RESUMEN
Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare thromboembolic complication of adenoviral-vectored SARS-CoV2 vaccines, mediated by antibodies directed against platelet factor 4 (PF4). Given their causal role in VITT, identification of the molecular composition of anti-PF4 antibodies is crucial for developing better diagnostics and treatments. Here, we utilised a novel proteomic workflow to analyse the immunoglobulin variable (IgV) region composition of anti-PF4 antibodies at the level of the secreted proteome. Serum anti-PF4 IgG antibodies from five patients with VITT triggered by ChAdOx1 nCoV-19 vaccination were affinity purified by PF4-coupled magnetic beads and sequenced by mass spectrometry. We revealed a single IgG heavy (H)-chain species paired with a single lambda light (L)-chain species in all five unrelated patients. Remarkably, all L-chains were encoded by the identical IGLV3-21*02 gene subfamily with identical L-chain third complementarity determining region (LCDR3) lengths. Moreover, striking stereotypic features were also identified in heavy-chains anti-PF4 antibodies characterised by identical HCDR3 length and homologous sequences. In summary, we unravelled the molecular signature of highly stereotyped clonotypic anti-PF4 antibodies, indicating shared pathways of antibody production in VITT patients. These discoveries are critical to understand the molecular basis of this serious condition and develop novel therapies aimed at removing pathogenic clones. KEY POINTSO_LIAnti-PF4 antibodies in VITT comprise highly stereotyped clonotype C_LIO_LIA single IGLV3-21*02 encoded light chain is found in unrelated patients C_LI
RESUMEN
The development of a safe and effective vaccine is a key requirement to overcoming the COVID-19 pandemic. Recombinant proteins represent the most reliable and safe vaccine approach but generally require a suitable adjuvant for robust and durable immunity. We used the SARS-CoV-2 genomic sequence and in silico structural modelling to design a recombinant spike protein vaccine (Covax-19). A synthetic gene encoding the spike extracellular domain (ECD) was inserted into a baculovirus backbone to express the protein in insect cell cultures. The spike ECD was formulated with Advax-SM adjuvant and first tested for immunogenicity in C57BL/6 and BALB/c mice. The Advax-SM adjuvanted vaccine induced high titers of binding antibody against spike protein that were able to neutralise the original wildtype virus on which the vaccine was based as well as the variant B.1.1.7 lineage virus. The Covax-19 vaccine also induced potent spike-specific CD4+ and CD8+ memory T-cells with a dominant Th1 phenotype, and this was shown to be associated with cytotoxic T lymphocyte killing of spike labelled target cells in vivo. Ferrets immunised with Covax-19 vaccine intramuscularly twice 2 weeks apart made spike receptor binding domain (RBD) IgG and were protected against an intranasal challenge with SARS-CoV-2 virus 2 weeks after the second immunisation. Notably, ferrets that received two 25 or 50g doses of Covax-19 vaccine had no detectable virus in their lungs or in nasal washes at day 3 post-challenge, suggesting the possibility that Covax-19 vaccine may in addition to protection against lung infection also have the potential to block virus transmission. This data supports advancement of Covax-19 vaccine into human clinical trials.
