Incorporation of CpG into a liposomal vaccine formulation increases the maturation of antigen-loaded dendritic cells and monocytes to improve local and systemic immunity.

Liposomal vaccine formulations incorporating stimulants that target innate immune receptors have been shown to significantly increase vaccine immunity. Following vaccination, innate cell populations respond to immune stimuli, phagocytose and process Ag, and migrate from the injection site, via the afferent lymphatic vessels, into the local lymph node. In this study, the signals received in the periphery promote and sculpt the adaptive immune response. Effector lymphocytes then leave the lymph node via the efferent lymphatic vessel to perform their systemic function. We have directly cannulated the ovine lymphatic vessels to detail the in vivo innate and adaptive immune responses occurring in the local draining lymphatic network following vaccination with a liposome-based delivery system incorporating CpG. We show that CpG induces the rapid recruitment of neutrophils, enhances dendritic cell-associated Ag transport, and influences the maturation of innate cells entering the afferent lymph. This translated into an extended period of lymph node shutdown, the induction of IFN-γ-positive T cells, and enhanced production of Ag-specific Abs. Taken together, the results of this study quantify the real-time in vivo kinetics of the immune response in a large animal model after vaccination of a dose comparable to that administered to humans. This study details enhancement of numerous immune mechanisms that provide an explanation for the immunogenic function of CpG when employed as an adjuvant within vaccines.

Cell recruitment and antigen trafficking in afferent lymph after injection of antigen and poly(I:C) containing liposomes, in aqueous or oil-based formulations.

After vaccination, innate cell populations transport antigen from the tissue, via the afferent lymphatic vessels, into the local lymph node where they provide critical signals for the generation of an adaptive immune response. The present study uses a unique lymphatic cannulation model to examine, in real time, changes in afferent lymph after injection of a liposome-based delivery system, incorporating diptheria toxoid (DT) and the innate stimulator, poly(I:C). There was a dramatic but temporal recruitment of innate cell populations over time, with neutrophils and monocytes peaking at 6h and 28h post vaccination respectively. The number of dendritic cells (DC) did not increase over the 198h time period, while lymphocytes were slightly elevated at the latest times, indicating the start of an adaptive response. Monocytes and neutrophils were the predominant cell types transporting antigen at the early time points while DC were the most dominant antigen-carrying cells after 78h, predominantly the Sirp-α(high) DC subtype. Resuspending liposomes in oil instead of aqueous solutions has recently been shown to dramatically increase the level and persistence of an immune response and forms the basis of the novel adjuvant formulations, Vaccimax© and Depovax©. In the present study, formulation of the DT and poly(I:C) containing liposomes in an oil carrier dramatically reduced antigen transport to the draining lymph nodes. Examination of the injection site revealed the creation of an ectopic lymphoid tissue with prominent antigen foci and organized lymphoid cells, providing a possible mechanism for the persistence of an immune response in liposome-in-oil adjuvant formulation. Together, the present studies demonstrate the real-time innate in vivo response to vaccination of two novel liposome-based adjuvant systems and the dramatic effect of different carrier formulations.