Influenza hemagglutinin and neuraminidase variants

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DESCRIPTION

Variants of influenza hemagglutinin and neuraminidase

Background of the invention

Vaccines against various evolving strains of influenza are important from a public health standpoint, as well as commercially, since numerous individuals become infected with different strains and types of influenza viruses each year. Infants, the elderly, and those with inadequate healthcare and immunocompromised individuals are at particular risk of death from such infections. The problem of influenza infections is compounded because novel influenza strains evolve rapidly and can spread between various species, thus necessitating the continued production of new vaccines.

Numerous vaccines that can produce a specific protective immune response to such different influenza virus / 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, although appropriate formulations of any of these types of vaccines can elicit a 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 has been done by the present inventors and co-workers in the production of influenza viruses, and fragments thereof, for the production of vaccines; see, for example, US 2004029251 and US 2005042229.

Due to the continued emergence (or reappearance) of different influenza strains, new influenza vaccines are continually desired. Typically, such vaccines are created using antigenic moieties from newly emerged virus strains, therefore polypeptides and polynucleotides from novel, newly emerged or newly re-emerged virus strains (especially antigenic gene sequences) are highly desired.

Webby at al (2004/04/03), The Lancet, vol. 363, pages 1099-1103, describes a use of reverse genetics for rapid influenza vaccine development.

The present disclosure provides new and / or freshly isolated influenza hemagglutinin and neuraminidase variants that are useful in the production of numerous types of vaccines as well as in research, diagnostics, etc. Numerous other benefits will become apparent upon review of the following.

Summary of the invention

The invention in its broadest sense is as defined in the independent claims.

It describes an isolated or recombinant polypeptide that is selected from: the polypeptides encoded by any one of the sequences of SEQ ID N ° 1 to SEQ ID N ° 10, any one of the polypeptides encoded by SEQ ID N ° 1 to SEQ ID No. 10; any one of the polypeptides of SEQ ID N ° 1 to SEQ ID N ° 20; only the open reading frames of the polypeptides of SEQ ID No. 11 to SEQ ID No. 20; alternative forms (eg, the mature form without the signal peptide, or without the 5 'and 3' sequences out of the open reading frame, or the sequences as expressed on the surface of a virus (eg, influenza)) of the polypeptides of SEQ ID NO: 11-20.

The polypeptides described herein optionally comprise fusion proteins, proteins with a leader sequence, a precursor polypeptide, proteins with a secretion signal or a localization signal, or proteins with an epitope tag, an E tag, or a tag. His epitope. The sequences described herein are also shown in Appendix 1 and in the sequence listings herein. The hemagglutinin sequences described herein may comprise sequences with modified polybasic cleavage sites (thereby allowing the virus to grow in eggs). The hemagglutinin polypeptide sequences of SEQ ID N^you 11-20 comprise the endogenous amino terminal signal peptide sequences, however, the hemagglutinin polypeptide sequences described herein also include the mature form (cleaved amino terminal signal peptide) of the hemagglutinin polypeptides. The cleavage sites of any hemagglutinin polypeptide sequence from any strain of influenza can be routinely predicted or measured using any of several methods in the art.

Also described herein is a composition with one or more polypeptides listed above, or fragments thereof. Also described herein are polypeptides to which a polyclonal antiserum generated against at least 1 antigen comprising at least one amino acid sequence described above, or a fragment thereof, specifically binds. Such antibodies specific for the polypeptides described above are also described herein. The polypeptides described herein are optionally immunogenic.

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 system are further described.

ES 2 525 672 T3 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 vehicle.

The invention includes recombinant influenza virus as characterized by the appended claims comprising one or more of the above polypeptides or polynucleotides, in addition to immunogenic compositions comprising an immunologically effective amount of such recombinant influenza virus. The recombinant influenza virus of the invention for use in a method of treatment by stimulating the immune system of an individual to produce a protective immune response against influenza virus, through the administration of an immunologically effective amount of such recombinant influenza virus in a physiologically acceptable vehicle is also part of the invention.

Also described herein is a composition of matter having two or more nucleic acids described above (eg, 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 nucleic acid (s) described above, (eg, mechanically, chemically, enzymatically with a restriction endonuclease / RNase / DNase, etc.). Other compositions include, for example, compositions produced by incubating one or more of the nucleic acid (s) described above in the presence of deoxyribonucleotide triphosphate and a thermostable nucleic acid polymerase.

Also described herein are cells that comprise at least one of the nucleic acids described above, or an amplified or excised product or fragment thereof. Such cells can optionally express a polypeptide encoded by such nucleic acid. Vectors (eg, plasmids, cosmids, phages, viruses, virus fragments, etc.) comprising any of the nucleic acids described above are further described herein. Such vectors may optionally comprise an expression vector. Preferred expression vectors include, but are not limited to, vectors comprising the pol I promoter and terminator sequences or vectors using both the pol I and pol II promoters "the pol I / pol II promoter system" (eg Zobel et al., Nucl. Acids Res. 1993,21: 3607; US 20020164770; Neumann et al., Proc. Natl. Acad. Sci. USA 1999, 96: 9345; Fodor et al., J Virol. 1999, 73: 9679; and US 20030035814). Cells transduced by such vectors are also described.

In some embodiments, the invention encompasses a virus (eg, influenza virus) comprising one or more nucleic acids described above (eg, encoding hemagglutinin and / or neuraminidase) as characterized by the appended claims. Immunogenic compositions comprising such a virus are also part of the present invention as characterized by the appended claims. The invention is a reassortant influenza virus, where the virus is a 6: 2 reassortant influenza virus and comprises 6 coding regions of A / Ann Arbor / 6/60 genes and 2 coding regions of genes that encode surface antigens of influenza virus, wherein the first of the two mentioned gene coding regions encodes an HA polypeptide comprising the amino acid sequence of SEQ ID No. 13. Methods as characterized by the appended claims of production of recombinant influenza virus by culturing a host cell harboring an influenza virus in a suitable culture medium under conditions that allow expression of the nucleic acid and isolation of the recombinant influenza virus of one or more of the host cell (s) or the medium are also part of the invention.

In other embodiments herein, the invention comprises immunogenic compositions having an immunologically effective amount of any of the recombinant influenza viruses described above as characterized by the appended claims. Other embodiments include the regrouped influenza virus of the invention for use in a method of treatment by 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 said virus (optionally in a physiologically effective vehicle).

Also described herein are methods of producing an isolated or recombinant polypeptide by culturing any previous host cell, in a suitable culture medium under conditions that allow expression of nucleic acid, and isolating the polypeptide from one or more of the host cells. or the medium in which the cells are grown.

Immunogenic compositions as characterized by the appended claims are also characteristic of the invention. For example, immunogenic compositions comprising any one or more viruses described above as characterized by the appended claims (for example, together with one or more pharmaceutically acceptable delivery component (s).

The regrouped influenza virus of the invention for use in a method of prophylactic treatment of a viral infection (eg, viral influenza) in a subject through the administration of any one or more viruses described above or immunogenic compositions as mentioned in the claims in an amount effective to produce an immunogenic response against viral infection is also part of the present invention.

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Subjects for such treatment can include mammals (eg, humans). Such reassortant virus of the invention for use in such methods may also be for in vivo administration to the subject as well as for in vitro or ex vivo administration to one or more cell (s) of the subject. Additionally, such reassortant virus of the invention for use in such methods may also comprise a virus composition and a pharmaceutically acceptable carrier for administration to the subject in an amount effective to prophylactically treat the viral infection.

Also disclosed herein are compositions of matter comprising nucleic acid sequences encoding hemagglutinin and / or neuraminidase polypeptides from one or more pandemic influenza strain (s) and nucleic acid sequences encoding one or more plus A / Ann Arbor / 6/60 polypeptide (s). Compositions of matter comprising nucleic acid sequences encoding hemagglutinin and / or neuraminidase polypeptides from one or more pandemic influenza strain (s) and nucleic acid sequences encoding one or more pandemic influenza strain (s) are further described herein. plus PR8 or A / Ann Arbor / 6/60 polypeptide (s). Such sequences may include those listed in the sequence listing herein. Additionally, these compositions include compositions of matter comprising sequences encoding the hemagglutinin and / or neuraminidase of one or more pandemic influenza strain (s) and nucleic acid sequences encoding a selected backbone strain in a reassortment. 6: 2. Such compositions described herein include sequences encoding hemagglutinin and neuraminidase selected from the sequence listing herein and a backbone strain, the backbone strain being PR8 or A / Ann Arbor / 6/60. Also described are compositions as described above in which the hemagglutinin comprises a modified polybasic cleavage site. The invention also includes a live attenuated influenza vaccine comprising such above compositions as characterized by the appended claims.

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 appendix and accompanying figures.

Brief description of the drawings

Figure 1: Shows engineered modifications to the HA gene of VN / 1203/2004 to remove the polybasic cleavage site.

Figure 2: Presents results showing that H5N1 ca reassortant viruses administered intranasally do not replicate in chickens.

Figure 3: Illustrates that H5N1 / AA ca vaccine candidates are not lethal to mice.

Figure 4: Illustrates that H5N1 ca 1997 and 2004 reassortant viruses are replication restricted in mice.

Figure 5: Illustrates that reassortant H5N1 / AA influenza viruses are restricted in replication in mouse lungs.

Figure 6: Shows the serum Ac HAI titers produced in mice after a single in dose of vaccine.

Figure 7: Shows the serum neutralizing Ab titers produced in mice after a single in dose of vaccine.

Figure 8: Illustrates that H5N1 ca reassortant viruses protect mice against lethal exposures with 50, 500, or 5000 LD50 wild-type H5N1 virus.

Figure 9: Illustrates the efficacy of protection against lung replication of homologous and heterologous H5N1 challenge viruses in mice.

Figure 10: Illustrates the efficacy of protection against replication of homologous and heterologous H5N1 challenge viruses in the upper respiratory tract of mice.

Figure 11: Illustrates the efficacy of protection conferred by the H5N1 ca 2004 vaccine against high-dose exposure (10⁵DICT5ü) with homologous or heterologous wt H5N1 viruses in mice.

Figure 12: Illustrates the efficacy of protection conferred by the H5N1 ca 1997 and 2003 vaccines against high-dose exposures (10⁵TCID50) with homologous or heterologous wild-type H5N1 viruses in mice.

Figure 13: Illustrates the efficacy of the protection conferred by the H5N1 ca 2004 vaccine against low or high dose exposures to the homologous wild-type H5N1 virus in mice.

Detailed description

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Definitions

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 relates. The following definitions supplement those in the art and refer to the present application and should not necessarily be attributed to any related or unrelated case, eg, any jointly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in practice to test the present invention, 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.

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.

An "amino acid sequence" is a polymer of amino acid residues (a protein, polypeptide, etc.) or a string of characters that represents a polymer of amino acids, depending on the context.

The terms and expressions "nucleic acid", "polynucleotide", "polynucleotide sequence" and "nucleic acid sequence" refer to polymers of single or double stranded deoxyribonucleotiodes or ribonucleotides, chimeras or analogs thereof, or a string of characters that represent such, depending on the context. As used herein, the term optionally includes naturally occurring nucleotide analog polymers having the essential nature of natural nucleotides because they hybridize to single stranded nucleic acids in a manner similar to naturally occurring nucleotides. (eg, peptide nucleic acids). Unless otherwise indicated, a particular nucleic acid sequence optionally encompasses complementary sequences in addition to the explicitly stated sequence. From any specified polynucleotide sequence, either the given nucleic acid or the complementary polynucleotide sequence (eg, the complementary nucleic acid) can be determined.

The term "nucleic acid" or "polynucleotide" also encompasses any physical chain of monomer units that can be matched to a nucleotide chain, including a nucleotide polymer (eg, a typical DNA or RNA polymer), PNAs, oligonucleotides modified (eg, oligonucleotides comprising bases that are not typical for biological DNA or RNA in solution, such as 2'-Omethylated oligonucleotides) and the like. A nucleic acid can be, for example, single-stranded or double-stranded.

A "subsequence" is any part of an entire sequence, up to and including the entire 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.

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, when a substituted amino acid has similar chemical or structural properties, for example, the substitution of isoleucine for leucine. Alternatively, a variant may have "non-conservative" changes, for example, the substitution of a glycine for a tryptophan. Analogous minor variation can also include amino acid deletion or insertion, or both. Guidance can be found to determine which amino acid residues can be substituted, inserted or deleted without eliminating biological or immunological activity using computer programs well known in the art, eg, DNASTAR software. Examples of conservative substitutions are also described herein.

The term "gene" is used broadly to refer to any nucleic acid associated with a biological function. Thus, genes include coding sequences and / or regulatory sequences required for their expression. The term "gene" applies to a specific genomic sequence, as well as a cDNA or mRNA encoded by that genomic sequence.

Genes also include unexpressed 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 the transcription of adjacent or nearby sequences. A "tissue specific" promoter or enhancer is one that regulates transcription in a specific cell type or tissue type, or types.

"Expression of a gene" or "expression of a nucleic acid" means the transcription of DNA into RNA (optionally including modification of RNA, eg alternative splicing), translation of RNA into a polypeptide (possibly including modification polypeptide, e.g., post-translational modification), or both transcription and translation, as the context indicates.

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An "open reading frame" or "ORF" is a possible translational reading frame of DNA or RNA (eg, of a gene), which can be 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.

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, proviruses, phagemids, transposons, artificial chromosomes, and the like, which 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 polylysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a conjugated DNA with liposomes or the like, which does not replicate autonomously. In many, but not all common cases, the vectors are plasmids.

An "expression vector" is a vector, such as a plasmid that can promote the expression as well as the replication of a nucleic acid incorporated therein. Typically, the nucleic acid to be expressed is "operably linked" to a promoter and / or enhancer, and is subject to transcriptional regulatory control by the promoter and / or enhancer.

A "bidirectional expression vector" is characterized by two alternative promoters oriented in the opposite direction relative to a nucleic acid located between the two promoters, such that expression can be initiated in both orientations resulting, for example, in transcription of both positive (+) or sense strand RNA, as well as negative (-) or antisense strand RNA.

A "polypeptide" is a polymer that comprises two or more amino acid residues (eg, 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.

In the context of the invention, the term "isolated" refers to a biological material, such as a virus, nucleic acid, or protein, that is substantially free of components that normally accompany or interact with it in its environment that it occurs naturally. The isolated biological material optionally comprises additional material not found with the biological material in its natural environment, for example a wild type virus or a cell. For example, if the material is in its natural environment, such as a cell, the material may be placed in a location in the cell (eg, 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.) is isolated if it is introduced by non-naturally occurring means at a locus in the genome (e.g. For example, a vector, such as a virus or plasmid vector, or amplicon) not native to that nucleic acid. Such nucleic acids are also called "heterologous" nucleic acids. An isolated virus, for example, is in an environment (eg, a cell culture system, or purified from cell culture) other than the native environment of the wild-type virus (eg, the nasopharynx of an infected individual).

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 parent viral source or strain. Similarly, the term "chimeric" or "chimera", when referring to a viral protein, indicates that the protein includes polypeptide components (ie, amino acid subsequences) derived from more than one parent viral source or strain.

The term "recombinant" indicates that the material (eg, a nucleic acid or protein) has been artificially or synthetically (unnaturally) altered by human intervention. The alteration can be made to the material within, or extracted from, its natural state or environment. Specifically, for example, 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 prepared by recombining nucleic acids, for example during cloning, DNA rearrangement or other procedures, or by chemical or other mutagenesis; a "recombinant polypeptide" or "recombinant protein" is a polypeptide or protein that is produced by the expression of a recombinant nucleic acid; and a "recombinant virus", for example a recombinant influenza virus, is produced by the expression of a recombinant nucleic acid.

The term "reassortant", when referring to a virus, indicates that the virus includes genetic and / or polypeptide components derived from more than one parent viral source or strain. For example, a 7: 1 reassortment includes 7 viral genomic segments (or gene segments) derived from a first parent virus, and a single complementary viral genomic segment, eg, encoding a hemagglutinin or neuraminidase. A 6: 2 reassortment includes 6 genomic segments, most commonly the 6 internal genes from a first parent virus, and two complementary segments, eg, hemagglutinin and neuraminidase, from a different parent virus.

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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 in which the nucleic acid can be incorporated into the genome of the cell (eg, chromosome , plasmid, plastid, or mitochondrial DNA), become an autonomous replicon, or be transiently expressed (eg, 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.

The term "host cell" means a cell that contains a heterologous nucleic acid, such as a vector, and supports replication and / or expression of the nucleic acid. The host cells can be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, bird, or mammalian cells, including human cells. Exemplary host cells may include, for example, Vero cells (African green monkey kidney), BHK cells (baby hamster kidney), primary chicken kidney cells (PCK), Madin-Darby canine kidney cells. (MDCK), Madin-Darby bovine kidney cells (MDBK), 293 cells (eg, 293T cells), and COS cells (eg, COS1, COS7 cells), etc.

An "immunologically effective amount" of influenza virus is an amount sufficient to enhance an individual's (eg, human) own immune response to subsequent exposure to influenza virus. Induced immunity levels can be monitored, eg, by measuring amounts of serum antibodies and / or neutralizing secretors, eg, by plaque neutralization, complement fixation, enzyme-linked immunosorbent or microneutralization assay.

A "protective immune response" against influenza virus refers to an immune response presented by an individual (eg, a human) that is protective against disease when the individual is subsequently exposed to and / or infected with such influenza virus. . In some cases, the influenza virus (for example, which circulates naturally) can still cause infection, but cannot cause a serious infection. Typically, the protective immune response results in detectable levels of host-generated serum and secretory antibodies that can neutralize virus from the same strain and / or subgroup (and possibly also from a subgroup and / or non-vaccine strain, different), in vitro and in vivo.

As used herein, an "antibody" is a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or immunoglobulin gene fragments. Recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as myriads of 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) building block comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having a "light" chain (approximately 25 kD) and a "heavy" chain (approximately 50-70 kD). The N-terminus of each chain defines a variable region of approximately 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 several well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide bonds in the hinge region to produce F (ab) '₂, an Fab dimer that is itself a light chain linked to VH-CH1 via a disulfide bond. The F (ab) '₂ can be reduced under mild conditions to break the disulfide bond in the hinge region thereby converting the dimer (Fab ')₂ in a Fab 'monomer. The Fab 'monomer is essentially a Fab with part of the hinge region (see, Fundamental Immunology, WE Paul, ed., Raven Press, NY (1999), for a more detailed description of other antibody fragments). Although various antibody fragments are defined as to the digestion of an intact antibody, one of skill will appreciate that such Fab 'fragments can be synthesized de novo either chemically or using recombinant DNA methodology. Thus, the term antibody, as used herein, includes antibodies or fragments either produced by modifying whole antibodies or synthesized de novo using recombinant DNA methodologies. Antibodies include, for example, polyclonal antibodies, monoclonal antibodies, single or multiple chain antibodies, including single chain Fv antibodies (sFv or scFv) in which a variable heavy chain and a variable light chain (directly or through peptide linker) to form a continuous polypeptide, and humanized or chimeric antibodies.

Influenza virus

The polypeptides and polynucleotides described herein, eg, SEQ ID NO: 1-20, are influenza HA and NA sequence variants. In general, influenza viruses consist of an inner ribonucleoprotein core containing a segmented single-stranded RNA genome and an outer lipoprotein envelope coated by a matrix protein. The influenza virus genome is composed of eight segments of linear (-) stranded ribonucleic acid (RNA), encoding the immunogenic proteins hemagglutinin (HA) and

ES 2 525 672 T3 neuraminidase (NA), and six inner core polypeptides: the nucleocapsid nucleoprotein (NP); matrix proteins (M); non-structural proteins (NS); and 3 RNA polymerase proteins (PA, PB1, PB2). During replication, 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 in ribonucleoprotein complexes that contain, in addition to RNA, NP and a polymerase complex (PB1, PB2, and PA).

Influenza is commonly grouped into the categories influenza A and influenza B. Influenza A and influenza B viruses each contain eight segments of negative polarity single-stranded RNA. The influenza A genome codes for eleven polypeptides. Segments 1-3 code for three polypeptides, which constitute an RNA-dependent RNA polymerase. Segment 1 codes for the PB2 protein of the polymerase complex. The remaining polymerase proteins PB1 and PA are encoded by segment 2 and segment 3, respectively. Furthermore, segment 1 of some influenza strains codes for a small protein, PB1-F2, produced from an alternative reading frame within the PB1 coding region. Segment 4 codes for the surface glycoprotein hemagglutinin (HA) involved in cell attachment and entry during infection. Segment 5 codes for the nucleocapsid nucleoprotein polypeptide (NP), the major structural component associated with viral RNA. Segment 6 codes for an envelope glycoprotein, neuraminidase (NA). Segment 7 codes for two matrix proteins, designated M1 and M2, which are translated from differentially spliced ​​mRNAs. Segment 8 codes for NS1 and NS2, two nonstructural proteins, which are translated from alternatively spliced ​​and spliced ​​mRNA variants. The eight segments of the influenza B genome code for 11 proteins. The three largest genes code for components of RNA polymerase, PB1, PB2, and PA. Segment 4 codes for the HA protein. Segment 5 codes for NP. Segment 6 codes for the NA protein and the NB protein. Both proteins, NB and NA, are translated from overlapping reading frames of a bicistronic mRNA. Influenza B segment 7 also codes for two proteins: M1 and BM2. The smaller segment codes for two products: NS1 is translated from full-length RNA, while NS2 is translated from a variant of splice mRNA.

Influenza virus vaccines

The sequences, compositions, and methods herein relate primarily, but not solely, to the production of influenza viruses for vaccines. Historically, influenza virus vaccines have been produced primarily in embryonated chicken eggs using selected virus strains or based on empirical predictions of relevant strains. More recently, reassortant viruses incorporating selected hemagglutinin and / or neuraminidase antigens have been produced in the context of an approved attenuated, temperature-sensitive master strain. Following culture of the virus through multiple passages in chicken eggs, influenza viruses are recovered and optionally inactivated, for example, using formaldehyde and / or β-propiolactone (or alternatively used in live attenuated vaccines). Thus, it will be appreciated that the HA and NA sequences (eg, SEQ ID NO: 1-20) are quite useful in the construction of influenza vaccines. The present disclosure includes viruses / vaccines that comprise HA and / or NA sequences from pandemic influenza strains (including when the HA sequences comprise modified polybasic cleavage sites such as the modifications described herein); and including when viruses / vaccines comprise a ca backbone such as A / AA / 6/60 or the PR8 backbone.

Attempts to produce reassortant and recombinant vaccines in cell culture have been hampered by the inability of some of the approved vaccine production strains to grow efficiently under conventional cell culture conditions. However, previous work by the inventors and their collaborators provided a vector system and methods for producing reassortant and recombinant viruses in culture, thus making it possible to rapidly produce vaccines corresponding to one or many selected antigenic strains of viruses, for example, strains either A or B, various subtypes or sub-strains, etc., for example, comprising the HA and / or NA sequences herein. See multiple plasmid system for influenza virus production, US 20044029251. Typically, cultures are maintained in a system, such as a cell culture incubator, under controlled CO2 and humidity, at constant temperature using a temperature regulator, such as a thermostat to ensure that the temperature does not exceed 35 ° C. Regrouped 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 (eg, HA and / or NA antigenic variants herein). Typically, master strains are selected based on desirable properties relevant to vaccine delivery. For example, for the production of vaccines, for example, for the production of a live attenuated vaccine, the master donor virus strain can be selected for an attenuated phenotype, cold adaptation and / or temperature sensitivity. As explained elsewhere herein, and, for example, in US 20044029251, the invention uses influenza A / Ann Arbor (AA) / 6/60 strain as a "backbone" upon which to add HA and / or NA genes (eg, such as the sequences listed herein, etc.) to create desired reassortant viruses as mentioned in the claims. Thus, for example, in a 6: 2 rearrangement, 2 genes (i.e., NA and HA) would be from the influenza strain (s) against which an immunogenic reaction is desired, whereas the other 6 genes would be from the Ann Arbor strain. The Ann Arbor virus is useful for its cold-adapted, attenuated, and temperature-sensitive attributes. Of course, it will be appreciated that

ES 2 525 672 T3 HA and NA sequences herein can be reassembled with several other virus genes or virus types (eg, several different "backbones" such as PR8, etc., that contain the other influenza genes present in a reassortment, specifically, genes that are neither HA nor NA).

Various embodiments herein may comprise live attenuated vaccines as mentioned in the claims, having the HA comprising an amino acid sequence of SEQ ID No. 13 and NA sequences herein, for pandemic influenza. A problem that arises from the growth of vaccine virus strains (for example, reassorted) in eggs is that avian strains (which may be implicated in pandemics) can destroy the eggs in which the vaccines are to be produced and thus, they are difficult to handle, produce, etc. through the use of traditional regrouped production (without plasmid rescue). Such avian strains are of interest as evidence indicates that they can result in human flu and possible pandemics. Thus, the use of plasmid rescue systems to create / manipulate influenza reassorts with pandemic strains such as various avian sequences (eg, HA and NA sequences herein) are quite desirable. It will be appreciated, however, that the current sequences can also be used with non-plasmid or traditional systems.

Waterfowl (among others) can be infected with influenza A viruses of the hemagglutinin 15 (HA) and neuraminidase 9 (NA) subtypes. Such birds can serve as a reservoir from which novel influenza subtypes can enter human populations and cause pandemics. The observation that avian influenza A H7N7 viruses infected humans in the Netherlands in 2003 and that avian H5N1 and H9N2 viruses previously infected humans in Hong Kong and China raises concerns that these (and other) subtypes have the same potential to cause pandemics. Therefore, vaccines are needed to prevent human infections with avian influenza A viruses. Recently, live attenuated influenza A virus vaccines against human influenza viruses were licensed in the United States. See above. Such vaccines are reassorted H1N1 and H3N2 viruses in which the cold-adapted virus (ca) internal protein genes of A / Ann Arbor (AA) / 6/60 (H2N2) confer the cold-adapted, attenuation, and sensitive phenotypes. at AA virus temperature ca in reassorted viruses (ie, those with the hemagglutinin and neuraminidase genes of the non-Ann Arbor strain). Plasmid-based reverse genetics and classical genetic reassortment techniques have been applied to generate reassortant viruses containing the hemagglutinin and neuraminidase genes of avian influenza A virus (H4H14 subtypes) and six internal gene segments of AA virus ca. Such reassorted viruses as mentioned in the claims are characteristics of the invention. See HA and NA gene sequences below. These viruses carry biological properties that are desirable in candidate vaccines because the phenotypes associated with AA ca viruses are present in reassortant viruses. The generation and evaluation of these reassortant viruses as vaccine seed viruses are important steps in pandemic preparedness. It is contemplated that clinical trials can establish the safety, infectivity and immunogenicity of such live attenuated pandemic vaccines. Methods of construction and use of such viruses and vaccines are also included. "Pandemic virus strains" as used herein is defined as a subtype of influenza strain A virus that is not circulating in the human population, which is declared to be a pandemic strain by the Centers for Disease Control or the World Health Organization or are generally recognized as such within the scientific community.

As described herein, antigenic sequences (eg, HA sequences) as well as viruses and vaccines of 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, for example, Li et al., J. of Infectious Diseases, 179: 1132-8, 1999. Such cleavage sites, in typical embodiments herein, for example, are modified or altered in their sequences compared to the wild-type sequences from which current sequences are derived (for example, to render cleavage useless or reduce the split therein, etc.). Such modifications / alterations may be different in different strains due to the various sequences of the cleavage sites in the wild-type sequences. For example, 4 polybasic residues (RRKK) at 326-329 of mature H5 (compared to wt) are normally deleted in sequences herein. See sequence listing and Figure 1. Polybasic cleavage sites can be modified in a number of ways. For example, the polybasic cleavage site can remove one amino acid at a time (eg one R removed, two R removed, RRK removed, or RRKK removed). Additionally, the amino acid residue directly upstream of the cleavage site can also be removed or altered (eg, from an R to a T, etc.); in addition, the nucleotides encoding the amino acid residue can also be modified directly after the cleavage site. See, for example, Figure 1 for an illustration of the cleavage site modification. Furthermore, influenza virus hemagglutinin polypeptide sequences comprise amino terminal signal peptide sequences, thus hemagglutinin polypeptide sequences include both the mature form (cleaved amino terminal signal peptide) of the hemagglutinin polypeptides and the pre-cleaved form. of hemagglutinin. The cleavage sites of any hemagglutinin polypeptide sequence from any strain of influenza can be routinely measured or predicted using any of several methods in the art.

The terms "temperature sensitive", "cold adapted" and "attenuated" as applied to viruses (typically used as vaccines or for vaccine production) optionally encompassing the present sequences are well known in the art. For example, the term "temperature sensitive" (ts) indicates, for example, that the virus

ES 2 525 672 T3 has a 100-fold or more reduction in titer at 39 ° C relative to 33 ° C for influenza A strains, or the virus has a 100-fold or more reduction in titer at 37 ° C relative to 33 ° C for influenza B strains. The term "cold adapted" (ca) indicates that the virus shows growth at 25 ° C within 100 times of its growth at 33 ° C, while the term "attenuated" (att) indicates that the virus replicates in the Ferrets upper respiratory tract but is not detectable in their lung tissues, and does not cause flu-like illness in the animal. It will be understood that viruses with intermediate phenotypes, that is, viruses that show reductions in the titer of less than 100 times at 39 ° C (for virus of strain A) or 37 ° C (for virus of strain B), or that present growth at 25 ° C which is greater than 100 times its growth at 33 ° C (for example, within 200 times, 500 times,

1000 times, 10,000 times less), and / or have reduced growth in the lungs relative to growth in the ferret upper respiratory tract (i.e. partially attenuated) and / or flu-like illness in the animal, are also useful viruses and can be used in conjunction with the HA and NA sequences herein.

Again, the HA and NA sequences described herein are used in such plasmid reassortant vaccines (and / or other ts, cs, ca and / or att viruses and vaccines).

Flumist ™

As mentioned above, there are numerous examples and types of influenza vaccines. An example influenza vaccine is FluMist ™ 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 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).

FluMist ™ vaccine strains contain, for example, HA and NA gene segments derived from the strains (eg wild-type strains) targeted by the vaccine along with six gene segments, PB1, PB2, PA, NP, M and NS, from a common master donor virus (MDV). The MDV for FluMist ™ Influenza A Strains (MDVA) was created by serial passage of the wild-type A / Ann Arbor / 6/60 (A / AA / 6/60) strain in chicken kidney tissue culture primary 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 ts phenotype is believed to contribute to the attenuation of the vaccine in humans by restricting its replication in all but the coldest regions of the respiratory system. 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 MDVA is not reversed upon passage through infected hamsters or in isolates extracted from children (for a recent review, see Murphy & Coelingh (2002) Principles underlying the development and use of live attenuated coldadapted influenza A and B virus vaccines Viral Immunol 15: 295-323).

Clinical studies in more than 20,000 adults and children involving 12 separate 6: 2 reassortant strains have shown that these vaccines are attenuated, safe and effective (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, attenuated intranasal influenza virus vaccine in healthy, working adults: a randomized controlled trial JAMA 282: 137-44). The reassortants carrying the six internal MDV-A genes and the two HA and NA gene segments of a wild-type virus (i.e., a 6: 2 reassortment) consistently maintain the ca, ts, and att phenotypes ( Maassab et al. (1982) Evaluation of a cold-recombinant influenza virus vaccine in ferrets J. Infect. Dis. 146: 780-900).

The production of such reassorted viruses using influenza B strains is more difficult, however recent work (see, for example, Multiple plasmid system for the production of Influenza virus, US 20044029251) has shown an eight plasmid system for the generation of influenza B virus completely from cloned cDNA. Methods for the production of live attenuated influenza A and B viruses suitable for vaccine formulations, such as live virus vaccine formulations useful for intranasal administration, were also shown.

The system and methods described above are useful for the rapid production in cell culture of recombinant and reassorted 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 (eg, SEQ ID No. 1-10 and the corresponding amino acids in SEQ ID No. 11-20), methods, etc. described herein are optionally used in conjunction with, or in combination with, such prior work involving, for example, reassorted influenza viruses for the production of vaccines to produce virus for vaccines.

ES 2 525 672 T3

Methods and compositions for the prophylactic administration of vaccines

The recombinant and reassortant viruses described herein (for example, those comprising the polynucleotides of SEQ ID N ° 1-10 or the polypeptides of SEQ ID N ° 11-20, or fragments thereof), in particular those cited in the claims, they can be administered prophylactically in an immunologically effective amount and in an appropriate vehicle or excipient to stimulate an immune response specific to one or more influenza virus strain (s) as determined by the HA and / or NA sequence. Typically, the carrier or excipient is a pharmaceutically acceptable carrier or excipient, such as sterile water, aqueous saline solution, aqueous buffered saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, ethanol, or combinations thereof. The preparation of such solutions that guarantee sterility, pH, isotonicity and stability is carried out according to protocols established in the art. Generally, a vehicle or excipient is selected to minimize allergic and other unwanted effects, and to suit the particular route of administration, eg, subcutaneous, intramuscular, intranasal, etc.

A related aspect of the invention provides the regrouped influenza virus as mentioned in the claims for use in methods of 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 (eg, comprising an HA and / or NA molecule as described herein) is administered to the individual in a physiologically acceptable vehicle.

Generally, the influenza viruses of the invention are administered in an amount sufficient to stimulate an immune response specific to one or more strain (s) of influenza virus (i.e., against the HA and / or NA strains described herein). . Preferably, the administration of influenza viruses elicits a protective immune response against such strains. Dosages and methods of eliciting a protective immune response against one or more strain (s) of influenza are known to those of skill in the art. See, for example, US Pat. No. 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 HID50 (human infectious dose), that is, about 105-108 pfu (plaque-forming units) per administered dose. Typically, the dose will be adjusted within this range based, for example, on age, physical condition, body weight, sex, diet, timing of administration, and other clinical factors. The prophylactic vaccine formulation is administered systemically, for example, by subcutaneous or intramuscular injection using a needle and syringe, or a needleless injection device. Alternatively, the vaccine formulation is administered intranasally, by either droplet, large particle aerosol (greater than about 10 microns), or upper respiratory tract spray. Although any of the above routes of administration results in a protective systemic immune response, intranasal administration confers the added benefit of eliciting mucosal immunity at the site of influenza virus entry. For intranasal administration, live attenuated virus vaccines are often preferred, for example, a recombinant or regrouped attenuated, cold adapted and / or temperature sensitive influenza virus. See above. Although stimulation of a protective immune response with a single dose is preferred, additional dosages may be administered, by the same or different route, to achieve the desired prophylactic effect.

Typically, the attenuated recombinant influenza virus of the present invention as used in a vaccine is sufficiently attenuated so that no symptoms of infection, or at least symptoms of severe infection, will occur in most immunized (or infected with) individuals. otherwise) with attenuated influenza virus. In some cases, the attenuated influenza virus can still produce symptoms of mild illness (eg, mild upper respiratory illness) and / or spread to unvaccinated individuals. However, its virulence is sufficiently suppressed that severe lower respiratory tract infections do not occur in the vaccinated or accidental host.

Alternatively, an immune response can be stimulated by targeting ex vivo or in vivo dendritic cells with influenza viruses comprising the sequences herein. For example, proliferating dendritic cells are exposed to viruses in a sufficient quantity and for a sufficient period of time to allow the capture of influenza antigens by the dendritic cells. The cells are then transferred to a subject to be vaccinated by conventional intravenous transplantation methods.

Although stimulation of a protective immune response with a single dose is preferred, additional dosages can be administered, by the same or a 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 may 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 severe influenza infection, such as, for example, healthcare workers, daycare 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. Induced immunity levels can be monitored, for example, by measuring the amounts of serum and secretory antibodies.

ES 2 525 672 T3 neutralizers, and dosages adjusted or vaccinations repeated as necessary to elicit and maintain desired levels of protection.

Optionally, the formulation for prophylactic administration of influenza viruses also contains one or more adjuvants to enhance the immune response against 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, Bacillus Calmette-Guerin (BCG ), Corynebacterium parvum and the synthetic adjuvant QS-21.

If desired, administration of prophylactic influenza virus vaccine can be performed in conjunction with administration of one or more immunostimulatory molecules. Immunostimulatory molecules include various cytokines, lymphokines, and chemokines with immunostimulatory, immunopotentiating, and pro-inflammatory activities, such as interleukins (eg, IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors (eg, granulocyte-macrophage (GM) colony stimulating factor (CSF)); and other immunostimulatory molecules, such as such as macrophage inflammatory factor, Flt3 ligand, B7.1; B7.2, etc. Immunostimulatory molecules can be administered in the same formulation as influenza viruses, or they can be administered separately.

Although vaccination of an individual with an attenuated influenza virus of a particular strain of a particular subgroup can induce cross protection against influenza viruses of different strains and / or subgroups, cross protection can be enhanced, if desired, by vaccinating the individual with viruses. attenuated influenza strains of at least two strains, for example, each representing a different subgroup. Additionally , vaccine combinations may optionally include pandemic vaccine mixtures (eg, those against pandemic influenza strains such as various avian strains, see, eg, sequences herein, or other pandemic strains) and non-pandemic strains. pandemics. Vaccine mixtures (or multiple vaccinations) may comprise components of human strains and / or non-human influenza strains (eg, avian and human, etc.). Similarly, the attenuated influenza virus vaccines of the present invention can optionally be combined with vaccines that induce protective immune responses against other infectious agents.

Polynucleotides

It is well known in the art that influenza virus HA and NA polynucleotide segments comprise both a coding region (encoding the ORF) and non-coding regions (NCR), both 5 'and 3' of the sequence that codes for HA and NA. Examples of these NCRs are shown in SEQ ID NO: 1-9 (outside of ORFs). It is also known that primers can be prepared for these NCRs to facilitate amplification of the entire HA and NA segments of influenza viruses. (See, eg, Hoffmann et al. Arch Virol. 2001 Dec; 146 (12): 2275-89). In addition, influenza HA and NA CRNs are known to increase the efficacy of reassembling. Therefore, the polynucleotide sequences of these NCRs are described herein. When the HA and NA segments of either pandemic strain are amplified, polynucleotide primers could be prepared and used to bind to conserved regions (eg, between related strains) of the HA and NA NCRs for amplification (eg, by RT -PCR).

The HA and NA polynucleotides of the virus of the invention, for example SEQ ID No. 3 or SEQ ID No. 4, are optionally used in several different capacities alternatively to, or in addition to, the vaccines described above. Other exemplary uses are described herein for illustrative purposes and not as limitations on the current range of uses. Different methods of construction, purification and characterization of the nucleotide sequences of the disclosure are also described herein. It is described herein that nucleic acids including one or more polynucleotide sequence (s) described herein are favorably used as probes for the detection of corresponding or related nucleic acids in a variety of contexts, such as in nucleic acid hybridization experiments, e.g., to find and / or characterize homologous influenza variants (e.g., homologous to the sequences herein, etc.) that infect other species or in different flu outbreaks, etc. The probes can be either DNA or RNA molecules, such as genomic or cloned DNA restriction fragments, cDNAs, PCR amplification products, transcripts, and oligonucleotides, and can range in length from oligonucleotides as short as about 10 nucleotides of length to full length sequences or cDNA in excess of 1 kb or more. For example, a probe includes a polynucleotide sequence or subsequence selected, for example, from SEQ ID No. 3 or SEQ ID No. 4, or sequences complementary thereto. Alternatively, polynucleotide sequences that are variants of one of the sequences designated above 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, in which a polynucleotide sequence corresponds identically to a first variant or / and the (s ) other (s) correspond identically to additional variants. Such pairs of oligonucleotide probes are particularly useful, for example, for specific hybridization experiments to detect polymorphic nucleotides or, for example,

ES 2 525 672 T3 example, to detect variants of influenza HA and NA homologous, for example, homologous to current HA and NA sequences, that infect other species or are present in different influenza outbreaks (for example, or temporarily and / or geographically different). In other applications, probes are selected that are more divergent, that is, probes are selected 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.

The probes described herein for example, as exemplified by sequences derived from the sequences herein, may also be used to identify additional useful polynucleotide sequences according to routine procedures in the art. One or more probes, as described above, are used to screen libraries of expression products or chromosomal segments (eg, expression libraries or genomic libraries) to identify clones that include sequences identical to, or with significant sequence similarity to , for example, one or more probe (s) of the sequences herein, ie, variants, homologues, etc. It will be understood that in addition to physical methods such as library screening, computer-aided bioinformatics approaches, eg, BLAST and other sequence homology search algorithms, and the like, can also be used to identify related polynucleotide sequences.

Oligonucleotide probes are optionally produced by a variety of methods well known to those of skill in the art. Most commonly, 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, for example, using an automated synthesizer, or as described in Needham-Van Devanter et al. (1984) Nucl Acids Res, 12: 6159-6168. Custom oligonucleotides can also be prepared and ordered from a variety of commercial sources known to those of skill. Oligonucleotide purification, when necessary, is typically performed by either native acrylamide gel electrophoresis or anion exchange HPLS 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 oligonucleotides can also be readily ordered from a variety of commercial sources known to those of skill.

In other circumstances, for example, relating to attributes of cells or organisms that express the polynucleotides and polypeptides described herein of the invention (for example, those harboring viruses comprising the sequences described herein), they favorably use probes that are polypeptides, peptides or antibodies. For example, isolated or recombinant polypeptides, polypeptide fragments, and peptides derived from any of the amino acid sequences described herein (eg, SEQ ID NO: 13-14) and / or encoded by described polynucleotide sequences are favorably used. herein, for example, selected from SEQ ID No. 1 to SEQ ID No. 10, to identify and isolate antibodies, for example, from phage display libraries, combinatorial libraries, polyclonal sera, and the like. Polypeptide fragments include a peptide or polypeptide that comprises 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 acid residues, or at least 70 contiguous amino acid residues, or at least 80 contiguous amino acid residues, or at least 90 contiguous amino acid residues, or at least 100 contiguous amino acid residues, or at least 125 contiguous amino acid residues, or at least 150 residues contiguous amino acid residues, or at least 175 contiguous amino acid residues, or at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues, or at least 350 contiguous amino acid residues, or at least 400 contiguous, or at least 450 contiguous, or at least 500 contiguous, or at least 550 contiguous of the amino acid sequence of an HA or NA polypeptide described herein (e.g., SEQ ID NO: 11- twenty). Also described herein are polynucleotides that encode said polypeptide fragments and antibodies that specifically bind to said polypeptides.

Antibodies specific to any polypeptide sequence or subsequence, for example, from SEQ ID No. 11 to SEQ ID No. 20, and / or encoded by polynucleotide sequences described herein, for example, selected from SEQ ID No. 1 to SEQ ID NO: 10, they are also valuable as probes for evaluating expression products, for example, from cells or tissues. Furthermore, antibodies are particularly suitable for evaluating the expression of proteins that comprise amino acid subsequences, for example, those given herein, or encoded by polynucleotide sequences described herein, for example, selected from the shown herein, in situ, in a tissue alignment, in a cell, tissue, or organism, for example, an organism infected by an unidentified influenza virus or similar. Antibodies can be directly labeled with a detectable reagent, or indirectly detected by labeling a secondary antibody specific for the heavy chain constant region (i.e., isotype) of the

ES 2 525 672 T3 specific antibody). Additional details regarding the production of specific antibodies are provided below.

Diagnostic tests

The nucleic acid sequences described herein can be used in diagnostic assays to detect influenza (and / or hemagglutinin and / or neuraminidase) in a sample, to detect hemagglutinin-like and / or neuraminidase-like sequences, and to detect differences. of strains in influenza clinical isolates using either chemically synthesized or recombinant polynucleotide fragments, eg, selected from the sequences herein. For example, fragments of the hemagglutinin and / or neuraminidase sequences comprising at least 10-20 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 samples.

The probes described herein, for example, as exemplified by unique subsequences selected from those provided herein, can also be used to identify additional useful polynucleotide sequences (such as to characterize additional influenza strains ) according to routine procedures in the art. One or more probes, as described above, are used to screen libraries of cloned viral nucleic acids or expression products (i.e., expression libraries or genomic libraries) to identify clones that include sequences identical to, or with sequence identity. significant with the sequences herein. In turn, each of these identified sequences can be used to prepare probes, including variant probe pairs or sets as described above. It will be understood that in addition to such physical methods such as library screening, computer aided bioinformatics approaches, eg, BLAST and other sequence homology search algorithms, and the like, can also be used to identify related polynucleotide sequences.

The probes described herein are particularly useful for detecting the presence and for determining the identity of influenza nucleic acids in cells, tissues, or other biological samples (eg, a nasal wash or bronchial wash). For example, the probes described herein are favorably used to determine whether a biological sample, such as a subject (eg, a human subject) or model system (such as a cultured cell sample) has been exposed to, or is you have infected with influenza, or particular strain (s) of influenza. Detection of hybridization of the selected probe with nucleic acids originating from (eg, 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 polynucleotide is selected. probe.

It will be appreciated that the design of the probe is influenced by the intended application. For example, when multiple allele-specific probe-target interactions are to be detected in a single assay, eg, on a single DNA chip, it is desirable to have similar melting temperatures for all probes. Accordingly, the lengths of the probes are adjusted so that the melting temperatures for all the probes in the array are closely similar (it will be appreciated that different lengths may be needed for different probes to achieve a particular Tm when different probes have different contents in GC). Although melting temperature is a primary consideration in probe design, other factors are optionally used to further fine-tune probe construction, such as selecting against primer self-complementarity and the like.

Vectors, promoters, and expression systems

Recombinant constructs incorporating one or more of the nucleic acid sequences described herein are also described herein. Such constructs optionally include a vector, eg, a plasmid, a cosmid, a phage, a virus, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), etc., in which one or more of the Polynucleotide sequences of SEQ ID No. 3 or for example to SEQ ID No. 4, or a subsequence thereof, etc., have been inserted, in a forward or reverse orientation. For example, the inserted nucleic acid can include a viral chromosomal sequence or cDNA that includes all or part of at least one of the polynucleotide sequences described herein. In one aspect, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable promoters and vectors are known to those of skill in the art and are commercially available.

The polynucleotides described herein can be included in any one of a variety of vectors suitable for generating sense or antisense RNA, and optionally, polypeptide (or peptide) expression products (eg, such a hemagglutinin and / or neuraminidase molecule as described herein). Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, eg, 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, fowlpox virus, pseudorabies, adenovirus, adeno-associated virus, retrovirus, and many others (eg, pCDL). Can

ES 2 525 672 T3 use any vector that can introduce genetic material into a cell and, if replication is desired, that can replicate in the relevant host.

In an expression vector, the HA and / or NA polynucleotide sequence of interest is physically arranged in proximity and orientation with an appropriate transcriptional control sequence (e.g., promoter, and optionally one or more enhancers) to direct the mRNA synthesis. That is, the polynucleotide sequence of interest is operably linked to an appropriate transcriptional control sequence. Examples of such promoters include: SV40 or LTR promoter, E. coli lac or trp promoter, lambda phage PL promoter, and other promoters known to control gene expression in prokaryotic or eukaryotic cells and their viruses.

A variety of promoters are suitable for use in expression vectors to regulate the transcription of influenza virus genome segments. In certain embodiments, the cytomegalovirus (CMV) DNA-dependent RNA polymerase II (Pol II) promoter is used. If desired, for example, to regulate conditional expression, other promoters that induce RNA transcription under specified conditions, or in specified cells or tissues, can be substituted. Numerous mammalian and viral promoters are available, eg, human, or can be isolated depending on the specific application contemplated. For example, alternative promoters derived from the genomes of animal and human viruses include promoters such as 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.

Transcription is optionally increased by including an enhancer sequence. Enhancers are typically normally short cis-acting DNA elements, eg, 10-500 bp, 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 viruses from eukaryotic cells. The enhancer can be cut and spliced ​​into the vector 5 'or 3' to the heterologous coding sequence, but is typically inserted 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 may also contain a ribosome binding site or an internal ribosome entry site (IRES) for the initiation of translation.

The vectors described herein 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 viral or eukaryotic DNA or cDNAs. The SV40 polyadenylation signal sequences can provide a bidirectional polyadenylation site that protects the transcription of strand (+) mRNA molecules from replication initiated by the Poly promoter of the (-) strand viral genome.

Furthermore, 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 the genes listed above, markers such as dihydrofolate reductase or neomycin resistance are suitable for selection in eukaryotic cell culture.

The vector containing the appropriate nucleic acid sequences as described above, as well as an appropriate promoter or control sequence, can be used to transform a host cell allowing expression of the protein. Although the vectors described herein can replicate in bacterial cells, it will most often be desirable to introduce them into mammalian cells, eg, Vero cells, BHK cells, MDCK cells, 293 cells, COS cells, or the like, for the purpose of expression.

As described elsewhere, the HA and NA sequences herein may be comprised within plasmids involved in plasmid rescue reassortment. See, for example, US 2004029251 and US 2005042229. For example, preferred expression vectors described herein include, but are not limited to, vectors comprising the pol I promoter and terminator sequences or vectors using both the pol I and pol II promoters, "the system "polI / polII promoters" (eg, Zobel et al., Nucl. Acids Res. 1993, 21: 3607; US 20020164770; Neumann et al., Proc. Natl. Acad. Sci. USA 1999, 96: 9345 ; Fodor et al., J. Virol. 1999, 73: 9679; and US 20030035814). The rearrangements produced include the HA and NA genes arranged with the other 6 influenza genes of the donor strain A / Ann Arbor / 6/60. Other backbone strains are described, for example, in US 20040137013 and US 0030147916.

Additional expression elements

Most frequently, the genome segment encoding the HA and / or NA protein of influenza viruses includes

ES 2 525 672 T3 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, may be employed, eg, an HA and / or NA protein. Again, in such a case, it is often desirable to include specific initiation signals that aid in efficient translation of the heterologous coding sequence. These signals can include, for example, the ATG start codon and adjacent sequences. To ensure translation of the entire insert, the start 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 cellular system in use.

If desired, polynucleotide sequences encoding additional expressed elements, such as signal sequences, secretory or localization sequences, and the like, can be incorporated into the vector, usually in frame with the polynucleotide sequence of interest, for example, to select as targets the expression of the polypeptide in a desired cell organelle, membrane, or compartment, or to direct the secretion of the polypeptide to the periplasmic space or cell culture medium. Such sequences are known to those of skill, and include secretory leader peptides, organelle targeting sequences (e.g., nuclear localization sequences, ER retention signals, mitochondrial transit sequences), membrane anchoring / localization sequences ( eg, termination transfer sequences, GPI anchor sequences), and the like.

When translation of a polypeptide encoded by a nucleic acid sequence described herein is desired, additional specific translation initiation signals can improve translation efficiency. These signals can include, for example, an ATG start codon and adjacent sequences, an IRES region, etc. In some cases, for example, full-length cDNA molecules or chromosomal segments are inserted that include a coding sequence that incorporates, for example, a polynucleotide sequence as described herein, a translation initiation codon, and associated sequence elements, in the appropriate expression vector simultaneously with the polynucleotide sequence of interest. In such cases, additional translational control signals are often not required. However, in cases where only a sequence encoding the polypeptide, or a portion thereof, is inserted, exogenous translational control signals, including, for example, an ATG start codon, are often provided to the expression of the relevant sequence. The start 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. Efficiency of expression can be enhanced by the inclusion of enhancers appropriate to the cellular system in use (see, for example, ScharfD. Et al. (1994) Results Probl Cell Differ 20: 125-62; Bittner et al. (1987) Methods in Enzymol 153: 516-544).

Recombinant virus production

Negative strand RNA viruses can be engineered and recovered using a recombinant reverse genetics approach (see, for example, US Patent No. 5,166,057 issued to Palese et al.). Such a 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 unsegmented negative-strand RNA viruses, for example rabies (Schnell et al. (1994) EMBO J. 13: 4195-4203) ; VSV (Lawson et al. (1995) Proc. Natl. Acad. Sci. USA 92: 4477-4481); mumps 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: 32723277; Dubin et al. (1997) Virology 235: 323-332); SV5 (He et al. (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 skilled in the art will be familiar with these and other similar techniques for producing influenza viruses comprising the HA and NA sequences described herein. Recombinant influenza viruses produced according to such methods are a feature of the disclosure, as recombinant influenza viruses comprising one or more nucleic acids and / or polypeptides described herein.

Cell culture and expression hosts

Also described herein are host cells that are introduced (transduced, transformed, or transfected) with vectors described herein, and the production of polypeptides described herein by recombinant techniques. Host cells are engineered (ie, transduced, transformed, or transfected) with a vector, such as an expression vector. 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.

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Most frequently, mammalian cells are used to grow the HA and NA molecules described herein. Suitable host cells for influenza virus replication include, for example, Vero cells, BHK cells, MDCK cells, 293 cells, and COS cells, including 293T cells, COS7 cells, or the like. Commonly, cocultures including two of the above cell lines, eg, MDCK cells and either 293T cells or COS at a ratio of, eg, 1: 1, are employed to improve replication efficiency. Typically, cells are grown in a conventional 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 of CO2 adequate to maintain neutral buffered pH (eg at pH 7.0 to 7.2). Optionally, the medium contains antibiotics to prevent bacterial growth, for example, penicillin, streptomycin, etc., and / or additional nutrients, such as L-glutamine, sodium pyruvate, non-essential amino acids, additional supplements to promote favorable growth characteristics , for example, trypsin, β-mercaptoethanol, and the like.

The engineered host cells can be cultured in conventional nutrient media modified as appropriate to activate promoters, select for transformants, or amplify inserted polynucleotide sequences. Culture conditions, such as temperature, pH, and the like, are normally those used above with the particular host cell selected for expression, and will be apparent to those skilled in the art and from the literature referenced herein, including, for example, For example, Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, 3^to edition, Wiley-Liss, New York and the bibliography mentioned therein. Other useful literature includes, for example, Paul (1975) Cell and Tissue Culture, 5th ed., Livingston, Edinburgh; Adams (1980) Laboratory Techniques in Biochemistry and Molecular Biology-Cell Culture for Biochemists, Work and Burdon (eds.) Elsevier, Amsterdam. Additional details regarding tissue culture procedures of particular interest in in vitro influenza virus production include, for example, Merten et al. (1996) Production of influenza virus in cell cultures for vaccine preparation. In Cohen and Shafferman (eds.) Novel Strategies in Design and Production of Vaccines. Additionally, variations in such procedures adapted to the present disclosure are readily determined through routine experimentation and will be familiar to those skilled in the art.

Cells for the production of influenza viruses (eg, having the HA and / or NA sequences of the disclosure) can be cultured in serum-free or serum-containing medium. In some cases, for example, for the preparation of purified viruses, it is normally desirable to grow the host cells under serum-free conditions. Cells can be cultured on a small scale, for example, less than 25 ml of medium, culture tubes or flasks or in large shake flasks, in roller bottles, or in microcarrier beads (for example, DEAE-dextran microcarrier beads, such such as Dormacell, Pfeifer & Langen; Superbead, Flow Laboratories; styrene-trimethylamine copolymer beads, such as Hillex, SoloHill, Ann Arbor) in flasks, flasks, 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 cell culture volume. For example, a single liter of medium can include more than 20 million microcarrier beads providing more than 8000 square centimeters of growth surface. For commercial virus production, for example vaccine production, it is often desirable to grow the cells in a bioreactor or fermenter. Bioreactors are available in volumes from less than 1 liter to in excess of 100 liters, eg, 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).

Regardless of culture volume, in many desired aspects of the present disclosure, it is important that cultures are maintained at an appropriate temperature, to ensure efficient recovery of recombinant and / or reassortant influenza viruses using temperature-dependent multiple plasmid systems (see , for example, Multiple plasmid system for influenza virus production, US 2004029251), heating virus solutions for filtration, etc. Typically, a regulator, for example, a thermostat, or other device is employed to detect and maintain the temperature of the cell culture system and / or other solution, to ensure that the temperature is at the correct level for the appropriate period (for example, virus replication, etc.).

In the description herein (eg, when reassorted viruses are to be produced from segments in vectors), vectors comprising influenza genome segments are introduced (eg, transfected) into host cells according to methods well known in the art. technique for introducing heterologous nucleic acids into eukaryotic cells, including, for example, calcium phosphate co-precipitation, electroporation, microinjection, lipofection and transfection using polyamine transfection reagents. For example, vectors, eg, 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 TransITLT1 polyamine transfection reagent (Mirus) according to the manufacturer's instructions with in order to produce regrouped viruses, etc. Thus, in one example, about 1 gg of each vector is introduced into a host cell population with about 2 gl of TransIT-LT1 diluted in 160 gl of medium, preferably serum-free medium, in a total volume of 200 gl. The DNA: transfection reagent mixtures are incubated at room temperature for

ES 2 525 672 T3 minutes followed by the addition of 800 gl of medium. The transfection mixture is added to the host cells, and the cells are cultured as described by other methods well known to those of skill in the art. Therefore, 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) are mixed with approximately 20 gl of TransIT-LT1 and transfected into host cells. Optionally, serum-containing medium is replaced prior to transfection with serum-free medium, eg Opti-MEM I, and incubated for 4-6 hours.

Alternatively, electroporation can be employed to introduce such vectors that incorporate 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 short, they are resuspended approximately 5 x 10⁶ Vero cells, for example, grown in Modified Eagle's Medium (MEM) supplemented with 10% Fetal Bovine Serum (FBS), in 0.4 ml of OptiMEM and placed in an electroporation cuvette. Twenty micrograms of DNA in a volume of up to 25 gl are added to the cells in the cuvette, which are then mixed gently by tapping. Electroporation is performed according to the manufacturer's instructions (eg BioRad Gene Pulser II with Capacitance Extender Plus attached) at 300 volts, 950 microfarads with a time constant between 28-33 ms. The cells are remixed by gently tapping and approximately 1-2 minutes after 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 plate 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. The final volume is approximately 3.5 ml. The cells are then incubated under conditions permissive for viral growth, eg, at about 33 ° C for cold adapted strains.

In mammalian host cells, various expression systems can be used, such as virus-based systems. In cases where an adenovirus is used as the expression vector, a coding sequence is optionally ligated into an adenovirus transcription / translation complex consisting of the late promoter and the tripartite leader sequence. Insertion into a nonessential E1 or E3 region of the viral genome will result in a viable virus that can express the polypeptides of interest in infected host cells (Logan and Shenk (1984) Proc Natl 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.

A host cell strain is optionally chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein as desired. Such protein modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing of the protein, which cleaves a precursor form to a mature form, is sometimes important to correct insertion, folding, and / or function. Additionally, the proper location within a host cell (eg, on the surface of the cell) is also important. Different host cells such as COS, CHO, BHK, MDCK, 293, 293T, COS7, etc. they have specific cellular machinery and characteristic mechanisms for such post-translational activities and can be chosen to ensure correct processing and modification of the present introduced foreign protein.

For the long-term, high-throughput production of recombinant proteins encoded by, or having subsequences encoded by, the polynucleotides described herein, stable expression systems are optionally used. For example, cell lines, stably expressing a polypeptide described herein, are transfected using expression vectors that contain viral origins of replication or endogenous expression elements and a selectable marker gene. For example, after vector introduction, cells are allowed to grow for 1-2 days in enriched medium before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows the growth and recovery of cells that successfully express the introduced sequences. Thus, resistant clusters of stably transformed cells, for example derived from a single cell type, can proliferate using tissue culture techniques appropriate for the cell type.

Host cells transformed with a nucleotide sequence encoding a polypeptide described herein are optionally cultured under conditions suitable for expression and recovery of the encoded protein from cell culture. 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 (for example, depending on fusion proteins that encode a membrane retention signal or the like) and / or the vector used.

Expression products corresponding to the nucleic acids described herein 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 cited below, 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.

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In bacterial systems, various expression vectors can be selected depending on the intended use for the expressed product. For example, when large amounts of a polypeptide or fragments thereof are needed for the production of antibodies, vectors that direct the high-level expression of fusion proteins that are easily purified are favorably employed. Such vectors include, but are not limited to, E. coli such as BLUESCRIPT (Stratagene), in which the coding sequence of interest, eg, sequences comprising those found herein, etc., can be ligated into the vector in-frame with sequences for amino terminal translation that are starts with methionine and the subsequent 7 beta-galactosidase residues that produce 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, various vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH can be used for the production of the desired expression products. For reviews, see Ausubel, cited below, and Grant et al., (1987); Methods in Enzymology 153: 516-544.

Nucleic acid hybridization

Comparative hybridization can be used to identify nucleic acids (eg, SEQ ID NO: 3-4) including conservative variations of nucleic acids described herein. This comparative hybridization method is a preferred method of distinguishing nucleic acids. Furthermore, target nucleic acids that hybridize to the nucleic acids represented by those shown herein under conditions of high, ultra-high, and ultra-ultra-high stringency are features of the disclosure. Examples of such nucleic acids include those with one or a few silent or conservative nucleic acid substitutions compared to a given nucleic acid sequence.

A test target nucleic acid is said to hybridize specifically to a probe nucleic acid when it hybridizes at least half as well to the probe as it does to the perfectly matched complementary target, i.e. with a signal-to-noise ratio at least half as high as probe-target hybridization under conditions where 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 with any of the unpaired nucleic acids.

Nucleic acids "hybridize" when they associate, usually in solution. Nucleic acids hybridize due to a variety of well-characterized physicochemical 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 nucleic acid hybridization 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 Ausubel, Sambrook, and Berger and Kimmel, all cited below. Hames and Higgins (1995) Gene Probes 1 IRL Press at Oxford University Press, Oxford, England, (Harnes and Higgins 1) and Hames and Higgins (1995) Gene Probes 2 IRL Press at Oxford University Press, Oxford, England (Harnes and Higgins 2) provide details on the synthesis, labeling, detection and quantification of DNA and RNA, including oligonucleotides.

An example of stringent hybridization conditions for the hybridization of complementary nucleic acids that 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, taking carry out X hybridization overnight. An example of stringent wash conditions comprises a wash with 0.2x SSC at 65 ° C for 15 minutes (see, Sambrook, cited below, for a description of the SSC buffer and other nucleic acid hybridization parameters). The high stringency wash is often preceded by a low stringency wash to remove the background probe signal. An example of a low stringency wash is 2x SSC at 40 ° C for 15 minutes. In general, a signal-to-noise ratio of 5x (or greater) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.

After hybridization, unhybridized nucleic acids can be removed by a series of washes, the stringency of which can be adjusted depending on the desired results. Low stringency wash conditions (eg, using higher salt concentration and lower temperature) increase sensitivity, but can produce non-specific hybridization signals and high background signals. Higher stringency conditions (eg, using lower salt concentration and higher temperature that is closer to the Tm) lower the background signal, usually with the specific signal remaining specifically. See, also, Rapley, R. and Walker, JM eds., Molecular Biomethods Handbook (Humana Press, Inc. 1998), "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 nucleic acid hybridization is found in Tijssen (1993), cited above, and in Hames and Higgins, 1 and 2. Stringent washing and hybridization conditions can easily be determined empirically for any test nucleic acid. For example, when determining highly stringent wash and hybridization conditions, the wash and hybridization conditions are gradually increased (by

ES 2 525 672 T3 example, increasing the temperature, decreasing the salt concentration, increasing the detergent concentration and / or increasing the concentration of organic solvents such as formalin in hybridization and washing), until a selected set of criteria. For example, the washing and hybridization 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 probe hybridization to a non-target. paired.

In general, a signal-to-noise ratio of at least 2x (or greater, for example, at least 5x, 10x, 20x, 50x, 100x, or more) 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 disclosure indicates relatively strong structural similarity to, for example, the nucleic acids provided in the sequence listings herein.

"Very stringent" conditions are selected to be equal to the thermal melting point (T_m) for a particular probe. The Tm is the temperature (at defined pH and ionic strength) at which 50% of the hybrid test sequence with a perfectly matched probe. For the purposes of the present disclosure, generally, "highly stringent" hybridization and wash conditions are selected to be approximately 5 ° C lower than the Tm for the specific sequence at a defined pH and ionic strength (as noted below , can also refer to highly stringent conditions in comparative terms). Target sequences that are closely related to or identical to the nucleotide sequence of interest (eg, "probe") can be identified under stringent or highly stringent conditions. Lower stringency conditions are appropriate for sequences that are less complementary.

"Ultra-high stringency" hybridization and wash conditions are those in which the stringency of the hybridization and wash conditions are increased until the signal-to-noise ratio for binding a probe to a complementary target nucleic acid is at least 10x so as high as that observed for hybridization to any unpaired target nucleic acid. A target nucleic acid that hybridizes to a probe under such conditions, with a signal-to-noise ratio of at least half that of the perfectly matched complementary target nucleic acid is said to bind to the probe under ultra-high stringency conditions.

When determining stringent or highly stringent hybridization and wash conditions (or even more stringent hybridization), the hybridization and wash conditions are gradually increased (e.g., increasing the temperature, decreasing the salt concentration, increasing the detergent concentration and / or increasing the concentration of organic solvents, such as formamide, in hybridization or washing), until a selected set of criteria is met. For example, hybridization and wash conditions are gradually increased until a probe comprising one or more polynucleotide sequences of the disclosure, eg, unique sequences or subsequences selected from those provided herein (eg, SEQ ID NO. 3-4) and / or complementary polynucleotide sequences, bind to a perfectly matched complementary target (again, a nucleic acid comprising one or more nucleic acid sequences or subsequences selected from those provided 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 probe hybridization to an unpaired 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 submission, and / or complementary polynucleotide sequences thereof), as desired.

Using the polynucleotides described herein, or subsequences thereof, novel target nucleic acids can be obtained. 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 SEQ ID NO: 3-4).

Similarly, even higher stringency levels 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 the hybridization and wash conditions are increased 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 higher than that observed for hybridization to any unpaired target nucleic acid. The particular signal will depend on the marker used in the relevant assay, eg, a fluorescent marker, a colorimetric marker, a radioactive marker, or the like. A target nucleic acid that hybridizes to a probe under such conditions, with a signal-to-noise ratio of at least half that of the perfectly matched complementary target nucleic acid, is said to bind to the probe under ultra-ultra-high stringency conditions.

Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy allowed by the genetic code.

Cloning, mutagenesis and expression of biomolecules of interest

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General texts describing molecular biology 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 (3^to Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2000 (“Sambrook”) and Current Protocols in Molecular Biology, FM 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, for example, the generation of HA and / or NA molecules, etc.

Various types of mutagenesis are also described herein, e.g., to produce and / or isolate, e.g., novel or freshly isolated HA and / or NA molecules and / or to further modify / mutate polypeptides (e.g., HA and / or NA molecules as in SEQ ID No. 13-14) described herein. They include, but are not limited to, random, site-directed point mutagenesis, homologous recombination (DNA swapping), mutagenesis using uracil-containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using incomplete double-stranded DNA, or the like. . Additional suitable methods include point mismatch repair, mutagenesis using repair-deficient host chains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, double chain break repair, and the like. Also described herein is mutagenesis, eg, involving chimeric constructs. Mutagenesis can be guided by known information from the naturally occurring molecule or the altered or mutated naturally occurring molecule, eg, by comparisons of sequence, physical properties, crystal structure, or the like.

The above texts and examples found herein describe these procedures, as do the following publications (and bibliography cited within them): Sieber, et al., Nature Biotechnology, 19: 456460 (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: 369374 (1996); 1. A. Lorimer, I. Pastan, Nucleic Acid Res 23, 3067-8 (1995); WPC 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 α-subunit of bovine rod outer segment guanine nucleotide-binding protein (transducin), Nucl Acids Res 14: 6361-6372 (1988); Sayers et al., YT 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 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, DMJ 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-siranded 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 delections, Nucl Acids Res 14: 5115 (1986); Mandecki, Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli: a method for site-specific mutagenesis, Proc Natl 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, Science 229: 1193-1201 (1985); 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: 8765-8787 (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: 9441-9456 (1984); Kramer et al., Point Mismatch Repair, Cell 38: 879-887 (1984); Nambiar et al., Total synthesis and cloning of a gene coding 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

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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 error correction problems with various methods of mutagenesis, gene isolation, expression, and others.

Oligonucleotides, eg, for use in mutagenesis eg by mutating or altering libraries of HA and / or NA molecules, are typically chemically synthesized according to the solid phase phosphoramidite triester method described by Beaucage and Caruthers, Tetrahedron Letts 22 ( 20): 1859-1862, (1981) for example using an automated synthesizer, as described in Needham-VanDevanter et al., Nucleic Acid Res, 12: 6159-6168 (1984).

Furthermore, essentially any nucleic acid can be adapted or ordered in a conventional manner from any of a variety of commercial sources, such as The Midland Certified Reagent Company (mcrc@oligos.com), The Great American Gene Company (www.genco.com ), ExpressGen Inc. (www.expressgen.com), Operon Technologies Inc. (Alameda, CA) and many others. Similarly, peptides and antibodies can also be custom-ordered from any of a variety of sources, such as PeptidoGenic (available at pkim@ccnet.com), HTI Bio-products, Inc. (www.htibio.com), BMA Biomedicals Ltd. (UK), Bio.Synthesis, Inc., and many others.

Also described herein are host cells and organisms comprising an HA and / or NA molecule or other polypeptide and / or nucleic acid of eg SEQ ID NO 3-4. Host cells are engineered (eg, transformed, transduced, or transfected) with the vectors described herein, 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 naked polynucleotide or a conjugated polynucleotide. Vectors are introduced into cells and / or microorganisms by conventional methods including electroporation (see, From et al., Proc Natl Acad Sci USA 82, 5824 (1985)), infection by viral vectors, high speed ballistic penetration by particles small with the nucleic acid either within the matrix of beads or small particles, or on the surface (Klein et al., Nature 327, 70-73 (1987)). Berger, Sambrook, and Ausubel provide a variety of suitable transformation methods. See above.

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 the present invention. Bacteria are grown to log phase and plasmids can be isolated within bacteria by a variety of methods known in the art (see, for example, Sambrook). In addition, a multitude of kits are commercially available for the purification of plasmids from bacteria (see, for example, EasyPrep ™, FlexiPrep ™, both from Pharmacia Biotech; StrataClean ™ from Stratagene; and, QIAprep ™ 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 terminator sequences, transcription and translation initiation sequences, and promoters useful for regulating the expression of the particular target nucleic acid. Vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences that allow replication of the cassette in eukaryotes, or prokaryotes, or both, (eg, 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: 73 (1987); Schneider, B., et al., Protein Expr Purif 6435: 10 (1995); Ausubel, Sambrook, Berger (all cited above). A catalog of bacteria and bacteriophages useful for cloning is provided, for example, by the ATCC, eg, The ATCC Catalog of Bacteria and Bacteriophages (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. Additional vectors useful with the sequences herein are illustrated above in the section relating to the production of influenza viruses for vaccines and the literature cited therein.

PRODUCTION AND RECOVERY OF POLYPEPTIDES

Following transduction of a suitable host strain or cell line and growth of the host cells to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature change or chemical induction) and the cells are cultured for a period of time. additional period. In some embodiments, a secreted polypeptide product, eg, an HA and / or NA polypeptide in the form of a secreted fusion protein, etc., is then recovered from the culture medium. In other embodiments, a virus particle of the invention is produced that contains an HA and / or NA polypeptide from the cell. Alternatively, cells can be harvested by centrifugation, altered by physical or chemical means, and the resulting crude extract can be retained for further purification. The eukaryotic or microbial cells used in the

ES 2 525 672 T3 protein expression can be altered by any convenient method, including freeze-thaw cycles, sonication, mechanical alteration, or use of cell lysis agents, or other methods well known to those of skill in the art. Additionally, cells expressing a polypeptide product of HA and / or NA described herein can be used without removing the polypeptide from the cell. In such situations, the polypeptide is optionally expressed on the cell surface and thus examined (eg, having HA and / or NA molecules eg comprising fusion proteins or the like) on the cell surface, etc.

Expressed polypeptides can be recovered and purified from recombinant cell cultures by any of a number of methods well known in the art, including ethanol or ammonium sulfate precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction, affinity chromatography (for example, using any of the labeling systems known to those skilled in the art), hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as desired, to complete the configuration of the mature protein. In addition, high performance liquid chromatography (HPLC) can be employed in the final purification steps. In addition to the literature referenced herein, a variety of purification methods are well known in the art, including, for example, those discussed in Sandana (1997) Bioseparation of Proteins, Academic Press, Inc .; and Bollag et al. (1996) Protein Methods, 2^to Wiley-Liss Edition, NY; Walker (1996) The Protein Protocols Handbook Humana Press, NJ, Harris and Angal (1990) Protein Purification Applications: A Practical Approach IRL Press at Oxford, Oxford, England; Harris and Angal, Protein Purification Methods: A Practical Approach | Rl 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.

When the expressed polypeptides are produced in viruses, the viruses are normally recovered from the culture medium, in which the infected (transfected) cells have been grown. Normally, the crude medium is rinsed prior to concentration of influenza viruses. Common methods include ultrafiltration, barium sulfate adsorption and elution, and centrifugation. For example, crude medium from infected cultures can first be clarified by centrifugation at, eg, 1000-2000 xg for a time sufficient to remove cellular debris and other large particulate matter, eg, between 10 and 30 minutes. Optionally, the clarified medium supernatant is then centrifuged to pellet influenza viruses, eg, at 15,000 xg, for approximately 3-5 hours. After suspension of the virus pellet in an appropriate buffer, such as STE (0.01 M Tris-HCl; 0.15 M NaCl; 0.0001 M EDTA) or phosphate buffered saline (PBS) at pH 7.4 , the virus is concentrated by density gradient centrifugation over sucrose (60% -12%) or potassium tartrate (50% -10%). Either continuous or stepped gradients are suitable, for example a sucrose gradient of between 12% and 60% in four steps of 12%. The gradients are centrifuged at a speed and for a time sufficient for the viruses to concentrate in a visible band for recovery. Alternatively, and for larger scale commercial applications, the virus is elutriated from density gradients using a zone centrifuge rotor in continuous mode. Sufficient additional details to guide an expert through the preparation of tissue culture influenza viruses are provided, for example, 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 US Patent No. 5,690,937. If desired, recovered viruses can be stored at -80 ° C in the presence of sucrose-phosphate-glutamate (SPG) as a stabilizer.

Alternatively, cell-free transcription / translation systems may be employed to produce polypeptides comprising a sequence or subsequence of amino acids or of, for example, the sequences provided herein such as SEQ ID NO: 13-14, or encoded by sequences polynucleotide, eg, SEQ ID NO: 3-4. Various 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.

In addition, polypeptides, or subsequences thereof, for example, subsequences comprising antigenic peptides, can be produced manually or 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 431A peptide synthesizer from Applied Biosystems (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

The expressed polypeptides described herein can contain one or more modified amino acids. The presence of modified amino acids can be advantageous in, for example, (a) increasing the

ES 2 525 672 T3 serum half-life of the polypeptide, (b) reducing / increasing the antigenicity of the polypeptide, (c) increasing the storage stability of the polypeptide, etc. The amino acid (s) are modified, for example, in conjunction with translation or post-translation during recombinant production (for example, N-terminal associated glycosylation in N-XS / T motifs during expression in cells of mammal) or are modified by synthetic means (eg, via PEGylation).

Non-limiting examples of a modified amino acid include a glycosylated amino acid, a sulfated amino acid, a prenylated amino acid (e.g., farnesylated, geranylgeranylated), an acetylated amino acid, an acylated amino acid, a pegylated 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 with, for example, lipid moieties or other organic derivatizing agents. There are a multitude of suitable bibliographic references to guide one of ordinary skill in amino acid modification in the literature. Example protocols are found in Walker (1998) Protein Protocols on CD-ROM Human Press, Towata, NJ.

Fusion proteins

Also described herein are fusion proteins comprising fusions of the sequences described herein (eg, encoding HA and / or NA polypeptides as provided by way of example by SEQ ID NO: 13 -14) with, for example, immunoglobulins (or parts thereof), sequences encoding for, for example, GFP (green fluorescent protein), or other similar markers, etc. Fusion proteins are optionally used for, for example, similar applications (including, for example, therapeutic, prophylactic, diagnostic, experimental applications, etc., as described herein) to those of the non-fusion proteins described. in the present document. In addition to fusion with immunoglobulin sequences and marker sequences, the proteins described herein are also optionally fused with, for example, sequences that allow sorting of fusion proteins and / or targeting of fusion proteins toward regions, specific cell types, etc.

Antibodies

The polypeptides described herein can be used to produce antibodies specific for the polypeptides provided herein and / or polypeptides encoded by the polynucleotides described herein and conservative variants thereof. Antibodies specific to the aforementioned polypeptides are useful, for example, for diagnostic and therapeutic purposes, for example, related to the activity, distribution and expression of target polypeptides.

Antibodies specific for the polypeptides described herein 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 a Fab expression library.

Polypeptides do not require biological activity for antibody production (eg, full-length functional hemagglutinin or neuraminidase 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 extensions of a polypeptide can be fused with another protein, such as keyhole limpet hemocyanin and an antibody raised against the chimeric molecule.

Numerous methods of producing polyclonal and monoclonal antibodies are known to those of skill in the art and can be adapted to produce antibodies specific for the polypeptides described herein and / or encoded by the polynucleotide sequence described herein etc. See, for example, 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 (4^to ed.) Lange Medical Publications, Los Altos, CA, and bibliographies cited therein; Goding (1986) Monoclonal Antibodies: Principles and Practice (2nd ed.) Academic Press, New York, NY; and Kohler and Milstein (1975) Nature 256: 495-497. Other suitable techniques for the preparation of antibodies include the selection of recombinant antibody libraries on 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 antisera and antibodies will usually bind with a KD of, for example, at least about 0.1 gM, at least about 0.01 gM or better, and usually at least about 0.001 pM or better.

For certain therapeutic applications, humanized antibodies are desirable. Detailed methods for the preparation of chimeric (humanized) antibodies can be found in US Patent 5,482,856. Additional details on humanization and other antibody production and engineering techniques can be found in Borrebaeck (ed.) (1995) Antibody Engineering, 2nd Edition, Freeman and Company, NY (Borrebaeck); McCafferty et al. (1996) Antibody Engineering, A Practical Approach IRL at Oxford

ES 2 525 672 T3

Press, Oxford, England (McCafferty), and Paul (1995) Antibody Engineering Protocols Humana Press, Towata, NJ (Paul). Additional details regarding specific procedures can be found, for example, in Ostberg et al. (1983), Hybridoma 2: 361-367, Ostberg, US Patent No. 4,634,664, and Engelman et al., US Patent No. 4,634,666.

Definition of polypeptides by immunoreactivity

The generation of antisera that specifically bind to the polypeptides described herein as well as polypeptides that bind by such antisera are features described herein.

For example, polypeptides (eg, HA and / or NA molecules) are described that specifically bind to or are specifically immunoreactive with an antibody or antisera raised against an immunogen comprising an amino acid sequence selected from one or more of the sequences. provided herein (eg, SEQ ID NO: 13-14), etc. To eliminate cross-reactivity with other homologs, the antibody or antiserum is extracted with the HA and / or NA molecules found in public databases at the time of presenting, for example, the "control" polypeptide (s). . When the other control sequences correspond to a nucleic acid, a polypeptide encoded by the nucleic acid is generated and used for antibody / antiserum extraction purposes.

In a typical format, the immunoassay uses a polyclonal antiserum that was raised against one or more polypeptides comprising one or more of the sequences corresponding to the sequences herein (eg, SEQ ID NO: 13-14), etc. or a substantial subsequence thereof (ie, at least about 30% of the full length sequence provided). The pool of potential polypeptide immunogens derived from the present sequences is collectively referred to as "immunogenic polypeptides" hereinafter. The resulting antiserum is optionally selected to have low cross-reactivity against control hemagglutinin and / or neuraminidase homologs and any such cross-reactivity is removed, for example, by immunosorption, with one or more of the control hemagglutinin and neuraminidase homologs, before the use of the polyclonal antiserum in the immunoassay.

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, a recombinant protein can be produced in a recombinant cell. An inbred variety of mice (used in this assay because the results are more reproducible due to the virtual genetic identity of the mice) are immunized with the immunogenic protein (s) in combination with a conventional adjuvant, such as Freund's adjuvant, and a standard mouse immunization protocol (see, eg, 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 antibody literature and comments can be found herein and may be applied in this case to define polypeptides by immunoreactivity. Alternatively, one or more synthetic or recombinant polypeptide (s) derived from the sequences disclosed herein are conjugated to a carrier protein and used as an immunogen.

Polyclonal sera are collected and titrated against the immunogenic polypeptide in an immunoassay, eg, a solid phase immunoassay with one or more of the immunogenic proteins immobilized on a solid support. Polyclonal antisera are selected with a titer of 10⁶ or greater are pooled and extracted with the control hemagglutinin and / or neuraminidase polypeptide (s) to produce extracted, pooled, titrated polyclonal antisera.

The extracted, pooled, titrated polyclonal antisera are tested for cross-reactivity against the control homolog (s) in a comparative immunoassay. In this comparative assay, discriminatory binding conditions are determined for the extracted, titrated, polyclonal antisera, resulting in at least about a 5-10-fold signal-to-noise ratio for the binding of the titrated polyclonal antisera. to immunogenic polypeptides, compared to binding to control homologs. That is, the stringency of the binding reaction is adjusted by the addition of non-specific competitors such as albumin or skimmed milk powder and / or by adjusting the saline conditions, temperature, and / or the like. These binding conditions are used in subsequent assays to determine whether a test polypeptide (a polypeptide that is compared to immunogenic polypeptides and / or control polypeptides) is specifically bound by pooled extracted polyclonal antisera. In particular, test polypeptides that show at least a 2-5x higher signal-to-noise ratio than control receptor homologues under discriminatory binding conditions, and at least about a ¹/ 2 signal to noise ratio compared to the immunogenic polypeptide (s), share substantial structural similarity with the immunogenic polypeptide compared to the known receptor, etc.

In another example, immunoassays in the competitive binding format are used for the detection of a test polypeptide. For example, as noted, cross-reactive antibodies are removed from the pooled antisera mixture by immunosorption with control polypeptides. The immunogenic polypeptide (s)

ES 2 525 672 T3 is then immobilized to a solid support which is exposed to the pooled extracted antisera. Test proteins are added to the assay to compete for binding to the pooled extracted antisera. The ability of the test protein (s) to compete for binding to the pooled extracted antisera compared to the immobilized protein (s) is compared to the ability of the polypeptide (s) ) immunogenic (s) added to the assay to compete for binding (immunogenic polypeptides compete effectively with immobilized immunogenic polypeptides for binding to pooled antisera). The percent cross-reactivity for the test proteins is calculated using standard calculations.

In a parallel assay, the ability of the control protein (s) to compete to bind the pooled extracted antisera is optionally determined compared to the ability of the immunogenic polypeptide (s) to compete for bind to antisera. Again, the percent cross-reactivity is calculated for the control polypeptide (s), using standard calculations. When the percent cross-reactivity is at least 5-10x as high for the test polypeptides compared to the control polypeptide (s) and / or when the binding of the test polypeptides is approximately in the range of binding Of the immunogenic polypeptides, the test polypeptides are said to specifically bind to the pooled extracted antisera.

In general, pooled and immunosorbed antisera can be used in a competitive binding immunoassay as described herein to compare any test polypeptide with the immunogenic polypeptide (s) and / or control. In order to make this comparison, the immunogenic, test and control polypeptides are assayed in a wide range of concentrations and the amount of each polypeptide required to inhibit 50% of the binding of the extracted antisera to, for example, is determined. example, an immobilized immunogenic, test or control protein, using standard techniques. If the amount of the test polypeptide required in the competitive assay is less than twice the amount of the immunogenic polypeptide that is required, then the test polypeptide is said to bind specifically to an antibody generated against the immunogenic protein, provided that the amount is at least about 510x as high as for the control polypeptide.

As a further determination of specificity, the pooled antiserum is optionally fully immunosorbed with the immunogenic polypeptide (s) (rather than the control polypeptide (s)) until little is detected. or no binding of the resulting immunogenic polypeptide extracted pooled antisera to the immunogenic polypeptide (s) used in immunosorption. This fully immunosorbed antiserum 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 ratio observed for the binding of fully immunosorbed antisera to the immunogenic polypeptide), then the test polypeptide is specifically bound by the antisera elicited by the immunogenic protein.

SEQUENCE VARIANTS OF NUCLEIC ACID AND POLIPEPTIDE

Silent variations

Due to the degeneracy of the genetic code, any of a variety of nucleic acid sequences encoding the polypeptides described herein are optionally produced, some of which may carry lower levels of sequence identity to the nucleic acid sequences and HA and NA polypeptide herein. Below is a table of typical codons specifying the genetic code, found in many biology and biochemistry texts.

Table 1

<td colspan="7">Codon table</td>

<td colspan="3">Amino acids</td><td colspan="4">Codon</td>

<td>To the girl</td><td>To</td><td>TO</td><td>GCA</td><td>GCC</td><td>GCG</td><td>GCU</td>

<td>Cysteine</td><td>Cys</td><td>C</td><td>UGC</td><td>UGU</td><td></td><td></td>

<td>Acid</td><td>aspartic</td><td>Asp</td><td>GAC</td><td>GAU</td><td></td><td></td>

<td>Acid</td><td>glutamic</td><td>Glu</td><td>GAA</td><td>GAG</td><td></td><td></td>

<td>Phenylalanine</td><td>Phe</td><td>F</td><td>UUC</td><td>UUU</td><td></td><td></td>

<td>Wisteria</td><td>Gly</td><td>G</td><td>GGA</td><td>GGC</td><td>GGG</td><td>GGU</td>

<td>Histidine</td><td>His</td><td>H</td><td>CAC</td><td>CAU</td><td></td><td></td>

<td>Isoleucine</td><td>Ile</td><td>I</td><td>AUA</td><td>AUC</td><td>AUU</td><td></td>

<td>Lysine</td><td>Lys</td><td>K</td><td>AAA</td><td>AAG</td><td></td><td></td>

ES 2 525 672 T3

<td>Leucine</td><td>Leu</td><td>L</td><td>UUA</td><td>UUG</td><td>CUA</td><td>CUC</td><td>CUG</td><td>CUU</td>

<td>Methionine</td><td>Met</td><td>M</td><td>AUG</td><td></td><td></td><td></td><td></td><td></td>

<td>Asparagine</td><td>Asn</td><td>N</td><td>AAC</td><td>AAU</td><td></td><td></td><td></td><td></td>

<td>Proline</td><td>Pro</td><td>P</td><td>CCA</td><td>CCC</td><td>CCG</td><td>CCU</td><td></td><td></td>

<td>Glutamine</td><td>Gln</td><td>Q</td><td>CAA</td><td>CAG</td><td></td><td></td><td></td><td></td>

<td>Arginine</td><td>Arg</td><td>R</td><td>AGA</td><td>AGG</td><td>CGA</td><td>CGC</td><td>CGG</td><td>CGU</td>

<td>Serine</td><td>To be</td><td>S</td><td>AGC</td><td>AGU</td><td>UCA</td><td>UCC</td><td>UCG</td><td>UCU</td>

<td>Threonine</td><td>Thr</td><td>T</td><td>HERE</td><td>ACC</td><td>ACG</td><td>ACU</td><td></td><td></td>

<td>Valine</td><td>Val</td><td>V</td><td>GUA</td><td>GUC</td><td>GUG</td><td>GUU</td><td></td><td></td>

<td>Tryptophan</td><td>Trp</td><td>W</td><td>UGG</td><td></td><td></td><td></td><td></td><td></td>

<td>Tyrosine</td><td>Tyr</td><td>Y</td><td>UAC</td><td>UAU</td><td></td><td></td><td></td><td></td>

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 code for the amino acid arginine. Thus, at each position in the nucleic acids described herein 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. U in an RNA sequence is understood to correspond to T in a DNA sequence.

Such "silent variations" are a kind of "conservatively modified variations" discussed below. One of skill will recognize that each codon in a nucleic acid (except ATG, which is normally the only codon for methionine, and TTG, which is normally the only codon for tryptophan) can be modified by conventional techniques to encode a functionally identical polypeptide. Accordingly, each silent variation of a nucleic acid encoding a polypeptide is implicit in any described sequence. The description, therefore, explicitly provides each and every possible variation of a nucleic acid sequence encoding a polypeptide described herein that could be made by selecting combinations based on possible codon choices. These combinations are made according to the conventional triplet genetic code (eg, as set forth in Table 1, or as commonly available in the art) as applied to the nucleic acid sequence encoding a polypeptide of hemagglutinin or neuraminidase described herein. All of these variations of each nucleic acid herein are specifically provided and described with the sequence in mind in combination with the genetic code. One skilled in the art is fully capable of making these silent substitutions using the methods herein.

Conservative variations

Due to the degeneracy of the genetic code, "silent substitutions" (ie, substitutions in a nucleic acid sequence that do not result in an alteration in an encoded polypeptide) are an implicit feature of each nucleic acid sequence described herein. document that codes for an amino acid.

"Conservative variations" of a particular nucleic acid sequence refer to those nucleic acids that code for identical amino acid sequences. Finally, the addition of sequences that 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.

Unique polypeptide and polynucleotide sequences

Also described herein is a nucleic acid comprising a unique subsequence in a nucleic acid selected from the sequence of HA and / or NA molecules disclosed herein, eg, SEQ ID NO: 3-4. The unique subsequence is unique compared to a nucleic acid corresponding to nucleic acids such as, for example, those found in GenBank or other similar public databases at the time of submission. The alignment can be done using, for example, the BLAST tool configured with default parameters. Any unique subsequence is useful, for example, as a probe to identify the nucleic acids described herein. See above.

Similarly, described herein is a polypeptide comprising a unique subsequence in a polypeptide selected from the sequence of HA and / or NA molecules disclosed herein, eg, SEQ ID NO: 13-14. . In this case, the unique subsequence is unique compared to a polypeptide corresponding to, for example, the amino acid corresponding to the polynucleotide sequences found in,

ES 2 525 672 T3 eg GenBank or other similar public databases at the time of filing.

In addition, target nucleic acids that hybridize under stringent conditions with a unique coding oligonucleotide that encodes a unique subsequence in a polypeptide selected from the HA and / or NA molecule sequences described herein are described, the subsequence being unique compared to a polypeptide corresponding to any of the control polypeptides (sequences of, for example, the nucleic acids corresponding to those found in, for example, GenBank or other similar public databases at the time of filing). Unique sequences are determined as above.

Sequence comparison, identity and homology

The terms "identical" or "identity" in percent, in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid or nucleotide residues that are equal, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (or other algorithms available to those of skill) or by visual inspection.

The term "substantially identical," in the context of two nucleic acids or polypeptides (eg, DNA encoding an HA or NA molecule, or the amino acid sequence of an HA or NA molecule) refers to two or more sequences or subsequences having nucleotide or amino acid residue identity of 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, when compared and aligned for maximum match, as measured using a sequence comparison algorithm or by visual inspection. Such "substantially identical" sequences are normally considered to be homologous, without reference to actual ancestors. Preferably, "substantial identity" exists along a region of amino acid sequences that is at least about 200 residues in length, more preferably along a region of at least about 250 residues, and most preferably the sequences are substantially identical across at least approximately 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 along the full length of the two sequences to be compared.

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, the test and reference sequences are entered into a computer, subsequence coordinates are designated if necessary, and the parameters of the sequence algorithm program are designed. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence (s) relative to the reference sequence, based on designated program parameters.

Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith &amp; Waterman, Adv Appl Math 2: 482 (1981), by the homology alignment algorithm of Needleman &amp; Wunsch, J Mol Biol 48: 443 (1970), by means of the search for similarity method of Pearson &amp; Lipman, Proc Natl Acad Sci USA 85: 2444 (1988), by means of 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., Cited above).

An example of an algorithm that is suitable for determining sequence similarity and percent sequence identity is the BLAST algorithm, which is described in Altschul et al., J Mol Biol 215: 403-410 (1990). Software to perform BLAST analyzes is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high-scoring sequence pairs (HSP) by identifying short words of length W in the problem sequence, which either correspond to or satisfy some threshold T-score of positive value when aligned with a word of the same length in a sequence in the database. T refers to the neighbor word score threshold (see, Altschul et al., Cited above). These initial neighbor word hits act as seeds to initiate searches to find longer HSPs that contain them. The word hits are then spread out in both directions along each sequence as much 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 mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The spread of word hits in each direction stops when: the cumulative alignment score falls below the amount X from its maximum achieved value; the cumulative score goes to zero or less, due to the accumulation of one or more negative scoring residue alignments; or the end of any sequence is reached. The parameters W, T and X of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses

ES 2 525 672 T3 as default parameters a word length (W) of 11, an expectation (E) of 10, a cut-off point of 100, M = 5, N = 4, and a comparison of both strings. For amino acid sequences, the BLASTP program uses as default parameters a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, Henikoff &amp; Henikoff (1989) Proc Natl Acad Sci USA 89: 10915).

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin &amp; Altschul, Proc Natl Acad Sci USA 90: 5873-5787 (1993)). A measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid with 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.

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. A tree can also be graphed showing the grouping relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng &amp; Doolittle (1987) J. Mol. Evol. 35: 351-360. The method used is similar to the method described by Higgins &amp; Sharp (1989) CABIOS5: 151-153. The program can align, for example, up to 300 sequences with 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 sequence clusters can be aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved through a series of progressive alignments, in pairs. The program can also be used to graph a dendrogram or tree representation of grouping relationships. The program runs by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison.

A further example of an algorithm that is suitable for multiple DNA or amino acid sequence alignments is the CLUSTALW program (Thompson, JD et al. (1994) Nucl. Acids. Res. 22: 4673-4680). CLUSTALW performs multiple pairwise comparisons between groups of sequences and brings them together in a multiple alignment based on homology. The gap opening and gap extension penalties can be, for example, 10 and 0.05, respectively. For amino acid alignments, the BLOSUM algorithm can be used as a matrix of protein weights. See, for example, Henikoffand Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 1091510919.

DIGITAL SYSTEMS

Further described are digital systems, eg, computers, computer-readable media, and embedded systems comprising character strings corresponding to sequence information herein for the isolated or recombinant nucleic acids and polypeptides herein, including, by example, the sequences shown herein, and the various silent substitutions and conservative substitutions thereof. Integrated systems may further include, for example, gene synthesis kits to obtain genes corresponding to character strings.

Various methods known in the art can be used to detect homology or similarity between different character strings (see above), or they can be used to perform other desirable functions such as controlling output files, providing the basis for making information displays including sequences and the like. Examples include BLAST, discussed above. Computer systems may include such programs, for example, in conjunction with one or more data file (s) or database (s) comprising (s) a sequence as indicated herein.

Thus, different types of homology and similarity of varying stringency and length between various HA or NA sequences or fragments, etc. can be detected and recognized. in the systems integrated in this document. For example, many homology determination methods have been designed for comparative analysis of biopolymer sequences, for "spell checking in word processing, and for data retrieval from various databases. With an understanding of the complementary pairwise double helix interactions between 4 major nucleobases in natural polynucleotides, models that simulate the hybridization of complementary homologous polynucleotide strands can also be used as the basis for sequence alignment or other operations typically performed on the strands. of characters corresponding to the sequences in this document (for example, word processing manipulations, construction of figures comprising sequence or subsequence character strings, output tables, etc.).

Therefore, conventional desktop applications such as data processing software can be adapted.

ES 2 525 672 T3 texts (for example, Microsoft Word ™ or Corel WordPerfect ™) and database software (for example, spreadsheet software such as Microsoft Excel ™, Corel Quattro Pro ™, or database programs such as Microsoft Access ™, Paradox ™, GeneWorks ™, or MacVector ™ or other similar programs) by entering a character string corresponding to one or more polynucleotides and polypeptides described herein (either nucleic acids or proteins, or both). For example, a system may include the above software that has the appropriate string information, for example, used in conjunction with a user interface (for example, a GUI in a conventional operating system such as a Windows system, Macintosh or LINUX) to manipulate character strings corresponding to the sequences in this document. As noted, specialized alignment programs such as BLAST can also be incorporated into the systems described herein for alignment of nucleic acids or proteins (or corresponding character strings).

The systems described herein typically include a digital computer with data sets entered into the software system comprising any of the sequences herein. The computer can be, for example, a PC (DOS ™ based machine, OS2 ™ WINDOWS ™, WINDOWSNT ™, WINDOWS95 ™, WINDOWS2000 ™, WINDOWS98 ™, LINUX compatible with an Intel x86 or Pentium processor, a MACINTOSH ™ based machine , Power PC or UNIX (for example, SUN ™ workstation) or other commercially available computer known to the expert. Software is available to align or otherwise manipulate sequences, or can be easily constructed by an expert using a conventional programming language such as Visualbasic, PERL, Fortran, Basic, Java, or the like.

Any controller or computer optionally includes a monitor which is often a cathode ray tube ("CRT") display, a flat panel display (eg, active matrix liquid crystal display, liquid crystal display), or others. . Computer circuitry is often housed in a box that 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 writable CD-ROM, and other common peripheral items. Input devices such as a keyboard or mouse optionally provide input by a user and for the user to compare the selection of sequences or otherwise manipulate them in the relevant computer system.

The computer typically includes appropriate software to receive instructions from the user, either in the form of user input in a set parameter field, for example in a GUI, or in the form of pre-programmed instructions, for example, pre-programmed for a variety of different specific operations. The software then converts these instructions into appropriate language to instruct the operation of, for example, appropriate transport mechanisms or controllers to carry out the desired operation. The software may also include output elements to control nucleic acid synthesis (eg, based on sequence or sequence alignment herein); sample comparisons for differential gene expression, or other operations.

KITS AND REAGENTS

A kit is also described. For example, a kit contains one or more nucleic acid (s), polypeptide (s), antibody (s), or cell line (s) described herein (for example, comprising, or with , an HA and / or NA molecule described herein). The kit may contain a diagnostic nucleic acid or polypeptide, for example, an antibody, probe set, for example, as a microarray of cDNA packaged in a suitable container, or another nucleic acid such as one or more vector (s) of expression. The kit may also further comprise one or more additional reagents, eg substrates, labels, primers, for labeling expression products, tubes and / or other accessories, reagents for collecting samples, buffers, hybridization chambers, coverslips, etc. The kit optionally further comprises a set of instructions or user manual detailing the preferred methods of using the kit components for the discovery or application of diagnostic kits, etc.

When used as directed, the kit can be used, for example, to assess a condition or pathology, to evaluate the effects of a pharmaceutical agent or other treatment intervention on the progression of a condition or pathology in a cell or organism, or for use as a vaccine, etc.

System kits incorporating the methods, composition, systems, and apparatus are further described herein. System kits optionally comprise one or more of the following: (1) an apparatus, system, system component, or apparatus component; (2) instructions for practicing the methods described herein, and / or for operating the apparatus or components of the apparatus herein and / or for using the compositions herein. The use of any apparatus, apparatus component, composition or kit is also described herein, for the implementation of any method or assay herein, and / or for the use of any apparatus or kit to put into practice any test or method in this document.

Additionally, the kits may include one or more translation system (s) as indicated above (for

ES 2 525 672 T3 example, a cell) with appropriate packaging material, containers to contain the kit components, instructional materials to practice the methods herein and / or the like. Similarly, products of translation systems (eg, proteins such as HA and / or NA molecules) may be provided in kit form, eg, with packages to contain kit components, instructional materials to put into practice the methods herein and / or the like.

To facilitate the use of the methods and compositions of the invention, any of the components and / or vaccine compositions, for example allantoic liquid reassortant virus, etc., and additional components, such as, buffer, cells, culture medium, useful for packaging and infection of influenza viruses for experimental or therapeutic vaccine purposes. Typically, the kit contains, in addition to the above components, additional materials that may include, for example, instructions for performing the methods of the invention, packaging material, and a container.

Examples

Construction and analysis of vaccines and H5N1 ca viruses

Various sequences were used herein comprising the HA / NA H5N1 sequences to create vaccines and influenza viruses. HA sequences in such vaccines were altered from wild type by removing the polybasic cleavage site within HA. HA / NA sequences were rearranged (in a 6: 2 rearrangement) with A / AA / 6/60 (an att virus, ca, see above).

Three HSN1 influenza strains 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 strains 97, 03, and 04 based on their year designations. The percentage similarity of the HA genes of these three strains is 95-96%. Figure 1 illustrates the modification of the polybasic cleavage site of an exemplary HA sequence, the 04 HA sequences, used to construct the viruses / vaccines. As stated above, various embodiments of the invention comprise sequences that have different regions of the polybasic cleavage site removed. See above.

As established, the modified H5N1 sequences (ie the modified 97, 03 and 04 genes) were used to construct viruses with 6: 2 reassortment with A / AA / 6/60. It will be appreciated and noted elsewhere herein that other desirable backbones could also have been used (eg, PR8, etc.).

In the 6: 2 rearrangements 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 derived from the parent virus A / AA / 6/60 ca. The reassortant viruses replicated up to 8.0-8.5 log10 TCID50 in eggs. However, it will be appreciated that a virus as claimed in which the logw TCID50 is between about 7.0 and about 9.0, between about 7.5 and 8.5, or between about 8.0 and 8, 5 is also part of the invention. The cleavage capacity of modified HA in the viruses constructed by endogenous proteases in vitro was limited and the viruses were trypsin-dependent (eg, from about 0.1 ug / ml to about 1.0 ug / ml) for growth . The constructed viruses were temperature sensitive in vitro.

H5N1 ca reassortant viruses (having modified HA genes 97, 03 or 04) were not highly pathogenic for chickens. For example, when 4-week-old white SPF Plymouth Rock chickens were inoculated intravenously with a 1:10 dilution of stock virus (10^8-8,75 DICTsü / ml) and were observed for 10 days, it was observed that 8 out of 8 chickens died within 1-2 days when wild-type H5N1 97, 03 and 04 were used, while 0 out of 8 chickens died when they were used. reassorted H5N1 viruses ca. As can be seen in FIG. 2, the H5N1 ca reassortant viruses administered intranasally did not replicate in chickens.

H5N1 / AA ca reassortants were also not lethal to mice. See Figure 3, which also shows the TCID50 for H5N1 wild type strains. Figure 4 shows that the H5N1 ca 1997 and 2004 reassortant viruses were restricted in replication in mice. Figure 5 shows that the H5N1 ca reassortant viruses were restricted in replication in mouse lungs.

Figure 6 shows a comparison of serum HAI antibody titers produced in mice after a single intranasal dose of vaccine (2003 ca ​​compared to 2003 wild type).

Figure 7 shows similar measurements, but using serum neutralizing antibody titers.

Figure 8 shows that 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 efficacy of protection against lung replication of homologous and heterologous H5N1 challenge viruses in mice. As can be seen, ca reassortant viruses replicated worse than wild-type viruses. Figure 10 shows related data using mouse upper respiratory apparatus. Those skilled in the art will be familiar with statements of

ES 2 525 672 T3 homologous and heterologous (e.g. testing whether the 2003 vaccine protects against a 2003 wild-type challenge (homologous) or whether a 2003 vaccine protects against a 1997 wild-type challenge ( heterologous), etc.).

Figure 11 shows the efficacy of protection conferred by the H5N1 ca 2004 vaccine against high-dose exposure (10⁵DICT5ü) with homologous or heterologous wild-type H5N1 viruses in mice. Figure 12 shows the efficacy of protection conferred by the H5N1 ca 1997 and 2003 vaccines against high-dose exposure (10⁵TCID50) with homologous or heterologous H5N1 wild-type viruses in mice. Figure 13 shows the efficacy of protection conferred by the H5N1 ca 2004 vaccine against low or high doses of exposure to homologous H5N1 wild-type viruses in mice. Figures 11-13 demonstrate that the vaccines tested could protect against other related viruses.

The current example demonstrates several points regarding H5N1 ca reassortant viruses / vaccines by way of example of the invention. The regrouped viruses ca 97, 03, and 04 were shown to have ts phenotype in vitro, loss of pathogenicity in chickens, and attenuation in mice. Attenuation is expected to be present in ferrets as well. The efficacy of protection and cross-protection against lethal challenge and systemic spread with wild-type viruses in mice was also demonstrated. Efficacy of protection and cross-protection against wild-type challenge virus replication in the respiratory tract of mice is also expected.

It is contemplated to use these viruses / vaccines (and the like) to determine if immunogenicity and efficacy improve after 2 doses of vaccine; to evaluate immunogenicity in non-human primates; to evaluate the attenuation and efficacy of the vaccine in ferrets; to determine the contribution of humoral and cellular immunity to the observed efficacy of vaccines produced in mice; to determine which residues of HA 2003 contribute to enhanced immunogenicity and introduce them into HA 1997 and 2004; and to determine the effects of deleting the multibasic amino acid cleavage site and gene constellation.

SEQUENCES

A / Vietnam / 1203/04

Nucleotide sequence of A / Vietnam / 1203/04 H5 (SEQ ID NO: 1) Full molecule length: 1767 nt

ES 2 525 672 T3

101

151

201

251

301

351

401

451

501

551

601

651

701

751

801

851

901

951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 agcaaaagca ttgcaatagt gcaaacaact tgttacacat gcgatctaga ggatggctcc atggtcttac caggggattt aaccattttg tgaagcctca cctttttcag acaataaaga gtgggggatt aaaacccaac ttggtaccaa gatggagttc agagtaatgg aaaggggact caccaagtgt acaatataca aacagattag tcgaggatta gaatggtaga gggtacgctg caataaggtc ttggaaggga aagatggaag ggttctcatg agaaccttta ctgggtaacg ggaaagtgta cgagactaaa atttaccaaa ggcaatcatg tacaatgcag acacccttgt ggggttcaat cagtcttgtt gcccaagaca cgacagagca tggagtgaag tcggaaaccc atagtggaga caatgactat agaaaattca ttaggggtga ggagctacaa aaatgtggta caccatccta cacctatatt gaatagctac ttctggacaa aaatttcatt caacaattat caaactccaa ccctctcacc tccttgcgac tttggagcta tggttggtat cagacaaaga aactcgatca atttaacaac acgggttcct gaaaatgaga cgacaaggtc gttgtttcga agaaatggaa aagagaggaa tactgtcaat gtagctggtc aatttgcatt ttctact ctgtcaaaat aaaagtgatc ggttgacaca tactggaaaa cctctaattt aatgtgtgac aggccaatcc gaagaattga gatcatcccc gctcagcatg tggcttatca taataccaac atgatgcggc tccgttggga tagatccaaa ttttaaagcc gctccagaat gaaaagtgaa tgggggcgat attggggaat tgggctcaga tagcaggttt gggtaccacc atccactcaa ttgacaaaat ttagaaagga agatgtctgg gaactctaga cgactacagc gttctatcat cgtatgacta ataagtggag ttattctaca tatccttatg taaatttgtg ggagaaaata agatttgcat ataatggaaa gaaacacaac tgagagattg gaattcatca agtcaatgac aacacctatt aaaagttctt tccataccag aaaagaacag caagaagatc agagcagaca catcaacact gtaaacgggc gaatgatgca atgcatacaa ttggaatatg aaactctagc gccccaaata aatagccctc tatagaggga atagcaatga aaggcaatag gaacactcag gaatagagaa acttataatg ctttcatgac ttagggataa aaatgtgata cccgcagtat taaaattgga gtggcgagtt gatgtgctcc agttcagatt gtgcttcttt tggttaccat agaacgttac gggaagctct tagcgtagct atgtgccgga ctctgttacc gagcagaata ggtccagtca ggaaagtcct tacataccca ttttggtact aagctctatc aaaccagaga aaagtggaag atcaacttcg aattgtcaag gtaactgcaa atgccattcc tgtgaaatca aaagagagac ggatggcagg gcaggggagt atggagtcac tttgaggccg tttaaacaag ctgaacttct tcaaatgtca tgcaaaggag atgaatgtat tcagaagaag atcaatagga ccctagcact aatgggtcgt gtagttaaaa

A / Vietnam / 1203/04 H5 amino acid sequence (SEQ ID No. 11) Whole molecule length: 564 aa

101 mekivllfai kkhngklcdl pvndlcypgd vslvksdqic igyhannste qvdtimeknv tvthaqdile dgvkplilrd csvagwllgn pmcdefinvp ewsyivekan fndyeelkhl lsrinhfeki qiipksswss heaslgvssa

151

201

251

301

351

401

451

501

551 cpyqgkssff aeqtklyqnp pndainfesn inssmpfhni fieggwqgmv mntqfeavgr dfhdsnvknl ypqyseearl wmcsngslqc rnwwlikkn ttyisvgtst gnfiapeyay hpltigecpk dgwygyhhsn efnnlerrie ydkvrlqlrd kreeisgvkl rici styptikrsy lnqrlvpria kivkkgdsti yvksnrlvla eqgsgyaadk nlnkkmedgf nakelgngcf esigiyqils nntnqedllv trskvngqsg mkseleygnc tglrnspqre estqkaidgv ldvwtynael iystvassla efyhkcdnec lwgihhpnda rmeffwtilk ntkcqtpmga trglfgaiag tnkvnsiidk lvlmenertl mesvrngtyd laimvaglsl

Nucleotide sequence of A / Vietnam / 1203/04 N1 (SEQ ID NO: 2) Whole molecule length: 1398 nt

ES 2 525 672 T3

101

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601

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951 1001 1051 1101 1151 1201 1251 1301 1351 agcaaaagca gggtcaatct gaacatgatc accaatctga gcttcagtaa ggctgtatac tgtttgttat actttctttt gactgtcaaa gtgaggctcc gcaagtgctt cccagacaat acactatcaa tgtgcatgtg taatggtcag ttaaatcagt tgttatccta tggctcaaat taggatatat ggaacaggta agggttttca gcactaattc actgaaacgg tgattggtca gactagattg cccaaagaga tgtaaatagt cattcaccat ggagttcaaa gtatggtaac tcaatatggg accaatcagc aattagcggg agtaaggaca aagagagccg tgactcaggg gacagaagcc ctccccatat gccatgatgg ggggctgtgg gagttggagg taaatggctc gcatcacata cgaattggat atgccggaga cggccatggg atgcagtgga gttgtggtcc tttaaatacg caggagcggc acagtagctt ggatatagcg cataagacct gcacaatttg gacactgtgg tgacaagtag atgaatccaa tggaatagtt tcagtcattc aatactaatt caattcatct acagtataag ttcatctcat agccttgctg ctcacagaac aactcaaggt caccagttgg ctgtattgaa aacaacatac ttgctttact agatcttcaa gctcctaatt aatcacatgt tatctttcaa gttttcggag ggtgtcctct gcaatggtgt tttgaaatga ttcagtgaaa ggagttttgt tgtttctggg gactagtggg gttggtcttg tttgttcaaa atcagaagat agcttaatgt aattcacaca ttcttactga ctttgcccca gatcggttcc gctcccactt aatgacaagc attaatgagt ttgagtct gt ttgacgattg atacaatggc tgagaactca gtaatgactg aatggaaaaa atcactatga gtgtgcaggg tcaaaatttg acaatccacg aacggggcat ctggatcggg tttgggatcc caagatatcg ccagcatcca ttgagttgat agcagcatat gccagacggt aaactccttg aataaccatc tacaaattgg gggaatcaac gaaagctgtg ttaacggatg aagggggatg ggaatgcaga actccaatgg tgtcctgtgg tgcttggtca gaatttctgg ataataacag agagtctgaa acggaccaag gggaaagtgg ggaatgctcc ataattggca gagtatcaaa ccccaatgat atggggtaaa agaaccaaaa aaatgggtgg tagcaataac gaactgacag cagagggcgg ctttttgtgg gctgagttgc tttctact

A / Vietnam / 1203/04 N1 amino acid sequence (SEQ ID NO: 12) Whole molecule length: 449 aa

101

151 mnpnqkiiti ntnfltekav fiscshlecr nsrfesvaws gsicmvtgiv asvklagnss tffltqgall asachdgtsw slmlqignmi lcpingwavy ndkhsngtvk ltigisgpdn siwvshsiht skdnsirigs drsphqfgevps cvitwpti cgwpti kyvpdtlps kgvitwpdnkvtvpdv

201

251

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401 nnilrtqese apnyhyeecs vfgdnprpnd femiwdpngw cfwvelirgr cacvngscft cypnageitc gtgscgpvss tetdssfsvk pkestiwtsg vmtdgpsngq vcrdnwhgsn ngaygvkgfs qdivaitdws ssisfcgvns ashkifkmek rpwvsfnqnl fkygngvwig gysgsfvqhp dtvgwswpdg gkwksveld eyqigyicsg rtkstnsrsg eltgldcirp aelpftidk

A / Hong Kong / 213/03

Nucleotide sequence of A / Hong Kong / 213/03 H5 (SEQ ID NO: 3) Length of entire molecule: 1767 nt

ES 2 525 672 T3 agcaaaagca 51 ttgcaatagt

101 gcaaacaact

151 tgttacacat 201 gcgatctaga 251 ggatggctcc 301 atggtcttac

351 caggggattt 401 aaccattttg 451 tgaagcctca 501 cctttttcag

551 acaataaaga 601 gtgggggatt 651 aaaacccaac

701 ttggtaccaa

751 gatggagttc Θ01 agagcaatgg

851 aaaggggact 901 caccaagtgt 951 acaatataca 1001 aacagattag 1051 tcgaggatta 1101 gaatggtaga 1151 gggtacgctg 1201 caataaggtc 1251 ttggaaggga 1301 aagatggaag 1351 ggttctcatg 1401 agaaccttta 1451 ctgggtaacg 1501 ggaaagtgta 1551 caagactaaa 1601 acttaccaaa 1651 ggcaatcatg 1701 tacaatgcag 1751 acacccttgt ggggttcaat cagtcttgtt gcccaagaca cgacagagca tggagtgaag tcggaaaccc atagtggaga caacgactat agaaaattca ttaggggtga ggagctacaa gaatgtggta caccatccta cacctacatt aaatagctac ttctggacaa aaatttcatt cagcaattat caaactccaa ccctctcacc tccttgcgac tttggagcta tggttggtat aactcgatca cagacaaaga atttaataac acggattcct gaaaatgaga cgacaaggtc gttgtttcga agaaacggaa aagagaggaa tactgtcaat gtagctggtc aatttgcatt ttctact ctgtcaaaat aaaagtgatc ggttgacaca tactggaaaa cctctaattt aatgtgtgac aggccaatcc gaagaattga gatcatcccc gctcagcatg tggcttatca taataccaac atgatgcggc tccgttggga tagatccaaa ttttaaaacc gctccagaat gaaaagtgaa tgggggcgat atcggggaat tgggctcaga tagcaggttt gggtaccacc atccactcaa ttgacaaaat ttagaaagga agatgtctgg gaactctaga cgactacagc gttctatcac cgtatgacta ataagtggag ttattctaca tatctttatg taaatttgtg ggagaaaata agatttgcat ataatggaaa gacacacaac tgagagattg gaattcatca agccaatgac aacacctatt aaaaattctt tccataccaa aaaagaacaa caagaagatc agagcagact catcaacact gtaaacgggc gaatgatgca atgcatacaa ttggaatatg aaactctagt gccccaaata aatagccctc tatagaggga atagcaatga aaggcaatag gaacactcag gaatagagaa acttataatg ctttcatgac ttagggataa aaatgtgata cccgcagtat taaaattgga gtggcgagtt gatgtgctcc agttcagatt gtgcttcttt tggttaccat agaacgttac gggaagctct tagtgtagct atgtgccgga ctctgttacc gagcagaata ggtccagtca ggaaagtcct tgcataccca ttttggtatt aggctctatc aaaccagaga aaaatggaag atcaacttcg aattgtcaag gtaactgcaa atgccattcc tgtgaaatca aaagagagac ggatggcagg gcaggggagt atggagtcac tttgaggccg tttaaacaag ctgaacttct tcaaatgtca tgcaaaggag atgaatgtat tcagaagaag gtcaatagga ccctagcact aatgggtcgt gtagttaaaa

A / Hong Kong / 213/03 H5 amino acid sequence (SEQ ID NO: 13) Whole molecule length: 564 aa

ES 2525672 T3 mekivllfai vslvksdqic igyhannste qvdtimeknv tvthaqdile kthngklcdl dgvkplilrd csvagwllgn pmcdefinvp ewsyivekan 101 fndyeelkhl pandlcypgd lsrinhfeki qiipknswss heaslgvssa 151 cpyqgkssff rnwwlikkn nayptikrsy nntnqedllv lwgihhpnda 201 aeqtrlyqnp ttyisvgtst lnqrlvpkia trskvngqng rmeffwtilk 251 pndainfesn gnfiapeyay kivkkgdsai 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 esigtyqils iystvassla1 rimvaglcsngs 55

Nucleotide sequence of A / Hong Kong / 213/03 N1 (SEQ ID No. 4) Length of entire molecule: 1458 nt 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 tgagaactca aacaacataa 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 agggttctca atggaataaa 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 cccaaagaga cagagggcgg gcacaatttg gactagtggg agcagcatat 1351 ccttttgtgg tgtaaatagt gacactgtgg gttggtcttg gccagacggt 1401 gctgagttgc cattcaccat tgacaagtag tttgttcaaa aaactccttg May 1451 tttctact

Amino acid sequence of A / Hong Kong / 213/03 N1 (SEQ ID No. 14)

Full molecule length: 469 aa

ES 2525672 T3 mnpnqkitti gsictrtvigiv slmlqignii siwvshsiqt gnqhqaepcn '51 qsiityennt wvnqtyvnis ntnfltekav asvtlagnss lcpisgwavy 101 skdngirigs kgdvfvirep fiscshlecr tffltqgall ndkhsngtvk 151 drsphrtlms cpvgeapspy nsrfeavaws asachdgtsw ltigisgpdn 201 gavavlkyng iitdtikswr nnimrtqese cacvngscft vmtdgpsngq 251 asykifriek gkwksaeln apnyhyeecs cypdageitc vcrdnwhgsn 301 rpwvsfnqnl eyrigyicsg vfgdnprpnd gtgscgpvsp kgaygikgfs 351 fkygngvwig rtkstnsrsg fémiwdpngw 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 / 491/97 H5 (SEQ ID No. 5) Complete molecule length: 1767 nt agcaaaagca ggggtataat ctgtcaaaat ggagaaaata gtgcttcttc ttgcaacagt cagccttgtt aaaagtgacc tggttaatttg

101 gcaaacaact agttgacaca cgacagagca ataatggaaa agaatgttac 151 tgttacacat gcccaagaca tactggaaag gacacacaac gggaagctct 201 gcgatctaaa tggagtgaag cctctgattt tgagggattg tagtgtagct 251 ggatggctcc tcggaaaccc tatgtgtgac atgtgccgga gaattcatca 301 atggtcttac atagtggaga aggccagtcc agccaatgac ctctgttatc 351 caacgactat cagggaattt gaagaactga aacacctatt gagcagaata 401 agaaaattca aaccattttg gataatcccc aaaagttctt ggtccaatca 451 tcaggggtga tgatgcctca gctcagcatg tccatacctt gggaggtcct 501 aaatgtggta cctttttcag 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 caaactccaa caccaagtgt tgggggcaat aaactctagt atgccattcc 951 acaacataca ccccctcacc atcggggaat tgtgaaatca gccccaaata 1001 aacagattag tccttgcaac tggactcaga aatacccctc aacgagagac 1051 tttggagcta gcgaggacta tagcaggttt tatagaggga ggatggcagg 1101 gaatggtaga tggttggtat gggtaccacc atagcaatga gcaggggagt 1151 ggatacgctg atccacacaa cagaccaaga aaggcaatag atggagtcac 1201 aactcgatca caataaggtc ttaacaaaat gaacactcag tttgaggccg 1251 atttaataac ttggaaggga ttggaaagga ggatagagaa tttaaacaag 1301 aaaatggaag acggattcct agatgtctgg acttacaatg ccgaacttct 1351 ggttctcatg gaaaatgaga gaactctaga tcaaatgtca ctttcatgac 1401 agaaccttta cgacaaggtc cgactacagc ttagggataa tgcaaaggag 1451 ctgggtaatg gttgtttcga attctatcac aaatgtgata acgaatgtat 1501 ggaaagtgta aaaaacggaa cgtatgacta cccgcagtat tcagaagaag 1551 caagactaaa cagagaggaa ataagtggag tacatacattga tactcggattg tacaaataattga tacataggatt 160

ES 2 525 672 T3

1651 ggcaatcatg gtagctggtc tatctttatg gatgtgctcc aatggatcgt 1701 tacaatgcag aatttgcatt taaatttgtg agttcagatt gtagttaaaa 1751 acacccttgt ttctact

A / Hong Kong / 491/97 H5 amino acid sequence (SEQ ID NO: 15) Length of entire molecule: 564 aa mekivlllat vslvksdqic igyhannste qvdtimeknv tvthaqdile rthngklcdl ngvkplilrd csvagwllgn pmcdefinvp ewsyivekas 101 fndyeelkhl pandlcypgn lsrinhfeki qiipksswsn hdassgvssa 151 cpylgrssff rnwwlikkn ssyptikrsy nntnqedllv lwgihhpnda 201 aeqtrlyqnp ttyisvgtst lnqrlvpeia trpkvngqsg rmeffwtilk 251 pndainfesn gnfiapeyay kivkkgdsti mkseleygnc ntkcqtpmga 301 inssmpfhni hpltigecpk yvksnrlvla tglrntpqre trglfgaiag 351 fieggwqgmv dgwygyhhsn eqgsgyaadq estqkaidgv tnkvnsiink 401 mntqfeavgr efnnlerrie nlnkkmedgf ldvwtynael lvlmenertl 451 dfhdsnvknl ydkvrlqlrd nakelgngcf efyhkcdnec mesvkngtyd 501 ypqyseearl nreeisgvkl esmgtyqils iystvassla laimvaglngl 551 wcmcsla laimvaglngsql1 wcmcsla laimvaglngl 551 wcmcs

Nucleotide sequence of A / Hong Kong / 486/97 N1 (SEQ ID No. 6) Full molecule length: 1401 nt agcaaaagca ggagtttaaa atgaatccaa ggatcaatct gcatggtagt tgggataatc

101 aaacacaata tcagtatggg tcagccacat

151 accagcctga accatgcaac caaagcatca

201 gcagcttcag tgacattagc gggcaattcc

251 atgggctata tacagcaagg acaatagtat

301 atgtgtttgt tataagagaa ccattcatct

351 agaacctttt tcttgaccca aggagcccta

401 tgggaccgtc aaagacagga gcccctatag

451 ttggtgaggc cccttcccca tacaactcaa

501 tcagcaagtg cttgccatga tggcattagt

551 cggtccggat aatggggctg tggctgtgtt

601 cagacaccat caagagttgg aggaacaaca

651 gaatgtgcat gtgtgaatgg ttcttgtttt

701 gagtaatgaa caggcctcat acaagatttt

751 tagtcaaatc agttgagttg aacgccccta

801 tcctgttatc ctgatgctgg cgaaatcaca

851 gcatggctcg aaccgaccat gggtgtcttt

901 aaataggata tatatgcagt ggggttttcg

951 gatgggacag gcagttgtgg tccagtgtct 1001 aaaagggttt tcatttaaat acggcaatgg 1051 aaagcactag ttccaggagc ggttttgaaa 1101 tggaccgaaa cagacagtag cttctcgttg 1151 aactgattgg tcaggataca gcgggagttt 1201 caggattaaa ttgcatgaga ccttgcttct 1251 aggcccaaag agaaaacaat ctggactagt 1301 tggtgtaaat agtgacactg tgggttggtc atcagaagat agcttgatgt aattaaaact atttttacac tctctctgcc aagaattggt catgctccca ttgaatgaca aactttaatg ggtttgagtc tggctaacaa gaaatacaat actgtaatga cactgaggac caagatagaa attatcatta tgtgtgtgca caatcagaat gagacagtcc cttaacggag tgtttggatc tgatttggga aagcaagaca tattcaacat gggttgaact gggagcagta ttggccagac aataaccatt tacaaattgg tggcacccaa tgagcaggct ctattagtgg tccaaagggg tttggaatgc agcattctaa agctgtcctg tgttgcttgg ttggaatttc ggcataataa gcaagagtct cagatggacc aaggggággg cgaggaatgc gggataattg ctggagtatc acgccccaat cgtatggagt gggagaacca tccaaatggg tcatagcgat ccagaactga aatcagaggg tatctttctg ggtgctgagt

ES 2 525 672 T3

1351 tgccatacac cattgacaag tagtttgttc aaaaaactcc ttgtttctac 1401 t

A / Hong Kong / 486/97 N1 amino acid sequence (SEQ ID N^s 16) Length of the entire molecule: 450 aa mnpnqkiiti gsicmwgii slmlqignti svwvshiikt whpnqpepcn qsinfyteqa aasvtlagns slcpisgwai yskdnsirig skgdvfvire 101 rtffltqgal pfiscshlec lndkhsngtv kdrspyrtlm scpvgeapsp 151 ynsrfesvaw sasachdgis wltigisgpd ngavavlkyn giitdtiksw 201 rnntlrtqes ecacvngscf tvmtdgpane qasykifkie kgrwksvel 251 napnyhyeec scypdageit cvcrdnwhgs nrpwvsfnqn leyqigyics 301 gvfgdsprpn dgtgscgpvs lngaygvkgf sfkygngvwi grtkstssrs 351 gfemiwdpng wtetdssfsl kqdiiaitdw sgysgsfiqii peltglncmr ₅ 401 pcfwvelirg rpkektiwts gssisfcgvn sdtvgwswpd gaelpytidk

A / Honq Konq / 491/97 (Ser211) (HA) + A / Honq Konq / 486/97 (NA)

Nucleotide sequence of A / Hong Kong / 491/97 (Ser211) H5 (SEQ ID N^s 7) Length of the entire molecule: 10 1767 nt agcaaaagca ggggtataat ctgtcaaaat ggagaaaata gtgcttcttc 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 caceatccta atgatgcggc agagcagaca aggctctatc

651 aaaacccaac cacctacatt tccgttggaa catcaacact gaaccagaga

701 ttggtttcag aaatagctac tagacccaaa gtaaacgggc aaagtggaag

751 aatggagttc ttctggacaa ttttaaagcc gaatgatgcc atcaatttcg

001 agagtaatgg aaatttcatt gctccagaat atgcatacaa aattgtcaag

851 aaaggggact caacaattat gaaaagtgaa ttggaatatg gtaactgcaa

901 caccaagtgt caaactccaa tgggggcaat aaactctagt atgccattcc

951 acaacataca ccccctcacc atcggggaat tgtgaaatca gccccaaata 1001 aacagattag tccttgcaac tggactcaga aatacccctc aacgagagac 1051 tttggagcta gcgaggacta tagcaggttt tatagaggga ggatggcagg 1101 gaatggtaga tggttggtat gggtaccacc atagcaatga gcaggggagt 1151 ggatacgctg atccacacaa cagaccaaga aaggcaatag atggagtcac 1201 aactcgatca caataaggtc ttaacaaaat gaacactcag tttgaggccg 1251 atttaataac ttggaaggga ttggaaagga ggatagagaa tttaaacaag 1301 aaaatggaag acggattcct agatgtctgg acttacaatg ccgaacttct

ES 2 525 672 T3

1351 ggttctcatg gaactctaga gaaaatgaga tcaaatgtca ctttcatgac agaaccttta 1401 cgacaaggtc cgactacagc ttagggataa tgcaaaggag 1451 ctgggtaatg gttgtttcga attctatcac aaatgtgata acgaatgtat 1501 aaaaacggaa ggaaagtgta cgtatgacta cccgcagtat tcagaagaag 1551 cagagaggaa caagactaaa ataagtggag taaaattgga atcaatggga 1601 acttaccaaa tactgtcaat ttattcaaca gtggcgagtt ccctagcact 1651 ggcaatcatg gtagctggtc tatctttatg gatgtgctcc aatggatcgt 1701 tacaatgcag aatttgcatt taaatttgtg agttcagatt gtagttaaaa 1751 acacccttgt ttctact

A / Hong Kong / 491/97 (Ser211) H5 (SEQ ID No. 17) amino acid sequence Complete molecule length: 564 aa mekivlllat vslvksdqic igyhannste qvdtimeknv tvthaqdile rthngklcdl ngvkplilrd csvagwllgn pmcdekasvp ewsyive

101 pandlcypgn fndyeelkhl lsrinhfeki qiipksswsn hdassgvssa

151 cpylgrssff rnwwlikkn ssyptikrsy nntnqedllv lwgihhpnda

201 aeqtrlyqnp ttyisvgtst lnqrlvseia trpkvngqsg rmeffwtilk

251 pndainfesn gnfiapeyay kivkkgdsti mkseleygnc ntkcqtpmga

301 inssmpfhni hpltigecpk yvksnrlvla tglrntpqre trglfgaiag

351 fieggwqgmv dgwygyhhsn eqgsgyaadq estqkaidgv tnkvnsiink

401 mntqfeavgr efnnlerrie nlnkkmedgf ldvwtynael lvlmenertl

451 dfhdsnvknl ydkvrlqlrd nakelgngcf efyhkcdnec mesvkngtyd

501 ypqyseearl nreeisgvkl esmgtyqils iystvassla laimvaglsl

551 wmcsngslqc rici

Nucleotide sequence of A / Hong Kong / 486/97 N1 (SEQ ID No. 8) Full molecule length: 1401 nt agcaaaagca ggagtttaaa atgaatccaa atcagaagat aataaccatt 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 ggggtettcg gagacagtcc acgccccaat

951 gatgggacag gcagttgtgg tccagtgtct cttaacggag cgtatggagt ₁₀ 1001 aaaagggttt tcatttaaat acggcaatgg tgtttggatc gggagaacca

ES 2 525 672 T3

1051 aaagcactag ttccaggagc ggttttgaaa tgatttggga tccaaatggg 1101 tggaccgaaa cagacagtag cttctcgttg aagcaagaca tcatagcgat 1151 aactgattgg tcaggataca gcgggagttt tattcaacat ccagaactga 1201 ttgcatgaga caggattaaa ccttgcttct gggttgaact aatcagaggg 1251 aggcccaaag agaaaacaat ctggactagt gggagcagta tatctttctg 1301 tggtgtaaat agtgacactg tgggttggtc ttggccagac ggtgctgagt 1351 tgccatacac cattgacaag tagtttgttc aaaaaactcc ttgtttctac 1401 t

A / Hong Kong / 486/97 N1 amino acid sequence (SEQ ID NO: 18) Length of entire molecule: 450 aa mnpnqkiiti gsicrnwgii slmlqignti svwvshiikt whpnqpepcn 51 qsinfyteqa aasvtlagns slcpisgwai yskdnsirig skgdvfvire 101 rtffltqgal pfiscshlec lndkhsngtv kdrspyrtlm scpvgeapsp 1S1 ynsrfesvaw sasachdgis wltigisgpd ngavavlkyn giitdtiksw 201 rnntlrtqes ecacvngscf tvmtdgpsne qasykifkie kgrwksvel 251 napnyhyeec scypdageit cvcrdnwhgs nrpwvsfnqn leyqigyics 301 gvfgdsprpn dgtgscgpvs Ingaygvkgf sfkygngvwi grtkstssrs 351 gfemiwdpng wtetdssfsl kqdiiaitdw sgysgsfiqh peltglncmr ₅ 401 pcfwvelirg rpkektiwts gssisfcgvn sdtvgwswpd gaelpytidk ca A / ck / Hong Kong / G9 / 97

Nucleotide sequence of ca A / ck / Hong Kong / G9 / 97 (SEQ ID No. 9) Length of the entire molecule: 1690 bp 0 ttaaccactc aagatggaag caataccact aataactata ctactagtag 51 taacagcaag caatgcagac aaaatctgca tcggctacca atcaacaaac 101 ccgtagacac tccacagaaa gctaacagaa aacaatgttc ctgtgacaca 151 ttgctccaca tgccaaagaa cagagcacaa tgggatgctg tgtgcaacaa 201 atctgggacg tcctcttatt ctagacactt gcaccattga aggactgatc 251 tatggcaacc cttcttgtga tctactgttg ggaggaagag aatggtccta 301 catcgtcgaa agaccatcgg ctgttaatgg aatgtgttac cccgggaatg 351 tagaaaacct agaggaacta aggtcatttt ttagttctgc tagttcctac 401 caaagaatcc agatctttcc agacacaatc tggaatgtgt cttacagtgg 451 aacaagcaaa attcattcta gcatgttcag 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 cattctttca ggtatgggca ggagagagcc acggaagaat cctgaagact 851 gatttaaaca gtggtagctg tgtagtgcaa tgtcaaacag aaagaggtgg 901 cttaaatact actttgccat tccacaatgt cagtaaatat gcatttggaa 951 atatgttgga actgcccaaa gtaaagagtc tcaaactggc agttggtctg 1001 aggaatgtgc ctgctagatc aagtagagga ctatttgggg ccatagctgg 1051 attcatagag ggaggttggt cagggctggt cgctggttgg tatgggttcc 1101 agcattcaaa tgatcaaggg gttggtatag ctgcagatag agactcaact

ES 2 525 672 T3

1151 caaagggcaa aacgtccaaa ttgacaaaat tagtcgataa gtgaataata 1201 aatgaacaag ttattgatca caatatgaaa gaggttgaaa tgaattcagc 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 / ck / Hong Kong / G9 / 97 H9 (SEQ ID NO: 19) Whole molecule length: 558 aa meaiplitil lwtasnadk icigyqstns tetvdtlten nvpvthakel lhtehngmlc atnlgrplil dtctiegliy gnpscdlllg grewsyiver

101 psavngmcyp gnvenleelr sffssassyq riqifpdtiw nvsysgtska

151 csdsfyrsmr wltqknnayp iqdaqytnnr gksilfmwgi nhpptdtaqt

201 nlytrtdttt svatedinrt fkpvigprpl vnglqgridy ywsvlkpgqt

251 lrvrsngnli apwyghilsg eshgrilktd Insgscwqc qtergglntt

301 lpfhnvskya fgncpkyvgv kslklavglr nvparssrgl fgaiagfieg

351 gwsglvagwy gfqhsndqgv giaadrdstq raidkitskv nnivdkmnkq

401 yeiidhefse venrlnminn kiddqiqdiw aynaellvll enqktldehd

451 anvnnlynkv kralgsname dgkgcfelyh kcddqcmeti rngtynrrky

501 keesrlerqk iegvkleseg tykiltiyst vasslviamg faaflfwams

551 ngscrcni

Nucleotide sequence of ca A / ck / Hong Kong / G9 / 97 N2 (SEQ ID NO 10) Full molecule length: 1428 bp aaatgaatcc aaatcagaag ataatagcaa ttggctctgt ttctctaact 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 caaccaaaca tttcatttgg agtgtgcata gcatggtcca gctcaagctg 551 ccatgatggg aaagcatggt tacatgtttg tgtcactggg gatgatagaa 601 atgcaacggc tagcaCcatt tatgatggga tacttgttga cagtattggt 651 tcatggtcta aaaacatcct cagaactcag gagtcagaat gcgtttgcat 701 caatggaacc tgtgcagtag taatgactga tggaagtgca tcaggaaggg 751 ctgacactag aatactattt attagagagg ggaaaattgc acacattagc 801 gaagtgctca ccattgtcag gcatgtggag gaatgctcct gttacccccg 851 atatccagaa gttagatgtg tttgcagaga caattggaag ggatccaata 901 ggcccgttct atatataaat atggcaaatt atagtattga ttccagttat

ES 2 525 672 T3

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 1401 atgcctatat aagctttcgc aattttag

Amino acid sequence of ca A / ck / Hong Kong / G9 / 97 N2 (SEQ ID NO: 20) Length of the entire molecule: 469 5 aa mnpnqkiiai gsvsltiati cllmqiaila ttmtlhfkqn ecinssnnqv vpcepiiier niteivhlns ttlekeicpk vadyrnwskp qcqitgfapf 101 skdnsirlsa ggdiwvtrep yvscglgkcy qfalgqgttl enkhsngtah 151 drtphrtllm nelgvpfhla tkqvciawss sschdgkawl hvcvtgddrn 201 atasiiydgi lvdsigswsk nilrtqesec vcingtcaw 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 sec id n ° designations

<td>SEQ ID No.</td><td>HA or NA</td><td>NAME OF THE STRAIN</td><td>Amino acids or nucleotides</td>

<td>SEQ ID No. 1</td><td>HA (H5)</td><td>ca A / Vietnam / 1203/04</td><td>Nucleotides</td>

<td>SEQ ID No. 2</td><td>NA (N1)</td><td>ca A / Vietnam / 1203/04</td><td>Nucleotides</td>

<td>SEQ ID No. 3</td><td>HA (H5)</td><td>ca A / Hong Kong / 213/03</td><td>Nucleotides</td>

<td>SEQ ID No. 4</td><td>NA (N1)</td><td>ca A / Hong Kong / 213/03</td><td>Nucleotides</td>

<td>SEQ ID No. 5</td><td>HA (H5)</td><td>ca A / Hong Kong / 491/97</td><td>Nucleotides</td>

<td>SEQ ID No. 6</td><td>NA (N1)</td><td>ca A / Hong Kong / 486/97</td><td>Nucleotides</td>

<td>SEQ ID No. 7</td><td>HA (H5)</td><td>ca A / Hong Kong / 491/97 (Ser211)</td><td>Nucleotides</td>

<td>SEQ ID No. 8</td><td>NA (N1)</td><td>ca A / Hong Kong / 486/97</td><td>Nucleotides</td>

<td>SEQ ID No. 9</td><td>HA (H9)</td><td>ca A / ck / Hong Kong / G9 / 97</td><td>Nucleotides</td>

<td>SEQ ID No. 10</td><td>NA (N2)</td><td>ca A / ck / Hong Kong / G9 / 97</td><td>Nucleotides</td>

<td>SEQ ID No. 11</td><td>HA (H5)</td><td>ca A / Vietnam / 1203/04</td><td>Amino acids</td>

<td>SEQ ID No. 12</td><td>NA (N1)</td><td>ca A / Vietnam / 1203/04</td><td>Amino acids</td>

<td>SEQ ID No. 13</td><td>HA (H5)</td><td>ca A / Hong Kong / 213/03</td><td>Amino acids</td>

<td>SEQ ID No. 14</td><td>NA (N1)</td><td>ca A / Hong Kong / 213/03</td><td>Amino acids</td>

<td>SEQ ID No. 15</td><td>HA (H5)</td><td>ca A / Hong Kong / 491/97</td><td>Amino acids</td>

<td>SEQ ID No. 16</td><td>NA (N1)</td><td>ca A / Hong Kong / 486/97</td><td>Amino acids</td>

<td>SEQ ID No. 17</td><td>HA (H5)</td><td>ca A / Hong Kong / 491/97 (Ser211)</td><td>Amino acids</td>

ES 2 525 672 T3

<td>SEQ ID No. 18</td><td>NA (N1)</td><td>ca A / Hong Kong / 486/97</td><td>Amino acids</td>

<td>SEQ ID No. 19</td><td>HA (H9)</td><td>ca A / ck / Hong Kong / G9 / 97</td><td>Amino acids</td>

<td>SEQ ID No. 20</td><td>NA (N2)</td><td>ca A / ck / Hong Kong / G9 / 97</td><td>Amino acids</td>

The disclosure further comprises the following points:

1. An isolated polypeptide, wherein said polypeptide is selected from the group consisting of:

a) a polypeptide encoded by a polynucleotide sequence of SEQ ID NO: 1-10;

b) a polypeptide of SEQ ID Nos. 11-20;

c) the mature form of the polypeptide of SEQ ID Nos. 11-20;

d) a polypeptide encoded by a polynucleotide sequence that hybridizes under high stringency conditions to a polynucleotide sequence encoding (a) (b) or (c); Y

d) a polypeptide having at least 90% sequence identity to the polypeptide of (b).

two. An immunogenic composition comprising an immunologically effective amount of at least one polypeptide of item 1.

3. An isolated antibody that specifically binds to the polypeptide of item 1.

Four. A method of stimulating the immune system of an individual to produce a protective immune response against influenza virus, the method comprising administering to the individual an immunologically effective amount of the polypeptide of item 1 in a physiologically acceptable vehicle.

5. A recombinant influenza virus comprising the polypeptide of item 1.

6. An immunogenic composition comprising an immunologically effective amount of the recombinant influenza virus of item 5.

7. A method of stimulating the immune system of an individual to produce a protective immune response against influenza virus, the method comprising administering to the individual an immunologically effective amount of the recombinant influenza virus of item 5 in a physiologically acceptable vehicle.

8. An isolated nucleic acid, wherein said nucleic acid is selected from the group consisting of:

a) a polynucleotide sequence of SEQ ID No. 1-10 or a complementary sequence thereof;

b) a polynucleotide sequence encoding a polynucleotide of SEQ ID Nos. 11-20 or a complementary sequence thereof;

c) a polynucleotide sequence that hybridizes under high stringency conditions to substantially the entire length of the polynucleotide sequence (a); Y

d) a polynucleotide sequence having at least 98% sequence identity to the polynucleotide sequence of (a).

9. An immunogenic composition comprising at least one of the nucleic acids of item 8.

10. A cell comprising at least one nucleic acid from item 8.

eleven. A vector comprising the nucleic acid of item 8.

12. The vector of item 12, wherein the vector is a plasmid, a cosmic, a phage, a virus or a fragment of a virus.

13. The vector of item 12, wherein the vector is an expression vector.

14. A cell comprising the vector of item 13.

fifteen. An influenza virus comprising one or more nucleic acids from item 8.

ES 2 525 672 T3

16. The virus of item 15, in which the virus is a regrouped virus.

17. An influenza virus with 6: 2 regrouping, wherein said virus comprises 6 coding regions of genes of A / Ann Arbor / 6/60 and 2 coding regions of genes that encode a polypeptide selected from the group consisting of: the polypeptides of SEQ ID N ° 11-20.

18. A method for producing a recombinant influenza virus, the method comprising:

culturing the cell of point 14 in a suitable culture medium under conditions that allow the expression of nucleic acid; and isolating the recombinant influenza virus from a cell population comprising said cell or the medium.

19. An immunogenic composition comprising an immunologically effective amount of the recombinant influenza virus of item 17.

twenty. A method of stimulating the immune system of an individual to produce a protective immune response against influenza virus, the method comprising administering to the individual an immunologically effective amount of the recombinant influenza virus of item 17 in a physiologically effective vehicle.

twenty-one. A method for producing an isolated or recombinant polypeptide, the method comprising: culturing the host cell of item 10 in a suitable culture medium under conditions that allow the expression of said nucleic acid; and isolating the polypeptide from one or more of the host cells or the medium.

22. A method of prophylactic or therapeutic treatment of a viral infection in a subject, the method comprising: administering to the subject a virus of item 17 in an amount effective to produce an immunogenic response against the viral infection.

2. 3. The method of point 22, in which the subject is a human being.

24. The immunogenic composition of item 19, wherein the hemagglutinin comprises a modified polybasic cleavage site.

25. A live attenuated influenza virus vaccine comprising the composition of item 19.

26. A split or killed virus vaccine comprising the composition of item 19.

27. A live attenuated influenza virus vaccine comprising the composition of item 24.

28. A split or killed virus vaccine comprising the composition of item 24.

29. A method for producing influenza virus in cell culture, the method comprising:

i) introducing into a population of host cells, a population of host cells that is capable of supporting replication of the influenza virus, a plurality of vectors comprising nucleic acid that encodes at least 6 internal segments of the genome of a first strain of influenza, in the that the first influenza strain is A / Ann Arbor / 6/60; and at least one genomic segment encoding an immunogenic influenza surface antigen from a second influenza strain, wherein said second strain is a pandemic influenza strain, ii) culturing the host cell population at a temperature equal to or lower than 35 ° C; and iii) recovering a plurality of influenza viruses.

30. The method of item 29, wherein the plurality of vectors comprise at least one isolated nucleic acid, wherein said nucleic acid is selected from the group consisting of:

a) a polynucleotide sequence of SEQ ID No. 1-10 or a complementary sequence thereof;

b) a polynucleotide sequence that encodes a polypeptide of SEQ ID NOS 11-20 or a complementary polynucleotide sequence thereof;

c) a polynucleotide sequence that hybridizes under high stringency conditions to substantially the entire length of the polynucleotide sequence (a); Y

d) a polynucleotide sequence that has a sequence identity of at least 98% to the sequence of