Role of nanoscale antigen organization on B-cell activation probed using DNA origami

Abstract

Vaccine efficacy can be increased by arraying immunogens in multivalent form on virus-like nanoparticles to enhance B-cell activation. However, the effects of antigen copy number, spacing and affinity, as well as the dimensionality and rigidity of scaffold presentation on B-cell activation remain poorly understood. Here, we display the clinical vaccine immunogen eOD-GT8, an engineered outer domain of the HIV-1 glycoprotein-120, on DNA origami nanoparticles to systematically interrogate the impact of these nanoscale parameters on B-cell activation in vitro. We find that B-cell signalling is maximized by as few as five antigens maximally spaced on the surface of a 40-nm viral-like nanoparticle. Increasing antigen spacing up to ~25-30 nm monotonically increases B-cell receptor activation. Moreover, scaffold rigidity is essential for robust B-cell triggering. These results reveal molecular vaccine design principles that may be used to drive functional B-cell responses.

Fig. 1.. Scaffolded DNA origami nanoparticles to control nanoscale organization of HIV immunogens.

(a) DNA-NPs were designed to self-assemble the eOD-GT8 antigen in a controlled manner, mimicking features of the eOD-GT8–60mer immunogen. (i) eOD-GT8–60mer protein NP; (ii) Icosahedral DNA-NP presenting 10 copies of eOD-GT8 (Ico-10x); and (iii) 6HB rod-like structure presenting two copies of eOD-GT8 (6HB-2x). Scale bars are 10 nm. (b) Both the icosahedral and 6HB structures were used to explore the (i) stoichiometry; (ii) inter-antigen distance, d1 and d2; and (iii) 1D versus 3D dimensionality of eOD-GT8 antigens presentation (DX: double-crossover).

Fig. 2.. Increasing antigen valency improves B cell responses to nanoparticle antigens up to a threshold.

(a) Folding of the two types of DNA-NPs (six-helix bundle, 6HB, and DNA icosahedron) that were designed and used in this study for 1D versus 3D presentation of antigens. TEM images show high folding yield and monodisperse DNA-NPs. (Scale bar: 40 nm) (b) Overview of the antigen conjugation protocol to attach eOD-GT8 antigens to the DNA-NPs using PNA single strands complementary to ssDNA overhangs on the DNA-NPs and characterization with electrophoresis. Shown are representative gel electrophoresis samples and fluorescence quantification of four different icosahedral DNA-NPs conjugated with eOD-GT8:PNA-AF647 (Ico-1x; Ico-5x; Ico-30x; Ico-60x)); (M, Molecular weight Marker; Sc, Scaffold; bp: base pair). Error bars represent standard deviation of the mean (n=3 biological replicates/group for fluorescence quantification). (c and d) DNA-NPs modified with eOD-GT8 activate IgM-BCR at both (c) 5 nM and (d) 0.5 nM eOD-GT8. Fluo-4 calcium probe fluorescence is shown in the top row (representative individual calcium traces), and average area-under-the-curve measurements for calcium signaling normalized to the maximum response of all samples in a repeat are shown in the bottom row. For (c) and (d) Error bars represent standard deviation of the mean (n=3 biological replicates/group). P-values are from a two-way ANOVA, paired Student’s t-test (*: p<0.05; **: p<0.01; ***: p<0.001. All P-values are available in the Supplementary Table 5). (The electron microscopy images in a are from 3 technical replicates [10 images per replicates] with similar results. The gels in b have been repeated 3 times (biological replicates) with similar results).

Fig. 3.. IgM-BCR response increases and then plateaus with increasing inter-antigen distance on a rigid scaffold.

(a) Area-under-the-curve total calcium signaling in glVRC01 B cells stimulated with DNA-NP eOD-GT8 dimers with inter-antigen distances between 7 nm and 80 nm at an antigen concentration of 5 nM (n=2 biological replicates/group). Fluo-4 AUC is normalized as in Figure 2, where error bars represent standard deviations of the mean. (b) Representative flow cytometry plots of 6H-2x-7nm, and 6HB-2x-28nm binding to antigen-specific B cells (left) after 30 min incubation at 4°C at a fixed antigen concentration of 5 nM. (Right) Quantitation of data from flow cytometry (MFI: Mean Fluorescence Intensity, (n=3 distinct biological replicates/group), where error bars represent standard deviations of the mean and the P-values are from a one-way ANOVA, followed by Tukey post hoc comparison test (***: p<0.001; NS: Not statistically significant, p=0.9999. All P-values are available in the Supplementary Table 6). (c) Total calcium release (Fluo-4 fluorescence) integrated over 7 minutes following antigen addition from cells stimulated with eOD-GT8 dimers attached to the flexible polymeric scaffolds (ssDNA or PEG), compared with rigid 6HB DNA-NP eOD-GT8 dimer structures at a fixed antigen concentration of 5 nM (n=3 distinct biological replicates/group). Fluo-4 AUC is normalized as in Figure 2, where error bars represent standard deviations of the mean and P-values are from a two-way ANOVA, paired Student’s t-test (*: p<0.05; **: p<0.01; ***: p<0.001. All P-values are available in the Supplementary Table 7). (L: Contour Length; Rg: Radius-of-Gyration).

Fig. 4.. Clustering of antigens on one face of an icosahedral DNA-nanoparticle.

(a) Fluo-4 calcium probe fluorescence versus time following addition of 5 nM eOD-GT8 antigen to glVRC01 B cells. Icosahedral (Ico) structures with varying inter-antigen distances are plotted in the same graph. (Representative individual calcium traces, n=3 biological replicates with similar results) (b) Total calcium signaling integrated over 6 min following antigen addition from cells stimulated with different Ico-5x structures presenting antigen at a total concentration of 5 nM eOD-GT8 (n=3 biological replicates/group). Fluo-4 AUC is normalized as in Figure 2, where error bars represent standard deviations of the mean and P-values are from a two-way ANOVA, paired Student’s t-test (*: p<0.05; **: p<0.01. All P-values are available in the Supplementary Table 8).

Fig. 5.. Confocal microscopic imaging of DNA-nanoparticles on Ramos B cells.

(a) Time series imaging of Ico-30x, 6HB-2x-28nm, and 6HB-2x-7nm shows surface binding and internalization into Ramos B-cells. Here, eOD-GT8 was fluorescently labelled with Alexa Fluor 647, VRC01 IgM-BCR was labeled with a FAb fragment conjugated to Janelia Fluor 549 prior to antigen addition, and actin was labeled with phalloidin Alexa Fluor 405 after cell fixation. Scale bar is 5 μm. This experiment has been performed twice (n=2 biological replicates) with similar results. (b) Total intensity of eOD-GT8 is highly correlated with intensity of IgM-BCR, confirming specific binding of NPs to the IgM-BCR and co-internalization. Numbers of cells analyzed are 15 (1 min), 23 (5 min), 16 (30 min), cells are from the same culture. (c) Ramos cells were labeled with an anti-phospho-Syk antibody after fixation and the total pSyk intensity per cell was determined. Numbers of cells analyzed are 11 (control), 58 (6HB-2x-7nm), 43 (6HB-2x-28nm), 56 (Ico-30x), and 56 (eOD-GT8–60mer), cells are from the same culture. (d) Internalized fraction of eOD was estimated by segmenting the cell surface using a phalloidin stain as detailed in Methods. Total internal eOD fluorescence was divided by total cellular eOD fluorescence on a cell-by-cell basis. Numbers of cells analyzed are 19 (6HB-2x-7nm), 23 (6HB-2x-28nm), and 15 (Ico-30x), cells are from the same culture. In (c) in (d), error bars denote the standard error of the mean fluorescence between cells with significance determined by a two-sided Student’s t-test (*: p=0.0314; **: p=0.0016;; NS: Not statistically significant, p= 0.3038 for 6HB-2x-28nm/Ico-30x, and p= 0.4153 for 6HB-2x-28nm/eOD-GT8–60mer). In (d), error bars denote the standard error of the mean fluorescence between cells with significance determined by Student’s t-test (*: p=0.0142; **: p=0.0074; NS: Not statistically significant, p=0.55). (All P-values are available in the Supplementary Table 9)