key: cord-0807827-lqz2hj2f
authors: Arora, Prerna; Zhang, Lu; Krüger, Nadine; Rocha, Cheila; Sidarovich, Anzhalika; Schulz, Sebastian; Kempf, Amy; Graichen, Luise; Moldenhauer, Anna-Sophie; Cossmann, Anne; Dopfer-Jablonka, Alexandra; Behrens, Georg M.N.; Jäck, Hans-Martin; Pöhlmann, Stefan; Hoffmann, Markus
title: SARS-CoV-2 Omicron sublineages show comparable cell entry but differential neutralization by therapeutic antibodies
date: 2022-05-06
journal: Cell Host Microbe
DOI: 10.1016/j.chom.2022.04.017
sha: 3defe18299452cea168f677acba5ed04ffc8576d
doc_id: 807827
cord_uid: lqz2hj2f

The Omicron variant of SARS-CoV-2 evades antibody-mediated neutralization with unprecedented efficiency. At least three Omicron sublineages have been identified, BA.1, BA.2 and BA.3, with BA.2 exhibiting increased transmissibility. However, it is currently unknown whether BA.2 differs from the other sublineages regarding cell entry and antibody-mediated inhibition. Here, we show that BA.1, BA.2 and BA.3 enter and fuse target cells with similar efficiency and in an ACE2-dependent manner. However, BA.2 was not efficiently neutralized by seven of eight antibodies used for COVID-19 therapy, including Sotrovimab, which robustly neutralized BA.1. In contrast, BA.2 and BA.3 but not BA.1 were appreciably neutralized by Cilgavimab, which may constitute a treatment option. Finally, all sublineages were comparably and efficiently neutralized by antibodies induced by BNT162b2 booster vaccination after previous two-dose homologous or heterologous vaccination. Collectively, the Omicron sublineages show comparable cell entry and neutralization by vaccine-induced antibodies but differ in susceptibility to therapeutic antibodies.

formation was observed between the Omicron sublineages, indicating that the mutations in the 143 respective S proteins do not alter the ability to fuse cells (Fig. 1G, H) . 144 Differential sensitivity of Omicron sublineages to neutralization by therapeutic 145 monoclonal antibodies. Recombinant monoclonal antibodies that bind to the S protein and 146 neutralize SARS-CoV-2 are successfully employed for COVID-19 therapy. Therefore, we 147 analyzed whether the Omicron sublineages differ in sensitivity to neutralization by such 148 therapeutic antibodies. Unspecific immunoglobulin did not interfere with particle entry while all 149 therapeutic antibodies efficiently neutralized B.1pp, as expected ( Fig. 2A-B ). In contrast, most 150 antibodies failed to neutralize the Omicron sublineages in keeping with mutations being located 151 in the respective epitopes ( Fig. 2A-B ) (Dong et al., 2021; Hansen et al., 2020; Jones et al., 2021; 152 Kim et al., 2021; Pinto et al., 2020; Shi et al., 2020) . However, differences in neutralization of the 153 sublineages were noted. Thus, BA.1pp were only appreciably neutralized by Sotrovimab and 154 neutralization was not as efficient as that measured for B.1pp (Fig. 2B) , potentially due to 155 mutations G339D and N440K in the BA.1 S protein ( Fig. 2A) . In contrast, BA.2pp were robustly 156 neutralized only by Cilgavimab and neutralization efficiency was lower than that detected for with the S proteins of BA.1 reported resistance against most antibodies (Carreno et al., 2022; 215 Dejnirattisai et al., 2022; Garcia-Beltran et al., 2022; Hoffmann et al., 2022; Rossler et al., 2022; 216 Schmidt et al., 2022; VanBlargan et al., 2022; Zhang et al., 2022) Limitations of the study 241 We present, to our knowledge, the first side-by-side analysis of the Omicron sublineages BA.1, 

-All data reported in this paper will be shared by the lead contact upon request.

-This paper does not report original code.

-Any additional information required to reanalyze the data reported in this paper is In order to test binding of the different S proteins to ACE2, 293T cells were seeded in 6-well 550 plates and transfected with expression plasmids for the respective SARS-CoV-2 S protein by calcium-phosphate precipitation. Cells transfected with empty plasmid served as a negative 552 control. At 24 h posttransfection, the medium was replaced. At 48 h posttransfection, the culture 553 medium was removed and cells were resuspended in PBS and transferred into 1.5 ml reaction 554 tubes before being pelleted by centrifugation All centrifugation steps were carried out at room 555 temperature at 600 x g for 5 min. Subsequently, the supernatant was aspirated and the cells were Giemsa solutions (each staining was performed for 30 min at room temperature). After each staining, cells were washed three times with deionized water and air-dried. Finally, S protein-576 driven cell-to-cell fusion was investigated by bright field microscopy using a Zeiss LSM800 577 confocal laser scanning microscope (Zeiss).

Quantitative cell-to-cell fusion assay 580 293T effector cells grown to ~75% confluency in 12-well plates were cotransfected with 581 expression plasmids for the respective S protein or empty vector (1.5 µg/well) and the beta- for additional 24 h. Next, beta-galactosidase substrate (Gal-Screen, Thermo Fisher Scientific) 591 was added (100 µl/well) and samples were incubated for 90 min in the dark at room temperature 592 before luminescence was recorded using a Hidex Sense plate luminometer (Hidex). The results on S protein-driven cell entry represent average (mean) data acquired from six to 596 twelve biological replicates, each conducted with four technical replicates. Transduction was 597 normalized against that measured for SARS-CoV-2 S B.1 (set as 1). Alternatively, transduction 598 was normalized against the background signal (luminescence measured for cells inoculated with particles bearing no viral glycoprotein; set as 1). For ACE2 binding analyzed by flow cytometry, 600 the average (mean) geometric mean channel fluorescence from six biological replicates is Transduction was normalized to samples without serum (= 0% inhibition). 

Recombinant Anti-ACE2 Neutralizing Antibody Sino Biological Cat#

RRID: N/A Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody

Thermo Fisher Scientific Cat# A-11013

produced from CRL-2700 mouse hybridoma cells) ATCC Cat# CRL-2700

Chemicals, peptides, and recombinant proteins Soluble human ACE2 (sol-hACE2-Fc) Laboratory of Stefan

 262 We thank B. J. Bosch, R. Cattaneo, G. Herrler, A. Maisner, S. Ludwig, T. Pietschmann and G.

Zimmer for providing reagents. We gratefully acknowledge the originating laboratories