In vitro and in vivo effects of Pelargonium sidoides DC. root extract EPs® 7630 and selected constituents against SARS-CoV-2 B.1, Delta AY.4/AY.117 and Omicron BA.2

Abstract

The occurrence of immune-evasive SARS-CoV-2 strains emphasizes the importance to search for broad-acting antiviral compounds. Our previous in vitro study showed that Pelargonium sidoides DC. root extract EPs^® 7630 has combined antiviral and immunomodulatory properties in SARS-CoV-2-infected human lung cells. Here we assessed in vivo effects of EPs^® 7630 in SARS-CoV-2-infected hamsters, and investigated properties of EPs^® 7630 and its functionally relevant constituents in context of phenotypically distinct SARS-CoV-2 variants. We show that EPs^® 7630 reduced viral load early in the course of infection and displayed significant immunomodulatory properties positively modulating disease progression in hamsters. In addition, we find that EPs^® 7630 differentially inhibits SARS-CoV-2 variants in nasal and bronchial human airway epithelial cells. Antiviral effects were more pronounced against Omicron BA.2 compared to B.1 and Delta, the latter two preferring TMPRSS2-mediated fusion with the plasma membrane for cell entry instead of receptor-mediated low pH-dependent endocytosis. By using SARS-CoV-2 Spike VSV-based pseudo particles (VSVpp), we confirm higher EPs^® 7630 activity against Omicron Spike-VSVpp, which seems independent of the serine protease TMPRSS2, suggesting that EPs^® 7630 targets endosomal entry. We identify at least two molecular constituents of EPs^® 7630, i.e., (-)-epigallocatechin and taxifolin with antiviral effects on SARS-CoV-2 replication and cell entry. In summary, our study shows that EPs^® 7630 ameliorates disease outcome in SARS-CoV-2-infected hamsters and has enhanced activity against Omicron, apparently by limiting late endosomal SARS-CoV-2 entry.

Keywords: COVID-19; EPs 7630; Pelargonium sidoides; SARS-CoV-2; coronavirus; cytokine storm; drug repurposing; immune modulation.

FIGURE 1

EPs^® 7630 has limited effects on virus replication in vivo. (A) Schematic treatment overview. Hamsters received Pelargonium sidoides root extract either orally (p.o.) or both orally and intranasally (p.o. + i. n.). On day 0 animals were infected with 10⁵ PFU SARS-CoV-2 B.1 variant euthanized on day 2, 4 and 7 after infection. (B) Genomic viral RNA copies and (C) PFU per swab. (D) Genomic viral RNA copies and (E) PFU in homogenized lung tissue. Statistical significance is indicated by (*) as determined by two-way ANOVA of the log-transformed data with Dunnett’s multiple comparison test. Asterisks are shown only for significantly different data sets. (*) = p < 0.05; (**) = p < 0.01; (***) = p < 0.001.

FIGURE 2

Histopathology reveals significant effects of EPs^® 7630 on lungs of SARS-CoV-2-infected hamsters. Histopathology of representative hematoxylin-and-eosin-stained, paraffin-embedded left lungs comparing the three groups (vehicle, EPs^® 7630 p. o., EPs^® 7630 i. n. and p. o.) in parameters bronchitis and edema. (A) Severe bronchitis in the control group (vehicle, left column), indicated by large amounts of neutrophils in the bronchial lumen (arrow) as well as bronchial epithelial cell necrosis. Delayed onset of bronchitis in the EPs^® 7630 i. n. and p. o. group (right column) with occurrence of neutrophil infiltration in bronchial lumina (arrow) by day 4 of infection. All groups show proliferative regenerative change of the bronchial epithelium with bronchial epithelial hyperplasia in the late stage (asterisk) by day 7 of infection (bottom row). (B) Perivascular and alveolar edema formation less prominent in the EPs^® 7630 i. n. and p. o. group in comparison to vehicle and EPs^® 7630 p. o. group (middle row, day 4 of infection). Prominent regenerative change in all groups during the late stage (day 7 of infection) with strong pneumocyte type 2 hyperplasia (hash symbol) (bottom row). Scale bar 100 µm for all pictures.

FIGURE 3

EPs^® 7630 delays bronchiolitis and limits lung edema in SARS-CoV-2-infected hamsters. Hamsters received Pelargonium sidoides root extract either orally (p.o.) or both orally and intranasally (p.o. + i. n.) as described in Figure 1. (A) Approximate lung area affected by inflammatory damage in percentage per group and time point. (B–D) Semi-quantitative scoring of pneumonia severity (B) bronchitis (C) and Edema (perivascular and alveolar) (D) for all groups and respective time points. Statistical significance is indicated by (*) as determined by two-way ANOVA of the data with Dunnett’s multiple comparison test. Asterisks are shown only for significantly different data sets. (*) = p < 0.05; (**) = p < 0.01; (***) = p < 0.001; (****) = p < 0.0001.

FIGURE 4

SARS-CoV-2 (B.1) propagation and inflammatory cytokine expression in human bronchial airway epithelial cells (bAEC). (A) Bronchial AEC were inoculated with SARS-CoV-2 (MOI = 0.005) with and without EPs^® 7630 (100 μg/mL) treatment at 37°C for 2 h. For sample collection, the apical side of the bAEC was incubated with 250 µL Mucilair medium for 20 min, which was subsequently removed and frozen at −80°C until analysis. Supernatants were analyzed by plaque assays between 0 and 72 h post-infection (B) or at 24 h post-infection using the Human Cytokine/Chemokine/Growth Factor Multiplex Assay (Merck Millipore) with the Luminex MAGPIX System according to the manufacturer’s instructions (C). Data are derived from n = 3 biological samples. Cell culture medium was used as the vehicle control. No statistical significance was observed for (B), as determined by two-way ANOVA with Tukey’s multiple comparison test on log-transformed data. Statistical significance for (C) was determined by paired t-tests. Asterisks are shown only for significantly different data sets. (*) = p < 0.05. ALI = air liquid interface.

FIGURE 5

Enhanced inhibition of SARS-CoV-2 variant BA.2 by EPs^® 7630 in human bronchial airway epithelial cells (bAEC). Nasal AEC (A, C) and bAEC (B, D) were inoculated with SARS-CoV-2 variant AY.4 (A, B) or BA.2 (C, D) using an MOI of 0.005 with and with EPs^® 7630 (100 μg/mL) treatment at 37°C for 2 h. For sample collection, the apical side of the AEC was incubated with 250 µL Mucilair medium for 20 min, which was subsequently removed and frozen at −80°C until analysis. Supernatants were analyzed by plaque assays between 0 and 72 h post-infection. Data are derived from n = 3 biological samples. Variant growth kinetics are shown in parallel with the B.1 growth kinetics previously depicted in Figure 4B. Cell culture medium was used as the vehicle control. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparison test on log-transformed data. Asterisks are shown when EPs^® 7630 treatment resulted in significantly different levels of Delta or Omicron PFU for given timepoints. (*) = p < 0.05; (**) = p < 0.01; (***) = p < 0.001; (****) = p < 0.0001.

FIGURE 6

Differential entry inhibition of SARS-CoV-2 variants by EPs^® 7630. All cells were pre-treated with the indicated compounds for 2 h pre-infection at 37°C. Infection of Calu-3, A549-ACE2, and A549-ACE2/TMPRSS2 cells with VSV-G as control (A), SARS-CoV-2-S VSVpp (SARS-CoV-2-S) from B.1 (B), variant Delta AY.117 (C), or variant Omicron BA.2 (D) was done in the presence of compounds for 30 min at 4°C at 300 g followed by 1-h incubation at 37°C. The medium was then replaced by DMEM containing the indicated compounds. DMSO was additionally used as a vehicle control. As positive controls, we applied 1 and 10 µM niclosamide (pH-dependent endosomal entry inhibitor) and 1 and 10 µM camostat mesylate (TMPRSS2 inhibitor). Cell lysates were prepared after 24 h and the luciferase signal was measured using a multi-mode 96-well plate reader. Statistical significance is indicated by (*) as determined by two-way ANOVA of the data with Dunnett’s multiple comparison test. Asterisks are shown only for significantly different data sets. (*) = p < 0.05; (**) = p < 0.01; (***) = p < 0.001; (****) = p < 0.0001.

FIGURE 7

Epigallocatechin, epigallocatechin gallate, and taxifolin inhibit SARS-CoV-2 B.1 propagation dose dependently. (A-B) Calu-3 cells were infected with SARS-CoV-2 (MOI = 0.0005) and treated with 7 defined low molecular weight constituents of EPs^® 7630 using 10 μg/mL, as well as DMSO as a vehicle control (A), and additionally for cpd C (epigallocatechin), cpd F (epigallocatechin gallate), and cpd G (taxifolin) in a dilution series of 0.5–10 μg/mL (B). Virus-containing supernatants were collected 24 h post-infection and viral titers were determined as PFU/mL by plaque titration assay. For both Calu-3 (C) and A549-ACE2 cells (D), compound toxicity was evaluated by performing a CellTiter Glo assay in a dilution range of 0.5–512 μg/mL for each compound and 0.5%–5.12% DMSO (to evaluate vehicle toxicity) at 24 h post-infection. Data are derived from n = 3 biological samples. Statistical significance is indicated by (*) as determined by two-way ANOVA of the data with Dunnett’s multiple comparison test. Asterisks are shown only for significantly different data sets. (*) = p < 0.05; (**) = p < 0.01; (***) = p < 0.001; (****) = p < 0.0001.

FIGURE 8

Epigallocatechin and epigallocatechin gallate inhibit predominantly endosomal-mediated SARS-CoV-2 entry showing enhanced activities for B.1 and Omicron BA.2. All cells were pre-treated with the indicated compounds for 2 h pre-infection at 37°C. Infection of Calu-3, A549-ACE2, and A549-ACE2-TMPRSS2 cells with VSV-G as control (A), SARS-CoV-2-S VSVpp (SARS-CoV-2-S) from B.1 (B), variant Delta AY.117 (C), or variant Omicron BA.2 (D) was done in the presence of compounds for 30 min at 4°C at 300 g followed by 1-h incubation at 37°C. The medium was then replaced by DMEM containing the indicated compounds. DMSO was additionally used as a vehicle control. For comparison, the EPs^® 7630 data from Figure 6 were included as all experiments were done in parallel. Cell lysates were prepared after 24 h and the luciferase signal was measured using a multi-mode 96-well plate reader. Statistical significance is indicated by (*) as determined by two-way ANOVA of the data with Dunnett’s multiple comparison test. Asterisks are shown only for significantly different data sets. (*) = p < 0.05; (**) = p < 0.01; (***) = p < 0.001; (****) = p < 0.0001.