Clin Vaccine Immunol. 2014 Jul;21(7):930-9. doi: 10.1128/CVI.00685-13. Epub 2014 Apr 30.

Diagnostic potential of monoclonal antibodies specific to the unique O-antigen of multidrug-resistant epidemic Escherichia coli clone ST131-O25b:H4.

Szijártó V, Lukasiewicz J, Gozdziewicz TK, Magyarics Z, Nagy E, Nagy G.

 

Abstract

The Escherichia coli lineage sequence type 131 (ST131)-O25b:H4 is a globally spread multidrug-resistant clone responsible for a great proportion of extraintestinal infections. Driven by the significant medical needs associated with this successful pathogenic lineage, we generated murine monoclonal antibodies (mAbs) against its lipopolysaccharide (LPS) O25b antigen in order to develop quick diagnostic tests. Murine monoclonal antibodies were generated by immunizing mice with whole killed nonencapsulated ST131-O25b E. coli cells and screening hybridoma supernatants for binding to purified LPS molecules obtained from an E. coli ST131-O25b clinical isolate. The mAbs selected for further study bound to the surface of live E. coli O25b strains irrespective of the capsular type expressed, while they did not bind to bacteria or purified LPS from other serotypes, including the related classical O25 antigen (O25a). Using these specific mAbs, we developed a latex bead-based agglutination assay that has greater specificity and is quicker and simpler than the currently available typing methods. The high specificities of these mAbs can be explained by the novel structure of the O25b repeating unit elucidated in this article. Based on comparative analysis by nuclear magnetic resonance (NMR) and mass spectrometry, the N-acetyl-fucose in the O25a O-antigen had been replaced by O-acetyl-rhamnose in the O25b repeating unit. The genetic determinants responsible for this structural variation were identified by aligning the corresponding genetic loci and were confirmed by trans-complementation of a rough mutant by the subserotype-specific fragments of the rfb operons.

PMID: 24789798

 

 

Antimicrob Agents Chemother. 2015 Jun;59(6):3109-16. doi: 10.1128/AAC.04494-14. Epub 2015 Mar 16.

Bactericidal Monoclonal Antibodies Specific to the Lipopolysaccharide O Antigen from Multidrug-Resistant Escherichia coli Clone ST131-O25b:H4 Elicit Protection in Mice.

Szijártó V1, Guachalla LM1, Visram ZC1, Hartl K1, Varga C1, Mirkina I1, Zmajkovic J1, Badarau A1, Zauner G1, Pleban C1, Magyarics Z1, Nagy E1, Nagy G2.

 

Abstract

The Escherichia coli sequence type 131 (ST131)-O25b:H4 clone has spread worldwide and become responsible for a significant proportion of multidrug-resistant extraintestinal infections. We generated humanized monoclonal antibodies (mAbs) that target the lipopolysaccharide O25b antigen conserved within this lineage. These mAbs bound to the surface of live bacterial cells irrespective of the capsular type expressed. In a serum bactericidal assay in vitro, mAbs induced >95% bacterial killing in the presence of human serum as the complement source. Protective efficacy at low antibody doses was observed in a murine model of bacteremia. The mode of action in vivo was investigated by using aglycosylated derivatives of the protective mAbs. The significant binding to live E. coli cells and the in vitro and in vivo efficacy were corroborated in assays using bacteria grown in human serum to mimic relevant clinical conditions. Given the dry pipeline of novel antibiotics against multidrug-resistant Gram-negative pathogens, passive immunization with bactericidal antibodies offers a therapeutic alternative to control infections caused by E. coli ST131-O25b:H4.

PMID: 25779571

 

Supplemental

Emergence of widespread antibiotic resistance is recognized as a serious threat to the healthcare system. Multi-drug resistant (MDR) bacterial infections are estimated to cause 2 million infections alone in the US causing 23,000 deaths annually (ANTIBIOTIC RESISTANCE THREATS in the United States, 2013, www.cdc.org). Current death toll is estimated to be 700,000 worldwide with a predicted significant growth reaching 10 million deaths, annually, by 2050.

Escherichia coli is a commensal bacterium, however, certain variants (pathotypes) can cause either intestinal or extraintestinal infections. Extraintestinal pathogenic E. coli (ExPEC) is responsible for urinary tract infections, bacteremia, intra-abdominal infections, and meningitis. Many ExPEC strains have become MDR in the past decades, often leading to therapeutic failure and consequently increased morbidity and mortality. Since MDR strains are usually compromised in their fitness and virulence characteristics, only a few clones – that successfully balance between being MDR and retained virulence – could have spread globally. Among these, the clonal lineage ST131-O25b:H4 is clearly the best example for a widespread successful clone. ST131 strains are disseminated globally and are responsible for a significant proportion (15-25%) of all extraintestinal E. coli infections. Among drug-resistant infections, contribution of ST131 strains is even more dominant: they represent roughly half of the ESBL-positive (strains expressing an extended-spectrum beta-lactamase) and the majority of quinolone resistant E. coli infections. Besides most beta-lactams and quinolones, ST131 strains are typically resistant to other classes of clinically relevant antibiotics, e.g. aminoglycosides, trimethoprim-sulphonamides. Moreover, emerging resistance to last resort antibiotics, such as carbapenems, colistin and tygecycline was also reported recently. On the other hand, the pipeline of novel classes of antibiotics is alarmingly dry (especially against Gram-negative bacteria), which necessitates the development of alternative antimicrobial drugs against MDR bacterial pathogens. We have aimed at developing humanized monoclonal antibodies (mAbs) targeting the MDR E. coli lineage ST131-O25b:H4. While mAbs are widely used in other fields of medicine, their use against infectious diseases until recently had not been justified with the availability of cheap and effective antibiotics. Upon entering to a potential post-antibiotic era, this view may need to be revisited.

We resolved the unique LPS O-antigen subunit structure of ST131-O25b:H4, which is by definition shared by all strains of this lineage. This polysaccharide structure is extremely abundant and located on the surface, and hence expected that O25b epitopes may be readily accessible for antibodies. Therefore, specific murine mAbs were generated by hybridoma fusions using mice immunized repeatedly with whole bacterial cells. These mAbs on one hand were assessed as diagnostic tools to identify O25b-expressing E. coli, and on the other hand, were tested for their therapeutic potency.

For the diagnostic application we coupled murine mAbs to latex beads that enabled agglutination of bacteria expressing the O25b antigen. The assay was validated on a broad panel of ST131 isolates collected from various geographical regions. Sensitivity and specificity of this agglutination tool was found to be superior to currently used methods used for the identification of ST131 strains. Importantly, the assay requires no manipulation (e.g. lysis or heat treatment) of bacterial cultures, can be performed within minutes and evaluated by the naked eye. Based on this, we envision that it could be routinely used in clinical microbiology laboratories to identify this MDR clonal lineage.

In order to test protective efficacy of O25b specific mAbs, the murine antibodies were subjected to humanization. Three candidates originating from different parental murine mAbs were tested extensively both in vitro and in vivo. The primary aim was to identify mAbs that elicit direct bactericidal activity, i.e. killing independent of immune cells. Antibody dependent complement-mediated bacterial killing activity was proven in a serum bactericidal assay. In agreement with this the mAbs could elicit protection in a lethal mouse model of E. coli bacteremia.

Based on the above, we propose considering O25b specific mAbs against infections caused by E. coli ST131 strains. Given the high specificity of mAbs to O25b antigen, the spectrum of protection is limited to E. coli expressing the corresponding O-type. Therefore, companion diagnostic that could identify relevant clinical isolates appropriate for the suggested therapy will need to be applied.

The importance of these studies is two-fold: humanized O25b specific mAbs provide a potential alternative therapeutic option against MDR infections caused by E. coli ST131-O25b:H4 strains. Complimentary to this, O25b mAbs could be used as diagnostic tools for different purposes: i) to determine the incidence of infections caused by the aforementioned MDR E. coli clone, ii) identify ST131 as causative agent in order to guide antibiotic therapy, and iii) to be used as rapid companion diagnostics to identify patients appropriate for prophylaxis/therapy by O25b specific mAbs.

 

Acknowledgements: the research work was substantially supported by the General Program of the Austrian Research Promotion Agency (FFG).

 

Contact:

Gabor Nagy MD, PhD

Director, Microbiology & In Vivo Models Department

Arsanis Biosciences GmbH

Helmut Qualtinger Gasse 2

1030 Vienna, Austria

www.arsanis.com

 

 

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