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Research Progress on Aluminum Adjuvants and Their Mechanisms of Action

What are Adjuvants?

Adjuvants, also known as immunomodulators or immune enhancers, are an additive to vaccines. Adjuvants are non-specific immune enhancers that enhance or alter the type of immune response to an antigen. Adjuvants can help antigens induce long-term effective specific immune responses in vivo, leading to higher vaccine efficacy and prolonged protection from immune responses. Adjuvants can also reduce the amount of antigen used, the cost of production and the number of immunizations.

Based on their chemical properties, adjuvants can be broadly classified into the following categories: Inorganic adjuvants such as aluminum adjuvants; emulsion-based adjuvants such as MF59 and AS03; water-soluble adjuvants such as saponins; adjuvants targeting pattern-recognition receptors such as CpG; and cytogenic adjuvants such as interleukins. Binding of adjuvants to antigens can increase the surface area of the antigen, and the biological effects include (1) deposit effect of antigen; (2) upregulation of cytokine and chemokine expression, and recruitment of immune cells at the injection site; (3) activation of inflammasome; and (4) carrier effects. On the one hand, in the presence of adjuvants, antigens are more likely to be efficiently processed and presented by antigen-presenting cells. On the other hand, adjuvants can alter the physical properties of antigens, leading to slower release of antigens in the body and prolonging the interaction between antigens and immune cells.

Hundreds of natural or synthetic compounds have been used in adjuvant research, but the number of adjuvants approved for use in vaccines remains limited. Aluminum hydroxide (AH) and aluminum phosphate (AP) still dominate the field of adjuvants for human vaccine formulations. However, despite the fact that aluminum adjuvants in vaccines have been used in enhancing vaccine immune responses for nearly decades, the molecular mechanisms of aluminum adjuvants are still not fully understood. Therefore, there is a need to deepen our understanding of the physical, chemical, and biological properties of these adjuvants to provide a theoretical basis for determining the quality attributes of each adjuvant and selecting the appropriate type of adjuvant as early as possible in the clinical development phase.

Type of Aluminum Adjuvants

The two main types of aluminum adjuvants currently included in licensed human vaccines are aluminum hydroxide and aluminum phosphate. The selection of AH adjuvants versus AP adjuvants in vaccine formulations is highly dependent on the nature of the antigen and the requirements for adsorption to achieve an optimal immune response.

Aluminum hydroxide adjuvants are prepared by adding sodium hydroxide to a solution of aluminum ions under carefully controlled conditions. Temperature, concentration and mixing rate are factors that affect the physicochemical properties of the adjuvant produced. Electron microscopy reveals that the AH adjuvant consists of nanoparticle fibers that form loose particulate aggregates.

Aluminum phosphate adjuvants are prepared by precipitating aluminum ions under alkaline conditions in the presence of phosphate. The addition of phosphate ions leads to the formation of aluminum hydroxyphosphate Al(OH)x(PO4)y, in which a percentage of the hydroxyl group is replaced by phosphate. AP adjuvants are amorphous, i.e., non-crystalline, because the incorporation of phosphate interferes with the crystallization process. AP adjuvants are primary spherical nanoparticles, approximately 50 nm in diameter, which typically form loose aggregates of particles.

Amorphous Aluminum Hydroxyphosphate Sulfate (AAHS) is an aluminum adjuvant  and used in a variety of commercial vaccines, including Recombivax, Gardasil, and Vaxelis. AAHS has a zero charge point of about 7, which means it does not carry a surface charge at neutral pH. Ultrastructurally, it consists of lamellar nanoparticles similar to AP and alum precipitated vaccines. Thus, AAHS can be considered a different form of AP adjuvant with a relatively low P:OH ratio. Some licensed vaccines, such as Twinrix and Infanrix Hexa, contain both AH and AP adjuvants. Vaccines are prepared by mixing selected antigens with AH or AP adjuvants followed by an optimal combination of adsorption components. Electron microscopy showed aggregates of nanofibers adjacent to or occasionally mixed with aggregates of lamellar nanoparticles, suggesting that the adjuvant maintains its predominantly particulate structure when mixed.

Physicochemical properties of Aluminum Adjuvant

There are a number of established assays that can be used to characterize aluminum adjuvants and ensure lot-to-lot consistency. Structural information can be obtained using X-ray diffraction (for AH adjuvants only), spectroscopy (FTIR, NMR, Raman) and transmission electron microscopy.

Granularity

Aluminum adjuvants typically consist of primary nanoparticles that form irregularly shaped aggregates ranging in size from 1 to 20 μm. Particle size and shape are important for the efficiency of particle uptake by immune cells through phagocytosis. Particle size can be determined using laser diffraction, dynamic light scattering, or microfluidic imaging. The surface charge of the aluminum adjuvant depends on the pH and as the electrostatic repulsion decreases, larger particle sizes are observed as the pH approaches the zero charge point. Similarly, the addition of NaCl masks the surface charge and enhances particle aggregation.

Surface charge

Aluminum ions on the surface of AH nanoparticles are coordinated with hydroxyl groups, which can accept or provide protons, depending on the pH of the dispersion medium. Thus, AH has a pH-dependent surface charge. It has a point zero charge (PZC) of 11.4 and is positively charged at neutral pH. For AP, a portion of the surface hydroxyl group is replaced by phosphate due to the higher affinity of aluminum for phosphate. Commercial AP adjuvants have a P:Al ratio of 1.1-1.15:1 and a PZC of about 5, which gives them a negative surface charge at neutral pH. Larger ratios of surface hydroxyl groups result in higher PZC.

Surface area

Based on water adsorption measured using gravimetric FTIR spectroscopy, the primary nanoparticles comprising the aluminum aggregates provide a very large surface area for the adjuvant, with the surface area of the AH adjuvant estimated to be 514 m2/g. The surface area can also be determined after nitrogen adsorption using the Brunauer-Emmett-Teller (BET) theory. Although these methods cannot be used for AP adjuvants, the ultrastructure of AP consisting of 50 nm nanoparticles suggests that AP also has a very large surface area.

Adsorption

The large surface area of aluminum adjuvants allows for high adsorption capacity to antigens, which can be used as a key tool for adjuvant characterization. Importantly, adsorption of antigen affects the quality and extent of the immune response and may enhance or reduce antigen stability. It should be noted that the dose of antigen in vaccine formulations is usually low and often well below the full adsorption capacity. The adsorption capacity is affected by the type of antigen, the buffer (pH, ionic strength, composition) and other excipients, including the presence of stabilizers or surfactants. The main mechanisms of adsorption are ligand exchange between phosphates on the antigen and surface hydroxyl groups on the adjuvant, as well as electrostatic and hydrophobic interactions.

Elemental composition

The presence of impurities in aluminum adjuvants can be determined using inductively coupled plasma mass spectrometry (ICP-MS). Differences in the type and amount of metal ions have been reported between AH adjuvants obtained from different manufacturers and from different batches, possibly due to differences in the sources of aluminum salts, chemicals, and water used in the production process. Some contaminants, such as copper, may affect the stability of adsorbed antigens.

The Mechanism and Analytical Characterization of the Binding of Aluminum Adjuvants to Antigens

Antigens can usually be adsorbed to aluminum adjuvants by hydrogen bonding, van der Waals forces, hydrophobic interactions, electrostatic attraction, and ligand exchange, with electrostatic attraction being the most common. Adsorption usually depends on electrostatic attraction when the adjuvant and antigen have opposite charges, respectively. Under neutral conditions, AP can adsorb positively charged antigen.

Ligand exchange is the strongest adsorption between antigen and adjuvant and may occur even if the adjuvant and antigen have opposite charges. When surfactants are present in vaccine formulations, hydrophobic interaction between antigen and adjuvant increases and electrostatic adsorption between antigen and adjuvant decreases. These effects are often necessary to induce protective immune responses against recombinant subunit antigens and protein toxins, and require the screening of suitable adjuvants based on the properties of the antigen.

Quality Control of Aluminum-adjuvanted Vaccines

First, the nature of the surface charge of aluminum-adsorbed antigens needs to be understood in order to predict the physical properties of vaccine suspensions;

Second, determining the rate and strength of adsorption of aluminum adsorbed antigens can help us to understand the ability of aluminum adjuvants to adsorb antigens, as well as their adsorption capacity under different conditions (e.g., different pH or ionic strengths) and long-term storage;

Determination of the dissociation kinetics of aluminum-adsorbed antigens can help us to understand the rate at which antigens dissociate from aluminum adjuvants as well as the stability of the dissociation under different conditions (e.g., different temperatures or times);

Finally, the immunogenicity of aluminum-adsorbed antigens needs to be assessed, which is usually determined by testing in animal models.

Deposit Effect

In most cases, the antigens used in vaccine products are weakly immunogenic and can be enhanced by combining them with adjuvants. After adsorption, the antigen accumulates on the surface and inside the adjuvant particles, helping the antigen to maintain its physical and chemical properties. Numerous studies have shown that antigens can bind to adjuvants and be released slowly at the injection site, continuously stimulating the immune system and enhancing the immune response. This is known as the deposit effect.

The reservoir effect at the vaccination site has long been recognized as one of the main mechanisms of action of immune adjuvants. The deposit effect is influenced by the physical properties (surface area, charge and morphological structure) of the aluminum adjuvant. For example, a larger surface area enhances antigen adsorption, promotes antigen storage, and facilitates antigen presentation to antigen-presenting cells (APCs). Antigens bound to aluminum adjuvants are not only favored for presentation to antigen-presenting cells, but are also slowly released into the tissues as the aluminum adjuvant breaks down, resulting in a delayed depletion of antigens and a prolonged stimulation of the immune system. In addition, the prolonged interaction interval between antigen and presenting cells also enhances the induced immune response.

Recruitment of Immune Cells

When injected intramuscularly, the aluminum adjuvant stimulates the body’s innate immunity. The major players in innate immunity include dendritic cells, macrophages, monocytes, neutrophils, eosinophils, basophils, mast cells, natural killer cells, interferon, and complement proteins.

Both AH and AP can activate immune system-associated pathways in monocytes, with a more pronounced immune response to AH in vitro compared to AP. In vitro and in vivo experiments have demonstrated that AH and AP can recruit different kinds of cells and trigger different immune responses after injection. AH and AP adjuvant antigens were injected into mouse muscle. Proteomic analysis of the injection sites showed that 67% of the epiregulin near the injection sites overlapped after injection of the two aluminum adjuvants, suggesting that the two adjuvants have some similarity in stimulating immune responses.

Enhanced Antigen Uptake by APC

A key step in the induction of the immune response is the uptake of antigen by the APC. Both antigens adsorbed by aluminum adjuvant and free antigens in the intercellular matrix can be taken up by APCs, but the former form particles that are more readily taken up by APCs. Adsorption between the antigen and the adjuvant keeps the antigen in high concentration at the injection site and releases it slowly, thus prolonging the uptake of the antigen by the APC and the effect of the antigen on the immune system.

AH was found to be superior to AP in assisting antigen presentation and processing. Antigen presentation and processing were down-regulated after 24 hours of AP incubation, at which time the AH incubation had not been down-regulated. However, injection of AP-containing antigen recruited monocytes/macrophages, which were strongly up-regulated after 48 h of incubation and only AP was present. This suggests that there are some biological differences between AH and AP and that the choice of these adjuvants should be carefully considered in vaccine formulations.

Activation of the Pre-signaling Pathway for NLRP3 Inflammation

Aluminum adjuvants can recruit leukocytes, promote dendritic cell differentiation, and accelerate local tissue inflammation independent of Toll-like receptors. Studies have shown that aluminum adjuvants can target nucleotide-binding oligomeric structural domains (NODs) such as NLRP3, which is a member of the inflammatory vesicle that recognizes danger signals delivered to cells. The primary role of macrophages is to phagocytose and process antigens. Aluminum adjuvants can induce and activate endogenous immune responses via NLRP3, which promotes the secretion of high levels of pro-inflammatory cytokines, such as IL-1β and IL-18, by macrophages.

Both AH and AP can promote IL-1β secretion by stimulating NLRP3 inflammasome, but AH can stimulate THP-1 cells to produce higher levels of IL-1β than AP, which may be related to the differences between these two aluminum adjuvants in terms of their structures, densities, surface charges, and charge densities.

Other Mechanisms of Immunostimulation

Research has found that AH can activate complement and induce granuloma formation and macrophage activation. In addition, dendritic cells can recruit aluminum-adsorbed antigen-antibody complexes, and complement factors can modulate receptors on B cells to form aluminum-adsorbed antigen-antibody complexes, thereby promoting immune responses. Thus, aluminum adjuvants can activate complement and further enhance the immune response via B cells and dendritic cells.

Major Factors Affecting the Immunogenicity of Aluminum Vaccines

Adsorption Ability

The strength of antigen adsorption is a major factor influencing the immune response. The adsorption of AP can be evaluated using two parameters: the maximum adsorption of a monolayer, characterized by the adsorption capacity; and the strength of adsorption, characterized by the adsorption coefficient. Typically, the immunogenicity of an antigen increases after adsorption onto an aluminum adjuvant, but the stability of the antigen decreases with increasing adsorption time. In addition, proteins adsorbed on the surface of solid particles are prone to defold and lose their secondary and tertiary structures, thereby exposing more hydrophobic groups and further enhancing their ability to bind to aluminum adjuvants. Therefore, the adsorption strength of aluminum adjuvants has an important impact on the immunogenicity, safety and stability of antigen-aluminum complexes.

P/Al

P/Al is the molar ratio of phosphorus to aluminum in aluminum adjuvants. It was shown that the strength of ligand-exchange adsorption could be altered by pretreating AH with phosphate to reduce the number of hydroxyl groups, and that immunoreactivity could be optimized in this way. For positively charged proteins, the amorphous nature of these compounds establishes a larger surface area and higher adsorption capacity.

PH and Ionic Strength

The pH and ionic strength of the solution can significantly alter the settling and suspension characteristics of mixtures for adjuvants and adsorption products, which can switch between flocculated and non-flocculated states. These particles exist as loose aggregates and have a higher deposition rate when they are clustered together. Deposits formed in this manner are looser, usually have a scaffold-like structure, and are easily resuspended. To date, there is a lack of clear understanding of the relationship between the suspension behavior of vaccine mixtures containing aluminum adjuvants and their immunogenicity. However, it has been demonstrated that sedimentation behavior severely affects the dispersion state of the mixture, which further affects the safety and stability of the vaccine.

Granularity

The two main factors controlling the performance of adjuvant suspensions are particle size and the charge of the dispersed particles. It is well known that particle size affects the settling rate of suspensions and that the particle size of an adjuvant is a determining factor in the properties of the adjuvant, e.g., the available adsorption surface area of an antigen. In turn, this affects the conformation of the antigen and may provide functional epitopes to the immune system of immunized individuals.

Antigen Type and Applied Dose

There are several binding mechanisms between antigens and APs, two of which, electrostatic attraction and ligand exchange, largely determine the adsorption and elution properties of vaccines. Thus, aluminum-adjuvanted vaccine efficacy also depends on the nature of the antigens in the vaccine and in the formulation. Antigenic properties that should be characterized in preformulation studies include PZC, the presence of accessible phosphate groups or phosphate-generating groups, and the effect of pH of the solution on chemical and conformational stability.

Next Generation of Aluminum Adjuvants

Aluminum adjuvants have contributed significantly to the success of vaccination in controlling infectious diseases. They are effective in enhancing the immune response to pathogens. Their protection against pathogens is dependent on an antibody-mediated immune response. Besides that, they are inexpensive and have a good safety profile. However, there are some limitations to the use of aluminum adjuvants in vaccines. For example, aluminum adjuvants are unable to support a robust cell-mediated immune response, which is necessary to induce protection against certain pathogens (e.g., Mycobacterium tuberculosis). Therefore, the next generation of aluminum adjuvants will primarily strive to convert the Th2 response into a more Th1 response. One approach is represented by nano-aluminum, which has a smaller particle size than conventional AH, as well as a change in the shape and crystallinity of the material. Studies on nano-aluminum have shown stronger and longer-lasting antibody responses, increased uptake of antigen by APCs, and a shift from a Th2 response to a Th1/Th17 response. Another approach is to combine aluminum adjuvants with other immunostimulants, especially TLR agonists.

The first combination of a TLR agonist and an aluminum adjuvant in an approved vaccine for human use was AS04, which consists of 3-O-deacetyl-4′-monophosphoryl lipid a (MPL) adsorbed on aluminum, and which is used in two GSK-developed vaccines, Cervarix and Fendrix, for the prevention of human papilloma virus (HPV) and hepatitis B virus (HBV), respectively. MPL is a detoxified version of lipopolysaccharide (LPS), an agonist of TLR4. Preclinical studies have shown that AS04 greatly enhanced antibody production and induced high levels of memory cells. Fendrix demonstrated these characteristics in humans, with the HBV vaccine containing the AS04 adjuvant exhibiting higher seroprotection rate and longer antibody responses than the aluminum salt alone.

The second example is AS37, a small molecule immunopotentiator (SMIP), an agonist of TLR7, adsorbed on aluminum hydroxide. Studies have shown that AS37 can be used in preclinical models with many different types of vaccine candidates. In a range of animal models, including primates, it converts the immune response to Th1 type, with an overall increase in the immunogenicity of the vaccine.

Finally, the aluminum combination adjuvant included the addition of AH with cytosine/guanosine oligodeoxyribonucleotide (CpG-ODN), CpG-ODN which is a TLR9 agonist and immunopotentiator approved for use in humans. Negatively charged CpG ODNs are easily adsorbed onto AH. Combined adjuvants of CpG ODNs plus AH have been applied to different antigens, including COVID-19 RBD subunits and hepatitis b surface antigen (HbsAg), and the results demonstrated that the aluminum-adjuvanted vaccine combined with CpG induced a stronger and more balanced immune response of Th1/Th2 cells and a higher antibody response.

Summary

Aluminum adjuvants have been used in billions of doses of vaccines over the last 100 years, primarily in children and adolescents. Despite their limitations, such as their relative inability to induce robust cell-mediated (Th1) immune responses and susceptibility to freezing, their documented safety and tolerability, as well as their low cost, continue to make this class of adjuvants a very attractive component of vaccines. Currently, aluminum adjuvants remain the gold standard for evaluating new and exploratory adjuvants. Recent advances in the biophysical properties of aluminum-adjuvanted vaccines have facilitated the development of new vaccine formulations and enabled quality control during production. Taking advantage of the high adsorption capacity of aluminum adjuvants, they will increasingly be used as a platform for the development of novel combinatorial adjuvants capable of driving the requisite immune response to specific pathogens. These advances will ensure that aluminum adjuvants remain the backbone of vaccine formulations for the foreseeable future.