Multicistronic vaccines and methods of making and using same

Abstract

The present disclosure provides multicistronic vaccines and methods for producing and using multicistronic vaccines to prevent infection or transmission by influenza and / or SARS-Cov-2 viruses in a subject, or to reduce the severity of disease caused by these viruses. The multicistronic vaccines of the present disclosure can be administered intramuscularly, intranasally, or via inhalation. In one specific embodiment, the present disclosure provides a recombinant adenovirus containing at least two different foreign oligonucleotides capable of stimulating an immune response in a subject. Each oligonucleotide independently contains an oligonucleotide encoding either an influenza or SARS-Cov-2 virus antigen.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 447,734, filed February 23, 2023, which is incorporated herein by reference in its entirety.

[0003] Reference to Electronic Sequence Listing

[0004] The contents of the following electronic sequence listing are incorporated herein by reference in their entirety: Name of XML file: OCU-000900PC.xml; File size: 46.286 bytes; Created:02-16-2024.

[0005] Field of Disclosure

[0006] The present disclosure relates to multicistronic vaccines and methods of making and using the same for preventing infection or transmission by, or reducing the severity of disease caused by, influenza and / or SARS-Cov-2 viruses in a subject. The multicistronic vaccines of the present disclosure can be administered intramuscularly, intranasally, or by inhalation. [Background technology]

[0007] The COVID-19 pandemic caused by SARS-CoV-2 continues to have a significant impact on healthcare and social systems worldwide. Because COVID-19 and influenza share many clinical and epidemiological features, optimal management of both respiratory diseases is of paramount importance, particularly among healthcare workers and high-risk groups. Understanding the dynamics of transmission between SARS-CoV-2 and influenza viruses is essential for effective preparation for ongoing co-circulation.

[0008] The average basic reproduction number (R0) for these two viruses is estimated to be 1.28 for influenza virus and 2.79 for SARS-CoV-2 virus. Without being bound by any theory, the difference in infectivity (R0) is likely due to herd immunity against influenza due to the implementation of national influenza vaccination policies, compared with the lack of pre-existing immunity against SARS-CoV-2. COVID-19 is likely to have a more pronounced impact on healthcare systems during seasons when influenza is most prevalent. Therefore, comprehensive control measures are needed to mitigate the impact of both SARS-CoV-2 and influenza virus, including transmission control of both diseases through combined vaccination. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] WO 2021 / 247567 Summary of the Invention [Problem to be solved by the invention]

[0010] Furthermore, most currently available influenza and COVID-19 vaccines are designed for intramuscular (IM) immunization. Unfortunately, IM vaccines do not appear to provide significant mucosal immunity, which is necessary for protection against upper respiratory tract infections. New omicron variants rely more heavily on nucleosome-mediated endocytosis than TMPRSS2 cleavage to infect cells. This shortens the incubation period from 4–12 days to 3.5 days, but this is insufficient to activate immune memory responses due to the shortened incubation period and mutations that allow escape from antibody neutralization. Inhaled vaccines using updated (bivalent / multivalent) spike antigens from the latest variants of concern (VOCs) not only generate protective local humoral and cellular immunity (IgA antibodies, CD4+ and CD8+ T cells) in the respiratory system, but also generate strong systemic immunity. [Means for solving the problem]

[0011] The present disclosure provides a multicistronic vaccine and methods for producing and using the same. One particular aspect of the present disclosure provides a recombinant adenovirus adapted for use in preventing infection or transmission by influenza and / or SARS-Cov-2 virus in a subject, or reducing the severity of disease caused by these viruses. The recombinant adenovirus comprises two or more oligonucleotides encoding antigens that stimulate different immune responses in the subject. In particular, the recombinant adenovirus comprises at least two different oligonucleotides, each independently selected from the group consisting of: (i) an oligonucleotide encoding influenza A virus hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof; (ii) an oligonucleotide encoding influenza A virus neuraminidase or an immunogenic portion, variant, mutant, or fragment thereof; (iii) an oligonucleotide encoding a hemagglutinin of a first influenza B virus or an immunogenic portion, variant, mutant, or fragment thereof; (iv) an oligonucleotide encoding a hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof of a second influenza B virus, wherein the second influenza B virus is a different strain from the first influenza B virus; (v) an oligonucleotide encoding the spike protein (S protein) of the first SARS-CoV-2 virus or an immunogenic portion, variant, mutant, or fragment thereof; and (vi) an oligonucleotide encoding an S protein or an immunogenic portion, variant, mutant, or fragment thereof of a second SARS-CoV-2 virus, wherein the second SARS-CoV-2 virus is a different strain from the first SARS-CoV-2 virus; and wherein the adenovirus of the recombinant adenovirus is selected from the group consisting of: (i) first, second, third, or fourth generation human adenovirus 5; (ii) rare-serotype adenoviruses; (iii) non-human adenoviruses, and (iv) combinations thereof.

[0012] It is understood that the oligonucleotides encoding the immune response-stimulating antigens are different from each other, thereby allowing for the translation, transcription, and / or expression of different immune response-stimulating antigens. In this way, introduction of a single recombinant adenovirus into a host can express more than one immune response-stimulating antigen in the host.

[0013] In some embodiments, the recombinant adenovirus is adapted to be introduced into host cells via intramuscular, intranasal, or inhalation routes.

[0014] In other embodiments, at least one of the oligonucleotides encodes at least one of the 18 hemagglutinin (HA) subtypes 1-18, or an immunogenic portion, variant, mutant, or fragment thereof. In other embodiments, at least one of the oligonucleotides encodes at least one of the ten neuraminidase (NA) subtypes 1-10, or an immunogenic portion, variant, mutant, or fragment thereof. In yet other embodiments, at least one of the oligonucleotides encodes B / YAMAGATA / 16 / 88 HA or an immunogenic portion, variant, mutant, or fragment thereof. In further embodiments, at least one of the oligonucleotides encodes B / Victoria / 2 / 87 HA or an immunogenic portion, variant, mutant, or fragment thereof. In yet other embodiments, at least one of the oligonucleotides encodes the S protein of the first SARS-CoV-2 virus selected from the group consisting of SARS-CoV-2 variants B.1.1.7, B.1.351, B1.1.28-P.1, B.1.617.2, B.1.1.529, BA.4, BA.5, BQ1.1, XBB.1.5, BA.2.75.2, and immunogenic portions, variants, mutants, or fragments thereof. In yet other embodiments, at least one of the oligonucleotides encodes the S protein of the second SARS-CoV-2 virus.

[0015] In another embodiment, the recombinant adenovirus comprises at least three different oligonucleotides. In yet another embodiment, the recombinant adenovirus comprises at least four different oligonucleotides. In yet another embodiment, the recombinant adenovirus comprises at least five different oligonucleotides. In a further embodiment, the recombinant adenovirus comprises at least six different oligonucleotides.

[0016] In some embodiments, the rare serotype adenovirus comprises Ad11, Ad26, Ad35, Ad48, Ad49, Ad50, or a combination thereof. In yet other embodiments, the non-human adenovirus comprises simian Ad36, bovine Ad3, canine Ad2, porcine Ad3, or a combination thereof. In one specific embodiment, the non-human adenovirus comprises simian Ad36.

[0017] In yet other embodiments, the recombinant adenovirus further comprises 5' and 3' inverted repeats (ITRs) and a packaging signal (ψ). In other embodiments, the recombinant adenovirus further comprises (i) a promoter, (ii) an enhancer, (iii) a polyadenylation moiety, (iv) an internal ribosome entry site (IRES), (v) a self-cleaving protein site, or (vi) a combination thereof. In some cases, the self-cleavage protein site comprises T2A, P2A, E2A, F2A, or a combination thereof. In other cases, the polyadenylation portion comprises a simian virus 40 (SV40) polyadenylation (polyA) portion, a bovine growth hormone (bGH) polyA portion, or a combination thereof.

[0018] In still other embodiments, the recombinant adenovirus further comprises a cytomegalovirus (CMV) promoter or enhancer, elongation factor 1a (EF1a), chicken beta actin (CBA) promoter, CAG promoter, or a combination thereof.

[0019] Another aspect of the present disclosure provides a plasmid comprising an adenoviral genome, wherein the adenoviral genome has been modified to include a transgene operably linked to an expression control sequence that directs transcription, translation, and / or expression in a host cell, and wherein the transgene comprises at least two different oligonucleotides, each of the oligonucleotides independently selected from the group consisting of: (i) an oligonucleotide encoding influenza A virus hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof; (ii) an oligonucleotide encoding influenza A virus neuraminidase or an immunogenic portion, variant, mutant, or fragment thereof; (iii) an oligonucleotide encoding a hemagglutinin of a first influenza B virus or an immunogenic portion, variant, mutant, or fragment thereof; (iv) an oligonucleotide encoding a hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof of a second influenza B virus, wherein the second influenza B virus is a different strain from the first influenza B virus; (v) an oligonucleotide encoding the spike protein (S protein) of the first SARS-CoV-2 virus or an immunogenic portion, variant, mutant, or fragment thereof; and (vi) An oligonucleotide encoding an S protein or an immunogenic portion, variant, mutant, or fragment thereof of a second SARS-CoV-2 virus, wherein the second SARS-CoV-2 virus is a different strain from the first SARS-CoV-2 virus.

[0020] In some embodiments, the genome is derived from an adenovirus selected from the group consisting of: (i) first, second, third, or fourth generation human adenovirus 5; (ii) adenoviruses of rare serotypes; (iii) non-human adenoviruses, and (iv) combinations thereof.

[0021] In yet another embodiment, the adenovirus is a non-human adenovirus. In some cases, the non-human adenovirus comprises simian Ad36, bovine Ad3, canine Ad2, porcine Ad3, or a combination thereof. In still other cases, the non-human adenovirus comprises simian Ad36. In one particular case, the genome of simian Ad36 lacks the native E1 locus and optionally also the E3 or E3B locus.

[0022] In a further embodiment, at least one of the oligonucleotides encodes at least one of the 18 hemagglutinin (HA) subtypes 1-18, or an immunogenic portion, variant, mutant, or fragment thereof. In other embodiments, at least one of the oligonucleotides encodes at least one of the ten neuraminidase (NA) subtypes 1-10, or an immunogenic portion, variant, mutant, or fragment thereof. In yet other embodiments, at least one of the oligonucleotides encodes B / YAMAGATA / 16 / 88 HA or an immunogenic portion, variant, mutant, or fragment thereof. In other embodiments, at least one of the oligonucleotides encodes B / Victoria / 2 / 87 HA or an immunogenic portion, variant, mutant, or fragment thereof. In yet other embodiments, at least one of the oligonucleotides encodes the S protein of the first SARS-CoV-2 virus selected from the group consisting of SARS-CoV-2 variants B.1.1.7, B.1.351, B1.1.28-P.1, B.1.617.2, B.1.1.529, BA.4, BA.5, BQ1.1, XBB.1.5, BA.2.75.2, and immunogenic portions, variants, mutants, or fragments thereof. In yet other embodiments, at least one of the oligonucleotides encodes the S protein of the second SARS-CoV-2 virus.

[0023] In another embodiment, the plasmid comprises at least three different oligonucleotides. In yet another embodiment, the plasmid comprises at least four different oligonucleotides. In a further embodiment, the plasmid comprises at least four different oligonucleotides. In another embodiment, the plasmid comprises at least five different oligonucleotides. In yet another embodiment, the plasmid comprises at least six different oligonucleotides.

[0024] Yet another aspect of the present disclosure provides a recombinant adenovirus (rAd) vector comprising an adenovirus genome modified to contain at least two different exogenous oligonucleotides operably linked to expression control sequences that direct transcription, translation, and / or expression in a host cell, and each of the exogenous oligonucleotides is independently selected from the group consisting of: (i) an oligonucleotide encoding influenza A virus hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof; (ii) an oligonucleotide encoding influenza A virus neuraminidase or an immunogenic portion, variant, mutant, or fragment thereof; (iii) an oligonucleotide encoding a hemagglutinin of a first influenza B virus or an immunogenic portion, variant, mutant, or fragment thereof; (iv) an oligonucleotide encoding a hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof of a second influenza B virus, wherein the second influenza B virus is a different strain from the first influenza B virus; (v) an oligonucleotide encoding the spike protein (S protein) of the first SARS-CoV-2 virus or an immunogenic portion, variant, mutant, or fragment thereof; and (vi) An oligonucleotide encoding an S protein or an immunogenic portion, variant, mutant, or fragment thereof of a second SARS-CoV-2 virus, wherein the second SARS-CoV-2 virus is a different strain from the first SARS-CoV-2 virus.

[0025] In some embodiments, the adenovirus of the recombinant adenovirus is selected from the group consisting of: (i) first, second, third, or fourth generation human adenovirus 5; (ii) adenoviruses of rare serotypes; (iii) non-human adenoviruses, and (iv) combinations thereof.

[0026] In yet another embodiment, the rAd vector further comprises the naturally occurring adenovirus (Ad) major serotype capsid proteins (hexon, penton base, and fiber) and four minor proteins (IIIa, VI, VIII, and IX). In other embodiments, the rAd vector is suitable for introduction into host cells via intramuscular, intranasal, or inhalation routes. In still other embodiments, the non-human adenovirus comprises simian Ad36, bovine Ad3, canine Ad2, porcine Ad3, or a combination thereof. In some instances, the non-human adenovirus comprises simian Ad36.

[0027] In yet other embodiments, the rAd vector contains 5' and 3' inverted repeats (ITRs) and a packaging signal (ψ). In other embodiments, the rAd vector further comprises (i) a promoter, (ii) an enhancer, (iii) a polyadenylation moiety, (iv) an internal ribosome entry site (IRES), (v) a self-cleaving protein site, or (vi) a combination thereof. In some cases, the self-cleaving protein site comprises T2A, P2A, E2A, F2A, or a combination thereof. In other cases, the polyadenylation portion comprises a simian virus 40 (SV40) polyadenylation (polyA) portion, a bovine growth hormone (bGH) polyA portion, or a combination thereof.

[0028] In further embodiments, the rAd vector further comprises a cytomegalovirus (CMV) promoter or enhancer, elongation factor 1a (EF1a), chicken beta actin (CBA) promoter, CAG promoter, or a combination thereof.

[0029] In some embodiments, at least one of the oligonucleotides encodes at least one of the 18 hemagglutinin (HA) subtypes 1-18, or an immunogenic portion, variant, mutant, or fragment thereof. In other embodiments, at least one of the oligonucleotides encodes at least one of the ten neuraminidase (NA) subtypes 1-10, or an immunogenic portion, variant, mutant, or fragment thereof. In yet other embodiments, at least one of the oligonucleotides encodes B / YAMAGATA / 16 / 88 HA or an immunogenic portion, variant, mutant, or fragment thereof. In yet other embodiments, at least one of the oligonucleotides encodes B / Victoria / 2 / 87 HA or an immunogenic portion, variant, mutant, or fragment thereof. In a further embodiment, at least one of the oligonucleotides encodes the S protein of the first SARS-CoV-2 virus selected from the group consisting of SARS-CoV-2 variants B.1.1.7, B.1.351, B1.1.28-P.1, B.1.617.2, B.1.1.529, BA.4, BA.5, BQ1.1, XBB.1.5, BA.2.75.2, and immunogenic portions, variants, mutants, or fragments thereof. In yet other embodiments, at least one of the oligonucleotides encodes the S protein of the second SARS-CoV-2 virus.

[0030] In other embodiments, the recombinant adenovirus comprises at least three different oligonucleotides, hi yet other embodiments, the recombinant adenovirus comprises at least four different oligonucleotides. In yet another embodiment, the recombinant adenovirus comprises at least four different oligonucleotides. In a further embodiment, the recombinant adenovirus comprises at least five different oligonucleotides. In yet another embodiment, the recombinant adenovirus comprises at least six different oligonucleotides.

[0031] In still other embodiments, the rare serotype adenovirus comprises Ad11, Ad26, Ad35, Ad48, Ad49, Ad50, or a combination thereof.

[0032] Another aspect of the present disclosure provides a pharmaceutical composition comprising any one of the recombinant adenoviral (rAd) vectors disclosed herein and a pharmaceutically acceptable excipient.

[0033] Yet another aspect of the present disclosure provides a method of administering an influenza or SARS-CoV-2 virus vaccine, wherein the vaccine is a multivalent vaccine comprising at least two different foreign oligonucleotides, the method comprising administering a therapeutically effective amount of a recombinant adenovirus (rAd) vector to a subject in need of such a vaccine, thereby eliciting an immune response in the subject, wherein the rAd vector comprises an adenovirus genome, and the adenovirus genome has been modified to include at least two different foreign oligonucleotides operably linked to expression control sequences that direct transcription, translation, and / or expression in a host cell, and each of the foreign oligonucleotides is independently selected from the group consisting of: (i) an oligonucleotide encoding influenza A virus hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof; (ii) an oligonucleotide encoding influenza A virus neuraminidase or an immunogenic portion, variant, mutant, or fragment thereof; (iii) an oligonucleotide encoding a hemagglutinin of a first influenza B virus or an immunogenic portion, variant, mutant, or fragment thereof; (iv) an oligonucleotide encoding a hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof of a second influenza B virus, wherein the second influenza B virus is a different strain from the first influenza B virus; (v) an oligonucleotide encoding the spike protein (S protein) of the first SARS-CoV-2 virus or an immunogenic portion, variant, mutant, or fragment thereof; and (vi) An oligonucleotide encoding an S protein or an immunogenic portion, variant, mutant, or fragment thereof of a second SARS-CoV-2 virus, wherein the second SARS-CoV-2 virus is a different strain from the first SARS-CoV-2 virus.

[0034] In some embodiments, the vaccine is administered to the subject via an intramuscular, intranasal, or inhalation route. In yet another embodiment, the vaccine is a bivalent vaccine. In yet another embodiment, the vaccine is a trivalent vaccine. In a further embodiment, the vaccine is a tetravalent vaccine. In a further embodiment, the vaccine is a pentavalent vaccine. In yet another embodiment, the vaccine is a hexavalent vaccine.

[0035] In yet other embodiments, the adenovirus of the rAd vector is selected from the group consisting of: (i) first, second, third, or fourth generation human adenovirus 5; (ii) adenoviruses of rare serotypes; (iii) non-human adenoviruses, and (iv) combinations thereof.

[0036] In yet other embodiments, the non-human adenovirus comprises simian Ad36, bovine Ad3, canine Ad2, porcine Ad3, or a combination thereof. In another embodiment, the non-human adenovirus comprises simian Ad36.

[0037] A further aspect of the present disclosure provides a method of reducing or preventing the incidence or lessening the severity of influenza or SARS-CoV-2 infection, said method comprising administering a therapeutically effective amount of any one of the recombinant adenoviruses disclosed herein, or any one of the recombinant adenoviral (rAd) vectors disclosed herein, or any pharmaceutical composition disclosed herein.

[0038] One particular aspect of the present disclosure provides a method for reducing or preventing the occurrence of, or lessening the severity of, an influenza virus infection, a SARS-CoV-2 virus infection, or a combination thereof in a subject. The method comprises administering to a subject a therapeutically effective amount of a polycistronic vaccine, wherein the polycistronic vaccine comprises a recombinant adenovirus (rAd) vector or a pharmaceutical composition comprising the rAd vector. The rAd vector can be any one of the rAd vectors disclosed herein. In one specific embodiment, the rAd vector comprises an adenovirus genome modified to include (i) a first exogenous oligonucleotide encoding at least one immune response-stimulating antigen of an influenza virus and (ii) a second exogenous oligonucleotide encoding at least one immune response-stimulating antigen of a SARS-CoV-2 virus. In some embodiments, the first foreign oligonucleotide is selected from the group consisting of: (i) an oligonucleotide encoding an influenza A virus hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof; (ii) an oligonucleotide encoding an influenza A virus neuraminidase or an immunogenic portion, variant, mutant, or fragment thereof; (iii) an oligonucleotide encoding a first influenza B virus hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof; (iv) an oligonucleotide encoding a second influenza B virus hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof, wherein the second influenza B virus is a different strain from the first influenza B virus; and (v) a combination of two or more of the oligonucleotides (i) to (iv). In yet other embodiments, the second foreign oligonucleotide is selected from the group consisting of: (vi) an oligonucleotide encoding the spike protein (S protein) or an immunogenic portion, variant, mutant, or fragment thereof of a first SARS-CoV-2 virus, (vii) an oligonucleotide encoding the S protein or an immunogenic portion, variant, mutant, or fragment thereof of a second SARS-CoV-2 virus, wherein the second SARS-CoV-2 virus is a different strain from the first SARS-CoV-2 virus, and (viii) a combination of the oligonucleotides of (vi) and (vii).

[0039] Moreover, in some embodiments, the vaccine is administered intranasally. [Brief explanation of the drawings]

[0040] [Figure 1] Figure 1 is a schematic diagram showing the hemagglutinin (HA) and neuraminidase (NA) proteins on the surface of the influenza virus and the spike protein on the surface of the SARS-CoV-2 virus. [Figure 2] Figure 2 is a prior art diagram of a transgene cassette for a SARS-CoV-2 vaccine. ChAd-Control has no transgene insert, and ChAd-SARS-CoV-2-S encodes a SARS-CoV-2-S protein with the two proline mutations shown. See WO 2021 / 247567, published December 9, 2021. [Figure 3] FIG. 3 is a schematic diagram of the wild-type human Ad5 vector used to prepare the rAd vectors of the present disclosure. [Figure 4] FIG. 4 is a schematic diagram of the first generation Ad5 vector used to prepare the rAd vectors of the present disclosure. [Figure 5] FIG. 5 is a schematic diagram of the second generation Ad5 vector used to prepare the rAd vectors of the present disclosure. [Figure 6] FIG. 6 is a schematic diagram of the third generation Ad5 vector used to prepare the rAd vectors of the present disclosure. [Figure 7] FIG. 7 is a schematic diagram of the fourth generation Ad5 vector used to prepare the rAd vectors of the present disclosure. [Figure 8] FIG. 8 is a schematic diagram showing the maximum transgene capacity of the first to fourth generation human Ad5 vectors. [Figure 9] FIG. 9 is a schematic diagram showing a comparison of the maximum transgene payload capacity of gutted human Ad5 vectors compared to first and second generation human Ad5 vectors. DETAILED DESCRIPTION OF THE INVENTION

[0041] The present disclosure is based, at least in part, on the development of recombinant non-human adenoviral vector compositions and immunogenic compositions thereof for treating or preventing coronavirus (COVID-19) and / or influenza infections. Additionally, the present disclosure provides methods of administering the compositions disclosed herein. The compositions disclosed herein provide immunity against coronavirus and / or influenza infection in a single dosage unit. In one specific embodiment, the present invention provides compositions for administering a vaccine via intramuscular, intranasal, or inhalation routes. The compositions of the present disclosure can be used to prevent infection or transmission by influenza and / or SARS-Cov-2 viruses or reduce the severity of the disease in a subject.

[0042] The term "antibody" refers to an immunoglobulin, antigen-binding fragment, or derivative thereof that specifically binds to and recognizes an analyte (antigen), such as a coronavirus S protein, an antigenic fragment thereof, or an antigen dimer or multimer. As used herein, the term "antibody" is used in the broadest sense to encompass a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof that retain binding affinity for the antigen. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SFI, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments produced by the modification of whole antibodies or synthesized de novo using recombinant DNA techniques (see, e.g., Kontermann and Dubel (Ed), Antibody Engineering, Vols. 1-2, 2nd Ed., Springer Press, 2010).

[0043] A "conservative" amino acid substitution is one that does not substantially affect or reduce the function of a protein, such as the ability of the protein to elicit an immune response when administered to a subject. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid. Furthermore, deletions or additions that alter, add, or delete a single amino acid or a small percentage of amino acids (e.g., less than 5%, in some embodiments less than 1%) in an encoded sequence are conservative mutations in which the resulting amino acid is replaced with a chemically similar amino acid.

[0044] The viral genome is capped, polyadenylated, and surrounded by nucleocapsid proteins. Coronavirus virions contain a viral envelope containing a type I fusion glycoprotein called the spike (S) protein (S protein). Most coronaviruses share a common genome organization, with the replicase gene contained in the 5' portion of the genome and the structural genes contained in the 3' portion of the genome.

[0045] Coronavirus spike (S) protein: A class I fusion glycoprotein initially synthesized as a precursor protein. Individual precursor S polypeptides form homotrimers and undergo glycosylation, signal peptide removal, and cleavage by cellular proteases in the Golgi apparatus to generate separate S1 and S2 polypeptide chains. These chains remain associated within the homotrimer as S1 / S2 protomers, resulting in a heterotrimer of trimers. The S1 subunit is located distal to the viral membrane and contains the receptor-binding domain (RBD) that mediates viral attachment to host receptors. The S2 subunit contains the fusion protein machinery, including a fusion peptide, two heptad repeats (HR1 and HR2), a central helix typical of fusion glycoproteins, a transmembrane domain, and a cytoplasmic tail domain.

[0046] The coronavirus spike (S) protein prefusion conformation is the structural conformation adopted by the ectodomain of the coronavirus S protein after it is processed into the mature coronavirus S protein in the secretion system and before triggering a fusion event that leads to the transition to the postfusion conformation of coronavirus S. The three-dimensional structure of an exemplary coronavirus S protein (HKU1-CoV) in the prefusion conformation is disclosed herein and provided by Kirchdoerfer et al., "Pre-fusion structure of a human coronavirus spike protein," Nature, 531:118-121, 2016 (incorporated herein by reference).

[0047] A coronavirus S ectodomain trimer "stabilized in the prefusion structure" contains one or more amino acid substitutions, deletions, or insertions relative to the native coronavirus S sequence, resulting in improved retention of the prefusion structure compared to a coronavirus S ectodomain trimer formed from the corresponding native coronavirus S sequence. The "stabilization" of the prefusion structure by the substitution, deletion, or insertion of one or more amino acids can be, for example, energetic stabilization (e.g., a reduction in the energy of the prefusion structure relative to the postfusion open structure) and / or kinetic stabilization (e.g., a reduction in the transition rate from the prefusion structure to the postfusion structure). Furthermore, stabilization of the coronavirus S ectodomain trimer in the prefusion structure can include increased resistance to denaturation compared to the corresponding native coronavirus S sequence. Methods for determining whether a coronavirus S ectodomain trimer is in the prefusion structure include, but are not limited to, negative stain electron microscopy and antibody binding assays using prefusion structure-specific antibodies.

[0048] Degenerate variant: In the context of this disclosure, a "degenerate variant" refers to a polynucleotide encoding a polypeptide that contains a degenerate sequence as a result of the genetic code. There are 20 naturally occurring amino acids, most of which are specified by multiple codons. Therefore, all degenerate nucleotide sequences that encode a peptide are included, as long as the amino acid sequence of the peptide encoded by the nucleotide sequence is unchanged.

[0049] In one example, the desired response is to inhibit, reduce, or prevent influenza and / or SARS-CoV-2 infection. The method need not completely eliminate, reduce, or prevent infection to be effective. For example, administration of an effective amount of immunogen can induce an immune response that reduces infection (e.g., as measured by infection of cells or the number or percentage of infected subjects) by a desired amount, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 100% (eliminating or preventing detectable infection) compared to an appropriate control.

[0050] Expression refers to the transcription or translation of a nucleic acid sequence. For example, a gene is expressed when DNA is transcribed into RNA or an RNA fragment, which may be processed into mRNA. A gene may also be expressed when mRNA is translated into an amino acid sequence, such as a protein or protein fragment. In certain instances, a heterologous gene is expressed when it is transcribed into RNA. In other instances, a heterologous gene is expressed when RNA is translated into an amino acid sequence. As used herein, the term "expression" refers to either transcription or translation. Control of expression can include controlling transcription, translation, RNA transport and processing, degradation of intermediate molecules such as mRNA, or the activation, inactivation, compartmentalization, or degradation of a specific protein molecule after its production, for example.

[0051] An expression control sequence refers to a nucleic acid sequence that controls the expression of a heterologous nucleic acid sequence to which it is operably linked. An expression control sequence is operably linked to a nucleic acid sequence if it controls and regulates the transcription and, if necessary, translation of the nucleic acid sequence. Thus, an expression control sequence can include an appropriate promoter, enhancer, transcription termination, an initiation codon (ATG) in front of a protein-encoding gene, splicing signals for introns, maintaining the correct reading frame of the gene to enable proper translation of mRNA, and a stop codon. The term "control sequence" includes at least components whose presence can affect expression and can also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. An expression control sequence can include a promoter.

[0052] A promoter is a minimal sequence sufficient to induce transcription. It also includes promoter elements sufficient to regulate promoter-dependent gene expression in a cell-type-specific, tissue-specific, or inducible manner by external signals or factors. Such elements can be located in the 5' or 3' region of the gene. Both constitutive and inducible promoters are included (see, e.g., Bitter et al., Methods in Enzymology 153:516-544, 1987). For example, when cloning in a bacterial system, inducible promoters such as pL, plac, ptrp, and ptac (ptrp-lac hybrid promoter) from bacteriophage lambda can be used. In one embodiment, when cloning in a mammalian cell system, promoters derived from the genome of a mammalian cell (e.g., metallothionein promoter) or mammalian virus promoters (e.g., retroviral long terminal repeat, adenovirus late promoter, vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques can also be used to transcribe nucleic acid sequences. Expression vector: A vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression are supplied by the host cell or an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate recombinant polynucleotides.

[0053] The term "heterologous" means derived from a different genetic source. A nucleic acid molecule heterologous to a cell, derived from a genetic source other than the cell in which it is expressed. In a specific, non-limiting example, a heterologous nucleic acid molecule encoding a recombinant coronavirus S protein is expressed in a cell, such as a mammalian cell. Methods for introducing heterologous nucleic acid molecules into cells or organisms are well known in the art and include, for example, electroporation, lipofection, particle gun acceleration, and nucleic acid transformation, including homologous recombination.

[0054] A host cell refers to a cell in which a vector can be propagated and its DNA expressed. The cell can be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parent cell because mutations may occur during replication. However, when the term "host cell" is used, such progeny are included.

[0055] An immune response is a reaction by cells of the immune system, such as B cells, T cells, and monocytes, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response"). In one embodiment, the immune response is a T cell response, such as a CD4+ or CD8+ response. In another embodiment, the immune response is a B cell response, resulting in the production of specific antibodies.

[0056] Nucleic acid molecules are polymeric forms of nucleotides and may include both sense and antisense strands of RNA, cDNA, and genomic DNA, as well as synthetic and mixed polymers thereof. Nucleotide refers to ribonucleotides, deoxynucleotides, or modified forms of either type of nucleotide. As used herein, the terms "nucleic acid molecule" are synonymous with "nucleic acid" and "polynucleotide." Nucleic acid molecules are typically at least 10 bases in length, unless otherwise specified. The term includes single- and double-stranded DNA. Polynucleotides may contain naturally occurring and / or modified nucleotides linked by naturally occurring and / or non-naturally occurring nucleotide linkages. "cDNA" refers to DNA that is complementary or identical to mRNA, in single- or double-stranded form. "Encoding" refers to the unique property of a particular nucleotide sequence within a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes with a defined nucleotide sequence (e.g., rRNA, tRNA, mRNA) or a defined amino acid sequence, and the biological properties resulting therefrom.

[0057] The term "operably linked" means that a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed into a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked nucleic acid sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

[0058] A polypeptide is a chain of amino acids, regardless of length or post-translational modifications (such as glycosylation or phosphorylation). The term "polypeptide" applies to amino acid polymers, including natural and unnatural amino acid polymers, as well as those in which one or more amino acid residues are unnatural amino acids (e.g., artificial chemical mimics of the corresponding natural amino acids). A "residue" refers to an amino acid or amino acid mimetic incorporated into a polypeptide by an amide bond or amide bond mimetic. A polypeptide has an amino terminus (N-terminus) and a carboxy terminus (C-terminus). "Polypeptide" is used interchangeably with peptide or protein and refers herein to a polymer of amino acid residues.

[0059] Prime-boost vaccination is an immunotherapy in which a first immunogenic composition (priming vaccine) is administered to a subject, followed by a second immunogenic composition (booster vaccine) to elicit an immune response. The priming vaccine and / or booster vaccine contain a vector (e.g., a viral vector, an RNA vector, or a DNA vector) expressing an antigen to which an immune response is directed. The booster vaccine is administered to a subject after the administration of the priming vaccine, and examples of appropriate time intervals between the administration of the priming vaccine and the booster vaccine, as well as examples of such time frames, are disclosed herein. In some embodiments, the priming vaccine, the booster vaccine, or both the priming vaccine and the booster vaccine further contain an adjuvant. In one non-limiting example, the priming vaccine is a DNA-based vaccine (or other vaccine based on gene delivery), and the booster vaccine is a protein subunit or protein nanoparticle-based vaccine.

[0060] A recombinant nucleic acid molecule is a molecule that has a sequence that is not found in nature, e.g., contains one or more nucleic acid substitutions, deletions, or insertions, and / or has a sequence that is created by an artificial combination of two separate sequence segments. This artificial combination can be achieved by chemical synthesis, but more commonly, by the artificial manipulation of separate segments of nucleic acid, e.g., by genetic engineering techniques. A recombinant virus is a virus whose genome includes a recombinant nucleic acid molecule. A recombinant protein is one that has a sequence that is not found in nature or has a sequence that is created by the artificial combination of two naturally separate sequence segments. In some embodiments, the recombinant protein is encoded by a heterologous (e.g., recombinant) nucleic acid that has been introduced into the genome of a host cell, such as a bacterial or eukaryotic cell, or a recombinant virus.

[0061] Sequence identity is the similarity between amino acid sequences, and is expressed in terms of the similarity between sequences, also called sequence identity.Sequence identity is often measured by percentage identity, and the higher the percentage, the higher the similarity between two sequences.Polypeptide homolog, ortholog or variant shows relatively high sequence identity when aligned by standard method.The method of aligning sequences for comparison is well known in the art. For various programs and alignment algorithms, see Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al., Computer Appls. in the Biosciences 8,155-65, 1992; and Pearson et al. al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990 presents a detailed review of sequence alignment methods and homology calculations.

[0062] Homologs and variants of a polypeptide (e.g., the S extracellular domain of a coronavirus) are typically characterized by at least about 75% sequence identity, e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, counted across the full-length alignment with the amino acid sequence of interest. Proteins with even greater similarity to the reference sequence will have increasing percentage identities, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, when assessed by this method. When comparing sequence identity across less than the entire sequence, homologs and variants typically share at least 80% sequence identity over short stretches of 10-20 amino acids, and may share at least 85%, or at least 90%, or 95% sequence identity, depending on the similarity to the reference sequence. Methods for determining sequence identity within such short regions are available on the internet at the NCBI website. As used herein, reference to "at least 90% identity" or similar terminology refers to "at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity" to a specified reference sequence.

[0063] A vaccine is a pharmaceutical composition that induces a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine induces an antigen-specific immune response against an antigen of a pathogen (e.g., a viral pathogen) or a cellular component correlated with pathology. A vaccine may include a polynucleotide (e.g., a nucleic acid encoding an antigen disclosed herein), a peptide or polypeptide (e.g., an antigen disclosed herein), a virus, a cell, or one or more cellular components. As a non-limiting example, a vaccine induces an immune response that reduces the severity of symptoms associated with SARS-CoV-2 virus and / or influenza virus infection and / or reduces viral load compared to a control. In another non-limiting example, a vaccine induces an immune response that reduces and / or prevents SARS-CoV-2 virus and / or influenza virus infection compared to a control.

[0064] A vector is an entity comprising a DNA or RNA molecule operably linked to a coding sequence for an antigen of interest and having a promoter capable of expressing the coding sequence. Non-limiting examples include naked or packaged (lipid and / or protein) DNA, naked or packaged RNA, subcomponents of viruses, bacteria, or other microorganisms that may be replication-incompetent, or viruses, bacteria, or other microorganisms that may be replication-competent. Vectors are sometimes referred to as constructs. A recombinant DNA vector is a vector that contains recombinant DNA. A vector may contain a nucleic acid sequence, such as an origin of replication, that enables replication in a host cell. A vector may also contain one or more selectable marker genes and other genetic elements known in the art. A viral vector is a recombinant nucleic acid vector that contains at least some nucleic acid sequences derived from one or more viruses.

[0065] Virus-like particles (VLPs) are non-replicating viral shells derived from various viruses. VLPs are typically composed of one or more viral proteins, such as, but not limited to, viral proteins called capsid, coat, shell, surface, or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can be spontaneously formed by recombinant expression of proteins in an appropriate expression system. Methods for producing specific VLPs are known in the art. The presence of VLPs after recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as electron microscopy and biophysical characterization. Furthermore, VLPs can be separated by known techniques, such as density gradient centrifugation, and can be identified by characteristic density bands. See, for example, Baker et al. (1991) Biophys. J. 60:1445-1456; and Hagensee et al. (1994) Virol. 68:4503-4505; Vincente, J Invertebr Pathol., 2011; Schneider-Ohrum and Ross, Curr. Top. Microbiol. Immunol, 354:53073, 2012.

[0066] The compositions of the present disclosure may include one or more active agents. In some embodiments, the active agent may be an agent for preventing, treating, or reducing the infectivity of influenza and / or SARS-CoV-2 virus infection. In some embodiments, treating a viral infection may include reducing the infectivity and / or transmissibility of the virus. In some embodiments, preventing a viral infection may include reducing the infectivity and / or transmissibility of the virus. The compositions of the present disclosure may include an active agent for preventing a viral infection, an active agent for treating a viral infection, an active agent for reducing the infectivity of a viral infection, or a combination thereof. The compositions of the present disclosure may further include a pharmaceutically acceptable excipient, carrier, or diluent. Additionally, the compositions of the present disclosure may include a preservative, solubilizer, stabilizer, wetting agent, emulsifier, sweetener, colorant, flavoring agent, salt (the substances of the present disclosure themselves may be provided in the form of a pharmaceutically acceptable salt), buffer, coating agent, or antioxidant.

[0067] The present invention relates to non-human adenoviral vector compositions and methods of using immunogenic compositions comprising an adenoviral vector and, optionally, one or more additional active ingredients, pharmaceutically acceptable carriers, diluents, excipients, or adjuvants for treating or preventing respiratory viral infections. In some embodiments, the use of simian adenoviral vectors can overcome the problem of heterologous vector cross-immunity seen with human adenoviral vector platforms (PMID: 32450106).

[0068] In one particular aspect of the present disclosure, an adenoviral vector is provided. Adenoviruses are non-enveloped viruses containing a nucleocapsid and a linear double-stranded DNA genome. The viral nucleocapsid contains a penton capsomer and a hexon capsomer. Each penton base has a unique fiber associated with it, which helps the virus attach to host cells via the coxsackie adenovirus receptor on the host cell surface. The adenovirus genome contains four early transcription units (E1, E2, E3, and E4), which primarily function as regulators and prepare the host cell for viral replication. The genome also contains five late transcription units (L1, L2, L3, L4, and L5), which encode structural proteins, including the penton (L2), hexon (L3), scaffold protein (L4), and fiber protein (L5), all of which are controlled by a single promoter. Each end of the genome contains an inverted terminal repeat (ITR) required for viral replication.

[0069] Adenoviruses offer many other advantages for clinical vaccine development. Their genomes are relatively small, well-characterized, and easy to manipulate. Deletion of a single transcription unit, E1, renders the virus replication-incompetent, increasing predictability and reducing side effects in clinical applications. Recombinant adenoviruses can accommodate relatively large transgenes, allowing for greater flexibility in subunit design and broader tropism, facilitating transgene delivery to a variety of cells and tissues. Important for clinical applications is the established methodology for scaling up the production and purification of recombinant adenoviruses to high titers. To date, subgroup C serotypes AdHu2 and AdHu5 have primarily been used as vectors. However, first-generation vaccine vectors based on AdHu5, a typical human adenovirus, demonstrated limited efficacy in clinical trials despite promising preclinical data. Subsequently, it was discovered that a large proportion of adults possess significant amounts of neutralizing antibodies against common human serotypes, such as AdHu2 and AdHu5, as a result of natural infection. Neutralizing antibodies can reduce the efficacy of viral vector vaccines by blocking virus entry into host cells and preventing delivery of targeted genes.

[0070] The compositions described herein include vectors that deliver heterologous molecules to cells for either therapeutic or vaccine purposes. As used herein, a vector can include any genetic element, such as, but not limited to, naked DNA, phage, transposon, cosmid, episome, plasmid, or virus. In some embodiments, such vectors include simian adenovirus DNA (e.g., SAdV-36) and a transgene. "Transgene" refers to the combination of a selected heterologous gene and other regulatory elements necessary to promote translation, transcription, and / or expression of the gene product in a host cell. In some embodiments, viral vectors can include adenovirus vectors expressing a gene encoding a coronavirus S protein. Adenoviruses of various origins, subtypes, or mixtures of subtypes can be used as the source of the viral genome for adenovirus vectors. Non-human adenoviruses (e.g., simian, chimpanzee, gorilla, avian, canine, ovine, or bovine adenoviruses) can also be used to generate adenovirus vectors. For example, simian adenovirus can be used as the source of viral genome for adenovirus vector.Simian adenovirus includes serotype 1, 3, 7, 11, 16, 18, 19, 20, 27, 33, 36, 38, 39, 48, 49, 50 or other simian adenovirus serotypes.Simian adenovirus can be represented by any suitable abbreviation known in the art, such as SV, SAdV, SAV, sAV, etc.In some examples, simian adenovirus vector is serotype 36 simian adenovirus vector.

[0071] Typically, SAdV-derived adenoviral vectors are designed so that a transgene is placed within a nucleic acid molecule containing other adenoviral sequences within a region unique to a selected adenoviral gene. If desired, the transgene can be inserted into an existing gene region to disrupt its function. Alternatively, the transgene can be inserted into a site of a partially or completely deleted adenoviral gene. For example, the transgene can be placed into a functional E1 deletion site or a functional E3 deletion site (or E3B), among other sites that may be selected. The term "functionally deleted" or "functional deletion" means that a sufficient amount of the gene region has been removed or damaged, for example, by mutation or modification, so that the gene region is no longer capable of producing a functional product of gene expression. If desired, the entire gene region can be deleted. In one embodiment, an adenoviral vector useful according to the present disclosure is the simian adenovirus SAd36, which has deletions in the E1 and E3B genes. In one aspect, the adenoviral vector has the nucleotide sequence of SEQ ID NO:3. In another embodiment, the adenoviral vector has a nucleic acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO:3.

[0072] For example, in the case of a production vector useful for generating recombinant viruses, the vector may contain a transgene and either the 5' end of the adenoviral genome, the 3' end of the adenoviral genome, or both the 5' and 3' ends of the adenoviral genome. The 5' end of the adenoviral genome contains 5' cis elements required for packaging and replication, namely, the 5' inverted repeat (ITR) sequence (which functions as an origin of replication) and the native 5' packaging enhancer domain (which contains sequences required for packaging of the linear Ad genome and the enhancer element of the E1 promoter). The 3' end of the adenoviral genome contains 3' cis elements (including the ITRs) required for packaging and encapsidation. Recombinant adenoviruses preferably contain both 5' and 3' adenoviral cis elements, with the transgene located between the 5' and 3' adenoviral sequences. Adenoviral vectors derived from simians (e.g., SAdV-36) may contain additional adenoviral sequences.

[0073] Preferably, these monkey-derived adenoviral vectors contain one or more adenoviral elements derived from the adenoviral genome. In one embodiment, the vector contains adenoviral sequences derived from an adenoviral serotype different from the adenoviral serotype that provides the ITRs. As defined herein, pseudotyped adenovirus refers to an adenovirus whose adenoviral capsid proteins are derived from a different adenovirus than the adenovirus that provides the ITRs. Chimeric or hybrid adenoviruses can be constructed using the adenoviruses described herein using techniques known to those skilled in the art. See, for example, US 7,291,498.

[0074] A transgene is a nucleic acid sequence that is heterologous to the vector sequences that flank it and that encodes a polypeptide, protein, or other product of interest. The nucleic acid coding sequence is operably linked to regulatory components in a manner that allows for the transcription, translation, and / or expression of the transgene in a host cell.

[0075] Like SARS-CoV-1 and the Middle East respiratory syndrome coronavirus (MERS-CoV), which causes Middle East respiratory syndrome (MERS), SARS-CoV-2 infects cells by binding to angiotensin-converting enzyme 2 (ACE2) on the cell surface. Briefly, ACE-2 is the functional receptor for SARS-CoV-1, SARS-CoV-2, MERS-CoV, and likely future SARS-CoV variants. ACE-2 is a critical component of the renin-angiotensin-aldosterone system (RAAS). ACE-2 converts angiotensin 2 to angiotensin 1-7. High levels of angiotensin 2 are associated with vasoconstriction, inflammation, and acute lung injury. ACE2 is expressed in various organs, including the lungs, heart, kidneys, liver, intestine, and other tissues. The SARS-CoV virus binds to ACE2 to enter cells.

[0076] The SARS-CoV-2 RNA genome is approximately 30,000 nucleotides long. The 5'-two-thirds encode nonstructural proteins that enable genome replication and viral RNA synthesis. The remaining one-third encodes structural proteins that form spherical virus particles, such as spike (S), envelope, membrane, and nucleoprotein (NP), as well as accessory proteins that control cellular responses. The S protein forms homotrimeric spikes on the virus particle and binds to the cell surface receptor angiotensin-converting enzyme 2 (ACE2), facilitating coronavirus entry into human cells. During the entry process, the S protein is sequentially cleaved to generate S1 and S2 fragments, and then S2 is further processed to generate the smaller S2' protein. The S1 protein contains the receptor-binding domain (RBD), and the S2 protein promotes membrane fusion. The structure of a soluble, stabilized pre-fusion form of the SARS-CoV-2 S protein has been elucidated by cryo-electron microscopy and revealed significant similarity to the S protein of SARS-CoV. This form of the S protein is being used as a vaccine target.

[0077] In virus classification, influenza viruses are negative-sense, single-stranded RNA viruses. The influenza virus genus is currently classified into the Orthomyxoviridae family (influenza virus A, influenza virus B, and influenza virus C). Each of these genera contains one influenza virus species. The influenza virus A genus consists of a single species, influenza A virus, which includes all influenza virus strains currently circulating in humans.

[0078] The various types of influenza A are classified based on two proteins on the surface of the virus: hemagglutinin (H) and neuraminidase (N). There are at least 18 different hemagglutinin subtypes and at least 11 different neuraminidase subtypes (H1-H18 and N1-N11, respectively). These include, but are not limited to, the H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2, and H10N7 serotypes. Influenza A subtypes (e.g., H1N1) are further divided into different genetic clades (e.g., 6B.1) and subclades (e.g., 6B.1A). Clades and subclades are genetically distinct but not necessarily antigenically distinct. Given the virus's tendency to reassort, there may be many more possible combinations of influenza A subtypes. Reassortment is the process by which influenza viruses exchange genetic segments. Reassortment occurs when two different influenza viruses simultaneously infect the same host and exchange genetic information.

[0079] The influenza B virus genus consists of a single species, influenza B virus, with only one currently known serotype, further divided into two lineages: B / Yamagata and B / Victoria. Influenza B viruses are almost exclusively human pathogens, but they are far less common and genetically diverse than influenza A strains. Due to this limited genetic diversity, most humans acquire some immunity to influenza B viruses early in life. However, the frequency of viral mutations is high enough to prevent most humans from acquiring lasting immunity, but not high enough to cause a pandemic infection with influenza B viruses across the human population. Like influenza A viruses, influenza B viruses are further divided into specific clades (e.g., V1A) and subclades (e.g., V1A1).

[0080] The influenza C genus also consists of a single species, influenza C virus, with only one known serotype. This serotype is known to infect both primates and pigs. Influenza C virus infection in humans is rare, but can cause illness ranging from mild to severe. However, because of rapid transmission between close contacts, epidemics are not uncommon in populations exposed to influenza C virus.

[0081] A fourth family of influenza viruses, influenza D, was first isolated in 2011 and identified in 2016.

[0082] Hemagglutinin (HA) and neuraminidase (NA) are two large glycoproteins located on the exterior of virus particles. HA is a lectin that mediates virus binding to and entry of the viral genome into target cells, while NA is involved in the release of progeny viruses from infected cells by cleaving the sugars attached to mature virus particles. Therefore, these proteins are targets for antiviral drugs. Furthermore, they are antigens that can be targeted by antibodies. Influenza A viruses are classified into subtypes based on antibody responses to HA and NA. These different types of HA and NA form the basis for distinguishing between H and N subtypes, such as H5N1. While 18 subtypes of HA and 11 subtypes of NA are known, only HA1, 2, and 3 and NA1 and 2 are commonly found in humans. Influenza A viruses in particular have many different serotypes, with over 144 possible "HN" serotypes resulting from mutations in these two proteins alone. Only a few of these combinations are likely to be consistently prevalent within susceptible populations.

[0083] Influenza viruses are causative agents of infectious respiratory diseases (commonly referred to as "influenza") that primarily infect humans and other vertebrates. Influenza is a highly contagious acute respiratory illness that has plagued humanity since ancient times. Infectious diseases are characterized by annual epidemics and periodic global pandemics. Influenza virus infections cause mild to severe symptoms and can be fatal. On average, 5-20% of the US population is infected with influenza each year, resulting in more than 200,000 hospitalizations and more than 36,000 deaths due to complications. Due to the high disease-related morbidity and mortality, influenza has significant direct and indirect socioeconomic impacts. Four pandemics have occurred in the past century, resulting in tens of millions of deaths worldwide.

[0084] In the case of influenza, the immunodominant HA and NA proteins protrude from the central capsid of the virus particle and tend to interact most strongly with the host's internal environment and dominate the host's immune response. Mutations occurring within the microbial genome protect the microbe from the host's immune system. These mutations are most commonly known to affect immunodominant antigens.

[0085] Non-immunodominant antigens are antigens that are capable of eliciting a host immune response but account for only a small proportion of the overall immune response. This is thought to occur because non-immunodominant antigens are at least partially protected from the host immune system, such as when the antigen is located in crevices or folds on the microbial surface or surrounded by protruding elements of the microorganism. In the case of influenza, non-immunodominant antigens located near the capsid surface are protected from the host immune system by the immunodominant HA and NA spikes that protrude from the surface. Non-immunodominant antigens tend to mutate less in response to host immune pressure than immunodominant antigens.

[0086] The CDC and leading authorities on global disease prevention recommend that the best way to prevent viral respiratory infections such as SARS and influenza is through regular vaccination. Traditional vaccines typically target a single virus. Therefore, separate vaccines are administered for influenza and SARS-CoV-2 viruses. Separate administration may lead to poor compliance and require a "drug holiday" between influenza and SARS-CoV-2 vaccine administration.

[0087] Regarding influenza vaccines, conventional vaccines target influenza's immunodominant proteins, the HA and NA antigens. These vaccines do not provide universal or 100% protection. Due to antigenic shift, influenza vaccines cannot provide universal protection or maintain efficacy over many years. The ineffectiveness of conventional vaccines is likely due, in part, to antigenic drift and mutations occurring within the antigenic portions of the HA and NA proteins (i.e., immunodominant antigens) that are most commonly recognized by the immune system. As a result, influenza is constantly developing resistance to current treatments, potentially leaving many people infected with influenza virus without an effective treatment. This scenario is particularly concerning for the H5N1 virus, which, despite its highly pathogenic nature, currently lacks a widely available commercial vaccine to immunize susceptible human populations.

[0088] Some embodiments of the present disclosure provide multicistronic (i.e., multivalent) vaccines, as well as methods for making and using the same. Thus, the compositions of the present disclosure can be used for vaccination against influenza virus and SARS-CoV-2 virus. Alternatively, the compositions of the present disclosure can target a combination of two or more different SARS-CoV-2 virus strains, two or more different influenza virus strains, or SARS. In certain embodiments, the compositions disclosed herein include at least one oligonucleotide encoding an immune response-stimulating antigen of the SARS-CoV-2 virus and at least one oligonucleotide encoding an immune response-stimulating antigen of an influenza virus. Thus, some compositions of the present disclosure provide simultaneous immunity or protection against both SARS-CoV-2 and influenza viruses.

[0089] The antigens and epitopes (i.e., immune stimulating antigens or immune response stimulating antigens) disclosed herein comprise or are derived from multiple antigenic regions (e.g., epitopes) of pathogens, such as influenza virus and SARS-CoV-2 virus, or different pathogens. The composite antigens of the present disclosure can include antigenic regions that represent a combination of all or part of two or more similar or dissimilar epitopes, or multiple immune response regions derived from one or more antigenic sources (e.g., epitopes of viral particles). These immunological regions are amino acid sequences or epitopes that represent sequences found in antigenic regions of pathogens or other antigens that are associated with infection or disease, or, importantly, with stimulating the immune system to provide protection against pathogens. Administration can be to an individual via injection (e.g., intradermal, intravenous, oral, nasal, intraperitoneal), or the like.

[0090] Some embodiments of the present disclosure relate to antigens of pathogens, such as influenza virus or SARS-CoV-2 virus antigens. As used herein, unless the context dictates otherwise, the terms "antigen" and "epitope" are used interchangeably herein to refer to an immune response-stimulating antigen produced or expressed by an oligonucleotide used in the vaccine composition of the present disclosure. An antigen may be a selected region of a virus (e.g., the S protein of the SARS-CoV-2 virus) known or believed to generate an effective immune response following administration. The peptide sequence of the antigen may include multiple immune response regions or epitopes of one or more pathogens, typically arranged artificially (i.e., not naturally occurring) along a single amino acid sequence or peptide. The multiple may include multiples of the same pathogen, typically not in the order in which they occur in nature (e.g., different epitopes of the influenza virus or different S proteins of the SARS-CoV-2 virus), or may include multiples of a variety of different epitopes, typically from one or more different viruses or different virus strains. The epitopes may be identical to known immune regions of a pathogen or may be entirely novel structures artificially constructed because they did not previously exist. Preferably, antigens produced by the vaccine compositions of the present disclosure elicit a protective immunogenic response in animals or mammals (e.g., humans) and stimulate both mucosal and systemic immune responses similar to those observed in natural infection. Preferably, the response includes a killer T cell (TC or CTL) response, a helper T cell (TH) response, macrophages (MP), and the production of specific antibodies in the inoculated subject.

[0091] Antigens of the present disclosure can also be obtained or derived from sequences of pathogens such as, for example, multiple or composite epitopes of proteins and / or polypeptides of coronaviruses, influenza viruses, or combinations thereof.

[0092] Antigens disclosed herein include those containing engineered "composite" epitopes. These epitopes are artificially created from two or more distinct epitopes, and the resulting composite antigens have physical and / or chemical properties that are distinct from or additional to the individual epitopes. These distinct epitopes may be highly conserved regions of the same epitope, possibly with slightly different sequences across different serotypes. Oligonucleotides can also be constructed to express conserved regions of different epitopes (e.g., influenza virus HA and NA, coronavirus S and M, or influenza virus HA and coronavirus S).

[0093] In some embodiments, the antigen may be a selected region of the influenza virus and / or SARS-CoV-2 virus that is known or believed to generate an effective immune response following administration. The peptide sequence of the antigen may include multiple artificially arranged immune response regions or epitopes of one or more viruses. The epitopes may be identical to known immune response regions of the virus, or may be artificially constructed with completely new structures that have never existed before. In a particular embodiment, the antigens of the present disclosure induce a protective immunogenic response in an animal, mammal, or subject (e.g., a human), stimulating both mucosal and systemic immune responses similar to those observed in natural infection. In some embodiments, the response includes killer T cell (TC or CTL) responses, helper T cell (TH) responses, macrophages (MPs), and the production of specific antibodies in the inoculated subject.

[0094] Antigens of the present disclosure can be obtained or derived from influenza virus and / or SARS-CoV-2 virus sequences, e.g., multiple or combined epitopes of proteins and / or polypeptides. Antigens may be constructed from conserved regions of different epitopes (e.g., influenza virus HA and NA, coronavirus S and M, or influenza virus HA and coronavirus S, etc.).

[0095] Antigens produced by expression of two or more oligonucleotides of the present disclosure include epitopes representing two or more epitopes that have epitope sequences similar only to the epitope sequence from which they are derived, where the epitopes are regions obtained or derived from viral proteins or peptides that elicit a strong immune response when administered to a subject. As used herein, the term "subject" refers to a mammal or animal, including but not limited to homo sapiens, monkeys, pigs, cows, cats, dogs, horses, and the like. The subject is often a human. In some embodiments, the potent response provides the subject with immunological protection against developing disease upon exposure to influenza virus and / or SARS-CoV-2 virus. In some embodiments, compositions of the present disclosure include oligonucleotides encoding immune response-stimulating antigens, wherein the antigens include a conserved region or two related epitopes (e.g., HA and HA, NA and NA, M and M, S protein and S protein, etc.), or conserved regions of two different epitopes (e.g., HA and NA, HA and M, NA and M, HA, NA, and S protein, HA and S protein, NA and S protein, etc.). The antigen may also contain different variable regions.

[0096] The antigen may include one or more T cell stimulatory epitopes, such as, for example, diphtheria toxoid, tetanus toxoid, polysaccharide, lipoprotein, or derivatives or any combination thereof (including fragments or variants thereof). Typically, at least one sequence of the antigen is contained within the same molecule as the T cell stimulatory epitope. In the case of a protein-based T cell stimulatory epitope, at least one repeat sequence of the composite antigen may be contained within, linked to, or otherwise associated with the T cell stimulatory epitope within the same polypeptide. In some embodiments, one or more T cell stimulatory epitopes are located at the N-terminus or C-terminus (or both) of the antigen.

[0097] In some embodiments, the antigen may be a peptide variant of a naturally occurring sequence. As used herein, the term "variant" refers to an antigen that contains one or more conservative substitutions. A "conservative substitution" is one in which one amino acid is substituted for another amino acid with similar properties, and one skilled in the art of peptide chemistry would predict that the secondary structure and hydropathic properties of the peptide will not be substantially altered. Amino acid substitutions can generally be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine, and tyrosine. Examples of amino acid substitutions that represent conservative changes include: (1) substitution of one or more alanine, proline, glycine, glutamine, aspartic acid, glutamic acid, aspartic acid, serine, or threonine residues with one or more residues from the same group; (2) substitution of one or more cysteine, serine, tyrosine, or threonine residues with one or more residues from the same group; (3) substitution of one or more valine, isoleucine, leucine, methionine, alanine, or phenylalanine residues with one or more residues from the same group; (4) substitution of one or more lysine, arginine, or histidine residues with one or more residues from the same group; and (5) substitution of one or more phenylalanine, tyrosine, tryptophan, or histidine residues with one or more residues from the same group. A variant may additionally or alternatively include non-conservative changes, for example, by substituting one of the amino acid residues in Group (1) with one of the amino acid residues in Group (2), Group (3), Group (4), or Group (5). A variant may also (or alternatively) be altered by, for example, the deletion or addition of amino acids that have minimal effect on the immunogenicity, secondary structure, and hydropathic properties of the peptide.

[0098] Another aspect of the present disclosure provides polynucleotides, including DNA, RNA (e.g., cRNA, mRNA), and PNA (peptide nucleic acid) constructs, encoding the antigens of the present disclosure. These polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA, or synthetic) or RNA molecules. Additional coding or non-coding sequences can, but are not necessarily, present within the polynucleotides of the present disclosure, and polynucleotides may, but are not necessarily, linked to other molecules and / or supporting materials. As will be appreciated by those skilled in the art, due to the degeneracy of the genetic code, numerous nucleotide sequences exist that encode a given primary amino acid sequence. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any naturally occurring gene. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically contemplated in the present disclosure. Polynucleotides encoding immunogenic peptides are typically used for in vitro or in vivo production of the peptide. Any polynucleotide can be further modified to enhance in vivo stability. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and / or 3' ends, the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages in the backbone, and / or the inclusion of non-traditional bases such as inosine, queosine, and valine, as well as acetyl, methyl, thio, and other modified forms of adenine, cytidine, guanine, thymine, and uridine.

[0099] The vaccine composition of the present disclosure includes genetic sequences for two or more antigens as cRNA, mRNA, or DNA, along with other necessary sequences that provide for intracellular expression of the antigens. When the recombinant nucleic acid is injected into a mammal, the antigens are produced in or on the cells, which the subject's immune system recognizes and generates a humoral or cellular response against the antigens and, in turn, the virus. Nucleic acid vaccines offer many advantages over conventional vaccines, including the ability to induce a general and complete immune response in a subject.

[0100] In some embodiments, the vaccine compositions of the present disclosure include a pharmaceutically acceptable excipient, such as, for example, an adjuvant. Adjuvants refer to vehicles used to enhance antigenicity. In some embodiments, adjuvants include suspensions of minerals (alum, aluminum hydroxide, or phosphates) to which antigens are adsorbed, or water-in-oil emulsions, for example, where the vaccine solution is emulsified in mineral oil. In some embodiments, the adjuvant used in the disclosed immunogenic compositions is a combination of lecithin and carbomer homopolymer (e.g., ADJUPLEX™ adjuvant, available from AdvancedBioAdjuvants, LLC; see also Wegmann, Clin Vaccine Immunol, 22(9):1004-1012, 2015). Additional adjuvants for use in the disclosed immunogenic compositions include QS21 purified plant extract, matrix M, MF59, ALFQ, ALFA, AS01, AS10b, and / or combinations, derivatives, and variants thereof. Immunostimulatory oligonucleotides (such as those containing CpG motifs) can also be used as adjuvants. Adjuvants include biological molecules ("biological adjuvants"), such as costimulatory molecules. Exemplary adjuvants include IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL, and Toll-like receptor (TLR) agonists (such as TLR-9 agonists). Additional information regarding adjuvants can be found, for example, in Singh (ed.) Vaccine Adjuvants and Delivery Systems. Wiley-Interscience, 2007. Adjuvants can be used in combination with the disclosed compositions. The formulation of pharmaceutically acceptable excipients and carrier solutions, as well as the development of appropriate dosages and treatment regimens for use with the particular compositions described herein in various treatment regimens, are well known to those skilled in the art. In one specific embodiment, the vaccine composition of the present disclosure includes ALFQ, which can be administered by IM, SQ, intradermal, or intranasal administration, or by any other method compatible with the dosage formulation, in an amount that is prophylactically or therapeutically effective, and preferably immunogenic. The dosage will vary depending on the subject being treated (e.g., the immune system's capacity for immune response, the desired level of protection, etc.). Suitable dosage ranges are on the order of several hundred micrograms (μg) of active ingredient per subject. Exemplary dosages include ranges of about 0.1 μg to 2000 μg (although higher amounts, e.g., about 1 to about 10 mg, are contemplated), about 0.5 μg to 1000 μg, about 1 μg to about 500 μg, or about 10 μg to about 100 μg. Suitable treatment regimens for initial and booster doses vary, but typically an initial dose is followed by an optional but preferred booster dose or other periodic dose.

[0101] The amount of vaccine composition and the time required for administration of such vaccine composition are within the knowledge of one of ordinary skill in the art having the benefit of the teachings of the present invention. Administration of a therapeutically, pharmacologically, and / or prophylactically effective amount of the disclosed vaccine composition may be achieved by a single administration. Alternatively, in some situations, multiple or sequential administrations of the immunogenic composition may be desirable over a relatively short or relatively long period of time, as may be determined by the skilled artisan supervising the administration of such compositions.

[0102] An effective dose ranges from about 1 μg to about 1 mg per subject. As an example, the vaccine dosage range is about 0.1 μg to about 10 mg per subject. However, it may be advisable to adjust the dosage depending on the amount of oligonucleotide administered. In any case, these ranges are merely guidelines, and those skilled in the art can deviate from these ranges using conventional administration techniques. The exact dosage can be determined by assessing the immunogenicity of the conjugate produced in an appropriate host, and an immunologically effective dose can be administered. An immunologically effective dose is one that stimulates the subject's immune system to establish an immune response to the immunogenic composition or vaccine. In some embodiments, a level of immunological memory sufficient to provide long-term protection against disease caused by influenza and / or SARS-CoV-2 virus infection is achieved. The vaccine of the present disclosure may be formulated with an adjuvant. "Long-term" preferably refers to a period of at least about 6 months or more, at least about 1 year or more, at least about 2 to 5 years or more, or at least about 2 to about 10 years or more.

[0103] In one particular aspect of the present disclosure, a recombinant adenovirus (rAd) is provided. The rAds of the present disclosure are adapted for use in preventing infection or transmission by, or reducing the severity of disease caused by, influenza virus and / or SARS-Cov-2 virus in a subject. In some embodiments, the recombinant adenovirus comprises an oligonucleotide that encodes an antigen that stimulates an immune response in a subject. In particular, the recombinant adenovirus comprises: (i) a first oligonucleotide encoding an influenza A virus hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof; (ii) a second oligonucleotide encoding influenza A virus neuraminidase or an immunogenic portion, variant, mutant, or fragment thereof; (iii) a third oligonucleotide encoding the hemagglutinin of the first influenza B virus or an immunogenic portion, variant, mutant, or fragment thereof; (iv) a fourth oligonucleotide encoding a hemagglutinin of a second influenza B virus or an immunogenic portion, variant, mutant, or fragment thereof; (v) a fifth oligonucleotide encoding the spike protein (S protein) of the first SARS-CoV-2 virus or an immunogenic portion, variant, mutant, or fragment thereof; and (vi) a sixth oligonucleotide encoding the S protein of a second SARS-CoV-2 virus or an immunogenic portion, variant, mutant, or fragment thereof; Each of the oligonucleotides independently comprises at least two different oligonucleotides.

[0104] In general, the oligonucleotide sequences required for influenza viruses and SARS-CoV-2 viruses (e.g., the oligonucleotides (i) to (vi) above) are available from the World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC). Thus, the compositions of the present disclosure can be modified as needed to provide vaccines against future dominant or emerging strains of influenza virus and / or SARS-CoV-2 virus.

[0105] In some embodiments, at least one of the oligonucleotides encodes the S protein of SARS-CoV-2 (e.g., a stabilized form of the S protein of SARS-CoV-2). In some embodiments, at least one of the oligonucleotides encodes at least a portion or immunogenic fragment of the SARS-CoV-2 viral spike protein (S protein), or is homologous to the SARS-CoV-2 viral spike protein portion of SEQ ID NO:2 (or an immunogenic portion or variant thereof) by at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or an immunogenic fragment or variant thereof having a sequence with 99.9% sequence identity, or encoding an immunogenic fragment or variant thereof having a sequence with at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO:2 with the K986P and V987P mutations or SEQ ID NO:2 with the D614G mutation. In some embodiments, at least one of the oligonucleotides encodes a spike protein of a SARS-CoV-2 variant, such as, but not limited to, a spike protein having a D80G, 144del, F157S, L5F, T95I, A67V, S477N, 144del, Q677H, A701V, F888L, T791I, T859N, D950H, E484Q, D614G, E484K, N501Y, D69-70, L452R, or K417N mutation or an RBD E484K mutation relative to SEQ ID NO:2.In some embodiments, at least one of the oligonucleotides encodes a spike protein derived from WA1 / 2020, B.1.1.7, B.1.351, B.1.1.28, P.1, B.1.427, B.1.526, B.1.526.1, B.1.525, P.2, B.1.617, B.1.617.1, B.1.617.2, B.1.617.3, B.1.429, or a variant of B.1.429. In other embodiments, the spike protein is further modified to be in a prefusion-stabilized form (e.g., having a double proline substitution between residues 1050 and 1069 or between residues 981 and 999).

[0106] The vectors disclosed herein also include the necessary conventional control elements operably linked to two or more oligonucleotides encoding immune response stimulating antigens in a manner that allows for transcription, translation, and / or expression in cells transfected with the plasmid vector or infected with the viruses produced according to the present disclosure. As will be appreciated, the term "operably linked" sequences includes both expression control sequences adjacent to a gene of interest and expression control sequences that act in trans or remotely to regulate the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if necessary, sequences that enhance secretion of the encoded product.

[0107] Numerous expression control sequences, including native, constitutive, inducible, and / or tissue-specific promoters, are known in the art and can be utilized. Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFIa promoter [Invitrogen].

[0108] Inducible promoters allow for the control of gene expression and can be regulated by the presence of exogenously supplied compounds, environmental factors such as temperature, or specific physiological states (e.g., acute phase, specific differentiation states of cells, only replicating cells). Inducible promoters and inducible systems are available from a variety of commercial sources, including Invitrogen, Clontech, and Ariad. Many other systems have been described and can be readily selected by those skilled in the art. For example, inducible promoters include the zinc-inducible sheep metallothionine (MT) promoter and the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter. Other inducible systems include the T7 polymerase promoter system [WO 98 / 10088], the ecdysone insect promoter [No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)], the tetracycline repressible system [Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)], and the tetracycline inducible system [Gossen et al., Science, 268:1766-1769 (1995); see also Flarvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)]. Other systems include FK506 dimers, castradiol-based VP16 or p65, diphenol murislerone, and RU486-inducible systems [Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)], and rapamycin-inducible systems [Magari et al., J. Clin. Invest., 100:2865-2872 (1997)]. Some inducible promoters increase in effectiveness over time. In such cases, inserting multiple repressors in tandem (e.g., TetR linked with an IRES) can enhance the effectiveness of the system. Alternatively, wait at least three days before screening for the desired function.To enhance the effectiveness of this system, expression of the desired protein can be increased using known means, such as the use of the Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE).

[0109] In other embodiments, the native promoter of the oligonucleotide is used. Native promoters may be preferred when it is desired to mimic native expression of the oligonucleotide (i.e., the transgene). Native promoters can be used when transgene expression needs to be controlled temporally or developmentally, tissue-specifically, or in response to specific transcriptional stimuli. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites, and Kozak consensus sequences, can also be used to mimic native expression. Another embodiment of the transgene comprises a transgene operably linked to a tissue-specific promoter. For example, when expression in skeletal muscle is desired, a promoter active in muscle must be used. This includes promoters from genes encoding skeletal β-actin, myosin light chain 2A, dystrophin, and muscle creatine kinase, as well as synthetic muscle promoters with higher activity than the native promoter. Other components of the vector may include an origin of replication. Selection of these and other promoter and vector elements is conventional, and numerous such sequences are available and well known to those of skill in the art (see, e.g., Sambrook et al., and the references cited therein). These vectors are generated using the techniques and sequences described herein in combination with techniques known to those of skill in the art. Such techniques include conventional cDNA cloning techniques as described in the literature (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY), use of overlapping oligonucleotide sequences from the adenoviral genome, polymerase chain reaction, and any suitable method that provides the desired nucleotide sequence.

[0110] In some embodiments, the adenoviral vectors of the present disclosure comprise at least one oligonucleotide encoding an immune response stimulating antigen expressed by the nucleic acid sequence of SEQ ID NO:1, or an oligonucleotide encoding an immune response stimulating antigen expressed by a nucleic acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO:1. In still other embodiments, the rAd vectors of the present disclosure contain at least 80%, 90%, or 100% homologous sequences of the spike protein portion of the SARS-CoV-2 virus of WA1 / 2020, B.1.1.7, B.1.351, B.1.1.28, P.1, B.1.427, B.1.526, B.1.526.1, B1.525, P2, B1.617, B1.617.1, B1.617.2, B1.617.3, B1.429, or a B.1.429 variant. and at least one oligonucleotide encoding an immune response stimulating antigen expressed by a nucleic acid sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity.

[0111] In yet other embodiments, the immune response stimulating antigen encoded by at least one oligonucleotide in the rAd vector has the protein sequence of SEQ ID NO:2. In still other embodiments, the immune response-stimulating antigen encoded by at least one oligonucleotide in the rAd vector has a protein sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO:2. In yet other embodiments, the immune response-stimulating antigen encoded by at least one oligonucleotide in the rAd vector is a variant of WA1 / 2020, B.1.1.7, B.1.351, B.1.1.28, P.1, B.1.427, B.1.526, B.1.526.1, B.1.525, P.2, B.1.617, B.1.617.1, B1.617.2, B1.617.3, B1.429, or B.1.429. The antibody has 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the spike protein of the SARS-CoV-2 virus of the HIV-1 strain.

[0112] In some embodiments, the recombinant adenovirus comprises (i) at least one oligonucleotide encoding influenza A or B hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof, and (ii) at least one oligonucleotide encoding the S protein of SARS-CoV-2 virus or an immunogenic portion, variant, mutant, or fragment thereof. In yet other embodiments, the recombinant adenovirus comprises (i) at least two oligonucleotides encoding influenza A or B hemagglutinin or immunogenic portions, variants, mutants, or fragments thereof, and (ii) at least one oligonucleotide encoding two different S proteins of the SARS-CoV-2 virus or immunogenic portions, variants, mutants, or fragments thereof.

[0113] However, in some embodiments, the adenovirus is selected from the group consisting of: (i) a first-, second-, third-, or fourth-generation human adenovirus 5 (see Figures 3-7, respectively); (ii) an adenovirus of a rare serotype; (iii) a non-human adenovirus; and (iv) a combination thereof.

[0114] In some embodiments, the recombinant adenovirus is adapted to transfect host cells via intramuscular, intranasal, or inhalation routes.

[0115] In one particular embodiment, the first oligonucleotide encodes at least one of the 18 hemagglutinin (HA) subtypes 1-18, or an immunogenic portion, variant, mutant, or fragment thereof. In another embodiment, the second oligonucleotide encodes at least one of the ten neuraminidase (NA) subtypes 1-10, or an immunogenic portion, variant, mutant or fragment thereof. In yet another embodiment, the third oligonucleotide encodes B / YAMAGATA / 16 / 88 HA or an immunogenic portion, variant, mutant, or fragment thereof. In yet another embodiment, the fourth oligonucleotide encodes B / Victoria / 2 / 87 HA or an immunogenic portion, variant, mutant, or fragment thereof. In a further embodiment, the fifth oligonucleotide encodes the S protein of one of the SARS-CoV-2 variants B.1.1.7, B.1.351, B1.1.28-P.1, B.1.617.2, B.1.1.529, BA.4, BA.5, BQ1.1, XBB.1.5, BA.2.75.2, and immunogenic portions, variants, mutants, or fragments thereof. In yet another embodiment, the sixth oligonucleotide encodes the S protein of a different SARS-CoV-2 variant than the fifth oligonucleotide.

[0116] In yet other embodiments, the recombinant adenovirus comprises at least three different oligonucleotides encoding three different immune response stimulating antigens, or is trivalent (ie, tricistronic). In other embodiments, the recombinant adenovirus comprises at least four different oligonucleotides, or is tetravalent (ie, quadricistronic). In yet other embodiments, the recombinant adenovirus comprises at least five different oligonucleotides, or is pentavalent (ie, pentacistronic). In a further embodiment, the recombinant adenovirus comprises at least six different oligonucleotides, or is hexavalent (ie, hexacistronic).

[0117] Exemplary adenoviruses useful in the present disclosure include, but are not limited to, first-, second-, third-, or fourth-generation human adenoviruses 5 (Figures 3-7, respectively), rare serotype adenoviruses, non-human adenoviruses, and combinations thereof. Examples of rare serotypes of adenovirus useful in the present invention include, but are not limited to, Ad11, Ad26, Ad35, Ad48, Ad49, Ad50, and combinations thereof. Exemplary non-human adenoviruses useful in the present disclosure include, but are not limited to, simian Ad36, bovine Ad3, canine Ad2, porcine Ad3, and combinations thereof. In one particular embodiment, the non-human adenovirus comprises simian Ad36.

[0118] In yet other embodiments, the recombinant adenovirus further comprises 5' and 3' inverted repeats (ITRs) and a packaging signal (ψ).

[0119] In yet other embodiments, the recombinant adenovirus further comprises (i) a promoter, (ii) an enhancer, (iii) a polyadenylation moiety, (iv) an internal ribosome entry site (IRES), (v) a self-cleaving protein site, or (vi) a combination thereof. In some embodiments, the self-cleaving protein site comprises T2A, P2A, E2A, F2A, or a combination thereof. In yet other embodiments, the polyadenylation moiety comprises a simian virus 40 (SV40) polyadenylation (polyA) moiety, a bovine growth hormone (bGH) polyA moiety, or a combination thereof.

[0120] In further embodiments, the recombinant adenovirus further comprises a cytomegalovirus (CMV) promoter or enhancer, elongation factor 1a (EF1a), chicken beta actin (CBA) promoter, CAG promoter, or a combination thereof.

[0121] In another embodiment of the present disclosure, a plasmid is provided comprising an adenoviral genome modified to contain a transgene operably linked to an expression control sequence that directs transcription, translation, and / or expression in a host cell. Typically, the transgene comprises at least two different oligonucleotides encoding antigens (e.g., antigenic proteins) that stimulate an immune response. Each of the oligonucleotides encoding antigens that stimulate an immune response is different and independently selected from the group consisting of: (i) an oligonucleotide encoding influenza A virus hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof; (ii) an oligonucleotide encoding influenza A virus neuraminidase or an immunogenic portion, variant, mutant, or fragment thereof; (iii) an oligonucleotide encoding a hemagglutinin of a first influenza B virus or an immunogenic portion, variant, mutant, or fragment thereof; (iv) an oligonucleotide encoding a hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof of a second influenza B virus, wherein the second influenza B virus is a different strain from the first influenza B virus; (v) an oligonucleotide encoding the spike protein (S protein) of the first SARS-CoV-2 virus or an immunogenic portion, variant, mutant, or fragment thereof; and (vi) An oligonucleotide encoding an S protein or an immunogenic portion, variant, mutant, or fragment thereof of a second SARS-CoV-2 virus, wherein the second SARS-CoV-2 virus is a different strain from the first SARS-CoV-2 virus.

[0122] In some embodiments, the genome is derived from an adenovirus selected from the group consisting of: (i) a first, second, third, or fourth generation human adenovirus 5, (ii) an adenovirus of a rare serotype, (iii) a non-human adenovirus, and (iv) a combination thereof. In a specific embodiment, the adenovirus is a non-human adenovirus.

[0123] Exemplary non-human adenoviruses that may be used include, but are not limited to, simian Ad36, bovine Ad3, canine Ad2, porcine Ad3, or combinations thereof. In one particular embodiment, the non-human adenovirus comprises simian Ad36. In another embodiment, the genome of simian Ad36 is deleted for its native E1 locus and, optionally, for its E3 or E3B locus.

[0124] In yet other embodiments, at least one of the oligonucleotides encodes at least one of the 18 hemagglutinin (HA) subtypes 1-18, or an immunogenic portion, variant, mutant, or fragment thereof. In yet other embodiments, at least one of the oligonucleotides encodes at least one of the ten neuraminidase (NA) subtypes 1-10, or an immunogenic portion, variant, mutant, or fragment thereof. In other embodiments, at least one of the oligonucleotides encodes B / YAMAGATA / 16 / 88 HA or an immunogenic portion, variant, mutant, or fragment thereof. In another embodiment, at least one of the oligonucleotides encodes B / Victoria / 2 / 87 HA or an immunogenic portion, variant, mutant, or fragment thereof. In yet other embodiments, at least one of the oligonucleotides encodes the S protein of SARS-CoV-2 variants B.1.1.7, B.1.351, B1.1.28-P.1, B.1.617.2, B.1.1.529, BA.4, BA.5, BQ1.1, XBB.1.5, BA.2.75.2, and immunogenic portions, variants, mutants, or fragments thereof. In yet other embodiments, at least one of the oligonucleotides encodes the S protein of a different SARS-CoV-2 variant.

[0125] The plasmid can contain oligonucleotides encoding at least 3, 4, 5, or 6 different antigens.

[0126] In yet another aspect of the present disclosure, there is provided a recombinant adenovirus (rAd) vector comprising an adenovirus genome modified to contain at least two different exogenous oligonucleotides operably linked to expression control sequences that direct transcription, translation, and / or expression in a host cell, and each of the exogenous oligonucleotides is independently selected from the group consisting of: (i) an oligonucleotide encoding influenza A virus hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof; (ii) an oligonucleotide encoding influenza A virus neuraminidase or an immunogenic portion, variant, mutant, or fragment thereof; (iii) an oligonucleotide encoding a hemagglutinin of a first influenza B virus or an immunogenic portion, variant, mutant, or fragment thereof; (iv) an oligonucleotide encoding a hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof of a second influenza B virus, wherein the second influenza B virus is a different strain from the first influenza B virus; (v) an oligonucleotide encoding the spike protein (S protein) of the first SARS-CoV-2 virus or an immunogenic portion, variant, mutant, or fragment thereof; and (vi) An oligonucleotide encoding an S protein or an immunogenic portion, variant, mutant, or fragment thereof of a second SARS-CoV-2 virus, wherein the second SARS-CoV-2 virus is a different strain from the first SARS-CoV-2 virus.

[0127] In some embodiments, the adenovirus is selected from the group consisting of: (i) a first, second, third, or fourth generation human adenovirus 5; (ii) an adenovirus of a rare serotype; (iii) a non-human adenovirus; and (iv) a combination thereof.

[0128] In yet another embodiment, the rAd vector further comprises the naturally occurring adenovirus major serotype capsid proteins (i.e., hexon, penton base, and fiber) and four minor proteins (i.e., IIIa, VI, VIII, and IX).

[0129] In some embodiments, the rAd vectors are suitable for transfecting host cells via intramuscular, intranasal, or inhalation routes.

[0130] In still other embodiments, the non-human adenovirus comprises simian Ad36, bovine Ad3, canine Ad2, porcine Ad3, or a combination thereof. In one specific embodiment, the non-human adenovirus comprises simian Ad36.

[0131] In a further embodiment, the rAd vector comprises 5' and 3' inverted repeats (ITRs) and a packaging signal (ψ).

[0132] In yet other embodiments, the rAd vector further comprises (i) a promoter, (ii) an enhancer, (iii) a polyadenylation moiety, (iv) an internal ribosome entry site (IRES), (v) a self-cleaving protein site, or (vi) a combination thereof. In one particular embodiment, the self-cleaving protein site comprises T2A, P2A, E2A, F2A, or a combination thereof. In another embodiment, the polyadenylation moiety comprises a simian virus 40 (SV40) polyadenylation (polyA) moiety, a bovine growth hormone (bGH) polyA moiety, or a combination thereof.

[0133] In yet other embodiments, the rAd vector further comprises a cytomegalovirus (CMV) promoter or enhancer, elongation factor 1a (EF1a), chicken beta actin (CBA) promoter, CAG promoter, or a combination thereof.

[0134] In other embodiments, at least one of the oligonucleotides of the rAd vector encodes at least one of the 18 hemagglutinin (HA) subtypes 1-18, or an immunogenic portion, variant, mutant, or fragment thereof. In yet other embodiments, at least one of the oligonucleotides of the rAd vector encodes at least one of the ten neuraminidase (NA) subtypes 1-10, or an immunogenic portion, variant, mutant, or fragment thereof. In yet other embodiments, at least one of the oligonucleotides of the rAd vector encodes B / YAMAGATA / 16 / 88 HA or an immunogenic portion, variant, mutant, or fragment thereof. In a further embodiment, at least one of the oligonucleotides of the rAd vector encodes B / Victoria / 2 / 87 HA or an immunogenic portion, variant, mutant, or fragment thereof. In yet other embodiments, at least one of the oligonucleotides of the rAd vector encodes the S protein of SARS-CoV-2 variants B.1.1.7, B.1.351, B1.1.28-P.1, B.1.617.2, B.1.1.529, BA.4, BA.5, BQ1.1, XBB.1.5, BA.2.75.2, and immunogenic portions, variants, mutants, or fragments thereof. In yet other embodiments, at least one of the oligonucleotides of the rAd vector encodes the S protein of a different strain of the SARS-CoV-2 virus.

[0135] In further embodiments, the recombinant adenovirus comprises at least three, at least four, at least five, or at least six different oligonucleotides encoding immunostimulatory antigens.

[0136] In one particular embodiment, the rare serotype adenovirus comprises Ad11, Ad26, Ad35, Ad48, Ad49, Ad50, or a combination thereof.

[0137] In a further aspect of the present disclosure, there is provided a pharmaceutical composition comprising any one of the recombinant adenoviral (rAd) vectors disclosed herein.

[0138] Yet another aspect of the present disclosure provides a method of administering an influenza or SARS-CoV-2 virus vaccine, wherein the vaccine is a multivalent vaccine comprising at least two different foreign oligonucleotides, the method comprising administering a recombinant adenovirus (rAd) vector to a subject in need of such a vaccine, wherein the rAd vector comprises an adenovirus genome, and the adenovirus genome has been modified to comprise at least two different foreign oligonucleotides operably linked to an expression control sequence that directs transcription, translation, and / or expression in a host cell, and wherein each of the foreign oligonucleotides is independently selected from the group consisting of: Each exogenous oligonucleotide is independently selected from the group consisting of: (i) an oligonucleotide encoding influenza A virus hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof; (ii) an oligonucleotide encoding influenza A virus neuraminidase or an immunogenic portion, variant, mutant, or fragment thereof; (iii) an oligonucleotide encoding a hemagglutinin of a first influenza B virus or an immunogenic portion, variant, mutant, or fragment thereof; (iv) an oligonucleotide encoding a hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof of a second influenza B virus, wherein the second influenza B virus is a different strain from the first influenza B virus; (v) an oligonucleotide encoding the spike (S) protein of the first SARS-CoV-2 virus or an immunogenic portion, variant, mutant, or fragment thereof; and (vi) An oligonucleotide encoding an S protein or an immunogenic portion, variant, mutant, or fragment thereof of a second SARS-CoV-2 virus, wherein the second SARS-CoV-2 virus is a different strain from the first SARS-CoV-2 virus.

[0139] In some embodiments, the vaccine is administered to the subject via an intramuscular, intranasal, or inhalation route. In yet other embodiments, the vaccine is a bivalent or bicistronic vaccine. In yet other embodiments, the vaccine is a trivalent or tricistronic vaccine. In further embodiments, the vaccine is a tetravalent or quadricistronic vaccine. In another embodiment, the vaccine is a pentavalent or pentacistronic vaccine. In yet another embodiment, the vaccine is a hexavalent or hexacistronic vaccine.

[0140] In yet other embodiments, the method uses an adenovirus selected from the group consisting of: (i) a first, second, third, or fourth generation human adenovirus 5; (ii) an adenovirus of a rare serotype; (iii) a non-human adenovirus; and (iv) a combination thereof. In some embodiments, the methods use a non-human adenovirus, including simian Ad36, bovine Ad3, canine Ad2, porcine Ad3, or a combination thereof. In one particular embodiment, the method uses simian Ad36.

[0141] Particular oligonucleotides that can be used in the compositions of the present disclosure include any prevalent variant sequences of influenza HA, NA proteins obtained from global surveillance agencies such as WHO and CDC. Generally, there are four types of influenza viruses: A, B, C, and D. Influenza A and B viruses typically cause seasonal (flu season) epidemics each winter in the United States. Of the four types of influenza viruses, only influenza A viruses cause influenza pandemics, which occur when different influenza A viruses spread efficiently among people with little or no immunity. In general, influenza B viruses change their genetic and antigenic characteristics more slowly than influenza A viruses. Influenza C viruses generally cause mild disease and are not thought to cause epidemics in humans. Influenza D viruses primarily infect cattle and are not known to infect humans.

[0142] In one particular embodiment, the composition of the present disclosure is a tetravalent influenza vaccine that uses antigens targeting four influenza strains (two type A (H1N1 and H3N2) and two type B (Victoria and Yamagata lineages)) that cause the majority of influenza cases worldwide.

[0143] Influenza virus: Influenza A viruses are classified into subtypes based on two proteins on the surface of the virus, hemagglutinin (HA) and neuraminidase (NA). See Figure 1 for an example. There are 18 HA subtypes (H1-H18) and 10 NA subtypes (N1-N10), with over 130 influenza A subtype combinations identified in nature. Currently, A(H1N1) and A(H3N2) are two subtypes of influenza A viruses that circulate regularly among humans (seasonal influenza). Influenza A subtypes can be further divided into different genetic clades and subclades based on the similarity of their HA gene sequences, helping influenza experts track the proportion of viruses of different clades circulating. Clades and subclades emerge as a result of the high mutation rate of influenza A viruses, potentially producing antigenically distinct viruses. This means that the host immune response generated by infection or vaccination against one type A virus will not neutralize antigenically distinct viruses and will not protect against other viruses. Therefore, laboratories around the world collect influenza samples, monitor antigenic drift of HA and NA in real time, and recommend the latest vaccine strains. Influenza B viruses are classified into two lineages, B / Yamagata and B / Victoria, rather than subtypes, and are further divided into specific clades and subclades. Influenza B viruses generally undergo slower genetic and antigenic changes than influenza A viruses. Recent surveillance data reveal that both influenza B viruses co-circulate in the United States and worldwide. However, the proportion of influenza B viruses belonging to each lineage varies by geographic location and season.

[0144] Based on World Health Organization (WHO) recommendations, the four main influenza candidate vaccine viruses (CVVs) currently in use (2022-2023) are: (i)A / Wisconsin / 588 / 2019 (H1N1) (ii) A / Darwin / 6 / 2021(H3N2) (iii)B / Austria / 1359417 / 2021 (B / Victoria strain) (iv)B / Phuket / 3073 / 2013 (B / Yamagata strain). Lists of prototype viruses for egg-grown, cell-culture-grown, and recombinant vaccines, as well as candidate vaccine viruses (CVVs) suitable for the production of human vaccines, are publicly available on the WHO website, and their sequences are available on the Global Initiative for Sharing Avian Influenza Data (GISAID).

[0145] SARS-CoV-2 Virus: The SARS-CoV-2 RNA genome is approximately 30,000 nucleotides long. Two-thirds of the genes encode nonstructural proteins that enable genome replication and viral RNA synthesis. The remaining one-third encodes structural proteins, such as the spike (S) (Figure 1), envelope, membrane, and nucleoprotein (NP), which form spherical viral particles, as well as accessory proteins that control cellular responses. The S protein forms homotrimeric spikes on the viral particle and binds to the cell surface receptor angiotensin-converting enzyme 2 (ACE2), facilitating coronavirus entry into human cells. The S proteins of SARS-CoV and SARS-CoV-2 are sequentially cleaved during the entry process to generate S1 and S2 fragments, and the S2 fragment is then further processed to generate the smaller S2' protein. The S1 protein contains the receptor-binding domain (RBD), and the S2 protein promotes membrane fusion. The structure of the soluble and stabilized prefusion form of the SARS-CoV-2 S protein has been elucidated by cryo-electron microscopy and reveals considerable similarity to the SARS-CoV S protein, which is recognized by potently neutralizing monoclonal antibodies and serves as a vaccine target.

[0146] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense, single-stranded RNA virus that was first isolated in late 2019 from patients with severe respiratory disease in Wuhan, China. SARS-CoV-2 is related to two other highly pathogenic respiratory viruses: SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). SARS-CoV-2 infection causes the clinical syndrome coronavirus 2019 (COVID-19), which can progress to respiratory failure and may present with cardiac pathology, gastrointestinal disease, coagulation disorders, or hyperinflammatory syndromes. The elderly, immunocompromised individuals, and individuals with certain comorbidities (e.g., obesity, diabetes, and hypertension) are at highest risk for death from COVID-19. SARS-CoV-2 has created a global emergency due to the rapid global spread of coronavirus disease 2019 (COVID-19). As of January 6, 2023, SARS-CoV-2 and its variants have infected more than 657 million people and killed 6.68 million, including 99.4 million cases and 1,082,265 deaths in the United States alone (covid19.who.int). With early SARS-CoV-2 variants, approximately 15% of infected patients developed pneumonia, and approximately 5% developed more severe symptoms, such as acute respiratory distress syndrome and multiple organ failure. The Omicron variant (B.1.1.529) is significantly more infectious (2-5 times more contagious) than the Delta variant (B.1.617.2), yet its case fatality rate is 2-5 times lower, significantly reducing adult morbidity, hospitalization, and the need for supplemental oxygen. However, pediatric hospitalization rates were higher during the Omicron peak. Globally monitored SARS-CoV-2 sequences shared by the Global Initiative on Sharing Influenza Data (GISAID) allow for real-time tracking of SARS-CoV-2 mutations and new variants. Omicron is currently the most prevalent variant worldwide, accounting for over 98% of viral sequences shared by GISAID since February 2022.Since being designated a VOC by the WHO on November 26, 2021, viruses comprising the Omicron complex have continued to evolve, giving rise to descendant lineages with distinct genetic constellations of mutations (BA.1, BA.1.1, BA.2, BA.3, BA.4, and BA.5). The persistent transmission of these VOCs has led to significant evolution within them. In light of this, the WHO has added a new category, "Omicron Subvariants Under Surveillance," to its Variant Tracking System to alert public health authorities worldwide to VOC lineages that may require priority attention and surveillance, including BF.7, BF.14, BQ.1, BQ.1.1, BA.2.75, BA4.6, XBB, and BA2.3.20. The primary purpose of this category is to investigate whether these lineages may pose an additional threat to global public health compared to other circulating viruses.

[0147] The rapid spread and prolonged nature of the COVID-19 pandemic and its associated morbidity, mortality, and destabilizing socioeconomic impacts have made the development and deployment of a SARS-CoV-2 vaccine an urgent global health priority. While multiple vaccines against SARS-CoV-2 are available, including mRNA, adenovirus, and protein-based vaccines, the rate of viral evolution and transmission remains high in the United States and around the world.

[0148] Most currently available vaccines, such as Johnson & Johnson's Ad26.COV2 and AstraZeneca's ChAdOx1 nCoV-19 adenoviral platforms, are administered intramuscularly. Therefore, questions remain about the ability of these vaccines to reduce both infection and severity, especially if they do not prevent or reduce upper respiratory tract infection. While many intramuscular vaccines prevent SARS-CoV-2-induced pneumonia in nonhuman primates, their efficacy against upper respiratory tract infection and possibly transmission remains unclear. For example, many intramuscularly (IM) administered vaccines have shown variable protection against upper respiratory tract infection and transmission in preclinical studies and have failed to induce substantial mucosal (immunoglobulin A [IgA]) immunity. This is important because more transmissible SARS-CoV-2 variants have substitutions in the spike protein that reduce the neutralizing effect of vaccine-induced sera. In addition to the negative impact on protection, reduced immunity to variants, combined with naturally declining levels of anti-S IgG in the respiratory mucosa, further selects for vaccine resistance in the upper respiratory tract and increases infection in the general population.

[0149] The spike (S) protein of the SARS-CoV-2 virus particle binds to the cell surface receptor angiotensin-converting enzyme 2 (ACE2) and facilitates the entry of the coronavirus into human cells. Because the S protein is essential for viral entry, it is a target for vaccine development and therapeutic antibody intervention. The SARS-CoV-2 S protein is cleaved to generate S1 and S2 fragments, and then S2 is further processed to generate the smaller S20 protein. The S1 protein contains the receptor-binding domain (RBD), and the S2 protein promotes membrane fusion. The spike protein in vaccines is stabilized in its pre-fusion form by the S-2P or hexapro mutation. A prefusion-stabilized form of the SARS-CoV-2 S protein, displaying the RBD in the "up" position (exposing the RBD to immune surveillance), is recognized by potent neutralizing monoclonal antibodies or protein inhibitors, resulting in strong systemic and mucosal immunity. The destabilized SARS-CoV-2 S protein prematurely refolds into a postfusion conformation, impairing immunogenicity.

[0150] The nucleotide sequence of the surface glycoprotein or spike (S) protein of the Wuhan-Hu-1 SARS-CoV-2 virus, along with the corresponding protein sequence, are disclosed in SEQ ID NO: 1 (accession code: YP_009724390.1) and SEQ ID NO: 2, respectively. Below are examples of mutations in the SARS-CoV-2 virus compared to the Wuhan-Hu-1 SARS-CoV02 virus. Alpha variant (B.1.1.7 - UK origin) S1 domain: deletions H69-V70, Y144, A570D, D614G, and P681H RBD:N501Y S2 domain: T716I, S982A, D1118H Beta variant (B.1.351 - South African origin) S1 domain: L18F, D80A, D215G, deletions L242-L244, D614G RBD: K417N,E484K,N501Y S2 domain: A701V Delta variant (B.1.617 - Indian origin) S1 domain: T19R, T95I, G142D, E156G, deletion (Δ) F157-R158, D614G, P681R RBD: L452R,T478K S2 Domain: D950N Omicron BA.1 variant (B.1.529 - BA.1) S1 domain: A67V,deletion(Δ)H69-V70,T95I,G142D,ΔV143-Y145,ΔN211,L212I,ins214EPE,T547K, D614G,H655Y,N679K,P681H RBD: G339D,S371L,S373P,S375F,K417N,N440K,G446S,S477N,T478K,E484A,Q493R,Q498R,N501Y,Y505H S2 domain: N764K, D796Y, N856K, Q954H, N969K, L981F Omicron BA.4 / BA.5 variant (B.1.529 - BA.4 / BA.5) S1 domain:T19I,deletion(Δ)L24-P26,A27S,ΔH69+V70,G142D, V213G,D614G,H655Y,N679K,P681H RBD: G339D,S371F,S373P,S375F,T376A,D405N,R408S,K417N,N440K,L452R,S477N,T478K,E484A,F486V,Q498R,N501Y,Y505H S2 domain: N764K, D796Y, Q954H, N969K

[0151] The vectors of the present invention include appropriate sequences operably linked to a coding sequence or ORF to promote expression of two or more immune response-stimulating antigens in a target host cell or subject. As used herein, the term "subject" refers to mammals such as Homo sapiens, dogs, cats, monkeys, horses, cows, pigs, etc. Typically, the subject is a human. "Operably linked" sequences include both expression control sequences, such as a promoter, that are contiguous with a coding sequence, and expression control sequences that act in trans or distally to control expression of a polypeptide product.

[0152] Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if necessary, sequences that enhance protein processing and / or secretion. A variety of expression control sequences, including native and non-native, constitutive, inducible and / or tissue-specific, are known in the art and can be utilized herein depending on the type of expression desired.

[0153] Eukaryotic expression control sequences typically include a promoter, an enhancer (such as those derived from immunoglobulin genes, SV40, or CMV), and a polyadenylation sequence, which may contain splice donor and acceptor sites. Polyadenylation sequences are usually inserted 3' to the coding sequence and 5' to the 3'-ITR sequence. Poly A sequences such as those derived from bovine growth hormone are sometimes used.

[0154] The promoter can be selected from a number of constitutive or inducible promoters capable of driving expression of the selected oligonucleotide.

[0155] The rAds used in this disclosure can be constructed and manufactured using materials and methods described herein and those known in the art. The methods typically used to produce any of the constructs of the present disclosure are conventional and include genetic engineering, recombinant engineering, and synthetic techniques readily understood by those of skill in the art.

[0156] The present disclosure also provides a pharmaceutical composition. The pharmaceutical composition comprises the adenovirus composition of the present disclosure as an active ingredient and at least one pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be a diluent, binder, filler, buffer, pH adjuster, disintegrant, dispersant, preservative, lubricant, taste masking agent, flavoring agent, or coloring agent. The amounts and types of excipients utilized to form a pharmaceutical composition can be selected according to known principles of pharmacy.

[0157] In one embodiment, the excipient may be a diluent. Non-limiting examples of suitable compressible diluents include microcrystalline cellulose (MCC), cellulose derivatives, cellulose powder, cellulose esters (i.e., mixed esters of acetate and butyrate), ethyl cellulose, methyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, corn starch, phosphorylated corn starch, pregelatinized corn starch, rice starch, potato starch, tapioca starch, starch-lactose, starch-calcium carbonate, sodium starch glycolate, glucose, fructose, lactose, lactose monohydrate, sucrose, xylose, lactitol, mannitol, maltitol, sorbitol, xylitol, maltodextrin, trehalose, dibasic calcium phosphate (anhydrous or dihydrate), tribasic calcium phosphate, calcium carbonate, and magnesium carbonate.

[0158] In another embodiment, the excipient may be a binder. Suitable binders include starch, pregelatinized starch, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamide, polyvinyloxoazolidone, polyvinyl alcohol, C 12 -C 18 These include, but are not limited to, fatty acid alcohols, polyethylene glycols, polyols, sugars, oligosaccharides, polypeptides, oligopeptides, and combinations thereof.

[0159] In another embodiment, the excipient may be a filler.Suitable fillers include, but are not limited to, carbohydrates, inorganic compounds, and polyvinylpyrrolidone.Non-limiting examples of fillers include dibasic and tribasic calcium sulfate, starch, calcium carbonate, magnesium carbonate, microcrystalline cellulose, dibasic calcium phosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc, modified starch, lactose, sucrose, mannitol, or sorbitol.

[0160] In yet another embodiment, the excipient may be a buffer. Representative examples of suitable buffers include, but are not limited to, phosphate, carbonate, citrate, Tris buffer, and buffered saline (e.g., Tris-buffered saline, phosphate-buffered saline).

[0161] In various embodiments, the excipient may be a pH adjusting agent, such as, by way of non-limiting example, sodium carbonate, sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid.

[0162] In a further embodiment, the excipient may be a disintegrant. The disintegrant may be non-effervescent or effervescent. Suitable examples of non-effervescent disintegrants include, but are not limited to, starches (e.g., corn starch, potato starch, pregelatinized starch and modified starches thereof), sweeteners, clays (bentonite), microcrystalline cellulose, alginates, sodium starch glycolate, gums (e.g., agar, guar gum, locust bean gum, karaya gum, pesitin, tragacanth gum). Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid and sodium bicarbonate in combination with tartaric acid.

[0163] In yet another embodiment, the excipient may be a dispersant or dispersion enhancer. Suitable dispersants include, but are not limited to, starch, alginic acid, polyvinylpyrrolidone, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isocrystalline silicates, and microcrystalline cellulose.

[0164] In another alternative embodiment, the excipient may be a preservative. Non-limiting examples of suitable preservatives include antioxidants (e.g., BHA, BHT, vitamin A, vitamin C, vitamin E, or retinyl palmitate, citric acid, sodium citrate), chelating agents (e.g., EDTA or EGTA), and antimicrobial agents (e.g., parabens, chlorobutanol, phenol).

[0165] In further embodiments, the excipient may be a lubricant. Non-limiting examples of suitable lubricants include minerals (e.g., talc and silica), fats (e.g., vegetable stearin, magnesium stearate, or stearic acid).

[0166] In yet another embodiment, the excipient may be a taste-masking agent, including cellulose ethers, polyethylene glycol, polyvinyl alcohol, copolymers of polyvinyl alcohol and polyethylene glycol, monoglycerides or triglycerides, acrylic polymers, mixtures of acrylic polymers and cellulose ethers, cellulose acetate phthalate, and combinations thereof.

[0167] In another embodiment, the excipient may be a flavoring agent, which may be selected from synthetic flavor oils and flavoring aromatics and / or natural oils, extracts from plants, leaves, flowers, fruits, and combinations thereof.

[0168] The weight fraction of the excipient or combination of excipients in the composition can be about 99% or less, about 97% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2%, or about 1% or less of the total weight of the composition.

[0169] The agents and compositions described herein can be formulated by any conventional method using one or more pharmaceutically acceptable carriers or excipients, for example, as described in Remington's Pharmaceutical Sciences (A.R. Gennaro, ed.), 21st Edition, ISBN: 0781746736 (2005), the contents of which are incorporated herein by reference. Such formulations include a therapeutically effective amount of a bioactive agent described herein, optionally in purified form, together with a suitable amount of carrier to provide a form suitable for administration to a subject.

[0170] The term "formulation" refers to the preparation of a drug in a form suitable for administration to a subject, such as a human. Thus, a "formulation" may include pharmaceutically acceptable excipients, including diluents or carriers. As used herein, the term "pharmaceutically acceptable" refers to a substance or ingredient that does not cause an unacceptable loss of pharmacological activity or unacceptable side effects. Examples of pharmaceutically acceptable ingredients include those listed in monographs in the United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Rockville, MD, 2005 ("USP / NF"), or more recent editions, and those listed in the FDA's continuously updated Ingredient Search online database. Other useful ingredients not listed in the USP / NF, etc., may also be used. The term "pharmaceutically acceptable excipient" as used herein may include any solvent, dispersion medium, coating, antibacterial and antifungal agent, isotonic agent, or absorption delaying agent. The use of such media and agents for pharmaceutical active substances is well known in the art (see generally Remington's Pharmaceutical Sciences (A.R. Gennaro, ed.), 21st Edition, ISBN: 0781746736 (2005)). Except where a conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated.

[0171] A "stable" formulation or composition can refer to a composition that has sufficient stability to be stored at a convenient temperature, such as from about 0°C to about 60°C, for a commercially reasonable period of time, such as at least about 1 day, at least about 1 week, at least about 1 month, at least about 3 months, at least about 6 months, at least about 1 year, or at least about 2 years.

[0172] The formulation must be suitable for the method of administration. The agents used in the present disclosure can be formulated by known methods for administration to a subject using several routes, including, but not limited to, parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ocular, buccal, and rectal. Individual agents can also be administered in combination with one or more additional agents, or together with other biologically active or biologically inactive agents. Such biologically active or biologically inactive agents may be in fluid or mechanical communication with the agent, or may be attached to the agent by ionic, covalent, van der Waals, hydrophobic, hydrophilic, or other physical forces.

[0173] Most intramuscularly (IM) administered vaccines for the SARS-CoV-2 virus have shown variable protection against upper respiratory tract infection and transmission in preclinical studies and have failed to induce substantial mucosal (immunoglobulin A [IgA]) immunity. Thus, in some embodiments, the compositions of the present disclosure are delivered intranasally, as more transmissible SARS-CoV-2 variants have been shown to have substitutions in the spike protein that reduce the neutralizing effect of vaccine-induced sera. In addition to the negative impact on protection, reduced immunity to the variant, combined with a natural decline in anti-S IgG levels in the respiratory mucosa, could further select for vaccine resistance in the upper respiratory tract, leading to increased infection in the general population. As used herein, the term "composition," when referring to a composition of the present disclosure, refers to any composition, rAd, recombinant viral vector, plasmid, pharmaceutical composition, or any other material disclosed herein that comprises two or more oligonucleotides disclosed herein.

[0174] Formulations containing compositions for intranasal delivery may have a pH corresponding to a physiologically acidic nasal pH. The physiologically acidic nasal pH value may depend on the function of the natural nasal mucosa. The compositions may have a pH of about 6.5±0.5 (5.9-7.3) or about 6.7±0.6 (5.3-7.6). The compositions may have a pH of about 3.8-7.7 (mean ± SD 5.7±0.9). Compositions for nasal administration may be in the weakly acidic range. The average pH may have an acidity of 5.7.

[0175] Effective delivery of therapeutic drugs via nasal administration must consider the reduced transport rate across the protective mucus layer of the nasal mucosa, in addition to drug loss due to binding to glycoproteins in the mucus layer. Normal mucus is a viscoelastic, gel-like substance composed of water, electrolytes, mucins, polymers, and exfoliated epithelial cells. It primarily protects the underlying mucosal tissue and functions as a lubricant. Mucus is secreted by secretory cells randomly distributed in the nasal and other mucosal epithelia. The structural unit of mucus is mucin. This glycoprotein is primarily responsible for the viscoelastic properties of mucus, although other polymers may also contribute to these properties. These polymers in airway mucus include locally produced secretory IgA, IgM, IgE, lysozyme, and bronchial transferrin, which also play important roles in host defense mechanisms.

[0176] The coordinate administration methods of the present disclosure optionally incorporate effective mucolytic or mucus-clearing agents that act to break down, thin, or remove mucus from intranasal mucosal surfaces to facilitate absorption and / or adsorption of intranasally administered biological therapeutic agents. In these methods, the mucolytic or mucus-clearing agents are coordinately administered as adjunct compounds to enhance intranasal delivery of the biologically active agent. Alternatively, an effective amount of a mucolytic or mucus-clearing agent is incorporated as a processing agent within the multi-processing methods of the present disclosure or as an additive within the combination formulations of the present disclosure to provide improved formulations that enhance intranasal delivery of biotherapeutic compounds by reducing the barrier effect of intranasal mucus.

[0177] Various mucolytic or mucus-clearing agents can be incorporated into the methods and compositions of the present disclosure. Mucolytic and mucus-clearing agents can often be classified based on their mechanism of action into groups such as proteases (e.g., pronase, papain) that cleave the protein core of mucin glycoproteins, sulfhydryl compounds that split the disulfide bonds of mucoproteins, and detergents (e.g., Triton X-100, Tween 20) that disrupt non-covalent bonds within mucus. Additional compounds in this context include, but are not limited to, bile salts and detergents (e.g., sodium deoxycholate, sodium taurodeoxycholate, sodium glycocholate, lysophosphatidylcholine).

[0178] The effectiveness of bile salts in causing structural disruption of mucus is in the order of deoxycholic acid > taurocholic acid > glycocholic acid. Other effective agents that reduce mucus viscosity or adhesiveness to enhance intranasal delivery according to the methods of the present disclosure include, for example, short-chain fatty acids, and mucolytic agents that act by chelation, such as N-acyl collagen peptides, bile acids, and saponins (the latter of which inhibits Ca, which plays an important role in maintaining the mucus layer structure). 2+ and / or Mg 2+(which function in part by chelating

[0179] Additional mucolytic agents for use within the disclosed methods and compositions include N-acetyl-L-cysteine ​​(ACS), a potent mucolytic agent that reduces both the viscosity and adhesiveness of bronchopulmonary mucus and has been reported to modestly increase the nasal bioavailability of human growth hormone (7.5-12.2%) in anesthetized rats. These and other mucolytic or mucus-clearing agents, typically in the concentration range of about 0.2-20 mM, contact the nasal mucosa in coordination with the administration of a biologically active agent to reduce the viscosity and / or elasticity of nasal mucus.

[0180] Still other mucolytic or mucus-clearing agents can be selected from a range of glycosidase enzymes that can cleave glycosidic bonds within mucus glycoproteins; representative examples of this class of enzymes are α-amylase and β-amylase, although their mucolytic effect may be limited. In contrast, bacterial glycosidases allow these microorganisms to penetrate the host's mucus layer.

[0181] The composition can be formulated into various dosage forms and can be administered in various ways to deliver a therapeutically effective amount of the active ingredient. Such compositions can be administered orally (e.g., inhalation) or parenterally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles, as needed. Local administration can also include transdermal administration, such as transdermal patches and iontophoresis devices. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, or intrasternal injection or infusion techniques. Pharmaceutical formulations are described, for example, in Gennaro, AR, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (18th ed., 1995), and Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Dekker Inc., New York, NY (1980). In certain embodiments, the composition can be a food supplement, or the composition can be a cosmetic.

[0182] For parenteral administration (including subcutaneous, intraocular, intradermal, intravenous, intramuscular, intraarticular, and intraperitoneal), formulations may be aqueous or oily. Aqueous solutions may contain a sterile diluent, such as water, saline, or a pharmaceutically acceptable polyol, such as glycerol or propylene glycol, or other synthetic solvent; an antibacterial and / or antifungal agent, such as benzyl alcohol, methylparaben, chlorobutanol, phenol, or thimerosal; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate, or phosphate; and / or an agent for adjusting osmotic pressure, such as sodium chloride, dextrose, or a polyhydric alcohol, such as mannitol or sorbitol. The pH of aqueous solutions may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Oily solutions or suspensions may further contain sesame oil, peanut oil, olive oil, or mineral oil. The compositions may be presented in unit-dose or multi-dose containers, for example, sealed ampoules or vials, and may be stored in a freeze-line (lyophilized) condition requiring only the addition of sterile liquid, for example, water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0183] Generally, the adenovirus composition is administered in a safe and effective amount, for example, an amount that provides a desired therapeutic effect to a subject while minimizing undesirable side effects. In various embodiments, an effective amount of the adenovirus composition described herein can substantially reduce the infectivity of the virus in a subject suffering from a viral infection. In some embodiments, an effective amount is an amount that can treat a respiratory viral infection. In some embodiments, an effective amount is an amount that can treat one or more symptoms associated with a respiratory viral infection.

[0184] The amount of the compositions described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending on the host being treated and the particular method of administration. It will be understood by those skilled in the art that the unit content of drug contained in an individual dosage of each dosage form need not itself constitute a therapeutically effective amount, but rather the required therapeutically effective amount can be achieved by administering multiple individual dosages.

[0185] The dosage of an adenoviral vector depends primarily on factors such as the condition to be treated, the patient's age, weight, and health condition, and may vary from patient to patient. For example, a therapeutically effective dosage of a viral vector for an adult human or animal is generally about 1 x 10 6 ~approx. 1x10 15 Particles, approx. 1x10 7 ~1x10 13 particles, or approximately 1x10 9 ~1x10 12 The concentration of virus particles ranges from about 100 pL to about 100 mL of carrier. Dosage varies depending on the size of the animal and the route of administration. For example, a suitable human or veterinary dose for intramuscular injection (for an animal weighing approximately 80 kg) is about 1 x 10 per mL per single site. 9 ~about 5x10 12 In another example, for oral formulations, a suitable dose for humans or animals is about 1 x 10 11 ~approx. 1x10 15 The dosage ranges from 100 to 1200 micrograms. Those skilled in the art can adjust these dosages depending on the route of administration and the therapeutic or vaccine application for which the recombinant vector is used. The frequency of administration can be determined by monitoring the expression level of the transgene or, in the case of an immunogen, the circulating antibody level. Other methods for determining the frequency of administration will be readily apparent to those skilled in the art.

[0186] Optionally, the method steps include co-administering an appropriate amount of a short-acting immunomodulator to the patient simultaneously with, before, or after administration of the viral vector. The selected immunomodulator is defined herein as an agent capable of inhibiting the formation of neutralizing antibodies against the recombinant vector of the present disclosure or inhibiting cytolytic T lymphocyte (CTL) elimination of the vector. The immunomodulator may interfere with the interaction between T helper subsets (THi or T^) and B cells to inhibit the formation of neutralizing antibodies. Alternatively, the immunomodulator may inhibit the interaction between THI cells and CTL, reducing the occurrence of CTL elimination of the vector. Various useful immunomodulators and dosages for their use are disclosed, for example, in Yang et al., J. Virol., 70(9) (September, 1996); International Patent Application PCT / US96 / 03035, all of which are incorporated herein by reference.

[0187] The specific therapeutically effective dosage level for a particular subject will depend upon a variety of factors, including the disease being treated and its severity, the activity of the specific compound used, the particular composition used, the subject's age, weight, general health, sex, diet, time of administration, route of administration, rate of excretion of the composition used, duration of treatment, drugs used in combination with or concomitantly with the particular compound used, and similar factors well known in the medical arts (see, e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4th ed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill / Appleton & Lange, ISBN 0071375503). For example, it is within the skill of those skilled in the art to start administering a composition at a dosage lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.If necessary, the effective daily dosage can be divided into multiple doses according to the purpose of administration.Thus, a single-dose composition can contain that amount or a fraction thereof to constitute a daily dosage.However, it will be understood that the total daily usage amount of the compounds and compositions of the present disclosure will be determined by the attending physician within the scope of sound medical judgment.

[0188] The viral composition can be administered as a single dose or over a treatment period. For example, one or more nanoparticle compositions can be administered daily, weekly, biweekly, or monthly. For acute disease treatment, the treatment period typically lasts at least several days. Depending on the specific condition, the treatment period can last from several days to several weeks. For example, the treatment period can last one, two, or three weeks. For chronic disease, the treatment period can last from several weeks to several months, or even a year or more.

[0189] Treatment according to the methods described herein can be administered before, concurrently with, or after conventional therapy for respiratory viruses.

[0190] The present disclosure encompasses pharmaceutical compositions comprising the recombinant adenoviruses disclosed above, which facilitate administration and promote the stability of the recombinant adenoviruses. For example, the recombinant adenoviruses of the present disclosure can be mixed with at least one pharmaceutically acceptable carrier or excipient, resulting in a pharmaceutical composition that can be appropriately and effectively administered (given) to a living subject, such as a suitable subject (i.e., a "subject in need of treatment" or a "subject in need of treatment").

[0191] The present disclosure also provides kits. Such kits include a drug or recombinant adenovirus described herein and, in certain embodiments, instructions for administration. Such kits facilitate the implementation of the methods described herein. When provided as a kit, the different components of the composition are packaged in separate containers and can be mixed immediately before use. Components include, but are not limited to, compositions and pharmaceutical formulations containing the nanoparticle compositions or recombinant adenovirus described herein. When the components are packaged separately, they can be packaged in a package or dispenser device that can accommodate one or more unit dosage forms containing the composition, if desired. The package can be constructed of metal or plastic foil, such as a blister pack. Packaging the components separately in this manner can allow for long-term storage, in some cases without compromising the activity of the components.

[0192] The compositions and methods described herein that utilize molecular biology protocols can follow a variety of standard techniques known in the art (e.g., Sambrook and Russell (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, CP 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expression Purif.41(1),207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10:3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10:0954523253)

[0193] Certain embodiments disclosed herein may be further limited in the claims by using the phrase "consisting of" or "consisting essentially of" rather than the phrase "comprising." When used in a claim, whether as filed or added by amendment, the transitional term "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional term "consisting essentially of" limits the scope of the claimed invention to the specified materials or steps and those that do not materially affect the basic and novel characteristics. The disclosed embodiments so claimed are essentially or expressly described and enabled herein.

[0194] Because the materials and methods described above can be varied in many ways without departing from the scope of this disclosure, it is intended that all matter contained in the above description and the following examples be interpreted as illustrative and not in a limiting sense.

[0195] Additional objects, advantages, and novel features of the present disclosure will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting. In the examples, procedures as practiced constructively are described in the present tense, while procedures carried out in the laboratory are shown in the past tense. [Example]

[0196] Construction of ChAd vector expressing SARS-CoV-2 spike proteinThe simian Ad36 vector (ChAd) was obtained from the Penn Vector Core at the University of Pennsylvania. The ChAd genome was engineered by deleting the E1 and E3B regions (GenBank: FJ025917.1; nucleotides 455–3026 and 30072–31869, respectively). A modified human cytomegalovirus (CMV) major immediate-early promoter sequence was integrated into the complementary DNA strand in place of the E1 gene, in a counterclockwise orientation. The CMV modification included the addition of two copies of the Tet operator 2 (TetO2) sequence (T-REx system) (5'-TCT CTA TCA CTGATA GGG AGA TCT CTA TCA CTG ATA GG GA-3', SEQ ID NO: 4) inserted in tandem between the TATA box and mRNA start (GenBank: MN920393, nucleotides 174211–174212). The SARS-CoV-2 spike (S) glycoprotein gene (encoding a prefusion-stabilizing mutant with two proline substitutions at residues K986 and V987, which stabilize the prefusion form of S) was cloned into the unique PmeI site under the control of the CMV-tetO2 promoter in the pSAd36 genomic plasmid, generating pSAd36-S. In parallel, a pSAd36 control carrying an empty CMV-tetO2 cassette without any transgene was also generated. The pSAd36-S and pSAd control plasmids were then digested with PacI restriction enzyme and transfected with T-REx. TM The viral genome was liberated for transfection into 293 cells (Invitrogen). The rescued replication-incompetent ChAd-SARS-CoV-2-S and ChAd-Control vectors were scaled up to 293 cells and purified by CsCl density gradient ultracentrifugation. The viral particle concentration in each vector preparation was measured spectrophotometrically at 260 nm as described.

[0197] Construction of ChAd vector expressing SARS-CoV-2 spike geneConstruction: Figure 2 is a schematic diagram showing one specific configuration of a ChAd vector for expressing the SARS-CoV-2 spike protein. Similar constructs can be used to include one or more SARS-CoV-2 (S) protein genes in the rAd vectors of the present disclosure.

[0198] Adenoviral vectors for nasal / inhaled or intramuscular vaccines PCT Application International Publication No. WO 2021 / 247567, incorporated herein in its entirety, describes a single-dose intranasally administered chimpanzee adenovirus (simian Ad-36)-based SARS-CoV-2 vaccine (ChAd-SARS-CoV-2-S) encoding a prefusion-stabilized S protein, which induced robust humoral, cellular, and mucosal immune responses and suppressed upper and lower respiratory tract infection in K18-hACE2 transgenic mice, hamsters, and non-human primates. See also Hassan et al., 2021, Cell Reports, 36, 109452. This vaccine differs from ChAdOx1 nCoV-19, a chimpanzee Ad-23-based SARS-CoV-2 vaccine that has progressed to human clinical trials (BBV154, Clinical Trial NCT04751682) and has been authorized for emergency use in some countries. The potential utility, dose-response, durability, and cross-protective activity of ChAd-SARS-CoV-2-S in mice, including its effects on upper and lower respiratory tract infection, were examined. Approximately 9 months after IN immunization, levels of neutralizing antibodies and anti-S protein IgG and IgA remained high in the serum of ChAd-SARS-CoV-2-S-vaccinated animals, and it inhibited infection by SARS-CoV-2 strains displaying the B.1.351 and B.1.1.28 spike proteins. K18-hACE2 transgenic mice, which are highly susceptible to COVID-19, were completely protected from upper and lower respiratory tract infection after infection with SARS-CoV-2 viruses displaying the B.1.351 spike protein.

[0199] Some embodiments of the present disclosure utilize adenoviral vectors to deliver a tetravalent influenza antigen (HA from the most recent recommended vaccine strain) and a bivalent SARS-CoV-2 (spike protein antigen from the recommended SARS-CoV-2 strain) as a combination influenza-COVID vaccine.

[0200] Schematic diagrams of WT and first- to fourth-generation Ad5 used to produce the rAD vectors of the present disclosure are shown in Figures 3-7, and are referred to herein as first-, second-, third-, and fourth-generation Ad5, respectively. The maximum transgene payload capacity of first- to fourth-generation human Ad5 vectors is shown in Figure 8. Gutted vectors can also be used to significantly improve the antigen delivery capacity of adenoviral vectors. See also Danthinne et al., Gene Therapy, 2000, 7, pp. 707-1714.

[0201] The foregoing discussion of the present disclosure has been presented for purposes of illustration and description. It is not intended to limit the present disclosure to the form disclosed herein. While the description of the present disclosure includes a description of one or more embodiments and certain variations and modifications, other variations and modifications are also within the scope of the present disclosure, e.g., as would be within the skill and knowledge of one of ordinary skill in the art after understanding the present disclosure. To the extent permitted, the invention is intended to include alternative embodiments, including alternative, interchangeable, and / or equivalent structures, functions, ranges, or steps to those set forth in the claims, regardless of whether such alternative, interchangeable, and / or equivalent structures, functions, ranges, or steps are disclosed herein, and is not intended to publicly dedicate patentable subject matter. All references cited herein are incorporated by reference in their entirety.

[0202] Drawing Terminology Influenza Virus Hemagglutinin Neuraminidase Spike protein Transgene Poly A Stabilized Spike PRIOR ART Human Adenovirus 1st Generation 2nd Generation 3rd Generation 4th Generation Maximum transgene capacity First generation Second generation Gutted Vectors: Vectors in which most of the viral genes have been deleted (Gutted Vectors)

Claims

1. 1. A recombinant adenovirus adapted for use in preventing infection or transmission by, or reducing the severity of, influenza and / or SARS-CoV-2 virus in a subject, said recombinant adenovirus comprising oligonucleotides encoding antigens that stimulate an immune response in the subject, said recombinant adenovirus comprising at least two different respective oligonucleotides selected from the group consisting of: (i) an oligonucleotide encoding influenza A virus hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof; (ii) an oligonucleotide encoding influenza A virus neuraminidase or an immunogenic portion, variant, mutant, or fragment thereof; (iii) an oligonucleotide encoding a hemagglutinin of a first influenza B virus or an immunogenic portion, variant, mutant, or fragment thereof; (iv) an oligonucleotide encoding a hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof of a second influenza B virus, wherein the second influenza B virus is a different strain from the first influenza B virus; (v) an oligonucleotide encoding the spike protein (S protein) of the first SARS-CoV-2 virus or an immunogenic portion, variant, mutant, or fragment thereof; and (vi) an oligonucleotide encoding an S protein or an immunogenic portion, variant, mutant, or fragment thereof of a second SARS-CoV-2 virus, wherein the second SARS-CoV-2 virus is a different strain from the first SARS-CoV-2 virus; and wherein the adenovirus of the recombinant adenovirus is: (i) first, second, third, or fourth generation human adenovirus 5; (ii) adenoviruses of rare serotypes; (iii) a non-human adenovirus, and (iv) combinations thereof; selected from the group consisting of Recombinant adenovirus.

2. 2. The recombinant adenovirus of claim 1, wherein the recombinant adenovirus is adapted to be introduced into host cells via intramuscular, intranasal, or inhalation routes.

3. The recombinant adenovirus of claim 1 or 2, wherein at least one of the oligonucleotides encodes at least one of the 18 hemagglutinin (HA) subtypes 1 to 18, or an immunogenic portion, variant, mutant, or fragment thereof.

4. The recombinant adenovirus of any one of claims 1 to 3, wherein at least one of the oligonucleotides encodes at least one of the ten neuraminidase (NA) subtypes 1 to 10, or an immunogenic part, variant, mutant, or fragment thereof.

5. The recombinant adenovirus of any one of claims 1 to 4, wherein at least one of the oligonucleotides encodes B / YAMAGATA / 16 / 88 HA or an immunogenic part, variant, mutant, or fragment thereof.

6. The recombinant adenovirus of any one of claims 1 to 5, wherein at least one of the oligonucleotides encodes B / Victoria / 2 / 87 HA or an immunogenic part, variant, mutant, or fragment thereof.

7. 7. The recombinant adenovirus of any one of claims 1-6, wherein at least one of the oligonucleotides encodes the S protein of the first SARS-CoV-2 virus selected from the group consisting of SARS-CoV-2 variants B.1.1.7, B.1.351, B1.1.28-P.1, B.1.617.2, B.1.1.529, BA.4, BA.5, BQ1.1, XBB.1.5, BA.2.75.2, and immunogenic portions, variants, mutants, or fragments thereof.

8. The recombinant adenovirus of any one of claims 1 to 7, wherein at least one of the oligonucleotides encodes the S protein of the second SARS-CoV-2 virus.

9. The recombinant adenovirus according to any one of claims 1 to 8, wherein the recombinant adenovirus comprises at least three different oligonucleotides.

10. The recombinant adenovirus according to any one of claims 1 to 9, wherein the recombinant adenovirus comprises at least four different oligonucleotides.

11. The recombinant adenovirus according to any one of claims 1 to 10, wherein the recombinant adenovirus comprises at least five different oligonucleotides.

12. The recombinant adenovirus according to any one of claims 1 to 11, wherein the recombinant adenovirus comprises at least six different oligonucleotides.

13. The recombinant adenovirus of any one of claims 1 to 12, wherein the rare serotype adenovirus comprises Ad11, Ad26, Ad35, Ad48, Ad49, Ad50, or a combination thereof.

14. The recombinant adenovirus of any one of claims 1 to 13, wherein the non-human adenovirus comprises simian Ad36, bovine Ad3, canine Ad2, porcine Ad3, or a combination thereof.

15. The recombinant adenovirus according to any one of claims 1 to 14, wherein the non-human adenovirus comprises simian Ad36.

16. The recombinant adenovirus according to any one of claims 1 to 15, further comprising 5' and 3' inverted repeat sequences (ITRs) and a packaging signal (ψ).

17. The recombinant adenovirus according to any one of claims 1 to 16, further comprising (i) a promoter, (ii) an enhancer, (iii) a polyadenylation moiety, (iv) an internal ribosome entry site (IRES), (v) a self-cleaving protein site, or (vi) a combination thereof.

18. 18. The recombinant adenovirus of claim 17, wherein the self-cleaving protein site comprises T2A, P2A, E2A, F2A, or a combination thereof.

19. 19. The recombinant adenovirus of claim 17 or 18, wherein the polyadenylation portion comprises a simian virus 40 (SV40) polyadenylation (polyA) portion, a bovine growth hormone (bGH) polyA portion, or a combination thereof.

20. The recombinant adenovirus according to any one of claims 1 to 19, further comprising a cytomegalovirus (CMV) promoter or enhancer, elongation factor 1a (EF1a), chicken beta actin (CBA) promoter, CAG promoter, or a combination thereof.

21. A plasmid comprising an adenoviral genome, said adenoviral genome modified to contain a transgene operably linked to an expression control sequence that directs transcription, translation, and / or expression in a host cell, and said transgene comprising at least two different oligonucleotides, each of said oligonucleotides comprising: (i) an oligonucleotide encoding influenza A virus hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof; (ii) an oligonucleotide encoding influenza A virus neuraminidase or an immunogenic portion, variant, mutant, or fragment thereof; (iii) an oligonucleotide encoding a hemagglutinin of a first influenza B virus or an immunogenic portion, variant, mutant, or fragment thereof; (iv) an oligonucleotide encoding a hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof of a second influenza B virus, wherein the second influenza B virus is a different strain from the first influenza B virus; (v) an oligonucleotide encoding the spike protein (S protein) of the first SARS-CoV-2 virus or an immunogenic portion, variant, mutant, or fragment thereof; and (vi) an oligonucleotide encoding an S protein or an immunogenic portion, variant, mutant, or fragment thereof of a second SARS-CoV-2 virus, wherein the second SARS-CoV-2 virus is a different strain from the first SARS-CoV-2 virus; A plasmid independently selected from the group consisting of:

22. The genome comprises: (i) first, second, third, or fourth generation human adenovirus 5; (ii) adenoviruses of rare serotypes; (iii) a non-human adenovirus, and (iv) combinations thereof; 22. The plasmid of claim 21, derived from an adenovirus selected from the group consisting of:

23. 23. The plasmid of claim 21 or 22, wherein the adenovirus is a non-human adenovirus.

24. The plasmid of any one of claims 21 to 23, wherein the non-human adenovirus comprises simian Ad36, bovine Ad3, canine Ad2, porcine Ad3, or a combination thereof.

25. The plasmid of any one of claims 21 to 24, wherein the non-human adenovirus comprises simian Ad36.

26. 26. The plasmid of any one of claims 21 to 25, wherein the genome of simian Ad36 lacks the native E1 locus and optionally also the E3 or E3B locus.

27. 27. The plasmid of any one of claims 21 to 26, wherein at least one of the oligonucleotides encodes at least one of the 18 hemagglutinin (HA) subtypes 1 to 18, or an immunogenic portion, variant, mutant, or fragment thereof.

28. 28. The plasmid of any one of claims 21 to 27, wherein at least one of the oligonucleotides encodes at least one of the ten neuraminidase (NA) subtypes 1 to 10, or an immunogenic portion, variant, mutant, or fragment thereof.

29. 29. The plasmid of any one of claims 21 to 28, wherein at least one of the oligonucleotides encodes B / YAMAGATA / 16 / 88 HA or an immunogenic portion, variant, mutant, or fragment thereof.

30. 30. The plasmid of any one of claims 21 to 29, wherein at least one of the oligonucleotides encodes B / Victoria / 2 / 87 HA or an immunogenic portion, variant, mutant, or fragment thereof.

31. 31. The plasmid of any one of claims 21-30, wherein at least one of the oligonucleotides encodes the S protein of the first SARS-CoV-2 virus selected from the group consisting of SARS-CoV-2 variants B.1.1.7, B.1.351, B1.1.28-P.1, B.1.617.2, B.1.1.529, BA.4, BA.5, BQ1.1, XBB.1.5, BA.2.75.2, and immunogenic portions, variants, mutants, or fragments thereof.

32. 32. The plasmid of any one of claims 21 to 31, wherein at least one of the oligonucleotides encodes the S protein of the second SARS-CoV-2 virus.

33. The plasmid of any one of claims 21 to 32, wherein the plasmid comprises at least three different oligonucleotides.

34. The plasmid of any one of claims 21 to 33, wherein the plasmid comprises at least four different oligonucleotides.

35. The plasmid of any one of claims 21 to 34, wherein the plasmid comprises at least four different oligonucleotides.

36. The plasmid of any one of claims 21 to 35, wherein the plasmid comprises at least five different oligonucleotides.

37. The plasmid of any one of claims 21 to 36, wherein the plasmid comprises at least six different oligonucleotides.

38. A recombinant adenovirus (rAd) vector comprising an adenovirus genome modified to contain at least two different exogenous oligonucleotides operably linked to expression control sequences that direct transcription, translation, and / or expression in a host cell, and each of the exogenous oligonucleotides comprises: (i) an oligonucleotide encoding influenza A virus hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof; (ii) an oligonucleotide encoding influenza A virus neuraminidase or an immunogenic portion, variant, mutant, or fragment thereof; (iii) an oligonucleotide encoding a hemagglutinin of a first influenza B virus or an immunogenic portion, variant, mutant, or fragment thereof; (iv) an oligonucleotide encoding a hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof of a second influenza B virus, wherein the second influenza B virus is a different strain from the first influenza B virus; (v) an oligonucleotide encoding the spike protein (S protein) of the first SARS-CoV-2 virus or an immunogenic portion, variant, mutant, or fragment thereof; and (vi) an oligonucleotide encoding an S protein or an immunogenic portion, variant, mutant, or fragment thereof of a second SARS-CoV-2 virus, wherein the second SARS-CoV-2 virus is a different strain from the first SARS-CoV-2 virus; A recombinant adenoviral (rAd) vector independently selected from the group consisting of:

39. The recombinant adenovirus comprises the following: (i) first, second, third, or fourth generation human adenovirus 5; (ii) adenoviruses of rare serotypes; (iii) a non-human adenovirus, and (iv) combinations thereof; 39. The rAd vector of claim 38, selected from the group consisting of:

40. 40. The rAd vector of claim 38 or 39, further comprising naturally occurring adenovirus (Ad) major serotype capsid proteins (hexon, penton base, and fiber) and four minor proteins (IIIa, VI, VIII, and IX).

41. The rAd vector of any one of claims 38 to 40, wherein the rAd vector is adapted for introduction into host cells via intramuscular, intranasal, or inhalation routes.

42. 42. The rAd vector of any one of claims 38 to 41, wherein the non-human adenovirus comprises simian Ad36, bovine Ad3, canine Ad2, porcine Ad3, or a combination thereof.

43. 43. The rAd vector of any one of claims 38 to 42, wherein the non-human adenovirus comprises simian Ad36.

44. The rAd vector of any one of claims 38 to 43, wherein the rAd vector comprises 5' and 3' inverted repeats (ITRs) and a packaging signal (ψ).

45. 45. The rAd vector of any one of claims 38 to 44, wherein the rAd vector further comprises (i) a promoter, (ii) an enhancer, (iii) a polyadenylation moiety, (iv) an internal ribosome entry site (IRES), (v) a self-cleaving protein site, or (vi) a combination thereof.

46. 46. ​​The rAd vector of claim 45, wherein the self-cleaving protein site comprises T2A, P2A, E2A, F2A, or a combination thereof.

47. 47. The rAd vector of claim 45 or 46, wherein the polyadenylation moiety comprises a simian virus 40 (SV40) polyadenylation (polyA) moiety, a bovine growth hormone (bGH) polyA moiety, or a combination thereof.

48. 48. The rAd vector of any one of claims 38 to 47, further comprising a cytomegalovirus (CMV) promoter or enhancer, elongation factor 1a (EF1a), chicken beta actin (CBA) promoter, CAG promoter, or a combination thereof.

49. 49. The rAd vector of any one of claims 38-48, wherein at least one of the oligonucleotides encodes at least one of the 18 hemagglutinin (HA) subtypes 1-18, or an immunogenic portion, variant, mutant, or fragment thereof.

50. 50. The rAd vector of any one of claims 38-49, wherein at least one of the oligonucleotides encodes at least one of the ten neuraminidase (NA) subtypes 1-10, or an immunogenic portion, variant, mutant, or fragment thereof.

51. 51. The rAd vector of any one of claims 38-50, wherein at least one of the oligonucleotides encodes B / YAMAGATA / 16 / 88 HA or an immunogenic portion, variant, mutant, or fragment thereof.

52. 52. The rAd vector of any one of claims 38-51, wherein at least one of the oligonucleotides encodes B / Victoria / 2 / 87 HA or an immunogenic portion, variant, mutant, or fragment thereof.

53. 53. The rAd vector of any one of claims 38-52, wherein at least one of the oligonucleotides encodes the S protein of the first SARS-CoV-2 virus selected from the group consisting of SARS-CoV-2 variants B.1.1.7, B.1.351, B1.1.28-P.1, B.1.617.2, B.1.1.529, BA.4, BA.5, BQ1.1, XBB.1.5, BA.2.75.2, and immunogenic portions, variants, mutants, or fragments thereof.

54. 54. The rAd vector of any one of claims 38-53, wherein at least one of the oligonucleotides encodes the S protein of the second SARS-CoV-2 virus.

55. The rAd vector of any one of claims 38 to 54, wherein the recombinant adenovirus comprises at least three different oligonucleotides.

56. 56. The rAd vector of any one of claims 38 to 55, wherein the recombinant adenovirus comprises at least four different oligonucleotides.

57. 57. The rAd vector of any one of claims 38 to 56, wherein the recombinant adenovirus comprises at least four different oligonucleotides.

58. 58. The rAd vector of any one of claims 38 to 57, wherein the recombinant adenovirus comprises at least five different oligonucleotides.

59. 59. The rAd vector of any one of claims 38 to 58, wherein the recombinant adenovirus comprises at least six different oligonucleotides.

60. 60. The rAd vector of any one of claims 38 to 59, wherein the rare serotype adenovirus comprises Ad11, Ad26, Ad35, Ad48, Ad49, Ad50, or a combination thereof.

61. A pharmaceutical composition comprising the recombinant adenoviral (rAd) vector of any one of claims 38 to 60 and a pharmaceutically acceptable excipient.

62. 1. A method of administering an influenza or SARS-CoV-2 virus vaccine, the method comprising administering a therapeutically effective amount of a recombinant adenovirus (rAd) vector to a subject in need of such a vaccine, thereby eliciting an immune response in the subject, wherein the rAd vector comprises an adenovirus genome, and the adenovirus genome has been modified to include at least two different exogenous oligonucleotides operably linked to expression control sequences that direct transcription, translation, and / or expression in a host cell, and wherein each of the exogenous oligonucleotides comprises: (i) an oligonucleotide encoding influenza A virus hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof; (ii) an oligonucleotide encoding influenza A virus neuraminidase or an immunogenic portion, variant, mutant, or fragment thereof; (iii) an oligonucleotide encoding a hemagglutinin of a first influenza B virus or an immunogenic portion, variant, mutant, or fragment thereof; (iv) an oligonucleotide encoding a hemagglutinin or an immunogenic portion, variant, mutant, or fragment thereof of a second influenza B virus, wherein the second influenza B virus is a different strain from the first influenza B virus; (v) an oligonucleotide encoding the spike protein (S protein) of the first SARS-CoV-2 virus or an immunogenic portion, variant, mutant, or fragment thereof; and (vi) an oligonucleotide encoding an S protein or an immunogenic portion, variant, mutant, or fragment thereof of a second SARS-CoV-2 virus, wherein the second SARS-CoV-2 virus is a different strain from the first SARS-CoV-2 virus; independently selected from the group consisting of:

63. 63. The method of claim 62, wherein the vaccine is administered to the subject via an intramuscular, intranasal, or inhalation route.

64. 64. The method of claim 62 or 63, wherein the vaccine is a bivalent vaccine.

65. 65. The method of any one of claims 62 to 64, wherein the vaccine is a trivalent vaccine.

66. 66. The method of any one of claims 62 to 65, wherein the vaccine is a tetravalent vaccine.

67. 67. The method of any one of claims 62 to 66, wherein the vaccine is a pentavalent vaccine.

68. 68. The method of any one of claims 62 to 67, wherein the vaccine is a hexavalent vaccine.

69. The adenovirus of the rAd vector is: (i) first, second, third, or fourth generation human adenovirus 5; (ii) adenoviruses of rare serotypes; (iii) a non-human adenovirus, and (iv) combinations thereof; The method of any one of claims 62 to 68, selected from the group consisting of:

70. 70. The method of any one of claims 62 to 69, wherein the non-human adenovirus comprises simian Ad36, bovine Ad3, canine Ad2, porcine Ad3, or a combination thereof.

71. 71. The method of any one of claims 62 to 70, wherein the non-human adenovirus comprises simian Ad36.

72. 62. A method of reducing or preventing the incidence or reducing the severity of influenza or SARS-CoV-2 infection, said method comprising administering a therapeutically effective amount of any one of the recombinant adenoviruses of any one of claims 1-20, or any one of the recombinant adenoviral (rAd) vectors of any one of claims 38-60, or the pharmaceutical composition of claim 61.

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