Compositions and methods for reducing viral or microbial load in the oral cavity - Patents.com

Abstract

Compositions and methods are disclosed for reducing viral load in the oral cavity, particularly coronavirus and influenza viral load. Also disclosed are compositions and methods for reducing bacterial load in the oral cavity.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 187,924, filed May 12, 2021, the entire contents of which are incorporated by reference herein as if set forth in full.

[0002] Subsidy details This invention was made with Government support under R01HL107904 awarded by the National Institutes of Health. The United States Government has rights in this invention.

[0003] The present invention relates to the field of viral infections and antiviral compositions. More specifically, the present invention provides compositions and methods that effectively reduce viral load in the oral cavity, thereby reducing viral infections, particularly SARS-CoV-2, influenza, and other infectious viruses. Also provided are compositions and methods for reducing bacterial and fungal loads in the oral cavity. [Background technology]

[0004] Several publications and patent documents are cited throughout this specification to describe the state of the art to which this invention pertains, all of which are incorporated herein by reference in their entirety.

[0005] Transmission of SARS-CoV-2 occurs through both droplet and aerosol transmission, the majority of which is associated with indoor exposure to symptomatic or asymptomatic infected individuals. To limit transmission, indoor aerosol concentrations must be reduced by wearing masks and physical distancing. In public buildings (e.g., classrooms, retail stores, restaurants, gyms, and houses of worship), 4–6 air changes per hour are recommended through outdoor ventilation, recirculated air through a minimum efficiency rating of 13 (MERV13), or passing air through HEPA filters (Allen, 2021). Although larger particles (>100 μm) can be retained by gravity, most people expel aerosols (<5 μm) that are more than 100 times smaller when talking, breathing, or coughing.

[0006] High SARS-CoV-2 viral loads are often detected in saliva. For respiratory viruses such as influenza, measles, and SARS-CoV2, highly infectious airborne droplets are the main source of transmission. Human papillomavirus, herpes simplex virus type 1, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus are transmitted orally and their replication in the oral epithelium is well known. The average RNA viral load in saliva is 7 × 10 6 For SARS-CoV-2 at copies / ml, 50 μm 2 Oral droplets from the mouth may contain at least one virus. High SARS-CoV-2 viral loads have been detected in the saliva of asymptomatic and symptomatic COVID-19 patients. In fact, the viral load in saliva correlates with the severity of COVID-19 symptoms, including loss of taste and smell, and the virus replicates in the salivary glands and oral mucosa. Thus, the oral mucosa and saliva appear to be high-risk routes of SARS-CoV-2 transmission. Summary of the Invention [Problem to be solved by the invention]

[0007] Currently, there are no compositions and methods that specifically and effectively remove viruses from the oral cavity and throat. It is an object of the present invention to address this need. [Means for solving the problem]

[0008] According to the present invention, a method for reducing the viral load in the oral cavity is disclosed. An exemplary method includes orally administering to a subject a therapeutically effective amount of a composition comprising a carrier and a capture molecule that binds to a protein or glucan (single or complex) on the surface of a virus, and the binding of the surface protein or glucan to the capture molecule captures the virus in the carrier, thereby reducing the viral load in the oral cavity. In certain embodiments, the reduction in the viral load in the oral cavity of the subject reduces the infection of the virus. In certain embodiments, the administration is performed before the subject is exposed to the virus (e.g., due to the use of personal protective equipment (PPE)). In other embodiments, the administration is performed after the subject is exposed to the virus. Preferably, the administration reduces the viral load, shortens the recovery time of the viral infection, eliminates or minimizes at least one complication of the viral infection, or reduces the viral infection. The virus includes any virus present in the oral cavity. Such viruses include, but are not limited to, coronaviruses, herpes viruses, papilloma viruses, influenza viruses, Epstein-Barr virus, cytomegalovirus, Hepatitis C virus, Zika virus, and other viruses that use the salivary gland as a reservoir.

[0009] In one embodiment of the invention, the virus comprises a coronavirus, such as at least one of an alphacoronavirus, a betacoronavirus, a gammacoronavirus, and a deltacoronavirus. In other embodiments, the coronavirus comprises at least one of MERS-CoV, SARS-CoV, and SARS-CoV-2.

[0010] In a particularly preferred embodiment, the virus is SARS-CoV-2.

[0011] In another preferred embodiment, the virus is SARS-CoV-2 and the capture molecule is ACE2 or CTB-ACE2, which binds to the spike protein on the viral surface and captures the virus onto the carrier, thereby reducing the viral load in the oral cavity.

[0012] In another embodiment, the virus is an alpha-influenza virus, including at least one of influenza A virus, influenza B virus, and influenza C virus. In this embodiment, the capture molecule can be an influenza virus A (IVA) blocking peptide that binds to portions of the influenza hemagglutinin (HA) and neuraminidase (NA) proteins on said influenza virus and captures influenza virus particles in said carrier. In a particular embodiment, the IVA blocking peptide comprises the virus-binding portions of the HA and neuraminidase proteins.

[0013] The carrier may be a chewing gum, a long-acting lozenge, or a tablet, hi a preferred embodiment, the carrier is a chewing gum.

[0014] The present invention also provides compositions useful for the methods disclosed above. An exemplary composition includes a carrier comprising an effective amount of a capture molecule having binding affinity for the surface protein or glucan (either alone or in complex) of a virus present in the oral cavity, which may or may not be transmitted by aerosolization, where the carrier is suitable for oral administration. The carrier may be a chewing gum, but may also be a long-acting lozenge or an oral tablet. In a preferred embodiment, the carrier is a chewing gum. In a particularly preferred embodiment of the composition, the virus to be treated is SARS-CoV-2, the capture molecule is ACE2 or CTB-ACE2, and the carrier is a chewing gum.

[0015] In another embodiment of the composition, the virus is influenza A virus, the capture molecule is an IVA blocking peptide and the carrier is chewing gum. In this embodiment, the IVA blocking peptide may comprise the virus binding portions of the HA and neuraminidase proteins.

[0016] In another embodiment of the composition, the virus is influenza A virus, SARS-CoV02, herpes virus or papilloma virus, the capture molecule is FRIL isolated from lablab bean powder and the carrier is chewing gum.

[0017] According to yet another embodiment, a method for reducing microorganisms, such as bacteria and fungi, in the oral cavity is disclosed. An exemplary method includes orally administering to a subject a therapeutically effective amount of a composition comprising a carrier and a capture molecule that binds to a protein on the surface of a microorganism, and the binding of the surface protein to the capture molecule captures the microorganism within the carrier, capturing the microbial load in the oral cavity. In certain embodiments, the reduction of the microbial load in the oral cavity of the subject reduces microbial infection.

[0018] The present invention also provides compositions useful for the methods disclosed above. Exemplary compositions include a carrier comprising an effective amount of a capture molecule having binding affinity for a surface protein of a microorganism present in the oral cavity, which may or may not be transmissible by aerosolization, and the carrier is suitable for oral administration. The carrier may be a chewing gum, but may also be a long-acting troche or an oral tablet. In certain embodiments, the microorganism is a bacterium, such as Streptococcus pyogenes, and the capture molecule is an antimicrobial peptide. Antimicrobial peptides suitable for this purpose include, but are not limited to, protegrin, retrocyclin defensin, PGLA (frog skin peptide), cecropin, apidaecin, melittin, MSI-99, bombinin, magainin, boceprevir, and telaprevir.

[0019] According to yet another embodiment, a method of reducing fungi, such as C. albicans, in the oral cavity is disclosed. An exemplary method includes orally administering to a subject a therapeutically effective amount of a composition comprising a carrier and an enzyme, such as a lipase. Lipids are membrane components important for fungal morphogenesis and hyphal elongation (Rella et al. 2016). In certain embodiments, reducing the fungal load in the oral cavity of the subject reduces fungal infection.

[0020] The present invention also provides compositions useful for the methods disclosed above. An exemplary composition includes a carrier and an enzyme, such as a lipase, where the carrier is suitable for oral administration. The carrier may be a chewing gum, but may also be a long-acting lozenge or an oral tablet. In a particular embodiment, the microorganism is a fungus, such as Candida albicans, and the capture molecule is a lipase. [Brief description of the drawings]

[0021] [Figure 1] Fresh weight (g) and ACE2 expression (mg / g dry weight) per plant at different ages from Aerofarms and Fraunhofer. ACE2 expression was quantified based on total protein (μg / μl) of homogenates and converted to mg / g of plant dry weight (mg / g DW). Values ​​are means ± SD (n=3). The percentage of ACE2 protein in total leaf protein is shown in blue above each column. Total leaf protein content (mg / g DW) of CTB-ACE2 plant material is shown in red above each column. Values ​​are means ± SD (n=8). Total amount of fresh biomass (Kg) harvested at different ages is shown in black above each column. Plant ages are relative to the date of germination. Despite the different growth conditions, biomass yield per plant was similar at 80-90 days but decreased as plants grew larger. However, the most dramatic difference was in the expression levels of CTB-ACE2. Maximum expression levels in plants grown at Aerofarms were <2 mg CTB-ACE2 / g dry weight, whereas expression levels at Fraunhofer USA ranged from 15 to 22 mg / g dry weight. [Figure 2A-2C]Neutralization of SARS-CoV-2 spike pseudotyped lentivirus. Luciferase-expressing spike pseudotyped lentivirus particles were infected into Vero cells. (Figure 2A) Raw luminescence values. (Figure 2B) Relative infectivity was determined by comparing luminescence of ACE2-gum to the corresponding placebo. Values ​​are shown as mean ± SD of n=6 for gum and n=3-4 for virus alone, infected cells, and non-virus control. Shown are relative infectivity mean ± SD of n=6 for gum treatment (p<0.0001 for all treatments as determined by two-tailed test) and n=3-4 for virus alone, infected cells, or non-infected cells. (Figure 2C) Luciferase-expressing SARS-CoV-2 spike glycoprotein pseudotyped virus was incubated with the indicated concentrations of ACE2-gum for 90 min at room temperature. After centrifugation, virus-containing supernatants were incubated with ACE2-expressing CHO cells for 72 h and viral infectivity was measured via luciferase. Data shown are representative of three independent experiments with N = 2-3 specimens. Bars represent condition means. Symbols represent means of duplicate or triplicate assays. Error bars represent SEM. p<0.01 ** p<0.001***, p<0.0001**** by Kruskal-Walis ANOVA. [Diagram 3] Effect of ACE2 on VSV spike particle entry: The relative inhibition of VSV-S particle entry after incubation with CTB-ACE2 and ACE2 gum powder was calculated to be statistically significant in all treatment groups compared to untreated VSV-S controls. Graphs in figure ** are representative of two replicates in two independent experiments. (student's t-test, p < 0.05) * = p <0.05, ** = p <0.01 [Figure 4A-4B] Neutralization of SARS-CoV-2 in NP swab specimens. (Figure 4A) Images of clinical NP swab specimens treated with different conditions. All images share the scale bar. (Figure 4B). Relative inhibition was determined by comparison of bubble counts in gum-treated and untreated samples. [Figure 5A-5D]Kinetic readings of ACE-2 in saliva and full-length ACE2 from plants. (Figure 5A- and 5B) ACE-2 activity measured by cleavage of fluorescent Mca-APK(Dnp) substrate in 10 control and 10 COVID (red) samples. Delta RFU was calculated by subtracting the data at time point 0 min from the data at time point 90 min. (Figure 5C) ACE-2 enzyme activity is expressed in enzyme units (mU / mg). Data were analyzed using Student's t-test. **: p< 0.0024 (Figure 5D) Interaction of full-length CTB-ACE2 with recombinant SARS-COV-2 spike protein. ACE2 activity was measured by cleavage of the fluorogenic Mca-APK(Dnp) substrate using 20 μg of CTB-ACE2 protein extract in the presence and absence of 10 μg of spike protein (SARS-COV-2 RBD, and SARS-COV-2 S1-S2); NC, negative control (substrate and buffer only). [Figure 6A-6C] Reduction of COVID-19 copies detected by ddPCR in chewing gum cultures: COVID-19 positive saliva samples were incubated with ACE2 powder gum. (Figure 6A) N1 target is specific for SARS-CoV-2, (Figure 6B) RP target is non-specific. N1 copy numbers are highly reduced by ACE2 chewing gum. (Figure 6C) Graph quantifying this data. [Figure 7A-7B] Figure 1. Blocking and neutralizing mechanisms of plant-derived SARS-CoV-2 virus capture proteins CTB-ACE2 and FRIL (Fig. 7A) Lettuce-based CTB-ACE2 pentameric insoluble microparticles contained in chewing gum tablets effectively bind to the viral spike protein, precipitate the SARS-CoV-2 virus, and block viral entry into human cells. (Fig. 7B) The homotetrameric plant-derived lectin FRIL (hyacinth bean) has potent anti-SARS-CoV-2 and anti-influenza activity by binding to complex N-glycans present on viral surface glycoproteins to form aggregates and sequester virions at the late endosome to prevent them from entering the nucleus. [Figure 8]Schematic diagram of the CTB-ACE2 chewing gum process from lab to clinic. Chronological sequence of the chewing gum manufacturing process from the creation and characterization of plants expressing CTB-ACE2, to the manufacture and characterization of the chewing gum, to the steps towards clinical development. Steps #1 to #11 are detailed in Daniell et al. 2020 and Daniell et al. 2021). Step #8 is described by Per Os Biosciences in US Patent 9,744,128. [Figure 9A-9B] Quantification of release and total dose of CTB-ACE2 gum tablets. (Figures 9A, 9B) Western blot analysis for immediate release of CTB-ACE2 without sonication. Data are mean ± SD; n=4. (Figures 9C, 9D) Western blot analysis for total dose quantification of CTB-ACE2. Data are mean ± SD; n=4. [Figure 10A-10B] Neutralization of SARS-CoV-2 omicron variants in NP swab samples by FRIL. (Figure 10A) Images of clinical NP / OP swab samples from patients #620 and #613 treated with FRIL bean powder (20, 40, 100 μg FRIL protein in 5, 10, 25 mg bean powder, respectively). (Figure 10B) Number of bubbles is quantified. All images share the same scale bar. Data were analyzed by one-way ANOVA test. There is a significant difference in microbubble count between untreated and FRIL bean powder at all concentrations (***p-value < 0.0001). [Figures 11A-11D]Neutralization of SARS-CoV-2 Delta and Omicron variants in NP swab specimens by CTB-ACE2 gum. (Fig. 11A) Images of clinical NP / OP swab specimens from patients #614, #615 treated with ACE2 gum (0.46 or 0.92 μg of CTB-ACE2 protein in 25 mg and 50 mg gum, respectively). (Fig. 11B) Quantification of the number of bubbles. Data were analyzed by one-way ANOVA test. There is a significant difference in the number of microbubbles between untreated Omicron strain and ACE2 gum at both concentrations (***p-value = 0.0001). All images share the same scale bar. (Fig. 11C) Images from clinical NP / OP swab specimens from patients #151, #153 and #155 treated with ACE2 gum (0.46 or 0.92 μg of CTB-ACE2 protein in 25 mg and 50 mg gum, respectively). (Figure 11D) The number of bubbles is quantified. Data was analyzed by one-way ANOVA test. There is a significant difference in the number of microbubbles between untreated Delta strain and ACE2 gum at both concentrations (***p value = 0.0009). [Figure 12] The capture efficacy of ACE2 chewing gum was evaluated in diagnostics by RAPID. Normalized charge transfer resistance values ​​in electrochemical impedance spectroscopy measurements of 20 SARS-CoV-2 clinical samples (left Y-axis) before and after exposure to 20 mg of ACE2 chewing gum. RNA copies μL-1 obtained from qPCR of infected samples before exposure to ACE2 chewing gum (right Y-axis). Numbers below samples are patient IDs. For other details, see Table 1. Specimen ID 593 was not genotyped. Specimens 595-609 are Delta and 613-631 are Omicron SARS-CoV-2 strains were identified based on whole genome sequencing or S gene targeting failure or collection date. RNA extraction was performed from 140 μl of patient specimens (1 / 50 μl shown) and RAPID was performed on 100 μl of patient specimens (Rct values ​​of 10 μl shown). [Fig. 13A-13F]FRIL plaque reduction neutralization assay. FRIL plaque reduction neutralization assay with H1N1 (A / California / 7 / 2009-X181), H3N2 (A / Singapore / INFMH-16 / 0019 / 2016), and HCoV-OC43 viruses. Viruses were preincubated with increasing amounts of purified FRIL protein or soluble extract of hyacinth bean powder in 100 μl for 1 h at 37 °C. Pretreated viruses were then added to cells, and plaque reduction assays were performed to quantitate plaque numbers at 28 h post-infection for the two Flu viruses and 5 days post-infection for HCoV-OC43 virus. Data represent the mean ± SD of plaque numbers obtained from two independent experiments. Because 4 mg of purified FRIL corresponds to 1 mg of FRIL bean powder, a higher input weight of FRIL bean powder (mg) was required to obtain a dose-response curve compared to purified FRIL (mg). For ease of comparison, 50% weight inhibition in each dose-response curve of FRIL protein was expressed in nanograms (ng). [Figure 14] FRIL plaque reduction neutralization assay. Vero FRIL inhibition at infectious doses of H1N1 (A / California / 7 / 2009 / X181) and H3N2 (A / Singapore / INFMB-16 / 0019 / 2016). [Figure 15A-15B] Containment of influenza virus by FRIL. (Figure 15A) Negative stain EM images of H1N1 (A / California / 7 / 2009-X181) cell culture virus alone (left panel) and virions aggregated with FRIL after incubation with 10 μg / mL and 150 μg / mL FRIL (right panel). Data are representative of two independent experiments. (Figure 15B) Negative stain EM images of sucrose gradient purified X181 virions alone (left panel) and aggregated X181 virions after mixing with 150 μg / mL FRIL (right panel). Scale bars: 100 nm and 500 nm, respectively. [Figure 16]CTB-ACE2 chewing gum study design. Phase I / II placebo-controlled, double-blind, randomized ACE2 or placebo gum study in which 13 gums were used for 4 days, 4 gums on days 1-3. Non-stimulating whole saliva samples will be collected before eating, drinking, or brushing teeth. Subjects will chew CTB-ACE2 chewing gum / placebo gum (study product containing 2 g of CTB-ACE2 or placebo) for 10 minutes and then immediately collect a 2-5 mL post-treatment saliva sample in a pre-labeled saliva collection tube. Viral load will be quantified by qPCR or protein (N or spike) quantification. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] As mentioned above, high SARS-CoV-2 viral loads are often detected in saliva (Li et al. Mol Oral Microbiol.2020 35(4):141-145). Indeed, salivary viral loads correlate with the severity of COVID-19 symptoms, including loss of taste and smell, and the virus replicates in the salivary glands and oral mucosa (Huang et al.2021 May;27(5):892-903). Thus, oral mucosa and saliva are high-risk routes of SARS-CoV-2 infection, and viral inactivation in the oral cavity may be an important strategy to reduce viral infectivity. Mouthwashes containing hydrogen peroxide and iodopovidone have activity against coronaviruses in vitro, but the overall magnitude and duration of control require further investigation due to the short contact time (Vergara-Buenaventura and Castro-Ruiz Brit J Oral Maxillofac Surg.58(8):924-927).

[0023] To test novel strategies for targeted virus reduction in the oral cavity, the primary site of viral replication, the protein CTB-ACE2, which captures the SARS-CoV-2 virus, was expressed in chloroplasts and clinical-grade plant material meeting FDA requirements was developed. See Figure 1. Chewing gum (2 grams) containing plant cells expressing CTB-ACE2 up to 17.2 mg ACE2 / g DW (11.7% leaf protein) had conventional gum-like physical properties, taste / flavor, and no protein was lost during gum compression. CTB-ACE2 gum efficiently inhibited (>95%) the entry of lentivirus-spiked or VSV-spiked pseudoviruses into Vero / CHO cells, as quantified by luciferase or red fluorescence. Incubation with CTB-ACE2 microparticles reduced SARS-CoV-2 virus counts in COVID-19 swab / saliva samples by more than 95% when assessed by microbubbles (femtomolar concentrations) or qPCR, demonstrating both virus capture and blocking of cell entry. COVID-19 saliva samples had low or no ACE2 activity compared to healthy controls (2582 vs. 50126 ΔRFU; 27 vs. 225 enzyme units), confirming the high susceptibility of infected patients to virus entry. CTB-ACE2 activity was completely inhibited by preincubation with SARS-COV-2 RBD, which could explain the reduced ACE2 activity in saliva of COVID-19 patients. Chewing gum containing a virus-capturing protein can reduce and minimize transmission to others, protecting patients from most oral virus reinfections.

[0024] The inventors surprisingly found that chewing gum containing ACE2 has the effect of minimizing infection by SARS-CoV-2 and reducing its infectivity by inhibiting its entry into human cells. Notably, these findings can be applied to other airborne viruses such as influenza and measles. For example, to treat influenza, chewing gum contains an IAV-blocking peptide that binds to the influenza virus, reduces its concentration in the oral cavity, and reduces the infectivity of influenza. Alternatively, chewing gum can contain FRIL, which is obtained from hyacinth bean powder. FRIL binds to the surface proteins of both SARS-CoV2 and influenza viruses.

[0025] SARS-CoV-2 and influenza can be transmitted both nasally and orally, but oral transmission is 3-5 orders of magnitude more likely than nasal transmission. We describe the reduction of both viruses using a viral capture protein (CTB-ACE2, FRIL) expressed in plant cells. The viral capture protein is delivered through chewing gum and optimally neutralizes the virus at the surface of the throat, the primary site of infection. In Omicron nasopharyngeal (NP) specimens, microbubbling counts (based on N-antigen) were significantly reduced by 20 μg FRIL (p<0.0001) and 0.925 μg CTB-ACE2 (p=0.0001). Of NP specimens from 20 patients infected with Delta or Omicron strains, viral loads in 17 specimens were below the detection level of spike protein by the RAPID assay. A dose-dependent 50% plaque reduction was observed with both purified FRIL and hyacinth bean powder against influenza strains H1N1, H3N2 and coronavirus HCoV-OC43 when 50-100 ng of FRIL was used. In electron micrographs, large, tightly packed clumps of overlapping influenza particles and FRIL proteins were observed at a FRIL concentration of 150 μg / mL, but not with untreated virus particles in cell culture or particles purified on sucrose gradients. Taken together, these results provide proof of principle that chewing gum can deliver oral virus clearing proteins in the oral cavity and pharynx, reducing infection and transmission. Indeed, since human papillomavirus, herpes simplex virus type I, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus are also orally transmitted, carriers containing molecules that bind to surface proteins of these viruses could also be advantageously used to clear the oral cavity and reduce viral infection.

[0026] CTB is a transmucosal carrier that facilitates oral delivery of therapeutic proteins by forming a pentameric structure and binding to the intestinal GM1 epithelial receptor (Daniell et al 2019 Biotechnol.Adv.37, 107413; Biomaterials.2020 233:119750; Plant Biotechnol.2021 J.19:430-447; Kwon and Daniell, Mol Ther.2016 24(8):1342-1350). CTB-ACE2 efficiently binds to both GM1 and ACE2 receptors, thus effectively inhibiting viral spike protein binding, especially in oral epithelial cells where both receptors are enriched (Xu et al. Int J Oral Sci. 2020 24;12(1):1-5.). Direct binding of ACE2 to SARS-CoV-2 spike protein captures viral particles and reduces infectivity. In accordance with this discovery, the present invention provides a composition comprising CTB-ACE2 in a carrier suitable for oral administration (e.g., chewing gum, long-acting lozenges, tablets, etc.) for inhibiting SARS-CoV-2 entry and viral transmission from the oral cavity. Of note, the tongue has a larger reservoir of ACE2 than cheek and gingival tissues (Xu et al.; 2020, supra). ACE2 chewing gum is a highly targeted approach to remove particles containing SARS-CoV-2 spikes, and when released exogenously by chewing, it is more persistent than mouthwashes to effectively clear SARS-CoV-2 viral load from the oral cavity.

[0027] In another application, oral SARS-CoV-2 reduction is needed for infected patients who require emergency treatment, especially dental procedures. CTB-ACE2 chewing gum could lower the viral load in such patients and better protect healthcare workers. In another approach, prophylactic consumption of CTB-ACE2 chewing gum in public could augment or replace mask wearing in situations where mask use is impossible or difficult, such as during meals. This strategy could also be employed to remove viruses other than SARS-CoV-2 from the oral cavity, as discussed above.

[0028] definition The expression sequences of native and codon-optimized CTB-ACE2 are described in US Patent 10,806,775, which is incorporated herein by reference. See Figures 21A and 21B.

[0029] As used herein, the term "administration" or "administering" of a composition to a subject includes any route of introducing or delivering a compound to a subject to perform its intended function. For the treatment of airborne infections, oral or nasal administration is preferred. For other treatments in which administration of CTB-ACE2 may be beneficial, other routes may be employed, such as parenteral (intravenous, intramuscular, intraperitoneal, subcutaneous), rectal, topical, etc. Administration or ingestion includes self-administration and administration by another person.

[0030] As used herein, the term "disease," "disorder," or "complication" refers to a deviation from the normal state in a subject.

[0031] As used herein, "infection" refers to the introduction and / or presence of a disease-causing or pathogenic organism in another organism, tissue, or cell.

[0032] As used herein, "antimicrobial peptides (AMPs)" refer to host defense peptides (HDPs) that are part of the innate immune response found in all living organisms. These peptides are potent, broad-spectrum antibiotics that have shown potential as novel therapeutic agents. Antimicrobial peptides have been demonstrated to kill gram-negative and gram-positive bacteria, enveloped viruses, fungi, and even transformed and cancer cells. Unlike most traditional antibiotics, antimicrobial peptides frequently destabilize biological membranes and can form transmembrane channels. They may also have the ability to enhance immunity by functioning as immunomodulators. AMPs suitable for use in the methods disclosed herein include, but are not limited to, protegrin, retrocyclin defensin, PGLA (frog skin), cecropin, apidaecin, melittin, MSI-99, bombinin, magainin, boceprevir, and telaprevir.

[0033] As used herein, "Flt3 receptor-interacting lectin" or "FRIL" refers to a legume lectin that shares 48% sequence identity with the well-known concanavalin A (ConA), has a similar b-prism type-II fold, and has one glycan-binding domain (CBD) per monomer. Previous studies suggest that FRIL is a glucose / mannose-specific lectin with a strong preference for the a-anomeric form, based on its affinity for the monosaccharides mannose, glucose, and N-acetylglucosamine.

[0034] As used herein, "lipase" refers to a family of enzymes that catalyze the hydrolysis of fats. Lipases can be advantageously used to entrap certain fungi on suitable carriers.

[0035] As used herein, "respiratory" refers to the system of cells and organs that function in respiration, and specifically respiratory organs, tissues and cells include the lungs, nose, nasal cavities, paranasal sinuses, nasopharynx, larynx, trachea, bronchi, bronchioles, respiratory bronchioles, alveolar ducts, alveolar sacs, alveoli, pneumocytes (type 1 and type 2), ciliated mucosal epithelium, mucosal epithelium, squamous epithelial cells, mast cells, goblet cells and intraepithelial dendritic cells.

[0036] An orally ingestible product according to the invention is any preparation or composition suitable for ingestion, nutrition or oral hygiene, intended to be introduced into the oral cavity of a human being, to remain there for a period of time and then to be swallowed (e.g. ingestible food) or removed again from the oral cavity (e.g. chewing gum or oral hygiene products). These products include any substance or product, in a processed, semi-processed or raw state, intended for human or animal ingestion. This also includes substances added during the manufacture, processing or processing of an orally ingestible product (e.g. active ingredients of extracts, nutrients, supplements, medicines, etc.) and intended to be introduced into the oral cavity of a human being.

[0037] As used herein, the terms "effective amount," "amount effective," and the like, refer to an amount effective, at dosages and for periods of time necessary, to achieve the desired result.

[0038] As used herein, the terms "inhibit" or "prevent" refer to preventing the worsening or development of clinical symptoms of a disease state in a subject who may be exposed to or is predisposed to the disease state, but who has not yet experienced or manifested symptoms of the disease state, e.g., inhibiting the onset of the disease.

[0039] As used herein, the term "expression" in the context of a gene or polynucleotide includes transcription of the gene or polynucleotide into RNA. This term may also, but does not necessarily, include the subsequent translation of the RNA into a polypeptide chain and assembly into a protein.

[0040] A plant remnant may include, but is not limited to, one or more molecules (proteins and fragments thereof, minerals, nucleotides and fragments thereof, plant structural components, etc.) derived from the plant in which the protein of interest is expressed. Thus, a composition of whole plant material (e.g., whole or part of the plant leaves, stems, fruits, etc.) or crude plant extract will certainly contain a high concentration of plant remnants, as will a composition containing a purified protein of interest having one or more detectable plant remnants. In a specific embodiment, the plant remnant is Rubisco.

[0041] In another embodiment, the present invention relates to an administrable composition for treating or inhibiting infection and / or infection by airborne viruses. In a particular embodiment, the composition comprises a therapeutically effective amount of ACE2, CTB-ACE2 for the treatment of Covid-19, or a combination thereof expressed by plants and plant residues. The composition of the present invention can also be used prophylactically to reduce the incidence of aerosol infection.

[0042] Importantly, this discovery can be utilized to inhibit infection and infectivity of other airborne viruses, such as influenza. In this embodiment, subjects ingest a chewing gum containing an influenza A virus (IAV) blocking peptide to reduce influenza viral load in the oral cavity.

[0043] Methods, vectors, and compositions for transforming plants and plant cells are taught, for example, in WO 01 / 72959; WO 03 / 057834; and WO 04 / 005467. WO 01 / 64023 describes the use of marker-free genetic constructs, each of which is incorporated herein by reference.

[0044] In certain embodiments, plant material (e.g., lettuce material) that contains chloroplasts capable of expressing ACE2, CTB-ACE2, or HA is homogenized, freeze-dried, and encapsulated. In a specific embodiment, the extract of the lettuce material is encapsulated. In another embodiment, the lettuce material is powdered before encapsulation. Other useful plants include, but are not limited to, edible plants such as tomatoes, carrots, and apples.

[0045] In some embodiments, the pharmaceutical composition for reducing viral load in the oral cavity can be formulated as a chewing gum. The formulation of the gum base varies widely depending on the particular product being prepared and the chewing and other sensory properties desired in the final product. By way of example, typical ranges of gum base ingredients include 5-80 wt% elastomeric compounds, 5-80 wt% natural and / or synthetic resins (elastomer plasticizers), 0-40 wt% wax, 5-35 wt% softeners other than wax, 0-50 wt% fillers, and 0-5 wt% other ingredients such as antioxidants, colorants, etc. The gum base can constitute about 5-95 wt%, often about 10-60 wt% or about 40-50 wt% of the total weight of the chewing gum.

[0046] In many cases, a buffer is used. Examples of buffers that can be used include Tris buffer, amino acid buffer, carbonates including monocarbonates, bicarbonates or sesquicarbonates, glycerates, phosphates, glycerophosphates, acetates, glyconates or citrates of alkali metals, such as trisodium citrate and tripotassium citrate, or ammonium, and mixtures thereof. Examples of other buffers include acetic acid, adipic acid, citric acid, fumaric acid, glucono-Δ-lactone, gluconic acid, lactic acid, malic acid, maleic acid, tartaric acid, succinic acid, propionic acid, ascorbic acid, phosphoric acid, sodium orthophosphate, potassium orthophosphate, calcium orthophosphate, sodium diphosphate, potassium diphosphate, calcium diphosphate, pentasodium triphosphate, pentaposodium triphosphate, sodium polyphosphate, potassium polyphosphate, carbonic acid, sodium carbonate, sodium bicarbonate, potassium carbonate, calcium carbonate, magnesium carbonate, magnesium oxide, or combinations thereof.

[0047] The buffering agents are to some extent microencapsulated or coated as granules with polymers and / or lipids that are less soluble in saliva than one or more of the buffering agents. Such microencapsulation controls the dissolution rate and extends the time frame of the buffering effect. The amount of buffering agent ranges from 0 to about 15%, and often from about 0.5 to about 10%, based on the total weight of the chewing gum.

[0048] Elastomers are used to give gum a rubber-like tack. Elastomers suitable for use in gum bases and gums can be natural or synthetic. Elastomer plasticizers can also be used to modify the hardness of the gum base. These specificities for the intermolecular chain interactions (plasticization) of elastomers, along with differences in softening points, change the hardness and completeness of compatibility of the gum when used as a base. This can expose more of the elastomer chains to the alkane chains of the wax.

[0049] The elastomers employed in the gum base may vary depending on a variety of factors, such as the type of gum base desired, the desired texture of the gum formulation, and other ingredients used in the formulation to produce the final chewing gum product. The elastomer may be any water-insoluble polymer known in the art, including gum polymers utilized in chewing gum and bubble gum. For example, suitable polymers for use in gum base include, but are not limited to, natural substances (plant derived), such as chicle gum, natural rubber, crown gum, nispero, rosidinha, jelutong, perillo, nigergutta, tunu, balata, gutta percha, reticapsi, sorva, gutta kei, and mixtures thereof. Examples of synthetic elastomers include, but are not limited to, styrene-butadiene copolymers (SBR), polyisobutylene, isobutylene-isoprene copolymers, polyethylene, polyvinyl acetate, and the like, and mixtures thereof.

[0050] Natural resins may be used in accordance with the present invention and may be natural rosin esters, referred to as ester gums, including glycerol esters of partially hydrogenated rosin, glycerol esters of polymerized rosin, glycerol esters of partially dimerized rosin, glycerol esters of tally oil rosin, pentaerythritol esters of partially hydrogenated rosin, methyl esters of rosin, partially hydrogenated methyl esters of rosin, pentaerythritol esters of rosin, synthetic resins such as terpene resins derived from α-pinene, β-pinene, and / or d-limonene, and natural terpene resins.

[0051] The resin may be selected from terpene resins, such as those derived from α-pinene, β-pinene, and / or d-limonene, natural terpene resins, glycerol esters of gum rosin, tall oil rosin, wood rosin, or derivatives such as the glycerol esters of partially hydrogenated rosin, the glycerol esters of polymerized rosin, the glycerol esters of partially dimerized rosin, the pentaerythritol esters of partially hydrogenated rosin, the methyl esters of rosin, the partially hydrogenated methyl esters of rosin, or the pentaerythritol esters of rosin, and combinations thereof.

[0052] Other chewing gum ingredients can be selected from bulk sweeteners, flavors, dry binders, tableting aids, anti-caking agents, emulsifiers, antioxidants, enhancers, absorption promoters, buffers, high intensity sweeteners, softeners, colorants, and combinations thereof. Non-limiting examples of emulsifiers include cyclodextrin, polyoxyethylene castor oil derivatives, polyoxyethylene alkyl ethers, macrogol alkyl ethers, block copolymers of ethylene and propylene oxide, polyoxyethylene alkyl ethers, polyoxyethylene glycols, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene (20) sorbitan monostearate, polyoxyethylene (20) sorbitan monooleate, polyoxyethylene stearate, sorbitan esters, diacetyl tartaric acid esters of monoglycerides, lactylated monoglycerides, and combinations thereof. The amount of emulsifiers often ranges from about 0.1% to about 25 wt% based on the total weight of the chewing gum.

[0053] Petroleum waxes aid in hardening of the finished gum made from the gum base as well as improving shelf life and texture. Wax crystal size affects flavor release. Waxes high in isoalkanes have smaller crystal sizes than waxes high in normal alkanes, especially those containing normal alkanes with 30 carbons or less. Small crystal sizes impede flavor escape from the wax compared to waxes with larger crystal sizes, slowing flavor release. Gum bases using normal alkane waxes have lower compatibility compared to gum bases using isoalkane waxes.

[0054] Petroleum waxes (refined paraffin and microcrystalline wax) and paraffin waxes are composed primarily of linear normal alkanes and branched isoalkanes, with the ratios of normal alkanes to isoalkanes varying.

[0055] Normal alkane waxes generally have carbon chain lengths greater than C-18, but rarely greater than C-30. In primarily normal alkane waxes, the branched and cyclic structures are located near the ends of the chain. Normal alkane waxes have a viscosity of <10 mm. 2 / s (100°C), and the number average molecular weight is <600 g / mol.

[0056] Isoalkane waxes generally have carbon lengths of C-30 or more. In isoalkane-based waxes, the branches and ring structures are randomly arranged along the carbon chain. The viscosity of isoalkane waxes is 10mm. 2 / s (100 °C) or more and a number average molecular weight of 600 g / mol or more. Synthetic waxes are not considered petroleum waxes because they are produced differently than petroleum waxes. Synthetic waxes include, but are not limited to, waxes containing branched alkanes and copolymerized with monomers such as propylene, polyethylene, and Fischer-Tropsch type waxes. Polyethylene wax is a synthetic wax containing alkane units of various lengths linked by ethylene monomers.

[0057] Waxes and fats are conventionally used in preparing chewing gum bases to modify the texture and soften the chewing gum base. Conventionally used natural and synthetic waxes and fats of any suitable type may be used, such as rice bran wax, polyethylene wax, petroleum wax (refined paraffin, microcrystalline wax), sorbitan monostearate, tallow, propylene glycol, paraffin, beeswax, carnauba wax, candelilla wax, cocoa butter, defatted cocoa powder, and any suitable fat, such as fully or partially hydrogenated vegetable oils, fully or partially hydrogenated animal fats, etc.

[0058] Antioxidants extend the shelf life and storage of gum bases, finished gums, or their respective ingredients, including fats, oils, and flavor oils. Antioxidants suitable for use in gum bases include butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), betacarotene, tocopherols, acidulants such as vitamin C, propyl gallate, other synthetic and natural types, or mixtures thereof.

[0059] The chewing gum may contain other conventional ingredients such as sweeteners, including bulk sweeteners, sugar sweeteners, sugar substitutes, artificial sweeteners, high intensity sweeteners, or combinations thereof. The bulk sweeteners may comprise from about 5 to about 95% by weight of the chewing gum, more typically from about 20 to about 80%, from about 30 to about 70%, or from about 30 to about 60% by weight.

[0060] Useful sugar sweeteners are saccharide-containing ingredients commonly known in the chewing gum art, including, but not limited to, sucrose, dextrose, maltose, dextrin, trehalose, D-tagatose, dry invert sugar, fructose, levulose, galactose, corn syrup solids, and the like, used alone or in combination.

[0061] Sorbitol can be used as a sweetener other than sugar. Other useful non-sugar sweeteners include, but are not limited to, other sugar alcohols such as mannitol, xylitol, hydrogenated starch hydrolysates, maltitol, isomalt, erythritol, lactitol, etc., used alone or in combination.

[0062] High intensity artificial sweeteners may also be used alone or in combination with the above sweeteners. Non-limiting examples of high intensity sweeteners include sucralose, aspartame, salts of acesulfame, alitame, saccharin and its salts, cyclamic acid and its salts, glycyrrhizin, dihydrochalcones, thaumatin, monellin, steriosides, and the like, used alone or in combination. To provide a longer lasting sweetness and flavor perception, it may be desirable to encapsulate or otherwise control the release of at least a portion of the artificial sweetener. Techniques such as wet granulation, wax granulation, spray drying, spray chilling, fluidized bed coating, preservation, encapsulation in yeast cells, fiber extrusion, and the like may be used to achieve the desired release characteristics. Encapsulation of the sweetener may also be achieved with another chewing gum ingredient, such as a resin compound.

[0063] The amount of artificial sweetener used varies widely depending on factors such as sweetener potency, release rate, sweetness desired in the product, level and type of flavor used, cost, etc. Artificial sweetener activity levels range from about 0.001 to about 8% by weight, and often from about 0.02 to about 8% by weight. When a carrier used for encapsulation is included, the amount of encapsulated sweetener used is proportionately higher. Combinations of sugar and / or non-sugar sweeteners can be used if desired.

[0064] The chewing gum and / or gum base may contain one or more fillers / texturizers, such as magnesium or calcium carbonate, sodium sulfate, ground limestone, silicate compounds such as magnesium or aluminum silicate, kaolin or clay, aluminum oxide, silicic acid oxide, talc, titanium dioxide, mono-, di-, or tri-calcium phosphate, cellulosic polymers such as wood, and combinations thereof.

[0065] Many other familiar chewing gum ingredients may be present, including, but not limited to, waxes, oils, softeners, fillers, flavors, antioxidants, emulsifiers, colorants, binders, acidulants, etc. The chewing gum may be provided with an outer coating, such as a hard coating, a soft coating, an edible film coating, or a combination thereof.

[0066] In some embodiments, the capture molecule that binds to the viral surface protein (e.g., CTB-ACE2, FRIL, IVA blocking peptide) is formulated with other components of the gum base so that the molecule is substantially uniformly contained in the gum base. The molecule is provided in various forms, for example, as a plant powder, freeze-dried leaves, or on an adsorbent such as finely divided silicic acid, amorphous silica, magnesium silicate, calcium silicate, kaolin, clay, crystalline aluminosilicate, macaloid bentonite, activated charcoal, alumina, hydroxylapatite, microcrystalline cellulose, or any combination thereof. In some approaches, the molecule can be encapsulated to achieve the desired controlled or sustained release. An example of a chewing gum that provides sustained release of nicotine is described in US2007 / 0014887, the disclosure of which is incorporated herein by reference. The chewing gum can be adapted for the purposes disclosed herein.

[0067] Similar release profiles can be achieved by oral dosage forms such as tablets, capsules, etc. For example, a tablet can have a core layer containing nicotine for sustained release and an outer layer containing CBD for immediate release. Other combinations are possible. For example, one or both layers can contain both CBD and nicotine, with each active ingredient being released in both an immediate and sustained release fashion.

[0068] The following materials and methods are provided to facilitate the practice of the methods disclosed herein.

[0069] Materials and Methods for Example 1 Saliva sample collection Saliva and nasopharyngeal and oropharyngeal swabs were collected from hospitalized patients with confirmed SARS-CoV-2 infection after informed consent under protocol #823392 approved by the University of Pennsylvania IRB. Saliva was spontaneously secreted by the patient, and oropharyngeal and nasopharyngeal samples were collected with flocked nylon swabs (Copan Diagnostics) and coeluted 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. Samples were stored at -80°C until use.

[0070] Fraunhofer, Aerofarms plant growth, biomass harvesting and freeze drying The Fraunhofer USA hydroponic system includes illuminated, multi-level growing shelves for growing transplastomic lettuce expressing CTB-ACE2. The growing trays contain rockwool, a lightweight hydroponic substrate made from fused basalt spun into fibers (Grodan, The Netherlands), as a support substrate. Rockwool is designed to retain approximately 80% of the nutrient solution and have approximately 15% voids and 5% fibers, allowing for good drainage and promoting rapid absorption of the nutrient solution by the plant's root system. The nutrient solution was supplemented with 200 ppm Peters Professional general-purpose fertilizer (ICL Fertilizers, Ohio) with a 20-10-20 ratio of nitrogen, phosphate, and potassium (potash), and 102 ppm Yarra Riva Calcinit (nitrate, ammonium, and calcium salt) (Yarra North America). This concentrated nutrient was supplied by a Dosatron in a 1:100 ratio with drinking water. Seedlings were spaced 81 / 2 inches apart and planted in a diagonal grid on the rockwool surface. Under temperature (24±2°C day / 20±2°C night) and humidity (60±10%) conditions, a photoperiod of 16 hours day and 8 hours night was used to measure the average irradiance of 368 μmols / m 2 The plants were illuminated in a growth chamber with LED lighting (Fluence SPYDRx) at 1.5 S. Airflow across the plants was approximately 1.2 m / s.

[0071] At Fraunhofer USA, transplanted lettuce was repeatedly harvested from plants at approximately 9 h (8–11 h) of the photoperiod on the 58th, 90th, 107th, and 120th days after sowing. Lettuce leaves were cut at the base of the petiole by workers wearing laboratory gloves, leaving the upper leaves intact, and the fresh weight of the harvested biomass was recorded. Leaves were rinsed with USP purified water and washed for approximately 5 min in a 200 ppm choline solution prepared with food-grade calcium hypochlorite, followed by three successive rinses with USP purified water, and the final choline concentration was measured using chlorine-sterilized test strips to confirm that the level was less than 4 ppm. Excess water was then removed from the surface of the leaves using a food-grade centrifugal lettuce dryer and transferred to polyvinyl zip-lock bags. The bags were frozen on dry ice and then stored at -60°C to -80°C. Frozen lettuce leaves expressing the CTB-ACE2 fusion protein were freeze-dried in a freeze dryer (Ultra50, SP Scientific, Stone Ridge, NY). The freeze dryer was thoroughly cleaned and disinfected after each use, and records of cycle parameters were collected. If not transferred directly to grinding, the material was placed in a storage bag with MiniPax® silica gel absorbent. The freeze-dried material was placed in a dark steel cabinet and stored in a storage room at room temperature. The freeze-dried leaves were ground in a grinder at single speed for one pass for 3 seconds (optimized to minimize cell disruption). The ground powder was passed through a 25 mesh (0.71 mm) hand sieve and filled into the final sterile container. Any material that did not pass the sieve was discarded. The material was placed in a sterile container (FDA approved) and stored in the dark at room temperature in a steel cabinet.

[0072] Transplastomic ACE2 lettuce grown at Fraunhofer was harvested 58, 90, 107, and 120 days after sowing, and those grown at Aerofarms were harvested 79 and 100 days after sowing. Leaves were cut from the base of the petiole with sterile gloved scissors, leaving the top 3–5 leaves intact. Leaves were transferred to polyvinyl zip-top bags. Bags were labeled with fresh weight, harvest date, and plant row and immediately frozen. Biomass stored in polyvinyl zip-top bags was stored at −60°C to −80°C. Frozen lettuce leaves expressing the CTB-ACE2 fusion protein were freeze-dried in a freeze dryer (Ultra50, SP Scientific, Stone Ridge, NY). The shelves were pre-cooled to -40°C, 15 shelves were loaded with 0.5 kg of frozen leaves each, and freeze-dryer probes were placed in the center of the plant material on every other shelf to monitor the product temperature. Each freeze-drying cycle started with the plant material being held at -40°C for 3 hours. This was followed by a three-stage drying cycle: the first stage was at -40°C for 10 minutes, the second stage increased in temperature to 25°C and lasted for 3260 minutes. Once the PVG / CM difference reached 43mTorr, the Ultra 50 moved to the third stage of drying, where the temperature was reduced to 22°C and held for 8 hours. After the cycle was completed, the plant material was removed, appropriately packed and weighed.

[0073] Moisture content Moisture content was measured by Karl Fischer titration, where available moisture reacts with iodine and sulfur dioxide to produce sulfur trioxide and hydrogen iodide. Briefly, samples were weighed, capped sample vials, and placed in a Metrohm 850 KF Thermoprep and heated to 150 °C. Water evaporated from the sample was pumped into a Metrohm 851 Titrando at a rate of 50 mL per minute. Percent moisture was calculated from the sample weight and the reaction power output, with Hydranal water standard (Honeywell) as the reference. Recent freeze dryers (Virtis Ultra 50) have a novel mechanism for evaluating and monitoring moisture content.

[0074] Bioburden After harvesting the biomass, the leaves were washed with a 200 ppm chlorine solution and rinsed three times with USP purified water. The excess water was then removed from the leaves and the tissue was frozen and stored. The tissue was then freeze-dried in a freeze dryer (Ultra50, SP Scientific, Stone Ridge, NY) and the moisture content was assessed, after which it was crushed and sieved again for moisture content and bioburden assessment. Bioburden was determined according to USP <61> (Microbiological Testing of Nonsterile Products: Microbial Enumeration Tests) and USP <62> The aerobic microbial and fungal loads were assessed by plating serial dilutions in duplicate on trypticase soy agar and Sabouraud's dextrose agar, respectively, and incubating at 30-35°C for 3-5 days or at 20-25°C for 5-7 days, respectively. For oral administration, the USP <1111> In accordance with (Microbiological testing of non-sterile products: Acceptance criteria for pharmaceutical preparations and pharmaceutical substances), the total number of aerobic microorganisms is ≦2×10 3 cfu / g, total yeast and mold count ≤ 2 x 10 2 The acceptance criterion was cfu / g.

[0075] Total protein extraction and quantification of ACE2 CTB-ACE2 expression levels were quantified by Western blot using CTB antibody and standards as previously described (Daniell et al., 2020) with appropriate modifications. Five milligrams of lyophilized plant powder was extracted with 500 μl of protein extraction buffer (PEB) containing 100 mM NaCl, 10 mM EDTA (pH 8), 200 mM Tris (pH 8), 100 mM DTT, 1X PIC, 2 mM PMSF, and 10 mM β-mercaptoethanol. Samples were incubated for 5 min with vortexing at 4 °C and centrifuged at 14,000 rpm for 10 min at 4 °C. The first supernatant was discarded and the pellet was suspended in 500 μl of PEB and incubated for 1 h at 4 °C with vortexing. The samples were then sonicated at 80% amplitude for 10 s on and 15 s off (3X) using a Sonicator 3000 (Misonix, Farmingdale, NY). Samples were then centrifuged at 14,000 rpm at 4°C for 10 min, and the second supernatant was discarded. The pellet was resuspended in 200 μl of PEB buffer and sonicated as above. Total protein concentration was quantified by Bradford assay (Bio-rad Laboratories, Hercules, CA), and homogenized proteins were heated at 70°C for 15 min in 1X Laemmli buffer and analyzed by 10% SDS-polyacrylamide PAGE. For each sample, 25 ng and 50 ng of total protein were loaded onto a Western blot gel, and anti-CTB antibody (1:10,000) (Gen Way Biotech, San Diego, CA), goat anti-rabbit IgG-HRP secondary antibody (1:4000) (Southern Biotechnology, Birmingham, AL), and precision plus protein standard (Bio-rad Laboratories, Hercules, CA) were used for all Western blot analyses.ACE2 protein was detected with Supersignal west Pico Chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA) on iBright Western Blot Imaging Systems (Thermo Fisher Scientific, Waltham, MA). ACE2 protein quantification was performed according to CTB standard concentration using iBright Analysis Software (Thermo Fisher Scientific, Waltham, MA).

[0076] ACE2 enzyme activity assay in plant extracts and saliva ACE2 activity was measured using saliva from SARS-CoV-19 infected and healthy subjects. Saliva samples were prepared at a 1:5 dilution. 75 μl of this prepared sample was added to a black 96-well microtiter plate containing 25 μl of ACE2 buffer (1 mol / L NaCl, 75 mmol / L Tris HCl, pH 7.5, 50 μmol / L ZnCl2) and 20 μmol / L of the ACE2-specific fluorescent peptide substrate VI Mca-APK(Dnp) (R&D Systems, Minneapolis, MN). Enzyme activity was recorded at 28°C for 90 min at 5 min intervals, with excitation at 340 nm and emission at 405 nm, with the optical position up and gain extended. After reading, the relative fluorescence units at each time point were plotted. ΔRFU was calculated by subtracting the data at time 0 min from the data at time 90 min. Total soluble protein in the remaining saliva samples was measured by Bradford assay using the standard Daniel Laboratory protocol. The final ACE-2 enzyme activity was calculated as pmol / min / mg (mU / mg) = Δpmol / 90min / mg total protein.

[0077] Microbubbling SARS-CoV-2 antigen assay 150 μL of patient specimen was added to each powder tube and vortexed. The tubes were then incubated at 4 °C for 1 h while rotating. After incubation, the tubes were vortexed again and spun at 14,000 RPM, 4 °C for 20 min. The tubes were collected and 120 μL of the supernatant was first mixed with a viral 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 samples were then tested by microbubbling SARS-CoV-2 antigen assay (Chen H. et al. 2019, 2021). Briefly, the sample solution (100 μL) was incubated with a suspension of 500,000 capture antibody-functionalized magnetic beads on a roller (12 rpm) for 30 min at room temperature. The beads were then separated by a magnet, washed three times with PBS buffer pH 7.4 containing 0.01% TWEEN 20, resuspended in 100 μL of 250 ng / mL biotinylated detection antibody in PBS containing 1% BSA, and left on a roller (12 rpm) at room temperature for 30 min. The beads were then separated by a magnet, washed three times with PBS buffer pH 7.4 containing 0.01% TWEEN 20, resuspended in 100 μL of 1 μg / mL NeutrAvidin functionalized PtNP in PBS containing 1% BSA, and left on a roller (12 rpm) at room temperature for 30 min. The beads were then separated by a magnet, 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 slurry was then added to the chamber of a microbubbling microchip. The microbubbling microchip was placed on a neodymium disk magnet for 1 min to pull the beads down to the bottom of the microchip. The microbubbles on the microbubbling microchip were imaged using an iPhone 11 or iPad with a uHandy mobile phone microscope (9x, focal length 5 mm, Aidmics Biotechnology Co., Taipei, Taiwan).

[0078] Lentivirus-spike neutralization assay Lentiviral particles were prepared in 293FT cells according to Crawford et al. (Crawford, Katharine HD, et al. "Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 spike protein for neutralization assays." Viruses 12.5 (2020): 513). Vero cells were cultured in a 96-well plate at 1.25x10 4 Cells / well were seeded overnight and cultured at 37°C in a humidified incubator with 5% CO2. ACE2 gum or the corresponding placebo was combined with 800 μL of growth medium containing enough virus particles to generate a signal at least 200 times higher than background luciferase activity. After gentle mixing for 1 h at 4°C, the supernatant was collected by centrifugation at 14,000 rpm for 20 min at 4°C, and 100 μL of the supernatant was added to each well, followed by 110 μL of fresh medium containing 5 μg / mL polybrene. After 24 h of treatment, cells were processed using the Bright-Glo Luciferase Assay System (Promega) according to the manufacturer's recommendations, and luminescence was measured on a microplate reader.

[0079] VSV-S pseudotype neutralization assay VSV-S pseudotyped particles were generated using the vesicular stomatitis virus (VSV) platform as previously described (1, 2). Briefly, pseudotyped VSV virions incorporating the SARS-CoV-2 Spike protein into the envelope were produced by transfecting HEK293T cells with the pCG1 SARS-CoV-2 S delta18 expression plasmid (kindly provided by Paul Bates and Stefan Pohlmann), encoding a codon-optimized SARS-CoV-2 S gene with an 18-residue truncation at the cytoplasmic tail (3). Thirty hours post-transfection, SARS-CoV-2 spike-expressing cells were transduced with VSVΔG-RFP pseudotype for 4 hours. The virus inoculum was removed and cells were washed twice with PBS 1x to remove unbound virus. Twenty-eight hours post-transduction, media containing VSV-S pseudotypes was harvested and clarified by centrifugation twice at 4,000 rpm for 15 min. The VSV-S pseudotypes were aliquoted and stored at −80°C until use in the ACE2 neutralization test.

[0080] Vero E6 cells were seeded at 1x10^4 cells per well in a 96-well plate and incubated overnight at 37°C. 100ul of VSV-S pseudotyped particles (~1.2x10 4 1000 ng of CTB-ACE2 or 5 mg, 10 mg, 20 mg, or 50 mg of ACE2 gum powder and rotated end-to-end for 30 min at 4°C. Samples were centrifuged at 14.8K 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 with shaking every 15 min. The virus inoculum was removed and cells were washed twice with PBS 1x to remove unbound viral particles. At 24 h post-transduction, red fluorescent protein (RFP) expression was visualized and quantified by fluorescence microscopy. RFP expression in each condition was measured in two technical replicates performed in two independent experiments. Percentage inhibition of VSV-S entry was calculated relative to untreated VSV-S control, and statistical significance was analyzed by student t-test.

[0081] ddPCR assay Saliva samples from 15 COVID-19 patients were collected by medical staff at the University of Pennsylvania. To avoid virus infection and false-positive results due to laboratory contamination, all experiments were performed in a BSL2-enhanced biosafety cabinet. 140 μl of saliva was mixed and incubated with 100 mg of ACE2 gum powder at room temperature for 1 h with rotation, followed by spinning at 14,000 rpm for 20 min. Total RNA was extracted from the supernatant using the QIAamp viral RNA mini kit (Qiagen) according to the manufacturer's instructions. ddPCR was performed in duplicate using the COVID-19 digital PCR detection kit (Biorad). All procedures followed the manufacturer's instructions for the QX200 Droplet Digital PCR System with Supermix Probes (Bio-Rad). This kit can detect regions of the nucleocapsid N1, N2 genes, and the Rnase P gene positive reference gene. Twenty-two microliters of each reaction mix was converted into droplets with a QX200 droplet generator (Bio-Rad). Droplet-split samples were transferred to 96-well plates, sealed, and cycled using a T100 Thermal Cycler (Bio-Rad) with the following cycling protocol: 95°C for 10 min (DNA polymerase activation), 94°C for 30 s (denaturation), 60°C for 1 min (annealing), repeated 40 times, followed by 4 infinite folds. The cycled plates were then transferred and read in the FAM and HEX channels using a QX200 reader (Bio-Rad). Data were analyzed using QuantaSoft analysis Pro 1.0.596 software (Bio-Rad).

[0082] The following examples are provided to illustrate various embodiments of the invention, but are not intended to limit the invention in any way. EXAMPLES

[0083] Example 1 Reducing oral virus transmission and infection using ACE2 or HA in chewing gum Fraunhofer USA to Produce Clinical-Grade ACE2 Plant at Aeropharms CTB-ACE2 plants were generated as reported in a previous publication (Daniell et al. 2020, supra). The same lot of seeds was provided to Fraunhofer and Aerofarms, and plants were grown as described in the methods section. Despite the different growth conditions, the biomass yield per plant was similar at 80–90 days, but decreased as the plants grew larger. The maximum expression in plants grown at Aerofarms was less than 2 mg CTB-ACE2 / g dry weight, whereas the expression levels at Fraunhofer USA were 15–22 mg / g dry weight, which seems to represent the highest expression levels achieved so far in modified leaves (Figure 1 and Table 1). However, regardless of the growth conditions, the highest expression was observed in the oldest plants, when they were harvested with the least biomass in both facilities. Thus, delaying the final harvest once could further improve the production and yield of protein formulations. Total protein on a dry weight basis was also higher in Fraunhofer (140-147mg / g DW) than in Aerofarms (93-129mg / g DW), suggesting that differences in nutrient solutions affect protein synthesis. High levels of expression are an important production indicator, as they reduce the amount of plant powder required for chewing gum or oral administration of therapeutic ACE2.

[0084] Table 1: Fresh harvest biomass, moisture content, total protein content, and ACE2 content of CTB-ACE2 plants grown at Fraunhofer and Aerofarms in relation to age and growing conditions. Values ​​for CTB-ACE2 expression are means ± SD (n = 3). Values ​​for total leaf protein are means ± SD (n = 8). [Table 1]

[0085] Chewing gum Per Os Biosciences (Hunt Valley, MD) prepared chewing gum tablets with plant powders using a compression method, whereas traditional gum manufacturing processes require high temperatures and extrusion / rolling, which results in variability in active ingredient concentration. The gum tablets contained 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%), and freeze-dried plant cells expressing ACE2 to provide the best flavor, taste, softness, and compression. The gum tablets chew and function exactly like traditional chewing gum based on their physical properties. The freeze-dried plant cells were milled with 5 and 10 pulses, releasing more proteins into the supernatant and retaining less of the intact plant cells that may require mechanical crushing by the teeth during chewing. The two concentrations of ACE2 gum powder were combined into 2-gram chewing gum tablets and a placebo gum that did not contain the CTB-ACE2 freeze-dried plant cells.

[0086] Neutralization assay using SARS-CoV-2 spike pseudotyped lentiviral particles CTB-ACE2 may effectively bind to the binding sites of GM1 and ACE2 receptors located in close proximity to the human cell surface, blocking viral entry into human cells. Therefore, we used SARS-CoV-2 pseudotyped lentivirus (also called lentiviral particles) to investigate the neutralizing effect of ACE2 gum on spike-mediated viral infection. Lentiviral particles pseudotyped with the viral spike protein and carrying a pseudovirus expressing a luciferase reporter gene were used to infect CHO cells expressing human ACE2. SARS-CoV-2 spike glycoprotein pseudotyped viruses expressing luciferase were incubated with the indicated concentrations of ACE2 gum at room temperature for 90 min. After centrifugation, the virus-containing supernatant was incubated with ACE2-expressing CHO cells for 72 h, and viral infectivity was measured by luciferase. As shown in Figure 2A, incubation of lentivirus with ACE2 gum significantly reduced luciferase activity in a dose-dependent manner, indicating that spike-mediated infection was effectively neutralized. Notably, a comparison of ACE2 gum treatment with the corresponding placebo showed that ACE2 significantly inhibited spike-mediated infection: for example, 50 mg of ACE2 gum was effective in reducing infectivity by approximately 95% compared to the corresponding placebo (Figure 2B).

[0087] Data shown in Figure 2C are representative of three independent experiments (N = 2-3 samples). Bars represent condition means, symbols represent means of duplicate or triplicate assays, error bars represent SEM. Incubation of pseudotyped lentivirus with CTB-ACE2 gum significantly reduced luciferase activity in a dose-dependent manner; the highest inhibition (p<0.0001****) was observed at 50 mg of CTB-ACE2 (Figures 2A, 2B and 2C). This confirms the ability of this protein to block lentiviral spike protein from entering CHO cells, either by direct binding to the spike protein or by binding of CTB-ACE2 to the ACE2 / GM1 receptor on CHO cells.

[0088] Entry of VSV-S pseudotypes is inhibited by recombinant CTB-ACE2 and ACE2 gum powder RFP-expressing VSV-S pseudotyped particles utilize the SARS-CoV-2 spike (S) protein to bind and enter cells. We assessed whether purified recombinant CTB-ACE2 protein or ACE2 gum powder binds to VSV-S particles and inhibits viral particle entry into Vero cells. Repeated experiments showed that the addition of CTB-ACE2 inhibited VSV-S entry by approximately 85% compared to untreated controls (Figure 3). Furthermore, incubation of VSV-S particles with increasing concentrations of ACE2 gum powder inhibited entry in a dose-dependent manner (Figure 3). Indeed, representative images from fluorescence microscopy highlight a clear significant difference in the number of cells expressing RFP between the control and the 20 mg ACE2 gum powder samples. Taken together, these data indicate that CTB-ACE2 protein and ACE2 gum powder capture VSV-S pseudotyped particles and reduce viral entry by either directly binding to the spike protein or by binding to the ACE2 / GM1 receptor on the Vero cell surface.

[0089] Evaluation of SARS-Cov-2 in swab samples using microbubbles and gum We assessed ACE2-Gum neutralization of viral particles in several de-identified nasopharyngeal (NP) swab samples from patients who tested positive for SARS-CoV-2 nucleic acid and nucleocapsid antigen. The microbubbling SARS-CoV-2 antigen assay detects SARS-CoV-2 nucleocapsid antigen at femtomolar concentrations. (See additional references in the references section, Chen et al. Ange Chemie 31, 14060-14066 (2019); Chen et al. MedRxiv doi:10.1101 / 2021.03.17.21253847.) The number and size of microbubbles correlate with the amount of nucleocapsid antigen in the sample. Samples from patient 1 and patient 2 were treated with 25 mg of gum per 150 μL of sample, and samples from patient 3 were treated with 50 mg of gum per 150 μL of sample. The samples were then tested using a microbubbling SARS-CoV-2 antigen assay. As shown in Figure 4, incubation of swab specimens with ACE-2 gum significantly reduced the number of microbubbles compared to the placebo gum and untreated groups. This indicates that ACE-2 gum reduced the amount of nucleocapsid antigen in patient samples. The reduction rates ranged from 70% to 89%. In contrast, there was less significant variation when comparing placebo gum with the untreated group (p value 0.892). ACE2 gum reduced the amount of nucleocapsid antigen in COVID-19 patient samples even at a very low concentration (25 mg), although each gum weighed 2 g. These results confirm that ACE2 directly binds to the spike protein of SARS-CoV-2 and is encapsulated into insoluble pentameric microparticles of CTB-ACE2, facilitating removal of bound viral particles by centrifugation.

[0090] ACE2 activity in saliva of controls and COVID-19 patients Saliva samples were collected from 10 COVID-19 patients (age range) and 10 healthy controls. ACE2 activity was significantly reduced in COVID-19 patients compared to controls (Figure 5A-C). ACE2 activity was observed to be significantly reduced in virus-infected saliva from 10 COVID-19 patients compared to healthy controls (2582 ± 439.82 vs 71356 ± 2116 ΔRFU, 27.63 ± 9.52 vs 257 ± 7.89 mU / mg enzyme activity units). The fluorescent cleavage product of ACE2 (Mca-YVADAPK) increased steadily up to 90 min in control saliva samples. Although all Covid-19 saliva samples showed similarly low and almost undetectable ACE2 activity, one sample stood out as an outlier with an enzymatic activity similar to that of control saliva (38504 ± 9688 ΔRFU; 236.4 ± 60.28 mU / mg enzymatic activity units), which is excluded in Fig. 4B,C. This patient was asymptomatic and did not develop COVID-19 according to his medical records, but PCR data confirmed the presence of SARS-CoV-2. Therefore, ACE2 activity in saliva is a strong biomarker to distinguish symptomatic from asymptomatic COVID-19 patients. The slight variation in ACE2 activity in healthy patients based on demographic data is currently under investigation in patients with different age groups and ethnic backgrounds.

[0091] The activity of full-length CTB-ACE2 is inhibited by SARS-COV-2 spike protein To investigate the mechanistic reasons for the reduced ACE2 activity in COVID-19 saliva, inhibition by SARS-COV-2 spike protein, we performed an in vitro enzymatic assay with full-length CTB-ACE2 in the presence or absence of SARS-COV-2 spike protein (RBD, S1-S2). CTB-ACE2 was extracted from CTB-ACE2 transplastomic lyophilized leaf powder, and 20 μg of protein extract was used for the fluorescence kinetic assay. The fluorescent cleavage product (Mca-YVADAPK) of the CTB-ACE2 protein extract gradually amplified up to 90 min, indicating that ACE2 was enzymatically active (Figure 5D). 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 RT (Figure 5D). Indeed, ACE2 activity was completely inhibited by pre-incubation (30 min) of 10 μg of SARS-COV-2 RBD. Taken together, the SARS-COV-2 spike protein binds to full-length ACE2 via the RBD. [4,5,6] , suggesting that it reduces ACE2 activity.

[0092] Assessment of SARS-CoV-2 debulking in saliva by ddPCR We evaluated the debulking of viral particles by ACE2 or placebo gum by ddPCR using several saliva samples collected from SARS-CoV-2 positive patients. Unlike the microbubble assay, which measures actual viral particles, ddPCR amplifies viral RNA and therefore does not measure the actual copies of viral RNA present in the patient. PCR amplification is used to increase the sensitivity of saliva tests but is not quantitative. In fact, PCR amplification has not yet been used to predict the severity of COVID-19. Considering these limitations of PCR methods, we evaluated this method to measure viral shedding in saliva. Despite the amplification, most samples tested showed a 2-4-fold reduction for placebo and more than 10-fold reduction for ACE2 gum, nearly to the minimum copy number that can be reliably measured by ddPCR. As shown in Figures 6A and 6B, incubation of saliva samples with ACE-2 gum significantly reduced SARS-Cov2 copies compared to the placebo gum and untreated groups. Most treated samples showed a reduction rate of more than 90%. See Figure 6C.

[0093] discussion In healthy human lungs, ACE2 is mainly expressed in type II alveolar epithelial cells, which produce surfactant to prevent alveolar collapse and have tight junctions that limit fluid exudation. ACE2 is an integral part of the renin-angiotensin system (RAS) and cleaves angiotensin II (Ang II), which causes vasoconstriction, inflammation, hypercoagulability, and fibrosis (Gheblawi et al. Circ Res.2020;126:1456-1474). , anti-inflammatory, and cytoprotective angiotensin 1-7 (Ang 1-7) peptides. Human ACE2 exists in both soluble (sACE2) and membrane-bound (mACE2) forms, with the latter being the most predominant (Rahman et al. Rev Med Virol.2021;1-12; Anand et al. Viruses 2020, 12, 1104; Batlle et al. 2020 Clinical Science 134:543-545). The low abundance and short lifespan of sACE2, as well as its entry into host cells as the SARS-CoV-2-sACE2 complex, are among the reasons that explain the lack of protection in patients with COVID-19 (Rahman et al. 2021, surpra). In the case of therapeutic ACE2, boosting infusion may help balance the RAS by compensating for lost sACE2 and preventing its downregulation in COVID-19 patients (Zoufaly et al., Lancet Respir Med. 2020;8:1154-1158), or in pulmonary hypertension models, oral ACE2 administration suppresses the development of pulmonary hypertension with reduced right ventricular (RV) hypertrophy, RV systolic pressure, total pulmonary resistance, and pulmonary artery remodeling (Daniell H et al., 2020, surpra; Shenoy et al., 2014 Hypertension 64, 1248-1259). In contrast to injected truncated (transmembrane) sACE2 (Zoufaly et al., 2020, supra), full-length oral CTB-ACE2 accumulates in the lungs at concentrations 10 times higher than in plasma upon oral administration of bioencapsulated plant cells (Daniell et al., 2020, 2021, supra), providing yet another approach to treat COVID-19 patients. Indeed, oral administration of protein pharmaceuticals bioencapsulated in plants reduces costs by eliminating prohibitively expensive fermentation, purification, cold chains for transportation / storage, and sterile injections (Daniell et al., 2019, supra; 2021, supra; Park et al., 2020 Biomaterials 233, 119591).

[0094] It was investigated whether ACE2 chewing gum could capture SARS-CoV-2 and clear the virus to reduce oral infection. Although SARS-CoV-2 entry into human cells via the ACE2 receptor has been widely reported, the requirement for the GM1 co-receptor has been less studied (Fantini et al. Int J Antimicrob Agents.2020;56:106020). Similarly, the published literature is largely focused on the ACE2 receptor, and the role of soluble ACE2 is less understood. Indeed, SARS-CoV-2 has a higher binding affinity with monomeric soluble ACE2 than other known coronaviruses (Anand et al., 2020, supra).

[0095] The experiments in this study were designed to: 1) show that CTB-ACE2 binds directly to the spike protein and captures the viral particles in the gum base or pellet after centrifugation; 2) CTB-ACE2 forms pentameric insoluble nanoparticles (which should facilitate capture of the viral particles); 3) SARS-CoV-2 requires both the ACE2 receptor and the GM1 co-receptor for cell entry - CTB-ACE2 is able to saturate both of these receptors due to the high affinity of CTB for the GM1 receptor, facilitating virus neutralization studies in Vero cells using VSV or lentiviruses engineered to express spike protein or block entry into human oral epithelial cells. Thus, sACE2 can compete with SARS-CoV-2 for the ACE2 receptor binding site, acting as a "decoy" and also directly bind to the spike protein of SARS-CoV-2, preventing its entry into human cells (Daniell et al. 2021, supra; Batle et al. 2020, supra). Indeed, recent studies have shown that pseudovirus-modified spike proteins damage vascular endothelial cells by downregulating ACE2 and consequently inhibiting mitochondrial function (Lei et al. Circulation Research 2021;128:1323-1326). Therefore, using ACE2 chewing gum to capture SARS-CoV-2 and reduce spike protein levels is of crucial therapeutic and preventive importance.

[0096] It should be noted that there is a placebo effect, the strength of which depends on the virus particle density tested in this study. In silico screening of 48 sugar alcohol compounds identified three sugar alcohol compounds (sorbitol, mannitol, and galactitol) with the highest binding affinity to viral proteins, and Ebola VP40 was found to have a high affinity for sorbitol, especially in terms of binding energy and number of hydrogen bond interactions (Nagarajan et al. 2019 Molecular Biology Reports 46:3315-3324). ACE2 chewing gum contains maltitol (20.4%) and 13% each of sorbitol and xylitol. Sugar-free chewing gum products from Wrigley's, Mondelaz, and Hershey's contain various combinations of xylitol, sorbitol, isomalt, and maltitol. Two major chewing gum brands, Stride (Mondelez) and Icebreakers (Hershey's), contain all four of these sugar alcohols, so while gum chewers during a pandemic may have some advantage in reducing viruses in the mouth, it is noteworthy that adding ACE2 to the gum base dramatically enhances capture and neutralization of viral particles.

[0097] Pseudovirus evaluation VSV particles expressing the S protein of SARS-CoV-2 can be used to accurately mimic the entry of this coronavirus into ACE2-expressing cells. Notably, this assay can be performed rapidly at the BSL-2 level, allowing for early evaluation of the efficacy of entry inhibitors. Here, we used this method to show that increasing concentrations of recombinant CTB-ACE2 and ACE2 gum powder effectively inhibited VSV-S particle entry compared to controls. Consistently, ACE2 gum powder also effectively inhibited infection by spike-pseudotyped lentiviral particles.

[0098] Chewing gum for preventing influenza infection Since the first influenza pandemic in 1918 and the 2005 / 2006 epidemic, WHO has established 149 national centers for influenza surveillance. In humans, influenza is an infectious respiratory disease. There are four types of influenza viruses: A (IAV), B (IVB), C (ICV), and D (IDV). Types A and B are the most common, with type A causing pandemics and seasonal epidemics. Influenza A and B viruses have eight linear segments of single-stranded RNA with a diameter of 80–120 nm and a mass of approximately 170–200,000 kDa (Paules & Subbarao, Lancet 2017, 390, 697-708.; Jang & Seong, 2017 Front.Cell Infect.Microbiol.9, 344). Nearly 40% of the influenza virus surface is covered by spike proteins, with 350 hemagglutinin (H subtype) and 70 neuraminidase (N subtype) spikes, and mutations affect the infectivity of the virus particles (Ksenofontov et al. Molekulyarnaya Biologiya, 2008, Vol. 42, No. 6, pp. 1078-1080). In China, epidemics of avian influenza virus (H7N9) occurred in 2013 and 2017. Influenza viruses utilize the sialic acid receptors of host cells and the receptor-binding domain of the HA protein. With each seasonal influenza, new influenza vaccines are developed for antigenically drifted variants (Krammer et al. Nat. Rev. Drug Discov.14 (3) (2015) 167-182). While vaccines against IAV are being developed, newly evolving avian / mammalian or bat-derived serotypes (H1-H18, N1-N11, Wu et al., Trends Microbiol. 22 (4) (2014) 183-191) pose a greater risk.Using the concepts described in this example for SARS-Cov-2 spike protein, influenza virus infections can be reduced during seasonal influenza by reducing the virus with modified IAV blocking peptides (or FRIL as disclosed in Example 2) in chewing gum. Such peptides can include portions of the HA and neuraminidase proteins. These compositions and methods will provide therapeutic and societal benefits by lowering the viral load in affected individuals.

[0099] Example 2 It eliminates different Corona (SARS-COV-2 Delta, Omicron, OC43) and Influenza (H1N1, H3N2) virus strains in the oral cavity by capturing molecules in chewing gum, thus reducing infection and transmission.

[0100] Oral diseases caused by microbial infections afflict 3.5 billion people worldwide. While bacteria and fungi colonize tooth surfaces and form sticky, refractory biofilms that cause severe caries, saliva is a major source of pathogens transmitted as droplets or aerosolized particles [1]. A recent concern is COVID-19, in which the salivary glands are the primary site of replication of SARS-CoV-2, leading to loss of taste and smell [2-5]. In addition, influenza, HPV, HSV1, EBV, and KSHV viruses are also transmitted orally and have well-known life cycles in the oral epithelium [6-11]. Each of these viruses possesses simple or complex glucans on their surface to which FRIL can bind.

[0101] SARS-CoV-2 continues to be the primary cause of infection spread among unvaccinated individuals, but vaccinated patients have similar peak viral loads to unvaccinated individuals and transmit the virus efficiently within households

[12] . Equally important is the evolutionary pattern of SARS-CoV-2, where antibody resistance mutations enhance the infectivity of the virus

[13] . SARS-CoV-2 is transmitted nasally and orally, with oral infections 3–5 orders of magnitude higher than nasal infections [14–28]. The airborne volume of saliva droplets in healthy subjects is 3–5 orders of magnitude higher than exhaled droplets. Speaking four words releases more viral particles than an entire hour of breathing, suggesting that a reduction in oral viral load may have a significant impact on viral transmission [14–28]. Therefore, new methods have been proposed to remove pathogens from the oral cavity and minimize infection.

[0102] Clinical evaluation of mouthwash in COVID-19 patients showed no statistically significant change in salivary viral load up to 2 hours after rinsing

[29] . SARS-CoV-2 was detected several weeks after the disappearance of symptoms [30-32], and qPCR may detect non-viable viral particles, as evidenced by the CDC's subsequent guidelines not to perform qPCR testing until 90 days after the onset of infection. ACE2 enzyme has been expressed in chloroplasts in previous studies to treat pulmonary hypertension, but is now moving to the clinic to treat COVID-19 patients. Furthermore, as explained above in Example 1, CTB-ACE2 chewing gum was able to significantly reduce (>95%) SARS-CoV-2 in saliva or swab samples of COVID-19 patients, measured by microbubbles or qPCR

[34] , by direct binding of the spike protein to soluble ACE2 (Figure 7A). Inhibition of spike protein engagement of ACE2 and GM1 receptors by CTB-ACE2 (Figure 7A) was assessed by inhibiting the uptake of lentivirus or VSV pseudotyped virions into Vero cells. In addition to the native human ACE2 enzyme used in this study, several mutants with higher affinity for SARS-CoV-2 have been developed and can be used as viral capture proteins [35,36].

[0103] SARS-CoV-2 and influenza are the most potent viruses that spread by aerosol infection and cause illness and death to the greatest number of people. The significance of the plant lectin FRIL is that it preferentially captures viruses that express complex N-glycans (Figure 7B) on their outer envelope [38,39]. Enveloped viruses express either high mannose, complex N-linked glycans, or noncomplex hybrid polysaccharides. Both SARS-CoV-2 and influenza (Flu) viruses have been shown to contain complex N-glycans on their envelope [38-41]. Influenza (Flu) is a particularly important respiratory virus that affects people worldwide

[42] . Infections are seasonal and mostly endemic, with high morbidity and mortality

[42] . Globally, influenza causes 500,000 deaths annually

[40] . Enveloped virus influenza vaccines are developed based on new strains that undergo small mutations (genetic drift)

[42] . Influenza becomes particularly life-threatening when segmented genome strands that separately code for human influenza HA and NA become mixed during infection of different species, such as birds and pigs (Genetic Shift)

[42] . The past 150 years have seen several major pandemics, the worst of which was in 1918, when 50 million people died and one-third of the world's population was infected [40,42]. H5N1 avian influenza emerged in China in 2013 and can infect humans directly and spread worldwide

[43] .

[0104] Based on the success of COVID Gum in debulking SARS-CoV-2 with the native human protein ACE2 (see Example 1), in this example, we investigated the containment efficacy in different strains of SARS-CoV-2 using microbubbling (N-antigen) or RAPID (spike protein) assays. In addition, we investigated the encapsulation mechanism of a virus-capturing plant protein lectin (FRIL), which may neutralize SARS-CoV-2 and influenza viruses, using plaque reduction assays and electron micrographs. Protein drugs have been provided as sterile injectables for 50 years, which requires cryogenic storage and transportation, reducing patient affordability and compliance. To address some of these challenges, Daniel's laboratory has developed an oral delivery system through encapsulation of protein drugs in plant cells [44-49]. Bioencapsulation in plant cells eliminates the challenges of cryogenic storage / transportation [50-53]. Plant cells are currently being developed as a novel strategy to disrupt biofilms to kill caries-causing pathogens

[50] , to clear SARS-CoV-2 in saliva to reduce reinfection and transmission

[34] , and to deliver protein drugs against pathogens that colonize the oral cavity. This method is particularly suitable for reducing the viral load in saliva and for cleaning the throat surface where most viral infections occur. Chewing gum has been used to deliver small molecules such as aspirin

[55] , caffeine

[56] , calcium carbonate

[57] , chlorhexidine

[58] , nicotine [59,60], and xylitol

[61] since 1928

[54] , but delivering proteins via gum poses additional challenges in their stability and release kinetics. For example, insulin contained in chewing gum tablets is largely degraded in gastric juices and was not tested in animal studies

[62] , whereas oral administration of insulin bioencapsulated in plant cells is possible

[63] . Similarly, proteins bioencapsulated in plant cells survive the gum manufacturing process (which requires high temperatures) and remain stable in chewing gum for several years [34,50].In this study, we optimize the protein release kinetics from chewing gum in order to initiate clinical trials of the protein in the oral cavity.

[0105] Materials and Methods for Example 2 Preparation of medical CTB-ACE2 lettuce drug protein for making chewing gum tablets CTB-ACE2 lettuce plant material was grown, washed and freeze-dried at Fraunhofer according to the procedure previously described in Example 1. Grinding of plant material was performed using a steel grinder (BioloMix Mill Grinder, Swing -700g) on ​​a sanitized bench in a clean room. All washed instruments were disinfected with 70% isopropyl alcohol (IPA) / ethanol to remove bioload attached to the surface. Forceps, aluminum foil sheets and sieves (USA standard sieve - ASTM E11 specification, No. 25, 710 μm) were autoclaved at 121 °C for 20 min. Freeze-dried leaves were placed on a sterile aluminum foil sheet, placed on a clean bench and the midrib was removed using pre-sterilized forceps. 10 grams of plants were weighed on a pre-sterilized balance and transferred to the grinder. Plants were ground for 12 seconds. Grinding time was carefully monitored using a Traceable Nano Timer (Fisher Scientific). The ground powder was aseptically sieved onto sterile aluminum foil using an ASTM E11 No. 25 710 μm sieve and transferred into a sterile Uline black container. Anything remaining on the sieve was discarded. The Uline container containing the material was stored in a steel cabinet at room temperature. USP <61> and <62> For bioburden evaluation, 100 mg of ground samples were aseptically removed into sterile containers for evaluation of total microbial and yeast and mold counts according to the protocol described above. The moisture content of the plant material used in the gum preparation was determined according to the protocol described above (Figure 8).

[0106] Preparation of CTB-ACE2 chewing gum Chewing gum tablets formulated with CTB-ACE2 botanical powder were manufactured by Per Os Biosciences (Hunt Valley, MD). This method preserves the efficacy of the active ingredient, whereas traditional gum manufacturing techniques routinely involve extrusion / rolling at high temperatures that can lead to variability in protein concentration and reduce efficacy. CTB-ACE2 gum tablets use the following excipients - gum base (24.46%), magnesium stearate (3.00%), maltitol (15.98%), xylitol (1.98%), sorbitol (20.93%), silicon dioxide (0.40%), isomalt (10.00%), stevia 99% (0.45%), and natural flavors (maltodextrin, dextrose, gum arabic, essential oils) to flavor the gum tablets and aid in compression. The gum thus produced containing 50 mg of plant powder (2 g / tablet) performs quite similarly to conventional gum available in the market in terms of physical properties. The gum tablets received from Per Os Biosciences were stored in Mylar bags to avoid moisture absorption. A few tablets were placed in a Uline black container and subjected to general testing, i.e. bioburden, moisture content, drug dose determination and release (Figure 8).

[0107] Quantification of total dose and release of CTB-ACE2 gum The total amount of CTB-ACE2 in 2 g gum tablets was examined by Western blotting. 100 mg of crushed gum powder was suspended in 500 μL of plant extraction buffer (100 mM NaCl; 10 mM EDTA; 200 mM Tris-HCl, pH 8.0; 0.05% (v / v) Tween-20; 1 x protease inhibitor cocktail; 0.1% SDS; 14 mM β-Mercapto-ethanol; 400 mM sucrose; and 2 mM Phenyl-methylsulfonyl fluoride (PMSF)) and incubated at 4 °C for 1 h with a vortex mixer. The samples were then sonicated for 6 cycles at 80% amplitude with a 10 s on and 15 s off sonicator 3000 (Misonix, Farmingdale, NY). Bradford assays for total protein quantification and immunoblot analysis for total CTB-ACE2 dosage quantification were performed according to protocols developed by the Daniel laboratory (see Example 1).

[0108] To assess the release of CTB-ACE2, 100 mg of crushed gum tablets were suspended in 500 μL of plant extraction buffer (PEB) (10 mM EDTA; 400 mM Sucrose; 100 mM NaCl; 0.05% (v / v) Tween-20; 0.1% SDS; 14 mM β-Mercapto-ethanol; 200 mM Tris-HCl, pH 8.0; 2 mM PMSF; and 1 x protease inhibitor cocktail) and incubated at 4 °C for 30 min with vortexing. This was followed by centrifugation at 750 g for 5 min at 4 °C. The supernatant fraction was kept on ice until analysis. The remaining pellet fraction was resuspended in PEB and sonicated at 80% amplitude for 3 cycles of 5 s on and 10 s off using a sonicator 3000 (Misonix, Farmingdale, NY). Bradford assays for total protein quantification and immunoblot analysis of CTB-ACE2 release were performed according to protocols developed by the Daniel laboratory.

[0109] Nasopharyngeal swab specimen preparation Oropharyngeal (OP) and nasopharyngeal (NP) swab specimens were collected using flocked nylon swabs (Copan Diagnostics) from patients admitted to the Hospital of the University of Pennsylvania with clinically confirmed COVID-19 infection. OP and NP swabs were coeluted in 1.5 ml of viral transport medium (VTM) as previously described

[64] , aliquoted, and stored frozen (-80 °C) prior to analysis. Informed consent was provided by all study participants under a protocol approved by the University of Pennsylvania IRB (protocol #823392). Viral quantification was performed by qPCR with N1 primers as previously described

[64] . Assignment of viral lineage (Table 2) was based on whole genome sequencing, assigned using Pangolin lineage as described

[65] , or based on S gene target failure in RT-PCR of patient clinical diagnostic samples, a marker for Omicron lineage [66, 67]. In some cases, omicron lineage assignment was based on nearly 100% omicron circulation within the local community at the time of sampling.

[0110] Table 2: Viral titer information for omicron variants in SARS-CoV-2 NP / OP specimens treated with FRIL and CTB-ACE2 antiviral capture proteins and assessed by microbubble SARS-CoV antigen assay and RAPID. [Table 2]

[0111] Microbubbling SARS-CoV-2 antigen assay Microbubble SARS-CoV-2 antigen assay was performed with clinical NP swab specimens as described above. Of four patient NP / OP samples for the omicron variant of SARS-CoV-2, two were tested with FRIL bean powder and the remaining one with CTB-ACE2 gum powder. Briefly, patient samples (150 μL) were incubated with different doses of FRIL bean powder (5, 10, and 25 mg containing 20, 40, and 100 μg of protein, respectively) and CTB-ACE2 gum (10, 25, and 50 mg containing 0.18, 0.46, and 0.92 μg, respectively) at 4 °C for 30 min. This was followed by centrifugation at 14000 rpm for 20 min at 4 °C. The thus-recovered supernatant (120 μL) was carefully collected in a separate tube without disrupting the pellet. The supernatant was first treated with lysis buffer 10% Tween 20 (100x, 1.2 μL) and 100x protease inhibitor cocktail (1.2 μL) and incubated for 30 min at room temperature. 100 μL of the lysed sample was incubated with a suspension of 500,000 capture antibody-functionalized magnetic beads in a 96-well plate and fixed for 30 min at room temperature by rotating (12 rpm). The 96-well plate was then placed on a magnet to separate the magnetic beads, and the wells were washed three times with wash buffer (0.05% Tween 20 in PBS buffer, pH 7.4) and then resuspended in 100 μL of 250 ng / mL biotinylated detection antibody in PBS containing 1% BSA. After incubation for 30 min at room temperature, the beads were washed three times in the same way and then resuspended in 100 μL of 1 μg / mL NeutrAvidin-functionalized platinum nanoparticles for 30 min at room temperature. The beads were then washed three times and finally resuspended in 100 μL of 30% H2O2. The magnetic bead mix containing immune sandwich complexes formed between magnetic beads / target protein / PtNps was transferred to a microwell array (14 μm x 14 μm, 7 μm deep, 100 x 100) containing previously designed [68,69] microbubbling microchips. These microchips were then placed on top of a neodymium disk magnet and exposed to an external magnetic field for 9 min, which pulled the beads down into the microwells.The microbubbles formed as a result of oxygen accumulation in the microwells catalyzed by PtNPs from H2O2 decomposition were imaged using an iPad and a uHandy mobile phone microscope (9x magnification, 5mm focal length; Aidmics Biotechnology, Taipei, Taiwan).

[0112] Rapid Assay Electrochemical sensors were prepared as previously described

[70] . Virus detection was performed using a SquidStat Plus (Admiral Instruments) potentiostat and electrochemical impedance spectroscopy (EIS). EIS measurements were performed as previously described

[70] . SARS-CoV-2 delta and omicron variant NP / OP patient swab specimens were heat inactivated at 56 °C for 1 h. For the RAPID assay, 150 μL of each sample was treated with 20 mg of CTB-ACE2 crushed gum powder at 4 °C under stirring for 1 h. After incubation, the samples were vortexed again and spun down at 14,000 rpm at 4 °C for 20 min. First, 10 μL of VTM (blank) was added to the working electrode and left for 2 min, after which 200 μL of redox probe was added to cover all electrodes (counter electrode, reference electrode, working electrode), and an EIS experiment was performed to obtain a blank signal. After analyzing the blank, the sensor was washed with PBS (pH 7.4) and 10 μL aliquots of the resulting supernatants of the CTB-ACE2 chewing gum samples before and after incubation and centrifugation were placed directly on the working electrode of the biosensor. The samples were removed after 2 min of exposure and carefully washed with PBS, after which 200 μL of the redox probe was added for EIS analysis.

[0113] cell line African green monkey kidney epithelial Vero E6 cells were cultured in 5% heat-treated fetal bovine serum (FBS, Sigma), 100 units / mL penicillin, 2 mM L-glutamine, 50 μg / mL gentamicin, 100 μg / mL streptomycin, 1.25 μg / mL amphotericin B (Fungizone), and 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, pH 7.2). Cells were cultured at 37 °C for 24 h at 4 °C for 1 h at 4 °C. 2 Madin-Darby Canine Kidney (MDCK) cells were cultured in Minimum Essential Medium Alpha (MEMα, Gibco) supplemented with 5% heat-treated fetal bovine serum (FBS, Sigma), 100 units / mL penicillin, 2 mM L-glutamine, 50 μg / mL gentamicin, 100 μg / mL streptomycin, 1.25 μg / mL amphotericin B (Fungizone), and 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.2).

[0114] Purification of FRIL protein Lablab purpureus bean powder was extracted with PBS buffer and dialyzed overnight against decreasing salt concentrations. The precipitate was then resuspended in 20 mM phosphate buffer (pH 8.0) and transferred to a Unosphare Q column (BioRad, Hercules, California). Bound proteins were extracted with different NaCl gradients (0–0.5 M). Fractions with the highest neutralization titers were pooled, concentrated, and loaded onto a Superdex s200 10 / 300GL size-exclusion column (GE, Boston, Massachusetts). Fractions with the highest neutralization titers were pooled and concentrated. Finally, bands representing FRIL were separated in the flow-through by Syberdon Blue affinity chromatography (Affi-Gel, BioRad) and separated from nonspecific bands of ~30 and 40 kDa.

[0115] Plaque reduction assay Influenza virus: Purified FRIL and Bean Powder, the source of FRIL, were evaluated for their ability to prevent infection with influenza virus strains H1N1 (A / California / 7 / 2009-X181) and H3N2 (A / Singapore / INFMH-16 / 0019 / 2016) using a quantitative virus plaque reduction assay. The assay was performed by co-incubating Flu strains (80 pfu) with increasing amounts of purified FRIL (0–3.2 μg) in serum-free medium or Bean Powder protein extract (0–2 mg) in PBS in 100 μL for 1 h at 37°C. Virus, FRIL, and powder were then added to and infected MDCK cells (~90% confluent) in 48-well plates, respectively. After 1 h of adsorption at 37°C, the virus mixture was aspirated and washed to remove unadsorbed Flu. Cells were covered with Avicel / methylcellulose and incubated for 28 h at 37°C, then fixed and immunostained with anti-Flu nucleoprotein antibody. Viral plaques were counted microscopically and dose-response curves were generated.

[0116] Coronavirus:OC43 was coincubated for 1 h with increasing amounts of purified FRIL (0–3.2 μg) in serum-free medium or protein extract of hyacinth bean powder (0–2 mg) in PBS. Vero cells were then infected by adsorption with 100 μL of the OC43 FRIL mixture for 1 h at 34°C in serum-free medium, and after 5 days at 37°C in culture medium containing heat-treated serum, cells were fixed and stained with 4% formaldehyde and 0.2% crystal violet. Viral plaques were counted microscopically and dose-response curves were generated.

[0117] Negative staining electron microscopy H1N1 virus culture: H1N1 (A / California / 7 / 2009-X181) virus and purified FRIL protein (10 μg / mL and 150 μg / mL) were co-incubated in HEPES buffer (50 nM, pH = 8.0) at 37°C for 60 min. H1N1 virus was cultured at 4 × 10 7 From a stock concentration of pfu / mL, dilute to 1 x 10 using HEPES buffer.6 The virus was diluted to a titer of pfu / mL. The virus and purified FRIL protein were precleared by centrifugation at 15,000 rpm for 5 min. After incubation, the samples were applied to glow-discharged carbon-coated 400-mesh copper grids. The carbon-coated grids were stained with 2% uranyl acetate and washed twice with 5 μL of diH2O. The viruses were then observed using a transmission electron microscope (FEI Tecnai T12) and a CMOS camera (Gatan Oneview, Pleasanton, California) at 100 kV. Images were captured at 42K magnification using Gatan Digital Micrographic software.

[0118] Purified H1N1 virus: For visualization by microscopy, virus was purified on a sucrose gradient. H1N1 (A / California / 7 / 2009-X181) virus was co-incubated with 150 μg / mL FRIL for 30 min at 37°C. Carbon-coated 400-mesh copper grids were glow discharged using PELCO easeGlow. TM The washing was performed using a 91000 Glow Discharge Cleaning System (Ted Pella Inc.). The grids were glow discharged for 30 seconds at a current of 15 mA. After incubation, the samples were diluted in PBS buffer and loaded onto carbon-coated grids. The grids were negatively stained with Nano-W (Nanoprobes, Yaphank, New York). Viruses were observed under an electron microscope (JEM-1400; JEOL, Peabody, MA) operated at 120 kV and equipped with a CCD camera (Gatan 895; Gatan, Pleasanton, CA). Images were taken at 2.6K and 5K magnifications with Gatan Digital Micrographic software.

[0119] statistical analysis Total dose quantification and release data for CTB-ACE2 were presented as mean ± SD. Data for microbubbling SARS-CoV-2 antigen assay were presented as mean ± SD. Statistical significance was determined using One-Way electrochemical impedance measurement as the average of three replicates for each condition. Graphs were generated and statistical tests were performed with GraphPad Prism 9.2. For plaque reduction assays, plaque numbers were quantified under a dissecting microscope. Half-maximal EC50 values ​​were determined by nonlinear regression fitting to a variable slope, four-parameter dose-response model using Prizm 6 software (GraphPad Software, LaJolla, CA).

[0120] result As pointed out in the introduction, oral transmission of SARS-CoV-2 is 3-5 orders of magnitude higher than nasal transmission [14-28]. Two years into the pandemic, there is still no FDA-approved quantitative detection method to end quarantine or return to work after a positive SARS-CoV-2 test. As evidenced by detection of SARS-CoV-2 weeks after symptoms have resolved, qPCR may detect nonviable viral particles [30-32], and CDC guidelines do not support qPCR testing up to 90 days after infection onset. To develop a quantitative antigen test and examine changes in the viral load of COVID-19 samples in response to viral capture proteins, this study utilizes two different approaches. We evaluate hyacinth bean powder containing CTB-ACE2 gum or viral capture protein-FRIL using microbubbling (N antigen) and RAPID (spike protein) assays (Figures 7A, 7B). Additionally, the impact of FRIL on influenza virus strains was evaluated with a plaque reduction assay. The mechanism of containment of FRIL was further studied using electron microscopy.The various steps involved in the creation of plants expressing CTB-ACE2, seed and biomass production, sterilization, freeze-drying, grinding / sieving, production of chewing gum from freeze-dried plant powder, evaluation of FDA requirements (e.g. moisture content, bioburden, drug dosage, stability), functional characterization, toxicity / pharmacokinetics studies, IND application, and clinical trial design are summarized in Figure 8.

[0121] Dose determination of CTB-ACE2 and its release from gum tablets during incubation: Western blot evaluation of the gum tablets revealed that the total dose of CTB-ACE2 was 352.45 ± 18.9 μg / 2 g tablet (Figure 9A, 9B). The release of CTB-ACE2 without sonication was 37 ± 1.0 μg / tablet (2 g) under optimized experimental conditions performed in the laboratory (Figure 9C, 9D). The grinding conditions were optimized to maximize the release of protein suitable for the chewing gum drug delivery system. This differs from orally administered protein drugs [49,63], which are bioencapsulated and therefore protected from degradation in the stomach by gastric juices and subsequently released in the intestine by digestion of the plant cell wall by microorganisms. Bioburden evaluation of the plant materials used to prepare the gum and gum tablets showed no microbial and fungal growth in compliance with FDA parameters. The moisture content of clinical grade CTB-ACE2 lettuce powder was found to be 5.5%, which meets the FDA requirements for orally administered plant powders. The gum tablets prepared in this way meet FDA specifications and can be used in clinical trials.

[0122] Microbubbling evaluation of SARS-CoV-2 in swab samples treated with CTB-ACE2 gum and FRIL bean protein The microbubbling SARS-CoV-2 antigen assay is designed to detect nucleocapsid (N) antigen at femtomolar concentrations

[68] . Due to limited sample volume, dose-dependent analysis was limited to three doses. For omicron variant NP samples from patients #620 and #613, the removal ability of FRIL bean protein was tested at doses of 20, 40 and 100 μg, respectively, and for patients #614 and #615, the removal ability of CTB-ACE2 gum was tested at doses of 0.18, 0.46 and 0.92 μg. Viral titers of the four omicron variant NP samples are shown in Table 1.

[0123] As can be seen in Figures 10A and 10B, FRIL protein can significantly reduce the number of microbubbles even at the lowest dose of 20 μg (p<0.0001).

[0124] CTB-ACE2 gum showed a dose-dependent decrease in microbubbles, with the 0.92 g treatment sample showing the lowest number of microbubbles (p=0.0001) (Figures 11A, 11B).

[0125] Comparing the doses of the antiviral capture proteins from both plants, it is interesting to note that CTB-ACE2 was effective at a dose of 0.46 μg, while FRIL showed a similar reduction effect at 20 μg. In our recent paper

[34] , we demonstrated the debulking effect of CTB-ACE2 against the delta mutant form of the virus, as seen in Figures 11C and 11D. The neutralizing ability of CTB-ACE2 is effective against both SARS-CoV-2 variants. These results confirmed that the SARS-CoV-2 debulking efficiency of the plant lectin FRIL is different from that of CTB-ACE2. While ACE2 directly binds to the spike protein of SARS-CoV-2 and encapsulates the virus in the pentameric insoluble particulate structure of CTB-ACE2

[34] , FRIL preferentially binds to complex-type N-glycans on viral glycoproteins that form aggregates

[38] . Both of these distinct mechanisms could be effective preventive measures to suppress viral infections, as they facilitate the removal of internalized viral particles. In all four swab samples, there was no positive correlation between the viral load measured by RT-PCR and the number of microbubbles, which depended on the presence or absence of N antigen (Table 1). The low number of microbubbles in patients with high viral loads (#620, #613) suggests that RT-PCR may be detecting N1 gene fragments that do not generate intact N1 antigen with antibody-binding epitopes

[69] .

[0126] Assessment of SARS-CoV-2 in clinical swab samples by RAPID after CTB-ACE2 gum treatment Our previously developed RAPID (Real-time Accurate Portable Impedimetric Detection) method [70,71] detects the binding of spike protein to the human receptor ACE2 using electrochemical impedance spectroscopy (EIS), and was used here to verify the capture effect of CTB-ACE2 chewing gum [72-76]. To identify optimal analytical conditions for the assay, based on previous studies using hundreds of clinical samples [70,71], we used the normalized R CT Response used:

number

[70] . Except for one sample (ID 593), the samples were genotyped based on whole genome sequencing, or target failure of the S gene, or collection date (Table 1, Figure 12). Samples 595-609 are the Delta strain of SARS-CoV-2, and 613-631 are the Omicron strain. RNA extraction for all samples was performed from the same volume of patient sample (140 μl), and the extracted RNA was resuspended and quantified by qPCR under similar conditions. No specific changes in viral load were observed between Delta and Omicron specimens, but viral loads differed significantly between patients. Patient samples (150 μl of sample) were incubated with CTB-ACE2 chewing gum and RAPID was performed on 10 μl aliquots of patient samples (Figure 12). R CTThe values ​​of vary widely among patient samples. CTB-ACE2 chewing gum was able to efficiently capture viral particles, reducing the viral concentration in all 20 analyzed samples (Figure 12). In 17 of these samples, no viral particles were detected by RAPID after incubation with CTB-ACE2 chewing gum, highlighting the ability of this material to capture viral particles down to very low SARS-CoV-2 SP concentration levels (below the LOD of RAPID).

[0127] FRIL potently neutralizes influenza viruses and coronaviruses Because FRIL has binding affinity for complex N-linked glycans, it is believed to be broadly effective against enveloped viruses expressing complex N-linked glycans, such as influenza virus, HBV, and HSV [79-81]. Here, we investigated the antiviral activities of purified FRIL protein and hyacinth bean powder against influenza H1N1 (A / California / 7 / 2009-X181) and H3N2 (A / Singapore / INFMH-16 / 0019 / 2016), as well as coronavirus HCoV-OC43, using plaque reduction assays (Figures S13 and S14).

[0128] In influenza plaque reduction assays, purified FRIL protein showed midpoint inhibition of 95 ng (in 100 μL processing volume) against H1N1 (Figure 13A) and 96 ng against H3N2 (Figure 13B). Interestingly, the antiviral potency of 76 ng of FRIL against OC43 was experimentally close to that of influenza virus, considering possible slight differences in viral titers and plaque numbers (Figure 13C). These results are consistent with previously reported 50% plaque reduction neutralization tests (PRNTs) against H1N1, H3N2, and hCoV-19 / Taiwan / NTU04 / 2020. 50 ) value

[38] .

[0129] Importantly, the antiviral potential of labium bean powder has not been studied so far. Compared to purified FRIL, labium bean powder has advantages related to affordability, availability, and enzyme stability, making it a better candidate for antiviral chewing gum. Therefore, we investigated the neutralization potential of labium bean powder containing 4 μg FRIL protein per mg of bean powder. The resulting concentration of total soluble labium bean protein was 149.28 μg per mg of starting labium bean powder. Plaque assays with soluble labium bean powder showed midpoint inhibition values ​​of 25 μg (estimated 100 ng FRIL released) against H1N1 (Figure 13D), 21 μg (84 ng FRIL) against H3N2 (Figure 13E), and 13 μg (52 ng FRIL) against OC43 (Figure 13F). More importantly, the estimated amount of solubilized FRIL released at midpoint inhibition is experimentally close to the amount of purified FRIL used for midpoint inhibition, considering possible slight differences in virus titers and plaque counts. For example, hyacinth bean powder reaches midpoint inhibition against H1N1 at 25 μg, estimated to release 3732.5 ng of solubilized protein and 100 ng of solubilized FRIL, whereas purified FRIL reaches midpoint inhibition at 95 ng of solubilized FRIL. This is significant in that the antiviral activity is mediated through solubilized FRIL, whereas hyacinth bean powder contains 370 times more other plant proteins. These data indicate that it is not necessary to purify FRIL from hyacinth bean powder and lay a logical foundation for the production of chewing gum with hyacinth bean powder.

[0130] In summary, for both influenza strains and OC43 coronavirus, less than 1 mg / mL of hyacinth bean powder (which releases 4 μg / mL of FRIL) can effectively inhibit all infections. Considering that the saliva volume before swallowing is 0.87 ml for men and 0.66 ml for women

[81] , it is estimated that less than 1 mg of hyacinth bean powder in chewing gum can effectively reduce different influenza virus strains and coronaviruses in saliva, thereby preventing infection. Such high potency FRIL bean powder allows for the production of FRIL-containing chewing gum that can effectively reduce the risk of infection for both influenza and coronavirus.

[0131] Mechanism of virus containment by FRIL soybean protein Unlike the removal mechanism of CTB-ACE2, FRIL captures virions by binding affinity to complex-type N-linked glycans on the viral envelope. We added unpurified influenza virus to 10 μg / mL FRIL and observed aggregates of captured virus particles using negative staining EM (Figure 15A). Influenza particles were bound to each other much closer by FRIL compared to untreated virus particles, and FRIL protein visibly surrounded the virus aggregates. Large, dense clusters of overlapping influenza particles and FRIL protein were observed at a FRIL concentration of 150 μg / mL, but not untreated virus particles (Figure 15A).

[0132] To better observe the aggregation of influenza particles, the protocol was performed using sucrose gradient purified virus and 150 μg / mL purified FRIL. Aggregates of influenza virus particles were observed surrounding the FRIL aggregates (Figure 15B). The amount of isolated influenza virus, which was observed in large amounts with untreated influenza virus, was significantly reduced by the addition of FRIL.

[0133] These images suggest that the glycan-binding domain (CBD) in each of the four monomers of FRIL may bind multiple virus particles, thereby encapsulating virions in large aggregates at 10-150 μg / mL of FRIL protein (Figure 15). Clustering of virus particles could explain the clearance mechanism of FRIL: engulfed influenza virus particles are neutralized during centrifugation and removed from the sample. In addition, other virus-capturing lectins MBL and SP-D have been reported to promote virus clearance by the immune system

[82] , and FRIL may have a similar effect. Given that the spike (S) protein present in the SARS-CoV-2 viral envelope encodes 22 complex-type N-linked glycan sites per monomer

[41] , we hypothesized that FRIL could capture and clear SARS-CoV-2 by binding to the N-linked glycan sites of the spike protein in a microbubble assay. Primarily, the FRIL protein released from the hyacinth bean powder captured SARS-CoV-2 virus particles and formed clumps during incubation with patient samples, and upon centrifugation, the entrapped virus particles were removed, resulting in a reduction in the number of microbubbles.

[0134] Advancing delivery of viral-trapping proteins by chewing gum from the lab to the clinic In vitro SARS-CoV-2 clearance is effective against various SARS-CoV-2 strains, including the highly transmissible Omicron strain, but the time required for saliva regrowth by SARS-CoV-2 is still unknown. The current CTB-ACE2 gum can be used for short periods (dental clinics, public transport, gatherings, restaurants, etc.), but data on viral load dynamics in saliva are needed to use CTB-ACE2 gum therapeutically to lower the viral load in COVID-19 patients. A Phase I / II placebo-controlled double-blind study of CTB-ACE2 chewing gum is planned to be conducted according to the study plan shown in Figure 16.

[0135] The primary safety analysis will be performed in all participants who received the study formulation and will be presented by age group. All solicited and unsolicited AEs will be summarized as frequency by group for all groups and percentages by age group, and presented with associated exact 95% Clopper-Pearson confidence intervals. For virological endpoints, SARS-CoV-2 RNA levels on days 1, 2, 3, and 4 will be compared between groups, separated by each scheduled measurement time, using nonparametric Wilcoxon rank sum tests and descriptive statistics. In addition, viral antigen (N or spike protein) will be quantified in saliva samples using the microbubble or RAPID assays described above. For the assessment of the clinical evolution of COVID 19, a severity ranking will be performed based on the area under the curve (AUC) of the daily total COVID-19-related symptom score over time.

[0136] conclusion Approximately one-quarter of the US population chews gum two to three times a week

[85] , mostly recreationally, and small molecule delivery is currently or has been used in the past to deliver aspirin, nicotine, and caffeine, or for oral hygiene and health promotion. Attempts to deliver therapeutic proteins, such as insulin, using chewing gum have failed thus far. Our data show that delivery of neutralizing pathogens to the oral or throat surfaces via chewing gum is effective in lowering viral load and reducing infection transmission. Most importantly, the plant-produced proteins show incredible stability in the gum during production at elevated temperatures and during long-term storage at ambient temperature. This study demonstrates the power of delivering virus-capturing proteins in chewing gum to reduce infection and transmission of SARS-CoV-2 and influenza viruses, and the potential to extend this platform to a variety of other orally transmitted viral, bacterial, and fungal diseases.

[0137] References JPEG2024518540000005.jpg201170JPEG2024518540000006.jpg218170JPEG2024518540000007.jpg228170JPEG2024518540000008.jpg225170JPEG2024518540000009.jpg221170JPEG2024518540000010.jpg222170JPEG2024518540000011.jpg220170JPEG2024518540000012.jpg219170JPEG2024518540000013.jpg218170Although the preferred embodiments of the present invention have been described and specifically exemplified above, the present invention is not limited to such embodiments. Various modifications may be made without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

1. Use of a therapeutically effective amount of a composition comprising a carrier having a capture molecule having affinity for a protein or glucan on the surface of a virus in the manufacture of a pharmaceutical composition for reducing viral load from the oral cavity of a subject, wherein the capture molecule binds to the surface protein or glucan, thereby capturing the virus within the carrier and reducing the viral load in the oral cavity.

2. The use described in claim 1, wherein the reduction is performed before or after exposure to a virus.

3. The use described in claim 1, wherein the composition shortens, eliminates or minimizes recovery time from at least one complication due to viral infection.

4. 2. The use of claim 1, wherein the virus is selected from coronavirus, influenza virus, herpes virus, cytomegalovirus, papilloma virus, Epstein-Barr virus, hepatitis virus, Zika virus, and HHV-7.

5. The use of claim 4, wherein the coronavirus comprises at least one of an alphacoronavirus, a betacoronavirus, a gammacoronavirus, a deltacoronavirus, MERS-CoV, SARS-CoV, and SARS-CoV-2.

6. The use according to claim 1, wherein the virus is SARS-CoV-2 and the capture molecule is ACE2 or CTB-ACE2, which captures the spike protein containing the virus in the carrier, thereby reducing the viral load in the oral cavity.

7. 2. The use of claim 1, wherein the virus is an alpha-influenza virus, including at least one of an influenza A virus, an influenza B virus, and an influenza C virus.

8. The use according to claim 7, wherein the capture molecule is an influenza virus A (IVA) blocking peptide that captures influenza virus particles in the carrier.

9. The use according to claim 8, wherein the IVA blocking peptide comprises a virus-binding portion of the HA and / or neuraminidase protein.

10. The use according to claim 7, wherein the capture molecule is FRIL, which captures influenza virus particles in the carrier.

11. The use according to any one of claims 1 to 10, wherein the carrier is a chewing gum, a long-acting lozenge, or a tablet.

12. A composition comprising an effective amount of a capture molecule having binding affinity for a protein glucan on the surface of a virus, the virus being optionally capable of infection by aerosolization, and the capture molecule being present in a carrier suitable for oral administration.

13. The composition of claim 12, wherein the carrier is a chewing gum, a long-acting lozenge, or a tablet.

14. a) the virus is SARS-CoV-2, the capture molecule is ACE2 or CTB-ACE2, the surface protein is a spike protein, and the carrier is chewing gum, or b) the capture molecule is FRIL, which binds to glucan on the surface of SARS-CoV-2; or c) the virus is an influenza A virus and the capture molecule is an IVA blocking peptide; or d) the virus is an influenza A virus, a herpes virus or a papilloma virus, and the capture molecule is a FRIL protein from lablab bean powder that binds to glucan on the surface of the virus; 14. The composition according to claim 12 or 13.

15. 15. The composition of claim 13 or 14, wherein the chewing gum comprises a gum base (28.2%) comprising maltitol (20.4%), sorbitol (13%), xylitol (13%), isomalt (13%), natural and artificial flavors, magnesium stearate (3%), silicon dioxide (0.43%), stevia (0.65%).

16. Use of a therapeutically effective amount of a composition comprising a carrier comprising a capture molecule having affinity for a microorganism, wherein binding of the microorganism to the capture molecule captures the microorganism within the carrier, thereby reducing the pathogenic microbial load in the oral cavity.

17. 17. The use of claim 16, wherein the microorganism is a bacterium or fungus, administration occurs before or after the subject is exposed to the bacterium or fungus, and optionally, the use shortens, eliminates or minimizes recovery time of at least one complication from bacterial or fungal infection, and the bacterium or fungus is optionally infectable by aerosolization.

18. 17. The use according to claim 16, wherein the bacterium is Streptococcus pyogenes, which causes streptococcal pharyngitis.

19. A composition comprising an effective amount of a capture molecule having binding affinity for a bacterium or fungus, wherein the bacterium or fungus is present in the oral cavity and is optionally capable of infection by aerosolization, and the capture molecule is present in a carrier suitable for oral administration.

20. 20. The composition of claim 19, wherein the carrier is a chewing gum, a long-acting lozenge, or a tablet, or the bacterium is S. pyogenes and the capture molecule is an antimicrobial peptide and the carrier is a chewing gum, or the fungus is C. albicans and the capture molecule is a lipase and the carrier is a chewing gum.

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