Single-Dose Vaccine Carrier for Modulation of Immune Response Mechanisms

Matt J. Kipper1, Jennifer Wilson2, Michael Wannemuehler2, and Balaji Narasimhan1

1Department of Chemical Engineering, Iowa State University2Department of Veterinary Microbiology and Preventive

Medicine, Iowa State University

68a – AIChE Annual Meeting, November 9, 2004

Controlled Release Improves on Conventional Administration Schedules

Conventional• Poor patient

compliance

• Poor concentration control

• Overdose potential

Therapeutic Range

Toxicity

No Activity

Time

Con

cen

trati

on

Controlled Release• Single dose

• Tailored release kinetics

• Targeted to specific organs tissues or cells

Many Controlled Release Formulations have been Marketed

Brand Name ApplicationRelease

timeDelivery

route

Anti-tumor Weeks Implant

Allergy 1 Day Oral

Contraceptive

1 Month Injection

Anti-depressant

1 Week Oral

Polyanhydrides are Excellent Candidates for Controlled Release

Poly(Sebacic anhydride) Poly(SA)

Poly[1,6-bis (p-carboxyphenoxy)hexane] Poly(CPH)

Hydrophobic Surface erosion

Mutually incompatible

Biocompatible degradation products

-COOH

Hydrolytically labile

Narasimhan and Kipper, Adv. Chem. Eng. (2004)

Polymer Chemistry and Microstructure Affects Erosion and Release Kinetics

TimeC

um

ula

tive M

ass

R

ele

ase

dShen, Kipper, Dziadul, Lim, Narasimhan, J. Controlled Release (2002)

Tailored release kinetics

Surface Erosion

Bulk Erosion

Combined

Current Vaccine Administration Schedules are Non-Ideal• >700,000 neonatal deaths world-wide

from tetanus• Conventional injection schedules

– Many injections– Patient compliance

• Controlled release technology– Single injection– Multiple formulations

• NIH Lists Single Dose Vaccines as #1 Grand Challenge in Global Health

http://www.grandchallengesgh.org/

Controlled Release Formulations Offer Several Advantages for Vaccines

• Polyester-based (PLGA) single-dose vaccines– Protective immunity

possible w/single-dose (Corradin, O’Hagan)

– Antibody titer and isotype/subclass similar to that in alum-based systems (Corradin)

– Acidic/aqueous microenvironment reduces antigenicity (Schwendeman, Langer)

• Polyanhydrides– Hydrophobic

microenvironment– Protein

stabilization– Modulated release

kinetics– Reduced acidity– No studies with

vaccine formulations

Two Immune Response Mechanisms Offer Different Protection

DC

Pathogen

Migration to draining

lymph node

MHC II

Th1 Th1 B Cell

Secreted Antibody

MHC I

TCR

Th2 Th2Cytotoxic T Cell MHC I

Infected Cell

Humoral (Th1) response

Cellular (Th2) response

Circulation

Circulation

TCR

Research Paradigm

Hydrophobic Hydrolyzable

Surface Erosion

Polymer/APC Interactions

Controlled Release

Antigen Stabilization

Cytokine Profile

Immune Activation

Novel Adjuvants for Single-Dose Vaccines

Goal

Engineer tetanus toxoid (TT)-loaded polyanhydride microspheres and study in vivo immune response

Microspheres Fabricated by W/O/O Double Emulsion

100mg polymer in 4ml MeCl25mg protein in 100l water

Emulsify, add 4ml of silicon oil (dropwise) saturated with MeCl2

Continue emulsifying, to form outer emulsionAdd to 300ml of n-heptane, stir three hours to allow MeCl2 to evaporate

Filter, rinse, dry

Non-Porous Microspheres Provide Extended Release Kinetics

20:80 • Microspheres

incubated in 0.1M phosphate buffer (pH 7.4)

• TT antigen concentration determined by BCA assay

50:50

• 3 C3He/OuJ mice per group

• IM injection (right quadriceps)

• Unencapsulated TT and blank 20:80 CPH:SA microspheres

• 50% cottonseed oil/saline emulsion

• ELISA for TT-specific IgG at 1:400 dilution

Polyanhydride Microspheres Induce Dose-Dependent Inhibition

Group

Polymer

TT

I 3mg 3g

II 1mg 3g

III 0.5mg 3g

IV 3mg 3g (day 3)

V 3mg 3g (opposite leg)

VI none 3g

0

0.5

1

1.5

2

2.5

1

Optical Denisty (A.U.)

I II III IV V VI

TT-Loaded Microspheres Provide Immunity

• 5 C3He/OuJ mice per group• Injected IM (right quadriceps) with 2%

loaded TT-loaded microspheres (0.5mg) and/or bolus of unencapsulated TT (0.5g)

• Bled weekly from saphenous vein• TT-specific IgG antibody titer

determined by ELISA

TT-Loaded Microspheres Provide Immunity

+ Bolus only

Blank microspheres

Blank plus bolus

TT microspheres

TT microspheres plus bolus

x Equivalent dose of TT

50:50 CPH:SA

20:80 CPH:SA

20:80TT Microspheres Provide High-Avidity Antibody

• 10–week serum samples tested by ELISA

• Sodium thiocyanate dissociates antibody from TT

• Avidity index is maximum molarity of NaSCN that results in <50% dissociation in 20-minute incubation

20:80 CPH:SA Microspheres Provide High-Avidity Antibody

20:80 CPH:SA provides high titer and high avidity protective immunity

0

1

2

3

VIII IX X XII XIII XIV XV

Avidity Index

VIII – 20:80 blank w/bolus

IX – 20:80TT

X – 20:80TT w/bolus

XII – 50:50 blank w/bolus

XIII – 50:50TT

XIV – 50:50TT w/bolus

XV – Equivalent TT dose

Immune Response Pathway can be Tuned by Microsphere Formulation

•IgG1 and IGg2a isotypes determined by ELISA

•Ratio of OD at 1:400 dilution

•IgG2a = Th1

•IgG1 = Th2

0

0.2

0.4

0.6

0.8

1

1.2

IX X XIII XIV Eq.Dose

Fraction of IgG1 + IgG2a titer

IgG2a

IgG1

20:80 50:50

How do 20:80 Microspheres Mediate Immune Response?

Immunogen-loaded

microspheres

DC

Migration to lymph node delayed by hydrophobic

adjuvant

Inflammatory cues wane

Antigen-driven (TH2) response

Microspheres degrade, releasing antigen

DC Migration to lymph node not

delayed

Antigen-driven (TH2) response

Unencapsulated immunogen

DCsMigration to

lymph node not delayed

Inflammatory cytokine context

results in balanced responseIL-12

Summary• Low polymer doses provide adjuvant effect• TT-Loaded microspheres provide immunity

– Preserved antigenicity– Sustained exposure to antigen provides

secondary immune response– Antibody titers and avidity show efficacy in

single-dose formulation

• Immune response mechanism can be modulated by altering formulation

Acknowledgements

Institute for Combinatorial Discovery, Iowa State University