A peer review file is available. Data availability The source data used to generate Figs.?1C4 and Supplementary Figs.?1C9 are provided in the Source Data file. the valency of the antigen displayed and on T cell help. Importantly, the immune sera do not contain boosted, class-switched antibodies against the DNA scaffold, in contrast to P-VLPs that elicit strong B cell memory space against both the target antigen and the scaffold. Therefore, DNA-VLPs enhance target antigen immunogenicity without generating scaffold-directed immunity and therefore offer an important alternative material for particulate vaccine design. Subject terms: Vaccines, Organizing materials with DNA, Immunological memory, Antibodies, Contamination Three-dimensional DNA origami constructs can be used to deliver vaccine antigens in a multi-valent form. Here the authors design a DNA origami system for SARS-CoV-2 proteins and characterize in mice the immune response and protective capacity of generated antibodies, finding that the construct itself is not immunogenic. Introduction Multivalent display of antigens on virus-like particles (VLPs) can improve the immunogenicity of subunit vaccines1C3. Nanoparticulate vaccines with diameters between 50 and 200?nm ensure efficient trafficking to secondary lymphoid organs, whereas particle diameters below 50?nm overcome undesired retention at the injection site and promote the penetration of B cell follicles4,5. In secondary lymphoid organs, multivalency promotes B cell receptor (BCR) crosslinking and signaling as well as BCR-mediated antigen uptake, thereby driving B cell activation and humoral immunity6C13. The importance of BCR signaling for the generation of antibody responses was initially acknowledged for thymus-independent (TI) antigens, particularly of the TI-2 class14C16. The multivalent display of these non-protein antigens induces BCR crosslinking in the absence of T cell help. The resultant antibody responses proceed through extrafollicular B cell pathways, with limited germinal center (GC) reactions, affinity maturation, and induction of B cell memory17,18. Multivalent antigen display also enhances BCR-mediated responses to thymus-dependent (TD) antigens, namely proteins8,9. In this context, follicular T cell help enables GC reactions to generate affinity-matured B cell memory that can be boosted or recalled upon antigen reexposure19C21. Consequently, the nanoscale business of antigens represents a well-established vaccine design principle not only for S18-000003 TI antigens, but also to elicit humoral immunity through the TD pathway1C3. Leveraging this design theory, protein-based virus-like particles (P-VLPs) have emerged S18-000003 as an important material platform for multivalent subunit vaccines22C38. P-VLPs enable the rigid display of TD antigens and have been used to investigate the impact of valency on B cell activation in vivo, suggesting early B cell activation and downstream humoral immune responses are improved for some antigens as valency increases8C10. However, control over antigen valency in P-VLPs is usually constrained S18-000003 to the constituent self-assembled protein scaffold subunits, rendering the investigation of antigen valency on humoral immunity challenging without simultaneously altering scaffold size, geometry, and protein composition9,10. Alternatively, if a constant protein scaffold geometry is used, then current approaches are limited to stochastically-controlled antigen valency and spatial positioning8,29,30,38. Furthermore, protein-based scaffolds themselves are TD antigens that elicit humoral immunity38C40. This potentially misdirects antibody responses from the target antigens of interest41,42, and might also lead to imprinting43 in which off-target, immunodominant epitopes distract from target epitopes of interest in generating de novo B cell memory. Finally, scaffold-directed immunological memory may CD96 also result in antibody-dependent clearance of the vaccine material, thereby limiting sequential or diversified immunizations with a given P-VLP44,45. We hypothesized that these limitations could be overcome by multivalent antigen display on a non-protein scaffold, which we could test by scaffolding a TD antigen on an icosahedral DNA origami nanoparticle that is a TI antigen. This platform provides unique access, compared with other materials, including proteins, to rationally designed?DNA-based VLPs?(DNA-VLPs) below the optimal 50?nm size-scale with scaffold-independent control over the valency and spatial business of antigen display46C51. While we as well as others have leveraged these VLPs in vitro to probe the nanoscale parameters of IgM recognition52 and BCR signaling.