FLUORESCENT ANTI-PNAG DEMONSTRATES SUPERIOR ACCUMULATION AT THE INFECTION SITE IN A MURINE MODEL OF S. AUREUS IMPLANT-ASSOCIATED OSTEOMYELITIS

Introduction & aim Implant-associated osteomyelitis (IAOM) is difficult to treat due to biofilm formation, which reduces bacterial metabolic activity and promotes antibiotic tolerance and immune evasion. Targeted delivery of cytotoxic compounds to bacteria can improve treatment while limiting off-target effects. We investigated the targeting potential of antibodies against biofilm-associated molecules (e.g., PNAG) and antibiotics in a murine model of S. aureus implant-associated osteomyelitis. Methods Antibodies against the following biofilm derived molecules ZD11, anti-Z22, anti-PNAG and anti-dPNAG were conjugated to sulfonatedcyanine 5 (sCy5). As controls, a polyclonal S. aureus-IgG antibody* and anti-FITCS* were also conjugated to sCy5. Likewise, Dalbavancin (0.01 mg/kg) and Vancomycin (0.01 mg/kg) were conjugated to sCy5. To investigate in vivo targeting, a murine model of S. aureus IAOM was used. 7 days post inoculation, mice(n=4) were injected with one of the fluorescent targeting agents. For each agent, sterile implant controls (n=4) were also investigated. At 1-, 6-, and 24-hours post-injection, mice were scanned using an IVIS Newton 7 imaging system, and total radiance (ph/s/sr) was quantified. 48 hours post-injection, the mice were euthanized, and IVIS imaging was performed again on the tibia before both the implant and the tibia were removed for microbiological or histological analysis. Results All infected mice (n=72) had comparable mean (sd) CFU levels from either implant (4.71 (0.83) log CFU/implant) or bone (6.89 (0.61) log CFU/bone). All sterile mice (n=72) remained sterile. Prior to injection of targeting agent, there were no significant difference in autofluorescence amongst either infected or sterile mice. Among the specific targeting agents, anti-PNAG demonstrated significant accumulation in infected (n=8) vs sterile mice (n=8), but only 24h and 48h post-injection (p= 0.021 and p=0.012, respectively). Both vancomycin and dalbavancin demonstrated significant accumulation at the site of infection 24 h after injection, independent of administration route. While total radiance was comparable, there was a trend towards increased radiance for Dalbavancin administered SC compared to vancomycin. (p=0.057). By day 9 post inoculation, histological analysis demonstrated extensive tissue damage, abundance of intracellular staphylococci and invasion of OLCN. Conclusion Among the investigated antibodies, only anti-PNAG demonstrated significant accumulation 24 hours after injection and remained detectable at 48 hours post injection. As PNAG is an important constituent of S. aureus biofilm matrices anti-PNAG holds promise as a targeting molecule. Further, dalbavancin demonstrated good targeting properties in a setting in which both intracellular staphylococci and invasion of the OLCN, highlighting its potential in novel applications. * Merck