Development of a Novel Medium Throughput Flow-Cytometry Based Micro-Neutralisation Test for SARS-CoV-2 with Applications in Clinical Vaccine Trials and Antibody Screening (preprint)

Quantifying neutralising capacity of circulating SARS-COV-2 antibodies is critical in evaluating protective humoral immune responses generated post-infection/post-vaccination. Here we describe a novel medium-throughput flow cytometry-based micro-neutralisation test to evaluate Neutralising Antibody (NAb) responses against live SARS-CoV-2 Wild Type and Variants of Concern (VOC) in convalescent/vaccinated populations. Flow Cytometry-Based Micro-Neutralisation Test (Micro-NT) was performed in 96-well plates using clinical isolates WT-B, WT-B.177.18 and/or VOCs Beta and Omicron. Plasma samples (All Ireland Infectious Diseases (AIID) Cohort) were serially diluted (8 points, half-log) from 1/20 and pre-incubated with SARS-CoV-2 (1h, 37{degrees}C). Virus-plasma mixture were added onto VERO E6/VERO E6 TMPRSS2 cells for 18h. Percentage infected cells was analysed by automated flow cytometry following trypsinisation, fixation and SARS-CoV-2 Nucleoprotein intracellular staining. Half-maximal Neutralisation Titres (NT50) were determined using four-parameter logistic regression. Our assay was compared to Plaque Reduction Neutralisation Test (PRNT) and validated against WHO anti-SARS-CoV-2 Immunoglobulin Standards. Using WHO Standards with low, medium or high anti-SARS-CoV-2 IgG, both Micro-NT and PRNT achieved comparable NT50 values. Micro-NT was found to be highly reproducible (inter-assay CV of 11.64%). Screening 190 convalescent samples and 11 COVID-19 naive controls (AIID cohort) we demonstrated that Micro-NT has broad dynamic range differentiating NT50s <1/20 to >1/5000. We could also characterise immune-escape VOC observing up to 10-fold reduction in NT50 against SARS-CoV-2 Beta variant. Our flow cytometry-based Micro-NT is a robust and reliable assay to quantify NAb titres, and has been selected as an endpoint in clinical trials. It has higher throughput (96 well format versus 12 well) and reduced infection time (18h vs 48-96h) compared to the gold standard PRNT.

Excessive inflammatory and metabolic responses to acute SARS-CoV-2 infection are associated with a distinct gut microbiota composition (preprint)

Protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and associated clinical sequelae requires well-coordinated metabolic and immune responses that limit viral spread and promote recovery of damaged systems. In order to understand potential mechanisms and interactions that influence coronavirus disease 2019 (COVID-19) outcomes, we performed a multi-omics analysis on hospitalised COVID-19 patients and compared those with the most severe outcome (i.e. death) to those with severe non-fatal disease, or mild/moderate disease, that recovered. A distinct subset of 8 cytokines and 140 metabolites in sera identified those with a fatal outcome to infection. In addition, elevated levels of multiple pathobionts and lower levels of protective or anti-inflammatory microbes were observed in the faecal microbiome of those with the poorest clinical outcomes. Weighted gene correlation network analysis (WGCNA) identified modules that associated severity-associated cytokines with tryptophan metabolism, coagulation-linked fibrinopeptides, and bile acids with multiple pathobionts. In contrast, less severe clinical outcomes associated with clusters of anti-inflammatory microbes such as Bifidobacterium or Ruminococcus, short chain fatty acids (SCFAs) and IL-17A. Our study uncovered distinct mechanistic modules that link host and microbiome processes with fatal outcomes to SARS-CoV-2 infection. These features may be useful to identify at risk individuals, but also highlight a role for the microbiome in modifying hyperinflammatory responses to SARS-CoV-2 and other infectious agents.

Risk of adverse outcomes with COVID-19 in the Republic of Ireland (preprint)

AimsTo compare the risk of adverse outcomes (i.e. hospital/intensive care admission, death) in population sub-groups during two periods of the COVID-19 pandemic in the Republic of Ireland. MethodsWe analysed routinely-collected, publicly-available data on 67,900 people with laboratory confirmed COVID-19 infection between 29th Feb to 14th Nov 2020. This period encompassed two waves of infection and two corresponding national lockdowns. For two observational periods covering each wave (W1, W2), each ending 17-19 days before implementation of high-level national restrictions, we segmented the population based on age and underlying clinical conditions. ResultsThe prevalence of laboratory confirmed COVID-19 was 1.4%. The risk of admission to hospital, admission to intensive care, and death was 7.2%, 0.9%, and 2.5%, respectively. Compared to younger confirmed cases, those aged [≥]65 y had increased risk of hospital admission (RR 5.61), ICU admission (RR 3.56), and death (RR 60.8). W2 was associated with more cases and fewer adverse events than W1. The risk of all adverse outcomes was reduced in W2 than in W1. ConclusionsOngoing responses should consider the variation in risk of adverse outcomes between specific sub-groups. These findings indicate the need to sustain the prevention, identification and management of noncommunicable diseases to reduce the burden of COVID-19.