Application of a new small RNA molecule, RNA-LZ-1, as a vaccine adjuvant

Abstract

Influenza A viruses are important pathogens that cause disease in a wide variety of species leading to economic losses and added burden on health care. Recently, outbreaks of avian influenza viruses in the human population have added to this growing risk of pandemic influenza viruses. Therefore, strategies to address the demands of higher efficiency, immunogenicity, and safety in influenza vaccines must be explored. One strategy which addresses efficiency and immunogenicity is to explore the addition of an adjuvant to influenza vaccines. Adjuvants are commonly used vaccine components which are included to induce a robust immune response leading to antigen sparing, a broader spectrum of protection and the elimination of a booster. Recently the retinoic acid-inducible gene-I (RIG-I) pathway, which is the natural defense against RNA viruses, has become an attractive target for adjuvant development. In this thesis, we sought to explore the possibility of a small RNA molecule, RNA- LZ-1, identified in our laboratory as a RIG-I agonist as an adjuvant. This project evaluated the induction of pro-inflammatory cytokines, chemokines, and interferons by RNA-LZ-1 in vitro and in vivo, and tested its ability to induce antigen-specific antibodies when paired with H5N1 and H7N9 inactivated whole influenza viruses. We hypothesized that due to RNA-LZ-1’s binding affinity for RIG-I it will induce a broad spectrum immune response and fortified adaptive response when used as an adjuvant. Overall our results showed that RNA-LZ-1 was capable of inducing activation of a diverse spectrum of inflammatory genes and proteins in vitro and in vivo. In human macrophages and lung epithelial cells RNA-LZ-1 induced significant levels of IFN-β protein and mRNA fold change. We also showed that as early as 3 hours post-injection, RNA-LZ-1 was capable of up-regulating immune genes with the effect dissipating by 48 hours post-injection. These immune genes were grouped as follows cytokines (IL-12α, IL-12β, IL-6, and IL-18), chemokines (MIP-1α, RANTES, MIP-2, MCP-1, and IP-10), and interferons (IFN-α, IFN-β, and IFN-ɣ). The overall immune response observed was skewed towards a Th1 response. In vivo, RNA-LZ-1 was able to induce protective antigen-specific antibodies against H5N1 in comparable levels to the proven VIDO triple adjuvant showing that RNA-LZ-1 is a strong candidate as an adjuvant for influenza vaccines.