Paul Pumpens. Latvian Biomedical Research and Study Centre, Riga

Transcription

1 Paul Pumpens Latvian Biomedical Research and Study Centre, Riga Bethesda, September 22, 2009

2 VLPs = icosahedrons (viral cores)

3 Icosahedron (viral core) with outer envelope (surface)

4 A typical example of an enveloped virus with icosahedric core: Hepatitis B virus isometric envelope icosahedric core (nucleocapsid)

5 Hepatitis B surface: RTS,S malaria vaccine

6 Oligonucleotides, DNA, RNA Proteins Peptides Drugs Hepatitis B core VLPs: Packaging Vaccines Drug delivery Gene therapy Chemotherapy

7 VLPs: Combination of Exposure and Packaging Oligonucleotides, DNA, RNA Proteins Peptides Drugs Vaccines Drug delivery Gene therapy Chemotherapy

8 VLPs: Nanotechnological Applications Nanomagnetic particles Physics Electronics

9 Types of VLPs empty non-infectious empty infectious replication-competent infectious replication-noncompetent

10 3D structure of the HBc platform

11

12 HBc: Electron microscopy 34 nm (T=4) and 30 nm (T=3)

13 HBc on the size scale of the most promising VLP candidates

14 Hepatitis B core: T=4 and T=3 31,2 nm Vaccines: HBV HCV Malaria influenza

15 Hepatitis B core as a platform HBV particles from blood recombinant HBc full-length recombinant HBc C-terminally truncated recombinant HBc C-terminally truncated with long C-terminal insertions recombinant HBc full-length with long C-terminal insertions recombinant HBc C-terminally truncated with internal (MIR) insertions

16 Structural and immunological features of the HBc protein B epitopes CTL epitopes monomer tetramer - asymmetric unit

17 HBc tetramer: putative insertion sites MIR: positions C-terminal: positions 144,161, 167, etc Wynne et al., 1999

18 HBc tetramer (surface): putative insertion sites MIR: positions C-terminal: positions 144,161, 167, etc Wynne et al., 1999

19 HBc dimer (chains A and B - surface): putative insertion sites MIR: positions C-terminal: positions

20 HBc dimer (chains A and B): putative insertion sites MIR: positions C-terminal: positions

21 Chimeric HBc dimer (chains A and B): successful insertion at the MIR MIR: insertion at the position 78 modelling by 3D-JIGSAW

22 Chimeric HBc dimer (A and B - surface): successful insertion at the MIR MIR: insertion at the position 78 modelling by 3D-JIGSAW

23 ProteinengineeringontheHBc

24 HBc VLPs: Expression Promoter HBc gene Virus-like particles (VLPs) - Escherichia coli - Pichia pastoris - Hansenula polymorpha

25 Construction of chimeric HBc VLPs: direct gene fusion INS INS INS HBc chimeric HBc

26 Construction of chimeric HBc VLPs: direct gene fusion chimeric HBc

27 Construction of Chimeric HBc VLPs: Chemical Coupling Martin F. Bachmann Zuerich

28 HBc: design DNA/RNA packaging: - gene therapy Surface elements: - vaccines - diagnostics - gene therapy

29 HBc VLPs: Types of chimeric particles Uniform Mosaic Mosaic multi-epitope

30 HBc: what and where to insert? Promoter Surface elements Surface elements Hidden elements

31 HBc: capacity Up to 50 foreign aa Up to 250 foreign aa Promoter Surface elements Surface elements Hidden elements Up to 800 foreign aa

32 HBc vectors

33 Main problems of the HBc: capacity and exposition VECTOR CLASSES: MIR full-length C-terminally truncated major immunodominant region (MIR) aa exposed: YES capacity:? rate of success:?

34 Main problems of the HBc: capacity and exposition VECTOR CLASSES: N-terminus full-length C-terminally truncated N-terminus exposed:? capacity:? rate of success:?

35 Main problems of the HBc: capacity and exposition VECTOR CLASSES: C-terminus full-length C-terminally truncated C-terminus exposed: NO capacity: HIGH rate of success: HIGH

36 Main problems of the HBc: capacity and exposition VECTOR CLASSES: C-terminus modified full-length C-terminus exposed: YES capacity: HIGH rate of success: HIGH

37 Main problems of the HBc: capacity and exposition VECTOR CLASSES: C-terminus modified full-length ~100 foreign aa C-terminus exposed: YES capacity: HIGH rate of success: HIGH

38 Main problems of the HBc: capacity and exposition VECTOR CLASSES: C-terminus modified full-length ~30 foreign aa C-terminus exposed: YES capacity: HIGH rate of success: HIGH

39 Purification of chimeric HBc derivatives

40 Purification of chimeric HBc derivatives Gel chromatography: Sepharose CL4B YIELDS: Escherichia coli mg of pure HBc from 1 l of culture yeast mg of pure HBc from 1 l of culture

41 Packaging of HBc particles

42 HBc vectors: packaging - CpG Chimeric HBc, which contains immunodominant T helper and CTL epitope Agarose gel electrophoresis, ethidium bromide staining: proteins were taken in equimolar concentrations Marker Full-length C-terminally truncated 1kbp M HBc183 HBc145 Chimeric HBc packed with CpG HBc161

43 HBc vectors: packaging magnetic nanoparticles

44 Vaccine candidates on the HBc basis

45 Vaccine candidates: HBV

46 Vaccine candidates: HCV

47 Vaccine candidates: Malaria ICC-1132

48 Vaccine candidates: flu (M2)

49 Vaccine candidates: flu (M2) chemical coupling more complicated fusions

50 Follow-up of VLP technologies CRC Press, 2002 CRC Press, 2008

51 VLP: Latvian Biomedical Research and Study Centre Tatjana Kozlovska Irīna Sominska Elmārs Grēns Kaspars Tārs Andris Zeltiņš Andris Kazāks Velta Ose Andris Dišlers Regīna Renhofa Indulis Cielēns Tatjana Voronkova Ivars Petrovskis Dace Skrastiņa Anna Zajakina Juris Jansons

52 Special thanks to: Rolf M. Zinkernagel Zuerich Wolfram H. Gerlich, Giessen Martin F. Bachmann Zuerich R. Anthony Crowther, Cambridge

53 Thank you for your attention