eLifePub Date : 2025-02-12DOI: 10.7554/eLife.101886
Ke Ning, Yuanyuan Xie, Wen Sun, Lingke Feng, Can Fang, Rong Pan, Yan Li, Ling Yu
{"title":"Non-destructive in situ monitoring of structural changes of 3D tumor spheroids during the formation, migration, and fusion process.","authors":"Ke Ning, Yuanyuan Xie, Wen Sun, Lingke Feng, Can Fang, Rong Pan, Yan Li, Ling Yu","doi":"10.7554/eLife.101886","DOIUrl":"10.7554/eLife.101886","url":null,"abstract":"<p><p>For traditional laboratory microscopy observation, the multi-dimensional, real-time, in situ observation of three-dimensional (3D) tumor spheroids has always been the pain point in cell spheroid observation. In this study, we designed a side-view observation petri dish/device that reflects light, enabling in situ observation of the 3D morphology of cell spheroids using conventional inverted laboratory microscopes. We used a 3D-printed handle and frame to support a first-surface mirror, positioning the device within a cell culture petri dish to image cell spheroid samples. The imaging conditions, such as the distance between the mirror and the 3D spheroids, the light source, and the impact of the culture medium, were systematically studied to validate the in situ side-view observation. The results proved that placing the surface mirror adjacent to the spheroids enables non-destructive in situ real-time tracking of tumor spheroid formation, migration, and fusion dynamics. The correlation between spheroid thickness and dark core appearance under light microscopy and the therapeutic effects of chemotherapy doxorubicin and natural killer cells on spheroids' 3D structure was investigated.</p>","PeriodicalId":11640,"journal":{"name":"eLife","volume":"13 ","pages":""},"PeriodicalIF":6.4,"publicationDate":"2025-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11820107/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143398653","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
eLifePub Date : 2025-02-12DOI: 10.7554/eLife.98152
Kira Breunig, Xuifen Lei, Mauro Montalbano, Gabriela D A Guardia, Shiva Ostadrahimi, Victoria Alers, Adam Kosti, Jennifer Chiou, Nicole Klein, Corina Vinarov, Lily Wang, Mujia Li, Weidan Song, W Lee Kraus, David S Libich, Stefano Tiziani, Susan T Weintraub, Pedro A F Galante, Luiz O Penalva
{"title":"SERBP1 interacts with PARP1 and is present in PARylation-dependent protein complexes regulating splicing, cell division, and ribosome biogenesis.","authors":"Kira Breunig, Xuifen Lei, Mauro Montalbano, Gabriela D A Guardia, Shiva Ostadrahimi, Victoria Alers, Adam Kosti, Jennifer Chiou, Nicole Klein, Corina Vinarov, Lily Wang, Mujia Li, Weidan Song, W Lee Kraus, David S Libich, Stefano Tiziani, Susan T Weintraub, Pedro A F Galante, Luiz O Penalva","doi":"10.7554/eLife.98152","DOIUrl":"10.7554/eLife.98152","url":null,"abstract":"<p><p>RNA binding proteins (RBPs) containing intrinsically disordered regions (IDRs) are present in diverse molecular complexes where they function as dynamic regulators. Their characteristics promote liquid-liquid phase separation (LLPS) and the formation of membraneless organelles such as stress granules and nucleoli. IDR-RBPs are particularly relevant in the nervous system and their dysfunction is associated with neurodegenerative diseases and brain tumor development. Serpine1 mRNA-binding protein 1 (SERBP1) is a unique member of this group, being mostly disordered and lacking canonical RNA-binding domains. We defined SERBP1's interactome, uncovered novel roles in splicing, cell division and ribosomal biogenesis, and showed its participation in pathological stress granules and Tau aggregates in Alzheimer's brains. SERBP1 preferentially interacts with other G-quadruplex (G4) binders, implicated in different stages of gene expression, suggesting that G4 binding is a critical component of SERBP1 function in different settings. Similarly, we identified important associations between SERBP1 and PARP1/polyADP-ribosylation (PARylation). SERBP1 interacts with PARP1 and its associated factors and influences PARylation. Moreover, protein complexes in which SERBP1 participates contain mostly PARylated proteins and PAR binders. Based on these results, we propose a feedback regulatory model in which SERBP1 influences PARP1 function and PARylation, while PARylation modulates SERBP1 functions and participation in regulatory complexes.</p>","PeriodicalId":11640,"journal":{"name":"eLife","volume":"13 ","pages":""},"PeriodicalIF":6.4,"publicationDate":"2025-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11820137/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143398685","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
eLifePub Date : 2025-02-12DOI: 10.7554/eLife.105929
Marjan Slak Rupnik
{"title":"Opportunity makes a hub or a leader.","authors":"Marjan Slak Rupnik","doi":"10.7554/eLife.105929","DOIUrl":"10.7554/eLife.105929","url":null,"abstract":"<p><p>Functional subpopulations of β-cells emerge to control pulsative insulin secretion in the pancreatic islets of mice through calcium oscillations.</p>","PeriodicalId":11640,"journal":{"name":"eLife","volume":"14 ","pages":""},"PeriodicalIF":6.4,"publicationDate":"2025-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11820101/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143398689","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles.","authors":"Xu Zheng, Shi Yu, Yanqiu Zhou, Kuai Yu, Yuhui Gao, Mengdan Chen, Dong Duan, Yunyi Li, Xiaoxian Cui, Jiabin Mou, Yuying Yang, Xun Wang, Min Chen, Yaming Jiu, Jincun Zhao, Guangxun Meng","doi":"10.7554/eLife.98593","DOIUrl":"10.7554/eLife.98593","url":null,"abstract":"<p><p>Innate immune responses triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection play pivotal roles in the pathogenesis of COVID-19, while host factors including proinflammatory cytokines are critical for viral containment. By utilizing quantitative and qualitative models, we discovered that soluble factors secreted by human monocytes potently inhibit SARS-CoV-2-induced cell-cell fusion in viral-infected cells. Through cytokine screening, we identified that interleukin-1β (IL-1β), a key mediator of inflammation, inhibits syncytia formation mediated by various SARS-CoV-2 strains. Mechanistically, IL-1β activates RhoA/ROCK signaling through a non-canonical IL-1 receptor-dependent pathway, which drives the enrichment of actin bundles at the cell-cell junctions, thus prevents syncytia formation. Notably, in vivo infection experiments in mice confirmed that IL-1β significantly restricted SARS-CoV-2 spread in the lung epithelium. Together, by revealing the function and underlying mechanism of IL-1β on SARS-CoV-2-induced cell-cell fusion, our study highlights an unprecedented antiviral function for cytokines during viral infection.</p>","PeriodicalId":11640,"journal":{"name":"eLife","volume":"13 ","pages":""},"PeriodicalIF":6.4,"publicationDate":"2025-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11820142/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143405629","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
eLifePub Date : 2025-02-12DOI: 10.7554/eLife.97029
Andrea Brenna, Micaela Borsa, Gabriella Saro, Jürgen A Ripperger, Dominique A Glauser, Zhihong Yang, Antoine Adamantidis, Urs Albrecht
{"title":"Cyclin-dependent kinase 5 (Cdk5) activity is modulated by light and gates rapid phase shifts of the circadian clock.","authors":"Andrea Brenna, Micaela Borsa, Gabriella Saro, Jürgen A Ripperger, Dominique A Glauser, Zhihong Yang, Antoine Adamantidis, Urs Albrecht","doi":"10.7554/eLife.97029","DOIUrl":"10.7554/eLife.97029","url":null,"abstract":"<p><p>The circadian clock enables organisms to synchronize biochemical and physiological processes over a 24 hr period. Natural changes in lighting conditions, as well as artificial disruptions like jet lag or shift work, can advance or delay the clock phase to align physiology with the environment. Within the suprachiasmatic nucleus (SCN) of the hypothalamus, circadian timekeeping and resetting rely on both membrane depolarization and intracellular second-messenger signaling. Voltage-gated calcium channels (VGCCs) facilitate calcium influx in both processes, activating intracellular signaling pathways that trigger <i>Period</i> (<i>Per</i>) gene expression. However, the precise mechanism by which these processes are concertedly gated remains unknown. Our study in mice demonstrates that cyclin-dependent kinase 5 (Cdk5) activity is modulated by light and regulates phase shifts of the circadian clock. We observed that knocking down Cdk5 in the SCN of mice affects phase delays but not phase advances. This is linked to uncontrolled calcium influx into SCN neurons and an unregulated protein kinase A (PKA)-calcium/calmodulin-dependent kinase (CaMK)-cAMP response element-binding protein (CREB) signaling pathway. Consequently, genes such as <i>Per1</i> are not induced by light in the SCN of Cdk5 knock-down mice. Our experiments identified Cdk5 as a crucial light-modulated kinase that influences rapid clock phase adaptation. This finding elucidates how light responsiveness and clock phase coordination adapt activity onset to seasonal changes, jet lag, and shift work.</p>","PeriodicalId":11640,"journal":{"name":"eLife","volume":"13 ","pages":""},"PeriodicalIF":6.4,"publicationDate":"2025-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11820109/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143398636","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
eLifePub Date : 2025-02-12DOI: 10.7554/eLife.98523
Giuliana Giamundo, Daniela Intartaglia, Eugenio Del Prete, Elena Polishchuk, Fabrizio Andreone, Marzia Ognibene, Sara Buonocore, Bruno Hay Mele, Francesco Giuseppe Salierno, Jlenia Monfregola, Dario Antonini, Paolo Grumati, Alessandra Eva, Rossella De Cegli, Ivan Conte
{"title":"Ezrin defines TSC complex activation at endosomal compartments through EGFR-AKT signaling.","authors":"Giuliana Giamundo, Daniela Intartaglia, Eugenio Del Prete, Elena Polishchuk, Fabrizio Andreone, Marzia Ognibene, Sara Buonocore, Bruno Hay Mele, Francesco Giuseppe Salierno, Jlenia Monfregola, Dario Antonini, Paolo Grumati, Alessandra Eva, Rossella De Cegli, Ivan Conte","doi":"10.7554/eLife.98523","DOIUrl":"10.7554/eLife.98523","url":null,"abstract":"<p><p>Endosomes have emerged as major signaling hubs where different internalized ligand-receptor complexes are integrated and the outcome of signaling pathways are organized to regulate the strength and specificity of signal transduction events. Ezrin, a major membrane-actin linker that assembles and coordinates macromolecular signaling complexes at membranes, has emerged recently as an important regulator of lysosomal function. Here, we report that endosomal-localized EGFR/Ezrin complex interacts with and triggers the inhibition of the Tuberous Sclerosis Complex (TSC complex) in response to EGF stimuli. This is regulated through activation of the AKT signaling pathway. Loss of Ezrin was not sufficient to repress TSC complex by EGF and culminated in translocation of TSC complex to lysosomes triggering suppression of mTORC1 signaling. Overexpression of constitutively active EZRIN<sup>T567D</sup> is sufficient to relocalize TSC complex to the endosomes and reactivate mTORC1. Our findings identify EZRIN as a critical regulator of autophagy via TSC complex in response to EGF stimuli and establish the central role of early endosomal signaling in the regulation of mTORC1. Consistently, Medaka fish deficient for Ezrin exhibit defective endo-lysosomal pathway, attributable to the compromised EGFR/AKT signaling, ultimately leading to retinal degeneration. Our data identify a pivotal mechanism of endo-lysosomal signaling involving Ezrin and its associated EGFR/TSC complex, which are essential for retinal function.</p>","PeriodicalId":11640,"journal":{"name":"eLife","volume":"13 ","pages":""},"PeriodicalIF":6.4,"publicationDate":"2025-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11820125/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143398582","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
eLifePub Date : 2025-02-12DOI: 10.7554/eLife.103068
Erli Jin, Jennifer K Briggs, Richard K P Benninger, Matthew J Merrins
{"title":"Glucokinase activity controls peripherally located subpopulations of β-cells that lead islet Ca<sup>2+</sup> oscillations.","authors":"Erli Jin, Jennifer K Briggs, Richard K P Benninger, Matthew J Merrins","doi":"10.7554/eLife.103068","DOIUrl":"10.7554/eLife.103068","url":null,"abstract":"<p><p>Oscillations in insulin secretion, driven by islet Ca<sup>2+</sup> waves, are crucial for glycemic control. Prior studies, performed with single-plane imaging, suggest that subpopulations of electrically coupled β-cells have privileged roles in leading and coordinating the propagation of Ca<sup>2+</sup> waves. Here, we used three-dimensional (3D) light-sheet imaging to analyze the location and Ca<sup>2+</sup> activity of single β-cells within the entire islet at >2 Hz. In contrast with single-plane studies, 3D network analysis indicates that the most highly synchronized β-cells are located at the islet center, and remain regionally but not cellularly stable between oscillations. This subpopulation, which includes 'hub cells', is insensitive to changes in fuel metabolism induced by glucokinase and pyruvate kinase activation. β-Cells that initiate the Ca<sup>2+</sup> wave (leaders) are located at the islet periphery, and strikingly, change their identity over time via rotations in the wave axis. Glucokinase activation, which increased oscillation period, reinforced leader cells and stabilized the wave axis. Pyruvate kinase activation, despite increasing oscillation frequency, had no effect on leader cells, indicating the wave origin is patterned by fuel input. These findings emphasize the stochastic nature of the β-cell subpopulations that control Ca<sup>2+</sup> oscillations and identify a role for glucokinase in spatially patterning 'leader' β-cells.</p>","PeriodicalId":11640,"journal":{"name":"eLife","volume":"13 ","pages":""},"PeriodicalIF":6.4,"publicationDate":"2025-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11820133/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143398588","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
eLifePub Date : 2025-02-12DOI: 10.7554/eLife.98110
Junhong Choi, Wei Chen, Hanna Liao, Xiaoyi Li, Jay Shendure
{"title":"A molecular proximity sensor based on an engineered, dual-component guide RNA.","authors":"Junhong Choi, Wei Chen, Hanna Liao, Xiaoyi Li, Jay Shendure","doi":"10.7554/eLife.98110","DOIUrl":"10.7554/eLife.98110","url":null,"abstract":"<p><p>One of the goals of synthetic biology is to enable the design of arbitrary molecular circuits with programmable inputs and outputs. Such circuits bridge the properties of electronic and natural circuits, processing information in a predictable manner within living cells. Genome editing is a potentially powerful component of synthetic molecular circuits, whether for modulating the expression of a target gene or for stably recording information to genomic DNA. However, programming molecular events such as protein-protein interactions or induced proximity as triggers for genome editing remains challenging. Here, we demonstrate a strategy termed 'P3 editing', which links <u>p</u>rotein-<u>p</u>rotein <u>p</u>roximity to the formation of a functional CRISPR-Cas9 dual-component guide RNA. By engineering the crRNA:tracrRNA interaction, we demonstrate that various known protein-protein interactions, as well as the chemically induced dimerization of protein domains, can be used to activate prime editing or base editing in human cells. Additionally, we explore how P3 editing can incorporate outputs from ADAR-based RNA sensors, potentially allowing specific RNAs to induce specific genome edits within a larger circuit. Our strategy enhances the controllability of CRISPR-based genome editing, facilitating its use in synthetic molecular circuits deployed in living cells.</p>","PeriodicalId":11640,"journal":{"name":"eLife","volume":"13 ","pages":""},"PeriodicalIF":6.4,"publicationDate":"2025-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11820115/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143398668","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
eLifePub Date : 2025-02-11DOI: 10.7554/eLife.98258
Ismail M Meraz, Mourad Majidi, Renduo Song, Feng Meng, Lihui Gao, Qi Wang, Jing Wang, Elizabeth J Shpall, Jack A Roth
{"title":"<i>NPRL2</i> gene therapy induces effective antitumor immunity in <i>KRAS/STK11</i> mutant anti-PD1 resistant metastatic non-small cell lung cancer (NSCLC) in a humanized mouse model.","authors":"Ismail M Meraz, Mourad Majidi, Renduo Song, Feng Meng, Lihui Gao, Qi Wang, Jing Wang, Elizabeth J Shpall, Jack A Roth","doi":"10.7554/eLife.98258","DOIUrl":"10.7554/eLife.98258","url":null,"abstract":"<p><p>Expression of <i>NPRL2/TUSC4</i>, a tumor-suppressor gene, is reduced in many cancers including NSCLC. Restoration of <i>NPRL2</i> induces DNA damage, apoptosis, and cell-cycle arrest. We investigated <i>NPRL2</i> antitumor immune responses in aPD1<sup>R</sup>/<i>KRAS/STK11<sup>mt</sup></i> NSCLC in humanized-mice. Humanized-mice were generated by transplanting fresh human cord blood-derived CD34 stem cells into sub-lethally irradiated NSG mice. Lung-metastases were developed from <i>KRAS/STK11<sup>mt</sup></i>/aPD1<sup>R</sup> A549 cells and treated with <i>NPRL2</i> w/wo pembrolizumab. <i>NPRL2</i>-treatment reduced lung metastases significantly, whereas pembrolizumab was ineffective. Antitumor effect was greater in humanized than non-humanized-mice. <i>NPRL2</i> + pembrolizumab was not synergistic in <i>KRAS/STK11<sup>mt</sup></i>/aPD1<sup>R</sup> tumors but was synergistic in <i>KRAS<sup>wt</sup></i>/aPD1<sup>S</sup> H1299. <i>NPRL2</i> also showed a significant antitumor effect on <i>KRAS<sup>mt</sup></i>/aPD1<sup>R</sup> LLC2 syngeneic-tumors. The antitumor effect was correlated with increased infiltration of human cytotoxic-T, HLA-DR<sup>+</sup>DC, CD11c<sup>+</sup>DC, and downregulation of myeloid and regulatory-T cells in TME. Antitumor effect was abolished upon in-vivo depletion of CD8-T, macrophages, and CD4-T cells whereas remained unaffected upon NK-cell depletion. A distinctive protein-expression profile was found after <i>NPRL2</i> treatment. <i>IFNγ, CD8b</i>, and <i>TBX21</i> associated with T-cell functions were significantly increased, whereas <i>FOXP3, TGFB1/B2</i>, and <i>IL-10RA</i> were strongly inhibited by <i>NPRL2</i>. A list of T-cell co-inhibitory molecules was also downregulated. Restoration of <i>NPRL2</i> exhibited significantly slower tumor growth in humanized-mice, which was associated with increased presence of human cytotoxic-T, and DC and decreased percentage of Treg, MDSC, and TAM in TME. <i>NPRL2</i>-stable cells showed a substantial increase in colony-formation inhibition and heightened sensitivity to carboplatin. Stable-expression of <i>NPRL2</i> resulted in the downregulation of MAPK and AKT-mTOR signaling. Taken-together, <i>NPRL2</i> gene-therapy induces antitumor activity on <i>KRAS/STK11<sup>mt</sup></i>/aPD1<sup>R</sup> tumors through DC-mediated antigen-presentation and cytotoxic immune-cell activation.</p>","PeriodicalId":11640,"journal":{"name":"eLife","volume":"13 ","pages":""},"PeriodicalIF":6.4,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11813225/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143390487","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Immunogenicity and safety of a live-attenuated SARS-CoV-2 vaccine candidate based on multiple attenuation mechanisms.","authors":"Mie Suzuki Okutani, Shinya Okamura, Tang Gis, Hitomi Sasaki, Suni Lee, Akiho Kashiwabara, Simon Goto, Mai Matsumoto, Mayuko Yamawaki, Toshiaki Miyazaki, Tatsuya Nakagawa, Masahito Ikawa, Wataru Kamitani, Shiro Takekawa, Koichi Yamanishi, Hirotaka Ebina","doi":"10.7554/eLife.97532","DOIUrl":"10.7554/eLife.97532","url":null,"abstract":"<p><p>mRNA vaccines against SARS-CoV-2 were rapidly developed and were effective during the pandemic. However, some limitations remain to be resolved, such as the short-lived induced immune response and certain adverse effects. Therefore, there is an urgent need to develop new vaccines that address these issues. While live-attenuated vaccines are a highly effective modality, they pose a risk of adverse effects, including virulence reversion. In the current study, we constructed a live-attenuated vaccine candidate, BK2102, combining naturally occurring virulence-attenuating mutations in the <i>NSP14</i>, <i>NSP1</i>, spike, and <i>ORF7-8</i> coding regions. Intranasal inoculation with BK2102 induced humoral and cellular immune responses in Syrian hamsters without apparent tissue damage in the lungs, leading to protection against a SARS-CoV-2 D614G and an Omicron BA.5 strains. The neutralizing antibodies induced by BK2102 persisted for up to 364 days, which indicated that they confer long-term protection against infection. Furthermore, we confirmed the safety of BK2102 using transgenic (Tg) mice expressing human ACE2 (hACE2) that are highly susceptible to SARS-CoV-2. BK2102 did not kill the Tg mice, even when virus was administered at a dose of 10<sup>6</sup> plaque-forming units (PFUs), while 10<sup>2</sup> PFU of the D614G strain or an attenuated strain lacking the furin cleavage site of the spike was sufficient to kill mice. These results suggest that BK2102 is a promising live-vaccine candidate strain that confers long-term protection without significant virulence.</p>","PeriodicalId":11640,"journal":{"name":"eLife","volume":"13 ","pages":""},"PeriodicalIF":6.4,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11813227/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143389884","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}