Wei Peng

Respiratory syncytial virus (RSV) remains a major cause of severe respiratory disease, and stabilization of the prefusion (preF) conformation of the F glycoprotein is central for vaccine development. Here, we report a structure-guided engineering strategy that enhances preF stability by reducing the hydrophobic cavity within the trimeric F protein. Targeted modifications at metastability-associated sites generated RVF-88, a disulfide-free stabilized preF immunogen that preserves key neutralizing epitopes, including antigenic site Ø, while exhibiting improved structural integrity and long-term storage stability. Formulated as an unadjuvanted bivalent vaccine, RVF-88 elicited potent neutralizing antibody responses and durable immune protection lasting up to 5 months in mice. Vaccination also protected both mice and cotton rats against RSV challenge. Structural analyses confirmed the intended cavity-reduction design, revealing a reduced apical hydrophobic cavity volume and surface area while maintaining the prefusion architecture. Together, these findings establish hydrophobic cavity reduction as a rational strategy for stabilizing prefusion RSV F and provide a promising next-generation vaccine candidate with improved stability and immunogenicity for further clinical development.

Neisseria meningitidis serogroup B (MenB) remains a major global cause of meningitis and septicemia. However, MenB vaccine development is hindered by antigenic diversity and manufacturing challenges associated with outer membrane vesicles (OMVs) or lipoproteins in resource-limited settings. Here, we engineered a high-purity circular RNA (circRNA) vaccine encoding a bacterial-derived, sequence-optimized fusion antigen of factor H binding protein (fHbp) and Neisserial heparin-binding antigen (NHBA) (fHbp-NHBA), which preserves key bactericidal epitopes in eukaryotic systems. The vaccine elicited potent functional antibody responses by serum bactericidal assay (SBA) using human complement and potent IFN-γ-secreting CD8 + T-cell responses. Crucially, the circRNA vaccine at a low dose provided complete protection and reduced bacteremia against lethal MenB challenge and demonstrated cross protection against a panel of prevalent strains in China. Both single- and two-dose regimens of the circRNA vaccine induced durable immune responses, and the two-dose immunization achieved strain coverage comparable to that of licensed MenB vaccines. Moreover, sera adoptively transferred from the circRNA-immunized group to neonatal rat pups dramatically reduced bacterial loads in blood. This study establishes a foundation for circRNA-based vaccines against bacterial pathogens.

Brown adipose tissue (BAT) dissipates energy through non-shivering thermogenesis and holds promise as a therapeutic target for obesity. The transcription factor Early B Cell Factor 2 (EBF2) is a key regulator of the thermogenic gene program, yet its underlying transcriptional regulatory mechanisms and potential for pharmacological targeting remain incompletely defined. Here, we identify a low-complexity C-terminal domain (CTD) within EBF2 that drives biomolecular condensation critical for thermogenic activation. Deletion of the CTD (ΔCTD) or mutation of conserved proline residues disrupts EBF2 phase separation without affecting genomic occupancy, thereby impairing brown adipocyte differentiation and thermogenic capacity both in vitro and in male mice. Remarkably, fusion of the intrinsically disordered region (IDR) of FUS to the EBF2 ΔCTD mutant fully rescues its function, whereas fusion with the MED1-IDR produces distinct transcriptional and phenotypic outcomes. Mechanistically, EBF2 condensates sequester the transcriptional repressor ZFP423 while excluding HDAC1, creating a permissive chromatin environment that promotes thermogenic gene expression. Finally, a phenotypic small-molecule screen targeting phase separation identifies compounds that modulate EBF2 condensate dynamics and thermogenic programming. Together, our findings reveal phase separation as an emerging regulatory mechanism underlying brown fat determination and suggest that targeting biomolecular condensation offers a potential therapeutic strategy for obesity.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in infants and the elderly worldwide. Although prophylactic monoclonal antibodies and RSV vaccines are available for preventing severe RSV infection, unmet medical need remains for an effective antiviral agent to treat patients who do not benefit from these interventions. Ziresovir (formerly AK0529) is a potent, selective, and orally bioavailable RSV fusion inhibitor with proved antiviral efficacy and clinical benefits. To understand the molecular mechanism of action, we computationally modeled ziresovir with the RSV fusion (F) protein. Here, we present a cryo-EM structure of the RSV F protein-ziresovir complex, elucidating the molecular interactions underlying the drug binding, revealing ziresovir specifically binds to the central cavity within the metastable prefusion conformation of RSV F protein. Leveraging this structural insight, we engineered site-directed RSV mutants guided by both the cryo-EM binding model and drug-resistant RSV variants for fusion inhibitors identified in vitro , and demonstrated that these resistant viruses do not replicate as efficient as wild-type RSV and indicated a fitness cost for viral escape from drug treatment. Collectively, these findings unveil the structural mechanism of ziresovir-mediated viral inhibition, providing a framework for developing the next-generation RSV fusion inhibitors.

Lonafarnib, an oral antiviral that targets the fusion glycoprotein of respiratory syncytial virus (RSV), has demonstrated efficacy in vitro and in vivo . However, because the RSV has evolved to become resistant to other fusion inhibitors, there is a concern that the same could occur for lonafarnib. Here, we identified resistance to lonafarnib in the RSV A2 strain and a recent clinical isolate, RSV ON1, via in vitro selection at scale. Cell‒cell fusion and recombinant live RSV analysis confirmed that the mutations at K394, K399, and T400 of the cysteine-rich region of the fusion protein mediate high-level resistance. Lonafarnib resistance mutations also confer cross-resistance to other fusion inhibitors of clinical interest. All-atom molecular dynamics simulations revealed that these resistance mutations confer reduced stability to the fusion protein, thereby diminishing its binding affinity with lonafarnib. To address this vulnerability proactively and increase the barrier to resistance development, we designed the first potent proteolysis-targeting chimera (PROTAC) fusion protein degrader, compound 0179841, which uses lonafarnib and cereblon as ligands. This PROTAC effectively inhibited RSV replication. Collectively, our findings indicate that RSV develops resistance to lonafarnib in the cysteine-rich region of the fusion protein. This work sheds light on the mechanisms by which RSV evolves resistance to lonafarnib and provides a foundation for the rational design of antivirals aimed at preventing resistance. IMPORTANCE: Respiratory syncytial virus (RSV) infection poses a substantial public health challenge. Resistance to several potent fusion inhibitors, which are currently in various stages of clinical development, can readily emerge. Through a drug repurposing screen, we identified lonafarnib as an RSV fusion inhibitor; however, concerns exist regarding the potential development of resistance. Here, large-scale in vitro selection experiments revealed specific mutations within the highly conserved cysteine-rich region of the fusion (F) protein that confer high-level lonafarnib resistance across diverse RSV strains. These resistance mutations also confer cross-resistance to other clinical-stage fusion inhibitors. Mechanistic investigations demonstrated that these mutations reduce F protein stability, thereby diminishing the binding affinity of lonafarnib. As a proof of concept for an alternative antiviral strategy, we rationally designed the first potent proteolysis-targeting chimera (PROTAC) F protein degrader, compound 0179841, by utilizing lonafarnib and cereblon ligands. This novel antiviral agent effectively inhibits RSV infection by inducing degradation of the F protein. This work elucidates the molecular basis of RSV resistance to lonafarnib and establishes a strategy for developing next-generation antivirals aimed at preempting resistance.

Integrated virtual and biological screening guided the design and synthesis of 50 novel pyrimido[1,2- a ]imidazole derivatives, leading to the discovery of potent Pks13-TE inhibitor 34 (IC ₅₀ = 0.23 μM).