Human Complement Inhibits Myophages against Pseudomonas aeruginosa.

Therapeutic bacteriophages (phages) are primarily chosen based on their in vitro bacteriolytic activity. Although anti-phage antibodies are known to inhibit phage infection, the influence of other immune system components is less well known. An important anti-bacterial and anti-viral innate immune system that may interact with phages is the complement system, a cascade of proteases that recognizes and targets invading microorganisms. In this research, we aimed to study the effects of serum components such as complement on the infectivity of different phages targeting Pseudomonas aeruginosa. We used a fluorescence-based assay to monitor the killing of P. aeruginosa by phages of different morphotypes in the presence of human serum. Our results reveal that several myophages are inhibited by serum in a concentration-dependent way, while the activity of four podophages and one siphophage tested in this study is not affected by serum. By using specific nanobodies blocking different components of the complement cascade, we showed that activation of the classical complement pathway is a driver of phage inhibition. To determine the mechanism of inhibition, we produced bioorthogonally labeled fluorescent phages to study their binding by means of microscopy and flow cytometry. We show that phage adsorption is hampered in the presence of active complement. Our results indicate that interactions with complement may affect the in vivo activity of therapeutically administered phages. A better understanding of this phenomenon is essential to optimize the design and application of therapeutic phage cocktails.

Monitoring phage-induced lysis of gram-negatives in real time using a fluorescent DNA dye.

Bacteriophages (phages) are viruses that specifically attack bacteria. Their use as therapeutics, which constitutes a promising alternative to antibiotics, heavily relies on selecting effective lytic phages against the pathogen of interest. Current selection techniques are laborious and do not allow for direct visualization of phage infection dynamics. Here, we present a method that circumvents these limitations. It can be scaled for high-throughput and permits monitoring of the phage infection in real time via a fluorescence signal readout. This is achieved through the use of a membrane-impermeant nucleic acid dye that stains the DNA of damaged or lysed bacteria and new phage progeny. We have tested the method on Pseudomonas aeruginosa and Klebsiella pneumoniae and show that an increase in fluorescence reflects phage-mediated killing. This is confirmed by other techniques including spot tests, colony plating, flow cytometry and metabolic activity measurements. Furthermore, we illustrate how our method may be used to compare the activity of different phages and to screen the susceptibility of clinical isolates to phage. Altogether, we present a fast, reliable way of selecting phages against Gram-negative bacteria, which may be valuable in optimizing the process of selecting phages for therapeutic use.

Inhibition of H1 and H5 Influenza A Virus Entry by Diverse Macrocyclic Peptides Targeting the Hemagglutinin Stem Region.

Influenza A viruses pose a serious pandemic risk, while generation of efficient vaccines against seasonal variants remains challenging. There is thus a pressing need for new treatment options. We report here a set of macrocyclic peptides that inhibit influenza A virus infection at low nanomolar concentrations by binding to hemagglutinin, selected using ultrahigh-throughput screening of a diverse peptide library. The peptides are active against both H1 and H5 variants, with no detectable cytotoxicity. Despite the high sequence diversity across hits, all tested peptides were found to bind to the same region in the hemagglutinin stem by HDX-MS epitope mapping. A mutation in this region identified in an escape variant confirmed the binding site. This stands in contrast to the immunodominance of the head region for antibody binding and suggests that macrocyclic peptides from in vitro display may be well suited for finding new druggable sites not revealed by antibodies. Functional analysis indicates that these peptides stabilize the prefusion conformation of the protein and thereby prevent virus-cell fusion. High-throughput screening of macrocyclic peptides is thus shown here to be a powerful method for the discovery of novel broadly acting viral fusion inhibitors with therapeutic potential.

Mechanisms and clinical importance of bacteriophage resistance.

We are in the midst of a golden age of uncovering defense systems against bacteriophages. Apart from the fundamental interest in these defense systems, and revolutionary applications that have been derived from them (e.g. CRISPR-Cas9 and restriction endonucleases), it is unknown how defense systems contribute to resistance formation against bacteriophages in clinical settings. Bacteriophages are now being reconsidered as therapeutic agents against bacterial infections due the emergence of multidrug resistance. However, bacteriophage resistance through defense systems and other means could hinder the development of successful phage-based therapies. Here, we review the current state of the field of bacteriophage defense, highlight the relevance of bacteriophage defense for potential clinical use of bacteriophages as therapeutic agents and suggest new directions of research.

Janus Dendrimers to Assess the Anti-HCV Activity of Molecules in Cell-Assays.

The use of nanocarriers has been revealed as a valid strategy to facilitate drug bioavailability, and this allows for expanding the drug libraries for the treatment of certain diseases such as viral diseases. In the case of Hepatitis C, the compounds iopanoic acid and 3,3',5-triiodothyroacetic acid (or tiratricol) were identified in a primary screening as bioactive allosteric inhibitors of viral NS3 protease, but they did not exhibit accurate activity inhibiting viral replication in cell-based assays. In this work, dendritic nanocarriers are proposed due to their unique properties as drug delivery systems to rescue the bioactivity of these two drugs. Specifically, four different amphiphilic Janus dendrimers synthesized by combining 2,2'-bis(hydroxymethyl)propionic acid (bis-MPA) and 2,2'-bis(glyciloxy)propionic acid (bis-GMPA) functionalized with either hydrophilic or lipophilic moieties at their periphery were used to entrap iopanoic acid and tiratricol. Interestingly, differences were found in the loading efficiencies depending on the dendrimer design, which also led to morphological changes of the resulting dendrimer aggregates. The most remarkable results consist of the increased water solubility of the bioactive compounds within the dendrimers and the improved antiviral activity of some of the dendrimer/drug aggregates, considerably improving antiviral activity in comparison to the free drugs. Moreover, imaging studies have been developed in order to elucidate the mechanism of cellular internalization.