PcrV-specific B cells in people with cystic fibrosis (pwCF).

A) Schematic of primary human B cells binding to PcrV tetramer reagent and decoy (left). Representative flow plot for B cells after enrichment. Cells binding only to the PcrV tetramer are indicated with the red box. B-C) Percentage (B) and number (C) of PcrV-specific B cells in pwCF (CF; n = 14) vs. control, blood bank donors (non-CF; n = 14). Statistics determined by 2-tailed Mann-Whitney tests. p = ***: <0.001, ****: <0.0001. Error bars represent mean and SD.

Generation of anti-PcrV mAbs derived from B cells isolated from a PA-infected CF donor.

A) Percentage of somatic hypermutation (SHM) detected in B cell receptor (BCR) sequences from cells of the indicated phenotype for CF donor 1. B) ELISAs showing PcrV binding using supernatants derived from 293T cells transfected with IgM expression plasmids containing the indicated BCR sequences. C) Area under the curve (AUC) for representative ELISAs of purified antibodies 408 and 411 expressed alternatively as IgM vs. IgG in a single experiment. D) AUC for representative ELISA of purified mAbs V2L2 and 411 expressed, alternatively as, IgG vs. IgM; and for the commercially sourced, clinical, bi-specific mAb, gremubamab.

Clinical characteristics of subjects from whom PcrV-specific BCR sequences were obtained.

ETI: elexacaftor/tezacaftor/ivacaftor triple therapy. Chronic infection is defined here as positive PA cultures in at least 2 of 3 consecutive years (Green et al., 2012; Rosenfeld et al., 2022).

pwCF-derived, germline, anti-PcrV-specific mAbs exhibit robust anti-PA activity in an in vivo mouse pneumonia model.

A) Schematic illustrating the experimental PA infection and mAb delivery protocol. B) Bacterial load in mouse lungs at 24 h post-infection for mice that received 20 µg intranasal dose of off-target, control IgG1 mAb (Ctl), indicated anti-PcrV mAbs, or diluent alone (PBS). Asterisks show significance in Dunn’s test versus animals treated with diluent only (PBS); p = *: <0.05.

High affinity anti-PcrV mAbs derived from memory B cells isolated from CF Donor 2.

A) Somatic hypermutation (%SHM) rates in BCR sequences from individual B cells with the indicated surface phenotype isolated from CF donor 2. Each circle represents a heavy or light chain sequence from a singly-sorted cell. B) Heatmap showing paired heavy (x-axis) and light (y-axis) V genes for 79 MBCs in donor 3. The color gradient depicts SHM for the heavy chains in each pairing. Clone numbers for BCRs subsequently expressed as mAbs are included above their corresponding box. C) PcrV binding for purified mAbs generated from five MBC BCRs (colored lines) screened in a single ELISA. The two Donor 1-derived mAbs with in vivo protective activity (408 and 411) are used as benchmarks for relative binding activity (dashed lines). The off-target control (anti-SARS-CoV-2 RBD) line appears hidden because it overlaps with the blue line (432).

mAbs derived from CF MBC BCR sequences control PA infection in a murine pneumonia model.

Lung bacterial load for mice treated intranasally with 20 µg of the indicated anti-PcrV mAb, or vehicle only (PBS). Combined data from two independent experiments is shown (n = 10 mice per condition). Asterisks show significance in Dunn’s test versus animals treated with the off-target control antibody (Ctl); p = *: <0.05, **: <0.01, ***: <0.001, n.s.: not significant.