Nanoscale antigen organization regulates binding to specific B-cells and B-cell activation

Abstract

The successes of vaccines in modern medicine diminished the morbidity and mortality of many pathogenic infections. Yet, difficulties remain in improving the immunogenicity of modern subunit vaccines. In addition, isolation of antigen-specific memory B cells that would elucidate the long-term efficacy of vaccines beyond using antibody titers as surrogates has been challenging due to the lack of specific and sensitive detection reagent. We sought to improve the binding and activation of B cells by presenting antigens in a multivalent manner on the surface of liposomes. Motivated by structural requirements originally defined for haptens triggering T-cell-independent stimulation of B cells, we investigated how the mode of antigen presentation, antigen density, particle size, and lipid mobility influence B cell receptor (BCR) crosslinking by multivalent antigen-bearing liposomes, and found that BCR binding is not only a function of antigen density, but also the spacing of antigens on a nanoscale- even for highly multivalent particles. We demonstrated high sensitivity in detecting antigen-specific B cells in vitro, as well as in detecting antigenspecific memory B cells in immunized mice. We first present a novel method of nanoclustering biotinylated antigens conjugated on liposomes with streptavidin, and examine the effect of nanoclustering on BCR binding and B cell response. The mere addition of streptavidin to otherwise 'unclustered' antigens displayed on liposomes increased binding of these particles to antigen-specific B-cells twofold and upregulated activation markers six fold while demonstrating a dose-sparing effect. A three-fold increase in the expression of the activation marker CD86 over soluble tetrameric antigen indicated that surface presentation on liposomes enhances the recognition of nanoclustered antigen by B cells. We then examined how nanoscale organization of antigens influences B cell responses for application to subunit vaccines, using well-defined peptide antigen multimers. Our experiments revealed that B cells bind to and respond to antigens in a valency-dependent manner, with a end-to-end distance spacing of approximately 11.8 nm required between antigens. In vivo immunization experiments demonstrated that higher antigen valencies elicited increased antigen titers and antibody avidity, as well as a responsive memory B cell population that proliferated more rapidly during secondary challenge, indicating a promising strategy for designing subunit vaccines of high immunogenicity. In conclusion, we demonstrated that multivalent presentation of antigens on liposomes enhanced BCR crosslinking and subsequent B cell activation. In addition, we showed that by systematically optimizing the structural requirements of nanoscale antigen organization, we are able to elicit robust B cell responses to low-affinity antigens.