Biology subjects

Lakerveld, A. J.

Publications and source records attributed to Lakerveld, A. J..

3 recordsLinked to original sources

bioRxiv2025

Respiratory syncytial virus nonstructural protein 1 inhibits production of cytokines and chemokines by differentiated primary nasal epithelial cells cultured at air-liquid interface

Respiratory syncytial virus (RSV) is a major cause of severe respiratory infections in children and older adults. Several vaccines have recently been approved, which are all based on the RSV fusion (F) protein. Among the strategies to induce a broader immune response is the development of live-attenuated RSV vaccines, for example by disabling nonstructural protein 1 (NS1). RSV NS1 has been found to modulate the host immune response by repression of interferon (IFN) production. However, functional characterization of this protein was mainly performed in immortalized cell lines, which do not necessarily represent the in vivo situation. Here, we assessed the effect of NS1 mutations on replication and host responses in differentiated primary human nasal epithelial cells (HNEC) cultured at air-liquid interface (ALI), which mimic several physiological properties of the human airways. Using a newly developed yeast-based RSV reverse genetics system, inactivating mutations in the NS1 gene were introduced. These mutations did not affect viral replication in Vero and A549 cells. However, in HNEC we observed a delayed replication of the NS1 mutants compared to the WT virus. Bulk RNA sequencing data from early post infection revealed a stronger antiviral signature in HNEC that were infected with NS1 mutants compared to WT, characterized by upregulation of IFN-{beta} and IFN-{lambda}1/2/3 and chemokines CXCL11 and CCL5. This signature was confirmed by analysis of the cytokines that were released into the HNEC basal medium. Finally, the basal medium containing these cytokines was used for an indirect immune cell migration assay. In this set-up, both WT and NS1 mutant viruses induce migration of mainly neutrophils. In conclusion, this study shows that RSV NS1 supports viral replication not only via inhibition of the production of IFNs, but also by reducing early chemokine production and secretion by epithelial cells. Furthermore, our data highlight the suitability of the ALI transwell model for preclinical assessment of live-attenuated vaccine candidates.

microbiology↗

bioRxiv2025

Pre-assembly of biomolecular condensate seeds drives RSV replication

During infection many RNA viruses, including respiratory syncytial virus (RSV), form specialized biomolecular condensates, inclusion bodies (IBs), where viral transcription and replication occur1-4. Paradoxically, high protein concentrations are typically required for condensate nucleation5, yet attaining sufficient protein levels in infection is thought to require IBs for viral transcription and replication. To uncover how viruses solve this paradox to establish IBs, we visualized early infection of RSV in real-time with single genomic viral ribonucleoprotein (vRNP) resolution. Our results reveal that IBs are nucleated from infecting vRNPs rather than de novo in the cytoplasm. IB nucleation further requires in-virion pre-assembly of viral protein-protein interaction networks on vRNPs to form pre-replication centers (PRCs). PRCs are potent condensate nucleation seeds due to their resistance to disassembly and efficient recruitment of newly-synthesized viral proteins. The high protein affinity of PRCs also results in increased polymerase complex association, allowing efficient viral transcription even in the absence of IBs. Together, these activities create a feed-forward loop that drives rapid IB formation. Intriguingly, PRC assembly depends on in-virion viral protein levels and is highly heterogeneous among virions, explaining cell-to-cell heterogeneity in infection progression, and identifying heterogeneous virions as the origin of infection heterogeneity. Together, our results show that in-virion pre-assembly of PRCs kick-starts viral condensate nucleation upon host-cell entry, and explains cell-to-cell heterogeneity in RSV infection.

microbiology↗

bioRxiv2019

NK cell activation by RSV-specific antibodies is decreased in infants with severe respiratory infections and correlates with Fc-glycosylation

BackgroundRespiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants and there is no vaccine available. In early life, the most important contributors to protection against infectious diseases are the innate immune system and maternal antibodies. However, the mechanisms by which antibodies can protect against RSV disease are incompletely understood, as both antibody levels and neutralization capacity correlate poorly with protection. We therefore asked whether antibody-mediated natural killer (NK) cell activation correlates with RSV disease.\n\nMethodsWe performed an observational case-control study including infants hospitalized for RSV infection (n=43, cases), hernia surgery (n=16, controls), or RSV-negative viral respiratory tract infections (n=18, controls). First, we determined RSV antigen-specific antibody levels in infant plasma using a multiplex immunoassay. Subsequently, we measured the capacity of these antibodies to activate NK cells. Finally, we assessed Fc-glycosylation of the RSV-specific antibodies by mass spectrometry.\n\nResultsWe found that RSV-specific maternal antibodies potently activate NK cells in vitro. While the concentrations of RSV-specific antibodies did not differ between cases and controls, antibodies from infants hospitalized for severe lower respiratory tract infections (RSV and/or other) induced significantly less NK cell interferon gamma production than those from uninfected controls. Furthermore, NK cell activation correlated with Fc-fucosylation of RSV-specific antibodies, but their glycosylation status did not significantly differ between cases and controls.\n\nConclusionsOur results suggest that Fc-dependent antibody function and quality, exemplified by NK cell activation and glycosylation, contribute to protection against severe RSV disease and warrant further studies to evaluate the potential of harnessing these activities to develop an effective vaccine.

immunology↗