Immunomodulation by targeting the junctional epithelium of the gingival sulcus

Abstract

The present invention includes methods of activating, modulating, and / or anergizing an immune response by targeting the junctional epithelium of the gingival sulcus.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 229,784, filed August 5, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates generally to the field of targeting immune responses, and more specifically to targeting the junctional epithelium (JE) of the gingival sulcus for vaccination against infectious pathogens, allergen immunotherapy, and immunomodulation against autoimmune diseases.

[0003] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH This invention was made with Government support under R01AI135197 and R01AI137846 awarded by the National Institutes of Health / NSF / DARPA. The Government has certain rights in the invention.

[0004] INCORPORATION-BY-REFERENCE OF MATERIAL FILED ON A COMPACT DISC none. [Background technology]

[0005] Without limiting the scope of the invention, its background is described in the context of immunization and allergen immunotherapy.

[0006] The eruption of teeth through the gingiva creates a breach in the otherwise continuous and uninterrupted human mucosal surface. To seal this discontinuity, gingival tissue attaches to each tooth via the junctional epithelium. The junctional epithelium attaches to the teeth and forms a seal between the oral cavity and the tissues below. The junctional epithelial seal is leaky and highly permeable because it is only a few cell layers thick with wide intercellular spaces between the cells. Gingival tissue beyond this area of ​​attachment forms the gingival sulcus. The high permeability of the junctional epithelium, a property not found elsewhere in the mucosal system, allows easy passage of commensal bacteria, potential pathogens and food allergens. The gingival crevice harbors an extensive network of immune cells, including both innate and adaptive immune cells, such as neutrophils, natural killer cells, macrophages, dendritic cells, CD4+ / CD8+ T cells, B cells and innate lymphocytes. This network helps to protect against constant irritation by microorganisms, allergens and food proteins and to generate an immune response.

[0007] Mucosal surfaces are the first point of contact with the environment, and so naturally they serve as the portal of entry for the majority of pathogens and allergens. For example, the coronavirus responsible for the current pandemic is primarily transmitted through the respiratory mucosa, HIV is primarily transmitted through the genital and gastrointestinal mucosa, pollen causing respiratory allergies initiates contact at the respiratory mucosa, and peanuts, a food allergen, initiates initial contact at the oral mucosa. It is recognized that strong mucosal and systemic immune responses are more effective in fighting infectious diseases compared to merely systemic immune responses, and have been widely reported in the literature. However, vaccine delivery by injection does not stimulate strong mucosal immunity, only strong systemic immunity. To generate strong mucosal immunity and strong systemic immunity, the vaccine must be delivered through a mucosal surface. However, simply placing the vaccine on a mucosal surface does not lead to efficient uptake, because mucosal surfaces are designed to keep substances out. For example, a mucosal delivery approach called sublingual immunotherapy (SLIT), in which an allergen is placed under the patient's tongue for about one minute to stimulate the oral mucosa, has attracted attention for the treatment of allergies. Recently, a tablet containing grass and pollen allergens utilizing the SLIT approach was approved by the FDA to treat allergic rhinitis caused by grass and pollen allergies. Because uptake through the sublingual mucosa is inefficient, SLIT requires approximately 50-100 times more allergen doses than injections, and there is a greater variability in patient responses. As another example, oral vaccination by ingesting a vaccine essentially helps to place the vaccine on the gastrointestinal mucosa, but is less effective because the highly acidic and enzyme-rich environment of the stomach can damage the vaccine, and the strong barrier provided by the gastrointestinal mucosa prevents efficient transport of the vaccine through the mucosal lining to the underlying tissue layers. Thus, it is understood that to obtain an efficient immune response, the mucosal barrier must be breached to deliver the molecule through the mucosal surface to the underlying tissues, and simply placing the molecule on the mucosal surface does not lead to an efficient immune response.Enhanced molecular uptake and delivery can be achieved using a variety of approaches, including encapsulating molecules into micro- and nanoparticles and relying on cell-mediated uptake of the particles, using chemicals to disrupt mucosal barriers to improve delivery, using infectious viruses and bacteria, altering pH, and mechanical methods.

[0008] One such prior art patent is U.S. Pat. No. 9,271,899, issued to Francois and entitled "Methods, Articles and Kits for Allergy Hyposensitization Therapy Via Oral Mucosa," which purportedly provides compositions and methods of use for desensitizing a subject to allergens via the oral mucosa region, specifically teaching targeting the vestibular mucosa to induce oral immune tolerance.

[0009] Despite these advances, there remains a need for novel immune targeting strategies that maximize antigen dosing to elicit robust immune responses. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] U.S. Pat. No. 9,271,899 Summary of the Invention [Means for solving the problem]

[0011] As embodied and broadly described herein, one aspect of the disclosure relates to a method of modulating an immune response in a subject (e.g., humans and pets such as dogs, cats, cows, pigs or other livestock) comprising delivering an effective amount of one or more antigens, immunogens, allergens or combinations thereof to the gingival sulcus, specifically targeting the junctional epithelium (JE), said amount being sufficient to activate or modulate an immune response. In one aspect, the one or more antigens, immunogens, allergens or combinations thereof are not delivered to the vestibular mucosa. In another aspect, modulating an immune response is activating or anergizing an immune response by targeting the junctional epithelium of the gingival sulcus. In another aspect, one or more antigens, immunogens, allergens or combinations thereof are provided to maximize delivery of the one or more antigens, immunogens, allergens or combinations thereof to the gingival sulcus. In another aspect, the method further comprises adding one or more agents that enhance the permeability of the one or more antigens to the gingival sulcus (GC). In another aspect, between 0.001% and 100% of the one or more antigens, immunogens, allergens, or combinations thereof are in a depot of the junctional epithelium (JE) of the gingival sulcus. In another aspect, the one or more antigens, immunogens, allergens, or combinations thereof are repeatedly provided to the junctional epithelium (JE) of the gingival sulcus. In another aspect, the delivery of the one or more antigens, immunogens, allergens, or combinations thereof to the JE is performed before or after eating or drinking. In another aspect, the one or more antigens, immunogens, allergens, or combinations thereof are provided 1, 2, 3, 4, 5, or 6 times daily or weekly. In another aspect, two or more antigens, immunogens, allergens, or combinations thereof are delivered to the junctional epithelium (JE) of the gingival sulcus. In another aspect, the one or more antigens, immunogens, allergens, or combinations thereof desensitizes the individual by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% to the one or more antigens, immunogens, allergens, or combinations thereof.In another aspect, the one or more antigens, immunogens, allergens, or combinations thereof desensitize the subject to between 0.1-100% to the one or more antigens, immunogens, allergens, or combinations thereof. In another aspect, delivery of the one or more antigens, immunogens, allergens, or combinations thereof to the JE occurs 0 hours, 0.1 hours, 0.2 hours, 0.3 hours, 0.4 hours, 0.5 hours, 0.6 hours, 0.7 hours, 0.8 hours, 0.9 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, or more, before the subject eats food, drinks water, or both. In another aspect, delivery of one or more antigens, immunogens, allergens, or combinations thereof to the JE occurs 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, or more hours after the subject eats food, drinks water, or both. In another aspect, the amount of antigen, immunogen, allergen, or combinations thereof delivered to the junctional epithelium is in the picogram to milligram range. In another aspect, the immune response is an immune response that is activated, regulated, or anergized.

[0012] As embodied and broadly described herein, one aspect of the disclosure includes: providing an effective amount of one or more antigens, immunogens, allergens, or combinations thereof to target the gingival sulcus, specifically the junctional epithelium (JE), wherein said amount is sufficient to elicit an immune response against one or more of said antigens, immunogens, allergens, or combinations thereof; wherein one or more of said antigens, immunogens, allergens, or combinations thereof are embedded in, coated on, or attached to a delivery device that targets the junctional epithelium of the gingival sulcus. In one aspect, the one or more antigens, immunogens, allergens, or combinations thereof are not delivered to the vestibular mucosa. In another aspect, eliciting an immune response is activating or anergizing an immune response by targeting the junctional epithelium of the gingival sulcus. In another aspect, the thickness of the delivery device is less than 5 mm, preferably less than 3 mm, more preferably less than 1 mm. In another aspect, the delivery device comprises a natural or synthetic polymer, an organic material, a metal, an inorganic material, or a combination thereof. In another aspect, the delivery device comprises a mucoadhesive layer or a hydrophobic layer or a hydrophilic layer, or a combination thereof. In another aspect, the delivery device comprises a microporous structure that allows diffusion of the antigen to the gingival sulcus. In another aspect, the device comprises an interdental brush or bristles that are systems / devices designed to reach the gingival sulcus. In another aspect, the amount of antigen, immunogen, allergen, or combination thereof delivered to the junctional epithelium ranges from picograms to milligrams. In another aspect, the one or more antigens, immunogens, allergens, or combination thereof desensitizes an individual to one or more of said antigens, immunogens, allergens, or combination thereof by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In another aspect, the one or more antigens, immunogens, allergens, or combinations thereof desensitizes the individual to the one or more antigens, immunogens, allergens, or combinations thereof by between 0.1-100%.In another aspect, delivery of one or more antigens, immunogens, allergens, or combinations thereof to the JE occurs 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, or more hours before the subject eats food, drinks water, or both. In another aspect, delivery of one or more antigens, immunogens, allergens, or combinations thereof to the JE occurs 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, or more hours after the subject eats food, drinks water, or both. In another aspect, increasing the immune response is making the immune response more activated, more regulated, or more anergized. In another aspect, the immune response targets at least one of: bacteria, viruses, fungi, protozoa, parasites, prions, toxins, cancer, allergies, or autoimmune diseases. In another aspect, the one or more antigens are selected from at least one of: proteins, peptides, deoxyribonucleic acid (DNA) oligonucleotides, ribonucleic acid (RNA) oligonucleotides, disrupted cells, intact cells, lipids, toxin variants, carbohydrates, virus-like particles, liposomes, attenuated live or killed natural or recombinant microorganisms, virosomes, polymeric / inorganic / organic micro- and nanoparticles, or immune stimulating complexes (ISCOMS). In another aspect, the one or more antigens comprise a peptide derived from a cancer cell or portion thereof selected from T-cell and B-cell lymphoproliferative disorders, ovarian cancer, pancreatic cancer, head and neck cancer, squamous cell carcinoma, gastrointestinal cancer, breast cancer, prostate cancer, or non-small cell lung cancer.In another aspect, the one or more antigens comprise a food allergen selected from peanuts, shellfish, egg proteins, milk proteins, legumes, nuts, or an airway allergen selected from dust mites or pollen. In another aspect, the one or more antigens are at least one of the antigens physically or chemically attached, adsorbed or immobilized to a dental floss or a thin device or strip / patch or an interdental brush of suitable thickness for placement in the gingival sulcus. In another aspect, the composition is selected from the group consisting of cytokines, chemokines, toll-like receptor ligands or activators, alum, muramyl dipeptides, pyridine, chitosan, saponin, oils, emulsions, bacterial cell wall extracts, bacterial proteins, cytoplasmic bacterial DNA or mimetics, viral RNA or mimetics, synthetic oligonucleotides, stimulator of interferon (IFN) genes (STING) agonists (2'3'-cGAMP, c-di-AMP, 2'3'-c-di-AM(PS)2 (Rp,RP), c-di-GMP, CL401, CL413, CL429, Flagellin, Imiquimod, LPS-EB, MPLA, ODN 1585, ODN 1826, ODN2006, ODN2395, pam3CSK4, poly(I:C), R848, TDB), natural polymers (poly-gamma-glutamic acid, chitosan, mannan, lipomannan, lentinan, dextran), synthetic polymers (poly-N-isopropylacrylamide, copolymers, block polymers, polyphosphazenes, polyelectrolytes, polyanhydrides, polymethacrylates, poly(lactic-co-glycolic acid), polycaprolactone, polyvinylpyrrolidone, cationic polymers), and combinations thereof. In another aspect, the one or more antigens activate the innate immune response, the adaptive immune response, or both.

[0013] As embodied and broadly described herein, one aspect of the disclosure relates to an immunization comprising an effective amount of one or more antigens, immunogens, allergens, or combinations thereof on a delivery device that targets the junctional epithelium of the gingival sulcus, where the amount of said one or more antigens, immunogens, allergens, or combinations thereof is sufficient to activate or modulate an immune response. In one aspect, the one or more antigens, immunogens, allergens, or combinations thereof are not delivered to the vestibular mucosa. In another aspect, modulating an immune response is activating or anergizing an immune response by targeting the junctional epithelium of the gingival sulcus. In another aspect, one or more antigens, immunogens, allergens, or combinations thereof are provided to maximize delivery of said one or more antigens, immunogens, allergens, or combinations thereof to the gingival sulcus. In another aspect, the immunization further comprises one or more agents that enhance the permeability of said one or more antigens, immunogens, allergens, or combinations thereof to the gingival sulcus (GC). In another aspect, between 0.001% and 100% of the one or more antigens, immunogens, allergens, or combinations thereof are present in the junctional epithelial (JE) reservoir of the gingival sulcus. In another aspect, the immunization further comprises a pharma- ceutically acceptable carrier, excipient, diluent, buffer, or salt.

[0014] In another aspect, one or more active agents (one or more antigens, immunogens, allergens or combinations thereof) are targeted to the junctional epithelium of the gingival sulcus, thereby enhancing the pharmacodynamics / pharmacokinetics of the active agent, thereby improving health.

[0015] As embodied and broadly described herein, one aspect of the disclosure relates to a method of making a floss containing a predetermined amount of one or more active agents, comprising: providing a floss; and depositing the active agent(s) on the floss in a pharmacologically acceptable carrier containing the predetermined amount of the active agent(s). In one aspect, the deposition method deposits the active agent(s) on a single adjacent portion of the floss or on two or more separate portions of the floss with the same or different spacing between each deposition area. In one aspect, the deposition method includes placing a droplet on the floss or using a pipette to drag and stretch the droplet over the floss to spread it over a certain distance / length on the floss or spray deposition or inkjet deposition or pipette based deposition or cartridge deposition or combinations thereof. In one aspect, the viscosity of the deposited material is 0.01 centipoise (cp), 1 cp, 10 cp, 100 cp, 1000 cp, 10000 cp, 100000 cp, 200000 cp, 300000 cp, 500000 cp, 1000000 or 100000000 cp. In one aspect, the deposition method is manual or automated or semi-automated or a combination thereof. In another aspect, deposition is on one side of the floss or on both sides of the floss. In one aspect, each adjacent deposit comprises the same active agent or a different active agent, or each adjacent deposit comprises the same active agent at a different concentration / amount; or wherein each adjacent deposit comprises a different active agent at a different concentration; or wherein each adjacent deposit is disposed on a different side / face of the adjacent deposit; or wherein each adjacent deposit is disposed on an opposite side of the floss from the adjacent deposit; or wherein each adjacent deposit comprises a different active agent from the previous adjacent deposit. In another aspect, the deposition of the different active agents is on one side of the floss or on both sides of the floss. In another aspect, the deposition of the active agent is on one side of the floss or on both sides of the floss, each side comprising the same or different concentrations / amounts of active agent. In one aspect, each adjacent deposit comprises the same or different active agents deposited on top of one another. In another aspect, the active agents are deposited on opposite sides at the same or different distances / lengths.In another aspect, each adjacent deposit contains an active agent that has a different solvent requirement for solubility (e.g., one active agent requires water as a solvent while the other requires an organic solvent); or each adjacent deposit is disposed on a different side / face of the adjacent deposit; or each adjacent deposit is disposed on an opposite side of the floss from the adjacent deposit; or each adjacent deposit contains a different active agent that has a different solvent requirement from the previous adjacent deposit that has a different solvent requirement. In another aspect, active agents with different solvent requirements are deposited on opposite sides at the same or different distances / lengths. In another aspect, active agents with the same solvent requirements are deposited overlapping at the same or different distances / lengths. In another aspect, active agents with different solvent requirements are deposited overlapping at the same or different distances / lengths. In another aspect, the floss is solid, unraveled, includes multiple strands, and is treated to be adhesive, or treated to adhere to a pharmacologically acceptable carrier, or treated to adhere to an active agent, or treated to adhere to an active agent and a pharmacologically acceptable carrier. In another aspect, the floss is treated to change its surface energy to facilitate the deposition process or to promote the formation of a uniform deposit. In another aspect, each adjacent deposit contains a dye or indicia that distinguishes adjacent deposits. In another aspect, the floss is not soaked with an active agent, a pharmacologically acceptable carrier, or both. In another aspect, the one or more active agents are selected from antigens, immunogens, allergens, or combinations thereof that are not delivered to the vestibular mucosa. In another aspect, the one or more active agents induce an immune response by targeting the junctional epithelium of the gingival sulcus, i.e., activate or anergize the immune response. In another aspect, the floss has a thickness of less than 5 mm, preferably less than 3 mm, and more preferably less than 1 mm. In another aspect, the floss contains a natural or synthetic polymer, an organic material, a metal, an inorganic material, or a combination thereof. In another aspect, the floss contains a mucoadhesive layer, a hydrophobic layer, a hydrophilic layer, or a combination thereof. In another aspect, the floss contains a microporous structure that allows diffusion of antigens to the gingival sulcus.In another aspect, the one or more active agents comprise an antigen, immunogen, allergen, or combination thereof, delivered to the junctional epithelium in an amount in the picogram to milligram range. In another aspect, the one or more active agents comprise an antigen, immunogen, allergen, or combination thereof, and desensitize the individual to said antigen, immunogen, allergen, or combination thereof by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In another aspect, the one or more active agents comprise an antigen, immunogen, allergen, or combination thereof, and desensitize the subject to said antigen, immunogen, allergen, or combination thereof by between 0.1-100%. In another aspect, the floss delivers an antigen, immunogen, allergen, or combination thereof to the JE, and the floss delivers said antigen, immunogen, allergen, or combination thereof to the JE at 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, or more hours after the subject eats food, drinks water, or both. In another aspect, the one or more active agents activate, modulate, or anergize the immune response. In another aspect, the one or more active agents induce an immune response that targets at least one of: bacteria, viruses, fungi, protozoa, parasites, prions, toxins, cancer, allergies, or autoimmune diseases. In another aspect, the one or more active agents comprise one or more antigens selected from at least one of: proteins, peptides, deoxyribonucleic acid (DNA) oligonucleotides, ribonucleic acid (RNA) oligonucleotides, disrupted cells, intact cells, lipids, toxin variants, carbohydrates, virus-like particles, liposomes, attenuated live or killed natural or recombinant microorganisms, virosomes, polymeric / inorganic / organic micro- and nanoparticles, or immune stimulating complexes (ISCOMS).In another aspect, the one or more active agents include an antigen comprising a peptide obtained from or a portion of a cancer cell selected from T-cell and B-cell lymphoproliferative disorders, ovarian cancer, pancreatic cancer, head and neck cancer, squamous cell carcinoma, gastrointestinal cancer, breast cancer, prostate cancer, or non-small cell lung cancer. In another aspect, the one or more active agents include an antigen which is a food allergen selected from peanuts, shellfish, egg protein, milk protein, legumes, nuts, or an airway allergen selected from dust mites or pollen. In another aspect, the one or more active agents include an antigen which is at least one of an antigen physically or chemically attached, adsorbed, or immobilized to a dental floss or a thin device or strip / patch of suitable thickness for placement in the gingival sulcus or an interdental brush. In another aspect, the floss contains a compound selected from the group consisting of cytokines, chemokines, toll-like receptor ligands or activators, alum, muramyl dipeptides, pyridine, chitosan, saponin, oils, emulsions, bacterial cell wall extracts, bacterial proteins, cytoplasmic bacterial DNA or mimetics, viral RNA or mimetics, synthetic oligonucleotides, stimulator of interferon (IFN) genes (STING) agonists (2'3'-cGAMP, c-di-AMP, 2'3'-c-di-AM(PS)2 (Rp,RP), c-di-GMP, CL401, CL413, CL429, Flagellin, Imiquimod, LPS-EB, MPLA, ODN 1585, ODN 1826, ODN2006, ODN2395, pam3CSK4, poly(I:C), R848, TDB), natural polymers (poly-gamma-glutamic acid, chitosan, mannan, lipomannan, lentinan, dextran), synthetic polymers (poly-N-isopropylacrylamide, copolymers, block polymers, polyphosphazenes, polyelectrolytes, polyanhydrides, polymethacrylates, poly(lactic-co-glycolic acid), polycaprolactone, polyvinylpyrrolidone, cationic polymers), and combinations thereof. In another aspect, the one or more active agents include one or more antigens, immunogens, allergens, or combinations thereof that activate the innate immune response, the adaptive immune response, or both.

[0016] As embodied and broadly described herein, one aspect of the disclosure relates to: a floss; and a method of making a floss comprising a predetermined amount of one or more active agents, comprising discontinuous deposition on the floss of one or more deposits of an active agent in a pharmacologically acceptable carrier, where each deposit comprises a known, predetermined amount of the active agent. In one aspect, each adjacent deposit comprises the same active agent or a different active agent, or each adjacent deposit comprises the same active agent at a different concentration; or where each adjacent deposit comprises a different active agent at a different concentration; or each adjacent deposit is disposed on a different side of the floss than the adjacent deposit; or each adjacent deposit is disposed on an opposite side of the floss from the adjacent deposit; or each adjacent deposit comprises a different active agent than the previous adjacent deposit. In another aspect, the floss is solid, unraveled, comprises multiple strands, and has been treated to be adhesive, has been treated to adhere to the pharmacologically acceptable carrier, or has been treated to adhere to the active agent. In another aspect, each adjacent drop or patch contains a dye or indicia that distinguishes adjacent deposits. In another aspect, the floss is not soaked with an active agent, a pharmacologically acceptable carrier, or both. In another aspect, the one or more active agents are selected from antigens, immunogens, allergens, or combinations thereof that are not delivered to the vestibular mucosa. In another aspect, the one or more active agents target the junctional epithelium of the gingival sulcus to trigger an immune response, i.e., activate or anergize the immune response. In another aspect, the floss has a thickness of less than 5 mm, preferably less than 3 mm, and more preferably less than 1 mm. In another aspect, the floss contains a natural or synthetic polymer, an organic material, a metal, an inorganic material, or a combination thereof. In another aspect, the floss contains a mucoadhesive layer, a hydrophobic layer, or a hydrophilic layer, or a combination. In another aspect, the floss contains a microporous structure that allows diffusion of the antigen to the gingival sulcus. In another aspect, the one or more active agents contain an amount of an antigen, an immunogen, an allergen, or a combination thereof that is delivered to the junctional epithelium in the picogram to milligram range.In another aspect, the one or more active agents comprise an antigen, immunogen, allergen, or a combination thereof, and desensitize the individual to said antigen, immunogen, allergen, or a combination thereof by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In another aspect, the one or more active agents comprise an antigen, immunogen, allergen, or a combination thereof, and desensitize the subject to said antigen, immunogen, allergen, or a combination thereof by between 0.1-100%. In another aspect, delivery of an antigen, immunogen, allergen or combination thereof to the JE by floss occurs 0 hours, 0.1 hours, 0.2 hours, 0.3 hours, 0.4 hours, 0.5 hours, 0.6 hours, 0.7 hours, 0.8 hours, 0.9 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours or more before the subject eats food, drinks water, or both. In another aspect, delivery of the antigen, immunogen, allergen, or combination thereof to the JE by the floss deposit occurs at 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, or more hours after the subject eats food, drinks water, or both. In another aspect, the one or more active agents activate, modulate, or anergize the immune response. In another aspect, the one or more active agents induce an immune response that targets at least one of: bacteria, viruses, fungi, protozoa, parasites, prions, toxins, cancer, allergies, or autoimmune diseases. In another aspect, the one or more active agents comprise one or more antigens selected from at least one of a protein, a peptide, a deoxyribonucleic acid (DNA) oligonucleotide, a ribonucleic acid (RNA) oligonucleotide, a disrupted cell, an intact cell, a lipid, a toxin variant, a carbohydrate, a virus-like particle, a liposome, an attenuated live microorganism or a killed natural or recombinant microorganism, a virosome, a polymeric / inorganic / organic micro- and nanoparticle, or an immune stimulating complex (ISCOMS).In another aspect, the one or more active agents include an antigen comprising a peptide obtained from a cancer cell or a portion thereof selected from T-cell and B-cell lymphoproliferative disorders, ovarian cancer, pancreatic cancer, head and neck cancer, squamous cell carcinoma, gastrointestinal cancer, breast cancer, prostate cancer, or non-small cell lung cancer. In another aspect, the one or more active agents include an antigen which is a food allergen selected from peanuts, shellfish, egg protein, milk protein, legumes, nuts, or an airway allergen selected from dust mites or pollen. In another aspect, the one or more active agents include an antigen which is at least one of an antigen physically or chemically attached, adsorbed, or immobilized to a dental floss or a thin device or strip / patch of suitable thickness for placement in the gingival sulcus or an interdental brush. In another aspect, the floss contains a compound selected from the group consisting of cytokines, chemokines, toll-like receptor ligands or activators, alum, muramyl dipeptides, pyridine, chitosan, saponin, oils, emulsions, bacterial cell wall extracts, bacterial proteins, cytoplasmic bacterial DNA or mimetics, viral RNA or mimetics, synthetic oligonucleotides, stimulator of interferon (IFN) genes (STING) agonists (2'3'-cGAMP, c-di-AMP, 2'3'-c-di-AM(PS)2 (Rp,RP), c-di-GMP, CL401, CL413, CL429, Flagellin, Imiquimod, LPS-EB, MPLA, ODN 1585, ODN 1826, ODN2006, ODN2395, pam3CSK4, poly(I:C), R848, TDB), natural polymers (poly-gamma-glutamic acid, chitosan, mannan, lipomannan, lentinan, dextran), synthetic polymers (poly-N-isopropylacrylamide, copolymers, block polymers, polyphosphazenes, polyelectrolytes, polyanhydrides, polymethacrylates, poly(lactic-co-glycolic acid), polycaprolactone, polyvinylpyrrolidone, cationic polymers), and combinations thereof. In another aspect, the one or more active agents include one or more antigens, immunogens, allergens, or combinations thereof that activate the innate immune response, the adaptive immune response, or both. [Brief description of the drawings]

[0017] For a more complete understanding of the features and advantages of the present invention, reference should now be made to the detailed description of the invention taken in conjunction with the accompanying drawings, in which: [Figure 1-1] ~ [Figure 1-3] Figures 1A-1F show the gingival sulcus and junctional epithelium: (Figure 1A) human mouth; (Figure 1B) structure of the gingival sulcus and junctional epithelium (JE); (Figure 1C) delivery of active agents to the junctional epithelium of the gingival sulcus and diffusion of active agents to the junctional epithelium and adjacent tissues over time; (Figure 1D) delivery of antigen molecules coated on floss and effect of flossing on mouse gingival tissue; and (Figure 1E) diffusion of rhodamine-conjugated ovalbumin (Ova, ovalbumin) in gingival tissue (ex-vivo). Flossing is performed by placing antigen-deposited floss around each incisor and flossing approximately 10-15 times to deposit the coated antigen at the gingival margin. (Figure 1F) Delivery efficiency of floss coated with fluorescein isothiocyanate (FITC, fluorescein isothiocyanate)-conjugated ovalbumin (Ova). [Figure 2-1] ~ [Figure 2-3]Figures 2A-G show the characterization of vaccine delivery and immune responses via floss. (Figure 2A) (1) Stereomicroscope photograph of coated floss, and (2) flossing procedure in mice. (Figure 2B) Vaccination schedule: Balb / c mice (n=5) were vaccinated by flossing the gums with antigen (ovalbumin (Ova), a model antigen)-deposited floss. The floss contained deposits of 25 μg Ova + / - 25 μg CpG (single-stranded oligodeoxynucleotide adjuvant), and mice were vaccinated weekly for up to 4 weeks in total. Mice treated with floss without any coating served as controls. Systemic immune response: Mice were bled on days 28 and 56, and serum anti-Ova antibody responses (1:12500 or 1:2500 dilution) were analyzed by enzyme-linked immunosorbent assay (ELISA). Figure 2(C)(1)-(3) Anti-Ova antibody responses on day 56 - Figure 2(C)(1) IgG, Figure 2(C)(2) IgG1 and Figure 2(C)(3) IgG2a. Individual mouse sera were used for analysis. Figure 2(D)(1)-(3) shows memory immune responses: Vaccinated mice were euthanized and bone marrow cells were harvested. Cells were cultured in triplicate at a concentration of 1 x 106 cells per well using RPMI medium supplemented with 10% fetal bovine serum and penicillin-streptomycin antibiotics. Supernatants of cultured cells were collected after 96 hours and anti-Ova responses were analyzed. Figure 2(D)(1)-(3) Anti-Ova responses in bone marrow cells - Figure 2(D)(1) IgG, Figure 2(D)(2) IgG1, Figure 2(D)(3) IgG2a. This result suggests that the response was not only local and systemic, but also induced a memory response so that the individual was better prepared for future exposure to the same antigen. Figure 2(E)(1)-(4) shows the mucosal immune response. On day 56, feces, nasal washes and lung washes were collected from vaccinated mice and mice treated with floss only. Anti-OVA Figure 2(E)(1) Fecal IgG (1:5 dilution), Figure 2(E)(2) Fecal IgA (1:5 dilution), Figure 2(E)(3) Nasal wash IgG (undiluted) and Figure 2(E)(4) Lung wash IgG (undiluted).Figure 2(F)(1)-(2) No significant amount of IgE was detected in either (1) serum or (2) bone marrow of mice vaccinated via floss, indicating that the target site, JE, does not sensitize individuals to the delivered Ag. Figure 2(G) Vaccinated mice were euthanized and splenocytes were harvested. Cells were cultured in triplicate at a concentration of 1 × 106 cells per well in RPMI medium supplemented with 10% fetal bovine serum and penicillin-streptomycin antibiotics and restimulated with Ova (200 μg / ml). Cultured cell supernatants were collected after 96 hours and analyzed for cytokine values. Figure 2(G) shows cytokine values ​​in splenocyte cultures, with Figure 2(G)(1) showing IFN-gamma and Figure 2(G)(2) showing IL-4. Data are expressed as mean ± SD. One-way ANOVA test was used to compare groups with different serum dilutions. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. [Diagram 3] Figure 3A-3C show floss for influenza vaccination. Figure 3(A) Vaccination schedule: Balb / c mice (n=10) were vaccinated with 10 μg or 25 μg of inactivated (Inac., inactivated) virus coated on floss, once each on days 0, 14, and 28, for a total of three times. Blood was collected from the mice on day 56, and anti-inactivated virus immune responses (1:800 or 1:50 dilution) were analyzed by ELISA. Figure 3(B)(1)-(3) Serum anti-inactivated virus Figure 3(B)(1) IgG, Figure 3(B)(2) IgG1, Figure 3(B)(3) IgG2a antibody responses on day 56. Virus challenge: On day 56, mice were challenged with 3×LD50 (50% lethal dose) of A / PR / 8 / 34 (H1N1) influenza virus. Individual mouse samples were used for analysis. Data are expressed as mean ± SD. Comparisons between groups were made using one-way ANOVA test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Figure 3(C)(1)-(2) The mice were observed daily for changes in body weight and severity of infection. Figure 3(C)(1) Percentage of body weight change, Figure 3(C)(2) Percentage survival rate after infection of vaccinated mice. n=5 mice in each group. [Figure 4-1] ~ [Figure 4-2] Figure 4A-4D show the delivery of the vaccine M2e-AuNP+CpG (MAC) via floss, a vaccine formulation consisting of gold nanoparticles (AuNP's) conjugated peptide (M2e) plus adjuvant (CpG), and characterization of the immune response. Figure 4(A)(1) shows a real micrograph of floss coated with M2e-AuNP+CpG, containing 56 μg AuNP's, 8.1 μg M2e, and 20 μg CpG (one dose), and Figure 4(A)(2) shows the flossing procedure of mice. Figure 4(B) Vaccination schedule: Balb / c mice (n=10) were vaccinated by flossing the gums with floss coated with the vaccine formulation [M2e-AuNP+CpG (MAC)] or by placing the vaccine formulation [M2e-AuNP+CpG (MAC)] under the tongue [sublingual immunotherapy (SLIT)]. Vaccine formulations (MAC) coated on floss or delivered via SLIT consisted of 56 μg AuNP's, 8.1 μg M2e and 20 μg CpG (single-stranded oligodeoxynucleotide adjuvant) and were used to vaccinate mice on days 0 and 21. Naive mice that did not receive treatment were treated as controls. Systemic immune response: Blood was collected from mice on days 21 and 42 and serum anti-M2e antibody responses (1:6400 dilution) were analyzed by enzyme-linked immunosorbent assay (ELISA). Figure 4C(1)-(3) Serum anti-M2e antibody responses on day 42 - Figure 4C(1) IgG, Figure 4C(2) IgG1 and Figure 4C(3) IgG2a. Individual mouse sera were used for analysis. Data are expressed as mean ± SD. Comparisons between groups were made using one-way ANOVA test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Virus challenge. (Fig. 4D(1)-(2)) On day 43, mice were challenged with 3×LD50 (50% lethal dose) of A / California / 07 / 2009 H1N1 virus. Mice were observed daily for weight change and severity of infection. Fig. 4D(1) Percentage of weight change, Fig. 4D(2) Percentage survival rate after infection of vaccinated mice. n=5 mice in each group. [Figure 5-1] ~ [Figure 5-2]Figures 5A-5E show the characterization of vaccine delivery and immune response via floss. (Figure 5A) Vaccination schedule: Balb / c mice (n=5) were vaccinated by flossing the gums with antigen (peanut extract (PE))-deposited floss. The floss was deposited with 25μg PE + / - 25μg CpG (single-stranded oligodeoxynucleotide adjuvant) and mice were vaccinated weekly for a total of up to 4 weeks. Mice treated with floss without any coating or deposit served as control. Systemic immune response: Mice were bled on days 28 and 56 and serum anti-PE antibody responses (1:12500 dilution) were analyzed by enzyme-linked immunosorbent assay (ELISA). Figures 5B(1)-(3) Serum anti-PE antibody responses on day 56 - Figure 5B(1) IgG, Figure 5B(2) IgG1 and Figure 5B(3) IgG2a. Individual mouse sera were used for analysis. (Figure 5C(1)-(3)) Memory immune response: Vaccinated mice were euthanized and bone marrow cells were harvested. Cells were cultured in triplicate at a concentration of 1 × 106 cells per well using RPMI medium supplemented with 10% fetal bovine serum and penicillin-streptomycin antibiotics. The supernatants of cultured cells were collected after 96 hours and anti-PE responses were analyzed. Anti-PE Figure 5C(1) IgG, Figure 5C(2) IgG1, Figure 5C(3) IgG2a. This result suggests that the response was not only local and systemic, but also that memory responses could be induced to better prepare individuals for future exposure to the same antigen (Ag). (Figure 5D) Mucosal immune response. On day 56, feces, nasal washes and lung washes were collected from vaccinated and naive mice. Anti-PE Figure 5D(1) Fecal IgG (1:5 dilution), Figure 5D(2) Fecal IgA (1:5 dilution), Figure 5D(3) Nasal wash IgG (undiluted), and Figure 5D(4) Lung wash IgG (undiluted). (Figure 5E(1)-(2)) No significant amount of IgE was detected in either Figure 5E(1) serum or Figure 5E(2) bone marrow of mice vaccinated via floss, indicating that the target site, the junctional epithelium, does not sensitize individuals to the delivered Ag. Data are presented as mean ± SD. One-way ANOVA test was used to compare groups with different serum dilutions.*p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. [Figure 6-1] ~ [Figure 6-2]Figure 6A-6D show the schedule of peanut allergen immunotherapy. (Figure 6A) Immunotherapy schedule: Balb / c mice (n=5) were sensitized by oral route [peanut extract (PE) 1mg + cholera toxin (CT) 15μg] for 5 consecutive weeks with 1 week interval. Mice were then vaccinated by flossing with antigen-coated floss. The floss was coated with PE 5μg + / - CpG (single-stranded oligodeoxynucleotide adjuvant) 5μg, and mice were vaccinated 3 times a week for a total of up to 3 weeks. Mice that did not receive any treatment after sensitization served as control (untreated). Blood was collected from mice 10 days after vaccination (PV). Eight weeks after vaccination, mice were challenged with PE allergen (500μg) by intraperitoneal route (IP), and then mice were euthanized and various tissues were collected. (Fig. 6B(1)-(3)) Serum anti-PE antibodies (1:12500 dilution) were confirmed by enzyme-linked immunosorbent assay (ELISA). Anti-PE 6B(1) IgG, Fig. 6B(2) IgG1 and Fig. 6B(3) IgG2a antibody responses 10 days after vaccination. Individual mouse sera were used for analysis. Data are presented as mean ± SD. One-way ANOVA test was used to compare between groups with different serum dilutions. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 and ns: not significant. PE induced anaphylaxis. (Fig. 6C)(1) Plasma MCPT-1 values ​​after IP challenge with PE. Histological analysis of intestinal tissue. (Fig. 6D) Eight weeks after vaccination, mice were challenged with PE allergen (500 μg) via intraperitoneal route. Mice were then euthanized and the small intestine was harvested from the proximal, mid and distal ends, fixed, dehydrated and embedded in paraffin wax for sectioning. Tissue sections were stained with hematoxylin and eosin (H&E) staining and sectioned for histology. Figure 6D(1) Number of eosinophils counted in each section obtained from mice in different treatment groups. Figure 6D(2) Brightfield image of H&E stained intestine with arrows indicating eosinophil infiltration.Individual mouse samples were used for analysis. Data are presented as mean ± SD. One-way ANOVA was used for comparison between groups. *p<0.05, **p<0.01, ***p<0.001 and ns: no significant difference. [Figure 7-1] ~ [Figure 7-2]Figures 7A-7D show airway allergen immunotherapy. (Figure 7A) Immunotherapy schedule: Balb / c mice (n=5) were sensitized by two intraperitoneal (IP) injections (Ova 25μg + alum 2mg (adjuvant)) with a one-week interval. Ten days after sensitization (PS), mice were challenged with Ova (50μg) via the intranasal route (IN) for three consecutive days to develop airway inflammation. Mice were then vaccinated by flossing with antigen-deposited floss. The floss was coated with Ova 25μg + / - CpG (single-stranded oligodeoxynucleotide adjuvant) 25μg, and mice were vaccinated three times a week for a total of up to three weeks. Mice that did not receive any treatment after sensitization served as controls (untreated). Blood was collected from mice 10 days after vaccination. On the 28th day after vaccination, mice were challenged with Ova allergen (50 μg) by intranasal route (IN) for three consecutive days, then mice were euthanized and various tissues were collected. Systemic immune response: (Figure 7B(1)-(4)) Serum anti-Ova Figure 7B(1) IgG, Figure 7B(2) IgG1, Figure 7B(3) IgG2a and Figure 7B(4) IgE antibody responses (1:12500 or 1:500 or 1:20 dilution) on the 10th day after vaccination analyzed by enzyme-linked immunosorbent assay (ELISA). (Figure 7C) Lung lavage analysis after challenge. Mice were then euthanized and mucosal secretions of lung lavage were collected. Figure 7C(1) Eosinophils and Figure 7C(2) Neutrophils cell counts in lung lavage - cells were counted by staining with Diff-stain kit and observing the cells under confocal microscope. Lung histological analysis: (Figure 7D) Mice were then euthanized and lungs were removed, fixed, washed, and sectioned for histology. Tissue sections were stained with periodic acid-Schiff (PAS) to stain mucus deposits or trichrome blue (TCB) to stain collagen deposits. Representative bright-field images of PAS-stained lungs (upper panels) and TCB-stained lungs (lower panels). Arrows in the upper panels point to mucus deposits and arrows in the lower panels point to collagen deposits. [Figure 8]FIG. 8 shows the deposition capabilities of peptides, nanoparticles, proteins, oligonucleotides, microparticles deposited on floss in different patterns, either as short or longer lengths, a single area deposition or multiple distinct areas deposition on only one side of the floss. [Figure 9] FIG. 9 shows the deposition potential of deposited water-soluble and water-insoluble materials including pollen grain particles. [Figure 10] Figure 10 shows the deposition potential and different deposition patterns of two different compounds as examples. One formulation is NHS-Rhodamine (fluorescent reagent) conjugated ovalbumin (protein) in water (referred to as "A"), and the second is gold nanoparticles and CpG (single-stranded DNA) conjugated M2e peptide in water (referred to as "B"). [Figure 11] FIG. 11 illustrates the deposition capabilities of multiple substances, here showing four different food colorings (blue, green, yellow, and red) deposited as four distinct moieties. [Figure 12] FIG. 12 shows the ability to coat both sides of the floss with different formulations. [Figure 13] FIG. 13 shows an example of an automatic coating device for coating the floss. [Figure 14] Figure 14 shows two examples of flosser system designs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Although the making and using of various embodiments of the invention are discussed in detail below, it should be understood that the invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not limit the scope of the invention.

[0019] To facilitate understanding of the present invention, a number of terms are defined below. Terms defined herein have meanings commonly understood by one of ordinary skill in the art to which the present invention pertains. Terms such as "a," "an," and "the" are not intended to refer to only one thing, but include general classes for which specific examples may be used for illustration. While the terms used herein are used to describe particular embodiments of the present invention, their usage is not intended to limit the invention, except as outlined in the claims.

[0020] U.S. Patent No. 9,271,899 (the '899 patent) is entitled "Methods, Articles and Kits for Allergy Hyposensitization via Oral Mucosa" and alleges administering allergy immunotherapy to an individual by targeting areas of the oral mucosa with a high ratio of dendritic cells to mast cells, particularly the vestibular mucosa. Specifically, the first paragraph of the description of the invention states, "The present invention relates to allergy immunotherapy targeting areas of the oral mucosa, such as areas with a high ratio of dendritic cells to mast cells, particularly the vestibular mucosa." The oral vestibule is a narrow slit-like portion of the mouth bounded by the gums and teeth on the inside and the cheeks and lips on the outside. While the patent focuses specifically on the vestibular mucosa, it notes that allergens may come into contact with other oral mucosal sites such as the gums / cheeks. The '899 patent alleges the need to provide a method to enhance delivery of allergens to desired ratios of dendritic cells to mast cells, specifically areas of the oral mucosa with a high ratio of dendritic cells to mast cells, and maximize contact time between the allergen and the oral vestibular tissue. Methods of allergen delivery include toothpaste, pouches, dental creams, mouthwashes, mouth sprays, and the like. The '899 patent discusses formulations containing between 1 picogram and 15 mg of allergen protein that can be administered via a pouch or other dental product. The '899 patent discusses making the formulation in the form of a toothpaste that contains the allergen. The '899 patent describes an example in which 2 grams of toothpaste may contain 1-10% allergen. Thus, according to this method, an individual would receive 2-200 mg of allergen. On the other hand, while the '899 patent asserts that a high ratio of dendritic cells to mast cells is important and suggests that the vestibular mucosa is one such desirable mucosa, the method described in the '899 patent does not teach how these mucosae, specifically the vestibular mucosa, are primarily targeted, nor does it mention the dose that would reach the cells of the vestibular tissue.

[0021] In contrast, the technology of the present invention specifically targets the junctional epithelium (JE) and delivers picograms to micrograms of allergen / vaccine molecules to the JE to generate an immune response. The JE is located at the bottom of the deepest recess of the gingival sulcus (sometimes also called the gingival sulcus or gingival groove or gum pocket). The JE is not freely exposed, but is attached to the hard tooth surface on one side and to the underlying soft connective tissue on the other side. In this way, the JE wraps around the tooth, forming an attachment band. The cells of the JE are not keratinized and have wide intercellular spaces. This wide intercellular space of the JE gives the JE a unique property of high permeability that is not found in other parts of the oral mucosa, including the cheek, lips, attached gingiva and vestibular mucosa. This degree of high permeability of the JE is even higher than that of the sublingual mucosa, which is currently considered to be the most permeable oral mucosal site. The inventors of the present invention recognized this uniqueness of JE and show herein that microgram amounts of antigen molecules were able to rapidly penetrate the JE and induce a robust immune response in mice. Thus, the present technology targets the JE because it is highly permeable, allowing efficient uptake of molecules into the underlying tissues, which helps generate a robust immune response. The present invention also directly compares immunogen (vaccine / allergen) targeting to the junctions with sublingual immunotherapy (SLIT) and demonstrates a significantly higher immune response by targeting the junctional epithelium compared to SLIT. In contrast, the '899 patent does not focus on permeability, and in fact, the permeability of the vestibular mucosa is lower than that of JE. The '899 patent further discusses the use of floss to deliver allergens. The '899 patent states that the allergen can be embedded in a coating layer or can be directly coated on the floss as a coating layer. The allergen is then detached or released from the floss, delivering the antigenic material to the vestibular mucosa. However, the '899 patent is silent as to where the allergens delivered by the floss are delivered.Furthermore, the '899 patent does not teach the junctional epithelium or its inherently high permeability, nor does the '899 patent speak of vaccination. In contrast, the present invention targets the junctional epithelium of the gingival sulcus, an area that has fewer dendritic cells than the vestibular mucosa, which is contrary to the teachings of the '899 patent. Dendritic cell numbers in human oral mucosa have been documented to be lower in the gingiva compared to vestibular, buccal, palatal and lingual tissues. Indeed, the highest numbers of LCs were found in vestibular, buccal, palatal and lingual tissues, while lower numbers were observed in the sublingual region and gingiva (Reference: "Dendritic cells of the oral mucosa", AH Hovav, Mucosal Immunology, 7, 27-37, 2014). Allam et al. Allergy 2008: 63: 720-727, a reference from the above patent on which the teachings of the '899 patent are based, also confirms that the gingiva has the highest number of mast cells, and therefore, according to the teachings of the '899 patent, the junctional epithelium would be less preferred / convenient for allergen immunotherapy. Furthermore, the present invention does not target the vestibular mucosa.

[0022] Contrary to the conventional wisdom that merely placing molecules on the mucosal surface of the oral mucosa does not lead to efficient immunomodulation, the present invention demonstrates that placing a substance on the junctional epithelium actually achieves a strong immunomodulatory response. No additional approach is required to weaken or destroy the mucosal barrier of the junctional epithelium, and the mere placement of small molecules such as deoxyribonucleic acid (DNA) or large molecules such as proteins and even nanoparticles and viruses on the junctional epithelium can result in a strong immune response. In fact, the junctional epithelium is rich in lymphatic vessels.

[0023] The present invention aims to administer antigens and allergens through the junctional epithelium of the gingival sulcus to obtain a strong systemic and mucosal immune response. Because the junctional epithelium is only 2 mm long and the gingival sulcus is 1-2 mm deep, we deposited antigens and / or allergens on dental floss to target and deposit them in the gingival sulcus for uptake via the junctional epithelium. Although we used floss to target the junctional epithelium, other approaches that can target the junctional epithelium can also be used. For example, a thin flat surface of similar dimensions to the gingival sulcus can be used. This flat surface can be coated with a substance that can be used to induce immune modulation, or a substance can be encapsulated on this flat surface. Using a mouse model, we have shown that floss can be coated with antigen / allergen solution, shown that mouse teeth can be flossed, and that this method is an effective form of antigen / allergen delivery. This new approach serves as a non-invasive, painless, and simple method of administering allergens and antigens for immune modulation. In the case of allergies, the immunization of the present invention serves to attenuate the allergic immune response and / or induce a protective immune response against allergens. Conversely, the present invention can also be used to generate an immune response against infectious diseases and other pathogens.

[0024] The present invention can be used with dental flosses known in the art. For example, dental flosses can be made as nylon dental flosses, where nylon is polymerized into a polymer that is then formed, pumped or extruded to form monofilaments or multifilaments. The polymer is cured and the monofilaments or multifilaments are combined to form one or more strands of dental floss. Dental flosses can be made from polytetrafluoroethylene (PTFE, polytetrafluoroethylene or TEFLON®), polypropylene, polyethylene, styrene-butadiene copolymers, and combinations thereof. After the polymer is formed, it can be melted and extruded into thin strands. See, for example, U.S. Patent No. 6,270,890, the relevant portions of which are incorporated herein by reference.

[0025] In one non-limiting example, nylon or PTFE is mixed with a basic amino acid (or salt thereof) and formed or extruded to form one or more filaments. In the case of multifilaments, these are typically twisted together to form dental floss. Alternatively, a single elongated string of floss such as PTFE can be formed. Denier of dental floss is often about 450 to about 1350, and in other examples, denier of floss is about 100 to about 900.

[0026] The dental floss is then deposited with the immunogens and / or allergens and / or antigens of the present invention, as known to those skilled in the art. For example, the dental floss is treated in a bath containing the antigens and / or allergens. The bath can contain one or more waxes that are attached to the floss, thereby causing the antigens and / or allergens to adhere to the floss. In one example, dental floss containing nylon or PTFE fibers is coated with antigens and / or allergens. Waxes or polymers, such as polyvinyl alcohol, polyvinyl acetate, etc., can be used to coat the antigens and / or allergens in, on, or around the dental floss. See, for example, U.S. Patent No. 6,289,904, the relevant portions of which are incorporated herein by reference.

[0027] In the case of filamentous dental floss, the antigens and / or allergens can be embedded in a bundle of thin filaments, such as nylon filaments, before, during, or even after the bundle is formed. The bundle may then also be optionally coated with a wax or polymer. The number of filaments can be about 2 to about 500, for example, about 2 to about 250, depending on the denier of the dental floss filaments. Often, the floss is formed by twisting and twisting the dental floss filaments about 1 to 5 turns per inch. The twisting provides integrity when the dental floss is placed on a spool and / or during subsequent handling. In immunization, the filaments of the dental floss unfold and spread over the tooth surface at the gingival junctional epithelium, thereby delivering the antigen and / or allergen immunization. The floss may also be formed from interlocking fibers. The dental floss product is preferably thick enough to fit between the teeth as well as reach the gingival junctional epithelium. When multifilaments are used, coatings can be applied before and / or after twisting, and usually after application of the antigens and / or allergens. Other additives can be applied to the dental floss to preserve the antigens and / or allergens, or to aid in the coating process, or to achieve sustained release of the antigens and / or allergens.

[0028] In addition, the dental floss may be coated with a liquid or solid flavoring. The flavoring may be spray dried in liquid or solid form. If a liquid flavoring is applied, the floss is typically dried before being wound onto a spool, either by air drying or by drying until hot, after which the floss is wound onto a spool.

[0029] As used herein, the term "antigen" refers to a molecule capable of initiating a humoral and / or cellular immune response in a recipient of the antigen. Antigens may be used in different contexts in the present invention, for example, but not limited to, as agents that generate an immune response to prevent or treat a disease or condition for which vaccination is an advantageous treatment, and / or as agents that anergize the immune response, i.e., the agent reduces the activation level of activated immune cells, and / or as agents that modulate the immune response to achieve a beneficial therapeutic effect in a subject. Antigens include any type of biomolecule, including, for example, simple intermediate metabolites, sugars, lipids, and hormones, as well as macromolecules such as peptides, polypeptides, complex carbohydrates, phospholipids, nucleic acids, and / or glycoproteins, or combinations thereof. Common categories of antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoan and other parasitic antigens, tumor antigens, and conversely, antigens involved in autoimmune diseases, allergies, and graft rejection, as well as various other antigens.

[0030] Examples of viral antigens disclosed herein include retroviral antigens, such as retroviral antigens from human immunodeficiency virus (HIV), such as gene products of the gag, pol, and env genes, Nef protein, reverse transcriptase, and other HIV components; coronavirus antigens, such as spike protein, nucleoprotein, and messenger RNA (mRNA); hepatitis virus antigens, such as the S, M, and L proteins of hepatitis B virus, pre-S antigen of hepatitis B virus, and other hepatitis, e.g., hepatitis A, B, and C, viral components, such as hepatitis C virus RNA; influenza virus antigens, such as hemagglutinin and neuraminidase, and other influenza virus components; measles virus antigens and other measles virus components, such as measles virus fusion protein; rubella virus antigens and other rubella virus components, such as proteins E1 and E2; rotavirus antigens and other rotavirus components; cytomegalovirus antigens, such as envelope glycoprotein B, and other cytomegalovirus antigen components; RSV Respiratory syncytial virus antigens such as fusion protein, M2 protein, and other respiratory syncytial virus antigen components; Herpes simplex virus antigens such as immediate early protein, glycoprotein D, and other herpes simplex virus antigen components; Varicella zoster virus antigen components such as gpI, gpII, and other varicella zoster virus antigen components; Japanese encephalitis virus antigens such as E, ME, ME-NS1, NS1, NS1-NS2A, 80%E protein, and other Japanese encephalitis virus antigen components; Rabies virus antigens such as rabies glycoprotein, rabies nucleoprotein, and other rabies virus antigen components, West Nile virus; Yellow fever; Tularemia; Hepatitis (viral; bacterial); Respiratory syncytial virus (RSV, respiratory syncytial virus); HPIV1 and HPIV3; adenovirus; smallpox, although for examples of additional viral antigens see Fundamental Virology, Second Edition, eds. Fields, BN and Knipe, DM, the relevant portions of which are incorporated herein by reference.(Raven Press, New York, 1991).

[0031] Other examples of antigens include picornaviruses, coronaviruses, togaviruses, flaviviruses, rhabdoviruses, paramyxoviruses, orthomyxoviruses, bunyaviruses, arenaviruses, reoviruses, retroviruses, papillomaviruses, parvoviruses, herpesviruses, poxviruses, hepadnaviruses, sponge viruses, influenza, herpes simplex virus types 1 and 2, measles, dengue, smallpox, polio, or HIV, in whole, heat-killed, or in part. Other antigens may be directed against pathogens such as trypanosomes, tapeworms, roundworms, helminths, malaria, etc. Specific examples of organisms, allergens, and nucleic acid and amino sequences used in vectors and ultimately as antigens in the present invention can be found in U.S. Pat. No. 6,541,011, the relevant portions of which are incorporated herein by reference, in particular in the table of organisms and specific sequences that may be used in the present invention.

[0032] Examples of bacterial antigens disclosed herein include, for example, pertussis toxin, filamentous hemagglutinin, pertactin, adenylate cyclase, and other pertussis bacterial antigen components; diphtheria bacterial antigens, such as diphtheria toxin or toxoid, and other diphtheria bacterial antigen components; tetanus bacterial antigens, such as tetanus toxin or toxoid, and other tetanus bacterial antigen components; streptococcal antigens, such as M protein, and other streptococcal antigen components; gram-negative bacillus bacterial antigens, such as lipopolysaccharide, and other gram-negative bacterial antigen components; Mycobacterium tuberculosis bacterial antigens, such as mycolic acid, heat shock protein 65 (HSP65), 30 kDa major secretory protein, antigen 85A, and other tuberculosis antigen components; Helicobacter pylori pylori antigenic components; pneumococcal bacterial antigens such as pneumolysin, pneumococcal capsular polysaccharides and other pneumococcal bacterial antigenic components; Haemophilus influenza bacterial antigens such as capsular polysaccharides and other Haemophilus influenza bacterial antigenic components; Bacillus anthracis antigens such as Bacillus anthracis protective antigen and other Bacillus anthracis antigenic components; and bacterial antigens such as Rickettsia bacterial antigens such as rompA and other Rickettsia bacterial antigenic components. Bacterial antigens as referred to herein include any other bacterial antigen such as Neisseria meningitidis; Streptococcus pneumoniae; Neisseria gonorrhoeae; Salmonella serovar Typhi; Shigella; Vibrio cholerae; Dengue fever; Encephalitis; Japanese encephalitis; Lyme disease; Yersinia pestis; mycobacterial antigens, mycoplasmal antigens, rickettsial antigens or chlamydial antigens.

[0033] Examples of fungal antigens for use in the present invention include, but are not limited to, Candida fungal antigen components; Histoplasma fungal antigens such as heat shock protein 60 (HSP60) and other Histoplasma fungal antigen components; Cryptococcus fungal antigens and other Cryptococcus fungal antigen components such as capsular polysaccharides; Coccidioides fungal antigens such as spherule antigens and other Coccidioides fungal antigen components; and Trichophyton antigens such as trichophytin and other Coccidioides fungal antigen components.

[0034] Examples of protozoan antigens and other parasitic antigens for use in the present invention include, but are not limited to, Plasmodium falciparum antigens and other Plasmodium antigen components, such as merozoite surface antigens, sporozoite surface antigens, circumsporozoite antigens, gametocyte / gamete surface antigens, blood stage antigen pf155 / RESA; Toxoplasma antigens and other Toxoplasma antigen components, such as SAG-1 and p30; Schistosoma antigens and other Schistosoma antigen components, such as glutathione-S-transferase and paramyosin; Leishmania major and other Leishmania antigens and other Leishmania antigen components, such as gp63, lipophosphoglycan and its associated proteins; and Trypanosoma cruzi antigens and other Trypanosoma antigen components, such as 75-77 kDa antigen and 56 kDa antigen.

[0035] Examples of tumor antigens used in the present invention include CEA, prostate specific antigen (PSA), HER-2 / neu, BAGE, GAGE, MAGE1 to 4, 6 and 12, MUC (Mucin) (e.g., MUC-1, MUC-2, etc.), GM2 and GD2 gangliosides, ras, myc, tyrosinase, MART (melanoma antigen), Pmel17 (gp100), GnT-V intron V sequence (N-acetylglucoaminyltransferase V intron V sequence), prostate Ca psm, PRAME (melanoma antigen), beta-catenin, MUM-1-B (melanoma ubiquitous mutant gene product), GAGE ​​(melanoma antigen) 1, BAGE (melanoma antigen) 2 to 10, c-ERB2 (Her2 / neu), Epstein-Barr Virus nuclear antigen (EBNA, Epstein-Barr Virus nuclear antigen), and the like. Examples of immunogenic molecules include, but are not limited to, autoantigens 1-6, gp75, human papilloma virus (HPV) E6 and E7, p53, lung resistance protein (LRP), Bcl-2, and Ki-67. In addition, the immunogenic molecule may be an autoantigen involved in the initiation and / or propagation of an autoimmune disease, the pathology of which is primarily due to the activity of antibodies specific for molecules such as CII, SLE, or MG expressed by the relevant target organ, tissue, or cell. In such diseases, it may be desirable to redirect an ongoing antibody-mediated (i.e., Th1 / Th17-type) immune response to the relevant autoantigen toward a cellular (i.e., Th2-type) immune response. Alternatively, it may be desirable to prevent the onset of, or reduce the level of, a Th1 / 17 response to an autoantigen by prophylactically inducing a Th2 response to the appropriate autoantigen in a subject who does not have, but is suspected of being susceptible to, the relevant autoimmune disease. Autoantigens that may be used include, but are not limited to, (a) for SLE, Smith protein, RNP ribonucleoprotein, and SS-A and SS-B proteins; and (b) for MG, acetylcholine receptor.Examples of other antigens involved in one or more autoimmune responses include, for example, type II collagen proteins / peptides, myelin oligodendrocyte glycoprotein (MOG), endogenous hormones such as luteinizing hormone, follicle-stimulating hormone, testosterone, growth hormone, prolactin, and other hormones.

[0036] Examples of antigens involved in autoimmune diseases, allergies, and graft rejection that can be used in the present invention include, for example, diabetes, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, and psoriatic arthritis), multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjogren's syndrome including dry keratoconjunctivitis secondary to Sjogren's syndrome, alopecia areata, allergic responses due to arthropod bite reactions, Crohn's disease, aphthous ulcers, iritis, conjunctivitis, keratoconjunctivitis, and ulcerative colitis. , asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug rash, leprosy reversal reaction, leprosy nodosum erythema, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Crohn's disease, Graves' ophthalmopathy, sarcoidosis, primary biliary cirrhosis, posterior uveitis, and interstitial pulmonary fibrosis.Examples of antigens involved in autoimmune diseases include type II collagen, type II collagen peptide (CII250-270), proteoglycan, citrullinated peptide antigen, vimentin, fibrinogen, α-enolase, peptidylarginine deiminase-4, insulin, islet antigen 2 (IA2), zinc transporter 8 (ZnT8), islet specific glucose-6-phosphatase catalytic subunit related protein (IGRP), chromogranin A (ChgA), islet amyloid polypeptide (IAPP), glutamic acid decarboxylase 65 (GAD65), natural DNA, myelin basic protein, myelin proteolipid protein, acetylcholine receptor components, thyroglobulin, and thyroid stimulating hormone (TSH) receptor. Examples of antigens involved in allergies include pollen antigens such as cedar pollen antigen, ragweed pollen antigen, and ryegrass pollen antigen, insect-derived antigens such as house dust mites (i.e., Der p1, Der p2, LTN-DP2-1, LTN-DPE-1), animal-derived antigens such as cockroach antigen (i.e., Bla g2), cat antigen (i.e., Fel d1), and dog antigen (i.e., Can f1), histocompatibility antigens, peanut antigens (Ar1 h1, Ara h2, Ara h3, Ara h6), milk antigens (i.e., Bos d11, Bos d4, Bos d6, Bos d8), egg proteins (i.e., Gal d2, Gal d3, Gal d4), shrimp antigens (i.e., tropomyosin), nuts (i.e., hazelnut Cor a 9, almond Pru du6), legumes (i.e., soybean Gly m6), antibiotics such as penicillin and cephalosporin, and other therapeutic drugs (insulin, epinephrine, etc.).Examples of antigens involved in transplant rejection include antigenic components of the graft transplanted into the transplant recipient, such as heart, lung, liver, pancreas, kidney and nerve graft components. The antigen may be a modified peptide ligand useful for treating autoimmune diseases. The antigen may be a crude or purified extract from allergy-causing substances, such as respiratory allergens (pollen, dust mites, insects, etc. and others), food allergens (peanuts, cashews, walnuts, soybeans, shellfish, etc. and others), venoms (bee venom, etc.) and other allergens.

[0037] As used herein, the terms "deposit," "depot," and "deposition" refer to the active agent being disposed on the floss in the form of one or more deposits spaced apart from adjacent deposits.

[0038] As used herein, the term "epitope" refers to a peptide or protein antigen and / or immunogenic allergen that contains a primary, secondary or tertiary structure similar to an epitope located in any of the multiple pathogen polypeptides encoded by the pathogen's DNA or RNA.

[0039] Antigens and / or epitopes are not limited to peptides, proteins and portions thereof, but may include genes, plasmids, vectors (viral, bacterial and non-viral), DNA, RNA, CRISPR molecules, mRNA, siRNA or other nucleotides, either individually or in combination. Pharmaceutically acceptable carriers and formulations may be used to stabilize these molecules, enhance the function of these molecules, or provide sustained release.

[0040] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier that causes no adverse effects to the subject to which it is administered (e.g., humans and pets, such as dogs, cats, cows, pigs or other farm animals, as well as non-domesticated animals, etc.). Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, dimethyl sulfoxide, and the like, and combinations thereof. In addition, if desired, the shot / vaccine can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and / or adjuvants which enhance the effectiveness of the vaccine.

[0041] Non-limiting examples of adjuvants that may be effective include, but are not limited to, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, MTP-PE, and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate, and cell wall skeleton, for example in a 2% squalene / Tween 80 emulsion. STING agonists (e.g., 2'3'-cGAMP, c-di-AMP, 2'3'-c-di-AM(PS)2 (Rp,RP), c-di-GMP, CL401, CL413, CL429, Flagellin, Imiquimod, LPS-EB, MPLA, ODN 1585, ODN 1826, ODN2006, ODN2395, ODN 1018, pam3CSK4, poly(I:C), R848, TDB). Other examples of adjuvants include dimethyldioctadecylammonium bromide (DDA), complete and incomplete Freund's adjuvant, QuilA, natural polymers (i.e., poly-gamma-glutamic acid, chitosan, mannan, lipomannan, lentinan, dextran), synthetic polymers (i.e., poly-N-isopropylacrylamide, copolymers, block polymers, polyphosphazenes, polyelectrolytes, polyanhydrides, polymethacrylates, poly(lactic acid-co-glycolic acid), polycaprolactone, polyvinylpyrrolidone, cationic polymers). In addition, immunomodulators such as lymphokines (e.g., IFN-gamma, IL-2, and IL-12) or synthetic IFN-gamma inducers such as polyI:C can be used in combination with the adjuvants described herein.

[0042] As used herein, the term "subject" refers to humans, pets (such as dogs, cats, cows, sheep, goats, horses, rabbits or pigs) or other farm animals or non-domesticated animals such as deer, buffalo or wild horses.

[0043] The junctional epithelium is at the bottom of the gingival sulcus, which is 1-2 mm deep in healthy gums. Furthermore, the apical tissue of the cervical cavity tightly holds the tooth, so only thin instruments, less than 1 mm, and preferably less than 500 μm, can enter the cavity. Thus, administration of substances to the gingival sulcus is not straightforward. To overcome this challenge, the present invention uses antigens and / or allergens deposited on dental floss. Dental floss is used by millions of people every day to clean the gingival sulcus, and the present invention describes that dental floss can be coated with antigens / allergens for targeted deposition in the gingival sulcus for uptake via the junctional epithelium. Dental floss offers the added advantage of being non-invasive, painless, and can be self-administered in the comfort of one's home. Dental floss should be taken as a non-limiting example of a system with the end goal of delivering substances to the junctional epithelium. Other approaches based on the principle of enabling and allowing the device to enter the sulcus to help target the junctional epithelium, such as tapes, films, strips, strings, threads, sutures, gels, hydrogels, polymers, gums, particles, or combinations thereof, are included in the present invention. These systems and devices can be inserted into the sulcus, but rather than entering the sulcus, they can also be placed on the apical side of the sulcus, where the molecules of interest then diffuse from the systems and devices into the sulcus and eventually reach the junctional epithelium and penetrate the tissue. These systems placed on the apical side of the sulcus can be designed to maximize the diffusion of the molecules into the sulcus while minimizing the loss of the molecules out and throughout the oral cavity. In one such approach, the delivery system can be coated with an impermeable layer on the side facing the opposite side of the sulcus.

[0044] To coat the floss, the inventors developed a simple manual coating method using a pipette to apply the material onto the floss. They selected Oral-B® Glide Pro Health Original Floss among five types of floss after a preliminary coating feasibility study. Using this method, the inventors were able to coat the floss with a variety of molecules, including proteins, small molecules, peptides, nanoparticles, single-stranded DNA oligonucleotides, and influenza viruses. Next, the inventors established the feasibility of flossing the teeth of mice. The inventors chose to floss the lower anterior incisors because of their ease of access. Flossing was performed while the mice were under anesthesia. The figure shows the incisors before, during, and after flossing. Imaging with a fluorescent stereomicroscope confirmed that the coated fluorescent ovalbumin (Ova) entered the junctional epithelium and passed through it to enter the gingival tissue within 30 minutes. We determined the percentage of Ova delivered to the gingival sulcus by quantifying Ova coated on the floss (M1) and Ova remaining on the floss after flossing (M2). For n=4 mice, the delivery efficiency ([M1-M2] / M1×100) was approximately 75%.

[0045] Immune response generated when an antigen or allergen is administered to the junctional epithelium. We coated floss with Ova (25μg Ova + / - 25μg CpG), peanut extract protein (PE) (25μg PE + / - 25μg CpG) or inactivated influenza virus (A / PR / 8 / 34(H1N1)) (25 / 10μg PR8) and administered 5 doses of Ova and PE once a week and 3 doses of influenza virus every other week. Serum samples taken on day 56 (day 0 means the day of first administration) clearly showed a strong stimulation of systemic IgG response to Ova, PE and PR8, and fecal analysis showed a rise in mucosal IgA and IgG. The response with the use of CpG was significantly higher, especially IgG2a, indicating a clear adjuvant effect of CpG.

[0046] To determine whether administration to the junctional epithelium would induce IgE production that could be indicative of an allergic response, serum IgE antibodies specific to Ova and PE were not significant and were comparable to mice given floss alone (uncoated), suggesting that floss-based targeting of the junctional epithelium does not induce allergies.

[0047] Influenza vaccine administered to the junctional epithelium is protective. Mice were given inactivated PR8 as a vaccine and challenged with 3 x 50% lethal doses. The figure shows that the mice were protected and showed minimal weight loss.

[0048] The present invention can be used to determine the efficacy of floss containing peanut extract (PE) or Ova in treating mice sensitized to peanut as a food allergy model or Ova in an airway allergy model. In both allergy models, sublingual immunotherapy (SLIT) can be used as a positive control. Recently, the FDA has also approved sublingual tablets for pollen allergy SLIT. SLIT requires placing large amounts of allergen under the tongue because the epithelium under the tongue is not very permeable. For peanut immunotherapy, peanut-sensitized mice were given 5 μg PE without CpG (Floss:PE) or with 5 μg CpG (Floss:PE+CpG) 9 times over a period of 3 weeks. Two control groups were added: the first group was sensitized mice that did not receive treatment (untreated), and the second group was naive mice that received an oral peanut challenge (naive mice that received only challenge). Mice were challenged with 500 μg PE intraperitoneally to evaluate the efficacy of the treatment. Floss provided better desensitization than untreated mice, as shown by lower clinical scores of allergic symptoms, less mast cell degranulation as quantified by the MCPT-1 marker, and less eosinophil infiltration in the mice's intestinal tissue after challenge, requiring fewer administrations (9) and lower doses. As expected, untreated peanut-sensitized mice showed a significant increase in MCPT-1 and eosinophils in the intestinal tissue. Naive mice that were challenged only showed no abnormal measurements after challenge. Similarly, for the Ova airway allergy model, Ova-sensitized mice were administered 25 μg of OVA without CpG (Floss:Ova) or with 25 μg of CpG (Floss:Ova+CpG) nine times over a period of three weeks. A control group was added: sensitized mice that did not receive any treatment (Naive). Mice were challenged by intranasal administration of 50 μg of Ova three times per day. The floss group had fewer inflammatory cells (eosinophils and neutrophils) and less mucus in their lungs. The control, untreated group showed significant inflammatory cell and mucus production.Mucus, a hallmark of an airway allergic response, was lower in the floss group, suggesting that a lower dose stimulated a better response.

[0049] To gain insight into the cellular response, splenocytes from mice given Ova were restimulated in vitro with Ova (note: these mice were part of a vaccination study, not an airway allergy). Cytokine profiles showed that both TH1 and TH2 effector responses were generated in mice given Ova+CpG. Bone marrow cells from these same mice without restimulation produced Ova-specific IgG, as did mice given PE+CpG (a vaccine study, not a food / airway allergy study). This indicates that the response is systemic and not just local, suggesting the generation of a memory response, but further studies are needed to confirm this.

[0050] These studies demonstrate that floss can be coated and used to target the junctional epithelium to generate systemic and mucosal immune responses. This method of administration also protected mice from a lethal influenza virus challenge and demonstrated hyposensitization in mouse models of airway allergy and peanut food allergy.

[0051] We manually coated the floss with the antigen / allergen containing solution using a pipette tip. To increase the reproducibility of coating and improve delivery efficiency, an automated coating approach using a computer controlled linear stage and liquid ejection system can be used. Using a floss coating machine, a specific length of floss can be coated with any antigen or allergen by simply changing the coating liquid vial. Other coating options include dip coating or spray coating or inkjet printing or pipette based coating or cartridge printing or combinations of these. Coating may require excipients such as thickeners or surface tension reducing agents to improve coating and delivery efficiency. Moreover, trehalose and other substances known to protect molecules from drying forces can be used to improve the stability of the molecules. Coating with nanoparticles and microparticles is also possible, as they can be coated with virus particles and therefore potentially enhance the immune response, as shown herein. Delivery efficiency can be evaluated and imaging can be used to characterize the coating.

[0052] Develop a new paradigm for peanut allergen immunotherapy. The mouth is the first place where food comes into contact with the body, and chewed food particles may enter the gingival sulcus and then cross the junctional epithelium to enter the tissues. It is therefore not surprising that the gingival immune network may play a major role in maintaining tolerance. Indeed, a proof-of-concept study using coated floss for peanut allergen immunotherapy reduced sensitization. This approach allows for the rapid development of allergen immunotherapy for peanut and other food allergens. Floss can also be used for peanut allergen immunotherapy, for example, by targeting the junctional epithelium. The effects of peanut allergen dose, frequency of flossing, use of adjuvants, use of particles to enhance phagocytosis and antigen processing, and delayed release coatings will be studied from an immunotherapy perspective.

[0053] Administration into the gingival sulcus can only occur after tooth eruption, which in humans occurs at 6-12 months. Although the proposed paradigm may not become mainstream in pediatric vaccines until infants are about 1 year of age, the amplified immune responses generated by the new paradigm will certainly impact and inform vaccine development, aiding cancer and HIV vaccines, and providing better treatments for allergies that are treated later in life for safety, and autoimmune diseases that often manifest in older age.

[0054] Figures 1A-1F show the oral route of immunization: (Figure 1A) human mouth; (Figure 1B) structure of the gingival sulcus and junctional epithelium (JE); (Figure 1C) delivery of active agent to the junctional epithelium of the gingival sulcus and diffusion of active agent to the junctional epithelium and adjacent tissues over time; (Figure 1D) delivery of antigen molecules coated on floss and effect of flossing on mouse gingival tissue; and (Figure 1E) diffusion of rhodamine-conjugated ovalbumin (Ova) in gingival tissue. Flossing was performed by placing antigen-deposited floss around each incisor and flossing 10 times to deposit the coated antigen at the gingival margin. (Figure 1F) Delivery efficiency of floss coated with fluorescein isothiocyanate (FITC)-conjugated ovalbumin (Ova).

[0055] Targeting the junctional epithelium of the gingival sulcus for vaccination against infectious pathogens. EXAMPLES

[0056] Floss-mediated delivery of ovalbumin (Ova) to the junctional epithelium (JE) induces strong systemic and mucosal antibody responses in mice (from a vaccine perspective).

[0057] Figures 2A-G show the characterization of vaccine delivery and immune response via floss. (Figure 2A) (1) Stereomicroscope photograph of coated floss and (2) flossing procedure in mice. (Figure 2B) Vaccination schedule: Balb / c mice (n=5) were vaccinated by flossing the gums with floss deposited with antigen (ovalbumin (Ova), a model antigen). The floss contained deposits of 25 μg Ova + / - 25 μg CpG (single-stranded oligodeoxynucleotide adjuvant) and mice were vaccinated weekly for up to 4 weeks in total. Mice treated with floss without any coating served as control. Systemic immune response: Mice were bled on days 28 and 56 and serum anti-Ova antibody responses (1:12500 or 1:2500 dilution) were analyzed by enzyme-linked immunosorbent assay (ELISA). Figures 2(C)(1)-(3) Anti-Ova antibody responses on day 56 - Figure 2(C)(1) IgG, Figure 2(C)(2) IgG1 and Figure 2(C)(3) IgG2a. Individual mouse sera were used for analysis. Figure 2(D)(1)-(3) shows memory immune responses: Vaccinated mice were euthanized and bone marrow cells were harvested. Cells were cultured at 1 × 10 per well in RPMI medium supplemented with 10% fetal bovine serum and penicillin-streptomycin antibiotics. 6The cells were cultured in triplicate at different concentrations. The supernatants of the cultured cells were collected after 96 hours and the anti-Ova response was analyzed. Figure 2(D)(1)-(3) Anti-Ova response in bone marrow cells - Figure 2(D)(1) IgG, Figure 2(D)(2) IgG1, Figure 2(D)(3) IgG2a. This result suggests that the response was not only local and systemic, but also induced a memory response so that the individual could be better prepared for future exposure to the same antigen (Ag). Figure 2(E)(1)-(4) shows the mucosal immune response. On day 56, feces, nasal washes and lung washes were collected from vaccinated mice and mice treated with floss only. Anti-Ova Figure 2(E)(1) Fecal IgG (1:5 dilution), Figure 2(E)(2) Fecal IgA (1:5 dilution), Figure 2(E)(3) Nasal wash IgG (undiluted), and Figure 2(E)(4) Lung wash IgG (undiluted). Figure 2(F)(1)-(2) No significant amounts of IgE were detected in either (1) serum or (2) bone marrow of mice vaccinated via floss, indicating that the target site, JE, does not sensitize individuals to the delivered Ag. Figure 2(G) Vaccinated mice were euthanized and splenocytes were harvested. 1 × 10 per well in RPMI medium supplemented with 10% fetal bovine serum and penicillin-streptomycin antibiotics. 6 Cells were cultured in triplicate at 100x the concentration of 0.1% IFN-gamma and 0.1% IL-4 in 100% spleen cells and restimulated with Ova (200 μg / ml). Cultured cell supernatants were harvested after 96 hours and analyzed for cytokine levels. Figure 2(G) shows cytokine levels in splenocyte cultures, with Figure 2(G)(1) showing IFN-gamma and Figure 2(G)(2) showing IL-4. Data are presented as mean ± SD. One-way ANOVA test was used to compare groups with different serum dilutions. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. EXAMPLES

[0058] Floss-mediated delivery of inactivated (Inac.) influenza virus to the junctional epithelium (JE) induces potent systemic antibody responses and protects mice from lethal challenge.

[0059] Figures 3A-C show the floss deposited for influenza vaccination. Figure 3(A) Vaccination schedule: Balb / c mice (n=10) were vaccinated three times with 10 μg or 25 μg of inactivated (Inac.) virus coated on floss, once each on days 0, 14, and 28. Mice were bled on day 56 and anti-inactivated virus immune responses (1:800 or 1:50 dilution) were analyzed by ELISA. Figure 3(B)(1)-(3) Serum anti-inactivated virus Figure 3(B)(1) IgG, Figure 3(B)(2) IgG1, Figure 3(B)(3) IgG2a antibody responses on day 56. Virus challenge: On day 56, mice were vaccinated with 3×LD 50 (50% lethal dose) of A / PR / 8 / 34(H1N1) influenza virus. Individual mouse sera were used for analysis. Data are expressed as mean ± SD. Comparisons between groups were performed using one-way ANOVA test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Figure 3(C)(1)-(2) Mice were observed daily for changes in body weight and severity of infection. Figure 3(C)(1) Percentage of body weight change, Figure 3(C)(2) Percentage survival rate after infection of vaccinated mice. n=5 mice in each group. EXAMPLES

[0060] Floss-mediated delivery of a universal influenza vaccine based on M2e gold nanoparticle (AuNP) conjugates to the junctional epithelium (JE) induces potent systemic antibody responses and protects mice from lethal challenge.

[0061] Figure 4A-4D show the delivery of the vaccine, M2e-AuNP+CpG (MAC), a vaccine formulation consisting of gold nanoparticles (AuNP's) conjugated peptide (M2e) plus adjuvant (CpG), via floss and characterization of the immune response. Figure 4(A)(1) Actual micrograph of floss coated with M2e-AuNP+CpG containing 56 μg AuNP's, 8.1 μg M2e, and 20 μg CpG (one dose) and Figure 4(A)(2) Flossing procedure of mice. Figure 4(B) Vaccination schedule: Balb / c mice (n=10) were vaccinated by flossing the gums with floss coated with the vaccine formulation [M2e-AuNP+CpG (MAC)] or by placing the vaccine formulation [M2e-AuNP+CpG (MAC)] under the tongue [sublingual immunotherapy (SLIT)]. Vaccine formulations (MAC) coated on floss or delivered via SLIT consisted of 56 μg AuNP's, 8.1 μg M2e and 20 μg CpG (single-stranded oligodeoxynucleotide adjuvant) and were used to vaccinate mice on days 0 and 21. Naive mice that did not receive treatment were treated as controls. Systemic immune response: Blood was collected from mice on days 21 and 42 and serum anti-M2e antibody responses (1:6400 dilution) were analyzed by enzyme-linked immunosorbent assay (ELISA). Figure 4C(1)-(3) Serum anti-M2e antibody responses on day 42 - Figure 4C(1) IgG, Figure 4C(2) IgG1 and Figure 4C(3) IgG2a. Individual mouse sera were used for analysis. Data are expressed as mean ± SD. Comparisons between groups were made using one-way ANOVA test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Virus challenge. (Fig. 4D(1)-(2)) On day 43, mice were challenged with 3×LD 50 The mice were challenged with 100 mg of A / California / 07 / 2009 H1N1 virus (50% lethal dose). The mice were monitored daily for changes in body weight and severity of infection. Figure 4D(1) shows the percentage change in body weight, and Figure 4D(2) shows the percentage survival rate after infection of vaccinated mice. n=5 mice in each group.

[0062] Targeting the junctional epithelium of the gingival sulcus for allergen-specific immunotherapy. EXAMPLES

[0063] Delivery of peanut extract (PE) via floss to the junctional epithelium (JE) induces strong systemic and mucosal antibody responses in mice (from a vaccine perspective).

[0064] Figures 5A-5E show the characterization of vaccine delivery and immune response via floss. (Figure 5A) Vaccination schedule: Balb / c mice (n=5) were vaccinated by flossing the gums with antigen (peanut extract (PE))-deposited floss. The floss was deposited with 25μg PE + / - 25μg CpG (single-stranded oligodeoxynucleotide adjuvant) and mice were vaccinated weekly for a total of up to 4 weeks. Mice treated with floss without any coating or deposit served as control. Systemic immune response: Mice were bled on days 28 and 56 and serum anti-PE antibody responses (1:12500 dilution) were analyzed by enzyme-linked immunosorbent assay (ELISA). Figures 5B(1)-(3) Serum anti-PE antibody responses on day 56 - Figure 5B(1) IgG, Figure 5B(2) IgG1 and Figure 5B(3) IgG2a. Individual mouse sera were used for analysis. (Figure 5C(1)-(3)) Memory immune response: Vaccinated mice were euthanized and bone marrow cells were harvested. The cells were cultured at 1 × 10 per well in RPMI medium supplemented with 10% fetal bovine serum and penicillin-streptomycin antibiotics. 6The cells were cultured in triplicate at different concentrations. The supernatants of the cultured cells were collected after 96 hours and the anti-PE response was analyzed. Anti-PE Figure 5C(1) IgG, Figure 5C(2) IgG1, Figure 5C(3) IgG2a. This result suggests that the response was not only local and systemic, but also induced a memory response so that the individual could be better prepared for future exposure to the same antigen (Ag). (Figure 5D) Mucosal immune response. On day 56, feces, nasal washes and lung washes were collected from vaccinated and naive mice. Anti-PE Figure 5D(1) IgG in feces (1:5 dilution), Figure 5D(2) IgA in feces (1:5 dilution), Figure 5D(3) IgG in nasal wash (undiluted) and Figure 5D(4) IgG in lung wash (undiluted). (Figure 5E(1)-(2)) No significant amount of IgE was detected in either the serum of mice vaccinated via floss (Figure 5E(1)) or in the bone marrow of mice vaccinated via floss (Figure 5E(2)), indicating that the target site, the junctional epithelium, does not sensitize the individual to the delivered Ag. Data are presented as mean ± SD. One-way ANOVA test was used to compare groups with different serum dilutions. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. EXAMPLES

[0065] Floss-mediated delivery of peanut extract (PE) to the junctional epithelium (JE) induces a strong systemic antibody response in mice (therapeutic regimen). Targeting the JE for immunotherapy of "food allergy".

[0066] Figure 6A-6D show the schedule of peanut allergen immunotherapy. (Figure 6A) Immunotherapy schedule: Balb / c mice (n=5) were sensitized by oral route [peanut extract (PE) 1mg + cholera toxin (CT) 15μg] for 5 consecutive weeks with 1 week interval. Mice were then vaccinated by flossing with antigen-coated floss. The floss was coated with PE 5μg + / - CpG (single-stranded oligodeoxynucleotide adjuvant) 5μg, and mice were vaccinated 3 times a week for a total of up to 3 weeks. Mice that did not receive any treatment after sensitization served as control (untreated). Blood was collected from mice on day 10 post-vaccination (PV). Eight weeks after vaccination, mice were challenged with PE allergen (500μg) by intraperitoneal route (IP), and then mice were euthanized and various tissues were collected. (Fig. 6B(1)-(3)) Serum anti-PE antibodies (1:12500 dilution) were confirmed by enzyme-linked immunosorbent assay (ELISA). Anti-PE 6B(1) IgG, Fig. 6B(2) IgG1 and Fig. 6B(3) IgG2a antibody responses 10 days after vaccination. Individual mouse sera were used for analysis. Data are presented as mean ± SD. One-way ANOVA test was used to compare between groups with different serum dilutions. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 and ns: no significant difference. PE induced anaphylaxis. (Fig. 6C)(1) Plasma MCPT-1 values ​​after IP challenge with PE. Histological analysis of intestinal tissue. (Fig. 6D) Eight weeks after vaccination, mice were challenged with PE allergen (500 μg) via intraperitoneal route. Mice were then euthanized and the small intestine was harvested from the proximal, middle and distal ends, fixed, dehydrated and embedded in paraffin wax for sectioning. Tissue sections were stained with hematoxylin and eosin (H&E) staining and sectioned for histological diagnosis. Figure 6D(1) Number of eosinophils counted in each section obtained from mice in different treatment groups. Figure 6D(2) Bright field images of H&E stained intestines with arrows indicating eosinophil infiltration. Individual mouse samples were used for analysis. Data are presented as mean ± SD. Groups were compared using one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001 and ns: no significant difference. EXAMPLES

[0067] Floss-mediated delivery of ovalbumin (Ova) to the junctional epithelium (JE) induces potent systemic antibody responses in mice (therapeutic regimen). Targeting the JE for immunotherapy of "airway allergy".

[0068] Figure 7A-7D show airway allergen immunotherapy. (Figure 7A) Immunotherapy schedule: Balb / c mice (n=5) were sensitized by two intraperitoneal (IP) injections (Ova 25μg + alum 2mg (adjuvant)) with a one-week interval. On day 10 post-sensitization (PS), mice were challenged with Ova (50μg) via the intranasal route (IN) for three consecutive days to develop airway inflammation. Mice were then vaccinated by flossing with antigen-deposited floss. The floss was coated with Ova 25μg + / - CpG (single-stranded oligodeoxynucleotide adjuvant) 25μg, and mice were vaccinated three times a week for a total of up to three weeks. Mice that did not receive any treatment after sensitization served as controls (untreated). Blood was collected from mice on day 10 post-vaccination. On the 28th day after vaccination, mice were challenged with Ova allergen (50 μg) by intranasal route (IN) for three consecutive days, then mice were euthanized and various tissues were collected. Systemic immune response: (Figure 7B(1)-(4)) Serum anti-Ova Figure 7B(1) IgG, Figure 7B(2) IgG1, Figure 7B(3) IgG2a and Figure 7B(4) IgE antibody responses (1:12500 or 1:500 or 1:20 dilution) on the 10th day after vaccination analyzed by enzyme-linked immunosorbent assay (ELISA). (Figure 7C) Lung lavage analysis after challenge. Mice were then euthanized and mucosal secretions of lung lavage were collected. Figure 7C(1) Eosinophils and Figure 7C(2) Neutrophils cell counts in lung lavage - cells were counted by staining with Diff-stain kit and observing the cells under confocal microscope. Lung histological analysis: (Figure 7D) Mice were then euthanized and lungs were removed, fixed, washed, and sectioned for histology. Tissue sections were stained with Periodic Acid-Schiff (PAS) to stain mucus deposits or Trichrome Blue (TCB) to stain collagen deposits. Representative brightfield images of PAS-stained lungs (upper panels) and TCB-stained lungs (lower panels). Arrows in the upper panels point to mucus deposits and arrows in the lower panels point to collagen deposits. EXAMPLES

[0069] How to deposit it on dental floss.

[0070] The majority of flosses available on the market are coated with a continuous coating of a substance (wax, flavoring, etc.). Currently, the entire length of the floss (hundreds of feet in a floss cartridge) is coated. These coatings are not adequately characterized and cannot be used for medical applications where it is important to deliver a known amount of agent, such as in the case of delivery of vaccines or drugs / therapeutic molecules where deviations from the recommended dose can be harmful or cause side effects.

[0071] The compositions and methods of the present invention allow for the coating of any molecule via a simple manner of fluid ejection. This method can be used to coat specific lengths of floss, specific surfaces of the floss, and even discrete locations with one or more active agents / molecules. This method can also be used to coat both sides if desired.

[0072] The present invention provides a novel method of coating dental floss. Substances (e.g., synthetic molecules or polymers, amino acids or polymers thereof, nucleotides or polymers thereof, lipids, carbohydrates, natural substances, antigens / allergens / adjuvants / drugs / combinations thereof) can be coated on the surface of the floss for delivery to the gum tissue. Delivery can have uses such as, for example, to modulate immune responses, systemic effects or local effects. The surface of the floss can be coated by depositing the biologic over a shorter or longer distance / length of the floss (deposition methods include depositing on a single adjacent portion of the floss or on two or more separate portions of the floss with the same or different spacing between each deposition area) (deposition methods include placing a drop on the floss or using a pipette to drag and stretch the drop over the floss to spread it over a certain distance / length on the floss or spray coating or inkjet printing or pipette-based coating or cartridge printing or combinations thereof) and allowing the coating to dry. To demonstrate the invention, we have demonstrated efficacy and proof-of-concept using antigens / allergens / peptides / microparticles / nanoparticles / single-stranded deoxyribonucleic acid (DNA). Various amounts of biologics can be coated onto the surface of the floss. The coated substances are easily delivered into the gum tissue by the simple act of flossing.

[0073] For purposes of medical use with coated floss, it is important that it possess the following properties: (a) the coating should be consistent over a short length of the coated floss to allow consistent delivery to the gum pocket by the user; (b) a known amount of formulation should be coated onto the floss; and / or (c) the coating should remain adhered to the surface until it is used for its intended purpose.

[0074] Flosses are often made of hydrophobic materials (such as TEFLON® or NYLON®), making it difficult to wet these surfaces using coating solutions. Due to the poor wetting, it is difficult to achieve a continuous and uniform coating on the floss. Many different solvents can be used to make the coating solution, but water is preferred for the biological material that needs to be coated onto the floss, and aqueous coating solutions are more difficult to coat onto the floss. However, non-aqueous solutions can also be used in the present invention, where the active agent is in a solvent that is insoluble (or partially soluble) in water, the active agent is deposited on the floss, and the solvent evaporates leaving the active agent behind.

[0075] Rather than creating a continuous coating, separate droplets can be deposited onto the floss surface. This reduces the need to spread the coating evenly over its length, allowing for reproducible coatings and patterns to be achieved. (1) A liquid dispense system (manual or automated or a combination of both) can be used to dispense onto the floss. For proof of concept, manual dispensing was used. (2) The surface of the floss can be made hydrophilic (e.g., by coating with a hydrophilic polymer, or by, e.g., oxygen plasma treatment, or other conventional surface treatment approaches that can change the surface energy of the floss surface to make the coating liquid more spreadable). (3) The coating liquid is placed on the surface of the floss. After a period of time and sufficient solvent has evaporated, the liquid on the floss can be mechanically spread. As some of the solvent evaporates, the viscosity of the coating liquid increases, improving its ability to spread on the floss.

[0076] The advantages of using a dispensing system (manual, automated, or a combination thereof) for deposition onto the floss are: (1) less loss of the formulation being deposited compared to spray / dip coating; (2) precise amounts can be deposited; (3) multiple deposition formulations can be deposited; and / or (4) even aqueous solutions can be deposited as droplets to form a uniform pattern, thus avoiding alteration of the floss surface.

[0077] Spray or dip coating can lead to wasteful consumption of materials. In contrast, using separate deposition methods on the surface of the floss results in little or minimal loss of materials. When depositing on the floss, precise control (e.g., when the goal is to deposit on small spots on the floss, such as less than 1 mm in length / diameter) is difficult to achieve. However, by ejecting fluid, even nanoliter to picoliter volumes can be easily deposited on the floss at known precise locations. By ejecting fluid, it is also easy to deposit different materials with small gaps between different deposition spots. This level of accuracy and precision is difficult with spray / dip coating. This approach could be used to develop and build deposition devices that can be installed in pharmacies, homes, or clinicians' offices. Furthermore, the proposed invention can be used to deposit active agents onto the floss that have different solvent requirements relative to their solubility (e.g., one active agent, an antigen, requires water as a solvent, while another active agent, an adjuvant, requires an organic solvent).

[0078] FIG. 8 shows the deposition capabilities of peptides, nanoparticles, proteins, oligonucleotides, microparticles deposited on floss in different patterns, either as short or longer lengths, a single area deposition or multiple distinct areas deposition on only one side of the floss.

[0079] FIG. 9 shows the deposition potential of deposited water-soluble and water-insoluble materials including pollen grain particles.

[0080] Figure 10 shows the deposition potential and different deposition patterns of two different compounds as examples. One formulation is NHS-Rhodamine (fluorescent reagent) conjugated ovalbumin (protein) in water (referred to as "A"), and the second is gold nanoparticles and CpG (single-stranded DNA) conjugated M2e peptide in water (referred to as "B").

[0081] FIG. 11 illustrates the deposition capabilities of multiple substances, here showing four different food colorings (blue, green, yellow, and red) deposited as four distinct moieties.

[0082] FIG. 12 shows the ability to coat both sides of the floss with different formulations.

[0083] 13 shows an example of an automated coating apparatus 10 for coating floss. The automated coating apparatus 10 includes a stand 12 that includes a controlled linear motion stage 14 that allows movement in one or two dimensions, in this embodiment shown with a two-dimensional stage having a back surface 16 to which a syringe assembly 18 is attached that controls the delivery of droplets 20 onto the floss 22.

[0084] Figure 14 shows two examples of flosser system designs.

[0085] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Further, the compositions of the invention can be used to effect the methods of the invention.

[0086] It is understood that the specific embodiments described herein are presented by way of example and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of the invention and are encompassed by the claims.

[0087] All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0088] When used in conjunction with the word "comprising" in the claims and / or specification, the use of the word "a" or "an" can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more than one." While the present disclosure supports the definition referring to alternatives only and "and / or," the use of the word "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or the alternatives are not mutually exclusive. Throughout this application, the word "about" is used to indicate that a value includes the inherent variation of error of the device, the variation that exists among the methods used to determine the value, or the subjects of the study.

[0089] As used in the specification and claims, the word "comprising" (and all forms of comprising, such as "comprise" and "comprises"), the word "having" (and all forms of having, such as "have" and "has"), the word "including" (and all forms of including, such as "includes" and "include"), or the word "containing" (and all forms of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In any embodiment of the compositions and methods provided herein, "comprising" may be replaced with "consisting essentially of" or "consisting of." As used herein, the phrase "consisting essentially of" requires the specified integers or steps as well as those that do not materially affect the personality or function of the claimed invention. As used herein, the term "consisting" is used to indicate the presence of only a recited integer (e.g., feature, element, property, quality, method / process step, or limitation) or group of integers (e.g., feature, element, property, quality, method / process step, or limitation).

[0090] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed before the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of A, B, C, AB, AC, BC, or ABC: and, where order is important in a particular context, at least one of BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations containing one or more repeats of an item or term are expressly included, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc. One of skill in the art will understand that typically there is no limit to the number of items or terms in any combination, unless otherwise clear from the context.

[0091] As used herein, without limitation, approximation words such as "about", "substantially" or "substantially" refer to a condition that is understood not necessarily to be absolute or complete when so modified, but that is considered close enough for a person of ordinary skill in the art to warrant specifying that the condition exists. The extent to which the description differs depends on how large a change can be made, such that a person of ordinary skill in the art will recognize that the modified feature still has the required properties and capabilities of the original feature. Generally, however, in accordance with the above discussion, numerical values ​​in this specification modified by approximation words such as "about" can vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12, or 15%.

[0092] Moreover, the section headings in this specification are provided in accordance with the suggestions of 37 CFR 1.77 or otherwise to facilitate the organization of this specification. These headings are not intended to limit or characterize the inventions defined in any claims that may arise from this disclosure. Specifically, and by way of example, the heading "Technical Field" is provided, but the claims of this application should not be limited by the words contained therein to describe the so-called technical field. Furthermore, the description of the technology in the "Background Art" section should not be construed as an admission that the technology is prior art to any invention in this disclosure. The "Summary of the Invention" should not be construed as characterizing the inventions defined in the claims arising herein. Furthermore, any reference in this disclosure to the singular "invention" should not be used to argue that there is only one novel feature in this disclosure. Multiple inventions may be defined based on the limitations of multiple claims arising from this disclosure, and thus, these claims define the inventions and their equivalents, thereby protecting the inventions and their equivalents. In all cases, the scope of these claims shall be considered on their own merits in light of this disclosure, and should not be limited by the headings set forth herein.

[0093] All compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes may be made in the compositions and / or methods and in the steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0094] In order to assist the Patent Office and any reader of any patent issued based on this application in interpreting the claims appended hereto, Applicant wishes to add that, unless the words "means for" or "step for" are expressly used in a particular claim, it is not intended that any of the appended claims invoke 35 U.S.C. § 112, section 6, 112(f), or the equivalent, as they exist as of the filing date of this application.

[0095] For each claim, each dependent claim may depend on both the independent claim and each preceding dependent claim, so long as the preceding claim provides a suitable antecedent for a term or element of the claim.

Claims

**Claim 1** A composition for modulating an immune response in a subject, comprising an effective amount of one or more antigens, immunogens, allergens, or combinations thereof, wherein the antigen, immunogen, allergen, or combination thereof is delivered to the gingival sulcus and the amount is sufficient to activate or modulate the immune response. **Claim 2** The composition according to claim 1, wherein the antigen, immunogen, allergen, or combination thereof is not targeted for delivery to the vestibular mucosa. **Claim 3** The composition according to claim 1, wherein modulating the immune response is activating or anergizing the immune response by targeting the junctional epithelium of the gingival sulcus. **Claim 4** The composition according to claim 1, wherein one or more antigens, immunogens, allergens, or combinations thereof are provided to maximize delivery of one or more of the antigens, immunogens, allergens, or combinations thereof to the gingival sulcus. **Claim 5** The composition according to claim 1, further comprising one or more agents that enhance the permeability of the antigen to the gingival sulcus (GC). **Claim 6** The composition according to claim 1, wherein 0.001% to 100% of one or more antigens, immunogens, allergens, or combinations thereof are in the reservoir of the junctional epithelium (JE) of the gingival sulcus. **Claim 7** The composition according to claim 1, wherein one or more antigens, immunogens, allergens, or combinations thereof are repeatedly provided to the junctional epithelium of the gingival sulcus. **Claim 8** The composition according to claim 7, wherein delivery of one or more antigens, immunogens, allergens, or combinations thereof to the junctional epithelium is performed before or after ingestion of food or drink. **Claim 9** The composition according to claim 1, wherein one or more antigens, immunogens, allergens, or combinations thereof are applied one or more times at a daily or weekly or monthly frequency such as once, twice, three times, four times, five times, or six times a day, or once, twice, three times, four times, five times, six times, or seven times a week, or once, twice, or three times a month. **Claim 10** The composition according to claim 1, wherein two or more antigens, immunogens, allergens, or combinations thereof are delivered to the junctional epithelium (JE) of the gingival sulcus. **Claim 11** The composition according to claim 1, wherein the antigen, immunogen, allergen, or a combination thereof desensitizes the subject to the antigen, immunogen, allergen, or a combination thereof by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

12. The composition according to claim 1, wherein the antigen, immunogen, or a combination thereof elicits an immune response in the subject to the antigen, immunogen, allergen, or a combination thereof between 0.1% and 100% and defends the subject from the antigen, immunogen, or a combination thereof.

13. The composition according to claim 10, wherein the delivery of the antigen, immunogen, allergen, or a combination thereof to the JE is performed 0 hours before, 0.1 hours before, 0.2 hours before, 0.3 hours before, 0.4 hours before, 0.5 hours before, 0.6 hours before, 0.7 hours before, 0.8 hours before, 0.9 hours before, 1 hour before, 2 hours before, 3 hours before, 4 hours before, 5 hours before, 6 hours before, 7 hours before, 8 hours before, or more before the subject eats food, drinks water, or does both.

14. The composition according to claim 10, wherein the delivery of the antigen, immunogen, allergen, or a combination thereof to the JE is performed 0 hours after, 0.1 hours after, 0.2 hours after, 0.3 hours after, 0.4 hours after, 0.5 hours after, 0.6 hours after, 0.7 hours after, 0.8 hours after, 0.9 hours after, 1 hour after, 2 hours after, 3 hours after, 4 hours after, 5 hours after, 6 hours after, 7 hours after, 8 hours after, or more after the subject eats food, drinks water, or does both.

15. The composition according to claim 10, wherein the amount of the antigen, immunogen, allergen, or a combination thereof delivered to the junctional epithelium ranges from picograms to milligrams.

16. The composition according to claim 1, wherein the immune response is an activated immune response, a regulatory immune response, or an anergic immune response.

17. The composition according to claim 1, wherein one or more antigens, immunogens, allergens, or a combination thereof are embedded in, coated on, or attached to a delivery device that targets the junctional epithelium of the gingival sulcus.

18. The composition according to claim 17, wherein the delivery device has a thickness of less than 5 mm, preferably less than 3 mm, more preferably less than 1 mm.

19. The delivery device, a composition according to claim 17, comprising a natural or synthetic polymer, an organic substance, a metal, an inorganic substance, or a combination thereof.

20. The delivery device, a composition according to claim 17, comprising a mucoadhesive layer or a hydrophobic layer or a hydrophilic layer or a combination thereof.

21. The delivery device, a composition according to claim 17, comprising a microporous structure that allows diffusion of the antigen into the gingival sulcus.

22. The device, a composition according to claim 17, comprising an interdental brush or bristles that are part of a system / device designed to reach the gingival sulcus.

23. The immune response, a composition according to claim 17, targeting at least one of bacteria, viruses, fungi, protozoa, parasites, prions, toxins, cancer, allergies, or autoimmune diseases.

24. One or more antigens, a composition according to claim 17, selected from at least one of proteins, peptides, deoxyribonucleic acid (DNA) oligonucleotides, ribonucleic acid (RNA) oligonucleotides, broken cells, intact cells, lipids, toxin variants, carbohydrates, virus-like particles, liposomes, attenuated live microorganisms or killed natural microorganisms or recombinant microorganisms, virosomes, polymer / inorganic / organic micro and nanoparticles, or immunostimulating complexes (ISCOMs).

25. The antigen, a composition according to claim 17, comprising a peptide obtained from cancer cells or a part thereof selected from T cell and B cell lymphoproliferative diseases, ovarian cancer, pancreatic cancer, head and neck cancer, squamous cell carcinoma, gastrointestinal cancer, breast cancer, prostate cancer, or non-small cell lung cancer.

26. The antigen, a composition according to claim 17, comprising a food allergen selected from peanuts, shellfish, egg protein, milk protein, beans, nuts, or an airway allergen selected from house dust mites or pollen.

27. The antigen, a composition according to claim 17, being at least one of an antigen physically or chemically attached, adsorbed, or fixed to dental floss or a thin device or strip / patch or interdental brush of an appropriate thickness for placement in the gingival sulcus.

28. The composition according to claim 17, further comprising one or more adjuvants selected from cytokines, chemokines, toll-like receptor ligands or activators, alum, muramyl dipeptide, pyridine, chitosan, saponin, oil, emulsion, bacterial cell wall extract, bacterial protein, cytoplasmic bacterial DNA or mimics, viral RNA or mimics, synthetic oligonucleotides, interferon (IFN) gene stimulator (STING) agonists (2’3’-cGAMP, c-di-AMP, 2’3’-c-di-AM(PS)2 (Rp,RP), c-di-GMP, CL401, CL413, CL429, Flagellin, Imiquimod, LPS-EB, MPLA, ODN 1585, ODN 1826, ODN2006, ODN2395, pam3CSK4, poly(I:C), R848, TDB), natural polymers (poly-γ-glutamic acid, chitosan, mannan, lipomannan, lentinan, dextran), synthetic polymers (poly-N-isopropylacrylamide, copolymer, block polymer, polyphosphazene, polyelectrolyte, polyanhydride, polymethacrylate, polylactic-co-glycolic acid copolymer, polycaprolactone, polyvinylpyrrolidone, cationic polymer), and combinations thereof.

29. The composition according to claim 17, wherein one or more antigens, immunogens, allergens, or combinations thereof activate the innate immune response, the adaptive immune response, or both.

30. The composition according to claim 1, further comprising one or more pharmaceutically acceptable carriers, excipients, diluents, buffers, or salts.

31. The step of preparing a dentifrice; and The step of depositing one or more deposits of an active agent in a pharmaceutically acceptable carrier on the dentifrice; A method of making a dentifrice comprising a predetermined amount of one or more active agents, wherein each said deposit comprises a known predetermined amount of said active agent.

32. Each adjacent deposit contains the same or different active agents, or each adjacent deposit contains the same active agent at different concentrations; or each adjacent deposit contains different active agents at different concentrations; or each adjacent deposit is arranged on a different surface from the adjacent deposit; or each adjacent deposit is arranged on the side opposite to the froth from the deposit; or each adjacent deposit contains a different active agent from the previous adjacent deposit, the method according to claim 31.

33. The method according to claim 31, wherein each adjacent deposit contains different active agents having different solvent requirements selected from an active agent soluble in an aqueous solvent and another active agent soluble in an organic solvent.

34. The method according to claim 31, wherein two or more active agents are deposited overlapping in the form of deposits of the same or different distances / lengths.

35. The method according to claim 33, wherein active agents having different solvent requirements are deposited on opposite sides at the same or different distances / lengths.

36. The method according to claim 34, wherein active agents having the same solvent requirements are deposited overlapping at the same or different distances / lengths.

37. The method according to claim 31, wherein active agents having different solvent requirements are deposited on opposite sides at the same or different distances / lengths.

38. The method according to claim 31, wherein the froth is solid, loose, contains a plurality of strands, is treated to be adhesive, is treated to adhere to a pharmacologically acceptable carrier, or is treated to adhere to an active agent.

39. The method according to claim 31, wherein each adjacent deposit contains a dye or a mark that distinguishes adjacent droplets or patches.

40. The method according to claim 31, wherein the froth is not immersed in an active agent, a pharmacologically acceptable carrier, or both.

41. The method according to claim 31, wherein one or more active agents are selected from an antigen, an immunogen, an allergen, an amino acid or its polymer, a nucleotide or its polymer, a lipid, a carbohydrate, a natural substance, a drug, or a combination thereof, and are not delivered to the vestibular mucosa.

42. The method according to claim 31, wherein one or more active agents target the junctional epithelium of the gingival sulcus to activate an immune response or cause an anergic immune response.

43. The method according to claim 31, wherein the plaque has a thickness of less than 5 mm, preferably less than 3 mm, more preferably less than 1 mm.

44. The method according to claim 31, wherein the plaque comprises a natural or synthetic polymer, an organic substance, a metal, an inorganic substance, or a combination thereof.

45. The method according to claim 31, wherein the plaque comprises a mucoadhesive layer or a hydrophobic layer or a hydrophilic layer or a combination thereof.

46. The method according to claim 31, wherein the plaque comprises a microporous structure that allows diffusion of the antigen into the gingival sulcus.

47. The method according to claim 31, wherein the one or more active agents comprise an antigen, an immunogen, an allergen, or a combination thereof in an amount delivered to the junctional epithelium ranging from picograms to milligrams.

48. The method according to claim 31, wherein the one or more active agents comprise an antigen, an immunogen, an allergen, or a combination thereof, and desensitize the subject to the antigen, the immunogen, the allergen, or a combination thereof by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

49. The method according to claim 31, wherein the one or more active agents comprise an antigen, an immunogen, an allergen, or a combination thereof, and desensitize the subject to the antigen, the immunogen, the allergen, or a combination thereof between 0.1% and 100%.

50. The method according to claim 31, wherein the one or more active agents activate, modulate, or anergize an immune response.

51. The method according to claim 31, wherein the one or more active agents elicit an immune response targeting at least one of bacteria, viruses, fungi, protozoa, parasites, prions, toxins, cancer, allergies, or autoimmune diseases.

52. The method according to claim 31, wherein the one or more active agents comprise one or more antigens selected from at least one of proteins, peptides, deoxyribonucleic acid (DNA) oligonucleotides, ribonucleic acid (RNA) oligonucleotides, broken cells, intact cells, lipids, toxin variants, carbohydrates, virus-like particles, liposomes, attenuated live microorganisms or killed native or recombinant microorganisms, virosomes, polymer / inorganic / organic micro- and nanoparticles, or immunostimulating complexes (ISCOMS).

53. The method according to claim 31, wherein one or more active agents comprise an antigen containing a peptide obtained from cancer cells or a part thereof selected from T-cell and B-cell lymphoproliferative diseases, ovarian cancer, pancreatic cancer, head and neck cancer, squamous cell carcinoma, gastrointestinal cancer, breast cancer, prostate cancer, or non-small cell lung cancer.

54. The method according to claim 31, wherein one or more active agents comprise an antigen containing a food allergen selected from peanuts, shellfish, egg protein, milk protein, beans, nuts, or an airway allergen selected from house dust mites or pollen.

55. The method according to claim 31, wherein one or two or more active agents comprise a drug such as insulin, epinephrine, steroid, stimulant.

56. The method according to claim 31, wherein one or more active agents comprise at least one antigen that is physically or chemically adhered, adsorbed, or fixed to dental floss, a thin device or strip / patch, or an interdental brush having a thickness suitable for placement in the gingival sulcus.

57. Cytokines, chemokines, toll-like receptor ligands or activators, alum, muramyl dipeptide, pyridine, chitosan, saponin, oil, emulsion, bacterial cell wall extract, bacterial protein, cytoplasmic bacterial DNA or mimics, viral RNA or mimics, synthetic oligonucleotides, interferon (IFN) gene stimulator (STING) agonists (2’3’-cGAMP, c-di-AMP, 2’3’-c-di-AM(PS)2 (Rp,RP), c-di-GMP, CL401, CL413, CL429, Flagellin, Imiquimod, LPS-EB, MPLA, ODN 1585, ODN 1826, ODN2006, ODN2395, pam3CSK4, poly(I:C), R848, TDB), natural polymers (poly-γ-glutamic acid, chitosan, mannan, lipomannan, lentinan, dextran), synthetic polymers (poly-N-isopropylacrylamide, copolymer, block polymer, polyphosphazene, polyelectrolyte, polyanhydride, polymethacrylate, lactic acid / glycolic acid copolymer, polycaprolactone, polyvinylpyrrolidone, cationic polymer), and one or more adjuvants selected from combinations thereof.

58. The method according to claim 31, wherein the viscosity of the deposit is 0.01 centipoise (cp), 1 cp, 10 cp, 100 cp, 1000 cp, 10000 cp, 100000 cp, 200000 cp, 300000 cp, 500000 cp, 1000000, or 100000000 cp.

59. Froth; and Deposits on the froth, which are one or more reservoirs of the active agent in a pharmacologically acceptable carrier; A froth containing a predetermined amount of one or more active agents, wherein each droplet or patch contains a known predetermined amount of the active agent. Said froth.

60. Each adjacent deposit contains the same active agent or different active agents, or each adjacent deposit contains the same active agent at different concentrations; or each adjacent deposit contains different active agents at different concentrations; or each adjacent deposit is arranged on a different surface from the adjacent deposit; or each adjacent deposit is arranged on the opposite side of the adjacent deposit of the froth; or each adjacent deposit contains a different active agent from the previous adjacent deposit, respectively. The froth according to claim 59.

61. The froth according to claim 59, wherein each adjacent deposit contains different active agents having different solvent requirements selected from one active agent soluble in an aqueous solvent and another active agent soluble in an organic solvent.

62. The froth according to claim 59, wherein two or more active agents are deposited overlapping in the form of droplets or patches at the same or different distances / lengths.

63. The froth according to claim 61, wherein active agents having different solvent requirements are deposited on opposite sides at the same or different distances / lengths.

64. The froth according to claim 62, wherein active agents having the same solvent requirements are deposited overlapping at the same or different distances / lengths.

65. The froth according to claim 59, wherein active agents having different solvent requirements are deposited on opposite sides at the same or different distances / lengths.

66. The froth according to claim 59, wherein the froth is solid, loose, contains a plurality of strands, is treated to have adhesiveness, is treated to adhere to a pharmacologically acceptable carrier, or is treated to adhere to an active agent.

67. The froth according to claim 59, wherein each adjacent droplet or patch contains a dye or mark that distinguishes adjacent deposits from each other.

68. The film according to claim 59, which is not immersed in an active agent, a pharmaceutically acceptable carrier, or both.

69. The film according to claim 59, wherein the one or more active agents are selected from an antigen, an immunogen, an allergen, an amino acid or a polymer thereof, a nucleotide or a polymer thereof, a lipid, a carbohydrate, a natural substance, a drug, or a combination thereof, and are not delivered to the vestibular mucosa.

70. The film according to claim 59, wherein the one or more active agents target the junctional epithelium of the gingival sulcus to activate an immune response or cause an immune response that anergizes.

71. The film according to claim 59, having a thickness of less than 5 mm, preferably less than 3 mm, more preferably less than 1 mm.

72. The film according to claim 59, comprising a natural or synthetic polymer, an organic substance, a metal, an inorganic substance, or a combination thereof.

73. The film according to claim 59, comprising a mucoadhesive layer, a hydrophobic layer, a hydrophilic layer, or a combination thereof.

74. The film according to claim 59, comprising a microporous structure that allows diffusion of an antigen into the gingival sulcus.

75. The film according to claim 59, wherein the one or more active agents comprise an antigen, an immunogen, an allergen, a drug, a small molecule, or a combination thereof in an amount delivered to the junctional epithelium in the range from picograms to milligrams.

76. The film according to claim 59, wherein the one or more active agents comprise an antigen, an immunogen, an allergen, or a combination thereof, and desensitize the subject by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% to the antigen, the immunogen, the allergen, or a combination thereof.

77. The film according to claim 59, wherein the one or more active agents comprise an antigen, an immunogen, or a combination thereof that causes an immune response in the subject in the range of 0.1 to 100% to the antigen, the immunogen, or a combination thereof.

78. The film according to claim 59, wherein the one or more active agents activate, modulate, or anergize an immune response.

79. The film according to claim 59, wherein the one or more active agents cause an immune reaction targeting at least one of bacteria, viruses, fungi, protozoa, parasites, prions, toxins, cancer, allergies, or autoimmune diseases.

80. The floss according to claim 59, wherein the one or more active agents comprise one or more antigens selected from at least one of proteins, peptides, deoxyribonucleic acid (DNA) oligonucleotides, ribonucleic acid (RNA) oligonucleotides, broken cells, intact cells, lipids, toxin variants, carbohydrates, virus-like particles, liposomes, attenuated live microorganisms or killed natural or recombinant microorganisms, virosomes, polymer / inorganic / organic micro- and nanoparticles, or immunostimulating complexes (ISCOMs).

81. The floss according to claim 59, wherein the one or more active agents comprise an antigen comprising a peptide obtained from cancer cells or a part thereof selected from T-cell and B-cell lymphoproliferative diseases, ovarian cancer, pancreatic cancer, head and neck cancer, squamous cell carcinoma, gastrointestinal cancer, breast cancer, prostate cancer, or non-small cell lung cancer.

82. The floss according to claim 59, wherein the one or more active agents comprise an antigen that is a food allergen selected from peanuts, shellfish, egg protein, milk protein, legumes, nuts, or an airway allergen selected from house dust mites or pollen.

83. The floss according to claim 59, wherein the one or more active agents comprise drugs such as insulin, epinephrine, steroids, stimulants.

84. The floss according to claim 59, wherein the one or more active agents comprise at least one antigen that is physically or chemically attached, adsorbed, or fixed to dental floss or a thin device or strip / patch or interdental brush having a thickness suitable for placement in the gingival sulcus.

85. One or more adjuvants selected from cytokines, chemokines, Toll-like receptor ligands or activators, alum, muramyl dipeptide, pyridine, chitosan, saponin, oil, emulsion, bacterial cell wall extract, bacterial protein, cytoplasmic bacterial DNA or mimics, viral RNA or mimics, synthetic oligonucleotides, interferon (IFN) gene stimulator (STING) agonists (2’3’-cGAMP, c-di-AMP, 2’3’-c-di-AM(PS)2 (Rp,RP), c-di-GMP, CL401, CL413, CL429, Flagellin, Imiquimod, LPS-EB, MPLA, ODN 1585, ODN 1826, ODN2006, ODN2395, pam3CSK4, poly(I:C), R848, TDB), natural polymers (poly-γ-glutamic acid, chitosan, mannan, lipomannan, lentinan, dextran), synthetic polymers (poly-N-isopropylacrylamide, copolymer, block polymer, polyphosphazene, polyelectrolyte, polyanhydride, polymethacrylate, lactic acid / glycolic acid copolymer, polycaprolactone, polyvinylpyrrolidone, cationic polymer), and combinations thereof, further comprising the frost according to claim 59.

86. The frost according to claim 59, wherein one or more active agents comprise one or more antigens, immunogens, allergens, or combinations thereof that activate the innate immune response, the adaptive immune response, or both.

87. The frost according to claim 59, wherein the viscosity of the deposit is 0.01 centipoise (cp), 1 cp, 10 cp, 100 cp, 1000 cp, 10000 cp, 100000 cp, 200000 cp, 300000 cp, 500000 cp, 1000000, or 100000000 cp.

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