Adjuvants Based on Synthetic Mycobacterial Cord Factor Analogues: Biophysical Properties of Neat Glycolipids and Nanoself-Assemblies with DDA

Abstract

Synthetic mycobacterial cord factor analogues, e.g., trehalose 6,6'-dibehenate (TDB), are highly promising adjuvants due to their strong immunopotentiating capabilities, but their biophysical properties have remained poorly characterized. Here, we report the synthesis of an array of synthetic TDB analogues varying in acyl chain length, degree of acylation, and headgroup display, which was subjected to biophysical characterization of neat nondispersed self-assembled nanostructures in excess buffer and as aqueous dispersions with cationic dimethyldioctadecylammonium (DDA) bromide. The array comprised trehalose mono- (TMX) and diester (TDX) analogues with symmetrically shortened acyl chains [denoted by X: arachidate (A), stearate (S), palmitate (P), myristate (Myr), and laurate (L)] and an analogue with a short hydrophilic polyethylene glycol (PEG) linker inserted between the trehalose headgroup of TDS and the acyl chains (PEG-TDS). All dispersions were liposomes, but in contrast to the colloidally stable and highly cationic TDX-containing liposomes, the zeta-potential was significantly reduced for DDA/TMX and DDA/PEG-TDS liposomes, suggesting a charge-shielding effect, which compromises the colloidal stability. An increased d-spacing was observed for the lamellar phase of neat TDB analogues in excess buffer (TDS < TMS < PEG-TDS), confirming that the charge shielding is caused by an extended molecular configuration of the more flexible headgroup. Differential scanning calorimetry showed highly cooperative phase transitions for all tested dispersions albeit the monoesters destabilized the lipid bilayers. Langmuir experiments demonstrated that incorporation of TDXs and PEG-TDS stabilized DDA monolayers due to improved hydrogen bonding and reduced intermolecular repulsions. In conclusion, data suggest that the DDA/TDS dispersions exhibit favorable physicochemical properties rendering these DDA/TDS liposomes an attractive vaccine adjuvant, and they emphasize that not only the receptor binding and immune activation but also the biophysical properties of immunopotentiator formulations should be collectively considered when designing adjuvants with optimal safety, efficacy, and storage stability.

Dual-Isotope SPECT/CT Imaging of the Tuberculosis Subunit Vaccine H56/CAF01: Induction of Strong Systemic and Mucosal IgA and T-Cell Responses in Mice Upon Subcutaneous Prime and Intrapulmonary Boost Immunization

“…CAF01 was prepared by using the thin film method combined with ultrasonication as previously described ( 44 ). The resulting unilamellar liposomes had an average hydrodynamic diameter of approximately 153 ± 17 nm, a PDI of 0.324 ± 0.048 (Figure 1A ), and a zeta-potential of 63.6 ± 11.7 ( n = 5, results not shown), which are in accordance with previously reported values for CAF01 prepared using the same method ( 44 – 46 ). The chelation with 18:0 PE-DTPA significantly decreased the size of the CAF01 liposomes (average size = 123 ± 11 nm, PDI = 0.270 ± 0.021, Figure 1A , zeta-potential = 69.9 ± 8.2, n = 8, results not shown).…”

Spray dried cubosomes with ovalbumin and Quil-A as a nanoparticulate dry powder vaccine formulation

“…The zeta potential of particles is a measure of their surface charge and is often used as an indicator of the colloidal stability of the dispersion (Heurtault et al, 2010;Kallerup et al, 2017). It is reported in the literature that as a rule of thumb, particles with zeta potential of magnitude greater than 30 mV will be colloidally stable and that a zeta potential of approximately 20 mV is indicative of short-term colloidal stability.…”

Water-soluble trehalose glycolipids show superior Mincle binding and signaling but impaired phagocytosis and IL-1β production

“…The distinct lack of IL-1β production in response to 4f and the other PEG-TGLs prompted us to consider the mechanism by which 4f and TDB lead to IL-1β production. Although we, and others, had previously determined that glycolipid presentation can influence the immune response to trehalose glycolipids ( Kallerup et al, 2017 ; Stocker et al, 2019 ), the molecular mechanisms for this were not investigated. Moreover, we had previously observed that short-chain trehalose glycolipids led to a less inflammatory immune response ( Khan et al, 2011 ; Khan et al, 2018 ), though had assumed that this might be related to poor Mincle binding.…”

“…Notwithstanding, the immune responses towards the mono-esters are generally poorer than those elicited by their diester counterparts ( Huber et al, 2016 ; Bird et al, 2018 ). Whether the delivery of water-soluble ligands with particulate adjuvants, such as alum ( Eisenbarth et al, 2008 ), or in liposomal formulations ( Kallerup et al, 2017 ), can be used to develop effective water-soluble Mincle ligands also remains to be seen.…”