key: cord-0716771-u0zazcrd authors: Hoffmann, Markus; Zhang, Lu; Krüger, Nadine; Graichen, Luise; Kleine-Weber, Hannah; Hofmann-Winkler, Heike; Kempf, Amy; Nessler, Stefan; Riggert, Joachim; Winkler, Martin Sebastian; Schulz, Sebastian; Jäck, Hans-Martin; Pöhlmann, Stefan title: SARS-CoV-2 mutations acquired in mink reduce antibody-mediated neutralization date: 2021-02-12 journal: bioRxiv DOI: 10.1101/2021.02.12.430998 sha: 0d8199eae744b725fea9b5a33ea31517e39a7cda doc_id: 716771 cord_uid: u0zazcrd Transmission of SARS-CoV-2 from humans to farmed mink was observed in Europe and the US. In the infected animals viral variants arose that harbored mutations in the spike (S) protein, the target of neutralizing antibodies, and these variants were transmitted back to humans. This raised concerns that mink might become a constant source of human infection with SARS-CoV-2 variants associated with an increased threat to human health and resulted in mass culling of mink. Here, we report that mutations frequently found in the S proteins of SARS-CoV-2 from mink were mostly compatible with efficient entry into human cells and its inhibition by soluble ACE2. In contrast, mutation Y453F reduced neutralization by an antibody with emergency use authorization for COVID-19 therapy and by sera/plasma from COVID-19 patients. These results suggest that antibody responses induced upon infection or certain antibodies used for treatment might offer insufficient protection against SARS-CoV-2 variants from mink. We employed previously described vesicular stomatitis virus-based reporter particles 97 bearing the SARS-CoV-2 S protein to study whether mutations observed in infected mink 98 modulate cell entry and its inhibition (Hoffmann et al., 2020) . The S protein from SARS-CoV-2 99 isolate hCoV-19/Wuhan/Hu-1/2019, which harbors an aspartic acid at amino acid position 614 100 (D614) (Korber et al., 2020) , was used as control and is subsequently referred to as wildtype 101 (WT). Further, an S protein of identical amino acid sequence but harboring a glycine at position 102 614 (D614G), was used as a reference for S protein variants containing the dominant D614G 103 mutation (Fig. 1D ). Finally, S proteins with mutations found in SARS-CoV-2 from mink were 104 analyzed as shown in figure 1D . Immunoblot analysis of S protein-bearing particles revealed that all mutations were 106 compatible with robust particle incorporation of the S protein and cleavage at the furin motif 107 located at the S1/S2 cleavage site (Fig. 1E) . Similarly, all S proteins efficiently utilized human 108 ACE2 upon directed expression in otherwise non-susceptible BHK-21 cells ( Fig. 2A) . Further, all 109 tested S proteins mediated entry into cell lines commonly used for SARS-CoV-2 research ( Fig. 110 2B), which were also readily transduced by control particles bearing VSV-G (SI Fig. S1 ). Substitution D614G, which is dominant in SARS-CoV-2 from humans (Korber et al., 2020) and 112 was also found in viruses from mink, increased the efficiency of S protein-driven entry, as We focused our analysis on mutation Y453F, since this mutation is located in the RBD, The presence of mutation Y453F alone or in combination with H69Δ and V70Δ did not 183 compromise S protein-mediated entry into human cells and its inhibition by soluble ACE2. However, entry into certain cell lines was reduced when Y453F was combined with H69Δ, 185 V70Δ, I692V and M1229I, as found in the S protein of the SARS-CoV-2 cluster 5 variant. This The following limitations of our study need to be considered. We employed pseudotyped 208 particles instead of authentic SARS-CoV-2 and we did not determine whether Y453F affects viral 209 inhibition by T cell responses raised against SARS-CoV-2. Further, we did not investigate whether Table. Summary of S protein sequences used for analysis and their respective sequence 420 information (related to Figure 1C ). 421 422 Figure S1 . Transduction of target cells (related to Figure 2B ). 423 Data presented in Figure 2B were normalized against the assay background (set as 1). Further, 424 transduction efficiency by pseudotype particles bearing VSV-G is shown. The relative difference in NT50 values between SARS-2-S harboring D614G alone or in 429 conjunction with Y453F was calculated (indicated as Fold difference with SARS-2-S D614G set 430 as 1). The median is indicated by a black line. Weekly operational update on COVID-19 -9 REGN-COV2 antibodies prevent and treat SARS-481 CoV-2 infection in rhesus macaques and hamsters Antibody cocktail to SARS-CoV-2 spike protein prevents 484 rapid mutational escape seen with individual antibodies Emergence of Y453F and Δ69-70HV 488 mutations in a lymphoma patient with long-term COVID-19 A vesicular stomatitis virus replicon-based bioassay 490 for the rapid and sensitive determination of multi-species type I interferon The IFITM proteins mediate cellular 493 resistance to influenza A H1N1 virus, West Nile virus, and dengue virus The glycoprotein of vesicular stomatitis virus promotes release of virus-like 496 particles from tetherin-positive cells Distinct conformational states of SARS-CoV-2 spike protein Intranasal Infection of Ferrets CoV-2 as a Model for Asymptomatic Human Infection Isolation and characterization of viruses related to the 505 SARS coronavirus from animals in southern China Transmission of SARS-CoV-2 in 508 Domestic Cats Studies in humanized mice and convalescent humans yield a 511 SARS-CoV-2 antibody cocktail Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor Mutations in the Spike Protein of Middle East Respiratory SARS-CoV-2 and the human-animal interface: outbreaks on mink farms Tracking Changes in SARS-CoV-2 Spike: 524 Evidence that D614G Increases Infectivity of the COVID-19 Virus A gene regulation system with four distinct expression levels Identifying SARS-CoV-2-related coronaviruses in Malayan 530 pangolins Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 533 receptor Severe acute respiratory syndrome coronavirus-like virus in 536 Chinese horseshoe bats Pandemic dooms Danish mink-and mink research Bats are natural reservoirs of SARS-like coronaviruses From People to Panthera: Natural SARS-542 CoV-2 Infection in Tigers and Lions at the Bronx Zoo Pathological Findings in SARS-CoV-2 Disease Outbreaks in Farmed Mink (Neovison vison) Infections in Engineered Human Tissues Using Clinical-Grade Soluble Human ACE2 SARS-CoV-553 2 infection in farmed minks, the Netherlands Transmission of SARS-CoV-2 on mink farms between humans and mink and back to humans Spike mutation D614G alters SARS-CoV-2 Safety and Efficacy of the BNT162b2 mRNA 563 Covid-19 Vaccine CORONAVIRUS DISEASE 2019 UPDATE CORONAVIRUS DISEASE 2019 UPDATE CORONAVIRUS DISEASE 2019 UPDATE CORONAVIRUS DISEASE 2019 UPDATE (468): ANIMAL CORONAVIRUS DISEASE 2019 UPDATE CORONAVIRUS DISEASE 2019 UPDATE (510): ANIMAL, MINK CORONAVIRUS DISEASE 2019 UPDATE (531): ANIMAL CORONAVIRUS DISEASE 2019 UPDATE (536): ANIMAL, USA Specific Immune Memory Persists after Mild COVID-19 BNT162b2 induces SARS-CoV-2-neutralising 585 antibodies and T cells in humans. medRxiv Characterization of the sialic acid binding activity of 588 influenza A viruses using soluble variants of the H7 and H9 hemagglutinins Detection of SARS-CoV-2 in a cat owned by a 592 COVID-19-affected patient in Spain Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS-595 coronavirus 2 Deep Mutational Scanning SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding. 599 Cell Robust neutralizing antibodies to SARS-602 CoV-2 infection persist for months Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2 Isolation of SARS-CoV-2-related coronavirus from Malayan pangolins A pneumonia outbreak associated with a new coronavirus of probable 611 bat origin Figure 1. Mink-specific spike protein variants are robustly expressed, proteolytically 623 processed and incorporated into viral particles European countries that have reported SARS-CoV-2 infection in mink. The mink-specific 625 spike (S) protein mutations under study are highlighted Summary of mink-specific S protein mutations found in human and mink SARS-CoV-2 627 isolates. Sequences were retrieved from the GISAID (global initiative on sharing all influenza 628 data) database. Legend: a = reference sequences, b = 36/219 sequences carry additional L452M 629 mutation Location of the mink-specific S Schematic illustration of the S protein variants under study and their transmission history Abbreviations: RBD = receptor binding domain, S1/S2 = border between the S1 and S2 subunits Rhabdoviral pseudotypes bearing the indicated S protein variants HA-epitope tag) or no viral glycoprotein were subjected to SDS-PAGE under reducing 638 conditions and immunoblot in order to investigate S protein processing and particle 639 incorporation. Detection of vesicular stomatitis virus matrix protein (VSV-M) served as loading 640 control. Black and grey circles indicate bands for unprocessed and processed Similar results were obtained in four separate experiments. blocked by soluble ACE2 and the protease inhibitor Camostat 645 (A) Rhabdoviral pseudotypes bearing the indicated S protein variants, VSV-G or no viral 646 glycoprotein were inoculated onto BHK-21 cells previously transfected with empty plasmid or 647 human angiotensin-converting enzyme Rhabdoviral pseudotypes bearing the indicated S protein variants, VSV-G (shown in SI 649 Figure 1) or no viral glycoprotein were Caco-2, A549-ACE2, Huh-7 (all human) or Vero76 (non-human primate) cells Rhabdoviral pseudotypes bearing the indicated S protein variants or VSV-G were 652 preincubated with different dilutions of a soluble hACE2 form fused to the Fc portion of human 653 immunoglobulin G (sol-hACE2-Fc) and subsequently inoculated onto Vero76 cells Calu-3 cells that were preincubated with different concentrations of Camostat. For all 656 panels: Transduction efficiency was quantified at 16 h postinoculation by measuring the activity 657 of virus-encoded luciferase in cell lysates. Presented are the normalized average (mean) data of 658 three biological replicates, each performed with technical quadruplicates. Error bars indicate the 659 standard error of the mean (SEM). Statistical significance was tested by one-(panels a and b) or 660 two-way (panels c and d) ANOVA with Dunnett's post-hoc test Figure 3. Y453F reduces neutralization by convalescent sera and monoclonal antibodies 664 (A) Rhabdoviral pseudotypes bearing the indicated spike (S) protein variants or VSV-G were 665 preincubated with different dilutions of serum (Pos Samples #1-6) or plasma (Pos samples #7-14) 666 from convalescent COVID-19 patients (serum from a healthy individual served as control Transduction efficiency was quantified at 16 668 h postinoculation by measuring the activity of virus-encoded luciferase in cell lysates. The top 669 left panel indicates the serum/plasma titers that lead to a 50% reduction in transduction efficiency 670 (neutralizing titer 50, NT50), which was calculated by a non-linear regression model. Data points 671 from identical serum/plasma samples are connected by lines (grey bars indicate the mean NT50 672 values for all positive samples) SARS-2-S harboring D614G alone or in conjunction with Y453F was analyzed by paired 674 student's t-test The experiment outlined in panel A was repeated using serial dilutions of human monoclonal 676 antibodies. For panels A and B: Presented are the normalized average (mean) data of a single 677 experiment performed with technical quadruplicates. Results were confirmed in a separate 678 experiment (due to limited sample material, only two technical replicates could be analyzed in the 679 confirmatory experiment for the serum samples shown in panel A)