Membrane vesicles containing multiple influenza antigens for vaccine

Abstract

Polyvalent influenza virus vaccines (MIVs), polyvalent immunogenic polypeptides (MIPs), and fusion proteins are described herein. Further methods for producing and using MIVs, MIPs, and fusion proteins are provided herein.

Description

Technical Field

[0001] Other references This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 491,005, filed on March 17, 2023, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing The sequence listing submitted via EFS in accordance with 37 CFR §1.52(e)(5) is incorporated herein by reference. The sequence listing XML file submitted via EFS includes the file "4377.1003 US.xml" created on March 14, 2024, with a size of 25,040 bytes.

Summary of the Invention

[0003] Summary of the Disclosure In some aspects of the present disclosure, a multivalent influenza virus vaccine (MIV) is provided, the MIV comprising: A-B-C; wherein A is a transport protein or a fragment thereof comprising at least domains X1 and X2, where X1 is a transmembrane domain polypeptide and X2 is a polypeptide that can be operably linked to B; B is a multivalent immunogenic polypeptide (MIP) comprising at least 5 influenza virus immunogenic epitopes selected from the group consisting of Y1, Y2, Y3, Y4, Y5, and Y6; C is a membrane vesicle derived from a genetically modified Gram-positive bacterium; wherein A and B are operably linked to produce a multivalent immunogenic transmembrane polypeptide; A-B is linked to C via the transmembrane domain X1, thereby forming the MIV; and the MIV can induce an immune response against at least 2 influenza strains when administered to a mammal.

[0004] In some aspects of this disclosure, a polyvalent influenza virus vaccine (MIV) is provided, the MIV comprising: a transport protein or a fragment thereof, wherein the transport protein comprises a transmembrane domain; a polyvalent immunogenic polypeptide (MIP) comprising at least five covalently linked influenza virus immunogenic epitopes; and a membrane vesicle derived from a genetically modified Gram-positive bacterium, wherein the transport protein of a) is operably linked to the MIP of b) to produce a polyvalent immunogenic transmembrane polypeptide; the polyvalent immunogenic transmembrane polypeptide is linked to the vesicle to form the MIV; and the MIV may induce an immune response to at least two influenza virus strains when administered to a mammal.

[0005] In some embodiments, the transport protein is an adhesin, an immunomodulatory compound, a protease or toxin or a fragment thereof. In some embodiments, the transport protein is ClyA. In some embodiments, ClyA contains an amino acid sequence that is at least about 80% identical to SEQ ID NO: 1.

[0006] In some embodiments, at least five influenza virus immunogenic epitopes are fused longitudinally. In some embodiments, the operable bond between A and B includes a covalent bond. In some embodiments, at least five influenza virus immunogenic epitopes are fused to the N- or C-terminus of a transport protein. In some embodiments, at least five influenza virus immunogenic epitopes are presented on the outside of the vesicle.

[0007] In some embodiments, the influenza virus is influenza A. In some embodiments, influenza A is human, pig, or avian. In some embodiments, avian is chicken, mute swan, quail, or mallard. In some embodiments, influenza A is an influenza subtype that is H1, H2, H3, H5, H6, H7, or H9. In some embodiments, the influenza virus is selected from the group consisting of H1N1, H1N2, H2N1, H3N2, H5N1, H5N2, H9N2, H7N9, H7N7, H7N3, H6N6, H6N2, and H6N1.

[0008] In some embodiments, at least five influenza virus immunogenic epitopes are different. In some embodiments, at least five influenza virus immunogenic epitopes are the same. In some embodiments, at least one of the at least five influenza virus immunogenic epitopes contains a consensus sequence or a sequence that does not exist in nature.

[0009] In some embodiments, MIP comprises an influenza A matrix protein 2 extracellular (M2e) peptide or a fragment thereof. In some embodiments, MIP comprises five M2e peptides. In some embodiments, MIP comprises six M2e peptides. In some embodiments, the M2e peptides comprise an amino acid sequence that is at least about 90% identical to any of SEQ ID NOs: 2-8. In some embodiments, the transport protein and MIP are linked using one or more linkers.

[0010] In some embodiments, one or more linkers include an array selected from the group consisting of (GS)n (sequence number: 12), (G2S)n (sequence number: 13), (G3S)n (sequence number: 14), (G4S)n (sequence number: 15), and (G)n (sequence number: 16), where n is an integer from 2 to 20. In some embodiments, one or more linkers include an array selected from the group consisting of (GGSGGD)n (sequence number: 17) or (GGSGGE)n (sequence number: 18), where n is an integer from 2 to 6. In some embodiments, one or more linkers include an array selected from the group consisting of (GGGSGGG)n (sequence number: 19), (GGGSGSGGGGS)n (sequence number: 20), and (GGGGGPGGGGP)n (sequence number: 21), where n is an integer from 1 to 3. In some embodiments, one or more linkers include sequences selected from the group consisting of (GX)n, (GGX)n, (GGGX)n, (GGGGX)n, and (GzX)n, where z is 1 to 20 and n is at least 8. In some embodiments, X is serine, aspartic acid, glutamic acid, threonine, or proline.

[0011] In some embodiments, Y1, Y2, Y3, Y4, Y5, and Y6 are selected from the group consisting of human M2e peptide, porcine M2e peptide, swan M2e peptide, chicken M2e peptide, and mallard M2e peptide. In some embodiments, the polyvalent immunogenic transmembrane polypeptide comprises an amino acid sequence having the formula ClyA-(M2e)5. In some embodiments, the polyvalent immunogenic transmembrane polypeptide comprises an amino acid sequence having the formula ClyA-(M2e)6. In some embodiments, the polyvalent immunogenic transmembrane polypeptide comprises an amino acid sequence having the formula ClyA-(M2e-1)-(M2e-2)-(M2e-3)-(M2e-4)-(M2e-5)-(M2e-6), where M2e-1 comprises human M2e peptide, M2e-2 comprises porcine M2e peptide, M2e-3 comprises swan M2e peptide, M2e-4 comprises chicken M2e peptide, M2e-5 comprises chicken M2e peptide, and M2e-6 comprises mallard duck M2e peptide. In some embodiments, the polyvalent immunogenic transmembrane polypeptide comprises an amino acid sequence having the formula ClyA-(M2e-1)-(M2e-2)-(M2e-3)-(M2e-4)-(M2e-5)-(M2e-6), where M2e-1 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 3, M2e-2 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 4, M2e-3 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 5, M2e-4 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 6, M2e-5 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 7, and M2e-6 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 8. In some embodiments, the polyvalent immunogenic transmembrane polypeptide comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO: 9.

[0012] In some aspects, Gram-positive bacteria include: Bifidobacterium longum, Bifidobacterium lactis, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium adolescentis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus fermentum, Lactococcus lactis, Streptococcus thermophilus, Lactococcus lactis, Lactococcus diacetylactis The group is selected from Lactococcus diacetylactis, Lactococcus cremoris, Lactobacillus bulgaricus, Lactobacillus helveticus, Lactobacillus delbrueckii, Escherichia coli, and combinations thereof. In some embodiments, the Gram-positive bacterium is Escherichia coli. In some embodiments, the Gram-positive bacterium is a probiotic bacterium. In some embodiments, the Gram-positive bacterium is Escherichia coli Nissle. In some embodiments, Escherichia coli Nissle includes one or more modifications in genes selected from the group consisting of: lpxL, lpxP, lpxM, crcA, eptA, lpxT, nlpl, recA, ompT, lon, lpxA, lpxB, lpxD, pagL, pagP, lpxE, MsbA, MsbB, gutQ, and KdsD. In some embodiments, *E. coli* Nissle includes one or more modifications in genes selected from the group consisting of:lpxL, lpxP, lpxM, crcA, eptA, lpxT, nlpl, recA, ompT, and lon.

[0013] In some embodiments, MIV can induce an immune response in mammals without the administration of an adjuvant.

[0014] In some aspects of this disclosure, a polyvalent immunogenic polypeptide (MIP) is provided that comprises at least five influenza virus immunogenic epitopes selected from the group consisting of Y1, Y2, Y3, Y4, Y5, and Y6.

[0015] In some embodiments, at least five influenza virus immunogenic epitopes are fused in a longitudinal order. In some embodiments, the MIP comprises an amino acid sequence having the formula (M2e-1)-(M2e-2)-(M2e-3)-(M2e-4)-(M2e-5)-(M2e-6), where M2e-1 comprises a human M2e peptide, M2e-2 comprises a pig M2e peptide, M2e-3 comprises a swan M2e peptide, M2e-4 comprises a chicken M2e peptide, M2e-5 comprises a chicken M2e peptide, and M2e-6 comprises a mallard M2e peptide. In some embodiments, MIP comprises an amino acid sequence having the formula (M2e-1)-(M2e-2)-(M2e-3)-(M2e-4)-(M2e-5)-(M2e-6), where M2e-1 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 3, M2e-2 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 4, M2e-3 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 5, M2e-4 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 6, M2e-5 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 7, and M2e-6 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 8.

[0016] In some aspects of this disclosure, a fusion protein is provided that includes a transport protein and a MIP comprising at least five influenza virus immunogenic epitopes selected from the group consisting of Y1, Y2, Y3, Y4, Y5, and Y6.

[0017] In some embodiments, the MIP is fused to the C-terminus of a transport protein. In some embodiments, the amino acid sequence has the formula X1-(M2e-1)-(M2e-2)-(M2e-3)-(M2e-4)-(M2e-5)-(M2e-6), where X1 comprises ClyA, M2e-1 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 3, M2e-2 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 4, M2e-3 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 5, M2e-4 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 6, M2e-5 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 7, and M2e-6 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 8. In some embodiments, the MIP or fusion protein of the present disclosure is provided, where the MIP comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO: 9.

[0018] In several aspects of this disclosure, nucleic acids encoding the MIP or fusion protein of this disclosure are provided.

[0019] In some aspects of this disclosure, vectors containing the nucleic acids of this disclosure are provided. In some aspects of this disclosure, bacterial cells containing the vectors of this disclosure are provided.

[0020] In some aspects of this disclosure, bacterial cells expressing the MIP or fusion protein of this disclosure are provided.

[0021] In several aspects of this disclosure, a method for producing a polyvalent influenza virus vaccine (MIV) is provided, the method comprising: a) culturing Escherichia coli Nissle expressing the transport protein and MIP of this disclosure, including one or more modifications; b) exposing the culture from step a) to air by providing about 5% to about 20% oxygen; and c) isolating the MIV from the supernatant of the culture.

[0022] In some embodiments, the particle size of the MIV is 180 nanometers (nm) to 200 nm when measured by dynamic light scattering (DLS). In some embodiments, at least about 50% of the bacterial-derived vesicles of the plurality of bacterial-derived vesicles are intact. In some embodiments, at least about 75% of the bacterial-derived vesicles of the plurality of bacterial-derived vesicles are intact. In some embodiments, at least about 90% of the bacterial-derived vesicles of the plurality of bacterial-derived vesicles are intact. In some embodiments, at least about 50% of the bacterial-derived vesicles of the plurality of bacterial-derived vesicles express MIP. In some embodiments, at least about 75% of the bacterial-derived vesicles of the plurality of bacterial-derived vesicles express MIP. In some embodiments, at least about 90% of the bacterial-derived vesicles of the plurality of bacterial-derived vesicles express MIP.

[0023] In some embodiments, step a) includes culturing Escherichia coli Nissle in a bacterial medium. In some embodiments, step a) includes culturing Escherichia coli Nissle in a mammalian cell medium.

[0024] In some aspects of the present disclosure, a pharmaceutical composition comprising the MIV of the present disclosure and a pharmaceutically acceptable excipient is provided.

[0025] In some aspects of the present disclosure, a method of treating a subject at risk of influenza is provided, the method comprising administering to the subject an appropriate amount of the pharmaceutical composition of the present disclosure. In some embodiments, the method does not include the step of administering an adjuvant to the subject.

Brief Description of the Drawings

[0026] Brief Description of the Drawings The present disclosure will be more fully understood by reference to the following drawings. [Figure 1] FIG. 1 shows a model of an exemplary fusion protein. [Figure 2]Figure 2 shows the viral strains and the resulting M2e peptides and the peptides present in 6xM2e or 4xM2e. Figure 2 discloses each of SEQ ID NOs: 2-8 in visual order. [Figure 3] Figure 3 shows the viral strains and the resulting M2e peptides. Figure 3 discloses each of SEQ ID NOs: 2-8 in visual order. [Figure 4A] Figures 4A and 4B show models of protein impairment of in silico determination of impaired proteins for M2e1x, M2e2x M2e3x M2e4x M2e5x and M2e6x. The Phyre2 web portal for protein modeling, prediction and analysis is used. [Figure 4B] Figures 4A and 4B show models of protein impairment of in silico determination of impaired proteins for M2e1x, M2e2x M2e3x M2e4x M2e5x and M2e6x. The Phyre2 web portal for protein modeling, prediction and analysis is used. [Figure 5] Figure 5A shows an exemplary SDS PAGE of purified ClyA-6M2e protein. Figure 5B shows analytical size exclusion chromatography of exemplary OMVs. [Figure 6] Figure 6 shows the design strategy of Escherichia coli Nissle strain. [Figure 7] Figures 7A and 7B VT-104 provided 100% protection in mice. Figure 7A The ClyA-M2 fusion protein with 4xM2e vaccine provided 100% protection. Figure 7B Serum from M2e-immunized mice provided 100% protection against lethal challenge with influenza. [Figure 8] Figures 8A and 8B Antibody titers correlate with protection. Figure 8A Mouse sera were analyzed for anti-M2e titers 8 weeks after the first vaccination. Figure 8B Linear regression of anti-M2e IgG2a logarithms converted the titers against the minimum percent of the original weight of rOMV-immunized mice during PR8 challenge. [Figure 9]Figure 9 shows the efficacy test designs for 4xM2e and 6xM2e ferret H5N1. Attacker strain: derived from chicken eggs; H5N1(+) vaccine: formalin / UV inactivated, derived from Vero cells. [Figure 10] Figure 10 shows the ferret survival curves illustrating the survival of VT-105-immunized ferrets against VT-104-immunized ferrets when attacked with H5N1. [Figure 11] Figure 11 shows a graph of antibody titers measured by ELISA in ferrets vaccinated with either VT-104 or VT-105. [Modes for carrying out the invention]

[0027] Detailed explanation In one embodiment, a polyvalent influenza virus vaccine (MIV), a polyvalent immunogenic polypeptide (MIP), and a fusion protein are described herein. Further methods for producing and using MIV, MIP, and fusion proteins are provided herein.

[0028] Various aspects of this disclosure are shown and described herein, but it will be apparent to those skilled in the art that such aspects are provided by illustration only. Many variations, changes, and substitutions can be conceived by those skilled in the art without departing from the disclosure. It should be understood that various substitutes for the aspects of the disclosure described herein may be used.

[0029] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in the subject matter claimed. Generally, the nomenclature used herein in relation to immunology, oncology, cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization techniques is well known and commonly used in the art. Units of measurement, unless otherwise defined, are consistent with The International System of Units (SI), NIST Special Publication 330, 2019 edition.

[0030] As used herein, all numbers or numerical ranges include all integers within or encompassing such ranges, as well as any values ​​or integers within such ranges, unless the context explicitly indicates otherwise. For example, a reference to the range 90–100% includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc. In another example, references to the range of 1 to 5,000 times include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times, etc., as well as 1.1, 1.2, 1.3, 1.4 or 1.5 times, etc., 2.1, 2.2, 2.3, 2.4 or 2.5 times, etc.

[0031] The terms used herein are intended to describe only specific aspects and are not intended to limit any aspect. Where used herein, the singular forms “a,” “an,” and “the” are intended to include the plural form unless the context explicitly indicates otherwise. Where used herein, the terms “comprises” and / or “comprising” identify the presence of the described features, integers, processes, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, processes, operations, elements, components, and / or groups thereof. Where used herein, the terms “and / or” include any and all combinations of one or more of the related enumerated items.

[0032] Unless otherwise specifically stated or evident from the context, the term “about” as used herein with respect to a number or range of numbers is understood to mean the number stated and ±10% of that number, or less than 10% of the listed lower limit and more than 10% of the listed upper limit of the values ​​enumerated for a range.

[0033] As used herein, the term “immunogenicity” means the ability of a substance to induce an immune response in a recipient. In some embodiments, an immune response is induced when the immune system of an organism or a particular type of immune cells is exposed to an immunogenic substance. The term “non-immunogenicity” means the absence or non-existence of an immune response to a substance that exceeds a detectable threshold. In some embodiments, when the immune system of an organism or a particular type of immune cells is exposed to a non-immunogenic substance, no immune response is detected. In some embodiments, non-immunogenic compositions, vectors or nucleic acids of the present disclosure are provided herein that do not induce an immune response exceeding a predetermined threshold when measured by an immunogenicity assay. In some embodiments, reduced immunogenic compositions, vectors or nucleic acids of the present disclosure are provided herein that induce a reduced immune response below a predetermined threshold when measured by an immunogenicity assay. For example, when using an immunogenicity assay to measure antibodies produced against an inflammatory marker, the non-immunogenic or reduced immunogenic compositions provided herein result in the production of antibodies or markers at a level below a predetermined threshold. For example, a given threshold is up to 1.5 times, 2 times, 3 times, 4 times, or 5 times the level of the antibody or marker produced by the control reference.

[0034] "Percent identity," "% identity," or "sequence identity" refers to the degree to which two sequences (nucleotides or amino acids) have the same residues at the same positions in their alignment. For example, "The nucleotide sequence is X% identical to sequence number Y" means the % identity of the nucleotide sequence to sequence number Y, and more specifically, that X% of the residues in the nucleotide sequence are identical to the corresponding residues in the sequence disclosed in sequence number Y. A sequence said to be X% identical to a reference sequence may contain more nucleotide or amino acid residues than those identified in the reference sequence, but it must contain the sequence corresponding to the reference sequence. In most cases, the sequence in question contains the sequence corresponding to all of a particular reference sequence. Generally, computer programs are used for such calculations. Exemplary programs for comparing and aligning pairs of sequences include ALIGN, FASTA, GAPBLAST, BLASTP, BLASTN, or GCG.

[0035] The term "plasmid" refers to an extrachromosomal factor that carries genes from a cell's chromosomes and can replicate independently. Plasmids can take the form of a circular double-stranded DNA molecule. Such factors may include self-replicating sequences of any origin, genomic integration sequences, phages or nucleotide sequences, and linear, circular, or supercoiled, single-stranded or double-stranded DNA or RNA. Exemplary plasmids include, but are not limited to, minicircle and doggybone plasmids.

[0036] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably to refer to nucleotides or analogs thereof in polymeric form of either deoxyribonucleotides or ribonucleotides, in single-stranded, double-stranded, or multi-stranded forms, of any length. Examples of polynucleotides considered include genes or fragments thereof. Exemplary polynucleotides include, but are not limited to, DNA, RNA, coding or non-coding regions of genes or gene fragments, loci (locus) as defined by ligation analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interference RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, cell-inactive polynucleotides such as cell-inactive DNA (cfDNA) and cell-inactive RNA (cfRNA), nucleic acid probes, and primers. When referring to T in polynucleotides, T means U (uracil) in RNA and T (thymine) in DNA. Polynucleotides can be exogenous or endogenous to cells and / or present in non-cellular environments. The term polynucleotide encompasses modified polynucleotides (e.g., modified skeletons, sugars, or nucleobases). Where present, modifications to the nucleotide structure are conferred before or after polymer assembly. Non-limiting examples of modifications include: 5-bromouracil, peptide nucleic acids, xeno nucleic acids, morpholino, locto nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to sugars), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7 guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, quosine, and wyosine. The sequence of nucleotides can be interrupted by non-nucleotide components.

[0037] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically acceptable to mammals, particularly humans or animal patients.

[0038] As used herein, the term “vector” includes nucleic acid vectors, e.g., DNA vectors, e.g., plasmids, RNA vectors, or other suitable replicons (e.g., viral vectors). Various vectors have been developed for the delivery of exogenous polynucleotides or polynucleotides encoding proteins into prokaryotic or eukaryotic cells. Examples of such expression vectors are disclosed, for example, in WO 1994 / 011026, concerning vectors suitable for the expression of the nucleic acid molecule of interest. Expression vectors suitable for use by the compositions and methods described herein include polynucleotide sequences and further sequence factors used for the expression of heterologous nucleic acid material (e.g., nucleic acid molecules) in cells, for example. Specific vectors used for the expression of nucleic acid molecules described herein include plasmids containing regulatory sequences such as promoter and enhancer regions that direct gene transcription. In some embodiments, compact bidirectional promoters do not include enhancers. Other useful vectors for the expression of nucleic acid molecular agents disclosed herein include polynucleotide sequences that increase the rate of translation of these polynucleotides or improve the stability or nuclear export of RNA resulting from gene transcription. These sequence factors include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRESs), and polyadenylation signals (polyA) to direct effective transcription of genes embodied on the expression vector. In some embodiments, expression vectors suitable for use by the compositions and methods described herein include a backbone polynucleotide encoding a marker for the selection of cells containing such vectors. Examples of suitable markers are genes encoding resistance to antibiotics, such as ampicillin, chloramphenicol, neomycin, zeosin, kanamycin, noseotricin, aminoglycosides, β-lactams, glycopeptides, macrolides, polypeptides, tetracyclines, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, chloramphenicol, or derivatives thereof.

[0039] The term "immunity" refers to the process of increasing a mammalian subject's response to an antigen, thereby improving its ability to resist or overcome infection and / or resist disease.

[0040] As used herein, the term "vaccination" refers to the introduction of a vaccine into the body of a subject, preferably a mammalian subject, such as a human.

[0041] Polyvalent influenza virus vaccine composition In one embodiment, a polyvalent influenza virus vaccine (MIV) and a polyvalent immunogenic polypeptide (MIP) are described herein.

[0042] In some embodiments, MIV comprises ABC, where i) A is a transport protein or fragment thereof comprising at least domains X1 and X2, where a) X1 is a transmembrane domain polypeptide and b) X2 is a polypeptide that can be operably linked to B; ii) B is a polyvalent immunogenic polypeptide (MIP) comprising at least five influenza virus immunogenic epitopes selected from the group consisting of Y1, Y2, Y3, Y4, Y5 and Y6; and iii) C is a membrane vesicle derived from a genetically modified Gram-positive bacterium; A and B are operably linked to produce a polyvalent immunogenic transmembrane polypeptide; AB are linked to C via the transmembrane domain X1, thereby forming MIV; and MIV, when administered to a mammal, can induce an immune response to at least two influenza strains.

[0043] In some embodiments, a polyvalent influenza virus vaccine (MIV) comprises: a) a transport protein or a fragment thereof, the transport protein comprising a transmembrane domain; b) a polyvalent immunogenic polypeptide (MIP) comprising at least five covalently linked influenza virus immunogenic epitopes; and c) a membrane vesicle derived from a genetically modified Gram-positive bacterium; the transport protein of a) is operably linked to the MIP of b) to produce a polyvalent immunogenic transmembrane polypeptide; the polyvalent immunogenic transmembrane polypeptide is linked to a vesicle to form the MIV; and the MIV, when administered to a mammal, may induce an immune response to at least two influenza virus strains.

[0044] In some embodiments, MIV can induce an immune response in mammals without the administration of an adjuvant.

[0045] immunogenic polypeptide In some embodiments, MIVs comprising immunogenic polypeptides or epitopes are described herein. In some embodiments, the MIV comprises at least five or more immunogenic epitopes. In some embodiments, the immunogenic epitopes are influenza virus immunogenic epitopes. In some embodiments, the influenza virus is influenza A. In some embodiments, influenza A is human, porcine, or avian. In some embodiments, avian is chicken, mute swan, quail, or mallard. In some embodiments, influenza A is an influenza subtype that is H1, H2, H3, H5, H6, H7, or H9. In some embodiments, the influenza virus is selected from the group consisting of H1N1, H1N2, H2N1, H3N2, H5N1, H5N2, H9N2, H7N9, H7N7, H7N3, H6N6, H6N2, and H6N1.

[0046] In some embodiments, at least five influenza virus immunogenic epitopes are different. In some embodiments, at least five influenza virus immunogenic epitopes are the same. In some embodiments, at least one of the at least five influenza virus immunogenic epitopes contains a consensus sequence or a sequence that does not exist in nature.

[0047] In some embodiments, at least five influenza virus immunogenic epitopes are fused longitudinally. In some embodiments, the operable linkage between A and B includes a covalent bond. In some embodiments, at least five influenza virus immunogenic epitopes are fused to the N- or C-terminus of a transport protein. In some embodiments, at least five influenza virus immunogenic epitopes are presented on the outside of the vesicle.

[0048] In some embodiments, MIP comprises an influenza A matrix protein 2 extracellular (M2e) peptide or a fragment thereof. In some embodiments, MIP comprises five M2e peptides. In some embodiments, MIP comprises six M2e peptides. In some embodiments, the M2e peptides comprise an amino acid sequence that is at least about 90% identical to any of SEQ ID NOs: 2-8. In some embodiments, Y1, Y2, Y3, Y4, Y5 and Y6 are selected from the group consisting of human M2e peptides, porcine M2e peptides, swan M2e peptides, chicken M2e peptides and mallard duck M2e peptides.

[0049] In some embodiments, the polyvalent immunogenic transmembrane polypeptide comprises an amino acid sequence having the formula ClyA-(M2e)5. In some embodiments, the polyvalent immunogenic transmembrane polypeptide comprises an amino acid sequence having the formula ClyA-(M2e)6. In some embodiments, the polyvalent immunogenic transmembrane polypeptide comprises an amino acid sequence having the formula ClyA-(M2e-1)-(M2e-2)-(M2e-3)-(M2e-4)-(M2e-5)-(M2e-6), where M2e-1 comprises human M2e peptide, M2e-2 comprises porcine M2e peptide, M2e-3 comprises swan M2e peptide, M2e-4 comprises chicken M2e peptide, M2e-5 comprises chicken M2e peptide, and M2e-6 comprises mallard duck M2e peptide. In some embodiments, the polyvalent immunogenic transmembrane polypeptide comprises an amino acid sequence having the formula ClyA-(M2e-1)-(M2e-2)-(M2e-3)-(M2e-4)-(M2e-5)-(M2e-6), where M2e-1 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 3, M2e-2 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 4, M2e-3 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 5, M2e-4 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 6, M2e-5 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 7, and M2e-6 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 8.

[0050] In some embodiments, the polyvalent immunogenic transmembrane polypeptide contains an amino acid sequence that is at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 9. In some embodiments, the polyvalent immunogenic transmembrane polypeptide contains an amino acid sequence that is at least 70% identical to SEQ ID NO: 9. In some embodiments, the polyvalent immunogenic transmembrane polypeptide contains an amino acid sequence that is at least 80% identical to SEQ ID NO: 9. In some embodiments, the polyvalent immunogenic transmembrane polypeptide contains an amino acid sequence that is at least 85% identical to SEQ ID NO: 9. In some embodiments, the polyvalent immunogenic transmembrane polypeptide contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 9. In some embodiments, the polyvalent immunogenic transmembrane polypeptide contains an amino acid sequence that is at least 70% identical to SEQ ID NO: 9. In some embodiments, the polyvalent immunogenic transmembrane polypeptide contains an amino acid sequence that is at least 91% identical to SEQ ID NO: 9. In some embodiments, the polyvalent immunogenic transmembrane polypeptide contains an amino acid sequence that is at least 92% identical to SEQ ID NO: 9. In some embodiments, the polyvalent immunogenic transmembrane polypeptide contains an amino acid sequence that is at least 93% identical to SEQ ID NO: 9. In some embodiments, the polyvalent immunogenic transmembrane polypeptide contains an amino acid sequence that is at least 94% identical to SEQ ID NO: 9. In some embodiments, the polyvalent immunogenic transmembrane polypeptide contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 9. In some embodiments, the polyvalent immunogenic transmembrane polypeptide contains an amino acid sequence that is at least 96% identical to SEQ ID NO: 9. In some embodiments, the polyvalent immunogenic transmembrane polypeptide contains an amino acid sequence that is at least 97% identical to SEQ ID NO: 9. In some embodiments, the polyvalent immunogenic transmembrane polypeptide contains an amino acid sequence that is at least 98% identical to SEQ ID NO: 9.In some embodiments, the polyvalent immunogenic transmembrane polypeptide comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 9.

[0051] transport proteins In some embodiments, a polyvalent influenza virus vaccine (MIV) is described herein, which comprises a transport protein (A) or a fragment thereof. In some embodiments, the transport protein is an adhesin, an immunomodulatory compound, a protease or a toxin or a fragment thereof.

[0052] In some embodiments, the transport protein is ClyA. In some embodiments, ClyA contains an amino acid sequence that is at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 1. In some embodiments, ClyA contains an amino acid sequence that is at least 70% identical to SEQ ID NO: 1. In some embodiments, ClyA contains an amino acid sequence that is at least 80% identical to SEQ ID NO: 1. In some embodiments, ClyA contains an amino acid sequence that is at least 85% identical to SEQ ID NO: 1. In some embodiments, ClyA contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 1. In some embodiments, ClyA contains an amino acid sequence that is at least 70% identical to SEQ ID NO: 1. In some embodiments, ClyA contains an amino acid sequence that is at least 91% identical to SEQ ID NO: 1. In some embodiments, ClyA contains an amino acid sequence that is at least 92% identical to SEQ ID NO: 1. In some embodiments, ClyA contains an amino acid sequence that is at least 93% identical to SEQ ID NO: 1. In some embodiments, ClyA contains an amino acid sequence that is at least 94% identical to SEQ ID NO: 1. In some embodiments, ClyA contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 1. In some embodiments, ClyA contains an amino acid sequence that is at least 96% identical to SEQ ID NO: 1. In some embodiments, ClyA contains an amino acid sequence that is at least 97% identical to SEQ ID NO: 1. In some embodiments, ClyA contains an amino acid sequence that is at least 98% identical to SEQ ID NO: 1. In some embodiments, ClyA contains an amino acid sequence that is at least 99% identical to SEQ ID NO: 1. In some embodiments, ClyA contains an amino acid sequence that is identical to SEQ ID NO: 1.

[0053] Polyvalent immunogenic polypeptide (MIP) In some embodiments, polyvalent immunogenic polypeptides (MIPs) comprising immunogenic polypeptides or epitopes are described herein.

[0054] In some embodiments, MIP comprises at least five immunogenic epitopes. In some embodiments, the immunogenic epitopes are influenza virus immunogenic epitopes. In some embodiments, the influenza virus is influenza A. In some embodiments, influenza A is human, pig, or avian. In some embodiments, avian is chicken, mute swan, quail, or mallard. In some embodiments, influenza A is an influenza subtype that is H1, H2, H3, H5, H6, H7, or H9. In some embodiments, the influenza virus is selected from the group consisting of H1N1, H1N2, H2N1, H3N2, H5N1, H5N2, H9N2, H7N9, H7N7, H7N3, H6N6, H6N2, and H6N1.

[0055] In some embodiments, the polyvalent immunogenic polypeptide (MIP) comprises at least five influenza virus immunogenic epitopes selected from the group consisting of Y1, Y2, Y3, Y4, Y5, and Y6.

[0056] In some embodiments, at least five influenza virus immunogenic epitopes are different. In some embodiments, at least five influenza virus immunogenic epitopes are the same. In some embodiments, at least one of the at least five influenza virus immunogenic epitopes contains a consensus sequence or a sequence that does not exist in nature.

[0057] In some embodiments, at least five influenza virus immunogenic epitopes are fused together in a longitudinal arrangement.

[0058] In some embodiments, MIP comprises an influenza A matrix protein 2 extracellular (M2e) peptide or a fragment thereof. In some embodiments, MIP comprises five M2e peptides. In some embodiments, MIP comprises six M2e peptides. In some embodiments, the M2e peptides comprise an amino acid sequence that is at least about 70% (e.g., at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) identical to any of SEQ ID NOs: 2-8. In some embodiments, Y1, Y2, Y3, Y4, Y5, and Y6 are selected from the group consisting of human M2e peptides, porcine M2e peptides, swan M2e peptides, chicken M2e peptides, and mallard duck M2e peptides. In some embodiments, MIP comprises an amino acid sequence having the formula (M2e-1)-(M2e-2)-(M2e-3)-(M2e-4)-(M2e-5)-(M2e-6), where M2e-1 comprises human M2e peptide, M2e-2 comprises porcine M2e peptide, M2e-3 comprises swan M2e peptide, M2e-4 comprises chicken M2e peptide, M2e-5 comprises chicken M2e peptide, and M2e-6 comprises mallard duck M2e peptide. In some embodiments, MIP comprises an amino acid sequence having the formula (M2e-1)-(M2e-2)-(M2e-3)-(M2e-4)-(M2e-5)-(M2e-6), where M2e-1 comprises an amino acid sequence that is at least about 70% identical to SEQ ID NO: 3, M2e-2 comprises an amino acid sequence that is at least about 70% identical to SEQ ID NO: 4, M2e-3 comprises an amino acid sequence that is at least about 70% identical to SEQ ID NO: 5, M2e-4 comprises an amino acid sequence that is at least about 70% identical to SEQ ID NO: 6, M2e-5 comprises an amino acid sequence that is at least about 70% identical to SEQ ID NO: 7, and M2e-6 comprises an amino acid sequence that is at least about 70% identical to SEQ ID NO: 8.

[0059] In some embodiments, the disclosure provides a fusion protein comprising a transport protein and a MIP containing at least five influenza virus immunogenic epitopes selected from the group consisting of Y1, Y2, Y3, Y4, Y5, and Y6. In some embodiments, the MIP is fused to the C-terminus of the transport protein. In some embodiments, the fusion protein comprises an amino acid sequence having the formula X1-(M2e-1)-(M2e-2)-(M2e-3)-(M2e-4)-(M2e-5)-(M2e-6), where X1 comprises ClyA, M2e-1 comprises an amino acid sequence that is at least approximately 70% identical to SEQ ID NO: 3, M2e-2 comprises an amino acid sequence that is at least approximately 70% identical to SEQ ID NO: 4, M2e-3 comprises an amino acid sequence that is at least approximately 70% identical to SEQ ID NO: 5, M2e-4 comprises an amino acid sequence that is at least approximately 70% identical to SEQ ID NO: 6, M2e-5 comprises an amino acid sequence that is at least approximately 70% identical to SEQ ID NO: 7, and M2e-6 comprises an amino acid sequence that is at least approximately 70% identical to SEQ ID NO: 8. In some embodiments, the MIP or fusion protein comprises an amino acid sequence that is at least approximately 80% identical to SEQ ID NO: 9.

[0060] In some embodiments, MIP comprises an influenza A matrix protein 2 extracellular (M2e) peptide or a fragment thereof. In some embodiments, MIP comprises five M2e peptides. In some embodiments, MIP comprises six M2e peptides. In some embodiments, the M2e peptides comprise an amino acid sequence that is at least approximately 80% identical to any of SEQ ID NOs: 2-8. In some embodiments, Y1, Y2, Y3, Y4, Y5 and Y6 are selected from the group consisting of human M2e peptides, porcine M2e peptides, swan M2e peptides, chicken M2e peptides and mallard duck M2e peptides. In some embodiments, MIP comprises an amino acid sequence having the formula (M2e-1)-(M2e-2)-(M2e-3)-(M2e-4)-(M2e-5)-(M2e-6), where M2e-1 comprises human M2e peptide, M2e-2 comprises porcine M2e peptide, M2e-3 comprises swan M2e peptide, M2e-4 comprises chicken M2e peptide, M2e-5 comprises chicken M2e peptide, and M2e-6 comprises mallard duck M2e peptide. In some embodiments, MIP comprises an amino acid sequence having the formula (M2e-1)-(M2e-2)-(M2e-3)-(M2e-4)-(M2e-5)-(M2e-6), where M2e-1 comprises an amino acid sequence that is at least approximately 80% identical to SEQ ID NO: 3, M2e-2 comprises an amino acid sequence that is at least approximately 80% identical to SEQ ID NO: 4, M2e-3 comprises an amino acid sequence that is at least approximately 80% identical to SEQ ID NO: 5, M2e-4 comprises an amino acid sequence that is at least approximately 80% identical to SEQ ID NO: 6, M2e-5 comprises an amino acid sequence that is at least approximately 80% identical to SEQ ID NO: 7, and M2e-6 comprises an amino acid sequence that is at least approximately 80% identical to SEQ ID NO: 8.

[0061] In some embodiments, the disclosure provides a fusion protein comprising a transport protein and a MIP containing at least five influenza virus immunogenic epitopes selected from the group consisting of Y1, Y2, Y3, Y4, Y5, and Y6. In some embodiments, the MIP is fused to the C-terminus of the transport protein. In some embodiments, the fusion protein comprises an amino acid sequence having the formula X1-(M2e-1)-(M2e-2)-(M2e-3)-(M2e-4)-(M2e-5)-(M2e-6), where X1 comprises ClyA, M2e-1 comprises an amino acid sequence that is at least approximately 80% identical to SEQ ID NO: 3, M2e-2 comprises an amino acid sequence that is at least approximately 80% identical to SEQ ID NO: 4, M2e-3 comprises an amino acid sequence that is at least approximately 80% identical to SEQ ID NO: 5, M2e-4 comprises an amino acid sequence that is at least approximately 80% identical to SEQ ID NO: 6, M2e-5 comprises an amino acid sequence that is at least approximately 80% identical to SEQ ID NO: 7, and M2e-6 comprises an amino acid sequence that is at least approximately 80% identical to SEQ ID NO: 8. In some embodiments, the MIP or fusion protein comprises an amino acid sequence that is at least approximately 80% identical to SEQ ID NO: 9.

[0062] In some embodiments, MIP comprises an influenza A matrix protein 2 extracellular (M2e) peptide or a fragment thereof. In some embodiments, MIP comprises five M2e peptides. In some embodiments, MIP comprises six M2e peptides. In some embodiments, the M2e peptides comprise an amino acid sequence that is at least approximately 90% identical to any of SEQ ID NOs: 2-8. In some embodiments, Y1, Y2, Y3, Y4, Y5 and Y6 are selected from the group consisting of human M2e peptides, porcine M2e peptides, swan M2e peptides, chicken M2e peptides and mallard duck M2e peptides. In some embodiments, MIP comprises an amino acid sequence having the formula (M2e-1)-(M2e-2)-(M2e-3)-(M2e-4)-(M2e-5)-(M2e-6), where M2e-1 comprises human M2e peptide, M2e-2 comprises porcine M2e peptide, M2e-3 comprises swan M2e peptide, M2e-4 comprises chicken M2e peptide, M2e-5 comprises chicken M2e peptide, and M2e-6 comprises mallard duck M2e peptide. In some embodiments, MIP comprises an amino acid sequence having the formula (M2e-1)-(M2e-2)-(M2e-3)-(M2e-4)-(M2e-5)-(M2e-6), where M2e-1 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 3, M2e-2 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 4, M2e-3 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 5, M2e-4 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 6, M2e-5 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 7, and M2e-6 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 8.

[0063] In some embodiments, the disclosure provides a fusion protein comprising a transport protein and a MIP containing at least five influenza virus immunogenic epitopes selected from the group consisting of Y1, Y2, Y3, Y4, Y5, and Y6. In some embodiments, the MIP is fused to the C-terminus of the transport protein. In some embodiments, the fusion protein comprises an amino acid sequence having the formula X1-(M2e-1)-(M2e-2)-(M2e-3)-(M2e-4)-(M2e-5)-(M2e-6), where X1 comprises ClyA, M2e-1 comprises an amino acid sequence that is at least approximately 90% identical to SEQ ID NO: 3, M2e-2 comprises an amino acid sequence that is at least approximately 90% identical to SEQ ID NO: 4, M2e-3 comprises an amino acid sequence that is at least approximately 90% identical to SEQ ID NO: 5, M2e-4 comprises an amino acid sequence that is at least approximately 90% identical to SEQ ID NO: 6, M2e-5 comprises an amino acid sequence that is at least approximately 90% identical to SEQ ID NO: 7, and M2e-6 comprises an amino acid sequence that is at least approximately 90% identical to SEQ ID NO: 8. In some embodiments, the MIP or fusion protein comprises an amino acid sequence that is at least approximately 80% identical to SEQ ID NO: 9.

[0064] In some embodiments, the MIV and / or MIP are designed in silico.

[0065] The advantages of in silico design of multiepitope fusion proteins may include, but are not limited to, higher genetic diversity of the influenza strains shown, improved magnitude of the immune response, improved quality of the antibody response, improved cross-strain protection, higher potential, and reduced toxicity associated with LPS.

[0066] In some embodiments, in silico-designed MIPs can be investigated in silico for the degree of post-folding impairment.

[0067] Damaged antigens may offer various advantages, though not limited to, improved efficiency of antibodies binding to damaged epitopes, greater ease of binding of damaged epitopes to antibodies, a larger, more actively preferred interface for contact with antibodies, more favorable enthalpic (i.e., electrostatic interaction) enrichment of polar contact residues, greater exposure to solvents than ordered / third-structure epitopes, and increased hydrogen bonding and salt crosslinking per antigen residue.

[0068] Membrane vesicles In some embodiments, a polyvalent influenza virus vaccine (MIV) comprising membrane vesicles is described herein. In some embodiments, the membrane vesicles are derived from Gram-positive bacteria. In some embodiments, the Gram-positive bacteria are: Bifidobacterium longum, Bifidobacterium lactis, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium adolescentis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus The group is selected from Russula rhamnosus, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus fermentum, Lactococcus lactis, Streptococcus thermophilus, Lactococcus lactis, Lactococcus diacetylactis, Lactococcus cremoris, Lactobacillus bulgaricus, Lactobacillus helveticus, Lactobacillus delbrueckii, Escherichia coli, and combinations thereof. In some embodiments, the Gram-positive bacteria are Escherichia coli. In some embodiments, the Gram-positive bacteria are probiotic bacteria. In some embodiments, the Gram-positive bacteria are Escherichia coli Nissle. In some embodiments, *E. coli* Nissle contains one or more modifications in genes selected from the group consisting of:lpxL, lpxP, lpxM, crcA, eptA, lpxT, nlpl, recA, ompT, lon, lpxA, lpxB, lpxD, pagL, pagP, lpxE, MsbA, MsbB, gutQ, and KdsD. In some embodiments, *E. coli* Nissle contains one or more modifications in genes selected from the group consisting of:lpxL, lpxP, lpxM, crcA, eptA, lpxT, nlpl, recA, ompT, and lon.

[0069] Linker array In some embodiments, a polyvalent influenza virus vaccine (MIV) is described herein, wherein the components of the MIV are linked using one or more linkers. In some embodiments, a transport protein and a polyvalent immunogenic polypeptide (MIP) are linked using one or more linkers.

[0070] In some embodiments, one or more linkers contain at least 5 to about 50 amino acids. In some embodiments, one or both of the first and second linkers contain about 5 to about 50 amino acids, about 5 to about 45 amino acids, about 5 to about 40 amino acids, about 5 to about 35 amino acids, about 5 to about 30 amino acids, about 5 to about 25 amino acids, about 5 to about 20 amino acids, about 5 to about 15 amino acids, about 5 to about 10 amino acids, about 10 to about 50 amino acids, about 15 to about 50 amino acids, about 20 to about 50 amino acids, about 25 to about 50 amino acids, about 30 to about 50 amino acids, about 35 to about 50 amino acids, about 40 to about 50 amino acids, or about 45 to about 50 amino acids.

[0071] In some embodiments, one or more linkers include an array selected from the group consisting of (GS)n (sequence number: 12), (G2S)n (sequence number: 13), (G3S)n (sequence number: 14), (G4S)n (sequence number: 15), and (G)n (sequence number: 16), where n is an integer from 2 to 20. In some embodiments, n is an integer from 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 4 to 20, 6 to 20, 8 to 20, 10 to 20, 12 to 20, 14 to 20, 16 to 20, or 18 to 20.

[0072] In some embodiments, one or more linkers include an array selected from the group consisting of (GGSGGD)n (sequence number: 17) or (GGSGGE)n (sequence number: 18), where n is an integer from 2 to 6.

[0073] In some embodiments, one or more linkers include an array selected from the group consisting of (GGGSGSGGGGS)n (sequence number: 20) and (GGGGGPGGGGP)n (sequence number: 21), where n is an integer from 1 to 3.

[0074] In some embodiments, one or more linkers include an array selected from the group consisting of (GGGSGGG)n (sequence number: 19), (GGGSGSGGGGS)n (sequence number: 20), and (GGGGGPGGGGP)n (sequence number: 21), where n is an integer from 1 to 3.

[0075] In some embodiments, one or more linkers include sequences selected from the group consisting of (GX)n, (GGX)n, (GGGX)n, (GGGGX)n, and (GzX)n, where z is 1 to 20 and n is at least 8. In some embodiments, z is 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 4 to 20, 6 to 20, 8 to 20, 10 to 20, 12 to 20, 14 to 20, 16 to 20, or 18 to 20. In some embodiments, X is serine, aspartic acid, glutamic acid, threonine, or proline.

[0076] In some embodiments, one or more linkers are GGGGGG (Sequence ID: 22).

[0077] Methods of use and manufacture In some embodiments, methods for the use and manufacture of polyvalent influenza virus vaccines (MIVs) are described herein.

[0078] In some embodiments, the MIP or fusion protein is encoded within a nucleic acid. In some embodiments, the disclosure provides a vector comprising a nucleic acid. In some embodiments, the disclosure provides a bacterial cell comprising a nucleic acid or vector. In some embodiments, the bacterial cell expresses the MIP or fusion protein of the disclosure.

[0079] In some embodiments, a method is provided for producing a polyvalent influenza virus vaccine (MIV), the method comprising: a) culturing Escherichia coli Nissle expressing one or more modified transport proteins and the MIP of the Disclosure; b) exposing the culture from step a) to air by providing about 5% to about 20% oxygen; and c) isolating the MIV from the supernatant of the culture.

[0080] In some embodiments, the particle size of the MIV produced by this method is 180 nanometers (nm) to 200 nm as measured by dynamic light scattering (DLS). In some embodiments, the particle size of the MIV produced by this method is at least or about 170, 175, 180, 185, 190, 195, 200, 205, 210 nm or greater than 210 nm. In some embodiments, the particle size of the MIV produced by this method is 170 nanometers (nm) to 210 nm, 170 nm to 200 nm, 180 nm to 190 nm, 180 nm to 210 nm, 180 nm to 200 nm, 180 nm to 190 nm, 190 nm to 210 nm, and 190 nm to 200 nm.

[0081] In some embodiments, at least about 50% of the bacterial vesicles in multiple bacterial vesicles are intact. In some embodiments, at least about 55% of the bacterial vesicles in multiple bacterial vesicles are intact. In some embodiments, at least about 60% of the bacterial vesicles in multiple bacterial vesicles are intact. In some embodiments, at least about 65% of the bacterial vesicles in multiple bacterial vesicles are intact. In some embodiments, at least about 75% of the bacterial vesicles in multiple bacterial vesicles are intact. In some embodiments, at least about 80% of the bacterial vesicles in multiple bacterial vesicles are intact. In some embodiments, at least about 85% of the bacterial vesicles in multiple bacterial vesicles are intact. In some embodiments, at least about 90% of the bacterial vesicles in multiple bacterial vesicles are intact. In some embodiments, at least about 95% of the bacterial vesicles in multiple bacterial vesicles are intact. In some embodiments, at least about 97% of the bacterial vesicles in multiple bacterial vesicles are intact. In some embodiments, at least about 99% of the bacterial vesicles in a group of bacterial vesicles are intact.

[0082] In some embodiments, at least about 50% of the bacterial vesicles in a group of bacterial vesicles express MIP. In some embodiments, at least about 55% of the bacterial vesicles in a group of bacterial vesicles express MIP. In some embodiments, at least about 60% of the bacterial vesicles in a group of bacterial vesicles express MIP. In some embodiments, at least about 65% of the bacterial vesicles in a group of bacterial vesicles express MIP. In some embodiments, at least about 70% of the bacterial vesicles in a group of bacterial vesicles express MIP. In some embodiments, at least about 75% of the bacterial vesicles in a group of bacterial vesicles express MIP. In some embodiments, at least about 80% of the bacterial vesicles in a group of bacterial vesicles express MIP. In some embodiments, at least about 85% of the bacterial vesicles in a group of bacterial vesicles express MIP. In some embodiments, at least about 90% of the bacterial vesicles in a group of bacterial vesicles express MIP. In some embodiments, at least about 95% of the bacterial vesicles in a group of bacterial vesicles express MIP. In some embodiments, at least about 97% of the bacterial vesicles in a group of bacterial vesicles express MIP. In some embodiments, at least about 99% of the bacterial vesicles in a group of bacterial vesicles express MIP.

[0083] In some embodiments, step a) includes culturing E. coli Nissle in a bacterial culture medium. In some embodiments, step a) includes culturing E. coli Nissle in a mammalian cell culture medium.

[0084] Pharmaceutical composition In some embodiments, pharmaceutical compositions comprising a polyvalent influenza virus vaccine are described herein. In some embodiments, pharmaceutical compositions comprising the MIV of the Disclosure and pharmaceutically acceptable excipients are provided.

[0085] The compositions or pharmaceuticals of this disclosure are in a form suitable for administration to individuals in need.

[0086] In certain embodiments, the pharmaceutically acceptable excipient compositions of this disclosure are appropriately selected from the group consisting of liquid excipients for injection, such as sterile water for injection; and aqueous solutions, such as saline solution.

[0087] Acceptable excipients are those that are physiologically tolerable to the subject being administered and that preserve the therapeutic properties of the compound being administered. Acceptable excipients and their formulations are generally described, for example, in Remington's Pharmaceutical Sciences (see above). One exemplary excipient is saline. The phrase “pharmaceutically acceptable excipient,” as used herein, means a pharmaceutically acceptable material, composition or vehicle, e.g., liquid or solid fillers, diluents, excipients, solvents or mounting materials, involved in the transport or delivery of the compound of the subject from one organ or part of the body administration site to another organ or part of the body, or in an in vitro assay system. Each excipient is acceptable in the sense that it is compatible with the other components of the formulation and does not harm the subject being administered. Acceptable excipients should not alter the specific activity of the compound of the subject.

[0088] In another embodiment, the pharmaceutical compositions disclosed herein further include acceptable additives to improve the stability of the compounds in the composition and / or to control the release rate of the composition. The acceptable additives do not alter the specific activity of the compound of the subject. Exemplary acceptable additives include, but are not limited to, sugars, e.g., mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose, and mixtures thereof. In some embodiments, the acceptable additives are combined with acceptable carriers and / or excipients, e.g., dextrose. Alternatively, exemplary acceptable additives include, but are not limited to, surfactants for increasing the stability of peptides and reducing gelation of the solution, e.g., polysorbate 20 or polysorbate 80. In some embodiments, the surfactant is added to the composition in an amount of 0.01% to 5% of the solution. The addition of such acceptable additives increases the stability and half-life of the composition during storage.

[0089] Nucleic acids or compositions in the form of suspensions, lyophilized and crystalline forms are also intended in the present invention; methods for preparing suspensions, lyophilized agents and crystalline forms are known to those skilled in the art.

[0090] In some embodiments, the pharmaceutical compositions disclosed herein are sterile. In some embodiments, the pharmaceutical compositions disclosed herein are sterilized by conventional, well-known sterilization techniques. For example, sterilization is readily achieved by filtration through a sterile filtration membrane. In some embodiments, the resulting solution is packaged for use or filtered under sterile conditions and lyophilized, and the lyophilized formulation is combined with a sterile solution before administration.

[0091] In some embodiments, freeze-drying is used to stabilize polypeptides for long-term storage, such as when polypeptides are relatively unstable in liquid compositions.

[0092] In some embodiments, certain excipients, such as polyols (e.g., mannitol, sorbitol, and glycerol); sugars (e.g., glucose and sucrose); and amino acids (e.g., alanine, glycine, and glutamic acid), act as stabilizers for lyophilized products. In some embodiments, polyols and sugars are also used to protect polypeptides from damage induced by lyophilization and to enhance their stability during storage in a dry state. In some embodiments, sugars are effective both during the lyophilization process and during storage. Other categories of molecules, such as monosaccharides and disaccharides as well as polymers like PVP, have also been reported as stabilizers for lyophilized products.

[0093] In some embodiments, for injection, the pharmaceutical compositions disclosed herein are powders suitable for reconstitution with the appropriate solutions described above. Examples of these include, but are not limited to, lyophilized, tumble-dried, or spray-dried powders, amorphous powders, granules, precipitates, or granular materials. For injection, the compositions optionally include stabilizers, pH adjusters, surfactants, bioavailability modifiers, and combinations thereof.

[0094] In some embodiments, sustained-release formulations are prepared. A suitable example of a sustained-release formulation is a semipermeable matrix of a solid hydrophobic polymer containing the pharmaceutical composition herein, the matrix in the form of a formed article, such as a film or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides (e.g., see U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, e.g., Lupron Depot TMExamples include (injectable microspheres composed of lactate-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyrate. Polymers such as ethylene-vinyl acetate and lactate-glycolic acid can release molecules over a period of 100 days, while certain hydrogels release proteins in a shorter time.

[0095] In some embodiments, the pharmaceutical compositions disclosed herein are designed to act briefly, release rapidly, act for a long time, or release continuously, as described herein. In one embodiment, the pharmaceutical compositions disclosed herein are formulated for controlled release or delayed release.

[0096] In some embodiments, the pharmaceutical composition is contained in a container, pack, or dispenser, along with instructions for administration.

[0097] In some embodiments, a method is provided for treating a subject at risk of contracting influenza, comprising the step of administering an appropriate amount of the pharmaceutical composition of the present disclosure. In some embodiments, the method does not involve the step of administering an adjuvant to the subject. [Examples]

[0098] Examples The following are examples of specific embodiments for carrying out this disclosure. These examples are provided for illustrative purposes only and are not intended to limit the scope of this disclosure in any way. While efforts are made to ensure accuracy with respect to the numbers used (e.g., quantity, temperature, etc.), some experimental errors and biases should be expected.

[0099] Example 1. Design of fusion protein and MIP This example describes the in silico design of a multi-epitope fusion protein containing five or six peptides from the extracellular domain (M2e) of the M2 protein derived from an influenza A antigen variant.

[0100] The average effectiveness of commercial influenza vaccines has fallen to less than 50% over the past 13 years. Most of these vaccines are inactivated influenza vaccines (IIVs) produced by purifying influenza viruses grown in chicken eggs, then isolating them with surfactants and subsequently chemically inactivating them. The immune response induced by these commercial vaccines is primarily enhanced against the variable domain of the major glycoprotein viral hemagglutinin (HA) on the surface of the virus, which changes seasonally (known as antigenic drift). While these antibodies neutralize viral infection, they are typically strain-specific, and vaccines on the market are limited in their ability to display and present multiple antigenic strain variants. Each year, in accordance with WHO recommendations, typically one or more novel strains are incorporated into the current seasonal vaccine, but often, despite all the best efforts involved, the strain recommendations do not match the prevailing virus. Given the poor performance of influenza vaccines on the market, there is an urgent need for improved or universal influenza vaccines.

[0101] M2 is a protein abundantly expressed on the surface of influenza-infected host cells and is an essential ion channel that enables the viral budding process. M2-specific antibodies disrupt the viral budding process and inhibit viral replication by targeting influenza-infected cells for killing by antibody-dependent cell-mediated cytotoxicity (ADCC). The ectodomain of influenza A matrix protein 2 (M2e) is a target antigen for a potential universal influenza A vaccine due to its remarkable conservation over time. To overcome its low immunogenicity, M2e requires a potential adjuvant, and as shown, alum adsorbed to the peptide alone improperly induces an immune response to influenza. Subsequently, a range of subunit fusion vaccines combining M2e with carrier proteins such as hepatitis B protein, keyhole limpet protein, and flagellin have been studied and developed, but many require the assistance of an incomplete Freund's adjuvant or further exogenous adjuvant such as MPL.

[0102] Because of antigenic variability among M2 ectodomains, multiple antigenic variants were used to help ensure that a specific immune response is potentially induced against all influenza strains, based on the respective gene sequences of known antigenic variants. The primary correlate of protective immunity was found to be M2-specific IgG antibodies that can provide protection when M2-specific IgG antibodies are passively transferred to naive animals.

[0103] In short, we designed a universal influenza vaccine candidate that contains multiple ectodomains of the M2 antigen, presented vertically as a ClyA fusion protein, and is displayed on the surface of bacterial exosome-like vesicles (recombinant outer membrane vesicles, rOMVs).

[0104] The designs included 6xM2e (VRT-105) and 4xM2e (VT-104). An exemplary design is shown in Figure 1. Details and differences between the 6xM2e (VRT-105) and 4xM2e (VT-104) designs are shown in Figure 2. The M2e variants, origin viruses, and corresponding amino acid sequences are shown in Figure 3. The M2e variants, representative viruses, and virus types are shown in Table 1, and the M2e sequences are shown in Table 2. [Table 1] [Table 2]

[0105] The degree of post-folding impairment of in silico-designed universal influenza vaccine candidate proteins M2e1x, M2e2x, M2e3x, M2e4x, M2e5x, and M2e6x was investigated using the Phyre2 web portal for protein modeling, prediction, and analysis. Kelly LA et al., Nature Protocols 10, 845-858 (2015).

[0106] Furthermore, lipid tethering induces a native-like conformation of damaged epitopes, thereby improving mAb binding.

[0107] The results show that the expected impairment increases with the length of the M2e fusion protein (Figures 4A and 4B).

[0108] Example 2. Production and Characterization of Expression Plasmids Construction of expression plasmids ClyA and four or six M2e sequences were linked together via a flexible linker.

[0109] The DNA encoding ClyA-6xM2e (483 amino acids) was cloned into a 3689 bp plasmid with optimized E. coli codon usage.

[0110] Custom plasmids were fabricated and characterized. The plasmids were transformed into *E. coli* DH10B, and transformants were selected on LB plates under kanamycin selection (50 μg / ml). Plasmids isolated from multiple isolates were subjected to restriction digestion using specific restriction enzymes. Plasmids exhibiting the expected agarose gel electrophoresis band pattern were selected for DNA sequencing, and clones were sequenced to confirm the insertion sequence. An exemplary SDS-PAGE gel showing expression of the ClyA-6M2e fusion protein is shown in Figure 5A. Analysis of the culture supernatant was performed by SDS-PAGE and analytical SEC in Figure 5B.

[0111] Example 3. Production and characterization of an E. coli cell bank. rOMV is derived from a foliative symbiotic strain of *E. coli* (Nissle 1917) that has been genetically modified to produce excessive vesicles (overgenerating rOMV), and acts as both a vaccine delivery vehicle and an adjuvant to induce robust immunity without biologically active lipopolysaccharide (LPS). The foliative symbiotic strain was selected due to its ability to induce a more robust immune response than other strains of *E. coli*. rOMV vaccines are nano-sized lipid vesicles that can harbor protein and polysaccharide antigens and arise constitutively from the outer membrane and periluminal space of Gram-negative bacteria. rOMV has been shown to be stable (2 years in solution at 5°C) and has the ability to be freeze-dried for increased thermal stability. Some rOMV vaccines are derived from a BSL-1 probiotic strain of *E. coli* (Nissle 1917) that is detoxified by structural remodeling of lipopolysaccharide (LPS; Nissle-4 has 4 acyl chains on its LPS instead of 6). This disruptive form of endotoxin does not induce a fever response in human blood and does not activate cells via mouse or human Toll-like receptor 4 (TLR4); a similar approach has shown an excellent safety profile.

[0112] In short, ClyA-6xM2e rOMV was recombinantly expressed using a genetically engineered derivative of the probiotic E. coli strain Nissle 1917 (EcN) serotype O6:K5:H1. Genetic engineering was performed to generate a cell line that (i) lacked the ability to synthesize the pyrogenic form of lipopolysaccharide (LPS) and (ii) produced vesicles (i.e., free rOMV) at a significantly higher rate than the parental strain. A complete description of the genetic engineering of the cell line is shown in Figure 6.

[0113] The knockout was constructed using a two-step process. During the first step, the gene targeted for the knockout was replaced with an antibiotic resistance cassette in which the tandem flippase recognition target (FRT) site was adjacent. Insertion clones were selected for antibiotic resistance and confirmed by PCR amplification of a specific locus reporting the exact insertion. Subsequently, the antibiotic resistance cassette was removed by introducing and activating plasmid-encoded flippase (FLP) recombination, which recognizes the tandem FRT site and deletes the cassette between these sites. The plasmid encoding FLP is temperature-sensitive and is subsequently corrected by culturing cells at 37°C, a temperature that supports cell proliferation rather than plasmid replication. Deletion of the inserted cassette was confirmed by PCR amplification of the targeted locus. The "clean" deletion after cassette deletion always results in a specific amplicon size compared to that derived from wild-type and insertional mutations. The modifications are shown in Table 3. [Table 3]

[0114] The strain was genetically engineered to naturally produce rOMV in high yield by excessively generating vesicles, thus avoiding the need for surfactant extraction used in other OMV vaccine preparations. Furthermore, heterologous protein antigens are targeted against the surface of rOMV using ClyA as the fusion and displaying protein. Together, this allows diverse antigens to be displayed on the surface of vesicles in the multimer complex. rOMV has an average diameter of 100 nm, ideal for efficient efflux to lymph nodes, uptake by antigen-presenting cells, and induction of a robust immune response. The resulting rOMV vaccine candidate is non-toxic, well-tolerated, and overcomes the poor immunogenicity of purified peptides in animal model studies.

[0115] Multiple epitopes can be represented and aligned in a morphological manner that allows for the induction of high levels of cross-reactive antibodies that may provide cross-protection against influenza strains in mice and ferrets.

[0116] Example 4. Animal efficacy study of OMV expressing the multimer M2e. Regarding the efficacy against influenza A strains, VT-104 and VT-105 were tested for efficacy in animal studies.

[0117] VT-104 was tested in multiple mouse models with influenza A strain A / PR8 / 34 attack. In each mouse model study, immunization was performed as an induction / accelerated immunization regimen including 0.2 μg of total M2 antigen followed by viral attack, separated by 4 weeks

[24] . As shown in one of the studies, all sham (PBS) control mice succumbed to infection by 10 days after attack, confirming a lethal attack dose (Figures 7A and 8B). Mice vaccinated with VT-104 showed the lowest morbidity among the vaccinated groups. Survival analysis showed that only M2e4xHet-OMV (VT-104) provided significantly better protection than the protection of the sham (PBS) vaccine (p<0.01). In contrast, when the M2e peptide was administered with alum, protection against lethal attack was not achieved, and only 25% of survivors (1 / 4) exhibited a robust rOMV response. Furthermore, the mean lung and tracheal viral titers of VT-104-vaccinated mice were approximately 10 times lower than those of sham (PBS) controls 6 days after attack. In addition, serum derived from VT-104-immunized mice provided 100% protection during passive transfer to naive mice.

[0118] Displaying the M2e peptide as a ClyA fusion protein on the surface of rOMV resulted in a significantly increased anti-M2e IgG2a titer relative to the IgG1 titer, achieving protection against influenza attack without further adjuvants (Figures 8A and 8B). A significant relationship was found in a linear regression analysis of logarithmically transitioned anti-M2e total IgG titer versus the minimum percentage of original weight (r²=0.403, p<0.05). Since the literature suggests that IgG2a is a correlate of protection for M2e, we analyzed IgG2a versus the minimum percentage of original weight and found an even stronger relationship (r²=0.477, p<0.01), although IgG1 titer versus the minimum percentage of original weight did not show a significant relationship (r²=0.228, p>0.05). The proposed mechanism of protection for M2e-based vaccines is the removal of infected cells via antibody-dependent cytotoxicity and phagocytosis (ADCC and ADCP), and IgG2a antibodies were shown to be necessary and sufficient to provide protection in mice. The results further support this proposed mechanism. Protection against weight loss during attack was found to be most significantly correlated with IgG2a antibody levels. The minimum IgG2a antibody titer among passively immunized mice was 8,000, adding strength to the reported minimum protective IgG2a antibody titer of 10,000. Overall, the data support the premise that OMV represents a promising adjuvant platform for the development of M2e-based universal influenza vaccines.

[0119] Example 5. Efficacy test of VT-104 and VT-105 in ferrets attacked with H5N1. The efficacy of VT-105 was compared to VT-104 in a ferret H5N1 influenza model. For this study, a total of 24 ferrets were divided into four groups, each containing 6 ferrets. Each group of ferrets was immunized with one of two rOMV-M2e vaccine candidates (M2 antigen, 85 μg), an inactivated H5N1 vaccine (VN / 1203 / 04) as a positive control, or empty rOMV acting as a negative control preparation. Ferrets received three rounds of immunization at 28-day intervals, and blood samples were collected to monitor antibody response. 30 days after the final immunization, all ferrets were attacked with a lethal dose of highly pathogenic (path) avian influenza virus (VN / 1203 / 04; H5N1). Details of the study are shown in Figure 9.

[0120] The results showed that H5N1 virus infection caused severe illness in negative control ferrets (Group 1), and due to the severe illness (extreme lethargy, neurological signs), the ferrets in this group did not survive the attack, and the remaining two ferrets were euthanized on day 7 after the attack (Figure 10). The positive (H5N1) control ferrets had a favorable outcome, achieving 100% survival. The VT-104 group had a survival rate of 33% (2 / 6), and the VT-105 group survived even better, achieving a survival rate of 66% (4 / 6).

[0121] Antisera were evaluated using M2e IgG ELISA to detect influenza-specific antibodies. Figure 11 shows the antibody titers in ferret serum over four test periods. As expected, all pre-immunized serum was below the assay cutoff (200). Serum from all four test groups was tested on day 84 (two weeks after the last immunization). At this point, 3 / 6 ferrets in the negative control group had low IgG titers (800–1,600), and the remaining 3 ferrets tested were below detection levels. For the H5N1 inactivated vaccine group, 5 / 6 ferrets had low to moderate IgG titers ranging from 400–6,400. In contrast, the 2 ferrets in the rOMV vaccine group that received the M2e antigen produced the highest titers. By day 84, the highest response group, VT-105(6xM2e), exhibited the strongest titer, ranging from 64,000 to 256,000.

[0122] While the present invention is specifically illustrated and described in relation to its preferred embodiments, it will be understood by those skilled in the art that various modifications in form and detail can be made in the present invention without departing from the scope of the invention as encompassed by the appended claims. [Table 4]

Claims

1. A polyvalent influenza virus vaccine (MIV), wherein the MIV is: ABC Includes, During the ceremony, i) A is at least domain X 1 and X 2 A transport protein or fragment thereof, which includes, here a) X 1 It is a transmembrane domain polypeptide, b) X 2 is a polypeptide that can be operably linked to B; ii) B is Y 1 Y 2 Y 3 Y 4 Y 5 and Y 6 and is a multivalent immunogenic polypeptide (MIP) comprising at least 5 influenza virus immunogenic epitopes selected from the group consisting of iii) C is a membrane vesicle derived from genetically modified Gram-positive bacteria. Including; A and B are operably linked to produce a multivalent immunogenic transmembrane polypeptide; AB is the transmembrane domain X 1 It is connected to C via this, thereby forming an MIV; MIV is a type of influenza virus that, when administered to mammals, can induce an immune response against at least two influenza strains.

2. A polyvalent influenza virus vaccine (MIV), wherein the MIV is: a) A transport protein or a fragment thereof, wherein the transport protein includes a transmembrane domain; b) A polyvalent immunogenic polypeptide (MIP) containing at least five covalently bound influenza virus immunogenic epitopes; c) Membrane vesicles derived from genetically modified Gram-positive bacteria Including; The transport protein a) is manipulatively linked to the MIP b) to produce a multivalent immunogenic transmembrane polypeptide; Multivalent immunogenic transmembrane polypeptides are linked to vesicles to form MIVs; MIV is a type of influenza virus that, when administered to mammals, can induce an immune response against at least two influenza virus strains.

3. The MIV according to claim 1 or 2, wherein the transport protein is an adhesin, an immunomodulatory compound, a protease, or a toxin or a fragment thereof.

4. The MIV according to claim 3, wherein the transport protein is ClyA.

5. The MIV according to claim 4, wherein ClyA comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO:

1.

6. The MIV according to any one of claims 1 to 5, wherein at least five influenza virus immunogenic epitopes are fused in a longitudinal arrangement.

7. The MIV according to any one of claims 1 to 5, wherein the operable link between A and B includes a covalent bond.

8. The MIV according to any one of claims 1 to 6, wherein at least five influenza virus immunogenic epitopes are fused to the N- or C-terminus of the transport protein.

9. The MIV according to any one of claims 1 to 8, wherein at least five influenza virus immunogenic epitopes are presented on the outside of the vesicle.

10. The MIV according to any one of claims 1 to 9, wherein the influenza virus is influenza A.

11. The MIV according to claim 10, wherein influenza A is human, swine, or avian.

12. The MIV according to claim 11, wherein the bird is a chicken, a whooper swan, a quail, or a mallard.

13. The MIV according to claim 10, wherein influenza A is an influenza subtype that is H1, H2, H3, H5, H6, H7, or H9.

14. The MIV according to claim 10, wherein the influenza virus is selected from the group consisting of H1N1, H1N2, H2N1, H3N2, H5N1, H5N2, H9N2, H7N9, H7N7, H7N3, H6N6, H6N2, and H6N1.

15. The MIV according to any one of claims 1 to 14, wherein at least five influenza virus immunogenic epitopes are different.

16. The MIV according to any one of claims 1 to 14, wherein at least five influenza virus immunogenic epitopes are the same.

17. The MIV according to any one of claims 1 to 16, wherein at least one of at least five influenza virus immunogenic epitopes comprises a consensus sequence or a sequence not naturally occurring.

18. The MIV according to any one of claims 1 to 16, wherein the MIP comprises an influenza A matrix protein 2 extracellular (M2e) peptide or a fragment thereof.

19. The MIV according to claim 18, wherein the MIP contains five M2e peptides.

20. The MIV according to claim 18, wherein the MIP contains six M2e peptides.

21. The MIV according to any one of claims 18 to 20, wherein the M2e peptide comprises an amino acid sequence that is at least about 90% identical to any of SEQ ID NOs: 2 to 8.

22. The MIV according to any one of claims 1 to 21, wherein the transport protein and the MIP are linked using one or more linkers.

23. The MIV according to claim 22, wherein one or more linkers include an array selected from the group consisting of (GS)n (sequence number: 12), (G2S)n (sequence number: 13), (G3S)n (sequence number: 14), (G4S)n (sequence number: 15), and (G)n (sequence number: 16), and n is an integer from 2 to 20.

24. The MIV according to claim 22, wherein one or more linkers include an array selected from the group consisting of (GGSGGD)n (sequence number: 17) or (GGSGGE)n (sequence number: 18), and n is an integer from 2 to 6.

25. The MIV according to claim 22, wherein one or more linkers include an array selected from the group consisting of (GGGSGGG)n (sequence number: 19), (GGGSGSGGGGS)n (sequence number: 20), and (GGGGGPGGGGP)n (sequence number: 21), where n is an integer from 1 to 3.

26. The MIV according to claim 22, wherein one or more linkers include sequences selected from the group consisting of (GX)n, (GGX)n, (GGGX)n, (GGGGX)n and (GzX)n, where z is 1 to 20 and n is at least 8.

27. The MIV according to claim 26, wherein X is serine, aspartic acid, glutamic acid, threonine, or proline.

28. Y 1 , Y 2 , Y 3 , Y 4 , Y 5 and Y 6 The MIV according to any one of claims 1 to 27, wherein the MIV is selected from the group consisting of human M2e peptide, porcine M2e peptide, swan M2e peptide, chicken M2e peptide, and mallard M2e peptide.

29. The polyvalent immunogenic transmembrane polypeptide is given by formula ClyA-(M2e) 5 The MIV according to any one of claims 18 to 27, comprising an amino acid sequence having the above.

30. The polyvalent immunogenic transmembrane polypeptide is given by formula ClyA-(M2e) 6 The MIV according to any one of claims 18 to 27, comprising an amino acid sequence having the above.

31. The MIV according to any one of claims 18 to 27, wherein the polyvalent immunogenic transmembrane polypeptide comprises an amino acid sequence having the formula ClyA-(M2e-1)-(M2e-2)-(M2e-3)-(M2e-4)-(M2e-5)-(M2e-6), where M2e-1 comprises human M2e peptide, M2e-2 comprises porcine M2e peptide, M2e-3 comprises swan M2e peptide, M2e-4 comprises chicken M2e peptide, M2e-5 comprises chicken M2e peptide, and M2e-6 comprises mallard duck M2e peptide.

32. The MIV according to any one of claims 18 to 27, wherein the polyvalent immunogenic transmembrane polypeptide comprises an amino acid sequence having the formula ClyA-(M2e-1)-(M2e-2)-(M2e-3)-(M2e-4)-(M2e-5)-(M2e-6), where M2e-1 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 3, M2e-2 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 4, M2e-3 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 5, M2e-4 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 6, M2e-5 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 7, and M2e-6 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO:

8.

33. The MIV according to any one of claims 1 to 32, wherein the polyvalent immunogenic transmembrane polypeptide comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO:

9.

34. Gram-positive bacteria include: Bifidobacterium longum, Bifidobacterium lactis, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium adolescentis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus MIV according to any one of claims 1 to 33, selected from the group consisting of Suh Johnsoni, Lactobacillus plantarum, Lactobacillus fermentum, Lactococcus lactis, Streptococcus thermophilus, Lactococcus lactis, Lactococcus diacetylactis, Lactococcus cremoris, Lactobacillus bulgaricus, Lactobacillus helveticus, Lactobacillus delbrueckii, Escherichia coli, and combinations thereof.

35. The MIV according to any one of claims 1 to 34, wherein the Gram-positive bacterium is Escherichia coli.

36. The MIV according to any one of claims 1 to 35, wherein the Gram-positive bacteria are probiotic bacteria.

37. The MIV according to any one of claims 1 to 36, wherein the Gram-positive bacterium is Escherichia coli Nissle.

38. The MIV according to claim 37, wherein Escherichia coli Nissle comprises one or more modifications in genes selected from the group consisting of: lpxL, lpxP, lpxM, crcA, eptA, lpxT, nlpl, recA, ompT, lon, lpxA, lpxB, lpxD, pagL, pagP, lpxE, MsbA, MsbB, gutQ, and KdsD.

39. The MIV according to claim 37, wherein Escherichia coli Nissle comprises one or more modifications in genes selected from the group consisting of lpxL, lpxP, lpxM, crcA, eptA, lpxT, nlpl, recA, ompT, and lon.

40. An MIV according to any one of claims 1 to 39, which can induce an immune response in mammals without the administration of an adjuvant.

41. Y 1 , Y 2 , Y 3 , Y 4 , Y 5 and Y 6 A polyvalent immunogenic polypeptide (MIP) comprising at least five influenza virus immunogenic epitopes selected from the group consisting of the following.

42. The MIP according to claim 41, wherein at least five influenza virus immunogenic epitopes are fused in a vertical arrangement.

43. The MIP according to claim 41 or 42, wherein the MIP comprises an amino acid sequence having the formula (M2e-1)-(M2e-2)-(M2e-3)-(M2e-4)-(M2e-5)-(M2e-6), where M2e-1 comprises human M2e peptide, M2e-2 comprises porcine M2e peptide, M2e-3 comprises swan M2e peptide, M2e-4 comprises chicken M2e peptide, M2e-5 comprises chicken M2e peptide, and M2e-6 comprises mallard duck M2e peptide.

44. The MIP according to claim 41 or 42, wherein the MIP comprises an amino acid sequence having the formula (M2e-1)-(M2e-2)-(M2e-3)-(M2e-4)-(M2e-5)-(M2e-6), where M2e-1 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 3, M2e-2 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 4, M2e-3 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 5, M2e-4 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 6, M2e-5 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 7, and M2e-6 comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO:

8.

45. Transport proteins and Y 1 , Y 2 , Y 3 , Y 4 , Y 5 and Y 6 A fusion protein comprising an MIP containing at least five influenza virus immunogenic epitopes selected from the group consisting of the following.

46. The fusion protein according to claim 45, wherein MIP is fused to the C-terminus of the transport protein.

47. formula 1 A fusion protein comprising an amino acid sequence having -(M2e-1)-(M2e-2)-(M2e-3)-(M2e-4)-(M2e-5)-(M2e-6), X 1 The fusion protein according to claim 45 or 46, wherein ClyA is included, M2e-1 contains an amino acid sequence that is at least about 90% identical to SEQ ID NO: 3, M2e-2 contains an amino acid sequence that is at least about 90% identical to SEQ ID NO: 4, M2e-3 contains an amino acid sequence that is at least about 90% identical to SEQ ID NO: 5, M2e-4 contains an amino acid sequence that is at least about 90% identical to SEQ ID NO: 6, M2e-5 contains an amino acid sequence that is at least about 90% identical to SEQ ID NO: 7, and M2e-6 contains an amino acid sequence that is at least about 90% identical to SEQ ID NO:

8.

48. The MIP according to claim 41 or the fusion protein according to any one of claims 45 to 47, wherein the MIP comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO:

9.

49. A nucleic acid encoding an MIP according to any one of claims 41 to 44 or a fusion protein according to any one of claims 45 to 47.

50. A vector comprising the nucleic acid described in claim 49.

51. A bacterial cell comprising the vector according to claim 50.

52. A bacterial cell expressing the MIP according to any one of claims 41 to 44 or the fusion protein according to any one of claims 45 to 47.

53. a) A step of culturing Escherichia coli Nissle expressing a transport protein and MIP according to any one of claims 1 to 40, comprising one or more modifications; b) A step of exposing the culture from step a) to air by providing approximately 5% to approximately 20% oxygen; and c) Steps to isolate MIV from the culture supernatant. A method for producing a polyvalent influenza virus vaccine (MIV) containing [the specified ingredient].

54. The method according to claim 53, wherein the particle size of the MIV is 180 nanometers (nm) to 200 nm when measured by dynamic light scattering (DLS).

55. The method according to claim 53, wherein at least about 50% of the bacterial vesicles of a plurality of bacterial vesicles are intact.

56. The method according to claim 53, wherein at least about 75% of the bacterial vesicles of a plurality of bacterial vesicles are intact.

57. The method according to claim 53, wherein at least about 90% of the bacterial vesicles of a plurality of bacterial vesicles are intact.

58. The method according to any one of claims 53 to 57, wherein at least about 50% of the bacterial vesicles of a plurality of bacterial vesicles express MIP.

59. The method according to any one of claims 53 to 57, wherein at least about 75% of the bacterial vesicles of a plurality of bacterial vesicles express MIP.

60. The method according to any one of claims 53 to 57, wherein at least about 90% of the bacterial vesicles of a plurality of bacterial vesicles express MIP.

61. The method according to any one of claims 53 to 60, wherein step a) comprises culturing Escherichia coli Nissle in a bacterial culture medium.

62. The method according to any one of claims 53 to 60, wherein step a) comprises culturing Escherichia coli Nissle in a mammalian cell culture medium.

63. A pharmaceutical composition comprising the MIV according to any one of claims 1 to 40 and a pharmaceutically acceptable excipient.

64. A method for treating a subject at risk of contracting influenza, comprising the step of administering an appropriate amount of the pharmaceutical composition described in claim 63 to the subject at risk of contracting influenza.

65. The method according to claim 64, which does not include the step of administering an adjuvant to a subject.

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