The Squalene Oil‐in‐Water Nanoemulsion Vaccine Carrier <scp>AddaVax</scp> Potentiates <scp>TLR</scp> ‐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.