Continuum electrostatic calculations of PF4 and PF4 in complex with fondaparinux show that the electronegative potential of PF4 is ablated in the presence of fondaparinux ( Fig. In purified ChAdOx1 and the AZD1222 vaccine preparation, we observed that PF4 binding to ChAdOx1 was strongly inhibited by preincubation with heparin ( Fig. In BD experiments, we observed that PF4 in complex with fondaparinux, an anticoagulant drug composed of the PF4-binding heparin pentasaccharide, significantly reduced the number of binding initiation events with the ChAdOx1 capsid surface (fig. We performed SPR and BD experiments to test what effect heparin binding to PF4 has on the ability of PF4 to bind ChAdOx1. Heparin is also a key component in the mechanism for HIT, binding to multiple copies of PF4 and forming aggregates with anti-PF4 antibodies that stimulate platelet activation via Fcγ Receptor IIa (RIIa).
Charge complementarity facilitates a ChAdOx1 complex with PF4 3D, are robust evidence that ChAdOx1 uses CAR as a primary receptor, not CD46 or DSG2. A similar analysis in CHO-BC1 cells, expressing CD46-BC1 isoform but no other known primary adenovirus receptors, determined that HAdV-B35, had a strong CD46 interaction, while HAdV-C5and ChAdOx1 fiber knobs did not have a measurable ability to prevent anti-CD46 antibody binding ( Fig. It is possible that very high HAdV-B35 fiber knob protein concentrations give a false signal via nonspecific CAR interactions in both this and the SPR experiments. HAdV-C5 showed that the strongest CAR-binding affinity followed by ChAdOx1.HAdV-B35 demonstrated weak CAR-binding affinity (IC 50 > 1000× higher) ( Fig. We validated these finding in a cellular context using antibody inhibition assays in CHO (Chinese Hamster Ovary)–CAR cells, which express CAR but are negative for other known primary adenovirus receptors. CAR is a high affinity ChAdOx1 fiber knob receptor Instead, the fiber knob was solved by crystallography. The trimeric fiber protein, consisting of a long flexible shaft terminating in the globular knob domain, was not modeled as only some poorly resolved portions of the shaft were visible, likely due to its flexibility (fig.
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Reconstruction of the full capsid atomic model results in minimal clashes between asymmetric units and reconstructs a full icosahedral capsid ( Fig. Hexon was well resolved, including the hypervariable regions (HVRs) on the exterior ( Fig. We observed partial densities for 12 copies of pVI in the base of the hexons that adopted two conformations, either wrapping around the hexon interior before extending into the capsid core or extending directly out of the base of the hexon to wrap over the top of pVIII ( Fig. Partial density was observed for pIIIa, concentrated near the base of the penton ( Fig. 1E), in line with the observations of other groups ( 16, 17). 1C) was well resolved, barring residues 103 to 163, for which we observed no signal ( Fig. These data confirm that PF4 is capable of forming stable complexes with clinically relevant adenoviruses, an important step in unraveling the mechanisms underlying TTS.
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. 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. 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.
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. Borad +22 authors +20 authors +15 authors fewer Authors Info & Affiliations Parker, Abhishek Singharoy, and Mitesh J. Nagalo, Lynda Coughlan, Scott Umlauf, Po-Lin Chiu, Pierre J. Truong, Magdalena Lipka-Lloyd, Petra Fromme, Josh Vermaas, Dewight Williams, LeeAnn Machiesky, Meike Heurich, Bolni M. Boyd, , Daipayan Sarkar, Alicia Teijeira-Crespo, , Chun Kit Chan, Emily Bates, Kasim Waraich, John Vant, Eric Wilson, , Chloe D.
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