Transcriptional profile in afferent lymph cells following vaccination with liposomes incorporating CpG.

Vaccine formulations incorporating innate immune stimulants are highly immunogenic; however, the biological signals that originate in the peripheral tissues at the site of injection and are transmitted to the local lymph node to induce immunity remain unclear. By directly cannulating the ovine afferent lymphatic vessels, we have previously shown that it takes 72 hr for mature antigen-loaded dendritic cells and monocytes to appear within afferent lymph following injection of a liposomal formulation containing the Toll-like receptor ligand CpG. In this present study, we characterize the global transcriptional signatures at this time-point in ovine afferent lymph cells as they migrate from the injection site into the lymphatics following vaccination with a liposome antigen formulation incorporating CpG. We show that at 72 hr post vaccination, liposomes alone induce no changes in gene expression and inflammatory profiles within afferent lymph; however, the incorporation of CpG drives interferon, antiviral and cytotoxic gene programmes. This study also measures the expression of key genes within individual cell types in afferent lymph. Antiviral gene signatures are most prominent in lymphocytes, which may play a significant and unexpected role in sustaining the immune response to vaccination at the site of injection. These findings provide a comprehensive analysis of the in vivo immunological pathways that connect the injection site with the local draining lymph node following vaccination.

Vaccination with liposomal poly(I:C) induces discordant maturation of migratory dendritic cell subsets and anti-viral gene signatures in afferent lymph cells.

Vaccine formulations administered in the periphery must activate naive immune cells within the lymph node. In this study, we have directly cannulated the ovine lymphatic vessels to investigate the cellular and molecular mechanisms that transfer information from the periphery into the local draining lymph node via the afferent lymph. Inclusion of poly(I:C) into a liposomal vaccine formulation enhances the neutrophil-associated inflammatory immune response in afferent lymph and increases antigen uptake by migratory dendritic cells (DCs). Interestingly, antigen positive migratory DCs undergo discordant maturation, with peak expression of CD86 at 4 h and CD80 at 48-72 h post vaccination. Afferent lymph monocytes up-regulate expression of genes related to inflammatory and anti-viral immune phenotypes following vaccination however show no differentiation into APCs prior to their migration to the local lymph node as measured by surface MHC II expression. Finally, this study reveals the addition of poly(I:C) increases systemic antigen-specific humoral immunity. These findings provide a detailed understanding of the real time in vivo immune response induced by liposomes incorporating the innate immune agonist poly(I:C) utilising a vaccination setting comparable to that administered in humans.

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.

Modulation of soluble and particulate antigen transport in afferent lymph by monophosphoryl lipid A.

Vaccine adjuvants stimulate the innate immune system and determine the outcome of the immune response induced. A better understanding of their action is therefore crucial to the development of new and safer vaccines. Monophosphoryl lipid A (MPL), a 'detoxified' version of lipolysaccharide, is a promising new adjuvant component in human vaccines. The present study uses an ovine lymphatic cannulation model to study cell recruitment and antigen transport from the injection site into the afferent lymph, and how this is modulated by co-injection with MPL. Compared with saline, MPL injections caused only minor variations in lymph flow and no difference in cell number migrating into the lymph. MPL did, however, cause a significantly increased recruitment of neutrophils and monocytes, but not dendritic cells (DC) into the lymph for the first 12 h. Soluble ovalbumin (OVA) antigen flowed freely into the lymph over a 24-h period and was slightly reduced at 6-9 h in the MPL-injected sites. OVA-coated fluorescent 1-µ beads were initially transported predominantly by neutrophils and, from 24 to 72 h, by DC. MPL induced an increased and more sustained transport of beads by neutrophils and monocytes although it did not increase the phagocytic capacity of these cells. In contrast to aluminium adjuvant, MPL did not increase bead transport by DC at the later time point. These studies provide important new insights in the in vivo action of different adjuvants and the initial events that set up an immune response after vaccination.

The kinetics of soluble and particulate antigen trafficking in the afferent lymph, and its modulation by aluminum-based adjuvant.

Aluminium adjuvants are potent enhancers of immune responses. Despite being a component in most human and animal vaccines, their specific mode of action remains elusive. We have used a sheep lymphatic cannulation model to directly measure the trafficking of soluble and particulate antigen in real-time from the site of injection. Aluminium adjuvant does not alter the kinetics of antigen flow from the site of injection; however it does reduce the amount of soluble antigen entering into afferent lymph. Large numbers of neutrophils, but not DCs, were recruited into the lymph in both saline and aluminium-injected sites and were predominantly responsible for the early uptake of particulate antigen into the lymphatic. Aluminium adjuvant did not significantly increase neutrophil uptake but markedly increased the subsequent uptake of particulate antigen by DCs from 48 to 72 h after antigen injection. Thus, the adjuvanticity of aluminium does not correlate with slow antigen release or increased cell recruitment, but with retention of antigen at the site of injection, and increased uptake of particulate antigen by mature migratory DCs after 24h.