Keywords: debulking, transmission, oral virus, topical delivery, plant cells, COVID chewing gum, SARS-CoV-2, angiotensin, virus trap protein, biomarker

Clinical-grade ACE2 plant biomass production at Fraunhofer USA and AeroFarms

CTB-ACE2 plants were created as reported in a previous publication.⁴³ Seeds from the same batch were grown at Fraunhofer USA and AeroFarms as described inmaterials and methods. While growth conditions were different, biomass yield per plant was similar at 80–90 days and declined with repeated harvests as plants grew older. However, total biomass harvested decreased dramatically at Fraunhofer from 48.3 kg on day 90 to 10 kg on day 120. At AeroFarms, total biomass decreased in tower 1 (higher far-red light) from 8.4 kg on day 79 to 1.6 on day 100, but two other towers (higher blue light) showed a moderate decrease in biomass (Figure 1 andTable 1). Biomass yields at Fraunhofer and AeroFarms (towers 2 and 3) were very similar based on fresh weight. Expression level of CTB-ACE2 was higher at Fraunhofer (17.2 ± 1.1 mg/g dry weight [DW] or 11.7% ACE2 in total leaf protein [TLP]), a significant improvement from a previous report⁴³ achieved by optimizing growth conditions, including increasing spacing and light intensity. At AeroFarms, tower 1 with higher far-red percentage showed higher expression (8.4 ± 0.1 mg/g DW or 8.8% TLP) than the other two towers. The reproducibility of ACE2 protein expression was evaluated in several independent trials of plants grown at Fraunhofer and AeroFarms, the statistical data of which is presented inTable 1. However, irrespective of growth conditions, the highest expression was observed in the oldest plants when the lowest biomass was harvested at both facilities (Figure 1 andTable 1). Therefore, delayed single final harvest could further improve protein drug production and yield. Total protein based on dry weight was also higher at Fraunhofer (140–147 mg/g DW) than plants grown at AeroFarms (93–129 mg/g DW), suggesting that the different nutrient solutions influence protein synthesis (Figures 1 andS1;Table 1). Higher level expression reduces the amount of plant powder required for chewing gum or oral delivery of therapeutic ACE2 and therefore is an important production metric.

Preparation of chewing gums

Chewing gum tablets containing ground plant powder were prepared by Per Os Biosciences (Hunt Valley, MD) by a compression process but not the traditional gum manufacturing process, which requires higher temperature and extrusion/rolling that introduces variability in the concentration of proteins. Placebo gum tablets contained the gum base (28.2%), maltitol (20.4%), sorbitol (13%), xylitol (13%), isomalt (13%), natural and artificial flavors, magnesium stearate (3%), silicon dioxide (0.43%), and stevia (0.65%) in order to offer the best flavor, taste, softness, and compression. The gum tablet (2 g weight) chews and performs exactly like conventional chewing gum based on physical characteristics. Freeze-dried plant cells were ground with five pulses to disrupt plant cells and readily release CTB-ACE2. The ACE2 gum tablet has all the components of the placebo gum but in addition includes 50 mg of CTB-ACE2 freeze-dried 5× ground plant cells. Evaluation of sum total of proteins in all fractions (supernatant and pellet) revealed an insignificant loss of CTB-ACE2 during the gum manufacturing process (Figure S2).

Debulking and blocking of viral entry using ACE2 chewing gum

CTB has been shown to be a transmucosal carrier and facilitates oral delivery of therapeutic proteins by forming a pentameric structure and binding to gut GM1 epithelial receptors.43,44,45,46 CTB-ACE2 has the potential to effectively bind to both the GM1 and ACE2 receptor-binding sites located in close proximity on the human cell surface and thereby prevent viral entry into human cells, especially via oral epithelial cells that are enriched with both receptors.⁴⁷ In addition, direct binding of ACE2 to the SARS-CoV-2 spike proteins could trap the virus particles and decrease infectivity (Figure 2). Pentameric insoluble microparticles^43,44 of CTB-ACE2 could facilitate removal of bound viral particles by centrifugation. Therefore, CTB-ACE2 chewing gum is evaluated in this study for its impact on entry and transmission of SARS-CoV-2 (Figure 2). In the oral cavity, tongue epithelial cells constitute a large reservoir of ACE2, even more so than buccal and gingival tissues.43,44,45,46 The ACE2 released upon mastication of the chewing gum serves as a novel approach to diminish virus infection.

Evaluation of SARS-CoV-2 in swab samples by microbubbles and gum

Several de-identified nasopharyngeal (NP) swab samples collected from patients positive for SARS-CoV-2 nucleic acid and nucleocapsid antigen were used to evaluate the neutralization of viral particles by ACE2 gum. The microbubble SARS-CoV-2 antigen assay detects the nucleocapsid antigen of SARS-CoV-2 at femtomolar concentration.^40,41 The number and size of microbubbles correlates with the amount of nucleocapsid antigen in the sample. Because of the limited volume of swab samples, a dose-dependent study on ACE2 gum could not be done using the same patient sample or in duplicates/triplicates. A dose-dependent study was therefore carried out using different patient samples. Samples of patient 1 and patient 2 were treated with 25 mg of gum per 150-μL sample, and the sample of patient 3 was treated with 50 mg of gum per 150-μL sample. Samples were then tested using the microbubble SARS-CoV-2 antigen assay. As shown inFigure 3, the incubation of swab samples with ACE2 gum significantly reduced the microbubble count (p = 0.004). In contrast, the placebo gum when compared with the untreated group had less significant variation (p = 0.892). ACE2 gum decreased the amount of nucleocapsid antigens in COVID-19 patient samples even at very low concentration (25 mg), whereas each gum weighs 2 g. These results confirm direct binding of ACE2 to the SARS-CoV-2 spike proteins and entrapment in pentameric insoluble microparticles of CTB-ACE2 facilitating removal of bound viral particles by centrifugation.

Neutralization assay with SARS-CoV-2 spike-pseudotyped lentiviral particles

CTB-ACE2 has the potential to effectively bind to both the GM1 and ACE2 receptor-binding sites located in close proximity on the human cell surface and thereby prevent viral entry into human cells. Therefore, we employed a SARS-CoV-2 pseudotyped lentivirus (also referred to as lentivirus particles) to determine the effectiveness of ACE2 gum in neutralizing spike-mediated viral infection. Lentiviral particles pseudotyped with the viral spike protein and harboring the pseudoviruses expressing a luciferase reporter gene were used to infect Chinese hamster ovary (CHO) cells expressing human ACE2.⁴⁸ SARS-CoV-2 spike glycoprotein pseudotyped viruses expressing luciferase were incubated with ACE2 gum at the indicated concentration for 90 min at room temperature. Following centrifugation, virus-containing supernatant was incubated with ACE2-expressing CHO cells for 72 h, and viral infectivity was measured via luciferase. Data shown inFigure 4 are representative of three independent experiments (N = 2–3 samples). Bars represent the condition mean, symbols represent the average of duplicate or triplicate assays, and error bars represent the SEM. The incubation of pseudotyped lentivirus with increasing amounts of CTB-ACE2 gum significantly reduced luciferase activities in a dose-dependent manner: at 50 mg of CTB-ACE2 the highest inhibition (p < 0.0001) was observed (Figure 4), confirming the ability of this protein to block entry of lentivirus spike protein into CHO cells either by direct binding to the spike protein or by binding of CTB-ACE2 to ACE2/GM1 receptors on CHO cells.

VSV-S pseudotype entry is inhibited by recombinant CTB-ACE2 and ACE2 gum powder

The red fluorescent protein (RFP)-expressing vesicular stomatitis virus spike (VSV-S) pseudotype particles utilize the SARS-CoV-2 spike (S) protein to bind and enter cells. We investigated the impact of purified recombinant CTB-ACE2 protein or ACE2 gum powder binding to VSV-S particles and inhibition of viral particle entry into Vero cells. In repeated experiments, we observed that VSV-S entry was inhibited with the addition of CTB-ACE2 by approximately 85% when compared with untreated controls (Figure 5, lane 2), confirming the antiviral effect of our recombinant protein on entry of the VSV-S pseudotypes. We also observed a dose-dependent inhibition of entry when VSV-S particles were incubated with increasing concentrations of our ACE2 gum powder (Figure 5, lanes 3–6). Indeed, representative images from fluorescent microscopy highlight clear and significant differences in the number of cells expressing RFP in control versus 20-mg ACE2 gum powder samples (Figure S3). Taken together, these data show that CTB-ACE2 protein and ACE2 gum powder traps the VSV-S pseudotype particles, thus decreasing viral entry either by binding directly to the spike protein or by binding to ACE2/GM1 receptors on the surface of Vero cells.

ACE2 activity in healthy control and COVID-19 patient saliva

After informed consent under protocol #823392 approved by the University of Pennsylvania (UPenn) IRB, saliva samples were obtained from hospitalized patients with confirmed SARS-CoV-2 infection and healthy volunteers confirmed to be free of SARS-CoV-2 infection. Results on analysis of ten samples each of healthy and COVID-19 (PCR positive) are presented inFigure 6. ACE2 activity was markedly reduced in COVID-19 patients compared with controls (Figures 6A–6C). We observed a drastic reduction in ACE2 activity in viral-laden saliva collected from ten COVID-19 patients compared with healthy individuals (2,582 ± 439.82 versus 50,126 ± 2,102, change in relative fluorescence units [ΔRFU]: 27.63 ± 9.52 versus 225 ± 30.82 mU/mg enzyme activity units). The fluorescent cleaved product of ACE2 (Mca-YVADAPK) steadily increased up to 90 min in control saliva samples. While all COVID-19 saliva samples showed a similar low and almost undetectable ACE2 activity, one sample stood out as an outlier with enzyme activity (38,504 ± 9,688 ΔRFU; 236.4 ± 60.28 mU/mg enzyme activity units) similar to control saliva (Figures 6A and 6B). This patient was identified as SARS-CoV-2 positive through routine screening of hospital admissions and had no symptoms of COVID-19 infection, although PCR confirmed virus in the saliva.

The activity of CTB-ACE2 is inhibited by SARS-CoV-2 spike protein

To investigate the mechanism of decreased ACE2 activity observed in COVID-19 saliva, we performedin vitro enzymatic assays using plant extract containing full-length CTB-ACE2 in the presence or absence of the SARS-CoV-2 spike protein (receptor-binding domain [RBD] S1-S2). The fluorescent cleaved product of CTB-ACE2 protein extracts (Mca-YVADAPK) increased up to 90 min, demonstrating that ACE2 was enzymatically active (Figure 6D). However, this activity was partially inhibited when CTB-ACE2 was pre-incubated with 10 μg of SARS CoV-2 S1-S2 spike protein for 30 min at room temperature (Figure 6D). In fact, ACE2 activity showed complete inhibition by pre-incubation (30 min) with 10 μg of SARS-CoV-2 RBD (Figure 6D). Collectively, this finding suggests that SARS-CoV-2 spike proteins bind directly to full-length ACE2 through the RBD and thus abolish ACE2 activity.

Evaluation by debulking of SARS-CoV-2 in saliva by ddPCR

Several de-identified saliva samples collected from patients positive for SARS-CoV-2 were used to evaluate debulking of the viral particles by ACE2 or placebo gum using droplet digital PCR (ddPCR). Unlike the microbubble assay in which actual viral particles are measured, ddPCR amplifies and then quantifies the viral RNA, and therefore actual copies of viral RNA present in patients is not directly measured. While PCR amplification is used to increase sensitivity of saliva tests, it is not quantitative. Therefore, PCR amplification has not yet been used to predict the severity of COVID-19 disease. Despite these limitations with the nucleic acid testing approach, we observed some reduction with the placebo and significant reduction with ACE2 gum in COVID-19 saliva samples, almost to the lowest number of copies that could be reliably measured by ddPCR (Figure 7).

Saliva sample collection

After informed consent under protocol #823392 approved by the UPenn IRB, saliva and swabs of the nasopharynx and oropharynx were obtained from hospitalized patients with confirmed SARS-CoV-2 infection. Saliva was produced by patients spontaneously, and oropharyngeal and nasopharyngeal samples were collected with flocked nylon swabs (Copan Diagnostics) and eluted together in 2 mL of viral transport medium. Saliva from healthy volunteers (confirmed SARS-CoV-2 negative) was collected following informed consent under protocol #842613 approved by the UPenn IRB. Specimens were stored at −80°C until use.

Plant growth at Fraunhofer USA, biomass harvest, and lyophilization

The hydroponic growth system at Fraunhofer USA includes multilevel growth racks illuminated to grow the transplastomic lettuce expressing CTB-ACE2. Growth trays contained rockwool, a lightweight hydroponic substrate that is manufactured by spinning molten basaltic rock into fibers, as the supporting substrate (Grodan, the Netherlands). Rockwool is designed to hold approximately 80% nutrient solution, with approximately 15% air space and 5% fibers, drains freely, and encourages rapid uptake of nutrient solution by plant root systems. The final nutrient solution for hydroponic growth comprised Peters Professional general purpose fertilizer at 200 parts per million nitrogen with a 20:10:20 ratio of nitrogen, phosphate, and potash (potassium oxide) (ICL Fertilizers, OH), and YaraLiva Calcinit (nitric acid, ammonium calcium salt) (Yara North America) at 102 parts per million calcium. Nutrient concentrates were supplied via Dosatrons at a 1:100 injection ratio with potable water. Seedlings were spaced at 8.5 inches on a diagonal grid on the rockwool surface. Plants were exposed to LED lighting (Fluence SPYDRx) at an average of 368 μmol/m²·S on a 16-h day/8-h night photoperiod under conditions of temperature (24°C ± 2°C day/20°C ± 2°C night) and humidity (60% ± 10%) in a growth room. Air flow across the plants was approximately 1.2 m/s.

At Fraunhofer, the transplastomic lettuce was repeatedly harvested from plants on days 58, 90, 107, and 120 post seeding, typically at approximately 9 (8–11) hours into the day portion of the photoperiod, while the AeroFarms grown plants were harvested 79 and 100 days post seeding. The leaves of lettuce plants were cut at the base of the petiole by operators wearing laboratory gloves, leaving the upper leaves intact on each plant, and the fresh weight of biomass harvested was recorded. Leaves were rinsed in USP purified water, washed for approximately 5 min in a 200 parts per million chlorine solution prepared using food-grade calcium hypochlorite, and rinsed three consecutive times in USP purified water, with final chlorine levels measured using chlorine sanitizer test strips to ensure levels were less than 4 parts per million. Excess water was then removed from leaf surfaces using a food-grade centrifugal lettuce drier before being transferred to polyvinyl zip-top bags. Bags were frozen on dry ice and then progressed to storage at ≤ −60°C to −80°C. Frozen lettuce leaves expressing CTB-ACE2 fusion proteins were freeze-dried in a lyophilizer (Ultra50; SP Scientific, Stone Ridge, NY).

Shelves were pre-cooled to −40°C and each of the 15 shelves loaded with frozen leaves that had been briefly pounded with a mallet and from which remaining sizable pieces of petiole had been removed (typically loading approximately 0.5 kg per shelf), with lyophilizer thermocouple probes placed in the middle of plant materials in every other tray to monitor product temperature. The lyophilization cycle was optimized to reduce the duration of the cycle time in order to maximize throughput, while achieving a sufficiently low moisture content of at least ≤10% and preferably ≤5% for further processing, with a preferred cycle comprising the following sequence. Lyophilization started with a freezing phase, holding plant materials at −40°C for 180 min at a vacuum of 300 mTorr. This was followed by a drying phase that comprised the following stages: holding at −40°C for 15 min at a vacuum of 300 mTorr, then ramping to −5°C over a period of 350 min followed by holding at −5°C for 180 min at a vacuum of 300 mTorr, then ramping to 25°C over a period of 300 min followed by holding at 25°C for at least 1,000 min at a vacuum of 300 mTorr, and finally holding at 25°C for a further 500 min at a vacuum of 300 mTorr. A PVG/CM of 43 mTorr needed to be reached before progression to the final holding step in the above cycle. After the cycle ended, plant materials were unloaded, packed, weighed, and assessed for moisture content. The lyophilizer was thoroughly cleaned and disinfected after each use and records collected on cycle parameters. If not proceeding directly to milling, the materials were placed into zip-top bags and then into light-tight storage bags. The freeze-dried materials were stored in dark cabinets at room temperature in a storage room. The lyophilized leaves were ground in a grinder (BioMix-700g) at single speed once for 3 s (optimized for minimal disrupted cells). The milled powder was poured over a 25-mesh (0.71 mm) hand sieve into the final sterile container for closure. Any material that did not pass through the sieve was discarded. The materials were stored in sterile containers (FDA approved) inside a steel cabinet for dark, room-temperature storage and assessed for bioburden.

Moisture content

Moisture content was determined by Karl Fischer titration, whereby available water reacts with iodine and sulfur dioxide to form sulfur trioxide and hydrogen iodide. In brief, samples were weighed, and sample vials capped and placed in a Metrohm 850 KF Thermoprep and heated to 150°C. Vaporized water from samples was pumped into a Metrohm 851 Titrando at a rate of 50 mL/min. Percentage of moisture was calculated from sample weight and reaction output, with Hydranal water standard (Honeywell) as a reference control. A recent model of freeze dryer (Virtis Ultra 50) has new gadgets to evaluate and monitor moisture content, which were not available in previous versions.

Bioburden

Following harvest of biomass, leaves were washed in a 200 parts per million chlorine solution with triple rinse in USP purified water. Excess water was then removed from leaves and the tissue frozen and stored. The tissue was then freeze-dried in a lyophilizer (Ultra50; SP Scientific), assessed for moisture content, ground, and sieved and again assessed for moisture content and for bioburden. Bioburden was evaluated according to USP <61> (microbiological examination of nonsterile products: microbial enumeration tests) and USP <62> (microbiological examination of nonsterile products: tests for specified microorganisms). Samples were assessed for aerobic microbial and fungal loads by plating serial dilutions in duplicate on each of trypticase soy agar and Sabouraud dextrose agar, respectively, and incubating for 3–5 days at 30°C–35°C or 5–7 days at 20°C–25°C, respectively. For oral delivery, acceptance criteria were set according to USP <1111> (microbial examination of nonsterile products: acceptance criteria for pharmaceutical preparations and substances for pharmaceutical use) at ≤ 2 × 10³ CFU/g for total aerobic microbial count and ≤2 × 10² CFU/g for total yeast and mold count.

Total protein extraction and quantification of ACE2

CTB-ACE2 expression was examined by western blots as described previously,³⁹ with suitable modifications. Ten milligrams of lyophilized plant powder was extracted with 500 μL of protein extraction buffer containing 100 mM NaCl, 10 mM EDTA (pH 8), 200 mM Tris (pH 8), 100 mM dithiothreitol, 1× protease inhibitor cocktail, 2 mM phenylmethylsulfonyl fluoride, and 10 mM β-mercaptoethanol. The homogenates were incubated for 1 h while vortexing at 4°C. This was followed by sonication of samples at 80% amplitude for 10 s on and 15 s off (6×) using a sonicator 3000 (Misonix, Farmingdale, NY). Bradford assay was used to quantify total protein content (Bio-Rad Laboratories, Hercules, CA). After adding 1× Laemmli buffer, homogenized proteins were heated at 70°C for 15 min on a hot plate and run on 10% SDS-PAGE. For each Fraunhofer and AeroFarms sample, two different concentrations of total protein were loaded on gels, Plant materials across different harvests, 58- to 120-day-old plants grown at Fraunhofer were compared with 79- to 107-day-old plants grown at AeroFarms.

For all immunoblot analyses, anti-CTB antibody (1:10,000) (Gen Way Biotech, San Diego, CA), goat antirabbit IgG-HRP secondary antibody (1:4,000) (Southern Biotechnology, Birmingham, AL), and the Precision Plus protein standard (Bio-Rad Laboratories) were used. The West Pico Chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA) was used to detect ACE2 proteins on iBright Western Blot Imaging Systems (Thermo Fisher Scientific). Different concentrations of CTB standard were used to quantify ACE2 protein using iBright Analysis Software (Thermo Fisher Scientific). Total protein content of plant samples was quantified in three technical replicates performed in eight independent experiments.

ACE2 enzyme activity assay in plant extract and saliva

ACE2 activity assay was performed using saliva of SARS-CoV-19 affected and healthy people. Saliva samples were prepared at 1:5 dilution. Seventy-five microliters of this prepared sample was added to a black 96-well microtiter plate containing 25 μL of ACE2 buffer (50 μmol/L ZnCl₂, 75 mmol/L Tris-HCl [pH 7.5], and 1 mol/L NaCl), and 20 μmol/L ACE2-specific fluorogenic peptide substrate VI Mca-APK(Dnp) (R&D Systems, Minneapolis, MN). The enzymatic activity was recorded for 90 min at 5-min intervals at 28°C with optic position top and gain extended; with excitation at 340 nm and emission at 405 nm. The relative fluorescence unit at each time point was plotted. ΔRFU was calculated by subtracting data of time point 0 min from the data of time point 90 min. Total soluble protein in the remaining saliva sample was determined by Bradford assay using standard Daniell lab protocol. The final ACE2 enzyme activity was calculated as pmol/min/mg (mU/mg) = Δpmol/90 min/mg total protein.

Microbubble SARS-CoV-2 antigen assay

Patient samples (150 μL) were added to each powder tube and vortexed. Tubes were then incubated at 4°C for 1 h while rotating. Following incubation, tubes were vortexed again, then spun at 14,000 rpm for 20 min at 4°C. Tubes were recovered and 120 μL of supernatant was first mixed with virus lysis buffer of 10% Tween 20 (100×, 1.2 μL) and 100× protease inhibitor cocktail (1.2 μL) and incubated at room temperature for 30 min. The lysed sample was then tested with the Microbubble SARS-CoV-2 Antigen Assay.^40,41 In brief, sample solutions (100 μL) were incubated with suspensions of 500,000 capture antibody functionalized magnetic beads, on a roller (12 rpm) at room temperature for 30 min. The beads were then separated using magnets and washed three times with PBS buffer (pH 7.4) containing 0.01% Tween 20, then resuspended in 100 μL of 250 ng/mL biotinylated detection antibody in PBS containing 1% BSA, on a roller (12 rpm) at room temperature for 30 min. The beads were then separated using magnets and washed three times with PBS buffer (pH 7.4) containing 0.01% Tween 20 and then resuspended in 100 μL of 1 μg/mL NeutrAvidin functionalized Pt nanoparticles in PBS containing 1% BSA, on a roller (12 rpm) at room temperature for 30 min. The beads were then separated using magnets and washed three times with PBS buffer (pH 7.4) containing 0.01% Tween 20 and resuspended in 100 μL of 30% H₂O₂. The magnetic bead slurries were then added into the chambers of the microbubbling microchips. The microbubbling microchips were placed on a neodymium disk magnet for 1 min to pull down the beads to the bottom of the microchips. Microbubbles on the microbubbling microchips were imaged using an iPhone 11 or an iPad with the uHandy mobile phone microscope (9×, 5 mm focusing length; Aidmics Biotechnology, Taipei, Taiwan).

Neutralization assay with SARS-CoV-2 spike-pseudotyped lentiviral particles

SARS-CoV-2 pseudovirus was produced using HEK293T cells transfected with a 1:1 ratio of IgE-SARS-CoV-2 S plasmid (Genscript) and pNL4-3.Luc.R-E⁻ plasmid (NIH AIDS reagent) using Gene Jammer (Agilent) as transfection reagent. Forty-eight hours post transfection, transfection supernatant was collected, enriched with fetal bovine serum (FBS) to 12% final volume, and stored at −80°C. SARS-Cov-2 pseudovirus neutralization assay was set up using D10 medium (DMEM supplemented with 10% FBS and 1× penicillin-streptomycin) in a 96-well format using huCHOAce2 cells (Creative Biolabs, catalog no. VCeL-Wyb019). For neutralization assay, 10,000 CHO-ACE2 cells were plated in 96-well plates and rested overnight at 37°C and 5% CO₂ for 24 h. CTB-ACE2 cells at specific weights were incubated with a fixed amount of SARS-Cov-2 pseudovirus for 90 min at room temperature in complete medium. After centrifugation at 15,005 rpm for 10 min, the supernatant of this mix was added to huCHOAce2 cells and allowed to incubate in a standard incubator (37% humidity, 5% CO₂) for 72 h. After 72 h, cells were lysed using the Britelite Plus luminescence reporter gene assay system (PerkinElmer, catalog no. 6066769), and relative light units were measured using a BioTek plate reader. Percent neutralization was calculated using GraphPad Prism 8.

VSV-S-pseudotyped neutralization assay

VSV-S pseudotype particles were generated using a VSV platform described previously.⁴² In brief, pseudotyped VSV virions that incorporate the SARS-CoV-2 spike protein into their envelopes were produced by transfection of HEK293T cells with pCG1 SARS-CoV-2 S delta18 expression plasmid encoding a codon optimized SARS-CoV-2 S gene with an 18-residue truncation in the cytoplasmic tail (kindly provided by Paul Bates and Stefan Pohlmann). At 30 h post transfection, the SARS-CoV-2 spike-expressing cells were transduced for 4 h with VSVΔG-RFP pseudotypes. The viral inoculum was removed, and the cells were washed twice with 1× PBS to remove unbound virus. At 28 h post transduction, the medium containing the VSV-S pseudotypes was harvested and clarified by centrifugation twice at 4,000 rpm for 15 min. VSV-S pseudotypes were aliquoted and stored at −80°C until use in ACE2 neutralization studies.

Vero E6 cells were seeded at 1 × 10⁴ cells per well in a 96-well plate and incubated overnight at 37°C. One hundred microliters of VSV-S pseudotype particles (∼1.2 × 10⁴ red fluorescent units) in medium were mixed with 250 ng of CTB-ACE2 or 5 mg, 10 mg, 20 mg, or 50 mg ACE2 gum powder and rotated end over end for 30 min at 4°C. Samples were centrifuged at 14,800 rpm for 20 min at 4°C to pellet the gum powder, and the supernatant was added to Vero cells for 1 h at 37°C, swirling every 15 min. Virus inoculum was removed, and the cells were washed twice with 1× PBS to remove unbound viral particles. At 24 h post transduction, RFP expression was visualized and quantified on a fluorescent microscope. RFP expression in each condition was measured in two technical replicates performed in two independent experiments. Inhibition of VSV-S entry was calculated relative to the untreated VSV-S control, and statistical significance was analyzed by Student’s t test.

ddPCR assay

Fifteen saliva specimens from COVID-19 patients were collected by UPenn under an IRB-approved protocol. Saliva (140 μL) was mixed and incubated with 100 mg of ACE2 or placebo gum powder for 1 h at room temperature while rotating, then spun at 14,000 rpm for 20 min. Manufacturer's instructions were followed while using the QIAamp viral RNA mini kit (Qiagen), and total RNA was extracted from the supernatant. ddPCR was performed in duplicates via the COVID-19 digital PCR detection kit (Bio-Rad). The QX200 Droplet Digital PCR System using Supermix probe (Bio-Rad) was used following the manufacturer's instructions. The kit allows detection of the regions of nucleocapside N1, N2 gene, and Rnase P gene positive reference gene. QX200 droplet generator (Bio-Rad) converted 22 μL of each reaction mix to droplets. Soon after transferring and sealing the droplet partitioned specimens to a 96-well plate, they were cycled in a T100 Thermal Cycler (Bio-Rad) following the protocol: DNA polymerase activation at 95°C for 10 min, denaturation at 94°C for 30s and final step of annealing at 60°C for 1 min, followed by 4-degree infinite hold. Using the QX200 reader (Bio-Rad) the cycled plate was read in the HEX and FAM channels. QuantaSoft analysis Pro 1.0.596 software (Bio-Rad) was used for data interpretation.

Author contributions

H.D. conceived this project, designed experiments, identified collaborators, and coordinated projects with different groups or institutions, interpreted data, and wrote/edited several versions of the manuscript. S.K.N. quantified CTB-ACE2 in chewing gum and performed ACE2 activity assays in saliva samples, and interpreted data. S.K.N., H.D., S.J.S., N.E., and SP Scientific experts optimized the recipe for the lyophilization cycles in Ultra50. N.E. freeze-dried plants grown at AeroFarms, and along with G.W., N.S., and P.K.G. quantified ACE2. Md.R.I. performed ACE2 assay in plant extract. At Fraunhofer USA, S.J.S. designed the growth conditions and contributed to the manuscript, R.M.-L. and B.J.G. contributed to the design of the growth process and optimization of the leaf washing protocol, and P.K. contributed to the design of the growth and lyophilization process. S.N.C. amd M.M. designed and performed ddPCR studies to evaluate SARS-CoV-2 copy number. M.M. seeded ACE2 plants and assisted with biomass growth, harvesting, processing, and lyophilization. J.G.-W. and R.G.C. contributed saliva samples. E.N.G., A.R.A., M.N., R.R., and D.B.W. performed and interpreted lentivirus spike neutralization assays. A.S.-M. and R.N.H. performed and interpreted VSV-spike pseudotype entry assays. P.W. and S.F. contributed nasopharyngeal swab samples, and performed and interpreted the microbubbling SARS-CoV-2 antigen assay. K.B.M. participated in discussions and edited the manuscript.

Declaration of interests

All authors declare no competing interests except the corresponding author (H.D.), who is a patentee in this field but has no specific financial conflict of interest to disclose.

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