In vitro, classical complement activation differs by disease severity and between SARS-CoV-2 antigens (preprint)

Antibodies specific for the spike glycoprotein (S) and nucleocapsid (N) SARS-CoV-2 proteins are typically present during severe COVID-19, and induced to S after vaccination. The binding of viral antigens by antibody can initiate the classical complement pathway. Since complement could play pathological or protective roles at distinct times during SARS-CoV-2 infection we determined levels of antibody-dependent complement activation along the complement cascade. Here, we used an ELISA assay to assess complement protein binding (C1q) and the deposition of C4b, C3b, and C5b to S and N antigens in the presence of anti-SARS-CoV-2 antibodies from different test groups: non-infected, single and double vaccinees, non-hospitalised convalescent (NHC) COVID-19 patients and convalescent hospitalised (ITU-CONV) COVID-19 patients. C1q binding correlates strongly with antibody responses, especially IgG1 levels. However, detection of downstream complement components, C4b, C3b and C5b shows some variability associated with the antigen and subjects studied. In the ITU-CONV, detection of C3b-C5b to S was observed consistently, but this was not the case in the NHC group. This is in contrast to responses to N, where median levels of complement deposition did not differ between the NHC and ITU-CONV groups. Moreover, for S but not N, downstream complement components were only detected in sera with higher IgG1 levels. Therefore, the classical pathway is activated by antibodies to multiple SARS-CoV-2 antigens, but the downstream effects of this activation may differ depending on the specific antigen targeted and the disease status of the subject. O_LISpike- and nucleocapsid-specific antibodies activate complement in vitro C_LIO_LIC1q binding correlates with IgG1 antibody levels C_LIO_LIGeneration of C4b, C3b and C5b relates to the antigen targeted and the patient group tested C_LI

Anti-PF4 levels of patients with VITT do not reduce 4 months following AZD1222 vaccination (preprint)

BackgroundAnti-Platelet Factor 4 (PF4) IgG antibodies that activate platelets via Fc{gamma}RIIa have been shown to be an important part of the pathophysiology of vaccine-induced immune thrombocytopenia and thrombosis (VITT). There is now extensive literature on its presentation and initial management. There is no literature however on what happens to these patients following discharge. MethodsWe collected clinical data and samples from seven patients presenting with VITT and followed them up for 82-145 days. We also collected clinical samples from them at last follow-up. Testing for anti-PF4/heparin antibodies was done using an anti-PF4/heparin enzymatic immunoassay. Flow Cytometry was used to look at Fc{gamma}RIIa levels on patient platelets. Light Transmission Aggregometry with patient serum and healthy donor / patient platelets was used to analyse platelet responsiveness, in the presence and absence of PF4. FindingsAll patients were discharged on direct oral anticoagulants. Two patients remain completely symptom free, three have ongoing headaches, two have residual neurological deficits. Two patients developed mild thrombocytopenia and worsening headache (but without cerebral venous sinus thrombosis) and were retreated, one of these with rituximab. All patients, except the one treated with rituximab, had similar anti-PF4 antibody titres at 80-120 days to their levels at diagnosis. Platelets from patients at follow-up had normal levels of Fc{gamma}RIIa and had normal responses to thrombin and collagen-related-peptide. Patient serum from diagnosis strongly activated healthy donor platelets in the presence of PF4. Serum from follow-up was much weaker at stimulating platelets, even in the presence of PF4. InterpretationThis study shows that despite similar PF4 antibody titres at diagnosis and during follow-up, there are further differences in patient serum, that are not apparent from currently used testing, that result in lower levels of platelet activation during the follow-up period. Further understanding of these factors are important in order to assess duration of anticoagulation for these patients. FundingThis work was supported by an Accelerator Grant (AA/18/2/34218) from the British Heart Foundation (BHF) and by a National Institute for Health Research (NIHR) grant. Key pointsO_LIPF4 antibody titres do not reduce up to 4-months post ChAdOx1 nCoV-19 in patients with VITT C_LIO_LIDespite similar PF4 antibody titres, diagnostic serum is more potent at activating platelets in the presence of PF4 than follow-up serum. C_LI

Platelet Activation by Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT) Patient Serum is Blocked by COX, P2Y12 and Kinase Inhibitors (preprint)

Background The novel coronavirus SARS-CoV-2 has caused a global pandemic. Vaccines are an important part of the response. Although rare, unusual thrombotic events and thrombocytopenia in recipients 4-16 days after vaccination with the AstraZeneca AZD1222 have been reported. This syndrome of vaccine-induced immune thrombotic thrombocytopenia (VITT) clinically resembles heparin induced thrombocytopenia (HIT), which is caused by platelet activating antibodies against platelet factor 4 (PF4). Here, we investigate the effect of serum from patients with VITT on platelet activation, and assess the ability of clinically available therapeutics to prevent platelet activation. Methods Aggregation responses of healthy donor washed platelets were assessed in response to serum from patients with VITT pre- and post-intravenous immunoglobulin (IVIg) treatment and in the presence of anti-Fc{gamma}RIIA blocking IV.3 F(ab), anti-platelet drugs and kinase inhibitors. Findings Four patients (21-48 years old) presented with thrombosis (three patients: cerebral venous sinus thrombosis, one patient: ischemic stroke) and thrombocytopenia 10-14 days after AZD1222 vaccination. All patients tested positive for anti-PF4 antibody despite no prior heparin exposure. Serum from patients with VITT, but not healthy donor controls, induced platelet aggregation, which was abrogated following IVIg treatment. Aggregation to patient sera was blocked by IV.3 F(ab) which targets Fc{gamma}RIIA, and inhibitors of Src, Syk and Btk kinases downstream of the receptor. Anti-platelet therapies indomethacin and ticagrelor also blocked aggregation. Interpretation In conclusion, serum from patients with VITT activates platelets via the Fc{gamma}RIIA, which can be blocked in vitro by anti-platelet therapies suggesting possible new therapeutic interventions for this rare syndrome. Funding This work was supported by an Accelerator Grant (AA/18/2/34218) from the British Heart Foundation (BHF).