Distinct oligomeric assemblies of STING induced by non-nucleotide agonists.

STING plays essential roles coordinating innate immune responses to processes that range from pathogenic infection to genomic instability. Its adaptor function is activated by cyclic dinucleotide (CDN) secondary messengers originating from self (2'3'-cGAMP) or bacterial sources (3'3'-CDNs). Different classes of CDNs possess distinct binding modes, stabilizing STING's ligand-binding domain (LBD) in either a closed or open conformation. The closed conformation, induced by the endogenous ligand 2'3'-cGAMP, has been extensively studied using cryo-EM. However, significant questions remain regarding the structural basis of STING activation by open conformation-inducing ligands. Using cryo-EM, we investigate potential differences in conformational changes and oligomeric assemblies of STING for closed and open conformation-inducing synthetic agonists. While we observe a characteristic 180° rotation for both classes, the open-LBD inducing agonist diABZI-3 uniquely induces a quaternary structure reminiscent but distinct from the reported autoinhibited state of apo-STING. Additionally, we observe slower rates of activation for this ligand class in functional assays, which collectively suggests the existence of a potential additional regulatory mechanism for open conformation-inducing ligands that involves head-to-head interactions and restriction of curved oligomer formation. These observations have potential implications in the selection of an optimal class of STING agonist in the context of a defined therapeutic application.

Neuraminidase-specific antibodies drive differential cross-protection between contemporary FLUBV lineages.

The two influenza B virus (FLUBV) lineages have continuously diverged from each other since the 1980s, with recent (post-2015) viruses exhibiting accelerated evolutionary rates. Emerging data from human studies and epidemiological models suggest that increased divergence in contemporary viruses may drive differential cross-protection, where infection with Yamagata lineage viruses provides limited immunity against Victoria lineage viruses. Here, we developed animal models to investigate the mechanisms behind asymmetric cross-protection between contemporary FLUBV lineages. Our results show that contemporary Victoria immunity provides robust cross-protection against the Yamagata lineage, whereas Yamagata immunity offers limited protection against the Victoria lineage. This differential cross-protection is driven by Victoria-elicited neuraminidase (NA)-specific antibodies, which show cross-lineage reactivity, unlike those from Yamagata infections. These findings identify a phenomenon in contemporary FLUBV that may help explain the recent disappearance of the Yamagata lineage from circulation, highlighting the crucial role of targeting NA in vaccination strategies to enhance cross-lineage FLUBV protection.

Long-lasting B cell convergence to distinct broadly reactive epitopes following vaccination with chimeric influenza virus hemagglutinins.

In a phase 1 clinical trial, a chimeric hemagglutinin (cHA) immunogen induced antibody responses against the conserved hemagglutinin (HA) stalk domain as designed. Here, we determined the specificity, function, and subsets of B cells induced by cHA vaccination by pairing single-cell RNA sequencing and B cell receptor repertoire sequencing. We have shown that the cHA-inactivated vaccine with a squalene-based adjuvant induced a robust activated B cell and memory B cell (MBC) phenotype against two broadly neutralizing epitopes in the stalk domain. The overall specificities of the acute plasmablast (PB) and MBC responses clonally overlapped, suggesting B cell convergence to these broadly protective epitopes. At 1 year post immunization, we identified that cHA vaccination reshaped the HA-specific MBC pool to enrich for stalk-binding B cells. Altogether, these data indicate the cHA vaccine induced robust and durable B cell responses against broadly protective epitopes of the HA stalk domain, in line with serological data.

Rational design of next-generation filovirus vaccines with glycoprotein stabilization, nanoparticle display, and glycan modification.

Filoviruses pose a significant threat to human health with frequent outbreaks and high mortality. Although two vector-based vaccines are available for Ebola virus, a broadly protective filovirus vaccine remains elusive. In this study, we evaluate a general strategy for stabilizing glycoprotein (GP) structures of Ebola, Sudan, and Bundibugyo ebolaviruses and Ravn marburgvirus. A 3.2 Å-resolution crystal structure provides atomic details for the redesigned Ebola virus GP, and cryo-electron microscopy reveals how a pan-ebolavirus neutralizing antibody targets a conserved site on the Sudan virus GP (3.13 Å-resolution), in addition to a low-resolution model of antibody-bound Ravn virus GP. A self-assembling protein nanoparticle (SApNP), I3-01v9, is redesigned at the N-terminus to allow the optimal surface display of filovirus GP trimers. Following detailed in vitro characterization, the lymph node dynamics of Sudan virus GP and GP-presenting SApNPs are investigated in a mouse model. Compared with soluble GP trimer, SApNPs show ~112 times longer retention in lymph node follicles, up-to-28 times greater presentation on follicular dendritic cell dendrites, and up-to-3 times stronger germinal center reactions. Functional antibody responses induced by filovirus GP trimers and SApNPs bearing wildtype and modified glycans are assessed in mice. Our study provides a foundation for next-generation filovirus vaccine development.

Local structural flexibility drives oligomorphism in computationally designed protein assemblies.

Many naturally occurring protein assemblies have dynamic structures that allow them to perform specialized functions. Although computational methods for designing novel self-assembling proteins have advanced substantially over the past decade, they primarily focus on designing static structures. Here we characterize three distinct computationally designed protein assemblies that exhibit unanticipated structural diversity arising from flexibility in their subunits. Cryo-EM single-particle reconstructions and native mass spectrometry reveal two distinct architectures for two assemblies, while six cryo-EM reconstructions for the third likely represent a subset of its solution-phase structures. Structural modeling and molecular dynamics simulations indicate that constrained flexibility within the subunits of each assembly promotes a defined range of architectures rather than nonspecific aggregation. Redesigning the flexible region in one building block rescues the intended monomorphic assembly. These findings highlight structural flexibility as a powerful design principle, enabling exploration of new structural and functional spaces in protein assembly design.