Biology subjects

Mykytyn, A. Z.

Publications and source records attributed to Mykytyn, A. Z..

12 recordsLinked to original sources

bioRxiv2023

Comparative Analysis of SARS-CoV-2 Antigenicity across Assays and in Human and Animal Model Sera

The antigenic evolution of SARS-CoV-2 requires ongoing monitoring to judge the immune escape of newly arising variants. A surveillance system necessitates an understanding of differences in neutralization titers measured in different assays and using human and animal sera. We compared 18 datasets generated using human, hamster, and mouse sera, and six different neutralization assays. Titer magnitude was lowest in human, intermediate in hamster, and highest in mouse sera. Fold change, immunodominance patterns and antigenic maps were similar among sera. Most assays yielded similar results, except for differences in fold change in cytopathic effect assays. Not enough data was available for conclusively judging mouse sera, but hamster sera were a consistent surrogate for human first-infection sera.

bioinformatics↗

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

The glycosylated extracellular domain of MUC1 protects against SARS-CoV-2 infection at the respiratory surface

Mucins play an essential role in protecting the respiratory tract against microbial infections but can also serve as binding sites for bacterial and viral adhesins. The heavily O-glycosylated gel-forming mucins MUC5AC and MUC5B eliminate pathogens by mucociliary clearance while transmembrane mucins MUC1, MUC4, and MUC16 can restrict microbial invasion at the apical surface of the epithelium. In this study, we determined the impact of host mucins and mucin glycans on SARS-CoV-2 epithelial entry. Human lung epithelial Calu-3 cells express the SARS-CoV-2 entry receptor ACE2 and high levels of glycosylated MUC1, but not MUC4 and MUC16, on their cell surface. The O-glycan-specific mucinase StcE specifically removed the glycosylated part of the MUC1 extracellular domain while leaving the underlying SEA domain and cytoplasmic tail intact. StcE treatment of Calu-3 cells significantly enhanced infection with SARS-CoV-2 pseudovirus and authentic virus, while removal of sialic acid and fucose from the epithelial surface did not impact viral entry. Both MUC1 and MUC16 are expressed on the surface of human air-liquid interface (ALI) differentiated airway organoids and StcE treatment led to mucin removal and increased levels of SARS-CoV-2 entry and replication. On the surface of Calu-3 cells, the transmembrane mucin MUC1 and ACE2 are often co-expressed and StcE treatment results in enhanced binding of purified spike protein and SARS-CoV-2 pseudovirus. This study points at an important role for glycosylated mucin domains as components of the host defense that can restrict SARS-CoV-2 infection. Author summarySARS-CoV-2, the virus that has caused the devastating COVID-19 pandemic, causes a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A fundamental understanding of host factors influencing viral entry is critical to elucidate SARS-CoV-2-host interactions and identify novel therapeutic targets. In this study, we investigated the role of host mucins and mucin glycans on SARS-CoV-2 entry into the airway epithelial cells. Mucins are a family of high molecular weight O-glycosylated proteins that play an essential role in protecting the respiratory tract against viral and bacterial infections. The gel-forming mucins MUC5AC and MUC5B clear pathogens by mucociliary clearance while transmembrane mucins MUC1, MUC4, and MUC16 can restrict or facilitate microbial invasion at the apical surface of the epithelium. The mucin-selective protease StcE specifically cleaves the glycosylated extracellular part of the mucins without perturbing the underlying domains. We show that removal of mucins from the surface of Calu-3 cells and primary airway epithelial cultures with StcE mucinase increases binding of the SARS-CoV-2 spike protein to the respiratory surface and greatly enhances infection. This study demonstrates the important role of glycosylated extracellular mucin domains as a host defense mechanism during SARS-CoV-2 entry. Future efforts should be focused on characterizing the role of specific soluble and transmembrane mucins during the different stages of SARS-CoV-2 infection.

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

Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection

The SARS-CoV-2 pandemic is continuing to disrupt personal lives, global healthcare systems and economies. Hence, there is an urgent need for a vaccine that prevents viral infection, transmission and disease. Here, we present a two-component protein-based nanoparticle vaccine that displays multiple copies of the SARS-CoV-2 spike protein. Immunization studies show that this vaccine induces potent neutralizing antibody responses in mice, rabbits and cynomolgus macaques. The vaccine-induced immunity protected macaques against a high dose challenge, resulting in strongly reduced viral infection and replication in upper and lower airways. These nanoparticles are a promising vaccine candidate to curtail the SARS-CoV-2 pandemic.

immunology↗

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 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↗