Influenza b virus mutants and uses thereof

Abstract

To provide a method for propagating a recombinant influenza B virus, and a host cell.SOLUTION: A method for propagating a recombinant influenza B virus comprising a mutant BM2 gene having a specific sequence comprises: contacting a host cell with the recombinant influenza B virus; and incubating the host cell for a sufficient time and under conditions suitable for viral replication; where the host cell is an MDCK cell or a Vero cell expressing functional BM2 or chimeric M2-BM2 proteins.SELECTED DRAWING: None

Description

Technical Field

[0001] (Cross - reference to related applications) This application claims priority to U.S. Application No. 62 / 463,994, filed Feb. 27, 2017 (incorporated herein by reference in its entirety).

Background Art

[0002] Influenza is a leading cause of death among adult Americans. Every year, approximately 36,000 people die from influenza and over 200,000 are hospitalized. Influenza is a highly contagious disease that spreads through coughing, sneezing, and direct physical contact with objects that carry the virus (such as doorknobs and telephones). The symptoms of influenza include, for example, extreme fatigue, headache, chills, and body aches, and about 50% of infected people 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 matches 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 to multiple viral antigens. Thus, the use of live virus overcomes the problem of virus antigen destruction 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 a recombinant influenza B virus having a mutant BM2 gene comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. In some embodiments, the mutation in the BM2 gene results in a failure of the virus to express the BM2 protein or causes the virus to express a truncated BM2 protein. In some embodiments, the mutant BM2 gene does not revert to a wild-type or non-wild-type sequence encoding a functional BM2 protein for at least 10 passages in an in vitro host cell line, where the host cell is modified to produce a functional version of the mutant gene, thereby providing the gene product in trans to the virus. In some embodiments, the recombinant virus elicits an immune response in a mammal infected with the virus. In some embodiments, the recombinant virus is non-pathogenic to a mammal infected with the virus. In some embodiments, the in vitro cell line comprises Mardin-Darby canine kidney (MDCK) cells or Vero cells. In one aspect, the present disclosure provides a composition comprising a recombinant influenza B virus having a mutant BM2 gene comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. In some embodiments, the mutation in the BM2 gene results in a failure of the virus to express the BM2 protein or causes the virus to express a truncated BM2 protein. In some embodiments, the virus elicits an immune response in a mammal infected with the virus. In some embodiments, the virus is non-pathogenic to a mammal infected with the virus. In some embodiments, the composition further comprises an adjuvant.

[0004] In one aspect, the present disclosure provides a method for propagating a recombinant influenza B virus, the method comprising contacting a host cell with a recombinant influenza virus comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; and incubating the host cell for a sufficient time and under conditions appropriate for viral replication, wherein the host cell is modified to produce a functional version of the influenza BM2 gene, thereby providing the viral gene product in a trans configuration to the virus. In some embodiments, the method further comprises isolating progeny virus particles. In some embodiments, the method further comprises formulating the virus particles into a vaccine. In some embodiments, the virus is unable to express the BM2 protein or expresses a truncated BM2 protein. In some embodiments, the virus elicits an immune response in a mammal infected with the virus. In some embodiments, the virus is non-pathogenic to a mammal infected with the virus. In some embodiments, the mutant BM2 gene does not revert to a wild-type or functional BM2 protein-encoding non-wild-type sequence in host cells for at least 10 passages. In some embodiments, the host cell is an MDCK cell or a Vero cell.

[0005] In one aspect, the present disclosure provides a method for propagating a recombinant influenza A virus, the method comprising contacting a host cell with a recombinant influenza A virus comprising SEQ ID NO: 33; and incubating the host cell for a sufficient time and under conditions appropriate for viral replication, wherein the host cell is an MDCK cell modified to produce a wild-type version of the influenza BM2 gene, thereby providing the BM2 gene product in a trans configuration to the virus. In one aspect, the present disclosure provides a method for growing a recombinant influenza A virus, the method comprising contacting a host cell with a recombinant influenza A virus comprising SEQ ID NO: 33; and incubating the host cell for a sufficient time and under conditions appropriate for virus replication, wherein the host cell is a Vero cell modified to produce a chimeric version of the influenza A M2 and an influenza B BM2 gene selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, thereby providing the virus with a chimeric M2:BM2 gene product in a trans configuration. In one aspect, the present disclosure provides a method for growing a recombinant influenza A virus, the method comprising contacting a host cell with a recombinant influenza A virus comprising SEQ ID NO: 33; and incubating the host cell for a sufficient time and under conditions appropriate for virus replication, wherein the host cell is a Vero cell modified to produce a codon-optimized version of the BM2 gene comprising SEQ ID NO: 27, thereby providing the virus with the BM2 gene product in a trans configuration. In one aspect, the present disclosure provides a recombinant influenza A virus comprising a mutant BM2 protein. In one aspect, the present disclosure provides a host cell for growing a recombinant influenza virus, wherein the host cell is a Vero cell modified to produce a gene product encoded by a cDNA sequence selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32. In some embodiments, the recombinant influenza virus is an influenza A virus comprising a mutant M2 gene shown in SEQ ID NO: 33. In some embodiments, the recombinant influenza virus is an influenza B virus comprising a mutant BM2 gene shown in SEQ ID NO: 4 or SEQ ID NO: 6, wherein the Vero cell is modified to produce a gene product encoded by SEQ ID NO: 27. BRIEF DESCRIPTION OF THE DRAWINGS

[0006]

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Mode for Carrying Out the Invention

[0007] 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 explicitly 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 intended to be limited to the number itself indicated in this specification, but rather are intended to refer to a range substantially within the recited range, provided that the scope of the 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 used unclearly to those skilled in the art, "about" will refer to plus or minus 10% of that term, conditional on the context in which the term is used.

[0008] As used herein, the term "attenuated" when used in connection with a virus refers to a virus that has a reduced virulence or pathogenicity compared to its non-attenuated counterpart, but still has viability or is alive. Typically, attenuation renders 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.

[0009] As used herein, the term "effective amount" or "therapeutically effective amount" or "pharmaceutically effective amount" refers to an amount sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount that results in the prevention of a disease, symptom and / or its manifestations. 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 compound administered. The effective amount will also depend on the stage, severity and type of the disease or symptom. One of ordinary skill in the art can determine an appropriate dosage regimen according to these requirements 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, such as vaccines) are administered.

[0010] 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 an in vitro, cultured cell (e.g., a CHO cell, a Vero cell, an MDCK cell, etc.). 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, for example, to enhance virus production. The enhancement of virus production can be, for example, by enhancing virus infection of the host cell and / or enhancing the virus growth rate. By way of non-limiting example, exemplary host cell modifications include: recombinant expression of a 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.

[0011] 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 of five BM2 incomplete influenza B BM2SR variants (e.g., BM2SR-1, BM2SR-2, BM2SR-3, BM2SR-4, BM2SR-5). In some embodiments, the immunogenic compositions described herein can be formulated for administration in a number of forms (i.e., formulated for "exposure" to a mammal). For example, in some embodiments, the immunogenic composition is formulated for oral, pulmonary, intravenous, intramuscular, subcutaneous, parenteral, nasal, or topical administration. The composition can also be formulated for special dosage forms. For example, in some embodiments, the immunogenic composition can be formulated as a liquid, a gel, an aerosol, an ointment, a cream, a lyophilized formulation, a powder, a cake, a tablet, or a capsule. In other embodiments, the immunogenic composition is formulated as a controlled release formulation, a sustained release formulation, a long term release formulation, a pulsatile release formulation, and a mixed immediate release formulation. In some embodiments, the immunogenic composition is provided as a liquid. In other embodiments, the immunogenic composition is provided in lyophilized form.

[0012] 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 transfer of a pathogen from one organism to another or from a contaminated surface to the organism).

[0013] 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 not to be associated with unwanted in vivo substances or to be substantially purified from unwanted in vivo substances that normally coexist. 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 for that compound or agent.

[0014] As used herein, the terms "variant", "mutation" and "variant" are used interchangeably and refer to a nucleic acid or polypeptide sequence that is different from the wild-type sequence. In some embodiments, the variant or variant sequence occurs naturally. In other embodiments, the variant or variant sequence is introduced recombinantly and / or chemically. In some embodiments, a mutation of a nucleic acid includes modifications (e.g., additions, deletions, substitutions) to an RNA and / or DNA sequence. In some embodiments, the modifications include chemical modifications (e.g., methylation) and can further include substitutions or additions with 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, a stop codon, or can introduce a splicing defect or splicing change. A nucleic acid mutation to a coding sequence can also result in conservative or non-conservative amino acid changes.

[0015] As used herein, the term "recombinant virus" refers to a virus that has been engineered in vitro, for example using recombinant nucleic acid technology, to introduce changes into the viral genome and / or to introduce changes into viral proteins. For example, in some embodiments, a recombinant virus can contain both wild-type (endogenous) nucleic acid sequences and variant sequences and / or exogenous nucleic acid sequences. Additionally or alternatively, in some embodiments, a recombinant virus can contain modified protein components, such as variant or variant matrix, hemagglutinin, neuraminidase, nucleoprotein, non-structural and / or polymerase proteins.

[0016] As used herein, the term "recombinant cell" or "modified cell" refers to a cell that has been manipulated in vitro, for example using recombinant nucleic acid technology, to introduce nucleic acid into the cell and / or modify the cellular nucleic acid. Examples of recombinant cells include prokaryotic or eukaryotic cells that harbor an exogenous plasmid, expression vector, etc., and / or cells that contain modifications (e.g., substitutions, mutations, insertions, deletions, etc.) in their cellular nucleic acids. Exemplary recombinant cells are cells that have been manipulated in vitro to stably express an exogenous protein (e.g., the viral BM2 protein).

[0017] 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 the host cell or release from the 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 the SR vaccine virus thus does not affect viral infection kinetics and antigen production as opposed to live attenuated vaccines.

[0018] 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 include mammals such as humans, along with mammals and birds that are either endangered and thus important, economically important (animals raised on farms for human consumption), and / or socially important to 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.

[0019] 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 than influenza B virus. Similarly, influenza A H1N1 is a different type of virus than 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" based on antigenic differences in two viral transmembrane proteins, hemagglutinin (HA) and neuraminidase (NA). Two closely related lineages of type B virus (B / Victoria and B / Yamagata) are examples of influenza B viruses circulating in humans.

[0020] The term "vaccine" is used herein to refer to a composition that, when administered to a subject animal, elicits or enhances immunity against a particular disease. In some embodiments, the vaccine includes a pharmaceutically acceptable adjuvant and / or a pharmaceutically acceptable carrier. As used herein, the term "vRNA" refers to RNA that includes the viral genome, which includes segmented or non-segmented viral genomes, both plus-strand and minus-strand viral genomes. The vRNA may be entirely endogenous and "wild-type" and / or may contain recombinant and / or mutant sequences.

[0021] As used herein, "BM2SR-1" refers to SEQ ID NO:1, a virus comprising SEQ ID NO:1, or a vaccine comprising a virus comprising SEQ ID NO:1, depending on the context in which it is used. For example, when describing the mutations of the BM2 gene shown in this specification, "BM2SR-1" refers to SEQ ID NO:1. As used herein, "BM2SR-2" refers to SEQ ID NO:2, a virus comprising SEQ ID NO:2, or a vaccine comprising a virus comprising SEQ ID NO:2, depending on the context in which it is used. For example, when describing the mutations of the BM2 gene shown in this specification, "BM2SR-2" refers to SEQ ID NO:2. As used herein, "BM2SR-3" refers to SEQ ID NO:3, a virus comprising SEQ ID NO:3, or a vaccine comprising a virus comprising SEQ ID NO:3, depending on the context in which it is used. For example, when describing the mutations of the BM2 gene shown in this specification, "BM2SR-3" refers to SEQ ID NO:3. As used herein, "BM2SR-4" refers to SEQ ID NO:4, a virus comprising SEQ ID NO:4, or a vaccine comprising a virus comprising SEQ ID NO:4, depending on the context in which it is used. For example, when describing the mutations of the BM2 gene shown in this specification, "BM2SR-4" refers to SEQ ID NO:4. As used herein, "BM2SR-5" refers to SEQ ID NO:5, a virus comprising SEQ ID NO:5, or a vaccine comprising a virus comprising SEQ ID NO:5, depending on the context in which it is used. For example, when describing the mutations of the BM2 gene shown in this specification, "BM2SR-5" refers to SEQ ID NO:5. As used herein, "BM2SR-0" refers to SEQ ID NO:6, a virus comprising SEQ ID NO:6, or a vaccine comprising a virus comprising SEQ ID NO:6, depending on the context in which it is used. For example, when describing the mutations of the BM2 gene shown in this specification, "BM2SR-0" refers to SEQ ID NO:6. Unless otherwise specified, "BM2SR" refers to BM2SR-0.

[0022] Influenza B virus A. General Introduction Influenza is a major cause of death in adult Americans. The causative agent of influenza is the Orthomyxoviridae family (Orthomyxoviridae ) is a virus, including influenza A virus, influenza B virus, and influenza C virus, and influenza B circulates almost exclusively in humans. Influenza B virus is 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 the 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 the influenza B virus. The total genome size is approximately 14,500 bases. The characteristic of genome fragmentation enables 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 channel) by a dicistronic mRNA (whose translation method is unique). The BM2 start codon overlaps with the BM1 stop codon (TAATG, a termination-start pentanucleotide motif). The BM2 protein is translated by this termination-start translation mechanism, unlike the M2 protein of the 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.

[0023] Both influenza A and B have antigenically evolved over time by the process of antigenic drift. In antigenic drift, 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, 16 different H antigens are known (H1 to H16), and 9 different N antigens (N1 to N9) are known. Each virus subtype has mutated into a variety of strains with various pathogenicity profiles. Some are pathogenic for one species but not for others, and some are pathogenic for 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 to date); and H9N2, H7N2, H7N3, H5N2, H10N7. Influenza B is not divided into subtypes, but there are two antigenically and genetically distinct lineages, and strains B / Victoria / 2 / 87 and B / Yamagata / 16 / 88, later named Victoria and Yamagata, are currently circulating in humans. 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.

[0024] B. Life Cycle and Structure The life cycle of influenza virus generally includes attachment to cell surface receptors, entry into cells and uncoating of viral nucleic acid, followed by replication of viral genes within cells. After synthesis of new copies of viral proteins and genes, these components are assembled into progeny virus particles, which subsequently exit the cell. Various viral proteins play roles in each of these processes. Influenza B particles are composed of a lipid envelope that encapsulates the viral core. The inside of the envelope is lined with the matrix protein (M1), while the outer surface is characterized by two types of glycoprotein spikes (hemagglutinin (HA) and neuraminidase (NA)). BM2 (transmembrane ion channel protein) is also part of the lipid envelope (see, for example, Figure 1).

[0025] 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 surface of host cells. This protein is also required for fusion between the viral membrane and the host cell membrane and subsequent internalization of virions by endocytosis. Neuraminidase (NA) (tetrameric type II membrane protein) is a sialidase that 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 surface of host cells and for prevention of aggregation of progeny by the binding activity of viral HA to other glycoproteins. Thus, the receptor-binding activity of HA and the receptor-destroying activity of NA probably act antagonistically to enable efficient replication of influenza.

[0026] Genome segments are packaged in the core of virus particles. RNP (RNA + nucleoprotein (NP)) exists as a helix with three viral polymerase polypeptides bound to each segment. The life cycle of the 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 (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-encloses 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.

[0027] C. Role of BM2 Protein As described above, three proteins (hemagglutinin (HA), neuraminidase (NA), and BM2) span the viral membrane. For influenza A, there is significant variability in the extracellular domains (ectodomains) of HA and NA, whereas the ectodomain of M2 is essentially invariant among influenza A viruses. 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 has undergone endocytosis, the virion-bound M2 ion channel (a 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 due to the 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 for the influenza virus life cycle because amantadine hydrochloride (which blocks M2 ion channel activity) has been shown to inhibit viral replication. The functional counterpart of influenza A virus M2 protein in influenza B virus is a type III transmembrane protein known as BM2. The mechanism for its translation requires a concatenation termination / restart event, described below.

[0028] III. BM2 Virus Mutants In one aspect, influenza B viruses retaining a mutant BM2 vRNA sequence are disclosed. Typically, such mutants lack BM2 ion channel activity, exhibit attenuated growth properties 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 a vaccine, provide protection against infection by the corresponding wild-type and / or other pathogenic viruses. In addition, the BM2 mutants disclosed herein are stable and do not mutate to express a functional BM2 polypeptide, regardless of the host cell used. Additionally or alternatively, in some embodiments, the BM1 proteins of these mutants are produced without a detectable change in their function. In some embodiments, a virus retaining a mutant BM2 nucleic acid sequence is unable to replicate in a host cell (wherein a wild-type virus would be able to grow). By way of example and not limitation, in some embodiments, a wild-type virus can grow, propagate, and replicate in cultured MDCK cells, CHO cells, and / or Vero cells, while the corresponding virus retaining the mutant BM2 sequence is unable to grow, replicate, or propagate beyond one cycle in the same type of cells.

[0029] As noted above, in some embodiments, the BM2 mutant virus is stable and does not mutate or revert to a non-wild-type sequence encoding a wild-type or functional BM2 protein in a host cell. For example, in some embodiments, the BM2 mutant virus is stable in a host cell 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 cell is an unmodified host cell. In other embodiments, the host cell is a modified host cell, e.g., an MDCK cell that expresses the BM2 protein (i.e., a BM2CK cell). In some embodiments, the BM2 variant comprises one or more nucleic acid substitutions and / or deletions. In some embodiments, the mutation localizes to a nucleic acid encoding one or more of the extracellular domain of the BM2 protein, the transmembrane domain of the BM2 protein, and / or the cytoplasmic tail of the BM2 protein. Additionally or alternatively, in some embodiments, one or more nucleic acid mutations result in one or more stop codons and / or one or more amino acid deletions of the BM2 peptide. In some embodiments, a virus having a mutant BM2 nucleic acid produces a non-functional BM2 polypeptide. In some embodiments, a virus having a mutant BM2 nucleic acid does not produce a BM2 polypeptide. In some embodiments, a virus having a mutant BM2 nucleic acid produces a truncated BM2 polypeptide.

[0030] As noted above, influenza B genomic segment 7 expresses two major polypeptides (BM1 matrix protein and BM2 proton channel) that are 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 M1 translation termination 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 BM2 protein is synthesized by a coupled translation termination - re - initiation mechanism of overlapping stop - start pentanucleotides in a bicistronic mRNA transcribed from RNA segment 7. The mRNA transcribed from RNA segment 7 contains a pentanucleotide (residues 769±773) where the AUG start codon for the BM2 protein overlaps the stop codon for the M1 protein (Horvath et al., EMBO Journal 9:2639 - 2647, 1990). The BM2 protein is synthesized by the coupled translation termination±initiation mechanism of pentanucleotides, which is influenced by the initiation and termination of the upstream M1 protein (Horvath et al., 1990). This process requires the proximity of the stop and re - start codons and a defined region of the mRNA upstream of the stop - start site (including a functionally essential stretch of bases complementary to helix 26 of 18S rRNA). This complementary region is located within the loop of a hairpin structure adjacent to the terminating ribosome and may play a role in the re - initiation process of BM2 protein expression. This secondary structure may have an additional role in the stabilization of segment 7 mRNA, and thus BM2 mutants that disrupt or affect the hairpin structure may decrease M1 protein expression in addition to the desired lack of BM2 expression.

[0031] Another region required for the replication and packaging of all eight genomic segments is the 5’ non-coding region (5’NCR) of the genomic RNA. Since influenza is a negative-strand RNA virus, the 5’NCR of the genome is the 3’NCR of the mRNA and cDNA. Furthermore, the structural requirements of the 5’NCR can also extend to the protein ORF itself. This has been shown for influenza A. That is, protein-coding silent mutations near the 3’ end of the mRNA of segment 4 (HA) (or near the genomic 5’NCR) (the codon changes but the amino acid composition of HA does not change) can completely block virus replication. Therefore, the internal region within the COOH terminus of M2 may be important for structure and efficient genomic packaging into virions. The requirement for BM2 in virus replication was revealed by constructing an influenza B virus with an exact deletion in the 109-amino acid BM2 ORF. This virus can replicate in M2CK cells (MDCK cells that constitutively express the BM2 protein). This MD deletion construct allows replication in MDCK lineage cells, while an overall deletion removes regulatory element portions, which could detrimentally affect the expression of the M1 protein, especially when restricted by other factors that can affect virus production. For example, influenza B virus grows to high titers in MDCK cells, while those viruses are highly restricted in Vero cells, in part due to low expression of the M1 protein (Nakamura, 1981). Thus, manipulation of the M segment may inadvertently have further effects on M1 expression. To address these drawbacks, a series of five novel BM2 null mutant constructs (BM2SR-1, BM2SR-2, BM2SR-3, BM2SR-4, and BM2SR-5) (Figure 2) based on segment 7 of B / Florida / 4 / 2006 were designed and subsequently synthesized as double-stranded DNA fragments. The fragments are suitable for standard in vitro gene assembly using standard techniques known in the art. The single-stranded mRNA or plus-sense DNA nucleotide sequences of the constructs are provided in Table 1: BM2SR-1 (SEQ ID NO: 1), BM2SR-2 (SEQ ID NO: 2), BM2SR-3 (SEQ ID NO: 3), BM2SR-4 (SEQ ID NO: 4), BM2SR-5 (SEQ ID NO: 5), and BM2SR-0 (SEQ ID NO: 6).

[0032]

Table 1-1

[0033]

Table 1-2

[0034]

Table 1-3

[0035]

Table 1-4

[0036]

Table 1-5

[0037] The BM2SR-0, BM2SR-1, and BM2SR-3 variants do not express the BM2 polypeptide. The BM2SR-2, BM2SR-4, and BM2SR-5 variants potentially could express a truncated polypeptide containing the first 11 amino acids of the BM2 protein, although this was not clearly shown. Table 2 provides the wild-type influenza B segment 7 showing the BM1 and BM2 coding sequences and the pentanucleotide motif in bold underline.

[0038] [Table 2]

[0039] Table 3 provides the amino acid sequences of wild-type BM1 and BM2 and the amino acid sequence of the BM1 M86V variant.

[0040] [Table 3-1]

[0041] [Table 3-2]

[0042] The first construct (referred to as BM2SR-1 (SEQ ID NO: 1)) is a complete deletion of the BM2 ORF as previously reported by Hatta et al. (Hatta, et al., J. Virol. 83(11): 5939-5942, 2009). The foregoing is identical to BM2SR-0 (SEQ ID NO: 6) but encodes a modern protein rather than the BM1 of B / Lee / 1940. Two antigenically and genetically distinct lineages of influenza B virus have co-circulated and caused disease in humans since at least 1988. The Victoria lineage of influenza virus was the dominant B strain circulating globally in the 1980s, and the Yamagata lineage became the dominant B virus in the early 1990s. Since 1991, viruses of the Victoria lineage have often been isolated and are almost entirely restricted to East Asia. The Victoria virus re-emerged in 2002, and both the Yamagata and Victoria lineages have coexisted since then. 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 to B / Lee / 40. The phylogenetic relationships are shown in FIGS. 17A and 17B.

[0043] The BM2SR-2 construct (SEQ ID NO: 2) restores the termination-initiation site of the BM1-BM2 pentanucleotide and the surrounding environment of the native 3'-sequence to improve BM1 expression and regulation. One 5-nucleotide sequence (TAATG) in the FluB mRNA encodes a ribosomal slippage sequence. The foregoing is referred to as the termination-initiation site and includes the stop codon TAA of the upstream BM1 ORF and the start codon ATG of the downstream frameshifted BM2 ORF. The surrounding environment of the native RNA sequences both 5' to 3' of this sequence contains elements that regulate the translation of BM1 protein expression. Restoration of the pentanucleotide requires restoration of the BM2 start codon and restoration in the native surrounding environment. Thus, translation is expected to re-initiate at the BM2 ATG site. An artificial sequence was inserted into BM2SR-2 to block the expression of all sequences 3' of this region. The foregoing encodes three tandem stop codons, one inserted into each potential translation open reading frame downstream.

[0044] The BM2SR-3 construct (SEQ ID NO: 3) is identical to BM2SR-2 but has one single mutation. The single mutation incorporates valine instead of methionine, which is unconditionally conserved at amino acid 86 of the influenza B M1 gene (M1 M86V). The M86V substitution is known to improve the growth of wild-type influenza B strains in Vero cells (N. Wressnigg et al., Vaccine 27:2851-2857, 2009). The BM2SR-4 construct (SEQ ID NO: 4) has a slightly smaller deletion of M2 of 90 bp, which is based on the observation that changes in the segment size of genomic RNA can have a negative impact on the stability of viral segments. This is due to sequences near the 5’ end of genomic RNA (present within the coding region of viral proteins) (Hatta et al., 2009). This would probably be the case for the M2 ORF located proximal to the 5' end of genomic segment 7 (3’ end of mRNA). Segment instability can increase the number of defective viral particles lacking a certain segment, causing a decrease in virus growth and a reduction in virus titer (Hutchinson et al., J.Virol.82:11869-11879, 2008). The 90 bp deletion in BM2SR-4 is less than 8% of the natural full length (1190 bp) of segment 7. The BM2SR-5 construct (SEQ ID NO: 5) is all of the following combinations of improvements to segment 7: a smaller deletion in M2 for segment stability, M1 translational regulation for M1 expression, and the M1 M86V mutation to improve virus titer and growth on a Vero cell substrate. The BM2SR-1 and BM2SR-3 mutants do not express the BM2 polypeptide. The BM2SR-2, BM2SR-4, and BM2SR-5 mutants may potentially express a truncated polypeptide containing the first 11 amino acids of the BM2 protein, however, said peptide has not yet been presented.

[0045] IV. Cellular Virus Production System A. Generation of "First Generation" Mutant Viruses and Virus Reverse Genetics Mutant viruses, such as those having a mutant BM2 nucleic acid, can be generated 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). Briefly, one or more plasmids encoding eight viral RNAs (corresponding to the eight influenza B segments) are transfected into a eukaryotic host cell. 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 BM2 protein contains the mutant BM2 nucleic acid sequence. In addition, the host cell is transfected with one or more expression plasmids encoding viral proteins (such as polymerase, nucleoprotein, and structural proteins) (including the wild-type BM2 protein). Transfection of the host cell with the viral RNA plasmid results in the synthesis of all eight influenza viral RNAs, one of which has the mutant BM2 sequence. The cotransfected 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 functional BM2 polypeptide with the mutant BM2 nucleic acid sequence but still incorporated into the viral lipid envelope.

[0046] For example, in the BM2SR mutants based on segment 7 of B / FL / 04 / 2006 and B / Lee / 1940 control, the BM2 deletion segment 7 was inserted into a standard influenza reverse genetics plasmid vector and used for the generation of vaccine candidate influenza B viruses having HA and NA derived from segments 4 and 6 of modern Victoria and Yamagata lineages. Segments 1, 2, 3, 5, and 8 encoding internal gene products (including but not limited to PB1, PB2, PA, NP, NS1, and NS2) were derived from B / Lee / 40. The recombinant virus was generated by chemical-mediated transfection into 293T cells. Recovery of the 293T-produced BM2-incomplete mutant virus was performed in Madin-Darby canine kidney (MDCK) cells that constitutively express the BM2 protein (i.e., BM2CK cells). Twelve BM2-incomplete influenza B BM2SR mutant viruses described in Table 4 were constructed. B / Lee / 40-0, B / FL / 04 / 2006-1, B / FL / 04 / 2006-2, B / FL / 04 / 2006-3, B / FL / 04 / 2006-4, and B / FL / 04 / 2006-5 shown in Table 4 correspond to BM2SR-0, BM2SR-1, BM2SR-2, BM2SR-3, BM2SR-4, and BM2SR-5, respectively.

[0047]

Table 4

[0048] 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 with in vitro-generated recombinant RNA molecules. The system was described by Enami and Palese (Enami and Palese, J. Virol. 65(5):2711-2713, 1991). As shown by Luytjes et al., viral RNA is synthesized in vitro, and the RNA transcript is coated with viral nucleoprotein (NP) and polymerase protein that function as biologically active RNP in transfected cells (Luytjes et al., Cell 59:1107-1113, 1989). 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 capsid encapsulation: The transcribed RNA is subsequently mixed with gradient-purified NP and polymerase protein (isolated from disrupted influenza virus) to form a biologically active RNP complex; 3) Transfection and rescue of the capsid-encapsulated RNA: The artificial ribonucleocapsid is 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 the transfected gene: Since both the helper virus and the transfectant containing the rescued gene are present in the culture supernatant, an appropriate selection system using antibodies is necessary 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 positive or negative selection.

[0049] In addition or alternatively, the same antibody can be used to 'capture' the helper virus and concentrate the transfectant. For example, the antibody can be used to coat the bottom of a tissue culture dish or to concentrate the transfectant in the supernatant or eluate using a column matrix. The transfectant virus can be identified and cloned by growth in BM2-expressing cells in a multi-well plate by limiting dilution, followed by replica plating. For example, 1 / 2 aliquots of a given well of a multi-well plate containing the growing virus can be used to infect MDCK cells and the other half to infect BM2 protein-expressing MDCK cells (i.e., BMCK cells). Both the transfectant virus and the helper virus will grow in BM2 protein-expressing MDCK cells. However, only the helper virus will grow in standard MDCK cells, allowing identification of the wells of the multi-well plate containing the transfectant. The transfectant virus can further be plaque purified in cells expressing the BM2 protein.

[0050] B. Propagating Virus Mutants 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 growth of influenza virus variants (e.g., influenza B virus variants) include, but are not limited to, a number of eukaryotic cells such as the following: Madin-Darby canine kidney cells (MDCK cells), 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). In addition or alternatively, in some embodiments, eukaryotic host cells are modified to enhance virus production, for example, by enhancing viral infection of the host cells and / or by 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 B virus. See, for example, U.S. Patent Publication No. 2010-0021499 and U.S. Patent No. 7,176,021. Thus, in some exemplary embodiments, Chinese hamster ovary cells (CHO cells) and / or Vero cells modified to express at least one copy of the 2,6-sialyltransferase gene (ST6GAL1) are used. 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 incorporated 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.

[0051] 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, thereby providing the gene to the virus in a trans configuration. For example, a viral strain having a mutant BM2 protein can exhibit an enhanced growth rate (e.g., greater virus production) when passaged in a host cell that produces the wild-type BM2 protein. In some embodiments, a viral strain having a mutant BM2 protein cannot grow or replicate in a cell that does not express the wild-type BM2 gene. Additionally, such host cells can slow or prevent viral reversion to a functional BM2 sequence. This is 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, a BM2 expression vector can be made by placing the following BM2 nucleic acid sequence (BM2 ORF sequence; i.e., from the start codon to the stop codon of “wild-type” BM2 (Table 5)) into a eukaryotic cell expression vector.

[0052]

Table 5-1

[0053]

Table 5-2

[0054] Subsequently, host cells (e.g., MDCK cells, Vero 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 BM2 expression by co-transfection with a detectable or selectable marker (e.g., hygromycin resistance) and / or by screening, for example, by indirect immunostaining using a BM2 antibody. BM2 expression can be determined by indirect immunostaining, flow cytometry, or ELISA. In some embodiments, cells and viral variants are cultured and grown by methods well known in the art. By way of non-limiting example, in some embodiments, host cells are grown in the presence of MEM supplemented with 10% fetal bovine serum. Cells expressing 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.

[0055] Disposable bioreactors have been developed for mammalian cells (with or without virus included) along with these strains. The advantages described above include faster equipment setup and reduced risk of cross-contamination. The cells described herein can be, for example, disposable bags (e.g., bags from Stedim, Bioeaze bags (SAFC Biosciences), HybridBag TM(Cellexus Biosystems), or a single-use bioreactor (HyClone), or a Celltainer (Lonza). The bioreactor can be of sizes 1L, 10L, 50L, 250L, 1000L. In some embodiments, the cells are maintained as a suspension in an optimized serum-free medium without animal products. The system can be a fed-batch system that can expand the culture in a single bag, for example, from 1L to 10L, or a perfusion system that allows a steady supply of nutrients while simultaneously avoiding the accumulation of potentially harmful by-products in the culture broth. For long-term storage, the mutant virus can be stored as a frozen stock.

[0056] V. BM2-Expressing Cell Lines A. Propagation of M2-Deficient Influenza A and BM2-Deficient Influenza B Strains on BM2-Expressing Cell Substrates As described above, BM2 is an essential membrane protein incorporated into the envelope of influenza B virions. BM2 has proton (H + ) ion channel activity, as shown by electrophysiological studies (Mould, et al., Dev.Cell 5:175-184, 2003). The influenza M2 protein is required for replication in both influenza A and influenza B viruses (Watanabe, S., et al.J.Virol.83:5947-5950, 2009). Deletion or mutation of the open reading frame (ORF) encoding the M2 ion channel within influenza A or influenza B genomic RNA segment 7 causes viral maturation defects that prevent the release of progeny particles from all infected cells.

[0057] Mutations that inactivate M2 can be complemented in trans by the M2 protein encoded within the genome of cell lines engineered to stably express influenza A or influenza B M2. Expression of the cDNAs for wild-type A / PR / 8 / 1934 M2 (SEQ ID NO: 23) and B / Lee / 40 BM2 (SEQ ID NO: 24) permits replication of M2-incomplete mutant A viruses (M2SR) and BM2-incomplete mutant B viruses (BM2SR). For example, this has been demonstrated in a modified Madin-Darby canine kidney (MDCK) cell line known as M2CK (FluA) cells, and in BM2CK (FluB) cells that utilize the strong synthetic CAG promoter, which can drive constitutive expression of high levels of A / PR / 8 M2 and B / Lee / 40 BM2 proteins, respectively (Miyazaki, J; Takaki, S; Araki, K; Tashiro, F; Tominaga, A; Takatsu, K; Yamamura, K (July 15, 1989); "Expression vector system based on the chicken beta-actin promoter directs efficient production of interleukin-5.". Gene. 79 (2): 269-77). To determine whether influenza A M2 and influenza B M2 proteins can complement M2-incomplete influenza A and / or BM2-incomplete B viruses, a standard 50% tissue culture infective dose (TCID 50 ) assay (WHO) can be used according to methods known in the art.

[0058] Accordingly, in some embodiments, the present disclosure provides a BM2CK cell line that supports the growth of both BM2-incomplete influenza B virus (i.e., BM2SR virus) and M2-incomplete influenza A virus (i.e., M2SR virus). In some embodiments, influenza A mutants with incomplete M2 are grown in BM2CK cells in which BM2 is complemented in trans. In some embodiments, the BM2CK cells described herein complement and support the growth of influenza A M2-incomplete M2SR virus strains. In some embodiments, influenza B mutants with incomplete BM2 are grown in M2CK cells in which M2 is complemented in trans. In some embodiments, the M2CK cells described herein complement and support the growth of influenza B M2-incomplete BM2SR virus strains.

[0059] B. Chimeric M2 Protein Rescue of influenza B BM2-incomplete BM2SR virus by reverse genetics (RG) requires that the BM2 protein be supplied in trans within the recipient cell substrate. However, 293T cells, which are most frequently used for high-efficiency RG, lack BM2 expression. Thus, BM2SR virus is typically generated in 293T by supplying a 13th plasmid encoding the BM2 protein along with the standard 12-plasmid influenza RG system encoding the replicase (4) and genomic RNA (8) as previously described. See, for example: Hatta & Kawaoka, J. Virol. 77:6050-6054, 2003; Neumann et al., Proc. Natl. Acad. Sci. USA 96:9345-9350, 1999.

[0060] The M2 protein can be divided into the following three domains: 1) extracellular or ectodomain; 2) transmembrane (TM) domain; and 3) intracellular cytoplasmic or endodomain (Figure 10). The extracellular domain of influenza A M2 is approximately 24 to 27 amino acids in length and has a known structure, encoding the 14C2 epitope compared to only 6 to 9 residues of the BM2 domain (it is unclear whether the domain is antigenic or has a well-defined structure (Cross, Nature Structural & Molecular Biology 16:1207-1209, 2009)). The first 9 amino acids of the influenza A M2 extracellular domain are identical to the N-terminus of the M1 protein as they are encoded by a shared exon that is alternatively spliced to form M2. Such an arrangement does not exist in influenza B, and thus BM1 and BM2 do not share protein sequence identity.

[0061] The transmembrane (TM) domains of both influenza A and B encode pH-dependent proton channels that are approximately 22-24 residues in length, the channels contain a canonical 19-amino acid structure and have histidine and tryptophan residues in the same spacing as that shown to be required for channel function. The TM domain of influenza A M2 is the target of amantadine (a therapeutic channel inhibitor), but BM2 is not. The endodomain of BM2 is significantly longer at 81 to 83 amino acids compared to approximately 47 to 51 of influenza A M2. Both endodomains are highly hydrophilic and have spatially separated highly charged acidic and basic regions. In fact, the BM2 endodomain has one of the largest dipole moments reported for a protein to date.

[0062] In some embodiments, the present disclosure provides chimeric fusions of three domains derived from influenza A M2 and influenza B BM2 arranged in various permutations as shown in FIG. 10. In some embodiments, the influenza A TM domain can be split into a subdomain encoding the amantadine-binding domain closer to the cell surface and the remainder of the TM domain. The domain structures of these variants are provided in FIG. 10. In some embodiments, two amino acid substitutions (S12L and H19C, each of which has been shown to abrogate BM2 H + -channel activity) are combined to provide a BM2 channel-null (BM2-null; BBB-null) variant.

[0063] The wild-type and chimeric amino acid sequences of influenza A / PR / 8 M2, influenza B / Lee / 40 BM2, and their chimeric fusion proteins are provided in Table 6.

[0064]

Table 6-1

[0065]

Table 6-2

[0066]

Table 6-3

[0067] In some embodiments, the present disclosure provides codon-optimized M2, BM2, and chimeric M2 proteins. The influenza A / PR / 8 M2, influenza B / Lee / 40 BM2, codon-optimized influenza A / PR8 / M2, codon-optimized influenza B / Lee / 40 BM2, and their codon-optimized chimeric proteins are provided in Table 7.

[0068]

Table 7-1

[0069]

Table 7-2

[0070]

Table 7-3

[0071]

Table 7-4

[0072] As described herein, in some embodiments, Vero cells are modified to produce: wild-type A M2 protein (SEQ ID NO: 14), wild-type B M2 protein (BBB (SEQ ID NO: 15)), or B M2 chimeric proteins (e.g., ABB (SEQ ID NO: 17), AAB (SEQ ID NO: 18), AA’B (SEQ ID NO: 19), BAB (SEQ ID NO: 20), BBA (SEQ ID NO: 21), and BA’B (SEQ ID NO: 22)). Methods for making expression plasmids and modified host cells (e.g., modified Vero cells) are both well known in the art. For example, a chimeric M2 fusion protein expression plasmid can be made by placing the nucleic acid sequence of the M2 fusion protein (e.g., codon-optimized BBA (SEQ ID NO: 32)) into a eukaryotic expression plasmid.

[0073] Subsequently, it can be transfected into Vero cells 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 BM2 expression by co-transfection with a detectable or selectable marker (e.g., hygromycin resistance) and / or by screening, for example, by indirect immunostaining using a BM2 antibody. BM2 expression can be determined by indirect immunostaining, flow cytometry, or ELISA.

[0074] In some embodiments, for the production of M2-incomplete influenza A M2SR and BM2-incomplete BM2SR vaccines, the performance of Vero host cells can be improved by the use and expression of M2 proteins (including chimeric fusions of M2 genes from various serotypes, influenza A and influenza B). For example, in some embodiments, Vero cells expressing A M2 and BM2 chimeric proteins (e.g., ABB (SEQ ID NO: 17), AAB (SEQ ID NO: 18), BAB (SEQ ID NO: 20), BBA (SEQ ID NO: 21)) support the growth of influenza A M2SR variants. In some embodiments, Vero cells expressing wild-type BM2 protein (BBB (SEQ ID NO: 15)) support the growth of influenza A M2SR variants. In some embodiments, Vero cells expressing wild-type BM2 protein (BBB (SEQ ID NO: 15)) support the growth of influenza BM2SR variants.

[0075] C. BM2 Vero Cells Support the Propagation of Influenza A M2SR Mutants Due to the ability of MDCK cells to support the rapid and high-titer replication of influenza virus, these cells are typically used for influenza research and development. However, it is difficult to obtain regulatory approval for any biological products derived from MDCK cells because the cells themselves have been shown to be neoplastic / oncogenic. On the other hand, Vero cells have been approved since the 1960s for the production of cell-based vaccines (e.g., in polio and MMR vaccines).

[0076] In some embodiments, the present disclosure provides Vero cells that can be used as production cells for the production of M2-deficient influenza A viruses. In some embodiments, the Vero cell line comprises a codon-optimized wild-type influenza B / Lee / 40 BM2 nucleotide sequence (SEQ ID NO: 27) that expresses the BM2 protein. This Vero cell line is referred to herein as BM2Vero4. In some embodiments, the M2-deficient influenza A virus is A / California / 07 / 2009pdm H1N1 M2SR (A / CA / 07), which comprises the H1 and N1 segments of A / California / 07 / 2009pdm. The cDNA sequence of the influenza A M2 virus mutant is provided in Table 8 below. In some embodiments, BM2Vero4 cells generate influenza A H1N1 M2SR virus at a level substantially equivalent to that of MDCK cells that express the influenza A version of the M2 protein.

[0077]

Table 8

[0078] The M2 polypeptide sequence generated from this mutant is as follows: MSLLTEVETPIRNEWGCRCNGSSD (SEQ ID NO: 34).

[0079] D. M2 Content of Influenza A M2SR Virions Produced by BM2 Vero Cells In some embodiments, the present disclosure provides, for example, an influenza A M2SR comprising an influenza A M2SR backbone expressing HA and NA derived from A / Brisbane / 10 (SEQ ID NO: 33), which cannot express the influenza A M2 protein but contains the BM2 protein, and a method for generating said virus as shown in the following examples. In some embodiments, the BM2 Vero cells are BM2Vero4 cells that express the wild-type B / Lee / 40 BM2 protein from a codon-optimized cDNA (SEQ ID NO: 27).

[0080] VI. Vaccines and Administration Methods A. Immunogenic Compositions / Vaccines There are various different types of vaccines, which can be made from the cell-based virus production system disclosed herein. The present disclosure includes, but is not limited to: 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 thereof. Thus, there are a number of vaccines that can provide a protective immune response specific to various influenza viruses, and appropriate formulations of any of these vaccine types can provide 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 in complex with a carrier component, such as 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.

[0081] In some embodiments, a complete virion vaccine is provided. The complete virion vaccine is concentrated by ultrafiltration and subsequently purified by zonal centrifugation or by chromatography. Typically, the virions are inactivated before or after purification, for example using formalin or beta-propiolactone. In some embodiments, a subunit vaccine is provided, which comprises a purified glycoprotein. Such a vaccine can be prepared as follows: using a virus suspension fragmented by treatment with a detergent, the surface antigen is purified, for example by ultracentrifugation. Thus, the subunit vaccine mainly also contains the HA protein and the NA. The detergent used can be a cationic detergent (for example cetyltrimethylammonium bromide), an anionic detergent (for example ammonium deoxycholate), or a non-ionic detergent (for example those commercialized under the name TRITON X100). Hemagglutinin can also be isolated after treatment of the virions with a protease (for example bromelin) and subsequently purified by standard methods.

[0082] 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 may include whole virus (WV) vaccines or subvirion (SV) (split) vaccines. WV vaccines contain intact inactivated viruses, while SV vaccines contain purified viruses that are disrupted with a detergent that lyses the lipid-containing viral envelope and subsequently the remaining virus is chemically inactivated.

[0083] 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. In some embodiments, attenuation is achieved in a single step by transferring the attenuation gene of 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, the protein product is not produced from the mutant viral nucleic acid.

[0084] In some embodiments, the vaccine comprises a recombinant influenza virus lacking the expression of a functional BM2 protein. In some embodiments, the mutant virus replicates well in cells expressing the BM2 protein but does not replicate in the corresponding wild-type cells and expresses viral proteins without producing infectious progeny virions.

[0085] 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 (whether inactivated or not) is treated with a lipid solvent (such as ethyl ether or chloroform) together with a detergent while stirring. 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.

[0086] The pharmaceutical compositions of the present invention suitable for nasal administration, vaccination, or parenteral or oral administration contain attenuated or inactivated influenza virus and may optionally further contain sterile aqueous or non-aqueous solutions, suspensions and emulsions. The compositions may further contain 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.

[0087] In some embodiments, preparations for parenteral administration may include sterile aqueous or non-aqueous solutions, suspensions, and / or emulsions. The foregoing may include adjuvants or excipients known in the art. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). A carrier or occlusive dressing can be used to enhance skin permeability and antigen absorption. Generally, liquid dosage forms for oral administration can include liposome solutions 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 (such as purified water). 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.

[0088] When the compositions of the present invention are used for administration to an individual, the compositions can further include salts, buffers, adjuvants, or other substances desired to improve the effectiveness of the compositions. For immunogenic compositions or vaccines, adjuvants (substances that enhance specific immunity) can be used. Typically, the adjuvant and the composition are mixed prior to presentation to the immune system or are presented separately but at the same site of the organism to be immunized.

[0089] Thus, the virus can be attenuated or inactivated according to known methods, formulated, and further administered as an immunogenic composition (e.g., a vaccine) to induce an immune response in animals (e.g., birds and / or mammals). Methods for determining whether such attenuated or inactivated vaccines maintain antigenicity similar to that of clinical isolates or high-growth strains derived therefrom are well known in the art. Such known methods include the following: 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 of 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.

[0090] B. Administration The immunogenic compositions (e.g., vaccines) disclosed herein can be administered via either the routes customarily used or recommended for vaccines (parenteral route, mucosal route), 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). The vaccine can be administered using a syringe or needleless 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, lung, vagina, or rectum.

[0091] 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 recovered and administered to a recipient suspected of being infected by at least one influenza virus strain.

[0092] In some embodiments, the immunogenic compositions (e.g., vaccines) disclosed herein comprise a plurality of different types of viruses or viral antigens, at least one of which comprises a mutated BM2 gene (e.g., a virus comprising BM2SR-1 (SEQ ID NO: 1), BM2SR-2 (SEQ ID NO: 2), BM2SR-3 (SEQ ID NO: 3), BM2SR-4 (SEQ ID NO: 4), or BM2SR-5 (SEQ ID NO: 5)). In some embodiments, the vaccine comprises a virus comprising a BM2SR-1 (SEQ ID NO: 1), BM2SR-2 (SEQ ID NO: 2), BM2SR-3 (SEQ ID NO: 3), BM2SR-4 (SEQ ID NO: 4), or BM2SR-5 (SEQ ID NO: 5) mutation, together with other viral components and / or genes expressing other viral components. In some embodiments, the vaccine (e.g., a virus comprising a BM2SR-1 (SEQ ID NO: 1), BM2SR-2 (SEQ ID NO: 2), BM2SR-3 (SEQ ID NO: 3), BM2SR-4 (SEQ ID NO: 4), or BM2SR-5 (SEQ ID NO: 5) mutation) comprises genes from other virus strains (e.g., including but not limited to HA and NA genes from other virus strains). In some embodiments, the vaccine comprises HA and NA genes from, for example, the B / Florida / 04 / 2006, B / Brisbane / 60 / 2008, B / Wisconsin / 01 / 2010, or B / Lee / 1940 virus.

[0093] The present invention thus includes methods for preventing or attenuating 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 attenuate a disease if administration of the vaccine results in a total or partial attenuation of the signs or symptoms of the disease, or a total or partial immunity of an individual to the disease.

[0094] At least one inactivated or attenuated influenza virus of the present invention, or a composition thereof, can be administered by any means that achieves the intended purpose using the pharmaceutical composition described above. For example, administration of such a composition can be by various 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 perfusion gradually over time. In some embodiments, the immunogenic compositions disclosed herein are by intramuscular or subcutaneous application.

[0095] In some embodiments, a regimen for preventing, suppressing, or treating influenza virus-related lesions includes administration of 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 1 week to about 24 months, or any period range or period value therebetween. In some embodiments, the influenza vaccines disclosed herein are administered annually.

[0096] According to the present invention, an "effective amount" of a vaccine composition is an amount sufficient to achieve the desired biological effect. In some embodiments, it is understood that the effective dosage will depend on the age, sex, health status and weight of the recipient, if any, the type of concomitant therapy, 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 will be understood and determined by those skilled in the art. The dosage of a live attenuated virus vaccine for an adult mammalian (e.g., human) is about 10 1 -10 10 plaque forming units (PFU / kg) or any numerical range or value therebetween. In some embodiments, the dosage of a live attenuated virus vaccine for an adult mammalian (e.g., human) is about 10 2 -10 10 plaque forming units (PFU / kg) or any numerical range or value therebetween. In some embodiments, the dosage of a live attenuated virus vaccine for an adult mammalian (e.g., human) is about 10 3 -10 10 plaque forming units (PFU / kg) or any numerical range or value therebetween. In some embodiments, the dosage of a live attenuated virus vaccine for an adult mammalian (e.g., human) is about 10 4 -10 10 plaque forming units (PFU / kg) or any numerical range or value therebetween. In some embodiments, the dosage of a live attenuated virus vaccine for an adult mammalian (e.g., human) is about 10 5 -10 10 plaque forming units (PFU / kg) or any numerical range or value therebetween. In some embodiments, the dosage of a live attenuated virus vaccine for an adult mammalian (e.g., human) is about 10 6 -10 10 plaque forming units (PFU / kg) or any value range therewithin. In some embodiments, the dosage of a live attenuated virus vaccine for an adult mammalian (e.g., human) is about 10 7-10 10 It can be in plaque forming units (PFU / kg) or any range of values 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 numerical range or value therebetween. 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 numerical range or value therebetween. 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 numerical range or value therebetween. In some embodiments, the dosage of the attenuated virus vaccine for an adult mammal (e.g., a human) is 10 10 PFU / kg can be exceeded. The dosage of the inactivated vaccine can be in the range of about 0.1 to 200, such as 50 μg of hemagglutinin protein. However, the dosage is a safe and effective amount determined by conventional methods using existing vaccines as a starting point.

[0097] C. Intradermal Delivery Live flu vaccines are conventionally delivered intranasally, mimicking the natural route of infection and promoting a response similar to the immune response to natural viral infection. Alternatively, methods of intradermal delivery are disclosed herein. The methods require the use of a novel microneedle device and utilize the immunological advantages of intradermal delivery. In some embodiments, an attenuated virus (e.g., a BM2 virus mutant) is used in the vaccine composition for intradermal administration. In some embodiments, a BM2 virus mutant (which does not produce infectious progeny virus) is provided as the vaccine. Thus, the potential for recombination with wild-type circulating influenza virus is substantially eliminated.

[0098] In the embodiments disclosed herein, intradermal delivery administers a 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 a 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 pro-inflammatory signals that enhance the immune response to the antigen introduced from the skin. Furthermore, the skin immune system generates antigen-specific antibodies and cellular immune responses. Intradermal delivery enables dose reduction of the vaccine. That is, when delivered intradermally, it may be effective with a lower dose of antigen 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 less painful compared to intramuscular injection with a conventional needle and syringe.

[0099] Micro-needle 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 micro-needle devices are fabricated by machining a stainless steel sheet by wet etching (Trinity Brand Industries, Georgia; SS 304 (thickness 50 μm)). In some embodiments, the individual micro-needles 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 micro-needles 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 BM2 mutant virus described herein). To achieve higher concentrations, the coating solution may 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 micro-needles can be determined by immersing the micro-needles in 200 μL of phosphate buffered saline (PBS) for 5 minutes and assaying for the antigen by methods known in the art.

[0100] The pharmaceutical composition of the present invention may further or additionally contain at least one chemotherapeutic compound, for example, for gene therapy, an immunosuppressive agent, an anti-inflammatory agent or an immunostimulant, or an antiviral agent. The foregoing includes, but is not limited to: gamma globulin, amantadine, guanidine, hydroxybenzimidazole, interferon-α, interferon-β, interferon-γ, tumor necrosis factor-α, thiosemicarbazone, methisazone, rifampin, ribavirin, pyrimidine analog, purine analog, foscarnet, phosphonoacetic acid, acyclovir, dideoxynucleoside, protease inhibitor, or ganciclovir. The composition may 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.

[0101] In some embodiments, the microneedle device used is made mainly of first-cut pieces of propylene and stainless steel, which are assembled into one by simple snap-fitting and heat-sealing. In some embodiments, the device is completely self-contained and includes a vaccine, a pump mechanism, an activation mechanism, and a microneedle 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 microneedles are inserted into the skin to start the pump mechanism, which pressurizes the primary drug container. When the spring mechanism applies sufficient pressure to the vaccine reservoir, the vaccine begins to flow from the microneedle array into the skin. In some embodiments, delivery of the vaccine dose is completed within about 2 minutes after activation of the device. After the injection is complete, the device is gently removed from the skin.

[0102] In some embodiments, a method for intradermal administration of an immunogenic composition (e.g., a vaccine) using a microneedle device is provided. In some embodiments, the microneedle device includes a piercing mechanism and an immunogenic composition layer, and the foregoing includes a plurality of microneedles capable of piercing the skin and further enabling administration of the immunogenic composition intradermally. In some embodiments, the method includes a step of weakening the piercing mechanism. In some embodiments, the immunogenic composition (e.g., a vaccine) includes a virus encoding a mutated BM2 protein to be expressed or a BM2 protein not to be expressed, wherein the expressed mutated BM2 protein includes or consists of an amino acid sequence encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. In some embodiments, first, the microneedle array is disposed inside the device housing, and the microneedles are extended by a button of the device upon actuation of a lever, thereby enabling injection of the vaccine solution into the skin.

[0103] Any desired substance can be delivered using the delivery device described herein. 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 subject animal. As used herein, the term "drug" is intended to include any substance delivered to a subject animal for any therapeutic, prophylactic, or medicinal purpose (e.g., vaccines, pharmaceuticals, nutrients, nutraceuticals, etc.). In certain such embodiments, the drug delivery device is configured to deliver an influenza vaccine. The embodiments contemplated herein relate primarily to devices configured to deliver substances transdermally. In some embodiments, the device can be configured to directly deliver a substance to an organ other than the skin.

Examples

[0104] The following embodiments are provided by way of example only and not limitation. Those skilled in the art will readily recognize a variety of non-deterministic parameters that can be changed or modified to obtain essentially the same or similar results. These examples should not be construed as limiting the scope of the present technology as defined in the appended claims.

[0105] [Example 1] Generation of BM2 Virus Mutants A series of five novel BM2 null mutant constructs (BM2SR-1, BM2SR-2, BM2SR-3, BM2SR-4, and BM2SR-5) (Figure 2) based on segment 7 of B / Florida / 4 / 2006 were designed and subsequently synthesized as double-stranded DNA fragments. The fragments are suitable for standard in vitro gene assembly using standard techniques known in the art. The single-stranded mRNA or plus-sense DNA nucleotide sequences of BM2SR-1 (SEQ ID NO: 1), BM2SR-2 (SEQ ID NO: 2), BM2SR-3 (SEQ ID NO: 3), BM2SR-4 (SEQ ID NO: 4), and BM2SR-5 (SEQ ID NO: 5) are provided in Table 1.

[0106] [Example 2] Generation and Culture of BM2 Mutant Viruses This example shows the culture of BM2SR mutants (including BM2SR-1 (SEQ ID NO: 1), BM2SR-2 (SEQ ID NO: 2), BM2SR-3 (SEQ ID NO: 3), BM2SR-4 (SEQ ID NO: 4) or BM2SR-5 (SEQ ID NO: 5)) based on segment 7 of B / Florida / 04 / 2006, and the B / Lee / 1940 control, BM2SR mutants based on deleted segment 7 of BM2. Mutant viruses (e.g., those carrying mutant BM2 nucleic acids) can be generated by plasmid-based reverse genetics as described by Neumann et al. (Neumann et al., Proc. Natl. Acad. Sci. USA 96:9345-9350, 1999). Briefly, one or more plasmids encoding eight viral RNAs corresponding to each of the eight influenza B segments were transfected into 293T host cells. Each viral RNA sequence was flanked by an RNA polymerase I promoter and an RNA polymerase I terminator. In particular, the viral RNA encoding the BM2 protein contained the mutant BM2 nucleic acid sequence. In addition, one or more expression plasmids encoding viral proteins (e.g., polymerase, nucleoprotein and structural proteins) (including wild-type BM2 protein) were transfected into the host cells. The plasmids were mixed with a transfection reagent (2 μL of TransIT™ LT1 (Mirus Bio, Madison, WI) per 1 μg of DNA) and incubated at room temperature for 15 - 30 minutes, then added to 1 x 10 6 293T cells. After 48 hours, the virus in the supernatant was serially diluted and inoculated onto BM2CK cells. Two to four days after inoculation, for the production of stock virus, the supernatant virus from the well of the final dilution showing a clear cytopathic effect (CPE) was inoculated onto BM2CK cells. The sequence of the M gene of the resulting virus was determined to confirm the gene and the intended mutation, and to ensure that no unwanted mutations were present.

[0107] The mutant BM2 virus was propagated and passaged as follows. BM2CK host cells were grown in MEM supplemented with 10% fetal bovine serum. The cells were infected at an MOI of 0.001 by washing with PBS and then allowing the virus to adsorb at 37°C. Virus growth medium containing trypsin / TPCK was added, and the cells were incubated for 2 - 3 days until a cytopathic effect was observed. Twelve BM2 incomplete influenza B BM2SR mutant viruses (described in Table 4) were constructed. B / FL / 4 / 2006-1, B / FL / 4 / 2006-2, B / FL / 4 / 2006-3, B / FL / 4 / 2006-4, and B / FL / 4 / 2006-5 shown in Table 4 correspond to BM2SR-1, BM2SR-2, BM2SR-3, BM2SR-4, and BM2SR-5, respectively.

[0108] [Example 3] Expression of BM2SR Virus Protein This example shows the proteins expressed by the twelve BM2 incomplete influenza B BM2SR viruses described in Table 4. Vero cells (200,000) were cultured in wells of TC-12 plates for 24 hours in MEM medium supplemented with 10% fetal calf serum (FCS). After washing twice with D-PBS, the twelve constructed B / Florida / 4 / 2006 BM2SR-1 to BM2SR-5 and B / Lee / 40 BM2SR mutant viruses, together with two wild-type virus controls B / Lee / 1940 and B / Florida / 4 / 2006, were used to infect the Vero cells at a high multiplicity of infection of 1.0. After incubation at 35°C in a 5% CO2 atmosphere for 6 hours to allow one round of replication, samples were collected for Western analysis.

[0109] Infected cells were gently lysed in 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1% Triton X-100 containing protease inhibitor. Nuclei and insoluble debris were pelleted by centrifugation at 14,000 x G. The clarified supernatant was denatured and further reduced prior to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (4-12% gradient acrylamide gel in MES buffer (Invitrogen)). The degraded proteins were transferred to a polyvinylidene difluoride (PVDF) membrane for 7 minutes using a semi-dry apparatus. The PVDF was blocked in phosphate buffered saline with Tween 20 (PBS-T) containing 3% non-fat dry milk (NFDM). Proteins were detected in 1% NFDM PBS-T with antisera specific for two influenza B antigens HA and M1, and the cellular housekeeping protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Influenza B M1 was detected with a primary antibody cocktail of three 1000:1 diluted monoclonal antibodies (sc-101353, sc-101408, sc-101409; Santa Cruz Biotechnology) and a 2000:1 diluted anti-mouse IgG secondary antibody-horseradish peroxidase (HRP) conjugate. Influenza B hemagglutinin protein (HA) was detected with a 2000:1 goat anti-HA polyclonal primary serum (NR-3120, BEI) and a 2000:1 secondary anti-goat IgG antibody-HRP conjugate. Control GAPDH was detected with a polyclonal primary antiserum against GAPDH (NB100-56875, Novusbio) and a 2000:1 secondary anti-rabbit IgG-HRP conjugate. Specific bands were detected using the chromogenic HRP substrate 3,3',5,5'-tetramethylbenzidine (TMB).

[0110] The results presented in Figure 4 show that the expression of influenza B / Lee / 40 M1 and HA is lower than that of B / FL / 4 / 2006. Similarly, the B / Lee / 40 BM2SR virus expresses low levels or undetectable M1 and HA proteins in both the B / Brisbane / 60 / 2008 and B / WI / 01 / 2010 HA:NA backgrounds. The change of the BM2SR segment in BM2SR-1 from B / Lee / 1940 to B / Florida / 4 / 2006 seems to increase the protein amounts of both M1 and HA. No increase was observed with the BM2SR-2 mutation. The M86V mutation of BM2SR-3 has a very large effect of increasing both M1 and HA expression. The BM2SR-4 mutation also improves the expression compared to the B / Lee / 40 control. The combination of the last BM2SR-5 mutations also brings about a large improvement in M1 and HA expression compared to the complete deletion strain. These results also show that the disruption of BM2 expression in the schematic diagram of Figure 2 can disrupt BM1 protein expression.

[0111] [Example 4] BM2SR Virus Replication This example shows the growth kinetics of the 12 BM2 incomplete influenza B BM2SR viruses described in Table 4. The viral growth kinetics of the constructed influenza BM2SR virus was tested in BM2Vero (a Vero-derived cell line that continuously expresses the BM2 protein of B / Lee / 40). BM2Vero cells were cultured in a P-060 dish in MEM medium containing 10% fetal bovine serum (FCS) for 24 hours. 1 microgram / mL of tosylphenylalanyl chloromethyl ketone-treated trypsin (T-TPCK) was added, and viral infection with 12 viruses was performed in triplicate at an MOI of 0.001. Furthermore, T-TPCK was added once a day for 3 days (on days 1, 2, and 3 after infection) (total addition of 4 μg / mL). The infectants were incubated at 5% CO2 and 35°C. At 2-day intervals (on days 2, 4, 6, and 8 after infection), the infected Vero cell tissue culture supernatant containing secreted influenza B virus was aseptically sampled, and aliquots were cryopreserved at -80°C. After the growth curve was completed, the titer of the frozen aliquots was measured by determining the standard 50% tissue culture infective dose (TCID 50 ) using BM2CK cells (MDCK cells expressing the BM2 protein). The standard World Health Organization hemagglutinin assay (WHO HA assay) was used to determine the productive infection at a given dilution. The mean and standard deviation of the triplicate TCID 50 measurements of each virus at each time point were calculated. The results are provided in Figures 5A and 5B.

[0112] The growth curves of the six viruses expressing HA and NA of B / Brisbane / 60 / 2008 provided in Figure 5 showed that the nearly full-length mutants BM2SR-4 and especially the M1 M86V mutants BM2SR-3 and BM2SR-5 directed faster viral growth and reached higher final titers. The M1 M86V mutation appeared to improve viral growth in the BM2Vwro4 cell substrate when combined with the BM2SR-2 mutation in the BM2SR-3 mutant and the BM2SR-4 mutation in the BM2SR-5 mutant. The best mutant, BM2SR-5, had a titer of 7.50 log10±0.1 log10 TCID 50The final titer per equivalent / mL was reached. The B / FL / 04 2006 BM2SR-3, -4, -5 mutations confer an improvement of more than 2 log10 to 5 log10 compared to the TCID 50 value (2.42 log10 ± 0.39 log10 TCID 50 / equivalent / mL) of the B / Lee / 1940 BM2SR strain when combined with B / Brisbane / 60 HA and NA. The growth of influenza B strains with HA and NA of B / Wisconsin / 01 / 2010 was more uniform (Figure 5B). A subtle improvement of about 10^0.5 TCID 50 / mL was observed for the BM2SR-2, BM2SR-3, and BM2SR-4 mutants on day 4 of the growth curve. This difference was lost by day 6 and the final titers were substantially similar. Thus, in the context of viruses containing HA and NA of B / Wisconsin / 01 / 2010, the engineered BM2SR constructs (especially BM2SR-4 and BM2SR-5) could improve viral growth kinetics only slightly but showed a smaller effect on the final virus yield. The performance of a given virus depends on the composition of the HA and NA of the strain.

[0113] [Example 5] Stability of BM2SR Variants To test the stability of the BM2 gene of BM2SR variants in wild-type cells, the BM2SR variants were passaged in BM2CK cells (MDCK cells expressing BM2 protein) together with wild-type MDCK cells (lacking BM2 protein expression). All BM2SR variants and wild-type influenza B virus (WT B / WI01) could be passaged in BM2CK cells without any mutations occurring for at least 10 passages (Figure 6A). However, the BM2SR variants could not be passaged in wild-type MDCK cells (Figure 6B).

[0114] [Example 6] BM2 Mutant Viruses Are Attenuated in Vivo Experiments were conducted to show that the BM2SR mutant virus is attenuated in vivo. Six-week-old female BALB / c mice were inoculated intranasally with one of the following mutants: BM2SR Bris60 and BM2SR Wisc01 (both of which contain the mutant M segment derived from B / Lee / 40 shown in SEQ ID NO: 6 (BM2SR-0)). The mutants were administered at a dose of 1.2 x 10 6 TCID 50 / mouse. The control mouse group was administered PBS. These mice were observed for 14 days after inoculation for changes in body weight and signs of infection. As shown in Figure 7, the mice inoculated with the BM2SR virus and PBS showed no clinical signs of infection and no decrease in body weight over 14 days. The changes in body weight between the groups were similar over 14 days. Collectively, the absence of clinical signs and the absence of body weight loss indicate that the BM2SR mutant virus is attenuated and non-pathogenic in mice.

[0115] [Example 7] BM2 Mutants Induce Antibodies against Influenza B Virus Challenge Tests were conducted to determine the antibody titers against two influenza B lineages (Yamagata and Victoria) in the serum samples of the mice described above. Serum samples were collected on days 7, 14, and 21 after prime inoculation, and on days 35, 42, and 49 after a second immunization on day 28. The anti-HA IgG antibodies 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 Figures 8A-8B. The figures show that both BM2SR mutants (containing the M segment derived from B / Lee / 40 with a deletion in M2 (SEQ ID NO: 6; BM2SR-0)) raised anti-influenza virus antibodies higher than the PBS control group against both antigens. Mice boosted with the BM2SR mutant virus had higher levels of anti-influenza HA antibodies after the second immunization than after the prime inoculation.

[0116] [Example 8] Propagation of M2-Deficient Influenza A and BM2-Deficient Influenza B Strains on BM2-Expressing Cell Substrates Experiments were conducted to test the ability of influenza A M2 and influenza B BM2 to complement M2-incomplete influenza A (A / CA / 07 M2SR and A / Brisbane / 10 M2SR (both containing the M2 variant shown in SEQ ID NO: 33)) and B (B / WI / 01 BM2SR-0 and B / Brisbane / 60 BM2SR-0) viruses. A standard 50% tissue culture infectious dose (TCID 50 ) assay (WHO) was used. A confluent monolayer of M2CK and BM2CK cells prepared in 96-well tissue culture plates was infected in quadruplicate with 0.2 mL of 10-fold serial dilutions of four influenza viruses. Of the four viruses, two dilutions were prepared from influenza A M2SR and two from the influenza B BM2SR virus strain. After incubation for 4 days at 35°C in a 5% CO2 atmosphere, the culture supernatant was tested for influenza replication using the cytopathic effect and hemagglutination assay (HA) (WHO) at the endpoint. The log 10 TCID 50 values for each infection of the two cell lines by the four viruses shown in Figure 9 were determined using the highest dilution factor at which influenza replication was observed from each of the replication infectants.

[0117] Surprisingly, the BM2CK cell substrate was able to complement both strains of influenza A M2-incomplete M2SR, similar to the serotype-matched M2CK cells (Figure 9). M2SR containing surface antigens derived from both of the common HA NA subtypes of human influenza, H1N1 and H3N2, were both able to replicate with log 10 TCID 50 values comparable to about 7.0. In contrast, the influenza A M2 protein of M2CK cells was able to support only slightly the replication of the BM2-incomplete influenza B BM2SR strains tested, with a log 50 about 3 logs lower than the TCID 10A titer was generated. This indicates that the influenza B M2 protein can substitute for the influenza A protein and can assist in the replication of two subtypes of influenza A virus. In contrast, the influenza A M2 protein does not appear to be able to substitute for the BM2 protein for influenza B replication.

[0118] [Example 9] Chimeric M2 Protein To test the function of the chimeric M2 protein, the fusion construct was inserted into a standard plasmid by typical gene assembly procedures. The standard plasmid contains a CMV IE promoter and a bovine growth hormone polyadenylation transcriptional control sequence for high-level expression in cultured mammalian cells. Wild-type M2 and BM2 cDNA expression plasmids were also constructed. In addition, the wild-type BM2 cDNA sequence was inserted into a pCAGGS promoter terminator plasmid for the highest possible level of expression even exceeding CMV. Using the plasmid, M2 function was supplied in reverse genetics (RG) of the influenza B BM2SR virus. The efficiency of RG was estimated by inoculating 1% of the 293T-RG produced virus into recipient BMCK cells in 96 independent wells for the recovery of BM2 incomplete progeny. After incubation at 35°C and 5% CO2 for 4 days, the BM2CK culture supernatant was tested for virus replication by a standard hemagglutination assay (WHO HA). The percentage of wells that were positive for WHO HA is shown in Figure 11.

[0119] The BM2 null mutation did not support RG, confirming that M2 proton channel activity is required for influenza replication. Wild-type influenza A M2 also did not support replication. Chimeric M2 proteins, ABB and AA’B (including the influenza A M2 ectodomain (M2e)), hardly supported RG as did wild-type BM2 or the BA’B mutant (lacking M2e), suggesting that the influenza A ectodomain is inhibitory. Collectively, the data indicate that the influenza B ectodomain is required for influenza B replication, while the influenza A transmembrane (TM) domain can substitute for the TM of BM2. Compared to the standard CMV promoter, increased expression by the pCAGGS vector improved the performance of wild-type BM2 cDNA and also showed that the total expression level of BM2 can also be a limitation to RG.

[0120] [Example 10] Ability of Wild-Type and Chimeric M2 Proteins to Express BM2 Protein When expressed from the cellular substrate genome, the M2 protein was tested for its ability to support the growth of M2-incomplete and BM2-incomplete viruses. neo ) gene (conferring resistance to the aminoglycoside inhibitor of protein synthesis (G418)), plasmids encoding M2 and chimeric mutants were chemically transfected into Vero cells. After culturing for 2 days, the transfected cells were isolated and replated in medium containing G418. The cells were cultured for several weeks under G418 selection, and clones in which the plasmid DNA was randomly integrated into the cell line genome were selected. Some portions of the G418-resistant clones would also contain functional copies of the integrated M2 chimeric mutant transgene. Subsequently, these functional clones can be tested for their ability to assist in virus selection. Replication can be tested by a pooled selection of G418-resistant clones or by individual clones selected and isolated using standard limiting dilution cloning (LDC) procedures. Analysis of the clone pool provides measurements of construct performance within a few weeks, while LDC takes much longer. However, several rounds of LDC are required to isolate an appropriate cell substrate for vaccine production that is regulated for human use.

[0121] After several rounds of selection and LDC using both wild-type and mutant cDNA plasmids, no appropriate clones expressing high levels of BM2 were isolated (data not shown). Western analysis showed that the detected amount of BM2 decreased rapidly after each passage of the cultured cells. Replication data of M2SR and BM2SR viruses in the initial passage clone pool suggested that the construct has function but stable expression of the construct by the cell genome is not established in Vero cells. Analysis of the gene sequences encoded by influenza virus showed that they are 60% A-T and that the virus codon usage frequency bias is significantly different from that of the host Vero. Furthermore, multiple sequence motifs capable of stopping or suppressing transcription were identified within the native virus gene coding region. This phenomenon is common when genes from one organism are transferred to another organism but may be underestimated in influenza virus. Influenza virus must replicate in human cells, but the virus does not utilize the same codon usage frequency pattern as the host.

[0122] To enhance the expression of wild-type M2, BM2, and chimeric variants, codon optimization of the coding sequences was carried out to remove negative regulatory motifs, bring the cDNA to approximately 50% A-T, and further change the influenza genes to the codon bias of Vero cells. These constructs were tested by chemical-mediated transfection of Vero cells. Two days after transfection, transient expression of the protein was assayed. Total cell protein extracts were analyzed using polyclonal antiserum specific for BM2. The data in Figure 12 show that much higher protein expression was obtained from the engineered transgene than previously obtained in transient assays of the native sequence (the native one is not shown). Notably, the BBA variant could not be detected because the anti-BM2 antiserum was directed against the BM2 endodomain.

[0123] [Example 11] Testing the Ability of Codon-Optimized Chimeric M2 Protein to Support the Propagation of M2-Deficient and BM2-Deficient Viruses The ability of codon-optimized chimeric M2 protein to support the growth of M2-incomplete and BM2-incomplete viruses was tested. A plasmid encoding the codon-optimized M2 chimeric variant together with the neomycin phosphotransferase ( neo ) gene (which confers resistance to the aminoglycoside inhibitor of protein synthesis (G418)) was chemically transfected into Vero cells. After culturing for 2 days, the transfected cells were isolated and replated in medium containing G418. The cells were cultured under G418 for 2 weeks, and clones in which the plasmid DNA had been randomly integrated into the cell line genome were selected. Some portions of the G418-resistant clones will also contain functional copies of the integrated M2 chimeric variant transgene. The BM2 and chimeric transfected and bulk-selected clone pools were infected with influenza A / Brisbane / 10 M2SR (containing the M2 variant shown in SEQ ID NO: 33) and influenza B / Brisbane / 60 BM2SR-0 (containing the BM2 variant shown in SEQ ID NO: 6) M2-incomplete viruses at an MOI of 0.01. Six days after infection, standard TCID 50Using the procedure, the viral titer of the culture supernatant was measured. The data are presented in Figure 13.

[0124] All of the M2 clone pools tested supported the growth of influenza A M2SR in Vero cells. Only the wild-type BM2(BBB) clone supported the growth of influenza B BM2SR. Thus, all three BM2 domains appear to be specific for influenza B BM2SR replication in Vero cells. A clone pool expressing the BBA mutant lacking the influenza A M2e and transmembrane (TM) domains but containing the M2 endodomain was found to be optimal for supporting the growth of influenza A M2SR. The BM2 endodomain was less effective, suggesting that the M2 endodomain has evolved to be somewhat specific for influenza A. The ABB and AAB chimeric mutants presented poorer performance than the BAB and BBA constructs, indicating that the influenza A ectodomain can be inhibitory to virus growth (and also to influenza A itself). The inhibitory region containing M2e (but not limited to M2e) can be removed and a modified domain that enhances replication and titer can be added. Therefore, the performance of the cell substrate producing the M2-incomplete influenza A M2SR virus and the BM2-incomplete BM2SR vaccine virus can be improved by using and expressing an engineered M2 protein containing a chimeric fusion of M2 genes derived from the completely different serotypes influenza A and influenza B.

[0125] [Example 12] Propagation of M2-Deficient Influenza A / California / 07 / H1N1pdm M2SR on BM2-Expressing Vero Cell Substrates A / California / 07 / 2009pdm H1N1 M2SR (A / CA / 07) is an M2 defective influenza A virus containing the H1 and N1 segments of A / California / 07 / 2009pdm and includes the M2 variant shown in SEQ ID NO: 33. Using this vaccine candidate virus that is non-replicable in normal cells, two cell lines that constitutively express two M2 protein types were infected. The first M2 cell substrate is the standard cell line M2CK of the MDCK lineage, which expresses the A / PR8 M2 protein (the protein is encoded by the nucleotide sequence shown in SEQ ID NO: 23). MDCK cells are typically used in influenza research and development because they are known to support rapid replication to high titers of influenza virus in culture. Unfortunately, since MDCK cells have been shown to be carcinogenic themselves, it is difficult for any biologic derived from MDCK to obtain regulatory approval. The second cell line, BM2Vero4, contains the codon-optimized wild-type influenza B / Lee / 40 BM2 protein (encoded by the nucleotide sequence shown in SEQ ID NO: 27), which was derived from Vero (African green monkey kidney cell line). Vero cell substrates have been used since the 1960s for the production of approved cell line vaccines (polio and MMR vaccines).

[0126] Viral infection was carried out at two multiplicity of infection (MOI) levels (0.01 and 0.001) in minimum essential medium (MEM) supplemented with 0.3% BSA and 1.0 μg / mL of trypsin-TPCK. Aliquots were withdrawn daily from day 2 to day 6 post-inoculation. The concentration of viable influenza A virus contained in the cell culture supernatant samples was assayed using a standard TCID 50 assay and incubated at 35°C, 5% CO2 for 4 days with influenza A M2SR permissive M2CK cells and BM2Verp4 cells. The results are presented in Figure 14. As expected, influenza A / CA / 07 / 2009pdm M2SR replicated well in M2CK cells and reached peak titers rapidly by day 2. Titers in Vero lines took 5 - 6 days to reach peak. The titer of A / CA / 07 in M2CK cells was log 10 Compared to titers of 7.00 and 6.33 obtained in M2CK cells, in BM2Vero cells it grew to titers of log 10 5.67 and 6.00. This was observed despite BM2Vero4 being derived from Vero cells, which are much less permissive for influenza replication compared to MDCK cells. Thus, codon - optimized influenza B / Lee / 40 BM2 protein (encoded by the nucleotide sequence shown in SEQ ID NO: 27) in Vero cells achieves influenza A H1N1 M2SR virus production at levels almost equivalent to those of R & D quality MDCK cells expressing the influenza A form of the M2 protein.

[0127] [Example 13A] Influenza BM2 Protein Can Provide the Ion Channel Activity Required for Virus Uncoating to Chimeric Influenza A M2SR Viruses M2SR does not express the M2 protein and thus cannot generate progeny virions, but M2SR performs de novo protein synthesis and generates viral antigens in normal cells. This example shows that the influenza B M2 protein can provide the ion channel activity required for viral uncoating to a chimeric influenza A M2SR virus (i.e., influenza A M2SR generated in a complementing BM2 cell line yields BM2 that is incorporated into the viral membrane). To actually demonstrate the above, the influenza A M2SR Hong Kong / 4801 / 2014 (H3N2) virus was passaged 3 times in A M2 - expressing M2VeroA cells, and the chimeric M2SR Hong Kong / 4801 / 2014 (H3N2) was passaged 3 times in influenza B BM2 - expressing BM2Vero cells. Subsequently, human A549, canine MDCK, or African green monkey Vero cells were inoculated with M2SR or chimeric M2SR virus at an MOI of 4 and cultured in medium without trypsin to ensure that the virus completed only one life cycle.

[0128] Coarse cytoplasmic extracts of the cells were performed at 6, 9, and 12 hours post-infection (p.i.). The cells were lysed in 50 mM Tris HCl (pH = 8.0), 150 mM NaCl, 1% Triton X-100 and then centrifuged (15,000 x G). The soluble proteins in the supernatant were combined with LDS sample buffer (Thermo Fisher Scientific, Waltham, MA) and 5 mM TCEP and heated at 70 °C for 10 minutes to denature. The proteins were separated on a 4-12% denaturing Bis-Tris NuPAGE polyacrylamide gel (Thermo Fisher Scientific, Waltham, MA) in MES buffer and transferred to a PVDF membrane. A prestained protein molecular weight standard was run for size comparison. The membrane was blocked with 1% (w / v) non-fat dry milk and incubated with a 2000:1 diluted polyclonal goat primary antiserum NR-3134 against influenza virus matrix protein (BEI Resources, NIAID, NIH), followed by incubation with a 3000:1 diluted horseradish peroxidase-conjugated anti-goat IgG (H+L) secondary antibody (SeraCare KPL, Milford, MA). Bands were visualized using a TMB membrane peroxidase substrate (SeraCare KPL, Milford, MA). Polyclonal antisera against the M1 matrix protein detected the expression of a 28 kDa molecular weight protein that was very similar for both M2SR and chimeric M2SR viruses, and showed approximately the same induction kinetics at about 9 hours in all three cell lines tested (Figure 15A).

[0129] [Example 13B] Only Influenza B BM2 Protein Was Detected and Influenza A M2 Protein Was Not Detected in Chimeric Influenza A M2SR Viruses Produced by Complementary BM2 Cell Lines Unless propagated in a helper cell line that expresses influenza A M2 protein or influenza B BM2 protein, M2SR cannot express the M2 protein and thus cannot generate progeny virions. This example shows that chimeric influenza A M2SR virus generated in a complementing BM2 cell line yields only BM2 protein incorporated into the viral membrane. M2SR virus propagated in a complementing M2 cell line contains only the M2 protein. To actually demonstrate this, the influenza A M2SR Brisbane / 10 / 2008 (H3N2) virus was passaged in influenza A M2-expressing M2CK cells, and the chimeric M2SR Brisbane / 10 / 2008 (H3N2) was passaged three times in influenza B BM2-expressing BM2Vero cells in MEM medium containing 0.3% BSA. The culture supernatant was clarified by centrifugation. The clarified culture fluid was treated with benzonase (Novagen EMD, Merck KGaA, Darmstadt, Germany) or SAN (Articzymes, Tromso, Norway) nuclease to reduce the viscosity by removing residual cell substrate DNA. The nuclease-treated culture fluid was concentrated 60-fold using a centrifugal concentrator (using a 100,000 nominal molecular weight cut-off regenerated cellulose membrane (Amicon, Merck KGaA, Darmstadt, Germany)). The viral protein in the supernatant was combined with LDS sample buffer (Thermo Fisher Scientific, Waltham, MA) and 5 mM TCEP and heated at 70 °C for 10 minutes to denature. The protein was separated on a 4-12% denaturing Bis-Tris NuPAGE polyacrylamide gel (Thermo Fisher Scientific, Waltham, MA) in MES buffer and transferred to three PVDF membranes. A prestained protein molecular weight standard was electrophoresed for size comparison. The membranes were blocked with 1% (w / v) non-fat dry milk and incubated with three different primary and secondary antibody pairs. The first membrane was incubated with a 2000:1 dilution of influenza A virus M2 protein monoclonal mouse antiserum 14C2 (Santa Cruz Biotechnology, Dallas, TX), followed by incubation with a 3000:1 dilution of horseradish peroxidase-conjugated anti-mouse IgG (H+L) secondary antibody (SeraCare KPL, Milford, MA).The second membrane was incubated with a 2000:1 dilution of influenza B virus BM2 protein polyclonal rabbit antiserum (Hatta-M, et al, J.Virol, 78(11): 5576-5583, 2004), followed by incubation with a 3000:1 dilution of horseradish peroxidase-conjugated anti-rabbit IgG(H+L) secondary antibody (SeraCare KPL, Milford, MA). The third membrane was incubated with a 2000:1 dilution of influenza virus matrix protein polyclonal goat antiserum NR-3134 (BEI Resources, NIAID, NIH), followed by incubation with a 3000:1 dilution of horseradish peroxidase-conjugated anti-goat IgG(H+L) secondary antibody (SeraCare KPL, Milford, MA). Bands were visualized using TMB membrane peroxidase substrate (SeraCare KPL, Milford, MA). The monoclonal anti-M2 antibody detected M2 in the influenza A M2SR virus preparation, and the influenza A M2 protein was not detected in the chimeric M2SR preparation. Conversely, the polyclonal anti-BM2 serum detected BM2 in the envelope of the chimeric virus, and BM2 was not detected in the standard M2SR virus. The polyclonal antiserum against the M1 matrix protein detected the PR8 M1 protein with a molecular weight of 28 kDal in both M2SR and chimeric M2SR viruses (Figure 15B).

[0130] [Example 14] Use of individual BM2 variants as vaccines To test whether BM2 mutant viruses can elicit an antibody response in mice, BM2 mutant viruses (BM2SR-4 viruses representing B / Yamagata and B / Victoria) were formulated as monovalent, trivalent, or quadrivalent vaccines together with influenza A H1N1 or H3N2 M22SR vaccines. Monovalent, trivalent, or quadrivalent vaccines were intranasally administered to 6-week-old BALB / c female mice at 10 6 to 10 7Mice were inoculated with doses in the range of TCID50 / mouse. The control mouse group was administered PBS. Serum samples were collected on day 14 after the prime inoculation. The anti-HA IgG antibody titers of the serum samples were determined by enzyme-linked immunosorbent assay (ELISA) against antigens representing the B / Victoria and B / Yamagata lineages. In addition, the anti-HA IgG antibody titers of the serum samples were determined by enzyme-linked immunosorbent assay (ELISA) against influenza A H1N1 and H3N2 antigens (Figures 16A and 16B). Both BM2SR vaccine components (representing the B / Victoria and B / Yamagata lineages) raised higher anti-influenza virus antibodies against influenza B antigens representing the two influenza B lineages in the multivalent formulation compared to the control PBS group (Figures 16C and 16D). These results indicate that the BM2SR vaccine elicits an antibody response against the HA of interest and that there is no interference between the monovalent components when formulated as a multivalent vaccine. In the multivalent formulation, an immune response occurs against each of the monovalent components.

[0131] To show that the BM2SR variant protects mice from lethal influenza B virus challenge as a monovalent, trivalent or tetravalent formulation, BALB / c female mice (N = 4) were challenged 22 days after inoculation with a lethal dose of a heterologous subtype influenza B virus (e.g., B / Malaysia / 2706 / 2004 virus (20 mouse 50% lethal dose (MLD 50 ). All mice vaccinated with BM2SR, trivalent and tetravalent vaccines survived the challenge and did not lose weight (Figures 16E and 16F). However, control mice administered only PBS lost weight and did not survive 7 days after the challenge. These results indicate that the BM2SR virus protects against influenza B virus challenge even when the virus does not match the vaccine component. Protection is provided by BM2SR in the multivalent formulation as well as in the monovalent formulation.

[0132] [Example 15] Immune responses and protective efficacy elicited by BM2SR in monovalent and tetravalent vaccine formulations in a ferret model Summary This example shows that the BM2SR vaccine can elicit an immune response in a ferret model in either monovalent or polyvalent formulations. That is, the protective immune response elicited by BM2SR is not subject to interference from other vaccine components when formulated as a tetravalent, and BM2SR also does not interfere with (i.e., suppress) the immune response of the other components. Twelve male ferrets were inoculated with BM2SR candidate virus at 1x10 7 TCID 50 (single value) or 4x10 7 TCID 50 As a control, one group of ferrets was administered OPTI-MEM as a placebo. TM A prime-boost vaccination regimen was used for each treatment group. Ferrets received a prime vaccine (day 0) and a boost vaccine 28 days later (day 28). Following each vaccination, ferrets were observed for mortality for 14 days after vaccination, and weights, temperatures, and clinical signs were measured daily. Serum was collected from all ferrets on days 21, 35, and 56 after vaccination to assess antibody levels over time. On day 70, all animals were 6 Ferrets were challenged intranasally with PFU of influenza A virus A / California / 07 / 2009 (H1N1pdm). Following challenge, ferrets were observed for mortality for 14 days post-inoculation, and body weight, temperature, and clinical signs were measured daily. Nasal washes were collected for virus titer from ferrets in each group (N=8) on days 1, 3, 5, and 7 post-challenge. In addition, serum was collected for analysis from surviving ferrets after challenge (day 82). Necropsies were performed on four ferrets per group three days after challenge (day 73). Organs were collected after challenge for viral load (titer) determination. No vaccine-related adverse events were observed in the 5 groups. After challenge, the placebo control group showed a weight loss (~15%). Weight loss was also observed in the monovalent BM2SR vaccine-inoculated group that did not antigenically match, but the decrease was less than that observed in the placebo group (~5 - 8%). Quadrivalent M2SR showed no significant weight loss after challenge.

[0133] B. Materials and methods Vaccine virus inoculation : As shown in Table 9, ferrets were inoculated intranasally with two doses of the monovalent BM2SR vaccine (1 dose 1x10 7 TCID 50 ) or two doses of the quadrivalent M2SR vaccine (1 dose 4x10 7 TCID 50 ). The frozen virus stock was thawed at room temperature for at least 10 minutes and then stored refrigerated (or on ice) until use. Ferrets were anesthetized with ketamine / xylazine and the virus dose was administered intranasally in a volume of 500 μL (250 μL / nare). Animals were observed daily for 7 days after each vaccination. Body weight, body temperature, and clinical signs were monitored for 7 days.

[0134]

Table 9

[0135] The BM2SR virus is a recombinant influenza B virus that does not express a functional BM2 protein, contains the BM2SR-0 variant containing SEQ ID NO: 11, and encodes the HA and NA of B / Brisbane / 60 / 2008 (Victoria) or B / Wisconsin / 01 / 2010 (Yamagata). Quadrivalent M2SR is composed of two M2SR and two BM2SR viruses encoding the HA and NA of H1N1, H3N2, B / Victoria, and B / Yamagata.

[0136] Animal handling: Male ferrets were purchased from a vendor (Triple F Farms), and 48 of these ferrets were used in the study. The animals were approximately 4 months old at the start of the study. It was certified by the supplier that the animals were healthy and had no antibodies against infectious diseases. Upon arrival, the animals were housed individually in hanging wire cages. The cages had a flat bottom and were suspended over a soiled pan lined with paper. The animal room and cages were cleaned prior to animal acceptance according to accepted animal handling practices and standard operating procedures. Certified Teklad Global Ferret Diet #2072 (Teklad Diets, Madison WI) and city of Chicago tap water were provided ad libitum and refreshed at least three times per week. Fluorescent lighting in the animal room was maintained on a 12-hour light / dark cycle. The temperature and relative humidity in the animal room were within the limits of the corresponding protocol and were in the ranges of 20.0 to 25.0 °C and 30 to 63%, respectively, during the study.

[0137] 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 the study. Following isolation, the ferrets were weighed and assigned to treatment groups using a computer-controlled randomization procedure based on weight (yielding similar group means) (ToxData™, version 2.1.E.11 (PDS Pathology Data Systems, Inc., Basel, Switzerland)). Within groups, all weights were within 20% of their mean. The 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.

[0138] Experimental design: To assess the immunogenicity and efficacy of the vaccines, ferrets were immunized with each of BM2SR or quadrivalent M2SR virus, or sham-immunized with medium (OPTI-MEM TM ). The body weight, body temperature, and clinical signs of the ferrets were monitored, and the immunological responses were evaluated. Forty-eight male ferrets (Triple F Farms, Sayre PA) (4 months old at the start of the study) were used in this study. Procedures for all animals were conducted in an animal biosafety level 2 facility in accordance with a protocol approved by the IIT Research Institute's Institutional Animal Care and Use Committee. Ferrets were monitored for 3 days before inoculation to measure body weight and establish a baseline body temperature. Temperature readings were recorded daily via a transponder (BioMedic data systems, Seaford, DE) implanted subcutaneously in each ferret. Blood was collected and tested for influenza antibodies before the start of the study. Pre-vaccination serum samples were treated with receptor-destroying enzyme (RDE) to remove non-specific inhibitors, followed by serial dilution and testing against a defined quantity of influenza A viruses (A / California / 07 / 2009-like (H1N1pdm), A / Switzerland / 9715293 / 2013 (H3N2)), influenza B viruses (B / Brisbane / 60 / 2008 (Victoria lineage) and B / Wisconsin / 01 / 2010 (Yamagata lineage)), and mixed with 0.5% chicken red blood cells or 0.75 - 1.0% guinea pig red blood cells. Antibody titers were defined by the lowest serum dilution that caused hemagglutination inhibition. Only ferrets with HAI (hemagglutination inhibition) titers less than 40 were considered seronegative and used in this study. The test animals were randomized and divided into 4 groups (12 ferrets / group) as shown in Table 9. One dose of 1x10 7 TCID 50 of 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 mock-inoculated intranasally on days 0 and 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 (-3 and -5 days) and on days 21, 35, and 56, and the serum was kept at -65°C until antibody titer measurement by ELISA and HAI assays.

[0139] C. Results The anti-HA IgG antibody titers of the serum samples were determined by enzyme-linked immunosorbent assay (ELISA) against 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 with 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 serum anti-HA antibodies against the corresponding antigen. The sera of the BM2SR immunization group showed the expected specificity and did not react with influenza A antigens (CA07-H1N1, Bris10-H3N2). More importantly, the quadrivalent M2SR group presented a significant increase in serum anti-HA antibodies against all four antigens (Figure 18), indicating that BM2SR is immunogenic when formulated as a quadrivalent vaccine and there is no interference between the components of the multivalent formulation. These data suggest that BM2SR and the quadrivalent M2SR viruses elicit a significant immune response in ferrets.

[0140] Serum samples were analyzed by hemagglutination inhibition (HAI) assay to demonstrate the functional activity of the antibodies detected by ELISA. 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 with 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°C ± 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 diluted 4-fold by two-fold dilutions (from 1:10 to 1:80) with phosphate-buffered saline (PBS) (in duplicate), 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 hemagglutination was scored. As shown in FIGS. 19A and 19B, all immunized ferrets showed significant HAI antibody titers against their corresponding test viruses. Quadrivalent M2SR showed significant HAI titers against all four test viruses. The placebo (naïve) group elicited no influenza-specific antibodies. The CDC has stated that a serum HAI antibody titer of 40 indicates at least a 50% reduction in the risk of influenza infection or disease in the population. Thus, these results suggest that the BM2SR virus elicits a protective immune response that is maintained when these viruses are formulated as a quadrivalent vaccine with the influenza A M2SR virus.

[0141] After challenge with A / California / 09 / 2009 (H1N1pdm), a 5 - 8% weight loss was observed in BM2SR - immunized animals on day 6 post - challenge. OPTI - MEM TM Ferrets (placebo group) showed the greatest weight loss (15%). The weight loss in vaccinated ferrets was dependent on the antigenicity of the vaccine. Ferrets administered with quadrivalent M2SR (including H1N1pdm M2SR) showed no significant weight loss. Ferrets administered with either BM2SR vaccine showed a ~5 - 8% weight loss. Nasal wash samples were collected from all ferrets on days 1, 3, 5, and 7 post - challenge and evaluated for the presence of the challenge virus by plaque assay in MDCK cells. Figure 20 shows that quadrivalent M2SR controlled the replication of the challenge virus. Placebo and monovalent BM2SR did not control the challenge virus, and at least 5 log of virus was detected for 5 days post - infection. The BM2SR vaccine did not control the influenza A challenge virus but showed a mitigating effect on disease progression (the mitigating effect is evidenced by less weight loss than the placebo group (data not shown)).

[0142] Furthermore, respiratory organs collected from 4 additional ferrets on day 3 post - infection showed control of the challenge virus. As shown in Figures 21A, 21B, and 21C, quadrivalent M2SR did not allow any replication of the challenge virus in the upper and lower respiratory tissues (e.g., turbinates, trachea, and lung tissue). In contrast, the challenge virus grew to high titers in the upper respiratory tissues (e.g., turbinates and trachea) of the other groups. In the lower airways (lungs), monovalent BM2SR vaccine controlled the challenge virus compared to the placebo group. These results suggest that quadrivalent M2SR prevented the establishment of influenza infection itself and that the BM2SR vaccine mitigated the severity of the infection.

[0143] D. Conclusions This example shows that intranasal administration of BM2SR and quadrivalent M2SR vaccine virus to ferrets was not associated with any vaccine-related adverse events (such as elevated body temperature, weight loss or clinical signs), and that it is useful as an intranasal influenza vaccine. These results indicate that BM2SR elicits an immune response in ferrets and, when formulated as a quadrivalent M2SR virus, elicits a protective immune response against each strain included in the multivalent formulation.

[0144] [Example 16] Influenza AOF growth in Vero cells Virus production : The M2SR and BM2SR-4 viruses were generated as previously described. For M2SR, influenza A virus RNA segments 1, 2, 3, 5, 8 from influenza A / Puerto Rico / 1934, and M2SR segment 7, and HA and NA virus RNA segments 4, 6 from influenza A / Hong Kong / 4801 / 2014 (H3N2) were used. For BM2SR, influenza B virus RNA segments 1, 2, 3, 5, 8 from influenza B / Yamagata / 1973 and segment 7 BM2SR-4 from B / Florida / 2006, and HA and NA virus RNA segments 4, 6 from influenza B / California / 12 / 2015 (Yamagata lineage) were used. Influenza cDNA was cloned with an RNA polymerase I expression cassette. The resulting plasmids were transfected into BM2Vero cells together with virus polymerase, NP and MP protein expression plasmids, and the virus released into the supernatant was amplified in BM2Vero cells.

[0145] Cell and virus culture: Madin - Darby canine kidney (MDCK) cells were maintained in MEM supplemented with 10% fetal bovine serum (FBS). M2CK and BM2CK cells (MDCK cell lines stably expressing influenza A M2 and BM2 proteins, respectively) were maintained at 37 °C, 5% CO2 in MEM supplemented with 10% FBS and 150 μg / mL of hygromycin B. Vero (African green monkey kidney) cells were grown either in MEM supplemented with 10% fetal bovine serum (FBS) or in OptiVero (animal - origin - free (AOF) medium formulated according to the manufacturer's (Invitria, Fort Collins, CO) recommendations). M2VeroA and BM2Vero cells (Vero cell lines stably expressing influenza A M2 and BM2 proteins, respectively) were maintained in OptiVero medium supplemented with 500 or 1000 μg / mL of G418 sulfate. Wild - type influenza A / Hong Kong / 4801 / 2014 (H3N2) was propagated in MDCK cells, and wild - type influenza B / CA / 12 / 2015 was propagated in Vero cells in MEM supplemented with 0.3% bovine serum albumin (BSA) and 1 μg / mL trypsin - TPCK. M2SR and BM2SR - 4 viruses were propagated in M2VeroA or BM2Vero cells using OptiVero medium and 1 μg / mL T - TPCK.

[0146] To directly compare the virus growth kinetics, virus growth curves were generated using the same AOF culture conditions for each combination of virus / cell line as follows. Vero, M2VeroA, or BM2Vero cells in the exponential growth phase were plated at 1,000,000 cells / 60 - mm tissue culture dish in AOF medium. After culturing for 24 hours in a humidified incubator at 37 °C, 5% CO2, 0.001 TCID of the virus was added to the Vero cell line in AOF medium. 50The test virus was infected at a multiplicity of infection (MOI) of three replicates per unit / cell. After incubation at 37 °C and 5% CO2 for 120 minutes, the virus inoculum was removed and fresh AOF medium containing 1 mg / mL of trypsin-TPCK was applied to the cells. The virus culture was carried out in a humidified incubator at 37 °C and 5% CO2. Aliquots were taken from the virus culture every 24 hours for 4 days and stored frozen at -80 °C for subsequent titer measurement analysis. The virus titer in the culture supernatant was determined by the 50% tissue culture infective dose (TCID 50 ) assay (M2CK cells that support the growth for M2SR virus, and BM2CK cells that support the growth for BM2SR4 virus samples were used). Wild-type virus samples were titrated in MDCK or M2-expressing M2CK and BM2CK cells. In wild-type Vero cells, virus titers exceeding the assay detection limit (log 10 TCID 50 / mL = 1.67) were not detected for M2-incomplete M2SR (Figure 22A) or BM2-incomplete BM2SR4 (Figure 22B) virus cultures. Influenza A M2SR HK4801 replicated with a growth curve similar to that of the wild-type A / Hong Kong / 2014 (H3N2) in homologous M2VeroA cells, and influenza B BM2SR4 CA12 replicated with a growth curve similar to that of the wild-type B / CA / 12 / 2015 in BM2Vero cells. Overexpression of M2 or BM2 did not affect the replication of wild-type influenza A or B viruses compared to replication in unmodified wild-type Vero cell lines.

[0147] Viral replication was not observed with the BM2SR-4 influenza B virus grown in the M2VeroA cell line (Figure 22B). In contrast, the influenza A M2SR virus did replicate in BM2Vero cells and showed kinetics very similar to those observed with influenza A M2SR grown in M2VeroA cells (Figure 22A). Thus, expression of the influenza B M2 protein in the BM2Vero cell line permits replication of the influenza A M2-incomplete M2SR virus strain, which is comparable to the growth kinetics of wild-type influenza A. In contrast, influenza A M2 does not substitute for the function of BM2 in influenza B replication.

[0148] [Example 17] Genetic stability of influenza A M2SR on a BM2 Vero cell substrate Virus production : The influenza A M2SR virus was generated using plasmid-based reverse genetics in M2VeroA cells. The M2SR HK4801 virus was generated using influenza A virus RNA segments 1, 2, 3, 5, 8 and M2SR segment 7 from influenza A / Puerto Rico / 1934, and HA and NA virus RNA segments 4, 6 from influenza A / Hong Kong / 4801 / 2014 (H3N2).

[0149] Influenza segment cDNA was cloned with a unidirectional RNA polymerase I expression cassette. The eight resulting RNA expression plasmids were co-transfected into M2 Vero A cells together with five protein expression plasmids (including three viral polymerase plasmids, an NP plasmid, and an M2 plasmid). The virus released into the supernatant was amplified by three rounds of virus passage in fresh M2 Vero A cells. After three passages, the virus titer was determined and the genetic identity was confirmed by sequencing the entire genome of the strain. Briefly, influenza A-specific cDNA reaction products were amplified using influenza A-specific PCR primers. Amplification products of all eight segments were subjected to dye terminator sequencing using segment-specific internal primers. Subsequently, influenza A M2SR HK4801 was further passaged 10 times in the BM2 protein-expressing BM2 Vero cell line. The M2 Vero A P3 / BM2 Vero P10 virus strain (which had been passaged in a total of 13 passages in Vero cell lines) was tested again for genetic identity by complete genome nucleotide sequencing. The nucleotide sequences obtained from the M2SR HK4801 M2 Vero A P3 virus strain generated by reverse genetics showed that all eight viral segment sequences were identical to those of the starting cDNA plasmids. After further 10 passages in the BM2 Vero cell line, the virus showed a very delicate adaptation. A total of four nucleotide substitutions were observed between the two strains. None of the observed changes were silent mutations, which means that none of the observed substitutions were expected to change the amino acid code of the adapted segment (Table 10). Therefore, these results first show that the M2SR HK4801 virus generated by reverse genetics in Vero cells is genetically stable because only four nucleotide substitutions were observed in the 13,588-base influenza A gRNA genome after a total of 13 rounds of virus amplification. Second, the BM2 Vero cell line expressing the BM2 protein provides all the required influenza A M2 functions and does not appear to show any excessive selection against the influenza A M2SR virus.This is because the genomic sequence was stably maintained after 10 passages of the virus, which was only assisted by the BM2 protein.

[0150]

Table 10

Claims

1. A recombinant influenza B virus having a mutant BM2 gene comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:

5.

2. The recombinant influenza B virus according to claim 1, wherein the mutation of the BM2 gene results in failure of viral BM2 protein expression or causes the virus to express a truncated BM2 protein.

3. The recombinant influenza B virus according to claim 1 or 2, wherein the mutant BM2 gene does not revert to a non-wild-type sequence encoding a wild-type or functional BM2 protein during at least 10 passages in an in vitro host cell line, where the host cell is modified to produce a functional version of the mutant gene, thereby providing the viral gene product in a trans manner.

4. The recombinant virus according to any one of claims 1 - 3, which elicits an immune response in a mammal infected with the virus.

5. The recombinant virus according to any one of claims 1 - 4, which is non-pathogenic to a mammal infected with the virus.

6. The recombinant virus according to claim 3, wherein the in vitro cell line comprises Madin - Darby canine kidney (MDCK) cells or Vero cells.

7. A composition comprising a recombinant influenza B virus having a mutant BM2 gene comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:

5.

8. The composition according to claim 7, wherein the mutation of the BM2 gene results in failure of viral BM2 protein expression or causes the virus to express a truncated BM2 protein.

9. The composition according to claim 7 or 8, wherein the virus elicits an immune response in a mammal infected with the virus.

10. The composition according to any one of claims 7 - 9, wherein the virus is non-pathogenic to a mammal infected with the virus.

11. The composition according to claim 7, further comprising an adjuvant.

12. A method for propagating a recombinant influenza B virus, comprising the steps of contacting a recombinant influenza virus comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 with a host cell; and incubating the host cell for a sufficient time and under conditions appropriate for virus replication, wherein the host cell is modified to produce a functional version of the influenza B M2 gene, thereby providing the virus with the gene product in a trans manner, said method.

13. The method according to claim 12, further comprising the step of isolating progeny virus particles.

14. The method according to claim 13, further comprising the step of formulating the virus particles into a vaccine.

15. The method according to claim 12, wherein the virus is unable to express the BM2 protein or expresses a truncated BM2 protein.

16. The method according to claim 12, wherein the virus elicits an immune response in a mammal infected with the virus.

17. The method according to claim 12, wherein the virus is non-pathogenic to a mammal infected with the virus.

18. The method according to claim 12, wherein the mutant BM2 gene does not revert to a non-wild-type sequence encoding a wild-type or functional BM2 protein during at least 10 passages of the host cell.

19. The method according to claim 12, wherein the host cell is an MDCK cell or a Vero cell.

20. A method for propagating a recombinant influenza A virus, comprising the steps of contacting a host cell with a recombinant influenza A virus comprising SEQ ID NO: 33; and incubating the host cell for a sufficient time and under conditions appropriate for virus replication, wherein the host cell is an MDCK cell modified to produce a wild-type version of the influenza B M2 gene, thereby providing the virus with the BM2 gene product in a trans manner.

21. A method for propagating a recombinant influenza A virus, comprising the steps of contacting a host cell with a recombinant influenza A virus comprising SEQ ID NO: 33; and incubating the host cell for a sufficient time and under conditions suitable for virus replication, wherein the host cell is a Vero cell modified to generate a chimeric version of an influenza B BM2 gene selected from the group consisting of influenza A M2 and SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, thereby providing the virus with a chimeric M2:BM2 gene product in a trans configuration.

22. A method for propagating a recombinant influenza A virus, comprising the steps of contacting a host cell with a recombinant influenza A virus comprising SEQ ID NO: 33; and incubating the host cell for a sufficient time and under conditions suitable for virus replication, wherein the host cell is a Vero cell modified to generate a codon-optimized version of the BM2 gene comprising SEQ ID NO: 27, thereby providing the virus with the BM2 gene product in a trans configuration.

23. A recombinant influenza A virus comprising a mutant BM2 protein.

24. A host cell for propagating a recombinant influenza virus, which is a Vero cell modified to generate a gene product encoded by a cDNA sequence selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO:

32.

25. The host cell according to claim 24, wherein the recombinant influenza virus is an influenza A virus comprising a mutant M2 gene shown in SEQ ID NO:

33.

26. The host cell according to claim 24, wherein the recombinant influenza virus is an influenza B virus comprising a mutant BM2 gene shown in SEQ ID NO: 4 or SEQ ID NO: 6, and the Vero cell is modified to generate a gene product encoded by SEQ ID NO: 27.

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