Compilation of results.

<p>Critical micelle concentration (<i>cmc</i> in µM units), effective charge (<i>z_eff</i>) of the peptide, partition coefficient of insertion (<i>K_ins</i>), effective partition coefficient (<i>K_eff</i>), molar enthalpy of partitioning ( in kJ/mol units), minimal inhibitory concentration (<i>MIC</i> in µM units) and the haemolytic potency is given as the concentration <i>H5</i> (in µM units) at which 5% haemolysis has been obtained. Membrane partitioning data for PAMPX, Leu8, Adec1, Adec8, Adec14 and OAMPX could not be analysed and these cells are marked with a dashed line.</p

Critical micelle concentration assessed via fluorescence spectroscopy.

<p>As an example, pyrene fluorescence excitation spectra are shown for a concentration sequence of Adec8 in (A). The formation of micelles is monitored via partitioning of pyrene into the hydrophobic cavity of the forming micelles. This induces the observed peak-shift of pyrene (A), which is quantified via the intensity ratio R = I₃₃₉/I₃₃₃ plotted in (B) as a function of peptide concentration. In the current work, the cmc is defined as the point where R deviates from the background signal.</p

Bactericidal potency of MPX and analogues.

<p>The degree of red blood cell haemolysis is plotted as a function of peptide concentration.</p

Structures of MPX and eight analogues.

<p>Ala1 and Ala14 have alanine substitution at position 1 or 14, and are analogues with reduced hydrophobicity. Leu8 has a leucine substitution in position 8 resulting in augmented hydrophobicity. Adec1, Adec8 and Adec14 have 2-amino-decanoic acid substitution in position 1, 8 or 14 respectively, and constitute the most hydrophobic analogues of MPX considered in this study. PAMPX and OAMPX are N^α-terminal propanoic and octanoic acid acyl analogues of MPX, respectively.</p

Partitioning of MPX, Ala1 and Ala14 onto POPC∶POPG (3∶1) LUVs studied via ITC at 37°C.

<p>In panel (A), heat traces of 25 mM LUVs injected into 20 µM peptide (19×2 µL injections) are shown. Panel (B) shows the corresponding heat of reaction, Q, as a function of number of injections. The solid lines in (B) represent fits to the data. In panel C, the change in potency of Ala1 and Ala14 for neutral or anionic lipid membranes measured relative to MPX, is evaluated via the ratio <i>K_ins</i>/<i>K_ins(MPX)</i> and <i>K_eff/K_eff(MPX)</i> respectively. The membrane charge selectivity of the peptide (ability to select between neutral and anionic lipid membranes) is assessed via the partitioning coefficient ratio <i>K_eff/K_ins</i> shown in (C).</p

Mode of peptide-membrane interaction evaluated using ITC at 37°C.

<p>Panel A–C shows the heat trace of 20 mM POPC∶POPG (3∶1) LUVs injected into 100 µM peptide (38×1 µL injections), except for OAMPX which was titrated using 8 mM LUVs. The corresponding heat of reaction (Q) and accumulated heat of reaction (Q_acc) are shown in panel D–F and G–I respectively. The arrows in panel D–F highlight the endpoint of the secondary process and the solid lines serve to guide the eye.</p

FACS gating strategy.

Despite development of effective SARS-CoV-2 vaccines, a sub-group of vaccine non-responders depends on therapeutic antibodies or small-molecule drugs in cases of severe disease. However, perpetual viral evolution has required continuous efficacy monitoring as well as exploration of new therapeutic antibodies, to circumvent resistance mutations arising in the viral population. We performed SARS-CoV-2-specific B cell sorting and subsequent single-cell sequencing on material from 15 SARS-CoV-2 convalescent participants. Through screening of 455 monoclonal antibodies for SARS-CoV-2 variant binding and virus neutralization, we identified a cluster of activated B cells highly enriched for SARS-CoV-2 neutralizing antibodies. Epitope binning and Cryo-EM structure analysis identified the majority of neutralizing antibodies having epitopes overlapping with the ACE2 receptor binding motif (class 1 binders). Extensive functional antibody characterization identified two potent neutralizing antibodies, one retaining SARS-CoV-1 neutralizing capability, while both bind major common variants of concern and display prophylactic efficacy in vivo. The transcriptomic signature of activated B cells harboring broadly binding neutralizing antibodies with therapeutic potential identified here, may be a guide in future efforts of rapid therapeutic antibody discovery.</div

In vitro analysis of lead mAbs binding.

(A) Ranking of purified mAbs from lowest IC50 pseudovirus neutralization value at the top (best neutralization) to highest IC50 at the bottom (poorest neutralization). Each antibody IC50 value obtained from triplicate point determinations of the dilution curve. Mesoscale binding values are shown for each mAb (supernatant) towards SARS-CoV-2 Spike, N-terminal domain (NTD) and receptor binding domain (RBD), as a heat-map. The binding determinations were performed once in duplicate. Colors indicate normalization from 0–100 within each column. (B) Mesoscale binding values for binding to the RBD of viral variants Alpha (N501Y, A570D), Beta (K417N, E484K, N501Y), Gamma (K417T, E484K, N501Y) and Delta (L452R), shown as fold change from SARS-CoV-2 RBD binding within each mAb individually. The binding determinations were performed once in duplicate. (C) Heat-map showing percentage ACE2 blocking for each mAb binding viral variant spike proteins (CoV-2, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2) and Omicron BA.1(B.1.1.529). The ACE2 blocking analysis was performed in duplicate determinations of a dilution curve.</p

Cryo-EM refinement.

Despite development of effective SARS-CoV-2 vaccines, a sub-group of vaccine non-responders depends on therapeutic antibodies or small-molecule drugs in cases of severe disease. However, perpetual viral evolution has required continuous efficacy monitoring as well as exploration of new therapeutic antibodies, to circumvent resistance mutations arising in the viral population. We performed SARS-CoV-2-specific B cell sorting and subsequent single-cell sequencing on material from 15 SARS-CoV-2 convalescent participants. Through screening of 455 monoclonal antibodies for SARS-CoV-2 variant binding and virus neutralization, we identified a cluster of activated B cells highly enriched for SARS-CoV-2 neutralizing antibodies. Epitope binning and Cryo-EM structure analysis identified the majority of neutralizing antibodies having epitopes overlapping with the ACE2 receptor binding motif (class 1 binders). Extensive functional antibody characterization identified two potent neutralizing antibodies, one retaining SARS-CoV-1 neutralizing capability, while both bind major common variants of concern and display prophylactic efficacy in vivo. The transcriptomic signature of activated B cells harboring broadly binding neutralizing antibodies with therapeutic potential identified here, may be a guide in future efforts of rapid therapeutic antibody discovery.</div

Demographics and clinical characteristics of included participants.

Demographics and clinical characteristics of included participants.</p