The SARS-CoV-2 Y453F mink variant displays a pronounced increase in ACE-2 affinity but does not challenge antibody neutralization

Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 from humans to animals has been reported for many domesticated species, including farmed minks. The identification novel spike gene mutations appearing in minks raised major concerns about potential immune evasion and challenges the global vaccine strategy. One genetic variant, known as “cluster five,” arose among Denmark resulted a complete shutdown world’s largest mink production. However, functional properties this new variant are not established. Here we present data on cluster-five which contains mutation resulting Y453F residue change receptor-binding domain (RBD) protein. Using an ELISA-based angiotensin-converting enzyme-2/RBD inhibition assay, show that does decrease established humoral immunity previously infected individuals or affect neutralizing antibody response mouse model based original Wuhan strain RBD antigens. biolayer interferometry analysis demonstrates it binds human enzyme-2 receptor with 4-fold higher affinity than strain, suggesting enhanced transmission capacity possible challenge viral control. These results also indicate rise frequency farms might be result fitness advantage conferred by adaptation rather evading responses. Experimental infection models have shown (SARS-CoV-2) is capable infecting wide range animal such cats, dogs, ferrets, hamsters (1Shi J. Wen Z. Zhong G. Yang H. Wang C. Huang B. Liu R. He X. Shuai L. Sun Zhao Y. P. Liang Cui et al.Susceptibility other SARS-coronavirus 2.Science. 2020; 368: 1016-1020Crossref PubMed Scopus (1026) Google Scholar, 2Halfmann P.J. Hatta M. Chiba S. Maemura T. Fan Takeda Kinoshita N. 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Zandvoort Eggo Funk al.Estimated transmissibility impact lineage B.1.1.7 England.medRxiv. 1-80https://doi.org/10.1101/2020.12.24.20248822Crossref There general lack evidence characterizing pathogenesis, properties, variants' memory Despite this, there speculations reducing convalescent could even current strategies Based presumptions, government November decided completely shut down all farms, at time represented fur production world. address biophysical characteristics immunity, expressed recombinant WT (WT, Wuhan-Hu-1/2019 isolate) “cluster-five” ectodomain. did alter potency sera exposed 2020, nor immunized prefusion-stabilized increase spreading potential. determine biological significance mutation, studied stability function thermal denaturation kinetics experiments (Fig. 1). ratio intrinsic fluorescence 350 330 nm over temperature gradient 35 95 °C, observed no significant differences inflection temperatures (53.43 53.37 °C Y453F, respectively), critical effect 1B). 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In models, case 18 different verified 453 engaged direct via tyr OH group therefore expected would either similar phenylalanine. our surprise, four-fold 453F (KD 3.85 precise reason remains established, residues interaction. consequence physiological avidity pronounced multimeric spike/ACE-2 indicative, caution should taken evaluating variant. Regardless, highlight essential points regarding clusters variants: dramatically emphasis placed emerging follows line UK short gained dominance London, mid-December 60% (28Kupferschmidt Mutant sets off alarms, importance unclear.Science (80-). https://doi.org/10.1126/science.abg2626Crossref yet Taken together, suggest evolution direction optimization nucleotide sequence corresponding C-terminal 10xHis-AviTag (SGSGHHHHHHHHHHGSGGLNDIFEAQKIEWHE) synthesized subcloned pcDNA3.4-TOPO expression vector GeneArt (Thermo Fisher Scientific, MA). Recombinant produced transiently transfecting Expi293 cells (Gibco, Thermo MA) according manufacturer’s recommendations. On day after transfection, supernatant collected, batch-purified incubating h room (RT) HisPur Ni-NTA Resin (1 ml sedimented resin/100 supernatant) Scientific) followed wash elution instructions. eluted subsequently applied HiLoad Superdex 16/600 200 pg size-exclusion chromatography column (Cytiva, equilibrated PBS. Purified buffer-exchanged biotinylated AviTag biotin ligase kit (Avidity, CO). (aa. 17–740) CD33 signal peptide IgG1 Twist Bioscience (San Francisco, CA) pTT5 vector. ACE-2-Fc 5 collected affinity-purified 5-ml MabSelect SuRe column, 26/600 (both Cytiva, production, purification, biotinylation analyzed Tycho NT.6 (NanoTemper technologies GmbH, Munich, Germany). subjected triplicates ramp 30 °C/min Protein monitored detected nm. Inflection representing discrete unfolding event calculated system 350:330 ratio. Binding performed Octet (Octet RED384) (ForteBio, 16-channel mode loaded anti-human Fc capture sensors (Pall Life Sciences, CA). Briefly 13 ?g/ml tips (500 s) (2Halfmann base (60 (3Sia association 5-point 2-fold serial dilutions starting 100 60 s), dissociation phase s). Two specific reference assigned each sensors, dipped running buffer during phases. sensor sensorgrams column-wise subtracted sample sensorgrams. 20-mM Hepes, 150-mM NaCl, 5-mM CaCl2, 0.1% BSA (IgG free), 0.03% Tween-20, pH 7.4. Association curves globally fitted 1:1 model. sera, house method Briefly, Nunc MaxiSorp microtiter plates (Invitrogen, coated overnight PBS (10.1-mM Na2HPO4, 1.5-mM KH2PO4, 2.7-mM KCl, 137-mM NaCl). next day, sera/mAbs incubated low-binding round-bottom solution (4 ng/ml) HS-strep-HRP (1:16,000 dilution) 0.05% Tween-20 (PBS-T) follows: 10% dilution, 8-point dilution 1.25% 6-point 20 ?g/ml. Afterward, sera/mAbs:RBD:HS-Strep-HRP transferred 15 min. developed TMB (Kem-En-Tec Diagnostics, Taastrup, Denmark) min stopped 0.3 M H2SO4. absorbance read 450 Between steps, washed thrice PBS-T. Unless otherwise stated, incubations orbital shaker RT. A patients study. participants (29Hansen Jarlhelt I. Hummelshøj Landsy Loftager Bjelke J.R. Egebjerg Sværke Jørgensen Iversen Bayarri-Olmos responses severity individuals.J. 206: 109-117Crossref (60) pool healthy before December 2019 negative use donor blood approved Regional Ethical Committee Capital Region approval ID: H-20028627. obtained Declaration Helsinki. experimental procedures Animal Experiments Inspectorate 2019-15-0201-00090. Data GraphPad Prism 9 (GraphPad Software, equation [inhibitor] normalized variable slope calculate (from non-neutralizing 100, respectively). estimated Mann-Whitney U (reported R2), two-tailed tests value p). Best-fit selected extra-sum-of-squares F test. <0.05 considered statistically significant. All contained article. authors declare they interest contents like thank Jais Rose Bjelke, Frederik Kryh Öberg, Per Franklin Nielsen Novo Nordisk A/S quality proteins Mss Camilla Xenia Holtermann Jahn, Sif Kaas Nielsen, Jytte Bryde Clausen, Laboratory Molecular Medicine excellent technical assistance. B.-O. conceived designed study; enabled production; B-O., R., J., experiments; B.-O., M-O. data; G., wrote paper inputs co-authors. final version manuscript.