bio-beads - PolyBatics

A NOVEL PARTICULATE VACCINE
UTILISING POLYESTER
NANOPARTICLES (BIO-BEADS)
An approach for the single step produc3on of an3gen displaying nanopar3cles…. The PolyBa+cs expression system produces engineered an+gen displaying nanopar+cles in microbial hosts in a single step. Produc3on strain development Glucose/Carbon source (R)-3-hydroxyacyl-CoA
Polyester
Fusion partner/ Polymer forming enzyme Bacterial cell antigen
Linker
synthase
Inser+on of the engineered gene into the host produces covalently a=ached an+gens displayed on the surface of the polyester core. Density 1.06 g /ml Size distribu3on 100-­‐600 nm pH stability 2.0 – 12.5 Temperature stability 160oC (for polymer) Electron backscaNering micrograph of lyophilized beads Advantages of PolyVax as vaccines   Par3cle’s polymer core is biocompa3ble and biodegradable   Simple -­‐ gene3c sequence of an3gen/protein   Versa3le   Different produc3on hosts (including GRAS Lactococcus)   Addi3onal display e.g. Immunomodulators on the same par3cle   Mul3plexed display – mul3valent vaccines   Can combine targe3ng proteins with an3gens   range of disease an3gens – tailor to disease   Mycobacterial an3gens, hepa33s C core an3gen, influenza an3gens   Produc3on   Scalable ultra low-­‐cost   Purifica3on rela3vely simple   One-­‐step produc3on of an3gen and carrier: no need to produce, purify and conjugate recombinant proteins – ultra low cost Bacterial host for an3gen display • 
• 
• 
E. coli Gram nega3ve bacteria e.g. E. coli Engineered to produce BIO-­‐BEADS which display Ag85A-­‐ESAT6 • 
Lactococcus lac-s • 
• 
• 
gram posi3ve food grade bacteria does not contain endotoxin recent uses   recombinant protein produc3on   vaccina3on trials –mucosal route • 
Engineered to produce BIO-­‐BEADS which display Ag85A-­‐ESAT6 • 
• 
Alterna3ve “Pathogen” hosts Beads will contain extra an3gens and molecules from HCP • 
  adjuvant proper3es   TLR agonists Under development Uptake of BIO-­‐BEADS by Dendri3c cells (DC) -­‐ TEM DC -­‐ no BIO-­‐BEADS DC + E. coli BIO-­‐BEADS DC+ L. lac+s BIO-­‐BEADS Mouse Trials
Challenge 0 2 4 15 6 20 weeks Vaccina3on responses • 
 
3 x vaccina3ons (subcut) 2 week intervals   BIO-­‐BEADS: +/-­‐ Ag85A-­‐ESAT-­‐6   E. coli and L. lac+s bacterial produc3on hosts   Bacille CalmeNe-­‐Guérin (BCG) posi3ve control (1x vaccina3on) Challenge 15 weeks afer first immunisa3on   M. bovis 50 organisms per lung via aerosol route Ag85A-ESAT6-specific IFN-γ responses in
spleen cultures
6
*
IFN-γ (ng/ml)
5
*
4
3
2
1
0
PBS
3 ug 10 ug 30 ug 90 ug 30 ug
Ag85A-ESAT6
beads HR1-2 Recombinant
HR1-2
HR1-2 HR1-2
rec
beads beads beads beads Ag85A/ESAT-6
HR1-2
protein
* Significantly greater than all other groups (P<0.01)
Serum antibody responses following vaccination
**
**
10000
**
1000
*
**
IgG1
IgG2c
* *
*
100
10
* Significantly greater than WT beads (P<0.05)
4
13
12
6
30 µg 90 µg 3 µg
30 µg 10 µg
Ag85A-ESAT6
Rec. Ag85AAg85A-ESAT6
BNPs
Ag85A-­‐ESAT6 beads beads
ESAT6
11
PBS
1
1
Parlane et al. 2009
PROTECTION TRIAL Culture of lungs and spleens from mice infected with M. bovis Lung M. bovis CFU (log10)
6.0 *
*
*
5.0 Spleen M.bovis CFU (log10)
6.0
7.0 5.0
*
**
4.0
4.0 3.0
3.0 PBS Ec AgE Ec WT Lc AE Lc WT recAgE BCG PBS Ec AgE Ec WT Lc AE Lc WT recAgE BCG * different to non-­‐vaccinated group (P<0.05)   Protec3ve immunity is induced in mice vaccinated with BIOBEADS displaying mycobacterial an3gens Ag85A-­‐ESAT-­‐6
11 IMMUNOGENICITY:
Vaccination
cytokine responses
6 weeks post vaccina3on Spleen cell response to Ag85A and ESAT-­‐6 pep3des Code Host An3gen Ec AgE E. coli Ag85A-­‐ESAT6-­‐PhaC Ec WT PhaC E. coli Lc AgE L. lac3s Ag85A-­‐ESAT6-­‐PhaC Lc WT PhaC L. lac3s recAgE E. coli Ag85A-­‐ESAT6 BCG -­‐ -­‐ PBS -­‐ -­‐ * different to non-­‐vaccinated group (P<0.05) IL-­‐4: not detected IL-­‐5 and IL-­‐10: <10pg/ml. No significant differences Parlane et al. CVI 2012, 19(1): 37 HepC bead: Immune
response analysis
Splenocytes restimulated with recombinant HepC core antigen
Conclusions
Following vaccina3on with PolyVax displaying an3gens (Ag85A-­‐ESAT6 fusion protein and HepC core an3gen):  IFN-­‐γ, IL-­‐17A, TNF-­‐α and IL-­‐6 were released post-­‐ vaccina3on  Significant protec3on (tuberculosis) was induced  Pathology correlated with protec3on data Future developments of PolyVax   BCG Prime-­‐BIOBEAD Boost   Mice   CaNle   Immunomodulator incorpora3on   Alterna3ve an3gens on BIOBEADS eg. TB10.4/Ag85B   BIOBEAD produc3on in alterna3ve hosts   Mycobacterium sp.   Other genera   Skin-­‐test reagents   Mul3-­‐valent vaccines