key: cord-0988149-tz2d3lff authors: Arora, Prerna; Kempf, Amy; Nehlmeier, Inga; Sidarovich, Anzhalika; Krüger, Nadine; Graichen, Luise; Moldenhauer, Anna-Sophie; Winkler, Martin S.; Schulz, Sebastian; Jäck, Hans-Martin; Stankov, Metodi V.; Behrens, Georg M. N.; Pöhlmann, Stefan; Hoffmann, Markus title: Increased lung cell entry of B.1.617.2 and evasion of antibodies induced by infection and BNT162b2 vaccination date: 2021-06-23 journal: bioRxiv DOI: 10.1101/2021.06.23.449568 sha: 6df93790de87f89d5bea2590e210b8d9f0f6baf8 doc_id: 988149 cord_uid: tz2d3lff The delta variant of SARS-CoV-2, B.1.617.2, emerged in India and has subsequently spread to over 80 countries. B.1.617.2 rapidly replaced B.1.1.7 as the dominant virus in the United Kingdom, resulting in a steep increase in new infections, and a similar development is expected for other countries. Effective countermeasures require information on susceptibility of B.1.617.2 to control by antibodies elicited by vaccines and used for COVID-19 therapy. We show, using pseudotyping, that B.1.617.2 evades control by antibodies induced upon infection and BNT162b2 vaccination, although with lower efficiency as compared to B.1.351. Further, we found that B.1.617.2 is resistant against Bamlanivimab, a monoclonal antibody with emergency use authorization for COVID-19 therapy. Finally, we show increased Calu-3-lung cell entry and enhanced cell-to-cell fusion of B.1.617.2, which may contribute to augmented transmissibility and pathogenicity of this variant. These results identify B.1.617.2 as an immune evasion variant with increased capacity to enter and fuse lung cells. we addressed this question using reporter particles pseudotyped with the SARS-CoV-2 spike (S) 73 protein, which are suitable tools to study SARS-CoV-2 neutralization by antibodies (Riepler et 74 al., 2020, Schmidt et al., 2020) . The S protein of B.1.617.2 harbors nine mutations in the surface unit, S1, of the S protein 98 and 1 mutation in the transmembrane unit, S2 ( Figure 1A -B). Mutations T19R, G142D, E156G, 99 F157Δ and R158Δ are located in the N-terminal domain of S1, which contains epitopes for Zhou et al., 2021). Finally, P681R might increase cleavage of S protein at the S1/S2 site while the 107 impact of D950N on S protein driven entry and its inhibition by antibodies is unknown. 108 We first asked whether B.1.617.2 S protein mediates robust entry into cell lines frequently 109 used for SARS-CoV-2 research, Vero (African green monkey, kidney), 293T (human, kidney), 110 Caco-2 (human, colon) and Calu-3 (human, lung). All cell lines express endogenous ACE2 and 111 Vero, Caco-2 and Calu-3 cells are often used for infection studies with authentic SARS-CoV-2. The B.1.617.2 S protein mediated entry into 293T and Vero cells with the same efficiency as WT 113 S protein while entry into Caco-2 (~1.5-fold) and Calu-3 cells (~2.0-fold) was augmented ( Figure 114 1C and Supplemental figure 1A ). The lung is the central target of SARS-CoV-2 but infection of 115 colon has also been reported, suggesting that B.1617.2 might have increased capacity to enter 116 target cells in these tissues. Finally, we did not detect increased ACE2 binding of B.1.617.2 S 117 protein ( Figure 1D ), suggesting that increased entry into Caco-2 and Calu-3 cells was not due to 118 augmented ACE2 binding. Besides its ability to drive fusion of viral and cellular membranes, the S protein is further 120 able to drive the fusion of neighboring cells, resulting in the formation of multinucleated giant 121 cells, so called syncytia, which have been observed in vitro following directed S protein CoV-2 B.1.617.2 (related to Figure 1) . 4 (A) Transduction data normalized against the assay background (related to Figure 1C ). The 5 experiment was performed as described in the legend of Figure 1C . Presented are the average 6 (mean) data from the same six biological replicates (each conducted with technical 7 quadruplicates) as presented in Figure 1C with the difference that transduction was normalized 8 against signals obtained from cells inoculated with particles bearing no viral glycoprotein 9 (background, set as 1). In addition, transduction data of particles bearing VSV-G are included. Error bars indicate the SEM. Figure 1E ). The experiment was performed 16 as described in the legend of Figure 1E . (D) Individual neutralization data for convalescent plasma (related to Figure 1F ). Pseudotype technical quadruplicates. For normalization, inhibition of S protein-driven entry in samples 23 without plasma was set as 0%. Error bars indicate the SD. The data were further used to 24 2 calculated the plasma/serum dilution that leads to 50% reduction in S protein-driven cell entry 25 (neutralizing titer 50, NT50; shown in Figure 1F ). (E) Individual neutralization data for vaccinee serum (related to Figure 1G ). Pseudotype particles inhibition of S protein-driven entry in samples without plasma was set as 0%. Error bars indicate 33 the SD. The data were further used to calculated the NT50 shown (shown in Figure 1G ). N-terminal domain antigenic mapping reveals a site of vulnerability 471 for SARS-CoV-2 Spike mutation D614G alters SARS-CoV-2 fitness & 479 MENACHERY, V. D. 2021b. The variant gambit: COVID-19's next move Safety and 487 Efficacy of the BNT162b2 mRNA Covid-19 Vaccine Comparison of Four SARS-CoV-2 Neutralization Assays Measuring SARS-CoV-2 neutralizing antibody activity using 496 pseudotyped and chimeric viruses A human neutralizing antibody targets the receptor-binding 501 site of SARS-CoV-2 CoV-2 variants of concern are emerging in India Complete map of 505 SARS-CoV-2 RBD mutations that escape the monoclonal antibody LY-CoV555 and its 506 cocktail with LY-CoV016 Neutralizing and protective human monoclonal antibodies recognizing the N-512 terminal domain of the SARS-CoV-2 spike protein Novel Coronavirus (COVID-19) Pneumonia in Two Patients With 515 Lung Cancer .617.2 S proteins was analyzed by two-tailed Students t-test Neutralization of SARS-CoV-2 WT, B.1.351 and B.1.617.2 S proteins by monoclonal 603 antibodies used for COVID-19 therapy SARS-CoV-2 or variant B.1.617.2 were incubated for 30 min at 37 °C in the presence of 605 escalating concentrations CoV-2 S protein-specific monoclonal antibody (please see Figure S1B) or an unrelated control 607 antibody (please see Figure S1C), before being inoculated onto Vero cells. Transduction 608 efficiency was quantified by measuring virus-encoded luciferase activity in cell lysates at 16-18 h 609 post transduction. Presented are the average (mean) data from a single biological replicate 610 (conducted with technical quadruplicates) for which transduction was normalized against samples 611 Transduction efficiency 616 was quantified by measuring virus-encoded luciferase activity in cell lysates at 16-18 h post-617 transduction and used to calculate the plasma dilution factor that leads to 50 % reduction in S 618 protein-driven cell entry (neutralizing titer 50, NT50) **; p ≤ 0.001, ***). Please see also Figure S1D The experiment was performed as described for panel F but this time serum from 623 Presented are the data from a 624 total of fifteen vaccinee sera (black lines indicate the median)