Table 1.
S. pneumoniae strains used in this study.
Table 2.
Binding spectrum of mouse anti-PspA mAbs.
Fig 1.
Epitope regions of lead anti-PspA mAbs.
To determine the epitope regions of lead mAbs, PspA fragments linked to maltose-binding protein (MBP-R1-R4) with overlapping regions were prepared. (A) The locations of MBP-R1-R4 in BAA-658 PspA are schematically depicted; superscripted, the amino acids (aa) of PspA encompassed by the fragments are indicated. Dotted lines mark overlapping fragment regions. CDR, Clade Defining Region; PRR, proline-rich region; NPB, non-proline block. (B) Binding of anti-PspA mAbs to MBP (negative control) and MBP-R1 to MBP-R4 was tested by ELISA. An anti-MBP antibody served as positive control. The table summarizes the data. +, mAb binding observed; -, binding not observed.
Fig 2.
Anti-PspA mAbs show activity in complement deposition and opsonophagocytic killing assays.
(A) Pneumococci were incubated with 0.25–5 μg/mL mouse isotype control or anti-PspA IgG2a mAbs and mouse serum. Bound C3 was detected with a FITC-labeled anti-mouse C3 antibody using flow cytometry. Histograms: FL-1 for isotype (tinted; gray lines) or the respective anti-PspA (black lines) mAbs. Note: 139G3 did not bind to ATCC-6305. (B) S. pneumoniae PJ-1324 cells were pre-opsonized with 1 μg/mL mouse IgG2a control or anti-PspA mAbs in 10% baby rabbit complement and 106 PMN-like HL-60 cells or vehicle. At the indicated times, CFU were enumerated. Samples were run in triplicate or quadruplicate and mean values + SD of one representative experiment of several performed are shown (***, p<0.0005; anti-PspA vs. isotype mAb, unpaired t-test).
Fig 3.
Activity of lead anti-PspA mAbs in mouse sepsis models with S. pneumoniae strains BAA-658, WU2 and PJ-1324.
4–6 h before i.p. or i.n. infection with S. pneumoniae, CD-1 or Swiss-Webster mice were pretreated i.p. with the indicated mAbs. In most experiments, heparinized tail vein blood was collected and bacterial CFU enumerated (Left panels). The results of one to two independent experiments are shown (dotted line = 100 CFU/mL, limit of detection) (*, p<0.05; CFU isotype vs. anti-PspA mAb treatment, unpaired t-test). Survival was followed for 13–14 days. Combined survival results of two to three independent experiments are shown (*, p<0.05; **, p<0.005; ***, p<0.0005, isotype vs. anti-PspA mAbs, Mantel-Cox test).
Fig 4.
Activity of lead anti-PspA mAbs in mouse sepsis models with S. pneumoniae strains NCTC-11905 and ATCC-6303.
4–6 h before i.p. or i.v. infection with S. pneumoniae, CD-1 mice were pretreated i.p. with the indicated mAbs. In most experiments, heparinized tail vein blood was collected and bacterial CFU enumerated (Left panels). The results of one to two independent experiments are shown (dotted line = 100 CFU/mL, limit of detection) (*, p<0.05; CFU isotype vs. anti-PspA mAb treatment, unpaired t-test). Survival was followed for 13–14 days. Combined survival results of two to three independent experiments are shown (*, p<0.05; **, p<0.005; ***, p<0.0005, isotype vs. anti-PspA mAbs, Mantel-Cox test).
Fig 5.
Combination treatment can potentially increase efficacy of anti-PspA mAbs and standard-of-care antibiotics.
(A-B) 24 h after i.p. infection with ~2.1–3.2x104 CFU of exponential phase PJ-1324 (~21- to 32-fold LD100), CD-1 mice were treated i.p. with 200 μL PBS alone (vehicle), or 200 μL PBS containing 100 μg isotype IgG2a mAb or 140csH1 ± 1 mg ceftriaxone (~50 mg/kg). Survival was followed for 15 days after infection. Combined results of three independent experiments are shown. *, p<0.05, Mantel-Cox test. (C) 30 min after i.p. infection with 3.2–6.5x107 CFU exponential phase BAA-658 (ERM resistant), groups of 4–5 female CD-1 mice were treated i.p. with 200 μl of PBS containing 300 μg of mouse isotype IgG2a antibody or 140csH1. Mice were immediately treated intragastrically with 200 μL PBS, 1.5% ethanol (vehicle) or 200 μL PBS, 1.5% ethanol, 1.5 mg/mL ERM (Dose: 15 mg/kg). Survival was monitored for 8 days. Combined results of three independent experiments are shown. *, p<0.05 vehicle + isotype vs. vehicle +140csH1; #, p<0.04 vehicle + 140csH1 vs. ERM + 140csH1; $, p<0.0001 ERM + isotype vs. ERM + 140csH1, Mantel-Cox test.
Fig 6.
Therapeutically administered 140csH1 improves outcome in mouse lung infection model with PJ-1324.
(A-B) 6 h or (C) 24 h after i.t. infection with 0.8–2.7x106 CFU of PJ-1324 in 50 μL PBS, CD-1 mice were treated i.p. with 200 μg isotype IgG2a mAb (open circles) or 140csH1 (closed circles). 24 h after infection, (A.) lung and (B.) tail vein blood CFU were determined (dotted line = detection limit of 100 CFU/mL). Combined results of three independent experiments are shown. *, p<0.04, isotype vs. 140csH1 treated mice, unpaired t-test. (C) Mice were treated 24 h after infection with isotype (open circles; n = 12 total) or 140csH1 (closed circles; n = 13 total). Survival was followed for 15 days after infection. Combined results of two independent experiments are shown. **, p<0.004, isotype vs. 140csH1 survival curve, Mantel-Cox test.
Fig 7.
Increased activity of human IgG1/IgG3 versions of chimeric anti-PspA antibodies in CDAs with human serum (WU2).
(A-B) 108 CFU/mL of highly encapsulated WU2 were incubated for 15 min in 2.5% unabsorbed human serum pool ± 5 μg/mL of control or chimeric anti-PspA antibodies. Subsequently, C3b deposition was detected with FITC-labeled anti-C3 antibody. Samples were run in triplicate and the FL-1 intensity of the cells measured by flow cytometry. (A) Average MFI values ± SD of one representative experiment of two performed with similar results are shown. ***, p<0.0005, vs. chimeric Fu+ and Fu- hIgG1 versions of 140H1, unpaired Student’s t-test. (B) FL-1 data for 10,000 bacterial particles
Fig 8.
Increased activity of human IgG1/IgG3 versions of chimeric anti-PspA antibodies in CDAs with human serum (BAA-658 and PJ-1324) and of non-fucosylated chimeric anti-PspA antibodies in OPAs with human phagocytes.
(A-B) 108 CFU/mL of BAA-658 and PJ-1324 were incubated for 15 min in 40% absorbed human plasma ± 5 μg/mL of control or chimeric anti-PspA antibodies. Subsequently, C3b deposition was detected with FITC-labeled anti-C3 antibody. Samples were run in triplicate and the FL-1 intensity of the cells measured by flow cytometry, using 20,000 analyzed particles per sample. (A) Average MFI values ± SD of one representative experiment of two performed with similar results are shown. ***, p<0.0005, vs. chimeric Fu+ and Fu- hIgG1 versions of 140H1, unpaired Student’s t-test. (C) OPAs. FITC-labeled D39 particles were incubated for 30 min (5:1) with human blood PMN ± 10 μg/mL isotype control or chimeric anti-PspA antibodies. Subsequently, 200 PMN/sample were analyzed by fluorescence microscopy for phagocytosis of pneumococci. Ethidium bromide was used to differentiate between intra- and extracellular pneumococci. Samples were run in quadruplicate and average values ± SD of one representative experiment of two performed with similar results are shown (**, p<0.005, vs. chimeric IgG1 version of 140H1, unpaired Student’s t-test).