Aluminium-based Adjuvants Research Articles

Adsorption and Desorption of Immune-Modulating Substances by Aluminium-Based Adjuvants: An Overlooked Feature of the Immune-Stimulating Mechanisms of Aluminium-Based Adjuvants.

Vaccine antigens are partly adsorbed onto aluminium-based adjuvant particles, forming an unstable corona. At the inoculation site, the corona will be restructured, and the adsorbed antigens will be released through replacement with biomolecules from the interstitial fluid of the recipient. Aluminium-based adjuvants (ABAs) carrying a corona of serum proteins as a model of particles with a pre-formed antigen corona were shown to adsorb several categories of cytokines and growth factors, as assessed from a protein array covering 18 different analytes. Out of the 18 analytes, 12 were shown to be adsorbed by the aluminium-based adjuvant Alhydrogel®, which had a pre-formed protein corona. The adsorption of TNF-α, IL-2, IL-4, IL-10, and IFN-γ was studied in detail. Among the studied cytokines, IL-2, IL-4, and IFN-γ, were adsorbed by Alhydrogel®. Adsorbed IFN-γ was further studied to show that the adsorption of IFN-γ did not denature the cytokine, and the cytokine could be desorbed from adjuvant particles in a biologically active form and in relevant amounts. The adsorption of immune-stimulating molecules onto ABAs at the administration site of a vaccine is a neglected event in the mode of action of aluminium-based adjuvants. This process may modulate the immune response with a profound impact on initiating the innate immune response and consequently the adaptive immune response.

Evaluation of Aluminium Hydroxide Nanoparticles as an Efficient Adjuvant to Potentiate the Immune Response against Clostridium botulinum Serotypes C and D Toxoid Vaccines.

Clostridium botulinum serotypes C and D cause botulism in livestock, a neuroparalytic disease that results in substantial economic losses. Vaccination with aluminium-based toxoid vaccines is widely used to control the spread of botulism. Aluminium-based adjuvants are preferred owing to their apparent stimulation of the immune responses to toxoid vaccines when compared to other adjuvants. The aim of our study was to evaluate aluminium hydroxide nanoparticles as a potential substitute for alhydrogel in the botulism bivalent vaccine. Botulism vaccines were formulated with either alhydrogel or nanoalum and comparative efficacy between the two formulations was conducted by evaluating the immune response in vaccinated guinea pigs. A significant increase in immunological parameters was observed, with the antibody titres higher in the serum of guinea pigs (20 IU/mL of anti-BoNT C/D) injected with nanoalum-containing vaccine than guinea pigs inoculated with the standard alhydrogel-containing vaccine (8.7 IU/mL and 10 IU/mL of anti-BoNT C and anti-BoNT D, respectively). Additionally, the nanoalum-containing vaccine demonstrated potency in a multivalent vaccine (20 IU/mL of anti-BoNT C/D), while the standard alhydrogel-containing vaccine showed a decline in anti-BoNT C (5 IU/mL) antibody titres.

Review: Unravelling the Role of DNA Sensing in Alum Adjuvant Activity.

Public interest in vaccines is at an all-time high following the SARS-CoV-2 global pandemic. Currently, over 6 billion doses of various vaccines are administered globally each year. Most of these vaccines contain Aluminium-based adjuvants (alum), which have been known and used for almost 100 years to enhance vaccine immunogenicity. However, despite the historical use and importance of alum, we still do not have a complete understanding of how alum works to drive vaccine immunogenicity. In this article, we critically review studies investigating the mechanisms of action of alum adjuvants, highlighting some of the misconceptions and controversies within the area. Although we have emerged with a clearer understanding of how this ubiquitous adjuvant works, we have also highlighted some of the outstanding questions in the field. While these may seem mainly of academic interest, developing a more complete understanding of these mechanisms has the potential to rationally modify and improve the immune response generated by alum-adjuvanted vaccines.

A prophylactic effect of aluminium-based adjuvants against respiratory viruses via priming local innate immunity

ABSTRACT Infection caused by respiratory viruses can lead to a severe respiratory disease and even death. Vaccination is the most effective way to prevent the disease, but it cannot be quickly applied when facing an emerging infectious disease. Here, we demonstrated that immunization with an aluminium-zinc hybrid particulate adjuvant (FH-001) alone, bearing great resemblance in morphology with commonly used aluminium-based adjuvants in vaccines, could quickly induce mice to generate a broadly protective immune response to resist the lethal challenge of influenza B viruses. Furthermore, a multi-omics-based analysis revealed that the alveolar macrophage and type I interferon pathway, rather than adaptive immunity and type II interferon pathway, were essential for the observed prophylactic effect of FH-001. More importantly, a similar protective effect was observed against influenza A virus strain A/Shanghai/02/2013(H7N9), A/California/04/2009(H1N1) and respiratory syncytial virus. Therefore, we introduced here a new and promising strategy that can be quickly applied during the outbreak of emerging respiratory viruses.

About the alleged toxicity of aluminium-based adjuvants in vaccines: All published studies should be taken into account

About the alleged toxicity of aluminium-based adjuvants in vaccines: All published studies should be taken into account

Aluminiodun txerto-laguntzaileen eraginaren azterketa sistemen biologiaren bidez

Hamarkadak dira aluminio konposatuak txertoen laguntzaile gisa erabiltzen direla albaitaritzan eta giza txertoetan. Laguntzaile horien erabilera oso hedatua egon arren, ez da ongi ezagutzen zein mekanismoren bidez eragiten dituzten ondorio onuragarriak eta, aldizka, kontrako erreakzioak eragin ditzakete. Sistemen biologiako tekniken bidez laguntzaile hauen sinadura molekularra ikertu da, gizakietan zein etxabereetan. Hainbat eragin-mekanismo proposatu dira aluminioaren sistema immunea pizteko ahalmena azaltzeko eta analisi transkriptomikoetan erlazionatutako geneak aurkitu dira. Batzuetan emaitzak kontraesanezkoak dira, aktibatzen den erantzun immunea desberdina izan daitekeelako egoeraren arabera. Aluminiogatzen tamainak, formak, propietate fisiko-kimikoek eta antigenoen absortzioak eragina dutela ematen du era berean. Laguntzaile hauek ez dira antigeno-garraiatzaile soilak, sistema immunea kitzikatzen duten arrisku-seinale endogenoak pizten baitituzte. Argitzeke dago oraindik haien eragin-mekanismo zehatza eta epe luzeko aktibazio immunitarioak sistema immunitarioaren gainestimulazioa eragin dezakeen.

COVID-19 vaccines: neutralizing antibodies and the alum advantage.

Achieving high levels of neutralizing antibodies to the spike protein of SARS-CoV-2 in a safe manner is likely to be crucial for an effective vaccine. Here, we propose that aluminium-based adjuvants might hold the key to this. Here, Peter Hotez and colleagues discuss the advantages of using an aluminium-based adjuvant in candidate COVID-19 vaccines.

β-glucan as a new tool in vaccine development.

Vaccination constitutes one of the major breakthroughs in human medicine. At the same time, development of more immunogenic vaccine alternatives to using aluminium-based adjuvants is one of the most important phases of vaccination development. Among different sources of carbohydrate polymers, including plants, microbes and synthetic sources tested, glucans were found to be the most promising vaccine adjuvant, as they alone stimulate various immune reactions including antibody production without any negative side effects. The use of glucan particles as a delivery system is a viable option based on the documented efficient antigen loading and receptor-targeted uptake in antigen-presenting cells. In addition to particles, soluble glucans can be used as novel hydrogels or as direct immunocyte-targeting delivery systems employing novel complexes with oligodeoxynucleotides. This review focuses on recent advances in glucan-based vaccine development from glucan-based conjugates to a glucan-based delivery and adjuvant platform.

The interaction of aluminium-based adjuvants with THP-1 macrophages in vitro: Implications for cellular survival and systemic translocation

Within clinical vaccinations, recombinant antigens are routinely entrapped inside or adsorbed onto the surface of aluminium salts in order to increase their immunological potency in vivo. The efficacy of these immunisations is highly dependent upon the recognition and uptake of these complexes by professional phagocytes and their subsequent delivery to the draining lymph nodes for further immunological processing. While monocytes have been shown to internalise aluminium adjuvants and their adsorbates, the role of macrophages in this respect has not been fully established. Furthermore, this study explored the interaction of THP-1 macrophages with aluminium-based adjuvants (ABAs) and how this relationship influenced the survival of such cells in vitro. THP-1 macrophages were exposed to low concentrations of ABAs (1.7 μg/mL Al) for a maximum of seven days. ABA uptake was determined using lumogallion staining and cell viability by both DAPI (4′,6-diamidino-2-phenylindole) staining and LDH (lactate dehydrogenase) assay. Evidence of ABA particle loading was identified within cells at early junctures following treatment and appeared to be quite prolific (>90% cells positive for Al signal after 24 h). Total sample viability (% LDH release) in treated samples was predominantly similar to untreated cells and low levels of cellular death were consistently observed in populations positive for Al uptake. It can thus be concluded that aluminium salts can persist for some time within the intracellular environment of these cells without adversely affecting their viability. These results imply that macrophages may play a role in the systemic translocation of ABAs once administered in the form of an inoculation.

Tracing Aluminium-based Adjuvants: Their Interactions with Immune Competent Cells and their Effect on Mitochondrial Activity

Background: Studies revealing the immune stimulatory properties of aluminium-based adjuvants (ABAs) have been impaired by the absence of simple and reliable methods of tracing the adjuvants and their effect on biochemical processes upon endocytosis. Objective: To verify that labelling of ABAs with lumogallion doesn’t affect the physicochemical properties of the adjuvant; tracing cellular interaction with aluminium adjuvants; explore their effect on metabolic activity upon endocytosis. Methods: Physicochemical characterization by Z-potential and size distribution of ABAs labelled with lumogallion. Cellular interactions with ABAs by flow cytometry and confocal microscopy. Metabolic activity explored by measuring transformation of tetrazolium into formazan. Results: No or minor change of zeta potential and average particle size of lumogallion labelled aluminium oxyhydroxide, AlO(OH) and aluminium hydroxyphosphate, Al(OH)x(PO4)y. Both phagocytosing and non-phagocytosing leukocytes became associated with ABAs at concentrations expected after in vivo administration of a vaccine. The ABAs were relatively toxic, affecting both lymphocytes and monocytes, and Al(OH)x(PO4)y was more toxic than AlO(OH). Endocytosed aluminium adjuvant particles were not secreted from the cells and remained intracellular throughout several cell divisions. The presence of ABAs increased the mitochondrial activity of the monocytic cell line THP-1 and peripheral monocytes, as based on the transformation of tetrazolium into formazan. Conclusion: Lumogallion labelled ABAs is a valuable tool tracing interactions between ABAs and cells. Labelled ABAs can be traced intracellularly and ABAs are likely to remain intracellular for a long period of time. Intracellular ABAs increase the mitochondrial activity and the presence of intracellular Al ions is suggested to cause an increased mitochondrial activity.

Intraperitoneal administration of aluminium-based adjuvants produces severe transient systemic adverse events in mice

Vaccines typically come with adjuvants that trigger the innate immune system in order to prepare best possible inflammatory conditions as to allow the adaptive immune system to become activated, generally for the induction of antibodies. The oldest approved and most abundant immunological adjuvants are salts of aluminium, which are also frequently used in animal models of immunisation and allergy desensitization. In rodents, the intraperitoneal administration of aluminium adjuvants is commonly performed and considered safe. In the current investigation, we show that intraperitoneal administration of aluminium adjuvants is associated with a dose-dependent hypothermic reaction within 10 min of the injection. The body temperature of mice dropped as much as 4 °C, and the clinical symptoms included apathy, hunched posture, and piloerection. The temperature normalised and other clinical manifestations disappeared within 60–80 min of the intraperitoneal aluminium injection, which caused strong infiltration of neutrophil and eosinophil granulocytes into the peritoneal cavity, a clinical manifestations typically associated with inflammasome activation. However, the observed reactions to aluminium adjuvants were independent of NALP3, caspase-1, and interleukin-1β, but dependent on histamine. Hence, aluminium adjuvants may have potential local and systemic side effects, which warrants further investigations into the nature of these side effects, but also into the possible implications on health in man.

Sequestering of damage-associated molecular patterns (DAMPs): a possible mechanism affecting the immune-stimulating properties of aluminium adjuvants

Aluminium-based adjuvants (ABAs) have been used in human and veterinary vaccines for decades, and for a long time, the adjuvant properties were believed to be mediated by an antigen depot at the injection site, prolonging antigen exposure to the immune system. The depot hypothesis is today more or less abandoned, and instead replaced by the assumption that ABAs induce an inflammation at the injection site. Induction of an inflammatory response is consistent with immune activation initiated by recognition of molecular patterns associated with danger or damage (DAMPs), and the latter are derived from endogenous molecules that normally reside intracellularly. When extracellularly expressed, because of damage, stress or cell death, a sterile inflammation is induced. In this paper, we report the induction of DAMP release by viable cells after phagocytosis of aluminium-based adjuvants. Two of the most commonly used ABAs in pharmaceutical vaccine formulations, aluminium oxyhydroxide and aluminium hydroxyphosphate, induced a vigorous extracellular expression of the two DAMP molecules calreticulin and HMGB1. Concomitantly, extracellular adjuvant particles adsorbed the DAMP molecules released by the cells whereas IL-1β, a previously reported inflammatory mediator induced by ABAs, was not absorbed by the adjuvants. Induction of extracellular expression of the two DAMP molecules was more prominent using aluminium hydroxyphosphate compared to aluminium oxyhydroxide, whereas the extracellular adsorption of the DAMP molecules was more pronounced with the latter. Furthermore, it is hypothesised how induction of DAMP expression by ABAs and their concomitant adsorption by extracellular adjuvants may affect the inflammatory properties of ABAs.

Neutrophil swarming and extracellular trap formation play a significant role in Alum adjuvant activity

There are over 6 billion vaccine doses administered each year, most containing aluminium-based adjuvants, yet we still do not have a complete understanding of their mechanisms of action. Recent evidence has identified host DNA and downstream sensing as playing a significant role in aluminium adjuvant (aluminium hydroxide) activity. However, the cellular source of this DNA, how it is sensed by the immune system and the consequences of this for vaccination remains unclear. Here we show that the very early injection site reaction is characterised by inflammatory chemokine production and neutrophil recruitment. Intravital imaging demonstrates that the Alum injection site is a focus of neutrophil swarms and extracellular DNA strands. These strands were confirmed as neutrophil extracellular traps due to their sensitivity to DNAse and absence in mice deficient in peptidylarginine deiminase 4. Further studies in PAD4−/− mice confirmed a significant role for neutrophil extracellular trap formation in the adjuvant activity of Alum. By revealing neutrophils recruited to the site of Alum injection as a source of the DNA that is detected by the immune system this study provides the missing link between Alum injection and the activation of DNA sensors that enhance adjuvant activity, elucidating a key mechanism of action for this important vaccine component.

Insight into the cellular fate and toxicity of aluminium adjuvants used in clinically approved human vaccinations.

Aluminium adjuvants remain the most widely used and effective adjuvants in vaccination and immunotherapy. Herein, the particle size distribution (PSD) of aluminium oxyhydroxide and aluminium hydroxyphosphate adjuvants was elucidated in attempt to correlate these properties with the biological responses observed post vaccination. Heightened solubility and potentially the generation of Al3+ in the lysosomal environment were positively correlated with an increase in cell mortality in vitro, potentially generating a greater inflammatory response at the site of simulated injection. The cellular uptake of aluminium based adjuvants (ABAs) used in clinically approved vaccinations are compared to a commonly used experimental ABA, in an in vitro THP-1 cell model. Using lumogallion as a direct-fluorescent molecular probe for aluminium, complemented with transmission electron microscopy provides further insight into the morphology of internalised particulates, driven by the physicochemical variations of the ABAs investigated. We demonstrate that not all aluminium adjuvants are equal neither in terms of their physical properties nor their biological reactivity and potential toxicities both at the injection site and beyond. High loading of aluminium oxyhydroxide in the cytoplasm of THP-1 cells without immediate cytotoxicity might predispose this form of aluminium adjuvant to its subsequent transport throughout the body including access to the brain.

IL-33 released by alum is responsible for early cytokine production and has adjuvant properties.

Human vaccines have used aluminium-based adjuvants (alum) for >80 years despite incomplete understanding of how alum enhances the immune response. Alum can induce the release of endogenous danger signals via cellular necrosis which elicits inflammation-associated cytokines resulting in humoral immunity. IL-33 is proposed to be one such danger signal that is released from necrotic cells. Therefore, we investigated whether there is a role for IL-33 in the adjuvant activity of alum. We show that alum-induced cellular necrosis results in elevated levels of IL-33 following injection in vivo. Alum and IL-33 induce similar increases in IL-5, KC, MCP-1, MIP-1α and MIP-1β; many of which are dependent on IL-33 as shown in IL-33 knockout mice or by using an IL-33-neutralizing recombinant ST2 receptor. Furthermore, IL-33 itself functions as an adjuvant that, while only inducing a marginal primary response, facilitates a robust secondary response comparable to that observed with alum. However, IL-33 is not absolutely required for alum-induced antibody responses since alum mediates similar humoral responses in IL-33 knockout and wild-type mice. Our results provide novel insights into the mechanism of action behind alum-induced cytokine responses and show that IL-33 is sufficient to provide a robust secondary antibody response independently of alum.

IL-33 drives alum-induced cytokine production and provides a robust secondary antibody response independently of alum (CCR4P.211)

Abstract For over 80 years, human vaccines have used aluminium-based adjuvants (alum) despite incomplete understanding of how alum enhances the immune response. One proposed mechanism is that alum induces cellular necrosis and release of endogenous danger signals. These signals induce inflammation-associated cytokines that lead to humoral immunity. IL-33 is proposed to be released from necrotic cells and can elicit TH2-biased cytokines. Therefore, we investigated a role for IL-33 in the adjuvant activity of alum. We show that alum induces cellular necrosis and elevates IL-33 levels shortly after intraperitoneal administration. Alum or IL-33 injection induces similar significant increases in IL-5, IL-6, KC, MCP-1 and MIP-1β. We establish that many of the alum-induced cytokines are dependent on IL-33 using IL-33 knockout mice or an IL-33-neutralizing recombinant ST2 receptor. Interestingly, IL-33 itself can function as an adjuvant. While IL-33 only induces a marginal primary response, it facilitates a secondary response comparable to alum and correlates with delayed splenic germinal center formation. Alum-induced antibody responses in IL-33 knockout mice are similar to those in wild-type mice, demonstrating that IL-33 is not absolutely required for the alum-induced humoral response. Our results provide novel insights into the mechanism of action behind alum-induced cytokine responses and show that IL-33 is sufficient to provide a robust secondary antibody response independently of alum.

Polysaccharides: Candidates of promising vaccine adjuvants.

Aluminium-based adjuvants remain the only adjuvants approved for human use in the USA for over 80 years because of alum's simplicity, tolerability, safety and cost-efficiency. Recent development of vaccines, especially the increasing applications of recombinant subunit and synthetic vaccines, makes aluminium adjuvants cannot stimulate enough immunity to the antigens, since aluminium adjuvants can only induce Th2 type immune responses. So, novel adjuvants are urgent to make up the disadvantages of aluminium adjuvants. However, some major hurdles need to be overcome, not only the scientific knowledge of adjuvants but also unacceptable side-effects and toxicity. A number of carbohydrate-based polysaccharides from plant, bacterial, yeast and synthetic sources can act as pathogen-associated molecular patterns (PAMPs) and recognize pattern recognition receptors (PRRs) on immune cells, followed by triggering innate immunity and regulating adaptive immunity. What is more, polysaccharides are safe and biodegradable without tissue deposits as observed in aluminium adjuvants. Therefore, polysaccharide-based compounds and formulations are potential vaccine adjuvant candidates. Here, we mainly review polysaccharide-based adjuvants investigated in recent years.

Aluminium-based adjuvants should not be used as placebos in clinical trials

Aluminium-based adjuvants should not be used as placebos in clinical trials

Alhydrogel ® adjuvant, ultrasonic dispersion and protein binding: A TEM and analytical study

Aluminium-based vaccine adjuvants have been in use since the 1920s. Aluminium hydroxide (alum) that is the chemical basis of Alhydrogel, a widely used adjuvant, is a colloid that binds proteins to the particular surface for efficient presentation to the immune system during the vaccination process. Using conventional TEM and cryo-TEM we have shown that Alhydrogel can be finely dispersed by ultrasonication of the aqueous suspension. Clusters of ultrasonicated aluminium hydroxide micro-fibre crystals have been produced (∼10–100 nm), that are significantly smaller than those present the untreated Alhydrogel (∼2–12 μm). However, even prolonged ultrasonication did not produce a homogenous suspension of single aluminium hydroxide micro-fibres. The TEM images of unstained and negatively stained air-dried Alhydrogel are very similar to those obtained by cryo-electron microscopy. Visualization of protein on the surface of the finely dispersed Alhydrogel by TEM is facilitated by prior ultrasonication. Several examples are given, including some of medical relevance, using proteins of widely ranging molecular mass and oligomerization state. Even with the smaller mass proteins, their presence on the Alhydrogel surface can be readily defined by TEM. It has been found that low quantities of protein tend to cross-link and aggregate the small Alhydogel clusters, in a more pronounced manner than high protein concentrations. This indicates that complete saturation of the available Alhydrogel surface with protein may be achieved, with minimal cross-linkage, and future exploitation of this treatment of Alhydrogel is likely to be of immediate value for more efficient vaccine production.

Adjuvants for the next generation of vaccines

The use of aluminium phosphate to enhance the immune response to vaccines was first described by Glenny and Pope in 19261, and for the ensuing 70 years aluminium-based adjuvants (alum) were the only adjuvants used in registered human vaccines. It is only in the last two decades that novel adjuvants have been utilised in the formulation of newly licensed human vaccines.