Viruses SARS-CoV-2 (WA-2020) and the widely transmitted Delta variant hCoV-19/USA/MD-HPO567/2021 were obtained from the National Institutes of Health (NIH, Bethesda, MD, USA) and subsequently grown in Vero cells and in V-TMPRESS-2 cells, respectively, at the Bioqual Laboratory. Human coronavirus (HCoV-229E) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in Vero cells.
Generation of Pseudovirus VSV-SARS-CoV-2. Single-cycle pseudoparticles were generated using a vesicular stomatitis virus (VSV) genome encoding the firefly luciferase reporter gene (Whitt, M.A. 2010) instead of the native viral glycoprotein (ΔG-VSV-Luc). Infectious virus particles were produced by transiently expressing the native VSV glycoprotein by transfection (G*ΔG-VSV-Luc). Briefly, HEK293T cells (American Type Culture Collection, ATCC, VA, USA) were transfected with a plasmid expressing the SARS-CoV-2 spike protein using Lipofectamine LTX reagent (Life Technologies, CA, USA); pSARS-CoV-2-Sdel18 with a C-terminal 18 amino acid truncation was shown to increase infection efficacy (Xiong, Y. et al. 2020). After 24 hours of transfection, the cells were infected with G*ΔG-VSV-Luc at a multiplicity of infection (MOI) of 3 and then incubated for 24 hours. Cell supernatants containing SARS-CoV-2 pseudotyped (VSV-SARS-CoV-2) virus were collected, centrifuged at 1,200 rpm for 5 minutes, filtered through a 0.2 micron filtering device, aliquoted, and frozen at -80°C. VSV-SARS-CoV-2 virus preparation was quantified using Vero cells (ATCC) seeded at 50,000 cells per well in clear bottom black 96-well plates and incubated for 24 hours at 37°C in minimum essential medium (MEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1X penicillin/streptomycin/L-glutamine. Cells were inoculated with a 1:2 dilution series of pseudotyped virus in 100 μl of MEM for 24 hours at 37 °C. Luciferase activity was quantified with 100 μl of Bright-Glo reagent (Promega, WI, USA), and relative light units (RLU) were read on a Cytation 5 Imaging Multi-Mode Reader (BioTek, CA, USA) plate reader. The dilution generating approximately 10,000 RLU was selected for experiments described below.
Preparation of ethyl lauroyl arginine hydrochloride (ELAH) in nasal spray formula: The stock solution contained 1.0 mg/ml of ELAH, glycerin (10.0%), polyvinyl pyrrolidone (1.0%), 1,2-hexanediol, PEG-40 hydrogenated castor oil, phenoxyethanol and cupric gluconate as preservatives, sodium hydroxide (qs), citric acid (qs) for buffer and deionized distilled water to 100%.
Neutralization of pseudovirus VSV-SARS-CoV-2 infectivity by anti-hyperimmune rat serum against the SARS-CoV-2 receptor binding domain: Vero cells were seeded in 96-well black, clear bottom tissue culture-treated plates (Corning, Corning, NY) at 50,000 cells/well in growth medium and incubated at 37°C for 24 hours. Cells were allowed to reach approximately 85% confluence.
VSV-SARS-CoV-2 (approximately 10,000 RUL) was mixed at a ratio of 1:1 with serial dilutions of hyperimmune serum against the receptor binding domain (RBD) of SARS-CoV-2 or with medium only (control) and incubated for 1 hour at 37°C. Vero cells were infected with VSV-SARS-CoV-2 treated with anti-hyperimmune serum or with untreated control virus and incubated for 24 hours at 37°C. Virus yield was determined by RLU counts. Luciferase activity was quantified with 100 μl of Bright-Glo reagent (Promega), and relative light units (RLU) were read on a Cytation 5 (BioTek, Winooski, VT) plate reader.
In vitro effect of ELAH on the replication of pseudovirus VSV-SARS-CoV-2 in Vero cells: Vero cells were seeded in 96-well black, clear bottom tissue culture-treated plates at 50,000 cells/well in DMEM for 24 hours at 37°C. Cells were allowed to reach approximately 85% confluence before the assay was performed. ELAH was diluted in medium to a 2X assay concentration and diluted 2-fold down in variable pressure scanning electron microscopy (VP-SFM) medium for an 8-point dilution curve in the assay beginning at 200 µg/ml. Pseudo VSV-SARS-CoV-2 particles were diluted 1:20 in media for an RLU count of 10,000 in the assay. Various dilutions of ELAH starting with 200 µg/ml were mixed 1:1 with VSV-SARS-CoV-2 in triplicate and incubated at 37°C for 1 hour. ELAH-treated and untreated control virus samples were used to infect Vero cells and incubated for 24 hours at 37°C. Virus yield was determined by adding Bright-Glo reagent (Promega-delete Promega). Plates were read immediately, and RLUs were quantitated using the Cytation 5 Cell Imaging Multi-Mode Reader. Exported values were analyzed using Microsoft Excell XLFit (5.5) software.
Animal Model Studies: Golden Syrian hamsters were chosen as the animal model for this study based on recent publications indicating that Syrian golden hamsters are a small animal model for COVID-19 (Tostanoski, L. H. et al. 2020, Imai, M. 2020). A total of 21 male and female golden Syrian hamsters (6-8 weeks old, approximately 100 g in weight) were purchased from Envigo RMS, Inc., Indianapolis, IN, USA. Animal acclimation and husbandry followed the procedures and practices outlined in the Institutional Animal Care and Use Committee (IACUC) Study Protocol. Housing and handling of the animals were performed in accordance with the animal welfare requirements and accreditations stated below.
The animal study was conducted in BIOQUAL’s animal facility (BIOQUAL, Inc. Rockville, MD, USA), which is the Approved Animal Welfare Assurance (OLAW) assured (A-3086-01), United States Department of Agriculture (USDA) registered (51R 036) and has full Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) Accreditation (File no. 624). In addition, BIOQUAL has Centers for Disease Control (CDC)/USDA approval for working with restricted BSL-2 and BSL-3 select agents and has approved ABSL/BSL-3 facilities and training for working with infectious agents under containment. Based on the final study plan, BIOQUAL prepared, submitted, and received approval for the IACUC protocol. BIOQUAL Study Directors of both Animal/Veterinary Services and Laboratory Services reviewed the IACUC protocol submission to ensure that all scheduled procedures were consistent with the approved final study plan. This nonclinical study was performed under the BIOQUAL IACUC approved protocol (Number: 20-153P) and was conducted in accordance with the study protocol and BIOQUAL Standard Operating Procedures (SOPs).
Experimental Design and Grouping: These experiments were conducted in collaboration with BIOQUAL and Merck research scientists.
Study 1: The retention of ELAH after a single administration followed by mock challenge was examined for 20 minutes. A total of six animals were divided equally into three groups. ELAH (50 µl/nare) was administered to each nare of all the animals. Ten minutes post-ELAH administration, animals in Group 1 received 50 µl per nare of mock infection consisting of Delbecco’s modified Eagle’s medium (DMEM) containing 2% fetal bovine serum (FBS). Fifteen minutes after ELAH administration, animals in Group 2 received the mock infection, and 20 minutes after ELAH administration, animals in Group 3 received the mock infection. Leakage of the solution was monitored for 10 min after each mock infection in all three groups.
Study 2: The in vitro effect of ELAH on the pathogenesis of SARS-CoV-2 to cause clinical disease in hamsters was examined. A total of 15 animals were equally divided into three groups of 5 each.
Group 1 Animals were challenged with SARS-CoV-2 (1x106 PFU/ml), treated in vitro with undiluted ELAH (1 mg/ml) diluted in DMEM and incubated at 37°C for 10 minutes. Following incubation, each animal was challenged nasally with 0.05 ml of treated virus/nare, corresponding to 2x104 PFU/nare) as described in detail below.
Group 2: Animals were challenged with SARS-CoV-2 and treated in vitro with ELAH as in group 1, with the exception that ELAH (1 mg/ml) was diluted 1:1 with phosphate buffered saline (PBS) prior to incubation with SARS-CoV-2 under similar experimental conditions as in Group 1.
Group 3 (control): The same concentration of SARS-CoV-2 (1x106 PFU/ml) was diluted in DMEM and incubated at 37°C for 10 minutes. Following incubation, each animal was challenged as described in Group 1.
Procedure for Hamsters Challenged with SARS-CoV-2: SARS-CoV-2 treated with ELAH or untreated control virus from the three animal groups described above was administered intranasally (IN) to anesthetized hamsters and performed in a BSL-3 laboratory. The administration of virus was conducted as follows: Using a calibrated pipettor, 0.05 ml of the viral inoculum was administered dropwise into each nare, that is, 0.1 ml/animal, while the animal's head was tilted back so that the nare was pointing toward the ceiling. The syringe was introduced into the first nare, slowly injected into the nasal passage, and then removed. This was repeated for the second nare. The animal’s head was tilted back for approximately 20 seconds and then returned to its housing unit and monitored until fully recovered.
Body weights were measured once daily during the 14-day challenge phase. The animals were monitored twice daily during the morning and afternoon for clinical symptoms of COVID-19 (ruffled fur, hunched posture, labored breathing) during the study period, starting on the day of SARS-CoV-2 challenge, and the information was recorded on BIOQUAL clinical observation forms and/or the Pristima® database. The raw data for the body weights and the clinical observations were made and recorded.
Collection of Oral Swabs for SARS-CoV-2 Replication Studies: Oral swabs were collected from control and SARS-CoV-2-infected animals during days 1 through 4 and on day 7 post challenge. Scheduled euthanasia and necropsies were carried out for each animal at the end of the experiment.
Specimen Processing for Viral RNA and Viral Segmented RNA Assays: For viral load assays of oral swabs, the samples were processed as follows. Upon collection, the swabs were placed into 1 ml of phosphate buffered saline (PBS) and then snap-frozen. Samples were then thawed, and an aliquot of the sample was used for RNA isolation following the manufacturer’s instructions (Qiagen, MD, USA)
Quantitation of SARS-CoV-2 RNA in Syrian Golden Hamsters: The qRT-PCR assay was used for quantitation of viral RNA from oral swabs using primers and a probe specifically designed to amplify and bind to a conserved region of the nucleocapsid gene of coronavirus (forward primer: 5’-GAC CCC AAA ATC AGC GAA AT-3’; reverse primer: 5’-TCT GGT TAC TGC CAG TTG AAT CTG-3’ and probe: 5’-FAM-ACC CCG CAT TAC GTT TGG TGG ACC-BHQ1-3’) as described previously (Baum, A. et al. 2020). Regeneron, a cocktail made of two noncompeting neutralizing human IgG 1 antibodies (REGN-CoV2) that target the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, thereby preventing viral entry into human cells via the angiotensin-converting enzyme 2 (ACE2) receptor. REGN-CoV-2 antibodies have been shown to prevent and treat SARS-CoV-2 infections in rhesus macaques and hamsters (Baum, A. et al. 2020). The signal was compared to a known standard curve and calculated to give copies/ml. For the qRT-PCR assay, viral RNA was first isolated from oral swabs using the Qiagen Min Elute virus spin kit (Qiagen, MD, USA). To generate a control for the amplification reaction, RNA was isolated from the SARS-CoV-2 virus using the same procedure as mentioned above. The amount of RNA was determined by O.D. reading at 260 nm (A260), using the estimate that 1.0 OD at A260 equals 40 µg/mL of RNA. With the number of known bases and the average base of RNA weighing 340.5 g/mole, the number of copies was then calculated, and the control was diluted accordingly. A final dilution of 108 copies per 3.0 µl was then divided into single-use aliquots of 10 µl and stored at -80°C. For the master mix preparation, 2.5 ml of 2X buffer containing Taq-polymerase, obtained from the TaqMan RT-PCR kit (Bioline, Meridian Biosciences, Inc., OH, USA), was added to a 15 ml tube. From the kit, 50 µl of RT and 100 µl of RNase inhibitor were also added. The primer pair at a 2 µM concentration was then added in a volume of 1.5 ml. Finally, 0.5 ml of water and 350 µl of the probe at a concentration of 2 µM were added, and the tube was vortexed. For the reactions, 45 µl of the master mix and 5 µl of the sample RNA were added to the wells of a 96-well plate. All samples were tested in triplicate. The plates were sealed with a plastic sheet. For control curve preparation, samples of the control RNA were prepared to contain 106 to 107 copies/3.0 µl. Eight (8) 10-fold serial dilutions of control RNA were prepared using RNase-free water by adding 5.0 µl of the control to 45 µl of water and repeating this for 7 dilutions. This generated a standard curve with a range of 1 to 107 copies/reaction. For amplification, the plate was placed in an Applied Biosystems 7500 Sequence detector and amplified using the following program: 48°C for 30 minutes, 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds, and 1 minute at 55°C. The number of copies of RNA/ml was calculated by extrapolation from the standard curve and multiplying by the reciprocal of the 0.2 ml extraction volume.
Subgenomic RNA quantitation: The quantitation of subgenomic RNA (sgRNA) was carried out by an RT-qPCR assay as described by Wölfel, R. et al (2020)).
The primers and probe selected from the N gene (Forward:
5’-CGATCTCTTGTAGATCTGTTCTC-3’; reverse: SG-N-R: 5’-GGTGAACCAAGACGCAGTAT-3’ and probe: 5’-FAM- TAACCAGAATGGAGAACGCAGTGGG -BHQ-3’) were similar to what was previously described (Li et al. 2021). The PCR signal obtained with the sample was compared to a known standard curve of plasmid containing the sequence of part of the messenger RNA and calculated to give copies/ml. To generate a control for the amplification reaction, a plasmid containing a portion of the N gene messenger RNA was used. A final dilution of 106 copies/3.0 µl was then divided into single-use aliquots of 10 µl and stored at -80°C until needed. The samples extracted for viral RNA were then amplified in duplicate to pick up sgRNA. Seven (7) 10-fold serial dilutions of control RNA were prepared by adding 5.0 µl of the control to 45 µl of water and repeating this for 7 dilutions, leading to the generation of a standard curve with a range of 1x 106 copies/reaction. For amplification, the plate was placed in an Applied Biosystems 7500 Sequence detector and amplified using the following program: 48°C for 30 minutes, 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds, and 1 minute at 55°C. A printout of the results is maintained in the laboratory notebook. The number of copies of RNA/ml was calculated by extrapolation from the standard curve and multiplying 0.2 ml of extracted volume.
In Vitro Treatment of SARS-CoV-2 Delta Variant with ELAH: The variant used in this study is a widely transmitted Delta variant, hCoV-19/USA/MD-HPO5647/2021. The antiviral effect of ELAH on the Delta variant was conducted in the Vero-TMPRSS2 cell line (NIH, Bethesda, MD) expressing TMPRSS-2 protein (Shutoku, M. et al. 2020). This cell line is highly susceptible to SARS-CoV-2 infection and is used for the isolation and propagation of SARS-CoV-2 variants.
The Delta variant (37 PFU) was incubated with 100 and 200 μl/ml of ELAH at 37°C for 10 minutes in a total volume of 600 µl of DMEM containing 10% FBS. As a control, the Delta variant was suspended in a total of 600 µl of DMEM. The antiviral activity of ELAH was then determined by TCID50 assay. Briefly, Vero-TMPRSS2 cells (25,000 cells/well) were plated in a 96-well plate in DMEM and incubated for 24 hours at 37°C. The medium was removed, 200 µl of the ELAH-Delta variant mixture or 200 µl of control virus was added to Vero-TMPRSS-2 cells in quadruplicate, and the plate was re-incubated for 60 minutes at 37°C. After 60 minutes of incubation, the ELAH virus inoculum and the control virus inoculum were removed from each well, and the cells were washed and replenished with 200 µl of fresh medium. Cultures were re-incubated at 37°C for 72 hours. The antiviral effect of ELAH on the Delta variant was determined by TCID50 assay in Vero-TMPRSS-2 cells as the dilution of virus showing 50% reduction of PFU at a given dilution.
Effect of ELAH on Human Coronavirus-Induced Cytopathic Effects in MRC-5 Cells by Bright Field Microscopy and Scanning Electron Microscopy: MRC-5 cells were seeded at 1x105 cells/ml in 4-chamber cell culture slides and incubated at 37°C for 4 days until approximately 85-90% confluency was obtained. Two concentrations of ELAH, 1 µg/ml and 10 µg/ml, in DMEM were added to the cells and incubated for 10 minutes at 37°C. Cell cultures treated with medium only were used as controls. ELAH was then removed from the cell cultures and infected with a 103 dilution of stock HCoV-229E (log10TCID50/ml 5.625), and cultures were re-incubated at 35°C for 2 hours. Similarly, cells not treated with ELAH were also infected with 229E. After an adsorption period of 2 hours, the cells were washed to remove unabsorbed virus, refed with medium and incubated for 48 hours at 37°C. Control cultures were treated in a similar manner. After 48 hours, chamber cell cultures were imaged via bright field microscopy at a magnification of X63.
Following bright field imaging, select samples were fixed and processed for scanning electron microscopy (SEM) imaging at the University of Wyoming per Methods and Materials. Samples were fixed with 1 ml glutaraldehyde for 2 hours and processed according to the procedure of Caldas et al. (2020). After the samples underwent fixation, they were placed in a Kinney Vacuum KSE-2A-M Evaporator under 10-4 Torr vacuum for 24 hours and then sputtered with a 5 nm thick gold coat using a Model 30000 Ladd Research Industries apparatus. SEM imaging was conducted at the Materials Characterization Laboratory, University of Wyoming, WY, USA. Secondary electron and backscattered electron images were collected on a Quanta 250 scanning electron microscope under 10-5 Torr vacuum using an accelerating voltage of 5 kV and spot sizes of 2 and 3. Electronic alignments on the electron gun (Gun Alignment, Final Lens Aperture Alignment, and Stigmator Alignment) were performed prior to imaging to optimize resolution.