CN102586199A - Defective influenza virus particles - Google Patents

Abstract

The invention relates to the field of influenza virus and the vaccination against flu. The invention provides a conditionally defective influenza virus particle having seven different influenza nucleic acid segments. The invention also provides a conditionally defective influenza virus particle lacking an influenza nucleic acid segment selected from the group of segments essentially encoding acidic polymerase (PA), the basic polymerase 1 (PB1 ) and the basic polymerase 2 (PB2). In particular, the invention provides defective influenza virus particles having seven different influenza nucleic acid segments and lacking an influenza nucleic acid segment essentially encoding acidic polymerase.; Furthermore, the invention provides use of a composition comprising a defective influenza virus particle according to the invention for the production of a pharmaceutical composition directed at generating immunological protection against infection of a subject with an influenza virus, and provides a method for generating immunological protection against infection of a subject with an influenza virus comprising providing a subject in need thereof with a composition comprising such defective influenza virus particle.

Description

Defective influenza virus particles

The present invention relates to influenza virus and to the field of the vaccine inoculation of influenza.

Influenza virus (orthomyxoviridae family) is the tunicary genomic minus-stranded rna virus of sectional (Taubenberger and Layne, Molecular Diagnosis Vol.6 No.42001) that has.According to their nucleoprotein and the significant antigenic difference between the stromatin, can they be divided into 2 types: one type comprises influenza A and B, the another kind of influenza C that comprises.These 3 Virus Types also exist pathogenic and difference genomic organization.Find that type A extensively is present in the warm blooded animal, but type B and C mainly are human pathogens.Through the hemagglutinin (HA) that stretches out from the virus particle surface and the antigen property of NA surface glycoprotein, further segment influenza A virus.At present, 15 kinds of HA and 9 kinds of NA hypotypes are arranged.Influenza A virus can infect the animal of broad variety, comprises bird, pig, horse, people and other Mammals.Aquatic bird is the natural reservoir (of bird flu viruses) of the influenza A of all known hypotypes, and possibly be the genetic material source of the popular influenza strain of people.

Be different from relevant paramyxovirus, influenza virus has sectional rna gene group.Influenza A and B virus have similar structure, and influenza C is more discrete.A and category-B virus all contain 8 at least a proteic gene fragments of discrete coding respectively, and the C type contains 7 discrete fragments, and it has made up the fragment 4 and 6 of A and category-B type.Influenza A and B virus are covered with 3 kinds of proteic protuberance: HA, NA and matrix 2 (M2).Influenza C virus only has a kind of surface glycoprotein.Every kind of influenza RNA fragment is rolled into shell by nucleoprotein (NP), forms ribonucleotide nucleoprotein (RNP) mixture.These 3 kinds of polymerase proteins link to each other with an end of RNP mixture.The RNP tunicle centers on, and described film contains the stromatin (matrix 1) as integral part.The phospholipid moiety of coating is derived from host cell membrane.In virion, also found unstructuredness albumen 2 (NS2).

The World Health Organization (WHO) about the influenza virus nomenclature the rule as follows.At first, specifying the type (A, B, or C) of virus, is host (if inhuman) then, discretely, separates number and isolating year (separating with oblique line).For influenza A, HA and NA hypotype in bracket, have been indicated.For example, the strain that is included in the trivalent vaccine in 2000 to 2001 recent seasons is: A/ Panama/2007/99 (H3N2), A/ New Caledonia/20/99 (H1N1), and B/Yamanashi/16/98.Since 1977, there have been 2 kinds of influenza A hypotypes to coexist as the mankind: H1N1 and H3N2.

Influenza virus can accumulate the point mutation in the reproduction process, because their RNA polymerase mixture does not have the check and correction activity.The amino acid whose sudden change that can change in the antigen part of surface glycoprotein can be that virus strain provides selective advantage through the immunity that escape is pre-existing in.Through combining the acceptor on some host cell, the HA molecular energy starts infection.Receptors bind can be stoped to the proteic antibody of HA, the subinfection again of same strain can be effectively prevented very much.Through antigenic drift (wherein the sudden change of round-robin HA gene can destroy antibodies at present) or antigenic shift (wherein virus obtains the HA of new subtype), HA can escape previously obtd immunity.Antigenic drift pressure is unequal in the HA molecule, and positive selection variation mainly occurs on the proteic spherical head of HA.These change accumulation degree in HA also greater than in NA.In other influenza proteins, change more slowly.Likewise, antigenic drift pressure is maximum in the influenza strain of people-adaptation, pig-with the strain of horse-adaptation in medium, minimum in the strain of bird-adaptation.

Because influenza virus has the sectional genome, 2 kinds not the coinfection of homophyletic in same host can cause generating the new influenza strain that reconfigures, it contains the segmental various combination of parental gene.Known 15 kinds of HA hypotypes are present in the wild bird, and the source of HA is provided, and said HA is for the mankind and Yan Shixin.The appearance of the influenza strain that contains new hypotype (through antigenic shift) in people's circulation, become 1957 with the reason of nearest 2 flu outbreaks of nineteen sixty-eight, and very likely be the reason of flu outbreak in 1918.For consistent with known appearance about epidemic influenza virus, epidemic strain must have and on antigenicity, is different from present popular HA; This HA haves no alternative but through in the mankind, circulating 60 to 70 years; And should virus must propagate in the human world.1957 and nineteen sixty-eight, the transformation of HA has caused being very popular, and under 2 kinds of situation, the HA of epidemic strain and bird strain are closely related.Although a pandemic absolute condition is that HA must change, other part that it be unclear that virus can the maybe necessary degree that changes.Only can obtain 1957 and be used for direct research, use the molecule archeology, characterize epidemic influenza virus in 1918 with the epidemic virus of nineteen sixty-eight.In nineteen fifty-seven, 3 genes are replaced by birds gene: HA, the subunit (PB1) of NA and polysaccharase mixture.In nineteen sixty-eight, HA and PB1 have only been replaced.

Through virus separate, blood clotting suppresses (HI) experiment, immunoassay detects antigen, NA in the serology experiment, checking secretory product is active or based on the experiment of molecule, can diagnose influenza infection specifically.Can be that saliva, Nasopharyngeal swabs or through port are gargled the nasopharynx washing lotion that buffered saline solution obtains with sample collection.The standard of influenza diagnosis is to carry out immunology after cultivating to characterize.Serological analysis can provide accurately, but retrospective influenza infection method because it need collect the serum of acute phase and convalesce phase.

Influenza virus can grow in the perhaps many tissue culture systems of the egg that contains embryo.The adding trypsin is used for the cutter activation of HA), can make influenza virus is middle breeding at Madin-Darby dog kidney (MDCK) cell with other.The main method of production of vaccine remains and in egg, cultivates influenza virus.The culture of clone is generally used for the initial gross separation of human influenza virus (type A and B).Can directly in the allantoic cavity of the egg that contains embryo, cultivate many human influenza viruses.Some influenza A and B virus need at first in amniotic cavity, to cultivate, and transfer in the allantoic cavity then.After culture separates, use immunoassay or immunofluorescence technique, identified most of influenza isolates definitely.The HA molecular energy of influenza virus combines to breathe the sialic acid residues on the cell surface, is beneficial to the entering of virus.

Utilize the external ability that makes RCA of influenza virus, can aspect antigen, characterize the influenza strain.Anti--HA antibody can suppress aggegation.Thereby it is to be used to one of standard method that characterizes the influenza strain that hemagglutination suppresses (HI) experiment.Whether relevant with recent vaccine strain on immunology HI experiment can be used for confirming sample strain (being cross reactivity).With a series of 2 times of dilutions, Xiang Kongzhong adds blood grouping serum, and the latter generally produces in ferret, the experimenter through observe to suspend with the agglutinative red corpuscle, experimental port is given a mark.In most of the cases, one group of serum is used to mate sample strain and the reference strain to vaccine, and in the process in any given influenza season, has mated most of sample strains through HI Success in Experiment ground.

About differentiating the antigen property of each virus strain, WHO provides criterion, and WHO cooperation center provides guide.According to the immunology pedigree, for example A/ Moscow/10/99 (H3N2)-appearance, A/ New Caledonia/20/99 (H1N1)-appearance and B/ Beijing/184/93-appearance virus, strain is classified to sample.For the sample strain that can not in HI experiment, characterize, laboratory worker must be inoculated them in the ferret, to generate the specific antiserum(antisera) of strain.After new antiserum(antisera) was ready to, the said for another example HI that carries out tested.If new serum shows significant difference (being normally defined 4 times of differences between sample and the vaccine strain) in cross reactivity, then it is integrated in the Routine Test Lab group, and is used to seek new popular strain.Thereby HI experiment is extremely important in the influenza virus follow-up work that vaccine strain is selected, and is the method for the most frequently used estimation antigenic drift.

Sequence contrast through each gene fragment can characterize the influenza strain hereditarily, and WHO criterion and WHO cooperation center provide the guide that comprises following segmental individual character about identification again: comprise the genomic RNA fragment of influenza; Can the encode influenza C viral nucleic acid fragment of the influenza A and the B viral nucleic acid fragment of nucleoprotein (NP), alkaline polymerization enzyme 1 (PB1), alkaline polymerization enzyme 2 (PB2), acid polysaccharase (PA), hemagglutinin (HA), neuraminidase (NA), stromatin (M1 and M2) and Nonstructural Protein (NS1 and NS2) and the nucleoprotein (NP) of encoding, alkaline polymerization enzyme 1 (PB1), alkaline polymerization enzyme 2 (PB2), hemagglutinin-neuraminidase appearance gp (HN), stromatin (M1 and M2) and Nonstructural Protein (NS1 and NS2).Contrast and differentiate the request of the reference strain of vaccine virus about antigen analysis, nucleotide sequence; Can so that send reference of WHO influenza and research cooperation center; 45 Poplar Road, Parkville, Victoria 3052; Australia (fax:+61 3 9,389 1881, web site:http//www.influenzacentre.org); The reference of WHO influenza and research cooperation center, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashi-Murayama, Tokyo 208-0011, Japan (fax:+81 42 5610812 or+81 42 5652498); The supervision of WHO influenza, epidemiology and control cooperation center, 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 the reference of WHO influenza and research cooperation center, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, England (fax:+44 208 906 4477).On the http://www.who.int/influenza of the website of WHO, can obtain up-to-date epidemiology information, on http://www.who.int/flunet, can obtain geographical information system(GIS), FluNet.

People are for the influence of influenza and prevent its health and the understanding of economic benefits progressively to improve, and more than ten years in past have been observed the application and the benefit of vaccine inoculation, and have occurred many Tamiflu significantly.As in the result of longer life expectancy of many countries; More people is in the danger of complication; The burden of the health care system in the sick process of influenza pandemic is admitted more widely; More frequent international travel has been created chance for the propagation of virus, although the appearance of product innovation has increased the selection of prevention and treatment disease.About 50 countries have the national influenza Immunization programme of government-funded, and can obtain vaccine in many other countries.Concrete recommendation about using vaccine is different, but comprise usually old individuality and since the year in the sick high-risk that is in serious disease of the chronic medical science that existed in the past greater than the individuality at 6 monthly ages immune.In some country, vaccine is used for reducing the propagation of influenza to the high risk population.In their scope of overall publilc health right of priority, member states need consider the benefit of influenza preventive activities.

Whether contain whole virus particles according to them, the virion (splitted vaccine) that part is broken or the envelope antigen (subunit vaccine) of purifying can be divided into several types with the vaccine of inactivation.That some subunit vaccine and adjuvant or delivery system is combined.

Few countries has been permitted the influenza vaccines of the attenuation of the work that is used for some target group.In the Russian Federation, 2 kinds of a kind of vaccine different preparations have been used for healthy grownup and children, extensive testing another kind of living vaccine.But, before can obtaining the attenuated vaccine that lives more widely, generally still do not recommend they are used for the influenza prevention.

In order to prevent and treat influenza, 2 types of antiviral drugs have been developed.The M2 suppressor factor is that amantadine and Rimantadine are limited to the treatment influenza A virus, and has reported that they can effectively prevent infection.Although two kinds of products all can cause some spinoffs, the more common tangible neurological side effects of amantadine.Recently, many countries have permitted neuraminidase inhibitor (for example zanamivir and Olympic Competition rice Wei) to be used to treat type A and B influenza, and have reported that they also are effective for prevention.In the patient who accepts 2 types of antiviral drugs, detected resistant mutant strain.Although it is not regarded as important public health problem as yet at present, if use these medicines very on a large scale, situation may change.

WHO is keeping global international supervision plan, and it moves under the cooperation of 110 national influenza centers that are positioned at 82 countries and 4 WHO influenzas reference that is positioned at Atlanta (U.S.), London (Britain), Melbourne (Australia) and Tokyo (Japan) and research cooperation center.These centers provide the early warning system about the strain that prevailing disease potential occurs having.This system is very important, if because influenza vaccines do not contain present round-robin strain, then its effect can reduce.WHO can issue the recommendation of forming about vaccine; As can (for example see that issue 9,2004 at the Weekly Epidemiological Record that publishes by the World Health Organization; 79; The 88th page or http://www.who.int/wer) in being seen, be distributed on the vaccine that use in the Northern Hemisphere February, be distributed on the vaccine that use in the SouthernHemisphere September.Because influenza is distinguished under the line and had not sure seasonal characteristic, EPDML consideration can influence the vaccine that is applicable to that country under the line uses these any (February or September) in recommending.

The influenza strain isolated that cooperation center submits to national center carries out antigen and genetic analysis.When observing the evidence of antigen variation, itself and epidemic data are compared, to estimate the epidemiology significance of this variant.Use before the vaccine inoculation with the human serum group of collecting afterwards, representational strain isolated and present vaccine strain are compared, can think that with definite present vaccine can be protected from these viruses.After the annual vaccine of publishing WHO is recommended, developed high growth strain, and offered the manufacturer as with reference to virus, generate the seed virus that is used for production of vaccine to assist.To the security of influenza vaccines and the test of effectiveness, comprise virally inactivated, microorganism sterilizing, be used for the antigen concentration that the measurement and confirming of the chemical reagent of break virus is recommended.Recommend vaccine can meet the requirement of WHO, still, state control office should ratify the specific vaccine virus in each country's use.Public health office of country is responsible for recommending the use of vaccine.In addition, WHO published recommendation about flu-prevention (see WER No.35,2002, pp.281-288).

Verified, present influenza vaccines can not be protected immunity (naive) individuality, and on the line and when the flu outbreak outburst occurring when the many individualities that never run into influenza infection, it is extremely important that this fact can become.Virus generally begins their life cycle through following method: be attached on the host cell surface acceptor, get into cell, make their viral nucleic acid shelling, subsequently the replication-competent virus genome.Behind viral protein that has synthesized new copy and gene, these components to be packed in the progeny virus particle, they withdraw from cell then.In the process of installation step, select its genomic nucleic acids in a large amount of viruses that progeny virus must be from be present in tenuigenin and the nucleus effectively.Viral genome is packed in the virus particle, typically comprise by the virus component of the cis acting sequence in the viral nucleic acid and discern, so-called " packaging signal ".Defining such signal is important for understanding viral life cycle, and for us the information that can be used for the virus vector of construction expression foreign protein is provided.In fact, use the transportation means of retrovirus, can pack the into process of progeny virus particle owing to the vRNA that very positively understands them to a great extent as the gene delivery carrier of expressing foreign protein.

Not too understand the genome packaging signal of other RNA viruses, hindered them as the application process of expressing and carrying the carrier of alien gene.For example; Influenza A virus is tunicary minus-stranded rna virus, and its sectional genome has the code capacity of nucleoprotein (NP), alkaline polymerization enzyme 1 (PB1), alkaline polymerization enzyme 2 (PB2), acid polysaccharase (PA), hemagglutinin (HA), neuraminidase (NA), stromatin (M1 and M2) and Nonstructural Protein (NS1 and NS2).

This virus has 2 kinds of transmembrane glycoproteins on coating, hemagglutinin (HA) and neuraminidase (NA).HA albumen can be incorporated into containing on the sialic acceptor on the host cell surface, and after receptor-mediated endocytosis, the fusion of mediation peplos and endosome film.On the contrary, NA albumen provides following mechanism to play a crucial role at later period of infection: remove sialyl from sialyloligosaccharide, thereby discharge newly assembled virus particle from cell surface, and stop the self aggregation of virus particle.In coating, comprise the segmental viral genome of 8 different virus RNA (vRNA) and closely be connected on nucleoprotein (NP) and the polymerase protein (PA, PB1, and PB2), form the ribonucleoprotein mixture.In order to generate infectious virus, need whole 8 (perhaps under the situation of C class C-type virus C: whole 7) functioning gene fragments.(US 5578473 for WO2004/094466, WO2003/091401 to have described a plurality of sudden changes in the pol gene; Fodor etc., J.Virol.77,5017-5020; 2003), it can change polymerase activity, perhaps changes polysaccharase in another way; But can not make it weaken functional in synthetic viral RNA, so that contain the virus reproducible not of the polysaccharase of this sudden change.In WO2004/094466, generated the infectious virus of PA gene with sudden change, confirmed to allow to produce and reclaim the benefit of selective system of the infectious virus of gene thus with sudden change.In WO2003/091401; Confirmed how to produce infectious virus, had the influenza virus that produces relevant ideal performance (the for example attenuation of temperature sensitivity or other type) with the vaccine virus of the attenuation of living so that produce with reclaiming with pol gene sudden change.In US55788473, proposed to change the specificity and its active pol gene fragment of reduction of multiple polysaccharase.But they are not used in reconstruct virus, say nothing of the virus that reconstruct has completely lost its polymerase activity.In addition, in above-mentioned application, all do not produce the defective particle of having lost their replication.As everyone knows, when influenza A virus goes down to posterity with high infection multiplicity, can generate defective virion, it lacks one or more functioning gene fragments.In such virion, because the mistake that influenza virus polymerase causes, one or more functional genes are replaced by defective interference (DI) gene fragment.Because high infection multiplicity and therefore cell infection surpass a virion, the virus that the defective that contains the virus of DI RNA is contained the functional gene of losing of complete copy compensates.Recently confirm that some sudden change in the acid pol gene can improve the efficient (Fodor 2003) that generates the virion with dcc gene.Be important to note that the generation of the defective virion in these experiments and complementation all take place randomly in these experiments.This process at random can limit the use of virion in practical application of DI RNA and conditionality defective.And, when using these disclosed methods to produce defective virion, except the virus of the conditionality defective of needs, also generated the reproducible virus of wild-type.This reproducible virus can be complete wild-type (helper virus) or mixed the rearrangement strain (reassortant) that forms by the heredity of helper virus and defective virus.No matter the wrapping process of the gene fragment of influenza virus is through at random or specific mechanism, all argues many years.Some evidences of these 2 kinds of selections have been described.The evidence of packing is at random; The accumulative virion has than the higher infectivity of accumulative virion not; And when the time with low infection mode (moi) cells infected culture; The cell of some infection can not be expressed a fragment, this two show that existence does not contain the virus particle of complete influenza virus gene group.Other evidence of packing is in experiment, to have produced and contained 9 segmental influenza viruses at random.

Support the reason of specificity wrapping process to be for one, although all gene fragments all ana be present in the virus stock solution used, they are present in different amounts and produce in the cell.In addition, when having generated defective interference (DI) particle, DI vRNA can replace its fragment that is derived from, and (defective interference particle is the virion that one of them gene fragment has bigger inside disappearance.When virus goes down to posterity with high moi, these particles can appear).At last, the efficient of virus particle formation increases along with the increase of gene fragment number.

Summary of the invention

Defective influenza virus particles (Mena I. etc. for example., J.Virol.70:5016-24 (1996); Neumann G. etc.; J.Virol.74:547-51 (2000) .) can be used as the vaccine candidate article; Because they can be induced to the antibody except HA and other viral protein the NA; If they can get into host cell, because they can also induce the cell immune response (for example helper cell, cytotoxic T cell) to virus except humoral response.Up to now, through transfection produced defective influenza virus particles (Mena I. etc., J.Virol.70:5016-24 (1996); Neumann G. etc., J.Virol.74:547-51 (2000) .), reduced and produced a large amount of this particulate possibilities.A replacement scheme of this method is that the virion of working condition property defective can duplicate them in some production system, but can not in normal cell or production system, duplicate.For this reason, modified the cell of production system, made it can produce one or more influenza virus genes or gene product, can the defective influenza virus particles of trans-complementation.The present invention discloses the trans-complementation of confirming of defective influenza virus particles first.In the laboratory, when the defective interference base that carries wild-type has compensated the defective interference influenza virus in the same cell because of segmental virus, observed the trans-complementation of influenza virus particles." natural system " of this trans-complementation can not be used to produce the influenza virus particles of definite conditionality defective.At first, this system requirements, a kind of (partly) defective virus is compensated by at least a (partly) reproducible virus, and this can cause all of a sudden generating complete infective virus.Secondly, because the different gene fragment is produced defective interference particle with random fashion, can not produce the virion of definite conditionality defective.

The influenza virus particles of conditionality defective in theory can be based on the disappearance of complete genome fragment or its part.If the packing of influenza virus gene group depends on all 8 segmental existence (this is many in question a kind of versions (seeing other place in this specification sheets)), the ability of producing the virion of definite conditionality defective through deletion complete genome fragment (with the gene product of trans production coding) can be restricted.If wrapping process requires the existence of all 8 gene fragments, whether need not know all gene fragments all to exist with the total length form, this makes that the production of virion of conditionality defective is further complicated.The present invention solves these problems.

The invention provides the method for the influenza virus particles that obtains the conditionality defective; It comprises: the first step, with first kind of suitable cell of following substances transfection, for example 293T cell: gene construct with inner disappearance; The nucleic acid deutero-p Δ PB2 that passes through inner deletion coding influenza polysaccharase that for example this paper provides; P Δ PB1, p Δ PA or pDIPA make described gene construct not produce to copy or the function polysaccharase of synthetic viral RNA thus; With the complementary influenza nucleic acids fragment of encoding influenza virus, for example the complementary construct of 7 A/WSN/33 that can encode (HW181-188, Hoffmann etc., 2000); With the expression plasmid that can in said cell, express said polysaccharase, the HMG-PB2 that provides of this paper for example, HMG-PB1, among the HMG-PA one; Proper time point after transfection, for example in 10-50 hour, preferably at about 20-30 hour, at least one virion of results from the supernatant of described first kind of cell; Second step is with second kind of suitable cell, the for example mdck cell of expression plasmid transfection that can in said cell, express said polysaccharase; The 3rd step is with the described second kind of cell of supernatant transfection that comprises at least one virion that obtains from described first kind of cell; The 4th step; Be included in proper time point after the transfection; For example 24-96 hour, preferred 48-72 hour; From the supernatant of described first kind of cell, gather in the crops at least one (now for the conditionality defective,, have the gene fragment that inside lacks) virion because they have been packed because the virus that generates lacks the gene fragment of the function polysaccharase that can express copy or synthetic viral RNA.

Preferably can make gene fragment can not produce the inside disappearance of functional protein, the gene fragment of virus packed in the virion but it must not stop.Preferably, these disappearances are calculated from 5 ' and 3 ' non-coding region respectively.For influenza A, for PA albumen, these preferably lack from the 5 '-Nucleotide that for example between Nucleotide 58 to 75, (does not contain end points) and begin, to the 3 '-Nucleotide end that between Nucleotide 27 to 50, (does not contain end points); For PB1 albumen, begin from the 5 '-Nucleotide that between Nucleotide 43 to 75, (does not contain end points), to the 3 '-Nucleotide end that between Nucleotide 24 to 50, (does not contain end points); For PB2 albumen, begin from the 5 '-Nucleotide that between Nucleotide 34 to 50, (does not contain end points), to the 3 '-Nucleotide end that between Nucleotide 27 to 50, (does not contain end points).More preferably, these disappearances: for PA albumen, begin, to the 3 '-Nucleotide end that between Nucleotide 27 to 100, (does not contain end points) from the 5 '-Nucleotide that between Nucleotide 58 to 100, (does not contain end points); For PB1 albumen, begin from the 5 '-Nucleotide that between Nucleotide 43 to 100, (does not contain end points), to the 3 '-Nucleotide end that between Nucleotide 24 to 100, (does not contain end points); For PB2 albumen, begin from the 5 '-Nucleotide that between Nucleotide 34 to 100, (does not contain end points), to the 3 '-Nucleotide end that between Nucleotide 27 to 100, (does not contain end points).Even more preferably, these disappearances: for PA albumen, begin, to the 3 '-Nucleotide end that between Nucleotide 27 to 150, (does not contain end points) from the 5 '-Nucleotide that between Nucleotide 58 to 150, (does not contain end points); For PB1 albumen, begin from the 5 '-Nucleotide that between Nucleotide 43 to 150, (does not contain end points), to the 3 '-Nucleotide end that between Nucleotide 24 to 150, (does not contain end points); For PB2 albumen, begin from the 5 '-Nucleotide that between Nucleotide 34 to 150, (does not contain end points), to the 3 '-Nucleotide end that between Nucleotide 27 to 150, (does not contain end points).More preferably, these disappearances: for PA albumen, begin, to the 3 '-Nucleotide end that between Nucleotide 27 to 175, (does not contain end points) from the 5 '-Nucleotide that between Nucleotide 58 to 175, (does not contain end points); For PB1 albumen, begin from the 5 '-Nucleotide that between Nucleotide 43 to 175, (does not contain end points), to the 3 '-Nucleotide end that between Nucleotide 24 to 175, (does not contain end points); For PB2 albumen, begin from the 5 '-Nucleotide that between Nucleotide 34 to 175, (does not contain end points), to the 3 '-Nucleotide end that between Nucleotide 27 to 175, (does not contain end points).Most preferably, these disappearances: for PA albumen, begin, to the 3 '-Nucleotide end that between Nucleotide 27 to 194, (does not contain end points) from the 5 '-Nucleotide that between Nucleotide 58 to 207, (does not contain end points); For PB1 albumen, begin from the 5 '-Nucleotide that between Nucleotide 43 to 246, (does not contain end points), to the 3 '-Nucleotide end that between Nucleotide 24 to 197, (does not contain end points); For PB2 albumen, begin from the 5 '-Nucleotide that between Nucleotide 34 to 234, (does not contain end points), to the 3 '-Nucleotide end that between Nucleotide 27 to 209, (does not contain end points).

In this article, complementary fragment is defined as the fragment that can cause a whole set of 8 gene fragments (for example influenza A virus).Thereby if fragment 1 has been used to produce defective fragment, then complementary (flawless) fragment is a fragment 2,3,4,5,6,7 and 8.If fragment 2 is defective, then complementary fragment is a fragment 1,3,4,5,6,7 and 8.The rest may be inferred.

Advantageously, the invention provides a kind of method, do not need or do not exist helper virus thus.

The invention provides isolating and the influenza virus particles conditionality defective; It lacks the functional influenza virus nucleic acid fragment (being also referred to as the influenza virus particles of conditionality defective in this article) that coding is selected from the polysaccharase of acid polysaccharase (PA), alkaline polymerization enzyme 1 (PB1) and alkaline polymerization enzyme 2 (PB2); Described particle can not generate polysaccharase maybe can not be as the source of polysaccharase; With copy or synthetic viral RNA, only and conditionally allow in the cell of the trans compensation of function polysaccharase, to generate reproducible virion thus.In addition, the invention provides the method for the influenza virus particles that obtains the conditionality defective, comprise, cell is provided through with the trans compensation of functional influenza virus polymerase.

In a preferred embodiment; Can in the cell of the similar nucleic acid fragment compensation that self lacks with this particle, duplicate according to particle of the present invention; For example lacking the segmental particle of functional influenza virus nucleic acid PA can duplicate at least providing or compensated in the segmental cell of functional influenza virus nucleic acid PA; Lacking the segmental particle of functional influenza virus nucleic acid PB1 can duplicate in the segmental cell of functional influenza virus nucleic acid PB1 at least is provided, and lacking the segmental particle of functional influenza virus nucleic acid PB2 can duplicate in the segmental cell of functional influenza virus nucleic acid PB at least is provided.In a preferred embodiment; The invention provides according to particle of the present invention; The influenza nucleic acids fragment that it has the VGP of encoding more preferably has the influenza nucleic acids fragment of the nucleoprotein (NP) of encoding, hemagglutinin (HA), neuraminidase (NA), stromatin (M1 and M2) and Nonstructural Protein (NS1 and NS2).In one embodiment, provide according to particle of the present invention, it has the influenza nucleic acids fragment that is derived from influenza A virus.In addition, provide according to particle of the present invention, it contains the nucleic acid of the influenza peptides of can not encoding.In addition, the invention provides isolated cells, it comprises according to particle of the present invention, and said cell does not contain wild-type influenza virus or helper virus, but preferably has been provided or has compensated influenza virus polymerase or its gene fragment of encoding.In a preferred embodiment, such cell is the 293T or the mdck cell of trans compensation.In one embodiment; The invention provides isolated cells; It comprises and lacks the segmental particle of functional influenza virus nucleic acid PA; Said cell does not contain wild-type influenza virus or helper virus, but at least has been provided or has compensated functional influenza virus nucleic acid PA fragment or functional PA.In another embodiment; The invention provides isolated cells; It comprises and lacks the segmental particle of functional influenza virus nucleic acid PB1; Said cell does not contain wild-type influenza virus or helper virus, but at least has been provided functional influenza virus nucleic acid PB1 fragment or functional PB1.In another embodiment; The invention provides isolated cells; It comprises and lacks the segmental particle of functional influenza virus nucleic acid PB2; Said cell does not contain wild-type influenza virus or helper virus, but at least has been provided or has compensated functional influenza virus nucleic acid PB2 fragment or functional PB2.In addition; The invention provides compsn; It comprises according to particle of the present invention or cell or is derived from according to the material of cell of the present invention and the such application of compsn in producing pharmaceutical composition, and described pharmaceutical composition can produce the immunoprotection that makes object avoid influenza infection.Therefore, the invention provides generation and make object avoid the method for the immunoprotection of influenza infection, comprise that the object to needs provides according to compsn of the present invention.In addition, the invention provides the application of influenza virus particles according to the present invention in producing compsn, described compsn is used for the nucleic acid of the influenza peptides of not encoding is delivered into cell.In addition; The invention provides the application of particle according to the present invention in producing pharmaceutical composition; Described compsn is used for the nucleic acid of the influenza peptides of not encoding is delivered into the cell of object; Be delivered into the method in cell or the object with the nucleic acid of the influenza peptides of will not encoding, comprise to described cell or object providing according to particle of the present invention.

The invention provides the influenza virus particles of conditionality defective; When comparing with its nature gene group; It lacks a functional influenza virus fragment, and it is: when comparing with wild-type or auxiliary A or category-B C-type virus C, have 7 (rather than 8) different functions property influenza nucleic acids fragments; Or when comparing, have 6 (rather than 7) different functions property influenza nucleic acids fragments with wild-type or auxiliary C class C-type virus C.When using a technical term " the conditionality defective " in this article, it includes but not limited to, virion, and one of them virogene fragment has bigger inside disappearance, and this causes expressing non-functional albumen from it.The production of infectious virus needs all albumen of all 8 gene fragments (for example influenza A virus) and their codings.Thereby; It self is defective containing the segmental virus of dcc gene: it can cells infected, and can be through a replicative cycle, because all viral proteins (for example all are present in this virus particle; When producing virus; Produce this albumen through expression plasmid) in, but in the cell that infects, do not generate infectious viral particle, because this virus can not be produced a kind of viral protein.But when the cell that infects can be expressed general albumen by defective gene fragment expression, defective virus can be duplicated in these cells, because all viral proteins all exist.Thereby these viruses are conditionality defectives: up to the cell with correct condition is provided, they just can duplicate (in this case, cell can be expressed the viral protein of not encoding because virus lacks gene fragment).

In addition, the invention provides the influenza virus particles of conditionality defective, it lacks the functional influenza virus nucleic acid fragment of the polysaccharase of encoding.In this article, the functional influenza virus nucleic acid fragment comprises the nucleic acid of ability encoding function property influenza proteins, and it allows to generate reproducible virus, and is essential.For example, influenza A virus is a minus-stranded rna virus, and it has the genome of 8-section.8 gene fragments, the 11 kinds of albumen of encoding; Gene fragment 1-8 can encode respectively alkaline polymerization enzyme 2 (PB2), alkaline polymerization enzyme 1 (PB1) and PB1-ORF2 (F2), acid polysaccharase (PA), hemagglutinin (HA); Nucleoprotein (NP), neuraminidase (NA), stromatin 1 and 2 (M1; M2) and non-structural protein 1 and 2 (NS1, NS2).The flank of the coding region of 8 gene fragments is the synthetic essential non-coding region (NCR) of viral RNA.Guard in all influenza A virus fragments at end 13 and 12 Nucleotide of 5 ' and 3 ' of virus genome RNA-end respectively, and be complementary partly, can be to form by the secondary structure of varial polymerases mixture identification.NCR can contain 60 other Nucleotide at the most, and they cannot not be conservative between 8 gene fragments, but are conservative relatively between different influenza viruses.Effectively the viral genome packing needs the flanking sequence in NCR and the coding region.Thereby the functional influenza virus nucleic acid fragment generates sequence (1 or 2 opening code-reading frame/fragment), mRNA, the viral RNA (vRNA) of reproducible virus by the potentiality with encoding function property influenza proteins, permission and transcribes required NCR with viral RNA complementary RNA (cRNA) and form with the packaging signal that is present in NCR and the flank encoding sequence.

Preferably, the influenza virus particles that lacks an influenza nucleic acids of described conditionality defective lacks the fragment of ability encoding function polysaccharase PA, PB1 or PB2.In addition, for the vaccine purpose, described particle preferably has the influenza nucleic acids fragment of the VGP of encoding.

In one embodiment, the invention provides the influenza A virus particle, it has 7 different influenza A nucleic acid fragments.Defective influenza virus particles according to the present invention can duplicate, though in suitable, uncompensated host animal or cell only once.

In the cell of suitable compensation, can duplicate a plurality of circulations according to particle of the present invention.Carry purpose for vaccine and gene; Defective particle can not be normally, infinite copy is a very big advantage in the cell of not trans compensation; Reduce the danger that vaccine virus spreads thus between the host, and reduced the danger that is returned to wild-type virus.

This is to use reverse genetics to produce defective influenza A virus first, and it only contains 7 functioning gene fragments, and can experience duplicating of 1 cycle, perhaps when having compensated defective gene fragment, can experience duplicating of a plurality of cycles when trans.In one embodiment, the invention provides the influenza virus particles of conditionality defective, it lacks the influenza nucleic acid fragment of the acid polysaccharase of basic coding (PA).Similar with the trans-complementation of PA, can predict the trans-complementation of other influenza virus gene.But owing to confirmed that the PA expression level is crucial not as the expression level of other influenza virus protein, PA is the preferred gene fragment of the polysaccharase group of disappearance, also can produce the virus that has lacked PB2 and PB1, can not trans compensation have lacked the virus of NP.In a preferred embodiment, the invention provides the influenza A virus particle of conditionality defective, it has 7 different influenza A nucleic acid fragments, and lacks the influenza A nucleic acid fragment of the acid polysaccharase of basic coding.For the vaccine purpose; Preferably the influenza A virus particle according to conditionality defective of the present invention has basic coding hemagglutinin (HA) and the proteic influenza A nucleic acid fragment of neuraminidase (NA), and these albumen are maximally related on the immunology for giving protection.In order to select the suitable gene fragment in the vaccine that is included in, gene fragment preferably is selected from the virus that is used for vaccine use that WHO recommends.Certainly, according to the HA and the NA hypotype of the influenza variant that will inoculate against, HA and NA hypotype can change.Most preferably; Production is according to the influenza virus particles of conditionality defective of the present invention; It has the influenza A nucleic acid fragment of basic 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), and " basic coding " refers to specifically that in this article functional protein is by separately gene fragment expression.Especially, maybe can encode and such particle is provided in the segmental isolated cells of functioning gene of PA having functional PA.In another embodiment; Influenza virus particles according to conditionality defective of the present invention is provided in this article; It has the influenza A nucleic acid fragment of basic 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), and " basic coding " refers to specifically that in this article functional protein is by separately gene fragment expression.Especially, maybe can encode and such particle is provided in the segmental isolated cells of functioning gene of PB1 having functional PB1.In another embodiment; Influenza virus particles according to conditionality defective of the present invention is provided in this article; It has the influenza A nucleic acid fragment of basic 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), and " basic coding " refers to specifically that in this article functional protein is by separately gene fragment expression.Especially, maybe can encode and such particle is provided in the segmental isolated cells of functioning gene of PB2 having functional PB2.In another embodiment; The invention provides according to particle of the present invention, it contains the nucleic acid of the influenza peptides of can not encoding in addition, for example; Coding is used to cause immunoreactive foreign protein or peptide, or it contains the nucleic acid of interfere cell or the function of pathogenic agent in cell.

In addition, the invention provides the cell that comprises according to influenza virus particles of the present invention.When the gene fragment of the required polysaccharase of basic coding not being provided for particle; Usefully consider to be provided the cell of suitable functional influenza virus polysaccharase, allow defective influenza virus particles in the cell of compensation like this, to duplicate a plurality of cycles.

In addition, the invention provides compsn, it comprises according to defective influenza virus particles of the present invention or cell or is derived from the material according to cell of the present invention; Such compsn can for example be used to produce and is intended to generate the pharmaceutical composition that makes object avoid the immunoprotection of influenza infection.In addition, the invention provides generation and make object avoid the method for the immunoprotection of influenza infection, comprise that the object to needs provides such compsn.Except the application of particle according to the present invention as vaccine or immune composition; Preferably such compsn is mixed with vaccine; Promptly through the hybrid virus particle or be derived from such particulate viral protein (division-vaccine) and suitable pharmaceutical carrier, salts solution or adjuvant (aluminium salt or other vehicle (seeing for example http://www.cdc.gov/nip/publications/pink/Appendices/A/Excipient .pdf.) for example commonly used for example.Influenza virus particles according to conditionality defective of the present invention also is to be used for the alternative carrier that foreign protein was carried and expressed to alien gene, because can functioning gene for example be inserted between 5 ' and 3 ' the PA sequence.Consider the invention provides obtain the conditionality defective, possibly contain method external or host's nucleic acid fragment or its segmental influenza virus particles; It comprises: the first step; With first kind of suitable cell of following substances transfection: through inside deletion can the encode proteic nucleic acid of influenza and the one or more gene constructs of deutero-; Make described gene construct can not produce functional protein thus, and can not stop the gene fragment of virus is packed into virion; Complementary influenza nucleic acids fragment with the ability encoding influenza virus; With can in said cell, express said proteic one or more expression plasmids; Proper time point after transfection, at least one virion of results from the supernatant of described first kind of cell; Second step is with in said cell, expressing second kind of suitable cell of said proteic one or more expression plasmid transfections; In the 3rd step, infect described second kind of cell with the supernatant that comprises at least one virion that obtains from described first kind of cell; The 4th step was included in and infects the back proper time point, at least one virion of results from the supernatant of described second kind of cell.Therefore; The invention provides the method for the influenza virus particles that obtains the conditionality defective; It comprises: with the suitable cell of following substances transfection: through inside deletion can encode nucleic acid and the one or more gene constructs of deutero-of influenza polysaccharase; Make the described gene construct can not production function polysaccharase thus, and can not stop the gene fragment of virus is packed into virion; Complementary influenza nucleic acids fragment with the ability encoding influenza virus; With the 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 said cell.The described method that obtains the influenza virus particles of conditionality defective 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, use the supernatant of the influenza virus particles that comprises the conditionality defective to infect described cell; The 3rd step was included in and infects the back proper time point, at least one virion of results from the supernatant of said cell; Or obtain the method for the influenza virus particles of conditionality defective; It comprises: the first step; With first kind of suitable cell of following substances transfection: through inside deletion can encode nucleic acid and the one or more gene constructs of deutero-of influenza polysaccharase; Make the described gene construct can not production function polysaccharase thus, and can not stop the gene fragment of virus is packed into virion; Complementary influenza nucleic acids fragment with the ability encoding influenza virus; With the 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 kind of cell; Second step is with the second kind of suitable cell of one or more expression plasmid transfections that can in said cell, express said polysaccharase; In the 3rd step, infect described second kind of cell with the supernatant that comprises at least one virion that obtains from described first kind of cell; The 4th step was included in and infects the back proper time point, at least one virion of results from the supernatant of described second kind of cell.In the method, described polysaccharase can be for example acid polysaccharase (PA), alkaline polymerization enzyme 1 (PB1) or alkaline polymerization enzyme 2 (PB2).Preferably; The invention provides through can the encode nucleic acid of influenza polysaccharase of inside deletion and cause the method for inner disappearance; For PA albumen; Described inner disappearance begins from the 5 '-Nucleotide that between the Nucleotide 58 to 207 by the non-coding region meter, (does not contain end points), to the 3 '-Nucleotide end that between the Nucleotide that is risen by the non-coding region meter 27 to 194, (does not contain end points); Perhaps, begin, to the 3 '-Nucleotide end that between the Nucleotide that rises by the non-coding region meter 24 to 197, (does not contain end points) from the 5 '-Nucleotide that between Nucleotide 43 to 246, (does not contain end points) by the non-coding region meter for PB1 albumen; Perhaps, begin, to the 3 '-Nucleotide end that between the Nucleotide that rises by the non-coding region meter 27 to 209, (does not contain end points) from the 5 '-Nucleotide that between Nucleotide 34 to 234, (does not contain end points) by the non-coding region meter for PB2 albumen.In another variant, with external fragment insert should the inside disappearance in.In addition; The invention provides method with the supernatant cells infected of the influenza virus particles that comprises the conditionality defective; Described cell can be expressed non-functional polysaccharase; For example acid polysaccharase (PA), alkaline polymerization enzyme 1 (PB1) or alkaline polymerization enzyme 2 (PB2) and the influenza particle that can obtain through method as herein described.For example, use gene construct and nucleic acid fragment cells transfected can express non-functional polysaccharase.More specifically; The invention provides influenza virus particles; It comprises one or more nucleic acid fragments that in fragment, have inner disappearance; Said disappearance makes fragment can not produce functional influenza polysaccharase, and can not stop the gene fragment of virus is packed in the virion, and wherein polysaccharase is selected from acid polysaccharase (PA), alkaline polymerization enzyme 1 (PB1) or alkaline polymerization enzyme 2 (PB2).Preferably; Inner disappearance: for PA albumen; Begin from the 5 '-Nucleotide that between Nucleotide 58 to 207, (does not contain end points), to the 3 '-Nucleotide end that between the Nucleotide that rises by the non-coding region meter 27 to 194, (does not contain end points) by the non-coding region meter; For PB1 albumen, begin from the 5 '-Nucleotide that between Nucleotide 43 to 246, (does not contain end points), to the 3 '-Nucleotide end that between the Nucleotide that rises by the non-coding region meter 24 to 197, (does not contain end points) by the non-coding region meter; For PB2 albumen, begin from the 5 '-Nucleotide that between Nucleotide 34 to 234, (does not contain end points), to the 3 '-Nucleotide end that between the Nucleotide that rises by the non-coding region meter 27 to 209, (does not contain end points) by the non-coding region meter.In a preferred embodiment, the invention provides according to particle of the present invention, it has the influenza nucleic acids fragment of the VGP of encoding.The present invention also provides according to particle of the present invention; It has the influenza nucleic acids fragment of the nucleoprotein (NP) of encoding, alkaline polymerization enzyme 1 (PB1), alkaline polymerization enzyme 2 (PB2), hemagglutinin (HA), neuraminidase (NA), stromatin (M1 and M2) and Nonstructural Protein (NS1 and NS2); Particle perhaps is provided; It has the influenza nucleic acids fragment of the nucleoprotein (NP) of encoding, acid polysaccharase (PA), alkaline polymerization enzyme 2 (PB2), hemagglutinin (HA), neuraminidase (NA), stromatin (M1 and M2) and Nonstructural Protein (NS1 and NS2); Particle perhaps is provided, and it has the influenza nucleic acids fragment of the nucleoprotein (NP) of encoding, acid polysaccharase (PA), alkaline polymerization enzyme 1 (PB1), hemagglutinin (HA), neuraminidase (NA), stromatin (M1 and M2) and Nonstructural Protein (NS1 and NS2).More specifically, the invention provides according to particle of the present invention, it has the influenza nucleic acids fragment that is derived from influenza A virus.The present invention also provides according to particle of the present invention, and it contains the nucleic acid of the influenza peptides of not encoding.In addition, the invention provides and comprise according to particulate cell of the present invention, more specifically, cell has contained one or more influenza virus polymerases, and wherein polysaccharase is selected from acid polysaccharase (PA), alkaline polymerization enzyme 1 (PB1) or alkaline polymerization enzyme 2 (PB2).In addition; The invention provides compsn; It comprises according to particle of the present invention or cell or is derived from the material according to cell of the present invention; Such compsn is intended to generate the application in the pharmaceutical composition of the immunoprotection that makes object avoid influenza infection and produces in production makes object avoid the method for the immunoprotection of influenza infection, comprises that the object to needs provides such compsn.In addition; The invention provides particle according to the present invention and be intended to the application in the pharmaceutical composition in the cell that nucleic acid with the influenza peptides of not encoding flows to object in production at application and the particle according to the present invention that production is intended in the compsn that nucleic acid with the influenza peptides of not encoding flows to cell.Such nucleic acid (being also referred to as external nucleic acid in this article) can encode alien gene or gene fragment, epitope that it is suitable that the latter can encode or albumen, the one section Nucleotide of perhaps can encoding, the transcribed nucleic acid in latter's interfere cell.In one embodiment, the invention provides influenza A virus particle according to the present invention and be intended to the application in the compsn in the cell that nucleic acid with the influenza peptides of not encoding is delivered into cell or object in production.In addition, the nucleic acid that the invention provides the influenza peptides of not encoding is delivered into the method in cell or the object, comprises defective influenza virus particles to said cell or described object is provided, and it contains with good grounds external nucleic acid of the present invention.

The accompanying drawing legend

The legend of Fig. 1

The production of the influenza A virus of conditionality defective and breeding.P Δ PA in the time of at first, and suitably or pDIPA transfection with the two-way plasmid of 7 A/PR/8/34 that can encode, pHMG-PA the 293T cell.After the transfection 48 hours, the supernatant of results cells transfected, and be used to inoculate to mdck cell and 24 hours mdck cells with the HMG-PA transfection in advance.The supernatant of virus replication male MDCK-PA cell is gone down to posterity 4 times in MDCK and MDCK-PA cell.

The legend of Fig. 2

The construct that is used for the virion of working condition property defective.Last figure has shown wild-type PA gene fragment.Non-coding region (NCR) and initiator codon have been indicated.Through digesting pHW183 with StuI, a kind of two-way plasmid (9) that contains the PA of A/WSN/33, and connection have again subsequently made up p Δ PA.5 ' 194 and 3 ' 207nts through with the PA gene fragment of A/PR/8/34 is cloned among the pSP72, has made up pDIPA.Then inset is shifted to two-way reverse genetics carrier.As described herein, made up p Δ PB1 and p Δ PB2.

The legend of Fig. 3

RT-PCR analyzes the PA gene fragment at supernatant rPR8-7, the existence among rPR8-Δ PA and the rPR8-DIPA.Make MDCK-PA the 4th generation supernatant pass 22 μ M strainers, and centrifugal concentrating.Subsequently, isolation of RNA, and use primer to the segmental non-coding region of PA, carry out RT-PCR.Use from the isolated RNA of wild-type A/PR/8/34 as contrast.Swimming lane 1:rPR8-7; Swimming lane 2:rPR8-Δ PA; Swimming lane 3rPR8-DIPA; Swimming lane 4: wild-type A/PR/8/34.The affinity tag size is indicated in the left side.

The legend of Fig. 4

Delete the major part of p Δ PA construct again, produced p Δ PA-2, p Δ PA-3, p Δ PA-4, p Δ PA-5.

Detailed Description Of The Invention

Embodiment 1

Generate defective influenza A virus particle from recombinant DNA

Influenza A virus is minus strand, sectional virus.Its genome is made up of 8 gene fragments.All 8 functioning gene fragments are that the production infectious virus is necessary, and promptly described infectious virus is reproducible virus, and it can duplicate in it has been generally acknowledged that the cell that suitable influenza virus is duplicated unlimited or several at least cycles.No matter the wrapping process of the gene fragment of influenza A virus is through at random or specific mechanism, all argues many years.Some evidences of these 2 kinds of selections have been described.The evidence of packing is at random; The accumulative virion has the infectivity (6) than the accumulative virion is not higher; And when the time with low moi cells infected culture; The cell of some infection can not be expressed a fragment (8), this two show that existence does not contain the virus particle of complete influenza virus gene group.Other evidence of packing is in experiment, to have produced and contained 9 segmental influenza viruses (4) at random.Bancroft and Parslow find, is derived from not competition (1) aspect the packaging virus particle between the vRNA of identical gene fragment.

Support the reason of specificity wrapping process to be for one, although all gene fragments all ana be present in the virus stock solution used, they are present in different amounts and produce in the cell (10).In addition, when having generated defective interference (DI) particle, DI vRNA can replace its fragment that is derived from (3), and (defective interference particle is the virion that one of them gene fragment has bigger inside disappearance.When virus goes down to posterity with high moi, these particles can occur, and think that the reason of its generation is the R638A sudden change [Fodor etc. of polysaccharase acidic protein; J.Virol.77,5017-5020,2003]).At last, the efficient of virus particle formation increases (5) along with the increase of gene fragment number.Fujii etc. have also confirmed NA fragment district, and it is that fragment is integrated into virus particle effectively is necessary, and this study group had also confirmed HA and NS district afterwards, and it advances virion for packing is important [Fujii, 2005#256; Watanabe, 2003#184].

Here, we have proposed the evidence of specificity packing.Only contain 7 segmental virions of functioning gene in order to produce, we must confirm to save which gene fragment, and do not hinder virus production.In view of the application of the virus of replication defective as vaccine arranged, do not save HA and NA, do not save MA or NS, because need 2 expression plasmids that separate yet.We have produced the virus that lacks pol gene.When the gene fragment of the trans compensation of expression plasmid of no use disappearance, we can not produce virus (table 1,2 and 3, rPR8-7ntc).Behind 7 gene fragments and the proteic plasmid transfection that can express with low-down titre usually by the gene fragment expression of disappearance, can produce virus (table 1,2 and 3, rPR8-7).Therefore, produced the gene fragment 1,2 of influenza virus A/WSN/33 and 3 deletion mutant, it carries the inside disappearance of 1032,528 and 1120 Nucleotide respectively.With these deletion mutant called afters p Δ PB2, p Δ PB1 and p Δ PA (see figure 2).(de Wit as previously mentioned; E., M.I.Spronken, T.M.Bestebroer; G.F.Rimmelzwaan; A.D.Osterhaus, and R.A.Fouchier.2004.Efficient generation and growth of influenza virus A/PR/8/34from eight cDNA fragments.Virus Res 103:155-61), with each and can the encode two-way construct (DeWit etc. of A/PR/8/34 of 7 complementary in the gene fragment of these disappearances; 2004) and suitable expression plasmid, transfection the 293T cell.After transfection 48 hours, the results supernatant.Subsequently, expression plasmid HMG-PB2 is used in (2) as previously mentioned, among HMG-PB1 or the HMG-PA one, transfection mdck cell.These cells transfected are inoculated the corresponding supernatant (see Fig. 1, it has explained experimental technique) to the 293T cell of transfection.Through the HA-experiment, confirmed the virus replication in these mdck cells.During beginning, in the mdck cell of untransfected, there is not virus replication.Using HMG-PB2, HMG-PB1 or HMG-PA transfection, be inoculated in the mdck cell in the corresponding supernatant, found virus replication.Then, from the influenza sequence library ( Www.flu.lanl.gov, registration number K00867) and on the basis of sequence of defective interference PA vRNA of influenza virus A/PR/8/34 of obtaining, we have cloned defective PA gene fragment.PCR-'s 5 ' 207nt and 3 ' 194nt of PA has increased, the clone advance to be derived from as previously mentioned (De Wit etc., 2004) in the bidirectional transcription vector of the pHW2000 (7) that modifies.The plasmid that obtains is called the pDIPA (see figure 2).Use pDIPA, the two-way construct transfection of HMG-PA and 7 remaining influenza virus A/PR/8/34 gene fragments of encoding 293T cell (see figure 2).After the transfection 48 hours, the results supernatant subsequently, was inoculated to this supernatant with the mdck cell of HMG-PA transfection 24 hours in advance.Inoculate back 72 hours, in the supernatant of these mdck cells, carry out the HA-experiment, discovery is a male, and this is indicating the virus replication in these cells.As determined through the HA-experiment, the mdck cell of inoculation untransfected can not cause virus production.Subsequently untransfected or transfection on the mdck cell of HMG-PA, the supernatant of the virion that contains the PA-defective of going down to posterity has produced identical result's (table 1).Up to the 4th generation, in mdck cell, produced virus with the HMG-PA transfection.Serial dilution the supernatant of MDCKp4, obtain the indication (indication) of virus titer, confirm that it is about 10 ₄TCID ₅₀/ ml.

The method steps that uses is: with construct p Δ PB2, p Δ PB1, p Δ PA; Among the p Δ NP one, the complementary construct of 7 A/PR/8/34 that can encode (De Wit etc., 2004) and HMG-PB2; HMG-PB1, (expression plasmid is described in for example Pleschka, S. among the HMG-PA; 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.; Available from A.Garcia-Sastre andP.Palese), transfection the 293T cell (about transfection method, see De Wit etc., 2004).After transfection 48 hours, the supernatant of the 293T cell of results transfection.When having generated when viral, they are present in the supernatant.Simultaneously, use expression plasmid HMG-PB2, HMG-PB1; Among the HMG-PA one (according to the deletion mutant that uses, so under the situation of using p Δ PB2, then use HMG-PB2 transfection mdck cell) transfection mdck cell (about transfection method; See Basler etc.; 2000),, the virus of producing to express this proteic gene fragment, because they have packed the gene fragment that contains inner disappearance because lacking.After transfection 24 hours, the mdck cell of transfection is inoculated the supernatant that obtains to 293T cell from transfection.When virus was present in the 293T supernatant, this virus can be duplicated in the mdck cell of transfection, and produced more virus.In inoculation back 72 hours, can collect supernatant once more.

In order to confirm that not generation can produce the PA or the DIPA reorganization of functional PA gene fragment, has isolated RNA from the supernatant of MDCKp4.At first, make supernatant pass 22 μ M strainers, and centrifugal concentrating.Subsequently, isolation of RNA, and use primer to the segmental non-coding region of PA, carry out RT-PCR.Use has confirmed that to the RT-PCR that the specific primer of PA vRNA carries out Δ PA and DIPA are through still stable after repeatedly going down to posterity.In the supernatant of the mdck cell that has infected the DIPA virion, the clear band of about 400bp has appearred, and in the supernatant of the mdck cell that has infected the virus that contains Δ PA, the band of 1100bp has appearred.In the supernatant of the mdck cell that has infected wild-type A/PR/8/34, the band (Fig. 3) of visible about 2300nt.These results show that Δ PAPR8 gene fragment has stably been packed in the virus particle.

In order to produce the virus that lacks PB2, with the gene fragment 2,3 of 7 ability encoding influenza virus A/PR/8/34; 4,5,6; 7 and 8 (de Wit, E., M.I.Spronken; T.M.Bestebroer, G.F.Rimmelzwaan, A.D.Osterhaus; With R.A.Fouchier.2004.Efficient generation and growth of influenza virus A/PR/8/34from eight cDNA fragments.Virus Res 103:155-61) two-way construct transfection 293T cell (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 plasmids.Proc Natl Acad Sci U S A97:6108-13.), caused the expression of vRNA and mRNA.Cotransfection can express the PB2 of A/PR/8/34, the plasmid (Pleschka of pHMG-PB2; 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 contrast, only transfection 7 two-way constructs of the A/PR/8/34 that can encode, saved pHMG-PB2.After the transfection 48 hours, the results supernatant, and inoculation give mdck cell or 24 hours in advance in the 100mm dish transfection mdck cell of pHMG-PB2 (MDCK-PB2).Inoculate back 3 days, use the indicator of turkey red corpuscle, tested the hemagglutination activity of the supernatant of the mdck cell of inoculating as virus production.In inoculated only transfection 7 gene fragments, do not have not detect virus in the cell of supernatant of 293T cell of transfection pHMG-PB2 (rPR8-7ntc, table 2).The MDCK-PB2 cell conditioned medium liquid of the 293T cell conditioned medium liquid of inoculated transfection 7 gene fragments and pHMG-PB2 is male.Subsequently, the rPR8-7 supernatant is gone down to posterity in MDCK and MDCK-PB2 cell.RPR8-7 can duplicate in the MDCK-PB2 cell, but can not (table 2) in mdck cell.We have then produced the 1032nt deletion mutant of the gene fragment 1 of influenza virus A/WSN/33, produced 344 amino acid whose disappearances (p Δ PB2, Fig. 2).As stated, produced the recombinant virus (Fig. 1) that contains Δ PB2 (rPR8-Δ PB2).In mdck cell, do not detect virus, and in the MDCK-PB2 cell of having inoculated rPR8-Δ PB2, detected virus.Go down to posterity behind the rPR8-Δ PB2, in mdck cell, do not have the viral evidence that generates, this and MDCK-PB2 cell be (table 2) on the contrary.

Also produced the virus that lacks PB1.With can encoding influenza virus A/PR/8/34 gene fragment 1,3,4,5,6,7 and 87 two-way construct transfections the 293T cell, caused the expression of vRNA and mRNA.Cotransfection can express the PB1 of A/PR/8/34 and the plasmid of pHMG-PB1.As contrast, only transfection 7 two-way constructs of the A/PR/8/34 that can encode, saved pHMG-PB1.After the transfection 48 hours, the results supernatant, and inoculation give mdck cell or 24 hours in advance in the 100mm dish transfection pHMG-PB1 (MDCK-PB1) mdck cell (2) (Fig. 1).Inoculate back 3 days, use the indicator of turkey red corpuscle, tested the hemagglutination activity of the supernatant of the mdck cell of inoculating as virus production.In inoculated only transfection 7 gene fragments, do not have not detect virus in the cell of supernatant of 293T cell of transfection pHMG-PB1 (rPR8-7ntc, table 3).The MDCK-PB1 cell conditioned medium liquid of the 293T cell conditioned medium liquid of inoculated transfection 7 gene fragments and pHMG-PB1 is male.Subsequently, the rPR8-7 supernatant is gone down to posterity in MDCK and MDCK-PB1 cell.RPR8-7 can duplicate in the MDCK-PB1 cell, but can not (table 3) in mdck cell.We have then produced the 528nt deletion mutant of the gene fragment 2 of influenza virus A/WSN/33, produced 178 amino acid whose disappearances (p Δ PB1, Fig. 2).As stated, produced the recombinant virus (Fig. 1) that contains Δ PB1 (rPR8-Δ PB1).In mdck cell, do not detect virus, and in the MDCK-PB1 cell of having inoculated rPR8-Δ PB1, detected virus.Go down to posterity behind the rPR8-Δ PB1, in mdck cell, do not have the viral evidence that generates, this and MDCK-PB1 cell be (table 3) on the contrary.

Thereby we can use the PB2 of aforesaid rna plymerase ii-driving, PB1 or PA expression plasmid, and through p Δ PB2 is provided, p Δ PB1, or p Δ PA/pDIPA construct and trans-complementation produce and lack fragment 1,2, or 3 virus.The virus of conditionality defective described here can only experience duplicating of 1 cycle in the cell of non-trans compensation, but can in the clone of trans-complementation, breed.This is to use reverse genetics to produce defective virus first, and it only contains 7 functioning gene fragments, and can experience duplicating of 1 cycle, perhaps when having compensated defective gene fragment, can experience duplicating of a plurality of cycles when trans.

The defective virion of producing with this mode is the vaccine candidate article, because they can experience duplicating of 1 cycle, and does not generate infectious virus.The result that this monocycle duplicates is that vaccine can be induced body fluid and cell immune response.Although these defective particles can not duplicate in normal cell,, a large amount of viruses are grown in expressing defective proteic clone for the production purpose.Verified as us, duplicating of a plurality of cycles can not influence viral genotype.Except the application of defective virion as vaccine, they also are candidate's carriers that foreign protein was carried and expressed to gene, because can functioning gene be inserted 5 ' and 3 ' PA, between PB2 or the PB1 sequence.This has also obtained the confirmation of (11) such as Watanabe, and they have replaced HA and NA with alien gene, still can generate virus.

The further brachymemma of p Δ PA

In addition, delete the more most of of p Δ PA construct, produced p Δ PA-2, p Δ PA-3, p Δ PA-4, p Δ PA-5 (Fig. 4).As previously mentioned (De Wit etc., 2004), with 7 two-way constructs of complementation (De Wit etc., 2004) of one of the gene fragment of these disappearances and the A/PR/8/34 that can encode and can express PA the expression plasmid transfection 293T cell.After the transfection 48 hours, the results supernatant.Subsequently, as previously mentioned (Basler, C.F., etc., 2000.Proc Natl Acad Sci U S A 97:12289-94.), with expression plasmid HMG-PA transfection mdck cell.Inoculated the cell of these cells transfected and untransfected with the supernatant of the 293T cell of transfection.Through the HA-experiment, detected the virus replication in these MDCK and the MDCK-PA cell.In the mdck cell of untransfected, there is not virus replication.In the mdck cell of the HMG-PA transfection of having inoculated any supernatant, found virus replication.Thereby all vRNA that are derived from these constructs have packed into virus particle (table 4).

Table 1. lacks recombinant influenza reassortant virus the duplicating in MDCK and MDCK-PA cell of complete PA gene fragment

Ntc: (in the 293T cell, not the having transfection pHMG-PA) of trans compensation not

Table 2. lacks recombinant influenza reassortant virus the duplicating in MDCK and MDCK-PB2 cell of complete PB2 gene fragment

Ntc: (in the 293T cell, not the having transfection pHMG-PB2) of trans compensation not

Table 3. lacks recombinant influenza reassortant virus the duplicating in MDCK and MDCK-PB1 cell of complete PB1 gene fragment

Ntc: (in the 293T cell, not the having transfection pHMG-PB1) of trans compensation not

Table 4. lacks recombinant influenza reassortant virus the duplicating in MDCK-PA and mdck cell of complete PA gene fragment

Embodiment 2

With defective recombinant virus vaccination

On the basis of the high throughput virus main chain (for example being derived from vaccine strain A/PR/8/34) of HA that contains relevant prevailing disease virus (for example A/ Moscow/10/99) and NA gene; As described herein, produced the recombinant virus that lacks functional PA, PB1 or PB2 gene of conditionality defective.Produced the virus of conditionality defective through transfection, wherein realized the polymerase protein expression through trans-complementation.Subsequently, in the virus that can stably express have increased in the suitable cell matrix (for example mdck cell or Vero cell) of relevant polysaccharase.Through centrifugal 10 minutes, clarified viral supernatant at 1000xg.Ultra centrifugal through in the 20-60% saccharose gradient, carrying out, concentrated with purifying virus, post precipitation is re-suspended in the phosphate buffered saline (PBS) (PBS).Use the 12.5%SDS-polyacrylamide gel of coomassie brilliant blue staining; The purity and the quantity of virus product have been confirmed; Through the infection of mdck cell and the mdck cell that can express relevant polysaccharase with anti--nucleoprotein monoclonal antibody dyeing, confirmed the virus titer of the virus of conditionality defective.Use atomizer, inoculate 1x10550% tissue-culture infective dose (TCID-50) for interior ground of mouse tracheae or nose interiorly.Use hemagglutination to suppress experiment, neuraminidase inhibition experiment, ELISA or virus neutralization experiment, confirmed HA, NA and inner proteic antibody titers before the vaccination and in the serum sample of collecting afterwards to influenza virus.Intracellular cytokine-expressing is measured in tetramer dyeing through flow cytometer, CD4 and CD8-positive cell, lysis activity, T-cell proliferation etc., and the cell immune response of the antigen-specific in the vaccinated animal has been carried out quantitatively.In 6 weeks after the vaccination, use 1x10 ⁶Influenza virus A/Moscow of TCID-50/10/99 or allos virus isolated strain are attacked vaccinated animal and control animal.After the attack, that collect nose from animal every day or throat swab sample, carried out 10 days, through quantitative PCR analysis or titration of virus, confirmed the amount of the virus that infection animal is discharged.Carry out quantitatively through the rising of antagonist titre; Confirmed the vaccine-induced humoral immunization that obtains; Through the rising of auxiliary and cytotoxic T-cell response is carried out quantitatively; Confirmed the vaccine-induced cellular immunization that obtains, through confirming to have confirmed immune aggregate level to the protection of the infection of challenge virus.

Reference

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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-specificmanner.Virology?216：326-37.

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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?A?virus?from?eight?plasmids.Proc?Natl?Acad?Sci?U?S?A?97：6108-13.

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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.

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