Ebolaviruses, including EBOV, SUDV, and BDBV, cause severe hemorrhagic fever, yet currently licensed monoclonal antibody (mAb) therapies against EBOV lack cross-species efficacy. While mAbs offer high specificity, favourable safety profiles, and durable serum persistence, their susceptibility to viral escape highlights the need for broader, more resilient antibody strategies. Bispecific antibodies (bsAbs), which concurrently target non-overlapping epitopes, have the potential to enhance neutralization potency, expand strain coverage, and mitigate mutation-driven resistance. In this study, we engineered three bsAb formats-CrossMab®, DVD-IgG, and IgG-ScFv-directed against distinct ebolavirus epitopes and systematically characterized their antiviral activities. All bsAbs exhibited potent neutralizing activity and conferred substantial protection in mouse challenge models. Notably, the IgG-ScFv format demonstrated the greatest improvements in neutralization potency and in vivo efficacy. These findings provide a framework for rational bsAb design and underscore their promise as next-generation immunotherapeutics capable of broad and durable protection against diverse ebolaviruses.

Traditional methods for engineering and sequence-fitness analysis of proteins in mammalian cells are limited by the time, cost, and labor associated with plasmid cloning and preparation. Here we present Microbe-Independent Deep Assembly and Screening (MIDAS), a deterministic platform for rapid protein variant expression and characterization in mammalian cells that bypasses microbial cloning by directly transfecting PCR-assembled genes. MIDAS enables high-quality sequence-fitness assessment of arbitrary mutational spaces, including truly deep saturation mutagenesis and combinatorial variant assembly, requiring less than one workday from initial PCR to cell transfection. Using MIDAS, we engineer a high-performance acetylcholine neurotransmitter bioluminescent indicator (ACh-NeuBI), achieving stepwise improvements in responsivity through linker engineering, single-site, and multi-site mutagenesis. We also apply MIDAS to engineer improved NanoLuc luciferase variants for multiple substrates, and to characterize the structural basis of mutational tolerance and substrate specificity. Thus, MIDAS is a versatile method for rapid plasmid-free protein engineering and sequence-fitness analysis in mammalian cells, offering a practical alternative to cloning-based approaches for many protein optimization and characterization tasks.

Current respiratory vaccines face two major obstacles: limited breadth of protection and insufficient induction of mucosal immunity. Here, we present a neonatal Fc receptor (FcRn)-targeted mucosal delivery strategy integrating computational antigen design to address both challenges. Using severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a model virus, we employed Epigraph to generate T cell epitope-optimized consensus sequences for the receptor-binding domain (RBD) and incorporated them into a modified human IgG1 Fc framework engineered for enhanced FcRn binding, yielding a single fusion antigen. In vitro, this antigen efficiently binds FcRn, facilitating epithelial transcytosis and prolonging mucosal retention. In vivo, it elicits robust cellular, humoral, and mucosal immune responses against SARS-CoV-2 and its major variants in both respiratory and systemic compartments, with evidence of tissue-resident memory responses. Following intranasal administration, the antigen conferred complete cross-protection against lethal challenge with representative SARS-CoV-2 and its variants. Notably, a low antigen dose of 0.2 μg in a two-dose regimen achieved full protection. Immunomics further revealed that this design induces a broader T and B cell repertoire. Collectively, this study establishes a generalizable framework that combines epitope-optimized antigen design with FcRn-mediated mucosal delivery, providing a promising strategy for broad-spectrum vaccine development against rapidly evolving respiratory viruses.

The ongoing epidemic of mpox highlights the urgency of developing novel and effective vaccines against mpox virus (MPXV). Circular RNA (circRNA) has emerged as a promising novel vaccine platform due to its high stability, resistance to exonuclease-mediated degradation, and potential to support prolonged antigen expression. Based on our lipid nanoparticle (LNP)-encapsulated circRNA vaccine platform, we developed two novel bi-antigen circRNA vaccines cirEV and cirMV, which respectively encode the combination of MPXV extracellular enveloped virion (EEV) antigens (A35R and B6R) or intracellular mature virion (IMV) antigens (A29L and M1R). The two MPXV circRNA vaccines alone or mixed as a combination vaccine successfully induce high levels of antigen-specific antibody responses and cellular immune responses against the MPXV antigens and protect mice from lethal vaccinia virus (VACV) challenge in a dose-dependent manner. Furthermore, both circRNA vaccines can provide complete long-term cross-protection against lethal VACV challenge at day 260 after first immunization, indicating the durability of vaccine-induced protective immunity. Notably, vaccine-induced cellular immune responses play a crucial role in immune protection and virus clearance in mice. Our study provides critical insights to understand the protective mechanisms underlying circRNA vaccines in an orthopoxvirus surrogate model, demonstrating that bi-antigen circRNA vaccines represent a promising multivalent vaccine platform.

Bioluminescent calcium sensors have unique uses in neuroscience and neuroengineering, enabling noninvasive imaging of neuronal activity and contactless activation of opsin-expressing neurons. However, the speed, range, and robustness of non-invasive imaging and rewiring or neuronal activity are limited by the radiance and dynamic range of existing bioluminescent calcium sensors. Here, we report the stepwise engineering of improved cyan-excitable red fluorescent proteins (mCyRFP4 and dCyRFP4), an improved red bioluminescent protein based on NanoLuc and CyRFP4 (Antares3), and an improved bioluminescent calcium sensor based on Antares3 (CaMBI3). Antares3 is 3-fold brighter than its predecessor, while CaMBI3 responds with an overall dynamic range of 24-fold, enabling high-sensitivity detection of calcium dynamics in muscle and neuronal tissues in vivo. Finally, a CaMBI3 variant and a red-shifted opsin ChRmine enabled red photon-assisted synaptic transmission in C. elegans. CaMBI3 thus facilitates genetically targeted non-invasive imaging and rewiring of neural activity in living animals.

Abstract Noninvasive imaging tools that enable real-time visualization of biological events in living subjects are highly valuable in biomedical research. Bioluminescence imaging (BLI), with its high sensitivity and low background, provides an ideal platform for developing molecular sensors to monitor intracellular signaling in vivo. Here, we report the development of a kinase-modulated bioluminescent indicator (KiMBI) for rapid, noninvasive pharmacodynamic (PD) assessment of Akt-targeted therapeutics, substantially reducing compound use and animal requirements. This ATP-independent reporter, based on NanoLuc luciferase, produces light upon administration of the brain-penetrant substrate cephalofurimazine (CFz9). Using KiMBI, we performed a structure-PD relationship analysis of the brain-active Akt inhibitor ipatasertib by designing and characterizing two novel analogs. One analog, ML-B01, exhibited robust Akt inhibition in both brain and peripheral tissues. Remarkably, capivasertib, ipatasertib, and ML-B01 all demonstrated prolonged PD effects that outlasted their pharmacokinetic (PK) profiles. Furthermore, KiMBI imaging revealed that the PD effect of an Akt-targeted proteolysis-targeting chimera (PROTAC) degrader persisted for more than three days following a single dose. Together, these results establish bioluminescence imaging with the Akt KiMBI as a sensitive and efficient method for real-time, longitudinal visualization of Akt inhibitor and degrader activity in vivo. This platform offers a powerful approach for early-stage drug optimization and for elucidating the pharmacodynamics of kinase-targeted therapies in live animals. Citation Format: Yan Wu, Chenzhou Hao, Chao Gao, Matt Hageman, Sungmoo Lee, Thomas A. Kirkland, Nathanael S. Gray, Yichi Su, Michael Z. Lin. Pharmacodynamics of Akt drugs revealed by a kinase-modulatedbioluminescent indicator withBBB-permeable substrate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2129.

Nairoviruses are emerging tick-borne pathogens for which effective antiviral therapies are currently unavailable. Although nucleoproteins (NPs) are essential for viral genome encapsulation and have been extensively characterized at the structural level, whether they perform additional functions during viral replication remains unclear. Here, we investigated the NP of the representative nairovirus Tacheng tick virus 1 (TcTV1). We found that the TcTV1 NP binds to nucleic acids in a sequence-independent manner and assembles into tetramer-based ribonucleoprotein complexes upon nucleic acid binding. This assembly process is accompanied by a pronounced conformational rearrangement that facilitates NP polymerization. In addition to its role in RNA encapsulation, TcTV1 NP exhibits intrinsic endonuclease activity that does not require metal ions and preferentially cleaves unstructured single-stranded RNA, while structured RNA substrates are largely resistant to cleavage. Functional analysis indicates that the stalk domain of NP plays a central role in coordinating RNA binding, oligomerization, and access to the nuclease-active site, thereby influencing whether an RNA molecule is protected or degraded. Finally, we identified a small-molecule compound that interferes with both RNA binding and nuclease activity by targeting a conserved functional region of nairovirus NP. Together, these results reveal an expanded functional repertoire of nairovirus NPs and suggest that NP-mediated RNA discrimination may contribute to viral replication. Our findings also support the feasibility of targeting NP for the development of antiviral drugs against emerging nairoviruses.

Due to a lack of complete mechanistic understanding, there are no specific therapeutics for necrotizing enterocolitis (NEC), a deadly gastrointestinal disease affecting premature newborns. While sodium butyrate offers intestinal protection, its efficacy is strictly dose-dependent, and the underlying mechanisms regulating intestinal epithelial cell (IEC) survival remain elusive. Here, using neonatal murine NEC models and human IECs, we demonstrate that moderate-dose butyrate (50 mg/kg) significantly attenuates mucosal injury and improves survival, whereas high-dose butyrate (250 mg/kg) exacerbates mortality by inducing complex cell death pathways including necroptosis and ferroptosis. Mechanistically, moderate butyrate suppresses glycolysis and intracellular lactate production, which restores expression of the translational regulator PABPC1; PABPC1 then binds to YB-1 mRNA to enhance YB-1 protein synthesis without altering transcription. Elevated YB-1 subsequently binds BCL-2 mRNA, increasing BCL-2 protein levels to inhibit intrinsic mitochondrial apoptosis. Functional validation shows that YB-1 knockdown abolishes butyrate-mediated protection and exacerbates TNF-α-induced apoptosis, while YB-1 overexpression rescues cell viability, and exogenous lactate supplementation reverses these effects by suppressing the PABPC1-YB-1 axis. These findings elucidate a novel Lactate-PABPC1-YB-1-BCL-2 signaling axis through which butyrate safeguards the intestinal epithelium, highlighting precise dosing and the modulation of epithelial translational control as promising therapeutic strategies for NEC.

Sudan (SUDV) and Bundibugyo (BDBV) ebolaviruses are major outbreak pathogens lacking approved vaccines or therapeutics. Research progress has been hampered by biosafety level 4 (BSL-4) restrictions. Here, we generated recombinant vesicular stomatitis viruses expressing SUDV or BDBV glycoproteins (VSV∆G-SUDV GP, VSV∆G-BDBV GP) and established lethal infection models in Type I interferon receptor knockout (IFNAR -/ -) mice under BSL-2 conditions. Both surrogate viruses induced rapid, dose-dependent mortality with median lethal dose (LD 5 0) values below 2 PFU. Infected mice developed high viral loads in the liver, spleen, and lungs, along with leukopenia, thrombocytopenia, and elevated transaminases indicating systemic infection and liver injury. Treatment with a single monoclonal antibody rEBOV-515 (mAb 515) completely protected mice from lethal challenge, reducing viral loads and restoring body weight. These findings establish reproducible surrogate models for studying SUDV and BDBV infection and provide an accessible experimental platform for evaluating GP-targeting therapeutic strategies under BSL-2 conditions.

Coronaviruses remain a challenge due to the limited or incomplete protection provided by existing vaccines, highlighting the need for improved antigen-based designs that can reduce mortality, block transmission, and provide long-lasting, broad-spectrum protection. In this study, we adapted artificial antibody strategies to display receptor-binding domains (RBDs) from representative human coronaviruses, utilizing an engineered human IgG1 framework modified at the Fab and Fc domains to support diverse antigen presentation and enhanced immunopotentiation. The results indicate that bivalent, tetravalent, and multivalent RBD constructs developed within this framework confer broad-spectrum immune protection against severe acute respiratory syndrome coronavirus 2 and other pathogenic coronaviruses. Moreover, Fc-mediated antigen delivery, primarily engaging the neonatal Fcγ receptor, enhances mucosal, cellular, and sustained immune responses. This underscores the versatility and practical utility of the modified IgG1 framework, based on artificial antibody strategies, for developing broad-spectrum mucosal vaccine antigens, representing promising vaccine candidates targeting human coronaviruses.

Developing a safe and effective vaccine is crucial to control the recent worldwide outbreaks of mpox. Here, building upon our previously established lipid nanoparticle (LNP)-encapsulated circRNA vaccine platform, we constructed two bivalent mpox virus (MPXV) circRNA vaccines: cirBA encoding a B6R-A29L tandem antigen and cirAM encoding an A35R-M1R fusion antigen. Both bivalent MPXV circRNA vaccines, whether administered alone or in combination (designated cirMix), could induce robust and durable MPXV antigen-specific humoral and cellular immune responses in mice, conferring complete protection against lethal vaccinia virus Tian Tan strain (VTT) challenge. Moreover, even at low doses (2 µg for cirBA and cirAM, 4 µg for cirMix), all circRNA vaccines could provide 100

Cholesterol (CHL) serves as a building block for membrane biogenesis and a precursor to oxysterols, steroid hormones, bile acids, and vitamin D. The lysosome serves as a major sorting station for low-density lipoproteins (LDLs), which carry dietary CHL, and it is also the cellular site where the master growth regulator, the protein kinase mechanistic Target of Rapamycin Complex 1 (mTORC1), is activated. Recently, the lysosomal transmembrane protein GPR155 was reported to signals CHL sufficiency to mTORC1 through sequestration of the GTPase-activating protein towards the Rags 1 (GATOR1). Although the recently reported structures of GPR155 have revealed the CHL binding site, how the signal is transduced from the CHL binding site to the soluble parts of GPR155 and GATOR1 remains unknown. Here, with our three cryo-EM structures of GPR155 captured in different conformations in complex with CHL, complemented by long-time scale molecular dynamics simulations, the dynamic rearrangement of different domains was observed. CHL binding induces a widening of the crevice between the transporter and GPCR domains. The extending helix preceding transmembrane helix (TM) 16, which was unresolved in other structures, acts as a linkage lever that transmits the rotation of the GPCR domain to the soluble parts of GPR155 in response to CHL binding. This work not only answers the question of how CHL is sensed by GPR155, but also addresses a more profound question: how the signal perceived by the TMs regions is transduced to the LED and DEP domains.

Negative-stranded segmented RNA viruses (NSVs) employ a cap-snatching mechanism for transcription, which makes cap-dependent endonuclease (CEN) an attractive target for drug development. Pathogenic arenaviruses pose a serious threat to humans, yet no approved treatments exist, underscoring the importance of discovering novel compounds targeting arenaviral CENs. Therefore, this study aimed to identify novel CEN inhibitors for arenaviruses and investigate their antiviral mechanisms. A high-throughput screening system based on enzymatic activity of CEN was established for discovering inhibitors of lymphocytic choriomeningitis virus (LCMV). Several hit compounds were screened from a vast natural product library, and then evaluated for both toxicity and inhibition through cellular and animal experiments. One candidate compound was finally identified, and its mechanism of action on CEN was elucidated through simulation analysis and biochemical studies. Moreover, its broad-spectrum effects were investigated among pathogenic arenaviruses as well as representative NSVs. Consequently, salvianolic acid A (SAA) from Salvia miltiorrhiza was identified as a promising compound that effectively inhibited LCMV infection and significantly reduced the viral load via intravenous administration. It was shown to bind to the active pocket of arenaviral CENs while chelating their metal ions through its acid carboxyl group, acting in a substrate-competitive manner. Additionally, SAA exhibited broad-spectrum inhibition of pathogenic arenaviruses as well as representative viruses from the order Bunyavirales. This study identified SAA as a novel CEN inhibitor, particularly for pathogenic arenaviruses, showcasing its promise for antiviral drug development.

Ferroptosis is an iron-dependent form of programmed cell death, which is characterized by iron overload and accumulation of lipid peroxidation. As a newly identified type of cell death, its involvement in poxvirus infection and pathogenesis remains unclear. Since MPXV shares biological and pathogenic similarities with other poxviruses, such as vaccinia virus (VACV), we used VACV-infected cell and mouse models to demonstrate that VACV infection induces ferroptosis both in vitro and in vivo. Inhibition of ferroptosis significantly reduce virus replication and alleviates the inflammatory response. Additionally, we observed that VACV infection upregulates prostaglandin-endoperoxide synthase 2 (PTGS2), which contributes to virus-triggered ferroptosis and inflammation. This study identifies a novel form of cell death triggered by poxvirus infection, shedding light on host-pathogen interactions and offering a potential therapeutic target for MPXV and other Orthopoxviruses.

Outer membrane vesicles (OMVs) are 20-400 nm in size, membrane-bound, and secreted by gram-negative bacteria. OMVs play important roles in processes such as toxin delivery and immune evasion. Although many studies have revealed the critical roles played by OMVs, their heterogeneity has limited our ability to attain a comprehensive understanding of their protein compositions and functions. Therefore, studying the compositions of heterogeneous OMVs subpopulations and their biological functions is important. Herein, we used ultracentrifugation combined with density-gradient centrifugation and quantitative proteomics to systematically separate, characterize, and comprehensively analyze OMVs secreted by Escherichia coli DH5α and Pseudomonas aeruginosa PAO1. First, crude OMVs extracts from both strains were obtained by ultracentrifugation and subjected to iodixanol density-gradient centrifugation to afford six fractions each. DH5α-OMVs and PAO1-OMVs particle-size distributions were then determined via nanoparticle tracking analysis, with average particle sizes of 131.0-161.0 and 140.0-169.0 nm determined for the two subpopulation, respectively. Vesicles were observed to have classical chattel structures by transmission electron microscopy. OMVs subpopulation distributions in the density-gradated fractions were determined by silver staining and protein immunoblotting, which also identified F1a-F4a and F1b-F5b as the effective DH5α-OMVs and PAO1-OMVs subpopulation fractions, respectively. We then identified 2388 and 905 proteins from the DH5α-OMVs and PAO1-OMVs subpopulation, respectively, and used k-means clustering and gene ontology (GO) enrichment analyses to reveal the heterogeneities of the various density subpopulations in terms of biological functions, such as energy metabolism, material transport and ribosome synthesis. Comparative analysis of the E. coli DH5α-OMVs and P. aeruginosa PAO1-OMVs subpopulations finally revealed that they exhibit different functional characteristics, despite sharing commonalities in their basic OMVs functions. The F1a DH5α-OMVs subpopulation was found to be enriched for functions related to amino-acid metabolism and protein synthesis, while the F2b PAO1-OMVs subpopulation exhibited significant biomolecule synthesis functions. This study revealed that bacterial OMVs subpopulations have distinct biological functions, which in turn provides a new theoretical basis for understanding the pathogenic mechanisms of bacteria and their interactions with the host, thereby expanding their biological applications.

Filoviruses, including Ebola and Marburg viruses, present significant global health challenges due to their high mortality rates. Despite the availability of several Ebola virus vaccines, none provide cross-protection against multiple filovirus species. In this study, we developed recombinant live attenuated measles virus-based vaccine candidates designed to express Ebola or Marburg virus glycoproteins and generate virus-like particles for pan-filovirus immunization. The administration of these candidates by intraperitoneal injection into IFNAR-/- mice elicited robust antibody and cellular immune responses specific to Ebola and Marburg virus glycoproteins, with virus-like particles expressing candidates inducing the strongest immunity. Importantly, all vaccines containing Ebola virus glycoproteins afforded complete protection against mouse-adapted Ebola virus; meanwhile, those with Marburg glycoproteins provided protection against lethal replication competent vesicular stomatitis virus-Marburg virus challenges. These findings support the potential of measles virus-based vectors and virus-like particles as promising platforms for the development of vaccines targeting multiple filoviruses.

Lung cancer is the most prevalent malignancy worldwide, with the majority of fatalities attributed to metastasis. Recent studies have demonstrated the pivotal role of extracellular vesicles (EVs) and glycoproteins in tumor progression. In this study, we compared the glycoproteome of EVs from 95C (low metastatic) and 95D (high metastatic) lung cancer cells to discover key targets in metastasis. Through coupling lectin affinity chromatography with quantitative proteomics, 1562 glycoproteins were identified. Compared to 95C EVs, 23 glycoproteins were significantly upregulated more than 20-fold in 95D EVs, including CDCP1, TNC, NCAM2, and ITGA4. CUB-domain containing protein 1 (CDCP1) was upregulated 143-fold in 95D EVs, which is significantly correlated with poor prognosis of lung cancer patients in the TCGA database. We subsequently performed site-specific glycoform profiling of CDCP1 using intact glycopeptide enrichment. Then we generated CDCP1 knockout (KO) 95D cell lines and revealed that the absence of CDCP1 reduced cell migration ability, which was also confirmed by EVs and cell co-culture experiments. We further performed Ti4+-IMAC-based phosphoproteomic analysis to investigate the changes in signaling pathways in CDCP1 KO cell lines. 147 differentially expressed phosphoproteins were revealed. Verification experiments confirmed that the levels of phosphorylated SRC and JUN proteins, markers of ErbB signaling pathway, were decreased 5.5-fold and 4.2-fold, respectively. Glycosylation site mutagenesis identified N339 and N386 as critical functional determinants of CDCP1. Collectively, our data demonstrate that glycoprotein CDCP1 was selectively packed into EVs and potentially contributed to cancer metastasis, which is a critical target for anti-metastasis research and cancer therapy.

Lassa virus (LASV) belongs to the Arenaviridae family and causes severe hemorrhagic fever in humans. Although many vaccine candidates for Lassa fever exist, no vaccines have been approved for clinical use currently. The precursor glycoprotein complex (GPC), which is expressed as a trimer on the viral surface, is the main target for vaccine development. However, it has been a significant challenge to elicit effective neutralizing antibodies against LASV. In this study, we designed and produced a prefusion GPC trimer antigen of LASV, named GPCv2. Based on the structural information of GPC, we made modifications by replacing the amino acid at position 328 with proline and appending the trimerization domain. This resulted in a highly expressed prefusion trimeric form of GPCv2 that retained important conformational epitopes and stimulated higher levels of neutralizing antibodies. Moreover, vaccination with GPCv2 protected mice from LASV pseudovirus challenge. Additionally, immune repertoire sequencing showed that the induced immune clones in the trimeric group were more convergent and has its own unique V-J pairing bias compared with monomeric group. These findings demonstrate the potential of GPCv2 as a promising candidate antigen for an effective vaccine against LASV.

[This corrects the article DOI: 10.1371/journal.ppat.1010686.].