, S., Vaughn, D.W., Kalayanarooj, S., Stephens, H.A., Chandanayingyong, D., Nisalak, A., Ennis, F.A., Rothman, A.L (2002) T cell responses to an HLA-B*07-restricted epitope on the dengue NS3 protein correlate with disease severity J Immunol 168, 59595965 Zivny, J., DeFronzo, M., Jarry, W., Jameson, J., Cruz, J., Ennis, F.A., Rothman, A.L (1999) Partial agonist effect influences the CTL response to a heterologous dengue virus serotype J Immunol 163, 2754-2760 Zivny, J., Kurane, I., Leporati, A.M., Ibe, M., Takiguchi, M., Zeng, L.L., Brinton, M.A., Ennis, F.A (1995) A single nine-amino acid peptide induces virus-specific, CD8+ human cytotoxic T lymphocyte clones of heterogeneous serotype specificities J Exp Med 182, 853-863 119 AUTHOR’S PUBLICATIONS PUBLICATIONS 2002-2005 Journal articles Khan AM, Lee XK, Tan AH, Zhang GL, Srinivasan KN, Tan TW, August JT, Brusic V (2005) Analysis of targets of immune responses in complete dengue viral genomes Bioinformatics (Manuscript in preparation) Khan AM, Srinivasan KN, Tan TW, August JT, Brusic V (2005) Computer-aided vaccine research Current computer aided-drug design (Manuscript in preparation) Zhang GL, Khan AM, Srinivasan KN, August JT, Brusic V (2005) Neural models for predicting viral vaccine targets J Bioinfo Comp Biol (In press) Zhang GL, Khan AM, Srinivasan KN, August JT, Brusic V (2005) MULTIPRED: a computational system for prediction of promiscuous HLA binding peptides Nucleic Acids Res 33 (Web Server issue), W172-W179 Siew JP, Khan AM, Tan PT, Koh JL, Seah SH, Koo CY, Chai SC, Armugam A, Brusic V, Jeyaseelan K (2004) Systematic analysis of snake neurotoxins functional classification using a data warehousing approach Bioinformatics 20, 3466-3480 Srinivasan KN, Zhang G, Khan AM, August JT, Brusic V (2004) Prediction of class I T-cell epitopes: evidence of presence of immunological hot spots inside antigens Bioinformatics 20, I297-I302 Lenffer J, Lai P, El Mejaber W, Khan AM, Koh JL, Tan PT, Seah SH, Brusic V (2004) CysView: protein classification based on cysteine pairing patterns Nucleic Acids Res 32 (Web Server issue), W350-W355 Bramachary M, Krishnan SPT, Koh JLY, Khan AM, Seah SH, Tan TW, Brusic V, Bajic VB (2004) ANTIMIC: a database of antimicrobial sequences Nucleic Acids Res 32, D586-D589 Fry BG, Wüster W, Kini RM, Brusic V, Khan A, Venkataraman D, Rooney AP (2003) Molecular evolution and phylogeny of snake venom three finger toxins Journal of Molecular Evolution 57, 110-129 [Zuckerkandl prize] 10 Tan PTJ, Khan AM, Brusic V (2003) Bioinformatics for venom and toxin sciences Briefings in Bioinformatics 4, 53-62 Conference articles 11 Koh JLY, Lee ML, Khan AM, Tan PTJ, Brusic V (2004) Duplicate Detection in Biological Data using Association Rule Mining 2nd European Workshop on Data Mining and Text Mining for Bioinformatics Pisa, Italy, September 24, 2004 12 Koh JLY, Krishnan SPT, Seah SH, Tan PT, Khan AM, Lee ML, Brusic V (2004) BioWare: A framework for bioinformatics data retrieval, annotation and publishing Search and Discovery in Bioinformatics 27th Annual International ACM SIGIR Conference on Research and Development in IR Sheffield, UK, July 29, 2004 13 Zhang G, Khan AM, Srinivasan KN, August JT, Brusic V (2003) Neural models for predicting viral vaccine targets Conference on Neuro-Computing and Evolving Intelligence 2003 (NCEI'2003) Auckland, New Zealand, November 20-21, 2003 120 Books 14 Brusic V, Khan AM (editors) Abstract book of the 3rd Asia-Pacific Bioinformatics Conference & Singapore Bioinformatics Week, World Scientific, Singapore, January 2005 (142 pages) 15 Khan AM (2002) Snake venom PLA2: bioinformatics approach, Honours Thesis, National University of Singapore, Singapore (123 pages) Oral presentations 16 Khan AM, Tan TW, Brusic V (2005) Antigenic diversity analysis of complete dengue viral genome: implications for vaccine design 3rd Asia-Pacific Bioinformatics Conference (APBC2005) Graduate Student Satellite Symposium NTU, Singapore, January 15, 2005 17 Srinivasan KN, Khan AM, August JT, Brusic V (2004) Analysis of T-cell epitope hotspots in dengue virus envelope protein 1st Asian Regional Dengue Research Network Meeting Bangkok, Thailand, October 18-20, 2004 18 Srinivasan KN, Zhang GL, Khan AM, August JT, Brusic V (2004) Prediction of class I T-cell epitopes: evidence of presence of immunological hot spots inside antigens 12th International Conference on Intelligent Systems for Molecular Biology, with the European Conference on Computational Biology (ISMB/ECCB2004) Glasgow, Scotland, July 31- August 4, 2004 19 Zhang G, Khan AM, Srinivasan KN, August JT, Brusic V (2003) Neural models for predicting viral vaccine targets Conference on Neuro-Computing and Evolving Intelligence 2003 (NCEI'2003) Auckland, New Zealand, November 20-21, 2003 20 Khan AM, Koh JLY, Tan PTJ, Brusic V (2003) Snake Venom Phospholipase A2: A Bioinformatics Approach Bioinformatics Symposium 2003 NUS, Singapore, August 15, 2003 21 Fry BG, Wuster W, Kini RM, Brusic V, Khan AM, Venkataraman D, Rooney A (2002) The three finger toxin toolkit Venoms to Drugs (V2D) meeting Heron Island, Australia, July 2002 Posters 22 Khan AM, Koh JLY, Veeramani A, Brusic V (2005) Antigenic analysis of viral genomes using a data warehousing approach 2nd International Immunoinformatics Symposium and Immunoinformatics workshop (IIMMS2005) Boston, USA, March 79, 2005 23 Khan AM, Tan TW, Brusic V (2005) Antigenic diversity analysis of complete dengue viral genome: implications for vaccine design 3rd Asia-Pacific Bioinformatics Conference (APBC2005) NUS, Singapore, January 17-21, 2005 24 Khan AM, Zhang GL, Srinivasan KN, August JT, Tan TW, Brusic V (2004) Analysis of antigenic hot-spots in dengue virus: A bioinformatics approach 1st Asian Regional Dengue Research Network Meeting Bangkok, Thailand, October 18-20, 2004 25 Khan AM, Tan TW, Brusic V (2004) Antigenic diversity analysis of complete dengue viral genome: Implications for vaccine design 1st Asian Regional Dengue Research Network Meeting Bangkok, Thailand, October 18-20, 2004 26 Khan AM, Seah SH, Tan TW, Brusic V (2004) VAGAT: mapping targets of immune response in complete viral genomes 8th NUS-NUH Annual Scientific Meeting NUS, Singapore, October 7, 2004 121 27 Khan AM, Tan TW, Brusic V (2004) Distribution of TAP binding peptide regions in epitope precursors targeted to major MHC class I supertypes 8th NUS-NUH Annual Scientific Meeting NUS, Singapore, October 7, 2004 28 Khan AM, Tan TW, Brusic V (2004) Antigenic Diversity Analysis Of Complete Dengue Viral Genome: Implications For Vaccine Design 8th NUS-NUH Annual Scientific Meeting NUS, Singapore, October 7, 2004 29 Brahmachary M, Krishnan SPT, Koh JLY, Khan AM, Seah SH, Tan TW, Brusic V, Bajic VB (2003) ANTIMIC: a database of antimicrobial sequences European Conference on Computational Biology (ECCB 2003) Paris, France, September 2730, 2003 30 Siew JP, Khan AM, Tan PT, Koh JL, Armugam A, Jeyaseelan K, Vladimir Brusic (2003) Systemization of snake venom neurotoxins based on structure and function 14th World Congress on Animal, Plant and Microbial Toxins Adelaide, Australia, September 14-19, 2003 122 LIST OF SUPPLEMENTARY MATERIALS Supplementary Material URL Description sdmc.i2r.a-star.edu.sg/asif/thesis/sup1.pdf sdmc.i2r.a-star.edu.sg/asif/thesis/sup2.pdf sdmc.i2r.a-star.edu.sg/asif/thesis/sup3.pdf sdmc.i2r.a-star.edu.sg/asif/thesis/sup4.pdf sdmc.i2r.a-star.edu.sg/asif/thesis/sup5.pdf sdmc.i2r.a-star.edu.sg/asif/thesis/sup6.pdf sdmc.i2r.a-star.edu.sg/asif/thesis/sup7.pdf sdmc.i2r.a-star.edu.sg/asif/thesis/sup8.pdf sdmc.i2r.a-star.edu.sg/asif/thesis/sup9.pdf 10 sdmc.i2r.a-star.edu.sg/asif/thesis/sup10.pdf Dengue virus epidemiology and cases in Singapore HLA polymorphism and supertypes MULTIPRED Errors and discrepancies found in DENV-1, -2, -3, and -4 data records collected from NCBI Entrez protein database Number of complete and incomplete protein sequences for each dengue serotype protein Antigenic diversity in whole protein sequences of each dengue serotype protein Predicted T-cell epitope consensus hot-spots specific to HLA-A2, -A3, and -DR supertypes for each dengue serotype protein and antigenic diversity analysis of the consensus hot-spots Conserved localization of hot-spots DENV-2 consensus hotspots for HLA-A2, -A3 and -DR supertypes and regions immunologically relevant to various combinations of the supertypes Dengue sequence data growth 123 [...]... identify peptide targets for vaccine formulation 9 Immunogenic is defined as capable of producing an immune response 14 2.1.3 Targets of immune responses in dengue viral genome The principal targets of cellular immune response to DENV are shown in Table 2.1 The T cell responses to DENV are directed against multiple viral proteins - both structural and nonstructural proteins (NS) (Rothman, 2004; Brinton et... a combination of bioinformatics and experimental approaches 5 1.1 Research issues investigated in this thesis Dengue virus diversity is, believed to be, increasing and so is the number of dengue variant sequences in public databases Formulation of a vaccine against dengue variants is difficult and challenging because of the ever increasing viral diversity and the large diversity of the human immune. .. application of a set of bioinformatics tools for comprehensive analyses of the DENV sequences This analysis aims to improve our understanding of immune response to DENV viral variants and aid in identification of peptide targets for vaccine formulations By assisting the selection of critical targets, this study helps reduce the number of essential experiments for mapping the targets of immune responses. .. strains of other serotypes Antigenic diversity of viruses, therefore, results in an increased pool of non -immune hosts and is a significant obstacle for the development of therapeutic and prophylactic vaccines (Gaschen et al., 2002) An ideal dengue vaccine must be effective in providing long-lasting immunity against multiple antigenic variants of all the four DENV serotypes Mapping targets of immune responses. .. number of known dengue sequences is analogous to the proverbial saying “finding needles in a haystack” This is because the natural prevalence of T-cell epitopes in a single protein sequence is approximately only 0.1-5% (Brusic and Zeleznikow, 1999) A detailed understanding of antigenic diversity will aid in identification of targets of immune responses for vaccine formulation against broad range of dengue. .. raises several intriguing questions, such as: 1 What is the relevance and the effect of viral diversity to identification of targets of immune responses? 2 How do vaccine developers deal with the increasing diversity of targets of immune responses (antigenic diversity)? Is it possible to identify a minimal set of targets that can represent antigenic diversity? 3 What are the implications of antigenic... immunity, an effective protective (including DHF/DSS protection) DENV vaccine must induce high-affinity T cells against all four DENV serotypes (Rothman, 2004) Hence, successful dengue vaccine must be capable of providing simultaneous protection to variants of all four serotypes (Edelman, 2005) Mapping targets of immune responses in DENVs helps improve our understanding of immune response to heterologous... immune responses in the proteome of dengue virus variants Mapping targets of immune response, using traditional experimental approaches is inefficient for large-scale screening of dengue sequences This thesis describes application of a systematic bioinformatics approach to map and analyse potential targets of cellular immune responses in DENV sequence data The specific goals of this work are: 6 1) Map potential... 2004) for immune responses and protection against pathogens The immune responses that mediate viral clearance contribute to shock (DSS) or DHF by enhancing lysis of dengue virus-infected cells (e.g virus-induced immunopathology) during secondary dengue virus infection (Rothman, 2004) The serotype causing secondary disease is always different than the serotype that induced immune responses during the... vaccine formulation? This thesis focuses on the study of antigenic diversity in order to gain insights into the relationship between DENV variability and host immune responses A detailed understanding of this relationship will aid in developing effective strategies for vaccine formulation against DENV To probe the questions posed above, it is necessary to first map the targets of immune responses in ... diversity Mapping targets of immune responses in viral antigens is an important pre-requisite to antigenic diversity analysis 18 2.3 Mapping targets of immune responses in dengue viral genomes. .. peptide targets for vaccine formulation Immunogenic is defined as capable of producing an immune response 14 2.1.3 Targets of immune responses in dengue viral genome The principal targets of cellular... can diminish discovery cost by 10-20 folds (De Groot et al., 2002; Kast et al., 1994) 2.3.3 Current status of mapping immune targets in dengue virus Mapping targets of immune responses using experimental