Evaluating the effect of formulation on the uptake of a Zika virus subunit vaccine candidate by antigen-presenting cells

BEBPA EUR Bioassay 2023 -- RNA vaccines, comprising a mRNA encapsulated in a lipid nanoparticle (LNP), are a new modality for rapid development of synthetic prophylactic and therapeutic vaccines. The LNP acts as a device to deliver the RNA payload into the cell cytoplasm for translation of the transgene; moreover, the precise formulation of cationic/ionisable lipid, phospholipid, PEGylated lipid and cholesterol can affect the LNP cellular interaction. Crucial to the mRNA delivery is cellular uptake and escape from the endosomal compartment. To evaluate the effects of formulation components on cell uptake, a cell-based assay using the fluorescent dye 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-labelled LNPs has been developed. Using a microfluidic production process, BODIPY-cholesterol has been incorporated into a range of lipid nanoparticle formulations as a potential screen for evaluating LNP formulations. A major issue with vaccination for Zika, Dengue and other flaviviruses is the potential for antibody-dependent enhancement (ADE) of disease, caused by the generation or boosting of infection-enhancing antibodies. To address this concern, a subunit vaccine is being developed against the Zika virus using a modified version of the envelope protein as the antigen, which has been modified with glycan residues to mask the fusion loop region of the protein. The fusion loop is a cross-reactive and immunodominant site strongly implicated in the generation of antibodies capable of ADE. With subunit vaccines generally, there is a need to formulate with an appropriate adjuvant to enhance the immunogenicity of the modified envelope protein. In this study, we have evaluated a range of adjuvants using flow cytometry and fluorescence microscopy and have determined the relative uptake by human Antigen-presenting cells (APCs).