MAJOR METABOLITES FROM HYPERICUM PERFORATUM L., HYPERFORIN AND HYPERICIN, ARE BOTH ACTIVE AGAINST HUMAN CORONAVIRUSES (preprint)

COVID-19 pandemic has highlighted the need of antiviral molecules against coronaviruses. Plants are an endless source of active compounds. In the current study, we investigated the potential antiviral effects of Hypericum perforatum L. Its extract contained two major metabolites belonging to distinct chemical classes, hypericin (HC) and hyperforin (HF). First, we demonstrated that HC inhibited HCoV-229E at the entry step by directly targeting the viral particle in a light-dependent manner. While antiviral properties have already been described for HC, the study here showed for the first time that HF has pan-coronavirus antiviral capacity. Indeed, HF was highly active against Alphacoronavirus HCoV-229E (IC50 value of 1.10 {micro}M), and Betacoronaviruses SARS-CoV-2 (IC50 value of of 0.24 to 0.98 {micro}M), SARS-CoV (IC50 value of 1.01 {micro}M) and MERS-CoV (IC50 value of 2.55 {micro}M). Unlike HC, HF was active at a post-entry step, most likely the replication step. Antiviral activity of HF on HCoV-229E and SARS-CoV-2 was confirmed in primary human respiratory epithelial cells. Furthermore, in vitro combination assay of HF with remdesivir showed that their association was additive, which was encouraging for a potential therapeutical association. As HF was active on both Alpha- and Betacoronaviruses, a cellular target was hypothesized. Heme oxygenase 1 (HO-1) pathway, a potential target of HF, has been investigated but the results showed that HF antiviral activity against HCoV-229E was not dependent on HO-1. Collectively, HF is a promising antiviral candidate in view of our results and pharmacokinetics studies already published in animal models or in human.

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.

The spike gene is a major determinant for the SARS-CoV-2 Omicron-BA.1 phenotype (preprint)

Variant of concern (VOC) Omicron-BA1 has achieved global predominance in early 2022. Therefore, surveillance and comprehensive characterization of Omicron-BA.1 in advanced primary cell culture systems and multiple animal models is urgently needed. Here, we characterized Omicron-BA.1 and recombinant Omicron-BA.1 spike gene mutants in comparison with VOC Delta in well-differentiated primary human nasal and bronchial epithelial cells in vitro, followed by in vivo fitness characterization in naive hamsters, ferrets and hACE2-expressing mice, and in immunized hACE2-mice. We demonstrate a spike mediated enhancement of early replication of Omicron-BA.1 in nasal epithelial cultures, but limited replication in bronchial epithelial cultures. In Syrian hamsters, Delta showed dominance over Omicron-BA.1 and in ferrets, Omicron-BA.1 infection was abortive. In mice expressing the authentic hACE2-receptor, Delta and a Delta spike clone also showed dominance over Omicron-BA.1 and an Omicron-BA.1 spike clone, respectively. Interestingly, in naive K18-hACE2 mice, we observed Delta spike-mediated increased replication and pathogenicity and Omicron-BA.1 spike-mediated reduced replication and pathogenicity, suggesting that the spike gene is a major determinant of both Delta and Omicron-BA.1 replication and pathogenicity. Finally, the Omicron-BA.1 spike clone was less well controlled by mRNA-vaccination in K18-hACE2-mice and became more competitive compared to the progenitor and Delta spike clones, suggesting that spike gene-mediated immune evasion is another important factor that led to Omicron-BA.1 dominance.