22:0 Trehalose: A Powerful Synthetic Adjuvant

Posted on July 22, 2022

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One of our most popular products through the first half of this year has been D-(+)-trehalose-6,6’-dibehenate (TDB), also known as 22:0 trehalose. TDB is a synthetic analog of the mycobacterial cord factor trehalose-6,6’-dimycolate (TDM). It is a strong adjuvant that induces strong Th1 and Th17 immune responses. TDB is commonly combined with dimethyldioctadecylammonium bromide (DDAB) to create a powerful adjuvant system capable of producing a cell-mediated immune response and strong antibody response.

How DDAB/TDB Promotes an Immune Response

TDB activates macrophages and dendritic cells, as well as interacts with the Mincle receptor. When combined with DDAB, DDAB/TDB promotes high interferon-gamma (IFN-γ) and interleukin-17 (IL-17) secretion. Another work published in 2021 reported the importance of interleukin-23 (IL-23) in protection after vaccination with a subunit vaccine adjuvanted with the DDAB/TDB system against Mycobacterium tuberculosis (Mtb). IL-23 was identified as a superior mediator of induced memory T-cell immunity (Ritter, et al., 2021).

The DDAB/TDB combination also results in a high IgG antibody production and results in an immune response that is desirable for a large number of disease targets.

Work has been done attempting to add Toll-like receptor (TLR) agonists to DDAB/TDB formulations to increase the immune response. In 2018, researchers from the University of Strathclyde and Statens Serum Institute published work that investigated the immune response of a resiquimod-lipid conjugate-DDAB/TDB liposome. They were successful in creating a DDAB/TDB-TLR7/8 formulation with similar physical characteristics to the DDAB/TDB formulation, but the immune response observed was not noticeably increased. So, they proved that conjugating TLR7/8 agonists to cationic liposomes was possible, but that the added complexity to formulate these conjugated liposomes was not worth the effort (Wilkinson, et al., 2018). Other agonists could therefore theoretically be added to this system to test increased immunogenicity via other mechanisms.

Making DDAB/TDB Liposomes

In the past, DDAB and TDB were incorporated into liposomal formulations via the hydration method which results in heterogenous large multilamellar vesicles (MLVs). High shear mixing and sonication could then be applied to create a homogenous small unilamellar vesicle (SUV). However, this process is difficult to scale up, so a new method was developed by the groups at Statens Serum Institute and Strathclyde University using microfluidics processing.

To create the DDAB/TDB liposomes, they used a Nanoassemblr Benchtop system from Precision Nanosystems. They prepared stock solutions of DDAB and TDB in isopropanol (IPA) at the desired concentrations of 20mg/mL and 2 mg/mL, respectively. Previous reports indicated that the ideal ratio of DDAB/TDB for an immunological response was a molar ratio of 8:1 (weight ratio of 5:1). Adjusting processing parameters such as the flow rate ratio (FRR) and the total flow rate (TFR) while keeping the molar ratio of DDAB/TDB constant, they achieved the smallest particle size (250 nm) and polydispersity index (PDI; 0.3) with a FRR of 3:1 and a TFR of 10 mL/min. Controlling the FRR and TFR, they were able to control the particle size of the liposomes between 1000-160 nm depending on processing parameters (Roces, Khadke, Christensen, and Perrie, 2019).

If you are interested in using a DDAB/TDB liposomal formulation in your vaccine research, Avanti offers high-purity DDAB and TDB. Get them today to enhance your adjuvanted vaccine research!

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