Small Molecule Potentiator of Adjuvant Activity Enhancing Survival to Influenza Viral Challenge

Abstract

As viruses continue to mutate the need for rapid high titer neutralizing antibody responses has been highlighted. To meet these emerging threats, agents that enhance vaccine adjuvant activity are needed that are safe with minimal local or systemic side effects. To respond to this demand, we sought small molecules that would sustain and improve the protective effect of a currently approved adjuvant, monophosphoryl lipid A (MPLA), a Toll-like receptor 4 (TLR4) agonist. A lead molecule from a high-throughput screen, (N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide, was identified as a hit compound that sustained NF-κB activation by a TLR4 ligand, lipopolysaccharide (LPS), after an extended incubation (16 h). In vitro, the resynthesized compound (2D216) enhanced TLR4 ligand-induced innate immune activation and antigen presenting function in primary murine bone marrow-derived dendritic cells without direct activation of T cells. In vivo murine vaccination studies demonstrated that compound 2D216 acted as a potent co-adjuvant when used in combination with MPLA that enhanced antigen-specific IgG equivalent to that of AS01B. The combination adjuvant MPLA/2D216 produced Th1 dominant immune responses and importantly protected mice from lethal influenza virus challenge. 2D216 alone or 2D216/MPLA demonstrated minimal local reactogenicity and no systemic inflammatory response. In summary, 2D216 augmented the beneficial protective immune responses of MPLA as a co-adjuvant and showed an excellent safety profile.

Small Molecule Calcium Channel Activator Potentiates Adjuvant Activity

“…We recently demonstrated that mice immunized with an inactivated influenza A virus adjuvanted with 2D216 in combination with another TLR4 ligand, MPLA, were protected from a lethal influenza challenge and had an increased antiviral neutralization titers. 34 These results indicated that a 2D216 plus MPLA adjuvanted vaccine induced an effective antibody response. The induction of antigen-specific IgG2c antibodies with 2D216 , but not with LPS alone, suggested that the mechanism of action of 2D216 differed from the canonical TLR4 signaling pathways.…”

“…The most widely used FDA-approved adjuvant, alum, preferentially induces T helper (Th)-2-associated antibody responses (IgG1 in mice), , but 2D216 with LPS also increased the IgG2c response (Th1 associated), indicating that LPS with 2D216 induced a more balanced immune response. We recently demonstrated that mice immunized with an inactivated influenza A virus adjuvanted with 2D216 in combination with another TLR4 ligand, MPLA, were protected from a lethal influenza challenge and had an increased antiviral neutralization titers . These results indicated that a 2D216 plus MPLA adjuvanted vaccine induced an effective antibody response.…”

“…Flow cytometry performed as previously described. 34 Enhanced T Cell Activation in a Mixed Lymphoid Reaction by 2D216 and Its Analogs. As 2D216 did not directly activate Jurkat T cells, we tested 2D216, 2D291, and 2E151 for their ability to enhance functional antigen presentation by murine bone marrow-derived dendritic cells (mBMDCs) (Figure S9).…”

“… 33 We selected one of these compounds ( 2D216 ) and found that it functioned as a co-adjuvant in mice and increased viral neutralizing titer sufficiently to protect mice from a lethal influenza challenge. 34 Here, we aimed to examine the mechanisms of action in human cells and evaluated a small set of analogs to enhance activation of human immune cells.…”

“…To improve the reliability of identifying hit compounds, we employed a statistical clustering method to identify compounds with conserved scaffolds and noted that eight aminothiazoles in the original library increased the NF-κB activation by LPS . We selected one of these compounds ( 2D216 ) and found that it functioned as a co-adjuvant in mice and increased viral neutralizing titer sufficiently to protect mice from a lethal influenza challenge . Here, we aimed to examine the mechanisms of action in human cells and evaluated a small set of analogs to enhance activation of human immune cells.…”