RU2420577C2 - Conditionally defective particle of influenza virus and methods of producing thereof (versions) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: disclosed is a conditionally defective particle of influenza virus with the absence of a nucleic acid segment of influenza virus selected from a group of segments, mainly coding acid polymerase (PA), basic polymerase 1 (PB1) and basic polymerase 2 (PB2). The conditionally defective particle of influenza virus is used for induction of influenza virus defence. Also, a method of producing such particles, a pharmaceutical composition containing such particles, its application and a method of induction of influenza virus defence are described.

EFFECT: higher efficacy.

27 cl, 4 dwg, 4 tbl, 2 ex

Description

The invention relates to the field of influenza virus and influenza vaccination.

Influenza viruses (Orthomyxoviridae) are negative-chain enveloped RNA-containing viruses with a segmented genome (Taubenberger and Layne, Molecular Diagnosis Vol. 6 No. 4 2001). They are divided into two genera based on significant antigenic differences between their nucleoproteins and matrix proteins: one includes influenza A and B, and the other consists of influenza C. Three types of viruses also differ in pathogenicity and genome organization. Type A has been identified in a wide range of warm-blooded animals, and types B and C are predominantly human pathogens. Influenza A viruses are further divided according to the antigenic characteristics of hemagglutinin (HA) and surface NA glycoproteins that are exposed on the surface of the virion. Currently, there are 15 subtypes of HA and nine subtypes of NA. Influenza A viruses infect a wide range of animals, including birds, pigs, horses, humans and other mammals. Waterfowl serves as a natural reservoir for all known influenza A subtypes and possibly serves as a source of genetic material for human pandemic influenza strains.

Unlike related paramyxoviruses, influenza viruses have a segmented RNA genome. Influenza A and B viruses have a similar structure, while influenza C differs more strongly. While each of type A and B viruses contains eight discrete gene segments encoding at least one protein each, type C contains seven discrete segments, combining segments 4 and 6 of types A and B. Influenza A and B viruses are coated with exposure to three proteins: HA, NA and matrix protein 2 (M2). Influenza C virus carries only one surface glycoprotein. Each influenza virus RNA segment is encapsulated in nucleoproteins (NP) to form ribonucleoprotein (RNP) complexes. Three polymerase proteins are associated with one end of the RNP complex. RNPs are surrounded by a matrix protein matrix (matrix protein 1) as an integral part. The phospholipid portion of the membrane originates from the host cell membrane. Also in the viral particle is non-structural protein 2 (NS2).

The World Health Organization (WHO) influenza nomenclature rules include the following. First, indicate the type of virus (A, B or C), then the host (if the host is not a person), the location of isolation, strain number and year of isolation (separated by slashes). For influenza A, the HA and NA subtypes are indicated in parentheses. For example, the strains included in the modern trivalent vaccine for the seasons 2000 to 2001 are: A / Panama / 2007/99 (H3N2), A / New Caledonia / 20/99 (H1N1), and B / Yamanashi / 16 / 98. Since 1977, two subtypes of influenza A have been circulating in humans together: H1N1 and H3N2.

Influenza viruses accumulate point mutations during replication, since their RNA polymerase complex does not have corrective activity. Mutations leading to a change in amino acids in the antigenic parts of surface glycoproteins can provide selective benefits for the viral strain, allowing it to elude existing immunity. The HA molecule initiates infection by binding to receptors on specific host cells. Antibodies to the HA protein prevent receptor binding and are very effective in preventing re-infection with the same strain. HA can elude the previously acquired immunity either by antigenic drift, in which mutations of the currently circulating HA gene disrupt antibody binding, or by antigenic shift, in which the virus acquires a new subtype HA. Antigenic drift pressure is not evenly distributed across the HA molecule, with positively selected changes predominantly occurring in the globular head of the HA protein. These changes also accumulate to a greater extent in HA than in NA. Changes in other flu virus proteins are slower. Similarly, antigenic drift pressure is highest in human-adapted strains of the influenza virus, medium in strains adapted for pigs and horses, and lowest in bird-adapted strains.

Since influenza viruses have a segmented genome, co-infection with two different strains of the same host can lead to the formation of new rearranged strains of the influenza virus containing different combinations of parent gene segments. In wild birds, fifteen HA subtypes are known to exist, and they provide a source of HA that are new to humans. The emergence in humans of a strain of the influenza virus with a new subtype due to an antigenic shift caused the last two influenza pandemics in 1957 and 1968 and was most likely the cause of the influenza pandemic in 1918. To be consistent with everything that is known about the appearance of pandemic influenza viruses, a pandemic strain must have HA antigenicity different from the antigenicity currently prevailing; this HA could not circulate among people for a period of 60 to 70 years; and the virus must be transmitted from person to person. Both in 1957 and in 1968, the pandemic occurred due to a shift in HA, and in both cases the HA pandemic strains were closely related to the bird strains. Although one of the absolute requirements for a pandemic is that HA must vary, the extent to which the remainder of the virus can or must change is unknown. Only pandemic viruses of 1957 and 1968 are available for direct investigation, and the pandemic influenza virus of 1918 is characterized using molecular archeology. In 1957, three genes were replaced with genes similar to those of bird viruses: HA, NA, and the polymerase complex subunit (PB1). In 1968, only HA and PB1 were substituted.

A specific diagnosis of influenza infection can be made through virus isolation, hemagglutination inhibition test (HI), antigen detection by immunoassay, serological tests, detection of NA activity in secretions or molecular analyzes. Samples can be collected in the form of sputum, nasopharyngeal swabs or nasopharyngeal swabs obtained by rinsing a buffered saline solution. The standard for the diagnosis of influenza is an immunological characteristic after cultivation. Serological analysis provides an accurate but retrospective method for influenza infection, as it requires the collection of serum in the acute phase and in the recovery phase.

Influenza viruses can be grown in chicken eggs with an embryo or in a number of tissue culture systems. Adding trypsin (to activate HA cleavage) allows the influenza virus to multiply in Madin-Darby dog kidney cells (MDCK) and other lines. The main way to get the vaccine is still the cultivation of influenza viruses in eggs. Cultivation in cell lines is usually used for the primary isolation of human influenza viruses (type A and B). Many human influenza viruses can be cultured directly in the allantoic cavity of eggs with the embryo. Some influenza A and B viruses need initial cultivation in the amniotic cavity and subsequent adaptation to the allantoic cavity. After isolation of the culture, many influenza virus isolates are finally identified using immunoassay or immunofluorescence. HA molecules of influenza viruses bind sialic acid residues on the surface of respiratory cells to enhance the penetration of the virus.

Influenza virus strains can be characterized antigenically, given the ability of influenza viruses to cause red blood cell agglutination in vitro. Antibodies to HA can inhibit agglutination. Thus, hemagglutination inhibition (HI) analysis is one of the standard methods for characterizing influenza virus strains. HI assays are used to determine whether reference strains are immunologically related (i.e., cross-reactive) with the latest vaccine strains. Typing sera, usually obtained from ferrets, are added to the wells in a series of two-fold dilutions, and laboratory staff evaluate the assayed wells by determining suspended red blood cells compared to clumped ones. In most cases, the serum panel is used to select exemplary strains for vaccine or reference strains and, during any given influenza season, the vast majority of exemplary strains are successfully selected using HI assays.

WHO makes recommendations, and WHO collaborating centers provide guidance on identifying the antigenic characteristics of specific viral strains. Exemplary strains are classified according to immunological lineage, for example, A / Moscow / 10/99 (H3N2), similar to A / New Caledonia 20/99 (H1N1) and similar to B / Beijing / 184/93 viruses. Sample strains for which it was not possible to characterize HI analysis, laboratory staff should inoculate ferrets to obtain strain-specific antisera. When a new antiserum is obtained, the HI assay is again performed as described. If the new serum shows significant gaps in cross-specificity (usually defined as the four-fold difference between the sample strain and the vaccine strain), it is embedded in a regular laboratory panel and used to search for new epidemic strains. Thus, the HI assay is crucial in monitoring the influenza virus to select a vaccine strain and the most commonly used method for evaluating antigenic drift.

Influenza virus strains can be characterized genetically by comparing sequences of specific gene segments, and again, WHO guidelines and WHO collaboration centers provide guidance on identifying the individual characteristics of RNA segments within the influenza virus genome; influenza A and B nucleic acid segments encoding nucleoprotein (NP), basic polymerase 1 (PB1), basic polymerase 2 (PB2), acid polymerase (PA), hemagglutinin (HA), neuraminidase (NA), matrix proteins (M1 and M2) and non-structural protein (NS1 and NS2), and the nucleic acid segments of the influenza virus C, encoding nucleoprotein (NP), basic polymerase 1 (PB1), basic polymerase 2 (PB2), hemagglutinin ruminidase glycoprotein (HN), matrix proteins (M1 and M2) and non-structural protein (NS1 and NS2). Requests for reference strains for antigenic analysis to compare nucleic acid sequences and to identify vaccine viruses can be sent to the WHO Collaborating Center for Reference and Research on Influenza, 45 Poplar Road, Parkville, Victoria 3052, Australia (fax: +61 3 9389 1881, web Website: http://www.influenzacentre.org ); WHO Collaborating Center for Reference and Research on Influenza, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashi-Murayama, Tokyo 208-0011, Japan (fax: +81 42 5610812 or +81 42 5652498); WHO Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Centers for Disease Control and Prevention, 1600 Clifton Road, Mail stop G16, Atlanta, GA 30333, United States of America (fax: +1 404 639 23 34) or WHO Collaborating Center for Reference and Research on Influenza, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, England (fax: +44 208 906 4477). Updated epidemiological information is available on the WHO website at http://www.who.int/influenza and the Geographic Information System, FluNet, http://www.who.int/fluent.

Awareness of the effects of influenza and the benefits to health and the economy of preventing it is increasing and over the last decade there has been a significant increase in the use and effectiveness of vaccination and a number of influenza medicines. As a result of longer life expectancy in many countries, significantly more people are at risk of complications, the burden on health care systems during influenza epidemics is more widely recognized, and more frequent international tourism creates favorable opportunities for the spread of the virus, despite the fact that the introduction of new products creates more opportunities for the prevention and treatment of the disease. More than 50 countries have national government-funded influenza immunization programs, and many others have a vaccine available. Specific recommendations for the use of the vaccine vary, but as a rule, they include annual immunization of elderly individuals and individuals older than 6 months with an increased risk of serious illness due to a preexisting chronic medical condition. In some countries, the vaccine is used to reduce the spread of influenza in people with increased medical risk. Member countries need to consider the benefits of influenza prevention actions in the context of their common public health priorities. Inactivated vaccines are classified into several types depending on whether they contain whole viral particles, partially destroyed viral particles (fragmented vaccines) or purified sheath antigens (subunit vaccines). Some subunit vaccines are combined with an adjuvant or delivery system.

Some countries have licensed live attenuated influenza vaccine for some target groups. In the Russian Federation, two different formulations of 1 vaccine are used for healthy adults and children, while other live vaccines have been extensively tested. However, while live attenuated vaccines are not widely available, they are generally not recommended for influenza prophylaxis.

For the prevention and treatment of influenza, two classes of antiviral agents have been developed. M2 inhibitors, amantadine and rimantadine are limited to the treatment of influenza A viruses, and have also been reported to be effective in preventing infection. Although both products cause some side effects, significant neurological side effects are more frequent for amantadine. In some countries, neuraminidase inhibitors such as zanamivir and oseltamivir have recently been licensed to treat types A and B influenza, and have been reported to be effective in prevention. In patients taking both classes of antiviral agents, resistant mutants have been identified. Although this fact is not currently considered an important public health problem, the situation may change if these medicines are used on a wide scale.

WHO supports the worldwide international control program, working in collaboration with 110 national influenza centers located in 82 countries, and 4 centers for collaboration with WHO on influenza counseling and research, located in Atlanta (United States of America), London (UK), Melbourne (Australia) ) and Tokyo (Japan). These centers provide an early warning system for developing strains with epidemic potential. This system is important because the effectiveness of influenza vaccines, if they do not contain currently circulating strains, is reduced. WHO publishes vaccine formulation guidelines as can be found in the Weekly Epidemiological Record (e.g. see issue 9, 2004, 79, page 88 or http://www.who.int/wer ) published by the World Health Organization in February for vaccines used in the northern hemisphere, and in September for vaccines used in the southern hemisphere. Since influenza has less defined seasonal features in equatorial areas, which of these recommendations (February or September) are suitable for vaccines for use in equatorial countries, epidemiological factors will influence.

Collaboration centers conduct antigenic and genetic analyzes of influenza isolates provided by national centers. Where signs of antigenic variability are detected, they are compared with epidemiological data to determine the epidemiological significance of the options. Representative isolates are compared with strains of currently used vaccines using human serum panels collected before and after vaccination to assess whether the currently used vaccines can be expected to protect against these viruses. Following the publication of WHO's annual vaccine guidelines, fast-growing strains are grown and provided to manufacturers as reference viruses, helping to produce progeny viruses for vaccine production. Tests for the safety and effectiveness of influenza vaccines include virus inactivation, microbiological sterility, quantification of the chemicals used to destroy the virus, and confirmation of the concentration of recommended antigens. It is recommended that vaccines be in accordance with WHO requirements, however, specific vaccine viruses used in each country must be approved by national regulatory authorities. National health authorities are responsible for vaccine recommendations. WHO has also published recommendations for influenza prevention (see WER No. 35, 2002, pp. 281-288).

It has already been shown that currently used influenza vaccines do not protect unexposed individuals, a fact that is of direct relevance in the event of a pandemic outbreak of influenza, in which case most individuals who have not experienced an influenza infection are at risk. Typically, viruses begin their life cycle by interacting with the surface receptors of the host cell, entering cells and releasing their viral nucleic acid, followed by replication of the viral genome. After the synthesis of new copies of viral proteins and genes, these components are assembled into descendant virions, which then leave the cell. During the assembly stage, the descendant virus must efficiently select its genomic nucleic acid from a large pool of viral and cellular nucleic acids located in the cytoplasm. The packaging of viral genomes in virions typically involves recognition by the viral components of the cis-acting sequence in viral nucleic acid, the so-called “packaging signal”. The identification of such signals is important for understanding the life cycle of viruses and provides us with information that can be used to construct viral vectors for the expression of foreign proteins. Indeed, the suitability of retroviruses as carriers for gene delivery vectors for the expression of foreign proteins can be largely explained by a well-founded knowledge of the process of packaging their vRNAs in descendant virions.

The genomic packaging signals of other RNA-containing viruses have not been adequately studied, which prevents their use as vectors for the expression and delivery of foreign genes. For example, influenza A virus is a negative-strand enveloped RNA virus whose segmented gene has the potential to encode nucleoprotein (NP), basic polymerase 1 (PB1), basic polymerase 2 (PB2), acid polymerase (PA), hemagglutinin ( HA), neuraminidase (NA), matrix proteins (M1 and M2) and non-structural protein (NS1 and NS2).

In this virus, two membrane-crossing glycoproteins, hemagglutinin (HA) and neuraminidase (NA), are present on the envelope. The HA protein binds to sialic acid-containing receptors on the surface of the host cell and mediates the fusion of the envelope of the virus with the endosomal membrane after receptor-mediated endocytosis. In contrast, the NA protein plays a key role in the final stage of infection by removing sialic acid from sialooligosaccharides, thereby releasing newly collected virions from the cell surface and preventing auto-aggregation of viral particles. In the envelope, the viral genome, containing eight different segments of viral RNA (vRNA), binds strongly to nucleoprotein (NP) and polymerase proteins (PA, PB1 and PB2), forming ribonucleoprotein complexes. All eight (or, in the case of type C virus, all seven) functional gene segments are necessary to produce an infectious virus. Various mutations in polymerase genes have been described (WO2004 / 094466, WO2003 / 091401, US 5578473, Fodor et al, J. Virol. 77, 5017-5020, 2003), which alter the polymerase activity or otherwise alter the polymerase, but do not contribute to the loss of it functionality in relation to the synthesis of viral RNA, with obtaining containing such mutated polymerase virus, unable to replicate. In WO2004 / 094466, infectious viruses with a mutated PA gene were obtained, thus demonstrating the advantages of a selection system allowing to obtain and repair an infectious virus with mutated genes. WO2003 / 091401 shows how to obtain an infectious virus with mutations in polymerase genes to provide for the production and recovery of an influenza virus with desirable properties suitable for producing an attenuated vaccine virus, such as temperature sensitivity or other types of attenuation. In US 55788473 proposed segments of polymerase genes, possibly changing the specificity and reducing the activity of various polymerases. However, they were not used to repair the virus, not counting the recovery of the virus, which completely lost the activity of its polymerases. In addition, none of the above applications received defective particles that lost their ability to replicate. It is well known that when influenza A viruses are subcultured with a high multiplicity of infection, defective viral particles are formed in which one or more functional gene segments are missing. In such viral particles, one or more functional genes are replaced by defective interfering (DI) gene segments due to errors made by influenza polymerase. Due to the high multiplicity of infection and, therefore, infection of cells with more than 1 virus particle, defects in viruses containing DI RNA are complemented by viruses containing intact copies of lost functional genes. It has recently been shown that certain mutations in the acid polymerase gene can increase the proliferation efficiency of viral particles with defective genes (Fodor 2003). It is important to note that the propagation of defective viral particles in these experiments and complementation occurred in such experiments randomly. This random process limits the use of DI RNA and conditionally defective viral particles for practical use. In addition, when defective viral particles are obtained using these published methods, in addition to the desired conditionally defective viruses, replicable wild-type viruses are generated. Such replicable viruses can be either completely wild-type viruses (helper virus) or recombinants obtained by genetic mixing of the helper virus with a defective virus. How the process of packaging gene segments of the influenza virus occurs, randomly or through a specific mechanism, has been discussed for many years. Experimental evidence for both options is described. The evidence for random packaging is that aggregated viral particles have greater infectivity than non-aggregated viral particles, and that when a cell culture is infected with low multiplicity of infection (MOI), expression of one of the segments is absent in some infected cells, which together indicates that there are virions that do not contain the whole genome of the influenza virus. Additional evidence of random packaging is that influenza viruses containing nine segments were experimentally obtained.

One of the arguments for the specific packaging process is that although all gene segments in the biomass of the virus are in equal amounts, they are in different amounts in the producing cells. In addition, when defective interfering (DI) particles are formed, the DI vRNA replaces the segment from which it is derived (A defective interfering particle is a viral particle in which a large internal deletion is present in one of the gene segments. These particles form when the virus is re-grafted at high MOI). Finally, the efficiency of virion formation increases with an increase in the number of gene segments.

SUMMARY OF THE INVENTION

Defective influenza virus particles (e.g., Mena I. et al., J. Virol. 70: 5016-24 (1996); Neumann G. et al., J. Virol. 74: 547-51 (2000)) as candidates for the creation of a vaccine, since they, in addition to HA and NA, induce antibodies to other viral proteins and if they are able to penetrate the host cell, since they, in addition to the humoral response, can induce a cellular immune response against the virus (for example, helper T cells, cytotoxic T cells). Until now, defective influenza virus particles have been obtained by transfection (Mena I. et al., J. Virol. 70: 5016-24 (1996); Neumann G. et al., J. Virol. 74: 547-51 (2000 )), reducing the possibility of obtaining large quantities of such particles. An alternative to this approach could be the production of viral particles that are conditionally defective, allowing them to replicate in a specific producing system, but not in normal cells or producing systems. To this end, the cells of the producing system can be modified to create the possibility of the formation of one or more genes or gene products of influenza viruses, providing transcomplementation of defective particles of influenza virus. The present invention describes for the first time a specific transcomplementation of defective influenza virus particles. In the laboratory, the transcomplementation of influenza virus particles is observed when defective interfering influenza viruses complement in the same cells with viruses carrying the wild-type version of the defective interfering gene segment. This "natural system" of trans complementation is unsuitable for producing certain conditionally defective particles of the influenza virus. First, the complementation of one (partially) defective virus with at least one (partially) replicable virus is necessary in this system, which can lead to the undesirable formation of a completely infectious virus. Secondly, since the formation of defective interfering particles occurs randomly for different gene segments, it is impossible to obtain certain conditionally defective viral particles.

Conditionally defective particles of the influenza virus can theoretically be obtained by removing whole gene segments or parts thereof. The ability to produce certain conditionally defective viral particles by removing entire gene segments (and obtaining the encoded gene product (s) in the transposition) may be limited if the packaging of the influenza virus genome is based on the presence of all 8 segments, which is the subject of much discussion (see elsewhere in this description). If the packaging process requires the presence of all 8 gene segments, it is not known whether all gene segments should be present in full size, which even further complicates the production of conditionally defective viral particles. The present invention solves these difficulties.

The invention relates to a method for producing a conditionally defective influenza virus particle, comprising the first step of transfecting a suitable first cell or cells, such as a 293T cell, with a gene construct with internal deletions, such as pΔPB2, pΔPB1, pΔPA or pDIPA, as provided herein, obtained by internal removal of a nucleic acid encoding an influenza polymerase, whereby said gene construct is incapable of forming a functional polymerase capable of copying or synthesizing a virus RNA, and complementing influenza virus nucleic acid segments encoding influenza virus, such as seven complementing constructs encoding A / WSN / 33 (HW181-188, Hoffmann et al., 2000), and an expression plasmid capable of expressing said polymerase in said cells, such as one of HMG-PB2, HMG-PB1, HMG-PA, as provided herein, and obtaining at least one viral particle from the supernatant of said first cell or cells, at a suitable point in time, such as in the range of 10 to 50, preferably approximately tionary from 20 to 30 hours after transfection; and a second step for transfecting a suitable second cell or cells, such as an MDCK cell, expressing a plasmid capable of expressing said polymerase in said cell; and a third step for transfecting said second cell or cells with a supernatant containing at least one viral particle obtained from said first cell; and the fourth stage, including obtaining at least one (now conditionally defective due to the fact that the resulting viruses lack a gene segment expressing a functional polymerase capable of copying or synthesizing viral RNA, since the gene segment with internal deletion is packed in them) a viral particle from the supernatant of said first cell or cells at a suitable point in time, such as from 24 to 96, preferably from 48 to 72 hours after transfection.

Preferred are internal deletions that render the gene segment incapable of producing a functional protein, but not so large as to impede the packaging of the gene segments of the virus into viral particles. Preferably, these deletions are counted, respectively, from 5 ′ and 3 ′ non-coding regions. For influenza A, such preferred deletions, for example, start from a 5'-nucleotide located between nucleotides 58 and 75, but not including them, and end on a 3'-nucleotide located between nucleotides 27 and 50, but not including them, for protein PA, start from the 5'-nucleotide located between nucleotides 43 and 75, but not including them, and end on the 3'-nucleotide located between nucleotides 24 and 50, but not including them, for protein PB1, start from 5'- a nucleotide located between nucleotides 34 and 50, but not including them, and end on a 3'-nucleotide, p positioned between nucleotides 27 and 50, but not including them, for protein PB2. More preferably, these deletions: start from a 5'-nucleotide located between nucleotides 58 and 100, but not including them, and end on a 3'-nucleotide located between nucleotides 27 and 100, but not including them, for the PA protein, start from 5'-nucleotide located between nucleotides 43 and 100, but not including them, and end on a 3'-nucleotide located between nucleotides 24 and 100, but not including them, for protein PB1, start from 5'-nucleotide located between nucleotides 34 and 100, but not including them, and end on a 3'-nucleotide located at dy nucleotides 27 and 100, but not including, for PB2 protein. Even more preferably, these deletions: start from a 5'-nucleotide located between nucleotides 58 and 150, but not including them, and end on a 3'-nucleotide located between nucleotides 27 and 150, but not including them, for the PA protein, begin from the 5'-nucleotide located between nucleotides 43 and 150, but not including them, and end on the 3'-nucleotide located between nucleotides 24 and 150, but not including them, for protein PB1, start from the 5'-nucleotide located between nucleotides 34 and 150, but not including them, and end on a 3'-nucleotide located m between nucleotides 27 and 150, but not including, for PB2 protein. Even more preferably, these deletions: start from the 5'-nucleotide located between nucleotides 58 and 175, but not including them, and end on the 3'-nucleotide located between nucleotides 27 and 175, but not including them, for the PA protein, begin from a 5'-nucleotide located between nucleotides 43 and 175, but not including them, and end on a 3'-nucleotide located between nucleotides 24 and 175, but not including them, for protein PB1, start from a 5'-nucleotide located between nucleotides 34 and 175, but not including them, and end on a 3'-nucleotide, located between nucleotides 27 and 175, but not including, for the PB2 protein. Most preferably, these deletions: start from a 5'-nucleotide located between nucleotides 58 and 207, but not including them, and end on a 3'-nucleotide located between nucleotides 27 and 194, but not including them, for the PA protein, start from 5'-nucleotide located between nucleotides 43 and 246, but not including them, and end on a 3'-nucleotide located between nucleotides 24 and 197, but not including them, for protein PB1, start from 5'-nucleotide located between nucleotides 34 and 234, but not including them, and terminate at the 3'-nucleotide located between nucleotides 27 and 209, but not including them, for the PB2 protein.

In this document, complementing segments are defined as segments leading to a complete set of eight gene segments, for example, influenza A virus. Thus, if segment 1 has already been used to produce a defective segment, complementing (non-defective) segments are segments 2, 3, 4 , 5, 6, 7, and 8. If 2 is defective, the complementing segments are segments 1, 3, 4, 5, 6, 7, and 8. And so on. Advantageously, the invention provides a method in which an helper virus is not required or not present.

The invention relates to an isolated and conditionally defective influenza virus particle with no functional segment of the influenza virus nucleic acid (also referred to as conditionally defective influenza virus particle) encoding a polymerase selected from the group of acidic polymerase (PA), basic polymerase 1 (PB1) and basic polymerase 2 (PB2), where the specified particle

unable to synthesize polymerase or serve as a source for polymerase synthesis for copying or synthesis of viral RNA, thus only and conditionally allowing the formation of replication virus particles in cells transformed with functional polymerase. In addition, the invention relates to a method for producing a conditionally defective particle of an influenza virus, which method comprises producing a cell by trans complementation with a functional polymerase of an influenza virus.

In a preferred embodiment, a particle of the invention is replicated in a cell complemented by a similar nucleic acid segment that is not present in the particle itself, for example a particle with no functional influenza virus nucleic acid segment PA is replicated in a cell that is provided with at least a functional virus nucleic acid segment influenza PA, a particle lacking the functional nucleic acid segment of the influenza virus PB1 is replicated in the cell, which is provided, by at least a segment of the functional nucleic acid of influenza virus PB1, a particle with no segment of the functional nucleic acid of influenza virus PB2 is replicated in the cell, which is provided with at least a segment of the functional nucleic acid of influenza virus PB, respectively. In a preferred embodiment, the invention relates to a particle of the invention with influenza virus nucleic acid segments encoding for viral glycoproteins, more preferably with influenza virus nucleic acid segments encoding for nucleoprotein (NP), hemagglutinin (HA), neuraminidase (NA), matrix proteins (M1 and M2) and a non-structural protein (NS1 and NS2). In one embodiment, a particle of the invention is provided with influenza virus nucleic acid segments derived from influenza A. A particle of the invention is also provided that contains a nucleic acid that does not encode an influenza peptide. The invention also relates to an isolated cell containing a particle of the invention, wherein the wild-type influenza virus or helper virus is absent in said cell, but preferably contains or complements an influenza virus polymerase or a gene segment coding for it. In a preferred embodiment, such a cell is a 293T transiently complemented cell or MDCK. In one embodiment, the invention relates to an isolated cell containing a particle lacking a functional influenza virus PA nucleic acid segment, wherein the wild-type influenza virus or helper virus is absent in said cell, but the cell is provided with or is complemented with at least a functional nucleic acid segment acid of influenza virus PA or functional PA. In another embodiment, the invention relates to an isolated cell containing a particle lacking a functional nucleic acid segment of the influenza virus PB1, wherein the wild-type influenza virus or helper virus is absent in said cell, but the cell is provided with or is complemented with at least a functional nucleic acid segment influenza virus PB1 or functional PB1. In yet another embodiment, the invention relates to an isolated cell containing a particle lacking a functional nucleic acid segment of the PB2 influenza virus, wherein the wild-type influenza virus or helper virus is absent in said cell, but the cell is provided with or is complemented with at least a functional nucleic acid segment acid of influenza virus PB2 or functional PB2. In addition, the invention relates to a composition containing a particle of the invention or a cell or product obtained from a cell of the invention, and to the use of such a composition for the preparation of a pharmaceutical composition intended to provide immunological protection against infection of a subject with influenza virus. Thus, the invention relates to a method for providing immunological protection against an infection of a subject with an influenza virus, comprising providing a composition of the invention to a subject in need thereof. The invention also relates to the use of an influenza virus particle according to the invention for preparing a composition for delivery to a cell of a nucleic acid that does not encode an influenza peptide. The invention also relates to the use of a particle according to the invention for the preparation of a pharmaceutical composition intended for delivery to a subject cell of a nucleic acid that does not encode an influenza peptide, and to a method for delivery to a cell or subject of a nucleic acid that does not encode an influenza peptide, comprising introducing into said cell or subject particles of the invention.

The invention relates to a conditionally defective particle of the influenza virus with the absence of one functional segment of the influenza virus compared to its natural genome, i.e.: compared to the wild-type virus or helper virus type A or B with seven (instead of eight) different functional segments influenza virus nucleic acid, or compared to wild-type virus or type C helper virus with six (instead of seven) different functional segments of the influenza virus nucleic acid. When the term “conditionally defective” is used herein, it includes, but is not limited to, viral particles, where one of the gene segments of the virus carries a large internal deletion resulting in a non-functional protein expressing therefrom. All eight gene segments, for example, influenza A virus and all the proteins encoded by them, are necessary to obtain an infectious virus. Thus, a virus containing a defective gene segment is itself defective: it can infect a cell and can go through one replication cycle, since all viral proteins are present in the virion (for example, this protein was obtained through an expression plasmid when the virus was received), but infectious viral particles do not form in infected cells, since the virus cannot synthesize one of the viral proteins. However, when cells expressing a protein that is normally expressed by a defective gene segment become infected, the defective virus can replicate in these cells since all viral proteins are present. Thus, these viruses are conditionally defective: they cannot replicate unless a cell with the correct condition is provided (in this case, a cell expressing a viral protein that is not encoded by the virus due to deletion in the gene segment).

In addition, the invention relates to a conditionally defective influenza virus particle with the absence of a functional nucleic acid segment of an influenza virus encoding a polymerase. In this document, the functional segment of the nucleic acid of the influenza virus includes a nucleic acid encoding a functional protein of influenza, allowing to obtain a replicating virus and necessary for it. For example, influenza A virus is an RNA-containing negative chain virus with an 8-segment genome. 8 gene segments encode 11 proteins; gene segments 1-8 encode basic polymerase 2 (PB2), basic polymerase 1 (PB1) and PB1-ORF2 (F2), acid polymerase (PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix proteins 1 and 2 (M1, M2) and non-structural proteins 1 and 2 (NS1, NS2), respectively. The coding regions of 8 gene segments are flanked by non-coding regions (NCR) necessary for the synthesis of viral RNA. The last 13 and 12 nucleotides at the 5'- and 3'-ends of the viral genomic RNA, respectively, are conserved in all segments of the influenza A virus and are partially complementary, forming a secondary structure recognized by the viral polymerase complex. NCRs may contain up to 60 additional nucleotides that are not conserved in 8 gene segments, but relatively conserved among various influenza viruses. NCR and flanking sequences in the coding regions may be necessary for efficient packaging of the viral genome. Thus, the functional segment of the nucleic acid of the influenza virus consists of a sequence with a coding potential for the functional protein of influenza, allowing the formation of a replication virus (1 or 2 open reading frames per segment), NCR necessary for the transcription of mRNA, viral RNA (vRNA) and RNA, complementary viral RNA (cRNA), and a packaging signal located in the NCR and flanking the coding sequences. Preferably, in the conditionally defective particle of the influenza virus with the absence of one of the nucleic acids of the influenza virus, there is no segment encoding a functional polymerase, which is PA, PB1 or PB2. In addition, for vaccination purposes, it is preferred that said particle carry a nucleic acid segment of the influenza virus (s) encoding for the viral glycoprotein (s).

In one embodiment, the invention relates to an influenza A virus particle with seven different segments of influenza A nucleic acid. Defective influenza virus particles of the invention are capable of replication, although only once, in suitable but not complementary animal or host cells. In suitable complemented cells, the particles of the invention can replicate for several cycles. For vaccination and gene delivery purposes, the great advantage is that defective particles cannot replicate indefinitely in normal non-trans complemented cells, thereby reducing the risk of spreading the vaccine virus from host to host and reducing the risk of reversal to wild-type virus.

For the first time using reverse genetics, defective influenza A viruses were obtained that contain only seven functional gene segments and which can undergo one replication cycle or several replication cycles when the defective gene segment is trans-complemented. In one embodiment, the invention relates to a conditionally defective influenza virus particle with no nucleic acid segment of the influenza virus, mainly encoding acid polymerase (PA). Like the transcomplementation of PA, one can imagine the transcomplementation of other genes of the influenza virus. However, since PA expression levels have been shown to be less critical than expression levels of other influenza virus proteins, PA represents the preferred gene segment from the polymerase group that is deleted, viruses with deletion of PB2 and PB1 can be obtained with the same success, and viruses with a deletion of NP cannot be complemented. In a preferred embodiment, the invention relates to a conditionally defective influenza A virus particle with seven different segments of the influenza A nucleic acid and the absence of the influenza A nucleic acid segment mainly encoding an acidic polymerase. For vaccination purposes, the preferred conditionally defective influenza A virus particle of the invention carries influenza A nucleic acid segments predominantly encoding hemagglutinin (HA) and neuraminidase (NA) proteins, these proteins are most immunologically relevant to provide protection. To select the appropriate gene segments for inclusion in the vaccine, it is preferred that the gene segments be selected from a virus recommended by WHO for use in vaccines. Of course, the HA and NA subtypes may vary depending on the HA and NA subtypes of the influenza variant against which a vaccine is desired. It is most preferable to obtain a conditionally defective influenza virus particle according to the invention with influenza A nucleic acid segments predominantly encoding nucleoprotein (NP), basic polymerase 1 (PB1), basic polymerase 2 (PB2), hemagglutinin (HA), neuraminidase (NA), matrix proteins (M1 and M2) and non-structural protein (NS1 and NS2), where mainly coding in this document in particular indicates that a functional protein is expressed from the corresponding gene segment. Such a particle is in particular provided in an isolated cell containing functional PA or a functional gene segment encoding PA. In another embodiment, provided herein is a conditionally defective influenza virus particle of the invention with influenza A nucleic acid segments mainly encoding nucleoprotein (NP), acid polymerase (PA), basic polymerase 2 (PB2), hemagglutinin (HA), neuraminidase ( NA), matrix proteins (M1 and M2) and non-structural protein (NS1 and NS2), where mainly coding in this document in particular indicates that a functional protein is expressed from the corresponding gene segment. Such a particle is in particular provided in an isolated cell containing functional PB1 or a functional gene segment encoding PB1. In another embodiment, provided herein is a conditionally defective influenza virus particle of the invention with influenza A nucleic acid segments mainly encoding nucleoprotein (NP), acid polymerase (PA), basic polymerase 1 (PB1), hemagglutinin (HA), neuraminidase ( NA), matrix proteins (M1 and M2) and non-structural protein (NS1 and NS2), where mainly coding in this document in particular indicates that a functional protein is expressed from the corresponding gene segment. Such a particle is in particular provided in an isolated cell containing functional PB2 or a functional gene segment encoding PB2. In another embodiment, the invention relates to particles of the invention further comprising a nucleic acid not encoding an influenza peptide, for example, encoding a foreign protein or peptide suitable for eliciting an immune response, or containing a nucleic acid capable of interfering with cellular functions or functions of a pathogenic microorganism in a cell.

In addition, the invention relates to a cell containing a particle of the influenza virus according to the invention. When the particle does not contain gene segments, basically encoding the necessary polymerase, it is suitable for treating a cell containing suitable functional influenza virus polymerase, providing multiple replication cycles of defective influenza virus particles in the cell thus complemented. The invention also relates to a composition comprising a defective particle of an influenza virus according to the invention or a cell or product obtained from a cell according to the invention; where such a composition can be used, for example, to obtain a pharmaceutical composition intended to provide immunological protection against infection of a subject with influenza virus. The invention also relates to a method for creating an immunological defense against an infection of a subject with an influenza virus, comprising providing such a composition to a subject in need thereof. In addition to using the particles of the invention as a vaccine or immunogenic composition, such compositions are preferably formulated as a vaccine, i.e. by mixing viral particles or viral proteins derived from such particles (subunit vaccines) with a suitable pharmaceutical carrier, such as saline or adjuvant (e.g. aluminum salt or other common excipient) (see, for example, http: //www.cdc .gov / nip / publications / pink / Appendices / A / Excipient.pdf ). Conditionally defective influenza virus particles according to the invention are also candidate vectors for the delivery of foreign genes and for the expression of a foreign protein, since a functional gene can be inserted, for example, between 5 ′ and 3 ′ PA sequences. In general, the invention relates to a method for producing a conditionally defective influenza virus particle, possibly containing a foreign nucleic acid segment or a host nucleic acid segment or a fragment thereof, comprising the first step of transfecting a suitable first cell or cells with one or more gene constructs obtained by internal deletion a nucleic acid encoding an influenza protein, as a result of which the indicated gene constructs are incapable of producing a functional protein and do not interfere with packaging virus gene segments into viral particles, and complementary influenza virus nucleic acid segments encoding an influenza virus, and one or more expression plasmids capable of expressing said proteins in said cell and obtaining at least one viral particle from the supernatant of said first cell or cells at a suitable point in time after transfection; and a second step for transfecting a suitable second cell or cells with one or more expression plasmids capable of expressing said proteins in said cell; and a third stage of infection of said second cell or cells with a supernatant containing at least one viral particle obtained from said first cell; and a fourth step comprising obtaining at least one viral particle from the supernatant of said second cell or cells at a suitable point in time after infection. Thus, the invention relates to a method for producing a conditionally defective particle of an influenza virus, comprising the step of transfecting a suitable cell or cells with one or more gene constructs obtained by internal deletion of a nucleic acid encoding influenza polymerase, as a result of which said gene constructs become incapable of production functional polymerase, but do not interfere with the packaging of gene segments of the virus into viral particles, and complementing nucleic acid segments of vir influenza ca encoding influenza virus, and one or more expression plasmids capable of expression of said polymerase in said cell, and obtaining at least one virus particle from the supernatant of said cell or cells in an appropriate time after infection. The method for producing a conditionally defective influenza virus particle includes the first step of transfecting a suitable cell or cells with one or more expression plasmids capable of expressing influenza polymerases in said cell; and a second stage of infection of said cell or cells with a supernatant containing conditionally defective influenza virus particles; and a third stage, comprising obtaining at least one viral particle from the supernatant of the specified cell or cells at a suitable time after infection, or a method for producing a conditionally defective influenza virus particle, comprising the first stage of transfection of a suitable first cell or cells of one or several gene constructs obtained by internal deletion of a nucleic acid encoding influenza polymerase, as a result of which these gene constructs become incapable of production functional polymerase, but do not interfere with the packaging of viral gene segments into viral particles, and with complementary influenza virus nucleic acid segments encoding the influenza virus, and one or more expression plasmids capable of expressing said polymerases in said cell and obtaining at least one a viral particle from the supernatant of said first cell or cells at a suitable point in time after transfection; and a second step for transfecting a suitable second cell or cells with one or more expression plasmids capable of expressing said polymerases in said cell; and a third stage of infection of said second cell or cells with a supernatant containing at least one viral particle obtained from said first cell; and the fourth stage, which includes obtaining at least one viral particle from the supernatant of the specified second cell or cells at a suitable point in time after infection. In the methods, said polymerases can be, for example, acid polymerase (PA), basic polymerase 1 (PB1) or basic polymerase 2 (PB2). Preferably, the invention relates to a method in which an internal deletion results from an internal deletion of an influenza polymerase-coding nucleic acid that starts from a 5'-nucleotide located between nucleotides 58 and 207, counting from but not including the non-coding region, and ends at 3 '-nucleotide located between nucleotides 27 and 194, counting from the non-coding region, but not including them, for the PA protein, alternatively starts from the 5'-nucleotide located between nucleotides 43 and 246, counting from region, but not including them, and ends on a 3'-nucleotide located between nucleotides 24 and 197, counting from the non-coding region, but not including them, for protein PB1, alternatively starts from a 5'-nucleotide located between nucleotides 34 and 234, counting from the non-coding region, but not including them, and ends on the 3'-nucleotide located between nucleotides 27 and 209, counting from the non-coding region, but not including them, for the PB2 protein. In another embodiment, a foreign fragment is inserted into this internal deletion. The invention further relates to a method in which a cell or cells for infection with a supernatant containing conditionally defective influenza virus particles already express non-functional polymerases such as acidic polymerase (PA), basic polymerase 1 (PB1) or basic polymerase 2 (PB2 ), and flu particles are provided by the method provided herein. For example, it is described herein that a cell or cells for transfection with gene constructs and nucleic acid segments already express non-functional polymerases. In particular, the invention relates to a particle of an influenza virus containing one or more nucleic acid segments with an internal deletion in a segment that makes the segment incapable of producing functional influenza polymerase but does not interfere with the packaging of the virus gene segments into viral particles, whereby the polymerase is selected from the group acid polymerase (PA), basic polymerase 1 (PB1) or basic polymerase 2 (PB2). Preferably, the internal deletion: starts from a 5'-nucleotide located between nucleotides 58 and 207, counting from the non-coding region, but not including them, and ends with a 3'-nucleotide located between nucleotides 27 and 194, counting from the non-coding region, but not including them, for the PA protein, it started from a 5'-nucleotide located between nucleotides 43 and 246, counting from the non-coding region, but not including them, and ended with a 3'-nucleotide located between nucleotides 24 and 197, counting from non-coding region, but not including them, for protein PB1 started from a 5'-nucleotide located between nucleotides 34 and 234, counting from the non-coding region, but not including them, and ended on a 3'-nucleotide located between nucleotides 27 and 209, counting from the non-coding region, but not including them , for protein PB2. In a preferred embodiment, the invention relates to a particle of the invention with influenza virus nucleic acid segments encoding viral glycoproteins. The invention also relates to a particle according to the invention with influenza virus nucleic acid segments encoding nucleoprotein (NP), basic polymerase 1 (PB1), basic polymerase 2 (PB2), hemagglutinin (HA), neuraminidase (NA), matrix proteins (M1 and M2 ) and non-structural protein (NS1 and NS2), or a particle with influenza virus nucleic acid segments encoding nucleoprotein (NP), acid polymerase (PA), basic polymerase 2 (PB2), hemagglutinin (HA), neuraminidase (NA), matrix proteins (M1 and M2) and non-structural protein (NS1 and NS2), or a particle with nucleic acid segments s influenza virus encoding the nucleoprotein (NP), polymerase acidic (PA), basic polymerase 1 (PB1), hemagglutinin (HA), neuraminidase (NA), matrix proteins (M1 and M2) and the nonstructural protein (NS1 and NS2). In particular, the invention relates to a particle according to the invention with influenza virus nucleic acid segments derived from influenza A. The invention also relates to a particle according to the invention containing a nucleic acid not encoding an influenza peptide. In addition, the invention relates to a cell containing a particle according to the invention, in particular to a cell containing one or more influenza virus polymerases, whereby the polymerase is selected from the group of acidic polymerase (PA), basic polymerase 1 (PB1) or basic polymerase 2 (PB2). In addition, the invention relates to a composition comprising a particle according to the invention or a cell or product obtained from a cell according to the invention, the use of such a composition for the preparation of a pharmaceutical composition intended to provide immunological protection against infection of a subject with influenza virus, and a method for creating immunological protection against infection a subject with an influenza virus, which comprises providing such a composition to a subject in need thereof. In addition, the invention relates to the use of a particle of the invention to obtain a composition intended for delivery to a cell of a nucleic acid that does not encode an influenza peptide, and the use of a particle of the invention to produce a pharmaceutical composition intended to deliver a nucleic acid that does not encode an influenza peptide to cells of a subject . Such a nucleic acid (also referred to herein as foreign nucleic acid) may encode a foreign gene or gene fragment encoding a suitable antigenic epitope or protein, or it may encode a portion of nucleotides capable of inhibiting transcription of nucleic acids in a cell. In one embodiment, the invention relates to the use of an influenza virus particle according to the invention for the preparation of a composition for delivery to a cell or cell of a subject of a nucleic acid that does not encode an influenza peptide. The invention further relates to a method for delivering a nucleic acid that does not encode an influenza peptide into a cell or subject, wherein the method comprises introducing into the said cell or the indicated subject a defective influenza virus particle containing the foreign nucleic acid of the invention.

Drawing captions

Signature to figure 1

Obtaining and propagating conditionally defective influenza virus A. First, 293T cells were transfected with 7 bidirectional plasmids encoding A / PR / 8/34, pHMG-PA and, if appropriate, pΔPA or pDIPA. 48 hours after transfection, transfected cell supernatants were collected and used to inoculate MDCK cells, and MDCK cells were transfected with HMG-PA 24 hours earlier. The supernatant of MDCK-PA cells, positive for virus replication, was plated on MDCK and MDCK-PA cells 4 times.

Signature to figure 2

Designs used to obtain conditionally defective viral particles. Above, the wild type PA gene segment is shown. Non-coding regions (NCR) and initiation codons are indicated. pΔPA was constructed by digestion of pHW183, a bidirectional plasmid containing PA A / WSN / 33 (9) Stu I, and subsequent re-ligation. pDIPA was constructed by cloning nucleotides between 5 ′ 194 and 3 ′ 207 of the PA A / PR / 8/34 gene segment in pSP72. The insert was then transfected into a bidirectional reverse genetics vector. pΔPB1 and pΔPB2 were constructed as described in the text.

Signature to figure 3

RT-PCR analysis for the presence of the PA gene segment in the supernatants rPR8-7, rPR8-ΔPA and rPR8-DIPA. Supernatants after 4 reseeding on MDCK-PA were passed through a 22 μm filter and concentrated by centrifugation. RNA was then isolated and RT-PCR was performed using primers complementary to the non-coding regions of the PA segment. RNA isolated from wild type A / PR / 8/34 was used as a control. Track 1: rPR8-7; lane 2: rPR8-ΔPA; lane 3 rPR8-DIPA; lane 4: A / PR / 8/34 wild type. Marker sizes are shown on the left.

Signature to figure 4

Additional enlarged parts of the pΔPA construct that have been removed resulting in pΔPA-2, pΔPA-3, pΔPA-4, pΔPA-5.

Detailed description

Example 1

Obtaining defective influenza virus particles from recombinant DNA

Influenza A virus is a segmented antisense virus. The genome consists of eight gene segments. All eight functional gene segments are necessary for the production of an infectious virus, i.e. a replication virus that is capable of unlimited or at least several replication cycles in cells, commonly considered suitable for replication of influenza virus. How the process of packaging gene segments of influenza A virus occurs, randomly or through a specific mechanism, has been discussed for many years. Experimental evidence for both options is described. The evidence for random packaging is that aggregated viral particles have greater infectivity than non-aggregated viral particles (6), and that when a cell culture is infected at low MOI, expression of one of the segments is absent in some infected cells (8), which together indicates that there are virions that do not contain the whole genome of the influenza virus. Additional evidence of random packaging is that experimentally obtained influenza viruses containing nine segments (4). Bancroft and Parslow revealed that there was no competition between vRNAs originating from the same gene segment for packaging in virion (1).

One of the arguments for the specific packaging process is that although all gene segments in the biomass of the virus are in equal amounts, they are in different amounts in the producing cells (10). In addition, when defective interfering (DI) particles are formed, the DI vRNA replaces the segment from which it is derived (3) (the defective interfering particle is a viral particle in which a large internal deletion is present in one of the gene segments. These particles are formed, when the virus is subcultured at high MOI and is thought to also form due to mutation of the R638A acid polymerase protein [Fodor et al; J. Virol. 77, 5017-5020, 2003]). Finally, the efficiency of virion formation increases with an increase in the number of gene segments (5). Fujii et al. also showed the NA segment region necessary for efficient insertion of the segment into the virion, and later the same group also showed the HA or NS region important or packing into the viral particle [Fujii, 2005 # 256; Watanabe, 2003 # 184].

In this document, the authors present evidence of specific packaging. To obtain viral particles containing only seven functional gene segments, it is necessary to determine which gene segment can be skipped without stopping the formation of the virus. In light of the use of the replication-defective virus as a vaccine, HA and NA should not be skipped, and neither MA nor NS, since in this case 2 separate expressing plasmids are necessary. The authors obtained a virus with no polymerase gene. The authors were unable to obtain the virus when the deleted gene segment was not transplemented with an expression plasmid (Tables 1, 2 and 3, rPR8-7ntc). The virus can be obtained by transfection of seven gene segments and a protein expressing plasmid normally expressed by the deleted gene segment at very low titers (Tables 1, 2 and 3, rPR8-7). Thus, deletion mutants of the gene segments 1, 2 and 3 of the influenza virus A / WSN / 33 were obtained, carrying an internal deletion of 1032, 528 and 1120 nucleotides, respectively. These deletion mutants were designated pΔPB2, pΔPB1 and pΔPA (see FIG. 2). 293T cells were transfected as previously described (de Wit, E., MI Spronken, TM Bestebroer, GF Rimmelzwaan, AD Osterhaus and RA Fouchier. 2004. Efficient generation and growth of influenza virus A / PR / 8/34 from eight cDNA fragments. Virus Res 103: 155-61) with each of these deleted gene segments and seven complementary bi-directional constructs encoding A / PR / 8/34 (De Wit et al, 2004) and the corresponding expression plasmid. 48 hours after transfection, supernatants were collected. Then, as described previously (2), MDCK cells of one of the expressing plasmids HMG-PB2, HMG-PB1 or HMG-PA were transfected. These transfected cells were inoculated with the appropriate supernatant of transfected 293T cells (see FIG. 1 for an explanation of the experimental procedure). Virus replication in these MDCK cells was determined by HA analysis. There was no initial virus replication in untransfected MDCK cells. Virus replication is indicated in MDCK cells transfected with HMG-PB2, HMG-PB1 or HMG-PA inoculated with the corresponding supernatant. Further, the authors cloned the defective PA gene segment based on the sequence of the defective interfering vRNA PA of the influenza virus A / PR / 8/34 obtained from the influenza virus sequence database ( www.flu.lanl.gov , accession number K00867). The region between the 5'-nucleotide 207 and the 3'-nucleotide 194 PA was amplified by PCR and cloned into a bidirectional transcription vector derived from pHW2000 (7), which was modified as described previously (De Wit et al., 2004). The resulting plasmid was called pDIPA (see FIG. 2). 293T cells were transfected with pDIPA, HMG-PA and 7 bidirectional constructs encoding the remaining gene segments of the influenza virus A / PR / 8/34 (see FIG. 2). The supernatant was collected 48 hours after transfection, and then MDCK cells, 24 hours previously infected with HMG-PA, were inoculated with this supernatant. 72 hours after inoculation, an HA analysis was performed on the supernatants of these MDCK cells and found to be positive, indicating virus replication in these cells. Inoculation of untransfected MDCK cells also did not lead to virus production, as determined by the HA assay. Subsequent inoculation of supernatants containing PA defective viral particles onto MDCK cells untransfected or transfected with HMG-PA led to the same result (Table 1). Prior to passage 4, the virus was produced in MDCK cells transfected with HMG-PA. The supernatant of MDCKp4 was subsequently diluted to obtain the titer of the virus, which was shown to be approximately 10 ⁴ TCID ₅₀ / ml.

The stages of the method used were: 293T cells were transfected (for the transfection protocol, see De Wit et al., 2004) of one of the constructs pΔPB2, pΔPB1, pΔPA, pΔNP, seven complementing constructs encoding A / PR / 8/34 (De Wit et al. , 2004) and one of HMG-PB2, HMG-PB1, HMG-PA (expression plasmids, for example, described in Pleschka, S., R. Jaskunas, OG Engelhardt, T. Zurcher, P. Palese and A. Garcia-Sastre 1996. A plasmid-based reverse genetics system for influenza A virus. J Virol 70: 4188-92; obtained from A. Garcia-Sastre and P. Palese). 48 hours after transfection, the supernatant of transfected 293T cells is harvested. When viruses are produced, they are present in the supernatant. Since that time, MDCK cells are transfected (for the transfection protocol, see Basler et al., 2000) with one of the expression plasmids HMG-PB2, HMG-PB1, HMG-PA (depending on the deletion mutant used, so if pΔPB2 is used, MDCK cells transfect HMG -PB2), since the produced viruses lack the gene segment expressing the protein, since the gene segment with internal deletion was packaged in them. 24 hours after transfection, transfected MDCK cells are inoculated with supernatants obtained from transfected 293T cells. When a virus is present in the 293T supernatant, this virus will replicate in transfected MDCK cells and more virus will form. This supernatant can be harvested again 72 hours after inoculation.

To prove that there was no recombination of PA or DIPA leading to a functional gene segment of PA, RNA was isolated from the MDCKp4 supernatant. First, supernatants were passed through a 22 μm filter and concentrated by centrifugation. Then RNA was isolated and RT-PCR was performed using primers complementary to the non-coding regions of the PA segment. RT-PCR performed with primers specific for PA vRNA showed that ΔPA and DIPA remained stable for several passages. A distinct band of approximately 400 bp appeared in the supernatant of MDCK cells infected with DIPA virus particles, a band of 1100 bp appeared in the supernatant of MDCK cells infected with the virus containing ΔPA In the supernatant of wild-type A / PR / 8/34 infected MDCK cells, a band of approximately 2300 bp is visible. (figure 3). These results indicate that the ΔPAPR8 gene segment is stably packaged in virions.

To obtain viruses without PB2, 293T cells were transfected with 7 bidirectional constructs (Hoffmann, E., G. Neumann, Y. Kawaoka, G. Hobom and RG Webster. 2000. A DNA transfection system for generation of influenza A virus from eight plasmids. Proc Natl Acad Sci USA 97: 6108-13.), Encoding the gene segments 2, 3, 4, 5, 6, 7 and 8 of the influenza virus A / PR / 8/34 (de Wit, E., MI Spronken, TM Bestebroer, GF Rimmelzwaan, AD Osterhaus and RA Fouchier. 2004. Efficient generation and growth of influenza virus A / PR / 8/34 from eight cDNA fragments. Virus Res 103: 155-61), which led to the expression of vRNA and mRNA. A plasmid expressing PB2 A / PR / 8/34, pHMG-PB2 (Pleschka, S., R. Jaskunas, OG Engelhardt, T. Zurcher, P. Palese and A. Garcia-Sastre. 1996. A plasmid-based reverse was co-transfected genetics system for influenza A virus. J Virol 70: 4188-92). As a control, only 7 bidirectional constructs encoding A / PR / 8/34 were transfected, excluding pHMG-PB2. Supernatants were collected 48 hours after transfection and were inoculated into MDCK cells or MDCK cells 24 hours previously transfected with pHMG-PB2 (MDCK-PB2) in 100 mm dishes. Three days after inoculation, the supernatant of the inoculated MDCK cells was tested for hemagglutinin activity using turkey red blood cells as an indicator of virus formation. No viruses were detected in cells inoculated with the supernatant of 293T cells transfected with only 7 gene segments without pHMG-PB2 (rPR8-7ntc, table 2). The supernatant of MDCK-PB2 cells inoculated with the supernatant of 293T cells transfected with 7 gene segments and pHMG-PB2 was positive. Then, the rPR8-7 supernatant was subcultured into MDCK and MDCK-PB2 cells. rPR8-7 was replicated in MDCK-PB2 cells, but not in MDCK cells (table 2). The authors then received a mutant with a deletion of 1032 nucleotides in the gene segment 1 of influenza virus A / WSN / 33, which led to a deletion of 344 amino acids (pΔPB2, figure 2). A recombinant virus was formed containing ΔPB2 (rPR8-ΔPB2) as described above (Fig. 1). The virus could not be detected in MDCK cells, while the virus was detected in MDCK-PB2 cells inoculated with rPR8-ΔPB2. After rPR8-ΔPB2 reseeding, there was no evidence of virus production in MDCK cells, unlike MDCK-PB2 cells (Table 2).

Also obtained viruses lacking PB1. 293T cells were transfected with 7 bidirectional constructs encoding the gene segments 1, 3, 4, 5, 6, 7 and 8 of the influenza virus A / PR / 8/34, which led to the expression of vRNA and mRNA. A plasmid expressing PB1 A / PR / 8/34, pHMG-PB1 was cotransfected. Only 7 bidirectional constructs encoding A / PR / 8/34 were transfected as a control, with the exception of pHMG-PB1. Supernatants were harvested 48 hours after transfection and inoculated into MDCK cells or MDCK cells 24 hours previously transfected with pHMG-PB1 (MDCK-PB1) in a 100 mm dish (2) (Fig. 1). Three days after inoculation, the supernatant of the inoculated MDCK cells was tested for hemagglutinin activity using turkey red blood cells as an indicator of virus formation. No viruses were detected in cells inoculated with the supernatant of 293T cells transfected with only 7 gene segments without pHMG-PB1 (rPR8-7ntc, table 3). The supernatant of MDCK-PB1 cells inoculated with the supernatant of 293T cells transfected with 7 gene segments and pHMG-PB1 was positive. Then, the rPR8-7 supernatant was subcultured into MDCK and MDCK-PB1 cells. rPR8-7 was replicated in MDCK-PB1 cells, but not in MDCK cells (table 3). The authors then received a mutant with a deletion of 528 nucleotides in the gene segment 2 of influenza virus A / WSN / 33, which led to a deletion of 178 amino acids (pΔPB1, figure 2). A recombinant virus was formed containing ΔPB1 (rPR8-ΔPB1) as described above (Fig. 1). The virus could not be detected in MDCK cells, while the virus was detected in MDCK-PB1 cells inoculated with rPR8-ΔPB1. After rPR8-ΔPB1 reseeding, there was no evidence of virus production in MDCK cells, unlike MDCK-PB1 cells (Table 3).

Thus, the authors were able to obtain viruses with the absence of segments 1, 2 or 3, by obtaining constructs pΔPB2, pΔPB1 or pΔPA / pDIPA and trans complementation using RNA polymerase II-directed expression plasmids PB2, PB1 or PA, as described above. The conditionally defective viruses described herein can go through only one replication cycle in cells without trans complementation, but they can be grown in trans complemented cell lines. This represents the first time that defective viruses are obtained using reverse genetics that contain only seven functional gene segments and which can undergo one replication cycle or several replication cycles when the defective gene segment is trans-complemented.

Defective viral particles thus obtained are candidates for vaccines since they can go through a single replication cycle without the formation of an infectious virus. The result of this single replication cycle is that the vaccine induces a humoral and cellular immune response. Despite the fact that these defective particles do not replicate in ordinary cells, large quantities of the virus can be grown in the cell line expressing the defective protein in order to obtain. As the authors showed, several replication cycles do not affect the genotype of the virus. In addition to using defective viral particles as a vaccine, they are also candidate vectors for gene delivery and for the expression of foreign proteins, since a functional gene can be inserted between the 5'- and 3'-sequences of PA, PB2 or PB1. It is also shown by Watanabe et al. (11), which replaced the foreign genes with both HA and NA and could still receive the virus.

Further shortening pΔPA

In addition, large removable parts of the pΔPA structure led to pΔPA-2, pΔPA-3, pΔPA-4, pΔPA-5 (Fig. 4). 293T cells were transfected as previously described (De Wit et al., 2004) with each of these deleted gene segments and seven complementary bi-directional constructs encoding A / PR / 8/34 (De Wit et al, 2004) and an expression plasmid expressing PA. Supernatants were harvested 48 hours after transfection. Then, MDCK cells were transfected as previously described (Basler, C. F., et al., 2000. Proc Natl Acad Sci USA 97: 12289-94) expressing the HMG-PA plasmid. These transfected cells and non-transfected cells were inoculated with supernatants of transfected 293T cells. Through HA analysis, virus replication was determined in these MDCK and MDCK-PA cells. In untransfected MDCK cells, virus replication was not observed. Virus replication was observed in MDCK cells transfected with HMG-PA inoculated with one of the supernatants. Thus, all vRNAs obtained from these constructs were packaged in virions (Table 4).

Table 1
Replication of recombinant influenza viruses A / PR / 8/34 with the absence of the original PA gene segment in MDCK and MDCK-PA cells Virus Hemagglutinating activity in supernatants Virus titer MDCK MDCK-PA TCID ₅₀ / ml 5 P1 P2 P3 P4 P1 P2 P3 P4 P1 P4 rPR8-7ntc ¹ - - - - - - - - - - rPR8-7 - - - - + + + + 5.610 ¹ <10 ¹ rPR8-deltaPA - - - - + + + + 3.110 ⁵ 3.1 · 10 ⁴ rPR8-DIPA - - - - + + + + 3.1 · 10 ⁴ 3.1 · 10 ⁴ rPR180-wt + + + + + + + + 1,0 · 10 ⁷ but. ntc: not trans-complemented (pHMG-PA was not transfected into 293T cells)

table 2
Replication of recombinant influenza viruses A / PR / 8/34 with the absence of the original PB2 gene segment in MDCK and MDCK-PB2 cells Virus Hemagglutinating activity in supernatants MDCK MDCK-PB2 p1 p2 p1 p2 rPR8-7ntc - - - - rPR8-7 - - + + rPR8-pΔPB2 - - + + rPR8 + + + + ntc: not trans-complemented (pHMG-PB2 was not transfected into 293T cells)

Table 3
Replication of recombinant influenza A / PR / 8/34 viruses with the absence of the original PB1 gene segment in MDCK and MDCK-PB1 cells Virus Hemagglutinating activity in supernatants MDCK MDCK-PB1 p1 p2 p1 p2 rPR8-7ntc - - - - rPR8-7 - - + + rPR8-pΔPB1 - - + + rPR8 + + + + ntc: not trans-complemented (pHMG-PB1 was not transfected into 293T cells)

Table 4
Replication of recombinant influenza viruses A / PR / 8/34 with the absence of the original PA gene segment in MDCK-PA and MDCK cells Virus containing Virus replication in MDCK-PA MDCK pΔPA + - pΔPA-2 + - pΔPA-3 + - pΔPA-4 + - pΔPA-5 + -

Example 2

Defective recombinant virus vaccination

A conditionally defective recombinant virus with no functional gene PA, PB1 or PB2 was obtained, as described herein, on the basis of a high-performance virus base (for example, obtained from vaccine strain A / PR / 8/34) with the HA and NA genes of a relevant epidemic virus (for example , A / Moscow / 10/99). A conditionally defective virus was obtained by transfection, while the expression of the polymerase protein was achieved by trans complementation. The virus was then amplified in a suitable cell substrate, such as MDCK cells or Vero cells stably expressing the relevant polymerase. The virus supernatant was purified by centrifugation for 10 min at 1000g. The virus was concentrated and purified by ultracentrifugation in a 20-60% sucrose gradient, precipitated and resuspended in phosphate-buffered saline (PBS). The purity and quantity of the virus preparation was confirmed using polyacrylamide gels with 12.5% SDS stained with Coomassie brilliant blue, and the virus titer of the conditionally defective virus was determined by infection of MDCK cells and MDCK cells expressing the corresponding polymerase and staining with monoclonal antibody to nucleoprotein. Mice were intratracheally or intranasally using a nebulizer inoculated with a 1 × 10 ⁵ 50% tissue culture infectious dose (TCID-50). Antibody titers for HA, NA, and internal influenza virus proteins in serum samples collected before and after vaccination are determined using hemagglutination inhibition assays, neuraminidase inhibition assays, ELISA or virus neutralization assays. The antigen-specific cellular immune response in vaccinated animals is quantified by measuring intracellular cytokine expression by flow cytometry, tetrameric staining of CD4 and CD8 positive cells, cytolytic activity, T-cell proliferation, etc. Vaccinated and control animals were stimulated 6 weeks after vaccination using 1 × 10 ⁶ TCID-50 influenza virus A / Moscow / 10/99 or a heterologous viral isolate. After stimulation in animals, nasal and pharyngeal swab samples were collected daily for 10 days, and the amount of virus secreted by infected animals was determined by quantitative PCR analysis or titration of the virus. Vaccine-induced acquired humoral immunity was determined by quantifying the increase in antibody titers, vaccine-induced acquired cellular immunity by increasing the responses of helper and cytotoxic T cells, and the overall level of immunity by confirming protection against infection during virus stimulation.

LINKS

1. Bancroft, S. T., and T. G. Parslow. 2002. Evidence for segment-nonspecific packaging of the influenza a virus genome. J Virol 76: 7133-9.

2. Basler, CF, X. Wang, E. Muhlberger, V. Volchkov, J. Paragas, HD Klenk, A. Garcia-Sastre, and P. Palese. 2000. The Ebola virus VP35 protein functions as a type IIFN antagonist. Proc Natl Acad Sci USA 97: 12289-94.

3. Duhaut, SD, and JW McCauley. 1996. Defective RNAs inhibit the assembly of influenza virus genome segments in a segment-specific manner. Virology 216: 326-37.

4. Enami, M., G. Sharma, C. Benham, and P. Palese. 1991. An influenza virus containing nine different RNA segments. Virology 185: 291-8.

5. Fujii, Y., H. Goto, T. Watanabe, T. Yoshida, and Y. Kawaoka. 2003. Selective incorporation of influenza virus RNA segments into virions. Proc Natl Acad Sci USA 100: 2002-2007.

6. Hirst, GK, and MW Pons. 1973. Mechanism of influenza recombination. II. Virus aggregation and its effect on plaque formation by so-called noninfective virus. Virology 56: 620-31.

7. Hoffmann, E., G. Neumann, Y. Kawaoka, G. Hobom, and RG Webster. 2000. A DNA transfection system for generation of influenza A virus from eight plasmids. Proc Natl Acad Sci USA 97: 6108-13.

8. Martin, K., and A. Helenius. 1991. Nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (M1) promotes export and inhibits import. Cell 67: 117-30.

9. Neumann, G., T. Watanabe, and Y. Kawaoka. 2000. Plasmid-driven formation of influenza virus-like particles. J Virol 74: 547-51.

10. Smith, GL, and AJ Hay. 1982. Replication of the influenza virus genome. Virology 118: 96-108.

11. Watanabe, T., S. Watanabe, T. Noda, Y. Fujii, and Y. Kawaoka. 2003 . Exploitation of Nucleic Acid Packaging Signals To Generate a Novel Influenza Virus-Based Vector Stably Expressing Two Foreign Genes. J Virol 77: 10575-10583.

Claims (49)

1. A method for producing a conditionally defective particle of an influenza virus, comprising the step of transfecting a suitable cell or cells with one or more gene constructs obtained by internal deletion of a nucleic acid encoding influenza polymerase, the gene constructs being unable to produce functional polymerase, but do not interfere with the packaging of the gene segments of the virus in viral particles
and complementary influenza virus nucleic acid segments encoding an influenza virus,
and one or more expression plasmids capable of expressing said polymerases in said cell,
and collecting at least one conditionally defective influenza virus particle from the supernatant of the indicated cell or cells at a suitable point in time after infection. 2. The method according to claim 1, where the internal deletion is the result of an internal deletion of a nucleic acid encoding an influenza polymerase:
starting with a 5'-nucleotide located between, but not including, nucleotides 58 and 207, counting from the non-coding region, and ending with a 3'-nucleotide, located between, but not including them, nucleotides 27 and 194, counting from the non-coding region RA protein starting with a 5'-nucleotide located between, but not including, nucleotides 43 and 246, counting from the non-coding region, and ending with a 3'-nucleotide, located between, but not including, nucleotides 24 and 197, counting from the non-coding region of the PB 1 protein starting with the 5'-nucleotide, located ennogo between, but not including, 34 and 234 nucleotides counting from the non-coding region, and terminating at the 3'-nucleotide situated between, but not including, 27 and 209 nucleotides counting from the noncoding region of the PB2 protein. 3. The method according to claim 1 or 2, where the specified polymerase is an acidic polymerase (RA). 4. The method according to claim 1 or 2, where the specified polymerase is a basic polymerase 1 (PB1). 5. The method according to claim 1 or 2, where the specified polymerase is a basic polymerase 2 (PB2). 6. A method of obtaining a conditionally defective particle of an influenza virus, comprising
the first stage of transfection of a suitable cell or cells with one or more expression plasmids capable of expressing influenza polymerases in said cell;
and a second stage of infection of said cell or cells with a supernatant containing conditionally defective influenza virus particles, as defined in claim 1, incapable of producing functional influenza polymerase;
and a third step comprising collecting at least one conditionally defective influenza virus particle from the supernatant of said cell or cells at a suitable point in time after infection. 7. The method according to claim 6, where a cell or cells that infect a supernatant containing conditionally defective particles of the influenza virus already express non-functional polymerases. 8. The method according to any one of claims 6 or 7, wherein said polymerase is an acidic polymerase (RA). 9. The method according to any one of claims 6 or 7, wherein said polymerase is basic polymerase 1 (PB1). 10. The method according to any one of claims 6 or 7, wherein said polymerase is basic polymerase 2 (PB2). 11. A method for producing a conditionally defective influenza virus particle, comprising
the first stage of transfection of a suitable first cell or cells with one or more gene constructs obtained by internal deletion of a nucleic acid encoding an influenza polymerase, while these gene constructs are unable to produce functional polymerase, but do not interfere with the packaging of virus gene segments into viral particles,
and complementary influenza virus nucleic acid segments encoding an influenza virus,
and one or more expression plasmids capable of expressing said polymerases in said cell,
and collecting at least one conditionally defective influenza virus particle from the supernatant of said first cell or cells at a suitable point in time after transfection;
and a second step for transfecting a suitable second cell or cells with one or more expression plasmids capable of expressing said polymerases in said cell;
and a third stage of infection of said second cell or cells with a supernatant containing at least one conditionally defective influenza virus particle obtained from said first cell;
and a fourth step comprising collecting at least one conditionally defective influenza virus particle from the supernatant of said second cell or cells at a suitable point in time after infection. 12. The method according to claim 11, where the specified polymerase is an acidic polymerase (RA). 13. The method according to claim 11, where the specified polymerase is a basic polymerase 1 (PB1). 14. The method according to claim 11, where the specified polymerase is a basic polymerase 2 (PB2). 15. The method according to claim 11, where the internal deletion is the result of an internal deletion of a nucleic acid encoding an influenza polymerase:
starting with a 5'-nucleotide located between, but not including, nucleotides 58 and 207, counting from the non-coding region, and ending with a 3'-nucleotide, located between, but not including them, nucleotides 27 and 194, counting from the non-coding region RA protein starting with a 5'-nucleotide located between, but not including, nucleotides 43 and 246, counting from the non-coding region, and ending with a 3'-nucleotide, located between, but not including, nucleotides 24 and 197, counting from the non-coding region of the PB1 protein starting with the 5'-nucleotide, located between, but not including, nucleotides 34 and 234, counting from the non-coding region, and ending on a 3'-nucleotide located between, but not including, nucleotides 27 and 209, counting from the non-coding region of the PB2 protein. 16. The method according to clause 15, where the cell or cells that are infected with a supernatant containing conditionally defective particles of the influenza virus, already express non-functional polymerases. 17. The method according to clause 15 or 16, where the specified polymerase is an acidic polymerase (RA). 18. The method according to clause 15 or 16, where the specified polymerase is a basic polymerase 1 (PB1). 19. The method according to clause 15 or 16, where the specified polymerase is a basic polymerase 2 (PB2). 20. The conditionally defective particle of the influenza virus obtained by the method according to any one of claims 1 to 19, for the induction of immune defense against the influenza virus. 21. The particle of claim 20 with influenza virus nucleic acid segments encoding viral glycoproteins. 22. The particle of claim 20 with influenza virus nucleic acid segments encoding nucleoprotein (NP), basic polymerase 1 (PB1), basic polymerase 2 (PB2), hemagglutinin (HA), neuraminidase (NA), matrix proteins (M1 and M2 ) and non-structural protein (NS1 and NS2). 23. The particle of claim 20 with influenza virus nucleic acid segments encoding nucleoprotein (NP), acid polymerase (RA), basic polymerase 2 (PB2), hemagglutinin (HA), neuraminidase (NA), matrix proteins (M1 and M2) and non-structural protein (NS1 and NS2). 24. The particle of claim 20 with influenza virus nucleic acid segments encoding nucleoprotein (NP), acid polymerase (RA), basic polymerase 1 (PB1), hemagglutinin (HA), neuraminidase (NA), matrix proteins (M1 and M2) and non-structural protein (NS1 and NS2). 25. The particle of claim 20 with influenza virus nucleic acid segments derived from influenza A. 26. A conditionally defective particle of the influenza virus containing one or more nucleic acid segments with an internal deletion in the segment that makes the segment incapable of producing functional influenza polymerase but does not prevent the packaging of the virus gene segments into viral particles, while the polymerase is selected from the group of acid polymerase (RA ), basic polymerase 1 (PB1) or basic polymerase 2 (PB2), for the induction of immune defense against influenza virus. 27. The particle of claim 26 with influenza virus nucleic acid segments encoding for viral glycoproteins. 28. The particle of claim 26 with influenza virus nucleic acid segments encoding nucleoprotein (NP), basic polymerase 1 (PB1), basic polymerase 2 (PB2), hemagglutinin (HA), neuraminidase (NA), matrix proteins (M1 and M2 ) and non-structural protein (NS1 and NS2). 29. The particle of claim 26 with influenza virus nucleic acid segments encoding nucleoprotein (NP), acid polymerase (RA), basic polymerase 2 (PB2), hemagglutinin (HA), neuraminidase (NA), matrix proteins (M1 and M2) and non-structural protein (NS1 and NS2). 30. The particle of claim 26 with influenza virus nucleic acid segments encoding nucleoprotein (NP), acid polymerase (RA), basic polymerase 1 (PB1), hemagglutinin (HA), neuraminidase (NA), matrix proteins (M1 and M2) and non-structural protein (NS1 and NS2). 31. The particle of claim 26 with influenza virus nucleic acid segments derived from influenza A. 32. A conditionally defective particle of the influenza virus containing one or more nucleic acid segments with an internal deletion in the segment that makes the segment incapable of producing functional influenza polymerase but does not prevent the packaging of the virus gene segments into viral particles, while the polymerase is selected from the group of acid polymerase (RA ), basic polymerase 1 (PB1) or basic polymerase 2 (PB2), and internal deletion:
starts with a 5'-nucleotide located between, but not including them, nucleotides 58 and 207, counting from the non-coding region, and ends with a 3'-nucleotide, located between, but not including them, nucleotides 27 and 194, counting from the non-coding region RA protein
starts with a 5'-nucleotide located between, but not including them, nucleotides 43 and 246, counting from the non-coding region, and ends with a 3'-nucleotide, located between, but not including them, nucleotides 24 and 197, counting from the non-coding region PB1 protein,
starts with a 5'-nucleotide located between, but not including them, nucleotides 34 and 234, counting from the non-coding region, and ends with a 3'-nucleotide, located between, but not including them, nucleotides 27 and 209, counting from the non-coding region PB2 protein, for the induction of immune defense against influenza virus. 33. The particle of claim 32 with influenza virus nucleic acid segments encoding for viral glycoproteins. 34. The particle of claim 32 with influenza virus nucleic acid segments encoding nucleoprotein (NP), basic polymerase 1 (PB1), basic polymerase 2 (PB2), hemagglutinin (HA), neuraminidase (NA), matrix proteins (M1 and M2 ) and non-structural protein (NS1 and NS2). 35. The particle of claim 32 with influenza virus nucleic acid segments encoding nucleoprotein (NP), acid polymerase (RA), basic polymerase 2 (PB2), hemagglutinin (HA), neuraminidase (NA), matrix proteins (M1 and M2) and non-structural protein (NS1 and NS2). 36. The particle of claim 32 with influenza virus nucleic acid segments encoding nucleoprotein (NP), acid polymerase (RA), basic polymerase 1 (PB1), hemagglutinin (HA), neuraminidase (NA), matrix proteins (M1 and M2) and non-structural protein (NS1 and NS2). 37. The particle of claim 32 with influenza virus nucleic acid segments derived from influenza A. 38. A conditionally defective particle of an influenza virus containing a nucleic acid that does not encode an influenza peptide to deliver a nucleic acid that does not encode an influenza peptide to a cell selected from the group consisting of:
particles obtained by the method according to any one of claims 1 to 19;
particles containing one or more nucleic acid segments with an internal deletion in a segment that makes the segment incapable of producing functional influenza polymerase, but does not prevent the packaging of virus gene segments into viral particles, while the polymerase is selected from the group of acid polymerase (RA), basic polymerase 1 ( PB1) or basic polymerase 2 (PB2);
particles containing one or more nucleic acid segments with an internal deletion in the segment, which makes the segment incapable of producing functional influenza polymerase, but does not prevent the packaging of the virus gene segments into viral particles, while the polymerase is selected from the group of acid polymerase (RA), basic polymerase 1 ( PB1) or basic polymerase 2 (PB2), and internal deletion:
starts with a 5'-nucleotide located between, but not including them, nucleotides 58 and 207, counting from the non-coding region, and ends with a 3'-nucleotide, located between, but not including them, nucleotides 27 and 194, counting from the non-coding region the RA protein begins with a 5'-nucleotide located between, but not including, nucleotides 43 and 246, counting from the non-coding region, and ends with a 3'-nucleotide, located between, but not including, nucleotides 24 and 197, counting from the non-coding region of the PB1 protein, begins with a 5'-nucleotide located between y, but not including, 34 and 234 nucleotides counting from the noncoding region, and ends at the 3'-nucleotide situated between, but not including, 27 and 209 nucleotides counting from the noncoding region of the PB2 protein. 39. A cell containing a particle according to any one of claims 20-37, producing said particles. 40. The cell of claim 39, comprising one or more influenza virus polymerases, wherein the polymerase is selected from the group of acidic polymerase (RA), basic polymerase 1 (PB1) or basic polymerase 2 (PB2). 41. A cell containing a particle according to claim 38, producing these particles. 42. A cell according to claim 41, containing one or more influenza virus polymerases, wherein the polymerase is selected from the group of acidic polymerase (RA), basic polymerase 1 (PB1) or basic polymerase 2 (PB2). 43. A pharmaceutical composition intended to induce an immune defense against an infection caused by an influenza virus in an individual containing a particle according to any one of claims 20 to 37, or a cell or a product obtained from a cell according to any one of claims 39 or 40. 44. The use of the composition according to item 43 to obtain a pharmaceutical composition intended for the induction of immune defense against infection caused by influenza virus in an individual. 45. A method of inducing an immune defense against an infection caused by an influenza virus in an individual, comprising administering to the individual an effective amount of a composition according to claim 43. 46. The use of a particle according to claim 38 to obtain a composition intended for delivery of a nucleic acid that does not encode an influenza peptide into a cell. 47. The use of a particle according to claim 38 for the preparation of a pharmaceutical composition for the delivery of a nucleic acid that does not encode an influenza peptide into cells of an individual. 48. A method for delivering a nucleic acid that does not encode an influenza peptide into a cell, comprising introducing into the indicated cell a defective influenza virus particle according to claim 38. 49. A method for delivering a nucleic acid that does not encode an influenza peptide to an individual, comprising administering to said individual a defective influenza virus particle according to claim 38.

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