Fig 1.
Phage panning output screening cascade.
More than 4000 colonies were picked for high throughput screening after phage panning, scFv.Fc conversion and transformation. Four clones including clones 1, 4, 5, and 6 were selected for further characterization.
Fig 2.
Anti-MrkA antibody binding is influenced by the antigen presentation format.
MrkA protein, either coated directly to the ELISA plate (left panel), or captured by streptavidin after biotinylation (right panel), was recognized differently by the anti-MrkA antibodies.
Table 1.
KD measurement in IgG format against a mixture of monomeric and multimeric MrkA.
Fig 3.
Mutational analysis of MrkA and mAb characterizations by Western blot.
3A. Western blot against full length MrkA and deletion mutants. Protein samples were resolved on a 4–12% SDS-PAGE gel under reducing condition and subjected to Western blot analysis by a murine anti-his mAb, KP3, and clones 1–6 IgGs as indicated underneath each blot. Sample arrangement is as follow: Lane 1, cell lysate from KP strain 43816DM; Lane 2, E.coli BL21 strain control; Lane 3–5 BL21 strain expressing his-tagged recombinant MrkA, MrkA with N terminal 40 amino acid deletion, and MrkA with C terminal 32 amino acid deletion, respectively. In the anti-his blot, a bracket indicates the positions of the monomeric, full length MrkA as well as the deletion mutants. 3B. Western blot analysis of various MrkA deletion mutants. MrkA deletion mutants 1 to 7 (del1-7) as indicated on the left were expressed in BL21 strain, resolved on a 4–12% SDS-PAGE gel under reducing condition, and subjected to Western blot detection by anti-his and KP3 antibodies as indicated. Lane arrangement: KP is the lysate prepared from 43816DM strain and E.coli represents lysate prepared from BL21 expressing the recombinant, his-tagged MrkA. 1–7 represent the mutants with corresponding numbers (del 1–7) described on the left. For both 3A and B, numbers to the left of the gel indicate the molecular weight in kDa.
Table 2.
KD measurement in Fab format against monomeric MrkA.
ND, not detectable; N/A, not applicable.
Fig 4.
Epitope binning was performed against three test article including KP3, clone 4 and 5 as described in the materials and method. There are three phases (I, II, and III) for each graph. In phase I biotin-labeled MrkA protein was loaded to the neutravidin probe. In phase II one of the mAbs (test articles) as indicated above each graph bound to the immobilized MrkA. In phase III different mAbs as indicated were mixed with the test article mAb and loaded to the probe.
Fig 5.
5A. The twelve peptides included in the peptide array (P 1-P 12): 5B. Upper panel: summary of the array analysis. Only the peptides showing any sign of binding activity by the anti-MrkA mAbs were included. “++++” indicates an ELISA signal 10 fold above background, “+++” 5 fold above background, “++” 2 fold above background and “+” marginally above background. KP3, KP16, 88D10, and 89E10 are mAbs identified from a previous study [11]. Lower panel: The two peptides (P 2 and P 5) showing positive binding are underlined in the context of MrkA. The two cysteines are circled.
Fig 6.
OPK activity is important for in vivo protective activity.
KP3-TM mutation was generated and tested in both in vitro OPK (4A) and in vivo challenge assay (4B). Significant reduction was seen in the OPK assay and a trend towards significance was seen in the in vivo challenge assay. R347 is a human IgG1 isotype control.
Fig 7.
Serotype-independent binding to KP strains by the new antibodies.
Flow cytometry experiment was used to gauge the binding of the four new antibodies against three WT KP strains of different serotype as indicated. KP3 serves as the positive control and R347 is a human IgG1 isotype control.
Fig 8.
Serotype independent OPK activity by the new antibodies.
Two KP strains with LPS serotypes O1 and O2 respectively were used in the OPK assay. All newly isolated antibodies displayed comparable OPK activities to that of KP3. R347 is a human IgG1 isotype control.
Fig 9.
The newly identified antibodies were tested in in vivo challenge models under both prophylaxis and therapeutic settings. In the prophylaxis setting antibodies were given 24 hours prior to KP challenge (7A), whereas in the therapeutic setting antibodies were given one hour after KP challenge (7B). There were eight animals in each group. R347 is a human IgG1 isotype control.
Fig 10.
No synergistic effect by antibody combinations in the therapeutic model.
KP3 was combined with either clone 1 or 5 in equal amount as indicated and tested in a therapeutic model. No significant improvement was observed. There were eight animals in each group. R347 is a human IgG1 isotype control.