Author Correction: Genome-wide CRISPR-Cas9 screening reveals ubiquitous T cell cancer targeting via the monomorphic MHC class I-related protein MR1.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genome-wide CRISPR-Cas9 screening reveals ubiquitous T cell cancer targeting via the monomorphic MHC class I-related protein MR1.

Human leukocyte antigen (HLA)-independent, T cell-mediated targeting of cancer cells would allow immune destruction of malignancies in all individuals. Here, we use genome-wide CRISPR-Cas9 screening to establish that a T cell receptor (TCR) recognized and killed most human cancer types via the monomorphic MHC class I-related protein, MR1, while remaining inert to noncancerous cells. Unlike mucosal-associated invariant T cells, recognition of target cells by the TCR was independent of bacterial loading. Furthermore, concentration-dependent addition of vitamin B-related metabolite ligands of MR1 reduced TCR recognition of cancer cells, suggesting that recognition occurred via sensing of the cancer metabolome. An MR1-restricted T cell clone mediated in vivo regression of leukemia and conferred enhanced survival of NSG mice. TCR transfer to T cells of patients enabled killing of autologous and nonautologous melanoma. These findings offer opportunities for HLA-independent, pan-cancer, pan-population immunotherapies.

Adenovirus serotype 5 hexon mediates liver gene transfer.

Adenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo.

The Fiber Knob Protein of Human Adenovirus Type 49 Mediates Highly Efficient and Promiscuous Infection of Cancer Cell Lines Using a Novel Cell Entry Mechanism.

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.

Pouring petrol on the flames: Using oncolytic virotherapies to enhance tumour immunogenicity.

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.

Superlensing microscope objective lens.

Conventional microscope objective lenses are diffraction limited; they cannot resolve subdiffraction features of a size smaller than 250-300 nm under white lighting condition. New innovations are required to overcome this limitation. In this paper, we propose and demonstrate a new superlensing objective lens that possesses a resolution of 100 nm, which is a two-times resolution improvement over conventional objectives. This is accomplished by integrating a conventional microscope objective lens with a superlensing microsphere lens using a customized lens adaptor. The new objective lens was successfully demonstrated for label-free super-resolution imaging of 100 nm features in engineering and biological samples, including a Blu-ray disk sample and adenoviruses. Our work opens a new door to develop a generic optical superlens, which may transform the field of optical microscopy and imaging.

Retargeting FX-binding-ablated HAdV-5 to vascular cells by inclusion of the RGD-4C peptide in hexon hypervariable region 7 and the HI loop.

Recent studies have generated interest in the function of human adenovirus serotype 5 (HAdV-5) hexon: factor X (FX) binding and subsequent hepatocyte transduction and interaction with the immune system. Here, we retargeted adenovirus serotype 5 vectors, ablated for FX interaction, by replacing amino acids in hexon HVR7 with RGD-4C or inserting the peptide into the fibre HI loop. These genetic modifications in the capsid were compatible with virus assembly, and could efficiently retarget transduction of the vector via the αvß3/5 integrin-mediated pathway, but did not alter immune recognition by pre-existing human neutralizing anti-HAdV-5 antibodies or by natural antibodies in mouse serum. Thus, FX-binding-ablated HAdV-5 can be retargeted but remain sensitive to immune-mediated attack. These findings further refine HAdV-5-based vectors for human gene therapy and inform future vector development.

The Immune System-A Double-Edged Sword for Adenovirus-Based Therapies.

Pathogenic adenovirus (Ad) infections are widespread but typically mild and transient, except in the immunocompromised. As vectors for gene therapy, vaccine, and oncology applications, Ad-based platforms offer advantages, including ease of genetic manipulation, scale of production, and well-established safety profiles, making them attractive tools for therapeutic development. However, the immune system often poses a significant challenge that must be overcome for adenovirus-based therapies to be truly efficacious. Both pre-existing anti-Ad immunity in the population as well as the rapid development of an immune response against engineered adenoviral vectors can have detrimental effects on the downstream impact of an adenovirus-based therapeutic. This review focuses on the different challenges posed, including pre-existing natural immunity and anti-vector immunity induced by a therapeutic, in the context of innate and adaptive immune responses. We summarise different approaches developed with the aim of tackling these problems, as well as their outcomes and potential future applications.

Identification of cross reactive T cell responses in adenovirus based COVID 19 vaccines.

Vaccination has proven to be a valuable tool to combat SARS-CoV-2. However, reports of rare adverse reactions such as thrombosis/thrombocytopenia syndrome after ChAdOx1 nCoV-19 vaccination have caused scientific, public and media concern. ChAdOx1 was vectorised from the Y25 chimpanzee adenovirus, which was selected due to low human seroprevalence to circumvent pre-existing immunity. In this study, we aimed to explore patterns of T-cell activation after SARS-CoV-2 COVID-19 vaccine exposure in vitro using PBMCs collected from pre-pandemic ChAdOx1 nCoV-19 naïve healthy donors (HDs), and ChAdOx1 nCoV-19 and Pfizer vaccinated controls. PBMCs were assessed for T-cell proliferation using the lymphocyte transformation test (LTT) following exposure to SARS-CoV-2 COVID-19 vaccines. Cytokine analysis was performed via intracellular cytokine staining, ELISpot assay and LEGENDplex immunoassays. T-cell assays performed in pre-pandemic vaccine naïve HDs, revealed widespread lymphocyte stimulation after exposure to ChAdOx1 nCoV-19 (95%), ChAdOx-spike (90%) and the Ad26.COV2. S vaccine, but not on exposure to the BNT162b2 vaccine. ICS analysis demonstrated that CD4+ CD45RO+ memory T-cells are activated by ChAdOx1 nCoV-19 in vaccine naïve HDs. Cytometric immunoassays showed ChAdOx1 nCoV-19 exposure was associated with the release of proinflammatory and cytotoxic molecules, such as IFN-γ, IL-6, perforin, granzyme B and FasL. These studies demonstrate a ubiquitous T-cell response to ChAdOx1 nCoV-19 and Ad26.COV2. S in HDs recruited prior to the SARS-CoV-2 pandemic, with T-cell stimulation also identified in vaccinated controls. This may be due to underlying T-cell cross-reactivity with prevalent human adenoviruses and further study will be needed to identify T-cell epitopes involved.

Ad5:Ad48 hexon hypervariable region substitutions lead to toxicity and increased inflammatory responses following intravenous delivery.

The development of adenoviral vectors for intravascular (i.v.) delivery will require improvements to their in vivo safety and efficacy. The hypervariable regions (HVRs) of the Ad5 hexon are a target for neutralizing antibodies, but also interact with factor X (FX), facilitating hepatocyte transduction. Ad48, a species D adenovirus, does not bind FX and has low seroprevalence. Therefore, it has been suggested that Ad5HVR48(1-7), a hexon-chimeric vector featuring the seven HVRs from Ad48, should display advantageous properties for gene therapy, by evading pre-existing Ad5 immunity and blocking FX interactions. We investigated the in vivo biodistribution of Ad5, Ad5HVR48(1-7), and Ad48 following i.v. delivery. Ad5HVR48(1-7) displayed reduced hepatocyte transduction and accumulation in Kupffer cells (KCs), but triggered a robust proinflammatory response, even at relatively low doses of vector. We detected elevated serum transaminases (48 hours) and increased numbers of periportal CD11b(+)/Gr-1(+) cells in the livers of Ad5HVR48(1-7)-treated animals following i.v., but not intramuscular (i.m.), delivery. In contrast, Ad48 did not elevate transaminases or result in the accumulation of CD11b(+)/Gr-1(+) cells. Collectively, these findings suggest that substantial hexon modifications can lead to unexpected properties which cannot be predicted from parental viruses. Therefore, refined mutations may be preferential for the successful development of targeted vector systems which require i.v. administration.

Oncolytic Viruses and Immune Checkpoint Inhibitors: The "Hot" New Power Couple.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer care and shown remarkable efficacy clinically. This efficacy is, however, limited to subsets of patients with significant infiltration of lymphocytes into the tumour microenvironment. To extend their efficacy to patients who fail to respond or achieve durable responses, it is now becoming evident that complex combinations of immunomodulatory agents may be required to extend efficacy to patients with immunologically "cold" tumours. Oncolytic viruses (OVs) have the capacity to selectively replicate within and kill tumour cells, resulting in the induction of immunogenic cell death and the augmentation of anti-tumour immunity, and have emerged as a promising modality for combination therapy to overcome the limitations seen with ICIs. Pre-clinical and clinical data have demonstrated that OVs can increase immune cell infiltration into the tumour and induce anti-tumour immunity, thus changing a "cold" tumour microenvironment that is commonly associated with poor response to ICIs, to a "hot" microenvironment which can render patients more susceptible to ICIs. Here, we review the major viral vector platforms used in OV clinical trials, their success when used as a monotherapy and when combined with adjuvant ICIs, as well as pre-clinical studies looking at the effectiveness of encoding OVs to deliver ICIs locally to the tumour microenvironment through transgene expression.

Capture and inactivation of viral particles from bioaerosols by electrostatic precipitation.

Infectious viral particles in bioaerosols generated during laparoscopic surgery place staff and patients at significant risk of infection and contributed to the postponement of countless surgical procedures during the COVID-19 pandemic causing excess deaths. The implementation of devices that inactivate viral particles from bioaerosols aid in preventing nosocomial viral spread. We evaluated whether electrostatic precipitation (EP) is effective in capturing and inactivating aerosolized enveloped and non-enveloped viruses. Using a closed-system model mimicking release of bioaerosols during laparoscopic surgery, known concentrations of each virus were aerosolized, exposed to EP and collected for analysis. We demonstrate that both enveloped and non-enveloped viral particles were efficiently captured and inactivated by EP, which was enhanced by increasing the voltage to 10 kV or using two discharge electrodes together at 8 kV. This study highlights EP as an effective means for capturing and inactivating viral particles in bioaerosols, which may enable continued surgical procedures during future pandemics.

Engineering Adenoviral Vectors with Improved GBM Selectivity.

Glioblastoma (GBM) is the most common and aggressive adult brain cancer with an average survival rate of around 15 months in patients receiving standard treatment. Oncolytic adenovirus expressing therapeutic transgenes represent a promising alternative treatment for GBM. Of the many human adenoviral serotypes described to date, adenovirus 5 (HAdV-C5) has been the most utilised clinically and experimentally. However, the use of Ad5 as an anti-cancer agent may be hampered by naturally high seroprevalence rates to HAdV-C5 coupled with the infection of healthy cells via native receptors. To explore whether alternative natural adenoviral tropisms are better suited to GBM therapeutics, we pseudotyped an HAdV-C5-based platform using the fibre knob protein from alternative serotypes. We demonstrate that the adenoviral entry receptor coxsackie, adenovirus receptor (CAR) and CD46 are highly expressed by both GBM and healthy brain tissue, whereas Desmoglein 2 (DSG2) is expressed at a low level in GBM. We demonstrate that adenoviral pseudotypes, engaging CAR, CD46 and DSG2, effectively transduce GBM cells. However, the presence of these receptors on non-transformed cells presents the possibility of off-target effects and therapeutic transgene expression in healthy cells. To enhance the specificity of transgene expression to GBM, we assessed the potential for tumour-specific promoters hTERT and survivin to drive reporter gene expression selectively in GBM cell lines. We demonstrate tight GBM-specific transgene expression using these constructs, indicating that the combination of pseudotyping and tumour-specific promoter approaches may enable the development of efficacious therapies better suited to GBM.

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.

A cluster of basic amino acids in the factor X serine protease mediates surface attachment of adenovirus/FX complexes.

Hepatocyte transduction following intravenous administration of adenovirus 5 (Ad5) is mediated by interaction between coagulation factor X (FX) and the hexon. The FX serine protease (SP) domain tethers the Ad5/FX complex to hepatocytes through binding heparan sulfate proteoglycans (HSPGs). Here, we identify the critical HSPG-interacting residues of FX. We generated an FX mutant by modifying seven residues in the SP domain. Surface plasmon resonance demonstrated that mutations did not affect binding to Ad5. FX-mediated, HSPG-associated cell binding and transduction were abolished. A cluster of basic amino acids in the SP domain therefore mediates surface interaction of the Ad/FX complex.

Requirements for receptor engagement during infection by adenovirus complexed with blood coagulation factor X.

Human adenoviruses from multiple species bind to coagulation factor X (FX), yet the importance of this interaction in adenovirus dissemination is unknown. Upon contact with blood, vectors based on adenovirus serotype 5 (Ad5) binds to FX via the hexon protein with nanomolar affinity, leading to selective uptake of the complex into the liver and spleen. The Ad5:FX complex putatively targets heparan sulfate proteoglycans (HSPGs). The aim of this study was to elucidate the specific requirements for Ad5:FX-mediated cellular uptake in this high-affinity pathway, specifically the HSPG receptor requirements as well as the role of penton base-mediated integrin engagement in subsequent internalisation. Removal of HS sidechains by enzymatic digestion or competition with highly-sulfated heparins/heparan sulfates significantly decreased FX-mediated Ad5 cell binding in vitro and ex vivo. Removal of N-linked and, in particular, O-linked sulfate groups significantly attenuated the inhibitory capabilities of heparin, while the chemical inhibition of endogenous HSPG sulfation dose-dependently reduced FX-mediated Ad5 cellular uptake. Unlike native heparin, modified heparins lacking O- or N-linked sulfate groups were unable to inhibit Ad5 accumulation in the liver 1h after intravascular administration of adenovirus. Similar results were observed in vitro using Ad5 vectors possessing mutations ablating CAR- and/or α(v) integrin binding, demonstrating that attachment of the Ad5:FX complex to the cell surface involves HSPG sulfation. Interestingly, Ad5 vectors ablated for α(v) integrin binding showed markedly delayed cell entry, highlighting the need for an efficient post-attachment internalisation signal for optimal Ad5 uptake and transport following surface binding mediated through FX. This study therefore integrates the established model of α(v) integrin-dependent adenoviral infection with the high-affinity FX-mediated pathway. This has important implications for mechanisms that define organ targeting following contact of human adenoviruses with blood.

Functional characterization of a 13-bp deletion (c.-1522_-1510del13) in the promoter of the von Willebrand factor gene in type 1 von Willebrand disease.

We have studied the effect of a 13-bp deletion in the promoter of the von Willebrand factor (VWF) gene in a patient with type 1 von Willebrand disease. The index case has a VWF:Ag of 0.49 IU/mL and is heterozygous for the deletion. The deletion is located 48 bp 5' of the transcription start site, and in silico analysis, electrophoretic mobility shift assays, and chromatin immunoprecipitation studies all predict aberrant binding of Ets transcription factors to the site of the deletion. Transduction of reporter gene constructs into blood outgrowth endothelial cells showed a 50.5% reduction in expression with the mutant promoter (n = 16, P < .001). A similar 40% loss of transactivation was documented in transduced HepG2 cells. A similar marked reduction of transgene expression was shown in the livers of mice injected with the mutant promoter construct (n = 8, P = .003). Finally, in studies of BOEC mRNA, the index case showed a 4.6-fold reduction of expression of the VWF transcript associated with the deletion mutation. These studies show that the 13-bp deletion mutation alters the binding of Ets (and possibly GATA) proteins to the VWF promoter and significantly reduces VWF expression, thus playing a central pathogenic role in the type 1 von Willebrand disease phenotype in the index case.

Biodistribution and retargeting of FX-binding ablated adenovirus serotype 5 vectors.

A major limitation for adenoviral transduction in vivo is the profound liver tropism of adenovirus type 5 (Ad5). Recently, we demonstrated that coagulation factor X (FX) binds to Ad5-hexon protein at high affinity to mediate hepatocyte transduction after intravascular delivery. We developed novel genetically FX-binding ablated Ad5 vectors with lower liver transduction. Here, we demonstrate that FX-binding ablated Ad5 predominantly localize to the liver and spleen 1 hour after injection; however, they had highly reduced liver transduction in both control and macrophage-depleted mice compared with Ad5. At high doses in macrophage-depleted mice, FX-binding ablated vectors transduced the spleen more efficiently than Ad5. Immunohistochemical studies demonstrated transgene colocalization with CD11c(+), ER-TR7(+), and MAdCAM-1(+) cells in the splenic marginal zone. Systemic inflammatory profiles were broadly similar between FX-binding ablated Ad5 and Ad5 at low and intermediate doses, although higher levels of several inflammatory proteins were observed at the highest dose of FX-binding ablated Ad5. Subsequently, we generated a FX-binding ablated virus containing a high affinity Ad35 fiber that mediated a significant improvement in lung/liver ratio in macrophage-depleted CD46(+) mice compared with controls. Therefore, this study documents the biodistribution and reports the retargeting capacity of FX binding-ablated Ad5 vectors in vitro and in vivo.

The aetiopathogenesis of vaccine-induced immune thrombotic thrombocytopenia.

In the new science emanating from the COVID-19 pandemic, effective vaccine development has made a huge difference and saved countless lives. Vaccine roll-out led to the identification of rare cases of severe thrombotic and thrombocytopenic problems in some recipients. This apparent coupling of thrombosis with haemorrhagic potentiation might seem baffling but the ensuing clinical investigation rapidly shed important light on its molecular mechanism. This review outlines the current understanding on the role of adenovirus-based platforms, the immunogenic triggers and the immunothrombotic response underlying vaccine-induced immune thrombotic thrombocytopenia.

Engineering Cancer Selective Virotherapies: Are the Pieces of the Puzzle Falling into Place?

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.