TOLL-like receptor (TLR) ligands activate both innate and adaptive immune cells, while modulating the cellular immune response. The outer membrane protein (OMP) from Neisseria meninigitidis, PorB, is a naturally occurring TLR2 ligand and functions as an adjuvant. Here, we demonstrate that PorB increases the level of OVA in the endo-/lysosomal cellular compartment of BMDCs, increases antigen presenting cell (APC) trafficking to draining lymph nodes, and enhances antigen cross-presentation. PorB is capable of mounting an antigen specific T cell response by efficiently stimulating antigen cross-presentation in vivo and in vitro assessed by BMDC OT-I cocultivation assays. The enhanced antigen cross-presentation and the increased APC recruitment to secondary lymphoid tissues expand the scope of known adjuvant effects of PorB on the immune system. Our findings lead to a better understanding of how TLR-ligand based adjuvants can alter and modulate immune responses.

Vaccines play a vital role in modern medicine. The development of novel vaccines for emerging and resistant pathogens has been aided in recent years by the use of novel adjuvants in subunit vaccines. A deeper understanding of the molecular pathways behind adjuvanticity is required to better select immunostimulatory molecules for use in individual vaccines. To this end, we have undertaken a study of the essential signaling pathways involved in the innate and adaptive immune responses to the Neisseria meningitidis outer membrane protein Porin B (PorB). We have previously demonstrated that PorB is an agonist of Toll-Like Receptor 2 (TLR2) and acts as an adjuvant in vaccines for protein, carbohydrate and lipopolysaccharide antigens using murine models. Here we demonstrate NFκB translocation following stimulation with PorB only occurs in the presence of TLR2. IL-6 and TNF-α secretion was shown to be MAPK dependent. Surface expression of activation markers on macrophages, including CD40, CD69, and CD86, was increased following PorB stimulation in vitro. Interestingly, some upregulation of CD54 and CD69 was still observed in macrophages obtained from TLR2 KO mice, indicating a possible non-TLR2 mediated activation pathway induced by PorB. In a murine vaccination model, using ovalbumin as the antigen and PorB as the adjuvant, a decreased antigen-specific IgG production was observed in TLR2 KO mice; adjuvant-dependent increased IgG production was entirely ablated in MyD88 KO mice. These observations demonstrate the importance of the above pathways to the adjuvant activity of PorB. The potential TLR2 independent effect is currently being explored.

The use of vaccines has led to tremendous decreases in disease burdens across the world. Many challenges remain in expanding vaccine coverage to new pathogens, however, a struggle further hampered by a lack of understanding into many of the fundamental processes through which vaccines elicit robust immunity. In this review we cover recent advances in the field of innate immunity and vaccinology that offer new insights into the reasons some vaccines may succeed or fail. We begin with the secreted cytokines that can influence the nature of the adaptive immune response, and how these may be tuned with the use of particular adjuvants. From there we cover dendritic cells, perhaps the key cell at the interface between innate and adaptive immunity. We discuss mechanisms for targeting specific subsets of dendritic cells, and the effects of this targeting. We further discuss additional modifications of the vaccine formulation to enhance interactions with innate immunity, including phagocytocis and antigen presentation. Finally, we step back to review recent advances in systems biology, and the ability of these new tools to provide deeper understanding of innate immune functions. We hope that this review will provide researchers with access to a breadth and depth of recent work that will allow for the rational design of novel vaccines to combat the most serious infectious diseases of today and tomorrow.

There are no approved therapeutics for the most deadly nonsegmented negative-strand (NNS) RNA viruses, including Ebola (EBOV). To identify new chemical scaffolds for development of broad-spectrum antivirals, we undertook a prototype-based lead identification screen. Using the prototype NNS virus, vesicular stomatitis virus (VSV), multiple inhibitory compounds were identified. Three compounds were investigated for broad-spectrum activity, and inhibited EBOV infection. The most potent, CMLDBU3402, was selected for further study. CMLDBU3402 did not show significant activity against segmented negative-strand RNA viruses suggesting proscribed broad-spectrum activity. Mechanistic analysis indicated that CMLDBU3402 blocked VSV viral RNA synthesis and inhibited EBOV RNA transcription, demonstrating a consistent mechanism of action against genetically distinct viruses. The identification of this chemical backbone as a broad-spectrum inhibitor of viral RNA synthesis offers significant potential for the development of new therapies for highly pathogenic viruses.

TLR ligands play an essential role in identifying threats to the innate immune system, which can then activate the adaptive arm. Our lab has worked with the N. meningitidis outer membrane protein Porin B (PorB), a TLR2 ligand, and shown that it increases the adaptive immune response as measured by antibody production and T cell activity. In examining the adjuvant activity of PorB we now investigate the ability of PorB to increase APC antigen uptake and migration to effector draining lymph nodes. Using an in vitro model with a fluor-linked antigen we show that PorB increases the quantity of the model antigen ovalbumin taken up by the APC in a rate dependent manner. We are in the process of characterizing the intracellular compartmentalization of the processed antigen by fluorescent microscopy of BMDM. In vivo experiments were conducted using a hock injection model and tracking the movement of the antigen through its fluorescent tag. Draining lymph nodes were examined through flow cytometry and immunofluorescence of frozen node sections. Using the latter technique, we are studying changes to lymph node morphology with the inclusion of the TLR ligand. Confirming the in vitro results, the addition of PorB to a vaccine injection increases the degree of APC migration to draining lymph nodes and the carriage of the antigen by these cells. With these results we have identified characteristics of the TLR2 ligand PorB that may account for some of its efficacy as a vaccine adjuvant.