Biology subjects

Saelens, X.

Publications and source records attributed to Saelens, X..

17 recordsLinked to original sources

bioRxiv2026

The breadth of protection mediated by anti-N2 neuraminidase antibody responses relies on cross-reactive and not cross-inhibiting antibodies

Neuraminidase (NA) inhibition (NAI) titers have been identified as an independent correlation of protection against influenza. Few studies, however, have investigated the breadth of NA-based immune protection. Previously, we have reported that N2 NAs derived from human H3N2 viruses that circulated between 2009 and 2017 can be subdivided into four antigenic groups. Here, we immunized mice with recombinant soluble tetrameric NA from H3N2 strains representing those four antigenic groups or passively transferred N2 NA immune serum into naive mice to evaluate the breadth of protection against a heterologous HxN2 influenza virus challenge. We show that the breadth of protection goes beyond the breadth of NAI but still requires the presence of cross-reactive antibodies. Interestingly, in the absence of cross-reactive antibodies, the immunization of DBA/2J mice with heterologous NA was associated with an early onset of disease upon challenge with the reassortant HxNind11 or H2N2 A/Singapore/1/1957.

immunology↗

bioRxiv2025

Identification of DksA as a novel pro-inflammatory mediator of Pseudomonas aeruginosa under conditions mimicking chronic cystic fibrosis lung infection

Chronic infection with Pseudomonas aeruginosa is a major driver of airway inflammation, which plays a central role in the progression of cystic fibrosis (CF) lung disease. During long-term colonization, P. aeruginosa adapts to the CF lung by downregulating virulence factors and adopting a biofilm-associated, mucoid lifestyle. Despite the expected reduction in immune activation due to these adaptations, excessive inflammation persists, a paradox that remains poorly understood. Our objective was to identify novel bacterial mediators sustaining persistent inflammation by P. aeruginosa in the CF lung. To this end, we analyzed clinical P. aeruginosa CF isolates, cultured them in synthetic CF sputum medium, and exposed 3D lung epithelial cell cultures to the resulting cell-free supernatants. There was considerable variability in pro-inflammatory activity among the isolates, with a subset of the isolates inducing strong IL-8 secretion by the 3D cells despite low production of known virulence factors. Comparative proteomics analysis of the cell-free supernatants of pro- and anti-inflammatory isolates revealed several mediators not previously linked to inflammation. Thirteen of these candidate pro-inflammatory mediators were selected for further analysis. Using P. aeruginosa transposon mutants lacking the respective mediators, DksA (a transcription factor) was confirmed as an immunomodulatory mediator in the 3D lung model. Finally, analysis of existing transcriptomes of P. aeruginosa in CF sputum, revealed that dksA was found to be one of the most strongly expressed genes in this patient population, highlighting the relevance of our findings. In conclusion, we identified a novel P. aeruginosa mediator that may contribute to CF airway inflammation.

microbiology↗

bioRxiv2025

Multi-segment mouse-adaptation of a recent B/Victoria-lineage virus independent from hemagglutinin and neuraminidase

Influenza B viruses (IBVs) contribute significantly to the annual influenza epidemics in human. Most IBV strains are non- or poorly pathogenic in mice, which are frequently used for vaccine studies. We describe the generation of a mouse-adapted IBV strain that retains pathogenicity in mice when carrying hemagglutinin (HA) and neuraminidase (NA) gene segments from a heterologous IBV strain. Serial passage of an influenza B reassortant virus, containing the HA and NA segments from B/Washington/02/2019 on a mouse-adapted B/Memphis/12/1997 backbone, resulted in the selection of an IBV that was highly pathogenic for mice. This mouse-adapted IBV strain had acquired non-synonymous mutations in 5 gene segments. Sequence analysis of the intermediate passages indicated that mutations in the matrix (M), polymerase acidic (PA), and polymerase basic 1 (PB1) gene segments appeared at passages 9 and 13, suggesting that these mutations contributed to the pathogenicity in mice. Mouse challenge studies with rescued reassortant viruses with one or multiple mutated gene segments, confirmed the importance of substitutions in the M and PA segments for pathogenicity. Using the novel mouse-adapted IBV backbone, we rescued reassortant viruses containing the HA and NA segments of B/Austria/1359417/2021 and demonstrated its increased pathogenicity in BALB/c mice compared to IBV rescued on the parental strain. This mouse-adapted IBV backbone provides a valuable tool for the study of IBV in mice.

immunology↗

bioRxiv2025

Rapid assay optimization by plug-and-play MAPPIT

Studying protein-protein interactions, which are crucial for all cellular processes in both health and disease, offers valuable insights into underlying mechanisms and reveals promising therapeutic targets. While efforts have been made to vastly increase the scale of MAPPIT assays, covering the full protein interactome remains challenging, in particular when considering the availability of different assay configurations. In this study, we expanded our Golden Gate modular cloning platform to enable rapid, customizable construct assembly for MAPPIT assays. We demonstrate the strength of this expanded toolbox for multiple distinct protein-protein interactions, including a MAPPIT assay for six different intracellular VHH targeting the anti-viral effector protein MXA.

synthetic biology↗

bioRxiv2025

Development of a General Purpose Targeted LC-MS Method for Accurate Quantification of the SARS-CoV-2 Spike Protein Expression

The COVID-19 pandemic has catalyzed interest in immuno-multiple reaction monitoring (immuno-MRM) methods, with the detection of peptides unique to the nucleocapsid protein in nasopharyngeal swabs. While current applications predominantly focus on disease biomarkers, the pandemic has unveiled new opportunities, namely for the quantification of antigen expression following mRNA vaccination. Here, we present an optimized immuno-MRM method for quantifying SARS-CoV-2 spike protein fusion peptide, SFIEDLLFNK, for several practical applications. The method is versatile, applicable to multiple biological matrices, including plasma, and can be extended to nasopharyngeal swabs. It also offers a high-precision tool for assessing protein expression following plasmid and mRNA transfection. Moreover, in parallel to enabling accurate antigen quantification, the flow-through can be used to determine the proteome profile of the infected cells, providing insights into the intracellular immune response. This dual capability supports the rapid optimization of mRNA vaccines, thereby driving advancements in vaccine development strategies.

immunology↗

bioRxiv2024

Structure-based Design of Chimeric Influenza Hemagglutinins to Elicit Cross-group Immunity

Antigenic variability among influenza virus strains poses a significant challenge to developing broadly protective, long-lasting vaccines. Current annual vaccines target specific strains, requiring accurate prediction for effective neutralization. Despite sequence diversity across phylogenetic groups, the hemagglutinin (HA) head domains structure remains highly conserved. Utilizing this conservation, we designed cross-group chimeric HAs that combine antigenic surfaces from distant strains. By structure-guided transplantation of receptor-binding site (RBS) residues, we displayed an H3 RBS on an H1 HA scaffold. These chimeric immunogens elicit cross-group polyclonal responses capable of neutralizing both base and distal strains. Additionally, the chimeras integrate heterotrimeric immunogens, enhancing modular vaccine design. This approach enables the inclusion of diverse strain segments to generate broad polyclonal responses. In the future, such modular immunogens may serve as tools for evaluating immunodominance and refining immunization strategies, offering potential to bridge and enhance immune responses in individuals with pre-existing immunity. This strategy holds promise for advancing universal influenza vaccine development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/628867v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@18dec5borg.highwire.dtl.DTLVardef@98587forg.highwire.dtl.DTLVardef@1d9fb2dorg.highwire.dtl.DTLVardef@1f97884_HPS_FORMAT_FIGEXP M_FIG Graphical abstract: Overview of cross-group RBS transplantation approachPhylogenetically diverse HA strains can be incorporated into chimeric immunogens by RBS transplantation. The chimera are evaluated for cross-reactivity to subtype-specific antibodies and the ability to elicit neutralizing antibodies to multiple strains. C_FIG

biochemistry↗

bioRxiv2024

The antigenic landscape of N1 neuraminidase in human influenza A virus strains isolated between 2009 and 2020

The clinical burden caused by influenza can be mitigated by the prophylactic use of seasonal influenza vaccines. Their immunogen composition is revised biannually to optimally match the antigenic drift of the hemagglutinin of circulating influenza virus strains. Antibodies directed against the influenza neuraminidase also correlate with protection against influenza, yet the antigenic evolution of influenza neuraminidase remains underexplored. To evaluate the antigenic diversity of N1 neuraminidase, we generated a panel of immune sera directed against 17 N1 neuraminidases derived from human H1N1 strains that were isolated between 2009 and 2020 and determined its neuraminidase inhibition titers against a panel of 15 HxN1 viruses. The resulting neuraminidase inhibition pattern revealed two antigenic groups that circulated in this period. A machine learning method identified K432E and I321V as key determinants of N1 neuraminidase antigenicity.

immunology↗

bioRxiv2024

Direct and ultrasensitive bioluminescent detection of intact respiratory viruses

Respiratory viruses such as SARS-CoV-2, influenza, and respiratory syncytial virus (RSV) represent pressing health risks. Rapid diagnostic tests for these viruses detect single antigens or nucleic acids, which do not necessarily correlate with the amount of intact virus. Instead, specific detection of intact respiratory virus particles may better assess the contagiousness of a patient. Here, we report GLOVID, a modular biosensor platform to detect intact virions against a background of free viral proteins in solution. Our approach harnesses the multivalent display of distinct proteins on the surface of a viral particle to template the reconstitution of a split luciferase, allowing specific, single-step detection of intact influenza A and RSV virions corresponding to 0.1 - 0.3 fM of genomic units. The protein ligation system used to assemble GLOVID sensors is compatible with a broad range of binding domains, including nanobodies, scFv fragments and cyclic peptides, which allows straightforward adjustment of the sensor platform to target different viruses.

bioengineering↗

bioRxiv2024

Fc-dependent protective efficacy of non-inhibitory antibodies targeting influenza A virus neuraminidase is limited by epitope availability

Antibodies targeting hemagglutinin and neuraminidase (NA) are key components of the adaptive immune response against influenza A virus (IAV). However, antigenic drift allows the virus to escape inhibition by such antibodies. In this study, we aimed to isolate antibodies with cross-subtype reactivity against human H1N1 and H3N2 IAVs from transgenic mice bearing genes encoding the human immunoglobulin variable regions. We immunized these mice with recombinant N1 and N2 NA proteins, presenting them either as unconjugated soluble proteins or conjugated to self-assembling protein nanoparticles. This approach yielded a panel of NA-specific monoclonal antibodies (mAbs) with various levels of intra-and inter-subtype reactivity for N1 and N2 NA. Three of these mAbs, which collectively recognize two distinct epitopes, were cross-reactive against N1 and N2 NAs in ELISA, but did not inhibit NA enzymatic activity. Two of these mAbs, 21H8 and 45D9, were selected for further characterization. These recognized different epitopes and induced Fc-mediated effector functions to varying extents. Prophylactic administration of 21H8, but not 45D9, protected mice against challenge with H1N1 IAV, while neither mAb protected against a H3N2 challenge. The observed protective efficacy correlated with the mAbs capacity, or lack thereof, to bind membrane-associated full-length NA. The introduction of Fc silencing mutations in mAb 21H8 resulted in an inability to activate NK cells or mediate phagocytosis in vitro and significantly reduced protection in vivo, indicating that the protective efficacy of mAb 21H8 is Fc-dependent. However, mAb 21H8 expressed with reduced core fucosylation of its Fc N-glycan, which specifically enhanced NK cell activation in vitro, failed to improve protection against H1N1 challenge in vivo. Future work is needed to decipher in more detail the mechanism of Fc-mediated protection against influenza via NA-specific antibodies and to identify the optimal strategies for their enhancement.

microbiology↗

bioRxiv2024

Mosaic display of stable hemagglutinin monomers induces broad immune responses

The nature of the interplay between immunity and viral variation is infinitely adaptive. Infection frequently induces immune responses against variation-prone epitopes, rather than against spatially hidden conserved epitopes. It thus remains a substantial challenge to elicit the immune responses to the conserved epitopes providing broad-spectrum immunity. We developed an approach of scaffold-mediated mosaic display to present monomeric influenza virus hemagglutinins (HAs), which exposes highly conserved stem and interface epitopes. Stable monomers were rationally engineered from H1 and H3 subtypes and B type HA trimers, with amino acid mutations at the monomer-monomer interface and for disulfide bond formation, and fused to a self-assembling scaffold, to generate a mosaic HA monomer-displaying nanoparticle, 3HA-np. Immunization with 3HA-np induced broadly neutralizing antibodies (bnAbs) in mice and ferrets and protected against challenges with H1N1 and H3N2 viruses. Competitive immunoassays revealed that 3HA-np induced high interface- and stem-binding Ab titers as compared to head Ab titers, indicating that the monomeric and mosaic nature of 3HA-np elicit cross-reactive Abs. Our results suggest that exposure of the hidden conserved epitope by monomer-displaying nanoparticles is a promising approach to generate a universal influenza vaccine.

bioengineering↗

bioRxiv2024

Yeast-based production platform for potent and stable heavy chain-only antibodies

Monoclonal antibodies are the leading drug of the biopharmaceutical market because of their high specificity and tolerability, but the current CHO-based manufacturing platform remains expensive and time-consuming leading to limited accessibility, especially in the case of diseases with high incidence and pandemics. Therefore, there is an urgent need for an alternative production system. In this study, we present a rapid and cost-effective microbial platform for heavy chain-only antibodies (VHH-Fc) in the methylotrophic yeast Komagataella phaffii (aka Pichia pastoris). We demonstrate the potential of this platform using a simplified single-gene VHH-Fc fusion construct instead of the conventional monoclonal antibody format, as this is more easily expressed in Pichia pastoris. We demonstrate that the Pichia-produced VHH-Fc fusion construct is stable and that a Pichia-produced VHH-Fc directed against the SARS-CoV-2 spike has potent SARS-CoV-2 neutralizing activity in vitro and in vivo. We expect that this platform will pave the way towards faster and cheaper development and production of broadly neutralizing single-chain antibodies in yeast.

microbiology↗

bioRxiv2023

The antigenic landscape of human influenza N2 neuraminidases from 2009 until 2017

Human H3N2 influenza viruses are subject to rapid antigenic evolution which translates into frequent updates of the composition of seasonal influenza vaccines. Despite these updates, the effectiveness of influenza vaccines against H3N2-associated disease is suboptimal. Seasonal influenza vaccines primarily induce hemagglutinin-specific antibody responses. However, antibodies directed against influenza neuraminidase (NA) also contribute to protection. Here, we analyzed the antigenic diversity of a panel of N2 NAs derived from human H3N2 viruses that circulated between 2009 and 2017. The antigenic breadth of these NAs was determined based on the NA inhibition (NAI) of a broad panel of ferret and mouse immune sera that were raised by infection and recombinant N2 NA immunization. This assessment allowed us to distinguish at least 4 antigenic groups in the N2 NAs derived from human H3N2 viruses that circulated between 2009 and 2017. Computational analysis further revealed that the amino acid residues in N2 NA that have a major impact on susceptibility to NAI by immune sera are in proximity of the catalytic site. Finally, a machine learning method was developed that allowed to accurately predict the impact of mutations that are present in our N2 NA panel on NAI. These findings have important implications for the renewed interest to develop improved influenza vaccines based on the inclusion of a protective NA antigen formulation.

immunology↗

bioRxiv2023

MX2 restricts HIV-1 and herpes simplex virus-1 by forming cytoplasmic biomolecular condensates that mimic nuclear pore complexes

Human myxovirus resistance 2 (MX2) can potently restrict HIV-1 and herpesviruses at a post-entry step by a process that requires MX2 interaction with the capsids of these viruses. The involvement of other host cell factors in this process, however, remains poorly understood. Here, we mapped the proximity interactome of MX2 revealing strong enrichment of phenylalanine-glycine (FG)-rich proteins related to the nuclear pore complex as well as proteins that are part of cytoplasmic ribonucleoprotein granules. MX2 interacted with these proteins to form multiprotein cytoplasmic biomolecular condensates that were essential for its anti-HIV-1 and -herpes simplex virus-1 (HSV-1) activity. MX2 condensate formation required the disordered N-terminal region of MX2 and its dimerization. Incoming HIV-1 and HSV-1 capsids associated with MX2 at these dynamic cytoplasmic biomolecular condensates. Our results demonstrate that MX2 forms cytoplasmic condensates that act as nuclear pore decoys, which trap capsids and induce premature viral genome release, and thereby interfere with nuclear targeting of HIV-1 and HSV-1.

microbiology↗

bioRxiv2023

Ultrapotent SARS coronavirus-neutralizing single-domain antibodies that bind a conserved membrane proximal epitope of the spike

Currently circulating SARS-CoV-2 variants have gained complete or significant resistance to all SARS-CoV-2-neutralizing antibodies that have been used in the clinic. Such antibodies can prevent severe disease in SARS-CoV-2 exposed patients for whom vaccines may not provide optimal protection. Here, we describe single-domain antibodies (VHHs), also known as nanobodies, that can broadly neutralize SARS-CoV-2 with unusually high potency. Structural analysis revealed their binding to a unique, highly conserved, membrane proximal, quaternary epitope in the S2 subunit of the spike. Furthermore, a VHH-human IgG1 Fc fusion, efficiently expressed in Chinese hamster ovary cells as a stable antibody construct, protected hamsters against SARS-CoV-2 replication in a therapeutic setting when administered systemically at low dose. This VHH-based antibody represents a new candidate anti-COVID-19 biologic that targets the Achilles heel of the viral spike.

microbiology↗

bioRxiv2022

Cleavage of cFLIP restrains cell death during viral infection and tissue injury and favors tissue repair

Cell death coordinates repair programs following pathogen attack and tissue injury. However, aberrant cell death can interfere with such programs and cause organ failure. cFLIP is a crucial regulator of cell death and a substrate of Caspase-8. Yet, the physiological role of cFLIP cleavage by Caspase-8 remains elusive. Here, we discovered an essential role for cFLIP cleavage in restraining cell death in different pathophysiological scenarios. Mice expressing a cleavage-resistant cFLIP mutant, CflipD377A, exhibited increased sensitivity to SARS-CoV-induced lethality, impaired skin wound healing and increased tissue damage caused by Sharpin deficiency. In vitro, abrogation of cFLIP cleavage sensitizes cells to TNF-induced necroptosis and apoptosis by favoring complex-II formation. Mechanistically, the cell death-sensitizing effect of the D377A mutation depends on Gln(Q)469. These results reveal a crucial role for cFLIP cleavage in controlling the amplitude of cell death responses occurring upon tissue stress, to ensure the execution of repair programs.

cell biology↗

bioRxiv2021

Ring Finger Protein 213 Assembles into a Sensor for ISGylated Proteins with Antimicrobial Activity

ISG15 is an interferon-stimulated, ubiquitin-like protein that can conjugate to substrate proteins (ISGylation) to counteract microbial infection, but the underlying mechanisms remain elusive. Here, we used a viral-like particle trapping technology to identify ISG15-binding proteins and discovered Ring Finger Protein 213 (RNF213) as an ISG15 interactor and cellular sensor of ISGylated proteins. RNF213 is a poorly-characterized, interferon-induced megaprotein that is frequently mutated in Moyamoya disease, a rare cerebrovascular disorder. We found that interferon induces ISGylation and oligomerization of RNF213 on lipid droplets, where it acts as a sensor for ISGylated proteins. We showed that RNF213 has broad antimicrobial activity in vitro and in vivo, counteracting infection with Listeria monocytogenes, herpes simplex virus 1 (HSV-1), human respiratory syncytial virus (RSV) and coxsackievirus B3 (CVB3), and we observed a striking co-localization of RNF213 with intracellular bacteria. Together, our findings provide novel molecular insights into the ISGylation pathway and reveal RNF213 as a key antimicrobial effector.

cell biology↗

bioRxiv2020

Structural Basis for Potent Neutralization of Betacoronaviruses by Single-domain Camelid Antibodies

The pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV-1) and COVID-19 coronavirus (SARS-CoV-2) have all emerged into the human population with devastating consequences. These viruses make use of a large envelope protein called spike (S) to engage host cell receptors and catalyze membrane fusion. Because of the vital role that these S proteins play, they represent a vulnerable target for the development of therapeutics to combat these highly pathogenic coronaviruses. Here, we describe the isolation and characterization of single-domain antibodies (VHHs) from a llama immunized with prefusion-stabilized coronavirus spikes. These VHHs are capable of potently neutralizing MERS-CoV or SARS-CoV-1 S pseudotyped viruses. The crystal structures of these VHHs bound to their respective viral targets reveal two distinct epitopes, but both VHHs block receptor binding. We also show cross-reactivity between the SARS-CoV-1 S-directed VHH and SARS-CoV-2 S, and demonstrate that this cross-reactive VHH is capable of neutralizing SARS-CoV-2 S pseudotyped viruses as a bivalent human IgG Fc-fusion. These data provide a molecular basis for the neutralization of pathogenic betacoronaviruses by VHHs and suggest that these molecules may serve as useful therapeutics during coronavirus outbreaks.

microbiology↗