Title: The Physical Chemistry and Biology of AluminumContaining Adjuvants
1The Physical Chemistry and Biology of
Aluminum-Containing Adjuvants
- Stanley Hem
- Harm HogenEsch
- Purdue University
2Historical Perspective
- Immunopotentiation by aluminum-containing
adjuvants discovered by Glenny in 1926 - Assumed that adsorption of antigen was important
as no antigen was in supernatant - Regulatory agencies require adsorption of the
antigen
3Structure and Properties
- Aluminum Hydroxide Adjuv.
- Aluminum oxyhydroxide
- AlO(OH)
- Surface OH groups
- IEP 11.4
- surface charge at pH 7.4
- Aluminum Phosphate Adjuv.
- Aluminum hydroxyphosphate
- Al(OH)m(PO4)n
- Surface OH and PO4 groups
- IEP 4-6
- - surface charge at pH 7.4
4Morphology
Primary particles AH fibers
AP plates
4.5 x 2.2 x 10 nm
50 nm diam. Aggregates (average
diameter) AH 17 µm
AP 3 µm
Vaccine 22, 1973, 2004
5Major Adsorption Mechanisms
- Electrostatic attraction
- Ligand exchange
6Electrostatic Attraction
- Choose adjuvant with opposite charge of antigen
- BSA (iep5) adsorbed by AH (iep11.4)
- Lysozyme (iep11) adsorbed by AP (iep5)
Vaccine, 9, 201, 1991
7Electrostatic Attraction
- Pretreat AH or AP with phosphate to modify iep
- AH (iep 11.4) with lysozyme (iep 11)
PDA J. Pharm. Sci. Technol. 51, 25, 1997
8Ligand Exchange
- PO4 displaces surface OH
- Ligand exchange occurs with phosphorylated
antigens - Strongest adsorption force
PO4 - Protein
PDA J. Pharm. Sci. Technol. 51, 25, 1997
9Ligand Exchange
- Pretreat AH with PO4
- Ovalbumin (iep5) contains 2 PO4
- Adsorbs by ligand exchange when AH is negatively
charged - Directly related to number of surface OH in
adjuvant
PDA J. Pharm. Sci. Technol. 51, 25, 1997
10Ligand Exchange
- Adsorption is directly related to degree of
phosphorylation of antigen
Vaccine 23, 1502, 2005
11Elution of Antigen
- Composition of IF different from formulation
- PO4
- Citrate
- Fibrinogen
AC/AH
DPAC/AH
- Elution usually observed when adsorption is by
electrostatic attraction
- Elution usually not observed when adsorption is
by ligand exchange
Vaccine 23, 1502, 2005
12Microenvironment pH
- pH of double layer of charged particles is
different from bulk pH - Negative surface attracts protons
- Positive surface attracts hydroxyls
13Microenvironment pH
- Antigens with pH-dependent immunogenicity or
stability include foot and mouth disease,
tetanus, influenza A, mycoplasma hyopneumoniae,
recombinant protective antigen, and Botulinum
Neurotoxin. - Adsorbed antigens degrade at a rate associated
with the microenvironment pH, not the bulk pH. - Optimize stability of antigen by modifying the
surface charge of the adjuvant to produce pH of
maximum stability in the microenvironment of the
surface.
14Is Adsorption Necessary?
15Experimental Design
- Develop vaccine formulation in which the antigen
was not adsorbed - Adsorption did not occur when vaccine was mixed
in vitro with interstitial fluid - Antibody titer determined in mice (8 per
treatment)
Vaccine 25, 825, 2007
16Alpha Casein Vaccine
- Cas
- 26,000 Daltons
- iep 4.6
- 8 phosphate groups
- AH
- iep 11.4
- adsorbs CAS by electrostatic attraction and
ligand exchange - AP
- iep 5
- repels CAS by electrostatics
- adsorbs CAS by ligand exchange
17Pretreat AP to Block Ligand Exchange
- Antigen Adjuvant Adsorbed
- CAS AH 100
- CAS AP
100 - DP-CAS AP 100
- DP-CAS 0.4 M PT-AP 15
- DP-CAS 0.5 M PT-AP
2 - DP-CAS 0.7 M PT-AP 0
- DP-CAS/0.5 M PT-AP was not adsorbed in either the
vaccine or when mixed in vitro with interstitial
fluid
18DP- CAS Antibody Study
-
-
- Potentiation by PT-AP without adsorption
19Immunopotentiation without Adsorption
- Ovalbumina
- Lysozymea
- Haemophilus Influenza Type Bb
- Anthrax (rPA)c
- a Vaccine 25, 825, 2007
- b Pediatr Int. 45, 314, 2003
- c Vaccine 23, 1993, 2005
20Monitor Loss of Antigen from Injection Site
- Use In-111, a gamma emitting radioisotope
- Half life 3 days
- Whole body imaging of rats
- Regions of interest drawn around injection site
211
p-SCN-Bn-DOTA
a-Casein
a-casein
DOTA-a-Casein
111In3
2
111In3
a-casein
Indium-111
111In-DOTA-a-Casein
22Reproducibility of SC Administration of AC
Adsorbed to AH
23Strength of Adsorption
- AP-adsorbed elutes more than AH-adsorbed
- AP-adsorbed is less strongly adsorbed
24Vaccines Studied
- Solution AC
- Non Adsorbed AC/PT-AP
- Adsorbed AC/AH
- Adsorbed AC/AP
25Percent 111In-DOTA-a-Casein Remaining at the
Injection Site - SC
AH-Adsorbed
AP-Adsorbed
Non-Adsorbed
Solution
26Antibody Titers
Significantly different than solution Significan
tly different than AH-adsorbed
27Comparisons
- Solution (928 GMT) and non-adsorbed were retained
the same (14,706 GMT) - AP-adsorbed (14,154 GMT) retained longer than
non-adsorbed (14,706 GMT) - AH-adsorbed (7,791 GMT) retained longer than AP
adsorbed (14,154 GMT)
28Conclusions
- Depot mechanism not supported
- Presence of Al-containing adjuvant produces
immunopotentiation - Immunopotentiation not derived from adsorption
and retention
29Can Adsorption Be Too Strong?
30Adsorption Forces in Vaccines
-
Adsorption Force Adsorptive - coefficient
- Antigen Adjuvant Electrostatic
Ligand exchange mL/mg - CAS AH attractive
strong 2400 - DP-CAS AH attractive
weak 410 - CAS PT-AH repulsive
weak 60 - DP-CAS PT-AH repulsive
very weak 1
Vaccine 25, 6618, 2007
31Geometric Mean Antibody Titers In Mice
2
Vaccine, 25, 6618, 2007
32T-Cell Activation
SI
Antigen concentration (µg/mL)
Vaccine, 25, 6618, 2007
33Hepatitis B Surface Antigen
- Contains phospholipids
- Adsorbs by ligand exchange
34HBsAg Binding Constant
- Pretreat AH with PO4 to reduce the number of
ligand exchange sites
35Degree of Elution
- In vitro elution in interstitial fluid was
inversely related to the adsorptive coefficient.
36HBsAg Antibody Titer in Mice
37HIV Antigen, gp140
- Contains phosphate groups
- Adsorbs by ligand exchange
38Gp140 Antibody Titer in Mice
Tightness of binding is more important at lower
doses.
39Optimum Formulation
- Characterize antigen
- Isoelectric point
- Contains phosphate or phospholipids?
- Effect of pH on chemical stability
40Optimum Formulation
- Characterize adjuvant
- Isoelectric point
- Surface OH vs. surface PO4
41Optimum Formulation
- Characterize antigen/adjuvant interaction
- Adsorbed, yes or no
- Strength of adsorption
- Elution in interstitial fluid
- Microenvironment pH
42Acknowledgements
- The graduate students who are the first authors
of the cited publications - Merck Research Laboratories
- General Chemical Performance Chemicals