1. Name of presentation Month 2009 Using layered double hydroxide nanoparticles conjugated with CpG to polarise immune responses from Th2 to Th1 bias Shiyu Yan, Bing Zhang, Wenyi Gu, Zhi Ping (Gordon) Xu Australian Institute for Bioengineering and Nanotechnology The University of Queensland

2. Name of presentation Month 2009 Outline 1.Background 2.Experimental Methods 3.Results and Discussion 4.Conclusion 5.Acknowledgements

3. Name of presentation Month 2009 Basic challenges to antigenic vaccines Low immunogenicity of purified tumour antigens Low efficacy of immune-stimulatory molecules Vaccine adjuvants are required to regulate and enhance the immune responses BACKGROUND

4. Name of presentation Month 2009 Current vaccine adjuvants AdjuvantsPractical Limitations Oil-in-water emulsions (e.g. MF59 and CFA) MF59 contains shark oil, unrenewable resource CFA contains mineral oil and arlacel, toxic for human use Monophospho ryl lipid A (MPL) Lengthy fermentation and purification Aluminium hydroxide phosphate (Alum) Antibody-mediated (Th2) response induction; Short duration of immune responses Strong inflammatory reaction BACKGROUND

5. Name of presentation Month 2009 Current vaccine adjuvants BACKGROUND 1.Simple formulation 2.Purely empirical 3.Unclear about antigen-adjuvant interactions 4.Unclear about how APCs interact with antigen through the adjuvant

6. Name of presentation Month 2009 TLR-mediated immune responses T Kawai1 and Akira (2006) “TLR signaling” Cell Death and Differentiation 13, 816–825 Immune-stimulatory biomolecules Toll-like receptor (TLR) ligands BACKGROUND

7. Name of presentation Month 2009 TLR-9 ligand CpG Interacting with TLR-9 in the endosome Promoting Th1 responses Low efficacy: –Rapid degradation –Ineffective delivery to the endosome BACKGROUND

8. Name of presentation Month 2009 BACKGROUND- MgAl-layered double hydroxide (LDH) In brucite layer (Mg(OH) 2 ), Mg 2+ is partially substituted with Al 3+, resulting in a positively charged layer. An anion enters the interlayer to keep the charge balanced. Cl -, NO 3 - A n- + + + + + + + + + + + + + + + + + + + + + + + + + +

9. Name of presentation Month 2009 Low cost Low toxicity Excellent biocompatibility Efficient carrier for delivering genes and drugs Design strategy of LDH-based vaccine

10. Name of presentation Month 2009 Hypothesis APC cell T cell A B

11. Name of presentation Month 2009 EXPERIMENTAL METHOD Day 0 Day 21Day 35 B16/F10/OVA melanoma cells (s.c.) Tumour OVA vaccines (subcutaneous, s.c.) OVA vaccines (s.c.)

12. Name of presentation Month 2009 Characterisation of LDH nanoparticles Particle size distribution (A) and TEM image (B) of Mg 2 Al-Cl-LDH nanoparticles in the suspension. A B Yan, Shiyu, Barbara E. Rolfe, Bing Zhang, Yousuf H. Mohammed, Wenyi Gu, and Zhi P. Xu. "Polarized immune responses modulated by layered double hydroxides nanoparticle conjugated with CpG." Biomaterials 35, no. 35 (2014): 9508-9516. RESULTS

13. Name of presentation Month 2009 Animal Expt: IV: 480 µg LDH + 60 µg OVA + 5 µg CpG V: 240 µg LDH + 60 µg OVA + 5 µg CpG OVA adsorption isotherm to Mg2Al-Cl-LDH nanoparticles Loading capacity of LDH for OVA RESULTS Yan, Shiyu, Barbara E. Rolfe, Bing Zhang, Yousuf H. Mohammed, Wenyi Gu, and Zhi P. Xu. "Polarized immune responses modulated by layered double hydroxides nanoparticle conjugated with CpG." Biomaterials 35, no. 35 (2014): 9508-9516.

14. Name of presentation Month 2009 Loading of OVA and CpG by LDH NPs 240 µg LDH + 60 µg OVA + 5 µg CpG

15. Name of presentation Month 2009 LDH adjuvant activity and CpG enhancement OVA-specific serum IgG1 production in C57BL/6 female mice at 35 days following immunisation with OVA coupled with LDH or Alum, with or without CpG. RESULTS *

16. Name of presentation Month 2009 OVA-specific serum IgG2a production in C57BL/6 female mice at 35 days following immunisation with OVA coupled with LDH or Alum, with or without CpG. LDH adjuvant activity and CpG enhancement RESULTS

17. Name of presentation Month 2009 Induction of the Th1-polarised immune response Serum IgG2a:IgG1 ratio 35 days after immunisation. RESULTS

18. Name of presentation Month 2009 Determination of tumour-infiltrating CD8+ T lymphocytes (TIL) on tumour tissue OVA Isotype controlLDH-CpG-OVA LDH-OVA RESULTS

19. Name of presentation Month 2009 LDH adjuvant effects on anti-tumour response to B16/F10/OVA melanoma. Tumour growth in mice (A) immunised against OVA using TLR ligand conjugated LDH nanoparticles; (B) comparison of LDH and Alum adjuvant activity on OVA-specific anti-tumour protective immunity. RESULTS A B Time after tumour inoculation (day) Tumour volume (mm 3 ) Yan, Shiyu, Barbara E. Rolfe, Bing Zhang, Yousuf H. Mohammed, Wenyi Gu, and Zhi P. Xu. "Polarized immune responses modulated by layered double hydroxides nanoparticle conjugated with CpG." Biomaterials 35, no. 35 (2014): 9508-9516.

20. Name of presentation Month 2009 LDH had high loading capacity of antigen and TLR ligand LDH as an adjuvant can induce a specific immune response to the model antigen (OVA) LDH co-delivery of OVA and CpG induces a switch from a Th2-biased to Th1-biased immune response Conclusions

21. Name of presentation Month 2009 Acknowledgements Organisations ARC Centre of Excellece for Functional Nanomaterials Australian Institute for Bioengineering and Nanotechnology The University of Queensland Research Grants ARC DP0879769 (ARF) and DP0559594 (APD) ARC FT120100813, UQ Excellence Awards Collaborators Dr Barbara Rolfe Dr Yousuf Mohammed Thank you for your attention