Introduction

Aluminium adjuvants have been used in practical vaccination for more than half a century to induce early, high-titre, long-lasting protective immunity. Billions of doses of aluminium-adjuvanted vaccines have been administered over the years and they are, at present, the most widely used adjuvants in both veterinary and human vaccines. In general, aluminium adjuvants are regarded as safe when used in accordance with current vaccination schedules^{1, 2}.

In human vaccinations, aluminium adjuvants have primarily been used in tetanus, diphtheria, pertussis and poliomyelitis vaccines as part of standard child vaccination programmes in many countries for approximately 50 years. Aluminium adjuvants have also been introduced into hepatitis A and hepatitis B virus vaccines. Other aluminium-adsorbed vaccines against, for example, anthrax, are available for special risk groups. In veterinary medicine aluminium adjuvants have been used in a large number of vaccine formulations against viral^{3, 4, 5, 6, 7} and bacterial^{8, 9, 10, 11} diseases, and in attempts to make antiparasite vaccines^{12, 13, 14, 15} (Table 1).

Glenny and coworkers were the first researchers to demonstrate the adjuvant effect of aluminium compounds in 1926. Glenny prepared a variety of diphtheria toxoid precipitates and investigated their immunogenicity. Among these were toxoids precipitated by the addition of potassium alum (KAl(SO₄)₂:12H₂O). Glenny observed that injecting the diphtheria toxoid as an alum precipitate led to a significant increase in the immune response against the toxoid^{16, 17}. Vaccines prepared in accordance with this principle have been used in practical vaccination and are referred to as alum-precipitated vaccines. Research showed that such preparations could be highly heterogenous, depending on which anions (such as bicarbonate, sulfate or phosphate) were present at the time of precipitation (e.g. as buffer constituents or growth media residues in the antigen solution)¹⁸. However, preformed aluminium-hydroxide hydrated gels have the ability to adsorb protein antigens from an aqueous solution and these gels can be preformed in a well-defined and standardized way¹⁹. Vaccine preparations based on this approach are called aluminium-adsorbed vaccines in contrast to alum-precipitated vaccines. Aluminium phosphate was introduced as an adjuvant somewhat later. In 1946, Ericsson²⁰ devised a method in which diphtheria toxoid was coprecipitated into a matrix of aluminium phosphate. Holt²¹, shortly after, demonstrated that preformed aluminium phosphate, prepared from equimolar amounts of aluminium chloride and trisodium phosphate, acted as an adsorbant and was adjuvant active with diphtheria.

Data on the use of alum-precipitated vaccines can be found in older literature²², but in practical vaccinations the adsorption onto preformed hydroxide and aluminium phosphate gels has now almost completely substituted the alum precipitation in vaccine preparations. A number of other aluminium compounds have been investigated as adjuvants, for example, aluminium hydrochloride^{23, 24} and aluminium silicate²⁵, but are not used in practical vaccinology. Occasionally the word ‘alum’ is used to describe both aluminium hydroxide and aluminium phosphate gels, but this is incorrect use of the terminology. Potassium alum, KAl(SO₄)₂:12H₂O, is in accordance with the chemical definition of an alum, whereas neither aluminium hydroxide nor aluminium phosphate are.

Preparation and analysis of aluminium adjuvants

Aluminium hydroxide and aluminium phosphate adjuvants are generally prepared by exposing aqueous solutions of aluminium ions, typically as sulfates or chlorides, to alkaline conditions in a well-defined and controlled chemical environment. Various soluble aluminium salts can be used for the production of aluminium hydroxide, but the experimental conditions, temperature, concentration and even the rate of the addition of the reagents, strongly influence the results^{26, 27}. Anions present at the time of preparation may coprecipitate and change the characteristics away from those of a ‘pure’ aluminium hydroxide. Aluminium phosphate gel can be seen as an example of such a preparation, where the soluble aluminium salts are exposed to alkaline conditions in the presence of phosphate ions.

Stanley Hem’s group at Purdue University has intensively studied the physicochemical nature of the aluminium hydroxide and aluminium phosphate gel preparations that have commonly been used as vaccine adjuvants for approximately 25 years. Using X-ray crystallography and infrared spectroscopy they demonstrated a boehmite-like (aluminium oxyhydroxide) pattern in preparations traditionally known as aluminium hydroxide, whereas commercialized aluminium phosphate gel adjuvant was identified as amorphous aluminium hydroxyphosphate²⁸. They were able to calculate an average primary crystallite size of 4.5 2.2 10 nm for the boehmite preparations²⁹.

Analytical methods

A number of physicochemical characteristics of the aluminium adjuvants can now be described using modern analytical methods.

Particle size distributions of the aluminium gel particles can be obtained using, for example, laser diffraction analysis. An example of this, obtained using Malvern equipment, is given in Figure 1. The size of the aluminium hydroxide particles is comparable to the size of microorganisms and is adequate for uptake by phagocytosis by APC.

Stanley Hem’s group has investigated the fine structure and crystallite size of aluminium adjuvants using X-ray diffraction and infrared spectroscopy. The X-ray diffraction band width measured at the half height at the 020 reflection (6.46Å), occasionally referred to as WHH₀₂₀, expresses the crystallite size²⁸. Infrared spectroscopy applied to aluminium hydroxide preparations reveals the structure known as boehmite²⁸.

The protein adsorption capacity of the adjuvant can be measured using a variety of analytical methods. In general, it is done by comparing the protein content in the aqueous phase of the antigen solution before and after adsorption onto the adjuvant. One of the classical methods, the Ramon flocculation test (named after the Frenchman Gaston Ramon who introduced the adjuvant concept around 1925), is still used to determine the adsorption of diphtheria and tetanus toxoid³⁰. If an antibody specific for the antigen you wish to adsorb is available, adsorption can be measured using immunoprecipitation techniques, by using either quantitative immunoelectrophoresis³¹ or single radial immunodiffusion³². Without the use of an antibody it can be tested spectrophotometrically (e.g. the BCA method)³³. However, it is important to note that contamination with the fine fraction of aluminium gel particles can disturb the spectrophotometrical readings by light-scatter effects. ELISA methods in which aluminium-adsorbed antigens can be used directly as the antigens in the ELISA have been designed³⁴. ELISA methods can also be applied for in vitro assessment of various viral antigens, that is, pseudorabies, porcine parvovirus and infectious bovine rhinotracheitis vaccines adsorbed onto aluminium hydroxide adjuvant³⁵.

Differential adsorption of complex mixtures of antigens can be measured using either immunoelectrophoresis or by HPLC. If an antiserum raised against the complex antigen mixture is available, a crossed 2-D immunoelectrophoresis will reveal if single components from the complex solution of proteins remain unadsorbed. To use this approach, the precipitation band pattern from an electrophoresis run on the complex antigen mixture prior to adsorption is compared with the bands of an electrophoresis run on the supernatant of the same mixture after adsorption. Unadsorbed components will retain their immunoprecipitation band pattern, whereas missing bands or a reduced height of the bands are indicative of complete or partial protein adsorption³¹. A comparison of HPLC chromatograms of the antigen-mixture liquid phase before and after adsorption may provide similar information.

The aluminium content in the final vaccine can be monitored using atomic adsorption spectrometry as devised by May et al³⁶.

Mechanisms of adjuvant activity

The immunostimulating effect of the traditional aluminium adjuvants is highly complex and must be attributed to several different mechanisms. In the past¹⁷, the function of a repository adjuvant was attributed to delayed clearing from the injection site and sustaining a gradual release of adsorbed antigen from the inoculated depot. Although it quickly became obvious that the gradual release explanation was inadequate, the physical adsorption of antigen onto the adjuvant is still considered to be a very important mechanism for the function of these adjuvants. Over the past two decades the role of T cells and cytokine profiles has also been described.

Antigen adsorption

Although the literature provides examples of publications where injection of adjuvant and unadsorbed antigen at distant sites leads to immunostimulation towards the antigen³⁷, this result is not consistent³⁸ and the nature of the antigen chosen in the research may provide part of the explanation for these differing conclusions. As a general rule the antigen should be adsorbed onto the adjuvant prior to immunization.

A soluble antigen may upon adsorption be presented to the immunocompetent cells in a ‘particulate’ manner that could facilitate antigen targeting (i.e. favour uptake by APC). It is likely that APC may be more efficient in antigen uptake by phagocytosis than by pinocytosis. There is experimental evidence of enhanced uptake of aluminium adsorbed tetanus toxoid when compared to the soluble toxoid by APC³⁹.

The physicochemical mechanisms behind the antigen adsorption itself are complex and depend on the nature of the individual antigen. Some mechanisms may predominate over others.

As a general guideline, for many protein antigens adsorption is best accomplished in the pH interval between the isoelectric point (IEP) of the protein antigen and the point of zero charge (PZC) of the aluminium adjuvant. This applies to both aluminium hydroxide and aluminium phosphate adjuvants. In this interval the adjuvant and the antigen will have opposite electrical charges, facilitating electrostatic attraction and adsorption (Figure 2). Seeber et al.⁴⁰ concluded that aluminium hydroxide should be superior to aluminium phosphate in adsorbing proteins with an acid IEP and vice versa for proteins with an alkaline IEP.

However, if the antigen contains phosphorylated groups ligand exchange between the antigen-associated phosphate and hydroxyl groups of the adjuvant may account for a high-affinity binding to the adjuvant. This is the case with HBsAg particles⁴¹ and has been shown in experiments using phosphorylated alpha-casein as the model antigen⁴².

The primary mechanisms responsible for adsorption have been explained, in part, by electrostatic attraction and, in part, by anionic ligand exchange⁴³. In addition, other intermolecular binding forces, including hydrophilic–hydrophobic interaction, may play a role in protein adsorption⁴⁴ and each binding force may play it’s role in a given antigen–adjuvant combination depending on the nature of the antigen and the chemical environment (pH, ionic strength, presence of surfactants etc.)^{45, 46, 47}.

T-cell reactivity, antibody subclasses and stimulation of cytokines

In the past it was often claimed that aluminium adjuvants were not ‘T-cell adjuvants’. But this conclusion was generally based on work in which a ‘T-cell adjuvant’ was primarily considered an adjuvant capable of inducing a delayed-type hypersensitivity (DTH) response. It is correct that aluminium adjuvants are not efficient DTH inducers in rodents⁴⁸. There is also very little evidence that aluminium adjuvants should be able to generate MHC class I restricted cytotoxic T cells; to date only a single report from Dillon et al.⁴⁹ using a recombinant influenza vaccine in mice. However, as early as the late 1970s the ability of aluminium adjuvants to induce eosinophilia was shown to require the presence of T cells⁵⁰ and the reaction profile of aluminium adjuvants is now known to include stimulation of CD4⁺ T cells⁵¹.

Studies examining the impact of interleukins in model systems were initiated almost 20 years ago and in recent times more studies have been conducted because of the introduction of gene-disrupted mice. Among the early observations in classical animal models was the demonstration that aluminium-adsorbed tetanus toxoid led to an increase in antigen-induced T-cell proliferation, apparently resulting from the increased release of IL-1³⁹. In contrast, IL-1 does not appear to be important in the augmentation of the primary antibody response in rabbits immunized with aluminium adjuvant⁵².

Grun and Maurer⁵¹ demonstrated that anti-IL-1 or anti-IL-4 were able to inhibit an antigen-specific T-cell proliferative response after immunization with aluminium adjuvant, but not if the mice were immunized with Freund’s Complete Adjuvant. However, the proliferative responses were inhibited by anti-CD4 antibody, regardless of the adjuvant used. This indicated that the proliferating CD4⁺ T cells from mice immunized using aluminium adjuvant were from the Th2 subset. Lindblad and coworkers⁵³ found a corresponding profile in C57BL/6J mice when they examined IgG2a and IgG1 subclass assays and reverse transcription–polymerase chain reaction (RT-PCR) for IL-4- and IL-10-specific mRNA in the regional draining lymph nodes at day 7 following vaccination with aluminium-adjuvanted vaccine.

Although it is now generally accepted that aluminium adjuvants are Th2 stimulators, it is interesting to note that a complex between Al(OH)₃ and IL-12 (Al(OH)₃/IL-12) induced a Th1 response, rather than a Th2 reponse, when used as an adjuvant⁵⁴ and the Th1-promoting effect of the Al(OH)₃/IL-12 complex was greatly augmented by the coadministration of exogenous IL-18⁵⁵.

A new line of research has been initiated with the introduction of gene-disrupted mice, which has facilitated studies examining the significance of interleukins.

In IL-4-gene-disrupted mice immunization with OVA + Al(OH)₃ gave IgG2a titres of a similar magnitude to OVA + FCA⁵⁶. Interestingly, the group immunized with OVA + Al(OH)₃ continued to produce IL-5 (a cytokine normally associated with the Th2 profile). In contrast, when the IL-4^-/- mice had been immunized with FCA, a similar stimulation of IL-5 was not observed (Figure 3). These results support the idea that the major role of aluminium-induced IL-4 in Th-subset stimulation is to downregulate the Th1 response.

The role of IL-18 in the adjuvant mechanisms of aluminium hydroxide and its effect on Th2 induction has been demonstrated⁵⁵. Brewer et al. revealed that IL-18-deficient mice immunized with OVA + Al(OH)₃ had reduced IL-4 production in lymph node cells compared to wild-type mice. However, if they added exogenous IL-18 the aluminium-induced Th2 response was not further enhanced. Although the aluminium adjuvant led to reduced IL-4 production in IL-18^-/- mice, this was not accompanied by a reduced level of serum anti-OVA IgG1. Apparently, there is a poor correlation between this particular antibody subclass and IL-4 production.

Stimulation of IgE

It is well established that aluminium adjuvants stimulate the production of IgE as part of the overall Th2 profile^{57, 58}. Although this has been considered a disadvantage, it has been difficult to demonstrate cases where vaccination with aluminium adjuvants has led to IgE-mediated allergy towards the vaccine antigen in practical vaccination. In contrast, aluminium adjuvants have been used to hyposensitize allergic patients for many years with good results.

There are some interesting similarities between the immune response (e.g. stimulation of IgE and eosinophilia) elicited by some helminthic parasites and the immune response following immunization with aluminium adjuvants that make these adjuvants interesting candidates for antiparasitic vaccines. A good vaccine against, for example, schistosomiasis would be of major importance in the health of people in developing countries. To date, however, only partial protection has been achieved in animal models^{59, 60}. In a special mouse hybrid (C57BL/6J DBA/2) F1, which is known to be a high-producer of IgE, it was possible to induce a significant degree of protection against schistosomiasis⁶⁰. The protected mice had high titres of specific IgE antibody after immunization using sonicated parasite antigens and aluminium hydroxide adjuvant. Mice immunized with sonicated antigen and FCA had low-level protection and lower IgE titres. Although the group immunized with FCA had high titres of specific total Ig⁶⁰, protection against schistosomiasis was still lower. This suggests a protective superiority of specific IgE against helminthic infections.

A very useful way to examine IgE/Th2 stimulation in rodent models is to immunize groups of animals using aluminium adjuvant versus Freund’s Complete Adjuvant, respectively, and assess the resulting antibody and cytokine profiles. Uede et al.^{61, 62} pioneered this line of research over two decades ago using keyhole limpet haemocyanin as the antigen. Their study revealed the involvement of glycosylation-enhancing factors and Fc R+ T cells in a dichotomous regulatory pathway in which aluminium adjuvant stimulated the synthesis of IgE and FCA supressed it^{61, 62}.

The introduction of gene-disrupted mice has led to more sophisticated applications of the aluminium versus FCA model for studying IgE regulation. Brewer and coworkers used the same approach to compare the adjuvant profiles of aluminium hydroxide versus FCA⁵⁶. They found that there was no IgE production in IL-4-gene-disrupted mice (IL-4^-/-) regardless of whether aluminium adjuvant or FCA was used as the adjuvant. This suggests that IL-4 is an essential prerequisite for the induction of IgE by aluminium adjuvants.

Dose–response considerations

The optimum dose of adjuvant is normally determined empirically in a pilot trial, but helpful guidelines are available in the literature. In veterinary vaccines there is no defined maximum limit for the allowed content of aluminium adjuvants and, in general, the dose is set with the aim of balancing efficacy and local reactogenicity. However, there are limitations for the content of aluminium allowed in vaccines for humans. These limits are 1.25 mg aluminium per dose in Europe⁶³, and in USA 0.85 mg aluminium per dose if determined by assay, 1.14 mg if determined by calculation and 1.25 mg if safety and efficacy data justifies it⁶⁴.

For the dose–response relationship of aluminium adjuvants in combination with bacterial antigens the immunomodulation observed may reflect a composite effect between the aluminium adjuvant and immunomodulatory and adjuvant active bacterial substances, such as LPS, trehalose dimycolate (‘cord factor’), muramyl peptides from peptidoglycans or CpG motifs from bacterial DNA⁶⁵.

Limitations to the applicability of aluminium adjuvants

One obvious limitation in the application of aluminium adjuvants lies in the clear Th2 profile of these adjuvants. A Th2-biased immune response is not likely to protect against diseases for which Th1 immunity and MHC class I restricted CTL are essential for protection (e.g. intracellular parasites or tuberculosis)⁵³. Another limitation lies in the fact that traditional aluminium-adsorbed vaccines are frost sensitive and thus not lyophilizable.

Aluminium adjuvants have failed to provide satisfactory augmentation of the immune response in a number of cases, including influenza and typhoid fever vaccines^{66, 67}. In some recent approaches to vaccine preparation, aluminium adjuvants have shown limitations in their applicability in vaccines based on small-size peptides⁶⁸. In some cases, for example, with foot-and-mouth disease virus peptides, the problem could be overcome by conjugating the peptide to a larger carrier molecule⁶⁹, while in others it could not^{70, 71}. Aluminium adjuvants have been tested in a few DNA vaccine formulations. In these cases it was revealed that aluminium hydroxide had an inhibiting effect, whereas aluminium phosphate adjuvant augmented the immune response against the antigen encoded by the nucleotide^{72, 73}. The high content of phosphate in the DNA molecule apparently gives a high binding affinity of the nucleotide to the aluminium hydroxide, which in turn prevents the RNA from getting access to and then translating the nucleotides into protein⁷³.

In vivo clearing of aluminium adjuvants

Mammalian organisms are constantly being exposed to aluminium compounds in the environment. As a consequence aluminium is found in the blood and serum of humans and animals whether or not they have been vaccinated using aluminium adjuvants. The major source of this aluminium is apparently oral intake with food and drinking water. Martyn et al.⁷⁴, based on a study in Britain, calculated the average daily intake of aluminium by humans to be 5–10 mg. Individuals with normal renal functions would excrete this aluminium uptake with the urine. In contrast to other metallic ions, such as Zn⁺⁺ and Mg⁺⁺, aluminium apparently does not act as an essential trace element or coenzyme in normal metabolism. The clearing in vivo of aluminium adjuvants has been investigated in rabbits using adjuvants prepared from the isotope ²⁶Al⁷⁵. Blood and urine excreted ²⁶Al was followed using accelerator mass spectroscopy for a period of 28 days. As early as 1 h after i.m. injection radioactive-labelled Al could be detected in the blood and approximately threefold more ²⁶Al was excreted from animals vaccinated with aluminium phosphate than those vaccinated with aluminium hydroxide. Thus, it appears that interstitial fluid containing organic acids with an -hydroxy carboxylic acid, which is able to chelate Al, react more readily with aluminium phosphate than with aluminium hydroxide⁷⁵. It is of interest that following injection of adjuvant containing 0.85 mg Al the normal plasma concentration of Al in rabbits (30 ng Al ml^-1) only rose approximately 2 ng Al ml^-1 during the experiment. Flarend⁷⁵ calculated that a similar Al dose injected into humans, provided similar clearing kinetics existed, would lead to an estimated increase of serum Al of only 0.04 ng Al ml^-1, which is equal to 0.8% above the normal level of approximately 5 ng Al ml^-1. Based on these figures it appears unlikely that the amount of aluminium administered via vaccination would contribute significantly to the general exposure of humans to aluminium or to the serum levels of aluminium.

Side-effects

Aluminium hydroxide and aluminium phosphate adjuvants have been used for more than half a century now and are generally regarded as safe when used according to current immunization schedules². In 1993 the United States the National Cooperative Vaccine Development Group on safety evaluation of vaccine adjuvants with the participation of FDA representatives concluded that ‘the extensive experience with this class of adjuvant for vaccine use has indicated that it is safe’⁷⁶.

There is no evidence that aluminium adjuvants themselves should be immunogenic and act as haptens, accordingly they are not likely to cause harmful immune complex reactions and observations of contact hypersensitivity reactions are rare^{2, 77}. The aluminium adjuvants are not in themselves pyrogenic and there is no evidence of carcinogenicity or teratogenicity attributed to their use. In contrast, aluminium hydroxide has a significant binding affinity to endotoxins that may reduce the reactogenicity of some adsorbed vaccines containing, for example, residual LPS.

Cases of local reactions have occasionally been reported⁷⁸. These may comprise swellings, indurations, erythemas and cutaneous nodules that can persist for up to 8 weeks and occasionally longer⁷⁹. These reports often describe cases of allergic patients undergoing hyposensitizing therapy, during which they receive a large number of injections of adsorbed allergenic extracts over a limited period of time. The aluminium adjuvants should be regarded as depot-forming or repository adjuvants. With these adjuvants the formation of an inflammatory focus attracting immunocompetent cells shortly after injection must be expected¹. After injection macrophages are attracted to the site to phagocytize and clear the inoculum. The local reaction may be negligible if the inoculum is rapidly dispersed from the injection site. However, if the inoculum resides for a prolonged period of time at the injection site (as is the case with repository adjuvants such as mineral adjuvants or water-in-oil emulsions) then in situ accumulation of phagocytic and immunocompetent cells may in some cases manifest itself as an inflammatory focus accompanied by a transient swelling, local irritation and redness. Some observations of aluminium-adsorbed vaccines giving rise to more local reactions than unadsorbed vaccines with plain toxoid⁸⁰ could, in part, be explained by the plain toxoid vaccine being dispersed from the injection site before a local reaction was established.

According to the danger model described by Matzinger⁸¹ it is conceivable that such an inflammatory focus, through disturbance of local tissue integrity, elicits danger signals and generates heat-shock proteins and pro-inflammatory cytokines. Such molecules may contribute to stimulation of the primary immune response against the antigen.

Any visible or palpable reaction at the injection site is in principle non grata because it hinders the obtaining of a hypothetical ‘ideal adjuvant’. However, it is important to realize that the mechanisms described are part of a normal functioning immune system. Hence, it may not be achievable to use repository adjuvants without temporarily inducing an inflammatory focus around the inoculum.

Vaccinations may be given subcutaneously or intramuscularly and the injection modus is not without importance with respect to local reactogenicity. When immunizing using the subcutaneous route the vaccine inoculum is introduced into a compartment with numerous sensory neurones (in contrast to the intramuscular compartment). In this case the introduction of a local inflammatory response may more easily give rise to irritation and itching reactions. In addition, a transient swelling, as a consequence of the inflammatory focus formed, may be more easily palpable through the skin. When immunizing using the intramuscular route a similar size swelling may be less easily visible and palpable because it is located in deeper lying tissue.

Whether or not adsorption onto aluminium adjuvants leads to increased or decreased vaccine reactogenicity is not clear^{80, 82}. However, Butler et al. found that adsorption onto aluminium hydroxide (Alhydrogel) significantly reduced the side-effects with combined diptheria–tetanus–pertussis vaccines⁸³. The binding affinity of LPS to aluminium hydroxide is well established and is much higher than the binding to aluminium phosphate⁸⁴, probably because of the phosphate content of LPS. It is conceivable that acute toxicity is reduced in adsorbed vaccines simply because of a delayed release of toxic vaccine constituents, such as pertussis toxin, peptidoglycans from Gram-negative cell walls or LPS from the injection site. Norimatsu showed that adsorption of LPS onto aluminium hydroxide prior to injection inhibited or mitigated systemic effects including trembling, transient leucopenia and elevated serum TNF that would otherwise be observed following i.m. injection of LPS in saline⁸⁵. In addition, the level of IL-6 after administration of LPS was reduced if the LPS was adsorbed to aluminium hydroxide prior to injection⁸⁴.

A number of observations of side-effects following vaccination with adjuvanted vaccines must, however, be attributed to the vaccine preservatives (e.g. thiomersal, betapropriolactone or formaldehyde) or to bacterial toxins from the antigen preparation⁸⁶.

Epilogue

Aluminium adjuvants have beyond doubt proven their efficiency in a large number of applications as vaccine adjuvants in both human and veterinary immunoprofylaxis with very few problems. In addition, they are relatively inexpensive, which may not be relevant from a scientific point of view, but may be of great importance in establishing effective vaccination programmes in developing countries where funding is limited.

When evaluating the profile of an adjuvant for possible new applications very few adjuvants can match the extremely comprehensive cohorts that are available for aluminium adjuvants with respect to records of efficacy and safety profiles, which after more than half a century of use reach the lifespan of humans.

There is still a lot of research to be done to get a clearer picture of the mode of action of aluminium adjuvants. Incubation of dendritic cells (DC) with aluminium adjuvant failed to activate the DC as judged by increased expression of MHC class II and costimulatory molecules. This indicates that aluminium adjuvants, in contrast to bacteria-derived adjuvants (e.g. LPS, CpG), are not directly addressing toll-like receptors⁸⁷. But, it would be an interesting working hypothesis if heat-shock proteins and pro-inflammatory cytokines could indeed be demonstrated to be a consequence of the local tissue disturbance created by the injected aluminium-adsorbed vaccine. As heat-shock proteins contain highly conserved molecular regions with structural similarities to bacterial components it is possible that interaction with toll-like receptors could take place in vivo as a secondary reaction derived from the local tissue disturbance.

Aluminium adjuvants have their limitations, especially with regard to their profound Th2 reactivity, which means that there are vaccines in which they will have little or no effect. However, in the future, modified formulations with a more balanced Th1–Th2 profile may become available for use in practical vaccinology along with more potent Th1 stimulators, such as monophosphoryl lipid A. The potential application of the Al(OH)₃/IL-12 complex^{54, 55} for use in vaccines for Th1 stimulation is yet to be explored in detail. The same is the case for another composite formulation, Algammulin (gamma inulin + aluminium hydroxide), which has been shown to give a more balanced immune response with stimulation of IgG2a isotypes in mice indicative of a mixed Th1–Th2 stimulation⁸⁸. It is also interesting to note that it appears to be possible to combine traditional aluminium-phosphate-adsorbed protein vaccines with DNA oligonucleotide vaccines resulting in a combination of the two different technologies in a single vaccine formulation⁷³. The potential of these as well as other approaches to expand the use of aluminium adjuvants into new vaccine applications requires further research.

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