Codelivery of SARS-CoV-2 Prefusion-Spike Protein with CBLB502 by a Dual-Chambered Ferritin Nanocarrier Potentiates Systemic and Mucosal Immunity.

Thousands of breakthrough infections are confirmed after intramuscular (i.m.) injection of the approved vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Two major factors might contribute to breakthrough infections. One is the emergence of mutant variants of SARS-CoV-2, and the other is that i.m. injection has an inefficient ability to activate mucosal immunity in the upper respiratory tract. Here, we devised a dual-chambered nanocarrier that can codeliver the adjuvant CBLB502 with prefusion-spike (pre-S) onto a ferritin nanoparticle. This vaccine enabled enhanced systemic and local mucosal immunity in the upper and lower respiratory tract. Further, codelivery of CBLB502 with pre-S induced a Th1/Th2-balanced immunoglobulin G response. Moreover, the codelivery nanoparticle showed a Th1-biased cellular immune response as the release of splenic INF-γ was significantly heightened while the level of IL-4 was elevated to a moderate extent. In general, the developed dual-chambered nanoparticle can trigger multifaceted immune responses and shows great potential for mucosal vaccine development.

Scaffold subunits support associated subunit assembly in the Chlamydomonas ciliary nexin-dynein regulatory complex.

The nexin-dynein regulatory complex (N-DRC) in motile cilia and flagella functions as a linker between neighboring doublet microtubules, acts to stabilize the axonemal core structure, and serves as a central hub for the regulation of ciliary motility. Although the N-DRC has been studied extensively using genetic, biochemical, and structural approaches, the precise arrangement of the 11 (or more) N-DRC subunits remains unknown. Here, using cryo-electron tomography, we have compared the structure of Chlamydomonas wild-type flagella to that of strains with specific DRC subunit deletions or rescued strains with tagged DRC subunits. Our results show that DRC7 is a central linker subunit that helps connect the N-DRC to the outer dynein arms. DRC11 is required for the assembly of DRC8, and DRC8/11 form a subcomplex in the proximal lobe of the linker domain that is required to form stable contacts to the neighboring B-tubule. Gold labeling of tagged subunits determines the precise locations of the previously ambiguous N terminus of DRC4 and C terminus of DRC5. DRC4 is now shown to contribute to the core scaffold of the N-DRC. Our results reveal the overall architecture of N-DRC, with the 3 subunits DRC1/2/4 forming a core complex that serves as the scaffold for the assembly of the "functional subunits," namely DRC3/5-8/11. These findings shed light on N-DRC assembly and its role in regulating flagellar beating.

The Structure of a Full-length Membrane-embedded Integrin Bound to a Physiological Ligand.

Increased ligand binding to integrin ("activation") underpins many biological processes, such as leukocyte trafficking, cell migration, host-pathogen interaction, and hemostasis. Integrins exist in several conformations, ranging from compact and bent to extended and open. However, the exact conformation of membrane-embedded, full-length integrin bound to its physiological macromolecular ligand is still unclear. Integrin αIIbß3, the most abundant integrin in platelets, has been a prototype for integrin activation studies. Using negative stain electron microscopy and nanodisc-embedding to provide a membrane-like environment, we visualized the conformation of full-length αIIbß3 in both a Mn(2+)-activated, ligand-free state and a Mn(2+)-activated, fibrin-bound state. Activated but ligand-free integrins exist mainly in the compact conformation, whereas fibrin-bound αIIbß3 predominantly exists in a fully extended, headpiece open conformation. Our results show that membrane-embedded, full-length integrin adopts an extended and open conformation when bound to its physiological macromolecular ligand.

Three-dimensional structure of the hepatitis B core antigen particle truncated at residue 154.

The three-dimensional structure of recombinant hepatitis B core antigen (HBcAg) particles truncated at residue 154 (HBcAg-154) was determined to 7.8 Å resolution by cryo-electron microscopy (cryoEM) and computer reconstruction. The capsid of HBcAg-154 is mainly constituted by α-helical folds, highly similar to that of HBcAg-149. The C-terminal region between residues 155 and 183 of the core protein is more crucial to the encapsidation of RNA, and the short C-terminal tail of HBcAg-154 results in a nearly empty capsid.

Structural comparisons of hepatitis B core antigen particles with different C-terminal lengths.

Core protein of hepatitis B virus (HBV) with various C-terminal lengths (residue 154, 164, 167 and 183) can self-assemble into recombinant hepatitis B core antigen (HBcAg) particles. To understand the RNA encapsidation mechanism of HBV, the three-dimensional structures of these particles were reconstructed by cryo-electron microscopy (cryoEM). Detailed structural comparisons showed that their capsid structures are highly similar, while the RNA content is increased upon the retention of more amino acid residues at the C-terminus of core protein, suggesting the crucial role of the basic C-terminal tail on determining the genome size.

The truncated virus-like particles of C6/36 cell densovirus: implications for the assembly mechanism of brevidensovirus.

The brevidensovirus is one of the smallest viruses in the world and the capsid of Aedes albopictus C6/36 cell densovirus (C6/36DNV) is the simplest and most compact capsid in brevidensovirus. To understand the assembly mechanism of icosahedral-virus capsid from this simplest model, we tried to express various lengths of virus proteins (VPs) of C6/36DNV in Bac-to-Bac system and evaluate their self-assembly capacities in insect Spodoptera frugiperda 9 (Sf9) cells. The result showed that the N-terminal GGSG sequence (residue 23-26), highly conserved glycine-rich region in Parvoviridae, and C-terminal GTGGVVTCMP (residue 344-353) were essential for capsid assembly, while the N-terminal nuclear localization signal, GTKRKR sequence (residue 15-20), was nonessential for the virus-like particles (VLPs) assembly, but did effect the formation of crystalline arrays in infected Sf9 cells. These information provided clues for how icosahedral-virus capsids formed and showed the potential of C6/36DNV-VLPs becoming a powerful nanoparticle vector.