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The Physical Chemistry and Biology of AluminumContaining Adjuvants

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Immunopotentiation by aluminum-containing adjuvants discovered by ... Ovalbumin (iep=5) contains 2 PO4. Adsorbs by ligand exchange when AH is negatively charged ... – PowerPoint PPT presentation

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Title: The Physical Chemistry and Biology of AluminumContaining Adjuvants


1
The Physical Chemistry and Biology of
Aluminum-Containing Adjuvants
  • Stanley Hem
  • Harm HogenEsch
  • Purdue University

2
Historical 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

3
Structure 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

4
Morphology
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
5
Major Adsorption Mechanisms
  • Electrostatic attraction
  • Ligand exchange

6
Electrostatic 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
7
Electrostatic 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
8
Ligand Exchange
  • PO4 displaces surface OH
  • Ligand exchange occurs with phosphorylated
    antigens
  • Strongest adsorption force

PO4 - Protein
PDA J. Pharm. Sci. Technol. 51, 25, 1997
9
Ligand 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
10
Ligand Exchange
  • Adsorption is directly related to degree of
    phosphorylation of antigen

Vaccine 23, 1502, 2005
11
Elution 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
12
Microenvironment pH
  • pH of double layer of charged particles is
    different from bulk pH
  • Negative surface attracts protons
  • Positive surface attracts hydroxyls

13
Microenvironment 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.

14
Is Adsorption Necessary?
15
Experimental 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
16
Alpha 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

17
Pretreat 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

18
DP- CAS Antibody Study
  • Potentiation by PT-AP without adsorption

19
Immunopotentiation 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

20
Monitor 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

21
1

p-SCN-Bn-DOTA
a-Casein
a-casein
DOTA-a-Casein
111In3
2

111In3
a-casein
Indium-111
111In-DOTA-a-Casein
22
Reproducibility of SC Administration of AC
Adsorbed to AH
23
Strength of Adsorption
  • AP-adsorbed elutes more than AH-adsorbed
  • AP-adsorbed is less strongly adsorbed

24
Vaccines Studied
  • Solution AC
  • Non Adsorbed AC/PT-AP
  • Adsorbed AC/AH
  • Adsorbed AC/AP

25
Percent 111In-DOTA-a-Casein Remaining at the
Injection Site - SC
AH-Adsorbed
AP-Adsorbed
Non-Adsorbed
Solution
26
Antibody Titers
Significantly different than solution Significan
tly different than AH-adsorbed
27
Comparisons
  • 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)

28
Conclusions
  • Depot mechanism not supported
  • Presence of Al-containing adjuvant produces
    immunopotentiation
  • Immunopotentiation not derived from adsorption
    and retention

29
Can Adsorption Be Too Strong?
30
Adsorption 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
31
Geometric Mean Antibody Titers In Mice
2
Vaccine, 25, 6618, 2007
32
T-Cell Activation
SI
Antigen concentration (µg/mL)
Vaccine, 25, 6618, 2007
33
Hepatitis B Surface Antigen
  • Contains phospholipids
  • Adsorbs by ligand exchange

34
HBsAg Binding Constant
  • Pretreat AH with PO4 to reduce the number of
    ligand exchange sites

35
Degree of Elution
  • In vitro elution in interstitial fluid was
    inversely related to the adsorptive coefficient.

36
HBsAg Antibody Titer in Mice
37
HIV Antigen, gp140
  • Contains phosphate groups
  • Adsorbs by ligand exchange

38
Gp140 Antibody Titer in Mice
Tightness of binding is more important at lower
doses.
39
Optimum Formulation
  • Characterize antigen
  • Isoelectric point
  • Contains phosphate or phospholipids?
  • Effect of pH on chemical stability

40
Optimum Formulation
  • Characterize adjuvant
  • Isoelectric point
  • Surface OH vs. surface PO4

41
Optimum Formulation
  • Characterize antigen/adjuvant interaction
  • Adsorbed, yes or no
  • Strength of adsorption
  • Elution in interstitial fluid
  • Microenvironment pH

42
Acknowledgements
  • The graduate students who are the first authors
    of the cited publications
  • Merck Research Laboratories
  • General Chemical Performance Chemicals
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