Improved adenoviral vectors for antigen delivery

Abstract

A modified adenoviral vector, lacking splice acceptor dinucleotides in a region of the vector and having improved expression is provided.

Description

PATENT Attorney Docket No.090402-1499752-002310PC IMPROVED ADENOVIRAL VECTORS FOR ANTIGEN DELIVERY CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] The present patent application claims benefit of priority to U.S. Provisional Patent Application No. 63 / 648,506, filed May 16, 2024, which is incorporated by reference for all purposes. BACKGROUND OF THE INVENTION

[0002] Replication-deficient adenoviruses can be used to deliver virus-expressed proteins to a subject. For example such vectors can be used as a delivery system, or “vector,” to carry nucleic acids encoding an antigen, i.e., a component of a different infectious agent (e.g., a different virus) that is known to generate a protective immune response, and an adjuvant, i.e., a molecule that stimulates the innate immune system. Exemplary replication-deficient adenoviral vectors that deliver these two components to the epithelial cells lining the mucosa of the small bowel have been described. See, e.g., WO 2015 / 127278. BRIEF SUMMARY OF THE INVENTION

[0003] Adenoviral vectors for use in expression heterologous polypeptides are provided. In some embodiments, the vector comprises a heterologous expression cassette comprising a first promoter operably linked to a nucleic acid encoding a payload polypeptide; a nucleic acid encoding an adenovirus pIX protein; and a polynucleotide from the heterologous expression cassette (i.e., the end of the expression cassette) to the nucleic acid encoding the pIX protein, wherein the polynucleotide is free of canonical splice acceptors comprising an AG dinucleotide sequence.

[0004] In some embodiments, the polynucleotide comprises GTTTTGCXYCXYCCGCCGCCGCC (SEQ ID NO:1), wherein XY represents a dinucleotide sequence other than AG. In some embodiments, X is C, G, or T or Y is A, C, or T. In some embodiments, X is C, G, or T and Y is A, C, or T. In some embodiments, X inone XY is C, G, or T and one Y the other XY is A, C, or T. In some embodiments, the polynucleotide comprises GTTTTGCTGCCGCCGCCGCCGCC (SEQ ID NO:2).

[0005] In some embodiments, the polynucleotide comprises CTAGAAATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGAA TCGATAGTACTAACATACGCTCTCCATCTCGAGCCTAAGCTTGTCGACTCGAAGA TCTGGGCGTGGTTAAGGGTGGGAAAGAATATATAAGGTGGGGGTCTTATGTAGT TTTGTATCTGTTTTGCXYCXYCCGCCGCCGCC (SEQ ID NO:3), wherein XY represents a dinucleotide sequence other than AG. In some embodiments, X is C, G, or T or Y is A, C, or T. In some embodiments, X is C, G, or T and Y is A, C, or T. In some embodiments, X in one XY is C, G, or T and one Y the other XY is A, C, or T. In some embodiments, the polynucleotide comprises CTAGAAATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGAA TCGATAGTACTAACATACGCTCTCCATCTCGAGCCTAAGCTTGTCGACTCGAAGA TCTGGGCGTGGTTAAGGGTGGGAAAGAATATATAAGGTGGGGGTCTTATGTAGT TTTGTATCTGTTTTGCTGCCGCCGCCGCCGCC (SEQ ID NO:4).

[0006] In some embodiments, the payload polypeptide is an antigenic protein; and wherein the expression cassette further comprises a second promoter operably linked to a nucleic acid encoding a toll-like receptor-3 (TLR-3) agonist. In some embodiments, the TLR-3 agonist is a dsRNA.

[0007] In some embodiments, the first promoter comprises the CMV promoter or the SV40 promoter.

[0008] In some embodiments, the first promoter is inducible. In some embodiments, the first promoter comprises a Tet operator.

[0009] In some embodiments, the first promoter and the second promoter have different sequences. I n some embodiments, the first promoter and second promoter are independently selected from a CMV promoter and a SV40 promoter. In some embodiments, the first promoter comprises a CMV promoter and the second promoter comprises an SV40 promoter. In some embodiments, the CMV promoter comprises a Tet operator, rendering the CMV promoter inducible.

[0010] In some embodiments, the vector lacks an adenoviral E1A coding sequence, rendering the vector replication deficient absent exogenous provision of the E1A protein.

[0011] In some embodiments, the adenoviral vector is an Adenovirus 5 vector.

[0012] In some embodiments, the payload polypeptide is selected from the group consisting of: a SARS-CoV2 spike protein (e.g., XBB 1.5, KP.2 or JN.1 variants), a SARS-CoV2 nucleoprotein, a Norovirus VP1 (e.g., from a GI and GII genogroup) protein, an Influenza A hemagglutinin (H1, H3, or H5) protein, an Influenza B hemagglutinin (Victoria or Yamagata) protein, an HPV E6 / E7 (e.g., HPV16 and 18) protein, and a Rotavirus VP4, VP6, or VP8 protein.

[0013] Also provided is a pharmaceutical composition comprising the adenoviral expression vector as described above or elsewhere herein. In some embodiments, the pharmaceutical composition is a tablet or a capsule.

[0014] Also provided are methods of expressing a payload protein in a subject. In some embodiments, the method comprises administering to the subject an effective amount of the vector as described above or elsewhere herein or the pharmaceutical composition as described above or elsewhere herein to a mammalian subject. In some embodiments, the administering is oral, intranasal, or mucosal. In some embodiments, the administering is oral delivery by swallowing a tablet or capsule. In some embodiments, the immune response is elicited in an alveolar cell, an absorptive enterocyte, a ciliated cell, a goblet cell, a club cells, dendritic cell, macrophage, and / or an airway basal cell of the subject. In some embodiments, the subject is a human.

[0015] Also provided is a method of making the adenoviral expression vector as described above or elsewhere herein. In some embodiments, the method comprises introducing a nucleic acid encoding the adenoviral expression vector into a cell that heterologously expresses the E1A protein; and harvesting the adenoviral expression vector from the cell. In some embodiments, the CMV promoter comprises a Tet operator, rendering the CMV promoter inducible. In some of these embodiments, the cell heterologously expresses a tetracycline repressor protein.DEFINITIONS

[0016] The term “chimeric” or “recombinant” as used herein with reference, e.g., to a nucleic acid, protein, or vector, indicates that the nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein. Thus, for example, chimeric and recombinant vectors include nucleic acid sequences that are not found within the native (non-chimeric or non- recombinant) form of the vector. A chimeric adenoviral expression vector refers to an adenoviral expression vector comprising a nucleic acid sequence encoding a heterologous polypeptide, such as a SARS-CoV-2 protein or a protein from a different target infectious agent.

[0017] The term “expression vector” refers to a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.

[0018] The term “promoter” refers to an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as many as several thousand base pairs from the start site of transcription. Promoters include constitutive and inducible promoters. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under environmental or developmental regulation. The term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0019] The term “TLR agonist” or “Toll-like receptor agonist” as used herein refers to a compound that binds and stimulates a Toll-like receptor including, e.g., TLR-2, TLR-3, TLR- 6, TLR-7, or TLR-8. TLR agonists are reviewed in MacKichan, IAVI Report. 9:1-5 (2005) and Abreu et al., J Immunol, 174(8), 4453-4460 (2005). Agonists induce signal transduction following binding to their receptor.

[0020] The term “TLR-3 agonist” or “Toll-like receptor 3 agonist” as used herein refers to a compound that binds and stimulates the TLR-3. TLR-3 agonists have been identified including double-stranded RNA, virally derived dsRNA, several chemically synthesized analogs to double-stranded RNA including polyinosine-polycytidylic acid (poly I:C) - polyadenylic-polyuridylic acid (poly A:U) and poly I:poly C, and antibodies (or cross-linking of antibodies) to TLR-3 that lead to IFN-beta production (Matsumoto, M, et al, Biochem Biophys Res Commun 24:1364 (2002), de Bouteiller, et al, J Biol Chem 18:38133-45 (2005)). In some embodiments, a TLR-3 agonist comprises a sequence of any one of SEQ ID NOS: 6- 14. In some embodiments, a TLR-3 agonist is a dsRNA.

[0021] The term “heterologous” when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0022] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

[0023] Unless otherwise indicated, a particular nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J.Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0024] The term “antigen” refers to a protein or part of a polypeptide chain that can be recognized by T cell receptors and / or antibodies. Typically, antigens are derived from bacterial, viral, or fungal proteins.

[0025] The term "immunogenically effective dose or amount” of the compositions of the present disclosure is an amount that elicits or modulates an immune response specific for the antigenic target protein expressed from the viral vector. Immune responses include humoral immune responses and cell-mediated immune responses. An immunogenic composition can be used therapeutically or prophylactically to treat or prevent disease at any stage. Humoral immune responses are generally mediated by cell free components of the blood, i.e., plasma or serum; transfer of the serum or plasma from one individual to another transfers immunity. Cell mediated immune responses are generally mediated by antigen specific lymphocytes; transfer of the antigen specific lymphocytes from one individual to another transfers immunity.

[0026] The term “therapeutic dose” or “therapeutically effective amount” or “effective amount” of a chimeric adenoviral vector or a composition comprising a chimeric adenoviral vector refers to an amount of the vector or composition comprising the vector which prevents, alleviates, abates, or reduces the severity of symptoms of diseases and disorders associated with the source of the SARS-CoV-2 protein (e.g., a SARS-CoV-2 virus).

[0027] The term “adjuvant” refers to a non-specific immune response enhancer. Suitable adjuvants include, for example, cholera toxin, monophosphoryl lipid A (MPL), Freund’s Complete Adjuvant, Freund’s Incomplete Adjuvant, Quil A, and Al(OH). Adjuvants can also be those substances that cause antigen-presenting cell activation and enhanced presentation of T cells through secondary signaling molecules like Toll-like receptors. Examples of Toll-like receptors include the receptors that recognize double-stranded RNA, bacterial flagella, LPS, CpG DNA, and bacterial lipopeptide (Reviewed recently in Abreu et al., J Immunol, 174(8), 4453-4460 (2005)).

[0028] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a correspondingnaturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

[0029] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline and O- phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0030] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0031] As used herein, the term "percent identity" or "percent identical," used in the context of nucleic acids or polypeptides, refers to a sequence that has at least 50% sequence identity with a reference sequence (e.g., a sequence provided herein). Alternatively, percent identity can be any integer from 50% to 100%. In some embodiments, a sequence is substantially identical to a reference sequence if the sequence has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the reference sequence as determined using the methods described herein; preferably BLAST using standard parameters, as described below. Percent identity may also be determined by manual alignment.

[0032] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparisonalgorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0033] A comparison window includes reference to a segment of any one of the number of contiguous positions, e.g., a segment of at least 10 residues. In some embodiments, the comparison window has from 10 to 600 residues, e.g., about 10 to about 30 residues, about 10 to about 20 residues, about 50 to about 200 residues, or about 100 to about 150 residues, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

[0034] Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol.215: 403-410 and Altschul et al. (1977) Nucleic Acids Res.25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0035] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, an amino acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test amino acid sequence to the reference amino acid sequence is less than about 0.01, more preferably less than about 10-5, and most preferably less than about 10-20.

[0036] “Codon-altered” coding sequences have at least one change from a natural coding sequence such that the codon-altered coding sequence has higher expression than the natural coding sequence in a particular cell, e.g., a human cell.

[0037] A “canonical splice donor / acceptor” refers to the originally-described and most common splice donor (GT) in combination with AG as a splice acceptor sequence. Thus “canonical splice acceptors comprising an AG dinucleotide sequence” refers an AG dinucleotide sequence in a transcript that follows a GT dinucleotide sequence. See, e.g., Burset, et al., Nucleic Acids Res. 2000 Nov 1; 28(21): 4364–4375. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG. 1 shows two nucleotide changes in adenoviral vectors, resulting in a large reduction in alternative splice variants (see Table in FIG. 1) being produced by the vector lacking the two splice acceptor sites. FIG. 1 discloses SEQ ID NO: 18.

[0039] FIG. 2 depicts an exemplary adenoviral vector for delivering antigenic proteins. The first promoter in some embodiments can be a CMV promoter, optionally with a tet operator. The second promoter can optionally be an SV40 promoter. FIG. 2 discloses SEQ ID NO: 18. DETAILED DESCRIPTION OF THE INVENTION

[0040] The inventors have discovered that exclusion of AG splice acceptor sites between a payload expression cassette and the initial coding sequence for the pIX protein in an adenoviral vector results in surprisingly better expression of the desired payload and reduction of various spurious splice variants. The resulting chimeric adenoviral vectors can be used to express one or more payloads. As shown in FIG. 2, in some embodiments, theadenoviral vector expresses two payloads, e.g., an RNA encoding an antigenic protein and a TLR-3 agonist, e.g., such as a dsRNA.

[0041] In some embodiments, the adenoviral vector is, for example, an adenovirus 5 (Ad5), which can include, for example, Ad5 with deletions of the E1 / E3 regions and Ad5 with a deletion of the E4 region. Other suitable adenoviral vectors include but are not limited to strains 2, orally tested strains 4 and 7, enteric adenoviruses 40 and 41, and other strains (e.g. Ad34) that are sufficient for delivering an antigen and eliciting an adaptive immune response to the transgene antigen (Lubeck et al., Proc Natl Acad Sci U S A, 86(17), 6763-6767 (1989); Shen et al., J Virol, 75(9), 4297-4307 (2001); Bailey et al., Virology, 202(2), 695-706 (1994)). In some embodiments, the adenoviral vector is a live, replication incompetent adenoviral vector (such as E1 and E3 deleted rAd5), live and attenuated adenoviral vector (such as the E1B55K deletion viruses), or a live adenoviral vector with wild-type replication.

[0042] One or more heterologous expression cassettes can be inserted in the adenoviral vector, e.g., in the location from which E1A was deleted, and inserted upstream of the adenoviral pIX coding sequence. The E1A-deleted Ad5 is a vector that is widely used, and a transgene cassette is frequently placed in this location. See, e.g., Hitt and Graham, Advances in Virus Research, Volume 55, 2000, Pages 479-505. Removal of one or more, optionally all, AG splice acceptor sites from the end of the heterologous expression cassette introduced into the adenoviral vector and the beginning of the coding sequence of pIX is provided for in this disclosure. As an example, in adenoviral Adv5 viruses the sequence just upstream of the pIX coding sequence is the sequence GTTTTGCAGCAGCCGCCGCCGCC (SEQ ID NO: 17), where two AG splice acceptor sites are shown by underlining. The vectors provided herein can include this sequence, but each of the two AG dinucleotide sequences are changed. Changing of an AG dinucleotide can comprise changing the A or G or both to a different nucleotide. This for example, the “A” in an AG dinucleotide can be changed to a C, G, or T, and / or the “G” in the AG dinucleotide can be changed to A, C, or T, any of which change the “AG” to a non-AG sequence. In embodiments in which more than one AG dinucleotide sequence is disrupted, the different AG dinucleotides can be disrupted by the same change (merely as an example, both As changes to Cs) or each AG can be changed in a different manner (again merely as an example, a first AG disrupted by changing the A to a C and a second AG disrupted by for example changing the A to a T or changing the G to a C).

[0043] Accordingly, in some embodiments, the adenoviral vector comprises a sequence lacking an AG dinucleotide upstream (e.g., directly upstream of the ATG) of the pIX coding sequence and after the heterologous expression cassette, the sequence of SEQ ID NO:1. An example of such SEQ ID NO:1 with a specific change in each AG is SEQ ID NO:2. In some embodiments, the adenoviral vector comprises between the heterologous expression cassette and the pIX coding sequence SEQ ID NO:3. An example of such SEQ ID NO:3 with a specific change in each AG is SEQ ID NO:4.

[0044] The transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells in vivo may be provided by viral sources. For example, commonly used promoters and enhancers are derived, e.g., from beta-actin, adenovirus, simian virus (SV40), and human cytomegalovirus (CMV). For example, vectors allowing expression of proteins under the direction of the CMV promoter, beta-actin promoter, SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, transducer promoter, or other promoters shown effective for expression in mammalian cells are suitable. Further viral genomic promoter, control and / or signal sequences may be used, provided such control sequences are compatible with the host cell chosen.

[0045] Various promoters can be used in the heterologous expression cassette of the chimeric adenoviral vectors described herein. In some embodiments, when the chimeric adenoviral vector expresses two or more nucleic acids, the promoters used to express the nucleic acids can be identical or different. For example, in some embodiments, a first promoter used to express an element (a) (e.g., an antigenic protein) and a second promoter used to express an element (b) (e.g., a TLR-3 agonist) can both be a CMV promoter, or the two promoters may be different, e.g., one promoter is a CMV promoter and the other promoter is a SV40 or beta-actin promoter. In other embodiments, when an element (c) is included, a third promoter can be identical or different from the first and / or second promoter. For example, the first promoter and the second promoter can both be a CMV promoter and the third promoter can be a beta-actin promoter (e.g., a human beta-actin promoter).

[0046] Expression cassettes to express polypeptides as described herein can contain additional regulatory elements such as a polyadenylation signal, e.g., bovine growth hormone polyadenylation signal, and other sequences to regulate expression, such as terminator sequences or RNA stability elements.

[0047] A heterologous expression cassette in the adenoviral vector as described herein can include, for example, a promoter and an operably-linked coding sequence for a protein (or alternatively encoding an non-coding RNA). The coding sequence of the protein can be codon-optimized for the target host of the vector, e.g., a human. In some embodiments, the encoded protein is an antigenic protein, e.g., that will act to induce an immune response in a human or non-human mammal or bird. Exemplary antigenic proteins can include, but are not limited to a for example, viral antigens, bacterial antigens, cancer antigens, fungal antigens, or parasite antigens.

[0048] Viral antigens may be derived from, for example, SARS-COV-2 ((e.g., the S, M, E or N protein, e.g., WO 2021 / 248017 and Magazine, et al., Viruses 2022 Mar 19;14(3):640, or any variant thereof including but not limited to the S protein variant XBB 1.5 (SEQ ID NO:5), KP.2 (SEQ ID NO:15) or JN.1 (SEQ ID NO:16)) or an antigenic fragment thereof. Particular examples of antigens that can be used can include those derived from norovirus (e.g., VP1, and sequences thereof as described in U.S. Patent No. 11, 433, 146, incorporated by reference) and Respiratory syncytial virus (RSV) (e.g., fusion protein (F) and sequences thereof as described in U.S. Patent No. 11, 433, 146, incorporated by reference). Other suitable antigens include those from the influenza virus (e.g., HA, NA, M1, NP), human immunodeficiency virus (HIV, e.g., gag, pol, env, etc.), human papilloma virus (HPV, e.g., capsid proteins such as L1), Venezuelan Equine Encephalomyelitis (VEE) virus, Epstein Barr virus, herpes simplex virus (HSV), human herpes virus, rhinoviruses, cocksackieviruses, enteroviruses, hepatitis A, B, C, E, and G (HAV, HBV, HCV, HEV, HGV e.g., surface antigen), mumps virus, rubella virus, measles virus, poliovirus, smallpox virus, rabies virus, and Varicella-zoster virus.

[0049] Suitable viral antigens also include viral nonstructural proteins, e.g., proteins encoded by viral nucleic acid that do not encode for structural polypeptides, in contrast to those that make capsid or the protein surrounding a virus. Non-structural proteins include those proteins that promote viral nucleic acid replication, viral gene expression, or post- translational processing, such as, for example, Nonstructural proteins 1, 2, 3, and 4 (NS1, NS2, NS3, and NS4, respectively) from Venezuelan Equine encephalitis (VEE), Eastern Equine Encephalitis (EEE), or Semliki Forest.

[0050] Other suitable viral antigens can include, e.g., human immunodeficiency virus (e.g., gag (p55 and p160), pol, env (gp120 and gp41) as set forth in Shiver et al. Nature 415(6869):331 (2002); the HIV genomic sequences set forth in Genbank Accession Nos.EF363127; EF363126; EF363125; EF363124; EF363123; EF363122; EF192592; and EF192591; the HIV gag sequences set forth in Genbank Accession Nos. EF396891; EF396890; EF396889; EF396888; EF396887; EF396886; EF396885; EF396884; EF396883; EF396882; EF396881; EF396880; EF396879; EF396878; EF396877; EF396876; EF39687; EF396874; EF396873; and EF396872; the HIV pol sequences set forth in Genbank Accession Nos. EF396810; EF396809; EF396808; EF396807; EF396806; EF396805; EF396804; EF396803; EF396802; EF396801; EF396800; EF396799; EF396798; EF396797; EF396796; EF396795; EF396794; EF396793; EF396792; and EF396791; and the HIV env sequences set forth in Genbank Accession Nos. 9: EF367234; EF367233; EF367232; EF367231; EF367230; EF367229; EF367228; EF367227; EF367226; EF367225; EF367224; and EF367223, human papilloma virus (e.g., capsid protein L1 as described in, e.g., Donnelly et al. J Infect Dis. 173:314 (1996) and the sequences set forth in Genbank Accession Nos. EF362755; EF362754; NC_001694; NC_001693; NC_001691; NC_001690; NC_005134; NC_001458; NC_001457; NC_001354; NC_001352; NC_001526; and X94164), Epstein Barr virus, herpes simplex virus, human herpes virus, rhinoviruses, cocksackieviruses, enteroviruses, hepatitis A, B, C, and E (e.g., hepatitis B surface antigen as described in e.g., Lubeck et al, PNAS USA 86:6763 (1989) and the sequences set forth in GenBank Accession Nos. AB236481; AB236471; AB206501; AB206489; AB206487; AB221788; AB221777; AB221773; AR933671;AR933670; AB236514; AB236513; AB236512; AB236511; AB236510; AB236509; AB236508; AB236507); hepatitis C NS5 (see, e.g., Genbank Accession Nos. X59609; DQ911563; S71627; S70787; S70786; S70341; S62220; S70790; S70789; S70788; and AB204642)), mumps virus, rubella virus, measles virus, poliovirus, smallpox virus , rabies virus, and Variella-zoster virus. Influenza antigens include, e.g., hemagluttinin (HA), matrix protein 1 (M1), and nucleoprotein (NP) (see, e.g., Donnelly, et al, Vaccine 15:865 (1997) and the influenza HA sequences set forth in Genbank Accession Nos. AB294219; AB294217; AB294215; AB294213; EF102944; EF102943; EF102942; EF102941; EF102940; EF102939; EF102938; EF102937; EF102936; EF102935; EF102934; EF102933; DQ643982; DQ464354; CY019432; CY019424; CY019416; CY019408; CY019400; CY019392; CY019384; CY019376; CY019368; CY019360; CY019352; EF124794; EF110519; EF110518; EF165066; EF165065; EF165064; and EF165063; the influenza M1 sequences set forth in Genbank Accession Nos. AB292791; CY019980; CY019972; CY019964; CY019956; CY019948; CY019940; CY019628; CY019652; CY019644; CY019932; CY019924; CY019916; CY019908; CY019900; CY019892; CY019884; CY019876;CY019868;CY019860; and the influenza NP sequences set forth in Genbank Accession Nos. AB292790; CY019461; CY019974; CY019966; CY019958; CY019950; CY019942; CY019630; CY019654; CY019646; CY019934; CY019926; CY019918 CY019910; CY019902; CY019894; CY019886; CY019878; CY019870; and CY019862.

[0051] Suitable viral antigens also include, e.g., viral nonstructural proteins. The term “viral nonstructural protein” as used herein refers to proteins encoded by viral nucleic acid that do not encode for structural polypeptides, such as those that make capsid or the protein surrounding a virus. Non-structural proteins include those proteins that promote viral nucleic acid replication and viral gene expression such as, for example, Nonstructural proteins 1, 2, 3, and 4 (NS1, NS2, NS3, and NS4, respectively) from Venezuelan Equine encephalitis (VEE), EEE, or Semliki Forest virus [Dubensky et al., J Virol, 70(1), 508-519 (1996); Petrakova et al J Virol 200579(12): 7597-608; U.S. Patent Nos.5,185,440; 5,739,026; 6,566,093; and 5,814,482. Several representative examples of suitable alphaviruses include Aura (ATCC VR-368), Bebaru virus (ATCC VR-600, ATCC VR-1240), Cabassou (Genbank Accession Nos. AF398387, ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCC VR-1241), Eastern equine encephalomyelitis virus (Genbank Accession Nos. AY705241, AY705240, ATCC VR-65, ATCC VR-1242), Fort Morgan (ATCC VR-924), Getah virus (ATCC VR- 369, ATCC VR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR-66), Mayaro virus (ATCC VR-1277), Middleburg (ATCC VR-370), Mucambo virus (ATCC VR-580, ATCC VR-1244), Ndumu (ATCC VR-371), Pixuna virus (ATCC VR-372, ATCC VR-1245), Ross River virus (ATCC VR-373, ATCC VR-1246), Semliki Forest (Genbank Accession Nos. AJ251359, ATCC VR-67, ATCC VR-1247), Sindbis virus (Genbank Accession Nos. J02363, ATCC VR-68, ATCC VR-1248), Tonate (ATCC VR-925), Triniti (ATCC VR-469), Una (ATCC VR-374), Venezuelan equine encephalomyelitis (ATCC VR-69), Venezuelan equine encephalomyelitis virus (Genbank Accession Nos. AY986475, AY973944, NC 001449, ATCC VR-923, ATCC VR-1250 ATCC VR-1249, ATCC VR-532), Western equine encephalomyelitis (ATCC VR-70, ATCC VR-1251, ATCC VR-622, ATCC VR-1252), Whataroa (ATCC VR-926), and Y-62-33 (ATCC VR-375).

[0052] Bacterial antigens may be derived from, for example, Staphylococcus aureus, Staphylococcus epidermis, Helicobacter pylori, Streptococcus bovis, Streptococcus pyogenes, Streptococcus pneumoniae, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium leprae, Corynebacterium diphtheriae, Borrelia burgdorferi, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium difficile, Salmonella typhi,Vibrio chloerae, Haemophilus influenzae, Bordetella pertussis, Yersinia pestis, Neisseria gonorrhoeae, Treponema pallidum, Mycoplasm sp., Neisseria 15ransducer15s, Legionella pneumophila, Rickettsia typhi, Chlamydia trachomatis, and Shigella dysenteriae, Vibrio cholera(e.g., Cholera toxin subunit B as set forth in Genbank Accession Nos. U25679; A09803; EF158842; X76391; AF390572; cholera toxin-coregulated pilus (TCP) as described in Wu et al., Infection and Immunity Vol. 69(12):7695 (2001) and as set forth in Genbank Accession Nos. NC_002505 and AE004169); Helicobacter pylorii (VacA as set forth in Genbank Accession Nos. AY848858; AF042737; AF042736; AF042735; AF042734; NC_000921; CagA as set forth in Genbank Accession Nos. AF043490; AF043489; AF043488; AF043487; NAP as set forth in Genbank Accession Nos. AF284121; AF284120; AF284119; AF284118; AF284117; AF284116; AB045143; AB045142; AF227081; AF227080; AF227079; AF227078; AF227077; AF227076; AF227075; AF227074; Hsp or catalase as set forth in Genbank Accession No. NC_000921; urease as set forth in Genbank Accession Nos. AM417610; AM417609; AM417608; AM417607; AM417606; AM417605; AM417604; AM417603; AM417602; AM417601; and AM417600; E. coli antigens as set forth in Genbank Accession Nos. NC_000913; U00096; NC_002655; BA000007; AE014075; including E. coli fimbrial antigens as set forth in Genbank Accession Nos. AB214865; AB214864; AB214863; AB214862; E. coli heat-labile enterotoxin as set forth in Genbank Accession Nos. X83966; V00275; X83966; J01646; V00275; M35581; M17873; M17874; K01995; M61015; M17894; M17101; K00433.

[0053] Parasite antigens may be derived from, for example, Giardia lamblia, Leishmania sp., Trypanosoma sp., Trichomonas sp., Plasmodium sp. (e.g., P. faciparum surface protein antigens such as pfs25 sequences as set forth in Genbank Accession Nos. XM_001347551; X07802; AF193769; AF179423; AF154117; and AF030628, pfs28 sequences as set forth in Genbank Accession No. L25843, pfs45 sequences as set forth in Genbank Accession Nos. EF158081; EF158079; EF158078; EF158076; EF158075; and EF158085, pfs84, pfs 48 / 45 sequences as set forth in Genbank Accession Nos. AF356146; AF356145; AF356144; AF356143; AF356142; AF356141; AF356140; AF356139; AF356138; AF356137; AF356136; AF356135; AF356134; AF356133; AF356132; AF356131; AF356130; AF356129; AF356128; AF356127, pfs 230 sequences as set forth in Genbank Accession Nos. NC_000910; XM_001349564; AE001393; L22219; L08135; and AF269242, P. vivax antigens such as Pvs25 sequences as set forth in Genbank Accession Nos. DQ641509; DQ641508; DQ641507; AY639972; AY639971; AY639970; AY639969; AY639968; AY639967; AY639966; and AY639965; and Pvs28 sequences as set forth in GenbankAccession Nos. AB033364; AB033363; AB033362; AB033361; AB033360; AB033359; AB033358; AB033357; AB033356; B033355; AB033354; AB033353; AB033352; AB033351; AB033350; AB033349; AB033348; AB033347; AB033346; and AB033345), Schistosoma sp., Mycobacterium tuberculosis (e.g., Ag85 sequences as set forth in Genbank Accession Nos. AX253506; AX253504; AX253502; and AX211309; MPT64, ESAT-6, CFP10, R8307, MTB-32 MTB-39, CSP, LSA-1, LSA-3, EXP1, SSP-2, SALSA, STARP, GLURP, MSP-1, MSP-2, MSP-3, MSP-4, MSP-5, MSP-8, MSP-9, AMA-1, Type 1 integral membrane protein, RESA, EBA-175, and DBA sequences as set forth in Genbank Accession Nos. BX842572; BX842573; BX842574; BX842575; BX842576; BX842577; BX842578; BX842579; BX842580; BX842581; BX842582;BX842583; BX842584 and NC_000962, HSP65 sequences as set forth in Genbank Accession Nos. AY299175; AY299174; AY299144; AF547886; and AF547885).

[0054] Cancer antigens include, for example, antigens expressed, for example, in colon cancer, stomach cancer, pancreatic cancer, lung cancer, ovarian cancer, prostate cancer, breast cancer, skin cancer (e.g., melanoma), leukemia, lymphoma, or myeloma, exemplary cancer antigens include, for example, HPV L1, HPV L2, HPV E1, HPV E2, placental alkaline phosphatase, AFP, BRCA1, Her2 / neu, CA 15-3, CA 19-9, CA-125, CEA, Hcg, urokinase-type plasminogen activator (Upa), plasminogen activator inhibitor.

[0055] Fungal antigens may be derived from, for example, Tinea pedis, Tinea corporus, Tinea cruris, Tinea unguium, Cladosporium carionii, Coccidioides immitis, Candida sp., Aspergillus fumigatus, and Pneumocystis carinii.

[0056] As noted above, the chimeric adenoviral vectors described herein (e.g., comprising a first promoter operably linked to a nucleic acid as described herein encoding an antigenic or other protein) can also include a nucleic acid comprising a second promoter operably linked to a nucleic acid encoding a toll-like receptor (TLR) agonist, which can serve as an effective adjuvant when administered in conjunction with viral vectors. For example, the chimeric adenoviral vectors of the disclosure can comprise an expression cassette comprising the following elements: (a) a first promoter operably linked to a nucleic acid encoding an antigenic protein; and (b) a second promoter operably linked to a nucleic acid encoding a toll- like receptor-3 (TLR-3) agonist. TLR agonists can be used to enhance the immune response to the SARS-CoV-2 protein. In some embodiments, TLR-3 agonists are used. In some embodiments, the TLR agonists described herein can be delivered simultaneously with the expression vector encoding an antigen of interest (e.g., a SARS-CoV-2 protein). In other embodiments, the TLR agonists can be delivered separately (i.e., temporally or spatially)from the expression vector encoding an antigen of interest (e.g., a SARS-CoV-2 protein). For example, the expression vector can be administered via a non-parenteral route (e.g., orally, intranasally, or mucosally), while the TLR agonist can be delivered by a parenteral route (e.g., intramuscularly, intraperitoneally, or subcutaneously).

[0057] In particular embodiments, a TLR-3 agonist is be used to stimulate immune recognition of an antigen of interest. TLR-3 agonists include, for example, short hairpin RNA, virally derived RNA, short segments of RNA that can form double-strands or short hairpin RNA, and short interfering RNA (siRNA). In one embodiment of the disclosure, the TLR-3 agonist is virally derived dsRNA, such as for example, a dsRNA derived from a Sindbis virus or dsRNA viral intermediates (Alexopoulou et al, Nature 413:732-8 (2001)). In some embodiments, the TLR-3 agonist is a short hairpin RNA. Short hairpin RNA sequences typically comprise two complementary sequences joined by a linker sequence. The particular linker sequence is not a critical aspect of the disclosure. Any appropriate linker sequence can be used so long as it does not interfere with the binding of the two complementary sequences to form a dsRNA.

[0058] In some embodiments, the TLR-3 agonist can comprise a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% (e.g., 85%, 87%, 89%, 91%, 93%, 95%, 97%, 99%, or 100%) identity to a sequence set forth in SEQ ID NOS:6-14. In certain embodiments, dsRNA that is a TLR-3 agonist does not encode a particular polypeptide, but produces a pro- inflammatory cytokine (e.g. IL-6, IL-8, TNF-alpha, IFN-alpha, IFN-beta) when contacted with a responder cell (e.g., a dendritic cell, a peripheral blood mononuclear cell, or a macrophage) in vitro or in-vivo.

[0059] In particular embodiments, the TLR agonist (e.g., TLR-3 agonist) described herein can be delivered simultaneously within the same the expression vector that encodes the antigenic protein. In other embodiments, the TLR agonist (e.g., TLR-3 agonist) can be delivered separately (i.e., temporally or spatially) from the expression vector that encodes the antigenic protein. In some cases when the TLR-3 agonist is delivered separately from the expression vector, the nucleic acid encoding the TLR-3 agonist (e.g., an expressed dsRNA) and the chimeric adenoviral vector comprising a nucleic acid encoding the antigenic protein can be administered in the same formulation. In other cases the nucleic acid encoding the TLR-3 agonist and the chimeric adenoviral vector comprising a nucleic acid encoding the antigenic protein can be administered in different formulations. When the nucleic acidencoding the TLR-3 agonist and the adenoviral vector comprising a nucleic acid encoding the XBB 1.5 protein are administered in different formulations, their administration may be simultaneous or sequential. For example, the nucleic acid encoding the TLR-3 agonist may be administered first, followed by the chimeric adenoviral vector (e.g., 1, 2, 4, 8, 12, 16, 20, or 24 hours, 2, 4, 6, 8, or 10 days later). Alternatively, the adenoviral vector may be administered first, followed by the nucleic acid encoding the TLR-3 agonist (e.g., 1, 2, 4, 8, 12, 16, 20, or 24 hours, 2, 4, 6, 8, or 10 days later). PHARMACEUTICAL COMPOSITIONS AND ROUTES OF ADMINISTRATION

[0060] An immunogenic pharmaceutical composition can contain a chimeric adenoviral vector described herein and a pharmaceutically acceptable carrier. Suitable carriers include, for example, water, saline, alcohol, a fat, a wax, a buffer, a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, or biodegradable microspheres (e.g., polylactate polyglycolate). Suitable biodegradable microspheres are disclosed, for example, in US Patent Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883. The immunogenic polypeptide and / or carrier expression vector can be encapsulated within the biodegradable microsphere or associated with the surface of the microsphere.

[0061] The ingredients in an immunogenic pharmaceutical composition are closely related to factors such as, but are not limited to, the route of administration of the immunogenic pharmaceutical composition, the timeline and / or duration of drug release, and the targeted delivery site. In some embodiments, a delayed release coating or an additional coating of the formulation can contain other film-forming polymers being non-sensitive to luminal conditions for technical reasons or chronographic control of the drug release. Materials to be used for such purpose includes, but are not limited to; sugar, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl acetate, hydroxypropyl cellulose, methylcellulose, ethylcellulose, hydroxypropyl methylcellulose, carboxymethylcellulose sodium and others, used alone or in mixtures.

[0062] Additives such as dispersants, colorants, pigments, additional polymers, e.g., poly(ethylacrylat, methylmethacrylat), anti-tacking and anti-foaming agents can be included into a coating layer. Other compounds may be added to increase film thickness and to decrease diffusion of acidic gastric juices into the core material. The coating layers can also contain pharmaceutically acceptable plasticizers to obtain desired mechanical properties. Suchplasticizers are for instance, but not restricted to, triacetin, citric acid esters, phthalic acid esters, dibutyl sebacate, cetyl alcohol, polyethylene glycols, glycerol monoesters, polysorbates or other plasticizers and mixtures thereof. The amount of plasticizer can be optimised for each formula, and in relation to the selected polymer(s), selected plasticizer(s) and the applied amount of said polymer(s).

[0063] Such immunogenic pharmaceutical compositions can also comprise non-immunogenic buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), suspending agents, thickening agents and / or preservatives. Alternatively, compositions of the present disclosure may be formulated as a lyophilate. Compounds may also be encapsulated within liposomes using well known technology.

[0064] Further, pharmaceutical compositions can be prepared to protect against stomach degradation such that the administered immunogenic biological agent reach the desired location. Methods for microencapsulation of DNA and drugs for oral delivery are described, e.g., in US2004043952. For the oral environment, several of these are available including the Eudragit and the TimeClock release systems as well as other methods specifically designed for adenovirus (Lubeck et al., Proc Natl Acad Sci U S A, 86(17), 6763-6767 (1989); Chourasia and Jain, J Pharm Pharm Sci, 6(1), 33-66 (2003)). In some embodiments, the Eudragit system can be used to deliver the chimeric adenoviral vector to the lower small intestine, e.g., the ilemum. See, e.g., WO 2015 / 127278.

[0065] In particular embodiments, the immunogenic composition is in the form of a tablet or capsule, e.g., in the form of a compressed tablet covered by enteric coating. In some embodiments, the immunogenic composition is encapsulated in a polymeric capsule comprising gelatin, hydroxypropylmethylcellulose, starch, or pullulan. In some embodiments, the immunogenic composition is in the form of microparticles less than 2 mm in diameter, e.g., each microparticle covered with enteric coating as described herein. In particular embodiments, the immunogenic composition in the form of a tablet, a capsule, or a microparticle can be orally administered. In some embodiments, site-specific delivery can be achieved via tablets or capsules that release upon an externally generated signal. Early models released for a high-frequency (HF) signal, as disclosed in Digenis et al. (1998) Pharm. Sci. Tech. Today 1:160. The original HF capsule concept has since been updated and the resultmarketed as InteliSite®. The updated capsule is a radio-frequency activated, non- disintegrating delivery system. Radiolabeling of the capsule permits the determination of the capsule location within a specific region of the GI tract via gamma scintigraphy. When the capsule reaches the desired location in the GI tract, external activation opens a series of windows to the capsule drug reservoir.

[0066] In some embodiments, the immunogenic composition can be enclosed in a radio- controlled capsule, so that the capsule is tracked and signaled once it reaches the delivery site. In some embodiments, the capsule is signaled at a given time after administration that corresponds to when the capsule is expected to arrive at the delivery site, with or without detecting.

[0067] The compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule or sponge that effects a slow release of compound following administration). Such formulations may generally be prepared using well known technology (see, e.g., Coombes et al. (1996) Vaccine 14:1429-1438). Sustained- release formulations may contain a polypeptide, polynucleotide or antibody dispersed in a carrier matrix and / or contained within a reservoir surrounded by a rate controlling membrane.

[0068] Carriers for use within such formulations are biocompatible and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release. Such carriers include microparticles of poly(lactide-co-glycolide), as well as polyacrylate, latex, starch, cellulose and dextran. Other delayed-release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound (see, e.g., WO 94 / 20078; WO 94 / 23701; and WO 96 / 06638). The amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.

[0069] In some embodiments, the immunogenic compositions are presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers are preferably hermetically sealed to preserve sterility of the formulation until use. In general, formulations can be stored as suspensions, solutions, or emulsions in oily or aqueous vehicles. Alternatively, an immunogenic composition may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use.

[0070] In some embodiments of targeted delivery, enteric coatings are used to shield substances from the low pH environment of the stomach and delay release of the enclosed substance until it reaches a desired target later in the digestive tract. Enteric coatings are known, and commercially available. Examples include pH-sensitive polymers, bio- degradable polymers, hydrogels, time-release systems, and osmotic delivery systems (see, e.g., Chourasia & Jain (2003) J. Pharm. Pharmaceutical Sci. 6:33).

[0071] In some embodiments, the targeted delivery site is the ileum. The pH of the gastrointestinal tract (GIT) progresses from very acidic in the stomach (pH ~2), to more neutral in the ileum (pH ~ 5.8-7.0). pH sensitive coatings can be used that dissolve in the ileum or just before the ileum. Examples include Eudragit® L and S polymers (threshold pH’s ranging from 5.5-7.0); polyvinyl acetate phthalate (pH 5.0), hydroxypropyl methylcellulose phthalate 50 and 55 (pH 5.2 and 5.4, respectively), and cellulose acetate phthalate (pH 5.0). Thakral et al. (2013) Expert Opin. Drug Deliv. 10:131 review Euragit® formulations for ileal delivery, in particular, combinations of L and S that ensure delivery at pH≤7.0. Crotts et al. (2001) Eur. J Pharm. Biol. 51:71 describe Eudragit® formulations with appropriate disintegration properties. Vijay et al. (2010) J. Mater. Sci. Mater. Med. 21:2583 review acrylic acid (AA)-methyl methacrylate (MMA) based copolymers for ileal delivery at pH 6.8.

[0072] For ileal delivery, the polymer coating typically dissolves at about pH 6.8 and allows complete release within about 40 min (see, e.g., Huyghebaert et al. (2005) Int. J. Pharm. 298:26). To accomplish this, a therapeutic substance can be covered in layers of different coatings, e.g., so that the outermost layer protects the substance through low pH conditions and is dissolved when the tablet leaves the stomach, and at least one inner layer that dissolves as the tablet passes into increasing pH. Examples of layered coatings for delivery to the distal ileum are described, e.g., in WO 2015 / 127278, WO 2016 / 200951, and WO 2013 / 148258.

[0073] Biodegradable polymers (e.g., pectin, azo polymers) typically rely on the enzymatic activity of microflora living in the GIT. The ileum harbors larger numbers of bacteria than earlier stages, including lactobacilli and enterobacteria.

[0074] Osmotic-controlled Release Oral delivery Systems (OROS®; Alza) is an example of an osmotic system that degrades over time in aqueous conditions. Such materials can bemanipulated with other coatings, or in varying thicknesses, to deliver specifically to the ileum (see, e.g., Conley et al. (2006) Curr. Med. Res. Opin. 22:1879).

[0075] Combination polymers for delivery to the ileum are reported in WO2000062820. Examples include Eudragit® L100-55 (25 mg / capsule) with triethyl citrate (2.4 mg / capsule), and Povidone K-25 (20 mg / tablet) followed by Eudragit® FS30D (30 mg / tablet). pH sensitive polymers can be applied to effect delivery to the ileum, as described above and, e.g., methacrylic acid copolymers (e.g., poly(methacylic acid-co-methyl methacrylate) 1:1), cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, cellulose acetate trimellitate, carboxymethyl ethyl- cellulose, shellac or other suitable polymer(s). The coating layer can also be composed of film-forming polymers being sensitive to other luminal components than pH, such as bacterial degradation or a component that has such a sensitivity when it is mixed with another film-forming polymer. Examples of such components providing delayed release to the ileum are polymers comprising azo bond(s), polysaccharides such as pectin and its salts, galactomannans, amylose and chondroitin, disulphide polymers and glycosides.

[0076] Components with varying pH, water, and enzymatic sensitivities can be used in combination to target a therapeutic composition to the ileum. The thickness of the coating can also be used to control release. The components can also be used to form a matrix, in which the therapeutic composition is embedded. See generally, Frontiers in DrugDiscovery (Bentham Science Pub. 2009) vol. 4.

[0077] In some embodiments of the present disclosure, in addition to or instead of the TLR agonist (e.g., TLR-3 agonist) encoded in the chimeric adenoviral vector, the compositions can further comprise additional adjuvants. Suitable adjuvants include, for example, the lipids and non-lipid compounds, cholera toxin (CT), CT subunit B, CT derivative CTK63, E. coli heat labile enterotoxin (LT), LT derivative LTK63, Al(OH)3, and polyionic organic acids as described in e.g., WO 04 / 020592, Anderson and Crowle, Infect. Immun. 31(1):413-418 (1981), Roterman et al., J. Physiol. Pharmacol., 44(3):213-32 (1993), Arora and Crowle, J. Reticuloendothel. 24(3):271-86 (1978), and Crowle and May, Infect. Immun. 38(3):932-7 (1982)). Suitable polyionic organic acids include for example, 6,6’-[3,3’-demithyl[1,1’- biphenyl]-4,4’-diyl]bis(azo)bis[4-amino-5-hydroxy-1,3-naphthalene-disulfonic acid] (Evans Blue) and 3,3’-[1,1’biphenyl]-4,4’-diylbis(azo)bis[4-amino-1-naphthalenesulfonic acid] (Congo Red). It will be appreciated by those of skill in the art that the polyionic organicacids may be used for any genetic vaccination method in conjunction with any type of administration.

[0078] Other suitable adjuvants include topical immunomodulators such as, members of the imidazoquinoline family such as, for example, imiquimod and resiquimod (see, e.g., Hengge et al., Lancet Infect. Dis. 1(3):189-98 (2001).

[0079] Additional suitable adjuvants are commercially available as, for example, additional alum-based adjuvants (e.g., Alhydrogel, Rehydragel, aluminum phosphate, Algammulin); oil based adjuvants (Freund’s Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.), Specol, RIBI, TiterMax, Montanide ISA50 or Seppic MONTANIDE ISA 720); nonionic block copolymer-based adjuvants, cytokines (e.g., GM-CSF or Flat3-ligand); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and Quil A. Cytokines, such as GM- CSF or interleukin-2, -7, or -12, are also suitable adjuvants. Hemocyanins (e.g., keyhole limpet hemocyanin) and hemoerythrins may also be used in the disclosure. Polysaccharide adjuvants such as, for example, chitin, chitosan, and deacetylated chitin are also suitable as adjuvants. Other suitable adjuvants include muramyl dipeptide (MDP, N acetylmuramyl L alanyl D isoglutamine) bacterial peptidoglycans and their derivatives (e.g., threonyl-MDP, and MTPPE). BCG and BCG cell wall skeleton (CWS) may also be used as adjuvants in the disclosure, with or without trehalose dimycolate. Trehalose dimycolate may be used itself (see, e.g., U.S. Pat. No. 4,579,945). Detoxified endotoxins are also useful as adjuvants alone or in combination with other adjuvants (see, e.g., U.S. Pat. Nos. 4,866,034; 4,435,386; 4,505,899; 4,436,727; 4,436,728; 4,505,900; and 4,520,019. The saponins QS21, QS17, QS7 are also useful as adjuvants (see, e.g., U.S. Pat. No. 5,057,540; EP 0362279; WO 96 / 33739; and WO 96 / 11711). Other suitable adjuvants include Montanide ISA 720 (Seppic, France), SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g., SBAS-2, SBAS-4 or SBAS-6 or variants thereof, available from SmithKline Beecham, Rixensart, Belgium), Detox (Corixa, Hamilton, Mont.), and RC-529 (Corixa, Hamilton, Mont.).

[0080] Within the pharmaceutical compositions provided herein, the adjuvant composition can be designed to induce, e.g., an immune response predominantly of the Th1 or Th2 type.High levels of Th1-type cytokines (e.g., IFN-gamma, TNF-alpha, IL-2 and IL-12) tend to favor the induction of cell mediated immune responses to an administered antigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction of humoral immune responses. Following oral delivery of a composition comprising an immunogenic polypeptide as provided herein, an immune response that includes Th1- and Th2-type responses will typically be elicited.

[0081] A composition comprising the chimeric adenoviral vector can be administered by any non-parenteral route (e.g., orally, intranasally, or mucosally via, for example, the vagina, lungs, salivary glands, nasal cavities, small intestine, colon, rectum, tonsils, or Peyer’s patches). The composition may be administered alone or with an adjuvant as described above. In particular embodiments, the immunogenic composition is administered orally in the form of a tablet or capsule. In further embodiments, the immunogenic composition is administered orally for targeted delivery in the ileum in the form of a tablet or capsule.

[0082] One aspect of the present disclosure involves using the immunogenic compositions described herein to elicit an antigen specific immune response towards a SARS-CoV-2 protein in a subject. In some embodiments, the immune response is elicited in an alveolar cell, an absorptive enterocyte, a ciliated cell, a goblet cell, a club cells, and / or an airway basal cell of the subject. As used herein, a “subject” refers to any warm-blooded animal, such as, for example, a rodent, a feline, a canine, or a primate, preferably a human. The immunogenic compositions can be used before the subject developed COVID-19 to prevent disease. The disease can be diagnosed using criteria generally accepted in the art. For example, viral infection can be diagnosed by the measurement of viral titer in a biological sample (e.g., a nostril swab or mucosal sample) from the subject. The immune response can be induced in a subject prior to infection by SARS-CoV-2 (e.g., to prevent a future information or reduce the resulting symptoms if infection later occurs) or during an active infection by SARS-CoV-2.

[0083] Frequency of administration of the immunogenic composition described herein, as well as dosage, will vary from individual to individual, and may be readily established using standard techniques. In some embodiments, between 1 and 10 (e.g., between 2 and 10, between 3 and 10, between 4 and 10, between 5 and 10, between 6 and 10, between 7 and 10, between 8 and 10, between 9 and 10, between 1 and 9, between 1 and 8, between 1 and 7, between 1 and 6, between 1 and 5, between 1 and 4, between 1 and 3, or between 1 and 2) doses may be administered over a 52 week period. In some embodiments, 2 or 3 doses areadministered at intervals of 1 month; or for example, 2-3 doses are administered every 2-3 months. It is possible that the intervals will be once a year for certain therapies. Booster vaccinations may be given periodically thereafter.

[0084] A suitable dose is an amount of a compound for example that, when administered as described above, is capable of promoting an anti-viral immune response, and is at least 10- 50% above the basal (i.e., untreated) level. Such response can be monitored by measuring the anti-viral antibodies in a patient or by vaccine-dependent generation of cytolytic T cells capable of killing, e.g., the patient’s virus-infected cells in vitro. Immunogenic responses can also be measured by detecting immunocomplexes formed between the immunogenic polypeptides and antibodies in body fluid which are specific for the immunogenic polypeptides. Samples of body fluid taken from an individual prior to and subsequent to initiation of therapy may be analyzed for the immunocomplexes. Briefly, the number of immunocomplexes detected in both samples can be compared. A substantial change in the number of immunocomplexes in the second sample (post-therapy initiation) relative to the first sample (pre-therapy) reflects successful therapy. Such vaccines should also be capable of causing an immune response that leads to prevention of the relevant target disease in vaccinated patients as compared to non-vaccinated patients.

[0085] Exemplary dosages can be measured in infectious units (I.U.). A replication- deficient recombinant Ad5 vector can be titered and quantified using I.U. units. This is accomplished through performance of an IU assay in the adherent human embryonic kidney (HEK) 293 cell line, which is permissive for growth of replication-deficient Ad5. HEK293 cells are plated in a 24-well sterile tissue culture plate and allowed to adhere. The viral material is diluted in sequential 10-fold dilutions and infected into individual wells of plated HEK293 cells in an appropriate number of replicates, usually in duplicate or triplicate. Infection is allowed to proceed via incubation for ~40-42 hours at 37C, 5% CO2. Cells are then fixed with methanol to allow permeability, washed, and blocked with a buffer solution containing bovine serum albumin (BSA). Cells are then incubated with a rabbit-derived primary antibody against the Ad5 hexon surface protein, washed, and probed again with an HRP-conjugated anti-rabbit secondary antibody. Infected cells are then stained via incubation with 3,3′-diaminobenzidine tetrahydrochloride (DAB) and hydrogen peroxide. Infected cells are visualized using phase-contrast microscopy and a dilution is chosen that exhibits discreet individual infection events – these are visible as darkly stained cells that are highly visible against the semi-transparent monolayer of uninfected cells. Total infected cells are countedper field-of-vision in at least ten fields-of-vision of the appropriate dilution. Viral titer can be calculated using the average number of these counts in conjunction with the total number of fields-of-vision for the objective lens / eyepiece magnification used and multiplying by the dilution factor used in the counts.

[0086] In some embodiments, the vaccines administered can have a dosage of 107-1011, e.g., 108-1011, 109-1011, 5x109-5x1010I.U. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.01 ml to about 10 ml for an injected vaccine, more typically from about 0.025 to about 7.5 ml, most typically from about 0.05 to about 5 ml. For a tablet or capsule final product, the size would be between 10 mg to 1000 mg, most typically between 100-400 mg. Those of skill in the art will appreciate that the dose size may be adjusted based on the particular patient or the particular disease or disorder being treated. EXAMPLES

[0087] Adenoviral (Adv5) vectors designated ED202 and GD102 have the same transgene coding for the rotavirus VP8 and VP6 separated by a ribosomal skipping T2A sequence. The two adenoviral vectors differ in only 2 nucleotides, where the potential splice acceptors just upstream of the pIX gene are present in ED202. See, e.g., FIG. 1. The two nucleotides were mutated (A4609T and A4612C) in GD102.

[0088] Both constructs were used to transduce human intestinal epithelial HIEC-6 cells and the RNA was extracted for nanopore sequencing. The ED202 transcripts contained splice variants that involve both 4609 and 4612 splice acceptor sites, as highlighted in the table (right). All the splice variants that involve these 2 splice acceptor sites are not present in the GD102 transcripts.

[0089] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. SEQUENCES SEQ ID NO:1GTTTTGCXYCXYCCGCCGCCGCC, wherein XY represents a dinucleotide sequence other than AG SEQ ID NO:2 GTTTTGCTGCCGCCGCCGCCGCC SEQ ID NO:3 CTAGAAATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGAA TCGATAGTACTAACATACGCTCTCCATCTCGAGCCTAAGCTTGTCGACTCGAAGA TCTGGGCGTGGTTAAGGGTGGGAAAGAATATATAAGGTGGGGGTCTTATGTAGT TTTGTATCTGTTTTGCXYCXYCCGCCGCCGCC, wherein XY represents a dinucleotide sequence other than AG SEQ ID NO:4 CTAGAAATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGAA TCGATAGTACTAACATACGCTCTCCATCTCGAGCCTAAGCTTGTCGACTCGAAGA TCTGGGCGTGGTTAAGGGTGGGAAAGAATATATAAGGTGGGGGTCTTATGTAGT TTTGTATCTGTTTTGCTGCCGCCGCCGCCGCC SEQ ID NO:5 XBB 1.5 Spike protein amino acid sequence MFVFLVLLPLVSSQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSN VTWFHAIHVSGTNGTKRFDNPALPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLI VNNATNVVIKVCEFQFCNDPFLDVYQKNNKSWMESEFRVYSSANNCTFEYVSQPFL MDLVGKEGNFKNLREFVFKNIDGYFKIYSKHTPINLERDLPQGFSALEPLVDLPIGINI TRFQTLLALHRSYLTPVDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDC ALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASV YAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEV SQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRSLRKSKLKPF ERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAP ATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTIDAVRD PQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVY STGSNVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKSHSRAGSVASQSIIAY TMSLGAENLVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLL QYGSFCTQLKRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKYFGGFNFSQILPDPSKPS KRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIA QYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQF NSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLVKQLSSKFGAISSVLNDILSRLD PPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFC GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNG THWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFK NHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWY IWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT * SEQ ID NO:6: TLR-3 agonist sequenceGAAACGATATGGGCTGAATACTTAAGTATTCAGCCCATATCGTTTC SEQ ID NO:7: TLR-3 agonist sequence CGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCGCCCTT AGATATCGTCGACGCCCAGCACCCCAAGGCGGCCAACGCCAAAACTCTCCCTCCT CCTCTTCCTCAATCTCGCTCTCGCTCTTTTTTTTTTTCGCAAAAGGAGGGGAGAGG GGGTAAAAAAATGCTGCACTGTGCGGCGAAGCCGGTGAGTGAGCGGCGCGGGGC CAATCAGCGTGCGCCGTTCCGAAAGTTGCCTTTTATGGCTCGAGCGGCCGCGGCG GCGCCCTATAAAACCCAGCGGCGCGACGCGCCACCACCGCCGAGACATCGATGA TATCTAAAGGGCGAATTCCTGCAGCCCGGGGGATCCACTAGTCTAGATGCATGCT CGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTTCAGCTGCGGATC CATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTT CGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGG TAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTA ATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACGG TATCGATAAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCACTAGTTTCTAG AAATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGGCGGCC GCCACCGCGGTGGAGCTATCGAATTCAAGCTTGTCGACTCGAAGATCCTAGACTA GTGGATCCCCCGGGCTGCAGGAATTCGCCCTTTAGATATCATCGATGTCTCGGCG GTGGTGGCGCGTCGCGCCGCTGGGTTTTATAGGGCGCCGCCGCGGCCGCTCGAGC CATAAAAGGCAACTTTCGGAACGGCGCACGCTGATTGGCCCCGCGCCGCTCACTC ACCGGCTTCGCCGCACAGTGCAGCATTTTTTTACCCCCTCTCCCCTCCTTTTGCGA AAAAAAAAAAGAGCGAGAGCGAGATTGAGGAAGAGGAGGAGGGAGAGTTTTGG CGTTGGCCGCCTTGGGGTGCTGGGCGTCGACGATATCTAAGGGCGAATTCGATAT CAAGCTTATCGATACCGTCGACCTCGAGGGGGGGCCCG SEQ ID NO:8: TLR-3 agonist sequence CGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCGCCCTT AGATATCGTCGACGCCCAGCACCCCAAGGCGGCCAACGCCAAAACTCTCCCTCCT CCTCTTCCTCAATCTCGCTCTCGCTCTTTTTTTTTTTCGCAAAAGGAGGGGAGAGG GGGTAAAAAAATGCTGCACTGTGCGGCGAAGCCGGTGAGTGAGCGGCGCGGGGC CAATCAGCGTGCGCCGTTCCGAAAGTTGCCTTTTATGGCTCGAGCGGCCGCGGCG GCGCCCTATAAAACCCAGCGGCGCGACGCGCCACCACCGCCGAGACATCGATGA TATCTAAAGGGCGAATTCCTGCAGCCCGGGGGATCCACTAGTCTAGATGCATGCT CGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTTCAGCTGCGGATC CATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTT CGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGG TAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTA ATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACGG TATCGATAAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCACTAGTTTCTAG AAATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGGCGGCC GCCACCGCGGTGGAGCTATCGAATTCAAGCTTGTCGACTCGAAGATCGTACACA GGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTTGGGCGTA ACCGAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGAGGAAGTGAAAT CTGAATAATTTTGTGTTACTCATAGCGCGTAATACTGGTACCGGGCCCCCCCTCG AGGTCGACGGTATCGATAAGCTTGATATCGAATTCGCCCTTAGATATCGTCGACG CCCAGCACCCCAAGGCGGCCAACGCCAAAACTCTCCCTCCTCCTCTTCCTCAATC TCGCTCTCGCTCTTTTTTTTTTTCGCAAAAGGAGGGGAGAGGGGGTAAAAAAATGCTGCACTGTGCGGCGAAGCCGGTGAGTGAGCGGCGCGGGGCCAATCAGCGTGCG CCGTTCCGAAAGTTGCCTTTTATGGCTCGAGCGGCCGCGGCGGCGCCCTATAAAA CCCAGCGGCGCGACGCGCCACCACCGCCGAGACATCGATGATATCTAAAGGGCG AATTCCTGCAGCCCGGGGGATCCACTAGTCTAGAACTAGTGGATCCCCCGGGCTG CAGGAATTCGATATCAAGCTTATCGATACCGTCGACCTCGAGGGGGGGCCCGGT ACCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAA CGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATC CCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCA ACAGTTGCGCAGCCTGAATGGCGAATGGATCCGCAGCTGAAGGGCGAATTCTGC AGATATCCATCACACTGGCGGCCGCTCGAGCATGCATCTAGAAATAAAATATCTT TATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGGCGGCCGCCACCGCGGTGGA GCTA SEQ ID NO:9: TLR-3 agonist sequence GATGGTGCTTCAAGCTAGTACTTAAGTACTAGCTTGAAGCACCATC SEQ ID NO:10: TLR-3 agonist sequence GATGGTGCTTCAAGCTAGTACGGATCCGTACTAGCTTGAAGCACCATC SEQ ID NO:11: TLR-3 agonist sequence GAAACGATATGGGCTGAATACGGATCCGTATTCAGCCCATATCGTTTC SEQ ID NO:12: TLR-3 agonist sequence CCTAATAATTATCAAAATGTGGATCCACATTTTGATAATTATTAGG SEQ ID NO:13: TLR-3 agonist sequence CCTAATAATTATCAAAATGTAATTACATTTTGATAATTATTAGG SEQ ID NO:14: TLR-3 agonist sequence GCCACTGCAGGAAACGATATGGGCTGAATACGGATCCGTATTCAGCCCATATCGT TTC SEQ ID NO:15 KP.2 SARS-COV2 stabilized spike protein MFVFLVLLPLVSSQCVMPLFNLITTTQSYTNSFTRGVYYPDKVFRSSVLHLTQDLFLP FFSNVTWFHAISGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLL IVNNATNVFIKVCEFQFCNDPFLDVYHKNNKSWMESESGVYSSANNCTFEYVSQPFL MDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPIIGRDFPQGFSALEPLVDLPIGINITR FQTLLALNRSYLTPGDSSSGWTAGAADYYVGYLQPRTFLLKYNENGTITDAVDCAL DPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNVTNLCPFHEVFNATTFASVYA WNRTRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIKGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKHSGNYDYWYRSLRKSKLKPFE RDISTEIYQAGNKPCKGKGPNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPAT VCGPKKSTNLVKNKCVNFNFNGLTGTGVLTKSNKKFLPFQQFGRDIVDTTDAVRDP QTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVSVAIHADQLTPTWRVYS TGSNVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKSRSRAGSVASQSIIAYT MSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQ YGSFCTQLKRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKYFGGFNFSQILPDPSKPSK RSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQ YTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNS AIGKIQDSLFSTASALGKLQDVVNHNAQALNTLVKQLSSKFGAISSVLNDILSRLDPP EAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCG KGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGT HWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQLELDSFKEELDKYFKN HTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYI WLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT SEQ ID NO:16 JN.1 SARS-COV2 stabilized spike protein MFVFLVLLPLVSSQCVMPLFNLITTTQSYTNSFTRGVYYPDKVFRSSVLHLTQDLFLP FFSNVTWFHAISGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLL IVNNATNVFIKVCEFQFCNDPFLDVYHKNNKSWMESESGVYSSANNCTFEYVSQPFL MDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPIIGRDFPQGFSALEPLVDLPIGINITR FQTLLALNRSYLTPGDSSSGWTAGAADYYVGYLQPRTFLLKYNENGTITDAVDCAL DPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNVTNLCPFHEVFNATRFASVYA WNRTRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIKGNEVSQ IAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKHSGNYDYWYRSFRKSKLKPFE RDISTEIYQAGNKPCKGKGPNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPAT VCGPKKSTNLVKNKCVNFNFNGLTGTGVLTKSNKKFLPFQQFGRDIVDTTDAVRDP QTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVSVAIHADQLTPTWRVYS TGSNVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKSRSRAGSVASQSIIAYT MSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQ YGSFCTQLKRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKYFGGFNFSQILPDPSKPSK RSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQ YTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNS AIGKIQDSLFSTASALGKLQDVVNHNAQALNTLVKQLSSKFGAISSVLNDILSRLDPP EAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCG KGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGT HWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQLELDSFKEELDKYFKN HTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYI WLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT **

Claims

WHAT IS CLAIMED IS:

1. An adenoviral expression vector, the vector comprising, a heterologous expression cassette comprising a first promoter operably linked to a nucleic acid encoding a payload polypeptide; a nucleic acid encoding an adenovirus pIX protein; and a polynucleotide from the heterologous expression cassette to the nucleic acid encoding the pIX protein, wherein the polynucleotide is free of canonical splice acceptors comprising an AG dinucleotide sequence.

2. The adenoviral expression vector of claim 1, wherein the polynucleotide comprises GTTTTGCXYCXYCCGCCGCCGCC (SEQ ID NO:1), wherein XY represents a dinucleotide sequence other than AG.

3. The adenoviral expression vector of claim 2, wherein X is C, G, or T or Y is A, C, or T.

4. The adenoviral expression vector of claim 2, wherein X is C, G, or T and Y is A, C, or T.

5. The adenoviral expression vector of claim 2, wherein X in one XY is C, G, or T and one Y in the other XY is A, C, or T.

6. The adenoviral expression vector of claim 1, wherein the polynucleotide comprises GTTTTGCTGCCGCCGCCGCCGCC (SEQ ID NO:2).

7. The adenoviral expression vector of claim 1, wherein the polynucleotide comprises CTAGAAATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGAA TCGATAGTACTAACATACGCTCTCCATCTCGAGCCTAAGCTTGTCGACTCGAAGA TCTGGGCGTGGTTAAGGGTGGGAAAGAATATATAAGGTGGGGGTCTTATGTAGT TTTGTATCTGTTTTGCXYCXYCCGCCGCCGCC (SEQ ID NO:3), wherein XY represents a dinucleotide sequence other than AG.

8. The adenoviral expression vector of claim 7, wherein X is C, G, or T or Y is A, C, or T.

9. The adenoviral expression vector of claim 7, wherein X is C, G, or T and Y is A, C, or T.

10. The adenoviral expression vector of claim 7, wherein X in one XY is C, G, or T and one Y the other XY is A, C, or T.

11. The adenoviral expression vector of claim 1, wherein the polynucleotide comprises CTAGAAATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGAA TCGATAGTACTAACATACGCTCTCCATCTCGAGCCTAAGCTTGTCGACTCGAAGA TCTGGGCGTGGTTAAGGGTGGGAAAGAATATATAAGGTGGGGGTCTTATGTAGT TTTGTATCTGTTTTGCTGCCGCCGCCGCCGCC (SEQ ID NO:4).

12. The adenoviral expression vector of any one of claims 1-11, wherein the payload polypeptide is an antigenic protein; and wherein the expression cassette further comprises a second promoter operably linked to a nucleic acid encoding a toll-like receptor-3 (TLR-3) agonist.

13. The adenoviral expression vector of claim 12, wherein the first promoter comprises the CMV promoter or the SV40 promoter.

14. The adenoviral expression vector of claims 12 or 15, wherein the first promoter is inducible.

15. The adenoviral expression vector of claim 14, wherein the first promoter comprises a Tet operator.

16. The adenoviral expression vector of any one of claims 12-15, wherein the TLR-3 agonist is a dsRNA.

17. The adenoviral expression vector of any one of claims 12-16, wherein the first promoter and the second promoter have different sequences.

18. The adenoviral expression vector of claim 17, wherein the first promoter and second promoter are independently selected from a CMV promoter and a SV40 promoter.

19. The adenoviral expression vector of claim 18, wherein the first promoter comprises a CMV promoter and the second promoter comprises an SV40 promoter.

20. The adenoviral expression vector of claim 19, wherein the CMV promoter comprises a Tet operator, rendering the CMV promoter inducible.

21. The adenoviral expression vector of any one of claims 1-20, wherein the vector lacks an adenoviral E1A coding sequence, rendering the vector replication deficient absent exogenous provision of the E1A protein.

22. The adenoviral expression vector of any one of claims 1-21, wherein the adenoviral vector is an Adenovirus 5 vector.

23. The adenoviral expression vector of any one of claims 1-22, wherein the payload polypeptide is selected from the group consisting of: a SARS-CoV2 spike protein (e.g., XBB 1.5, KP.2 or JN.1 variants), a SARS-CoV2 nucleoprotein, a Norovirus VP1 (e.g., from a GI and GII genogroup) protein, an Influenza A hemagglutinin (H1, H3, or H5) protein, an Influenza B hemagglutinin (Victoria or Yamagata) protein, an HPV E6 / E7 (e.g., HPV16 and 18) protein, and a Rotavirus VP4, VP6, or VP8 protein.

24. A pharmaceutical composition comprising the adenoviral expression vector of any one of claims 1-22.

25. The pharmaceutical composition of claim 24, wherein the pharmaceutical composition is a tablet or a capsule.

26. A method of expressing a payload protein in a subject, the method comprising administering to the subject an effective amount of the vector of any one of claims 1-22 or the pharmaceutical composition of any one of claims 24-25 to a human subject.

27. The method of claim 26, wherein the administering is oral, intranasal, or mucosal.

28. The methods of claim 26, wherein the administering is oral delivery by swallowing a tablet or capsule.

29. The method of any one of claims 26-28, wherein the immune response is elicited in an alveolar cell, an absorptive enterocyte, a ciliated cell, a goblet cell, a club cells, dendritic cell, macrophage, and / or an airway basal cell of the subject.

30. A method of making the adenoviral expression vector of claim 21, the method comprising, introducing a nucleic acid encoding the adenoviral expression vector into a cell that heterologously expresses the E1A protein; and harvesting the adenoviral expression vector from the cell.

31. The method of claim 30, wherein the CMV promoter comprises a Tet operator, rendering the CMV promoter inducible and the cell heterologously expresses a tetracycline repressor protein.

Images