DOI: 10.15129/aa2ce969-38a9-49bd-95d4-6727155ab5cd
title: Data for: "Lipid conjugation of TLR7 agonist Resiquimod ensures co-delivery with the liposomal Cationic Adjuvant Formulation 01 (CAF01) but does not enhance immunopotentiation compared to non-conjugated Resiquimod+CAF01"

description: Pattern recognition receptors, including the Toll-like receptors (TLRs), are important in the induction and activation of two critical arms of the host defence to pathogens and microorganisms; the rapid innate immune response (as characterised by the production of Th1 promoting cytokines and type 1 interferons) and the adaptive immune response. Through this activation, ligands and agonists of TLRs can enhance immunotherapeutic efficacy. Resiquimod is a small (water-soluble) agonist of the endosome-located Toll-like receptors 7 and 8 (TLR7/8). However due to its molecular attributes it rapidly distributes throughout the body after injection. To circumvent this, these TLR agonists can be incorporated within delivery systems, such as liposomes, to promote the co-delivery of both antigen and agonists to antigen presenting cells. In this present study, resiquimod has been chemically conjugated to a lipid to form a lipid-TLR7/8 agonist conjugate which can be incorporated within immunogenic cationic liposomes composed of dimethyldioctadecylammonium bromide (DDA) and the immunostimulatory glycolipid trehalose 6,6’ – dibehenate (DDA:TDB). This DDA:TDB-TLR7/8 formulation offers similar vesicle characteristics to DDA:TDB (size and charge) and offers high retention of both resiquimod and the electrostatically adsorbed TB subunit antigen Ag85B-ESAT6-Rv2660c (H56). Following immunisation through the intramuscular (i.m.) route, these cationic liposomes form a vaccine depot at the injection site. However, immunisation studies have shown that this biodistribution does not translates into notably increased antibody nor Th1 responses at the spleen and draining popliteal lymph node. This work demonstrates that the conjugation of TLR7/8 agonists to cationic liposomes can promote co-delivery but the immune responses stimulated do not merit the added complexity considerations of the formulation.

Figure 1. Conjugation of DSPE to Resiquimod. Step 1 includes the linker formation with SA and in step 2 the succinylated 1,2-distearoyl-sn-glycero-phosphoethanolamine (DSPE) is conjugated to resiquimod in a Mitsunobu reaction.

Figure 2. Characterisation of liposome product. The vesicle size and PDI (A) and zeta potential (B) of the DDA:TDB liposomes with and without the addition of H56 antigen (DDA:TDB and DDA:TDB:H56), and either mixed with resiquimod (DDA:TDB:Res) or with resiquimod conjugated to the liposome (DDA:TDB-Res). All results represent mean ± SD of 3 independent liposome batches.

Figure 3. Antigen and Agonist loading and retention on liposomes; A) shows the Antigen (H56) and Agonist (resiquimod) loading on the three liposome formulations; B) shows the antigen retention with the three liposome formulations over 98 h; C) shows resiquimod retention when conjugated to the liposomes. All results represent mean ± SD of 3 independent liposome batches.

Figure 4. Biodistribution of liposomes and agonist. Liposome (A, C) and agonist dose retention (B, D) at the site of injection and draining lymph node respectively following i.m. injection of either DDA:TDB, DDA:TDB:Res or DDA:TDB-Res (all adsorbing H56 antigen) or resiquimod alone (negative control). The proportion of 3H or 125I radionucleotides at the injection site and draining lymph node as a percentage of the initial dose were calculated. Results represent the mean ± SD of four mice. 

Figure 5. H56-antigen specific antibody responses in the blood sera (IgG, IgG1, IgG2b). Blood was collected at day 46 from mice immunised with H56 in combination with either DDA:TDB, DDA:TDB:Res or DDA:TDB-Res. As negative controls, resiquimod and H56 antigen were injected alone. Mice received 3 injections with 2-week intervals. Results represent the mean of 5 mice per experimental group ± SD.

Figure 6. Cytokine production (IFN-γ, IL-17, IL-2, IL-5, IL-6 and IL-10) from cultured restimulated spleen (A) and popliteal lymph node (B) cells derived from mice immunised with H56 in combination with either DDA:TDB, DDA:TDB:Res, DDA:TDB-Res. As negative controls, resiquimod and H56 antigen were injected alone. Mice received 3 injections with 2-week intervals and cells were obtained 3 weeks post the final immunisation. Cells were restimulated for 72 hrs in the presence of H56 (at 5 μg/mL). Cytokines were detected from spleen (A) and popliteal lymph node (B) cell supernatants and measured using sandwich ELISAs. Results represent the mean of 5 mice per group ± SD.