A Novel Adenovirus (Adv)-Vaccinated Murine Model Simulates the Prothrombotic Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT) Clinical Disorder

Background: VITT has emerged as a rare (10^-5 to 10^-6 vaccinated individuals), but life-threatening side-effect of post-AdV-based COVID-19 vaccination, characterized by rapidly progressive catastrophic thrombosis, thrombocytopenia and high levels of platelet-activating anti-platelet factor 4 (PF4) antibodies (Abs). While the disorder resembles heparin-induced thrombocytopenia (HIT) in that both anti-PF4 Abs activate platelets via FcγRIIA in the presence of PF4, the Abs differ in their requirements for heparin, i.e. HIT Abs require heparin, but VITT Abs do not. Moreover, alanine substitutions in PF4 revealed distinct antigenic sites in these two prothrombotic disorders. We observed that the defined VITT antigenic site, unlike the HIT antigenic site, is conserved on a second platelet-specific chemokine structurally similar to PF4, neutrophil activating peptide 2 (NAP2) and its precursor platelet-basic protein (PBP) and proposed that PBP as well as PF4 may be able to induce VITT.

Objective: We have now generated a murine model of VITT (mVITT) to better understand the pathogenesis of this immune disorder and to examine whether PBP may be involved in this process.

Methods: Transgenic FcγRIIA mice (n>5/group) that lack mPF4 (PF4^KO), but express human (h) PF4 were injected intramuscularly with 5x10⁹ viral particles/mouse of ChadOx1-nCoV-19 (“nCoV-19 AdV”) leading to expression of the COVID-19 spike protein or control ChadOx-GFP, (“GFP AdV”) expressing the green fluorescent protein. To augment mVITT Ab production, mice also received CpG oligodeoxynucleotides (ODN 2395 or “ODN”, 10µg/mouse i.p for 5 days) as an adjuvant. In these latter experiments, ODN only- and carrier alone- (physiologic buffered saline, “PBS”) injected mice served as controls. The clinical course, blood counts and ELISA for Abs to spike protein, hPF4 and mPBP/NAP2 were followed once a week and organs were harvested for histology at the endpoint.

Results: Only mice that received the nCoV-19 developed anti-spike protein Abs. In the studies without ODN, 1 out of the 20 mice injected with either nCoV-19 or GFP AdV developed progressive thrombocytopenia, massive thrombi in the lungs, heart and brain, and died at day 43, one day after receiving a second dose of GFP. This mouse developed a high titer of anti-mNAP2 Abs, but no significant increase in anti-hPF4 (Table 1).

In the studies with ODN adjuvant, 2 out of the 5 mice injected with nCoV-19 AdV and one of the 5 mice injected with GFP AdV developed thrombocytopenia and died at days 14, 39 and 40 after the 1^st dose and prior to being able to receive a second dose. Gross histology of the brains showed areas of infarction and/or hemorrhage. All three affected mice had raised anti-hPF4 Abs (Table 1). Further characterization of these mice revealed newly formed anti-hPF4 Abs that were heparin independent. Moreover, two monoclonal (mo) Abs, a humanized version of KKO (G4 KKO) that only binds to the HIT antigenic site and 1E12 shown to bind to the VITT antigenic site, were tested for their ability to block the binding of mVITT Ab to hPF4. Only 1E12 blocked that binding (Figure 1). None of the ODN or PBS control mice developed thrombocytopenia or died (Table 1).

Conclusions: We have developed a murine model to simulate the development of VITT that supports AdV:chemokine complex formation as being key to the development of Abs and the prothrombotic state in VITT. This model indicates that anti-PBP/NAP2 Abs can develop after AdV immunization. We also show that the inclusion of an oligonucleotide adjuvant switched antigenic targeting to hPF4. The anti-hPF4 Ab did not develop when adjuvant alone was given, and the resultant Abs clearly bound as VITT-like Abs and not HIT Abs. The basis for the apparent ability of ODN to switch which chemokine is immunogenic will need future study. This model system may be useful in designing AdV that do not induce VITT as backbone for vaccines in the future as well.