(719e) Single-Cell Analysis of Specific B Cell Binding and Uptake of Peptide-Targeted Liposomes for Vaccine Formulations

Lipid-based nanoparticles have been widely characterized for the delivery of small molecules, peptides and proteins. Liposomes containing surface-bound antigens are similarly being developed as vaccines and have been shown to induce strong immune responses when compared to soluble antigens by facilitating cross-presentation and enhancement of humoral responses. Yet so far no studies have focused on how specific liposome characteristics modulate immune responses at the single cell level to enhance CD4+ T cell help and CD8+ CTLs through interaction and activation of specific B cells. Using a 3-83Ig transgenic mouse model, we evaluated the role of liposome composition on the direct binding and internalization of liposomes with surface-displayed peptide antigens to antigen-specific B cells. Initial studies were focused on the role of surface PEGylation. We hypothesized that while surface PEGylation has been shown to improve liposome stability and prolong circulation in vivo, that it may hinder binding and internalization through specific BCRs. Unilamellar PEGylated liposomes containing maleimide-functionalized PEG-lipids were synthesized by membrane extrusion, followed by post-synthesis conjugation to cysteine-containing antigenic peptides (p31 and p0 peptides with high and no binding affinities, respectively). Through flow cytometry analysis of liposomes with 10, 5, and 1% PEG-DSPE and 5% MPB (0% PEG) but identical antigen density, we found that 5% PEG liposomes bound with the highest intensity to B cells. There was, however, only a slight difference in the total number of B cells with bound liposomes between 10, 5, and 1% PEG liposomes. Only in the case of 0% PEG liposomes, where the peptide antigen was anchored directly to the bilayer surface, was binding significantly reduced, likely due to steric hinderance. Surprisingly, binding and internalization were inversely related, with 5% PEG liposomes having only 13% internalization after 22 hours at 37^oC compared to almost 75 and 90% internalization of 1 and 0% PEG liposomes, respectively. Future studies will focus on the relationship between internalization and B cell activation by assessing B cell proliferation, Ig class switch, presentation of activation markers, and the ability to present helper peptides to CD4+ T cells. We believe the results obtained in these studies will further the development and understanding of effective particle based vaccines.