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. Author manuscript; available in PMC: 2022 Feb 1.
Published in final edited form as: Cell Rep. 2022 Jan 11;38(2):110217. doi: 10.1016/j.celrep.2021.110217

Figure 6. Differentially glycosylated nanoparticles accumulate in follicles in a mannose density-dependent manner.

Figure 6.

(A) Design models of glycosylated I53–50 nanoparticles with either 240 glycans (left) or 120 glycans (right) displayed on the particle. The left particle is assembled with 20 glycosylated I53–50A trimeric subunits (protein in gray and glycans in green) and 12 non-glycosylated I53–50B pentameric subunits (orange) to display 240 glycans on the particle. The right particle is assembled with 10 glycosylated and 10 non-glycosylated I53–50A trimeric subunits, and 12 non-glycosylated I53–50B pentameric subunits to display 120 glycans. The two-component nature of I53–50 particles (i.e., each particle is composed of 20 trimers and 12 pentamers) enabled titration of glycan densities on the particle through varying the molar ratio of non-glycosylated to glycosylated I53–50A trimeric subunits; glycosylation of I53–50A trimers was either native or high-mannose for each particle formulation.

(B) Mean glycan distances calculated from the particle structure for NPs with titrated levels of total glycans.

(C) BLI analysis of serially glycosylated I53–50 nanoparticles binding to immobilized recombinant murine MBL2 as a function of I53–50 nanoparticle concentration.

(D) The apparent dissociation constant (KD) of immobilized murine MBL2 binding to each I53–50 high-mannose glycoform was determined by BLI analysis using a global 1:1 binding model applied to the three highest I53–50 concentrations.

(E and F) C57Bl/6 mice (n = 5/group) were immunized with 5 μg I53–50 high-mannose glycan variants and saponin adjuvant. Shown are average intensity Z projections through 360 μm of cleared draining lymph nodes harvested on days 3 and 7 (E, blue, CD35; red, I53–50; scale bars denote 500 μm), and quantification of the percent I53–50 signal found within follicles (F). Error bars indicate SEM; points represent average values between paired draining lymph nodes from one animal; *, p <0.05; ****, p <0.0001, ns = not significant by one-way ANOVA followed by Tukey post hoc test.

(G) Absolute counts of germinal center B cells and antigen-specific germinal center B cells from WT and MBL KO mice 12 days after immunization with 5 μg I53–50 and saponin adjuvant. Error bars indicate SEM; *, p <0.05; ns = not significant by one-way ANOVA followed by Tukey post hoc test.