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.

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.

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.

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.

Cellular and molecular characterization of immune responses elicited by influenza virus infection and seasonal vaccination have informed efforts to improve vaccine efficacy, breadth, and longevity. Here, we use negative stain electron microscopy polyclonal epitope mapping (nsEMPEM) to structurally characterize the humoral IgG antibody responses to hemagglutinin (HA) from human patients vaccinated with a seasonal quadrivalent flu vaccine or infected with influenza A viruses. Our data show that both vaccinated and infected patients had humoral IgGs targeting highly conserved regions on both H1 and H3 subtype HAs, including the stem and anchor, which are targets for universal influenza vaccine design. Responses against H1 predominantly targeted the central stem epitope in infected patients and vaccinated donors, whereas head epitopes were more prominently targeted on H3. Responses against H3 were less abundant, but a greater diversity of H3 epitopes were targeted relative to H1. While our analysis is limited by sample size, on average, vaccinated donors responded to a greater diversity of epitopes on both H1 and H3 than infected patients. These data establish a baseline for assessing polyclonal antibody responses in vaccination and infection, providing a context for future vaccine trials and emphasizing the need for further characterization of protective responses toward conserved epitopes.

The COVID-19 pandemic underscores the need to prepare for future emerging coronavriuses (CoVs) by understanding the principles behind effective CoV vaccine design such as protective immunity and antibody responses. To study which epitopes and subdomains contribute to in vivo protection, we utilized the prefusion-stabilized spike protein of MERS-CoV, MERS S-2P, as a vaccine immunogen. Vaccination with MERS S-2P elicited both receptor-binding domain (RBD)- and non-RBD-specific antibodies, including N-terminal domain (NTD)-specific G2-and CDC2-A2-like antibodies. Intriguingly, the immunogen MERS S-2P_ΔRBD, MERS S-2P with the RBDs removed, protects comparably to S1 and S-2P immunogens against MERS-CoV challenge. Moreover, passive transfer studies of polyclonal IgG from MERS S-2P immunized mice depleted of subdomain-specific antibodies demonstrated that non-RBD antibodies protected more than non-NTD antibodies. Altogether, these findings illustrate that in-vivo protection is not solely driven by RBD-specific antibodies and highlights the importance of targeting non-RBD sites in future CoV vaccine designs.

Chimeric hemagglutinins (cHA) appear to be promising for the design and development of universal influenza vaccines. Influenza A group 1 cHAs, cH5/1, cH8/1, and cH11/1, comprising an H1 stem attached to either an H5, H8, or H11 globular head, have been used sequentially as vaccine immunogens in human clinical trials and induced high levels of broadly protective antibodies. Using X-ray crystallography and negative-stain electron microscopy, we determined structures of cH5/1, cH8/1, and cH11/1 HAs in their apo (unliganded) and antibody Fab-bound states. Stem-reactive antibodies 3E1 and 31.b.09 recognize their cognate epitopes in cH5/1, cH8/1, and cH11/1 HAs. However, with cH5/1, the head domains are rotated by 35 to 45° around the threefold axis of the HA trimer compared to native HA with a more splayed-open conformation at the stem base. cH11/1 with 3E1 is structurally more native-like but resembles cH5/1 with 31.b.09, whereas cH8/1 with 31.b.09 exhibited a range of closed-to-open stem configurations with some separation of head and stem domains. Furthermore, all of these group 1 cHAs effectively bound a broad head trimer interface antibody and other broad stem antibodies. Thus, the cHAs exhibit structural plasticity without compromising the stem and head trimer interface epitopes for elicitation of influenza A group 1 cross-reactive antibodies.

H1N1 influenza viruses are responsible for both seasonal and pandemic influenza. The continual antigenic shift and drift of these viruses highlight the urgent need for a universal influenza vaccine to elicit broadly neutralizing antibodies (bnAbs). Identification and characterization of bnAbs elicited in natural infection and immunization to influenza virus hemagglutinin (HA) can provide insights for development of a universal influenza vaccine. Here, we structurally and biophysically characterize four antibodies that bind to a conserved region on the HA membrane-proximal region known as the anchor epitope. Despite some diversity in their VH and VK genes, the antibodies interact with the HA through germline-encoded residues in HCDR2 and LCDR3. Somatic mutations on HCDR3 also contribute hydrophobic interactions with the conserved HA epitope. This convergent binding mode provides extensive neutralization breadth against H1N1 viruses and suggests possible countermeasures against H1N1 viruses.

Background We report findings from an experimental medicine study of rationally designed prefusion stabilised native-like HIV envelope glycoprotein (Env) immunogens, representative of global circulating strains, delivered by sequential intramuscular injection. Methods Healthy adult volunteers were enrolled into one of five groups (A to E) each receiving a different schedule of one of two consensus Env immunogens (ConM SOSIP, ConS UFO, either unmodified or stabilised by chemical cross-linking, followed by a boost with two mosaic Env immunogens (Mos3.1 and Mos3.2). All immunogens were co-formulated with liposomal Monophosphoryl-Lipid A (MPLA) adjuvant, and volunteers were followed up for 28 days post final Mosaic booster injection. Participants gave written informed consent to join the study. The study is registered on ClinicalTrials.gov ID NCT03816137. Findings Fifty-one participants (men n = 23 and women n = 28) aged 18–55 were enrolled. The seroconversion rate against Env was 100% with all participants having measurable anti-Env IgG antibodies after their second injection and throughout the study. Neutralisation was detected against the ConM pseudovirus in sera of those who had received both ConM and ConS immunogens. However, this activity was limited in breadth and was neither boosted nor broadened in those receiving the Mos3.1 and Mos3.2 immunogens. Neutralising antibody function correlated with binding to V1/V3 and V5 epitopes and peaked after the third injection. Interpretation Rationally designed prefusion-stabilised native-like Env trimers are robustly immunogenic in a prime-boost schedule. When given alone they are insufficient to induce neutralising antibody titres of significant breadth, but they represent potentially valuable polishing immunogens after germline-targeting. Funding European Aids Vaccine initiative (EAVI2020) received funding from EU Horizon 2020, grant number 681137. Structural studies were supported by the Bill and Melinda Gates Foundation (INV-002916).

Vaccination strategies against HIV-1 aim to elicit broadly neutralizing antibodies (bnAbs) using prime-boost regimens with HIV envelope (Env) immunogens. Epitope mapping has shown that early antibody responses are directed to easily accessible nonneutralizing epitopes on Env instead of bnAb epitopes. Autologously neutralizing antibody responses appear upon boosting, once immunodominant epitopes are saturated. Here, we use electron microscopy–based polyclonal epitope mapping (EMPEM) to elucidate how repeated immunization with HIV Env SOSIP immunogens results in the generation of Ab2α anti-idiotypic antibodies in rabbits and rhesus macaques. We present the structures of six anti–immune complex antibodies and find that they target idiotopes composed of framework regions of antibodies bound to Env. Examination of cryo–electron microscopy density enabled prediction of sequences for an anti–immune complex antibody, the paratope of which is enriched with aromatic amino acids. This work sheds light on current vaccine development efforts for HIV, as well as for other pathogens in which repeated exposure to antigen is required.