Persistence of Ad26.COV2.S-associated VITT and specific detection of VITT antibodies

ABSTRACT

Background: Some COVID-19 vaccinated individuals develop anti-platelet factor 4 (PF4) antibodies that cause thrombocytopenia and thrombosis;a rare syndrome referred to as vaccine-induced immune thrombotic thrombocytopenia (VITT). Currently, information on the characteristics and persistence of anti-PF4 antibodies that cause VITT after Ad26.COV2.S vaccination is limited, and available PF4-polyanion enzyme-linked immunosorbent assays (ELISAs) and functional diagnostic assays fail to differentiate Ad26.COV2.S and ChAdOx1 nCoV-19-associated VITT from similar clinical disorders, namely heparin-induced thrombocytopenia (HIT) and spontaneous HIT. Aim(s) Evaluate the persistence of anti-PF4 antibodies in Ad26. COV2.S-associated VITT and correlate findings with clinical and laboratory variables such as thrombosis and platelet counts. Develop/ investigate laboratory tools that differentiate VITT antibodies from HIT and spontaneous HIT. Method(s) Blood samples from VITT and HIT patient cohorts were tested in antigen-based and functional assays and correlated with clinical and laboratory features. Result(s) While Ad26.COV2.S-associated VITT patients were strongly positive in PF4-polyanion ELISAs;they were frequently negative in the serotonin release assay (4 of 8 tested patients were negative). In contrast, the PF4-dependent p-selectin expression assay (PEA) that uses PF4-treated platelets consistently diagnosed Ad26.COV2.S-associated VITT. Most Ad26.COV2.S-associated VITT antibodies persisted for >5 months in PF4-polyanion ELISAs, while the PEA became negative earlier. Two patients had otherwise unexplained mild persistent thrombocytopenia (140-150,000/ mul) six months after acute presentation. No recurrence of thrombosis was noted. Additionally, a novel un-complexed PF4 ELISA specifically differentiated VITT secondary to Ad26.COV2.S and ChAdOx1 nCoV-19 vaccination, from spontaneous HIT and HIT (Fig 1A-PF4/ polyanion ELISA;Fig 1B-Un-complexed PF4 ELISA;closed black circles-Ad26. COV2.S-associated VITT;closed red circle-ChAdOx1 nCoV-19-associated VITT;***p < 0.001;****p < 0.0001). Its specificity was further confirmed by testing commonly-encountered HIT-suspected patient samples that are PF4/polyanion ELISA-positive but negative in functional assays (1A-1B). Conclusion(s) Ad26.COV2.S-associated VITT antibodies are persistent, and the un-complexed PF4 ELISA appears to be both sensitive and specific for VITT diagnosis.