Screening workflow and donor selection

(A) Schematic representation of the workflow from patient selection to evaluation of T3SS-blocking activity. (B) ELISA results indicating that sera of donors 16 and 25 possess the highest Ab titers against recombinant PcrV and PscF. (C) ExoS-Bla translocation blocking activity of serum 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.

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.

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. IgGs from donor 25, as well as mAbs P5B3 and P3D6 block ExoS-Bla injection, while the control mAb VRCO1 is inefficient. While P3D6 displays significant inhibition only towards V1, P5B3 displays dose-dependent inhibition on all five variants (see also Supplementary Figure 1).

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) Both P3D6 and P5B3 mAbs significantly reduce pore formation at 100 µg/mL. (B, C) Only P3D6 exhibits a significant dose-response inhibitory effect on cytotoxicity towards J774 cells with 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. Note that the bound Fab clashes with a neighboring PcrV protomer, precluding the formation of the pentamer.

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 (normalized J774 macrophage cytotoxicity) for three mAbs.

(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 Galan 2021). Fab P3D6 binds to a PcrV monomer but clashes with adjacent protomers in the context of the pentamer, while Fab MEDI3902 clashes with other Fab molecules. Fab 30-B8 binds to both the PcrV monomer and all pentamer protomers without clashes. 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 ExoS-Bla 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. (A and B) Both mAbs P5B3 (A) and P3D6 (B) display dose-response inhibition with the strain expressing the V1 variant, with respective IC50 of 96 and 3.7 µg/mL. (C to F) Monoclonal Ab P5B3 displays dose-response inhibition with the strain expressing the V2, V3, V4 and V5 variants with respective IC50 of 206, 192, 212 and 426 µg/mL. P3D6 had no effect on strains expressing variants 2, 3, 4 and 5 (see Fig. 3B).

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.