(545h) 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 design considerations modulate immune responses at the single cell level to enhance CD4+ T cell help and antibody production through interaction and activation of specific B cells. Using an antigen-specific 3-83 Ig 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. Initially we focused on the role of surface PEGylation and antigen tethering. We hypothesized that while surface PEGylation has been shown to improve liposome stability in serum and enhance lymph node targeting in vivo, it may hinder binding and internalization through specific B cell receptors. Our studies showed that unilamellar PEGylated liposomes containing maleimide-functionalized PEG-lipids conjugated to cysteine-containing antigenic peptides (p31 and p0 peptides with high and no binding affinities, respectively) bound with varying intensities to B cells as a function of PEGylation (10-0% PEG): 5% PEG liposomes bound with the highest intensity to B cells, while 0% PEG liposomes containing peptide antigen anchored directly to the bilayer surface had significantly reduced binding as a result of steric hindrance. These conditions as well as a third condition (5% PEG with surface anchored peptide antigen which resulted in even lesser binding compared to 0% PEG liposomes) were evaluated for their ability to activate antigen-specific B cells in vitro. B cell activation was assessed at the single-cell level using flow cytometry to quantify the up-regulation of early B cell activation markers, such as CD69, CD86, CD40, and MHCII. As predicted, 5% PEG liposomes with tethered p31 resulted in the strongest antigen-specific B cell activation, while the weakest binding 5% PEG liposomes with surface anchored p31 resulted in the lowest levels of activation comparable to unstimulated cells. The incorporation of TLR4 agonist, monophosphoryl lipid A (MPLA), into the liposome bilayer significantly enhanced early activation (day 1) and helped sustain activation and B cell viability at later times (up to 4 days) in an antigen-specific manner. However, MPLA incorporation into liposomes interestingly provided no enhancement on B cell binding despite the presence of TLR4 on the surface of B cells, signifying MPLA-triggered activation is dependent on specific antigen-mediated uptake of liposomes by B cells. Finally, we evaluated the effectiveness of these 3 liposome conditions to induce antigen-specific humoral responses in naïve mice. Antigen-specific antibody titers in mice immunized with antigen-bearing liposomes containing MPLA were found to correlate directly to B cell binding profiles, with 5% PEG liposomes with tethered p31 resulting in titers ~log₁₀-fold higher than 5% PEG liposomes with surface anchored p31. Current studies are focused on understanding the relationship between B cell binding and activation and the ability of these B cells to present helper peptides to CD4+ T cells, as well as how these liposomes can be utilized to probe and expand low-frequency antigen-specific B cells in spleens and lymph nodes post-immunization. We believe the results obtained in these studies will further the development and understanding of effective particle-based peptide vaccines.