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

The emergence and rapid spread of SARS-CoV-2 variants may impact vaccine efficacy significantly. 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 assessed. 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.

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

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 Omicron's 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.

Extended Shedding and Enhanced Fitness of the SARS-CoV-2 Variant of Concern B.1.1.7 in Human Organoid Systems (preprint)

A new phase of the COVID-19 pandemic has started as SARS-CoV-2 variants are emerging globally, raising concerns for increased transmissibility. Early 2021 the B.1.1.7 (or Alpha) variant, became the dominant variant globally and epidemiological data suggests this variant spreads faster than its ancestors. However, this does not prove that a variant is intrinsically phenotypically different, let alone more transmissible or fit. Therefore, rapid phenotyping of SARS-CoV-2 variants of concern is urgently needed. We found that airway, intestinal and alveolar organoids infected with the B.1.1.7 variant produced higher levels of infectious virus late in infection compared to its 614G-containing ancestor. The B.1.1.7 variant also had a clear fitness advantage in human airway organoids. In alveolar organoids, the B.1.1.7 variant induced lower levels of innate immunity. These findings suggest that the B.1.1.7 variant is phenotypically different from its ancestor and may explain why this clade has spread rapidly across the globe.Funding Information: This work was supported by Netherlands Organization for Health Research and Development (10150062010008; B.L.H.), PPP allowance (LSHM19136; B.L.H.). This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 874735. Declaration of Interests: H.C. is inventor on patents held by the Royal Netherlands Academy of Arts and Sciences that cover organoid technology. H.C.’s full disclosure is given at https://www.uu.nl/staff/JCClevers. All other authors have nothing to declare. Ethics Approval Statement: The Medical Ethical Committee of the Erasmus MC Rotterdam granted permission for this study (METC 2012-512). The study was approved by the UMC Utrecht (Utrecht, The Netherlands) ethical committee and was in accordance with the Declaration of Helsinki and according to Dutch law. This study is compliant with all relevant ethical regulations regarding research involving human participants.

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

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.

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

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.

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

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.

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

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.

Susceptibility of rabbits to SARS-CoV-2 (preprint)

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.

SARS-CoV-2 Productively Infects Human Gut Enterocytes (preprint)

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

Comparative Pathogenesis Of COVID-19, MERS And SARS In A Non-Human Primate Model (preprint)

A novel coronavirus, SARS-CoV-2, was recently identified in patients with an acute respiratory syndrome, COVID-19. To compare its pathogenesis with that of previously emerging coronaviruses, we inoculated cynomolgus macaques with SARS-CoV-2 or MERS-CoV and compared with historical SARS-CoV infections. In SARS-CoV-2-infected macaques, virus was excreted from nose and throat in absence of clinical signs, and detected in type I and II pneumocytes in foci of diffuse alveolar damage and mucous glands of the nasal cavity. In SARS-CoV-infection, lung lesions were typically more severe, while they were milder in MERS-CoV infection, where virus was detected mainly in type II pneumocytes. These data show that SARS-CoV-2 can cause a COVID-19-like disease, and suggest that the severity of SARS-CoV-2 infection is intermediate between that of SARS-CoV and MERS-CoV. One Sentence SummarySARS-CoV-2 infection in macaques results in COVID-19-like disease with prolonged virus excretion from nose and throat in absence of clinical signs.