KR20230113532A - Vaccine composition for mucosal immune response - Google Patents

Abstract

A vaccine composition comprising a lyophilized adenovirus-based expression vector and a stabilizing compound such as aragonite is provided. Further provided is a composition comprising a solid dosage form prepared from aragonite for loading and delivery of a vaccine composition.

Description

Vaccine composition for mucosal immune response

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims 35 U.S.C. claims to U.S. Provisional Application Serial No. 63/104,770, filed October 23, 2020, and U.S. Provisional Application Serial No. 63/082,907, filed September 24, 2020; Claims the benefit of priority under §119(e), each of which is incorporated herein by reference.

technology field

The present disclosure relates generally to the field of vaccine compositions. More specifically, the present disclosure relates to solid dosage forms of vaccines for effective administration and manufacture, as well as vaccine compositions with improved stability and ease of administration.

Reference to Sequence Listing

This application is a sequence listing text file created on October 22, 2021, "8774-16-PCT_Sequence_Listing_ST25.txt," file size 30,000 bytes (B) for amino acid sequences and/or nucleic acid sequences submitted concurrently with this application. Include references. The foregoing sequence listing is 37 C.F.R. Pursuant to §1.52(e)(5), the entire text is hereby incorporated by reference.

Vaccines can be prepared by allowing cultures to stand to weaken the virulence of an infecting organism. More recently, however, modern recombinant techniques have been used to produce viral vectors capable of producing antigenic proteins and expressing antigenic proteins for a variety of infectious organisms.

Regardless of the method or technology used to produce a vaccine, the ability to store a vaccine while maintaining potency has been a continuing challenge. Currently, an important part of maintaining vaccine efficacy relies on an expensive chain of refrigerated storage systems. To alleviate the need for these costly supply chains, manufacturers have tried adding chemical stabilizers to vaccines. However, these fillers are often toxic and cause allergic reactions.

Besides stability, vaccination protocols suffer from challenges related to the route of delivery. Many vaccines are administered via injections that many people dislike.

Vaccines are traditionally delivered by intramuscular, intradermal or subcutaneous injection. Although such injections can elicit a strong systemic immune response, the potency to trigger a mucosal immune response is variable and often weak or undetectable, especially for subunit vaccines. Antibodies produced by antigen-specific cytotoxic T cells (CTL) and B cells in the draining lymph nodes treated with the injected vaccine can migrate to other organs in the body, but they can be transferred to various mucosal tissues (e.g., genitals, intestines, respiratory tract). ) is often limited or impossible due to inadequate homing mucosal receptors and chemotaxis. However, the nasal route, also considered the parenteral route of immunization, can trigger superior mucosal immune responses in the respiratory tract, genital tract and intestinal tract that share some interconnections, which are more accessible when vaccines are delivered to mucosal sites. Thus, these parenteral vaccines can provide protection against mucosal pathogens in some cases.

Most pathogens (e.g. COVID19) enter the body through mucosal tissues (oral, respiratory, genital and intestinal) and many of them replicate only in mucosal tissues, and mucosal vaccination is innate at local and distal mucosal sites. Both (eg, NK cells) and adaptive (T and B cells) immune responses can be induced to optimally induce front-line defense.

Even considering currently approved vaccines against coronavirus disease 2019 (COVID-19), rapid and global-scale vaccine distribution and administration remain impeded. Global immunization has not yet been achieved with currently approved vaccines due to a number of factors including manufacturing and/or storage costs and requirements.

Mucosal vaccine delivery (via the buccal, sublingual, nasal, buccal or vaginal mucosa) is receiving increasing attention as a means of inducing a systemic immune response as well as a local and distant antibody immune response. Additionally, mucosal vaccine delivery by solid dosage form (e.g., buccal/sublingual tablet, oral tablet or capsule, vaginal insert) has the potential for mass immunization, patient compliance, ease of use, product shelf life stability, cold-chain independence and It can provide some of the same benefits. In addition, mucosal vaccine delivery may be suitable for patients with needle phobia and allow the patient to self-administer the vaccine with appropriate instructions. The buccal/sublingual route has been used for many years to deliver drugs and small molecules to the bloodstream, but its application as a vehicle for mucosal delivery of vaccines has not been previously developed.

Solid dosage forms including powders, tablets or capsules for drug delivery require disintegration and release of the active ingredient (eg vaccine) at the time of administration. At the same time, dosage forms loaded with active ingredients must be stable during transportation and storage from the time of production to the time of administration. As such, the use of solid dosage forms requires stability of the active ingredient loaded therein, which readily disintegrates in aqueous solutions (eg, water, saliva or mucosal fluids).

Therefore, there is a need for a vaccine composition with improved vaccine coverage effectiveness with improved stability and ease of administration. There is also a need for improved solid vaccine dosage forms that provide a compatible platform for vaccine loading, are stable prior to disintegration (administration), readily disintegrate, have low manufacturing costs, and are easy to administer for effective global immunization. The present disclosure addresses this need and provides additional advantages as well.

One embodiment relates to a composition comprising a filler comprising a carbonite mineral and a lyophilized adenoviral vector, wherein the adenoviral vector comprises a nucleic acid molecule encoding at least a portion of a heterologous protein.

In one aspect, the adenoviral vector is derived from a type 5 adenovirus, said adenovirus having deletions of the E1, E2b and E3 regions.

In various embodiments, the heterologous protein is of viral origin.

In various embodiments, the heterologous protein is 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 viruses.

In various embodiments, the heterologous protein is 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 another embodiment, the heterologous protein is at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 95% SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 97%, or even 100% identical.

In various embodiments, adenoviral vectors have a residual moisture content of 0.5% to 5%. In one aspect, the adenoviral vector has less than 5% residual moisture. In another embodiment, the adenoviral vector has less than 3% residual moisture.

In various embodiments, carbonite minerals have an orthorhombic lattice. In one aspect, the carbonite mineral is selected from the group consisting of aragonite, serousite, strontianite, witherite and rutherfordin. In one aspect, the carbonite mineral is aragonite.

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

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

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.

Another embodiment relates to a capsule comprising a composition as disclosed herein. In one aspect, the capsule is enterically coated. In another aspect, the capsule comprises an alginate.

Another embodiment relates to a solid dosage form for delivering a vaccine composition by oral, sublingual or buccal administration of the vaccine composition, the solid dosage form comprising a plurality of aragonite particles impregnated with carbon dioxide (CO ₂ ) arago night composition; and a biocompatible polymer and a disintegrant mixed with the aragonite composition; The solid dosage form further comprises a vaccine composition, and the solid dosage form is a powder, tablet or capsule.

In one aspect, the solid dosage form further comprises at least one excipient and is formulated to form a lozenge.

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

In various embodiments of the solid dosage form, the biocompatible polymer is polylactic acid (PLA), polyethylene, polystyrene, polyvinylchloride, polyamide 66 (nylon), polycaprolactam, polycaprolactone, acrylic polymer, 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.

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

In various embodiments of the solid dosage form, the disintegrant is starch, modified cellulose gum, insoluble cross-linked 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 polymorphic forms thereof.

In one aspect of the solid dosage form, the disintegrant is pea starch.

In one aspect of the solid dosage form, the vaccine composition comprises a recombinant viral expression construct encoding a viral protein or fragment thereof. In one aspect, the viral protein or fragment thereof corresponds to a coronavirus protein or fragment thereof. In another embodiment, the coronavirus protein or fragment thereof is a SARS-CoV2 virus binding protein. In one aspect, the recombinant ACE2 protein has at least 85% sequence identity to SEQ ID NO:5. In another aspect, the recombinant ACE2 protein comprises the sequence of SEQ ID NO:6. In another embodiment, the recombinant ACE2 protein has at least one mutation selected from T27F, T27W, T27Y, D30E, H34E, H34F, H34K, H34M, H34W, H34Y, D38E, D38M, D38W, Q24L, D30L, H34A, and D355L. include

In one aspect of the solid dosage form, the vaccine composition includes an adenovirus expression construct.

Another embodiment relates to a method of manufacturing a solid dosage form for loading a vaccine, the method comprising providing an aragonite composition comprising a plurality of aragonite particles impregnated with carbon dioxide (CO ₂ ); 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.

In one aspect of the method of preparing the solid dosage form, mixing the aragonite composition with the biocompatible polymer and disintegrant comprises hot melt extrusion.

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

In another embodiment of the method of preparing the solid dosage form, the solid dosage form is a tablet and the method further comprises compressing the solid dosage form.

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

In another embodiment of the method of making the solid dosage form, the plurality of aragonite particles have an average particle size of 100 nm to 1 mm.

In another embodiment of the method of making the solid dosage form, the biocompatible polymer is polylactic acid (PLA), polyethylene, polystyrene, polyvinylchloride, polyamide 66 (nylon), polycaprolactam, polycaprolactone, acrylic polymer, acrylic Ronitrile butadiene styrene, polybenzimidazole, polycarbonate, polyphenylene oxide/sulfide, polypropylene, Teflon, polylactic acid, aliphatic polyesters such as polyhydroxybutyrate, poly-3-hydroxybutyrate (P3HB), poly Hydroxyvalerate, polyhydroxybutyrate-polyhydroxyvalerate copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), polyglyconate, poly(dioxanone) and mixtures thereof is selected from

In another aspect of the method of making the solid dosage form, the biocompatible polymer is PLA. In another embodiment of the solid dosage form, the biocompatible polymer is Eudragit L30 D-55 (Evonik).

In another embodiment of the method of preparing the solid dosage form, the disintegrant is starch, modified cellulose gum, insoluble cross-linked 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 polymorphic forms thereof.

In another embodiment of the method of preparing the solid dosage form, the disintegrant is pea starch.

In one aspect of the method of making the solid dosage form, the vaccine composition comprises a recombinant viral expression construct encoding a viral protein or fragment thereof. In one aspect, the viral protein or fragment thereof corresponds to a coronavirus protein or fragment thereof. In another embodiment, the coronavirus protein or fragment thereof is a SARS-CoV2 virus binding protein. In one aspect, the recombinant ACE2 protein has at least 85% sequence identity to SEQ ID NO:5. In another aspect, the recombinant ACE2 protein comprises the sequence of SEQ ID NO:6. In another embodiment, the recombinant ACE2 protein has at least one mutation selected from T27F, T27W, T27Y, D30E, H34E, H34F, H34K, H34M, H34W, H34Y, D38E, D38M, D38W, Q24L, D30L, H34A, and D355L. include

In one aspect of the method of preparing the solid dosage form, the vaccine composition includes an adenovirus expression construct.

Embodiments discussed in the context of a method and/or composition described herein may be used in connection with any other method or composition described herein. Thus, embodiments directed to one method or composition may be applied to other methods and compositions.

Other objects, features and advantages will become apparent from the detailed description that follows. However, it should be understood that the detailed description and specific examples, while indicating specific implementations, are provided by way of example only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

1 shows the CaCO ₃ chemical structures and symmetries for calcite, aragonite and vaterite, respectively, as indicated.
2A is a photograph of a biocompatible bioplastic aragonite composition containing 40% aragonite formed by three-dimensional (3D) printing of extruded filaments, in accordance with an embodiment of the present disclosure.
2B is a photograph of a biocompatible bioplastic aragonite composition containing 40% aragonite formed by three-dimensional (3D) printing of extruded filaments, in accordance with an embodiment of the present disclosure.
3A to 3D show the results of a lyophilized adenoviral vector (Ad5) of known viral titer loaded into capsules with aragonite or lactose. Infectious units/gram (without acid (FIG. 3A) or with acid (FIG. 3C)) or percent virus recovery (without acid (FIG. 3B) or with acid (FIG. 3D)) were obtained using the following aragonite or lactose formulations. Indicated: Aragonite Coating (C) 1 (0.357 g dry, powder, pH 8.81), Aragonite (C) 5 (0.425 g dry, powder, pH 8.84); Lactose uncoated (NC) 4 (0.8023 g, liquid, pH 2.14), aragonite (NC) 6 (0.545 g, paste, pH 7.78), aragonite (NC) 7 (0.629 g, paste, pH 7.79) and Aragonite (NC) 8 (0.801 g, paste, pH 7.00). Samples blended with aragonite maintained >/= 7 pH when exposed to acid. Each bar is one capsule.

The following passages describe the various aspects in more detail. Each aspect may be combined with any other aspect or aspects unless clearly indicated to the contrary. Specifically, any feature indicated as preferred or advantageous may be combined with any other feature of the feature indicated as preferred or advantageous.

Although certain features of the disclosure are described in the context of separate implementations, they may also be provided in combination in a single implementation. Conversely, various features of the disclosure that are described in the context of a single implementation can also be provided individually or in any suitable subcombination. All combinations of the disclosed embodiments are specifically included in this disclosure and are disclosed herein as if each and every combination were individually and explicitly disclosed. Moreover, all subcombinations are also specifically included in this disclosure and are disclosed herein as if each and every subcombination were individually and explicitly disclosed.

This disclosure is not limited to the specific implementations described herein. The terminology used herein to describe specific embodiments is not intended to be limiting. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is an admission that the present disclosure is not entitled to antedate such publications by virtue of prior disclosure. In addition, publication dates provided may differ from actual publication dates and may need to be independently verified. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by the lay vaccine scientist. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the vaccines described herein, the preferred methods and materials are now described.

As disclosed herein, vaccine compositions are provided in a form that is stable and easy to administer. More specifically, lyophilization of a recombinant virus-based vaccine and combining the vaccine with appropriate stabilizing compounds results in a composition that is stable and can be packaged for stable storage, transport and easy administration. Thus, a composition of the present disclosure can be prepared by lyophilizing an adenoviral vector and combining the adenoviral vector with a carbonite mineral such as aragonite. Thus, compositions of the present disclosure generally include a lyophilized adenoviral vector and a carbonite mineral such as aragonite.

A subject of the present invention is a solid dosage form (eg, self-administration) that is stable during storage, readily disintegrates in an aqueous solution upon mucosal administration (eg, sublingual or buccal administration) or for ingestion. Further included are compositions and methods for producing dosage forms of vaccines using aragonite to form (eg, powders, tablets or capsules).

Specifically, the present subject matter relates to an aragonite composition prepared from a plurality of aragonite particles, wherein the aragonite particles can be loaded into a vaccine composition to provide a solid dose vaccine in the form of a powder, tablet, or capsule. In an exemplary embodiment, the vaccine composition immunizes against a coronavirus. More specifically, a vaccine composition is a recombinant viral expression construct for expressing a corresponding antigen for a relevant infection/disease. Preferably, the recombinant construct is an adenoviral construct expressing an antigenic coronavirus protein or protein fragment.

In particular, as described in more detail herein, the use of aragonite in currently contemplated solid dosage forms enables cost effective manufacture and easy administration of stable vaccine compositions. As such, currently contemplated vaccines in powder, tablet or capsule form can be mass-produced and easily transported. In addition, the rapid disintegration of the solid dosage form (eg, 30 seconds or less) allows for self-administration. For example, vaccines in powder form may be packaged in individual dose packages. Exemplary packaging of the dosage form powder is similar to the packaging around a TWININGS ^® tea bag. The vaccine powder may be opened and dissolved in water or a suitable liquid beverage for ingestion (eg from a drinking water container or dropper) and the liquid released vaccine may be administered orally. Additionally, for vaccines in powder, tablet, or capsule form, if the dosage form disintegrates upon contact with an aqueous fluid upon sublingual or buccal administration, human saliva disintegrates the dosage form, leaving the vaccine composition inside the oral mucosa for absorption. will be emitted with In a further embodiment, tablets may be formed from compressed vaccine powder. Tablets may be compressed into any suitable shape, for example round or cubed. Tablet forms may also be prepared with additional excipients (eg, flavorings and gelatin) to form lozenges.

Aragonite (e.g. oocyte aragonite) is one of the purest forms of natural precipitated calcium carbonate. Referring to Figure 1, aragonite has a crystalline morphology of orthorhombic, bipyramidal, and characteristically needle-shaped crystals, distinguishing itself from calcite and vaterite. Aragonite can be recrystallized and/or reformed in various forms, so it can be used for various purposes by using the mechanical and chemical properties of calcium carbonate minerals. Aragonite particles as disclosed herein are solid materials having regular (eg, spherical or ovoid) or irregular shapes. Aragonite particles as used herein have an average particle size of 100 nm to 1 mm. Methods of milling aragonite particles are described in US 16/858,548, PCT/US20/29949, the entire contents of which are incorporated herein by reference. For example, a method of milling aragonite particles of 2.5 to 3.5 microns to a clean top size is disclosed. Net top size means that few particles larger than 3.5 microns in size are produced using the disclosed milling method with a classifier set in the 2.5 to 3.5 micron size range. Thus, aragonite particles as disclosed herein using the method of US 16/858,548, PCT/US20/29949 have a purer top size than conventional GCC.

The adsorption capacity of aragonite depends on three parameters: (1) surface charge (also known as “ζ (zeta) potential”); (2) surface area/void ratio; and (3) a function of particle solubility. By accurately measuring these three parameters, it is possible to determine which substances will adsorb on the surface of aragonite particles under given conditions. In particular, the zeta potential of aragonite increases the stability of surfactants such as glycerol and sorbitol.

In addition, aragonite naturally has a large number of measurable pores in particles less than 2 nm in diameter (ie highly “microporous”) (see EP 2719373). As such, the aragonite platform strongly holds the active ingredient particles together, allowing the loaded aragonite to be formulated into a solid dosage form, such as a powder, tablet or capsule.

Advantageously, untreated aragonite has a neutral pH (7.8 to 8.2), natural hydrophilic properties, electronic charge (zeta potential), and already formed nitrogen pairings with amino acids and proteins. Without wishing to be bound by any one theory, these advantageous properties of aragonite provide aragonite metastability at ambient conditions. More specifically, aragonite particles naturally contain an amino acid content of about 2 to 3%, most of which is aspartic acid (about 25 to 30%) and glutamic acid (about 8 to 10%), which renders the aragonite surface hydrophilic. am. See Mitterer, 1972, Geochimic et Cosmochimica Acta , 36: 1407-1422. Thus, in some embodiments the vaccine composition (eg recombinant adenovirus) is directly coupled to the natural untreated surface of the aragonite particles.

Currently, calcium carbonate available on the market is processed as or from ground calcium carbonate (GCC), precipitated calcium carbonate (PCC) (synthesized), and/or limestone production. The products produced are commodity grades with different properties. To obtain a pure particle size distribution (PSD) top size and low retention, most companies utilize wet grinding processes with either high-density solids or low-density solids. However, the products and these processes are neither biological nor environmentally benign. As used herein, aragonite refers to naturally occurring aragonite having an orthorhombic, bipyramidal and characteristically acicular crystalline morphology distinct from GCC, PCC and limestone.

One embodiment is a lyophilized adenoviral vector; and a filler comprising or consisting of a carbonite mineral, wherein the adenoviral vector comprises a nucleic acid molecule encoding at least a portion of the heterologous protein. An "adenoviral vector" is a genomic adenovirus lacking one or more genes required for adenoviral replication in unmodified mammalian cells. "Adenovirus"("Ad" for short) refers to a group of non-enveloped double-stranded DNA viruses with a diameter of about 60 to 110 nm from the family Adenoviridae . The adenoviral vectors disclosed herein can be classified into four genera of the adenoviridae (e.g., aviadenovirus, mastadenovirus, atadenovirus and may be derived from any adenovirus among iadenoviruses . In humans, most adenovirus infections are asymptomatic and not associated with neoplastic disease. The most extensively characterized Ad serotypes are serotype 2 (Ad2) and serotype 5 (Ad5). In one aspect, the adenoviral vector used herein is an Ad2 vector. In another aspect, the adenoviral vector is an Ad5 vector.

The adenovirus dsDNA genome is about 36 kb in length. The genome contains two sets of genes: early region genes E1A, E1B, E2, E3 and E4 that are transcribed before DNA replication; and late region genes L1 to L5, which are transcribed and expressed at high levels after initiation of DNA replication. Early region genes are required to enhance viral replication and viral DNA replication by activating transcription of other viral regions and altering the host cell environment. The E1A transcription unit encodes two major E1a involved in the regulation of viral transcription. The two major E1bs are involved in stimulation of viral mRNA transport, blocking E1A-induced apoptosis and blocking host mRNA transport. The E2b gene encodes viral polymerase and terminal protein precursors.

As used herein, “activity,” “function,” and the like refer to the ability of a molecule to do something. For example, adenovirus E2a binds to and modulates the activity of numerous cellular factors to induce host cells into S phase. Each that binds cellular factors, modulates their activity, and induces cells into S phase can be considered an E1a activity. As a result of lacking one or more activities, adenoviral vectors are unable to replicate in unmodified mammalian cells ("replication defective"). As used herein, "unmodified" mammalian cells lack DNA encoding adenoviral proteins. Replication defective adenoviral vectors are able to replicate in helper cells. A helper cell is a mammalian cell that has DNA encoding a protein that provides, in trans , one or more activities required for adenovirus replication that are missing from the adenovirus vector.

Modifications to the adenoviral genome (also referred to as mutations) that result in the creation of adenoviral vectors can be made at any location(s) of the genome, as long as the modification removes at least one function required for replication in unmodified cells. . Preferred modifications result in the removal of functions that can be provided in trans in helper cells. Useful modifications to the adenoviral genome include modifications that result in the deletion of at least some or all of the adenoviral genes, such that the resulting adenoviral vector is incapable of producing functional proteins with the activity required for replication. An adenoviral vector that lacks an activity associated with a protein is referred to as "null" for that protein or activity and may be designated [protein-] (eg, [E2b-]).

Exemplary adenoviral proteins required for replication in unmodified mammalian cells include, but are not limited to E1a, E1b, E2a and E2b. In one aspect, the adenoviral vector comprises a modification of the sequence encoding E1a. In one aspect, the adenoviral vector comprises a modification of the sequence encoding E1b. In one aspect, the adenoviral vector comprises a modification of the sequence encoding E2a. In one aspect, the adenoviral vector comprises a modification of the sequence encoding E2b. In one aspect, the adenoviral vector comprises a deletion of the E1 gene region. In one aspect, the adenoviral vector comprises a deletion of the E1a gene region. In one aspect, the adenoviral vector comprises a deletion of the E1b gene region. In one aspect, the adenoviral vector comprises a deletion of the E2 gene region. In one aspect, the adenoviral vector comprises a deletion of the E2a gene region. In one aspect, the adenoviral vector comprises a deletion of the E2b gene region. In one aspect, the adenoviral vector comprises a deletion of the E1 gene region and the E2 gene region. In one aspect, the adenoviral vector comprises a deletion of one or more gene regions selected from the group consisting of an E1a gene region, an E1b gene region, an E2a gene region, and an E2b gene region. In one aspect, the adenoviral vector lacks one or more activities associated with E1a. In one aspect, the adenoviral vector lacks one or more activities associated with E1b. In one aspect, the adenoviral vector lacks one or more activities associated with E2a. In one aspect, the adenoviral vector lacks one or more associated with E2b. In one aspect, the adenoviral vector lacks one or more activities associated with one or more proteins selected from the group consisting of E1a, E1b, E2a and E2b. In one aspect, the adenovirus vector is [E1a-] and/or [E1b-] and/or [E2a-] and/or [E2b-].

As used herein, "heterologous" refers to a molecule derived from an organism different from the organism being referenced, or a protein derived from the same kind of organism as the organism in which it is expressed, although heterologous proteins are customary for the tissue context in which they are expressed. It is expressed to an extent that is not A molecule can be a protein or a nucleic acid sequence (ie, RNA or DNA). For example, a heterologous nucleic acid sequence of a recombinant viral vector refers to the fact that the heterologous nucleic acid sequence is derived from an organism other than the basic virus used to construct the recombinant viral vector. As a further example, a heterologous nucleic acid sequence in an adenoviral vector refers to the fact that the heterologous nucleic acid sequence is from an organism other than an adenovirus. Similarly, a protein heterologous to an adenoviral vector refers to the fact that the heterologous protein is derived from an organism other than an adenovirus.

In one aspect, at least a portion of the heterologous protein is an immunogenic portion. As used herein, "immunogenicity" refers to the ability of a particular protein portion to elicit an immune response against a particular protein, or a protein comprising an amino acid sequence with a high degree of identity to a heterologous protein. According to the present disclosure, amino acid sequences with a high degree of identity are 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 contiguous amino acid sequences. The heterologous protein may be selected from the group consisting of neoepitopes, viral proteins and bacterial proteins. Heterologous proteins include adenovirus, herpesvirus, papillomavirus, polyomavirus, hepadnavirus, parvovirus, astrovirus, calicivirus, picornavirus, coronavirus, flavivirus, togavirus, hefevirus, retrovirus viruses, orthomyxoviruses, arenaviruses, bunyaviruses, filoviruses, paramyxoviruses, rhabdoviruses, reoviruses, influenza viruses, and poxviruses.

In certain embodiments, the protein is a coronavirus protein. Alphacoronavirus and betacoronavirus only infect mammals. Gammacoronaviruses and deltacoronaviruses infect birds, but some of them can also infect mammals. Alphacoronaviruses and betacoronaviruses usually cause respiratory illness in humans and gastroenteritis in animals. The highly pathogenic viruses SARS-CoV, MERS-CoV and SARS-CoV-2 cause severe respiratory syndrome in humans, while the other four human coronaviruses (HCoV-NL63, HCoV-229E, HCoV-OC43 and HKU1) are immunocompetent. Although they can cause mild upper respiratory illness in their host, some of the four human coronaviruses can cause severe infection in infants, children and the elderly.

Coronavirus is the largest single positive-stranded RNA virus with a genome of 27 to 32 kb. The genome is packed inside a helical capsid formed by the nucleocapsid protein (N) and surrounded by an additional envelope. There are at least three structural proteins associated with viral envelope: membrane protein (M) and envelope protein (E), which are involved in viral assembly, and spike protein (S), which mediate viral entry into the host cell. Some coronaviruses also encode the envelope-associated hemagglutinin-esterase protein (HE). Among these structural proteins, S forms large protrusions on the surface of the virus, giving the coronavirus a crowned appearance. Besides mediating viral entry, S determines host range and tissue tropism. S also induces host immune responses.

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

In one aspect, the heterologous protein is N from a coronavirus selected from the group consisting of SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-NL63, HCoV-229E, HCoV-OC43, HKU1. In one aspect, the heterologous protein is SARS-CoV-2 N. In one aspect, the heterologous protein is N from SARS. In one aspect, the heterologous protein is N from MERS.

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

In one aspect, the heterologous protein is E from a coronavirus selected from the group consisting of SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-NL63, HCoV-229E, HCoV-OC43, HKU1. In one aspect, the heterologous protein is E from SARS-CoV-2. In one aspect, the heterologous protein is E from SARS. In one aspect, the heterologous protein is E from MERS.

In one aspect, the heterologous protein is S from a coronavirus selected from the group consisting of SARS-CoV-2, MERS-CoV, SARS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, HKU1. In one aspect, the heterologous protein is S from SARS-CoV-2. In one aspect, the heterologous protein is S from SARS. In one aspect, the heterologous protein is S from MERS.

In one aspect, the heterologous protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or more relative to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, or contain 100% identical amino acid sequences. In one aspect, the at least one heterologous protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4. In one aspect, at least a portion of the heterologous protein comprises at least 6 contiguous amino acid residues from an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4.

Another example of a virus from which heterologous proteins can be obtained is an influenza virus. The protective immune response to the influenza virus is primarily directed against the viral hemagglutinin (HA) protein, a glycoprotein on the surface of the virus responsible for interacting with host cell receptors. The influenza virus HA protein represents an attractive target for eliciting an immune response by vaccination. The heterologous protein may be derived from an influenza virus such as, but not limited to, human influenza virus and avian influenza virus. The heterologous protein may be influenza HA, an epitope thereof, an immunogenic portion thereof, or a variant thereof. Any influenza HA, its epitope, part or variant thereof, as long as the HA protein, its epitope, part or variant thereof induces an immune response, preferably induces a protective immune response against influenza virus, is an adenovirus of the present disclosure. Can be used in viral vectors. Examples of useful influenza HA proteins, epitopes thereof, fragments thereof and variants thereof are disclosed in US 2010/0074916, US 2011/0171260, US 2011/0177122 and US 2014/0302079, the entire contents of which are incorporated herein by reference. included

In one aspect, 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.

Lyophilization is the process of removing water from a material (eg, an adenoviral vector) using freezing temperatures and low pressure. In exemplary lyophilization, the material to be lyophilized is cooled below its triple point, generally between -50°C and -80°C. Once the material is frozen, the surrounding air pressure is reduced and enough heat is added to sublimate the ice. In the second drying step, additional heat is applied to remove unfrozen water molecules. When complete, the lyophilized material has a residual moisture content of less than 5%, typically less than 3%, and typically in the range of about 0.5% to about 3%. Lyophilization methods are described in US 7,888,097, the disclosure of which is incorporated herein by reference. In one aspect, a lyophilized composition of the present disclosure has a residual moisture of less than 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%. In one aspect, a lyophilized composition of the present disclosure has a residual moisture of about 0.5% to about 5%. In one aspect, a lyophilized composition of the present disclosure has a residual moisture of about 0.5% to about 3%. In one aspect, a lyophilized composition of the present disclosure has a residual moisture of about 0.5% to about 1%. As used herein with reference to residual moisture content, “about” refers to a variation of 10% or less in the stated value.

As used herein, a "filler" is one or more compounds added to a composition to increase the mass or volume of the composition. Exemplary fillers commonly used for administration to humans and animals include, but are not limited to, lactose, sucrose, magnesium stearate, glucose, mannitol, starch, dextrose, maltodextrin, maltitol, vegetable cellulose, and the like. Because some individuals are sensitive to certain compounds, fillers present in the compositions of the present disclosure may include lactose, sucrose, magnesium stearate, glucose, mannitol, sorbitol, starch, dextrose, maltodextrin, maltitol, and vegetable cellulose. One or more compounds selected from the group may be missing. In one aspect, the composition is lactose free. In one aspect, the composition lacks mannitol. In one aspect, the composition lacks sorbitol. In one aspect, the composition lacks starch.

Given the sensitivity issues discussed above, carbonite minerals have desirable properties, making them excellent fillers. Carbonite minerals contain the carbonate ion CO ₃ ^2- . Examples of carbonite fillers include, but are not limited to, calcite, vaterite, aragonite, cerucite, strontianite, witherite, and rutherpodin. In one aspect, the filler comprises or consists of a compound selected from the group consisting of calcite, vaterite, aragonite, selucite, strontianite, witherite and rutherpodin. In one aspect, the filler comprises a carbonite mineral having an orthorhombic lattice. In one aspect, the filler comprises or consists of a compound selected from the group consisting of aragonite, cerucite, strontianite, witherite and rutherpodin. In one aspect, the filler comprises or consists of aragonite. In one aspect, the filler comprises or consists of cerucite. In one aspect, the filler comprises or consists of strontianite. In one aspect, the filler comprises or consists of rutherpodin.

In addition to the foregoing components, the compositions of the present disclosure may, but need not, include, for example, stabilizers, sweeteners, buffers, binders, carriers, diluents, adjuvants, and additional components that act as pharmaceutically active compounds. . Examples of additional ingredients include, but are not limited to, sodium chloride, potassium chloride, sodium citrate, sodium phosphate, sucrose, dimethylglycine, methylsulfonylmethane, and yeast lysate. In one aspect, the composition includes one or more of stabilizers, sweeteners, buffers, binders, carriers, diluents, adjuvants and pharmaceutically active compounds. In one aspect, 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. .

The present disclosure includes compositions formulated for ease of administration to a subject. Accordingly, one embodiment of the present disclosure is a capsule comprising a composition disclosed herein. In one embodiment, the capsule contains a filler comprising or consisting of a carbonite mineral and a composition comprising or consisting of a lyophilized adenoviral vector, wherein the adenoviral vector comprises a nucleic acid sequence encoding at least a portion of a heterologous protein. . Capsules can be made of one or more materials including, but not limited to, cellulose, gelatin, and alginates. In one aspect the capsule comprises cellulose. In one aspect, the capsule includes alginate. In one aspect the capsule contains gelatin. In one aspect, the capsule is enterically coated.

One embodiment is a composition comprising a filler comprising aragonite and a lyophilized adenoviral vector, wherein the adenoviral vector is replication defective due to lack of E1 and E2b activity, and the adenoviral vector is SARS-CoV, MERS-CoV , SARS-CoV-2, HCoV-NL63, HCoV-229E, HCoV-OC43, HKU1, wherein the coronavirus protein is selected from the group consisting of coronavirus N protein, coronavirus M protein, coronavirus E protein, and coronavirus S protein.

One embodiment is a method of making a composition disclosed herein. In one aspect, the method comprises lyophilizing an adenoviral vector comprising a nucleic acid molecule encoding at least a portion of a heterologous protein; and combining the adenoviral vector with a filler comprising or consisting of a carbonite mineral.

In a further embodiment the composition is encapsulated in a capsule suitable for administration to a subject. In one aspect, the adenoviral vector is any of the four genera of the adenoviridae (e.g., Aviadenovirus , Mastadenovirus , Artadenovirus , and Cyadenovirus ), along with any serotype of each species. is derived from In one aspect, the adenoviral vector is derived from a serotype 2 adenovirus. In one aspect, the adenoviral vector is derived from a serotype 5 adenovirus.

In one aspect, the lyophilized adenoviral vector has a residual moisture of less than 5%, less than about 4%, less than about 3%, less than about 2% or less than about 1%. In one aspect, the lyophilized adenoviral vector has a residual moisture of about 0.5% to about 5%. In one aspect, the lyophilized adenoviral vector has a residual moisture of about 0.5% to about 3%.

In one embodiment, the filler comprises one or more compounds selected from the group consisting of lactose, sucrose, magnesium stearate, glucose, mannitol, sorbitol, starch, dextrose, maltodextrin, maltitol and vegetable cellulose. In one embodiment, the filler lacks one or more compounds selected from the group consisting of lactose, sucrose, magnesium stearate, glucose, mannitol, sorbitol, starch, dextrose, maltodextrin, maltitol and vegetable cellulose. In one aspect, the filler lacks lactose. In one aspect, the filler lacks mannitol. In one aspect, the filler lacks sorbitol. In one aspect, the filler lacks starch.

In one aspect, the composition includes one or more of a stabilizer, a sweetener, a buffer, a binder, a carrier, a diluent, an adjuvant, and a pharmaceutically active compound. In one aspect, the composition includes one or more compounds selected from the group consisting of sodium chloride, potassium chloride, sodium citrate, sodium phosphate, sucrose, dimethylglycine, methylsulfonylmethane, and yeast lysate.

In one aspect, the capsule contains one or more materials selected from the group consisting of cellulose, gelatin and alginates. In one aspect the capsule comprises cellulose. In one aspect, the capsule includes alginate. In one aspect the capsule contains gelatin. In one aspect, the capsule is enterically coated.

Methods of vaccinating an individual against an infectious organism are disclosed herein. These methods include administering to a subject one or more times a composition disclosed herein, or a capsule containing a composition disclosed herein. In one aspect, a composition comprises a filler comprising or consisting of a carbonite mineral and a lyophilized adenoviral vector, wherein the adenoviral vector comprises a nucleic acid sequence encoding at least a portion of a heterologous protein from an infectious organism. In one aspect, the individual is at risk of exposure to an infectious organism. Such individuals may occasionally have been exposed to an infectious agent, or they may be previously exposed and yet asymptomatic.

The dosage, as well as the route and frequency of administration of the compositions and capsules containing the compositions described herein will vary from individual to individual and from disease to disease, and can be readily established using standard techniques. In general, the composition is administered intravenously, oral, parenteral, intraarterial, cutaneous, subcutaneous, intramuscular, topical, intracranial, intraorbital, intraocular, intravitreal, intraventricular, intracapsular, intraspinal, intracisternal, 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.

The method of administering a composition of the present disclosure will depend on factors such as the age, weight and physical condition of the patient being treated, and the disease or condition being treated. Thus, a skilled operator will be able to select the best dosage method for the patient on a case-by-case basis.

One embodiment is a kit. A kit may include, for example, an adenoviral vector of the present disclosure, a nucleic acid molecule for constructing an adenoviral vector of the present disclosure, a filler of the present disclosure, a composition of the present disclosure, and/or a capsule of the present disclosure. can A kit may also include related components such as, but not limited to, media, buffers, labels, containers, vials, syringes, and instructions for use of the kit.

Solid Dosage Forms for Vaccine Compositions:

As disclosed herein, dosage forms (also referred to as solid dosage forms) include aragonite with inherently stable properties that can be directly coupled with vaccine compositions. In an exemplary embodiment, a contemplated dosage form comprises aragonite impregnated (ie, coupled) with carbon dioxide (CO ₂ ) prior to addition of the vaccine composition (see EP 2719373 and US 2020/0155458). In a further embodiment, contemplated dosage forms include aragonite with biocompatible polymers and/or disintegrants that have been mixed and processed with aragonite prior to addition of the vaccine composition. Typically, aragonite is impregnated with CO ₂ and mixed with both a biocompatible polymer and a disintegrant prior to adding the vaccine composition. More commonly, aragonite is impregnated with CO ₂ , mixed with a biocompatible polymer and disintegrant prior to addition of the vaccine composition, and formed into a solid form (eg compressed) (see EP 2719373 and US 2020/0155458). .

The vaccine composition is loaded (eg, mixed) onto a contemplated aragonite solid dosage form as disclosed herein (eg, optionally impregnated with CO ₂ , a biocompatible polymer, and/or a disintegrant). ). Solid dosage forms contemplated may be compressed before or after loading of the vaccine composition. Typically, the solid dosage form is compressed (eg, compressed) before the vaccine composition is loaded thereon. In an exemplary embodiment, the compressed (ie compressed) solid dosage form with the vaccine composition loaded thereon is ground to form a powder, tablet or capsule.

Compression of the solid dosage form before or after loading of the vaccine composition is performed using a compression force of 5 to 500 kN. Preferably, compression of the solid dosage form before or after loading of the vaccine composition is performed using a compression force of 6 to 300 kN, most preferably 8 to 200 kN. More preferably, compression of the solid dosage form before or after loading of the vaccine composition is performed using a compression force of 8 to 100 kN, 8 to 50 kN, or 8 to 28 kN.

In an exemplary embodiment, CO ₂ -coupled aragonite is mixed with at least one biocompatible polymer. Typically, the weight ratio of CO ₂ -coupled aragonite to biocompatible polymer is about 95:5 to 5:95. In a further embodiment, the biocompatible polymer is a hot melt extruded biocompatible polymer. Exemplary biocompatible polymers include polylactic acid (PLA), polyethylene, polystyrene, polyvinylchloride, polyamide 66 (nylon), polycaprolactam, polycaprolactone, acrylic polymers, acrylonitrile butadiene styrene, polybenzimidazole, poly carbonate, polyphenylene oxide/sulfide, polypropylene, teflon, polylactic acid, aliphatic polyesters such as polyhydroxybutyrate, poly-3-hydroxybutyrate (P3HB), polyhydroxyvalerate, polyhydroxybutyrate-poly hydroxyvalerate copolymers, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), polyglyconates, poly(dioxanone), and mixtures thereof. Preferably the biocompatible polymeric resin is PLA. Preferably the biocompatible polymer is Eudragit L30 D-55 (Evonik).

In certain embodiments, the weight ratio of the plurality of aragonite particles (ie, the aragonite composition with or without carbon dioxide) to the biocompatible polymer is between about 95:5 and 5:95. Preferably, the weight ratio of the plurality of 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 the plurality of aragonite particles to the biocompatible polymer is about 50:50.

In a further embodiment, the CO ₂ -coupled aragonite and biocompatible polymer also include a disintegrant mixed therein (eg, engineered). Examples of suitable disintegrants include starch (eg pea starch), modified cellulose gum, insoluble cross-linked polyvinylpyrrolidone, starch glycolate, microcrystalline cellulose, pregelatinized starch, sodium carboxymethyl starch, low-substituted hydroxyl propyl cellulose, homopolymers of N-vinyl-2-pyrrolidone, alkyl-, hydroxyalkyl-, carboxyalkyl-cellulose esters, alginates, and microcrystalline cellulose and its polymorphic forms.

Mixing of the plurality of aragonite particles with the biocompatible polymer and, optionally, the disintegrant is accomplished using any conventional hot melt extrusion method. For example, hot melt extrusion can be performed on a twin screw hot melt extruder with a perforated die (eg Three-Tec, ZE9 20602, Switzerland). Compounding and extrusion methods for aragonite and bioplastic compositions, including filament production, are described, for example, in PCT/US20/45451, the entire contents of which are incorporated herein by reference. Additionally, extruded filament compositions made of bioplastics, aragonite and optionally disintegrants can be formed into useful or suitable shapes using 3D printing. Referring to FIGS. 2A and 2B , an exemplary bioplastic aragonite (containing 40% aragonite) composition was blended and extruded to make filaments, which were then processed using 3D printing to form the aragonite structure as shown.

Additional excipients may also be added to the solid dosage form as determined by manufacturing and packaging needs. Additional excipients are ion exchange resins, gums, chitin, chitosan, clay, gellan gum, cross-linked polyacrylic copolymers, agar, gelatin, dextrin, acrylic acid polymers, carboxymethylcellulose sodium/calcium, hydroxypropyl methylcellulose phthalate, shellac or mixtures thereof, lubricants, internal wound lubricants, external lubricants, impact modifiers, plasticizers, waxes, stabilizers, pigments, colorants, flavoring agents, taste masking agents, flavoring agents, sweeteners, texture improvers, binders, diluents, film formers, adhesives, buffers, adsorbents, odor masking agents, and mixtures thereof.

In particular, the solid dosage forms contemplated are loaded with vaccine compositions for oral, sublingual or buccal administration. Loading or mixing the vaccine composition into a solid dosage form (eg, CO ₂ -coupled aragonite already mixed with a biocompatible polymer and disintegrant) can be accomplished by any conventional methodology. For example, the vaccine composition can be loaded into a solid dosage form in a mixer (eg, tumbling mixer) or blender.

In an exemplary embodiment, a contemplated solid dosage form is loaded with a vaccine composition for inducing immunity against a virus. As will be readily appreciated by those skilled in the art, there are a wide variety of vaccine types depending on the disease/infection (eg virus) to be immunized. For example, if the pathogenic virus is a coronavirus (e.g., SARS-CoV, MERS-CoV, SARS-CoV-2 as well as human coronavirus NL63/HCoV-NL63), the vaccine can inhibit all or all of the coronavirus ACE2 protein. It may be a recombinant expression vector encoding a portion. On the other hand, when the pathogenic virus is poliovirus, the vaccine may be a recombinant expression vector encoding all or part of the CEA protein. In another example, when the pathogenic virus is the HIV virus, the vaccine may be a recombinant expression vector encoding all or part of gp120. Similar considerations apply of course to all other types of pathogenic viruses (eg influenza virus, rhinovirus, enterovirus, echovirus, herpes virus, etc.).

In an exemplary embodiment, the vaccine composition is a SARS-CoV2 vaccine (eg, an adenovirus construct) and is disclosed in US 16/880,804 and US 63/016,048, the entire contents of which are incorporated herein by reference. , soluble ACE2 protein coupled to an immunoglobulin Fc portion to form an ACE2-Fc hybrid construct, which may also include a J-chain portion. In another exemplary embodiment, the SARS-CoV2 vaccine (eg, adenovirus construct) comprises a mutant variant of the recombinant soluble ACE2 protein (eg SEQ ID NO: 6), wherein the mutant variant is described in US 63/022,146 at least one mutated amino acid residue (e.g., substitution by). In another exemplary embodiment, a SARS-CoV2 vaccine (eg, an adenoviral construct) is a U.S. 16/883,263 and U.S. 63/009,960 (the entire contents of which are incorporated herein by reference), CoV2 spike protein fused to CoV2 nucleocapsid protein or endosome targeting sequence (N-ETSD).

Preferably, the dosage forms contemplated are, for example, U.S. 16/880,804, the entire contents of which are incorporated herein by reference, into a vaccine composition comprising a recombinant expression vector (eg, adenovirus) encoding a recombinant ACE2 protein. 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 to SEQ ID NO:5.

In additional or alternative embodiments, the contemplated dosage form is T27F, T27W, T27Y, D30E, H34E, H34F, H34K, H34M, H34W, A vaccine composition comprising a recombinant expression vector (eg, adenovirus) encoding a recombinant ACE2 variant mutant comprising H34Y, D38E, D38M, D38W, Q24L, D30L, H34A, and/or D355L is loaded into the vaccine composition.

In an alternative embodiment, the aragonite particle surface can be treated to modify the bonding surface. For example, as disclosed in US 16/858,548 and PCT/US20/29949, treatment with stearic acid (ie octadecanoic acid) provides a hydrophobic surface. For protein loading, aragonite is treated with phosphoric acid to form a lamellar structure. Additional conjugation techniques for coupling reactive groups to the amino acid surface of aragonite are known in the art, for example as described in Bioconjugate Techniques, Third Edition, Greg T. Hermanson, Academic Press, 2013.

As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, a nucleic acid molecule refers to one or more nucleic acid molecules. As such, the terms "one", "an", "one or more" and "at least one" may be used interchangeably. Similarly, the terms "comprising" ("including") and "having" may be used interchangeably. The claims may be drawn to exclude any optional elements. As such, these statements are intended to serve as antecedent to any use of exclusive terms such as "only", "only", etc. in connection with recitation of a claim element or use of a "negative" limitation.

The invention is explained in more detail with 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 in a way limited to the following examples, but rather should be construed to include any and all modifications that become apparent as a result of the teachings provided herein.

Example

Example 1

This example demonstrates that uncoated aragonite formulations result in good virus recovery (pH stability and retention of solids (i.e., paste)), whereas non-coated lactose formulations result in less virus recovery and thus turn liquid and become acidic. prove

Capsule Formulation:

Lyophilized adenoviral vectors (Ad5) of known viral titer were loaded into capsules at 1×10 ⁹ infectious units (IU)/capsule. Lactose or aragonite was loaded to a final total weight of 550 mg/capsule. Capsules were sealed under moisture-controlled conditions and optionally coated with the anionic copolymer L30 D-55 and triethyl citrate (TEC).

Infectious titer determined by hexon assay:

E.C7 cells are seeded in 12 well plates at approximately 5.0 x 10 ⁵ cells per well and incubated at 37±2° C. for at least 2 hours. The hAd5 construct is serially diluted in 1X DMEM (Dulbecco's Modified Eagle's Medium). 2-4 hours after inoculation, inoculate 100 μL per well of the diluted test article in triplicate. Adenovirus type 5 (Ad5) reference material procured from the American Type Culture Collection (ATCC) is a positive control and is processed in the same way. A negative control uses 100 μL of diluent alone and inoculates 4 wells. Plates are incubated at 37°C ± 2°C for 42 hours.

Hexon immunostaining:

Plates are then fixed with cold methanol for 10 minutes, rinsed with 1X DPBS (Dulbeco acid buffered saline) and assayed by Hexon immunostaining. Mouse anti-hexon antibody solution (0.5 mL) is added to each well and incubated at 37°C ± 2°C for 60 ± 6 minutes. After washing the plate, 0.5 mL of rat anti-mouse antibody solution is added to the wells and incubated at 37°C ± 2°C for 60 ± 6 minutes. After washing, freshly prepared DAB (diaminobenzidine) working solution is added and incubated for 10 minutes at room temperature. Aspirate the DAB and add 1.0 mL of 1X PBS (Phosphate Buffered Saline) to each well.

Stained cells are visualized by capturing and counting images of the whole well using a light microscope at 10X objective 4 h after stroma development. The average number of positive cells/colony per well is calculated and the infection titer is determined by the formula:

Infectious units/mL = (average positive cells/well) x dilution factor x 10.

The results are shown in Figures 3a to 3d. Data are presented in infectious units/gram (without acid (FIG. 3A) or with acid (FIG. 3C)) or percent virus recovery (with encapsulation without acid treatment (FIG. 3B) or encapsulation with acid treatment (FIG. 3B)) using aragonite or lactose formulations. Fig. 3d) shows.The sample of aragonite compounding when exposed to acid maintains the pH of >/=7.Recovered sample mass (g) is shown in table 1 below.The final result is powder, liquid or It was a paste The pH was determined 2 minutes after resuspension and read 3 times.

sample mass recovered compounding agent Recovered sample mass (g) form pH Aragonite (C) 1 0.357 dry powder 8.81 Aragonite (C) 5 0.425 dry powder 8.84 Lactose (NC) 4 0.8023 Liquid 2.14 Aragonite (NC) 6 0.545 paste 7.78 Aragonite (NC) 7 0.629 paste 7.79 Aragonite (NC) 8 0.801 paste 7.00

Aspects of the embodiments may also be modified to utilize concepts from various patents, patent applications, and publications as necessary to provide additional embodiments.

These and other changes may be made to the implementations in light of the above detailed description. In general, the language used in the following claims should not be construed as limiting the claims to the specific embodiments disclosed in the specification and claims, but to all possible embodiments along with the full scope of equivalents given to such claims. should be construed as including Accordingly, the claims are not limited by this disclosure.

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

SEQUENCE LISTING <110> Nant Holdings IP, LLC NantCell Inc. Soon-Shiong, Patrick Gabitzsch, Elizabeth Peykov, Victor <120> VACCINE COMPOSITIONS FOR MUCOSAL IMMUNE RESPONSE <130> 8774-16-PCT <140> not yet assigned <141> 2021-10-22 <150> 63/104,770 <151 > 2020-10-23 <150> 63/082,907 <151> 2020-09-24 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 419 <212> PRT <213> SARS-CoV-2 virus <400> 1 Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr 1 5 10 15 Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg 20 25 30 Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn 35 40 45 Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu 50 55 60 Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro 65 70 75 80 Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Arg Ala Thr Arg Arg Ile Arg Gly 85 90 95 Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr 100 105 110 Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp 115 120 125 Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp 130 135 140 His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln 145 150 155 160 Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser 165 170 175 Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn 180 185 190 Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Lys Arg Thr Ser Pro Ala 195 200 205 Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu 210 215 220 Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln 225 230 235 240 Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys 245 250 255 Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln 260 265 270 Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp 275 280 285 Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile 290 295 300 Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile 305 310 315 320 Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala 325 330 335 Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu 340 345 350 Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro 355 360 365 Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln 370 375 380 Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu 385 390 395 400 Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser 405 410 415 Thr Gln Ala <210> 2 <211> 222 <212> PRT <213> SARS-CoV-2 virus <400> 2 Met Ala Gly Ser Asn Gly Thr Ile Thr Val Glu Glu Leu Lys Lys Leu 1 5 10 15 Leu Glu Gln Trp Asn Leu Val Ile Gly Phe Leu Phe Leu Thr Trp Ile 20 25 30 Cys Leu Leu Gln Phe Ala Tyr Ala Asn Arg Asn Arg Phe Leu Tyr Ile 35 40 45 Ile Lys Leu Ile Phe Leu Trp Leu Leu Trp Pro Val Thr Leu Ala Cys 50 55 60 Phe Val Leu Ala Ala Val Tyr Arg Ile Asn Trp Ile Thr Gly Gly Ile 65 70 75 80 Ala Ile Ala Met Ala Cys Leu Val Gly Leu Met Trp Leu Ser Tyr Phe 85 90 95 Ile Ala Ser Phe Arg Leu Phe Ala Arg Thr Arg Ser Met Trp Ser Phe 100 105 110 Asn Pro Glu Thr Asn Ile Leu Leu Asn Val Pro Leu His Gly Thr Ile 115 120 125 Leu Thr Arg Pro Leu Leu Glu Ser Glu Leu Val Ile Gly Ala Val Ile 130 135 140 Leu Arg Gly His Leu Arg Ile Ala Gly His His Leu Gly Arg Cys Asp 145 150 155 160 Ile Lys Asp Leu Pro Lys Glu Ile Thr Val Ala Thr Ser Arg Thr Leu 165 170 175 Ser Tyr Tyr Lys Leu Gly Ala Ser Gln Arg Val Ala Gly Asp Ser Gly 180 185 190 Phe Ala Ala Tyr Ser Arg Tyr Arg Ile Gly Asn Tyr Lys Leu Asn Thr 195 200 205 Asp His Ser Ser Ser Ser Asp Asn Ile Ala Leu Leu Val Gln 210 215 220 <210> 3 <211> 75 <212> PRT <213> SARS-CoV-2 virus <400> 3 Met Tyr Ser Phe Val Ser Glu Glu Thr Gly Thr Leu Ile Val Asn Ser 1 5 10 15 Val Leu Leu Phe Leu Ala Phe Val Val Phe Leu Leu Val Thr Leu Ala 20 25 30 Ile Leu Thr Ala Leu Arg Leu Cys Ala Tyr Cys Cys Asn Ile Val Asn 35 40 45 Val Ser Leu Val Lys Pro Ser Phe Tyr Val Tyr Ser Arg Val Lys Asn 50 55 60 Leu Asn Ser Ser Arg Val Pro Asp Leu Leu Val 65 70 75 <210> 4 <211> 1273 <212> PRT <213> SARS-CoV-2 virus <400> 4 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 Tyr Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665 670 Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala 675 680 685 Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser 690 695 700 Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile 705 710 715 720 Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val 725 730 735 Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu 740 745 750 Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr 755 760 765 Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln 770 775 780 Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe 785 790 795 800 Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser 805 810 815 Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly 820 825 830 Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp 835 840 845 Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu 850 855 860 Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly 865 870 875 880 Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile 885 890 895 Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr 900 905 910 Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn 915 920 925 Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala 930 935 940 Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn 945 950 955 960 Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val 965 970 975 Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln 980 985 990 Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val 995 1000 1005 Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn 1010 1015 1020 Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys 1025 1030 1035 Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 1040 1045 1050 Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val 1055 1060 1065 Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His 1070 1075 1080 Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn 1085 1090 1095 Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln 1100 1105 1110 Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val 1115 1120 1125 Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro 1130 1135 1140 Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn 1145 1150 1155 His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn 1160 1165 1170 Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu 1175 1180 1185 Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu 1190 1195 1200 Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu 1205 1210 1215 Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met 1220 1225 1230 Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys 1235 1240 1245 Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro 1250 1255 1260 Val Leu Lys Gly Val Lys Leu His Tyr Thr 1265 1270 <210> 5 <211> 805 <212> PRT <213> Homo sapiens <400> 5 Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala 1 5 10 15 Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe 20 25 30 Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp 35 40 45 Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn 50 55 60 Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala 65 70 75 80 Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln 85 90 95 Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Ser Val Leu Ser Glu Asp Lys 100 105 110 Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser 115 120 125 Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu 130 135 140 Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu 145 150 155 160 Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu 165 170 175 Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg 180 185 190 Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 195 200 205 Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu 210 215 220 Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu 225 230 235 240 His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile 245 250 255 Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 260 265 270 Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys 275 280 285 Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala 290 295 300 Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu 305 310 315 320 Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro 325 330 335 Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly 340 345 350 Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp 355 360 365 Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala 370 375 380 Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe 385 390 395 400 His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys 405 410 415 His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn 420 425 430 Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly 435 440 445 Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe 450 455 460 Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met 465 470 475 480 Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr 485 490 495 Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe 500 505 510 Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala 515 520 525 Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile 530 535 540 Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu 545 550 555 560 Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala 565 570 575 Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe 580 585 590 Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr 595 600 605 Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu 610 615 620 Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met 625 630 635 640 Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu 645 650 655 Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val 660 665 670 Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro 675 680 685 Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile 690 695 700 Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn 705 710 715 720 Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln 725 730 735 Pro Pro Val Ser Ile Trp Leu Ile Val Phe Gly Val Val Met Gly Val 740 745 750 Ile Val Val Gly Ile Val Ile Leu Ile Phe Thr Gly Ile Arg Asp Arg 755 760 765 Lys Lys Lys Asn Lys Ala Arg Ser Gly Glu Asn Pro Tyr Ala Ser Ile 770 775 780 Asp Ile Ser Lys Gly Glu Asn Asn Pro Gly Phe Gln Asn Thr Asp Asp 785 790 795 800 Val Gln Thr Ser Phe 805 <210> 6 <211> 594 <212 > PRT <213> Artificial Sequence <220> <223> Soluble ACE2 mutant constructs <220> <221> MISC_FEATURE <222> (4)..(4) <223> Can be Gln or Leu <220> <221> MISC_FEATURE <222> (7)..(7) <223> Can be Thr, Phe, Trp, or Tyr <220> <221> MISC_FEATURE <222> (10)..(10) <223> Can be Asp, Glu , Leu <220> <221> MISC_FEATURE <222> (14)..(14) <223> Can be His, Glu, Phe, Lys, Met, Trp, Tyr, or Ala <220> <221> MISC_FEATURE <222 > (18)..(18) <223> Can be Asp, Glu, Met, or Phe <220> <221> MISC_FEATURE <222> (335)..(335) <223> Can be Asp or Leu <400 > 6 Ile Glu Glu Xaa Ala Lys Xaa Phe Leu Xaa Lys Phe Asn Xaa Glu Ala 1 5 10 15 Glu Xaa Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr Asn Thr 20 25 30 Asn Ile Thr Glu Asn Val Gln Asn Met Asn Asn Ala Gly Asp Lys 35 40 45 Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln Met Tyr Pro 50 55 60 Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu Gln Ala Leu 65 70 75 80 Gln Gln Asn Gly Ser Ser Ser Val Leu Ser Glu Asp Lys Ser Lys Arg Leu 85 90 95 Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly Lys Val 100 105 110 Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro Gly Leu 115 120 125 Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu Trp Ala 130 135 140 Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg Pro Leu Tyr 145 150 155 160 Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn His Tyr 165 170 175 Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn Gly Val 180 185 190 Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp Val Glu His 195 200 205 Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala Tyr Val 210 215 220 Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro Ile Gly 225 230 235 240 Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe Trp Thr 245 250 255 Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn Ile Asp 260 265 270 Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg Ile Phe 275 280 285 Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn Met Thr 290 295 300 Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly Asn Val Gln 305 310 315 320 Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly Xaa Phe 325 330 335 Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu Thr Ala 340 345 350 His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala Ala Gln 355 360 365 Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu Ala Val 370 375 380 Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu Lys Ser 385 390 395 400 Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr Glu Ile 405 410 415 Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu Pro Phe 420 425 430 Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly Glu Ile 435 440 445 Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg Glu Ile 450 455 460 Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys Asp Pro 465 470 475 480 Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg Tyr Tyr 485 490 495 Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys Gln Ala 500 505 510 Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn Ser Thr 515 520 525 Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys Ser Glu 530 535 540 Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys Asn Met Asn 545 550 555 560 Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp Leu Lys 565 570 575 Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp Ser Pro 580 585 590 Tyr Ala

Claims (53)

A composition comprising a filler comprising a carbonite mineral and a lyophilized adenoviral vector, wherein the adenoviral vector comprises a nucleic acid molecule encoding at least a portion of a heterologous protein. The composition of claim 1 , wherein the adenoviral vector is derived from a type 5 adenovirus, and the adenovirus has deletions of 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 method of 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 viruses. , composition. 5. The composition of claim 4, wherein the heterologous protein is from SARS-CoV-2. The composition according to claim 5, wherein the heterologous protein is a spike (S) protein, a nucleocapsid (N) protein or a membrane (M) protein. 7. The method of claim 6, wherein the heterologous protein is at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 95%, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 at least 97% or even 100% identical, composition. 8. The composition according to any one of claims 1 to 7, wherein the adenovirus vector has a residual moisture content of 0.5% to 5%. 9. The composition of claim 8, wherein the adenoviral vector has a residual moisture of less than 5%. 10. The composition of claim 9, wherein the adenoviral vector has a residual moisture 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 of claim 11, wherein the carbonite mineral is selected from the group consisting of aragonite, serousite, strontianite, witherite and rutherpodin. 13. The composition of claim 12, wherein the carbonite mineral is aragonite. 14. The method of any one of claims 1 to 13, wherein the filler is selected from the group consisting of lactose, sucrose, magnesium stearate, glucose, mannitol, sorbitol, starch, dextrose, maltodextrin, maltitol and vegetable cellulose. A composition comprising one or more compounds. 15. The method of any one of claims 1 to 14, wherein the filler is selected from the group consisting of lactose, sucrose, magnesium stearate, glucose, mannitol, sorbitol, starch, dextrose, maltodextrin, maltitol and vegetable cellulose. A composition lacking one or more compounds. The method of any one of claims 1 to 15, wherein the composition is one selected from the group consisting of sodium chloride, potassium chloride, sodium citrate, sodium phosphate, sucrose, dimethylglycine, glycine, methylsulfonylmethane and yeast lysate A composition containing the above compounds. A capsule comprising the composition of any one of claims 1 to 16. 18. The capsule of claim 17, wherein the capsule is enteric coated. 19. The capsule according to claim 17 or 18, wherein the capsule comprises alginate. A solid dosage form for delivering a vaccine composition by oral, sublingual or buccal administration of the vaccine composition, said solid dosage form comprising:
an aragonite composition comprising a plurality of aragonite particles impregnated with carbon dioxide (CO ₂ ); and
Biocompatible polymer and disintegrant mixed with the aragonite composition
including,
The solid dosage form further comprises a vaccine composition,
The solid dosage form, wherein the solid dosage form is a powder, tablet or capsule. 21. The solid dosage form of claim 20, wherein the solid dosage form further comprises at least one excipient and is formulated to form a lozenge. 21. The solid dosage form of claim 20, wherein the plurality of aragonite particles have an average particle size of 100 nm to 1 mm. 23. The method of any one of claims 20-22, wherein the biocompatible polymer is polylactic acid (PLA), polyethylene, polystyrene, polyvinylchloride, polyamide 66 (nylon), polycaprolactam, polycaprolactone, acrylic polymer. , 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 these A solid dosage form selected from mixtures of 24. The solid dosage form of claim 23, wherein the biocompatible polymer is PLA. 25. The method according to any one of claims 20 to 24, wherein the disintegrant is starch, modified cellulose gum, insoluble cross-linked polyvinylpyrrolidone, starch glycolate, microcrystalline cellulose, pregelatinized starch, sodium carboxymethyl starch, low -substituted hydroxypropyl celluloses, homopolymers of N-vinyl-2-pyrrolidone, alkyl-, hydroxyalkyl-, carboxyalkyl-cellulose esters, alginates, and microcrystalline cellulose and its polymorphic forms. form. 26. The solid dosage form of claim 25, wherein the disintegrant is pea starch. 27. A solid dosage form according to any one of claims 20 to 26, wherein the powder, the tablet and the capsule disintegrate in aqueous solution in less than 30 seconds. 28. The solid dosage form of any one of claims 20-27, wherein the vaccine composition comprises a recombinant viral expression construct encoding a viral protein or fragment thereof. 29. The solid dosage form of claim 28, wherein the viral protein or fragment thereof corresponds to a coronavirus protein or fragment thereof. 30. The solid dosage form of claim 29, wherein the coronavirus protein or fragment thereof is a SARS-CoV2 virus binding protein. 31. The solid dosage form of claim 30, wherein the SARS-CoV2 virus binding protein comprises a recombinant ACE2 protein. 32. The solid dosage form of claim 31, wherein the recombinant ACE2 protein has at least 85% sequence identity to SEQ ID NO:5. 33. The solid dosage form of claim 32, wherein the recombinant ACE2 protein comprises the sequence of SEQ ID NO:6. 34. The method of claim 33, wherein the recombinant ACE2 protein is at least one selected from T27F, T27W, T27Y, D30E, H34E, H34F, H34K, H34M, H34W, H34Y, D38E, D38M, D38W, Q24L, D30L, H34A, and D355L. A solid dosage form comprising a mutagenesis. 35. The solid dosage form of any one of claims 20-34, wherein the vaccine composition comprises an adenovirus expression construct. A method of manufacturing a solid dosage form for loading a vaccine comprising:
providing an aragonite composition comprising a plurality of aragonite particles impregnated with carbon dioxide (CO ₂ );
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
How to include. 37. The method of claim 36, wherein mixing the aragonite composition with the biocompatible polymer and disintegrant comprises hot melt extrusion. 38. The method of claim 36 or 37, wherein the solid dosage form is a powder, tablet or capsule. 39. The method of claim 38, wherein the solid dosage form is a tablet and the method further comprises compressing the solid dosage form. 40. The method of any one of claims 36-39, wherein the aragonite composition and biocompatible polymer have a weight ratio of 95:5 to 5:95. 41. The method of any one of claims 36-40, wherein the plurality of aragonite particles have an average particle size of 100 nm to 1 mm. 42. The method of any one of claims 36-41, wherein the biocompatible polymer is polylactic acid (PLA), polyethylene, polystyrene, polyvinylchloride, polyamide 66 (nylon), polycaprolactam, polycaprolactone, acrylic polymer. , 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 these selected from mixtures of 43. The method of any one of claims 36-42, wherein the biocompatible polymer is PLA. 44. The method of any one of claims 36 to 43, wherein the disintegrant is starch, modified cellulose gum, insoluble cross-linked polyvinylpyrrolidone, starch glycolate, microcrystalline cellulose, pregelatinized starch, sodium carboxymethyl starch, low -substituted hydroxypropyl celluloses, homopolymers of N-vinyl-2-pyrrolidone, alkyl-, hydroxyalkyl-, carboxyalkyl-cellulose esters, alginates, and microcrystalline cellulose and polymorphic forms thereof. 45. The method of any one of claims 36-44, wherein the disintegrant is pea starch. 46. The method of any one of claims 36-45, wherein the vaccine composition comprises a recombinant viral expression construct encoding a viral protein or fragment thereof. 47. The method of claim 46, wherein the viral protein or fragment thereof corresponds to a coronavirus protein or fragment thereof. 48. The method of claim 47, wherein the coronavirus protein or fragment thereof is a SARS-CoV2 virus binding protein. 49. The method of claim 48, wherein the SARS-CoV2 virus binding protein comprises a recombinant ACE2 protein. 50. The method of claim 49, wherein the recombinant ACE2 protein has at least 85% sequence identity to SEQ ID NO:5. 51. The method of claim 50, wherein the recombinant ACE2 protein comprises the sequence of SEQ ID NO:6. 52. The method of claim 51, wherein the recombinant ACE2 protein is at least one selected from T27F, T27W, T27Y, D30E, H34E, H34F, H34K, H34M, H34W, H34Y, D38E, D38M, D38W, Q24L, D30L, H34A, and D355L. A method comprising a mutation. 53. The method of any one of claims 36-52, wherein the vaccine composition comprises an adenovirus expression construct.