Immune stealth-driven O2 serotype prevalence and potential for therapeutic antibodies against multidrug resistant Klebsiella pneumoniae

Emerging multidrug-resistant bacteria are a challenge for modern medicine, but how these pathogens are so successful is not fully understood. Robust antibacterial vaccines have prevented and reduced resistance suggesting a pivotal role for immunity in deterring antibiotic resistance. Here, we show the increased prevalence of Klebsiella pneumoniae lipopolysaccharide O2 serotype strains in all major drug resistance groups correlating with a paucity of anti-O2 antibodies in human B cell repertoires. We identify human monoclonal antibodies to O-antigens that are highly protective in mouse models of infection, even against heavily encapsulated strains. These antibodies, including a rare anti-O2 specific antibody, synergistically protect against drug-resistant strains in adjunctive therapy with meropenem, a standard-of-care antibiotic, confirming the importance of immune assistance in antibiotic therapy. These findings support an antibody-based immunotherapeutic strategy even for highly resistant K. pneumoniae infections, and underscore the effect humoral immunity has on evolving drug resistance

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

Staphylococcus aureus drives expansion of low-density neutrophils in diabetic mice

Diabetic individuals are at considerable risk for invasive infection by Staphylococcus aureus, however, the mechanisms underlying this enhanced susceptibility to infection are unclear. We observed increased mortality following i.v. S. aureus infection in diabetic mice compared with nondiabetic controls, correlating with increased numbers of low-density neutrophils (LDNs) and neutrophil extracellular traps (NETs). LDNs have been implicated in the inflammatory pathology of diseases such as lupus, given their release of large amounts of NETs. Our goal was to describe what drives LDN increases during S. aureus infection in the diabetic host and mechanisms that promote increased NET production by LDNs. LDN development is dependent on TGF-β, which we found to be more activated in the diabetic host. Neutralization of TGF-β, or the TGF-β–activating integrin αvβ8, reduced LDN numbers and improved survival during S. aureus infection. Targeting S. aureus directly with MEDI4893*, an α toxin–neutralizing monoclonal antibody, blocked TGF-β activation, reduced LDNs and NETs, and significantly improved survival. A comparison of gene and protein expression in high-density neutrophils and LDNs identified increased GPCRs and elevated phosphatase and tensin homolog (PTEN) in the LDN subset. Inhibition of PTEN improved the survival of infected diabetic mice. Our data identify a population of neutrophils in infected diabetic mice that correlated with decreased survival and increased NET production and describe 3 therapeutic targets, a bacterial target and 2 host proteins, that prevented NET production and improved survival

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

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

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

Anti-MrkA antibody binding is influenced by the antigen presentation format.

<p>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.</p