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.

Distinct and overlapping immunological responses to SARS-CoV-2 and Mycobacterium tuberculosis identified by single-cell RNA-seq of co-infected whole blood (preprint)

Introduction: COVID-19 and tuberculosis (TB) exhibit similar symptomatic presentation and clinical parameters. Common underlying immunological mechanisms also highlight potential routes of immunopathogenic interaction between these diseases during co-infection. To explore immunological similarities, differences and interactions, single-cell RNA-seq (scRNA-seq) was performed on whole blood infected with Mycobacterium tuberculosis (Mtb), SARS-CoV-2, or both pathogens. Methods: Whole blood from four healthy adults, were subjected to ex vivo infection with Mtb and/or SARS-CoV-2 ancestral strain, or were maintained in an uninfected state, for 24 or 96 hours. At each timepoint, for each condition, the four biological replicates were captured, fixed and cryopreserved to be processed for scRNA-seq as a single batch. Following quality control filtering, genotype-based demultiplexing was performed to obtain data from each biological replicate for pseudobulk differential expression analysis. Results: Thirteen distinct clusters of cells were identified based on marker gene expression. Profound differences in the proportions of monocytes, T cells and neutrophils were observed between infection conditions and timepoints. The greatest divergence between pathogen responses occurred within myeloid cells at early timepoints of infection. Co-infection had the greatest synergistic effect 24 hours post-infection with 238 immunological pathways uniquely enriched, including IFN-{gamma} and TNF production, whilst by 96 hours post-infection there was a large overlap of 182 shared pathways between Mtb, SARS-CoV-2 and co-infection. SARS-CoV-2-only infection resulted in widespread cell death by 96 hours post-infection, while Mtb-only and co-infected samples remained enriched for monocyte, T cell and NK cell signatures, sharing negative regulation of extrinsic apoptotic signalling. Distinct from Mtb, SARS-Co-V-2 had unique regulating of {beta} T cell activation and differentiation at both time points. Conclusion: These data provide a high-resolution characterisation of distinct and overlapping immunological responses generated by SARS-CoV-2 and Mtb when a single infection or co-infection occurs. This sheds light on the potential effects of novel or existing host-directed therapies that target these pathways, which is particularly crucial for settings where dual presentation is common.

Cohort Profile: A longitudinal Victorian COVID-19 cohort (COVID PROFILE) (preprint)

Purpose: The COVID PROFILE cohort is a longitudinal clinical study based in Victoria Australia, which was established to understand immunity to SARS-CoV-2 in a low transmission population setting and to identify immunological markers of long-term immunity and immune-dysregulation after both infection and vaccination. Additionally, this cohort was established as a biobank resource for researchers to address other health-related immunological questions. Participants: We enrolled 178 adult community members, including household contacts, who had either recovered from a SARS-CoV-2 infection or were SARS-CoV-2 naive. Only participants 18 years of age or older and, in the case of female participants, non-pregnant women at the time of enrollment were included in the study. Detailed COVID-19 clinical data, vaccination status, medical history and demographics was collected. Findings to date: At enrollment, we found that 87.8% of COVID-19 recovered individuals were seropositive with detectable levels of anti-SARS-CoV-2 IgG antibodies. Seronegative COVID-19 recovered individuals included asymptomatic individuals or participants that were enrolled more than 12 months after their COVID-19 diagnosis. Except for one individual who was seropositive at baseline despite a previous SARS-CoV-2 PCR negative diagnosis, all household contacts and other community members enrolled as SARS-CoV-2 PCR negative, were seronegative for all SARS-CoV-2-specific antibodies tested. The infection rate (re-infection or new infection) during 24 months of the study was 42.7%, as determined by either rapid antigen tests, PCRs or serology screens. Of the SARS-CoV-2 recovered participants, 32.6% reported ongoing symptoms at enrollment of which 47% had already experienced ongoing symptoms for more than 12 weeks. Future Plans: COVID PROFILE will be used to comprehensively understand temporal immunity to SARS-CoV-2 and COVID-19 vaccines and to understand the impact of host immunological composition on such immunity and symptom severity. Additionally, studies focusing on understanding immunity following breakthrough infections and immunological risk factors that contribute towards development of long COVID are planned.