c Acinetobacter baumannii, a Gram-negative multidrug-resistant (MDR) bacterium, is now recognized as one of the more common nosocomial pathogens. Because most clinical isolates are found to be multidrug resistant, alternative therapies need to be developed to control this pathogen. We constructed a bacteriophage genomic library based on prophages induced from 13 A. baumannii strains and screened it for genes encoding bacteriolytic activity. Using this approach, we identified 21 distinct lysins with different activities and sequence diversity that were capable of killing A. baumannii. The lysin (PlyF307) displaying the greatest activity was further characterized and was shown to efficiently kill (>5-log-unit decrease) all tested A. baumannii clinical isolates. Treatment with PlyF307 was able to significantly reduce planktonic and biofilm A. baumannii both in vitro and in vivo. Finally, PlyF307 rescued mice from lethal A. baumannii bacteremia and as such represents the first highly active therapeutic lysin specific for Gram-negative organisms in an array of native lysins found in Acinetobacter phage. Members of Acinetobacter are soil bacteria that frequently colonize the human skin without harm (1). However, in environments in which individuals are immunocompromised or suffer from a variety of wounds (e.g., in hospital settings or on battlefields), Acinetobacter baumannii can cause severe life-threatening infections (2-4). Symptoms of A. baumannii infections range from mild skin wounds and urinary tract infections to more severe conditions, including pneumonia, meningitis, and sepsis (5). A. baumannii is now one of the most common causes of hospital-acquired pneumonia (2) and sepsis; while not common (only 1.3% of all sepsis cases), it is associated with mortality rates of up to 58% (6).One of the main threats from A. baumannii is the high rate of resistance to antibiotics commonly used to treat Gram-negative infections. More than 80% of Acinetobacter species are considered to be multidrug resistant (MDR) (i.e., resistant to at least three classes of antibiotics), resulting in infections with poor clinical outcomes, including high rates of morbidity and death, prolonged hospital stays, and substantial health care expenses (3, 7). In addition, several strains of pan-drug-resistant A. baumannii have been isolated, showing resistance to a wide variety of clinically used antibiotics (8). A. baumannii is also capable of surviving treatments with detergents and disinfectants, dehydration, and UV radiation and thus is difficult to eradicate from surfaces in hospital environments (9, 10). The organism not only is intrinsically resistant to many antibiotics (owing to ␤-lactamases, weak membrane permeability, and efficient efflux systems) but also can readily acquire foreign plasmids and is considered to have a high degree of genetic plasticity (11). Outbreaks caused by MDR Acinetobacter have been reported from hospitals worldwide; more recently, they have become a serious problem in military medical facilities (4). One of ...

Hepatitis C virus (HCV) establishes a chronic infection in the majority of exposed individuals and can cause cirrhosis and hepatocellular carcinoma. The role of antibodies directed against HCV in disease progression is poorly understood. Neutralizing antibodies (nAbs) can prevent HCV infection in vitro and in animal models. However, the effects of nAbs on an established HCV infection are unclear. Here, we demonstrate that three broadly nAbs, AR3A, AR3B and AR4A, delivered with adeno-associated viral (AAV) vectors can confer protection against viral challenge in humanized mice. Furthermore, we provide evidence that nAbs can abrogate an ongoing HCV infection in primary hepatocyte cultures and in a human liver chimeric mouse model. These results showcase a novel therapeutic approach to interfere with HCV infection exploiting a previously unappreciated need for HCV to continuously infect new hepatocytes in order to sustain chronicity.

Acinetobacter baumannii is a Gram-negative bacterial pathogen responsible for a range of nosocomial infections. The recent rise and spread of multidrug-resistant A. baumannii clones has fueled a search for alternative therapies, including bacteriophage endolysins with potent antibacterial activities. A common feature of these lysins is the presence of a highly positively charged C-terminal domain with a likely role in promoting outer membrane penetration. In the present study, we show that the C-terminal amino acids 108 to 138 of phage lysin PlyF307, named P307, alone were sufficient to kill A. baumannii (>3 logs). Furthermore, P307 could be engineered for improved activity, the most active derivative being P307 SQ-8C (>5-log kill). Both P307 and P307 SQ-8C showed high in vitro activity against A. baumannii in biofilms. Moreover, P307 SQ-8C exhibited MICs comparable to those of levofloxacin and ceftazidime and acted synergistically with polymyxin B. Although the peptides were shown to kill by disrupting the bacterial cytoplasmic membrane, they did not lyse human red blood cells or B cells; however, serum was found to be inhibitory to lytic activity. In a murine model of A. baumannii skin infection, P307 SQ-8C reduced the bacterial burden by ϳ2 logs in 2 h. This study demonstrates the prospect of using peptide derivatives from bacteriophage lysins to treat topical infections and remove biofilms caused by Gram-negative pathogens.

In the absence of an exogenous ligand, the hemoglobins from the cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 coordinate the heme group with two axial histidines (His46 and His70). These globins also form a covalent linkage between the heme 2-vinyl substituent and His117. The in vitro mechanism of heme attachment to His117 was examined with a combination of site-directed mutagenesis, NMR spectroscopy, and optical spectroscopy. The results supported an electrophilic addition with vinyl protonation being the rate-determining step. Replacement of His117 with a cysteine demonstrated that the reaction could occur with an alternative nucleophile. His46 (distal histidine) was implicated in the specificity of the reaction for the 2-vinyl group as well as protection of the protein from oxidative damage caused by exposure to exogenous H2O2.

Hepatitis B virus causes chronic infections in 250 million people worldwide. Chronic hepatitis B virus carriers are at risk of developing fibrosis, cirrhosis, and hepatocellular carcinoma. A prophylactic vaccine exists and currently available antivirals can suppress but rarely cure chronic infections. The study of hepatitis B virus and development of curative antivirals are hampered by a scarcity of models that mimic infection in a physiologically relevant, cellular context. Here, we show that cell-culture and patient-derived hepatitis B virus can establish persistent infection for over 30 days in a self-assembling, primary hepatocyte co-culture system. Importantly, infection can be established without antiviral immune suppression, and susceptibility is not donor dependent. The platform is scalable to microwell formats, and we provide proof-of-concept for its use in testing entry inhibitors and antiviral compounds.