CN102586199A - Defective influenza virus particles - Google Patents

Abstract

The present invention relates to the field of influenza virus and vaccination against influenza. The present invention provides conditionally deficient influenza virions having 7 different influenza nucleic acid segments. The invention also provides a conditionally deficient influenza virion lacking an influenza nucleic acid segment selected from the group consisting of a segment substantially encoding acid polymerase (PA), alkaline polymerase 1 (PB1 ), and alkaline polymerase 2 (PB2). More specifically, the present invention provides defective influenza virions having 7 distinct influenza nucleic acid segments and lacking an influenza nucleic acid segment substantially encoding an acid polymerase. In addition, the invention provides the use of a composition comprising a defective influenza virus particle according to the invention for the manufacture of a pharmaceutical composition intended to generate immune protection of a subject against influenza virus infection, and for the production of immune protection in a subject A method of immunoprotection against influenza virus infection comprising providing to a subject in need thereof a composition comprising such defective influenza virus particles.

Description

defective influenza virus particles

The present invention relates to the field of influenza viruses and vaccination against influenza.

Influenza viruses (Orthomyxoviridae) are enveloped, negative-strand RNA viruses with a segmented genome (Taubenberger and Layne, Molecular Diagnosis Vol. 6 No. 42001). Based on the significant antigenic differences between their nucleoproteins and matrix proteins, they can be divided into 2 classes: one class includes influenza A and B, and the other class includes influenza C. There were also differences in pathogenicity and genome organization among the 3 virus types. Type A was found to be widespread in warm-blooded animals, but types B and C were mainly human pathogens. Influenza A viruses are further subdivided by antigenic characterization of the hemagglutinin (HA) and NA surface glycoproteins protruding from the virion surface. Currently, there are 15 HA and 9 NA subtypes. Influenza A viruses can infect a wide variety of animals, including birds, pigs, horses, humans and other mammals. Waterfowl are the natural reservoirs of all known subtypes of influenza A and may be the source of genetic material for circulating influenza strains in humans.

Unlike the related paramyxoviruses, influenza viruses have a segmented RNA genome. Influenza A and B viruses have a similar structure, while influenza C is more discrete. Both type A and B viruses contain 8 discrete gene segments each encoding at least one protein, and type C contains 7 discrete segments that combine segments 4 and 6 of types A and B. Influenza A and B viruses are covered with overhangs of 3 proteins: HA, NA, and matrix 2 (M2). Influenza C viruses have only one surface glycoprotein. Each influenza RNA segment is encapsulated by nucleoprotein (NP) to form a ribonucleotide nucleoprotein (RNP) complex. These three polymerase proteins are attached to one end of the RNP complex. The RNP is surrounded by a membrane that contains the matrix protein (matrix 1 ) as a constituent. The phospholipid portion of the envelope is derived from the host cell membrane. Within the virus particle, nonstructural protein 2 (NS2) is also found.

The World Health Organization (WHO) rules for influenza virus nomenclature are as follows. First, specify the type of virus (A, B, or C), then the host (if non-human), place of isolation, number of isolates, and year of isolation (separated by a slash). For influenza A, the HA and NA subtypes are indicated in parentheses. For example, the strains included in the recent 2000-2001 season trivalent vaccine are: A/Panama/2007/99 (H3N2), A/New Caledonia/20/99 (H1N1), and B/Yamanashi /16/98. Since 1977, 2 influenza A subtypes have coexisted in humans: H1N1 and H3N2.

Influenza viruses are able to accumulate point mutations during replication because their RNA polymerase complexes are not proofreading active. Mutations that alter amino acids in the antigenic portion of surface glycoproteins can provide a selective advantage to virus strains by evading pre-existing immunity. By binding to receptors on certain host cells, HA molecules can initiate infection. Antibodies against the HA protein prevent receptor binding and are very effective in preventing reinfection with the same strain. HA can evade previously acquired immunity through antigenic drift (in which mutations in currently circulating HA genes disrupt antibody binding) or antigenic shift (in which viruses acquire new subtypes of HA). Antigenic drift pressure was unequal among HA molecules, and positive selection changes mainly occurred in the spherical head of HA protein. These changes also accumulated to a greater extent in HA than in NA. Changes occurred more slowly in other influenza proteins. Likewise, antigenic drift pressure was greatest in human-adapted influenza strains, moderate in pig- and horse-adapted strains, and minimal in avian-adapted strains.

Because influenza viruses have segmented genomes, co-infection of 2 different strains in the same host can result in the generation of new recombined influenza strains that contain different combinations of parental gene segments. Fifteen HA subtypes are known to exist in wild birds and provide a source of HA that is new to humans. The emergence in human circulation of influenza strains containing novel subtypes (via antigenic shift) has been the cause of the last 2 influenza pandemics in 1957 and 1968, and most likely the 1918 influenza pandemic. To be consistent with what is known about the emergence of pandemic influenza viruses, the circulating strain must have an HA that is antigenically different from currently circulating; the HA must not have been circulating in humans for 60 to 70 years; and the virus must be spread among humans. In 1957 and 1968, shifts in HA resulted in pandemics, and in both cases the HA of the circulating strains was closely related to the avian strains. Although an absolute condition for a pandemic is that HA must change, it is unclear to what extent other parts of the virus can or must change. Only the 1957 and 1968 pandemic viruses were available for direct study, using molecular archaeology, to characterize the 1918 pandemic influenza virus. In 1957, 3 genes were replaced by avian genes: HA, NA, and a subunit of the polymerase complex (PB1). In 1968, only HA and PB1 were replaced.

Influenza infection can be specifically diagnosed by virus isolation, hemagglutination inhibition (HI) assays, immunoassays to detect antigens, serological assays, verification of NA activity in secretions, or molecular-based assays. Specimens can be collected as saliva, nasopharyngeal swabs, or nasopharyngeal washes obtained by gargling with buffered saline solution. The standard for influenza diagnosis is immunological characterization after culture. Serological analysis provides an accurate, but traceable method of influenza infection because it requires the collection of acute and convalescent sera.

Influenza viruses can be grown in embryonated eggs or in many tissue culture systems. The addition of trypsin (for cleavage activation of HA) allowed influenza virus propagation in Madin-Darby canine kidney (MDCK) cells and other lines. The main method of vaccine production is still to grow influenza virus in eggs. Cultures of cell lines are commonly used for primary isolation of human influenza viruses (types A and B). Many human influenza viruses can be cultured directly in the allantoic cavity of embryonated eggs. Some influenza A and B viruses require first culture in the amniotic cavity and then translocation into the allantoic cavity. Following culture isolation, most influenza isolates were positively identified using immunoassays or immunofluorescence. The HA molecule of influenza virus can bind to sialic acid residues on the surface of respiratory cells to facilitate virus entry.

The ability of influenza viruses to agglutinate erythrocytes in vitro allows influenza strains to be characterized antigenically. Anti-HA antibody can inhibit agglutination. Thus, the hemagglutination inhibition (HI) assay is one of the standard methods used to characterize influenza strains. The HI assay can be used to determine whether a sample strain is immunologically related (ie, cross-reactive) to a recent vaccine strain. Typed serum, typically produced in ferrets, is added to the wells in a series of 2-fold dilutions, and the experimental wells are scored by the experimenter by observing suspended and agglutinated red blood cells. In most cases, a panel of sera was used to match the sample and reference strains against the vaccine, and most sample strains were successfully matched by HI experiments over the course of any given influenza season.

Guidelines are provided by WHO and guidelines are provided by WHO Collaborating Centers regarding the antigenic characterization of individual virus strains. According to immunological pedigrees, such as A/Moscow/10/99(H3N2)-like, A/New Caledonia/20/99(H1N1)-like, and B/Beijing/184/93-like viruses, the Sample strains were classified. For sample strains that cannot be characterized in HI experiments, laboratory workers must inoculate them into ferrets to generate strain-specific antisera. When new antiserum was ready, the HI assay was performed as described again. If the new sera exhibited a significant difference in cross-reactivity (usually defined as a 4-fold difference between sample and vaccine strains), it was integrated into routine laboratory groups and used to search for new epidemic strains. Thus, the HI assay is very important in influenza virus surveillance for vaccine strain selection and is the most commonly used method for estimating antigenic drift.

Influenza strains can be genetically characterized by sequence alignment of individual gene segments, and WHO Guidelines and WHO Collaborating Centers provide guidance on identifying individual properties that contain: RNA segments comprising the influenza genome; encoding nucleoprotein (NP) , alkaline polymerase 1 (PB1), alkaline polymerase 2 (PB2), acid polymerase (PA), hemagglutinin (HA), neuraminidase (NA), matrix proteins (M1 and M2) and non- Influenza A and B virus nucleic acid fragments of structural proteins (NS1 and NS2), and encoding nucleoprotein (NP), basic polymerase 1 (PB1), basic polymerase 2 (PB2), hemagglutinin-neuraminic acid Influenza C virus nucleic acid fragments of the enzyme-like glycoprotein (HN), matrix proteins (M1 and M2), and nonstructural proteins (NS1 and NS2). Requests for antigenic analysis, comparison of nucleic acid sequences and identification of reference strains of vaccine viruses may be addressed to the WHO Collaborating Center for Influenza Reference and Research, 45 Poplar Road, Parkville, Victoria 3052, Australia (fax: +61 3 9389 1881, web site: http://www.influenzacentre.org); WHO Collaborating Center for Influenza Reference and Research, 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 Influenza Surveillance, Epidemiology and Control, 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 Influenza Reference and Research, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, England (fax: +44 208 906 4477). The latest epidemiological information is available on the WHO website at http://www.who.int/influenza and the geographic information system, FluNet, at http://www.who.int/flunet.

Awareness of the impact of influenza and the health and economic benefits of preventing it is gradually increasing, the use and benefits of vaccination have been observed over the past decade and notably the emergence of many anti-influenza drugs. As a result of longer life expectancy in many countries, more people are at risk of complications, the burden on health care systems during influenza epidemics is more widely recognized, and more frequent international travel has contributed to the spread of the virus Opportunities have been created, although the emergence of new products has increased the options for preventing and treating diseases. About 50 countries have government-funded national influenza immunization programs, and the vaccine is available in many others. Specific recommendations regarding the use of the vaccine vary, but generally include annual immunization for elderly individuals and those older than 6 months of age who are at high risk of severe disease due to pre-existing chronic medical conditions. In some countries, vaccines are used to reduce the spread of influenza to high-risk groups. Within their overall public health priorities, Member States need to consider the benefits of influenza prevention activities.

Inactivated vaccines can be classified into several types depending on whether they contain whole virions, partially broken virions (split vaccines) or purified envelope antigens (subunit vaccines). Some subunit vaccines have been combined with adjuvants or delivery systems.

A few countries have licensed live attenuated influenza vaccines for certain target groups. In the Russian Federation, 2 different formulations of one vaccine have been administered to healthy adults and children, and another live vaccine has been extensively tested. However, until live attenuated vaccines become more widely available, they generally remain not recommended for influenza prevention.

For the prevention and treatment of influenza, 2 classes of antiviral drugs have been developed. The M2 inhibitors, amantadine and rimantadine, are limited to the treatment of influenza A virus, and they have been reported to be effective in preventing infection. Although both products cause some side effects, amantadine is more commonly associated with significant neurological side effects. Recently, neuraminidase inhibitors such as zanamivir and oseltamivir have been licensed in many countries for the treatment of influenza types A and B, and they have been reported to be effective for prevention as well. Resistance mutants have been detected in patients receiving class 2 antivirals. While it is not currently considered a significant public health concern, that could change if these drugs are used on a very large scale.

WHO maintains a global international surveillance program with 110 National Influenza Centers in 82 countries and four WHO Influenza Reference and Research Collaborations in Atlanta (USA), London (UK), Melbourne (Australia) and Tokyo (Japan) operated in cooperation with the Centre. These centers provide an early warning system for the emergence of strains with epidemic potential. This system is important because influenza vaccines are less effective if they do not contain currently circulating strains. WHO issues recommendations on vaccine composition, as can be seen in the Weekly Epidemiological Record published by the World Health Organization (see, for example, issue 9, 2004, 79, p. 88 or http://www.who.int/wer) Yes, the vaccine for use in the northern hemisphere was released in February, and the vaccine for use in the southern hemisphere was released in September. Because influenza has less defined seasonal characteristics in equatorial regions, epidemiological considerations can influence which of these recommendations (February or September) are appropriate for vaccines used in equatorial countries.

The collaborating centers performed antigenic and genetic analysis on the influenza isolates submitted by the national centres. When evidence of an antigenic change is observed, it is compared to epidemiological data to estimate the epidemiological significance of the variant. Using human serogroups collected before and after vaccination, representative isolates were compared with current vaccine strains to determine whether current vaccines could be expected to protect against these viruses. Following publication of WHO's annual vaccine recommendations, high-growth strains were developed and provided to manufacturers as reference viruses to aid in the generation of seed viruses for vaccine production. Tests for the safety and efficacy of influenza vaccines, including virus inactivation, microbiological sterilization, measurement of chemical agents used to destroy the virus, and confirmation of recommended antigen concentrations. Recommended vaccines can meet WHO requirements, however, national control agencies should approve specific vaccine viruses for use in each country. National public health agencies are responsible for recommending the use of vaccines. In addition, WHO has published recommendations on influenza prevention (see WER No. 35, 2002, pp. 281-288).

It has been demonstrated that current influenza vaccines do not protect naive individuals, a fact that becomes very important when many individuals who have never encountered influenza infection are at risk and an influenza pandemic breaks out. Viruses generally begin their life cycle by binding to host cell surface receptors, entering the cell, uncoating their viral nucleic acid, and subsequently replicating the viral genome. After new copies of viral proteins and genes are synthesized, these components are packaged into progeny virions, which then exit the cell. During the assembly step, the progeny virus must efficiently select its genomic nucleic acid from the multitude of viral and cellular nucleic acids present in the cytoplasm. Packaging of the viral genome into virions typically includes viral components recognized by cis-acting sequences in the viral nucleic acid, the so-called "packaging signal". Defining such signals is important for understanding the viral life cycle and provides us with information that can be used to construct viral vectors that express foreign proteins. Indeed, the use of retroviruses as vehicles for gene delivery vehicles expressing foreign proteins can be attributed in large part to a very precise understanding of the process by which their vRNA is packaged into progeny virions.

Poor understanding of the genome packaging signals of other RNA viruses hinders their application as vectors for the expression and delivery of foreign genes. For example, influenza A virus is an enveloped, negative-strand RNA virus whose segmented genome has nucleoprotein (NP), basic polymerase 1 (PB1), basic polymerase 2 (PB2), acid polymerase ( PA), hemagglutinin (HA), neuraminidase (NA), matrix proteins (M1 and M2) and nonstructural proteins (NS1 and NS2).

The virus has two transmembrane glycoproteins on the envelope, hemagglutinin (HA) and neuraminidase (NA). The HA protein binds to sialic acid-containing receptors on the surface of host cells and, following receptor-mediated endocytosis, mediates the fusion of the viral envelope with the endosomal membrane. Conversely, the NA protein provides a mechanism that plays a key role late in infection by removing sialic acid from sialooligosaccharides, thereby releasing newly assembled virions from the cell surface and preventing virion self-aggregation. Within the envelope, the viral genome, comprising eight distinct viral RNA (vRNA) segments, is tightly linked to nucleoprotein (NP) and polymerase proteins (PA, PB1, and PB2), forming a ribonucleoprotein complex. In order to generate an infectious virus, all 8 (or in the case of type C viruses: all 7) functional gene segments are required. Multiple mutations in the polymerase gene have been described (WO2004/094466, WO2003/091401, US 5578473, Fodor et al., J. Virol. 77, 5017-5020, 2003), which alter the polymerase activity, or in another Altering the polymerase in a way that does not render it less functional in synthesizing viral RNA so that viruses containing such a mutated polymerase cannot replicate. In WO2004/094466, infectious viruses with mutated PA genes were generated, thereby demonstrating the benefit of a selection system allowing the production and recovery of infectious viruses with mutated genes. In WO2003/091401 it was demonstrated how to produce infectious virus with mutations in the polymerase gene in order to produce and recover live attenuated vaccine virus with desirable properties (e.g. temperature sensitivity or other types of attenuation) influenza virus. In US55788473, polymerase gene fragments that can change the specificity and reduce the activity of various polymerases are proposed. However, they have not been used to reconstitute a virus, let alone a virus that has completely lost its polymerase activity. Furthermore, in none of the above mentioned applications were defective particles produced which had lost their ability to replicate. It is well known that when influenza A viruses are passaged at high multiplicity of infection, defective virions are produced that lack one or more functional gene segments. In such virions, one or more functional genes are replaced with defective interfering (DI) gene segments due to errors made by the influenza virus polymerase. Due to the high multiplicity of infection, and thus cells infected with more than one virus particle, the deficiency of viruses containing DI RNA is compensated by viruses containing intact copies of the lost functional gene. It was recently demonstrated that certain mutations in the acid polymerase gene increase the efficiency of production of virus particles with the defective gene (Fodor 2003). It is important to note that both generation and complementation of defective virus particles occurred randomly in these experiments. This stochastic process limits the practical use of DI RNA and conditionally deficient viral particles. Furthermore, when defective virus particles are produced using these disclosed methods, wild-type replication-competent virus is produced in addition to the desired conditional defective virus. Such replication-competent viruses may be completely wild-type (helper virus) or reassortants formed by genetic mixing of helper and defective viruses. The packaging of gene segments of influenza viruses, whether by stochastic or specific mechanisms, has been debated for many years. Some evidence for these 2 options has been described. Evidence for random packaging is that aggregated virions are more infective than unaggregated virions, and when cell cultures are infected at a low infectivity (moi), some infected cells fail to express a fragment, both of which Indicates the presence of virions that do not contain the complete influenza virus genome. Additional evidence for random packaging is that influenza viruses containing nine segments have been produced experimentally.

One argument in favor of a specific packaging process is that although all gene segments are present in equal amounts in the virus stock, they are present in varying amounts in the producer cells. In addition, when a defective interfering (DI) particle is generated, the DI vRNA replaces the segment it was derived from (a defective interfering particle is a virus particle in which a gene segment has a large internal deletion. These particles appear when the moi is passaged). Finally, the efficiency of virion formation increases with the number of gene segments.

Summary of the invention

Defective influenza virus particles (such as Mena I. et al., J.Virol.70:5016-24 (1996); Neumann G. et al., J.Virol.74:547-51 (2000).) can be used as Vaccine candidates, because they induce antibodies against other viral proteins besides HA and NA, if they can enter host cells, because they can induce a cellular immune response against the virus in addition to the humoral response (e.g. helper T cells, cytotoxic T cells). To date, defective influenza virus particles have been produced 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 producing large quantities of such particles. An alternative to this approach is to produce virions that are conditionally defective such that they replicate in certain production systems, but not in normal cells or production systems. To this end, the cells of the production system are modified to produce one or more influenza virus genes or gene products that complement defective influenza virus particles in trans. The present invention discloses for the first time the defined trans-complementation of defective influenza virions. Trans-complementation of influenza virions has been observed in the laboratory when viruses carrying a wild-type defective interfering gene segment complement the defective interfering influenza virus in the same cell. This "natural system" of trans complementation cannot be used to produce defined conditionally deficient influenza virions. First, the system requires that a (partially) defective virus be compensated by at least one (partially) replicable virus, which can lead to the unintentional generation of fully infectious viruses. Second, because different gene segments produce defective interfering particles in a random fashion, definitive conditionally defective virions cannot be produced.

Conditionally defective influenza virions could theoretically be based on the deletion of entire gene segments or parts thereof. If packaging of the influenza virus genome is dependent on the presence of all 8 segments (this is a version of many debates (see elsewhere in this specification)), by deleting the entire gene segment (and producing the encoded gene product in trans) The ability to produce virus particles with defined conditional defects is limited. If the packaging process requires the presence of all 8 gene segments, it is not known whether all gene segments are required to be present in full-length form, which further complicates the production of conditionally deficient virions. The present invention has solved these problems.

The invention provides a method for obtaining conditionally deficient influenza virus particles, comprising: a first step, transfecting a suitable first cell, such as a 293T cell, with a gene construct having an internal deletion, such as provided herein pΔPB2, pΔPB1, pΔPA or pDIPA derived by internal deletion of nucleic acid encoding influenza polymerase, thereby rendering said genetic construct incapable of producing a functional polymerase capable of copying or synthesizing viral RNA; and complementary influenza viruses encoding influenza viruses Nucleic acid fragments, such as seven complementary constructs capable of encoding A/WSN/33 (HW181-188, Hoffmann et al., 2000); and expression plasmids capable of expressing said polymerase in said cells, such as HMG provided herein - one of PB2, HMG-PB1, and HMG-PA; at a suitable time point after transfection, such as within 10-50 hours, preferably within about 20-30 hours, from the supernatant of the first cell Harvest at least one viral particle in the liquid; second step, transfect suitable second kind of cell, for example MDCK cell, with the expression plasmid that can express described polymerase in described cell; The supernatant of at least one virus particle obtained from the first cell is transfected into the second cell; the fourth step includes at a suitable time point after transfection, such as 24-96 hours, preferably 48-72 hours, Harvest at least one (now conditionally deficient because the resulting virus lacks a gene segment capable of expressing a functional polymerase for copying or synthesizing viral RNA from the supernatant of said first cell because they package a gene segment with an internal missing gene segments) virus particles.

An internal deletion that renders the gene segment incapable of producing functional protein is preferred, but it must not prevent packaging of the viral gene segment into viral particles. Preferably, these deletions are calculated from the 5' and 3' non-coding regions, respectively. For influenza A, for the PA protein, these preferred deletions start, for example, from the 5'-nucleotide located between nucleotides 58 to 75 (excluding endpoints) to between nucleotides 27 to 50 (excluding end point); for the PB1 protein, from the 5'-nucleotide located between nucleotides 43 to 75 (not including the end point) to the 5'-nucleotide located between nucleotides 24 to 50 ( End of 3'-nucleotide excluding endpoint); for PB2 protein, from 5'-nucleotide located between nucleotides 34 to 50 (excluding endpoint) to nucleotides located between nucleotides 27 to 50 3'-nucleotide ends between (excluding endpoints). More preferably, these deletions: for the PA protein, starting from the 5'-nucleotide located between nucleotides 58 to 100 (exclusive) to located between nucleotides 27 to 100 (exclusive) end of the 3'-nucleotide; for the PB1 protein, from the 5'-nucleotide located between nucleotides 43 to 100 (excluding endpoints) to the 5'-nucleotide located between nucleotides 24 to 100 (excluding end point); for the PB2 protein, from the 5'-nucleotide located between nucleotides 34 to 100 (excluding the end point) to the 5'-nucleotide located between nucleotides 27 to 100 ( 3'-nucleotide end without endpoint). Even more preferably, these deletions: for the PA protein, from the 5'-nucleotide located between nucleotides 58 to 150 (exclusive) to nucleotides located between nucleotides 27 to 150 (exclusive) ) at the end of the 3'-nucleotide; for the PB1 protein, from the 5'-nucleotide located between nucleotides 43 to 150 (excluding the endpoint) to the 5'-nucleotide located between nucleotides 24 to 150 (excluding end at the 3'-nucleotide inclusive); for the PB2 protein, from the 5'-nucleotide located between nucleotides 34 to 150 (exclusive) to between nucleotides 27 and 150 3'-nucleotide end of (excluding endpoint). More preferably, these deletions: for the PA protein, starting from the 5'-nucleotide located between nucleotides 58 to 175 (exclusive) to located between nucleotides 27 to 175 (exclusive) end of the 3'-nucleotide; for the PB1 protein, from the 5'-nucleotide located between nucleotides 43 to 175 (excluding endpoints) to the 5'-nucleotide located between nucleotides 24 to 175 (excluding end point); for the PB2 protein, from the 5'-nucleotide located between nucleotides 34 to 175 (excluding the end point) to the 5'-nucleotide located between nucleotides 27 to 175 ( 3'-nucleotide end without endpoint). Most preferably, these deletions: for the PA protein, from the 5'-nucleotide located between nucleotides 58 to 207 (exclusive) to nucleotides located between nucleotides 27 to 194 (exclusive) end of the 3'-nucleotide; for the PB1 protein, from the 5'-nucleotide located between nucleotides 43 to 246 (excluding the endpoint) to the 5'-nucleotide located between nucleotides 24 to 197 (excluding end) at the 3'-nucleotide end); for the PB2 protein, from the 5'-nucleotide located between nucleotides 34 to 234 (excluding the end point) to the 5'-nucleotide located between nucleotides 27 to 209 ( 3'-nucleotide end without endpoint).

Herein, a complementary segment is defined as a segment that results in a complete set of 8 gene segments (eg of influenza A virus). Thus, if fragment 1 has been used to produce a defective fragment, the complementary (non-defective) fragments are fragments 2, 3, 4, 5, 6, 7 and 8. If fragment 2 is defective, the complementary fragments are fragments 1, 3, 4, 5, 6, 7 and 8. So on and so forth.

Advantageously, the present invention provides a method whereby a helper virus is not required or present.

The present invention provides isolated and conditionally deficient influenza virus particles lacking the functionality encoding a polymerase selected from the group consisting of acid polymerase (PA), basic polymerase 1 (PB1) and basic polymerase 2 (PB2) Influenza nucleic acid fragments (also referred to herein as conditionally deficient influenza virions) that are incapable of producing or serving as a source of polymerase for copying or synthesizing viral RNA, whereby only and conditionally allows the generation of replicable virus particles in cells that are trans-compensated with a functional polymerase. In addition, the present invention provides methods for obtaining conditionally deficient influenza virus particles comprising providing cells by trans-compensation with a functional influenza virus polymerase.

In a preferred embodiment, a particle according to the invention is capable of replicating in a cell that is compensated for by a similar nucleic acid fragment that the particle itself lacks, for example a particle lacking a PA fragment of a functional influenza virus nucleic acid may at least have provided or compensated for the function Particles lacking the PB1 fragment of functional influenza virus nucleic acid can replicate in cells that have provided at least the PB1 fragment of functional influenza virus nucleic acid, and particles lacking the PB2 fragment of functional influenza virus nucleic acid can Replication occurs in cells that have provided at least the PB segment of functional influenza virus nucleic acid. In a preferred embodiment, the present invention provides a particle according to the present invention having an influenza virus nucleic acid segment encoding a viral glycoprotein, more preferably an influenza virus encoding nucleoprotein (NP), hemagglutinin (HA), Influenza virus nucleic acid fragments of neuraminidase (NA), matrix proteins (M1 and M2) and nonstructural proteins (NS1 and NS2). In one embodiment there is provided a particle according to the invention having an influenza virus nucleic acid segment derived from an influenza A virus. In addition, there is provided a particle according to the invention comprising a nucleic acid which does not encode an influenza peptide. In addition, the invention provides isolated cells comprising particles according to the invention, which cells do not contain wild-type influenza virus or helper virus, but have preferably been provided or complemented with influenza virus polymerase or the gene encoding it fragment. In a preferred embodiment, such cells are trans-compensated 293T or MDCK cells. In one embodiment, the invention provides isolated cells comprising particles lacking a functional influenza virus nucleic acid PA segment, which cells do not contain wild-type influenza virus or helper virus, but have at least been provided or compensated for a functional Influenza virus nucleic acid PA fragment or functional PA. In another embodiment, the invention provides isolated cells comprising particles lacking the PB1 fragment of functional influenza virus nucleic acid, said cells not containing wild-type influenza virus or helper virus, but having at least been provided with functional influenza virus Viral nucleic acid PB1 fragment or functional PB1. In another embodiment, the invention provides isolated cells comprising particles lacking a functional influenza virus nucleic acid PB2 fragment, said cells not containing wild-type influenza virus or helper virus, but at least provided or compensated for Functional influenza virus nucleic acid PB2 fragment or functional PB2. In addition, the invention provides compositions comprising particles or cells according to the invention or substances derived from cells according to the invention, and the use of such compositions for the manufacture of pharmaceutical compositions capable of Immunological protection against influenza virus infection in a subject is produced. Accordingly, the present invention provides a method of producing immune protection against influenza virus infection in a subject comprising providing a composition according to the invention to a subject in need thereof. In addition, the present invention provides the use of influenza virions according to the invention for the production of compositions for the delivery into cells of nucleic acids not encoding influenza peptides. In addition, the present invention provides the use of particles according to the invention for the manufacture of pharmaceutical compositions for the delivery of nucleic acids not encoding influenza peptides into cells of a subject, and for the delivery of nucleic acids not encoding influenza peptides A method for introducing into a cell or a subject, comprising providing said cell or subject with a particle according to the invention.

The present invention provides a conditionally deficient influenza virion that, when compared to its native genome, lacks a functional influenza segment that: 7 when compared to wild-type or helper type A or B virus 4 (instead of 8) different functional influenza virus nucleic acid segments, or 6 (instead of 7) different functional influenza virus nucleic acid segments when compared to wild-type or helper type C virus. When the term "conditionally defective" is used herein, it includes, but is not limited to, viral particles in which a viral gene segment has a large internal deletion which results in the expression of a non-functional protein from it. Production of infectious virus requires all eight gene segments (eg of influenza A virus) and all proteins they encode. Thus, a virus containing a defective gene segment is itself defective: it can infect cells and can go through a replication cycle because all viral proteins are present in the virion (e.g., when a virus is produced, the protein is produced by an expression plasmid ), but no infectious virus particles are produced in infected cells because the virus cannot produce a viral protein. However, when infected cells are capable of expressing proteins normally expressed by the defective gene segment, the defective virus can replicate in these cells because all viral proteins are present. Thus, these viruses are conditionally defective: they cannot replicate until cells are provided with the correct conditions (in which case the cells express viral proteins that are not encoded because the virus lacks a gene segment).

In addition, the present invention provides conditionally deficient influenza virus particles that lack a functional influenza virus nucleic acid segment encoding a polymerase. As used herein, functional influenza virus nucleic acid fragments comprise nucleic acids encoding functional influenza proteins that allow and are required for the production of replicable viruses. For example, influenza A virus is a negative-strand RNA virus with an 8-segment genome. 8 gene fragments can encode 11 kinds of proteins; gene fragments 1-8 can respectively encode alkaline polymerase 2 (PB2), alkaline polymerase 1 (PB1) and PB1-ORF2 (F2), acid polymerase (PA), Hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix proteins 1 and 2 (M1, M2) and nonstructural proteins 1 and 2 (NS1, NS2). The coding regions of the eight gene segments are flanked by non-coding regions (NCRs) necessary for viral RNA synthesis. The terminal 13 and 12 nucleotides at the 5' and 3'-ends of the viral genomic RNA, respectively, are conserved among all influenza A virus segments and are partially complementary to form a nucleotide that is recognized by the viral polymerase complex. secondary structure. The NCR can contain up to 60 other nucleotides that are not conserved among the 8 gene segments but are relatively conserved among different influenza viruses. Efficient viral genome packaging requires NCRs and flanking sequences in the coding region. Thus, functional influenza virus nucleic acid fragments consist of sequences (1 or 2 open reading frames/fragments) that have the potential to encode functional influenza proteins, allowing the generation of replicable viruses, mRNA, viral RNA (vRNA) and viral RNA The NCR required for complementary RNA (cRNA) transcription consists of packaging signals present in the NCR and flanking coding sequences.

Preferably, said conditionally deficient influenza virus particle lacking an influenza virus nucleic acid lacks a fragment encoding a functional polymerase PA, PB1 or PB2. Additionally, for vaccine purposes, the particles preferably have an influenza virus nucleic acid fragment encoding a viral glycoprotein.

In one embodiment, the invention provides an influenza A virion having 7 different influenza A nucleic acid segments. A defective influenza virus particle according to the invention is capable of replicating, albeit only once, in a suitable, uncompensated host animal or cell.

In suitably compensated cells, the particles according to the invention can replicate for multiple cycles. For vaccine and gene delivery purposes, the inability of defective particles to replicate indefinitely in normal, non-trans-compensated cells is a great advantage, thereby reducing the risk of vaccine virus spreading between hosts and reducing reversion to Danger of wild-type virus.

This is the first time that reverse genetics has been used to produce a defective influenza A virus that contains only seven functional gene segments and can undergo one cycle of replication, or when the defective gene segment is compensated in trans, can undergo Multiple cycles of replication. In one embodiment, the invention provides a conditionally deficient influenza virion lacking an influenza nucleic acid segment substantially encoding acid polymerase (PA). Similar to the trans complementation of PA, trans complementation of other influenza virus genes can be envisioned. However, since PA expression levels have been shown to be less critical than those of other influenza virus proteins, PA is the preferred gene segment for the deleted polymerase group, and viruses lacking PB2 and PB1 can also be produced, and viruses lacking NP cannot be transcompensated . In a preferred embodiment, the present invention provides conditionally deficient influenza A virions having 7 different influenza A nucleic acid segments and lacking an influenza A nucleic acid segment substantially encoding an acid polymerase. For vaccine purposes, preferred conditionally deficient influenza A virions according to the invention have influenza A nucleic acid fragments encoding essentially the hemagglutinin (HA) and neuraminidase (NA) proteins that are immunologically important for conferring protection. most relevant. In order to select a suitable gene segment to be included in the vaccine, the gene segment is preferably selected from viruses recommended by WHO for vaccine use. Of course, the HA and NA subtypes can vary depending on the HA and NA subtypes of the influenza variants to be vaccinated against. Most preferably, conditionally deficient influenza virions according to the invention are produced that essentially encode nucleoprotein (NP), alkaline polymerase 1 (PB1), alkaline polymerase 2 (PB2), hemagglutinin (HA ), neuraminidase (NA), matrix proteins (M1 and M2) and nonstructural proteins (NS1 and NS2) of influenza A nucleic acid fragments, "basic coding" herein specifically refers to the expression of functional proteins by the respective gene fragments . In particular, such particles are provided in isolated cells having functional PA or a functional gene segment encoding PA. In another embodiment, there is provided herein a conditionally deficient influenza virion according to the invention having essentially encoded nucleoprotein (NP), acid polymerase (PA), alkaline polymerase 2 (PB2), Influenza A nucleic acid fragments of hemagglutinin (HA), neuraminidase (NA), matrix proteins (M1 and M2) and nonstructural proteins (NS1 and NS2), "basic coding" refers specifically herein to functional proteins composed of The respective gene fragments are expressed. In particular, such particles are provided in isolated cells having functional PB1 or a functional gene segment encoding PB1. In another embodiment, there is provided herein a conditionally deficient influenza virion according to the present invention, which essentially encodes nucleoprotein (NP), acid polymerase (PA), alkaline polymerase 1 (PB1 ), Influenza A nucleic acid fragments of hemagglutinin (HA), neuraminidase (NA), matrix proteins (M1 and M2) and nonstructural proteins (NS1 and NS2), "basic coding" refers specifically herein to functional proteins composed of The respective gene fragments are expressed. In particular, such particles are provided in isolated cells having functional PB2 or a functional gene segment encoding PB2. In another embodiment, the invention provides a particle according to the invention which additionally contains a nucleic acid which does not encode an influenza peptide, for example, encodes a foreign protein or peptide for eliciting an immune response, or which contains a nucleic acid capable of interfering with a cell or pathogen in Functional nucleic acids in cells.

In addition, the invention provides cells comprising an influenza virus particle according to the invention. When the particle is not provided with a gene segment essentially encoding the required polymerase, it is useful to consider cells that have been provided with a suitable functional influenza polymerase, allowing defective influenza virus particles to replicate in such compensated cells multiple cycles.

In addition, the invention provides compositions comprising defective influenza virus particles or cells according to the invention or substances derived from cells according to the invention; such compositions can be used, for example, for the production of Pharmaceutical composition for immune protection of influenza virus infection. In addition, the present invention provides methods of producing immune protection against influenza virus infection in a subject comprising providing such compositions to a subject in need thereof. In addition to the use of the particles according to the invention as vaccines or immunological compositions, such compositions are preferably formulated as vaccines by mixing viral particles or viral proteins derived from such particles (split-vaccine) with the appropriate drug Carriers, such as saline solutions or adjuvants (such as commonly used aluminum salts or other excipients (see for example http://www.cdc.gov/nip/publications/pink/Appendices/A/Excipient.pdf.). According to this The conditionally deficient influenza virus particles of the invention are also alternative vectors for foreign gene delivery and expression of foreign proteins, since functional genes can be inserted, for example, between the 5' and 3' PA sequences. Considering that the present invention provides Method for conditionally deficient influenza virus particles that may contain foreign or host nucleic acid fragments or fragments thereof, comprising: in a first step, transfecting a suitable first cell with one or more genetic constructs derived from nucleic acids, thereby rendering said genetic constructs incapable of producing functional proteins and preventing the packaging of viral gene segments into viral particles; and complementary influenza virus nucleic acid segments encoding influenza viruses and one or more expression plasmids capable of expressing the protein in the cell; at a suitable time point after transfection, harvesting at least one viral particle from the supernatant of the first cell; the second step, transfecting suitable second cells with one or more expression plasmids capable of expressing said protein in said cells; The supernatant infects the second cell; the fourth step includes harvesting at least one virus particle from the supernatant of the second cell at a suitable time point after infection.Therefore, the present invention provides A method for obtaining conditionally deficient influenza virions comprising: transfecting suitable cells with one or more genetic constructs derived by internal deletion of a nucleic acid encoding influenza polymerase, thereby rendering said A genetic construct that does not produce a functional polymerase and does not prevent the packaging of a viral gene segment into a viral particle; and a complementary influenza virus nucleic acid segment that encodes an influenza virus; and one or that expresses said polymerase in said cell A plurality of expression plasmids; at a suitable time point after transfection, at least one virus particle is harvested from the supernatant of the cell. The method for obtaining conditionally defective influenza virus particles includes: the first step, using Transfecting suitable cells with one or more expression plasmids capable of expressing influenza polymerase in the cells; second step, infecting the cells with the supernatant containing conditionally deficient influenza virus particles; third step, Including harvesting at least one virus particle from the supernatant of the cells at a suitable time point after infection; or obtaining conditionally deficient influenza virus particles, which includes: a first step, transfecting a suitable The first cell: one or more genetic constructs derived by internal deletion of nucleic acid encoding influenza polymerase, thereby making said genetic construct The body cannot produce a functional polymerase and cannot prevent the packaging of the gene segment of the virus into the virus particle; and can encode a complementary influenza virus nucleic acid segment of the influenza virus; and can express one or more expression of the polymerase in the cell plasmid; at a suitable time point after transfection, at least one virus particle is harvested from the supernatant of the first cell; in the second step, one or more polymerases capable of expressing the polymerase in the cell are used An expression plasmid is transfected into a suitable second cell; the third step is to infect the second cell with a supernatant comprising at least one virus particle obtained from the first cell; the fourth step includes At a suitable time point after infection, at least one virus particle is harvested from the supernatant of said second cell. In this method, the polymerase may be, for example, acid polymerase (PA), alkaline polymerase 1 (PB1 ) or alkaline polymerase 2 (PB2). Preferably, the present invention provides a method for causing an internal deletion by internal deletion of a nucleic acid encoding influenza polymerase. For the PA protein, the internal deletion is located between nucleotides 58 to 207 from the non-coding region ( starting at the 5'-nucleotide excluding the endpoint) and ending at the 3'-nucleotide located between nucleotides 27 and 194 (excluding the endpoint) from the noncoding region; or for the PB1 protein, from Starting from the 5'-nucleotide located between nucleotides 43 to 246 (excluding endpoints) from the non-coding region to 5'-nucleotides located between nucleotides 24 to 197 (excluding Endpoint) at the end of the 3'-nucleotide; or for the PB2 protein, from the 5'-nucleotide located between nucleotides 34 to 234 (excluding the endpoint) from the non-coding region, to the 5'-nucleotide located between The 3'-nucleotide ends between nucleotides 27 and 209 (excluding endpoints) from the non-coding region. In another variant, a foreign segment is inserted into the internal deletion. In addition, the present invention provides methods for infecting cells with supernatants comprising conditionally deficient influenza virus particles, said cells already expressing non-functional polymerases, such as acid polymerase (PA), alkaline polymerase 1 (PB1) or alkaline polymerase 2 (PB2), and influenza particles obtainable by the methods described herein. For example, cells to be transfected with gene constructs and nucleic acid fragments already express a non-functional polymerase. More specifically, the invention provides influenza virions comprising one or more nucleic acid segments having internal deletions in the segment that render the segment incapable of producing a functional influenza polymerase and that do not prevent the packaging of the viral gene segments Into the virus particle, wherein the polymerase is selected from acid polymerase (PA), basic polymerase 1 (PB1) or basic polymerase 2 (PB2). Preferably, the internal deletion: for the PA protein, starting from the 5'-nucleotide located between nucleotides 58 to 207 (excluding the endpoint) from the non-coding region to the 5'-nucleotide located from the non-coding region 3'-nucleotide ends between nucleotides 27 and 194 (excluding endpoints); Beginning with the '-nucleotide and ending at the 3'-nucleotide located between nucleotides 24 and 197 (excluding endpoints) from the non-coding region; for the PB2 protein, starting from the non-coding region Starting from the 5'-nucleotide between nucleotides 34 to 234 (not including the endpoint) of the 3'-core located between nucleotides 27 to 209 (not including the endpoint) from the non-coding region nucleotide ends. In a preferred embodiment, the invention provides a particle according to the invention having an influenza virus nucleic acid segment encoding a viral glycoprotein. The present invention also provides particles according to the present invention, which have the ability to encode nucleoprotein (NP), basic polymerase 1 (PB1), basic polymerase 2 (PB2), hemagglutinin (HA), neuraminidase (NA), matrix proteins (M1 and M2) and nonstructural proteins (NS1 and NS2) of influenza virus nucleic acid fragments, or provided particles, which have the ability to encode nucleoprotein (NP), acid polymerase (PA), basic Influenza virus nucleic acid fragments of polymerase 2 (PB2), hemagglutinin (HA), neuraminidase (NA), matrix proteins (M1 and M2) and nonstructural proteins (NS1 and NS2), or provided particles, which It has the ability to encode nucleoprotein (NP), acid polymerase (PA), basic polymerase 1 (PB1), hemagglutinin (HA), neuraminidase (NA), matrix protein (M1 and M2) and nonstructural Influenza virus nucleic acid fragments of proteins (NS1 and NS2). More specifically, the invention provides a particle according to the invention having an influenza virus nucleic acid segment derived from an influenza A virus. The invention also provides a particle according to the invention comprising a nucleic acid that does not encode an influenza peptide. In addition, the invention provides cells comprising particles according to the invention, more particularly cells already containing one or more influenza virus polymerases, wherein the polymerases are selected from the group consisting of acid polymerase (PA), alkaline polymerase 1 ( PB1) or alkaline polymerase 2 (PB2). In addition, the invention provides compositions comprising particles or cells according to the invention or substances derived from cells according to the invention, such compositions being useful in the manufacture of a medicament intended to generate immune protection of a subject against influenza virus infection Use of the compositions, and methods of producing immune protection against influenza virus infection in a subject, comprises providing such compositions to a subject in need thereof. In addition, the present invention provides the use of the particles according to the invention in the production of compositions intended to deliver nucleic acids not encoding influenza peptides to cells, and the use of particles according to the invention in the production of nucleic acids not encoding influenza peptides. Application of the pharmaceutical composition to the cells of a subject. Such nucleic acid (also referred to herein as foreign nucleic acid) may encode a foreign gene or gene fragment that encodes a suitable antigenic epitope or protein, or may encode a stretch of nucleotides that interferes with the transcription of the nucleic acid in the cell . In one embodiment, the invention provides the use of an influenza A virosome according to the invention for the manufacture of a composition intended to deliver a nucleic acid not encoding an influenza peptide into a cell or into a cell of a subject. In addition, the present invention provides a method of delivering a nucleic acid that does not encode an influenza peptide into a cell or a subject, comprising providing said cell or said subject with a defective influenza virus particle containing a foreign nucleic acid according to the present invention.

Figure legend

Legend to Figure 1

Production and propagation of conditionally deficient influenza A virus. First, 293T cells were transfected with seven bidirectional plasmids encoding A/PR/8/34, pHMG-PA and pΔPA or pDIPA as appropriate. Forty-eight hours after transfection, supernatants of transfected cells were harvested and used to inoculate MDCK cells and MDCK cells transfected with HMG-PA 24 hours earlier. The supernatant of virus replication positive MDCK-PA cells was passaged 4 times in MDCK and MDCK-PA cells.

Legend to Figure 2

Constructs for the production of conditionally deficient virus particles. The upper panel shows the wild-type PA gene fragment. Noncoding regions (NCRs) and initiation codons are indicated. pΔPA was constructed by digesting pHW183, a bidirectional plasmid (9) containing the PA of A/WSN/33, with StuI and subsequent religation. pDIPA was constructed by cloning the 5'194 and 3'207 nts of the PA gene fragment of A/PR/8/34 into pSP72. The insert is then transferred to a bidirectional reverse genetics vector. pΔPB1 and pΔPB2 were constructed as described herein.

Legend to Figure 3

RT-PCR analysis of the presence of PA gene fragments in the supernatants rPR8-7, rPR8-ΔPA and rPR8-DIPA. MDCK-PA passage 4 supernatant was passed through a 22 μM filter and concentrated by centrifugation. Subsequently, RNA was isolated and RT-PCR was performed using primers targeting the non-coding region of the PA fragment. RNA isolated from wild-type A/PR/8/34 was used as a control. Lane 1: rPR8-7; Lane 2: rPR8-ΔPA; Lane 3 rPR8-DIPA; Lane 4: wild-type A/PR/8/34. Marker sizes are indicated on the left.

Legend to Figure 4

A larger portion of the pΔPA construct was then deleted, resulting in pΔPA-2, pΔPA-3, pΔPA-4, pΔPA-5.

Detailed description of the invention

Example 1

Generation of defective influenza A virus particles from recombinant DNA

Influenza A viruses are negative-strand, segmented viruses. Its genome consists of 8 gene segments. All eight functional gene segments are necessary for the production of infectious virus, ie, a replicable virus capable of replicating for unlimited or at least several cycles in cells generally considered suitable for influenza virus replication. The packaging of gene segments of influenza A viruses, whether by stochastic or specific mechanisms, has been debated for many years. Some evidence for these 2 options has been described. Evidence for random packaging is that aggregated virions are more infective than unaggregated virions (6), and when cell cultures are infected at low moi, some infected cells fail to express a fragment (8), which Both indicate the presence of virions that do not contain the complete influenza virus genome. Additional evidence for random packaging is that influenza viruses containing 9 segments have been experimentally produced (4). Bancroft and Parslow found that there was no competition between vRNAs derived from the same gene segment for packaging virions (1).

One argument in favor of a specific packaging process is that although all gene segments are present in equal amounts in the virus stock, they are present in variable amounts in the producer cells (10). Additionally, when a defective interfering (DI) particle is generated, the DI vRNA replaces the segment from which it was derived (3) (a defective interfering particle is a virion in which a gene segment has a large internal deletion. When These particles appear when the virus is passaged at high moi and are thought to be due to the R638A mutation of the acidic protein of the polymerase [Fodor et al; J. Virol. 77, 5017-5020, 2003]). Finally, the efficiency of virion formation increases with the number of gene segments (5). Fujii et al. also confirmed the NA fragment region, which is necessary for the efficient integration of fragments into virions, and later the research group also confirmed the HA and NS regions, which are important for packaging into virions [Fujii, 2005 #256 ; Watanabe, 2003 #184].

Here we present evidence for specific packaging. In order to produce virus particles containing only seven functional gene segments, we had to determine which gene segments could be omitted without hindering virus production. In view of the use of replication-defective viruses as vaccines, HA and NA were not omitted, nor were MA or NS, as 2 separate expression plasmids were required. We produced viruses that lacked the polymerase gene. When the missing gene segment was not compensated in trans with an expression plasmid, we could not produce virus (Tables 1, 2 and 3, rPR8-7ntc). After transfection of the 7 gene segments and a plasmid expressing at very low titers the protein normally expressed by the deleted gene segment, virus could be produced (Tables 1, 2 and 3, rPR8-7). Therefore, deletion mutants of gene segments 1, 2 and 3 of influenza A/WSN/33 carrying internal deletions of 1032, 528 and 1120 nucleotides, respectively, were generated. These deletion mutants were named pΔPB2, pΔPB1 and pΔPA (see Figure 2). As previously described (de Wit, E., MISpronken, TMBestebroer, GFRimmelzwaan, ADOsterhaus, and RAFouchier. 2004. Efficient generation and growth of influenza virus A/PR/8/34 from eight cDNA fragments. Virus Res 103:155-61) , 293T cells were transfected with each of these deleted gene segments and seven complementary bidirectional constructs encoding A/PR/8/34 (DeWit et al., 2004) and appropriate expression plasmids. 48 hours after transfection, the supernatant was harvested. Subsequently, MDCK cells were transfected with one of the expression plasmids HMG-PB2, HMG-PB1 or HMG-PA as previously described (2). These transfected cells were inoculated to the corresponding supernatants of transfected 293T cells (see Figure 1, which explains the experimental method). Virus replication in these MDCK cells was determined by HA-assay. Initially, there was no virus replication in untransfected MDCK cells. Viral replication was found in MDCK cells transfected with HMG-PB2, HMG-PB1 or HMG-PA, inoculated into the corresponding supernatants. Next, based on the sequence of the defective interfering PA vRNA of influenza virus A/PR/8/34 obtained from the influenza sequence database ( www.flu.lanl.gov , accession number K00867), we cloned the defective PA gene fragment. The 5'207nt and 3'194nt of PA were PCR-amplified and cloned into a bidirectional transcription vector derived from pHW2000 (7) modified as described previously (De Wit et al., 2004). The resulting plasmid was called pDIPA (see Figure 2). 293T cells were transfected with pDIPA, HMG-PA and seven bidirectional constructs encoding the remaining influenza A/PR/8/34 gene segments (see Figure 2). Forty-eight hours after transfection, the supernatant was harvested, and subsequently, MDCK cells transfected with HMG-PA 24 hours earlier were inoculated to the supernatant. 72 hours after inoculation, the HA-assay was performed on the supernatant of these MDCK cells and found to be positive, indicating viral replication in these cells. Inoculation of untransfected MDCK cells also failed to result in virus production as determined by HA-assay. Subsequent passaging of supernatants containing PA-deficient virus particles on MDCK cells untransfected or transfected with HMG-PA produced the same results (Table 1). Up to passage 4, virus was produced in MDCK cells transfected with HMG-PA. The supernatant of MDCKp4 was serially diluted to give an indication of the virus titer, which was confirmed to be about 10 ₄ TCID ₅₀ /ml.

The method steps used were: with one of the constructs pΔPB2, pΔPB1, pΔPA, pΔNP, 7 complementary constructs encoding A/PR/8/34 (De Wit et al., 2004) and HMG-PB2, HMG- PB1, one of the HMG-PA (expression plasmids described in e.g. Pleschka, S., R. Jaskunas, O.G. 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), transfected 293T cells (see De Wit et al., 2004 for the transfection method). 48 hours after transfection, supernatants of transfected 293T cells were harvested. When viruses were produced, they were present in the supernatant. At the same time, MDCK cells were transfected with one of the expression plasmids HMG-PB2, HMG-PB1, HMG-PA (according to the deletion mutant used, so in the case of pΔPB2, HMG-PB2 was used to transfect MDCK cells) (For the transfection method, see Basler et al., 2000), because the viruses produced lacked the gene segment capable of expressing the protein because they packaged the gene segment containing the internal deletion. 24 hours after transfection, transfected MDCK cells were inoculated with supernatant obtained from transfected 293T cells. When the virus was present in the 293T supernatant, the virus replicated in the transfected MDCK cells and more virus was produced. 72 hours after inoculation, the supernatant can be collected again.

RNA was isolated from the supernatant of MDCKp4 in order to confirm that no PA or DIPA recombination occurred that would result in a functional PA gene fragment. First, the supernatant was passed through a 22 μM filter and concentrated by centrifugation. Subsequently, RNA was isolated and RT-PCR was performed using primers targeting the non-coding region of the PA fragment. RT-PCR using primers specific for PA vRNA confirmed that ΔPA and DIPA were stable over multiple passages. A clear band of about 400 bp appeared in the supernatant of MDCK cells infected with DIPA virus particles, and a band of 1100 bp appeared in the supernatant of MDCK cells infected with ΔPA-containing virus. In the supernatant of MDCK cells infected with wild-type A/PR/8/34, a band of about 2300 nt was seen (Fig. 3). These results indicated that the ΔPAPR8 gene fragment was stably packaged into virions.

In order to produce viruses lacking PB2, seven gene segments 2, 3, 4, 5, 6, 7 and 8 (de Wit, E., M.I. Spronken, T.M. Bestebroer, 293T cells (Hoffmann, E., G.Neumann, Y.Kawaoka, G.Hobom, and R.G.Webster.2000.A DNA transfection system for generation of influenza A virus from eight plasmamids.Proc Natl Acad Sci U S A97:6108-13.), leading to Expression of vRNA and mRNA. Plasmids (Pleschka, S., R. Jaskunas, O.G. Engelhardt, T. Zurcher, P. Palese, and A. Garcia-Sastre. 1996 . Aplasmid-based reverse genetics system for influenza A virus. J Virol70: 4188-92.). As a control, only seven bidirectional constructs encoding A/PR/8/34 were transfected, omitting pHMG-PB2. 48 hours after transfection, the supernatant was harvested and inoculated into MDCK cells or MDCK cells transfected with pHMG-PB2 (MDCK-PB2) in a 100 mm dish 24 hours in advance. Three days after inoculation, supernatants of inoculated MDCK cells were tested for hemagglutination activity using turkey erythrocytes as an indicator of virus production. No virus was detected in cells inoculated with the supernatant of 293T cells transfected with only 7 gene segments but not with pHMG-PB2 (rPR8-7ntc, Table 2). MDCK-PB2 cell supernatant inoculated with 293T cell supernatant transfected with 7 gene fragments and pHMG-PB2 was positive. Subsequently, rPR8-7 supernatants were passaged in MDCK and MDCK-PB2 cells. rPR8-7 was able to replicate in MDCK-PB2 cells, but not in MDCK cells (Table 2). We next produced a 1032nt deletion mutant of gene segment 1 of influenza virus A/WSN/33, resulting in a deletion of 344 amino acids (pΔPB2, Figure 2). A recombinant virus containing ΔPB2 (rPR8-ΔPB2) was produced as described above (Fig. 1). No virus was detected in MDCK cells, whereas virus was detected in MDCK-PB2 cells inoculated with rPR8-ΔPB2. After passaging rPR8-ΔPB2, there was no evidence of virus production in MDCK cells, in contrast to MDCK-PB2 cells (Table 2).

Viruses lacking PB1 were also produced. 293T cells were transfected with seven bidirectional constructs encoding gene segments 1, 3, 4, 5, 6, 7 and 8 of influenza virus A/PR/8/34, resulting in the expression of vRNA and mRNA. Plasmids expressing A/PR/8/34 PB1 and pHMG-PB1 were co-transfected. As a control, only the seven bidirectional constructs encoding A/PR/8/34 were transfected, omitting pHMG-PB1. 48 hours after transfection, the supernatant was harvested and inoculated into MDCK cells or MDCK cells transfected with pHMG-PB1 (MDCK-PB1) in a 100 mm dish 24 hours in advance (2) (Figure 1). Three days after inoculation, supernatants of inoculated MDCK cells were tested for hemagglutination activity using turkey erythrocytes as an indicator of virus production. No virus was detected in cells inoculated with the supernatant of 293T cells transfected with only 7 gene segments but not with pHMG-PB1 (rPR8-7ntc, Table 3). MDCK-PB1 cell supernatants inoculated with 293T cell supernatants transfected with 7 gene fragments and pHMG-PB1 were positive. Subsequently, rPR8-7 supernatants were passaged in MDCK and MDCK-PB1 cells. rPR8-7 was able to replicate in MDCK-PB1 cells, but not in MDCK cells (Table 3). We next generated a 528nt deletion mutant of gene segment 2 of influenza virus A/WSN/33, resulting in a 178 amino acid deletion (pΔPB1, Figure 2). A recombinant virus containing ΔPB1 (rPR8-ΔPB1 ) was produced as described above ( FIG. 1 ). No virus was detected in MDCK cells, whereas virus was detected in MDCK-PB1 cells inoculated with rPR8-ΔPB1. After passaging rPR8-ΔPB1, there was no evidence of virus production in MDCK cells, in contrast to MDCK-PB1 cells (Table 3).

Thus, we have been able to produce fragments lacking 1, 2, or 3 viruses. The conditionally deficient virus described here can undergo only 1 cycle of replication in non-trans-complemented cells, but can propagate in trans-complemented cell lines. This is the first time that reverse genetics has been used to produce a defective virus that contains only seven functional gene segments and can undergo one cycle of replication, or multiple cycles when the defective gene segment is compensated in trans. Periodic replication.

Defective virus particles produced in this manner are vaccine candidates because they can undergo 1 cycle of replication without generating infectious virus. As a result of this single-cycle replication, the vaccine induces both humoral and cellular immune responses. Although these defective particles cannot replicate in normal cells, large quantities of virus can be grown in cell lines expressing the defective protein for production purposes. As we have demonstrated, multiple cycles of replication do not affect viral genotype. In addition to the use of defective virus particles as vaccines, they are also candidate vectors for gene delivery and expression of foreign proteins, since functional genes can be inserted between the 5' and 3' PA, PB2 or PB1 sequences. This was also confirmed by Watanabe et al. (11), who replaced HA and NA with foreign genes and still produced virus.

Further truncation of pΔPA

In addition, larger portions of the pΔPA construct were deleted, resulting in pΔPA-2, pΔPA-3, pΔPA-4, pΔPA-5 (Figure 4). As previously described (De Wit et al., 2004), with one of these deleted gene segments and seven complementary bidirectional constructs encoding A/PR/8/34 (De Wit et al., 2004) and PA-expressing The expression plasmid was transfected into 293T cells. 48 hours after transfection, the supernatant was harvested. Subsequently, MDCK cells were transfected with the expression plasmid HMG-PA as previously described (Basler, C.F., et al., 2000. Proc Natl AcadSci US A 97:12289-94.). These transfected and untransfected cells were inoculated with supernatants of transfected 293T cells. Virus replication in these MDCK and MDCK-PA cells was examined by HA-assay. There was no viral replication in untransfected MDCK cells. Viral replication was found in HMG-PA transfected MDCK cells inoculated with either supernatant. Thus, all vRNAs derived from these constructs were packaged into virions (Table 4).

Table 1. Replication of recombinant influenza A/PR/8/34 viruses lacking the complete PA gene segment in MDCK and MDCK-PA cells

ntc: not trans-compensated (no transfection of pHMG-PA in 293T cells)

Table 2. Replication of recombinant influenza A/PR/8/34 viruses lacking the complete PB2 gene segment in MDCK and MDCK-PB2 cells

ntc: not trans-compensated (no transfection of pHMG-PB2 in 293T cells)

Table 3. Replication of recombinant influenza A/PR/8/34 viruses lacking the complete PB1 gene segment in MDCK and MDCK-PB1 cells

ntc: not trans-compensated (no transfection of pHMG-PB1 in 293T cells)

Table 4. Replication of recombinant influenza A/PR/8/34 viruses lacking the complete PA gene segment in MDCK-PA and MDCK cells

Example 2

Vaccination with defective recombinant virus

Based on a high-throughput viral backbone (e.g. derived from the vaccine strain A/PR/8/34) containing the HA and NA genes of a relevant epidemic virus (e.g. A/Moscow/10/99), as described herein , conditionally deficient recombinant viruses lacking functional PA, PB1 or PB2 genes were produced. Conditionally deficient viruses were produced by transfection in which polymerase protein expression was achieved by trans-complementation. Subsequently, the virus is amplified in a suitable cell matrix (eg, MDCK cells or Vero cells) stably expressing the relevant polymerase. Viral supernatants were clarified by centrifugation at 1000 xg for 10 minutes. Viruses were concentrated and purified by ultracentrifugation in a 20-60% sucrose gradient, pelleted, and resuspended in phosphate buffered saline (PBS). The purity and quantity of the virus preparation was confirmed using a 12.5% SDS-polyacrylamide gel stained with Coomassie brilliant blue, by infection of MDCK cells and MDCK cells expressing the relevant polymerase and staining with anti-nucleoprotein monoclonal antibodies, Viral titers of conditionally deficient viruses were determined. Mice were inoculated with 1x1050% tissue-culture infectious dose (TCID-50) intratracheally or intranasally using a nebulizer. Antibody titers against HA, NA and internal proteins of influenza virus were determined in serum samples collected before and after vaccination using hemagglutination inhibition assay, neuraminidase inhibition assay, ELISA or virus neutralization assay. Measurement of intracellular cytokine expression, antigen-specific cellular immune responses in vaccinated animals by flow cytometry, tetramer staining of CD4 and CD8-positive cells, cytolytic activity, T-cell proliferation, etc. Quantification was performed. Six weeks after vaccination, vaccinated and control animals were challenged with ^1x106 TCID-50 of influenza A/Moscow/10/99 or heterologous virus isolates. Nasal or throat swab samples were collected from animals daily for 10 days after challenge, and the amount of virus shed by infected animals was determined by quantitative PCR analysis or virus titration. The resulting vaccine-induced humoral immunity was determined by quantifying the rise in antibody titers, the resulting vaccine-induced cellular immunity was determined by quantifying the rise in helper and cytotoxic T-cell responses, and by determining the Protection against infection by the challenge virus determines the overall level of immunity.

references

1. Bancroft, C.T., and T.G.Parslow. 2002. Evidence forsegment-nonspecific packaging of the influenza a virus genome. J Virol76: 7133-9.

2.Basler, C.F., X.Wang, E.Muhlberger, V.Volchkov, J.Paragas, H.D.Klen k, A.Garcia-Sastre, and P.Palese.2000.The Ebola virus VP35protein functions as a type I IFN antagonist .Proc Natl Acad Sci U S A97:12289-94.

3. Duhaut, S.D., and J.W.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. Aninfluenza 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 U S A 100:2002-2007.

6. Hirst, G.K., and M.W.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 R.G. Webster. 2000. A DNA transfection system for generation of influenza Avirus from eight plasmamids. Proc Natl Acad Sci U S A 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-drivenformation of influenza virus-like particles. J Virol 74: 547-51.

10. Smith, G.L., and A.J.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 (19)

1. compsn, the material that it comprises the virion of conditionality defective or comprises cell or be derived from said cell, described virion are the virions that is selected from following group each, and described cell comprises the virion that is selected from following group each:

(1) through virion that following method obtained: a kind of method that obtains the influenza virus particles of conditionality defective; It comprises: the first step; With the first suitable cell of following substances transfection: through in the proteic nucleic acid of coding influenza, carrying out one or more gene constructs that inner deletion generates; Make described gene construct can not produce functional protein thus, the said gene fragment of said virus is not packed into virion but do not hinder; Complementary influenza nucleic acids fragment with encoding influenza virus; With can in said cell, express said proteic one or more expression plasmids; Proper time point after infection, at least one virion of results from the supernatant of described first cell; Second step is with in said cell, expressing the second suitable cell of said proteic one or more expression plasmid transfections; The 3rd step is with said second cell of supernatant transfection that comprises at least one virion that obtains from said first cell; The 4th step, proper time point after infection, at least one virion of results does not wherein use helper virus in said method from the supernatant of described second cell;

(2) through virion that following method obtained: a kind of method that obtains the influenza virus particles of conditionality defective; It comprises: with the suitable cell of following substances transfection: one or more gene constructs that the nucleic acid through inside deletion coding influenza polysaccharase generates; Make described gene construct can not produce the functional polyalkylene synthase thus, the said gene fragment of said virus is not packed into virion but do not hinder; Complementary influenza nucleic acids fragment with encoding influenza virus; With one or more expression plasmids that can in said cell, express said polysaccharase; Proper time point after infection, at least one virion of results does not wherein use helper virus in said method from the supernatant of said cell;

(3) through virion that following method obtained: a kind of method that obtains the influenza virus particles of conditionality defective, it comprises: the first step, with can be in said cell the suitable cell of one or more expression plasmid transfections of expression of influenza polysaccharase; In second step, infect said cell with the defined supernatant that comprises the influenza virus particles of conditionality defective in above-mentioned (2); The 3rd step, proper time point after infection, at least one virion of results does not wherein use helper virus in said method from the supernatant of said cell;

(4) through virion that following method obtained: a kind of method that obtains the influenza virus particles of conditionality defective; It comprises: the first step; With the first suitable cell of following substances transfection: one or more gene constructs that the nucleic acid through inside deletion coding influenza polysaccharase generates; Make described gene construct can not produce the functional polyalkylene synthase thus, the said gene fragment of said virus is not packed into virion but do not hinder; Complementary influenza nucleic acids fragment with encoding influenza virus; With one or more expression plasmids that can in said cell, express said polysaccharase; Proper time point after transfection, at least one virion of results from the supernatant of described first cell; Second step is with the second suitable cell of one or more expression plasmid transfections that can in said cell, express said polysaccharase; In the 3rd step, infect said second cell with the supernatant that comprises at least one virion that obtains from said first cell; The 4th step, proper time point after infection, at least one virion of results does not wherein use helper virus in said method from the supernatant of described second cell.

(5) a kind of influenza virus particles; It comprises one or more nucleic acid fragments; In said fragment, has inner disappearance; Thereby make the influenza polysaccharase that said fragment can not systematic function property, but do not hinder the said gene fragment of said virus is not packed into virion, wherein said polysaccharase is selected from acid polysaccharase (PA), alkaline polymerization enzyme 1 (PB1) or alkaline polymerization enzyme 2 (PB2).

(6) a kind of influenza virus particles; It comprises one or more nucleic acid fragments; In said fragment, have inner disappearance, thereby make the influenza polysaccharase that said fragment can not systematic function property, the said gene fragment of said virus is not packed into virion but do not hinder; Wherein said polysaccharase is selected from acid polysaccharase (PA), alkaline polymerization enzyme 1 (PB1) or alkaline polymerization enzyme 2 (PB2); And wherein said inner disappearance is as follows: for PA albumen, from being begun by 5 '-Nucleotide between the Nucleotide 58 to 207 of non-coding region meter, to the end of 3 '-Nucleotide between the Nucleotide that is risen by the non-coding region meter 27 to 194; For PB1 albumen, from beginning, to the end of 3 '-Nucleotide between the Nucleotide that rises by the non-coding region meter 24 to 197 by 5 '-Nucleotide between the Nucleotide 43 to 246 of non-coding region meter; For PB2 albumen, from beginning, to finish to 3 '-Nucleotide between the Nucleotide that rises by the non-coding region meter 27 to 209 by 5 '-Nucleotide between the Nucleotide 34 to 234 of non-coding region meter, above-mentioned Nucleotide interval does not comprise end points.

2. according to the compsn described in the claim 1; Wherein for each method in (2) and (4); Wherein the inside disappearance that generates of the nucleic acid through inside deletion coding influenza polysaccharase is for as follows: for PA albumen; From beginning, to the end of 3 '-Nucleotide between the Nucleotide that rises by the non-coding region meter 27 to 194 by 5 '-Nucleotide between the Nucleotide 58 to 207 of non-coding region meter; For PB1 albumen, from beginning, to the end of 3 '-Nucleotide between the Nucleotide that rises by the non-coding region meter 24 to 197 by 5 '-Nucleotide between the Nucleotide 43 to 246 of non-coding region meter; For PB2 albumen, from beginning, to finish to 3 '-Nucleotide between the Nucleotide that rises by the non-coding region meter 27 to 209 by 5 '-Nucleotide between the Nucleotide 34 to 234 of non-coding region meter, above-mentioned Nucleotide interval does not comprise end points.

3. according to the compsn of claim 1; Wherein for each the method in (3) to (4); The said cell that wherein will infect with the supernatant of the influenza virus particles that contains the conditionality defective can be expressed polysaccharase, and wherein said polysaccharase is corresponding to the polysaccharase of the non-functional in the virion of said defective.

4. according to the compsn of claim 3; The said cell that wherein will infect with the supernatant of the influenza virus particles that contains the conditionality defective can the expression of influenza polysaccharase, and wherein said influenza polysaccharase is corresponding to the polysaccharase of the non-functional in the influenza virus particles of said conditionality defective.

5. according to the compsn of claim 1, wherein for each the method in (2) to (4), wherein said polysaccharase is acid polysaccharase (PA), alkaline polymerization enzyme 1 (PB1) or alkaline polymerization enzyme 2 (PB2).

6. according to each described compsn among the claim 2-4, wherein said polysaccharase is acid polysaccharase (PA), alkaline polymerization enzyme 1 (PB1) or alkaline polymerization enzyme 2 (PB2).

7. want 1 compsn according to right, wherein said virion has the influenza nucleic acids fragment of coding VGP.

8. according to the compsn of claim 1 or 7, wherein said virion has the influenza nucleic acids fragment of coding nucleoprotein (NP), alkaline polymerization enzyme 1 (PB1), alkaline polymerization enzyme 2 (PB2), hemagglutinin (HA), neuraminidase (NA), stromatin (M1 and M2) and Nonstructural Protein (NS1 and NS2).

9. according to the compsn of claim 1 or 7, wherein said virion has the influenza nucleic acids fragment of coding nucleoprotein (NP), acid polysaccharase (PA), alkaline polymerization enzyme 2 (PB2), hemagglutinin (HA), neuraminidase (NA), stromatin (M1 and M2) and Nonstructural Protein (NS1 and NS2).

10. according to the compsn of claim 1 or 7, wherein said virion has the influenza nucleic acids fragment of coding nucleoprotein (NP), acid polysaccharase (PA), alkaline polymerization enzyme 1 (PB1), hemagglutinin (HA), neuraminidase (NA), stromatin (M1 and M2) and Nonstructural Protein (NS1 and NS2).

11. according to the compsn of claim 1 or 7, wherein said virion has the influenza nucleic acids fragment from influenza virus A.

12. according to the compsn of claim 1 or 7, wherein said virion has the nucleic acid of the influenza peptides of not encoding.

13. compsn according to claim 1, the cell that is wherein contained in the compsn has one or more influenza virus polymerases, and wherein said polysaccharase is selected from acid polysaccharase (PA), alkaline polymerization enzyme 1 (PB1) or alkaline polymerization enzyme 2 (PB2).

14. be intended to produce the application in the pharmaceutical composition of the immunoprotection that makes object avoid influenza infection in production according to each compsn in claim 1-11 and 13.

Make object avoid the method for the immunoprotection of influenza infection 15. produce, to comprise to the object that these needs are arranged providing according to each compsn in claim 1-11 and 13.

16. the defined virion of claim 12 is intended to the application in the compsn that nucleic acid with the influenza peptides of not encoding flows to cell in production.

17. the defined virion of claim 12 is intended to the application in the pharmaceutical composition of cell that nucleic acid with the influenza peptides of not encoding flows to object in production.

18. the nucleic acid of the influenza peptides of will not encoding flows to the method for cell, being included as said cell provides claim 12 defined defective influenza virus particles.

19. the nucleic acid of the influenza peptides of will not encoding flows to the method for object, being included as said object provides claim 12 defined defective influenza virus particles.

Images