Association of a vaccine adjuvant with endogenous HDL increases lymph uptake and dendritic cell activation.

Vaccines are a powerful health intervention but there is still an unmet need for effective preventative and therapeutic vaccines for many diseases such as cancer and infections. Interstitial (e.g. subcutaneous (SC)) injection in nano-sized carriers such as high density lipoproteins (HDLs) can improve the access of vaccine subunit antigens or adjuvants to target immune cells in the lymphatics and potentiate vaccination responses such as cytotoxic T lymphocyte (CTL) responses (Kuai et al., 2016, 2018; Qian et al., 2016). Here we examined how cholesterol conjugation to the vaccine adjuvant CpG, and incorporation into HDL, changes lymphatic absorption and association with, and processing by, dendritic cells (DCs), ultimately influencing adjuvant efficacy. We investigated the lymphatic disposition of cholesterol conjugated CpG incorporated into HDL (HDL(Chol-CpG-Cy5)) relative to free cholesterol conjugated CpG (Chol-CpG-Cy5) and unconjugated CpG (free CpG-Cy5) after SC administration in rats and mice. HDL (Chol-CpG-Cy5) and Chol-CpG-Cy5 differentially altered CpG absorption into lymph vs. blood, but surprisingly resulted in similarly higher LN accumulation relative to free CpG. The mechanism of access of Chol-CpG-Cy5 into lymph might be partly due to association with endogenous HDL at the injection site followed by transport into lymph in association with the HDL. To measure CpG association with and processing by DCs and the strength of the immune response, mice were vaccinated with free ovalbumin (OVA) co-administered with the different CpG constructs. There were significant changes in DC activation that were reflective of the trend in LN accumulation at 24 h post-vaccination. However, T cell responses at 24 h and 7 days post-vaccination were not significantly different across the CpG groups although the response was less variable for Chol-CpG-Cy5 compared to free CpG Cy5 and also HDL(Chol-CpG-Cy5) - despite similar LN accumulation with the latter. Overall, our data indicate that cholesterol conjugation and incorporation into HDL increases adjuvant lymph disposition and DC activation.

Targeted delivery of mycophenolic acid to the mesenteric lymph node using a triglyceride mimetic prodrug approach enhances gut-specific immunomodulation in mice.

The mesenteric lymph nodes (MLN) are a key site for the generation of adaptive immune responses to gut-derived antigenic material and immune cells within the MLN contribute to the pathophysiology of a range of conditions including inflammatory and autoimmune diseases, viral infections, graft versus host disease and cancer. Targeting immunomodulating drugs to the MLN may thus be beneficial in a range of conditions. This paper investigates the potential benefit of targeting a model immunosuppressant drug, mycophenolic acid (MPA), to T cells in the MLN, using a triglyceride (TG) mimetic prodrug approach. We confirmed that administration of MPA in the TG prodrug form (MPA-TG), increased lymphatic transport of MPA-related species 83-fold and increased MLN concentrations of MPA >20 fold, when compared to MPA alone, for up to 4 h in mice. At the same time, the plasma exposure of MPA and MPA-TG was similar, limiting the opportunity for systemic side effects. Confocal microscopy and flow cytometry studies with a fluorescent model prodrug (Bodipy-TG) revealed that the prodrug accumulated in the MLN cortex and paracortex at 5 and 10 h following administration and was highly associated with B cells and T cells that are found in these regions of the MLN. Finally, we demonstrated that MPA-TG was significantly more effective than MPA at inhibiting CD4+ and CD8+ T cell proliferation in the MLN of mice in response to an oral ovalbumin antigen challenge. In contrast, MPA-TG was no more effective than MPA at inhibiting T cell proliferation in peripheral LN when mice were challenged via SC administration of ovalbumin. This paper provides the first evidence of an in vivo pharmacodynamic benefit of targeting the MLN using a TG mimetic prodrug approach. The TG mimetic prodrug technology has the potential to benefit the treatment of a range of conditions where aberrant immune responses are initiated in gut-associated lymphoid tissues.