Immunogenic composition against influenza

Abstract

To provide compositions and methods related to mutant viruses, and in particular, mutant influenza viruses.SOLUTION: The mutant virus disclosed herein includes mutant M2 sequences and mutant BM2 sequences, and are useful in immunogenic compositions, e.g., as tetravalent vaccines. Further disclosed herein are methods, compositions, and cells for propagating the viral mutants, and methods, devices, and compositions related to vaccination.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] (Cross - reference to related applications) This application claims priority to U.S. Patent Application No. 62 / 464,019, filed on February 27, 2017. The entire content of the said application is incorporated herein by reference.

Background Art

[0002] Influenza is a major cause of death among American adults. Every year, approximately 36,000 people die from influenza and over 200,000 are hospitalized. Influenza is a highly contagious disease and is spread by coughing, sneezing, and direct physical contact with objects (such as doorknobs and telephones) that carry the virus. The symptoms of influenza include, for example, severe fatigue, headache, chills, and body aches. Approximately 50% of infected individuals are asymptomatic but still contagious. Immunization is 50 - 60 percent effective in preventing influenza in healthy people under 65 years of age as long as the antigenicity of the circulating virus strain is compatible with that of the vaccine. Vaccination is the primary method of preventing influenza, and both live - attenuated and inactivated (killed) virus vaccines are currently available. Live virus vaccines (typically administered intranasally) can activate all phases of the immune system and stimulate an immune response against a number of viral antigens. Thus, the use of live virus overcomes problems related to the destruction of viral antigens that can occur during the preparation of inactivated virus vaccines. In addition, the immunity generated by live virus vaccines is generally more persistent, effective, and cross - reactive than inactivated virus - induced immunity, and live virus vaccines have lower manufacturing costs than inactivated virus vaccines. However, the mutations of attenuated viruses are often unclear and there is concern about reversion.

Summary of the Invention

[0003] In one aspect, the present disclosure provides an immunogenic composition. The composition is a multivalent composition and includes recombinant viruses derived from at least two influenza virus strains. In some embodiments, the multivalent composition comprises the following: a) at least one engineered attenuated influenza A M2-incomplete recombinant virus (the engineered influenza A virus comprises a mutant M2 gene comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3); and b) at least one engineered attenuated BM2-incomplete recombinant virus (the engineered influenza B virus comprises a mutant BM2 gene comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11). In some embodiments, the at least one influenza A virus is selected from the group of H1N1 and H3N2 subtypes, and the at least one influenza B virus is selected from the group of B / Yamagata and B / Victoria lineages.

[0004] In some embodiments, the multivalent composition comprises a recombinant virus selected from the group consisting of: a) two engineered attenuated influenza A M2-deficient viruses selected from the group of H1N1 and H3N2 subtypes (the A M2-deficient virus comprises a mutated M2 gene comprising SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3), and two engineered attenuated influenza B M2-deficient viruses selected from the group of B / Yamagata and B / Victoria lineages (the B M2-deficient virus comprises a mutated B M2 gene comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11); b) two engineered attenuated influenza A M2-deficient viruses selected from the group of H1N1 and H3N2 subtypes (the A M2-deficient virus comprises a mutated M2 gene comprising SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3), and one engineered attenuated influenza B M2-deficient virus selected from the group of B / Yamagata and B / Victoria lineages (the B M2-deficient virus comprises a mutated B M2 gene comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11); c) one engineered attenuated influenza A M2-deficient virus selected from the group of H1N1 and H3N2 subtypes (the A M2-deficient virus comprises a mutated M2 gene comprising SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3), and two engineered attenuated influenza B M2-deficient viruses selected from the group of B / Yamagata and B / Victoria lineages (the B M2-deficient virus comprises a mutated B M2 gene comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11);and d) one engineered live attenuated influenza A M2-incomplete virus selected from the group of H1N1 and H3N2 subtypes (the A M2-incomplete virus comprises a mutated M2 gene comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3), and one engineered live attenuated influenza B M2-incomplete virus selected from the group of B / Yamagata and B / Victoria lineages (the B M2-incomplete virus comprises a mutated BM2 gene comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11);

[0005] In some embodiments, the multivalent composition is a tetravalent composition comprising: a) i) two engineered live attenuated influenza A viruses consisting of H1N1 having a mutated M2 gene comprising SEQ ID NO: 1, and ii) H3N2 having a mutated M2 gene comprising SEQ ID NO: 1; and b) i) B / Victoria having a mutated BM2 gene comprising SEQ ID NO: 9 or SEQ ID NO: 11, and ii) two engineered live attenuated influenza B viruses consisting of B / Yamagata having a mutated BM2 gene comprising SEQ ID NO: 9 or SEQ ID NO: 11. In some embodiments, the multivalent composition is a tetravalent composition comprising: a) i) two engineered live attenuated influenza A viruses consisting of H1N1 having a mutated M2 gene comprising SEQ ID NO: 1, and ii) H3N2 having a mutated M2 gene comprising SEQ ID NO: 1; and b) one engineered live attenuated influenza B virus selected from the group consisting of i) B / Victoria having a mutated BM2 gene comprising SEQ ID NO: 9 or SEQ ID NO: 11, and ii) B / Yamagata having a mutated BM2 gene comprising SEQ ID NO: 9 or SEQ ID NO: 11.

[0006] In some embodiments, the immunogenic composition of the present disclosure further comprises a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition of the present disclosure further comprises a pharmaceutically acceptable adjuvant. In some embodiments, the immunogenic composition of the present technology is formulated for intranasal or intradermal administration. In one aspect, the present disclosure provides a method of stimulating an immune response against influenza A and influenza B, the method comprising administering to a subject animal in need thereof a multivalent immunogenic composition comprising: a) at least one engineered attenuated influenza A M2-incomplete recombinant virus (the engineered influenza A virus comprises a mutated M2 gene comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3); and b) at least one engineered attenuated B M2-incomplete recombinant virus (the engineered influenza B virus comprises a mutated BM2 gene comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11). In some embodiments, the at least one influenza A virus is selected from the group consisting of H1N1 and H3N2 subtypes, and the at least one influenza B virus is selected from the group consisting of B / Yamagata and B / Victoria lineages.

[0007] In some embodiments, the multivalent immunogenic composition comprises a recombinant virus selected from the group consisting of: a) two engineered attenuated influenza A M2-deficient viruses selected from the group of H1N1 and H3N2 subtypes (the A M2-deficient virus comprises a mutated M2 gene comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3), and two engineered attenuated influenza B M2-deficient viruses selected from the group of B / Yamagata and B / Victoria lineages (the B M2-deficient virus comprises a mutated BM2 gene comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11); b) two engineered attenuated influenza A M2-deficient viruses selected from the group of H1N1 and H3N2 subtypes (the A M2-deficient virus comprises a mutated M2 gene comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3), and one engineered attenuated influenza B M2-deficient virus selected from the group of B / Yamagata and B / Victoria lineages (the B M2-deficient virus comprises a mutated BM2 gene comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11); c) one engineered attenuated influenza A M2-deficient virus selected from the group of H1N1 and H3N2 subtypes (the A M2-deficient virus comprises a mutated M2 gene comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3), and two engineered attenuated influenza B M2-deficient viruses selected from the group of B / Yamagata and B / Victoria lineages (the B M2-deficient virus comprises a mutated BM2 gene comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11);and d) one engineered live attenuated influenza A M2-incomplete virus selected from the group of H1N1 and H3N2 subtypes (said A M2-incomplete virus contains a mutated M2 gene comprising SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3), and one engineered live attenuated influenza B M2-incomplete virus selected from the group of B / Yamagata and B / Victoria lineages (said B M2-incomplete virus contains a mutated BM2 gene comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11);

[0008] In some embodiments, the multivalent immunogenic composition is a quadrivalent composition comprising: a) i) two engineered live attenuated influenza A viruses consisting of H1N1 having a mutated M2 gene comprising SEQ ID NO: 1, and ii) H3N2 having a mutated M2 gene comprising SEQ ID NO: 1; and b) i) B / Victoria having a mutated BM2 gene comprising SEQ ID NO: 9 or SEQ ID NO: 11, and ii) two engineered live attenuated influenza B viruses consisting of B / Yamagata having a mutated BM2 gene comprising SEQ ID NO: 9 or SEQ ID NO: 11. In some embodiments, the multivalent immunogenic composition is a quadrivalent composition comprising: a) i) two engineered live attenuated influenza A viruses consisting of H1N1 having a mutated M2 gene comprising SEQ ID NO: 1, and ii) H3N2 having a mutated M2 gene comprising SEQ ID NO: 1; and b) one engineered live attenuated influenza B virus selected from the group consisting of i) B / Victoria having a mutated BM2 gene comprising SEQ ID NO: 9 or SEQ ID NO: 11, and ii) B / Yamagata having a mutated BM2 gene comprising SEQ ID NO: 9 or SEQ ID NO: 11. In some embodiments, the immunogenic composition of the present disclosure further comprises a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition of the present disclosure further comprises a pharmaceutically acceptable adjuvant. In some embodiments, the immunogenic composition of the present disclosure is formulated for intranasal or intradermal administration.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0010] I. Definitions The following terms are used in this specification and their definitions are provided for guidance. As used in this specification, the singular forms “a,” “an,” and “the” refer to both the singular and the plural unless clearly stated otherwise to refer to only the singular. The term “about” and the use of ranges generally (whether or not modified by the term about) are not limited to the number itself indicated in this specification, but are intended to refer to a range substantially within the recited range, provided that the scope of the present invention is not departed from. As used in this specification, “about” is understood by those skilled in the art and will vary to some extent depending on the context in which the term is used. When the term is not clearly used to those skilled in the art, depending on the context in which the term is used, “about” will refer to plus or minus 10% of that term exactly. As used in this specification, the term “attenuated” when used with a virus refers to a virus that has a reduced virulence or pathogenicity compared to a non-attenuated counterpart virus, but still has survival activity or is alive. Typically, attenuation makes an infectious agent, such as a virus, less harmful or less virulent to the animal being infected compared to a non-attenuated virus. This is in contrast to a killed or completely inactivated virus.

[0011] As used herein, the terms "effective amount" or "therapeutically effective amount" or "pharmaceutically effective amount" refer to an amount sufficient to achieve the desired therapeutic and / or prophylactic effect, e.g., an amount that results in the prevention of a disease, condition and / or its symptoms. In the context of a therapeutic or prophylactic application, the amount of the composition administered to a subject animal will depend on the type and severity of the disease, as well as the characteristics of the individual, such as general health, age, sex, weight and tolerance to the administered agent. The effective amount will also depend on the stage, severity and type of the disease or condition. One of ordinary skill in the art can determine the appropriate dosage according to these and other requirements. In some embodiments, multiple doses are administered. Additionally or alternatively, in some embodiments, multiple therapeutic compositions or compounds (e.g., immunogenic compositions, e.g., vaccines) are administered.

[0012] As used herein, the term "host cell" refers to a cell in which a pathogen (e.g., a virus) can replicate. In some embodiments, the host cell is a cultured cell in vitro. Non-limiting examples of such host cells include, but are not limited to, CHO cells, Vero cells, and MDCK cells. Additionally or alternatively, in some embodiments, the host cell is present in vivo (e.g., a cell of an infected vertebrate (e.g., a bird or a mammal)). In some embodiments, the host cell can be modified, e.g., to enhance virus production. The enhancement of virus production can be, for example, by enhancing virus infection of the host cell and / or enhancing virus propagation. By way of example and not limitation, exemplary host cell modifications include: recombinant expression of the 2-6 linked sialic acid receptor on the cell surface of the host cell, and / or recombinant expression in the host cell of a protein that has been deleted or inactivated in the pathogen or virus.

[0013] The term "immunogenic composition" is used herein to refer to a composition that elicits an immune response in a mammal exposed to the composition. In some embodiments, the immunogenic composition comprises at least one M2-incomplete mutant influenza A M2SR strain (e.g., A / California / 07 / 2009 (H1N1) (comprising an M2SR mutant comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3), A / Brisbane / 10 / 2007 (H3N2) (comprising an M2SR mutant comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3)). In some embodiments, the immunogenic composition comprises at least one BM2-incomplete mutant influenza B BM2SR strain (e.g., B / Brisbane / 60 / 2008 (Victoria) (comprising a BM2SR mutant comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11), B / Wisconsin / 01 / 2010 (Yamagata) (comprising a BM2SR mutant comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11)). In some embodiments, the immunogenic composition comprises A / California / 07 / 2009 (H1N1) (comprising an M2SR mutant comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3), A / Brisbane / 10 / 2007 (H3N2) (comprising an M2SR mutant comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3), B / Brisbane / 60 / 2008 (Victoria) (comprising a BM2SR mutant comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11), and B / Wisconsin / 01 / 2010 (Yamagata) (comprising a BM2SR mutant comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11), which is formulated as a quadrivalent vaccine.

[0014] In some embodiments, the immunogenic compositions described herein can be formulated for administration in a number of forms (i.e., formulated for "exposure" to, for example, a mammal). For example, in some embodiments, the immunogenic compositions are prepared for oral, pulmonary, intravenous, intramuscular, subcutaneous, parenteral, nasal, or topical administration. The compositions can also be formulated for particular dosage forms. For example, in some embodiments, the immunogenic compositions can be formulated as liquids, gels, aerosols, ointments, creams, lyophilized formulations, powders, cakes, tablets, or capsules. In other embodiments, the immunogenic compositions are formulated as controlled release formulations, sustained release formulations, long acting release formulations, pulsatile release formulations, and mixed immediate release formulations. In some embodiments, the immunogenic compositions are provided as liquids. In other embodiments, the immunogenic compositions are provided in lyophilized form.

[0015] As used herein, the term "infected" refers to having a disease or pathogen (e.g., a virus). Infection can be either intentional (e.g., by administering a virus or pathogen (e.g., by vaccination)) or unintentional (e.g., by natural movement of a pathogen from one organism to another or from a contaminated surface to the organism). As used herein, the terms "isolated" and / or "purified" refer to preparing, isolating and / or purifying a nucleic acid (e.g., a vector or plasmid), polypeptide, virus or cell in vitro so as to be free of unwanted in vivo substances or to be substantially purified from unwanted in vivo substances that are normally present together. For example, in some embodiments, an isolated virus preparation is obtained by in vitro culture and propagation and is substantially free of other infectious agents. As used herein, "substantially free of" means below the level of detection when standard detection methods for the particular compound (e.g., unwanted nucleic acids, proteins, cells, viruses, infectious agents, etc.) are used.

[0016] As used herein, the terms "variant", "mutation" and "variety" are used interchangeably and refer to a nucleic acid or polypeptide sequence that differs from the wild-type sequence. In some embodiments, the variant or variety sequence occurs naturally. In other embodiments, the variant or variety sequence is introduced recombinantly and / or chemically. In some embodiments, a mutation of a nucleic acid comprises a modification (e.g., addition, deletion, substitution) to an RNA and / or DNA sequence. In some embodiments, the modification comprises a chemical modification (e.g., methylation) and can further comprise substitution or addition of natural and / or non-natural nucleotides. A mutation of a nucleic acid can be a silent mutation (e.g., a change in one or more nucleic acids that encodes the same amino acid as the wild-type sequence), or can result in a change in the encoded amino acid, or result in a stop codon, or can introduce a splicing defect or a splicing change. A nucleic acid mutation to a coding sequence can also result in conservative or non-conservative amino acid changes.

[0017] As used herein, the term "recombinant virus" refers to a virus that has been engineered in vitro, for example using recombinant nucleic acid technology, such that a change has been introduced into the viral genome and / or a change has been introduced into a viral protein. For example, in some embodiments, a recombinant virus can comprise both wild-type (endogenous) nucleic acid sequences and variant and / or exogenous nucleic acid sequences. Additionally or alternatively, in some embodiments, a recombinant virus can comprise a modified protein component, such as a variant or variety matrix, hemagglutinin, neuraminidase, nucleoprotein, non-structural and / or polymerase protein. As used herein, the term "recombinant cell" refers to a cell that has been manipulated in vitro, e.g., using recombinant nucleic acid technology, such that a nucleic acid has been introduced into the cell and / or the cellular nucleic acid has been modified. Examples of recombinant cells include prokaryotic or eukaryotic cells that harbor an exogenous plasmid, expression vector, etc., and / or cells that contain a modification (e.g., substitution, mutation, insertion, deletion, etc.) in their cellular nucleic acid (e.g., in the cell genome). Exemplary recombinant cells are cells that have been manipulated in vitro to stably express an exogenous protein (e.g., the viral M2 protein).

[0018] As used herein, the term "single replication (SR) virus" refers to a virus that has a defect in a virion protein that functions in viral entry into a host cell or release from a host cell. For example, M2SR described herein belongs to a novel class of single replication (SR) virus vaccines as opposed to classical live attenuated influenza vaccines. SR viruses have a defect in a virion protein (e.g., the flu M2 ion channel protein) that functions in viral entry or release and is essential for viral growth but does not affect viral genome replication. In contrast, traditional live attenuated virus vaccines contain multiple mutations in the viral replication machinery, resulting in a highly attenuated phenotype. The mechanism of SR vaccine viruses, therefore, does not affect the viral infection kinetics and antigen production as opposed to live attenuated vaccines.

[0019] As used herein, the terms "subject animal" and "patient" are used interchangeably and refer to an animal (e.g., a member of any vertebrate species). The methods and compositions that are the subject of the present disclosure are particularly useful for warm-blooded vertebrates (including mammals and birds). Exemplary subject animals can include mammals, e.g., humans, as well as mammals and birds that are endangered and thus important, economically important (animals raised on farms for human consumption), and / or socially important for humans (animals maintained as pets or in zoos). In some embodiments, the subject animal is a human. In some embodiments, the subject animal is not a human.

[0020] As used herein, the terms "type" and "strain" when used with a virus are used interchangeably and are generally used to refer to viruses having different characteristics. For example, influenza A virus is a different type of virus from influenza B virus. Similarly, influenza A H1N1 is a different type of virus from influenza A H2N1, H2N2, and H3N2. Additionally or alternatively, in some embodiments, different types of viruses, such as influenza A H2N1, H2N2, and H3N2, can be referred to as "subtypes". The term "vaccine" as used herein refers to a composition that is administered to a subject animal to produce or enhance immunity against a particular disease. In some embodiments, the vaccine comprises a pharmaceutically acceptable adjuvant and / or a pharmaceutically acceptable carrier.

[0021] As used herein, the term "vRNA" refers to RNA that contains a viral genome, and the viral genome includes segmented or non-segmented viral genomes, similar to plus-strand and minus-strand viral genomes. The vRNA may be completely endogenous and "wild-type" and / or may contain recombinant sequences and / or variant sequences. The term "virulence" as used herein refers to the relative ability of a pathogen to cause disease. The term "attenuated virulence", or "attenuated pathogenicity", as used herein refers to the attenuation of the relative ability of a pathogen to cause disease. For example, attenuated virulence or attenuated pathogenicity indicates a virus that, when exposed to a subject animal, elicits an immune response but is weakened such that it does not cause the disease or results in a less severe form of the disease, a shorter duration, a decrease in the incidence, or a delay in the onset of the disease.

[0022] As used herein, "M2SR" refers to a once-replicated (SR) M2 defective recombinant influenza virus. The exemplary MS2R influenza viruses described herein, depending on the context in which they are used, include SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3; viruses comprising SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3; vaccines comprising viruses comprising SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3. For example, in the description of the M2 gene mutations presented herein, "M2SR" refers to SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3. In particular, "M2SR-1" refers to SEQ ID NO: 1, "M2SR-2" refers to SEQ ID NO: 2, and "M2SR-3" refers to SEQ ID NO: 3. When describing the viral component of a vaccine, "M2SR" refers to a recombinant influenza virus that does not express a functional M2 protein. When describing a vaccine, "M2SR" refers to a vaccine comprising an M2SR recombinant virus. As used herein, "M2SR virus" encompasses a recombinant influenza virus that does not express a functional M2 protein. In some embodiments, the M2SR virus contains genes of other influenza viruses. In some embodiments, the virus contains the HA and NA genes of influenza A / Brisbane / 10 / 2007-like A / Uruguay / 716 / 2007 (H3N3). In some embodiments, the M2SR virus contains the HA and NA genes of the A / California / 07 / 2009 (CA07) (H1N1pdm) virus.

[0023] As used herein, "BM2SR" refers to a once-replicated (SR) BM2-incompletely recombinant influenza virus. Exemplary BM2SR influenza viruses described herein include, depending on the context in which they are used, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and / or SEQ ID NO:11; viruses comprising SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and / or SEQ ID NO:11; vaccines comprising viruses comprising SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and / or SEQ ID NO:11. For example, in the description of BM2 gene mutations presented herein, "BM2SR" refers to SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and / or SEQ ID NO:11. In particular, "BM2SR-1" refers to SEQ ID NO:6, "BM2SR-2" refers to SEQ ID NO:7, "BM2SR-3" refers to SEQ ID NO:8, "BM2SR-4" refers to SEQ ID NO:9, "BM2SR-5" refers to SEQ ID NO:10, and "BM2SR-0" refers to SEQ ID NO:11. When describing the viral component of a vaccine, "BM2SR" refers, by way of example and not limitation, to a recombinant influenza virus that has the internal genes of B / Lee / 40 (nucleoprotein (NP), polymerase genes (PA, PB1, PB2), non-structural (NS1 and NS2), NB, matrix (BMI)) but does not express a functional BM2 protein. When describing a vaccine, "BM2SR" refers to a vaccine comprising a BM2SR recombinant virus.

[0024] As used herein, "BM2SR virus" encompasses recombinant influenza viruses that have the internal genes of B / Lee / 40 (nucleoprotein (NP), polymerase genes (PA, PB1, PB2), non-structural (NS1 and NS2), matrix (BMI)), but do not express a functional BM2 protein, either alone or together with other viral components and / or genes encoding other viral components. In some embodiments, the BM2SR virus contains genes of other influenza viruses. In some embodiments, the virus contains the HA and NA genes of influenza B / Brisbane / 60 / 2008-like B / Brisbane / 60 / 2008 (B Victoria lineage). In some embodiments, the BM2SR virus contains the HA and NA genes of a B / Wisconsin / 1 / 2010-like (B Yamagata lineage) virus. In some embodiments, the BM2SR virus has the internal genes (NP, PA, PB1, PB2, NS1 and NS2, BM2) of recent influenza B viruses.

[0025] II. Influenza A Virus and Influenza B Virus A. General Introduction Influenza is a leading cause of death among adult Americans. The causative agents of influenza are viruses of the family Orthomyxoviridae ( Orthomyxoviridae ), which include influenza A virus, influenza B virus, and influenza C virus. Influenza A virus is a negative-strand RNA virus with an envelope. The genome of the influenza A virus is contained in eight single-stranded (non-paired) RNAs, and its complementary strand encodes 11 proteins (HA, NA, NP, M1, M2, NS1, NEP, PA, PB1, PB1-F2, PB2). The total genome size is approximately 14,000 bases. The characteristic of genome fragmentation enables the exchange of all genes between different virus strains when cells coexist. The eight RNA segments are as follows: 1) HA encodes hemagglutinin (about 500 molecules of hemagglutinin are required for the formation of one virion); 2) NA encodes neuraminidase (about 100 molecules of neuraminidase are required for the formation of one virion); 3) NP encodes nucleoprotein; M encodes two proteins (M1 and M2) by using different reading frames derived from the same RNA segment (about 3,000 molecules of M1 are required for the formation of one virion); 5) NS encodes two proteins (NS1 and NEP) by using different reading frames derived from the same RNA segment; 6) PA encodes RNA polymerase; 7) PB1 encodes RNA polymerase and PB1-F2 protein (inducing apoptosis) by using different reading frames derived from the same RNA segment; 8) PB2 encodes RNA polymerase.

[0026] Influenza B virus is also a negative-strand RNA virus with an envelope. The genome of the influenza B virus is contained in eight single-stranded (non-paired) RNAs, and its complementary strand encodes 11 proteins. Of these proteins, the following nine are also found in influenza A virus: three RNA-dependent RNA polymerase subunits (PB1, PB2, and PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix protein (M1 or BM1), and two non-structural proteins (NS1 and NS2 (also known as NEP)). Two proteins (NB and BM2) are specific to influenza B virus. The total genome size is approximately 14,500 bases. The property of genome segmentation allows for the exchange of all genes (a process known as reassortment) between different virus strains when cells coexist. The eight RNA segments (numbered in descending order of length) are as follows: Segments 1, 2, and 3 encode PB1, PB2, and PA, respectively, which are RNA polymerase subunits. Segment 4 encodes HA (hemagglutinin). Segment 5 encodes NP (nucleoprotein). Segment 6 encodes both NB (NB protein, whose function is unknown) and NA. Segment 7 encodes both BM1 (matrix protein) and BM2 (ion exchange channel) by a bicistronic mRNA, and its translation method is unique. The BM2 start codon overlaps with the BM1 stop codon (TAATG, a termination-initiation 5-nucleotide motif). The BM2 protein is translated by this termination-initiation translation mechanism, unlike the M2 protein of influenza A virus (translated from a spliced mRNA). Segment 8 encodes both NS1 and NEP by using different reading frames derived from the same RNA segment.

[0027] Both influenza A and B antigenically evolve over time by antigenic drift, in which mutations in the hemagglutinin (HA) protein enable the virus to escape existing human immunity and persist in the human population. There are several subtypes of influenza A, named according to the number of H (type of hemagglutinin) and the number of N (type of neuraminidase). Currently, there are 16 different known H antigens (H1 to H16) and 9 different known N antigens (N1 to N9). Each virus subtype has mutated into diverse strains with various pathogenicity profiles. Some are pathogenic to one species but not to others, and some are pathogenic to multiple species. Exemplary influenza A virus subtypes identified in humans include, but are not limited to: H1N1 (which caused the "Spanish flu" and the 2009 swine flu pandemic); H2N2 (which caused the "Asian flu" in the late 1950s); H3N2 (which caused the Hong Kong flu in the late 1960s); H5N1 (which is considered a threat of a global influenza pandemic due to its spread in the mid-2000s); H7N7; H1N2 (which has been epidemic in humans and swine so far); and H9N2, H7N2, H7N3, H5N2, H10N7.

[0028] Since at least 1988, two antigenically and genetically distinct lineages of influenza B viruses have been co-circulating and causing disease in humans. The Victoria lineage of influenza viruses was the dominant B strain circulating globally in the 1980s, and the Yamagata lineage became dominant in the early 1990s. Since 1991, the isolation frequency of Victoria lineage viruses has declined and has been almost completely limited to East Asia. The Victoria virus reappeared in 2002, and since then both the Yamagata and Victoria lineages have coexisted. The evolutionary relationships of influenza B viruses isolated from 1940 to 2016 indicate that the BM1 and BM2 proteins of modern isolates are more closely related to each other than those of B / Lee / 40.

[0029] Influenza viruses have a standard nomenclature, which includes the virus type, the species from which the virus was isolated (if non-human), the location where it was isolated, the isolate number, the year of isolation, and, for influenza A viruses only, the HA and NA subtypes. Thus, B / Yamagata / 16 / 88 was the isolate number 16 of a human influenza B virus isolated in Yamagata (Japan) in 1988. Several influenza A variants have been identified and named as follows: based on the known isolate to which they are most similar (and thus presumed to share a lineage, e.g., Fujian-like influenza virus), based on their typical host (e.g., human influenza virus), based on their subtype (e.g., H3N2), based on their pathogenicity (e.g., LP (low pathogenicity)). Thus, an influenza caused by a virus similar to isolate A / Fujian / 411 / 2002 (H3N2) can be called Fujian influenza, human influenza, and H3N2 influenza. In addition, influenza variants are sometimes named based on the species (host) in which the strain has become endemic or adapted. The major variants named using this convention are avian influenza, human influenza, swine influenza, equine influenza, and canine influenza. Variants are also named based on their pathogenicity in poultry, especially chickens, e.g., low pathogenic avian influenza (LPAI) and highly pathogenic avian influenza (HPAI).

[0030] B. Life Cycle and Structure The life cycle of the influenza virus generally includes attachment to cell surface receptors, entry into the cell and uncoating of the viral nucleic acid, followed by replication of the viral genes within the cell. After synthesizing new copies of the viral proteins and genes, these components are assembled into progeny virus particles, which then exit the cell. Various viral proteins play roles in each of these steps. Influenza A particles are composed of a lipid envelope that encapsulates the viral core. The inside of the envelope is lined with matrix protein (M1), while the outer surface is characterized by two types of glycoprotein spikes (hemagglutinin (HA) and neuraminidase (NA)). M2 (transmembrane ion channel protein) is also part of the lipid envelope (see, for example, Figure 1). Influenza B particles also contain a similar structure.

[0031] The HA protein (trimeric type I membrane protein) is required for binding to sialyl oligosaccharides (oligosaccharides containing terminal sialic acid linked to galactose) on glycoproteins or glycolipids on the host cell surface. This protein is also required for fusion between the viral and host cell membranes and subsequent internalization of the virion by endocytosis. Neuraminidase (NA) (tetrameric type II membrane protein) is a sialidase, which cleaves terminal sialic acid residues from host cell glycoconjugates as well as HA and NA, and is thus recognized as a receptor-destroying enzyme. This sialidase activity is required for efficient release of progeny virions from the host cell surface and for prevention of aggregation of progeny by the binding activity of the viral HA to other glycoproteins. Thus, the receptor-binding activity of HA and the receptor-destroying activity of NA probably act antagonistically to allow efficient replication of influenza.

[0032] Genome segments are packaged in the core of the viral particle. RNPs (RNA + nucleoprotein (NP)) exist in a helical shape together with three viral polymerase polypeptides bound to each segment. The life cycle of influenza virus begins with the binding of HA to sialic acid-containing receptors on the surface of host cells, followed by receptor-mediated endocytosis (Figure 1). The low pH of the late endosome initiates a conformational shift in HA, thereby exposing the N-terminus of the HA2 subunit (the so-called fusion peptide). The fusion peptide initiates the fusion of the viral membrane and the endosomal membrane, and the matrix protein (M1) and the RNP complex are released into the cytoplasm. The RNP consists of the nucleoprotein (NP) (which capsid-envelops the vRNA) and the viral polymerase complex (formed by the PA, PB1, and PB2 proteins). The RNP is transported to the nucleus, where transcription and replication occur. The RNA polymerase complex catalyzes three different reactions: (1) the synthesis of mRNA with a 5’ cap and a 3’ polyA structure, (2) the synthesis of full-length complementary RNA (cRNA), and (3) the synthesis of genomic vRNA using cDNA as a template. The newly synthesized vRNA, NP, and polymerase proteins are subsequently assembled into RNP, exported from the nucleus, transported to the plasma membrane, and budding of progeny virus particles occurs at the plasma membrane. The neuraminidase (NA) protein removes sialic acid from sialyl oligosaccharides, thus releasing newly assembled virions from the cell surface and further preventing self-aggregation of virus particles, thereby playing a role in the late stage of infection. The assembly of the virus requires protein-protein interactions and protein-vRNA interactions, but the nature of these interactions remains mostly unknown.

[0033] C. Roles of M2 and BM2 Proteins As described above, three proteins (hemagglutinin (HA), neuraminidase (NA) and M2) span the influenza A virus membrane. The extracellular domains (ectodomains) of HA and NA have significant variability, while the ectodomain of M2 is essentially unchanged among influenza A viruses. The M2 ion channel protein does not affect viral genome replication but is essential for virus growth. Single replication (SR) viruses are defective in virion proteins (e.g., influenza A M2 or influenza B M2 ion channel proteins) that function in virus entry or release. In contrast, traditional live attenuated virus vaccines contain multiple mutations in the viral replication machinery, resulting in a highly attenuated phenotype. Without being bound by theory, in influenza A virus, the M2 protein (which has ion channel activity) is thought to function early in the viral life cycle between host cell penetration and viral RNA uncoating. Once the virion undergoes endocytosis, the virion-bound M2 ion channel (homotetrameric helix bundle) allows protons to flow from the endosome into the virion interior, disrupting the acid-labile M1 protein-ribonucleoprotein complex (RNP) interaction and thereby facilitating RNP release into the cytoplasm. In addition, in some influenza strains in which HA is cleaved intracellularly (e.g., A / fowl plagues / Rostock / 34), the M2 ion channel is thought to raise the pH of the trans-Golgi network and prevent the conformational change of HA caused by low pH conditions within this compartment. It has also been shown that the M2 transmembrane domain itself can function as an ion channel. The M2 protein ion channel activity is thought to be essential in the influenza virus life cycle because amantadine hydrochloride (which blocks M2 ion channel activity) has been shown to inhibit virus replication. However, the requirement for this activity in influenza A virus replication has not been directly demonstrated. The functional counterpart of the influenza A virus M2 protein in influenza B virus is a type III transmembrane protein known as BM2.

[0034] D. M2 and BM2 Virus Variants as Vaccines M2SR belongs to a novel class of single replication (SR) viral vaccines. SR viruses are defective in virion proteins that function in viral entry or release (e.g., the flu M2 ion channel protein). Said proteins do not affect viral genome replication but are essential for viral growth. In contrast, traditional live attenuated vaccines contain multiple mutations in the viral replication machinery, resulting in a highly attenuated phenotype. Thus, two different vaccine virus mechanisms affect viral infection kinetics and antigen production, which in turn affects the induction of defense and immune responses. Replication-deficient viruses provide a unique viral vaccine form. Said form combines the safety of attenuated viral vaccines and the immunogenicity of live viral vaccines by enabling the intracellular expression of viral gene products to efficiently present antigens via the MHC class I and class II pathways. Single replication viruses can also activate Toll-like receptors and other innate immune response pathways, thereby functioning as their own adjuvants. In addition, these viruses can be used as tools to probe the function of the immune system. These mutant viruses are defective in virion proteins that function after viral assembly. The virus grows in complementing cells that express the lost gene product. In normal cells, the replication cycle occurs normally and progeny virions are produced. However, these virions are non-infectious and thus infection does not spread to secondary cells.

[0035] III. M2 and BM2 Virus Variants In one aspect, influenza A viruses that retain a mutant M2 vRNA sequence are disclosed. Typically, such mutants lack M2 ion channel activity, exhibit attenuated growth characteristics in vivo, are unable to produce infectious progeny, and are non-pathogenic or exhibit attenuated pathogenicity in infected target animals. In another aspect, influenza B viruses that retain a mutant BM2 vRNA sequence are disclosed. Typically, such mutants lack BM2 ion channel activity, exhibit attenuated growth characteristics in vivo, are unable to produce infectious progeny, and are non-pathogenic or exhibit attenuated pathogenicity in infected target animals. The mutant viruses are immunogenic and, when used as vaccines, provide protection against infection by the corresponding wild-type and / or other pathogenic viruses. In addition, the M2 and BM2 mutants disclosed herein are stable and do not mutate to express functional M2 or BM2 polypeptides regardless of the host cell used. Additionally or alternatively, in some embodiments, the M1 protein of these mutants is produced without a detectable change in its function. In some embodiments, viruses that retain a mutant M2 or BM2 nucleic acid sequence are unable to replicate in host cells (where wild-type viruses would be able to grow). By way of illustration and not limitation, in some embodiments, wild-type viruses are able to grow, propagate, and replicate in cultured MDCK cells, CHO cells, and / or Vero cells, while the corresponding viruses that retain a mutant M2 or BM2 sequence are unable to grow, replicate, or propagate in the same type of cells.

[0036] As noted above, in some embodiments, the M2 or BM2 mutant viruses are stable and do not mutate or revert to non-wild-type sequences encoding wild-type or functional M2 or BM2 proteins. For example, in some embodiments, the M2 or BM2 mutant viruses are stable in host cells for 2 passages, 3 passages, 5 passages, 10 passages, 12 passages, 15 passages, 20 passages, 25 passages, or more than 25 passages. In some embodiments, the host cells are non-modified host cells. In some embodiments, the host cells transAny mammalian cell that stably provides in a manner. In other embodiments, the host cell is a modified host cell (e.g., MOCK or Vero cell) that expresses the M2 or BM2 protein.

[0037] In some embodiments, the M2 or BM2 variant contains one or more nucleic acid substitutions and / or deletions. In some embodiments, the mutation is localized to the nucleic acid encoding one or more of the extracellular domain of the M2 or BM2 protein, the transmembrane domain of the M2 or BM2 protein, and / or the cytoplasmic tail of the M2 or BM2 protein. Additionally or alternatively, in some embodiments, one or more nucleic acid mutations result in a splice variant of M2 or BM2, one or more stop codons and / or one or more amino acid deletions. In some embodiments, a virus having a mutant M2 or BM2 nucleic acid produces a non-functional M2 or BM2 polypeptide. In some embodiments, a virus having a mutant M2 or BM2 nucleic acid does not produce an M2 or BM2 polypeptide. In some embodiments, a virus having a mutant M2 or BM2 nucleic acid produces a truncated M2 or BM2 polypeptide.

[0038] Three exemplary and non-limiting M2 virus variants (M2SR-1, M2SR-2, and M2SR-3) are provided in Tables 1-3 below. In these tables, lowercase letters correspond to the M2 sequence, uppercase letters correspond to the M1 sequence and non-coding regions, and mutant sequences (e.g., stop codons, splice defects) are bolded and underlined. The underlined (lowercase) bases of the M2SR-2 variant indicate the region deleted in the M2SR-1 and M2SR-3 variants. Italicized lowercase bases include the M and M2 overlap regions.

[0039] [Table 1]

[0040] The M2 polypeptide sequence generated from this variant is as follows: MSLLTEVETPIRNEWGCRCNGSSD (SEQ ID NO: 4)

[0041]

Table 2

[0042] The M2 polypeptide sequence is not generated from this variant.

[0043]

Table 3

[0044] The M2 polypeptide sequence is not generated from this variant. The wild-type M1 and M2 coding sequences are provided in Table 4 below.

[0045]

Table 4

[0046] Exemplary non-limiting BM2SR virus variants (BM2SR-1, BM2SR-2, BM2SR-3, BM2SR-4, BM2SR-5 and BM2SR-0) are provided in Table 5 below.

[0047]

Table 5-1

[0048]

Table 5-2

[0049]

Table 5-3

[0050]

Table 5-4

[0051]

Table 5-5

[0052] Influenza B genomic segment 7 expresses two major polypeptides (BM1 matrix protein and BM2 proton channel) required by the virus for replication. Expression of the BM1 and BM2 polypeptides is partially regulated by a pentanucleotide motif translational slippage site that is present at the junction between the BM1 and BM2 ORFs. The pentanucleotide motif (TAATG) contains both the TAA stop codon for the end of M1 translation and the ATG start codon for M2 initiation in the alternate -1 reading frame. This pentanucleotide motif and flanking sequences have been shown to be important for the regulation of M1 protein expression. The wild - type influenza B segment 7 showing the BM1 and BM2 coding sequences and the pentanucleotide motif underlined in bold is provided in Table 6 below.

[0053]

Table 6-1

[0054]

Table 6-2

[0055] IV. Cell-Based Virus Production System A. Production of "First Generation" Mutant Viruses Mutant viruses, such as those having a mutant M2 nucleic acid, can be produced by the plasmid-based reverse genetics described by Neumann et al. (Neumann et al., Generation of influenza A viruses entirely from clone cDNAs, Proc. Natl. Acad. Sci. USA 96:9345-9350, 1999 (the entire disclosure of which is incorporated herein by reference)). Mutant viruses, such as those having a mutant BM2 nucleic acid, can be produced by similar means. Briefly, eukaryotic host cells are transfected with one or more plasmids encoding the eight viral RNAs. Each viral RNA sequence is flanked by an RNA polymerase I promoter and an RNA polymerase I terminator. In particular, the viral RNA encoding the M2 protein contains the mutant M2 nucleic acid sequence. In addition, the host cells are transfected with one or more expression plasmids encoding viral proteins (e.g., polymerase, nucleoprotein, and structural proteins) (including the wild-type M2 protein). Transfection of the host cells with the viral RNA plasmids results in the synthesis of all eight influenza virus RNAs, one of which has the mutant M2 sequence. The co-transfected viral polymerase and nucleoprotein assemble the viral RNA into functional vRNA, which is replicated and transcribed, and ultimately forms an infectious influenza virus having a mutant M2 nucleic acid sequence but still having a functional M2 polypeptide incorporated into the viral lipid envelope.

[0056] Another method for producing the "first generation" of mutant viruses involves a ribonucleoprotein (RNP) transfection system that allows for the exchange of influenza virus genes by in vitro-generated recombinant RNA molecules. The system was described by Enami and Palese (Enami and Palese, High-efficiency formation of influenza virus transfectants, J. Virol. 65(5):2711-2713, which is hereby incorporated by reference in its entirety). As shown by Luytjes et al., viral RNA is synthesized in vitro, and the RNA transcripts are coated with viral nucleoprotein (NP) and polymerase proteins that function as biologically active RNPs in transfected cells (Luytjes et al., Amplification, expression, and packaging of a foreign gene by influenza virus, Cell 59:1107-1113, which is hereby incorporated by reference in its entirety).

[0057] The RNP transfection method can be divided into four steps: 1) RNA preparation: Plasmid DNA encoding influenza virus segments is transcribed into minus-sense RNA in an in vitro transcription reaction; 2) RNA encapsidation: The transcribed RNA is subsequently mixed with gradient-purified NP and polymerase proteins (isolated from disrupted influenza virus) to form biologically active RNP complexes; 3) Transfection and rescue of encapsidated RNA: The artificial ribonucleocapsids are transfected into cells. The cells are pre-infected with a helper influenza virus containing various genes derived from the virus to be rescued, and the helper virus will amplify the transfected RNA; 4) Selection of transfected genes: Since both the helper virus and the transfectants containing the rescued genes are present in the culture supernatant, an appropriate selection system using antibodies is required for the isolation of the virus carrying the transfected gene. This selection system enables the generation of novel transfectant influenza viruses with specific biological and molecular characteristics. Subsequently, antibody selection against the target surface protein can be used for either positive or negative selection.

[0058] For example, a transfectant or mutant virus containing an M2 gene that does not express the M2 protein can be propagated in a suitable mammalian cell line that has been modified to stably express wild-type functional M2 protein. To prevent or inhibit the replication of helper virus that expresses wild-type M2 gene (and thus expresses M2e protein on the membrane surface), antibodies against M2e can be used. Such antibodies are commercially available and the antibodies will inhibit the replication of helper virus and allow the growth and concentration in the supernatant of transfectants / mutant viruses containing mutant M2. Inhibition of influenza virus growth by M2e antibodies has been previously described in: Influenza A virus M2 protein: monoclonal antibody restriction of virus growth and detection of M2 in virions, J Virol 62:2762-2772, 1988; and Treanor et al, Passively transferred monoclonal antibody to the M2 protein inhibits influenza A virus replication in mice, J. Virol. 64:1375-1377, 1990.

[0059] In addition or alternatively, the same antibodies can be used to 'capture' helper virus and concentrate transfectants. For example, the antibodies can be used to coat the bottom of tissue culture dishes or to concentrate transfectants in the supernatant or eluate using a column matrix. The transfectant virus can be propagated in M2-expressing cells of a multiwell plate by limiting dilution and subsequently identified and cloned by creating replica plates. For example, one-half aliquots of wells of a multiwell plate containing the propagated virus can be used to infect MDCK cells, and the other half can be used to infect MDCK cells expressing the M2 protein. Both the transfectant virus and the helper virus will grow in M2 protein-expressing MDCK cells. However, only the helper virus will grow in standard MDCK cells, allowing identification of the wells of the multiwell plate containing the transfectant. The transfectant virus can further be plaque purified in cells expressing the M2 protein.

[0060] B. Propagating Virus Variants In some embodiments, the virus variants described herein are maintained and passaged in host cells. By way of illustration and not limitation, exemplary host cells suitable for the propagation of influenza virus variants (e.g., influenza A virus variants) include, but are not limited to, a number of eukaryotic cells such as the following: Madin-Darby canine kidney cells (MDCK), monkey cells (e.g., African green monkey cells (e.g., Vero cells), CV-1 cells, and rhesus monkey kidney cells (e.g., LLC-MK2 cells)), bovine cells (e.g., MDBK cells), porcine cells, ferret cells (e.g., mink lung cells), BK-1 cells, rodent cells (e.g., Chinese hamster ovary cells), human cells, such as fetal human kidney cells (e.g., PER-C6™), 293T human fetal kidney cells, and avian cells (including fetal fibroblasts). Additionally or alternatively, in some embodiments, eukaryotic host cells are modified to enhance viral production, for example, by enhancing viral infection of the host cells and / or enhancing the viral growth rate. For example, in some embodiments, host cells are modified to express or have enhanced expression of 2,6-linked sialic acid, enabling more efficient and effective infection of these cells by mutant or wild-type influenza A virus. See, e.g., U.S. Patent Publication No. 2010-0021499 and U.S. Patent No. 7,176,021 (which are hereby incorporated by reference in their entirety). Thus, in some exemplary embodiments, Chinese hamster ovary cells (CHO cells) and / or Vero cells are used, which are modified to express at least one copy of the 2,6-sialyltransferase gene (ST6GAL1). By way of illustration and not limitation, Homo sapiens ( Homo sapiens)The ST6 beta-galatosamide alpha-2,6-sialyltransferase gene sequence (represented by accession number BC040009.1) is an example of the ST6Gal gene that can be integrated and expressed in CHO cells. One or more polynucleotides encoding a functional ST6GalI gene product can be engineered into cells. That is, cells stably transformed to express 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 copies of the ST6GalI gene can be used. A single expression cassette can contain one or more copies of the ST6GalI gene that can be expressed. The gene is operably linked to regulatory elements (such as promoters, enhancers and terminators) and polyadenylation signals and can promote the expression of the ST6GalI gene or its copies. Alternatively, a single expression cassette can be engineered to express one copy of the ST6GalI gene and multiple expression cassettes can be incorporated into the host cell genome. Thus, in some embodiments, at least one ST6GalI gene is incorporated into the genome of the host cell and the cell can express the ST6GalI gene and its enzyme protein product. Depending on the copy number, a single host cell can express many functional ST6GalI gene proteins.

[0061] Suitable vectors for cloning, transfection and generation of stable modified cell lines are well known in the art. A non-limiting example includes the pcDNA3.1 vector (Invitrogen). Additionally or alternatively, in some embodiments, eukaryotic host cells are modified to produce the wild-type version of the mutant viral gene, whereby the gene is provided to the virus in a trans configuration. For example, a viral strain having a mutant M2 protein can exhibit an enhanced growth rate (e.g., greater virus production) when passaged in host cells that produce the wild-type M2 protein. In some embodiments, a viral strain having a mutant M2 protein is unable to grow or replicate in cells that do not express the wild-type M2 gene. Additionally, such host cells can slow or prevent viral reversion to a functional M2 sequence, because, for example, there is no selective pressure for reversion in such hosts. Methods for making both expression vectors and modified host cells are well known in the art. For example, an M2 expression vector can be made by placing the following M2 nucleic acid sequence (M2 ORF sequence; this is from the start codon to the stop codon of “wild-type” M2 (Table 7)) into a eukaryotic cell expression vector. A similar method can be utilized for BM2 (the sequence of BM2 is provided in Table 7 below).

[0062]

Table 7

[0063]

Table 8

[0064] Subsequently, host cells (e.g., MDCK cells) can be transfected by methods known in the art, for example, using commercially available reagents and kits (e.g., TransIT™ LT1 (Mirus Bio, Madison, WI)). By way of non-limiting example, cells can be selected and tested for M2 expression by co-transfection with a detectable or selectable marker (e.g., hygromycin resistance) and / or by screening, for example, by indirect immunostaining using an M2 antibody. M2 expression can be determined by indirect immunostaining, flow cytometry, or ELISA.

[0065] By way of non-limiting example, 293 human fetal kidney cells and Madin-Darby canine kidney (MDCK) cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and minimum essential medium (MEM) containing 5% newborn bovine serum, respectively. All cells were maintained in 5% CO 2It was maintained at 37°C below. Hygromycin-resistant MDCK cells that stably express the M2 protein derived from A / Puerto Rico / 8 / 34 (H1N1) were established by co-transfecting plasmid pRHyg (containing the hygromycin resistance gene) and plasmid pCAGGS / M2 (expressing the full-length M2 gene) at a ratio of 1:1. Stable MDCK cell clones expressing M2 (M2CK) were selected by screening in a medium containing 0.15 mg / mL hygromycin (Roche, Mannheim, Germany) using indirect immunostaining with anti-M2 (14C2) monoclonal antibody (Iwatsuki et al., JVI, 2006, vol.80, No.1, p.5233-5240). M2CK cells were cultured in MEM supplemented with 10% fetal bovine serum and 0.15 mg / mL hygromycin. In M2CK cells, the expression level and distribution of M2 were similar to those in virus-infected cells (data not shown). BM2 expression BM2CK cells can be prepared in a similar manner, and furthermore, Vero cells expressing M2 or BM2 can also be prepared in a similar manner.

[0066] In some embodiments, cells and virus variants are cultured and grown by methods well known in the art. By way of illustration and not limitation, in some embodiments, host cells are grown in the presence of MEM supplemented with 10% fetal bovine serum. Cells expressing M2 or BM2 are infected at an MOI of 0.01 by adsorbing the virus at 37°C after washing with PBS. In some embodiments, a virus growth medium containing trypsin / TPCK is added, and the cells are incubated for 2 - 3 days until a cytopathic effect is observed.

[0067] Along these lines, disposable bioreactors have been developed for mammalian cells (with or without inclusion of viruses). The advantages described above include more rapid setup of equipment and reduced risk of cross-contamination. The cells described herein can be cultured, for example, in disposable bags (e.g., bags from Stedim, Bioeaze bags (SAFC Biosciences), HybridBag TM (Cellexus Biosystems), or single-use bioreactors (HyClone), or Celltainer (Lonza)). The bioreactors can be of 1L, 10L, 50L, 250L, 1000L size formats. In some embodiments, the cells are maintained as a suspension in an optimized serum-free medium and free of animal products. The system can be a fed-batch system that can expand the culture, for example, in a single bag from 1L to 10L, or a perfusion system that allows for a steady supply of nutrients and simultaneously avoids the accumulation of potentially harmful by-products in the culture medium. For long-term storage, the mutant virus can be stored as a frozen stock.

[0068] V. Vaccines and Administration Methods A. Immunogenic Compositions and Vaccines There are various different types of vaccines, and they can be made from the cell-dependent virus production systems disclosed herein. The present disclosure includes (but is not limited to) the following: the manufacture and production of live attenuated virus vaccines, single replication vaccines, replication-defective vaccines, viral vector vaccines, inactivated virus vaccines, whole virus vaccines, split virus vaccines, virosome virus vaccines, virus surface antigen vaccines, and combinations of the foregoing. Thus, there are numerous vaccines that can elicit a protective immune response specific to various influenza viruses, and appropriate formulations of any of these vaccine types can elicit an immune response, such as a systemic immune response. Live attenuated virus vaccines also have the advantage of being able to stimulate local mucosal immunity in the airway. In some embodiments, the vaccine antigens used in the compositions described herein are "direct" antigens. That is, they are not administered as DNA, but are the antigens themselves. Such vaccines can include: whole viruses or only parts of viruses, such as, but not limited to, viral polysaccharides (either alone or as a complex with a carrier component, e.g., a carrier protein), live attenuated whole microorganisms, inactivated microorganisms, recombinant peptides and proteins, glycoproteins, glycolipids, lipopeptides, synthetic peptides, or disrupted microorganisms in the case of vaccines referred to as "split" vaccines.

[0069] In some embodiments, whole virion vaccines are provided. Whole virion vaccines are concentrated by ultrafiltration and subsequently purified by zonal centrifugation or by chromatography. Typically, the virions are inactivated, for example, using formalin or beta-propiolactone, either before or after purification. In some embodiments, subunit vaccines are provided, which contain purified glycoproteins. Such vaccines can be prepared as follows: using a virus suspension fragmented by treatment with a detergent, the surface antigens are purified, for example, by ultracentrifugation. Thus, subunit vaccines mainly also contain the HA protein and NA. The detergents used can be cationic detergents (e.g., cetyltrimethylammonium bromide), anionic detergents (e.g., ammonium deoxycholate), or non-ionic detergents (e.g., those commercialized under the name TRITON X100). Hemagglutinin can also be isolated after treatment of the virions with a protease and subsequently purified by standard methods.

[0070] In some embodiments, a split vaccine is provided, which comprises virions that have been subjected to treatment with an agent that lyses lipids. The split vaccine can be prepared as follows: An aqueous suspension of the purified virus obtained as described above is treated (whether inactivated or not) while stirring with a lipid solvent (such as ethyl ether or chloroform) together with a detergent. Lysis of the viral envelope lipids results in fragmentation of the virus particles. The aqueous phase containing the split vaccine is recovered. The latter consists mainly of hemagglutinin and neuraminidase (together with their native lipid environment removed), and the core or its degradation products. Subsequently, if inactivation has not yet been carried out, the remaining infectious particles are inactivated.

[0071] In some embodiments, an inactivated influenza virus vaccine is provided. In some embodiments, the inactivated vaccine is produced by inactivating the virus using known methods, such as, but not limited to, formalin or β-propiolactone treatment. Inactivated vaccine types that can be used in the present invention can include whole virus (WV) vaccines or subvirion (SV) (split) vaccines. WV vaccines contain intact inactivated virus, while SV vaccines contain purified virus that has been disrupted with a detergent that lyses the lipid-containing viral envelope and subsequently the remaining virus is chemically inactivated. In addition or alternatively, in some embodiments, a live attenuated influenza virus vaccine is provided. Such a vaccine can be used for the prevention or treatment of influenza virus infection according to known method steps.

[0072] In some embodiments, attenuation is achieved in a single step by transferring an attenuating gene from an attenuated donor virus to an isolate or a recombinant virus according to known methods (see, for example, Murphy, Infect. Dis. Clin. Pract. 2, 174, 1993). In some embodiments, the virus is attenuated by mutations in one or more viral nucleic acid sequences (resulting in a mutant virus). For example, in some embodiments, the mutant viral nucleic acid sequence encodes a defective protein product. In some embodiments, the protein product has a reduced function or no function. In other embodiments, no protein product is produced from the mutant viral nucleic acid.

[0073] The single replication viruses described herein can be formulated and administered according to known methods as immunogenic compositions (e.g., as vaccines) to induce an immune response in animals (e.g., birds and / or mammals). Methods for determining whether such attenuated or inactivated vaccines maintain an antigenicity similar to that of clinical isolates or high-growth strains derived therefrom are well known in the art. Such known methods include: using antiserum or antibodies to remove viruses expressing antigenic determinants of the donor virus; chemical selection (e.g., amantadine or rimantadine); HA and NA activity and inhibition; and screening DNA (e.g., probe hybridization or PCR) to confirm that the donor gene encoding the antigenic determinant (e.g., HA or NA gene) or other variant sequences (e.g., M2) is not present in the attenuated virus. See, for example, Robertson et al., Giornale di Igiene e Medicina Preventiva, 29, 4, 1988; Kilbourne, Bull. M2 World Health Org., 41, 643 (1969); and Robertson et al., Biologicals, 20, 213, 1992. In some embodiments, the vaccine comprises a single replication influenza virus lacking the expression of functional M2 protein. In some embodiments, the mutant virus replicates well in cells expressing the M2 protein but does not replicate in the corresponding wild-type cells and expresses viral proteins without producing infectious progeny virions.

[0074] Pharmaceutical compositions of the present technology suitable for intranasal, intradermal, inoculation, or parenteral or oral administration include attenuated or inactivated influenza virus and may optionally further include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. The compositions may further include adjuvants or excipients known in the art. See, for example: Berkow et al., The Merck Manual, 15th edition, Merck and Co., Rahway, N.J., 1987; Goodman et al., eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, Eighth Edition, Pergamon Press, Inc., Elmsford, N.Y., 1990; Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, Third Edition, ADIS Press, LTD., Williams and Wilkins, Baltimore, Md., 1987; and Katzung, ed., Basic and Clinical Pharmacology, Fifth Edition, Appleton and Lange, Norwalk, Conn., 1992.

[0075] In some embodiments, the liquid preparation for nasal delivery can take the form of a solution or a suspension and can include conventional excipients such as tonicity adjusters (e.g., sodium chloride, dextrose, or mannitol); preservatives (e.g., benzalkonium chloride, thimerosal, or phenylethyl alcohol); and other formulation agents (e.g., suspending agents, buffers, stabilizers, and / or dispersants).

[0076] In some embodiments, the preparation for parenteral administration can include sterile aqueous or non-aqueous solutions, suspensions, and / or emulsions. The foregoing can include adjuvants or excipients known in the art. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). A carrier or occlusive dressing can be used to enhance skin permeability and enhance antigen absorption. Generally, the liquid dosage form for oral administration can include a liposome solution containing the liquid dosage form. Forms suitable for suspension liposomes include emulsions, suspensions, solutions, syrups, and elixirs, which include inert diluents commonly used in the art. In addition to the inert diluent, compositions such as those described above can also include adjuvants, wetting agents, emulsifying and suspending agents, or sweetening, flavoring, or aromatic agents.

[0077] When the composition of the present invention is used for administration to an individual, the composition can further include salts, buffers, adjuvants, or other substances desired to improve the effectiveness of the composition. For vaccines, adjuvants (substances that enhance specific immunity) can be used. Typically, the adjuvant and the composition are mixed before presentation to the immune system or are presented separately but at the same site of the organism to be immunized. In some embodiments, the present disclosure provides a multivalent immunogenic composition comprising a virus derived from at least two influenza viruses. In some embodiments, the multivalent immunogenic composition comprises: (a) at least one engineered attenuated influenza A M2 defective recombinant virus (the engineered influenza A virus comprises a mutated M2 gene comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3); and (b) at least one engineered attenuated influenza B M2 defective recombinant virus (the engineered influenza B virus comprises a mutated BM2 gene comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11). In some embodiments, the influenza A virus is selected from the group consisting of H1N1 and H3N2 subtypes, and the influenza B virus is selected from the group consisting of B / Yamagata and B / Victoria lineages.

[0078] In some embodiments, the present disclosure provides a quadrivalent immunogenic composition comprising: the following two M2-defective influenza A M2SR viruses: A / California / 07 / 2009 (H1N1) and A / Brisbane / 10 / 2007 (H3N2) (both of which comprise the M2SR-1 variant comprising SEQ ID NO: 1); and the following two BM2-defective influenza B BM2SR viruses: B / Brisbane / 60 / 2008 (Victoria) and B / Wisconsin / 01 / 2010 (Yamagata) (both of which comprise the BM2SR-0 variant comprising SEQ ID NO: 11).

[0079] In some embodiments, the present disclosure provides a method of stimulating an immune response against influenza A and influenza B. The method includes administering to a subject animal in need thereof a multivalent immunogenic composition comprising at least one influenza A strain and at least one influenza B strain. In some embodiments, the multivalent immunogenic composition comprises: a) at least one engineered attenuated influenza A M2-incomplete recombinant virus (the engineered influenza A virus comprises a mutated M2 gene comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3); and b) at least one engineered attenuated BM2-incomplete recombinant virus (the engineered influenza B virus comprises a mutated BM2 gene comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11). In some embodiments, the influenza A virus is selected from the group consisting of H1N1 and H3N2 subtypes, and the influenza B virus is selected from the group consisting of B / Yamagata and B / Victoria lineages.

[0080] In some embodiments, the present disclosure provides a method of stimulating an immune response against influenza A and influenza B. The method includes administering to a subject animal in need thereof an immunogenic composition comprising: the following two M2-incomplete influenza A M2SR viruses: A / California / 07 / 2009 (H1N1) comprising the M2SR-1 variant comprising SEQ ID NO: 1 and A / Brisbane / 10 / 2007 (H3N2) comprising the M2SR-1 variant comprising SEQ ID NO: 1; and the following two BM2-incomplete influenza B BM2SR viruses: B / Brisbane / 60 / 2008 (Victoria) comprising the BM2SR-0 variant comprising SEQ ID NO: 11 and B / Wisconsin / 01 / 2010 (Yamagata) comprising the BM2SR-0 variant comprising SEQ ID NO: 11. In some embodiments, this immunogenic composition is formulated as a quadrivalent influenza vaccine.

[0081] In some embodiments, the immunogenic composition formulated as a tetravalent influenza vaccine described herein exhibits attenuated virulence. For example, in some embodiments, mice infected with the tetravalent vaccine have an extended post-infection mean lifespan after influenza A challenge compared to mice infected with only a monovalent influenza B vaccine. In some embodiments, mice infected with the tetravalent vaccine have an extended post-infection mean lifespan after influenza B challenge compared to mice infected with only a monovalent influenza A vaccine.

[0082] The pharmaceutical composition of the present invention may further or additionally contain at least one chemotherapeutic compound, for example, for gene therapy. The foregoing includes, but is not limited to, immunosuppressive agents, anti-inflammatory agents or immunostimulants, or antiviral agents, gamma globulin, amantadine, guanidine, hydroxybenzimidazole, interferon-α, interferon-β, interferon-γ, tumor necrosis factor-α, thiosemicarbazone, methylthiazone, rifampin, ribavirin, pyrimidine analogs, purine analogs, foscarnet, phosphonoacetic acid, acyclovir, dideoxynucleosides, protease inhibitors, or ganciclovir. The composition can also contain variable but small amounts of endotoxin-free formaldehyde and preservatives, and these substances have been found to be safe and not contribute to undesirable effects in the organisms to which the composition of the present invention is administered.

[0083] B. Administration The immunogenic compositions (e.g., vaccines) disclosed herein can be administered via either the routes customarily used or recommended for vaccines (parenteral routes, mucosal routes), and can further be in a variety of forms (such as injectable or sprayable liquids, or formulations that are lyophilized or dried by atomization or air-drying, etc.). The vaccine can be administered using a syringe or needle-free injector for intramuscular, subcutaneous, or intradermal injection. The vaccine can also be administered by using a nebulizer capable of delivering a dry powder or liquid spray to the mucosa, regardless of the nose, lungs, vagina, or rectum. The vaccines disclosed herein can confer resistance to one or more influenza strains by passive or active immunization. In active immunization, an inactivated or attenuated vaccine composition is prophylactically administered to a host (e.g., a mammal), and the host's immune response to the administration defends against infection and / or disease. In the case of passive immunization, the elicited antiserum is collected and administered to a recipient suspected of having an infection caused by at least one influenza virus strain.

[0084] The present invention thus includes a method of preventing or alleviating a disease or disorder (e.g., infection by at least one influenza virus strain). As used herein, a vaccine can be said to prevent or alleviate a disease if administration of the vaccine results in an overall or partial alleviation of the signs or symptoms of the disease, or an overall or partial immunity of the individual to the disease. At least one inactivated or attenuated influenza virus, or a composition thereof, of the present invention can be administered by any means that achieves the intended purpose using the pharmaceutical compositions described above. For example, administration of such a composition can be by a variety of parenteral routes, such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, oral, or transdermal routes. Parenteral administration can be carried out by bolus injection or by slow perfusion over time. In some embodiments, the immunogenic compositions disclosed herein are by intramuscular or subcutaneous application.

[0085] In some embodiments, a regimen for preventing, suppressing, or treating influenza virus-related lesions comprises administering an effective amount of the vaccine composition described herein, which is administered as a single treatment or repeated as a booster or booster dose over a period of from one week to about 24 months or a range of values thereof. In some embodiments, the influenza vaccines disclosed herein are administered annually.

[0086] According to the present technology, an "effective amount" of a vaccine composition is an amount sufficient to achieve the desired biological effect. It is understood that in some embodiments, the effective dosage will depend on the age, sex, health status and weight of the recipient, if any, the type of concomitant treatment, the frequency of treatment, and the nature of the desired effect. The ranges of effective dosages provided below are not intended to be limiting and present exemplary dosage ranges. Thus, in some embodiments, the dosage will be adjusted according to the individual subject animal as would be understood and determined by one of ordinary skill in the art. The dosage of a live attenuated virus vaccine for a mammalian (e.g., human) adult is about 10 1 -10 10 plaque forming units (PFU / kg) or any range therein. In some embodiments, the dosage of a live attenuated virus vaccine for a mammalian (e.g., human) adult is about 10 2 -10 10 plaque forming units (PFU / kg) or any range therein. In some embodiments, the dosage of a live attenuated virus vaccine for a mammalian (e.g., human) adult is about 10 3 -10 10 plaque forming units (PFU / kg) or any range therein. In some embodiments, the dosage of a live attenuated virus vaccine for a mammalian (e.g., human) adult is about 10 4 -10 10 plaque forming units (PFU / kg) or any range therein. In some embodiments, the dosage of a live attenuated virus vaccine for a mammalian (e.g., human) adult is about 10 5 -10 10It can be plaque forming units (PFU / kg) or any value range therein. In some embodiments, the dosage of the attenuated virus vaccine for an adult mammal (e.g., a human) is about 10 6 -10 10 It can be plaque forming units (PFU / kg) or any value range therein. In some embodiments, the dosage of the attenuated virus vaccine for an adult mammal (e.g., a human) is about 10 7 -10 10 It can be plaque forming units (PFU / kg) or any value range therein. In some embodiments, the dosage of the attenuated virus vaccine for an adult mammal (e.g., a human) is about 10 8 -10 10 It can be plaque forming units (PFU / kg) or any value range therein. In some embodiments, the dosage of the attenuated virus vaccine for an adult mammal (e.g., a human) is about 10 9 -10 10 It can be plaque forming units (PFU / kg) or any value range therein. In some embodiments, the dosage of the attenuated virus vaccine for an adult mammal (e.g., a human) is about 10 9 -10 10 It can be plaque forming units (PFU / kg) or any value range therein. In some embodiments, the dosage of the attenuated virus vaccine for an adult mammal (e.g., a human) can exceed 10 10 plaque forming units (PFU / kg). The dosage of the inactivated vaccine can be about 0.1 to 200, such as half of 50 μg, of the hemagglutinin protein. However, the dosage is a safe and effective amount determined by conventional methods using existing vaccines as a starting point.

[0087] C. Intradermal Delivery Live attenuated influenza vaccines are conventionally delivered intranasally, mimicking the natural route of infection and promoting an immune response similar to that of natural viral infection. Alternatively, methods of intradermal delivery are disclosed herein. The methods require the use of novel microneedle devices and take advantage of the immunological benefits of intradermal delivery. In some embodiments, attenuated viruses (e.g., M2 and / or BM2 virus mutants) are used in vaccine compositions for intradermal administration. In some embodiments, M2 and BM2 virus mutants (which do not give rise to infectious progeny virus) are provided in a tetravalent vaccine. Thus, any risk of recombination with wild-type circulating influenza virus is substantially eliminated.

[0088] In the embodiments disclosed herein, intradermal (intracutaneous) delivery administers the vaccine to the skin. In some embodiments, intradermal delivery is performed using a microneedle delivery device. As disclosed herein, intradermal delivery has a number of advantages. For example, the immunogenicity of the vaccine is enhanced by activating the immunological potential of the skin immune system. The vaccine has direct access to the skin's potent antigen-presenting dendritic cells (i.e., epidermal Langerhans cells and dermal dendritic cells). Skin cells generate proinflammatory signals, which enhance the immune response to antigens introduced through the skin. Additionally, the skin immune system generates antigen-specific antibodies and cellular immune responses. Intradermal delivery allows for a reduction in the dose of the vaccine. That is, when delivered intradermally, a lower dose of antigen may be effective considering the above requirements. Furthermore, since the vaccine is delivered to the skin from the microneedle array of the device, the risk of accidental needle stick is reduced, and intradermal vaccine delivery by the microneedle array is relatively painless compared to intramuscular injection with conventional needles and syringes.

[0089] Microneedle devices are known in the art and include, for example, those described in published U.S. patent applications 2012 / 0109066, 2011 / 0172645, 2011 / 0172639, 2011 / 0172638, 2011 / 0172637, and 2011 / 0172609. The microneedle devices are fabricated by machining a stainless steel sheet by wet etching (Trinity Brand Industries, Georgia; SS 304 (50 μm thick)). In some embodiments, the individual microneedles have a length of about 500 μm to 1000 μm (e.g., about 750 μm) and a width of about 100 μm to 500 μm (e.g., about 200 μm). Subsequently, a vaccine can be applied to the microneedles as a coating. By way of illustration and not limitation, the coating solution can include 1% (w / v) sodium carboxymethylcellulose salt (low viscosity, USP grade; Carbo-Mer, San Diego CA), 0.5% (w / v) Lutrol F-68 NF (BASF, Mt. Olive, NJ), and an antigen (e.g., 5 ng / mL of soluble HA protein; live attenuated virus, such as the M2 and BM2 mutant viruses described herein). To achieve higher concentrations, the coating solution can be evaporated at room temperature (about 23° C.) for 5 to 10 minutes. The coating can be performed by a dip coating process. The amount of vaccine per row of microneedles can be determined by submerging the microneedles in 200 μL of phosphate buffered saline (PBS) for 5 minutes and assaying for the antigen by methods known in the art.

[0090] In some embodiments, the micro - needle device used is mainly made of first - cut pieces of propylene and stainless steel, which are assembled together by simple snap - fits and heat - sealing. In some embodiments, the device is completely self - contained and includes a vaccine, a pump mechanism, an activation mechanism, and a micro - needle unit. These components are hidden within a plastic cover. With this device, vaccine injection is initiated by pressing an activation button. Simultaneously with pressing the button, the micro - needles are inserted into the skin to start the pump mechanism, which pressurizes a primary drug container. When the spring mechanism applies sufficient pressure to the vaccine reservoir, the vaccine begins to flow from the micro - needle array into the skin. In some embodiments, the delivery of the vaccine dose is completed within about 2 minutes after the device is activated. After the injection is complete, the device is gently removed from the skin.

[0091] In some embodiments, a method for intradermal administration of an immunogenic composition (e.g., a quadrivalent vaccine) using a micro - needle device is provided. In some embodiments, the micro - needle device includes a piercing mechanism and an immunogenic composition layer, which includes a plurality of micro - needles capable of piercing the skin and further enabling the intradermal administration of the immunogenic composition. In some embodiments, the immunogenic composition (e.g., a quadrivalent vaccine) includes a virus encoding a nucleic acid sequence for a mutant M2 and BM2 protein that is expressed or an M2 and BM2 protein that is not expressed, where the expressed mutant M2 protein includes or consists of an amino acid sequence encoded by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and further, the BM2 protein includes or consists of an amino acid sequence encoded by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. In some embodiments, first, the micro - needle array is disposed inside the device housing, and the micro - needles are extended by a button of the device upon actuation of a lever, thereby enabling the injection of the vaccine solution into the skin.

[0092] Using the delivery device described herein, any desired substance can be delivered. In certain embodiments, the substance to be delivered is a drug, and the delivery device is a drug delivery device configured to deliver the drug to a target animal. As used herein, the term "drug" is intended to include any substance delivered to a target animal for any therapeutic, prophylactic, or medicinal purpose (e.g., vaccines, pharmaceuticals, nutrients, nutraceuticals, etc.). In certain embodiments, the delivery device is configured to deliver a flu vaccine. The embodiments discussed herein primarily relate to devices configured to deliver substances transdermally. In some embodiments, the device may be configured to directly deliver the substance to an organ other than the skin.

Example

[0093] As described above, the present application provides a novel tetravalent immunogenic composition, said composition comprising influenza A and influenza B mutant strains useful for eliciting an immune response in mammals against influenza A and influenza B. The following embodiments are presented to illustrate methods of eliciting an immune response using mutants formulated as a multivalent vaccine, and methods of testing the attenuation of the virulence of multivalent formulations. The following embodiments are presented by way of illustration and not limitation. Those skilled in the art will recognize a variety of non-deterministic parameters that can be changed or modified to obtain essentially the same or similar results. These embodiments should in no way be construed as limiting the scope of the technology as defined in the appended claims.

[0094] [Example 1] The BM2SR variant formulated as a quadrivalent vaccine elicits an antibody response against influenza B virus Experiments were conducted to show that the BM2SR mutant virus elicits an antibody response when formulated as a quadrivalent vaccine. The following four monovalent vaccines were formulated together: A / California / 07 / 2009 (H1N1) containing the M2SR-1 mutant containing SEQ ID NO: 1, A / Brisbane / 10 / 2007 (H3N2) containing the M2SR-1 mutant containing SEQ ID NO: 1, B / Brisbane / 60 / 2008 (Victoria) containing BM2SR-0 containing SEQ ID NO: 11, and B / Wisconsin / 01 / 2010 (Yamagata) containing BM2SR-0 containing SEQ ID NO: 11. 1x10 6 TCID 50 of each were mixed together and each quadrivalent dose per mouse was 4x10 6 TCID 50 . The sequences of each of the M2SR-1 and BM2SR-0 mutant constructs are provided in Tables 1 and 5.

[0095] Six-week-old female BALB / c mice were inoculated intranasally with the quadrivalent formulation at a dose of 4x10 6 TCID 50 / mouse. The control mouse group was given PBS. Serum samples were collected on days 7, 14, and 21 after the first inoculation, and on days 35, 42, and 49 after the second immunization on day 28. The anti-HA IgG antibody titers of the serum samples were determined by enzyme-linked immunosorbent assay (ELISA) against B / Wisconsin / 01 / 2010 and B / Brisbane / 60 / 2008. The humoral response is shown in Figure 2. Figure 2 shows that the quadrivalent MS2R and BMS2R vaccines produced higher influenza virus antibodies against both influenza B antigens, which are two influenza B lineages (B / Bris / 60 and B / Wisc / 01), than the control PBS group. Mice boosted with the quadrivalent vaccine had higher levels of anti-influenza HA antibodies after the second immunization than after the first administration. These results indicate that each monovalent BM2SR vaccine can elicit an antigen-specific response in the quadrivalent formulation.

[0096] [Example 2] The M2SR and BM2SR variants formulated as a multivalent vaccine elicit an antibody response against influenza A and influenza B viruses A. The BM2SR variant formulated as a multivalent vaccine elicits an antibody response against influenza B virus Experiments were conducted to show that the BM2SR mutant virus elicits an antibody response when formulated as a monovalent, bivalent, trivalent, or tetravalent vaccine using influenza A H1N1 or H3N2 M2SR vaccines. Table A shows how the various formulations of the following four monovalent M2SR and BM2SR vaccines were formulated together: A / California / 07 / 2009 (H1N1) (containing the M2SR-1 mutant containing SEQ ID NO: 1), A / Brisbane / 10 / 2007 (H3N2) (containing the M2SR-1 mutant containing SEQ ID NO: 1), B / Brisbane / 60 / 2008 (Victoria) (containing BM2SR-0 containing SEQ ID NO: 11), B / Wisconsin / 01 / 2010 (Yamagata) (containing BM2SR-0 containing SEQ ID NO: 11).

[0097]

Table A

[0098] Six-week-old female BALB / c mice were inoculated intranasally with monovalent, bivalent, trivalent, or tetravalent vaccines at the doses shown in Table A. The control mouse group was given PBS. Serum samples were collected on days 7, 14, and 21 after the first inoculation, and on days 35, 42, and 49 after the second immunization on day 28. The anti-HA IgG antibody titers of the serum samples were determined by enzyme-linked immunosorbent assay (ELISA) against B / Wisconsin / 01 / 2010 and B / Brisbane / 60 / 2008. The humoral response is shown in FIGS. 3A-3B. The figures show that both BMS2R vaccine components (Bris60 and WI01) produced higher influenza virus antibodies against both influenza B antigens of the two influenza B strains of the multivalent formulation than the control PBS group. These results indicate that there is no interference between the monovalent components when formulated as a multivalent vaccine.

[0099] B. The M2SR variant formulated as a multivalent vaccine elicits an antibody response against influenza A virus Experiments were conducted to show that the M2SR mutant virus elicits an antibody response when formulated as a monovalent, bivalent, trivalent, or tetravalent vaccine using influenza B Yamagata or Victoria lineage BM2SR vaccines. Table A shows how the various formulations of the following four monovalent M2SR and BM2SR vaccines were formulated together: A / California / 07 / 2009 (H1N1) (including the M2SR-1 mutant containing SEQ ID NO: 1), A / Brisbane / 10 / 2007 (H3N2) (including the M2SR-1 mutant containing SEQ ID NO: 1), B / Brisbane / 60 / 2008 (Victoria) (including BM2SR-0 containing SEQ ID NO: 11), B / Wisconsin / 01 / 2010 (Yamagata) (including BM2SR-0 containing SEQ ID NO: 11). Six-week-old female BALB / c mice were inoculated intranasally with the monovalent, bivalent, trivalent, or tetravalent vaccines at the doses shown in Table A. The control mouse group was given PBS. Serum samples were collected on days 7, 14, and 21 after the first inoculation, and on days 35, 42, and 49 after the second immunization on day 28. The anti-HA IgG antibody titers of the said serum samples were determined by enzyme-linked immunosorbent assay (ELISA) against A / California / 07 / 2009 (H1N1) and A / Brisbane / 10 / 2007 (H3N2). The humoral response is shown in FIGS. 3C-3D. The figures show that both influenza A MS2R vaccine components (H1N1 and H3N2) produced higher influenza virus antibodies against both influenza A antigens of the H1N1 and H3N2 subtypes in the multivalent formulation than the control PBS group. These results indicate that there is no interference between the monovalent components when formulated as a multivalent vaccine.

[0100] [Example 3] The BM2SR variant protects mice from lethal influenza B virus challenge as a monovalent or quadrivalent formulation BALB / c female mice (N = 8) were challenged on day 49 after the first inoculation (3 weeks after the booster) with a lethal dose of B / Malaysia / 2506 / 2004 virus (20 mouse 50% lethal dose (MLD 50 )) as shown in Figures 4A and 4B, all mice inoculated with BM2SR and the quadrivalent vaccine survived the challenge and their body weights did not decrease. However, control mice given only PBS had a decrease in body weight and did not survive 9 days after the challenge day. Lungs were obtained on day 4 after the challenge, and the virus titer was determined in MDCK cells by plaque assay. As shown in Figure 4C, the lung virus titers of BM2SR- and quadrivalent vaccine-inoculated mice were below the detection limit, while naive control PBS mice had high virus titers, indicating that BM2SR and the quadrivalent vaccine conferred cross-protection and restricted the replication of the challenge virus.

[0101] [Example 4] The quadrivalent M2SR vaccine protects mice from lethal influenza A virus challenge BALB / c female mice (N = 8) were challenged on day 49 after the first inoculation (3 weeks after the booster) with a lethal dose of A / Aichi / 02 / 1968 (H3N2) virus (40 mouse 50% lethal dose (MLD 50 )) as shown in Figures 5A and 5B, all mice inoculated with the monovalent H1N1 or H3N2 M2SR and the quadrivalent M2SR vaccine survived the challenge, with a transient decrease in body weight but complete recovery. However, control mice given only PBS had a decrease in body weight and did not survive 8 days after the challenge day. Lungs were obtained on day 4 after the challenge, and the virus titer was determined in MDCK cells by plaque assay. As shown in Figure 5C, the lung virus titers of M2SR monovalent- and quadrivalent vaccine-inoculated mice were at least 1 log lower than those of naive control PBS mice, indicating that the M2SR monovalent and quadrivalent M2SR vaccines conferred cross-protection and restricted the replication of the challenge virus (which did not match any of the vaccine components).

[0102] [Example 5] The BM2SR variant formulated as a quadrivalent vaccine elicits an antibody response against influenza B virus Experiments were conducted to show that the BM2SR mutant virus elicits an antibody response when formulated as a quadrivalent vaccine. The following four monovalent vaccines were formulated together: H1N1 influenza A virus containing the M2SR-1 mutant containing SEQ ID NO: 1, H3N2 influenza A virus containing the M2SR-1 mutant containing SEQ ID NO: 1, Victoria lineage influenza B virus containing the BM2SR-4 mutant containing SEQ ID NO: 9, and Yamagata lineage influenza B virus containing the BM2SR-4 mutant containing SEQ ID NO: 9. 0.2 - 1 x 10 6 TCID 50 of each were mixed together and each quadrivalent dose per mouse was made approximately 3 x 10 6 TCID 50 . The sequences of each of the M2SR-1 and BM2SR-4 mutant constructs are provided in Tables 1 and 5. Six-week-old female BALB / c mice were inoculated intranasally with the quadrivalent formulation at a dose of approximately 3 x 10 6 TCID 50 / mouse. The control mouse group was given PBS. Serum samples were collected 14 days after the first inoculation. The anti-HA IgG antibody titers of the serum samples were determined by enzyme-linked immunosorbent assay (ELISA) against influenza antigens (B / Victoria and B / Yamagata). As shown in FIGS. 6A and 6B, the quadrivalent MS2R and BMS2R vaccines produced higher influenza virus antibodies against both influenza B antigens, which are two influenza B lineages, than the control PBS group. These results indicate that each monovalent BM2SR vaccine can elicit an antigen-specific response in the quadrivalent formulation.

[0103] [Example 6] The M2SR and BM2SR variants formulated as a quadrivalent vaccine elicit an antibody response against influenza A and influenza B viruses A. The BM2SR variant formulated as a multivalent vaccine elicits an antibody response against influenza B virus Experiments were conducted to show that the BM2SR mutant virus elicits an antibody response when formulated as a monovalent, trivalent, or tetravalent vaccine using influenza A H1N1 or H3N2 M2SR vaccines. The following four monovalent M2SR and BM2SR vaccines were formulated together: H1N1 (containing the M2SR-1 mutant containing SEQ ID NO: 1), H3N2 (containing the M2SR-1 mutant containing SEQ ID NO: 1), B / Victoria lineage (containing the BM2SR-4 mutant containing SEQ ID NO: 9), B / Yamagata (containing the BM2SR-4 mutant containing SEQ ID NO: 9). Six-week-old female BALB / c mice were inoculated intranasally with monovalent, trivalent, or tetravalent vaccines. The control mouse group was given PBS. Serum samples were collected 14 days after the first inoculation. The anti-HA IgG antibody titers of the serum samples were determined by enzyme-linked immunosorbent assay (ELISA) against both influenza B antigens. As shown in FIGS. 7C and 7D, both BM2SR vaccine components were higher against the influenza B antigen, which is one of the two influenza B lineages of the multivalent formulation, than the control PBS group. These results indicate that there is no interference between the monovalent components when formulated as a multivalent vaccine.

[0104] B. The M2SR variant formulated as a multivalent vaccine elicits an antibody response against influenza A virus Experiments were conducted to show that the M2SR mutant virus elicits an antibody response when formulated as a monovalent, trivalent, or tetravalent vaccine using influenza B Yamagata or Victoria lineage BM2SR vaccines. The following four monovalent M2SR and BM2SR vaccines were formulated together: H1N1 (containing the M2SR-1 mutant containing SEQ ID NO: 1), H3N2 (containing the M2SR-1 mutant containing SEQ ID NO: 1), B / Victoria lineage (containing the BM2SR-4 mutant containing SEQ ID NO: 9), B / Yamagata (containing the BM2SR-4 mutant containing SEQ ID NO: 9). Six-week-old female BALB / c mice were inoculated intranasally with monovalent, trivalent, or tetravalent vaccines. The control mouse group was given PBS. Serum samples were collected 14 days after the first inoculation. The anti-HA IgG antibody titers of the serum samples were determined by enzyme-linked immunosorbent assay (ELISA) against H1N1 and H3N2 influenza A viruses. As shown in FIGS. 7A and 7B, both influenza A M2SR vaccine components (H1N1 and H3N2) produced higher anti-influenza virus antibodies against both influenza A antigens of the H1N1 and H3N2 subtypes in the multivalent formulation than the control PBS group. These results indicate that there is no interference between the monovalent components when formulated as a multivalent vaccine.

[0105] [Example 7] The BM2SR-4 variant protects mice from lethal influenza B virus challenge as a monovalent, trivalent or quadrivalent formulation On day 22 after inoculation, BALB / c female mice (N = 4) were challenged with a lethal dose of a heterologous subtype influenza B virus, B / Malaysia / 2506 / 2004 virus (20 mouse 50% lethal dose (MLD 50 ). All mice inoculated with the BM2SR-4 monovalent, trivalent, and tetravalent vaccines survived the challenge (FIG. 8B), and there was no weight loss (FIG. 8A). However, control mice given only PBS lost weight and did not survive the challenge. These results indicate that the monovalent BM2SR-4 vaccines (each different from the challenge virus), the trivalent, and the tetravalent vaccines confer cross-protection against the challenge virus. These results indicate that there is no interference between the monovalent components in the multivalent formulation.

[0106] [Example 8] The quadrivalent M2SR vaccine protects mice from lethal influenza A virus challenge On day 22 after inoculation, BALB / c female mice (N = 4) were challenged with a lethal dose of a heterologous influenza A virus, such as A / Aichi / 02 / 1968 (H3N2) virus (40 mouse 50% lethal dose (MLD 50)(challenged with)). All mice vaccinated with trivalent MS2R (containing M2SR-1 variants containing SEQ ID NO: 1, including H1N1, H3N2 (containing M2SR-1 variants containing SEQ ID NO: 1), and B / Yamagata (containing BM2SR-4 variants containing SEQ ID NO: 9)), or tetravalent M2SR vaccine (containing H1N1 (containing M2SR-1 variants containing SEQ ID NO: 1), H3N2 (containing M2SR-1 variants containing SEQ ID NO: 1), B / Victoria lineage (containing BM2SR-4 variants containing SEQ ID NO: 9), and B / Yamagata (containing BM2SR-4 variants containing SEQ ID NO: 9)) had more surviving individuals after challenge (Figure 9B), transiently lost weight but began to recover on day 7 (Figure 9A). However, control mice given only PBS lost weight and did not survive 7 days after challenge. These results indicate that trivalent and tetravalent M2SR / BM2SR vaccines confer cross-protection against challenge viruses that do not match any of the vaccine components.

[0107] [Example 9] Immune responses and protective efficacy elicited by monovalent M2SR and BM2SR and tetravalent M2SR vaccines in the ferret model A. Summary This example shows that the immune responses elicited by the tetravalent M2SR vaccine are similar to those of the monovalent M2SR and BM2SR vaccines in the ferret model. That is, the tetravalent M2SR does not show interference and elicits protective immune responses against each of the components. Each of the M2SR and BM2SR candidate viruses was administered intranasally to 12 male ferrets at a dose level of 1x10 7 TCID 50 (monovalent) or 4x10 7 TCID 50 (tetravalent). As a control, OPTI-MEM was given to one group of ferrets TMIt was administered as a placebo control. A prime-boost vaccination regimen was used for each group. Ferrets were administered the prime vaccine (day 0) and the boost vaccine (day 28) 28 days later. After each vaccination, the ferrets were observed for 14 days post-vaccination for mortality, and body weight, body temperature, and clinical signs were examined daily. Serum was collected from all ferrets on days 21, 35, and 56 post-vaccination to evaluate antibody levels over time. All animals were intranasally challenged with 1x10 6 PFU of A / California / 07 / 2009 (H1N1 pdm) on day 70. Following the challenge, the ferrets were monitored for 14 days post-challenge for mortality, and body weight, body temperature, and clinical signs were examined daily. For viral titers, nasal washes were collected from ferrets in each group (N = 8) on days 1, 3, 5, and 7 post-challenge. In addition, serum was collected from surviving ferrets for analysis on day 82 post-challenge. Necropsies were performed on 4 ferrets per group 3 days after the challenge (day 73). Organs were collected for determination of the post-challenge viral load (titer). Vaccine-related adverse effects were not observed in the 5 groups. After the challenge, the placebo control group showed a decrease in body weight (about 15%). A decrease in body weight was also observed in the antigenically mismatched monovalent H3N2 M2SR and BM2SR vaccine-inoculated groups, but the decrease was less than that observed in the placebo group. Quadrivalent M2SR and H1N1 pdm M2SR showed no significant body weight loss after the challenge. B. Materials and methods Vaccine virus inoculation : As shown in Table B, ferrets were intranasally administered either a monovalent M2SR or BM2SR vaccine at a dose of 1x10 7 TCID 50 twice or a quadrivalent M2SR vaccine at a dose of 4x10 7 TCID 50It was administered intranasally twice. One vial of the frozen stock was thawed at room temperature for at least 10 minutes and subsequently stored refrigerated (or on ice) until use. Ferrets were anesthetized with ketamine / xylazine and the viral dose was administered intranasally in a volume of 500 μL (250 μL in each nostril). The animals were observed daily for 7 days after each vaccination. Body weight, body temperature, and clinical signs were monitored for 7 days.

[0108]

Table B

[0109] The MS2R virus is a recombinant influenza A virus that does not express a functional M2 protein, said virus comprising the M2SR-1 variant comprising SEQ ID NO: 1 and encoding the HA and NA genes of A / Brisbane / 10 / 2007-like A / Uruguay / 716 / 2007 (H3N2) or A / California / 07 / 2009 (H1N1pdm). The BMS2R virus is a recombinant influenza B virus that does not express a functional BM2 protein, said virus comprising the BM2SR-0 variant comprising SEQ ID NO: 11 and encoding the HA and NA of B / Brisbane / 60 / 2008 (Victoria) or B / Wisconsin / 01 / 2010 (Yamagata). The quadrivalent M2SR is composed of two M2SR and two BM2SR viruses encoding the HA and NA of H1N1, H3N2, B / Victoria, and B / Yamagata.

[0110] Animals and animal management: Eighty male ferrets were purchased from a vendor (Triple F Farms), and 72 of these ferrets were used in the study. At the start of the study, the animals were approximately 4 months of age. The animals were healthy and certified by the supplier as not having antibodies to infectious diseases. Upon arrival, the animals were housed singly in suspended wire cages with slatted floors (suspended over pans of paper for excrement). The animal rooms and cages were cleaned and sanitized prior to animal acceptance and were in accordance with accepted animal husbandry practices and related standard operating procedures. Feed (Certified Teklad Global Ferret Diet #2072 (Teklad Diets, Madison WI)) and Chicago city tap water were provided ad libitum and refreshed at least three times weekly. Fluorescent lighting in the animal rooms was maintained on a 12-hour light / dark cycle. Animal room temperature and relative humidity were within the limits of the relevant protocol and ranged from 20.0 to 25.0 °C and 30 to 63%, respectively, during the study.

[0111] Animal isolation and randomization : The ferrets were isolated for 7 days and observed daily prior to randomization. Based on daily observations indicating that the animals were in generally good health, these ferrets were released from isolation for randomization and study. Following isolation, the ferrets were weighed and assigned to treatment groups using a computer-controlled randomization procedure based on weight (yielding similar group mean values) (ToxData™, version 2.1.E.11 (PDS Pathology Data Systems, Inc., Basel, Switzerland)). Within groups, all weights were within 20% of their mean. Animals selected for the study were given permanent identification numbers by ear tags and transponders, and individual cage cards also identified the study animals by individual number and group. The assigned identification numbers were unique within this study.

[0112] Experimental design : To assess vaccine efficacy, ferrets were immunized with each of the M2SR, BM2SR, or quadrivalent M2SR viruses, or with medium (OPTI-MEMTM were pseudo-immunized by One dose of 1x10 7 TCID 50 of M2SR or BM2SR was administered intranasally to the ferrets on days 0 and 28, or one dose of 4x10 7 TCID 50 of quadrivalent M2SR was administered on days 0 and 28. The control group received OPTI-MEM TMThey were intranasally mock - inoculated on day 0 and day 28. For 14 days after inoculation, the body temperature, body weight and clinical signs of the ferrets were monitored daily. The nasal wash samples were kept at - 65°C. Blood was collected before inoculation (days - 3 and - 5) and on days 21, 35 and 56, and the serum was kept at - 65°C until antibody titer measurement by ELISA and HAI assays.

[0113] C. Results The anti - HA IgG antibody titers of serum samples against the following were determined by enzyme - linked immunosorbent assay (ELISA): A / Brisbane / 10 / 2007 (H3N2), A / California / 07 / 2009 (H1N1pdm), B / Wisconsin / 01 / 2010 (Yamagata lineage), and B / Brisbane / 60 / 2008 (Victoria lineage). Briefly, ELISA plates were coated with the recombinant HA protein of each strain, blocked with bovine serum albumin (BSA), and the samples were applied. Ferret IgG antibodies were detected by horseradish peroxidase - labeled anti - ferret IgG goat antibody (KPL, Inc., Gaithersburg, MD) and SureBlue TMB (KPL, Inc.) substrate. As expected, ferrets in each immunization group showed a significant increase in anti - HA antibodies in serum against their corresponding antigen. More importantly, the quadrivalent M2SR group presented a significant increase in anti - HA antibodies in serum against all four antigens (Figure 10), indicating that there was no interference between the components of the multivalent formulation. These data suggest that M2SR, BM2SR, and quadrivalent M2SR viruses elicit a significant immune response in ferrets.

[0114] To demonstrate the functional activity of the antibodies detected by ELISA, serum samples were analyzed by hemagglutination inhibition (HAI) assay. Serum samples were treated with receptor-destroying enzyme (RDE) (Denka Seiken, Tokyo, Japan) to remove non-specific hemagglutination-inhibiting substances. RDE was reconstituted according to the manufacturer's instructions. Serum was diluted 1:3 in RDE and incubated in a water bath at 37 °C ± 2 °C for 18 - 20 hours. After adding an equal volume of 2.5% (v / v) sodium citrate, the samples were incubated in a water bath at 56 ± 2 °C for 30 ± 5 minutes. After RDE treatment, 0.85% NaCl was added to each sample at a final serum dilution of 1:10. Subsequently, the diluted samples were serially diluted 4-fold twice with phosphate-buffered saline (PBS) in duplicate (from 1:10 to 1:80) and then incubated with 4 hemagglutination units of A / Brisbane / 10 / 2007 (H3N2), A / California / 07 / 2009 (H1N1pdm), B / Wisconsin / 01 / 2010 (Yamagata lineage), and B / Brisbane / 60 / 2008 (Victoria lineage) influenza viruses. After incubation, 0.5% turkey red blood cells were added to each sample and incubated for 30 ± 5 minutes. Subsequently, the presence or absence of agglutination was scored.

[0115] As shown in Figures 11A and 11B, all M2SR immunized ferrets showed significant HAI titers against their corresponding test viruses. The placebo (naïve) group elicited no influenza-specific antibodies. The CDC has stated that an HAI antibody titer of 40 is associated with at least a 50% reduction in the risk of influenza infection or disease in the population. Therefore, these results suggest that the M2SR and BM2SR viruses elicit a protective immune response that is maintained when these viruses are formulated together as a quadrivalent vaccine.

[0116] After challenge with A / California / 09 / 2009 (H1N1pdm), 5 - 8% weight loss was observed in all animals on day 6 post - challenge. Throughout the 14 - day observation period, the animals' weights remained lower than their initial weights, but OPTI - MEM TM The administered ferrets (placebo group) had the greatest weight loss (15%). The weight loss in vaccinated ferrets was dependent on the antigenicity of the vaccine. Ferrets administered with a matched H1N1 pdm M2SR or quadrivalent M2SR (including H1N1 pdm M2SR) showed no significant weight loss. Ferrets administered either a heterologous H3N2 M2SR or BM2SR vaccine showed a weight loss of approximately 5 - 8%.

[0117] Nasal wash samples were collected on days 1, 3, 5, and 7 post - challenge and assayed for the presence of challenge virus by plaque assay in MDCK cells. Figure 12 shows that the quadrivalent M2SR controls virus replication in a manner similar to the monovalent homologous H1N1 pdm M2SR. Placebo and BM2SR monovalent vaccines were unable to control the challenge virus, and at least 5 log of virus was detected up to 5 days post - infection. The heterologous H3N2 M2SR group did not eliminate the challenge virus similar to the homologous H1N1 and quadrivalent M2SR, but partially controlled virus replication compared to the placebo group.

[0118] Respiratory organs collected from 4 ferrets on day 3 post - infection showed control of the challenge virus. H1N1 M2SR and quadrivalent M2SR did not permit any replication of the challenge virus in the upper and lower respiratory tissues (turbinates, trachea, lung), as shown in Figures 13A, 13B, and 13C. In contrast, the challenge virus grew to higher titers in the upper respiratory tissues (turbinates and trachea) of the other groups. In the lower airways (lung), the monovalent M2SR vaccine controlled the challenge virus compared to the placebo group. These results suggest that homologous and quadrivalent M2SR prevent the establishment of influenza infection, and unrelated M2SR vaccines reduce the severity of infection.

[0119] D. Conclusions This example shows that intranasal administration of the tetravalent M2SR vaccine was not associated with any vaccine-related adverse effects (such as fever, weight loss, or clinical signs). These results indicate that the tetravalent M2SR virus elicits a protective immune response against each strain included in the multivalent formulation and is useful as an intranasal influenza vaccine.

Claims

1. A composition for use in the manufacture of a medicament for the treatment of influenza, comprising a trivalent immunogenic composition comprising three recombinant influenza viruses, said viruses being: (i) an engineered attenuated H1N1 virus having a mutated M2 gene comprising SEQ ID NO: 1; (ii) an engineered attenuated H3N2 virus having a mutated M2 gene comprising SEQ ID NO: 1; and (iii) an engineered attenuated B / Victoria virus having a mutated BM2 gene comprising SEQ ID NO:

9.

2. The composition according to claim 1, wherein the trivalent immunogenic composition further comprises a pharmaceutically acceptable carrier.

3. The composition according to claim 1 or 2, wherein the trivalent immunogenic composition further comprises a pharmaceutically acceptable adjuvant.

4. The composition according to any one of claims 1 - 3, wherein the trivalent immunogenic composition is formulated for intranasal or intradermal administration.

5. A composition for use in the manufacture of a medicament for the treatment of influenza, comprising a trivalent immunogenic composition comprising three recombinant influenza viruses, said viruses being: (i) an engineered attenuated H1N1 virus having a mutated M2 gene comprising SEQ ID NO: 1; (ii) an engineered attenuated H3N2 virus having a mutated M2 gene comprising SEQ ID NO: 1; and (iii) an engineered attenuated B / Victoria virus having a mutated BM2 gene comprising SEQ ID NO:

11.

6. The composition according to claim 5, wherein the trivalent immunogenic composition further comprises a pharmaceutically acceptable carrier.

7. The composition according to claim 5 or 6, wherein the trivalent immunogenic composition further comprises a pharmaceutically acceptable adjuvant.

8. The composition according to any one of claims 5 - 7, wherein the trivalent immunogenic composition is formulated for intranasal or intradermal administration.

9. A method for preparing an immunogenic composition, comprising the step of combining: (i) an engineered attenuated H1N1 virus having a mutated M2 gene comprising SEQ ID NO: 1; (ii) an engineered attenuated H3N2 virus having a mutated M2 gene comprising SEQ ID NO: 1; and (iii) an engineered attenuated B / Victoria virus having a mutated BM2 gene comprising SEQ ID NO:

9.

10. A method for preparing an immunogenic composition comprising the step of combining: (i) an engineered attenuated H1N1 virus having a mutant M2 gene comprising SEQ ID NO: 1; (ii) an engineered attenuated H3N2 virus having a mutant M2 gene comprising SEQ ID NO: 1; and (iii) an engineered attenuated B / Victoria virus having a mutant BM2 gene comprising SEQ ID NO:

11. **Claim 11** (a) A step of growing an engineered attenuated H1N1 virus having a mutant M2 gene comprising SEQ ID NO: 1 by contacting a host cell with the virus and incubating the host cell under conditions that promote virus replication, wherein the host cell is modified to produce the wild-type influenza M2 gene, whereby the gene product is provided to the virus in a trans manner; (b) A step of growing an engineered attenuated H3N2 virus having a mutant M2 gene comprising SEQ ID NO: 1 by contacting a host cell with the virus and incubating the host cell under conditions that promote virus replication, wherein the host cell is modified to produce the wild-type influenza M2 gene, whereby the gene product is provided to the virus in a trans manner; (c) A step of growing an engineered attenuated B / Victoria virus having a mutant BM2 gene comprising SEQ ID NO: 9 or 11 by contacting a host cell with the recombinant virus and incubating the host cell under conditions that promote virus replication, wherein the host cell is modified to produce the wild-type influenza BM2 gene, whereby the gene product is provided to the virus in a trans manner; and (d) A step of combining the grown viruses of (a) to (c) to form an immunogenic composition A method for preparing an immunogenic composition comprising the above steps.

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