Emily A. Bates

Adenoviruses are widely used as vectors for subunit vaccines and oncolytic therapies. Efficient vectors must infect target cells and deliver therapeutic transgenes at high levels. Species D adenoviruses, such as human adenovirus type 10 (HAdV-D10), are promising candidates due to low seroprevalence in humans. Here, we present the cryo-electron microscopy structure of the HAdV-D10 capsid alongside transcriptomic profiling of infected cells to inform vector development. The fiber shaft, essential for cell entry, was resolved at 4.6 Å, revealing a previously uncharacterized ‘umbrella’ motif. Viral transcript analysis using an ORF-centric pipeline uncovered key differences from HAdV-C5, including abundant expression of a transcript encoding a protein equivalent to mature protein VII. These findings highlight the importance of detailed vector characterization prior to clinical translation and support the advancement of HAdV-D10 as a next-generation platform for gene delivery and vaccine development.

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.

We observe previously unknown interactions between clinically important adenovirus vector capsids, platelet factor 4, and CAR.

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. Summary: We present the structure of the ChAdOx1 viral vector, derived from chimpanzee adenovirus Y25 at 4.2Å resolution ¹ . 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 clots ² , 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 vaccination ^4–6 . 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.