Abstract

Vaccines based on protein antigens have numerous advantages over inactivated pathogens, including easier manufacturing and improved safety. However, purified antigens are weakly immunogenic, as they lack the spatial organization and the associated 'danger signals' of the pathogen. Formulating vaccines as nanoparticles enhances the recognition by antigen presenting cells, boosting the cell-mediated immune response. This study describes a nano-precipitation method to obtain stable protein nanoaggregates with uniform size distribution without using covalent cross-linkers. Nanoaggregates were formed via microfluidic mixing of ovalbumin (OVA) and lipids in the presence of high methanol concentrations. A purification protocol was set up to separate the nanoaggregates from OVA and liposomes, obtained as byproducts of the mixing. The nanoaggregates were characterized in terms of morphology, ζ-potential and protein content, and their interaction with immune cells was assessed in vitro. Antigen-specific T cell activation was over 6-fold higher for nanoaggregates compared to OVA, due in part to the enhanced uptake by immune cells. Lastly, a two-dose immunization with nanoaggregates in mice induced a significant increase in OVA-specific CD8