Elucidating the bioinorganic chemistry of aluminium-based adjuvants: The influence of physicochemical characteristics upon events following simulated in vitro vaccination

Despite their essential role and widespread use within clinical vaccinations, the bioinorganic chemistry of aluminium salts at the injection site remains elusive due to a lack of information concerning their physicochemical properties. Such information is essential in order to understand how these materials interact with the physiological environment and potentiate an immunological response, which is still not fully understood. These properties were extensively studied herein in order to elucidate the relationship between adjuvant physicochemistry and the events occurring post vaccination. While characterisation of zeta potential and surface functionality were both undertaken, experiments focused upon the determination of novel compositional information regarding the PSD of aluminium adjuvants. This approach involved the use of optimised DLS, TEM and filtration-GFAAS. Following characterisation, the cellular uptake and toxicological impact of aluminium salts upon model phagocytic cells (THP-1) was evaluated using lumogallion tracing and a live/dead assay respectively. Alhydrogel and Adju-Phos presented as micron sized, negatively charged aggregates in biological fluid with predominant populations existing 5.6μm respectively following 1hr exposure. Generation of Al3+ was limited within this environment (<1μg/mL over 72hrs) and both adjuvants were visible within the cytoplasm of phagocytes following 24hrs exposure, although the uptake of particles <2.7μm was considered preferable. Adju-Phos induced higher toxicity at aluminium concentrations used clinically (100μg/mL) and promoted higher levels of metabolic activity and chemokine production (MCP-1 & MIP-1α), which were attributed to its enhanced intracellular solubility and bioavailability. These studies have shown that surface functionality, solubility and particle size, but not zeta potential play a significant role in the toxicological and immunological response to aluminium salts in vitro. These parameters should be considered in future studies attempting to elucidate the biological mechanisms involved in adjuvant immunopotentiation and those dedicated to the development of safer and more efficient vaccine adjuvants

Intracellular tracing of amyloid vaccines through direct fluorescent labelling

Abstract Alzheimer’s disease is a debilitating neurodegenerative condition that progressively causes synaptic loss and major neuronal damage. Immunotherapy utilising Aβ as an active immunogen or via passive treatment utilising antibodies raised to amyloid have shown therapeutic promise. The migratory properties of peripheral blood-borne monocytes and their ability to enter the central nervous system, suggests a beneficial role in mediating tissue damage and neuroinflammation. However, the intrinsic phagocytic properties of such cells have pre-disposed them to internalise misfolded amyloidogenic peptides that could act as seeds capable of nucleating amyloid formation in the brain. Mechanisms governing the cellular fate of amyloid therefore, may prove to be key in the development of future vaccination regimes. Herein, we have developed unequivocal and direct conformation-sensitive fluorescent molecular probes that reveal the intracytoplasmic and intranuclear persistence of amyloid in a monocytic T helper 1 (THP-1) cell line. Use of the pathogenic Aβ42 species as a model antigen in simulated vaccine formulations suggested differing mechanisms of cellular internalisation, in which fibrillar amyloid evaded lysosomal capture, even when co-deposited on particulate adjuvant materials. Taken collectively, direct fluorescent labelling of antigen-adjuvant complexes may serve as critical tools in understanding subsequent immunopotentiation in vaccines directed against amyloidosis and wider dementia

X-Ray Absorption Spectroscopy Measurements of Cu-ProIAPP Complexes at Physiological Concentrations

The amyloidogenic islet amyloid polypeptide (IAPP) and the associated pro-peptide ProIAPP1&ndash;48 are involved in cell death in type 2 diabetes mellitus. It has been observed that interactions of this peptide with metal ions have an impact on the cytotoxicity of the peptides as well as on their deposition in the form of amyloid fibrils. In particular, Cu(II) seems to inhibit amyloid fibril formation, thus suggesting that Cu homeostasis imbalance may be involved in the pathogenesis of type 2 diabetes mellitus. We performed X-ray Absorption Spectroscopy (XAS) measurements of Cu(II)-ProIAPP complexes under near-physiological (10 &mu;M), equimolar concentrations of Cu(II) and peptide. Such low concentrations were made accessible to XAS measurements owing to the use of the High Energy Resolved Fluorescence Detection XAS facility recently installed at the ESRF beamline BM16 (FAME-UHD). Our preliminary data show that XAS measurements at micromolar concentrations are feasible and confirm that ProIAPP1&ndash;48-Cu(II) binding at near-physiological conditions can be detected

In-depth mechanistic analysis including high-throughput RNA sequencing in the prediction of functional and structural cardiotoxicants using hiPSC cardiomyocytes

Cardiotoxicity remains one of the most reported adverse drug reactions that lead to drug attrition during pre-clinical and clinical drug development. Drug-induced cardiotoxicity may develop as a functional change in cardiac electrophysiology (acute alteration of the mechanical function of the myocardium) and/or as a structural change, resulting in loss of viability and morphological damage to cardiac tissue. Non-clinical models with better predictive value need to be established to improve cardiac safety pharmacology. To this end, high-throughput RNA sequencing (ScreenSeq) was combined with high-content imaging (HCI) and Ca2+ transience (CaT) to analyze compound-treated human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Analysis of hiPSC-CMs treated with 33 cardiotoxicants and 9 non-cardiotoxicants of mixed therapeutic indications facilitated compound clustering by mechanism of action, scoring of pathway activities related to cardiomyocyte contractility, mitochondrial integrity, metabolic state, diverse stress responses and the prediction of cardiotoxicity risk. The combination of ScreenSeq, HCI and CaT provided a high cardiotoxicity prediction performance with 89% specificity, 91% sensitivity and 90% accuracy. Overall, this study introduces mechanism-driven risk assessment approach combining structural, functional and molecular high-throughput methods for pre-clinical risk assessment of novel compounds.</p

Unraveling the enigma: elucidating the relationship between the physicochemical properties of aluminium-based adjuvants and their immunological mechanisms of action

Abstract Aluminium salts are by far the most commonly used adjuvants in vaccines. There are only two aluminium salts which are used in clinically-approved vaccines, Alhydrogel® and AdjuPhos®, while the novel aluminium adjuvant used in Gardasil® is a sulphated version of the latter. We have investigated the physicochemical properties of these two aluminium adjuvants and specifically in milieus approximating to both vaccine vehicles and the composition of injection sites. Additionally we have used a monocytic cell line to establish the relationship between their physicochemical properties and their internalisation and cytotoxicity. We emphasise that aluminium adjuvants used in clinically approved vaccines are chemically and biologically dissimilar with concomitantly potentially distinct roles in vaccine-related adverse events