key: cord-1048172-56x3r9ad
authors: Lamers, Mart M.; Breugem, Tim I.; Mykytyn, Anna Z.; Wang, Yiquan; Groen, Nathalie; Knoops, Kèvin; Schipper, Debby; van der Vaart, Jelte; Koopman, Charlotte D.; Zhang, Jingshu; Wu, Douglas C.; van den Doel, Petra B.; Bestebroer, Theo; GeurtsvanKessel, Corine H.; Peters, Peter J.; Muraro, Mauro J.; Clevers, Hans; Wu, Nicholas C.; Haagmans, Bart L.
title: Human organoid systems reveal in vitro correlates of fitness for SARS-CoV-2 B.1.1.7
date: 2021-05-03
journal: bioRxiv
DOI: 10.1101/2021.05.03.441080
sha: dce71ad21eb96988ab6c94fe13f124325de91e34
doc_id: 1048172
cord_uid: 56x3r9ad

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 Summary British SARS-CoV-2 variant (clade B.1.1.7) infects organoids for extended time and has a higher fitness in vitro.

The ongoing COVID-19 pandemic is causing an immense global health crisis, affecting all parts 44 of society. As many countries have started the rollout of vaccines, several SARS-CoV-2 variants 45 have emerged that raise concerns for increased transmissibility and escape from immunity (1). 46 The B.1.1.7 variant, also known as the British variant or VOC-202012/01, now has become the 47 dominant variant in Europe, and is increasing in frequency in Asia and the American continent 48 (2). Epidemiological data suggest that this variant is 35-100% more transmissible than the 49 ancestral lineage and associated with higher viral loads (3-6). As epidemiological studies are 50 easily confounded by public health measures or founder effects, they should preferably be 51 complemented by experimental studies on viral fitness and infectiousness. However, correlates 52 of fitness or infectiousness for respiratory viruses -let alone SARS-CoV-2 -are ill-defined and 53 complicated by the lack of relevant experimental systems to compare virus strains. 5 hours post-infection. Therefore, we assessed the replication kinetics of the B.1.1.7 and Bavpat-1 120 virus in air-liquid interface (ALI) human airway organoids differentiated using Pneumacult 121 medium as described before (22) by sampling the organoids until day 10 post-infection from the 122 apical side. As the RNA copies/PFU ratio of both viruses was equal on Calu-3 cells (Fig. 1I) , we 123 used viral titers determined on Calu-3 cells by plaque assay to equalize virus input at a low To investigate whether SARS-CoV-2 is genetically stable on 2D airway organoids, we deep-152 sequenced the apical washes from day 10 of the replication curves. For both the Pneumacult and 153 BMP2 differentiated airway organoids we did not observe any indication of cell culture 154 adaptation for Bavpat-1 and B.1.1.7 ( Fig. S3; Fig. S4 ). Whereas the human airway epithelium is highly relevant to virus transmission, alveolar cells can 160 be used to model the severe respiratory disease caused by SARS-CoV-2. Therefore, we 161 established human alveolar type 2 (AT2) cell organoids using a method adapted from Youk and 162 colleagues (2020) (Fig. S5 ). Messenger RNA sequencing indicated that the cultured alveolar 163 organoids expressed typical AT2 marker genes (e.g. LPCAT1, SFTPB, SFTPC), but not airway 164 marker genes (e.g. KRT5, TP63, MUC5AC, SCGB1A1, FOXJ1) ( Fig. 4A; Fig S6) . Notably, 165 AT2 cells expressed BMP4, which was shown to inhibit transdifferentiation into AT1 cells (30). 166 The expression of surfactant protein C (SFTPC) was confirmed by immunofluorescent staining 167 (Fig. 4B ). AT2 organoids also contained cells that could be stained with the mature human AT2 168 cell surface marker antibody HTII-280 ( Fig 4B) . In line with the mRNA data, we did not detect 169 any expression of the airway basal cell marker TP63 (Fig 3A; Fig. S7A (Fig. 4F ), but the infectious virus titers were slightly higher for B.1.1.7 at late time 178 points (not significant). This discrepancy between viral RNA and infectious virus is likely to be 179 caused by a higher peak titer for Bavpat-1 at 2 days post-infection and by the fact that in this 3D 180 model the virus/RNA cannot be collected by daily washing of the cells. As viral spread between 181 3D organoids is limited, 3D systems may not be ideal for long replication kinetics experiments. 182 Therefore, we cultured the AT2 cells in transwell inserts at 2D air-liquid interface and repeated Sanger data (Fig. 6F) . Furthermore, Sanger sequencing analysis results were confirmed using a 221 different analysis software, the Synthego ICE indel frequency analysis tool, commonly used for 222 analyzing CRISPR indel frequencies (Fig. S11F ). An important question related to variants such as the B.1.1.7 clade is whether they are associated 280 with increased health risks and mortality (4, 5, 49). In our study we did not observe major 281 differences in the host response between B.1.1.7 and Bavpat-1-infected organoids suggesting that 282 an altered host response is unlikely to drive potential differences in disease severity. 283 Interestingly, Frampton and colleagues noted a trend towards a ~2 day longer time to DNAJA4  LRTOMT  KRT5  CRIP1  SCGB1A1  RSPH1  DHRS9  PIFO  TPPP3  C12orf75  SERPINB3  C1orf194  C9orf116  MORN2  BBOF1  CETN2  MARCKS  PROM1  CCDC17  DNAI1  C20orf85  IGFBP7  DNALI1  SLPI  SPACA9  SOX2  CAPS  TSPAN1  CAPSL  ALDH1A1  SPA17  BMP4  SCGB3A2  CD63  NPC2  MLPH  CTSH  SFTPB  SPINK5  SFTA2  LPCAT1  CADM1  RNASE1  ATP11A LGMN HPGD SFTPC SFTPA1 SFTPA2 ADGRF5 DNAJA4  LRTOMT  KRT5  CRIP1  SCGB1A1  RSPH1  DHRS9  PIFO  TPPP3  C12orf75  SERPINB3  C1orf194  C9orf116  MORN2  BBOF1  CETN2  MARCKS  PROM1  CCDC17  DNAI1  C20orf85  IGFBP7  DNALI1  SLPI  SPACA9  SOX2  CAPS  TSPAN1  CAPSL  ALDH1A1  SPA17  BMP4  SCGB3A2  CD63  NPC2  MLPH  CTSH  SFTPB  SPINK5  SFTA2  LPCAT1  CADM1  RNASE1  ATP11A LGMN The first method used commercially available Pneumacult-ALI medium (complete base medium 40 with 1X maintenance supplement; Stemcell) as described before (22 1 and 2) AT2_Uninfected_1   AT2_Uninfected_2  AT2_Uninfected_3  AT2_WT_3  AT2_WT_1  AT2_WT_2   RARRES3  TRIM14  MDK  DDX60  IRF9  HIST1H1C  MT2A  RTP4  XAF1  IFI44  IFI27  BST2  IFI6  IFI44L  HLA−B  HERC6  CFB  EPSTI1  PLSCR1  MX1  ISG15  OAS2  SAMD9L  IFITM1  IFIT1  MX2  IRF7  SP110  USP18  C1R  MT−RNR1  EIF4B  RPS6  RPL15  EEF1B2  RACK1  SFTPC  RPS3A  EEF1A1  RPS15A  EIF3L AT2_Uninfected_3  AT2_WT_3  AT2_WT_1  AT2_WT_2   TRIM14  MDK  DDX60  IRF9   MT2A  RTP4  XAF1  IFI44  IFI27  BST2  IFI6  IFI44L  HLA−B  HERC6  CFB  EPSTI1  PLSCR1  MX1  ISG15  OAS2  SAMD9L  IFITM1  IFIT1  MX2  IRF7  SP110  USP18  C1R  MT−RNR1  EIF4B  RPS6  RPL15  EEF1B2  RACK1  SFTPC  RPS3A  EEF1A1  RPS15A  EIF3L 

The variant gambit: COVID-19's next move

Data, disease and diplomacy: GISAID's innovative contribution to global 298 health

Early transmissibility assessment of the N501Y 300 mutant strains of SARS-CoV-2 in the United Kingdom

Changes in symptomatology, reinfection, and transmissibility associated with the 303 SARS-CoV-2 variant B.1.1.7: an ecological study

Genomic characteristics and clinical effect of the emergent SARS-CoV-2 B.1.1.7 305 lineage in London, UK: a whole-genome sequencing and hospital-based cohort study

Assessing transmissibility of SARS-CoV-2 lineage B.1.1.7 in England

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

The B1.351 and P.1 variants extend SARS-CoV-2 host range to mice. bioRxiv

SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum 313 from acutely infected, hospitalized COVID-19 patients. bioRxiv

Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture 315 adaptation

SARS-CoV-2 entry into human airway organoids is serine protease-mediated and 317 facilitated by the multibasic cleavage site

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Submitted Manuscript: Confidential Template revised

SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation 321 and cytopathology

Modeling Development and Disease with Organoids

Organogenesis in a dish: modeling development and disease using 324 organoid technologies

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Using brain organoids to understand Zika virus-328 induced microcephaly

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VOC2020212/01) is not accounted for by a replicative advantage in primary airway cells or 373 antibody escape 374

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Human alveolar type 2 (AT2) organoids express AT2 -but not 375 airway -marker genes. (A) Heatmap of normalized expression values scaled across samples of 376 selected airway and alveolar marker genes in bronchiole and AT2 organoids

MUC5AC, SCGB1A1 and FOXJ1 are markers for ionocytes, basal cells, goblet cells, club cells 379 and ciliated cells, respectively

AT2) organoids do not express the 381 basal cell marker TP63. (A-B) Immunofluorescent staining of AT2 organoids (A) and 382 bronchiolar organoids (B) for the basal cell marker TP63 (yellow). Nuclei are stained with 383 hoechst (cyan) and actin is stained with phalloidin (white)

A-F) Overview of an intact alveolar type 2 (AT2) organoid showing 386 cytoplasmic lamellar bodies (LB) (B and C) and secreted lamellar bodies (D and E) that organize 387 into tubular myelin (TM). (F) Apical microvilli (MV) are predominant on the AT2 cells. Scale 388 bars indicate 20 µm (A), 1 µm (B and F)

2D plated alveolar type 2 (AT2) cells stained for HTII-280

Scale bar depicts 20 µm. 392 Supplementary figure 10. Host mRNA responses to SARS-CoV-2 B1.1.7 and Bavpat-1 393 infection in alveolar type 2 (AT2) organoids. (A-B) E gene expression (A) and viral RNA 394 copies (B) in AT2 organoids infected with Bavpat-1 and B.1.1.7 at 72 h p.i.. (C-D) Heatmap of 395 differentially expressed genes in response to Bavpat-1 (C) or B.1.1.7 (D) infection. Heatmaps 396 show normalized expression of genes across samples. (E) Volcano plot depicting differentially 397 expressed genes between

The names of the top 10 genes with the lowest Padj are shown

A-B) Relative 402 frequencies of Bavpat-1 and B.1.1.7 RNAs were assessed by RT-PCR and Sanger sequencing 403 targeting the P681H mutation starting with an initial inoculum ratio of 1:1 (A) or

7), and a cumulative multiplicity of infection (moi) of 0.1 in airway donor 2

D) These relative frequencies were used to calculate the relative replicative fitness at a 1:1 (C) 406 and

RNAs was assessed by RT-PCR and Sanger sequencing targeting the 3675-77 deletion starting 408 with an initial inoculum ratio of 1:1, and a cumulative moi of 0.1 in airway donor 1. Sanger 409 sequences were analysed with either QSVanalyser (Insilicase) (E) or Synthego ICE CRISPR 410 indel frequency analysis software (F). The data in E and F was generated from the same 411 competition experiment as Figure 6E and F. D p.i. = days post infection

was removed to establish an air-liquid interface. Cells were differentiated at the air-liquid 49 interface for 3-4 weeks. Medium was replaced every 4-5 days. 50 Adult human lung tissue for airway and alveolar stem cell (see below) isolation was 51 obtained from non-tumour lung tissue obtained from patients undergoing lung resection. Lung 52 tissue was obtained from residual, tumor-free, material obtained at lung resection surgery for 53 lung cancer. The Medical Ethical Committee of the Erasmus MC Rotterdam granted permission 54 for this study (METC 2012-512) . 55 56 Alveolar cell isolation, culture and differentiation 57 Human adult alveolar stem cells were isolated using a protocol adapted from Lamers and 58 colleagues (2020). Distal lung pieces were chopped into 2 x 2 mm pieces, washed twice with 59 3 cold PBS and incubated in 100% dispase (Corning), supplemented with 10 µM Y-27632 60 (MedChemExpress) for 30-60 min at 37°C. Next, cells were dissociated from the tissue by 61 pipetting using a 5 ml serological pipet and subsequently using a P1000 micropipette. Cells were 62 then strained using a 100 µm cell strainer (Falcon) and washed twice in cold AdDF+++. Red 63 blood cells were lysed using red blood cell lysis buffer (Roche), after which cells were again 64 washed twice with cold AdDF+++. Cells were pelleted, incubated on ice for 2 min, and plated in 65 growth factor reduced basement membrane extract (BME; Cultrex). After BME solidification, 66 alveolar medium (recipe (see below) adapted from Youk and colleagues (27) µM; Tocris), and Primocin (1X; Invivogen). Y-27632 (10 µM) was added for the first 5 days. 73 Medium was replaced every 4-5 days. 74 To sort AT2 cells, organoids were digested using TrypLE Express and washed in 75 AdDF++ twice before incubating the cells in Lysotracker (Thermofisher) for 20 min at 37°C. 76 Next, cells were incubated in FACS buffer (2mM EDTA, 2.5% bovine serum albumin (BSA) in 77 PBS) on ice for 5 min, stained with the AT2 marker antibody HTII-280 (1:40; Terrace Biotech) 78 on ice for 15 min, and with goat anti-mouse IgM Alexa Fluor 488 (1:400; Invitrogen) for 5 min. 79 Cells were then washed once in FACS buffer and HTII-280-high and Lysotracker-high cells 80 were sorted using a FACS Aria cell sorter (BD biosciences) into AdDF+++ containing 10 µM Y-81 27632. Cells were pelleted, incubated on ice for 2 min and plated in BME domes in alveolar 82 medium. Y-27632 (10 µM) was added for the first 5 days. Cells were incubated at 37°C in a 83 humidified CO2 incubator and medium was replaced every 4-5 days. 84 Sorted and unsorted AT2 organoids were split every 2-3 weeks at a 1:3-1:8 ratio. 85 Organoids were dissociated to small clumps using TrypLE. 86 For 2D experiments, sorted AT2 organoids were dissociated using TrypLE, washed in 87 AdDF+++ twice, and plated on BME-coated transwell inserts in alveolar medium containing Y-88 27632 (10 µM). Inserts were coated with BME at a 1:100 dilution in PBS at 37°C for 30 min. 89 Cells were incubated at 37°C in a humidified CO2 incubator for 4 days (until a confluency of 90 ~80% was reached), after which the medium was replaced with AdDF+++ supplemented with 91 4 10% FBS. In this medium, the cells grew to confluency within 3 days. Next, the medium in the 92 top compartment was removed to establish an air-liquid interface and the medium in the bottom 93 compartment was replaced for AdDF+++ without FBS. These cells were used for infections 94 directly. 95 All centrifugation steps were performed at 400 xg for 3 min. For the analysis using Synthego ICE, sanger sequences were uploaded and analysed using a 252 Bavpat-1 control sequence as reference and TTGATACTAGTTTG as a guide to generate allele 253 percentages of B.1.1.7 (3675-3677 del)) and Bavpat-1 (no deletion) present in the sample. Next, 254 allele percentages were used to calculate the relative replicative fitness as described above. The 255 lower limit of detection for the Synthego ICE analysis was set at 1% allele frequency. CoV-2 B.1.1.7 and Bavpat-1 are genetically stable on Pneumacult differentiated 2D human airway organoids. Full genome frequency plots of virus variants in apical washes of pneumacult-differentiated 2D airway organoids at day 10 post-infection for both the Bavpat-1 and B.1.1.7 isolate (three replicates each). In all plots variants with a frequency >10% are depicted.