Refolding the HCV E2 glycoprotein to enhance immunogenicity

Transcription

1 Refolding the HCV E2 glycoprotein to enhance immunogenicity Lilian Phu, Rob Center, Pantelis Poumbourious, Heidi Drummer Viral Entry and Vaccines Laboratory 1

2 E2 glycoprotein E2 RBD Mediates viral entry The RBD contains 17 cysteines that form 8 intramolecular disulfides (HVR1 and HVR2 not shown) Kong L, Giang E, Nieusma T, Kadam RU, Cogburn KE, Hua Y, Dai X, Stanfield RL, Burton DR, Ward AB, Wilson IA, Law M Hepatitis C virus E2 envelope glycoprotein core structure. Science 342: Variable regions are involved in immune evasion HVR1: Immunodominant Elicits type-specific NAbs Occludes CD81 binding sites and bnab epitopes CD81 binding sites are highly conserved 2

3 Delta123 (Delta3) HVR1 HVR2 igvr E2 RBD L L Delta3 Delta3 A recombinant protein with the variable regions removed Contains all 17 cysteines Native conformation of E2 and CD81 binding ability are retained McCaffrey K, Gouklani H, Boo I, Poumbourios P, Drummer HE The variable regions of hepatitis C virus glycoprotein E2 have an essential structural role in glycoprotein assembly and virion infectivity. J Gen Virol 92:

4 G1a (strain H77c) Delta3 1 2 dimer Delta3 oligomers Non-reducing non-delta3 Reducing size Delta3 oligomerises into different disulfide-linked forms: Monomers 47 kda Dimers 97 kda High molecular weight () species kda An aggregate of 5-50 Delta3 subunits Coomassie stained gels 4

5 G1a (strain H77c) Delta3 1 2 dimer Monomers vs. Monomer: High yields and pure Elicits type-specific NAbs : Low yields and prone to contamination Elicits high titres of bnabs Directs Ab response to the neutralising face of E2 An attractive vaccine candidate size Percent neutralization G1a G2a G3a G4a G5a G6a G7a Genotype 5

6 G1a (strain H77c) Delta3 1 2 dimer Monomers vs. Monomer: High yields and pure Elicits type-specific NAbs : Low yields and prone to contamination Elicits high titres of bnabs Directs Ab response to the neutralising face of E2 An attractive vaccine candidate size How to increase the yield and purity of Delta3? 6

7 Refolding s into Delta3 Aim To increase the yield and purity of Delta3 Method Protein refolding Used to refold misfolded protein aggregates into functional lower-order species Higher-order species Lower-order species 7

8 Refolding s into Delta3 Hypothesis: Delta3 is formed by intermolecular disulfide bonds between surface exposed cysteines 1) Partial Reduction Addition of reducing agent Examples: Dithiothreitol (DTT) Glutathione Tris(2-carboxyethane)phosphine Β-mercaptoethanol Monomeric Delta3 8

9 Refolding s into Delta3 Hypothesis: Delta3 is formed by intermolecular disulfide bonds between surface exposed cysteines 1) Partial Reduction Addition of reducing agent To generate free sulfhydryls Monomeric Delta3 9

10 Refolding s into Delta3 Hypothesis: Delta3 is formed by intermolecular disulfide bonds between surface exposed cysteines 2) Re-oxidation Removal of reducing agent Monomeric Delta3 10

11 Refolding s into Delta3 Hypothesis: Delta3 is formed by intermolecular disulfide bonds between surface exposed cysteines 2) Re-oxidation Removal of reducing agent To form intermolecular disulfide bonds Delta3 11

12 Refolding conditions 1) Reducing agents: Dithiothreitol (DTT) Reduced L-glutathione (GSH) Tris(2-carboxyethyl)phosphine (TCEP) β-mercaptoethanol (βme) 2) Concentration of reducing agent 3) Concentration of ic D123 4) Incubation time: 0.5h, 1h, 2h, 24h 5) Temperature: 4 C, room temperature, 37 C 6) Refolding methods: Slow dilution Oxidised L-glutathione (GSSG) Bismaleimidoethane (BMOE) crosslinkers 12

13 1 dimer 2 1 dimer 2 Refolding using the glutathione redox system Simultaneous addition of reduced and oxidised glutathione The glutathione redox system promotes: Re-conjugation of free sulfhydryls Disulfide rearrangement 97% 45% 55% Purified s before glutathione treatment Purified s after glutathione treatment 13

14 m A u m A u m A u dimer dimer 2 dimer 2 1 Refolding using dithiothreitol (DTT) Addition of DTT to purified ic Delta3, followed by slow dilution % 50% % 35% % 39% m L 1 round 2 rounds 3 rounds m L m L 14

15 Antigenic Characterisation Neutralising Abs H C H C V 1 M A b O D O D O D MAb24 HC R e c ip ro c a l D ilu tio n R e c ip ro c a l D ilu tio n R e c ip ro c a l D ilu tio n Neutralising Ab epitopes are similarly exposed on ic and refolded Delta3 Non-neutralising Abs C B H 4 B C B H 4 G M A b O D O D O D R e c ip ro c a l D ilu tio n R e c ip ro c a l D ilu tio n R e c ip ro c a l D ilu tio n Non-neutralising Ab epitopes are more occluded on refolded Delta3 15

16 Summary Refolding Improved Delta3 yield by 50% Need to assess purity and stability Antigenicity Conformation of refolded Delta3 occludes non-neutralising antibody epitopes Immunogenicity Next step Efficient Delta3 production presents a promising pathway for the development of a prophylactic vaccine for hepatitis C 16

17 Acknowledgements Viral Entry and Vaccines Laboratory: Associate Professor Heidi Drummer Co-head and supervisor Dr. Andy Poumbourios Co-head Dr. Rob Center Supervisor Dr. Patricia Vietheer Irene Boo Joey McGregor Jun Gu Annamarie Laumaea Hannah King David Harrison Conference organisers for scholarship Vani Geetha 17