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Yoon Sung Nam1 Jeong Heon Yu1

1, KAIST, Daejeon, , Korea (the Republic of)

The natural selection of resistant bacteria has led to serious antibiotic resistance. Phage therapy has gained increasing attention as an alternative to antibiotics because of high specificity and low inherent toxicity. Importantly, the directed evolution of phage provides an opportunity to overcome the resistance of bacteria. In contrast to chemical compounds, phages can be amplified in bacteria, so the number of phages can increase with the increasing number of host bacteria, effectively suppressing the growth of the bacterial population. However, the use of phage for in vivo anti-bacterial treatments is limited due to the rapid elimination from the body by reticuloendothelial (RES) system clearance. Polyethylene glycol (PEG) and other polymers can be employed, but the steric barrier by the PEG chains can significantly reduce the intrinsic infectivity of phages. Also, any chemical modification is applied to the first generation of phages; i.e., the amplified phages do not have PEG on their surface. In this work, we report the prolonged blood circulation and dramatically increased therapeutic efficacy of lytic phages through the immunological cloaking based on the expression of a self-peptide on the major capsid. The stealth self-peptide was originally suggested through the computational simulations of human CD47 (hCD47) interacting with signal regulatory protein-α (SIRPα) on macrophages and imparting a “do-not-eat-me” signal to avoid phagocytosis (Rodriguez et al., Science, 339, 971). Our results show that the self-peptide expressing T7 phage (Self-T7) suppresses up to 70 % phagocytosis of mouse macrophages compared to the wild-type T7 phage (WT-T7). Real-time in vivo image analysis demonstrates that the Self-T7 exhibits a markedly longer blood circulation compared to the WT-T7. Accordingly, the in vivo anti-bacterial effects of the phage was dramatically increased in a mouse model of E.coli infection of the intestines, as indicated by the high survival rate of mice by the intraperitoneal or intravenous injection of the Self-T7. The WT-T7 and PEG-modified T7 phage showed limited therapeutic effects: the WT-T7 increased the survival time from two days to four days, and the PEG-T7 showed a large variation in the survival rate and no complete recovery. In contrast, all of the mice treated with the Self-T7 were returned to normal conditions and showed no symptoms in 5 days. Plaque assay for the liquid from the infected intestines showed that no single bacteria remained in the mice treated with the Self-T7. The mice treated with WT-T7 and PEG-T7 also the exhibited significantly reduced numbers of plaques compared to non-treated mice, indicating the antibacterial activity of T7 phages. This work demonstrated that the immunological cloaking of lytic phages through genetic expression of a self-peptide is an effective means to improve the in vivo anti-bacterial efficacy of phage through suppressed phagocytosis and prolonged blood circulation.

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