Mechanisms of action of polyphosphazene-based adjuvants in porcine monocytes

Abstract

Adjuvants are compounds that enhance immune responses to antigens present in a vaccine. They are particularly important in subunit vaccines; without adjuvants, these vaccines are often poorly immunogenic. A novel adjuvant platform developed at VIDO-InterVac is comprised of CpG-ODN or poly I:C, innate defense regulator peptides, and a new class of adjuvant called polyphosphazene. The polyphosphazenes have demonstrated a great potential as a safe and effective adjuvant. In particular, the polysphosphazenes poly[di(carboxylatophenoxy)-phosphazene] PCPP and poly[di(sodium carboxylatoethylpehnoxy)-phosphazene](PCEP) have been used in numerous animal studies where they not only have been shown to enhance the quality and quantity of the adaptive immune response, but also were shown to induce parenteral and mucosal immune responses with many different antigens, demonstrating their versatility. However the mechanisms by which the polyphosphazenes stimulate the innate immune response are only partially understood. Antigen presenting cells (APCs) are capable of facilitating the uptake of antigen and directing the immune response. Based upon the proposed mechanism of action of another adjuvant, we chose to investigate whether porcine monocytes could be induced to secrete pro-inflammatory cytokines IL-1 and IL-18 in response to stimulation with polyphosphazenes PCEP and PCPP. The release of these cytokines is thought to be mediated by the Nod-Like Receptors (NLRs), which are cytosolic pattern recognition receptors expressed in APCs. It is suggested that these receptors act in conjunction with TLR transcription pathways to control caspase-1 and release associated pro-inflammatory cytokines IL-1and IL-18 (Kawai and Akira, 2011). We first investigated the relative gene expression of three Nod-like receptor genes: nod1, nod2 and nlrp3 in various populations of porcine peripheral blood mononuclear cells (PBMCs) and found that monocytes, dendritic cells and B cells express increased relative levels of these receptors as compared to T cells. Subsequently, we evaluated the relative NLR expression in several porcine mucosal and lymphoid tissues and observed genes to be most significantly expressed in nasal mucosa, bronchial mucosa, and lung while limited in tissues associated with Peyer’s patches, jejunal wall. Both the mesenteric lymph node and bronchial lymph node exhibited similar patterns and levels of expression of nod1, nod2 and nlrp3. To characterize the activation of NOD1, NOD2 and NLRP3 receptors in response to stimulation with polyphosphazenes, porcine monocytes were stimulated with PCEP or PCPP in both the presence and absence of a second signal (poly I:C and CpG-ODN, TLR-7 and -9 agonists respectively). We found that PCEP and PCPP alone did not significantly upregulate nod1, nod2 and nlrp3, nor genes for cell activation markers such as CD80 and CD86. However monocytes cultured with the combination of CpG-ODN, Poly I:C and PCPP appeared to moderately express IL-18, CD80 and CD86. The secretion of pro-inflammatory cytokines from cultured monocytes was determined with Enzyme Linked Immunosorbent Assays (ELISA). It was found that IL-1was secreted in significantly higher quantities in the supernatant of cells stimulated with both polyphosphazene and TLR ligands, as opposed to those cultured with polyphosphazene alone. Assays for IL-18, IL-6, IL-10 and IL-12 did not detect a significant presence of these proteins in the supernatant. Furthermore, we found that a soluble caspase inhibitor did not significantly reduce the production of IL-1by monocytes, and was likely attributable to cell death at high concentrations. Taken together, these results suggest that porcine monocytes, B cells and dendritic cells express elevated levels of the NLRs as compared to T cells. Additionally, areas of the respiratory tract appear to express increased levels of these receptors relative to mucosal and lymphoid tissues of the gastrointestinal tract. Neither PCPP or PCEP alone were capable of inducing significant production of IL-1 or IL-18 by cultured monocytes, however stimulation of these cells with a combination of CpG-ODN, poly I:C and polyphosphazene resulted in the secretion of IL-1.