Figures for <b>Synergistic Combination of a</b><b>n</b><b>Anti-PcrV Antibody and Polymyxin B Rescues Lethal </b><b>Pseudomonas aeruginosa</b><b>Infections</b>

Hemolysis was quantified by measuring the optical density at OD405 of the supernatant post-centrifugation to calculate the inhibition rate (%). (E) Survival curves of mice under lethal P. aeruginosa challenge after antibody administration. NC-IgG,negative control immunoglobulin G.Survival curves of C57BL/6N mice (n = 4) following intravenous challenge with 7.2×107 cfu/mL of P. aeruginosa strain PA.103753.

Mice were administered a single 5 mg/kg dose of candidate mAbs via tail vein injection, and survival was monitored post-infection. Statistical analysis was performed using the log-rank test (**P < 0.01, by the Long-Rank test.). Data are representative of two independent experiments.</p><p dir="ltr"><b>Figure 2</b>.

High-Affinity PcrV binding and broad hemolysis inhibition by mAb-81C8.</p><p dir="ltr">(A) Bio-layer interferometry (BLI) test forreal-time binding-dissociation kinetics of mAb-81C8 interacting with gradient-diluted PcrVL6F/A9G/S21P/S225R, PcrVL6F/S21P/S225, PcrVL6F/A9G/S21P/S225K, PcrVL6F/A9G/S21P/L33M/S225R and PcrVH106Y protein (6.25–400 nM). Antibody was immobilized onto FAB2G biosensors, followed by an association step with PcrV for 120 s and a dissociation step in running buffer for 600 s.

Kinetic constants derived using a 1:1 binding model revealed that mab-81C8 bound to wild-type PcrV (PcrV-WT) with a KD of 1.843 ×10-10M and maintained comparable sub-nanomolar affinity across all mutated proteins, exhibiting $K_D$ values of 1.171×10-10M for PcrVL6F/A9G/S21P/S225R, 8.957×10-11M for PcrVL6F/S21P/S225K, 1.957×10-10M for PcrVL6F/A9G/S21P/S225K, 2.363×10-10M for PcrVL6F/A9G/S21P/L33M/S225R, and 3.175×10-10M for PcrVH106Y. </p><p dir="ltr">(B)Dose-dependent inhibition curves of mAb-81C8 against the hemolytic activity of 11 exoU⁺ clinical P. aeruginosa isolates.

Diluted mAb-81C8 was co-incubated with bacteria (7.5×106 cfu/well）and 5% rabbit erythrocytes at 37℃ for 1.5h. Hemolysis was quantified by measuring supernatant OD405 post-centrifugation to determine the inhibition rate (%). Data represent the mean of 3 independent experiments.</p><p dir="ltr">Figure 3.

MAb-81C8 confers strong therapeutic protection against P. aeruginosa bloodstream infection.(A)Survival curves of female C57BL/6N mice (n=6) following intravenous challenge with 3.6×108 cfu/mL（200μl） of P. aeruginosa. Antibodies diluted in sterile PBS were administered via tail vein 1h prior to infection. Survival was monitored for 168h post-infection. **P < 0.01, P>0.05,by the Log-Rank test.

NC-IgG,negative control immunoglobulin G(data represent three independent experiments).(B)Representative hematoxylin and eosin (H&E) staining of kidney and lung tissues from NC-IgG and mAb-81C8 treatment groups against PA.103753, Scale bar=500 μm(n=3,data represent three independent experiments).(C) Quantification of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α, IL-10) in serum and lung homogenates of PA.103753 infected mice by ELISA.

Mice were administered 10 mg/kg of mAb-81C8 via tail vein injection prior to intravenous challenge with 6×107 CFU/mouse of P. aeruginosa strain PA.103753. *P < 0.05, ***P < 0.001,P>0.05. (n=6,data represent three independent experiments).</p><p dir="ltr">Figure 4. MAb-81C8 demonstrates high protective efficacy against lethal P. aeruginosa pneumonia.(A)Intratracheal challenge with a lethal dose of exoU⁺ strain PA.103753 yields dose-dependent survival variations in C57BL/6N mice (n = 6).

Mice were prophylactically administered varying doses of mAb-81C8 (3, 1, or 0.5 mg/kg) or a negative control antibody (NC-IgG, 3 mg/kg) via the tail vein 1 h prior to infection, and monitored up to 336 h. Statistical significance was determined using the Log-rank test. **P < 0.01,P>0.05.(B) Representative histopathological images of lung tissue sections stained with hematoxylin and eosin (H&E) at 24 h post-infection (n=3).

Upper panels: NC-IgG-treated group (5 mg/kg) displaying severe parenchymal damage, widespread alveolar structural collapse, and massive inflammatory cell infiltration. Lower panels: mAb-81C8-treated group (5 mg/kg) exhibiting well-preserved alveolar architecture and markedly reduced inflammatory cell recruitment. Scale bars: 500 μm(data represent three independent experiments).(C) Local and systemic cytokine profiles quantified by ELISA at 24 h post-infection (n=6).

Concentrations of IL-6, IL-1β, TNF-α, and IL-10 were measured in the circulatory system (Serum, upper panels) and target organs (Lung homogenates, lower panels). Statistical analysis was performed using a two-tailed T-test.

• P < 0.05, **P < 0.01, P>0.05(data represent three independent experiments).</p><p dir="ltr">Figure 5. Synergistic combination of mAb-81C8 and Polymyxin B enhances in vivo efficacy while minimizing antibiotic dosing against P. aeruginosa bloodstream infection.(A) Comparison of low-dose combination (1 mpk mAb-81C8 + 1 mpk PMB) versus 2 mpk single agents against exoU+ type PA.103753（7.2×107 CFU/mouse）(n=6,data represent three independent experiments). (B) Combinatorial therapy using mAb-81C8 (3, 1.5, or 0.75 mg/kg) and a fixed PMB dose (5 mg/kg) confers dose-dependent protection against exoU⁺ strain PA.103753（7.2×107 CFU/mouse）(n=6,data represent three independent experiments).(C) Monotherapy outcomes of mAb-81C8 at 5 and 15 mpk Dose-dependent protection of mAb-81C8 (3, 1.5, or 0.75 mpk) combined with a fixed dose of PMB (5 mpk) against exoS+ type PA.100142（7.2×107 CFU/mouse）(n=6,data represent three independent experiments).(D) Complete survival rescue (100%) achieved by combining 30 mpk mAb-81C8 with 5 mpk PMB against exoS+ type PA.100142（7.2×107 CFU/mouse）(n=6,data represent three independent experiments).(E) High resistance profile observed under 10 mpk mAb-81C8 monotherapy against exoS+ type PA.100491（7.2×107 CFU/mouse）(n=6,data represent three independent experiments).Reversal of resistance and 100% survival rate achieved by the combination of 30 mpk mAb-81C8 and 5 mpk PMB against exoS+ type PA.100491（7.2×107 CFU/mouse）.*P < 0.05,
• *P < 0.01, P>0.05.Statistical analysis by the Log-Rank test(n=6,data represent three independent experiments).</p><p dir="ltr">Figure 6. Hu81C8-H1L3 exhibits binding affinity and in vitro inhibition comparable to mAb-81C8, along with strong in vivo efficacy.(A) Bio-layer interferometry (BLI) test for real-time binding-dissociation kinetics of mAb-81C8 and Hu81C8-H1L3 interacting with gradient-diluted PcrV4-17/128-249 protein (6.25–400 nM). Antibody was immobilized onto FAB2G biosensors, followed by an association step with PcrV for 120 s and a dissociation step in running buffer for 600 s. Kinetic constants derived using a 1:1 binding model revealed that mAb-81C8 binds with an association rate constant kon of 2.646×105 Ms-1, a dissociation rate constant koff of 4.115×10-5 s-1, and an equilibrium dissociation constant KD of 1.555×10-10 M ;Hu81C8-H1L3 binds with an association rate constant kon of 3.741×105 Ms-1, a dissociation rate constant koff of 1.216×10-4 s-1, and an equilibrium dissociation constant KD of 3.252×10-10 M. The humanized antibody exhibited a binding affinity comparable to that of its chimeric progenitor.(B) Inhibition curves of mAb-81C8 and Hu81C8-H1L3 against the hemolytic activity of exoU⁺ clinical P. aeruginosa isolate PA.103753. Serially diluted antibody was co-incubated with bacteria (7.5×106 CFU/well) and 5% rabbit erythrocytes at 37℃. Hemolysis was quantified by measuring supernatant OD405 post-centrifugation to determine the inhibition rate (%). Hu81C8-H1L3 retained potent in vitro blocking potency equivalent to the parental chimeric antibody mAb-81C8.(C) Dose-dependent survival curves of C57BL/6N mice (n = 6) treated with Hu81C8-H1L3 (1, 3, or 5 mg/kg) or NC-IgG (5 mg/kg) following lethal challenge with P. aeruginosa strain PA.103753 (6×107 CFU/mouse). Survival was monitored up to 168 h post-infection. **P < 0.01. NC-IgG by the Log-rank test. NC-IgG, negative control immunoglobulin G (data represent three independent experiments).(D) Female C57BL/6N mice（n=6）were administered 10 mg/kg of Hu81C8-H1L3 via tail vein injection prior to intravenous challenge with 6×107 CFU/mouse of P. aeruginosa strain PA.103753. At 24 h post-infection, lung tissues were harvested to quantify bacterial load. Statistical analysis was performed using Student's t-test (***P < 0.001). Data are representative of three independent experiments.(E) Quantification of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α, IL-10) in serum and lung homogenates of PA.103753 infected mice by ELISA. Mice were administered 10 mg/kg of Hu81C8-H1L3 via tail vein injection prior to intravenous challenge with 6×107 CFU/mouse of P. aeruginosa strain PA.103753. *P < 0.05, ***P < 0.001,P>0.05. (n=6,data represent three independent experiments).</p><p dir="ltr">Figure 7. Hu81C8-H1L3 constricts the T3SS translocon channel via steric hindrance and suppresses ExoU release. (A) Hydrodynamic diameter of a 60 mM raffinose solution (1.188nm) measured by dynamic light scattering (DLS).(B) Evaluation of the physical effect of 60 mM raffinos alone, showing no independent inhibitory effect on P. aeruginosa PA.103753-mediated hemolysis in vitro. (C) The addition of 60 mM raffinos significantly increases the hemolysis inhibition rate when PA.103753 is co-incubated with Hu81C8-H1L3 at varying concentrations (100, 6.25 and 0.0977 μg).All experiments were performed independently (n = 3). Statistical analysis was conducted using multiple t-test, *P < 0.05;
• *P < 0.01,P > 0.05.(D) Inhibition of T3SS-mediated exoU toxin secretion by Hu81C8-H1L3 in P. aeruginosa. SDS-PAGE analysis of culture supernatants from P. aeruginosa strain PA.103753. M: Protein molecular weight marker; Lane 1: Uninduced culture supernatant; Lane 2: Induced culture supernatant.(E) Lanes 4–6: Induced culture supernatants co-cultured with 1, 0.5, and 0 mg of Hu81C8-H1L3, respectively; Lanes 3 and 7: Recombinant exoU protein (1 µg) as positive control. Co-culture with Hu81C8-H1L3 concentration-dependently suppressed the secretion of the exoU toxin protein. Data are representative of at least two independent experiments.</p>