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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., T.G.C. was funded by Knowledge Economy Skills Scholarships (KESS 2) PhD studentship award (Reference 515374) and Medical Research Council (MRC) Confidence in Concept award (Reference 520464), both to A.L.P.: 515374. A.J. acknowledges funding from the Ministerio de Ciencia e Innovacion PID2020-118436GB-I00, FP7-ICT-610730 (EVOPROG), BBSRC BB/P020615/1 (EVO-ENGINE), EPSRC-BBSRC BB/M017982/1 (WISB Centre), and the departmental allocation from the School of Life Sciences (University of Warwick).

Purpose:Broadly expressed, highly differentiated tumor-associated antigens (TAA) can elicit antitumor immunity. However, vaccines targeting TAAs have demonstrated disappointing clinical results, reflecting poor antigen selection and/or immunosuppressive mechanisms.Experimental Design:Here, a panel of widely expressed, novel colorectal TAAs were identified by performing RNA sequencing of highly purified colorectal tumor cells in comparison with patient-matched colonic epithelial cells; tumor cell purification was essential to reveal these genes. Candidate TAA protein expression was confirmed by IHC, and preexisting T-cell immunogenicity toward these antigens tested.Results:The most promising candidate for further development is DNAJB7 [DnaJ heat shock protein family (Hsp40) member B7], identified here as a novel cancer-testis antigen. It is expressed in many tumors and is strongly immunogenic in patients with cancers originating from a variety of sites. DNAJB7-specific T cells were capable of killing colorectal tumor lines in vitro, and the IFNγ+ response was markedly magnified by control of immunosuppression with cyclophosphamide in patients with cancer.Conclusions:This study highlights how prior methods that sequence whole tumor fractions (i.e., inclusive of alive/dead stromal cells) for antigen identification may have limitations. Through tumor cell purification and sequencing, novel candidate TAAs have been identified for future immunotherapeutic targeting.

Supplemental Figure I. Homology modelling of the Ad5NULL-A20 complex; Supplement II. Biodistribution study: Heat-map images of luminescence intensity ex vivo; Supplement II. Biodistribution study: Immunohistochemistry on formalin-fixed, paraffinembedded liver sections; Supplementary Figure IV. Pilot study: Tumour localisation and take rate in NOD/SCID mice; Supplement II. Biodistribution study: Immunohistochemistry on formalin-fixed, paraffinembedded; liver sections; Supplementary Figure IV. Pilot study: Tumour localisation and take rate in NOD/SCID mice.

Supplementary Figures 1-7

Purpose: Virotherapies are maturing in the clinical setting. Adenoviruses (Ad) are excellent vectors for the manipulability and tolerance of transgenes. Poor tumor selectivity, off-target sequestration, and immune inactivation hamper clinical efficacy. We sought to completely redesign Ad5 into a refined, tumor-selective virotherapy targeted to αvβ6 integrin, which is expressed in a range of aggressively transformed epithelial cancers but nondetectable in healthy tissues.Experimental Design: Ad5NULL-A20 harbors mutations in each major capsid protein to preclude uptake via all native pathways. Tumor-tropism via αvβ6 targeting was achieved by genetic insertion of A20 peptide (NAVPNLRGDLQVLAQKVART) within the fiber knob protein. The vector's selectivity in vitro and in vivo was assessed.Results: The tropism-ablating triple mutation completely blocked all native cell entry pathways of Ad5NULL-A20 via coxsackie and adenovirus receptor (CAR), αvβ3/5 integrins, and coagulation factor 10 (FX). Ad5NULL-A20 efficiently and selectively transduced αvβ6+ cell lines and primary clinical ascites-derived EOC ex vivo, including in the presence of preexisting anti-Ad5 immunity. In vivo biodistribution of Ad5NULL-A20 following systemic delivery in non–tumor-bearing mice was significantly reduced in all off-target organs, including a remarkable 107-fold reduced genome accumulation in the liver compared with Ad5. Tumor uptake, transgene expression, and efficacy were confirmed in a peritoneal SKOV3 xenograft model of human EOC, where oncolytic Ad5NULL-A20–treated animals demonstrated significantly improved survival compared with those treated with oncolytic Ad5.Conclusions: Oncolytic Ad5NULL-A20 virotherapies represent an excellent vector for local and systemic targeting of αvβ6-overexpressing cancers and exciting platforms for tumor-selective overexpression of therapeutic anticancer modalities, including immune checkpoint inhibitors. Clin Cancer Res; 24(17); 4215–24. ©2018 AACR.

Human adenoviruses (HAdV) are widespread pathogens causing infections of the respiratory and gastrointestinal tracts, genitourinary system and the eye. Species D (HAdV-D) are the most diverse species and cause both gastrointestinal tract infections and epidemic keratoconjunctivitis (EKC). Despite being significant pathogens, HAdV-D are understudied and knowledge around basic mechanisms of cell infection is lacking. Sialic acid (SA) usage has been proposed as a major mechanism of cell infection for EKC causing HAdV-D. Here, we provide apo state crystal structures for fiber knob proteins of 7 previously undetermined HAdV-D, and provide crystal structures of HAdV-D25, HAdV-D29 and HAdV-D53 knob proteins bound to 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. Together, these data indicate an important role for SA engagement in the tropism of many HAdV-D and may facilitate the development of suitable antivirals to control EKC outbreaks.

The presence of infectious viral particles in bioaerosols generated during laparoscopic surgery places surgical staff at significant risk of infection and represents a major cause of nosocomial infection. These factors contributed to the postponement and cancellation of countless surgical procedures during the early stages of the ongoing COVID-19 pandemic, causing backlogs, increased waiting times for surgical procedures and excess deaths indirectly related to the pandemic. The development and implementation of devices that effectively inactivate viral particles from bioaerosols would be beneficial in limiting or preventing the spread of infections from such bioaerosols. Here, we sought to evaluate whether electrostatic precipitation (EP) is a viable means to capture and inactivate both non-enveloped (Adenovirus) and enveloped (SARS-CoV-2 Pseudotyped Lentivirus) viral particles present in bioaerosols. We developed a closed-system model to mimic the release of bioaerosols during laparoscopic surgery. Known concentrations of each virus were aerosolised into the model system, exposed to EP using a commercially available system (UltravisionTM, Alesi Surgical Limited, UK) and collected in a BioSampler for analysis. Using qPCR to quantify viral genomes and transduction assays to quantify biological activity, we show that both enveloped and non-enveloped viral particles were efficiently captured and inactivated by EP. Both capture and inactivation could be further enhanced when increasing the voltage to 10kV, or when using two Ultravision™discharge electrodes together at 8kV. This study highlights EP as an efficient means for capturing and inactivating viral particles present in bioaerosols. The use of EP may limit the spread of diseases, reducing nosocomial infections and potentially enable the continuation of surgical procedures during periods of viral pandemics.HighlightsBioaerosols released from patients during surgery have the potential to facilitate viral spread.Ultravision™technology works via the process of electrostatic precipitation.Electrostatic precipitation can be manipulated to capture and inactivate aerosolised viral particles, preventing viral spread.Electrostatic precipitation is effective against both enveloped and non-enveloped viral particles.Electrostatic precipitation represents a viable means to reduce nosocomial infections.Graphical Abstract

Advances in gene therapy, synthetic biology, cancer genomics, and patient-derived cancer models have expanded the repertoire of strategies for targeting human cancers using viral vectors. Novel capsids, synthetic promoters, and therapeutic payloads are being developed and assessed through approaches such as rational design, pooled library screening, and directed evolution. Ultimately, the goal is to generate precision-engineered viruses that target different facets of tumor cell biology, without compromising normal tissue and organ function. In this study, we briefly review the opportunities for engineering cancer selectivity into viral vectors at both the cell extrinsic and intrinsic level. Such stringently tumor-targeted vectors can subsequently act as platforms for the delivery of potent therapeutic transgenes, including the exciting prospect of immunotherapeutic payloads. These have the potential to eradicate nontransduced cells through stimulation of systemic anticancer immune responses, thereby side-stepping the inherent challenge of achieving gene delivery to the entire cancer cell population. We discuss the importance of using advanced primary human cellular models, such as patient-derived cultures and organoids, to enable rapid screening and triage of novel candidates using disease-relevant models. We believe this combination of improved delivery and selectivity, through novel capsids and promoters, coupled with more potent choices for the combinations of immunotherapy-based payloads seems capable of finally delivering innovative new gene therapies for oncology. Many pieces of the puzzle of how to build a virus capable of targeting human cancers appear to be falling into place.

Background Diffuse midline glioma, formerly DIPG (diffuse intrinsic pontine glioma), is the deadliest pediatric brainstem tumor with median survival of less than one year. Here, we investigated (i) whether direct delivery of adenovirus-expressing cluster of differentiation (CD)40 ligand (Ad-CD40L) to brainstem tumors would induce immune-mediated tumor clearance and (ii) if so, whether therapy would be associated with a manageable toxicity due to immune-mediated inflammation in the brainstem. Methods Syngeneic gliomas in the brainstems of immunocompetent mice were treated with Ad-CD40L and survival, toxicity, and immune profiles determined. A clinically translatable vector, whose replication would be tightly restricted to tumor cells, rAd-Δ24-CD40L, was tested in human patient–derived diffuse midline gliomas and immunocompetent models. Results Expression of Ad-CD40L restricted to brainstem gliomas by pre-infection induced complete rejection, associated with immune cell infiltration, of which CD4+ T cells were critical for therapy. Direct intratumoral injection of Ad-CD40L into established brainstem tumors improved survival and induced some complete cures but with some acute toxicity. RNA-sequencing analysis showed that Ad-CD40L therapy induced neuroinflammatory immune responses associated with interleukin (IL)-6, IL-1β, and tumor necrosis factor α. Therefore, to generate a vector whose replication, and transgene expression, would be tightly restricted to tumor cells, we constructed rAd-Δ24-CD40L, the backbone of which has already entered clinical trials for diffuse midline gliomas. Direct intratumoral injection of rAd-Δ24-CD40L, with systemic blockade of IL-6 and IL-1β, generated significant numbers of cures with readily manageable toxicity. Conclusions Virus-mediated delivery of CD40L has the potential to be effective in treating diffuse midline gliomas without obligatory neuroinflammation-associated toxicity.

Description, We observe previously unknown interactions between clinically important adenovirus vector capsids, platelet factor 4, and CAR., Vaccines derived from chimpanzee adenovirus Y25 (ChAdOx1), human adenovirus type 26 (HAdV-D26), and human adenovirus type 5 (HAdV-C5) are critical in combatting the severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic. As part of the largest vaccination campaign in history, ultrarare side effects not seen in phase 3 trials, including thrombosis with thrombocytopenia syndrome (TTS), a rare condition resembling heparin-induced thrombocytopenia (HIT), have been observed. This study demonstrates that all three adenoviruses deployed as vaccination vectors versus SARS-CoV-2 bind to platelet factor 4 (PF4), a protein implicated in the pathogenesis of HIT. We have determined the structure of the ChAdOx1 viral vector and used it in state-of-the-art computational simulations to demonstrate an electrostatic interaction mechanism with PF4, which was confirmed experimentally by surface plasmon resonance. These data confirm that PF4 is capable of forming stable complexes with clinically relevant adenoviruses, an important step in unraveling the mechanisms underlying TTS.

Oncolytic viruses possess the ability to infect, replicate and lyse malignantly transformed tumour cells. This oncolytic activity amplifies the therapeutic advantage and induces a form of immunogenic cell death, characterized by increased CD8 + T‐cell infiltration into the tumour microenvironment. This important feature of oncolytic viruses can result in the warming up of immunologically ‘cold’ tumour types, presenting the enticing possibility that oncolytic virus treatment combined with immunotherapies may enhance efficacy. In this review, we assess some of the most promising candidates that might be used for oncolytic virotherapy: immunotherapy combinations. We assess their potential as separate agents or as agents combined into a single therapy, where the immunotherapy is encoded within the genome of the oncolytic virus. The development of such advanced agents will require increasingly sophisticated model systems for their preclinical assessment and evaluation. In vivo rodent model systems are fraught with limitations in this regard. Oncolytic viruses replicate selectively within human cells and therefore require human xenografts in immune‐deficient mice for their evaluation. However, the use of immune‐deficient rodent models hinders the ability to study immune responses against any immunomodulatory transgenes engineered within the viral genome and expressed within the tumour microenvironment. There has therefore been a shift towards the use of more sophisticated ex vivo patient‐derived model systems based on organoids and explant co‐cultures with immune cells, which may be more predictive of efficacy than contrived and artificial animal models. We review the best of those model systems here., Oncolytic virotherapies have immense potential to ‘heat up’ otherwise immunologically ‘cold’ tumour types. In this review, the capacity of virotherapies to synergize with or express a range of types of immunotherapies is explored. Optimizing such combinations in preclinical models is complex, and we therefore review the most clinically relevant rodent and ex vivo human models available for such assessment.

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 αvβ6 integrin-selective peptide, A20, to target αvβ6-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 αvβ6 integrin into the DG loop of HAdV-D10 fiber knob. Assays in αvβ6+ cancer cell lines demonstrated significantly increased transduction mediated by αvβ6-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 αvβ6 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 αvβ6 integrin-selective peptide resulted in HAdV-D10.A20, with significant potential for clinical translation.

Adenoviruses represent exceptional candidates for wide-ranging therapeutic applications, from vectors for gene therapy to oncolytics for cancer treatments. The first ever commercial gene therapy medicine was based on a recombinant adenovirus vector, while most recently, adenoviral vectors have proven critical as vaccine platforms in effectively controlling the global coronavirus pandemic. Here, we discuss factors involved in adenovirus cell binding, entry, and trafficking; how they influence efficiency of adenovirus-based vectors; and how they can be manipulated to enhance efficacy of genetically modified adenoviral variants. We focus particularly on endocytosis and how different adenovirus serotypes employ different endocytic pathways to gain cell entry, and thus, have different intracellular trafficking pathways that subsequently trigger different host antiviral responses. In the context of gene therapy, the final goal of the adenovirus vector is to efficiently deliver therapeutic transgenes into the target cell nucleus, thus allowing its functional expression. Aberrant or inefficient endocytosis can impede this goal, therefore, it should be considered when designing and constructing adenovirus-based vectors.

The human adenovirus phylogenetic tree is split across seven species (A–G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of pre-existing immunity detected across screened populations. However, many aspects of the basic virology of species D—such as their cellular tropism, receptor usage, and in vivo biodistribution profile—remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49)—a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry, but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting, whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells, and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen, whilst avoiding liver interactions, such as intravascular vaccine applications.

Vaccine-induced immune thrombotic thrombocytopenia (VITT), or thrombotic thrombocytopenic syndrome (TTS), has caused global concern. VITT is characterized by thrombosis and thrombocytopenia following COVID-19 vaccinations with the AstraZeneca ChAdOx1 nCov-19 and the Janssen Ad26.COV2.S vaccines. The clinical features of VITT include thrombosis, typically cerebral venous thrombosis, and severe thrombocytopenia developing 5 to 24 days following first dose of vaccine, with elevated D-dimer, and antibodies specific to platelet factor 4 (PF4), signifying platelet activation. As of June 1, 2021, over 1.93 billion COVID-19 vaccine doses had been administered worldwide. Currently, 467 VITT cases (0.000024%) have been reported across the UK, Europe, Canada and Australia. Clinically, VITT presents similarly to a rare autoimmune condition called “spontaneous/autoimmune heparin Induced Thrombocytopenia” (HIT) without prior heparin exposure. Guidance on diagnosis and management of VITT has been reported but the pathogenic mechanism of VITT is not fully elucidated. A definite causal relationship with the vaccine material is yet to be confirmed. To date, it is established that IgG antibodies recognizing PF4 activate platelets through FcγRIIA, however it remains unclear what triggers production of these antibodies. The fact that VITT, has only been described in association with adenoviral vector-based DNA virus vaccines, but not mRNA/lipid-based vaccines, raises the likelihood that the syndrome is somehow linked to the vector or other constituents in the vaccine preparation. Here, we propose and discuss potential mechanisms in relation to adenovirus induction of VITT. We discuss adenovirus immunogenicity and interactions with platelets and other host proteins, the role of PF4 and platelet activation. Whilst confirming a single mechanism underpinning VITT is challenging, we provide insights and clues into areas warranting investigation into the mechanistic basis of VITT, highlighting the unanswered questions. Further research is required to help solidify a pathogenic model for this condition.

We previously developed a refined, tumor-selective adenovirus, Ad5NULL-A20, harboring tropism ablating mutations in each major capsid protein, to ablate all native means of infection. We incorporated a 20-mer peptide (A20) in the fiber knob for selective infection via αvβ6 integrin, a marker of aggressive epithelial cancers. Methods: To ascertain the selectivity of Ad5NULL-A20 for αvβ6-positive tumor cell lines of pancreatic and breast cancer origin, we performed reporter gene and cell viability assays. Biodistribution of viral vectors in mice harboring xenografts with low, medium, and high αvβ6 levels was quantified by qPCR for viral genomes 48 h post intravenous administration. Results: Ad5NULL-A20 vector transduced cells in an αvβ6-selective manner, whilst cell killing mediated by oncolytic Ad5NULL-A20 was αvβ6-selective. Biodistribution analysis following intravenous administration into mice bearing breast cancer xenografts demonstrated that Ad5NULL-A20 resulted in significantly reduced liver accumulation coupled with increased tumor accumulation compared to Ad5 in all three models, with tumor-to-liver ratios improved as a function of αvβ6 expression. Conclusions: Ad5NULL-A20-based virotherapies efficiently target αvβ6-integrin-positive tumors following intravenous administration, validating the potential of Ad5NULL-A20 for systemic applications, enabling tumor-selective overexpression of virally encoded therapeutic transgenes.

Adenovirus derived vectors, based on chimpanzee adenovirus Y25 (ChAdOx1) and human adenovirus type 26 are proving critical in combatting the 2019 SARS-CoV-2 pandemic. Following emergency use authorisation, scale up in vaccine administration has inevitably revealed vaccine related adverse effects; too rare to observe even in large Phase-III clinical trials. These include vaccine-induced thrombotic thrombocytopenia (VITT), an ultra-rare adverse event in which patients develop life-threatening blood clots 5-24 days following vaccination.To investigate vector-host interactions of ChAdOx1 underpinning VITT we solved the structure of the ChAdOx1 capsid by CryoEM, and the structure of the primary receptor tropism determining fiber-knob protein by crystallography. These structural insights have enabled us to unravel key protein interactions involved in ChAdOx1 cell entry and a possible means by which it may generate misplaced immunity to platelet factor 4 (PF4), a protein involved in coagulation.We use in vitro cell binding assays to show that the fiber-knob protein uses coxsackie and adenovirus receptor (CAR) as a high affinity binding partner, while it does not form a stable interface with CD46. Computational simulations identified a putative mechanism by which the ChAdOx1 capsid interacts with PF4 by binding in the spaces between hexon proteins, with downstream implications for the causes of VITT.SummaryWe present the structure of the ChAdOx1 viral vector, derived from chimpanzee adenovirus Y25 at 4.2Å resolution1. ChAdOx1 is in global use in the AstraZeneca vaccine, ChAdOx1 nCoV-19/AZD-1222, to combat the SARS-CoV-2 coronavirus pandemic. Recently observed, rare, adverse events make detailed mechanistic understanding of this vector key to informing proper treatment of affected patients and the development of safer viral vectors.Here, we determine a primary mechanism ChAdOx1 uses to attach to cells is coxsackie and adenovirus receptor (CAR), a protein which is identical in humans and chimpanzees. We demonstrate the vector does not form a stable CD46 interaction, a common species B adenovirus receptor, via its primary attachment protein.Further, we reveal the surface of the ChAdOx1 viral capsid has a strong electronegative potential. Molecular simulations suggest this charge, together with shape complementarity, are a mechanism by which an oppositely charged protein, platelet factor 4 (PF4) may bind the vector surface. PF4 is a key protein involved in the formation of blood clots2, and the target of auto-antibodies in heparin-induced immune thrombotic thrombocytopenia (HITT)3, an adverse reaction to heparin therapy which presents similarly to vaccine-induced immune thrombotic thrombocytopenia (VITT), a rare complication of ChAdOx1 nCoV-19 vaccination4–6. We propose a mechanism in which the ChAdOx1-PF4 complex may stimulate the production of antibodies against PF4, leading to delayed blood clot formation, as observed in VITT.

Early phase clinical trials have demonstrated good therapeutic index for oncolytic adenoviruses in patients with solid tumours when administered intratumorally, resulting in local tumour elimination. Entrapment and binding of adenovirus to erythrocytes, blood factors, and neutralising antibodies have prevented efficient systemic delivery and targeting of distant lesions in the clinic. We previously generated the novel replication-selective Ad-3∆-A20T to improve tumour targeting by increasing the viral dose at distant sites. Here, we developed a protocol to directly radiolabel the virus for rapid and sensitive detection by single-photon emitted computed tomography (SPECT/CT) providing a convenient method for determining biodistribution following intravenous administration in murine models. Longitudinal whole-body scans, demonstrated efficient viral uptake in pancreatic Suit-2 and Panc04.03 xenografts with trace amounts of 125I-Ad-3∆-A20T up to 48 h after tail vein delivery. Hepatic and splenic radioactivity decreased over time. Analysis of tissues harvested at the end of the study, confirmed potency and selectivity of mutant viruses. Ad-3∆-A20T-treated animals showed higher viral genome copy numbers and E1A gene expression in tumors than in liver and spleen compared to Ad5wt. Our direct radiolabeling approach, allows for immediate screening of novel oncolytic adenoviruses and selection of optimal viral genome alterations to generate improved mutants.

Adenoviruses are powerful tools experimentally and clinically. To maximize efficacy, the development of serotypes with low preexisting levels of immunity in the population is desirable., The human adenovirus (HAdV) phylogenetic tree is diverse, divided across seven species and comprising over 100 individual types. Species D HAdV are rarely isolated with low rates of preexisting immunity, making them appealing for therapeutic applications. Several species D vectors have been developed as vaccines against infectious diseases, where they induce robust immunity in preclinical models and early phase clinical trials. However, many aspects of the basic virology of species D HAdV, including their basic receptor usage and means of cell entry, remain understudied. Here, we investigated HAdV-D49, which previously has been studied for vaccine and vascular gene transfer applications. We generated a pseudotyped HAdV-C5 presenting the HAdV-D49 fiber knob protein (HAdV-C5/D49K). This pseudotyped vector was efficient at infecting cells devoid of all known HAdV receptors, indicating HAdV-D49 uses an unidentified cellular receptor. Conversely, a pseudotyped vector presenting the fiber knob protein of the closely related HAdV-D30 (HAdV-C5/D30K), differing in four amino acids from HAdV-D49, failed to demonstrate the same tropism. These four amino acid changes resulted in a change in isoelectric point of the knob protein, with HAdV-D49K possessing a basic apical region compared to a more acidic region in HAdV-D30K. Structurally and biologically we demonstrate that HAdV-D49 knob protein is unable to engage CD46, while potential interaction with coxsackievirus and adenovirus receptor (CAR) is extremely limited by extension of the DG loop. HAdV-C5/49K efficiently transduced cancer cell lines of pancreatic, breast, lung, esophageal, and ovarian origin, indicating it may have potential for oncolytic virotherapy applications, especially for difficult to transduce tumor types. IMPORTANCE Adenoviruses are powerful tools experimentally and clinically. To maximize efficacy, the development of serotypes with low preexisting levels of immunity in the population is desirable. Consequently, attention has focused on those derived from species D, which have proven robust vaccine platforms. This widespread usage is despite limited knowledge in their basic biology and cellular tropism. We investigated the tropism of HAdV-D49, demonstrating that it uses a novel cell entry mechanism that bypasses all known HAdV receptors. We demonstrate, biologically, that a pseudotyped HAdV-C5/D49K vector efficiently transduces a wide range of cell lines, including those presenting no known adenovirus receptor. Structural investigation suggests that this broad tropism is the result of a highly basic electrostatic surface potential, since a homologous pseudotyped vector with a more acidic surface potential, HAdV-C5/D30K, does not display a similar pantropism. Therefore, HAdV-C5/D49K may form a powerful vector for therapeutic applications capable of infecting difficult to transduce cells.

Simple Summary If harnessed appropriately, oncolytic viruses offer significant potential as anti-cancer agents. Such virotherapies can be engineered to replicate inside cancerous cells, stimulating the immune system, spreading daughter virions to surrounding cells and producing additional anticancer agents as a by-product of infection. To achieve this necessitates deep understanding of the biology of the virus and tumour cell, to tailor viruses from naturally pathogenic agents into refined, tumour selective “precision virotherapies” suitable for clinical translation. Here, we focus on the adenovirus, which in its pathogenic form causes transient and mild ocular, respiratory or gastrointestinal tract infections, depending on the serotype. We highlight advances that have been made in refining adenovirus to ablate natural means of infection and the strategies that have been employed to engineer viral tropism and selectivity for tumour cells. Further advances in these strategies will be required to deliver fully bespoke and efficacious precision virotherapies to the clinic. Abstract 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.

The human adenovirus (HAdV) phylogenetic tree is diverse, divided across seven species and comprising over 100 individual types. Species D HAdV are rarely isolated with low rates of pre-existing immunity, making them appealing for therapeutic applications. Several species D vectors have been developed as vaccines against infectious diseases where they induce robust immunity in pre-clinical models and early phase clinical trials. However, many aspects of the basic virology of species D HAdV, including their basic receptor usage and means of cell entry, remain understudied.Here, we investigated HAdV-D49, which previously has been studied for vaccine and vascular gene transfer applications. We generated a pseudotyped HAdV-C5 presenting the HAdV-D49 fiber knob protein (HAdV-C5/D49K). This pseudotyped vector was efficient at infecting cells devoid of all known HAdV receptors, indicating HAdV-D49 uses an unidentified cellular receptor. Conversely, a pseudotyped vector presenting the fiber knob protein of the closely related HAdV-D30 (HAdV-C5/D30K), differing in four amino acids to HAdV-D49, failed to demonstrate the same tropism. These four amino acid changes resulted in a change in isoelectric point of the knob protein, with HAdV-D49K possessing a basic apical region compared to a more acidic region in HAdV-D30K. Structurally and biologically we demonstrate that HAdV-D49 knob protein is unable to engage CD46, whilst potential interaction with CAR is extremely limited by extension of the DG loop. HAdV-C5/49K efficiently transduced cancer cell lines of pancreatic, breast, lung, oesophageal and ovarian origin, indicating it may have potential for oncolytic virotherapy applications, especially for difficult to transduce tumour types.ImportanceAdenoviruses are powerful tools experimentally and clinically. To maximise efficacy, the development of serotypes with low pre-existing levels of immunity in the population is desirable. Consequently, attention has focussed on those derived from species D, which have proven robust vaccine platforms. This widespread usage is despite limited knowledge in their basic biology and cellular tropism.We investigated the tropism of HAdV-D49, demonstrating it uses a novel cell entry mechanism that bypasses all known HAdV receptors. We demonstrate, biologically, that a pseudotyped HAdV-C5/D49K vector efficiently transduces a wide range of cell lines, including those presenting no known adenovirus receptor. Structural investigation suggests that this broad tropism is the result of a highly basic electrostatic surface potential, since a homologous pseudotyped vector with a more acidic surface potential, HAdV-C5/D30K, does not display a similar pan-tropism. Therefore, HAdV-C5/D49K may form a powerful vector for therapeutic applications capable of infecting difficult to transduce cells.

Background The COVID‐19 global pandemic has resulted in a plethora of guidance and opinion from surgical societies. A controversial area concerns the safety of surgically created smoke and the perceived potential higher risk in laparoscopic surgery. Methods The limited published evidence was analysed in combination with expert opinion. A review was undertaken of the novel coronavirus with regards to its hazards within surgical smoke and the procedures that could mitigate the potential risks to healthcare staff. Results Using existing knowledge of surgical smoke, a theoretical risk of virus transmission exists. Best practice should consider the operating room set‐up, patient movement and operating theatre equipment when producing a COVID‐19 operating protocol. The choice of energy device can affect the smoke produced, and surgeons should manage the pneumoperitoneum meticulously during laparoscopic surgery. Devices to remove surgical smoke, including extractors, filters and non‐filter devices, are discussed in detail. Conclusion There is not enough evidence to quantify the risks of COVID‐19 transmission in surgical smoke. However, steps can be undertaken to manage the potential hazards. The advantages of minimally invasive surgery may not need to be sacrificed in the current crisis., The COVID‐19 pandemic is unprecedented and raises concerns regarding transmission of the virus within surgical smoke. This review summarizes the current evidence and provides advice for its management. Bad for your health?

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.

Purpose: Virotherapies are maturing in the clinical setting. Adenoviruses (Ad) are excellent vectors for the manipulability and tolerance of transgenes. Poor tumor selectivity, off-target sequestration, and immune inactivation hamper clinical efficacy. We sought to completely redesign Ad5 into a refined, tumor-selective virotherapy targeted to αvβ6 integrin, which is expressed in a range of aggressively transformed epithelial cancers but nondetectable in healthy tissues. Experimental Design: Ad5NULL-A20 harbors mutations in each major capsid protein to preclude uptake via all native pathways. Tumor-tropism via αvβ6 targeting was achieved by genetic insertion of A20 peptide (NAVPNLRGDLQVLAQKVART) within the fiber knob protein. The vector's selectivity in vitro and in vivo was assessed. Results: The tropism-ablating triple mutation completely blocked all native cell entry pathways of Ad5NULL-A20 via coxsackie and adenovirus receptor (CAR), αvβ3/5 integrins, and coagulation factor 10 (FX). Ad5NULL-A20 efficiently and selectively transduced αvβ6+ cell lines and primary clinical ascites-derived EOC ex vivo, including in the presence of preexisting anti-Ad5 immunity. In vivo biodistribution of Ad5NULL-A20 following systemic delivery in non–tumor-bearing mice was significantly reduced in all off-target organs, including a remarkable 107-fold reduced genome accumulation in the liver compared with Ad5. Tumor uptake, transgene expression, and efficacy were confirmed in a peritoneal SKOV3 xenograft model of human EOC, where oncolytic Ad5NULL-A20–treated animals demonstrated significantly improved survival compared with those treated with oncolytic Ad5. Conclusions: Oncolytic Ad5NULL-A20 virotherapies represent an excellent vector for local and systemic targeting of αvβ6-overexpressing cancers and exciting platforms for tumor-selective overexpression of therapeutic anticancer modalities, including immune checkpoint inhibitors. Clin Cancer Res; 24(17); 4215–24. ©2018 AACR.

Metastatic pancreatic ductal adenocarcinomas (PDAC) are incurable due to the rapid development of resistance to all current therapeutics. Oncolytic adenoviral mutants have emerged as a promising new strategy that negates such resistance. In contrast to normal tissue, the majority of PDACs express the αvβ6 integrin receptor. To exploit this feature, we modified our previously reported oncolytic adenovirus, AdΔΔ, to selectively target αvβ6 integrins to facilitate systemic delivery. Structural modifications to AdΔΔ include the expression of the small but potent αvβ6-binding peptide, A20FMDV2, and ablation of binding to the native coxsackie and adenovirus receptor (CAR) within the fiber knob region. The resultant mutant, Ad5-3Δ-A20T, infected and killed αvβ6 integrin–expressing cells more effectively than the parental wild-type (Ad5wt) virus and AdΔΔ. Viral uptake through αvβ6 integrins rather than native viral receptors (CAR, αvβ3 and αvβ5 integrins) promoted viral propagation and spread. Superior efficacy of Ad5-3Δ-A20T compared with Ad5wt was demonstrated in 3D organotypic cocultures, and similar potency between the two viruses was observed in Suit-2 in vivo models. Importantly, Ad5-3Δ-A20T infected pancreatic stellate cells at low levels, which may further facilitate viral spread and cancer cell elimination either as a single agent or in combination with the chemotherapy drug, gemcitabine. We demonstrate that Ad5-3Δ-A20T is highly selective for αvβ6 integrin–expressing pancreatic cancer cells, and with further development, this new and exciting strategy can potentially be extended to improve the systemic delivery of adenoviruses to pancreatic cancer patients. Mol Cancer Ther; 17(2); 575–87. ©2018 AACR.

Background: The prognosis of patients with peritoneal metastases is poor. Treatment options are limited because systemically delivered chemotherapy is not usually effective in this type of disease. Pressurised intraperitoneal aerosolised chemotherapy (PIPAC) is a recently developed alternative technology for delivering intraperitoneal chemotherapy, potentially enhancing treatment efficacy. Here, we assess the feasibility of pressurised intraperitoneal aerosolised virotherapy (PIPAV) to deliver a different class of anticancer agents, oncolytic adenoviruses, in vitro and in vivo. Methods: Adenoviral vectors expressing reporter genes green fluorescence protein (Ad5.GFP) or firefly luciferase (Ad5.Luc) were subject to pressurised aerosolisation. The ability of the virus to survive PIPAV was assessed in vitro and in vivo by monitoring reporter gene activity. Wistar rats subjected to PIPAV were assessed for any adverse procedure related events. Results: In vitro transduction assays demonstrated that Ad5 retained viability following pressurised aerosolisation and could transduce permissive cells equally effectively as non-aerosolised control vector. PIPAV was well tolerated in rats, although minimal transduction was observed following intraperitoneal administration. Conclusions: PIPAV appears viable and well tolerated, though in vivo efficacy requires further optimisation.

Purpose: Broadly expressed, highly differentiated tumor-associated antigens (TAA) can elicit antitumor immunity. However, vaccines targeting TAAs have demonstrated disappointing clinical results, reflecting poor antigen selection and/or immunosuppressive mechanisms. Experimental Design: Here, a panel of widely expressed, novel colorectal TAAs were identified by performing RNA sequencing of highly purified colorectal tumor cells in comparison with patient-matched colonic epithelial cells; tumor cell purification was essential to reveal these genes. Candidate TAA protein expression was confirmed by IHC, and preexisting T-cell immunogenicity toward these antigens tested. Results: The most promising candidate for further development is DNAJB7 [DnaJ heat shock protein family (Hsp40) member B7], identified here as a novel cancer-testis antigen. It is expressed in many tumors and is strongly immunogenic in patients with cancers originating from a variety of sites. DNAJB7-specific T cells were capable of killing colorectal tumor lines in vitro, and the IFNγ+ response was markedly magnified by control of immunosuppression with cyclophosphamide in patients with cancer. Conclusions: This study highlights how prior methods that sequence whole tumor fractions (i.e., inclusive of alive/dead stromal cells) for antigen identification may have limitations. Through tumor cell purification and sequencing, novel candidate TAAs have been identified for future immunotherapeutic targeting.

Human adenovirus type 26 uses sialic acid as a primary cellular receptor—structural insights for this phase 3 vaccine vector., Adenoviruses are clinically important agents. They cause respiratory distress, gastroenteritis, and epidemic keratoconjunctivitis. As non-enveloped, double-stranded DNA viruses, they are easily manipulated, making them popular vectors for therapeutic applications, including vaccines. Species D adenovirus type 26 (HAdV-D26) is both a cause of EKC and other diseases and a promising vaccine vector. HAdV-D26–derived vaccines are under investigation as protective platforms against HIV, Zika, and respiratory syncytial virus infections and are in phase 3 clinical trials for Ebola. We recently demonstrated that HAdV-D26 does not use CD46 or Desmoglein-2 as entry receptors, while the putative interaction with coxsackie and adenovirus receptor is low affinity and unlikely to represent the primary cell receptor. Here, we establish sialic acid as a primary entry receptor used by HAdV-D26. We demonstrate that removal of cell surface sialic acid inhibits HAdV-D26 infection, and provide a high-resolution crystal structure of HAdV-D26 fiber-knob in complex with sialic acid.

Adenoviruses are clinically important agents. They cause respiratory distress, gastroenteritis, and epidemic keratoconjunctivitis (EKC). As non-enveloped, double stranded DNA viruses, they are easily manipulated, making them popular vectors for therapeutic applications, including vaccines. Species D adenovirus serotype 26 (HAdV-D26) is both a cause of EKC and other disease, and a promising vaccine vector. HAdV-D26 derived vaccines are under investigation as protective platforms against HIV, Zika, RSV infections and are in Phase-III clinical trials for Ebola.We recently demonstrated that HAdV-D26 does not utilise CD46 or desmoglein 2 as entry receptors, whilst the putative interaction with Coxsackie and Adenovirus Receptor (CAR) is low affinity and unlikely to represent the primary cell receptor.Here, we definitively establish sialic acid as the primary entry receptor utilised by HAdV-D26. We demonstrate removal of cell surface sialic acid inhibits HAdV-D26 infection and provide a high-resolution crystal structure of HAdV-D26 fiber-knob in complex with sialic acid.

Adenoviruses (Ad) are commonly used both experimentally and clinically, including oncolytic virotherapy applications. In the clinical area, efficacy is frequently hampered by the high rates of neutralizing immunity, estimated as high as 90% in some populations that promote vector clearance and limit bioavailability for tumor targeting following systemic delivery. Active tumor targeting is also hampered by the ubiquitous nature of the Ad5 receptor, hCAR, as well as the lack of highly tumor-selective targeting ligands and suitable targeting strategies. Furthermore, significant off-target interactions between the viral vector and cellular and proteinaceous components of the bloodstream have been documented that promote uptake into non-target cells and determine dose-limiting toxicities. Novel strategies are therefore needed to overcome the obstacles that prevent efficacious Ad deployment for wider clinical applications. The use of less seroprevalent Ad serotypes, non-human serotypes, capsid pseudotyping, chemical shielding and genetic masking by heterologous peptide incorporation are all potential strategies to achieve efficient vector escape from humoral immune recognition. Conversely, selective vector arming with immunostimulatory agents can be utilized to enhance their oncolytic potential by activation of cancer-specific immune responses against the malignant tissues. This review presents recent advantages and pitfalls occurring in the field of adenoviral oncolytic therapies.