Science 349, 1301C1306. [PubMed] [Google Scholar] 70. promote the shut trimeric condition, we utilized Rosetta to optimize the hydrophobic interfaces between your RBDs while staying away from immediate mutation of residues approached by trimer-interface-directed antibodies27,29 (Statistics 1A and ?and1B).1B). A style formulated with the designed disulfide and four mutations, which we called TH-2heptad (S203L/N210D/R212V/E216I; Body 1B), was scaled up and purified by SEC effectively, which verified its general trimeric condition (Body S1A). In contrast to previous constructs, TH-2heptad did not bind to FluA-20, while a monomeric RBD containing the same trimer interface mutations, NC99-RBD-TI, bound as well as the wild-type NC99-RBD (Figure 1C). C05 binding was unaffected by the trimeric interface mutations, as expected. These results demonstrate that the trimer interface mutations help to stabilize the RBDs in a closed trimeric state, preventing FluA-20 access, but do not inherently disrupt the FluA-20 epitope. We assembled TH nanoparticles by mixing purified TH-2heptad trimer with the complementary pentameric component I53_dn5A (Figure 1D)70 and purified them using SEC (Figures S1B and S1C). Cryo-electron microscopy (cryo-EM) 2D class averages of the nanoparticles revealed clear density for trimeric antigens on the surface of the nanoparticle (Figure 1E). The clarity with which the THs were resolved relative to the icosahedral nanoparticle scaffold sharply contrasts with previously described nanoparticle immunogens that used flexible genetic linkers62,72C77 and indicates rigid attachment to I53_dn5. Removal of the disulfide between E107C and C220 in the heptad domain abolished secretion, establishing that the combination of this disulfide and the trimer interface mutations was required for TH formation. Taken together, our Fidarestat (SNK-860) BLI and cryo-EM data establish that TH-2heptad-I53_dn5 rigidly displays closed trimeric HA heads as intended. Design and characterization of TH nanoparticles with varied antigen spacing To enable investigation of the effects of antigen spacing on immunogenicity, we designed two new sequences that modified solely the heptad domain while retaining its rigidity. These were TH-1heptad, with one fewer heptad Fidarestat (SNK-860) domain than TH-2heptad, and TH-6heptad, with four additional heptad domains that substantially lengthen the extendable region (Figure 2A). These constructs aimed to alter antigen-antigen spacing while keeping fixed the angle between the symmetry axes of neighboring antigens. We also designed a fourth bobblehead construct that introduced a glycine- and serine-based linker between the 2heptad domain and I53_dn5B to introduce flexibility between the antigens and I53_dn5 while maintaining trimeric closure of the RBDs. Finally, we included a control component, HA-I53_dn5B, which features the full NC99 HA ectodomain fused to I53_dn5B in the same manner as we previously reported.72 Each of these novel nanoparticle components were secreted from HEK293F cells and purified via IMAC and SEC (Figure S1A). All of the proteins, including the original monohead component, showed similar binding to the RBS-directed mAb C0522 and the lateral patch-targeting mAb Ab6649,35 demonstrating the preservation of these epitopes across all designs (Figure 2B). By contrast, FluA-20 binding was high Fidarestat (SNK-860) only for the monohead, with very slight binding to TH-6heptad and HA-I53_dn5B and no binding to any of the other constructs, indicating stable RBD closure. As described above for TH-2heptad, the novel components were assembled with the I53_dn5A pentamer to form nanoparticles and purified via SEC (Figure S1B). The SEC chromatograms, SDS-PAGE (Figure S1C), dynamic light scattering (DLS; Figure 2C), and negative-stain EM (nsEM; Figure 2D) all indicated that the nanoparticles formed the intended assemblies with high Rabbit Polyclonal to MMP1 (Cleaved-Phe100) homogeneity. The expected differences in size and antigen Fidarestat (SNK-860) spacing between the three TH nanoparticles were evident in the DLS and nsEM data, respectively. The lack of significant amounts of residual, unassembled component during SEC indicated efficient assembly for all TH nanoparticles, bobblehead-I53_dn5, and HA-I53_dn5, whereas the monohead nanoparticle assembled less efficiently (Figure S1B; peak at ~16 mL). All TH nanoparticles showed melting temperatures that were similar to HA-I53_dn5 but higher than monohead-I53_dn5 (Figure S1D), suggesting that conformational stabilization of the RBDs in a trimeric state also reinforces their thermal stability. Open in a separate window Figure 2. Fidarestat (SNK-860) Design and characterization of trihead nanoparticles with varied antigen spacing(A) Models of trihead nanoparticles with various extension domains, monohead-I53_dn5, and HA-I53_dn5. The models are colored blue, gray, and orange, respectively, for HA-derived segments, I53_dn5B/GCN4, and I53_dn5A. (B) BLI of trihead nanoparticle.
