Voltage-gated T-type calcium channel blockers reduce apoptotic body-mediated SARS-CoV-2 cell-to-cell spread and subsequent cytokine storm (preprint)

SARS-CoV-2 typically utilises host angiotensin-converting enzyme 2 (ACE2) as a cellular surface receptor and host serine protease TMPRSS2 for the proteolytic activation of viral spike protein enabling viral entry. Although macrophages express low levels of ACE2, they are often found positive for SARS-CoV-2 in autopsied lungs from COVID-19 patients. As viral-induced macrophage inflammation and overwhelming cytokine release are key immunopathological events that drives exacerbated tissue damage in severe COVID-19 patients, insights into the entry of SARS-CoV-2 into macrophages are therefore critical to understand COVID-19 pathogenesis and devise novel COVID-19 therapies. Mounting evidence suggest that COVID-19 pathogenesis is associated with apoptosis, a type of programmed cell death that often leads to the release of numerous large extracellular vesicles (EVs) called apoptotic bodies (ApoBDs). Here, we showed that ApoBDs derived from SARS-CoV-2-infected cells carry viral antigens and infectious virions. Human monocyte-derived macrophages readily efferocytosed SARS-CoV-2-induced ApoBDs, resulting in SARS-CoV-2 entry and pro-inflammatory responses. To target this novel ApoBD-mediated viral entry process, we screened for ApoBD formation inhibitors and discovered that T-type voltage-gated calcium channel (T-channel) blockers can inhibit SARS-CoV-2-induced ApoBD formation. Mechanistically, T-channel blockers impaired the extracellular calcium influxes required for ApoBD biogenesis. Importantly, blockade of ApoBD formation by T-channel blockers were able to limit viral dissemination and virus-induced macrophage inflammation in vitro and in a pre-clinical mouse model of severe COVID-19. Our discovery of the ApoBD-efferocytosis-mediated viral entry reveals a novel route for SARS-CoV-2 infection and cytokine storm induction, expanding our understanding of COVID-19 pathogenesis and offering new therapeutic avenues for infectious diseases.

mRNA vaccines encoding membrane-anchored receptor-binding domains of SARS-CoV-2 mutants induce strong humoral responses and can overcome immune imprinting (preprint)

To address the limitations of whole-spike COVID vaccines, we explored mRNA vaccines encoding membrane-anchored receptor-binding domain (RBD-TMs), each a fusion of a variant RBD, the transmembrane (TM) and cytoplasmic tail (CT) fragments of the SARS-CoV-2 spike protein. In naive mice, RBD-TM mRNA vaccines against ancestral SARS-CoV-2, Beta, Delta, Delta-plus, Kappa, Omicron BA.1 or BA.5, all induced strong humoral responses against the target RBD. Multiplex surrogate viral neutralization (sVNT) assays indicated broad neutralizing activity against a range of variant RBDs. In the setting of a heterologous boost, against the background of exposure to ancestral whole spike vaccines, sVNT studies suggested that RBD-TM vaccines were able to overcome the detrimental effects of immune imprinting. Omicron BA.1 and BA.5 RBD-TM booster vaccines induced serum antibodies with 12 and 22-fold higher neutralizing activity against the target RBD than their equivalent whole spike variants. Boosting with BA.1 or BA.5 RBD-TM provided good protection against more recent variants including XBB and XBB.1.5. Each RBD-TM mRNA is 28% of the length of its whole-spike equivalent. This advantage will enable tetravalent mRNA vaccines to be developed at well-tolerated doses of formulated mRNA.