Broad sialic acid usage amongst species D human adenovirus.

Human adenoviruses (HAdV) are widespread pathogens causing usually mild infections. The Species D (HAdV-D) cause gastrointestinal tract infections and epidemic keratoconjunctivitis (EKC). Despite being significant pathogens, knowledge around HAdV-D mechanism of cell infection is lacking. Sialic acid (SA) usage has been proposed as a cell infection mechanism for EKC causing HAdV-D. Here we highlight an important role for SA engagement by many HAdV-D. We provide apo state crystal structures of 7 previously undetermined HAdV-D fiber-knob proteins, and structures of HAdV-D25, D29, D30 and D53 fiber-knob proteins in complex with SA. Biologically, we demonstrate that removal of cell surface SA reduced infectivity of HAdV-C5 vectors pseudotyped with HAdV-D fiber-knob proteins, whilst engagement of the classical HAdV receptor CAR was variable. Our data indicates variable usage of SA and CAR across HAdV-D. Better defining these interactions will enable improved development of antivirals and engineering of the viruses into refined therapeutic vectors.

Pseudotyping Bacteriophage P2 Tail Fibers to Extend the Host Range for Biomedical Applications.

Bacteriophages (phages) represent powerful potential treatments against antibiotic-resistant bacterial infections. Antibiotic-resistant bacteria represent a significant threat to global health, with an estimated 70% of infection-causing bacteria being resistant to one or more antibiotics. Developing novel antibiotics against the limited number of cellular targets is expensive and time-consuming, and bacteria can rapidly develop resistance. While bacterial resistance to phage can evolve, bacterial resistance to phage does not appear to spread through lateral gene transfer, and phage may similarly adapt through mutation to recover infectivity. Phages have been identified for all known bacteria, allowing the strain-selective killing of pathogenic bacteria. Here, we re-engineered the Escherichia coli phage P2 to alter its tropism toward pathogenic bacteria. Chimeric tail fibers formed between P2 and S16 genes were designed and generated through two approaches: homology- and literature-based. By presenting chimeric P2:S16 fibers on the P2 particle, our data suggests that the resultant phages were effectively detargeted from the native P2 cellular target, lipopolysaccharide, and were instead able to infect via the proteinaceous receptor, OmpC, the natural S16 receptor. Our work provides evidence that pseudotyping P2 is feasible and can be used to extend the host range of P2 to alternative receptors. Extension of this work could produce alternative chimeric tail fibers to target pathogenic bacterial threats. Our engineering of P2 allows adsorption through a heterologous outer-membrane protein without culturing in its native host, thus providing a potential means of engineering designer phages against pathogenic bacteria from knowledge of their surface proteome.

Development of a low-seroprevalence, αvß6 integrin-selective virotherapy based on human adenovirus type 10.

Oncolytic virotherapies (OV) hold immense clinical potential. OV based on human adenoviruses (HAdV) derived from HAdV with naturally low rates of pre-existing immunity will be beneficial for future clinical translation. We generated a low-seroprevalence HAdV-D10 serotype vector incorporating an αvß6 integrin-selective peptide, A20, to target αvß6-positive tumor cell types. HAdV-D10 has limited natural tropism. Structural and biological studies of HAdV-D10 knob protein highlighted low-affinity engagement with native adenoviral receptors CAR and sialic acid. HAdV-D10 fails to engage blood coagulation factor X, potentially eliminating "off-target" hepatic sequestration in vivo. We engineered an A20 peptide that selectively binds αvß6 integrin into the DG loop of HAdV-D10 fiber knob. Assays in αvß6+ cancer cell lines demonstrated significantly increased transduction mediated by αvß6-targeted variants compared with controls, confirmed microscopically. HAdV-D10.A20 resisted neutralization by neutralizing HAdV-C5 sera. Systemic delivery of HAdV-D10.A20 resulted in significantly increased GFP expression in BT20 tumors. Replication-competent HAdV-D10.A20 demonstrated αvß6 integrin-selective cell killing in vitro and in vivo. HAdV-D10 possesses characteristics of a promising virotherapy, combining low seroprevalence, weak receptor interactions, and reduced off-target uptake. Incorporation of an αvß6 integrin-selective peptide resulted in HAdV-D10.A20, with significant potential for clinical translation.

Hitting the Target but Missing the Point: Recent Progress towards Adenovirus-Based Precision Virotherapies.

More people are surviving longer with cancer. Whilst this can be partially attributed to advances in early detection of cancers, there is little doubt that the improvement in survival statistics is also due to the expansion in the spectrum of treatments available for efficacious treatment. Transformative amongst those are immunotherapies, which have proven effective agents for treating immunogenic forms of cancer, although immunologically "cold" tumour types remain refractive. Oncolytic viruses, such as those based on adenovirus, have great potential as anti-cancer agents and have seen a resurgence of interest in recent years. Amongst their many advantages is their ability to induce immunogenic cell death (ICD) of infected tumour cells, thus providing the alluring potential to synergise with immunotherapies by turning immunologically "cold" tumours "hot". Additionally, enhanced immune mediated cell killing can be promoted through the local overexpression of immunological transgenes, encoded from within the engineered viral genome. To achieve this full potential requires the development of refined, tumour selective "precision virotherapies" that are extensively engineered to prevent off-target up take via native routes of infection and targeted to infect and replicate uniquely within malignantly transformed cells. Here, we review the latest advances towards this holy grail within the adenoviral field.

Identification of folate receptor α (FRα) binding oligopeptides and their evaluation for targeted virotherapy applications.

Oncolytic virotherapies (OV) based on human adenoviral (HAdV) vectors hold significant promise for the treatment of advanced ovarian cancers where local, intraperitoneal delivery to tumour metastases is feasible, bypassing many complexities associated with intravascular delivery. The efficacy of HAdV-C5-based OV is hampered by a lack of tumour selectivity, where the primary receptor, hCAR, is commonly downregulated during malignant transformation. Conversely, folate receptor alpha (FRα) is highly expressed on ovarian cancer cells, providing a compelling target for tumour selective delivery of virotherapies. Here, we identify high-affinity FRα-binding oligopeptides for genetic incorporation into HAdV-C5 vectors. Biopanning identified a 12-mer linear peptide, DWSSWVYRDPQT, and two 7-mer cysteine-constrained peptides, CIGNSNTLC and CTVRTSAEC that bound FRα in the context of the phage particle. Synthesised lead peptide, CTVRTSAEC, bound specifically to FRα and could be competitively inhibited with folic acid. To assess the capacity of the elucidated FRα-binding oligopeptides to target OV to FRα, we genetically incorporated the peptides into the HAdV-C5 fiber-knob HI loop including in vectors genetically ablated for hCAR interactions. Unfortunately, the recombinant vectors failed to efficiently target transduction via FRα due to defective intracellular trafficking following entry via FRα, indicating that whilst the peptides identified may have potential for applications for targeted drug delivery, they require additional refinement for targeted virotherapy applications.

Complete Genome Sequence of Peste des Petits Ruminants Virus from Georgia, 2016.

We report here the complete genome sequence of a peste des petits ruminants virus (PPRV) from the first outbreak of the disease in Georgia in January 2016. Genome sequencing was performed using Illumina next-generation sequencing technology in conjunction with Sanger sequencing. This PPRV/Georgia/Tbilisi/2016 genome sequence clustered within lineage IV PPRV viruses.