The Squalene Oil-in-Water Nanoemulsion Vaccine Carrier AddaVax Potentiates TLR-Induced B-Cell Responses

For vaccination, the mechanisms of action of antigens and of immunostimulatory pathogen-associated molecular pattern (PAMP) adjuvants are better understood than the roles of the depot, carrier, and other formulation-related influences. Here, we carried out cytometric studies of vaccine formulations and the effect of adjuvant formulations on B-cell responses. In particular, the AddaVax squalene oil-in-water nanoemulsion formulated with MPLA and CpG, termed IVAX-1, which is particularly effective in eliciting influenza hemagglutinin (HA)-specific mono-reactive or dual-reactive antibodies, was used as the model adjuvant to explore the requirements for generation of these B-cell antibody responses in vitro and in vivo. Using an in vitro inducible germinal center (GC) reaction, we found that AddaVax-mediated pre-assembly of B-cell stimuli for antigen, TLR agonists, CD40 ligand, and cytokines enhanced antibody class switching compared to aqueous buffer solutions. Covalent HA labeling was used to provide high-fidelity detection of mono- and dual-reactive B cells. In immunized mice, MPLA and CpG together enhanced the numbers of total and HA-specific plasma cells relative to each alone. Interestingly, titration of green fluorescent protein (GFP) and fluorescent HAs delivered in AddaVax nanoemulsion revealed that > 50% of input antigens are adsorbed onto the surface of these nanoparticles at the level of ~50-200 protein antigens per nanoparticle. Likewise, several protein antigens, including HAs, a bacterial outer membrane protein, CBU1910 from Coxiella burnetii , and GFP, were all found to associate with AddaVax emulsion nanoparticles in flow cytometry assays. Finally, the AddaVax carrier was more potent in inducing total and dual-reactive antigen-reactive B cells compared to a liposomal carrier, the Immunosome-NTA(Ni). These data cumulatively indicate that concentrated oil-in-water nanoemulsions are effective carriers for molecular adjuvants and antigen in extemporaneous formulations of research-grade protein subunit vaccines.