key: cord-0816890-qmhg0rm3
authors: Daniell, Henry; Nair, Smruti K.; Esmaeili, Nardana; Wakade, Geetanjali; Shahid, Naila; Ganesan, Prem Kumar; Islam, Md Reyazul; Shepley-McTaggart, Ariel; Feng, Sheng; Gary, Ebony N.; Ali, Ali R.; Nuth, Manunya; Cruz, Selene Nunez; Graham-Wooten, Jevon; Streatfield, Stephen J.; Montoya-Lopez, Ruben; Kaznica, Paul; Mawson, Margaret; Green, Brian J.; Ricciardi, Robert; Milone, Michael; Harty, Ronald N.; Wang, Ping; Weiner, David B.; Margulies, Kenneth B.; Collman, Ronald G.
title: Debulking SARS-COV-2 in saliva using angiotensin converting enzyme 2 in the chewing gum to decrease oral virus transmission and infection
date: 2021-11-11
journal: Mol Ther
DOI: 10.1016/j.ymthe.2021.11.008
sha: 8a34c1000328d9ccf597ec237ebdcea7e03c0dc5
doc_id: 816890
cord_uid: qmhg0rm3

To advance a novel concept of debulking virus in the oral cavity, the primary site of viral replication, virus trapping proteins CTB-ACE2 were expressed in chloroplasts and clinical grade plant material was developed to meet FDA requirements. Chewing gum (2 grams) containing plant cells expressed CTB-ACE2 up to17.2 mg ACE2/g DW (11.7% leaf protein) have physical characteristics, taste/flavor like conventional gums and no protein was lost during gum compression. CTB-ACE2 gum efficiently (>95%) inhibited entry of Lentivirus-Spike or VSV-Spike pseudovirus into Vero/CHO cells, when quantified by luciferase or red fluorescence. Incubation of CTB-ACE2 microparticles reduced SARS-CoV- 2 virus count in COVID-19 swab/saliva samples >95%, when evaluated by microbubbles (femtomolar concentration) or qPCR, demonstrating both virus trapping and blocking of cellular entry. COVID-19 saliva samples showed low or undetectable ACE2 activity when compared to healthy individuals (2582 vs 50126 ΔRFU; 27 vs 225 enzyme units), confirming greater susceptibility of infected patients for viral entry. CTB-ACE2 activity was completely inhibited by pre-incubation with SARS-COV-2 RBD, offering an explanation for reduced saliva ACE2 activity among COVID-19 patients. Chewing gum with virus trapping proteins offers a general affordable strategy to protect patients from most oral virus reinfections through debulking or minimizing transmission to others.

linked in large part with indoor exposure of infected individuals, symptomatic or asymptomatic.

Controlling transmission has involved reduction of concentrations of indoor aerosols largely through masking and physical distancing. In public buildings (classrooms, retail shops, restaurants, gyms, Churches, etc.), air exchanges through filters to decrease transmission 1 . Most people emit > 100 times smaller aerosols (<5 μm) during talking, breathing or coughing 2 . Less than 10% of the global population is currently vaccinated, only to be made worse by the shortage of the SARS-CoV-2 vaccines and evolving new strains with higher viral load and greater transmission [3] [4] [5] [6] [7] [8] . With A high SARS-CoV-2 viral load is often detected in saliva 9 . Highly contagious airborne droplets are the major cause of transmission in respiratory viruses like influenza, measles, and SARS-CoV-2 [10] [11] [12] [13] . Human Papillomavirus, Herpes Simplex virus type 1, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus are orally transmitted and their life cycle in the oral epithelium is well known 14, 15 . In SARS-CoV-2 with saliva average load of 7 × 10 6 copies of RNA virus per ml, an oral fluid droplet of 50 µm 2 could contain at least one virion 2, 14 . High SARS-CoV-2 viral loads are detected in saliva of both asymptomatic and symptomatic COVID-19 patients 16, 17 .

In fact, salivary viral burden correlates with the severity of COVID-19 symptoms including the loss of taste and smell, and the virus replicates in salivary glands and oral mucous membranes 18 .

Thus, the oral mucous membranes and saliva appear to be a high-risk route for SARS-CoV-2 transmission and viral inactivation within the oral cavity could be an important strategy to reduce J o u r n a l P r e -p r o o f viral infectivity at source. COVID-19 patients have low ACE2 activity due to RAAS dysregulation and this causes respiratory stress [19] [20] [21] and injected ACE2 restores health in COVID-19 patients 22 . ACE receptor traps have large binding interfaces capable of blocking the entire receptor interface, thereby facilitating inhibition of different SARS-CoV-1 and SARS-CoV-2 variants 23, 24 . In the absence of any FDA approved receptor traps as antiviral biologics 25 , several traps using soluble ACE 2 as nasal spray have been recently developed 23 . While nasal sprays could help in reducing viral load in the nose, additional approaches are needed to decrease viral load in the saliva because salivary glands are the primary sites of SARS-CoV-2 replication 9, 17, 18 . That salivary glands serve as a reservoir of replication for viruses causing highly prevalent diseases such as Epstein-Barr virus, Herpes simplex, HHV-7, cytomegalovirus, Hepatitis C, and Zika virus is a known fact [26] [27] [28] 29 .

Newly evolving strains have higher viral load in saliva and greater transmission [4] [5] [6] [7] . The viral load of people infected by the delta variant is 1260 times higher than individuals infected with previous strains 4 . The CDC has reported a higher basic reproduction rate -R0 = 5-8 for the delta variant that estimates to 60,466,176 infections as opposed to R0 of 2.79 of the ancestral strain 30 , which translates into an estimated 9536 infections 31 . The delta variant is 40 to 60 percent more contagious than the previously dominant alpha strain 31, 32 . High viral density, transmissibility, of these variants along with the increase in replication potential and serial viral shedding 33 , warrants development of novel approaches to curb viral loads in saliva.

Mouth wash with antimicrobial agents have short period of contact 32 . Therefore, in this study we explore longer duration of contact using the chewing gum topical delivery approach.

SARS-CoV-2 utilizes ACE2 and GM1 co-receptors to enter human cells [34] [35] [36] [37] [38] . Therefore, in this study, we explore receptor binding/blocking proteins CTB-ACE2 chewing gum to minimize J o u r n a l P r e -p r o o f transmission and decrease infectivity by binding directly to the spike protein to trap virus particles and saturating both ACE2/GM1 receptors located in close proximity on the surface of human oral epithelial cells. Furthermore, we explore impact of SARS-CoV-2 on ACE2 activity in saliva and potential role as biomarker to distinguish symptomatic from asymptomatic COVID-19 patients.

In addition to prophylactic protection against COVID in general social settings or restaurants, the ACE2 gum could be used as a rapid means of reducing SARS-CoV-2 from oral cavity of infected patients requiring dental procedures. This general concept could be extended to minimize infection or transmission of most oral viruses.

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 in the methods section.

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 moderate decrease in biomass (Figure 1 , Table 1) .

Biomass yields at Fraunhofer and AeroFarms (tower 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 DW or 11.7% ACE2 in total leaf protein, TLP), a significant improvement from previous report 43 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 ± 0.1mg/g DW or 8.8%TLP) than other two towers. The reproducibility of ACE2 protein expression was evaluated J o u r n a l P r e -p r o o f in several independent trials of plants grown at Fraunhofer and AeroFarms and statistical data is presented in Table 1 . However, irrespective of growth conditions, highest expression was observed in oldest plants when lowest biomass was harvested at both facilities (Figure 1 , Table   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 (Figure 1, Table 1 , Figure S1 ). 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.

Chewing gum tablets containing ground plant powder were prepared by Per Os Biosciences (Hunt Valley, MD) by 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%), 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 the 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. ACE2 gum tablet has all components of the placebo gum but in addition includes 50 mg of CTB-ACE2 freeze-dried 5X ground plant cells. Evaluation of sum total of proteins in all fractions (supernatant and pellet) revealed an insignificant loss of CTB-ACE2 during gum manufacturing process ( Figure S2 ).

Debulking and blocking of viral entry using ACE2 chewing gum J o u r n a l P r e -p r o o f 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. 

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) in order 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 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 minutes at room temperature. Following centrifugation, virus-containing supernatant was incubated with ACE2expressing CHO cells for 72hrs, and viral infectivity was measured via luciferase. Data shown in 

The RFP-expressing 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 inhibit 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 to untreated controls ( 

To investigate the mechanism of decreased ACE2 activity observed in COVID-19 saliva, in vitro enzymatic assays were performed using plant extract containing full length CTB-ACE2 in the presence or absence of the SARS-COV-2 spike protein (RBD, S1-S2). The fluorescent cleaved product of CTB-ACE2 protein extracts (Mca-YVADAPK) increased up to ninety minutes, demonstrating the 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 mins at RT ( Figure 6D ). In fact, ACE2 activity showed complete inhibition by preincubation (30 min) with 10 µg SARS-COV-2 RBD ( Figure 6D ). Collectively, this finding suggests that SARS-COV-2 spike proteins bind directly to the full-length ACE2 through the RBD and thus abolished ACE2 activity.

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 ddPCR.

Unlike the microbubble assay where actual viral particles are measured, ddPCR amplified and then J o u r n a l P r e -p r o o f quantified 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 hasn't 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 ).

The oral cavity is an important portal of entry for SARS-CoV-2 virus and plays particularly significant role in the transmission of infection or continued reinfection. The heterogeneity of the oral mucosa at a cellular level and presence of several ubiquitous receptors including ACE2 and GM1 facilitate entry of oral pathogens 47, [49] [50] [51] . Saliva plays a vital role in transmission of infection among critically ill patients and can also compromise oral tissues 52 . Reducing the viral load in saliva should limit the risk of transmission from a potential carrier [53] [54] [55] and may help reduce severity of COVID-19 disease by minimizing reinfection because salivary glands is the primary site of SARS-CoV-2 replication. Therefore, we explore the ability of CTB-ACE2 chewing gum to trap SARS-CoV-2 to debulk virus from saliva in preclinical studies that provide a foundation of clinical testing designed to reduce oral viral load and transmission.

ACE2 is an integral part of the renin-angiotensin system (RAS), and cleaves angiotensin II (Ang II), which causes vasoconstriction, inflammation, hypercoagulation and fibrosis 19 to produce the anti-inflammatory, cytoprotective angiotensin 1-7 (Ang 1-7) peptide. Human ACE2 exists in both the soluble (sACE2) and membrane associated ACE2 (mACE2) forms, the latter being the most predominant form 20, 21, 56 . Infusion could supplement the lost sACE2 and help balance RAS by prevention of downregulation in COVID-19 patients 22 . In pulmonary hypertension (PH) disease J o u r n a l P r e -p r o o f with disease symptoms similar to COVID-19, oral ACE2 results attenuates PH with decreases in right ventricular (RV) hypertrophy, RV systolic pressure, total pulmonary resistance and pulmonary artery remodeling 43, 57 . In contrast to injected truncated (transmembrane deleted) sACE2 22 full length oral CTB-ACE2 accumulates in the lungs at 10-fold higher concentrations than in the plasma upon oral delivery of bioencapsulated plant cells 43, 44 offering yet another approach to treat COVID-19 patients.

Utilizing concepts described above, ACE2 chewing gum is investigated here to trap SARS-CoV-2 to debulk virus and reduce oral transmission. While entry of SARS-CoV-2 into human cells through the ACE2 receptor has been widely reported 34 , the requirement for GM1 co-receptor or direct binding of the spike protein to soluble ACE2 has received less attention. In fact, SARS-CoV-2 has greater binding affinity to monomeric soluble ACE2 than other known coronaviruses 21 . CTB-ACE2 saturation of both ACE2/GM1 receptors located in close proximity 35, 36 , enabled virus neutralization studies in Vero cells using VSV or Lentivirus engineered to express spike proteins or could prevent entry into human oral epithelial cells. Therefore, sACE2 could compete for receptor binding site with SARS-CoV-2 and act as "decoy" and also directly bind to SARS-CoV-microbubble counts at femtomolar concentration 40, 41 , confirming ACE2 gum dramatically decreased the amount of nucleocapsid antigens. Unlike the microbubble assay where actual viral particles are measured, ddPCR quantifies viral RNA, which may not necessarily be associated with virions and has been shown to persist after clearance of active infection 58, 59 . Despite these limitations with the nucleic acid testing approach, we observed significant reduction with ACE2 gum in COVID-19 saliva samples, almost to the lowest number of copies that could be reliably measured by ddPCR.

Our studies observed that placebo gum without ACE2 had some antiviral activity, depending on viral particle density. In previous studies, in silico screening of 48 sugar alcohol compounds sorbitol, mannitol, and galactitol showed maximum binding to viral proteins, especially Ebola VP40 60 . ACE2 chewing gum contains maltitol (20.4%) and 13% each of sorbitol and xylitol, similar to commercial chewing gums and could explain this placebo effect. In addition to demonstrating debulking of SARS-CoV-2 using several evaluation criteria, in this study we demonstrate feasibility of clinical grade plant biomass that can be prepared to meet FDA criteria.

We followed guidelines based on FDA approval of Ara h proteins expressed in peanut cells for treatment of peanut allergy by oral delivery of plant cells 61, 62 . Importantly, total aerobic microbial count and total yeasts and molds count in batches used for preparation of ACE chewing gum were within acceptance criteria established by the FDA for a non-aqueous drug product being delivered by the oral route. Furthermore, moisture content was less than 10%, as reported in the peanut allergy clinical trials (NCT02635776). Indeed, oral delivery of protein drugs bioencapsulated in plant cells reduces production and delivery costs by elimination of prohibitively expensive fermentation, purification, cold chain maintenance for transportation/storage and sterile injections 44, 45, 63 . Most recently, the Daniell lab also developed the chewing gum expressing lipase, dextranase, mutanase to disrupt dental biofilm and kill pathogenic bacteria and fungi 64 . In chewing gum tablets formulated with freeze dried plant cells, GFP was stable up to three years at ambient temperature and was efficiently released in a time dependent manner in a mechanical chewing simulator device, suggesting feasibility of therapeutic gum for topical drug delivery. In this study, 90% of protein release from the chewing gum was observed using chewing simulator machine (Pickering Laboratories) and artificial saliva 64 (male, femalewhite/black), five in their 50s (4 females -2 white/2 black; 1 male -black) and three in their 60s (male/white). All COVID patients were tested positive and PCR data is provided (Supplementary Table 1 ). All Covid-19 saliva samples showed low or almost undetectable ACE2 activity (50% below zero and others 10-40 mU/mg). However, one patient was asymptomatic based on the patient chart and didn't develop the COVID-19 disease, although PCR data showed presence of SARS-CoV-2. Therefore, saliva ACE2 activity could serve as a biomarker to distinguish symptomatic from asymptomatic COVID-19 patients. The decrease in ACE2 activity observed in saliva could be due to downregulation of RAS47-48 68, 69 . Alternatively or in addition, SARS-CoV-2 spike protein which has high affinity to soluble ACE2 could bind directly and thus decrease ACE2 activity 21 . Indeed, this is strongly supported by our studies where RBD binds with much higher affinity than S1/S2 spike protein, fully inhibiting ACE2 activity. [73] [74] [75] [76] [77] . Therefore, reducing the viral load using the ACE2 chewing gum will not only help in mitigating the severity of COVID-19 variants but also limit the risk of transmission from a potential carrier [53] [54] [55] . While current chewing gum used the native full length human ACE2 protein, future studies could explore engineered ACE proteins that have 170fold higher affinity to SARS-CoV-2 spike protein to bind to evolving variants 23,24 and efficiently block infection or transmission.

In summary, this is the first report of using a biomaterial to debulk SARS-CoV-2 in saliva.

Transmission of SARS-CoV-2 is an urgent concern around the globe due to emerging variants, inadequate vaccination, and limitation of current containment methods. This report offers a novel and affordable concept to reduce SARS-CoV-2 reinfection through saliva, minimize oral aerosol transmission and offers patients time to build immunity in countries where vaccines are not available or affordable. In particular, this is a novel approach to protect individuals at home and also healthcare workers from patients without masks during oral procedures and dental cleaning.

There are no examples of FDA approved antiviral biologic traps, making this a novel approach.

In addition, we utilize virus quantitation without any nucleic acid amplification by using recently developed microbubble technology, capable of detecting viral surface protein signals. ACE2 activity is also quantified for the first time in human saliva with striking reductions demonstrated J o u r n a l P r e -p r o o f among COVID-19 patients. Demonstration that the viral spike protein binds avidly to soluble ACE2 provides a mechanism for in vitro saliva debulking with CTB-ACE2 in chewing gum. Direct correlation of ACE2 activity inhibition to SARS-CoV-2 infection, and ability to distinguish asymptomatic from symptomatic COVID-19 PCR positive patients offers unique ability to identify a potential new biomarker. Recent reports show that the Delta variant has >1,000 fold her viral load in saliva and therefore it is important to debulk SARS-CoV-2 in saliva 3, 4 . While masks can prevent transmission to others, they don't protect reinfection of infected individuals. Therefore, chewing gum as a biomaterial offers novelty and practical applications during the current pandemic.

After informed consent under protocol #823392 approved by the University of Pennsylvania 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 media. 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.

The hydroponic growth system at Fraunhofer USA includes multilevel growth racks Shelves were pre-cooled to -40 ºC and each of the fifteen 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 ºC for 180 minutes at a vacuum of 300 mTorr. This was followed by a drying phase that comprised the following stages: holding at -40 ºC for 15 minutes at a vacuum of 300 mTorr, then ramping to -5 ºC over a period of 350 minutes followed by holding at -5 ºC for 180 minutes at a vacuum of 300 mTorr, then ramping to 25 ºC over a period of 300 minutes followed by holding at 25 ºC for at least 1000 minutes at a vacuum of 300 mTorr, and finally holding at 25 ºC for a further 500 minutes 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 three seconds (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 J o u r n a l P r e -p r o o f 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 was determined by Karl Fischer titration by which 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 150ºC. Vaporized water from samples was pumped into a Metrohm 851 Titrando at a rate of 50 mL per minute. Percent 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 was not available in previous versions.

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, Stone Ridge, NY), 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 30-35 ºC or 5-7 days at 20-25 ºC, respectively. For oral delivery, acceptance criteria were set according to USP <1111> (microbial examination of nonsterile products: acceptance criteria for J o u r n a l P r e -p r o o f pharmaceutical preparations and substances for pharmaceutical use) at ≤ 2×10 3 cfu/g for total aerobic microbial count and ≤ 2×10 2 cfu/g for total yeasts and molds count.

CTB-ACE2 expression was examined by western blots as described previously 39 

One hundred and fifty µL patient samples were added to each powder tube and vortexed.

Tubes were then incubated at 4 °C for one hour while rotating. Following incubation, tubes were incubator (37% humidity, 5% CO2) for 72h. Post 72h, cells were lysed using britelite plus luminescence reporter gene assay system (Perkin Elmer Catalog no. 6066769) and RLU were measured using the Biotek plate reader. Percent neutralization was calculated using GraphPad Prism 8. wells.

VSV-S pseudotype particles were generated using a vesicular stomatitis virus (VSV) platform described previously 42 experiments. Inhibition of VSV-S entry was calculated relative to the untreated VSV-S control and statistical significance was analyzed by student t-test.

Fifteen saliva specimens from COVID-19 patients were collected were collected by the University of Pennsylvania under an IRB approved protocol. Saliva (140 µl) were mixed and incubated with 100 mg ACE2 or placebo gum powder for 1h 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 (Biorad). QX200 Droplet Digital PCR System using supermix probe (Bio-Rad) was used by following the manufacturer's instructions. The kit allows the 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. Data were analyzed using the Student's t-test; While no statistical significance was found between untreated and placebo gum (p value = 0.892), significant difference was seen with ACE2 gum treated group (p value = 0.004). Standard deviation could not be added to individual samples due to limited volume of COVID-19 patient swab samples to run duplicate or triplicate biological replicates for three treatment conditions. 

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Oral delivery of novel human IGF-1 bioencapsulated in lettuce cells promotes musculoskeletal cell proliferation, differentiation and diabetic fracture healing

Affordable oral health care: dental biofilm disruption using chloroplast made J o u r n a l P r e -p r o o f enzymes with chewing gum delivery

Maximum Bite Force Analysis in Different Age Groups

Maximum voluntary molar bite force in subjects with normal occlusion

Master Regulator Analysis of the SARS-CoV-2/Human Interactome

COVID-19: the role of excessive cytokine release and potential ACE2 down-regulation in promoting hypercoagulable state associated with severe illness

Quantitation of the glycoprotein spike area on the surface of enveloped viruses

Advances in the development of influenza virus vaccines

A Carbohydrate-Binding Protein from the Edible Lablab Beans Effectively Blocks the Infections of Influenza Viruses and SARS-CoV-2

Characterization of SARS-CoV-2 different variants and related morbidity and mortality: a systematic review

SARS-CoV-2 variants, spike mutations and immune escape

Kinetic reading of ACE-2 in saliva and plant derived full length ACE2. (A,B) ACE-2 activity determined by cleavage of fluorogenic Mca-APK (Dnp) substrate in 10 control

COVID (red) samples. RFU was calculated by subtracting data of timepoint 0 minutes from data of timepoint 90 minutes. Data were analyzed using the Student's t-test

0019 (C) ACE-2 enzyme activity presented as enzyme units (mU/mg). Data were analyzed using the Student's ttest

Interaction of full length CTB-ACE2 with or without recombinant SARS-COV-2 spike proteins. ACE2 activity was measured using 20 µg of CTB-ACE2 protein extracts by cleavage of fluorogenic Mca-APK (Dnp) substrate in the presences and absence of 10 µg spike proteins (SARS-COV-2 RBD, and SARS-COV-2 S1-S2). NC, negative control

Figure 7: Reduction of COVID-19 copies detected by ddPCR in chewing gum incubation

COVID-19 positive Saliva samples were incubated with ACE2 powder gum. N1 target is specific to SARS-CoV-2 and is reduced by ACE2 chewing gum

With large majority of the unvaccinated global population and outbreaks of variants with higher viral load, new methods are needed to decrease SARS-CoV-2 transmission/infection. Daniell et al report chewing gum containing plant cells expressing the angiotensin converting enzyme (ACE2) efficiently traps SARS-CoV-2 in COVID-19 patient saliva or swab samples

J o u r n a l P r e -p r o o f

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