Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) cause human respiratory diseases and are major targets for vaccine development. In this study, we design uncleaved prefusion-closed (UFC) trimers for the fusion protein (F) of both viruses by examining mutations critical to F metastability. For RSV, we assess four previous prefusion F designs, including the first and second generations of DS-Cav1, SC-TM, and 847A. We then identify key mutations that can maintain prefusion F in a native-like, closed trimeric form (up to 76%) without introducing any interprotomer disulfide bond. For hMPV, we develop a stable UFC trimer with a truncated F₂-F₁ linkage and an interprotomer disulfide bond. Dozens of UFC constructs are characterized by negative-stain electron microscopy (nsEM), x-ray crystallography (11 RSV-F structures and one hMPV-F structure), and antigenic profiling. Using an optimized RSV-F UFC trimer as bait, we identify three potent RSV neutralizing antibodies (NAbs) from a phage-displayed human antibody library, with a public NAb lineage targeting sites Ø and V and two cross-pneumovirus NAbs recognizing site III. In mouse immunization, rationally designed RSV-F and hMPV-F UFC trimers induce robust antibody responses with high neutralizing titers. Our study provides a foundation for future prefusion F-based RSV and hMPV vaccine development.

Filoviruses pose a significant threat to human health due to frequent outbreaks and high mortality. Although two vector-based vaccines are available for Ebola virus, a broadly protective filovirus vaccine remains elusive. Here, we evaluate a general strategy for stabilizing glycoproteins (GP) from Ebola, Sudan, and Bundibugyo orthoebolaviruses, as well as Ravn orthomarburgvirus. A 3.2 Å crystal structure provides atomic-level details of the redesigned Ebola virus GP, while cryo-electron microscopy reveals how a pan-orthoebolavirus neutralizing antibody targets a conserved site on the stabilized Sudan virus GP (3.13 Å resolution), along with 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 enable optimal surface display of filovirus GP trimers. Following detailed in vitro characterization, we examine the lymph node dynamics of Sudan virus GP and GP-presenting SApNPs in mice. Compared with the soluble trimer, SApNPs exhibit ~112-fold longer retention in lymph node follicles, up to 28-fold greater presentation on follicular dendritic cell dendrites, and up to 3-fold stronger germinal center reactions. Functional antibody responses induced by filovirus GP trimers and SApNPs bearing wild-type or modified glycans are assessed in mice. This study provides a foundation for next-generation filovirus vaccine development.

The development of an effective and broadly protective influenza vaccine against circulating and emerging strains remains elusive. In this study, we evaluated a potentially universal influenza vaccine based on single-component self-assembling protein nanoparticles (1c-SApNPs) presenting the conserved matrix protein 2 ectodomain (M2e) from influenza A and B viruses (IAV and IBV, respectively). We previously designed a tandem antigen comprising three IAV M2e domains of human, avian/swine, and human/swine origins (termed M2ex3). The M2ex3-presenting 1c-SApNPs conferred complete protection in mice against sequential lethal challenges with H1N1 and H3N2. To broaden this protection to cover IBVs, we designed a series of antigens incorporating different arrangements of three IAV M2e domains and three copies of IBV M2e. Tandem repeats of IAV and IBV (termed influenza A-B) M2e arrayed on the I3-01v9a 60-mer 1c-SApNP, when formulated with an oil-in-water emulsion adjuvant, generated greater M2e-specific immunogenicity and protective efficacy than the soluble influenza A-B M2e trimer, indicated by higher survival rates and reduced weight loss post-challenge. Importantly, one of the influenza A-B M2e SApNP constructs elicited 100% protection against a lethal influenza A/Puerto Rico/8/1934 (H1N1) challenge in mice and 70% protection against a lethal influenza B/Florida/4/2006 (Yamagata lineage) challenge, the latter of which has not been reported in the literature to date. Our study thus provides a promising M2e-based single-component universal vaccine candidate against the two major types of influenza virus circulating in humans.

Hepatitis C virus (HCV) is a leading cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma worldwide. Development of an E1E2-based HCV vaccine has been hindered by the difficulty of producing a soluble E1E2 (sE1E2) antigen that faithfully recapitulates the native virion-associated heterodimer. Guided by cryo-electron microscopy (cryo-EM) structures, we engineer genotype 1a H77 sE1E2 by truncating the E1 and E2 stems (Cut₁), deleting a putative fusion peptide–containing region in E1 (Cut₂), and stabilizing the heterodimer using diverse scaffolds. All H77 sE1E2.Cut₁₊₂ scaffolds exhibit native-like E1–E2 association and strong binding to the broadly neutralizing antibody (bNAb) AR4A. A genotype 1a HCV-1 sE1E2.Cut₁₊₂ variant scaffolded by a modified SpyTag/SpyCatcher (SPYΔN) is selected for in vitro and in vivo characterization, as well as further construct refinement. The structure of this HCV-1 sE1E2 construct in complex with bNAbs is determined by cryo-EM and negative-stain EM (nsEM), with an nsEM-based strategy established for antibody epitope mapping. HCV-1 sE1E2.Cut₁₊₂.SPYΔN is displayed on self-assembling protein nanoparticles (SApNPs) to enhance immunogenicity. The HCV-1 sE1E2.Cut₁₊₂.SPYΔN heterodimer and SApNPs bearing wildtype or modified glycans are evaluated in mice, alongside E2 core-based immunogens for comparison. Together, these results establish a framework for advancing E1E2-based HCV vaccines toward clinical development.

Current influenza vaccines primarily target hemagglutinin (HA), the major viral surface glycoprotein and principal determinant of neutralizing antibody (NAb) responses. However, antigenic drift and shift, together with HA’s intrinsic metastability and low-pH sensitivity, limit broad and durable vaccine protection. Here, we stabilize HA in its prefusion-closed conformation through structure-guided amino acid substitutions. Targeting a conserved residue in the HA2 central triple helix—N95 in influenza A and Q95 in influenza B—provides a core design principle for modulating HA metastability across influenza A subtypes and both influenza B lineages, although the effects vary across viral groups. Using H1 CA09 and H3 HK68 as representative strains, we display stabilized HA trimers on 24-mer ferritin and 60-mer multilayered single-component self-assembling protein nanoparticles (SApNPs). In mice, HA-presenting SApNPs exhibit prolonged retention in lymph node follicles and elicit more robust germinal center responses compared with soluble trimers. Stabilized HA trimers and SApNPs induce functional antibody responses and confer varying levels of protection against homologous, heterologous, and cross-lineage viral challenges. Glycan modification enhances NAb induction or protection in some settings. Together, these findings provide mechanistic insights into HA metastability and establish a rational design framework for next-generation HA-based influenza vaccines.