MXPA05012712A - High titer recombinant influenza viruses for vaccines and gene therapy - Google Patents

Abstract

The invention provides a composition useful to prepare high titer influenza viruses, e.g., in the absence of helper virus, which includes a sequence from a high titer influenza virus isolate.

Description

INFLUENZA VIRUS RECO BINANTES OF HIGH TITULATION FOR VACCINES AND GENIUS THERAPY BACKGROUND OF THE INVENTION Negative sense RNA viruses are classified into seven families. { Rhabdoviridae, Paramyxoviridae, Filoviridae, Bornaviridae, Orthomyxoviridae, Bunyaviridae and Arenaviridae) which include common human pathogens, such as respiratory syncytial virus, influenza virus, measles virus and Ebola virus, as well as animal viruses with the greatest economic impact in the poultry industries and livestock (for example, Newcastle disease virus and Rinderpest virus). The first four families are characterized by non-segmented genomes, while the last three have genomes comprising six to eight, three, or two RNA segments of negative sense, respectively. The common feature of negative sense RNA viruses is the negative polarity of their RNA genome; that is, viral RNA (vRNA) is complementary to mRNA and therefore is not infectious in itself. To initiate viral transcription and replication, vRNA has to be transcribed into a non-inverse mRNA or cRNA, respectively, by the viral polymerase complex and the nucleoprotein; for influenza A virus, the viral polymerase complex comprises all three polymerase proteins REP .: 168297 PB2, PB1 and PA. During viral replication, the cRNA serves as a template for the synthesis of new vRNA molecules. For all negative-strand RNA viruses, the non-coding regions at both the 5 'and 3' ends of the vRNA and cRNA are critical for the transcription and replication of the viral genome. Unlike cellular or viral mRNA transcripts, both cRNA and vRNA are neither blocked at the 5 'end nor polyadenylated at the 3' end. The basic functions of many viral proteins have been elucidated biochemically and / or in the context of viral infection. However, reverse genetics systems have dramatically increased our knowledge of segmented and unsegmented negative strand RNA viruses with respect to viral replication and pathogenicity, as well as the development of live attenuated virus vaccines. Reverse genetics, as the term is used in molecular virology, is defined as the generation of viruses that possess a genome derived from cloned cDNA molecules (for a review, see Neumann et al., 2002). To initiate viral replication of negative-strand RNA viruses, vRNA or cRNA molecules must be coexpressed with the polymerase complex and the nucleoprotein. Rabies virus was the first unsegmented negative sense RNA virus that was generated entirely from cloned cDNA: Schnell et al. , (1994) generated recombinant rabies viruses by cotransfection of a cDNA construct that encoded full-length protein and cRNA expression constructs for L, P and N proteins, all under the control of the T7 RNA polymerase promoter. Infection with recombinant vaccinia virus, which provided T7 RNA polymerase resulted in the generation of infectious rabies virus. In this T7 polymerase system, the primary transcription of the full length cRNA under the control of the T7 RNA polymerase resulted in an unblocked cRNA transcript. However, three guanidine nucleotides, which form the optimal start sequence for T7 RNA polymerase, were bound at the 5 'end. To create an authentic 3 'end of the cRNA transcript that is essential for a productive infectious cycle, the hepatitis delta ribozyme sequence (HDVRz) was used for an exact autocatalytic cut-off at the 3 'end of the cRNA transcript. Since the initial report by Schnell et al. , (1994), several inverse genetics systems using similar techniques led to the generation of many unsegmented negative strand RNA viruses (Conzelmann, 1996; Conzelmann, 1998; Conzelmann et al. , nineteen ninety six; Marriott et al. , 1999; Muñoz et al. , 2000; Nagai 1999; Neumann et al. , 2002; Roberts et al. , 1998; Rose, 1996). Refinements of the original rescue procedure included the expression of T7 RNA polymerase from stably transfected cell lines (Radecke et al., 1996) or from protein expression plasmids (Lawson et al., 1995), or shock procedures thermal to increase rescue efficiencies (Parks et al., 1999). Based on the T7 polymerase system, Bridgen and Elliott (1996) created the Bunyamwera virus (Bunyaviridae family) from cloned cDNA molecules and demonstrated the possibility of artificially generating a negative sense RNA virus segmented by the T7 system. polymerase In 1999, a reverse genetics technique based on plasmids based on cellular RNA polymerase I was generated for the generation of influenza A virus completely segmented from cloned cDNA molecules (Fodor et al., 1999; Neumann et al. Kawaoka, 1999). RNA polymerase I, a nucleolar enzyme, synthesizes ribosomal RNA which, like the RNA of the influenza virus, does not contain 5'-end blocking structures or polyA 3 '. Transcription of RNA polymerase I from a construct containing an influenza viral cDNA, flanked by promoter and terminator sequences of RNA polymerase I, resulted in the synthesis of influenza vRNA (Fodor et al., 1999; Neumann and Kawaoka, 1999; Neumann and Kawaoka, 2001; Pekosz et al. , 1999). The system was highly efficient, producing more than 108 infectious virus particles per ml of supernatant of cells transfected with plasmid 48 hours after transfection. What is required is a method for preparing high titration orthomyxoviruses such as influenza A virus, completely from cloned cDNA molecules.

BRIEF DESCRIPTION OF THE INVENTION The invention provides an isolated and / or purified nucleic acid molecule (polynucleotide) encoding at least one of the proteins of a high titre influenza virus, eg, titers of more than 109 / ml , for example more than 1010 / ml, or a portion thereof, or the complement of the nucleic acid molecule. In one embodiment, the isolated and / or purified nucleic acid molecule encodes HA, NA, PB1, PB2, PA, NO, M or NS, or a portion thereof that has substantially the same activity as a corresponding polypeptide encoded by a of SEQ ID No: 1-8. As used herein, substantially the same activity "includes an activity that is about 0.1%, 1%, 10%, 30%, 50%, 90%, for example, up to 100% or more, or a level of proteins. detectable that is approximately 80%, 90% or more, the activity or level of proteins, respectively, of the corresponding full-length polypeptide In one embodiment, the isolated and / or purified nucleic acid molecule encodes a polypeptide that is substantially same as, for example, the one having at least 80%, for example, 90%, 92%, 95%, 97% or 99%, identity of contiguous amino acid sequences with, a polypeptide encoded by one of SEQ ID No In one embodiment, the isolated and / or purified nucleic acid molecule comprises a nucleotide sequence that is substantially the same as, for example, has at least 50%, eg, 60%, 70%, 80 % or 90% or more identity of nucleic acid sequences contiguous with, one of SEQ ID No: 1-8, or the plement thereof, and, in one embodiment, also encodes a polypeptide having at least 80%, eg, 90%, 92%, 95%, 97% or 99% identity of contiguous amino acid sequences with a polypeptide encoded by one of SEQ ID No: 1-8. In one embodiment, the isolated and / or purified nucleic acid molecule encodes-for a polypeptide with one or more, for example, 2, 5, 10, 15, 20 or more, conservative amino acid substitutions, eg, conservative substitutions of up to 10% or 20% of the residues, relative to a polypeptide encoded by one of SEQ ID No: 1-8. "Conservative amino acid substitutions refer to the exchange capacity of residues having similar side chains, for example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine and isoleucine.; a group of amino acids that have aliphatic hydroxyl side chains is serine and threonine; A group of amino acids having side chains containing amide is asparagine and glutamine; a group of amino acids that have aromatic side chains is phenylalanine, tyrosine and tryptophan; A group of amino acids that have basic side chains is lysine, arginine and histidine and a group of amino acids that have a side chain that contains sulfur is cysteine and methionine. The conservative amino acid substitution groups are: valine-leucine-isoleucine; phenylalanine tyrosine; lysine-arginine; alanine-valine; glutamic-aspartic and asparagine-glutamine. In another embodiment, the isolated and / or purified nucleic acid molecule of the invention or the complement thereof, hybridized to one of SEQ ID NO: 1-8, or the complement thereof, under conditions of low severity, severity moderate or severe. For example, the following conditions can be used: 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50 ° C with 2X SSC wash, 0.1% SDS at 50 ° C (low severity) , very desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50 ° C with IX wash of SSC, 0.1% SDS at 50 ° C (moderate severity), most desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50 ° C with 0.5X SSC wash, 0.1% SDS at 50 ° C (severe), preferably in 7% sodium dodecyl sulfate. % (SDS), 0.5 M NaP04, lmM EDTA at 50 ° C with washing in 0.1X SSC, 0.1% SDS at 50 ° C (more severe), most preferably in 7% sodium dodecylsulfate (SDS) , 0.5 M NaP04, lmM EDTA at 50 ° C with 0.5X SSC wash, 0.1% SDS at 65 ° C (too severe). In one embodiment, the nucleic acid molecule of the invention encodes a polypeptide that is substantially the same as, for example, has at least 50%, eg, 60%, 70%, 80% or 90% or more identity of nucleic acid sequences contiguous with, one of SEQ ID NO: 1-8, and preferably 'have substantially the same activity as a corresponding full-length polypeptide encoded by one of SEQ ID NO: 1-8. The nucleic acid molecule of the invention can be used to express influenza proteins, to prepare chimeric genes, for example, with other viral genes including other influenza virus genes, and / or to prepare recombinant viruses. In this manner, the invention also provides isolated polypeptides, recombinant viruses and host cells contacted with the nucleic acid molecules or recombinant virus of the invention.

The invention also provides at least one of the following isolated and / or purified vectors: a vector comprising a promoter operably linked to an influenza A virus cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus cDNA PB1 linked to a transcription termination sequence, a vector comprising a promoter operably linked to a cDNA of influenza virus PB2 linked to a transcription termination sequence, a vector comprising a promoter operably linked to a HA influenza virus cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus NP cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an NA influenza virus cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to a cDNA of influenza virus M linked to a transcription termination sequence, a vector comprising a promoter operably linked to a cDNA of influenza virus NS linked to a transcription termination sequence, wherein at least one vector comprises sequences encoding HA, NA, PB1, PB2, PA, NP, M, NS, or a portion thereof, which have substantially the same activity as a corresponding polypeptide encoded by one of SEQ ID NO: 1-8, for example, a sequence encoding a polypeptide with at least 80% amino acid identity with a polypeptide encoded by one of SEQ ID N? : 1-8. Optionally, two vectors can be employed in place of the vector comprising a promoter operably linked to a cDNA of influenza virus M linked to a transcription termination sequence, eg, a vector comprising a promoter operably linked to a cDNA of Ml influenza virus linked to a transcription termination sequence and a vector comprising a promoter operably linked to an M2 influenza virus cDNA linked to a transcription termination sequence. The invention provides isolated and purified vectors or plasmids, which are expressed or encoded influenza virus proteins, or express or encode influenza vRNA, both native and recombinant vRNA. Preferably, the vectors comprise influenza cDNAs, for example, for example, influenza A DNA (e.g., any influenza A gene including any of the HA or 9 NA subtypes), B or C (see chapters 45 and 46). of Fields Virology (Fields et al., (eds.), Lippincott-Raven Publ., Philadelphia, PA (1996), which are specifically incorporated by way of reference herein), although it is envisioned that genes of any organism can used in the vectors or methods of the invention The cDNA may be in sense orientation or anti-sense in relation to the promoter Thus, a vector of the invention may encode a protein (sense) or vRNA (anti-sense) of influenza virus Any suitable transcription or promoter termination sequence can be used to express a protein or peptide, for example, a viral protein or peptide, a protein or peptide from a non-viral pathogen or a therapeutic protein or peptide. The invention provides a composition comprising a plurality of influenza virus vectors of the invention. In one embodiment of the invention, the composition comprises: a) at least two vectors selected from a vector comprising a promoter operably linked to an influenza virus PA cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus cDNA PBl linked to a transcription termination sequence, a vector comprising a promoter operably linked to a cDNA of influenza virus PB2 linked to a transcription termination sequence, a vector comprising a promoter operably linked to a cDNA of influenza A virus linked to a terminator sequence of transcription, a vector comprising a promoter operably linked to a cDNA of influenza virus NP linked to a transcription termination sequence, a vector comprising a promoter operably linked to a cDNA of NA virus bound to a transcription termination sequence, a vector comprising a promoter operably linked to an M influenza virus cDNA linked to a transcription termination sequence and a vector comprising a promoter operably linked to a virus cDNA of the inf luence NS linked to a transcription termination sequence, wherein at least one vector comprises a omotor operably linked to a nucleic acid molecule of the invention linked to a transcription termination sequence and b) at least two vectors selected from a vector coding for influenza A virus, a vector coding for influenza virus PBl , a vector coding for influenza virus PB2 and a vector coding for influenza virus NP. Optionally, the vectors of b) include one or more vectors encoding NP, NS, M, eg, Ml and M2, HA or NA. Preferably, the vectors encoding viral proteins further comprise a transcription termination sequence.

In another embodiment, the composition comprises: a) at least two vectors selected from a vector comprising a promoter operably linked to an influenza virus PA cDNA linked to a transcription termination sequence, a vector comprising a linked promoter operably to an influenza virus cDNA PB1 linked to a transcription termination sequence, a vector comprising a promoter operably linked to a cDNA of influenza virus PB2 linked to a transcription termination sequence, a vector comprising a promoter operably linked to a HA influenza virus cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus NP cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to a cDNA of influenza virus NA and NB linked to a sec transcription termination order, a vector comprising a promoter operably linked to a cDNA of influenza virus M linked to a transcription termination sequence, a vector comprising a promoter operably linked to a cDNA of linked influenza virus NS to a transcription termination sequence and a vector comprising a promoter operably linked to an influenza virus BM2 cDNA operably linked to a nucleic acid molecule of the invention linked to a transcription termination sequence; and b) at least two vectors selected from a vector coding for influenza virus PA, a vector coding for influenza virus PB1, a vector coding for influenza virus PB2 and a vector coding for influenza virus. influenza NP. Optionally, the vectors of b) include one or more vectors encoding NP, NS, M, HA or NA. Preferably, the vectors encoding viral proteins further comprise a transcription termination sequence. A composition of the invention may also comprise an open reading frame or gene of interest, for example, a foreign gene encoding an immunogenic peptide or protein useful as a vaccine. In this manner, another embodiment of the invention comprises a composition of the invention as described above in which one of the vectors is replaced with, or the composition further comprises, a vector comprising a promoter linked to sequences of influenza viruses. 5 'which optionally include 5' influenza virus coding sequences or a portion thereof, linked to a desired nucleic acid sequence, eg, a desired cDNA, linked to 3 'influenza virus sequences that include optionally 3 'influenza virus coding sequences or a portion thereof, linked to a transcription termination sequence. Preferably, the desired nucleic acid sequence such as a cDNA is in an antisense orientation. The introduction of this composition into a host cell permissive for the replication of influenza viruses results in a recombinant virus comprising vRNA which corresponds to vector sequences. The promoter in this vector for the production of vRNA can be a promoter of RNA polymerase I, an RNA polymerase II promoter, an RNA polymerase III promoter, a T7 promoter and a T3 promoter, and optionally the vector comprises a terminator sequence of transcription such as a transcription termination sequence of RNA polymerase I, a transcription termination sequence of RNA polymerase II, a transcription termination sequence of RNA polymerase III, or a ribozyme. In one embodiment, the vector comprising the desired nucleic acid sequence comprises a cDNA of interest. The cDNA of interest, whether in a vector for the production of vRNAs or proteins, can code for an immunogenic epitope, such as a useful epitope in a cancer therapy or vaccine, or a peptide or polypeptide useful in gene therapy. When viruses are prepared, the vector or plasmid comprising the gene or cDNA of interest can substitute a vector or plasmid for an influenza viral gene or can be in addition to vectors or plasmids for all influenza viral genes. A plurality of the vectors of the invention can be physically linked or each vector can be present in a single or other plasmid, for example, linear nucleic acid delivery vehicle. The transcription or promoter termination sequence in a virus or vRNA protein expression vector can be the same or different in relation to the promoter or any other vector. Preferably, the vector or plasmid expressing influenza vRNA comprises a promoter suitable for expression in at least one particular host cell, for example, host cells of birds or mammals such as canine, feline, equine, bovine, ovine or of primate, including human cells, or preferably, for expression in more than one host. In one embodiment, one or more vectors for the production of vRNAs comprise a promoter including, but not limited to, an RNA polymerase I promoter, eg, a human RNA polymerase I promoter, an RNA polymerase II promoter, an RNA polymerase III promoter, a T7 promoter or a T3 promoter. Preferred transcription termination sequences for vRNA vectors include, but are not limited to, a transcription termination sequence of RNA polymerase I, a transcription termination sequence of RNA polymerase II, a transcription termination sequence of RNA polymerase III or a ribozyme. Ribozymes within the scope of the invention include, but are not limited to, Tetrahymena ribozymes, RNase P, hammerhead ribozymes, pin ribozymes, hepatitis ribozyme, as well as synthetic ribozymes. In one embodiment, at least one vector for vRNA comprises an RNA polymerase II promoter linked to a ribozyme sequence linked to viral coding sequences linked to other ribozyme sequences, optionally linked to a transcription termination sequence of RNA polymerase II . In one embodiment, at least two and preferably more, for example, 3, 4, 5, 6, 7 or 8 vectors for the production of vRNAs comprise an RNA polymerase II promoter, a first ribozyme sequence, which is 5 'to a sequence corresponding to viral sequences that include viral coding sequences, which is 5' to a second sequence of ribozymes, which is 5 'to a transcription termination sequence. Each RNA polymerase II promoter in each vRNA vector may be the same or different than the RNA polymerase II promoter in any other vRNA vector. Similarly, each ribozyme sequence in each vRNA vector may be the same or different as the ribozyme sequences in any other vRNA vector. In one embodiment, the ribozyme sequences in a single vector are not the same. The invention also provides a method for preparing influenza viruses. The method comprises contacting a cell with a plurality of the vectors of the invention, for example, sequentially or simultaneously, for example, using a composition of the invention, in an amount effective to produce infectious influenza virus. The invention also includes isolating virus from a cell contacted with the composition. Thus, the invention also provides isolated viruses, as well as a host cell contacted with the composition or virus of the invention. In another embodiment, the invention includes contacting the cell with one or more vectors, either RNAV or protein production vectors, rather than with other vectors, either RNAV or protein production vectors. The method of the invention allows easy handling of influenza virus, for example, by introducing attenuation mutations in the viral genome. In addition, because influenza viruses induce strong humoral and cellular immunity, the invention broadly increases these viruses as vaccine vectors, particularly in view of the availability of natural variants of the. viruses, which can be used sequentially, allowing repetitive use for gene therapy. The methods for producing viruses described herein, which do not require infection with helper viruses, are useful in studies of viral mutagenesis, and in the production of vaccines (eg, for AIDS, influenza, hepatitis B, hepatitis C, rhinoviruses, filoviruses, malaria, herpes and foot-and-mouth disease) and vectors for gene therapy (eg, for cancer, AIDS, adenosine deaminase, muscular dystrophy, ornithine transcarbamylase deficiency, and central nervous system tumors). Thus, a virus is provided for use in medical therapy (e.g., for a therapy with vaccine or genes). The invention also provides a method for immunizing an individual against a pathogen, for example, a bacterium, virus or parasite, or a malignant tumor. The method comprises administering to the individual an amount of at least one isolated virus of the invention, optionally in combination with an adjuvant, effective to immunize the individual. The virus comprises vRNA comprising a polypeptide encoded by the pathogen or a tumor-specific polypeptide. A method is also provided for increasing or increasing the expression of an endogenous protein in a mammal having an indication or disease characterized by a reduced amount or a lack of the endogenous protein. The method comprises administering to the mammal an amount of an isolated virus of the invention effective to increase or increase the amount of the endogenous protein in the mammal. Preferably, the mammal is a human. BRIEF DESCRIPTION OF THE FIGURES Figures 1A-1B is a schematic diagram of established reverse genetics systems. In the transfection method with RNP (Fig. 1A), purified NP and polymerase proteins are assembled in RNPs with the use of vRNA synthesized in vitro. The cells are transfected with RNPs, followed by infection with helper virus. In the RNA polymerase I method (Fig. IB), a plasmid containing the RNA polymerase I promoter, a cDNA encoding the vRNA to be rescued, and the RNA polymerase I terminator are transferred into cells. Intracellular transcription by RNA polymerase I produces synthetic vRNA, which is packaged in progeny virus particles after infection with helper virus. With both methods, transfectant viruses (ie, those containing RNA derived from cloned cDNA), are selected from the population of helper viruses. Figure 2 is a schematic diagram of the generation of RNA polymerase I constructs. CRNA molecules derived from influenza viruses were amplified by PCR, digested with SSIΔBI and cloned in the BsmBl sites of the vector pHH21 (thesis of E. Hoffmann, Ph.D. Justus, Liebig-University, Giessen, Germany), which contains the human RNA polymerase I (P) promoter and mouse RNA polymerase I (T) terminator. The thymidine nucleotide towards the 5 'end of the terminator sequence (* T) represents the 3' end of the influenza viral RNA. The influenza A virus sequences are shown in bold letters (SEQ ID No: 29-40). Figure 3 is a proposed reverse genetics method for generating segmented negative sense RNA viruses. Plasmids containing the cDNA of the RNA polymerase a promoter for each of the eight viral RNA segments, and the RNA polymerase I terminator are transfected into cells together with protein expression plasmids. Although infectious viruses can be generated with plasmids that express PA, PBl, PB2 and NP, the expression of all the remaining structural proteins (shown in brackets) increases the efficiency of virus production depending on the virus generated. Figure 4 shows the titration of several influenza viruses.

DETAILED DESCRIPTION OF THE INVENTION Definitions As used herein, the terms "isolated and / or purified" refer to the preparation, isolation and / or in vitro purification of a vector, plasmid or virus of the invention, such that is not associated with substances in vivo, ie substantially purified from substances in vitro. An isolated virus preparation is generally obtained by in vitro culture and propagation and is substantially free of other infectious agents. As used herein "substantially free" means below the level of detection for a particular infectious agent using standard detection methods for that agent. A "recombinant" virus is one that has been engineered in vitro, for example, using recombinant DNA techniques, to introduce changes in the viral genome. As used herein, the term "recombinant nucleic acid" or "recombinant DNA sequence or segment" refers to a nucleic acid, for example, to DNA, that has been derived or isolated from a source, which may be subsequently chemically altered in vi tro, in such a way that its sequence does not occur naturally, or corresponds to sequences that occur naturally that are not placed as if they were placed in the native genome. An example of DNA "derived" from a source would be a DNA sequence that was identified as a useful fragment, and which was then chemically synthesized in essentially pure form. An example of this DNA "isolated" from a source would be a useful DNA sequence that would be removed or removed from the source by chemical means, that is, by the use of restriction endonucleases, in such a way that it could be manipulated further, by example, amplified, for use in the invention, by genetic engineering methodology. Replication of influenza viruses Influenza A viruses possess a genome of eight negative-strand viral RNA molecules (vRNA molecules) that code for a total of ten proteins. The life cycle of the influenza virus begins with the binding of hemagglutinin (HA) to receptors containing sialic acid on the surface of the host cell, followed by receptor-mediated endocytosis. The low pH in late endosomes triggers a conformational shift in HA, thus exposing the N-terminus of the HA2 subunit (the so-called fusion peptide). The fusion peptide initiates fusion of the viral and endosomal membrane, and the matrix protein (Ml) and RNP complexes are released into the cytoplasm. The RNPs consist of the nucleoprotein (NP), which encapsida vRNA, and the viral polymerase complex, which is formed by the proteins PA, PBl and PB2. The RNPs are transported to the interior of the nucleus, where transcription and replication take place. The RNA polymerase complex catalyzes three different reactions: synthesis of an mRNA with a 5 'end block and a 3' polyA structure, of a full length complementary RNA (cRNA), and of genomic vRNA using the cDNA as a template. The newly synthesized vRNA, NP and polymerase proteins are then assembled into RNPs, exported from the nucleus, and transported to the plasma membrane, where the sprouting of progeny virus particles occurs. The neuraminidase (NA) protein plays a crucial role late in the infection by removing sialic acid from sialiloligosaccharides, thus releasing freshly assembled virions from the cell surface and preventing self-aggregation of virus particles. Although the assembly of viruses includes protein-protein and vRNA-protein interactions, the nature of these interactions is largely unknown. Although influenza B and C viruses are structurally and functionally similar to the influenza A virus, there are some differences. For example, influenza B virus does not have an M2 protein with ion channel activity. Similarly, influenza C virus does not have an M2 protein with ion channel activity. However, the CM1 protein is likely to have this activity. The activity of an ion channel protein can be measured by methods well known in the art, see, for example, Holsinger et al. , (1994) and WO 01/79273.

Influenza cell lines and viruses that can be used in the present invention According to the present invention, any cell that supports efficient replication of influenza viruses can be employed in the invention, including mutant cells that express reduced or decreased levels of one or more sialic acids which are receptors for influenza virus. The viruses obtained by the methods can be made a reclassifying virus. Preferably, the cells are continuous cell lines certified, or certifiable by the WHO. The requirements for certifying these cell lines include characterization with respect to at least one of genealogy, growth characteristics, immunological markers, tumorigenicity of virus susceptibility and storage conditions, as well as by tests on animals, eggs and cell cultures. This characterization is used to confirm that the cells are free of detectable upstream agents. In some countries, cariology may also be required. In addition, tumorigenicity is preferably tested in cells that are at the same passage level as those used for vaccine production. The virus is preferably purified by a process that has been shown to give consistent results, before being inactivated or attenuated for vaccine production (see, eg, World Health Organization, 1982). It is preferred to establish a complete characterization of the cell lines that will be used, so that suitable tests can be included to verify the purity of the final product. The data that can be used for the characterization of a cell to be used in the present invention include a) information about its origin, derivation and passing history; b) information on their growth and morphological characteristics; c) results of tests of upstart agents; d) distinctive characteristics such as biochemical, immunological and cytogenetic patterns which allow cells to be easily recognized among other cell lines and e) test results for tumorigenicity. Preferably, the level of passage, or duplication of the population, of the host cell is as low as possible. It is preferred that the virus produced in the cell be highly purified prior to the formulation for therapy with vaccines or genes. In general, purification procedures will result in the extensive removal of cellular DNA, other cellular components, and upstart agents. Procedures that degrade or extensively denature DNA can also be used. See, for example, Mizrahi, 1990.

Vaccines A vaccine of the invention may comprise immunogenic proteins including glycoproteins of any pathogen, for example, an immunogenic protein of one or more bacteria, viruses, yeasts or fungi. Thus, in one embodiment, the influenza viruses of the invention can be vaccine vectors for influenza viruses or other viral pathogens including but not limited to lentiviruses such as HIV, hepatitis B virus, hepatitis C virus, herpes viruses such as CMV or HSV or foot-and-mouth disease virus. A complete virion vaccine is concentrated by ultrafiltration and then purified by zonal centrifugation or by chromatography. It is inactivated before or after purification using formalin or beta-propiolactone, for example. A subunit vaccine comprises purified glycoproteins. This vaccine can be prepared as follows: using fragmented viral suspensions by treatment with detergents, the surface antigens are purified, by ultra-centrifugation for example. The subunit vaccines then contain mainly HA protein, and also NA. The detergent used can be a cationic detergent for example, such as hexadecyltrimethylammonium bromide (Bachmeyer, 1975), an anionic detergent such as ammonium deoxycholate (Laver &; Webster, 1976) or a non-ionic detergent such as that marketed under the name TRITON X100. The hemagglutinin can also be isolated after treatment of the virions with a protease such as bromelain, and then purified by a method such as that described by Grand and Skehel (1972). A divided vaccine comprises virions that have been subjected to treatment with agents that dissolve lipids. A divided vaccine can be prepared as follows: an aqueous suspension of the purified virus obtained as mentioned above, inactivated or not, is treated, under agitation, by lipid solvents such as ethyl ether or chloroform, associated with detergents. The dissolution of the lipids of the envelope or viral capsid results in the fragmentation of the viral particles. The aqueous phase containing the divided vaccine is recovered, consisting mainly of hemagglutinin and neuraminidase with its original lipid environment removed, and the core or its degradation products. Then the residual infectious particles are inactivated if this has not already been done.

Inactivated vaccines The inactivated influenza virus vaccines of the invention are provided by inactivating replicated viruses of the invention using known methods, such as, but not limited to, formalin or β-propiolactone treatment. The types of inactivated vaccine that can be used in the invention can include whole virus vaccines (WV) or subvirion (SV) vaccines (divided). The WV vaccine is intact and inactivated, while the SV vaccine contains purified virus broken with detergents that solubilize the viral envelope containing lipids, followed by the chemical inactivation of residual virus. In addition, vaccines that can be used include those containing the isolated HA and NA surface proteins, which are referred to as surface antigens or subunit vaccines. In general, responses to SV vaccines and surface antigen (ie, purified HA or NA) are similar. An experimental inactivated WV vaccine containing an NA antigen immunologically related to the epidemic virus and an unrelated HA seems to be less effective than conventional vaccines (Ogra et al., 1977). Inactivated vaccines containing both relevant surface antigens are preferred.

Live attenuated virus vaccines Live attenuated influenza virus vaccines can also be used to prevent or treat infection with influenza viruses, according to known method steps. Attenuation is preferably achieved in a single step by transferring attenuated genes from an attenuated donor virus to a replicated isolate or virus reclassified according to known methods (see, eg, Murphy, 1993). Since resistance to influenza A virus is mediated by the development of an immune response to HA and NA glycoproteins, the genes encoding these surface antigens must come from reclassified viruses or high growth clinical isolates. The attenuated genes are derived from the attenuated parent. In this approach, the genes that confer preferential attenuation do not code for the HA and NA glycoproteins. Otherwise, these genes could not be transferred to reclassifiers that carry the surface antigens of the clinical virus isolate. Many donor viruses have been evaluated to verify their ability to reproducibly attenuate influenza viruses. As a non-limiting example, donor virus (ca) cold-adapted A / Ann Arbor (AA) / 6/60 (H2N2) can be used for the production of attenuated vaccines (see, for example, Edwards, 1994; Murphy, 1993). In addition, live and attenuated reclasificant virus vaccines can be generated by coupling the donor virus ca with a virulent replicated virus of the invention. The reclassifying progeny are then selected at 25 ° C, (restrictive for the replication of virulent viruses), in the presence of an H2N2 antiserum, which inhibits the replication of the viruses carrying the surface antigens of the ca donor virus (A / AA / 6/60 (H2N2) attenuated A large series of reclassifiers H1N1 and H3N2 have been evaluated in humans and have been found to be satisfactory: (a) infectious, (b) attenuated for immunologically primed and immunologically primed children, (c) immunogenic and (d) genetically stable The immunogenicity of ac reclassifiers is parallel to their level of replication, thus the acquisition of the six transferable genes of the ca-acifer virus by new wild type viruses has reproducibly attenuated these viruses for used in vaccinating susceptible adults and children Other attenuation mutations can be introduced into influenza virus genes by site-directed mutagenesis to rescue virus in fecciosos that carry these mutant genes. Attenuation mutations can be introduced into non-coding regions of the genome, as well as into coding regions. These attenuation mutations can also be introduced into genes other than the HA or NA gene, for example the PB2 gene of polymerase (Subbarao et al., 1993). Thus, new donor viruses carrying attenuation mutations introduced by site-directed mutagenesis can also be generated, and these new donor viruses can be used in the reduction of live attenuated reclassifying H1N1 and H3N2 vaccine candidates in a manner analogous to that described above for the AC donor virus A / AA / 6/60. Similarly, other known and suitable attenuated donor strains can be reclassified with influenza viruses of the invention to obtain attenuated vaccines suitable for use in vaccination of mammals (Enami et al., 1990; Muster et al. , 1991; Subbarao et al. , 1993). It is preferred that these attenuated viruses maintain the genes of the viruses encoding antigenic determinants substantially similar to those of the original clinical isolates. This is due to the purpose that the attenuated vaccine should provide substantially the same antigenicity as the original clinical isolate of the virus, while at the same time lacking infectivity to the extent that the vaccine causes a minimal change in the induction of a serious pathogenic condition in the vaccinated mammal. The virus can then be attenuated or inactivated, formulated and administered, according to known methods, such as a vaccine to induce an immune response in an animal, for example, a mammal. Methods for determining whether these attenuated or inactivated vaccines have maintained antigenicity similar to that of the clinical isolate or high growth strain derived therefrom are well known in the art. These known methods include the use of antisera or antibodies to eliminate viruses that express antigenic determinants of the donor virus; chemical selection (for example, amantadine or rimantidine); activity and inhibition of HA and NA; and DNA screening (such as probe hybridization or PCR) to confirm that donor genes encoding antigenic determinants (e.g., HA or NA genes) are not present in attenuated viruses. See, for example, Robertson et al. , 1988; Kilbourne, 1969; Aymard-Henry et al. , 1985; Robertson et al. 1992 Pharmaceutical Compositions The pharmaceutical compositions of the present invention, suitable for inoculation or for parenteral or oral administration, comprise attenuated or inactivated influenza viruses, optionally further comprising sterile aqueous or non-aqueous solutions, suspensions and emulsions. The compositions may further comprise auxiliary agents or excipients, such as those known in the art. See, for example, Berkow et al. , 1987; Avery 's Drug Treatment, 1987; Osol, 1980; Katzung, 1992. The composition of the present invention is generally presented in the form of individual doses (unit doses). Conventional vaccines generally contain about 0.1 to 200 μg, preferably 10 to 15 μg of haemagglutinin from each of the strains that come into their composition. The vaccine forming the main constituent of the vaccine composition of the invention may comprise a virus of type A, B or C, or any combination thereof, for example, at least two of the three types, at least two of subtypes different, at least two of the same type, at least two of the same subtype, or isolated or different reclassifiers. The type A human influenza virus includes the subtypes H1N1, H2N2 and H3N2. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and / or emulsions, which may contain auxiliary agents or excipients known in the art. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. Vehicles or occlusive dressings can be used to increase the permeability of the skin and improve the absorption of antigens. The liquid dosage forms for oral administration may generally comprise a liposome solution containing the liquid dosage form. Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups and elixirs containing inert diluents commonly used in the art, such as purified water. Apart from the inert diluents, these compositions may also include adjuvants, wetting agents, emulsifying and suspending agents, or sweetening, flavoring or perfuming agents. See, for example, Berkow et al. , 1992; Avery 's 1987; Osol, 1980; and Katzung, 1992. When a composition of the present invention is used for administration to an individual, it may further comprise salts, pH regulators, adjuvants or other substances which are desirable to improve the effectiveness of the composition. For vaccines and adjuvants, substances that can increase a specific immune response can be used. Normally, the adjuvant and the composition are mixed before presentation to the immune system, or they are presented separately, but in the same place of the organism that is being immunized. Examples of suitable materials for use in vaccine compositions are provided in Osol (1980). Heterogeneity in a vaccine can be provided by mixing replicated influenza viruses for at least two strains of influenza virus, such as 2-50 strains or any scale or value there. Strains of influenza A or B viruses having a modern antigenic composition are preferred. In accordance with the present invention, vaccines can be provided for variations in a single strain of an influenza virus, using techniques known in the art. A pharmaceutical composition according to the present invention may further comprise or additionally at least one chemotherapeutic compound, for example, for gene therapy, immunosuppressants, anti-inflammatory or immunity enhancing agents, and for vaccines, chemotherapeutics which include, but are not limited to, gamma globulin, amantadine, guanidine, hydroxybenzimidazole, interferon-a, interferon-beta, interferon?, tumor necrosis factor alpha, thiosemicarbazonas, metizazona, raffle pin, ribavirin, a pyrimidine analogue, a purine analogue, foscarnet, phosphonoacetic acid, acyclovir, dideoxynucleosides, a protease inhibitor or ganciclovir. See, for example, Katzung (1992), and references cited there on pages 798-800 and 680-681, respectively. The composition may also contain variable but small amounts of formaldehyde free of endotoxins, and preservatives, which have been found to be safe and which do not contribute to undesirable effects in the organism to which the composition is administered.

Pharmaceutical Purposes The administration of the composition (or the antisera that it develops) can be either for a "prophylactic" or "therapeutic" purpose. When provided prophylactically, the compositions of the invention which are vaccines, are provided before any symptoms of a pathogenic infection become manifest. The prophylactic administration of the composition serves to prevent or attenuate any subsequent infection. When provided prophylactically, the compositions for gene therapy of the invention are provided before any symptom of a disease becomes manifest. Prophylactic administration of the compositions serves to prevent or attenuate one or more symptoms associated with the disease. When provided therapeutically, an attenuated or inactivated viral vaccine is provided after detection of a symptom of a real infection. The therapeutic administration of the compounds serves to attenuate any real infection. See, for example, Berkow et al. , 1992; Avery, 1987 and Katzung, 1992. When provided therapeutically, a composition for gene therapy is administered after the detection of a symptom or indication of the disease. The therapeutic administration of the compounds serves to attenuate a symptom or indication of that disease. Thus, an attenuated or inactivated vaccine composition of the present invention can then be provided either before the onset of the infection (to thereby prevent or attenuate an early infection) or after the onset of a real infection. Similarly, for gene therapy, the composition may be provided before any symptom of a disorder or disease manifests, or after one or more symptoms are detected. A composition is said to be "pharmacologically acceptable" if its administration can be tolerated by a recipient patient. It is said that this agent is administered in a "therapeutically effective amount" if the amount administered is physiologically significant. A composition of the present invention is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient, for example, it increases at least one primary or secondary humoral or cellular immune response against at least one strain of a virus. the infectious influenza. The "protection" provided does not have to be absolute, that is, influenza infection does not have to be totally prevented or eradicated, if there is a statistically significant improvement compared to a control population or group of patients. Protection may be limited to mitigating the severity or rapidity of onset of symptoms of infection with influenza virus.

Pharmaceutical Administration A composition of the present invention can confer resistance to one or more pathogens, for example, one or more strains of influenza virus, either by passive immunization or active immunization. In active immunization, an inactivated or attenuated live vaccine composition is administered prophylactically to a host (e.g., a mammal) and the host immune response to administration protects against infection and / or disease. For passive immunization, developed antisera can be recovered and administered to a recipient suspected of having an infection caused by at least one strain of influenza virus. A composition for gene therapy of the present invention can produce prophylactic or therapeutic levels of the desired gene product by active immunization. In one embodiment, the vaccine is provided to a female mammal (on or before pregnancy or delivery), under conditions of sufficient time and amount to cause the production of an immune response that serves to protect both the female and the fetus or newly born (through passive incorporation of antibodies through the placenta or into breast milk). The present invention then includes methods for preventing or attenuating a disorder or disease, for example, an infection by at least one strain of pathogen. As used herein, a vaccine is said to prevent or attenuate a disease if its administration results in either total or partial attenuation (i.e., suppression) of a symptom or condition of the disease, or total or partial of the individual to the disease. As used herein, a gene therapy composition is said to prevent or attenuate a disease if its administration results in either total or partial attenuation (i.e., suppression) of a symptom or condition of the disease, or immunity. total or partial of the individual to the. disease. At least one inactivated or attenuated influenza virus, or composition thereof of the present invention can be administered by any means that achieves the desired purposes, using a pharmaceutical composition as described above. For example, administration of this composition can be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, oral or transdermal routes. Parenteral administration can be by bolus injection or by gradual perfusion over time. A preferred way to use a pharmaceutical composition of the present invention is by intramuscular or subcutaneous application. See, for example, Berkow et al. , -1992; Avery, 1987 and Katzung, 1992. A typical regimen for preventing, suppressing or treating a pathology related to influenza virus, comprises administering an effective amount of a vaccine composition as described herein, administered as a single treatment. , or repeated as an increase or reinforcement dose, for a period of up to and including between one week and approximately 24 months, or any scale or value therein. According to the present invention, an "effective amount" of a composition is one that is sufficient to achieve a desired biological effect. It is understood that the effective dose will depend on the age, sex, health and weight of the recipient, type of concurrent treatment, if any, frequency of treatment and the nature of the desired effect. The different dose scales provided below are not designed to limit the invention and represent preferred dose scales. However, the most preferred dose will be that which is designed for the individual subject, as understood and can be determined by one skilled in the art. See, for example, Berkow et al. , 1992; Avery's, 1987 and Katsung, 1992. The dose of an attenuated virus vaccine for a mammalian (eg, human) or aviary adult organism can be about 103-107 plaque forming units (PFU) / kg, or any scale or value in it. The dose of inactivated vaccine may vary from about 0.1 to 200, eg, 50 μg of hemagglutinin protein. However, the dose should be a safe and effective amount as determined by conventional methods, using existing vaccines as a starting point. The dose of immunoreactive HA in each dose of replicated virus vaccine can be standardized to contain an appropriate amount, for example, 1-50 μg or any scale or value therein, or the amount recommended by the United States Public Health Service. United (PHS), which is normally 15 μg per component for children over 3 years of age, and 7.5 μg per component for older children < 3 years old. The amount of NA can also be standardized, however, this glycoprotein can be labile during purification and storage in processors (Kendal et al., 1980). Each 0.5 ml dose of vaccine preferably contains about 1-50 trillion virus particles, and preferably 10 trillion particles.

The invention will be further described by means of the following examples.

Example 1 Materials and methods Cells and viruses. 293T human embryonic kidney cells and Madin-Darby canine kidney cells (MSCK) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and in medium Modified Eagle (MEM) containing 5% newborn calf serum, respectively. All cells were maintained at 37 ° C in 5% C02. Influenza virus A / WSN / 33 (H1N1) and A / PR / 8/34 (H1N1) were propagated in 10-day-old eggs. Construction of plasmids To generate RNA polymerase I constructs, cloned cDNA molecules derived from viral RNA A / WSN / 33 or A / PR / 8/34 were introduced between the promoter and terminator sequences of RNA polymerase I. Briefly, the cloned cDNA molecules were amplified by PCR with primers containing BsitBl sites, digested with BsiriBl and cloned in the Bs BX sites of the ve pHH21 containing the human RNA polymerase I promoter and the human RNA polymerase I terminator, separated by the BspiBI sites (figure 2) . The PB2, PBl, PA, HA, NP, NA, M and NS genes of strain A / WSN / 33 were amplified by PCR with the use of the following plasmids: pSCWPB2, pGW-PBl and pSCWPA (all obtained from Dr Debi Nayak at the University of California at Los Angeles) and pWH17, pWNP152, pT3WNA15 (Castrucci et al., 1992), pGT3WM and pWNSl, respectively. The PBl gene of influenza virus A / PR / 8/34 was amplified by the use of pcDNA774 (PBl) (Pérez et al., 1992) as a template. See figure 6 for the sequences of the primers. To ensure that the genes were free of unwanted mutations, fragments derived from PCR were sequenced with a self-sequencer (Applied Biosystem Inc. CA, USA) according to the protocol recommended by the manufacturer. The cDNA molecules encoding HA, NP, NA and Ml genes of virus A / WSN / 33 were cloned as described in (Huddleston et al., 1982) and subcloned into the eukaryotic expression ve pCAGGS / MCS (controlled by the chicken ß-actin promoter) (Niwa et al., 1991), resulting in pEWSN-HA, pCAGGS-WSN-NP0-14, pCAGGS-WNA15 and pCAGGS-WSN-Ml-2 / l, respectively. The M2 and NS2 genes of virus A / PR / 8/34 were amplified by PCER and cloned into pCAGGS / MCS, producing pEP24c and pCA-NS2. Finally, pcDNA774 (PBl), pcDNA762 (PB2) and pcDNA (787 (PA) were used to express PB2, PBl and PA proteins under the control of the cytomegalovirus promoter (Pérez et al., 1998).

Generation of infectious influenza particles. 293T cells (1 x 10s) were transfected with a maximum of 17 plasmids in different amounts with the use of Trans IT LT-1 (Pnavera, Madison, Wisconsin) according to the manufacturer's instructions. Briefly, DNA and transfection reagent were mixed (2 μl of Trans IT LT-1 per μg of DNA), incubated at room temperature for 45 minutes and added to the cells. Six hours later, the DNA transfection reagent mixture was replaced with Opti-MEM (Gibco / BRL, Gaithersburg, Maryland) containing 0.3% bovine serum albumin and 0.01% fetal calf serum. At different times after transfection, the supernatant viruses were harvested and titered on MDCK cells. Since no helper virus was required by this procedure, the recovered transfectant viruses were analyzed without plaque purification. Determination of the percentage of cells transfected with plasmids that produce viruses. Twenty-four hours after transfection, the 293T cells were dispersed with 0.02% EDTA in individual cells. The cell suspension was then diluted tenfold and transferred to confluent monolayers of MDCK cells in 24-well plates. The viruses were detected by the haemagglutination assay.

Immuno staining assay. Nine hours after infection with influenza virus, the cells were washed twice with phosphate buffered saline (PBS) and fixed with 3.7% paraformaldehyde (PBS) for 20 minutes at room temperature. Then, they were treated with 0.1% Triton X-100 and processed as described by Neumann et al. , (1997).

Results Generation of infectious viruses by expression driven with plasmids from viral RNA segments, three polymerase subunits and NP protein. Although transfection of the cells with a mixture of RNPs extracted from purified virions results in infectious influenza particles, this strategy is not likely to be efficient when used with eight RNPs generated in vitro and different. To produce infectious influenza virus completely from cDNA molecules, eight viral RNPs were generated in vivo. In this manner, plasmids containing cDNA molecules were prepared for the full length viral RNA molecules of virus A / WSN / 33, flanked by the human RNA polymerase I promoter and the mouse RNA polymerase I terminator. In principle, the transfection of these eight plasmids into eukaryotic cells should result in the synthesis of the eight influenza vRNA molecules. The proteins PB2, PB1, PA and NP, generated by cotransfection of protein expression plasmids, must then assemble the vRNA molecules into functional RNPv molecules that are replicated and transcribed, finally forming infectious influenza viruses (figure 3). 1 x 10s 293T cells were transfected with protein expression plasmids (1 μg of pcDNA762 (PB2), 1 μg of pcDNA774 (PBl), 1 μg of pcDNA787 (PA) and 1 μg of pCAGGS-WSN-NP0 / 14) and 1 μg of each of the following RNA polymerase I plasmids (pPolI-WSN-PB2, pPolI-WSN-PBl, pPolI-WSN-PA, pPolI-WSN-HA, pPolI-WSN-NP, pPolI-WSN-NA, pPolI-WSN-M and pPolI-WSN-NS). The decision to use a reduced amount of pcDNA787 (PA) was based on previous observations (Mena et al., 1996), and data about the optimal conditions for the generation of virus-like particles (VLPs) (data not shown). Twenty-four hours after transfection of 293T cells, 7 x 103 pfu of virus per ml were found in the supernatant (experiment 1, table 1), demonstrating for the first time the ability of reverse genetics to produce influenza A virus completely at from plasmids.

Table 1 Sets of plasmids used to produce influenza viruses from cloned cDNA * * 293T cells were transfected with the indicated plasmids. Twenty-four (Experiments 1 and 2) or forty-eight hours (Experiments 3-8) later, the virus titration in the supernatant was determined in MDCK cells. + Unless otherwise indicated, the plasmids were constructed with cDNA molecules representing the RNA molecules of A / WSN / 33 virus. Efficiency of influenza virus production with the co-expression of all structural proteins. Although the expression of the viral NP and polymerase proteins is sufficient for generation driven by influenza virus plasmids, it was possible that the efficiency could be improved. In previous studies, the expression of all structural proteins of influenza viruses (PB2, PB1, PA, HA, NP, NA, M1, M2 and NS2) resulted in VLPs that contained an artificial vRNA that encoded a reporter gene from chloramphenicol acetyltransferase (Mena et al., 1996). Thus, the availability of the complete complement of structural proteins, instead of only those required for the replication and transcription of viral RNA, could improve the efficiency of virus production. For this purpose, 293T cells were transfected with optimal amounts of viral protein expression plasmids (as judged by the production of VLP, unpublished data): 1 μg of pcDNA762 (PB2), 1 μg of pcDNA774 (PBl), 1 μg of pcDNA787 (PA) and 1 μg of pCAGGS-WSN-NPO / 14 and pCAGGS-WNA-15; 2 μg of pCAGGS-WSN-Ml-2/1; 0.3 μg of pCA-NS2 and 0.03 μg of pEP24c (for M2), together with 1 μg of each RNA polymerase plasmid I (Experiment 2, table 1). A second set of cells was transfected with the same set of RNA polymerase I plasmids, with the exception of the PBl gene, for which pPolI-PR / 8/34-PB1 was replaced in an effort to generate a reclassifying virus, together with plasmids expressing only PA, PBl, PB2 and NP (Experiment 3, table 1) 'or those expressing all the structural proteins of influenza (Experiment 4, table 1). The WSN virus yields did not differ appreciably 24 hours later (experiments 1 and 2, table 1) or 36 hours after (data not shown) after transfection. However, a more than 10 fold increase in virus yields with PR / 8/34-PB1 was found when all the viral structural proteins of influenza were provided (experiments 3 and 4, table 1). Negative controls, which lacked one of the plasmids for the expression of PA proteins, PBl, PB2 of NP proteins, did not produce any virus (experiments 5-8, table 1). In this way, depending on the virus generated, the expression of all the structural proteins of influenza A virus appreciably improved the efficiency of the reverse genetics method. Next, the kinetics of virus production after cell transfection was determined using the set of plasmids used to generate a virus with the gene A / PR / 8/34-PB1. In two of three experiments, the virus was first detected 24 hours after transfection. The degree measured at that time, > 103 pfu / ml, had increased to > 106 pfu / ml 48 hours after transfection (table 2). To calculate the percentage of cells transfected with plasmids that produced viruses, 293T cells were treated with EDTA (0.02%) 24 hours after transfection to disperse the cells, and then limitation dilution studies were carried out. In this experiment, no free virus was found in the culture supernatant at this time point. The results indicated that one in 103.3 cells was generating infectious virus particles.

Table 2 Kinetics of virus production after transfection of plasmids in 293T cells * * The 293T cells were transfected with eight RNA polymerase I plasmids that encoded for A / WSN / 33 virus genes with the exception of the PBl gene, which is derived from the A / PR / 8/34 virus, and nine expression plasmids from proteins as those described in the text. At different time points, the inventors named viruses in the culture supernatant in MDCK cells. ND = not elaborated.

Recovery of influenza virus containing the FLAG epitope in the NA protein. To verify that the new inverse genetics system allowed the introduction of mutations in the genome of influenza A virus, a virus containing a FLAG epitope (Castrucci et al., 1992) was generated in the NA protein. 293T cells were transfected with an RNA polymerase I plasmid (pPolI-WSN-NA / FL79) containing a cDNA encoding both the NA protein and a FLAG epitope at the bottom of the protein, along with the RNA polymerase I required and the protein expression plasmids. To confirm that the recovered virus PR-8-WSN-FL79) indeed expressed the NA-FLAG protein, immunostaining assays were carried out on cells infected with wild type virus PR8-WSN-FL79 or A / WSN / 33. A monoclonal antibody to the FLAG epitope detected cells infected with PR8-WSN-FL79, but not those infected with wild-type virus. The recovery of the PR8-WSN-FL79 virus was efficient as well as that for the unlabelled wild type virus (data not shown). These results indicate that the new inverse genetics system allows introducing mutations in the genome of influenza A virus. Generation of infectious influenza viruses that contain mutations in the PA gene. To produce viruses that possess mutations in the PA gene, two silent mutations were introduced creating new recognition sequences for restriction endonucleases (Bspl20I at position 846 and PvuII at position 1281 of mRNA). Previously, no, it was possible to modify this gene through inverse genetics, due to the lack of a reliable selection system. Transfectant viruses PA-T846C and PA-A1284 were recovered. The recovered transfectant viruses were biologically cloned by two consecutive dilutions of limitation. To verify that the viruses recovered were in fact transfectants with mutations in the PA gene, CDNA for the PA gene was obtained by PCR-reverse transcriptase. PA-T846C and PA-A1284C viruses had the expected mutations within the PA gene, as evidenced by the presence of newly introduced restriction sites. PCR of the same viral samples and primers without the reverse transcription step could not produce any product (data not shown), indicating that the PA cDNA was in fact originated from vRNA instead of the plasmid used to generate the viruses . These results illustrate how viruses with mutated genes can be produced and recovered without the use of helper viruses.

Discussion The reverse genetics systems described herein allow someone to efficiently produce influenza A virus completely from cloned cDNA molecules. Bridgen and Elliott (1996) also used reverse genetics to generate a Bunyamwera virus (family Bunyaviridae), but it contains only three segments of negative sense RNA, and the efficiency of its production was low, 102 pfu / 107 cells. Although virus yields differed between the experiments, consistently > 103 pfu / 106 cells were observed for influenza virus, which contains eight segments. There are several explanations for the high efficiency of the reverse genetics system described above in the present. Instead of producing RNPs in vitro (Luytjes et al., 1989), RNPs were generated in vivo through the intracellular synthesis of vRNA molecules using RNA polymerase I and through the expression driven by plasmids of the viral polymerase proteins and NP. Likewise, the use of 293T cells, which are easily transfected with plasmids (Goto et al., 1997), ensured that a large population of cells received all the plasmids necessary for virus production. In addition, the large number of transcripts produced by RNA polymerase I, which is among the most abundantly expressed enzymes in growing cells, probably contributed to the overall efficiency of the system. These characteristics led to a corresponding abundant number of vRNA transcripts and to adequate amounts of viral protein for encapsidation of vRNA, formation of RNPs in the nucleus and export of these complexes to the cell membrane, where new viruses are assembled and released. The previously established reverse genetics systems (Enami et al., 1992, Neumann et al., 1994, Luytjes et al., 1989, Pleschka et al., 1996) require infection with ancillary viruses and therefore methods of selection that allow a small number of transcriptionists to be removed from a vast number of helper viruses. These strategies have been used to generate influenza viruses that possess one of the following genes derived from cDNA: PB2 (Subbarao et al., 1993), HA (Enami et al., 1991; Horimoto et al., 1994), NP (li et al., 1995), NA (Enami et al., 1990), M (Castrucci et al., 1995; Yasuda et al. , 1994) and NS (Enami et al., 1991). Most selection methods, except for those applicable to HA and NA genes, are based on growth temperature, host scale restriction or drug sensitivity, thus limiting the utility of inverse genetics for functional analysis of gene products. Even with the HA and NA genes, for which selection systems driven by reliable antibodies are available, it is difficult to produce viruses with prominent growth defects. In contrast, the reverse genetics system described herein does not require helper viruses and allows someone to generate transfectants with mutations in any segments of the gene or with various growth defects. Having the technology to introduce any viable mutation in the genome of influenza A virus makes it possible for researchers to resolve a number of long-time aspects, such as the nature of regulatory sequences in untranslated regions of the viral genome, structural relationships, function of viral proteins and the molecular basis of the restriction of host range and viral pathogenicity. Although inactivated influenza vaccines are available, their efficacy is sub-optimal due partly to their limited ability to develop responses to local cytotoxic T cells and IgA. The chemical tests of cold-adapted live influenza vaccines now suffer from doubts that these vaccines are optimally attenuated, so that they do not cause influenza symptoms, but still induce protective immunity (reviewed in Keitel & amp; amp; amp;; Stone, 1998). However, preliminary results indicate that these live virus vaccines will not be significantly more effective than the best inactivated vaccine (reviewed in Keitel &Piedra, 1998), leaving room for further improvement. One possibility would be to modify a cold-adapted vaccine with the reverse genetics system described above. Alternatively, one can start from a search by using reverse genetics to produce a "master" influenza A strain with several attenuation mutations in the genes encoding internal proteins. The most intriguing application of the reverse genetics system described here could be based on the rapid production of live and attenuated virus vaccines in cases of suspected pandemics involving new HA or NA subtypes of influenza viruses. This new reverse genetics system will likely increase the use of influenza viruses as vaccine vectors. Viruses can be manipulated to express foreign proteins or immunogenic epitopes in addition to viral influenza proteins. One could, for example, generate viruses with foreign proteins as a ninth segment (Enami et al., 1991) and use them as live vaccines. Not only do influenza viruses stimulate humoral immune responses mediated by strong cells, but they also produce a wide variety of HA and NA proteins from virion surfaces (eg, 15 HA subtypes and 9 NA subtypes and their epidemic variants), allowing a repeated immunization of the same target population. Influenza VLPs that possess an artificial vRNA that codes for a reporter gene have been produced by expressing viral structural proteins and vRNA with the vaccinia-T7 polymerase system (Mena et al., 1996). Using inverse genetics, VLPs containing vRNA molecules that encode proteins required for the transcription and replication of vRNAs (ie, PA, PBl, PB2 and NP) as well as vRNA molecules encoding proteins of interest can now be generated. These VLPs could be useful gene delivery vehicles. Importantly, their lack of genes coding for viral structural proteins would ensure that infectious viruses were not produced after therapy with VLP genes. Since the influenza virus genome is not integrated into the host chromosome, the VLP system might be suitable for gene therapy in situations that require only short-term cell transduction (eg, for the treatment of cancer). ). In contrast to adenovirus vectors (Kovesdi et al., 1997), influenza VLPs could contain both HA and NA variants, allowing repeated treatment of target populations. The Ortho yxoviridae family comprises influenza A, B and C viruses, as well as the recently classified Thogotoviruses. The strategy for generating infectious influenza A viruses completely from cloned cDNA molecules described herein would apply to any orthomyxovirus, and perhaps to other negative sense RNA viruses also segmented (e.g., Bunyaviridae, Aernaviridae). The ability to manipulate the viral genome without chemical limitations has profound implications in the study of viral life cycles and their regulation, the function of viral proteins and the molecular mechanisms of viral pathogenicity.

Example 2 To develop a reverse genetics system for influenza A / Puerto Rico / 8/34, viral RNA was extracted from the allantoic fluid of A / Puerto Rico / 8/34 (H1N1) of Madison high growth variant (PR8HG), using the RNeasy team Mini (Qiagen) according to the manufacturer's protocol. He CDNA was synthesized using MMLV-Rtease (Promega) and Uni primer 12. The cDNA molecules were amplified overnight by PCR using the following: PBl primer sets: Ba PB1-1 and PB1-1735R (front fragment) and PB1-903 and Ba-PB1-2341R (later fragment) Ba-PBl-1 CACACACGGTCTCCGGGAGCGAAAGCAGGCA (SEQ IDN0.9) 173PB1-1735R GGGTTTGTATT GTGTGTCACC (SEQ E) NO: 10) 233? B1-903 CCAGGACACK? AAAT rcrTTCAC (SEQIDNO: ll) Ba-PB1-2341R CACACAGGTX? "CCGATTAGTAGAAAC AG 3CATTT (SEQ ID NO: 12) PB2: Ba PB2-1 and PB2-1260R (frontal fragment) and WSN PB2 seq-2 and Ba-PB2-2341R (posterior fragment) Ba-PB2-1 CACACAGGTCTCCGGGAGCGAAAGCAGGTC (SEQ IDN0: 13) B2 126OR CACA < ^ CXÍTCTXXATCATACAATCCIUGGG (SEQ ID NO: 14) WSN PB2 seq-2 CTCCTCTGATGGTGGCATAC (SEQ ID NG'aS) B & -PB2-2341R CACACAGGTCTCCTATTAGTAGAAACAAGGTCGTTT (SEQ ID N0: 16) PA: Bm-PA-1 CACACACGTCTCCGGGAGCGAAAGCAGGTAC (SEQ IDN0: 17) Bm-PA-2233R CACACACGTCTCCTATTAGTAGAAACAAGGTACTT (SEQ ID NO: 18) HA: Bm-HA-1: CACACACGTCTCCGGGAGCAAAAGCAGGGG (SEQ ID NO: 19) Bm-NS-890R: CACACACGTCTCCTATTAGTAGAAACAAGGGTGTTTT (SEQ ID NO: 20) NP: Bm-NP-1 CACACACGTCTeCGGGAGCAAAAGCAGGGTA (SEQ TDNO: 21) Bm-NP-1565R C ^ CACACGTt rCCTATTAGTAGAAACAAGGGTATTTTT (SEQ ID N022) NA: NA-1 Ba-: CACACAGGTCTCCGGGAGCAAAAGCAGGAGT (SEQ IDNO-23) Ba-NA-1413R: CACACAGGTCTGGTATTAGTAGAAACAAGGAGT G T (SEQ IDNO: 24)? * Bm-M -1 CACAC ^ CGTCTCX: GK IAGCAAAAGCAGGTAG (SEQ OO: 25) Bm-M-I027R (^ CACACGTCrCCTATTAGTAGAAACAAGGTAGTTTTT (SEQ ID N026) NS: Bm-NS-1 CACACA < ^ TCTCCGGGAGCAAAAGCAGGGTG (SEQ ID: 27) Bm-NS- 890R CACAC ACGTCTCCTATTAGTAGAAAC AAGGGTGTTTT (SEQ ID NO: 28) DNA polymerase: pfu Native DNA polymerase (Statagene) The PCR products were separated by gel electrophoresis and extracted from agarose gel using a gel extraction kit (Qiagen). extracted genes were ligated into a shaded vector pT7Blue (Novagen) using a Takara ligation kit, version II (Takara) After 5 hours, the ligated genes were transformed into JM109 (PB2, M and NS genes) or DH5alpha (PA, PBl and NP) Six colonies for each gene were cultured in TB for 8 hours, the plasmids were ex brought from the culture of bacteria and four clones per gene were sequenced. The PA, NP, M and NS genes in pT7Blue were excised by Bsm Bl enzyme (New England Biolabs). The PBl gene was excised by Bsa I (New England Biolabs). The excised genes were ligated overnight with pPolIR vector containing the human RNA polymerase I promoter and mouse RNA polymerase I terminator which had been digested with Bsm Bl. The frontal fragment of the PB2 gene in pT7Blue was excised by Bsr Gl (New England Biolabs) and Bam Hl (Roche) and the subsequent fragment was excised by Bsr Gl (New England Biolabs) and Spe I (Roche) The excised fragments were mixed and digested by Bsa I. After 6 hours, the digested genes were purified using a PCR purification kit (Qiagen) and ligated overnight between the Bsm I sites of the pPoTLR vector. - The PBl genes , PA, NP, M and linked NS-pPolIR were used to transform JM109 (M and NS genes) or DHSalpha (PBl, PA and NP genes) overnight. Colonies of transformed bacteria were grown in LB overnight. The ligated PB2-pPolIR was used to transform JM109 overnight. The plasmids were extracted from the bacterial cultures and gene inserts were confirmed by enzyme digestion. Colonies of bacteria transformed by PB2-pPolIR were cultured in LB for 8 hours. The plasmids were then extracted and the insertion of genes was confirmed by enzymatic digestion. All pPolI constructs were sequenced to ensure they did not contain unwanted mutations. The pPolIR constructs for PR8HG were transfected into 293T human embryonic kidney cells with constructions A / WSN / 33 (WSN) -HA and NA, A / Hong Kong / 483/97 (HK) -Haavir and NA, or A / Kawasaki / 01 (Kawasaki) -HA and NA Poli and four protein expression constructs for the polymerase and NP proteins of A / WSN / 33 The supernatants of transfected 293T cells were serially diluted (not diluted to 10"7) and infected in the allantoic cavities of nine-day-old embryonated chicken eggs.The allantoic fluids of the infected eggs were harvested and their virus titers were harvested. tested by HA assay (Table 3) Table 3 HA-positive samples (viruses with WSN-HA NA at 10 and viruses with undiluted HK-HAavir NA) were serially diluted from 10 ~ 2 to 10-8 and 100 μl of each dilution was infected in embryonic chicken eggs . The allantoic fluids of the infected eggs were harvested and their virus titers tested by HA assay (table 4). 50% of infectious egg dose (EID50) of A / Puerto Rico / 8/34 (H1N1) prepared from plasmids was 1010.33 / ml and the HA titration was 1: 3200. A recombinant virus that had the HA and NA genes of A / Hong Kong / 213/2003 (H5N1) and the rest of the influenza A virus genes of PR8HG was prepared. The titre of the recombinant virus was from? 10-67 EID50 / ml and the HA titration was 1: 1600. Table 4 PR8 gene sequences: Eá AGCGAAAGCA GGTACTGATC CAAAATGGAA GATTTTGTGC GACAATGCTT CAATCCGATG ATTGTCGAGC TTGCGGAAAA AACAATGAAA GAGTATGGGG AGGACCTGAA AATCGAAACA AACAAATTTG CAGCAATATG CACTCACTTG GAAGTATGCT TCATGTATTC AGATTTTCAC TTCATCAATG AGCAAGGCGA GTCÁATAATC GTAGAACTTG GTGATCCAAA TGCACiTTTG AAGCACAGAT TTGAAATAAT CGAGGGAAGA GATCGCACAA TGGCCTGGAC AGTAGTAAAC AGTATTTGCA ACACTACAGG GGCTGAGAAA CCAAAGTTTC TACCAGATTT GTATGATTAC AAGGAGAATA GATTCATCGA AATTGGAGTA ACAAGGAGAG AAGTTCACAT ATACTATCTG GAAAAGGCCA ATAAAATTAA ATCTGAGAAA ACACACATCC ACATITTCTC GTTCACTGGG GAAGAAATGG CCACAAAGGC AGACTACACT CTCGATGAAG AAAGCAGGGC TAGGATCAAA ACCAGACTAT TCACCATAAG ACAAGAAATG GCCAGCAGAG GCCTCTGGGA TTCCTTTCGT CAGTCCGAGA GAGGAGAAGA GACAATTGAA GAAAGGTTTG AAATCACAGG AACAATGCGC AAGCTTGCCG ACCAAAGTCT CCCGCCGAAC TTCTCCAGCC TTGAAAATTT TAGAGCCTAT GTGGATGGAT TCGAACCGAA CGGCTACATT GAGGGCAAGC TGTCTCAAAT GTCCAAAGAA GTAAATGCTA GAATTGAACC 5 TTTTTTGAAAACAACACCAC GACX ^ srrAG ACTTCCGAAT GGGCCTCCCT GTTCTCAGCG GTCCAAATTC CTGCTGATGG ATGCCTTAAA ATTAAGCATT j GAGGACCCAA GTCATGAAGG AGAGGGAATA CCGCTATATG ATGCAATCAA ATGCATGAGA AC ATTCTTTG GATGGAAGGA ACCCAATGTT GTTAAACCAC ACGAAAAGGG AATAAATCCA AATTATCTTC TGTCATGGAA GCAAGTACTG GC AGAACTGC AGGACATTGA GAATGAGGAG AAAATTCC AA 10 AGACTAAAAA TATGAAGAAA ACAAGTCAGC TAAAGTGGGC ACTTGGTGAG AACATGGCAC CAGAAAAGGT AGACTTTGAC GACTGTAAAG ATGTAGGTGA TTTGAAGCAA TATGATAGTG ATGAACCAGA ATTGAGGTCG CTTGCAAGTT GGATTCAGAA TGAGTrTAACAAGGCATGCGAACTGACAGATrCAAGCTGG '. • ATAGAGCTCG ATGAGATTGG AGAAGATGTG GCTCCAATTG AACACATTGC AAGCATGAGA 15 AGGAATTATT TCACATCAGA GGTGTCTCAC TGCAGAGCCA CAGAATACAT AATGAAGGGA GTGTACATCA ATACTGCCTT GCTTAATGCA TCTTGTGCAG CAATGGATGA TTTCCAAT AT ATTCCAATGA TAAGCAAGTG TAGAACTAAG GAGGGAAGGC GAAAGACCAA CTTGTATGGT TTCATCATAA AAGGAAGATC CCACTTAAGG AATGACACCG ACGTGGTAAA CTTTGTGAGC ATGGAGTTTT 20 CTCTCACTGA CCCAAGACTT GAACCACATA AATGGGAGAA GTACTGTGTT CTTGAGATAG GAGATATGCT TATAAGAAGT GCCATAGGCC AGGTTTCAAG GCCCATGTTC TTGTATGTGA GAACAAATGG AACCTCAAAA ATTAAAATGA AATGGGGAAT GGAGATGAGG CGTTGCCTCC TCCAGTCACT TCAACAAATT GAGAGTATGA TTGAAGCTGA GTCCTCTGTC AAAGAGAAAG ACATGACCAA 25 AGAGTTCTTT GAGAACAAAT CAGAAACATG GCCC ATTGGA GAGTCCCCCA AAGGAGTGGA GGAAAGTTCC ATTGGGAAGG TCTGCAGGAC TTTATTAGCA AAGTCGGTAT TCAACAGCTT GTATGCATCT CCACAACTAG AAGGATTTTC AGCTGAATCA AG AAACTGC TTCTT ATCGT TCAGGCTCTT AGGGACAACC c TGGAACCTGG GACCTTTGAT CTTGGGGGGC TATATGAAGC ° AATTGAGGAG TGCCTGATTA ATGATCCCTG GGTTTTGCTT AATGCTTCTT GGTTCAACTC CTTCCTTACA CATGCATTGA GTTAGTTGTG GCAGTGCTAC TATTTGCTAT CCATACTGTC CAAAAAAGTA CCTTGTTTCT ACT (SEQ IDN0: 1) PBl 0 AGCGAAAGCA GGCAAACCATTTGAATGGAT GTCAATCCGA CCTTACTTTT CTTAAAAGTG CCAGCACAAA ATGCTATAAG CACAACTTTC CCTTATACTG GAGACCCTCC TTACAGCCAT GGGACAGGAA CAGGATACAC CATGGATACT GTCAACAGGA CAC ATCAGTA CTCAGAAAAG GGAAGATGGA CAACAAACAC CGAAACTGG? GCACCGCAAC TCAACCCGAT TGATGGGCCA CTGCCAGAAG ACAATGAACC AAGTGGTTAT GCCCAAACAG ATTGTGTATT 5 GGAGGCGATG GCTTTCCTTG AGGAATCCCA TCCTGGTATT TTTGAAAACT CGTGTATTGA AACGATGGAG GTTGTTCAGC AAACACGAGT AGACAAGCTG ACACAAGGCC GACAGACCTA TGACTGGACT CTAAATAGAA ACCAACCTGC TGCAACAGCA TTGGCCAACA CAATAGAAGT GTTCAGATCA AATGGCCTCA CGGCCAATGA GTCTGGAAGG CTCATAGACT TCCTTAAGGA TGTAATGGAG TCAATGAACA AAGAAGAAAT GGGGATCACA ACTCATTTTC 0 AGAGAAAGAG ACGGGTGAGA GACAATATGA CTAAGAAAAT GATAACACAG AGAACAATGG GTAAAAAGAA GCAGAGATTG AACAAAAGGA GTTATCTAAT TAGAGCATTG ACCCTGAACA CAATGACCAA AGATGCTGAG AGAGGGAAGC TAAAACGGAG AGCAATTGCA ACCCCAGGGA TGCAAATAAG GGGGTTTGTA TACTTTGTTG 5 AGACACTGGC AAGGAGTATA TGTGAGAAAC TTGAACAATC AGGGTTGCCA GTTGGAGGCA ATGAGAAGAA AGCAAAGTTG GCAAATGTTG TAAGGAAG? T- GATGACCAAT TCTCAGGACA (XGAACITTC TTTCACCATC ACTGGAGATA ACACCAAATG GAACGAAAAT CAGAATCCTC GGATGTTTTT GGCCATGATC AC? TATATX3ACCAGAAATCAGCCCGAATGG TTCAGAAATG TTCTAAGTAT TGCTCC AATA ATGTTCTC AA ACAAAATGGC GAGACTGGGA AAAGGGTATA TGTTTGAGAG CAAGAGTATG AAACTTAGAA CTCAAATACC TGCAGAAATG CTA 3CAAG; ATCGATrrGAAATATpCAATGATTCAACAA GAAAGAAGAT TGAAAAAATC CGACCGCTCT TAATAGAGGG GACTGCATCA TTGAGCCCTG GAATGATGAT GGGCATGTTC AATATGTTAA GCACTGTATT AGGCGTCTCC ATCCTGAATC TTGGACAAAA GAGATACACC AAGACTACTT ACTGGTGGGA TGGTCTTCAA TCCTCTGACG ATTTTGCTCT GATTGTGAAT GCACCCAATC ATGAAGGGAT TCAAGCCGGA GTCGACAGGT TTTATCGAAC CTGTAAGCTA CTTGGAATCA ATATGAGC AA GAAAAAGTCT TAC ATAAACA GAACAGGTAC ATTTGAATTC ACAAGTTTTT TCTATCGTTA TGGGTTTGTT GCCAATTTCA GC ATGGAGCT TCCCAGTTTT GGGGTGTCTG GGATC AACGA GTCAGCGGAC ATGAGTATTG GAGTTACTGT CATCAAAAAC AATATGATAA ACAATGATCT TGGTCCAGCA ACAGCTCAAA TGGCCCTTCA GTTGTTCATC AAAGATTACA GGTACACGTA CCGATGCCAT ATAGGTGACA CACAAATACA AACCCGAAGA TCATTTGAAA TAAAGAAACT GTGGGAGCÁA ACCCGTTCCA AAGCTGGACT GCTGGTCTCC GACGGAGGCC CAAATTTATA CAACATTAGA AATCTCCACA TTCCTGAAGT CTGCCTAAAA TGGGAATTGA TGGATGAGGA TTACCAGGGG CGTTTATGCA ACCCACTGAA CCCATTTGTC AGCCATAAAG AAATTGAATC AATGAACAAT GCAGTGATGA TGCCAGCACA TGGTCCAGCC AAAAACATGG AGTATGATGC TGTTGCAACA ACACACTCCT GGATCCCCAA AAGAAATCGA TCCATCTTGA ATACAAGTCA AAGAGGAGTA CTTGAGGATG AACAAATGTA CCAAAGGTGC TGCAATTTAT TTGAAAAATT CTTCCCCAGC AGTTCATACA GAAGAOCAGT CGGGATATCC AGTATGGTGG AGGCTATGGT TTCCAGAGCC CGAATTG TG CACGGATGGATTTCGAATCT GGAAGGATAA AGAAAGAAGA GTTCACTGAG ATCATGAAGATCTGTTCCAC CATTGAAGAG CTCAGACGGC AAAAATAGTG AATTTAGCTT GTCCTTCATG AAAAAATGCC TTGTTTCTAC T (SEQ ID NO?) PB2 AGCGAAAGC A GGTCAATTAT ATTCAATATG GAAAGAATAA AAGAACTACG AAATCTAATG TCGCAGTCTC GCACCCGCGA GATACTCACA AAAACCACCG TGGACCATAT GGCCATAATC AAGAAGTACA CATCAGGAAG ACAGGAGAAG AACCCAGCAC TTAGGATGAA ATGGATGATG GCAATGAAAT ATCCAATTAC AGCAGACAAG AGGATAACGG AAATGATTCC TGAGAGAAAT GAGCAAGGAC AAACTTTATG G AGTAAAATG AATGATGCCG GATCAGACCG AGTGATGGTA TC ACCTCTGG CTGTGAC ATG GTGGAATAGG AATGGACCAA TAACAAATAC AGTTCATTAT CCAAAAATCT ACAAAACTTA TTTTGAAAGA GTCGAAAGGC TAAAGCATGG AACCTTTGGC CCTGTCCATT TTAGAAACCA AGTCAAAATA CGTCGGAGAG TTGACATAAA TCCTGGTCAT GCAGATCTCA GTGCCAAGGA GGC AC AGGAT GTAATC ATGG AAGTTGTTTT CCCTAACGAA GTGGGAGCCA GGATACTAAC ATCGGAATCG CAACTAACGA TAACCAAAGA GAAGAAAGAA GAACTCCAGG ATTGCAAAAT TTCTCCTTTG ATGGTTGCAT ACATGTTGGA GAGAGAACTG GTCCGCAAAA CGAGATTCCT CCCAGTGGCT GGTGGAACAA GCAGTGTGTA CATTGAAGTG TTGCATTTGA CTCAAGGAAC ATGCTGGGAA CAGATGTATA CTCCAGGAGG GG'AAGTGAGG AATGATGATG TTGATCAAAG CTTGATTATT GCTGCTAGGA ACATAGTGAG AAGAGCTGCA GTATCAGCAG ATCCACTAGC ATCTTTATTG GAGATGTGCC ACAGCACACA GATTGGTGGA ATTAGGATGG TAGACATCCT TAGGCAGAAC CCAACAGAAG AGCAAGCCGT GGATATATGC AAGGCTGCAA TGGGACTGAG AATTAGCTCA TCCTTCAGTT TTGGTGGATT CACATTTAAG AGAACAAGCG GATCATCAGT CAAGAGAGAG GAAGAGGTGC TTACGGGCAA TCTTCAAACA TTGAAGATAA GAGTGCATGA GGGATATGAA GAGTTCACAA TGGTTGGGAG AAGAGCAACA GCCATACTCA GAAAAGCAAC CAGGAGATTG ATTC AGCTGA TAGTGAGTGG GAGAGACGAA C AGTCGATTG CCGAAGCAAT AATTGTGGCC ATGGTATTTT CACAAGAGGA TTGTATGATA AAAGCAGTCA GAGGTGATCT GAATTTCGTC AATAGGGCGA ATCAACGATT GAATCCTATG CATCAACTTT TAAGACATTT TCAGAAGGAT GCGAAAGTGC TTTTTCAAAA TTGGGGAGTT GAACCTATCG ACAATGTGAT GGGAATGATT GGGATATTGC CCGACATGAC TCCAAGCATC GAGATGTCAA TGAGAGGAGT GAGAATCAGC AAAATGGGTG TAGATGAGTA CTCCAGCACG GAGAGGGTAG TGGTGAGCAT TGACCGTTTT TTGAGAATCC GGGACCAACG AGGAAATGTA CTACTGTCTC CCGAGGAGGT CAGTGAAACA CAGGGAACAG AGAAACTGAC AATAACTTAC TCATCGTCAA TGATGTGGGA GATTAATGGT CCTGAATCAG TGTTGGTCAA TACCTATCAA TGGATCATCA GAAACTGGGA AACTGTTAAA ATTCÁGTGGT CCCAGAACCC TACAATGCTA TACAATAAAA TGGAATTTGA ACCATTTCAG TCTTTAGTAC CTAAGGCC AT TAGAGGCCAA TACAGTGGGTTTGTAAGAAC TCTGTTCCAA CAAATGAGGG ATGTGCTTGG GACATTTGAT ACCGCACAGA TAATAAAACT TCTTCCCTTC GCAGCCGCTC CACCAAAGCA AAGTAGAATG CAGTTCTCCT CATTTACTGT GAATGTGAGG GGATCAGGAA TGAGAATACT TGTAAGGGGC AATTCTCCTG TATTCAACTA TAACAAGGCC ACGAAGAGAC TCACAGTTCT CGGAAAGGAT GCTGGCACTT TAACTGAAGA CCCAGATGAA GGCACAGCTG GAGTGGAGTC CGCTGTTCTG AGGGGATTCC TCATTCTGGG CAAAGAAGAC AAGAGATATG GGCCAGCACT AAGC ATCAAT GAACTGAGCA ACCTTGCGAA AGGAGAG AAG GCTAATGTGC TAATTGGGCA AGGAGACGTG GTGTTGGTAA TGAAACGGAA ACGGGACTCT AGCATACTTA CTGACAGCCA GACAGCGACC AAAAGAATTC GGATGGCCAT CAATTAGTGT CGAATAGTTT AAAAACGACC TTGTTTCTAC T (SEQIDNO: 3) NP AGCAAAAGCA GGGTAGATAA TCACTCACTG AGTGACATCA AAATCATGGC GTCTCAAGGC ACCAAACGAT CTTACGAACA GATGGAGACT GATGGAGAAC GCCAGAATGC CACTGAAATC AGAGCATCCG TCGGAAAAAT GATTGGTGGA ATTGGACGAT TCTACATCCA AATGTGCACC GAACTCAAAC TCAGTGATTA TGAGGGACGG TTGATCCAAA ACAGCTTAAC AATAGAGAGA ATGGTGCTCT CTGCTTTTGA CGAAAGGAGA AATAAATACC 'TTGAAGAACA TCCCAGTGCG GGGAAAGATC CTAAGAAAAC TGGAGGACCT ATATACAGGA GAGTAAACGG AAAGTGGATG AGAGAACTCA TCCTTTATGA CAAAGAAGAA ATAAGGCGAA TCTGGCGCCAAGCTAATAAT GGTGACGATG CAACGGCTGG TCTGACTCAC ATGATGATCT GGCATTCCAA TTTGAATGAT GCAACTTATC AGAGGACAAG AGCTCTTGTTCGCACCGGAA TGGATCCCAG GATGTGCTCT CTGATGCAAG GTTCAACTCT CCCTAGGAGG TCTGGAGCCG CAGGTGCTGC AGTCAAAGGA GTTGGAACAA TGGTGATGGA ATTGGTCAGA ATGATCAAAC GTGGGATCAA TGATCGGAAC TTCTGGAGGG GTGAGAATGG ACGAAAAACA AGAATTGCTT ATGAAAGAAT GTGCAACATT CTCAAAGGGA AATTTCAAAC TGCTGCACAA AAAGCAATGA TGGATCAAGT GAGAGAGAGC CGGAACCCAG GGAATGCTGA GTTCGAAGAT CrcACTTTTC TAGCACGGTC TGCACTCATA TTGAGAGGGT CGGTTGCTCA CAAGTCCTGC CTGCCTGCCT GTGTGTATGG ACCTGCCGTA GCCAGTGGGT ACGACTTTGA AAGGGAGGGA TACTCTCTAG TCGGAATAGA CCCTTTCAGA CTGCrTCAAA ACAGCCAAGT GTACAGCCTA ATCAGACCAA ATGAGAATCC AGCACACAAG AGTCAACTGG TGTGGATGGC ATGCCATTCT GCCGCATTTG AAGATCTAAG AGTATTAAGC TTCATCAAAG GGA (^ AAGK.}. TGCGCCCAAGAGGGAAGCGGTCCACTAGAGG AGTTCAAATT GCTTCCAATG AAAATATGGA GACTATGGAA TCAAGTACAC TTGAACTGAG AAGCAGGTAC TGGGCCATAA GGACCAGAAG TGGAGGAAAC • ACCAATCAAC AGAGGGCATC TGCGGGCCAA ATCAGCATAC AACCTACGTT CTCAGTACAG AGAAATCTCC CTTTTGACAG AACAACCATT ATGGCAGC AT TCAATGGGAA TACAGAGGGG AGAACATCTG ACATGAGGAC CGAAATCATA AGGATGATGG AAAGTGCAAG ACCAGAAGAT GTGTCTTTCC AGGGGCGGGG AGTCTTCGAG CTCTCGGACG AAAAGGCAGC GAGCCCGATC GTGCCTTCCT TTGACATGAG TAATGAAGGA TCpTATITCT TCGGAGACAA TGC AGAGGAG TACGACAATT AAAGAAAAAT ACCCTTGTTT CTACT (SEQ IDNO: 4) AGCAAAAGCA GGTAGATATT GAAAGATGAG TCTTCTAACC GAGGTCGAAA CGTACGTACT CTCTATCATC CCGTCAGGCC CCCTCAAAGC CGAGATCGCA CAGAGACTTG AAGATGTCTT TGCAGGGAAG AACACCGATC TTGAGGTTCT CATGGAATGG CTAAAGACAA GACCAATCCT GTCACCTCTG ACTAAGGGGA TTTTAGGATT TGTGTTCACG CTCACCGTGC CCAGTGAGCG AGGACTGCAG CGTAGACGCT TTGTCCAAAA TGCCCTTAAT GGGAACGGGG ATCCAAATAA CATGGACAAA GCAGTTAAAC TGTATAGGAA GCTCAAGAGG GAGATAACAT TCCATGGGGC CAAAGAAATC TCACTCAGTT ATTCTGCTGG TGCACTTGCC AGTTGTATGG GCCTCATATA CAACAGGATG GGGGCTGTGA CCACTGAAGT GGCATTTGGC CTGGTATGTG CAACCTGTGA ACAGATTGCT GACTCCCAGC ATCGGTCTCA TAGGCAAATG GTGAC AACAA CCAATCCACT AATCAGACAT GAGAACAGAA TGGTTTTAGC CAGCACTAC A GCTAAGGCTA TGGAGCAAAT GGCTGGATCG AGTGAGCAAG CAGCAGAGGC CATGGAGGTT GCTAGTCAGG CTAGACAAAT GGTGCAAGCG ATGAGAACCA TTGGGACTCA TCCTAGCTCC AGTGCTGGTC TGAAAAATGA TCTTCTTGAA AATTTGCAGG CCTATCAGAA ACGAATGGGG GTGCAGATGC AACGGTTCAA GTGATCCTCT CACTATTGCC GCAAATATCA TTGGGATCTT GCACTTGACA TTGTGGATTC TTGATCGTCT TTTGGTCAAA TGCATTTACC GTCGCT TAA ATACGGACTG AAAGGAGGGC CTTCTACGGA AGGAGTGCCA AAGTCTATGA GGGAAGAATA TCGAAAGGAA C AGCAGAGTG CTGTGGATGC TGACGATGGT CATTTTGTCA GCATAGAGCT GGAGTAAAAA ACTACCTTGT TTCTACT (SEQ ID NO: 5) DK AGC AAAAGCA GGGTGACAAA AACATAATGG ATCCAAACÁC TGTGTCAAGC TTTCAGGTAG ATTGCTTTCT TTGGCATGTC CGCAAACGAG TTGCAGACCA AGAACTAGGC GATGCCCCAT TCCTTGATCG GCTTCGCCGA GATCAGAAAT CCCTAAGAGG AAGGGGCAGT ACTCTCGGTC TGGACATCAA GACAGCCACA CGTGCTGGAA AGCAGATAGT GGAGCGGATT CTGAAAGAAG AATCCGATGA GGCACTTAAA ATGACCATGG CCTCTGTACC TGCGTCGCGT TACCTAACTG ACATGACTCT TGAGGAAATG TCAAGGGACT GGTCCATGCT CATACCCAAG CAGAAAGTGG CAGGCCCTCT TTGTATC AGA ATGGACCAGG CGATCATGGA TAAGAACATC ATACTGAAAG CGAACTTCAG TGTGATTTTT GACCGGCTGG AGACTCTAAT ATTGCTAAGG GCTTTCACCG AAGAGGGAGC AATTGTTGGC GAAATTTCAC CATTGCCTTC TCTTCCAGGA CATACTGCTG AGGATGTCAA AAATGCAGTT GGAGTCCTCA TCGGAGGACT TGAATGGAAT GATAACACAG TTCGAGTCTC TGAAACTCTA CAGAGATTCG CTTGGAGAAG CAGTAATGAG AATGGGAGAC CTCCACTCAC TCCAAAACAG AAACGAGAAA TGGCGGGAAC AATTAGGTCA GAAGTTTGAA GAAATAAGAT GGTTGATTGA AGAAGTGAGA CACAAACTGA AGATAACAGÁGAATAGTTTT GAGCAAATAA CATTTATGCA AGCCTTACAT CTATTGCTTG AAGTGGAGCA AGAGATAAGA ACTTTCTCGT GTCAGCTTATTTAGTACTAAAAAA (^ CCCTTGTTTCTACT (SEQ1DN0: 6) HA AGCAAAAGCAGGGGAAAATAAAAACAACCAAAATGAAGGCAAACCT ACTX3GTCX ratTATGTGCACTTGC ^ ^ ^ GCTGCAGAT GCAGACACAATATGTATAGGCTACCATGCGAACAATTCAACCGACAC TGTTGACACAGTACTCGAGAAGAATGTGACAGT GACACACTCTGTTAACCTGCTCGAAGACAGCCA GTAGATTAAAAGGAATAGCCCCACTACAATTGG GGAAATGTAACATCGC GATGGCTCTTGGGAAACCCAGAATGCGAC CCACTGCTTCCAGTGAGATCATGGTCCTACATT GTAGAAACAO! AAACTCTGAGAATGGAATATGTTATCCAGGAGATTT CATCGACTATGAGGAGCTGAGGGAGCAATTGAG CTCAGTGTCATCATTCGAAAGATTCGAAATATTTCCCAAAGAAAGCT CATGGCCCAACCACAACACAAACGGAGTAACGG CAGCATGCTCCCATGAGGGGAAAAGCAGTT1TTACAGAAATITGCTA TGGCTGACGGAGAAGGAGGGCTCATACCCAAAG. ,.: CTGAAAAATTCGGATGTGAACAAAAAAGGGAAAGAAGTCCTTGTACT GTGGGGTATTCATCACCCGCCTAACAGTAAGGA ACAACAGAATCTC ATCAGAATGAAAATGCTTATGGCTCTGTAGTGA CTTCAAATTATAACAGGAGATTTACCCCGGAAA TAGCAGAAAGACCCAAAGTAAGAGATCAAGCTGGGAGGATGAACTA TTACTGGACCTTGCTAAAACCCGGAGACACAATA ATATGTGAGGCAAATGGAAATCTAATAGCACCAATGTATGCTTTCGC ACTGAGTAGAGGCTTTGGGTCCGGCATCATCAC CTCAAACGCATCAATGCATGAGTGTAACACGAAGTGTCAAACACCCC TGGGAGCTATAAACAGCAGTCTCCCTTACCAGA ATATACACCCAGTCACAATAGGAGAGTGCCCAAAATACGTCAGGAGT GCCAAATTGAGGATGGTTACAGGACTAAGGAAC ATGCX: GTCCATTCAATCCAGAGGTCTATTTGGAGCCATTGCCGGTTTT ATTGAAGGGGGATGGACTGGAATGATAGATGG ATGGTATGGTTATCATCATCAGAATGAACAGGGATCAGGCTATGCAG O ATCAAAAAAGCACACAAAATGCCATTAACG GGATTACAAACAAGGTGAACACTGTTATCGAGAAAATGAACATTCAA TTCACAGCTGTGGGTAAAGAATTCAACAAATTA GAAAAAAGGATGGAAAATTGAAATAAAAAAGTTGATGATGGATTTCT GGACATTTGGACATATAATGCAGAATTGTTAGT TCJGACTGGAAAATGAAAGGACTCTGGATTTCCATGACTCAAATGTGA AGAATCTGTATGAGAAAGTAAAAAGCCAATTAA AGAATAATGCCAAAGAAATCGGAAATGGATGTTTTGAGTTCTACCAC AAGTGTGACAATGAATGCATGGAAAGTGTAAGA AATGGGACTTATG ATGATCCCAAATATTCAGAAGAGTCAAAGTTGAA CAGGGAAAAGGTAGATGGAGTGAAATTGGAATC AATGGGGATCTATCAGATTCTGGCGATCTACTCAACTGTCGCCAGTTC ACTGGTGCTTITGGTCTT CIX3GGGGCAATCA GTTICGGGATGTGTTCGAATGGATCTITGCAGTGCAGAATATGCATCT GAGATTAGAATTTCAGAGATATGAGGAAAAAC ACX C? TGTTTCTACT (SEQ ID N0: 7) AGCAAAAGCAGGGGT1TAAAATGAATCCAAATCAGAAAATAATAAC CATTGGATCAATGTGTCGGGTAGTCGGACTAATT AGCCTAATATIX? AAATAGGGAATATAATCRCAATATGGATTAGCCA TrcAATTCAAACrGGAAGTCAAAACCATACTGG AATATGCAAC < ^ AAACATCA1TACCTATAAAAATAGCACCTGGGTAA AGGACACAACTTCAGTGATATTAACCGGCAATT CATCTCTITGTCCCATCCGTGGGTGGGCTATATACAGCAAAGACAAT AGCATAAGAATTGGTTCCAAAGGAGACGTTTTT GTCAT ^ ^ GAGAGCO itTATTI TGTICTC TTTTTTCTGA (X < _ \ AGGTGCCTTACTGAATGA CAAGCATTCAAGTGGGACTGTTAAGGACIAGAAGCCCTTATAGGGCCT TAATGAGCTGCCCTGTCGGTGAAGCTCCGTCCC CGTAC TTCAAGATITGAATCGGTTGCITGGTÍ ^^ ^ GCAAGTGCATGTC ATGATGGCATGGGCTGGCTAACAATCGGAATT TCAGGTCCAGATAATGGAGCAGTGGCGGTÁTTAAAATACAACGGCAT AATAACTGAAACCATAAAAAGTTGGAGGAAGAA AATATTGAGGACACAAGAGTCTGAATGTGCCTGTGTAAATGGTJCAT GTTTTACTATAATGACTGATGGCCCGAGTGATG GGCTGGC (CGTACAAAATTRTCAAGATCGAAAAGGGGAAGGTTACT AAATCAATAGAGTTGAATGCACCTAATTCTCAC TATGAGGAATGTTCCTGTTACCCTGATACCGGCAAAGTGATGTGTGT GTG AGAGACAATTGGCATGGTTCGAACCGGCC ATGGGTGTCTTTCGATCAAAACCTGGATTATCAAATAGGATACATCT GCAGTGGGGTTTTCGGTGACAACCCGCGTCCCG AAGATGGAACAGGCAGCTGTGGTCCAGTGTATGTTGATGGAGCAAAC GGASTAAAGGGATTTTCATATAGGTATGGTAAT GGTGTTTGGATAGGAAGGACCAAAAGTCACAGTTCCAGACATGGGTT TGAGATGATTTGGGATCCTAATGGATGGACAGA GACTGATAGTAAGTTCTCTGTGAGGCAAGATGTTGTGGCAATGACTG ATTGGTCAGGGTATAGCGGAAGTTTCGTTCAAC ATCCTGAGSRAACAGGGCTAGACTGTATGAGGCCGTGCTTCTGGGTT GAATTAATCAGGGGACGACCTAAAGAAAAAACA ATCTGGACTAGTGCGAGCAGCATTTCT1TTTGTGGCGTGAATAGTGAT ACTGTAGATTGGTCTTGGCCAGACGGTGCTGA GTTGCCATTCAGCATTGAC ^^ AGTAGTCTGTTCAAAAAACTCCTTGTTT CTACT (SEQ IDN0: 8) Example 3 Influenza A / Hong Kong / 213/2003 virus (H5N1, HK213) is replicated systemically in chickens, causing lethal infection. In addition, this virus is lethal to chicken embryos. Thus, although its surface proteins are highly related to currently circulating pathogenic avian influenza viruses, HK213 can not be used as a vaccine strain since attempts to culture it in embryonated chicken eggs result in the production of allantoic fluid. of poor quality. In addition, the use of this highly virulent virus in the production of vaccines is not safe for those who elaborate the vaccines. To test the possibility of using A / pR / 8/34 as a master vaccine acepase, the hemagglutinin gene cut site (HA) of HK213 (which contains several basic amino acids) was mutated from a virulent to an anti-virulent phenotype (from RERRRKKR (SEQ ID No: 9) to -ETR). A virus containing the mutated HA gene produced localized and non-lethal infection in chickens. In addition, the mutated virus was not lethal to the embryos of the chickens. Thus, the growth of the virus mutated in embryonated eggs produced high quality allantoic fluid, and in this attenuated form, the virus is safe for vaccine producers.

A recombinant virus containing the mutated genes of neuraminidase (NA) and HA of HK213, and all the remaining genes of virus A / PR / 8/34 (H1N1, HG-PR8) of high titration (Example 2), which grows 10 times better than other strains of A / PR / 8/34 PR8 in eggs (1010EID50 / ml, HA titration: 1: 8,000), was generated in embryonated chicken eggs. This recombinant virus, which expresses surface proteins related to the pathogenic avian influenza virus currently circulating, grew to high titers in embryonated chicken eggs (figure 4). Thus, replacement of the HA and NA genes of HG-PR8 with those of the currently circulating strain of influenza virus resulted in a vaccine strain that can be produced safely, and demonstrates the use of PR8-HG as a Master vaccine strain. References Avery 's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, ADIS Press, Ltd., Williams and Wilkins, Baltimore, MD (1987). Aymard-Henry et al. , Virology: A Practical Approach, Oxford IRL Press, Oxford, 119-150 (1985). Bachmeyer, Intervirologv, 5: 260 (1975). Berkow et al, eds., The Merck Manual, 16th edition, Merck & Co., Rahway, NJ (1992).

Bridgen et al, Proc. Nati Acad. Sci. U. S. A, 93: 15400 (1996). Castrucci et al., J. Virol. 66: 4647 (1992). Castrucci et al., J. Viral., 69: 2725 (1995). Conzelmann et al., J. Gen. Virol. , x-, 381 (1996). Conzelmann et al., Trends Microbiol, 4: 386 (1996). Conzelmann, Annu. Rev. Genet., 32: 123 (1998). Cozelmamz et al., J. Viral., 68: 713 (1994). Edwards, J. Infect Dis. , j: 68 (1994). Enami et al., J. Viral .. 65: 2711 (1991). Enami et al., Proc. Nat] Acad. Sci. U.S.A., 87: 3802 (1990). Enami et al., Virologv, 185,291 (1991). Fodor et al., J. Virl., 73: 9679 (1999). Goto et al., Virology, 238: 265 (1997). Grand and Skehel, Nature. New Biologv, 238: 145 (1972). Hatta et al., Science 293: 1840 (2001). Horimoto et al., J. Virol, 68: 3120 (1994). Huddleston et al., Nucí. Acids Res., 10: 1029 (1982). Keitel et al., In Textbook of Influenza, eds. Nickolson, K. G., Webster, R-G, and Hay, A. (Blackwell, Oxford), pp. 373-390 (1998). Kendal et al., Infect. Immunity, 29: 966 (1980).

Kilbourne, Bull. M2 World Health Org., 41: 653 (1969) Kovesdi et al., J. Curr. Ovin. Biotechnol., 8: 583 (1997) Laver & Webster, Virology, 69: 511 (1976). Lawson et al., Proc. Nati Acad. Sci. U. S. A., 92: 4477 (1995). Li et al., Virus Res .. 37: 153 (1995). Luytjes et al., Cell, 59: 1107 (1989). Marriott et al., Adv. Virus Res., 53: 321 (1999). Mena et al., J. Viral., 70: 5016 (1996). Mizrahi, (ed.), Viral Vaccines, Wiley-Liss, New York, 39-67 (1990). Murphy, Infect Dis. Clin. Pract., 2: 174 (1993). Muster et al., Proc. Nati Acad. Sci. USA, 88: 5177 (1991). Muñoz et al., Antiviral Res., 46:91 (2000). Nagai et al., MicrobioL ImmunoL, 43: 613 (1999). Nagai, Rev. Med. Virol., 2:83 (1999). Neumann et al., Adv. Virus Res., 53: 265 (1999). Neumann et al., J. Gen. Virol., 83: 2635 (2002). Neumarm et al., J. ViroL, 71: 9690 (1997). Neumann et al., Proc. Nati Acad. Sci. U. S. A, 96: 9345 (1999). Neumann et al., Virology, 202: 477 (1994).

Neumann et al., Virology, 287: 243 (2001). Niwa et al-, Gen, 108: 193 (1991). Ogra et al., J. Infect Dis., 134: 499 (1977). Osol (ed.), Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 1324-1341 (1980). Parks et al.,. Virol. 73: 3560 (1999). Pekosz et al., Proc. Nati Acad. Sci. U. S. A, 96.8804 (1999). Pérez et al., Virology, 249: 52 (1998). Pleschka et al., J. ViroL. 70: 4188 (1996). Radecke et al., EMBO J., .4: 5773 (1995). Roberts et al., Virology. 247: 1 (1998). Robertson et al., Biologicals, 20: 213 (1992). Robertson et al., Giornale di Igiene e Medicina Preventive, 29: 4 (1988). Rose, Proc. Nati Acad. Sci. U. S. A, 93: 14998 (1996). Schnell et al., EMBO J., 13 -.4195 (1994). Subbarao et al., J. Virol. 67: 7223 (1993). World Health Organization TSR No. 673 (1982). All publications, patents and patent applications are incorporated herein by reference.

Although the foregoing description of this invention has been described in connection with certain preferred embodiments thereof, and many details have been established for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional modalities and that certain of the details described herein can be varied considerably without departing from the basic principles of the invention. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (42)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. An isolated polynucleotide characterized in that it comprises a nucleic acid segment encoding an influenza virus HA, NA, PB1, PB2, PA, NP, M, NS or a portion thereof, which has substantially the same activity as a corresponding encoded polypeptide by one of SEQ ID No: 1-8, or the complement of the nucleic acid segment.
2. 2. An isolated polynucleotide characterized in that it comprises a nucleic acid segment encoding an influenza virus HA, NA, PB1, PB2, PA, NP, M or NS having substantially the same amino acid sequence as a corresponding polypeptide encoded by a of SEQ ID No: 1-8, or the complement of the nucleic acid segment.
3. 3. The isolated polynucleotide according to claim 1 or 2, characterized in that the isolated polynucleotide has substantially the same nucleotide sequence as one of SEQ ID NO: 1-8 or the complement thereof.
4. 4. The isolated polynucleotide according to claim 1 or 2, characterized in that the isolated polynucleotide hybrid under conditions of moderate severity to one of SEQ ID NO: 1-8 or the complement thereof.
5. 5. The isolated polynucleotide according to claim 1 or 2, characterized in that it also comprises a promoter. The isolated polynucleotide according to claim 5, characterized in that the promoter is an RNA polymerase I promoter, an RNA polymerase II promoter, an RNA polymerase III promoter, a T3 promoter or a T7 promoter. The isolated polynucleotide according to claim 1 or 2, characterized in that the polynucleotide encodes a polypeptide with one or more conservative substitutions in relation to a corresponding polypeptide encoded by one of SEQ. ID No: 1-8. 8. A composition comprising a plurality of influenza virus vectors, characterized in that it comprises a) at least two vectors selected from a vector comprising a promoter operably linked to a cDNA of influenza virus PA linked to a sequence of transcription termination, a vector comprising a promoter operably linked to an influenza virus cDNA PB1 linked to a transcription termination sequence, a vector comprising a promoter operably linked to a cDNA of influenza virus PB2 linked to a transcription termination sequence, a vector comprising a promoter operably linked to a cDNA of influenza virus HA linked to a transcription termination sequence, a vector comprising a promoter operably linked to a cDNA of bound NP influenza virus to a transcription termination sequence, a vector comprising a linked promoter or The invention relates to a NA influenza virus cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus M cDNA linked to a transcription termination sequence, or a vector comprising a promoter operably linked to an NS influenza virus cDNA linked to a transcription termination sequence, wherein at least one vector comprises a promoter operably linked to the polynucleotide according to claim 1 or 2 linked to a terminator sequence of transcription and b) at least two vectors selected from a vector comprising a promoter operably linked to a DNA segment encoding an influenza virus PA, a vector comprising a promoter operably linked to a cDNA of influenza virus PBl, a vector comprising a promoter operably linked to a segment of DNA encoding vir us of influenza PB2, or a vector comprising a promoter operably linked to a segment of DNA that codes for influenza virus NP, and optionally also selected from a vector comprising a promoter operably linked to a DNA segment coding for influenza virus HA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus Ml, a vector comprising a promoter operably linked to a DNA segment encoding influenza M2 virus or a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NS2, wherein optionally at least one vector comprises a promoter operably linked to the polynucleotide according to claim 1 or 2. 9. A composition comprising a plurality of vectors of influenza virus, characterized in that it comprises a) at least two vectors selected from a vector or comprising a promoter operably linked to an influenza virus PA cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus cDNA PBl linked to a transcription termination sequence , a vector comprising a promoter operably linked to a cDNA of influenza virus PB2 linked to a transcription termination sequence, a vector comprising a promoter operably linked to a cDNA of influenza A virus linked to a terminator sequence of transcription, a vector comprising a promoter operably linked to a cDNA of influenza virus NP linked to a transcription termination sequence, a vector comprising a promoter operably linked to a cDNA of influenza virus for NB and NA bound to a transcription termination sequence, a vector comprising an operable linked promoter The invention relates to an influenza virus M cDNA linked to a transcription termination sequence, or a vector comprising a promoter operably linked to an NS influenza virus cDNA linked to a transcription termination sequence, wherein minus one vector comprises a promoter operably linked to the polynucleotide according to claim 1 or 2 linked to a transcription termination sequence and b) at least two vectors selected from a vector comprising a promoter operably linked to a DNA segment encoding for a PA influenza virus, a vector comprising a promoter operably linked to an influenza virus cDNA PB1, a vector comprising a promoter operably linked to a DNA segment coding for influenza virus PB2, or a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NP, and optionally selected from a vector comprising a promoter operably linked to a DNA segment coding for influenza virus HA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NA and NB , a vector comprising a promoter operably linked to a DNA segment coding for influenza virus M, or a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NS2, wherein optionally at least one vector comprises a promoter operably linked to the polynucleotide according to claim 1 or 2. 10. The composition according to claim 8 or 9, characterized in that the HA is a HA type A. 11. The composition in accordance with claim 8 or 9, characterized in that the HA is a type B HA. 12. The composition according to claim 8 or 9, characterized in that a p The vectority of the vectors of a) comprises an RNA polymerase I promoter or an RNA polymerase II promoter. 13. The composition according to claim 12, characterized in that the RNA polymerase I promoter is a human RNA polymerase I promoter. 1 . The composition according to claim 8 or 9, characterized in that all vectors of a) comprise an RNA polymerase II promoter. 15. The composition according to claim 8 or 9, characterized in that each vector of a) is in a separate plasmid. 16. The composition according to claim 8 or 9, characterized in that each vector of b) is in a separate plasmid. The composition according to claim 8 or 9, characterized in that each of the vectors of b) further comprises an RNA transcription termination sequence. 18. The composition according to claim 8 or 9, characterized in that it further comprises a vector comprising a promoter linked to sequences of 5 -influenza viruses that comprise sequences coding for 5'-influenza viruses linked to a cDNA. of interest linked to 3 'influenza virus sequences comprising "non-coding sequences of influenza virus 3' linked to a transcription termination sequence. 19. The composition according to claim 18, characterized in that the cDNA of interest is in the sense orientation. 20. The composition according to claim 18, characterized in that the cDNA of interest is in the antisense orientation. The composition according to claim 18, characterized in that the cDNA of interest comprises an open reading frame that encodes an immunogenic polypeptide or peptide of a pathogen or a therapeutic polypeptide or peptide. 22. A method for preparing influenza viruses, characterized in that it comprises: contacting a cell with the composition according to any of claims 8 to 21 in an amount effective to produce infectious influenza viruses. 23. The method according to claim 22, characterized in that it further comprises isolating the virus. 24. A virus characterized in that it is obtained by the method according to claim 22. 25. A method for preparing a gene delivery vehicle, characterized in that it comprises: contacting cells with the composition according to any of claims 8 to 21 in an effective amount to produce influenza virus, and isolate the virus. 26. A virus characterized in that it is obtained by the method according to claim 25. 27. A cell characterized in that it is contacted with the composition according to any of claims 8 to 21. 28. A cell characterized in that it is infected with the virus according to claim 24 or 26. 29. A vector characterized in that it comprises a promoter and a nucleic acid segment comprising nucleic acid sequences coding for an influenza virus protein, wherein the virus protein of influenza is an influenza virus PA that has substantially the same activity as the polypeptide encoded by SEQ ID N0: 1, an influenza virus PB1 having substantially the same activity as the polypeptide encoded by SEQ ID NO: 2, an influenza virus PB2 having substantially the same activity as the polypeptide encoded by SEQ ID NO: 3, an influenza virus NP having substantially the same activity as the polypeptide encoded by SEQ ID NO:, an influenza virus HA having substantially the same activity as the polypeptide encoded by SEQ ID NO: 7, an influenza virus NA having substantially the same activity than the polypeptide encoded by SEQ ID NO: 8, an influenza virus M cDNA having substantially the same activity as the polypeptide encoded by SEQ ID NO: 5 and / or an influenza virus NS having substantially the same activity than the polypeptide encoded by SEQ ID NO: 6. 30. A method for preparing influenza viruses, characterized in that it comprises contacting a cell with a vector comprising a promoter operably linked to a cDNA of bound PA influenza virus. to a transcription termination sequence, a vector comprising a promoter operably linked to a cDNA of influenza virus PBl linked to a sequence of termination transcription ion, a vector comprising a promoter operably linked to a cDNA of influenza virus PB2 linked to a transcription termination sequence, a vector comprising a promoter operably linked to a cDNA of influenza virus HA linked to a transcription termination sequence, a vector comprising a promoter operably linked to a cDNA of Influenza NP virus linked to a transcription termination sequence, a vector comprising a promoter operably linked to a cDNA of Influenza NA virus bound to a transcription termination sequence, a vector comprising a promoter operably linked to a cDNA of influenza virus M linked to a transcription termination sequence, a vector comprising a promoter operably linked to a cDNA of influenza virus NS linked to a transcription termination sequence, a vector comprising a promoter r operably linked to a DNA segment encoding an influenza PA virus, a vector comprising a promoter operably linked to a segment of DNA encoding influenza virus PB1, a vector comprising a promoter operably linked to a segment of DNA that codes for influenza virus PB2, and a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NP, to thereby produce an infectious virus, wherein the promoter in at least one vector comprising a viral cDNA comprises a promoter. operably linked to the polynucleotide according to claim 1 or 2 linked to a transcription termination sequence. 31. The method according to claim 30, characterized in that it further comprises a vector comprising a promoter operably linked to a DNA segment coding for influenza virus HA, a vector comprising a promoter operably linked to a DNA segment. coding for influenza virus NA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus Ml, a vector comprising a promoter operably linked to a DNA segment encoding a virus of the M2 influenza and a vector comprising a promoter operably linked to a DNA segment encoding influenza NS2 virus. 32. The method according to claim 30 or 31, characterized in that it further comprises a vector comprising a promoter linked to 5 'influenza virus sequences comprising non-coding sequences of 5' influenza viruses linked to a cDNA of interest or a fragment thereof linked to 3 'influenza virus sequences comprising non-coding sequences of 3' influenza viruses linked to a transcription termination sequence. 33. The method according to claim 32, characterized in that the cDNA of interest comprises an open reading frame that encodes an immunogenic polypeptide or peptide of a pathogen or a therapeutic polypeptide or peptide. 34. The method according to claim 32, characterized in that the cDNA of interest is in the sense orientation. 35. The method of compliance with the claim 32, characterized in that the cDNA of interest is in the antisense orientation. 36. The method according to claim 32, characterized in that the polynucleotide is not one that codes for an HA that corresponds to the polypeptide encoded by SEQ ID NO: 7 and / or is not one that codes for an NA that corresponds to the encoded polypeptide by SEQ ID NO: 8. 37. The method according to any of claims 30 to 36, characterized in that it further comprises isolating the virus. 38. A virus characterized in that it is obtained by the method according to any of claims 30 to 37. 39. A cell characterized in that it is contacted with the virus according to claim 38. 40. A cell characterized by being infected with the virus according to claim 38. 41. A method for immunizing an individual against a pathogen, characterized in that it comprises administering to the individual an amount of the virus according to claim 38 effective to immunize the individual. 42. An isolated influenza virus characterized in that it comprises a polynucleotide corresponding to the polynucleotide according to any of claims 1 to 7.