The studies were carried out in accordance with all relevant guidelines and regulations. These guidelines, as outlined in the instructions to authors, were followed. All animal experiments were conducted in a facility approved by FDA ,States and local authorities and the conduct of such studies were done after obtaining approval from independent committees as indicated in the method section
Recombinant VSV: rVSV-dG SARS-CoV-2 S:
The recombinant vesicular stomatitis virus (rVSV) whose glycoprotein gene (G) has been deleted is used as the base platform for IBT Bioservices’s pseudo type-based neutralization assay (Whitt 2010). The VSV-G glycoprotein is transiently expressed by transfection to produce virus particles. To create a pseudotype virus, VSV-G is substituted with the SARS-CoV-2 spike protein lacking the last eighteen amino acids of the cytoplasmic domain. The resulting virus, rVSV-ΔG SARS-CoV-2 S, also expresses firefly luciferase and can be handled at biosafety level 2 (BSL-2). Infection efficiency was measured by quantification of luciferase activity reading the relative light units (RLU). Briefly, rVSV-ΔG SARS-CoV-2 S was preincubated with and without ELAH along with SARS-CoV-2 seropositive rat sample (IBT Bioservices)used as internal assay control. Rats serum was obtained after immunizing rats in house with SAR-CoV-2 spike protein and added to Vero cells .. After 24-hours infection, firefly luciferase activity was measured and the 50% inhibitory dose (ID50) defined as the reciprocal of the serologic reagent dilution that caused a 50% reduction in RLUs compared to virus control wells was determined.
Formula for nasal spray:
The formula contains 0.1% ELAH, glycerin, xylitol (moisturizing agents), 1,2-hexanediol, polyvinyl pyrrolidone, PEG-40 hydrogenated castor oil, phenoxyethanol and cupric gluconate as preservatives, citric acid for buffer and deionized distilled water (COVIXYL-V).
Vero cells were seeded at 60,000 cells/well in 96-well flat bottom black cell culture plates in Dulbecco’s modified Eagle medium (DMEM) containing 10% serum and incubated overnight. Four dilutions of ELAH were prepared in 1% serum medium at two times (2X) the final intended concentration.
Virus dilution and ELAH/virus mix preincubation:
rVSV-SARS-CoV-2 S was diluted 1:10 in 1% serum medium to obtain a final dilution of 1:20 in 2.5 ml and 175 µl of virus inoculum was then mixed to 175 µl of each ELAH concentrations for 350 µl final; 350 µl of virus only was also prepared. All mixtures were incubated for 1 hour at 37°C and 5% CO2. All the medium was removed from the 96-well black plates, and 100 µl of each virus/TA (testing article) mixture was added in triplicate to the Vero cells. One hundred microliters of virus only and 100 µl of 1% serum medium were also added to a minimum of 6 wells and incubated for 24 hours at 37°C and 5% CO2.
Firefly luciferase readout:
100 µl of Bright-Glo reagent was added to each well as instructed by the manufacturer. Plates were read immediately in our luminometer, and the relative light unit (RLU) was measured.
Animal Model Studies:
The Syrian golden hamster was chosen as the animal model for this study based on in-house data and recent publications indicating that SARS-CoV-2 productively replicates in this model and that aspects of COVID-19 are recapitulated (Tostanoski, L, H,2020 and others). A total of 21 male and female Golden Syrian hamsters (6-8 weeks old, approximately 100 g of weight) were purchased from Envigo (Indianapolis, IN, barrier 202C). The animals were received in good condition. Animal acclimation and husbandry followed the procedures and practices outlined in the IACUC Study Protocol.
ARRIVE guidelines for animal USE: We estimated number of animal for the study using the guidelines full compliance with AAALAC accreditation (File no 624) which meets all national and international guidelines for the use of animal for experimentation. Minimum number were used to comply with guidelines and achieve statistical power. “We insured that all methods reported are in accordance with ARRIVE guidelines(https://arrivegudelines.org)”. This compliance was verified by Bioqual study Directors of both animal/veterinary services (IACUC protocol number:20-153P)
The animal study was conducted in BIOQUAL’s animal facility, BIOQUAL’s facilities are OLAW assured (A-3086-01), USDA registered (51R 036), and have Full AAALAC Accreditation (File no. 624). Additionally, BIOQUAL has 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. Housing and handling of the animals were performed in accordance with the animal welfare requirements and accreditations stated above. 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 Institutional Animal Care and Use Committee approved Protocol (IACUC Protocol Number: 20-153P) and was conducted in accordance with the Study Protocol and BIOQUAL Standard Operating Procedures (SOPs).
Experimental design and grouping:
A study design was prepared in collaboration with BIOQUAL and Merck research scientists and finalized in a study protocol prior to the start of the study. In Study 1, retention of ELAH after a single administration followed by mock challenge was examined over time, while in Study 2, hamsters challenged with virus treated in vitro either with medium or with ELAH were evaluated for infection and clinical outcome.
Study 1 was conducted with a total of six animals divided equally into three groups. ELAH (50 µl per nare) was administered into each nare of the animals. Each animal then received 50 µl of DMEM containing 2% FBS per nostril after 10 min for Group 1, 15 min for Group 2 and 20 min for Group 3 to mimic mock infection. Leakage of solution was monitored for 10 min after each mock infection.
For Study 2, a total of 15 animals divided equally into three groups were used. Group 1 animals were challenged with virus treated in vitro with undiluted ELAH; Group 2 animals were challenged with virus treated in vitro with diluted ELAH (dose diluted 1:1 in sterile PBS), and Group 3 animals were challenged with virus treated in vitro with medium alone. Each treatment was performed at 37°C in 5% CO2 for 10 min. Daily weights and BID observations during challenge periods SD 1, 2, 3, 4, 5, 6, 7, 10 and 14.
Preparation and Administration Procedures:
For Study 1, ELAH-COVIXYL-V solution was applied directly followed by DMEM containing 2% FBS. For Study 2, a vial of SARS-CoV-2 virus was thawed, and the virus was divided into groups:
Group 1: Virus stock (0.2 ml) was diluted to 1.8 ml with medium by adding 1.6 ml of medium and 0.2 ml of ELAH-COVIXYL-V and incubated at 37°C in a 5% CO2 incubator for 10 min. Each animal was challenged nasally with 0.1 ml of virus (0.05 ml per nare).
Group 2: ELAH-COVIXYL-V (0.5 ml) was diluted to 1 ml with 0.5 ml of sterile PBS. Virus stock (0.2 ml) was diluted to 1.8 ml with medium by adding 1.6 ml of medium and 0.2 ml of diluted ELAH-COVIXYL-V (1:1) and incubated at 37°C in a 5% CO2 incubator for 10 min. Each animal was challenged nasally with 0.1 ml of virus (0.05 ml per nare).
Group 3 (Control): Virus stock (0.2 ml) was diluted to 2 ml by adding 1.8 ml of medium and incubated at 37°C in a 5% CO2 incubator for 10 min. Each animal was challenged nasally with 0.1 ml of virus (0.05 ml per nare).
Challenge of hamsters with SARS-CoV-2:
ELAH- or medium-treated SARS-CoV-2 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 nostril, 0.1 ml per animal while the animal's head was tilted back so that the nostrils were pointing toward the ceiling. syringe into the first nostril and slowly the inoculum into the nasal passage, and then removed. This was repeated for the second nostril. 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 challenge phase. The animals were monitored twice daily during the morning and afternoon for signs of COVID-19 disease (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.
Only oral swabs were collected on study days 1, 2, 3, 4, 5, 6, 7, 10 and 14 post challenge as per the study protocol. Scheduled euthanasia and necropsies were carried out for each nare.
Specimen Processing for viral RNA and viral subgenomic RNA assays:
For viral load assays of oral swabs, the samples were processed. Upon collection, the swabs were placed into 1 ml of 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, Cat. No. 57704).
Viral RNA quantitation:
The qRT-PCR assay was used for quantitation of viral RNA from the oral swabs using the primers and a probe specifically designed to amplify and bind to a conserved region of 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 elsewhere (Baum et al. REGN-COV2 antibodies prevent and treat SARS-CoV-2 infection in rhesus macaques and hamsters. science.sciencemag.org/cgi/content/full/science. abe2402/DC1). The signal was compared to a known standard curve and calculated to give copies per mL. For the qRT-PCR assay, viral RNA was first isolated from oral swabs using the Qiagen Min Elute virus spin kit (cat. no. 57704). To generate a control for the amplification reaction, RNA was isolated from the applicable COVID virus stock using the same procedure. The amount of RNA was determined from an O.D. reading at 260, using the estimate that 1.0 OD at A260 equals 40 µg/mL of RNA. With the number of bases known 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 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 cat# BIO-78005), 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 are 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 per 3 µL. Eight (8) 10-fold serial dilutions of control RNA were prepared using RNAse-free water by adding 5 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 per mL was calculated by extrapolation from the standard curve and multiplying by the reciprocal of 0.2 mL extraction volume.
Subgenomic RNA quantitation:
The method used for quantitation of subgenomic mRNA measured by an RT-qPCR assay was similar to what was described elsewhere (Wölfel R., Corman V.M., and others (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 per 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 per 3 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 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 1 to 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 per ml was calculated by extrapolation from the standard curve and multiplying 0.2 mL of extracted volume.
The effect of pretreatment of MRC-5 cells with ELAH on the replication of human coronavirus 229E:
A human lung fibroblast MRC-5 (ATCC® CCL-171™) cell line grown in Eagle’s Minimum Essential Medium (EMEM) containing 2% fetal bovine serum and human coronavirus 229E (ATCC® VR-740™) was used in these experiments.
Preliminary experiments were conducted to determine the cytotoxicity of ELAH on MRC-5 cell cultures. Serial 10-fold dilutions of ELAH starting with a stock solution containing 0.08% or 800 µg/ml ELAH or cell medium only as a control were added to MRC-5 cell cultures and incubated for 6 days. Cytotoxicity screening using bright field imaging was conducted to determine the lowest noncytotoxic concentration of ELAH in MRC-5 cell cultures under these experimental conditions.
To assess the replication of human coronavirus 229E in MRC-5 cells pretreated with ELAH, the following experiment was conducted. Noncytotoxic concentrations of ELAH were added to MRC-5 cell cultures at 37°C for 10 minutes. The culture medium containing unbound ELAH was removed from treated cell cultures, and human coronavirus 229E was added to the cells and incubated at 35°C for an additional 2 h for the virus to adsorb to the cells. The virus inoculum was removed, and the cultures were washed with medium and reincubated for 4 days at 35°C. Appropriate controls, medium alone, were also included in the experiment. Virus yield from cultures pretreated with ELAH and control nontreated cells was assayed for virus yield by TCID50, and virus-induced cytopathic effect (CPE) was determined by bright field imaging using an Olympus BX63 microscope and Olympus cellsSens Dimension software of the ELAH-treated and control MRC-5 cell cultures.
Inhibition of cytopathic effect by human coronavirus 229E in MRC-5 cells pretreated with ELAH as assessed by bright field 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 10^3 dilution of stock human coronavirus 229E (log10TCID50/ml 5.625), and cultures were reincubated at 35°C for 2 hours. Similarly, cells not treated with ELAH were also infected with 229E. After a 2-hour adsorption period, the unadsorbed virus was removed, and the cells were washed, refed with medium and incubated for 48 hours at 35°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. Samples for scanning electron microscopy were fixed with 1 ml glutaraldehyde for 2 hours and processed according to Caldas et al. (2020). SEM imaging was conducted at the University of Wyoming’s Materials Characterization Laboratory. After 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. 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.