Biology subjects

Lebbink, R. J.

Publications and source records attributed to Lebbink, R. J..

10 recordsLinked to original sources

bioRxiv2026

RSM01, an extended half-life RSV monoclonal antibody with a high barrier to resistance

Respiratory syncytial virus (RSV) causes substantial infant morbidity and mortality, particularly in low- and middle-income countries (LMICs). RSM01 is a long-acting RSV monoclonal antibody (mAb) in development for LMICs. To date, RSM01 resistant mutants have not been able to be generated in vitro. We systematically assessed the in vitro resistance barrier of RSM01 relative to licensed RSV mAbs and the susceptibility of a panel of global contemporary strains to RSM01. The emergence of mAb-resistant mutant (MARM) was assessed for RSM01, nirsevimab, and palivizumab. Triple plaque-purified RSV-A2 and RSV-B1 were serially passaged under mAb pressure to generate MARMs, which were phenotyped and genotyped. Moderate resistance was defined as > 3-fold and high resistance > 30-fold increase in IC50 compared with parental strains. The susceptibility of contemporary clinical strains from South Africa, Argentina, and the Netherlands to RSM01 was tested in a neutralisation assay. RSV-A and B lab strains developed high resistance to palivizumab and nirsevimab, while RSM01 pressure induced moderate resistance. No MARMs demonstrated a fitness advantage; one RSM01 MARM incurred fitness costs. RSM01 potently neutralized global contemporary RSV-A and B strains. In conclusion, in vitro RSM01 resistance was infrequent and moderate, suggesting a high resistance barrier in vitro. Laboratory-selected MARMs may not directly predict clinical escape, but the confirmation of the high barrier to resistance of RSM01 is encouraging and provides an alternative in the event that resistance is observed with widespread use of current licensed anti-RSV mAbs. One Sentence SummaryIn vitro RSV resistance to RSM01 is infrequent and moderate, suggesting a high barrier to resistance.

microbiology↗

bioRxiv2025

Rab27-dependent egress of SARS-CoV-2 via secretory amphisomes

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, hijacks host cellular machinery to replicate and spread. Understanding how SARS-CoV-2 reorganizes host cell architecture to accommodate this is essential for elucidating its pathogenesis and identifying therapeutic targets. While the molecular mechanisms of SARS-CoV-2 entry are well characterized, the pathways governing viral egress remain incompletely understood. Conventional approaches such as transcriptomics and electron microscopy have provided valuable insights but lack the combined spatial and molecular resolution needed to map these processes within intact cells. Here, we apply Ten-fold Robust Expansion Microscopy (TREx) to visualize SARS-CoV-2-induced remodeling of the endolysosomal system in multiciliated cells of primary human airway epithelial tissue. This approach reveals Golgi fragmentation and the formation of enlarged virus-containing organelles. Analysis of endolysosomal markers in Vero E6 cells shows that these structures are positive for CD63, Rab7, and LC3, consistent with amphisome identity. Moreover, pharmacological inhibition of Rab27-dependent amphisome-plasma membrane fusion with Nexinhib20 reduces viral infection, implicating secretory autophagy as a pathway for SARS-CoV-2 egress. These findings establish expansion microscopy as a powerful tool for spatial virology and uncover a Rab27-mediated amphisome fusion mechanism as a druggable route for SARS-CoV-2 release.

cell biology↗

bioRxiv2024

Small molecule-directed differentiation of submerged-cultured human nasal airway epithelia for respiratory disease modelling

Submerged cultures of undifferentiated or transformed epithelial cells are widely used in respiratory research due to their ease of use and scalability. However, these systems fail to capture the cellular diversity of the human airway epithelium. In this study, we developed an in vitro model where cryopreserved human nasal epithelial cells, collected by brushings, are differentiated under submerged conditions on standard plastic cultureware. By applying small-molecule inhibitors targeting Notch and BMP signaling, we achieved efficient differentiation of cultures containing basal, secretory, and ciliated cells. This approach supports scalable culturing of both 2D epithelial monolayers and 3D organoids, validated as (personalized) disease models for primary ciliary dyskinesia, cystic fibrosis, and respiratory syncytial virus infection. This model offers a cost-effective, scalable platform that combines the simplicity of traditional cultures with the cellular complexity of the human airway epithelium, providing a valuable tool for respiratory disease research.

cell biology↗

bioRxiv2023

Proinsulin degradation and presentation of a proinsulin B-chain autoantigen involves ER-associated protein degradation (ERAD)-enzyme UBE2G2

Type 1 diabetes (T1D) is characterized by HLA class I-mediated presentation of autoantigens on the surface of pancreatic {beta}-cells. Recognition of these autoantigens by CD8+ T cells results in the destruction of pancreatic {beta}-cells and, consequently, insulin deficiency. Most epitopes presented at the surface of {beta}-cells derive from the insulin precursor molecule proinsulin. The intracellular processing pathway(s) involved in the generation of these peptides are poorly defined. In this study, we show that a proinsulin B-chain antigen (PPIB5-14) originates from proinsulin molecules that are processed by ER-associated protein degradation (ERAD) and thus originate from ER-resident proteins. Furthermore, screening genes encoding for E2 ubiquitin conjugating enzymes, we identified UBE2G2 to be involved in proinsulin degradation. These results indicate that insulin-derived peptides, presented by HLA-class I molecules at the cell surface, originate from ER-resident proinsulin that has been dislocated to the cytosol for subsequent degradation. These insights into the pathway involved in the generation of insulin-derived peptides emphasize the importance of proinsulin processing in the ER to T1D pathogenesis and identify novel targets for future therapies that may cure or even prevent T1D. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=172 HEIGHT=200 SRC="FIGDIR/small/545333v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@131d5org.highwire.dtl.DTLVardef@6eb4b3org.highwire.dtl.DTLVardef@196674org.highwire.dtl.DTLVardef@1957af6_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

The SARS-CoV-2 accessory factor ORF7a downregulates MHC class I surface expression

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 500 million infections and more than six million deaths worldwide. Although the viral genomes of SARS-CoV-1 and SARS-CoV-2 share high sequence homology, the clinical and pathological features of COVID-19 differ profoundly from those of SARS. It is apparent that changes in viral genes contribute to the increased transmissibility of SARS-CoV-2 and pathology of COVID-19. Cytotoxic T lymphocytes play a key role in the elimination of virus-infected cells, mediated by recognition of virus-derived peptides that are presented on MHC class I molecules. Here, we show that SARS-CoV-2 can interfere with antigen presentation thereby evading immune surveillance. SARS-CoV-2 infection of monkey and human cell lines resulted in reduced cell-surface expression of MHC class I molecules. We identified a single viral gene product, the accessory factor open reading frame 7a (ORF7a), that mediates this effect. ORF7a interacts with HLA class I molecules in the ER, resulting in ER retention or impaired HLA heavy chain (HC) trafficking to the Golgi. Ultimately, these actions result in reduced HLA class I surface expression on infected cells. Whereas ORF7a from SARS-CoV-2 reduces surface HLA class I levels, the homologous ORF7a from the 2002 pandemic SARS-CoV-1 did not, suggesting that SARS-CoV-2 ORF7a acquired the ability to downregulate HLA-I during evolution of the virus. We identified a single amino acid in the SARS-CoV-1 ORF7a luminal domain that, upon mutating to the corresponding SARS-CoV-2 ORF7a sequence, induced a gain-of-function in HLA surface downregulation. By abrogating HLA class I antigen presentation via ORF7a, SARS-CoV-2 may evade host immune responses by inhibiting anti-viral cytotoxic T cell activity, thereby contributing to the pathology of COVID-19.

microbiology↗

bioRxiv2022

Structure and mechanism of a novel cytomegaloviral DCAF mediating interferon antagonism

Human cytomegalovirus (CMV) is a highly relevant and ubiquitously distributed human pathogen. Its rodent counterparts such as mouse and rat CMV serve as common infection models. Here, we conducted the first global proteome profiling of rat CMV-infected cells and uncovered a pronounced loss of the transcription factor STAT2, which is crucial for interferon signalling. Deletion mutagenesis documented that STAT2 is targeted by the viral protein E27. Cellular and in vitro analyses showed that E27 exploits host-derived Cullin4-RING ubiquitin ligases (CRL4) to induce poly-ubiquitylation and proteasomal degradation of STAT2. A cryo-electron microscopic structure determination revealed how E27 mimics molecular surface properties of cellular CRL4 substrate receptors called DDB1- and Cullin4-associated factors (DCAFs) to displace them from the catalytic core of CRL4. Moreover, structural analyses elucidated the mechanism of STAT2 recruitment and indicate that E27-binding additionally disturbs STAT2-dependent interferon signalling by occupying its IRF9 binding interface. For the first time, these data provide structural insights into cytomegalovirus-encoded interferon antagonism and establish an atomic model for STAT2 counteraction by CRL4 misappropriation with important implications for viral immune evasion.

biochemistry↗

bioRxiv2021

Optical nanoscopy reveals SARS-CoV-2-induced remodeling of human airway cells

A better understanding of host cell remodeling by the coronavirus SARS-CoV-2 is urgently needed to understand viral pathogenesis and guide drug development. Expression profiling and electron microscopy have frequently been used to study virus-host interactions, but these techniques do not readily enable spatial, sub-cellular and molecular analysis of specific cellular compartments. Here, we use diffraction-unlimited fluorescence microscopy to analyze how SARS-CoV-2 infection exploits and repurposes the subcellular architecture of primary human airway cells. Using STED nanoscopy, we detect viral entry factors along the motile cilia of ciliated cells and visualize key aspects of the viral life cycle. Using Tenfold Robust Expansion (TREx) microscopy, we analyze the extensively remodeled three-dimensional ultrastructure of SARS-CoV-2-infected ciliated cells and uncover Golgi fragmentation, emergence of large and atypical multivesicular bodies enclosing viral proteins, ciliary clustering, and remodeling of the apical surface. These results demonstrate a broadly applicable strategy to study how viruses reorganize host cells with spatial and molecular specificity and provide new insights into SARS-CoV-2 infection in primary human cell models.

cell biology↗

bioRxiv2020

Inhibitory CD200-receptor signaling is rewired by type I interferon

CD200 Receptor 1 (CD200R) is an established inhibitory immune receptor that inhibits TLR-induced cytokine production through Dok2 and RasGAP. RasGAP can be cleaved under certain conditions of mild cellular stress. We found that in the presence of cleaved RasGAP, CD200R loses its capacity to inhibit rpS6 phosphorylation. Furthermore, IFN pre-stimulation of human mononuclear cells results in increased amounts of cleaved RasGAP. Coherently, upon pretreatment with increasing concentrations of IFN, CD200R gradually shifts from an inhibitor to a potentiator of TLR7/8-induced IFNG mRNA production. In peripheral blood mononuclear cells from Systemic Lupus Erythematosus (SLE) patients, a prototypic type I IFN disease, we found an increased proportion of cleaved RasGAP compared to healthy controls. In line with this, in subsets of SLE patients the inhibitory function of CD200R is lost or converted to a potentiating signal for IFNG mRNA production. Thus, our data show that type I IFN rewires CD200R signaling and suggest that this cell-extrinsic regulation of signaling could contribute to perpetuation of inflammation in SLE.

immunology↗

bioRxiv2019

Genetic editing of SEC61, SEC62, and SEC63 abrogates human cytomegalovirus US2 expression in a signal peptide-dependent manner

Newly translated proteins enter the ER through the SEC61 complex, via either co- or post-translational translocation. In mammalian cells, few substrates of post-translational SEC62- and SEC63-dependent translocation have been described. Here, we targeted all components of the SEC61/62/63 complex by CRISPR/Cas9, creating knock-outs or mutants of the individual subunits of the complex. We show that functionality of the human cytomegalovirus protein US2, which is an unusual translocation substrate with a low-hydrophobicity signal peptide, is dependent on expression of not only SEC61, -{beta}, and -{gamma}, but also SEC62 and SEC63, suggesting that US2 may be a substrate for post-translational translocation. This phenotype is specific to the US2 signal peptide.

cell biology↗

bioRxiv2018

A genome-wide haploid genetic screen for essential factors in vaccinia virus infection identifies TMED10 as regulator of macropinocytosis

Vaccinia virus is a promising viral vaccine and gene delivery candidate, and has historically been used as a model to study poxvirus-host cell interactions. We employed a genome-wide insertional mutagenesis approach in human haploid cells to identify host factors crucial for vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to Modified Vaccinia Virus Ankara (MVA). Deep-sequencing analysis of virus-resistant cells identified host factors involved in heparan sulfate synthesis, Golgi organization, and vesicular protein trafficking. We validated EXT1, TM9SF2 and TMED10 (TMP21/p23/p24{delta}) as important host factors for vaccinia virus infection. The critical role of EXT1 in heparan sulfate synthesis and vaccinia virus infection was confirmed. TM9SF2 was validated as a player mediating heparan sulfate expression, explaining its contribution to vaccinia virus infection. In addition, TMED10 was found to be crucial for virus-induced plasma membrane blebbing and phosphatidylserine-induced macropinocytosis, suggesting that TMED10 regulates actin cytoskeleton remodelling necessary for virus infection.\n\nImportancePoxviruses are large DNA viruses that can infect a wide range of host species. A number of these viruses are clinically important to humans, including variola virus (smallpox) and vaccinia virus. Since the eradication of smallpox, zoonotic infections with monkeypox virus and cowpox virus are emerging. Additionally, poxviruses can be engineered to specifically target cancer cells, and are used as vaccine vector against tuberculosis, influenza, and coronaviruses.\n\nPoxviruses rely on host factors for most stages of their life cycle, including attachment to the cell and entry. These host factors are crucial for virus infectivity and host cell tropism. We used a genome-wide knock-out library of host cells to identify host factors necessary for vaccinia virus infection. We confirm a dominant role for heparin sulfate in mediating virus attachment. Additionally, we show that TMED10, previously not implicated in virus infections, modulates the host cell membrane to facilitate virus uptake.

microbiology↗