Biology subjects

Lamers, M. M.

Publications and source records attributed to Lamers, M. M..

16 recordsLinked to original sources

bioRxiv2026

An integration-free CRISPR platform for genetic dissection of respiratory virus host dependencies in human nasal epithelium

Respiratory viruses, including coronaviruses, generally initiate infection in the upper respiratory tract, where mechanistic studies of host-virus interactions require models amenable to genetic manipulation. Human nasal air-liquid interface (ALI) cultures are realistic models of the nasal epithelium, but robust CRISPR/Cas9 engineering workflows for this primary cell system have been lacking. Here, we established an integration-free CRISPR/Cas9 workflow for genetic engineering of human nasal basal cells, which are subsequently differentiated into gene-edited ALI cultures. GFP-based sorting of RNP-transfected nasal basal cells substantially increases editing, achieving knockout efficiencies of 70-90% across gene targets (B2M, SLC35A1, ACE2, and TMPRSS2). Genetic disruption of ACE2 markedly reduces infection by SARS-CoV-2 and hCoV-NL63, confirming its important role for both viruses in the human nasal epithelium. TMPRSS2 knockout substantially reduces replication of both viruses, demonstrating SARS-CoV-2 depending on TMPRSS2 in the human nasal epithelium and providing genetic evidence in primary human cell cultures that hCoV-NL63 relies on TMPRSS2. This platform enables systematic genetic perturbation in a physiologically relevant model of the primary site of respiratory virus infection, validates ACE2 and TMPRSS2 as targets for medical countermeasures against SARS-CoV-2 and hCoV-NL63, and provides a versatile approach to identify host dependency factors for both circulating and emerging respiratory viruses.

microbiology↗

bioRxiv2025

Clarifying the priming requirement and activator specificity for the NLRP3 inflammasome in human keratinocytes in vitro

While NLRP3 has been extensively studied in myeloid cells, its existence and regulation in epithelial cells, including keratinocytes, are unclear. In fact, whether human keratinocytes express a functional NLRP3 inflammasome at all remains a matter of debate in the inflammasome field. Here, we provide additional evidence that NLRP3 is repressed in human keratinocytes cultured under non-inflammatory conditions but can be sharply induced by interferon-{gamma} (IFN{gamma})--but not lipopolysaccharide (LPS). In this IFN{gamma}-primed state, not all established NLRP3 activators are specific to NLRP3. We report that nigericin-driven keratinocyte pyroptosis occurs via both NLRP1 and NLRP3, whereas Staphylococcus aureus -hemolysin (Hla) exclusively and nonredundantly activates NLRP3, even though both require K+ efflux. Furthermore, in the presence of T cells, certain virulent S. aureus strains can cause NLRP3-dependent pyroptotic death in keratinocytes in vitro through the cooperative actions of superantigens (SAgs) and Hla. In summary, our findings establish the strict inducibility and functional relevance of the NLRP3 inflammasome in non-myeloid, epithelial cells in vitro. These results resolve conflicting reports and position keratinocytes as a context-specific, non-hematopoietic cellular model for studying NLRP3 activation in host-microbe interactions at barrier tissues. KEY FINDINGSO_LIAdditional evidence that NLRP3 is absent in resting, nonstimulated human keratinocytes in vitro C_LIO_LIIFN{gamma}, but not LPS, is a potent priming signal for NLRP3 in human keratinocytes in vitro C_LIO_LIIn IFN{gamma}-primed keratinocytes, S. aureus -hemolysin (Hla) selectively activates NLRP3, whereas nigericin activates both NLRP1 and NLRP3 in vitro C_LIO_LISAg and Hla kill keratinocytes via NLRP3-driven pyroptosis in the presence of T cells in vitro C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/669639v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@4996f4org.highwire.dtl.DTLVardef@155a2a9org.highwire.dtl.DTLVardef@12a21a0org.highwire.dtl.DTLVardef@105bc15_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

The pro-inflammatory response to influenza A virus infection is fueled by endothelial cells

Morbidity and mortality from influenza are associated with high levels of systemic inflammation. Endothelial cells have been shown to play a key role in this systemic inflammatory response during severe influenza A virus (IAV) infections, despite the fact that these are rarely infected in humans. However, how endothelial cells contribute to these systemic inflammatory responses is unclear. To investigate this, we developed a transwell-system in which airway organoid-derived differentiated human lung epithelial cells at the apical side were co-cultured with primary human lung microvascular endothelial cells (LMEC) at the basolateral side. We compared the susceptibility of endothelial cells to pandemic H1N1 virus isolated in 2009 and seasonal H1N1 and H3N2 virus isolated in 2019, and assessed the associated immune responses. Despite the detection of IAV nucleoprotein in LMEC monocultures, there was no evidence for productive infection. In epithelial-endothelial co-cultures, abundant IAV infection of epithelial cells resulted in the breakdown of the epithelial barrier, but infection of LMECs was rarely detected. Furthermore, we observed a significantly higher secretion of pro-inflammatory cytokines in LMECs when co-cultured with IAV-infected epithelial cells, compared to LMEC monocultures exposed to IAV. Taken together, our data show that endothelial cells are abortively infected by IAV, but can fuel the inflammatory response. As endothelial cells are a prominent cell type in the lung, it is possible that they play an important role in the systemic inflammatory response during IAV infections.

microbiology↗

bioRxiv2022

Pulmonary lesions following inoculation with the SARS-CoV-2 Omicron BA.1 (B.1.1.529) variant in Syrian golden hamsters

The Omicron BA.1 (B.1.1.529) SARS-CoV-2 variant is characterized by a high number of mutations in the viral genome, associated with immune-escape and increased viral spread. It remains unclear whether milder COVID-19 disease progression observed after infection with Omicron BA.1 in humans is due to reduced pathogenicity of the virus or due to pre-existing immunity from vaccination or previous infection. Here, we inoculated hamsters with Omicron BA.1 to evaluate pathogenicity and kinetics of viral shedding, compared to Delta (B.1.617.2) and to animals re-challenged with Omicron BA.1 after previous SARS-CoV-2 614G infection. Omicron BA.1 infected animals showed reduced clinical signs, pathological changes, and viral shedding, compared to Delta-infected animals, but still showed gross- and histopathological evidence of pneumonia. Pre-existing immunity reduced viral shedding and protected against pneumonia. Our data indicate that the observed decrease of disease severity is in part due to intrinsic properties of the Omicron BA.1 variant.

microbiology↗

bioRxiv2022

Omicron BA.1 and BA.2 are antigenically distinct SARS-CoV-2 variants

The emergence and rapid spread of SARS-CoV-2 variants may impact vaccine efficacy significantly1. The Omicron variant termed BA.2, which differs genetically substantially from BA.1, is currently replacing BA.1 in several countries, but its antigenic characteristics have not yet been assessed2,3. Here, we used antigenic cartography to quantify and visualize antigenic differences between SARS-CoV-2 variants using hamster sera obtained after primary infection. Whereas early variants are antigenically similar, clustering relatively close to each other in antigenic space, Omicron BA.1 and BA.2 have evolved as two distinct antigenic outliers. Our data show that BA.1 and BA.2 both escape (vaccine-induced) antibody responses as a result of different antigenic characteristics. Close monitoring of the antigenic changes of SARS-CoV-2 using antigenic cartography can be helpful in the selection of future vaccine strains.

microbiology↗

bioRxiv2022

An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants

The ongoing evolution of SARS-CoV-2 has resulted in the emergence of Omicron, which displays striking immune escape potential. Many of its mutations localize to the spike protein ACE2 receptor-binding domain, annulling the neutralizing activity of most therapeutic monoclonal antibodies. Here we describe a receptor-blocking human monoclonal antibody, 87G7, that retains ultrapotent neutralization against SARS-CoV-2 variants including the Alpha, Beta, Gamma, Delta and Omicron (BA.1/BA.2) Variants-of-Concern (VOCs). Structural analysis reveals that 87G7 targets a patch of hydrophobic residues in the ACE2-binding site that are highly conserved in SARS-CoV-2 variants, explaining its broad neutralization capacity. 87G7 protects mice and/or hamsters against challenge with all current SARS-CoV-2 VOCs. Our findings may aid the development of sustainable antibody-based strategies against COVID-19 that are more resilient to SARS-CoV-2 antigenic diversity. One sentence summaryA human monoclonal antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants

microbiology↗

bioRxiv2022

SARS-CoV-2 Omicron efficiently infects human airway, but not alveolar epithelium

In late 2021, the highly mutated SARS-CoV-2 Omicron variant emerged, raising concerns about its potential extensive immune evasion, increased transmissibility and pathogenicity. Here, we used organoids of the human airways and alveoli to investigate Omicrons fitness and replicative potential in comparison with earlier SARS-CoV-2 variants. We report that Omicron replicates more rapidly in the airways and has an increased fitness compared to the early 614G variant and Delta. In contrast, Omicron did not replicate productively in human alveolar type 2 cells. Mechanistically, we show that Omicron does not efficiently use TMPRSS2 for entry or spread through cell-cell fusion. Altogether, our data show that Omicron has an altered tropism and protease usage, potentially explaining its higher transmissibility and decreased pathogenicity.

microbiology↗

bioRxiv2021

A CRISPR/Cas9 genetically engineered organoid biobank reveals essential host factors for coronaviruses

Rapid identification of host genes essential for virus replication may expedite the generation of therapeutic interventions. Genetic screens are often performed in transformed cell lines that poorly represent viral target cells in vivo, leading to discoveries that may not be translated to the clinic. Intestinal organoids (IOs) are increasingly used to model human disease and are amenable to genetic engineering. To discern which host factors are reliable anti-coronavirus therapeutic targets, we generate mutant clonal IOs for 19 host genes previously implicated in coronavirus biology. We verify ACE2 and DPP4 as entry receptors for SARS-CoV/SARS-CoV-2 and MERS-CoV respectively. SARS-CoV-2 replication in IOs does not require the endosomal Cathepsin B/L proteases, but specifically depends on the cell surface protease TMPRSS2. Other TMPRSS family members were not essential. The newly emerging coronavirus variant B.1.1.7, as well as SARS-CoV and MERS-CoV similarly depended on TMPRSS2. These findings underscore the relevance of non-transformed human models for coronavirus research, identify TMPRSS2 as an attractive pan-coronavirus therapeutic target, and demonstrate that an organoid knockout biobank is a valuable tool to investigate the biology of current and future emerging coronaviruses.

molecular biology↗

bioRxiv2021

Human organoid systems reveal in vitro correlates of fitness for SARS-CoV-2 B.1.1.7

A new phase of the COVID-19 pandemic has started as several SARS-CoV-2 variants are rapidly emerging globally, raising concerns for increased transmissibility. As animal models and traditional in vitro systems may fail to model key aspects of the SARS-CoV-2 replication cycle, representative in vitro systems to assess variants phenotypically are urgently needed. We found that the British variant (clade B.1.1.7), compared to an ancestral SARS-CoV-2 clade B virus, produced higher levels of infectious virus late in infection and had a higher replicative fitness in human airway, alveolar and intestinal organoid models. Our findings unveil human organoids as powerful tools to phenotype viral variants and suggest extended shedding as a correlate of fitness for SARS-CoV-2. One-Sentence SummaryBritish SARS-CoV-2 variant (clade B.1.1.7) infects organoids for extended time and has a higher fitness in vitro.

microbiology↗

bioRxiv2021

Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation

Virus propagation methods generally use transformed cell lines to grow viruses from clinical specimens, which may force viruses to rapidly adapt to cell culture conditions, a process facilitated by high viral mutation rates. Upon propagation in VeroE6 cells, SARS-CoV-2 may mutate or delete the multibasic cleavage site (MBCS) in the spike protein that facilitates serine protease-mediated entry into human airway cells. We report that propagating SARS-CoV-2 on the human airway cell line Calu-3 - that expresses serine proteases - prevents MBCS mutations. Similar results were obtained using a human airway organoid-based culture system for SARS-CoV-2 propagation. Thus, in-depth knowledge on the biology of a virus can be used to establish methods to prevent cell culture adaptation.

microbiology↗

bioRxiv2020

The SARS-CoV-2 multibasic cleavage site facilitates early serine protease-mediated entry into organoid-derived human airway cells

After the SARS-CoV outbreak in 2003, a second zoonotic coronavirus named SARS-CoV-2, emerged late 2019 in China and rapidly caused the COVID-19 pandemic leading to a public health crisis of an unprecedented scale. Despite the fact that SARS-CoV-2 uses the same receptor as SARS-CoV, transmission and pathogenesis of both viruses seem to be quite distinct. A remarkable feature of the SARS-CoV-2 spike is the presence of a multibasic cleavage site, which is absent in the SARS-CoV spike. The viral spike protein not only attaches to the entry receptor, but also mediates fusion after cleavage by host proteases. Here, we report that the SARS-CoV-2 spike multibasic cleavage site increases infectivity on differentiated organoid-derived human airway cells. Compared with SARS-CoV, SARS-CoV-2 entered faster into the lung cell line Calu-3, and more frequently formed syncytial cells in differentiated organoid-derived human airway cells. Moreover, the multibasic cleavage site increased entry speed and plasma membrane serine protease usage relative to endosomal entry using cathepsins. Blocking serine protease activity using the clinically approved drug camostat mesylate effectively inhibited SARS-CoV-2 entry and replication in differentiated organoid-derived human airway cells. Our findings provide novel information on how SARS-CoV-2 enters relevant airway cells and highlight serine proteases as an attractive antiviral target. Significance StatementHighly pathogenic coronaviruses have spilled from animals to humans three times in the past two decades. Late 2019, SARS-CoV-2 emerged in China and was declared a pandemic by March 2020. The other two highly pathogenic coronaviruses, SARS-CoV and MERS-CoV, emerged in 2002 and 2012, respectively, but did not attain sustained human-to-human transmission. Given the high diversity of coronaviruses in animals, urbanization and increased air travel, future coronavirus pandemics are likely to occur intermittently. Identifying which factors determine pandemic potential and pathogenicity are therefore of key importance to global health. Additionally, there is an urgent need to rapidly translate fundamental knowledge to the clinic, a process that is expedited through the use of relevant cell culture systems.

microbiology↗

bioRxiv2020

Susceptibility of rabbits to SARS-CoV-2

Transmission of severe acute respiratory coronavirus-2 (SARS-CoV-2) between livestock and humans is a potential public health concern. We demonstrate the susceptibility of rabbits to SARS-CoV-2, which excrete infectious virus from the nose and throat upon experimental inoculation. Therefore, investigations on the presence of SARS-CoV-2 in farmed rabbits should be considered.

microbiology↗

bioRxiv2020

SARS-CoV-2 neutralizing human antibodies protect against lower respiratory tract disease in a hamster model

Effective clinical intervention strategies for COVID-19 are urgently needed. Although several clinical trials have evaluated the use of convalescent plasma containing virus-neutralizing antibodies, the effectiveness has not been proven. We show that hamsters treated with a high dose of human convalescent plasma or a monoclonal antibody were protected against weight loss showing reduced pneumonia and pulmonary virus replication compared to control animals. However, a ten-fold lower dose of convalescent plasma showed no protective effect. Thus, variable and relatively low levels of virus neutralizing antibodies in convalescent plasma may limit their use for effective antiviral therapy, favouring concentrated, purified (monoclonal) antibodies.

microbiology↗

bioRxiv2020

SARS-CoV-2 Productively Infects Human Gut Enterocytes

COVID-19, caused by SARS-CoV-2, is an influenza-like disease with a respiratory route of transmission, yet clinical evidence suggests that the intestine may present another viral target organ. Indeed, the SARS-CoV-2 receptor angiotensin converting enzyme 2 (ACE2) is highly expressed on differentiated enterocytes. In human small intestinal organoids, enterocytes were readily infected by SARS-CoV and SARS-CoV-2 as demonstrated by confocal- and electron-microscopy. Consequently, significant titers of infectious viral particles were measured. mRNA expression analysis revealed strong induction of a generic viral response program. We conclude that intestinal epithelium supports SARS-CoV-2 replication. One Sentence SummarySARS-CoV-2 infection of enterocytes in human small intestinal organoids

cell biology↗

bioRxiv2020

TARGETED PROTEOMICS FOR THE DETECTION OF SARS-COV-2 PROTEINS.

The rapid, sensitive and specific detection of SARS-CoV-2 is critical in responding to the current COVID-19 outbreak. In this proof-of-concept study, we explored the potential of targeted mass spectrometry based (MS) proteomics for the detection of SARS-CoV-2 proteins in both research samples and clinical specimens. First, we assessed the limit of detection for several SARS-CoV-2 proteins by parallel reaction monitoring (PRM) MS in infected Vero E6 cells. For tryptic peptides of Nucleocapsid protein, the limit of detection was in the mid-attomole range (9E-13 g). Next, this PRM methodology was applied to the detection of viral proteins in various COVID-19 patient clinical specimens, such as sputum and nasopharyngeal swabs. SARS-CoV-2 proteins were detected in these samples with high sensitivity in all specimens with PCR Ct values <24 and in several samples with higher CT values. A clear relationship was observed between summed MS peak intensities for SARS-CoV-2 proteins and Ct values reflecting the abundance of viral RNA. Taken together, these results suggest that targeted MS based proteomics may have the potential to be used as an additional tool in COVID-19 diagnostics.

biochemistry↗

bioRxiv2020

SARS-CoV-2 is transmitted via contact and via the air between ferrets.

SARS-CoV-2, a coronavirus that newly emerged in China in late 2019 1,2 and spread rapidly worldwide, caused the first witnessed pandemic sparked by a coronavirus. As the pandemic progresses, information about the modes of transmission of SARS-CoV-2 among humans is critical to apply appropriate infection control measures and to slow its spread. Here we show that SARS-CoV-2 is transmitted efficiently via direct contact and via the air (via respiratory droplets and/or aerosols) between ferrets. Intranasal inoculation of donor ferrets resulted in a productive upper respiratory tract infection and long-term shedding, up to 11 to 19 days post-inoculation. SARS-CoV-2 transmitted to four out of four direct contact ferrets between 1 and 3 days after exposure and via the air to three out of four independent indirect recipient ferrets between 3 and 7 days after exposure. The pattern of virus shedding in the direct contact and indirect recipient ferrets was similar to that of the inoculated ferrets and infectious virus was isolated from all positive animals, showing that ferrets were productively infected via either route. This study provides experimental evidence of robust transmission of SARS-CoV-2 via the air, supporting the implementation of community-level social distancing measures currently applied in many countries in the world and informing decisions on infection control measures in healthcare settings 3.

microbiology↗