Characterising the immune response to liposomal adjuvant formulations: a systems biology approach
thesisposted on 27.02.2017, 02:27 by Neeland, Melanie Rose
Despite the remarkable success of vaccination, there are a range of human and veterinary diseases that are in need of new vaccines. The rational design of vaccines against these diseases relies on increased understanding of the immunological mechanisms that contribute to vaccine immunity, the development of novel vaccine delivery systems and the characterisation of immune stimulants that are able to increase vaccine efficacy. Vaccine formulations incorporating stimulants that target innate immune receptors have been shown to significantly increase vaccine induced immunity. When incorporated into liposome-based delivery systems, the TLR ligands CpG and poly(I:C) are able to induce protective, long lasting cellular and humoral immune responses in mice. However, the cellular targets of these liposomal adjuvant formulations and the in vivo mechanisms of immune induction remain to be elucidated. The early immune response to vaccination is characterised by activation of cells present at the injection site and their subsequent migration to the local lymph node via the afferent lymphatics. The immunological signals received by innate cells at the peripheral injection site are conveyed to lymphocytes in the local lymph node, leading to the generation of an adaptive immune response where antigen specific lymphocytes emigrate via the efferent lymphatics to perform their tailored effector function. Examination of the afferent and efferent lymphatic compartments during the innate and adaptive phases of an immune response permits the quantification and characterisation of the in vivo biological mechanisms triggered following vaccination. By directly cannulating the ovine lymphatic vessels, the results of this thesis demonstrate that the addition of poly(I:C) or CpG to a liposomal vaccine formulation enhances the immediate inflammatory response at the site of injection, improves antigen uptake by innate cell populations and induces genetic signatures associated with interferon-mediated antiviral immune responses in afferent lymph. The liposomal adjuvant formulations also increased the production of antigen-specific antibodies in the circulation following vaccine challenge. The results additionally show that CpG and poly(I:C) target distinct pathways in afferent lymph to induce their immunological effects, where CpG uniquely increased dendritic-cell associated antigen transport and induced the maturation of monocytes and dendritic cells 72h after injection. CpG also induced the persistence of gene programs involved in cell migration, intracellular DNA sensing and cytotoxic immunity in afferent lymph at this time point. These immunological effects were not observed with liposomal poly(I:C) or liposomes alone. This further translated into an extended period of lymph node cell shut down, the induction of IFNγ positive T cells in efferent lymph and enhanced production of antigen-specific antibodies after injection of liposomal CpG when compared to liposomal poly(I:C) and liposomes alone. The development of a preliminary mathematical model of DC trafficking and T cell activation in the local lymph node showed that all liposomal formulations induce a sufficient number of antigen positive DCs to scan the T cell receptor repertoire and that the adjuvanted formulations induce at least a four-fold excess of antigen positive DCs than required. This model was further utilised to simulate the effect of reducing antigen dose, revealing the optimal number of antigen positive DCs entering the lymph node for an effective immune response and the contribution of DC migration kinetics on vaccine efficacy. The work presented within this thesis provides a comprehensive analysis of the real time in vivo kinetics of cell migration, antigen uptake and gene expression induced by the innate adjuvants poly(I:C) and CpG when incorporated into a liposome-based delivery system. The results demonstrate that liposomal vaccine formulations require the addition of adjuvants to enhance their immunogenicity and that poly(I:C) and CpG target distinct pathways in the lymphatic system to induce their immunological effects. This work quantifies the immunological signals that connect the peripheral injection site with the local draining lymph node, revealing that the cellular and transcriptional immune response induced by adjuvants at the site of injection influences adaptive and memory immune outcomes. Collectively, this body of research enhances our understanding of the complex immune response to vaccination and quantifies the in vivo immune mechanisms induced following injection with liposomal adjuvant formulations in a vaccination setting comparable to that administered to humans.