Name of presentation Month 2009 Using layered double hydroxide nanoparticles conjugated with CpG to polarise immune responses from Th2 to Th1 bias Shiyu

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


Outline

1. Background

2. Experimental Methods

3. Results and Discussion

4. Conclusion

5. Acknowledgements


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


Current vaccine adjuvants

Adjuvants Practical Limitations

Oil-in-water emulsions (e.g. MF59 and CFA)

MF59 contains shark oil, unrenewable resourceCFA contains mineral oil and arlacel, toxic for human use

Monophosphoryl 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


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


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


TLR-9 ligand

CpG

• Interacting with TLR-9 in the endosome

• Promoting Th1 responses

• Low efficacy: – Rapid degradation– Ineffective delivery to the endosome

BACKGROUND


BACKGROUND- MgAl-layered double hydroxide (LDH)

In brucite layer (Mg(OH)2), Mg2+ is partially substituted with Al3+, resulting in a positively charged layer. An anion enters the interlayer to keep the charge balanced.

Cl-, NO3-

An-

+ +++

+

++

++++

++

+ +++

+

++

++++

++


Low cost

Low toxicity

Excellent biocompatibility

Efficient carrier for delivering genes and drugs

Design strategy of LDH-based vaccine


Hypothesis

APC cell

T cell

A

B


EXPERIMENTAL METHOD

Day 0

Day 21 Day 35

B16/F10/OVA melanoma cells (s.c.)

Tumour

OVA vaccines(subcutaneous, s.c.)

OVA vaccines (s.c.)


Characterisation of LDH nanoparticles

Particle size distribution (A) and TEM image (B) of Mg2Al-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


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



Loading of OVA and CpG by LDH NPs

240 µg LDH + 60 µg OVA + 5 µg CpG


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

*


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


Induction of the Th1-polarised immune response

Serum IgG2a:IgG1 ratio 35 days after immunisation.

RESULTS


Determination of tumour-infiltrating CD8+ T lymphocytes (TIL) on tumour tissueOVA

Isotype controlLDH-CpG-OVA

LDH-OVA

RESULTS


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

A B

Time after tumour inoculation (day) Time after tumour inoculation (day)

Tum

ou

r vo

lum

e (

mm

3)

Tum

ou

r vo

lum

e (

mm

3)



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


Acknowledgements

Organisations

ARC Centre of Excellece for Functional NanomaterialsAustralian Institute for Bioengineering and NanotechnologyThe University of Queensland

Research Grants

ARC DP0879769 (ARF) and DP0559594 (APD)ARC FT120100813, UQ Excellence Awards

CollaboratorsDr Barbara RolfeDr Yousuf Mohammed

Thank you for your attention

Documents

Name of presentation Month 2009 Using layered double hydroxide nanoparticles conjugated with CpG to polarise immune responses from Th2 to Th1 bias Shiyu