I., Hintze Butoconazole B., Hung L. S with equilibrium dissociation constants (KD) of 7, 7, 12, and 16 nM, respectively (fig. S1, G and H). S2P6 also bound to HKU1 S, albeit Butoconazole with reduced affinity (KD ~120 nM) (fig. S1G). Collectively, these data demonstrate that S2P6 cross-reacts with all human-infecting betacoronaviruses. To evaluate the neutralization potency and breadth of S2P6, we investigated its ability to inhibit entry of authentic SARS-CoV-2 into Vero-E6 cells in the presence or absence of TMPRSS2, as this protease activates fusion with the cytoplasmic membrane in cultured lung cells (and subgenera. Peptide Butoconazole mapping experiments using 15-nucleotide oligomer linear overlapping peptides revealed that all five mAbs bound to peptides containing the SARS-CoV-2 motif F1148KEELDKYF1156 located in Butoconazole the S2 subunit stem helix (Fig. 1H and fig. S2A). This region is strictly conserved in SARS-CoV, is highly conserved among other betacoronaviruses, and overlaps with the epitopes of the B6 (Fig. 1I) and 28D9 mouse mAbs (axis indicates the percentage of monocytes double-positive for anti-CD14 (monocyte) marker and PKH67. (D) Lysis of SARS-CoV-2 S stably transfected CHO cells by mAbs in the presence of complement. S309 was included as positive control; S309-GRLR with diminished FcR binding capacity and an unrelated mAb (neg mAb) were used as negative controls. (E) Syrian hamsters were administered with the indicated amount of S2P6 mAb harboring either a hamster (Hm-S2P6) or a human (Hu-S2P6) constant region before intranasal challenge with prototypic SARS-CoV-2 (Wuhan-1 related). An irrelevant mAb (MGH2 against CSP) at 20 mg/kg was used as negative control (CTRL) (< 0.05; **< 0.01; Mann-Whitney test. We evaluated the prophylactic activity of S2P6 against challenge with the prototypic (Wuhan-1 related) SARS-CoV-2 in a Syrian hamster model (= 88), COVID-19 convalescent (C; = 72), vaccinees immune (VI; = 9), and vaccinees na?ve (VN; = 37) plasma Abs (diluted 1:10) to immobilized betacoronavirus stem helix peptides analyzed by ELISA. A cutoff of 0.7 was determined on the basis of the signal of prepandemic samples and binding to uncoated ELISA plates (horizontal dashed line). The fraction of samples for which binding above the cutoff was detected is indicated. (B to G) Analysis of memory B cell specificities for betacoronavirus stem helix peptides. Each dot represents a well containing oligoclonal B cell supernatant screened for the presence of stem helix peptide binding IgG Hoxd10 Abs using ELISA. Samples obtained from 21 COVID-19 convalescent individuals [(B) to (D)] and 16 vaccinees [(E) to (G)]. Pairwise reactivity comparison is shown for SARS-CoV/-2 and OC43 [(C) and (F)] and SARS-CoV/-2 and HKU1 [(D) and (G)]. Cultures cross-reactive with at least three peptides are highlighted in color. A cutoff of 0.4 is indicated by a horizontal dashed line. The fraction of wells for which binding above the cutoff was detected is indicated. (H and I) Binding to stem helix peptides of S2P6 (H) harboring mature (SH/SK), fully germline-reverted (UCA/UCA), germline-reverted heavy chain paired with mature light chain (UCA/SK), mature heavy chain paired with germline-reverted light chain (SH/UCA), and of P34D10, P34G12, and P34E3 (I) harboring either mature (SH/SK) or germline-reverted (UCA/UCA) sequences. Next, we investigated the frequencies of stem helixCspecific memory B cells among 21 convalescent and 17 vaccinated individuals using a clonal analysis based on in vitro polyclonal stimulation (for the refinement of macromolecular crystal structures. Acta Cryst. D67, 355C367 (2011). 10.1107/S0907444911001314 [PMC free article] [PubMed] [CrossRef] [Google Scholar] 77. Liebschner D., Afonine P. V., Baker M. L., Bunkczi G., Chen V. B., Croll T. I., Hintze B., Hung L. W., Jain S., McCoy A. J., Moriarty N. W., Oeffner R. D., Poon B. K., Prisant M. G., Read R. J., Richardson J. S., Richardson D. C., Sammito M. D., Sobolev O. V., Stockwell D. H., Terwilliger T. C., Urzhumtsev A. G., Videau L. L., Williams C. J., Adams P. D., Macromolecular structure determination using X-rays, neutrons and electrons: Recent developments in Phenix. Acta.