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.

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

“…Therefore, lumogallion has high specificity and selectivity for the binding and detection of aluminium, respectively. Such properties have rendered the fluorophore invaluable for the unequivocal identification of aluminium in both cells [ 7 , 65 , 144 , 153 , 162 , 163 ] and tissues [ 164 – 166 ].…”

“…Recently, the use of direct-fluorescent labelling has allowed for the conformation and intracellular fate of a model amyloid-β 42 (Aβ 42 ) peptide antigen to be followed in a cellular-based model of vaccination [ 163 ]. Therein, simulated vaccines were prepared adjuvanted with lumogallion-labelled ABAs that were subsequently co-cultured with THP-1 cells.…”

“…These aberrant misfolded deposits are suggested to break down into neurotoxic oligomeric Aβ species, triggering a progressive cascade of deleterious effects [ 171 ]. Thioflavin T (ThT) binds to mature amyloid fibrils in a β-pleated sheet conformation that thereby allowed for the detection and intracellular fate of the antigen to be assessed [ 163 ].…”

“…This new approach to directly label the moiety of interest suggests that Aβ 42 is endocytosed by THP-1 cells through micropinocytosis [ 174 ] of which its co-formulated ABA (Alhydrogel ® , Adju-Phos ® or Imject ® Alum) is processed via the alternative pathway of autophagy into late-stage autolysosomes [ 163 ]. The ability of Aβ to evade or disrupt lysosomal capture has recently been implicated in separate studies in both monocytic [ 175 ] and neuronal cell lines [ 168 , 174 ].…”

The size of micro-crystalline tyrosine (MCT®) influences its recognition and uptake by THP-1 macrophagesin vitro

“…35 How particulate adjuvants facilitate the polarisation and potentiation of the immune response during vaccination is yet to be fully elucidated; however, postulated mechanisms include the induction of pro-inammatory cytokine secretion through activation of the NALP-3 inammasome, [36][37][38][39] depot formation 35,40,41 and improved antigenic cross-presentation through enhanced recognition and uptake by antigen presenting cells (APCs). [42][43][44][45][46] Recent literature has highlighted the importance of particulate physicochemistry with regards to the subsequent biological activity of adjuvants administered as colloidal suspensions (see review 47 ). Properties which signicantly inuence the adjuvanticity of such materials include solid-state structure, particle size, particle shape and zeta potential.…”