Introduction

[0001] This patent application claims the benefit of priority from U.S. Provisional Serial Number 63/073,614, filed September 2, 2020, the content of which is incorporated herein by reference in its entirety.

[0002] This invention was made with government support under grant no. AI106700 awarded by the National Institutes of Health. The government has certain rights in this invention.

Background

[0003] The covalent modification of individual bases on mRNA transcripts has emerged as a potentially critical mechanism for the post-transcriptional regulation of gene expression. Analysis of the cellular epitranscriptome, defined as internal single nucleotide modifications that do not alter the mRNA sequence, has identified at least ten different modifications, of which the most prevalent is the addition of a methyl group to the N ⁶ position of adenosine, referred to as m ⁶ A. m ⁶ A is added co-transcriptionally to nuclear pre- mRNAs by a protein complex composed minimally of the methyltransferase METTL3 and two co-factors, METTL14 and WTAP, known collectively as m ⁶ A "writers." Once mRNAs have entered the cytoplasm, they encounter three m ⁶ A "reader" proteins called YTHDF1, YTHDF2 and YTHDF3, which are thought to mediate many of the phenotypic effects exerted by m ⁶ A.

[0004] In addition to the important role played by m ⁶ A in regulating cellular mRNA function, m ⁶ A has also been detected on every viral mRNA transcript examined so far including mRNAs encoded by several cytoplasmic RNA viruses. The first virus found to express mRNAs bearing internal m ⁶ A residues was influenza A virus, wherein eight m ⁶ As were detected by biochemical analysis of the HA mRNA segment (Krug, et al. (1976) J. Virol. 20(1):45—53; Narayan, et al. (1987) Mol. Cell. Biol. 7 (4):1572-5). However, these m ⁶ A residues were not mapped and no examination of how m ⁶ A affects influenza gene expression or replication has been reported.

[0005] Global inhibition of m ⁶ A addition by depleting intracellular levels of the methyl donor S-adenosylmethionine with 3-deazaadenosine (DAA) has been shown to inhibit influenza A virus (Bader, et al. (1978) Virology 89(2):494- 505; Fustin, et al. (2013) Cell 155(4):793-806). In addition, mutant forms of influenza A virus in which eight prominent m ⁶ A sites on the HA mRNA/cRNA plus strand, or nine m ⁶ A sites on the HA vRNA minus strand, have been prepared and shown to express lower levels of HA mRNA and be significantly less pathogenic when introduced into mice (Courtney, et al. (2017) Cell Host Microbe 22(3):377-386.e5).

Summary of the Invention

[0006] This invention provides a modified nucleic acid molecule encoding influenza virus hemagglutinin, wherein said nucleic acid molecule includes (a) a C->T mutation in the third position of codon 433 of the hemagglutinin; (b) an A- >T mutation in the first position of codon 436 of the hemagglutinin; or (c) a combination of (a) and (b). In some embodiments, the modified nucleic acid molecule encodes a modified hemagglutinin protein having a Thr436Ser amino acid substitution. A vector and host cell harboring the modified nucleic acid molecule are also provided as is a modified influenza virus hemagglutinin protein having a Thr436Ser amino acid substitution. Vaccines composed of the modified nucleic acid molecule or modified influenza virus hemagglutinin protein are also embraced by this invention as is a method of the production of influenza virus in a mammalian cell.

Brief Description of the Drawings

[0007] FIG. 1 shows vRNA and mRNA HA expression levels after a single replication cycle of wild-type and mutant 2 virus as determined by RT-PCR.

[0008] FIG. 2 shows infectious viral titers (Pfu) after a single replication cycle of wild-type and mutant 2 virus. ** P<0.01 vs PR8 wild-type, one-way ANOVA.

[0009] FIG. 3 shows relative vRNA and mRNA HA expression levels after multiple replication cycles of wild-type and mutant 2 virus as determined by RT-PCR.

[0010] FIG. 4 shows infectious viral titers (Pfu) after multiple replication cycles of wild-type and mutant 2 virus. [0011] FIG. 5 shows viral lung titers (Panel A) and percent active infection area (Panel B) in mice infected with wildtype and mutant 2 virus at days 2, 4 and 6 post-infection.

[0012] FIG. 6 shows cytokine and chemokine levels in mice infected with wild-type and mutant 2 virus.

Detailed Description of the Invention

[0013] It has now been found that mutation of specific sites associated with adenosine methylation (m6A) results in increases in vRNA, mRNA, and protein expression over time compared to wild-type levels in a human cell line. These increases occur within a single replication cycle and are maintained over multiple replication rounds. Increases in viral RNA and protein expression also result in a significant increase in infectious titers, immunological responses and lethality in a mouse model. Advantageously, increasing yield in human cell lines can reduce the cost of vaccine production in mammalian cells. Accordingly, this invention provides novel influenza HA nucleic acid molecules and proteins with selected mutations at m ⁶ A, which are of use in the production of influenza virus vaccines, in particular in mammalian cells.

[0014] Influenza viruses are members of the orthomyxoviridae family and are classified into three major distinct types (A, B, and C), based on antigenic differences between their nucleoprotein (NP) and matrix (M) protein. Influenza virions are composed of an internal ribonucleoprotein core (a helical nucleocapsid) containing a single-stranded, segmented RNA genome, and an outer lipoprotein envelope lined inside by a matrix protein (Ml). The segmented genome of influenza A virus includes eight molecules of linear, negative polarity, single-stranded RNAs that encode the RNA-dependent RNA polymerase proteins (PB2, PB1 and PA) and nucleoprotein (NP), which form the nucleocapsid; the matrix and ion channel proteins (Ml, M2); two surface glycoproteins (hemagglutinin (HA) and neuraminidase (NA)); and nonstructural proteins (NS1 and NS2) in addition to other accessory proteins. Transcription and replication of the genome takes place in the nucleus and assembly takes place at the plasma membrane. [0015] Hemagglutinin. HA is a viral surface glycoprotein comprising approximately 560 amino acids and representing 25% of the total virus protein. HA is responsible for adhesion of the viral particle to, and its penetration into, a host cell in the early stages of infection. There are 18 known HA subtypes, categorized as an Hl, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17 or H18 subtype. An exemplary wild-type influenza A virus HA protein has the amino acid sequence:

[0016] In some embodiments, the influenza A virus HA protein shares at least 75% (e.g., any number between 75% and 100%, inclusive of, e.g., 70%, 80%, 85%, 90%, 95%, 99%, and 100%) identity to an amino acid sequence of SEQ ID NO:1. Representative nucleic acid sequences encoding wild-type influenza A virus HA proteins are presented in Table 1.

TABLE 1

[0017] In some aspects of this invention, the nucleic acid molecule encoding the HA protein lacks one or more N6-methyl- adenosine (m ⁶ A) nucleotides. In particular aspects, the nucleic acid molecule encoding the HA protein has been modified to include a C->T mutation in the third position of the codon at position 433 of the HA protein amino acid sequence (i.e., mutation of the codon GAC to GAT); an A->T mutation in the first position of codon Thr436 of the HA protein amino acid sequence; or a combination thereof, thereby eliminating the m ⁶ A and providing a significant increase in HA RNA expression in mammalian cells. In other aspects, the nucleic acid molecule encoding the HA protein has been modified to include a C->T mutation in the third position of the codon at position 433 of the HA protein amino acid sequence of SEQ ID N0:l; an A->T mutation in the first position of codon Thr436 of the HA protein amino acid sequence of SEQ ID NO:1; or a combination thereof. With reference to SEQ ID NO:1, other aspects of this invention include a nucleic acid molecule encoding a modified HA protein that has an amino acid substitution at amino acid residue 436. In some aspects, this invention provides a nucleic acid molecule encoding a modified HA protein that has an amino acid substitution at amino acid residue 436 of SEQ ID NO:1. In certain aspects, the nucleic acid molecule of the invention encodes a modified HA protein having a Thr436Ser amino acid substitution.

[0018] For the purposes of this invention, a "nucleic acid molecule" refers to a single-stranded or double-stranded deoxyribonucleotide or ribonucleotide polymer, chimera or analogue thereof, or a character string representing such, depending on context. The term "nucleic acid molecule" encompasses any physical string of monomer units that can be corresponded to a string of nucleotides, including a polymer of nucleotides (e.g., a typical DNA or RNA polymer), PNAs, modified oligonucleotides (e.g., oligonucleotides comprising bases that are not typical to biological RNA or DNA in solution, such as 2'-O-methylated oligonucleotides), and the like. A nucleic acid can be e.g., single-stranded or doublestranded. A nucleic acid molecule is deemed to be "modified" when the sequence has been altered by one or more nucleotides with respect to a reference sequence. Similarly, a "modified" or "variant" protein has an amino acid sequence that has been altered by one or more amino acids with respect to a reference sequence (e.g., a wild-type protein).

[0019] Cleavage of the virus HAO precursor into the HA1 and HA2 subfragments is a necessary step for the virus to infect a cell. Thus, cleavage is required to convert new virus particles in the host cells into virions capable of infecting new cells. Cleavage is known to occur during transport of the integral HAO membrane protein within the infected cell as well as extracellularly . In the course of transport, hemagglutinin undergoes a series of co- and post- translational modifications which can include proteolytic cleavage of the precursor HA into the amino-terminal fragment HA1 and the carboxy terminal HA2. One of the primary difficulties in growing influenza strains in primary tissue culture or established cell lines arises from the requirement for proteolytic cleavage activation of the influenza hemagglutinin in the host cell.

[0020] Although it is known that an uncleaved HA can mediate attachment of the virus to its sialic acid-containing receptors on a cell surface, it is not capable of the next step in the infectious cycle, which is fusion. It has been reported that exposure of the hydrophobic amino terminus of the HA2 by cleavage is required so that it can be inserted into the target cell, thereby forming a bridge between virus and target cell membrane. This process is followed by fusion of the two membranes and release of the viral genome into the target cell.

[0021] Proteolytic activation of HA involves cleavage at an arginine residue by a trypsin-like endoprotease, which is often an intracellular enzyme that is calcium dependent and has a neutral pH optimum. Since the activating proteases are cellular enzymes, the infected cell type determines whether the HA is cleaved. The HA of the mammalian influenza viruses and the nonpathogenic avian influenza viruses are susceptible to proteolytic cleavage only in a restricted number of cell types. On the other hand, HA of pathogenic avian viruses among the H5 and H7 subtypes are cleaved by proteases present in a broad range of different host cells. Thus, there are differences in host range resulting from differences in hemagglutinin cleavability which are correlated with the pathogenic properties of the virus.

[0022] The differences in cleavability are due to differences in the amino acid sequence of the cleavage site of the HA. Sequence analyses show that the HA1 and HA2 fragments of the HA molecule of the non-pathogenic avian and all typical mammalian influenza viruses are linked by a single arginine. In contrast, the pathogenic avian strains have a sequence of several basic amino acids at the cleavage site with the common denominator being lysine-arginine or arginine-arginine, e.g., RRRK. The hemagglutinins of all influenza viruses are cleaved by the same general mechanism resulting in the elimination of the basic amino acids. [0023] Neuraminidase. NA is a second membrane glycoprotein of the influenza A viruses. The presence of viral NA has been shown to be important for generating a multi-faceted protective immune response against an infecting virus. NA is a 413 amino acid protein encoded by a gene of 1413 nucleotides. Eleven different NA subtypes have been identified in influenza viruses (Nl, N2, N3, N4, N5, N6, N7, N8, N9, N10 and Nil). NA is involved in the destruction of the cellular receptor for the viral HA by cleaving terminal sialic acid carbohydrate moieties on the surfaces of infected cells. NA also cleaves sialic acid residues from viral proteins, preventing aggregation of viruses. Using this mechanism, it is hypothesized that NA facilitates release of viral progeny by preventing newly formed viral particles from accumulating along the cell membrane, as well as by promoting transportation of the virus through the mucus present on the mucosal surface. NA is an important antigenic determinant that is subject to antigenic variation. Representative strains and nucleic acid sequences encoding wild-type influenza A virus NA proteins are presented in and Table 2.

TABLE 2

[0024] Internal Genes of Influenza. In addition to the surface proteins HA and NA, influenza virus includes six additional internal genes, which give rise to eight or more different proteins, including but not limited to polymerase genes FBI, PB2 and PA, matrix proteins Ml and M2, nucleoprotein (NP), and non-structural (NS) proteins NS1 and NS2.

[0025] In order to be packaged into progeny virions, viral RNA is transported from the nucleus as a ribonucleoprotein complex composed of the three influenza virus polymerase proteins, the nucleoprotein (NP), and the viral RNA, in association with the influenza virus matrix 1 (Ml) protein and nuclear export protein. The Ml protein that lies within the envelope functions in assembly and budding.

[0026] A limited number of M2 proteins are integrated into the virions. They form tetramers having H+ ion channel activity, and, when activated by the low pH in endosomes, acidify the inside of the virion, facilitating its uncoating. [0027] NS1 protein, a nonstructural protein, has multiple functions, including regulation of splicing and nuclear export of cellular mRNAs as well as stimulation of translation. The major function of NS1 seems to be to counteract the interferon activity of the host, since an NS1 knockout virus was viable although it grew less efficiently than the parent virus in interferon-nondefactive cells.

[0028] NS2 protein has been detected in virus particles. The average number of NS2 proteins in a virus particle was estimated to be 130-200 molecules. An in vitro binding assay shows direct protein-protein contact between Ml and NS2. NS2- M1 complexes were also detected by immunoprecipitation in virus-infected cell lysates. The NS2 protein, known to exist in virions, plays a role in the export of RNP from the nucleus through interaction with Ml protein.

[0029] Representative strains and nucleic acid sequences encoding wild-type influenza A virus PB1, PB2, and PA proteins are presented in Table 3 and representative strains and nucleic acid sequences encoding wild-type influenza A virus matrix protein (MP), NP, and NS proteins are presented in and Table 4.

TABLE 3

TABLE 4 [0030] Reverse Genetics and Reassortant Viruses. Techniques to isolate and modify specific nucleic acids and proteins are well-known to those of skill in the art. In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 1989; DNA Cloning. A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1985); Transcription and Translation (B. D. Hames & S. J. Higgins, eds. 1984); Immobilized Cells And Enzymes (IRL Press, 1996); Ausubel, F. M. et al. (eds.). Current Protocols in Molecular Biology. John Wiley & Sons, Inc., 1994. These techniques include site- directed mutagenesis employing oligonucleotides with altered nucleotides for generating PCR products with mutations.

[0031] In one aspect, the present invention includes a method for the production of influenza virus, which includes the steps of (a) transfecting host cells with a modified nucleic acid molecule encoding an HA protein as described herein along with nucleic acid segments NA, PB1, PB2, PA, MP, NP, and NS; (b) incubating the host cells under conditions that allow for influenza virus to be produced; and (c) isolating the influenza virus from the host cells. The general methodology for the production of influenza viruses and vaccines thereof by reverse genetics methodologies as described herein and known in the art.

[0032] The mechanism of influenza viral RNA transcription is unique. The 5' cap from cellular mRNAs is cleaved by a viral endonuclease and used as a primer for transcription by the viral transcriptase. Six of eight RNA segments are transcribed into mRNAs in a monocistronic manner and translated into HA, NA, NP, PB1, PB2, and PA. By contrast, two RNA segments are each transcribed to two mRNAs by splicing. For both the M and NS genes, coding mRNAs are translated in different reading frames, generating Ml and M2 proteins and NS1 and NS2 proteins, respectively. Increased concentration of free NP triggers the shift from mRNA synthesis to complementary RNA (cRNA) and viral RNA (vRNA) synthesis. Newly synthesized vRNAs are encapsidated with NP in the nucleus, where they function as templates for secondary transcription of viral mRNAs.

[0033] Reverse-genetics systems have allowed the manipulation of the influenza viral genome (Palese, et al.

(1996) Proc. Natl. Acad. Sci. USA 93:11354-58; Neumann & Kawaoka (1999) Adv. Virus Res. 53:265; Neumann, et al. (1999) Proc. Natl. Acad. Sci. USA 96:9345; Fodor, et al. (1999) J. Virol. 73:9679). Reverse genetics in the influenza virus context is a mechanism by which negative sense RNA is engineered into cDNA for recombinant preparation of organisms having negative strand RNA genomes. The reverse genetics technique involves the preparation of synthetic recombinant viral RNAs that contain the non-coding regions of the negative strand virus essential for the recognition of viral RNA by viral polymerases and for packaging signals necessary to generate a mature virion. The recombinant RNAs are synthesized from a recombinant DNA template and reconstituted in vitro with purified viral polymerase complex to form recombinant ribonucleoproteins (RNPs) which can be used to transfect cells. See US 6,022,726 and US 6,001,634, incorporated herein by reference in their entireties.

[0034] These recombinant methods allow for the production of influenza virus types with specific alterations to the polypeptide amino acid sequence. For example, an HA molecule containing a desired substitution may be part of a recombinant influenza virus. In one method, the recombinant influenza virus is made through a genetic engineering method such as the "plasmid only" system (Hoffmann, et al. (2002) Vaccine 20:3165,).

[0035] In another method for generating a recombinant virus, an eight plasmid system is used, wherein the negative sense RNAs are expressed from a Pol I promoter and the coexpression of the polymerase complex proteins result in the formation of infectious influenza A virus (Hoffmann, et al. (2000) Proc. Natl. Acad. Sci. USA 97:6108-13). This technology allows the rapid production of chimeric vaccines from cDNA for use in the event of an influenza pandemic, and provides the capability to attenuate pathogenic strains (Subbarao, et al. (2003) Virology 305:192-200), while eliminating the need to screen reassortant viruses for the 6:2 configuration (i.e., 6 internal genes and 2 HA and NA genes (one of each gene)). See also US 7,037,707.

[0036] In some embodiments, the reassortant viruses are prepared using the method of Palese et al. ((1996) Proc. Natl. Acad. Sci. USA 93:11354-58), which describes the use of a helper virus system to generate genetically engineered virus. In one embodiment, the virus is generated using an influenza helper virus method. For example, to construct a 6:2 reassortant, an attenuated VN1203 virus could be used as a helper virus to introduce the HA and NA from a second strain. Selection of the transfectant virus is carried out using neutralizing antibodies against the helper HA or NA proteins. [0037] With knowledge of internal genes and HA and NA genes from influenza virus strains, it will be appreciated that, in an alternative embodiment, polynucleotides encoding these genes are synthesized by techniques well know and routinely practiced in the art.

[0038] In another embodiment, influenza virus of other influenza A subtypes and influenza B viruses are useful in the methods and compositions of the invention. For example, influenza A virus having any HA subtype is contemplated, including any of the Hl to H18 subtypes. In still a further embodiment it is contemplated that an influenza virus having any of NA subtypes N1 to Nil is useful for the invention.

[0039] In certain embodiments, it is contemplated that when generating a reassortant, the HA and NA subtype are derived from the same strain, and the backbone is derived from an influenza virus of the same subtype. For example, it is contemplated that any of the following influenza A subtypes are useful in the invention; H1N1, H2N1, H3N1, H4N1, H5N1, H6N1, H7N1, H8N1, H9N1, H10N1, H11N1, H12N1, H13N1, H14N1, H15N1, H16N1; H1N2, H2N2, H3N2, H4N2, H5N2, H6N2, H7N2, H8N2, H9N2, H10N2, H11N2, H12N2, H13N2, H14N2, H15N2, H16N2; H1N3, H2N3, H3N3, H4N3, H5N3, H6N3, H7N3, H8N3, H9N3, H10N3, H11N3, H12N3, H13N3, H14N3, H15N3, H16N3; H1N4, H2N4, H3N4, H4N4, H5N4, H6N4, H7N4, H8N4, H9N4, H10N4, H11N4, H12N4, H13N4, H14N4, H15N4, H16N4; H1N5, H2N5, H3N5, H4N5, H5N5, H6N5, H7N5, H8N5, H9N5, H10N5, H11N5, H12N5, H13N5, H14N5, H15N5, H16N5; H1N6, H2N6, H3N6, H4N6, H5N6, H6N6, H7N6, H8N6, H9N6, H10N6, H11N6, H12N6, H13N6, H14N6, H15N6, H16N6; H1N7, H2N7, H3N7, H4N7, H5N7, H6N7, H7N7, H8N7, H9N7, H10N7, H11N7, H12N7, H13N7, H14N7, H15N7, H16N7; H1N8, H2N8, H3N8, H4N8, H5N8, H6N8, H7N8, H8N8, H9N8, H10N8, H11N8, H12N8, H13N8, H14N8, H15N8, H16N8; H1N9, H2N9, H3N9, H4N9, H5N9, H6N9, H7N9, H8N9, H9N9, H10N9, H11N9, H12N9, H13N9, H14N9, H15N9, and H16N9. Influenza A viruses of the following subtypes have been identified previously, H1N1, H2N2, H1N2, H3N2, H3N8, H4N6, H5N1, H5N2, H5N3, H5N9, H6N1, H6N2, H6N5, H7N1, H7N7, H8N4, H9N2, H10N7, H11N6, H12N5, H13N6, H14N5, H15N8, H15N9, H16N3. [0040] The instant modified HA nucleic acids and proteins are of use in any virus previously disclosed in the art, e.g. _r any viruses (Influenza A and influenza B) -previously disclosed or produced, e.g., using backbones such as A/Puerto Rico/8/34 (H1N1), A/Ann Arbor/6/60 (H2N2) and B/Ann Arbor/1/66, having internal genes from one strain and the HA and NA genes from a different strain, and in any prior publications referenced herein, including but not limited to: US 4,552,758, US 7,037,707, US 7,601,356, US 7,566,458, US 7,527,800, US 7,510,719, US 7,504,109, US 7,465,456, US 7,459,162; US 2009/0297554, US 2009/0246225, US 20090208527, US 2009/0175909, US 2009/0175908, US 2009/0175907, US 2009/0136530, US 2008/0069821, US 2008/0057081, US 2006/0252132, US 2006/0153872, US 2006/0110406, US 2005/0158342, US 2005/0042229, US 2007/0172929, WO 2017/070620, WO 2008/157583, WO 2008/021959, WO 2007/048089, WO 2006/098901, WO 2006/063053, WO 2006/041819, WO 2005/116260, WO 2005/116258, WO 2005/115448, WO 2005/062820, WO 2003/091401 and any viruses identified therein as useful for the FLUMIST™ vaccine, which may contain internal genes from one influenza A subtype and HA and NA genes of the same subtype in a recombinant virus. All such documents are incorporated by reference herein in their entirety.

[0041] Cells Lines. Typical influenza viruses are adapted for growth in chicken eggs but the expense for maintaining the egg cultures is significantly greater than growing virus in cell culture. Conventional chicken embryo cell (CEC) cultures have been used in attempts to grow influenza virus for vaccine, but these provide only some of the protease activities of a whole chicken embryo and, hence, allow replication of a limited range of influenza virus strains. Standard procedures for preparation of CEC cultures involve removal of the head and inner organs and multiple trypsinization steps. These procedures result specifically in the loss of brain, heart, lung, liver and kidney cells, which have been shown to replicate a number of influenza strains (Scholtissek, et al. (1988) J. Gen. Virol. 69:2155-2164). Standard procedures thus result in a highly selected cell population consisting mainly of fibroblasts, which are limited in terms of the virus strains that they can support. [0042] Improvements in influenza virus production have been achieved in both chicken cultures and in mammalian cell lines. For Instance, it has been reported that the limited replication of several influenza A strains in standard cell cultures could be ameliorated by the addition of trypsin to the tissue culture medium. For example, trypsin addition significantly increases the infectivity of various strains grown in CEC cultures (Lazarowitz, et al. (1975) Virology 68:440-454). In addition, Stieneke-Grober, et al. ((1992) EMBO J. 11:2407-2414) have identified the HA activating enzyme in MDBK cells as a furin-like protease. Such enzymes have been isolated from human and mouse tissues and constitute a new family of eukaryotic subtilisin-like endoproteases. Vero cells adapted for improved viral growth and vaccine production are described in US 6,146,873 and Kistner, et al. ((1998) Vaccine 16:960-8). Additional mammalian cell lines useful for culture and growth of virus for use in vaccines include, but are not limited to, MRC-5, MRC-9, Lederle 130, Chang liver and WI-38 (human fibroblast); U937 (human monocyte); Vero and CV-1 (African Green monkey): IMR-90 and IMR-91 (human lung fibroblast having characteristics of smooth muscle), MDCK (Madin Darby canine kidney), MDBK (Madin Darby bovine kidney), HEK 293T (human embryonic kidney), H9, CEM and CD4-expressing HUT78 (human T cell): PerC6 (human retinoblast); BHK-21 cells (baby hamster kidney), BSC (monkey kidney cell); and LLC-MK2 (monkey kidney). In accordance with the present invention, an HA2 protein encoded by a nucleic acid molecule modified to include an A->T mutation in the first position of the codon for Thr436 thereby eliminating m ⁶ A at this position provides a significant increase in HA RNA expression in mammalian cells. Therefore, in particular aspects, a host cell of the invention is a mammalian host cell.

[0043] Vaccines. It is contemplated that a desired virus strain obtained from cell-culture preparation is used to produce a vaccine. Many types of viral vaccines are known, including but not limited to, attenuated, inactivated, subunit, and split vaccines.

[0044] Attenuated vaccines are live viral vaccines that have been altered in some manner to reduce pathogenicity and no longer cause disease. Attenuated viruses are produced in several ways, including growth in tissue culture for repeated generations and genetic manipulation to mutate or remove genes involved in pathogenicity. For example, in one embodiment, viral genes and/or proteins identified as involved in pathogenicity or involved in the disease manifestation, are mutated or changed such that the virus is still able to infect and replicate within a cell, but it cannot cause disease. An example of this is to mutagenize the HA1/HA2 cleavage site. Attenuation of virus has also been successful by insertion of a foreign epitope into a viral gene segment, for example the NA gene (Castrucci, et al. (1992) J. Virol. 66:4647-4653), thereby interfering with the normal function of the genome. Viruses are also attenuated using cold adaptation methods well-known in the art. See, for example, Maassab, et al. ((1999) Rev. Med. Virol. 9:237-44), which discusses methods to attenuate type A and Type B influenza virus, and Ghendon, et al. ((2005) Vaccine 23:4678- 84), which describe a cold adapted influenza virus that grows in MDCK cells.

[0045] Additional methods to attenuate a virus include construction of a reassortant virus lacking the NS1 gene. See for example US 6,468,544, US 6,573,079, US 6,669,943, US 6,866,853, US 2003/0157131 and US 2004/0109877, which disclose an attenuated virus lacking a functional NS1 gene. The NS1 gene may be completely deleted or partially deleted or altered by mutation such that there is no functional expression of the NS1 gene in the virus. Virus particles lacking the NS1 gene demonstrate an attenuated phenotype compared to wild-type virus.

[0046] After production of the attenuated virus, the vaccine is prepared using standard methods. The virus is purified using standard methods known in the art, for example using size exclusion chromatography, high-speed (ultra)centrifugation or sucrose gradients.

[0047] Subunit vaccines are killed vaccines. Production of subunit vaccine involves isolating a portion of the virus that activates the immune system. In the case of influenza, subunit vaccines have been prepared using purified HA and NA, but any mixture of viral proteins is used to produce a subunit vaccine. Generally, the viral protein, such as HA is extracted from recombinant virus forms and the subunit vaccine is formulated to contain a mixture of these viral proteins from different strains.

[0048] Split vaccines are killed vaccines produced by treating an enveloped virus with detergent to solubilize the proteins in the envelope. In the case of influenza virus, HA and NA become solubilized. In one embodiment, nonionic detergents are used for producing split vaccines. Examples of non-ionic detergents, include, but are not limited to, Nonanoyl~N-Methylfucamide (Mega 9), Triton.™ X-100, Octylglucoside, Digitonin, C12E8, Lubrol, NONIDET® P-40, and polysorbate (for example polysorbate 20, 80 or 120).

[0049] Inactivated viral vaccines are prepared by inactivating the harvested virus and formulating it using known methods for use as a vaccine to induce an immune response in a mammal. Inactivation is carried out using agents including but not limited to formaldehyde, UV irradiation, glutaraldehyde, binary ethyleneimine (BEI), and betapropiolactone. Inactivating agents are used at a concentration high enough to inactivate substantially all viral particle in the solution. By way of example and without limitation, virus inactivation with gamma irradiation is described in US 6,254,873; inactivation with formalin is described in US 6,254,873 and US 6,635,246; inactivated with formaldehyde has been described for JE-VAX®, Japanese encephalitis virus vaccine (Couch, et al. (1997) J. Infect. Dis. 176(Suppl 1):538-44); photodynamic inactivation by visible light (Wallis, et al. (1963) J. Immunol. 91:677-682); inactivation with UV light (WO/2008/039494); chlorine inactivation; inactivation with water-insoluble, hydrophobic polycations, e.g. _z N,N-dodecyl methyl-polyethylenimine (PEI) (Halder, et al. (2006) Proc. Natl. Acad. Sci. USA 103:17667- 17671); thermal inactivation (Thomas, et al. (2007) J. Food Protect. 70:674-680); inactivation with betapropiolactone is described in European Pharmacopoeia 5.0; and inactivation by binary ethylenimine is described in US 6,803,041. It is contemplated that during the inactivation step, purification of subunits, and/or splitting is performed before or after purification of the virus from cell culture. For example, production of an inactivated virus vaccine, may involve removal of cellular material, inactivation of virus, purification and solubilization of the viral envelope. In one embodiment, a reassortant virus described herein is grown and isolated from Vero cells as described in Kistner, et al. ((2007) Vaccine 25:6028-36).

[0050] In some embodiments, the nucleic acid molecule (e.g., mRNA) of this invention may be used as the vaccine, wherein the vaccine is optionally formulated in a lipid nanoparticle (e.g., a lipid nanoparticle composed of a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid). See, e.g., WO 2017/070620 or WO 2017/070622.

[0051] A vaccine is then prepared using standard adjuvants and vaccine preparations known in the art. Adjuvants include, but are not limited to, saponin, non-ionic detergents, vegetable oil, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanins, and potentially useful human adjuvants such as N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, N- acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (l'-2'- dipalmitoyl-s-n-glycero-3-hydroxyphosphoryloxy )-ethylamine, BCG (bacille Calmette-Guerin) Corynebacterium parvum, ISCOMs, nano-beads, squalene, and block copolymers, which are contemplated for use alone or in combination. ISCOMs or Immune Stimulating Complexes are a novel vaccine delivery system and are unlike conventional adjuvants (Morein, et al. (1984) Nature 308:457-460). An ISCOM is formed in two ways: (1) the antigen is physically incorporated in the structure during its formulation; or (2) an ISCOM-matrix does not contain antigen but is mixed with the antigen of choice by the enduser prior to immunization. After mixing, the antigens are present in solution with the ISCOM-matrix but are not physically incorporated into the structure. [0052] In one embodiment, the adjuvant is an oil-in-water emulsion. Oil-in-water emulsions are well known in the art and have been suggested to be useful as adjuvant compositions (EP 399843; WO 95/17210, US 2008/0014217). Ideally, the oil is present in an amount of 0.5% to 20% (final concentration) of the total volume of the antigenic composition or isolated virus, at an amount of 1.0% to 10% of the total volume, or in an amount of 2.0% to 6.0% of the total volume. In some embodiments, oil-in-water emulsion systems useful as adjuvant have a small oil droplet size. For example, the droplet sizes will be in the range 120 to 750 nm, or from 120 to 600 nm in diameter.

[0053] In order for any oil-in-water composition to be suitable for human administration, the oil phase of the emulsion system includes a metabolizable oil. The oil may be any vegetable oil, fish oil, animal oil or synthetic oil, which is not toxic to the recipient and is capable of being transformed by metabolism. Nuts, seeds, and grains are common sources of vegetable oils. Synthetic oils are also part of this invention and can include commercially available oils such as NEOBEE® and others. A particularly suitable metabolizable oil is squalene. Squalene (2,6,10,15,19,23- Hexamethyl-2,6,10,14,18,22-tetracosahexaene) is an unsaturated oil which is found in large quantities in sharkliver oil, and in lower quantities in olive oil, wheat germ oil, rice bran oil, and yeast, and is a particularly suitable oil for use in this invention. Squalene is a metabolizable oil by virtue of the fact that it is an intermediate in the biosynthesis of cholesterol (Merck index, 10th Edition, entry no. 8619). Exemplary oils useful for an oil in water emulsion, include, but are not limited to, sterols, tocols, and alphatocopherol. [0054] In additional embodiments, immune system stimulants are added to the vaccine and/or pharmaceutical composition. Immune stimulants include: cytokines, growth factors, chemokines, supernatants from cell cultures of lymphocytes, monocytes, or cells from lymphoid organs, cell preparations and/or extracts from plants, cell preparation and, or extracts from bacteria (e.g., BCG, mycobacterium, Corynebacterium), parasites, or mitogens, and novel nucleic acids derived from other viruses, or other sources (e.g. double stranded RNA, CpG) block co-polymers, nano-beads, or other compounds known in the art, used alone or in combination .

[0055] Particular examples of adjuvants and other immune stimulants include, but are not limited to, lysolecithin; glycosides (e.g., saponin and saponin derivatives such as QUIL-A® (QS7 and QS21) or GPI-0100); cationic surfactants (e.g., DDA); quaternary hydrocarbon ammonium halogenides; pluronic polyols; polyanions and polyatomic ions; polyacrylic acids, non-ionic block polymers (e.g., PLURONIC® F-127); and 3D-MPL (3 de-O-acylated monophosphoryl lipid A). See, e.g., US 2008/0187546 and US 2008/0014217.

[0056] Pharmaceutical Formulations and Administration. The administration of the vaccine composition is generally for prophylactic purposes. The prophylactic administration of the composition serves to prevent or attenuate any subsequent infection. A "pharmacologically acceptable" composition is one tolerated by a recipient patient. It is contemplated that an effective amount of the vaccine is administered. An "effective amount" is an amount sufficient to achieve a desired biological effect such as to induce enough humoral or cellular immunity. This may be dependent upon the type of vaccine, the age, sex, health, and weight of the recipient. Examples of desired biological effects include, but are not limited to, production of no symptoms, reduction in symptoms, reduction in virus titer in tissues or nasal secretions, complete protection against infection by influenza virus, and partial protection against infection by influenza virus.

[0057] A vaccine or composition of the present invention is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient that enhances at least one primary or secondary humoral or cellular immune response against at least one strain of an infectious influenza virus. The vaccine composition is administered to protect against viral infection. The "protection" need not be absolute, i.e., the influenza infection need not be totally prevented or eradicated, if there is a statistically significant improvement compared with a control population or set of patients. Protection may be limited to reducing the severity or rapidity of onset of symptoms of the influenza virus infection.

[0058] In one embodiment, an attenuated or inactivated vaccine composition of the present invention is provided either before the onset of infection (so as to prevent or attenuate an anticipated infection) or after the initiation of an actual infection, and thereby protects against viral infection.

[0059] In one aspect, methods of the invention include a step of administration of a pharmaceutical composition. The virus, antigenic composition or vaccine is administered in any means known in the art, including via inhalation, intranasally, orally, and parenterally. Examples of parental routes of administration include intradermal, intramuscular, intravenous, intraperitoneal and subcutaneous administration.

[0060] Ideally, influenza vaccine administration is based on the number of hemagglutinin units (HAU) per dose. One HAU is defined as the quantity of antigen required to achieve 50% agglutination in a standard hemagglutinin assay with chicken red blood cells. Avian influenza virus vaccines as described herein are effective in formulations comprising HA units (HAU) between about 10 ng and about 1 pg, between about 20 ng and about 500 ng, between about 50 ng and about 250 ng, between about 75 ng and about 200 ng, about 100 ng, about 125 ng, about 150 ng, or about 175 ng. In a related embodiment, a vaccine composition composed of an inactivated virus includes an amount of virus corresponding to about 0.1 to about 200 pg of hemagglutinin protein/ml, or any range or value therein. In a related embodiment, a vaccine composition of the present invention includes from about 10 ² to 10 ⁹ plaque forming units (PFU)/ml, or any range or value therein, where the virus is attenuated. In some embodiments, the vaccine composition includes about 10 ² , about 10 ³ , about 10 ⁴ , about 10 ⁵ , about 10 ⁶ , about 10 ⁷ , about 10 ⁸ or about 10 ⁹ PFU/ml. It is further contemplated that the vaccine composition includes from 10 ² to about 10 ⁴ PFU/ml, from about 10 ⁴ to about 10 ⁶ PFU/ml, or from about 10 ⁶ to about 10 ⁹ PFU/ml.

[0061] In another aspect. inactivated flu vaccine is quantified by a single radial diffusion (3RD) assays (see Kistner, et al. (2007) Vaccine 25:6028-36; Wood, et al. (1997) J. Biol. Stand. 5:237-247) and expressed in micrograms hemagglutinin (per ml or per dose). In one embodiment, the dose of a seasonal vaccine is 15 pg per strain, 45 pg in total in three dosages. For (pre)pandemic vaccines the dose typically depends on the adjuvant. In one aspect, the dose range is 1 pg to 15 pg per vaccine, and in some preparations, up to 75 pg per vaccine are useful. In one embodiment, the vaccine dose is administered at a dose from 1 pg to 100 pg HA. In a further embodiment, the vaccine comprises an HA content of 1 pg to 30 pg per vaccine. In related embodiments, the vaccine dose is administered at 1 μg, at 3 pg, at 5 pg, at 7.5 pg, at 10 pg, at 12.5 pg, at 15 pg, at 20 pg, at 25 pg, or at 30 pg HA, or in any amount up to 100 pg as necessary. It is contemplated that, in some embodiments, the dose of vaccine is adjusted based on the adjuvant used for vaccine preparation.

[0062] Accordingly, single vaccine dosages include those having about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 65 μg, about 70 μg, about 75 μg, about 80 μg, about 85 μg, about 90 μg, about 95 μg, about 100 μg, and more than 100 μg hemagglutinin provided in single or multiple dosages at the same or different amount of hemagglutinin.

[0063] It is further contemplated that in certain embodiments, the virus or antigenic composition is administered in doses including similar HA units or pfu as contemplated for vaccine administration.

[0064] When administered as a solution, the vaccine is prepared in the form of an aqueous solution. Such formulations are known the art and are prepared by dissolution of the antigen and other appropriate additives in the appropriate solvent. Such solvents include water, saline, ethanol, ethylene glycol, and glycerol, for example. Suitable additives include certified dyes and antimicrobial preservatives, such as thimerosal (sodium ethylmercuithiosalicylate) . Such solutions may be stabilized using standard methods, for example, by addition of partially hydrolyzed gelatin, sorbitol, or cell culture medium and may be buffered using standard methods, using, for example reagents such as sodium hydrogen phosphate, sodium dihydrogen, phosphate, potassium hydrogen phosphate and/or potassium dihydrogen phosphate or Tris. Liquid formulations may also include suspensions and emulsions. The preparation of suspensions includes, for example, using a colloid mill, and emulsions include, for example, using a homogenizer.

[0065] The following non-limiting examples are provided to further illustrate the present invention.

Example 1: Identification and Mutation of Conserved m6A Sites in HA mRNA Sequences

[0066] Nucleic acid sequences encoding HA from multiple influenza virus strains were analyzed for m6A sites using a computational predictor of mammalian m6A site named SRAMP (Zhou, et al. (2016) Nucleic Acids Res. 44(10):e91). The aligned HA sequences were from viruses isolated from avian, swine, and human over the past 80 years. This analysis identified three clusters of m ⁶ A sites with cluster 1 located at nucleotide positions 764, 774, 785 and 795; cluster 2 located at nucleotide positions 1331 and 1338; and cluster 3 located at nucleotide positions 1380 and 1395. To determine the effects of m ⁶ A modifications at these clusters on viral replication and infectivity, nucleotide site mutations were performed to remove the m ⁶ A on the HA mRNA sequence. In particular, nucleotides 764, 774, 785, 795 (mutant 1); nucleotides 1331, 1338 (mutant 2); and nucleotides 1380, 1395 (mutant 3) of clusters 1, 2 and 3 were respectively mutated to remove the sites for m ⁶ A modifications. In light of the mutations at nucleotides 774, 1338 and 1380, the resulting HA proteins included threonine to serine mutations at the corresponding positions in the HA protein sequence. [0067] Reverse genetics was used to generate viruses using the mutant 2 HA gene sequence. In particular, seven influenza gene plasmids corresponding to A/Puerto Rico/8/1934 (Matrix, PB1, PB2, PA, NS, NP, NA) were transfected into a co-culture of MDCK and 293T cells along with a mutant 2 HA plasmid. Supernatant was harvested from transfected cells 48 hours later and injected into embryonic chicken eggs to generate mutant virus stock. In vitro single (4 to 8 hours at multiplicity of infection (MOI) 10) and multiple replication cycles (12 to 48 hours at MOI 0.1) were conducted using a human lung epithelial cell line (A549). Nuclear and cytoplasmic RNA was extracted from A549 cells; vRNA and mRNA HA, matrix and PB1 gene expression levels were determined by RT-PCR; and RNA levels were normalized to wild-type levels at 4 hours (single replication cycle) or 12 hours (multiple replication cycles). In addition, supernatant was harvested at 8 hours from A549 cells infected at MOI 10 and infectious viral titers were measured by plaque assay.

[0068] The results of this analysis indicated that the cluster 2 mutant (mutant 2) showed a significant increase in HA RNA expression over the course of a single replication round (FIG. 1). Notably, mutant 2 RNA was synthesized in the nucleus and successfully trafficked to the cytoplasm. As with HA RNA expression, the cluster 2 mutant (mutant 2) showed a significant increase in matrix and PB1 RNA expression over the course of a single replication round, thereby demonstrating that the mutations in the HA gene not only affected HA gene expression but also other non-mutated influenza genes. Consistent with an increase in RNA expression, the cluster 2 mutant (mutant 2) showed a significant increase in HA and matrix protein levels over the course of a single replication round. Further, cluster 2 mutant (mutant 2) showed a significant increase in infectious viral titers over the course of a single replication round (FIG. 2) thereby demonstrating that increased gene expression of cluster 2 mutant also resulted in an increase in infectious viral titer over a single replication cycle.

[0069] Analysis of HA and matrix expression levels after multiple replication cycles indicated that cluster 2 mutant (mutant 2) showed a significant increase in RNA expression over the course of multiple replication rounds thereby demonstrating that increased gene expression was sustained over time (FIG. 3). Consistent with an increase in RNA expression, the cluster 2 mutant (mutant 2) showed a significant increase in HA and matrix protein levels over the course of multiple replication rounds as well as a significant increase in infectious viral titers (FIG. 4).

[0070] To further characterize the mutant 2 virus, 7-week- old Balb/CJ mice were infected with 10 ⁴ median tissue culture infectious dose (TCIDso) or 10 ³ TCID50 of wild-type or mutant 2 virus. Mice were weighed daily for 14 days and euthanized at human endpoints. The results of this analysis indicated that compared to wild-type, which had a lethal dose 50 (LD50) of 1.10E+04, the mutant 2 virus exhibited increased lethality (LD50 of 1.40E+03) at the same dose as compared to wild-type. [0071] Viral lung titers were also analyzed by infecting 7- week-old Balb/CJ mice with 10 ⁴ TCID50 of virus and harvesting lung tissue at days 2, 4, and 6 days post-infection. The lung tissue was collected in 500 pL of phosphate buffered saline (PBS), homogenized, and centrifuged a 500 x g for 20 minutes. The supernatant was collected and viral titers in the lysates were determined by TCID50 on MDCK monolayers. This analysis indicated that the mutant 2 viral titers peaked earlier at 2 days post-infection compared to 4 days post-infection for the wild-type virus and mutant 2 viral titers peaked at a higher level compared to wild-type virus (FIG. 5, Panel A). Lung tissue was also dissected to assess active infection area. Lungs were inflated with 1 mL of 10% neutral-buffered formalin and then placed in 3 mL of formalin for 72 hours to complete fixation. Lungs were embedded in paraffin blocks and sectioned onto glass slides, and serial tissue sections were stained with haematoxylin and eosin for histology. Histologic grading of lesions was completed by a pathologist blinded to treatment group. Pulmonary lesions were assigned scores based on the basis of their severity and extent as follows: 0, no lesions; 1, minimal, focal to multifocal, barely detectable; 15, mild, multifocal, small but conspicuous; 40, moderate, multifocal, prominent; 80, marked, multifocal coalescing, lobar; 100, severe, diffuse, with extensive disruption of normal architecture and function. The results of this analysis indicated that mutant 2 virus spread more extensively in the lung (FIG. 5, panel B), which was consistent with increased mortality.

[0072] For immunological characterization of the mutant 2 virus, A549 cells were infected at an MOI of 3; cells were harvested at 3, 6, 9, 12, 24, and 48 hours post-infection; and RNA was extracted using a Qiagen RNeasy Kit. cDNA was prepared for quantitative PCR (qPCR) using the Fluidigm Biomark system. Data were collected using the Fluidigm Biomark data collection software and normalized to wild-type levels at 3 hours post-infection. Two-way ANOVA was performed for statistical analysis. This analysis indicated that levels of key type I interferons (ISG15, IFIT1) were significantly increased in cells infected with mutant 2 virus compared to wild-type virus. Increases in ISG15 and IFIT1 expression occurred at the onset of infection and were sustained for at least a minimum of 24 hours after infection. In addition, Type III interferon (IFNL2) and key inflammatory cytokine (TNFA) and chemokine (CXCL10) RNA levels were significantly increased in cells infected with mutant 2 virus compared to wild-type virus. Overall, mutant 2 virus induced a stronger immunological response compared to wild-type in human epithelial cells.

[0073] Further immunological characterization of the mutant 2 virus was carried out in vivo. Mice were infected with 10 ⁴ TCID50mutant 2 virus or wild-type. At 2 days post-infection, mouse lungs were harvested and flash-frozen in liquid nitrogen. Lungs were then homogenized in 500 μL of PBS and lysate was collected after centrifugation at 500 x g for 20 minutes. Cytokine and chemokine levels were measured using Milliplex Mouse Cytokine/Chemokine Magnetic Premixed 32 plex Bead Panel immunoassay per manufacture's protocol. The results of this analysis indicated that the mutant 2 virus induced a significant increase in the production of cytokines and chemokines at 2 days post-infection compared to wild-type virus (FIG. 6). Therefore, mutant 2 virus infection leads to an increased immunological response in mice early in infection compared to wild-type virus.