WO2015200770A1 - Composition and method for reducing zoonotic infectious diseases - Google Patents

Composition and method for reducing zoonotic infectious diseases Download PDF

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Publication number
WO2015200770A1
WO2015200770A1 PCT/US2015/037925 US2015037925W WO2015200770A1 WO 2015200770 A1 WO2015200770 A1 WO 2015200770A1 US 2015037925 W US2015037925 W US 2015037925W WO 2015200770 A1 WO2015200770 A1 WO 2015200770A1
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Prior art keywords
composition
agent
substrate
embodiments
method
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PCT/US2015/037925
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French (fr)
Inventor
Douglas Steven ZATECHKA
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Us Biologic, Inc
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Priority to US201462017699P priority Critical
Priority to US62/017,699 priority
Application filed by Us Biologic, Inc filed Critical Us Biologic, Inc
Publication of WO2015200770A1 publication Critical patent/WO2015200770A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/20Pills, tablets, discs, rods
    • A61K9/28Dragees; Coated pills or tablets, e.g. with film or compression coating
    • A61K9/2806Coating materials
    • A61K9/282Organic compounds, e.g. fats
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; THEIR TREATMENT, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
    • A23K10/00Animal feeding-stuffs
    • A23K10/10Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; THEIR TREATMENT, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
    • A23K10/00Animal feeding-stuffs
    • A23K10/10Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; THEIR TREATMENT, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
    • A23K40/00Shaping or working-up of animal feeding-stuffs
    • A23K40/20Shaping or working-up of animal feeding-stuffs by moulding, e.g. making cakes or briquettes
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; THEIR TREATMENT, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
    • A23K40/00Shaping or working-up of animal feeding-stuffs
    • A23K40/30Shaping or working-up of animal feeding-stuffs by encapsulating; by coating
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/0225Spirochetes, e.g. Treponema, Leptospira, Borrelia
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/025Enterobacteriales, e.g. Enterobacter
    • A61K39/0258Escherichia
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/09Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/0053Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/0056Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/51Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/54Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541Mucosal route
    • A61K2039/542Mucosal route oral/gastrointestinal
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/55Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552Veterinary vaccine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/48Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005Wall or coating material
    • A61K9/5015Organic compounds, e.g. fats, sugars
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/48Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5073Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals having two or more different coatings optionally including drug-containing subcoatings
    • A61K9/5078Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals having two or more different coatings optionally including drug-containing subcoatings with drug-free core

Abstract

The presently disclosed subject matter relates to a composition and method of using the composition for oral delivery of a bioactive agent to a subject. More particularly, the presently disclosed subject matter relates to a composition comprising an effective amount of at least one bioactive agent layered over a substrate and a method of reducing zoonotic infectious disease by administering the composition to a subject. The presently disclosed subject matter further relates to a method of preparing the composition.

Description

COMPOSITION AND METHOD FOR

REDUCING ZOONOTIC INFECTIOUS DISEASES

RELATED APPLICATIONS

|βββΙ] This application claims priority to U.S. Provisional Application Serial No. 62/017,699, filed June 26, 2014, the entire disclosure of which is incorporated herein by ibis reference,

TECHNICAL FIELD

[00021 The presently disclosed subject matter relates to a composition and method of using the composition for oral delivery of a bioactive agent to a subject. More particularly, the presently disclosed subject matter relates to a composition comprising a substrate and an elective amount of at least one bioactive agent layered over the substrate and a method of reducing zoonotic infectious disease. The presentl disclosed subject matter further relates to a method of preparing the composition.

BACKGROUND

[0003] Controlling zoonotic infectious diseases and antimicrobial resistance contributes to the reduction of disease itansraissibility. Current strategies to control zoonotic infectious diseases includehe deployment of pesticides as a means to eliminate the vector from the enzootic cycle. However, the use of pesticides presents with toxic off-target, effects upon the subject and environment. The use of prophylactic and therapeutic antibiotics, concomitantly, has inadvertently led to the evolution of antimicrobial-resistant strains of infectious agents being maintained in the zoonotic cycle. Further, while susceptible host prophylactic or therapeutic agent campaigns often employ parenteral administration, such administrative methods pose cost and logistics challenges. Further, compared to parenteral methods, orally delivered prophylactic or therapeutic agents are manufactured more cost effectively, offer a significant ease of use, and cause fewer side effects.

SUMMARY

[0004} This summary describes several embodiments of the presently disclosed subject matter, and, in many cases, lists variations and permutations of these embodiments. This Summary is merel exemplary of the numerous and varied embodiments. Mention of one or more representative features of i a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently- disclosed subject matter, whether listed in this Summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.

(0005] The presently disclosed subject matter relates to a composition and method of using the composition for oral delivery of a bioactive agent to a subject More particularly, the presently disclosed subject matter relates to a composition comprising a substrate and an effective amount of at least one bioactive agent layered o ver the substrate and a method of reducing zoonotic infectious disease. In some embodiments, the presently disclosed subject matter provides a method to reduce antimicrobial resistance by administering the composition, to a subject.

(0006] In some embodiments of the presently disclosed subject matter, a composition is provided. The composition includes a substrate, an effective amount of at least one bioactive agent layered over the substrate, and a cross-linking agent In some embodiments, the at least one bioactive agent is stabilized in a stabilizer under conditions facilitating anhydrobiosis. In some embodiments, the composition is prepared by the steps of f! ) stabilizing at least one bioactive agent in a stabilizer, (2) applying the stabilized at least one bioactive agent to a surface of a substrate, (3) cross-linking the stabilized at least one bioactive agent by applying a cross-linking agent to the substrate, and (4) drying under forced air at ambient temperatures in a range of temperatures. In some embodiments, the temperature is between about 2<fC to about 35°C.

(0007] Further provided in some embodiments, the composition further includes a coating on the exterior surface of the composition, in some embodiments, the coating on the exterior surface is a shellac coating. In some embodiments, the coating is an enteric coating.

(0008] In some embodiments, the substrate has a mean diameter of from about 1 0 μγα to about 2 cm. In some embodiments, the stabilizer includes, but not limited to, a hydrocolloid polymer or plasticizing sugar, in some embodiments, the plasticizing sugar includes non-limiting examples of sucrose trehalose. In some embodiments, a non-limiting example of hydroeoHoid polymer is sodium alginate. In some embodiment, the cross-linking agent is a calcium salt. Examples of cross-linking agent includes, but not limited to, calcium lactate, calcium chloride, calcium sulfate, calcium carbonate, calcium acetate, or calcium ascorbaie.

(0009] in some embodiments of the presently disclosed subject matter, examples of th substrate includes, but not limited to pellet, a che vable, a bead, and a powder, in some embodiments, the bead includes a non-limiting example of a micro ysiaiime cellulose bead, in some embodiments, the substrate comprises a plant-based or earthen-based substance, in some embodiments, the earthen-based substance includes but not limited to soil or water. In some embodiments, the bioactive agent is a .recombinant whole-cell bacteria engineered to express one or more antigens. In some embodiments, the whole-cell bacteria includes, but not limited to, a l cfobocHlus iolcudes L. acidophilus. I. hrevis, L. c ei, L. crispatus, L.f rmeni m, L g ss rl L plantarum, L reuieri, L rhamnzosus, aud i, saiivarms. In some embodiments, the one or more antigens are one or more Borreiia burgdorferi antigens.

[0001.0] Further provided, in some embodiments, the substrate is in an amount of about 85% to about 99% w w of the composition. In some embodiments, the substrate is about 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% and about 99% w/w of the composition of the presently disclosed subject matter. In some embodiments, the stabilizer is in an amount of about 1 % to about 15% w/w of the composition. The stabilizer is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1 %, 12%, 13%, 14%, and about 15% w/w of the composition as disclosed herein. In some embodiments, the effective amount of the bioaciive agent is an immuaogenically effective amount with the minimal immunizing dosage (MID) of about 5x10"' CFU to about 5x10' CFU, in some embodiments, the MID is about 5x.l 0s CFt!, 5x10* CFU, 5xi05 CFU, 5x 10s CFt!, and about 5x i07 CFU. in some embodiments, cross-linking agent is in an amount of about 0.5% to about 7.5% w/w of the composition. The cross-linking agent is about 0.5%, 1%, 1.5%, 2%, 2,5%, 3%, 3.5%, 4%,, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, and about 7.5% w/w of the composition. Further, in some embodiments, the coating is in an amount of about i .5% to about 22.5% w/w of the composition. The coating is about 1.5%,, 2%, 2.5%, 3%, 3,5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8,5%, 9%,, 9.5%, 10%, 10.5%, 1 1 , 1 1,5%, 12%, 1 2.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15,5%, 16%, 1 6.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21 %, 21.5%, 22%, and about 22.5% of the composition as disciosed herein.

00011 ] Further provided, in some embodiments of the presently disciosed subject matter, is a composition for oral delivery of an bioaciive agent. The composition includes a substrate, an effective amount of at least one bioaciive ageni coated or layered on a substrate, and a cross-linking agent to facilitate the encapsulation of the antigenic in the stabilizer on the surface of the substrate, in some embodiments, the at least one bioactive agent is stabilized in. a stabilizer selected from a group consisting of a hydrocoHoid polymer, a plasticizing sugar and a trehalose. In some embodiments, the bioactive agent is an antigenic agent.

[00012] Still further, in some embodiments, a method of preparing a composition for oral delivery of an bioactive agent is provided. The method includes the steps of (1 ) stabilizing at least one antigenic agent in a stabilizer; (2) coating the stabilized at least one antigenic agent on to a substrate; (3) applying a cross-linking agent; (4) cross-linking to facilitate gelation or encapsulation of antigenic agent; and (5) drying under forced air at an ambient temperature. In some embodiments, the temperature is in a range of between about 20°C to about 35°C. the temperature is about 20°C, 2 PC, 22°C, 23 °C,

24°C, 25*C, 26°C, 27°C, 28X, 29°C, 3(PC, 31 °C, 32°C, 33°C, 34°C, and about 3S°C. In some embodiments, a fan drives the ambient temperatures. In some embodiments, the method further include a step of coating with a confectionary glaze layer on the exterior surface for moisture barrier or flavored attractant. In some embodiments, the method further includes a step of coaling with a shellac layer on the exterior surface for moisture barrier.

[00013] The presently disclosed subject matter, in some embodiments, provides a method of controlling zoonotic infectious diseases by vaccinating a subject in need thereof. The method includes orally administering to the subject a composition as disclosed herein. .

100014] Further provided, in some embodiments, is a method of controlling zoonotic infectious diseases by vaccinating a subject in need thereof. The method includes orally administering to the subject a composition. The composition includes an effective amount of at least one bioactive agent layered over the substrate, wherein the at least one bioactive agent is stabilized in a stabilizer under conditions facilitating anhydrobiosis, and a cross-linking agent.

[00015] The presently disclosed subject matter, in some embodiments, provides a method of controlling zoonotic infectious diseases by vaccinating a subject in need thereof The method comprises adding a composition directly to a water supply in a suspension suitable for drinking. The composition includes an effective amount of at least one bioactive agent layered over the substrate, wherein the at least one bioactive agent is stabilized in a stabilizer under conditions facilitating anhydrobiosis, and a cross-linking agent.

[00016] In some embodiments, the subject is a reservoir host of the zoonotic infectious disease cycle, in some embodiments, the subject is a susceptible host of the zoonotic infectious disease. In some embodiments, the subject is a xenodiagnostie carrier. In some embodiments, the subject is an arthropod or insect. In. some embodiments, the subject is a mammal. In some embodiments, the mammal is a feral animal includes one or more of a mouse, a chipmunk, a squirrel, a shrew, a vole, a rat, a raccoon, an opossum, a skunk, a rabbit, and a deer. In some embodiments, the subject is a bird, in some embodiments, the subject is fish. In some embodiments, the subject is a domesticated or companion animal. In some embodiments, the domesticated animal comprises one or more of a dog, a eat, a cow, and a horse.

[00017] Advantages of the presently disclosed subject matter will become evident to those of ordinary skill in the art after a study of the description. Figures, and. non-limiting Examples in this document,

BRIEF DESCRIPTION OF THE .DRAWINGS

[00018] Figure 1 is an image of a restriction digest of pET e plasmid vec tored ospA .

[00019] Figure 2 is an image of DMA base-pair ladder standards.

[00020] Figure 3 is an image of the results of sequencing analysis of plasmid-vectored ospA in the forward direction. [00021] Figure 4 is an image of the results of sequencing analysis of piasmid-vectored ospA in the reverse direction.

[00022] Figure 5 is an image of the Western blotting analy sis for Os A protein expression from ospA -vectored E. coll.

[00023] Figure 6 is an image of protein molecular weight marker standards.

100024] Figure ? is a diagram showing vaccine coating, crosslhiking, and layering process.

[00025] Figure 8 is a diagram showing a current operational example for employment of drum- coating processing and formulation,

(00026) Figure 9 is a presentation of the shelf life of the vaccine embodiment disclosed herei as assayed by CPU counts generated from vaccine, as a function of storage time.

[00027] Figure 10 represents ami-OspA antibody titer load in response to vaccine administration employing ospA -vectored E. col

(00028] Figure 11 represents the efficacy of the embodiment disclosed herein as aligned with the studies establishing vaccine efficacy. Data establish the threshold levels of sero-protecfive OspA antibody liters equating to vaccine efficacy.

BRIEF DESCMP iON OF THE SEQUENCE LISTING

[00029] SEQ I» NO: 1 is T? Promoter Primer sequence.

[00030] SEQ ID NO: 2 is T7 Terminator Primer sequence.

[00031] SEQ ID NO: 3 is a nucleic acid sequence otospA gene reference sequence.

DESCRIPTION O EXEMPLARY EMBODIMENTS

[00032] The details of one or more embodiments of the presently disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In ease of conflict the specification of this document, including definitions, will control,

[00033] Each example is provided by way of explanation of the present disclosure and is not a limitation thereon. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the teachings of the present disclosure without departing from the scope of the disclosure. For instance, features illustrated or described as pan of one embodiment can be used with another embodiment to yield a still further embodiment. (00034] While the terms used herein are believed to be well understood by one of ordinary skill in the ait, definitions are set forth herein to facilitate explanation of the presently disclosed subject matter.

[00035] Unless defined otherwise, alt technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently-disclosed subject matter belongs. Although any methods, devices, and materials similar or equivalent to those described herein, can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are now described.

(00036] Following long-standing patent law convention, the terms "a", "an", and "the" refer to "one or more" when used in this application, including the claims. Thus, for example, reference to "a ceil" includes a plurality of such cells, and so forth.

[00037] A!i references to singular characteristics or limitations of the present disclosure shall include the corresponding plural charaeierisiic(s) or limitaiionfs) and vice versa, unless otherwise specified or clearly implied to the contrary by the context in which the reference is made.

(00038] All combinations of method or process steps as used herein can be performed in an order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.

[00039] The methods and compositions of the present disclosure, including components thereof, can comprise, consist of, or consist essentially of the essential elements and limitations of the embodiments described herein, as well as any additional or optional components or limitations described herein or otherwise useful.

(00040] Unless otherwise indicated, alt numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are t be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[00041 ] As used herein, the term "about," when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments -A-20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments .*.!%, in some embodiments 0.5%, and in some embodiments ±0.1% from, the specified amount, as such variations are appropriate to perform the disclosed method.

[00042 ] As used herein, ranges can be expressed as from "about" one particular value, and/or to

"about" another particular value. It is also understood that there are a number of values disclosed herein, and that each vaioe is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed, it is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and .1 are also disclosed.

[00043] The presently disclosed subject matter relates to a composition, and method of using the composition for oral delivery of a biologically active agent to a subject. More particularly, the presently disclosed subject matter relates to a composition comprising a substrate and an effective amount of at least one biologically active agent stabilized by iayered encapsulation over the sobstrate and a method of reducing zoonotic infections disease by administering the composition to a subject. The presently disclosed subject matter further relates to a method of preparing the composition.

[00044] In some embodiments of the presently disclosed subject matter, a composition is provided. The composition includes a substrate, an effective amount of at least one bioactive agent layered over the substrate, and cross-linking agent. In some embodiments, the at least one bioactive agent is stabilized in a stabilizer under conditions facilitating anhydrobiosis.

j 0045] The term "bioactive agent" or "biologically active agent" refers to an substance that is of medical or veterinary therapeutic, prophylactic or diagnostic utility, in some embodiments, the bioactive agent includes a therapeutic agent. As used herein, a therapeutic agent refers to a bioactive agent that, when administered to a patient, will cure, or at least relieve to some extent, one or more symptoms of, a disease or disorder, in some embodiments, bioactive agent includes a prophylactic agent. As used herein, a prophylactic agent refers to a bioactive agent that, when administered to a patient either prevents the occurrence of a disease or disorder or, if administered subsequent to a therapeutic agent, prevents or retards the recurrence of the disease or disorder. In some embodiments, bioactive agent refer to antigens that elicit an immune response, or proteins that can modulate the immune system, to enhance therapeutic potential. In some embodiments, the administration of the biologically active antigenic agent can elicit an immune response that is either prophylactic to prevent disease contraction and transmission, or therapeutic to resolve existing disease infection.

[00046] In certain embodiments, the composition comprises a substrate and an effective amount of at least one active agent coated or layered over the substrate. As used herein, the term "substrate" refers to a solid support composition, such as a carrier, onto which may he applied the vaccine composition. Non-limiting examples of the substrate include a pellet, a ehewable, a bead such as microcrystalHne cellulose (MCC).

[00047] In some embodiments, the substrate has a mean diameter of from about 100 pm to about

2 cm. in some embodiments, the composition may be of a range of size between about 100 μιη (MCC) to about 2 cm (as in pellets) to accommodate consumption by target animal species. In some embodiments, the stabilizer includes, but not limited to, a hydrocolioid polymer or a plasticizing sugar. In some embodiments, the plasticizing sugar includes non-limiting examples of sucrose trehalose. In some embodiments, a non-limiting example of hydroeolloid polymer is sodium alginate. In some embodiment, the cross-linking agent is a calcium salt. Examples of cross-linking agent includes, but not limited to, calcium lactate, calcium chloride, calcium sulfate, calcium carbonate, calcium acetate, or calcium aseorbate.

[00048] Further, in some embodiments of the presently disclosed subject matter, examples of the substrate includes, but not limited to pellet a ehewable, a bead, and a powder. In some embodiments, the bead includes a non-limiting example of a macrocrystalline cellulose bead. In some embodiments, the substrate comprises a plant-based or earthen-based substance, in some embodiments, the earthen- based substance includes but not limited to soil or water. In some embodiments, the bioaciive agent is a recombinant whole-cell bacteria engineered to express one or more antigens.

[00049] As used herein, "whole-cell bacieria" refers to bacterial cells, maintained under conditions that retain the bacterial cellular structural integrity, thai is, whole-cell structural integrity and antigenicity, as a bioaciive carrier for the stable presentation of antigen in certain embodiments. Conditions favorable for such whole-cell structural integrit will be defined, as "stabilized." In certain embodiments, whole cells will be maintained as stable, not to be broken down into cellular fragments and/or oilier biological material and/or organelles. In maintaining the stabilized whole-ceil structural architecture, some embodiments may encompass "active formulations," defined as live whole-cell bacterial units; other embodiments may encompass "inactive formulations," defined as killed whole-cell bacterial units.

[00050] In some embodiments, the whole-cell bacteria includes anhydrobioiic preparations of £ coli, while in other embodiments, the whole-cell bacteria includes, but not limited to, a laetobaeillus includes L. acidophilus, L. brevis, L, casei, L. crispatus, L fermentum, L gasset L pkmtan/m, L. r uteri, L rhammosm, and I. salivarius. In some embodiments, the whole-cell bacterial is a recombinant bacteria engineered to express one or more antigens, in some embodiments, the recombinant bacteria are engineered to express at least one outer surface protein of Borrelia burgdorferi for use as Lyme disease vaccines. (See U.S. Pat. No, 8,821 ,893, which is incorporated herein by reference in its entirely). In some embodiments, the recombinant bacteria are lyophili .ed/free .e-diied.

[00051 ] In some embodiments, the substrate further includes, but not limited to, a plant and/or forage material to include grass, herbaceous legumes, tree legumes, silage, or crop residues to include grains such as corn or soybean stover, or other earthen-based substance, such as soil, compost, or addition directly to water. In certain embodiments, the substrate i edible, and appropriate to be fed to animals in a composition with a vaccine formulation. In some embodiments, the substrate may comprise a dried pellet or kibble, such as a particle generated by compressing original .material, which may be broken up upon mastication into particulate material; and/or a chewable particle, soft and pliable in nature, such that it is not readily broken up or reduced to particulate matter upon mastication but may be readily dissolved; micro-crystalline cellulose beads or other subsirate for employment in the generation and appiicaiion of vaccine m powdered formulation for administration via nasal inhalation, or to be administered directl io water as an oral administration via drinking; a plant; a food-source, such as a food source that is available in the wild; and/or another earthen substance, soti or other onto which may be applied the active vaccine composition, which is ihen dried for stability, or water into which may be applied the active vaccine composition for consumption by drinking.

| 052] In some embodiments, non-limiting examples of the active vaccine agent include whole- cell bacteria as a biological carrier of the antigenic agent, In some embodiments, the active agent is osmoticaliy pre-conditioned for anhydrobiosis and stabilization. As used herein, the term "osmoiica!fy pre-conditioned" refers to the use of specific solutes employed to physically stabilize and protect membranes and proteins in intact bacteria prior to drying to desiccation. Non-limiting osmotic pre- eonditioners include plasticiziiig agents such as sugars, to include sucrose and/or trehalose, or hydroxyeetoine. As used herein,, ihe term "anhydrobiosis" refers to the physical state of biological tolerance to desiccation. Biological desiccation serves to maintain a biologically active composition, without water, thereby enhancing shelf life stability for extended vaccine potency,

f 000533 In some embodiments, the active agent is stabilized in a stabilizer. Non-limiting stabilizers incorporate the use of hydrocoJloids. As used herein, the term "hydrocoHoid" will refer to any materia! of the colloid family of hydrophiiic polymers dispersed in aqueous solution, Bydrocolloids present, in some embodiments, as small particles of about 1 to about 1000 nm in diameter, and serve to encapsulate and stabilize biological material. A hydrocoHoid of the present disclosure may include, but is not limited to, agar, alginate, carrageenan, chitosan, gelatin, and/or gum.

[00054] Suitable hydroeolloids further include one or more natural and synthetic polymers, which form colloidal solutions in aqueous systems. Suitable hydrocolloids include polyvinyl pyrrolidones; starch; polysaccharides, such as aiginic acid, sodium alginate, and calcium alginate; cellulose and cellulose derivatives, such as ethy!cellu!ose, methyl cellulose, hydroxypropylmethyl cellulose (HPMC), hydroxy-propyl cellulose (HPC), and carboxymeihylcel!ulose (CMC); polyethylene glycol (PEG); or mixtures thereof

[00055] In some embodiments, certain hydrocoHoid polymers, such as sodium alginate, may be cross-linked in the presence of a calcium salt. Cross-linking in the presence of a divalent cation such as calcium refers to the capacit to structurally link the polymeric bonds of the hydrocoHoid polymer, sodium alginate, to calcium, to generate a polymer of calcium alginate cross-linked bonds; calcium ions replace the sodium ions in the alginate polymer. Cross-linking facilitates ihe stabilized encapsulation of the active vaccine agent.

[000561 In some embodiments, ihe composition further includes a coating on the exterior surface of the composition, in some embodiments, the coating on the exterior surface is a shellac coating, in some embodiments, the coating is an enteric coating. In some embodiments, the coating on the exterior surface of the substrate is sequentially applied in layers as a top-dressing and comprises an enteric coaling ooce cross-linked. In some embodiments, the substrate is in n amount of about 85% to about 99% w w of the composition. In some embodiments, the stabilizer is in an amount of about 1% to about ! 5% w/w of the composition, in some embodiments, the effective amount of the bioactive agent is an tmniunogenically effective amount with the minimal immunizing dosage (MID) of about 5x10'' CPU to about 5x10'' CFU. In some embodiments, cross-linking agent is in an amount of about 0,5% to about 7,5% w/w of the composition. Further, in some embodiments, the coating is in an amount of about 1.5% to about 22,5% w/w of the composition.

[00057] Further, regardless of the particular mode and timing of administration used in accordance with the methods of the presently disclosed subject matter, the bacterium, including the bacterium-based compositions and antigenic agents described herein, are typically administered in an amount effective to achieve the desired response (i.e., protection against the antigenic agents). As such, the term "effective amount" is used herein to refer to an amount of the therapeutic composition sufficient to produce a measurable biological response (e.g., an immune response against BorreUa burgdorferi infection), in this regard, in some embodiments, the term "therapeutically effective" is used interchangeably herein with the phrase "immunogemcaHy effective" to refer to an amount of whole-cell bacteria expressing the at least one or more antigenic agent of the presently-disclosed subject matter sufficient to induce an effective immune response in a host against a virulent bacterium. Actual dosage levels of active ingredients in a therapeutic composition of the presently disclosed subject matter (e.g., the bacterium) can be varied so as to administer an amount of a composition thai is effective to achieve the desired therapeutic response for a particular subject and/or application. Of course. She selected dosage level and amount of the bacterium and the other components of such a composition will depend upon a variety of factors including the activity of the bacterium, formulation, the route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated. Preferably, a minimal dose is administered, and dose is escalated in the absence of dose-limiting toxicity to a minimally effective amount. Determination and adjustment of a therapeutically effective dose, as well as evaluation of when and how to make such adjustments, are known to those of ordinary skill in the art. As used herein, for example, in some embodiments, the therapeutic effective dose of the at least one antigenic agent is of a minimum immunizing dosage (MID) as measured in active antigen-expressing colony forming bacterial units (CFU) of about 5x M" to about 5x10" CPU .

[00058] In some embodiment, the composition disclosed in the present application further includes an adjuvant. As used herein, the term "adjuvant", or "adjuvantized", will refer to any material capable of enhancing a vaccine-induced immune response in an animal. In some embodiments, compositions embodying whole-ceil bacterial units, molecularly engineered antigenic fusion proteins, to or biochemical immunomodulators present as natural adjuvants. Whole E. colt bacterial eel! units are immunogenically reactive through the presentation of lipopoiysaccharide (LPS); LPS is a ligand for activating the Toll-like receptor 4 (TLR4), essentia! for the immono-surveillance of Gram-negative bacterial infections and ihe activation of the innate immune system (Flanagan et al., J. Endotoxin Res. 6:481 ., 1996). The ammo-terminai 22 ammo acids of OspA account for the lipidated moiety of the protein (the OspA lipoprotein) and presents as a natural immunogenic hydrophobic signal peptide capable of the induction of pro-inflammatory cytokines (Erdile ei al, Infect. Immun. 61 :81 , 1993). In some embodiments, the OspA lipoprotein can be fused in-frame and proximal with other antigenic proteins as an expressed molecular adjuvant fusion protein construct. Preparations employing the use of cholera toxin (CT), when mixed with the whole E. call bacterial eel! units serve as a potent, biochemical immunomodulators on enteric mucosal immune responses (Bowman and lements, Infect. Immun. 69: 1 528, 2001).

J000S9] In some embodiments of the presently disclosed subject matter, a composition for oral delivery of an bioaetive agent is provided. The composition includes a substrate, an effective amount of at least one bioaetive agent coated or layered on a substrate, and a cross-Unking agent to facilitate the encapsulation of the antigenic in the stabilizer on the surface of the substrate. In some embodiments, the at least one bioaetive agent is stabilized in a stabilizer selected from a group consisting of a hydrocoHoid polymer, a plasticizing sugar and a treha!ose. In some embodiments, the bioaetive agent is an antigenic agent.

[00060] Still further, in some embodiments, a method of preparing a composition for oral delivery of an bioaetive agent is provided. The method includes the steps of stabilizing at least one antigenic agent in a stabiliser; coating the stabilized at least one antigenic agent on to a substrate; applying a cross-linking agent; cross-linking to facilitate gelation or encapsulation of antigenic agent; and drying under forced air at a ambient temperature, in some embodiments, th temperature is in a range of between about 20°C to about 35°C. In some embodiments, a tan drives the ambient temperatures. In some embodiments, the methods further include a step of coating with a confectionary glaze layer on the exterior surface for moisture barrier or flavored aitractant. In some embodiments, the method further includes a ste of coating with, a shellac layer on the exterior surface for moisture barrier.

100061] Current methods for generating calcium-alginate encapsulated biological materials require the generation of hydrogel or calcium-alginate beads. Bead-encapsulated biological materials are generated by the pressurized dispensing of sodium alginate into a volume of caltitvm salt, a process employing specific encapsulation equipment (an encapsnlator) (Mazziteili et al, . Biomat. AppL 23 : 123, 2008). Calcium-alginate beads generated by an encapsulator can be harvested and dried for downstream application. The bead format does not reader itself beneficial or efficient for the uniform application onto defined substrates for targeted distribution and administration. Id.

[00062] Examples of encapsulation of antigens for oral administration include the following issued patents. For example, U.S. Pat, No. 5,352,448 describes the formulation and generation of hydrogels that can be loaded with antigen for oral administration as a hard, glassy bead. Additionally, U.S. Pat. No, 5,900,238 similarly describes the formulation and generation, of antigen-encapsulated hydrogel microbeads for mucosal vaccination. Further examples incksde WIPO Pat. No, WO 2013/096883, which presents the methods for generating a spray-dried microencapsulated biological moieties and chemicals in polymers cross-linked by multivalent ions,

[00063] The presently disclosed subject matter provides a method of preparing a composition for oral delivery of a biologically active antigenic agent. The method includes, for example, the steps of: stabilizing by osmotic conditioning at least one antigenic agent coating the at least one antigenic agent onto a substrate employing a sodium alginate suspension as a liquid carrier for layered application, cross-linking by a secondary layering of a calcium salt to facilitate layered gelation via caichim-aiginate encapsulation of the antigenic carrier, and air drying under forced air ambient temperatures yielding a layered anhydrohiotic preparation of the biologicall active antigenic agent. In some embodiments, the methods of the present disclosure include a step of coating the antigenic carrier and/or the substrate with a glaze layer on the exterio surface to provide a moisture barrier and/or flavored attractant Further, in some embodiments, the methods of the present disclosure include a step of coating a shellac layer on the exterior surface of the antigenic carrier and/or of the substrate to provide a moisture barrier. As such, Employment of the more simplified sequential spray coating and layering application of the encapsulated biological materials provides an efficient and commercially viable method for the applying stabilized biologically-active materials as layered coatings over a substrate. Encapsulated layering onto substrates provides a carrier method for targeted distribution of the biologically active agent.

[00064] In some embodiments, the presently disclosed subject matter relates to composition and methods for the stable expression of antigens fo mucosa! administration, more particularly, relates to oral administration to a subject such as a mammal.

[00065] In some embodiments of the presently disclosed subject matter, the active vaccine agent is first osmotically pre-conditioned in about 500 mM to about 625 roM sucrose and/or about 500 mM to about 625 niM hydroxyectoine. Pre-conditioned active agent is then mixed into a matrix of about .1 .0% to about 1.5% sodium alginate in suspension with about 500 mM to about 625 mM sucrose, which is then applied onto the surface of the substrate by spray coating. A secondary layering of about 200 raM to about 225 mM concentration of a calcium salt is applied onto the surface of the substrate by spray coating, in some embodiments, the secondary layer is applied to the surface of the substrate about I second to about 60 seconds after application of the first layer. As used in the presently disclosed subject matter, the calcium sail is calcium lactate. In some embodiment, ihe calcium salt can be calcium chloride, calcium sulfate, calcium carbonate, calcium acetate, or calcium aseorbate.

[00066] As used herein, the term "shellac" refers, in some embodiments, to an external edible glaze, resin, and/or coating applied externally to a vaccine-coated substrate, in some embodiments, a shellac coating comprises a moisture harrier that is useful, for example, in the deployment of an orally administered vaccine, enhancing the vaccine shelf life and stability,

100067] The presently disclosed subject matter, in some embodiments, provides a method of controlling oonotic infectious diseases by vaccinating a subject in need thereof The method includes orally administering to the subject a composition as disclosed herein, in some embodiments, the presently disclosed subject matter relates to a method of orally administered biological-based vaccines utilizing antigen-expression systems comprising vectored bacteri that, upon consumption, elicit an immune response in targeted mammalian populations.

{00068] The present disclosure also relates to methods of stabilizing the active biological agent/component of a vaccine such that antigenic activity may be preserved and measured as a dosage; thai the dosage maintains antigenicity for absorption by the immunoiogicaiiy-active GALT (gut- associated lymphoid tissue) or NALT (naso-pharyngeai-associated lymphoid tissue); and that the immunological response yields a reaction to the vaccine that is sustained, measurable, and prophylactic or therapeutic in the level of sero- responsiveness as assayed by antibody titers. Antibody titers are transmissible as prophylacticaliy active compounds to neutralize targeted zoonotic incidence of infection.

{00069] Related to a methods of use for she presently disclosed subject matter, animal mucosal surfaces present a principle entry sites for many infectious agents. Consequently, mucosal immunity offers an initial line of defense against infectious agents. The mucosal immune response interferes with the infectious process by hindering pathogen, attachment to the mucosal epithelium, neutralizing viral and bacterial agents, and providing the means by which io remove pathogens through phagocytosis. The mucosal-associated immune system functions to prevent microbial penetration and infection through the internal regions of ihe animal. Because of the extensive immunological nature associated with the mucosal immune system, the region is also effectively targeted for vaccine administration to elicit prophylactic immunoresponsi veness against several pathogens (Chen and Cerutti, Immunii tt ;479, 20.10; Fujkuyama et al., Expert Rev Vaccines 11 :367, 2012; Neutra and .ozl.owski, Nature Rev, Immunol, 6: 148, 2006; Ogra et af, Clin. Microbiol. Rev. 14:430, 2001 ; Woodrow et aL Aim . Rev. Biomed. Eng. 14: 17, 2012).

[00070] The local immune response is most effectively induced in response to direct application of antigens to the mucosal surface. The site-specific presentation of antigens in native conformation, however, presents a challenge in teens of the available administrative carriers and the toxicity associated wit the antigens. Therefore, local mu mity may be induced via the stimulation of the niucosa-associated lymphoid tissite (MALT), n integrated network of immunologically active mucosal tissue. Exposure by antigen at the mucosal surfaces of the gut or long triggers the complex migration of lymphocytes to ail mucosal regions where production of antigenic-responsive antibodies are produced. Antigenic exposure further induces a population of memory lymphocytes, which serve to generate antibodies in response to subsequent exposure by the same antigen. Hence, the MALT presents as a target for effective vaccination, by triggering the local immune response (Chen and Cerutti, Immunu mA- l^, 2010; Fujkuyama et a!,, Expert Rev Vaccines 11:367, 2012; Neutra and o/iowski, Nature Rev. Immunol. 6:148, 2006; Ogra et al, Clin. Microbial. Rev, 14:430, 200.1; Woodrow et a!., Ann . Rev. Biomed. Eng. 14:17, 2012).

(00071) Of the immunologically active mucosal surfaces, the MALI" of the gut-associated lymphoid tissues (GALT) in the intestines presents with the greatest accumulation of lymphoid tissue.

G ALT' contains populations of functional T and B lymphocytes in conjunction with antigen-presenting accessory ceils. Specifically, the B lymphocyte population of the GALT comprises a significant subset of cells committed to the generation of the immunoglobulin A (IgA) class of antibodies. As a neutralizing antibody, the localized availability of IgA of the GALT provides an effective means of preventing invading pathogens from attaching and penetrating the epithelial layer of the mucosal surfaces. This form of inimnnoresponsiveness differs from that of the systemic lymphoid tissues where the IgA antibody class is not effectively induced through the conventional intramuscular (IM or subcutaneous (SubQ) methods of immunization administration (Chen and Cerutti, Immunity 33:479, 2010; Fujkuyama et af. Expert Rev Vaccines 11 :367, 2012; Neutra and Kozlowski, Nature Rev. Immunol. 6:1.48, 2006; Ogra et al., Clm. Microbiol Rev. .14:430, 200.1 ; Woodrow et a!., Amu. Rev. Biomed. Eng. 14:17, 2012).

(00072) An epithelial cell layer of the GALT separates the underlying fymphoid. layer of the mucosa from the lumen of the gut. Interspersed within the epithelial layer are accessory cells with a committed function for antigen presentation. Such accessor}'' cells actively sample luminal antigenic samples, internalizing the samples for processing and presentation to ihe adjacent lymphoid cells. Presentation and exposure of antigen to the GALT iniiiaies the clonal expansion of anti en-specific B and T iymphocytes. The igA-commilted B !ymphohiasts consequently migrate through the -mesenteric lymph nodes, by means to trigger an enhanced specific immune response in all mucosal sites, including the intestinal tract, nasopharyngeal and respiratory tracts, lung, oral cavity, ocular regions, mammary gland, and genitourinary tract. Therefore, immuno-stimulation of the GALT through oral vaccination may consequently result in the prevention of infectious diseases at a variety of mucosal surfaces (Chen and Cerutti, lmimtiofy33:47 , 2010; Fujkuyama et al., Expert Rev Vaccines 11:367, 2012; Neutra and Koz!owskh Nature Rev. Immunol. 6:148, 2006; Ogra et al.„ Clin. Microbial. Rev. 14:430, 2001; Woodro et al., A m. Rev. Bkmml Eng. 1 : 17, 2012).

(00073] In accordance with an embodiment of the present disclosure, animals are vaccinated via oral administration wish hydroeoiioid-siabilized'eneapsulaied whole-ceil earners coated onto edible delivery substrates.

j 00074] Successful and efficacious orally administered, vaccines require stability of the antigen as it passes through the digestive tract. As functional proteins dependent upon conformational structure, the configuration of antigens can be denatured under the digestive process of the gut. Enteric drug- delivery systems have been developed to protect, the pharmaceutically active compounds for passage to the intestine. Moreover, hydroeoiloids are useful for whole-cell encapsulation and/ r for maintenance of cellular enzymatic reaction potential. In some embodiments of the present disclosure, hydroeoiloids are employed as an enteric coaiing(s) for stabilizing the whole-cell antigen for effective presentation to the MALT. Hydroeoiloids, such as alginates, offer a hydrophilic gel-network stabilisatio matrix that allow protection of encapsulated biologies for effective passage to targeted mucosal, tissues, such as the GALT.

[000753 In some embodiments, the presently disclosed subject matter provides a method for effectively stimulating a mucosal immune response to presented antigen. Specifically, the present disclosure relates, in certain embodiments, to the use of hydiOCol!oid-stabili ed biological carriers, such as whole-cell bacteria, as vehicles for the expression of antigens in native conformation. This presently disclosed subject matter furthe relates to methods of completing the stabilized biological carriers in combination with compositions of substrates that facilitate delivery' of the antigens to the mucosal- associated lymphoid tissues, via oral, and/or nasal administration.

[00076] Furthe provided, in som embodiments, is a method of controlling zoonotic infectious diseases by vaccinating a subject in need thereof. The method includes orally administering to the subject a composition. The composition includes an effeciive amount of at least one bioactive agent layered over the substrate, wherein the at least one bioactive agent is stabilized in a stabilizer under conditions facilitating anhydrobiosis, and a cross-linking agent

[00077] The presently disclosed subject matter, in some embodiments, provides a method of controlling zoonotic infectious diseases by vaccinating a subject in need thereof. The method comprises adding a composition directly to a water supply in a suspension suitable for drinking. The composition includes an effective amount of at. least one bioactive agent layered over the substrate, wherein the at least one bioactive agent is stabilized in a stabilizer under conditions facilitating anhydrobiosis, and a cross-Unking agent.

[00078] In some embodiments, the presently disclosed subject matter provides an oral vaccine composition for reducing vector-borne and/or other zoonotic infectious diseases. As used herein, the term "zoonotic" or "zoonosis" refers to an infectious disease that may be transmitted between species. In some embodiments, infectious disease transmission may be facilitated by a disease-transmitting vector, to include, but not limited to an insect (mosquito or other) and/or arthropod (tick or other). In some embodiments, zoonotic disease is vector-borne.

[00079} As used herein, "vectors" are living organisms that can transmit infectious diseases between humans or from animals to humans. Many of these vectors are bloodsucking insects, which ingest disease-producing microorganisms during a blood meal from an infected host (human or animal) and later inject it into a new host during their subsequent blood meal. In some embodiments, the vector- borne zoonotic disease is Lyme disease. "Vector-borne diseases" are illnesses caused by pathogens and parasites in human populations. 'Vector-borne and other types of zoonotic infectious disease include, but are not limited to Artaplasmosis, Babesiosis, Ehrlichiosis, Rocky Mountain Spotted Fever, Dengue, or Malaria.

j 0080] la some embodiments of the presently disclosed subject matter, a method of controlling zoonotic infectious disease by vaccinating a subject in need thereof is provided. The method comprises orally administering to the subject a composition comprising a substrate and an effective amount of at least one active agent coated or layered over the substrate. In some embodiments, the subject is a reservoir host of the zoonotic infectious disease cycle. In some embodiments, the subject is a susceptible host of the zoonotic infectious disease cycle. In some embodiments, the subject is a xenodiagnostic carrier. Non-limiting examples of the subject include an arthropod, an insect, a .mammal, a bird, a fish, and/or a domesticated or companion animal, in some embodiments, the mammal includes a feral animal including one or more of a mouse, a chipmunk, a squirrel, a shrew, a vole, a rat, a raccoon, an opossum, a skunk, a rabbit, and a deer. In some embodiments, the subject is a domesticated or companion animal. In some embodiments, the domesticated animal including one or more of a dog, a cat, a cow, and a horse. In some embodiments, the subject' is a bird. In some embodiments, the subject is fis

[00081 } As used herein, the term "reservoir host" refers to an organism harboring infectious disease microorganisms or pathogens, but presenting as asymptomatic. Specifically, as referred to herein, the reservoir host serves in the transmission cycle of zoonosis by harboring infectious agents that may be transmitted to other reservoir hosts, or subsequent susceptible hosts, in some embodiments, a reservoir host may transmit an amount of an infectious agent to, for example, a feeding vector, which ma be transmitted to a subsequent reservoir host and/or subsequent susceptible host.

[000St2j As used herein, the terra "susceptible host" refers to an individual organism that is predisposed, vulnerable, and/or receptive to disease. Specifically, as referred to herein, the susceptible host is receptive to the disease transmitted by a disease-harboring vector carrier. Specifically, as referred to herein, the teen "xenodiagnostic carrier" may be employed to describe a diagnostic method to doe eiH the presence of infectious disease-presenting microorganisms, or pathogens., by exposing potentially infectious tissue to a naive vector, and then assaying the vector for the presence (ingested) of the same infectious agent(s).

[00083] The presently disclosed subject matter is further illustrated by the specific but non- limiting examples provided herein. Moreover, the following examples may include compilations of data that are representative of data gathered at various times during the course of development and experimentation related to the present disclosure.

[00084] Methods and results disclosed herein are provided to report an analysis of vaccine efficacy. An efficacious vaccine is defined as one that induces a sustained immune response based upon a designated administration protocol, lmmun.oresponsiven.ess is dependent, upon the presentation of an antigen to the immune system. For the present disclosure, antigenic presentation considers the utility of a biological system, a maintained bacterial whole-cell carrier whereby said antigen is stably expressed on the cell surface for effective presentation to the immune system upon administration. Bacterial carrier systems further present as natural adjuvants for use in the enhancement of the vaccine- induced inimone response. The following testing measures are employed to assay for the effectiveness of the vaccine; ihe embodiment below encompasses the candidate reservoir-targeted vaccine for Lyme disease. Testing involves assaying for bacterial strain identity and purity, p!asmid sequence based upon restriction digestion and sequencing, and protein expression following the standard operating procedures for the sterility testing of modified live biological products in accord with 9 CFR 113.27(b). Vaccine efficacy validation and testing further follows that which is in accord with VSM 800.202.

EXAMPLE 1

Vaccine Biologic Purity and Identity

[00085] This study relates to methods for confirmation identity and purity of B. coli antigenic carrier bacterial culture.

[00086] In this study, to assay for the viable/active culture re uired for the potency and efficacy of the vaccine, each of 10 bottles containing 40 mL Fluid Thioglycolate was inoculated with 0.2 mL from the QOm ~ 1.2 culture sample vial. The bottles were incubated for 14 days at 30 - 35°C. Additionally, each of 10 bottles containing 40 mL Trypticase Soy Broth was inoculated with 0.2 mL from the sample vial. The bottles were incubated for .1 days at 20 - 25°C. At the conclusion of the 14- day incubaiion period all 20 bottles displayed growth. No extraneous growth was observed in any of the bottles when examined macroscopic-ally.

[00087} From each bottle, 0.2 mL of sample was plated onto Trypticase Soy Agar with 5% sheep blood and incubated for 24 hours at 37 ± 2°C After the 24-hour incubation, the colony morphology for each sample vial was uniform and consistent with that of E. coll Microscopic examination revealed a uniform gram-negative rod consistent with thai of E. coll

:t? }θβ088] The identity of the microorganism was confirmed as E. coii using a 96 well Biolog Mkrostation System, following the procedures for the Identification of Microorganisms. The Biolog MicrosiatioH System identified the organism as E. coii with an 80.4% likelihood score.

EXAMPLE 2

Vaccine Antigen Expression Stability

[0008 ] I he following study relates further to the methods for confirming plasmid stability and isolation from culture, identified as E. coii per that which is outlined in Example 1

(00890) For 2 different sample vials, A and 8, a .10 fold dilution series was made using 1% bacio peptone. From the 10" dilution, 0.1 ml, was transferred onto LB plates containing 50 pg mL Kanamycm and incubated for 24 hours at 37 ± 2¾C, For both A and B sample plates, three culture tubes containing 1 .0 mL of LB broth with i O jig mL Kanamycm were inoculated using isolated colonies and incubated for 1 hours with continuous, low level agitation at 37 ± 2°C. The tubes were then cenfrifuged at 8000 rpm (x 4300g) for 3 minutes to pellet the cells. The Qiagen Spin MiniPrep Kit. was used to isolate plasmid DNA from the ceil cultures. The plasmid DMA yield was quantified using the NanoVue spec ophotometer. The samples with the highest plasmid yield, Ai and B L 53.5 pg/.roL and 83.0 pg mL respectively, were selected for additional analysis. The isolated plasmid was then used for both sequencing and restriction digest analysis to verity the OspA genes successful transformation into the E. coii.

[00091 ] Restriction Digest of Isolated Plasmid. The isolated OspA plasmid sample A I and .81 were digested with BamHl and del as presented in Table 1 (These samples were incubated at 37 ± 2°C for 1 hour).

TABLE 1

Restriction Digest Dilution Scheme

Figure imgf000019_0001

[00092] An undigested isolated OspA plasmid sample of A I and Bl were diluted as presented in Table 2.

TABLE 2

Undigested OspA Dilution Scheme OspA Piasmid Sample (AI or 81 ) 10 μί

Dl water 40 μΐ

[0ββ(>3| One ί 1 ,0) μΕοί the 1 kb ladder was diluted with 9 μΕ of Di water. GFive (5.0) }tL of the superceded DNA ladder was diluted with 5 μ.Ε of DI water. Ten (10.0) Τ of each sample was added to 2 μ.Ε of 6X loading dye. Twel ve (12) μΐ, of each sample was loaded onto a 1% agarose gel + 4 μL of Ethidiom Bromide as presented in Table 3.

TABLE 3

SDS PAGE Lane Ke

Figure imgf000020_0001

[00094] The gel was run at 100 V for 90 minutes with 0.5'X TBE ÷ 24 μ£ of Ethidiom Bromide, The gel was analyzed on Transillominator with UV light. Acceptance criteria requires that ( I) the OspA band most align with the 1 kb ladder band at 1.0 kb. The Plasmid backbone must align, with the ladders just above the 4 kb band; (2) the undigested samples must have a band slightly above the 5 kb band on the ladders; and (3) bands were present and at the appropriate size (See Table 4 and Figures I and 2). The result was satisfactory.

TABLE 4

Estimated Lengths of DNA Fragments after Digestion

Figure imgf000020_0002

[00095] Sequencing T7 region of Plasmid. The primers used in. the sequencing of the T? region of the expression plasmid construct included (I) the T7 Promoter Primer of sequence: TAATACGACTCAC A AGGG (SEQ ID NO: 1 ), and (2) the T? Terminator Primer of sequence: GCTAGTTATTGCTCAGCGG (SEQ ID NO: 2). isoiaied plasmids and T7 Primers were sent to Iowa State to be sequenced in both the forward and reverse directions using the Sanger method. Usin Blast- software found online at http: fbIast.ncbi.nfm mh.gov/. a Nucleotide Blast alignment was conducted against the subject sequences (Sbjet) obtained from Iowa State against a known OspA gene reference sequence (Query) found at http:/Vwww jicbi.nlffl.nih.gov/gene/1 1 435? fSEQ ΪΙ) NO: 3), Results demonstrated thai the .forward and reverse orientatio sequences had an identity score of 100% (Figure 3) and 99% (Figure 4) respectively.

EXAMPLE 3

Methods for Assaying Vaccine Protein Antigen Expression

[00096] The demonstration of plasmid stability and expression is validated via western blot analysis for assay of the expressed protein product, OspA. Protein extraction for western blot analysis followed with an expansion of the ospA -vectored E. coli cultures. B. coii samples were inoculated into 10 ml, of LB + Broth containing 100 itg mL Kanamycin and incubated overnight with continuous, low level agitation at 37 ± 2°C. The sample was centrifuged at. 8000 rpm (x 4300g) for 3 minutes to pelletize the ceils. The supernatant was then discarded. The -pellet was resuspended in i ml. of ice cold PBS. The sample was centrifuged at 8000 rp.m for 3 minutes to pelietize the cells. The supernatant was then discarded. The pellet was resuspended in 1 mL of ice cold PBS and transferred to a clean 1.5 mL microcentrifuge tube. The sample was centrifuged at 8000 rpm (x 4300g) for 3 minutes to pelleiize the cells. The supernatant was then discarded. The pellet was resuspended in 1 mL of an ice cold 20mM tris-HCl solution and allowed to stand on ice for 30 minutes as a cell lysis step. The resulting cell lysaie was centrifuged at i 0,000 rpm (x 6?20g) for 1 minutes. The supernatant containing the soluble protein fraction was transferred into a clean microcentrifuge tube. The cell pellet containing the insoluble protein, fraction was resuspended in. I ml, of an ice-cold 20mM tris-HCl solution. Both soluble and insoluble protein fractions wer used for western blot analysis.

[00097] Western blot. After protein was extracted, samples were prepared as presented in Table 5.

TABLE 5

Protein Reducing Agent + Buffer Reaction

Figure imgf000021_0001

[0009$} Samples were heated at 7 CC for 10 minutes on a heat block. Ten ( 10) μ-L of each sample was loaded onto a 4-1.2% B.is-Tris Gel as presented in Table 6.

TABLE 6 Western blot Lane Key

Figure imgf000022_0001

[06099] The gel was ran at 220v for 30 minules on the Bio Rad gel system in I X Novex MES SDS Running Buffer ÷ 1 mL Antioxidant. The PVDF transfer membrane, filter paper and pads were soaked in IX NuPAGE Transfer Buffer (75 mL 20X NuPAGE Transfer Buffer ÷ .1.5 mL antioxidant +

150 m.L Methanol + 1273.5 mL Dl water). The gel was prepared with the transfer membrane and ran at 30V for approximately 1 hour. The OspA protein-transferred PVDF membrane was activated with Methanol, for 2 minutes at room temperature. The membrane was dispensed into IX TBST (100 mL TBS 4- 900 mL Dl water ÷ 500 pL Tween 20) -f- 3% Skim milk into reservoir and rocked the membrane for 1 hour. The solution was discarded and the OspA MAb LA2.2.1 hybridoma sup (100 pL OspA MAb + 9.9 mL IX TBST) + 1% skim milk was then added and the membrane rocked for 1 hour at room temperature. The membrane was then washed with 1 X TBST three times. The membrane was rocked for 5 minutes at each washing. The secondary antibody (goat anti-mouse IgG AP) (Dilution 1 ; 1000) in 1 TBST + 1% skim milk was added and rocked with the membrane for 30 minutes at room temperature. The membrane was again washed with I TBST three times. The membrane was rocked for 5 minutes at each washing. Five (5.0) mL of BCiP/NBT membrane phosphatase substrate was then added. This solution was allowed to develop the membrane and the reaction was stopped with the addition of water. The membrane was dried at room temperature. The acceptance criterion requires that a band must be present at the violet 30398 Dal ions band on the MWM. Results demonstrated that a band was present at the 30398 Daiton for each sample (See Figures 5 and 6). The result was satisfactory.

[OOOlOOj E. co!i testing positive for stable piasmid expression of OspA protein antigen,, meets the criteria necessary for use as the biological whole cell carrier of vaccine antigen and can be coated onto suitable substrate for administration to target animals.

EXAMPLE 4

Composition of Suitable Vaccine Carrier Substrate Material

[000101 j In some embodiments, the substrates of the present disclosure are of a pelletked type, extruded, and/or of a non- or low-mitritionai value. Further, said substrates raay comprise ground corn, wheat middlings, cane molasses, calcium carbonate, animai fat, such as porcine fat preserved with butylated hydroxyanisole (BHA), dehulled soybean meal, ground oats, salt, dehydrated alfalfa meal whole wheat, ground soybean hulls, fish meal, dried beet pulp, dicalcmm phosphate, wheat germ, corn gluten meat and soybean oil- In certain embodiments, ie compositions of the present disclosure include extruded pellets; and in some embodiments, the extruded pellets are of high pellet durability index (PDl 90) and can be, for example, spheroid or cylindrical in shape. The high durability and spheroid shape of said pellet substrates allow pel Sets to maintain integrity and shape while being coated with vaccine in an efficient commercial drum tumbling coating process.

EXAMPLE 5

Composition of Vaccine Stabilization and Coating Matrix

(099102] in some embodiments, to accommodate vaccine for the coating process useful in the methods of the present disclosure, whole-ceil bacterial biomass is first scaled up under standard fermentation methods to a cell density of entry into the stationary phase. Culture conditions supporting the growth of the whole-cell bacteria! biomass follow those as presented by Studier (2005), for growth of cultures harboring the pET9c piasmicl expression vector. Briefly, a single-colony isolate of the ospA- veeiored BL2 i(DE3) coli is grown on Kan t- selection solid media. Isolates are individuall grown overnight in 3 niLs of a TBY+Kan liquid selection media. The liquid culture is then passaged at a ratio of 1 : 1000 into TBY+Kan induction media to induce the expression of the T7 promoter driving expression of the ospA gene, in some embodiments, the biomass of the present disclosure undergoes hollow-fiber filtration for liquid media reduction to generate wet-cell paste (WCP). Reduction in liquid media involves washing the WCP to a suspension with PBS. To maintain a whole-cell antigen carrier as the metric for dictating the minimal immunizing dosage (MID) the biomass is stabilized.

[000103 ( As used herein, PBS refers to phosphate-buffered saline, without the divalent cations of calcium and/or magnesium.

(0001043 As used herein, the stabilization matrix useful in the methods of the present disclosure refers to a solution of a final concentration of 500 mM to 625 mM sucrose fully dissolved in PBS. The stabilization matrix serves as a (1 ) cryo-stabilization matrix for cryopreservation of the biological composition, and/or as a (2) matrix solution to osmoiically stabilize the whole-cell bacterial cells under the down-stream encapsulation processing and drying conditions of anhydrobiosis. In some embodiments, the use of a final concentration of ! mM trehalose is employed as the sugar used in the stab ί li zation ma tr i.x .

(0001 OS] In some embodiments, the stabilization process ensues by slowly mixing the biomass-

PBS suspension 1 :1 with a 1 mM to 1.25 mM concentrated solution of stabilization matrix at 4°C, yielding a final concentration at 500 mM to 625 mM sucrose in PBS. This embodiment yields the osmoiically stabilized liquid vaccine component. The liquid vaccine component can be used immediately as the composition applied io suitable substrate material, or frozen down to -80°C under coating can be performed.

)000106) In some embodimenis, sodium alginate is used as an. encapsulation platform for stabilizing viable celts under conditions of anhydrobiosis. in certain embodiments, the methods described in reierences such as the Standard Guide for Immobilization or Encapsulation of Living Ceils or Tissue in Alginate Gels (ASI'M, Designation F231 -10, 2010, reference provided by FMC Biopolymer) and thai which is outlined according to Smidsrod and Skjak-Braek, Trends. Biolechnol, 8:71, 1990, are followed for the preparation of the sodium alginate mixture.

[000107) An example is presented of the linear chemical equation representing the crosslinking reaction: 2Na-ASginaie ·*· CaC¾ Ca~Algimue ÷ 2NaCl. in some embodiments, hydrocoHoid polymers present as enteric polymers. In some embodimenis, polymeric stabilization of some or all of the components of the compositions of the present disclosure may protect the components from the digestive pH of the stomach, thereby conserving th components for targeted release in the gut. For example, in certain embodiments, an orally administered biological vaccine composition comprises one or more ingredieots/componeois thai lias been poSymericaily stabilized. In some embodiments, a composition of the present disclosure comprises sodium alginate that has been cross-linked with calcium,

J 000108) in some embodiments, an aqueous suspension of sodium alginate, such as a 1 to 4% suspension, is prepared in a solution of SOOmM to 625roM sucrose in distilied de-ionized water. The suspension is mixed for about 6 hours by agitation at room temperature, or until fully dissolved yielding a suspension of a sodium alginate-sucrose syrup. The osmoticaliy-stabil ized liquid vaccine component is then gently mixed with the alginate solution at room temperature. The liquid vaccine - sodium alginate mixture is then applied onto spheroid animal food pellets. The semi-dry vaccine - sodium alginate coating is then subsequently coated with a sprayed mixture of l O mM to 225mM calcium 'lactate (up to 7% solution, as a source tor crosslinking multivalent ion with enhanced taste; a 5% to 7% solution of calcium chloride may also be used) as a cross-linking agent to facilitate the generation of a calcium-alginaie micro-encapsulated bacterial cell layer upon the pellet substrates, as illustrated in the schematic in Figure 7. Caieium-alginate cross-linked/encapsulaiion maintains the viability and immunogenic integrity of the bacterial-based vaccine antigen.

EXAMPLE 6

Methodology for Spray Coating Stabilized 'Vaccine as a Top-dressing onto

Substrate Composition

00 109| Spray drying is a common practice for the stable drying, for enhanced shelf life, of biological materials. The prior art includes the application of technologies for the spray drying of biological materials for collection and utilization downstream. Biological stability is enhanced through the process of encapsulation whereby said biological, materia! is in composition with a hydrocolloid polymer and then mixed with a cross-linking compound. Spray-dried encapsulated biological material is then collected for utilization. The present invention introduces the application of spraying a biological material, in composition with a hydrocolloid or other encapsulation composition, onto a substrate material. The substrate material serves as a solid-support carrier onto which the spray-coated biological material may be subsequently sprayed with (1) cross-linking agents to facilitate encapsulation, and (2) moisture barrier glazes or shellacs, flavoring or scented baiting attractants. The coated pellets can then, be subjected to a gentle drying process whereby the coated materials may be dried.

ΙΘ0011Θ] Substrate materials of spheroid, or "cleaned" pellet substrates facilitate a "fluid" tumbling process during a coating-drum tumbling process. Pellet substrates are fed into a coating drum at a given rate to effectively yield appropriate dose coverage when spray-coated. Pellets are gently and. uniformly rolled/tumbled within the drum while concurrently being spray-coated with the sucrose- stabilized/alginate-encapsulated bacteria. Spraying is tightly controlled (can be electrostatically delivered) and automatically linked to the tumbling duration to ensure even coaling coverage, and proper dosage equating to the designated MID as measured in colony forming units (CFU) per pellet. The secondary spray coating facilitates the crosslinking reaction as reacted by calcium. An optional tertiary coating of a final outer sealant, of a confectionary glaze and/or pharmaceutical shellac in combination with an aitractant fiavor additive facilitates free-flowing pellets for handling, storage, and distribution; flavor additive provides palatable atiractam" to target animal reservoir hosts of Lyme disease. Drying occurs while tumbling, or by spreading product across a high surface-area drying table. Process yields high throughput under continuous batch operation.

JOOOl l lI In som embodiments, the employment of a method for centrifugaUy applying the biological material in composition with a hydrocolloid or other encapsulation composition, and subsequent cross-linking agent as coated matrix onto the surface of the pellets is considered using centrifugal batch coating technology. As illustrated in Figure 8 and used herein in the present invention disclosure, the use of centrifugal, coating equipment is employed for the high throughput commercial operation of efficiently coating tile substrate pellet (herein, ''pellets") material. The pellets (101 ) are held in bulk (batch hopper, 106) for entry into the coating drum (1.07) where they are gently folded and agitated under constant revolution, as driven by a standard motorized system (108). Biological material in composition with the sodium alginate 'hydrocolloid stabilization matrix (Reagent A, 102) (or other encapsulation composition) is maintained in reserve (109) and in suspension via an onboard motor- driven mixer (1 10-1 1 1). Similarly, calcium lactate crosslinking agents (Reagent B, 103) and moisture barriers are also held in reserve (1 13). Both reagents are added in succession (115) via a peristaltic pumping process (1 12, .1 .14) and coated onto the folding pellets through an aioniizaiion process ( 104) facilitated via dispensing onto a separate motorized (1 17) spinning centrifugal disc (1 16) positioned central to the revolutions of the folding substrate, thereby evenly coating the pellets ( 105) with the designated MID.

EXAMPLE 7

Methods for Demonstrating Vaccine Stability

[600112] An effective shelf life of the embodiment presented herein is essential to accommodate distribution schedules and campaigns. Stabilized vaccine-coated pellets present as water-insoluble articles for oral administration, entericaliy stable for effectively administering the vaccine to the GALT. Stabiiiiy/siieli-liie analyses were conducted on dried, vaccine coated pellets. Briefly, each individual vaccine pellet test article was rehydrated in a 4 mL volume of 55 mM sodium citrate solution of HEP'ES buffered saline, at a pH of 7.4. Volumes were incubated at 35eC for 1 hour under agitation, 100 ΐ. of the pellet supernatant, containing reieased de-encapsulated viable OspA-vectored whole cell E. co!i was plated out on a TB+ a agar plate and cultured overnight for the assessment of colony growth. Resulting colonies were calculated as approximate CPU per mL representative of the colonies release upon rehydration from each, vaccine-coated peliei; the resultant CPU counts were evaluated as a surrogate marker of the vaccine stability and decay (shelf life.) over time. This procedure was employed weekly upon, post-production vaccine-coaied pellets for up io 1.0 weeks. Results as presented in Figure 9 suggest that the viability of the active vaccine component, the £ colt antigenic carrier, maintain stable and viable, with a decay factor of 2 over an 8- week assay period.

EXAMPLE 8

Methods for Demonstrating Vaccine Efficacy

[000113] Vaccine efficacy is determined based upon ability to induce sero-responsiveness in an animal following administration of antigen. The employment of an indirect enzyme-linked immunoabsorbant assay (ELISA) for antibody titers raised against an antigen or mitriunogen is a surrogate marker for the vaccine efficacy. As used herein, the use of the indirect ELISA methodology is qualified for the detection of serum antibodies directed against OspA. The assay is conducted wherein a purified OspA target capture antigen protein is coated onto a solid support. Primary antibodies in the form of an OspA-specific monoclonal antibody (positive control, MAb LA2.2), or

OspA serum antibodies (test subject) are then allowed to bind to the target antigen. A secondary antibody conjugate specific to the primary antibody is then added, and the reaction is developed to indicate the relative levels of serum OspA-specific responsive antibodies. The procedural follow with the use of flat-bottom wells. The purified protein is then diluted to a final concentration 0.5 to 2 pg/niL in coating buffer ( 10 ml. of coating buffer solution for one plate - 96 wells), and to each well is dispensed at a volume of 100

Figure imgf000027_0001
an 8-channel pipette). The plate is covered and incubated at room temperature, with no shaking, for 1 h or at 4 C overnight (ideally). The solution is discarded and dried out on a paper towel 300 μΕ of blocking buffer is added (PBST + 1% BSA) to each well The plate is again covered and incubated at 37 C (so shake) for 1 h. The plate is washed 3x for each well with 300 μί, 1 x PBST. To each well is added 100 μΐ. of diluted serum sample (1 : 100, 1:500) in blocking/sample diluent; for control wells, a MAb standard curve (500, 250, 125, 62.5, 1.25 ng mL) is added. The plate is covered and incubated at room temperature for I h. The solution is discarded and again dried out on a paper towel. Each well of the plate is washed 4 x with 300 μΐ, Ix PBST (for ELISA). Dilute Bt-MAb purified protein at a final concentration 0.3 g/m.L in blocking sample diluent (make 10 rnL of coating buffer solution for one plate - 96 wells) is dispensed at 100 ,uL in each well (using an 8-ehanuel pipette). The plate is covered and incubated at room temperature for 1 h, after which the solution is discarded and the plate dried on a paper towel. Each well is washed 4 x with 300 uX I PBST (for ELISA). Nentravidio conjugated HRP is diluted 1:1.000 in blocking/sample diluent and added to each well at a volume of 100 μL. The plate is covered and incubated at room temperature for 30 min, alter which the solution is discarded and the plate dried out on a paper towel The plate is washed 4 x in each well with 300 μL Ix PBST (for ELISA). 100 μΙ of SureBlue (K.PL) substrate is added to each well (the positive samples will turn blue). The plate is covered and incubated at room temperature between 30 min and 1 h. The absorhance is read at 650 nm for blue color and 450 nm for yellow color after addition of 100 ΐ. of stop solution (K.PL).

(000114) As disclosed herein, the vaccine efficacy, or sero-protection is considered by assessing OspA-specific serum antibody titers equivalent to an OD450 value as assayed by ELISA. Early optimization studies conducted on the core vaccine technology formulated as presented in Riche et al., Clin. Vaccine Immunol 18; 1809, 201 1, and also disclosed herein (as referenced in US 8,821,893, incorporate by reference in its entirety) sought to define the sero-responsiveness in mice in response to oral vaccine administration as measured by the qualified ELISA methods. A vaccine "unit" represents a dose, and equates to approximately log 5x1 * CFU. Multiple dosages equating to 0, 6, 12, and 30 units were administered weekly (days 0, 14, 28, and 42} to mice for up to 6 weeks. As presented in Figure 10, the sero-response peaks around an OD450 serum antibody titer equivalent of approximately 1.5. For commercial application, it is considered relevant that a minimum number of dosages be applied in order to generate the most efficacious sero-response. thereby defining the MID. From the results presented in Figure 10, a weekly dosage of up to 6 dosages total was considered for the commercial e volution of the vaccine technology application disclosed in the present subject matter; the data demonstrate that an OD450 serum antibody titer equivalent to approximately 0.6 is obtained after about 21 days of vaccine administration at a dosase of 6 units/week. [00011 Si Experimental evidence has further demonstrated efficacy of the core vaccine technology when administered orally across multiple mouse species. Study summaries are outlined in Table 7,

TABLE 7

Summary of Vaccine Efficacy

Figure imgf000028_0001

[000116} Study 1 is a laboratory-based Borrelia challenge study,