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Polymeric Immunoglobulin G Scaffold (PIGS) Dengue Fusion Proteins as Vaccine Candidates St George’s University of London Dr Miyoung Kim June 17, 2015 Dengue: a potentially lethal disease that affects 50-100 million people a year Aedes mosquito Epidemiology Transmitted to humans Asia Dengue Virus Africa South America Dengue clinical symptoms * Dengue fever * Dengue hemorrhagic fever * Dengue shock syndrome Oceania There are currently no licensed vaccines and substantial vector control efforts have not stopped its rapid emergence and global spread. Vaccine challenge: • There are four antigenically distinct serotypes of dengue virus (65-70% homology) • An effective tetravalent dengue vaccine could be achieved by using a combination of all 4 serotypes or by using a consensus sequence from the 4 serotypes Currently leading vaccine candidate: Sanofi Pasteur’s vaccine Tetravalent dengue vaccine: live attenuated yellow fever virus delivering each of the 4 serotypes/antigens Phase 2: year 2012 The vaccine was partially effective but failed to protect against serotype 2; however, due to small sample size, the vaccine was allowed to enter a phase III trial Phase 3: year 2014 Sanofi Pasteur announces a successful phase III trial in Latin America. Plans to apply for license in 2015. Currently leading vaccine candidate: Sanofi Pasteur’s vaccine Difficult and expensive to make Raise some safety issues An alternative, cheaper and safer vaccine would be desirable and this is the main objective in our approach Aim of the study E (domain I, II, III) : viral attachment, entry to host cells EDIII domain: critical epitopes that elicit serotype-specific Abs E domain III (ED III) of dengue virus is poorly immunogenic and needs an appropriate adjuvant and multiple immunizations To generate a cE vaccine based on a fusion protein with polymeric IgG Scaffold (cE-PIGS) To demonstrate the adjuvanticity of the cE-PIGS fusion protein through Fcγ receptor binding and complement activation To demonstrate immunogenicity of cE-PIGS in mice Schematic cE-PIGS cE CH 1 H C H3 C H2 μtp mIgG2a Cys237,240,242 Cys274 Cys340 Cys390 Cys456 Cys : Intra-monomeric disulfide bonds Cys239,242 Cys274 Cys340 Cys390 Cys456 hIgG1 Cys575 : Inter-monomeric disulfide bonds. May form a disulphide bridge with J-chain Antigen-Presenting Cell Dengue cE Complement receptor mediated phagocytosis FcR binding C1q binding Fc receptor mediated phagocytosis June 17, 2015 Partial CγH1 H CγH2 CγH3 μ-tp : IgM tail-piece (tp) that participates in polymerization through a penultimate cysteine residue Plant expression of cE-PIGS pEAQ pTRAk.2 SAR 35x2/TEV5’UTR pA35S 35/RNA2 5’ UTR RNA2 3’ UTR J chain : small protein that links up monomeric IgM or IgA molecules into higher structures cE-mPIGS cE-hPIGS Monomer IgM Hexamer + J chain Pentamer IgA Expression of mouse cE-mPIGS in WT-Nicotiana benthamiana Reducing Non-reducing α -Mouse α -Dengue Expression of human cE-PIGS in ∆XF-Nicotiana benthamiana Reducing Non-reducing α-Human α-Dengue Verification of cE-hPIGS∆XF glycovariant mPIGS in Wild type N.benthamiana hPIGS in ∆XF N. benthamiana (absence of xylose & α-1,3-fucose) (Steinkellner, BOKU, Vienna) Reducing α -human α-plant glycan Analysis of purified cE-PIGS fusion protein on SDS-PAGE cE-mPIGS cE-hPIGS * SIgA: 385 kDa Non-reducing Polymer: ~480 kDa Dimer: 160 kDa Monomer: 80kDa Reducing Single chain cE-PIGS formation determined by Zetasizer measurements IgG 150 kDa PIGS 80 kDa Functional characterization by C1q complement binding assay cE-mPIGS cE-hPIGS Monocyte binding by FACS analysis Mouse J774 Human U937 Immunization protocol Balb/c mice (mPIGS) Human CD64 Tg mice (hPIGS) 0W 2W 4W 1st-immunization 2nd -immunization 3rd-immunization Subcutaneous injection of PIGS PIGS injected with/without Alum Antibody & T cell response analyzed Analysis of mPIGS-induced IgG responses, their neutralization potential Cellular immune responses of mPIGS Cytokine response T cell proliferation Evaluation of hPIGS specific IgG responses in sera of CD64 mice bias Skewing towards TH1 (IgG2a) Mixed TH1/TH2 Summary and conclusions Human and mouse IgM-like poly-IgG scaffold (PIGS) molecules incorporating consensus E domain III of dengue envelope glycoprotein (cE) have been generated and expressed in tobacco plants Both human and mouse cE-PIGS bound to C1q component of complement and to Fcγ receptor-bearing cells In vivo studies confirmed: Bacterially expressed cE is not immunogenic without an adjuvant; cE-PIGS induced a strong dengue cE specific IgG response in the absence of adjuvant which was equivalent to cE with alum. cE-mPIGS induced cellular immune responses in immunised mice above the controls. Initial studies showed neutralization of DV4 by mPIGS-immunised sera Future studies will focus on further characterisation of induced immune responses and neutralization of all 4 dengue serotypes Acknowledgments Dr J.R. del Valle Pro. J. K-C. Ma Dr R. Reljic Dr C. van Dolleweerd Dr M. Paul Mrs T Guerra Mr Gil Diogo