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

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 Jackson Emanuel 1 , 2† , Jan Papies 1 , 2† , Celine Galander 1 , 2† , Julia M. Adler 3 , Nicolas Heinemann 1 , 2 , Kathrin Eschke 3 , Sophie Merz 4 , Hannah Pischon 4 , Ruben Rose 5 , Andi Krumbholz 5 , 6 , Žarko Kulić 7 , Martin D. Lehner 7 , Jakob Trimpert 3‡ and Marcel A. Müller 1 , 2 * ‡ 1 Institute of Virology, Charité—Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany, 2 German Center for Infection Research (DZIF), Partner Site Charité, Berlin, Germany, 3 Institut für Virologie, Freie Universität Berlin, Berlin, Germany, 4 IDEXX Laboratories, Kornwestheim, Germany, 5 Institute for Infection Medicine, Kiel University and University Hospital Schleswig-Holstein, Kiel, Germany, 6 Labor Dr. Krause und Kollegen MVZ GmbH, Kiel, Germany, 7 Preclinical R&D, Dr. Willmar Schwabe GmbH and Co. KG, Karlsruhe, Germany 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-CoV2 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 SARSCoV-2 entry. OPEN ACCESS EDITED BY Thomas Brendler, PlantaPhile ® , United States REVIEWED BY Klaus Peter Latté, Independent researcher, Germany Benjamin Kirchweger, University of Vienna, Austria *CORRESPONDENCE Marcel A. Müller, [email protected] † These authors have contributed equally to this work and share first authorship ‡ These authors have contributed equally to this work RECEIVED 29 April 2023 ACCEPTED 11 July 2023 PUBLISHED 26 July 2023 CITATION Emanuel J, Papies J, Galander C, Adler JM, Heinemann N, Eschke K, Merz S, Pischon H, Rose R, Krumbholz A, KulićŽ, Lehner MD, Trimpert J and Müller MA (2023), 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. Front. Pharmacol. 14:1214351. doi: 10.3389/fphar.2023.1214351 COPYRIGHT © 2023 Emanuel, Papies, Galander, Adler, Heinemann, Eschke, Merz, Pischon, Rose, Krumbholz, Kulić, Lehner, Trimpert and Müller. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Pharmacology frontiersin.org01 TYPE Original Research PUBLISHED 26 July 2023 DOI 10.3389/fphar.2023.1214351 KEYWORDS SARS-CoV-2, coronavirus, Pelargonium sidoides, EPs 7630, drug repurposing, immune modulation, COVID-19, cytokine storm 1 Introduction Since its introduction into humans in late 2019 and global spread throughout 2020, SARS-CoV-2 has become endemic in the human population and remains an important public health challenge. Successively emergent SARS-CoV-2 variants, including Delta (e.g., AY.4, AY.117) and Omicron (e.g., BA.1, BA.2, BA2.75, XBB.1.5), have propertiesthatconferresistance to existing antiviral therapies. Specifically, vaccines and monoclonal antibody therapies, which previously elicited strong neutralization of SARS-CoV-2, show significantly reduced neutralization of currently circulating variants (Davis et al., 2021;Edara et al., 2022;Tada et al., 2022; Wang et al., 2023). Remaining methods for controlling SARS-CoV-2/COVID-19 show varying degrees of efficacy (Mohamed et al., 2022), and may be affected by the emergence of future SARS-CoV-2 variants. This emphasizes the continuing need for new or re-purposed, broad-acting antiviral therapeutics that can overcome viral resistance. COVID-19 can result in severe inflammatory responses and immunological dysregulations. The release of pro-inflammatory cytokines and infiltration of immune cells into the lungs are key characteristics of severe COVID-19-associated acute respiratory distress syndrome (Pelaia et al., 2020;Hu et al., 2021;Hönzke et al., 2022). Several COVID-19-related immune markers were proposed as potential druggable targets in the treatment of COVID-19, including members of the CXC chemokine family that promote immune cell chemotaxis to the site of infection (Didangelos, 2020;Gudowska-Sawczuk and Mroczko, 2022). Therapeutics which reduce the host pro-inflammatory response by limiting the release of cytokines, such as IL-8, CXCL9, and IP-10 (Zhao et al., 2020; Callahan et al., 2021;Matsushima et al., 2022) may therefore reduce excessive immune cell infiltration of the lungs and improve host respiratory function. Anti-SARS-CoV2 therapeutics should ideally combine antiviral and immunomodulatory properties, so that both the virus and the symptoms/disease can be effectively targeted with a single treatment. Pelargonium sidoides DC. is a medicinal plant indigenous to South Africa (Brendler and van Wyk, 2008). In a previous study, we characterized the P. sidoides DC. root extract EPs ® 7630, and found that it has both antiviral and anti-inflammatory effects on SARS-CoV-2 infection in vitro (Papies et al., 2021), making it a suitable candidate for further preclinical in vivo model-based investigations. The Syrian hamster is a widely accepted model that allows to fully evaluate COVID-19 and its effects on disease pathology connected to a dysregulated immune response (Osterrieder et al., 2020). However, detailed investigations of immunomodulatory effects in hamster models are challenging due to a lack of validated immunological tools. Sophisticated human respiratory in vitro models might serve as a complementary model to investigate putative antiviral and anti-inflammatory effects of immunomodulatory compounds during SARS-CoV-2 infections at the actual site of replication. EPs ® 7630, a proprietary hydroethanolic extract of P. sidoides DC. (Geraniaceae) roots, is the active principle in herbal medicinal products used for the treatment of respiratory tract infections such as acute bronchitis or common cold (Matthys et al., 2003;Chuchalin et al., 2005; Kamin et al., 2010a;Kamin et al., 2010b;Riley et al., 2019). It is composed of carbohydrates, minerals, peptides, purine derivatives, highly substituted benzopyranones, and oligoand polymeric prodelphinidins (Schötz et al., 2008). Subfractionation of multicomponent entities for pharmacological testing can help to distinguish antiviral and immunomodulatory effects of plant extracts and can be carried out following different strategies. EPs ® 7630 subfractions tested by Papies et al. were generated by ultrafiltration, generating fractions containing molecules of different molecular sizes. In contrast, an orthogonal strategy for subfractionation involves selecting single characteristic molecules of EPs ® 7630 representing different natural product classes. For example, umckalin and umckalin sulfate are characteristic TABLE 1 EPs ® 7630 small molecules. Compound (cpd) Name Content in EPs ® 7630 (m/m) Substance class cpd A umckalin ~0.2–0.8% in batch 878 a benzopyranone cpd B umckalin sulfate ~0.2–0.8% Σ= 0.75% benzopyranone cpd C (−)-epigallocatechin <0.3% catechin cpd D prodelphinidin B1 <0.7% prodelphinidin cpd E prodelphinidin B4 <0.7% prodelphinidin cpd F (−)-epigallocatechin gallate not analyzed b catechin cpd G (+)-taxifolin ~0.1% (Kulićet al., 2022)flavonoid a As specified in the certificate of analysis for the batch. The contents of the catechins and prodelphinidins were calculated from isolation yields (data not shown). b (−)-epigallocatechin gallate has been described for a 50% methanolic Pelargonium sidoides extract (Savickiene et al., 2018). Quantification of gallic acid after hydrolysis of ca. 0.01% in EPs® 7630 may correspond to a possible content of ca. <0.027% epigallocatechin gallate. Frontiers in Pharmacology frontiersin.org02 Emanuel et al. 10.3389/fphar.2023.1214351 representatives of benzopyranones (coumarins) found in EPs ® 7630. Taxifolin sulfate and other flavonoid sulfates were recently discovered as genuine constituents in P. sidoides root extract EPs ® 7630 (Kulićet al., 2022). Taxifolin has been identified in silico as a potential inhibitor of SARS-CoV2protease(Fischer et al., 2020) and RNA-dependent RNA polymerase (Kandeel et al., 2020). In addition, catechins such as gallocatechin, epigallocatechin and epigallocatechin gallate were identified in EPs ® 7630. Epigallocatechins from green tea, for example, were previously shown to inhibit SARS-CoV-2 and other CoV entry (Henss et al., 2021;Liu et al., 2021;Ohishi et al., 2022). Furthermore, EPs ® 7630 contains prodelphinidin B1 (Epigallocatechin-4β→8-gallocatechin) and prodelphinidin B4 (Gallocatechin-4α→8-epigallocatechin) which are dimeric prodelphinidins covering different stereochemical configurations of this substance class. Although multiple subfractions of EPs ® 7630 were found to contribute to its activity (Papies et al., 2021), the relative contribution of individual molecular components of EPs ® 7630 has not yet been determined. Since SARS-CoV-2 variant Omicron has been shown to prefer an altered cellular entry mechanism compared to preceding variants (Meng et al., 2022;Willett et al., 2022), it may be the case that entry-targeting compounds exhibit differential activity against SARS-CoV2 variants. Initial reports suggest that Omicron exhibits less TMPRSS2-dependent plasma membrane fusion and favors TMPRSS2-independent late endosomal entry (Meng et al., 2022;Willett et al., 2022). Thus, examining the degree to which EPs ® 7630 and its components inhibit the entry of phenotypically different SARS-CoV-2 variants provides a method of studying the antiviral mechanism of EPs ® 7630 in greater detail. 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 10 5 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. Frontiers in Pharmacology frontiersin.org03 Emanuel et al. 10.3389/fphar.2023.1214351 2 Materials and methods 2.1 Pelargonium sidoides DC. Extract EPs ® 7630 and individual constituents For all experiments, a sample of a production batch (EXCh. 878) of EPs ® 7630, a dried extract of P. sidoides DC. roots (1: 8–10), extraction solvent: ethanol 11% (w/w) was used. 80% of the roots used for the aforementioned production batch were collected from wild plant populations and 20% were harvested from plantations in South Africa. Prior to extraction, the dried plant material was tested in an array of DNA-based and phytochemical methods to confirm the quality and identity of the herbal material. Pharmacognosy was done by the quality control department of Dr. Willmar Schwabe GmbH and Co. KG. Voucher specimens of every lot are deposited in the Department of Pharmacognosy to be retained for 10 years. Chemical fingerprinting of the used EPs ® 7630 batch according to the Consensus statement on the Phytochemical Characterization of Medicinal Plant extracts (Heinrich et al., FIGURE 2 Histopathology reveals significant effects of EPs ® 7630 on lungs of SARS-CoV-2-infected hamsters. Histopathology of representative hematoxylinand-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. Frontiers in Pharmacology frontiersin.org04 Emanuel et al. 10.3389/fphar.2023.1214351 2022) by three different methods (NMR, HPLC, GPC) was already carried out in our previous study (Papies et al., 2021). Umckalin (7-Hydroxy-5,6-dimethoxy-2H-1-benzopyran-2one, cpd A) (Table 1) and umckalin sulfate (5,6-Dimethoxy-7- (sulfooxy)-2H-1-benzopyran-2-one, cpd B) were isolated as described previously (Schötz et al., 2008). (−)-Epigallocatechin (cpd C) was purchased from Interchim S.A., France. Prodelphinidin B1 (Epigallocatechin-4β→8-gallocatechin, cpd D) and prodelphinidin B4 (Gallocatechin-4α→8epigallocatechin, cpd E) were isolated by fractionation of the low molecular weight prodelphinidin fraction containing dimers and trimers, as previously described (Schötz and Nöldner, 2007). The single compounds were purified by dissolving the aforementioned dimer/trimer fraction in methanol to a concentration of 12% (w/v) and loading the solution on a Toyopearl HW-40S column (length = 45 cm, diameter = 2.5 cm) preconditioned with methanol, which was previously saturated with N 2 gas. Fractions were eluted from the resin by an isocratic N 2 -saturated methanol flow. Fractions were checked by thin layer chromatography and pooled to yield the single compounds. (−)-Epigallocatechin gallate (cpd F) was purchased from TCI Deutschland GmbH, Germany. (+)-Taxifolin (cpd G) was purchased from Merck KGaA, Darmstadt, Germany. The purity of the purchased compounds were taken from the vendors’ specifications and had an >98% HPLC purity. The purity of the isolated substances was checked by 1 H-NMR spectroscopy (Supplementary Figure S1), using the same instrument as described previously (Papies et al., 2021). Apart from residual solvents (water, ethanol), no additional impurities could be detected for umckalin and umckalin sulfate in the NMR spectra. For prodelphinidin B1 and B4, some minor impurities from oligo-/polymeric prodelphinidins were detectable as signal bulges below the sharp signals of the respective pure dimer, in addition to residual solvent (water). Thus, the purity of all substances can be considered suitable for the study. Test solutions were prepared as follows: EPs ® 7630 was suspended in DMEM for a stock concentration of 2 mg/mL, and serially diluted in assay medium to achieve the required working concentration. All compounds listed in Table 1 were prepared by suspension in DMSO for stock concentrations of 10 mg/mL, and serially diluted in assay medium to achieve the required working concentration. DMSO vehicle controls contained an equivalent amount of DMSO to the amount of DMSO in wells treated with the highest concentration of compound in each assay. 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. Frontiers in Pharmacology frontiersin.org05 Emanuel et al. 10.3389/fphar.2023.1214351 2.2 Ethics statement In vivo experiments were performed in the biosafety level three (BSL-3) facility at the Institut für Virologie, Freie Universität Berlin, Germany. Animal work was approved and executed in compliance with all applicable institutional, national and international regulations (Landesamt für Gesundheit und Soziales Berlin, permit number 0086/20). 2.3 Animal husbandry Syrian hamsters (Mesocricetus auratus; breed RjHan:AURA) were purchased from Janvier Labs at 10 weeks of age. The animals were kept in individually ventilated cages (IVCs) in groups of 1–3hamstersandhad 1 week to get used to the housing conditions. Food and water were offered ad libitum. During the experiment, the cage temperature was constantly between 22°Cand24 °C with a relative humidity between 40% and 55%. 2.4 Infection experiments Syrian hamsters were randomly assigned into groups of 9 animals (40%–60% female hamsters per group). Intranasal infection with 10 5 plaque forming units (PFU) of SARS-CoV-2 (BetaCoV/Munich/BavPat1/2020) in 60 µL minimal essential medium (MEM) was performed under general anesthesia. EPs ® 7630 was applied in strawberry syrup orally at a dose of 50 mg/kg body weight twice daily. One treatment group received the first dosage of EPs ® 7630 1 day before infection, while the second therapeutic and vehicle treatment group were started on the day of infection. The vehicle group received strawberry syrup without EPs ® 7630. The treatment group that started on the day of infection additionally received EPs ® 7630 intranasally at 5 mg/mL together with the virus inoculum (60 µL total volume). The rationale to include an additional intranasal administration of EPs ® 7630 in one of the treatment groups was based on results from our previous study (Papies et al., 2021). In that study fractionation of EPs ® 7630 demonstrated highest antiviral activity in fractions containing oligomeric proanthocyanidins with expected low oral bioavailability. We assumed that local administration at the site of infection to bypass low systemic bioavailability could increase antiviral activity. We decided to include a single intranasal administration concomitantly with the virus inoculum as a first proof-of-principle approach to assess whether topical mucosal administration holds any promise as a future development option. Infected hamsters were checked twice daily for development of clinical symptoms and body weight loss. Euthanasia was scheduled on day 2, 4 and 7 after infection. Animals were anesthetized with medetomidine (0.15 mg/kg body weight), midazolam (2 mg/kg body 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. Frontiers in Pharmacology frontiersin.org06 Emanuel et al. 10.3389/fphar.2023.1214351 weight), and butorphanol (2.5 mg/kg body weight) prior to euthanasia. Lungs, serum, EDTA blood and oropharyngeal swabs were collected to conduct virological and histopathological analysis. 2.5 RNA extraction and qPCR RNA was extracted from oropharyngeal swabs and 25 mg homogenized lung tissue using innuPREP Virus DNA/RNA Kit (Analytic Jena, Jena, Germany) according to the manufacturer’s instructions. NEB Luna universal Probe One-Step RT-qPCR Kit (New England Biolabs, Ipswich, MA, United States) was used to perform qPCR with cycling conditions of 10 min at 55°Cfor reverse transcription, 3 min at 94°C for activation of the enzyme, and 40 cycles of 15 s at 94°Cand30sat58 °ConaqTower G3 cycler (Analytic Jena, Jena, Germany) in sealed qPCR 96-well plates. To monitor virus growth, SARS-CoV-2 RNA was quantified in cell culture supernatants by RT-qPCR targeting the SARS-CoV-2 E gene, as described previously (Corman et al., 2020). 2.6 Plaque assay for in vivo experiments To quantify replication-competent infectious virus, titrations were performed from 50 mg lung tissue and oropharyngeal swabs. For sample preparation, swabs were thawed, kept in virus transport medium (PBS with 25 mg/L enrofloxacin and 10 mg/L voriconazole) for 30 min and vortexed 3 times during incubation. The organ samples were homogenized in a bead mill procedure with ceramic beads (Analytic Jena). Thereafter, 10fold serial dilutions were prepared starting from −1to−6and plated on VeroE6 cells grown in 12-well plates. The plates were incubated for 2 h at 37°C and subsequently overlaid with MEM medium containing 1.5% carboxymethylcellulose sodium (Sigma Aldrich, St. Louis, MO, United States). The plates were fixed with 4% PBS-buffered formaldehyde solution 72 h after infection. 0.75% methylene blue was used to visualize and manually count plaques. The assay-specific limit of detection is 10 PFU/ 50 mg tissue. All titration experiments were performed in duplicate wells. For samples without detectable plaques, a value of 5 PFU, corresponding to half the assay limit of detection was assigned to allow log-transformation of data. 2.7 Histopathology The left lung lobe was prepared for histopathological examination as previously described (Osterrieder et al., 2020). After careful preparation, it was fixed in PBS-buffered 4% formaldehyde solution for 48 h, embedded in paraffin and cut at 2μm thickness. Subsequently, the slides were stained with hematoxylin and eosin (H&E) as previously published (Bertzbach et al., 2021). 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°Cfor2h. 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. Frontiers in Pharmacology frontiersin.org07 Emanuel et al. 10.3389/fphar.2023.1214351 2.8 Cell lines Calu-3 (ATCC HTB-55), VeroFM (ATCC CCL-81), A549-ACE2, A549-ACE2-TMPRSS2 (Widera et al., 2021) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acids, and 1% sodium pyruvate at 37°Cand5%CO 2 . VeroE6 (ATCC CRL-1586) and VeroE6-TMPRSS2 (NIBSC 100978) cells were cultured in minimal essential medium (MEM) containing 10% fetal bovine serum, 100 IU/mL penicillin G, and 100 μg/mL streptomycin. For VeroE6-TMPRSS2 cell culture (NIBSC 100978), the medium also contained 1,000 μg/mL geneticin (G418) to select for cells expressing TMPRSS2. The cells were incubated at 37°Cand5%CO 2 . All cell lines were cultivated under sterile laboratory conditions and tested for simian virus 5 and mycoplasma contamination as described previously (Biesold et al., 2011). 2.9 Virus strains and infection The SARS-CoV-2 strain Munich/2020/984 was isolated from a respiratory swab obtained from the early 2020 Munich patient cohort (GenBank: MT270101; GISAID: EPI_ISL_406862). The Delta 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 gfollowed 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. Frontiers in Pharmacology frontiersin.org08 Emanuel et al. 10.3389/fphar.2023.1214351 AY.4 variant was isolated from a patient in Cotonou, Benin in July 2021 (GISAID: EPI_ISL_4566935) (Yadouleton et al., 2022). The Omicron BA.2 variant was isolated from a patient in Schleswig-Holstein, Germany in January 2022 (GISAID: EPI_ISL_9553926). Stocks for animal experimentation were generated on VeroE6TMPRSS2 cells and titrated on VeroE6 cells. Prior to animal infection, all virus stocks were stored at −80°C. Stocks for in vitro experimentation were both generated and titrated on VeroE6 cells. For SARS-CoV-2 infection of cell cultures, between 2 × 10 5 and 3 × 10 5 cells per mL were seeded in 6-well plates or 24-well plates. After 24 h, cells were infected with SARS-CoV-2 in a serum-free medium. After 1 h, virus dilutions were removed, and the wells were washed twice with PBS and refilled with DMEM (supplemented as described previously). Samples were taken at the indicated time points. The full sequence identity of B.1, Delta AY.4, and Omicron BA.2 SARS-CoV-2 stocks for in vitro experiments was confirmed with NGS and RT-PCR/Sanger sequencing, and can be made available upon request. Lineage assignment was verified with the 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. Frontiers in Pharmacology frontiersin.org09 Emanuel et al. 10.3389/fphar.2023.1214351 Bertzbach, L. D., Vladimirova, D., Dietert, K., Abdelgawad, A., Gruber, A. D., Osterrieder, N., et al. (2021). SARS-CoV-2 infection of Chinese hamsters (Cricetulus griseus) reproduces COVID-19 pneumonia in a well-established small animal model. Transbound. Emerg. Dis. 68, 1075–1079. doi:10.1111/tbed.13837 Biesold, S. 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