A High-Affinity Native Human Antibody Disrupts Biofilm from Staphylococcus aureus Bacteria and Potentiates Antibiotic Efficacy in a Mouse Implant Infection Model

Abstract

Many serious bacterial infections are difficult to treat due to biofilm formation, which provides physical protection and induces a sessile phenotype refractory to antibiotic treatment compared to the planktonic state. A key structural component of biofilm is extracellular DNA, which is held in place by secreted bacterial proteins from the DNABII family: integration host factor (IHF) and histone-like (HU) proteins. A native human monoclonal antibody, TRL1068, has been discovered using single B-lymphocyte screening technology. It has low-picomolar affinity against DNABII homologs from important Gram-positive and Gram-negative bacterial pathogens. The disruption of established biofilm was observedin vitroat an antibody concentration of 1.2 μg/ml over 12 h. The effect of TRL1068in vivowas evaluated in a murine tissue cage infection model in which a biofilm is formed by infection with methicillin-resistantStaphylococcus aureus(MRSA; ATCC 43300). Treatment of the established biofilm by combination therapy of TRL1068 (15 mg/kg of body weight, intraperitoneal [i.p.] administration) with daptomycin (50 mg/kg, i.p.) significantly reduced adherent bacterial count compared to that after daptomycin treatment alone, accompanied by significant reduction in planktonic bacterial numbers. The quantification of TRL1068 in sample matrices showed substantial penetration of TRL1068 from serum into the cage interior. TRL1068 is a clinical candidate for combination treatment with standard-of-care antibiotics to overcome the drug-refractory state associated with biofilm formation, with potential utility for a broad spectrum of difficult-to-treat bacterial infections.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Figures

FIG 1

ELISA binding of TRL1068 to IHF alpha/HU homologs from diverse bacterial species. Two unrelated bacterial, one viral, and seven mammalian proteins (including BSA) with different tags also were tested for binding to TRL1068. No binding was detected up to 6 nM for the 10 unrelated proteins.

FIG 2

Epitope mapping. (A) Full-length S. aureus HU sequence. A series of overlapping 15-mers, offset by 3, were assessed for binding by TRL1068 by semiquantitative ELISA, with the positive peptides denoted in red. (B) High conservation of the epitope in Gram-positive and Gram-negative bacterial species. Yellow denotes full identity across all species, blue denotes partial identity, and green denotes conservative substitutions. (C) Alanine substitution for each amino acid in turn (parental sequence shown below peptide number) was tested for binding to TRL1068 in a semiquantitative ELISA. Results are shown as fold change over the level of the parent peptide. (D) Key residues (in red) from the peptide binding experiments. Dotted lines indicate hydrogen bonds in the crystal structure of HU from S. aureus bound to DNA (24). (E) Location of the epitope mapped on the crystal structure. The HU dimer is shown in green with the peptide 19-20 region in blue.

FIG 3

In vitro biofilm disruption. Bacterial biofilms were formed on conical plastic pegs in 96-well format (MBEC assay by Innovotech, Inc.). Pegs were treated with vehicle (left) or TRL1068 (1.2 μg/ml; right) for 12 h. Adherent bacteria were visualized by scanning electron microscopy. Marked reductions in biofilm were observed for both S. aureus and P. aeruginosa.

FIG 4

In vivo efficacy in a murine tissue cage infection model. Four in vivo studies were conducted with different durations of treatment. (A and B) In the one-day treatment study, saline was used as a negative control. Two routes of administration were used for TRL1068: direct injection into the tissue cage and i.p.; as there were no significant differences between the two, the pooled data from both are plotted in blue. (A) 1-Log reduction after a single dose of DAP plus TRL1068 on day 2 (left) (P < 0.01) but rebound on day 3 (right), attributed to insufficient DAP to eradicate the continuously released bacteria. (B) 1-Log reduction (P < 0.02) in adherent bacteria inside the cage released by sonication after the explantation of the cage. (C and D) In the three-day treatment study, saline was used as a negative control. Panels C and D show a greater reduction in both planktonic and adherent bacteria than in the 1-day study; however, the results did not reach statistical significance. (E and F) In the five-day treatment study, an IgG1 isotype control antibody was used as a negative control (indistinguishable from saline controls used previously). (E) 1.5-Log reduction (P < 0.01) and 2-log reduction (P < 0.001) in planktonic bacteria observed for day 4 and day 6, respectively. (F) 3-Log reduction in adherent bacteria on day 6 (P < 0.01). The 5-day treatment study was repeated but with the additional administration of TRL1068 24 h prior to infection. At day 6, both planktonic (G) and adherent (H) bacteria within the cage were eradicated.

FIG 5

Meta-analysis of planktonic bacterial reduction in the tissue cage fluid. (A) Dispersion, in CFU/ml, on day 1 prior to treatment for three separate experiments. (B) Consolidation of day 4 data from the 3-day and 5-day treatment studies to yield a larger number of mice per group, compensating for the higher variability in the 3-day study. This meta-analysis shows highly significant reduction in planktonic bacteria with TRL1068 plus DAP versus DAP alone.