Among the tested compounds, 6i exhibited the most potent anti-proliferative activity towards NSCLC, CNS, Ovarian, Prostate, Colon, Melanoma, Breast, and Renal cancers with good to weak cytostatic activity and non-lethal actions. 6i demonstrated higher selectivity for cancer than normal cells. 6i could significantly increase the accumulation of S-phase cells during the cell cycle distribution of cancer cells with high potency in the induction of apoptosis. The results unveiled that 6i probably acts through dual inhibition of DHFR and TS enzymes . Docking studies of 6i displayed that N1-p-bromophenyl and C3-Methyl groups participate in substantial hydrophobic interactions. The drug-likeness features inferred that 6i met the acceptance criteria of Pfizer. Taking together, 6i could be a promising prototype for further optimization as an effective anticancer drug.A novel series of multifunctional pyrazolopyrimidine-based glutamate analogs ( In this context, the nonclassical antifolates such as nolatrexed and piritrexim (PTX) were designed as they are more lipophilic and non-polyglutamatable agents,in vitro anti-proliferative activity against the most sensitive tumour and normal cell lines to investigate its selectivity towards cancer cells. In addition, cell cycle analysis and apoptosis were also examined. Finally, the in-silico molecular modelling and ADMET analysis were reported.On the basis of isosterism and combination principles, as shown in 6a–l were prepared as cited in 1a,b to 5-amino-4-cyano-pyrazole derivatives 2a–f using arylhydrazine derivatives,2a–f using different aliphatic acids with a catalytic amount of phosphorous oxychloride afforded the precursors pyrazolopyrimidine-4-ones 3a–l5 was obtained from the chloroacetylation of glutamate anilinic derivative 4 with chloroacetyl chloride. Finally, the desired analogues 6a–n were synthesised through N5-alkylation of the precursor pyrazolopyrimidine-4-ones 3a–l with diethyl glutamate chloroacetamide intermediate 5.The Glu-based pyrazolopyrimidine analogues 7a and 7b were prepared through alkaline hydrolysis of the corresponding diethyl ester analogues 6c and 6d, respectively. On the other hand, the Biginelli reaction was utilised to synthesise the 1,4-dihydropyrimidine derivatives 9a–c through S-alkylation of 2-thioxo-1,4-dihydropyrimidine intermediates 8a–c with chloroacetamide glutamate derivative 5 as depicted in Moreover, as shown in 6a–l, 7a,b, and 9a–c have been screened towards a full panel of 59 cancer cells at NCI’s Developmental Therapeutics Program in vitro anticancer activity using sulforhodamine B (SRB) assay at 10 µM as a single dose to evaluate cell growth and viability according to the procedure of Drug Evaluation Branch,The growth percentage (G%) and cell viability of the herein target compounds Figures S1–S17  where the value recorded is the growth relative to that of both the negative control and zero-time number of cells in which both growth inhibition  and lethality  percentages can be identified. The growth inhibition means percentages (MGI%) for the examined target analogues towards the different treated 59 cancer cells were presented in Different types of tumours – colon, NSCLC, leukaemia, CNS, renal, breast, ovarian, melanoma, and prostate cancers – were investigated as a full panel of 59 cell lines, and the attained results are cited in 6k failed to demonstrate MGI%, while compounds 6a–h, 6j, 6 l, 7a,b, and 9a showed weak anticancer activity with MGI% ranging from 1.93% to 11.40%. Compounds 9b and 9c demonstrated moderate anticancer activity, with MGI% equal to 33.89% and 36.88%, respectively, while compound 6i exerted the significant mean growth inhibition percentage of 62.03% among the herein examined series. Superiorly, compound 6i emerged as the most efficient anticancer analogue eliciting high potency with broad-spectrum anticancer activity against all the herein subsets of cell panels, except against the A498  cell line (The attained results disclosed that compound ell line .6i against the herein examined 59 cell panel are presented in 6i exhibited excellent anti-proliferative activities towards Breast (MDA-MB-468 and MCF7), CNS (SF-295 and SF-268), NSCLC (NCI-H23), Ovarian (OVCAR-3 and OVCAR-8), Prostate (DU-145 and PC-3) and Renal (786-0) cancer cell lines with GI% of 92.44, 97.72, 89.78, 85.11, 87.19, 96.83, 87.86, 85.62, 91.71, and 82.93%, respectively. In addition, compound 6i displayed good inhibitory activity against Breast cancer (HS 578 T and BT-549), CNS cancer (SNB-19), Colon cancer , Leukaemia (CCRF-CEM), Melanoma , NSCLC  and Ovarian cancer (OVCAR-4) cell lines with GI% equals to 75.80, 66.64, 61.23, 70.04, 71.69, 62.17, 70.05, 79.78, 78.49, 75.54, 74.15, 63.82, 78.29, 78.12, 71.36, and 66.09, correspondingly. Furthermore, compound 6i demonstrated weak inhibitory activity against CNS cancer (SF-539 and U251), Colon cancer (HT29), Leukaemia (RPMI-8226 and MOLT-4), Melanoma (MDA-MB-435), NSCLC (NCI-H522), Ovarian cancer (NCI/ADR-RES) and Renal cancer (SN12C) cell lines with GI% of 54.23, 53.30, 59.90, 53.52, 52.51, 57.10, 52.34, 51.78, and 57.69, respectively. Moreover, the growth inhibitory percentages of 6i towards the other cell lines ranged from 9.83 to 48.82%. It is worth underlining that the lethality (L) of compound 6i was detected against some cell lines such as NSCLC (NCI-H226 and HOP-62), Renal (RXF 393), and Melanoma (SK-MEL-5) cancer cell lines with L% of 37.32, 8.41, 22.73, and 1.38%, respectively. Therefore, compound 6i exerted a remarkable anticancer profile in the One-Dose assay towards the herein tumour screening panel.The inhibitory growth percentages (GI%) of compound 6i (NSC: 838397) exhibited significant growth inhibition properties in the One-Dose (10 µM) screening assay; hence it was chosen for further evaluation at five-dose levels (0.01–100 µM) towards the herein examined tumour cell panelsFigures S53–S56 in Supporting Information). This assay evaluates GI50, TGI, and LC50 parameters, as cited in 50 values, the mean graph midpoints (MG-MID) for both full NCI 59 cell panel and subpanel cancer cell lines were evaluated, affording a median potency parameter for the examined compound 6i, as depicted in Remarkably, compound 6i displayed potent anticancer activity at a single-digit micromolar concentration towards NSCLC , CNS cancer (SF-268), Colon cancer (HCT-116), Ovarian cancer (OVCAR-8), Melanoma (LOX IMVI), Prostate cancer (DU-145 and PC-3) Renal cancer (786-0), and Breast cancer (BT-549 and MCF7) cells with GI50 spanning in the range: 3.93–9.85 µM as shown in 6i showed good to weak anti-proliferative action against the residual herein examined cell lines with GI50 spanning in the interval from 10.10 to 71.00 µM, except for Leukaemia (HL-60(TB), K-562, MOLT-4, and CCRF-CEM) and Melanoma (M14) cancer cell lines which had GI50 more than 100 µM. While compound 6i revealed good to weak cytostatic action towards 41 of the herein examined tumour cell lines with TGI spanning in the range: 16.50–89.10 µM, it had no impact (TGI more than 100 µM) towards Leukaemia (HL-60(TB), CCRF-CEM, MOLT-4, K-562, SR, and RPMI-8226), Colon , CNS (U250), Melanoma (M14 and UACC-257), Ovarian (OVCAR-8), Renal (SN12C and 786-0), and Breast (T-47D and BT-549) cancer cells .Interestingly, the target compound er cells .6i was found to be a non-lethal compound (LC50 > 100 µM) towards most of the herein examined cancer cells, except for NSCLC , Colon (COLO 205), CNS , Melanoma , Renal , Ovarian (OVCAR-4), Breast (MDA-MB-231/ATCC and MCF7) and Prostate (PC-3) cancer cell lines with LC50 interval: 47.0–96.1 µM  of the most efficient analogue 6a,c,e,g,i and 7a  would decrease their anti-proliferative effects while in compound 6k (afforded 6 l), the inhibitory activity was increased by 8.18%. Secondly, the inhibitory activity of 6a,b, 6i, and 6l indicated that introducing substitution to the C3, especially methyl group, could significantly increase the inhibitory effects against the cancer cell lines. Thirdly, increasing lipophilicity at N1 by installing a p-bromophenyl substituent would significantly increase the inhibitory activity as in 6i. Fourthly, the inhibitory activity of 7a and 7b showed that hydrolysis of carboxylate ester of benzoyl glutamate tail of 6c and 6d would not improve the inhibitory effects. In turn, they could moderately decrease the inhibitory activities against most cancer cell lines evidencing that the ester prodrug form is more potent. Fifthly, replacing the pteridine isosteric 6-5 fused system, pyrazolopyrimidine, with 1,4-dihydropyrimidine core would significantly increase the inhibitory effect in compounds 9b and 9c. Also, the inhibitory effect of 9a, 9b, and 9c indicated that installing p-bromophenyl substituent at C4 in a pyrimidine ring could significantly increase the inhibitory effects against most of the cancer cells as in 6i, demonstrating the importance of this moiety on the anti-proliferative activity. Finally, 6i established better anti-proliferative effects among the tested analogues against NCI-59 cancer cells. In brief, we could conclude that bridging of pyrazolopyrimidine core at C5 with diethyl glutamate tail through acetamide linker and installation of a more lipophilic group at N1 and a hydrophobic group at C3 along with no substitution at C6 could potentially impact the anti-proliferative activity  about these compounds could be summarised as follows. Firstly, the introduction of the methyl group at C6 of activity .6i were investigated towards PC-3 (Prostate cancer), MCF-7 (Breast cancer), OVCAR-3 (Ovarian Cancer), as well as HSF  cells, using MTT assay6i exhibited potent growth inhibitory activity, with IC50 values of 4.40 ± 0.26, 12.42 ± 0.62, and 11.52 ± 0.58 µM against MCF-7, PC-3, and OVCAR-3, correspondingly. In addition, compound 6i had about seven-fold, four-fold, and two-fold dwindled efficiency compared to MTX towards MCF-7, PC-3, and OVCAR-3 cancer cells, respectively. Furthermore, the cytotoxicity of 6a and MTX in normal fibroblast cells (HSF) was also studied, and the selectivity index (SI) was calculated. The results hinted that 6i elicited stronger cytotoxicity and higher selectivity for cancer than normal cells.The future growth-inhibiting effects of the most active derivative 6i. The assay was performed as reported elsewhere6i showed inhibitory potency of an IC50 of 2.41 µM compared to MTX (IC50 = 0.11 µM). In contrast, the TS inhibition activity was comparable to that of 5-FU .To identify the effect on the targeted enzymes, hDHFR and TS inhibition assay was performed on the most potent anticancer analogue 6i, in MCF-7 cells which were treated with 6i at 10 µM for 48 h along with an untreated DMSO control, washed with PBS, then fixed with 70% ice-cold ethanol for 12 h followed by staining with propidium iodide (PI), and analysed by flow cytometry,6i from 24.95% (DMSO control)  to assess its effect on cell apoptosis. As shown in 6i and DMSO control, respectively. The obtained results hinted that compound 6i could induce apoptotic pathways. However, the ratio of apoptosis inferred that the anti-proliferative effect of compound 6i was largely dependent on apoptosis.Again, MCF-7 cells were treated with 6i inside the active site of both hDHFR (PDB ID: 1U72) and TS (PDB ID: 1JU6),6i to validate the docking results and rationalise the predictive protein-ligand interactions within the active site,Supplementary Material), the docking pose of the co-crystallised ligand showing interactions similar to those existing in the crystal structure, and the hydrophobic interactions were roughly the same.Molecular modelling analyses were accomplished by OpenEye® Scientific software (ver. 2021.2.1) to investigate the docking mode of the most active analogue 6i (grey) showed an overlay with the cocrystallised ligand MTX (green) in the hDHFR active site6i displayed only hydrophobic interactions: alkyl, pi-pi T-shaped, and pi-alkyl. In contrast, there are three hydrogen bonds for MTX in the corresponding binding site. The NH of the acetamide bridge showed a hydrogen bond with Asp21, while the benzoyl moiety demonstrated pi-alkyl hydrophobic interactions with Pro61:A and Pro26:A residues. The benzoylglutamate tail of 6i formed two hydrogen bonds from the γ-carboxylate group with Asn64:A and Lys63:A residues, which differ from MTX′s. Also, compound 6i showed a FRED chemgauss4 score of −5.06, while MTX had a value of −13.38. The overlay of the docked pose of 6i with the crystal structure of MTX indicated that 6i established, to some extent, a similar binding mode with that of MTX, especially the formation of tortuous “L” structure, which allows tightly fitting in the active site of DHFR. This offered a plausible rationale for why 6i showed the best anti-cancer profile among the series tested, as established by NCI screening and molecular mechanisms. Furthermore, the molecular docking results infer that the hydrophobic interactions or hydrogen bonds of 6i were weaker than MTX′s. Consequently, 6i could be an inhibitor of DHFR but should be less potent than MTX. Overall, the Fred docking results proposed modifications or extensions on the N7 and benzoyl moiety as they projected towards the protein cavity.As seen in 6i (grey) with the crystal structure of PMX (green) in the human TS active site6i forms van der Waals attractions in the benzoylglutamate region. The binding pose of 6i indicates that the pyrazolopyrimidine motif and benzoylglutamate tail form a tortuous “L” structure through the acetamide bridge region and spatially stacked well with the docked pose of PMX. Furthermore, the modelling study implies that 6i could be an inhibitor of TS but should be less potent than PMX, which is aligned with the results of inhibitory enzyme assays. Overall, the Fred docking results proposed modifications or extensions on the N1, N7, benzoyl moiety, and γ-carboxylic ester group as they project towards the protein cavity.6i were calculated using ADMETlab 2.0 predictor,6i revealed two violations from Lipinski’s rule while Pfizer accepts 6i criteria as a good prodrug candidate. The ADMET prediction data of compound 6i are cited in 6i has a good synthetic accessibility score (SA score 3.14), a high GIT absorption rate since the human intestinal absorption (HIA) is 90% or more, and moderate solubility in water that helps in solubilisation in GIT fluids before absorption process as indicated by the estimated log S value (−5.07). In addition, the tested compound 6i demonstrated more than 20% bioavailability, as indicated by the F20% result, while the plasma protein binding was 91.72%. Also, it is expected to be a non-inhibitor of CYP1A2, CYP2C19, and CYP2D6 enzymes, suggesting a low probability of causing drug-drug interaction. Furthermore, carcinogenicity and acute toxicity tests showed that compound 6i is neither carcinogenic nor toxic, evidencing the good ADMET profile.Drug-likeness and pharmacokinetics ADMET properties for the most active compound 1H and 13C NMR in deuterated solvents DMSO-6d and CDCl3 using the residual peaks at δH = 2.50 ppm and δH = 7.27 ppm as an internal standard for 1H NMR spectra. In contrast, δC = 39.51 ppm and δC = 77.23 ppm were used for 13C NMR spectra, respectively. The multiplicity of signals was verified as singlet (s), doublet (d), doublet of doublets (dd), triplet (t), quartette (q), multiplet (m), or broad (br). The J-coupling constants (in Hz) were recorded. For reaction monitoring, silica gel 60 F254 pre-coated plates (Merck) were used for TLC using UV light (254 nm) for visualisation. The purification processes of the prepared analogues were accomplished by either recrystallisation from an appropriate solvent or chromatographic separation utilising silica gel for column (150–250 μm). All melting points were determined on an electro thermal melting point apparatus  and were uncorrected. Mass spectrum was carried out on Direct Inlet part to mass analyser in Thermo Scientific GC-MS model ISQ . Vario elemental analyser III  was used for analysing C, H, and N for all designed analogues. The findings agreed with the proposed structures within ±0.4% of the theoretical values. Dry solvents and chemicals were purchased from commercial suppliers and used as received. Compounds 2a–f,,3a–e,,,3g–l,,,58a–cBruker Avance III 400 MHz HD FT-high resolution-NMR spectrometer was used for recording the spectra of 2a–f (0.006 mol) in aliphatic acid (18 ml), POCl3 (1.2 ml) was added instantaneously. The mixture was refluxed for 2 h3a–l in good yields.To a suspension of 1-aryl-5-amino-4-cyanopyrazoles 3a–l (0.001 mol) in DMF (2 ml), diethyl (4-(2-chloroacetamido)benzoyl)glutamate 5 , anhydrous K2CO3 , and sodium iodide  were added. The reaction was heated at 65° for 1–2 h as monitored by TLC6a–l.To a solution of pyrazolopyrimidinone derivatives 6c,d (0.002 mol) in 5 ml aqueous sodium hydroxide (1 mol/l) was stirred at 25 °C for 2 h (TLC monitoring)7a,b.A suspension of 8a–c (0.001 mol) in DMF (2 ml), diethyl (4-(2-chloroacetamido)benzoyl)glutamate 5 , anhydrous potassium carbonate , and sodium iodide  were added. The reaction mixture was stirred at room temperature for 8 h, as indicated by TLC9a–c.To a stirred solution of 2-thioxo-1,4-dihydropyrimidine intermediates 3f) and target compounds  were fully characterised in the Supplementary Material.The key intermediate (6i was screened further at 0.01–100 µM (Five-Dose Screen). The screening procedures were performed according to NCI-Drug Evaluation Branch protocol using SRB protein assay, as described previously,The designed analogues were submitted to NCI-Chemotherapeutic Agents Repository  for screening on a panel of 59 cancer cell lines. All compounds were initially examined at 10 µM concentration (Single-Dose Screen), and then the most efficient analogue 6 cells/cm2; 100 µL/well) and incubated for 12 h at 37 °C and CO2 (5%), then serial dilutions of compound 6i were added and incubated for 48 h using DMSO control (0.5% V/V). MTT -2,5-diphenyl-tetrazolium bromide; Cat. No. M-5655) 5 mg/mL was then added. After 4 h incubation, the formed MTT formazan crystals were solubilised by 10% Triton X-100 plus 0.1 N HCl in anhydrous isopropanol. The absorbance was read using a Bioline plate reader at λ570–630 nm6i towards HSF cells was also determined as follows,The MTT assay was carried out according to the procedure cited earlier,2 (5%). After 12 h incubation with serum-free medium, they were treated with 10 μM of 6i, incubated for 48 h, then washed twice with 1× PBS, trypsinised and centrifuged for 5 min (500 xg) at 4 °C. And then the cell pellet, in ice-cold 1× PBS, was fixed with absolute ethanol at −20 °C for 2 h, washed with 1× PBS ice-cold solution and finally stained with propidium iodide (PI)/RNase  for 0.5 h at 25 °C in the dark. flow cytometry determined the DNA content in cell phases using BD FACS Calibur,The cell cycle assay was carried out according to the previously reported6 cells/ml) were treated with either 10 µM of 6i or DMSO (NC) for 24 h. After that, the cells were trypsinised, collected, washed, and stained with PE-Annexin-V  in the presence of PIThe MCF-7 cells (106i and enzyme control. An assay buffer (100 µl) was used as a Background Control. Then, 40 μL of diluted NADPH in assay buffer (40-fold dilution) was added and mixed well. At 25 °C in the dark, the plate was incubated for 15 min. After that, the diluted substrate in assay buffer at 15-fold dilution (60 μL) was added to assigned wells. Instantaneously, the absorbance was read at 340 nm for 10–20 min at 25 °C in kinetic mode by Robonik ELISA plate reader. Finally, the activity and relative inhibition percentages were measured using the equations indicated in the assay kitDHFR inhibition assay kit  supplied with recombinant hDHFR was utilised in this assay. The assay was performed as instructed in assay kit). Briefly, in a standard black well plate (Matrix 4318), water (12 µl), 10 × Buffer (3 µl), 10 × dTMP (3 µl), 10 × ATP (3 µl) and 10 × kinase (3 µl) were mixed well. After 2 min, 10 × MUK Reagent A (3 µl) and 10 × MUK Reagent B (3 µl). The mixture was then incubated at 37 °C for 1 h, and 1 × fluorescence dye (30 µl) was added. Finally, the fluorescence intensity was read at 535 nm with excitation at 485 nm utilising a Robonik ELISA plate reader.The assay was performed as instructed in the assay kit  was used for molecular docking studies6a–l, 7a,b, and 9a–c were designed and synthesised as potential antifolate agents. The NCI-59 Screening assay displayed that compounds 6i and 9a–c showed the best MGI% among the series investigated. At the NCI five-dose assay, compound 6i exhibited potent anti-proliferative activity against the herein tumour screening panel with GI50 spanning in the range: 3.93–9.85 µM. Compound 6i revealed good to weak cytostatic activity with TGI spanning in the range: 16.50–89.10 µM, and it was found to be a non-lethal compound (LC50 > 100 µM) towards most of the herein examined cancer cell lines. Moreover, compound 6i elicited broad-spectrum anticancer activity with effective subpanel MG-MID interval: 6.63–68.03 µM and promising full panel MG-MID of 20.45 µM with moderate selectivity towards Prostate cancer cell lines. The target analogue 6i, as the most potent derivative, demonstrated high selectivity towards cancer cell lines with IC50 values equal to 4.40, 12.42, and 11.50 against MCF-7, PC-3, and OVCAR-3 cells, respectively. Furthermore, compound 6i showed potent hDHFR inhibitory activity with an IC50 equal to 2.41 µM compared to MTX (IC50 = 0.11 µM), while the inhibitory activity towards TS was 8.88 µM compared to 5-FU (IC50 = 3.6 µM). The target compound 6i probably acts through dual inhibition of DHFR and TS enzymes. Compound 6i could significantly arrest the cells at the S-phase of the cell cycle and potentially induce apoptosis. The in silico molecular docking studies implied that compound 6i had an overlay with the crystal structure of MTX and PMX in their active sites of DHFR and TS, respectively, forming a tortuous “L” structure through the acetamide bridge region. The drug-likeness and ADMET analysis revealed that 6i met the Pfizer acceptance criteria. These results thoroughly delineate 6i as a good lead compound and multi-targeted antifolate agent for further study.In this research, a novel series of multifunctional pyrazolopyrimidine and 1,4-dihydropyrimidine Glu-based derivatives Click here for additional data file.


Nuclear magnetic resonance studies show a Pro–VPg complex interaction and VPg tertiary structure; however, information regarding structural changes of the Pro–VPg complex during interaction is lacking. Here, we solved a full Pro–VPg 3D structure of ryegrass mottle virus (RGMoV) that demonstrates the structural changes in three different conformations due to VPg interaction with Pro. We identified a unique site of VPg interaction with Pro that was not observed in other sobemoviruses, and observed different conformations of the Pro β2 barrel. This is the first report of a full plant Pro crystal structure with its VPg cofactor. We also confirmed the existence of an unusual previously unmapped cleavage site for sobemovirus Pro in the transmembrane domain: E/A. We demonstrated that RGMoV Pro in cis activity is not regulated by VPg and that in trans, VPg can also mediate Pro in free form. Additionally, we observed Ca2+ and Zn2+ inhibitory effects on the Pro cleavage activity.Sobemoviruses encode serine-like 3C proteases (Pro) that participate in the processing and maturation of other virus-encoded proteins. Its  Solemoviridae, genus Sobemovirus, and infects monocotyledons, such as Italian ryegrass (Lolium multiflorum), cocksfoot (Dactylis glomerata), wheat (Triticum aestivum), barley (Hordeum vulgare), oats (Avena sativa), rye , and Italian millet   rather than viral Pro [32–35)[D/E](X61–62)TXXGXSG  or G-250  staining.WB was performed on a semi-dry Western blot device transferred to an Amersham Protran 0.45 mm nitrocellulose (NC) blotting membrane  at 52 mA for 45 min. For a protein with 6xH-tag detection on the NC membrane, the H-Tag Antibody HRP Conjugate Kit  was used according to the manufacturer’s protocol.Samples for mass spectrometry analysis were prepared as follows: 2 µL of purified protein (1 mg/mL) was mixed with 2 µL of 2% trifluoroacetic acid and 2 µL 2,5-dihydroxyacetophenone  matrix solution , and 1 µL was spotted onto an MTP Anchor Chip 400/384TF  and left to crystallize. The sample analysis was performed on an “Autoflex” MALDI-TOF MS . A mix of mass calibration standards I and II or peptide molecular mass calibration standard II  was used for mass calibration.cm (13.7 µM) in a 100 µL reaction volume. These results were confirmed by a time-course experiment in which samples were withdrawn from the reaction mixture at different time points  at 37 °C, and immediately stopped with 4xLaemmli sample buffer  followed by sample heating at 95 °C for 10 min. Samples were analyzed in 15 well 1.0 mm Bolt 4–12% Bis-Tris Plus mini protein gels  and stained with Coomassie blue G-250.For the in vitro cleavage experiments, all proteins were used in equal micromolar amounts (1:1:1). The micromolar concentrations were calculated according to Δ50Procm were transferred to 20 mM Tris-HCl (pH 8.0) and concentrated to 9.38 mg/mL, 2.9 mg/mL, and 18.1 mg/mL concentrations, respectively. All Pro variants were crystallized using the sitting drop method in 0.4 + 0.4 μL or 1 + 1 μL setups. The initial screening was performed using JCSG+, Structure screens 1 and 2, and Pact Premier screens . Δ117Pro crystals grew within 2–3 weeks, and the final crystallization conditions were 0.1 M HEPES (pH 7.5), 22.5% m/v PEG3350, 0.22 M LiSO4 and 3% DMSO. Δ117Procm crystals grew within one week, and the final crystallization conditions were 0.18 M ammonium sulphate, 22% PEG8000, and 0.1 M sodium cacodylate (pH 6.5). Δ117Pro-E/A-VPg crystals grew within 3–4 months in 0.1 M MIB, pH 9.0, and 25% PEG1500 conditions. Crystals were soaked in a cryoprotectant (30% glycerol in mother liquid) and frozen in liquid nitrogen. Diffraction data were collected at the MX 14.1 beamline in the BESSY II synchrotron electron storage ring operated by the Helmholtz-Zentrum Berlin . Δ117Pr12121 with one chain in the asymmetric unit. The Δ117Procm crystals had a space group C2 with one chain in the asymmetric unit.The data were indexed with MOSFLM  and scalcm structures were determined in MOLREP using coordinates from the determined Δ117Pro-E/A-VPg structure. The models were further built manually with Coot [cm).The Δ117Pro-E/A-VPg structure was determined by molecular replacement using MOLREP , using aith Coot , and refith Coot  and phenith Coot . Data pr


The effects of these mutations on viral infection and transmission and the efficacy of vaccines and therapies remains poorly understood. Here we demonstrate that recently emerged BQ.1.1 and XBB.1.5 variants bind host ACE2 with high affinity and promote membrane fusion more efficiently than earlier Omicron variants. Structures of the BQ.1.1, XBB.1 and BN.1 RBDs bound to the fragment antigen-binding region of the S309 antibody (the parent antibody for sotrovimab) and human ACE2 explain the preservation of antibody binding through conformational selection, altered ACE2 recognition and immune evasion. We show that sotrovimab binds avidly to all Omicron variants, promotes Fc-dependent effector functions and protects mice challenged with BQ.1.1 and hamsters challenged with XBB.1.5. Vaccine-elicited human plasma antibodies cross-react with and trigger effector functions against current Omicron variants, despite a reduced neutralizing activity, suggesting a mechanism of protection against disease, exemplified by S309. Cross-reactive RBD-directed human memory B cells remained dominant even after two exposures to Omicron spikes, underscoring the role of persistent immune imprinting.Currently circulating SARS-CoV-2 variants have acquired convergent mutations at hot spots in the receptor-binding domain Convergent mutations in hot spots of the spike proteins of currently circulating SARS-CoV-2 Omicron variants increase the binding affinity for the host receptor and promote more efficient fusion with host cell membranes. As a result, repeated waves of infections driven by successive lineages  occurred globally, including in individuals who had received multiple COVID-19 vaccine doses.The emergence of the SARS-CoV-2 Omicron (B.1.1.529) variant at the end of 2021 marked a new phase of the COVID-19 pandemic10. Owing to convergent evolution, currently circulating Omicron variant lineages independently acquired identical or similar amino acid mutations at key antigenic sites in the RBD and in the N-terminal domain (NTD), relative to their presumed BA.2 and BA.5 ancestors1. The BA.2.75.2 lineage increased in frequency in multiple countries (such as India) and has the RBD mutations D339H, R346T, G446S, N460K, F486S and R493Q relative to BA.2  and addition of the G252V mutation in S yielded XBB.1, which has D339H, R346T, L368I, V445P, G446S, N460K, F486S, F490S and R493Q RBD substitutions relative to BA.2  = 12.8 nM and 13.7 nM, respectively), indicating that the additional BQ.1.1 mutations, which map outside of the ACE2-binding interface, do not influence receptor engagement  relative to BA.2, results from the R493Q reversion, as G446S has a negligible effect and F486S has a deleterious effect on ACE2 engagement12. ACE2 bound to the XBB.1 RBD with an affinity similar to that of the Wu RBD  whereas it bound more tightly to the XBB.1.5 RBD (Kd = 26.8 nM), owing to substitution of a serine for a proline at S residue 486 enhancing receptor engagement13. We observed a similar ranking of these variant RBDs using surface plasmon resonance (SPR) to determine ACE2 binding affinities  Fig. . We meas3. We observed slower and reduced fusogenicity for the BA.5, BA.2 and BA.1 S glycoproteins compared with Wu-G614 and even more so relative to Delta S15  pseudotyped with S glycoproteins into Vero E6-TMPRSS2 cells  and HEK293T-ACE2 cells . The serine protease (TMPRSS2) inhibitors camostat and nafamostat potently blocked entry of Wu-G614 and Delta S VSV in Vero E6-TMPRSS2 cells, but had a limited effect on the Omicron variants  route as opposed to the plasma membrane (TMPRSS2-mediated) routents Fig. . Recipronts Fig. . Furthernts Fig. . This in12, possibly owing to the removal of the positively charged arginine side chain restoring a network of local interactions similar to that made with the Wu RBD21  probably reduces binding to LY-CoV1404, owing to a loss of van der Waals interactions  structures for each RBD bound to the human ACE2 ectodomain and to the fragment antigen-binding (Fab) region of the S309 antibody  and CDRH3 Fig. . The S30RBD Fig. . The S37res Fig. . Residueres Fig. . These f29. The binding affinity of S309 against Omicron variant RBDs decreased up to around 160-fold, primarily owing to faster dissociation rates compared with the Wu RBD. For the BN.1 RBD, however, the S309 Fab exhibited a decrease of around 100-fold in the on-rate compared with that of the Wu RBD, resulting in an approximately 370-fold decrease in affinity  E108 (as resolved in PDB entry 7TN05), as (1) the BN.1 RBD harbouring the T356K reversion bound S309 similarly to the BA.2.75.2 RBD ; and (2) sotrovimab did not neutralize BA.2 S(K356T) VSV pseudovirus  salt bridge is the main mechanism of dampened binding affinity to and neutralization of BN.1 and that the newly introduced N354 glycan has a minimal effect on S309.On the basis of the cryo-EM visualization of S309 binding to the BQ.1.1 and XBB.1 RBDs and the fact that sotrovimab remains the only therapeutic antibody with in vitro neutralizing activity against currently circulating variants, we investigated the binding kinetics and affinity of the S309 Fab to the immobilized Wu, Delta, BA.1, BA.2, BA.2.75.2, BQ.1, BQ.1.1, BN.1, XBB.1, XBB.1.5, CH.1.1 and Wu-E340A RBDs using SPR  D93. Nevertheless, we found that the sotrovimab IgG cross-reacted with full-length, cell-surface-expressed BQ.1.1, XBB.1 and XBB.1.5 S trimers to a similar or greater degree than those observed for BA.2 S, and to a lesser extent with BN.1 S  escape mutant or with the S309(G236R/L328R) (hereafter S309-GRLR) Fc mutant, which cannot engage human Fcγ receptors31  using primary natural killer effector cells and antibody-dependent cellular phagocytosis (ADCP) with ExpiCHO-S target cells expressing SARS-CoV-2 S of the different Omicron variants at their surface. Sotrovimab efficiently promoted ADCC and ADCP of cells expressing Wu-D614, BA.2, BQ.1.1, XBB.1, XBB.1.5 and BN.1 S in a concentration- and Fc-dependent manner, but did not do so with the BA.2-E340A S  1 day before challenge with BQ.1.1  to Syrian hamsters challenged with XBB.1.5 reduced weight loss and viral burden  at doses of 3, 10 or 30 mg kg1.1 Fig. . S309 ad1.1 Fig. , consistses Fig. . Moreoveden Fig. , with a 4 vaccinated); (ii) vaccinated 3 times with Wu monovalent S mRNA vaccine and then 1 time with Wu/BA.5 bivalent S mRNA vaccine, with no known infection ; (iii) infected in 2020 and subsequently vaccinated 3 to 4 times with Wu monovalent S mRNA vaccine and then 1 time with Wu/BA.5 bivalent S mRNA vaccine ; and (iv) vaccinated with Wu monovalent S mRNA vaccine before experiencing a breakthrough infection with Omicron BA.1, BA.2, BA.2.12.1 or BA.5, followed by a vaccination with the Wu/BA.5 bivalent S mRNA vaccine . We also studied 4 additional cohorts from Switzerland, where a Wu/BA.1 bivalent S mRNA booster was available: (v) vaccinated 3 times with Wu monovalent S mRNA vaccine, with no known infection (Wu3 mono); (vi) vaccinated 3 times with Wu monovalent S mRNA vaccine after pre-Omicron infection (pre-Omicron–Wu3 mono); (vii) vaccinated 3 times with Wu monovalent S mRNA vaccine and then 1 time with Wu/BA.1 bivalent S mRNA vaccine, with no known infection (Wu/BA.1 biv); and (viii) vaccinated 3 times with Wu monovalent S mRNA vaccine and then 1 time with Wu/BA.1 bivalent S mRNA vaccine, with a BA.1 or a BA.2 breakthrough infection (Omicron BT–Wu/BA.1 biv).Vaccination represents a main line of defence against SARS-CoV-2, and recent mRNA vaccine updates have led to the administration of bivalent formulations. To assess the effects of the constellation of S mutations in the currently dominant variants on vaccine-elicited antibody responses, we quantified plasma neutralizing activity using VSV pseudotyped with Wu-G614, BA.1, BA.5, BQ.1.1, XBB.1, XBB.1.5 or BA.2.75.2 S. We compared plasma from individuals obtained 15–30 days after vaccination or PCR-confirmed breakthrough infection in 8 cohorts: (i) vaccinated 4 times with the Wu monovalent S mRNA vaccine, with no known infection  who received the Janssen adenovirus-vectored (Ad26.COV2.S) COVID-19 vaccine rather than the Moderna (mRNA-1273) or Pfizer (BNT162b2) mRNA COVID-19 vaccines as their primary vaccine series), pre-Omicron infected–Wu/BA.5 bivalent vaccinated, and Omicron BT–Wu/BA.5 bivalent vaccinated subjects (cohorts i–iv) by measuring the frequency of Wu RBD-binding, pooled Omicron  RBD-binding, and both Wu and pooled Omicron  RBD-binding cross-reactive MBCs by flow cytometry. Individuals who were exposed to Wu S only (Wu4 vaccinated) exhibited the highest frequency of Wu RBD-binding MBCs (mean frequency: 25.8%) and the lowest frequency of cross-reactive MBCs (mean frequency: 71.1%) of the four cohorts , they had few Omicron-specific  MBCs (mean: 5.6%), similar to individuals who received only the bivalent booster. MBCs cross-reactive with the Wu RBD and the Omicron  RBD pool were further enriched (mean frequency: 81.1%) in this cohort compared with the cohort vaccinated with Wu/BA.5 bivalent vaccine without Omicron BT infection  populations in the Wu40.We then assessed whether MBCs recognizing the Omicron  RBD pool could bind the BQ.1.1 RBD Fig. . Most MBWe next determined whether the bivalent boosters formulated against BA.5 or BA.1 differentially affected the composition of the RBD-binding MBC population Fig. . We asseThus, two exposures to Omicron S were not sufficient to overcome the immune imprinting induced by repeated exposures to Wu S, but instead mostly enriched for MBCs cross-reacting with multiple RBD variants. These results concur with the broader plasma nAb responses that we observed upon bivalent mRNA vaccination compared with monovalent Wu mRNA vaccination.30. Our findings illustrate the interplay of immune evasion, fusogenicity and ACE2 binding affinity driving SARS-CoV-2 evolution.Here we report that recently emerged SARS-CoV-2 Omicron variants show unprecedented immune evasion, reducing nAb titres up to ~150-fold for XBB.1 and XBB.1.5. BQ.1.1, XBB.1.5 and BA.2.75.2 retain high ACE2 binding affinity, similar to earlier Omicron variants, whereas XBB.1 has a lower affinity, similar to that of the Wu RBD. Although XBB.1 and XBB.1.5 are the most immune-evasive of these Omicron variants, the reduced affinity of XBB.1 for ACE2 relative to other co-circulating strains may have hindered its spread. The enhanced ACE2 binding affinity of the more recently emerged XBB.1.5 variant, which harbours the S486P RBD mutation (relative to XBB.1), may explain the current rapid spread of this variant41. Sotrovimab retains in vitro effector functions against BA.2 and conferred Fc-dependent protection in the lungs of mice infected with BA.242, in line with the low rate of hospitalization and death during the BA.2 and BA.5 waves in patients treated with sotrovimab45. Here we show that sotrovimab triggered in vitro effector functions against all Omicron variants assessed at levels similar to that observed with Wu. Prophylactic administration of S309 (the sotrovimab parent antibody) protected mice against BQ.1.1 challenge with a contribution of effector functions, and protected hamsters against XBB.1.5 challenge, despite a reduced in vitro neutralizing activity against these variants. Our observation that vaccine-elicited polyclonal plasma antibodies cross-reacted and promoted ADCC upon recognition of the BA.1, BA.5, BQ.1.1 and XBB.1 S glycoproteins concur with observations made with previous Omicron variants47 and further hint at a protective role for broadly reactive antibodies with effector functions. Our findings suggest that the erosion of nAb titres is associated with an increased frequency of breakthrough infections, and the persistence of cross-reactive antibodies mediating effector functions may contribute to protection against severe COVID-19.ADCC and ADCP are Fc-mediated effector functions that can promote virus clearance and enhance adaptive immune responses in vivo, independently of direct viral neutralization. Indeed, both neutralizing and binding antibody titres were reported as correlates of protection in a phase 3 clinical study49, whereas plasma cells obtained after the subsequent antigenic exposure (through vaccination) were subtype-specific . Similarly, Omicron breakthrough infections of Wu-vaccinated subjects primarily recall cross-reactive MBCs specific for epitopes shared by multiple SARS-CoV-2 variants rather than priming naive B cells that recognize Omicron RBD-specific epitopes34. We observed an unexpectedly small number of MBCs specific for Omicron RBDs (and not cross-reacting with the Wu RBD) even after two exposures to Omicron S antigens, including after Wu/BA.5 or Wu/BA.1 bivalent mRNA vaccination. This may be owing to strong immune imprinting resulting from repeated Wu-like S exposures, and possible antigenic dominance of the Wu S antigen in bivalent vaccines50. However, relative to monovalent Wu mRNA vaccination, bivalent Wu/BA.5 mRNA vaccination results in enrichment for MBCs that are cross-reactive with vaccine-matched and mismatched RBD variants.Immune imprinting—also known as ‘original antigenic sin’—describes how the first exposure to a virus shapes the immunological outcome of subsequent exposures to antigenically related strains. For instance, antibodies secreted by plasmablasts obtained one to two weeks after infection with the antigenically shifted H1N1 influenza virus (formerly known as swine flu) that caused the 2009 flu pandemic were recalled from pre-existing, cross-reactive MBCs52, or generated in-house 53. None of the cell lines used were authenticated or tested for mycoplasma contamination. SARS-CoV-2 isolates used in this study were obtained through BEI Resources, NIAID, NIH: . Viruses were propagated and titrated on Vero E6-TMPRSS2 cells in house. The genomic sequences of all strains were confirmed by Sanger and/or next generation sequencing.Cell lines used in this study were obtained from ATCC (HEK293T and Vero E6), Thermo Fisher Scientific , Takara (Lenti-X 293T cells), a gift from J. Bloom (HEK293T-ACE2)Samples from cohorts v–viii along with those from patients undergoing dialysis (DP) kidney transplant recipients (KTR) and healthcare workers (HCW) were obtained from SARS-CoV-2 convalescent and vaccinated individuals under study protocols approved by the local institutional review boards . PBMCs for effector function experiments were collected from healthy human donors under the informed consent and authorization of the Comitato Etico of Canton Ticino (Switzerland). All donors provided written informed consent for the use of blood and blood derivatives  for research. Sera and PBMCs from cohorts i–iv were obtained from the HAARVI study approved by the University of Washington Human Subjects Division Institutional Review Board (STUDY00000959). Demographic data for these individuals is presented in Supplementary Tables 54. The full-length BA.2.75.2 and XBB.1 S constructs containing a 21-amino-acid C-terminal deletion were codon optimized, synthesized, and inserted the HDM vector by Genscript. The full-length BQ.1.1 S construct containing a 21-amino acid C-terminal deletion was generated by mutagenesis of the BA.4/5 S construct and the full-length XBB.1.5 containing a 21-amino-acid C-terminal deletion was generated by mutagenesis of the XBB.1 S construct by Genscript.The full-length Wu/G614, Delta, BA.1, BA.2, and BA.4/5 S constructs with a 21-amino-acid C-terminal deletion used for pseudovirus assays were previously described elsewhereS expression plasmids used for the generation of VSV pseudoviruses harbour the following mutations. BA.1: A67V, Δ69-70, T95I, G142D, Δ143–145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F; BA.2: T19I, L24-, P25-, P26-, A27S, G142D, V213G, G339D, S371L, S373P, S375F, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K; K417N, N440K, G446S, N460K, S477N, T478K, E484A, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K; BA.2.75.2: T19I, L24-, P25-, P26-, A27S, G142D, K147E, W152R, F157L, I210V, V213G, G257S, G339H, R346T, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, G446S, N460K, S477N, T478K, E484A, F486S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, D1199N; BQ.1: T19I, L24-, P25-, P26-, A27S, Δ69-70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, K444T, L452R, N460K, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K; BQ.1.1: T19I, L24-, P25-, P26-, A27S, Δ69-70, G142D, V213G, G339D, R436T, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, K444T, L452R, N460K, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K; BF.7: T19I, L24-, P25-, P26-, A27S, Δ69-70, G142D, V213G, G339D, R436T, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K; XBB.1: T19I, L24-, P25-, P26-, A27S, V83A, G142D, Y144-, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486S, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K; XBB.1.5: T19I, L24-, P25-, P26-, A27S, V83A, G142D, Y144-, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K; CH.1.1: T19I, del24–26, A27S, G142D, K147E, W152R, F157L, I210V, V213G, G257S, G339H, R346T, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, K444T, G446S, L452R, N460K, S477N, T478K, E484A, F486S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K; BN.1: T19I, del24–26, A27S, G142D, K147E, W152R, F157L, I210V, V213G, G257S, G339H, R346T, K356T, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, G446S, N460K, S477N, T478K, E484A, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K.55. The BA.4/5 RBD construct containing an N-terminal BM40 secretion tag and a C-terminal octa-histidine tag followed by flexible linker and Avi tag was previously described elsewhere3. The BA.2.75.2, BQ.1.1, XBB.1, and XBB.1.5 RBD constructs containing an N-terminal BM40 secretion tag and a C-terminal octa-histidine tag followed by flexible linker and Avi tag were codon optimized, synthesized, and inserted into the pcDNA3.1(+) vector by Genscript. The boundaries of the construct are N-328RFPN331 and 528KKST531-C. The monomeric human ACE2 ectodomain (residues 19–615) construct used for BLI contains an N-terminal signal peptide and a 10x His tag and was synthesized and inserted into pTwist-CMV by Twist Bioscience.For BLI and cryo-EM, the SARS-CoV-2 Wu RBD construct containing an N-terminal mu-phosphatase secretion signal and a C-terminal octa-histidine tag followed by flexible linker and Avi tag was previously described elsewhereNC_045512.2 with an N-terminal signal peptide and a C-terminal 8×His–Avi Tag or thrombin cleavage site–8×His–Avi tag. The ACE2 construct used for SPR and cryo-EM, encodes for residues 19–615 from Uniprot entry Q9BYF1 with a C-terminal thrombin cleavage site–TwinStrep–10×His–GGG tag, and N-terminal signal peptide.For SPR, SARS-CoV-2 RBD plasmids encoding residues 328–531 of the S protein from GenBank 4 with some modifications to evaluate cohorts v–viii, DP, KTP and HCW, and monoclonal antibodies. Lenti-X 293T cells (Takara) were seeded in 15-cm2 dishes at a density of 10 × 106 cells per dish and the following day were transfected with 25 µg of S expression plasmid with TransIT-Lenti  according to the manufacturer’s instructions. One day after transfection, cells were infected with VSV-luc (VSV-G) with a multiplicity of infection (MOI) of 3 for 1 h, rinsed three times with PBS containing Ca2+ and Mg2+, then incubated for an additional 24 h in complete medium at 37 °C. The cell supernatant was clarified by centrifugation, aliquoted, and frozen at −80 °C.Replication-defective VSV pseudovirus expressing SARS-CoV-2 Wu and variant S were generated as previously described3 to evaluate cohorts i–iv. In brief, HEK293T cells were split into poly-d-lysine-coated 15-cm plates and grown overnight until they reached approximately 70–80% confluency. The cells were washed 3 times with Opti-MEM (Gibco) and transfected with either the Wu-G614, Delta, BA.1, BA.2, BA.4/5, BA.2.75.2, BQ.1.1, XBB.1, or XBB.1.5 S constructs using Lipofectamine 2000 (Life Technologies). After 4–6 h, the medium was supplemented with an equal volume of DMEM supplemented with 20% FBS and 2% penicillin-streptomycin. The cells were incubated for 20–24 h, washed 3 times with DMEM, and infected with VSVΔG-luc. Two hours after VSVΔG-luc infection, the cells were washed an additional five times with DMEM. The cells were grown in DMEM supplemented with anti-VSV-G antibody  for 18–24 h, after which the supernatant was harvested and clarified by low-speed centrifugation at 2,500g for 10 min. The supernatant was then filtered (0.45 μm) and some virus stocks were concentrated 10 times using a 30-kDa centrifugal concentrator (Amicon Ultra). The pseudotyped viruses were then aliquoted and frozen at −80 °C.Pseudotyped VSV was produced as previously described2+ and Mg2+ was added to cells. Plates were incubated for 15 min at room temperature and then analysed on the Synergy-H1 (BioTek). The average relative light units (RLU) of untreated infected wells (MAX RLUave) were subtracted by the average of MIN RLU (MIN RLUave) and used to normalize percentage of neutralization of individual RLU values of experimental data according to the following formula: (1 − (RLUx – MIN RLUave)/(MAX RLUave – MIN RLUave)) × 100. Data were analysed with Microsoft Excel (v16) and Prism (v.9.1.0). IC50 values were calculated from the interpolated value from the log(inhibitor) versus response, using variable slope (four parameters) non-linear regression with an upper constraint of <100. Each neutralization experiment was conducted on at least two independent experiments—that is, biological replicates—in which each biological replicate contains a technical duplicate or triplicate.For cohorts v–viii, DP, KTP and HCW, and monoclonal antibodies, Vero E6 cells were grown in DMEM supplemented with 10% FBS and seeded into white-walled 96-well plates  at a density of 20,000 cells per well. The next day, monoclonal antibodies were serially diluted in pre-warmed complete medium, mixed with pseudoviruses and incubated for 1 h at 37 °C in round bottom polypropylene plates. Medium from cells was aspirated and 50 µl of pseudovirus–monoclonal antibody complexes were added to cells, which were then incubated for 1 h at 37 °C. An additional 100 µl of pre-warmed complete medium was then added on top of complexes and cells were incubated for an additional 16–24 h. Conditions were tested in duplicate or triplicate wells on each plate and 6–8 wells per plate contained untreated infected cells  value) and uninfected cells  value). Virus–monoclonal antibody-containing medium was then aspirated from cells and 50 or 100 µl of a 1:2 dilution of SteadyLite Plus (PerkinElmer) or Bio-Glo (Promega) in PBS with Ca50 were determined from the normalized data points using a [inhibitor] versus normalized response–variable slope model using at least two technical repeats to generate the curve fits. At least two biological replicates with two distinct batches of pseudovirus were conducted for each sample.For cohorts i–iv, Vero E6-TMPRSS2 were split into white-walled, clear-bottom 96-well plates (Corning) and grown overnight until they reached approximately 70% confluency. Plasma was diluted in DMEM starting at a 1:10 dilution and serially diluted in DMEM at a 1:3 dilution thereafter. Pseudotyped VSV was diluted at a 1:25 to 1:100 ratio in DMEM and an equal volume was added to the diluted plasma. The virus–plasma mixture was incubated for 30 min at room temperature and added to the Vero E6-TMPRSS2 cells. After two hours, an equal volume of DMEM supplemented with 20% FBS and 2% penicillin-streptomycin was added to the cells. After 20–24 h, ONE-Glo EX (Promega) was added to each well and the cells were incubated for 5 min at 37 °C. Luminescence values were measured using a BioTek Synergy Neo2 plate reader. Luminescence readings from the neutralization assays were normalized and analysed using GraphPad Prism 9.1.0. The RLU values recorded from uninfected cells were used to define 100% neutralization and RLU values recorded from cells infected with pseudovirus without plasma were used to define 0% neutralization. IDVero E6-TMPRSS2 cells were seeded into black-walled, clear-bottom 96-well plates at 20,000 cells per well and cultured overnight at 37 °C. The next day, 9-point fourfold serial dilutions of monoclonal antibodies were prepared in growth medium (DMEM + 10% FBS). The different SARS-CoV-2 strains were diluted in infection medium (DMEM + 2% BSA) at a final MOI of 0.01 plaque-forming units per cell, added to the monoclonal antibody dilutions and incubated for 30 min at 37 °C. Medium was removed from the cells, monoclonal antibody–virus complexes were added and incubated at 37 °C for 18 h (WA-1 and XBB.1.5) or 24 h (BA.2). Cells were fixed with 4% PFA , permeabilized with Triton X-100  and stained with an antibody against the viral nucleocapsid protein  followed by a staining with the nuclear dye Hoechst 33342  and a goat anti-rabbit Alexa Fluor 647 antibody . Plates were imaged on a Cytation5 plate reader. Whole well images were acquired (12 images at 4× magnification per well) and nucleocapsid-positive cells were counted using the manufacturer’s software.Vero E6-TMPRSS2 or HEK293T-ACE2 were split into white-walled, clear-bottom 96-well plates (Corning) at a density of 18,000 or 36,000 cells, respectively, and grown overnight. The following day, the growth medium was removed and, for assays conducted with Vero E6-TMPRSS2, the cells washed once with DMEM. The cells were incubated for 2 h with DMEM containing 50 µM of Camostat (Sigma), Nafamostat (Sigma), E64d (Sigma), or 0.5% DMSO. All three protease inhibitors were dissolved in DMSO to a concentration of 10 mM and diluted in DMEM. The protease inhibitors were removed and pseudovirus diluted 1:50 or 1:200 in DMEM was added to the cells. After 2 h, an equal volume of DMEM supplemented with 20% FBS and 2% penicillin-streptomycin was added to the cells. After 20–24 h, ONE-Glo EX (Promega) was added to each well and the cells were incubated for 5 min at 37 °C. Luminescence values were measured using a BioTek Synergy Neo2 plate reader. Luminescence readings from the neutralization assays were normalized and analysed using GraphPad Prism 9.1.0. The RLU values recorded from uninfected cells were used to define 0% infectivity and RLU values recorded from cells incubated with 0.5% DMSO only and infected with pseudovirus were used to define 100% infectivity. Twelve technical replicates were performed for each inhibitor and pseudovirus and at least two biological replicates with two distinct batches of pseudovirus were conducted.3. In brief, cells were grown to a density of 3 × 106 cells per ml and transfected using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific). Three to five days post-transfection, proteins were purified from clarified supernatants using HisTrap HP affinity columns (Cytiva) and washed with ten column volumes of 20 mM imidazole, 25 mM sodium phosphate pH 8.0, and 300 mM NaCl before elution on a gradient to 500 mM imidazole, 25 mM sodium phosphate pH 8.0, and 300 mM NaCl. Proteins were buffer exchanged into 20 mM sodium phosphate pH 8 and 100 mM NaCl and concentrated using centrifugal filters (Amicon Ultra) before being flash frozen.SARS-CoV-2 RBDs and human ACE2 were produced and purified from Expi293F cells as previously described2 with kifunensine added to 10 µM. Cell culture supernatant was collected eight days post-transfection, supplemented with buffer to a final concentration of 80 mM Tris-HCl pH 8.0, 100 mM NaCl, and then incubated with BioLock (IBA) solution. ACE2 was purified using a 5-ml StrepTrap HP column (Cytiva) followed by isolation of the monomeric ACE2 by size-exclusion chromatography using a Superdex 200 Increase 10/300 GL column (Cytiva) pre-equilibrated in PBS.For cryo-EM, recombinant was expressed in ExpiCHO-S cells at 37 °C and 8% CORecombinant S309 Fab used for cryo-EM was expressed by ATUM Bio using HEK293-derived suspension cells (lacking the N55Q mutation introduced for improving its manufacturability), purified using CaptureSelect IgG-CH1 resin and buffer exchanged into PBS (ATUM Bio).2. Transfections were performed using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific). Cell culture supernatants were collected 4–5 days after transfection and supplemented with 10× PBS to a final concentration of 2.5× PBS . SARS-CoV-2 RBDs were purified by IMAC using Cobalt or Nickel resin followed by buffer exchange into PBS using Amicon centrifugal filters (Milipore Sigma) or by size-exclusion chromatography using a Superdex 200 Increase 10/300 GL column (Cytiva). For SPR binding measurements, recombinant ACE2  was expressed in Expi293F cells at 37 °C and 8% CO2. Transfection was performed using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific). Cell culture supernatant was collected 7–8 days after transfection, supplemented to a final concentration of 80 mM Tris-HCl pH 8.0, 100 mM NaCl, and then incubated with BioLock solution (IBA GmbH). ACE2 was purified using a 1-ml StrepTrap HP column (Cytiva) followed by isolation of the monomeric ACE2 by size-exclusion chromatography using a Superdex 200 Increase 10/300 GL column (Cytiva) pre-equilibrated in PBS. Recombinant S309 Fab used for SPR binding studies was produced in either ExpiCHO-S cells and purified using a Capture Select CH1-XL MiniChrom Column (Thermo Fisher), followed by buffer exchange into PBS using a HiPrep 26/10 Desalting Column (Cytiva) or in HEK293 suspension cells, purified using CaptureSelect IgG-CH1 resin and buffer exchanged into PBS (ATUM Bio).Proteins were expressed in Expi293F cells (Thermo Fisher Scientific) at 37 °C and 8% CO−1 in 10X Octet kinetics buffer (Sartorius) and loaded onto pre-hydrated Streptavidin biosensors to a 1 nm total shift. The loaded tips were dipped into a 1:3 dilution series of monomeric human ACE2 starting at 900 nM or 300 nM for 300 s followed by dissociation in 10× kinetic buffer for 300 s. All steps of the affinity measurements using BLI were carried out at 30 °C with a shaking speed of 1,000 rpm. The resulting data were baseline subtracted and affinity measurements were calculated using a 1:1 global fit binding model with Octet Data Analysis HT software v12.0. Binding curves were plotted using GraphPad Prism 9.1.0.BLI was used to assess binding of SARS-CoV-2 RBDs to human ACE2 using an Octet Red96 (Sartorius) and the Octet Data acquisition v11.1. Biotinylated Wu, BA.4/5, BA.2.75.2, BQ.1.1, XBB.1, and XBB.1.5 RBDs were diluted to a concentration of 5 ng µl51) antibody by intraperitoneal injection one day before intranasal inoculation with 104 FFU of BQ.1.1. Weight was recorded daily, and animals were euthanized on day 6 after virus inoculation.Mouse studies were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee at the Washington University School of Medicine (assurance number A3381–01). Virus inoculations were performed under anaesthesia that was induced and maintained with ketamine hydrochloride and xylazine, and all efforts were made to minimize animal suffering. Heterozygous K18-hACE2 C57BL/6 J mice (strain: 2B6.Cg-Tg(K18-ACE2)2Prlmn/J) were obtained from The Jackson Laboratory. All animals were housed in groups of 3 to 5 and fed standard chow diets. The photoperiod was 12 h on/12 h off dark/light cycle. The ambient animal room temperature was 21 °C, controlled within ±1 °C and the room humidity was 50%, controlled within ±5%. Eight- to ten-week-old female K18-hACE2 mice were administered indicated doses of S309 or isotype control  of 5–6 weeks of age (average weight 60–80 grams) were purchased from Janvier Laboratories and handled under specific pathogen-free conditions. The animals were housed and manipulated in isolators in a Biosafety level-3 facility, with ad libitum access to water and food. Before manipulation, animals underwent an acclimation period of one week. Twenty-four hours before infection, the hamsters received an intraperitoneal injection of different concentrations of the monoclonal antibodies S309 , or the control isotype MPE8 . Animal infection was performed as previously described56. In brief, the animals were anaesthetized with an intraperitoneal injection of 200 mg kg−1 ketamine  and 10 mg kg−1 xylazine , and 100 µl of physiological solution containing 6 × 104 plaque-forming units of SARS-CoV-2/Omicron_XBB.1.5  was then administered intranasally to each animal (50 µl per nostril). Mock-infected animals received the physiological solution only. Infected and mock-infected hamsters were housed in separated isolators and were followed-up daily, for four days, when the body weight and the clinical score were noted. At day 4 post-inoculation, the animals were euthanized with an excess of anaesthetics (ketamine and xylazine) and exsanguination. Blood samples were collected by cardiac puncture; after coagulation, the tubes were centrifuged at 2,000g during 10 min at 4 °C, the serum was collected and frozen at −80 °C until further analyses. The lungs were collected, weighted and frozen at −80 °C until further analyses.All hamster experiments were performed according to the French legislation and in compliance with the European Community Council Directives  and according to the regulations of Pasteur Institute Animal Care Committees. The Animal Experimentation Ethics Committee (CETEA 89) of the Institut Pasteur approved this study (200023) before experiments were initiated. Hamsters were housed by groups of 4 animals and manipulated in class III biosafety cabinets in the Pasteur Institute animal facilities accredited by the French Ministry of Agriculture for performing experiments on live rodents. All animals were handled in strict accordance with good animal practice. Male golden Syrian hamsters  on the Kingfisher Flex extraction robot (Thermo Fisher Scientific). RNA was reverse transcribed and amplified using the TaqMan RNA-to-CT 1-Step Kit (Thermo Fisher Scientific). Reverse transcription was carried out at 48 °C for 15 min followed by 2 min at 95 °C. Amplification was accomplished over 50 cycles as follows: 95 °C for 15 s and 60 °C for 1 min. Copies of total (genomic and subgenomic) SARS-CoV-2 N gene RNA in samples were determined using a previously published assay57. In brief, a TaqMan assay was designed to target a highly conserved region of the N gene . This region was included in an RNA standard to allow for copy number determination down to 10 copies per reaction. The reaction mixture contained final concentrations of primers and probe of 500 and 100 nM, respectively.Mouse tissues were weighed and homogenized with zirconia beads in a MagNA Lyser instrument (Roche Life Science) in 1 ml of DMEM medium supplemented with 2% heat-inactivated FBS. Tissue homogenates were clarified by centrifugation at approximately 10,000−1 for 20 s, incubation at 4 °C for 2 min, and new homogenization at 4.0 m s−1 for 20 s. The tubes were centrifuged at 10,000g for 1 min at 4 °C. Afterwards, 125 µl of the tissue homogenate supernatant were mixed with 375 μl of Trizol LS  and the total RNA was extracted using the Direct-zol RNA MiniPrep Kit . The presence of SARS-CoV-2 RNA in these samples was evaluated by one-step quantitative PCR with reverse transcription in a final volume of 12.5 μl per reaction in 384-wells PCR plates using a thermocycler . In brief, 2.5 μl of RNA were added to 10 μl of a master mix containing 6.25 μl of 2× reaction mix, 0.2 μl of MgSO4 (50 mM), 0.5 μl of Superscript III RT/Platinum Taq Mix (2 UI µl−1) and 3.05 μl of nuclease-free water containing the nCoV_IP2 primers  at a final concentration of 400 nM, and the nCoV_IP2 probe (5′-FAM-AGATGTCTTGTGCTGCCGGTA-3′-TAMRA) at a final concentration of 200 nM. The amplification conditions were as follows: 55 °C for 20 min, 95 °C for 3 min, 50 cycles of 95 °C for 15 s and 58 °C for 30 s, and a last step of 40 °C for 30 s. Viral load quantification (expressed as RNA copy number per mg of tissue) was assessed by linear regression using a standard curve of six known quantities of RNA transcripts containing the RdRp sequence (ranging from 107 to 102 copies).For hamster studies, frozen lung fragments were weighted and homogenized with 1 ml of ice-cold DMEM  supplemented with 1% penicillin/streptomycin  in Lysing Matrix M 2 ml tubes  using the FastPrep-24 system , and the following scheme: homogenization at 4.0 m s5 cells per well in 24-well tissue culture plates. The following day, medium was removed and replaced with 200 μl of material to be titrated diluted serially in DMEM supplemented with 2% FBS. One hour later, 1 ml of methylcellulose overlay was added. Plates were incubated for 72 h, and then fixed with 4% paraformaldehyde  in PBS for 20 min. Plates were stained with 0.05% (w/v) crystal violet in 20% methanol and washed twice with distilled, deionized water.Vero E6-TMPRSS2-ACE2 cells were seeded at a density of 1 × 1050) per mg tissue58.Lung tissues were homogenized as described above for measurement of viral RNA. To quantify infectious SARS-CoV-2 particles, lung homogenates titrations were performed on confluent Vero E6 cells in 96- well plates. Viral titres were expressed as 50% tissue culture infectious dose  plasmids  harbouring the Δ19 C-terminal truncation. S-encoding plasmids were diluted in cold OptiPRO SFM , mixed with ExpiFectamine CHO Reagent  and added to cells. Transfected cells were then incubated at 37 °C with 8% CO2 with an orbital shaking speed of 250 rpm  for 24 to 48 h. Transiently transfected ExpiCHO-S cells were harvested and washed twice in wash buffer . Cells were counted and distributed into round bottom 96-well plates  and incubated with serial dilutions of mAb starting at 10 μg ml−1. Alexa Fluor 647-labelled Goat anti-human IgG secondary antibody  was prepared at 2 μg ml−1 and added onto cells after two washing steps. Cells were then washed twice and resuspended in wash buffer for data acquisition at Ze5 cytometer (Bio-Rad).ExpiCHO-S cells were seeded at 6 × 10ADCC assays were performed using ExpiCHO-S cells transiently transfected with SARS-CoV-2 S glycoproteins  as target cells. Natural killer cells were isolated from fresh blood of healthy donors using the MACSxpress WB NK cell isolation kit, human . Target cells were incubated with titrated concentrations of monoclonal antibody for 10 min and then with primary human natural killer cells at an effector to target ratio ranging from 6:1 to 9:1. ADCC was measured using the LDH release assay (Cytotoxicity Detection Kit (LDH) ) after 4 h incubation at 37 °C.+ monocytes. After 20 min, cells were washed and fixed with 4% paraformaldehyde before acquisition on a ZE5 Cell Analyzer (Bio-Rad). Data were analysed using FlowJo software (v10.8.1). The percentage ADCP was calculated as the percentage of monocytes (CD3−CD19–CD14+ cells) positive for PKH67.ADCP assays were performed using ExpiCHO-S cells transiently transfected with SARS-CoV-2 S glycoproteins and labelled with PKH67 (Sigma-Aldrich) as target cells. PMBCs from healthy donors were labelled with CellTrace Violet (Invitrogen) and used as source of phagocytic effector cells. Target cells  were incubated with titrated concentrations of monoclonal antibody for 10 min and then mixed with PBMCs . The next day, cells were stained with APC-labelled anti-CD14 antibody (BD Pharmingen), BV605-labelled anti-CD16 antibody (Biolegend), BV711-labelled anti-CD19 antibody (Biolegend), PerCP/Cy5.5-labelled anti-CD3 antibody (Biolegend), APC/Cy7-labelled anti-CD56 antibody (Biolegend) for the identification of CD142 with a luminometer using the Bio-Glo-TM Luciferase Assay Reagent according to the manufacturer’s instructions (Promega).Antibody-dependent activation of human FcγRIIIa by plasma antibodies was quantified using a bioluminescent reporter assay. ExpiCHO-S cells transiently expressing full-length SARS-CoV-2 S from Wu-D614, BA.5, BQ.1.1 or XBB.1 (target cells) were incubated with serial dilutions of plasma from immune donors. After a 20-min incubation, Jurkat reporter cells stably expressing FcγRIIIa V158 and a NFAT-driven luciferase reporter gene (effector cells) were added at an effector to target ratio of 6:1. Signalling was quantified by the luciferase signal produced via activation of the NFAT pathway. Luminescence was measured after 22 h of incubation at 37 °C with 5% CONatural killer cell-mediated ADCC induced by plasma antibodies was measured as described for ADCC except that ExpiCHO-S cells transiently expressing full-length SARS-CoV-2 S from Wu-D614, BA.1, BA.5, BA.2.75.2, BQ.1.1 or XBB.1 (target cells) were incubated with plasma from immune donors at a single dilution (1:200).−1 R848 (3 M) and 1,000 U ml−1 human recombinant IL-2 at 37 °C and 5% CO2, as previously described59. After 10 days, the cell culture supernatants were collected for ELISA analysis.Replicate cultures of total unfractionated PBMCs obtained from SARS-CoV-2 infected and/or vaccinated individuals were seeded in 96 U-bottom plates (Corning) in RPMI1640 supplemented with 10% fetal calf serum (Hyclone), sodium pyruvate, MEM non-essential amino acids, stable glutamine, 2-mercaptoethanol, penicillin-streptomycin, kanamycin and transferrin. MBC stimulation and differentiation was induced by adding 2.5 μg ml−1 in PBS pH 7.2. Plates were then blocked with PBS 1% BSA  and subsequently incubated with serial dilutions of monoclonal antibodies for 1 h at room temperature. After 4 washing steps with PBS 0.05% Tween-20 (PBS-T) , goat anti-human IgG-AP secondary antibody  was added and incubated for 1 h at room temperature. Plates were then washed four times with PBS-T and 4-nitrophenyl phosphate  substrate was added. After 30 min incubation, absorbance at 405 nm was measured using a plate reader (BioTek) and data were plotted using Prism GraphPad 9.1.0. To test plasma and MBC-derived antibodies, Spectraplate-384 with high protein binding treatment (custom made from PerkinElmer) were coated overnight at 4 °C with 3 µg ml−1 of different SARS-CoV-2 RBDs (produced in house) and S trimers  in PBS pH 7.2 or PBS alone as control. Plates were subsequently blocked with Blocker Casein (1%) in PBS  supplemented with 0.05% Tween-20 . The coated plates were incubated with diluted B cell supernatant for 1 h at room temperature. Plates were washed with PBS containing 0.05% Tween-20 (PBS-T), and binding was revealed using secondary goat anti-human IgG-AP . After washing, pNPP substrate  was added and plates were read at 405 nm after 1 h or 30 min.Ninety-six half-area well plates  were coated overnight at 4 °C with 25 μl of sarbecovirus RBDs prepared at 5 μg ml−1 and incubated for 30 min at 37 °C. The mix was added for 30 min to ELISA 384-well plates  pre-coated overnight at 4 °C with 4 µg ml−1 human ACE2 (produced in house) in PBS. Plates were washed with PBS containing 0.05% Tween-20 (PBS-T), and RBD binding was revealed using secondary goat anti-mouse IgG-AP  or Streptavidin-AP (Jackson ImmunoResearch). After washing, pNPP substrate  was added and plates were read at 405 nm after 1 h.MBC culture supernatants were diluted in PBS and mixed with SARS-CoV-2 Wu RBD mouse Fc-tagged antigen  or with biotinylated BQ.1.1 or XBB.1 RBDs (Acrobiosystems) at a final concentration of 20 ng ml23, S3H3 for domain C/SD160 and S2P6 for the stem helix61) were biotinylated using the EZ-Link NHS-PEO solid phase biotinylation kit (Pierce). Labelled monoclonal antibodies were tested for binding to Wu-G614, BQ.1.1 and XBB.1 S by ELISA and the optimal concentration of each monoclonal antibody to achieve 80% maximal binding was determined. Plasma samples were serially diluted and added to ELISA 96-well plates (Corning) pre-coated overnight at 4 °C with 1 µg ml−1 of S (Acrobiosystems) in PBS. After 30 min, biotinylated anti-S monoclonal antibodies were added at the concentration achieving 80% maximal binding and the mixture was incubated at room temperature for 30 min. Plates were washed and antibody binding was revealed using alkaline phosphatase-comjugated streptavidin (Jackson ImmunoResearch). After washing, pNPP substrate (Sigma-Aldrich) was added and plates were read at 405 nm. The percentage of inhibition was calculated as follow: (1 − (absorbance of sample − absorbance of negative control)/(absorbance of positive control − absorbance of negative control)) × 100.Human anti-S monoclonal antibodies  to bring the total reaction volume to 50 μl. The reaction was incubated at room temperature overnight and used for SPR and mass intact mass spectrometry.Twenty micrograms of purified BN.1 RBD was combined with 2 µl PNGase F  and 5 µl of 10× GlycoBuffer 3and Hm/z data to protein average mass.Four micrograms of PNGase F-treated BN.1 RBD was used for each injection on the liquid chromatography–mass spectrometry (LC–MS) system to acquire intact mass spectrometry signal after separation of protease and protein by liquid chromatography (Agilent PLRP-S reversed phase column). Thermo MS (Q Exactive Plus Orbitrap) was used to acquire intact protein mass under denaturing conditions. BioPharma Finder 3.2 software was used to deconvolute the raw Peptide mapping with LC–MS was used to profile the site-specific glycosylation sites on BN.1 RBD. Glycopeptides containing only one specific glycan were achieved by selectively digesting with chymotrypsin protease. Twenty micrograms of each digest product (peptide with a single glycan) was analysed by LC–MS (Agilent AdvanceBio peptide mapping column and Thermo Q Exactive Plus Orbitrap MS). Peptide mapping data were analysed on Biopharma Finder 3.2 data analysis software.−1  were used for surface capture of His–Avi tag containing RBDs. Running buffer was HBS-EP+ pH 7.4 (Cytiva) and measurements were performed at 25 °C. Experiments were performed with a fourfold dilution series of monomeric S309 Fab or ACE2 at 300, 75, 18.8 and 4.7 nM and were run as single-cycle kinetics. Data were double reference-subtracted and fit to a binding model using Biacore Insight software (v4.0.8.20368). The 1:1 binding model was used to determine the kinetic parameters. 2–14 replicates were performed for each ligand (RBDs) and analyte (ACE2 or S309 Fab) pair. For BN.1 RBD–S309 Fab binding, due to a low binding signal because of a slow association rate constant, a constant Rmax calculated from a control analyte was applied to calculate the kinetic parameters. Kd values are reported as the average of all replicates with the corresponding standard deviation . CM5 chips with covalently immobilized anti-Avi polyclonal antibody diluted to a final concentration of 25 µg ml3. In brief, Vero E6-TMPRSS2-GFP11 cells were split into 96-well, glass bottom, black-walled plates (CellVis) at a density of 36,000 cells per well. BHK-21-GFP1–10 cells were split into 6-well plates at a density of 1 × 106 cells per well. The following day, the growth medium was removed and replaced with DMEM containing 10% FBS and 1% penicillin-streptomycin and the cells were transfected with 4 µg of S protein using Lipofectamine 2000. Twenty-four hours after transfection, BHK-21-GFP1–10 cells expressing the S protein were washed three times using FluoroBrite DMEM (Thermo Fisher) and detached using an enzyme-free cell dissociation buffer (Gibco). The Vero E6-TMPRSS2-GFP11 were washed 3 times with FluoroBrite DMEM and 9,000 BHK-21-GFP1–10 cells were plated on top of the Vero E6-TMPRSS2-GFP11 cells. The cells were incubated at 37 °C and 5% CO2 in a Cytation 7 plate Imager (BioTek) and both bright-field and GFP images were collected every 30 min for 18 h. Fusogenicity was assessed by measuring the area showing GFP fluorescence for each image using Gen5 Image Prime v3.11 software.Cell–cell fusion assays using a split-GFP system was conducted as previously described6 transiently transfected BHK-21-GFP1–10 cells were collected by centrifugation at 1,000g for 5 min. The cells were washed once with PBS and fixed with 2% paraformaldehyde. The cells were washed twice with flow staining buffer  and labelled with 25 µg ml−1 S2L20, an NTD-directed antibody that recognizes all currently and previously circulating SARS-CoV-2 variants, for 45 min. The cells were washed three times with flow staining buffer and labelled with a PE-conjugated anti-human IgG Fc antibody (Thermo Fisher) for 30 min. The cells were washed an additional three times and resuspended in flow staining buffer. The labelled cells were analysed using a BD FACSymphony A3. Cells were gated on singleton events and a total of 10,000 singleton events were collected for each sample. The fraction of S-positive cells was determined in FlowJo 10.8.1 by gating singleton events for the mock transfected cells on PE intensity.To measure surface expression of the variant SARS-CoV-2 S protein, 1 × 10RBD–streptavidin tetramers conjugated to fluorophores were generated by incubating biotinylated Wu, BA.1, BA.2, BA.4/5 or BQ.1.1 with streptavidin at a 4:1 molar ratio for 30 min at 4 °C. Excess free biotin was then added to the reaction to bind any unconjugated sites in the streptavidin tetramers. The RBD-streptavidin tetramers were washed once with PBS and concentrated with a 30-kDa centrifugal concentrator (Amicon). An additional streptavidin tetramer conjugated to biotin only was generated and included in the staining.g for 5 mins at 4 °C and washed twice with PBS. The cells were then stained with Zombie Aqua dye  for 30 min at room temperature after which the cells were washed twice with FACS staining buffer . The cells were then stained with antibodies for CD20-PECy7 (BD), CD3-Alexa eFluor780 (Thermo Fisher), CD8-Alexa eFluor780 (Thermo Fisher), CD14-Alexa eFluor780 (Thermo Fisher), CD16-Alexa eFluor780 (Thermo Fisher), IgM-Alexa Fluor 647 (BioLegend), IgD-Alexa Fluor 647 (BioLegend), and CD38-Brilliant Violet 785 (BioLegend), all diluted 1:200 in Brilliant Stain Buffer (BD), along with the RBD-streptavidin tetramers for 30 min at 4 °C. The cells were washed three times, resuspended in FACS staining buffer, and passed through a 35-µm filter. The cells were examined using a BD FACSAria III and FACSDiva for acquisition and FlowJo 10.8.1 for analysis. Single live CD20+CD3−CD8−CD14−CD16−IgMloIgDloCD38loRBD+ cells were sorted based on reactivity to the Omicron and Wu RBDs into RNAlater and stored at −80 °C.Approximately 5 to 15 million PMBCs were collected 5–72 days post-vaccination for individuals who received either the Wu monovalent mRNA booster or Wu/BA.5 bivalent mRNA booster. The cells were collected by centrifugation at 1,000−1 BQ.1.1 RBD–ACE2–S309 were loaded onto freshly glow-discharged R 2/2 UltrAuFoil grids62, prior to plunge freezing using a vitrobot Mark IV (Thermo Fisher Scientific) with a blot force of 0 and 6 s blot time at 100% humidity and 22 °C. Data were acquired using an FEI Titan Krios transmission electron microscope operated at 300 kV and equipped with a Gatan K3 direct detector and Gatan Quantum GIF energy filter, operated in zero-loss mode with a slit width of 20 eV. For BQ.1.1 RBD–ACE2-S309 data set, automated data collection was carried out using Leginon v3.463 at a nominal magnification of 105,000× with a pixel size of 0.843 Å and stage tilt angle of 0° and 30°. 6,487 micrographs were collected with a defocus range comprised between −0.5 and −2.5 μm. For XBB.1 RBD–ACE2–S309 complex, samples were prepared using a Vitrobot Mark IV (Thermo Fisher Scientific) with R 2/2 UltrAuFoil grids and a Chameleon (SPT Labtech) with self-wicking nanowire Cu R1.2/0.8 holey carbon grids. For XBB.1 RBD–ACE2-S309 data set, 6,355 micrographs from UltrAuFoil grids were collected with a defocus range comprised between −0.2 and −3 μm and stage tilt angle of 0° and 30° and 2,889 micrographs from chameleon grids were collected with a defocus range comprised between −0.2 and −3 μm without tilting the stage. For BN.1 RBD–ACE2–S309 complex, samples were prepared using a Vitrobot Mark IV (Thermo Fisher Scientific) with R 2/2 UltrAuFoil grids, manual blotting/plunging with C-flat holey thick carbon grids and Chameleon (SPT Labtech) with self-wicking nanowire Cu R1.2/0.8 holey carbon grids. For BN.1 RBD–ACE2–S309 data set, 3,822 micrographs from UltrAuFoil grids, 2,000, micrographs from chameleon grids and 1,915 micrographs from C-flat holey thick carbon grids were collected with a defocus range comprised between −0.2 and −3.5 μm and stage tilt angle of 0° and 30°. The dose rate was adjusted to 15 counts per pixel per s, and each movie was acquired in super-resolution mode fractionated in 75 frames of 40 ms. Movie frame alignment, estimation of the microscope contrast transfer function parameters, particle picking, and extraction were carried out using Warp64 (v1.0.9).Cryo-EM grids of BQ.1.1 RBD–ACE2–S309, XBB.1 RBD–ACE2–S309 or BN.1 RBD–ACE2–S309 complex were prepared fresh after purification by size-exclusion chromatography. For BQ.1.1 RBD–ACE2–S309 complex, 3 μl of 0.25 mg ml65 (v4.2.2) to select well-defined particle images. These selected particles were subjected to two rounds of 3D classification with 50 iterations each  using Relion67 (v3.1) with an initial model generated with ab-initio reconstruction in cryoSPARC. 3D refinements were carried out using non-uniform refinement68 along with per-particle defocus refinement in CryoSPARC. Selected particle images were subjected to the Bayesian polishing procedure69 implemented in Relion before performing another round of non-uniform refinement in cryoSPARC followed by per-particle defocus refinement and again non-uniform refinement. To further improve the density of the BQ.1.1 RBD and XBB.1 RBD, the particles were subjected to focus 3D classification without refining angles and shifts using a soft mask encompassing the ACE2, RBD and S309 variable domains using a tau value of 60 in Relion. To further improve the density of the BN.1 RBD, the particles were subjected to cryoSPARC heterogeneous refinement. Particles belonging to classes with the best resolved local density were selected and subjected to non-uniform refinement using cryoSPARC. Local resolution estimation, filtering, and sharpening were carried out using CryoSPARC. Reported resolutions are based on the gold-standard Fourier shell correlation (FSC) with 0.143 criterion and Fourier shell correlation curves were corrected for the effects of soft masking by high-resolution noise substitution71.Two rounds of reference-free 2D classification were performed using cryoSPARC72 (v1.17.1) and Coot73 (v0.9.6) were used to fit atomic models into the cryo-EM maps. RBD, ACE2 and S309 Fab models were refined and relaxed using Rosetta using sharpened and unsharpened maps75.UCSF ChimeraP < 0.05, and P values are indicated with: NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001. ED50, 80% of the maximum binding response (BD80), ID50 and IC50 titres were calculated from the interpolated value from the log(agonist) and the log(inhibitor), versus response using variable slope (four parameters) non-linear regression. Data were plotted and analysed with GraphPad Prism software (version 9.1.0).All statistical tests were performed as described in the indicated figure legends using Prism v9.1.0. The number of independent experiments performed are indicated in the relevant figure legends. Comparisons of means between multiple groups of unpaired data were made with Kruskal–Wallis rank test and corrected with Dunn’s test. Statistical significance is set as Further information on research design is available in the Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-023-06487-6.Supplementary FiguresThis file contains Supplementary Figures 1-6.Reporting SummarySupplementary TablesThis file contains Supplementary Tables 1-8.Source Data Fig. 3


Exposure to ionizing radiation (IR) poses risks to the expanding interest in human space exploration; in atomic bomb survivors and radiological professionals, IR exposure has been linked to GI diseases, including colon cancer. Here, we assessed the expression of multiple stem cell markers in premalignant tumors and adjacent normal mucosa after low- and high-linear energy transfer (LET) irradiation. Our results revealed a noticeable correlation between increased levels of stemness markers and β-catenin activation in premalignant tumors, which was more prominent in tumors induced after exposure to high-LET radiation compared to low-LET induced tumors. Our findings also highlight the complex relationship between radiation types and stem cell phenotypes and their potential influence on carcinogenesis processes, which has significant implications for cancer risk assessments.Apc1638N/+ mice. Using the same model system, i.e., Apc1638N/+ mice, here, we report qualitative differences in the cellular phenotype of low- and high-LET radiation-induced GI tumors. Stem cell (SC) phenotypes were identified using BMI1, ALDH1, CD133, DCLK1, MSI1, and LGR5 markers in low (γ-rays)- and high (56Fe)-LET radiation-induced and spontaneous tumors. We also assessed the expression of these markers in the adjacent normal mucosa. All six of these putative SC markers were shown to be overexpressed in tumors compared to the adjacent normal intestinal tissue. A differential SC phenotype for spontaneous and radiogenic intestinal tumors in Apc1638N/+ mice was observed, where the ALDH1, BMI1, CD133, MSI1, and DCLK1 expressing cells were increased, while LGR5 expressing cells were decreased in 56Fe-induced tumors compared to γ-ray-induced and spontaneous tumors. Furthermore, higher β-catenin activation  was observed in 56Fe-induced tumors compared to γ and spontaneous tumors. Since differential tumor cell phenotype along with activated β-catenin may very well affect malignant progression, our findings are relevant to understanding the higher carcinogenic risk of high-LET radiation. This study has implications for the assessment of GI-cancer risk among astronauts, as well as for the estimation of secondary cancer risk among patients receiving hadron therapy, considering that our results indicate increased stemness properties after radiation.Estimation of cancer risk among astronauts planning to undertake future deep-space missions requires understanding the quantitative and qualitative differences in radiogenic cancers after low- and high-LET radiation exposures. Previously, we reported a multifold higher RBE for high-LET radiation-induced gastrointestinal (GI) tumorigenesis in  Apc)-mutant mouse models of gastrointestinal (GI) tumorigenesis (Min/+Apc and Apc1638N/+ mice), we have reported a higher RBE for intestinal tumorigenesis, and higher incidence of carcinoma after HZE and simulated GCR exposure, relative to low-LET radiation [Astronauts are likely to receive a significant cumulative radiation dose from galactic cosmic radiation (GCR) during long-duration deep space missions. Particularly, the high atomic number and energy (HZE) particles present in GCR are considered a major threat to astronauts’ health as they can penetrate through spacecraft shielding and are densely ionizing due to their high linear energy transfer (LET) characteristics . Exposuradiation ,5,6,7. Fadiation . HoweverIntestinal stem cells (ISC) are considered the cell of origin for tumor initiation ,11. IR-i56Fe-ion) radiation-induced tumors (adenomas) in Apc1638N/+ mice. We also compared the SC phenotypes in the adjacent normal tissue. Here, we demonstrate that HZE-induced tumors display higher SC heterogeneity and enhanced oncogenic β-catenin expression, which has implications for understanding the higher risk of carcinoma after space radiation exposure.This study investigates the SC phenotypes of spontaneous, low-LET, and high-LET  or γ-rays (137Cs source), and an age-matched control group received no radiation. The mice were sacrificed by CO2 asphyxiation 150 days after being irradiated. The small intestine was surgically removed, cleaned with buffered saline, fixed with formalin, processed for sectioning (5 μm thickness), and hematoxylin/eosin (H&E) staining. H&E-stained tissue slides were scored for premalignant (adenoma) and malignant tumor (carcinoma) by a board-certified pathologist.Six- to eight-week-old male escribed ,7,9. AllAt least 15 premalignant tumors per group were used to perform immunostaining experiments as described earlier [The immunofluorescence experiment, t-test and a graphical presentation of the data show mean ± standard error of the mean (SEM). All data were statistically analyzed using and a p-value of 0.05 or less was applied for the detection of statistically significant differences.To determine the specificity of the immunostaining, appropriate controls were run in parallel with the experimental samples. In each section, randomly chosen fields of vision for tumor lesions and normal mucosa were acquired by an observer blinded to the treatment groups using cellSens Entry v1.15  for immunohistochemistry and immunofluorescence. Representative images including their scales were equally and proportionately reduced in size for figure compaction. For each treatment group, images were taken of the entire benign adenoma lesion and adjacent normal mucosa at 10× magnification. The respective SC marker expression in immunohistochemically stained digitalized images was analyzed by a semi-quantitative (DAB pixel density) method using ImageJ2 software fiji version: 2.14.0/1.54f . Average56Fe 3.77 ± 0.75 (mean ± SEM), indicating a decreasing trend in LGR5 expression after radiation exposure (p = 0.083) and 56Fe (p = 0.13) compared to the control. The average DAB pixel density for the spontaneous tumor tissue was 10.5 ± 2.04 (mean ± SEM), γ-rays 6.7 ± 0.64 (mean ± SEM), and 56Fe 5.0 ± 0.98 (mean ± SEM), also indicating a decreasing trend in LGR5 expression after radiation exposure. The decrease in level was insignificant for γ-rays (p = 0.82); however, it was significant in 56Fe (p = 0.026) compared to spontaneous tumors  was observed, suggesting insignificant change after radiation 56Fe (p = 0.1) or γ-rays (p = 0.8). However, change was significant in 56Fe compared with γ-rays (p = 0.027). Analyzing the average DAB pixel density in spontaneous tumors was 7.85 ± 0.94 (mean ± SEM), γ-rays 5.61 ± 0.6 (mean ± SE), and 56Fe 9.64 ± 0.70 (mean ± SE), implying insignificant change after γ-rays (p = 0.065) or 56Fe (p = 0.24). These results indicate a significantly higher level of CD133 in the spontaneous tumors (p = 0.02) and 56Fe (p = 0.04) compared to adjacent normal mucosa (56Fe 2.78 ± 0.94 (mean ± SEM) (56Fe 6.95 ± 0.94 (mean ± SEM). The results indicate a higher level of MSI1 in the tumors than in the normal mucosa of all treatment groups; however, the difference is particularly significant in 56Fe (p = 0.012). The data showed an increase in MSI1 in γ-rays compared to the control, while a decrease in response to 56Fe was observed  F, indicaobserved F.56Fe 1.07 ± 0.172 (mean ± SEM), indicating an increase in DCLK1 positivity with high LET. The increase was significant for 56Fe (p = 0.006) compared to the control normal mucosa. DCLK1 expression in the spontaneous tumors was 0.8 ± 0.172 (mean ± SEM), γ-rays 1.13 ± 0.19 (mean ± SEM), and 56Fe 2.08 ± 0.3 (mean ± SEM), depicting an increasing order of expression with 56Fe. The increased tumor DCLK1 expression was significant in 56Fe relative to γ-rays (p = 0.012) or to spontaneous (p < 0.001) tumors (p = 0.039) and 56Fe (p = 0.006).The staining of DCLK1 expression mostly showed membrane and cytoplasmic staining in tumors and normal mucosa . DCLK1-p) tumors C. Moreov56Fe 5.5 ± 0.75 (mean ± SEM), whereas in the spontaneous tumors, it was 4.05 ± 1.03 (mean ± SEM), γ-rays 5.4 ± 0.88 (mean ± SEM), and 56Fe 6.7 ± 0.85 (mean ± SEM), indicating higher expression after radiation exposure  compared to the control  and normal intestine  relative to the spontaneous or unirradiated normal mucosa, respectively. This relative increase is the most pronounced of all of the SC markers tested  and nuclear β-catenin in tumors. We found that in normal mucosa, positive cells were present as single or multiple cells in the crypts, while in tumors, there was an overexpression of the markers . Analysir tissue B,D. Addir tissue A. Interes tumors C.Apc-mutant mice has also been reported during intestinal tumor development [Apc1638N/+ mice intestinal tumors. Notably, high-LET IR exposure resulted in a significant increase in SC markers  in tumors as well as in the normal intestinal mucosa, relative to low-LET exposure. Contrary to high-LET, MSI1 expression was highest after low-LET IR exposure. A LET-dependent decrease in LGR5 expression and increase in DCLK1 expression was also noted  and β-catenin in space radiation-induced intestinal premalignant tumors and adjacent normal mucosa in the