Aluminium adjuvants, typically referred to as 'alum', are the most commonly used adjuvants in human and animal vaccines worldwide, yet the mechanism underlying the stimulation of the immune system by alum remains unknown. Toll-like receptors are critical in sensing infections and are therefore common targets of various adjuvants used in immunological studies. Although alum is known to induce the production of proinflammatory cytokines in vitro, it has been repeatedly demonstrated that alum does not require intact Toll-like receptor signalling to activate the immune system. Here we show that aluminium adjuvants activate an intracellular innate immune response system called the Nalp3 (also known as cryopyrin, CIAS1 or NLRP3) inflammasome. Production of the pro-inflammatory cytokines interleukin-1beta and interleukin-18 by macrophages in response to alum in vitro required intact inflammasome signalling. Furthermore, in vivo, mice deficient in Nalp3, ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) or caspase-1 failed to mount a significant antibody response to an antigen administered with aluminium adjuvants, whereas the response to complete Freund's adjuvant remained intact. We identify the Nalp3 inflammasome as a crucial element in the adjuvant effect of aluminium adjuvants; in addition, we show that the innate inflammasome pathway can direct a humoral adaptive immune response. This is likely to affect how we design effective, but safe, adjuvants in the future.

Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants.

Aluminum compounds as vaccine adjuvants

The peptide binding specificities of HLA-DRB1*0401, DRB1*0101, and DRB1*0701 have been analyzed by the use of large collections of synthetic peptides corresponding to naturally occurring sequences. The results demonstrated that nearly all peptides binding to these DR molecules bear a motif characterized by a large aromatic or hydrophobic residue in position 1 (Y, F, W, L, I, V, M) and a small, noncharged residue in position 6 (S, T, C, A, P, V, I, L, M). In addition, allele-specific secondary effects and secondary anchors were defined, and these parameters were utilized to derive allele-specific motifs and algorithms. By the combined use of such algorithms, peptides capable of degenerate DRB1*0101, DRB1*0401, and DRB1*0701 binding were identified. Additional experiments utilizing a panel of quantitative assays specific for nine additional common DR molecules identified a large set of DR molecules, which includes at least the DRB1*0101, DRB1*0401, DRB1*0701, DRB5*0101, DRB1*1501, DRB1*0901, and DRB1*1302 allelic products, characterized by overlapping peptide-binding repertoires. These results have implications for understanding the molecular interactions involved in peptide-DR binding, as well as the genetic and structural basis of MHC polymorphism. These results also have potential practical implications for the development of epitope-based prophylactic and therapeutic vaccines.

Several Common HLA-DR Types Share Largely Overlapping Peptide Binding Repertoires

Alum is the only adjuvant approved for routine use in humans, although the basis for its adjuvanticity remains poorly understood. We have recently shown that alum activates caspase-1 and induces secretion of mature IL-1β and IL-18. In this study we show that, in human and mouse macrophages, alum-induced secretion of IL-1β, IL-18, and IL-33 is mediated by the NLR (nucleotide-binding domain leucine-rich repeat-containing) protein NLRP3 and its adaptor ASC, but not by NLRC4. Other particulate adjuvants, such as QuilA and chitosan, induce inflammasome activation in a NLRP3-dependent fashion, suggesting that activation of the NLRP3-inflammasome may be a common mechanism of action of particulate adjuvants. Importantly, we demonstrate that Ag-specific Ab production elicited by vaccines that contain alum is significantly impaired in NLRP3-deficient mice. Our results demonstrate for the first time a role for the NLRP3-inflammasome during development of the immune response elicited by alum-enhanced vaccination and suggest that therapeutic intervention aimed at NLRP3 may improve adjuvant efficacy.

Cutting Edge: Inflammasome activation by Alum and Alum’s adjuvant effect are mediated by NLRP3

Adjuvants are vaccine additives that stimulate the immune system without having any specific antigenic effect of itself In this study we show that alum adjuvant induces the release of IL-1beta from macrophages and dendritic cells and that this is abrogated in cells lacking various NALP3 inflammasome components The NALP3 inflammasome is also required in vivo for the innate immune response to OVA in alum The early production of IL-1beta and the influx of inflammatory cells into the peritoneal cavity is strongly reduced in NALP3-deficient mice The activation of adaptive cellular immunity to OVA-alum is initiated by monocytic dendritic cell precursors that induce the expansion of Ag-specific T cells in a NALP3-dependent way We propose that, in addition to TLR stimulators, agonists of the NALP3 inflammasome should also be considered as vaccine adjuvants

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Cutting Edge: Alum Adjuvant Stimulates Inflammatory Dendritic Cells through Activation of the NALP3 Inflammasome

Adjuvants--a balance between toxicity and adjuvanticity.

Aluminium adjuvants—in retrospect and prospect

Stability of aluminium-containing adjuvants to autoclaving.

Influence of elemental impurities in aluminum hydroxide adjuvant on the stability of inactivated Japanese Encephalitis vaccine, IXIARO®

Are mineral adjuvants triggering TLR2/TLR4 on dendritic cells by a secondary cascade reaction in vivo through the action of heat shock proteins and danger signals?

Erik B. Lindblad

Papers

Adjuvants--a balance between toxicity and adjuvanticity.

Aluminium adjuvants—in retrospect and prospect

Stability of aluminium-containing adjuvants to autoclaving.

Influence of elemental impurities in aluminum hydroxide adjuvant on the stability of inactivated Japanese Encephalitis vaccine, IXIARO®

Are mineral adjuvants triggering TLR2/TLR4 on dendritic cells by a secondary cascade reaction in vivo through the action of heat shock proteins and danger signals?