Minimal PEGylation Improves Pharmacokinetics, Immunocompatibility, and Efficacy of a Lytic Bacteriophage against Multidrug-Resistant Acinetobacter baumannii

Multidrug-resistant (MDR) Acinetobacter baumannii presents a critical therapeutic challenge due to its extensive antibiotic resistance and the paucity of effective alternatives. This study evaluated whether minimal PEGylation could enhance the pharmacokinetic performance, immune compatibility, and antibacterial efficacy of the lytic phage vB_AbaSt_W16 in an immunocompetent murine model of systemic infection. The phage vB_AbaSt_W16 was conjugated with methoxy polyethylene glycol succinimidyl ester (mPEG-S-NHS, MW 5000) at a low concentration (4.2 pM), experimentally defined as the minimal PEGylation level. PEGylation efficiency, infectivity, adsorption, and replication kinetics were characterized in vitro, and serum and intracellular stability were assessed using mouse or human serum and RAW 264.7 macrophages. In vivo pharmacokinetics and therapeutic efficacy were examined in BALB/c mice challenged intraperitoneally with MDR A. baumannii KBN10P02782, while immune responses were profiled by cytokine quantification and antiphage IgG enzyme-linked immunosorbent assay (ELISA). PEGylated vB_AbaSt_W16 retained infectivity and adsorption capacity while markedly improving pharmacokinetics, showing a 2.7- to 3.7-fold increase in half-life, a >200-fold reduction in systemic clearance, and a >1000-fold increase in the area under the plasma concentration (AUC_0-t) relative to the wild-type (WT) phage. The PEGylated phage remained detectable for up to 96 h and achieved complete bacterial clearance within 72-96 h. Immune profiling revealed attenuated proinflammatory cytokine responses and reduced antiphage IgG titers, indicating diminished Th1/Th2 activation. These effects were phage-specific, as the structurally related vB_AbaSi_W9 (a siphovirus) exhibited no comparable improvements following PEGylation. Collectively, minimal PEGylation of vB_AbaSt_W16 enhanced circulation time, immune evasion, and infection control without impairing infectivity. This strategy offers a phage-compatible, structure-informed approach to overcoming key translational barriers in systemic phage therapy and establishes a quantitative framework for optimizing PEGylation in future bacteriophage therapeutics.