An Open Label Study to Evaluate Innate Immune Responses Induced by a Pattern Recognition Receptor Agonist, Poly-ICLC (Hiltonol), in Healthy Volunteers

This protocol will take a systems biology approach to understand in humans a major new area of vaccine biology: the mechanism whereby adjuvants, acting on defined pattern recognition receptors [PRR], enhance T and B cell immunity. Our focus is on synthetic double stranded RNA, or poly IC and its more RNase resistant form, called poly ICLC, which is available for studies in humans. The PRRs are the cytosolic helicase, MDA-5, and the endosomal toll like receptor, TLR3. The Steinman lab has pioneered in mice and monkeys that dsRNA is a superior adjuvant for T cell mediated immunity relative to several PRR agonists (Longhi et al., 2009; Stahl-Hennig et al., 2009). A major mechanism is that poly IC is a superior inducer of systemic type I interferon [IFN], which in turn acts on type I IFN receptors [IFNAR] to mature immune stimulatory function of dendritic cells [DCs]. An adaptive Th1 type T cell response is induced, but it is independent of IL-12 and IFN-g.

Multiparameter approaches now provide the means to understand adjuvant action. We hypothesize that DCs undergo changes that are adjuvant-specific and then link innate to select forms of adaptive immunity. Transcriptional arrays of splenic DCs in mice show that dsRNA induces a massive response with ~1000 splenic DC genes changing >2 fold in 4 hrs. These changes are driven primarily via type I IFN, produced systemically via MDA-5 in non-bone marrow derived cells and then acting on DC IFNAR. IFNAR mediate most changes termed "DC maturation" and acquisition of immune stimulating activity, e.g., high CD86, CD40, IL-15, and mechanisms to restore homeostasis. In other words, IFN rather than PRR per se, accounts for the bulk of the poly IC response in DCs.

In a recently completed phase I study (protocol MAC-682), poly ICLC was found to be safe and well tolerated when administered as a single dose of 1.6 mg subcutaneously (s.c.) and intranasally (i.n.) to healthy volunteers. Preliminary innate immune response data shows, similarly to preclinical studies in mice, marked upregulation of gene expression in whole PBMCs following s.c injection of poly ICLC. At day 1 following poly ICLC injection, over 2,000 genes are upregulated (> 1.3 fold change of expression at day 1 versus at baseline) and these responses are specific to the study drug. Importantly, gene changes are homogenous among the 8 volunteers who received poly ICLC. The top upregulated genes are interferon-stimulated genes as it would be predicted since poly ICLC is known to induce type I interferons. In addition, genes associated with dendritric cell (DC) activation such as CD40 and CD86 are also upregulated at day 1 after poly ICLC injection, as well as genes involved in signaling pathways such as IRF 5, IRF 7 and STAT1. Poly ICLC induced secretion of small amounts of types I and II interferons in plasma and these levels peaked at day 2 post injection. Lastly, FACS analysis of PBMCs demonstrated that poly ICLC induced upregulation of activation markers on different subsets of blood dendritic cells. Evaluation of later timepoints are currently taking place but analyses of samples from day 7 show that genes involved in the generation of T and B cell responses are upregulated and the early interferon-stimulated genes are trending down to baseline levels of expression. These results so far show that subcutaneous injection of poly ICLC led to systemic innate immune responses, dominated by the induction of type I interferons. Assays have been performed in whole PBMCs however genetic expression patterns of different subsets of blood leukocytes in response to poly ICLC remain to be defined. We now propose to extend the analysis of systemic immune responses after poly ICLC to the characterization of its transcriptional effects on specific FACS sorted blood cell types, such as DCs, monocytes, NK cells as well as T and B cells. By analyzing individual cell subsets we expect to better dissect how poly ICLC modulates innate immune responses that can in turn affect adaptive immune responses when given in combination with an antigen.

Significant regulation of interferon genes (both type I IFNs and IFN-gamma) was not evident by gene array analysis of whole PBMC's, despite measurement of small amounts of both IFN-alpha and IFN-gamma in plasma. It may be that the platform used was not sensitive enough to detect regulation of IFN genes and we plan to perform RT-PCR to verify these findings. However, it may be that when we restrict our analysis to peripheral blood, we miss the early events that take place following poly ICLC administration. In order to understand how poly ICLC activates different cell populations, it is important to characterize its immunoregulatory effects both locally and systemically.

The cutaneous immune environment is particularly amenable to PRR ligand modulation as evidenced by imiquimod, which acts on TLR 7. Indeed, use of imiquimod to stimulate immune responses against both infectious agents (HPV) and malignancy (squamous and basal cell carcinoma) has been documented. Migratory DCs, which traffic from the skin to the skin draining LN, have been shown to cross prime immune reponses to self and viral antigens. As such, it is possible that skin DCs may migrate into skin draining LNs or blood following poly ICLC administration. However analysis of blood populations alone will likely miss the window of immune alteration if these events are occuring locally in the skin. Analysis of genetic expression and of cellular infiltrates at the site of poly ICLC injection, both early on (at 6 hours) and when skin infiltration is evident clinically (at day 1), will likely add to our understanding of its adjuvant effects. Genomic expression profiles of skin samples have been successfully used for disease classification and to predict response to treatment in skin diseases such as psoriasis and squamous cell carcinoma (Zaba L et al., 2007; Suarez-Farinas et al., 2010). Analysis of genomic expression profile in skin after poly ICLC may prove useful to understand how innate immune responses are initiated by PRRs ligands and perhaps by other vaccine adjuvants.