Lovatt, C. | Search bioRxiv

bioRxiv2025

Investigating endothelial cell transduction and hexon:PF4 binding of ChAdOx1 in the context of VITT

BackgroundVaccines against SARS-CoV2 have been essential in controlling COVID-19 related mortality and have saved millions of lives. Adenoviral (Ad) based vaccines have played an integral part in this vaccine campaign, with licensed vaccines based on the simian Y25 isolate (Vaxzevria, Astrazeneca) and human Ad type 26 (Jcovden, Janssen) widely adopted. As part of the largest global vaccination programme ever undertaken, ultrarare thromboembolic events have been described in approximately 1:200,000 vaccinees administered with Ad based SARS-CoV2 vaccines. ObjectivesThe mechanism underpinning these adverse events remain to be completely delineated, but is characterised by elevated autoantibodies against PF4 which, in complex with PF4, cluster, bind FC{gamma}RIIa on platelets and induce thrombus formation. Here we investigated the ability of ChAdOx1 to transduce and activate endothelial cells. MethodsUsing protein sequence alignment tools and in vitro transduction assays, the ability of ChAdOx1 to infect endothelial cells was assessed. Furthermore, the ability of ChAdOx1 infection to activate endothelial cells was determined. Finally, using surface plasmon resonance we assessed the electrostatic interactions between the ChAdOx1 hexon and PF4. Results and ConclusionsDespite lacking the primary cell entry receptor, Coxsackie and Adenovirus Receptor (CAR), ChAdOx1 efficiently transduced endothelial cells in a CAR-independent manner. This transduction did not result in endothelial cell activation. Purified hexon protein from ChAdOx1 preps did, however, bind PF4 with a similar affinity to that previously reported for the whole ChAdOx1 capsid. These data confirm the need to develop non-PF4 binding adenoviral capsids to reduce the potential adverse events associated with VITT.

immunology↗

bioRxiv2025

Memory engram synapse 3D molecular architecture visualized by cryoCLEM-guided cryoET

Memory is incorporated into the brain as physicochemical changes to engram cells. These are neuronal populations that form complex neuroanatomical circuits, are modified by experiences to store information, and allow for memory recall. At the molecular level, learning modifies synaptic communication to rewire engram circuits, a mechanism known as synaptic plasticity. However, despite its functional role on memory formation, the 3D molecular architecture of synapses within engram circuits is unknown. Here, we demonstrate the use of engram labelling technology and cryogenic correlated light and electron microscopy (cryoCLEM)-guided cryogenic electron tomography (cryoET) to visualize the in-tissue 3D molecular architecture of engram synapses of a contextual fear memory within the CA1 region of the mouse hippocampus. Engram cells exhibited structural diversity of macromolecular constituents and organelles in both pre- and postsynaptic compartments and within the synaptic cleft, including in clusters of membrane proteins, synaptic vesicle occupancy, and F-actin copy number. This engram to tomogram approach, harnessing in vivo functional neuroscience and structural biology, provides a methodological framework for testing fundamental molecular plasticity mechanisms within engram circuits during memory encoding, storage and recall.

neuroscience↗