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Cationic liposomes prepared from dimethyldioctadecylammonium bromide (DDAB) and trehalose 6,6'-dibehenate (TDB) are strong liposomal adjuvants. As with many liposome formulations, within the laboratory DDAB:TDB is commonly prepared by the thin-film method, which is difficult to scale-up and gives high batch-to-batch variability. In contrast, controllable technologies such as microfluidics offer robust, continuous, and scale-independent production. Therefore, within this study, we have developed a microfluidic production method for cationic liposomal adjuvants that is scale-independent and produces liposomal adjuvants with analogous biodistribution and immunogenicity compared to those produced by the small-scale lipid hydration method. Subsequently, we further developed the DDAB:TDB adjuvant system to include a lymphatic targeting strategy using microfluidics. By exploiting a biotin-avidin complexation strategy, we were able to manipulate the pharmacokinetic profile and enhance targeting and retention of DDAB:TDB and antigen within the lymph nodes. Interestingly, redirecting these cationic liposomal adjuvants did not translate into notably improved vaccine efficacy.

A wide range of studies have shown that liposomes can act as suitable adjuvants for a range of vaccine antigens. Properties such as their amphiphilic character and biphasic nature allow them to incorporate antigens within the lipid bilayer, on the surface, or encapsulated within the inner core. However, appropriate methods for the manufacture of liposomes are limited and this has resulted in issues with cost, supply, and wider scale application of these systems. Within this chapter we explore manufacturing processes that can be used for the production of liposomal adjuvants, and we outline new manufacturing methods can that offer fast, scalable, and cost-effective production of liposomal adjuvants.

A prerequisite for vaccine-mediated induction of CD8(+) T-cell responses is the targeting of dendritic cell (DC) subsets specifically capable of cross-presenting antigen epitopes to CD8(+) T cells. Administration of a number of cationic adjuvants via the intraperitoneal (i.p.) route has been shown to result in strong CD8(+) T-cell responses, whereas immunization via e.g. the intramuscular (i.m.) or subcutaneous (s.c.) routes often stimulate weak CD8(+) T-cell responses. The hypothesis for this is that self-drainage of the adjuvant/antigen to the lymphoid organs, which takes place upon i.p. immunization, is required for the subsequent activation of cross-presenting lymphoid organ-resident CD8α(+) DCs. In contrast, s.c. or i.m. immunization usually results in the formation of a depot at the site of injection (SOI), which hinders the self-drainage and targeting of the vaccine to cross-presenting CD8α(+) DCs. We investigated this hypothesis by correlating the biodistribution pattern and the adjuvanticity of the strong CD8(+) T-cell inducing liposomal cationic adjuvant formulation 09 (CAF09), which is composed of dimethyldioctadecylammonium bromide/monomycoloyl glycerol liposomes with polyinosinic:polycytidylic acid electrostatically adsorbed to the surface. Biodistribution studies with radiolabeled CAF09 and a surface-adsorbed model antigen [ovalbumin (OVA)] showed that a significantly larger fraction of the vaccine dose localized in the draining lymph nodes (dLNs) and the spleen 6h after i.p. immunization, as compared to after i.m. immunization. Studies with fluorescently labelled OVA+CAF09 demonstrated a preferential association of OVA+CAF09 to DCs/monocytes, as compared to macrophages and B cells, following i.p. immunization. Administration of OVA+CAF09 via the i.p. route did also result in DC activation, whereas no DC activation could be measured within the same period with unadjuvanted OVA and OVA+CAF09 administered via the s.c. or i.m. routes. In the dLNs, the highest level of activated, cross-presenting CD8α(+) DCs was detected at 24h post immunization, whereas an influx of activated, migrating and cross-presenting CD103(+) DCs to the dLNs could be measured after 48h. This suggests that the CD8α(+) DCs are activated by self-draining OVA+CAF09 in the lymphoid organs, whereas the CD103(+) DCs are stimulated by the OVA+CAF09 at the SOI. These results support the hypothesis that the self-drainage of OVA+CAF09 to the draining LNs is required for the activation of CD8α(+) DCs, while the migratory CD103(+) DCs may play a role in sustaining the subsequent induction of strong CD8(+) T-cell responses., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)