Methods and compositions for universal influenza vaccination using an adenoviral vector

Abstract

An adenoviral vector with E1 and E3 regions removed and expressing a first nucleoprotein of an influenza A virus and / or a second nucleoprotein of an influenza B virus, with or without the presence of the Autophagy-Inducing Peptide C5 (AIP-C5) from the CFP10 protein of Mycobacterium tuberculosis, compositions comprising same, and methods of use for general vaccination against heterosubtypic influenza viruses.

Description

70855-02METHODS AND COMPOSITIONS FOR UNIVERSAL INFLUENZA VACCINATION USING AN ADENOVIRAL VECTORPRIORITY

[0001] This application claims the benefit of U.S. Provisional Application Number 63 / 715,784 filed on November 4, 2024, the entirety of which is hereby incorporated by reference herein.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under AI059374 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] This section introduces aspects that may help facilitate a better understanding of the disclosure. Accordingly, these statements are to be read in this light and are not to be understood as admissions about what is or is not prior art.

[0004] Seasonal influenza viruses, including A / H1N1 and A / H3N2 influenza A viruses (lAVs), as well as Yamagata lineage and Victoria lineage influenza B viruses (IBVs), cause around 3-5 billion cases resulting in 290,000 to 650,000 deaths annually. Influenza viruses are known for continuous antigenic changes due to the immune pressure and faulty genome replication system. This antigenic drift lowers the efficacy of seasonal influenza vaccines.

[0005] There are various conventional trivalent or quadrivalent seasonal influenza vaccines that target the immunodominant hypervariable epitopes in the head domain of hemagglutinin (HA), which can induce neutralizing antibodies and provide protection from influenza infection. The effectiveness of seasonal influenza vaccines primarily relies on the antigenic identity of the vaccine viral strains with the circulating strains. Vemula et al., Vaccine approaches conferring cross-protection against influenza viruses, Expert Review of Vaccines 16: 1141-1154 (2017). However, the antigenic mismatch between circulating and vaccine strains occurs frequently due to rapid viral evolution limiting the breadth of protection and vaccine efficacy. Yamayoshi & Kawaoka, Current and future influenza vaccines, Nature Medicine 25: 212-222 (2019); Liu et al., A universal dual mechanism immunotherapy for the treatment of influenza virus infections. Nature Communications 11: 5597 (2020). In addition, several zoonotic avian influenza viruses, e.g., A / H5N1, A / H7N7, A / H7N9, or A / H9N2 viruses, pose a significant pandemic risk since seasonal influenza vaccines often fail to protect against the distinct avian influenza viruses. Shi & Gao, Emerging H5N8 avian influenza viruses, Science 372(6544): 784-786 (2021); Watanabe et al., Characterization of H7N0 influenza A viruses isolated from humans, Nature 501: 551-55570855-02(2013); Bi et al.. The time is now: a call to contain H9N2 avian influenza viruses. The Lancet Microbe 3(11): e804-e805 (2022); Cohen, Worries about bird flu in U.S. cattle intensify, Science 384: 12-13 (2024). Therefore, there is a critical need to develop an effective universal influenza vaccine for the rapidly evolving seasonal and pandemic influenza viruses.

[0006] Influenza viruses continue to pose a significant threat to human health worldwide. Approximately one billion human infections, 3 to 5 million severe cases, and 300,000 to 500,000 deaths occur every year despite the availability of influenza vaccines. Influenza viruses are known for continuous antigenic changes due to the immune pressure and faulty genome replication system. This antigenic drift lowers the efficacy of seasonal influenza vaccines.

[0007] Besides seasonal influenza viruses (e.g., H1N1, H3N2, and influenza B), reports of human infections with either low or highly pathogenic avian influenza (HP Al) A viruses of H5, H7, and H9 subtypes underscore the public health threat and pandemic potential posed by these avian influenza viruses (AIV). Since their emergence in Asia over two decades ago, HP Al H5N1 viruses have spread to over sixty countries on three continents and are endemic among poultry in southeast Asia and Africa. Additionally, H9N2 infections are enzootic among poultry globally and sporadically infect humans, whereas both low and highly pathogenic AIVs of H7 subtype (e.g., H7N2, H7N3 and H7N7) continue to cause sporadic outbreaks. In 2013, a new AIV strain of the H7N9 subtype unexpectedly emerged in China and has since caused more than 1,568 human infections and 616 deaths as of 27 May 2021. Although human-to-human transmission has been limited, AIVs continue to produce variants. The genetic reassortment of the avian and human / porcine influenza viruses or gene mutations can result in virus replication in the upper respiratory tract of humans and generate novel pandemic influenza viruses as happened in the 2009 pandemic.

[0008] Antigenic drift in seasonal influenza viruses can substantially limit the duration of immunity conferred by infection or vaccination and is the reason influenza vaccine components are updated every year. The success of seasonal influenza vaccines is mainly dependent on the match between the vaccine constituents and the circulating strains, antigenic distance, attack rate and pre-existing antibodies. Antigenic shift, whether due to genomic reassortment between two or more influenza A viruses or adaptation of avian or swine influenza virus in humans, can lead to successful person-to-person transmission and, ultimately, an influenza pandemic. To address the issue of antigenic drift and antigenic shift in influenza A viruses, a universal influenza vaccine is needed.SUMMARY

[0009] Immunogenic compositions are provided. In certain embodiments, the immunogenic70855-02 compositions are bivalent or monovalent. In certain embodiments, the immunogenic compositions are polyvalent.

[0010] The immunogenic composition can comprise a nucleoprotein (NP) of an influenza A virus (IAV) and / or a NP of an influenza B virus (IBV), and a pharmaceutically acceptable carrier. Optionally, the immunogenic composition can further comprise at least 21 amino acid residues of an Autophagy-Inducing Peptide C5 (AIP-C5) from a CFP10 protein of Mycobacterium tuberculosis or a functional fragment thereof. The immunogenic composition can be cross- protective against at least one IAV and / or at least one IBVwhen administered to a subject. In certain embodiments, when administered to a subject, the immunogenic composition (e.g, bivalent immunogenic composition) confers general immunogenicity protection against the subtypes of viruses selected from the group consisting of Hl, H3, H5, H7, H9, B / Y amagata, and B / Victoria strains.

[0011] The AIP-C5 from a CFP10 protein of the immunogenic composition (e , bivalent immunogenic composition) can be or comprise SEQ ID NO: 9 (or at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 9). In certain embodiments, the AIP-C5 from a CFP 10 protein comprises at least 90% sequence identity to SEQ ID NO: 9.

[0012] The immunogenic composition can be formulated to be administered intranasally or orally (e.g, mucosally) or intramuscularly (e.g, systemically), for example. The composition can be formulated as an aerosol spray.

[0013] In certain embodiments, the immunogenic compositions comprise at least 80% sequence identity to SEQ ID NO: 6. and a pharmaceutically acceptable carrier, wherein the immunogenic composition is cross-protective against at least one IAV and at least one IBV when administered to a subj ect.

[0014] In certain embodiments, administration of the immunogenic compositions described herein to a subject induces CD8+T-cell responses cross-reactive to at least one influenza A subtype or / and at least one influenza B lineage.

[0015] Human, bovine, chimpanzee, or any other species adenoviral (Ad) vectors are also provided. In certain embodiments, the Ad vector comprises a polynucleotide sequence that encodes at least: a first NP from an IAV or a functional fragment thereof; and a second NP from an IBV or a functional fragment thereof. The polynucleotide sequence can further encode one or more immunogenic domains of an influenza virus other than a NP.

[0016] The polynucleotide sequence of the Ad vectors hereof can further encode AIP-C5 from the CFP 10 protein oiM. tuberculosis or a functional fragment thereof. The AIP-C5 can comprise 21 amino acid residues. In certain embodiments, the AIP-C5 has or comprises at least 80%70855-02 sequence identity to the amino sequence set forth in SEQ ID NO: 9.

[0017] In certain embodiments, the polynucleotide sequence has at least 80% sequence identify to the nucleic acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 7. In certain embodiments, the Ad vector further comprises one or more promoters.

[0018] The Ad vector can be replication-defective and suitable for expression in a mammalian cell. In certain embodiments, at least El and E3 regions are deleted from the Ad vector. In certain embodiments, the El region or the E3 region is deleted from the Ad vector. For example, and without limitation, the polynucleotide sequence of the first NP from an IAV or the functional fragment thereof can be inserted in the deleted El region of the Ad vector, and / or the second NP from an IBV or the functional fragment thereof can be inserted in the deleted El region of the Ad vector.

[0019] The Ad vector can be a bovine Ad (BAd). In certain embodiments, the Ad vector is a BAd type 3 (BAd3).

[0020] Isolated nucleic acid molecules are also provided. In certain embodiments, such an isolated nucleic acid molecule comprises sequences encoding: (a) an NP of an IAV; (b) an NP of an IBV; and (c) optionally, at least 21 amino acid residues of an AIP-C5 peptide from M. tuberculosis or a functional fragment thereof. The sequences of the isolated nucleic acid can be codon-optimized for mammalian expression.

[0021] In certain embodiments, the sequences of (a) (the NP of an IAV) and (b) (an NP of an IBV) are fused in-frame and separated by a linker peptide. The isolated nucleic acids hereof can have, for example, at least 80% sequence identify to SEQ ID NO: 5 or SEQ ID NO: 7.

[0022] Isolated polypeptides are also provided. In certain embodiments, the isolated polypeptide comprises (a) an NP sequence of an IAV; (b) an NP sequence of an IBV; and (c) optionally, a sequence encoding an AIP-C5 peptide fromM tuberculosis or a functional fragment thereof. Such isolated polypeptides can elicit cross-reactive T-cell responses against IAV and / or IBV when administered to a subject.

[0023] Methods of eliciting an immune response in a subject against an influenza virus using the compositions. Ad vectors, or isolated polypeptides hereof are also provided. A method of eliciting an immune response in a subject against an influenza virus, for example, can comprise administering to said subject an effective amount of an immunogenic composition of hereof, an Ad vector hereof, or an isolated polypeptide hereof. Said administration can be. for example, intranasal or oral. In certain embodiments, said composition, Ad vector, or isolated polypeptide is administered as an aerosol spray. In certain embodiments, administration is intramuscular, mucosal, or otherwise systemic.

[0024] The method can provide a general immunogenicity protection to the subject against70855-02 various subtypes of viruses selected from the group consisting of Hl, H3, H5, H7, H9, B / Yamagata. and B / Victoria strains. The immune response can comprise a dose-dependent increase in both humoral and cell-mediated immunity in the subject. The subject can be a human. The subject can be a mammal. The subject can be a bovine.

[0025] Methods of inducing cross-protective immune response against influenza A and influenza B viruses in a subject are also provided. In certain embodiments, such a method comprises administering to the subject an effective amount of an immunogenic composition described herein, an Ad vector described herein, or an isolated polypeptide described herein. Administration can be intranasal, intramuscular, mucosal, or oral. In certain embodiments, the composition, Ad vector, or isolated polypeptide is administered as an aerosol spray. The methods hereof can provide general immunogenicity protection to the subject against various subtypes of viruses selected from the group consisting of Hl, H3, H5, H7, H9, B / Y amagata, and B / Victoria strains. In certain embodiments, the immune response comprises a dose-dependent increase in both humoral and cell-mediated immunity in the subject. The immune response can be cross- protective immunity against at least IAV and IAB. In certain embodiments, administration of the composition, Ad vector, or isolated polypeptide results in reduction of viral load in the subject following heterologous influenza challenge of the subject.

[0026] For all methods of providing general immunogenicity protection or inducing cross- protective immune response, or otherwise eliciting an immune response described herein, the subject can be a mammal. The subject can be human.

[0027] Methods of producing an immunogenic composition - or manufacturing a medicament comprising an immunogenic composition - are also provided. In certain embodiments, such a method comprises: introducing a recombinant Ad vector into a host cell that can support replication of the Ad vector, wherein the Ad vector comprises any Ad vector described herein: and culturing the host cell in a culture medium under conditions that allow for the expression of the first and second NPs. The method can further comprise isolating or purify ing the expressed first and second NPs from the host cell or culture medium. In certain embodiments, the method is performed in vitro or in ovo.

[0028] Cell lines are also provided. In certain embodiments, the cell line is capable of being infected by an Ad vector described herein and / or comprises an isolated nucleic acid described herein, such that the infected cells of the cell line can replicate a genome of the Ad vector and / or express the isolated nucleic acid. In certain embodiments, cell lines are provided that comprise an isolated recombinant Ad vector hereof.

[0029] Cells are also provided that comprise an isolated recombinant Ad vector described herein, and / or an isolated nucleic acid described herein. Expression of the isolated nucleic acid by70855-02 the cell can, for example, result in production of a NP of an IAV and / or an IBV.

[0030] Immunogenic compositions for use in eliciting an immune response in a subject are also provided. The immune response can be against an influenza virus, for example. The immunogenic composition can be any of those described herein.BRIEF DESCRIPTION OF DRAWINGS

[0031] The disclosed embodiments and other features, advantages, and aspects contained herein, and the matter of attaining them, will become apparent in light of the following detailed description of various exemplary embodiments of the present disclosure. Such detailed description will be better understood when taken in conjunction with the accompanying drawings.

[0032] FIGS. 1A-1D comprises diagrammatic representations of adenoviral vector constructs and associated experimental protocols. In FIG. 1A, a diagram illustrates expression cassettes containing the nucleoprotein (NP) gene derived from influenza A, B, or a combination of A and B viruses. These cassettes incorporate an Autophagy -Inducing Peptide C5 (AIP-C5) and are utilized for generating bovine adenovirus type 3 (BAd3) or human adenovirus type 5 (HAd5) vectors. The resultant vectors, referred to as BAd or HAd vectors, are listed. Key components include CMV (cytomegalovirus), PA (bovine grow th hormone polyadenylation signal), and ITR (inverted terminal repeats). FIG. IB depicts the expression of C5-NP in each adenoviral vector as confirmed through immunoblotting analyses, where cell extracts infected with empty vectors serve as negative controls. FIG. 1C presents a schematic of an immunogenicity study involving 8-week-old BALB / c mice. The mice receive intranasal (i.n.) vaccination with the BAd or HAd vectors. Three weeks post-inoculation, the mice are euthanized, and samples from blood, lungs, spleen, and mediastinal lymph nodes (MLN) are collected to assess humoral and cell-mediated immune responses. FIG. ID outlines a protection study schematic, wherein mice vaccinated with BAd vectors are challenged with homosubtypic and heterosubtypic influenza viruses. The study monitors lung viral titer, weight loss, and survival rates to evaluate the efficacy of protection.

[0033] FIGS. 2A-2P and 3A-3P present graphical representations of NP-specific antibody response data in serum and lung washes for BALB / c mice subsequent to immunization with adenovirus-vectored vaccines described herein. In particular, 8-week-old BALB / c mice (n=5) were administered intranasally (i.n.) with 5 x 10 7 plaque-forming units (PFU) of BAd or HAd vectors as delineated in FIG. 1C. Sera and lung washes were harvested three weeks following immunization. Serum samples underwent ELISA analysis for NP / A-specific (FIGS. 2A-2D and 3A-3D) andNP / B-specific (FIGS. 2E-2H and 3E-3H) IgG, IgGl, IgG2a, and IgA titers. Similarly, lung washes were assessed via enzyme-linked immunosorbent assay (ELISA) for NP / A-specific (FIGS. 2I-2L and 3I-3L) and NP / B-specific (FIGS. 2M-2P and 3M-3P) IgG, IgGl, IgG2a, and IgA titers. The data key for FIGS. 2A-2P and 3A-3P is as follow s: 1 - BAd-AElE3; 2 - BAd-C5-70855-02NP / A; 3 - BAd-C5-NP / B; 4 - BAd Bivalent; 5 - BAd-C5-NP / A+B; 6 - HAd-AElE3; 7 - HAd- C5-NP / A; 8 - HAd-C5-NP / B; 9 - HAd Bivalent; 10 - HAd-C5-NP / A+B.

[0034] The ELISA data are represented as the area under the curve (AUC), w ith cut-off values determined by the average of blank wells. Each data point symbolizes an individual animal, with error depicted as the standard deviation (SD). Data analysis was conducted using two-way analysis of variance (ANOVA) accompanied by Dunnett’s post hoc test. Statistical significance for intravector comparison is indicated relative to the empty vector. Symbols denoting significance levels are as follows: * for p < 0.05; ** for p < 0.01; *** for p < 0.001; **** for p < 0.0001. For intervector comparison: # denotes significance at p < 0.05; ## at p < 0.01; and ### at p < 0.001. The term "bivalent" refers to the co-inoculation with vectors expressing C5-NP / A and C5-NP / B.

[0035] FIG. 4 illustrates the flow cytometry gating strategy for analyzing NP -specific cellular immune responses in BALB / c mice immunized with the adenoviral vector-based vaccines described herein.

[0036] FIGS. 5A-5L present graphical representations of NP-specific cellular immune response data obtained from BALB / c mice immunized with the adenoviral vectored vaccines disclosed herein. Eight- week-old BALB / c mice (n=5) received i.n. immunization with 5 x 10 7 PFU of BAd or HAd vectors, as detailed in FIG. 1C. Splenocytes were harvested three weeks following immunization and exposed to NP / A or NP / B overlapping peptide libraries. Cytokine expression by CD4+ or CD8+ T cells was quantified using flow' cytometry. The graphs illustrate percentages of NP / A- (FIGS. 5A-5F) or NP / B- (FIGS. 5G-5L) specific CD4+ or CD8+ T cells expressing interferon-gamma (IFN-y), interleukin-2 (IL-2), or TNF-a. Each data point signifies an individual animal, with error represented as SD. Statistical analysis was conducted using two-way ANOVA with Dunnett’s post-hoc test for intra-vector comparisons, with significance levels indicated as compared to the empty' vector. Symbols denote significance: n.s. (non-significant); * (p < 0.05); ** (p < 0.01); *** (p < 0.001); **** (p < 0.0001). For inter-vector comparisons, symbols are used: # (p < 0.05); ## (p < 0.01); ### (p < 0.001). The term "bivalent" refers to co-inoculation with vectors expressing C5-NP / A and C5-NP / B. The data key for FIGS. 5A-5L is as follows: 1 - BAd- AE1E3; 2 - BAd-C5-NP / A; 3 - BAd-C5-NP / B; 4 - BAd Bivalent; 5 - BAd-C5-NP / A+B; 6 - HAd-AElE3; 7 - HAd-C5-NP / A; 8 - HAd-C5-NP / B; 9 -HAd Bivalent; 10 - HAd-C5-NP / A+B.

[0037] FIGS. 6A-6N comprise graphs illustrating data pertaining to NP-specific CD8+ T cell responses in BALB / c mice immunized with the adenovirus-vectored vaccines described herein. Post-immunization, splenocytes, mediastinal lymph node (MLN) cells, and lung mononuclear (MN) cells were harvested from the immunized mice (n=5) three weeks following inoculation. These cells w ere stimulated using NP / A147-155 or NP / B167-174 peptides. The quantification of NP-specific cytokine-expressing CD8+ T cells was executed via enzyme-linked immunospot70855-02(ELISpot) assays. The graphs represent the numbers of NP / A- (FIGS. 6A-6F) and NP / B- (FIGS. 6G-6L) specific CD8+ T cells displaying IFN-y or IL-2 indicators within splenocytes, MLN cells, or lung MN cells. Fortetramer assays, splenocytes underwent staining withNP / A147-155-PE and NP / B166-174-FITC tetramers. The percentages of NP / A147-155- (FIG. 6M) and NP / B 166- 174- (FIG. 6N) tetramer-recognizing CD8+ T cells are presented. Each symbol denotes an individual animal, with error margins expressed as SD. Data analysis utilized two-way ANOVA with Dunnett’s post-hoc test. Statistical significance in intra-vector comparisons is indicated relative to the empty vector: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. For inter-vector comparisons: #, p < 0.05; ##, p < 0.01; ###, p < 0.001. “Bivalent” refers to co-inoculation with vectors expressing C5-NP / A and C5-NP / B. The data key for FIGS. 6A-6N is as follows: 1 - BAd- AE1E3; 2 - BAd-C5-NP / A; 3 - BAd-C5-NP / B; 4 - BAd Bivalent; 5 - BAd-C5-NP / A+B; 6 - HAd-AElE3; 7 - HAd-C5-NP / A; 8 - HAd-C5-NP / B; 9 -HAd Bivalent; 10 - HAd-C5-NP / A+B.

[0038] FIG. 7A illustrates a flow cytometry gating strategy for the tetramer assay conducted in BALB / c mice immunized with the Ad vectored vaccines described herein.

[0039] FIG. 7B presents representative diagrams for quantifying the percentage of NP / A147- 155 and NP / B167-174 tetramers recognizing CD8+T cells.

[0040] FIG. 8 comprises a table indicating the percent amino acid identity between the NP of influenza A and influenza B viruses utilized in the vaccine vectors described herein, compared with influenza A and influenza B viruses employed in the challenge studies in animals described herein.

[0041] FIG. 9 depicts a phylogenetic tree of influenza A and B viruses, including those from which NP gene sequences were utilized for generating vaccine vectors, alongside influenza A and B viruses used for mouse study challenges. The phylogenetic tree was generated using MEGA 11 software based on amino acid sequences available from the National Center for Biotechnology Information (NCBI). Numbers on each branch indicate bootstrap values derived from 100 resampling data.

[0042] FIG. 10 displays graphs of lung viral titers in immunized BALB / c mice post-challenge with various influenza viruses. Three weeks post-immunization with BAd vectors, as referenced in FIG. ID, mouse groups (n=5) were challenged with 5 mLD50 (50% mouse lethal dose) of A / Puerto Rico / 8 / 1934(HlNl) (subpart A of FIG. 10), A / Hong Kong / 1 / 68(H3N2) (subpart B of FIG. 10), or B / Florida / 04 / 2006 (Yamagata lineage) (subpart C of FIG. 10), or 100 mID50 of B / Brisbane / 60 / 2008 (Victoria lineage) (subpart D of FIG. 10), A / chukar / MN / 14951- 7 / 1998(H5N2) (subpart E of FIG. 10), A / goose / Nebraska / 17097 / 2011(H7N9) (subpart F of FIG. 10), or A / Hong Kong / 1073 / 1999(H9N2) (subpart G of FIG. 10). Lung specimens were harvested for viral titration three days post-challenge. Data are presented as LoglO TCID50 (50% tissue70855-02 culture infectious dose), with a detection limit of 0.5 LoglO TCID50. Each symbol represents an individual subject, with error bars indicating SD. Data evaluation utilized one-way ANOVA with Tukey’s post-hoc test. * Denotes significance at p < 0.05; ** at p < 0.01; *** at p < 0.001; **** at p < 0.0001. "Bivalent" refers to co-inoculation with vectors expressing C5-NP / A and C5-NP / B.

[0043] FIG. 11 illustrates graphs showing morbidity and mortality in immunized BALB / c mice post-challenge with seasonal or avian influenza viruses. Mouse groups (n=5) vaccinated with a single BAd vector dose were challenged with 5 mLD50 of A / Puerto Rico / 8 / 1934(HlNl), A / Hong Kong / 1 / 68(H3N2), or B / Florida / 04 / 2006 (Yamagata lineage). Morbidity, represented by weight loss (subparts A-C of FIG. 11), and mortality, represented by survival rates (subparts D and E of FIG. 11), were monitored for 14 days post-challenge. Mice exhibiting w eight loss exceeding 20% w ere euthanized and recorded as deceased. Data depict mean values with error bars indicating SD. "Bivalent" denotes co-inoculation with vectors expressing C5-NP / A and C5-NP / B.

[0044] FIGS. 12A-12I pertain to a study involving ferrets, focusing on NP-specific immune responses in ferrets immunized with a BAd vectored vaccine. The key shown in FIG. 12C is relevant to FIGS. 12B-I.

[0045] FIG. 12A outlines the study design, where 6-month-old specific pathogen-free (SPF) ferrets (n=3) were vaccinated intranasally twice with BAd-C5-NP / A + BAd-C5-NP / B (Bivalent) at intervals of three weeks. Blood samples were obtained for assessing humoral immune responses three weeks post-prime and booster vaccinations. Three w eeks following the second dose, ferrets faced challenges with 1 x 10 4 TCID50 of A / Puerto Rico / 8 / 1934(HlNl), 1 x 10 3 TCID50 of A / Hong Kong / 1 / 68(H3N2), or 1 x 10A5 TCID50 of B / Florida / 04 / 2006 (Yamagata lineage). Body temperature monitoring and collection of throat and nasal swabs occurred daily for five days postchallenge. Splenocytes and peripheral blood mononuclear cells (PBMCs) were gathered for analyzing cellular immune responses, and lungs were collected for viral titration. Serum NP / A- specific (FIG. 12B) and NP / B-specific (FIG. 12C) IgG titers were quantified via ELISA. NP- specific IFN-y-expressing T cells in the spleen and blood were tracked by ELISpot post-challenge with virus strains as mentioned above. Numbers of NP / A- and NP / B-specific T cells in splenocytes (FIGS. 12D-12F) or PBMCs (FIGS. 12G-12I) are presented. Each symbol denotes an individual animal, with errors shown as SD. Data were assessed using a t wo- tai led unpaired t-test. * Indicates significance at p < 0.05; ** at p < 0.01; *** at p < 0.001; **** at p < 0.0001.

[0046] FIG. 13 presents body temperature data of ferrets (n=6) pre- and post-vaccination, particularly the temperatures from one day preceding vaccination through five days postvaccination with either the empty BAd vector or BAd bivalent. The dashed line at 40 °C indicates fever in ferrets. Data depict mean body temperature with the SD.

[0047] FIGS. 14A-14H relate to the assessment of protection efficacy in ferrets vaccinated with70855-02BAd vectored vaccines following a challenge with seasonal influenza virus, with data labeled with a circle (•) being AE1E3 embodiments and data labeled with a triangle (A) being bivalent embodiments. More specifically, three weeks post-booster immunization with BAd vectors, ferrets (n=3) were challenged with 1 x 10A4 TCID50 of A / Puerto Rico / 8 / 1934(HlNl), 1 x 10A3 TCID50 of A / Hong Kong / 1 / 68(H3N2). or 1 x 1OA5 TCID50 of B / Flonda / 04 / 2006 (Yamagata lineage). Body temperature data, commencing three days pre-challenge until five days postchallenge, are illustrated (FIGS. 14A-14C), with the dashed line at 40 °C indicating fever in ferrets. Throat swabs collected from days 0-5 post-challenge were quantified using a TCID50 assay for viral load analysis (FIGS. 14D, 14E). Data are present as mean TCID50 / mL with SD, with a detection limit of 1 LoglO TCID50. Lung specimens were collected five days postchallenge and titrated via TaqMan real-time reverse transcription polymerase chain reaction (qRT- PCR) assay (FIGS. 14F-14H). Data are depicted as copy numbers, wi th a detection limit of 500 copy numbers. Each symbol denotes an individual animal, and errors are presented as SD. Data analysis was conducted using a two-tailed unpaired t-test. * Signifies significance at p < 0.05; ** at p < 0.01; *** at p < 0.001. "Bivalent" indicates co-inoculation with BAd-C5-NP / A and BAd- C5-NP / B.

[0048] Modifications and alternative forms are viable for the present disclosure; however, exemplary embodiments are depicted in the drawings and described in detail herein.SEQUENCE LISTING

[0049] The present application includes a Sequence Listing submitted electronically in XML format, which is hereby incorporated by reference in its entirety. The Sequence Listing XML file, created November 4, 2025, is named ' 0855-02_SequenceListing_f04NOV2025.xml?’ and is 29,165 bytes in size. The Sequence Listing complies with WIPO Standard ST.26, and the information contained in the XML file is identical that provided in the written sequence listing below, in accordance with 37 C.F.R. § 1.821(f).

[0050] SEQ ID NO: 1 is a fusion construct of A / Shanghai / 02 / 2013(H7N9)-AIP-C5 [C5-NP / A]: ATGGCAGCCCAGGCAGCTGTGGTCCGCTTCCAAGAAGCTGCCAATAAACAAAAGCA GGAGCTGGATGGACCAGGACCTGGAGCTACAAATTTCTCCTTGCTCAAGCAAGCAG GGGATGTCGAAGAAAATCCAGGGCCGGGACCAGGAATGGCTTCCCAGGGCACAAAG AGGTCTTACGAGCAGATGGAGACCGGCGGAGAGAGACAGAACGCCACAGAGATCA GAGCTAGCGTGGGACGGATGGTGTCCGGAATCGGCCGCTTCTACATCCAGATGTGCA CCGAGCTGAAGCTGTCCGACAACGAGGGCCGGCTGATCCAGAACTCCATCACAATC GAGCGCATGGTGCTGTCTGCCTTTGACGAGAGGAGAAACAGATACCTGGAGGAGCA70855-02ACGGCAAGTGGGTGAGAGAGCTGATCCTGTACGATAAGGAGGAGATCAGACGGATC TGGCGGCAGGCCAACAACGGAGAGGACGCCACCGCTGGCCTGACACACCTGATGAT CTGGCACAGCAACCTGAACGACGCCACCTACCAGCGCACAAGGGCTCTGGTGAGGA CCGGAATGGATCCCAGAATGTGCTCCCTGATGCAGGGCTCTACACTGCCTCGCAGGT CCGGAGCTGCTGGAGCTGCTGTGAAGGGAATCGGCACCATGGTCATGGAGCTGATCAGAATGATCAAGCGGGGCATCAACGATCGCAACTTCTGGAGGGGAGAGAACGGCAGA CGGACCCGCATCGCCTACGAGAGGATGTGCAACATCCTGAAGGGCAAGTTTCAGAC AGCCGCTCAGAGGGCCATGATGGACCAGGTGAGAGAGTCCCGGAACCCCGGAAACG CTGAGATCGAGGATCTGATCTTCCTGGCTCGGTCTGCTCTGATCCTGAGGGGAAGCGTGGCTCACAAGTCCTGCCTGCCAGCTTGCGTGTACGGACTGGCCGTGGCTTCTGGCT ACGACTTTGAGCGGGAGGGATACAGCCTGGTGGGCATCGATCCCTTCCGCCTGCTGC AGAACTCTCAGGTGTTTAGCCTGATCAGGCCAAACGAGAACCCCGCCCACAAGAGC CAGCTGGTGTGGATGGCTTGTCACTCCGCCGCTTTCGAGGACCTGCGGGTGAGCTCCTTTATCCGCGGCACCAGGATGGTGCCTAGGGGACAGCTGAGCACAAGAGGCGTGCA GATCGCCTCCAACGAGAACATGGAGGCTATGGATTCTAACACCCTGGAGCTGAGAAG CCGGTACTGGGCTATCAGGACCAGGAGCGGCGGAAACACAAACCAGCAGAGGGCTT CTGCTGGACAGGTGAGCGTGCAGCCTACCTTCTCCGTGCAGCGGAACCTGCCATTTGAGCGCGCCACAATCATGGCCGCTTTCACCGGAAACACAGAGGGCAGAACCTCTGAC ATGCGGACAGAGATCATCCGCATGATGGAGTCCGCCAGGCCAGAGGACGTGAGCTTC CAGGGAAGAGGCGTGTTTGAGCTGAGCGACGAGAAGGCTACAAACCCCATCGTGCC TTCTTTCGATATGAACAACGAGGGAAGCTACTTCTTTGGCGACAACGCCGAGGAGTA CGATAACTGA.

[0051] SEQ ID NO: 2 is an amino acid sequence encoded by SEQ ID NO: 1:MAAQAAVVRFQEAANKQKQELDGPGPGATNFSLLKQAGDVEENPGPGPGMASQGTK RSYEQMETGGERQNATEIRASVGRMVSGIGRFYIQMCTELKLSDNEGRLIQNSITIERMV LSAFDERRNRYLEEHPSAGKDPKKTGGPIYRRRDGKWVRELILYDKEEIRRIWRQANNG EDATAGLTHLMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGIGTMVMELIRMIKRGINDRNFWRGENGRRTRIAYERMCNILKGKFQTAAQRAMM DQVRESRNPGNAEIEDLIFLARSALILRGSVAHKSCLPACVYGLAVASGYDFEREGYSLV GIDPFRLLQNSQVFSLIRPNENPAHKSQLVWMACHSAAFEDLRVSSFIRGTRMVPRGQL STRGVQIASNENMEAMDSNTLELRSRYWAIRTRSGGNTNQQRASAGQVSVQPTFSVQRNLPFERATIMAAFTGNTEGRTSDMRTEIIRMMESARPEDVSFQGRGVFELSDEKATNPIV PSFDMNNEGSYFFGDNAEEYDN*. SEQ ID NO: 2 can comprise a CMV or other promoter at its N-terminus and / or a bGH poly A tail at its C-terminus.

[0052] SEQ ID NO: 3 is a fusion construct of B / Yamagata / 16 / 1988 (Yamagata lineage)-AIP-70855-02C5 [C5-NP / B]:ATGGCAGCCCAGGCAGCTGTGGTCCGCTTCCAAGAAGCTGCCAATAAACAAAAGCAGGAGCTGGATGGACCAGGACCTGGAGCTACAAATTTCTCCTTGCTCAAGCAAGCAGGGGATGTCGAAGAAAATCCAGGGCCGTCTAATATGGACATTGACGGCATCAACACAGGAACAATTGATAAAACTCCTGAGGAAATCACATCTGGGACCTCGGGCACAACGAGACCCATCATCCGGCCAGCCACTCTGGCTCCACCATCCAACAAAAGGACCAGAAATCCTTCACCTGAGCGTGCGACCACGTCAAGTGAGGCTGATGTGGGCAGAAAGACCCAGAAGAAACAGACCCCCACCGAAATTAAAAAATCCGTCTACAACATGGTTGTCAAGCTAGGGGAGTTCTATAACCAGATGATGGTGAAGGCTGGATTAAATGATGACATGGAGCGGAATTTGATCCAGAACGCCCACGCGGTCGAGAGAATTCTTCTCGCTGCGACGGACGATAAGAAAACAGAGTTTCAAAAAAAAAAGAATGCCCGAGACGTGAAGGAGGGAAAGGAAGAAATAGACCACAACAAGACTGGAGGCACTTTCTACAAAATGGTAAGAGATGACAAGACAATCTACTTCAGCCCCATCCGTATAACTTTCCTGAAAGAAGAGGTAAAGACCATGTATAAGACCACTATGGGGTCAGACGGATTCTCAGGGCTAAACCACATTATGATCGGACATTCTCAGATGAATGACGTCTGCTTCCAGCGGAGCAAAGCCTTAAAGCGAGTGGGACTTGACCCTTCCCTCATAAGCACATTTGCAGGTTCTACCTTGCCGAGAAGGTCTGGAGCCACTGGGGTTGCCATTAAAGGGGGCGGGACCTTGGTAGTTGAAGCTATCCGCTTTATCGGTCGGGCTATGGCTGACCGGGGCTTATTAAGGGATATCAAAGCAAAAACTGCATACGAGAAGATCCTCCTGAACTTAAAGAACAAGTGCTCTGCTCCACAGCAGAAGGCTCTGGTGGACCAGGTGATCGGGAGCAGGAACCCAGGCATTGCCGACATCGAAGACCTGACACTGCTGGCCAGGAGCATGGTCGTGGTCAGGCCCAGCCTTGCCAGCAAAGTTGTTCTACCAATCTCCATATATGCCAAAATTCCTCAGCTCGGCTTTAATGTGGAGGAGTACAGTATGGTGGGTTATGAGGCCATGGCGCTCTACAACATGGCTACCCCAGTCTCTATACTTCGCATGGGGGATGATGCCAAGGACAAGAGCCAACTGTTTTTCATGTCCTGTTTTGGTGCGGCCTATGAAGATCTCCGAGTGCTGTCGGCATTGACGGGCACAGAGTTTAAGCCTCGTAGTGCACTGAAGTGCAAGGGCTTCCATGTGCCCGCAAAGGAGCAGGTGGAGGGAATGGGTGCTGCTCTGATGAGCATTAAGCTGCAGTTCTGGGCCCCCATGACTCGCTCAGGCGGCAATGAAGTGGGTGGTGATGGCGGCTCTGGCCAGATCTCATGTTCCCCTGTGTTTGCCGTAGAACGACCGATCGCTCTTTCGAAGCAGGCCGTGAGGCGGATGCTGTCCATGAACATTGAGGGCCGAGATGCAGATGTTAAAGGGAACCTACTGAAGATGATGAACGACAGTATGGCCAAGAAAACCAATGGGAATGCATTTATTGGAAAAAAGATGTTTCAGATCAGTGACAAAAACAAAACAAACCCCGTCGAAATTCCTATAAAGCAAACAATTCCCAACTTCTTCTTTGGAAGAGACACTGCCGAGGATTATGACGATCTGGACTACTGA.

[0053] SEQ ID NO: 4 is an amino acid sequence encoded by SEQ ID NO: 3:70855-02MAAQAAVVRFQEAANKQKQELDGPGPGATNFSLLKQAGDVEENPGPSNMDIDGINTG TIDKTPEEITSGTSGTTRPIIRPATLAPPSNKRTRNPSPERATTSSEADVGRKTQKKQTPTEI KKSVYNMVVKLGEFYNQMMVKAGLNDDMERNLIQNAHAVERILLAATDDKKTEFQK KKNARDVKEGKEEIDHNKTGGTFYKMVRDDKTIYFSPIRITFLKEEVKTMYKTTMGSD GFSGLNHIMIGHSQMNDVCFQRSKALKRVGLDPSLISTFAGSTLPRRSGATGVAIKGGG TLVVEAIRFIGRAMADRGLLRDIKAKTAYEKILLNLKNKCSAPQQKALVDQVIGSRNPGI ADIEDLTLLARSMVVVRPSLASKVVLPISIYAKIPQLGFNVEEYSMVGYEAMALYNMAT PVSILRMGDDAKDKSQLFFMSCFGAAYEDLRVLSALTGTEFKPRSALKCKGFHVPAKE QVEGMGAALMSIKLQFWAPMTRSGGNEVGGDGGSGQISCSPVFAVERPIALSKQAVRR MLSMNIEGRDADVKGNLLKMMNDSMAKKTNGNAFIGKKMFQISDKNKTNPVEIPIKQTIPNFFFGRDTAEDYDDLDY*. SEQ ID NO: 4 can comprise a CMV or other promoter at its N-terminus and / or a bGH poly A tail at its C-terminus.

[0054] SEQ ID NO: 5 is a fusion construct of fusion of NP / A and NP / B with AIP-C5 [C5- NP / A+B]:ATGGCCGCCCAGGCCGCCGTGGTGAGGTTTCAGGAGGCCGCCAACAAGCAGAAGCA GGAGCTGGACGCCACAAACTTCAGCCTGCTCAAGCAGGCCGGCGACGTGGAAGAG AACCCAGGCCCTGGGCCTGGCATGGCCTCCCAGGGCACCAAGCGCTCCTACGAGCA GATGGAGACCGGCGGGGAAAGACAGAACGCTACTGAGATCAGGGCCAGCGTGGGC CGGATGGTGAGCGGAATTGGCCGGTTTTACATCCAGATGTGCACCGAGCTGAAGCTG TCCGACAATGAAGGCCGGCTCATCCAGAATAGCATCACCATCGAGAGAATGGTGCTG TCCGCCTTTGACGAGAGAAGGAATAGATATCTGGAGGAGCACCCTAGCGCCGGCAA GGACCCTAAAAAGACCGGCGGCCCTATCTATAGGAGGAGGGACGGCAAGTGGGTGA GGGAGCTGATCCTGTACGACAAGGAGGAGATTAGGCGGATTTGGCGCCAGGCTAAC AATGGAGAGGATGCAACAGCCGGACTGACTCACCTGATGATTTGGCACAGCAATCTG AACGACGCCACATACCAGAGAACCCGCGCCCTGGTGAGGACCGGGATGGACCCCAG GATGTGCAGCCTGATGCAGGGGAGCACACTGCCTAGGCGGTCCGGCGCCGCTGGCG CAGCCGTGAAGGGAATCGGGACTATGGTAATGGAGCTGATCCGGATGATTAAGAGGG GCATCAACGATCGCAACTTCTGGAGGGGAGAGAACGGCAGGAGGACCCGGATCGCT TACGAGCGCATGTGTAATATCCTGAAGGGCAAGTTTCAGACAGCCGCTCAGAGAGCC ATGATGGACCAGGTGAGGGAGTCCAGAAACCCCGGCAACGCCGAGATCGAGGATCT GATCTTTCTGGCCAGGTCCGCCCTGATCCTGAGGGGCTCTGTGGCCCATAAGAGTTG TCTGCCAGCTTGTGTGTACGGCCTCGCCGTGGCCAGCGGCTATGACTTTGAGCGGGA GGGCTATTCTCTGGTGGGGATTGATCCTTTCCGCCTGCTGCAGAACTCCCAGGTGTTC TCCCTGATCAGGCCAAACGAGAATCCCGCCCATAAGAGCCAGCTGGTGTGGATGGCT70855-02ATGGTGCCTCGCGGACAGCTGAGCACACGCGGCGTGCAGATCGCCAGTAACGAGAACATGGAGGCCATGGACTCTAATACACTCGAGCTGAGGAGCCGGTACTGGGCTATCAGGACCAGATCTGGCGGCAACACCAATCAGCAGAGGGCCTCTGCTGGACAGGTGTCTGTGCAGCCTACTTTCAGTGTTCAGCGCAACCTGCCTTTCGAGAGAGCCACCATCATGGCCGCCTTTACCGGCAACACAGAGGGCAGAACCTCCGATATGAGGACCGAGATCATCAGAATGATGGAGTCCGCCAGGCCCGAGGACGTGAGCTTCCAGGGTAGGGGCGTGTTCGAACTGTCTGATGAGAAGGCCACCAACCCTATTGTGCCTTCTTTTGATATGAACAATGAGGGATCTTACTTCTTCGGGGACAATGCCGAGGAGTACGACAACGCCACTAACTTCAGCCTCCTGAAACAGGCCGGCGATGTCGAGGAGAATCCCGGCCCAGGCCCCGGCAGTAACATGGACATTGACGGCATTAATACCGGGACCATCGACAAGACCCCTGAGGAGATTACATCCGGGACATCTGGCACTACCAGACCAATTATCAGGCCCGCCACACTGGCCCCACCCAGCAATAAGAGAACACGGAATCCTTCTCCAGAGAGAGCCACCACGAGCAGCGAGGCCGACGTGGGAAGGAAAACCCAGAAGAAGCAGACCCCAACAGAAATCAAGAAGAGCGTGTATAACATGGTGGTGAAGCTGGGGGAGTTCTATAATCAGATGATGGTGAAGGCCGGCCTGAACGACGACATGGAGCGGAACCTGATCCAGAACGCCCACGCTGTGGAGAGAATCCTGCTGGCCGCCACCGATGATAAGAAAACCGAGTTCCAGAAGAAGAAGAATGCTCGGGATGTGAAGGAGGGCAAAGAGGAGATCGACCACAATAAGACAGGGGGAACCTTTTACAAGATGGTGCGCGATGATAAGACAATCTATTTCAGCCCAATCCGCATCACCTTCCTGAAGGAGGAGGTCAAGACCATGTACAAGACCACAATGGGCTCCGATGGGTTTAGCGGCCTGAACCATATCATGATCGGCCATAGTCAGATGAACGACGTGTGCTTTCAGAGATCTAAAGCCCTGAAAAGGGTGGGCCTCGACCCTAGCCTGATCTCTACCTTCGCCGGCAGCACCCTCCCTAGGAGGAGCGGAGCTACCGGAGTGGCCATCAAGGGCGGCGGCACTCTGGTGGTGGAGGCAATCAGGTTCATTGGGAGGGCCATGGCCGATAGAGGACTGCTGAGGGATATCAAAGCCAAGACCGCCTATGAGAAGATCCTGCTGAACTTGAAGAACAAGTGTTCCGCCCCACAGCAGAAGGCTCTGGTGGACCAGGTTATCGGAAGCAGGAATCCTGGGATCGCTGACATCGAGGACCTGACTCTGCTGGCCAGGAGCATGGTCGTGGTGAGACCCTCCCTGGCCAGCAAGGTGGTGCTGCCTATCTCCATCTACGCCAAGATCCCCCAGCTGGGCTTTAACGTGGAGGAGTACTCTATGGTGGGCTATGAGGCCATGGCTCTGTACAACATGGCAACCCCCGTGAGCATCCTGAGGATGGGCGATGACGCTAAGGACAAGAGCCAGCTGTTCTTCATGAGCTGCTTTGGAGCTGCTTATGAGGACCTGAGGGTGCTGTCTGCCCTGACCGGCACAGAGTTTAAGCCAAGGAGCGCCCTGAAGTGCAAGGGCTTCCACGTGCCTGCTAAGGAGCAGGTGGAGGGAATGGGCGCCGCCCTGATGAGCATCAAGCTGCAGTTCTGGGCCCCCATGACCAGAAGCGGCGGGAACGAGGTGGGCGGCGACGGCGGCTCCGGCCAGATTAGCTGCTCCCCAGTGTTCGCCGTGGAACGGCC70855-02GCGACGCTGACGTGAAGGGAAACCTGCTGAAGATGATGAATGATAGCATGGCCAAA AAGACCAACGGCAACGCCTTCATCGGCAAGAAGATGTTTCAGATCAGCGACAAGAA CAAGACAAATCCCGTGGAGATCCCTATCAAGCAGACCATCCCTAACTTCTTCTTCGGC CGCGACACCGCCGAGGACTACGACGACCTGGACTACTGA.

[0055] SEQ ID NO: 6 is an amino acid sequence encoded by SEQ ID NO: 5:MAAQAAVVRFQEAANKQKQELDATNFSLLKQAGDVEENPGPGPGMASQGTKRSYEQ METGGERQNATEIRASVGRMVSGIGRFYIQMCTELKLSDNEGRLIQNSITIERMVLSAFD ERRNRYLEEHPSAGKDPKKTGGPIYRRRDGKWVRELILYDKEEIRRIWRQANNGEDATA GLTHLMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGIGT MVMELIRMIKRGINDRNFWRGENGRRTRIAYERMCNILKGKFQTAAQRAMMDQVRES RNPGNAEIEDLIFLARSALILRGSVAHKSCLPACVYGLAVASGYDFEREGYSLVGIDPFRLLQNSQVFSLIRPNENPAHKSQLVWMACHSAAFEDLRVSSFIRGTRMVPRGQLSTRGVQI ASNENMEAMDSNTLELRSRYWAIRTRSGGNTNQQRASAGQVSVQPTFSVQRNLPFERA TIMAAFTGNTEGRTSDMRTEIIRMMESARPEDVSFQGRGVFELSDEKATNPIVPSFDMNN EGSYFFGDNAEEYDNATNFSLLKQAGDVEENPGPGPGSNMDIDGINTGTIDKTPEEITSG TSGTTRPIIRPATLAPPSNKRTRNPSPERATTSSEADVGRKTQKKQTPTEIKKSVYNMVVK LGEFYNQMMVKAGLNDDMERNLIQNAHAVERILLAATDDKKTEFQKKKNARDVKEG KEEIDHNKTGGTFYKMVRDDKTIYF SPIRITFLKEEVKTMYKTTMGSDGFS GLNHIMIGH SQMNDVCFQRSKALKRVGLDPSLISTFAGSTLPRRSGATGVAIKGGGTLVVEAIRFIGRA MADRGLLRDIKAKTAYEKILLNLKNKCSAPQQKALVDQVIGSRNPGIADIEDLTLLARS MVVVRPSLASKVVLPISIYAKIPQLGFNVEEYSMVGYEAMALYNMATPVSILRMGDDAK DKSQLFFMSCFGAAYEDLRVLSALTGTEFKPRSALKCKGFHVPAKEQVEGMGAALMSI KLQFWAPMTRSGGNEVGGDGGSGQISCSPVFAVERPIALSKQAVRRMLSMNIEGRDADV KGNLLKMMNDSMAKKTNGNAFIGKKMFQISDKNKTNPVEIPIKQTIPNFFFGRDTAEDY DDLDY*. SEQ ID NO: 6 can comprise a CMV or other promoter at its N-terminus and / or a bGH poly A tail at its C-terminus.

[0056] SEQ ID NO: 7 is a fusion construct of NP / A+B-AIP-C5 with two promoters [C5- NP / A+B]:ATGGCAGCCCAGGCAGCTGTGGTCCGCTTCCAAGAAGCTGCCAATAAACAAAAGCA GGAGCTGGATGGACCAGGACCTGGAGCTACAAATTTCTCCTTGCTCAAGCAAGCAG GGGATGTCGAAGAAAATCCAGGGCCGGGACCAGGAATGGCTTCCCAGGGCACAAA GAGGTCTTACGAGCAGATGGAGACCGGCGGAGAGAGACAGAACGCCACAGAGATCAGAGCTAGCGTGGGACGGATGGTGTCCGGAATCGGCCGCTTCTACATCCAGATGTG CACCGAGCTGAAGCTGTCCGACAACGAGGGCCGGCTGATCCAGAACTCCATCACAA70855-02CACCCTTCTGCTGGAAAGGATCCAAAGAAGACCGGAGGACCAATCTACCGGCGCAGGGACGGCAAGTGGGTGAGAGAGCTGATCCTGTACGATAAGGAGGAGATCAGACGGATCTGGCGGCAGGCCAACAACGGAGAGGACGCCACCGCTGGCCTGACACACCTGATGATCTGGCACAGCAACCTGAACGACGCCACCTACCAGCGCACAAGGGCTCTGGTGAGGACCGGAATGGATCCCAGAATGTGCTCCCTGATGCAGGGCTCTACACTGCCTCGCAGGTCCGGAGCTGCTGGAGCTGCTGTGAAGGGAATCGGCACCATGGTCATGGAGCTGATCAGAATGATCAAGCGGGGCATCAACGATCGCAACTTCTGGAGGGGAGAGAACGGCAGACGGACCCGCATCGCCTACGAGAGGATGTGCAACATCCTGAAGGGCAAGTTTCAGACAGCCGCTCAGAGGGCCATGATGGACCAGGTGAGAGAGTCCCGGAACCCCGGAAACGCTGAGATCGAGGATCTGATCTTCCTGGCTCGGTCTGCTCTGATCCTGAGGGGAAGCGTGGCTCACAAGTCCTGCCTGCCAGCTTGCGTGTACGGACTGGCCGTGGCTTCTGGCTACGACTTTGAGCGGGAGGGATACAGCCTGGTGGGCATCGATCCCTTCCGCCTGCTGCAGAACTCTCAGGTGTTTAGCCTGATCAGGCCAAACGAGAACCCCGCCCACAAGAGCCAGCTGGTGTGGATGGCTTGTCACTCCGCCGCTTTCGAGGACCTGCGGGTGAGCTCCTTTATCCGCGGCACCAGGATGGTGCCTAGGGGACAGCTGAGCACAAGAGGCGTGCAGATCGCCTCCAACGAGAACATGGAGGCTATGGATTCTAACACCCTGGAGCTGAGAAGCCGGTACTGGGCTATCAGGACCAGGAGCGGCGGAAACACAAACCAGCAGAGGGCTTCTGCTGGACAGGTGAGCGTGCAGCCTACCTTCTCCGTGCAGCGGAACCTGCCATTTGAGCGCGCCACAATCATGGCCGCTTTCACCGGAAACACAGAGGGCAGAACCTCTGACATGCGGACAGAGATCATCCGCATGATGGAGTCCGCCAGGCCAGAGGACGTGAGCTTCCAGGGAAGAGGCGTGTTTGAGCTGAGCGACGAGAAGGCTACAAACCCCATCGTGCCTTCTTTCGATATGAACAACGAGGGAAGCTACTTCTTTGGCGACAACGCCGAGGAGTACGATAACTGA / ATGGCAGCTCAAGCAGCGGTAGTCCGCTTTCAAGAAGCCGCGAACAAACAAAAACAGGAGCTCGACGGCCCTGGGCCAGGGGCAACTAACTTCTCCCTTCTTAAACAGGCTGGCGATGTGGAAGAGAACCCTGGCCCGAGCAACATGGACATTGATGGCATCAACACGGGGACCATCGATAAAACACCAGAGGAAATTACATCGGGCACCAGCGGCACAACTCGCCCCATCATCAGACCGGCCACTCTGGCACCACCCAGTAACAAGAGGACCAGAAATCCCAGCCCTGAGCGGGCTACTACCTCTTCTGAGGCTGACGTAGGGAGAAAAACCCAGAAGAAGCAAACCCCCACCGAGATCAAAAAGTCTGTGTACAATATGGTCGTTAAGCTGGGAGAGTTCTACAACCAGATGATGGTAAAAGCTGGACTCAATGACGACATGGAAAGGAATCTGATCCAGAATGCTCATGCAGTTGAGAGAATACTACTTGCTGCTACTGATGACAAGAAGACCGAGTTTCAGAAAAAGAAAAATGCAAGGGACGTGAAGGAAGGAAAGGA70855-02GACAATATACTTCAGCCCAATACGGATTACCTTTCTGAAAGAAGAGGTGAAGACTATG TACAAGACGACTATGGGGAGCGATGGATTTTCTGGCTTGAACCACATTATGATTGGCC ATTCGCAAATGAATGATGTCTGCTTCCAGAGGAGTAAGGCGCTCAAACGAGTGGGTC TGGATCCATCACTTATCTCAACATTTGCTGGGTCGACACTGCCTCGACGGAGTGGTGC AACTGGGGTGGCAATTAAAGGAGGTGGTACTTTAGTGGTTGAAGCCATTCGATTCAT TGGACGTGCGATGGCCGATCGCGGCCTCCTTAGGGACATCAAGGCCAAGACAGCCTA CGAGAAGATTTTACTCAACCTGAAGAACAAGTGTTCAGCTCCGCAGCAGAAGGCTC TGGTGGACCAAGTAATAGGTAGTCGGAATCCTGGAATTGCAGATATTGAGGACCTTAC CTTACTGGCCAGATCTATGGTTGTGGTGCGCCCCTCCTTGGCATCCAAGGTGGTCTTA CCAATCTCTATCTATGCCAAAATCCCCCAGTTGGGGTTCAACGTGGAAGAATATTCAA TGGTCGGTTATGAGGCCATGGCGTTGTACAACATGGCGACACCTGTCTCTATCCTGAG AATGGGCGACGACGCCAAGGACAAGTCCCAGCTATTCTTCATGTCCTGTTTTGGTGC CGCCTATGAAGACCTGAGAGTGCTCAGTGCACTCACCGGTACAGAATTCAAGCCTCG ATCCGCACTGAAATGCAAGGGGTTCCACGTCCCGGCAAAAGAGCAGGTGGAGGGCA TGGGAGCCGCCCTGATGAGCATCAAACTGCAGTTCTGGGCTCCTATGACTCGTTCTG GAGGAAATGAAGTGGGAGGGGATGGGGGAAGCGGCCAGATAAGCTGCAGCCCTGTA TTCGCCGTTGAGAGGCCCATAGCTCTATCCAAGCAGGCCGTGCGGAGAATGTTGTCC ATGAACATCGAAGGCCGGGATGCCGACGTCAAGGGCAATCTACTGAAGATGATGAAC GACAGCATGGCTAAAAAGACGAATGGGAATGCCTTTATCGGGAAAAAAATGTTTCAG ATCAGTGATAAGAACAAAACCAACCCAGTTGAAATCCCTATCAAGCAGACGATTCCA AATTTCTTTTTTGGAAGGGACACCGCCGAGGATTATGATGATCTCGACTACTGA.

[0057] SEQ ID NO: 8 is an amino acid sequence encoded by SEQ ID NO: 9:MAAQAAVVRFQEAANKQKQELDGPGPGATNFSLLKQAGDVEENPGPGPGMASQGTKR SYEQMETGGERQNATEIRASVGRMVSGIGRFYIQMCTELKLSDNEGRLIQNSITIERMVL SAFDERRNRYLEEHPSAGKDPKKTGGPIYRRRDGKWVRELILYDKEEIRRIWRQANNGE DATAGLTHLMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVK GIGTMVMELIRMIKRGINDRNFWRGENGRRTRIAYERMCNILKGKFQTAAQRAMMDQV RESRNPGNAEIEDLIFLARSALILRGSVAHKSCLPACVYGLAVASGYDFEREGYSLVGIDP FRLLQNSQVFSLIRPNENPAHKSQLVWMACHSAAFEDLRVSSFIRGTRMVPRGQLSTRG VQIASNENMEAMDSNTLELRSRYWAIRTRSGGNTNQQRASAGQVSVQPTFSVQRNLPFE RATIMAAFTGNTEGRTSDMRTEIIRMMESARPEDVSFQGRGVFELSDEKATNPIVPSFDM NNEGSYFFGDNAEEYDN* SEQ ID NO: 8 can comprise a CMV or other promoter at its N- terminus and / or a SV40 poly A tail at its C-terminus.(reverse translation direction) bGH poly A tail|*YDLDDYDEATDRGFFFNPITQKIPIEVPNTKNKDSIQFMKKGIFANGN70855-02TKKAMSDNMMKLLNGKVDADRGEINMSLMRRVAQKSLAIPREVAFVPSCSIQGSGGDG GVENGGSRTMPAWFQLKISMLAAGMGEVQEKAPVHFGKCKLASRPKFETGTLASLVRL DEYAAGFCSMFFLQSKDKADDGMRLISVPTAMNYLAMAEYGVMSYEEVNFGLQPIKA YISIPLVVKSALSPRVVVMSRALLTLDEIDAIGPNRSGIVQDVLAKQQPASCKNKLNLLIK EYATKAKIDRLLGRDAMARGIFRIAEVVLTGGGKIAVGTAGSRRPLTSGAFTSILSPDLGV RKLAKSRQFCVDNMQSHGIMIHNLGSFGDSGMTTKYMTKVEEKLFTIRIPSFYITKDDR VMKYFTGGTKNHDIEEKGEKVDRANKKKQFETKKDDTAALLIREVAHANQILNREMD DNLGAKVMMQNYFEGLKVVMNYVSKKIETPTQKKQTKRGVDAESSTTAREPSPNRTR KNSPPALTAPRIIPRTTGSTGSTIEEPTKDITGTNIGDIDMNSPGPNEEVDGAQKLLSFNTA GPGPGDLEQKQKNAAEQFRVVAAQAAM |SV40 promoter | (SEQ ID NO: 13)

[0058] SEQ ID NO: 9 is an amino acid sequence for an Autophagy -Inducing Peptide C5 (AIP- C5) from the CFP10 protein of Mycobacterium tuberculosis'.AAQAAVVRFQEAANKQKQELD.

[0059] SEQ ID NO: 10 is the DNA sequence that encodes SEQ ID NO: 9:ATGGCCGCTCAGGCCGCTGTGGTGAGATTCCAGGAGGCCGCTAACAAGCAGAAGCA GGAGCTGGAC.

[0060] SEQ ID NO: 11 is an amino acid sequence for NP147 peptide (H-2Kd-restricted CTL epitope for NP): TYQRTRALV.

[0061] SEQ ID NO: 12 is an amino acid sequence for NP / B166-174 peptide: FSPIRITFL.DETAILED DESCRIPTION

[0062] The present disclosure generally pertains to a universal influenza vaccine. Specifically, it relates to methods and compositions for vaccination (e.g., an effective general vaccination) that induces protective immunity' against both influenza A viruses (lAVs) and influenza B viruses (IBVs). These methods and compositions can include an adenoviral vector (Ad vector), such as a BAd vector, which expresses both the nucleoprotein (NP) of an IAV (e.g., H7N9) and the NP of an IBV. This can occur with or without the inclusion of the Autophagy' -Inducing Peptide C5 (AIP- C5) from the CFP10 protein of Mycobacterium tuberculosis. In particular embodiments, modifications include the removal of El and E3 regions from the Ad vector. Furthermore, certain compositions can comprise one or more immunogenic domains, such as HA. HA2, or M2e, optionally with AIP-C5 expression from a CFP10 protein of M. tuberculosis. Additionally, pharmaceutical compositions and corresponding methods of use are also described.

[0063] Candidate vaccines can be developed for individual influenza strains; however, it remains impractical to prepare extensive vaccine stocks for each potential pandemic virus. The specific nature of a pandemic influenza virus is generally identified at the onset of the pandemic70855-02(not beforehand). Thus, development of a universal influenza vaccine capable of providing sufficient protection against seasonal influenza A viruses (e.g., H1N1 and H3N2), potential pandemic avian influenza A viruses (e.g, H5N1, H7N7, H7N9, and H9N2), and / or influenza B viruses would be advantageous.

[0064] Influenza A virus is extensively distributed in nature and can infect various birds and mammals. Influenza viruses are enveloped RNA viruses with genomes comprising 11 different proteins: one NP, three polymerase proteins (PA, PB1, and PB2), two matrix proteins (Ml and M2), three nonstructural proteins (NS1, NS2, and PB1-F2), and eight single-stranded RNA segments encoding two external glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Viruses are classified based on differences in antigenic structures of HA and NA proteins, with unique combinations representing distinct virus subtypes, further classified into influenza virus strains such as IAV and IBV.

[0065] Influenza B virus (IBV) strains exhibit less antigenic variation as compared to type A (IAV) strains. Two genetically and antigenically distinct IBV strains are represented by B / Yamagata / 16 / 88 (B / Yamagata) and B / Victoria / 2 / 87 (B / Victoria). Although the disease spectrum caused by influenza B is generally milder than that of IAV, severe illness requiring hospitalization from IBV infection remains frequently observed.

[0066] Seasonal influenza viruses undergo mutations that can result in altered antigenicity. Certain avian influenza viruses, including A / H5N1, A / H7N9, and A / H9N2, pose significant risks of evolving into pandemic strains. Other lAVs also present credible threats to human health. See Zhang et al., Clinical, epidemiological and virological characteristics of the first detected human case of avian influenza A(H5N6) virus, Infection, Genetics & Evolution: J Molecular Epidemiology & Evolutionary Genetics in Infectious Diseases 40: 236-242 (2016); Fouchier et al., Avian influenza A virus (H7N7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome, Proceedings of the National Academy of Sciences USA 101: 1356- 1361 (2004). Recent avian A / H5N1 influenza virus cases (clade 2.3.4.4b) infecting cattle and transmission to cattle, poultry, and cats further underscore the potential threat of avian influenza viruses. Burrough et al., Highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus infection in domestic dairy cattle and cats, Emerging Infectious Diseases 30(7): 1335-1343 (2024). Developing a broadly protective or universal influenza vaccine that offers improved protection as compared to current seasonal or pandemic vaccines is crucial globally. Relatively conserved influenza antigens, including chimeric HA, HA2 stem, M2e, or NP, have been evaluated for broadly protective vaccines with varying success. See Nachbagauer et al., A chimeric hemagglutinin-based universal influenza virus vaccine approach induces broad and long-lasting immunity in a randomized, placebo-controlled phase I trial, Nature Medicine 27: 106-114 (2021);70855-02Yassine et al.. Hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection, Nature Medicine 21: 1065-1070 (2015); Saelens, The role of matrix protein 2 ectodomain in the development of universal influenza vaccines, Journal of Infectious Diseases 219: S68-S74 (2019); Leroux-Roels et al., Immunogenicity, safety, and preliminary efficacy evaluation of OVX836, a nucleoprotein-based universal influenza A vaccine candidate: a randomized, double-blind, placebo-controlled, phase 2a trial, The Lancet Infectious Diseases 23: 1360-1369 (2023).

[0067] In addition to (or in concert with) mutations, immunodominance can limit the efficacy of influenza vaccines, particularly concerning IBV strains, by directing the immune response toward specific dominant viral epitopes rather than the full range of potential targets. For instance, a typical vaccine administration can prompt an immune response against prominent or dominant epitopes on the virus. However, IBV's antigenic drift can lead to reduced vaccine efficacy against the IBV strain.

[0068] Relatively conserved proteins or domains of the influenza virus, such as NP, HA stem domain, or matrix-2 (M2), can stimulate broadly protective immune responses. See Sayedahmed et al., Development of NP-based universal vaccine for influenza A viruses. Vaccines 12(2): 157 (2024); Lu et al., Production and stabilization of the trimetric influenza hemagglutinin stem domain for potentially broadly protective influenza vaccines, Proceedings of Nat ’I Academy Sciences USA 111: 125-130 (2014); Pardi et al., Development of a pentavalent broadly protective nucleoside-modified mRNA vaccine against influenza B viruses, Nature Communications 13: 4677 (2022). Additionally, Ad vector-based vaccines have demonstrated potential for developing effective vaccines against several pathogens, including the Ebola virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for human use. See Zhu et al., Immunogenicity and safety of a recombinant adenovirus type-5-vectored COVID- 19 vaccine in healthy adults aged 18 years or older: a randomized, double-blind, placebo-controlled, phase 2 trial, Lancet (London, England) 396: 479-488 (2020); Zhu et al., Safety and immunogenicity of a recombinant adenovirus type-5 vector-based Ebola vaccine in healthy adults in Sierra Leone: a single-center, randomized, double-blind, placebo-controlled, phase 2 trial, Lancet (London, England) 389: 621-628 (2017). Ad vector-based influenza vaccines expressing HA (the principal viral protein responsible for binding to host cell receptors), NA, NP, matrix protein 1 (Ml), matrix protein 2 (M2), or immunogenic domains or epitopes have shown promise in providing protection against influenza viruses in experimental animals or human clinical trials. Moreover, Ad vector immunity can be addressed by using less prevalent Human Ads (HAds) or nonhuman Ads as vaccine platforms.

[0069] The influenza virus internal protein, namely NP, is relatively conserved across multiple subtypes of the influenza virus and serves as an inducer of heterosubtypic CD8+cytotoxic T70855-02 lymphocyte (CTL) responses following infection. See Yewdell et al., Influenza A virus nucleoprotein is a major target antigen for cross-reactive anti -influenza A virus cytotoxic T lymphocytes, Proceedings Nat’l Academy Sciences USA 82: 1785-1789 (1985). For example, NP / A has about 91% to about 97.5% homology among lAVs used for challenge studies, but about 37% homology with IBVs (FIG. 8). Similarly. NP / B showed about 99% homology between IBVs used for challenge studies but 37.5% homology with lAVs (FIG. 8). Previous studies indicated that influenza-specific CD8+CTLs can provide cross-influenza subtype protection, resulting in rapid viral clearance and reduced disease severity. See van de Sandt et al., Challenging immunodominance of influenza-specific CD8(+) T cell responses restricted by the risk-associated HLA-A*68:01 allomorph, Nature Communications 10: 5579 (2019); Valkenburg et al., Acute emergence and reversion of influenza A virus quasi-species within CD8+T cell antigenic peptides, Nature Communications 4: 2663 (2013). The antibody-dependent cellular cytotoxicity (ADCC) and complement lysis mediated by NP-specific antibodies can also confer cross-subtype reactivity. See Jegaskanda et al., Induction of H7N9-cross-reactive antibody-dependent cellular cytotoxicity antibodies by human seasonal influenza A viruses that are directed toward the nucleoprotein, J Infectious Diseases 215: 818-823 (2017); Jia et al., Influenza antibody breadth and effector functions are immune correlates from acquisition of pandemic infection of children, Nature Communications 15: 3210 (2024). However, the NP-specific humoral immune response alone is insufficient to provide full protection. See Vanderven et al., Poor protective potential of influenza nucleoprotein antibodies despite w ide prevalence, Immunology & Cell Biology 100: 49- 60 (2022). The compositions, vectors, vaccines, platforms, and methods described herein overcome this limitation by inducing both an NP-specific humoral immune response and a robust NP-specific cell-mediated immune (CMI) response, which enhances protection against various influenza subty pes (as compared to conventional approaches), including both IAV and IBV.

[0070] The present antigens, compositions, Ad vectors, and methods leverage NP as a target for a universal influenza vaccine effective against both IAV and IBV strains. The Ad vector-based vaccine platform described herein can induce robust humoral and cellular immune responses, providing an advantageous vaccine delivery system for intracellular pathogens as compared to conventional vaccine platforms. See Elkashif et al., Adenoviral vector-based platforms for developing effective vaccines to combat respiratory viral infections, Clinical & Translational Immunology 10: el345 (2021). A critical limitation of traditional HAd vectors is preexisting vector immunity, which can significantly interfere with the efficiency of HAd vector vaccines. See Fausther-Bovendo & Kobinger, Pre-existing immunity against Ad vectors: humoral, cellular, and innate response, what's important?, Human Vaccines & Immunotherapeutics 10: 2875-2884 (2014); Seregin & Amalfitano, Overcoming pre-existing adenovirus immunity by genetic70855-02 engineering of adenovirus-based vectors. Expert Opinion on Biological Therapy 9: 1521-1531 (2009). Alternatively, non-human Ad vectors such as those from simian, bovine, porcine, ovine, canine, or avian origin can be developed to circumvent the preexisting HAd immunity. See Ahi et al., Adenoviral vector immunity’: its implications and circumvention strategies, Current Gene Therapy 11: 307-320 (201 1). Previously, chimpanzee Ad (ChAd) vector-based vaccines against malaria, Ebola virus, or HIV were evaluated in clinical trials, and the SARS-CoV-2 vaccine (ChAdOxl nCoV-19) was licensed in several countries, indicating the effectiveness of nonhuman Ad vectors. See Tapia et al., Use of ChAd3-EBO-Z Ebola virus vaccine in Milian and US adults, and boosting of Malian adults with MVA-BN-Filo: a phase 1. single-blind, randomized trial, a phase lb, open-label and double-blind, dose-escalation trial, and a nested, randomized, doubleblind, placebo-controlled trial, The Lancet, Infectious Diseases 16: 31-42 (2016); Ewer et al., Protective CD8+T-cell immunity to human malaria induced by chimpanzee adenovirus-MVA immunization, Nature Communications 4 2836 (2013); Hayton et al., Safety and tolerability of conserved region vaccines vectored by plasmid DNA, simian adenovirus and modified vaccinia virus Ankara administered to human immunodeficiency virus type 1 -uninfected adults in a randomized, single-blind phase 1 trial, PloS One: el01591 (2014); Mendonca et al., Adenoviral vector vaccine platforms in the SARS-CoV-2 pandemic, NPJ Vaccines 6: 97 (2021).

[0071] A rare but severe side effect of COVID-19 vaccines, especially Ad-based SARS-CoV-2 vaccines, is known as vaccine-induced immune thrombocytopenia (VITT) due to interactions of Ad hexon and / or SARS-CoV-2 S protein with platelet factor 4 (PF4) protein. See Cines & Bussel, SARS-CoV-2 vaccine-induced immune thrombotic thrombocytopenia, New England J Medicine 384: 2254-2256 (2021); Toh et al., The aetiopathogenesis of vaccine-induced immune thrombotic thrombocytopenia, C linical Medicine (London, England) 22: 140-144 (2022).

[0072] However, the exact mechanism of VITT is unclear. Ad-based vaccines are typically not advised for use in individuals with high levels of anti-PF4 antibodies or inherent issues with blood clotting. See Marcucci & Marietta, Vaccine-induced thrombotic thrombocytopenia: the elusive link between thrombosis and adenovirus-based SARS-CoV-2 vaccines. Internal & Emergency Medicine 16: 1113-1 119 (2021). Since natural infections with HAds are not known to cause VITT, the mucosal use of Ad vectored vaccines may not trigger this rare adverse reaction. The potential binding of the bovine Ad (BAd) vector with PF4 and other blood factors should be investigated.

[0073] Accordingly, in some embodiments, a vaccine platform is provided that comprises an Ad vector. The Ad vector comprises a nucleic acid encoding an immunogenic composition as described herein, wherein expression of the immunogenic composition in a host cell or subject elicits general immunogenicity’ protection and / or a cross-protective immune response against multiple subtypes of influenza A and influenza B viruses.70855-02

[0074] As used herein, ‘'general” refers to a broad-spectrum capability to induce an immune response across multiple virus subtypes. As used herein, conferring “immunogenicity protection” refers to eliciting a protective immune response (e.g., cellular and / or humoral and preferably both) in a subj ect.

[0075] As used herein, the term “cross-protective” refers to the ability of an immune response, immunogenic composition, or vaccine to confer protection against infection or disease caused by two or more distinct but related pathogens, strains, subtypes, or lineages. In the context of influenza viruses, a “cross-protective” immune response or vaccine elicits immunity that provides protection against heterologous influenza virus strains, including different subtypes of influenza A viruses (e.g., Hl, H3, H5, H7, H9) and / or distinct lineages of influenza B viruses (e.g., B / Yamagata and B / Victoria). A cross-protective response can include, but is not limited to, induction of antibodies, T-cells, or other immune effectors that recognize conserved viral epitopes shared among different influenza subtypes or lineages. Cross-protection can be measured by any suitable assay known in the art, including heterologous viral challenge studies, reductions in viral load, decreased morbidity' or mortality’ following infection, or in vitro assays demonstrating antibody cross-reactivity' or T-cell cross-reactivity to antigens from multiple influenza strains.

[0076] In other embodiments, the immunogenic composition (e.g., a vaccine) is provided as a standalone formulation that does not require delivery by a viral vector. Such a composition can comprise, for example, purified recombinant NPs of influenza A and influenza B viruses, optionally linked to at least 21 amino acid residues of an AIP-C5 peptide or a functional fragment thereof, formulated with a pharmaceutically acceptable carrier and, optionally, an adjuvant. When administered to a subject (e.g., intranasally, orally, or intramuscularly), the composition can elicit general immunogenicity protection and / or cross-protective immune response that confers protection against multiple influenza vims subtypes, including but not limited to Hl, H3, H5, H7, and H9 influenza A strains, and B / Yamagata, and B / Victoria influenza B lineages.

[0077] Accordingly, the present disclosure encompasses both vector-based and vector-free vaccine embodiments, each capable of eliciting cross-protective immunity against at least one influenza A strain and at least one influenza B vims lineage.

[0078] The Ad vectors hereof can comprise a polynucleotide sequence that encodes at least a first NP from an IAV or a functional fragment thereof, and at least a second NP from an IBV or a functional fragment thereof.

[0079] As used herein, the term '‘functional fragment” refers to a portion or sub-sequence of a full-length polypeptide, peptide, or nucleic acid that retains at least one biological activity or immunological property’ of the corresponding full-length molecule. In the context of the present disclosure, a functional fragment of an influenza nucleoprotein (NP) or an Autophagy-Inducing70855-02Peptide C5 (AIP-C5) includes, but is not limited to, a sequence that maintains the ability to elicit an immune response (e.g., an immunogenic epitope), induce or enhance autophagy, promote antigen processing or presentation, or confer cross-protective immunity7when administered to a subject. Functional fragments can include contiguous or non-contiguous amino acid residues, conservative variants, or fusion constructs that preserve at least one relevant biological or immunogenic function of the parent sequence.

[0080] As used herein, the term “immunogenic fragment” refers to a specific ty pe of functional fragment that retains the immunogenic activity of the full-length polypeptide. An immunogenic fragment is a portion or sub-sequence of a full-length polypeptide or peptide that is capable of eliciting an immune response in a subject. Such fragments can comprise, for example, an epitope or antigenic determinant recognized by B cells and / or T cells, and can induce humoral, cellular, or mucosal immune responses. Immunogenic fragments can include contiguous or non-contiguous amino acid residues derived from an influenza NP or from an AIP-C5 sequence, and can be naturally occurring, recombinant, or synthetic. In some embodiments, an immunogenic fragment retains at least about 70%, 80%, 90%, 95%, 98%, 99%, or greater sequence identity to a corresponding region of the full-length NP or AIP-C5 sequence while preserving its ability7to elicit a detectable immune response.

[0081] Human Ads (HAds) are well-known in the art (as described above) and can be constructed to include one or more of the components described herein. Alternatively, nonhuman Ads, such as chimpanzee, simian, ovine, avian, murine, porcine, or bovine Ad vectors (e.g. , ChAd, Sad, OAd, AAd, Mad, PAd, or BAd vectors) can be used. Typically, the Ad is a replicationdefective Ad that is incapable of multiple cycles of transcription and translation of the inserted genes in human cells. The replication-defective Ad vectors can have deletions in one or more genes (or regions) involved in replication, including one or more of an El region, an E3 region, an E2 region, and / or an E4 region, thereby rendering the vector incapable of replication within normal host cells. For instance, a replication-defective Ad vector can have a deletion in an El region, an E3 region, an E2 region, an E4 region, or a combination of any or all of the foregoing.

[0082] In certain embodiments, the Ad vector is a BAd vector. BAd vector-based influenza vaccines can induce stronger CMI responses than conventional HAd vector-based vaccines. See Sayedahmed et al., A bovine adenoviral vector-based H5N1 influenza-vaccine provides enhanced immunogenicity and protection at significantly low dose, Molecular Therapy. Methods & Clinical Development 10: 210-222 (2018). Prolonged persistence and higher proinflammatory chemokine and cytokine expression have also been observed in mice inoculated with BAd vectors, as compared to HAd vectors. See Sharma et al., Comparative analysis of vector biodistribution, persistence, and gene expression following intravenous delivery of bovine, porcine and human70855-02 adenoviral vectors in a mouse model. Virology 386: 44-54 (2009); Sharma et al.. Evaluation of innate immunity and vector toxicity following inoculation of bovine, porcine or human adenoviral vectors in a mouse model, Virus Research 153: 134-142 (2010). Further, the present investigators have previously demonstrated that the intranasal vaccination of mice with the HAd vector expressing NP of an H7N9 virus could provide protection against a broad panel of lAVs. See Sayedahmed et al. (2024). Additionally, potential binding of the BAd vector with PF4 and other blood factors can be advantageous (e.g., to facilitate mucosal administration).

[0083] In certain embodiments, the BAd vector is replication-defective and can bypass preexisting H Ad-neutralizing antibodies or cellular immunity. See Reddy et al., Replicationdefective bovine adenovirus type 3 as an expression vector, J Virology 73: 9137-9144 (1999); Tandon et al., Sequential administration of bovine and human adenovirus vectors to overcome vector immunity in an immunocompetent mouse model of breast cancer, Virus Research 163: 202-211 (2012). The use of BAd vectors can enhance innate immunity and induce higher levels of CMI responses as compared to conventional HAd delivery systems. Further, BAd vectors can exhibit improved biodistribution to the lungs as compared to HAd vector systems, which can further enhance their potential for respirator}' vaccine deliver}'.

[0084] In certain embodiments, the Ad vector is a BAd type 3 (BAd3) vector. BAd3 vectors can be engineered as a replication-defective gene expression vector to evade preexisting Ad vector immunity. See Reddy et al. (1999), supra,' Singh et al., Bovine adenoviral vector-based H5N1 influenza vaccine overcomes exceptionally high levels of pre-existing immunity against human adenovirus, Molecular Therapy: J Am Soc 'y Gene Therapy 16: 965-971 (2008). Unlike many HAd or ChAd vectors that use a coxsackievirus and adenovirus receptor (CAR) for cellular entry, BAd3 utilizes a sialic acid receptor for virus entry, making it suitable for nasal and / or mucosal delivery. See Li et al., Bovine adenovirus serotype 3 utilizes sialic acid as a cellular receptor for virus entry. Virology 392: 162-168 (2009).

[0085] In certain embodiments, the Ad vector can comprise a polynucleotide sequence that encodes a NP of an IAV. In certain embodiments, the Ad vector comprises a polynucleotide sequence that encodes a NP of an IBV. In further embodiments, the Ad vector comprises polynucleotide sequences that encode both an IAV NP and an IBV NP and, optionally, further includes more than one promoter to drive expression of each NP independently. The Ad vector can also comprise a polynucleotide sequence that encodes one or more additional immunogenic domains or epitopes of an influenza virus other than NP, such as an M2e domain or a conserved region of HA or NA.

[0086] The Ad vector can be suitable for expression in a mammalian cell. Suitability for expression refers to the inclusion of one or more regulator}- elements and sequence features that70855-02 enable transcription and translation of the encoded antigen(s) in mammalian host cells. Such features can include, without limitation, a promoter active in mammalian cells (e.g., CMV immediate early, EFla, RSV LTR, SV40, or UbC), a Kozak consensus sequence to enhance translation initiation, an intron to improve mRNA stability, and a polyadenylation signal for transcript termination. In some embodiments, the polynucleotide sequences encoding the influenza NPs and / or the AIP-C5 are codon-optimized for mammalian expression to improve translational efficiency. The Ad vector can further include enhancer elements and transcriptional control regions suitable for expression in a wide range of mammalian cells, including but not limited to human, bovine, ovine, porcine, murine, canine, or primate cells.

[0087] In certain embodiments, the Ad vector is capable of expressing the encoded immunogens in vivo following administration to a mammalian subject, such as a human or veterinary subject, or in vitro following transduction of mammalian cells for antigen production. Mammalian cell lines suitable for in vitro expression or vector propagation can include, for example, human embryonic kidney (HEK293 or 293T) cells, PER.C6 cells, Madin-Darby bovine kidney (MDBK) cells, Vero cells, or other complementing cell lines that provide deleted adenoviral gene functions in trans e.g., El or E3 proteins). In certain embodiments, the Ad vector can also be suitable for expression in primary' mammalian cells, including epithelial or dendritic cells, following mucosal or parenteral administration.

[0088] In certain embodiments, the Ad vector comprises one or more promoters operably linked to the polynucleotide sequences encoding the IAV NP, the IBV NP, or both. In some embodiments, a single promoter drives expression of a bicistronic or polycistronic transcript encoding both the IAV NP and the IBV NP, optionally separated by a self-cleaving peptide sequence (e.g., a P2A, T2A, or F2A element) or an internal ribosome entry site (IRES). In other embodiments, the Ad vector comprises multiple promoters, such as two distinct promoters, each operably linked to a separate transcriptional cassette encoding the IAV NP or the IBV NP. In yet other embodiments, the Ad vector comprises multiple expression cassettes, such as two distinct cassettes, each comprising a promoter operably linked to a polynucleotide encoding either the IAV NP or the IBV NP. The Ad vector and / or its expression cassettes can further include regulatory elements that enhance expression, such as enhancers, polyadenylation sequences, and transcriptional termination sites.

[0089] The promoters can be constitutive, inducible, or tissue-specific, and can be derived from viral or cellular sources, including but not limited to cytomegalovirus (CMV), elongation factor 1 -alpha (EFla), Rous sarcoma virus (RSV), simian virus 40 (SV40), or human ubiquitin C (UbC) promoters. In certain embodiments, selection of the promoter or combination of promoters is used to modulate expression levels and optimize immunogenicity of the encoded immunologic70855-02 proteins.

[0090] To further enhance T-cell immunity directed against NP, the polynucleotide sequence of the Ad vector can further encode an Autophagy -Inducing Peptide (AIP) C5 (AIP-C5) derived from the secreted CFP10 protein of Mycobacterium tuberculosis (Mtb), or a functional fragment thereof. In certain embodiments, the AIP-C5 comprises 21 -amino acids.

[0091] Inclusion of the C5-AIP sequence with the NP genes of influenza A and influenza B viruses can enhance both T-cell-mediated and humoral immune responses, thereby broadening the protective efficacy of an Ad vector-based universal influenza vaccine hereof. For example, when incorporated into the Ad vectors hereof, AIP-C5 can upregulate genes associated with the autophagy pathway and act as a molecular adjuvant by promoting autophagy, enhancing antigen processing and presentation, and strengthening downstream immune responses. Indeed, incorporation of AIP-C5 has been show n to increase NP-specific CD8+T-cell responses when C5- NP constructs are delivered via a HAd vector. See Sayedahmed et al. (2023), supra.

[0092] Autophagy is a conserved cellular process that degrades unwanted cytoplasmic components, including damaged organelles and macromolecules, while recycling nutrients to maintain cellular homeostasis. In addition to its housekeeping functions, autophagy contributes to both innate and adaptive immunity by degrading intracellular bacteria, parasites, and viruses, and by facilitating immune signaling pathways. See Levine & Deretic, Unveiling the roles of autophagy in innate and adaptive immunity, Nature Reviews Immunology 7: 767-777 (2007); Paludan et al., Endogenous MHC class II processing of viral nuclear antigen after autophagy. Science 307: 593-596 (2005); Schmid & Miinz, Innate and adaptive immunity through autophagy, Immunity 27 : 11-21 (2007).

[0093] Autophagy can modulate antigen presentation through both major histocompatibility complex (MHC) class I (MHC-I) and class II (MHC-II) pathways during infection with intracellular pathogens. See Paludan et al. (2005), supra and English et al., Autophagy7enhances the presentation of endogenous viral antigens on MHC class I molecules during HSV-1 infection, Nature Immunology 10: 480-487 (2009). Moreover, autophagy can facilitate antigen processing and presentation by dendritic cells, a property that can be harnessed to enhance vaccine efficacy. See Levine et al., Autophagy' in immunity' and inflammation, Nature 469: 323-335 (2011); Jagannath et al., Autophagy enhances the efficacy of BCG vaccine by increasing peptide presentation in mouse dendritic cells, Nature Medicine 15: 267-276 (2009).

[0094] The AIP-C5, derived from the secreted CFP10 protein of M. tuberculosis, has been shown to enhance CMI responses against M. tuberculosis , SAR.S-CoV-2, and influenza virus when expressed in an Ad vector-based vaccine platform. See Sayedahmed et al. (2024), supra-, Sayedahmed et al.. Impact of an autophagy -inducing peptide on immunogenicity and protection70855-02 efficacy of an adenovirus-vectored SARS-CoV-2 vaccine. Molecular Therapy, Methods & Clinical Development 30: 194-207 (2023); Khan et al., A recombinant bovine adenoviral mucosal vaccine expressing mycobacterial antigen-85B generates robust protection against tuberculosis in mice. Cell Reports Medicine 2: 100372 (2021).

[0095] Accordingly, the Ad vector (e.g., BAd vector) can comprise a polynucleotide sequence that encodes AIP-C5 from the CFP 10 protein of M. tuberculosis (e.g. , comprising at least 21 amino acid residues of AIP-C5), which may be inserted, for example, into a deleted El region of the vector genome.

[0096] The Ad vector can have a mutation (e.g.. a deletion, insertion, inversion, or substitution) in an El region, an E2 region, an E3 region, and / or an E4 region of the vector genome. In certain embodiments, at least the El region and the E3 region of the Ad vector are deleted to render the vector replication-defective. In certain embodiments, a polynucleotide sequence of a NP is inserted into the deleted El region. In other embodiments, a polynucleotide sequence encoding at least an AIP-C5 (e.g., derived from the CFP 10 protein of m. tuberculosis) or a functional fragment thereof is inserted into a deleted El region of the Ad vector. In additional embodiments, a polynucleotide sequence encoding both the influenza NPs and the AIP-C5 peptide, or functional fragment thereof, is inserted into the deleted El region of the Ad vector.

[0097] Deletion of the E3 region can further increase the cloning capacity of the Ad vector genome, permitting the insertion of larger or multiple heterologous antigen-encoding sequences such as those encoding IAV and IBV NPs, AIP-C5, or combinations thereof. In addition, removal of the E3 region, which normally encodes immunomodulatory7proteins that inhibit host immune responses, can enhance the safety7and immunogenicity of the vector by reducing interference with host antiviral mechanisms. The resulting El- and E3-deleted Ad vector can be replicationdefective in normal mammalian cells but can be propagated in complementing cell lines that provide the deleted functions in trans (e.g., cells expressing Ad El proteins). Accordingly, such El / E3-deleted vectors can serve as safe and efficient delivery platforms for expressing one or more influenza antigens and for eliciting cross-protective immune responses in vivo.

[0098] In certain embodiments, the AIP-C5 comprises the amino acid sequence set forth in SEQ ID NO: 9. The AIP-C5 can comprise an amino acid sequence having at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%. at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or greater sequence identity7to the amino acid sequence set forth in SEQ ID NO: 9, and / or a polynucleotide sequence encoding the AIP-C5 having at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 91%, at least at or about 92%. at least at or about 93%, at least at or about70855-0294%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or greater sequence identity to the nucleotide sequence set forth in SEQ ID NO: 10.

[0099] As used herein, the terms ‘“sequence identity” and “percent identity” refer to the degree of sequence similarity between two nucleic acid or amino acid sequences when optimally aligned to maximize matching residues. Sequence alignment refers to the process of arranging two or more sequences to achieve the highest level of correspondence between identical or functionally conserved nucleotides or amino acids, optionally allowing for the introduction of gaps to account for insertions or deletions. In certain embodiments, sequence alignment can be performed manually or using computer algorithms that are well known in the art, including but not limited to BLAST, ClustalW, or the GAP program in the GCG (Genetics Computer Group) software package, using default parameters unless otherwise specified.

[0100] The “identity fraction” for a sequence aligned with a reference sequence is calculated as the number of identical residues in positions of alignment (excluding gaps introduced into the reference sequence) divided by the full-length of the reference sequence. The “‘percent identity” is the identity fraction multiplied by 100. Unless otherwise specified, sequence identity values described herein refer to pairwise alignments performed over the entire length of the reference sequence.

[0101] In certain embodiments, the Ad (e.g. , B Ad) vector comprises a polynucleotide sequence comprising SEQ ID NO: 1, which encodes a NP of IAV fused to an AIP-C5 derived from the CFP10 protein of M. tuberculosis (e.g., at least 21 amino acid residues from AIP-C5), or a functional fragment thereof (e.g., an immunogenic epitope). In certain embodiments, the polynucleotide has at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or greater sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0102] The peptide encoded by SEQ ID NO: 1, or by a polynucleotide sequence having an acceptable variation thereof, can comprise or consist of the amino acid sequence set forth in SEQ ID NO: 2. In certain embodiments, the peptide encoded by SEQ ID NO: 1 (or by an acceptable nucleotide sequence variant thereof) comprises or consist of the amino acid sequence set forth in SEQ ID NO: 2, or has at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or greater sequence identity to the amino acid sequence set forth in SEQ70855-02ID NO: 2.

[0103] In certain embodiments, the Ad vector comprises a polynucleotide sequence comprising SEQ ID NO: 3, which encodes a NP of IBV fused to an AIP-C5 derived from the CFP10 protein ofM. tuberculosis (e.g., at least 21 amino acid residues from AIP-C5), or a functional fragment thereof (e.g.. an immunogenic epitope). In certain embodiments, the peptide encoded by SEQ ID NO: 3 (or an acceptable nucleotide sequence variant thereof) comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4, or has at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%. at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or greater sequence identity to the amino acid sequence set forth in SEQ ID NO: 4.

[0104] In certain embodiments, the Ad vector comprises a polynucleotide sequence comprising SEQ ID NO: 5. which encodes both an NP of IAV and an NP of IBV, fused to an AIP-C5 derived from the CFP10 protein of M. tuberculosis (e.g., at least 21 amino acid residues from AIP-C5), or a functional fragment thereof (e.g. , an immunogenic epitope). In certain embodiments, the peptide encoded by SEQ ID NO: 5 (or an acceptable nucleotide sequence variant thereof) comprises or consist of the amino acid sequence set forth in SEQ ID NO: 6, or has at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or greater sequence identity to the amino acid sequence set forth in SEQ ID NO: 6.

[0105] In certain embodiments, the Ad vector comprises a polynucleotide sequence comprising or consisting of SEQ ID NO: 7, which encodes a fusion construct containing a NP of IAV and a NP of IBV, fused with an AIP-C5 derived from the CFP10 protein of M. tuberculosis (e.g., at least 21 amino acid residues from AIP-C5), or a functional fragment thereof (e.g., an immunogenic epitope). The Ad vector can additionally comprise two promoters that independently drive expression of the IAV NP and the IBV NP (e.g., C5-NP / A+B configuration).

[0106] The Ad vector can comprise or consist of a polynucleotide sequence having at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 85%, at least at or about 88%, at least at or about 90%, at least at or about 92%. at least at or about 95%, at least at or about 98%, at least at or about 99%, or greater sequence identity to any one of SEQ ID NOs: 1, 3, 5, or 7.

[0107] The Ad vectors described herein can be utilized directly as vaccine delivery platforms or as production systems for generating influenza antigens that are subsequently formulated into vaccines. These vectors express conserved influenza antigens, such as NPs of IAV and IBV,70855-02 optionally fused to an AIP-C5 peptide or a functional fragment thereof, to elicit broad and durable immune responses. The antigens produced from such vectors can be formulated with suitable carriers, excipients, or adjuvants to produce immunogenic compositions capable of inducing cross-protective immunity against multiple influenza subtypes.

[0108] Also provided are isolated nucleic acid molecules and corresponding polypeptides useful in the preparation of Ad vectors or immunogenic compositions described herein. In certain embodiments, an isolated nucleic acid molecule is provided that comprises sequences encoding: (a) aNP of an IAV; (b) a NP of an IBV: and (c) optionally, at least 21 amino-acid residues of an AIP-C5 derived from the CFP 10 protein of M tuberculosis (Mtb), or a functional fragment thereof (e.g., an immunogenic epitope). In some embodiments, the nucleic acid molecule is codon- optimized for mammalian expression to enhance translation efficiency in human or veterinary host cells.

[0109] In certain embodiments, the sequences encoding the IAV NP and IBV NP are fused inframe and optionally separated by a linker peptide that provides structural flexibility and facilitates independent folding or processing of the NP antigens. Exemplary linkers can include flexible glycine-serine-rich sequences, such as (G4S)n, where n is an integer from 1 to 3, or other biologically compatible linker sequences know n in the art.

[0110] In some embodiments, the isolated nucleic acid molecule has at least at or about 80%. at least at or about 85%, at least at or about 90%, at least at or about 95%, at least at or about 98%, at least at or about 99%, or greater sequence identity to SEQ ID NO: 5 or SEQ ID NO: 7, or encodes a polypeptide having at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 95%, at least at or about 98%, at least at or about 99%. or greater aminoacid sequence identity to the corresponding amino-acid sequences set forth in SEQ ID NO: 5 or SEQ ID NO: 7. Such nucleic acids can be inserted into a viral or non-viral vector, expressed in a mammalian cell, or used directly in a DNA or RNA vaccine formulation.[OHl] Also provided are isolated polypeptides comprising: (a) an NP sequence of an influenza A virus (IAV); (b) an NP sequence of an influenza B virus (IBV); and (c) optionally, an AIP-C5 peptide from AT. tuberculosis or a functional fragment thereof. The isolated polypeptides can elicit cross-reactive T-cell and / or antibody responses against both IAV and IBV when administered to a subject, thereby providing cross-protective immunity. In certain embodiments, the polypeptides are produced recombinantly in a mammalian, insect, or bacterial expression system and can be formulated with a pharmaceutically acceptable carrier for use in vaccine compositions.

[0112] Accordingly, immunogenic compositions (e.g., vaccines) are also provided that are cross-protective against at least lAVs and IBVs when administered to a subject. These compositions can include, but are not limited to, formulations containing one or more Ad vectors70855-02 described herein, or influenza virus antigens produced using such vectors (e.g., BAd-C5-NPA + BAd-C5-NP / B, or BAd-C5-NP / A+B).

[0113] In certain embodiments, the immunogenic composition comprises a NP of IAV and a NP of IBV. and a pharmaceutically acceptable carrier. In certain embodiments, the immunogenic composition further comprises at least 21 amino acid residues of AIP-C5 from a CFP10 protein ofM. tuberculosis or a functional fragment thereof. As used herein, the term “composition” refers to any product comprising more than one ingredient, including one or more influenza vims antigens, vectors, carriers, adjuvants, excipients, or other components formulated together for administration to a subject. In certain embodiments, the immunogenic composition comprises a NP of an H7N9 influenza virus with or without a sequence encoding at least 21 amino acid residues of AIP-C5 derived from a CFP10 protein of M. tuberculosis and a pharmaceutically acceptable carrier.

[0114] In certain embodiments, administration (e.g. , of an effective amount) of an immunogenic composition hereof to a subject induces CD8+T-cell responses cross-reactive to at least one influenza A subtype and at least one influenza B subtype. The immunogenic composition can be bivalent. As used herein, the term “bivalent” refers to an immunogenic composition that comprises two distinct antigens or immunological targets and is capable of inducing immune responses against both. In the context of the present disclosure, a bivalent immunogenic composition includes, for example, an NP of an IAV and an NP of an IBV, formulated with a pharmaceutically acceptable carrier. In certain embodiments, the immunogenic composition further comprises at least 21 amino-acid residues of an AIP-C5 derived from the CFP10 protein of M. tuberculosis (Mtb), or a functional fragment thereof (e.g., an immunogenic epitope). Such AIP-C5 sequences can enhance both humoral and cellular immune responses, thereby broadening the cross-protective efficacy of the bivalent vaccine formulation.

[0115] In certain embodiments, the immunogenic composition is bivalent, meaning that it induces immune responses in the subject against two distinct antigens or immunological targets (e.g., IAV NP and IBV NP). In certain embodiments, the immunogenic composition is monovalent. In other embodiments, the immunogenic composition is trivalent, including three distinct antigens or targets (for example, two influenza A subtypes and one influenza B lineage), or polyvalent, including four or more distinct influenza antigens or viral components. Polyvalent compositions can elicit immune protection against multiple heterologous or heterosubtypic influenza strains, lineages, or subtypes, thereby providing broad or “universal” influenza immunity.

[0116] The NP (or functional fragment thereof) can be expressed using any of the Ad vectors described herein, such as HAd or BAd vectors. The NP (or functional fragment thereof) can70855-02 comprise or consist of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, or 8, or a variant thereof having at least at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 95%, at least at or about 98%, at least at or about 99%, or greater sequence identity thereto. The AIP-C5 can be encoded by a polynucleotide sequence set forth in SEQ ID NO: 9 or an acceptable variant thereof. In certain embodiments, the AIP-C5 derived from a CFP 10 protein comprises at or about at least 90% sequence identity to SEQ ID NO: 9.

[0117] The immunogenic compositions can be formulated in unit dosage form and can include one or more pharmaceutically acceptable carriers, adjuvants, diluents, excipients, and / or vehicles, or combinations thereof. As used herein, the term “administering’" (and variants thereof) includes all routes and methods of introducing the antigen(s) or compositions to a subject, including, without limitation, oral (p.o.), intravenous (i.v.), intramuscular (i.m.), subcutaneous (s.c.), transdermal, inhalation (e.g., intranasal (i.n.)), buccal, intraocular, sublingual, vaginal, rectal, or other mucosal or parenteral routes.

[0118] The term “adjuvant” refers to any substance that enhances, specifically or non- specifically, an immune response to an antigen. Non-limiting examples of adjuvants suitable for use with the compositions and methods described herein include cholera toxin B subunit, flagellin, human papillomavirus LI or L2 proteins, herpes simplex virus glycoprotein D (gD), complement C4 binding protein, Toll-like receptor (TLR) ligands such as TLR4 ligands, interleukin-1 beta (IL- 1P), lysolecithin, pluronic polyols, polyanions, oil-in-water emulsions, dinitrophenol, iscomatrix, and liposome-polycation-DNA complexes. In some embodiments, the Ad vector provides an inherent adjuvant effect, and no additional adjuvant is required.

[0119] The immunogenic compositions can further comprise pharmaceutically acceptable salts, particularly where the composition comprises a live vaccine prepared using Ad vectors provided herein. As used herein, “salts” includes buffered salts and other conventional, non-toxic salts such as sodium, potassium, or aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate). Additional exemplary salts include those derived from inorganic acids (e.g., hydrochloric, hydrobromic, sulfuric, phosphoric, nitric) or organic acids (e.g., acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, fumaric, benzoic, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, methanesulfonic, toluenesulfonic, sulfanilic, 2-acetoxybenzoic, oxalic, and isethionic acids).

[0120] The immunogenic compositions can be formulated as pharmaceutical compositions for administration to a mammalian subject (e.g., a human patient) via any suitable route. Illustrative forms include intranasal, subcutaneous, oral, or aerosol formulations. In certain embodiments, the immunogenic composition is administered intranasally, subcutaneously, orally, or as an aerosol spray. In certain embodiments, the immunogenic compositions are formulated to be administered70855-02 intranasally, or orally (e.g., mucosally). In certain embodiments, the immunogenic composition is formulated as an aerosol spray. The compositions can be systemically administered in combination with a pharmaceutically acceptable vehicle.

[0121] The relative percentages of components of the composition can vary depending on the formulation and can include about 1% to about 99% by weight of one or more active antigens, in combination with carriers, binders, excipients, disintegrants, lubricants, sweeteners, or other conventional ingredients. The amount of active antigen or vector in the composition can be selected to provide an effective dosage level when administered to a subject.

[0122] Exemplary formats for oral administration include tablets, capsules, elixirs, and syrups. Exemplary routes for parenteral administration include intravenous, intraarterial, intraperitoneal, epidural, intraurethral, intramuscular, and subcutaneous routes. Parenteral administration can be achieved by needle or microneedle injection, needle-free injector systems, or infusion devices.

[0123] Parenteral formulations are typically aqueous or sterile non-aqueous solutions or dispersions containing the active ingredient(s) and pharmaceutically acceptable excipients such as salts, carbohydrates, and buffering agents, preferably at a pH of about 3 to about 9. In certain embodiments, the compositions can be provided as sterile non-aqueous solutions or as dried (e.g., lyophilized) powders to be reconstituted with a suitable vehicle such as sterile, pyrogen-free water. Parenteral administration can also employ formulations containing liposomes or other delivery systems to enhance antigen uptake and immune response. When the antigen is poorly soluble, solubilizers such as ethanol or surfactants can be included.

[0124] The pharmaceutical dosage forms suitable for injection, intranasal administration, or infusion can include sterile aqueous solutions or dispersions, or sterile powders for reconstitution. The dosage form should be sterile, fluid, and stable under conditions of manufacture and storage. The carrier or vehicle can include water, electrolytes, sugars, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycols), vegetable oils, non-toxic glyceryl esters, or mixtures thereof. Fluidity can be maintained by inclusion of surfactants, control of particle size, or formation of liposomes. Sterile solutions can be prepared by dissolving or dispersing the immunogenic compositions in the desired solvent, followed by filtration or sterilization. Lyophilization or vacuum-drying methods can be used to produce sterile powders for reconstitution prior to administration.

[0125] The dosage administered to a subject will depend upon several factors, including the method of administration, the specific viral strain or antigen targeted, the presence or absence of active infection, whether prophylactic or therapeutic immunization is intended, and patientspecific factors such as age, body weight, health status, and genetic profile. In certain embodiments, pharmacogenomic data (e.g., the subject’s genotype affecting pharmacokinetic or70855-02 immunologic response) can also inform the dosage used. The inclusion of AIP-C5 or other molecular adjuvants can enhance immunogenicity, thereby allowing reduced antigen doses to achieve effective immune protection.

[0126] The Ad vectors, isolated nucleic acids, isolated polypeptides, and immunogenic compositions described herein can be useful for a variety of purposes, including both the production of influenza antigens and the induction of protective immune responses in a subj ect. For example, the Ad vectors and nucleic acid constructs can be employed to produce influenza virus antigens in vitro, in vivo, or in ovo, either for subsequent formulation into vaccines or for direct use as vaccine delivery platforms. The isolated polypeptides disclosed herein, which correspond to conserved influenza antigens such as NPs of lAVs and IBVs, optionally fused to an AIP-C5 or a functional fragment thereof, can also be used directly as immunogens in vaccine formulations or as reference antigens in analytical assays. Collectively, these Ad vectors, nucleic acids, and polypeptides can elicit broad, cross-protective immune responses encompassing multiple influenza subtypes and lineages.

[0127] Accordingly, the present disclosure provides methods for generating or enhancing immunogenicity in a subject against heterologous or heterosubtypic influenza viruses, and methods for eliciting an immune response in a subject through administration of the Ad vectors, isolated nucleic acids, isolated polypeptides, or immunogenic compositions described herein. In certain embodiments, the immune response induces a humoral immune response, a CMI response, or both, thereby promoting cross-protective immunity against multiple influenza A and B subtypes and lineages. The disclosed methods can be applied prophylactically to prevent influenza infection or therapeutically to augment or restore immune protection in previously exposed, infected, vaccinated, or immunocompromised subjects. In some embodiments, the methods further comprise administering the Ad vector, nucleic acid, or polypeptide composition in an effective amount to induce durable memory T-cell and / or antibody responses.

[0128] In certain embodiments, a method of inducing a cross-protective immune response against a heterosubtypic influenza virus in a subject comprises administering to the subject an effective amount of any of the immunogenic compositions or Ad vectors described herein. Administration of such an effective amount can provide general immunogenicity protection (e.g., cross-protection) to the subject against multiple influenza virus subtypes, including both IAV and IBV strains.

[0129] As used herein, the term '‘effective amount” refers to that quantity of active antigen (or fragment or epitope thereof), compound, or pharmaceutical agent that elicits a detectable immune response (e.g., secretory', humoral, and / or cellular protective immunity) in a subject, such as a mammal, that is reactive with one or more targeted disease-producing viral trains. The term70855-02■‘protective immunity" refers to an immune response induced by a vaccine or immunization regimen that prevents, delays, or reduces the severity of disease caused by a viral strain (e.g., an influenza virus), or diminishes or eliminates disease symptoms.

[0130] In one aspect, an effective amount is the quantity of antigen (or epitope thereof), compound or pharmaceutical agent sufficient to produce a measurable change in an immune response indicator between pre- and post-administration samples from a subject. Immune response indicators include, without limitation, antibody titers or specificity as measured by assays such as enzyme-linked immunoassay (ELISA), virus-neutralization assay, hemagglutination inhibition, ELISpot assay, flow cytometry, immunoprecipitation, Ouchterlony immunodiffusion, Western blot, antigen array binding assays, or cytotoxicity assays.

[0131] The total dosage and administration schedule of the compositions and / or vectors described herein can be determined by the attending physician, veterinarian, or clinician within the scope of sound medical judgment. The specific effective dose level for any subject will depend on a variety of factors, including the disorder being prevented or treated, its severity, the activity of the specific compound or antigen, the formulation, the route and timing of administration, and subject-specific variables such as age, weight, health status, sex, and metabolic rate. The presence of additional adjuvants or immune enhancers, such as AIP-C5, can also influence dosing, as AIP- C5 -mediated enhancement of T-cell responses can permit a reduced antigen dose while maintaining protective efficacy.

[0132] Also provided are methods of eliciting an immune response against an influenza virus in a subject. Such methods can comprise administering to the subject an effective amount of any immunogenic composition hereof or any Ad vector described herein. In particular embodiments, the Ad vector comprises a BAd3 vector that encodes at least a first NP of an IAV and a second NP of an IBV. In some embodiments, the BAd3 vector further encodes an AIP-C5 peptide or a functional fragment thereof.

[0133] Methods of inducing cross-protective immune response against at least IAV and IBV in a subject are also provided. Such methods can comprise administering to the subject an effective amount of any immunogenic composition hereof or any Ad vector described herein.

[0134] In the methods described herein, administration of an effective amount of the immunogenic composition, Ad vector, isolated nucleic acid, or isolated polypeptide can induce a dose-dependent increase in both humoral and CMI responses in the subject. Such administration can result in elevated antigen-specific antibody titers, enhanced T-cell responses e.g., CD4+and CD8+effector and memory responses), and increased cytokine production associated with antiviral immunity. In certain embodiments, administration of the composition, vector, or antigenic construct also leads to a measurable reduction in viral load following heterologous70855-02 influenza challenge, demonstrating cross-protective efficacy against multiple influenza subtypes and lineages.

[0135] For methods described herein comprising an administration step, the antigens and compositions can be administered in a single dose, or via a combination of multiple dosages, which can be administered by any suitable means, contemporaneously, simultaneously, sequentially, or separately. Where the dosages are administered in separate dosage forms, the number of dosages administered per day for each antigen or composition can be the same or different. The antigen and / or composition dosages can be administered via the same or different routes of administration. The antigens or compositions can be administered according to simultaneous or alternating regimens, at the same or different times during the therapy, concurrently in divided or single forms.

[0136] Depending upon the route of administration, a wide range of permissible dosages are contemplated herein, including doses falling in the range from about 106to 1011virus particles (VP) / kg. The dosages may be single or divided and may be administered according to a wide variety of protocols, including q.d. (once a day), b i d. (twice a day), t.i.d. (three times a day), or even every7other day, once a week, once a month, once a quarter, and the like. In each of these cases it is understood that the effective amounts described herein correspond to the instance of administration, or alternatively to the total daily, weekly, month, or quarterly dose, as determined by the dosing protocol.

[0137] In addition to the illustrative dosages and dosing protocols described herein, an effective amount of any one or a mixture of the compounds described herein can be determined by the attending diagnostician or physician using known techniques and / or by observ ing results obtained under analogous circumstances. In determining the effective amount or dose, a number of factors are considered by the attending diagnostician or physician, including, but not limited to the species of mammal, including human, its size, age, and general health, the specific disease or disorder involved, the degree of or involvement or the severity of the disease or disorder, the response of the individual patient, the particular compound administered, the mode of administration, the bioavailability characteristics of the preparation administered, the dose regimen selected, the use of concomitant medication, and other relevant circumstances.

[0138] Further provided are methods for producing influenza antigens, an immunogenic composition described herein, and / or manufacturing a medicament comprising any of the immunogenic compositions, isolated nucleic acids, or isolated polypeptides described herein. In certain embodiments, influenza antigens are produced by replicating or expressing an Ad vector or isolated nucleic acid construct in a suitable host cell, wherein the vector or nucleic acid comprises at least one polynucleotide sequence encoding a polypeptide having at least at or about70855-0280%, at least at or about 85%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or greater aminoacid sequence identity to any one of SEQ ID NOs: 2, 4, 6, or 8. The resulting influenza antigen can include a NP antigen derived from an Hl, H3, H5, H7. or H9 influenza A strain, or from an influenza B strain, or a functional fragment thereof (e.g, one or more immunogenic epitopes).

[0139] In certain embodiments, the Ad vector or isolated nucleic acid comprises sequences encoding at least SEQ ID NOs: 2 and 4, or a plurality of influenza antigens, including without limitation SEQ ID NO: 6 or SEQ ID NOs: 2 and 4. In additional embodiments, the influenza antigens or recombinant polypeptides produced according to these methods can be purified and formulated with a pharmaceutically acceptable carrier, excipient, or adjuvant to yield an immunogenic composition or vaccine suitable for administration to a subject.

[0140] In certain embodiments, influenza virus antigens are produced by introducing an Ad vector (e.g. , any of the Ad vectors described herein) or an isolated nucleic acid construct described herein into a host cell or culture medium that can support replication of the vector and / or expression of the encoded influenza antigens. In additional embodiments, the isolated nucleic acids or corresponding expression plasmids can be transfected, electroporated, or otherwise delivered into suitable eukaryotic or prokaryotic cells to produce the encoded polypeptides. Suitable host cells can include, without limitation, mammalian cells that provide one or more adenoviral gene functions deleted from the vector genome (e.g., El and / or E3), such as human embry onic kidney (HEK293 or 293T) cells, PER.C6 cells, Vero cells, or Madin-Darby bovine kidney (MDBK) cells, or other complementing cell lines capable of supporting Ad vector propagation and / or heterologous gene expression. In some embodiments, the host cell comprises a heterologous nucleic acid that complements replication functions absent in the vector (e.g, a nucleic acid encoding one or more E-region proteins deleted from the Ad genome). Cells suitable for supporting vector growth or recombinant expression can accommodate replication or transcription of different Ad serotypes or plasmid systems with distinct species tropisms.

[0141] Such cells are then cultured in a culture medium under conditions that allow’ for expression of a first NP from an IAV and a second NP from an IBV. Such methods can be performed in vitro or in ovo and can further include isolating the expressed antigen or vector and formulating it into a vaccine composition with one or more pharmaceutically acceptable carriers or adjuvants.

[0142] In additional embodiments, the host cell can be a non-complementing mammalian cell or a primary7cell used for transient expression of influenza antigens without vector replication. The host cells can be cultured using standard cell-culture methods known in the art, such as in70855-02 monolayer or suspension formats, and in various media formulations optimized for adenoviral replication or antigen expression. Exemplary culture media include, but are not limited to, Dulbecco’s Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Eagle’s MEM, RPMI 1640, or serum-free formulations optimized for viral or recombinant protein production, such as OptiPRO™. Ex-Cell™. VP-SFM, or Pro293™. In certain embodiments, the medium can be supplemented with fetal bovine serum (FBS), newborn calf serum, horse serum, or other suitable serum substitutes, typically at concentrations ranging from about 2% to about 10%. In other embodiments, chemically defined or protein-free media formulations are employed to facilitate large-scale or Good Manufacturing Practice (GMP) production.

[0143] The medium can further contain salts, amino acids, vitamins, glucose or other carbon sources, buffering agents, and antibiotics (e.g., penicillin, streptomycin) to maintain sterility' and support optimal cell viability7. In some embodiments, additives such as glutamine or glutamine substitutes (e.g., GlutaMAX™) are included to enhance viral or protein yield. The cells can be maintained under standard culture conditions (e.g, at about 35-38°C, 5-7% CO?, and 85-95% humidity) and may be grown in monolayer, suspension, or microcarrier-based systems. In certain embodiments, bioreactor or spinner-flask systems are employed for scale-up, enabling high- density culture and efficient production of Ad vectors, recombinant nucleic acids, or influenza antigens.

[0144] Following replication or expression, the Ad vector, recombinant nucleic acid, or expressed influenza antigen can be isolated and purified using conventional techniques such as density-gradient centrifugation (e.g., cesium chloride or iodixanol gradients), chromatography (e.g, ion exchange, affinity, or size-exclusion), tangential-flow filtration, or other purification methods known in the art. The purified Ad vector, recombinant nucleic acid, or expressed influenza antigen can then be formulated with a pharmaceutically acceptable carrier, excipient, or adjuvant to yield an immunogenic composition or vaccine suitable for administration to a subject.

[0145] Cell lines are also provided that express the antigens and / or Ad vectors described herein. In certain embodiments, a cell line is provided that allows an Ad vector described herein to infect cells of the line and replicate a genome of the Ad vector. The Ad vector genome can include, for example, a polynucleotide sequence encoding at least a first NP from an IAV or a functional fragment thereof, and a second NP from an IBV or a functional fragment thereof. In some embodiments, the genome further comprises a polynucleotide sequence encoding an AIP-C5 derived from the CFP10 protein of M. tuberculosis (Mtb). A cell comprising any isolated recombinant Ad vector described herein is also encompassed by the present disclosure.

[0146] Those skilled in the art will recognize that numerous modifications can be made to the specific implementations described above. The implementations should not be limited to the70855-02 limitations described. Other implementations may be possible. Indeed, while the concepts of the present disclosure are illustrated and described in detail in the description herein, results in the description are to be considered as exemplary7and not restrictive in character; it being understood that only the illustrative embodiments are shown and described, and that all changes and modifications that come within the spirit of the disclosure are desired to be protected.

[0147] It is intended that the scope of the present methods and compositions be defined by the following claims. However, it must be understood that this disclosure may be practiced otherwise than is specifically explained and illustrated without departing from its spirit or scope. It should be understood by those skilled in the art that various alternatives to the embodiments described herein may be employed in practicing the claims without departing from the spirit and scope as defined in the following claims.

[0148] Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting. Further, information that is relevant to a section heading may occur within or outside of that particular section.

[0149] All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages betw een this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0150] Certain Definitions

[0151] As used herein, the following terms and phrases shall have the meanings set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary7skill in the art, including those w orking in molecular biology7, virology7, immunology7, and vaccine development.

[0152] The term “about' ’ allows for a degree of variability in a value or range, for example, within ±10%, ±5%, or ±1% of a stated value or of a stated limit of a range, unless otherwise indicated.

[0153] The term “substantially” allows for a degree of variability in a value, range, or characteristic, for example, at least about 90%, 95%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or greater of a stated value or limit, such that the specified property or relationship is functionally maintained.

[0154] The terms “a,” “an,” and “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is generally used to mean a nonexclusive “or” (i.e., “A or B” means “A, B, or both”). In addition, unless expressly stated otherwise, all70855-02 terminology and phraseology employed herein is for descriptive purposes only and not for limitation.

[0155] The term “pharmaceutically acceptable carrier” is art-recognized and refers to any pharmaceutically acceptable material, composition, or vehicle - such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material - that facilitates the formulation, stability, or delivery of an active ingredient in a manner compatible with administration to a subject. Each carrier must be “acceptable” in the sense of being compatible with the active component(s) and not injurious to the subject. Exemplary' pharmaceutically acceptable carriers include, but are not limited to: (1) sugars e.g., lactose, glucose, sucrose); (2) starches (e.g, com starch, potato starch); (3) cellulose and its derivatives (e.g., sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate); (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients (e.g., cocoa butter, suppository waxes); (9) oils (e.g., peanut, cottonseed, safflower, sesame, olive, com, soybean); (10) glycols (e.g. propylene glycol); (11) polyols (e.g. glycerin, sorbitol, mannitol, polyethylene glycol); (12) esters (e.g, ethyl oleate, ethyl laurate); (13) agar; (14) buffering agents (e.g, magnesium hydroxide, aluminum hydroxide, phosphate buffers); (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer’s solution; (19) ethyl alcohol; and (20) other non-toxic, biocompatible substances commonly used in pharmaceutical formulations.

[0156] The terms “patient” and “subject” are used interchangeably and include any vertebrate, such as a mammal (including a human), laboratory animal (e.g, rodent, rabbit, nonhuman primate), domestic animal (e.g, dog, cat), agricultural animal (e.g, cow, horse, pig, sheep, goat), or wild animal in captivity' (e.g., elephant, panda, lion, giraffe, dolphin, whale). Preferably, the subject is a human.

[0157] The terms “cell” and “cell culture” are used interchangeably and include progeny of a parent cell or culture. It is understood that progeny may not be precisely identical in DNA content due to spontaneous or induced mutations, provided the progeny retain the relevant biological activity or function of the parent cell.

[0158] The term “gene” refers to a functional nucleic acid unit that encodes a protein, polypeptide, or peptide, including coding sequences for influenza antigens such as the nucleoprotein (NP), Matrix Protein 2 ectodomain (M2e), or hemagglutinin stem domain (HA2). The term encompasses genomic DNA, complementary DNA (cDNA), synthetic sequences, oligonucleotides, probes, and functional fragments thereof, whether naturally occurring or recombinant. “Purified” genes, nucleic acids, proteins, and the like refer to these entities when identified and separated from at least one contaminating nucleic acid or protein with which they are ordinarily associated.

[0159] The term “immunization’' refers to the process of inducing an immune response -70855-02 humoral, cellular, or both - that confers protective immunity against an influenza antigen (or a functional fragment thereof), either before or after exposure of the subj ect to the corresponding pathogen.

[0160] The terms '’immunogen" and ‘ mmunogenic’' refer to an antigen or antigenic composition capable of initiating lymphocyte activation and eliciting an antigen-specific immune response in a subject. An immunogen therefore includes any molecule containing one or more epitopes that stimulate secretory, humoral, and / or cell-mediated immune responses.

[0161] The terms “protein,"’ “polypeptide,"’ and “peptide"’ are used interchangeably (unless otherwise noted) and refer to chains of amino acids joined via peptide bonds, regardless of length, post-translational modification, or biological origin.

[0162] The term “vector” refers to a nucleic acid molecule or delivery system capable of transferring a DNA or RNA segment from one cell to another. The term includes both cloning vectors and expression vectors, the latter comprising regulatory elements (e.g.. promoters, enhancers, polyadenylation signals) operably linked to a coding sequence to permit expression in a host cell. In the context of the present disclosure, the term encompasses adenoviral vectors (e.g. , human or bovine Ad vectors), as well as other viral or non-viral delivery systems capable of delivering one or more influenza antigens or immunogenic sequences to a subject. Non-limiting examples include plasmid DNA, mRNA, liposomal carriers, nanoparticles, bacteria, protozoa, and cells (e.g., homologous or heterologous) that are live, live-attenuated, heat-killed, chemically inactivated, or recombinant. The skilled artisan will readily recognize the type of “vector” referenced herein based on the description of the materials and methods used and described in the present disclosure.EXAMPLES

[0163] The following examples sen e to illustrate the present disclosure. The examples are not intended to limit the scope of the claimed invention.Materials & Methods

[0164] Cells and Viruses

[0165] BHH / F5, BHH / F5-I-SceI (BHH / F5 cell line that expresses I-Scel endonuclease), BHH2C (bovine-human hybrid clone 2C), human embryonic kidney (HEK) 293 (CRL-1573, ATCC), 293Cre (293 cell line that expresses Cre recombinase), and Madin-Darby canine kidney (MDCK) (CCL-34, ATCC) cell lines were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Coming, Coming, NY) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT), gentamycin (50 mg / mL, Thermo Fisher Scientific, Waltham, MA) and penicillin (100U / mL)-streptomycin (100 pg / rnL) solution (Gibco, Thermo Fisher Scientific) at 37 °C in a 5% CO2 incubator, van Olphen & Mittal, Development and characterization of bovine x human70855-02 hybrid cell lines that efficiently support the replication of both wild-type bovine and human adenoviruses and those with El deleted, J Virology 76: 5882-5892 (2002); Sayedahmed et al., Current use of adenvirus vectors and their production methods, Methods in Molecular Biology 1937: 155-175 (2019).

[0166] The influenza A viruses (lAVs) [A / Puerto Rico / 8 / 1934(HlNl), A / Hong Kong / 1 / 68(H3N2), A / chukkar / MN / 14951-7 / 1998(H5N2),A / goose / Nebraska / 17097 / 2011(H7N9), and A / Hong Kong / 1073 / 1999(H9N2)] and the influenza B viruses (IBVs) [B / Florida / 04 / 2006 (Yamagata lineage), B / Brisbane / 60 / 2008 (Victoria lineage)] were grown in 10-day-old specific pathogen-free (SPF) embryonated chicken eggs (AVS Bio, Norwich, CT) for 72 h at 33 °C. After the incubation, the eggs were cooled at 4 °C overnight. The allantoic fluid was harvested and centrifuged at 3000 x g for 10 min at 4 °C to pellet debris. The supernatant virus stocks were aliquoted and stored at -80 °C. The virus stocks were titrated in the MDCK cells or embryonated chicken eggs (AVS Bio) by TCID50 (50% tissue culture infectious dose) assay.

[0167] Mice

[0168] All animal experiments were performed with approvals of the Institutional Biosafety Committee (IBC) and the Institutional Animal Care and Use Committee (IACUC) in a USDA- approved BSL-2+facility’ using 8-week-old BALB / c mice (Jackson Laboratory, Bar Harbor, ME, USA), unless otherwise indicated.

[0169] Statistical Data Analysis

[0170] The bar graph data in the Figures are displayed as individual values, with the average expressed as the mean. The line graph data are presented as means. The error for both graphs is represented by the standard deviation (SD) or standard error of the mean (SEM). The antibody responses were calculated by area under the curve (AUC). Significance in humoral or T cell immune responses in mice was analyzed by two-way ANOVA follow ed by a multiple comparison test to investigate the difference between the vaccine and control groups and between the two vector platforms.

[0171] Significance in lung viral titer in mice was analyzed by one-way ANOVA followed by multiple comparisons. The tw o-tailed unpaired t-test analyzed the significance of the data from the ferret study. The details for each assay, including the number of animals, median or mean values, and comparison methods, are indicated in the figure legends. Data were defined as statistical significance at p <0.05, and all statistical analyses were applied using GraphPad Prism 10. No data points were excluded from the analysis. Sufficient power (5 mice / group; 3 ferrets / group) w as used in most experiments to ensure the significance of differences between the groups.70855-02Example 1Development and Characterization of BAd or HAd Vectors Expressing C5-NP / A or C5-NP / B

[0172] The NP genes of A / Shanghai / 02 / 2013(H7N9) [NP / A] and B / Yamagata / 16 / 1988 (Y amagata lineage) [NP / B] were selected to represent the two types of influenza viruses and used for generating BAd and HAd vectors. The NP gene sequences of A / Shanghai / 02 / 2013(H7N9) [NP / A] or B / Yamagata / 16 / 1988 (Yamagata lineage) [NP / B] were acquired from the NCBI GenBank (Accession numbers: YP_009118476 and AAA82969.1, respectively).

[0173] Constructs were designed that contained the NP / A, NP / B, or a fusion of NP / A and NP / B (NP / A+B) gene cassette linked to AIP-C5 (FIG. 1A). Briefly, the sequence representing AIP-C5 (AAQAAVVRFQEAANKQKQELD (SEQ ID NO: 9)) was added at the 5’ end of the NP gene sequence to yield the C5-NP / A and C5-NP / B gene cassettes. The C5-NP / A and C5-NP / B gene cassettes were fused to generate the C5-NP / A+B gene cassette. All gene constructs were codon- optimized for rodents and synthesized commercially (Genscript. Piscataway, NJ). The gene construct was under the control of the human cytomegalovirus (HCMV) promotor and the bovine growth hormone (BGH) polyadenylation signal.

[0174] BAd vectors, BAd-C5-NP / A, BAd-C5-NP / B, and BAd-C 5 -NP / A+B, cartying the C5- NP / A, C5-NP / B, or C5-NP / A+B gene cassette, respectively, were generated by I-Scel-mediated cleavage of the genomic plasmid. More specifically, BAd vectors containing the C5-NP / A. C5- NP / B, or C5-NP / A+B in the El region were generated by I-scel-mediated release of the infectious BAd vector genome following transfection of BHH / F5-I-SceI cells with the El & E3 deleted BAd genomic plasmid containing the foreign gene cassette in the El region.

[0175] The BAd vector showed cytopathic effects 7-10 days post-transfection of BHH / F5-I- scel cells. The resultant BAd vectors, BAd-C5-NP / A (containing C5-NP / A), BAd-C5-NP / B (containing C5-NP / B), and BAd-C5 -NP / A+B (containing C5 -NP / A+B), were grown in BHH / F5 cells and purified by cesium chloride density gradient ultracentrifugation as described in Sayedahmed et al. (2019), supra. The vector stocks were titrated by plaque assay in BHH / F5-I- Scel cells. Sayedahmed et al. (2018), supra.

[0176] HAd vectors, HAd-C5-NP / A, HAd-C5-NP / B, and HAd-C5-NP / A+B, carrying the C5- NP / A, C5-NP / B, or C5-NP / A+B gene cassette, respectively, were also generated. However, HAd vectors were produced by Cre-recombinase site-specific recombination in 293 Cre cells as described in Sayedahmed et al. (2019). supra. The resultant HAd vectors, HAd-C5-NP / A (containing C5-NP / A), HAd-C5-NP / B (containing C5-NP / B), and HAd-C5-NP / A+B (containing C5-NP / A+B) were grown in 293 cells and purified by cesium chloride density gradient ultracentrifugation and titrated in BHH2C cells as described previously. Sayedahmed et al. (2018), supra,' Sayedahmed et al. (2019), supra.70855-02

[0177] Empty vectors, BAd-AElE3 (BAd-5 El and E3 deleted empty vector) or HAd-AElE3 (HAd-5 El and E3 deleted empty vector), were also generated in accordance with Sayedahmed et al. (2019), supra to serve as negative controls (i.e., an empty vector control).

[0178] The presence of the correct gene cassette in each vector was initially verified by restriction enzyme analysis of the genomic DNA followed by sequencing. For confirmation of protein expression in vector-infected cells, BAd-AElE3-, BAd-C5-NP / A-, BAd-C5-NP / B-, or BAd-C 5 -NP / A+B -infected BHH / F5 [bovine-human hybrid clone 3 (BHH3) cells expressing E1B region of BAd3], cell pellets for BAd vectors, and HAd-AElE3-, HAd-C5-NP / A-, HAd-C5- NP / B-, or HAd-C 5 -NP / A+B -infected 293 cell pellets were used for immunoblotting using NP / A- or NP / B-specific monoclonal antibody.

[0179] Briefly, BHH / F5 cells were infected with BAd vectors, and HEK 293 cells were infected with HAd vectors at a multiplicity of infection (MOI) 2 PFU per cell. Vector-infected cells were harvested 48 hours post-infection, and cell pellets were processed for immunoblotting, as explained previously in Sayedahmed et al (2018). supra. The membrane was incubated with the anti-H7N9 NP antibody (rabbit monoclonal, 1 :3,000, Sino Biological, Wayne, PA, 11675-R707) or anti-Pan Influenza B NP antibody (rabbit monoclonal, 1:5,000; Sino Biological, 40438-R016) as the primary antibodies. The horseradish peroxidase (HRP)-conjugated goat anti-rabbit (polyclonal, 1 : 10,000, Thermo Fisher Scientific, 65-6120) was used as a secondary antibody. An HRP-conjugated anti-(3-actin antibody (1 :5,000, Thermo Fisher Scientific, MA5-15739-HRP) was also used to detect the housekeeping gene as a marker for an equal amount of protein in each well. The blots were developed with SuperSignal West Pico PLUS Chemiluminescent Substrate Solution (Thermo Fisher Scientific), and images were captured with Azure 280 imaging system (Azure Biosystem, Dublin, CA, USA).

[0180] The presence of an approximately 60 kDa band in BAd-C5-NP / A-, BAd-C5-NP / B-, BAd-C 5 -NP / A+B-, HAd-C5-NP / A-, HAd-C5-NP / B-, or HAd-C 5 -NP / A+B -infected cell extracts suggested the expression of the target protein (FIG. IB), and cross-reactivity was not observed between NP / A and NP / B.Example 2Vaccination of Mice with BAd Vectors Elicited Robust Humoral Immune Responses

[0181] The experimental plan used for immunogenicity and protection studies is outlined (FIG. 1C). 8-week-old BALB / c mice (Jackson Laboratory. Bar Harbor, ME, USA) were anesthetized with ketamine-xylazine and each group was inoculated i n. once with 5 x lO7PFU of BAd [BAd- AE1E3 (empty vector), BAd-C5-NP / A, BAd-C5-NP / B, BAd Bivalent (BAd-C5-NP / A + BAd- C5-NP / B), or BAd-C5-NP / A+B] or HAd [HAd-AElE3 (empty' vector), HAd-C5-NP / A, HAd-C5- NP / B, HAd Bivalent (HAd-C5-NP / A + HAd-C5-NP / B), or HAd-C5-NP / A+B] vectors. Three70855-02 weeks post-vaccination, serum samples and lung washes were collected to monitor NP / A- or NP / B-specific antibody titers using an enzyme-linked immunosorbent assay (ELISA).

[0182] The ELISA protocol used was as described in Sayedahmed et al. (2023), supra. Briefly, 96-well Immulon plates (Immulon 2HB flat bottom; Thermo Fisher Scientific) were coated with 2 pg / mL recombinant NP / A [A / Brisbane / 10 / 2007(H3N2)] (International Reagent Resource, Manassas, VA, USA, FR-480) or NP / B [B / Florida / 4 / 2006] (Sino Biological, 40438-V08B) in 50 niM Na2CC>3 (pH 9.6; 50 pL) overnight at 4 °C. The wells were blocked with 1 x PBS + 2% bovine serum albumin (BSA) for 2 hours at 4 °C. Mice serum or lung wash samples were ten-fold serially diluted in dilution buffer (PBS + 0.5% BSA), added to the wells, and incubated for 2 hours at room temperature. HRP-conjugated anti-mouse IgG (l :10,000, Invitrogen, 31430), IgGi (1:5,000, Invitrogen, PAI-74421), IgG2a (1 :2,000, Invitrogen, A10685) or IgA (1:3,000, Invitrogen, 62- 6720) were used as secondary antibodies. Similarly, ferret sera samples were ten-fold serially diluted in dilution buffer, added to the wells, and incubated for 2 hours at room temperature. HRP- conjugated anti-ferret IgG (1: 10,000, Novus Biologicals, NB7224) was used as a secondary antibody. Plates were developed with 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific), and the reaction was stopped with 2N sulfuric acid. The optical density at 450 nm (OD450) was measured by SpectraMax i3x microplate reader (Molecular Device, San Jose, CA, USA). The average OD value of blank wells was set for each plate as a cut-off value and used to calculate the area under the curve (AUC) as the readout using GraphPad Prism version 10.0.

[0183] In the serum samples, high levels of IAV NP-specific IgG, IgGi, IgG2a and IgA titers were found in the mice immunized with BAd (BAd-C5-NP / A, BAd Bivalent, or BAd-C5- NP / A+B) or HAd (HAd-C5-NP / A, HAd Bivalent, or HAd-C5-NP / A+B) vectors (FIGS. 2A-D and FIGS. 3A-D) Serum from groups immunized with control vectors and vectors expressing NP / B yielded titers close to the background. Similarly, high levels of IBV NP-specific IgG, IgGi and IgGza titers were observed in the mice immunized with BAd (BAd-C5-NP / B, BAd Bivalent, or BAd-C5-NP / A+B) or HAd (HAd-C5-NP / B, HAd Bivalent, or HAd-C5-NP / A+B) vectors (FIGS. 2E-H and FIGS. 3E-H). serum from mice immunized with control vectors and vectors expressing NP / A yielded titers close to the background.

[0184] Overall, the findings demonstrate the superiority of BAd vectors, which induced comparatively higher levels of NP / A- or NP / B-specific antibody titers in serum compared to the corresponding HAd vector formulations.

[0185] The lung washes were then analyzed to determine the development of NP-specific mucosal antibody responses. High levels of IAV NP-specific IgG, IgGi, IgG2a, and IgA titers were observed in the mice immunized with BAd (BAd-C5-NP / A, BAd Bivalent, or BAd-C5-NP / A+B) or HAd (HAd-C5-NP / A, HAd Bivalent, vectors (FIGS. 2I-L and FIGS.70855-023I-L). Similarly, high levels of IBV NP-specific IgG and IgG2a titers were found in the mice immunized with BAd (BAd-C5-NP / B, BAd Bivalent, or BAd-C5-NP / A+B) or HAd (HAd-C5- NP / B, HAd Bivalent, or HAd-C5-NP / A+B) vectors (FIGS. 2M-P and FIGS. 3M-P) However, only the BAd-C5-NP / B and BAd Bivalent induced a significantly higher IBV NP-specific IgA titer, supporting that the BAd vector induced better mucosal immunity than HAd vectors. Overall, BAd vectors induced comparatively higher levels of NP / A- or NP / B-specific antibody titers in lung washes compared to the corresponding HAd vector formulations.Example 3Vaccination of Mice with BAd Vectors Induced Strong Cellular Immune Responses

[0186] Mouse groups were immunized with BAd (BAd-AElE3, BAd-C5-NP / A, BAd-C5- NP / B, BAd Bivalent, or BAd-C5-NP / A+B) or HAd (HAd-AElE3, HAd-C5-NP / A, HAd-C5- NP / B, HAd Bivalent, or HAd-C5-NP / A+B) vectors. Three weeks post-vaccination, splenocytes, mediastinal lymph node (MLN) cells, and lung mononuclear (MN) cells were collected to determine CMI responses by flow cytometry, ELISpot, or tetramer assay.

[0187] Initially, splenocytes were stimulated with NP / A or NP / B peptide library to evaluate the expression of IFN-y, IL-2, or TNF-a in CD4+or CD8+T cells by flow cytometry7. More specifically, splenocytes (5 x 106cells per sample) in complete Roswell Park Memorial Institute 1640 (RPMI-1640) medium (Coming) were stimulated with anti-mouse CD28 (1 : 1,000; BD Biosciences, 556620), and influenza NP / A peptide array (BEI Resources, Manassas, VA, NR- 50714) or influenza NP / B peptide array (BEI Resources, NR-36045) at 1 pg / mL per peptide for 6 hours at 37 °C in a 5% CO2 incubator. The protein transport inhibitor cocktail (Invitrogen, Thermo Fisher Scientific) was added to each sample after 1 hour of stimulation. Similarly, splenocytes were stimulated with 0.5 mg / mL phorbol 12-myristate 13-acetate (PMA) (Sigma- Aldrich, St. Louis, MO) and 1 mg / mL ionomycin (Sigma-Aldrich) as a positive control. After stimulation, cells were washed with fluorescence-activated cell sorting (FACS) buffer (1% FBS in PBS) and stained for 10 minutes in the dark at 4 °C with the LIVE / DEAD Fixable Near-IR Dead Cell Stain Kit (Invitrogen, L34975). Samples were incubated in the Fc blocker (Rat anti-mouse CD16 / CD32, Invitrogen, 14-0161-82) for 10 minutes in the dark at 4 °C and washed once with FACS buffer. The samples were then stained with surface-staining monoclonal antibodies: anti-CD3 FITC (Invitrogen, 11-0032-82), anti-CD4 PE-Cy7 (Invitrogen, 25-0042-82), and anti-CD8 APC (Invitrogen, 17-0081-82) for 30 minutes at 4 °C. After surface staining, cells were washed with FACS buffer twice and permeabilized and fixed using Cytofix / Cytoperm Fixation / Permeabilization Kit (BD Biosciences, Franklin Lakes, NJ). Cells were washed once with permeabilization buffer and then incubated with anti-IFN-y PE (Invitrogen, 12-7311-82), anti-IL-2 BV421 (BD Biosciences, 562914), and anti-TNF-a PerCP-Cy5.5 (BD Biosciences,70855-02560679) for 30 minutes in dark at 4 °C. Samples were washed twice in FACS buffer and then resuspended in FACS buffer. Samples were processed for flow cytometry using LSRFortessa X- 20 Cell Analyzer (BD Biosciences) and analyzed with FlowJo vlO software. The gating strategy is shown in FIG. 4.

[0188] Following stimulation of splenocytes with NP / A peptide library, reasonably high levels of IFN-y expression were observed in the CD4+(FIG. 5A) and CD8+(FIG. 5D) T cells from groups immunized with vectors expressing C5-NP / A compared to the control group. Low levels of stimulation for IL-2 expression were observed in the CD4+(FIG. 5B) and CD8+(FIG. 5E) T cells, while there was no noticeable expression of TNF-a in the CD4+(FIG. 5C) and CD8+(FIG. 5F) T cells. Following stimulation of splenocytes with NP / B peptide library, reasonably high levels of IFN-y expression were observed in the CD4+(FIG. 5G) and CD8+(FIG. 5J) T cells from groups immunized with vectors expressing C5-NP / B compared to the control group. Low levels of stimulation for IL-2 expression were observed in the CD4+(FIG. 5H) and CD8+(FIG. 5K) T cells, while there was no noticeable expression of TNF-a in the CD4+(FIG. 51) and CD8 (FIG. 5L) T cells. Animals immunized with BAd or HAd vectors yielded similar levels of IFN-y or IL-2 expressing CD4+or CD8+T cells.

[0189] Detection of a high percentage of IFN-y+CD8+T cells in splenocytes from immunized animal groups led to a further investigation of the vaccine-induced CD8+T cell responses in splenocytes, MLN cells, and lung MN cells by ELISpot. Splenocytes, MLN cells, and lung MN cells were stimulated with NPA147-155 and NPB167-174 (known NPA- or NPB-CTL epitope, respectively), and numbers of IFN-y or IL-2 expressing CD8+T cells were counted.

[0190] More specifically, the assay was conducted as described in Sayedahmed et al. (2024), supra. Briefly, the multiscreen plates (MilliporeSigma, Burlington, MA) were coated with unconjugated anti-mouse IFN-y or IL-2 mAbs (Mabtech, Cincinnati, OH, USA, 3321-3, 3441-3) overnight at 4 °C and then blocked with blocking buffer (PBS + 1% BSA). Mouse splenocytes, MLN cells, or lung MN cells were incubated with 5 pg / mL NP / A147-155 peptide (TYQRTRALV (SEQ ID NO: 11)) (MHC-I H-2Kd-restricted CTL epitope for NP / A) (67) or NP / B166-174 peptide (FSPIRITFL (SEQ ID NO: 12)) (MHC-I H-2Dd-restncted CTL epitope for NP / B) (67) for 48 hours at 37 °C in a 5% CO2 incubator. Then, the plates were washed with PBST and incubated with biotin-conjugated anti-mouse IFN-y or IL-2 mAbs (Mabtech, 3321-6, 3441-6) overnight at 4 °C. The plates were washed with PBST the next day and incubated w ith Streptavidin- ALP enzyme conjugate (Mabtech, 3310-10) for 30 minutes at room temperature. The plates were developed with BCIP / NBT-plus substrate (Mabtech, 3650-10), and the spot-forming units (SFU) were enumerated using AID iSpot Advanced Imaging Device (Autoimmun Diagnostika GmbH, Strassberg, Germany).70855-02

[0191] Following stimulation with the NPA147-155 peptide, high numbers of IFN-y or IL-2 expressing CD8+T cells in splenocytes, MLN, and lung MN cells (FIGS. 6A-F) from mouse groups immunized with BAd or HAd vaccine formulations expressing C5-NP / A were observed. Similarly, following stimulation with the NPB166-174 peptide, high numbers of IFN-y or IL-2 expressing CD8+T cells in splenocytes, MLN. and lung MN cells (FIGS. 6G-L) from mouse groups immunized with BAd or HAd vaccine formulations expressing C5-NP / B were noticed.

[0192] To further investigate the development of NP / A- or NP / B-specific CD8+T cells in splenocytes, PE-labeled NP / A147-155 or FITC-labeled NP / B166-174 tetramer was used in a tetramer assay. More specifically, the NP / A147-155 -PE and NP / B166-174 -FITC tetramers were generated by the NIH Tetramer Core Facility at Emory University, Atlanta, GA. Splenocytes (5 x 106cells per sample) were incubated with both tetramers at 5 pg / mL in FACS buffer for 2 hours at 37 °C. After tetramer staining, samples were incubated with the LIVE / DEAD Fixable Near-IR Dead Cell Stain Kit (Invitrogen) for 10 minutes in the dark at 4 °C and washed once with FACS buffer. Then, FACS buffer containing anti-CD4 PE-Cy7 (Invitrogen, 25-0042-82) and anti-CD8 APC (Invitrogen, 17-0081-82) were added, followed by incubation at 4 °C for 30 minutes. Samples were washed twice in FACS buffer and then resuspended in FACS buffer. Samples were processed for flow cytometry using LSRFortessa X-20 Cell Analyzer (BD Biosciences) and analyzed with FlowJo vlO software. The gating strategy and representative diagram are shown in FIG. 7

[0193] A significantly higher percentage of CD8+T cells recognizing the NPA147-155 epitope was observed in the mice immunized with BAd or HAd vaccine formulations containing C5-NP / A (FIG. 6M). Similarly, a significantly higher percentage of CD8+T cells recognizing the NPB166- 174 epitope was observed in the mice immunized with BAd or HAd vaccine formulations containing C5-NP / B (FIG. 6N). There were comparable higher percentages of NP / A- or NP / B- specific CD8+T cells in animal groups immunized with BAd vector formulations than in HAd vector immunized groups.Example 4Vaccination of Mice with BAd Vectors Provided Comprehensive Protection Against Homosubtypic or Heterosubtypic lAVs andlBVs

[0194] The immunogenicity study in mice showed that BAd vectors provided similar or higher levels of NP-specific humoral and CMI responses as compared to the HAd vectors. Therefore, BAd vector formulations were evaluated for protection efficacy against seasonal and potentially pathogenic lAVs and IBVs in mice.

[0195] Mice groups were immunized i.n. once with 5 xlO7PFU of BAd-AElE3 (empty vector), BAd-C5-NP / A, BAd-C5-NP / B. BAd Bivalent, or BAd-C5-NP / A+B. At three weeks post-70855-02 vaccination, the groups were challenged with either 5 mLDso (50% mouse lethal dose)) of IAV A / Puerto Rico / 8 / 1934(HlNl), IAV A / Hong Kong / 1 / 68 (H3N2) or IBV B / Florida / 04 / 2006 (Yamagata lineage) or 100 mIDso (50% mice infectious dose) of IAV A / chukkar / MN / 14951- 7 / 1998(H5N2). A / goose / Nebraska / 17097 / 2011(H7N9), IAV A / Hong Kong / 1073 / 1999(H9N2) or IVB B / Brisbane / 60 / 2008 (Victoria lineage). The NP amino acid sequence identity percentages between the NP / A or NP / B used for the BAd vectors, and lAVs and IBVs used for the challenge are show n in FIG. 8, and a phylogenetic tree of NP of lAVs and IBVs used are shown in FIG. 9.

[0196] The protection efficacy was evaluated by monitoring lung virus titers or morbidity and mortality depending on lAVs or IBVs pathogenesis in mice. More specifically, for the lethal challenge, morbidity (weight loss) and mortality were monitored for 14 days post-challenge, and the animals that lost > 20% of their initial body weight were humanely euthanized. The lungs were collected three days post-challenge for non-lethal and lethal challenges to titrate lung viral loads.

[0197] For the influenza virus titration, the assay was conducted following the protocol described in Sayedahmed et al. (2024), supra. The lung samples were homogenized with tissue grinders (Thermo Fisher Scientific) in PBS. Samples were ten-fold serially diluted in DMEM containing 4 pg / mL of TPCK-Trypsin (Thermo Fisher Scientific), added to the plates, and incubated at 37 °C for 48 hours. The incubated medium was used for hemagglutination assay with 0.5% turkey red blood cells (RBCs) to determine the infection status. The viral titers in TCIDso were calculated using the Reed-Muench method.

[0198] The levels of viral RNA w ere determined through influenza virus matrix gene-specific TaqMan real-time reverse transcription polymerase chain reaction (qRT-PCR) assay. Ward et al., Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement, J Clinical Virology: The Official Publication of Pan American Soc’y for Clinical Virology 29: 179-188 (2004). The RNA was extracted from the stock viruses using an RNA Extraction Kit (New England Biolabs, Ipswich, MA) and amplified the targeted segment by RT-PCR assay. The concentration of the targeted segment was quantified, converted to copy numbers, and serially diluted in a 10-fold serial dilution from 1010to 101copy numbers. Then, each dilution and sample underwent the TaqMan qRT-PCR (Integrated DNA Technologies, Coralville, IA) assay to generate the standard curve betw een the Ct value and copy numbers. Based on the standard curve, the samples that gave a Ct value were analyzed for copy numbers.

[0199] There were significant reductions in lung virus titers of H1N1, H3N2, H5N2, H7N9, and H9N2 in mouse groups immunized with BAd vector formulations expressing C5-NP / A (FIGS. 10A-B and 10E-G). Similarly, the mice vaccinated with BAd vector formulations expressing C5- NP / B significantly decreased the lung viral titers following challenge with the Victoria or Yamagata lineage of influenza B virus (FIGS. 10C and 10D). In addition, no morbidity (weight70855-02 loss) or mortality was observed in groups immunized with BAd vector formulations expressing C5-NP / A following challenge with H1N1 (FIGS. 11A and 11D) or H3N2 (FIGS. 11B and HE). In contrast, the control groups displayed severe weight loss and 100% mortality'.

[0200] Furthermore, groups immunized with BAd-C5-NP / B or BAd Bivalent conferred complete protection against B / Florida / 04 / 2006 from morbidity and mortality, while BAd- NP / A+B provided partial protection (FIGS. 11C and HF). Overall, the monovalent immunization of BAd-NP / A or NP / B resulted in ty pe-specific protection. Immunization with the BAd Bivalent vaccine significantly inhibited the viral infection and conferred comprehensive protection against heterosubtypic influenza A and B viruses, including the seasonal and potentially pathogenic avian influenza viruses.

[0201] Previous studies demonstrated that administering HAd vector expressing NP from either lAVs or IBVs via the mucosal route can protected against heterologous lAVs or IBVs in mice, respectively. Sayedahmed et al. (2024), supra,' Kim et al., Single mucosal vaccination targeting nucleoprotein provides broad protection against two lineages of influenza B virus, Antiviral Research 163: 19-28 (2019). This protection is primarily attributed to the NP-specific CD8 cellular immunity toward conserved epitopes stimulated by influenza vaccines, which can play a vital role in broad cross-reactive immunity across distinct influenza viruses. Grant et al., Broad CD8(+) T cell cross-recognition of distinct influenza A strains in humans, Nature Communications 9: 5427 (2018).Example 5Immunogenicity and Protection Efficacy of BAd Bivalent (BAd-C5-NP / A \ BAd-C5-NPfB) Vaccine in Ferrets

[0202] Ferrets are a relevant model for studying influenza virus pathogenesis and evaluating influenza vaccine efficacy because the infection process closely mimics that in humans. Belser et al., Complexities in ferret influenza virus pathogenesis and transmission models, Microbiology & Molecular Biology Reviews 80: 733-744 (2016). Like humans, ferrets share lung physiology and sialylated glycan receptor distribution within the respiratory tract. Jayaraman et aL. Decoding the distribution of glycan receptors for human-adapted influenza A viruses in ferret respiratory tract, PLoS One 7: e27517 (2012). Additionally, the viral attachment and pathology' patterns for both humans and zoonotic influenza viruses in the upper and lower respiratory tracts are similar between ferrets and humans. Belser et al., A guide for the use of the ferret model for influenza virus infection. Am J Pathology 190: 11-24 (2020). Therefore, ferrets are commonly considered the best mammalian model for studying the pathogenicity7and transmissibility7of influenza viruses. Belser et al., Pathogenesis and transmission of avian influenza A (H7N9) virus in ferrets and mice, Nature 501 : 556-559 (2013).70855-02

[0203] Building on the successful immunogenicity and protection outcomes in mice described above, the vaccine efficacy of the BAd Bivalent vaccine formulation was evaluated in the ferret model. The study design for ferrets is depicted in FIG. 12A.

[0204] Briefly. 6-month-old female SPF ferrets (Marshall BioResources, North Rose, NY) were anesthetized with isoflurane and inoculated i.n. twice at three-week intervals with 5 x 107PFU of BAd-AElE3 (empty vector) or BAd Bivalent.

[0205] Blood samples were collected at three and six weeks after the first vaccination. At three weeks after the booster inoculation, ferrets were challenged with 1 x lO4TCID50 of A / Puerto Rico / 8 / 1934 (H1N1), 1 xlO3TCID50 of A / Hong Kong / 1 / 68(H3N2), or 1 xlO5TCID50 of B / Florida / 04 / 2006 (Y). Body temperatures and clinical signs were monitored, and throat swab samples were collected daily for 5 days. Ferrets were euthanized 5 days post-challenge, the lungs were collected to quantify influenza viral copy number, and the blood and spleen were collected for evaluating CMI by ELISpot.

[0206] Notably, none of the ferrets exhibited any clinical signs after vaccination (FIG. 13), indicating the safety of the vectored vaccine. Blood samples collected after the prime and the booster immunizations were used to monitor the development of NP-specific humoral immune responses.

[0207] The virus titration assay and subsequent viral copy quantification was conducted as previously described in Example 4 above. The swab samples were kept in PBS. Samples were ten-fold serially diluted in DMEM containing 4 pg / mL of TPCK-Trypsin (Thermo Fisher Scientific), added to the plates, and incubated at 37 °C for 48 hours. The incubated medium was used for hemagglutination assay with 0.5% turkey RBCs to determine the infection status. The viral titers in TCID50 were calculated using the Reed-Muench method. Significant levels of NP / A- or NP / B-specific IgG titers were observed, with a marked increase in antibody titers following the booster immunization (FIGS. 12B and 12C).

[0208] As noted above, three weeks post the booster immunization, the ferret groups were challenged with A / Puerto Rico / 8 / 1934(HlNl), A / Hong Kong / 1 / 68(H3N2), or B / Florida / 04 / 2006 (Y amagata lineage), and splenocytes and peripheral blood mononuclear cells (PBMCs) were isolated five days post-challenge to monitor the development of CMI responses using an ELISpot assay according to the protocols described herein. In particular, anti-ferret IFN-y mAbs (Mabtech, 3112-3 3112-6) were used in the ELISpot assay for the ferret study. The PBMCs were isolated from the blood samples using the Lymphocyte Separation Medium (Coming). Splenocytes or PBMCs were incubated with 1 pg / mL influenza NP / A peptide array (BEI Resources, NR-50714) or influenza NP / B peptide array (BEI Resources, NR-36045) for 48 hours at 37 °C in a 5% CO270855-02 incubator. Plates were processed for IFN-y ELISpot assay as mentioned above for mouse samples.

[0209] There were significantly higher IFN-y-expressing cells in splenocytes and PBMCs from BAd Bivalent immunized groups following stimulation with either NP / A orNP / B peptide library compared to the empty vector control group. (FIGS. 12D and 121). Interestingly, the increase observed in IFN-y expressing cells in splenocytes and PBMCs from BAd Bivalent immunized groups following stimulation with either NP / A or NP / B peptide libraries depended on the type of influenza virus used for the challenge.

[0210] Following the influenza virus challenge, body temperature and other clinical signs were monitored as noted above. Throat swabs were collected daily for five days, and the lungs were collected from all animals five days post-challenge. The swab samples were kept in the PBS.

[0211] Animals inoculated with the empty vector showed fever with either H1N1 or H3N2 influenza infection. In contrast, no significant temperature increases were observed in the BAd Bivalent immunized groups (FIGS. 14A and 14B).

[0212] Additionally, in the throat samples, virus titration and quantification of viral copy numbers was performed as described above in the other studies herein. In the BAd Bivalent immunized groups, viral titers in the throat swabs were reduced by 20-100-fold compared to control groups after challenge with H1N1 or H3N2 influenza virus (FIGS. 14D and 14E). Furthermore, significant reductions in lung viral genome copy numbers were observed in BAd Bivalent immunized animal groups five days post-challenge with H1N1, H3N2, or B / Florida / 04 / 2006 compared to control groups (FIGS. 14F and 14H).

[0213] In sum, significant increases in NP-specific humoral and CMI responses resulted in substantial protection against seasonal influenza viruses [A / H1N1, A / H3N2, or B / Florida / 04 / 2006 (Y)J. Significant reductions in lung and throat swab viral titers and clinical signs demonstrated the protection efficacy. Previous studies have shown that IBVs produce relatively lower viral titers and milder lung histopathology7than lAVs in ferrets and humans. Kim et al., Influenza B vims cases milder pathogenesis and weaker inflammatory responses in ferrets than influenza A vims, Viral Immunology 22: 423-430 (2009). To distinguish the difference between vaccinated and control groups, a higher viral titer IBV challenge may be needed to trigger more apparent clinical signs and viral load.

[0214] Overall, these results demonstrate that immunization with the BAd Bivalent vaccine conferred protective immunity against H1N1, H3N2, or B / Florida / 04 / 2006 influenza virus in the ferret model.

Claims

70855-02CLAIMS1. An immunogenic composition comprising: a nucleoprotein (NP) of an influenza A virus (IAV) and a NP of an influenza B virus (IBV); and a pharmaceutically acceptable carrier; wherein the immunogenic composition is cross-protective against at least one IAV and at least one IBV when administered to a subject.

2. The immunogenic composition of claim 1. further comprising at least 21 amino acid residues of an Autophagy -Inducing Peptide C5 (AIP-C5) from a CFP10 protein of Mycobacterium tuberculosis or a functional fragment thereof.

3. The immunogenic composition of claim 1, wherein, when administered to a subject, the composition confers general immunogenicity protection against the subtypes of viruses selected from the group consisting of Hl, H3, H5, H7, H9, B / Yamagata, and B / Victoria strains.

4. The immunogenic composition of claim 2. wherein the AIP-C5 from a CFP10 protein comprises at least 90% sequence identity to SEQ ID NO: 9.

5. The immunogenic composition of any one of the foregoing claims formulated to be administered intranasally or orally (e.g, mucosally) or intramuscularly (e.g, systemically).

6. The immunogenic composition of any one of the foregoing claims formulated as an aerosol spray.

7. The immunogenic composition of any one of claims 1-4, wherein the immunogenic composition is bivalent or monovalent.

8. The immunogenic composition of any one of claims 1-4, wherein the immunogenic composition is polyvalent.

9. The immunogenic composition of claim 1 comprising a polypeptide with at least 80% sequence identity' to SEQ ID NO: 6.70855-0210. The immunogenic composition of claim 1, wherein administration to a subject induces CD8+ T-cell responses cross-reactive to at least one influenza A subtype or / and at least one influenza B lineage.

11. A human, chimpanzee or bovine adenoviral (Ad) vector comprising a polynucleotide sequence that encodes at least: a first nucleoprotein (NP) from an influenza A virus (IAV) or a functional fragment thereof; and a second NP from an influenza B virus (IBV) or a functional fragment thereof.

12. The Ad vector of claim 11, wherein the polynucleotide sequence further encodes one or more immunogenic domains of an influenza virus other than aNP13. The Ad vector of claim 11, wherein the polynucleotide sequence further encodes Autophagy -Inducing Peptide C5 (AIP-C5) from the CFP10 protein of Mycobacterium tuberculosis or a functional fragment thereof.

14. The Ad vector of claim 13, wherein the AIP-C5 comprises 21 amino acid residues and / or has at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:9.

15. The Ad vector of claim 13, wherein the polynucleotide sequence has at least 80% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 7.

16. The Ad vector of any one of claims 11-14, further comprising one or more promoters.

17. The Ad vector of any one of claims 5-10 that is replication-defective and suitable for expression in a mammalian cell.

18. The Ad vector of claim 11, wherein at least El and E3 regions are deleted.

19. The Ad vector of claim 18, wherein the polynucleotide sequence of the first NP from an IAV or the functional fragment thereof is inserted in the deleted El region of the Ad vector, and / or the second NP from an IBV or the functional fragment thereof is inserted in the70855-02 deleted El region of the Ad vector.

20. The Ad vector of any one of claims 11-15, 18, and 19, wherein the Ad vector is a bovine Ad (BAd).

21. The Ad vector of any one of claims 11-15, 18, and 19, wherein the Ad vector is a BAd type 3 (BAd3).

22. An isolated nucleic acid molecule comprising sequences encoding:(a) an NP of an IAV,(b) an NP of an IBV, and(c) optionally, at least 21 amino acid residues of an AIP-C5 peptide from M. tuberculosis or a functional fragment thereof.

23. The isolated nucleic acid of claim 22, wherein the sequences are codon-optimized for mammalian expression.

24. The isolated nucleic acid of claim 22, wherein the sequences of (a) and (b) are fused in-frame and separated by a linker peptide.

25. The isolated nucleic acid of claim 22 having at least 80% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 7.

26. An isolated polypeptide comprising:(a) an NP sequence of an IAV,(b) an NP sequence of an IBV, and(c) optionally, a sequence encoding an AIP-C5 peptide from M. tuberculosis or a functional fragment thereof; wherein the polypeptide elicits cross-reactive T-cell responses against both IAV and IBV when administered to a subj ect.

27. A method of eliciting an immune response in a subject against an influenza virus, comprising administering to said subject an effective amount of an immunogenic composition of any one of claims 1-10, an adenoviral (Ad) vector of any one of claims 11-21, or an isolated polypeptide of claim 26.70855-0228. A method of inducing cross-protective immune response against influenza A and influenza B viruses in a subject, comprising administering to the subject an effective amount of an immunogenic composition according to any one of claims 1-10, an adenoviral (Ad) vector according to any one of claims 11-21, or an isolated polypeptide of claim 26.

29. The method of claim 27 or claim 28, wherein said administration is intranasal, intramuscular, mucosal, or oral.

30. The method of claim 27 or claim 28, wherein said composition, Ad vector, or isolated polypeptide is administered as an aerosol spray.

31. The method of claim 27 or claim 28, wherein said method provides general immunogenicity protection to the subject against vanous subtypes of viruses selected from the group consisting of Hl, H3, H5, H7, H9, B / Yamagata, and B / Victoria strains.

32. The method of claim 27 or claim 28, wherein the immune response comprises a dose-dependent increase in both humoral and cell-mediated immunity in the subject.

33. The method of any one of claims 27-32, wherein the subject is a human.

34. The method of claim 27 or claim 28, wherein the immune response is cross- protective immunity against at least influenza A and influenza B.

35. The method of claim 27 or claim 28, wherein administration of the composition, Ad vector, or isolated polypeptide results in reduction of viral load in the subject following heterologous influenza challenge of the subject.

36. A method of manufacturing a medicament comprising an immunogenic composition, the method comprising: introducing a recombinant adenoviral (Ad) vector into a host cell that can support replication of the Ad vector, wherein the Ad vector comprises a vector of any one of claims 11- 21; and culturing the host cell in a culture medium under conditions that allow for the expression of the first and second NPs.70855-0237. The method of claim 36, further comprising isolating or purifying the expressed first and second NPs from the host cell or culture medium.

38. The method of claim 36, wherein the method is performed in vitro or in ovo.

39. A cell line capable of being infected by an adenoviral (Ad) vector according to any one of claims 11-21, and / or comprising an isolated nucleic acid according to any one of claims 22-25, wherein the cell line supports replication of the genome of the Ad vector and / or expression of the isolated nucleic acid.

40. A cell comprising an isolated recombinant Ad vector according to any one of claims 11-21, and / or the isolated nucleic acid according to any one of claims 22-25.

41. The cell line of claim 39 or the cell line of claim 40, wherein expression of the isolated nucleic acid results in production of aNP of an IAV and / or an IBV.

42. An immunogenic composition for use in eliciting an immune response in a subject, the immune response against an influenza virus, the immunogenic composition comprising any of the immunogenic composition of any one of claims 1-10.

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