Protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-related emergent zoonotic coronaviruses is urgently needed. We made homotypic nanoparticles displaying the receptor binding domain (RBD) of SARS-CoV-2 or co-displaying SARS-CoV-2 RBD along with RBDs from animal betacoronaviruses that represent threats to humans (mosaic nanoparticles with four to eight distinct RBDs). Mice immunized with RBD nanoparticles, but not soluble antigen, elicited cross-reactive binding and neutralization responses. Mosaic RBD nanoparticles elicited antibodies with superior cross-reactive recognition of heterologous RBDs relative to sera from immunizations with homotypic SARS-CoV-2–RBD nanoparticles or COVID-19 convalescent human plasmas. Moreover, after priming, sera from mosaic RBD–immunized mice neutralized heterologous pseudotyped coronaviruses as well as or better than sera from homotypic SARS-CoV-2–RBD nanoparticle immunizations, demonstrating no loss of immunogenicity against particular RBDs resulting from co-display. A single immunization with mosaic RBD nanoparticles provides a potential strategy to simultaneously protect against SARS-CoV-2 and emerging zoonotic coronaviruses.

Protection against SARS-CoV-2 and SARS-related zoonotic coronaviruses with pandemic potential is urgently needed. To evaluate immunization strategies, we made nanoparticles displaying the receptor-binding domain (RBD) of only SARS-CoV-2 (homotypic nanoparticles) or co-displaying the SARS-CoV-2 RBD along with RBDs from animal betacoronaviruses that represent threats to humans (mosaic nanoparticles; 4-8 distinct RBDs). Mice immunized with RBD-nanoparticles, but not soluble antigen, elicited cross-reactive antibody binding and neutralization responses, confirming increased immunogenicity from multimerization. Mosaic-RBD-nanoparticles elicited antibodies with superior cross-reactive recognition of heterologous RBDs compared to sera from immunizations with homotypic SARS-CoV-2-RBD-nanoparticles or antibodies from COVID-19 convalescent human plasmas. Moreover, sera from mosaic-RBD-immunized mice neutralized heterologous pseudotyped coronaviruses equivalently or better after priming than sera from homotypic SARS-CoV-2-RBD-nanoparticle immunizations, demonstrating no loss of immunogenicity against any particular RBD resulting from co-display. Thus, a single immunization with mosaic-RBD-nanoparticles provides a potential strategy to simultaneously protect against SARS-CoV-2 and emerging zoonotic coronaviruses.

GM1 has generally been considered as the major receptor that binds to cholera toxin subunit B (CTB) due to its low dissociation constant. However, using a unique nanocube sensor technology, we have shown that CTB can also bind to other glycolipid receptors, fucosyl-GM1 and GD1b. Additionally, we have demonstrated that GM2 can contribute to CTB binding if present in a glycolipid mixture with a strongly binding receptor (GM1/fucosyl-GM1/GD1b). This hetero-multivalent binding result was unintuitive because the interaction between CTB and pure GM2 is negligible. We hypothesized that the reduced dimensionality of CTB-GM2 binding events is a major cause of the observed CTB binding enhancement. Once CTB has attached to a strong receptor, subsequent binding events are confined to a 2D membrane surface. Therefore, even a weak GM2 receptor could now participate in second or higher binding events because its surface reaction rate can be up to 104 times higher than the bulk reaction rate. To test this hypothesis, we altered the surface reaction rate by modulating the fluidity and heterogeneity of the model membrane. Decreasing membrane fluidity reduced the binding cooperativity between GM2 and a strong receptor. Our findings indicated a new protein-receptor binding assay, that can mimic complex cell membrane environment more accurately, is required to explore the inherent hetero-multivalency of the cell membrane. We have thus developed a new membrane perturbation protocol to efficiently screen receptor candidates involved in hetero-multivalent protein binding.

Double-stranded ladderphanes 2, 3, and 5 having cubane, cuneane, and cyclooctatetraene linkers are synthesized by ring-opening metathesis polymerization (ROMP) of the corresponding bisnorbornene monomers 10, 11, and 13, respectively. Attempts to polymerize the corresponding tricyclooctadiene-linked bisnorbornene 12 are not successful, starting monomer being recovered. Polymer with this tricyclic diene linker 4 is obtained from the rhodium-catalyzed isomerization of 2. The scanning tunneling microscopic (STM) image of 2 shows an ordered pattern on the graphite surface by self-assembly.

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