Open Access

The mechanisms of action of vaccines containing aluminum adjuvants: an in vitro vs in vivo paradigm


Received: 15 December 2014

Accepted: 8 April 2015

Published: 16 April 2015


Adjuvants such as the aluminum compounds (alum) have been dominantly used in many vaccines due to their immunopotentiation and safety records since 1920s. However, how these mineral agents influence the immune response to vaccination remains elusive. Many hypotheses exist as to the mode of action of these adjuvants, such as depot formation, antigen (Ag) targeting, and the induction of inflammation. These hypotheses are based on many in vitro and few in vivo studies. Understanding how cells interact with adjuvants in vivo will be crucial to fully understanding the mechanisms of action of these adjuvants. Interestingly, how alum influences the target cell at both the cellular and molecular level, and the consequent innate and adaptive responses, will be critical in the rational design of effective vaccines against many diseases. Thus, in this review, mechanisms of action of alum have been discussed based on available in vitro vs in vivo evidences to date.


Alum internalization Depot Antigen targeting Inflammasome Innate and adaptive

1. Vaccines

Since the age of Edward Jenner, vaccines have revolutionized public health worldwide, successfully saving the lives of millions of people from infectious diseases such as diphtheria, Haemophilus influenzae type b (Hib) infection, hepatitis B viral infection, tetanus, measles, mumps, neonatal tetanus, pertussis, pneumococcal infection, rubella, and serogroup C meningococcal infection (Rappuoli et al. 2002; WHO 2010b). It has been about 30 years since the World Health Organization (WHO) announced the complete control and eradication of smallpox, achieved through the widespread application of the smallpox vaccine (Bonanni and Santos 2011). With increasing vaccine coverage, the eradication of polio is also nearly complete (WHO 2010a, b). This can be explained by the 99% reduction in the number of polio cases since 1988, leaving only Nigeria, Pakistan, and Afghanistan as polio-endemic countries (WHO 2014) (, Accessed on 4 February, 2015). Therefore, vaccine discovery has been one of the greatest achievements and one of the most economic and safe interventions of biomedical science.

While vaccines are one of the most successful scientific breakthroughs, the underlying immunology requires further research. The success of a vaccine depends on the quality, magnitude, and duration of the generated adaptive immune response following vaccination. To initiate an adaptive immune response, a number of signals are required by naïve T cells. Among these signals, signal 1 is the vaccine-derived, peptide antigen (Ag) bound to major histocompatibility (MHC) class II and class I displayed on the surface of antigen presenting cells (APCs) (Mueller et al. 1989; Watts 1997; Nelson et al. 1997). Signal 2 is also known as ‘costimulation’ and importantly, together with signal 1, induces immune response. Signal 2 involves cross-linking of CD28 and other receptors on the T cell by costimulatory molecules such as B7-1 (CD80), B7-2 (CD86), and other ligands expressed by the APC. Signal 3 is provided by cytokines and is delivered from the APC to the T cell that determines its differentiation into an effector cell. Both Signal 2 and signal 3 are provided to T cells by activated and matured APCs like dendritic cells (DCs). Mature DCs are able to induce T cell clonal expansion and prime immune responses (Reis e Sousa and Germain 1995; Reis e Sousa 2006) and are thus central to the understanding of vaccines.

DCs undergo maturation processes when they receive specific cues from their environment, such as exposure to toll-like receptor (TLR) ligands, necrosis, inflammatory soluble factors (cytokines), T cell ligands (such as CD40 ligands), and disruption of homotypic contacts between immature DCs (Reis e Sousa 2006; Trombetta and Mellman 2005; Sauter et al. 2000). DC maturation involves changes in both location and phenotype of DC, turning it from a cell specialized in surveillance into a potent activator of naïve T cell. DC maturation is characterized by the appearance of dendritic processes, the increased expression of MHCII molecules, costimulatory molecules, and chemokine receptor 7 (CCR7) (Yanagihara et al. 1998; Sallusto et al. 1999; Huang et al. 2000), and the production of cytokines. In this context, the MHCII molecules present Ag, costimulatory molecules contribute to activate the T cells, the CCR7 chemokine receptor mediates migration of the cells to the draining lymph node (DLN), and cytokines are involved in a variety of functions, e.g. cellular trafficking to vaccine-injected sites and DLNs, T cell activation, and T cell polarization (Figure 1).
Figure 1

Current understanding of immunology of vaccines containing alum adjuvants in vivo. Alum immunization leads to recruitment of neutrophil, natural killer cell, macrophage, eosinophil, and immature DC at the injection site. Immature DCs take up soluble Ag released from alum or particulate Ags mixed in alum in the subcutaneous areas and migrate towards draining lymph node (DLN). Soluble Ag can reach to DLN without help of DCs. In T cell area (paracortex), soluble Ags leaked out of conduits are taken up by resident DCs. DCs present Ag to the naïve T cells or transfer Ag to the resident DCs that present Ag to those T cells. In addition, B cells are capable of binding to this Ag with their surface immunoglobulins. B cells undergo activation, produce effector B cells (eB cells), and rapidly differentiate in plasma cells (PCs). Plasma cells produce low-affinity antibodies (LAb). B cells also migrate to B cell follicle. On the other hand, as a result of CD8 and CD4 T cell activation, effector CD8 (eCD8) T cells and effector CD4 (eCD4) T cells are produced. The eCD4 polarizes into T helper (Th) 1, 2, 17 or T follicular helper (Tfh) cells though alum induces development of mostly Th2 and Tfh cells. Tfh or Th2 may reach to the border of B cell follicle to activate B cells that produce eB cells and then PCs. The PCs consequently produce and secrete high-affinity antibody (HAb). Few alum-fed DCs and eCD4 may travel to efferent lymph vessels to reach into the distant LN in vivo. Ag: antigen, HEV: high endothelial venule, PVS: perivenular space (see text for explanation).

In the LN, DCs located around the T cell entry site express Epstein-Barr virus induced receptor ligand chemokine (ELC) or chemokine ligand 9 (CCL19) and secondary lymphoid tissue chemokine (SLC) or CCL21 that enhance DC-T cell attraction. In the LN paracortex, B cells bind Ags with their surface immunoglobulins and become activated. As a result, they rapidly differentiate in plasma cells and produce low-affinity antibody (Ab). DC-T cell interaction in the paracortex leads to Ag-specific CD4+ T cell activation, expansion, and polarization into T helper (Th) 1, 2, and 17, and T follicular helper (Tfh) cells. Th2 and Tfh cells initiate germinal center reaction where these cells and follicular DCs (FDCs) provide strong activating signals to B cells. The activated B cells produce plasma cells that secrete Ag-specific high-affinity Abs (Figure 1).

The advancing field of biomedical science has increased the practical and theoretical understanding of pathogen biology, molecular biology, biochemistry, immunology, and biotechnology. Therefore, vaccine development has progressed from trial and error- based empirical vaccine studies towards more rational and reductionist approaches (Van Regenmortel 2004). Nevertheless, these approaches have had limited success in developing effective vaccines against emerging diseases like Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS) and re-emerging diseases like tuberculosis (TB) and malaria. This may be due to a number of factors, such as rapid clearance from the body, poor recognition by the immune system, and failure to adequately stimulate appropriate immune cells (Edelman 2002; Edelman and Tacket 1990). Therefore, while appropriate and safe Ag discovery has been the main target of vaccinology, in recent years the development of adjuvants (a term derived from the Latin ‘to help’) that increase the immunogenicity of the Ags is also gaining equal importance. Adjuvants are used in vaccines to reduce the dose of vaccines, to induce the particular protective response (CD4 vs CD8 and Th1 vs Th2) and to enhance a broad immune response, suggesting adjuvants are essential to vaccine design and development. Therefore, designing a potent adjuvant is key to vaccine development.

2. Alum

Alum has dominated all the adjuvants currently approved and licensed in the world despite a great deal of interest in developing novel adjuvants. This adjuvant was first used by Alexander T. Glenny who prepared potassium aluminum sulphate or alum {KAl(SO4)2}-adjuvanted vaccines by co-precipitation with Diphtheria Toxoid (DT) dissolved in carbonate buffer (Glenny and Sudmersen 1921; Glenny et al. 1926, 1931). Due to the problems in manufacturing reproducibility (Marrack et al. 2009), the technique of alum precipitation has been substituted by the adsorption of vaccines onto preformed aluminum hydroxide (AH) or alhydrogel (chemically crystalline aluminum oxyhydroxide) and aluminum phosphate (AP) or adju-phos gels (chemically amorphous aluminum hydroxyphosphate) (Baylor et al. 2002; Shirodkar et al. 1990). Aluminum compounds such as aluminum chloride, aluminum silicate, algammulin (gamma inulin plus AH), cesium alum (CA), Imject alum (IA) (AH plus magnesium hydroxide) have sometimes been substituted for AH and AP adjuvants in experimental studies (Lindblad 2004a, b; Lindblad and Schonberg 2010; Flach et al. 2011; Kool et al. 2008a, b; Marichal et al. 2011). Although these compounds are often generically referred to as ‘alum’, alum is in fact a distinct chemical composition (Hem et al. 2007). Importantly, few of these compounds have been used to address their comparative roles in the induction of immune responses in vivo (Cain et al. 2013).

While these adjuvants have been in continuous use in human vaccines for about 90 years, their mechanisms of action have remained elusive. A number of alum-induced effects may contribute to the improved immunogenicity of vaccines, however, in many cases these effects are only partially described or lack clear causal association with adjuvant function.

3. Mechanisms of action: in vitro vs in vivo paradigm

Adjuvant biologists have hypothesized that adjuvants work by ‘depot formation’, ‘Ag targeting’, and ‘inflammation’. These hypotheses are based on evidence from in vitro studies, with few in vivo validation studies. This is because the study of vaccine adjuvants remains largely empirical, despite our updated knowledge and understanding of immunology. Reductionist approaches, such as analyzing adjuvant effects on key immune system cells in vitro will help define the features of adjuvants that are critical for their function, and greatly enhance our understanding of the mechanisms involved. However, adjuvants ultimately have complex interactions with their environment at the interface of immunology, physiology, and anatomy in vivo. In vitro a single cell may exhibit different behaviors under different experimental conditions, therefore understanding how cells behave in vivo and what interactions they have with their environment will be crucial to fully understanding the mode of action of adjuvants. A number of reviews have been published relating to the mechanisms of action of alum (Gupta 1998; Gupta et al. 1995; Lindblad 2004a, b; Brewer 2006; De Gregorio et al. 2008; Marrack et al. 2009; Reed et al. 2009; Mbow et al. 2010; Hogenesch 2012; Awate et al. 2013; Reed et al. 2013; Kool et al. 2012), however, in vitro and in vivo data has never been fully compared and evaluated. Thus, this review compares in vitro and in vivo studies examining the mode of action of adjuvants.

3.1 Challenging the theory of ‘depot formation’

The depot hypothesis is the earliest proposed mechanism for adjuvant action, hypothesized by Glenny, Buttle and Stevens in 1931 after working on DT-precipitated in alum (Glenny et al. 1931). They excised a portion of skin containing the site of injection from guinea pigs 3 days after administration of alum-precipitated DT or soluble DT. They then homogenized the skin and injected the emulsion into naïve guinea-pigs. The alum-precipitated DT-recipients were successfully immunized whereas, the DT-recipients (controls) groups were not, as measured by anti-toxin titers. This experiment led them to generate a hypothesis that the slow elimination of alum-precipitated Ags over a long period of time from single injection site may enhance both primary and secondary stimulation resulting in the associated enhanced Ab titers (Glenny et al. 1931). Similarly, Harrison proved this hypothesis by transferring the alum nodules from one guinea pig into another guinea pig (Harrison 1935). White and colleagues suggested that depot causes persisting inflammation that stimulates immune cells within the regional LNs, and induction of local granuloma that recruit Ab-producing plasma cells (White et al. 1955). Consequent studies suggested that Ag was detected for 2 – 3 weeks in alumina gel-triggered granulomas (Osebold 1982). It was realized that strong adsorption to an adjuvant may ensure a high localized concentration of Ag for a period of time (Harrison 1935; White et al. 1955), that may be sufficient to allow Ag uptake and activation of APCs like DCs (HogenEsch 2002).

Hypothetically, depot theory might be explained on the basis of alum’s role in a strong binding strength with Ag. This results in retention of Ag at the injection site, and in slow release of Ag in vivo. Aluminum adjuvants have been shown to adsorb various proteins via either electrostatic interaction, ligand exchange, or via hydrophilic-hydrophobic interaction and each binding interaction depends on the nature of the Ag, pH, ionic strength, and presence of surfactants (Lindblad 2004a; Hem and HogenEsch 2007). As a result of adsorption, the soluble Ags change into particulate form (Lindblad 2004a, b). Therefore, compared with soluble Ags, particulates efficiently interact with APCs resulting in enhanced phagocytosis. As well as going through the internalization process, the soluble Ags may be trapped inside the irregular aggregates of large sized (1 – 10 μm) AH particles formed by fibrous primary particles (Powell et al. 1995). These primary particles in the aggregates are loosely associated and are readily degraded (Morefield et al. 2005) with the subsequent release of Ags in tissue culture media in vitro (Heimlich et al. 1999). The release is observed in similar manner in vivo because aluminum-containing adjuvants are rapidly chelated and solubilized by alpha-hydroxycarboxylic acids such as citric acid, lactic acid, and malic acid in the interstitial fluid, absorbed into the tissues, and finally eliminated in the urine (Hem 2002; Seeber et al. 1991). As release of Ags or elution of alum in media (in vitro) or interstitial fluid (in vivo) is a time-dependent phenomenon, initial Ag uptake by APCs may include adsorbed and trapped Ags in alum aggregates via phagocytosis. This is then followed by macropinocytosis of soluble protein released from alum aggregates (Romero Mendez et al. 2007). The elution of Ags from the adjuvant surface is crucial in vivo because residential LN APCs like DCs pinocytose the eluted Ags from the injection sites (Figure 1). Though macropinocytosis and presentation of Ag by residential LN DC is not sufficient to induce an immune response in vivo (Itano et al. 2003), a second wave of presentation of Ag by DCs originating from the injection site interact with naïve T cells in the DLN inducing an effective immune response. Thus, retention of Ag for phagocytosis by injection site DCs and sustained release of Ag for macropinocytosis by LN DCs are critical for the efficient Ag presentation in this situation (Lu and Hogenesch 2013). However, the retention of Ag and its slow release have been shown dispensable to alum adjuvanticity in the context of an enhanced Ab response in vivo (Romero Mendez et al. 2007; Noe et al. 2010; Flach et al. 2011). In these contexts, the adsorption and elution rates have been quantified using Ovalbumin (OVA) mixed in AH or Phosphate-treated AH (PTAH) (adsorption: 91%, complete elution in interstitial fluid: 4 hours), Dephosphorylated alpha casein (DPAC) mixed in AH (Adsorption: 100%; complete elution in interstitial fluid: <6 hour) or Phosphate-treated AH (PTAH-B) (Adsorption: 40%, complete elution in interstitial fluid: <1 hour) (Iyer et al. 2003). Considering these in vitro data, the authors injected these vaccine preparations subcutaneously in mice in which they observed an enhanced IgG titer in vivo independent of adsorption and elution characteristics of Ags (Iyer et al. 2003). It, therefore, suggests that strong adsorption might be detrimental to physical and chemical properties of vaccines (Estey et al. 2009; Vessely et al. 2009). Nevertheless a slight interaction between alum and Ag is necessary, for example, a favorable immune response has been observed after an interaction between alum and anthrax Ag (Watkinson et al. 2013) or hepatitis B Ag (Egan et al. 2009). The necessity for interaction between alum and Ag has been also proved by other experiments in which no immune response was generated following separate Ag/alum injections (Chang et al. 2001) though quantitatively higher Ab titer was obtained in one similar experiment (Flebbe and Braley-Mullen 1986). Therefore, the suggestion by the WHO recommending >80% of DT and tetanus toxoid (TT) Ags should be adsorbed (WHO 1977) and by the United States minimum requirements recommending >75% DT and TT Ags should be adsorbed (Anonymous 1956) onto aluminum adjuvants may not be right for other vaccine candidates. These are, however, in vitro recommendations and therefore have no bearing on what happens in vivo.

The Ag retention hypothesis, was not substantiated by subcutaneous (s.c.) immunization of mice with radioactively (14C)-labeled TT adsorbed to AP adjuvant (Gupta et al. 1996). Subsequently, a study was conducted to show the relation between Ag retention at the site of injection and Ab titers in rat sera 5 weeks after primary immunization and 2 weeks after boost (Noe et al. 2010). In this study, rats were injected with 111In-labelled alpha casein (IDCAS) Ag adsorbed to AH or IDCAS adsorbed to AP or non-adsorbed IDCAS Ag formulated in phosphate-treated AP (PTAP) or IDCAS solution by s.c. route. They observed Ag retention in the following order IDCAS + AH > IDCAS + AP > PTAP = IDCAS and the Ab titers in the order PTAP = IDCAS + AP > IDCAS + AH> > IDCAS suggesting an inverse correlation between retention and Ab titer (Noe et al. 2010). This study and that of de Veer and colleagues summarized that alum reduces the amount of soluble Ag entering afferent vessels, although their adjuvanticity was not found to be correlated with slow Ag release (de Veer et al. 2010; Noe et al. 2010). Thus, in vivo dispensability of strong adsorption, retention, and slow release of Ag by alum in Ab response indicates that other mechanisms may exist in alum adjuvanticity.

While the depot effect was considered as the predominant effect of alum, resulting in the sustained release of Ags over time (Cox and Coulter 1997), this role has been questioned since the 1950s (Holt 1950; Gupta RK 1996; Noe et al. 2010; de Veer et al. 2010; Hutchison et al. 2011). The depot effect was first challenged by Holt, who observed no change in Ab titers in response to DT following excision of the alum-precipitated injection site after 7 or more days (Holt 1950).

Recently, by using the E alpha green fluorescence protein (EαGFP)/YAe system, Hutchison and colleagues failed to observe activation of Ag-specific T cells transferred into recipient mice later than 5 days post-immunization with OVA + alum (Hutchison et al. 2011). When this Ag is internalized by DCs, EαGFP is degraded and the Eα peptide is presented by I-Ab MHC class II molecules on the cell surface. These p:MHCII complexes can be detected by staining the cells with YAeAb because this Ab can efficiently bind the complex of Eα(52–68) and I-AbMHCII (Rudensky et al. 1992; Rudensky et al. 1991; Rush and Brewer 2010; Ghimire et al. 2012). Therefore, the YAeAb recognizes the same epitope-MHC complex as the T cell receptor (Figure 2). This system allows assessment of Ag uptake/degradation and, in combination with the Y-AeAb Ag presentation in situ (Rush and Brewer 2010; Ghimire et al. 2012) (Figure 2). Notably, Hutchison’s studies further demonstrated a lack of Ag persistence and presentation in APC populations in the DLN (Hutchison et al. 2011). Remarkably, B cells were the first APCs to present Eα:MHCII complexes within 6–12 hours after immunization, then, cDCs presented these complexes within 12–24 hours following alum + EαGFP immunization, and pDCs presented Ags within 48–72 hours after alum administration in DLNs in vivo (Hutchison et al. 2011). No difference was observed in Ag uptake and presentation by B cells, cDCs, and pDCs after injection site ablation 2 hours following EαGFP administration, suggesting no role for the depot effect in alum adjuvanticity (Hutchison et al. 2011).
Figure 2

EαGFP:YAe system can be used to assess antigen (Ag) uptake, processing, and presentation by DCs in the presence of alum in vitro and in vivo. (A): When EαGFP is taken up by DCs, it is processed and presented in the context of Eα(52–68):MHCII complexes. (B): The Eα(52–68):MHCII complexes can be bound by YAe antibody (Ab). (C): T cell receptor (TCR) of TEa mice sees the same complexes what YAe sees. Alum enhances Ag uptake, reduces degradation, and eventually increases presentation by DCs in vitro. This adjuvant also enhances the expression of CD86, CD80, and CD40 molecules by DCs in vitro. Arrow shows Ag processing path.

3.2 The alternative theory of ‘Ag targeting’

Landsteiner explained the mechanisms of action of alum in the context of slow absorption and delayed removal of Ag (White et al. 1955), suggesting that the ‘particulate’ nature of these adjuvants would favor phagocytosis by macrophages and subsequent activation. Further proof of Ag targeting by resident APCs was provided by the uptake of both Ags and AP by injection site macrophages following 7 days post-immunization of rabbits (White et al. 1955). This was a seminal study leading to the hypothesis of a mechanism of ‘Ag targeting’ for alum adjuvants. Ag targeting was defined in 3 distinct phases; the first phase involves the accumulation of cells at the injection sites, the second phase comprises of the induction of signal 1 and maturation signals by APCs, and the third phase includes the migration of Ag-loaded APCs into DLN.

3.2.1 Alum induces chemokine- and cytokine- triggered APC recruitment at the injection site

Immunization with alum induces recruitment of various subsets of leukocytes such as neutrophils, eosinophils, macrophages, monocytes, and DCs at the injection site in a time-dependent manner (Mosca et al. 2008; Kool et al. 2008a; Lu and Hogenesch 2013) (Figure 1). This recruitment is associated with an enhanced expression of mRNAs for chemokines, cytokines, and cell adhesion molecules (Mosca et al. 2008; Kool et al. 2008a) and their secretions (Kool et al. 2008a; McKee et al. 2009; Korsholm et al. 2010), and enhanced production of complement cascade (Ramanathan et al. 1979). Various data on spatial and temporal recruitment of inflammatory cells has been acquired; this effect on cellular recruitment may be associated with the formulation of alum, Ag, and route of delivery. It has been recorded that following alum or alum + TT immunization, lymphoid tissue containing macrophages, epithelioid cells, and collagenous fibers is produced from 2 to 20 weeks (Goto and Akama 1982). Notably, neutrophils have been shown to migrate to the injection site at 6 hours (Lu and Hogenesch 2013), (Kool et al. 2008a), 24 hours (Calabro et al. 2011), and until 72 hours (Goto and Akama 1982) postimmunization by alum. Neutrophil trafficking is associated with increased expression of macrophage inflammatory protein 2 (MIP2) or chemokine (C-X-C) motif ligand 2 (CXCL2) and keratinocyte chemoattractant (KC) or CXCL1 (Kool et al. 2008a) that is enhanced via proteolytic cleavage of the extracellular matrix (Sadik et al. 2011). Depletion of neutrophils increased activation of Ag-specific T cell and the magnitude of the Ab response following s.c. immunization with AH + lysozyme (Yang et al. 2010), thus suggesting that neutrophils compete with DCs and macrophages for Ag and thus interfere with Ag presentation (Yang et al. 2010). In contrast, depletion of neutrophils with an anti-Ly6G Ab did not affect the magnitude and isotype of the Ab response to intramuscular (i.m.) immunization with AH + OVA (Lu and Hogenesch 2013). These different results are probably related to vaccine formulations and different route of immunization.

Macrophage trafficking to the injection site has been investigated for many years (White et al. 1955; Lu and Hogenesch 2013) with the peak of infiltration at day 7 (Lu and Hogenesch 2013) post alum immunization. The trafficking may be associated with an increased expression of CCL2 and CCL4 at the injection sites (Lu and Hogenesch 2013). Similar patterns of expression of chemokines by human monocytes have been reported in vitro in response to alum (Seubert et al. 2008) thus validating in vivo data. However, disappearance of macrophages from the alum-injected site has been shown in few studies (Kool et al. 2008a; McKee et al. 2009) and the different data may be associated with different protocol of immunization and adjuvant formulations. Importantly, the role of macrophages in alum adjuvanticity has been queried as systemic depletion of macrophages did not affect Ab response following intraperitoneal (i.p.) alum injection (McKee et al. 2009) and even enhanced the Ab response following s.c. alum immunization (Mitchell et al. 2012).

Similarly, the numbers of eosinophils increase (Calabro et al. 2011) within 24 hours (Kool et al. 2008a; Korsholm et al. 2010; McKee et al. 2008), and constitute about 25% of inflammatory cells when they peak around day 6 (Walls 1977; Lu and Hogenesch 2013) post alum injection. Eosinophil recruitment may be related to interleukin (IL)-5 and histamine release from mast cells, unidentified factors secreted by macrophages (McKee et al. 2009) and the secretion of eosinophil chemotactic protein or eotaxin-1 (CCL11) (Kool et al. 2008a) and CCL24 molecules following secondary immunization (Lu and Hogenesch 2013; McKee et al. 2009). Following immunization, mast cell numbers decreased at the site of injection probably due to mast cell granulation and cell death (McKee et al. 2009). Despite the fact that mast cells are the primary source of IL-5, IL-16, granulocyte-colony stimulating factor (G-CSF), KC, and MIP2 and that mast cells in concert with macrophages induce the secretion of IL-1β, IL-1 receptor antagonist (Ra), IL-6, and eotaxin; cell depletion studies suggest that these cells are not required for alum-mediated responses in vivo (McKee et al. 2009). The role of CCL11 is controversial with its receptor, CCR3, expressed on murine eosinophils, CCL11 has been shown to be dispensable in eosinophil recruitment following alum immunization (McKee et al. 2009). Despite eosinophils being the primary sources of IL-4 and possibly contributing to B cell priming and Ab production, their absence did not influence the quality or magnitude of the Ab responses to alum-adjuvanted vaccines in vivo (McKee et al. 2009).

Monocyte and DC trafficking at the injection site is particularly important as APC interactions are at the interface of the innate and adaptive immune response. The trafficking of monocytes is linked to the various signals derived from neutrophil (Soehnlein and Lindbom 2010). The notion that inflammatory monocytes can differentiate into DCs en route to the DLNs suggests that subsequent Ag-specific immune response occurs following immunization (Geissmann et al. 2003). It has been recorded that DCs are actively recruited to the injection sites from day 1 to day 7 postimmunization (Mosca et al. 2008; Kool et al. 2008a; McKee et al. 2009; Calabro et al. 2011; Lu and Hogenesch 2013). Depletion of DCs nearly completely abolished T cell responses and Ab production (Kool et al. 2008a) indicating DCs are critical for the alum-mediated enhanced immune responses in vivo. Recently, CD8α+ DCs have been considered to prime CD8+ T cells via cross-presentation following alum immunization. In this context, in absence of CD8α+ DCs, other unknown subsets of DCs have been shown in priming CD4+ T cells (MacLeod MK 2011).

3.2.2 Alum enhances uptake and presentation of Ags by APCs

Alum adjuvants are reported to enhance the Ag uptake capacity of macrophages (White et al. 1955) and DCs (Kool et al. 2008a) in vivo and human peripheral blood mononuclear cells (PBMCs) (Mannhalter et al. 1985) and DCs (Morefield et al. 2005; Flach et al. 2011; Ghimire et al. 2012) in vitro. To illustrate alum’s role in enhancing Ag targeting efficiency of DCs in vitro, the EαGFP/YAe system has been used (Figure 2). This system has allowed the impact of AH on the intracellular Ag depot that provides a sustained release of Ag onto MHCII molecules and prolonged Ag presentation (Ghimire et al. 2012) to be assessed (Figure 2). Although the data is not validated by in vivo experiments (Hutchison et al. 2011), other factors such as DC’s potentiality to ingest alum particle and further consequences should be evaluated. In vitro experiment showed phagocytosis of alum particles by macrophages and consequently their phagosomal rupture (Hornung et al. 2008). Similar experiment by Flach and colleagues suggested that alum binds DC plasma membrane lipids in cholesterol- and cellular motility-dependent manner. Subsequently, the lipid sorting occurs resulting in enhanced Ag delivery to the cell without alum internalization, a process called abortive phagocytosis (Flach et al. 2011). In the same study, alum particles were identified inside DCs (Flach et al. 2011) and named them ‘confounding DCs’, indicating that DCs are efficient in phagocytic uptake of particulates. The in vitro experiments have been validated by in vivo observations, for example, alum crystals have been identified within s.c. injection-site granulomas by histochemistry, electron microscopy, X-ray microanalysis, and atomic absorption spectrophotometry (Frost et al. 1985; Miliauskas et al. 1993; Fawcett and Smith 1984). They have been identified inside macrophages (Rimaniol et al. 2004; Morefield et al. 2008), multinucleated giant cells (Morefield et al. 2008), and MHCII+ mononuclear cells, most probably the DC (Lu and Hogenesch 2013), and recently within a T helper 1 cell line (THP-1) (Mold et al. 2014) indicating these APCs actively engulf alum in vivo.

While influences on p:MHCII presentation by APCs in the presence of alum has been described in vitro (Ghimire et al. 2012) and in vivo (Hutchison S 2011), there is little data available on p:MHCI presentation. Understanding the effects on p:MHCI presentation is critically important particularly because alum has been reported to act as a poor cytotoxic T lymphocyte (CTL)-responding adjuvant which would be crucial for immune responses for intracellular pathogens like HIV, Plasmodium, and Mycobacterium tuberculosis. Importantly, one in vitro study of alum excluded class 1 processing and presentation in macrophages (Rimaniol et al. 2004). Few in vivo experiments have shown MHC class I-restricted CD8 T cell responses elicited by alum adjuvants. However, the induction of long-lasting protective CD8+ CTL response in mice primed and boosted with a highly purified recombinant influenza protein vaccine formulated in alum adjuvants has been reported (Dillon et al. 1992). The levels of CTL responses induced were similar to that obtained by an i.p. boost of Ag without alum adjuvant. Thus the vaccine containing alum induced CD8+ T cell response, but the alum itself did not necessarily contribute to the CD8+ T cell response (Dillon et al. 1992). This notion has been further clarified by subsequent experiments (McKee et al. 2009; MacLeod et al. 2011). McKee and colleagues observed both CD4+ and CD8+ T cell activation in the presence of alum (McKee et al. 2009). Ag (Kb/SIINFEKL tetramer)-specific CD8+ T cells and the consequently the development of long-lived memory cells have been uniquely shown in vivo following alum immunization (MacLeod et al. 2011). This study also addressed the generation of type 1 cytokine response and the consequent CTL function and the significant protection from influenza A challenge via alum-primed CD8+ T cells in vivo only in the presence of additional adjuvant called monophosphoryl lipid (MPL) (MacLeod et al. 2011). It further suggested that alum enhanced the generation of long-lived CD8+ T memory cells and MPL enhanced CTL differentiation indicating this type of vaccine formulation would be applicable to enhancing p:MHCII-primed Th1- and p:MHCI-primed-CD8+ T cell responses required for killing intracellular pathogens (MacLeod et al. 2011). It is therefore interesting to note that varying levels of adjuvanticity of alum have been reported in vivo depending on the protein or peptide used, and the addition of cytokines such as IL-12, and other TLR-agonists such as saponin (Newman et al. 1992), CpG and polyriboinosinicpolyribocytidylic acid (Chuai et al. 2013). Importantly, the use of inflammatory cytokines such as IL-12 in alum has been shown to induce Th1 responses (Jankovic et al. 1997) with synergistic enhancement when coadministered with IL-18 (Pollock et al. 2003). Enhanced levels of interferon-gamma (IFN-λ) following immunization with Bordetella pertussis toxin mixed in alum (Toellner et al. 1998) were also reported. Interestingly, vaccination with recombinant hepatitis B serum Ag (HBsAg) adsorbed to AH induced the production of viral-specific CTL induction, Th1 induction, and Ab production (Rahman et al. 2000). Notably, the effects on CD4+ and CD8+ T cell response can be explained on the basis of respective upregulation of cluster of both MHCII genes (H2-Aa, H2-Ea, and H2-Eb1) and MHCI genes (H2-Q, H2-K, H2-T, H2-D) at the injection site by alum after 4 days and 1 – 2 days of post injection (Mosca et al. 2008) respectively. These experiments suggest that alum, as a particulate adjuvant (Cox and Coulter 1997), may have the potential to modulate processing of the protein Ags via the MHCI pathway for the CTL induction via cross presentation (Munks et al. 2010; Carbone and Bevan 1990). This may be possible if exogenous Ags are released into the cytosol from the endosomes by alternative mechanism, for example, by the release of Ags into the cytosol following damage to the phagosome as discussed later. This type of particulate delivery is also important in delivering both Ag and adjuvant into the same cell to enhance immune responses (Pashine et al. 2005; Bramwell and Perrie 2005).

3.2.3 Alum enhances APC activation and maturation

Studies have suggested that aluminum-containing compounds have direct effects on DC activation as measured by expression of MHCII and costimulatory molecules (Figure 2). Ulanova et al. reported IL-4-dependent increased level of MHCII, CD86, CD83, IL-1α, IL-1β, tumor necrosis factor (TNF), IL-4, and IL-6 in human PBMCs which later acquired a DC morphology (Ulanova et al. 2001). Similarly, Rimaniol and colleagues showed that alum can trigger monocyte differentiation into myeloid DCs in an IL-4 dependent pathway (Rimaniol et al. 2004). Alum was shown to induce a dose dependent decrease CD80 expression and increase CD86 and CD40 expression in human PBMC-derived macrophages 48 hours post treatment (Rimaniol et al. 2004). This study also suggested that alum (AlOOH) can directly act on already differentiated macrophages and can change them to mature, specialized Ag presenting macrophage in an IL-4 independent manner (Rimaniol et al. 2004). In contrast to these studies, Sun and Brewer did not observe any significant increase in MHCII or costimulatory molecule expression on murine CD11c positive bone marrow-derived DCs (BMDCs) 24 hours post alum treatment (Sun et al. 2003). Sokolovska and colleagues in 2007 reported an increase in CD86 and CD80 expression on murine BMDCs (Sokolovska et al. 2007), while Seubert and colleagues observed an enhanced expression of CD86, MHCII, CD71, CD83, and CCR7 molecules and decreased expression of CD80 and CD1a molecules on DCs derived from the CD14-positive monocytes (Seubert et al. 2008). An enhanced CCL2, CCL3, CCL4, CXCL8, MHCII, CD86, CD71, CD54 and decreased CD14 molecule expression was also observed accompanied by the increased granularity of the monocytes derived from human PBMCs (Seubert et al. 2008). In this study a time-dependent increase in CD86 and decrease in CD80 molecules was noted on the surface of monocytes derived from human PBMCs. Flach and colleagues did not find any effect of CA (CA at 5 mg/mL concentration) on CD86, CD80, and CD40 expression on BMDCs derived from C57BL/6 mice 24 hours post treatment (Flach et al. 2011). The different patterns of costimulatory molecules induction by alum in these in vitro studies may be due to differences in experimental design, including variation in the dose of alum, duration of stimulation and host strains used. The differences in host strain is particularly important as the expression of maturation markers such as CD86, CD40, and Stat4, an IL-12-inducing gene, are quantitatively higher in spleen-derived DCs of C57BL/6 compared with BALB/c mice (Liu et al. 2002). Further in vivo studies should be conducted to address these issues in alum adjuvanticity.

3.2.4 Alum aids recruitment of Ag loaded APCs to the lymphoid tissue in vivo

It is believed that following alum immunization, inflammatory Ly6C+CD11b+monocytes take up Ag, differentiate into CD11c+MHCIIDCs in a myeloid differentiation primary response gene 88 (MYD88)-dependent manner in vivo (Kool et al. 2008a) validating the in vitro differentiation of PBMCs into DC morphology (Rimaniol et al. 2004; Seubert et al. 2008; Ulanova et al. 2001). These DCs become long-lived after ingesting alum (Hamilton et al. 2000) and slow down alum solubilization (Verdier et al. 2005; Gherardi et al. 2001; Authier et al. 2006). These DCs subsequently gain enhanced inflammatory signals such as IL-1β, TNF-α, and IL-6 that induce their trafficking to DLN (Kool et al. 2008a). Nevertheless, the latter two cytokines have been shown dispensable for alum adjuvanticity (Brewer et al. 1998). Thus, IL-1β may principally play a role in DC mobilization to DLNs by decreasing E-cadherin mRNA expression in DCs resulting in the detachment of cells from neighboring cells and matrix components (Jakob and Udey 1998). In the LNs, DCs can transfer Ag material to a large network of distant LN APCs (Carbone et al. 2004) and can induce activation of innate immune system in distant organs (Wang et al. 2013). That is why alum has been reported to induce the immune responses in distant LN and spleen (Kool et al. 2008a).

Although isotopic 26Al-enriched alum was detected in LNs up to 28 days (Flarend et al. 1997), the kinetics of alum-fed APC migration and the fate of this adjuvant was explained by Khan and colleagues (Khan et al. 2013). Following s.c. or i.m. alum injection, alum-fed APCs were recruited into DLN from 1 hour to 21 days (peaking at 4 days) post alum administration, and into spleen from 1 hour to 90 days (peaking at 21 days) post alum administration in chemokine-dependent manner (Khan et al. 2013). The s.c. route of administration was superior to i.m. administration in recruiting alum-positive cells to the DLNs (Khan et al. 2013). Gr1+ cells are the major subsets that engulf alum particles, but the kinetics of alum-engulfed Gr1+ and Gr1 recruitment to the DLN decreases with time. This phenomenon may be explained by dilution of particles by cell division (Kabashima et al. 2005) or particle transmission to other cells (Angeli et al. 2006).

Following migration to the DLN, alum-loaded DCs migrate to the bloodstream via thoracic duct and ultimately they can migrate distant organs such as spleen (Khan et al. 2013). In the spleen, DCs present Ags to the splenic T cells (Cavanagh et al. 2005). Interestingly, following 6 injections, increased accumulation of Gr1+/CD11b+ cells was noted in the spleens of alum-immunized mice, compared to the spleens of alum adsorbed to protein-immunized mice (HBsAg-anti-HBs), suggesting alum alone triggers effective inflammatory responses in vivo (Wang et al. 2013).

3.2.4 Alum generates a strong default Th2 response

In vitro studies have shown that alum potentially induces the production of IL-1β and IL-18 from DCs and the consequent expansion and differentiation of naïve CD4+ T cells into Th2 cells and promote the production of Abs (Sokolovska et al. 2007). It has been experimentally shown that IL-4 and IL-13 are not necessary for alum to enhance Th2 responses but these cytokines strongly suppress Th1 responses, thus alum’s role in regulating Th2 response may be mediated via Th1 suppression (Brewer et al. 1996; Brewer et al. 1999). Although IL-4 has been shown to direct both CD4+ and CD8+ T cells to produce Th2 cytokines in vitro, only CD4+ T cells produce this cytokine in alum-mediated response in vivo (Serre et al. 2010). Alum’s induction and enhancement of Th2 responses has been validated by in vivo experiments. Alum-adjuvanted Ag contact with CD4+ T cells promotes migration to the B cell follicles and the development of a central memory CD4+ T cells. These CD4+ T cells undergo expansion and migrate from DLN and to distant LNs not exposed to Ag (Luther et al. 2007; Serre et al. 2009). In the distant LNs, these CD4+ T memory cells migrate to B cell follicles and produce IL-4 for the Th2 responses (Luther et al. 2007; Serre et al. 2009). Alum has also been suggested to enhance Ab responses via various molecules such as IL-4 (Grun and Maurer 1989; Jordan et al. 2004; Serre et al. 2011; Cunningham et al. 2004a; Pai et al. 2004; Zhu et al. 2010; Lindblad et al. 1997), IL-1 (Grun and Maurer 1989), IL-25 (Serre et al. 2008), IL-6 (Serre et al. 2008), IL-10 (Lindblad et al. 1997), IL-13 (Serre et al. 2011), GATA-3 molecules (Cunningham et al. 2004b; Pai et al. 2004; Zhu et al. 2010) and CXCR5 chemokines (Serre et al. 2011). These molecules probably trigger induction of Ab-forming cells, germinal center B cell responses through inflammation (Cain et al. 2013). Interestingly, induction of Th2 cytokines in vivo in absence of IL-4 or STAT6 signaling (Cunningham et al. 2002; Cunningham et al. 2004a, b; van Panhuys et al. 2008) suggests that an alternative pathway of in vivo Th2 induction exists in alum adjuvanticity. An enhanced Ab response has been addressed in detail in vivo by Zlatkovic and colleagues (Zlatkovic et al. 2013). They hypothesized that immunization of mice with alum adjuvanted inactivated purified flavivirus tick-borne encephalitis particle leads to the fixation of the flexible protein subunits in a certain configuration because alum present within the LN may mask certain epitopes of incompletely desorbed Ag that results in an enhanced and high-specific and high-avidity Ab response (Zlatkovic et al. 2013).

3.3 The theory of ‘inflammation’

The use of adjuvants in vaccination is usually associated with some degree of injection site inflammation, and this process is considered an essential part of adjuvant function (Qin et al. 2009). This is consistent with the ‘Danger Theory’ of immune activation as proposed by Polly Matzinger in 1994 (Matzinger 1994). According to this theory, initiation of the immune response is not dependent on microbial recognition, but rather on the ability of pathogens or other agents such as adjuvants to cause tissue damage. The danger signals released from damaged tissues then have the capacity to drive inflammation and initiate an adaptive immune response (Matzinger 1994). This provides an important mechanistic theory for alum adjuvants with many reports of inflammatory effects at the injection site and the induction of danger signals from cells following alum interaction. For example, nodule or granuloma formation in humans and animals following alum injection has been reported from the 1930s to present day (Glenny 1931; Harrison 1935; Farago 1940; Holt 1950; White et al. 1955; Munks et al. 2010; Lu and Hogenesch 2013; Vogelbruch et al. 2000; Bordet et al. 2001; Chong et al. 2006; Rock et al. 2010; Marsee et al. 2008). The development of alum granuloma is independent of the route of immunization and occurs from a few days to several years (e.g., up to >12 years) following immunization, supporting the hypothesis that vaccines containing alum lead to a short-term inflammatory effect in a normal environment as well as long-term inflammatory effects in a pathological environment, at the site of injection (Gherardi et al. 2001; Kool et al. 2008a). It has been shown that alum induces uric acid or monosodium urate (MSU) crystal as a danger signal (Kool et al. 2008a). Subsequently, other signals such as heat shock protein 70 (HSP70) (Wang et al. 2012), and deoxyribonucleic acid (DNA) (Marichal et al. 2011; McKee et al. 2013) have been illustrated as inducers of alum-mediated immune responses, indicating that the mechanisms by which alum particles induce inflammation is central to understanding its adjuvant properties.

AH is able to induce caspase-1 activation and trigger the release of IL-1β and IL-18 by both human and mouse DCs via caspase-1 activation in a MyD88-independent fashion in vitro (Li et al. 2007).The use of BM mononuclear cells from NLRP3/ and wildtype mice demonstrated lipopolysaccharide (LPS) is not required for the alum (AH, IA)-mediated caspase-1 activation via NLRP3 signaling in vitro, however, LPS is necessary for pro-ILβ expression (Li et al. 2008). The experiments showed that caspase-1 activation and processing and secretion of IL-1β in response to alum is mediated by the NLRP3-inflammasome in both mouse and THP-1 cell line confirming the role of NLRP3 in alum adjuvanticity (Li et al. 2008). By taking wild type and LPS-primed NLRP3/or apoptosis-associated speck-like protein containing C-terminal caspase-recruitment domain knockout (ASC/) macrophages, it has been shown that alum (AH) mediates IL-1β secretion via NLRP3 inflammasome in an adenosine triphosphate (ATP)-independent manner in vitro (Franchi and Nunez 2008). It has been suggested that alum alone is insufficient to trigger caspase-1 activation because it requires priming or costimulation with LPS. However, i.p. immunization of NLRP3/ mice with human serum albumin (HSA, a T cell dependent Ag) and alum did not reduce the significant production of anti-HSA Ab in vivo (Franchi and Nunez 2008). Likewise McKee and colleagues, using DT or alum (AH), proved that the absence of caspase-1 or NLRP3 was not associated with altered CD4+ or CD8+ T cell responses, Th2 induction or Ag-specific IgG1 production in vivo (McKee et al. 2009). In contrast to these studies, it was shown that Ag-specific IgG1 and IL-5 production was reduced following immunization of NLRP3-deficient mice with OVA adsorbed to alum (IA) or HSA adsorbed to alum (Eisenbarth et al. 2008) indicating alum adjuvanticity is dependent on the NLRP3 pathway. This study illustrated a reduced innate immune response, specifically reduced airway eosinophilia, in the absence of NALP3 signalling, indicating a significant potential contribution to the adjuvanticity of alum (Eisenbarth et al. 2008). Similarly, in vitro studies using macrophages from NALP3/ and ASC/ mice suggest that the NALP3 molecule is required in alum-mediated caspase-1-triggered pro-IL-1β processing (Kool et al. 2008b). In vivo studies using NALP3/ mice suggest that alum activates DCs, increasing the expression of CD86 and MHCII molecules, and their trafficking to DLN in an IL-1β dependent manner, resulting in increased T cell division, and also increases the levels of OVA-specific IgE and IgG2c. Studies have also shown that alum activates caspase-1 in MyD88−/−, indicating no role of TLRs in alum-mediated inflammasome activation in vitro (Kool et al. 2008b). In contrast, the same groups have recorded that MyD88 molecules are necessary for the alum-mediated recruitment of inflammatory monocytes to the DLN in vivo (Kool et al. 2008a). In vitro studies using uricase to neutralize the alum-released uric acid resulted in only a minor effect on IL-1β processing suggesting no key role for this molecule in alum-mediated NALP3 activation (Kool et al. 2008b). However, an in vivo murine model using uricase to neutralize uric acid prior to alum immunization demonstrated reduced inflammatory monocyte trafficking to the DLN and consequently reduced T cell division (Kool et al. 2008a). This, in contrast to in vitro study, suggests that releasing uric acid acts a danger signal and contributes alum (IA) adjuvanticity (Kool et al. 2008a). Notably, the difference in the results in these labs may be associated with the differences in experimental design, such as the genetic background of the mice used, formulations of alum used, different routes of immunization, contamination with TLR agonists, and incomplete characterization of the alum induced immune response (De Gregorio et al. 2008; Lambrecht et al. 2009).

Recently, it has been shown that Ag-specific IgE is reduced in prostaglandin E2 (PGE2) synthase-deficient mice and that alum induces the production of the PGE2 by the Syk and p38MAP kinase pathway in an inflammasome-independent fashion (Kuroda et al. 2011). In this context, the immunoreceptor tyrosine-based activation motif (ITAM)-syk-phosphoinositide 3-kinase delta (PI3Kδ) pathway activates cytosolic phospholipase A2 (cPLA2) via p38 mitogen-activated protein kinase (MAPK) that results in the release of arachidonic acid (AA) from membrane lipids. AA is then converted into PGE2 by cyclooxygenases (COX)-2 and microsomal prostaglandin E synthase (mPGES)-1 molecule. The secreted PGE2 has the capability to trigger Th2 responses (Figure 3).
Figure 3

Hypotheses of NALP3 activation via inflammatory pathway. (A1): Alum activates XOR and induces the secretion of uric acid (UA). (A2): Alum-adsorbed antigen (Ag) is internalized by DC via actin-polymerization pathway. (A3): Alum interacts with lipid raft of DC membrane and activates ITAM-Syk pathway to internalize Ag. (A4): Alum-induced production of monosodium urate (MSU) crystals may be internalized by DC in CD16/32-dependent pathway. (B): Alum in phagosome enhances maturation into phagolysosome and release of Al3+ and OH. (C): Al3+ inhibits the cathepsin L activity and consequently the generated stress induces the rupture of phagolysosomes. (C1): The leaked Ag may bind with MHCI molecule in cytoplasm. (C2): Nanometer-sized Al3+ ion precipitates in the cytosol. (C3): The released K+ will be exported outside and extracellular adenosine tri-phosphate (ATP) will enter inside via P2X7 molecule. (C4): Cathepsin molecule will be released in the cytosol. (C5): MSU is released in cytosol. (D): Reactive oxygen species (ROS) is generated. The ROS will activate various molecules such as (D1) unknown or (D2) Heat shock protein (HSP)70 that can trigger nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling and induces the secretion of IL-1β (D3). (E): Mitochondrion becomes activated and generates ROS (E1) and mitochondrial deoxyribonucleic acid (DNA) (E2). (F): C3, C4, C5, D1, D2, E1, or E2 may activate nucleotide-binding oligomerization domain family-like receptor pyrin domain containing 3 (NALP3) and consequently AC-1 (G). (H): Necrosis-induced release of double stranded (ds)DNA enters cytoplasm and initiates toll-like receptor (TLR)9–interferon regulatory factor (IRF)-3 signaling (I). (J): MSU molecules directly activate myeloid differentiation (MYD)88 signaling. (K): Pro-IL-1β and IL-18 may be transcripted from nucleus. (L): Active caspase (AC)-1 induces the secretion of their matured forms. (M): immunoreceptor tyrosine-based activation motif (ITAM)-Syk-PI3Kδ pathway activates secretion of prostaglandin E2 (PGE2). (N): Alum activates complement.

3.3.1 Molecular mechanism of alum particles on inflammation

Inflammation is a component of the innate immune response that is comprised many proteins such as of nucleotide-binding oligomerization domain family-like receptor pyrin domain containing 3 or NLR protein 3 (NLRP3 or NALP3) inflammasome of macrophages and DCs (Martinon et al. 2007; Schroder and Tschopp 2010; Martinon et al. 2009). NALP3 consists of a domain present in neuronal apoptosis inhibitor protein (NAIP); MHC class II transactivator (CIITA), incompatibility locus protein from Podospora anserine (HET-E) and telomerase-associated protein (NACHT domain), and NACHT-associated domain (NAD) in between amino-terminus pyrin domain (PYD) and C-terminus leucine-rich repeat (LRR) (Schroder and Tschopp 2010). Notably, all the damage signals (Figure 3) activate the PYD of NALP3 that subsequently binds with the PYD of ASC. Consequently, the C-terminal caspase-recruitment domain (CARD) of ASC binds with CARD of pro-form of caspase-1, apoptosis-related cysteine peptidase (CASP1) domain that undergoes autocleavage at p20 or p10 sites. This results in the formation of the active caspase-1 p10/20 tetramer that processes the pro-forms of cytokines such as IL-1β into mature, active conformations and induces their secretion via unknown pathway (Agostini et al. 2004; Dinarello 2010; Schroder and Tschopp 2010). Similar mechanisms may occur during maturation and secretion of IL-18 (Martinon et al. 2009; Martinon et al. 2007). In contrast to the maturation process of IL-1β and IL-18, that of IL-33 is different. Although the secretion of mature IL-33 was triggered by alum treatment in THP-1 cells, and depended on ASC and NLRP3 (Li et al. 2008), caspase-1 processing actually causes inactivation of IL-33, rather than its activation (Cayrol and Girard 2009). The processing of caspase-1 is less effective in cleaving IL-33 compared with that of apoptosis-associated caspases (AAC) such as caspase-3 and caspase-7. However, the AAC-mediated IL-33 proteolysis has capacity to reduce IL-33 biological activity in vitro and in vivo (Luthi et al. 2009). Alum therefore induces the release of biologically active IL-33 as an endogenous danger signal via necrosis. In addition, it induces cleaving of IL-33 leading inactivation of its pro-inflammatory properties via apoptosis.

Alum’s role in the inflammasome (NLRP3) activation and inflammation is first mediated by enhanced phagocytosis (Hornung et al. 2008) followed by lysosomal acidification, resulting in targeting and activation of proteolytic enzymes found in lysosomes (Re 2011) (Figure 3). Consequently this induces stress (Pollock et al. 1995) leading to increased levels of the reactive oxygen species (ROS) such as the superoxide anion (O2 .-) and hydrogen peroxide (H2O2), resulting in the rupture of the lysosome. Rupture of the lysosomal compartment by alum or crystals has been shown to be sufficient to activate the NALP3 molecule (Hornung et al. 2008).

The generation of O2 .- and H2O2 occurs via phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase cytochrome b system (Martinon 2010; Holmstrom and Finkel 2014). Notably, the impact of alum and other particulates on phagocytic NADPH system has been studied in detail (Hornung et al. 2008). The importance of NADPH has been demonstrated in murine models, with mice deficient in this enzyme failing to produce phagocytic O2 .- and becoming hypersuceptible to various pathogens (Pollock et al. 1995). It has also been found that ROS triggers dissociation of thioredoxin interacting protein (TXNIP) that interacts with NLRP3 molecule from thioredoxin (Zhou et al. 2010). TXNIP deficient mice have also been shown to lack NLRP3 inflammasome activation and consequently lack of IL-1β secretion (Zhou et al. 2010). In contrast to this result, macrophages obtained from the mice lacking gp91phox, a phagosomal NADPH cytochrome b, retain activation of the NALP3 inflammasome in response to silica crystals, MSU, ATP, and poly (dA:dT) (Hornung et al. 2008). This suggests that the phagosomal respiratory-burst oxidase system is not necessarily involved in NALP3 activation (Hornung et al. 2008). This evidence is supported by the fact that the zymosan, a TLR2 agonist did not induce activation of the inflammasome in macrophages in spite of its potent phagocytic and ROS inducing properties (Hise et al. 2009; Joly et al. 2009). Thus, the notion that ROS may generate a specific but unknown ligand that can either activate NLRP3 or can modify this inflammasome or associated proteins (Dostert et al. 2009; Dostert et al. 2008; Martinon 2010) is being questioned.

In the same way, following lysosomal breakdown, HSP70 and K+, cathepsin molecules and MSU are released in the cytosol (Holmstrom and Finkel 2014; Pollock et al. 1995; Hornung et al. 2008). Alum has been shown to generates stress-triggered HSP70 molecules in CD11c+ DCs (Wang et al. 2012). Alum induced the cell surface and intracellular expression of HSP70 that activates either NF-κB pathway or NLRP3 inflammasomes (Wang et al. 2012). HSP70, an important molecular target for vaccines due to its role in enhancing Ag uptake and presentation by DCs (Basu et al. 2001), has been shown to enhance innate and adaptive immune response in vivo (Wang et al. 2012). This role has been confirmed in a murine model immunizing BALB/c with the mixture of alum (AH) in the presence of phenylethyne sulphonamide, an inhibitor of HSP70 (Wang et al. 2012).

Cathepsin B enzyme has also been shown to be released in the cytosol from the lysosome in vitro (Hornung et al. 2008). This enzyme causes pyroptosis, the NLRP3-mediated cell death that is inhibited when a cathepsin B inhibitor is used (Hornung et al. 2008). This suggests that cathepsin B is involved in NLRP3 activation. However, inflammasome activation was not affected in cathepsin B-deficient mice (Dostert et al. 2008). It is therefore possible that alum may contribute via other cathepsin molecules still present in cathepsin B-deficient mice (Felbor et al. 2002).

Following alum internalization, cellular perforation occurs resulting in the efflux of the potassium ion concentration (K+) channel via P2X7-pannexin-1 signaling and the consequent caspase-1 activation (Martinon 2010; Kanneganti et al. 2007). However, P2X7 and pannexin have been shown to be redundant in NLRP3 activation by MSU or alum (Re 2011), indicating the selective role of K+ efflux in inflammation.

In parallel to the effects on the phagosome, in vitro experiment using THP-1 showed increased mitochondrial activity following alum treatment, without any disruption to the mitochondria (Ohlsson et al. 2013). This has questioned the generally accepted theory that mitochondrial dysfunction may occur, releasing danger signals, such as oxidized mitochondrial DNA and ROS, into the cytosol (Nakahira et al. 2011; Shimada et al. 2012). This danger signal theory had been generalized as the secretion of inflammatory cytokines and mitochondrial ROS in NLRP3−/− and ASC−/− cells were reduced, indicating that mitochondria can sense intracellular stress and promote NLRP3 activation in a ROS-dependent manner (Zhou et al. 2011). In vitro model containing primary phagocytic cells such as DCs and macrophages rather than cell lines such as THP-1 may have more physiological relevant readouts and thus may be more informative in unraveling the mechanisms of action of alum.

3.3.2 Induction of immune responses via the release of DNA and induction of complement molecules

In vitro studies have shown that AH enhances immature and murine bone marrow-derived macrophage (BMDM) survival and induces DNA synthesis, and enhances proliferative response to granulocyte macrophage colony stimulating factor (GMCSF) and colony stimulating factor (CSF)-1 (Hamilton et al. 2000). In contrast to this study, alum has also been shown to trigger necrosis of cells resident at the alum-injection sites in vivo (McKee et al. 2013; Cain et al. 2013; Marichal et al. 2011). The released DNA has been shown to trigger the TLR9-interferon regulatory factor (IRF)3-dependent pathway, activate inflammatory DCs, and enhance Th2 responses (Figure 3). In addition, the DNA has potentiality to activate IRF3-independent pathways, activate Tfh responses, and enhance B cell responses accompanied by IgE isotype switching and IgG1 production (Marichal et al. 2011). Although Marichal et al., demonstrated reduced DC migration from the peritoneal cavity to the DLN following deoxyribonuclease (DNase) treatment and i.p. alum injection, in a murine model, McKee et al., reported no effect on accumulation of, or expression of costimulatory proteins on Ag-loaded DCs in DLNs of stimulator of interferon genes (STING) mice following i.m. immunization (McKee et al. 2013). The latter groups found that DNase inhibited prolonged Ag presentation and DC-T cell interactions after i.m. injection in these mice. Thus demonstrating that host DNA introduced into the cytoplasm with the activation of STING pathway of Ag-bearing DCs enhanced p:MHCII presentation and DC-T cell interactions (McKee et al. 2013). Other factors such as histone proteins may be involved in alum mediated innate immune responses (Munks et al. 2010). Following i.p. or i.m. immunization, alum accumulates into nodules that are held together by host chromatin, released from inflammatory cells (Munks et al. 2010). The DNA released in this deposited chromatin may contribute to the adjuvanticity of alum in vivo.

AH has been shown to activate complement via the lectin pathway that results in the production of anaphylatoxins and complement component (C) 3b (Ramanathan et al. 1979). This effect is dependent on plaminogen for C3 activation. Importantly, alum binds complement or prothrombin incubated with plasma depending on the incubation temperature (Polley and Nachman 1975). The surface of aluminum has been shown to bind C3 (Arvidsson et al. 2007). However, complement deposition on AH has not been detected in a study (Tengvall et al. 1998). The role of complement in vitro has been validated by an in vivo experiment in which complement receptor-deficient mice had an impaired response to alum adsorbed to Ag injection (Chen et al. 2000). In vitro data has shown that AH activates the lectin pathway, alternative pathway, and classical pathway; however, it has the greatest effect on the alternative complement pathway. The alum provides a surface for complement activation, Ag opsonization, and Ag can be released via interactions with complement receptors (Guven et al. 2013) (Figure 3).

4. Conclusions and future directions

Aluminum compounds are the only adjuvants widely approved for use in human vaccines due to their safety record, ease of preparation, stability and immunostimulatory effects (Tritto et al. 2009; O'Hagan and De Gregorio 2009; Mbow et al. 2010). Applications include incorporation in vaccines that are successfully used against human papillomavirus (AH in Cervarix) and hepatitis B virus (AP in Fendrix), indicating the significant contribution of alum in mixed adjuvant preparation. Adjuvant development is undoubtedly a long process that must address regulatory, safety, and economic concerns, during clinical and preclinical development. Therefore, despite our incomplete understanding of the mode of action of aluminum based adjuvants, in the absence of suitable alternative adjuvants, alum is the sole candidate for use in human vaccines in the future. Considering the widespread use of alum, further comparative in vitro and in vivo should be undertaken to establish the mode of action due to the conflicting in vitro and in vivo data published. It is difficult to translate the in vitro data to the dynamic immune system essential for vaccine development, thus physico-chemical characteristics established in vitro and in vivo data regarding its role in inflammation can be exploited for future vaccine development. A clear causal association of alum in triggering immune responses via cellular death and or enhancing the quality, duration, and magnitude of T- and B- cell responses will make a significant contribution to the rational design of effective and safe vaccines and development of new adjuvants for future use.



I acknowledge Professor James Brewer, University of Glasgow, Scotland and Dr. Karen Jane Bryson, The Roslin Institute, University of Edinburgh, Scotland for their advice and critical evaluation of the manuscript.

Authors’ Affiliations

Division of Veterinary and Primate Health, Global Primate Network
Department of Zoology, Birendra Multiple Campus, Tribhuvan University


  1. Agostini L, Martinon F, Burns K, McDermott MF, Hawkins PN, Tschopp J (2004) NALP3 forms an IL-1beta-processing inflammasome with increased activity in Muckle-Wells autoinflammatory disorder. Immunity 20(3):319–325, doi:S1074761304000469View ArticleGoogle Scholar
  2. Angeli V, Ginhoux F, Llodra J, Quemeneur L, Frenette PS, Skobe M, Jessberger R, Merad M, Randolph GJ (2006) B cell-driven lymphangiogenesis in inflamed lymph nodes enhances dendritic cell mobilization. Immunity 24(2):203–215, doi:S1074-7613(06)00102-6View ArticleGoogle Scholar
  3. Anonymous (1956) United States Minimum Requirements. Tetanus and Diphtheria Toxoids Combined Precipitated, Adsorbed (For Adult Use), vol Amendment No. 1. US Department of Health, Education and Welfare, National Institute of Health, BethesdaGoogle Scholar
  4. Arvidsson S, Askendal A, Tengvall P (2007) Blood plasma contact activation on silicon, titanium and aluminium. Biomaterials 28(7):1346–1354, doi:S0142-9612(06)00951-3View ArticleGoogle Scholar
  5. Authier FJ, Sauvat S, Christov C, Chariot P, Raisbeck G, Poron MF, Yiou F, Gherardi R (2006) AlOH3-adjuvanted vaccine-induced macrophagic myofasciitis in rats is influenced by the genetic background. Neuromuscul Disord 16(5):347–352. doi:S0960-8966(06)00036-8Google Scholar
  6. Awate S, Babiuk LA, Mutwiri G (2013) Mechanisms of action of adjuvants. Front Immunol 4:114, doi:10.3389/fimmu.2013.00114View ArticleGoogle Scholar
  7. Basu S, Binder RJ, Ramalingam T, Srivastava PK (2001) CD91 is a common receptor for heat shock proteins gp96, hsp90, hsp70, and calreticulin. Immunity 14(3):303–313View ArticleGoogle Scholar
  8. Baylor NW, Egan W, Richman P (2002) Aluminum salts in vaccines–US perspective. Vaccine 20(Suppl 3):S18–23View ArticleGoogle Scholar
  9. Bonanni P, Santos JI (2011) Vaccine evolution, vol 1. Perspectives in Vaccinology. Elsevier, Understanding Modern VaccinesGoogle Scholar
  10. Bordet AL, Michenet P, Cohen C, Arbion F, Ekindi N, Bonneau C, Kerdraon R, Coville M (2001) [Post-vaccination granuloma due to aluminium hydroxide]. Ann Pathol 21(2):149–152. doi:MDOI-AP-04-2001-21-2-0242-6498-101019-ART7Google Scholar
  11. Bramwell VW, Perrie Y (2005) Particulate delivery systems for vaccines. Crit Rev Ther Drug Carrier Syst 22(2):151–214View ArticleGoogle Scholar
  12. Brewer JM (2006) (How) do aluminium adjuvants work? Immunol Lett 102(1):10–15View ArticleGoogle Scholar
  13. Brewer JM, Conacher M, Satoskar A, Bluethmann H, Alexander J (1996) In interleukin-4-deficient mice, alum not only generates T helper 1 responses equivalent to freund's complete adjuvant, but continues to induce T helper 2 cytokine production. Eur J Immunol 26(9):2062–2066View ArticleGoogle Scholar
  14. Brewer JM, Conacher M, Gaffney M, Douglas M, Bluethmann H, Alexander J (1998) Neither interleukin-6 nor signalling via tumour necrosis factor receptor-1 contribute to the adjuvant activity of Alum and Freund's adjuvant. Immunology 93(1):41–48View ArticleGoogle Scholar
  15. Brewer JM, Conacher M, Hunter CA, Mohrs M, Brombacher F, Alexander J (1999) Aluminium hydroxide adjuvant initiates strong antigen-specific Th2 responses in the absence of IL-4- or IL-13-mediated signaling. J Immunol 163(12):6448–6454Google Scholar
  16. Cain DW, Sanders SE, Cunningham MM, Kelsoe G (2013) Disparate adjuvant properties among three formulations of “alum”. Vaccine 31(4):653–660. doi:S0264-410X(12)01658-1Google Scholar
  17. Calabro S, Tortoli M, Baudner BC, Pacitto A, Cortese M, O'Hagan DT, De Gregorio E, Seubert A, Wack A (2011) Vaccine adjuvants alum and MF59 induce rapid recruitment of neutrophils and monocytes that participate in antigen transport to draining lymph nodes. Vaccine 29(9):1812–1823View ArticleGoogle Scholar
  18. Carbone FR, Bevan MJ (1990) Class I-restricted processing and presentation of exogenous cell-associated antigen in vivo. J Exp Med 171(2):377–387View ArticleGoogle Scholar
  19. Carbone FR, Belz GT, Heath WR (2004) Transfer of antigen between migrating and lymph node-resident DCs in peripheral T-cell tolerance and immunity. Trends Immunol 25(12):655–658. doi:S1471-4906(04)00293-5Google Scholar
  20. Cavanagh LL, Bonasio R, Mazo IB, Halin C, Cheng G, van der Velden AW, Cariappa A, Chase C, Russell P, Starnbach MN, Koni PA, Pillai S, Weninger W, von Andrian UH (2005) Activation of bone marrow-resident memory T cells by circulating, antigen-bearing dendritic cells. Nat Immunol 6(10):1029–1037. doi:ni1249Google Scholar
  21. Cayrol C, Girard JP (2009) The IL-1-like cytokine IL-33 is inactivated after maturation by caspase-1. Proc Natl Acad Sci U S A 106(22):9021–9026View ArticleGoogle Scholar
  22. Chang M, Shi Y, Nail SL, HogenEsch H, Adams SB, White JL, Hem SL (2001) Degree of antigen adsorption in the vaccine or interstitial fluid and its effect on the antibody response in rabbits. Vaccine 19(20–22):2884–2889. doi:S0264410X00005594Google Scholar
  23. Chen Z, Koralov SB, Gendelman M, Carroll MC, Kelsoe G (2000) Humoral immune responses in Cr2−/− mice: enhanced affinity maturation but impaired antibody persistence. J Immunol 164(9):4522–4532. doi:ji_v164n9p4522 [pii]Google Scholar
  24. Chong H, Brady K, Metze D, Calonje E (2006) Persistent nodules at injection sites (aluminium granuloma) -- clinicopathological study of 14 cases with a diverse range of histological reaction patterns. Histopathology 48(2):182–188. doi:HIS2312Google Scholar
  25. Chuai X, Chen H, Wang W, Deng Y, Wen B, Ruan L, Tan W (2013) Poly(I:C)/alum mixed adjuvant priming enhances HBV subunit vaccine-induced immunity in mice when combined with recombinant adenoviral-based HBV vaccine boosting. PLoS One 8(1):e54126, doi:10.1371/journal.pone.0054126 PONE-D-12-35157View ArticleGoogle Scholar
  26. Cox JC, Coulter AR (1997) Adjuvants–a classification and review of their modes of action. Vaccine 15(3):248–256View ArticleGoogle Scholar
  27. Cunningham AF, Fallon PG, Khan M, Vacheron S, Acha-Orbea H, MacLennan IC, McKenzie AN, Toellner KM (2002) Th2 activities induced during virgin T cell priming in the absence of IL-4, IL-13, and B cells. J Immunol 169(6):2900–2906View ArticleGoogle Scholar
  28. Cunningham AF, Khan M, Ball J, Toellner KM, Serre K, Mohr E, MacLennan IC (2004a) Responses to the soluble flagellar protein FliC are Th2, while those to FliC on Salmonella are Th1. Eur J Immunol 34(11):2986–2995, doi:10.1002/eji.200425403View ArticleGoogle Scholar
  29. Cunningham AF, Serre K, Toellner KM, Khan M, Alexander J, Brombacher F, MacLennan IC (2004b) Pinpointing IL-4-independent acquisition and IL-4-influenced maintenance of Th2 activity by CD4 T cells. Eur J Immunol 34(3):686–694, doi:10.1002/eji.200324510View ArticleGoogle Scholar
  30. De Gregorio E, Tritto E, Rappuoli R (2008) Alum adjuvanticity: Unraveling a century old mystery. Eur J Immunol 38(8):2068View ArticleGoogle Scholar
  31. de Veer M, Kemp J, Chatelier J, Elhay MJ, Meeusen EN (2010) The kinetics of soluble and particulate antigen trafficking in the afferent lymph, and its modulation by aluminum-based adjuvant. Vaccine 28(40):6597–6602View ArticleGoogle Scholar
  32. Dillon SB, Demuth SG, Schneider MA, Weston CB, Jones CS, Young JF, Scott M, Bhatnaghar PK, LoCastro S, Hanna N (1992) Induction of protective class I MHC-restricted CTL in mice by a recombinant influenza vaccine in aluminium hydroxide adjuvant. Vaccine 10(5):309–318View ArticleGoogle Scholar
  33. Dinarello CA (2010) IL-1: discoveries, controversies and future directions. Eur J Immunol 40(3):599–606, doi:10.1002/eji.201040319View ArticleGoogle Scholar
  34. Dostert C, Petrilli V, Van Bruggen R, Steele C, Mossman BT, Tschopp J (2008) Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica. Science 320(5876):674–677. doi:1156995Google Scholar
  35. Dostert C, Guarda G, Romero JF, Menu P, Gross O, Tardivel A, Suva ML, Stehle JC, Kopf M, Stamenkovic I, Corradin G, Tschopp J (2009) Malarial hemozoin is a Nalp3 inflammasome activating danger signal. PLoS One 4(8):e6510, doi:10.1371/journal.pone.0006510View ArticleGoogle Scholar
  36. Edelman R (2002) The development and use of vaccine adjuvants. Mol Biotechnol 21(2):129–148View ArticleGoogle Scholar
  37. Edelman R, Tacket CO (1990) Adjuvants. Int Rev Immunol 7(1):51–66View ArticleGoogle Scholar
  38. Egan PM, Belfast MT, Gimenez JA, Sitrin RD, Mancinelli RJ (2009) Relationship between tightness of binding and immunogenicity in an aluminum-containing adjuvant-adsorbed hepatitis B vaccine. Vaccine 27(24):3175–3180, doi:S0264-410X(09)00471-XView ArticleGoogle Scholar
  39. Eisenbarth SC, Colegio OR, O'Connor W, Sutterwala FS, Flavell RA (2008) Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature 453(7198):1122–1126View ArticleGoogle Scholar
  40. Estey T, Vessely C, Randolph TW, Henderson I, Braun LJ, Nayar R, Carpenter JF (2009) Evaluation of chemical degradation of a trivalent recombinant protein vaccine against botulinum neurotoxin by LysC peptide mapping and MALDI-TOF mass spectrometry. J Pharm Sci 98(9):2994–3012, doi:10.1002/jps.21543View ArticleGoogle Scholar
  41. Farago FC (1940) Alum-Precipitated Toxoid in Diphtheria. Duration of immunity after one-shot method. The Lancet 236(6099):68–70View ArticleGoogle Scholar
  42. Fawcett HA, Smith NP (1984) Injection-site granuloma due to aluminum. Arch Dermatol 120(10):1318–1322View ArticleGoogle Scholar
  43. Felbor U, Kessler B, Mothes W, Goebel HH, Ploegh HL, Bronson RT, Olsen BR (2002) Neuronal loss and brain atrophy in mice lacking cathepsins B and L. Proc Natl Acad Sci U S A 99(12):7883–7888, doi:10.1073/pnas.112632299View ArticleGoogle Scholar
  44. Flach TL, Ng G, Hari A, Desrosiers MD, Zhang P, Ward SM, Seamone ME, Vilaysane A, Mucsi AD, Fong Y, Prenner E, Ling CC, Tschopp J, Muruve DA, Amrein MW, Shi Y (2011) Alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity. Nat Med 17(4):479–487. doi:nm.2306Google Scholar
  45. Flarend RE, Hem SL, White JL, Elmore D, Suckow MA, Rudy AC, Dandashli EA (1997) In vivo absorption of aluminium-containing vaccine adjuvants using 26Al. Vaccine 15(12–13):1314–1318. doi:S0264-410X(97)00041-8Google Scholar
  46. Flebbe LM, Braley-Mullen H (1986) Immunopotentiating effects of the adjuvants SGP and Quil A. I. Antibody responses to T-dependent and T-independent antigens. Cell Immunol 99(1):119–127View ArticleGoogle Scholar
  47. Franchi L, Nunez G (2008) The Nlrp3 inflammasome is critical for aluminium hydroxide-mediated IL-1beta secretion but dispensable for adjuvant activity. Eur J Immunol 38(8):2085–2089, doi:10.1002/eji.200838549View ArticleGoogle Scholar
  48. Frost L, Johansen P, Pedersen S, Veien N, Ostergaard PA, Nielsen MH (1985) Persistent subcutaneous nodules in children hyposensitized with aluminium-containing allergen extracts. Allergy 40(5):368–372View ArticleGoogle Scholar
  49. Geissmann F, Jung S, Littman DR (2003) Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity 19(1):71–82. doi:S1074761303001742 [pii]Google Scholar
  50. Gherardi RK, Coquet M, Cherin P, Belec L, Moretto P, Dreyfus PA, Pellissier JF, Chariot P, Authier FJ (2001) Macrophagic myofasciitis lesions assess long-term persistence of vaccine-derived aluminium hydroxide in muscle. Brain 124(Pt 9):1821–1831View ArticleGoogle Scholar
  51. Ghimire TR, Benson RA, Garside P, Brewer JM (2012) Alum increases antigen uptake, reduces antigen degradation and sustains antigen presentation by DCs in vitro. Immunol Lett 147(1–2):55–62. doi:S0165-2478(12)00159-9Google Scholar
  52. Glenny AT, Sudmersen HJ (1921) Notes on the Production of Immunity to Diphtheria Toxin. J Hyg (Lond) 20(2):176–220View ArticleGoogle Scholar
  53. Glenny AT, Pope CG, Waddington H, Wallace U (1926) Immunological notes. XXIII The antigenic value of toxoid precipitated by potassium alum. J Pathol 29:38–49Google Scholar
  54. Glenny AT, Buttle GAH, Stevens MF (1931) Rate of disappearance of diphtheria toxoid injected into rabbits and guinea pigs: toxoid precipitated with Alum. J Pathol Bacteriol 34:267–275View ArticleGoogle Scholar
  55. Goto N, Akama K (1982) Histopathological studies of reactions in mice injected with aluminum-adsorbed tetanus toxoid. Microbiol Immunol 26(12):1121–1132View ArticleGoogle Scholar
  56. Grun JL, Maurer PH (1989) Different T helper cell subsets elicited in mice utilizing two different adjuvant vehicles: the role of endogenous interleukin 1 in proliferative responses. Cell Immunol 121(1):134–145View ArticleGoogle Scholar
  57. Gupta RK (1998) Aluminum compounds as vaccine adjuvants. Adv Drug Deliv Rev 32(3):155–172. doi:S0169-409X(98)00008-8Google Scholar
  58. Gupta RK, Rost BE, Relyveld E, Siber GR (1995) Adjuvant properties of aluminum and calcium compounds. Pharm Biotechnol 6:229–248View ArticleGoogle Scholar
  59. Gupta RK, Chang AC, Griffin P, Rivera R, Siber GR (1996) In vivo distribution of radioactivity in mice after injection of biodegradable polymer microspheres containing 14C-labeled tetanus toxoid. Vaccine 14(15):1412–1416, doi:S0264410X96000734View ArticleGoogle Scholar
  60. Guven E, Duus K, Laursen I, Hojrup P, Houen G (2013) Aluminum hydroxide adjuvant differentially activates the three complement pathways with major involvement of the alternative pathway. PLoS One 8(9):e74445, doi:10.1371/journal.pone.0074445 PONE-D-13-23096View ArticleGoogle Scholar
  61. Hamilton JA, Byrne R, Whitty G (2000) Particulate adjuvants can induce macrophage survival, DNA synthesis, and a synergistic proliferative response to GM-CSF and CSF-1. J Leukoc Biol 67(2):226–232Google Scholar
  62. Harrison WT (1935) Some Observations on the Use of Alum Precipitated Diphtheria Toxoid. Am J Public Health Nations Health 25(3):298–300View ArticleGoogle Scholar
  63. Heimlich JM, Regnier FE, White JL, Hem SL (1999) The in vitro displacement of adsorbed model antigens from aluminium-containing adjuvants by interstitial proteins. Vaccine 17(22):2873–2881, doi:S0264410X99001267View ArticleGoogle Scholar
  64. Hem SL (2002) Elimination of aluminum adjuvants. Vaccine 20(Suppl 3):S40–43View ArticleGoogle Scholar
  65. Hem SL, HogenEsch H (2007) Aluminum-containing adjuvants: Properties. Formulations and Use. Vaccine Adjuvants and Delivery Systems. John Wiley & Sons, Inc., New JerseyGoogle Scholar
  66. Hem SL, Johnston CT, HogenEsch H (2007) Imject Alum is not aluminum hydroxide adjuvant or aluminum phosphate adjuvant. Vaccine 25(27):4985–4986View ArticleGoogle Scholar
  67. Hise AG, Tomalka J, Ganesan S, Patel K, Hall BA, Brown GD, Fitzgerald KA (2009) An essential role for the NLRP3 inflammasome in host defense against the human fungal pathogen Candida albicans. Cell Host Microbe 5(5):487–497. doi:S1931-3128(09)00143-7Google Scholar
  68. HogenEsch H (2002) Mechanisms of stimulation of the immune response by aluminum adjuvants. Vaccine 20(Suppl 3):S34–39View ArticleGoogle Scholar
  69. Hogenesch H (2012) Mechanism of immunopotentiation and safety of aluminum adjuvants. Front Immunol 3:406, doi:10.3389/fimmu.2012.00406Google Scholar
  70. Holmstrom KM, Finkel T (2014) Cellular mechanisms and physiological consequences of redox-dependent signalling. Nat Rev Mol Cell Biol 15(6):411–421, doi:nrm3801View ArticleGoogle Scholar
  71. Holt LB (1950) Developments in diphtheria prophylaxis. Heinemann, London, United KingdomGoogle Scholar
  72. Hornung V, Bauernfeind F, Halle A, Samstad EO, Kono H, Rock KL, Fitzgerald KA, Latz E (2008) Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nat Immunol 9(8):847–856View ArticleGoogle Scholar
  73. Huang FP, Platt N, Wykes M, Major JR, Powell TJ, Jenkins CD, MacPherson GG (2000) A discrete subpopulation of dendritic cells transports apoptotic intestinal epithelial cells to T cell areas of mesenteric lymph nodes. J Exp Med 191(3):435–444View ArticleGoogle Scholar
  74. Hutchison S, Benson RA, Gibson VB, Pollock AH, Garside P, Brewer JM (2011) Antigen depot is not required for alum adjuvanticity. FASEB J doi:fj.11-184556Google Scholar
  75. Itano AA, McSorley SJ, Reinhardt RL, Ehst BD, Ingulli E, Rudensky AY, Jenkins MK (2003) Distinct dendritic cell populations sequentially present antigen to CD4 T cells and stimulate different aspects of cell-mediated immunity. Immunity 19(1):47–57View ArticleGoogle Scholar
  76. Iyer S, HogenEsch H, Hem SL (2003) Relationship between the degree of antigen adsorption to aluminum hydroxide adjuvant in interstitial fluid and antibody production. Vaccine 21(11–12):1219–1223View ArticleGoogle Scholar
  77. Jakob T, Udey MC (1998) Regulation of E-cadherin-mediated adhesion in Langerhans cell-like dendritic cells by inflammatory mediators that mobilize Langerhans cells in vivo. J Immunol 160(8):4067–4073Google Scholar
  78. Jankovic D, Caspar P, Zweig M, Garcia-Moll M, Showalter SD, Vogel FR, Sher A (1997) Adsorption to aluminum hydroxide promotes the activity of IL-12 as an adjuvant for antibody as well as type 1 cytokine responses to HIV-1 gp120. J Immunol 159(5):2409–2417Google Scholar
  79. Joly S, Ma N, Sadler JJ, Soll DR, Cassel SL, Sutterwala FS (2009) Cutting edge: Candida albicans hyphae formation triggers activation of the Nlrp3 inflammasome. J Immunol 183(6):3578–3581. doi:jimmunol.0901323Google Scholar
  80. Jordan MB, Mills DM, Kappler J, Marrack P, Cambier JC (2004) Promotion of B cell immune responses via an alum-induced myeloid cell population. Science 304(5678):1808–1810, doi:10.1126/science.1089926304/5678/1808View ArticleGoogle Scholar
  81. Kabashima K, Banks TA, Ansel KM, Lu TT, Ware CF, Cyster JG (2005) Intrinsic lymphotoxin-beta receptor requirement for homeostasis of lymphoid tissue dendritic cells. Immunity 22(4):439–450. doi:S1074-7613(05)00092-0Google Scholar
  82. Kanneganti TD, Lamkanfi M, Kim YG, Chen G, Park JH, Franchi L, Vandenabeele P, Nunez G (2007) Pannexin-1-mediated recognition of bacterial molecules activates the cryopyrin inflammasome independent of Toll-like receptor signaling. Immunity 26(4):433–443. doi:S1074-7613(07)00211-7Google Scholar
  83. Khan Z, Combadiere C, Authier FJ, Itier V, Lux F, Exley C, Mahrouf-Yorgov M, Decrouy X, Moretto P, Tillement O, Gherardi RK, Cadusseau J (2013) Slow CCL2-dependent translocation of biopersistent particles from muscle to brain. BMC Med 11:99. doi:1741-7015-11-99Google Scholar
  84. Kool M, Petrilli V, De Smedt T, Rolaz A, Hammad H, van Nimwegen M, Bergen IM, Castillo R, Lambrecht BN, Tschopp J (2008a) Cutting edge: alum adjuvant stimulates inflammatory dendritic cells through activation of the NALP3 inflammasome. J Immunol 181(6):3755–3759View ArticleGoogle Scholar
  85. Kool M, Soullie T, van Nimwegen M, Willart MA, Muskens F, Jung S, Hoogsteden HC, Hammad H, Lambrecht BN (2008b) Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells. J Exp Med 205(4):869–882View ArticleGoogle Scholar
  86. Kool M, Fierens K, Lambrecht BN (2012) Alum adjuvant: some of the tricks of the oldest adjuvant. J Med Microbiol 61(Pt 7):927–934. doi:jmm.0.038943-0Google Scholar
  87. Korsholm KS, Petersen RV, Agger EM, Andersen P (2010) T-helper 1 and T-helper 2 adjuvants induce distinct differences in the magnitude, quality and kinetics of the early inflammatory response at the site of injection. Immunology 129(1):75–86, doi:IMM3164View ArticleGoogle Scholar
  88. Kuroda E, Ishii KJ, Uematsu S, Ohata K, Coban C, Akira S, Aritake K, Urade Y, Morimoto Y (2011) Silica crystals and aluminum salts regulate the production of prostaglandin in macrophages via NALP3 inflammasome-independent mechanisms. Immunity 34(4):514–526. doi:S1074-7613(11)00128-2Google Scholar
  89. Lambrecht BN, Kool M, Willart MA, Hammad H (2009) Mechanism of action of clinically approved adjuvants. Curr Opin Immunol 21(1):23–29View ArticleGoogle Scholar
  90. Li H, Nookala S, Re F (2007) Aluminum hydroxide adjuvants activate caspase-1 and induce IL-1beta and IL-18 release. J Immunol 178(8):5271–5276. doi:178/8/5271Google Scholar
  91. Li H, Willingham SB, Ting JP, Re F (2008) Cutting edge: inflammasome activation by alum and alum's adjuvant effect are mediated by NLRP3. J Immunol 181(1):17–21View ArticleGoogle Scholar
  92. Lindblad EB (2004a) Aluminium adjuvants–in retrospect and prospect. Vaccine 22(27–28):3658–3668, doi:10.1016/j.vaccine.2004.03.032S0264410X04002750View ArticleGoogle Scholar
  93. Lindblad EB (2004b) Aluminium compounds for use in vaccines. Immunol Cell Biol 82(5):497–505View ArticleGoogle Scholar
  94. Lindblad EB, Schonberg NE (2010) Aluminum adjuvants: preparation, application, dosage, and formulation with antigen. Methods Mol Biol 626:41–58, doi:10.1007/978-1-60761-585-9_4View ArticleGoogle Scholar
  95. Lindblad EB, Elhay MJ, Silva R, Appelberg R, Andersen P (1997) Adjuvant modulation of immune responses to tuberculosis subunit vaccines. Infect Immun 65(2):623–629Google Scholar
  96. Liu T, Matsuguchi T, Tsuboi N, Yajima T, Yoshikai Y (2002) Differences in expression of toll-like receptors and their reactivities in dendritic cells in BALB/c and C57BL/6 mice. Infect Immun 70(12):6638–6645View ArticleGoogle Scholar
  97. Lu F, Hogenesch H (2013) Kinetics of the inflammatory response following intramuscular injection of aluminum adjuvant. Vaccine 31(37):3979–3986. doi:S0264-410X(13)00769-X [pii]10.1016/j.vaccine.2013.05.107Google Scholar
  98. Luther SA, Serre K, Cunningham AF, Khan M, Acha-Orbea H, MacLennan IC, Toellner KM (2007) Recirculating CD4 memory T cells mount rapid secondary responses without major contributions from follicular CD4 effectors and B cells. Eur J Immunol 37(6):1476–1484, doi:10.1002/eji.200636573View ArticleGoogle Scholar
  99. Luthi AU, Cullen SP, McNeela EA, Duriez PJ, Afonina IS, Sheridan C, Brumatti G, Taylor RC, Kersse K, Vandenabeele P, Lavelle EC, Martin SJ (2009) Suppression of interleukin-33 bioactivity through proteolysis by apoptotic caspases. Immunity 31(1):84–98View ArticleGoogle Scholar
  100. MacLeod MK, McKee AS, David A, Wang J, Mason R, Kappler JW, Marrack P (2011) Vaccine adjuvants aluminum and monophosphoryl lipid A provide distinct signals to generate protective cytotoxic memory CD8 T cells. Proc Natl Acad Sci U S A 108(19):7914–7919, doi:1104588108View ArticleGoogle Scholar
  101. Mannhalter JW, Neychev HO, Zlabinger GJ, Ahmad R, Eibl MM (1985) Modulation of the human immune response by the non-toxic and non-pyrogenic adjuvant aluminium hydroxide: effect on antigen uptake and antigen presentation. Clin Exp Immunol 61(1):143–151Google Scholar
  102. Marichal T, Ohata K, Bedoret D, Mesnil C, Sabatel C, Kobiyama K, Lekeux P, Coban C, Akira S, Ishii KJ, Bureau F, Desmet CJ (2011) DNA released from dying host cells mediates aluminum adjuvant activity. Nat Med 17(8):996–1002View ArticleGoogle Scholar
  103. Marrack P, McKee AS, Munks MW (2009) Towards an understanding of the adjuvant action of aluminium. Nat Rev Immunol 9(4):287–293. doi:nri2510Google Scholar
  104. Marsee DK, Williams JM, Velazquez EF (2008) Aluminum granuloma after administration of the quadrivalent human papillomavirus vaccine. Report of a case. Am J Dermatopathol 30(6):622–624, doi:10.1097/DAD.0b013e318185a69100000372-200812000-00019View ArticleGoogle Scholar
  105. Martinon F (2010) Signaling by ROS drives inflammasome activation. Eur J Immunol 40(3):616–619, doi:10.1002/eji.200940168View ArticleGoogle Scholar
  106. Martinon F, Gaide O, Petrilli V, Mayor A, Tschopp J (2007) NALP inflammasomes: a central role in innate immunity. Semin Immunopathol 29(3):213–229, doi:10.1007/s00281-007-0079-yView ArticleGoogle Scholar
  107. Martinon F, Mayor A, Tschopp J (2009) The inflammasomes: guardians of the body. Annu Rev Immunol 27:229–265, doi:10.1146/annurev.immunol.021908.132715View ArticleGoogle Scholar
  108. Matzinger P (1994) Tolerance, danger, and the extended family. Annu Rev Immunol 12:991–1045View ArticleGoogle Scholar
  109. Mbow ML, De Gregorio E, Valiante NM, Rappuoli R (2010) New adjuvants for human vaccines. Curr Opin Immunol 22(3):411–416View ArticleGoogle Scholar
  110. McKee AS, MacLeod M, White J, Crawford F, Kappler JW, Marrack P (2008) Gr1+IL-4-producing innate cells are induced in response to Th2 stimuli and suppress Th1-dependent antibody responses. Int Immunol 20(5):659–669, doi:dxn025View ArticleGoogle Scholar
  111. McKee AS, Munks MW, MacLeod MK, Fleenor CJ, Van Rooijen N, Kappler JW, Marrack P (2009) Alum induces innate immune responses through macrophage and mast cell sensors, but these sensors are not required for alum to act as an adjuvant for specific immunity. J Immunol 183(7):4403–4414View ArticleGoogle Scholar
  112. McKee AS, Burchill MA, Munks MW, Jin L, Kappler JW, Friedman RS, Jacobelli J, Marrack P (2013) Host DNA released in response to aluminum adjuvant enhances MHC class II-mediated antigen presentation and prolongs CD4 T-cell interactions with dendritic cells. Proc Natl Acad Sci U S A 110(12):E1122-1131. doi:1300392110Google Scholar
  113. Miliauskas JR, Mukherjee T, Dixon B (1993) Postimmunization (vaccination) injection-site reactions. A report of four cases and review of the literature. Am J Surg Pathol 17(5):516–524View ArticleGoogle Scholar
  114. Mitchell LA, Henderson AJ, Dow SW (2012) Suppression of vaccine immunity by inflammatory monocytes. J Immunol 189(12):5612–5621, doi:jimmunol.1202151View ArticleGoogle Scholar
  115. Mold M, Eriksson H, Siesjo P, Darabi A, Shardlow E, Exley C (2014) Unequivocal identification of intracellular aluminium adjuvant in a monocytic THP-1 cell line. Sci Rep 4:6287. doi:srep06287Google Scholar
  116. Morefield GL, Sokolovska A, Jiang D, HogenEsch H, Robinson JP, Hem SL (2005) Role of aluminum-containing adjuvants in antigen internalization by dendritic cells in vitro. Vaccine 23(13):1588–1595View ArticleGoogle Scholar
  117. Morefield GL, Tammariello RF, Purcell BK, Worsham PL, Chapman J, Smith LA, Alarcon JB, Mikszta JA, Ulrich RG (2008) An alternative approach to combination vaccines: intradermal administration of isolated components for control of anthrax, botulism, plague and staphylococcal toxic shock. J Immune Based Ther Vaccines 6:5. doi:1476-8518-6-5Google Scholar
  118. Mosca F, Tritto E, Muzzi A, Monaci E, Bagnoli F, Iavarone C, O'Hagan D, Rappuoli R, De Gregorio E (2008) Molecular and cellular signatures of human vaccine adjuvants. Proc Natl Acad Sci U S A 105(30):10501–10506View ArticleGoogle Scholar
  119. Mueller DL, Jenkins MK, Schwartz RH (1989) Clonal expansion versus functional clonal inactivation: a costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy. Annu Rev Immunol 7:445–480View ArticleGoogle Scholar
  120. Munks MW, McKee AS, Macleod MK, Powell RL, Degen JL, Reisdorph NA, Kappler JW, Marrack P (2010) Aluminum adjuvants elicit fibrin-dependent extracellular traps in vivo. Blood 116(24):5191–5199View ArticleGoogle Scholar
  121. Nakahira K, Haspel JA, Rathinam VA, Lee SJ, Dolinay T, Lam HC, Englert JA, Rabinovitch M, Cernadas M, Kim HP, Fitzgerald KA, Ryter SW, Choi AM (2011) Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome. Nat Immunol 12(3):222–230. doi:ni.1980Google Scholar
  122. Nelson CA, Vidavsky I, Viner NJ, Gross ML, Unanue ER (1997) Amino-terminal trimming of peptides for presentation on major histocompatibility complex class II molecules. Proc Natl Acad Sci U S A 94(2):628–633View ArticleGoogle Scholar
  123. Newman MJ, Wu JY, Gardner BH, Munroe KJ, Leombruno D, Recchia J, Kensil CR, Coughlin RT (1992) Saponin adjuvant induction of ovalbumin-specific CD8+ cytotoxic T lymphocyte responses. J Immunol 148(8):2357–2362Google Scholar
  124. Noe SM, Green MA, HogenEsch H, Hem SL (2010) Mechanism of immunopotentiation by aluminum-containing adjuvants elucidated by the relationship between antigen retention at the inoculation site and the immune response. Vaccine 28(20):3588–3594View ArticleGoogle Scholar
  125. O'Hagan DT, De Gregorio E (2009) The path to a successful vaccine adjuvant–'the long and winding road'. Drug Discov Today 14(11–12):541–551View ArticleGoogle Scholar
  126. Ohlsson L, Exley C, Darabi A, Sanden E, Siesjo P, Eriksson H (2013) Aluminium based adjuvants and their effects on mitochondria and lysosomes of phagocytosing cells. J Inorg Biochem 128:229–236. doi:S0162-0134(13)00208-0Google Scholar
  127. Osebold JW (1982) Mechanisms of action by immunologic adjuvants. J Am Vet Med Assoc 181(10):983–987Google Scholar
  128. Pai SY, Truitt ML, Ho IC (2004) GATA-3 deficiency abrogates the development and maintenance of T helper type 2 cells. Proc Natl Acad Sci U S A 101(7):1993–1998, doi:10.1073/pnas.0308697100View ArticleGoogle Scholar
  129. Pashine A, Valiante NM, Ulmer JB (2005) Targeting the innate immune response with improved vaccine adjuvants. Nat Med 11(4 Suppl):S63–68View ArticleGoogle Scholar
  130. Polley MJ, Nachman RL (1975) Ultrastructural lesions on the surface of platelets associated with either blood coagulation or with antibody-mediated immune injury. J Exp Med 141(6):1261–1268View ArticleGoogle Scholar
  131. Pollock JD, Williams DA, Gifford MA, Li LL, Du X, Fisherman J, Orkin SH, Doerschuk CM (1995) Dinauer MC Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production. Nat Genet 9(2):202–209, doi:10.1038/ng0295-202View ArticleGoogle Scholar
  132. Pollock KG, Conacher M, Wei XQ, Alexander J, Brewer JM (2003) Interleukin-18 plays a role in both the alum-induced T helper 2 response and the T helper 1 response induced by alum-adsorbed interleukin-12. Immunology 108(2):137–143View ArticleGoogle Scholar
  133. Powell M, Newman M, Hem S, White J (1995) Structure and Properties of Aluminum-Containing Adjuvants. In: Vaccine Design, vol 6. Pharmaceutical Biotechnology. Springer, US, pp 249–276Google Scholar
  134. Qin H, Cha SC, Neelapu SS, Lou Y, Wei J, Liu YJ, Kwak LW (2009) Vaccine site inflammation potentiates idiotype DNA vaccine-induced therapeutic T cell-, and not B cell-, dependent antilymphoma immunity. Blood 114(19):4142–4149View ArticleGoogle Scholar
  135. Rahman F, Dahmen A, Herzog-Hauff S, Bocher WO, Galle PR, Lohr HF (2000) Cellular and humoral immune responses induced by intradermal or intramuscular vaccination with the major hepatitis B surface antigen. Hepatol 31(2):521–527. doi:S0270913900417696Google Scholar
  136. Ramanathan VD, Badenoch-Jones P, Turk JL (1979) Complement activation by aluminium and zirconium compounds. Immunology 37(4):881–888Google Scholar
  137. Rappuoli R, Miller HI, Falkow S (2002) The Intangible Value of Vaccination. Science 297(5583):937–939, doi:10.1126/science.1075173View ArticleGoogle Scholar
  138. Re F (2011) Inflammasome Activation by Pathogenic Crystals and Particles. In: PÃtrilli V, Martinon F (eds) Couillin I. The Inflammasomes. Progress in Inflammation Research, Springer Basel, pp 85–99Google Scholar
  139. Reed SG, Bertholet S, Coler RN, Friede M (2009) New horizons in adjuvants for vaccine development. Trends Immunol 30(1):23–32View ArticleGoogle Scholar
  140. Reed SG, Orr MT, Fox CB (2013) Key roles of adjuvants in modern vaccines. Nat Med 19(12):1597–1608, doi:nm.3409View ArticleGoogle Scholar
  141. Reis e Sousa C (2006) Dendritic cells in a mature age. Nat Rev Immunol 6(6):476–483View ArticleGoogle Scholar
  142. Reis e Sousa C, Germain RN (1995) Major histocompatibility complex class I presentation of peptides derived from soluble exogenous antigen by a subset of cells engaged in phagocytosis. J Exp Med 182(3):841–851View ArticleGoogle Scholar
  143. Rimaniol AC, Gras G, Verdier F, Capel F, Grigoriev VB, Porcheray F, Sauzeat E, Fournier JG, Clayette P, Siegrist CA, Dormont D (2004) Aluminum hydroxide adjuvant induces macrophage differentiation towards a specialized antigen-presenting cell type. Vaccine 22(23–24):3127–3135View ArticleGoogle Scholar
  144. Rock KL, Latz E, Ontiveros F, Kono H (2010) The sterile inflammatory response. Annu Rev Immunol 28:321–342, doi:10.1146/annurev-immunol-030409-101311View ArticleGoogle Scholar
  145. Romero Mendez IZ, Shi Y, HogenEsch H, Hem SL (2007) Potentiation of the immune response to non-adsorbed antigens by aluminum-containing adjuvants. Vaccine 25(5):825–833, doi:S0264-410X(06)01036-XView ArticleGoogle Scholar
  146. Rudensky A, Preston-Hurlburt P, Hong SC, Barlow A, Janeway CA Jr (1991) Sequence analysis of peptides bound to MHC class II molecules. Nature 353(6345):622–627View ArticleGoogle Scholar
  147. Rudensky A, Preston-Hurlburt P, al-Ramadi BK, Rothbard J, Janeway CA Jr (1992) Truncation variants of peptides isolated from MHC class II molecules suggest sequence motifs. Nature 359(6394):429–431, doi:10.1038/359429a0View ArticleGoogle Scholar
  148. Rush CM, Brewer JM (2010) Tracking dendritic cells in vivo. Methods Mol Biol 626:169–185View ArticleGoogle Scholar
  149. Sadik CD, Kim ND, Luster AD (2011) Neutrophils cascading their way to inflammation. Trends Immunol 32(10):452–460, doi:S1471-4906(11)00114-1View ArticleGoogle Scholar
  150. Sallusto F, Palermo B, Lenig D, Miettinen M, Matikainen S, Julkunen I, Forster R, Burgstahler R, Lipp M, Lanzavecchia A (1999) Distinct patterns and kinetics of chemokine production regulate dendritic cell function. Eur J Immunol 29(5):1617–1625View ArticleGoogle Scholar
  151. Sauter B, Albert ML, Francisco L, Larsson M, Somersan S, Bhardwaj N (2000) Consequences of cell death: exposure to necrotic tumor cells, but not primary tissue cells or apoptotic cells, induces the maturation of immunostimulatory dendritic cells. J Exp Med 191(3):423–434View ArticleGoogle Scholar
  152. Schroder K, Tschopp J (2010) The inflammasomes. Cell 140(6):821–832. doi:S0092-8674(10)00075-9Google Scholar
  153. Seeber SJ, White JL, Hem SL (1991) Solubilization of aluminum-containing adjuvants by constituents of interstitial fluid. J Parenter Sci Technol 45(3):156–159Google Scholar
  154. Serre K, Mohr E, Toellner KM, Cunningham AF, Granjeaud S, Bird R, MacLennan IC (2008) Molecular differences between the divergent responses of ovalbumin-specific CD4 T cells to alum-precipitated ovalbumin compared to ovalbumin expressed by Salmonella. Mol Immunol 45(13):3558–3566View ArticleGoogle Scholar
  155. Serre K, Mohr E, Toellner KM, Cunningham AF, Bird R, Khan M, MacLennan IC (2009) Early simultaneous production of intranodal CD4 Th2 effectors and recirculating rapidly responding central-memory-like CD4 T cells. Eur J Immunol 39(6):1573–1586, doi:10.1002/eji.200838922View ArticleGoogle Scholar
  156. Serre K, Mohr E, Gaspal F, Lane PJ, Bird R, Cunningham AF, Maclennan IC (2010) IL-4 directs both CD4 and CD8 T cells to produce Th2 cytokines in vitro, but only CD4 T cells produce these cytokines in response to alum-precipitated protein in vivo. Mol Immunol 47(10):1914–1922. doi:S0161-5890(10)00090-8Google Scholar
  157. Serre K, Mohr E, Benezech C, Bird R, Khan M, Caamano JH, Cunningham AF, Maclennan IC (2011) Selective effects of NF-kappaB1 deficiency in CD4(+) T cells on Th2 and TFh induction by alum-precipitated protein vaccines. Eur J Immunol 41(6):1573–1582, doi:10.1002/eji.201041126View ArticleGoogle Scholar
  158. Seubert A, Monaci E, Pizza M, O'Hagan DT, Wack A (2008) The adjuvants aluminum hydroxide and MF59 induce monocyte and granulocyte chemoattractants and enhance monocyte differentiation toward dendritic cells. J Immunol 180(8):5402–5412View ArticleGoogle Scholar
  159. Shimada K, Crother TR, Karlin J, Dagvadorj J, Chiba N, Chen S, Ramanujan VK, Wolf AJ, Vergnes L, Ojcius DM, Rentsendorj A, Vargas M, Guerrero C, Wang Y, Fitzgerald KA, Underhill DM, Town T, Arditi M (2012) Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis. Immunity 36(3):401–414. doi:S1074-7613(12)00044-1Google Scholar
  160. Shirodkar S, Hutchinson RL, Perry DL, White JL, Hem SL (1990) Aluminum compounds used as adjuvants in vaccines. Pharm Res 7(12):1282–1288View ArticleGoogle Scholar
  161. Soehnlein O, Lindbom L (2010) Phagocyte partnership during the onset and resolution of inflammation. Nat Rev Immunol 10(6):427–439, doi:nri2779View ArticleGoogle Scholar
  162. Sokolovska A, Hem SL, HogenEsch H (2007) Activation of dendritic cells and induction of CD4(+) T cell differentiation by aluminum-containing adjuvants. Vaccine 25(23):4575–4585View ArticleGoogle Scholar
  163. Sun H, Pollock KG, Brewer JM (2003) Analysis of the role of vaccine adjuvants in modulating dendritic cell activation and antigen presentation in vitro. Vaccine 21(9–10):849–855View ArticleGoogle Scholar
  164. Tengvall P, Askendal A, Lundstrom I (1998) Studies on protein adsorption and activation of complement on hydrated aluminium surfaces in vitro. Biomaterials 19(10):935–940. doi:S0142-9612(98)00005-2Google Scholar
  165. Toellner KM, Luther SA, Sze DM, Choy RK, Taylor DR, MacLennan IC, Acha-Orbea H (1998) T helper 1 (Th1) and Th2 characteristics start to develop during T cell priming and are associated with an immediate ability to induce immunoglobulin class switching. J Exp Med 187(8):1193–1204View ArticleGoogle Scholar
  166. Tritto E, Mosca F, De Gregorio E (2009) Mechanism of action of licensed vaccine adjuvants. Vaccine 27(25–26):3331–3334View ArticleGoogle Scholar
  167. Trombetta ES, Mellman I (2005) Cell biology of antigen processing in vitro and in vivo. Annu Rev Immunol 23:975–1028View ArticleGoogle Scholar
  168. Ulanova M, Tarkowski A, Hahn-Zoric M, Hanson LA (2001) The Common vaccine adjuvant aluminum hydroxide up-regulates accessory properties of human monocytes via an interleukin-4-dependent mechanism. Infect Immun 69(2):1151–1159View ArticleGoogle Scholar
  169. van Panhuys N, Tang SC, Prout M, Camberis M, Scarlett D, Roberts J, Hu-Li J, Paul WE, Le Gros G (2008) In vivo studies fail to reveal a role for IL-4 or STAT6 signaling in Th2 lymphocyte differentiation. Proc Natl Acad Sci U S A 105(34):12423–12428. doi:0806372105Google Scholar
  170. Van Regenmortel MH (2004) Reductionism and complexity in molecular biology. Scientists now have the tools to unravel biological and overcome the limitations of reductionism. EMBO Rep 5(11):1016–1020. doi:7400284Google Scholar
  171. Verdier F, Burnett R, Michelet-Habchi C, Moretto P, Fievet-Groyne F, Sauzeat E (2005) Aluminium assay and evaluation of the local reaction at several time points after intramuscular administration of aluminium containing vaccines in the Cynomolgus monkey. Vaccine 23(11):1359–1367. doi:S0264-410X(04)00688-7Google Scholar
  172. Vessely C, Estey T, Randolph TW, Henderson I, Cooper J, Nayar R, Braun LJ, Carpenter JF (2009) Stability of a trivalent recombinant protein vaccine formulation against botulinum neurotoxin during storage in aqueous solution. J Pharm Sci 98(9):2970–2993, doi:10.1002/jps.21498 2009View ArticleGoogle Scholar
  173. Vogelbruch M, Nuss B, Korner M, Kapp A, Kiehl P, Bohm W (2000) Aluminium-induced granulomas after inaccurate intradermal hyposensitization injections of aluminium-adsorbed depot preparations. Allergy 55(9):883–887Google Scholar
  174. Walls RS (1977) Eosinophil response to alum adjuvants: involvement of T cells in non-antigen-dependent mechanisms. Proc Soc Exp Biol Med 156(3):431–435View ArticleGoogle Scholar
  175. Wang Y, Rahman D, Lehner T (2012) A comparative study of stress-mediated immunological functions with the adjuvanticity of alum. J Biol Chem 287(21):17152–17160. doi:M112.347179Google Scholar
  176. Wang XY, Yao X, Wan YM, Wang B, Xu JQ, Wen YM (2013) Responses to multiple injections with alum alone compared to injections with alum adsorbed to proteins in mice. Immunol Lett 149(1–2):88–92. doi:S0165-2478(12)00242-8Google Scholar
  177. Watkinson A, Soliakov A, Ganesan A, Hirst K, Lebutt C, Fleetwood K, Fusco PC, Fuerst TR, Lakey JH (2013) Increasing the potency of an alhydrogel-formulated anthrax vaccine by minimizing antigen-adjuvant interactions. Clin Vaccine Immunol 20(11):1659–1668, doi:CVI.00320-13View ArticleGoogle Scholar
  178. Watts C (1997) Capture and processing of exogenous antigens for presentation on MHC molecules. Annu Rev Immunol 15:821–850View ArticleGoogle Scholar
  179. White RG, Coons AH, Connolly JM (1955) Studies on antibody production. III The alum granuloma. J Exp Med 102(1):73–82View ArticleGoogle Scholar
  180. WHO (1977) Manual for the production and control of vaccines - Tetanus Toxoid. BLG/UNDP/77.2 Rev. 1. World Health Organization, Geneva. Available in
  181. WHO (2010a) Polio vaccines and polio immunization in the pre-eradication era: WHO position paper. Wkly Epidemiol Rec 85(23):213–228Google Scholar
  182. WHO (2010b) WHO vaccine-preventable diseases:monitoring system, 2010 global summary. Organisation, World HealthGoogle Scholar
  183. WHO (2014) Poliomyelitis. Accessed 4 February, 2015
  184. Yanagihara S, Komura E, Nagafune J, Watarai H, Yamaguchi Y (1998) EBI1/CCR7 is a new member of dendritic cell chemokine receptor that is up-regulated upon maturation. J Immunol 161(6):3096–3102Google Scholar
  185. Yang CW, Strong BS, Miller MJ, Unanue ER (2010) Neutrophils influence the level of antigen presentation during the immune response to protein antigens in adjuvants. J Immunol 185(5):2927–2934. doi:jimmunol.1001289Google Scholar
  186. Zhou R, Tardivel A, Thorens B, Choi I, Tschopp J (2010) Thioredoxin-interacting protein links oxidative stress to inflammasome activation. Nat Immunol 1(2):136–140. doi:ni.1831Google Scholar
  187. Zhou R, Yazdi AS, Menu P, Tschopp J (2011) A role for mitochondria in NLRP3 inflammasome activation. Nature 469(7329):221–225. doi:nature09663Google Scholar
  188. Zhu J, Yamane H, Paul WE (2010) Differentiation of effector CD4 T cell populations (*). Annu Rev Immunol 28:445–489. doi:10.1146/annurev-immunol-030409-101212Google Scholar
  189. Zlatkovic J, Tsouchnikas G, Jarmer J, Koessl C, Stiasny K, Heinz FX (2013) Aluminum hydroxide influences not only the extent but also the fine specificity and functional activity of antibody responses to tick-borne encephalitis virus in mice. J Virol 87(22):12187–12195, doi:JVI.01690-13View ArticleGoogle Scholar


© Ghimire; licensee Springer. 2015

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.