CA2822895A1 - Influenza hemagglutinin and neuraminidase variants - Google Patents

Abstract

Polypeptides, polynucleotides, methods, compositions, and vaccines comprising (avian pandemic) influenza hemagglutinin and neuraminidase variants are provided. Specifically, the invention relates to a 6:2 reassortant influenza virus which comprises a genome segment encoding an HA polypeptide comprising the amino acid sequence of SEQ ID NO:15.

Description

INFLUENZA HEMAGGLUTININ AND NEURAMINIDASE VARIANTS
This application is a division of Canadian Application Serial No. 2,568,015 filed May 20, 2005 (parent application).
It should be understood that the expression "the present invention" or the like used in this specification encompasses not only the subject matter of this divisional application, but that of the parent application also.
The subject matter of this divisional application is directed to subject matter relating to a reassortant influenza virus, which comprises 6 internal genome segments from one or more donor influenza viruses other than A/Ann Arbor/6/60, a first genome segment and a second genome segment encoding surface antigens, wherein the first genome segment encodes an HA polypeptide comprising the amino acid sequence of SEQ ID NO:15.
BACKGROUND OF THE INVENTION
[0001] Vaccines against various and evolving strains of influenza are important from a community health stand point, as well as commercially, since each year numerous individuals are infected with different strains and types of influenza virus. Infants, the elderly, those without adequate health care and immuno-compromised persons are at special risk of death from such infections. Compounding the problem of influenza infections is that novel influenza strains evolve readily and can spread amongst various species, thereby necessitating the continuous production of new vaccines.
100021 Numerous vaccines capable of producing a protective immune response specific for such different and influenza viruses/virus strains have been produced for over 50 years and include whole virus vaccines, split virus vaccines, surface antigen vaccines and live attenuated virus vaccines. However, while appropriate formulations of any of these vaccine types are capable of producing systemic immune response, live attenuated virus vaccines have the advantage of also being able to stimulate local mucosal immunity in the respiratory tract. Considerable work in the production of influenza viruses, and fragments thereof, for production of vaccines has been done by the present inventors and co-workers;

see, e.g., U.S. Application Nos. 60/420,708, filed October 23, 2002;
60/574,117, filed May 24, 2004; 10/423,828, filed April 25, 2003; 60/578,962, filed June 12, 2004; and 10/870,690 filed June 16, 2004.
[0003] Because of the continual emergence (or re-emergence) of different influenza strains, new influenza vaccines are continually desired. Such vaccines typically are created using antigenic moieties of the newly emergent virus strains, thus, polypeptides and polynucleotides of novel, newly emergent, or newly re-emergent virus strains (especially sequences of antigenic genes) are highly desirable.
[0004] The present invention provides new and/or newly isolated influenza hemagglutinin and neuraminidase variants that are capable of use in production of numerous types of vaccines as well as in research, diagnostics, etc. Numerous other benefits will become apparent upon review of the following.
la -v. A, zuuniiioz::,6 CA 02822895 2013-06-07 1 I
LUU:VU 1 I I LY
SUMMARY OF THE INVENTION
[00051 In some aspects herein, the invention comprises an isolated or recombinant polypeptide that is selected from: the polypeptides encoded by any one of the sequences of SEQ ID NO:1 through SEQ ID NO:10, any one of the polypeptides encoded by SEQ
ID
NO:1 through SEQ ID NO:10; any one of the polypeptides of SEQ ID NO:11 through SEQ
ID NO:20; only the open reading frame of the polypeptides of SEQ ID NO:11 through SEQ
ID NO:20; alternative (e.g., the mature form without the signal peptide, or without the 5' and 3' sequences outside of the open reading frame, or the sequences as expressed on the surface of a virus (e.g., influenza)) forms of the polypeptide of SEQ ID NO:11-20; any polypeptide that is encoded by a polynucleotide sequence which hybridizes under highly stringent conditions over substantially the entire length of a polynucleotide sequence of SEQ ID NO:1 through SEQ ID NO:10; any polypeptide that is encoded by a polynucleotide sequence which hybridizes under highly stringent conditions to a polynucleotide sequence of SEQ ID NO:1 through SEQ ID NO:10; and, a fragment of any of the above wherein the sequence comprises a hemagglutinin or neuraminidase polypeptide, or a fragment of a hemagglutinin or neuraminidase polypeptide, preferably where the fragments generate an antibody that specifically binds a full length polypeptide of the invention.
In various embodiments, the isolated or recombinant polypeptides of the invention are substantially identical to about 300 contiguous amino acid residues of any of the above polypeptides. In yet other embodiments, the invention comprises isolated or recombinant polypeptides, that comprise an amino acid sequence that is substantially identical over at least about 350 amino acids; over at least about 400 amino acids; over at least about 450 amino acids; over at least about 500 amino acids; over at least about 502 amino acids; over at least about 550 amino acids; over at least about 559 amino acids; over at least about 565 amino acids; or over at least about 566 amino acids contiguous of any of the above polypeptides. In some embodiments, the polypeptide sequence (e.g., as listed in the sequence listing herein) =
comprises less than 565, 559, etc. amino acids. In such embodiments, the shorter listed polypeptides optionally comprise less than 565, 559, etc. amino acids. In yet other embodiments, the polypeptides of the invention optionally comprise fusion proteins, proteins with a leader sequence, a precursor polypeptide, proteins with a secretion signal or *I' kJ LAM.71/ 1101,00 CA 02822895 2013-06-07 1 /1..ThLtIll:V111 I
a localization signal, or proteins with an epitope tag, an E-tag, or a His epitope tag. In still other embodiments, the invention comprises a polypeptide comprising a sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8%, or at least 99.9%
sequence identity to at least one polypeptide listed above. The sequences of the invention are algo shown in Appendix 1 and in the sequence listings herein. The hemagglutinin sequences of the invention can comprise both those sequences with unmodified and modified polybasic cleavage sites (thereby allowing growth of the viruses in eggs). The hemagglutinin polypeptide sequences of SEQ ID NOS:11-20 comprise the endogenous amino terminal signal peptide sequences, however, the hemagglutinin polypeptide sequences of the invention also include the mature (amino terminal signal peptide cleaved) form of the hemagglutinin polypeptides. The cleavage sites of any hemagglutinin polypeptide sequence of any influenza strain can be routinely measured or predicted using any number of methods in the art.
[0006] In other aspects, the invention comprises a composition with one or more polypeptide listed above, or fragments thereof. The invention also includes polypeptides that are specifically bound by a polyclonal antisera raised against at least 1 antigen that comprises at least one amino acid sequence described above, or a fragment thereof. Such antibodies specific for the polypeptides described above are also features of the invention.
The polypeptides of the invention are optionally immunogenic.
[0007] The invention also encompasses immunogenic compositions comprising an immunologically effective amount of one or more of any of the polypeptides described above as well as methods for stimulating the immune system of an individual to produce a protective immune response against influenza virus by administering to the individual an =
immunologically effective amount of any of the above polypeptides in a physiologically acceptable carrier.
[0008] Additionally, the invention includes recombinant influenza virus that comprises one or more of the polypeptides or polynucleotides above, in addition to immunogenic compositions comprising an immunologically effective amount of such recombinant influenza virus. Methods for stimulating the immune system of an individual to produce a protective immune response against influenza virus, through administering an -;,-0 2005/116258 CA 02822895 2013-06-07 inununologically effective amount of such recombinant influenza virus in a physiologically .* acceptable carrier are also part of the invention.
. ' [0009] In other aspects, the invention comprises an isolated or recombinant nucleic . acid that is selected from: any one of the polynucleotide sequences SEQ ID NO:1 through SEQ ID NO.10 (or complemental y sequences thereof), any one of the polynucleotide sequences encoding a polypeptide of SEQ ID NO:11 through SEQ ID NO:20 (or complementary polynucleotide sequences thereof), a polynucleotide sequence which hybridizes under highly stringent conditions over substantially the entire length of any of the above polynucleotide sequences, and a polynucleotide sequence comprising all or a = 10 fragment of any of such polynucleotide sequences wherein the sequence preferably encodes a hemagglutinin or neuraminidase polypeptide or a fragment of a hemagglutinin or neuraminidase polypeptide. The invention also includes an isolated or recombinant nucleic acid that encodes an amino acid sequence which is substantially identical over at least about 300 amino acids of any of the above nucleic acids, or over at least about 350 amino acids;
over at least about 400 amino acids; over at least about 450 amino acids; over at least about 500 amino acids; over at least about 502 amino acids; over at least about 550 amino acids;
over at least about 559 amino acids; over at least about 565 amino acids; or over at least about 566 amino acids of any of the above nucleic acids. Again, in situations wherein the amino acid is less than, e.g., 566, 565, 559, etc. in length (e.g., see, Sequence Listing) then it should be understood that the length is optionally less than 566, 565, 559, etc. The invention also includes any of the above nucleic acids that comprise a hemagglutinin or neuraminidase polypeptide, or one or more fragments of one or more hemagglutinin or = neuraminidase polypeptide. Other aspects of the invention include isolated or recombinant nucleic acids that encode a polypeptide (optionally a hemagglutinin or neuraminidase polypeptide) whose sequence has at least 98% identity, at least 98.5%
identity, at least 99%
identity, at least 99.2% identity, at least 99.4% identity, at least 99.6%
identity, at least 99.8% identity, or at least 99.9% identity to at least one of the above described polynucleotides. The invention also includes isolated or recombinant nucleic acids encoding a polypeptide of hemagglutinin or neuraminidase produced by mutating or recombining one or more above described polynucleotide sequences. The polynucleotide sequences of the invention can optionally comprise one or more of, e.g., a leader sequence, VVJLl7l10/ Il/A...10 CA 02822895 2013-06-07 r /
l/0.411110/l11 Z,7 a precursor sequence, or an epitope tag sequence or the like, and can optionally encode a fusion protein (e.g., with one or more additional nucleic acid sequences).
[00101 In yet other embodiments, the invention comprises a composition of matter having two or more above described nucleic acids (e.g., a library comprising at least about 2, 5, 10, 50 or more nucleic acids). Such compositions can optionally be produced by cleaving one or more above described nucleic acid (e.g., mechanically, chemically, enzymatically with a restriction endonuclease/RNAse/DNAse, etc.). Other compositions of the invention include, e.g., compositions produced by incubating one or more above described nucleic acid in the presence of deoxyribonucleotide triphosphates and a thermostable nucleic acid polymerase.
[0011] The invention also encompasses cells comprising at least one of the above =
described nucleic acids, or a cleaved or amplified fragment or product thereof. Such cells can optionally express a polypeptide encoded by such nucleic acid. Other embodiments of the invention include vectors (e.g., plasmids, cosmids, phage, viruses, virus fragments, etc.) comprising any of above described nucleic acids. Such vectors can optionally comprise an expression vector. Preferred expression vectors of the invention include, but are not limited to, vectors comprising pol I promoter and terminator sequences or vectors using both the pol I and pol II promoters "the poll/polII promoter system" (e.g., Zobel et al., Nucl. Acids Res.
1993, 21:3607; US20020164770; Neumann et al., Proc. Natl. Acad. Sci. USA 1999, 96:9345; Fodor et al., J. Virol. 1999, 73:9679; and US20030035814). Cells transduced by such vectors are also within the current invention.
[00121 In some embodiments, the invention encompasses a virus (e.g., an influenza virus) comprising one or more above described nucleic acids (e.g., encoding hemagglutinin and/or ncuraminidase), or one or more fragments thereof. Immunogenic compositions comprising such virus are also part of the current invention. Such viruses can comprises a reassortment virus such as a 6:2 reassortment virus (e.g., comprising 6 gene encoding regions from one or more donor virus and 2 gene encoding regions from one or more above described nucleotide sequence (or one or more fragment thereof) which can optionally comprise hemagglutinin and/or neuraminidase). Reassortment viruses (optionally live viruses) of the invention can include donor viruses that are one or more of, e.g., cold-sensitive, cold-adapted, or an attenuated. For example, reassortment viruses can comprise e.g., A/Ann Arbor/6/60, PR8, etc. Reassortment viruses of the invention may alternatively t/ L0(1:1/1162M CA 02822895 2013-06-07 PCT/US2005/9177i9 exclude A/Ann Arbor/6/60. One preferred embodiment of the invention is a reassortant influenza virus, wherein the virus is a 6:2 reassortment influenza virus and comprises 6 gene =
encoding regions from A/Ann Arbor/6/60 and 2 gene encoding regions that encode a polypeptide selected from the group consisting of: the polypeptides of SEQ ED
NOS :11-20.
= 5 In an alternative embodiment, a reassortant influenza virus of the invention includes a 6:2 reassortment influenza virus, wherein said virus comprises 6 gene encoding regions from one or more donor viruses other than A/Ann Arbor/6/60 and 2 gene encoding regions that encode a polypeptide selected from the group consisting of: the polypeptides of SEQ ID
= NOS:11-20. In another alternative embodiment, a reassortant influenza virus of the invention includes a 6:2 reassortment influenza virus, wherein said virus comprises 6 gene encoding regions from one or more donor viruses other than A/Ann Arbor/6/60 and 2 gene encoding regions, wherein the 2 gene encoding regions are HA or NA
polypeptides from any pandemic influenza strain. Methods of producing recombinant influenza virus through culturing a host cell harboring an influenza virus in a suitable culture medium under conditions permitting expression of nucleic acid and, isolating the recombinant influenza = virus from one or more of the host cell or the medium are also part of the invention.
[0013] In other embodiments herein, the invention comprises immunogenic compositions having an immunologically effective amount of any of the above described recombinant influenza virus. Other embodiments include methods for stimulating the immune system of an individual to produce a protective immune response against influenza virus by administering to the individual an immunologically effective amount of any of the recombinant influenza virus described above (optionally in a physiologically effective carrier).
[0014] Other aspects of the invention include methods of producing an isolated or recombinant polypeptide by culturing any host cell above, in a suitable culture medium under conditions permitting expression of nucleic acid and, isolating the polypeptide from one or more of the host cells or the medium in which is the cells are grown.
[0015] Immunogenic compositions are also features of the invention. For example, immunogenic compositions comprising one or more of any of the polypeptides and/or nucleic acids described above and, optionally, an excipient such as a pharmaceutically acceptable excipient or one or more pharmaceutically acceptable administration component.
= Immunogenic compositions of the invention can also comprise any one or more above '=VV Ll1110/ 1 ItILOO CA 02822895 2013-06-07 1 /
I.MLUMVUI / /279 described virus as well (e.g., along with one or more pharmaceutically acceptable administration component).
[00161 Methods of producing immunogenic responses in a subject through = administration of an effective amount of any of the above viruses (or immunogenic compositions) to a subject are also within the current invention.
Additionally, methods of prophylactic or therapeutic treatment of a viral infection (e.g., viral influenza) in a subject through administration of any one or more above described virus (or immunogenic compositions) in an amount effective to produce an immunogenic response against the viral infection are also part of the current invention. Subjects for such treatment can include mammals (e.g., humans). Such methods can also comprise in vivo administration to the subject as well as in vitro or ex vivo administration to one or more cells of the subject.
Additionally, such methods can also comprise administration of a composition of the virus and a pharmaceutically acceptable excipient that are administered to the subject in an amount effect to prophylactically or therapeutically treat the viral infection.
[0017] In other aspects the invention includes compositions of matter comprising nucleic acid sequences encoding hemagglutinin and/or neuraminidase polypeptides of one or more pandemic influenza strain and nucleic acid sequences encoding one or more polypeptide of A/Ann Arbor/6/60. Additionally, the invention includes compositions of matter comprising nucleic acid sequences encoding hemagglutinin and/or neuraminidase polypeptides of one or more pandemic influenza strain and nucleic acid sequences encoding one or more polypeptide of PR8 or A/Ann Arbor/6/60. Such sequences can include those listed in the Sequence Listing herein. Additionally, preferred embodiments of the invention include compositions of matter comprising sequences encoding hemagglutinin and/or neuraminidase of one or more pandemic influenza strain and nucleic acid sequences encoding a selected backbone strain in a 6:2 reassortment. Such compositions preferably include sequences encoding the hemagglutinin and neuraminidase selected from the Sequence Listing herein and a backbone strain, wherein the backbone strain is PR8 or A/Ann Arbor/6/60. The invention also includes such compositions as described above wherein the hemagglutinin comprises a modified polybasic cleavage site. The invention also includes live attenuated influenza vaccine comprising such above compositions.

[0018] These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and appendix.
[0018a] Specific aspect of the invention include:
- a reassortant influenza virus, wherein said virus comprises 6 internal genome segments from one or more donor influenza viruses other than A/Ann Arbor/6/60, a first genome segment and a second genome segment encoding surface antigens, wherein the first genome segment encodes an HA polypeptide comprising the amino acid sequence of SEQ ID
NO:15;
- an immunogenic composition comprising an immunologically effective amount of the reassortant influenza virus as described herein in a carrier or diluent;
- the reassortant influenza virus as described herein for use in stimulating the immune system of a subject to produce a protective immune response against influenza virus, wherein the reassortant influenza virus is to be administered to the subject in an immunologically effective amount and in a physiologically effective carrier;
- the reassortant influenza virus as described herein for use in prophylactic treatment of a viral infection in a subject, wherein the virus is in an amount effective to produce an immunogenic response against the viral infection;
- a split virus or killed virus vaccine comprising the immunogenic composition as described herein;
- a live attenuated influenza vaccine comprising the composition as described herein;
- a method for producing influenza viruses in cell culture, the method comprising: i) introducing into a population of host cells, which population of host cells is capable of supporting replication of influenza virus, a plurality of vectors comprising nucleic acid sequences corresponding to at least 6 internal genome segments from one or more influenza donor virus other than A/Ann Arbor/6/60, a first genome segment and a second genome segment encoding immunogenic influenza surface antigens, wherein the first genome segment encodes a hemagglutinin polypeptide comprising the amino acid sequence of SEQ ID
NO: 15; ii) culturing the population of host cells at a temperature less than or equal to 35 C;
and iii) recovering a plurality of influenza viruses from the cultured cells;
and -a reassortant influenza virus as described herein for use in immunizing a subject against an influenza virus infection.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1: Shows modifications engineered into the HA gene of to remove the polybasic cleavage site.
[0020] Figure 2: Displays results showing that intranasally administered H5N1 ca reassortant viruses do not replicate in chickens.
[0021] Figure 3: Illustrates that the H5N1/AA ca vaccine candidates are not lethal to mice.
[0022] Figure 4: Illustrates that the 1997 and 2004 H5N1 ca reassortant viruses are restricted in replication in mice.
[0023] Figure 5: Illustrates that the reassortant H5N1/AA ca influenza viruses are restricted in replication in lungs of mice.
[0024] Figure 6: Shows the serum HAI Ab titers elicited in mice following a single i.n. dose of vaccine.
[0025] Figure 7: Shows serum neutralizing Ab titers elicited in mice following a single i.n. dose of vaccine.
[00261 Figure 8: Illustrates that H5N1 ca reassortant viruses protect mice from lethal challenges with 50,500 or 5000 LD50 of wild-type H5N1 viruses.
8a [0027] Figure 9: Illustrates the efficacy of protection from pulmonary replication of homologous and heterologous H5N1 challenge viruses in mice.
[0028] Figure 10: Illustrates the efficacy of protection from replication of homologous and heterologous H5N1 challenge viruses in the upper respiratory tract of mice.
[0029] Figure 11: Illustrates the efficacy of protection conferred by 2004 H5N1 ca vaccine against high dose (105TCID50) challenge with homologous or heterologous H5N1 wt viruses in mice.
8b 'VV 1-1 LIMO/ 111.11LDO CA 02822895 2013-06-07 rt_iivazuruzvui [00301 Figure 12: Illustrates the efficacy of protection conferred by 1997 and 2003 H5N1 ca vaccines against high dose (105TCID50) challenges with homologous or heterologous H5N1 wild-type viruses in mice.
[0031] Figure 13: Illustrates the efficacy of protection conferred by 2004 H5N1 ca vaccine against low or high doses of homologous H5N1 wild-type virus challenges in mice.
DETAILED DESCRIPTION
[0032] The present invention includes polypeptide and polynucleotide sequences of influenza hemagglutinin and neuraminidase as well as vectors, compositions and the like comprising such sequences and methods of their use. Additional features of the invention are described in more detail herein.
DEFINITIONS
[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. The following definitions supplement those in the art and are directed to the current application and are not necessarily to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.
Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
[0034] As used in this specification and the appended claims, the singular forms "a,"
"an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a virus" includes a plurality of viruses; reference to a "host cell"
includes mixtures of host cells, and the like.
[0035] An "amino acid sequence" is a polymer of amino acid residues (a protein, =
polypeptide, etc.) or a character string representing an amino acid polymer, depending on context.

viO 2005/116258 CA 02822895 2013-06-07 [0036] The terms "nucleic acid," "polynucleotide,"
"polynucleotide sequence" and "nucleic acid sequence" refer to single-stranded or double-stranded deoxyribonucleotide or ribonucleotide polymers, chimeras or analogues thereof, or a character string representing such, depending on context. As used herein, the term optionally includes polymers of analogs of naturally occurring nucleotides having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids). Unless otherwise indicated, _a particular nucleic acid sequence of this invention optionally encompasses complementary sequences in addition to the sequence explicitly indicated. From any specified polynucleotide sequence, either the given nucleic acid or the complementary polynucleotide sequence (e.g., the complementary nucleic acid) can be determined.
[0037] The term "nucleic acid" or "polynucleotide" also 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'-0-methylated oligonucleotides), and the like. A
nucleic acid can be e.g., single-stranded or double-stranded.
[0038] A "subsequence" is any portion of an entire sequence, up to and including =
the complete sequence. Typically, a subsequence comprises less than the full-length sequence. A "unique subsequence" is a subsequence that is not found in any previously determined influenza polynucleotide or polypeptide sequence = [0039] The term "variant" with respect to a polypeptide refers to an amino acid sequence that is altered by one or more amino acids with respect to a reference sequence.
The variant can have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine.
Alternatively, a variant can have "nonconservative" changes, e.g., replacement of a glycine = with a tryptophan. Analogous minor variation can also include amino acid deletion or insertion, or both. Guidance in determining which amino acid residues can be substituted, = inserted, or deleted without eliminating biological or immunological activity can be found using computer programs well known in the art, for example, DNASTAR software.
Examples of conservative substitutions are also described herein.

VV LIMO/ 11UL-10 rt- 1 /
IJOLl/k/ZI/l/1 / 1 .V7 [0040] The term "gene" is used broadly to refer to any nucleic acid associated with a biological function. Thus, genes include coding sequences and/or the regulatory sequences required for their expression. The term "gene" applies to a specific genomic sequence, as well as to a cDNA or an mRNA encoded by that genomic sequence.
[0041] Genes also include non-expressed nucleic acid segments that, for example, form recognition sequences for other proteins. Non-expressed regulatory sequences include "promoters" and "enhancers," to which regulatory proteins such as transcription factors bind, resulting in transcription of adjacent or nearby sequences. A "tissue specific"
promoter or enhancer is one that regulates transcription in a specific tissue type or cell type, or types.
[0042] "Expression of a gene" or "expression of a nucleic acid" means transcription of DNA into RNA (optionally including modification of the RNA, e.g., splicing), translation of RNA into a polypeptide (possibly including subsequent modification of the polypeptide, e.g., post-translational modification), or both transcription and translation, as indicated by the context.
[0043] An "open reading frame" or "ORF" is a possible translational reading frame of DNA or RNA (e.g., of a gene), which is capable of being translated into a polypeptide.
That is, the reading frame is not interrupted by stop codons. However, it should be noted that the term ORF does not necessarily indicate that the polynucleotide is, in fact, translated into a polypeptide.
[0044] The term "vector" refers to the means by which a nucleic acid can be .
propagated and/or transferred between organisms, cells, or cellular components. Vectors include plasmids, viruses, bacteriophages, pro-viruses, phagemids, transposons, artificial chromosomes, and the like, that replicate autonomously or can integrate into a chromosome of a host cell. A vector can also be a naked RNA polynucleotide, a naked DNA
polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like, that is not autonomously replicating. In many, but not all, common embodiments, the vectors of the present invention are plasmids.
[0045] An "expression vector" is a vector, such as a plasmid that is capable of promoting expression, as well as replication of a nucleic acid incorporated therein.

,O 2005/116258 CA 02822895 2013-06-07 Typically, the nucleic acid to be expressed is "operably linked" to a promoter and/or enhancer, and is subject to transcription regulatory control by the promoter and/or enhancer.
." [0046] A "bi-directional expression vector" is characterized by two alternative promoters oriented in the opposite direction relative to a nucleic acid situated between the two promoters, such that expression can be initiated in both orientations resulting in, e.g., transcription of both plus (+) or sense strand, and negative (-) or antisense strand RNAs.
[0047] A "polypeptide" is a polymer comprising two or more amino acid residues (e.g., a peptide or a protein). The polymer can optionally comprise modifications such as glycosylation or the like. The amino acid residues of the polypeptide can be natural or non-natural and can be unsubstituted, unmodified, substituted or modified.
[0048] In the context of the invention, the term "isolated" refers to a biological = material, such as a virus, a nucleic acid or a protein, which is substantially free from components that normally accompany or interact with it in its naturally occurring environment. The isolated biological material optionally comprises additional material not = 15 found with the biological material in its natural environment, e.g., a cell or wild-type virus.
For example, if the material is in its natural environment, such as a cell, the material can have been placed at a location in the cell (e.g., genome or genetic element) not native to such material found in that environment. For example, a naturally occurring nucleic acid (e.g., a coding sequence, a promoter, an enhancer, etc.) becomes isolated if it is introduced by non-naturally occurring means to a locus of the genome (e.g., a vector, such as a plasmid or virus vector, or amplicon) not native to that nucleic acid. Such nucleic acids are also referred to as "heterologous" nucleic acids. An isolated virus, for example, is in an environment (e.g., a cell culture system, or purified from cell culture) other than the native environment of wild-type virus (e.g., the nasopharynx of an infected individual).
[0049] The term "chimeric" or "chimera," when referring to a virus, indicates that the virus includes genetic and/or polypeptide components derived from more than one = parental viral strain or source. Similarly, the term "chimeric" or "chimera," when referring to a viral protein, indicates that the protein includes polypeptide components (i.e., amino acid subsequences) derived from more than one parental viral strain or source.
[0050] The term "recombinant" indicates that the material (e.g., a nucleic acid or protein) has been artificially or synthetically (non-naturally) altered by human intervention.

-YV Ll/t/Z1/11OLZIC. CA 02822895 2013-06-07 rt. I /
UOLl/l/D/U1 The alteration can be performed on the material within, or removed from, its natural environment or state. Specifically, e.g., an influenza virus is recombinant when it is produced by the expression of a recombinant nucleic acid. For example, a "recombinant nucleic acid" is one that is made by recombining nucleic acids, e.g., during cloning, DNA
shuffling or other procedures, or by chemical or other mutagenesis; a "recombinant polypeptide" or "recombinant protein" is a pOlypeptide or protein which is produced by expression of a recombinant nucleic acid; and a "recombinant virus", e.g., a recombinant influenza virus, is produced by the expression of a recombinant nucleic acid.
[0051] The term "reassortant," when referring to a virus, indicates that the virus includes genetic and/or polypeptide components derived from more than one parental viral strain or source. For example, a 7:1 reassortant includes 7 viral genomic segments (or gene segments) derived from a first parental virus, and a single complementary viral genomic segment, e.g., encoding a hemagglutinin or neuraminidase of the invention. A
6:2 reassortant includes 6 genomic segments, most commonly the 6 internal genes from a first parental virus, and two complementary segments, e.g., hemagglutinin and neuraminidase, from a different parental virus.
[0052] The term "introduced" when referring to a heterologous or isolated nucleic acid refers to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid can be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA). The term includes such methods as "infection,"
"transfection," "transformation" and "transduction." In the context of the invention a variety of methods can be employed to introduce nucleic acids into cells, including electroporation, calcium phosphate precipitation, lipid mediated transfection (lipofection), etc.
[0053] The term "host cell" means a cell that contains a heterologous nucleic acid, such as a vector, and supports the replication and/or expression of the nucleic acid. Host cells can be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, avian or mammalian cells, including human cells. Exemplary host cells can include, e.g., Vero (African green monkey kidney) cells, BHK (baby hamster kidney) cells, primary chick kidney (PCK) cells, Madin-Darby Canine Kidney (MDCK) cells, Madin-= 2005/116258 CA

Darby Bovine Kidney (MDBK) cells, 293 cells (e.g., 293T cells), and COS cells (e.g., . , COSI, COS7 cells), etc.
[0054] An "immunologically effective amount" of influenza virus is an amount ; sufficient to enhance an individual's (e.g., a human's) own immune response against a = 5 subsequent exposure to influenza virus. Levels of induced inummity can be monitored, e.g., by measuring amounts of neutralizing secretory and/or serum antibodies, e.g., by plaque = neutralization, complement fixation, enzyme-linked immunosorbent, or microneutralization .=
assay.
[0055] A "protective immune response" against influenza virus refers to an immune response exhibited by an individual (e.g., a human) that is protective against disease when the individual is subsequently exposed to and/or infected with such influenza virus. In some = instances, the influenza virus (e.g., naturally circulating) can still cause infection, but it cannot cause a serious infection. Typically, the protective immune response results in = detectable levels of host engendered serum and secretory antibodies that are capable of neutralizing virus of the same strain and/or subgroup (and possibly also of a different, non-vaccine strain and/or subgroup) in vitro and in vivo.
=
=
[0056] As used herein, an "antibody" is a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, = 20 alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad = immunoglobulin variable region genes. Light chains are classified as either kappa or = lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. A
typical immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 IcD) and one "heavy" chain (about 50-70 k.D). The N-terminus of each chain defines a = variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively. Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases.
Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CH1 by W) Luuzvliozzvs CA 02822895 2013-06-07 Yl_, 1 / 1 ///
a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab')2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with part of the hinge region (see, Fundamental Immunology, W.E. Paul, ed., Raven Press, N.Y. (1999), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab' fragments may be synthesized de novo either chemically or by utilizing recombinant DNA
methodology.
Thus, .the term antibody, as used herein, includes antibodies or fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA
methodologies. Antibodies include, e.g., polyclonal antibodies, monoclonal antibodies, multiple or single chain antibodies, including single chain Fv (sFy or scFv) antibodies in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide, and humanized or chimeric antibodies.
INFLUENZA VIRUS
[0057] The polypeptides and polynucleotides of the invention, e.g., SEQ ID
NO: 1-20, are variants of influenza HA and NA sequences. In general, influenza viruses are made up of an internal ribonucleoprotein core containing a segmented single-stranded RNA
genome and an outer lipoprotein envelope lined by a matrix protein. The genome of influenza viruses is composed of eight segments of linear (-) strand ribonucleic acid (RNA), encoding the immunogenic hemagglutinin (HA) and neuraminidase (NA) proteins, and six internal core polypeptides: the nucleocapsid nucleoprotein (NP); matrix proteins (M); non-structural proteins (NS); and 3 RNA polymerase (PA, PB1, PB2) proteins. During replication, the genomic viral RNA is transcribed into (+) strand messenger RNA and (-) strand genomic cRNA in the nucleus of the host cell. Each of the eight genomic segments is packaged into ribonucleoprotein complexes that contain, in addition to the RNA, NP and a polymerase complex (PB1, PB2, and PA).
100581 Influenza is commonly grouped into influenza A and influenza B
categories.
Influenza A and influenza B viruses each contain eight segments of single stranded RNA
with negative polarity. The influenza A genome encodes eleven polypeptides.
Segments 1-3 encode three polypeptides, making up a RNA-dependent RNA polymerase. Segment encodes the polymerase complex protein PB2. The remaining polymerase proteins PB1 and w0 2005/116258 CA 02822895 2013-06-07 PA are encoded by segment 2 and segment 3, respectively. In addition, segment 1 of some influenza strains encodes a small protein, PB1-F2, produced from an alternative reading =
frame within the PB1 coding region. Segment 4 encodes the hemagglutinin (HA) surface glycoprotein involved in cell attachment and entry during infection. Segment 5 encodes the nucleocapsid nucleoprotein (NP) polypeptide, the major structural component associated with viral RNA. Segment 6 encodes a neuraminidase (NA) envelope glycoprotein.
_ Segment 7 encodes two matrix proteins, designated M1 and M2, which are translated from differentially spliced mRNAs. Segment 8 encodes NS I and NS2, two nonstructural proteins, which are translated from alternatively spliced mRNA variants. The eight genome = 10 segments of influenza B encode 11 proteins. The three largest genes code for components of the RNA polymerase, PB1, PB2 and PA. Segment 4 encodes the HA protein.
Segment 5 encodes NP. Segment 6 encodes the NA protein and the NB protein. Both proteins, NB and NA, are translated from overlapping reading frames of a bicistronic mRNA.
Segment 7 of influenza B also encodes two proteins: M1 and BM2. The smallest segment encodes two products: NS1 is translated from the full length RNA, while NS2 is translated from a spliced mRNA variant.
INFLUENZA VIRUS VACCINES
= 100591 The sequences, compositions and methods herein are primarily, but not solely, concerned with production of influenza viruses for vaccines.
Historically, influenza virus vaccines have primarily been produced in embryonated hen eggs using strains of virus selected or based on empirical predictions of relevant strains. More recently, reassortant = viruses have been produced that incorporate selected hemagglutinin and/or neuraminidase = antigens in the context of an approved attenuated, temperature sensitive master strain.
Following culture of the virus through multiple passages in hen eggs, influenza viruses are recovered and, optionally, inactivated, e.g., using formaldehyde and/or P-propiolactone (or alternatively used in live attenuated vaccines). Thus, it will be appreciated that HA and NA
sequences (e.g., SEQ ID NO: 1-20) are quite useful in constructing influenza vaccines. The current invention includes viruses/vaccines comprising HA and/or NA sequences of =
pandemic influenza strains (including wherein the HA sequences comprise modified polybasic cleavage sites such as the modifications described herein); and including wherein the viruses/vaccines comprise a ca backbone such as A/AA/6/60 or the backbone of PRS.

ZdUlf.), I ju./.00 [0060] Attempts at producing recombinant and reassortant vaccines in cell culture have been hampered by the inability of some of the strains approved for vaccine production to grow efficiently under standard cell culture conditions. However, prior work by the inventors and their coworkers provided a vector system, and methods for producing recombinant and reassortant viruses in culture, thus, making it possible to rapidly produce vaccines corresponding to one or many selected antigenic strains of virus, e.g., either A or B
strains, various subtypes or substrains, etc., e.g., comprising the HA and/or NA sequences herein. See, Multi-Plasmid System for the production of Influenza virus, U.S.
Application No. 60/420,708, filed October 23, 2002, U.S. Application No. 10/423,828, filed April 25, 2003 and U.S. Application 60/574,117 filed May 24, 2004. Typically, the cultures are maintained in a system, such as a cell culture incubator, under controlled humidity and CO2, at constant temperature using a temperature regulator, such as a thermostat to insure that the temperature does not exceed 35 C. Reassortant influenza viruses can be readily obtained by introducing a subset of vectors corresponding to genomic segments of a master influenza virus, in combination with complementary segments derived from strains of interest (e.g., HA and/or NA antigenic variants herein). Typically, the master strains are selected on the basis of desirable properties relevant to vaccine administration. For example, for vaccine production, e.g., for production of a live attenuated vaccine, the master donor virus strain may be selected for an attenuated phenotype, cold adaptation and/or temperature sensitivity.
As explained elsewhere herein and, e.g., in U.S. Patent Application No.

10/423,828, etc., various embodiments of the invention utilize A/Ann Arbor (AA)/6/60 influenza strain as a "backbone" upon which to add HA and/or NA genes (e.g., such as those sequences listed herein, etc.) to create desired reassortant viruses. Thus, for example, in a 6:2 reassortant, 2 genes (i.e., NA and HA) would be from the influenza strain(s) against which an immunogenic reaction is desired, while the other 6 genes would be from the Ann Arbor strain, or other backbone strain, etc. The Ann Arbor virus is useful for its cold adapted, attenuated, temperature sensitive attributes. Of course, it will be appreciated that the HA
and NA sequences herein are capable of reassortment with a number of other virus genes or virus types (e.g., a number of different "backbones" such as PR8, etc., containing the other Influenza genes present in a reassortant, namely, the non-HA and non-NA genes [0061] Various embodiments herein can comprise live attenuated vaccines, having the HA and/or NA sequences herein, for pandemic influenza. Such vaccines typically = 11 0 2005/116258 CA 02822895 2013-06-07 PCT/US2005/017729 comprise, e.g., the HA and/or NA sequences of SEQ ID NO: 11-20, or their corresponding nucleotides of SEQ ID NO: 1-10. One problem arising from growth of vaccine virus strains =
(e.g., reassortants) in eggs is that avian strains (which can be involved in pandemics) can kill the eggs in which the vaccines are to be produced and are, thus, hard to manipulate, produce, etc. through use of traditional (non-plasmid rescue) reassortant production. Such avian strains are of interest since evidence indicate; they can result in influenza in humans and possible pandemics. Thus, use of plasmid-rescue systems to create/manipulate influenza reassortants with pandemic strains such as various avian sequences (e.g., the HA
and NA sequences herein) are quite desirable and are features of the invention. It will be appreciated, however, that the current sequences are also capable of use with non-plasmid or traditional systems.
[0062]
Aquatic birds (among others) can be infected by influenza A viruses of hemagglutinin (HA) and 9 neuraminidase (NA) subtypes. Such birds can serve as a reservoir from which novel influenza subtypes can be introduced into human populations and cause pandemics. The observation that avian H7N7 influenza A viruses infected humans in The Netherlands in 2003 and avian H5N1 and H9N2 viruses infected humans in Hong Kong and China earlier, raise concerns that these (and other) subtypes have the potential to cause pandemics. Thus, vaccines are needed to prevent human infections with avian influenza A viruses. Live, attenuated influenza A virus vaccines against human = 20 influenza viruses were recently licensed in the United States. See above. Such vaccines are reassortant H1N1 and H3N2 viruses in which the internal protein genes of A/Ann Arbor (AA)/6/60 (H2N2) cold adapted (ca) virus confer the cold adapted, attenuation and temperature sensitive phenotypes of the AA ca virus on the reassortant viruses (i.e., the ones having the hemagglutinin and neuraminidase genes from the non-Ann Arbor strain).
Classical genetic reassortment and plasmid-based reverse genetics techniques have been applied to generate reassortant viruses that contain the hemagglutinin and neuraminidase genes from avian influenza A viruses (H4-H14 subtypes) and six internal gene segments =
from the AA ca virus. Such reassortant viruses are features of the invention.
See the HA
and NA gene sequences below. These viruses bear biological properties that are desirable = 30 in candidate vaccines because the phenotypes associated with the AA ca virus are present in the reassortant viruses. The generation and evaluation of these reassortant viruses as seed viruses for vaccines are important steps in pandemic preparedness. It is contemplated that WO 200:1/116258 CA 02822895 2013-06-07 /US2005/(117729 clinical trials can establish the safety, infectivity and immunogenicity of such live attenuated pandemic vaccines. Other embodiments of the invention include reassortant viruses (e.g., those used in vaccines) comprising pandemic antigenic genes HA
and/or NA
from, e.g., avian, porcine, etc., pandemic virus strains in addition to those listed herein, to produce pandemic vaccines which are created through plasmid-rescue reassortment (e.g., reassortment with A/Ann Arbor 6/60 (i.e., A/AAJ6/60), PR8, etc. Methods of construction and use of such viruses and vaccines are also included. "Pandemic virus strains" as used herein is defined as an influenza strain A virus subtype that it is not circulating in the human population, that is declared to be a pandemic strain by the Centers for Disease Control or the 100631 In various embodiments herein, the antigenic sequences (e.g., the HA
sequences) as well as viruses and vaccines from such viruses comprise modified polybasic cleavage sites. Some highly pathogenic avian pandemic influenza strains comprise multiple basic amino acid cleavage sites within hemagglutinin sequences. See, e.g., Li et al., J. of Infectious Diseases, 179:1132-8, 1999. Such cleavage sites, in typical embodiments herein, are, e.g., modified or altered in their sequences in comparison to the wild-type sequences from which the current sequences are derived (e.g., to disable the cleavage or reduce the cleavage there, etc.). Such modifications/alterations can be different in different strains due hemagglutinin polypeptide sequences of the invention include both the mature (amino terminal signal peptide cleaved) form of the hemagglutinin polypeptides and the pre-cleaved .$.
form of hemagglutinin. The cleavage sites of any hemagglutinin polypeptide sequence of = any influenza strain can be routinely measured or predicted using any number of methods in the art.
=[00641 The terms "temperature sensitive," "cold adapted" and "attenuated"
as =
applied to viruses (typically used as vaccines or for vaccine production) which optionally encompass the current sequences, are well known in the art. For example, the term "temperature sensitive" (ts) indicates, e.g., that the virus exhibits a 100 fold or greater reduction in titer at 39 C relative to 33 C for influenza A strains, or that the virus exhibits a 100 fold or greater reduction in titer at 37 C relative to 33 C for influenza B strains. The term "cold adapted" (ca) indicates that the virus exhibits growth at 25 C
within 100 fold of its growth at 33 C, while the term "attenuated" (att) indicates that the virus replicates in the upper airways of ferrets but is not detectable in their lung tissues, and does not cause influenza-like illness in the animal. It will be understood that viruses with intermediate phenotypes, i.e., viruses exhibiting titer reductions less than 100 fold at 39 C (for A strain viruses) or 37 C (for B strain viruses), or exhibiting growth at 25 C that is more than 100 =
fold than its growth at 33 C (e.g., within 200 fold, 500 fold, 1000 fold, 10,000 fold less), and/or exhibit reduced growth in the lungs relative to growth in the upper airways of ferrets (i.e., partially attenuated) and/or reduced influenza like illness in the animal, are also useful viruses and can be used in conjunction with the HA and NA sequences herein.
100651 Again, the HA and NA sequences of the current invention are optionally utilized in such plasrnid reassortment vaccines (and/or in other ts, cs, ca, and/or att viruses = and vaccines). However, it should be noted that the HA and NA sequences, etc. of the = invention are not limited to specific vaccine compositions or production methods, and can, thus, be utilized in substantially any vaccine type or vaccine production method which utilizes strain specific HA and NA antigens (e.g., any of SEQ ID NO: 11-20 or the corresponding nucleotides in SEQ ID NO: 1-10).
= FLUMISTTm [0066] As mentioned previously, numerous examples and types of influenza vaccine exist. An exemplary influenza vaccine is FluMistTm which is a live, attenuated vaccine that protects children and adults from influenza illness (Belshe et al. (1998) The efficacy of live = attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine in children N Engl J

VV LUU:71/1.10LNS CA 02822895 2013-06-07 ft- I/ U
SL00:1/01 / IZJ
Med 338:1405-12; Nichol et al. (1999) Effectiveness of live, attenuated intranasal influenza virus vaccine in healthy, working adults: a randomized controlled trial JAMA
282:137-44).
In typical, and preferred, embodiments, the methods and compositions of the current invention are preferably adapted to/used with production of FluMistTm vaccine.
However, it will be appreciated by those skilled in the art that the sequences, methods, compositions, etc. herein are also adaptable to production of similar or even different viral vaccines.
100671 FluMistTm vaccine strains contain, e.g., HA and NA gene segments derived from the strains (e.g., wild-type strains) to which the vaccine is addressed along with six.
gene segments, PB1, PB2, PA, NP, M and NS, from a common master donor virus (IvIDV).
The HA sequences herein, thus, are part of various FluMistTm formulations. The MDV for influenza A strains of FluMistTm (MDV-A), was created by serial passage of the wild-type A/Ann Arbor/6/60 (A/AA/6/60) strain in primary chicken kidney tissue culture at successively lower temperatures (Maassab (1967) Adaptation and growth characteristics of influenza virus at 25 degrees C Nature 213:612-4). MDV-A replicates efficiently at 25 C
(ca, cold adapted), but its growth is restricted at 38 and 39 C (ts, temperature sensitive).
Additionally, this virus does not replicate in the lungs of infected ferrets (att, attenuation).
The Is phenotype is believed to contribute to the attenuation of the vaccine in humans by restricting its replication in all but the coolest regions of the respiratory tract. The stability of this property has been demonstrated in animal models and clinical studies.
In contrast to the ts phenotype of influenza strains created by chemical mutagenesis, the ts property of MDV-A does not revert following passage through infected hamsters or in shed isolates from children (for a recent review, see Murphy & Coelingh (2002) Principles underlying the development and use of live attenuated cold-adapted influenza A and B
virus vaccines Viral Immunol 15:295-323).
[0068] Clinical studies in over 20,000 adults and children involving 12 separate 6:2 reassortant strains have shown that these vaccines are attenuated, safe and efficacious (Belshe et al. (1998) The efficacy of live attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine in children N Engl J Med 338:1405-12; Boyce et al.
(2000) Safety and immunogenicity of adjuvanted and unadjuvanted subunit influenza vaccines administered intranasally to healthy adults Vaccine 19:217-26; Edwards et al.
(1994) A
randomized controlled trial of cold adapted and inactivated vaccines for the prevention of influenza A disease J Infect Dis 169:68-76 ; Nichol et al. (1999) Effectiveness of live, = 40 2005/116258 CA

attenuated intranasal influenza virus vaccine in healthy, working adults: a randomized . controlled trial JAMA 282:137-44). Reassortants carrying the six internal genes of MD V-, A and the two HA and NA gene segments of a wild-type virus (i.e., a 6-.2 reassortant) consistently maintain ca, ts and att phenotypes (Maassab et al. (1982) Evaluation of a cold-õ
recombinant influenza virus vaccine in ferrets J. Infect. Dis, 146:780-900).
[0069] Production of such reassorted virus using B strains of influenza is more difficult, however, recent work (see, e.g., Multi-Plasmid System for the Production of Influenza Virus, U.S. Application No. 60/420,708, filed October 23, 2002, U.S.
Application No. 10/423,828, filed April 25, 2003, and U.S. Application No. 60/574,117, filed May 24, 2004) has shown an eight plasmid system for the generation of influenza B
virus entirely from cloned cDNA. Methods for the production of attenuated live influenza A
and B virus = suitable for vaccine formulations, such as live virus vaccine formulations useful for intranasal administration were also shown.
= [0070] The system and methods described previously are useful for the rapid production in cell culture of recombinant and reassortant influenza A and B
viruses, = including viruses suitable for use as vaccines, including live attenuated vaccines, such as vaccines suitable for intranasal administration. The sequences (e.g., SEQ ID
NO: 1-10 and = the corresponding amino acids in SEQ ID NO: 11-20), methods, etc. of the current invention, are optionally used in conjunction with, or in combination with, such previous = 20 work involving, e.g., reassorted influenza viruses for vaccine production to produce viruses for vaccines.
METHODS AND COMPOSITIONS FOR PROPHYLACTIC ADMINISTRATION OF
VACCINES
100711 As stated above, alternatively, or in addition to, use in production of FluMistTm vaccine, the current invention can be used in other vaccine formulations. In = general, recombinant and reassortant viruses of the invention (e.g., those comprising = polynucleotides of SEQ ID NO:1-10 or polypeptides of SEQ ID NO:11-20, or fragments = thereof) can be administered prophylactically in an immunologically effective amount and in an appropriate carrier or excipient to stimulate an immune response specific for one or more strains of influenza virus as determined by the HA and/or NA sequence.
Typically, the carrier or excipient is a pharmaceutically acceptable carrier or excipient, such as sterile WU LUM1/110.LN5 CA 02822895 2013-06-07 rt. / U /1.13/l/
1 / I Mr water, aqueous saline solution, aqueous buffered saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, ethanol, or combinations thereof. The preparation of such solutions insuring sterility, pH, isotonicity, and stability is effected according to protocols established in the art. Generally, a carrier or excipient is selected to minimize allergic and other undesirable effects, and to suit the particular route of administration, e.g., subcutaneous, intramuscular, intranasal, etc.
10072] A related aspect of the invention provides methods for stimulating the -immune system of an individual to produce a protective immune response against influenza virus. In the methods, an immunologically effective amount of a recombinant influenza virus (e.g., comprising an HA and/or NA molecule of the invention), an immunologically effective amount of a polypeptide of the invention, and/or an immunologically effective amount of a nucleic acid of the invention is administered to the individual in a physiologically acceptable carrier.
100731 Generally, the influenza viruses of the invention are administered in a quantity sufficient to stimulate an immune response specific for one or more strains of influenza virus (i.e., against the HA and/or NA strains of the invention).
Preferably, administration of the influenza viruses elicits a protective immune response to such strains.
Dosages and methods for eliciting a protective immune response against one or more influenza strains are known to those of skill in the art. See, e.g., USPN
5,922,326; Wright et al., Infect. Immun. 37:397-400 (1982); Kim et al., Pediatrics 52:56-63 (1973); and Wright et al., J. Pediatr. 88:931-936 (1976). For example, influenza viruses are provided in the range of about 1-1000 HLD50(human infectious dose), i.e., about 105 - 108 pfii (plaque forming units) per dose administered. Typically, the dose will be adjusted within this range based on, e.g., age, physical condition, body weight, sex, diet, time of administration, and other clinical factors. The prophylactic vaccine formulation is systemically administered, e.g., by subcutaneous or intramuscular injection using a needle and syringe, or a needle-less injection device. Alternatively, the vaccine formulation is administered intranasally, either by drops, large particle aerosol (greater than about 10 microns), or spray into the upper respiratory tract. While any of the above routes of delivery results in a protective systemic immune response, intranasal administration confers the added benefit of eliciting mucosal immunity at the site of entry of the influenza virus. For intranasal administration, attenuated live virus vaccines are often preferred, e.g., an attenuated, cold adapted and/or w02005/116258 CA 02822895 2013-06-07 temperature sensitive recombinant or reassortant influenza virus. See above.
While stimulation of a protective immune response with a single dose is preferred, additional dosages can be administered, by the same or different route, to achieve the desired prophylactic effect.
[0074] Typically, the attenuated recombinant influenza of this invention as used in a vaccine is sufficiently attenuated such that symptoms of infection, or at least symptoms of serious infection, will not occur in most individuals immunized (or otherwise infected) with the attenuated influenza virus. In some instances, the attenuated influenza virus can still be capable of producing symptoms of mild illness (e.g., mild upper respiratory illness) and/or of dissemination to unvaccinated individuals. However, its virulence is sufficiently abrogated such that severe lower respiratory tract infections do not occur in the vaccinated or incidental host.
100751 Alternatively, an immune response can be stimulated by ex vivo or in vivo targeting of dendritic cells with influenza viruses comprising the sequences herein. For =
example, proliferating dendritic cells are exposed to viruses in a sufficient amount and for a sufficient period of time to permit capture of the influenza antigens by the dendritic cells.
The cells are then transferred into a subject to be vaccinated by standard intravenous transplantation methods.
[0076] While stimulation of a protective immune response with a single dose is . .
preferred, additional dosages can be administered, by the same or different route, to achieve = the desired prophylactic effect. In neonates and infants, for example, multiple administrations may be required to elicit sufficient levels of immunity.
Administration can continue at intervals throughout childhood, as necessary to maintain sufficient levels of protection against wild-type influenza infection. Similarly, adults who are particularly susceptible to repeated or serious influenza infection, such as, for example, health care workers, day care workers, family members of young children, the elderly, and individuals with compromised cardiopulmonary function may require multiple immunizations to establish and/or maintain protective immune responses. Levels of induced immunity can be monitored, for example, by measuring amounts of neutralizing secretory and serum = 30 antibodies, and dosages adjusted or vaccinations repeated as necessary to elicit and maintain = desired levels of protection.

[0077] Optionally, the formulation for prophylactic administration of the influenza viruses also contains one or more adjuvants for enhancing the immune response to the influenza antigens. Suitable adjuvants include: complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, bacille Calmette-Guerin (BCG), Colynebacterium parvum, and the synthetic adjuvant QS-21.
[0078] If desired, prophylactic vaccine administration of influenza viruses can be performed in conjunction with adthiriitratión of one or More immunostimulatory molecules. Immunostimulatory molecules include various cytokines, lymphokines and chemokines with immunostimulatory, immunopotentiating, and pro-inflammatory activities, such as interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors (e.g., granulocyte-macrophage (GM)-colony stimulating factor (CSF)); and other immunostimulatory molecules, such as macrophage inflammatory factor, F1t3 ligand, B7.1;
B7.2, etc. The immunostimulatory molecules can be administered in the same formulation as the influenza viruses, or can be administered separately. Either the protein (e.g., an HA
and/or NA polypeptide of the invention,e.g., any of SEQ ID NO: 11-20) or an expression vector comprising a nucleic acid (e.g., any of SEQ ID NO: 1-10) encoding the protein can be administered to produce an immunostimulatory effect.
[0079] The above described methods are useful for therapeutically and/or prophylactically treating a disease or disorder, typically influenza, by introducing a vector of the invention comprising a heterologous polynucleotide encoding a therapeutically or prophylactically effective HA and/or NA polypeptide (or peptide) or HA and/or NA RNA
(e.g., an antisense RNA or ribozyme) into a population of target cells in vitro, ex vivo or in vivo. Typically, the polynucleotide encoding the polypeptide (or peptide), or RNA, of interest is operably linked to appropriate regulatory sequences as described above in the sections entitled "Expression Vectors" and "Additional Expression Elements."
Optionally, more than one heterologous coding sequence is incorporated into a single vector or virus.
For example, in addition to a polynucleotide encoding a therapeutically or prophylactically active HA and/or NA polypeptide or RNA, the vector can also include additional therapeutic or prophylactic polypeptides, e.g., antigens, co-stimulatory molecules, cytokines, antibodies, etc., and/or markers, and the like.

w02005/116238 CA 02822895 2013-06-07 [0080] Although vaccination of an individual with an attenuated influenza virus of a =
particular strain of a particular subgroup can induce cross-protection against influenza virus of different strains and/or subgroups, cross-protection can be enhanced, if desired, by = vaccinating the individual with attenuated influenza virus from at least two strains, e.g., = 5 each of which represents a different subgroup. Additionally, vaccine combinations can optionally include mixes of pandemic vaccines (e.g., those against pandemic influenza strains such as various avian strains, see, e.g., the sequences herein, or other pandemic strains) and non-pandemic strains. Vaccine mixtures (or multiple vaccinations) can = comprise components from human strains and/or non-human influenza strains (e.g., avian and human, etc.). Similarly, the attenuated influenza virus vaccines of this invention can optionally be combined with vaccines that induce protective immune responses against other infectious agents.
POLYNUCLEOTIDES OF THE INVENTION
100811 It is well known in the art that the HA and NA polynucleotide segments of influenza = viruses comprise both a coding region (encoding the ORF) and noncoding regions (NCRs), both 5' and 3' of the HA and NA coding sequence. An example of these NCRs are shown in SEQ ID NOS:1-9 (outside of the ORFs). It is also known that primers can be made to these NCRs to facilitate amplification of the entire HA and NA segments of influenza virus.
.=
(see, e.g., Hoffmann et al. Arch Virol. 2001 Dec;146(12):2275-89). Further, it is known that the NCRs of the HA and NA of influenza may increase the efficiency of acheiving reassortants. Therefore, the polynucleotide sequences of these NCRs (including fragments and variants (e.g., at least about 60%, or at least 70%, or at least 80%, or at least 90%, or at least about 91% or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 98.5%, or at least about 98.7%, or at least about 99%, or at least about 99.1%, or at least about 99.2%, or at least about 99.3%, or at least about 99.4%, or at least about = 99.5%, or at least about 99.6% or at least about 99.7%, or at least about 99.8%, or at least about 99.9% identity) thereof) are within the scope of this invention. When amplifying the HA and NA segments of any pandemic strain, one could make and use polynucleotide primers to bind conserved (e.g., among related strains) regions of the HA and NA NCRs for amplification (e.g., by RT-PCR). In one embodiment, HA and NA polynucleotides of the ,L17.1.A1,1...,1l1 .1 IL, invention include both the NCR and ORF of the HA and NA sequences (including fragments and variants (e.g., at least about 60%, or at least 70%, or at least 80%, or at least 90%, or at least about 91% or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 98.5%, or at least about 98.7%, or at least about 99%, or at least about = 99.1%, or at least about 99.2%, or at least about 99.3%, or at least about 99.4%, or at least about 99.5%, or at least about 99.6% or at least about 99.7%, or at least about 99.8%, or at least about 99.9%) thereof) of pandemic virus strains. In alternative embodiments, the HA
and NA polynucleotides of the invention exclude the NCR, but include the ORF
(including fragments and variants (e.g., at least about 60%, or at least 70%, or at least 80%, or at least 90%, or at least about 91% or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 98.5%, or at least about 98.7%, or at least about 99%, or at least about 99.1%, or at least about 99.2%, or at least about 99.3%, or at least about 99.4%, or at least about 99.5%, or at least about 99.6% or at least about 99.7%, or at least about 99.8%, or at least about 99.9% thereof)) of the HA and NA sequences of pandemic virus strains (e.g., SEQ ID NOS: 1-9).
100821 The HA and NA polynucleotides of the invention, e.g., SEQ
ID NO:1 through SEQ ID NO:10, and fragments thereof, are optionally used in a number of different capacities alternative to, or in addition to, the vaccines described above.
Other exemplary uses are described herein for illustrative purpose and not as limitations on the actual range of uses. Different methods of construction, purification, and characterization of the nucleotide sequences of the invention are also described herein. In some embodiments, nucleic acids including one or more polynucleotide sequence of the invention are favorably used as probes for the detection of corresponding or related nucleic acids in a variety of contexts, such as in nucleic hybridization experiments, e.g., to find and/or characterize homologous influenza variants (e.g., homologues to the sequences herein, etc.) infecting other species or in different influenza outbreaks, etc. The probes can be either DNA or RNA molecules, such as restriction fragments of genomic or cloned DNA, cDNAs, PCR
amplification products, transcripts, and oligonucleotides, and can vary in length from oligonucleotides as short as about 10 nucleotides in length to full length sequences or cDNAs in excess of 1 kb or more. For example, in some embodiments, a probe of the invention includes a polynucleotide sequence or subsequence selected, e.g., from among w02005/116258 CA 02822895 2013-06-07 SEQ D NO: 1 through SEQ ID NO: 10, or sequences complementary thereto.
Alternatively, polynucleotide sequences that are variants of one of the above-designated : =
sequences are used as probes. Most typically, such variants include one or a few conservative nucleotide variations. For example, pairs (or sets) of oligonucleotides can be selected, in which the two (or more) polynucleotide sequences are conservative variations of each other, wherein one polynucleotide sequence corresponds identically to a first variant or and the other(s) corresponds identically to additional variants. Such pairs of oligonueleotide probes are particularly useful, e.g., for specific hybridization experiments to detect polymorphic nucleotides or to, e.g., detect homologous influenza HA and NA
variants, e.g., homologous to the current HA and NA sequences, infecting other species or present in different (e.g., either temporally and/or geographically different) influenza outbreaks. In other applications, probes are selected that are more divergent, that is probes that are at least about 91% (or about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 98.5%, about 98.7%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, or about 99.6% or more about 99.7%, about 99.8%, about 99.9% or more) identical are selected.
100831 The probes of the invention, e.g., as exemplified by sequences derived from the sequences herein, can also be used to identify additional useful polynucleotide sequences according to procedures routine in the art. In one set of embodiments, one or more probes, as described above, are utilized to screen libraries of expression products or chromosomal segments (e.g., expression libraries or genomic libraries) to identify clones that include sequences identical to, or with significant sequence similarity to, e.g., one or more probe of the sequences herein, i.e., variants, homologues, etc. It will be understood that in addition to such physical methods as library screening, computer assisted bioinformatic approaches, e.g., BLAST and other sequence homology search algorithms, and the like, can also be used for identifying related polynucleotide sequences.
Polynucleotide sequences identified in this manner are also a feature of the invention.
[0084] Oligonucleotide probes are optionally produced via a variety of methods well . known to those skilled in the art. Most typically, they are produced by well known synthetic methods, such as the solid phase phosphoramidite triester method described by Beaucage and Caruthers (1981) Tetrahedron Letts 22(20):1859-1862, e.g., using an automated synthesizer, or as described in Needham-Van Devanter et al. (1984) Nucl Acids LILI11.1:11(11 / ILY
Res, 12:6159-6168. Oligonucleotides can also be custom made and ordered from a variety of commercial sources known to persons of skill. Purification of oligonucleotides, where necessary, is typically performed by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson and Regnier (1983) J Chrom 255:137-149.
The sequence of the synthetic oligonucleotides can be verified using the chemical degradation method of Maxam and Gilbert (1980) in Grossman and Moldave (eds.) Academic Press, New York, Methods in Enzymology 65:499-560. Custom oligos can also easily be ordered from a variety of commercial sources known to persons of skill.
100851 In other circumstances, e.g., relating to attributes of cells or organisms expressing the polynucleotides and polypeptides of the invention (e.g., those harboring virus comprising the sequences of the invention), probes that are polypeptides, peptides or antibodies are favorably utilized. For example, isolated or recombinant polypeptides, polypeptide fragments and peptides derived from any of the amino acid sequences of the invention (e.g., SEQ ID NO: 11-20) and/or encoded by polynucleotide sequences of the invention, e.g., selected from SEQ ID NO: 1 through SEQ ID NO: 10, are favorably used to identify and isolate antibodies, e.g., from phage display libraries, combinatorial libraries, polyclonal sera, and the like. Polypeptide fragments of the inventions include a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, or at least 10 contiguous amino acid residues, or at least 15 contiguous amino acid residues, or at least 20 contiguous amino acid residues, or at least 25 contiguous amino acid residues, or at least 40 contiguous amino acid residues, or at least 50 contiguous amino acid residues, or at least 60 contiguous amino residues, or at least 70 contiguous amino acid residues, or at least contiguous 80 amino acid residues, or at least contiguous 90 amino acid residues, or at least contiguous 100 amino acid residues, or at least contiguous 125 amino acid residues, or at least 150 contiguous amino acid residues, or at least contiguous 175 amino acid residues, or at least contiguous 200 amino acid residues, or at least contiguous 250 amino acid residues, or at least contiguous 350, or at least contiguous 400, or at least contiguous 450, or at least contiguous 500, or at least contiguous 550 amino acid residues of the amino acid sequence an HA or NA polypeptide of the invention (e.g., SEQ ID
Nos: 11-20). Polynucleotides encoding said polypeptide fragments and antibodies that specifically bind said polypeptides are also preferred embodiments of the invention.

`IV 0 2005/116258 CA 02822895 2013-06-07 [0086] Antibodies specific for any a polypeptide sequence or subsequence, e.g., of SEQ ID NO: 11 through SEQ ID NO: 20, and/or encoded by polynucleotide sequences of the invention, e.g., selected from SEQ ID NO: 1 through SEQ ID NO: 10, are likewise valuable as probes for evaluating expression products, e.g., from cells or tissues. In addition, antibodies are particularly suitable for evaluating expression of proteins comprising amino acid subsequences, e.g., of those given herein, or encoded by polynucleotides sequences of the invention, e.g., selected from those shown herein, in situ, = in a tissue array, in a cell, tissue or organism, e.g., an organism infected by an unidentified influenza virus or the like. Antibodies can be directly labeled with a detectable reagent, or detected indirectly by labeling of a secondary antibody specific for the heavy chain constant region (i.e., isotype) of the specific antibody. Additional details regarding production of specific antibodies are provided below.
Diagnostic Assays [0087] The nucleic acid sequences of the present invention can be used in diagnostic assays to detect influenza (and/or hemagglutinin and/or neurarninidase) in a sample, to detect hemagglutinin-like and/or neuraminidase-like sequences, and to detect strain differences in clinical isolates of influenza using either chemically synthesized or recombinant polynucleotide fragments, e.g., selected from the sequences herein. For example, fragments of the hemagglutinin and/or neuraminidase sequences comprising at least between 10 and 20 nucleotides can be used as primers to amplify nucleic acids using polymerase chain reaction (PCR) methods well known in the art (e.g., reverse transcription-PCR) and as probes in nucleic acid hybridization assays to detect target genetic material = such as influenza RNA in clinical specimens.
[0088] The probes of the invention, e.g., as exemplified by unique subsequences selected from those given herein, can also be used to identify additional useful polynucleotide sequences (such as to characterize additional strains of influenza) according to procedures routine in the art. In one set of preferred embodiments, one or more probes, as described above, are utilized to screen libraries of expression products or cloned viral nucleic acids (i.e., expression libraries or genomic libraries) to identify clones that include sequences identical to, or with significant sequence identity to the sequences herein. In turn, each of these identified sequences can be used to make probes, including pairs or sets of variant probes as described above. It will be understood that in addition to such physical VV kJ Ll11131110Z:NS 1 /
UJZt/U:71/(J1 / LLY

methods as library screening, computer assisted bioinformatic approaches, e.g., BLAST and other sequence homology search algorithms, and the like, can also be used for identifying related polynucleotide sequences.
= 100891 The probes of the invention are particularly useful for detecting the presence and for determining the identity of influenza nucleic acids in cells, tissues or other biological samples (e.g., a nasal wash or bronchial lavage). For example, the probes of the invention are favorably utilized to determine whether a biological sample, such as a subject (e.g., a human subject) or model system (such as a cultured cell sample) has been exposed .to, or become infected with influenza, or particular strain(s) of influenza.
Detection of hybridization of the selected probe to nucleic acids originating in (e.g., isolated from) the biological sample or model system is indicative of exposure to or infection with the virus (or a related virus) from which the probe polynucleotide is selected.
[0090] It will be appreciated that probe design is influenced by the intended application. For example, where several allele-specific probe-target interactions are to be detected in a single assay, e.g., on a single DNA chip, it is desirable to have similar melting temperatures for all of the probes. Accordingly, the lengths of the probes are adjusted so that the melting temperatures for all of the probes on the array are closely similar (it will be appreciated that different lengths for different probes may be needed to achieve a particular Tn, where different probes have different GC contents). Although melting temperature is a primary consideration in probe design, other factors are optionally used to further adjust probe construction, such as selecting against primer self-complementarity and the like.
Vectors, Promoters and Expression Systems [0091] The present invention includes recombinant constructs incorporating one or more of the nucleic acid sequences described herein. Such constructs optionally include a vector, for example, a plasmid, a cosmid, a phage, a virus, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), etc., into which one or more of the polynucleotide sequences of the invention, e.g., comprising any of SEQ ID NO:
1 through SEQ ID NO:10, or a subsequence thereof etc., has been inserted, in a forward or reverse orientation. For example, the inserted nucleic acid can include a viral chromosomal sequence or cDNA including all or part of at least one of the polynucleotide sequences of the invention. In one embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of = WO 2005/116258 CA

suitable vectors and promoters are known to those of skill in the art, and are commercially available.
[0092] The polynucleotides of the present invention can be included in any one of a variety of vectors suitable for generating sense or antisense RNA, and optionally, polypeptidc (or peptide) expression products (e.g., a hcmagglutiniri and/or neuraminidase molecule of the invention, or fragments thereof). Such vectors include chromosomal, = nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;
bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of = plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, pseudorabies, adenovirus, adeno-associated virus, retroviruses and many others (e.g., pCDL). Any vector that is capable of introduCing genetic material into a cell, and, if replication is desired, which is replicable in the relevant host can be used.
[0093] In an expression vector, the HA and/or NA
polynucleotide sequence of interest is physically arranged in proximity and orientation to an appropriate transcription control sequence (e.g., promoter, and optionally, one or more enhancers) to direct mRNA
synthesis. That is, the polynucleotide sequence of interest is operably linked to an appropriate transcription control sequence. Examples of such promoters include: LTR or SV40 promoter, E. coli lac or trp promoter, phage lambda PL promoter, and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
[00941 A variety of promoters are suitable for use in expression vectors for regulating transcription of influenza virus genome segments. In certain embodiments, the cytomegalovirus (CMV) DNA dependent RNA Polymerase II (Pol II) promoter is utilized.
If desired, e.g., for regulating conditional expression, other promoters can be substituted which induce RNA transcription under the specified conditions, or in the specified tissues or cells. Numerous viral and mammalian, e.g., human promoters are available, or can be isolated according to the specific application contemplated. For example, alternative promoters obtained from the genomes of animal and human viruses include such promoters as the adenovirus (such as Adenovirus 2), papilloma virus, hepatitis-B virus, polyoma virus, and Simian Virus 40 (SV40), and various retroviral promoters. Mammalian promoters include, among many others, the actin promoter, immunoglobulin promoters, heat-shock promoters, and the like.

GM" 01 1 loLoo CA 02822895 2013-06-07 I-A. I /
U3LtIl/D/1/1 I I LY
[0095] Transcription is optionally increased by including an enhancer sequence.
=
Enhancers are typically short, e.g., 10-500 bp, cis-acting DNA elements that act in concert with a promoter to increase transcription. Many enhancer sequences have been isolated from mammalian genes (hemoglobin, elastase, albumin, alpha-fetoprotein, and insulin), and eukaryotic cell viruses. The enhancer can be spliced into the vector at a position 5' or 3' to the heterologous coding sequence, but is typically inserted at a site 5' to the promoter.
Typically, the promoter, and if desired, additional transcription enhancing sequences are chosen to optimize expression in the host cell type into which the heterologous DNA is to be introduced (Scharf et al. (1994) Heat stress promoters and transcription factors Results Probl Cell Differ 20:125-62; Kriegler et al. (1990) Assembly of enhancers, promoters, and splice signals to control expression of transferred genes Methods in Enzymol 185: 512-27).
Optionally, the amplicon can also contain a ribosome binding site or an internal ribosome entry site (IRES) for translation initiation.
[0096] The vectors of the invention also favorably include sequences necessary for the termination of transcription and for stabilizing the mRNA, such as a polyadenylation site or a terminator sequence. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. In one embodiment, the SV40 polyadenylation signal sequences can provide a bi-directional polyadenylation site that insulates transcription of (+) strand mRNA molecules from the Poll promoter initiating replication of the (-) strand viral genome.
[0097] In addition, as described above, the expression vectors optionally include one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, in addition to genes previously listed, markers such as dihydrofolate reductase or neomycin resistance are suitable for selection in eukaryotic cell culture.
[0098] The vector containing the appropriate nucleic acid sequence as described above, as well as an appropriate promoter or control sequence, can be employed to transform a host cell permitting expression of the protein. While the vectors of the invention can be replicated in bacterial cells, most frequently it will be desirable to introduce them into mammalian cells, e.g., Vero cells, BHK cells, MDCK cell, 293 cells, COS cells, or the like, for the purpose of expression.
=

Dit2266-4 CA 02822895 2013-06-07 [00991 As described elsewhere, the HA and NA sequences herein, in various embodiments, can be comprised within plasmids involved in plasmid-rescue reassortment.
See, e.g., U.S. Application Nos. 60/420,708, filed October 23, 2002;
60/574,117, filed May 24, 2004; 10/423,828, filed April 25, 2003; 60/578,962, filed June 12,2004;
and 10/870,690 filed June 16, 2004; and US20020164770. For example, preferred expression vectors of the invention include, but are not limited to, vectors comprising pol I promoter and terminator sequences or vectors using both the pal I
and pol II promoters "the polI/polII promoter system" (e.g., Zobel et al., Nucl. Acids Res.
1993, 21:3607; US20020164770; Neumann et al., Proc. Natl. Acad. Sci. USA 1999, 96:9345; Fodor et al., J. Virol. 1999,73:9679; and US20030035814). The reassortants produced can include the HA and NA genes arranged with the 6 other influenza genes from the A/Ann Arbor/6/60 donor strain (and/or derivatives and modifications thereof), the PR8 donor strain backbone, the A/Leningrad/17 donor strain backbone, etc. Other backbone strains are described, for example, in 20040137013 and 20030147916.
Additional Expression Elements [0100] Most commonly, the genome segment encoding the influenza virus HA
and/or NA protein includes any additional sequences necessary for its expression, including translation into a functional viral protein. In other situations, a minigene, or other artificial construct encoding the viral proteins, e.g., an HA and/or NA protein, can be employed.
Again, in such case, it is often desirable to include specific initiation signals that aid in the efficient translation of the heterologous coding sequence. These signals can include, e.g., the ATG initiation codon and adjacent sequences. To insure translation of the entire insert, the initiation codon is inserted in the correct reading frame relative to the viral protein.
Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers appropriate to the cell system in use.
[0101] If desired, polynucleotide sequences encoding additional expressed elements, such as signal sequences, secretion or localization sequences, and the like can be incorporated into the vector, usually, in-frame with the polynucleotide sequence of interest, e.g., to target polypeptide expression to a desired cellular compartment, membrane, or organelle, or to direct polypeptide secretion to the periplasmic space or into the cell culture a =

VVUI ARI:)/11blJti CA 02822895 2013-06-07 PUT/US2005/017729 media. Such sequences are known to those of skill, and include secretion leader peptides, organelle targeting sequences (e.g., nuclear localization sequences, ER
retention signals, mitochondrial transit sequences), membrane localization/anchor sequences (e.g., stop transfer sequences, GPI anchor sequences), and the like.
101021 Where translation of a polypeptide encoded by a nucleic acid sequence of the invention is desired, additional translation specific initiation signals can improve the efficiency of translation. These signals can include, e.g., an ATG initiation codon and adjacent sequences, an TRES region, etc. In some cases, for example, full-length cDNA
molecules or chromosomal segments including a coding sequence incorporating, e.g., a polynucleotide sequence of the invention (e.g., as in the sequences herein), a translation initiation codon and associated sequence elements are inserted into the appropriate expression vector simultaneously with the polynucleotide sequence of interest.
In such cases, additional translational control signals frequently are not required.
However, in cases where only a polypeptide coding sequence, or a portion thereof, is inserted, exogenous translational control signals, including, e.g., an ATG initiation codon is often provided for expression of the relevant sequence. The initiation codon is put in the correct reading frame to ensure transcription of the polynucleotide sequence of interest. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers appropriate to the cell system in use (see, e.g., Scharf D. et al. (1994) Results Probl Cell Differ 20:125-62; Bittner et al. (1987) Methods in Enzymol 153:516-544).
Production of recombinant virus [0103] Negative strand RNA viruses can be genetically engineered and recovered using a recombinant reverse genetics approach (see, e.g., USPN 5,166,057 to Palese et al.).
Such method was originally applied to engineer influenza viral genomes (Luytjes et al.
(1989) Cell 59:1107-1113; Enami et al. (1990) Proc. Natl. Acad. Sci. USA
92:11563-11567), and has been successfully applied to a wide variety of segmented and nonsegmented negative strand RNA viruses, e.g., rabies (Schnell et al. (1994) EMBO J. 13:
4195-4203); VSV (Lawson et al. (1995) Proc. Natl. Acad. Sci. USA 92: 4477-4481);
measles virus (Radecke et al.(1995) EMBO J. 14:5773-5784); rinderpest virus (Baron &
Barrett (1997) J. Virol. 71: 1265-1271); human parainfluenza virus (Hoffman &
Banerjee (1997) J. Virol. 71: 3272-3277; Dubin et al. (1997) Virology 235:323-332); SV5 (He et al.

.W0 2005/116258 CA 02822895 2013-06-07 (1997) Virology 237:249-260); canine distemper virus (Gassen et al. (2000) J.
Virol.
74:10737-44); and Sendai virus (Park et al. (1991) Proc. Natl. Acad. Sci. USA
88: 5537-5541; Kato et al. (1996) Genes to Cells 1:569-579). Those of skill in the art will be familiar with these and similar techniques to produce influenza virus comprising the HA
and NA
=
sequences of the invention. Recombinant influenza viruses produced according to such methods are also a feature of the invention, as are recombinant influenza virus comprising one or more nucleic acids and/or polypeptides of the invention.
Cell Culture and Expression Hosts 101041 The present invention also relates to host cells that are introduced (transduced, transformed or transfected) with vectors of the invention, and the production of polypeptides of the invention by recombinant techniques. Host cells are genetically engineered (i.e., transduced, transformed or transfected) with a vector, such as an expression vector, of this invention. As described above, the vector can be in the form of a plasmid, a viral particle, a phage, etc. Examples of appropriate expression hosts include: bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurium; fungal cells, such as Saccharomyces cerevisiae, Pichia pastoris, and Neurospora crassa; or insect cells such as ,Drosophila and Spodoptera frugiperda.
[0105] Most commonly, mammalian cells are used to culture the HA and NA
molecules of the invention. Suitable host cells for the replication of influenza virus include, e.g., Vero cells, BRK cells, MDCK cells, 293 cells and COS cells, including 293T cells, COS7 cells or the like. Commonly, co-cultures including two of the above cell lines, e.g., MDCK cells and either 293T or COS cells are employed at a ratio, e.g., of 1:1, to improve replication efficiency. Typically, cells are cultured in a standard commercial culture medium, such as Dulbecco's modified Eagle's medium supplemented with serum (e.g., 10%
fetal bovine serum), or in serum free medium, under controlled humidity and = concentration suitable for maintaining neutral buffered pH (e.g., at pH
between 7.0 and 7.2).
Optionally, the medium contains antibiotics to prevent bacterial growth, e.g., penicillin, streptomycin, etc., and/or additional nutrients, such as L-glutamine, sodium pyruvate, non-essential amino acids, additional supplements to promote favorable growth characteristics, e.g., trypsin, p-mercaptoethanol, and the like.
= [0106] The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the =

inserted polynucleotide sequences. The culture conditions, such as temperature, pH and the like, are typically those previously used with the particular host cell selected for expression, and will be apparent to those skilled in the art and in the references cited herein, including, e.g., Freshney (1994) Cllture of Animal Cells, a Manual of Basic Technique, 3rd edition, Wiley- Liss, New York and the references cited therein. Other helpful references include, e.g., Paul (1975) Cell an_sl Tissue attire, 5th ed., Livingston, Edinburgh;
Adams (1980) Laboratory Techniques in Biochemistry and Molecular Biology-Cell Culture for Biochemists, Work and Burdon (ed.'s.) Elsevier, Amsterdam. Additional details regarding tissue culture procedures of particular interest in the production of influenza virus in vitro include, e.g., Merten et al. (1996) Production of influenza virus in cell cultures for vaccine = preparation. in Cohen and Shafferman (eds.) /srovel Strategies in Desism and Production of Vaccines. Additionally variations in such procedures adapted to the present invention are readily determined through routine experimentation and will be familiar to those skilled in the art.
101071 Cells for production of influenza virus (e.g., having the HA and/or NA
sequences of the invention) can be cultured in serum-containing or serum free medium. In some case, e.g., for the preparation of purified viruses, it is typically desirable to grow the host cells in serum free conditions. Cells can be cultured in small scale, e.g., less than 25 ml medium, culture tubes or flasks or in large flasks with agitation, in rotator bottles, or on microcarrier beads (e.g., DEAE-Dextran microcarrier beads, such as Donnacell, Pfeifer &
Langen; Superbead, Flow Laboratories; styrene copolymer-tri-methylamine beads, such as Hillex, SoloHill, Ann Arbor) in flasks, bottles or reactor,cultures.
Microcarrier beads are small spheres (in the range of 100-200 microns in diameter) that provide a large surface area for adherent cell growth per volume of cell culture. For example a single liter of medium can include more than 20 million microcarrier beads providing greater than 8000 square centimeters of growth surface. For commercial production of viruses, e.g., for vaccine production, it is often desirable to culture the cells in a bioreactor or fermenter. Bioreactors are available in volumes from under 1 liter to in excess of 100 liters, e.g., Cyto3 Bioreactor (Osmonics, Minnetonka, MN); NBS bioreactors (New Brunswick Scientific, Edison, NJ);
laboratory and commercial scale bioreactors from B. Braun Biotech International (B. Braun Biotech, Melsungen, Germany).

WO 2005/116258 CA 02822895 2013-06-07 1-1_, 1 / U
PU:I/l/1 / I LY
[0108] Regardless of the culture volume, in many desired aspects of the current invention, it is important that the cultures be maintained at an appropriate temperature, to insure efficient recovery of recombinant and/or reassortant influenza virus using temperature dependent multi plasmid systems (see, e.g., Multi-Plasmid System for the Production of Influenza Virus, U.S. Application No. 60/420,708õ filed October 23, 2002, U.S. Application No. 10/423,828, filed April 25, 2003, and U.S. Application No.
60/574,117, filed May 24, 2004), heating of virus solutions for filtration, etc. Typically, a regulator, e.g., a thermostat, or other device for sensing and maintaining the temperature of the cell culture system and/or other solution, is employed to insure that the temperature is at the correct level during the appropriate period (e.g., virus replication, etc.).
[0109] In some embodiments herein (e.g., wherein reassorted viruses are to be produced from segments on vectors) vectors comprising influenza genome segments are introduced (e.g., transfected) into host cells according to methods well known in the art for introducing heterologous nucleic acids into eukaryotic cells, including, e.g., calcium phosphate co-precipitation, electroporation, microinjection, lipofection, and transfection employing polyamine transfection reagents. For example, vectors, e.g., plasmids, can be transfected into host cells, such as COS cells, 293T cells or combinations of COS or 293T
cells and MDCK cells, using the polyamine transfection reagent TransIT-LT1 (Minis) according to the manufacturer's instructions in order to produce reassorted viruses, etc.
Thus, in one example, approximately 1 g of each vector is introduced into a population of host cells with approximately 2 .1 of TransIT-LT1 diluted in 160 p.1 medium, preferably serum-free medium, in a total volume of 200 1. The DNA:transfection reagent mixtures are incubated at room temperature for 45 minuets followed by addition of 800 I of medium. The transfection mixture is added to the host cells, and the cells are cultured as described via other methods well known to those skilled in the art.
Accordingly, for the production of recombinant or reassortant viruses in cell culture, vectors incorporating each of the 8 genome segments, (PB2, PB1, PA, NP, M, NS, HA and NA, e.g., of the invention) are mixed with approximately 20 ill TransIT-LT1 and transfected into host cells.
Optionally, serum-containing medium is replaced prior to transfection with serum-free medium, e.g., Opti-MEM I, and incubated for 4-6 hours.
[0110] Alternatively, electroporation can be employed to introduce such vectors incorporating influenza genome segments into host cells. For example, plasmid vectors incorporating an influenza A or influenza B virus are favorably introduced into Vero cells using electroporation according to the following procedure. In brief, approximately 5 x 106 Vero cells, e.g., grown in Modified Eagle's Medium (MEM) supplemented with 10%
Fetal Bovine Serum (FBS) are resuspended in 0.4 ml OptiMEM and placed in an electroporation cuvette. Twenty micrograms of DNA in a volume of up to 25 1.11 is added to the cells in the cuvette, which is then mixed gently by tapping. Electroporation is performed according to the manufacturer's instructions (e.g., BioRad Gene Pulser II with Capacitance Extender Plus connected) at 300 volts, 950 microFarads with a time constant of between 28-33 msec.
The cells are remixed by gently tapping and approximately 1-2 minutes following electroporation 0.7 ml MEM with 10% FBS is added directly to the cuvette. The cells are then transferred to two wells of a standard 6 well tissue culture dish containing 2 ml MEM, 10% FBS. The cuvette is washed to recover any remaining cells and the wash suspension is divided between the two wells. Final volume is approximately 3.5 mL. The cells are then incubated under conditions permissive for viral growth, e.g., at approximately 33 C for cold adapted strains.
[0111] In mammalian host cells, a number of expression systems, such as viral-based systems, can be utilized. In cases where an adenovirus is used as an expression vector, a coding sequence is optionally ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence.
Insertion in a nonessential El or E3 region of the viral genome will result in a viable virus capable of expressing the polypeptides of interest in infected host cells (Logan and Shenk (1984) Proc Nat! Acad Sci 81:3655-3659). In addition, transcription enhancers, such as the rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.
[01121 A host cell strain is optionally chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the protein include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing, which cleaves a precursor form into a mature form, of the protein is sometimes important for correct insertion, folding and/or function. Additionally proper location within a host cell (e.g., on the cell surface) is also important. Different host cells such as COS, CHO, BHK, MDCK, 293, 293T, COS7, etc. have specific cellular machinery and =
vv0 2005/116258 CA 02822895 2013-06-07 = PCT/US2005/017729 characteristic mechanisms for such post-translational activities and can be chosen to ensure the correct modification and processing of the current introduced, foreign protein.
[0113] For long-term, high-yield production of recombinant proteins encoded by, or ..= having subsequences encoded by, the polynucleotides of the invention, stable expression systems are optionally used. For example, cell lines, stably exptessing a polypeptide of the invention, are transfected using expression vectors that contain viral origins of replication or endogenous expression elements and a selectable marker gene. For example, following the introduction of the vector, cells are allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences. Thus, resistant clumps of stably transformed cells, e.g., derived from single cell type, can be proliferated using tissue culture techniques appropriate to the cell type.
[0114] Host cells transformed with a nucleotide sequence encoding a polypeptide of = 15 the invention are optionally cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture. The cells expressing said protein can be sorted, isolated and/or purified. The protein or fragment thereof produced by a recombinant = cell can be secreted, membrane-bound, or retained intracellularly, depending on the sequence (e.g., depending upon fusion proteins encoding a membrane retention signal or the like) and/or the vector used.
[0115] Expression products corresponding to the nucleic acids of the invention can also be produced in non-animal cells such as plants, yeast, fungi, bacteria and the like. In = addition to Sambrook, Berger and Ausubel, all infra, details regarding cell culture can be = found in Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley &
Sons, Inc. New York, NY; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg . = New York) and Atlas and Parks (eds.) The Handbook of Microbiological Media (1993) =
CRC Press, Boca Raton, FL.
[0116] In bacterial systems, a number of expression vectors can be selected depending upon the use intended for the expressed product. For example, when large quantities of a polypeptide or fragments thereof are needed for the production of antibodies, WO 2U05/116258 CA 02822895 2013-06-07 litThzUuNui vectors that direct high-level expression of fusion proteins that are readily purified are favorably employed. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the coding sequence of interest, e.g., sequences comprising those found herein, etc., can be ligated into the vector in-frame with sequences for the amino-terminal translation initiating methionine and the subsequent 7 residues of beta-galactosidase producing a catalytically active beta galactosidase fusion protein; pIN vectors (Van Heeke & Schuster (1989) J Biol Chem 264:5503-5509); pET vectors (Novagen, Madison WI); and the like. Similarly, in the yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH can be used for production of the desired expression products. For reviews, see Ausubel, infra, and Grant et al., (1987);
Methods in Enzymology 153:516-544.
Nucleic Acid Hybridization 101171 Comparative hybridization can be used to identify nucleic acids (e.g., SEQ
ID NO: 1-10) of the invention, including conservative variations of nucleic acids of the invention. This comparative hybridization method is a preferred method of distinguishing nucleic acids of the invention. In addition, target nucleic acids which hybridize to the nucleic acids represented by, e.g., those shown herein under high, ultra-high and ultra-ultra-high stringency conditions are features of the invention. Examples of such nucleic acids include those with one or a few silent or conservative nucleic acid substitutions as compared to a given nucleic acid sequence.
[01181 A test target nucleic acid is said to specifically hybridize to a probe nucleic acid when it hybridizes at least one-half as well to the probe as to the perfectly matched complementary target, i.e., with a signal to noise ratio at least one-half as high as hybridization of the probe and target under conditions in which a perfectly matched probe binds to a perfectly matched complementary target with a signal to noise ratio that is at least about 5x-10x as high as that observed for hybridization to any of the unmatched target nucleic acids.
[01191 Nucleic acids "hybridize" when they associate, typically in solution. Nucleic acids hybridize due to a variety of well-characterized physico-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. Numerous protocols for nucleic acid hybridization are well known in the art. An extensive guide to the 4i0 2005/116258 CA 02822895 2013-06-07 hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology¨Hybridization with Nucleic Acid Probes part I
;= chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," (Elsevier, New York), as well as in Ausubel, Sambrook, and Berger and Kimmel, all below. Hames and Higgins (1995) Gene Probes 1 IRL Press at Oxford University Press, Oxford, England, (Hames and Higgins 1) and Hames and Higgins (1995) Gene Probes 2 :
IRL Press at Oxford University Press, Oxford, England (Hames and Higgins 2) provide details on the synthesis, labeling, detection and quantification of DNA and RNA, including oligonucleotides.
[01201 An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formalin with 1 mg of heparin at 42 C, with the hybridization being carried out overnight. An example of stringent wash conditions comprises a 0.2x SSC wash at 65 C for 15 minutes (see, Sambrook, infra for a description of SSC buffer and other nucleic acid hybridization parameters). Often the high stringency . = wash is preceded by a low stringency wash to remove background probe signal. An example low stringency wash is 2x SSC at 40 C for 15 minutes. In general, a signal to noise ratio of 5x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
= 20 [0121] After hybridization, unhybridized nucleic acids can be removed by a series of washes, the stringency of which can be adjusted depending upon the desired results. Low stringency washing conditions (e.g., using higher salt and lower temperature) increase sensitivity, but can produce nonspecific hybridization signals and high background signals.
= Higher stringency conditions (e.g., using lower salt and higher temperature that is closer to the Tm) lower the background signal, typically with primarily the specific signal remaining.
See, also, Rapley, R. and Walker, J.M. eds., Molecular Biomethods Handbook (Humana = Press, Inc. 1998).
[0122] "Stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993), supra, and in Hames and =

W LURK)/ 1 IDL35 CA 02822895 2013-06-07 U3LUU:MUI I /LY
Higgins, 1 and 2. Stringent hybridization and wash conditions can easily be determined empirically for any test nucleic acid. For example, in determining highly stringent hybridization and wash conditions, the hybridization and wash conditions are gradually increased (e.g., by increasing temperature, decreasing salt concentration, increasing detergent concentration and/or increasing the concentration of organic solvents such as = formalin in the hybridization or wash), until a selected set of criteria is met. For example, the hybridization and wash conditions are gradually increased until a probe binds to a perfectly matched complementary target with a signal to noise ratio that is at least 5x as high as that observed for hybridization of the probe to an unmatched target.
, [0123] In general, a signal to noise ratio of at least 2x (or higher, e.g., at least 5x, 10x, 20x, 50x, 100x, or more) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Detection of at least stringent hybridization between two sequences in the context of the present invention indicates relatively strong structural similarity to, e.g., the nucleic acids of the present invention provided in the sequence listings herein.
[0124] "Very stringent" conditions are selected to be equal to the thermal melting point (TO for a particular probe. The 'I'm is the temperature (under defined ionic strength and pH) at which 50% of the test sequence hybridizes to a perfectly matched probe. For the purposes of the present invention, generally, "highly stringent" hybridization and wash conditions are selected to be about 5 C lower than the Tn., for the specific sequence at a defined ionic strength and pH (as noted below, highly stringent conditions can also be referred to in comparative terms). Target sequences that are closely related or identical to the nucleotide sequence of interest (e.g., "probe") can be identified under stringent or highly stringent conditions. Lower stringency conditions are appropriate for sequences that are less complementary.
[0125] "Ultra high-stringency" hybridization and wash conditions are those in which the stringency of hybridization and wash conditions are increased until the signal to noise ratio for binding of a probe to a perfectly matched complementary target nucleic acid is at least 10x as high as that observed for hybridization to any unmatched target nucleic acids.
A target nucleic acid which hybridizes to a probe under such conditions, with a signal to noise ratio of at least one-half that of the perfectly matched complementary target nucleic acid is said to bind to the probe under ultra-high stringency conditions.

rii0 2005/116258 CA 02822895 2013-06-07 PCT/US2005/01772) = [0126] In determining stringent or highly stringent hybridization (or even more stringent hybridization) and wash conditions, the hybridization and wash conditions are gradually increased (e.g., by increasing temperature, decreasing salt concentration, increasing detergent concentration and/or increasing the concentration of organic solvents, =
such as formamide, in the hybridization or wash), until a selected set of criteria are met. For . .
example, the hybridization and wash conditions are gradually increased until a probe comprising one or more polynucleotide sequences of the invention, e.g., sequences or unique subsequences selected from those given herein (e.g., SEQ JD NO: 1-10) and/or complementary polynucleotide sequences, binds to a perfectly matched complementary target (again, a nucleic acid comprising One or more nucleic acid sequences or subsequences selected from those given herein and/or complementary polynucleotide sequences thereof), with a signal to noise ratio that is at least 2x (and optionally 5x, 10x, or 100x or more) as = high as that observed for hybridization of the probe to an unmatched target (e.g., a = polynucleotide sequence comprising one or more sequences or subsequences selected from known influenza sequences present in public databases such as GenBank at the time of filing, and/or complementary polynucleotide sequences thereof), as desired.
[0127] Using the polynucleotides of the invention, or subsequences thereof, novel target nucleic acids can be obtained; such target nucleic acids are also a feature of the invention. For example, such target nucleic acids include sequences that hybridize under stringent conditions to a unique oligonucleotide probe corresponding to any of the polynucleotides of the invention, e.g., SEQ ID NO: 1-10)..
= [0128] Similarly, even higher levels of stringency can be determined by gradually increasing the hybridization and/or wash conditions of the relevant hybridization assay. For example, those in which the stringency of hybridization and wash conditions are increased = 25 until the signal to noise ratio for binding of the probe to the perfectly matched complementary target nucleic acid is at least 10X, 20X, 50X, 100X, or 500X or more as = = high as that observed for hybridization to any unmatched target nucleic acids. The particular signal will depend on the label used in the relevant assay, e.g., a fluorescent label, a colorimetric label, a radioactive label, or the like. A target nucleic acid which hybridizes to a probe under such conditions, with a signal to noise ratio of at least one-half that of the perfectly matched complementary target nucleic acid is said to bind to the probe under = ultra-ultra-high stringency conditions and are also features of the invention.

=
[0129] Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
=
Cloning, Mutagenesis and Expression of Biomolecules of Interest [0130] General texts which describe molecular biological techniques, which are applicable to the present invention, such as cloning, mutation, cell culture and the like, include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al., Molecular Cloning - A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2000 ("Sambrook") and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2002) ("Ausubel")). These texts describe mutagenesis, the use of vectors, promoters and many other relevant topics related to, e.g., the generation of HA and/or NA
molecules, etc.
[0131] Various types of mutagenesis are optionally used in the present invention, e.g., to produce and/or isolate, e.g., novel or newly isolated HA and/or NA
molecules and/or to further modify/mutate the polypeptides (e.g., HA and NA molecules as in SEQ
ID NO:

11-20) of the invention. They include but are not limited to site-directed, random point mutagenesis, homologous recombination (DNA shuffling), mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA or the like. Additional suitable methods include point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, double-strand break repair, and the like. Mutagenesis, e.g., involving chimeric constructs, is also included in the present invention. In one embodiment, mutagenesis can be guided by known information of the naturally occurring molecule or altered or mutated naturally occurring molecule, e.g., sequence, sequence comparisons, physical properties, crystal structure or the like.
[0132] The above texts and examples found herein describe these procedures as well as the following publications (and references cited within): Sieber, et al., Nature Biotechnology, 19:456-460 (2001); Ling et al., Approaches to DNA mutagenesis:
an .

overview, Anal Biochem 254(2): 157-178 (1997); Dale et al., Oligonucleotide-directed random mutagenesis using the phosphorothioate method, Methods Mol Biol 57:369-(1996); I. A. Lorimer, I. Pastan, Nucleic Acids Res 23, 3067-8 (1995); W. P.
C. Stemmer, Nature 370, 389-91 (1994); Arnold, Protein engineering for unusual environments, Current Opinion in Biotechnology 4:450-455 (1993); Bass et al., Mutant Trp repressors with new DNA-binding specificities, Science 242:240-245 (1988); Fritz et al., Oligonucleotide-directed construction of mutations: a gapped duplex DNA procedure without enzymatic reactions in vitro, Nucl Acids Res 16: 6987-6999 (1988); Kramer et al., Improved enzymatic in vitro reactions in the gapped duplex DNA approach to oligonucleotide-directed construction of mutations, Nucl Acids Res 16: 7207 (1988); Sakamar and Khorana, Total synthesis and expression of a gene for the a-subunit of bovine rod outer segment guanine nucleotide-binding protein (transducin), Nucl Acids Res 14: 6361-6372 (1988);
Sayers et al., Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis, Nucl Acids Res 16:791-802 (1988); Sayers et al., Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidium bromide, (1988) Nucl Acids Res 16: 803-814; Carter, Improved oligonucleotide-directed mutagenesis using M13 vectors, Methods in Enzymol 154: 382-403 (1987); Kramer & Fritz Oligonucleotide-directed construction of mutations via gapped duplex DNA, Methods in Enzymol 154:350-367 (1987); Kunkel, The efficiency of oligonucleotide directed mutagenesis, in Nucleic Acids & Molecular Biology (Eckstein, F.
and Lilley, D.M.J. eds., Springer Verlag, Berlin)) (1987); Kunkel et al., Rapid and efficient site-specific mutagenesis without phenotypic selection, Methods in Enzymol 154, 367-382 (1987); Zoller & Smith, Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template, Methods in Enzymol 154:329-350 (1987); Carter, Site-directed mutagenesis, Biochem J 237:1-7 (1986);
Eghtedarzadeh & Henikoff, Use of oligonucleotides to generate large deletions, Nucl Acids Res 14: 5115 (1986); Mandecki, Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coll.- a method for site-specific mutagenesis, Proc Nat! Acad Sci USA, 83:7177-7181(1986); Nakamaye & Eckstein, Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis, Nucl Acids Res 14: 9679-9698 (1986); Wells et al., Importance of hydrogen-bond formation in stabilizing the transition state of subtilisin, Phil Trans R
Soc Lond A 317:
415-423 (1986); Botstein & Shortle, Strategies and applications of in vitro mutagenesis, --- = CA 02822895 2013-06-07 Science 229:1193-12041985); Carter et al., Improved oligonucleotide site-directed mutagenesis using M13 vectors, Nucl Acids Res 13: 4431-4443 (1985); Grundstrom et al., Oligonucleotide-directed mutagenesis by microscale 'shot-gun' gene synthesis, Nucl Acids Res 13: 3305-3316 (1985); Kunkel, Rapid and efficient site-specific mutagenesis without phenotypic selection, Proc Natl Acad Sci USA 82:488-492 (1985); Smith, In vitro mutagenesis, Ann Rev Genet 19:423-462(1985); Taylor et al., The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA, Nucl Acids Res 13:
8749-8764 (1985); Taylor et al.,. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA, Nucl Acids Res 13: 8765-(1985); Wells et al., Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites, Gene 34:315-323 (1985); Kramer et al., The gapped duplex DNA
approach to oligonucleotide-directed mutation construction, Nucl Acids Res 12:

(1984); Kramer et al., Point Mismatch Repair, Cell 38:879-887 (1984); Nambiar etal., Total synthesis and cloning of a genecoding for the ribonuclease S protein, Science 223:
1299-1301(1984); Zoller & Smith, Oligonucleotide-directed mutagenesis of DNA
fragments cloned into M13 vectors, Methods in Enzymol 100:468-500 (1983); and Zoller &
Smith, Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any DNA fragment, Nucl Acids Res 10:6487-6500 (1982). Additional details on many of the above methods can be found in Methods in Enzymol Volume 154, which also describes useful controls for trouble-shooting problems with various mutagenesis, gene isolation, expression, and other methods.
[0133] Oligonucleotides, e.g., for use in mutagenesis of the present invention, e.g., mutating libraries of the HA and/or NA molecules of the invention, or altering such, are typically synthesized chemically according to the solid phase phosphoramidite triester method described by Beaucage and Caruthers, Tetrahedron Letts 22(20):1859-1862, (1981) e.g., using an automated synthesizer, as described in Needham-VanDevanter et al., Nucleic , Acids Res, 12:6159-6168 (1984).
[0134] In addition, essentially any nucleic acid can be custom or standard ordered from any of a variety of commercial sources, such as The Midland Certified Reagent Company 3112 West Cuthbert Avenue, Midland, Texas, USA (mcrc@oligos.com), The Great American Gene Company 100 Papercraft Park, Pittsburgh, PA, USA
(www.genco.com), ExpressGen Inc. Chicago, IL, USA (www.expressgen.com), Operon Technologies Inc.
(Alatneda,CA) and many others. Similarly, peptides and antibodies can be custom ordered from any of a A

variety of sources, such as PeptidoGenic (available at pkim@ccnet.com), HTI
Bio-products, Inc. (www.htibio.com), BMA Biomedicals Ltd. (U.K.), Bio.Synthesis, Inc., and many others.
[0135] The present invention also relates to host cells and organisms comprising a HA and/or NA molecule or other polypeptide and/or nucleic acid of the invention, e.g., SEQ
ID NO:1-20. Host Cells are genetically engineered (e.g., transformed, transduced or transfected) with the vectorsof this invention, which can be, for example, a cloning vector or an expression vector. The vector can be, for example, in the form of a plasmid, a bacterium, a virus, a nakedpolynucleotide, or a conjugated polynueleotide. The vectors are introduced into cells and/or microorganisms by standard methods including electroporation (see, From et al., Proc Natl Acad Sci USA 82, 5824 (1985), infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327, 70-73 (1987)).
Berger, Sambrook, and Ausubel provide a variety of appropriate transformation methods.
See, above.
101361 Several well-known methods of introducing target nucleic acids into bacterial cells are available, any of which can be used in the present invention. These include: fusion of the recipient cells with bacterial protoplasts containing the DNA, electroporation, projectile bombardment, and infection with viral vectors, etc. Bacterial cells can be used to amplify the number of plasmids containing DNA constructs of this invention. The bacteria are grown to log phase and the plasmids within the bacteria can be isolated by a variety of methods known in the art (see, for instance, Sambrook). In addition, a plethora of kits are commercially available for the purification of plasmids from bacteria, (see, e.g., EasyPrepTM, FlexiPrepTM, both from Pharmacia Biotech;
StrataCleanTM, from Stratagene; and, QIAprepTM from Qiagen). The isolated and purified plasmids are then further manipulated to produce other plasmids, used to transfect cells or incorporated into related vectors to infect organisms. Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular target nucleic acid. The vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems. Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or optionally both. See, Giliman & Smith, Gene 8:81 (1979); Roberts, et al., Nature, 328:731 (1987); Schneider, B., etal., Protein Expr Purif 6435:10 (1995); Ausubel, Sambrook, Berger (all supra). A catalogue of Bacteria and Bacteriophages useful for cloning is provided, e.g., by the ATCC, e.g., The ATCC Catalogue of Bacteria and Bacteriophage (1992) Ghema et al. (eds.) published by the ATCC. Additional basic procedures for sequencing, cloning and other aspects of molecular biology and underlying theoretical considerations are also found in Watson et al. (1992) Recombinant DNA Second Edition Scientific American Books, NY. See, above. Further vectors useful with the sequences herein are illustrated above in the section concerning production of influenza virus for vaccines and the references cited therein.
POLYPEPTIDE PRODUCTION AND RECOVERY
[0137] Following transduction of a suitable host cell line or strain and growth of the host cells to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. In some embodiments, a secreted polypeptide product, e.g., a HA and/or NA
polypeptide as in a secreted fusion protein form, etc., is then recovered from the culture medium. In other embodiments, a virus particle containing a HA and/or a NA
polypeptide of the invention is produced from the cell. Alternatively, cells can be harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. Eukaryotic or microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, or other methods, which are well know to those skilled in the art. Additionally, cells expressing a HA
and/or a NA
polypeptide product of the invention can be utilized without separating the polypeptide from the cell. In such situations, the polypeptide of the invention is optionally expressed on the cell surface and is examined thus (e.g., by having HA and/or NA molecules (or fragments thereof, e.g., comprising fusion proteins or the like) on the cell surface bind antibodies, etc.
Such cells are also features of the invention.
[01381 Expressed polypeptides can be recovered and purified from recombinant cell cultures by any of a number of methods well known in the art, including ammonium sulfate N.1 GOO
rl_,1/1.J.JLIA/3/1.11//ZY

.=.=
or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity ,= chromatography (e.g., using any of the tagging systems known to those skilled in the art), hydroxylapatite chromatography, and lectin chromatography. Protein refolding steps can be used, as desired, in completing configuration of the mature protein. Also, high performance liquid chromatography (HPLC) can be employed in the final purification steps.
In addition =
to the references noted herein, a variety of purification methods are well known in the art, including, e.g., those set forth in Sandana (1997) Bioseparation of Proteins, Academic = Press, Inc.; and Bollag et al. (1996) Protein Methods, 2" Edition Wiley-Liss, NY; Walker = 10 (1996) The Protein Protocols Handbook Humana Press, NJ, Harris and Angal (1990) = Protein Purification Applications: A Practical Approach 1RL Press at Oxford, Oxford, England; Harris and Angal Protein Purification Methods: A Practical Approach IRL Press at Oxford, Oxford, England; Scopes (1993) Protein Purification: Principles and Practice 3rd =
Edition Springer Verlag, NY; Janson and Ryden (1998) Protein Purification:
Principles, High Resolution Methods and Applications, Second Edition Wiley-VCH, NY; and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ.
[0139]
When the expressed polypeptides of the invention are produced in viruses, the viruses are typically recovered from the culture medium, in which infected (transfected) == cells have been grown. Typically, crude medium is clarified prior to concentration of =
influenza viruses. Common methods include ultrafiltration, adsorption on barium sulfate = and elution, and centrifugation. For example, crude medium from infected cultures can first be clarified by centrifugation at, e.g., 1000-2000 x g for a time sufficient to remove cell debris and other large particulate matter, e.g., between 10 and 30 minutes.
Optionally, the clarified medium supernatant is then centrifuged to pellet the influenza viruses, e.g., at 15,000 x g, for approximately 3-5 hours. Following resuspension of the virus pellet in an appropriate buffer, such as STE (0.01 M Tris-HCI; 0.15 M NaCI; 0.0001 M EDTA) or = phosphate buffered saline (PBS) at pH 7.4, the virus is concentrated by density gradient =
centrifugation on sucrose (60%-12%) or potassium tartrate (50%-10%). Either continuous or step gradients, e.g., a sucrose gradient between 12% and 60% in four 12%
steps, are suitable. The gradients are centrifuged at a speed, and for a time, sufficient for the viruses to concentrate into a visible band for recovery. Alternatively, and for most large-scale ===
commercial applications, virus is elutriated from density gradients using a zonal-centrifuge 714elflI4,/ I I 4, rotor operating in continuous mode. Additional details sufficient to guide one of skill A
through the preparation of influenza viruses from tissue culture are provided, e.g., in Furminger. Vaccine Production, in Nicholson et al. (eds.) Textbook of Influenza pp. 324-332; Merten et al. (1996) Production of influenza virus in cell cultures for vaccine preparation, in Cohen & Shafferman (eds.) Novel Strategies in Design and Production of Vaccines pp. 141-151, and United States Patent No. 5,690,937. If desired, the recovered viruses can be stored at -80 C in the presence of sucrose-phosphate-glutamate (SPG) as a stabilizer [0140] Alternatively, cell-free transcription/translation systems can be employed to = 10 produce polypeptides comprising an amino acid sequence or subsequence of, e.g., the sequences given herein such as SEQ ID NO: 11-20, or encoded by the polynucleotide sequences of the invention, e.g., SEQ ID NO: 1-10. A number of suitable in vitro transcription and translation systems are commercially available. A general guide to in vitro transcription and translation protocols is found in Tymms (1995) In vitro Transcription and Translation Protocols: Methods in Molecular Biology Volume 37, Garland Publishing, NY.
[0141] In addition, the polypeptides, or subsequences thereof, e.g., subsequences comprising antigenic peptides, can be produced manually or by using an automated system, by direct peptide synthesis using solid-phase techniques (see, Stewart et al.
(1969) Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco; Merrifield J (1963) J
Am Chem Soc 85:2149-2154). Exemplary automated systems include the Applied Biosystems Peptide Synthesizer (Perkin Elmer, Foster City, CA). If desired, subsequences can be chemically synthesized separately, and combined using chemical methods to provide full-length polypeptides.
Modified Amino Acids [0142] Expressed polypeptides of the invention can contain one or more modified amino acids. The presence of modified amino acids can be advantageous in, for example, (a) increasing polypeptide serum half-life, (b) reducing/increasing polypeptide antigenicity, (c) increasing polypeptide storage stability, etc. Amino acid(s) are modified, for example, co-translationally or post-translationally during recombinant production (e.g., N-linked glycosylation at N-X-S/T motifs during expression in mammalian cells) or modified by synthetic means (e.g., via PEGylation).

Z00:1/110Z:125 CA 02822895 2013-06-07 [01431 Non-limiting examples of a modified amino acid include a glycosylated amino acid, a sulfated amino acid, a prenlyated (e.g., farnesylated, geranylgeranylated) *- amino acid, an acetylated amino acid, an acylated amino acid, a PEG-ylated amino acid, a . =
biotinylated amino acid, a carboxylated amino acid, a phosphorylated amino acid, and the like, as well as amino acids modified by conjugation to, e.g., lipid moieties or other organic = derivatizing agents. References adequate to guide one of skill in the modification of amino acids are replete throughout the literature. Example protocols are found in Walker (1998) Protein Protocols on CD-ROM Human Press, Towata, NJ.
Fusion Proteins [0144] The present invention also provides fusion proteins comprising fusions of the sequences of the invention (e.g., encoding HA and/or NA polypeptides as exampled by SEQ
1D NO: 11-20) or fragments thereof with, e.g., immunoglobulins (or portions thereof), sequences encoding, e.g., GFP (green fluorescent protein), or other similar markers, etc.
Nucleotide sequences encoding such fusion proteins are another aspect of the invention.
Fusion proteins of the invention are optionally used for, e.g., similar applications (including, e.g., therapeutic, prophylactic, diagnostic, experimental, etc. applications as described herein) as the non-fusion proteins of the invention. In addition to fusion with inununoglobulin sequences and marker sequences, the proteins of the invention are also optionally fused with, e.g., sequences which allow sorting of the fusion proteins and/or targeting of the fusion proteins to specific cell types, regions, etc.
Antibodies [0145] The polypeptides of the invention can be used to produce antibodies specific = for the polypeptides given herein and/or polypeptides encoded by the polynucleotides of the invention, e.g., those shown herein, and conservative variants thereof.
Antibodies specific for the above mentioned polypeptides are useful, e.g., for diagnostic and therapeutic purposes, e.g., related to the activity, distribution, and expression of target polypeptides.
[0146] Antibodies specific for the polypeptides of the invention can be generated by = methods well known in the art. Such antibodies can include, but are not limited to, polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments and fragments produced by an Fab expression library.

V V V LAO CA 02822895 2013-06-07 'IVA
I Le!
=
[0147] Polypeptides do not require biological activity for antibody production (e.g., full length functional hemagglutinin or neurarninidase is not required).
However, the polypeptide or oligopeptide must be antigenic. Peptides used to induce specific antibodies typically have an amino acid sequence of at least about 4 amino acids, and often at least 5 or 10 amino acids. Short stretches of a polypeptide can be fused with another protein, such as keyhole limpet hemocyanin, and antibody produced against the chimeric molecule.
[0148] Numerous methods for producing polyelonal and monoclonal antibodies are known to those of skill in the art, and can be adapted to produce antibodies specific for the polypeptides of the invention, and/or encoded by the polynucleotide sequences of the invention, etc. See, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY; Paul (ed.) (1998) Fundamental Immunology, Fourth Edition, Lippincott-Raven, Lippincott Williams & Wilkins; Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY; Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, CA, and references cited therein;
Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press, New York, NY;
and Kohler and Milstein (1975) Nature 256: 495-497. Other suitable techniques for antibody preparation include selection of libraries of recombinant antibodies in phage or similar vectors. See, Huse et al. (1989) Science 246: 1275-1281; and Ward, et al. (1989) Nature 341: 544-546. Specific monoclonal and polyclonal antibodies and antisera will usually bind with a KD of, e.g., at least about 0.1 p.M, at least about 0.01 p.M or better, and, typically and at least about 0.001 111\,4 or better.
[0149] For certain therapeutic applications, humanized antibodies are desirable.
Detailed methods for preparation of chimeric (humanized) antibodies can be found in U.S.
Patent 5,482,856. Additional details on humanization and other antibody production and engineering techniques can be found in Borrebaeck (ed.) (1995) Antibody Engineering, 2"d Edition Freeman and Company, NY (Borrebaeck); McCafferty et al. (1996) Antibody Engineering, A Practical Approach IRL at Oxford Press, Oxford, England (McCafferty), and Paul (1995) Antibody Engineering Protocols Humana Press, Towata, NJ
(Paul).
Additional details regarding specific procedures can be found, e.g., in Ostberg et al. (1983), Hybridoma 2: 361-367, Ostberg, U.S. Pat. No. 4,634,664, and Engelman et al., U.S. Pat.
No. 4,634,666.

%-/ LIMO/ 1 10L,10 CA 02822895 2013-06-07 Ft 1 /
UNZUlt_1/01 / /29 DefiningPolypeptides by Immunoreactivity [01501 Because the polypeptides of the invention provide a variety of new polypeptide sequences (e.g., comprising HA and NA molecules), the polypeptides also provide new structural features which can be recognized, e.g., in immunological assays.
The generation of antisera which specifically bind the polypeptides of the invention, as well as the polypeptides which are bound by such antisera, are features of the invention.
101511 For example, the invention includes polypeptides (e.g., HA and NA
molecules) that specifically bind to or that are specifically immunoreactive with an antibody or antisera generated against an immunogen comprising an amino acid sequence selected from one or more of the sequences given herein (e.g., SEQ ID NO: 11-20), etc.
To eliminate cross-reactivity with other homologues, the antibody or antisera is subtracted with the HA and/or NA molecules found in public databases at the time of filing, e.g., the "control" polypeptide(s). Where the other control sequences correspond to a nucleic acid, a polypeptide encoded by the nucleic acid is generated and used for antibody/antisera subtraction purposes.
[01521 In one typical format, the immunoassay uses a polyclonal antiserum which =
was raised against one or more polypeptide comprising one or more of the sequences corresponding to the sequences herein (e.g., SEQ ID NO: 11-20), etc. or a substantial subsequence thereof (i.e., at least about 30% of the full length sequence provided). The set of potential polypeptide immunogens derived from the present sequences are collectively referred to below as "the immunogenic polypeptides." The resulting antisera is optionally selected to have low cross-reactivity against the control hemagglutinin and/or neuraminidase homologues and any such cross-reactivity is removed, e.g., by immunoabsorbtion, with one or more of the control hemagglutinin and neuraminidase homologues, prior to use of the polyclonal antiserum in the immunoassay.
[0153] In order to produce antisera for use in an immunoassay, one or more of the immunogenic polypeptides is produced and purified as described herein. For example, recombinant protein can be produced in a recombinant cell. An inbred strain of mice (used in this assay because results are more reproducible due to the virtual genetic identity of the mice) is immunized with the immunogenic protein(s) in combination with a standard adjuvant, such as Freund's adjuvant, and a standard mouse immunization protocol (see, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor _ Publications, New York, for a standard description of antibody generation, immunoassay formats and conditions that can be used to determine specific immunoreactivity).
Additional references and discussion of antibodies is also found herein and can be applied here to defining polypeptides by immunoreactivity. Alternatively, one or more synthetic or recombinant polypeptide derived from the sequences disclosed herein is conjugated to a carrier protein and used as an immunogen.
[01541 Polyclonal sera are collected and titered against the immunogenic polypeptide in an immunoassay, for example, a solid phase immunoassay with one or more of the immunogenic proteins immobilized on a solid support. Polyclonal antisera with a titer of 106 or greater are selected, pooled and subtracted with the control hemagglutinin and/or neuraminidase polypeptide(s) to produce subtracted pooled titered polyclonal antisera.
[01551 The subtracted pooled titered polyclonal antisera are tested for cross reactivity against the control homologue(s) in a comparative immunoassay. In this comparative assay, discriminatory binding conditions are determined for the subtracted titered polyclonal antisera which result in at least about a 5-10 fold higher signal to noise ratio for binding of the titered polyclonal antisera to the immunogenic polypeptides as compared to binding to the control homologues. That is, the stringency of the binding reaction is adjusted by the addition of non-specific competitors such as albumin or non-fat dry milk, and/or by adjusting salt conditions, temperature, and/or the like.
These binding conditions are used in subsequent assays for determining whether a test polypeptide (a polypeptide being compared to the immunogenic polypeptides and/or the control polypeptides) is specifically bound by the pooled subtracted polyclonal antisera. In particular, test polypeptides which show at least a 2-5x higher signal to noise ratio than the control receptor homologues under discriminatory binding conditions, and at least about a 'A
signal to noise ratio as compared to the immunogenic polypeptide(s), shares substantial structural similarity with the immunogenic polypeptide as compared to the known receptor, etc., and is, therefore a polypeptide of the invention.
[0156] In another example, immunoassays in the competitive binding format are used for detection of a test polypeptide. For example, as noted, cross-reacting antibodies are removed from the pooled antisera mixture by immunoabsorbtion with the control polypeptides. The immunogenic polypeptide(s) are then immobilized to a solid support f= .
which is exposed to the subtracted pooled antisera. Test proteins are added to the assay to compete for binding to the pooled subtracted antisera. The ability of the test protein(s) to .f. compete for binding to the pooled subtracted antisera as compared to the immobilized protein(s) is compared to the ability of the immunogenic polypeptide(s) added to the assay = 5 to compete for binding (the immunogenic polypeptides compete effectively with the immobilized immunogenic polypeptides for binding to the pooled antisera). The percent cross-reactivity for the test proteins is calculated, using standard calculations.
[01571 In a parallel assay, the ability of the control protein(s) to compete for binding = to the pooled subtracted antisera is optionally determined as compared to the ability of the = 10 immunogenic polypeptide(s) to compete for binding to the antisera.
Again, the percent = cross-reactivity for the control polypeptide(s) is calculated, using standard calculations.
Where the percent cross-reactivity is at least 5-10x as high for the test polypeptides as compared to the control polypeptide(s) and or where the binding of the test polypeptides is approximately in the range of the binding of the immunogenic polypeptides, the test = 15 polypeptides are said to specifically bind the pooled subtracted antisera.
[01581 in general, the immunoabsorbed and pooled antisera can be used in a competitive binding immunoassay as described herein to compare any test polypeptide to = the immunogenic and/or control polypeptide(s). In order to make this comparison, the immunogenic, test and control polypeptides are each assayed at a wide range of 20 concentrations and the amount of each polypeptide required to inhibit 50% of the binding of = the subtracted antisera to, e.g., an immobilized control, test or immunogenic protein is determined using standard techniques. If the amount of the test polypeptide required for binding in the competitive assay is less than twice the amount of the immunogenic polypeptide that is required, then the test polypeptide is said to specifically bind to an 25 antibody generated to the immunogenic protein, provided the amount is at least about 5-10x as high as for the control polypeptide.
[01591 As an additional determination of specificity, the pooled antisera is optionally fully immunosorbed with the immunogenic polypeptide(s) (rather than the control polypeptide(s)) until little or no binding of the resulting immunogenic polypeptide 30 subtracted pooled antisera to the immunogenic polypeptide(s) used in the immunosorbtion is detectable. This fully immunosorbed antisera is then tested for reactivity with the test polypeptide. If little or no reactivity is observed (i.e., no more than 2x the signal to noise CA 02822895 2013-06-07 / ij 'Vol ky j ratio observed for binding of the fully immunosorbed antisera to the immunogenic polypeptide), then the test polypeptide is specifically bound by the antisera elicited by the immunogenic protein.
NUCLEIC ACID AND POLYPEPTIDE SEQUENCE VARIANTS
[0160] As described herein, the Inventionprovides for nucleic acid polynucleotide sequences and polypeptide amino acid sequences, e.g., hemagglutinin and neuraminidase sequences, and, e.g., compositions and methods comprising said sequences.
Examples of said sequences are disclosed herein (e.g., SEQ ID NO: 1-20). However, one of skill in the art will appreciate that the invention is not necessarily limited to those sequences disclosed herein and that the present invention also provides many related and unrelated sequences with the functions described herein, e.g., encoding a HA and/or a NA molecule.
[0161] One of skill will also appreciate that many variants of the disclosed sequences are included in the invention. For example, conservative variations of the .
disclosed sequences that yield a functionally identical sequence are included in the invention. Variants of the nucleic acid polynucleotide sequences, wherein the variants hybridize to at least one disclosed sequence, are considered to be included in the invention.
Unique subsequences of the sequences disclosed herein, as determined by, e.g., standard sequence comparison techniques, are also included in the invention.
Silent Variations [0162] Due to the degeneracy of the genetic code, any of a variety of nucleic acid sequences encoding polypeptides and/or viruses of the invention are optionally produced, some which can bear lower levels of sequence identity to the HA and NA nucleic acid and polypeptide sequences herein. The following provides a typical codon table specifying the genetic code, found in many biology and biochemistry texts.

WV ZUU:V1162:121 CA 02822895 2013-06-07 ez:
Table 1 Codon Table , Amino acids Codon Alanine Ala A GCA GCC GCG GCU
= Cysteine Cys C UGC UGU
Aspartic acid Asp D GAC GAI I
= Glutamic acid Glu E GAA GAG
Phenylalanine Phe F UUC UUU
Glycine Gly G GGA GGC GGG _ GGU
Histidine His H CAC CAU
Isoleucine Ile I AUA AUC AUU
=
=
Lysine Lys K AAA AAG
Leucine Leu L UUA UUG CUA CUC CUG CUU
Methionine . Met M AUG
Asparagine Asn N AAC AAU
Proline Pro P CCA CCC CCG CCU
Glutamine Gin Q CAA CAG
= Arginine Arg R AGA AGG CGA CGC CGG CGU
Serine Ser S AGC AGU UCA UCC UCG UCU
Threonine Thr T ACA ACC ACG ACU
Valine Val V GUA GUC GUG GUU
Tryptophan Tq W UGG
Tyrosine Tyr Y UAC UAU
[01631 The codon table shows that many amino acids are encoded by more than one = codon. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine. Thus, at every position in the nucleic acids of the invention where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described above without altering the encoded polypeptide. It is understood that U in an RNA sequence corresponds to T in a DNA sequence.
[01641 Such "silent variations" are one species of "conservatively modified variations," discussed below. One of skill will recognize that each codon in a nucleic acid (except ATG, which is ordinarily the only codon for methionine, and TTG, which is ordinarily the only codon for tryptophan) can be modified by standard techniques to encode a functionally identical polypeptide. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in any described sequence. The invention, therefore, explicitly provides each and every possible variation of a nucleic acid sequence encoding a polypeptide of the invention that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code (e.g., as set forth in Table 1, or as is commonly available in the art) as =

VV .41.1110"'' CA 02822895 2013-06-07 rk, U3LUUD/1/1 / I LY
applied to the nucleic acid sequence encoding a hemagglutinin or a neuraminidase polypeptide of the invention. All such variations of every nucleic acid herein are specifically provided and described by consideration of the sequence in combination with the genetic code. One of skill is fully able to make these silent substitutions using the methods herein.
Conservative variations [0165] Owing to the degeneracy of the genetic code, "silent substitutions" (i.e., substitutions in a nucleic acid sequence which do not result in an alteration in an encoded polypeptide) are an implied feature of every nucleic acid sequence of the invention which encodes an amino acid. Similarly, "conservative amino acid substitutions," in one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties, are also readily identified as being highly similar to a disclosed construct such as those herein. Such conservative variations of each disclosed sequence are a feature of the present invention.
[0166] "Conservative variations" of a particular nucleic acid sequence refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or, where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences, see, Table 2 below. One of skill will recognize that individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 4%, 3%, 2% or 1%) in an encoded sequence are "conservatively modified variations" where the alterations result in the deletion of an amino acid, addition of an amino acid, or substitution of an amino acid with a chemically similar amino acid. Thus, "conservative variations" of a listed polypeptide sequence of the present invention include substitutions of a small percentage, typically less than 5%, more typically less than 4%, 3%, 2% or 1%, of the amino acids of the polypeptide sequence, with a conservatively selected amino acid of the same conservative substitution group. Finally, the addition of sequences which do not alter the encoded activity of a nucleic acid molecule, such as the addition of a non-functional sequence, is a conservative variation of the basic nucleic acid.
101671 Table 2 -- Conservative Substitution Groups 1 Alanine (A) Serine (S) Threonine (T) :
2 Aspartic acid (D) Glutamic acid (E) 3 Asparagine (N) Glutamine (Q) 4 Arginine (R) Lysine (K) Isoleucine (I) Leucine (L) Methionine (M) Valine (V) 6 Phenylalanine (F) Tyrosine (Y) Tryptophan (W) Unique Polypeptide and Polynucleotide Subsequences 101681 In one aspect, the invention provides a nucleic acid which comprises a unique subsequence in a nucleic acid selected from the sequence of HA and NA
molecules 5 disclosed herein, e.g., SEQ ID NO: 1-10. The unique subsequence is unique as compared to a nucleic acids corresponding to nucleic acids such as, e.g., those found in GenBank or other similar public databases at the time of filing. Alignment can be performed using, e.g., = BLAST set to default parameters. Any unique subsequence is useful, e.g., as a probe to identify the nucleic acids of the invention. See, above.
101691 Similarly, the invention includes a polypeptide which comprises a unique subsequence in a polypeptide selected from the sequence of HA and NA molecules = disclosed herein, e.g., SEQ ID NO: 11-20. Here, the unique subsequence is unique as compared to a polypeptide corresponding to, e.g., the amino acid corresponding to = polynucleotide sequences found in, e.g., GenBank or other similar public databases at the time of filing.
10170] The invention also provides for target nucleic acids which hybridize under stringent conditions to a unique coding oligonucleotide which encodes a unique subsequence in a polypeptide selected from the sequences of HA and NA
molecules of the invention wherein the unique subsequence is unique as compared to a polypeptide corresponding to any of the control polypeptides (sequences of, e.g., the nucleic acids corresponding to those found in, e.g., GenBank or other similar public databases at the time of filing). Unique sequences are determined as noted above.
Sequence comparison, identity, and homology [0171] The terms "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that lik.71,MII.J/1/1 I 1,7 are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (or other algorithms available to persons of skill) or by visual inspection.
[0172] The phrase "substantially identical," in the context of two nucleic acids or polypeptides (e.g., DNAs encoding a HA or NA molecule, or the amino acid sequence of a HA or NA molecule) refers to two or more sequences or subsequences that have at least about 90%, preferably 91%, most preferably 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. Such "substantially identical" sequences are typically considered to be "homologous," without reference to actual ancestry. Preferably, "substantial identity" exists over a region of the amino acid sequences that is at least about 200 residues in length, more preferably over a region of at least about 250 residues, and most preferably the sequences are substantially identical over at least about 300 residues, 350 residues, 400 residues, 425 residues, 450 residues, 475 residues, 480 residues, 490 residues, 495 residues, 499 residues, 500 residues, 502 residues, 559 residues, 565 residues, or 566 residues, or over the full length of the two sequences to be compared.
[0173] For sequence comparison and homology determination, typically one sequence acts as a reference sequence to which test sequences are compared.
When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
[0174] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv App! Math 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J Mol Biol 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc Nat! Acad Sci USA
85:2444 (1988), by computerized implementations of algorithms such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by visual inspection (see generally, Ausubel et al., supra):
[0175] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J Mol Biol 215:403-410 (1990). Software for performing BLAST
analyses is publicly available through the National Center for Biotechnology Information 8600 Rockville Pike, Bethesda MD, USA
(www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (see, Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSIJ1V162 scoring matrix (see, Henikoff &
Henikoff (1989) Proc Natl Acad Sci USA 89:10915).
[0176] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc Natl Acad Sci USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a , . =
= 4 VV L.A.1 lu.,-00 CA 02822895 2013-06-07 1 Ill.1,11-AW.,1,J.1 1 14, =
reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
= 101771 Another example of a useful sequence alignment algorithm is PILEUP.
PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment -method of Feng & Doolittle (1987) J. Mol_ Evol. 35:351-360. The method used is similar to the method described by Higgins & Sharp (1989) CABIOS5:151-153. The program can align, e.g., up to 300 sequences of a maximum length of 5,000 letters. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster can then be aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences can be aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments.
The program can also be used to plot a dendogram or tree representation of clustering relationships. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison.
[01781 An additional example of an algorithm that is suitable for multiple DNA, or amino acid, sequence alignments is the CLUSTALW program (Thompson, J. D. et al.
(1994) Nucl. Acids. Res. 22: 4673-4680). CLUSTALW performs multiple pairwise comparisons between groups of sequences and assembles them into a multiple alignment based on homology. Gap open and Gap extension penalties can be, e.g., 10 and 0.05 respectively. For amino acid alignments, the BLOSUM algorithm can be used as a protein weight matrix. See, e.g., Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci.
USA 89:
10915-10919.
DIGITAL SYSTEMS
101791 The present invention provides digital systems, e.g., computers, computer readable media and integrated systems comprising character strings corresponding to the sequence information herein for the nucleic acids and isolated or recombinant polypeptides herein, including, e.g., the sequences shown herein, and the various silent substitutions and WO LUUJ/11()2Z18 CA 02822895 2013-06-07 conservative substitutions thereof. Integrated systems can further include, e.g., gene synthesis equipment for making genes corresponding to the character strings.
[0180] Various methods known in the art can be used to detect homology or similarity between different character strings (see, above), or can be used to perform other desirable functions such as to control output files, provide the basis for making presentations of information including the sequences and the like. Examples include BLAST, discussed supra. Computer systems of the invention can include such programs, e.g., in conjunction with one or more data file or data base comprising a sequence as noted herein.
[0181] Thus, different types of homology and similarity of various stringency and length between various HA or NA sequences or fragments, etc. can be detected and recognized in the integrated systems herein. For example, many homology determination methods have been designed for comparative analysis of sequences of biopolymers, for spell-checking in word processing, and for data retrieval from various databases. With an understanding of double-helix pair-wise complement interactions among 4 principal nucleobases in natural polynucleotides, models that simulate annealing of complementary homologous polynucleotide strings can also be used as a foundation of sequence alignment or other operations typically performed on the character strings corresponding to the -= sequences herein (e.g., word-processing manipulations, construction of figures comprising sequence or subsequence character strings, output tables, etc.).
.=
[0182] Thus, standard desktop applications such as word processing software (e.g., Microsoft WOrdTM or Corel WordPerfectTM) and database software (e.g., spreadsheet software such as Microsoft ExcelTM, Corel Quattro Pr0TM, or database programs such as Microsoft AccessTM, ParadoxTM, GeneWorksTM, or MacVectorTM or other similar programs) can be adapted to the present invention by inputting a character string corresponding to one or more polynucleotides and polypeptides of the invention (either nucleic acids or proteins, or both). For example, a system of the invention can include the foregoing software having the appropriate character string information, e.g., used in conjunction with a user interface (e.g., a GUI in a standard operating system such as a Windows, Macintosh or LINUX
system) to manipulate strings of characters corresponding to the sequences herein. As noted, specialized alignment programs such as BLAST can also be incorporated into the 1,1 LA11/..,/
CA 02822895 2013-06-07 J l/ 1J /
/
systems of the invention for alignment of nucleic acids or proteins (or corresponding character strings).
[01831 Systems in the present invention typically include a digital computer with data sets entered into the software system comprising any of the sequences herein. The computer can be, e.g., a PC (Intel x86 or Pentium chip- compatible DOSTM, WINDOWSTM WINDOWSNTTm, WINDOWS95TM, WINDOWS2000TM, WINDOWS98TM, LINUX based_machine, a MACINTOSH", Power PC, or a UNIX based (e.g., SUNTM
work station) machine) or other commercially available computer that is known to one of skill. Software for aligning or otherwise manipulating sequences is available, or can easily be constructed by one of skill using a standard programming language such as Visualbasic, PERL, Fortran, Basic, Java, or the like.
[0184] Any controller or computer optionally includes a monitor which is often a cathode ray tube ("CRT") display, a flat panel display (e.g., active matrix liquid crystal display, liquid crystal display), or others. Computer circuitry is often placed in a box which includes numerous integrated circuit chips, such as a microprocessor, memory, interface circuits, and others. The box also optionally includes a hard disk drive, a floppy disk drive, a high capacity removable drive such as a writeable CD-ROM, and other common peripheral elements. Inputting devices such as a keyboard or mouse optionally provide for input from a user and for user selection of sequences to be compared or otherwise manipulated in the relevant computer system.
[01851 The computer typically includes appropriate software for receiving user instructions, either in the form of user input into a set parameter fields, e.g., in a GUI, or in the form of preprogrammed instructions, e.g., preprogrammed for a variety of different specific operations. The software then converts these instructions to appropriate language for instructing the operation, e.g., of appropriate mechanisms or transport controllers to carry out the desired operation. The software can also include output elements for controlling nucleic acid synthesis (e.g., based upon a sequence or an alignment of sequences herein), comparisons of samples for differential gene expression, or other operations.
KITS AND REAGENTS
[01861 The present invention is optionally provided to a user as a kit. For example, a kit of the invention contains one or more nucleic acid, polypeptide, antibody, or cell line IT 1J Ltith)f Iluthoo CA 02822895 2013-06-07 YU 1 / UbLUU:71/t11 / /J
=
described herein (e.g., comprising, or with, a HA and/or NA molecule of the invention).
The kit can contain a diagnostic nucleic acid or polypeptide, e.g., antibody, probe set, e.g., = .
as a cDNA micro-array packaged in a suitable container, or other nucleic acid such as one or more expression vector. The kit can also further comprise, one or more additional reagents, e.g., substrates, labels, primers, for labeling expression products, tubes and/or other accessories, reagents for collecting samples, buffers, hybridization chambers, cover slips, etc. The kit optionally further comprises an instruction set or user manual detailing preferred methods of using the kit components for discovery or application of diagnostic _=
sets, etc.
[0187] When used according to the instructions, the kit can be used, e.g., for evaluating a disease state or condition, for evaluating effects of a pharmaceutical agent or = other treatment intervention on progression of a disease state or condition in a cell or organism, or for use as a vaccine, etc.
[0188] In an additional aspect, the present invention provides system kits embodying the methods, composition, systems and apparatus herein. System kits of the invention optionally comprise one or more of the following: (1) an apparatus, system, system component or apparatus component; (2) instructions for practicing methods described herein, and/or for operating the apparatus or apparatus components herein and/or for using the compositions herein. In a further aspect, the present invention provides for the use of any apparatus, apparatus component, composition or kit herein, for the practice of any method or assay herein, and/or for the use of any apparatus or kit to practice any assay or method herein.
= [0189] Additionally, the kits can include one or more translation system as noted above (e.g., a cell) with appropriate packaging material, containers for holding the components of the kit, instructional materials for practicing the methods herein and/or the like. Similarly, products of the translation systems (e.g., proteins such as HA and/or NA
molecules) can be provided in kit form, e.g., with containers for holding the components of = the kit, instructional materials for practicing the methods herein and/or the like.
101901 To facilitate use of the methods and compositions of the invention, any of the vaccine components and/or compositions, e.g., reassorted virus in allantoic fluid, etc., and additional components, such as, buffer, cells, culture medium, useful for packaging and WJ LutrzylityL:vs CA 02822895 2013-06-07 11 I.J.31.Allrolv /
=
infection of influenza viruses for experimental or therapeutic vaccine purposes, can be packaged in the form of a kit. Typically, the kit contains, in addition to the above components, additional materials which can include, e.g., instructions for performing the methods of the invention, packaging material, and a container.
EXAMPLES
Construction and Analysis of H5N1 ca Viruses and Vaccines [0191] Various sequences herein comprising H5N1 HA/NA sequences were used to create influenza viruses and vaccines. The HA sequences in such vaccines were altered from wild-type by removal of the polybasic cleavage site within the HA. The HAJNA
sequences were reassorted (in a 6:2 reassortment) with A/AA/6/60 (an att, ca virus, see above).
[0192] Three strains of H5N1 influenza were used in this example:
A/VN/1203/2004, A/HK/491/97, and A/HK/213/2003. Such strains are also referred to within this example as the '97, '03, and '04 strains based on their year designations. The percent similarity of the HA genes of such three strains is 95-96%. Figure 1 illustrates modification of the polybasic cleavage site of an exemplary HA sequence, the '04 HA
sequences, used to construct the viruses/vaccines. As stated previously, various embodiments of the invention comprise sequences which have differing regions of the polybasic cleavage site removed. See above.
101931 As stated, the modified H5N1 sequences (i.e., the modified '97, '03, and '04 genes) were used to construct 6:2 reassortant viruses with A/AA/6/60. It will be appreciated, and is pointed out elsewhere herein, that other desirable backbones could also have been used (e.g., PR8, etc.).
[01941 In the 6:2 reassortants of this example, the HA and NA gene sequences were derived from the wild type parent virus and the remaining genes were characterized by sequence analysis as derived from the A/AA/6/60 ca parent virus. The reassorted viruses replicated to 8.0-8.5 1og10TC1D50 in eggs. However, it will be appreciated that other embodiments wherein the log10TC1D50 comprises from about 7.0 to about 9.0, from about 7.5-8.5, or from about 8.0-8.5 are also claimed within the invention. The cleavability of the modified HA in the constructed viruses by endogenous proteases was restricted in vitro and 0 Mb/11625N CA 02822895 2013-06-07 the viruses were dependent on tryp sin (e.g., from about 0.1 ug/ml to about 1.0 ug/ml) for growth. The constructed viruses were temperature sensitive in vitro.
;
101951 The H5N1 ca reassortant viruses (having the modified '97, '03, or '04 HA
genes) were not highly pathogenic for chickens. For example, when 4-week-old SPF white Plymouth Rock chickens were inoculated intravenously with a 1:10 dilution of stock virus (108-875 TCID50/m1) and observed for 10 days, it was observed that 8 out of 8 chickens died within 1-2 days when wild-type '97, '03, and '04 H5N1 were used, while 0 of 8 chickens =
died when the H5N1 ca reassortant viruses were used. As can be seen in Figure 2, the intranasally administered H5N1 ca reassortant viruses did not replicate in chickens.
101961 The H5N1/AA ca reassortants were also not lethal for mice. See Figure 3, which also shows the TOD50 for the H5N1 wild-type strains. Figure 4 shows that the 1997 and 2004 H5N1 ca reassortant viruses were restricted in replication in mice.
Figure 5, shows that the H5N1 ca reassorted viruses are restricted in replication in lungs of mice.
[01971 A comparison of the serum HAI antibody titers elicited in mice following a single intranasal dose of vaccine (2003 ca as compared against 2003 wild-type), is shown in Figure 6.
Figure 7 shows similar measurements, but using serum neutralizing antibody titers.
[01981 Figure 8 displays that the H5N1 ca reassortant viruses protect mice from lethal challenge with 50, 500, or 5,000 LD50 of wild-type H5N1 virus. Figure 9 shows the õ
efficacy of protection from pulmonary replication of homologous and heterologous H5N1 challenge viruses in mice. As can be seen, the ca reassortants replicated less well than the wild-type viruses did. Figure 10 shows related data using upper respiratory tracts of mice.
Those of skill in the art will be familiar with homologous and heterologous challenges (e.g., = testing whether 2003 vaccine protects against a 2003 wild-type challenge (homologous) or whether a 2003 vaccine protects against a 1997 wild-type challenge (heterologous), etc.).
[01991 Figure 11 shows efficacy of protection conferred by 2004 H5N1 ca vaccine against high dose (105TCID50) challenge with homologous or heterologous H5N1 wild-type viruses in mice. Figure 12 shows efficacy of protection conferred by 1997 and ca vaccines against high dose (105TCID50) challenge with homologous or heterologous H5N1 wild-type viruses in mice. Figure 13 shows efficacy of protection conferred by 2004 = H5N1 ca vaccine against low or high doses of homologous H5N1 wild-type virus challenge .=

in mice. Figures 11- 13 demonstrate that the tested vaccines could protect against Other related viruses.
=
[02001 The current example demonstrates several points concerning exemplary H5N1 ca reassortant viruses/vaccines Of the invention. The modified ca reassortant '97, '03, and '04 viruses were shown to have in vitro ts phenotype, loss of pathogenicity in chickens and attenuation in mice. It is expected that attenuation is also present in ferrets.
Efficacy of protection and cross-protection against lethal challenge and systemic spread with wild-type viruses in mice was also shown. Efficacy of protection and cross-protections against replication of wild-type challenge viruses in the respiratory tract of mice is also expected.
102011 It is contemplated to use these (and similar) viruses/vaccines to determine whether immunogenticy and efficacy is improved following 2 doses of vaccine;
to assess immunogenicity in non-human primates; to assess attenuation and vaccine efficacy in ferrets; to determine the contribution of humoral and cellular immunity to observed efficacy of the produced vaccines in mice; to determine which residues of the 2003 HA
contribute to enhanced irnrnunogenicity and introduce them into 1997 and 2004 HAs; and to determine the effects of deleting the multibasic amino acid cleavage site and of the gene constellation.
102021 While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, all the techniques and apparatus described above may be used in various combinations. See also U.S. provisional application nos. 60/574,553 filed May 24, 2004 and 60/657,554, filed February 28, 2005.

, SEQUENCES
A/Vietnam/1203/04 Nucleotide Sequence of A/Vietnam/1203/04 H5 (SEQ 1D NO:1) Entire molecule length: 1767 nt 1 agcaaaagca ggggttcaat ctgtcaaaat ggagaaaata gtgcttcttt 51 ttgcaatagt cagtcttgtt aaaagtgatc agatttgcat tggttaccat 101 gcaaacaact cgacagagca ggttgacaca ataatggaaa agaacgttac 151 tgttacacat gcccaagaca tactggaaaa gaaacacaac gggaagctct 201 gcgatctaga tggagtgaag cctctaattt tgagagattg tagcgtagct 251 ggatggctcc tcggaaaccc aatgtgtgac gaattcatca atgtgccgga 301 atggtcttac atagtggaga aggccaatcc agtcaatgac ctctgttacc 351 caggggattt caatgactat gaagaattga aacacctatt gagcagaata 401 aaccattttg agaaaattca gatcatcccc aaaagttctt ggtccagtca 451 tgaagcctca ttaggggtga gctcagcatg tccataccag ggaaagtcct 501 cctttttcag aaatgtggta tggcttatca aaaagaacag tacataccca 551 acaataaaga ggagctacaa taataccaac caagaagatc ttttggtact 601 gtgggggatt caccatccta atgatgcggc agagcagaca aagctctatc 651 aaaacccaac cacctatatt tccgttggga catcaacact aaaccagaga 701 ttggtaccaa gaatagctac tagatccaaa gtaaacgggc aaagtggaag 751 gatggagttc ttctggacaa ttttaaagcc gaatgatgca atcaacttcg 801 agagtaatgg aaatttcatt gctccagaat atgcatacaa aattgtcaag 851 aaaggggact caacaattat gaaaagtgaa ttggaatatg gtaactgcaa 901 caccaagtgt caaactccaa tgggggcgat aaactctagc atgccattcc 951 acaatataca ccctctcacc attggggaat gccccaaata tgtgaaatca 1001 aacagattag tccttgcgac tgggctcaga aatagccctc aaagagagac 1051 tcgaggatta tttggagcta tagcaggttt tatagaggga ggatggcagg 1101 gaatggtaga tggttggtat gggtaccacc atagcaatga gcaggggagt 1151 gggtacgctg cagacaaaga atccactcaa aaggcaatag atggagtcac 1201 caataaggtc aactcgatca ttgacaaaat gaacactcag tttgaggccg 1251 ttggaaggga atttaacaac ttagaaagga gaatagagaa tttaaacaag 1301 aagatggaag acgggttcct agatgtctgg acttataatg ctgaacttct 1351 ggttctcatg gaaaatgaga gaactctaga ctttcatgac tcaaatgtca 1401 agaaccttta cgacaaggtc cgactacagc ttagggataa tgcaaaggag 1451 ctgggtaacg gttgtttcga gttctatcat aaatgtgata atgaatgtat 1501 ggaaagtgta agaaatggaa cgtatgacta cccgcagtat tcagaagaag 1551 cgagactaaa aagagaggaa ataagtggag taaaattgga atcaatagga 1601 atttaccaaa tactgtcaat ttattctaca gtggcgagtt ccctagcact 1651 ggcaatcatg gtagctggtc tatccttatg gatgtgctcc aatgggtcgt 1701 tacaatgcag aatttgcatt taaatttgtg agttcagatt gtagttaaaa = 1751 acacccttgt ttctact Amino acid sequence of ANietnam/1203/04 H5 (SEQ ID NO: 11) Entire molecule length: 564 aa 1 mekivllfai vslvksdqic igyhannste qvdtimeknv tvthaqdile 51 kkhngklcdl dgvkplilrd csvagwllgn pmcdefinvp ewsyivekan 101 pvndlcypgd fndyeelkhl lsrinhfeki qiipksswss heaslgvssa lJ1/l/J/t/1 / /
151 cpyqgkssff rnvvwlikkn styptikrsy nntnqedllv lwgihhpnda 201 aeqtklyqnp ttyisvgtst lnqrlvpria trskvngqsg rmeffwtilk 251 pndainfesn gnfiapeyay kivkkgdsti mkseleygnc ntkcqtpmga 301 inssmpfhni hpltigecpk yvksnrlvla tglrnspqre trglfgaiag 351 fieggwqgmv dgwygyhhsn eqgsgyaadk estqkaidgv tnkvnsiidk 401 mntqfeavgr efnnlerrie nlnkkmedgf ldvwtynael lvlmenertl 451 dfhdsnvknl ydkvrlqlrd nakelgngcf efyhkcdnec mesvrngtyd 501 ypqyseearl kreeisgvkl esigiyqils iystvassla laimvagls1 551 wmcsngslqc ridi Nucleotide Sequence of AJVietnam/1203/04 Ni iSEQ ED NO: 2) Entire molecule length: 1398 nt 1 agcaaaagca ggagttcaaa atgaatccaa atcagaagat aataaccatc 51 gggtcaatct gtatggtaac tggaatagtt agcttaatgt tacaaattgg 101 gaacatgatc tcaatatggg tcagtcattc aattcacaca gggaatcaac 151 accaatctga accaatcagc aatactaatt ttcttactga gaaagctgtg 201 gcttcagtaa aattagcggg caattcatct ctttgcccca ttaacggatg 251 ggctgtatac agtaaggaca acagtataag gatcggttcc aagggggatg 301 tgtttgttat aagagagccg ttcatctcat gctcccactt ggaatgcaga 351 actttctttt tgactcaggg agccttgctg aatgacaagc actccaatgg 401 gactgtcaaa gacagaagcc ctcacagaac attaatgagt tgtcctgtgg 451 gtgaggctcc ctccccatat aactcaaggt ttgagtctgt tgcttggtca 501 gcaagtgctt gccatgatgg caccagttgg ttgacgattg gaatttctgg 551 cccagacaat ggggctgtgg ctgtattgaa atacaatggc ataataacag 601 acactatcaa gagttggagg aacaacatac tgagaactca agagtctgaa 651 tgtgcatgtg taaatggctc ttgctttact gtaatgactg acggaccaag 701 taatggtcag gcatcacata agatcttcaa aatggaaaaa gggaaagtgg 751 ttaaatcagt cgaattggat gctcctaatt atcactatga ggaatgctcc 801 tgttatccta atgccggaga aatcacatgt gtgtgcaggg ataattggca 851 tggctcaaat cggccatggg tatctttcaa tcaaaatttg gagtatcaaa 901 taggatatat atgcagtgga gttttcggag acaatccacg ccccaatgat 951 ggaacaggta gttgtggtcc ggtgtcctct aacggggcat atggggtaaa 1001 agggttttca tttaaatacg gcaatggtgt ctggatcggg agaaccaaaa 1051 gcactaattc caggagcggc tttgaaatga tttgggatcc aaatgggtgg 1101 actgaaacgg acagtagctt ttcagtgaaa caagatatcg tagcaataac 1151 tgattggtca ggatatagcg ggagttttgt ccagcatcca gaactgacag 1201 gactagattg cataagacct tgtttctggg ttgagttgat cagagggcgg 1251 cccaaagaga gcacaatttg gactagtggg agcagcatat ctttttgtgg 1301 tgtaaatagt gacactgtgg gttggtcttg gccagacggt gctgagttgc 1351 cattcaccat tgacaagtag tttgttcaaa aaactccttg tttctact Amino acid sequence of ANietnam/1203/04 N1 (SEQ ID NO: 12) Entire molecule length: 449 aa 1 mnpnqkiiti gsicmvtgiv slmlqignmi siwvshsiht gnqhqsepis 51 ntnfltekav asvklagnss lcpingwavy skdnsirigs kgdvfvirep 101 fiscshlecr tffltqgall ndkhsngtvk drsphrtlms cpvgeapspy 151 nsrfesvaws asachdgtsw ltigisgpdn gavavlkyng iitdtikswr 201 nnilrtgese cacvngscft vmtdgpsngq ashkifkmek gkvvksveld 251 apnyhyeecs cypnageitc vcrdnwhgsn rpwvsfnqnl eygigyicsg = 301 vfgdnprpnd gtgscgpvss ngaygvkgfs fkygngvwig rtkstnsrsg 351 femiwdpngw tetdssfsvk gdivaitdws gysgsfvqhp eltgldcirp 401 cfwvelirgr pkestiwtsg ssisfcgvns dtvgwswpdg aelpftidk AThiong Kong/213/03 Nucleotide Sequence of A/Hong Kong/213/03 H5 fSEQ ID NO: 3) Entire molecule length: 1767 nt 1 agcaaaagca ggggttcaat ctgtcaaaat ggagaaaata gtgcttcttt 51 ttgcaatagt cagtcttgtt aaaagtgatc agatttgcat tggttaccat 101 gcaaacaact cgacagagca ggttgacaca ataatggaaa agaacgttac 151 tgttacacat gcccaagaca tactggaaaa gacacacaac gggaagctct 201 gcgatctaga tggagtgaag cctctaattt tgagagattg tagtgtagct 251 ggatggctcc tcggaaaccc aatgtgtgac gaattcatca atgtgccgga 301 atggtcttac atagtggaga aggccaatcc agccaatgac ctctgttacc = 351 caggggattt caacgactat gaagaattga aacacctatt gagcagaata 401 aaccattttg agaaaattca gatcatcccc aaaaattctt ggtccagtca = 451 tgaagcctca ttaggggtga gctcagcatg tccataccaa ggaaagtcct 501 cctttttcag gaatgtggta tggcttatca aaaagaacaa tgcataccca 551 acaataaaga ggagctacaa taataccaac caagaagatc ttttggtatt 601 gtgggggatt caccatccta atgatgcggc agagcagact aggctctatc 651 aaaacccaac cacctacatt tccgttggga catcaacact aaaccagaga = 701 ttggtaccaa aaatagctac tagatccaaa gtaaacgggc aaaatggaag 751 gatggagttc ttctggacaa ttttaaaacc gaatgatgca atcaacttcg 801 agagcaatgg aaatttcatt gctccagaat atgcatacaa aattgtcaag 851 aaaggggact cagcaattat gaaaagtgaa ttggaatatg gtaactgcaa 901 caccaagtgt caaactccaa tgggggcgat aaactctagt atgccattcc 951 acaatataca ccctctcacc atcggggaat gccccaaata tgtgaaatca = 1001 aacagattag tccttgcgac tgggctcaga aatagccctc aaagagagac 1051 tcgaggatta tttggagcta tagcaggttt tatagaggga ggatggcagg 1101 gaatggtaga tggttggtat gggtaccacc atagcaatga gcaggggagt 1151 gggtacgctg cagacaaaga atccactcaa aaggcaatag atggagtcac 1201 caataaggtc aactcgatca ttgacaaaat gaacactcag tttgaggccg 1251 ttggaaggga atttaataac ttagaaagga gaatagagaa tttaaacaag 1301 aagatggaag acggattcct agatgtctgg acttataatg ctgaacttct 1351 ggttctcatg gaaaatgaga gaactctaga ctttcatgac tcaaatgtca = 40 1401 agaaccttta cgacaaggtc cgactacagc ttagggataa tgcaaaggag 1451 ctgggtaacg gttgtttcga gttctatcac aaatgtgata atgaatgtat 1501 ggaaagtgta agaaacggaa cgtatgacta cccgcagtat tcagaagaag 1551 caagactaaa aagagaggaa ataagtggag taaaattgga gtcaatagga 1601 acttaccaaa tactgtcaat ttattctaca gtggcgagtt ccctagcact = 45 1651 ggcaatcatg gtagctggtc tatctttatg gatgtgctcc aatgggtcgt 1701 tacaatgcag aatttgcatt taaatttgtg agttcagatt gtagttaaaa 1751 acacccttgt ttctact Amino acid sequence of A/Hong Kong/213/03 H5 (SEQ ID NO: 13) = 50 Entire molecule length: 564 aa 1 mekiv11fai vslvksdqic igyhannste qvdtimeknv tvthaqdile 51 kthngklcdl dgvkplilrd csvagwllgn pmcdefinvp ewsyivekan 101 pandlcypgd fndyeelkhl lsrinhfeki qiipknswss heaslgvssa 151 cpyqgkssff rnvvwlikkn nayptikrsy nntnqedllv 1wgihhpnda 201 aeqtrlygnp ttyisvgtst ingrlvpkia trskvngqng rmeffwtilk 251 pndainfesn gnfiapeyay kivkkgdsai mkseleygnc ntkcqtpmga 301 inssmpfhni hpltigecpk yvksnrlvla tglrnspqre trglfgaiag 351 fieggwqgmv dgwygyhhsn eggsgyaadR estqkaidgv tnkvnsiidk 401 mntqfeavgr efnnlerrie nlnkkmedgf ldvwtynael lvlmenertl 451 dfhdsnvknl ydkvrlqlrd nakelgngcf efyhkcdnec mesvrngtyd 501 ypqyseearl kreeisgvkl esigtyqi1S- iystvassla laimvagls1 551 wmcsngslqc rici Nucleotide Sequence of A/Hong Kong/213/03 Ni (SEQ ID NO: 4) Entire molecule length: 1458 nt 1 agcaaaagca ggagttcaaa atgaatccaa atcagaagat aacaaccatt 51 ggatcaatct gtatggtaat tggaatagtt agcttgatgt tacaaattgg 101 gaacataatc tcaatatggg ttagtcattc aattcaaaca gggaatcaac 151 accaggctga accatgcaat caaagcatta ttacttatga aaacaacacc 201 tgggtaaacc agacatatgt caacatcagc aataccaatt ttcttactga 251 gaaagctgtg gcttcagtaa cattagcggg caattcatct ctttgcccca 301 ttagtggatg ggctgtatac agtaaggaca acggtataag aatcggttcc 351 aagggggatg tgtttgttat aagagagccg ttcatctcat gctcccactt 401 ggaatgcaga actttctttt tgactcaggg agccttgctg aatgacaagc 451 attctaatgg gaccgtcaaa gacagaagcc ctcacagaac attaatgagt 501 tgtcccgtgg gtgaggctcc ttccccatac aactcgaggt ttgagtctgt 551 tgcttggtcg gcaagtgctt gtcatgatgg cactagttgg ttgacaattg 601 gaatttctgg cccagacaat ggggctgtgg ctgtattgaa atacaatggc 651 ataataacag acactatcaa gagttggagg aacaacataa tgagaactca 701 agagtctgaa tgtgcatgtg taaatggctc ttgctttact gttatgactg 751 atggaccaag taatgggcag gcttcataca aaatcttcag aatagaaaaa 801 gggaaagtag ttaaatcagc cgaattaaat gcccctaatt atcactatga 851 ggagtgctcc tgttatcctg atgctggaga aatcacatgt gtgtgcaggg 901 ataactggca tggctcaaat cggccatggg tatctttcaa tcaaaatttg 951 gagtatcgaa taggatatat atgcagtgga gttttcggag acaatccacg 1001 ccccaatgat gggacaggca gttgtggtcc ggtgtcccct aaaggggcat 1051 atggaataaa agggttctca tttaaatacg gcaatggtgt ttggatcggg 1101 agaaccaaaa gcactaattc caggagcggc tttgaaatga tttgggatcc 1151 aaatggatgg actggtacgg acagtaattt ttcagtaaag caagatattg 1201 tagctataac cgattggtca ggatatagcg ggagttttgt ccagcatcca 1251 gaactgacag gattagattg cataagacct tgtttctggg ttgagctaat 1301 cagagggcgg cccaaagaga gcacaatttg gactagtggg agcagcatat 1351 ccttttgtgg tgtaaatagt gacactgtgg gttggtcttg gccagacggt 1401 gctgagttgc cattcaccat tgacaagtag tttgttcaaa aaactccttg 1451 tttctact Amino acid sequence of A/Hong Kong/213/03 Ni (SEQ ID NO: 14) PC1'/US2005/017729 Entire molecule length: 469 aa 1 mnpnqkitti gsicmvigiv slmlqignii siwvshsiqt gnqhqaepcn 51 qsiityennt wvnqtyvnis ntnfltekav asvtlagnss 1cpisgwavy 101 skdngirigs kgdvfvirep fiscshlecr tff1tqgall ndkhsngtvk 151 drsphrtlms cpvgeapspy nsrfesvaws asachdgtsw ltigisgpdn 201 gavavlkyng iitdtikswr nnimrtqese cacvngscft vmtdgpsngq = 251 asykifriek gkvvksaeln apnyhyeecs cypdageitc vcrdnwhgsn = 301 rpwvsfnqnl eyrigyicsg vfgdnprpnd gtgscgpvsp kgaygikgfs 351 fkygngvwig rtkstnsrsg femiwdpngw tgtdsnfsvk qdivaitdws 401 gysgsfvqhp eltgldcirp cfwvelirgr pkestiwtsg ssisfcgvns 451 dtvgwswpdg aelpftidk =
A/Hong Kong/491/97 (HA) + A/Hong Kong/486/97 (NA) Nucleotide Sequence of A/Hong Kong/491/971I5 (SEQ ID NO: 5) -, Entire molecule length: 1767 rit 1 agcaaaagca ggggtataat ctgtcaaaat ggagaaaata gtgcttcttc =
20 51 ttgcaacagt cagccttgtt aaaagtgacc agatttgcat tggttaccat = 101 gcaaacaact cgacagagca agttgacaca ataatggaaa agaatgttac = 151 tgttacacat gcccaagaca tactggaaag gacacacaac gggaagctct 201 gcgatctaaa tggagtgaag cctctgattt tgagggattg tagtgtagct 251 ggatggctcc tcggaaaccc tatgtgtgac gaattcatca atgtgccgga 301 atggtcttac atagtggaga aggccagtcc agccaatgac ctctgttatc = 351 cagggaattt caacgactat gaagaactga aacacctatt gagcagaata 401 aaccattttg agaaaattca gataatcccc aaaagttctt ggtccaatca 451 tgatgcctca tcaggggtga gctcagcatg tccatacctt gggaggtcct .N4 501 cctttttcag aaatgtggta tggcttatca aaaagaacag tagctaccca . 551 acaataaaga ggagctacaa taataccaac caagaagatc ttttggtact 601 gtgggggatt caccatccta atgatgcggc agagcagaca aggctctatc 651 aaaacccaac cacctacatt tccgttggaa catcaacact gaaccagaga 701 ttggttccag aaatagctac tagacccaaa gtaaacgggc aaagtggaag 751 aatggagttc ttctggacaa ttttaaagcc gaatgatgcc atcaatttcg 801 agagtaatgg aaatttcatt gctccagaat atgcatacaa aattgtcaag 851 aaaggggact caacaattat gaaaagtgaa ttggaatatg gtaactgcaa = 901 caccaagtgt caaactccaa tgggggcaat aaactctagt atgccattcc 951 acaacataca ccccctcacc atcggggaat gccccaaata tgtgaaatca 1001 aacagattag tccttgcaac tggactcaga aatacccctc aacgagagac 1051 gcgaggacta tttggagcta tagcaggttt tatagaggga ggatggcagg = 1101 gaatggtaga tggttggtat gggtaccacc atagcaatga gcaggggagt 1151 ggatacgctg cagaccaaga atccacacaa aaggcaatag atggagtcac 1201 caataaggtc aactcgatca ttaacaaaat gaacactcag tttgaggccg 1251 ttggaaggga atttaataac ttggaaagga ggatagagaa tttaaacaag 1301 aaaatggaag acggattcct agatgtctgg acttacaatg ccgaacttct 1351 ggttctcatg gaaaatgaga gaactctaga ctttcatgac tcaaatgtca =
1401 agaaccttta cgacaaggtc cgactacagc ttagggataa tgcaaaggag 1451 ctgggtaatg gttgtttcga attctatcac aaatgtgata acgaatgtat =
= 1501 ggaaagtgta aaaaacggaa cgtatgacta cccgcagtat tcagaagaag 1551 caagactaaa cagagaggaa ataagtggag taaaattgga atcaatggga 1601 acttaccaaa tactgtcaat ttattcaaca gtggcgagtt ccctagcact = 74 _ 1651 ggcaatcatg gtagctggtc tatctttatg gatgtgctcc aatggatcgt = 1701 tacaatgcag aatttgcatt taaatttgtg agttcagatt gtagttaaaa 1751 acacccttgt ttctact Amino acid sequence of A/Hong Kong/491/97 H5 iSEQ I1) NO: 15) Entire molecule length: 564 aa 1 mekivlllat vs1vksdqic igyhannste qvdtimeknv tvthaqdile 51 rthngklcdl ngvkplilrd csvagwllgn pmcdefinvp ewsyivekas 101 pandlcypgn fndyeelkhl lsrinhfeki qiipksswsn hdassgvssa 151 cpylgrssff rnvvw1ikkn ssyptikrsy nntnqedllv lwgihhpnda 201 aeqtrlyqnp ttyisvgtst lnqr1vpeia trpkvngqsg rmeffwtilk 251 pndainfesn gnfiapeyay kivkkgdsti mkseleygnc ntkcqtpmga 301 inssmpfhni hpltigecpk yvksnrlvla tglrntpqre trglfgaiag 351 fieggwqgmv dgwygyhhsn eggsgyaadq estqkaidgv tnkvnsiink 401 mntqfeavgr efnnlerrie nlnkkmedgf ldvwtynael lvlmenertl 451 dfhdsnvknl ydkvrlqlrd nakelgngcf efyhkcdnec mesvkngtyd 501 ypqyseearl nreeisgvkl esmgtyqils iystvasbla laimvagls1 551 wmcsngslqc ridi Nucleotide Sequence of A/Hong Kong/486/97 Ni (SEQ ID NO: 6) Entire molecule length: 1401 nt 1 agcaaaagca ggagtttaaa atgaatccaa atcagaagat aataaccatt 51 ggatcaatct gcatggtagt tgggataatc agcttgatgt tacaaattgg 101 aaacacaata tcagtatggg tcagccacat aattaaaact tggcacccaa 151 accagcctga accatgcaac caaagcatca atttttacac tgagcaggct 201 gcagcttcag tgacattagc gggcaattcc tctctctgcc ctattagtgg 251 atgggctata tacagcaagg acaatagtat aagaattggt tccaaagggg 301 atgtgtttgt tataagagaa ccattcatct catgctccca tttggaatgc 351 agaacctttt tcttgaccca aggagcccta ttgaatgaca agcattctaa 401 tgggaccgtc aaagacagga gcccctatag aactttaatg agctgtcctg 451 ttggtgaggc cccttcccca tacaactcaa ggtttgagtc tgttgcttgg 501 tcagcaagtg cttgccatga tggcattagt tggctaacaa ttggaatttc 551 cggtccggat aatggggctg tggctgtgtt gaaatacaat ggcataataa 601 cagacaccat caagagttgg aggaacaaca cactgaggac gcaagagtct 651 gaatgtgcat gtgtgaatgg ttcttgtttt actgtaatga cagatggacc 701 gagtaatgaa caggcctcat acaagatttt caagatagaa aaggggaggg 751 tagtcaaatc agttgagttg aacgccccta attatcatta cgaggaatgc 801 tcctgttatc ctgatgctgg cgaaatcaca tgtgtgtgca gggataattg 851 gcatggctcg aaccgaccat gggtgtcttt caatcagaat ctggagtatc 901 aaataggata tatatgcagt ggggttttcg gagacagtcc acgccccaat 951 gatgggacag gcagttgtgg tccagtgtct cttaacggag cgtatggagt = 1001 aaaagggttt tcatttaaat acggcaatgg tgtttggatc gggagaacca 1051 aaagcactag ttccaggagc ggttttgaaa tgatttggga tccaaatggg 1101 tggaccgaaa cagacagtag cttctcgttg aagcaagaca tcatagcgat = 1151 aactgattgg tcaggataca gcgggagttt tattcaacat ccagaactga 1201 caggattaaa ttgcatgaga ccttgcttct gggttgaact aatcagaggg 1251 aggcccaaag agaaaacaat ctggactagt gggagcagta tatctttctg 1301 tggtgtaaat agtgacactg tgggttggtc ttggccagac ggtgctgagt WV Irk.
UOLI.M.3/1/1 / /Ga, 1351 tgccatacac cattgacaag tagtttgttc aaaaaactcc ttgtttctac 1401 t Amino acid sequence of A/Hong Kong/486/97 Ni (SEQ ID NO: 16) Entire molecule length: 450 aa 1 mnpngkiiti gsicmvvgii slmlqignti svwvshiikt whpnqpepcn 51 qsinfytecia aasvtlagns slcpisgwai yskdnsirig skgdvfvire 101 pfiscshlec rtffltqgal lndkhsngtv kdrspyrtlm scpvgeapsp 151 ynsrfesvaw sasachdgis wltigisgpd ngavavlkyn giitdtiksw = 201 rnntlrtqes ecacvngscf tvmtdgpsne qasykifkie kgrvvksvel 251 napnyhyeec scypdageit cvcrdnwhgs nrpwvsfnqn leyqigyics 301 gvfgdsprpn dgtgscgpvs lngaygvkgf sfkygngvwi grtkstssrs 351 gfemiwdpng wtetdssfs1 kgdiiaitdw sgysgsfiqh peltglncmr 401 pcfwvelirg rpkektiwts gssisfcgvn sdtvgwswpd gaelpytidk = 20 A/Hong Kong/491/97 (Ser211) (HA) + A/Hong Kong/486/97 (NA) Nucleotide Sequence of A/Hong Kong/491/97 (Ser211) H5 ISEQ 1D NO: 7) = Entire molecule length: 1767 nt 1 agcaaaagca ggggtataat ctgtcaaaat ggagaaaata gtgcttcttc 51 ttgcaacagt cagccttgtt aaaagtgacc agatttgcat tggttaccat 101 gcaaacaact cgacagagca agttgacaca ataatggaaa agaatgttac 151 tgttacacat gcccaagaca tactggaaag gacacacaac gggaagctct 201 gcgatctaaa tggagtgaag cctctgattt tgagggattg tagtgtagct 251 ggatggctcc tcggaaaccc tatgtgtgac gaattcatca atgtgccgga 301 atggtcttac atagtggaga aggccagtcc agccaatgac ctctgttatc 351 cagggaattt caacgactat gaagaactga aacacctatt gagcagaata 401 aaccattttg agaaaattca gataatcccc aaaagttctt ggtccaatca = 451 tgatgcctca tcaggggtga gctcagcatg tccatacctt gggaggtcct 501 cctttttcag aaatgtggta tggcttatca aaaagaacag tagctaccca 551 acaataaaga ggagctacaa taataccaac caagaagatc ttttggtact = 601 gtgggggatt caccatccta atgatgcggc agagcagaca aggctctatc 651 aaaacccaac cacctacatt tccgttggaa catcaacact gaaccagaga 701 ttggtttcag aaatagctac tagacccaaa gtaaacgggc aaagtggaag 751 aatggagttc ttctggacaa ttttaaagcc gaatgatgcc atcaatttcg 801 agagtaatgg aaatttcatt gctccagaat atgcatacaa aattgtcaag 851 aaaggggact caacaattat gaaaagtgaa ttggaatatg gtaactgcaa 901 caccaagtgt caaactccaa tgggggcaat aaactctagt atgccattcc 951 acaacataca ccccctcacc atcggggaat gccccaaata tgtgaaatca 1001 aacagattag tccttgcaac tggactcaga aatacccctc aacgagagac 1051 gcgaggacta tttggagcta tagcaggttt tatagaggga ggatggcagg = 1101 gaatggtaga tggttggtat gggtaccacc atagcaatga gcaggggagt 1151 ggatacgctg cagaccaaga atccacacaa aaggcaatag atggagtcac 1201 caataaggtc aactcgatca ttaacaaaat gaacactcag tttgaggccg 1251 ttggaaggga atttaataac ttggaaagga ggatagagaa tttaaacaag 1301 aaaatggaag acggattcct agatgtctgg acttacaatg ccgaacttct %JUL,' 114-1351 ggttctcatg gaaaatgaga gaactctaga ctttcatgac tcaaatgtca = 1401 agaaccttta cgacaaggtc cgactacagc ttagggataa tgcaaaggag 1451 ctgggtaatg gttgtttcga attctatcac aaatgtgata acgaatgtat 1501 ggaaagtgta aaaaacggaa cgtatgacta cccgcagtat tcagaagaag 1551 caagactaaa cagagaggaa ataagtggag taaaattgga atcaatggga 1601 acttaccaaa tactgtcaat ttattcaaca gtggcgagtt ccctagcact 1651 ggcaatcatg gtagctggtc tatctttatg gatgtgctcc aatggatcgt 1701 tacaatgcag aatttgcatt taaatttgtg agttcagatt gtagttaaaa 1751 acacccttgt ttctact Amino acid sequence of A/Hong Kong/491/97 (Ser211) H5 iSEQ ID NO: 17) Entire molecule length: 564 aa 1 mekivlllat vslvksdqic igyhannste qvdtimeknv tvthaqdile 51 rthngklcdl ngvkplilrd csvagwllgn pmcdefinvp ewsyivekas 101 pandlcypgn fndyeelkhl lsrinhfeki qiipksswsn hdassgvssa 151 cpylgrssff rnvvwlikkn ssyptikrsy nntnqedllv lwgihhpnda 201 aeqtrlyqnp ttyisvgtst lnqrlvseia trpkvngqsg rmeffwtilk 251 pndainfesn gnfiapeyay kivkkgdsti mkseleygnc ntkcqtpmga 301 inssmpfhni hpltigecpk yvksnrlvla tglrntpqre trg1fgaiag 351 fieggwqgmv.dgwygyhhsn eggsgyaadq estqkaidgv tnkvnsiink 401 mntqfeavgr efnnlerrie nlnkkmedgf ldvwtynael lvlmenertl 451 dfhdsnvknl ydkvrlqlrd nakelgngcf efyhkcdnec mesvkngtyd 501 ypqyseearl nreeisgvkl esmgtyqils iystvassla laimvagls1 551 wmcsngslqc ridi Nucleotide Sequence of A/Hong Kong/486/97 Ni LSEQ ID NO: 8) Entire molecule length: 1401 nt 1 agcaaaagca ggagtttaaa atgaatccaa atcagaagat aataaccatt 51 ggatcaatct gcatggtagt tgggataatc agcttgatgt tacaaattgg 101 aaacacaata tcagtatggg tcagccacat aattaaaact tggcacccaa 151 accagcctga accatgcaac caaagcatca atttttacac tgagcaggct 201 gcagcttcag tgacattagc gggcaattcc tctctctgcc ctattagtgg 251 atgggctata tacagcaagg acaatagtat aagaattggt tccaaagggg 301 atgtgtttgt tataagagaa ccattcatct catgctccca tttggaatgc 351 agaacctttt tcttgaccca aggagcccta ttgaatgaca agcattctaa 401 tgggaccgtc aaagacagga gcccctatag aactttaatg agctgtcctg 451 ttggtgaggc cccttcccca tacaactcaa ggtttgagtc tgttgcttgg 501 tcagcaagtg cttgccatga tggcattagt tggctaacaa ttggaatttc 551 cggtccggat aatggggctg tggctgtgtt gaaatacaat ggcataataa 601 cagacaccat caagagttgg aggaacaaca cactgaggac gcaagagtct 651 gaatgtgcat gtgtgaatgg ttcttgtttt actgtaatga cagatggacc 701 gagtaatgaa caggcctcat acaagatttt caagatagaa aaggggaggg 751 tagtcaaatc agttgagttg aacgccccta attatcatta cgaggaatgc 801 tcctgttatc ctgatgctgg cgaaatcaca tgtgtgtgca gggataattg = 851 gcatggctcg aaccgaccat gggtgtcttt caatcagaat ctggagtatc 901 aaataggata tatatgcagt ggggttttcg gagacagtcc acgccccaat 951 gatgggacag gcagttgtgg tccagtgtct cttaacggag cgtatggagt 1001 aaaagggttt tcatttaaat acggcaatgg tgtttggatc gggagaacca VW LW:), itoLz+zs CA 02822895 2013-06-07 rk...1/uShoUNu1//ZY
1051 aaagcactag ttccaggagc ggttttgaaa tgatttggga tccaaatggg 1101 tggaccgaaa cagacagtag cttctcgttg aagcaagaca tcatagcgat 1151 aactgattgg tcaggataca gcgggagttt tattcaacat ccagaactga 1201 caggattaaa ttgcatgaga ccttgcttct gggttgaact aatcagaggg 1251 aggcccaaag agaaaacaat ctggactagt gggagcagta tatctttctg 1301 tggtgtaaat agtgacactg tgggttggtc ttggccagac ggtgctgagt 1351 tgccatacac cattgacaag tagtttgttc aaaaaactcc ttgtttctac 1401 t Amino acid sequence of A/Hong Kong/486/97 Ni ISEQ-ID NO: 18) Entire molecule length: 450 aa =
=
1 mnpnqkiiti gsicmvvgii slmlqignti svwvshiikt whpnqpepcn 51 gsinfytega aasvtlagns sicpisgwai yskdnsirig skgdvfvire 101 pfiscshlec rtffltqgal lndkhsngtv kdrspyrtlm scpvgeapsp 151 ynsrfesvaw sasachdgis wltigisgpd ngavavlkyn giitdtiksw 201 rnntlrtges ecacvngscf tvmtdgpsne qasykifkie kgrvvksvel 251 napnyhyeec scypdageit cvcrdnwhgs nrpwvsfnqn leyqigyics 301 gvfgdsprpn dgtgscgpvs ingaygvkgf sfkygngvwi grtkstssrs 351 gfemiwdpng wtetdssfsl kgdiiaitdw sgysgsfigh peltglncmr 401 pcfwvelirg rpkektiwts gssisfcgvn sdtvgwswpd gaelpytidk ea A./ck/Hong Kong/G9/97 Nucleotide sequence of ca A/c1c/Hong Kong/G9/97 (SEQ ID NO: 9) Entire molecule length: 1690 bp 1 ttaaccactc aagatggaag caataccact aataactata ctactagtag = 51 taacagcaag caatgcagac aaaatctgca tcggctacca atcaacaaac 101 tccacagaaa ccgtagacac gctaacagaa aacaatgttc ctgtgacaca 151 tgccaaagaa ttgctccaca cagagcacaa tgggatgctg tgtgcaacaa 201 atctgggacg tcctcttatt ctagacactt gcaccattga aggactgatc 251 tatggcaacc cttcttgtga tctactgttg ggaggaagag aatggtccta 301 catcgtcgaa agaccatcgg ctgttaatgg aatgtgttac cccgggaatg = 35 351 tagaaaacct agaggaacta aggtcatttt ttagttctgc tagttcctac 401 caaagaatcc agatctttcc agacacaatc tggaatgtgt cttacagtgg 451 aacaagcaaa gcatgttcag attcattcta caggagcatg agatggttga 501 ctcaaaagaa caacgcttac cctattcaag acgcccaata cacaaataat 551 agaggaaaga gcattctttt catgtggggc ataaatcacc cacctaccga 601 tactgcacag acaaatctgt acacaaggac tgacacaaca acaagtgtgg 651 caacagaaga tataaatagg accttcaaac cagtgatagg gccaaggccc 701 cttgtcaatg gtctgcaggg aagaattgat tattattggt cggtattgaa 751 accaggtcag acattgcgag taagatccaa tgggaatcta atcgctccat 801 ggtatgggca cattctttca ggagagagcc acggaagaat cctgaagact = 45 851 gatttaaaca gtggtagctg tgtagtgcaa tgtcaaacag aaagaggtgg 901 cttaaatact actttgccat tccacaatgt cagtaaatat gcatttggaa 951 actgcccaaa atatgttgga gtaaagagtc tcaaactggc agttggtctg 1001 aggaatgtgc ctgctagatc aagtagagga ctatttgggg ccatagctgg = 1051 attcatagag ggaggttggt cagggctggt cgctggttgg tatgggttcc 1101 agcattcaaa tgatcaaggg gttggtatag ctgcagatag agactcaact 1151 caaagggcaa ttgacaaaat aacgtccaaa gtgaataata tagtcgataa 1201 aatgaacaag caatatgaaa ttattgatca tgaattcagc gaggttgaaa 1251 atagactcaa tatgatcaat aataagattg atgaccagat acaagacata 1301 tgggcatata acgctgaatt gctagtgctg cttgaaaacc agaaaacact 1351 cgatgagcat gatgcgaatg taaacaatct atataacaaa gtgaagaggg 1401 cactgggttc caatgcaatg gaagatggga aaggatgttt cgagctatac 1451 cataaatgtg atgatcagtg catggagaca attcggaacg ggacctataa 1501 caggaggaag tataaagagg aatcaagact agaaagacag aaaatagaag 1551 gggtcaagct ggaatctgaa ggaacttaca aaatcctcac catttattcg 1601 actgtcgcct catctcttgt gattgcaatg gggtttgctg ccttcttgtt 1651 ctgggccatg tccaatggat cttgcagatg caacatttga Amino acid sequence of ca A/c1c/Hong KonWG9/97 H9 (SEQ ID NO: 19) Entire molecule length: 558 aa 1 meaiplitil lvvtasnadk icigyqstns tetvdtlten nvpvthakel 51 lhtehngmlc atnigrplil dtctiegliy gnpscdllig grewsyiver 101 psavngmcyp gnvenleelr sffssassyq riclifpdtiw nvsysgtska 151 csdsfyrsmr wltqknnayp igdaqytnnr gksilfmwgi nhpptdtaqt 201 nlytrtdttt svatedinrt fkpvigprpl vnglqgridy ywsvlkpgqt 251 lrvrsngnli apwyghilsg eshgrilktd lnsgscvvqc qtergglntt 301 lpfhnvskya fgncpkyvgv kslklavglr nvparssrgl fgaiagfieg 351 gwsglvagwy gfqhsndqgv giaadrdstq raidkitskv nnivdkulnkg 401 yeiidhefse venrinminn kiddgiqdiw aynaellvll enqktldehd 451 anvnnlynkv kralgsname dgkgcfelyh kcddqcmeti rngtynrrky 501 keesrlerqk iegvkleseg tykiltiyst vasslviamg faaflfwams 551 ngscrcni Nucleotide sequence of ca A/etc/Hong Kong/G9/97 N2 (SEQ 1D NO: 10) Entire molecule length: 1428 bp 1 aaatgaatcc aaatcagaag ataatagcaa ttggctctgt ttctctaact 51 attgcgacaa tatgcctcct catgcagatt gctatcttag caacgactat 101 gacactacat ttcaagcaga atgaatgcat caactcctcg aataatcaag 151 tagtgccatg tgaaccaatc ataatagaaa ggaacataac agagatagtg 201 catttgaata gtactacctt agagaaggaa atttgtccta aagtagcaga 251 ctacaggaat tggtcaaaac cacaatgtca aatcacaggg ttcgctcctt 301 tctccaagga caattcaatt aggctctccg caggtggaga tatttgggtg 351 acaagagaac cttatgtatc gtgcggtctt ggtaaatgtt atcaatttgc 401 acttgggcag ggaaccactt tggagaacaa acactcaaac ggcacagcac 451 atgatagaac tcctcataga acccttttaa tgaatgagtt gggtgttccg 501 tttcatttgg caaccaaaca agtgtgcata gcatggtcca gctcaagctg 551 ccatgatggg aaagcatggt tacatgtttg tgtcactggg gatgatagaa = 601 atgcaacggc tagcatcatt tatgatggga tacttgttga cagtattggt 651 tcatggtcta aaaacatcct cagaactcag gagtcagaat gcgtttgcat 701 caatggaacc tgtgcagtag taatgactga tggaagtgca tcaggaaggg 751 ctgacactag aatactattt attagagagg ggaaaattgc acacattagc 801 ccattgtcag gaagtgctca gcatgtggag gaatgctcct gttacccccg 851 atatccagaa gttagatgtg tttgcagaga caattggaag ggatccaata 901 ggcccgttct atatataaat atggcaaatt atagtattga ttccagttat CA 02822895 2013-06-07 rµ.., u3huv...liu ILY
951 gtgtgctcag gacttgttgg cgacacacca agaaatgatg ataggtctag 1001 cagcagcaac tgcagagatc ctaataacga gagaggggcc ccaggagtaa 1051 aagggtgggc ctttgacaat ggaaatgaca tttggatggg aagaacaatc 1101 aaaaaggatt cgcgctcagg ttatgagact ttcagggtca ttggtggttg 1151 gaccactgct aattccaagt cacagataaa tagacaagtc atagttgaca 1201 gtgataactc gtctgggtat tctggtatct tctctgttga aggcaaaagc 1251 tgcatcaaca ggtgttttta cgtggagttg ataagaggaa gaccaaagga 1301 gactagggtg tggtggactt caaatagcat cattgtattt tgtggaactt = 1351 caggtaccta tggaacaggc tcatggcctg atggggcgaa tatcaatttc = 10 1401 atgcctatat aagctttcgc aattttag Amino acid sequence of ca A/ck/Hong Kong/G9/97 N2 (SEQ ID NO: 20) = Entire molecule length: 469 aa 1 mnpnqkiiai gsvsltiati cllmqiaila ttmtlhfkqn ecinssnnqv 51 vpcepiiier niteivhlns ttlekeicpk vadyrnwskp qcqitgfapf 101 skdnsirlsa ggdiwvtrep yvscglgkcy qfalgqgttl enkhsngtah 151 drtphrtllm nelgvpfhla tkqvciawss sschdgkawl hvcvtgddrn 201 atasiiydgi lvdsigswsk nilrtqesec vcingtcavv mtdgsasgra 251 dtrilfireg kiahisplsg saqhveecsc yprypevrcv crdnwkgsnr 301 pvlyinmany sidssyvcsg lvgdtprndd rssssncrdp nnergapgvk 351 gwafdngndi wmgrtikkds rsgyetfrvi ggwttansks qinrqvivds 401 dnssgysgif svegkscinr cfyvelirgr pketrvwwts nsiivfcgts 451 gtygtgswpd ganinfmpi *

SUMMARY OF SEQ ID NO DESIGNATIONS
SEQ ID NO HA or NA Amino Acid or STRAIN NAME Nucleotide SEQ ID NO: 1 HA (H5) ca ANietnam/1203/04 Nucleotide SEQ ID NO: 2 NA (Ni) ca ANi etnam/1203/04 Nucleotide SEQ ID NO: 3 -HA (H5) ca AJHong Nucleotide Kong/213/03 SEQ ID NO: 4 _NA (Ni) ca A/Hong Nucleotide Kong/213/03 SEQ ID NO: 5 HA (H5) ca A/Hong Nucleotide - - Kong/491/97 SEQ ID NO: 6 NA (Ni) ca AJHong Nucleotide Kong/486/97 SEQ ID NO: 7 HA (H5) ca A/Hong Nucleotide Kong/491/97 (Ser211) SEQ ID NO: 8 NA (Ni) ca A/Hong Nucleotide Kong/486/97 SEQ ID NO: 9 HA (H9) ca Aick/Hong Nucleotide Kong/G9/97 SEQ ID NO: 10 NA (N2) ca A/ck/Hong Nucleotide Kong/G9/97 SEQ ID NO: 11 HA (H5) ca A/Vietnam/1203/04 Amino Acid SEQ ID NO: 12 NA (N1) ca A/Vietnam/1203/04 Amino Acid SEQ ID NO: 13 HA (H5) ca A/Hong Amino Acid Kong/213/03 SEQ ID NO: 14 NA (Ni) ca A/Hong Amino Acid Kong/213/03 SEQ ID NO: 15 HA (H5) ca A/Hong Amino Acid Kong/491/97 SEQ ID NO: 16 NA (Ni) ca A/Hong Amino Acid Kong/486/97 SEQ ID NO: 17 - HA (H5) ca A/Hong Amino Acid Kong/491/97 (Ser211) SEQ ID NO: 18 NA (N1) ca A/Hong Amino Acid Kong/486/97 SEQ ID NO: 19 HA (H9) ca A/ck/Hong Amino Acid Kong/G9/97 SEQ ID NO: 20 - NA (N2) ca A/c1dHong Amino Acid Kong/G9/97 SEQUENCE LISTING
<110> MedImmune Vaccines, Inc et al.
<120> INFLUENZA HEMAGGLUTININ AND NEURAMINIDASE VARIANTS
=
<130> 54286-4D1 <150> 60/574,553 <151> 2004-05-25 <150> 60/657,554 <151> 2005-02-28 <160> 20 <170> PatentIn version 3.3 <210> 1 <211> 1767 <212> DNA
<213> Influenza virus <400> 1 agcaaaagca ggggttcaat ctgtcaaaat ggagaaaata gtgcttcttt ttgcaatagt 60 cagtcttgtt aaaagtgatc agatttgcat tggttaccat gcaaacaact cgacagagca 120 ggttgacaca ataatggaaa agaacgttac tgttacacat gcccaagaca tactggaaaa 180 gaaacacaac gggaagctct gcgatctaga tggagtgaag cctctaattt tgagagattg 240 tagcgtagct ggatggctcc tcggaaaccc aatgtgtgac gaattcatca atgtgccgga 300 atggtcttac atagtggaga aggccaatcc agtcaatgac ctctgttacc caggggattt 360 caatgactat gaagaattga aacacctatt gagcagaata aaccattttg agaaaattca 420 gatcatcccc aaaagttctt ggtccagtca tgaagcctca ttaggggtga gctcagcatg 480 tccataccag ggaaagtcct cctttttcag aaatgtggta tggcttatca aaaagaacag 540 tacataccca acaataaaga ggagctacaa taataccaac caagaagatc ttttggtact 600 gtgggggatt caccatccta atgatgcggc agagcagaca aagctctatc aaaacccaac 660 cacctatatt tccgttggga catcaacact aaaccagaga ttggtaccaa gaatagctac 720 tagatccaaa gtaaacgggc aaagtggaag gatggagttc ttctggacaa ttttaaagcc 780 gaatgatgca atcaacttcg agagtaatgg aaatttcatt gctccagaat atgcatacaa 840 aattgtcaag aaaggggact caacaattat gaaaagtgaa ttggaatatg gtaactgcaa 900 caccaagtgt caaactccaa tgggggcgat aaactctagc atgccattcc acaatataca 960 ccctctcacc attggggaat gccccaaata tgtgaaatca aacagattag tccttgcgac 1020 tgggctcaga aatagccctc aaagagagac tcgaggatta tttggagcta tagcaggttt 1080 tatagaggga ggatggcagg gaatggtaga tggttggtat gggtaccacc atagcaatga 1140 gcaggggagt gggtacgctg cagacaaaga atccactcaa aaggcaatag atggagtcac 1200 caataaggtc aactcgatca ttgacaaaat gaacactcag tttgaggccg ttggaaggga 1260 atttaacaac ttagaaagga gaatagagaa tttaaacaag aagatggaag acgggttcct 1320 agatgtctgg acttataatg ctgaacttct ggttctcatg gaaaatgaga gaactctaga 1380 ctttcatgac tcaaatgtca agaaccttta cgacaaggtc cgactacagc ttagggataa 1440 tgcaaaggag ctgggtaacg gttgtttcga gttctatcat aaatgtgata atgaatgtat 1500 ggaaagtgta agaaatggaa cgtatgacta cccgcagtat tcagaagaag cgagactaaa 1560 aagagaggaa ataagtggag taaaattgga atcaatagga atttaccaaa tactgtcaat 1620 ttattctaca gtggcgagtt ccctagcact ggcaatcatg gtagctggtc tatccttatg 1680 gatgtgctcc aatgggtcgt tacaatgcag aatttgcatt taaatttgtg agttcagatt 1740 gtagttaaaa acacccttgt ttctact 1767 <210> 2 <211> 1398 <212> DNA
<213> Influenza virus <400> 2 agcaaaagca ggagttcaaa atgaatccaa atcagaagat aataaccatc gggtcaatct 60 gtatggtaac tggaatagtt agcttaatgt tacaaattgg gaacatgatc tcaatatggg 120 tcagtcattc aattcacaca gggaatcaac accaatctga accaatcagc aatactaatt 180 ttcttactga gaaagctgtg gcttcagtaa aattagcggg caattcatct ctttgcccca 240 ttaacggatg ggctgtatac agtaaggaca acagtataag gatcggttcc aagggggatg 300 tgtttgttat aagagagccg ttcatctcat gctcccactt ggaatgcaga actttctttt 360 tgactcaggg agccttgctg aatgacaagc actccaatgg gactgtcaaa gacagaagcc 420 ctcacagaac attaatgagt tgtcctgtgg gtgaggctcc ctccccatat aactcaaggt 480 ttgagtctgt tgcttggtca gcaagtgctt gccatgatgg caccagttgg ttgacgattg 540 gaatttctgg cccagacaat ggggctgtgg ctgtattgaa atacaatggc ataataacag 600 acactatcaa gagttggagg aacaacatac tgagaactca agagtctgaa tgtgcatgtg 660 taaatggctc ttgctttact gtaatgactg acggaccaag taatggtcag gcatcacata 720 agatcttcaa aatggaaaaa gggaaagtgg ttaaatcagt cgaattggat gctcctaatt 780 atcactatga ggaatgctcc tgttatccta atgccggaga aatcacatgt gtgtgcaggg 840 ataattggca tggctcaaat cggccatggg tatctttcaa tcaaaatttg gagtatcaaa 900 taggatatat atgcagtgga gttttcggag acaatccacg ccccaatgat ggaacaggta 960 gttgtggtcc ggtgtcctct aacggggcat atggggtaaa agggttttca tttaaatacg 1020 gcaatggtgt ctggatcggg agaaccaaaa gcactaattc caggagcggc tttgaaatga 1080 tttgggatcc aaatgggtgg actgaaacgg acagtagctt ttcagtgaaa caagatatcg 1140 tagcaataac tgattggtca ggatatagcg ggagttttgt ccagcatcca gaactgacag 1200 gactagattg cataagacct tgtttctggg ttgagttgat cagagggcgg cccaaagaga 1260 gcacaatttg gactagtggg agcagcatat ctttttgtgg tgtaaatagt gacactgtgg 1320 gttggtcttg gccagacggt gctgagttgc cattcaccat tgacaagtag tttgttcaaa 1380 aaactccttg tttctact 1398 <210> 3 <211> 1767 <212> DNA
<213> Influenza virus <400> 3 agcaaaagca ggggttcaat ctgtcaaaat ggagaaaata gtgcttcttt ttgcaatagt 60 cagtcttgtt aaaagtgatc agatttgcat tggttaccat gcaaacaact cgacagagca 120 ggttgacaca ataatggaaa agaacgttac tgttacacat gcccaagaca tactggaaaa 180 gacacacaac gggaagctct gcgatctaga tggagtgaag cctctaattt tgagagattg 240 tagtgtagct ggatggctcc tcggaaaccc aatgtgtgac gaattcatca atgtgccgga 300 atggtcttac atagtggaga aggccaatcc agccaatgac ctctgttacc caggggattt 360 caacgactat gaagaattga aacacctatt gagcagaata aaccattttg agaaaattca 420 gatcatcccc aaaaattctt ggtccagtca tgaagcctca ttaggggtga gctcagcatg 480 tccataccaa qgaaagtcct cctttttcag gaatgtggta tggcttatca aaaagaacaa 540 tgcataccca acaataaaga ggagctacaa taataccaac caagaagatc ttttggtatt 600 gtgggggatt caccatccta atgatgcggc agagcagact aggctctatc aaaacccaac 660 cacctacatt tccgttggga catcaacact aaaccagaga ttggtaccaa aaatagctac 720 tagatccaaa gtaaacgggc aaaatggaag gatggagttc ttctggacaa ttttaaaacc 780 gaatgatgca atcaacttcg agagcaatgg aaatttcatt gctccagaat atgcatacaa 840 aattgtcaag aaaggggact cagcaattat gaaaagtgaa ttggaatatg gtaactgcaa 900 caccaagtgt caaactccaa tgggggcgat aaactctagt atgccattcc acaatataca 960 ccctctcacc atcggggaat gccccaaata tgtgaaatca aacagattag tccttgcgac 1020 tgggctcaga aatagccctc aaagagagac tcgaggatta tttggagcta tagcaggttt 1080 tatagaggga ggatggcagg-gaatggtaga tggttggtat gggtaccacc atagcaatga 1140 gcaggggagt gggtacgctg cagacaaaga atccactcaa aaggcaatag atggagtcac 1200 caataaggtc aactcgatca ttgacaaaat gaacactcag tttgaggccg ttggaaggga 1260 atttaataac ttagaaagga gaatagagaa tttaaacaag aagatggaag acggattcct 1320 agatgtctgg acttataatg ctgaacttct ggttctcatg gaaaatgaga gaactctaga 1380 ctttcatgac tcaaatgtca agaaccttta cgacaaggtc cgactacagc ttagggataa 1440 tgcaaaggag ctgggtaacg gttgtttcga gttctatcac aaatgtgata atgaatgtat 1500 ggaaagtgta agaaacggaa cgtatgacta cccgcagtat tcagaagaag caagactaaa 1560 aagagaggaa ataagtggag taaaattgga gtcaatagga acttaccaaa tactgtcaat 1620 ttattctaca gtggcgagtt ccctagcact ggcaatcatg gtagctggtc tatctttatg 1680 gatgtgctcc aatgggtcgt tacaatgcag aatttgcatt taaatttgtg agttcagatt 1740 gtagttaaaa acacccttgt ttctact 1767 <210> 4 <211> 1458 <212> DNA
<213> Influenza virus <400> 4 agcaaaagca ggagttcaaa atgaatccaa atcagaagat aacaaccatt ggatcaatct 60 gtatggtaat tggaatagtt agcttgatgt tacaaattgg gaacataatc tcaatatggg 120 ttagtcattc aattcaaaca gggaatcaac accaggctga accatgcaat caaagcatta 180 ttacttatga aaacaacacc tgggtaaacc agacatatgt caacatcagc aataccaatt 240 ttcttactga gaaagctgtg gcttcagtaa cattagcggg caattcatct ctttgcccca 300 ttagtggatg ggctgtatac agtaaggaca acggtataag aatcggttcc aagggggatg 360 tgtttgttat aagagagccg ttcatctcat gctcccactt ggaatgcaga actttctttt 420 tgactcaggg agccttgctg aatgacaagc attctaatgg gaccgtcaaa gacagaagcc 480 ctcacagaac attaatgagt tgtcccgtgg gtgaggctcc ttccccatac aactcgaggt 540 ttgagtctgt tgcttggtcg gcaagtgctt gtcatgatgg cactagttgg ttgacaattg 600 gaatttctgg cccagacaat ggggctgtgg ctgtattgaa atacaatggc ataataacag 660 acactatcaa gagttggagg aacaacataa tgagaactca agagtctgaa tgtgcatgtg 720 taaatggctc ttgctttact gttatgactg atggaccaag taatgggcag gcttcataca 780 aaatcttcag aatagaaaaa gggaaagtag ttaaatcagc cgaattaaat gcccctaatt 840 atcactatga ggagtgctcc tgttatcctg atgctggaga aatcacatgt gtgtgcaggg 900 ataactggca tggctcaaat cggccatggg tatctttcaa tcaaaatttg gagtatcgaa 960 taggatatat atgcagtgga gttttcggag acaatccacg ccccaatgat gggacaggca 1020 gttgtggtcc ggtgtcccct aaaggggcat atggaataaa agggttctca tttaaatacg 1080 gcaatggtgt ttggatcggg agaaccaaaa gcactaattc caggagcggc tttgaaatga 1140 tttgggatcc aaatggatgg actggtacgg acagtaattt ttcagtaaag caagatattg 1200 tagctataac cgattggtca ggatatagcg ggagttttgt ccagcatcca gaactgacag 1260 gattagattg cataagacct tgtttctggg ttgagctaat cagagggcgg cccaaagaga 1320 gcacaatttg gactagtggg aggagcatat ccttttgtgg tgtaaatagt gacactgtgg 1380 gttggtcttg gccagacggt gctgagttgc cattcaccat tgacaagtag tttgttcaaa 1440 aaactccttg tttctact 1458 <210> 5 <211> 1767 <212> DNA
<213> Influenza virus <400> 5 agcaaaagca gqggtataat ctgtcaaaat ggagaaaata gtgcttcttc ttgcaacagt 60 cagccttgtt aaaagtgacc agatttgcat tggttaccat gcaaacaact cgacagagca 120 agttgacaca ataatggaaa agaatgttac tgttacacat gcccaagaca tactggaaag 180 gacacacaac gggaagctct gcgatctaaa tggagtgaag cctctgattt tgagggattg 240 tagtgtagct ggatggctcc tcggaaaccc tatgtgtgac gaattcatca atgtgccgga 300 atggtcttac atagtggaga aggccagtcc agccaatgac ctctgttatc cagggaattt 360 caacgactat gaagaactga aacacctatt gagcagaata aaccattttg agaaaattca 420 gataatcccc aaaagttctt ggtccaatca tgatgcctca tcaggggtga gctcagcatg 480 tccatacctt,gggaggtcct cctttttcag aaatgtggta tggcttatca aaaagaacag 540 tagctaccca acaataaaga ggagctacaa taataccaac caagaagatc ttttggtact 600 gtgggggatt caccatccta atgatgcggc agagcagaca aggctctatc aaaacccaac 660 cacctacatt tccgttggaa catcaacact gaaccagaga ttggttccag aaatagctac 720 tagacccaaa gtaaacgggc aaagtggaag aatggagttc ttctggacaa ttttaaagcc 780 gaatgatgcc atcaatttcg agagtaatgg aaatttcatt gctccagaat atgcatacaa 840 aattgtcaag aaaggggact caacaattat gaaaagtgaa ttggaatatg gtaactgcaa 900 caccaagtgt caaactccaa tgggggcaat aaactctagt atgccattcc acaacataca 960 ccccctcacc atcggggaat gccccaaata tgtgaaatca aacagattag tccttgcaac 1020 tggactcaga aatacccctc aacgagagac gcgaggacta tttggagcta tagcaggttt 1080 tatagaggga ggatggcagg gaatggtaga tggttggtat gggtaccacc atagcaatga 1140 gcaggggagt ggatacgctg cagaccaaga atccacacaa aaggcaatag atggagtcac 1200 caataaggtc aactcgatca ttaacaaaat gaacactcag tttgaggccg ttggaaggga 1260 atttaataac ttggaaagga ggatagagaa tttaaacaag aaaatggaag acggattcct 1320 agatgtctgg acttacaatg ccgaacttct ggttctcatg gaaaatgaga gaactctaga 1380 ctttcatgac tcaaatgtca agaaccttta cgacaaggtc cgactacagc ttagggataa 1440 tgcaaaggag ctgggtaatg gttgtttcga attctatcac aaatgtgata acgaatgtat 1500 ggaaagtgta aaaaacggaa cgtatgacta cccgcagtat tcagaagaag caagactaaa 1560 cagagaggaa ataagtggag taaaattgga atcaatggga acttaccaaa tactgtcaat 1620 ttattcaaca gtggcgagtt ccctagcact ggcaatcatg gtagctggtc tatctttatg 1680 gatgtgctcc aatggatcgt tacaatgcag aatttgcatt taaatttgtg agttcagatt 1740 gtagttaaaa acacccttgt ttctact 1767 <210> 6 <211> 1401 <212> DNA
<213> Influenza virus <400> 6 agcaaaagca ggagtttaaa atgaatccaa atcagaagat aataaccatt ggatcaatct 60 gcatggtagt tgggataatc agcttgatgt tacaaattgg aaacacaata tcagtatggg 120 tcagccacat aattaaaact tggcacccaa accagectga accatgcaac caaagcatca 180 atttttacac tgagcaggct gcagcttcag tgacattagc gggcaattcc tctctctgcc 240 ctattagtgg atgggctata tacagcaagg acaatagtat aagaattggt tccaaagggg 300 atgtgtttgt tataagagaa ccattcatct catgctccca tttggaatgc agaacctttt 360 tcttgaccca aggagcccta ttgaatgaca agcattctaa tgggaccgtc aaagacagga 420 gcccctatag aactttaatg agctgtcctg ttggtgaggc cccttcccca tacaactcaa 480 ggtttgagtc tgttgcttgg tcagcaagtg cttgccatga tggcattagt tggctaacaa 540 ttggaatttc cggtccggat aatggggctg tggctgtgtt gaaatacaat ggcataataa 600 cagacaccat caagagttgg aggaacaaca cactgaggac gcaagagtct gaatgtgcat 660 gtgtgaatgg ttcttgtttt actgtaatga cagatggacc gagtaatgaa caggcctcat 720 acaagatttt caagatagaa aaggggaggg tagtcaaatc agttgagttg aacgccccta 780 attatcatta cgaggaatgc tcctgttatc ctgatgctgg cgaaatcaca tgtgtgtgca 840 gggataattg gcatggctcg aaccgaccat gggtgtcttt caatcagaat ctggagtatc 900 aaataggata tatatgcagt ggggttttcg gagacagtcc acgccccaat gatgggacag 960 gcagttgtgg tccagtgtct cttaacggag cgtatggagt aaaagggttt tcatttaaat 1020 acggcaatgg tgtttggatc gggagaacca aaagcactag ttccaggagc ggttttgaaa 1080 tgatttggga tccaaatggg tggaccgaaa cagacagtag cttctcgttg aagcaagaca 1140 tcatagcgat aactgattgg tcaggataca gcgggagttt tattcaacat ccagaactga 1200 caggattaaa ttgcatgaga ccttgcttct gggttgaact aatcagaggg aggcccaaag 1260 agaaaacaat ctggactagt gggagcagta tatctttctg tggtgtaaat agtgacactg 1320 tgggttggtc ttggccagac ggtgctgagt tgccatacac cattgacaag tagtttgttc 1380 aaaaaactcc ttgtttctac t 1401 <210> 7 <211> 1767 <212> DNA
<213> Influenza virus <400> 7 agcaaaagca ggggtataat ctgtcaaaat ggagaaaata gtgcttcttc ttgcaacagt 60 cagccttgtt aaaagtgacc agatttgcat tggttaccat gcaaacaact cgacagagca 120 agttgacaca ataatggaaa agaatgttac tgttacacat gcccaagaca tactggaaag 180 gacacacaac gggaagctct gcgatctaaa tggagtgaag cctctgattt tgagggattg 240 tagtgtagct ggatggctcc tcggaaaccc tatgtgtgac gaattcatca atgtgccgga 300 atggtcttac atagtggaga aggccagtcc agccaatgac ctctgttatc cagggaattt 360 caacgactat gaagaactga aacacctatt gagcagaata aaccattttg agaaaattca 420 gataatcccc aaaagttctt ggtccaatca tgatgcctca tcaggggtga gctcagcatg 480 tccatacctt gggaggtcct cctttttcag aaatgtggta tggcttatca aaaagaacag 540 tagctaccca acaataaaga ggagctacaa taataccaac caagaagatc ttttggtact 600 gtgggggatt caccatccta atgatgcggc agagcagaca aggctctatc aaaacccaad 660 cacctacatt tccgttggaa catcaacact gaaccagaga ttggtttcag aaatagctac 720 tagacccaaa gtaaacgggc aaagtggaag aatggagttc ttctggacaa ttttaaagcc 780 gaatgatgcc atcaatttcg agagtaatgg aaatttcatt gctccagaat atgcatacaa 840 aattgtcaag aaaggggact caacaattat gaaaagtgaa ttggaatatg gtaactgcaa 900 caccaagtgt caaactccaa tgggggcaat aaactctagt atgccattcc acaacataca 960 ccccctcacc atcggggaat gccccaaata tgtgaaatca aacagattag tccttgcaac 1020 tggactcaga aatacccctc aacgagagac gcgaggacta tttggagcta tagcaggttt 1080 tatagaggga ggatggcagg gaatggtaga tggttggtat gggtaccacc atagcaatga 1140 gcaggggagt ggatacgctg cagaccaaga atccacacaa aaggcaatag atggagtcac 1200 caataaggtc aactcgatca ttaacaaaat gaacactcag tttgaggccg ttggaaggga 1260 atttaataac ttggaaagga ggatagagaa tttaaacaag aaaatggaag acggattcct 1320 agatgtctgg acttacaatg ccgaacttct ggttctcatg gaaaatgaga gaactctaga 1380 ctttcatgac tcaaatgtca agaaccttta cgacaaggtc cgactacagc ttagggataa 1440 tgcaaaggag ctgggtaatg gttgtttcga attctatcac aaatgtgata acgaatgtat 1500 ggaaagtgta aaaaacggaa cgtatgacta cccgcagtat tcagaagaag caagactaaa 1560 cagagaggaa ataagtggag taaaattgga atcaatggga acttaccaaa tactgtcaat 1620 ttattcaaca gtggcgagtt ccctagcact ggcaatcatg gtagctggtc tatctttatg 1680 gatgtgctcc aatggatcgt tacaatgcag aatttgcatt taaatttgtg agttcagatt 1740 gtagttaaaa acacccttgt ttctact 1767 <210> 8 <211> 1401 <212> DNA
<213> Influenza virus <400> 8 agcaaaagca ggagtttaaa atgaatccaa atcagaagat aataaccatt ggatcaatct 60 gcatggtagt tgggataatc agcttgatgt tacaaattgg aaacacaata tcagtatggg 120 tcagccacat aattaaaact tggcacccaa accagcctga accatgcaac caaagcatca 180 atttttacac tgagcaggct gcagcttcag tgacattagc gggcaattcc tctctctgcc 240 ctattagtgg atgggctata tacagcaagg acaatagtat aagaattggt tccaaagggg 300 atgtgtttgt tataagagaa ccattcatct catgctccca tttggaatgc agaacctttt 360 tcttgaccca aggagcccta ttgaatgaca agcattctaa tgggaccgtc aaagacagga 420 gcccctatag aactttaatg agctgtcctg ttggtgaggc cccttcccca tacaactcaa 480 ggtttgagtc tgttgcttgg tcagcaagtg cttgccatga tggcattagt tggctaacaa 540 ttggaatttc cggtccggat aatggggctg tggctgtgtt gaaatacaat ggcataataa 600 cagacaccat caagagttgg aggaacaaca cactgaggac gcaagagtct gaatgtgcat 660 gtgtgaatgg ttcttgtttt actgtaatga cagatggacc gagtaatgaa caggcctcat 720 acaagatttt caagatagaa aaggggaggg tagtcaaatc agttgagttg aacgccccta 780 attatcatta cgaggaatgc tcctgttatc ctgatgctgg cgaaatcaca tgtgtgtgca 840 gggataattg gcatggctog aaccgaccat gggtgtcttt caatcagaat ctggagtatc 900 aaataggata tatatgcagt ggggttttcg gagacagtcc acgccccaat gatgggacag 960 gcagttgtgg tccagtgtct cttaacggag cgtatggagt aaaagggttt tcatttaaat 1020 acggcaatgg tgtttggatc gggagaacca aaagcactag ttccaggagc ggttttgaaa 1080 tgatttggga tccaaatggg tggaccgaaa cagacagtag cttctcgttg aagcaagaca 1140 tcatagcgat aactgattgg tcaggataca gcgggagttt tattcaacat ccagaactga 1200 caggattaaa ttgcatgaga ccttgcttct gggttgaact aatcagaggg aggcccaaag 1260 agaaaacaat ctggactagt gggagcagta tatctttctg tggtgtaaat agtgacactg 1320 tgggttggtc ttggccagac ggtgctgagt tgccatacac cattgacaag tagtttgttc 1380 aaaaaactcc ttgtttctac t 1401 <210> 9 <211> 1690 <212> DNA
<213> Influenza virus <400> 9 ttaaccactc aagatggaag caataccact aataactata ctactagtag taacagcaag 60 caatgcagac aaaatctgca tcggctacca atcaacaaac tccacagaaa ccgtagacac 120 gctaacagaa aacaatgttc ctgtgacaca tgccaaagaa ttgctccaca cagagcacaa 180 tgggatgctg tgtgcaacaa atctgggacg tcctcttatt ctagacactt gcaccattga 240 aggactgatc tatggcaacc cttcttgtga tctactgttg ggaggaagag aatggtccta 300 catcgtcgaa agaccatcgg ctgttaatgg aatgtgttac cccgggaatg tagaaaacct 360 agaggaacta aggtcatttt ttagttctgc tagttcctac caaagaatcc agatctttcc 420 agacacaatc tggaatgtgt cttacagtgg aacaagcaaa gcatgttcag attcattcta 480 caggagcatg agatggttga ctcaaaagaa caacgcttac cctattcaag acgcccaata 540 cacaaataat agaggaaaga gcattctttt catgtggggc ataaatcacc cacctaccga 600 tactgcacag acaaatctgt acacaaggac tgacacaaca acaagtgtgg caacagaaga 660 tataaatagg accttcaaac cagtgatagg gccaaggccc cttgtcaatg gtctgcaggg 720 aagaattgat tattattggt cggtattgaa accaggtcag acattgcgag taagatccaa 780 tgggaatcta atcgctccat ggtatgggca cattctttca ggagagagcc acggaagaat 840 cctgaagact gatttaaaca gtggtagctg tgtagtgcaa tgtcaaacag aaagaggtgg 900 cttaaatact actttgccat tccacaatgt cagtaaatat gcatttggaa actgcccaaa 960 atatgttgga gtaaagagtc tcaaactggc agttggtctg aggaatgtgc ctgctagatc 1020 aagtagagga ctatttgggg ccatagctgg attcatagag ggaggttggt cagggctggt 1080 cgctggttgg tatgggttcc agcattcaaa tgatcaaggg gttggtatag ctgcagatag 1140 agactcaact caaagggcaa ttgacaaaat aacgtccaaa gtgaataata tagtcgataa 1200 aatgaacaag caatatgaaa ttattgatca tgaattcagc gaggttgaaa atagactcaa 1260 tatgatcaat aataagattg atgaccagat acaagacata tgggcatata acgctgaatt 1320 gctagtgctg cttgaaaacc agaaaacact cgatgagcat gatgcgaatg taaacaatct 1380 atataacaaa gtgaagaggg cactgggttc caatgcaatg gaagatggga aaggatgttt 1440 cgagctatac cataaatgtg atgatcagtg catggagaca attcggaacg ggacctataa 1500 caggaggaag tataaagagg aatcaagact agaaagacag aaaatagaag gggtcaagct 1560 ggaatctgaa ggaacttaca aaatcctcac catttattcg actgtcgcct catctcttgt 1620 gattgcaatg gggtttgctg ccttcttgtt ctgggccatg tccaatggat cttgcagatg 1680 caacatttga 1690 <210> 10 <211> 1428 <212> DNA
<213> Influenza virus <400> 10 aaatgaatcc aaatcagaag ataatagcaa ttggctctgt ttctctaact attgcgacaa 60 tatgcctcct catgcagatt gctatcttag caacgactat gacactacat ttcaagcaga 120 atgaatgcat caactcctcg aataatcaag tagtgccatg tgaaccaatc ataatagaaa 180 ggaacataac agagatagtg catttgaata gtactacctt agagaaggaa atttgtccta 240 aagtagcaga ctacaggaat tggtcaaaac cacaatgtca aatcacaggg ttcgctcctt 300 tctccaagga caattcaatt aggctctccg caggtggaga tatttgggtg acaagagaac 360 cttatgtatc gtgcggtctt ggtaaatgtt atcaatttgc acttgggcag ggaaccactt 420 tggagaacaa acactcaaac ggcacagcac atgatagaac tcctcataga acccttttaa 480 tgaatgagtt gggtgttccg tttcatttgg caaccaaaca agtgtgcata gcatggtcca 540 gctcaagctg ccatgatggg aaagcatggt tacatgtttg tgtcactggg gatgatagaa 600 atgcaacggc tagcatcatt tatgatggga tacttgttga cagtattggt tcatggtcta 660 aaaacatcct cagaactcag gagtcagaat gcgtttgcat caatggaacc tgtgcagtag 720 taatgactga tggaagtgca tcaggaaggg ctgacactag aatactattt attagagagg 780 ggaaaattgc acacattagc ccattgtcag gaagtgctca gcatgtggag gaatgctcct 840 gttacccccg atatccagaa gttagatgtg tttgcagaga caattggaag ggatccaata 900 ggcccgttct atatataaat atggcaaatt atagtattga ttccagttat gtgtgctcag 960 gacttgttgg cgacacacca agaaatgatg ataggtctag cagcagcaac tgcagagatc 1020 ctaataacga gagaggggcc ccaggagtaa aagggtgggc ctttgacaat ggaaatgaca 1080 tttggatggg aagaacaatc aaaaaggatt cgcgctcagg ttatgagact ttcagggtca 1140 ttggtggttg gaccactgct aattccaagt cacagataaa tagacaagtc atagttgaca 1200 gtgataactc gtctgggtat tctggtatct tctctgttga aggcaaaagc tgcatcaaca 1260 ggtgttttta cgtggagttg ataagaggaa gaccaaagga gactagggtg tggtggactt 1320 caaatagcat cattgtattt tgtggaactt caggtaccta tggaacaggc tcatggcctg 1380 atggggcgaa tatcaatttc atgcctatat aagctttcgc aattttag 1428 <210> 11 <211> 564 <212> PRT
<213> Influenza virus <400> 11 Met Glu Lys Ile Val Leu Leu Phe Ala Ile Val Ser Leu Val Lys Ser Asp Gin Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gin Val Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gin Asp Ile Leu Glu Lys Lys His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val Glu Lys Ala Asn Pro Val Asn Asp Leu Cys Tyr Pro Gly Asp Phe Asn Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu Lys Ile Gin Ile Ile Pro Lys Ser Ser Trp Ser Ser His Glu Ala Ser Leu Gly Val Ser Ser Ala Cys Pro Tyr Gin Gly Lys Ser Ser Phe Phe Arg Asn Val Val Trp Leu Ile Lys Lys Asn Ser Thr Tyr Pro Thr Ile Lys Arg Ser Tyr Asn Asn Thr Asn Gin Glu Asp Leu Leu Val Leu Trp Gly Ile His His Pro Asn Asp Ala Ala Glu Gin Thr Lys Leu Tyr Gin Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gin Arg Leu Val Pro Arg Ile Ala Thr Arg Ser Lys Val Asn Gly Gin Ser Gly Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly Asn Cys Asn Thr Lys Cys Gin Thr Pro Met Gly Ala Ile Asn Ser Ser Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser Pro Gin Arg Glu Thr Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Gin Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn Glu Gin Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gin Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp Lys Met Asn Thr Gin Phe Glu Ala Val Gly Arg Glu Phe Asn Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Leu Gin Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gin Tyr Ser Glu Glu Ala Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Ile Tyr Gin Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu Ala Ile Met Val Ala Gly Leu Ser Leu Trp Met Cys Her Asn Gly Ser Leu Gin Cys Arg Ile Cys Ile <210> 12 <211> 449 <212> PRT
<213> Influenza virus <400> 12 Met Asn Pro Asn Gin Lys Ile Ile Thr Ile Gly Ser Ile Cys Met Val Thr Gly Ile Val Ser Leu Met Leu Gin Ile Gly Asn Met Ile Ser Ile Trp Val Ser His Ser Ile His Thr Gly Asn Gin His Gin Ser Glu Pro Ile Ser Asn Thr Asn Phe Leu Thr Glu Lys Ala Val Ala Ser Val Lys Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Asn Gly Trp Ala Val Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu Cys Arg Thr Phe Phe Leu Thr Gin Gly Ala Leu Leu Asn Asp Lys His Ser Asn Gly Thr Val Lys Asp Arg Ser Pro His Arg Thr Leu Met Ser Cys Pro Val Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Thr Ser Trp Leu Thr Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr Asn Gly Ile Ile Thr Asp Thr Ile Lys Ser. Trp Arg Asn Asn Ile Leu Arg Thr Gin Glu Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr Val Met Thr Asp Gly Pro Ser Asn Gly Gin Ala Ser His Lys Ile Phe Lys Met Glu Lys Gly Lys Val Val Lys Ser Val Glu Leu Asp Ala Pro Asn Tyr His Tyr Glu Glu Cys Ser Cys Tyr Pro Asn Ala Gly Glu Ile Thr Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val Ser Phe Asn Gin Asn Leu Glu Tyr Gin Ile Gly Tyr Ile Cys Ser Gly Val Phe Gly Asp Asn Pro Arg Pro Asn Asp Gly Thr Gly Ser Cys Gly Pro Val Ser Ser Asn Gly Ala Tyr Gly Val Lys Gly Phe Ser Phe Lys Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Thr Asn Ser Arg Ser Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp Ser Ser Phe Ser Val Lys Gin Asp Ile Val Ala Ile Thr Asp Trp Ser Gly Tyr Ser Gly Ser Phe Val Gin His Pro Glu Leu Thr Gly Leu Asp Cys Ile Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Lys Glu Ser Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val Asn Ser Asp Thr Val Gly Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro Phe Thr Ile Asp Lys <210> 13 <211> 564 <212> PRT
<213> Influenza virus <400> 13 Met Glu Lys Ile Val Leu Leu Phe Ala Ile Val Ser Leu Val Lys Ser Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asp Phe Asn Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu Lys Ile Gln Ile Ile Pro Lys Asn Ser Trp Ser Ser His Glu Ala Ser Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Lys Ser Ser Phe Phe Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asn Ala Tyr Pro Thr Ile 165 170 ' 175 Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Arg Leu Tyr Gln Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Asn Gly Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile Val Lys Lys Gly Asp Ser Ala Ile Met Lys Ser Glu Leu Glu Tyr Gly Asn Cys Asn Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser Pro Gln Arg Glu Thr Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Leu Gin Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gin Tyr Ser Glu Glu Ala Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Thr Tyr Gin Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu Ala Ile Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly Ser Leu Gin Cys Arg Ile Cys Ile <210> 14 <211> 469 <212> PRT
<213> Influenza virus <400> 14 Met Asn Pro Asn Gin Lys Ile Thr Thr Ile Gly Ser Ile Cys Met Val Ile Gly Ile Val Ser Leu Met Leu Gin Ile Gly Asn Ile Ile Ser Ile 20 25 =30 Trp Val Ser His Ser Ile Gin Thr Gly Asn Gin His Gin Ala Glu Pro Cys Asn Gin Ser Ile Ile Thr Tyr Glu Asn Asn Thr Trp Val Asn Gin 50 55 ' 60 Thr Tyr Val Asn Ile Ser Asn Thr Asn Phe Leu Thr Glu Lys Ala Val Ala Ser Val Thr Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Ser Gly Trp Ala Val Tyr Ser Lys Asp Asn Gly Ile Arg Ile Gly Ser Lys Gly Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu Cys Arg Thr Phe Phe Leu Thr Gin Gly Ala Leu Leu Asn Asp Lys His Ser Asn Gly Thr Val Lys Asp Arg Ser Pro His Arg Thr Leu Met Ser Cys Pro Val Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser 165 170 175 ' Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Thr Ser Trp Leu Thr Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr Asn Gly Ile Tie Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Ile Met Arg Thr Gin Glu Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr 225 ' 230 235 240 =
Val Met Thr Asp Gly Pro Ser Asn Gly Gin Ala Ser Tyr Lys Ile Phe Arg Ile Glu Lys Gly Lys Val Val Lys Ser Ala Glu Leu Asn Ala Pro Asn Tyr His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Ala Gly Glu Ile Thr Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val Ser Phe Asn Gin Asn Leu Glu Tyr Arg Ile Gly Tyr Ile Cys Ser Gly Val Phe Gly Asp Asn Pro Arg Pro Asn Asp Gly Thr Gly Ser Cys Gly Pro Val Ser Pro Lys Gly Ala Tyr Gly Ile Lys Gly Phe Ser Phe LYs Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Thr Asn Ser Arg Ser Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Gly Thr Asp Ser Asn Phe Ser Val Lys Gin Asp Ile Val Ala Ile Thr Asp Trp Ser Gly Tyr Ser Gly Ser Phe Val Gin His Pro Glu Leu Thr Gly Leu Asp Cys Ile Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Lys Glu Ser Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val Asn Ser Asp Thr Val Gly Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro Phe Thr Ile Asp Lys <210> 15 <211> 564 <212> PRT
<213> Influenza virus <400> 15 Met Glu Lys Ile Val Leu Leu Leu Ala Thr Val Ser Leu Val Lys Ser Asp Gin Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gin Val Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gin Asp Ile Leu Glu Arg Thr His Asn Gly Lys Leu Cys Asp Leu Asn Gly Val Lys Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val Glu Lys Ala Ser Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu Lys Ile Gin Ile Ile Pro Lys Ser Ser Trp Ser Asn His Asp Ala Ser Ser Gly Val Ser Ser Ala Cys Pro Tyr Leu Gly Arg Ser Ser Phe Phe Arg Asn Val Val Trp Leu Ile Lys Lys Asn Ser Ser Tyr Pro Thr Ile 165 = 170 175 Lys Arg Ser Tyr Asn Asn Thr Asn Gin Glu Asp Leu Leu Val Leu Trp Gly Ile His His Pro Asn Asp Ala Ala Glu Gin Thr Arg Leu Tyr Gin Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gin Arg Leu Val Pro Glu Ile Ala Thr Arg Pro Lys Val Asn Gly Gin Ser Gly Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly Asn Cys Asn Thr Lys Cys Gin Thr Pro Met Gly Ala Ile Asn Ser Ser Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Thr Pro Gin Arg Glu Thr Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Gin Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn Glu Gin Gly Ser Gly Tyr Ala Ala Asp Gin Glu Ser Thr Gin Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asn Lys Met Asn Thr Gin Phe Glu Ala Val Gly Arg Glu Phe Asn Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Leu Gin Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Lys Asn ' Gly Thr Tyr Asp Tyr Pro Gin Tyr Ser Glu Glu Ala Arg Leu Asn Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Met Gly Thr Tyr Gin Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu Ala Ile Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly Ser Leu Gin Cys Arg Ile Cys Ile <210> 16 <211> 450 <212> PRT
<213> Influenza virus <400> 16 Met Asn Pro Asn Gin Lys Ile Ile Thr Ile Gly Ser Ile Cys Met Val Val Gly Ile Ile Ser Leu Met Leu Gin Ile Gly Asn Thr Ile Ser Val Trp Val Ser His Ile Ile Lys Thr Trp His Pro Asn Gin Pro Glu Pro Cys Asn Gin Ser Ile Asn Phe Tyr Thr Glu Gin Ala Ala Ala Ser Val Thr Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Ser Gly Trp Ala Ile 65 70 75. 80 Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu Cys Arg Thr Phe Phe Leu Thr Gin Gly Ala Leu Leu Asn Asp Lys His Ser Asn Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser Cys Pro Val Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Ile Ser Trp Leu Thr Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr Asn Gly Ile Ile Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Thr Leu Arg Thr Gin Glu Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr Val Met Thr Asp Gly Pro Ser Asn Glu Gin Ala Ser Tyr Lys Ile Phe Lys Ile Glu Lys Gly Arg Val Val Lys Ser Val Glu Leu Asn Ala Pro Asn Tyr His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Ala Gly Glu Ile Thr Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val Ser Phe Asn Gin Asn. Leu Glu Tyr Gin Ile Gly Tyr Ile Cys Ser Gly Val Phe Gly Asp Ser Pro Arg Pro Asn Asp Gly Thr Gly Ser Cys Gly Pro Val Ser Leu Asn Gly Ala Tyr Gly Val Lys Gly Phe Ser Phe Lys Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Thr Ser Ser Arg Ser Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp Ser Ser Phe Ser Leu Lys Gin Asp Ile Ile Ala Ile Thr Asp Trp Ser Gly Tyr Ser Gly Ser Phe Ile Gin His Pro Glu Leu Thr Gly Leu Asn Cys Met Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Lys Glu Lys Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val Asn Ser Asp Thr Val Gly Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro Tyr Thr Ile Asp Lys <210> 17 <211> 564 <212> PRT
<213> Influenza virus <400> 17 Met Glu Lys Ile Val Leu Leu Leu Ala Thr Val Ser Leu Val Lys Ser Asp Gin Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gin Val Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gin Asp Ile Leu Glu Arg Thr His Asn Gly Lys Leu Cys Asp Leu Asn Gly Val Lys 50 55 , 60 Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val Glu Lys Ala Ser Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu Lys Ile Gin Ile Ile Pro Lys Ser Ser Trp Ser Asn His Asp Ala Ser Ser Gly Val Ser Ser Ala Cys Pro Tyr Leu Gly Arg Ser Ser Phe Phe Arg Asn Val Val Trp Leu Ile Lys Lys Asn Ser Ser Tyr Pro Thr Ile Lys Arg Ser Tyr Asn Asn Thr Asn Gin Glu Asp Leu Leu Val Leu Trp , Gly Ile His His Pro Asn Asp Ala Ala Glu Gin Thr Arg Leu Tyr Gin Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gin Arg Leu Val Ser Glu Ile Ala Thr Arg Pro Lys Val Asn Gly Gin Ser Gly Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly Asn Cys Asn Thr Lys Cys Gin Thr Pro Met Gly Ala Ile Asn Ser Ser Met Pro She His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Thr Pro Gin Arg Glu Thr Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Gin Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn Glu Gin Gly Ser Gly Tyr Ala Ala Asp Gin Glu Ser Thr Gin Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asn Lys Met Asn Thr Gin Phe Glu Ala Val Gly Arg Glu Phe Asn Asn Leu Glu Arg Arg Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Leu Gin Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg Leu Asn Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Met Gly Thr Tyr Gin Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu Ala Ile Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly Ser Leu Gin Cys Arg Ile Cys Ile <210> 18 <211> 450 <212> PRT
<213> Influenza virus <400> 18 Met Asn Pro Asn Gin Lys Ile Ile Thr Ile Gly Ser Ile Cys Met Val Val Gly Ile Ile Ser Leu Met Leu Gin Ile Gly Asn Thr Ile Ser Val Trp Val Ser His Ile Ile Lys Thr Trp His Pro Asn Gin Pro Glu Pro Cys Asn Gin Ser Ile Asn Phe Tyr Thr Glu Gin Ala Ala Ala Ser Val Thr Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Ser Gly Trp Ala Ile Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu Cys Arg Thr Phe Phe Leu Thr Gin Gly Ala Leu Leu Asn Asp Lys His Ser Asn Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser Cys Pro Val Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Ile Ser Trp Leu Thr Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr Asn Gly Ile Ile Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Thr Leu Arg Thr Gin Glu Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr Val Met Thr Asp Gly Pro Ser Asn Glu Gin Ala Ser Tyr Lys Ile Phe Lys Ile Glu Lys Gly Arg Val Val Lys Ser Val Glu Leu Asn Ala Pro Asn Tyr His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Ala Gly Glu Ile Thr Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val Ser Phe Asn Gin Asn Leu Glu Tyr Gin Ile Gly Tyr Ile Cys Ser Gly Val Phe Gly Asp Ser Pro Arg Pro Asn Asp Gly Thr Gly Ser Cys Gly Pro Val Ser Leu Asn Gly Ala Tyr Gly Val Lys Gly Phe Ser Phe Lys Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Thr Ser Ser Arg Ser Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp Ser Ser Phe Ser Leu Lys Gin Asp Ile Ile Ala Ile Thr Asp Trp Ser Gly Tyr Ser Gly Ser Phe Ile Gin His Pro Glu Leu Thr Gly Leu Asn Cys Met Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Lys Glu Lys Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val Asn Ser Asp Thr Val Gly Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro Tyr Thr Ile Asp Lys <210> 19 <211> 558 <212> PRT
<213> Influenza virus <400> 19 Met Glu Ala Ile Pro Leu Ile Thr Ile Leu Leu Val Val Thr Ala Ser Asn Ala Asp Lys Ile Cys Ile Gly Tyr Gin Ser Thr Asn Ser Thr Glu Thr Val Asp Thr Leu Thr Glu Asn Asn Val Pro Val Thr His Ala Lys Glu Leu Leu His Thr Glu His Asn Gly Met Leu Cys Ala Thr Asn Leu Gly Arg Pro Leu Ile Leu Asp Thr Cys Thr Ile Glu Gly Leu Ile Tyr Gly Asn Pro Ser Cys Asp Leu Leu Leu Gly Gly Arg Glu Trp Ser Tyr Ile Vol Glu Arg Pro Ser Ala Val Asn Gly Met Cys Tyr Pro Gly Asn Val Glu Asn Leu Glu Glu Leu Arg Ser Phe Phe Ser Ser Ala Ser Ser Tyr Gin Arg Ile Gin Ile Phe Pro Asp Thr Ile Trp Asn Val Ser Tyr Ser Gly Thr Ser Lys Ala Cys Ser Asp Ser Phe Tyr Arg Ser Met Arg Trp Leu Thr Gin Lys Asn Asn Ala Tyr Pro Ile Gin Asp Ala Gin Tyr Thr Asn Asn Arg Gly Lys Ser Ile Leu Phe Met Trp Gly Ile Asn His Pro Pro Thr Asp Thr Ala Gin Thr Asn Leu Tyr Thr Arg Thr Asp Thr Thr Thr Ser Val Ala Thr Glu Asp Ile Asn Arg Thr Phe Lys Pro Val Ile Gly Pro Arg Pro Leu Val Asn Gly Leu Gin Gly Arg Ile Asp Tyr Tyr Trp Ser Val Leu Lys Pro Gly Gin Thr Leu Arg Val Arg Ser Asn Gly Asn Leu Ile Ala Pro Trp Tyr Gly His Ile Leu Ser Gly Glu Ser His Gly Arg Ile Leu Lys Thr Asp Leu Asn Ser Gly Ser Cys Val Val Gin Cys Gin Thr Glu Arg Gly Gly Leu Asn Thr Thr Leu Pro Phe His Asn Val Ser Lys Tyr Ala Phe Gly Asn Cys Pro Lys Tyr Val Gly Val Lys Ser Leu Lys Leu Ala Vol Gly Leu Arg Asn Val Pro Ala Arg Ser Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Ser Gly Leu Val Ala Gly Trp Tyr Gly Phe Gin His Ser Asn Asp Gin Gly Vol Gly Ile Ala Ala Asp Arg Asp Ser Thr Gin Arg Ala Ile Asp Lys Ile Thr Ser Lys Val Asn Asn Ile Val Asp Lys Met Asn Lys Gin Tyr Glu Ile Ile Asp His Glu Phe Ser Glu Vol Glu Asn Arg Leu Asn Met Ile Asn Asn Lys Ile Asp Asp Gin Ile Gin Asp Ile Trp Ala Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Gin Lys Thr Leu Asp Glu His Asp Ala Asn Val Asn Asn Leu Tyr Asn Lys Val Lys Arg Ala Leu Gly Ser Asn Ala Met Glu Asp Gly Lys Gly Cys Phe Glu Leu Tyr His Lys Cys Asp Asp Gin Cys Met Glu Thr Ile Arg Asn Gly Thr Tyr Asn Arg Arg Lys Tyr Lys Glu Glu Ser Arg Leu Glu Arg Gin Lys Ile Glu Gly Val Lys Leu Glu Ser Glu Gly Thr Tyr Lys Ile Leu Thr Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Ile Ala Met Gly Phe Ala Ala Phe Leu Phe Trp Ala Met Ser Asn Gly Ser Cys Arg Cys Asn Ile <210> 20 <211> 469 <212> PRT
<213> Influenza virus <400> 20 Met Asn Pro Asn Gin Lys Ile Ile Ala Ile Gly Ser Val Ser Leu Thr Ile Ala Thr Ile Cys Leu Leu Met Gin Ile Ala Ile Leu Ala Thr Thr Met Thr Leu His Phe Lys Gin Asn Glu Cys Ile Asn Ser Ser Asn Asn Gin Val Vol Pro Cys Glu Pro Ile Ile Ile Glu Arg Asn Ile Thr Glu Ile Val His Leu Asn Ser Thr Thr Leu Glu Lys Glu Ile Cys Pro Lys Val Ala Asp Tyr Arg Asn Trp Ser Lys Pro Gin Cys Gin Ile Thr Gly 85 90 95 , Phe Ala Pro Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly Asp Ile Trp Val Thr Arg Glu Pro Tyr Val Ser Cys Gly Leu Gly Lys Cys Tyr Gin Phe Ala Leu Gly Gin Gly Thr Thr Leu Glu Asn Lys His Ser Asn Gly Thr Ala His Asp Arg Thr Pro His Arg Thr Leu Leu Met Asn Glu Leu Gly Val Pro Phe His Leu Ala Thr Lys Gin Vol Cys Ile Ala Trp Ser Ser Ser Ser Cys His Asp Gly Lys Ala Trp Leu His Val Cys Val Thr Gly Asp Asp Arg Asn Ala Thr Ala Ser Ile Ile Tyr Asp Gly Ile Leu Val Asp Ser Ile Gly Ser Trp Ser Lys Asn Ile Leu Arg Thr Gin Glu Ser Glu Cys Vol Cys Ile Asn Gly Thr Cys Ala Val Val Met Thr Asp Gly Ser Ala Ser Gly Arg Ala Asp Thr Arg Ile Leu Phe Ile Arg Glu Gly Lys Ile Ala His Ile Ser Pro Leu Ser Gly Ser Ala Gin His Val Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Glu Val Arg Cys Vol Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Vol Leu Tyr Ile Asn Met Ala Asn Tyr Ser Ile Asp Ser Ser Tyr Val Cys Ser Gly Leu Val Gly Asp Thr Pro Arg Asn Asp Asp Arg Ser Ser Ser Ser Asn Cys Arg Asp Pro Asn Asn Glu Arg Gly Ala Pro Gly Vol Lys Gly Trp Ala Phe Asp Asn Gly Asn Asp Ile Trp Met Gly Arg Thr Ile Lys Lys Asp Ser Arg Ser Gly Tyr Glu Thr Phe Arg Val Ile Gly Gly Trp Thr Thr Ala Asn Ser Lys Ser Gin Ile Asn Arg Gln-Val Ile Val Asp Ser Asp Asn Ser Ser Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly Lys Ser Cys Ile Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Pro Lys Glu Thr Arg Val Trp Trp Thr Ser Asn Ser Ile Ile Val Phe Cys Gly Thr Ser Gly Thr Tyr Gly Thr Gly Ser Trp Pro Asp Gly Ala Asn Ile Asn Phe Met Pro Ile

Claims (17)

1. A reassortant influenza virus, wherein said virus comprises 6 internal genome segments from one or more donor influenza viruses other than A/Ann Arbor/6/60, a first genome segment and a second genome segment encoding surface antigens, wherein the first genome segment encodes an HA polypeptide comprising the amino acid sequence of SEQ ID
NO:15.

2. The reassortant influenza virus of claim 1, wherein the second genome segment encodes an NA polypeptide comprising the amino acid sequence of SEQ ID
NO:16.

3. The reassortant influenza virus of claim 1 or 2, wherein said one or more donor influenza viruses have one or more of the following properties: temperature-sensitivity, cold-adaption, or attenuated.

4. The reassortant influenza virus of claim 1 or 2, wherein said one or more donor viruses are PR8.

5. The reassortant influenza virus of claim 1 or 2, wherein said one or more donor viruses are A/Leningrad/17.

6. An immunogenic composition comprising an immunologically effective amount of the reassortant influenza virus of any one of claims 1 to 5 in a carrier or diluent.

7. The reassortant influenza virus of any one of claims 1 to 5 for use in stimulating the immune system of a subject to produce a protective immune response against influenza virus, wherein the reassortant influenza virus is to be administered to the subject in an immunologically effective amount and in a physiologically effective carrier.

8. The reassortant influenza virus of any one of claims 1 to 5 for use in prophylactic treatment of a viral infection in a subject, wherein the virus is in an amount effective to produce an immunogenic response against the viral infection.

9. A split virus or killed virus vaccine comprising the immunogenic composition of claim 6.

10. The reassortant influenza virus for use according to claim 7 or 8, wherein said virus is killed or inactivated.

11. A live attenuated influenza vaccine comprising the composition of claim 6.

12. A method for producing influenza viruses in cell culture, the method comprising:
i) introducing into a population of host cells, which population of host cells is capable of supporting replication of influenza virus, a plurality of vectors comprising nucleic acid sequences corresponding to at least 6 internal genome segments from one or more influenza donor virus other than A/Ann Arbor/6/60, a first genome segment and a second genome segment encoding immunogenic influenza surface antigens, wherein the first genome segment encodes a hemagglutinin polypeptide comprising the amino acid sequence of SEQ ID
NO: 15;
ii) culturing the population of host cells at a temperature less than or equal to 35 C; and iii) recovering a plurality of influenza viruses from the cultured cells.

13. The method of claim 12, wherein the second genome segment encodes an NA
polypeptide comprising the amino acid sequence of SEQ ID NO:16.

14. The method of claim 12 or 13, wherein the six internal genome segments from the one or more donor viruses encode one or more phenotypic attributes selected from the group consisting of: attenuated, cold-adapted and temperature sensitivity.

15. A reassortant influenza virus of any one of claims 1 to 5 for use in immunizing a subject against an influenza virus infection.

16. The reassortant influenza virus of claim 1, wherein the first genome segment comprises the nucleotide sequence of SEQ ID NO:5.

17. The reassortant influenza virus of claim 1, wherein the second genome segment comprises the nucleotide sequence of SEQ ID NO:6.