Flock House virus VLPs as a tool in structure-based vaccine design. Malaria VLP Development Workshop September 23, 2009

Transcription

1 Flock House virus VLPs as a tool in structure-based vaccine design Malaria VLP Development Workshop September 23, 2009

2 Flock House virus X-ray structure solved at 2.8 Å resolution Particle diameter 35 nm T=3 capsid T=3 icosahedral symmetry, 180 coat protein subunits Grows to VERY high titers in Drosophila cells (1 mg purified virus from a single 10 cm tissue culture dish in 2 days) Particles are very stable (ph 4-9; 6M Urea, detergents, DMSO, 65 ºC) Virus-like particles can be generated in a high yielding expression system genetically very simple; bipartite genome encodes only three proteins; viral RNAs have been cloned and sequenced, cdnas available

3 Flock House virus an attractive system for engineering new properties into the capsid that make it potentially useful in biotechnology, nanotechnology and materials synthesis T=3 capsid Potential applications: Antitoxins Vaccines Tumor imaging and destruction

4 The virulence of B. anthracis is largely a result of the expression and action of three proteins: Protective antigen (PA, 83kD) Edema factor (EF, adenylate cyclase, 89 kd) Lethal factor (LF, zinc protease, 90 kd)

5 Inhalational anthrax: Presents with flu-like symptoms Difficult to diagnose early Antibiotic treatment not sufficient once toxins have accumulated Need for antitoxins Current anthrax vaccine (Biothrax; AVA): B. anthracis culture supernatant of ill-defined composition Side effects, requires adjuvant Lengthy dosing schedule (6 injections followed by annual boosters) 2nd generation vaccine Based on recombinant PA Requires adjuvant Requires several injections Stability issues Need for 3rd generation vaccine that provides rapid, potent protection against inhalational anthrax

6 The virulence of B. anthracis is largely a result of the expression and action of three proteins: Protective antigen (PA, 83kD) Edema factor (EF, adenylate cyclase, 89 kd) Lethal factor (LF, zinc protease, 90 kd)

7 A decoy to mop up the toxin PA83 FHV CMG2 CMG2

8 Structure of the extracellular domain of CMG2 N C 188 amino acids approx. 20 kd Lacy et al. PNAS101:6367

9 aa 206 aa 264 Structure of Flock House virus

10 Schematic diagram showing the insertion of CMG2 VWA in the FHV coat protein Chimera 206 α β+ γ Cleavage site N363 - A FHV-Nterm CMG2 VWA -Ala-Glu- FHV-Cterm Chimera FHV-Nterm CMG2 VWA FHV-Cterm

11 SDS PAGE and electron-micrographs of chimeric and wild type FHV VLPs QTar Wt Å

12 CELL INTOXICATION ASSAY 1. Incubate FHV-CMG2 chimera with PA+LF-DTA at room temp. for min. 2. Add mixture to CHO cells and incubate for 48 hours at 37 C. 3. Remove medium. 4. Add Cell Titer Glow reagent to determine cell viability. Luciferase ATP + D-luciferin + O 2 AMP + PP i + oxyluciferin + CO 2 + light

13 Cell intoxication assay Construct IC50 scmg nM (± 0.87) FHV CMG nM (± 0.61) FHV CMG nM (± 0.36)

14 Structure of the PA-CMG2 Complex PA 20 PA 83 PA 63 CMG2 Santelli et al., Nature 905:430

15 CryoEM reconstructions of wt FHV and FHV-CMG2 chimeras wt FHV chimera 206 chimera 264

16 Pseudoatomic models of FHV-CMG2 chimeras Chimera 206 Chimera 264 FHV capsid CMG2 VWA cryo-em density

17 Pseudoatomic models of FHV-CMG2 chimeras Chimera 206 Chimera 264 FHV capsid CMG2 VWA

18 In silico model of PA83 bound to surface of FHV CMG2 chimeras Chimera 206 Chimera 264 FHV capsid CMG2 VWA PA

19 Idea: Use FHV-CMG2 particles decorated with PA83 as an immunogen to raise antibodies against PA Polyvalent display of PA83 should induce stronger, more rapid response than monomeric PA which is used in current and 2nd generation vaccine Use of adjuvant may not be necessary

20 Immunization studies with PA-decorated FHV-CMG2 particles PBS control Male Harlan Sprague Dawley rats; 4 rats per group. Two sub-cutaneous doses (2.5 μg PA83 or equivalent): primary and booster 3 weeks later. No adjuvants. Serum collected before priming, 3 weeks after priming, and 4 weeks after the boost. Serum was tested for antibodies by ELISA.

21 1.4 anti-pa83 (1:1000) * * FHV-264-PA FHV-264 PA 83 PBS 0.2 OD units anti-fhv (1:1000) * 0.25 anti-cmg2 (1:100) Pre-bleed week 3 week7

22 Lethal toxin challenge of rats immunized with PA83 or FHV-CMG2-PA83 FHV-CMG2-PA83 group

23 Antibody response after a single injection of 5 μg PA83 or equivalent anti-pa83 FV-268-PA (O.D) Ab response to PA Pre bleed Week 3 FHV-268 PA 83 PBS Lethal toxin challenge of rats immunized with PA83 or FHV-CMG2-PA83 FHV-CMG2-PA83 group

24 Summary: 180 copies of CMG2 were displayed on the surface of FHV Chimeric particles inhibited anthrax toxin in vitro and in vivo FHV-CMG2 VLPs complexed with PA83 elicited potent neutralizing antibody response The antibody response protected rats from lethal toxin challenge after a single immunization w/o adjuvant after 4 weeks The VLP platform represents a dually acting reagent for treatment and protection against anthrax

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26 Ig-like domain FHV CP

27 FHV/Ig-like chimera

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29 Using VNI-264 platform to develop new vaccines Immunogen Adapter Anthrax vaccine Vaccines against other pathogens and toxins

30 Expanding the utility of the FHV platform Domain 1 of PA Ricin A chain Receptor binding domain of botulinum neurotoxin A wt PA PA-RTA PA-Bont/A

31 Expanding the utility of the FHV platform wt PA PA-RTA PA-Bont/A

32 Acknowledgments Anette Schneemann Arno Venter Nick Brunn Darly Manayani Marc Siladi Vijay Reddy Mari Manchester Diane Thomas Mark Yeager Kelly Dryden John Young (Salk) John Marlett Jonah Reiny Heather Scobie NIH funding Flock House, New Zealand