A Humanized Monoclonal Antibody Potentiates Killing of Diverse Biofilm-Forming Respiratory Tract Pathogens by Antibiotics

Abstract

New strategies to treat diseases wherein biofilms contribute significantly to pathogenesis are needed as biofilm-resident bacteria are highly recalcitrant to antibiotics due to physical biofilm architecture and a canonically quiescent metabolism, among many additional attributes. We, and others, have shown that when biofilms are dispersed or disrupted, bacteria released from biofilm residence are in a distinct physiologic state that, in part, renders these bacteria highly sensitive to killing by specific antibiotics. We sought to demonstrate the breadth of ability of a recently humanized monoclonal antibody against an essential biofilm structural element (DNABII protein) to disrupt biofilms formed by respiratory tract pathogens and potentiate antibiotic-mediated killing of bacteria released from biofilm residence. Biofilms formed by six respiratory tract pathogens were significantly disrupted by the humanized monoclonal antibody in a dose- and time-dependent manner, as corroborated by CLSM imaging. Bacteria newly released from the biofilms of 3 of 6 species were significantly more sensitive than their planktonic counterparts to killing by 2 of 3 antibiotics currently used clinically and were now also equally as sensitive to killing by the 3 rd antibiotic. The remaining 3 pathogens were significantly more susceptible to killing by all 3 antibiotics. A humanized monoclonal antibody directed against protective epitopes of a DNABII protein effectively released six diverse respiratory tract pathogens from biofilm residence in a phenotypic state that was now as, or significantly more, sensitive to killing by three antibiotics currently indicated for use clinically. These data support this targeted, combinatorial, species-agnostic therapy to mitigate chronic bacterial diseases.

Crumbling the Castle: Targeting DNABII Proteins for Collapsing Bacterial Biofilms as a Therapeutic Approach to Treat Disease and Combat Antimicrobial Resistance

“…Using an acute otitis media model in chinchillas challenged with nontypeable Haemophilus influenzae (NTHI), NRel bacteria generated upon rapid biofilm collapse showed an increased expression of genes associated with cell envelope biogenesis, translation, and ribosomal structure and biogenesis, consistent with the observed sensitization to amoxicillin-clavulanate [ 54 ]. The transient and unique phenotype of NRel bacteria from rapidly collapsed biofilms accounts for previous in vitro [ 39 , 45 , 49 , 51 , 53 , 54 , 61 ] and in vivo [ 50 , 59 ] observations in multiple bacterial species of enhanced killing by common first-line antibiotics when used in combination with anti-DNABII antibodies. Although the degree of sensitization of NRel bacteria to different classes of antibiotics may differ, we have observed no instances where NRel bacteria are less sensitive than planktonic bacteria.…”

Monoclonal antibodies that target extracellular DNABII proteins or the type IV pilus of nontypeable Haemophilus influenzae (NTHI) worked additively to disrupt 2-genera biofilms

“…Thereby, incubation with a monoclonal antibody that targets the DNABII proteins induces an equilibrium shift that rapidly destabilizes the eDNA lattice, resulting in collapse of the biofilm matrix with concomitant release of the resident bacteria. This anti-DNABII monoclonal antibody has been effective against biofilms built by many diverse pathogens we’ve tested to date, and thus we consider it to be more of a species-agnostic therapeutic [ 10 , 18 , 26 , 32 , 55 , 60 ]. Both monoclonals release bacteria from biofilm-residence, by either active dispersal or passive disruption [ 10 , 44 , 45 , 53 , 55 ].…”

“…To collect bacteria that had been newly released from biofilm residence (NRel) by either α-rsPilA or α-DNABII, we used a previously described methodology [ 32 , 45 ]. Briefly, 150 μl of the antibody and NRel-containing medium was carefully removed from the well so as not to disturb any remaining biofilm.…”

“…To this end, selected single species and 2-genera biofilms were allowed to form in 8-well chambered coverglass as described, a P. aeruginosa PAO1 reporter that incorporated an mCherry-expressing plasmid pCJ5.2 and NTHI #86-028NP which expresses GFP under the control of the ompP2 promoter [ 40 ] were used. After 16 h, biofilms were gently washed with DPBS to remove non-adherent bacteria, fixed with 1.6% paraformaldehyde, 2.5% glutaraldehyde, 4% acetic acid in 0.1 M phosphate buffer, pH 7.4, [ 19 , 32 , 43 , 53 , 55 ], then imaged with a Zeiss 800 CLSM. To calculate co-localization in the CLSM images, a multi-step process with BiofilmQ v0.2.2 [ 20 , 29 ] was used.…”

Monoclonal Antibodies for Bacterial Pathogens: Mechanisms of Action and Engineering Approaches for Enhanced Effector Functions

“…Biofilms constitute bacterial communities that adhere to abiotic surfaces using a self-made extracellular matrix composed of proteins, polysaccharides, and extracellular DNA. Due to their biophysical architecture and quiescent metabolism [ 46 ], biofilms can protect bacteria from host immunity [ 47 ] and antibiotic therapy [ 48 ], thus playing a major role in the survival of bacterial pathogens. Biofilm formation is mediated by membrane-bound protein and carbohydrate factors, which act as adhesins that mediate cellular attachment to abiotic surfaces [ 49 ].…”