The Structure of ChAdOx1/AZD-1222 Reveals Interactions with CAR and PF4 with Implications for Vaccine-induced Immune Thrombotic Thrombocytopenia

Abstract

Adenovirus derived vectors, based on chimpanzee adenovirus Y25 (ChAdOx1) and human adenovirus type 26 are proving critical in combating the 2019 SARS-CoV-2 pandemic. Following emergency use authorization, 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.

Comparative analysis of ChAdOx1 nCoV-19 and Ad26.COV2.S SARS-CoV-2 vector vaccines

“…While we con rmed the previously observed interaction of PF4 with ChAdOx1 nCoV-19, PF4 did not form distinct complexes with Ad26.COV2.S or puri ed ChAdOx1 nCoV-19 virion preparations, highlighting the role of impurities in ChAdOx1 nCoV-19 for the observed interactions 6 . Consistent with our ndings in DLS, a recent study using cryo-electron microscopy of ChAdOx1 nCoV-19 established a possible electrostatic interaction of the positively charged PF4 and the negatively charged adenoviral hexon polypeptide 10 as a likely functional basis for the interactions. We now show that this interaction can be e ciently abrogated by purifying the intact adenoviral vector from the vaccine formulation.…”

Cardiovascular, neurological, and pulmonary events following vaccination with the BNT162b2, ChAdOx1 nCoV-19, and Ad26.COV2.S vaccines: An analysis of European data

“…As far as we know, the differences between the vaccines are as follows: 1) The type of adenovirus is different (human Ad26 and chimpanzee adenovirus in the AD26 and ChA vaccines, respectively); 2) the human cell lines by which adenovirus are produced are different: T-REx-293 cells for ChA and PER.C6 TetR cells for AD26 [ 44 ]; 3) ChA contains a large number of host cell proteins differently from AD26 [ 10 , 44 , 45 ]; 4) chimpanzee adenovirus has a stronger negative charge than human AD26 [ 40 ]. Molecular simulations suggest that the chimpanzee adenovirus charge, combined with aspects of the virus shape, could allow it to bind to the positively charged PF4 protein [ 46 ]; 5) The number of viral particle in the AD26 vaccine is 3.3-fold higher than that in the ChA vaccine [ 47 , 48 ], possibly implying a higher spike protein and adenoviral protein burden. 6) EDTA is present in the ChA vaccine but not in the AD26 vaccine [ 47 , 48 ], and it has been hypothesized that it favors the development of inflammation at the vaccine inoculation site.…”

Shooting at a Moving Target—Effectiveness and Emerging Challenges for SARS-CoV-2 Vaccine Development

“…This PF4-dependent thrombotic disorder is now referred to as vaccine-induced immune thrombotic thrombocytopenia (VIIT) and is characterized by thrombosis in unusual regions of cerebral sinuous veins and lung or splanchnic veins with mild-to-severe thrombocytopenia. Structural studies of ChAdOx1 suggested that the electronegative potential of the viral capsid could lead to interaction with PF4, leading to complex formations and antibody production, as observed in HIIT [152]. The probability to develop VIIT has been calculated to be one per 250,000 vaccinees.…”