Anti-MrkA Monoclonal Antibodies Reveal Distinct Structural and Antigenic Features of MrkA.

Antibody therapy against antibiotics resistant Klebsiella pneumoniae infections represents a promising strategy, the success of which depends critically on the ability to identify appropriate antibody targets. Using a target-agnostic strategy, we recently discovered MrkA as a potential antibody target and vaccine antigen. Interestingly, the anti-MrkA monoclonal antibodies isolated through phage display and hybridoma platforms all recognize an overlapping epitope, which opens up important questions including whether monoclonal antibodies targeting different MrkA epitopes can be generated and if they possess different protective profiles. In this study we generated four anti-MrkA antibodies targeting different epitopes through phage library panning against recombinant MrkA protein. These anti-MrkA antibodies elicited strong in vitro and in vivo protections against a multi-drug resistant Klebsiella pneumoniae strain. Furthermore, mutational and epitope analysis suggest that the two cysteine residues may play essential roles in maintaining a MrkA structure that is highly compacted and exposes limited antibody binding/neutralizing epitopes. These results suggest the need for further in-depth understandings of the structure of MrkA, the role of MrkA in the pathogenesis of Klebsiella pneumoniae and the protective mechanism adopted by anti-MrkA antibodies to fully explore the potential of MrkA as an efficient therapeutic target and vaccine antigen

Dimerization of Vaccinia Virus VH1 Is Essential for Dephosphorylation of STAT1 at Tyrosine 701*

The gene product of Vaccinia virus gene H1, VH1, is the first identified dual specificity phosphatase (DSP). The human genome encodes 38 different VH1-like DSPs, which include major regulators of signaling pathways, highly dysregulated in disease states. VH1 down-regulates cellular antiviral response by dephosphorylating activated STAT1 in the IFN-γ/STAT1 signaling pathway. In this report, we have investigated the molecular basis for VH1 catalytic activity. Using small-angle x-ray scattering (SAXS), we determined that VH1 exists in solution as a boomerang-shaped dimer. Targeted alanine mutations in the dimerization domain (aa 1–27) decrease phosphatase activity while leaving the dimer intact. Deletion of the N-terminal dimer swapped helix (aa 1–20) completely abolishes dimerization and severely reduces phosphatase activity. An engineered chimera of VH1 that contains only one active site retains wild-type levels of catalytic activity. Thus, a dimeric quaternary structure, as opposed to two cooperative active sites within the same dimer is essential for VH1 catalytic activity. Together with laforin, VH1 is the second DSP reported in literature for which dimerization via an N-terminal dimerization domain is necessary for optimal catalytic activity. We propose that dimerization may represent a common mechanism to regulate the activity and substrate recognition of DSPs, often assumed to function as monomers

K_D measurement in IgG format against a mixture of monomeric and multimeric MrkA.

<p>K_D measurement in IgG format against a mixture of monomeric and multimeric MrkA.</p

Mutational analysis of MrkA and mAb characterizations by Western blot.

<p>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.</p

Phage panning output screening cascade.

<p>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.</p

Serotype-independent binding to KP strains by the new antibodies.

<p>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.</p