Influenza hemagglutinin and neuraminidase variants

ES 2 394 033 T3

DESCRIPTION

Influenza hemagglutinin and neuraminidase variants

Background of the invention

Vaccines against various strains and evolutions of influenza are important from a community health standpoint, as well as commercially, since each year many individuals are infected with different strains and types of influenza virus. Babies, the elderly, those without adequate health care, and immunocompromised people are at special risk of death from such infections. What makes up the problem with influenza infections is that new strains of influenza are easily generated and can spread between various species, thus necessitating the continued production of new vaccines.

Numerous vaccines capable of producing a specific protective immune response to such different viral and influenza viruses / strains have been produced for over 50 years and include whole virus vaccines, split virus vaccines, surface antigen vaccines, and live virus vaccines. attenuated. However, although the appropriate formulations of any of these types of vaccine are capable of producing a systemic immune response, live attenuated virus vaccines have the advantage of also being capable of stimulating local mucosal activity in the respiratory tract. Considerable work has been done in the production of influenza viruses, and fragments thereof, for vaccine production by the present inventors and co-workers; see, for example, US 2004 029 251 and US 2005 042 229.

Because of the continued emergence (or resurgence) of different strains of influenza, new influenza vaccines are continually desired. Such vaccines are typically created using antigenic residues from newly emerged viral strains, therefore polypeptides and polynucleotides from newly emerged, or newly resurgent viral strains (especially antigenic gene sequences) are highly desirable.

Webby et al. 2004 describes the production of a reference virus against the 2004 H5N1 viruses circulating in Asia. The reference virus described in Webby et al. 2004 is based on a structure A / Puerto Rico / 8/34 comprising an HA sequence derived from A / Hong Kong / 213/03 in which the polybasic amino acids that are associated with high virulence have been removed.

Subbarao et al. 2003 describes a H5N1 regrouped influenza virus based on an A / Puerto Rico / 8/34 structure and a HA and NA sequence of A / HK / 491/97, where the multibasic cleavage site in the HA sequence was mutagenized.

Hien et al. 2004 describes the clinical characteristics and preliminary epidemiological findings in patients with confirmed cases of avian influenza A / H5N1) in Vietnam in December 2003 and January 2004.

The weekly epidemiological registry published by the World Health Organization in 2004 describes five laboratory-confirmed human cases of avian influenza A H5N1 all from Hanoi and obtained in the first month of the year.

Wareing et al. 2001 compared the immunogenicity of the Russian cold adapted donor strains A / Leningrad / 134/17/57 and A / Leningrad / 134/47/57 and the United States strain A / Ann Arbor / 6/60-ca in mice with their respective wild-type parental viruses.

The present disclosure provides new and / or freshly isolated influenza hemagglutinin and neuraminidase variants that are capable of being used 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 is encompassed by the independent claims. This document describes an isolated or recombinant polypeptide that is selected from: the polypeptides encoded by any one of the sequences of for example SEQ ID No: 1 or SEQ ID No: 2, any one of the encoded polypeptides for example SEQ ID No : 1 or SeQ ID No: 2 any one of the polypeptides of SEQ ID No: 11 or SEQ ID No: 12; only the open reading frame of the polypeptides of SEQ ID No: 11 or SEQ ID No: 12; 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 expressed on the surface of a virus (eg, influenza)) of the polypeptide SEQ ID No: 11-12. 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 epitopic tag, an E tag, or an epitopic His tag. . The sequences described in this document are also shown in Appendix 1 and in the sequence listing of this document. The hemagglutinin sequences described herein comprise sequences with modified polybasic cleavage sites (thereby allowing the virus to grow in eggs). The hemagglutinin polypeptide sequences of SEQ ID No: 11-12 comprise the endogenous amino-terminal signal peptide sequences, however, the

ES 2 394 033 T3 hemagglutinin polypeptide sequences described herein also include the mature form (cleaved amino-terminal signal peptide) of hemagglutinin polypeptides. 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.

Also described herein is a composition with one or more polypeptides listed above, or fragments thereof. Further described are polypeptides that specifically bind by a polyclonal antiserum raised against at least one antigen comprising at least one amino acid sequence described above, or a fragment thereof. Such antibodies specific for the polypeptides described above are also described herein. The polypeptides described herein are optionally immunogenic.

Further described are immunogenic compositions comprising an immunologically effective amount of one or more of any of the polypeptides described above as well as methods for stimulating an individual's immune system to produce a protective immune response against influenza virus by administering an immunologically effective amount to the individual. of any of the above polypeptides in a physiologically acceptable carrier.

The invention includes the recombinant influenza virus 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 said recombinant influenza virus. The recombinant influenza virus of the invention for use in methods to stimulate the immune system of an individual to produce a protective immune response against the influenza virus, through the administration of an immunologically effective amount of said recombinant influenza virus in a physiologically vehicle acceptable are also part of the invention.

Also disclosed herein is a composition of matter having two or more of the 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 cleavage of one or more nucleic acids 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 acids 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 a cleaved or amplified fragment or product thereof. These can optionally express a polypeptide encoded by said nucleic acid. Additionally described vectors (eg, plasmids, cosmids, phages, viruses, viral fragments, etc.) comprising any of the nucleic acids described above. Such vectors may optionally comprise an expression vector. Preferred expression vectors include, but are not limited to, vectors comprising the pol I promoter and termination sequences or vectors using both the pol I and pol II promoters and the pol II / pol III promoter system (eg, Zobel et al. , Nucl. Acids Res. 1993, 21: 3607; US20020164770; Neumann et al., Proc. Natl. Acad. Sci. United States of America 1999, 96: 9345; Fodor et al., J. Virol. 1999, 73: 9679; and US20030035814). Cells transduced by said vectors are also described.

In some embodiments, the invention encompasses a virus (eg, influenza virus) comprising one or more of the nucleic acids described above (eg, encoding hemagglutinin and / or neuraminidase) characterized by the appended claims. Immunogenic compositions comprising said virus are also part of the present invention characterized by the appended claims. Said viruses are a reassortant virus, such as a 6: 2 reassortant virus (for example, comprising 6 coding regions of genes from one or more different donor viruses from A / Ann Arbor / 6/60 and at least one region encoding a gene of one or more of the nucleotide sequences described above that encodes hemagglutinin A comprising SEQ ID No: 11 or a hemagglutinin comprising SEQ ID No: 11 and neuraminidase characterized by the appended claims. The reassortant (optionally live) viruses of the invention may include donor viruses that are one or more of, for example, cold sensitive, cold adapted, or attenuated. For example, reassortant viruses can comprise eg PR8, etc. Regrouped viruses of the invention exclude A / Ann Arbor / 6/60. The methods characterized by the appended claims for producing recombinant influenza viruses through the cultivation of a host cell that harbors an influenza virus in a suitable culture medium under conditions that allow the expression of nucleic acid and, the isolation of the recombinant influenza virus from a or more of the host cells 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 characterized by the appended claims. Other embodiments include recombinant influenza viruses of the invention for use in methods of stimulating the immune system of an individual to elicit a protective immune response against influenza virus by administering to the individual an immunologically effective amount of

ES 2 394 033 T3 any of the recombinant influenza viruses characterized by the appended claims (optionally in a physiologically effective vehicle).

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

The immunogenic compositions characterized by the appended claims are also characteristics of the invention. For example, immunogenic compositions comprising any one or more of the above described viruses characterized by the appended claims (for example, together with one or more pharmaceutically acceptable delivery components).

The recombinant virus of the invention for use in methods of eliciting immunogenic responses in a subject through the administration of an effective amount of any of the foregoing viruses (or immunogenic compositions) to a subject is also within the present invention. Additionally, the recombinant virus of the invention for use in methods of prophylactic treatment of a viral infection (eg, viral influenza) in a subject through the administration of any one or more of the viruses described above (or immunogenic compositions) in an amount effective to elicit an immunogenic response against viral infection is also part of the present invention. Subjects for such treatment can include mammals (eg, humans). Said recombinant virus of the invention for use in said methods can also be for in vivo administration to the subject as well as for in vitro or ex vivo administration to one or more cells of the subject. Additionally, said recombinant virus of the invention for use in said methods may also comprise a virus composition and a pharmaceutically acceptable excipient 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 the hemagglutinin and / or neuraminidase polypeptides of one or more pandemic influenza strains and nucleic acid sequences encoding one or more A / Ann Arbor / polypeptides. 6/60. Compositions of matter comprising nucleic acid sequences encoding the hemagglutinin and / or neuraminidase polypeptides of one or more pandemic influenza strains and nucleic acid sequences encoding one or more PR8 or A / Ann Arbor / 6 polypeptides are further described. / 60. Such sequences may include those listed in the sequence listing of this document. Additionally, these compositions include compositions of matter comprising sequences encoding the hemagglutinin and / or neuraminidase of one or more pandemic influenza strains and nucleic acid sequences encoding a selected structural strain in a 6: 2 rearrangement. Said composition described in this document includes sequences that encode hemagglutinin and neuraminidase selected from the sequence list of this document and a structural strain, where the structural strain is PR8 or A / Ann Arbor / 6/60. Compositions described above are also described where the hemagglutinin comprises a modified polybasic cleavage site. The invention also includes a live attenuated influenza vaccine comprising said above compositions characterized by the appended claims.

These and other objects and features of the invention will become fully apparent when the following detailed description is read in conjunction with the accompanying figures and the appendix.

Brief description of the drawings

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

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

Figure 3: Illustrates that the H5N1 / AA ca vaccines are not lethal to mice.

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

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

Figure 6: Shows serum HAI Ab 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.

ES 2 394 033 T3

Figure 8: Illustrates that H5N1 ca reassortant viruses protect mice from lethal challenges with 50, 500, or 5000 LD50 of 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 2004 H5N1 ca vaccine against high dose challenge (105TCID50) with homologous or heterologous wt H5N1 viruses in mice.

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

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

Detailed description

Definitions

Unless otherwise defined, 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 belongs. The following definitions supplement those of the art and refer to the present application and should not necessarily be imputed to any related or unrelated case, for example, to any patent or application of the same owner as the present one. Although any methods and materials similar or equivalent to those described herein may be used in practice to test the present invention, preferred materials and methods are described herein. Accordingly, the terminology used in this document is for the purpose of describing particular embodiments only, and is not intended to be limiting.

As used in this specification and in the appended claims, the singular forms a, an and the, include plural referents unless the context clearly indicates otherwise. Thus, for example, a reference to a virus includes a plurality of viruses; a 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 nucleic acid, polynucleotide, polynucleotide sequence and nucleic acid sequence refer to single or double stranded deoxyribonucleotide or ribonucleotide polymers, chimeras or analogs thereof, or a character string representing this, depending on the context. As used herein, the term optionally includes naturally-occurring nucleotide analog polymers that have the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally-occurring nucleotides (e.g., peptidonucleic acids ). Unless otherwise indicated, a particular nucleic acid sequence optionally encompasses complementary sequences in addition to the explicitly stated sequence. It can be determined for any specified polynucleotide sequence, the given nucleic acid or the complementary polynucleotide sequence (eg, the complementary nucleic acid).

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

A subsequence is any portion of a complete 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, where a substituted amino acid has similar structural or chemical properties, for example, replacement of leucine with isoleucine. Alternatively, a variant can have non-conservative changes, for example, the replacement of a glycine with a tryptophan. An analogous minor variation can also include an amino acid deletion or insertion, or both. Guidelines can be found to determine which remains

ES 2 394 033 T3 amino acids can be substituted, inserted, or deleted without eliminating biological or immunological activity using computer programs well known in the art, for example, DNASTAR software. Examples of conservative substitutions are also described in this document.

The term gene is widely used to refer to any nucleic acid associated with a biological function. Therefore, genes include coding sequences and / or regulatory sequences necessary for their expression. The term gene applies a specific genomic sequence, as well as a cDNA, to an 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 said regulatory proteins such as transcription factors bind, causing the transcription of adjacent or nearby sequences. A tissue-specific promoter or enhancer is one that regulates transcription in a specific tissue or cell type or types.

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

An open reading frame or ORF is a possible translational reading frame of DNA or RNA (eg, of a gene), which has the ability to be translated into a polypeptide. That is, the reading frame is not interrupted by stop codons. However, it should be appreciated 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 spread and / or transfer between organisms, cells, or cellular components. Vectors include plasmids, viruses, bacteriophages, proviruses, phagemids, transposons, artificial chromosomes, and the like, which replicate autonomously and 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 chain, a DNA or RNA conjugated to polylysine, a DNA or RNA conjugated to a peptide, a DNA conjugated to a liposome, or the like, that does not replicate autonomously. In many common cases, but not all, the vectors are plasmids.

An expression vector is a vector, such as a plasmid that is capable of promoting 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 opposite directions relative to the nucleic acid located between the two promoters, so that expression can be initiated in both orientations causing, for example, RNA transcription of both the positive strand (+) or sense, such as negative string (-) or antisense.

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, a nucleic acid or a protein, that is substantially free of components that normally accompany or interact with it in its environment of origin. natural. The isolated biological material optionally comprises additional material not found with the biological material in its natural environment, for example, a wild-type cell or virus. For example, if the material is in its natural environment, such as a cell, the material may have been located in a location in the cell (eg, genome or genetic element) not native to said material found in that environment. For example, a naturally occurring nucleic acid (eg, a coding sequence, a promoter, an enhancer, etc.) becomes isolated if it is introduced by a non-naturally occurring means at a locus in the genome (eg, a vector, such as a plasmid or viral vector, or amplicon) not native to that nucleic acid. Such nucleic acids are also referred to as heterologous nucleic acids. An isolated virus, for example, is in an environment (eg, a cell culture system, or purified from cell culture) different from 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 generic and / or polypeptide components derived from more than one parental viral strain or source. Similarly, the term "chimeric" or "chimera", when referring to a viral protein, indicates that the protein includes polypeptide components (i.e., amino acid subsequences) derived from more than one parental viral strain or source.

ES 2 394 033 T3

The term "recombinant" indicates that the material (eg, a nucleic acid or protein) has been altered artificially or synthetically (not naturally) by human intervention. The alteration can be made to the material in, or removed from, its natural environment or state. 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 made recombinant by recombining nucleic acids, for example, during cloning, DNA stripping, or other procedures, or by chemical or other mutagenesis; a "recombinant polypeptide or recombinant protein" is a polypeptide or protein that is produced by expression of a recombinant nucleic acid; and a recombinant virus, eg, a recombinant influenza virus, is produced by the expression of a recombinant nucleic acid.

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

The term "introduced" when referring to a heterologous or isolated nucleic acid refers to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid can be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or Mitochondrial DNA), converted to an autonomous replicon, or transiently expressed (eg, transfected mRNA). The term includes methods such 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 (MDBK) cells, 293 cells (eg, 293T cells), and COS cells (eg, COSI, COS7 cells), etc.

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

A "protective immune response against influenza virus" refers to an immune response displayed by an individual (eg, a human) that is protective against disease when the individual is subsequently exposed to and / or infected with said influenza virus. In some cases, the influenza virus (for example, naturally circulating) can still cause infection, but cannot cause a serious infection. Typically, the protective immune response elicits detectable levels of host-generated serum and secretory antibodies that are capable of neutralizing virus from the same strain and / or subgroup (and possibly also from a different non-vaccine strain and / or subgroup). 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 a myriad of immunoglobulin variable region genes. Light chains are classified as 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 structural unit (antibody) 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, a Fab dimer that is itself a light chain linked to VH-CH1 by a bond. disulfide. The F (ab) '2 can be reduced under mild conditions to break the disulfide bond in the hinge region thereby converting the F (ab)' 2 dimer to 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 fragments of

ES 2 394 033 T3 antibody). Although various antibody fragments are defined in terms of the digest of an intact antibody, one of ordinary skill in the art will appreciate that such Fab 'fragments can be synthesized de novo chemically or using recombinant DNA methodology. Thus, the term antibody, as used herein, includes antibodies or fragments produced by modifying whole or de novo synthesized antibodies using recombinant DNA methodologies. Antibodies include, for example, polyclonal antibodies, monoclonal antibodies, multiple or single chain antibodies, including single chain Fv antibodies (sFv or scFv) in which a variable heavy chain and a variable light chain are linked together (directly or through of a peptide linker) to form a continuous polypeptide, and humanized or chimeric antibodies.

Influenza virus

The peptides and polynucleotides described herein, eg, SEQ ID Nos: 1-20, are variants of the influenza HA and NA sequences. In general, influenza viruses are composed of an inner ribonucleoprotein core that contains a segmented single-stranded RNA genome and an outer lipoprotein envelope coated by a matrix protein. The genome of influenza viruses are composed of eight straight-chain (-) ribonucleic acid (RNA) segments, which encode the immunogenic proteins hemagglutinin (HA) and neuraminidase (NA), and six internal core polypeptides: the nucleocapsid nucleoprotein ( NP); matrix protein (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 of 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 encodes eleven polypeptides. Segments 1-3 encode three polypeptides, which make up an RNA-dependent RNA polymerase. Segment 1 encodes the PB2 polymerase complex protein. The remaining PB1 and PA polymerase proteins are encoded by segment 2 and segment 3, respectively. In addition, segment 1 of some influenza strains encodes a small protein, PB1-F2, produced from an alternative reading frame within the coding region of PB1. Segment 4 encodes the surface glycoprotein hemagglutinin (HA) involved in cell adhesion and entry during infection. Segment 5 encodes the nucleocapsid nucleoprotein polypeptide (NP), the major structural component associated with viral RNA. Segment 6 encodes a neuraminidase envelope glycoprotein (NA). Segment 7 encodes two matrix proteins, designated M1 and M2, which are translated from mRNA with differential splicing. Segment 8 encodes NS1 and NS2, two nonstructural proteins, which are translated from alternative spliced ​​mRNA variants. The eight genomic segments of influenza B encode 11 proteins. The three largest genes encode components of RNA polymerase, PB1, PB2, and PA. Segment 4 encodes the HA protein. Segment 5 encodes NP. Segment 6 encodes the NA protein and the NB protein. Both proteins, NB and NA, are translated from overlapping reading frames of a bicistronic mRNA. Influenza B segment 7 also encodes two proteins: M1 and BM2. The smaller segment encodes two products: NS1 is translated from full-length RNA, while NS2 is translated from a splice variant 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. After 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 Nos: 1-20) are quite useful for constructing influenza vaccines. The present disclosure includes viruses / vaccines that comprise HA or HA and NA sequences from pandemic strains of influenza (including when the HA sequences comprise modified polybasic cleavage sites such as the modifications described herein); and including when viruses / vaccines comprise a ca structure such as the PR8 structure.

Attempts to produce recombinant and reassortant 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 recombinant and reassortant viruses in culture, thus making it possible to rapidly produce vaccines corresponding to one or many selected antigenic strains of virus, for example, strains A or B, various subtypes of substrains, etc., for example, comprising the hA and / or NA sequences herein. See, the Multiplasmid System for the production of influenza virus, US 2004029251. Typically, cultures are maintained in a system, such as a

ES 2 394 033 T3 cell culture incubator, in controlled humidity and CO2, 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 administration. 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. It will be appreciated that the Ha and Na sequences of this document are capable of reassortment with various viral genes or viral types (eg, several different structures such as PR8, etc., which contain the other influenza genes present in a reassortment, namely genes not HA and not NA).

Various embodiments of this document may comprise live attenuated vaccines indicated in the claims, having the HA and / or NA sequences of SEQ ID No: 11 or SEQ ID No: 12 of this document, for pandemic influenza. Said vaccines comprise the HA or HA and NA sequences of SEQ ID Nos: 11-12, or their corresponding nucleotides of for example SEQ ID Nos: 1-2. One problem that arises from culturing viral vaccine strains (for example, reassorted) in eggs is that avian strains (which may be implicated in pandemics) can kill the eggs in which vaccines have to be produced and are therefore difficult to manipulate, produce, etc. through the use of traditional regrouping production (non-plasmid rescue). These avian strains are of interest as evidence indicates that they can cause influenza in humans and possible pandemics. Therefore, 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) is quite desirable. It will be appreciated, however, that the present sequences are also capable of use with non-plasmid or traditional systems.

Aquatic birds (among others) can be infected by influenza A viruses of 15 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes. These birds can serve as reservoirs from which new subtypes of influenza can enter human populations and cause pandemics. The observation that avian influenza A H7N7 viruses infected humans in the Netherlands in 2003 and avian H5N1 and H9N2 viruses previously infected humans in Hong Kong and China raises concern that these (and other) subtypes have the potential to cause pandemics. Therefore, vaccines must prevent human infections with avian influenza A viruses. Live attenuated influenza A virus vaccines against human influenza viruses were recently licensed in the United States. See above. These vaccines are reassorted H1N1 and H3N2 viruses in which the genes for the internal proteins of A / Ann Arbor (AA) / 6/60 (H2N2) cold adapted virus (ca) confer the cold adapted attenuation phenotypes and are sensitive at virus temperature ca AA in reassorted viruses (ie, those with the non-Ann Arbor strain hemagglutinin and neuraminidase genes). Classical genetic reassortment and plasmid-based reverse genetics techniques have been applied to generate reassortant viruses characterized in the appended claims. The generation and evaluation of these reassortant viruses as vaccine seed viruses are important steps in pandemic preparations. It is contemplated that clinical trials can establish the safety, effectiveness, and immunogenicity of such live attenuated pandemic vaccines. The methods of construction and use of such viruses and vaccines are also included. Pandemic viral strains as used herein are defined as a subtype of influenza strain A virus that is not circulating in the human population, which is declared a pandemic strain by the Centers for Disease Control or the World Organization for Disease Control and Prevention. Health or are generally known as such within the scientific community.

As described herein, antigenic sequences (eg, HA sequences) as well as viruses and vaccines of said viruses comprise modified polybasic cleavage sites. Some highly pathogenic strains of avian pandemic influenza comprise multiple basic amino acid cleavage sites within hemagglutinin sequences. See, for example, Li et al., J. of Infectious Diseases, 179: 1132-8, 1999. Said cleavage sites, in typical embodiments herein, are modified or altered, for example, in their sequences compared to the wild-type sequences from which current sequences are derived (for example, to disable cleavage or reduce cleavage there, 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, typically four polybasic residues (RRKK) are deleted at 326-329 from mature H5 in sequences herein (compared to wt). See sequence listing and Figure 1. Polybasic cleavage sites can be modified in a number of ways. For example, the polybasic cleavage site can be removed 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.); furthermore, the nucleotides encoding the amino acid residue directly after the cleavage site can also be modified. 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, therefore hemagglutinin polypeptide sequences include both the mature form (cleaved amino-terminal signal peptide) of 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.

ES 2 394 033 T3

The terms temperature sensitive, cold adapted and attenuated apply to viruses (typically used as vaccines or for the production of vaccines) optionally encompassing the present sequences are well known in the art. For example, the term temperature sensitive (ts) indicates, for example, that the virus shows a factor 100 or greater reduction in titer at 39 ° C relative to 33 ° C for influenza A strains, or that virus shows a factor 100 or greater reduction in titer at 37 ° C relative to 33 ° C for influenza B strains. the expression cold adapted (c) indicates that the virus shows growth at 25 ° C within a factor of 100 of its growth at 33 ° C, while the term attenuated (att) indicates that the virus replicates in the upper respiratory tract ferrets but is not detectable in their lung tissues, and they do not cause influenza-like illness in the animal. It will be understood that viruses with intermediate phenotypes, that is, viruses that show decreases in titers of less than 100 times at 39 ° C (for strain A virus) or 37 ° C (for strain B virus), or that show growth at 25 ° C that is more than 100 times their growth at 33 ° C (for example, within factor 200, factor 500, factor 1000, factor 10,000 less), and / or show reduced growth in the lungs relative to growth in the ferret's upper respiratory tract (i.e. partially attenuated) and / or reduced influenza-like disease 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 in other ts, cs, ca, and / or att viruses and vaccines).

Methods and compositions for the prophylactic administration of vaccines

The recombinant and reassortant viruses of the invention (for example, those comprising polynucleotides of, for example, SEQ ID Nos: 1-2, polypeptides of SEQ ID Nos: 11-12), can be administered prophylactically in an immunologically effective amount and in an appropriate vehicle or excipient to stimulate a specific immune response for one or more strains of influenza virus 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, aqueous buffered saline, aqueous dextrose solutions, aqueous glycerol solutions, ethanol, or combinations thereof. The preparation of said solutions that ensures 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 undesirable effects, and to suit the particular route of administration, eg, subcutaneous, intramuscular, intranasal, etc.

A related aspect of the invention provides the recombinant influenza virus of the invention 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 strains of influenza virus (ie, against the HA and / or NA strains described herein). Preferably, the administration of influenza viruses elicits a protective immune response against said strains. Dosages and methods for eliciting a protective immune response against one or more strains of influenza are known to those of skill in the art. See, for example, USPN 5,922,326; Wright et al., Infect. Immun. 37: 397-400 (1982); Kim et al., Pediatrics 52: 5663 (1973); and Wright et al., J. Pediatr. 88: 931-936 (1976). For example, influenza viruses are delivered 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 on, for example, age, physical condition, body weight, sex, diet, time 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 drop, large particle aerosol (greater than about 10 microns), or spray into the upper respiratory tract. Although none of the above routes of delivery elicit 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, an attenuated cold adapted and / or temperature sensitive recombinant or regrouped 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 a different route, to achieve the desired prophylactic effect.

Typically, the attenuated recombinant influenza virus of this invention used in a vaccine is sufficiently attenuated so that symptoms of infection, or at least symptoms of severe infection, do not occur in the majority of immunized (or otherwise infected) individuals. ) with attenuated influenza virus. In some cases, the attenuated influenza virus may still be capable of producing symptoms of mild illness (eg, mild upper respiratory illness) and / or of spreading to unvaccinated individuals. However, its virulence is sufficiently abridged that serious lower respiratory tract infections do not occur in the vaccinated or accidental host.

ES 2 394 033 T3

Alternatively, an immune response can be stimulated by ex vivo or in vivo targeting of dendritic cells with influenza viruses comprising the sequences herein. For example, proliferating dendritic cells are exposed to viruses in a sufficient 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 may 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 infection with wild-type influenza. Likewise, adults who are particularly susceptible to repeated or severe influenza infection, such as, for example, healthcare workers, day care workers, family members of young children, the elderly, and individuals with compromised cardiopulmonary function may requiring multiple immunizations to establish and / or maintain protective immune responses. Levels of induced immunity can be monitored, for example, by measuring the amounts of neutralizing serum and secretory antibodies, and dosages can be 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 of influenza antigens. Such adjuvants include: Freund's complete adjuvant, Freund's incomplete 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 QS21.

If desired, administration of the prophylactic influenza virus vaccine can be performed in conjunction with the administration of one or more immunostimulatory molecules. Immunostimulatory molecules include various cytokines, lymphokines, and chemokines with immunostimulatory, immunopotentiating, and proinflammatory 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 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 may induce cross protection against an influenza virus of different strains and / or subgroups, cross protection can be enhanced, if desired, by vaccinating the individual with viruses. attenuated influenza of at least two strains, for example, each of which represents 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. . Vaccine mixtures (or multiple vaccinations) may comprise components of human and / or non-human influenza strains (eg, avian and human, etc.). Also, the attenuated influenza virus vaccines of this invention can optionally be combined with vaccines to 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 HA and NA coding sequence. An example of these NCRs is shown in SEQ ID Nos: 1-9 (outside of ORFs). It is also known that primers can be prepared for these NCRs to facilitate amplification of the full HA and Na segments of the influenza virus. (See, for example, Hoffmann et al. Arch Virol. 2001 Dec; 146 (12): 2275-89). Furthermore, it is known that influenza HA and NA NCRs can increase the efficacy of regrouping. Therefore, the polynucleotide sequences of these NCRs are described herein. When amplifying the HA and NA segments of any pandemic strain, polynucleotide primers could be prepared and used to bind to conserved regions (e.g., between related strains) of the HA and nA NCRs for amplification (e.g., by RT- PCR).

The HA and NA polynucleotides of the virus of the invention, for example SEQ ID No: 1 and SEQ ID No: 2, are optionally used in several different competitions alternative to, or in addition to, the vaccines described above. Other exemplary uses are described herein for illustrative purposes and not as limitations on the actual range of uses. Different methods of construction, purification, and characterization of the nucleotide sequences of the disclosure are also described herein. In this document it is disclosed that nucleic acids including one or more polynucleotide sequences described above are favorably used as probes for the detection of corresponding or related nucleic acids in a variety of

ES 2 394 033 T3 contexts, such as in nucleic acid hybridization experiments, for example, to find and / or characterize homologous influenza variants (eg, homologous to the sequences of this document, etc.) infecting other species or in different outbreaks of influenza, etc. Probes can be DNA or RNA molecules, such as cloned or genomic DNA restriction fragments, cDNAs, PCR amplification products, transcripts, and oligonucleotides, and can vary in length from oligonucleotides as short as about 10 nucleotides in length. to full length sequences or cDNA in excess of one kb or more. For example, a probe includes a polynucleotide sequence or subsequence selected, for example, from sEq ID No: 1 or SEQ ID No: 2, or sequences complementary thereto. Alternatively, polynucleotide sequences that are variants of one of the sequences designated above as probes are used. More typically, such variants include one or a few conservative nucleotide variations. For example, pairs (or series) of oligonucleotides can be selected, in which the two (or more) polynucleotide sequences are conservative variations of each other, where a polynucleotide sequence corresponds identically to a first variant and the other or others correspond in a similar way. identical to additional variants. Such pairs of oligonucleotide probes are particularly useful, for example, for specific hybridization experiments to detect polymorphic nucleotides or to, for example, detect homologous variants of influenza HA and NA, for example, homologous to the present HA and NA sequences, that infect other species or are present in different (eg, temporally and / or geographically different) outbreaks of influenza. In other applications, probes that are more divergent are selected, that is, probes that are at least about 91% (or about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 98.5%, about 98.7%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, or about 99.6% or more about 99.7%, about 99.8%, about 99.9% or more) identical.

The probes described herein, for example, exemplified by sequences derived from the sequences herein, can also be used to identify additional useful polynucleotide sequences in accordance with routine procedures in the art. One or more probes are used, as described above, 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 probes of the sequences herein, ie, variants, homologues, etc. 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.

Oligonucleotide probes are optionally produced by a variety of methods well known to those of skill in the art. More typically, they are produced by well-known synthetic methods, such as the solid phase phosphoramidite triester method described by Beaucage and Caruthers (1981) Tetrahedron Letts 22 (20): 1859-1862, for example, using an automated synthesizer, or as described in Needham-Van Devanter et al. (1984) Nucl Acids Res, 12: 6159-6168. Oligonucleotides can also be custom made and ordered from a variety of commercial sources known to those of skill in the art. Oligonucleotide purification, when necessary, is typically performed by native acrylamide gel electrophoresis or anion exchange HPLC as described in Pearson and Regnier (1983) J Chrom 255: 137-149. The sequence of the synthetic oligonucleotides can be verified using the chemical degradation method of Maxam and Gilbert (1980) in Grossman and Moldave (eds.) Academic Press; New York, Methods in Enzymology 65: 499-560. Custom oligos can also be readily ordered from a variety of commercial sources known to those of skill in the art.

In other circumstances, for example, in relation to the attributes of cells or organisms that express the polynucleotides and polypeptides described herein (for example, those harboring viruses comprising the sequences described herein), 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 (for example, SEQ ID Nos: 11-12) and / or encoded by polynucleotide sequences described herein are favorably used, for example, selected from SEQ ID No: 1 or SEQ ID No: 2, 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 comprising an amino acid sequence of at least 5 contiguous amino acid residues, or at least 10 contiguous amino acid residues; or at least 15 contiguous amino acid residues, or at least 20 contiguous amino acid residues, or at least 25 contiguous amino acid residues, or at least 40 contiguous amino acid residues, or at least 50 contiguous amino acid residues, or at least 60 contiguous amino acid residues, or at least 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 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, or at least 400 contiguous, or at least 450 contiguous, or at least 500 contiguous, or at least 550 contiguous amino acid residues of the amino acid sequence of the HA or NA polypeptide described herein (for example, SEQ ID Nos: 11-12). The polynucleotides that encode said

ES 2 394 033 T3 polypeptide fragments, and antibodies that specifically bind to such polypeptides are also described herein.

Antibodies specific for any polypeptide sequence or subsequence, for example, of SEQ ID No: 11 or SEQ ID No: 12, and / or encoded by the polynucleotide sequences of for example SEQ ID No: 1 or SEQ ID No: 2 , 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 comprising amino acid subsequences, for example, those given herein, or encoded by polynucleotide sequences shown herein, in situ, in a tissue array, in a cell. , tissue or organism, for example, an organism infected by an unidentified influenza virus or the like. Antibodies can be directly labeled with a detectable reagent, or detected directly by labeling a secondary antibody specific for the heavy chain constant region (ie, isotype) of the 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 sequences and / or neuraminidase-like sequences, and to detect differences in strains in influenza clinical isolates using recombinant or chemically synthesized 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 sequences selected from those given herein, can also be used to identify additional useful polynucleotide sequences (such as to characterize additional strains of influenza) according to routine procedures in The technique. 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 in this document. In turn, each of these identified sequences can be used to prepare probes, including variant probe pairs or arrays 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 has been exposed to. become infected with influenza, or a particular strain or strains of influenza. Detection of hybridization of the probe selects 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 probe polynucleotide is selected.

It will be appreciated that the design of the probe is influenced by the intended application. For example, when multiple allele-target specific probe interactions have 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 series are very 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 of GC). Although melting temperature is a primary consideration in probe design, other factors are optionally used to further adjust probe construction, such as selection 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, for example a plasmid, a cosmid, a phage, a virus, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), etc., into which one or more has been inserted more of the polynucleotide sequences, for example, of SEQ ID No: 1 or SEQ ID No: 2, or a subsequence thereof, etc., 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 example, the construct further comprises regulatory sequences, including, for example

Example ES 2 394 033 T3, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available.

Polynucleotides 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, a hemagglutinin and / or neuraminidase molecule described herein) . Such vectors include chromosomal, nonchromosomal, and synthetic DNA sequences, eg, derived from 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). Any vector that is capable of introducing genetic material into a cell, and, if replication is desired, that is replicable in the relevant host can be used.

In an expression vector, the HA and / or NA polynucleotide sequence of interest is physically arranged in proximity and orientation to 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: the LTR or SV40 promoter, the lac or trp promoter from E. coli, the Pl promoter from phage lambda, and other promoters known to control gene expression in prokaryotic or eukaryotic cells or their viruses.

A variety of promoters are suitable for use in expression vectors to regulate the transcription of influenza virus genomic 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 tissues or cells, can be substituted. Numerous mammalian and viral promoters are available, eg from humans, or can be isolated according to the specific application contemplated. For example, alternative promoters derived from the genomes of animal and human viruses include promoters such as the promoter from 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 short, eg, 10-500 bp, cis-acting DNA elements that act in concert with a promoter to increase transcription. Many enhancer sequences have been isolated from mammalian genes (hemoglobin, elastase, albumin, alpha-fetoprotein, and insulin), and viruses from eukaryotic cells. The enhancer can be spliced ​​into the vector 5 'or 3' to the heterologous coding sequence, but typically is inserted 5 'to the promoter. Typically, the promoter, and if desired, additional transcription enhancer sequences are chosen to optimize expression in a host cell type into which heterologous DNA has to be introduced (Scharf et al (1994) Heat stress promoters and transcription factors Results Probl Cell Differ 20: 12562; 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 usually available from 5 'and occasionally 3' untranslated regions of eukaryotic or viral DNA or cDNA. The SV40 polyadenylation signal sequences can provide a bidirectional polyadenylation site that isolates the transcription of (+) strand mRNA molecules from the PolI promoter that initiates replication of the (-) strand viral genome.

In addition, as described above, expression vectors optionally include one or more selection marker genes to provide a phenotypic trait for selection of transformed host cells, in addition to the previously listed genes, markers such as dihydrofolate reductase or resistance to neomycin are suitable for selection in eukaryotic cell culture.

The vector containing the appropriate nucleic acid sequence as described above, as well as an appropriate promoter or control sequence, can be used to transform a host cell to allow expression of the protein. Although the vectors described herein can replicate in bacterial cells, it will more often be desirable to introduce them into mammalian cells, eg, Vero cells, BHK cells, MDCK cells, 239 cells, COS cells, or the like, for expression purposes.

As described elsewhere, the HA and NA sequences herein may be comprised within plasmids involved in plasmid rescue reassortment. See, for example, US 2004 029251 and US 2005 042229. 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 polI / polIII promoter system (e.g. , Zobel et al., Nucl. Acids Res. 1993, 21: 3607; US20020164770; Neumann et al., Proc. Natal.

ES 2 394 033 T3

Acad. Sci. USA 1999, 96: 9345; Fodor et al., J. Virol. 1999, 73: 9679; and US20030035814). The regroups produced can include the HA and NA genes arranged with the other 6 influenza genes of the donor strain A / Ann Arbor / 6/60 (and / or derivatives and modifications thereof), the structure of the donor strain PR8, the donor strain structure A / Leningtad / 17, etc. Other structural strains are described, for example, in US20040137013 and US20030147916.

Additional elements of expression

More commonly, the genomic segment encoding the influenza virus HA and / or NA protein includes any additional sequences necessary for its expression, including translation into a functional viral protein. In other situations, a minigene, or other artificial construct encoding the viral proteins, 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 transcript elements and start 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 phase with the polynucleotide sequence of interest, for example, to direct expression of the polypeptide to a desired cell compartment, membrane, or organelle, or to direct secretion of the polypeptides to the periplasmic space or into the cell culture medium. Such sequences are known to those of skill in the art, and include secretory leader peptides, organelle targeting sequences (eg, nuclear localization sequences, ER retention signals, mitochondrial transit sequences), a membrane (eg, stop 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 that include an encoding sequence incorporating, for example, a polynucleotide sequence described herein, a translation start codon, and associated sequence elements are inserted into the appropriate expression vector simultaneously with the polynucleotide sequence of interest. In such cases, additional translation control signals are often not required. However, in cases where only a polypeptide coding sequence, or a portion thereof, is inserted, exogenous translational control signals are often provided including, for example, an ATG start codon for expression of the sequence. relevant. The start codon is placed in the correct reading frame to ensure transcription of the polynucleotide sequence of interest. Exogenous transcriptional elements and start 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 (see, for example, Scharf D. 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, eg, USPN 5,166,057 to Palese et al.). This method was originally applied to design 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 virus (Schnell et al. (1994) EMBO J. 13: 4195 -4203); VSV (Lawson et al. (1995) Proc. Natl. Acad. Sci. USA 92: 4477-4481); measles virus (Radecke et al. (1995) EMBO J. 14: 5773-5784); rinderpest virus (Baron and Barrett (1997) J. Virol. 71: 1265-1271); human parainfluenza virus (Hoffman and Banerjee (1997) J. Virol. 71: 3272-3277; 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 similar techniques for producing an influenza virus comprising the HA and NA sequences described herein. Recombinant influenza viruses produced according to such methods are a feature of the disclosure as are 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 genetically engineered (i.e.

ES 2 394 033 T3 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.

More commonly, 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. Typically, co-cultures including two of the above cell lines are employed, eg, MDCK cells and 293T or COS cells at a ratio, eg, 1: 1, to improve replication efficiency. Typically, cells are grown in standard commercial culture medium, such as Dulbecco's Modified Eagle's Medium supplemented with serum (eg, 10% fetal bovine serum), or in serum-free medium, at controlled humidity and CO2 concentration. suitable for maintaining the buffered pH neutral (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 typically those previously used with the particular host cell selected for expression, and will be apparent to those skilled in the art and in the references cited herein, including, for example , Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, 3^to edition, Wiley-Liss, New York and references cited in that document. Other helpful references include, 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. Furthermore, variations in such procedures adapted to the present disclosure are readily determined through routine experimentation and will be known 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-containing or serum-free medium. In some cases, for example, for the preparation of purified viruses, it is typically desirable to grow the host cells under serum-free conditions. Cells can be cultured on a small scale, eg, less than 25 ml of medium, culture tubes or flasks, or in large shake flasks; in roller bottles, or in carrier microbeads (e.g. DEAE-Dextran carrier microbeads, such as Dormacell, Pfeifer, and Langen; Superbead, Flow Laboratories; styrene-trimethylamine copolymer beads, such as Hillex, SoloHill, Ann Arbor) in flasks, flasks or reactor cultures. Carrier microbeads 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 carrier microbeads providing more than 8,000 square centimeters of growing area. 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 below 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 reassorted influenza viruses using temperature-dependent multi-plasmid systems (see, eg Multi-Plasmid System for the Production of Influenza Virus, US 2004 029251), 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 of this document (for example, when reassembled viruses are to be produced from segments in vectors) vectors comprising segments of the influenza genome are introduced (for example, transfected) into host cells according to well-known methods. in the art 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 cells.

ES 2 394 033 T3 hosts, such as COS cells, 293T cells or combinations of COS or 293T cells and MDCK cells, using the TransIT-LT1 polyamine transfection reagent (Mirus) according to the manufacturer's instructions to produce reassorted viruses, etc. Thus, in one example, approximately 1 µg of each vector is introduced into a host cell population with approximately 2 µl of TransIT-LT1 diluted in 160 µl of medium, preferably serum-free medium, in a total volume of 200 µl. The DNA: transfection reagent mixtures are incubated at room temperature for 45 minutes followed by the addition of 800 µl 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 genomic segments (PB2, PB1, PA, NP, M, NS, HA and NA) are mixed with approximately 20 µl of TransIT -LT1 and are 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 genomic 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 about 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 µl are added to the cells in the cuvette, which is then mixed gently by tapping. Electroporation is performed according to manufacturer's instructions (eg BioRad Gene Pulser II with Capacitance Extender Plus attached) at 300 volts, 950 microFaraday, and with a time constant between 28-33 msec. These cells are remixed by 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 adenoviral transcription / translation complex consisting of the late promoter and the tripartite leader sequence. Insertion into a non-essential E1 or E3 region of the viral genome will produce a viable virus capable of expressing 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 modifications of the protein include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing, which cleaves a precursor form into a mature form, of the protein, is sometimes important for proper insertion, folding, and / or function. In addition, proper localization within a host cell (eg, on the cell surface) 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 said post-translational activities and can be chosen to ensure the correct modification and processing of the present foreign protein introduced.

For 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 containing viral origins of replication or endogenous expression elements and a selection marker gene. For example, after vector introduction, cells are allowed to grow for 1-2 days in enriched medium before switching to selective medium. The purpose of the selection marker is to confer resistance to selection, and its presence allows the cultivation and recovery of cells that successfully express the introduced sequences. Thus, resistant clumps of stably transformed cells, for example derived from a single cell type, can be proliferated 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 intracellularly retained, depending on the sequence (for example, depending on fusion proteins that encode a membrane retention signal or the like) and / or the vector used.

ES 2 394 033 T3

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 infra, details regarding cell culture can be found in Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, NY; Gamborg and Phillips (eds.) (1995) Plant Cell. Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL.

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 high-level expression of easily purified fusion proteins are favorably used. Such vectors include, but are not limited to, multifunctional 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 translation initiation methionine -terminal and subsequent 7 beta-galactosidase residues encoding a catalytically active beta-galactosidase fusion protein; pIN vectors (Van Heeke and Schuster (1989) J Biol Chem 264: 5503-5509); pET vectors (Novagen, Madison WI); and the like. Also, in Saccharomyces cerevisiae yeast, 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, infra, 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 Nos: 1-2), including conservative variations of nucleic acids described herein. This comparative hybridization method is a preferred method for distinguishing nucleic acids. Furthermore, target nucleic acids that hybridize to the nucleic acids represented by those shown herein under high, ultra-high and ultra-ultra-high stringency conditions are characteristic 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 higher than that observed for hybridization to any of the unpaired target nucleic acids.

Nucleic acids hybridize when they associate, typically 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 below. Hames and Higgins (1995) Gene Probes 1 IRL Press at Oxford University Press, Oxford, England, (Hames and Higgins 1) and Hames and Higgins (1995) Gene Probes 2 IRL Press at Oxford University Press, Oxford, England (Hames and Higgins 2) provide details on the synthesis, labeling, detection and quantification of DNA and RNA, including oligonucleotides.

An example of stringent hybridization conditions for 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, with hybridization performed during one night. An example of stringent wash conditions comprises a 0.2x SSC wash at 65 ° C for 15 minutes (see, Sambrook, infra for a description of SSC buffer and other nucleic acid hybridization parameters). High stringency wash is often preceded by low stringency wash to remove background signal from the probe. 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 salinity and lower temperature) increase sensitivity, but can produce non-specific hybridization signals and high background signals. Conditions of greater stringency (for example, using lower salinity and higher temperature that is closer to the Tm) decrease the background signal, typically leaving mainly the specific signal. See, also, Rapley, R. and Walker, JM eds., Molecular Biomethods Handbook (Humana Press, Inc. 1998).

ES 2 394 033 T3

Stringent hybridization wash conditions in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different in different environmental parameters. Extensive guidance for nucleic acid hybridization is found in Tijssen (1993), supra, and in Hames and Higgins, 1 and 2. Stringent hybridization and wash conditions can easily be determined empirically for any test nucleic acid. For example, in determining highly stringent hybridization and wash conditions, the wash and hybridization conditions are gradually increased (e.g., increasing the temperature, decreasing the salt concentration, increasing the detergent concentration, and / or increasing the solvent concentration. organics such as formalin on hybridization or wash), until a selected set of criteria is met. For example, the hybridization and wash conditions are gradually increased until a probe binds to a fully 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 listing herein.

Very stringent conditions are selected to match the thermal melting point (Tm) for a particular probe. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the test sequence hybridizes to a perfectly matched probe. For the purposes of the present 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 ionic strength and pH (as indicated below, conditions highly stringent can also be mentioned in comparative terms). Target sequences that are closely related 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 hybridization and wash conditions are increased until the signal-to-noise ratio for binding a probe to a perfectly matched complementary target nucleic acid is at least 10x as high as that seen 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.

In the determination of stringent or highly stringent conditions of hybridization (or even more stringent hybridization) and washing, the hybridization and washing conditions are gradually increased (for example, 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 washing conditions are gradually increased until a probe comprising one or more polynucleotide sequences of the description, for example, unique sequences or subsequences selected from those given herein (for example, SEQ ID Nos: 12) and / or complementary polynucleotide sequences, binds to a perfectly matched complementary target (again, a nucleic acid comprising one or more nucleic acid sequences or subsequences selected from those given herein and / or complementary polynucleotide sequences of the themselves), 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 presentation, and / or complementary polynucleotide sequences thereof), as desired.

Using the polynucleotides described herein or subsequences thereof, new 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, for example, SEQ ID Nos: 1-2).

Also, 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 probe binding to perfectly matched complementary 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 in

ES 2 394 033 T3 said 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 happens, 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

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 issues related to, for example, the generation of HA and / or NA molecules, etc.

Various types of mutagenesis are also described herein, eg, to produce and / or isolate, eg, new or freshly isolated HA and / or NA molecules and / or to further modify / mutate polypeptides (eg, HA molecules and NA as in SEQ ID Nos: 11-12) described herein. They include, but are not limited to, site-directed random mutagenesis, homologous recombination (DNA carryover), mutagenesis using uracil-containing templates, oligonucleotide-directed mutagenesis, phosphorothioate modified DNA mutagenesis, mutagenesis using gap duplex DNA, or the like. Additional suitable methods include point mismatch repair, mutagenesis using repair deficient host strains, restriction selection and restriction purification, deletion mutagenesis, total gene synthesis mutagenesis, double strand break repair, and the like. Also described herein is mutagenesis, eg, involving chimeric constructs. Mutagenesis may be guided by known information from the naturally occurring molecule or altered or mutated naturally occurring molecule, eg, sequence, sequence comparisons, physical properties, crystal structure, or the like.

The above texts and examples found in this document describe these procedures as well as the following publications (and references cited therein): Sieber, et al., Nature Biotechnology, 19: 436-460 (2001); Ling et al., Approaches to DNA mutagenesis: an overview, Anal Biochem 254 (2): 157-178 (1997); Dale et al., Oligonucleotide-directed random mutagenesis using the phosphorothioate method, Methods Mol Biol 57: 369-374 (1996); IA Lorimer, I. Pastan, Nucleic Acids 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., Oligonucleotided-directed construction of mutations: a gapped duplex DNA procedure without enzymatic reactions in vitro, Nucl Acids Res 16: 6987-6999 (1988); Kramer et al., Improved enzymatic in vitro reactions in the gapped duplex DNA approach to oligonucleotide-directed construction of mutations, Nucl Acids Res 16: 7207 (1988); Sakamar and Khorana, Total synthesis and expression of a gene for the a-subunit of bovine rod outer segment guanine nucleotide-binding protein (transducin), Nucl Acids Res 14: 6361-6372 (1988); Sayers et al., 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 the presence of ethidium bromide, (1988) Nucl Acids Res 16: 803-814; Carter, Improved oligonucleotide-directed mutagenesis using M13 vectors, Methods in Enzymol 154: 382-403 (1987); Kramer and Fritz Oligonucleolide-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 and Smith, Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template, Methods in Enzymol 154: 329-350 (1987); Carter, Site-directed mutagenesis, Biochem J 237: 1-7 (1986); Eghtedarzadeh and Henikoff, Use of oligonucleotides to generate large deletions, Nucl Acids Res 14: 5115 (1986); Mandecki, Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli: a method for site-specific mutagenesis, Proc Natl Acad Sci USA, 83: 7177-7181 (1986); Nakamaye and Eckstein, Inhibition of restriction endonuclease Nci I c / eavage 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 and 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

ES 2 394 033 T3 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: 94441-9456 (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 and Smith, Oligonucleotide-directed mutagenesis of DNA fragments cloned in M13 vectors, Methods in Enzymol 100: 468-500 (1983); and Zoller and Smith, Oligonucleotidedirected mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any DNA fragment, Nucl Acids Res 10: 6487-6500 (1982). Additional details of many of the above methods can be found in Methods in Enzymol Volume 154, which also describes controls useful for troubleshooting various methods of mutagenesis, gene isolation, expression, and other methods.

Oligonucleotides, eg, for use in mutagenesis, eg, mutating libraries of HA and / or NA molecules, or altering them, 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 Acids Res. 12: 6159-6168 (1984).

Additionally, essentially any custom or conventional nucleic acid can be ordered 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. Additionally, custom peptides and antibodies can be ordered from any of a variety of sources, such as PeptidoGenic (available from 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, eg, SEQ ID Nos: 1-2. Host cells are engineered (eg, transformed, transduced, or transfected with the vectors described herein, which may 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 small particles with nucleic acid within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327, 70-73 (1987)). Berger, Sambrook, and Ausubel provide a variety of appropriate transformation methods. See, above.

Several well-known methods are available for introducing target nucleic acids into bacterial cells, 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 amount of plasmids containing DNA constructs of this invention. Bacteria are grown to log phase and plasmids within bacteria can be isolated by a variety of methods known in the art (see, for example, Sambrook). In addition, a plethora 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 transcriptional and translational terminators, transcriptional 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 and Smith, Gene 8:81 (1979); Roberts, et al., Nature, 328: 731 (1987); Schneider, B., et al., Protein Expr Purif 6435: 10 (1995); Ausubel, Sambrook, Berger (all above). A catalog of bacteria and bacteriophages useful for cloning is provided, for example, by the ATCC, eg, The ATCC Catalog of Bacteria and Bacteriophage (1992) Gherna 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 useful vectors with the sequences of this document have been illustrated above in the section regarding the production of influenza viruses for vaccines and the references cited therein.

Polypeptide production and recovery

After transduction of a suitable host cell line or strain and culturing the host cells to an appropriate cell density, the selected promoter is induced by an appropriate medium (eg, temperature change or chemical induction) and the cells are cultured. for an additional period. In

In some embodiments, then a secreted polypeptide product, eg, an HA and / or NA polypeptide as in a secreted fusion protein form, etc., is recovered from the culture medium. In other embodiments, a viral particle of the invention containing an HA comprising the amino acid sequence of SEQ ID No: 11 and a NA polypeptide is produced from the cell. Alternatively, cells can be harvested by centrifugation, altered by physical or chemical means, and the resulting crude extract retained for further purification. Eukaryotic or microbial cells employed in protein expression can be altered by any convenient method, including sonication by freeze-thaw cycles, mechanical alteration, or use of cell lysis agents, or other methods, which are well known to those skilled in the art. The technique. Furthermore, cells expressing a HA and / or a NA polypeptide product 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 examined in this way (for example, having HA and / or NA molecules (or for example, comprising fusion proteins or the like) on the antibody-binding 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 references noted herein, a variety of purification methods are well known in the art, including, for example, those set forth 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 in Oxford, Oxford, England; Harris and Angal Protein Purification Methods: A Practical Approach iRL Press in 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 typically recovered from the culture medium, in which the infected (transfected) cells have been cultured. Typically, 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 cleared medium supernatant is then centrifuged to pellet influenza viruses, eg, at 15,000 xg, for about 3-5 hours. After resuspension 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 centrifugation in a density gradient over sucrose (60% -12%) or potassium tartrate (50% -10%). Continuous or step gradients are suitable, for example a sucrose gradient 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 most large-scale commercial applications, the virus is elutriated from density gradients using a zonal centrifuge rotor operating in continuous mode. Sufficient additional detail is provided to guide those skilled in the art through the preparation of influenza viruses from tissue culture, for example, in Furminger. Vaccine Production, in Nicholson et al. (eds.) Textbook of Influenza p. 324-332; 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 p. 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 can be employed to produce polypeptides comprising an amino acid sequence or subsequence of, for example, the sequences given herein such as SEQ ID Nos: 11-12, or encoded by the sequences polynucleotides of for example SEQ ID Nos: 1-12. Several suitable in vitro transcription and translation systems are commercially available. A general guide to in vitro transcription and translation protocols is found in Tymms (1995) In vitro Transcription and Translation Protocols: Methods in Molecular Biology Volume 37, Garland Publishing, NY.

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 Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City, CA). If desired, subsequences can be chemically synthesized separately, and combined using chemical methods to provide full-length polypeptides.

ES 2 394 033 T3

Modified amino acids

The expressed polypeptides described herein can contain one or more modified amino acids. The presence of modified amino acids may be advantageous in, for example, (a) increasing the 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, eg, co-translationally or post-translationally, during recombinant production (eg, N-linked glycosylation in NXS / T motifs during expression in mammalian cells) or are modified by synthetic means (eg, by PEGylation).

Non-limiting examples of a modified amino acid include a glycosylated amino acid, a sulfated amino acid, a prenylated amino acid (eg, farnesylated, geranylgeranylated), an acetylated amino acid, an acylated amino acid, a PEG-ylated amino acid, a biotinylated amino acid, a carboxylated amino acid, a phosphorylated amino acid, and the like, as well as amino acids modified by conjugation with, for example, lipid moieties or other organic derivatizing agents. The literature is replete with suitable references to guide those skilled in the art in amino acid modification. Examples of 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 (for example, encoding HA and / or NA polypeptides exemplified by SEQ ID Nos: 11-12) with, for example, immunoglobulins (or parts thereof), sequences encoding, 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. described herein) as non-fusion proteins described herein. 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 to cell types. specific, regions, etc.

Antibodies

The polypeptides described herein can be used to produce antibodies specific for the polypeptides given herein and / or polypeptides encoded by the polynucleotides described herein and conservative variants thereof. Antibodies specific to the aforementioned polypeptides are useful, eg, for diagnostic and therapeutic purposes, eg, 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 stretches of a polypeptide can be fused with another protein, such as keyhole limpet hemocyanin, and antibodies raised against the chimeric molecule.

Numerous methods for 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 sequences 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 references 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 screening of recombinant antibody libraries in phage vectors or the like. See, Huse et al. (1989) Science 246: 1275-1281; and Ward, et al. (1989) Nature 341: 544-546. Specific monoclonal and polyclonal antibodies and antisera will usually bind with a Kd of, for example, at least about 0.1 pM, at least about 0.01 pM or better, and typically and 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 Pat.

ES 2 394 033 T3

5,482,856. Additional details on humanization and other antibody production and design techniques can be found in Borrebaeck (ed.) (1995) Antibodv Engineering. 2^to Edition Freeman and Company, NY (Borrebaeck); McCafferty et al. (1996) Antibody Engineering. A Practical Approach IRL in Oxford Press, Oxford, England (McCafferty), and Paul (1995) Antibody Engineering Protocols Human 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

This document also describes the generation of antisera that specifically bind to the polypeptides described herein as well as the polypeptides that bind by said antisera.

For example, polypeptides (eg, HA and NA molecules) are disclosed that specifically bind to or are specifically immunoreactive with an antibody or antiserum raised against an immunogen comprising an amino acid sequence selected from one or more of the sequences given herein. document (eg SEQ ID Nos: 11-12), etc. To eliminate cross-reactivity with other homologues, the antibody or antiserum is subtracted with the HA and / or NA molecules found in public databases at the time of submission, eg, 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 subtraction 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 Nos: 11-12), etc. or a substantial subsequence thereof (ie, at least about 30% of the full length sequence provided). The series of potential polypeptide immunogens derived from the present sequences are collectively referred to below as the immunogenic polypeptides. The resulting antiserum is optionally selected to be cross-reactive against the hemagglutinin and / or neuraminidase control homologs and any such cross-reactivity is removed, for example, by immunosorption, with one or more of the hemagglutinin and neuraminidase control homologs, before the use of the polyclonal antiserum in the immunoassay.

To produce antisera for use in an immunoassay, one or more of the immunogenic polypeptides are produced and purified as described herein. For example, recombinant protein can be produced in a recombinant cell. An inbred strain of mice (used in this assay because it is found to be more reproducible due to the virtual generic identity of the mice) is immunized with the immunogenic protein (s) in combination with a conventional adjuvant, such as Freund's adjuvant, and a standard protocol. immunization of mice (see, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a conventional description of antibody generation, immunoassay formats and conditions that can be used to determine specific immunoreactivity). Additional references and an analysis of antibodies are also found herein and may be applied herein to define polypeptides by immunoreactivity. Alternatively, one or more synthetic or recombinant polypeptides derived from the sequences described 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 combined and subtracted with the control hemagglutinin and / or neuraminidase polypeptides to produce subtracted pooled titer polyclonal antisera.

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

ES 2 394 033 T3

In another example, immunoassays in the competitive binding format are used for the detection of a test polypeptide. For example, as indicated, cross-reactive antibodies are removed from the pooled antiserum mixture by immunosorption with the control polypeptides. The immunogenic polypeptide (s) are then immobilized on a solid support which is exposed to the pooled subtracted antisera. Test proteins are added to the assay to compete for binding to the pooled subtracted antisera. The ability of the test protein (s) to compete for binding with the pooled subtracted antisera compared to the immobilized protein (s) is compared to the ability of the immunogenic polypeptide (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 for binding to the pooled subtracted antisera is optionally determined compared to the ability of the immunogenic polypeptide (s) to compete for binding to the antisera. Again, the percent cross-reactivity for the control polypeptide (s) is calculated using standard calculations. 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 the immunogenic polypeptide binding, The test polypeptides are said to specifically bind the pooled subtracted antisera.

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

As an additional determination of specificity, the pooled antisera are optionally fully immunosorbed with the immunogenic polypeptide (s) (rather than the control polypeptide (s)) until little or no binding of the resulting immunogenic polypeptide-subtracted pooled antiserum is detected with 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 binding of the fully immunosorbed antiserum to the immunogenic polypeptide), then the test polypeptide is specifically bound by the antiserum produced by the immunogenic protein. .

Nucleic acid and polypeptide sequence variants

Silent variants

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

ES 2 394 033 T3

Table 1

<td colspan="9">Codon table</td>

<td colspan="3">Amino acids</td><td colspan="6">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></td><td></td>

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

<td>Aspartic acid</td><td>Asp</td><td>D</td><td>GAC</td><td>GAU</td><td></td><td></td><td></td><td></td>

<td>Glutamic acid</td><td>Glu</td><td>AND</td><td>GAA</td><td>GAG</td><td></td><td></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></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></td><td></td>

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

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

<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 encode the amino acid arginine. Thus, at any 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. It is understood that U in an RNA sequence corresponds to T in a DNA sequence.

These silent variations are a kind of conservatively modified variations, discussed below. One skilled in the art 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 genetic code of triplets (eg, as set forth in Table 1, or as commonly available in the art) applied to the nucleic acid sequence encoding a hemagglutinin or neuraminidase polypeptide described in this document. All of these variations of each nucleic acid in this document are specifically provided and described by consideration of the sequence in combination with the genetic code. One of ordinary skill in the art is fully capable of making these silent substitutions using the methods in this document.

Conservative variations

Due to the degeneracy of the genetic code, silent substitutions (i.e., substitutions in a nucleic acid sequence that do not cause an alteration in an encoded polypeptide) are an implicit feature of each nucleic acid sequence described herein that encodes an amino acid. .

Conservative variations of a particular nucleic acid sequence refer to those nucleic acids that encode 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 sub-sequences

Also described herein is a nucleic acid comprising a unique subsequence in a nucleic acid selected from the sequence of HA and NA molecules described herein, eg, SEQ ID Nos: 1-2. The unique subsequence is unique compared to nucleic acids 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 performed using, for example, BLAST set to default parameters.

ES 2 394 033 T3

Any unique subsequence is useful, for example, as a probe to identify the nucleic acids described herein. See, above.

Also described is a polypeptide comprising a unique subsequence in a polypeptide selected from the sequence of hA and NA molecules described herein, eg, SEQ ID Nos: 11-12. Here, the unique subsequence is unique compared to a polypeptide corresponding to, for example, amino acids corresponding to polynucleotide sequences found in, for example, GenBank or other similar public databases at the time of submission.

Further described are 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 NA molecule sequences described herein where the unique subsequence is 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 submission). Unique sequences are determined as above.

Sequence comparison identity and homology

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

The term "substantially identical," in the context of two or more 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 at least about 90%, preferably 91%, more 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 greater identity of nucleotides or amino acid residues, compared and aligned for maximum match, as measured using a sequence comparison algorithm or by visual inspection. Such substantially identical sequences are typically considered to be homologous, without reference to an actual lineage. Preferably, there is substantial identity over a region of the amino acid sequences that is at least about 200 residues in length, more preferably over a region of at least about 250 residues, and more preferably the sequences are substantially identical over at least about 300 residues. , 350 remains, 400 remains, 425 remains, 450 remains, 475 remains, 480 remains, 490 remains, 495 remains, 499 remains, 500 remains, 502 remains, 559 remains, 565 remains, or 566 residues, or over 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, the subsequence coordinates are designated, if necessary, and the sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence (s) relative to the reference sequence, based on the designated program parameters.

Optimal sequence alignment can be performed for comparison, for example, by the local homology algorithm of Smith and Waterman, Adv Appl Math 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J Mol Biol 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc Natl Acad Sci USA 85: 2444 (1988), by computerized implementations of algorithms such as GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by visual inspection (see broadly, Ausubel et al., Supra).

An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J Mol Biol 215: 403-410 (1990). Software for running BLAST analysis 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 query sequence, which match or satisfy some positive valued threshold value T when they are aligned with a word of the same length in a sequence of database. T is known as the threshold of the neighbor word value (see, Altschul et al., Supra). 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 for as long as the cumulative alignment value can be increased. Cumulative values ​​are calculated using, for nucleotide sequences, the parameters M (reward value for a pair of mating residues; always&gt; 0) and N (penalty value for mating residues; always &lt;0). For amino acid sequences, a titration matrix is ​​used to calculate the cumulative value. The word extension hits in each

ES 2 394 033 T3 addresses are interrupted when: the cumulative alignment value decreases by the amount X from its maximum achieved value; the cumulative value reaches zero or below, due to the accumulation of one or more negative-rated residue alignments; or when the end of any sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defects a word length (W) of 11, a forecast (E) of 10, a limit of 100, M = 5, N = -4, and a comparison of both strings . For amino acid sequences, the BLASTP program uses as defects a word length (W) of 3, a forecast (E) of 10, and the BLOSUM62 titration matrix (see, Henikoff and 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 and Altschul, Proc Natl Acad Sci USA 90: 5873-5787 (1993)). A measure of similarity provided by the BLAST algorithm is the smallest summation probability (P (N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a 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 much more 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. You can also represent a tree showing the grouping relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (1987) J. Mol. Evol. 35: 351-360. The method used is similar to the method described by Higgins and 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 group of two aligned sequences. This group can then be aligned to the next most related sequence or group of aligned sequences. Two groups of sequences can be aligned by a simple extension of the pairwise alignment of two individual sequences. Final alignment is achieved by a series of progressive pairwise alignments. The program can also be used to represent a dendrogram or tree representation of the 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 sequence alignments, or amino acids is the CLUSTALW program (Thompson, JD et al. (1994) Nucl. Acids. Res. 22: 4673-4680). CLUSTALW performs multiple pairwise comparisons between sets of sequences and assembles them into a multiple alignment based on homology. The Open Gap 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 protein weight matrix. See, for example, Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 1091510919.

Digital systems

Further described herein are digital systems, eg, computers, computer-readable media, and embedded systems comprising character strings corresponding to the sequence information of this document for the isolated or recombinant nucleic acids and polypeptides of this document, including , for example, the sequences shown herein, and the various silent substitutions and conservative substitutions thereof. Integrated systems may additionally include, for example, a gene synthesis kit to prepare genes corresponding to the 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 supra. Computer systems may include such programs, for example, together with one or more data files or databases comprising a sequence 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 embedded systems of 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 retrieval of data from various databases. As an understanding of complementary double-helix pairwise interactions between 4 major nucleobases in wild-type polynucleotides, models that stimulate hybridization of complementary homologous polynucleotide chains can also be used as a basis for sequence alignment or other operations typically performed on character strings corresponding to the sequences in this document (for example, word processing manipulations, construction of figures comprising sequence or subsequence character strings, output tables, etc.).

ES 2 394 033 T3

Thus, conventional desktop applications such as word processing software (eg Microsoft Word ™ or Corel Word-Perfect ™) and database software (eg spreadsheet software such as Microsoft Excel ™) can be adapted. , Corel Quattro Pro ™, or database programs such as Microsoft Access ™, Paradox ™, Gene Works ™, or Mac Vector ™ or other similar programs) by entering a character string corresponding to one or more polynucleotides and polypeptides described herein (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 on a conventional operating system such as a Windows, Macintosh, or LINUX system. ) 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 nucleic acid or protein (or corresponding character strings) alignment.

The systems described in this document typically include a digital computer with data sets entered into the software system comprising any of the sequences in this document. The computer can be, for example, a PC (system based on Intel x86 or Pentium chip- compatible DOS ™, OS2 ™ WINDOWS ™ WINDOWSNT ™, WINDOWS95 ™, WINDOWS2000 ™, WINDOWS98 ™, LINUX, a MACINTOSH ™, Power PC, or a UNIX-based system (eg, SUN ™ workstation) or other commercially available computer that is known to one of ordinary skill in the art. Software to align or otherwise manipulate sequences is available, or can be easily constructed by one of ordinary skill in the art 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, an 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 for a user and for user selection of sequences to be compared or otherwise manipulated in the relevant computer system.

The computer typically includes appropriate software to receive instructions from the user, in the form of input by a user in a series of parameter fields, 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 the appropriate language to instruct the operation, for example, of appropriate mechanisms or transport controllers to perform the desired operation. The software may also include output elements to control nucleic acid synthesis (eg, based on a sequence or sequence alignment of this document), sample comparisons for differential gene expression, or other operations.

Kits and reagents

A kit is also described in this document. For example, a kit contains one or more nucleic acids, polypeptides, antibodies, or cell lines described herein (eg, comprising, or with, an HA and / or NA molecule described herein). The kit may contain a diagnostic nucleic acid or polypeptide, eg, antibody, set of probes, eg, as a micro-set of cDNA packaged in a suitable container, or other nucleic acid such as one or more expression vectors. The kit may also additionally comprise one or more additional reagents, for example substrates, labels, primers, for the labeling of 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 for using the kit components for discovery or application of diagnostic sets, etc.

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

System kits incorporating the methods, composition, systems, and apparatus of this document 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 apparatus components of this document and / or for using the compositions of this document. It also describes the use of any apparatus, component of apparatus, composition or kit of this document, for the practice of any method or test of this document, and / or for the use of any device or kit for the practice of any test or method of this document.

In addition, kits can include one or more translation systems as indicated above (eg, a cell) with appropriate packaging material, containers to house kit components, instructional materials to practice the methods of this document. and / or the like. Likewise, the products of

ES 2 394 033 T3 translation systems (e.g. proteins such as HA and / or NA molecules) can be provided in kit form, e.g. with containers to house kit components, instructional materials to practice the methods of this document and / or the like.

To facilitate the use of the methods and compositions of the invention, any of the components and / or vaccine compositions can be packaged, for example, virus reassembled in allantoic fluid, etc., and additional components, such as, buffer, cells, medium. culture, useful for packaging and infection of influenza viruses for experimental or therapeutic vaccine purposes, in kit form. 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 H5N1 ca viruses and vaccines

Various sequences in this document comprising HA / NA H5N1 sequences were used to create influenza viruses and vaccines. The HA sequences in these vaccines were altered from the wild type by elimination of the polybasic cleavage site within the HA. The HA / NA sequences were reassorted (in a 6: 2 reassortment) with A / AA / 6/60 (an att virus, ca, see above).

Three H5N1 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 in this example as the '97, '03, and '04 strains based on their years of designation. The percentage of 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 HA '04 sequences, used to construct the viruses / vaccines. As previously indicated, various embodiments of the invention comprise sequences that have different regions of the polybasic cleavage site removed. See above.

As indicated, the modified H5N1 sequences (ie the '97, '03, and '04 modified) were used to construct 6: 2 reassortant viruses with A / AA / 6/60. It will be appreciated, and noted elsewhere in this document, that other desirable structures could also be used (eg, PR8, etc.).

In the 6: 2 pools of this example, the HA and NA gene sequences were obtained from the wild-type parental virus and the remaining genes were characterized by sequence analysis derived from the parental virus A / AA / 6/60 ca. Regrouped viruses replicated at 8.0-8.5 log10TCID₅0 in eggs. However, it will be appreciated that a claimed virus in which log10TCID₅0 ranging from about 7.0 to about 9.0, about 7.5-8.5, or about 8.0-8.5 is also part of the invention. The cleavage capacity of modified HA in the viruses constructed by endogenous proteases was restricted in vitro and the viruses were trypsin-dependent (eg, from about 0.1 pg / ml to about 1.0 pg / ml) for culture . The constructed viruses were sensitive to temperature in vitro.

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

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

A comparison of serum HAI antibody titers produced in mice after a single intranasal dose of vaccine (2003 ca ​​compared to 2003 wild-type) is shown in Figure 6. Figure 7 shows similar measurements, but using titers of serum neutralizing antibodies.

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, the ca reassortants replicated less well than the wild-type viruses. Figure 10 shows related data using mouse upper respiratory tracts. Those skilled in the art will be familiar with both homologous and heterologous exposures (for example, testing whether a 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 a 2004 H5N1 ca vaccine against high-dose exposure (10⁵TCID50) with wild-type H5N1 homologous or heterologous viruses in mice. Figure 12 shows the effectiveness of

ES 2 394 033 T3 protection conferred by H5N1 ca 1997 and 2003 vaccines against high dose exposure (10⁵TCID₅₀) with wild-type H5N1 homologous and heterologous viruses in mice. Figure 13 shows the efficacy of protection conferred by the 2004 H5N1 ca vaccine against low or high doses of challenge with wild-type H5N1 homologous virus in mice. Figures 11-13 demonstrate that the tested vaccines could protect against other related viruses.

The present example demonstrates several points regarding exemplary H5N1 ca reassortant viruses / vaccines of the invention. The reassorted '97, '03, and '04 ca viruses 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. Efficacy of protection and cross-protection against lethal exposure and systemic spread with wild-type viruses in mice is also shown. Efficacy of protection and cross-protection against replication of wild-type challenge viruses in the respiratory tract of mice is also expected.

It is contemplated to use these (and similar) viruses / vaccines to determine if immunogenicity and efficacy are improved after 2 doses of vaccine; evaluate immunogenicity in non-human primates; evaluate the attenuation and efficacy of the vaccine in ferrets; determine the contribution of humoral and cellular immunity to the observed efficacy of vaccines produced in mice; determine which HA 2003 residues contribute to enhanced immunogenicity and introduce them into HA 1997 and 2004; and determining 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) Whole molecule length: 1767 nt agcaaaagca ggggttcaat ctgtcaaaat ggagaaaata gtgcttcttt ttgcaatagt cagtcttgtt aaaagtgatc agatttattgcat

101 gcaaacaact cgacagagca ggttgacaca ataatggaaa agaacgttac

1S1 tgttacacat gcccaagaca tactggaaaa gaaacacaac gggaagctct

201 gcgatctaga tggagtgaag cctctaattt tgagagattg tagcgtagct

251 ggatggctcc tcggaaaccc aatgtgtgac gaattcatea atgtgccgga

301 atggtcttac atagtggaga aggccaatcc agtcaatgac ctctgttacc

351 caggggattt caatgactat gaagaattga aacacctatt gageagaata

401 aaccattttg agaaaattca gatcatcccc aaaagttctt ggtccagtca

451 tgaagcctca ttaggggtga gctcagcatg tccataccag ggaaagtcct

501 cctttttcag aaatgtggta tggcttatca aaaagaacag tacataccca

551 acaataaaga ggagctacaa taataccaac caagaagatc ttttggtact

601 gtgggggatt caccatccta atgatgcggc agagcagaca aagetetate

651 aaaacccaac cacctatatt tccgttggga catcaacact aaaccagaga

701 ttggtaccaa gaatagctac tagatccaaa gtaaacgggc aaagtggaag

751 gatggagttc ttctggacaa ttttaaagcc gaatgatgca atcaacttcg

801 agagtaatgg aaatttcatt gctccagaat atgcatacaa aattgtcaag

851 aaaggggact caacaattat gaaaagtgaa ttggaatatg gtaactgcaa

901 caccaagtgt caaactccaa tgggggcgat aaactctagc atgccattcc

951 acaatataca ccctctcacc attggggaat tgtgaaatca gccccaaata 1001 aacagattag tccttgcgac tgggctcaga aatagccctc aaagagagac 1051 tttggagcta tcgaggatta tagcaggttt tatagaggga ggatggcagg 1101 gaatggtaga tggttggtat gggtaccacc atagcaatga gcaggggagt 1151 gggtacgctg atccactcaa cagacaaaga aaggcaatag atggagtcac 1201 aactcgatca caataaggtc ttgacaaaat gaacactcag tttgaggccg 1251 atttaacaac ttggaaggga ttagaaagga gaatagagaa tttaaacaag 1301 aagatggaag acgggttcct agatgtctgg acttataatg ctgaacttct 1351 ggttctcatg gaaaatgaga gaactctaga ctttcatgac tcaaatgtca 1401 agaaccttta cgacaaggtc cgactacagc ttagggataa tgcaaaggag 1451 ctgggtaacg gttgtttcga gttctatcat aaatgtgata atgaatgtat 1501 agaaatggaa ggaaagtgta cgtatgaeta cccgcagtat tcagaagaag 1551 aagagaggaa cgagactaaa taaaattgga ataagtggag atcaatagga 1601 atrtaccaaa tactgtcaat ttatectaea gtggegagtt ccctagcact 1651 ggcaatcatg gtagctggtc tatccttacg gatgtgctcc aatgggtcgt 1701 tacaatgcag aatttgcatt taaatttgtg agtteagatt gtagttaaaa 1751 acacccttgt ttctact

ES 2 394 033 T3

A / Vietnam / 1203/04 H5 amino acid sequence (SEQ ID No: 11) Complete molecule length: 564 aa mekivllfai vslvksdqic igyhannste qvdtimeknv tvthaqdile kkhngklcdl dgvkplilrd csvagwllgn pmcdefinvp endweylivecykan 101 pvrinrinvp fndyeylivecykanBskivski BndweylfegSkiBskiSpg lsvagwlfek

151 cpyqgkssff rnwwlikkn styptikrsy nntnqedllv lwgihhpnda 201 aeqtklyqnp ttyisvgtst lnqrlvpria trskvngqsg rmeffwtilk 251 pndainfesn gnfiapeyay kivkkgdeti mkseleygnc ntkeqtpmga 301 inssmpfhni hpltigecpk yvksnrlvla tglrnspqre trglfgaiag 351 fieggwqgtnv dgwygyhhsn eqgsgyaadk estqkaidgv tnkvnsiidk 401 mntqfeavgr efnnlerrie nlnkkmedgf ldvwtynael lvlmenertl 451 dfhdsnvknl ydkvrlqlrd nakelgngcf efyhkcdnec mesvrngtyd 501 kreeisgvkl ypqyseearl esigiyqils iystvassla laimvaglsl 551 wmcsngslqc rici

Nucleotide sequence of A / Vietnam / 1203/04 N1 (SEQ ID No: 2) Full molecule length: 1398 nt agcaaaagca gggtcaatct

101 gaacatgatc

151 accaatctga

201 gcttcagtaa

251 ggctgtatac

301 tgtttgttat

351 actttctttt

401 gactgtcaaa

451 gcgaggctcc

501 gcaagtgctt

551 cccagacaat

601 acactatcaa

651 tgtgcatgtg

701 taatggtcag

751 ttaaatcagt

801 tgttatccta

851 tggctcaaat

901 taggatatat

951 ggaacaggta agggttttca 1001 1051 1101 gcactaattc actgaaacgg tgattggtca 1151 1201 1251 gactagattg cccaaagaga tgtaaatagt 1301 1351 cattcaccat ggagctcaaa gtatggtaac tcaatatggg accaatcagc aattagcggg agtaaggaca aagagagccg tgactcaggg gacagaagcc ctccccatat gccatgatgg ggggctgtgg gagttggagg taaatggctc gcatcacata cgaattggat atgccggaga cggccacggg atgcagtgga gttgtggtcc tttaaatacg caggagcggc acagtagcct ggatatagcg cacaagacct 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 ttgagtctgt ttgacgattg atacaatggc tgagaactca gtaatgactg aatggaaaaa atcactatga gtgtgcaggg tcaaaatttg acaatccácg aacggggcat ctggatcggg tttgggatcc caagatatcg ccagcatcca ttgagttgat agcagcatat

<img file="ES2394033T3_D0001.tif" />

aaactccttg aataaccatc tacaaattgg gggaatcaac gaaagctgtg ttaacggatg aagggggatg ggaatgcaga actceaatgg 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

ES 2 394 033 T3 mnpnqkiiti gsicmvtgiv slmlqignmi siwvshsiht gnqhqsepis ntnfltekav asvklagnss lcpingwavy skdnsirigs kgdvfvirep 101 fiscshlecr tffltqgall ndkhsngtvmspy narwsitwng cgpvgertlfesky lcpvgertlfesky dgvgertlfesky lcpvgertlfesky lcpvgertlfesky 151

201 nnilrtqese cacvngscft vmtdgpsngq ashkifkmek gkwksveld

251 apnyhyeecs cypnageitc vcrdnwhgsn rpwvsfnqnl eyqigyicsg

301 vfgdnprpnd gtgscgpvss ngaygvkgfs fkygngvwig rtkstnsrsg

351 femiwdpngw tetdssfsvk qdivaitdws gysgsfvqhp eltgldcirp

401 cfwvelirgr pkestiwtsg ssisfcgvns dtvgwswpdg aelpftidk

A / Honq Konq / 213/03

Nucleotide sequence of A / Hong Kong / 213/03 H5 (SEQ ID No: 3) Whole molecule length: 1767 nt agcaaaagca ggggttcaat cCgtcaaaat ggagaaaata gtgcttcttt ttgcaatagt cagtettgtt aaaagtgatc agatbactgcat

101 gcaaacaact cgacagagca ggttgacaca ataatggaaa agaacgttac

151 tgttacacat gcccaagaca tactggaaaa gacacacaac gggaagctct

201 gcgatctaga tggagtgaag cctctaattt tgagagattg tagtgtagct

251 ggatggctcc tcggaaaccc aatgtgtgac gaattcatca atgtgccgga

301 atggtcttzac atagtggaga aggccaatcc agccaatgac ctctgttacc

351 caggggattt caacgactat gaagaattga aacacctatt gagcagaata

401 aaccattttg agaaaattca gatcatcccc aaaaattctt ggtccagtca

451 tgaagcctca ttaggggtga gctcagcatg tccataccaa ggaaagtcct

501 cctttttcag gaatgtggta tggcttatca aaaagaacaa tgcataccca

551 acaataaaga ggagctacaa taataccaac caagaagatc ttttggtatt

601 gtgggggatt caccatccta atgatgcggc agagcagact aggctctatc

651 aaaacccaac cacctacatt tccgttggga catcaacact aaaccagaga

701 ttggtaccaa aaatagctac tagatccaaa gtaaacgggc aaaatggaag

751 gatggagttc ttctggacaa ttttaaaacc gaatgatgca atcaacttcg

801 agagcaatgg aaatttcatt gctccagaat atgcatacaa aattgtcaag

851 aaaggggact cagcaattat gaaaagtgaa etggaatatg gtaactgcaa

901 caccaagtgt caaactccaa tgggggcgat aaactctagt atgccattcc

951 acaacataca ccctctcacc atcggggaat gccccaaata tgtgaaatca

1001 aacagattag tccttgcgac tgggctcaga aatagccctc aaagagagac

1051 tcgaggatta tttggagcta tagcaggctt tatagaggga ggatggcagg

1101 gaatggtaga tggttggtat gggtaccacc atagcaatga gcaggggagt

1151 gggtacgctg cagacaaaga atccactcaa aaggcaatag atggagtcac

1201 caataaggtc aactcgatca ttgacaaaat gaacactcag tttgaggccg

1251 ttggaaggga atttaataac ttagaaagga gaatagagaa tttaaacaag

1301 aagatggaag acggattcct agatgtctgg acttataatg ctgaacttct

1351 ggttctcatg gaaaatgaga gaactctaga ctttcatgac tcaaacgcca

1401 agaaccttta cgacaaggtc cgactacagc ttagggataa tgcaaaggag

1451 ctgggtaacg gttgtttcga gttctatcac aaatgtgaca atgaatgtac

1S01 ggaaagtgta agaaacggaa cgtatgacta cccgcagtat tcagaagaag

1551 caagactaaa aagagaggaa ataagtggag taaaattgga gtcaatagga

1601 acttaccaaa tactgtcaat ttattctaca gtggcgagtt ccctagcact

1651 ggcaatcatg gtagctggtc tatetteatg gatgtgctcc aatgggtcgt

1701 tacaatgcag aatttgcatt taaatttgtg agtteagatt gtagttaaaa 1751 acacccttgt ttctact

Nucleotide sequence of A / Hong Kong / 213/03 H5 (SEQ ID No: 13) Whole molecule length: 564 aa

ES 2394033 T3 mekivllfai vslvksdqic igyhannste qvdtimeknv tvthaqdile kthngklcdl dgvkplilrd csvagwllgn pmcdefinvp ewsyivekan 101 fndyeelkhl pandlcypgd lsrinhfeki qiipknswss heaelgvssa 151 cpyqgkssff rnwwlikkn nayptikrsy nntnqedllv lwgihhpnda 201 aeqtrlyqnp ttyisvgtst Inqrlvpkia trskvngqng rmeffwtilk 251 pndainfesn gnfiapeyay kivkkgdsai mkseleygnc ntkcqtpmga 301 insampfhni hpltigecpk yvkanrlvla tglrnspqre trglfgaiag 351 fieggwqgmv dgwygyhhsn eqgggyaadk estqkaidgv tnkvnsiidk 401 mntqfeavgr efnnlerrie nlnkkrnedgf ldvwtynael lvlmenertl 451 dfhdsnvknl ydkvrlqlrd nakelgngcf efyhkcdnec mesvrngtyd 501 ypqyseearl kreeisgvkl esigtyqila iystvassla la laqimvaglsngl 55

A / Hong Kong / 213/03 N1 amino acid sequence (SEQ ID No: 4) Whole molecule length: 1458 nt agcaaaagca ggagttcaaa 51 ggatcaatct gtatggtaat

101 gaacataatc tcaatatggg

151 accaggctga accatgcaat 201 tgggtaaacc agacatatgt 251 gaaagctgtg gcttcagtaa 301 ttagtggatg ggctgtatac 351 aagggggatg tgtttgttat 401 ggaatgeaga actttctttt

451 attctaatgg gaccgtcaaa

501 tgtcccgtgg gcgaggctcc

551 tgcttggtcg gcaagtgctt 601 gaatttccgg cccagacaat 651 ataataacag acactatcaa 701 agagtctgaa tgtgcatgtg 751 atggaccaag taatgggcag 601 gggaaagtag ttaaatcagc

651 ggagtgctcc tgttatcctg

901 ataactggca tggctcaaat

951 gagtatcgaa taggatatat 1001 gggacaggca ccccaatgat 1051 agggttctca atggaataaa 1101 agaaccaaaa gcactaattc 1151 aaatggatgg actggtaegg 1201 cgattqgtca tagctataac 1251 gaactgacag gattagattg 1301 cccaaagaga cagagggcgg 1351 ccttttgtgg tgtaaatagt 1401 gctgagccgc cattcaccat 1451 tttctact atgaatccaa atcagaagat aacaaccatt tggaatagtt agcttgatgt tacaaattgg ttagtcattc aattcaaaca gggaatcaac caaagcatta ttacttatga aaacaacacc caacatcagc aataccaatt ttcttactga cattagcggg caattcatct agtaaggaca ccttgcccca acggtataag aatcggttcc aagagagccg ttcatctcat gctcccactt tgactcaggg agccttgctg aatgacaagc gacagaagcc ctcacagaac attaatgagt ttccccatac aactcgaggt ttgagtctgt gtcatgatgg cactagttgg ttgacaattg ggggctgtgg ctgtattgaa atacaatggc gagttggagg aacaacataa tgagaaccca taaatggctc ttgctttact gttatgactg gcttcataca aaatcttcag aatagaaaaa cgaattaaat gcccctaatt atcactatga atgctggaga aatcacatgt gtgtgcaggg cggccatggg tatctttcaa tcaaaatttg atgcagtgga gttttcggag acaatccacg gttgtggtcc ggtgtcccct aaaggggcat tttaaatacg gcaatggtgt ttggatcggg caggagcggc tttgaaatga tttgggatcc acagtaattt ttcagtaaag caagatattg ggatataqcg ggagttttgt ccagcatcca cataagacct tgtttctggg ttgagctaat gcacaatgactg gactagtcagg ttcagtaaag caagatattg gactaggtcaggt

A / Hong Kong / 213/03 N1 amino acid sequence (SEQ ID No: 14) Whole molecule length: 469 aa

ES 2394033 T3 tnnpnqkitci gsicmvigiv elmlqignii siwvshsiqC gnqhqaepcn SI qsiityennt wvnqtyvnis ntnfltekav asvtlagnss lcpiegwavy 101 skdngirigs kgdvfvirep fiscshlecr tffltqgall ndkhsngtvk 151 drsphrtlms cpvgeapspy nsrfesvaws asachdgtsw ltigisgpdn 201 gavavlkyng iitdtikswr nnimrtqese cacvngscft vmtdgpsngq 251 asykifriek gkwksaeln apnyhyeecs cypdageitc vcrdnwhgsn 301 rpwvafnqnl eyrigyicsg vfgdnprpnd gtgscgpvsp kgaygikgfs 351 fkygngvwig rtkstnsrsg femíwdpngw tgcdsnfsvk qdivaitdws 401 gysgsfvqhp eltgldcirp cfwvelirgr pkestiwtsg ssisfcgvns 451 dtvgwewpdg aelpftidk

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

A / Hong Kong / 491/97 H5 nucleotide sequence (SEQ ID No: 5) Whole molecule length: 1767 nt agcaaaagca ggggtataat ctgccaaaat ggagaaaata gtgcttcttc ttgcaacagt cagccttgtt aaaagtgacc tggttaatttg

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 atggtctcac atagtggaga aggccagtcc agccaacgac ctctgttatc

351 cagggaattt caacgacCat gaagaactga aacacctatt gagcagaata

401 aaccattttg agaaaattca gataatcccc aaaagttctt ggtccaatca

451 tgatgcctca tcaggggtga gctcagcatg tccatacctt gggaggtcct

501 cctttttcag aaatgtggta tggcttatca aaaagaacag tagctaccca

551 acaataaaga ggagctacaa taataccaac caagaagatc ttttggtact

601 gtgggggatt caccatccta atgatgcggc agagcagaca aggctctatc

651 aaaacccaac cacctacatt tccgttggaa catcaacact gaaccagaga

701 ttggctccag aaatagctac tagacccaaa gtaaacgggc aaagtggaag

751 aacggagctc ttctggacaa ttccaaagcc gaatgacgcc accaatttcg

801 agagtaatgg aaatttcatt gctccagaat atgcatacaa aattgtcaag

851 aaaggggact caacaattat gaaaagtgaa ttggaatatg gtaactgcaa

901 caccaagtgt caaactccaa Cgggggcaat aaactctagt atgccattcc

951 acaacataca ccccctcacc atcggggaat gccccaaata cgtgaaatca

1001 aacagattag tccttgcaac tggactcaga aataccccEc aacgagagac

1051 gcgaggacta tttggagcta tagcaggCtC tatagaggga ggatggcagg

1101 gaatggtaga tggttggtat gggcaccacc atagcaatga gcaggggagt

1151 ggatacgctg cagaccaaga atccacacaa aaggcaatag atggagtcac

1201 caataaggtc aactcgatca ttaacaaaat gaacactcag tttgaggccg

1251 ttggaaggga atttaataac ttggaaagga ggatagagaa tttaaacaag

1301 aaaatggaag acggattcct agatgtctgg acttacaatg ccgaacttct

1351 ggttctcatg gaaaatgaga gaactctaga ctttcatgac tcaaatgtca

1401 agaaccttta cgacaaggtc cgactacagc ttagggataa tgcaaaggag

1451 ctgggtaatg gttgtttcga attctatcac aaatgtgata acgaatgtat

1501 ggaaagtgta aaaaacggaa cgtatgacta cccgcagtat tcagaagaag

1551 caagactaaa cagagaggaa ataagtggag taaaattgga atcaatggga

1601 acetaccaaa tactgtcaat ttattcaaca gtggcgagtt ccctagcact

1651 ggcaatcatg gtagcxggtc 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) Whole molecule length: 564 aa

ES 2394033 T3 mekivlllat valvksdqic ígyhannste qvdtimeknv tvthaqdile rthngklcdl ngvkplilrd csvagwllgn pmcdefinvp ewsyivekas 101 fndyeelkhl pandlcypgn 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 iystvasBl1 wcmicicsl 55

A / Hong Kong / 486/97 H5 nucleotide sequence (SEQ ID No: 6) 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

2S1 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 ttggaattCc

551 cggtccggat aatggggctg tggctgtgtt gaaatacaat ggcataataa

601 cagacaccat caagagttgg aggaacaaca cactgaggac gcaagagtct

651 gaatgtgcat gtgtgaatgg tCcttgtttt actgcaatga cagatggacc

701 gagtaatgaa caggcctcat acaagatttt caagatagaa aaggggaggg

751 tagtcaaatc agttgagttg aacgccccta attatcatta cgaggaatgc

S01 tcctgttatc ctgatgctgg cgaaatcaca tgtgtgtgca gggataattg

651 gcatggctcg aaccgaccat gggtgtcCtt caatcagaat ctggagtatc

901 aaataggata tatatgcagt ggggttttcg gagacagtcc acgccccaat

951 gatgggacag gcagttgtgg tccagtgtct cttaacggag cgtatggagt

1001 aaaagggttt tcatttaaat acggcaatgg tgtttggatc gggagaacca

1051 aaagcactag ttccaggagc ggttttgaaa tgatttggga tccaaatggg

1101 tggaccgaaa cagacagtag cttctcgtEg aagcaagaca úcatagcgat

1151 aactgattgg tcaggataca gcgggagttt tattcaacat ccagaactga

1201 caggattaaa ttgcatgaga ccttgcttct gggttgaact aatcagaggg

1251 aggcccaaag agaaaacaat ctggactagt gggagcagta tatctttctg

1301 tggtgtaaat agtgacactg tgggttggtc ttggccagac ggtgctgagt

1351 tgccatacac cattgacaag tagtttgttc aaaaaactcc ttgtttctac 1401 t

A / Hong Kong / 486/97 N1 amino acid sequence (SEQ ID No: 16) Whole molecule length: 450 aa mnpnqkiiti gsicmwgii slmlqignti svwvshiikt whpnqpepcn qsinfyteqa aaevtlagns slcpisgwai yskdvnsirvig skgwai

101 pfiscshlec rtffltqgal lndkhsngtv kdrspyrtlm scpvgeapsp

151 ynsrfesvaw sasachdgis wltigisgpd ngavavlkyn giitdtiksw

201 rnntlrtqes ecacvngscf tvmtdgpHne qasykifkie kgrwksvel

251 napnyhyeec scypdageit cvcrdnwhgs nrpwvsfnqn leyqigyics

301 gvfgdsprpn dgtgscgpve lngaygvkgf sfkygngvwi grtkstssrs

351 gfemiwdpng wtetdssfsl kqdiiaitdw sgysgsfiqh peltglncmr

401 pcfwvelirg rpkektiwta gsBÍsfcgvn sdtvgwBwpd gaelpytidk

ES 2 394 033 T3

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 No: 7) Whole molecule length: 1767 nt

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 agcaaaagca ttgcaacagt gcaaacaact tgttacacat gcgatctaaa ggatggctcc atggtcttac cagggaattt aaccattttg tgatgcctca cctttttcag acaataaaga gtgggggatt aaaacccaac ttggtttcag aatggagttc agagtaatgg aaaggggact caccaagtgt acaacataca aacagattag gcgaggacta gaatggtaga ggatacgctg caataaggtc ttggaaggga aaaatggaag ggggtataat cagccttgtt gcccaagaca cgacagagca tggagtgaag tcggaaaccc atagtggaga caacgactat agaaaattca aaatgtggta tcaggggtga ggagctacaa caccatccta cacctacatt aaatagctac ctctggacaa aaatttcatt caacaattat caaactccaa ccccctcacc tccttgcaac tttggagcta tggttggtat cagaccaaga aactcgatca atttaataac acggattcct ctgtcaaaat aaaagtgacc agttgacaca tactggaaag cctctgattt tatgtgtgac aggccagtcc gaagaactga gataatcccc gctcagcatg tggcttatea taataccaac atgatgcggc tccgttggaa tagacccaaa ttttaaagcc gctccagaac gaaaagtgaa tgggggcaat atcggggaat tggactcaga tagcaggttt gggtaccacc atccacacaa ttaacaaaat ttggaaagga agatgtctgg ggagaaaata agatttgcat ataatggaaa gacacacaac tgagggattg gaattcatca agccaatgac aacacctatt aaaagttctt tccatacctt aaaagaacag caagaagatc agagcagaca catcaacact gtaaacgggc gaatgatgcc atgcatacaa ttggaatatg aaactctagt gccccaaata aatacccccc tatagaggga acagcaacga aaggcaatag gaacactcag ggatagagaa acttacaatg gtgcttcttc tggttaccat agaatgttac gggaagctct tagtgtagct atgtgccgga ctctgttatc gagcagaata ggtccaatca gggaggtcct tagctaccca ttttggtacc aggctctatc gaaccagaga aaagtggaag atcaatttcg aattgtcaag gtaactgcaa atgccattcc tgtgaaatca aacgagagac ggatggcagg gcaggggagt atggagtcac tttgaggccg tttaaacaag ccgaacttct

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

A / Hong Kong / 491/97 (Ser211) H5 amino acid sequence (SEQ ID No: 17) Length of entire molecule: 564 aa mekivlllat rthngklcdl 101 pandlcypgn 151 cpylgrssff 201 aeqtrlyqnp 251 pndainfeen 301 inssmpfhni 351 fieggwqgrnv 401 mntqfeavgr 451 dfhdsnvknl 501 ypqyseearl 551 wmcsngslqc velvksdqic ngvkplilrd fndyeelkhl rnwwl ikkn ttyisvgtst gnfiapeyay hpltigecpk dgwygyhhsn efnnlerrie ydkvrlqlrd nreeisgvkl rici igyhannste csvagwllgn lsrinhfeki ssyptikrsy Inqrlvseia kivkkgdsti yvksnrlvla eqgsgyaadq nlnkkmedgf nakelgngcf esmgtyqils qvdtimeknv pmcdefinvp qiipksswsn nntnqedllv trpkvngqsg mkseleygnc tglrntpqre estqkaidgv ldvwtynael e fyhkcdnec iystvassla tvthaqdile ewsyivekas hdassgvesa lwgihhpnda rmeffwtilk ntkcqtpmga trglfgaiag tnkvnsiink lvlmenertl la mesitnvagls

ES 2 394 033 T3

Nucleotide sequence of A / Hong Kong / 486/97 N1 (SEQ ID No: 8) Full molecule length: 1401 nt ageaaaagea ggatcaatct 101 aaacacaata 151 accagcctga 201 gcagcttcag 251 atgggctata

301 atgtgtttgt

351 agaacctttt

401 tgggaccgtc

451 ttggtgaggc

501 tcagcaagtg

551 cggtccggat

601 cagacaccat

651 gaatgtgcat

701 gagtaatgaa

751 tagtcaaatc

801 tcctgttatc

851 gcatggctcg

901 aaataggaca

951 gatgggacag 1001 aaaagggttt ggagtttaaa gcatggtagt tcagtatggg accatgcaac tgacattagc tacagcaagg tataagagaa tcttgaccca cccttcccca aaagacagga cttgccatga aatggggctg caagagttgg gtgtgaatgg caggcctcat agttgagttg ctgatgctgg aaccgaccat tatatgeagt gcagttgtgg tcatttaaat atgaatccaa tgggataatc tcagccacat eaaagcatca gggcaattcc acaatagtat cca.tt-ca.tcE. aggagcccta gcccctatag tacaactcaa tggcattagt tggctgtgtt aggaacaaca ttcttgtttt acaagatttt aacgccccta cgaaatcaca gggtgtcttt ggggttttcg tccagtgtct acggcaatgg atcagaagat agcttgatgt aattaaaact atttttacac tctctctgcc aagaattggt oat.gcC.ccc to ttgaatgaca aactttaatg ggtttgagtc tggctaacaa gaaatacaat actgtaatga cactgaggac attatcatta caagatagaa tgtgtgtgca caatcagaat gagacagtcc cttaacggag tgtttggatc aataaccatfc tacaaattgg tggcacccaa tgagcaggct ctattagtgg tccaaagggg tttggaatgc ageattetaa agctgtcctg tgttgcttgg ttggaatttc ggcataataa gcaagagtct cagatggacc aaggggaggg cgaggaatgc gggataattg ctggagtatc acgccccaat cgtatggagt gggagaacca

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) Whole molecule length: 450 aa mnpnqkiiti qsinfyteqa 101 pfiscshlec 151 ynsrfesvaw 201 rnntlrtqes 251 napnyhyeec 301 gvfgdsprpn 351 gfemiwdpng 401 pcfwvelirg gsicnrwgii aasvtlagns rtffltqgal sasachdgis ecacvngscf scypdageit dgtgscgpvs wtetdesfsl rpkektiwts slmlqignti slcpisgwai lndkhsngtv wltigisgpd tvmtdgpsne cvcrdnwhgs lngaygvkgf kqdiiaitdw gssisfcgvn svwvshiikt ygkdnsirig kdrspyrtlm ngavavlkyn qasykifkie nrpwvsfnqn sfkygngvwi sgysgsfiqh sdtvgwswpd whpnqpepcn akgdvfvire scpvgeapsp giitdtiksw kgrwksvel leyqigyics grtkstssrs peltglncmr gaelpytidk ca A / ck / Honq Konq / G9 / 97

Nucleotide sequence of ca A / ck / Hong Kong / G9 / 97 (SEQ ID No: 9) Length of the entire molecule: 1690 bp

ES 2 394 033 T3 ttaaccactc aagatggaag caataccact aataactata ctactagtag taacageaag caatgcagac aaaatctgca tcggctacca atcaacaaac

101 tccacagaaa ccgtagacac gctaacagaa aacaatgttc ctgtgacaca

151 tgceaaagaa cagagcacaa ttgctccaca tgggatgctg tgtgcaacaa 201 atctgggacg tcctcttatt ctagacactt gcaccattga aggactgatc 251 tatggcaacc cttcttgtga tctactgttg ggaggaagag aatggcccta 301 catcgtcgaa agaccatcgg ctgttaacgg aatgtgttac cccgggaatg 351 tagaaaacct agaggaacta aggtcatttt ttagttctgc tagttcctac 401 caaagaatcc agatctttcc agacacaatc tggaatgtgt cttacagtgg 451 aacaagcaaa gcatgttcag atteatteta 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 acactgcgag taagatccaa tgggaatcta atcgctccat 801 ggtatgggca cattctttca ggagagagee acggaagaat cctgaagact 8S1 gatttaaaca gtggtagctg tgtagtgcaa tgtcaaacag aaagaggtgg

801 cttaaatact actttgccat tccacaatgt cagtaaatat gcactcggaa

951 actgcccaaa atatgttgga gtaaagagtc tcaaactggc agttggtctg 1001 aggaatgtgc ctgctagatc aagtagagga ctatttgggg ccatagctgg 1051 attcatagag ggaggttggt cagggctggt cgctggtctg 1001 aggaatgtgc ctgctagatc aagtagagga ctatttgggg ccatagctgg 1051 attcatagag ggaggttggt cagggctggt cgctggtctg 1001 aggaatgtgcact ctgctagatc aaggcattgagga ctatttgggg ccatagctgg 1051 attcatagag ggaggttggt cagggctggt cgctggttatt1 tatcagtgtggaact 110

1151 caaagggcaa aacgtccaaa ttgacaaaat tagtcgataa gtgaataata 1201 caatatgaaa aatgaacaag ttattgatca tgaatteage gaggttgaaa 1251 atagactcaa tatgatcaat aataagattg atgaccagat acaagacata 1301 tgggcatata acgctgaatt gctagtgctg cttgaaaacc agaaaacact 1351 egatgageac 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 eatctcttgt gattgcaatg gggtttgctg ccttcttgtt 1651 ctgggccatg tccaatggac cttgcagatg caacatttga

Amino acid sequence of ca A / ck / Hong Kong / G9 / 97 H9 (SEQ ID No: 19) Length of entire molecule: 558 meaiplitil aa lhtehngmlc 101 psavngmcyp 151 csdsfyrsmr 201 nlytrtdttt 251 lrvrsngnli 301 lpfhnvskya 351 gweglvagwy 401 yeiidhefse 451 anvnnlynkv 501 keesrlerqk 551 ngsereni lwtasnadk atnlgrplil gnvenleelr wl tqknnayp svatedinrt apwyghi1sg fgncpkyvgv gfqhsndqgv venrlnminn kralgsname iegvkleseg icigyqstns dtctiegliy sffssassyq iqdaqytnnr fkpvigprpl eshgrilktd kslklavglr giaadrdstq kiddqiqdiw dgkgcfelyh tykiltiyst tetvdtlten gnpscdlllg riqifpdtiw gkailfmwgi vnglqgridy lnsgscwqc nvparssrgl raidkitskv aynaellvll kcddqcmeti vasslviamg nvpvthakel grewsyiver nvsysgtska nhpptdtaqt ywsvlkpgqt qtergglntt fgaiagfieg nnivdkmnkq enqktldehd rngtynrrky faaflfwams

Nucleotide sequence of ac A / ck / Hong Kong / G9 / 97 N2 (SEQ ID No: 10) Length of the entire molecule: 1428 bp

ES 2394033 T3 aaatgaatcc aaatcagaag ataatagcaa ttggctctgt ttctctaact attgcgacaa tatgcctcct catgcagatt gctatcttag caacgactat 101 gacactacat ttcaagcaga atgaatgcat caactcctcg aataatcaag 151 tagtgccatg tgaaccaatc ataatagaaa ggaacataac agagatagtg 201 gtactacctt catttgaata atttgtccta agagaaggaa aagtagcaga 251 ctacaggaat tggtcaaaac cacaatgtca aatcacaggg ttcgctcctt 301 tctccaagga caactcaatt aggctctccg caggtggaga tatttgggtg 351 acaagagaac cttatgtatc gtgcggtctt ggtaaatgtt atcaatttgc 401 acctgggcag ggaaccactt tggagaacaa acactcaaac ggcacagcac 451 atgatagaac tcctcataga acccttttaa tgaatgagtt gggtgttccg 501 caaccaaaea tttcatttgg agtgtgcata gcatggtcca gctcaagctg 551 ccatgatggg aaagcatggt tacatgtttg tgtcactggg gatgatagaa 601 atgcaacggc tagcatcatt tatgatggga tacttgttga cagtattggt 651 tcatggtcta aaaaeatcct cagaactcag gagtcagaat gcgttcgcat 701 caatggaacc tgtgcagtag taatgactga tggaagtgca tcaggaaggg 751 ctgaeactag aatactattt attagagagg ggaaaattgc acacattagc 801 gaagtgctca ccattgtcag gcatgtggag gaatgctcct gttacccccg 881 atatccagaa gttagatgtg tttgcagaga caattggaag ggatccaata 901 ggcccgttct atatataaat acggcaaatt atagtattga ttccagttat

951 gtgtgctcag gacttgttgg cgacacacca agaaatgatg ataggtctag 1001 cagcagcaac tgcagagatc ctaataacga gagaggggcc ccaggagtaa 1051 aagggtgggc ctttgacaat ggaaatgaca tttggatggg aagaacaatc 1101 aaaaaggatt cgcgctcagg ttatgagacC ttcagggtca ttggtggttg 1151 gaccactgct aattccaagt cacagataaa tagacaagtc atagttgaca 1201 gtgataactc gtctgggtat tctggtatct tctctgttga aggcaaaagc 1251 tgcatcaaca ggtgttttta cgtggagttg ataagaggaa gaccaaagga 1301 gactagggtg cggtggactc 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) Whole molecule length: 469 aa mnpnqkiiai vpcepiiier 101 skdnsirlsa 151 drtphrtllm 201 atasiiydgi gsvsltiati niteivhlna ggdiwvtrep nelvdsvpfwshla lgvdsvpfwshla

251 dtrilfireg kiahisplsg

301 pvlyinmany

351 gwafdngndi

401 dnssgysgi £

451 gtygtgswpd sidssyvcsg wmgrtikkds svegkscinr ganinfmpi cllmqiaila ttlekeicpk yvscglgkcy tkqvciawBS nilrtqesec saqhveecsc lvgdtpmdd rsgyetfrvi cfyvelirgr ttmtlhfkqn vadymwskp qf algqgttl sschdgkawl vcingtcaw yprypevrcv rssssncrdp ggwttansks pketrvwwts ecinssnnqv qcqitgf APF enkhsngtah hvcvtgddm mtdgsasgra crdnwkgsnr nnergapgvk qinrqvivds neiivfcgts

ES 2 394 033 T3

Summary of SEQ ID No. designations

<td>SEQ ID No</td><td>HA or NA</td><td>NAME OF THE STRAIN</td><td>Amino acid or nucleotide</td>

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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