Screening workflow and donor selection

(A) Schematic representation of the workflow from patient selection to evaluation of T3SS-blocking activity. (B) Patients sera (1/50 dilution) were tested in ELISA against recombinant PcrV and PscF. (C) ExoS-Bla translocation blocking activity of serum IgGs from donors 16 and 25. (D) (top) scheme of depletion experiment of specific Abs on either PscF-or PcrV–loaded columns. (bottom) blocking activity of depleted sera for both donors. The dots and bars represent the means and standard-deviations of experimental triplicates. The curves correspond to the modeled log-logistic dose-response curves. The dashed lines represent the mean of normalized ExoS-bla injection in the absence of Ab.

Selection of B cells from donors 16 and 25.

(A) B cells sorting and isolation using PscF and PcrV baits. (B) Table summarizing the EC50 values of selected Abs obtained by ELISA and the percentage of inhibition of ExoS-Bla injection into epithelial cells at 100 µg/mL. ExoS-Bla inhibitions were compared using ANOVA and “No inhibition” means an absence of significant difference with the control (adjusted p-values > 0.05). The P5B3 and P3D6 mAbs exhibited differences with the control (no Ab) with adjusted p-values < 0.001.

mAbs P5B3 and P3D6 activity on PcrV variants.

(A) PcrV variability in clinical strains. The most variable position (225) can either be Ser, Arg or Lys. Representative strains are indicated when available (PAO1 for V1, CHA for V2, PA14 for V3 and PA103 for V4). (B) Inhibition of ExoS-Bla activity following infection of A549 epithelial cells with P. aeruginosa expressing the V1 variant. Normalized ExoS-Bla injection values in the presence of 100 µg/mL Abs were compared to the control (no Ab) using ANOVA. Pairwised t-test significance is indicated by the symbols *, **, *** and **** for adjusted p-values below 0.05, 0.01, 0.001 and 0.0001, respectively. The absence of symbol corresponds to adjusted p-values > 0.05.

mAb P3D6 efficiently inhibits the PopB/PopD translocation pore.

J774 macrophages were infected with P. aeruginosa strain (PAO1, V1) deprived of all three T3SS toxins. The cell death (cytotoxicity) resulting from insertion of the translocon pore was measured by propidium iodide incorporation and normalized to the wild-type strain without addition of mAbs. (A) Normalized cytotoxicity values in the presence of mAb at 100 µg/mL. Specific mAbs were compared to the control (no Ab) using ANOVA. Pairwised t-test significance is indicated by the symbol ****, meaning p-values below 0.0001. (B, C) Dose-response analysis with mAb concentrations ranging from 0.01 to 100 µg/mL. The circles, the black line and the grey area represent the experimental values, the log-logistic modeled dose-response curve and the 95% confidence interval, respectively. The dashed lines represent the mean of normalized cytotoxicity in the absence of Ab.The Ab concentration is presented in logarithmic scale. No black curve nor grey area is displayed for P5B3 because no dose-response could be modeled. In contrast, P3D6 exhibits an IC50 of 11.8 µg/mL.

Structure of Fab P3D6 in complex with PcrV*.

(A) Crystal structure of Fab P3D6 in complex with PcrV*. Fab P3D6 is shown in brown, while PcrV* is in orange. Contacts are made between PcrV* and an interaction platform formed by both HC and LC of P3D6. (B) Closeup of the interaction between PcrV* and P3D6, with the latter being shown as an electrostatic surface where acidic regions are shown in red, and basic in blue. Side (C) and top (D) views of the modeled PcrV pentamer, in light blue) onto which the structure shown in (A) was overlaid.

Functional and structural comparisons between anti-PcrV mAbs.

(A) Dose-dependent inhibition of T3SS in two functional assays reflecting toxin injection (ExoS-Bla injection) and translocon assembly (J774 macrophage cytotoxicity) for three mAbs. The circles, the dark lines and the light-colored areas represent the experimental values, the log-logistic modeled dose-response curves and the 95% confidence intervals, respectively. The Ab concentration is presented in logarithmic scale. No red curve nor red area is displayed for MEDI3902 because no dose-response could be modeled. (B) Structures of PcrV-Fab complexes in the context of a PcrV monomer, as well as of a pentamer modeled based on the cryo-EM structure of SipD (Guo and Galán, 2021). The N– and C-termini of PcrV are indicated in all structures, which are referenced in the main text.

Sequence conservation of V and J regions of selected mAbs compared to germline.

Percentage (%) of identity was obtained by aligning variable region sequences on IMGT database (https://www.imgt.org).

Competition between A) anti-PscF mAbs and B) anti-PscF mAbs.

The indicated IC50 values correspond to the concentration of competitor mAbs necessary to obtain half of the signal generated by the biotinylated mAbs without competitor. ND corresponds to a non-detectable competition.

Affinities of anti-PcrV mAbs for PcrV.

The reported values correspond to the average of the measurements obtained from two independent experiments (n=2). Standard Deviations were calculated by the BLI analysis software.

Data collection, phasing and structure refinement statistics

Bacterial strains and plasmids

Dose-dependent inhibition by mAbs P5B3 and P3D6 of ExoS-Bla injection from strains expressing five PcrV variants.

Inhibition of ExoSBla activity following infection of A495 epithelial cells with P. aeruginosa expressing the V1 (A and B), V2 (C), V3 (D), V4 (E) or V5 (F) variants, with mAbs concentration ranging from 0.01 to 100 µg/mL. The circles, the black line and the grey area represent the experimental values, the log-logistic modeled dose-response curve and the 95% confidence interval, respectively. The dashed lines represent the mean of normalized ExoS-Bla injection in the absence of antibody. The Ab concentration is presented in logarithmic scale. Monoclonal Ab P5B3 displays dose-response inhibition with the strains expressing the V1, V2, V3, V4 and V5 variants with respective IC50s of 96, 206, 192, 212 and 426 µg/mL (no statistically significant difference). P3D6 (B) displays a dose-response inhibition with the strain expressing the V1 variant, with an IC50 of 3.7 µg/mL, significantly different from the one of P5B3 against the same variant (p-value = 0.015).

Dose-dependent inhibition by mAbs 30-B8 of ExoS-Bla injection from strains expressing five PcrV variants.

Inhibition of ExoS-Bla activity following infection of A495 epithelial cells with P. aeruginosa expressing the V1, V2, V3, V4 or V5 variants, with mAbs concentration ranging from 0.001 to 1 µg/mL. Monoclonal Ab 30-B8 displays dose-response inhibition with V1, V2, V3, V4 and V5 variants with respective IC50 of 21.3, 12.8, 11.2, 13.0, 10.5 ng/mL. The dark lines and the light-colored areas represent the log-logistic modeled dose-response curve and the 95% confidence interval, respectively. The dashed lines represent the mean of normalized ExoS-Bla injection in the absence of antibody. The Ab concentration is presented in logarithmic scale.

Interactions between PcrV* and Fab P3D6.

Chain codes correspond to: Fab HC (chain A), LC (chain C) and PcrV* (chain E). CDR H1, H2 and H3 of the heavy chain, as well as CDR L1 and L3 of the light chain, contribute to PcrV* binding. Interaction diagram generated with LigPlot+ v.2.9.9 (Lakowski & Swindells 2011, https://www.ebi.ac.uk/thornton-srv/software/LigPlus/). Hydrogen bonds are represented by green dashed lines and residues involved in hydrophobic contacts are indicated by spoked arcs.