Alu dsRNAs as adjuvants for influenza vaccines

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Despite medical progress, infectious diseases remain one of the leading causes of death worldwide. Vaccines are one of the most effective tools to prevent infectious diseases. Generation of immunity has two components. The first is the unique chemical structure of the antigen recognized by T and B cell receptors allowing clonal expansion and differentiation of cells of the adaptive immune system. The second is a ‘danger signal’ that stimulates the innate immune system via pattern recognition receptors (PRRs) that recognize conserved pathogen-associated molecular patterns (PAMPS) absent from eukaryotic cells. In general, the most effective viral vaccines are live attenuated viruses. Inactivated virus vaccines also confer immunity but offer less protection and require multiple immunizations. Subunit vaccines, such as viral protein antigens, are less useful due to poor immunogenicity. However, there are clear advantages to subunit vaccines in terms of cost, uniformity of production, stability, ability to control the type of immunity that is generated, and far superior safety profiles, but subunit vaccines require effective adjuvants to generate strong immunity. Examples in clinical use include mineral salts (Alum), oil-in-water emulsions, and a mineral salt-TLR4 agonist combination. Other PRR agonists as components of adjuvants are in various stages of clinical trials. A limitation to use of PAMPS as vaccine adjuvants is toxicity. In general terms, signaling through PRRs activates two major transcriptional cascades, interferon regulatory factor (IRF) and nuclear factor-kappa B (NF-kB) signaling culminating in expression of genes encoding proteins to both inhibit pathogen replication and strongly activate the immune system. Recent evidence argues that it may be possible to retain adjuvant activity of a PAMP and alleviate toxicity by combining a PAMP (CpG) with an inhibitor of the NF-kB signaling path. A limitation to this strategy derives from inherent serious mechanism-based toxicity of systemic inhibition of NF-kB signaling. Autoimmune disease generates a kind of ‘danger signal’; referred to as an ‘interferon signature’ in which many genes induced by interferons are elevated in patients in apparent absence of infection. We found that the ‘interferon signature’ in multiple sclerosis (MS) results from markedly increased levels of endogenous double-stranded Alu elements (Alu dsRNA) and showed that Alu dsRNAs stimulate strong IRF and NF-kB activation. Alu dsRNAs complexed with nanoparticles (AluJb/NP) have potent anti-tumor activity, in vivo, demonstrating their immunostimulatory activity. We identified Alu dsRNA elements that are strong activators of both IRF and NF-kB signaling and those that are strong activators of only IRF signaling but not NF-kB. Thus, our hypothesis to test is that we can design and deliver RNA adjuvants that mimic endogenous Alu elements that preferentially activate IRF responses and minimally agonize NF-kB signaling to enhance immune responses and improve tolerability of influenza subunit vaccines.