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.


vaccinations.
6 DAPI-fluorescence reveals extracellular genetic material from THP-1 cells in the presence of Adju-Phos ® Supplementary Figure 5 Representative DAPI staining of agar-paraffin embedded (2 μm sections) THP-1 cells co-cultured in the presence of 2.5 μg/mL Adju-Phos ® (Brenntag Biosector, Denmark). Cell sections were incubated for 24 h in 100 μM lumogallion, 50 mM PIPES, pH 7.4. Slides were mounted with ProLong ® Gold Antifade Reagent with DAPI. DAPI-staining is depicted by a blue fluorescence emission. White arrows indicate extracellular genetic material and the magnified insert shows a close-up of DAPI-stained cell nuclei. Magnification X 400, scale bar: 50 μm.

Supplementary Tables
Supplementary Table 1 The zeta potential values of native aluminium salts upon dilution into physiological saline (0.9% NaCl) to 0.25mg/mL Al. All measurement were conducted at 25°C and errors are expressed as ± SD of 5 individual replicates.

Graphite furnace atomic absorption spectroscopy of aluminium adjuvant filtrates
Experimental values were derived from the means of three machine replicates per sample provided that the relative standard deviation (RSD) did not exceed 10%. In the event that RSD values exceeded acceptable limits a fresh experimental sample was re-analysed using v/v heat-inactivated foetal bovine serum (certified US origin) (both from Fisher Scientific, Invitrogen). 100μg/mL gentamicin was added from a cell culture certified 10mg/mL stock solution in ultrapure water, to prevent microbial growth. Cell suspensions were passaged to a maximum of 1 x 10 6 cells / mL in TC-treated canted and vented T25 flasks, prior to subculturing into T75 flasks (both from VWR, Corning ® ). All cells were cultured at 37°C in a humidified atmosphere, containing 5% CO 2 . Cells were counted using a haemocytometer and their viability confirmed (prior to the addition of ABA), using the Trypan blue exclusion test.

Cytotoxicity assay (Live/dead staining)
Cells designated as dead cell controls were terminated using 70% MeOH for 10 minutes before washing, re-suspension and application to the plate. Prior to plating, estimates of cell replicates.

Agar-paraffin embedding of THP-1 cells
The preparation and staining of isolated THP-1 cell sections was performed using established methods described elsewhere 16 . Briefly, PFA fixed THP-1 cells co-cultured in the absence or presence of an ABA were pre-embedded into 5% w/v molten agar in BEEM ® capsules (Elektron, Technology, Agar Scientific) and the blocks dehydrated fully through a graded ethanol series from 30 -100% v/v. For paraffin embedding, the resultant dehydrated agar-cell blocks were cleared by immersion into Histo-Clear (National Diagnostics, USA) for 20 min, with one change of fresh Histo-Clear half way through. Blocks were subsequently infiltrated with paraffin wax maintained at 60 o C, for 35 min. 2μm cell sections were prepared by use of a Leica RM2025 rotary microtome and the agar-cell sections were rehydrated back into ultrapure water via reverse infiltration, prior to staining.

Modified agar-paraffin protocol for the preparation of THP-1 cells co-cultured with
Imject TM Alum.
The agar-paraffin embedding protocol employed for the sectioning of THP-1 cells necessitated modification for their successful sectioning in the presence of Imject™ Alum.
Upon the addition of molten agar to fixed cells, the centrifugation phase used to pellet the block was found to produce dense deposits of the ABA upon final sectioning at 2 μm. As a result, staining of the rehydrated sections with lumogallion for 24 h produced excessive extracellular fluorescence of the ABA. This was particularly problematic at the highest concentrations of 50.0 and 100.0 μg/mL of Imject™ Alum. Therefore, following the addition of molten agar to cells and prior to setting, cell treatments were vortexed at high speed to suspend the cells throughout the block. This was found to considerably reduce extracellular ABA material deposited, allowing for individual cells to be distinguished via DAPI-nucleic staining.

Electron micrograph acquisition
Samples for TEM were viewed on a JEOL 1230 transmission electron microscope operated at 100.0 kV (spot size 1), equipped with a Megaview III digital camera from Soft Imaging Systems (SIS). An activated field emission of 10μA was used which increased the standing current to 67 -68μA during operation. Images were obtained on the iTEM universal TEM imaging platform software. Measurements of intracellular ABA particles were made using the Cell D software package and the final editing of electron micrographs for publication was achieved using Photoshop (Adobe systems Inc. USA).

Statistical analyses
Data were analysed for statistical significance using Graph Pad Prism software. ANOVA with repeated measures followed by Tukey post hoc tests were used where multiple