Vaccine composition for mucosal immune response

Abstract

The present invention provides a vaccine composition with improved stability and ease of administration, as well as a solid dosage form of the vaccine for efficient administration and manufacture. [Solution] A vaccine composition is provided comprising a freeze-dried adenovirus-based expression vector and a stabilizing compound such as aragonite. Further provided is a composition comprising a solid dosage form made from aragonite for loading and delivery of the vaccine composition.

Description

Technical Field

[0001] Cross-reference of related applications This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 104,770, filed October 23, 2020, and U.S. Provisional Patent Application No. 63 / 082,907, filed September 24, 2020, each of which is incorporated herein by reference in its entirety.

[0002] The present disclosure generally relates to the field of vaccine compositions. In particular, the present disclosure relates to vaccine compositions having improved stability and ease of administration, as well as solid dosage forms of vaccines for efficient administration and manufacture.

[0003] Sequence listing reference This application includes a reference to an amino acid sequence and / or nucleic acid sequence submitted herewith simultaneously with this specification as a Sequence Listing text file "8774-16-PCT_Sequence_Listing_ST25.txt", created on October 22, 2021, with a file size of 30,000 bytes (B). The foregoing Sequence Listing is incorporated herein by reference in its entirety in accordance with 37 C.F.R. § 1.52(e)(5).

Background Art

[0004] Vaccines can be made by leaving a culture to attenuate the virulence of an infectious microorganism. However, more recently, modern recombinant techniques have been used to produce viral vectors that produce antigen proteins and can express antigen proteins against various infectious microorganisms.

[0005] Regardless of the methods or technologies used to manufacture vaccines, a persistent challenge is the ability to store vaccines while maintaining their efficacy. Currently, a crucial part of maintaining vaccine efficacy relies on expensive refrigeration systems. To reduce the need for such expensive supply chains, manufacturers have attempted to add chemical stabilizers to vaccines. However, such excipients are often toxic and can trigger allergic reactions.

[0006] Vaccination protocols have problems not only with stability but also with delivery routes. Many vaccines are administered by injection, which is not preferred by many people.

[0007] Vaccines are traditionally delivered by intramuscular, intradermal, or subcutaneous injection. While these injections can produce a potent systemic immune response, their effectiveness in inducing mucosal immune responses varies, and is often weak or undetectable, especially for subunit vaccines. Antibodies produced by antigen-specific cytotoxic T cells (CTLs) and B cells can migrate from the influx-region lymph nodes treated with the injected vaccine to various organs of the body, but their migration to various mucosal tissues (e.g., genital, intestinal, respiratory) is often limited or impossible due to inadequate homing mucosal receptors and chemotaxis. However, the intranasal route, also considered a parenteral immunization pathway, can induce a good mucosal immune response in the respiratory, genital, and intestinal tracts, which share several interconnections, and this is more achievable when the vaccine is delivered to a mucosal site. Thus, such parenteral vaccines can, in some cases, provide protection against mucosal pathogens.

[0008] Most pathogens (e.g., COVID-19) enter the body through mucosal tissues (oral cavity, respiratory tract, genital tract, and intestinal tract), and since many pathogens replicate only in mucosal tissues, mucosal vaccination can optimally induce first-line protection by inducing both innate immune responses (e.g., NK cells) and adaptive immune responses (T cells and B cells) at local and distal mucosal sites.

[0009] Even considering the currently approved COVID-19 vaccines, rapid and global distribution and administration of vaccines remains challenging. Global immunization has not yet been achieved with currently approved vaccines due to numerous factors, including manufacturing and / or storage costs and other requirements.

[0010] Mucosal vaccine delivery (via the buccal, sublingual, nasal, oral, or vaginal mucosa) is gaining increasing interest as a means of inducing local and distal antibody immune responses as well as systemic immune responses. In addition, mucosal vaccine delivery in solid dosage forms (e.g., buccal / sublingual tablets, oral tablets or capsules, or vaginal inserts) can offer several advantages, including the potential for herd immunization, patient compliance, ease of use, product shelf-life stability, and cold chain-independent performance. Furthermore, mucosal vaccine delivery may be suitable for patients with needle phobia, who can self-administer the vaccine after receiving appropriate instructions. While the buccal / sublingual route has been used for many years to deliver drugs and small molecules into the bloodstream, its application as a means of mucosal vaccine delivery has not been conventionally developed.

[0011] Solid dosage forms for drug delivery, including powders, tablets, or capsules, require disintegration and release of the active ingredient (e.g., vaccine) upon administration. Simultaneously, the dosage form containing the active ingredient must remain stable during transport and storage from manufacturing to administration. Therefore, the use of solid dosage forms requires the stability of the active ingredient, which is supported in the solid form and then readily disintegrates in an aqueous solution (e.g., water, saliva, or mucus). [Overview of the project] [Problems that the invention aims to solve]

[0012] Therefore, there is a need for a vaccine composition that offers improved stability and ease of administration, thereby enhancing the effectiveness of vaccination rates. Furthermore, there is a need for an improved solid vaccine dosage form that serves as a suitable platform for vaccine loading, is stable before disintegration (administration), disintegrates easily, has low manufacturing costs, and is easy to administer for effective global immunization. This disclosure addresses these needs and also provides further benefits. [Means for solving the problem]

[0013] One embodiment relates to a composition comprising a freeze-dried adenovirus vector and an excipient containing a carbonite mineral, wherein the adenovirus vector comprises a nucleic acid molecule encoding at least a portion of a heterologous protein.

[0014] In one embodiment, the adenovirus vector is derived from adenovirus type 5, and the adenovirus has deletions in the E1, E2b, and E3 regions.

[0015] In various forms, heterologous proteins originate from viruses.

[0016] In various embodiments, heterologous proteins are derived from viruses selected from the group consisting of SARS-CoV-2, MERS-CoV, SARS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, HKU1, and influenza viruses.

[0017] In various embodiments, the heterologous protein is derived from SARS-CoV-2. In some embodiments, the heterologous protein is a spike (S) protein, a nucleocapsid (N) protein, or a membrane (M) protein. In yet another embodiment, the heterologous protein is at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 97%, or even 100% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0018] In various embodiments, the adenovirus vector has a residual moisture content of 0.5% to 5%. In one embodiment, the adenovirus vector has a residual moisture content of less than 5%. In yet another embodiment, the adenovirus vector has a residual moisture content of less than 3%.

[0019] In various embodiments, carbonite minerals have an orthorhombic lattice. In one embodiment, the carbonite mineral is selected from the group consisting of aragonite, celsite, strontianite, wizerite, and rasafordine. In one embodiment, the carbonite mineral is aragonite.

[0020] In various embodiments, the excipient comprises one or more compounds selected from the group consisting of lactose, sucrose, magnesium stearate, glucose, mannitol, sorbitol, starch, dextrose, maltodextrin, maltitol, and plant cellulose.

[0021] In various embodiments, the excipient lacks one or more compounds selected from the group consisting of lactose, sucrose, magnesium stearate, glucose, mannitol, sorbitol, starch, dextrose, maltodextrin, maltitol, and plant cellulose.

[0022] In various embodiments, the composition comprises one or more compounds selected from the group consisting of sodium chloride, potassium chloride, sodium citrate, sodium phosphate, sucrose, dimethylglycine, glycine, methylsulfonylmethane, and yeast lysate.

[0023] Another embodiment relates to a capsule comprising the composition disclosed herein. In one aspect, the capsule is enteric-coated. In yet another aspect, the capsule comprises alginate.

[0024] Another embodiment is a solid dosage form for the delivery of a vaccine composition by oral, sublingual or buccal administration, comprising an aragonite composition comprising a plurality of aragonite particles impregnated with carbon dioxide (CO2), a biocompatible polymer and a disintegrant mixed with the aragonite composition, further comprising the vaccine composition, and being a powder, tablet or capsule.

[0025] In one aspect, the solid dosage form further comprises at least one additive and is formulated to form a lozenge.

[0026] In another aspect of the solid dosage form, the plurality of aragonite particles have an average particle size of 100 nm to 1 mm.

[0027] In various embodiments of the solid dosage form, the biocompatible polymer is selected from polylactic acid (PLA), polyethylene, polystyrene, polyvinyl chloride, polyamide 66 (nylon), polycaprolactam, polycaprolactone, acrylic polymer, acrylonitrile butadiene styrene, polybenzimidazole, polycarbonate, polyphenylene oxide / sulfide, polypropylene, Teflon®, polylactic acid, aliphatic polyester, such as polyhydroxybutyrate, poly-3-hydroxybutyrate (P3HB), polyhydroxyvalerate, polyhydroxybutyrate-polyhydroxyvalerate copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), polyglyconate, poly(dioxanone), and mixtures thereof.

[0028] In one embodiment of the solid dosage form, the biocompatible polymer is PLA. In one embodiment of the solid dosage form, the biocompatible polymer is Eudragit L30 D-55 (Evonik).

[0029] In various embodiments of the solid dosage form, the disintegrant is selected from starch, modified cellulose gum, insoluble crosslinked polyvinylpyrrolidone, starch glycolate, microcrystalline cellulose, pregelatinized starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, homopolymers of N-vinyl-2-pyrrolidone, alkyl-, hydroxyalkyl-, carboxyalkyl-cellulose esters, alginates, and microcrystalline cellulose and their polymorphs.

[0030] In one embodiment of the solid dosage form, the disintegrant is pea starch.

[0031] In one embodiment of the solid dosage form, the vaccine composition comprises a recombinant viral expression construct encoding a viral protein or a fragment thereof. In one embodiment, the viral protein or fragment thereof corresponds to a coronavirus protein or a fragment thereof. In yet another embodiment, the coronavirus protein or fragment thereof is a SARS-CoV-2 virus-binding protein. In one embodiment, the recombinant ACE2 protein has at least 85% sequence identity with SEQ ID NO: 5. In yet another embodiment, the recombinant ACE2 protein comprises the sequence of SEQ ID NO: 6. In yet another embodiment, the recombinant ACE2 protein comprises at least one mutation selected from T27F, T27W, T27Y, D30E, H34E, H34F, H34K, H34M, H34W, H34Y, D38E, D38M, D38W, Q24L, D30L, H34A, and D355L.

[0032] In one embodiment of the solid dosage form, the vaccine composition comprises an adenovirus expression construct.

[0033] Another embodiment relates to a method for preparing a solid dosage form for carrying a vaccine, comprising the steps of: providing an aragonite composition comprising a plurality of aragonite particles impregnated with carbon dioxide (CO2); mixing the aragonite composition with a biocompatible polymer and a disintegrant to form a solid dosage form; and adding a vaccine composition to the solid dosage form.

[0034] In one embodiment of a method for producing a solid dosage form, the step of mixing an aragonite composition with a biocompatible polymer and a disintegrant includes hot-melt extrusion.

[0035] In another embodiment of the method for preparing a solid dosage form, the solid dosage form is a powder, a tablet, or a capsule.

[0036] In yet another embodiment of the method for producing a solid dosage form, the solid dosage form is a tablet, and the method further includes the step of compacting the solid dosage form.

[0037] In another embodiment of the method for preparing a solid dosage form, the aragonite composition and the biocompatible polymer are in a weight ratio of 95:5 to 5:95.

[0038] In yet another embodiment of the method for preparing a solid dosage form, the plurality of aragonite particles have an average particle size of 100 nm to 1 mm.

[0039] In another embodiment of the method for producing a solid dosage form, the biocompatible polymer is selected from polylactic acid (PLA), polyethylene, polystyrene, polyvinyl chloride, polyamide 66 (nylon), polycaprolactam, polycaprolactone, acrylic polymer, acrylonitrile butadiene styrene, polybenzimidazole, polycarbonate, polyphenylene oxide / sulfide, polypropylene, Teflon®, polylactic acid, aliphatic polyester, such as polyhydroxybutyrate, poly-3-hydroxybutyrate (P3HB), polyhydroxyvalerate, polyhydroxybutyrate-polyhydroxyvalerate copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), polyglyconate, poly(dioxanone), and mixtures thereof.

[0040] In yet another embodiment of the method for preparing the solid dosage form, the biocompatible polymer is PLA. In yet another embodiment of the solid dosage form, the biocompatible polymer is Eudragit L30 D-55 (Evonik).

[0041] In yet another embodiment of the method for producing a solid dosage form, the disintegrant is selected from starch, modified cellulose gum, insoluble crosslinked polyvinylpyrrolidone, starch glycolate, microcrystalline cellulose, pregelatinized starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, homopolymers of N-vinyl-2-pyrrolidone, alkyl-, hydroxyalkyl-, carboxyalkyl-cellulose esters, alginates, and microcrystalline cellulose and their polymorphs.

[0042] In yet another embodiment of the method for preparing a solid dosage form, the disintegrant is pea starch.

[0043] In one embodiment of a method for preparing a solid dosage form, the vaccine composition comprises a recombinant viral expression construct encoding a viral protein or a fragment thereof. In one embodiment, the viral protein or fragment thereof corresponds to a coronavirus protein or a fragment thereof. In yet another embodiment, the coronavirus protein or fragment thereof is a SARS-CoV-2 virus-binding protein. In one embodiment, the recombinant ACE2 protein has at least 85% sequence identity with SEQ ID NO: 5. In yet another embodiment, the recombinant ACE2 protein comprises the sequence of SEQ ID NO: 6. In yet another embodiment, the recombinant ACE2 protein comprises at least one mutation selected from T27F, T27W, T27Y, D30E, H34E, H34F, H34K, H34M, H34W, H34Y, D38E, D38M, D38W, Q24L, D30L, H34A, and D355L.

[0044] In one embodiment of a method for preparing a solid dosage form, the vaccine composition comprises an adenovirus expression construct.

[0045] Embodiments discussed in relation to the methods and / or compositions described herein may be used in relation to any other methods or compositions described herein. Thus, embodiments relating to one method or composition may also be applicable to other methods and compositions.

[0046] Other purposes, features, and advantages will become apparent from the following detailed description. However, naturally, the detailed description and specific examples are given only as examples, illustrating specific embodiments, for various modifications and variations within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawing]

[0047] [Figure 1]As shown, the chemical structure and symmetry of CaCO3 are shown for calcite, aragonite, and vaterite, respectively. [Figure 2A] This is a photograph of a biocompatible bioplastic aragonite composition containing 40% aragonite, formed by three-dimensional (3D) printing of an extruded filament according to an embodiment of the present disclosure. [Figure 2B] This is a photograph of a biocompatible bioplastic aragonite composition containing 40% aragonite, formed by three-dimensional (3D) printing of an extruded filament according to an embodiment of the present disclosure. [Figure 3-1] The results for lyophilized adenovirus vectors (Ad5) of known viral titers, filled into capsules with either aragonite or lactose, are shown. The infection units / gram (without acid (Figure 3A) or with acid (Figure 3C)) or percentage of virus recovery (without acid (Figure 3B) or with acid (Figure 3D)) for the following aragonite or lactose formulations are shown: Aragonite, coated (C)1 (0.357g dry, powder, pH 8.81), Aragonite (C)5 (0.425g dry, powder, pH 8.84), Lactose, uncoated (NC)4 (0.8023g, liquid, pH 2.14), Aragonite (NC)6 (0.545g, paste, pH 7.78), Aragonite (NC)7 (0.629g, paste, pH 7.79), and Aragonite (NC)8 (0.801g, paste, pH 7.00). Samples formulated with aragonite were maintained at pH > / = 7 when exposed to acid. Each bar represents one capsule. [Figure 3-2] Continuation of Figure 3-1. [Figure 3-3] Continuation of Figure 3-2. [Figure 3-4] Continuation of Figure 3-3. [Modes for carrying out the invention]

[0048] The following sections describe various embodiments in more detail. Unless explicitly stated otherwise, each embodiment can be combined with any one or more other embodiments. In particular, any feature shown to be favorable or advantageous can be combined with any other feature shown to be favorable or advantageous.

[0049] Certain features of this disclosure are described in the content of separate embodiments, but may also be provided in combination in a single embodiment. Conversely, various features of this disclosure described in the content of a single embodiment may also be provided separately or in any suitable partial combination. All combinations of the disclosed embodiments are expressly encompassed by this disclosure and are disclosed herein as if any combination were individually and expressly disclosed. In addition, all partial combinations are also expressly encompassed by this disclosure and are disclosed herein as if any such partial combination were individually and expressly disclosed.

[0050] This disclosure is not limited to the specific embodiments described herein. Technical terms used herein solely to describe specific embodiments are not intended to limit the scope. Publications mentioned herein are presented only in relation to their disclosures prior to the filing date of this application. This specification does not assume that this disclosure is not preceded by such publications on the grounds of prior disclosure. Furthermore, the presented publication dates may differ from the actual publication dates, which may need to be verified individually. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials that the publications refer to in relation thereto.

[0051] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by vaccine scientists. Any methods and materials similar to or equivalent to those described herein may also be used in the implementation or testing of the vaccines disclosed herein, but preferred methods and materials are described below.

[0052] As disclosed herein, vaccine compositions are provided in a stable and easily administered form. More specifically, by lyophilizing a recombinant virus-derived vaccine and combining the vaccine with a suitable stabilizing compound, a composition is produced that is stable and can be packaged for stable storage, transport, and easy administration. For this purpose, the compositions of the disclosure can be produced by lyophilizing an adenovirus vector and combining the adenovirus vector with a carbonite mineral such as aragonite. Thus, the compositions of the disclosure generally comprise a lyophilized adenovirus vector and a carbonite mineral such as aragonite.

[0053] The subject further includes compositions and methods for producing vaccine dosage forms using aragonite to form solid dosage forms (e.g., powders, tablets, or capsules) that are stable during storage, easily administered (e.g., self-administered), and readily disintegrate when administered mucosally (e.g., sublingual or buccal) or in aqueous solutions for ingestion.

[0054] In particular, the subject matter of the invention relates to an aragonite composition made of a plurality of aragonite particles, the aragonite particles capable of carrying a vaccine composition, resulting in a solid-form vaccine in the form of a powder, tablet, or capsule. In exemplary embodiments, the vaccine composition provides immunity against coronavirus. More specifically, the vaccine composition is a recombinant virus expression construct for expressing an antigen corresponding to the relevant infection / disease. Preferably, the recombinant construct is an adenovirus construct expressing the antigen protein or antigen protein fragment of coronavirus.

[0055] In particular, as further detailed herein, the use of aragonite in currently envisioned solid dosage forms enables the cost-effective production and easy administration of stable vaccine compositions. Therefore, vaccines in the currently envisioned powder, tablet, or capsule forms can be mass-produced and easily transported. Furthermore, the rapid disintegration of solid dosage forms (e.g., 30 seconds or less) allows for self-administration. For example, vaccines in powder form can be packaged in individual dose packaging. Exemplary packaging for powder dosage forms is similar to packaging such as TWININGS® tea bags. Vaccine powders can be opened and dissolved in water or a suitable liquid beverage (e.g., from a beverage container or dropper) for ingestion, thereby allowing oral administration of the vaccine released into the liquid. In addition, considering that vaccines in powder, tablet, or capsule forms disintegrate when in contact with aqueous fluids, sublingually, or buccally, human saliva disintegrates the dosage form, thereby releasing the vaccine composition into the oral mucosa for absorption. In further embodiments, tablets can be formed from compressed vaccine powder. The tablets can be compressed into any suitable shape, such as round or cubic. The tablet form can also be prepared with additional additives (e.g., flavorings and gelatin) to form lozenges.

[0056] Aragonite (e.g., oocyte aragonite) is one of the purest forms of naturally precipitated calcium carbonate. With respect to Figure 1, aragonite has orthorhombic, bipyramidal, and characteristic acicular crystalline forms, thus distinguishing it from calcite and vaterite. Aragonite can be processed, recrystallized, and / or reformed into various shapes, and can therefore be used for a variety of purposes that utilize the mechanical and chemical properties of calcium carbonate minerals. The aragonite particles disclosed herein are solids having regular (e.g., spherical or oval) or irregular shapes. As used herein, aragonite particles have an average particle size of 100 nm to 1 mm. Methods for grinding aragonite particles are described in U.S. Patent Application Publication 16 / 858,548 and International Patent Application PCT / U.S. Patent Application Publication 20 / 29949, the entire contents of which are incorporated herein by reference. For example, a method for grinding aragonite particles to a clean top size of 2.5 to 3.5 microns is disclosed. The clean top size means that when produced using the disclosed grinding method in a classifier set to a size range of 2.5 to 3.5 microns, there are virtually no particles larger than 3.5 microns. Therefore, the aragonite particles disclosed herein using the method described in U.S. Patent Application Publication 16 / 858,548 and International Application PCT / U.S. Patent Application Publication 20 / 29949 have a cleaner top size than conventional GCC.

[0057] The adsorption capacity of aragonite is determined by three parameters: (1) surface charge (also known as "zeta potential"), (2) surface area / void ratio, and (3) particle solubility. By accurately measuring these three parameters, it is possible to determine which materials will adsorb to the surface of aragonite particles under given conditions. In particular, the zeta potential of aragonite enhances the stability of surfactants such as glycerol and sorbitol.

[0058] Furthermore, aragonite naturally possesses numerous measurable pores less than 2 nm in diameter within its particles (i.e., a highly microporous structure). See, for example, European Patent No. 2719373. Therefore, the aragonite platform strongly grips active ingredient particles, while simultaneously enabling the supported aragonite to be formulated into solid dosage forms—for example, powders, tablets, or capsules.

[0059] Advantageously, untreated aragonite has a neutral pH (7.8–8.2), natural hydrophilicity, an electronic charge (zeta potential), and already formed nitrogen-amino acid and protein pairings. While not bound by any theory, these advantageous properties of aragonite make it metastable under ambient conditions. More specifically, aragonite particles naturally contain approximately 2–3% amino acids, the majority of which are aspartic acid (approximately 25–30%) and glutamic acid (approximately 8–10%), which make the aragonite surface hydrophilic. See, for example, Mitterer, 1972, Geochimic et Cosmochimica Acta, 36:1407–1422. Therefore, in some embodiments, a vaccine composition (e.g., recombinant adenovirus) is directly bonded to the surface of natural, untreated aragonite particles.

[0060] Currently, calcium carbonate available on the market is produced as heavy calcium carbonate (GCC), precipitated calcium carbonate (PCC) (synthetic), and / or as limestone products or processed from these. The products produced are of different commodity grades with varying properties. To obtain a clean maximum particle size distribution (PSD) and to keep residues low, most companies utilize wet grinding processes, either high-solids or low-solids. However, these products and processes are neither biogenic nor environmentally friendly. As used herein, aragonite refers to natural aragonite that has orthorhombic, bipyramidal, and characteristic acicular crystalline forms, distinct from GCC, PCC, and limestone.

[0061] One embodiment is a composition comprising or consisting of a freeze-dried adenovirus vector and an excipient containing or consisting of a carbonite mineral, wherein the adenovirus vector comprises a nucleic acid molecule encoding at least a portion of a heterologous protein. An "adenovirus vector" is an adenovirus whose genome lacks one or more genes necessary for adenovirus replication in unmodified mammalian cells. An "adenovirus" (abbreviated as "Ad") refers to a group of non-enveloped, double-stranded DNA viruses belonging to the family adenoviridae, with a diameter of approximately 60-110 nm. The adenovirus vectors disclosed herein may be derived from any of the four genera of the family Adenoviridae (e.g., aviadenovirus, mastadenovirus, atadenovirus, and siadenovirus), as well as any serotype of each species of adenovirus. In humans, most adenovirus infections are asymptomatic and not associated with neoplastic diseases. The most well-characterized Ad serotypes are serotype 2 (Ad2) and serotype 5 (Ad5). In one embodiment, the adenovirus vector used herein is an Ad2 vector. In another embodiment, the adenovirus vector is an Ad5 vector.

[0062] The adenovirus dsDNA genome is approximately 36 kb long. Its genome contains two sets of genes: early region genes E1A, E1B, E2, E3, and E4, which are transcribed before DNA replication, and late region genes L1-L5, which are transcribed after the initiation of DNA replication and expressed at high levels. The early region genes are necessary for activating transcription in other regions of the virus, altering the host cell environment to enhance viral replication, and replicating viral DNA. The E1A transcription unit encodes two major E1a genes involved in viral transcriptional regulation. Two major E1b genes are involved in stimulating viral mRNA transport, blocking E1A-induced apoptosis, and blocking host mRNA transport. The E2b genes encode viral polymerases and terminal protein precursors.

[0063] As used herein, “activity” and “function” refer to the ability of a molecule to perform a certain action. For example, adenovirus E2a binds to numerous cellular factors and modulates their activity, thereby leading host cells into the S phase. Each of these actions—binding to cellular factors, modulating their activity, and leading cells into the S phase—can be considered E1a activity. As a result of the absence of one or more activities, an adenovirus vector cannot replicate in unmodified mammalian cells (“replication-deficient”). As used herein, “unmodified” mammalian cells lack the DNA encoding the adenovirus protein. A replication-deficient adenovirus vector can replicate in helper cells. Helper cells are mammalian cells that have DNA encoding proteins that provide—trans-—one or more activities necessary for adenovirus replication that the adenovirus vector cannot perform.

[0064] Modifications (also called mutations) of the adenovirus genome that result in the generation of an adenovirus vector can be made at any location in the genome, as long as the modification eliminates at least one function necessary for replication in the unmodified cell. A preferred modification is the elimination of a function that can consequently be provided trans into helper cells. Useful modifications of the adenovirus genome include modifications that result in the deletion of at least some or all of the adenovirus genes so that the resulting adenovirus vector cannot produce a functional protein with the activity necessary for replication. An adenovirus vector lacking protein-related activity is called "null" with respect to that protein or activity and can be represented as [protein-] (e.g., [E2b-]).

[0065] Examples of adenovirus proteins required for replication in unmodified mammalian cells include, but are not limited to, E1a, E1b, E2a, and E2b. In one embodiment, the adenovirus vector includes a modification in the sequence encoding E1a. In one embodiment, the adenovirus vector includes a modification in the sequence encoding E1b. In one embodiment, the adenovirus vector includes a modification in the sequence encoding E2a. In one embodiment, the adenovirus vector includes a modification in the sequence encoding E2b. In one embodiment, the adenovirus vector includes a deletion in the E1 gene region. In one embodiment, the adenovirus vector includes a deletion in the E1a gene region. In one embodiment, the adenovirus vector includes a deletion in the E1b gene region. In one embodiment, the adenovirus vector includes a deletion in the E2 gene region. In one embodiment, the adenovirus vector includes a deletion in the E2a gene region. In one embodiment, the adenovirus vector includes a deletion in the E2b gene region. In one embodiment, the adenovirus vector contains deletions in the E1 gene region and the E2 gene region. In one embodiment, the adenovirus vector contains deletions in one or more gene regions selected from the group consisting of the E1a gene region, the E1b gene region, the E2a gene region and the E2b gene region. In one embodiment, the adenovirus vector lacks one or more activities related to E1a. In one embodiment, the adenovirus vector lacks one or more activities related to E1b. In one embodiment, the adenovirus vector lacks one or more activities related to E2a. In one embodiment, the adenovirus vector lacks one or more activities related to E2b. In one embodiment, the adenovirus vector lacks one or more activities related to one or more proteins selected from the group consisting of E1a, E1b, E2a and E2b. In one embodiment, the adenovirus vector is [E1a-] and / or [E1b-] and / or [E2a-] and / or [E2b-].

[0066] As used herein, “heterogeneous” refers to a molecule derived from an organism different from the organism being referenced, or a protein derived from the same type of organism in which the protein is expressed, in which case the heterogeneous protein is expressed to an extent unusual for the context of the tissue in which it is expressed. The molecule may be a protein or a nucleic acid sequence (i.e., RNA or DNA). For example, heterogeneous nucleic acid sequence in a recombinant viral vector means that the heterogeneous nucleic acid sequence originates from an organism other than the base virus used to construct the recombinant viral vector. As a further example, heterogeneous nucleic acid sequence in an adenovirus vector means that the heterogeneous nucleic acid sequence originates from an organism other than the adenovirus. Similarly, a protein heterogeneous with an adenovirus vector means that the heterogeneous protein originates from an organism other than the adenovirus.

[0067] In one embodiment, at least a portion of the heterologous protein is an immunogenic portion. As used herein, “immunogenicity” means the ability of a particular protein portion to induce an immune response to a protein containing an amino acid sequence that has a high degree of identity with a particular protein or heterologous protein. According to this disclosure, a high degree of identity amino acid sequence includes a sequence of amino acids that is at least 80% identical, at least 85% identical, at least 87% identical, at least 90% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical. The heterologous protein can be selected from the group consisting of neoepitopes, viral proteins, and bacterial proteins. The heterologous proteins may be derived from viruses selected from the group consisting of adenoviruses, herpesviruses, papillomaviruses, polyomaviruses, hepadnaviruses, parvoviruses, astroviruses, caliciviruses, picornaviruses, coronaviruses, flaviviruses, togaviruses, hepeviruses, retroviruses, orthomyxoviruses, arenaviruses, bunyaviruses, filoviruses, paramyxoviruses, rhabdoviruses, reoviruses, influenza viruses, and poxviruses.

[0068] In certain embodiments, the protein is a coronavirus protein. Alpha-coronaviruses and beta-coronaviruses infect only mammals. Gamma-coronaviruses and delta-coronaviruses infect birds, but some can also infect mammals. Alpha-coronaviruses and beta-coronaviruses typically cause respiratory illness in humans and gastroenteritis in animals. Highly pathogenic viruses, SARS-CoV, MERS-CoV, and SARS-CoV-2, cause severe respiratory syndrome in humans, while four other human coronaviruses (HCoV-NL63, HCoV-229E, HCoV-OC43, and HKU1) induce mild upper respiratory illness in immunocompetent hosts, although some of them cause severe infections in infants, young children, and the elderly.

[0069] Coronaviruses are the largest single-stranded positive-sense RNA viruses, possessing a genome of 27–32 kb. The genome is packed within a helical capsid formed by the nucleocapsid protein (N), further surrounded by an envelope. The viral envelope is associated with at least three structural proteins: membrane proteins (M) and envelope proteins (E) involved in viral construction, as well as the spike protein (S) that mediates viral entry into host cells. Some coronaviruses also encode an envelope-related hemagglutinin-esterase protein (HE). Of these structural proteins, S forms a large protrusion from the viral surface, conferring the crown-like appearance of coronaviruses. In addition to mediating viral entry, S determines host morphology and tissue tropism. S also triggers a host immune response.

[0070] The N, M, E, and S proteins are good candidates for developing anti-coronavirus vaccines because they are located on the outside of the viral particle. In one embodiment, the heterologous protein is derived from a coronavirus selected from the group consisting of SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-NL63, HCoV-229E, HCoV-OC43, and HKU1. In one embodiment, the heterologous protein is derived from SARS-CoV-2. In one embodiment, the heterologous protein is derived from SARS. In one embodiment, the heterologous protein is derived from MERS.

[0071] In one embodiment, the heterologous protein is N derived from a coronavirus selected from the group consisting of SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-NL63, HCoV-229E, HCoV-OC43, and HKU1. In one embodiment, the heterologous protein is SARS-CoV-2 N. In one embodiment, the heterologous protein is N derived from SARS. In one embodiment, the heterologous protein is N derived from MERS.

[0072] In one embodiment, the heterologous protein is M derived from a coronavirus selected from the group consisting of SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-NL63, HCoV-229E, HCoV-OC43, and HKU1. In one embodiment, the heterologous protein is M derived from SARS-CoV-2. In one embodiment, the heterologous protein is M derived from SARS. In one embodiment, the heterologous protein is M derived from MERS.

[0073] In one embodiment, the heterologous protein is E derived from a coronavirus selected from the group consisting of SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-NL63, HCoV-229E, HCoV-OC43, and HKU1. In one embodiment, the heterologous protein is E derived from SARS-CoV-2. In one embodiment, the heterologous protein is E derived from SARS. In one embodiment, the heterologous protein is E derived from MERS.

[0074] In one embodiment, the heterologous protein is S derived from a coronavirus selected from the group consisting of SARS-CoV-2, MERS-CoV, SARS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, and HKU1. In one embodiment, the heterologous protein is S derived from SARS-CoV-2. In one embodiment, the heterologous protein is S derived from SARS. In one embodiment, the heterologous protein is S derived from MERS.

[0075] In one embodiment, the heterologous protein contains an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 1, SEQ ID NOs: 2, SEQ ID NOs: 3, and SEQ ID NOs: 4. In one embodiment, at least one heterologous protein contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4. In one embodiment, at least a portion of the heterologous protein contains at least six consecutive amino acid residues from an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4.

[0076] Another example of a virus from which heterologous proteins can be obtained is the influenza virus. The protective immune response against the influenza virus primarily targets viral hemagglutinin (HA) proteins (glycoproteins on the surface of the virus that are responsible for interacting with host cell receptors). Influenza virus HA proteins are ideal targets for inducing an immune response through vaccination. Heterologous proteins may be derived from influenza viruses, including but not limited to human influenza viruses and avian influenza viruses. Heterologous proteins may be influenza HA, its epitope, its immunogenic moiety, or its variants. Any HA protein, its epitope, its moiety, or its variant can be used in the adenovirus vectors of this disclosure, insofar as it induces an immune response, preferably a protective immune response against the influenza virus. Examples of useful influenza HA proteins, their epitopes, fragments, and variants are disclosed in U.S. Patent Publication No. 2010 / 0074916, U.S. Patent Publication No. 2011 / 0171260, U.S. Patent Publication No. 2011 / 0177122, and U.S. Patent Publication No. 2014 / 0302079, the entire contents of which are incorporated herein by reference.

[0077] In one embodiment, the heterologous protein is a therapeutic protein. Examples of therapeutic proteins include, but are not limited to, antibodies, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic proteins.

[0078] Freeze-drying is a process in which water is removed from a substance (e.g., an adenovirus vector) using freezing temperatures and low pressure. In an exemplary freeze-drying process, the material to be freeze-dried is cooled to below its triple point, typically -50°C to -80°C. Once the material is frozen, the ambient air pressure is reduced, and sufficient heat is applied to sublimate the ice. In a second drying stage, additional heat is applied to remove any unfrozen water molecules. Upon completion, the freeze-dried material has a residual moisture content of less than 5%, typically less than 3%, and usually in the range of about 0.5% to about 3%. A freeze-drying method is described in U.S. Patent No. 7,888,097, which is incorporated herein by reference. In one embodiment, the freeze-dried composition of this disclosure has a residual moisture content of less than 5%, about 4%, about 3%, about 2%, or less than 1%. In one embodiment, the freeze-dried composition of this disclosure has a residual moisture content of about 0.5% to about 5%. In one embodiment, the freeze-dried composition of the Disclosure has a residual moisture content of about 0.5% to about 3%. In one embodiment, the freeze-dried composition of the Disclosure has a residual moisture content of about 0.5% to about 1%. As used herein with respect to residual moisture content, "about" means a variation of no more than 10% from the mentioned figure.

[0079] As used herein, “excipient” is one or more compounds added to a composition to increase its mass or bulk. Examples of excipients commonly used for administration to humans and animals include, but are not limited to, lactose, sucrose, magnesium stearate, glucose, mannitol, starch, dextrose, maltodextrin, maltitol, and plant cellulose. Because some individuals are sensitive to certain compounds, the excipients present in the compositions of this disclosure may lack one or more compounds selected from the group consisting of lactose, sucrose, magnesium stearate, glucose, mannitol, sorbitol, starch, dextrose, maltodextrin, maltitol, and plant cellulose. In one embodiment, the composition lacks lactose. In one embodiment, the composition lacks mannitol. In one embodiment, the composition lacks sorbitol. In one embodiment, the composition lacks starch.

[0080] Considering the sensitivity issues mentioned above, carbonite minerals possess desirable properties and therefore make excellent excipients. Carbonite minerals contain carbonate ions, CO3 2- It contains. Examples of carbonite excipients include, but are not limited to, calcite, vaterite, aragonite, celsite, strontianite, wizerite, and rasafordine. In one embodiment, the excipient contains or consists of compounds selected from the group consisting of calcite, vaterite, aragonite, celsite, strontianite, wizerite, and rasafordine. In one embodiment, the excipient contains a carbonite mineral having an orthorhombic lattice. In one embodiment, the excipient contains or consists of compounds selected from the group consisting of aragonite, celsite, strontianite, wizerite, and rasafordine. In one embodiment, the excipient contains or consists of aragonite. In one embodiment, the excipient contains or consists of celsite. In one embodiment, the excipient contains or consists of strontianite. In one embodiment, the excipient contains or consists of rasafordine.

[0081] In addition to the components described above, the compositions of this disclosure may, but are not required, include additional components that act as, for example, stabilizers, sweeteners, buffers, binders, carriers, diluents, auxiliaries, and pharmaceutically active compounds. Examples of additional components include, but are not limited to, sodium chloride, potassium chloride, sodium citrate, sodium phosphate, sucrose, dimethylglycine, methylsulfonylmethane, and yeast lysate. In one embodiment, the composition comprises one or more stabilizers, sweeteners, buffers, binders, carriers, diluents, auxiliaries, and pharmaceutically active compounds. In one embodiment, the composition comprises one or more compounds selected from the group consisting of sodium chloride, potassium chloride, sodium citrate, sodium phosphate, sucrose, dimethylglycine, methylsulfonylmethane, triethyl citrate (TEC), and yeast lysate.

[0082] This disclosure encompasses compositions formulated for easy administration to an organism. Accordingly, one embodiment of this disclosure is a capsule comprising the compositions disclosed herein. In one embodiment, the capsule contains a composition comprising or comprising a lyophilized adenovirus vector and an excipient comprising or comprising a carbonite mineral, wherein the adenovirus vector comprises a nucleic acid sequence encoding at least a portion of a heterologous protein. The capsule may be made from one or more materials comprising, but not limited to, cellulose, gelatin, and alginate. In one embodiment, the capsule comprises cellulose. In one embodiment, the capsule comprises alginate. In one embodiment, the capsule comprises gelatin. In one embodiment, the capsule is enterically coated.

[0083] One embodiment is a composition comprising a lyophilized adenovirus vector and an excipient containing aragonite, wherein the adenovirus vector lacks E1 and E2b activity and is therefore replication-deficient, and the adenovirus vector comprises a nucleic acid molecule encoding at least a portion of a protein derived from a coronavirus selected from the group consisting of SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-NL63, HCoV-229E, HCoV-OC43, and HKU1, and the coronavirus protein is selected from the group consisting of coronavirus N protein, coronavirus M protein, coronavirus E protein, and coronavirus S protein.

[0084] One embodiment is a method for preparing the compositions disclosed herein. In one embodiment, the method comprises the steps of freeze-drying an adenovirus vector containing a nucleic acid molecule encoding at least a portion of a heterologous protein, and combining the adenovirus vector with an excipient containing or consisting of a carbonite mineral.

[0085] In further embodiments, the composition is encapsulated in capsules suitable for administration to an individual. In one embodiment, the adenovirus vector is derived from any of the four genera of the family Adenoviridae (e.g., Aviadenovirus, Mastadenovirus, Atadenovirus, and Siadenovirus), plus any serotype of each species of adenovirus. In one embodiment, the adenovirus vector is derived from serotype 2 adenovirus. In one embodiment, the adenovirus vector is derived from serotype 5 adenovirus.

[0086] In one embodiment, the freeze-dried adenovirus vector has a residual moisture content of less than 5%, less than about 4%, less than about 3%, less than about 2%, or less than 1%. In one embodiment, the freeze-dried adenovirus vector has a residual moisture content of about 0.5% to about 5%. In one embodiment, the freeze-dried adenovirus vector has a residual moisture content of about 0.5% to about 3%.

[0087] In one embodiment, the excipient comprises one or more compounds selected from the group consisting of lactose, sucrose, magnesium stearate, glucose, mannitol, sorbitol, starch, dextrose, maltodextrin, maltitol, and plant cellulose. In one embodiment, the excipient lacks one or more compounds selected from the group consisting of lactose, sucrose, magnesium stearate, glucose, mannitol, sorbitol, starch, dextrose, maltodextrin, maltitol, and plant cellulose. In one embodiment, the excipient lacks lactose. In one embodiment, the excipient lacks mannitol. In one embodiment, the excipient lacks sorbitol. In one embodiment, the excipient lacks starch.

[0088] In one embodiment, the composition comprises one or more stabilizers, sweeteners, buffers, binders, carriers, diluents, auxiliaries, and pharmaceutically active compounds. In one embodiment, the composition comprises one or more compounds selected from the group consisting of sodium chloride, potassium chloride, sodium citrate, sodium phosphate, sucrose, dimethylglycine, methylsulfonylmethane, and yeast lysate.

[0089] In one embodiment, the capsule comprises one or more materials selected from the group consisting of cellulose, gelatin, and alginate. In one embodiment, the capsule comprises cellulose. In one embodiment, the capsule comprises alginate. In one embodiment, the capsule comprises gelatin. In one embodiment, the capsule is enterically coated.

[0090] A method for vaccinating an individual against an infectious microorganism is disclosed herein. This method comprises the step of administering to an individual one or more times a composition disclosed herein or a capsule containing a composition disclosed herein. In one embodiment, the composition comprises a lyophilized adenovirus vector and an excipient comprising or consisting thereof a carbonite mineral, wherein the adenovirus vector comprises a nucleic acid sequence encoding at least a portion of a heterologous protein derived from an infectious microorganism. In one embodiment, the individual is at risk of exposure to the infectious microorganism. Such an individual may be one that is potentially exposed to the infectious agent at any time, or may have been previously exposed but still does not have symptoms of infection.

[0091] The routes, frequency, and dosage of administration of the compositions and capsules containing these compositions vary from individual to individual and disease to disease, and can be easily established using standard techniques. Generally, the compositions can be administered by routes including intravenous, oral, parenteral, intra-arterial, transdermal, subcutaneous, intramuscular, topical, intracranial, intraorbital, transocular, intravitreous, intraventricular, intra-articular, intraspinal, intracisional, intraperitoneal, intranasal, aerosol, central nervous system (CNS) administration, and suppository administration (for vaginal or rectal delivery). Generally, capsules containing the compositions disclosed herein are administered orally.

[0092] The method of administering the compositions of this disclosure will depend on factors such as the age, weight, and health status of the patient being treated, as well as the disease or condition being treated. Therefore, those skilled in the art can individually select the most appropriate method of administration for each patient.

[0093] One embodiment is a kit. The kit may include, for example, the adenovirus vector of the Disclosure, nucleic acid molecules for constructing the adenovirus vector of the Disclosure, excipients of the Disclosure, compositions of the Disclosure and / or capsules of the Disclosure. The kit may also include relevant components, such as, but are not limited to, a medium, buffer, label, container, vial, syringe and instructions for using the kit.

[0094] Solid dosage forms of vaccine compositions: As disclosed herein, the dosage form (also called a solid dosage form) comprises an intrinsically stable aragonite that can be directly bonded to a vaccine composition. In exemplary embodiments, the intended dosage form comprises aragonite impregnated with carbon dioxide (CO2) (i.e., bonded) before the addition of the vaccine composition. See, for example, European Patent No. 2719373 and U.S. Patent Application Publication 2020 / 0155458. In further embodiments, the intended dosage form comprises aragonite with a biocompatible polymer and / or disintegrant that has been mixed and treated with the aragonite before the addition of the vaccine composition. Typically, the aragonite is impregnated with CO2 and mixed with both the biocompatible polymer and the disintegrant before the addition of the vaccine composition. More typically, the aragonite is impregnated with CO2 and mixed with the biocompatible polymer and the disintegrant and formed into a solid form (e.g., compressed) before the addition of the vaccine composition. For example, see European Patent No. 2719373 and U.S. Patent Application Publication No. 2020 / 0155458.

[0095] The vaccine composition is supported (e.g., mixed) on an intended solid dosage form of aragonite (e.g., optionally impregnated with CO2, a biocompatible polymer, and / or a disintegrant) as disclosed herein. The intended solid dosage form can be compressed before or after the support of the vaccine composition. Typically, the solid dosage form is compressed (e.g., densified) before the support of the vaccine composition. In exemplary embodiments, the compressed (i.e., densified) solid dosage form on which the vaccine composition is supported is crushed to form a powder, tablet, or capsule.

[0096] With regard to densifying the solid dosage form before or after loading the vaccine composition, a compressive force of 5 to 500 kN is used. Preferably, the densification of the solid dosage form before or after loading the vaccine composition is carried out using a compressive force of 6 to 300 kN, most preferably 8 to 200 kN. More preferably, the densification of the solid dosage form before or after loading the vaccine composition is carried out using a compressive force of 8 to 100 kN, 8 to 50 kN, or 8 to 28 kN.

[0097] In exemplary embodiments, CO2-linked aragonite is mixed with at least one biocompatible polymer. Typically, the weight ratio of CO2-linked aragonite to biocompatible polymer is about 95:5 to 5:95. In further embodiments, the biocompatible polymer is a hot-melt extruded biocompatible polymer. Examples of biocompatible polymers include polylactic acid (PLA), polyethylene, polystyrene, polyvinyl chloride, polyamide 66 (nylon), polycaprolactam, polycaprolactone, acrylic polymers, acrylonitrile butadiene styrene, polybenzimidazole, polycarbonate, polyphenylene oxide / sulfide, polypropylene, Teflon®, polylactic acid, aliphatic polyesters such as polyhydroxybutyrate, poly-3-hydroxybutyrate (P3HB), polyhydroxyvalerate, polyhydroxybutyrate-polyhydroxyvalerate copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), polyglyconate, poly(dioxanone), and mixtures thereof. Preferably, the biocompatible polymer resin is PLA. Preferably, the biocompatible polymer is Eudragit L30 D-55 (Evonik).

[0098] In certain embodiments, the weight ratio of multiple aragonite particles (i.e., aragonite composition with or without carbon dioxide) to the biocompatible polymer is about 95:5 to 5:95. Preferably, the weight ratio of multiple aragonite particles to the biocompatible polymer is about 80:20 to 20:80, more preferably 70:30 to 30:70, and most preferably 60:40 to 40:60. For example, the weight ratio of multiple aragonite particles to the biocompatible polymer is about 50:50.

[0099] In further embodiments, CO2-linked aragonites and biocompatible polymers also include disintegrants mixed therein (e.g., treated disintegrants). Examples of suitable disintegrants include starch (e.g., pea starch), modified cellulose gum, insoluble crosslinked polyvinylpyrrolidone, starch glycolate, microcrystalline cellulose, pregelatinized starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, homopolymers of N-vinyl-2-pyrrolidone, alkyl-, hydroxyalkyl-, carboxyalkyl-cellulose esters, alginates, and microcrystalline cellulose and their polymorphs.

[0100] The mixing of multiple aragonite particles, a biocompatible polymer, and optionally a disintegrant is carried out using any conventional hot-melt extrusion method. For example, hot-melt extrusion can be performed using a twin-screw hot-melt extruder with a perforated die (e.g., Three-Tec, ZE9 20602, Switzerland). Methods for compounding and extruding aragonite and bioplastic compositions, including filament production, are described, for example, in International Patent Application PCT / US Patent Application Publication No. 20 / 45451, the entirety of which is incorporated herein by reference. Furthermore, extruded filament compositions made of bioplastics, aragonite, and optionally a disintegrant can be formed into useful or suitable shapes using 3D printing. With respect to Figures 2A and 2B, an exemplary bioplastic aragonite (containing 40% aragonite) composition was compounded, extruded to produce filaments, and then processed using 3D printing to form aragonite structures as shown.

[0101] Additional additives may also be added to the solid dosage form if determined by the needs of manufacturing and packaging. Examples of additional additives include ion exchange resins, gums, chitin, chitosan, clay, gellan gum, cross-linked polariline copolymers, agar, gelatin, dextrin, acrylic acid polymers, sodium / calcium carboxymethylcellulose, hydroxypropyl methylcellulose phthalate, shellac or mixtures thereof, lubricants, internal phase lubricants, external phase lubricants, impact resistance modifiers, plasticizers, waxes, stabilizers, pigments, colorants, fragrances, flavoring agents, sweeteners, mouthfeel modifiers, binders, diluents, film-forming agents, adhesives, buffers, adsorbents, odor masking agents and mixtures thereof.

[0102] In particular, the intended solid dosage form is designed to support the vaccine composition for oral, sublingual, or buccal administration. The vaccine composition can be supported or mixed onto a solid dosage form (e.g., CO2-linked aragonite already mixed with a biocompatible polymer and disintegrant) by any conventional method. For example, the vaccine composition can be supported onto a solid dosage form in a mixer (e.g., a tumble mixer) or blender.

[0103] In exemplary embodiments, the intended solid dosage form is supported with a vaccine composition for inducing immunity against a virus. As will be readily apparent to those skilled in the art, there are a wide variety of vaccine types depending on the disease / infection (e.g., virus) being immunized. For example, if the pathogenic virus is a coronavirus (e.g., SARS-CoV, MERS-CoV, SARS-CoV-2, and human coronavirus NL63 / HCoV-NL63), the vaccine may be a recombinant expression vector encoding all or part of the coronavirus ACE2 protein. On the other hand, if the pathogenic virus is a poliovirus, the vaccine may be a recombinant expression vector encoding all or part of the CEA protein. In yet another example, if the pathogenic virus is an HIV virus, the vaccine may be a recombinant expression vector encoding all or part of gp120. Similar determinations naturally apply to all other types of pathogenic viruses (e.g., influenza viruses, rhinoviruses, enteroviruses, echoviruses, herpesviruses, etc.).

[0104] In exemplary embodiments, the vaccine composition is a SARS-CoV-2 vaccine (e.g., an adenovirus construct) comprising a soluble ACE2 protein ligated to the Fc portion of an immunoglobulin, and possibly also comprising a J-chain portion, forming an ACE2-Fc hybrid construct. This is disclosed in U.S. Patent Application Publication 16 / 880,804 and U.S. Patent Application 63 / 016,048, both of which are incorporated herein by reference in their entirety. In other exemplary embodiments, the SARS-CoV-2 vaccine (e.g., an adenovirus construct) comprises a mutant variant of recombinant soluble ACE2 protein (e.g., SEQ ID NO: 6), the mutant variant having at least one mutant amino acid residue (e.g., by substitution) that enhances the binding affinity of the ACE2 protein to the RBD protein domain of the SARS-CoV-2 spike protein. This is disclosed in U.S. Patent Application 63 / 022,146, which is incorporated herein by reference in its entirety. In another exemplary embodiment, a SARS-CoV-2 vaccine (e.g., an adenovirus construct) comprises a CoV-2 nucleocapsid protein or CoV-2 spike protein (N-ETSD) fused to a target sequence in an endosome. This is disclosed in U.S. Patent Application Publication No. 16 / 883,263 and U.S. Patent Application No. 63 / 009,960, both of which are incorporated herein by reference in their entirety.

[0105] Preferably, the intended dosage form is supported by a vaccine composition comprising a recombinant expression vector (e.g., adenovirus) encoding a recombinant ACE2 protein, as disclosed in, for example, U.S. Patent Application Publication No. 16 / 880,804 (the entirety of which is incorporated herein by reference). In a typical embodiment, the vaccine composition is a recombinant human ACE2 protein having at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 5.

[0106] In additional or alternative embodiments, the intended dosage form is supported by a vaccine composition comprising a recombinant expression vector (e.g., adenovirus) encoding a recombinant soluble ACE2 protein (e.g., SEQ ID NO: 6) or a variant of a recombinant ACE2 variant including T27F, T27W, T27Y, D30E, H34E, H34F, H34K, H34M, H34W, H34Y, D38E, D38M, D38W, Q24L, D30L, H34A and / or D355L with respect to SEQ ID NO: 2.

[0107] In another embodiment, the surface of aragonite particles may be treated to modify the binding surface. For example, treatment with stearic acid (i.e., octadecanoic acid), as disclosed in U.S. Patent Application Publication 16 / 858,548 and International Application PCT / U.S. Patent Application Publication 20 / 29949, results in a hydrophobic surface. For protein support, treatment of aragonite with phosphoric acid forms a layered structure. Additional coupling techniques for coupling reactive groups to the amino acid surface of aragonite are known in the art, for example, as disclosed in Bioconjugate Techniques, Third Edition, Greg T. Hermanson, Academic Press, 2013.

[0108] As used herein, the singular forms “one (a),” “one (an),” and “it” include multiple referents unless the context clearly negates this. For example, a (a) nucleic acid molecule refers to one or more nucleic acid molecules. For this reason, the terms “one (a),” “one (an),” “one or more,” and “at least one” can be used interchangeably. Similarly, the terms “include,” “contain,” and “have” can be used interchangeably. Claims may be constructed to exclude any optional element. For this reason, this statement serves as a prior art for the use of exclusive terms such as “exclusively,” “simply,” or “negative” limitations with respect to the enumeration of elements of the claims.

[0109] The present invention will be further described in detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Accordingly, the present invention should not be construed as being limited in any way to the following examples, but rather as encompassing all variations that become apparent as a result of the teachings provided herein. [Examples]

[0110] [Example 1] This example demonstrates that the uncoated aragonite formulation yields good virus recovery (pH stability and retention of hardness (i.e., paste)), while the uncoated lactose formulation yields less virus recovery, resulting in a liquid state and acidity.

[0111] Capsule formulation: A lyophilized adenovirus vector (Ad5) of known viral titer is encapsulated in 1 x 10⁻¹ capsules. 9 The capsules were filled with infectious units (IU) per capsule. Either lactose or aragonite was added to achieve a final total weight of 550 mg / capsule. The capsules were sealed under moisture-controlled conditions and optionally coated with the anionic copolymer L30 D-55 and triethyl citrate (TEC).

[0112] Determination of infectivity titer by hexone assay: E.C7 cells were placed in a 12-well plate at a rate of approximately 5.0 × 10⁴ 5 Seed cells per well and incubated at 37±2°C for at least 2 hours. Serially diluted the hAd5 construct in 1×DMEM (Dulbecc's modified Eagle medium). 2–4 hours after seeding, inoculated 100 μL / well of the diluted test in triple repeats. Adenovirus type 5 (Ad5) reference material supplied from the American Type Culture Collection (ATCC) was used as a positive control and treated similarly. A negative control was inoculated into four wells using 100 μL of the diluent alone. Incubated the plate at 37±2°C for 42 hours.

[0113] Hexone immunohistochemical staining: Next, fix the plate with cold methanol for 10 minutes, rinse with 1× DPBS (Dulbeccio phosphate buffered saline), and then assay by immunostaining for hexone. Add 0.5 mL of mouse anti-hexone antibody solution to each well and incubate at 37°C ± 2°C for 60 ± 6 minutes. Wash the plate, then add 0.5 mL of rat anti-mouse antibody solution to each well and incubate at 37°C ± 2°C for 60 ± 6 minutes. After washing, add a freshly prepared working solution of DAB (diaminobenzidine) and incubate at room temperature for 10 minutes. Aspirate the DAB and add 1.0 mL of 1× PBS (phosphate buffered saline) to each well.

[0114] Stained cells are visualized using a light microscope with a 10x objective lens 4 hours after substrate color development, and an image of the entire well is obtained and counted. The average number of positive cells / colonies per well is calculated, and the infectivity titer is calculated using the following formula: Infection units / mL = (average positive cells / well) × dilution factor × 10 It will be decided by [the specified method].

[0115] The results are shown in Figures 3A-3D. The data represent either infection units / gram (without acid (Figure 3A) or with acid (Figure 3C)) or percentage of virus recovery (without post-encapsulation acid treatment (Figure 3B) or with post-encapsulation acid treatment (Figure 3D)) using either an aragonite-containing or lactose-containing formulation. Aragonite-containing samples maintained a pH > / = 7 when exposed to acid. The recovered sample mass (g) is shown in Table 1 below. The final result was either a powder, liquid, or paste. pH was determined 2 minutes after resuspension and read three times.

[0116] [Table 1]

[0117] The embodiments may be modified as necessary to provide further embodiments by adopting concepts from various patents, applications, and publications.

[0118] In light of the description detailed above, these and other modifications to the embodiments may be made. In general, the terms used in the following claims should not be construed as limiting the claims to the specific embodiments and claims disclosed herein, but rather as encompassing all possible embodiments, along with the entire scope of equivalents to which such claims are granted. Thus, the claims are not limited by this disclosure.

[0119] Each publication or patent cited herein is incorporated herein by reference in its entirety.

[0120] [Sequence List] TIFF2026123111000003.tif223158TIFF2026123111000004.tif223158TIFF2026123111000005.tif223158TIFF2026123111000006.t if223158TIFF2026123111000007.tif223158TIFF2026123111000008.tif223158TIFF2026123111000009.tif223158TIFF20261231110 00010.tif223158TIFF2026123111000011.tif223158TIFF2026123111000012.tif223158TIFF2026123111000013.tif223158TIFF202 6123111000014.tif223158TIFF2026123111000015.tif223158TIFF2026123111000016.tif223158TIFF2026123111000017.tif223158

Claims

1. A composition comprising a freeze-dried adenovirus vector and an excipient containing a carbonite mineral, wherein the adenovirus vector comprises a nucleic acid molecule encoding at least a portion of a heterologous protein.

2. The composition according to claim 1, wherein the adenovirus vector is derived from adenovirus type 5, and the adenovirus has deletions in the E1, E2b, and E3 regions.

3. The composition according to claim 1 or 2, wherein the heterologous protein is derived from a virus.

4. The composition according to claim 3, wherein the heterologous protein is derived from a virus selected from the group consisting of SARS-CoV-2, MERS-CoV, SARS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, HKU1, and influenza virus.

5. The composition according to claim 4, wherein the heterologous protein is derived from SARS-CoV-2.

6. The composition according to claim 5, wherein the heterogeneous protein is a spike (S) protein, a nucleocapsid (N) protein, or a membrane (M) protein.

7. The composition according to claim 6, wherein the heterologous protein is identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 by at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 97%, or further 100%.

8. The adenovirus vector has a residual moisture content of 0.5% to 5% according to any one of claims 1 to 7.

9. The composition according to claim 8, wherein the adenovirus vector has a residual moisture content of less than 5%.

10. The composition according to claim 9, wherein the adenovirus vector has a residual moisture content of less than 3%.

11. The composition according to any one of claims 1 to 10, wherein the carbonite mineral has an orthorhombic lattice.

12. The composition according to claim 11, wherein the carbonite mineral is selected from the group consisting of aragonite, celsite, strontianite, wizerite, and rasafordine.

13. The composition according to claim 12, wherein the carbonite mineral is aragonite.

14. The composition according to any one of claims 1 to 13, wherein the excipient comprises one or more compounds selected from the group consisting of lactose, sucrose, magnesium stearate, glucose, mannitol, sorbitol, starch, dextrose, maltodextrin, maltitol, and plant cellulose.

15. The composition according to any one of claims 1 to 14, wherein the excipient lacks one or more compounds selected from the group consisting of lactose, sucrose, magnesium stearate, glucose, mannitol, sorbitol, starch, dextrose, maltodextrin, maltitol, and plant cellulose.

16. A composition according to any one of claims 1 to 15, comprising one or more compounds selected from the group consisting of sodium chloride, potassium chloride, sodium citrate, sodium phosphate, sucrose, dimethylglycine, glycine, methylsulfonylmethane, and yeast lysate.

17. A capsule comprising the composition according to any one of claims 1 to 16.

18. The capsule according to claim 17, which is enterically coated.

19. A capsule according to claim 17 or 18, comprising alginate.

20. A solid dosage form for the delivery of the vaccine composition by oral, sublingual, or buccal administration, An aragonite composition containing multiple aragonite particles impregnated with carbon dioxide (CO2), A biocompatible polymer and a disintegrant mixed with the aragonite composition Includes, The vaccine composition further comprises, A solid dosage form, such as a powder, tablet, or capsule.

21. The solid dosage form according to claim 20, further comprising at least one additive and being formulated to form a lozenge.

22. The solid dosage form according to claim 20, wherein the plurality of aragonite particles have an average particle size of 100 nm to 1 mm.

23. The biocompatible polymer is selected from polylactic acid (PLA), polyethylene, polystyrene, polyvinyl chloride, polyamide 66 (nylon), polycaprolactam, polycaprolactone, acrylic polymer, acrylonitrile butadiene styrene, polybenzimidazole, polycarbonate, polyphenylene oxide / sulfide, polypropylene, Teflon®, polylactic acid, aliphatic polyester, for example, polyhydroxybutyrate, poly-3-hydroxybutyrate (P3HB), polyhydroxyvalerate, polyhydroxybutyrate-polyhydroxyvalerate copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), polyglyconate, poly(dioxanone), and mixtures thereof, as described in any one of claims 20 to 22.

24. The solid dosage form according to claim 23, wherein the biocompatible polymer is PLA.

25. The solid dosage form according to any one of claims 20 to 24, wherein the disintegrant is selected from starch, modified cellulose gum, insoluble crosslinked polyvinylpyrrolidone, starch glycolate, microcrystalline cellulose, pregelatinized starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, homopolymer of N-vinyl-2-pyrrolidone, alkyl-, hydroxyalkyl-, carboxyalkyl-cellulose esters, alginate, and microcrystalline cellulose and their polymorphs.

26. The solid dosage form according to claim 25, wherein the disintegrant is pea starch.

27. The solid dosage form according to any one of claims 20 to 26, wherein the powder, the tablet, and the capsule disintegrate in an aqueous solution in less than 30 seconds.

28. The solid dosage form according to any one of claims 20 to 27, wherein the vaccine composition comprises a recombinant viral expression construct encoding a viral protein or a fragment thereof.

29. The solid dosage form according to claim 28, wherein the viral protein or fragment thereof corresponds to a coronavirus protein or fragment thereof.

30. The solid dosage form according to claim 29, wherein the coronavirus protein or fragment thereof is a SARS-CoV2 virus-binding protein.

31. The solid dosage form according to claim 30, wherein the SARS-CoV2 virus-binding protein comprises recombinant ACE2 protein.

32. The solid dosage form according to claim 31, wherein the recombinant ACE2 protein has at least 85% sequence identity with SEQ ID NO:

5.

33. The solid dosage form according to claim 32, wherein the recombinant ACE2 protein comprises the sequence of SEQ ID NO:

6.

34. The solid dosage form according to claim 33, wherein the recombinant ACE2 protein comprises at least one mutation selected from T27F, T27W, T27Y, D30E, H34E, H34F, H34K, H34M, H34W, H34Y, D38E, D38M, D38W, Q24L, D30L, H34A, and D355L.

35. The vaccine composition comprises an adenovirus expression construct, in the solid dosage form according to any one of claims 20 to 34.

36. A method for producing a solid dosage form for carrying a vaccine, A step of providing an aragonite composition comprising multiple aragonite particles impregnated with carbon dioxide (CO2), The steps include: mixing the aragonite composition with a biocompatible polymer and a disintegrant to form the solid dosage form, and The step of adding the vaccine composition to the solid dosage form. A method that includes this.

37. The method according to claim 36, wherein the step of mixing the aragonite composition with the biocompatible polymer and the disintegrant comprises hot-melt extrusion.

38. The method according to claim 36 or 37, wherein the solid dosage form is a powder, a tablet, or a capsule.

39. The method according to claim 38, wherein the solid dosage form is a tablet, and the method further comprises the step of compacting the solid dosage form.

40. The method according to any one of claims 36 to 39, wherein the aragonite composition and the biocompatible polymer are in a weight ratio of 95:5 to 5:

95.

41. The method according to any one of claims 36 to 40, wherein the plurality of aragonite particles have an average particle size of 100 nm to 1 mm.

42. The method according to any one of claims 36 to 41, wherein the biocompatible polymer is selected from polylactic acid (PLA), polyethylene, polystyrene, polyvinyl chloride, polyamide 66 (nylon), polycaprolactam, polycaprolactone, acrylic polymer, acrylonitrile butadiene styrene, polybenzimidazole, polycarbonate, polyphenylene oxide / sulfide, polypropylene, Teflon®, polylactic acid, aliphatic polyester, such as polyhydroxybutyrate, poly-3-hydroxybutyrate (P3HB), polyhydroxyvalerate, polyhydroxybutyrate-polyhydroxyvalerate copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), polyglyconate, poly(dioxanone), and mixtures thereof.

43. The method according to any one of claims 36 to 42, wherein the biocompatible polymer is PLA.

44. The method according to any one of claims 36 to 43, wherein the disintegrant is selected from starch, modified cellulose gum, insoluble crosslinked polyvinylpyrrolidone, starch glycolate, microcrystalline cellulose, pregelatinized starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, homopolymer of N-vinyl-2-pyrrolidone, alkyl-, hydroxyalkyl-, carboxyalkyl-cellulose esters, alginates, and microcrystalline cellulose and their polymorphs.

45. The method according to any one of claims 36 to 44, wherein the disintegrant is pea starch.

46. The method according to any one of claims 36 to 45, wherein the vaccine composition comprises a recombinant virus expression construct encoding a viral protein or a fragment thereof.

47. The method according to claim 46, wherein the viral protein or fragment thereof corresponds to a coronavirus protein or fragment thereof.

48. The method according to claim 47, wherein the coronavirus protein or fragment thereof is a SARS-CoV2 virus-binding protein.

49. The method according to claim 48, wherein the SARS-CoV2 virus-binding protein comprises recombinant ACE2 protein.

50. The method according to claim 49, wherein the recombinant ACE2 protein has at least 85% sequence identity with SEQ ID NO:

5.

51. The method according to claim 50, wherein the recombinant ACE2 protein comprises the sequence of SEQ ID NO:

6.

52. The method according to claim 51, wherein the recombinant ACE2 protein comprises at least one mutation selected from T27F, T27W, T27Y, D30E, H34E, H34F, H34K, H34M, H34W, H34Y, D38E, D38M, D38W, Q24L, D30L, H34A, and D355L.

53. The method according to any one of claims 36 to 52, wherein the vaccine composition comprises an adenovirus expression construct.

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