Multi-scale characterization of antigen-polymerized immune complexes underlying thrombotic pathologies triggered by adenoviral-vectored vaccines

Project summary Immunothrombosis is a critical element of intravascular immunity, but its dysregulation or malfunction leads to a range of thrombotic disorders including stroke and disseminated intravascular coagulation. The massive vaccination campaign during the recent COVID-19 pandemic brought to light a novel immunothrombotic pathology, a relatively rare but extremely dangerous side effect of adenoviral (Ad) vectored vaccines, which is now known as vaccine-induced immune thrombotic thrombocytopenia (VITT). Although COVID-19 is no longer a global health threat, the close association of VITT with a specific delivery vector raises the specter of other Ad- vectored vaccines also eliciting this deadly side effect, a grave prospect given the popularity of this platform. VITT has been linked to the emergence of autoantibodies recognizing a cognate chemokine, platelet factor 4 (PF4), but the specific mechanism underlying this pathology remains elusive. Understanding its molecular mechanism and etiology is critical for addressing the currently unmet need to design rational therapeutic and prophylactic strategies targeting VITT. It will also go a long way towards filling the gaps in understanding the delicate interplay between the beneficial and deleterious effects of immunothrombosis and provide the urgently needed ammunition to suppress the latter without sacrificing the former. We will use a combination of experimental and modeling tools to study VITT emergence and progression on different scales, ranging from micro- (formation of platelet-activating immune complexes) to macroscale (thrombi formation). We have already obtained a complete amino acid sequence of the pathogenic VITT antibody and produced its recombinant copy (RVT1) in quantities sufficient for both biophysical and biological investigations. On the microscale, we will use mass spectrometry and other biophysical tools to study the architecture and biological properties of the immune complexes composed of PF4 and RVT1. On the macro-scale, we will use these complexes to study thrombi initiation and formation using in vitro models based on microfluidic devices mimicking vascular environments relevant for VITT pathogenesis (e.g., cerebral venous vasculature). Bridging the micro- and macro-scales will allow us to elucidate the detailed mechanism of VITT progression by understanding how the disease outcome is modulated by the physical and biochemical properties of its molecular triggers. It will also provide a unique opportunity to address another enigmatic feature of VITT - its frequent localization within the cerebral venous sinuses. Lastly, correlating the amino acid sequences of the pathogenic antibodies and the germline sequences for a set of VITT patients will reveal the etiology of this disease, enabling the design of effective prophylactic and monitoring strategies. The proposed research will be carried out by an interdisciplinary team comprising chemists and biophysicists (Dr. Kaltashov's lab at UMass-Amherst), hematologists and molecular biologists (Dr. Nazy's lab at McMaster University School of Medicine) and biomedical engineers (Dr. Jiménez' lab at UMass-Amherst).