relationship between physical and chemical properties of aluminum-containing adjuvants and...

Review

10.1586/14760584.6.5.685 © 2007 Future Drugs Ltd ISSN 1476-0584 685www.future-drugs.com

Relationship between physicaland chemical properties of aluminum-containing adjuvants and immunopotentiationStanley L Hem† and Harm HogenEsch

†Author for correspondencePurdue University, Industrial and Physical Pharmacy Department, West Lafayette, IN 47907, USATel.: +1 765 494 1451Fax: +1 765 494 [email protected]

KEYWORDS: adsorption mechanism, aluminum hydroxide adjuvant, aluminum phosphate adjuvant, antigen trapping, antigen uptake, dendritic cell maturation, isoelectric point, recruitment of antigen-presenting cells, T-cell activation, T-cell differentiation

Aluminum-containing adjuvants are an important component of many vaccines because they safely potentiate the immune response. The structure and properties of aluminum hydroxide adjuvant, aluminum phosphate adjuvant and alum-precipitated adjuvants are presented in this review. The major antigen adsorption mechanisms, electrostatic attraction and ligand exchange, are related to the adjuvant structure. The manner by which aluminum-containing adjuvants potentiate the immune response is related to the structure, properties of the adjuvant and adsorption mechanism. Immunopotentiation occurs through the following sequential steps: inflammation and recruitment of antigen-presenting cells, retention of antigen at the injection site, uptake of antigen, dendritic cell maturation, T-cell activation and T-cell differentiation.

Expert Rev. Vaccines 6(5), 685–698 (2007)

Aluminum-containing adjuvants, such as alumi-num hydroxide adjuvant and aluminum phos-phate adjuvant, contribute to the efficacy ofmany vaccines by potentiating the immuneresponse. This effect was first noted by Glennyet al. in 1926 [1]. Research since 1990 has eluci-dated many aspects of the physical and chemicalproperties of aluminum-containing adjuvantsand has related these properties to their mecha-nism of immunopotentiation. This review willfirst describe the chemistry of aluminum-con-taining adjuvants. The sequence of steps thatlead to increased antibody production will thenbe presented, along with the physicochemicalproperty of the adjuvant that is associated witheach step. Our hope is that understanding howthe properties of aluminum-containing adju-vants influence the adjuvant effect will lead tooptimal vaccine formulations.

Chemistry of aluminum-containing adjuvantsStructureThe names of the two forms of aluminum-con-taining adjuvants, aluminum hydroxide adjuvantand aluminum phosphate adjuvant, do not cor-rectly describe the adjuvant structures [2]. X-ray

diffraction and infrared spectroscopy identifyaluminum hydroxide adjuvant as crystalline alu-minum oxyhydroxide, AlO(OH). Aluminumphosphate adjuvant is amorphous to x-rays butits infrared spectrum identifies it as aluminumhydroxide in which phosphate has substitutedfor some hydroxyls. It is correctly termedamorphous aluminum hydroxyphosphate,Al(OH)x(PO4)y. Unlike aluminum hydroxideadjuvant, it is not a stoichiometric compound.Rather, the degree of phosphate substitution forhydroxyl depends on the reactants and methodof preparation.

When AlK(SO4)2 is used as the source of alu-minum cations in alum-precipitated vaccines, theresulting adjuvant is amorphous aluminumhydroxysulfate, as some sulfate anions substitutefor hydroxyls [2]. If a phosphate anion is presentat the time of precipitation, phosphate will sub-stitute some hydroxyls and the adjuvant will beamorphous aluminum hydroxyphosphate sulfate.

Surface groups & isoelectric point

All of the surface groups in aluminum hydrox-ide adjuvant are hydroxyls that are coordi-nated to aluminum. The surface groups of

CONTENTS

Chemistry of aluminum-containing adjuvants

Mechanisms of immunostimulation

Expert commentary & five-year view

Financial & competing interests disclosure

Key issues

References

Affiliations

http://www.future-drugs.com

k.rowland

Text Box

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Hem & HogenEsch

686 Expert Rev. Vaccines 6(5), (2007)

aluminum phosphate adjuvant are a mixture of hydroxyls andphosphates. When a hydroxyl is coordinated to a metal, such asaluminum, the hydroxyl is termed a metallic hydroxide and hasdifferent properties from an alcohol [3]. Metallic hydroxyls canaccept a proton and exhibit a positive charge or donate a pro-ton and exhibit a negative charge. Thus, similar to proteins,metallic hydroxides have an isoelectric point (IEP). The IEP ofaluminum hydroxide adjuvant, which is chemically AlO(OH),is 11.4 and is positively charged at the pH of interstitial fluid(pH 7.4). The IEP of aluminum hydroxide adjuvant can bemodified by exposure to phosphate anions [4]. This may occurthrough pretreatment of the adjuvant with a phosphate solu-tion or the use of a phosphate buffer in the vaccine formula-tion. FIGURE 1 shows that the IEP of aluminum hydroxide adju-vant is very sensitive to the replacement of surface hydroxyls byphosphate. The IEP may decrease from 11.4 to as low as 4,depending on the concentration of phosphate that the adjuvanthas been exposed to.

The IEP of aluminum phosphate adjuvant also depends onthe degree of phosphate substitution for hydroxyl (FIGURE 2) [5].The IEP approaches 4.0 at the highest levels of phosphatesubstitution. It is interesting to note, that extrapolation to aP:Al ratio of zero yields an IEP of 9.6, which is the IEP of alu-minum hydroxide, Al(OH)3. Commercial aluminumphosphate adjuvants have an IEP of approximately 5.0 [6].

Zhao and Sitrin developed a method for determining theavidity of phosphorylated proteins for aluminum-containingadjuvants [7]. They used the term ‘phosphophilicity’ todescribe the ability of aluminum-containing adjuvants toattract phosphate or phospho-groups of organic molecules. Itappears that the phosphophilicity of an adjuvant is related tothe number of surface hydroxyl groups that are available forligand exchange.

Morphology

Aluminum hydroxide adjuvant and aluminum phosphate adju-vant are composed of very small primary particles [8]. However,the primary particles form aggregates that are the functioningunits in vaccines. The primary particles of aluminum hydroxide

adjuvant are fibers with average dimensions of4.5 × 2.2 × 10 nm (FIGURE 3). The nanometer dimensions of theprimary particles give rise to a surface area of approximately500 m2/g [9]. The aggregates are porous and have irregularshapes that range from 1 to 20 µm in diameter.

The primary particles of aluminum phosphate adjuvant areplate-like with a diameter of approximately 50 nm and formporous aggregates that range from 1 to 20 µm (FIGURE 4).Although the surface area of aluminum phosphate adjuvant hasnot been reported, the 50-nm primary particles suggest that thesurface area approaches that of aluminum hydroxide adjuvant.

The morphology of aluminum-containing adjuvants contrib-utes to the uniform distribution of antigen in vaccines. Theaggregates readily deaggregate during mixing and fragments thenreaggregate to uniformly distribute adsorbed antigen throughoutthe aluminum-containing vaccine [10]. Thus, even though quan-tities of antigen as low as 10 µg are combined with quantities ofadjuvant up to 0.85 mg Al during the production of a vaccine,the nature of the adjuvant aggregates provides a mechanism touniformly distribute the antigen as long as adequate mixingprocedures are followed.

The weak association of the particles forming the aggregatesmust be considered when measuring the size of the aggregates.Since the aggregates are readily disassociated by mixing, it must beremembered that size measurements made when shear was appliedto the adjuvant reflect a disassociated system. Thus, different siz-ing techniques may give different results, depending on the shearconditions during measurement. The maximum size of the aggre-gates can only be determined when the adjuvant is not exposed toshear. Thus, image analysis of photographs of undisturbed sam-ples may be used to determine aggregate size [10]. If necessary, dilu-tion of the adjuvant, should be done using previously harvestedsupernatant solution because changes in the composition of theliquid phase may cause changes in the aggregates.

Adsorption mechanismsGlenny et al. mixed diphtheria toxoid with a solution of alumand added base to produce a precipitate [1]. When administeredto guinea pigs, the alum-precipitated vaccine provided greater

Figure 1. Relationship between phosphate adsorption by aluminum hydroxide adjuvant and the isoelectric point. Reprinted with permission from [4].

0

2

4

6

8

10

12

0 1 2 3 4 5 6Phosphate adsorbed (mg/mg Al)

Iso

elec

tric

po

int

Figure 2. Effect of phosphate substitution for hydroxyl on the isoelectric point of aluminum phosphate adjuvant. Reprinted with permission from [5].

3

4

5

6

7

8

9

10

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80P/Al in solid

Iso

elec

tric

po

int

Aluminum-containing vaccine adjuvants

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protection against a challenge than a solution of diphtheria tox-oid. Analysis of the vaccine revealed that none of the diphtheriatoxoid was in the supernatant. This observation led to the beliefthat the antigen must be adsorbed to the aluminum-containingadjuvant in order to achieve immunopotentiation.

Owing to the complex structure of antigens, it is not sur-prising that a number of attractive mechanisms may contrib-ute to their adsorption. These mechanisms include electro-static attraction, hydrogen bonding, hydrophobicinteractions, ligand exchange and van der Waals forces [11]. Itis likely that several mechanisms contribute to adsorption in avaccine formulation. However, in our experience, two of thesemechanisms, if present, predominate and largely define theadsorption/elution performance of the vaccine. These twomechanisms are electrostatic attraction and ligand exchangeand will be discussed in detail in this review.

Electrostatic attraction

Electrostatic attraction arises when the antigen and adjuvanthave opposite charges. For example, lysozyme (IEP 11.0)was adsorbed by aluminum phosphate adjuvant (IEP 5.0) atpH 7.4 but not by aluminum hydroxide adjuvant (IEP 11.4)[12]. Similarly, albumin (IEP 4.8) was adsorbed by aluminumhydroxide adjuvant at pH 7.4 but not by aluminumphosphate adjuvant.

The ionic strength of the vaccine reduces the contributionsof the surface charge. Therefore, electrostatic attraction isfavored by a media having a low ionic strength. Lysozyme wasadsorbed by aluminum phosphate adjuvant at pH 7.4 in a0.06 M NaCl solution. Adsorption was reduced greatly in a0.15 M NaCl solution (isotonic) and was not observed in0.25 M NaCl [13]. It may be desirable to use a polyol ratherthan sodium chloride to adjust the tonicity of a vaccine in

which the antigen is adsorbed by electrostatic attraction. TheIEP of both aluminum hydroxide adjuvant and aluminumphosphate adjuvant may be adjusted by treatment of the adju-vant with a phosphate solution, examples of which were givenpreviously under ‘Surface groups and isoelectric point’.

Ligand exchange

Phosphate binds more strongly to aluminum than hydroxyl [3].Thus, the addition of phosphate anions to aluminumhydroxide, Al(OH)3, will result in an increase in the pH asthe added phosphates displace surface hydroxyls. Owing tothe fact that both aluminum hydroxide adjuvant and alumi-num phosphate adjuvant contain surface hydroxyls, bothadjuvants can undergo ligand exchange with phosphorylatedantigens, although aluminum hydroxide adjuvant has thegreatest number of potential adsorption sites. An antigenmay contain free phosphate groups or have phosphate groupsgenerated by hydrolysis of phospholipids that are associatedwith cell membranes, as occurs with hepatitis B surface anti-gen (HBsAg) or hydrolysis of polyribosylribitolphosphate(Haemophilus influenzae antigen).

Ligand exchange is the strongest adsorption force and canoccur even when electrostatic repulsion exists between theantigen and the adjuvant. One approach to control thestrength of adsorption by ligand exchange is to modify thenumber of phosphate groups on the antigen. This was illus-trated by a study in which the molar ratio of phosphate inovalbumin increased from 1.8 to 3.2 by conjugation withphosphoserine or reduced to 1.2 or 0.14 by treatment withpotato acid phosphatase [14]. Ovalbumin (IEP 5.0) is repelled

Figure 3. Transmission electron photomicrograph of aluminum hydroxide adjuvant at a magnification of 100,000×. Reprinted with permission from [8].

Figure 4. Transmission electron photomicrograph of aluminum phosphate adjuvant at a magnification of 100,000×. Reprinted with permission from [8].

Hem & HogenEsch


electrostatically by aluminum phosphate adjuvant (IEP 5.0)at pH 7.4. TABLE 1 shows that the percentage of the dose ofovalbumin adsorbed onto aluminum phosphate adjuvant wasdirectly related to the degree of phosphorylation of ovalbu-min. The hyperphosphorylated ovalbumin was adsorbedcompletely, most likely by ligand exchange, in spite of electro-static repulsion. The degree of adsorption decreased as thephosphorylation decreased until no adsorption was observedfor the ovalbumin that contained virtually no phosphate.Electrostatic repulsion was the predominant antigen–adjuvantforce in this vaccine.

The strength of adsorption by ligand exchange can also becontrolled by pretreating the adjuvant with phosphate toreduce the number of surface hydroxyls available for ligandexchange. This was illustrated by an experiment in which alu-minum hydroxide adjuvant was treated with five concentra-tions of phosphate [4]. Langmuir adsorption isotherms weredetermined for the adsorption of ovalbumin by the adjuvants.The adsorptive capacity (amount of ovalbumin adsorbed atmonolayer coverage) and the adsorptive coefficient (thestrength of the adsorption force) were inversely related to thephosphate content of the aluminum hydroxide adjuvant(TABLE 2). Accordingly, the amount of ovalbumin adsorbed andthe strength of the adsorption force decreased as fewer ligandexchange sites were available on the surface of the aluminumhydroxide adjuvant.

Understanding whether electrostatic attraction or ligandexchange is the predominant adsorption mechanism is impor-tant because elution upon exposure to interstitial fluid dependson the adsorption mechanism. The vaccine comes in contactwith interstitial fluid following intramuscular or subcutaneousadministration. Components of interstitial fluid, such as phos-phate anion [15], citrate anion [16] or fibrinogen [17], have beenfound to elute a previously adsorbed antigen. Antigensadsorbed onto aluminum-containing adjuvants by electrostaticattraction elute more readily when mixed in vitro with inter-stitial fluid than antigens adsorbed by ligand exchange [18].FIGURE 5 shows that the four samples of ovalbumin presented inTABLE 1 exhibited different elution profiles when mixed withinterstitial fluid. The hyperphosphorylated sample that was

adsorbed by ligand exchange did not elute at all, whereas thedephosphorylated sample adsorbed by electrostatic attractioneluted completely.

The importance of electrostatic attraction and ligandexchange that was identified in the adsorption of model pro-teins has been confirmed in vaccines. Anthrax recombinant pro-tective antigen has been shown to adsorb to aluminum hydrox-ide adjuvant predominantly by electrostatic attraction [19].Adsorption of heavy chain fragments of botulinum neuro-toxin serotype A, which has an IEP of 9.2, by aluminumhydroxide adjuvant (IEP 11.4) required pretreatment withphosphate, indicating that the electrostatic adsorption forcepredominated [20]. By contrast, serotype B had a high affinityto aluminum hydroxide adjuvant even below pH 7.0, whenboth antigen and adjuvant were positively charged. Adsorp-tion was not due to hydrophobic interactions as ethylene gly-col had minimal effect on adsorption. The authors con-cluded that adsorption of serotype B was due to ligandexchange [20]. HBsAg is adsorbed onto aluminum hydroxideadjuvant by ligand exchange, due to the phosphate groupsarising from the lipid bilayer of the antigen [21].

Adsorption onto aluminum-containing adjuvants has beenshown to alter the tertiary structure of lysozyme, ovalbumin andbovine serum albumin and reduce their thermal stability [11,22].The authors suggested that the changes may make theadsorbed proteins more susceptible to proteolytic processingand, thereby, enhance antigen presentation. This hypothesisfor an additional mechanism by which aluminum-containingadjuvants enhance the immune response requires furtherstudy. For example, do the altered proteins return to theirnative conformation when they elute, which may occur uponintramuscular or subcutaneous administration? Proteins thatadsorb to aluminum-containing adjuvants by electrostaticattraction, such as lysozyme and bovine serum albumin, havebeen found to elute readily when exposed to interstitial fluid[18]. In addition, aluminum-containing adjuvants have beenfound to potentiate the immune response to Haemopilus influ-enzae type b [23], anthrax recombinant antigen [24], lysozyme,ovalbumin, and α-casein [8] in vaccines in which the antigenwas not adsorbed. The secondary structure of six model pro-teins, including ovalbumin, was not altered by adsorption toaluminum hydroxide adjuvant [25].

PropertiesMicroenvironment pH & antigen stability

The surface charge of a particle suspended in aqueous mediainfluences the distribution of ions in the water surroundingthe particle. This principle is known as the Gouy–Chapmandouble layer theory. A negatively charged particle will prefer-entially attract cations, including protons, into the doublelayer surrounding the particle. Positively charged particleswill preferentially attract anions, including hydroxyls, intothe double layer. Thus, the pH of the microenvironment sur-rounding a charged particle will be different from the bulkpH. This principle applies to aluminum-adjuvanted vaccines.

Table 1. Effect of phosphorylation of ovalbumin on adsorption by aluminum phosphate adjuvant.

Ovalbumin sample PO4:OVA molarratio

Adsorption (%)

P-OVA 3.20 99

OVA 1.80 38

DP-OVA 1 1.20 26

DP-OVA 2 0.14 0

DP-OVA: dephosphorylated ovalbumin; OVA: ovalbumin; P-OVA: hyperphosphorylated ovalbumin.Adapted with permission from [14].



The adsorbed antigen is exposed to the pH of the doublelayer and any pH-dependent reactions occur at a rate associ-ated with the double layer pH rather than the bulk pH. Therate of acid-catalyzed hydrolysis of glucose-1-phosphate thatwas adsorbed onto aluminum hydroxide adjuvant was signifi-cantly slower than the rate of hydrolysis of glucose-1-phos-phate in solution at the same bulk pH [26]. The positivelycharged aluminum hydroxide adjuvant attracted anions,including hydroxyls, into the double layer. Thus, theadsorbed glucose-1-phosphate experienced a higher pH in thedouble layer and the rate of acid-catalyzed hydrolysis wasslower than expected based on the bulk pH. It was concludedthat the microenvironment pH of aluminum hydroxide adju-vant was approximately two units higher than the bulk pH.The chemical stability of adsorbed antigens that degrade bypH-dependent mechanisms can be optimized by modifyingthe surface charge of the adjuvant to produce the pH of max-imum stability in the double layer. Antigens that have beenreported to have pH-dependent immunogenicity or stabilityinclude foot and mouth disease viral antigen, tetanus toxoid,influenza A virus and Mycoplasma hyopneumoniae [26].

Adjuvant stabilityAluminum hydroxide adjuvant exhibits broad x-ray diffrac-tion bands that indicate a low degree of order [2,9]. In fact, alu-minum hydroxide adjuvant corresponds to a mineral that isknown as poorly crystalline boehmite. It is likely that furtherorder develops as the adjuvant ages. Likewise, the amorphousstructure of aluminum phosphate adjuvant suggests that fur-ther order develops during aging. A study that monitored theaging of these two adjuvants at room temperature confirmedthe expected behavior [27].

Hydroxy-aluminum compounds develop order by thesequential deprotonation and dehydration reactions shownbelow.

These reactions join aluminum ions by double hydroxidebridges. Protons are released every time a double hydroxidebridge forms. Thus, the development of order is accompaniedby a decrease in both pH and surface area.

Typical data for the stability of aluminum hydroxide adjuvantand aluminum phosphate adjuvant are shown in TABLE 3. Thewidth-at-half-height (WHH) is a measure of the sharpness ofthe principal x-ray diffraction band and is inversely related tothe degree of order. The WHH, pH and bovine serum albuminadsorption capacity of aluminum hydroxide adjuvant alldecreased during a 15-month aging period at room temperature.Likewise, the pH and lysozyme adsorptive capacity of alumi-num phosphate adjuvant decreased during the same agingperiod. All of the observed changes are associated with the devel-opment of order. It is advisable to use freshly prepared adjuvantsto produce vaccines, as well as to store the adjuvant at 4°C.

Aluminum-containing adjuvants or vaccines containingthese adjuvants must not be frozen as freezing produces irre-versible coagulation [6] and loss of potency [28]. Coagulationduring freezing is a general phenomena of suspensions that isinversely related to the rate of freezing. Maa et al. used thisobservation to prepare powders for epidermal immunizationby spray freeze drying [29,30]. A vaccine was prepared byadsorbing HBsAg to aluminum hydroxide adjuvant. The vac-cine was atomized into liquid nitrogen at -196°C and the pow-der collected. When the powder was reconstituted, the physi-cal properties and immunogenicity in mice were the same asthe unfrozen vaccine. However, aluminum adjuvant-contain-ing vaccines that freeze at environmental or freezer conditionsare irreversibly damaged.

The sequential reactions responsible for the development oforder during aging at room temperature also occur duringautoclaving [31]. As shown in TABLE 4, the WHH, pH and pro-tein adsorptive capacity all decreased during a 30-min auto-claving period. Therefore, sterilization procedures that mini-mize exposure to elevated temperatures, such as rotating the

Table 2.Relationship between the surface phosphate concentration of aluminum hydroxide adjuvant and the adsorption of ovalbumin.

Adjuvant Phosphate adsorbed (mg/mg Al) Adsorptive capacity*(mg/mg Al)

Adsorptive coefficient‡ (ml/mg)

1 0.00 2.7 86

2 0.03 2.4 62

3 0.12 2.0 43

4 0.21 1.7 19

5 0.35 1.3 11

6 0.60 0.8 7

*Amount adsorbed at monolayer coverage.‡Strength of the adsorption force.Adapted with permission from [4].

Al OH2( )63+ Al OH( ) OH2( )2+

5→ H++

2Al OH( ) OH2( )52+ Al2 OH( )2 OH2( )4+

8→ 2H2O+

Hem & HogenEsch


containers to produce more uniform heating during auto-claving should be used. Repeated autoclaving of the sameadjuvant should be avoided.

Solubility

The solubility of both aluminum hydroxide adjuvant andaluminum phosphate adjuvant is pH dependent [32]. Mini-mum solubility occurs between pH 5 and 7. Both adjuvantsare more soluble in acidic or basic media. However, amor-phous aluminum phosphate adjuvant is more soluble thancrystalline aluminum hydroxide adjuvant.

It is known that α-hydroxycarboxylic acids are good chela-tors of metal ions and are used to solubilize hydroxy-alumi-num compounds. Solutions of sodium citrate at neutral pHsolubilize both aluminum hydroxide adjuvant and aluminumphosphate adjuvant, although aluminum phosphate adjuvantis more soluble [16]

The solubility properties of the aluminum-containingadjuvants have several practical applications. One approachto elute an antigen that is adsorbed onto an aluminum-con-taining adjuvant is to dissolve the adjuvant by treating the

vaccine with a solution of sodium citrate at neutral pH. If theantigen is stable at acidic pH conditions, dissolution of theadjuvant can be enhanced by treatment with a citrate solutionat pH 4–5.

Interstitial fluid contains at least three α-hydroxycarboxylicacids (citric, lactic and malic acids). For this reason, alumi-num-containing adjuvants are solubilized when exposed tointerstitial fluid following intramuscular or subcutaneousadministration. The elimination of aluminum hydroxideadjuvant and aluminum phosphate adjuvant following intra-muscular injection was studied in rabbits [33]. The aluminum-containing adjuvants were prepared by precipitation in thepresence of a very small amount of 26AlCl3. Thus, the adju-vants contained 26Al in addition to the natural 27Al. Accelera-tor mass spectrometry was used to quantify the concentrationof 26Al in blood and urine samples collected over a 28-daystudy period. 26Al was present in the first blood sample at 1 hfor both adjuvants indicating that dissolution begins uponadministration. The area under the 26Al blood level curveindicates that three-times more aluminum was adsorbed fromaluminum phosphate adjuvant than from aluminum hydrox-ide adjuvant. Three-times more 26Al from aluminum phos-phate adjuvant than from aluminum hydroxide adjuvant wasalso excreted. The study demonstrated that dissolution of theadjuvants in interstitial fluid begins shortly after intramuscu-lar administration and that the aluminum is eliminated byexcretion. In rabbits, the normal plasma aluminum concen-tration of 30 ng/ml was only increased by approximately2 ng/ml upon administration of the human dose (0.85 mg Al)of aluminum phosphate adjuvant or aluminum hydroxideadjuvant. It also indicates one reason for the excellent safetyrecord of aluminum-containing adjuvants.

Mechanisms of immunostimulationVaccines are most commonly administered by injection intoskeletal muscle or subcutaneous tissue. The initiation of theimmune response is thought to occur in the draining lymphnodes that may be separated from the injection site by a con-siderable distance. Although some aluminum-containingmaterial can be found in the draining lymph node followinginjection of aluminum-adjuvanted vaccines [34], it is likelythat the effect of aluminum-containing adjuvants on cells

Figure 5. Effect of phosphate content of ovalbumin on elution from aluminum hydroxide adjuvant upon exposure to interstitial fluidat 37°C. OVA: Ovalbumin.Reprinted with permission from [14].

Time (h)

Elu

tio

n (

%)

100

80

60

40

20

00 5 10 15 20 25

X

PO4/OVA = 3.2 PO4/OVA = 1.2

PO4/OVA = 1.8 PO4/OVA = 0.14

Table 3. Stability at room temperature.

Aluminum hydroxide adjuvant Aluminum phosphate adjuvant

Time (months) WHH°2Θ pH BSA adsorptive capacity(mg/mg Al)

pH Lysozyme adsorptivecapacity (mg/mg Al)

0 4.2 6.3 2.9 6.5 0.8

6 3.8 6.2 2.8 6.3 0.7

15 3.5 6.2 2.6 6.3 0.5

BSA: Bovine serum albumin; WHH: Width-at-half height of principle x-ray diffraction band.Adapted with permission from [27].



and tissues at the injection site is critical for the stimulationof the immune response. This is supported by the fact thatremoval of the injection site of alum-precipitated diphtheria tox-oid within 4 days interfered with the generation of the immuneresponse [35]. Dendritic cells are antigen-presenting cells(APCs) that are pivotal to the initiation of the immuneresponse [36]. They are located in lymphoid and nonlymphoidtissues. Here, the effect of aluminum-containing adjuvants onthe recruitment of dendritic APCs to the injection site, theretention of antigens at the injection site, the uptake of vaccineantigens and maturation of dendritic cells and the ability of den-dritic cells to induce differentiation of antigen-specificlymphocytes are discussed.

Inflammation & recruitment of antigen-presenting cellsThe induction of inflammation by aluminum-containing adju-vants is important for the recruitment of APCs and the releaseof cytokines and other mediators that induce maturation andactivation of dendritic cells. This inflammatory response hasnot been characterized at the molecular level, but a few histo-logical studies report local edema and necrosis of muscle fibersfollowed by infiltration of neutrophils at 24–72 h after intra-muscular injection of aluminum-adjuvanted vaccines [37,38].Others have reported that 4 days after injection, up to 25% ofthe infiltrating cells were eosinophils [39]. Monocytic cells beginto appear at approximately 72 h and eventually give rise togranulomatous inflammation [37,38,40]. It is likely that thesemonocytic cells contain dendritic cell precursors that differenti-ate into immature dendritic cells following their exit from theblood circulation [41].

Retention of antigen at the injection siteSince the original studies by Glenny et al. [42], the formation ofan antigen depot that slowly releases the antigen over time hasbeen considered the principal mechanism by which aluminum-containing adjuvants enhance the immune response. A depotmechanism based on continuous release of the antigen over aprolonged period of time (weeks or months) is no longer tena-ble and aluminum-containing adjuvants produce many othereffects that contribute to immunopotentiation [43]. However,since histologic studies suggest that it takes 3 days after injec-tion of the vaccine for newly recruited mononuclear cells toarrive, retention of the antigen for this period would enhancethe uptake and presentation of the antigen.

Antigens may be retained at the injection site either by beingadsorbed by the aluminum-containing adjuvant or by beingtrapped in the void spaces within the adjuvant aggregates(FIGURES 3 & 4). Antigens that are adsorbed by ligand exchangeprobably remain adsorbed following administration, whereasantigens that are adsorbed by electrostatic attraction may elutefairly rapidly upon contact with interstitial fluid. However, theeluted antigen may be trapped within the adjuvant aggregatesand remain at the injection site. Aluminum-containing adju-vants were found to potentiate the immune response to elec-trostatically adsorbed antigens that elute upon exposure tointerstitial fluid, as well as phosphorylated antigens that wereadsorbed by ligand exchange and remain adsorbed whenexposed to interstitial fluid [44]. Aluminum phosphate adjuvantalso potentiated the immune response to antigens that werenot adsorbed in the vaccine formulation or when mixedin vitro with interstitial fluid [8]. Despite of the lack of adsorp-tion, the vaccines induced a strong antibody response uponinjection into mice. Soluble antigens can reach the draininglymph node within 30 min and a rapid diffusion rate of anti-gens from the injection site would negate the interaction of theantigen with newly recruited APCs. Microscopic imaging ofthe nonadsorbed vaccines in vitro showed that the antigenswere associated with the aluminum phosphate adjuvant aggre-gates [8]. It was speculated that the antigens were trapped in thevoid space of the adjuvant aggregates and that this ‘antigentrapping’ sufficiently delays antigen diffusion from the injec-tion site to allow uptake of the retained antigens by influx ofAPCs. Studies with anthrax recombinant protective antigenalso concluded that adsorption to aluminum phosphateadjuvant was not essential for immunopotentiation [24].

Two studies have measured the amount of antigen remainingat the injection site. One study measured the distribution ofradioactivity in mice after subcutaneous administration of14C-labeled tetanus toxoid [45]. The percentage of radioactivityremaining at the injection site after 1 h following administra-tion of a solution of tetanus toxoid or tetanus toxoid adsorbedonto aluminum phosphate adjuvant was 22 and 55%, respec-tively. A second study used x-ray fluorescence to monitor theconcentration of iodinated serum albumin at the site of thesubcutaneous injection in rats of a solution of antigen or anti-gen mixed with Imject Alum® [46]. The half-life for the disap-pearance of iodine x-rays from the injection site was 6.5 and12 h, respectively.

Table 4. Effect of autoclaving at 121°C for 30 min.

Aluminum hydroxide adjuvant Aluminum phosphate adjuvant

WHH pH Albumin adsorptivecapacity (mg/mg Al)

pH Lysozyme adsorptivecapacity (mg/mg Al)

Before autoclaving 3.8 6.0 3.1 7.5 1.6

After autoclaving 3.7 5.8 3.0 6.7 1.3

WHH: Width-at-half height.Adapted with permission from [31].

Hem & HogenEsch


DNA vaccines provide a system in which vaccine antigensare expressed de novo in tissues. Aluminum phosphate adju-vant strongly enhanced the antibody response to antigensexpressed following DNA immunization, whereas addition ofaluminum hydroxide adjuvant decreased the immuneresponse [47–49]. These contrasting effects of the aluminum-containing adjuvants were inversely related to the degree ofadsorption of the DNA plasmids. Aluminum phosphate adju-vant did not adsorb the DNA plasmid, whereas aluminumhydroxide adjuvant completely adsorbed the plasmid, pre-sumably by electrostatic and ligand exchange mechanisms.Interestingly, the aluminum phosphate adjuvant enhanced theimmune response when injected 3 days prior, simultaneouslywith or 3 days after injection of the DNA plasmids [47]. Theadjuvant did not affect antigen expression following plasmidinjection. These experiments demonstrate further that alumi-num-containing adjuvants can enhance the immune responseindependently of antigen adsorption.

Uptake of vaccine antigensNaive B and T cells do not circulate efficiently through non-lymphoid extravascular tissues and are mostly restricted to theblood circulation and secondary lymphoid tissues [50,51]. Theactivation of naive B and T cells by their cognate antigensoccurs in the lymphoid tissues and requires the translocation ofantigen from the nonlymphoid injection site to the draininglymph node. Antigen-specific B-cell receptors (immunoglobu-lins) interact with antigens in their native conformation,whereas the T-cell receptors αβ expressed on CD4+ and CD8+

T cells interact with antigenic peptides bound by MHC mole-cules. Some antigens can move to the lymph node by diffusion ininterstitial fluid and subsequently, via the directional lymph flowin afferent lymph vessels. The afferent lymph vessels open intothe subcapsular sinus of the lymph node. Penetration of solubleantigens into the cortex of the lymph node is limited to relativelysmall molecules (<4 nm molecular radius) by the barrier formedby sinus endothelial cells and fibroblastic reticular cells [52].However, resident dendritic cells and macrophages can take uplarger antigens and carry them into the cortex for presentationto B and T cells. The proportion of antigen in aluminum-adjuvanted vaccines that diffuses via interstitial and lymphfluid to the lymph node depends on the adsorptive capacityand adsorptive coefficient, as well as on the rate of desorptionfollowing injection and exposure to interstitial fluid [18].

An alternative mechanism for translocation of vaccine anti-gens from the injection site to the lymph node is intracellu-larly following uptake by dendritic cells. Dendritic cells takeup antigens from the local microenvironment via three proc-esses: macropinocytosis, receptor-mediated endocytosis andphagocytosis [53]. Aluminum-containing adjuvants enhancedthe uptake of adsorbed antigens by human monocytes andmouse dendritic cells in vitro [54,55]. An in vitro study of theuptake of antigens by dendritic cells revealed that phagocyto-sis of aluminum-containing adjuvant with adsorbed antigenwas more efficient than uptake of soluble antigen [55]. The

size of the aluminum-containing adjuvant aggregates was animportant factor in the uptake of antigen. The α-casein thatadsorbed to aluminum phosphate adjuvant exhibited signifi-cantly greater uptake than α-casein adsorbed to aluminumhydroxide adjuvant. The mean aggregate size of aluminumphosphate and aluminum hydroxide adjuvants were 3 and17 µm, respectively. Dendritic cells have a diameter ofapproximately 10 µm. When the aluminum hydroxide adju-vant was pretreated with two concentrations of phosphate,the mean aggregate size decreased. Flow cytometry data pre-sented in FIGURE 6 showed that the uptake of fluorescentlylabeled α-casein was inversely related to the size of theadjuvant aggregates.

Dendritic cells with internalized antigens migrate via affer-ent lymphatics to the draining lymph node where theypresent antigenic peptides to T cells.

Dendritic cell maturationThe dendritic cells in nonlymphoid tissues are immature andnot effective in activation of naive T cells. In response toinfections and tissue damage, dendritic cells undergo a matu-ration process that is characterized by increased expression ofcostimulatory molecules, in particular CD80 and CD86.They also increase the expression of the chemokine receptorCCR7 that is essential for the migration of dendritic cells tothe draining lymph nodes. This maturation process is acti-vated by engagement of receptors, including Toll-like recep-tors (TLRs), by highly conserved pathogen-associated mole-cules [56]. A partially overlapping group of receptorsrecognizes endogenous ligands (alarmins), such as highmobility group box 1, heat shock proteins and uric acid,released during cell necrosis and inflammation [57]. Giventheir nature as mineral salts, it is unlikely that aluminumhydroxide and aluminum phosphate adjuvants wouldenhance the immune response through activation of TLRs.Indeed, the adjuvant effect of aluminum-containing adju-vants was not affected by genetic inactivation of MyD88 andTRIF, two critical adaptor molecules in the TLR signalingpathway [58,59]. Nevertheless, aluminum adjuvants have aneffect on the phenotype and function of APCs. Incubation ofhuman peripheral blood mononuclear cells and macrophageswith aluminum hydroxide adjuvant resulted in increasedexpression of CD86 [60–62]. Similarly, incubation of mousebone marrow-derived dendritic cells with either aluminumhydroxide or aluminum phosphate adjuvant increased theexpression of CD80 and CD86 [63]. In addition to directlystimulating dendritic cell maturation, aluminum-containingadjuvants may further enhance the expression of costimula-tory molecules through the induction of inflammation andrelease of alarmins.

Antigen presentationA model system of ovalbumin and ovalbumin-specificD011.10 T cells recently demonstrated that aluminum-con-taining adjuvants enhance the ability of dendritic cells to



activate CD4+ T cells [63]. This effect was observed with bothintact ovalbumin and the ovalbumin peptide that binds to theMHC II molecule recognized by D011.10 T cells, but doesnot require uptake and processing. The study suggests that alu-minum-containing adjuvants not only enhance antigen uptakebut also the efficiency of antigen presentation consistent withincreased expression of costimulatory molecules.

Although adsorbed antigens are more efficiently taken upby dendritic cells, too tight adsorption may interfere withthe immune response [64]. As discussed previously, antigenswith multiple phosphate groups are strongly adsorbed to alu-minum hydroxide adjuvant through the ligand exchangemechanism. Phosphate treatment of the adjuvant reduces thenumber of hydroxyl groups available for ligand exchange anddecreases the strength of the binding. This was used to inves-tigate the effect of strength of adsorption on the immuneresponse [64]. Mice were immunized with antigen/adjuvantcombinations with decreasing strength of adsorption and theprimary antibody response was measured after 3 weeks. Theantibody titers were inversely correlated to the strength ofadsorption, as measured by the adsorptive coefficient, andthe antigen/adjuvant combination with the strongest bind-ing failed to induce an antibody response (TABLE 5). To deter-mine if this was associated with an effect on T-cell activation,antigen-specific T-cell proliferation was performed. No acti-vated antigen-specific T cells were found in the spleens ofmice immunized with the antigen/adjuvant combinationwith the strongest binding, indicating that tight bindinginterferes with the processing and/or presentation of antigenby dendritic cells.

Dendritic cells can process antigens not only for presenta-tion in complex with MHC II molecules to CD4+ T cells,but also for presentation by MHC I molecules to CD8+

T cells. The efficiency of this cross-presentation of antigensdepends on the nature and physical form of the antigen and isnegligible for most aluminum-adjuvanted vaccines. No cyto-toxicity to influenza virus-infected cells was observed follow-ing immunization with two doses of recombinant influenzavirus protein with aluminum hydroxide adjuvant [65]. Only asubsequent injection with nonadjuvanted protein induced aCD8+ cytotoxic T-lymphocyte (CTL) response. Similarly,immunization with ovalbumin adsorbed to aluminumhydroxide adjuvant did not induce a CTL response [66,67].Interestingly, IFN-γ-secreting CD8+ T cells were detected fol-lowing immunization of HBsAg combined with either alumi-num hydroxide and aluminum phosphate adjuvant, but therewas no evidence of cytolytic activity [49]. It is not clearwhether this effect is specific to HBsAg or can also beobserved with other antigens.

T-cell differentiationFollowing activation, CD4+ T cells differentiate into effectorcells that provide help for the cell-mediated immune response(Th1 cells) or the humoral immune response (Th2 cells). TheTh1 cells are characterized by the secretion of IFN-γ and the

Th2 cells by the secretion of IL-4 and IL-5. Although alumi-num-adjuvanted vaccines can induce IFN-γ-secreting T cells,the immune response is predominantly a Th2 response. Themechanism underlying the Th2 bias of this immune responseis not well understood.

Upon arrival in the draining lymph node, mature dendriticcells provide T cells not only with antigen (signal 1) and cos-timulatory signals (signal 2), but also with instructions fordifferentiation into Th1 versus Th2 cells (signal 3) [68]. Thenature of signal 3 depends on the microenvironment inwhich the dendritic cell was activated. For example, engage-ment of TLRs by microbial molecules increases expression ofcostimulatory molecules (signal 2) and induces secretion ofIL-12 (signal 3), which promotes Th1 differentiation. As men-tioned previously, aluminum-containing adjuvants do not acti-vate TLRs and, consistent with this notion, did not induce secre-tion of IL-12 [63]. However, incubation of dendritic cells withaluminum-containing adjuvants induced the secretion of IL-1βand IL-18 [63]. The secretion of these cytokines, both members ofthe IL-1 superfamily, involves two steps that are regulated inde-pendently: the transcription of mRNA and translation into activeprecursors, pro-IL-1β and pro-IL-18 that accumulate in the cyto-plasm of the cells and, then, the activation of caspase-1 resultingin proteolytic cleavage of the precursors into active moleculesthat are released from the cell [69]. Aluminum-containing adju-vants did not induce mRNA expression of IL-1β and IL-18 butthe secretion of IL-1β and IL-18 was abrogated by caspase-1inhibitors [63]. This indicates that aluminum-containing adju-vants activate the second step and suggests that there iseither sufficient baseline transcription of IL-1β and IL-18mRNA or induction of mRNA during inflammation at theinjection site. Others have reported that aluminum-contain-ing adjuvants induced the secretion of IL-1β and IL-18, butonly when coincubated with TNF or lipopolysaccharide(LPS) [70].

Dendritic cells incubated with aluminum-containing adju-vants induced the differentiation of naive CD4+ T cells intoIL-4- and IL-5-secreting CD4+ T cells. The aluminum-con-taining adjuvants also induced expression of IFN-γ by CD4+

Table 5. Relationship between the strength of the adsorption force as measured by the adsorptive coefficient and geometric mean antibody titer.

Vaccine Adsorptive coefficient(ml/mg)

Geometric meanantibody titer

CAS/AH 2410 100

CAS/PT-AH 420 1900

DP-CAS/AH 60 23,600

DP-CAS/PT-AH 1 51,000

AH: Aluminum hydroxide adjuvant; CAS: α-casein; DP-CAS: Dephosphorylated α-casein; PT-AH: Phosphate-treated aluminum hydroxide adjuvant.Adapted with permission from [64].

Hem & HogenEsch


T cells, but at a lower level than LPS [63]. Neutralization ofIL-1β and IL-18 or addition of a caspase-1 inhibitor pre-vented the differentiation of CD4+ T cells into Th2 cells [67].This suggests that aluminum-containing adjuvants induceIL-1β and IL-18 which function as signal 3 in the activationof CD4+ T cells.

In summary, aluminum-containing adjuvants enhance theimmune response by direct and indirect effects on dendriticcells. The adjuvants retain the antigen at the site of injectionand induce a local inflammatory response that probably resultsin recruitment of monocytic precursors of dendritic cells. Directeffects include increased antigen uptake, increased antigen pres-entation, increased expression of costimulatory molecules andinduction of Th2 differentiation of CD4+ T cells.

Expert commentary & five-year viewAluminum-containing adjuvants have a long history of use invaccines and are safe, effective and inexpensive. Althoughnovel adjuvants are being studied extensively, the aluminum-containing adjuvants will continue to be an essential compo-nent of vaccine formulations. In fact, the latest vaccines to belicensed against human papilloma virus and H5N1 influenzavirus are formulated with aluminum-containing adjuvants.During the next 5 years, focusing research in four main areaswill lead to improved performance of aluminum-adjuvantedvaccines. These include greater emphasis on preformulationstudies to fully characterize the antigen; improved under-standing of the events that occur at the injection site; theeffect of aluminum-containing adjuvants on the processes bywhich dendritic cells produce antigenic peptides; and theinteraction between aluminum-containing adjuvants andother immunomodulatory compounds aimed at developingadjuvants with enhanced efficacy.

With the increasing use of well-defined subunit vaccines,more emphasis should be placed on characterizing both physi-cal and chemical properties of the antigen before formulation

studies begin. The value of comprehensive preformulationstudies is recognized in traditional dosage forms and will beequally important for vaccine formulations. Peek and cowork-ers have demonstrated a systematic, three-step approach in theformulation of ricin toxin A-chain [71] and erythrocyte-bindingantigen (EBA-175 RII-NG) [72]:

• Evaluate the stability of the antigen as a function of temperatureand pH;

• Screen stabilizers for the antigen that can be included in theformulation;

• Determine adsorption and desorption characteristics of theantigen to aluminum-containing adjuvants. This approachis an efficient application of current understanding ofaluminum-containing adjuvant properties.

The chemical stability of antigens needs to be studied moreextensively in the future. Understanding the effect of pH on therates of degradation reactions, such as deamidation, hydrolysis,and oxidation is essential to vaccine development. If the anti-gen exhibits pH-dependent chemical stability, the vaccine for-mulation should be designed so that the adsorbed antigen expe-riences the optimum pH in the microenvironment of theadjuvant. This may require the adjustment of the surfacecharge of the adjuvant to produce the pH of maximum stabilityin the microenvironment of the adjuvant.

Preformulation testing of the antigen should also includedetection and enumeration of phosphate groups that canadsorb to the aluminum-containing adjuvant by ligandexchange. It will also be important to determine if the antigencontains structures that can expose phosphate groups duringaging of the vaccine. For example, the hydrolysis of phospholi-pids produces phosphate groups that can then be adsorbed byligand exchange. This information will enable the developmentof a vaccine formulation that produces the desired adsorptionduring the shelf-life of the vaccine. Studies to date have shownthat ligand exchange can be controlled by modifying thenumber of surface hydroxyl groups on the adjuvant.

In vivo studies are needed to improve understanding of thecompeting events that occur at the injection site, such as dif-fusion of the antigen away from the site and infiltration ofdendritic cells to the site due to inflammation. Just asadsorbed antigen is retained at the injection site, unadsorbedor eluted antigen may also be retained by entrapment in thevoid spaces of the adjuvant aggregates. The ratio of antigen toadjuvant, as well as aggregate size, may affect the extent ofantigen trapping. We anticipate that the emphasis in vaccineformulation will move from the state of adsorption of theantigen in the vaccine to optimizing the competing eventsthat occur at the injection site.

The effect of aluminum-containing adjuvants on theprocessing of antigens in dendritic cells needs to be investi-gated. Recent evidence indicates that processing within den-dritic cells is inhibited if the antigen is adsorbed too stronglyby the aluminum-containing adjuvant.

Figure 6. Relationship between the degree of antigen internalization by dendritic cells and the mean diameter of the adjuvant aggregates in cell culture media. Redrawn with permission from [55].

0

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80

100

120

140

160

0 5 10 15 20Mean diameter (µm)

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Y F

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Can the antigen be processed while it is adsorbed to theadjuvant particle or must the antigen elute in order for pro-teases to produce the antigenic peptides? Emphasis will movefrom simply determining if the antigen is adsorbed by thealuminum-containing adjuvant to measuring both the extentand strength of adsorption. The surface charge and numberof surface hydroxyl groups of the adjuvant can be modifiedby pretreatment with phosphate to produce the desireddegree and strength of adsorption.

Several recent studies have explored the possibility of com-bining aluminum-containing adjuvants with other immuno-modulatory compounds to create novel adjuvants that are safebut induce an enhanced and possibly more effective immuneresponse. Examples are CpG oligodeoxynucleotides (ODNs),monophosphoryl lipid A (MPL) and IL-12. The ODNs bindTLR9 and induce a Th1-biased immune response [73]. Combi-nation of ODN with aluminum hydroxide adjuvant induced astronger antibody response to HBsAg than either compoundby itself, and the antibodies were both IgG1 and IgG2a indicat-ing a mixed Th1/Th2 response [74]. A similar synergism wasreported for aluminum phosphate adjuvant [75]. MPLA is aderivative of LPS and, similar to LPS, activates cells via theTLR4/MD2 complex. However, activation of TLR4 by MPLis more selective than LPS resulting in reduced toxicity [76].The combination of MPL with aluminum hydroxide adjuvant,known as AS04, induces a stronger immune response toHBsAg and recombinant human papilloma virus antigens thanaluminum hydroxide adjuvant alone [77,78]. In another study,MPL with aluminum phosphate adjuvant did not significantlyenhance the immune response over aluminum phosphate adju-vant in a nine-valent pneumococcal vaccine in children [79].The interaction between MPL and the aluminum-containingadjuvants, such as degree and strength of adsorption, was not

reported in these studies. IL-12 is a cytokine that directs thedifferentiation of CD4+ T cells to Th1 cells. Combination ofaluminum hydroxide adjuvant with IL-12 significantlyenhanced the antibody response and induced a shift toward aTh1 response in comparison with aluminum hydroxide adju-vant alone [80]. The IL-12 was adsorbed completely to theadjuvant, however, the strength of adsorption and elution ininterstitial fluid were not examined. In another study, IL-12adsorbed onto aluminum phosphate adjuvant induced astronger antibody response to HBsAg and Th1 shift in compar-ison with aluminum phosphate adjuvant only [75]. Althoughboth aluminum hydroxide and aluminum phosphate adjuvantscompletely adsorbed the IL-12 in the formulation, IL-12induced a stronger response combined with aluminum phos-phate adjuvant than with aluminum hydroxide adjuvant [75].These examples suggest that a better understanding of theinteraction between aluminum-containing adjuvants and otherimmunomodulatory compounds may lead to new adjuvantsystems that are safe and effective.

Even though aluminum-containing adjuvants have been usedsince 1926, great potential exists for improved performance ifthe vaccine formulation incorporates new insights obtainedthrough these suggested studies.

Financial & competing interests disclosureThe authors have no relevant affiliations or financial involve-ment with any organization or entity with a financial interest inor financial conflict with the subject matter or materials dis-cussed in the manuscript. This includes employment, consultan-cies, honoraria, stock ownership or options, expert testimony,grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of thismanuscript.

Key issues

• Aluminum-containing adjuvants adsorb antigens predominantly via electrostatic and ligand-exchange mechanisms.

• The isoelectric point of aluminum-containing adjuvants depends on the ratio of surface hydroxyl groups to surface phosphate groups.

• Adsorption by ligand exchange can be controlled by adjusting the ratio of surface hydroxyl groups to surface phosphate groups on the adjuvant or by adjusting the number of phosphate groups in the antigen.

• The degree of antigen adsorption may change when the vaccine comes into contact with interstitial fluid following administration.

• Antigen that is not adsorbed or is eluted following administration may be trapped in the void spaces of the adjuvant aggregates and retained at the injection site.

• Strong adsorption of antigens to aluminum-containing adjuvants may interfere with antigen presentation to T cells and the humoral immune response.

• Aluminum-containing adjuvants directly activate dendritic cells to secrete IL-1β and IL-18. These cytokines play a role in the differentiation of CD4+ T cells into Th2 cells.

• The pH at the surface of the adjuvant may be different from the bulk pH. The adsorbed antigen will undergo chemical degradation reactions at a rate associated with the microenvironment pH rather than the bulk pH.

Hem & HogenEsch


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Affiliations

• Stanley L Hem, PhD

Professor of Physical Pharmacy, Purdue University, Industrial and Physical Pharmacy Department, West Lafayette, IN 47907, USATel.: +1 765 494 1451Fax: +1 765 494 [email protected]

• Harm HogenEsch, DVM, PhD

Professor of Comparative Pathobiology, Purdue University, Department of Comparative Pathobiology, West Lafayette, IN 47907, USATel.: +1 765 494 0596Fax: +1 765 494 [email protected]

relationship between physical and chemical properties of aluminum-containing adjuvants and...

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