Interactions of Spike-RBD of SARS-CoV-2 and Platelet Factor 4: New Insights in the Etiopathogenesis of Thrombosis.

The rare but dangerous adverse events evidenced after massive vaccination against SARS-CoV-2 are represented by thrombosis and thrombocytopenia. The patients diagnosed with severe COVID-19 may develop a pro-thrombotic state with a much higher frequency, thus we decided to investigate the role of Spike protein (the only common product of the two conditions) or the anti-Spike antibodies in the etiopathogenesis of thrombosis. A pathogenic Platelet Factor 4 (PF4)-dependent syndrome, unrelated to the use of heparin therapy, has been reported after the administration of vaccines in the patients manifesting acute thrombocytopenia and thrombosis. Thus, we aimed at shedding light on the structural similarities of Spike of SARS-CoV-2 and PF4 on their eventual biochemical interactions and on the role of their specific antibodies. The similarities between PF4 and Spike-RBD proteins were evaluated by a comparison of the structures and by testing the cross-reactivity of their specific antibodies by ELISA assays. We found that the anti-Spike antibodies do not recognize PF4, on the contrary, the anti-PF4 antibodies show some cross-reactivity for Spike-RBD. More interestingly, we report for the first time that the PF4 and Spike-RBD proteins can bind each other. These data suggest that the interaction of the two proteins could be involved in the generation of anti-PF4 antibodies, their binding to Spike-RBD, which could lead to platelets aggregation due also to their high expression of ACE2.

Novel human neutralizing mAbs specific for Spike-RBD of SARS-CoV-2.

Among the therapies against the pandemic SARS-CoV-2 virus, monoclonal Antibodies (mAbs) targeting the Spike glycoprotein represent good candidates to interfere in the Spike/ACE2 interaction, preventing virus cell entry. Since anti-spike mAbs, used individually, might be unable to block the virus entry in the case of resistant mutations, we designed an innovative strategy for the isolation of multiple novel human scFvs specific for the binding domain (RBD) of Spike. By panning a large phage display antibody library on immobilized RBD, we obtained specific binders by eluting with ACE2 in order to identify those scFvs recognizing the epitope of Spike interacting with its receptor. We converted the novel scFvs into full size IgG4, differently from the previously isolated IgG1 mAbs, to avoid unwanted potential side effects of IgG1 potent effector functions on immune system. The novel antibodies specifically bind to RBD in a nanomolar range and interfere in the interaction of Spike with ACE2 receptor, either used as purified protein or when expressed on cells in its native conformation. Furthermore, some of them have neutralizing activity for virus infection in cell cultures by using two different SARS-CoV-2 isolates including the highly contagious VOC 202012/01 variant and could become useful therapeutic tools to fight against the SARS-CoV-2 virus.