Anti-platelet factor 4 antibodies causing VITT do not cross-react with SARS-CoV-2 spike protein

Abstract

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe adverse effect of ChAdOx1 nCoV-19 COVID-19 vaccine (Vaxzevria) and Janssen Ad26.COV2.S COVID-19 vaccine, and it is associated with unusual thrombosis. VITT is caused by anti-platelet factor 4 (PF4) antibodies activating platelets through their FcγRIIa receptors. Antibodies that activate platelets through FcγRIIa receptors have also been identified in patients with COVID-19. These findings raise concern that vaccination-induced antibodies against anti-SARS-CoV-2 spike protein cause thrombosis by cross-reacting with PF4. Immunogenic epitopes of PF4 and SARS-CoV-2 spike protein were compared using in silico prediction tools and 3D modeling. The SARS-CoV-2 spike protein and PF4 share at least 1 similar epitope. Reactivity of purified anti-PF4 antibodies from patients with VITT was tested against recombinant SARS-CoV-2 spike protein. However, none of the affinity-purified anti-PF4 antibodies from 14 patients with VITT cross-reacted with SARS-CoV-2 spike protein. Sera from 222 polymerase chain reaction-confirmed patients with COVID-19 from 5 European centers were tested by PF4-heparin enzyme-linked immunosorbent assays and PF4-dependent platelet activation assays. We found anti-PF4 antibodies in sera from 19 (8.6%) of 222 patients with COVID-19. However, only 4 showed weak to moderate platelet activation in the presence of PF4, and none of those patients developed thrombotic complications. Among 10 (4.5%) of 222 patients who had COVID-19 with thrombosis, none showed PF4-dependent platelet-activating antibodies. In conclusion, antibodies against PF4 induced by vaccination do not cross-react with the SARS-CoV-2 spike protein, indicating that the intended vaccine-induced immune response against SARS-CoV-2 spike protein is not the trigger of VITT. PF4-reactive antibodies found in patients with COVID-19 in this study were not associated with thrombotic complications.

Graphical abstract

Figure 1.

IgG antibodies in patients with COVID-19 or VITT against anti-SARS-CoV-2 spike protein and PF4. (A) Individual OD results of sera tested by ELISA. Error bars are medians with interquartile ranges (indicated by red lines). Graph shows sera of patients with COVID-19 (n = 20), patients with VITT (n = 24), and PF4-affinity-purified (blue stars) IgG or PF4-heparin affinity-purified (^) IgG from VITT sera (n = 14). The 14 sera used for affinity purification of anti-PF4 IgG are indicated by green filled circles. All sera and the respective affinity-purified anti-PF4 IgG fractions were tested against SARS-CoV-2 S1 domain, RBD-SD1 domain, spike full-length ectodomain, PF4, and PF4-heparin complexes. Sera of patients with COVID-19 reacted with the spike protein and its S1 and RBD domains but not with PF4 or PF4-heparin complexes. VITT sera reacted with spike protein epitopes and PF4, but reactions were strongest with PF4-heparin complexes, whereas the affinity-purified anti-PF4 antibody fraction reacted with PF4 and PF4-heparin complexes but not with the spike protein or its S1 and RBD-SD1 domains. All negative controls (n = 15) gave negative results (supplemental Figure 3). The positive controls in the experiment with affinity-purified antibody for binding of antibodies to the S1 domain, the RBD domain, and the spike protein were positive (data not shown). (B) As a control to show that the affinity-purified anti-PF4 antibodies could still activate platelets, we incubated 75 µL washed platelets in Tyrodes buffer with PF4 (10 µg/mL) and added 10 µL of the affinity-purified antibodies, which reacted in the same manner as the original serum. Results of 14 affinity-purified antibody fractions are shown. Twelve showed strong platelet activation in the presence of PF4. Two antibody fractions still reacted positively by PF4-heparin ELISA but no longer activated platelets, most likely as a result of an antibody yield that was too low after affinity purification.

Figure 2.

PF4-heparin ELISA OD and platelet count in patients with COVID-19. (A) Results of PF4-heparin ELISA OD are given in relation to the WHO Severity Score of COVID-19 disease in 222 patients with COVID-19. ELISA cutoff, 0.5 OD. The 10 patients who developed thrombosis are indicated by open circles. Solid symbols indicate the 212 patients without thrombosis. There was no correlation between WHO severity score of COVID-19 disease and reactivity on the PF4-heparin ELISA. (B) Platelet counts in COVID-19 patients with and without thrombosis.