Article Figures & Data
Figures
- FIG. 1.
Potential MAb binding sites on IsdB. IsdB-specific MAbs were reacted with IsdB muteins using Western blotting. MAb binding was disrupted by the accumulation of amino acid mutations in the muteins (Table 2). Amino acids required for binding, as defined by the amino acid mutations which lead to MAb loss of binding, are indicated above the bars. The potential binding site for each MAb is lined up below the corresponding segment of the wild-type IsdB. Vertical lines in the MAb binding site represent amino acid mutations (see Table 2).
- FIG. 2.
Survival of BALB/c mice passively immunized to IsdB and then challenged with S. aureus. Mice were injected i.p. with 0.3 to 0.5 mg of IsdB-specific MAb (solid line), irrelevant isotype control MAb (dashed line), or PBS (dotted line) 20 to 24 h prior to i.v. injection of S. aureus Becker bacteria, as described in Materials and Methods. Data shown are the mean at each time point (error bars show 95% confidence intervals). (A) Mice (five independent experiments, a combined total of 50 per group) were injected i.p. with test MAb 2H2.B8, control MAb, or PBS. In the comparison of test MAb 2H2.B8 and the control MAb, survival was enhanced by the test MAb (P < 0.03; log rank Mantel Cox test). (B) Mice (four independent experiments, a combined total of 40 per group) were injected i.p. with test MAb 2H2.BE11, control MAb, or PBS. In the comparison of test MAb 2H2.BE11 and the control MAb, survival was enhanced by the test MAb (P < 0.01; log rank Mantel Cox test). (C) Mice (four independent experiments, a combined total of 40 per group) were injected i.p. with test MAb 13C7.BC1, control MAb, or PBS. In the comparison of test MAb 13C7.BC1 and the control MAb, survival was enhanced by the test MAb (P < 0.01; log rank Mantel Cox test).
- FIG. 3.
MAb concentrations in blood after S. aureus challenge. Mice (22 per group) were injected i.p. with 0.5 mg of either IsdB-specific MAb 13C7.BC1 (•) or isotype control MAb 6G5.A8 (▪) 20 h prior to an i.v. sublethal challenge with S. aureus Becker. At time zero of the challenge, two mice were sacrificed and blood collected to measure the amount of MAb in the sera at the time of challenge. At each time point indicated, four mice per group were sacrificed and the titer of 13C7.BC1 or 6G6.A8 in sera determined. Titers were calculated based on a standard curve, and concentrations were normalized to those at time zero as described in Materials and Methods. The amount of 13C7.BC1 remaining in the mouse serum after 4 days was statistically less than the amount of control MAb 6G5.A8 remaining in the mouse serum (P < 0.01).
Tables
- TABLE 1.
Murine MAbs to IsdB obtained after two separate immunization/selection protocols
MAb IgG1 isotype Isolation protocola ELISA resultb IgG2b isotype MAbc 2H2.B8 1 ++ 2H2.BE11 8H6.E11d 1 ++ 7H2.C11 1 ++ 2E12.A8 2 ++ 8A8.B4 2 + 3G11.D5 2 + 13G11.C11 2 + 13G11.BF3 13C7.D12 2 ++ 13C7.BC1 1G3.B3 2 ++ 1G3.BD4 9H3.E4 2 ++ 3B7.G8 2 ++ 3G12.A4 2 ++ ↵ a MAbs were obtained from mice immunized using one of the two methods described in Materials and Methods.
↵ b Binding of MAb to recombinant IsdB in ELISA. ++, higher level of specific binding; +, lower level of specific binding.
↵ c MAbs were class switched from IgG1 to IgG2b by using limiting dilution as described in Materials and Methods.
↵ d 8H6.E11 was the only isotype IgG2a MAb; all other MAbs were of the IgG1 isotype.
- TABLE 2.
Western blot analysis of MAb binding to IsdB muteins
MAb Groupa Specific amino acid point mutations which cumulatively disrupted binding of MAb to the mutein 2H2.B8 I 130, 139, 180, 182, 217, 226, 454 8A8.B4 I 130, 157-159, 184, 454 13G11.C11 I 157-159, 184, 267, 400, 425 1G3.B3 9H3.E4 II 130, 157-159, 184, 454 3G11.D5 III 157-159, 184 13C7.D12 2E12.A8 IV 130, 296, 454 ↵ a Grouping for each MAb was determined by Western blot analysis of IsdB mutein binding.
- TABLE 3.
Summary of IsdB-specific MAb grouping, based on evaluation of IsdB binding sites
Grouping method Group I Group II Group III Group IV Mutein binding by Western blot 2H2.B8 1G3.B3 3G11.D5 13C7.D12 8A8. B4 9H3.E4 2E12.A8 13G11.C11 SPR analysis (exp. 1) 2H2.B8 1G3.B3 13C7.D12 8A8.B4 9H3.E4 2E12.A8 3G11.D5 13G11.C11 SPR analysis (exp. 2) 2H2.B8 1G3.B3 13C7.D12 13G11.C11 Native IsdB binding by flow cytometry 2H2.B8 1G3.B3 13G11.D5 13C7.D12 8A8.B4 9H3.E4 2E12.A8 3G11.D5 - TABLE 5.
OPK activities of IsdB-specific MAbsa
Irrelevant MAb isotype controlb MAb Group No. of CFU (% survival)c after 2 h of incubation with indicated amount (μg/ml)of MAb 100 50 20 0d 10B4.H4 (IgG1) 161, 163 110, 161 113, 111 189, 200 2H2.B8 I 74, 60 (41) 58, 64 (45) 65, 126 (85) 6G6.A8 (IgG2b) 153, 139 145, 170 94, 89 2H2.BE11 I 107, 70 (61) 88, 79 (53) 101, 111 (116) 1G3.BD4 II 139, 178 (109) 121, 128 (79) 98, 107 (112) 13G11.BF3 III 130, 155 (98) 114, 125 (76) 92, 83 (96) 13C7.BC1 IV 98, 147 (84) 111, 117 (72) 68, 90 (86) ↵ a The OPK reaction mixture was MAb, complement, HL60 cells, and S. aureus Becker bacteria.
↵ b 10B4H4 and 6G6.A8 murine MAbs were used as non-S. aureus binding isotype controls.
↵ c Data are replicates from two identical reaction mixtures. Percent survival is the mean number of CFU from tubes with test MAb divided by the mean number of CFU from tubes with control MAb, times 100, at 2 h. A percent survival value of >100 indicates bacterial growth during the 2-h assay incubation period. Representative data from one of two experiments with equivalent results are shown.
↵ d The numbers of CFU at time zero for the assay control (complement, HL60 cells, S. aureus Becker bacteria, and 0 μg MAb) replicates were 122 and 171.
- TABLE 6.
Ex vivo opsonization and protection by IsdB-specific MAbsa
Irrelevant MAb isotype control or nonprotein control MAb Group No. of tests Total no. of mice surviving (n = 5/test) Total % of mice surviving 10B4.H4, IgG1 8 4/40 10 2H2.B8 I 8 39/40 98b 6G6.A8, IgG2b 5 2/25 8 2H2.BE11 I 3 14/15 93b 1G3.BD4 II 1 1/5 20c 13G11.BF3 III 1 2/5 40c 13C7.BC1 IV 2 0/10 0c PBS 3 3/15 20
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
-
Supplemental methods and results.
Table S1. Matrix footprint evaluation of anti-IsdB MAbs.
Table S2. Western blot data with IsdB muteins.
Table S3. Mutein sequences.
Legends for Fig. S1 through S4.
MS Word document, 111K. - Supplemental file 2
-
Fig. S1. Pairwise MAb binding to IsdB, by SPR sensorgram.
MS PowerPoint file, 56K. - Supplemental file 3
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Fig. S2. SPR binding of IsdB-specific MAbs to sequestered IsdB.
MS PowerPoint file, 61K. - Supplemental file 4
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Fig. S3. ELISA titration of murine IsdB MAb binding to recombinant IsdB.
MS PowerPoint file, 113K. - Supplemental file 5
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Fig. S4. Pairwise binding of MAbs to S. aureus expressing IsdB.
MS PowerPoint file, 60K.
- Supplemental file 1
-
Supplemental methods and results.
