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.

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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.

Background:The self-resistant mechanism for holomycin has been elusive. Results: The holomycin gene cluster was identified and characterized in Y. ruckeri and the presence of hom12 shown to be crucial for holomycin resistance. Conclusion: Y. ruckeri is a holomycin producer and recruits an RNA methyltransferase for self-resistance. Significance: Our work has demonstrated the hitherto unknown resistant mechanism during holomycin production.

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. Ad5-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 and was assessed. The tropism-ablating triple mutation completely blocked all native cell entry pathways of Ad5-A20 via coxsackie and adenovirus receptor (CAR), αvβ3/5 integrins, and coagulation factor 10 (FX). Ad5-A20 efficiently and selectively transduced αvβ6 cell lines and primary clinical ascites-derived EOC , including in the presence of preexisting anti-Ad5 immunity. biodistribution of Ad5-A20 following systemic delivery in non-tumor-bearing mice was significantly reduced in all off-target organs, including a remarkable 10-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 Ad5-A20-treated animals demonstrated significantly improved survival compared with those treated with oncolytic Ad5. Oncolytic Ad5-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. .

The licensing of talimogene laherparepvec (T-Vec) represented a landmark moment for oncolytic virotherapy, since it provided unequivocal evidence for the long-touted potential of genetically modified replicating viruses as anti-cancer agents. Whilst T-Vec is promising as a locally delivered virotherapy, especially in combination with immune-checkpoint inhibitors, the quest continues for a virus capable of specific tumour cell killing via systemic administration. One candidate is oncolytic adenovirus (Ad); it’s double stranded DNA genome is easily manipulated and a wide range of strategies and technologies have been employed to empower the vector with improved pharmacokinetics and tumour targeting ability. As well characterised clinical and experimental agents, we have detailed knowledge of adenoviruses’ mechanisms of pathogenicity, supported by detailed virological studies and in vivo interactions. In this review we highlight the strides made in the engineering of bespoke adenoviral vectors to specifically infect, replicate within, and destroy tumour cells. We discuss how mutations in genes regulating adenoviral replication after cell entry can be used to restrict replication to the tumour, and summarise how detailed knowledge of viral capsid interactions enable rational modification to eliminate native tropisms, and simultaneously promote active uptake by cancerous tissues. We argue that these designer-viruses, exploiting the viruses natural mechanisms and regulated at every level of replication, represent the ideal platforms for local overexpression of therapeutic transgenes such as immunomodulatory agents. Where T-Vec has paved the way, Ad-based vectors now follow. The era of designer oncolytic virotherapies looks decidedly as though it will soon become a reality.

Adenovirus based vectors are of increasing importance for wide ranging therapeutic applications. As vaccines, vectors derived from human adenovirus species D serotypes 26 and 48 (HAdV-D26/48) are demonstrating promising efficacy as protective platforms against infectious diseases. Significant clinical progress has been made, yet definitive studies underpinning mechanisms of entry, infection, and receptor usage are currently lacking. Here, we perform structural and biological analysis of the receptor binding fiber-knob protein of HAdV-D26/48, reporting crystal structures, and modelling putative interactions with two previously suggested attachment receptors, CD46 and Coxsackie and Adenovirus Receptor (CAR). We provide evidence of a low affinity interaction with CAR, with modelling suggesting affinity is attenuated through extended, semi-flexible loop structures, providing steric hindrance. Conversely, in silico and in vitro experiments are unable to provide evidence of interaction between HAdV-D26/48 fiber-knob with CD46, or with Desmoglein 2. Our findings provide insight into the cell-virus interactions of HAdV-D26/48, with important implications for the design and engineering of optimised Ad-based therapeutics.

29Adenovirus based vectors are of increasing importance for wide ranging therapeutic applications. As 30 vaccines, vectors derived from human adenovirus species D serotypes 26 and 48 (HAdV-D26/48) are 31 demonstrating promising efficacy as protective platforms against infectious diseases. Significant 32 clinical progress has been made, yet definitive studies underpinning mechanisms of entry, infection, 33 and receptor usage are currently lacking. Here, we performed structural and biological analysis of 34 the receptor binding fiber-knob protein of HAdV-D26/48, reporting crystal structures, and modelling 35 putative interactions with two previously suggested attachment receptors, CD46 and Coxsackie and 36 Adenovirus Receptor (CAR). We provide evidence of a low affinity interaction with CAR, with 37 modelling suggesting affinity is attenuated through extended, semi-flexible loop structures, 38 providing steric hindrance. Conversely, in silico and in vitro experiments are unable to provide 39 evidence of interaction between HAdV-D26/48 fiber-knob with CD46, or with Desmoglein 2. Our 40 findings provide new insight to the cell-virus interactions of HAdV-D26/48, with important 41 implications for the design and engineering of optimised Ad-based therapeutics. 42 ARTICLE 43Adenoviruses are increasingly important vectors for wide ranging therapeutic interventions, from 44 gene delivery and oncolytic agents to platforms for vaccine applications 1-3 . As vaccine vectors, their 45 use clinically has been popularised by their excellent safety profile coupled with their ability to 46 induce robust cellular and humoral immunogenicity in humans 4 . Phylogenetically, the human 47 adenoviruses (HAdV's) are diverse, subdivided across 7 species, A-G 5 , based classically on serological 48 cross-reactivity, receptor usage, haemagglutination properties and, more recently, phylogenetic 49 sequence similarity 6,7 . 50Most experimental and clinical studies have focussed on the well-studied species C adenovirus, 51HAdV-C5. Although potently immunogenic, the efficacy of vaccines based on HAdV-C5 appears 52 hampered by high seroprevalence rates in humans, and enthusiasm for their use as clinical vaccine 53 platforms has been dampened by the well-publicised failure of the MERCK sponsored STEP vaccine 54 trial. This trial, to evaluate an HAdV-C5-based HIV vaccine encoding HIV gag/pol/nef antigens, was 55 abandoned due to apparent lack of efficacy upon 1 st term analysis. The study also identified a non-56 significant trend towards increased HIV acquisition in a specific high-risk, uncircumcised subset of 57 patients who also had high levels of baseline pre-existing neutralising antibodies to HAdV-C5 8,9 . As a 58 result, attention has switched from HAdV-C5 based vectors towards the development of alternative 59 adenoviral serotypes with lower rates of pre-existing immunity. Most notably, vectors under 60 development include those based on species D serotypes including HAdV-D26, which has entered 61Phase-III clinical trials as an Ebola v...