CN1347458A - Production of attenuated negative stranded RNA virus vaccines from cloned nucleotide sequences - Google Patents

Abstract

Attenuated, recombinant negative stranded RNA viruses suitable for vaccine use are produced from one or more isolated polynucleotide molecules encoding the virus. A recombinant genome or antigenome of the subject virus is modified to encode a mutation within a recombinant protein of the virus at one or more amino acid positions(s) corresponding to a site of an attenuating mutation in a heretologous, mutant negative stranded RNA virus. A similar attenuating mutation as identified in the heterologous negative stranded RNA virus is thus incorporated at a corresponding site within the recombinant virus to confer an attenuated phenotype on the recombinant virus. The attenuating mutation incorporated in the recombinant virus may be identical or conservative in relation to the attenuating mutation identified in the heterologous, mutant virus. By the transfer of mutations into recombinant negative stranded RNA viruses in this matter, candidate vaccine viruses are engineered to elicit a desired immune response against a subject virus in a host susceptible to infection thereby.

Description

Prepare attenuated negative stranded RNA virus vaccines from clone's nucleotide sequence

Background technology

Minus-stranded rna virus includes the single negative strand viruses (Mononegavirales) of the important and high-destructive pathogenic agent of various objectives.Human pathogen in this group comprises rabies virus (RaV), Measles virus (MeV), mumps virus (MuV), respiratory syncytial virus (RSV) and several genotypic parainfluenza viruses (PIV).With RSV is example, because it causes pneumonia and bronchitis disease one-year-old in interior baby, this pathogen hazard is positioned on other all microbial pathogenes.In the children that are hospitalized for treatment because of respiratory tract disease, by accounting for more than 1/5 that RSV causes, RSV causes estimating at 91,000 patients in 1 year in the U.S. and is in hospital and 4,500 death.Human PIV virus (for example, HPIV1, HPW2 and HPIV3) also causes very serious disease in the crowd, it can cause bronchitis, heavy breathing larynx and pneumonia, especially in the infant.Karron etc., J.Infect.Dis.172:1445-50 (1995); Collins etc., " parainfluenza virus " (Parainfluenza Viruses), p.1205-1243.In editors' such as B.N.Fields Fields Virology, the third edition, the first roll, Lippincott-Raven press, Philadelphia (1996); Murphy etc., virus research (Virus Res.), 11:1-15 (1988).Infect in the colony of being in hospital due to the human PIV virus and occupy near 20% in infant's respiratory tract infection.

Although also develop several effective vaccines with antagonism RSV and PIV through the research of many decades, also not have to obtain not only safely but also effective vaccine to reduce the M ﹠ M of serious rsv infection.Need develop equally effective vaccine or the improvement vaccine resist other important single negative strand viruseses.The obstacle that hinders the living vaccine development of anti-minus-stranded rna virus is the difficulty that averages out between attenuation and immunogenicity.The genetic stability of attenuated virus is a problem equally.In the evolution of RSV vaccine, also run into difficulties such as the relatively poor and virion unstable of RSV growth property in cell cultures.Another feature of rsv infection is to induce the immunity of generation and the not exclusively protection infection of generation subsequently.The reason that produces this situation is a lot, comprise immunity system relative poor efficiency aspect the surface-limited virus infection of the chamber of respiratory tract, local mucous membrane immunity short-term, reach virus replication widely fast, among the infant since immunity system immature due to the immune response of attenuation, because the immunosuppression of mother's serum antibody due to the placenta transmission, and some feature such as the proteic high glycosylation of G of virus.Such equally just as discussed below, RSV exists with the form of two antigenic subtype A and B, resists the immunity of one of them and renders a service lower to another hypotype.

In the twentieth century middle period sixties, the virus vaccines of formalin-inactivated is tested as the vaccine of anti-RSV, but does not succeed in the protection of anti-rsv infection or disease, and in fact subsequently virus infection presents the symptom of deterioration.(Kim etc., Am.J.Epidemiol., 89:422-434 (1969), Chin etc., Am.J.Epidemiol., 89:449-463 (1969); Kapikian etc., Am.J.Epidemiol., 89:405-421 (1969)).Recently, the RSV developing vaccines has focused on the attenuation RSV mutant strain.Friedewald etc., J.Amer.Med.Assoc.204:690-694 (1968) have reported a kind of cold mutant strain that goes down to posterity (cpRSV) of RSV, and it is become candidate vaccine by abundant attenuation.This mutant strain is compared with wild-type (wt) parental virus, demonstrates the ability 26 ℃ of growths of slight increase, but it duplicates and does not both have temperature sensitivity also not have tangible acclimatization to cold.Yet, but the cold mutant strain attenuation that goes down to posterity is used for the grownup.Although by the infant of rsv infection (for example, seropositive individuality), cpRSV is attenuated satisfactorily and has immunogenicity for, cpRSV still keeps the low-level toxicity for the seronegativity baby upper respiratory tract.

Similarly, people such as Gharpure, Journal of Virology (J.Virol.) 3:414-421 (1969) have reported temperature sensitivity RSV (tsRSV) mutant strain as valuable candidate vaccine.A kind of mutant strain ts-1 is widely used among laboratory and the volunteer.Mutant produces symptomless infection and causing immunity in the volunteer that grows up attacked the generation resistance to wild-type virus after 45 days.Baby that the contrast sero-reaction is positive and the children that suffer symptomless infection, the baby that sero-reaction is negative presents rhinitis sign and other slight symptom.In addition, the stability of ts phenotype is detected, although show the virus of part or all of temperature sensitivity disappearance account for the virus that can regain from vaccine than small proportion, and be not associated with disease sign except that slight coryza.

These and other studies show that specific cold go down to posterity and temperature sensitivity RSV strain causes slight genius morbi by insufficient attenuation and vaccine inoculation person; especially seronegative baby; and the super attenuation of other quilt and can not duplicate the immunne response that causes protectiveness fully; (Wright etc.; Infect.Immun., 37:397-400 (1982)).In addition, the genetic instability of candidate vaccine mutant strain causes the disappearance of its temperature sensitivity phenotype, has further hindered effective RSV developing vaccines.Usually referring to, Hodes etc., Proc.Soc.Exp.Biol.Med.145:1158-1164 (1974), McIntosh etc., Pediatr.Res.8:689-696 (1974), and Belshe etc., J.Med.Virol., 3:101-110 (1978).

Abandoned by for example cold the going down to posterity of uncertain biological method and made up the method for suitable attenuation RSV strain, the investigator tests subgroup vaccine candidate strain by the RSV envelope glycoprotein of purifying.Glycoprotein is induced the resistance to rsv infection in the lung of cotton mouse, Walsh etc., J.Infect.Dis.155:1198-1204 (1987), it is active that but antibody has very weak neutralization, and can cause the reinforcement of disease with the subgroup vaccine immunity rodent of purifying, this makes the people associate the RSV vaccine of before having handled (Murphy etc., Vaccine 8:497-502 (1990)).

The vaccine based on the vaccine virus recombinant chou of expressing F or G envelope glycoprotein has been carried out research.These recombinant chous are expressed can not be by the rsv glycoprotein that differentiates from real viral counterpart, with vaccine-RSV F and G recombinant chou through the rodent that skin infects show high-caliber in the specific antibody of virus infectivity.In fact, cotton mouse infects with vaccine F recombinant chou can stimulate resistance almost completely that RSV is duplicated at lower respiratory tract and the tangible resistance in the upper respiratory tract.Olmsted etc., NAS's journal (Proc.Natl.Acad.Sci.USA) 83:7462-7466 (1986).Yet; with vaccine F and G recombinant chou immunity chimpanzee; protection (the Collins etc. that in the upper respiratory tract, almost do not provide anti-RSV to attack; Vaccine8:164-168 (1990)); differ before and after its protection that in lower respiratory tract, provides (Crowe etc., vaccine (Vaccine) 11:1395-1404 (1993)).

The unsuccessful of biological attenuated vaccine strain, subgroup vaccine and other vaccine development strategy at RSV and other minus-stranded rna virus makes that pressing for a kind of new method develops new vaccine, particularly the method for artificial recombination candidate vaccine adds the heredity variation, has the attenuation RSV recombinant chou alive of new phenotypic characteristic with production.Yet, to the manipulation of the geneome RNA of RSV and other sense-rna virus handle be still so far very difficult.Unstable, genome complexity and the gene product structure of the bad growth of non-infectious, the virus of exposed geneome RNA that major obstacle in this respect comprises these viruses in tissue culture, tediously long replication cycle, virosome be difficult to control.

With PIV3 is example, and two attenuation candidate vaccines alive have obtained special concern, this one of them candidate vaccine be ox PIV3 (BPIV3) strain it is relevant on antigenicity with HPIV3, and it has shown the opposing HPIV3 that watches for animals.BPIV3 is an attenuation, inheritance stability, and be to have immunogenic (Karron etc., J.Inf.Dis.171:1107-14 (1995a) in human infant; Karron etc., J.Inf.Dis.172:1445-1450, (1995b)). another PIV3 candidate vaccine JS cp45, be JS wild-type (wt) strain of HPIV3 cold-adapted mutant (Karron etc., (1995b), the same; Belshe etc., J.Med.Virol.10:235-42 (1982)).This work attenuation, cold going down to posterity (cp) PIV3 candidate vaccine show temperature sensitivity (ts), acclimatization to cold (ca) and have stable attenuation (att) phenotype behind the virus replication in vivo.Cp45 virus can produce the active of anti-human PIV3 in laboratory animal and it is an attenuation, inheritance stability, and in seronegative human infant, have immunogenicity (Hall etc., virus research 22:173-184 (1992); Karron etc., J.Infect.Dis.172 (6): 1445-1450 (1995b), the same).

Recombinant DNA technology makes that regaining level that infectious minus-stranded rna virus, genetic manipulation virus clone make up new candidate vaccine and its attenuation of Fast estimation and phenotypic stability from cDNA becomes that possible (summary is referring to Conzelmann, J.Gen.Virol.77:381-89 (1996); Palese etc., Proc.Natl.Acad.Sci.U.S.A.93:11354-58, (1996)).In this literary composition, reported in the presence of must viral protein by the antigenomic RNA of cDNA coding recombinate rescue communicable respiratory syncytial virus (RSV), human parainfluenza virus 3 (HPIV3), rabies virus (RaV), vesicular stomatitis virus (VSV), Measles virus (MeV), Sendai virus (Sev), rinderpest virus, S type simian virus and ox RSV (referring to, for example, Garcin etc., EMBO are (1995) J.14:6087-6094; Lawson etc., NAS's journal (Proc.Natl.Acad.Sci.U.S.A.) .92:4477-81 (1995); Radecke etc., EMBO are (1995) J.14:5773-5784; Schnell etc., EMBO are (1994) J.13:4195-203; Whelan etc., the journal .92:8388-92 of NAS (1995); Hoffman etc., Journal of Virology (JVirol.) 71:4272-4277 (1997); Kato etc., Genes to Cells 1:569-579 (1996), Roberts etc., virusology (Virology) 247 (1), 1-6 (1998); Baron etc., Journal of Virology (J Virol.) .71:1265-1271 (1997); International Application No. WO 97/06270; Collins etc., the journal 92:11563-11567 of NAS (1995); Durbin etc., virusology (Virology) 235:323-332 (1997); Patent application U.S.No.08/892,403, apply on July 5th, 1997, (corresponding to International Application No. WO 98/02530, right of priority is a U.S. Provisional Application 60/047,634 (applying on May 23rd, 1997), 60/046,141 (applying on May 9th, 1997) and 60/021,773 (applying on July 15th, 1996)); Juhasz etc., J.Virol.71 (8): 5814-5819 (1997); Virology237:249-260 such as He (1997); Whitehead etc., virusology 247 (2): 232-9 (1998a); Whitehead etc., Journal of Virology 72 (5): 4467-4471 (1998b); Jin etc., virusology 251:206-214 (1998); Bucholz etc., Journal of Virology 73:251-259 (1999); And Whitehead etc., Journal of Virology 73:(4) 3438-3442 (1999), described document all is incorporated herein for referencial use).

Although use the reverse genetics means with reclaim and modify reorganization, minus-stranded rna virus is feasible, the exigence in this area still be to use other instrument and method with produce safe and efficient vaccine to alleviate because single negative strand viruses such as RSV, PIV and other pathogenic agent to healthy grievous injury.Other challenges as herein described are difficulty of obtaining the candidate vaccine of appropriate attenuation, immunogenicity and inheritance stability.For reaching this purpose, the method for existing discriminating attenuation sudden change and introducing attenuation sudden change in recombinant virus must further improve and improvement.Surprisingly, the present invention has realized this purpose and additional advantage as mentioned below is provided.

Summary of the invention

The invention provides new method and composition with design and produce be suitable for attenuation that vaccine uses, recombinant negative strand rna virus.According to the method described in the present invention, recombinant negative strand rna virus produce should virus from one or several separated coding polynucleotide.This can finish by one or more polynucleotide molecules that coexpression in cell or cell-free system is encoded this virus recombination group or anti-genome and produced infectious virus and the necessary viral protein of particle thereof.

The recombination group of theme virus and anti-genome are through modifying with the sudden change of encoding, it changes at one of a recombinant viral proteins or several amino acid positions, and these sites are corresponding to the attenuation mutational site at an allos, sudden change minus-stranded rna virus.Sudden change is therefore in adoptable or multiple mode " transfer ", to give this recombinant virus attenuation phenotype.By this way, candidate vaccine virus is by modified recombinant, and to cause immune response, described immune response can be resisted selected minus-stranded rna virus in being easy to by the host of theme virus infection.

Aspect related to the present invention some, isolating polynucleotide molecule and carrier are provided, its attenuation of can encoding, reorganization negative strand viruses genome or anti-genome.The sudden change relevant with above-mentioned aspect, that one of theme genome or anti-genome encoding take place on the amino acid position corresponding to the attenuation mutational site in allogenic, the sudden change minus-stranded rna virus in the viral recombinant protein of selecting.

The invention provides equally and comprise method and composition that as above suddenly change, attenuation, recombinant negative strand rna virus, with prevention and treatment by the caused infection of theme virus.

In a preferred embodiment of the invention, recombinant negative strand rna virus can be a respiratory syncytial virus (RSV), parainfluenza virus (PIV) or Measles virus.Be that heterology, sudden change minus-stranded rna virus can be a heterology RSV, human parainfluenza virus (HPIV1, HPIV2, HP1V3), ox PIV (BPIV), Sendai virus (SeV), Avian pneumo-encephalitis virus (NDV), SV 41 virus (SV5), mumps virus (MuV), Measles virus (MeV), canine distemper virus (CDV), rabies virus (RaV) or vesicular stomatitis virus (VSV) with each embodiment all is associated.

The attenuation sudden change is introduced in the proteic corresponding site of a heterology that different target proteins should be of value in a minus-stranded rna virus, and itself and the method for the invention adapt.In single negative strand viruses order, five target proteins are all strict conservative and demonstrate the sequence identity of appropriateness to height in the specific region.Particularly, all members of this order have five proteic homologies bunch: a nucleocapsid protein (N), a nucleocapsid phosphorprotein (P), a non-glycosylated matrix (M) albumen, at least one surface glycoprotein (HN, H or G) and a big polysaccharase (L) albumen.These albumen all are to be applicable to change one or more conserved residues on the site in the attenuation mutational site of differentiating in the albumen at recombinant virus to introduce the target protein of attenuation sudden change in corresponding to the strain of heterology mutated viruses.

The present invention more detailed aspect, can other albumen of target, it is confined to only by section special in single negative strand viruses, subfamily, genus or albumen that kind had.For example, all members of Paramyxoviridae have two kinds of surface glycoproteins at least, HN (or H or G) and F.A rich cysteine protein V is all arranged in the nearly all member of Respirovirus, Rubulavirus and Morbillivirus.Homology C albumen of the same coding of Respirovirus and Measles virus.The hypotype of Pneumovirus and Rubulaviruses (SV 41 virus (SVS) and mumps virus (MuV)) all has homology surface glycoprotein SH.In Pneumovirus (comprise ox, sheep and billy goat RSV and mouse Pneumovirinae---also can be described as mouse RSV here), several other albumen, promptly NS1, NS2, M2 (ORF1) and M2 (ORF2) they are conservative propertys.The birds Pneumovirinae lacks NS1 and NS2 but has M2 (ORF1), M2 (ORF2) and SH albumen.Aforesaid each albumen provides a useful target, and it can have allos transfer attenuation sudden change between the proteic taxonomical group of same target.

In this manual, method of the present invention is based on the identification to attenuation sudden change in first minus-stranded rna virus.On the subject amino acid position in mark mutational site, be identified as this sudden change of mutant with respect to wild-type sequence, provide with different virus in homologous protein carry out the correlated mark of sequence, described different virus is to be used for recombinant attenuated target viral.The attenuation sudden change can be known in advance, also can differentiate by mutagenesis and reverse genetics technology, and these technology are used to produce and characterising biological is learned the mutated viruses of going up acquisition.As a kind of selection, interested attenuation sudden change can for example be produced again and be characterized by site-directed mutagenesis and traditional screening method.

The sudden change of each attenuation that in the minus-stranded rna virus strain, identifies all provide with the strain of one or more allos negative strand viruses in homologous protein carry out the correlated mark of sequence.In this manual, can analyze existing sequence contrast, perhaps traditional sequence control methods can be used to carry out the sequence comparative analysis, to identify corresponding proteins zone and amino acid position between albumen that carries the attenuation sudden change and different virus homologous protein, wherein said virus is the target recombinant virus that is used for attenuation.The residue that suddenlys change when one or more mark attenuations is changed by wild-type, the corresponding amino acid position of this wild-type in target viral albumen guarded, the genome of target viral and anti-genome through recombinant modified for coding monoamino-acid disappearance, replace or insert, with change the conserved residues in the target viral albumen and therefore in recombinant virus, give with merit, the attenuation phenotype.

Make up in the appropriate design method of attenuation recombinant negative strand rna virus strain at this, the wild-type characteristic in the amino-acid residue site of the mark attenuation sudden change in a minus-stranded rna virus may be strict conservative, also may be to have carried out the conservative property replacement in the proteic corresponding amino-acid residue of target viral site.Like this, with respect to the wild-type residue, the proteic corresponding residue of target viral can be an identity, perhaps may be to be that conservative property is relevant in the structure of amino acid side chain group and function aspects, changes by the attenuation sudden change at wild-type residue described in the allos mutated viruses.Under any situation, the same merit attenuation of recombinant virus can come the coded amino acid disappearance, replaces or insert to change the method for the invention such as conservative property residue to realize by recombination group or the anti-genome of modifying target viral.In this manual, preferred modifying factor group or anti-genome are with the change of coding conservative property residue, the change that it conservatively suddenlys change corresponding to the attenuation that is marked with in the allos mutated viruses.For example, replace mark mutational site in the mutated viruses strain, then can carry out such replacement in corresponding recombinant virus residue site if compare amino acid with corresponding wild type sequence.Preferred described replacement with respect to the replacement residue in the mutated viruses albumen be identity or conservative property.Yet, may on the natural amino acid residue position of non-conservation sudden change, change with respect to the replacement residue of mutain equally (for example, by using arbitrary other amino acid to destroy or damage the characteristic and the function of wild-type residue).With disappearance or insert under the situation of sudden change of mark, these can be transferred in the recombinant virus with corresponding disappearance or insertion and go.Yet the aminoacid sequence and the concrete size of the protein fragments of disappearance or insertion can change.

In another aspect of this invention, except the attenuation phenotype of expection, introduce the sudden change of going in the reorganization negative strand viruses other phenotypes can also be provided, like this, except the attenuation phenotype, introduce typical case's sudden change of going in the recombinant viral proteins and can also give (sp) or (hr) phenotype of host range restricted type temperature sensitivity (ts), acclimatization to cold (ca), little plaque type, perhaps growth or immunogenic change.

In a typical embodiments of the present invention, a ts or non-ts sudden change occur in the big polysaccharase L albumen of negative strand viruses, for example HPIV3.(for example, RSV) be found, it also can be arbitrary virus, and it has the protein structure of the conservative property relevant with the PIV3 that sudden change interested is arranged in the strain of an allos sudden change minus-stranded rna virus in an attenuation sudden change.In detailed embodiment more, the attenuation sudden change in the strain of allos mutated viruses includes an amino acid replaces, and it is the replacement on the phenylalanine in proteic 521 sites of human RSV cpts530 (ATCC VR 2452) L.

In another typical embodiments, a ts or the sudden change of non-ts attenuation occur in the recombinant protein of parainfluenza virus (PIV) for example human PIV1 (HPIV1), human PIV2 (HPIV2), human PIV3 (HPIV3), ox PIV (BPIV) or mouse PIV (MPIV or Sendai virus).Recombination group or anti-genome are modified with the amino acid in N, the P, C, D, V, M, F, HN or the L albumen that are coded in recombinant virus and are replaced, lack or insert.Sudden change can produce a ts or non-ts attenuation phenotype in recombinant virus.Preferably, the attenuation sudden change in the allos sudden change minus-stranded rna virus is corresponding to the sudden change of the sudden change HPIV3 strain JS cp45 that biologically obtains, and its preferably replacement of the amino acid in the L albumen of HPIV3 JS cp45.Typical in this manual sudden change is included in the replacement of proteic 942 tyrosine of L of HPIV3 strain JS cp45, proteic 992 the leucic replacements of the L of HPIV3 strain JS cp45, and/or the amino acid on proteic 1558 Threonines of the L of HPIV3 strain JS cp45 is replaced.

But replace at the amino acid that other sudden changes that the allos sudden change minus-stranded rna virus strain that is used for making up reorganization PIV of the present invention is found include in the F albumen of a HPIV3 strain JS cp45.In one embodiment, the attenuation in allos virus strain sudden change includes the replacement of proteic 420 Isoleucine of the F of HPIV3 strain JS cp45.As a kind of selection, the attenuation sudden change also can comprise the replacement of the L-Ala that the F of HPIV3 strain JS cp45 is proteic 450.Equally, the attenuation of reorganization PIV can suddenly change by the same merit of modifying the attenuation sudden change that reorganization PIV genome or anti-genome take place in RSV to encode and realize.In the described below such embodiment, the attenuation sudden change that is taken place among the RSV includes the replacement of proteic 521 phenylalanines of L of a RSV.PIV genome or anti-genome have been modified the change of the conservative property residue that changes with the residue that conservatively suddenlys change corresponding to mark attenuation in allos RSV mutant strain of encoding.In one embodiment, sudden change occurs in the reorganization HPIV3 albumen and comprises that the amino acid on proteic 456 phenylalanines of L that occur in HPIV3 strain JS cp45 replaces.

But the other PIV candidate vaccine of the present invention can be finished with the sudden change of merit with the middle attenuation sudden change that takes place of coding and Sendai virus (SeV) by modifying reorganization PIV genome or anti-genome.In a following embodiment, the attenuation sudden change includes the replacement of 0 phenylalanine of C protein 17 of SeV.PIV genome or anti-genome are modified with the change of a conservative property ground corresponding to the conservative property residue of the residue change of the sudden change of mark attenuation in allos SeV mutant strain of encoding.In one embodiment, the amino acid of the phenylalanine that takes place in proteic 164 sites of the C of HPIV3 takes place and includes in sudden change in reorganization HPIV3 albumen.

In other typical embodiments, a ts or the sudden change of non-ts attenuation take place in the recombinant protein of Measles virus (MeV).The allos sudden change minus-stranded rna virus that the attenuation sudden change wherein takes place can be respiratory syncytial virus (RSV), human parainfluenza virus (HPIV) 1, HPIV2, HPIV3, ox PIV (BPIV), Sendai virus (SeV), Avian pneumo-encephalitis virus (NDV), SV 41 virus (SV5), mumps virus (MuV), Measles virus (MeV), canine distemper virus (CDV), rabies virus (RaV) or vesicular stomatitis virus (VSV).More suitable is that the allos mutated viruses is HPIV3 JS cp45.In detailed embodiment more, the attenuation sudden change that is taken place among the HPIV3 JS cp45 includes at 992 leucines of L albumen or at the amino acid of 942 tyrosine replaces.

But in the other embodiments provided by the present invention, recombinant negative strand rna virus is an embedded virus.More detailed condition is, this virus has a recombination group or anti-genome, and it includes part or all genome or the anti-genome with the heterologous gene of other minus-stranded rna virus strains, hypotype or kind or gene fragment bonded minus-stranded rna virus strain, kind or a hypotype.The attenuation sudden change can randomly occur in by described heterologous gene or coded albumen or the albumen zone of gene fragment.Aspect preferred, embedded virus is the HN and at least one gene of F glycoprotein gene or the full gene group or the anti-genomic PIV of gene fragment bonded PIV kind, hypotype or a strain that have with allos PIV kind, hypotype or strain.In another embodiment, embedded virus is a RSV, and wherein recombination group or anti-genome have gene or the gene fragment from F, the G of allos RSV kind, hypotype or a strain or SH glycoprotein gene.Preferred situation is that F and G glycoprotein gene are replaced by F and G glycoprotein gene among the human RSV hypotype B under the background condition of the A of RSV hypotype among the human RSV hypotype A.

In other aspects of the present invention, recombinant negative strand rna virus is by further being modified or attenuation recombinating genome or anti-genomic other changes.In one embodiment, genome or anti-genome are further modified, and with one or more additional attenuation sudden changes of encoding, the sudden change minus-stranded rna virus strain that it has biologically been obtained is adopted.For example, in a recombinant RSV incorporate, genome or anti-genome are modified with coding is present in biologically in the group of the sudden change RSV strain that obtains at least one up to whole attenuation sudden changes.So-called group includes (ATCC VR 2450), cpts RSV 248/404 (ATCC VR 2454), cpts RSV248/955 (ATCC VR 2453), cpts RSV 530 (ATCC VR 2452), cpts RSV530/1009 (ATCC VR 2451), cpts RSV 530/1030 (ATCC VR 2455), RSV B-1 cp52/2B5 (ATCC VR 2542) and RSV B-1 cp-23 (ATCC VR2579).In addition, in a reorganization PIV, at least one the attenuation sudden change in all being present in HPIV3 JS cp45 of recombination group or anti-genome encoding.Preferred situation is that at least one attenuation sudden change as herein described is stablized by the variation of a plurality of Nucleotide in the specificity codon of encoding mutant.

In other aspects of the present invention, recombinant negative strand rna virus by specially at one be selected from growth characteristics, attenuation, temperature sensitivity, acclimatization to cold, little plaque size, host range is restricted or immunogenic change in phenotypic alternation nucleotide modification and further modified or attenuation.For example, in RSV, recombination group or anti-genome can comprise the modification of SH, NS1, NS2 or G gene, for example elimination of genetically deficient or genetic expression.The nucleotide modification of other in RSV and other minus-stranded rna virus is included in nucleotide deletion, insertion, increase or the rearrangement in the middle of cis acting regulating and controlling sequence or recombination group or the anti-genome.

In other related fields, the invention provides an isolating recombinant negative strand rna virus, it has been attenuated and has caused immune response in the host to theme virus infection susceptible.This virus packets contains recombination group or anti-genome and produces the necessary viral protein of infectious viral particle of RNA viruses.Recombination group or anti-genome are modified, with in the viral recombinant protein of encoding corresponding to the sudden change on the amino acid position of the amino acid position of the attenuation sudden change of being differentiated in the allos sudden change minus-stranded rna virus.This sudden change by introducing, provides an attenuation phenotype for this recombinant virus in recombinant protein.

The present invention equally also provides isolating polynucleotide molecule, the recombination group or the anti-genome of a recombinant negative strand rna virus of its coding.Recombination group or anti-genome are modified equally, occur in viral recombinant protein to encode one corresponding to the sudden change on the amino acid position of the amino acid position of attenuation sudden change in the allos sudden change minus-stranded rna virus.This sudden change by introducing, provides an attenuation phenotype for this recombinant virus in recombinant protein.At related aspect, the expression vector that is provided includes the transcripting promoter that an operability connects, recombination group or anti-genomic polynucleotide molecule and a transcription terminator of the above-mentioned recombinant negative strand rna virus of coding.

The present invention equally also provides a kind of method, and it can excite individual immunity system to produce the immunoprotection reaction that anti-minus-stranded rna virus infects to induce.This method comprises and gives acceptable carrier on individual aforesaid attenuation recombinant negative strand rna virus of enough measuring with the immunology epipodium and the physiology.In other related fields, an immunogenic composition is provided, it is induced and produces the immune response that anti-minus-stranded rna virus infects.Said composition comprises acceptable carrier on the attenuation reorganization negative strand viruses of the present invention of q.s on the immunology and the physiology.Attenuation reorganization negative strand viruses is RSV, PIV or Measles virus preferably.

The brief description of legend

A among Fig. 1 group provides the aminoacid sequence contrast across the L polymerase protein of RSV Phe-521 (with the arrow note) of a respiratory syncytial virus (SEQ W NO.44), parainfluenza virus 3 (SEQ ID NO.45), Measles virus (SEQ ID NO.46) and celestial platform (SEQID NO.47) virus etc.A consensus sequence (SEQ ID NO.48) results from the accurate coupling of each at least three residue in position.Indicated the first amino acid whose Position Number of cited sequence here.Conservative property residue in all four viruses is all indicated with runic.The beneath residue of drawing horizontal line is (registration number is respectively P26676, P24658, P35341, Q88434, Y09630, U65312, X05399, AF017149, P41357) of guarding equally in PIV2, canine distemper virus, Hsiung DA virus 41, Hsiung DA virus 5, bird Pneumovirinae, Avian pneumo-encephalitis virus, Hendra virus and rinderpest virus L polymerase protein.456 amino acids residues are corresponding to 521 phenylalanine takes place to the position of leucine sudden change in RSV cpts530 in the L polysaccharase of PIV3.

B among Fig. 1 group provides an expression to be introduced in the F456L sudden change among the PIV3 rwt and the synoptic diagram of cp45 sudden change.The corresponding position of the cp45 sudden change of introducing indicates that with (*) corresponding position of F456L sudden change is indicated with (◆).

C group among Fig. 1 has shown a nucleotide sequence (justice), and its coding F456L sudden change (SEQ ID NO.50) is compared to wt sequence (SEQ ID NO.49).The PIV3 nucleotide sequence according to the anti-genome numbering of rwt completely at wt virus shows that mutant nucleotide sequence is following to be shown.The change of Nucleotide represents that with underscore the codon that carries the F456L sudden change is represented with runic.The Xmnl restriction endonuclease recognition site of being introduced is represented with italic in mutant nucleotide sequence.

Fig. 2 describes is duplicating of rcp45 in the chimpanzee upper respiratory tract and rcp45-456.In nasopharynx (NP) wiping sample average virus titer be after infection the named date from using mouthful rcp45-456, n=6; Perhaps ■, rcp45 n=4 infected animals.Specifying the statistically-significant difference between the virus to be respectively: (a) P＜0.005; (b) p＜0.05; (c) p＜0.025; Si Shi t check.(d) detectability≤0.5log ₁₀TCID ₅₀/ ml.

What Fig. 3 described is the organization chart (not proportionally) of HPIV3 P/C/D/V ORF.Three of P mRNA read frames and be shown as (+1 ,+2 and+3), simultaneously, P, C, D and V ORF show by rectangle.The rna editing site is shown as a vertical line, and has shown its sequence motif and be numbered according to its nucleotide position in the anti-genome sequence of complete HPIV3.

A group is described among Fig. 3 is the organization chart of the P mRNA that do not edit.This sequence that contains the translation initiation site of P and C ORF is shown (SEQ ID NO.52), and according to complete anti-genome sequence it is numbered.P, C in complete anti-genome sequence, D and V ORF nucleotide site are: P, 1784-3595; C, 1794-2393; D, 2442-2903; V, 2792-3066.With respect to P mRNA, the AUG of the ORF of open P is in the 80-82 site.

The B group has shown an organization chart that contains the P mRNA through editing of two non-template residue G (GG) (SEQ ID NO.53) insertion in editor site (SEQ ID NO.51) among Fig. 3.So just changed and read register so that the upstream termination and the D ORF at frame+3 places of P ORF at frame+2 places merges.The chimeric protein of gained contains 241 amino acid of the coded N-terminal of promising P ORF, described P ORF be the fusions of 131 amino acid of the coded C-terminal of D ORF.

Fig. 4 has shown the radioimmunoprecipitation test that proof C albumen is expressed in rF164S.The cytolysis thing of path (a) 35S-mark uses polyclone C specificity rabbit anti-serum to carry out immunoprecipitation.22kD band corresponding to C albumen (hollow arrow) clearly is present in rJS and the rF164S solute.Cytolysis thing in path (b) uses two mixtures that are specific to the proteic monoclonal antibody of HPIV3 HN to carry out immunoprecipitation.Be present in each viral throw out corresponding to the 64kD of HN albumen (filled arrows) band, this determines that they are the HPIV3 and the albumen of expressing similar level really.Simulaed path demonstrates the solute available from the tissue culture of non-infected cells.

Fig. 5 has shown with parental virus rJS and has compared, the result of many recursive copyings of recombination mutation virus rF164S.Virus titer is with TCID ₅₀/ ml represents and it is the average result of parallel two duplicate samples.

The explanation of specific embodiments

The method of the invention provides an attenuation recombinant negative strand rna virus that is suitable for as the vaccine use.Recombinant negative strand rna virus produce should virus from one or more separated coding polynucleotide molecule.The polynucleotide molecule of the production of recombinant virus by one or more coding following substances in cell or cell-free system carries out coexpression to be realized: (i) Bing Du recombination group or anti-genome and (ii) produce infectious virus or the necessary basic viral protein of subviral particle.

The recombination group or the anti-genome of theme recombinant virus are modified, and with the sudden change on the one or more amino acid positions that are coded in viral recombinant protein, it is corresponding to the attenuation mutational site in allos sudden change minus-stranded rna virus.Therefore attenuation sudden change in the allos minus-stranded rna virus is " transferred " a corresponding site that enters recombinant virus, thereby gives recombinant virus attenuation phenotype.Identity is comparatively compared in the sudden change of shifting with the attenuation of being differentiated sudden change in the allos mutated viruses or conservative property, although the replacement of non-conservative type also can be used.By in recombinant negative strand rna virus, introducing the sudden change of shifting by this way, design candidate vaccine virus, to cause the immune response of in host, resisting theme virus of an expection to this infection type virus susceptible.

The invention provides a new example, be used for based on discriminating reasonableness design attenuated vaccine virus in the sudden change of allos minus-stranded rna virus attenuation.Attenuation sudden change in allos virus is decided to be one or disappearance, replacement or the insertion of several amino acid in interested allos mutated viruses albumen, for example, by traditional Nucleotide between mutated viruses and non-attenuation parental virus thereof or amino acid comparison.In this manual, parental virus is generally the strain that biologically obtains, and it is a wild-type, is such for the attenuation phenotype at least.Yet the strain of part attenuation mutated viruses also can be used, and wherein additional attenuation sudden change can wait by the polymorphism of induced mutations or nature and realize.In addition, the attenuation sudden change parental virus that can be introduced into and be mapped subsequently wherein, it comprises the virus that the virus of artificial generation is for example provided according to known reverse genetics method by the cDNA virus clone.

Like this, the sudden change of the attenuation in the allos minus-stranded rna virus, differentiated can be previously known or can produce or discriminating by traditional mutagenesis and/or reverse genetics technology.These technology can be used to produce or characterising biological is learned the mutated viruses of going up acquisition, or produce and characterize the sudden change of interested attenuation again, for example and together, to differentiate the attenuation derivative in conjunction with traditional screening method by site-directed mutagenesis wild-type or non-attenuation mutated viruses cDNA clone.(summary can be referring to Conzelmann, J.Gen.Virol.77:381-89 (1996) for whole single negative strand viruses to be used for infectious virus clone's the recovery and the reverse genetics method of genetics operation; Palese etc., Proc.Natl.Acad.Sci.U.S.A.93:11354-58, (1996)) representational viral colony be known.

For example, reported from the antigenomic RNA of cDNA-coding and saved at rabies virus (RaV), vesicular stomatitis virus (VSV), Measles virus (MeV) and Sendai virus (SeV) rinderpest (Baron etc., J.Virol.71:1265-1271 (1997)) and simian virus S (He etc., Virology 237:249-260 (1997), referring to page 5) the infection type virus clone, the antigenomic RNA of described cDNA-coding and basic viral protein coexpression are to produce infectivity, described basic viral protein is nucleocapsid N, phosphorprotein P, big polymerase L (referring to, for example, Garcin etc., EMBO are (1995) J.14:6087-6094; Lawson etc., Proc.Natl.Acad.Sci.U.S.A.92:4477-81 (1995); Radecke etc., EMBO are (1995) J.14:5773-5784; Schnell etc., EMBO are (1994) J.13:4195-203; Whelan etc., Proc.Natl.Acad.Sci.U.S.A.92:8388-92 (1995); And international open WO 97/06270, described document all is incorporated herein by reference).

Rescue respiratory syncytial virus (RSV) system of the antigenomic RNA by developing novel use cDNA coding is finished, described antigenomic RNA and nucleocapsid N, phosphorprotein P, big polymerase L and the previous M2 ORF1 gene prod coexpression that does not characterize (referring to, U.S. Patent application No.08/720,132 (applying date is on September 27th, 1996, it is a U.S. Provisional Application No.60/007,083 (applying date is September 27 nineteen ninety-five)) continuation application, and U.S. Patent application No.08/892,403 (applying date is on July 15th, 1997, it is corresponding to the international application No.WO 98/02530 that announces and be U.S. Provisional Application No.60/047,634 (applying date is on May 23rd, 1997), 60/046,141 (applying date is on May 9th, 1997) and 60/021, the part continuation application of 773 (applying date is on July 15th, 1996) etc., described document all is incorporated herein by reference).These inventions comprise the explanation to the representative construct that is used to produce infectious recombinant RSV incorporate, and described reorganization RNV comprises the recombinant RSV incorporate clone who contains from the attenuation sudden change of the RSV mutant strain that biologically obtains.One of them such construct is the recombinant virus clone who contains the attenuation sudden change of RSV mutant strain cpts530 (being named as the D53-530-site).Be used for that RSV clone reclaims and other of the composition of reorganization operation and method are described referring to Collins etc., Proc.Natl.Acad.Sci.USA 92:11563-7 (1995); Juhasz etc., vaccine 17:1416-1424 (1999); Juhasz etc., J.Virol.71 (8): 5814-5819 (1997); Whitehead etc., Virology247 (2): 232-9 (1998a); Whitehead etc., J.Virol.72 (5): 4467-4471 (1998b); And Whitehead etc., J.Virol.73:(4) 3438-3442 (1999), described document all is incorporated herein by reference.

In another important embodiment, the rescue of infectious parainfluenza virus (PIV) can use equally the antigenomic RNA with N, P and L albumen coexpression of cDNA coding realize (referring to, U.S. Patent application No.09/083,793 (Mays 22 1998 date of application), it is corresponding to the patent application WO 98/53078 of public publication and be U.S. Provisional Application No.60/047,575 (Mays 23 1997 date of application) and 60/059,385 part continues, and described document all is incorporated herein by reference).These bibliographys comprise the following plasmid that is used to produce infectious PIV clone: p3/7 (131) (ATCC 97990); P3/7 (131) 2G (ATCC 97989); And p218 (131) (ATCC 97991); Each all is deposited in American type culture collection (ATCC) according to the clause of budapest treaty, 10801 Boulevard universities, Manassas, Virginia 20110-2209.

The development that is used for the reverse genetics system of minus-stranded rna virus recovery and operation allows concrete analysis and the mapping of attenuation sudden change, to develop special useful candidate vaccine at theme virus.Up to now, shift the attenuation sudden change by recombinant technology in single negative strand viruses order, it is not tested or finish.But, the for example representational kind of the attenuation mutant relative broad range of being differentiated such as RSV, PIV, Measles virus and other single negative strand viruses, it is used as the affluent resources of determining can be used for allogenic sudden change in the methods of the invention with the strong instrument that is provided by the reverse genetics means.

Attenuation sudden change in the allos minus-stranded rna virus can differentiate that it has been introduced in the allos recombinant virus of the present invention and has gone in the mutant strain that biologically obtains.By known mutagenesis program, in the parent plant of wild-type or part attenuation, the sudden change of this theme can natural existence or is introduced into.For example, the strain of attenuation mutated viruses can be by producing through adding the chemical mutagen chemomorphosis in the process of virus in cell cultures, produce to introduce the growth limitation sudden change by being chosen in the virus that goes down to posterity under the suboptimal temperature, perhaps by select with its of mutagenic compound mutagenesis can in cell culture, produce little plaque (sp) or the viral virus of temperature sensitivity (ts) produce (referring to, for example, United States Patent (USP) 08/327,263, described document is incorporated herein by reference).

" mutant strain of Huo Deing biologically " is meant any without mutated viruses that recombinant means produced here.Like this, biologically the mutant strain of Huo Deing just comprise have with relevant wild-type sequence at the different naturally occurring mutant strain of genome, for example, part attenuation sudden change PIV strain.Equally, biologically the mutant strain of Huo Deing also comprises available from arbitrary parental virus strain and without the mutant strain of recombinant means especially induced mutations and selection technology.

A well-known technical program that is used for producing the minus-stranded rna virus that biologically obtains relates to makes a wild-type or part attenuation mutant go down to posterity at a cell culture with the temperature that reduces gradually.Be example with RSV for example, wild-type virus is generally cultivated under 34-37 ℃ the temperature nearly.The part attenuation mutant then suboptimal temperature for example under 20-26 ℃ in cell culture through going down to posterity and producing (for example, elementary bovine kidney cells).Like this, cp mutant strain or other part attenuated strains be ts-l or spRSV for example, the effectively growth by in MRC-5 or Vero cell, going down to posterity at low temperatures, and described temperature is low to moderate about 20-24 ℃, preferably 20-22 ℃.The described sudden change RSV that selects in the cold process that goes down to posterity, the part of comparing attenuation parent plant has been eliminated any toxicity remaining in the strain of deriving fully.

As a kind of selection, concrete sudden change can be gone by wild-type or the chemomorphosis of part attenuation parent plant are introduced in the virus that biologically obtains, for example, introduce the ts sudden change or be in the virus of ts in virus, introduce other ts sudden change, it is enough to increase attenuation the derive degree and/or the stable ts phenotype of strain attenuation.The method of going in the minus-stranded rna virus is introduced in ts sudden change to be included in mutagen for example 5-floxuridine or 5 FU 5 fluorouracil is approximately 10 in its concentration ^-3To 10 ^-5M, preferably about 10 ^-4Carry out virus replication under the existence of M, virus is exposed in the nitrosoguanidine that concentration is approximately 100 μ g/ml, according to J Virol.3:414-421 (1969) such as common for example Gharpure and Richardson etc., the described step of J.Med.Virol.3:91-100 (1978) is carried out.Other chemical mutagen also can be used to.Attenuation can come from an almost sudden change of the ts in all virogenes, has been found to be high conservative polysaccharase (L) gene although be specially adapted to the target of this purpose.

The temperature sensitivity level of in typical attenuation mutated viruses used in the present invention strain, duplicating by under the permissive temperature relatively duplicate and the temperature of extreme limit under duplicate and determine.100 times of the virus replication of comparing with duplicating under permissive temperature minimizings or more lowest temperature are defined as by temperature.In the laboratory animal and the mankind, typical case sudden change RSV strain duplicating and toxicity approx with mutant strain by the temperature correlation connection.Have 39 ℃ limit by the duplicating of mutant strain of temperature by appropriateness, and have 38 ℃ duplicate obviously by the mutant strain of temperature that relatively poor and symptom disease mainly is limited to the upper respiratory tract.And have 35 to 37 ℃ of viruses by temperature generally is complete attenuation in the mankind.Like this, the mutant strain RSV (being the ts type) that biologically obtains of attenuation used in the present invention, its scope by temperature is approximately 35 to 39 ℃, and preferably 35 to 38 ℃.The ts sudden change is joined the virus that can produce the several times attenuation in the part attenuation mutant, and it can be used for vaccine composition of the present invention.

The RSV strain of many attenuations as candidate vaccine is by repeatedly using chemical mutagen to be developed (for example, Connors etc., Virology 208:478-484 (1995) through introducing multiple mutation in the virus of the cold attenuation that goes down to posterity at it; Crowe etc., Vaccine 12:691-699 (1994a); And Crowe etc., Vaccine 12:783-790 (1994b), described document all is incorporated herein by reference).In suitable rodent or orangutan class animal model and in adult humans and baby, measure the mutant strain that biologically obtains, show that in these candidate vaccine strains some are inheritance stabilities, high immunogenicity and attenuation.Equally also relevant for the description of PIV and the strain of the similar attenuation mutated viruses of some other minus-stranded rna virus strains of the present invention (referring to United States Patent (USP) 08/892,403 and corresponding International Application No. WO 98/02530 thereof; United States Patent (USP) 09/083,793 and corresponding International Application No. WO 98/53078 thereof, described document all is incorporated herein by reference).

According to the method described in the present invention, the nucleotide sequence analysis of attenuation mutated viruses strain can be used to draw the attenuation mutation map with concrete Nucleotide and amino acid change, and described analysis relates to DNA or the aminoacid sequence of comparison mutant strain and parental virus strain such as wild-type or part attenuated strain.Often be that these changes relate to the replacement of discrete amino acid, but other sudden changes of carrying out the conservative property transfer relate to multiple amino acid replacement, aminoacid insertion or disappearance according to the method for the invention, and equally also the conserved regions at target protein takes place by large-scale the change.

By using above-mentioned reverse genetics means, Nucleotide and amino acid whose change can be introduced into the virus of a cDNA coding that before had been described in the strain of attenuation mutated viruses, so just allow the technician to distinguish unrecorded occurrent sudden change and are used for the sudden change of target phenotypic alternation.In this, the theme sudden change is introduced individually with entering in infectious RSV cloned genes group or the anti-genome with different array modes and is gone.This process further identifies the sudden change that is used for target signatures such as attenuation, temperature sensitivity, acclimatization to cold, little plaque size, host range be restricted in conjunction with the mensuration of parent and derivative viral phenotypic characteristic.

Like this, the sudden change of differentiating and mapping provides the source that enriches of candidate's sudden change that is used to use allos transfer method design recombinant negative strand rna virus as herein described.The typical explanation of this attenuation sudden change is referring to United States Patent (USP) 08/892,403 and corresponding International Application No. WO 98/02530 thereof in discriminating and the description minus-stranded rna virus; And U.S. Patent application 08/083,793 and corresponding International Application No. WO 98/53078 thereof.These and some other bibliographys that are incorporated herein provide " catalogue " of an attenuation sudden change, and described attenuation sudden change candidate is used for entering the allos virus clone according to the method for the invention transfer.

For example, U.S. Patent application 08/892,403 and corresponding International Application No. WO 98/02530 thereof have been described cold going down to posterity (cp) and temperature sensitivity (ts) mutant strain (Pneumovirinae of RSV; Pneumovirus), the typical mutant strain that comprises called after cpts RSV 248 (ATCC VR 2450), cpts RSV248/404 (ATCC VR 2454), cpts RSV 248/955 (ATCC VR 2453), cptsRSV 530 (ATCC VR 2452), cpts RSV 530/1009 (ATCC VR 2451), cpts RSV 530/1030 (ATCC VR 2455), RSV B-1 cp52/2B5 (ATCC VR2542) and RSV B-1 cp-23 (ATCC VR 2579).A classical group of attenuation sudden change is mapped and is described in the mutant strain that these biologically obtain, and the concrete Nucleotide that is included among the big pol gene L changes, and its result is parent's residue/sequence location Phe ₅₂₁, Gln ₈₃₁, Met ₁₁₆₉And Tyr ₁₃₂₁Deng the amino acid whose replacement in place, replace as attenuation: Leu replaces Phe ₅₂₁, Leu replaces Gln ₈₃₁, Val replaces Met ₁₁₆₉, Asn replaces Tyr ₁₃₂₁Each such sudden change occurs in the L albumen of high conservative and give a ts phenotype in mutated viruses.Yet, other sudden changes are differentiated in RSV and other some minus-stranded rna virus and other some conservative property albumen, it has given some attenuation phenotypes, comprise ts or non-ts attenuation phenotype, as cold going down to posterity cited herein (cp), little plaque (sp), acclimatization to cold (ca) or the strain of host range restricted (hr) mutated viruses.

Like this, be used to introduce other typicalness sudden changes of reorganization negative strand viruses by paramyxovirus, human PIV3 (the paramyxovirus subfamily of discriminating according to the method for the invention for not really being correlated with; Respirovirus).Biologically among (the cold type that goes down to posterity) HPIV3 mutated viruses strain JS cp45 of Huo Deing (referring to United States Patent (USP) 08/083,793 and corresponding International Application No. WO 98/53078 thereof, described document is incorporated herein by reference), such one group of sudden change is differentiated and is characterized.Mapping and sporting of having characterized are coded in the parent's residue/sequence site Tyr of attenuation sudden change polysaccharase L gene in this strain ₉₄₂, Leu ₉₉₂, and/or Thr ₁₅₅₈The change of the Nucleotide that the ts of place attenuation amino acid is replaced.In typical JS cp45 sudden change L albumen, Tyr ₉₄₂By Ile is replaced, Leu ₉₉₂By Phe is replaced, Thr ₁₅₅₈By lie is replaced.These sudden changes have all successfully been introduced in the different PIV recombinant chous, comprise called after r942, r992, r1558, r942/992, r992/1558, r942/1558 and r942/992/1558, wherein use the single and combinatorial mutagenesis of numeral.

Another typical sudden change has been mapped and has been characterized by in F in HPIV3 and the C albumen coded amino acid and replaced in HPIV3 JS cp45.These comprise the sudden change that the non-ts attenuation amino acid of coding is replaced, and described replacement is positioned at the proteic JS HPIV3 of the C parent residue/site Ile of P gene ₉₆On, Ile for example ₉₆By Thr is replaced.Other typical case's sudden changes of being differentiated in the F albumen of HPIV3 are coded in parent's residue/site Ile ₄₂₀And Ala ₄₅₀The amino acid at place is replaced, for example Ile ₄₂₀For Val replaces, Ala ₄₅₀By Thr is replaced.

What in this way can be identified equally is the attenuation sudden change of non-coding region in a negative strand viruses gene.For example, the attenuation sudden change can comprise the change of single or several bases in the gene homing sequence, and for example the attenuation base at 7605 Nucleotide places is replaced in RSV M2 gene homing sequence.When these sudden changes were arranged in the conservative property nucleotide site of allos minus-stranded rna virus, it also can be suitable for shifting between the allos group according to the method for the invention.

Like this, each attenuation sudden change of differentiating in minus-stranded rna virus provides one homologous protein carried out the correlated index of sequence in one or several allos negative strand viruseses.In order to put into practice this one side of the present invention, can analyze the existing sequence contrast, perhaps use traditional sequence control methods carrying out sequence relatively, in a minus-stranded rna virus, carry the albumen of differentiating the attenuation sudden change and one different and be corresponding protein zone and amino acid position between the homologous protein of the virus that is used for recombinant attenuated target viral to differentiate.The focus of this technology is to differentiate and that is to say one or more and the relevant residue of the viral attenuation sudden change of first (allos), and it is by the residue from a parental array change, and wherein the parent lacks mutant phenotype.Contrast it through sequence and whether be determined that the parental array of mutant strain is a conservative property, this determines by discriminating existence identical at corresponding amino acid position or the conservative amino acid residue in target (reorganization) viral protein.Generally speaking, the wild-type sequence element of conservative property will occur in proteic conservative property zone like this, but isolating residue and amino-acid residue structure also are extensively conservative in different single negative strand viruses taxonomical groups and the target that is used in heterology between the allos RNA viruses equally and shifts the attenuation sudden change (wherein all or part of carry the conservative property sequential element that sudden change changes be replicated or import recombinant virus, to produce a novel attenuation redundant organism) can be provided.

Different conservative property albumen is introduced in a different recombinant virus for the attenuation sudden change that will find in an allos minus-stranded rna virus or produce in the present invention a useful target is provided in single negative strand viruses.These can give the credit in single negative strand viruses the function outstanding especially between the different taxonomical groups and the conservative property of structure.In this order, five target proteins are generally conservative, its homologous protein available from relation ancestors' street virus far away for generally acknowledging.These albumen generally demonstrate medium to high sequence identity, especially have the concrete zone or the structural domain of common function feature.Say a bit that clearly all known minus-stranded rna virus all have and contain nucleocapsid protein (N), a nucleocapsid phosphorprotein (P), a non-glycosylated matrix (M) albumen, at least one surface attachment glycoprotein (HN, H or G) and the proteic homologous protein group of a big polysaccharase (L).Each all shows the sequential element of conservative property these albumen, it is for by transforming the useful target that identical between target viral and allos parental virus one or several conservative property residues shift the attenuation sudden change, this sudden change derivative or construct are differentiated, demonstrated the amino acid whose change of a certain attenuation phenotype of influence.Albumen in addition is also fixed by target in this way, and it only has at certain section, subfamily, the genus of single negative strand viruses or between planting.

Single negative strand viruses order comprises filovirus section, Paramyxoviridae, Beaune Viraceae (Bornaviridae) section and rhabdoviridae, it all comprises and has the genomic virus of architomy (monopartite) strand RNA, referring to Pringle, Arch Virol.117:137-140 (1991).A taxonomic summary comprises type concrete in the discriminating of section/subfamily/genus and each group by Pringle in this group, and Arch Virol.142 (11): 2321-2326 (1997) provides.The classification of a representational classification of single negative strand viruses and an extended pattern of Paramyxoviridae is listed in the table 1.The common trait of the gene structure that is occurred in this three coe virus and the linear strand RNA genome has proved that it is as a purpose reasonableness, especially consider such fact, genetic recombination is rare, all reflects the continuity in the heredity after all in these viruses and in the phenotype that occurs the subsequently relation.The strand RNA animal virus list negative strand viruses order-rhabdoviridae of the non-segmentation of table 1.

Vesiculovirus genus (vesicular stomatitis virus)

Lyssavirus (rabies virus)

Ephemerovirus belongs to (bovine ephemeral fever virus)

The respiratory syncytial virus classification paramyxovirus subfamily Respirovirus of other genus-filovirus section (Ebola virus, Marburg virus)-Beaune Viraceae (Beaune disease virus)-Paramyxoviridae in the member of Paramyxoviridae and appointment taxonomical group

-Sendai virus (parainfluenza virus type 1,murine)

-human parainfluenza virus 1 type

-human parainfluenza virus 3 types

-bovine parainfluenza virus 3 type Morbillivirus

-Measles virus

-canine distemper virus

-rinderpest virus

-whale (dolphin) Measles virus

-sea dog distemper virus

-pest-des-petits-ruminating animal virus

-Hendra virus (the new Australian pathogenic agent of identifying) Rubulavirus belongs to

-mumps virus

-SV 41 virus (canine parainfluenza virus 2 types)

-human parainfluenza virus 2 types

-human parainfluenza virus 4 types

-birds parainfluenza virus (comprising Avian pneumo-encephalitis virus)

-pig Rubulavirus

-SV 41 virus type

-Mapuera virus Pneumovirinae Pneumovirus

-human airway syncytial virus (hypotype A and B)

-bovine respiratory syncytial virus

-sheep and billy goat respiratory syncytial virus

-pneumonia of mice virus birds Pneumovirus 1

-birds Pneumovirinae (before being Turkey Rhinotracheitis Virus) 1 prepares to distribute one independently to belong to, and is still unnamed

Beaune Viraceae and filovirus section represent with single genus.The Beaune Tobamovirus still contains a single kind (Beaune disease virus).According to viral nucleotide sequences and antigenicity difference and adhere to (G) proteic differential expression, in belonging to, wire Tobamovirus (type species is a Marburg virus) identifies four kinds.Rhabdoviridae comprises 5 genus: vesiculovirus genus, Lyssavirus, Ephemerovirus, Cytorhabdovirus and Nucleorhabdovirus, except sequence and antigenic specificity, it is the existence by host range, supplementary gene and endogenous multiplication site and differentiated also.Paramyxoviridae has two subfamilies; The paramyxovirus subfamily that three genus are arranged, described three genus are that Respirovirus (HPIV 1 is type strain), Morbillivirus (type species is MeV) and Rubulavirus belong to (MuV is a type species), and Pneumovirinae (human RSV is a type species) and a calculated additional kind, it comprises the birds Pneumovirinae.

In all members of this purpose, 5-10 gene wherein arranged, and transcription initiation is in 3 ' terminal promotor of a single supposition, it is by the dependent rna polymerase promoter of a viral RNA.Except the exception of some Pneumovirinaes, gene order all is strict conservative.At Pringle, among Fig. 1 of Arch Virol.142 (11): 2321-2326 (1997), 16 kinds of viruses to not equal, subfamily of representative and genus in single negative strand viruses are carried out the gene pairs ratio, and wherein said gene is divided and is grouped in described different taxonomical groups has homology.Like this, VSV has showed MIN 5 genes; Nucleoprotein (N), phosphorprotein (P), stromatin (M), attachment protein (G) and polymerase protein (L).Ebola virus and 8 members in the rhabdoviridae in Beaune disease virus in the Beaune Viraceae, the sick genus of the wire section have showed basic five gene models (N-P-M-G-L), in four viruses in Ebola virus and eight rhabdovirus, this pattern increases by insert one or more genes between G and L, perhaps among three in remaining four rhabdovirus, increase by between P and M, inserting one or more genes.It is more complicated that paramyxovirus in the paramyxovirus subfamily is showed, 5 basic gene models by the multiple encoding of genetic information in the P gene and between M and attachment protein (H or HN) the additional envelope protein gene F of insertion increase, in some Rubulaviruses, also make its raising by SH.All paramyxovirus all have two surface glycoprotein HN (or H or G) and F at least.Nearly all Respirovirus, Rubulavirus and Morbillivirus member have a richness half Guang thuja acid albumen V.Respirovirus and the Measles virus homology C albumen of also encoding.Pneumovirinae and Rubulaviruses subgroup (SV 41 virus (SV5) and mumps virus (MuV)) all have homology surface glycoprotein SH.Several additional albumen that (comprise that ox, sheep and billy goat RSV and pneumonia of mice virus----also are called mouse RSV here) in Pneumovirus, NS1, NS2, M2 (ORF1) and M2 (ORF2) are conservative propertys.The paramyxovirus of Pneumovirinae shows the unique difference with basic model, is apparent that most to have additional gene.In birds, people and mouse Pneumovirinae, M2 gene two albumen of in different reading frames, encoding, the both is to bringing about tangible results at vitro synthesized RNA.In the mankind and mouse Pneumovirinae, the unstructuredness albumen NS1 of two coding unknown function and two assignments of genes gene mapping of NS2 are between 3 ' leader and N.The birds Pneumovirinae approaches basic model, lacks 3 ' terminal NS gene of two uniquenesses and remains with the paramyxovirus gene order of standard, except having the M2 gene inserts.The conservative property mode annunciations of this genome structure develops by the expansion of intergenic region and by gene replication rather than from outside introducing genetic information.Pringle，Semin.Virol.8：49-57，1997。

As mentioned above, method of the present invention is based on the discriminating of attenuation sudden change in first minus-stranded rna virus, and this sudden change positions by the sequence that compares mutant strain and wild strain, to carry out the discriminating to the subject amino acid position in mark mutational site.Preferably, these sudden changes are introduced among the recombinant virus clone of non-attenuation or part attenuation, change with the theme on the proving fact to have produced an attenuation phenotype.The host of typical case's attenuation sudden change also will be provided in this article, and some other attenuation sudden change all is easy to differentiate according to described known mutagenesis of this specification sheets and reverse genetics technology.Each attenuation of differentiating sudden change provides one to carry out the correlated index of sequence in a minus-stranded rna virus, and this contrast is meant that the sequence of homologous protein in the strain of one or more allos negative strand viruses compares.

For the several aspects that realize that the present invention is above-mentioned, can analyze the existing sequence contrast, also can use traditional sequence control methods carrying out the sequence comparative analysis, to differentiate corresponding proteins zone and the amino acid position between the homologous protein of the target recombinant virus of attenuation mutain and other attenuation.For example wild-type conservative property feature is (promptly from the parent at the proteic corresponding amino acid position of target viral when the residue of one or more mark attenuations sudden change, identity or show as conservative property related amino acid residue) change, target viral genome or anti-genome with amino acid whose disappearance, replacement or the insertion identical or conservative property of encoding, also therefore in recombinant virus produce a homology attenuation phenotype with the conservative property residue that changes in the target viral protein through recombinant modified.

By sequence to when analyzing to realize several aspect of the present invention, it focuses on homology " correspondence " gene, gene fragment, albumen and/or protein structure domain, and wherein polynucleotide or amino acid canonical sequence are used as a defined sequence so that a correlated basis of statistical sequence to be provided.For example, this sequence can be a defined cDNA or gene fragment, whole cDNA or gene order or albumen or the sequence of a part wherein.

In general, a canonical sequence, be used for determining corresponding gene or gene fragment, it generally all has at least 20 Nucleotide on length, the most at least 25 Nucleotide, often be length, and for protein and protein fragments, have 20 amino acid whose length at least at 50 Nucleotide.Since all can (1) containing a sequence, two polynucleotide or aminoacid sequence (that is to say, the part of whole polynucleotide or protein sequence), it is similar between two polynucleotide or albumen, and (2) may further include a sequence, it is different between two (or more) polynucleotide or protein sequence, the polynucleotide of two (or more) or the contrast between the protein sequence generally realize by " comparison window " that compares two subject nucleotide sequences so, with the sequence area of differentiating and relatively having identity or similarity." comparison window " of this paper effect, mean at least 20 the successive Nucleotide on the conceptual or the fragment of amino acid position, one of them sequence can be compared with at least 20 continuous nucleotides or amino acid whose canonical sequence, when the ideal comparison of carrying out two sequences, the polynucleotide wherein or the part of aminoacid sequence in comparison window, can comprise its compare with canonical sequence (it does not comprise increases or disappearance) 20% or the sequence of lower ratio increase or disappearance (being breach).

The ideal comparison that is used to contrast the sequence of comparison window can be passed through Smith﹠amp; The regional homology algorithm of WatermanAdv.Appl.Math.2:482 (1981) is operated, perhaps Needleman﹠amp; Wunsch, the homology contrast algorithm of J.Mol.Biol.48:443 (1970) is operated, and perhaps passes through Pearson﹠amp; Lipman, the similarity searching method of Proc.Natl.Acad.Sci.USA 85:2444 (1988) is operated (described document all is incorporated herein by reference), perhaps implement (GAP by the computerize of these algorithms, BESTFIT, FASTA and TFASTA are (in 7.0 editions Wisconsin genetics software packages, genetics computer group, 575 Science Dr., Madison, WI), it is incorporated herein by reference) carry out, perhaps carry out through checking, the best comparison of selecting then to produce (that is to say, make the sequence similarity per-cent of comparison window the highest) by diverse ways.

Here employed phrase " sequence identity " means two polynucleotide or protein sequence is identical (that is to say, based on the comparison of Nucleotide-Nucleotide or residue-residue) in comparison window." sequence identity per-cent " calculates by following steps: compare two correlated sequences of the best in comparison window, determine that the identical nucleic acid base (for example in two sequences, A, T, C, G, U or I) or the position number of amino-acid residue to draw the number of matched position, divided by the total number of positions in the comparison window (size of window just), the result multiply by 100 and just draws sequence identity per-cent with the number of matched position.

The employed phrase of this paper two polynucleotides of " basic identity " expression or aminoacid sequence are having 20 at least, often be to have at least 85% in the comparison window of 25-50 Nucleotide or amino acid position at least, preferred 90-95%, sometimes 99% or the feature of higher sequence identity per-cent, wherein sequence identity per-cent can by on comparison window relatively canonical sequence calculate with can including disappearance or increase its 20% or still less comparative sequences that comes to canonical sequence.Canonical sequence can be the part of a big sequence.

Structural element in being applied to albumen and albumen, phrase " sequence identity " means the sequence that has one or more same amino acid in corresponding site.Phrase " sequence similarity " means two sequences and for example has identical one or more conservative property related amino acid in conservative property replacement site in corresponding site.Phrase " basic sequence identity " means two peptide sequences, when carrying out the suitableeest contrast, for example service routine GAP or BESTFIT also use the breach weight of acquiescence, has 85% sequence identity at least, preferably in 90% sequence identity, be more preferably 95% or higher sequence identity (99% sequence identity is for example arranged).Phrase " basic similarity " means two peptide sequences and has corresponding sequence similarity per-cent.

The sequence relation of conservative property even when but amino-acid residue in the corresponding site of two sequences is not the identity structural relation of difference a conservative amino acid, exist.In this manual, conservative amino acid is replaced and is referred to the general interchangeability of the amino-acid residue with similar side chain.For example, one group of amino acid with aliphatic side chains is glycine, L-Ala, Xie Ansuan, leucine and Isoleucine; One group of amino acid with aliphatic hydroxyl side chain is Serine and Threonine; One group of amino acid with amide containing side chain is l-asparagine and glutamine; One group of amino acid with aromatic series side chain is phenylalanine, tyrosine and tryptophane; One group of amino acid with basic side chain is Methionin, arginine and Histidine; One group of amino acid with sulfur-containing side chain is halfcystine and methionine(Met).Preferred conservative amino acid replacement group is: Xie Ansuan-leucine-Isoleucine, phenylalanine-tyrosine, Methionin-arginine, L-Ala-Xie Ansuan and l-asparagine-glutamine.20 amino acid whose steric isomers of tradition (for example, D-amino acid), non-natural amino acid is α for example, and α-two substituted amino acids, N-alkyl amino acid, lactic acid and other non-traditional amino acid also can be the suitable compositions of polypeptide of the present invention.Non-traditional amino acid whose example comprises: 4-oxyproline, Gla, ε-N; N; N-trimethyl lysine, ε-N-ethanoyl Methionin, O-phosphoserine, N-ethanoyl Serine, N-formyl radical methionine(Met), 3-Methyl histidine, 5-oxylysine, ω-N-methylarginine and some other similar amino acid and imino-acid (for example, 4-oxyproline).And amino acid can be modified by glycosylation, phosphorylation etc.

In order to promote to implement the above-mentioned all respects of the present invention, molecule development history with reference to some known conservative property viral proteins in single negative strand viruses order, in this point, carried out more extensive studies, its detailed evaluation the albumen dependency on more accurate molecular level.These researchs comprise for the comparing one by one of the proteic sequence of conservative property in single negative strand viruses, have made the collection of illustrative plates of the separation residue of these proteic conservative property structural domains, sequential element and the restriction more accepted extensively.Each of these conservative property protein structure domains, sequential element and restricted residue has promoted enforcement of the present invention by providing from the strain of allos attenuation mutated viruses conservative property to shift the useful target that the attenuation sudden change goes to the different viruses of recombinating according to the method for the invention.

For example, Poch etc., J.Gen.Virol.5:1153-62, (1990) (be incorporated herein by reference) comparison of 5 proteic deduced amino acid of L that a detailed single negative strand viruses is provided, it comprises the L albumen of rhabdovirus (V.SV and RaV) and paramyxovirus (SeV, NDV and MeV).Conventional control methods (being incorporated herein by reference) has disclosed from the L albumen of different virus section has showed that along the overwhelming majority of its length a very homology of high level---it has thick-and-thin amino acid in conservative region, and it is separated for some zones of guarding relatively.Like this, these Different L albumen from the allos minus-stranded rna virus have occupied the structure of the conservative property of a connection function area.For example, L albumen comprises the central zone of a high conservative, and it is considered to contain RNA synthetic avtive spot.Other conservative property structural area, motif and sequential element comprise strict conservative isolating residue, are differentiated that equally also some of them distribute and are considered to for polymerase activity along the conservative property central section is vital.The sequential element of these conservative propertys of in L albumen, being differentiated (referring to, Poch etc., J.Gen.Virol.5:1153-62, (1990) and EMBO such as Poch (12) 3867-3874 J.8, (1989), Fig. 1 particularly, described document is incorporated herein by reference), the details that provided have produced with attenuation sudden change wherein and the allos parent plant L protein sequence of mapping is compared.Like this, use these and other order-checking row method and data, comprise order-checking row method and data in the publication that this paper quotes, it can measure at an easy rate whether a residue that indicates the attenuation sudden change is changed from parent plant such as wild strain, it has pointed out the high possibility that the attenuation of being differentiated suddenlys change and goes in the genome that successfully occurs in target recombinant virus or the anti-genome in allos virus in the proteic corresponding amino acid position of the purpose virus L identity that is conservative property (that is to say, identity or be conservative property related amino acid residue representative).

Further detailed description about the structure conservative property of the proteic autoploid of Different L in single negative strand viruses is Stec etc., and Virology 183:273-287 (1991) (described document is incorporated herein by reference) provides.These sequential analyses are based on 3 paramyxovirus of having announced (from two in the Respirovirus (PIV3 and SeV), in Rubulavirus (NDV) virus one), paramyxovirus of Morbillivirus (MeV) Tobamovirus and two virus from rhabdovirus (RaV and VSV) section (referring to, Schubert etc. for example, 1985; Shioda etc., 1986; Yusoff etc., 1987; Blumberg etc., 1988; Galinski etc., 1988; And Teart etc., 1988, described document all is incorporated herein by reference) L gene and proteic sequence and other sequence information and comparison at RSV are provided.These results have confirmed description about relevant sequence conservation in paramyxovirus and rhabdoviridae such as Poch (the same), and it also identifies other conservative property structural domain, motif and sequential element in addition.

In brief, the discovery of Stec etc. relies in traditional control methods, and it is very useful in the present invention in other similar methods.Specifically, contrast such as Stec heterology minus-stranded rna virus sequence, wherein use Wilbur and Lipman Proc.Natl.Acad.Sci.USA80:726-730 accepted methods such as (1983), use the breach punishment of disappearance and 12 and 6 and " protein sequence and structure collection of illustrative plates " (M.O.Dayhoff edits), Vol.5, Suppl.3, pp.345-352 Natl.Biomed.Res.Found., Silver Spring, the similarity of the Dayhoff among the MD (1978) etc. gets sub matrix (described document all is incorporated herein by reference).Similarity gets sub-system and is used at RSV and to make up the contrast of paired spherical point matrix between some other minus-stranded rna virus L albumen of listing in table 1.By these methods, clear and definite sequence relevant range is found between the L albumen of the L of RSV albumen and paramyxovirus of very not being correlated with and rhabdovirus.Such as shown in Figure 3, the sequence relevant range between the L of RSV albumen and some other L albumen is on same straight line and concentrates on the zone of approaching amino, may occupy about 1/5 of molecule.Also put down in writing (Blumberg etc., 1988, Galinski etc., 1988 in the sequence contrast in the L albumen of the same pattern of these sequence similarities in some other paramyxovirus and rhabdovirus; Teart etc., 1988, described document all is incorporated herein by reference), comprising proteic five tunnel contrasts of SeV, MeV, NDV, RaV and VSV L, it is finished by (1989, the same) such as Poch.

In seven tunnel control methodss of (the same) such as Stec, RSV L albumen is determined that the N-terminal that contains nearly 70 amino-acid residues of comparing with other minus-stranded rna virus extends and the C-terminal tack of about 100 amino-acid residues.Yet, this variation does not influence the reliability of control methods discriminating conservative property structural element and is considered to belong to such fact, the L albumen of RSV is by L gene and its upstream adjacent part, be M2 gene (being called 22K in the past) (Collins etc., 1987, described document is incorporated herein by reference) lap part coding.

The invention of Stec etc. (the same) has proved the short segmental existence of a part in the L albumen of allos minus-stranded rna virus, and these fragments almost can be defined between the different taxonomical groups and guard.These are bordering on same fragment and equally also demonstrate conservative property in the L of RSV albumen, and it is positioned at the high conservative zone that is shown in Fig. 4 (Stec etc., described document is incorporated herein by reference, referring to the sequence in the box of packing into).Amino acid whose identity changes by 30% to 80% in seven kinds of albumen in these six fragments.A large amount of residues in the fragment of being differentiated are stablized constant in seven kinds of different minus-stranded rna virus that compare.In addition, it should be noted that when replacement takes place in these conservative property fragments, much is that conservative property is replaced.Just as discussed previously like that, pointing out them in these fragments of each member's camber identity of two Viraceaes is most important for the proteic function of L.

More detailed is, four different polysaccharase primitives (underscore marks and named and is A-D) are equally also contained in the proteic high conservative of the L zone that compares as described Fig. 4 such as Stec between the different taxonomical groups in single negative strand viruses, itself and Poch etc., (1989), the same primitive unanimity of differentiating.The zone of these and four polysaccharase motif homologys is found between proteic 696 to 887 amino acids of the L of RSV.Three (A-C) in four elements is consistent with the proteic high conservative fragment of the L of above-mentioned paramyxovirus and rhabdovirus.The conservative property of D fragment in these viruses is relatively poor, but it contains the glycine (at RSV, being respectively K-886 and G-877) of a constant Methionin and a conservative property, and it is the feature of this motif.

The proteic similarly detailed sequence that is present in the minus-stranded rna virus for other is open to when analyzing, and it decides albumen as target in the present invention is very useful.In this point, Barr etc., J.Gen.Virol.72:677-685 (1991) has analyzed sequence conservation in the N albumen among the allos member in single negative strand viruses, and described document is incorporated herein by reference.Especially this studies show that the high-caliber amino acid identity (60%) (referring to Fig. 7, described document is incorporated herein by reference) between the proteic predicted amino acid sequence of the N at RSV and PVM.Amino-acid residue 1 to 150 and 150 to 393 contains 38% and 74% identity respectively, yet residue 245 to 315 contains 68 identical amino acid (96% identity) in 71 amino acid.These proteic high conservatives with at the N of RSV and PVM albumen be relevant this of serology unanimity (Gimenez etc., 1984 as a result; Ling﹠amp; Pringle, 1989).

PVM and more other single negative strand viruses members' the proteic aminoacid sequence of N carry out computer matrix more also disclosed conservative property the zone (Barr etc., the same, Fig. 5).Same sequence conservation in this manual also occurs in the N of PVM and Ebola virus albumen.Further, from the member's of the more population-wide of the minus-stranded rna virus of non-segmentation the proteic wetting ability collection of illustrative plates of N at paramyxovirus and Measles virus (Galinski etc., 1985, described document is incorporated herein by reference) between the zone of sequence similarity maximum be similar each other.About 180 amino acid from each proteic aminoterminal 130 to 170 residue, form alternative hydrophilic area and hydrophobic region.Hydrophilic these zones of the secondary structure of these aminoacid sequences (Garnier etc., 1978, described document is incorporated herein by reference) prompting conservative property may begin but it ends at an a high proportion of beta sheet and corner with a high proportion of a-spiral.These conservative property albumen of these Notes of Key Datas can have a similar overlay structure in similar hydrophilic region.

What concentrate on the conservative property hydrophilic region uses known program CLUSTAL (Higging﹠amp to representational N albumen; Sharp, Gene 33:237-244 (1988), described document is incorporated herein by reference) the further arrangement research done point to still for additional conservative property protein function district, structural motif and isolating conservative amino acid residue (referring to, Barr etc., Fig. 7, box A, B and C).Learn that from this research it has found that the conservative property between the N of SeV, VSV and PVM albumen is very high in definite zone of half (Fig. 7, box C) of the C-terminal of PVM sequence.This zone differentiates in DIAGON that equally it is comparing between Ebola virus and PVM in less than 99 window.Two short similarity zones are detected in this sequence equally, and each all contains water repellent region, and it is that institute at interval for the basic aminoacids (K or R: box A and B, in Fig. 7) of single a, conservative property.In the N of other paramyxovirus and rhabdovirus albumen, corresponding similar area is arranged also.In the identity level in these zones shown in the table 1 of Barr etc.Although in a Viraceae a high proportion of identity is arranged, high-caliber similarity also can confirm in these zones of these Viraceaes, when especially being thought of as the conservative property replacement.

Helping of the invention process equally is the independently sequence contrast that confirms the high-caliber molecule conservative property of N albumen in single negative strand viruses allos member.For example, Parks etc., Virus Res.22:259-279, (1992), described document is incorporated herein by reference, and has described the area of computer aided contrast from the proteic aminoacid sequence of N of 10 different paramyxovirus.Shown in Figure 3 as in this bibliography contains a big sequence conservation at N albumen near the obvious zone (SV5 residue 323-340) of intermediary one in the N of different taxonomical groups albumen, it comprises a constant hydrophobicity primitive F-X ₄-Y-X ₄-S-Y-A-M-G (wherein X is arbitrary residue).The zone of another high conservative, it is rich in electronegative L-glutamic acid and aspartic acid, is found in the proteic C-terminal of N zone (SV5 residue 455-469).

In the other research of N protein molecular conservative property, Miyahara etc., Arch.Virol.124:255-268 (1992), described document is incorporated herein by reference, the corresponding sequence that has compared HPIV-1 N albumen and 12 other paramyxovirus, described 12 paramyxovirus are HPIV-2 (Yuasa etc., Virology 179:777-784 (1990)), HPIV3 (Galinski etc., Virology 149:139-151 (1986)), HPIV-4A and 4B (Kondo etc., Virology174:1-8 (1990)), SV5 and SV41 (Tsurudome etc., J.Gen.Virol.72:2289-2292 (1991)), MuV (Elango, Virus Res.12:77-86 (1989)), SeV (Morgan etc., Virology 135:279-287 (1984)), PIV-3 (Sakal etc., Nucleic AcidsRes.﹠amp; : 2927-2944 (1987)), NDV (Ishida etc., Nucleic Acids Res.14:6551-6564 (1986)), MeV and (CDV) (Rozenblatt etc., J.Virol.53:684-690 (1985)) (described document all is incorporated herein by reference).

Shown in Figure 3 as Miyahara etc., the N gene of HPIV1 and SeV have shown homology very widely; Nucleotide and amino acid whose identity are respectively 70.8% and 87.8%.The N albumen of HPIV-1 and HPIV-3 (63.1%) reach with BPIV-3 (63.3%) and demonstrate very high amino acid identity equally, and it is lower with the identity of following virus, with NDV (20.9%), MeV (20.5%), CDV (19.6%), SV41 (18.6%), SV5 (17.9%), HPIV-4A (17.9%), HPIV-4B (17.5%), HPIV-2 (17.5%) and MuV (17.1%)." hinge " of a proteolytic enzyme susceptibility is found the binding site that is positioned at two proteic definite zones of SeV K; Described zone is (1) amino terminal region, its direct and RNA interaction and (2) C-terminal zones, it is positioned at the surface (Heggeness etc. that make up nucleocapsid, Virology 114:555-562 (1981), described document is incorporated herein by reference).

As Miyahara etc. further described, 26 amino-acid residues are conservative propertys in all paramyxovirus N albumen, and the N district of a conservative property is found between the proteic amino acid sites 260-360 of N.In addition, 13 in 40 glycine 37 and 13 proline(Pro) is conservative property in the N of HPIV-1 and SV albumen, points out out these Protein S eV to maintain a common tertiary structure.

Miyahara HPIV-1 M albumen and 13 other paramyxovirus (referring to, Fig. 4 for example, Miyahara etc.) in compared the M protein sequence equally.These allos viruses demonstrate the high conservative of M protein sequence element equally.For example, HPIV-1 and SeV demonstrate the Nucleotide of M and the level of amino acid identity is respectively 72.6% and 88.4%.HPIV-1 demonstrates equally with HPIV-3 (65.7%) and reaches and the high-caliber conservative property of BPIV-3 (65.4%), and shows the moderate conservative property with MeV (36.4%), CDV (34.6%) and RPV (36.7%).24 in 5 halfcystines of among the HPIV-1 all, 22 proline(Pro) 20 and 25 glycine is conservative property in SeV, and wherein nearly all these also are conservative property in PIV-3.These find that the proteic tertiary structure of prompting M may be a conservative property in HPIV-1, SeV, HPIV-3 and BPIV-3.In addition, 14 amino acid are conservative propertys in all paramyxovirus M albumen relatively.

Be disclosed when analyzing for proteic other sequences of P, P albumen is to be a useful especially target during sudden change allos general conservative property and in the present invention shifts equally in single negative strand viruses.Referring to, for example, Kondo etc., Virology 178:321-326 (1990), described document is incorporated herein by reference.In this manual, the P albumen of known SeV (Giorgi etc., Cell 35:82-836 (1983); Neubert, Nucleic Acids Res.17:10-101 (1989), described document is incorporated herein by reference) and PIV3 (Luk etc., Virology153:318-325 (1986), described document is incorporated herein by reference) size is very approximate, has 568 and 603 amino acids respectively.For example among MuV, SV5, PIV-2 and the NDV, the P gene is encoded respectively and is contained 391,392,395 and 395 amino acid whose albumen in different taxonomical groups.(Takeuchi etc., J.Gen.Virol.69:2043-2049 (1988); Cell 54:891-902 (1988) such as Thomas; And Sato etc., Virus Res.7:241-255 (1987)).The P albumen of MeV and CDV is median size (Barret etc., Virus Res.3:367-372 (1985; Bellini etc., J.Virol.53:908-919 (1985).

By Kondo etc., the P specific regions in Virology 178:321-326 (1990) all allos virus relatively can both be arranged comparison (described document is incorporated herein by reference) according to the conventional method.Significant conservative property structure is found and is positioned at PIV4A, PIV4B, SV5, PIV-2, MuV, NDV, MeV, CDV, PIV-3 and SeV (referring to Fig. 2 of Kondo etc., described document is incorporated herein by reference).Like this, the research of this research and some other relevant P protein molecular theories of evolution identifies additional conservative property albumen zone, structural motif, amino acid fragment and separation residue, it is compared in the mutational site and is used for introducing the target of recombinant vaccine strain from the attenuation sudden change of allos mutated viruses with assessment.

Provide the further sequence that can promote realization of the present invention to when analyzing, it is used to all albumen in single negative strand viruses.In brief, Yuasa etc., Virology179:777-784 (1990), described document is incorporated herein by reference, be to identify the conservative property structural element in the N gene of PIV-4A, PIV-4B, MuV, NDV, MeV, PIV-3, BPIV-3, SeV and RSV (referring to, Fig. 2 and 4 for example) 3 ' gene end and the mankind and non-human paramyxovirus.Kawano etc., Virology 174:308-313 (1990) provide the proteic analysis of molecules of HN and the sequence contrast of eight kinds of allos paramyxovirus (referring to, Fig. 2 for example).Tsurudome etc., J.Gen.Virol.72:2289-2292 (1991) provide the sequence of HPIV-2, a SV5 and SV41 (referring to, Fig. 3 for example) to when analyzing.Spriggs etc., 1986, identify structure conservative property sequence in the F albumen of Virology 152:241-251 (1986) in allos paramyxovirus (comprising RSV).Higuchi etc., J.Gen.Virol.73:1005-1010 (1992) (referring to, Fig. 4 for example), Kawano etc., Nuc.Acids.Res.19 (10): 2739-2746 (1991) (referring to for example Fig. 2 and 6), Muhlberger etc., Virology 187:534-547 (1992) (referring to for example Fig. 4 and 6), and Ogawa etc., J.Gen.Virol.73:2743-2750 (1992) (referring to for example Fig. 3) has described for the L albumen and 3 ' and 5 ' the non-encoding gene group end in one in the single negative strand viruses big taxonomical group set.A more comprehensive viewpoint, it is included in the citations that the molecule conservative property in N, P, C, L, M, HN, F, SH, V, D and other ORF and the gene prod of single negative strand viruses describes in detail and provides for institutes such as Collins, Fields Virology, editors such as Fields, the third edition, the 41st chapter: 1205-1241, Lippincott-Raven, Philadelphia (1996).These researchs all are incorporated herein by reference, especially comprise its according to prompting of the present invention as the arrangement of the discriminating that is used for attenuation sudden change introduced on the target locating point that recombinant vaccine virus goes the conservative property structural element on the locating point really to figure when.

The present invention more detailed aspect, the recombinant negative strand rna virus strain of attenuation is transformed into an embedded virus by the sudden change transfer that will differentiate in allos virus, for example, chimeric RSV or PIV virus.The nucleotide sequence of chimeric negative strand viruses of the present invention and other a plurality of virus strain or the nucleotide sequence of hypotype are recombinated, to produce an infective embedded virus or subviral particle.By this way, the processing of being recombinated of candidate vaccine virus strain with in mammalian hosts, comprises and causes an immune response in human or the inhuman primate.Can cause an immune response or cause a multiple reaction at specific virus hypotype or strain according to embedded virus of the present invention at multiple virus subtype or strain.

In a typical embodiment, the embedded virus that contains the attenuation sudden change also can comprise the heterologous gene or the gene fragment of allos virus as mentioned above, it is added into or introduces recombination group or anti-genome in the theme virus, for example, produce a chimeric RSV genome or anti-genome by replacement from the corresponding sequence of allos RSV.An embedded virus of the present invention so just comprises from part or all " acceptor " genome of a virus strain or subtype virus or anti-genome and from the additional of a different virus strain or hypotype or " donor " gene or the gene fragment of replacing.

Of the present invention preferred aspect, chimeric attenuated virus is RSV, it comprises part or all human RSV A or B subtype virus genome or anti-genome and comes from the A hypotype of different human RSV or the heterologous gene or the gene fragment of B hypotype.In order to produce this recombinant virus, come from the allos donor gene of a RSV strain or hypotype or gene fragment in conjunction with or replace with as main chain and be used for inserting or increasing acceptor gene group or anti-genomic donor gene or gene fragment.Like this, acceptor gene group or anti-genome as a kind of carrier with input or expression alien gene or gene fragment, to produce a chimeric RSV, structure that its performance makes new advances and/or phenotypic characteristic.Preferably, when its phenotype with the acceptor of corresponding unmodified and/or donor is compared, increase or the replacement in selected acceptor RSV strain of heterologous gene or gene fragment produces new phenotype, for example phenotypic alternation of attenuation, growth alteration, immunogenic variation or other expections.

Typical attenuated chimeric RSV is developed and describes, and it includes human RSV B hypotype glycoprotein gene F and G, and it is compared with RSV A subtype gene group and has replaced corresponding F and G glycoprotein gene.This typical mosaic is further modified, and to contain the attenuation sudden change, it is selected from (i) a group and characterizes temperature sensitive acidic amino acid replacement Gln ₈₃₁To Leu and Tyr ₁₃₂₁Sudden change to Asn; (ii) the Nucleotide of the temperature sensitivity in the gene homing sequence of gene M 2 is replaced; And (iii) available from Cys319 to Tyr and His among amino acid replacement Va1267 to Ile and the RSV pol gene L in the sign RSV N gene of the cold RSV that goes down to posterity ₁₆₉₀Attenuation sudden change group to Tyr; Or (iv) lack the SH gene (referring to, for example Application No. 09/291,894).Preferably situation is, these and other embedded virus example contains at least two kinds of attenuations sudden changes, and it can be from identical or different mutated viruses strain.

Typical attenuated chimeric PIV has been developed and has described it equally and contained heterologous sequence from HPIV-1 and HPIV-3, with the attenuation sudden change that from PIV3 mutant strain cp45, obtains, referring to United States Patent (USP) 09/083,793, date of application is on May 22nd, 1998 (corresponding international application no is WO 98/53078) and priority application thereof, and the applying date is on May 23rd, 1997, and application number is 60/047,575, described document all is incorporated herein by reference.Recently, the sudden change of all attenuation of in cp45, differentiating, in F albumen, all successfully be introduced in the HPIV3-1 mosaic of attenuation reorganization.

The introducing of alloimmunization originality albumen, structural domain and antigenic determinant is in order to produce a chimeric minus-stranded rna virus, and it is particularly useful for produce new immune response in an immune host.In the acceptor gene group of a different virus strain or hypotype or anti-genome, increase or replace with the immunogenic gene that comes from a donor virus strain or hypotype or gene fragment and will produce one directly at the immune response of donor strain or hypotype, acceptor strain or hypotype or anti-simultaneously confession acceptor strain or hypotype.In order to reach this purpose, embedded virus can be fabricated equally, to express a chimeric protein, for example, one has the immunogenic protein of tenuigenin tail and/or membrane spaning domain, and it is specific for a virus strain or hypotype, and merges with the extracellular region territory of a different virus.The other typical recombinant chou of this type can be expressed the repetitive proteins zone, for example repeats the immunogenicity zone.

Though it is useful increasing or replace full gene (comprising cis-acting elements and coding region) generally speaking in a mosaic gene group or anti-genome, it also is useful only shifting a part of interested donor gene.More generally, for example cis acting controlling element and gene intervening sequence there is no need to use the donor gene coding region to shift to non-coding Nucleotide.In addition, many gene fragments provide useful donor polynucleotide, in order to be included in the embedded virus that has new and useful characteristic in mosaic gene group or the anti-genome with expression.Like this, the heterologous gene fragment can advantageously be encoded from the selected proteic tenuigenin afterbody of a virus, be striden film functional zone or extracellular domain, antigen decision site or zone, binding site or zone, avtive spot or contain the zone etc. of avtive spot.These and other gene fragment can be added or replace corresponding gene fragment in other virus to produce a new chimeric recombinant chou, for example expresses tenuigenin afterbody with viral glycoprotein that merges with another viral glycoprotein extracellular region and/or the recombinant chou of striding the chimeric protein of film functional zone.Useful in this genomic fragment is approximately from 15-35 Nucleotide (the little functional regions of these gene fragment proteins encoded) antigen decision site for example, to about 50,75,100,200-500 and 500-1,500 or more Nucleotide scope (albumen zone or structural domain that the gene fragment coding is big).

In order to make up the embedded virus of the attenuation sudden change that carries transfer, in whole or background genome partly or anti-genome, can increase or replace with heterologous gene to form a mosaic gene group or anti-genome.Described sudden change can be present in heterologous gene (that is, donor gene) or the gene fragment with one or more additional sudden changes, maybe can be introduced in all or part of acceptor gene group or the anti-genome " background " and go.With the embedded virus by the replacement generation is example, (for example use from the albumen of the selection of a virus or albumen zone, a tenuigenin tail, stride film functional zone or extracellular domain, antigen decision site or zone, binding site or zone, avtive spot or contain the zone etc. of avtive spot) replace in different genomes or anti-genome corresponding " correspondence " gene or gene fragment and compare with wild-type or parent plant to have and expect the new recombinant strain of phenotypic alternation to produce one.Just as used herein, " correspondence " gene, gene fragment, albumen or albumen zone refer to two allogenic corresponding polynucleotide, the source is included in the different genes in single kind or the strain or the different variants of same gene, is included in allele variant or kind in different virus strain or the hypotype.

Corresponding gene or gene fragment generally have moderate structural similarity at least.For example, the corresponding gene fragment common associated protein structural domain of can encoding, tenuigenin structural domain for example, membrane spaning domain or extracellular domain, antigen decision site or zone, binding site or zone or the like.Typical situation is that they can have a common biological function.For example, for the coded protein structure domain of corresponding gene fragment can provide an identical membrane spaning domain, a special combination is active, an immunology recognition site etc.The corresponding gene or the gene fragment that are used for constructing in the present invention attenuated chimeric virus can comprise the set not of the same race with different sizes and sequence difference.Yet the selection of corresponding gene or gene fragment relies sequence identity basic between the theme counterpart, just as described above.In this manual, the polynucleotide canonical sequence of a selection is as all or part of of sequence in donor or acceptor gene group or the anti-genome.This canonical sequence is used to one sequence contrast basis sequencing row really is provided.For example, canonical sequence can be the definite cDNA or the fragment of gene, or complete cDNA or gene order.

Well-known method based on cDNA is very useful for structure one big group contains the attenuation sudden change of being differentiated in allos virus reorganization embedded virus or subviral particle.These recombinant precursors provide the attenuation and the immunogenicity feature of the improvement that is used for vaccine reagent.Resistance to an attenuation phenotypic reversion is arranged in the desired phenotypic alternation of this specification sheets, the raising of attenuation in the host environment of culture or selection, the immunogenicity feature (for example, determine by the immune response that increases, reduces or cause), the pirate recordings of selected viral product or the just adjusting of translating or negative adjusting etc.

In other aspects of the present invention, the attenuation recombinant virus of the attenuation sudden change that contains in allos virus to be differentiated by the attenuation phenotype introducing one or more signs and change other attenuations sudden changes and further modified.These sudden changes can produce again and test the attenuation effect according to the mutagenesis strategy of reasonableness design.As a kind of selection, the attenuation point mutation is differentiated in as a RSV or PIV ts or cp mutant strain at the mutant strain that biologically obtains, is introduced into then in the attenuation recombinant virus of the present invention and goes.Can be an embedded virus by the recombinant strain of further attenuation like this.

Be used to introduce further attenuation sudden change among the RSV that biologically obtains of vaccine strain can natural existence or by as the above-mentioned and described known induced-mutation technique of USSN 08/327,263 (described document is incorporated herein by reference) be introduced in the wild strain and go.

" biologically obtain " mutated viruses and mean its arbitrary mutated viruses that does not produce by recombinant means.Like this, the mutated viruses strain of Huo Deing biologically can be any minus-stranded rna virus strain, hypotype or kind, for example, naturally occurring RSV or PIV, it has the genome sequence of a sudden change, or RSV or PIV, and it has and comes from one and change with reference to the genome of wild-type sequence, for example, has the sudden change that characterizes the attenuation phenotype.Similarly, the virus that biologically obtains comprises by (especially) induced mutations and the select procedure mutant strain available from a parent plant.

The further attenuation sudden change of being differentiated has as mentioned above been entered in one " catalogue " then as scheduled by separately or in conjunction with introducing regulating the suitable attenuation of candidate vaccine virus to one by editor, immunogenicity, to the genetic resistance of reversing from the attenuation phenotype or the like the level that as was expected.Preferably is, recombination mutation virus of the present invention is at least a by introducing, and more preferably two or more discriminatings is from the attenuation point mutation of such catalogue and be attenuated, described catalogue is, for example, RSV mutant strain group is cpts RSV 248 (ATCC VR 2450) for example, cptsRSV 248/404 (ATCC VR 2454), cpts RSV 248/955 (ATCC VR 2453), cpts RSV 530 (ATCC VR 2452), cpts RSV 530/1009 (ATCC VR 2451), cpts RSV 530/1030 (ATCC VR 2455), RSV B-1 cp52/2B5 (ATCC VR2542), and RSV B-1 cp-23 (ATCC VR 2579) etc.Other sudden change can be from of the same race or xenogenesis is viral, the virus that comprises the attenuation sudden change with the ts, the cp that have been differentiated or non--ts or non--cp, described attenuation sudden change is for example differentiated in little plaque (sp), acclimatization to cold (ca) or host range restricted (hr) mutant strain.The attenuation sudden change can for example be selected in the cis acting regulating and controlling sequence at the coding region or the non-coding region of gene.For example, attenuation sudden change can be included in the change of one or more bases in the gene homing sequence, and for example the single base on 7605 of the M2 homing sequence Nucleotide is replaced in RSV.By this way, the attenuation of reorganization candidate vaccine can very accurately be calculated, and goes with the patient who is used for one or more classifications, comprises seronegative infant.The possibility of producing virus from cDNA allows these to be suddenlyd change separately or with the conventional full length cDNA clone of introducing of different selected combinations, can be easy to determine to contain the phenotype of introducing the recombinant virus of having saved that suddenlys change subsequently.

By differentiate and introduce with the expection phenotype for example the attenuation sudden change of cp or ts phenotypic correlation enter an infective recombinant virus clone, the invention provides one other or be close to the site-specific sex modification that suddenlys change of differentiating.Although many attenuations that result from the virus that biologically obtains sport monistic amino acid and replace, other " locus specificity " sudden change also be directed in the recombinant virus of the present invention by recombinant technology goes.As described herein, locus specificity sudden change from 1-3 up to about 5-15 or the Nucleotide that more changed (for example comprises, change from wild-type sequence, change, or from parent's recombinant clone that carries out mutagenesis, change from a mutant strain of selecting) insertion, replacement, disappearance or rearrangement.Such locus specificity sudden change can be introduced in the selected attenuation sudden change or be arranged in its zone.As a kind of selection, sudden change can be introduced in other different zone in virus clone, for example or the nucleotides sequence that approaches cis acting regulating and controlling sequence or proteins encoded avtive spot, binding site, immunogenicity antigenic determinant etc. list.Locus specificity virus mutation strain generally keeps the attenuation phenotype of an expection, but may demonstrate the phenotypic characteristic of abundant change, and itself and attenuation have nothing to do, and for example, immunogenic raising or scope increase, grow and improve.The further example of the locus specificity mutant of expection comprises the recombinant virus of the stable coding mutation in the codon that contains additional sign attenuation sudden change.Under some possibility situations, two or more Nucleotide replacements are introduced on the codon of sign attenuation amino acid change in parent's mutant strain or recombinant clone, have therefore produced one and have had the recombinant strain that counter-rotating from the attenuation phenotype is had genetic resistance.In other embodiments, upstream (according to the N-terminal direction of coded viral protein) or downstream (the C-end direction) of deciding nucleotide site with respect to target replaced, increases, lacks or be binned in to locus specificity Nucleotide, for example, from 1-3,5-10 until 15 Nucleotide or more 5 ' or 3 ', for example, to make up or to excise an already present cis acting regulating and controlling sequence.

Except single or multiple point sudden change or locus specificity sudden change, the change of attenuation recombinant virus of the present invention comprises the rearrangement of disappearance, insertion, replacement or whole gene or gene fragment.These sudden changes (for example can change in acceptor or donor gene group or anti-genome base in a small amount, from 15-30 base, until 35-50 base or more), or big Nucleotide zone (for example 50-100,100-300,300-500, a 500-1000 base), its character that depends on change (that is to say, the base of small number can be changed, to insert or to lack an immunogenicity antigenic determinant or change a little gene fragment, and when relating to big base zone, big gene or gene fragment are increased, replace, lack or reset.

On the other hand, the invention provides the sudden change of CDNA in coming from the reorganization negative strand viruses of allos virus, for example replenish the sudden change that relates to the addition type of identical or different gene in the recombinant virus of further modifying in cp and the ts mutant strain.So, viral protein can optionally be changed according to expression amount, or by all or part of increase, disappearance, replacement, rearrangement, combine to produce a recombinant attenuated virus separately or with other expection modification, it has other novel vaccine feature.

Like this, except available from the sudden change of the attenuation of allos virus mutation strain, the present invention provides some additive methods equally, and it is used for attenuation and based on the modification of the reorganization candidate vaccine of recombinant technology.Corresponding to this point of the present invention is that different changes can result from coding recombination group or anti-genomic isolating polynucleotide sequence.More specific is, in order in reorganization PIV, to obtain the structure and the phenotypic alternation of expection, the present invention allows such modification to introduce: lack, replace, introduce or reset a selected Nucleotide or from parental gene group or anti-genomic most Nucleotide, and in parental gene group or anti-genome, take place whole gene or gene fragment disappearance, replacement, introducing or rearrangement.

The expection of attenuation recombinant virus is modified general selected to characterize the phenotypic alternation of an expection, viral growth for example, and temperature sensitivity causes the ability of host immune response, change of attenuation or the like.These changes can be in donor or acceptor realize by for example parent clone's mutagenesis in genome or the anti-genome, with disappearance, introduce or reset a special genes or gene region (for example, proteins encoded structural region for example tenuigenin, stride the gene fragment of film or born of the same parents' ectodomain, immunogenicity antigenic determinant, calmodulin binding domain CaM, avtive spot or the like).The genes involved here can comprise arbitrary virogene, for example, and 3 '-NS1-NS2-N-P-M-SH-G-F-M2-L-5 ' (is example with RSV) and from other viral heterologous gene.

What be provided equally is modification in candidate's recombiant vaccine, its change or eliminated selected expression of gene, for example by in selected RSV encoding sequence, introducing a terminator codon, change the position of gene with respect to the promotor of operability connection, introduce a upstream atg start codon to change the speed of expressing, (for example modify, by changing the site, change already present sequence, or use a heterologous sequence to replace already present sequence) transcribe signal to change phenotype (temperature limitation of for example, growing, transcribing or the like.)。Various other disappearance, replacement, increase and rearrangements also are provided, and these all are characterized in the change of duplicating quantity and quality of selected gene transcription, selected proteic translation, virus.

The attenuation sudden change of analyzing and merging other types enters the ability of going in the reorganization candidate vaccine and extends to a set very widely, and it hits and fixes on variation in the recombinant clone.For example, the disappearance of SH gene has produced one and has had the new phenotypic characteristic recombinant RSV incorporate of (it comprises the raising of growing) in RSV.Like this, a SH genetically deficient (or other dispensable gene or gene fragment disappearance) in recombinant RSV incorporate of the present invention, the sudden change with one or more additional sign attenuation phenotypes in a recombinant virus combines.These sudden changes for example are one or more point mutation from allos virus, and it is optionally replenished by one or more further attenuation sudden changes from the attenuation mutant that biologically obtains.In some embodiments, the SH gene of RSV or NS2 genetically deficient and combine the cp of one or more cpts248/404 of being selected from, cpts530/1009, cpts530/1030 or other selected RSV mutant strains and/or the ts sudden change has the recombinant RSV incorporate that the resistance that improves viral yield, strengthen attenuation, phenotypic reversion etc. belongs to the effects that combination was produced of different sudden changes to produce one.

In the representative instance in the RSV clone that SH reduces, the viral genome of being modified is 14,825 nt length, and it compares less 398 Nucleotide with wild-type.By causing the similar sudden change that can reduce the genome size, for example at genomic other coding region or the non-coding region of RSV, for example in P, M, F and M2 gene, the invention provides several method that is easy to obtain and experiment materials, it is used to improve the growth of chimeric RSV.

In addition, according to the method for the invention, other multiple genetics change also can result from a recombination group or anti-genome to be introduced into an infective attenuated virus particle.Other heterologous gene or gene fragment (for example available from different virus gene or different virus strain or type) can be by all or part of insertions, gene order is changed, remove the gene overlap sequence, genomic promoter is replaced by the corresponding site of anti-genome, portion gene is removed or replaces, sometimes even full gene disappearance.Different or additional modification in the sequence can be used to promote operation, for example in the insertion of the restriction site of different intergenic region territories or other site uniquenesses.Untranslated gene order disappearance is to increase the possibility that exogenous array inserts.

Genetic modification in attenuation reorganization candidate vaccine virus strain equally also is provided in the present invention, and gene or gene fragment are not removed in its change or eliminated an expression of selecting gene or gene fragment simultaneously from recombinant clone.For example, these are by introducing a terminator codon in a selected encoding sequence, change the site of a gene or introduce a upstream and start codon to change the speed of its expression, or change and to transcribe signal and realize to change phenotype (temperature limitation of for example, growing, transcribing etc.).

Preferred in this manual sudden change comprises the sudden change towards the cis acting signal, and it can be differentiated, for example, and by the little genomic mutation analysis of virus.For example the insertion of leader sequence, non-transcribed tail region and flanking sequence or deletion analysis can be differentiated viral promotors and transcribe signal and a series of and rna replicon are provided or have transcribed the relevant sudden change of change of minimizing degree.The saturation mutagenesis of cis acting signal (each position is modified to each selectable Nucleotide thus) identifies many sudden changes equally, and it has reduced duplicating or transcribing of (perhaps, increasing in both cases) RNA.Any one of these sudden changes can be inserted in the anti-genome of an into reorganization as herein described or the genome and go.

Trans-acting albumen and cis acting RNA sequence use anti-completely genome cDNA is estimated and operated and it uses virus little genome as auxiliary (referring to, Grosfeld etc. for example, J.Virol.69:5677-5686 (1995), described document is incorporated herein by reference), its state that relies on helper virus is very useful in the feature of describing these mutant strains, and these mutant strains are suppressed very much and can not reclaim in duplicating the infective virus of dependent/non-dependent.

Other sudden changes of recombinant virus of the present invention relate to the replacement of genome 3 ' end, and it is replaced by coming from anti-genomic corresponding site.Its with rna replicon and the change in transcribing be relevant.In addition, intergenic region (Collins etc., Proc.Natl.Acad.Sci.USA83:4594-4598 (1986), described document is incorporated herein by reference) can be prescinded or the content of prolongation or sequence changes and naturally occurring gene overlap (Collins etc., Proc.Natl.Acad.Sci.USA 84:5134-5138 (1987), described document is incorporated herein by reference) can be removed or change over different intergenic regions by method as herein described.

In another typical embodiment; specific protein is rsv protein protectiveness F and the antigenic expression level of G for example; can be designed to effectively translation is corresponding to by using one, and be fitted into codon among the synthetic CDNA and replace natural codon and be increased.In this manual, the selection of display password is a vital factor (Haas etc., Current Biol.6:315-324 (1996)) for the proteic translation skill of mammalian virus.What the F of coding RSV and the codon of the proteic mRNA of G were selected determines that shown this selection with consistent than weak expression, described F and G albumen are main protective antigen.Like this, by recombination method of the present invention, codon selects to be improved the expression with the raising that realizes selected gene.

In another typical embodiments, the sequence (preferably including the Nucleotide of one-3 position) around a translation initiation site of a selected virogene is modified, its separately or unite the introducing of a upstream initiator codon, thereby by at the plus or minus regulation and control of translation to regulate the genetic expression of attenuation recombinant virus.

At one more in the detailed embodiment, the RSV expression of gene of attenuation can be regulated and control by changing a selected gene transcription GS signal of virus.In another embodiment, the GS signal of NS2 is modified with the sudden change that comprises a definition (for example, 404 (M2) sudden change described below) thereby is added a ts and limit in the duplicating of virus.Another attenuation RSV clone can be introduced into modification one and transcribe the GE signal in addition.For example, RSV clone can be produced, and it has NS1 and NS2 gene GE signal a replacement or sudden change for the N gene, and its result is to read over the minimizing of mRNA level and from the increase of the protein expression of downstream gene.The recombinant virus of gained will be showed the raising of growth power, the increase of plaque size, and condition is that the growth characteristics that the modification by cis acting regulating and controlling sequence in the RSV genome changes RSV only are an example.

In another embodiment, the proteic particular expression of G of the RSV of attenuation reorganization is enhanced by modifying G mRNA.G albumen is expressed as a kind of film mating type and a kind of secreted protein, and the latter begins translation by the initiation site a G translation open reading frame and expressed.Secreted form approximately can account for half in the G albumen of expression.The excision in internal start site (for example, by sequence variation, disappearance or the like) combines to produce the G protein expression change of expection separately or with the change of upstream initiation site sequence.The excision of G protein excretion form will improve the immunoreactive quality of host at typical chimeric RSV equally, because the proteic soluble form of G is considered to inveigle and catches neutralizing antibody and work.Equally, resolvability G albumen is also relevant with immunopathologic raising, because its reaction to the Th2 deflection has preferential hormesis.

In another embodiment, attenuation recombinant viral genome expression levels is modified at transcriptional level, on the other hand, the position of a selected gene can change to a more promotor-proximal or away from the site of promotor in the RSV gene mapping, and gene will be by effective expression respectively more or less thus.According to this point, the expression regulation of specific gene can be done, and produces the genetic expression that the wild-type level of comparing reduces or increase twice, is typically 4 times, until 10 times or more.In one embodiment, the NS2 gene of RSV (being positioned at second in the RSV gene mapping) is replaced by SH gene (coming the 6th) in this position, has produced the expression decreased of the NS2 of an expection.The selected RSV expression of gene that takes place because the position changes improves and can be done, and goes up to 10 times or more, often with the corresponding minimizing that produces an expression level on complimentary positions in the gene of replacing.

In another typical embodiments, virogene can be separately or and other together by swivel base to the recombinant viral genome collection of illustrative plates more near promotor or more become estranged on the site of promotor, to realize the higher of gene expression dose respectively or than low value.These and other swivel base change to produce one and has the new clone that the attenuation phenotype for example relates to the decrement expression etc. of selected viral protein in the rna replicon.

The present invention more detailed aspect, the attenuation recombinant virus can further be modified on translation skill by the NS2 expression of gene of eliminating virogene such as RSV, and not missing gene or gene fragment, for example, by two tandem translation stop codon are introduced in the translation open reading frame (ORF).One of them selected gene of the live virus of Chan Shenging is reticent in translation skill like this, and does not lack this gene.These " rejecting " viral forms show poor growth in tissue culture and plaque diminishes.Like this, method and composition of the present invention provides a kind of additional, novel attenuation sudden change, and it can be eliminated not is the expression of main viral protective antigen or the necessary virogene of viral growth.In this manual, " rejecting " the viral phenotype that produces under the situation of not missing gene or gene fragment also can produce by deletion mutagenesis as described herein, thereby has got rid of the sudden change of answer target protein synthetic correcting property effectively.The method that produces these and other rejecting in this area be know altogether (as Kretzschmar etc., Virology 216:309-316 (1996); Radecke etc., Virology 217:418-412 (1996); And Kato etc., EMBO is (1987) J.16:178-587; And Schneider etc., Virology 277:314-322 (1996) is described like that, and described document all is incorporated herein by reference).

Infectious recombinant virus clone of the present invention can make to improve immunogenicity and to induce the immunoprotection of the higher level that is better than wild-type or parent's recombinant virus induced reaction to react according to method and composition disclosed herein equally.For example, from an allos virus strain or type for example the immunogenicity antigenic determinant of PIV can join a recombinant clone for example among the RSV, change by Nucleotide suitable in coding mosaic gene group or anti-genomic polynucleotide sequence and realize.In addition, also can operate virus to increase or elimination (for example, by aminoacid insertion, replacement or disappearance) immunogenicity antigenic determinant relevant with expection or unexpected immune response.

In the method for the invention, additional gene or gene fragment can be inserted into or a close acceptor gene group or anti-genome.These genes are can be with acceptor gene modulated or be under independent one group of control of transcribing signal.Interested gene comprises the gene of the Codocyte factor (for example, IL-2 to IL-15, especially IL-2, IL-6 and IL-12 etc.), the gene of coding gamma-interferon and the proteic gene that coding is rich in the complementary cell antigen determinant of T.These additional albumen or expressed as an independent albumen are perhaps expressed as the mosaic that for example obtains another copy of SH from a viral protein.So just provide and improved and the quantity of raising antiviral immunity reaction and the ability of quality.

In one embodiment of the invention, foreign gene or gene fragment, or be that non-coding nucleotide sequence is inserted in the recombinant virus genomes and goes sometimes, result cause having the expection of the genome length of additional expection phenotype effect to increase.The genome length result who increases makes viral attenuation, and the degree of attenuation partly depends on the length of insertion.In addition, from allos to certain albumen of recombinant attenuated virus of the present invention for example cytokine expression cause viral further attenuation because of protein-active.This has been verified in IL-2 expresses in vaccine virus (for example Flexner etc., Nature 33:-259-62 (1987)), equally in IFN-also expection analogue appears.

The disappearance, insertion, replacement and other sudden changes that relate to the change of whole virogene or gene fragment in recombinant virus of the present invention produce the candidate vaccine of high stability, and it is particularly important in immunosuppressed individuals.Many this change results cause the attenuation of vaccine strain, and other will characterize the dissimilar of expection phenotypic alternation.For example, certain virogene is known can encode with the special interferential albumen of host immune (referring to, for example, Kato etc., EMBO.J.16:578-87 (1997), described document is incorporated herein by reference).Eliminate this gene in the recombinant vaccine virus, expection can reduce toxicity and pathogenic and/or raising immunogenicity.

Additional sudden change can further make recombinant virus attenuation of the present invention, it comprises the introducing of heterologous gene or cis-acting elements, its host range restriction and other are provided some be suitable for the expection phenotype that vaccine uses.In one embodiment, among the human RSV of the selected introducing of ox RSV sequence, this is based on the known aspect of ox RSV structure and function, as, for example, Pastey etc., J.Gen.Virol.76:193-197 (1993); Pastey etc., Virus Res.29:195-202 (1993); Zamora etc., J.Gen.Virol.73:737-741 (1992); Mallipeddi etc., J.Gen.Virol.74:2001-2004 (1993); Mallipeddi etc., J.Gen.Virol.73:2441-2444 (1992); And Zamora etc., it is such that Virus Res.24:115-121 (1992) is provided, and described document all is incorporated herein by reference, and it is with disclosed herein consistent.In another embodiment of the invention, set up for introduction into the interested sudden change among the chimeric RSV, imitate the non-pathogenic strain (corresponding to the RSV in the mouse of human RSV) of a tissue culture adaptation type mouse Pneumovirinae, it lacks a proteic tenuigenin afterbody of G (Randhawa etc., Virology 207:240-245 (1995)).Therefore, in one aspect of the invention, one or more human rsv glycoprotein F, G and the tenuigenin of SH and/or stride diaphragm area, in the attenuation recombinant RSV incorporate, use the corresponding sequence of allos to add, lack, modify or (for example replace, from F, G and the proteic tenuigenin of SH of a mouse Pneumovirinae or stride the sequence of diaphragm area), to obtain the attenuation of expection.In other embodiments, the attachment region of inferring at the proteic cleavage site of F or G albumen or near the nucleotide sequence in this site can by point mutation, site-specific sexually revise or the change of full gene or gene fragment modify with to virus in tissue culture growth and/or infectious and pathogenic a new influence arranged.

Except above-mentioned modification, can use also that different or additional modification promotes operation in the virus clone, for example in the insertion of different genes transcribed spacer or other single restriction sites in site to recombinant vaccine virus.The untranslated gene order can be removed to improve the ability that exogenous array inserts.

In another aspect of this invention, composition (for example, isolating polynucleotide and contain the virus genomic carrier of recombinant negative strand rna with sudden change of being differentiated in allos virus) is provided to produce an isolating attenuation infectious virus.Use these compositions and method, infectious virus and subviral particle result from the viral protein of a recombination group or anti-genome and selection, for example, a nucleocapsid (N) albumen, a nucleocapsid phosphorprotein (P), big (L) polymerase protein and one RNA polymerase elongation factor (at RSV).At related aspect of the present invention, provide composition and method in recombinant virus, to introduce aforementioned structure and phenotypic alternation, to produce the vaccine virus of an infectivity, attenuation.

By as a reference known method above a series of will be aforementioned definite sudden change introduce in an infective recombinant virus and go.Here " infections clone " means cDNA or its product (synthetic or obtain through other approach), and it can be transcribed to enter in a genome or the antigenomic RNA and go, and it can be as a template to produce infectious virus or subviral particle.Like this, can be introduced in genome or the anti-genomic cDNA copy by the certain sudden change of conventional art (for example site-directed mutagenesis) and go.Use anti-genome or genome cDNA subfragment to make up a complete as described herein anti-genome or a genome cDNA, it has the advantage that can be operated separately in each zone (little cDNA compare big easy handling), and is easy to be fitted among the complete cDNA and goes.Like this, complete anti-genome or genome cDNA, or its subfragment can be used as a template to carry out the mutagenesis that oligonucleotide instructs.This can for example use the breadboard Muta-gene  of Bio-Rad test kit (Richmond by way of a single stranded plasmid form, CA) or use a double-stranded plasmid directly as the template Chameleon mutagenesis kit of Stratagene (La Jolla, method CA) or use to contain the oligonucleotide primer of relevant sudden change or the method for template is operated for example by polymerase chain reaction.The subfragment of a sudden change is fitted into complete an anti-genome or a genome cDNA then.Many other induced-mutation techniques are known and be suitable for using producing a sudden change interested in anti-genome or genome cDNA, and sudden change can change from the segmental replacement of big cDNA that changing to of single nucleosides contained one or more genes or genome area.

Like this, in an illustrative embodiment, introduce sudden change available from the Muta-gene phagemid mutagenesis kit of Bio-Rad company by using.Encode a part of RSV genome or anti-genomic cDNA entered among the plasmid pTZ18U by the clone, and is used to transform CJ236 cell (Life Technologies, Inc.).As recommending, handbook prepares phasmid reagent.Carry out mutagenesis by Nucleotide and design oligonucleotide in a genome or a change of anti-genomic expection site introducing.The plasmid that contains genome that genetics changes or anti-genomic fragment is amplified then and the fragment of suddenling change is introduced again in the genome of total length or the anti-genomic clone and gone.

The present invention equally also provides the method that contains the infectious recombinant virus of attenuation of the attenuation sudden change of differentiating from for example one or more cDNA of one or more isolating polynucleotide in the allos virus that obtains that produces.Coding one theme viral genome or an anti-genomic cDNA be fabricated with in born of the same parents or external with must the viral protein coexpression to produce a for example infective RSV.Here " anti-genome " means an isolating just polynucleotide molecule, and it is as the synthetic genomic template of progeny virus.Preferably is to make up a cDNA, it is the synthetic template of a just genome, it is corresponding to intermediate RNA that duplicates or anti-genome, thereby minimizing and coding help to transcribe the hybridization that the just transcript of the proteic complementary sequence of the generation of duplicating nucleocapsid carries out.For this purpose of the present invention, the genome of recombinant virus or anti-genome only need to contain these genes or wherein necessary part, so that coded infective virus or subviral particle have infectivity.Further, gene or its part can provide by the polynucleotide molecule more than, that is to say, provide a gene by complementary or similar approach from an independent nucleic acid molecule.

Here recombinant virus means the complete virus or the subviral particle of similar virus, and it directly or indirectly is derived from a recombinant expression system or obtains from consequent virus or subviral particle breeding.Recombinant expression system has a recombinant expression vector, it contains its aggregate that has at least one genetic elements or start to control the element of making usefulness in the viral gene expression process of transcriptional units that an operability connects, for example, a promotor, transcribe structure or the encoding sequence that enters viral RNA for one, suitable transcripting starting or terminator sequence.

In order to produce infectious viral particle the genome of expressing or the anti-genome from cDNA, produce the nucleocapsid that can carry out rna replicon according to currently known methods expressing gene group or anti-genome with (i), and (ii) make offspring's nucleocapsid carry out rna replicon and transcribe.Transcribe the albumen that other are provided and caused large-scale an infection by what the genome nucleocapsid carried out.As a kind of replacement, other can produce by coexpression for the necessary viral protein of extensive infection.

The anti-genome of virus can be fabricated and use in the present invention, for example, by assembling clone's cDNA fragment, the anti-completely genome of its set form representative, the polymerase chain reaction (PCR of the reverse transcription copy by using viral messenger RNA(mRNA) and geneome RNA; Referring to United States Patent (USP) 4,683,195 and 4,683,202 and the PCR handbook: practice and using method guide (PCR Protocols:A Guide to Methods and Applications), editors such as Innis, Academic Press, San Diego (1990), described document is incorporated herein by reference).For example, contain anti-genome or wherein the cDNA of a part be fitted into a suitable expression vector, for example plasmid or the various cosmid that gets, phage or dna viral vector.Carrier can and/or insert the synthetic polylinker and modify by mutagenesis, and described polylinker contains and is designed to promote the single restriction site that assembles.Under some situation as RSV, the use of special carrier (pBR322) can the stable virus sequence, and it may also can keep nucleotide deletion or insertion in other cases.In addition, the breeding of plasmid can be promoted that to avoid imagineering or insertion, (for example, closing on 4499 Nucleotide in RSV) can take place in other cases for it by the selection of specific bacterial strain (for example DH10B).

In some embodiments of the present invention, encode a pair of generation one for transcribe, the necessary proteic complementary sequence of replication-competent virus nucleocapsid can provide by one or more helper viruses.These helper viruses can be wild-type or mutant strain.Preferred situation is that helper virus can distinguish because of phenotypic difference from the virus of encoding for cDNA.For example, expection provides a monoclonal antibody, and it with helper virus immune response takes place but the virus of not encoding with CDNA reacts.Such antibody can be neutralizing antibody.In some embodiments, antibody can be used to affinity chromatography, to isolate helper virus from recombinant virus.In order to help obtaining of these antibody, can in cDNA, introduce sudden change so that an antigen diversity to be provided from helper virus, for example RSV HN or F glycoprotein gene.

In viral genome or anti-genome, can insert or lack many Nucleotide to produce the attenuated virus clone of a modification.Member in paramyxovirus and the Morbillivirus generally observes one " six principles ", that is to say that genome (or little genome) only duplicates the most effectively (being considered to the needs of nucleotide residue with respect to the proteic accurate spacing of nucleocapsid N) when its length of nucleotides is 6 multiple

Make up coding contains the recombinant negative strand rna virus of the sudden change of differentiating in allos virus the method for replaceability of cDNA comprise use improvement the PCR condition (for example, as described in Cheng etc., Proc.Natl.Acad.Sci.USA 91:5695-5699 (1994), described document is incorporated herein by reference) carry out reverse transcription with the quantity that reduces cDNA component subunit to one or two fragment.Can use in another embodiment different promotors (for example, T3, SP6) or different ribozyme (for example, the ribozyme of hepatitis D virus).It can be used to propagation different dna vector (for example cosmid), to carry big genome or anti-genome.

The isolating polynucleotide (for example cDNA) of the viral protein of the viral genome of encoding separately or jointly or anti-genome and necessity are inserted into the cell that can support effective virus infection to produce by transfection, electroporation, mechanical insertion, transduction or similar approach and for example go in HEp-2, FRhL-DBS2, MRC and the Vero cell.The transfection of isolating polynucleotide passage be directed in the cultured cells goes, by transfection (Wigler etc., the Cell 14:725 (1978) of for example calcium phosphate mediation; Corsaro and Pearson, Somatic Cell Genetics 7:603 (1981); Graham and Van der Eb, Virology 52:456 (1973)), electroporation (Neumann etc., EMBO is (1982) J.1:841-845), transfection (Ausubel etc. (editor) the Current Protocols in Molecular BiologJohn Wiley and Sons of DEAE-dextran mediation, Inc., NY (1987), transfection (the Hawley-Nelson etc. of cationic lipid mediation, Focus 15:73-79 (1993)) or a commercially available transfection reagent, for example, LipofectACE  (Life Technologies) (following each bibliography all is incorporated herein by reference).

Viral protein is that one or more expression vectors are coded, and described expression vector can be identical or different with the carrier of encoding gene group or anti-genome and various combination thereof.Additional albumen can be comprised as scheduled that its carrier for the carrier of oneself or the necessary viral protein of encoding is coded.The proteic expression that genome or anti-genome reach from infectious plasmid can obtain, for example by each cDNA under the regulation and control of T7 rna polymerase promoter, its back also can by infect with a T7 RNA polymerase expression system, transfection or transduction obtain, described expression system, vaccine virus MVA strain recombinant chou for example, it expresses T7 RNA polymerase (Wyatt etc., Virology, 210:202-205 (1995), described document is incorporated herein by reference).Viral protein and/or T7 RNA polymerase can obtain from the mammalian cell that transforms or obtain by preformed mRNA or proteic transfection equally.

As a kind of selection, genome or anti-genomic synthetic can be external finishing in the bonded transcription and translation reaction, transfection subsequently enters cell.Perhaps, genome or antigenomic RNA also can externally synthesize, and transfection enters the cell of expressing viral protein then.

In order to select candidate vaccine virus, the standard of survival, attenuation and immunogenicity can be determined in accordance with known methods according to the present invention.The virus that becomes vaccine of the present invention probably must be kept survival, has stable attenuation phenotype, in the host of immunity, can duplicate (even in very low level), and effectively cause enough immune responses and resist serious the catching that causes by wild-type virus.In this manual; virus of the present invention is not only alive and than former mutant strain attenuation; and the mutant strain of research before comparing; it is more stable on the genetics in vivo-can excite protective immune response and can expand this protection by multiple modification in some cases; for example; induce and resist reaction not of the same race, subtype virus; perhaps by different immune-bases antiserum(antisera) immunoglobulin secretion ability for example, cellular immunization and analogue thereof or the like are protected.

In order to breed the recombinant negative strand rna virus that carries the sudden change of in allos virus, being differentiated, can use the clone that some allow viral growth.The desirable clone that is used to breed the attenuation mutated viruses strain of using as vaccine comprises DBS-FRhL-2, MRC-5 and Vero cell.The high yield of RSV often can by use epithelial cell line for example the Vero cell obtain.Cell with virus different infection scopes for example from about 0.001 to 1.0 or inoculate and cultivate allowing under the condition of virus replication, for example in approximately 30-37 ℃ and about 3-5 days or longer so that virally reach enough titre.Virus shifts out from cell culture and separates from cellular component by known separation techniques is for example centrifugal, and can use this area known technology to be further purified to accomplish the end in view.

The recombinant virus of attenuation described herein can by in many known and received bodies or extracorporeal mode test with confirm its enough attenuation, to the resistance of phenotypic reversion and the immunogenicity used as vaccine.In external experimentation, the temperature sensitivity of the tested virus replication of the virus of modification (for example, a multiple attenuation, that biologically obtain or recombinant virus) is the ts phenotype, and little plaque phenotype.The virus of modifying is for further testing in the animal model of virus infection.The animal model of many RSV of being used for has been described and has been animal model (Animal Models ofRespiratory Syncytial Virus Infection) the Merieux fund press of editor's such as Meignier respiratory syncytial virus infection, (1991), described document is incorporated herein by reference) summarize.The cotton mouse model of a rsv infection is a United States Patent (USP) 4,800,078 and Prince etc., Virus Res, 3:193-206 (1985) (described document is incorporated herein by reference) describes, and can be used for reasonably inferring in human or inhuman primates attenuation and effect wherein.In addition, a primate model that uses the rsv infection of chimpanzee is used for reasonableness and infers its attenuation and validity the mankind, and it is described J.Med.Virol.3:91-100 (1978) by Richardson etc.; Wright etc., immunology of infection (Infect.Immun.) 37:397-400 (1982); Crowe etc., vaccine 11:1395-1404 (1993), described document all is incorporated herein by reference.The animal model of some other minus-stranded rna virus that is used for wide range is all known.Attenuation level and immunogenic dependency available from data in these animal models and recombinant negative strand rna virus are all generally accepted.For example, the medical effect of RSV neutralizing antibody has been shown with subsequently use monkey or has humanly used the experiment that rsv infection carried out that very high cognation is arranged in the cotton mouse that infects.In fact, cotton mouse demonstrates it for carry out rational reliable infected monkey, chimpanzee and the human experiment substitute of immunotherapy for use RSV neutralizing antibody.For example, the quantity of RSV neutralizing antibody is relevant with the curative effect in cotton mouse, it can be measured by the level of this antibody in the serum of treatment animal and (that is to say, among the serum RSV and titre be 1: 302 to 1: 518), it is consistent with (being 1: 539 titre) or (it is 1: 877) confirmed in human infant or child scope in monkey.Curative effect in the cotton mouse can reduce by 100 times or the (Prince etc. that confirm by virus titer in lung more, J.Virol.61:1851-1854), reduced by 50 times (Hemming etc., J.Infect.Dis.152:1083-1087 (1985)) and in monkey, observe a curative effect for lung's virus titer.At last, baby who is in hospital causing bronchitis or pneumonia because of serious rsv infection or the curative effect in the small children can confirm by the remarkable increase of oxygenation in treatment colony and the remarkable minimizing of RSV recyclable amount from treatment patient's the upper respiratory tract.(Hemming etc., Antimicrob.Agents Chemother.31:1882-1886 (1987)).Therefore, based on these research, cotton mouse is formed a relevant model, is used for predicting the successful property of chimeric or non-chimeric RSV vaccine the infant.Other rodents comprises that mouse will be very useful because these animals allow duplicating of RSV and have temperature (Wright etc. in the more subhuman body equally, J.Infect.Dis.122:501-512 (1970) and Anderson etc., J.Gen.Virol.71:(1990)).Similarly model for other minus-stranded rna virus be obtainable and widely known to.

With aforementioned consistent and based on following embodiment, the present invention provides the separated infection attenuated virus composition that vaccine uses that is used for equally.The virus as a component of vaccine of this attenuation is isolating and the form of purifying.Here " separated " refers to RSV, and it is not to be in the natural surroundings of wild-type virus, for example in the nasopharynx in an infected individuals.More general situation is that " separated " means and comprise the component of attenuated virus as cell cultures and other people worker's medium.For example, the RSV of attenuation of the present invention its can be by being produced in the infectious cell culture, from cell culture, separate and be added into a stablizer.

Vaccine of the present invention contains the recombinant negative strand rna virus as the immune significant quantity of an activeconstituents, and it carries the sudden change of being differentiated as described herein in an allos virus.Recombinant virus can directly be used as vaccine form or lyophilized form.Freeze-drying virus generally is kept at about 4 ℃.When prepare using, freeze-drying virus is at stable solution salt or contain SPG, Mg for example ⁺⁺And HEPES, there is or do not have adjuvant, these will be further described below.That biologically obtain or can utilize on the physiology acceptable carrier and/or adjuvant to introduce among the host through the virus of recombinant modified.Useful carrier is known in this area, and it comprises, for example, and water, damping fluid, 0.4% salt solution, 0.3% glycine, hyaluronic acid etc.The liquor of gained can packaged use or freeze-drying use, freeze-dried reagent uses as mentioned above in conjunction with a sterile liquid before administration.Composition can comprise pharmaceutically useful ancillary component with near acceptable condition on the physiology, for example pH adjustment and buffer reagent, ooze degree of rising conditioning agent, wetting agent etc.For example, sodium-acetate, Sodium.alpha.-hydroxypropionate, sodium-chlor, Repone K, calcium chloride, mono laurate dehydration sorb ester, triethanolamine oleate etc.The acceptable adjuvant comprises incomplete freund's adjuvant, aluminum phosphate, aluminium hydroxide or alum, and it is this area material in common knowledge.Preferred adjuvants comprises Stimulon equally ^TMQS-21 (Aquila biopharmaceutical company, Worchester, MA), MPL ^TM(3-0-deacylated tRNA monophosphoryl lipid A; RIBI immunochemistry research company, Hamilton, MT), and IL-12 (genetic institute, Cambridge, MA).

Use that the attenuated virus vaccine composition is as described herein to carry out immunity through aerosol, drops, oral, local or other approach, host's immunity system reacts to vaccine, and its generation is specific to for example antibody of RSV F and/or G glycoprotein of one or more viral proteins.The result of immunity is that the host becomes one for theme virus infection generation at least a portion or whole immune resistibility or extremely serious advancings of disease of opposing moderate.

Vaccine of the present invention can comprise attenuated virus, and it causes for example immune response of RSV A or B or anti-a plurality of strains or hypotype of anti-single virus strain or antigenic subtype.In this manual, a plurality of strains or hypotype are resisted in the immune response that can cause monospecific immune response or polyspecific of chimeric virus.Embedded virus with different immunogenicity features also can be incorporated in the vaccine mixture or in a combined treatment and give separately, to cause the immunoprotection reaction of a more efficiently anti-RSV strain or anti-a plurality of RSV strains or hypotype.

The host who is given vaccine can be the Mammals of viral to theme arbitrarily or its related viral infections susceptible, and it can produce a kind of at the antigenic protective immunological reaction of vaccine virus.Therefore, Shi Yi host comprises the mankind, non-human primates, ox, horse, pig, sheep, billy goat, rabbit, rodent or the like.Correspondingly, the invention provides the production method of the vaccine that different people and domestic animal are used.

The vaccine composition that contains attenuation recombinant virus of the present invention is administered at " immune significant quantity " (being enough to cause or improve the immune response ability of individual anti-theme virus at this dosage) susceptible or be among the patient of high risk of theme virus infection down.With the mankind is example, give attenuated virus of the present invention according to the human vaccine immunity program of having set up, for example, for RSV, referring to Wright etc., Infect Immun.37:397-400 (1982), Kim etc., Pediatrics52:56-63 (1973), and Wright etc., J.Pediatr.88:931-936 (1976), described document all is incorporated herein by reference.In brief, adult or child in nasal cavity by drops by the RSV of immunoprophylaxis significant quantity, it is usually on a physiology in acceptable diluent or the carrier, and cumulative volume is 0.5ml.Compare with parenteral immunization nonreplicative vaccine, it has advantage simple and safety.It provides the direct stimulation to local respiratory immunity equally, and local respiratory immunity plays an important role in anti-RSV.Say that further this inoculation pattern has been avoided the immunosuppressive effect from mother's RSV specific serum antibody effectively, this is more common in age the children.Equally, the antigenic parenteral of RSV gives relevant with immunological disease originality complication sometimes, but but never finds this situation in live virus.

In all situations, the accurate amount of the attenuated virus vaccine that is given and the number of times that gives and time will determine according to character of patient body state, body weight, the mode that gives, reagent etc.Dosage range is generally from about 10 ³To about 10 ⁶Plaque forming unit (PFU) or more virus/patient, more common is from about 10 ⁴To 10 ⁵Virus/the patient of PFU dosage.In each case, vaccine necessarily provides the attenuated virus of the present invention of sufficient amount with the antiviral immune response of effective initiation, for example, as complement in conjunction with in, the thrombocyte and/or enzyme linked immunosorbent assay and other methods determined.In this point, the symptom of individual same monitored upper respiratory disease.As for to the giving of chimpanzee, the attenuated virus vaccine causes the level of growth of vaccine in nasopharynx to reduce by 10 times or more than wild-type, or compares to be similar to the level of incomplete attenuated virus and reduce by 10 times or more.

In newborn infant or child; need the multiple mode that gives to cause the immune response of capacity; for rsv infection; should give in 1st month in birth; and give at interval in whole infancy; for example two months, six months, 1 year or 2 years, thus the protective reaction that keeps the anti-enough levels of natural (wild-type) rsv infection.Identical is; especially concerning the adult's individuality that for example hygiene care workman, daily treatment workman, immature kinsfolk, old man, cardio-pulmonary function weaken that is easy to superinfection or serious rsv infection, need multiple immunity to set up and/or the maintenance protective immunological reaction.The level of immunity can be monitored by the quantity of measuring neutrality secretion and serum antibody, and the words that are necessary are carried out dose titration and repeated inoculation to reach the protection level of expection.Further, different vaccine virus can be designated as and give different acceptor colonies.For example, the recombinant chou of expressing a cytokine or being rich in the proteic structure of T cell antigen determinant, the infant that compares concerning the adult has more advantage.Combine with another strain of expression or the antigenic virus of subtype virus to realize the immunoprotection reaction of anti-multiple strain or hypotype according to the vaccine that the present invention produced.As a kind of replacement, vaccine virus can contain that the protective antigen determinant of multiple virus strain or hypotype is as described herein to be advanced among another clone to go with processing.

When different vaccine viruses was used, it can be given simultaneously with the form of mixture generally speaking, but it also can be given respectively.For example, only in the difference of aminoacid sequence about 11%, this similarity is to use RSV or F antigen immune and using the immunoreactive basis of viewed cross protection in the allos strain infected animals as its difference of the F glycoprotein of two RSV hypotypes.Like this, use a strain immunity can avoid the infection of the not homophyletic of identical or different hypotype.Yet very possible situation is more preferably to have G or the F glycoprotein of RSV antigenic subtype A or B in vaccine.

Attenuation recombiant vaccine of the present invention causes an immunoreactive generation, and it can resist serious disease for example pneumonia and bronchitis in wild strain virus infection subsequently.Although natural propagation virus still can cause infecting, disease symptoms but has very big minimizing, and this is because immunity reaches the cause that infects the resistance that may improve subsequently because of wild-type strain.After the inoculation, reaching in serum that the host produces and the secretory antibody can detected level, and it can neutralize homology (or same hypotype) wild-type virus in vivo and in vitro.In a lot of cases, equally also can the neutralize wild-type virus of difference or non-vaccine hypotype of host's antibody.

Preferred attenuated virus of the present invention toxicity when comparing with the wild-type virus of natural propagation in human body has had attenuation significantly.Embedded virus by attenuation sufficiently so that the symptom that in most immune bodies, infects no longer take place.In some cases, attenuated virus still can be disseminated in the non-immune individuality.Yet, its toxicity had a greatly reduced quality so that immunity or accidental host in very serious lower respiratory infection takes place.

The attenuation degree of vaccine virus of the present invention can be determined by the comparison that for example is determined in immune host's respiratory tract the quantity that has virus and produces quantity in wild strain or other have been assessed as the attenuated virus of candidate vaccine virus strain.For example, attenuation RSV of the present invention in high susceptibility host for example the degree of the restriction of duplicating in the upper respiratory tract in the chimpanzee compare higherly with the level of wild strain virus replication, for example duplicate and reduce 10 to 1000-doubly.Equally, in the chimpanzee upper respiratory tract, the levels of replication of attenuation RSV vaccine strain should be lower than RSV A2 ts-1 mutant strain, and it had been proved to be incomplete attenuation in the past in seronegative human infant.In order further to reduce the development of the rhinorrhea relevant with virus replication in the upper respiratory tract, ideal candidate vaccine virus duplicate the level that in last lower respiratory tract, be in restriction.Yet, attenuated virus of the present invention can be enough infective in the mankind and have immunogenic, in the inoculation individuality, to produce the immunity protective reaction.Definite method of RSV level is known in this area altogether in an infectious host's nasopharynx.Washing by breathing or nasopharyngeal secretions obtains sample, and means quantitative measurment virus wherein in tissue culture by experiment, referring to, for example, .J.Med.Virology 1:157-162 (1977) such as Belshe, Friedewald etc., J.Amer.Med.Assoc.204:690-694 (1968); Oharpure etc., J.Virol.3:414-421 (1969); And Wright etc., Arch.Ges.Virusforsch.41:238-247 (1973), described document all is incorporated herein by reference.These viruses for example are easy in the nasopharynx of chimpanzee determined at host animal.The method of other the toxicity of determining additional minus-stranded rna virus and quantity levels is by known to extensively and be easy to implement.

Invention disclosed herein further specifies by following embodiment, provides these embodiment to illustrate and unrestricted the present invention for example.In order to reach the purpose of this specification sheets, all publications that this paper quoted and patent documentation are introduced the present invention in full because of a variety of causes.

Embodiment 1 is positioned to the sudden change of the attenuation differentiated in HPIV3 JS cp45 and RSV cp530 and the Sendai virus on the conservative property sequential element in the allos minus-stranded rna virus

Present embodiment has proved that the sudden change of being differentiated can be navigated to the corresponding conservative amino acid sequence in the allos virus in single negative strand viruses order at an easy rate in a sudden change minus-stranded rna virus strain.These sudden changes are characterised in that the structural modification corresponding to parental array, this parental array is conservative property (identity or conservative property) in one or more allos minus-stranded rna virus, and these sudden changes provide the candidate who introduces sudden change in the recombinant virus with parent's protein sequence element.

For illustration this point of the present invention, analyze a series of known sudden changes among the HPIV3 mutant strain JS cp45.These a series of sudden changes are included in the parent's residue or the sequence site Tyr of polysaccharase L gene ₉₄₂, Leu ₉₉₂And/or Thr ₁₅₅₈On ts attenuation amino acid replace.More detailed condition is, the L albumen of JS cp45 mutant strain shows attenuation sudden change, wherein parent's Tyr ₉₄₂By His is replaced, Leu ₉₉₂By Phe is replaced, and/or Thr ₁₅₅₈By Ile is replaced (referring to, U.S. Patent application 08/083,793 and corresponding International Application No. WO 98/53078 thereof, described document is incorporated herein by reference).Consistent with preferred aspect of the present invention, these sudden changes are not only that collection of illustrative plates is compared with parental array and differentiated this variation, and equally at PIV recombinant chou (r942, r992, r1558, r942/992, r992/1558, r942/1558 and r942/992/1558) in successfully show this feature, single ground or mutually combine with the effect of checking attenuation and recovery in clone's infectious virus.

Other typical sudden changes are also estimated in HPIV3 JS cp45 mutant strain, this mutant strain known at HPIV3 F and C albumen in coding attenuation amino acid replace.These sudden changes are included in the proteic parent's residue of JS HPIV3 C/site Ile ₉₆The non-ts attenuation acidic amino acid at place is replaced, for example, and by Ile ₉₆Replacement to Thr.Another typical case's sudden change in the F of HPIV3 albumen is coded in parent's residue or site Ile ₄₂₀And Ala ₄₅₀Replacement, for example by Ile ₄₂₀To Val and Ala ₄₅₀Replacement to Thr.

Each attenuation sudden change of differentiating in HPIV3 provides the correlated index of sequence for homologous protein in other negative strand viruses strain.Be illustration this point of the present invention, use traditional sequence contrast to be positioned at the sudden change differentiated among the L and F albumen among the HPIV3 corresponding site to a series of allos minus-stranded rna virus (HPIV1, Sendai virus, HPIV2, BPIV3, MeV and RSV) according to above-mentioned listed method.As with shown in the table 2, this research has disclosed quite significantly, the parental array element of the selected mutant strain quite high homology of comparing with the wild-type sequence of allos virus.This result is very astonishing, because the conservative property that sequence is evolved has important function owing to it, and it predictedly may produce even more important phenotype effect, for example viability or communicable decline, and it is more even more important than single attenuation sudden change.Table 2: the sequence contrast HPIV3 cp45 F ORF I420V and the A450T near the zone the attenuation sudden change of in HPIV3 cp45 F and L albumen, being differentiated

* HPIV3 407 ¹The active ingredient and the additive that grind are very carefully mixed.Obtain suspension concentrate, can prepare the suspension of any desired concn from it by dilute with water.Biological activity embodiment; Embodiment B 1: the herbicide effect of plant before emerging in plastic containers standard soil upper seeding wheel unifacial leaf and dicotyledonous test plant.After planting immediately with the form spray test material of aqeous suspension (with the formulation preparation of embodiment F 1) (500 premium on currency / ha) to produce the dosage of 2kg active substance / ha.Then under top condition; Test plant is cultivated in the greenhouse.Behind 3 trial periods in week; Divide nine grades of (1 = damages fully; and 9 = does not have effect) evaluation experimental results.1-4 (especially being 1-3) means good in extraordinary effect.Table B1, the role of pre-emergence test plants oats millet mustard chickweed active ingredient 1.015 1211 1.016 1111 1.018 1213 1.019 1111 1.021 14 11 1.039 1293 1.060 3331 1.075 2111 1.076 2243 1.078 3111 1.079 3141 1.081 223 11.108 216 41.112 213 11.113 223 11.121 4 21 11.128 111 11.134 111 11.136 121 11.137 211 11.140 221 11.141 111 11.150 235 11.161 111 11.191 113 11.235 221 11.237 111 11.240 221 11.241 141 11.259 211 91.278 223 51.282 631 31.293 2442 when appropriate embodiment according to F2-F8 obtained when compounds of formula I prepared the same results. Embodiment B 2, Herbicide effect after emerging...

* HPIV3, 923, NPNWMQYASL...IPASVGGFNYMAMSRCFVRNIGDPSVAALAD, (SEQ, ID, NO.9) BPIV3, 923, NIHWMQYASL...IPASVGGFNYMAMSRCFVRNIGDPTVAALAD, (SEQ, ID, NO.10) sendai virus, 923, GKNWLRCAVL...IPANVGGFNYMSTSRCFVRNIGDPAVAALAD, (SEQ, ID, NO.11) HPIV2, 927, HPRLISRIVL...LPSQLGGLNYLACSRLFNRNIGDPLGTAVAD, (SEQ, ID, NO.12) measles virus, 923, NNDLLIRMAL...LPAPIGGMNYLNMSRLFVRNIGDPVTSSIAD, (SEQ, ID, NO.13) RSV, 968, LDNIDTALTLYMNLPMLFGGGDPNLLYRSFYRRTPDFLTEAIVH, (SEQ, ID, NO.14) HPIV3, cp45, L, ORF, L992FHPIV3, 973, LDRSVLYRIMNQEPGESSFLDWASDPYSCNLPQSQNITTMIKNITA, (SEQ, ID, NO.15) BPIV3, 973, LDRGVLYRIMNQEPGESSFLDWASDPYSCNLPQSQNITTMIKNITA, (SEQ, ID, NO.16) sendai virus, 973, LDKQVLYRVMNQEPGDSSFLDWASDPYSCNLPHSQSITTIIKNITA, (SEQ, ID, NO.17) HPIV2, 977, LESWILYNLLARKPGKGSWATLAADPYSLNQEYLYPPTTILKRHTQ, (SEQ, ID, NO.18) measles virus, 973, MPEETLHQVMTQQPGDSSFLDWASDPYSANLVCVQSITRLLKNITA, (SEQ, ID, NO.19) RSV, 1036, LNKFLTCIITFDKNPNAEFVTLMRDPQALGSERQAKITSEINRLAV, (SEQ, ID, NO.20) HPIV3, cp45, L, ORF, T1558I

* HPIV3 1537 HPKVFKRFWDCGVLNPIYGPNTASQDQIKLALSICEYSLDLFMREWL (SEQ ID NO.21) BPIV3 1537 HPKVFKRFWDCGVLNPIYGPNTASQDQVKLALSICEYSLDLFMREWL (SEQ ID NO.22) of Sendai virus 1537 HPKIFKRFWNAGVVEPVYGPNLSNQDKILLALSVCEYSVDLFMHDWQ (SEQ ID NO.23) HPIV2 1543 HPKLLRRAMNLDIITPIHAPYLASLDYVKLSIDAIQWGVKQVLADLS (SEQ ID NO.24) measles virus 1535 HPKIYKKFWHCGIIEPIHGPSLDAQNLHTTVCNMVYTCYMTYLDLLL (SEQ ID NO .25) RSV 1584 EQKVIKYILSQDASLHRVEGCHSFKLWFLKRLNVAEFTVCPWVVNID (SEQ ID NO.26)^*Be illustrated in the amino acid that has suddenlyd change among the HPIV3 cp45.Underscore is illustrated in amino-acid residue total in the different virus. ¹The first amino acid whose position of sequence shown in the full-length proteins.

Look back contrast as shown in table 2, obvious each L that analyzes and F sudden change has changed the parental array element of a basic conservative property, its with shown in virus taxis group's corresponding site identity or conservative property amino-acid residue exist for mark.For example, the strain of cp45 L protein mutation is with the Tyr in parent's residue ₉₄₂Change into His, it has very high conservative property, for example in Sendai virus, HPIV2, HPIV3, BPIV3 and the MeV L albumen (table 2) in the reservation of the locational identical tyrosine residues of corresponding wild-type.Similarly, Leu in the cp45 L sudden change ₉₉₂By Phe is replaced, it is positioned to parent's residue or the site that keeps identity between Sendai virus, HPIV2, HPIV3, BPIV3 and MeV L albumen.Two sudden changes of being differentiated in cp45 F albumen are with Ile ₄₂₀Become Val and with Ala ₄₅₀Become Thr, show the same conservative property residue/site between HPIV3 and BPIV3 equally, and that further keep same conservative property in HPIV1 is Ile ₄₂₀Become the sudden change parent residue of Val.The conservative property sequential element corresponding to the corresponding site of the attenuation differentiated among HPIV3 JS cp45 sudden change in addition is as shown in table 2.

The heterologous sequence contrast of carrying out other is to estimate the conservative property of the typical R SV attenuation sudden change of differentiating in mutant strain RSV cp530.This mutant strain is characterised in that the extremely leucic replacement of 521 site phenylalanines at cp530 L polysaccharase, and wherein sudden change occurs in the big conservative property structural domain of this albumen.Just as shown in table 3, at Phe ₅₂₁The sudden change at place demonstrates conservative property equally, as each the reservation (table 3 of same conservative property alanine residue in corresponding wild type site among 13 the subject classification groups that coexist; Fig. 1, the A group).Table 3: the conservative property benzene gene pool near the contrast virus sequence of the L polysaccharase sequence the PIV3 PHE-456 (*) in different paramyxovirus kinds

The registration number of first amino L-Ala

the position of acid, position PIV3, 431, NAYGSNSAISYENAVDYYQSFIGIKFNKFIEPQLDEDLTIY, (SEQ, ID, NO.27), 456, Z11575RSV, 496, YYKLNTYPSLLELTERDLIVLSGLRFYREFRLPKKVDLEMI, (SEQ, ID, NO.28), 521, the P28887 measles virus, 423, NAQASGEGLTHEQCVDNWKSFAGVKFGCFMPLSLDSDLTMY, (SEQ, ID, NO.29), 448, the P35975 sendai virus, 431, NAQGSNTAISYECAVDNYTSFIGFKFRKFIEPQLDEDLTIY, (SEQ, ID, NO.30), 456, Q06996PIV2, 434, EFQHDNAEISYEYTLKHWKEISLIEFRKCFDFDPGEELSIF, (SEQ, ID, NO.31), 459, P26676CDV, 423, NAHASGEGITYSQCIENWKSFAGIRFKCFMPLSLDSDLTMY, (SEQ, ID, NO.32), 448, P24658SV41, 435, ELHHDNSEISYEYTLRHWKELSLIEFKKCFDFDPGEELSIF, (SEQ, ID, NO.33), 460, P35341PDV, 423, NACVSGEGITYSQCVENWKSFAGIKFRCFMPLSLDSDLTMY, (SEQ, ID, NO.34), 448, Y09630Hendra virus, 430, RLKNSGESLTVDDCVKNWESFCGIQFDCFMELKLDSDLSMY, (SEQ, ID, NO.35), 455, AF017149SV5, 433, ELMNDNTEISYEFTLKHWKEVSLIKFKKCFDADAGEELSIF, (SEQ, ID, NO.36), 458, the Q88434 rinderpest virus, 423, NAQASGEGLTYEQCVDNWKSFAGIRFGCFMPLSLDSDLTMY, (SEQ, ID, NO.37), 448, P41357APV, 431, YMNAKTYPSNLELCVEDFLELAGISFCQEFYVPSQTSLEMV, (SEQ, ID, NO.38), 456, U65312NDV, 427, QLHADSAEISHDIMLREYKSLSALEFEPCIEYDPVTNLSMF, (SEQ, ID, NO.39), 452, X05399 runic amino acid is conservative in all viruses of analyzing are planted

The heterologous sequence contrast of also carrying out other is to estimate the ability of differentiating the conservative property structural element in the corresponding site of known attenuation sudden change.In this embodiment, attenuation sudden change of differentiating in proteic 170 sites of the C of Sendai virus and allos virus HPIV-1, HPIV-3 and BPIV-3 (referring to, table 4; The first residue site in corresponding sequence indicates numeral) the proteic corresponding sequence of C carries out collection of illustrative plates contrast.In addition, these sudden changes characterize by the parent's residue of identity in different taxonomical groups or the change of sequence site upper amino acid.Table 4

164HPIV3??144??MKLERWIRTLLRGKCDNLQMFQARYQEVMTYLQQNKVETVIMEEAWNLSVHLIQDQ ^*????(SEQ?ID?NO.40)BPIV3??144??MKLERWIRTLLRGKCDNLKMFQSRYQEVMPFLQQNKMETVMMEEAWNLSVHLIQDIPA ^*??(SEQ?ID?NO.41)SeV????150??MKTERWLRTLIRGEKTKLKDFQKRYEEVHPYLMKEKVEQIIMEEAWSLAAHIVQE ^*?????(SEQ?ID?NO.42)HPIV-1?150??MKTERWLRTLIRGKKTKLRDFQKRYEEVHPYLMMERVEQIIMEEAWKLAAHIVQE ^*?????(SEQ?ID?NO.43)

170

All these discoveries have proved the astonishing conservative property of attenuation mutant corresponding positions point sequence.Based on this, reasonable design scheme of the present invention is developed, go to import in attenuation sequence variation to the different recombinant virus of in an allos virus strain, being differentiated, for example, by recombination group or anti-genomic identity or conservative property change, to produce an attenuation recombinant clone.The development of the practice of these schemes can further directly be proved by the following examples.

The allos of the HPIV3 candidate vaccine of example II attenuation sudden change from RSV cpts530 L to reorganization shifts

Have between the allos taxonomical group of single negative strand viruses the attenuation sudden change corresponding to the discriminating like this on the site of conservative property structural element, the notion that the allos of attenuation sudden change ride through system generation boundary shifts can use important disease virus RSV to test as model.In this manual, present embodiment has proved attenuation (att) sudden change of differentiating in RSV (virus in the Pneumovirus in Paramyxoviridae), can be transferred and enter PIV3, and it is a member in the Respirovirus of very not being correlated with.These viruses are represented two kinds of different subfamilies in Paramyxoviridae.It is respectively Pneumovirinae and paramyxovirus subfamily.

More clear and definite is, an attenuation sudden change in first allos virus (RSV cpts530), its sequence that has changed parent RSV at a locating point (the proteic Phe521 of RSV L) really of suddenling change is to characterize the attenuation phenotype, and it has been positioned to the identity conserved residues in a proteic corresponding site of the L at selected target viral HPIV3 (table 3) (F456L).The therefore tested potential target that the allos transfer is carried out in sudden change between RSV and HPIV3 as attenuation of so conservative wild-type HPIV3 sequential element.

In order to finish this transfer, part or all of conservative property sequential element that carries sudden change preferably is replicated and imports recombinant virus to produce a novel attenuation redundant organism.Though it is preferred that the identity of will suddenling change is copied into a strain recombinant virus, and does not require and reach this fidelity of reproduction.On the other hand, parent who differentiates like this or wild-type residue can lack or be replaced by aminoacid insertion on the mutational site of containing one or more and the residue that the sudden change residue is irrelevant as a target.Preferably is, when theme sudden change has been labeled one or more amino acid and replaces, in recombinant virus parent clone, replaced by one or more residues relevant with the replaceability residue conservative property of in mutant nucleotide sequence, differentiating corresponding to the residue on the site in mutational site.More suitable is to be replaced by one or more identity residues that are present in the mutant nucleotide sequence corresponding to the residue on the site in mutational site in recombinant virus parent clone.

Like this, in this embodiment, the phenylalanine that 521 amino acid sites of the L polysaccharase of cpts530 take place to leucic sudden change is described to represent ts and attenuation (att) phenotype.Several is not that to have disclosed this phenylalanine be conservative on a large scale to proteic this regional sequence contrast of very relevant paramyxovirus (table 3) L.Use reverse genetics, 456 sites in the L of wild-type PIV3 albumen with the phenylalanine of merit by mutagenesis to leucine (F456L).The virus of gained, called after r456L is ts (plaque form be 40 ℃ by temperature), and it is not to be that duplicating in lower respiratory tract compared with the wild-type of recombinating (rwt) PIV3 and to have been reduced 10 times at the hamster upper respiratory tract.This result shows that the phenylalanine that is transferred to leucine sudden change is not the similar level that demonstrates temperature sensitive and attenuation in the very relevant paramyxovirus at two.

Equally in the present embodiment, confirmed that the F456L sudden change has been incorporated in two rPIV3 candidate vaccine virus strain, one has at L albumen (rcp45 _L) in three cp45 ts missense mutation, another carries in vivo all the 15 kinds of cp45 sudden changes (rcp45) in the further attenuated virus.More particularly, F456L sudden change is introduced in the reorganization PIV3 candidate vaccine of two latest developments and goes, and one is rcp45, the recombinant forms of the PIV3 candidate vaccine cp45 of Huo Deing biologically, and another is rcp45 _L, three amino acid that the cp45 that wherein only deposits sports in L albumen are replaced (disclosed international application 98/53078; Skiadopoulos etc., J.Viro1.72 (3): 1762-8 (1998); Skiadopoulos etc., J.Viro1.73 (2): 1374-1381, (1999), described document all is incorporated herein by reference).The F456L sudden change is increased to rcp45 or rcp45 _LIn improved attenuation in its body and to the sensitivity levels of temperature.Rcp45-456 is that immunogenicity and phenotype are stable.Rcp45 _L-456 and the copy limit of rcp45-456 virus in hamster be its rcp45 _LAnd the 100-1000 of rcp45 parent plant doubly.Use rcp45-456 to carry out the resistance that the PIV3 challenge virus is duplicated that immune induction produces a moderate level.In chimpanzee, it has restrictedly increased by 5 times to rcp45-456 compared with rcp45 in respiratory tract, and induces the PIV3 specific serum antibody of that match a, moderate to high level.Rcp45 that separates from chimpanzee and rcp45-456 virus keep its temperature sensitivity level of importing virus separately duplicating of two weeks in the time limit, thereby have proved the stability of its phenotype.

Therefore, corresponding to the RSVF521L mutant strain, the F456L sudden change is shifted and is entered in the cp45 recombinant virus, and its result is increasing considerably of a recombinant virus attenuation, has proved the availability of the sudden change of this transfer for the attenuation of adjusting the PIV candidate vaccine well.Shift the ability of representing the sudden change of differentiating in the allos paramyxovirus strain of different subfamilies in the present embodiment, greatly strengthened the possibility of development of new paramyxovirus.Virus and cell

RPIV3 JS wt (referring to rwt here), rcp45 _LReach rcp45 virus and be used as contrast in the present embodiment, these viruses are set forth in previous international application 98/53078; Durbin etc., Virology 235:323-332 (1997); Skiadopoulos etc., J.Virol.72 (3): 1762-8 (1998); Skiadopoulos etc., J.Virol.73 (2): 1374-1381 (1999), described document all is incorporated herein by reference).RPIV3 is incubated at ape and monkey LLC-MK2 cell (ATCCCCL 7.1), and it before was described (referring to Durbin etc., Virology 235:323-332,1997; Hall etc., Virus Res.22 (3): 173-184,1992; Skiadopoulos etc., J.Virol.72 (3): 1762-8 (1998), described document is incorporated herein by reference).Modified vaccine virus Ankara (MVA-T7) (Wyatt etc., Virology 210 (1): 202-205 (1995)), it expresses the T7 polysaccharase, and it provides for Linda Wyatt and Bernard Moss.HEp-2 (ATCCCCL 23) and LLC-MK2 cell are stored in OptiMEMI (the Life Technologies that is supplemented with 2%FBS and gentamicin sulphate (50 μ g/mL), Gaithersburg, MD) in, or be kept among the EMEM that is supplemented with 10%FBS, gentamicin sulphate (50 μ g/ml) and 4mM glutamine.The structure of point mutation in rwt

Subgene group fragment (Durbin etc., Virology 235:323-332,1997 of anti-genome cDNA clone p3/7 (131) 2G+ of the PIV3 JS wt of infectious virus before be used to reclaim; Skiadopoulos etc., J.Virol.72 (3): 1762-8,1998; J.Virol.73 such as Skiadopoulos (2): 1374-1381,1999), contain 7437 to 11312 Nucleotide of PIV3 (Xho I-Sph I), cloned in the pUC19 carrier by standard molecular biological technique, this carrier is modified to accept this gene fragment.CDNA utilizes the TransformerMutagenesis test kit, and (Clontech CA) introduces two point mutation as previously mentioned and has carried out modifying (Skiadopoulos etc., J.Virol.72 (3): 1762-8,1998; Skiadopoulos etc., J.Virol.73 (2): 1374-1381,1999).These two changes are on position 10011 (T to C) and 10013 (C to G), and its complete just sequence according to the rPIV3 antigenomic RNA is numbered.The change of F456L codon and eclipsed, reticent XmnI site have so just been introduced as a mark (as Fig. 1).Through mutagenesis, the restriction enzyme enzyme fragment is entered full-length clone p3/7 (131) 2G+ by complete order-checking and direct clone, or enters in the redundant organism that carries the cp45 sudden change, has wherein used the molecule clone technology of standard.Carry the recovery of the reorganization PIV3 of F456L sudden change

Carry the anti-genomic cDNA redundant organism of total length and three vector plasmid pTM (N), pTM (P no C) and pTM (the L) (Durbin etc. of F456L sudden change, Virology235:323-332,1997) in 6 hole flat boards, use LipofectACE (Life Technologies, MD) remove (Costar in the transfected HEp-2 of the entering individual layer, MA), described individual layer is infected by MVA-T7, as previously mentioned (Durbin etc., Virology 235:323-332 (1997); Skiadopoulos etc., J.Virol.72 (3): 1762-8 (1998).Plasmid pTM (P noC) is a kind of form (Virology 235:323-332 (1997) such as Durbin) of the preceding pTM that has described (P), and wherein C ORF has been modified, and its translation initiation codon fades to ACG (methionine(Met) becomes Threonine) from AUG like this.Through 32 ℃ of incubations 4 days, the transfection product is transfused to the LLC-MK2 cell in the T-25 flask into, and it was at 32 ℃ of following incubation 4-8 days.Clarifying culture supernatant is carried out three-wheel plaque purification (Durbin etc., the Virology 235:323-332 (1997) of LLC-MK2 cell as previously mentioned; Hall etc., Virus Res.22 (3): 173-184 (1992); J.Virol.72 such as Skiadopoulos (3): 1762-8 (1998)).Each biology clone's recombinant virus is amplified twice under 32 ℃ in the LLC-MK2 cell, the virus that is produced is made further signature analysis.By polyethylene glycol precipitation concentrating virus (Mbiguino and Menezes, J.Virol.Methods 31:161-170 (1991)) from the clarification substratum, use TRIzol reagent (Life Technologies) to extract viral RNA (vRNA).Use Superscript II increase in advance system (Life Technologies, Inc.) and any six conjuncted primer reverse transcription vRNA.Advantage cDNA PCR test kit (Clontech, CA) and be used to amplified fragments at the justice of PIV3 genome different piece and antisense primer and be used for restriction enzyme digestion and/or sequential analysis.The PCR fragment is analyzed by order-checking or restriction enzyme digestion, and wherein the enzyme of the Restriction Enzyme that uses is cut recognition site and produced or excise in the building process that suddenlys change.The plaque that carries the rPIV3 of F456L sudden change under temperature that allow and restriction forms efficient (EOP)

35 ℃ to 41 ℃ of 32 ℃ and temperature ranges in LLC-MK2 cell monolayer culture (about six days of incubation), just as described above, the temperature sensitivity level that the external plaque of control group and recombinant virus forms is determined (Hall etc., Virus Res.22 (3): 173-184,1992).Remove the methylcellulose gum coating and use guinea-pig red blood cell absorption to carry out plaque counting later on, what perhaps can replace is that the viral plaque that is present in the individual layer is differentiated by two the anti-HN mouse of PIV3 specificity monoclonal antibodies 101/1 and 454/11 that use the immune peroxidase staining that dilutes 1: 250 times.(Murphy etc., Vaccine 8 (5): 497-502 (1990); Murphy etc., (1990); Van Wyke Coelingh, Winter and Murphy, Virology 143 (2): 569-582, (1985), described document all is incorporated herein by reference).Estimate the att phenotype of rPIV3 mutated viruses in the hamster

(Charles River Laboratories, NY) (it is seronegative for PIV3) is inoculated in nasal cavity to the Golden Syrian hamster in 4 ages in week, uses to contain 10 ^6.0TCID ₅₀Rwt or the 0.1ml L15 medium of sudden change one of rPIV3.After infection the 4th day, kill hamster, collect its lung and concha, virus is measured (Durbin etc., Virology 235:323-332,1997 as previously mentioned; Skiadopoulos etc., J.Virol.72 (3): 1762-8,1998).Average log under each group hamster calculated 32 ℃ ₁₀TCID ₅₀/ gram.The immunogenicity of rcp45-456 and efficient in the hamster

Three groups of each five hamster via intranasal application are inoculated (i) L15 medium (placebo) with 0.1ml, (ii) 10 ^6.0TCID ₅₀Rcp45 or (iii) 10 ^6.0TCID ₅₀Rcp45-456.Hamster was got blood in the 42nd day before infection and after infecting, the blood clotting of serum antagonism PIV3 suppresses (HAI) antibody titers and is determined (van Wyke Coelingh, Winter and Murphy, 1985) as previously mentioned.At the 44th day, by giving 10 in the nose ^6.0TCID ₅₀Rwt and attack hamster collects its lung and concha after four days, the rwt titre in these tissue homogenates is determined as mentioned above.Estimate the att phenotype of the rPIV3 mutated viruses in the chimpanzee

For RSV and PIV3 is the male and female chimpanzee of seronegative youth, and body weight 6.6-10.0kg is positioned in pairs and also raises (Crowe etc., 1994a in the big glass isolator ware as previously mentioned; Hall etc., J.Infect.Dis.167:958-962 (1993)).Use one milliliter and contain 10 ^6.0TCID ₅₀Rcp45 or rcp45-456 inoculum infect chimpanzee by (T) in (IN) in the nose and the tracheae in each site and organize.After the virus inoculation, nasopharynx wiping and lavage of trachea sample are collected, and are used for virus quantitatively (Hall etc., 1993 as previously mentioned; Kairon etc., 1997).From 1-10 and 13 days collection nasopharynx wipe samples, collected the lavage of trachea sample at the 2nd, 4,6,8 and the 10th days.Estimate the degree of rhinorrhea every day, and (0=there is not rhinorrhea to distribute the 0-4 value; The 1=trace; The 2=trace; The 3=moderate; 4=is serious).Rcp45 and rcp45-456 virus are estimated with two different tests according to identical program.In experiment 1, two chimpanzees use rcp45 to inoculate and 4 use rcp45-456 to inoculate, in experiment 2, each virus gives two animals simultaneously.Carrying out second group of test is in order to verify the difference of two viral growths that observed in first group of experiment.These two groups of data are to distinguish according to viral growth and immunogenicity, and they lump together and average.Accept 10 from four groups by IN and IT approach ⁴TCID ₅₀The data (Hall etc., 1993) as previously mentioned of animal of PIV3 wt JS strain, listing these data is in order to compare.The copy feature of rPIV in chimpanzee

Viral load in nasopharynx wiping and lavage of trachea thing is determined under 32 ℃ in the LLC-MK2 individual layer and is represented as log ₁₀TCID ₅₀/ ml is as preceding described (Crowe etc., 1994a; Hall etc., 1993).Virus in chimpanzee nasopharynx and the lavage of trachea sample is cultivated under 32 ℃ in 24 hole flat boards in the LLC-MK2 individual layer up to cytopathic effect widely and is detected.The level of these rPIV3 isolate temperature sensitivities is by determining on the LLC-MK2 individual layer plaque as mentioned above and form efficient and being estimated.All researchs of using chimpanzee sample or isolate to carry out comprise an antibiotic cocktail that contains clindamycin (100 μ g/ml), Ciprofloxacin (100 μ g/ml), gentamicin (100 μ g/ml) and amphotericin B (2.5 μ g/ml).The F456L mutant strain enters among the rwt and brings the ts phenotype to it as a result

As mentioned above, in the mouse respiratory tract, duplicating, RSV cpts530 be ts and attenuation (Crowe etc., 1994b).Replacement on the phenylalanine in the L of RSV cpts530 albumen 521 sites has brought temperature sensitivity (39 ℃ of temperature of ending for plaque formation) and attenuation (duplicate and reduce 10 times) (Crowe etc., 1994b in the mouse upper respiratory tract; Juhasz etc., 1997).The 521 site phenylalanines that the proteic sequence contrast of the L that 13 kinds of different paramyxovirus comprise RSV and PIV3 has disclosed at RSV L are high conservative (tables 3; Fig. 1, the A group).With HPIV3 is example, and corresponding amino acid occurs in 456 sites in PIV3 L polysaccharase.RSV (521) to PIV3 (456) in the difference in site of this residue be consistent with previous viewed result, described result is RSV L albumen nearly 70 the amino acid whose N-terminals of comparing with the paramyxovirus of other genus and extends (Stec etc., Virology 183:273-287 (1991)).In order to determine whether proteic 456 site mutation of L can provide ts and the att phenotype that is similar to allos RSV cpts530 mutant strain among the PIV3, and the phenylalanine in rwt 456 sites is leucine (F456L) by mutagenesis directly.This coding changes the replacement that is designed to relate to two Nucleotide, and this is in order to reduce the possibility that it changes wt into.Equally, by the Xmn I restriction endonuclease recognition site (Fig. 1, C group) of introducing a silence, this sudden change is labeled.This sudden change is introduced in the infectious rwt cDNA plasmid of total length, and recombinant virus is recovered (Durbin etc. as previously mentioned, Virology 235:323-332 (1997), Skiadopoulos etc., J.Virol.72 (3): 1762-8 (1998) and Skiadopoulos etc., J.Virol.73 (2): 1374-138l (1999)).Based on the RT-PCR of the vRNA of purifying and through restricted enzyme cutting analysis and order-checking, the r456 of recovery _LMutant strain (Fig. 1, B group) is proved has Xmn I mark and F456L sudden change.F456L sudden change be incorporated into give among the rwt one 40 ℃ by temperature (table 5), be higher than 1 ℃ of RSV cpts530, having proved that a ts sudden change that occurs in the Pneumovirinae can be transferred that to enter one be not to go in the very relevant Respirovirus, is not identical ts phenotype although give it similar.Table 5: control group virus and carry virus temperature sensitivity under permission and non-permissive temperature in the LLC-MK2 cell of F456L sudden change

Average log under assigned temperature ₁₀Pfu/ml reduces value ^aVirus is at 32 ℃ following 35 ℃ 36 ℃ 37 ℃ 38 ℃ 39 ℃ 40 ℃ 41 ℃

Average titer Rwt 7.5-0.2 0.0 0.0-0.1 0.1 0.3 0.8r456 _L7.3--0.3 0.2 0.6 2.5 〉=5.5rcp45 _L7.3--0.5 1.9 4.3 〉=6.0-rcp45 _L-456 6.5 2.5 〉=4.5 〉=5.2 〉=5.2 〉=5.2 〉=5.2-rcp45,7.8 0.6 1.0 1.4 2.4 〉=5.6 〉=6.8-rcp45-456 7.7 0.2 1.0 2.2 〉=4.9 〉=7.3 〉=6.9-

^aInoculate back 6 days and by immunoperoxidase staining plaque is counted in the temperature of setting, institute's column data is the mean value of three groups or more experiments, the value that runic marks shows the limit temperature that it is minimum, plaque forms efficient and reduces 100 times with this understanding, and this temperature is confirmed as " by temperature " that plaque forms.By deducting the reduction that titre value under the assigned temperature draws titre the titre value under permissive temperature (32 ℃).In reorganization rcp45 base attenuated virus, introduce the F456L sudden change and improved its temperature sensitivity

Three concrete amino acid that cp45 has in L albumen are replaced, and it before had been shown as main definite factor (Skiadopoulos etc., J.Virol.72 (3): the 1762-8 (1998) of its ts and att phenotype.Cp45 contains other the 12 kinds of sudden changes outward of L albumen equally, and it comprises ts and att sudden change (Skiadopoulos etc., J.Virol.73 (2): 1374-1381 (1999).F456L sudden change is introduced into rcp45 and enters equally in the virus strain that only carries three rcp45 L transgenation removes (rcp45 _L).Do like this be for be defined as temperature sensitivity that F456L sudden change characterized whether can add by other three kinds of cp45 L sudden changes sign or be the temperature sensitivity that whole sudden change characterized of 15 cp45.Be rcp45 significantly _L-456 have proved the very big raising (table 5) of temperature sensitivity, and it has one 35 ℃ the temperature of ending, and rcp45 _LIt is 39 ℃.The rcp45-456 mutant strain has an intermediary by 37 ℃ of temperature, and it is compared with 38 ℃ of rcp45.Although the temperature sensitivity that is characterized by the F456L sudden change is additional to by rcp45 _LAnd the ts that sudden change characterized of rcp45, the effective scope of these two kinds of viruses but is complete difference.Like this, the rcp45-456 that contains relatively large sudden change has the rcp45 of ratio _L-456 lower temperature sensitivities.The evidence of complex interactions is observed in the rPIV3 virus of the various combination that contains the cp45 sudden change by other in the cp45 ts sudden change, although produce the basis of these effects and not exclusively understand (Skiadopoulos etc., J.Virol.72 (3): 1762-8 (1998) and Skiadopoulos etc., J.Virol.73 (2): 1374-1381 (1999).The immunogenicity of the rPIV3 that suddenlys change in the hamster and duplicating

Use 10 ^6.0TCID ₅₀Rwt or use one of another severe sudden change rPIV3, comprise carry separately or with the virus of other cp45 specific mutant (table 6) bonded F456L sudden change, by IN inoculation hamster.After 4 days, collect lung and concha, determine the levels of replication that each is viral.Independent F456L sudden change is for r456 _LThe effect of duplicating an appropriateness, the result is to be replicated in the concha 10 times reduction and no tangible copy limit in lung.The level of attenuation is similar to the RSV cpts530 mutant strain (Crowe etc., Vaccine12 (8), 691-699 (1994a)) in the mouse upper respiratory tract.Surprising is that the F456L sudden change joins arbitrary rcp45 _LOr rcp45 result produces and duplicates remarkable restricted recombinant strain in the upper respiratory tract.It shows the attenuation of these mutated viruses of F456L sudden change can the enhancing greatly, duplicating from then influence the upper respiratory tract hamster.Table 6: rwt and rcp45 redundant organism the duplicating in the hamster respiratory tract of containing the F456L sudden change ^a

Average virus titer (the log in the specified tissue ₁₀TCID ₅₀/ g ± S.E. ^b) viral hamster quantity concha lung rwt 10 6.9 ± 0.1 6.3 ± 0.4r456 _L10 6.0 ± 0.1 5.7 ± 0.5rcp45 _L10 3.8 ± 0.2 1.9 ± 0.2rcp45 _L-456 5≤1.5 ± 0≤1.5 ± 0rc,p45 10 4.9 ± 0.2 2.1 ± 0.2rcp45-456 5≤1.5 ± 0 1.6 ± 0.1

^aTo give hamster 10 in the 0.1mL inoculum intranasal ^6.0TCID ₅₀Virus.Collect lung and concha after 4 days and measure virus titer down at 32 ℃.The mean value of two groups of experiments.

^bS.E.: standard error

In order to determine whether that rcp45-456 shifts mutant strain and causes an immune response, seronegative hamster is given 10 through IN ^6.0TCID ₅₀Rcp45-456, rcp45 or L15 substratum, after 42 days, collect serum sample, used rwt to attack animal at the 44th day then.Use the immunity that rcp45-456 or rcp45 did to cause the HAI antibody of moderate respectively, and induce the resistance (table 7) that the rwt challenge virus is duplicated to high titre.Infecting the level of the antibody brought out and resistance by rcp45-456 is lower than and is brought out by rcp45.Low-level immune response reaches the remarkable reduction that is reflected virus replication level in the hamster respiratory tract by the protection that rcp45-456 brought out.Table 7: infect hamster with rcp45 and rcp45-456 and induce the resistivity that it is attacked rwt ^a

Average attack rwt titre ^b(log ¹⁰TCID ₅₀/ g ± S.E.): viral concha lung HAI titre ^cL15 control group 5.6 ± 0.1 4.8 ± 0.2＜2.0 ± 0Rcp45＜1.5 ± 0＜1.5 ± 0 10.4 ± 0.2rcp45-456 3.1 ± 0.6 2.1 ± 0.6 4.8 ± 0.9 of log2 ± S.E) ^aThe group intranasal administration 10 of 5 hamsters ^6.0TCID ₅₀Virus, each animal 0.1mL inoculum.Inoculate back 44 days, use 10 ^6.0TCID ₅₀Rwt attack animal.Collect lung and concha after 4 days. ^bViral load in each tissue sample is determined down at 32 ℃ in the LLC-MK2 cell. ^cPromptly attacked the hemagglutination inhibition antibody titre of the back 2 days anti-PIV3 of serum on the 42nd day.S.E: standard error.In the serum of HAI mean value log2 titre reciprocal before infection＜2.The rcp45-456 mutant strain in chimpanzee than rcp45 attenuation more

Because the rcp45-456 mutant strain shows excessive attenuation in hamster, we attempt to determine its duplicating and the immunogenicity level in chimpanzee, and chimpanzee is inhuman primates, and itself and human close source property are nearer.Two groups independently in the replication experiment, chimpanzee uses 10 through IN or IT respectively ^6.0TCID ₅₀The rcp45-456 or the rcp45 in/site inoculate, and infect in 10 and 13 day time limit of back and collect lavage of trachea and nasopharynx wipe samples respectively, and the virus titer in each sample is determined.Rcp45-456 and rcp45 the duplicating in last lower respiratory tract of comparing with PIV3 wt (table 8) all is strict restriction, and it shows that each mutated viruses strain duplicating all in chimpanzee is attenuated.As the above (table 6), it is exactly to reduce virus replication above 2500 times in the upper respiratory tract of hamster that the F456L sudden change is increased to the effect of going among the cp45.On the contrary, when assessing in chimpanzee, virus replication reduces only 5 times (table 8 and Fig. 2) in last lower respiratory tract.The difference of above-mentioned two groups of experiments is observed.One of them test, two animals received rcp45 and four accept rcp45-456, and each virus all gives two animals in another group experiment.Table 8: for duplicating in the lower respiratory tract on chimpanzee, rcp45-456 shows more attenuation compared with rcp45

Virus replication

Nasopharynx wiping liquid lavage of trachea liquid is used to infect moving animal dosage ^aTitre in spite of illness ^bTitre in spite of illness ^bAverage virus numbers (the fate hydrorrhea degree of the poison of log10 poison that flows out average peak nose thing

TCID ₅₀/ ml) number number rcp45-456 6 6.0 6 3.4 ± 0.1 ^c3 1.2 ± 04 0.7 ± 0.3 0.2 ± 0.1 ^dRcp45 4 6.0 4 4.1 ± 0.3 ^c3 1.9 ± 0.5 1.8 ± 0.6 1.0 ± 0.3 ^dPIV3 ^e4 6.0 4 6.3 ± 0.5 4 5.2 ± 0.8 3.8 ± 0.5 3.3 ± 0.5

^aVirus with specified amount on each site is administered in nose and the tracheae in the 1ml inoculum.

^bAverage peak virus titer (log ₁₀TCID ₅₀/ ml).Virus titer passes through TCID under 32 ℃ in the LLC-MK2 cell ₅₀Determine.

^cHas significant difference between the designated value statistically; P＜0.025 (Si Shi t check).

^dHas significant difference between the designated value statistically; P＜0.01 (Si Shi t check).

^ePIV3 wild-type JS strain (from the data of Hall etc., 1992).

The contrast of flowing out virus in the upper respiratory tract every day demonstrates rcp45-456 virus and reach a lower peak value virus titer with respect to rcp45, but the speed of its minimizing but is (Fig. 2) very slowly.This kind pattern before had been shown it and had been the RSV mutant strain feature (Prince etc., Infect.Immun.26 (3): 1009-13,1979) that the attenuation level increases in non-human primates.The increase of this kind attenuation level is reflected in the remarkable reduction (table 8) of average peak Rhinorrhea degree equally.Therefore, the result of F456L sudden change introducing rcp45 is the remarkable increase of viral attenuation in the chimpanzee respiratory tract.In chimpanzee, duplicate the maintenance of the ts phenotype of back rcp45 and rcp45-456

Use rcp45 and rcp45-456 to infect chimpanzee respectively, determined from the level of the temperature sensitivity of the isolate that wherein obtains, be used for determining whether to keep after two recombinant chous duplicate their ts phenotype in high admissibility host.In view of having obtained a large amount of isolates, isolate is estimated respectively from two study group.The isolate of testing two animals of 1 is analyzed, and the result is as shown in table 9, and the situation of all animals is shown in table 10.The level of same control group virus suspended substance temperature sensitivity is 1 ℃ difference (table 10) in two groups of experiments, demonstrates to have experimental variation in analysis.For example, the temperature of not observing plaque in experiment in 1 for rcp45-456 and rcp45 is respectively 38 ℃ and 39 ℃, is 39 ℃ and 40 ℃ (table 10) and test 2.Sometimes observe the level of experimental variation.The most important thing is that the difference of difference between rcp45-456 and rcp45 that plaque forms by temperature in all tests remains 1 ℃.Because there is experimental variation in these two groups tests, they are described respectively in table 10.Each isolate keeps the ts phenotype, there is no obvious difference with control group input virus in the whole reproduction process of the temperature sensitivity level of isolate virus in the chimpanzee respiratory tract.Percentage that it should be noted that the isolate plaque formation of rcp45-456 is lower than the rcp45 isolate at 38 ℃ and 39 ℃, and its temperature sensitivity that is presented at the higher level of external viewed rcp45-456 is duplicated in vivo in the back and still kept.Table 9: in the seronegativity chimpanzee, duplicate back rcp45 and rcp45-456 and all keep its ts phenotype: from the sample analysis of two animals of experiment 1 and from the comparison viral isolates or the contrast isolate of the control group of two experiments ^cVirus titer under design temperature ^d(log ₁₀Pfu/ml)

Organize 32 ℃ of 36 ℃ of 37 ℃ of 38 ℃ 39 ℃ 40 ℃ rcp45-456 virus N P 1 7.1＜3.7＜2.7＜0.7＜0.7＜0.7 (animal #1614) NP 2 7.3＜3.7＜2.7＜0.7＜0.7＜0.7 of fate

NP????????????3??????7.7???＜3.7???＜2.7????＜0.7????＜0.7????＜0.7

NP????????????4??????8.0?????4.4?????4.2????＜0.7????＜0.7????＜0.7

NP????????????5??????7.8?????4.3?????4.2????＜0.7????＜0.7????＜0.7

NP????????????6??????7.3?????4.2?????4.0????＜0.7????＜0.7????＜0.7

NP????????????7??????6.9?????3.7?????3.0????＜0.7????＜0.7????＜0.7

NP????????????8??????7.7???＜3.7?????3.0????＜0.7????＜0.7????＜0.7

NP????????????9??????7.5?????5.3?????4.8????＜0.7????＜0.7????＜0.7

NP???????????10??????7.8?????5.4?????4.2????＜0.7????＜0.7????＜0.7

TL????????????2??????7.8?????4.7?????2.7????＜0.7????＜0.7????＜0.7

TL 4 7.4 4.0＜2.7＜0.7＜0.7＜0.7rcp45 NP 1 7.0 ND 4.7 4.2＜0.7＜0.7 (animal #1616) NP 2 7.5 ND 4.4 3.7＜0.7＜0.7

NP????????????3??????7.3?????ND??????3.7????＜0.7????＜0.7????＜0.7

NP????????????4??????7.4?????ND??????4.5????＜0.7????＜0.7????＜0.7

NP????????????5??????6.3?????ND??????4.0????＜0.7????＜0.7????＜0.7

NP????????????6??????7.2?????ND??????4.9??????3.4????＜0.7????＜0.7

NP????????????7??????5.8?????ND????＜3.7????＜0.7????＜0.7????＜0.7

NP????????????8??????5.9?????ND??????4.5??????2.2????＜0.7????＜0.7

NP???????????13??????6.9?????ND??????4.3??????3.4????＜0.7????＜0.7

TL????????????2??????7.5?????5.3?????4.3??????3.3????＜0.7????＜0.7

TL 4 7.2 5.7 4.5＜0.7＜0.7＜0.7rcp45-456 ^a8.4 6.0 4.6＜0.7＜0.7＜0.7rcp45 ^a8.0 7.0 5.8 3.4＜0.7＜0.7rwta ^a7.2 7.2 7.3 7.0 6.9 7.0rcp45-456 ^b8.3 7.0 5.0 3.0＜0.7＜0.7rcp45 ^b7.8 7.5 6.3 4.8 2.3＜0.7rwt ^b7.8 7.6 7.4 7.5 7.4 7.2 ^aThe control group viral suspension, research #1. ^bThe control group viral suspension, research #2 (referring to table 10). ^cAfter wiping of chimpanzee nasopharynx and lavage of trachea sample go down to posterity, collect viral isolates under 30 ℃ in the LLC-MK2 cell. ^dIn LLC-MK2 monolayer culture thing, under specified temperature, measure viral isolates 6 days, carry out plaque counting by using anti-HPIV3 HN monoclonal antibody to carry out immunoperoxidase staining.ND=does not detect.Table 10: duplicate back rcp45 and rcp45-456 and all keep its temperature sensitivity phenotype in the seronegativity chimpanzee: the summary of two groups of experiments is used to infect 36 ℃ of 37 ℃ of 38 ℃ of 39 ℃ 40 ℃ (size of animal) total quantitys experiments 1 of viral isolates of the percentage ratio animal of the isolate with detected viral plaque under design temperature

rcp45(2)??????20 ^a?????100??????95?????70???????0????????0

Rcp45-456 (4) 42 ^b83 60 000 experiments 2

rcp45(2)??????25 ^c?????100?????100????100??????72????????0

Rcp45-456 (2) 22 ^d100 86 91 50 as described in material and the method and the isolate that obtains, and plaque forms forms efficient by temperature by the plaque under design temperature and determine (referring to materials and methods and table 9). ^aTotal quantity comprises 18 nasopharynx isolates and 2 lavage of trachea isolates ^bTotal quantity comprises 39 nasopharynx isolates and 3 lavage of trachea isolates ^cTotal quantity comprises 20 nasopharynx isolates and 5 lavage of trachea isolates ^dTotal quantity comprises 21 nasopharynx isolates and 1 lavage of trachea isolate

Aforesaid result has proved the sudden change among the RSV cpts530, and promptly in the replacement of parent's phenylalanine in proteic 521 sites of L, it has given the temperature sensitivity and the attenuation of similar level when it imports in the proteic corresponding site of L (456) of PIV3.RSV and PIV3 represent different subfamilies in Paramyxoviridae, it is named as Pneumovirinae and paramyxovirus subfamily respectively.That the L albumen of this two papova has is clear and definite, crucial serial correlation (Stec etc. on the statistics, Virology 183:273-287 (1991)), especially closing on aminoterminal 540-570 amino acid whose zone (the 422-938 amino acids of RSV and the 357-896 amino acids of PIV3), it comprise four high conservatives the polysaccharase primitive (referring to Poch etc., J.Gen.Virol.5:1153-62 (1990); And Stec etc., Virology183:273-287 (1991)).Sudden change on 521 of RSV and PIV3 456 described herein is in this zone, but at about 175 residue places, described first conservative property primitive upstream such as Poch.More particularly, this residue is strict conservative, and all has a leucine residue to be positioned at carboxyl terminal 12 positions (table 1, A group) of this position in 13 allos paramyxovirus of test in this research.Although its strict conservative property, successfully do not transfer on the proteic conservative property of the HPIV3 L position if identified 521 L sudden change and this sudden change before in the RSVcpts530 mutant strain, can not predict from 2233 sites of the proteic aminoacid sequence of PIV3 L that then this kind residue is that mutagenesis is to produce the appropriate site of conditioned lethal property attenuation mutant.Consider these results, very might expand method of the present invention, to comprise not homophyletic of single negative strand viruses, a large amount of attenuation sudden change (Bukreyev etc., J.Virol.71 (12), the 8973-8982 (1997) that especially in paramyxovirus, differentiate; Garcin etc., Virology238 (2): 424-431 (1997); Juhasz etc., Vaccine 17:1416-1424 (1999); Juhasz etc., J.Virol.71 (8): 5814-5819 (1997); Kato etc., EMBO are (3) J.16: 578-587 (1997a); Kato etc., J.Virol.71 (10): 7266-7272 (1997b); Kondo etc., J.Biol.Chem.268 (29): 21924-21930 (1993); Kurotani etc., Genes Cells 3 (2): 111-124 (1998); Skiadopoulos etc., J.Virol.72 (3): 1762-8 (1998); Whitehead etc., J.Virol.73:871-877 reaches: 3438-3442 (1999); Whitehead etc., Virology 247 (2): 232-239 (1998a); Whitehead etc., J.Virol-73 (2): 871-877 (1999b); Whitehead etc., J.Virol.72 (5): 4467-4471 (1998b), described document all is incorporated herein by reference).

This discovery provides novel PIV candidate vaccine and can carry out the transfer of the attenuation sudden change from RSV to other paramyxovirus equally, and wherein sudden change is positioned at the parent's residue or the site of a conservative property.Illustrate further in this manual, the chimeric PIV3 recombinant chou that the 521 L sudden change of RSV has just been developed recently by the suitability input, it has hemagglutinin-neuraminidase (HN) and the F gene of JS wt PIV3, it is replaced (Tao etc. by those of PIV1, J.Virol.72 (4), 2955-2961 (1998), described document is incorporated herein by reference).One or more addition mutations that this chimeric recombinant chou can be found by being combined in the cp45 mutant strain and by further attenuation, wherein these sudden changes have been done as a whole (occurring in except three kinds of sudden changes in F and the HN gene) and have been introduced among a PIV3-1 infectivity, attenuation, the chimeric clone, and this clone carries and carried out PIV1 HN and the F gene replaced in a JS wt PIV3 background.

The attenuation sudden change of EXAMPLE III in Sendai virus C albumen shifted to enter in the reorganization candidate HPIV3 vaccine by allos and gone

The allos that present embodiment has been described a known attenuation sudden change in Sendai virus (SeV) the C albumen shifts, it is characterized in that at the replacement (Itoh etc. of 170 site phenylalanines (F) to Serine (S), J.Gen.Virol.78:3207-3215 (1997), described document is incorporated herein by reference), described attenuation sudden change is transferred to a corresponding site among the reorganization HPIV3 clone.Illustrated as reaching as mentioned above in table 4, F170 parental array element is positioned to same conservative sequence site/residue F164 in HPIV3 C albumen, and this element is also guarded in SeV and BPIV-3.

The FI70S sudden change of SeV is transferred by introducing one point mutation in the C of HPIV3 ORF and enters among the reorganization HPIV3 virus strain rF164S, and corresponding amino acid change is the changes of 164 sites from phenylalanine (F) to Serine (S).The rF164S recombinant strain of gained has the attenuation that makes us more startled than it in lower respiratory tract in the upper respiratory tract.This pattern is opposite with the sudden change of viewed temperature sensitivity attenuation, includes this novel sudden change thus and be proved to be that to reduce in the toxicity residual in the upper respiratory tract be useful in vaccine virus reorganization, that live attenuation.The same immunoprotection reaction that produces anti-wild-type HPIV3 of rF164S recombinant strain.

The P of HPIV3 and C albumen are isolating from mRNA, eclipsed ORF translates (Fig. 3).Although P albumen of all paramyxovirus coding, only C albumen of Respirovirus and Morbillivirus coding.From C ORF expressed proteins quantity different virus different and its reach external importance of duplicating in vivo also can be different.Sendai virus (SeV) has been expressed four albumen that independence is initial: C ', C, Y1 and Y2 from C ORF, and its translation initiation site also is this order (Curran etc., Enzyme44:244-9 (1990) in mRNA; Lamb etc., p.181-214, D.Kingsbury (editor), TheParamyxoviruses, Plenum Press, New York (1991), and HPIV3 and Measles virus (MeV) are only expressed a single C albumen (Bellini etc., J Virol.53:908-19, (1985); Sanchez etc., Virology 147:177-86 (1985); Spriggs etc., J.Gen.Virol.67:2705-2719 (1986)).

Extreme poor efficiency (Kurotani etc. when wherein the reorganization SeV that lives of the strain that is eliminated of all four C-derived proteins is found in replication in vitro, Genes Cells 3:111-124 (1998)), yet indivedual proteic removings of C have complicated effect (Cadd etc., J Virol.70:5067-74 (1996); Curran etc., Virology 189:647-56 (1992); Latorre etc., J Virol.72:5984-93 (1998)).

The SeV that carries a reorganization of simple point mutation is attenuated in mouse, the result of described simple point mutation is to replace at proteic 170 one phenylalanine of C (F) to the amino acid of Serine (S), but it duplicates not weakened (Garcin etc., Virology 238:424-431 (1997) in cell culture; J.Gen.Virol.78:3207-15 such as Itoh (1997)).Form contrast with SeV, the Measles virus (MeV) of a C-minimizing is duplicated (Radecke etc. effectively in the Vero cell, Virology 217:418-21 (1996)), although it demonstrates duplicate restricted and only shows to a certain degree attenuation (Escoffier etc., J.Virol.73:1695-8 (1999) in vivo in human peripheral blood cells; Valsamakis etc., J.Virol.72:7754-61 (1998)).

Different MeV and the change of duplicating of SeV C mutant strain in animal show that this albumen is a potential target for introducing useful attenuation sudden change in the attenuation HPIV3 vaccine of living in exploitation.In the present embodiment, use the reverse genetics method to introduce a sudden change in the C of HPIV3 ORF, it is in the change of a F-S of 164 amino acids generation, and it changes corresponding to the F on 170 amino acids in the allos attenuation SeV mutant strain → S.Cell and virus

HEp-2 and ape and monkey LLC-MK2 monolayer cell culture be maintained at OptiMEM 1 (Life Technologies, Gaithersburg, MD) in, it is supplemented with 2% foetal calf serum, gentamicin sulphate (50 μ g/mL) and 4mM glutaminate.Vaccine strain Ankara (MVA) recombinant virus of modifying, it expresses bacteriophage t7 rna polymerase, for L.Wyatt and B.Moss (Wyatt etc., Virology 210:202-205 (1995)) provide.JS wild-type (wt) strain of PIV3 and attenuation ts thereof the strain JS cp45 that derives breeds in the LLC-MK2 cell, as described (Hall etc., Virus Res.22:173-184 (1992)) like that.cDNA

{ full length cDNA clone of p3/7 (131) 2G} was described (the Genebank registration number is #Z11575) (Durbin etc., Virology 235:323-332 (1997)) to the whole anti-genome of 15462 nt of coding JS wt virus in the past.This clone be used as the sudden change cDNA that 164 phenylalanines to the Serine that makes up a coding C ORF changes template (table 11, Fig. 3).PmlI to the BainHI fragment of p3/7 (131) 2G (the anti-genomic nt 1215-3903 of PIV3) is entered plasmid pUC119{pUC119 (PmlI-BamHI) by subclone }, it has been modified to the PmlI site that comprises in the polyclone zone.Then, use the method (Kunkel etc., Methods Enzymol.154:367-382,1987) of Kunkel to go up site-directed mutagenesis in C ORF, to introduce sudden change at pUC119 (PmlI-BamHI).

Table 11: in rPIV3, introduce Nucleotide and change to produce rF164S virus

The nucleotide sequence of wild-type/sudden change ¹RPIV3 amino acid is replaced SEQ.ID

NO.rF164S Phe-164 to Ser 2276-GCA AAT GTT CCA AGC GAG ATA TC 5

GCA?AAT?GTC?CCA?AGC?GAG?ATA?TC????6

¹Shown the regional nucleotide sequence of sudden change, and itself and wild-type (wt) sequence have been compared.First Nucleotide in the sequence is numbered according to its position in complete antigenomic RNA.The sequence of intercepting is the P open reading frame, and the Nucleotide that underscore marks is at the codon of HP103 on 164 amino acids of C ORF.Be coded in the structure of the anti-genome cDNA (rF164S) of the virus that has the F164S sudden change among the C ORF

Use the mutagenic primer of on 2284 Nucleotide of the anti-genome of total length, introducing A to G change to realize in the phenylalanine (F) on 164 amino acids of C ORF to the change of Serine (S).This sudden change also is reticent in P ORF.PmlI to the BamHI fragment of full-length clone all checks order by it, with existing of the sudden change that confirms to be introduced, and confirms that other sudden changes are unexpected and introduces.This fragment is cloned back respectively then among full-length clone p3/7 (131) 2G and is gone, and (Durbin etc., Virology 235:323-332 (1997)) as previously mentioned is to produce anti-genome cDNA clone.Retrieve mutant strain from the recombinant C F164s of cDNA

The anti-genome cDNA of total length that carries C F164S sudden change uses LipofectACE (Life Technologies) transfection to advance six orifice plate (Costar with vector plasmid { pTM (N), pTM (P no C) and pTM (L) }, Cambridge, MA) in the HEp-2 cell on, and infect (Durbin etc. with MVA-T7 as previously mentioned, Virology 235:323-332,1997).PTM (P no C) is (Durbin etc. of identity with pTM (P) plasmid as previously mentioned, Virology234:74-83 (1997)), except the C transcription initiation site is sported the ACG by ATG, it makes first amino acid among the C ORF become Threonine by methionine(Met).Through 32 ℃ of following incubations 3 days, the transfection product goes down to posterity culture transferring in the T25 flask on the fresh LLC-MK2 cell monolayer and in 5 days (referring to pass 1 generation) of 32 ℃ of following incubations then.The viral load that is present in the F164S product is determined by the plaque titre in LLC-MK2 monolayer culture thing, and plaque its carry out immunoperoxidase staining and show (Durbin etc., Virology 235:323-332 (1997)) with PIV3 HN-specific monoclonal antibody as previously mentioned.

Be present in the F164S recombinant virus that passes in the 1 generation product supernatant liquor then at LLC-MK2 plaque purification three times (Hall etc., Virus Res.22:173-184 (1992)) as described above.Under 32 ℃, be amplified the LLC-MK2 cell from the biology recombinant clone virus that plaque purification obtained for the third time and be used for the virus that further characterizes for twice with generation.The sequential analysis of the recombinant virus that is reclaimed

Virus from clarifying substratum from the cells infected individual layer through polyethylene glycol precipitation concentrate as previously mentioned (Durbin etc., Virology:323-332 (1997)).Use TRIzol reagent according to handbook recommended program purifying RNA (Life Technologies).RT-PCR uses Advantage RT-for-PCR test kit, and (CA) program of recommending according to handbook is operated for Clontech, Palo Alto.Except ThermoScript II was rejected from reaction reagent, control reaction all was identical, and it has confirmed that the PCR product is separately available from viral RNA and be not available from possible impurity cDNA plasmid.Use primer to produce the PCR fragment, it covers the anti-genomic Nucleotide 1595-3104 of total length position.This fragment comprises whole C, D and the VORF of recombinant virus.The PCR product of gained use then the analysis of circulation di-deoxynucleoside acid sequence check order (New England Biolabs, Beverly, MA).Carry out analysis of protein by immunoprecipitation

Two PIV3 C-specific antiseras double antigenic peptide (MAP) technology by use and induce generation from rabbits, its at two kinds of different C peptides (Research Genetics, Huntsville, AL).These two polypeptide cover C Argine Monohydrochloride zone 30-44 and 60-74.Eight copies of each C protein polypeptide are positioned over a branch carrier in the heart and use freund's adjuvant to be injected in (two rabbits of each polypeptide injection) in the rabbit respectively.2 and 4 weeks using specified MAP booster immunization and getting blood in the 4th, 8 and 10 weeks, each antiserum(antisera) is used as immunogenic C peptide with very high titre identification, and through radioimmunoprecipitation method (RIPA) deposit C polypeptide from the HPIV3 cells infected.

The T25 cell monolayer of LLC-MK2 cell is to use rF164S, reorganization JS wt virus (rJS) to infect at 5 o'clock or simulated to infect and at 32 ℃ of incubations in infection multiplicity (MOI).Infected back 24 hours, and use to remove that methionine(Met) DMEM (Life Technologies) washed monolayer cell and 10uCi/ μ l's ³⁵The S methionine(Met) was being cultivated additional 6 hours down except that methionine(Met) DMEM (Life Technologies).Collecting cell, wash three times, and be resuspended to 1ml RIPA damping fluid 1% (w/v) sodium deoxycholate, 1% (v/v) TritonX-100,0.2% (w/v) SDS, 150mM NaCl, 50mM Tris-HCl, pH7.4}, freeze-thaw and under 6500XG aggegation become bead.Extraction liquid of cell is transferred and enters in the fresh microcentrifugation test tube and the mixture of two C antiserum(antisera)s (each 5 μ l) is added into each sample and continue is mixed under 40 degree and carried out incubation in two hours.10 μ l of mAb 454/11 and 101/1 mixture, the HN glycoprotein of its identification HPIV3 (van Wyke Coelingh etc., J Virol.61:1473-1477 (1987)) is added in each sample to confirm that the virus that reclaims is HPIV3 really.(MO) suspension continues to mix at 4 ℃ to each sample and spends the night to carry out the precipitation of immunocomplex 10%A albumen sepharose 4B by adding 200 μ l subsequently for Sigma, St.Louis.Each sample is by sex change, reduces and (San Diego CA) goes up the method for recommending according to handbook and analyzes for NuPAGE, Novex at the 4-12% polyacrylamide gel.Gel is dried and is used autoradiographic technique analysis.Many recursive copyings of rPIV3

Individual layer LLC-MK2 cell in the T25 flask with rF164S or rJS under 0.1MOI parallel two parts infect, and 32 ℃ at 5%CO ₂Following incubation.250 μ l samples were removed at interval according to 24 hours from each flask, after continuous 5 days, were freezed by instantaneous then.The fresh culture of a same volume is used for changing at each time point.(its in 96 hole flat boards 32 ℃ of following incubations 7 days) measures each sample in the LLC-MK2 cell monolayer.By red blood corpuscle absorption detection virus and with log ₁₀TCID ₅₀/ ml carries out record.Experimentation on animals

The gold Syria hamster in every group of 21 4-6 age in week uses each animal 10 ⁵The rF164S of PFU, rJS, cp45 (the JS wt of the attenuation alive of Huo Deing virus redundant organism biologically) or the 0.1ml EMEM (Life Technologies) of respiratory syncytial virus (RSV) carry out intranasal vaccination.After inoculation the 3rd, 4 and 5 day, 5 European mouse in each group were accepted RSV except those, were killed entirely and collected lung and concha.Concha and lung by homogeneity so that (Quality Biologicals, Gaithersburg prepare one 10% or 20%w/v suspension and this sample in MD) and be rapidly frozen at L-15 respectively.Virus in the sample (32 ℃ of following incubations 7 days) in 96 hole flat boards of the cell monolayer of LLC-MK2 is carried out titration.By red blood corpuscle absorption detection virus and according to 5 every group hamster computation of mean values log every day ₁₀TCID ₅₀/ gram.In each group, after inoculation, collected serum in remaining six hamster on the the 0th and 28 day.Each viral serum antibody response is suppressed (HAI) test by blood clotting estimate (van Wyke Coelingh etc., Virology 143:569-582 (1985)) as previously mentioned.Remaining hamster in the 28th day each group comprises the mouse that those use the RSV immunity, uses 10 ⁶The PIV3 JS wt virus that biologically obtains of pFU is carried out nasal cavity and is attacked.The 4th day kill animals collected its lung and concha after attack, and schedule of operation as mentioned above.The viral load of attacking in the sample is as above determined.

Cercopithecus aethiops (AGM) 4 animal per groups uses 10 ⁶The rF164S of PFU or JS wt or cp45 carry out inoculation (Durbin etc., Vaccine 16:1324-30 (1998)) in nasal cavity or the segmental bronchus as the research in rhesus monkey not long ago.Inoculate and collect the nasopharynx wipe samples back 12 days continuous every days and the 2nd, 4,6,8 and 10 day collection lavage of trachea sample after inoculation.These samples are by freezing and be stored in-70 ℃ and be collected up to all samples rapidly.Virus in the sample is carried out titration on 7 days LLC-MK2 cell monolayer of 32 ℃ of incubations in 96 orifice plates.Detect virus by red blood corpuscle absorption, to computation of mean values log every day ₁₀TCID ₅₀/ milliliter.Collected the serum of each monkey at the 0th and 28 day, determine PIV3 HAI antibody response the experimental infection that carries out with different mutant strains and PIV3 wild-type (JS).After inoculation the 28th day, AGM used 10 ⁶The PIV3 wild strain virus that biologically obtains of PFU is attacked with inoculation in 1ml nasal cavity and the tracheae.After attack, collected the nasopharynx wipe samples on the 0th, 1,2,4,6,8,10 and 12 day, the 2nd, 4,6,8 and 10 day collection lavage of trachea sample after attack.Sample is rapidly frozen, and stores, and as above carries out virus titer and measures.The recovery of the rF164S of recombination mutation strain as a result

The anti-genome cDNA of HPIV3 is produced with encoding mutant virus strain rF164S, and wherein C protein 16 4 amino acids residues are changed into S by F.Sudden change is that silence is gone up in translation in overlapping P ORF (table 11).

Anti-genome cDNA and the transfected together HEp2 cell that enters of three PIV3 vector plasmids { pTM (P no C), pTM (N), pTM (L) } use the MVA while transfectional cell of expressing the T7 RNA polymerase.The organic efficiency that contains the rPIV3 of C ORF sudden change is compared with the HEp-2 cell culture of similar use p3/7 (131) 2G transfection, p3/7 (131) 2G expresses the genomic plasmid of total length PIV3, before from wherein reclaiming the JSwt PIV3 (rJS) (Durbin etc., Virology 235:323-332 (1997)) that obtains recombinating.After 3 days, collect transfected cell at 32 ℃ of following incubations, supernatant liquor by culture transferring to the T25 flask on the fresh LLC-MK2 cell monolayer, at 32 ℃ of following incubations 5 days (passing for 1 generation).At 32 ℃ after following 5 days, the LLC-MK2 cell monolayer of rJS and rF164S demonstrates the cytopathogenic effect effect (CPE) of 3-4+.By plaque separate carried out 3 round-robin biology clone after, the recombination mutation strain is amplified twice and is used for further sign to produce viral suspension in the LLC-MK2 cell.

For the virus that confirms to reclaim is the rF164S mutant strain of expection really, clone's virus uses a pair of primer to analyze by RT-PCR, can the increase dna fragmentation of the anti-genomic Nucleotide 1595-3104 scope of HPIV3 of this primer comprises the part of the P gene that contains C, D and V ORF.The generation of PCR product depends on the introducing of RT, and its indication product is not from RNA and from impurity cDNA.The RT-PCR product is carried out nucleotide sequence analysis existing with the sudden change that confirms to be introduced.The sudden change of being introduced is proved and is present in the RT-PCR fragment of crossing over 1595-3104 position Nucleotide, and its amplification is from clone's recombinant chou, and other accidental sudden changes and undiscovered.

Compare rJS wt virus and rF164S mutated viruses by radioimmunoprecipitation test (RIPA).24 to 30 hours incubations in the presence of 35S first sulphur thuja acid behind the infected and self-infection of cell are prepared the cytolysis thing.The total protein of use same amount and anti-C and anti-HN antibody carry out incubation, and antibody is incorporated in in the albumin A agar beads then.RJS and rF164S encode respectively HN and C albumen (Fig. 4).The C albumen expressed for rF164S also has good similarity (Fig. 4) with the quantity of comparing same size by the C albumen of parent plant rJS expression and express.RF164S duplicating in cell culture

The double culture of LLC-MK2 cell monolayer use rF164S or rJS MOI be infect for 0.1 time and incubation following 5 days at 32 ℃.From the substratum of each culture at per 24 hours interval samplings and this material by titration subsequently to estimate the levels of replication of each virus in cell culture.About the speed of virus production and final titre (Fig. 5) with and improving the ability of duplicating under the temperature, duplicate and its parent plant rJS wt of rF164S not have significantly to distinguish substantially.RF164S's duplicates in the hamster

The strain of rF164S mutated viruses further compares the ability that this virus is duplicated in the lower respiratory tract in hamster with rJS wt parent plant.The group of hamster is used rF164S or cp45, and promptly the candidate vaccine that biologically obtains is with each animal 10 ⁵PFU carries out intranasal vaccination.Be compared to rJS, being replicated in of rF164S be reduced in the upper respiratory tract 100-150 doubly and its amount that in the lower respiratory tract of hamster, is reduced surpass 10 times (table 12).The hamster of using rFI64S and rJS to infect for HPIV3, have one clearly antibody response and its demonstrate the limitation in height (table 13) of duplicating to the PIV3 challenge virus.Table 12:rF164S virus is attenuation in the lower respiratory tract on hamster

Metainfective average virus titer (log ₁₀TCID ₅₀/ g ± S.E. ²) the peak value virus titer

The 3rd day the 4th day the 5th day (log ₁₀TCID ₅₀The virus of/g ± S.E.) ¹Nasopharynx lung nasopharynx lung nasopharynx lung nasopharynx lung cp45 4.7 ± 0.2 2.7 ± 0.2 5.0 ± 0.5 2.8 ± 0.5 5.4 ± 0.3 1.6 ± 0.2 5.4 ± 0.3 2.8 ± 0.5

(C) ³????????(C) ³rF164S??4.0±0.2????5.3±0.3????4.7±0.3????5.4±0.2????4.4±0.6????4.3±0.1????4.7±0.3????5.4±0.2

(B)?????????(B)RJS?????6.7±0.3????6.8±0.5????7.4±0.2????6.6±0.4????6.6±0.2????7.2±0.2????7.4±0.2????7.2±0.2

(A)?????????(A)

¹The group of each 5 hamster uses 10 ⁵The setting virus of pfu is carried out intranasal vaccination.Cp45 is the virus that biologically obtains, and other is a recombinant virus.

²Standard error.

³Use the mean value in each hurdles of different letters to have significant difference (a=0.05), it uses the many gaps tests of Duncan to determine, does not have a significant difference and those have a same letter.Table 13:rF164S has immunogenicity and can resist the reaction immune serum HAI antibody titers mean P IV3 titre of attack to attacking of PIV3 wt virus in hamster ²(log ₁₀TCID ₅₀/ g) ± S.E. virus ¹(mean value log reciprocal ₂± S.E.) nasopharynx lung RF164S 10.8 ± 0.2＜1.5 ± 0.0＜1.2 ± 0.0Cp45 11.8 ± 0.3＜1.5 ± 0.0＜1.2 ± 0.0rJS 12.1 ± 0.2＜1.5 ± 0.0＜1.2 ± 0.0RSV＜2.0 ± 0.0 7.0 ± 0.2 4.7 ± 0.0

¹Show the virus that was used for each 6 hamster group of immunity at the 0th day.

²At the 28th day, all hamsters were got blood to determine serum HAI antibody titers and to use 10 ⁶The JS wt HPIV3 that PFU biologically obtains attacks.Lung and concha were collected in attack in back 4 days.RF164S duplicating in primate

For attenuation phenotype and the protection effect of further estimating rF164S, this recombination mutation strain is given in a group of 4 AGM, and its duplicating with cp45 and JSwt in last lower respiratory tract compares.Be reduced 100 times or more in the upper respiratory tract that is replicated in AGM of rF164S.The attenuation of this virus in the upper respiratory tract compared with cp45 (table 14), and rF164S is subjected to moderate (＜10 times) restriction in the AGM lower respiratory tract.This recombinant virus is induced a HAI antibody (table 14) to HPIV3.The JS wt virus that this immune AGM used 106PFU biologically to obtain at the 28th day is attacked by IN and IT.The animal of having accepted rF164S or cp45 candidate vaccine can be subjected to protecting fully with opposing challenge virus duplicating in the lower respiratory tract on AGM.Table 14:rF164S in cercopithecus aethiops be attenuation and bring out serum HAI antibody response

The average peak titre was the 28th day serum average peak titre

(log ₁₀TCID ₅₀/ g) ± S.E. ²(the log of HAI antibody titers challenge virus ₁₀TCID ₅₀/ g) ± S.E. ⁴Virus ¹Nasopharynx tracheae animal (mean value nasopharynx tracheae reciprocal

Wiping lavation quantity log ₂± S.E.) ³Wiping lavation rF164S 4.3 ± 0.3 5.8 ± 0.2 4 12.5 ± 0.3＜0.5 ± 0.0＜0.5 ± 0.0

(C) ⁵????????(B) ⁵cp45????5.1±0.4????5.3±0.1????4?????12.0±0.6??????????＜0.5±0.0?????????＜0.5±0.0

(B)?????????(C)JSwt????6.3±0.2????6.6±0.1????6?????13.2±0.2

(A)?????????(A)

¹Used 10 at the 0th day ⁶The appointment virus of PFU is by inoculating AGM in intranasal vaccination and the tracheae.

²Average variance.

³The 0th day average serum HAI antibody titers (mean value log2 reciprocal) is ± 2.0.

⁴Used in the 28th day 106 PFU HPIV3 JS wild-type virus by nose in and the tracheae inside fire attack hit animal.

⁵Use the mean value in each alphabetical hurdle of difference to have significant difference (α=0.05), it uses the many gaps tests of Duncan to determine, and the mean value of use same letter does not have significant difference.

⁶Two animals received reorganization JS wt virus rJS in this group, 4 biological obtainable JS wt viruses of animals received.The peak averaging titre of these two viruses does not have difference (being 6.4 pairs 6.3, is 6.6 pairs 6.7) in lower respiratory tract in the upper respiratory tract.Because the peak value titre does not have difference, so these groups are combined further analysis.

Brief overview the foregoing description, F170S sudden change in the SeV strain is by making virus growth (Garcin etc. in the LLC-MK2 simian cells, Virology 238:424-431 (1997), and Itoh etc., J.Gen.Virol.78:3207-15 (1997)) and carry out biological production.Confirmed its enhancing although have the reorganization SeV of F170S sudden change at replication in vitro, it but is limitation in height that this mutant strain duplicates in mouse, as duplicating of SeV C '/C mutant strain is the (Gamin etc. that weaken, Virology 238:424-431 (1997) and Latorre etc., J Virol.72:5984-93,1998)).Because the C albumen of SeV and HPIV3 only has 38% homology, it is shocking that very the F170S sudden change of finding SeV is input to HPIV3 and causes attenuation in the body.

HPIV3 recombinant strain rF164S, its 170 sites corresponding to SeV carry the amino acid whose sudden change of HPIV3, but its replication in vitro and unrestricted in hamster and AGM on be obvious attenuation in the lower respiratory tract.The compare comparable serum antibody response of wt PIV3 infection-induced and its of rF164S protects hamster and AGM opposing wt HPIV3 to attack fully.These results show that the F164S sudden change is very useful for the attenuated vaccine alive of the anti-HPIV3 of exploitation.

The preservation of biomaterial

* HPIV3 1537 HPKVFKRFWDCGVLNPIYGPNTASQDQIKLALSICEYSLDLFMREWL (SEQ ID NO.21) BPIV3 1537 HPKVFKRFWDCGVLNPIYGPNTASQDQVKLALSICEYSLDLFMREWL (SEQ ID NO.22) of Sendai virus 1537 HPKIFKRFWNAGVVEPVYGPNLSNQDKILLALSVCEYSVDLFMHDWQ (SEQ ID NO.23) HPIV2 1543 HPKLLRRAMNLDIITPIHAPYLASLDYVKLSIDAIQWGVKQVLADLS (SEQ ID NO.24) measles virus 1535 HPKIYKKFWHCGIIEPIHGPSLDAQNLHTTVCNMVYTCYMTYLDLLL (SEQ ID NO .25) RSV 1584 EQKVIKYILSQDASLHRVEGCHSFKLWFLKRLNVAEFTVCPWVVNID (SEQ ID NO.26)...

Although in order to be expressly understood that the present invention at length is described above-mentioned invention with legend and embodiment, clearly, to modification of the present invention and improve all in the protection domain of claims.

Sequence <110> U.S. Government Department of Health and Human Services <120> was prepared from the nucleotide sequence of the cloned attenuated negative-strand RNA virus vaccines <130> NIH-0160 <150> 60/129, 006 <151> 1999-04-13 <150> PCT/US00/09695 <151> 2000-04-12 <160> 53 <170> PatentIn version 3.1 <210> 1 <211> 53 <212> PRT <213> human parainfluenza virus 3 <400> 1 Gln Gly Val Lys Ile Ile Thr His Lys Glu Cys Ser Thr Ile Gly Ile 1 5 10 15 Asn Gly Met Leu Phe Asn Thr Asn Lys Glu Gly Thr Leu Ala Phe Tyr 20 25 30 Thr Pro Asn Asp Ile Thr Leu Asn Asn Ser Val Ala Leu Asp Pro Ile 35 40 45 Asn Ile Ser Ile Glu 50 <210> 2 <211> 53 <212> PRT <213> Bovine parainfluenza virus 3 <400> 2 Gln Gly Ile Lys Ile Ile Thr His Lys Glu Cys Gln Val Ile Gly Ile 1 5 10 15 Asn Gly Met Leu Phe Asn Thr Asn Arg Glu Gly Thr Leu Ala Thr Tyr 20 25 30 Thr Phe Asp Asp Ile Ile Leu Asn Asn Ser Val Ala Leu Asn Pro Ile 35 40 45 Asp Ile Ser Met Glu 50 <210> 3 <211> 53 <212> PRT <213> human parainfluenza virus 1 <400> 3 Arg Gly Val Thr Phe Leu Thr Tyr Thr Asn Cys Gly Leu Ile Gly Ile 1 5 10 15 Asn Gly Ile Glu Leu Tyr Ala Asn Lys Arg Gly Arg Asp Thr Thr Arg 20 25 30 Gly Asn Gln Ile Ile Lys Val Gly Pro Ala Val Ser Ile Arg Pro Val 35 40 45 Asp Ile Ser Leu Asn 50 <210> 4 <211> 53 <212> PRT <213> human parainfluenza virus 2 <400> 4 Gln Gly Ile Ser Ile Ile Asp Ile Lys Arg Cys Ser Glu Met Met Leu 1 5 10 15 Asp Thr Phe Ser Phe Arg Ile Thr Ser Thr Phe Asn Ala Thr Tyr Val 20 25 30 Thr Asp Phe Ser Met Ile Asn Ala Asn Ile Val His Leu Ser Pro Leu 35 40 45 Asp Leu Ser Asn Gln 50 <210> 5 <211> 23 <212> DNA <213> human parainfluenza virus 3 <400> 5 gcaaatgttc caagcgagat atc 23 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> a nucleotide sequence encoding F164S mutation <400> 6 gcaaatgtcc caagcgagat atc 23 <210> 7 <211> 53 <212> PRT <213> respiratory syncytial virus <400> 7 Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val 1 5 10 15 Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr 20 25 30 Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro 35 40 45 Ser Asp Gln Phe Asp 50 <210> 8 < 211> 53 <212> PRT <213> Measles virus <400> 8 Lys Ile Leu Thr Tyr Ile Ala Ala Asp His Cys Pro Val Val Glu Val 1 5 10 15 Asn Gly Val Thr Ile Gln Val Gly Ser Arg Arg Tyr Pro Asp Ala Val 20 25 30 Tyr Leu His Arg Ile Asp Leu Gly Pro Pro Ile Ser Leu Glu Arg Leu 35 40 45 Asp Val Gly Thr Asn 50 <210> 9 <211> 41 <212> PRT <213> human parainfluenza virus 3 <400> 9 Asn Pro Asn Arg Met Gln Tyr Ala Ser Leu Ile Pro Ala Ser Val Gly 1 5 10 15 Gly Phe Asn Tyr Met Ala Met Ser Arg Cys Phe Val Arg Asn Ile Gly 20 25 30 Asp Pro Ser Val Ala Ala Leu Ala Asp 35 40 < 210> 10 <211> 41 <212> PRT <213> Bovine parainfluenza virus 3 <400> 10 Asn Ile His Trp Met Gln Tyr Ala Ser Leu Ile Pro Ala Ser Val Gly 1 5 10 15 Gly Phe Asn Tyr Met Ala Met Ser Arg Cys Phe Val Arg Asn Ile Gly 20 25 30 Asp Pro Thr Val Ala Ala Leu Ala Asp 35 40 <210> 11 <211> 41 <212> PRT <213> Sendai virus <400> 11 Gly Leu Asn Trp Leu Arg Cys Ala Val Leu Ile Pro Ala Asn Val Gly 1 5 10 15 Gly Phe Asn Tyr Met Ser Thr Ser Arg Cys Phe Val Arg Asn Ile Gly 20 25 30 Asp Pro Ala Val Ala Ala Leu Ala Asp 35 40 <210> 12 <211> 41 <212> PRT <213> human parainfluenza Virus 2 <400> 12 His Pro Arg Leu Ile Ser Arg Ile Val Leu Leu Pro Ser Gln Leu Gly 1 5 10 15 Gly Leu Asn Tyr Leu Ala Cys Ser Arg Leu Phe Asn Arg Asn Ile Gly 20 25 30 Asp Pro Leu Gly Thr Ala Val Ala Asp 35 40 <210> 13 <211> 41 <212> PRT <213> Measles virus <400> 13 Asn Asn Asp Leu Leu Ile Arg Met Ala Leu Leu Pro Ala Pro Ile Gly 1 5 10 15 Gly Met Asn Tyr Leu Asn Met Ser Arg Leu Phe Val Arg Asn Ile Gly 20 25 30 Asp Pro Val Thr Ser Ser Ile Ala Asp 35 40 <210> 14 <211> 44 <212> PRT <213> respiratory syncytial virus <400> 14 Leu Asp Asn Ile Asp Thr Ala Leu Thr Leu Tyr Met Asn Leu Pro Met 1 5 10 15 Leu Phe Gly Gly Gly Asp Pro Asn Leu Leu Tyr Arg Ser Phe Tyr Arg 20 25 30 Arg Thr Pro Asp Phe Leu Thr Gln Ala Ile Val His 35 40 <210> 15 <211> 46 <212> PRT <213> human parainfluenza virus 3 <400> 15 Leu Asp Arg Ser Val Leu Tyr Arg Ile Met Asn Gln Glu Pro Gly Glu 1 5 10 15 Ser Ser Phe Leu Asp Trp Ala Ser Asp Pro Tyr Ser Cys Asn Leu Pro 20 25 30 Gln Ser Gln Asn Ile Thr Thr Met Ile Lys Asn Ile Thr Ala 35 40 45 <210> 16 <211> 46 <212> PRT <213> Bovine parainfluenza virus 3 <400> 16 Leu Asp Arg Gly Val Leu Tyr Arg Ile Met Asn Gln Glu Pro Gly Glu 1 5 10 15 Ser Ser Phe Leu Asp Trp Ala Ser Asp Pro Tyr Ser Cys Asn Leu Pro 20 25 30 Gln Ser Gln Asn Ile Thr Thr Met Ile Lys Asn Ile Thr Ala 35 40 45 <210 > 17 <211> 46 <212> PRT <213> Sendai virus <400> 17 Leu Asp Lys Gln Val Leu Tyr Arg Val Met Asn Gln Glu Pro Gly Asp 1 5 10 15 Ser Ser Phe Leu Asp Trp Ala Ser Asp Pro Tyr Ser Cys Asn Leu Pro 20 25 30 His Ser Gln Ser Ile Thr Thr Ile Ile Lys Asn Ile Thr Ala 35 40 45 <210> 18 <211> 46 <212> PRT <213> human parainfluenza virus 2 <400> 18 Leu Gly Ser Trp Ile Leu Tyr Ash Leu Leu Ala Arg Lys Pro Gly Lys 1 5 10 15 Gly Ser Trp Ala Thr Leu Ala Ala Asp Pro Tyr Ser Leu Asn Gln Glu 20 25 30 Tyr Leu Tyr Pro Pro Thr Thr Ile Leu Lys Arg His Thr Gln 35 40 45 <210> 19 <211> 46 <212> PRT <213 > measles virus <400> 19 Met Pro Glu Glu Thr Leu His Gln Val Met Thr Gln Gln Pro Gly Asp 1 5 10 15 Ser Ser Phe Leu Asp Trp Ala Ser Asp Pro Tyr Ser Ala Asn Leu Val 20 25 30 Cys Val Gln Ser Ile Thr Arg Leu Leu Lys Asn Ile Thr Ala 35 40 45 <210> 20 <211> 46 <212> PRT <213 > respiratory syncytial virus <400> 20 Leu Asn Lys Phe Leu Thr Cys Ile Ile Thr Phe Asp Lys Asn Pro Asn 1 5 10 15 Ala Glu Phe Val Thr Leu Met Arg Asp Pro Gln Ala Leu Gly Ser Glu 20 25 30 Arg Gln Ala Lys Ile Thr Ser Gly Ile Asn Arg Leu Ala Val 35 40 45 <210> 21 <211> 47 <212> PRT <213> human parainfluenza virus 3 <400> 21 His Pro Lys Val Phe Lys Arg Phe Trp Asp Cys Gly Val Leu Asn Pro 1 5 10 15 Ile Tyr Gly Pro Asn Thr Ala Ser Gln Asp Gln Ile Lys Leu Ala Leu 20 25 30 Ser Ile Cys Glu Tyr Ser Leu Asp Leu Phe Met Arg Glu Trp Leu 35 40 45 <210> 22 <211> 47 <212> PRT <213> Bovine parainfluenza virus 3 <400> 22 His Pro Lys Val Phe Lys Arg Phe Trp Asp Cys Gly Val Leu Asp Pro 1 5 10 15 Ile Tyr Gly Pro Asn Thr Ala Ser Gln Asp Gln Val Lys Leu Ala Leu 20 25 30 Ser Ile Cys Glu Tyr Ser Leu Asp Leu Phe Met Arg Glu Trp Leu 35 40 45 < 210> 23 <211> 47 <212> PRT <213> Sendai virus <400> 23 His Pro Lys Ile Phe Lys Arg Phe Trp Asn Ala Gly Val Val Glu Pro 1 5 10 15 Val Tyr Gly Pro Asn Leu Ser Asn Gln Asp Lys Ile Leu Leu Ala Leu 20 25 30 Ser Val Cys Glu Tyr Ser Val Asp Leu Phe Met His Asp Trp Gln 35 40 45 <210> 24 <211> 47 <212> PRT <213> human parainfluenza virus 2 <400> 24 His Pro Lys Leu Leu Arg Arg Ala Met Asn Leu Asp Ile Ile Thr Pro 1 5 10 15 Ile His Ala Pro Tyr Leu Ala Ser Leu Asp Tyr Val Lys Leu Ser Ile 20 25 30 Asp Ala Ile Gln Trp Gly Val Lys Gln Val Leu Ala Asp Leu Ser 35 40 45 <210> 25 <211> 47 <212> PRT <213> the measles virus <400 > 25 His Pro Lys Ile Tyr Lys Lys Phe Trp His Cys Gly Ile Ile Glu Pro 1 5 10 15 Ile His Gly Pro Ser Leu Asp Ala Gln Asn Leu His Thr Thr Val Cys 20 25 30 Asn Met Val Tyr Thr Cys Tyr Met Thr Tyr Leu Asp Leu Leu Leu 35 40 45 <210> 26 <211> 47 <212> PRT <213> respiratory syncytial virus <400> 26 Glu Gln Lys Val Ile Lys Tyr Ile Leu Ser Gln Asp Ala Ser Leu His 1 5 10 15 Arg Val Glu Gly Cys His Ser Phe Lys Leu Trp Phe Leu Lys Arg Leu 20 25 30 Asn Val Ala Glu Phe Thr Val Cys Pro Trp Val Val Asn Ile Asp 35 40 45 <210> 27 <211> 41 <212> PRT <213> human parainfluenza virus 3 <400> 27 Asn Ala Tyr Gly Ser Asn Ser Ala Ile Ser Tyr Glu Asn Ala Val Asp 1 5 10 15 Tyr Tyr Gln Ser Phe Ile Gly Ile Lys Phe Asn Lys Phe Ile Glu Pro 20 25 30 Gln Leu Asp Glu Asp Leu Thr Ile Tyr 35 40 <210> 28 <211> 41 <212> PRT <213> Respiratory syncytial virus <400> 28 Tyr Tyr Lys Leu Asn Thr Tyr Pro Ser Leu Leu Glu Leu Thr Glu Arg 1 5 10 15 Asp Leu Ile Val Leu Ser Gly Leu Arg Phe Tyr Arg Glu Phe Arg Leu 20 25 30 Pro Lys Lys Val Asp Lys Glu Met Ile 35 40 <210> 29 <211> 41 <212> PRT <213> Measles virus <400> 29 Asn Ala Gln Ala Ser Gly Glu Gly Leu Thr His Glu Gln Cys Val Asp 1 5 10 15 Asn Trp Lys Ser Phe Ala Gly Val Lys Phe Gly Cys Phe Met Pro Leu 20 25 30 Ser Leu Asp Ser Asp Leu Thr Met Tyr 35 40 <210> 30 <211> 41 <212> PRT <213> sendai virus <400> 30 Asn Ala Gln Gly Ser Asn Thr Ala Ile Ser Tyr Glu Cys Ala Val Asp 1 5 10 15 Asn Tyr Thr Ser Phe Ile Gly Phe Lys Phe Arg Lys Phe Ile Glu Pro 20 25 30 Gln Leu Asp Glu Asp Leu Thr Ile Tyr 35 40 <210> 31 <211> 41 <212> PRT <213> human parainfluenza virus 2 <400> 31 Glu Phe Gln His Asp Asn Ala Glu Ile Ser Tyr Glu Tyr Thr Leu Lys 1 5 10 15 His Trp Lys Glu Ile Ser Leu Ile Glu Phe Arg Lys Cys Phe Asp Phe 20 25 30 Asp Pro Gly Glu Glu Leu Ser Ile Phe 35 40 <210> 32 <211> 41 <212> PRT <213> canine distemper virus <400> 32 Asn Ala His Ala Ser Gly Glu Gly Ile Thr Tyr Ser Gln Cys Ile Glu 1 5 10 15 Asn Trp Lys Ser Phe Ala Gly Ile Arg Phe Lys Cys Phe Met Pro Leu 20 25 30 Ser Leu Asp Ser Asp Leu Thr Met Tyr 35 40 <210> 33 <211> 41 <212> PRT <213> Simian virus 41 <400> 33 Glu Leu His His Asp Asn Ser Glu Ile Ser Tyr Glu Tyr Thr Leu Arg 1 5 10 15 His Trp Lys Glu Leu Ser Leu Ile Glu Phe Lys Lys Cys Phe Asp Phe 20 25 30 Asp Pro Gly Glu Glu Leu Ser Ile Phe 35 40 <210> 34 <211> 41 <212> PRT <213> Seal distemper virus <400> 34 Asn Ala Cys Val Ser Gly Glu Gly Ile Thr Tyr Ser Gln Cys Val Glu 1 5 10 15 Asn Trp Lys Ser Phe Ala Gly Ile Lys Phe Arg Cys Phe Met Pro Leu 20 25 30 Ser Leu Asp Ser Asp Leu Thr Met Tyr 35 40 <210> 35 <211> 41 <212> PRT <213> Hendra virus <400> 35 Arg Leu Lys Asn Ser Gly Glu Ser Leu Thr Val Asp Asp Cys Val Lys 1 5 10 15 Asn Trp Glu Ser Phe Cys Gly Ile Gln Phe Asp Cys Phe Met Glu Leu 20 25 30 Lys Leu Asp Ser Asp Leu Ser Met Tyr 35 40 <210> 36 <211> 41 <212> PRT <213> Simian virus 5 <400> 36 Glu Leu Met Asn Asp Asn Thr Glu Ile Ser Tyr Glu Phe Thr Leu Lys 1 5 10 15 His Trp Lys Glu Val Ser Leu Ile Lys Phe Lys Lys Cys Phe Asp Ala 20 25 30 Asp Ala Gly Glu Glu Leu Ser Ile Phe 35 40 <210> 37 <211> 41 <212> PRT <213> rinderpest virus <400> 37 Asn Ala Gln Ala Ser Gly Glu Gly Leu Thr Tyr Glu Gln Cys Val Asp 1 5 10 15 Asn Trp Lys Ser Phe Ala Gly Ile Arg Phe Gly Cys Phe Met Pro Leu 20 25 30 Ser Leu Asp Ser Asp Leu Thr Met Tyr 35 40 <210> 38 <211> 41 <212> PRT <213> Avian metapneumovirus <400> 38 Tyr Met Asn Ala Lys Thr Tyr Pro Ser Asn Leu Glu Leu Cys Val Glu 1 5 10 15 Asp Phe Leu Glu Leu Ala Gly Ile Ser Phe Cys Gln Glu Phe Tyr Val 20 25 30 Pro Ser Gln Thr Ser Leu Glu Met Val 35 40 <210> 39 <211> 41 <212> PRT <213> Newcastle disease virus <400> 39 Gln Leu His Ala Asp Ser Ala Glu Ile Ser His Asp Ile Met Leu Arg 1 5 10 15 Glu Tyr Lys Ser Leu Ser Ala Leu Glu Phe Glu Pro Cys Ile Glu Tyr 20 25 30 Asp Pro Val Thr Asn Leu Ser Met Phe 35 40 <210> 40 <211> 56 <212> PRT <213> human parainfluenza virus 3 <400> 40 Met Lys Leu Glu Arg Trp Ile Arg Thr Leu Leu Arg Gly Lys Cys Asp 1 5 10 15 Asn Leu Gln Met Phe Gln Ala Arg Tyr Gln Glu Val Met Thr Tyr Leu 20 25 30 Gln Gln Asn Lys Val Glu Thr Val Ile Met Glu Glu Ala Trp Asn Leu 35 40 45 Ser Val His Leu Ile Gln Asp Gln 50 55 <210> 41 <211> 58 <212> PRT <213> Bovine parainfluenza virus 3 <400> 41 Met Lys Leu Gly Arg Trp Ile Arg Thr Leu Leu Arg Gly Lys Cys Asp 1 5 10 Asn Leu Lys Met Phe Gln Ser Arg Tyr Gln Gly Val Met Pro Phe Leu 20 25 30 Gln Gln Asn Lys Met Glu Thr Val Met Met Glu Glu Ala Trp Asn Leu 35 40 45 Ser Val His Leu Ile Gln Asp Ile Pro Ala 50 55 <210> 42 <211> 55 <212> PRT <213> sendai virus <400> 42 Met Lys Thr Glu Arg Trp Leu Arg Thr Leu Ile Arg Gly Glu Lys Thr 1 5 10 15 Lys Leu Lys Asp Phe Gln Lys Arg Tyr Glu Glu Val His Pro Tyr Leu 20 25 30 Met Lys Glu Lys Val Glu Gln Ile Ile Met Glu Glu Ala Trp Ser Leu 35 40 45 Ala Ala His Ile Val Gln Glu 50 55 <210> 43 <211> 55 <212> PRT <213> human parainfluenza virus 1 <400> 43 Met Lys Thr Glu Arg Trp Leu Arg Thr Leu Ile Arg Gly Lys Lys Thr 1 5 10 15 Lys Leu Arg Asp Phe Gln Lys Arg Tyr Glu Glu Val His Pro Tyr Leu 20 25 30 Met Met Glu Arg Val Glu Gln Ile Ile Met Glu Glu Ala Trp Lys Leu 35 40 45 Ala Ala His Ile Val Gln Glu 50 55 <210> 44 <211> 41 <212> PRT <213> respiratory syncytial virus <400> 44 Tyr Tyr Lys Leu Asn Thr Tyr Pro Ser Leu Leu Glu Leu Thr Glu Arg 1 5 10 15 Asp Leu Ile Val Leu Ser Gly Leu Arg Phe Tyr Arg Glu Phe Arg Leu 20 25 30 Pro Lys Lys Val Asp Leu Glu Met Ile 35 40 <210> 45 <211 > 41 <212> PRT <213> human parainfluenza virus 3 <400> 45 Asn Ala Tyr Gly Ser Asn Ser Ala Ile Ser Tyr Glu Asn Ala Val Asp 1 5 10 15 Tyr Tyr Gln Ser Phe Ile Gly Ile Lys Phe Asn Lys Phe Ile Glu Pro 20 25 30 Gln Leu Asp Glu Asp Leu Thr Ile Tyr 35 40 <210> 46 <211> 41 <212> PRT <213> Measles virus <400> 46 Asn Ala Gln Ala Ser Gly Glu Gly Leu Thr His Glu Gln Cys Val Asp 1 5 10 15 Asn Trp Lys Ser Phe Ala Gly Val Lys Phe Gly Cys Phe Met Pro Leu 20 25 30 Ser Leu Asp Ser Asp Leu Thr Met Tyr 35 40 <210> 47 <211> 41 <212> PRT <213> Sendai virus <400> 47 Asn Ala Gln Gly Ser Asn Thr Ala Ile Ser Tyr Glu Cys Ala Val Asp 1 5 10 15 Asn Tyr Thr Ser Phe Ile Gly Phe Lys Phe Arg Lys Phe Ile Glu Pro 20 25 30 Gln Leu Asp Glu Asp Leu Thr Ile Tyr 35 40 <210> 48 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Consensus Sequence <400> 48 Asn Ala Ser Glu Val Asp Ser Phe Gly Lys Phe Phe Leu Asp Asp Leu 1 5 10 15 Thr Tyr <210> 49 <211> 12 <212> DNA <213> human parainfluenza virus 3 <400> 49 ttcaataaat tc 12 <210> 50 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> F456L mutant nucleotide sequence encoding <400> 50 ctgaataaat tc 12 <210> 51 <211> 11 <212> DNA <213> human parainfluenza virus 3 <400> 51 aaaaaagggg g 11 <210 > 52 <211> 20 <212> DNA <213> human parainfluenza virus 3 <400> 52 gttgatggaa agcgatgcta 20 <210> 53 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> modified The P mRNA editing sites <400> 53 aaaaaagggg ggg 13 ...

Claims (126)

1. method of producing described minus-stranded rna virus from the separated polynucleotide molecule of the separated attenuation recombinant negative strand rna of one or more codings virus, it comprises:

The one or more expression vectors of coexpression in a cell or cell-free system, the polynucleotide molecule that it contains one or more coding recombination groups or anti-genome and produces the necessary basic viral protein of infectious viral particle of described recombinant negative strand rna virus, described recombination group or anti-genome are modified, with in the recombinant protein that is coded in described recombinant virus corresponding to the sudden change on the amino acid position of the amino acid position of the attenuation sudden change of in allos sudden change minus-stranded rna virus, being differentiated, it gives described recombinant virus attenuation phenotype by introducing in described recombinant protein.

2. the method for claim 1 is characterized in that described recombinant negative strand rna virus is respiratory syncytial virus (RSV).

3. method as claimed in claim 2 is characterized in that described RSV is human RSV hypotype A, human RSV hypotype B, ox RSV, mouse RSV or bird Pneumovirinae.

4. method as claimed in claim 2 is characterized in that described allos, sudden change minus-stranded rna virus are allos RSV, human parainfluenza virus (HPIV) 1, HPIV2, HPIV3, ox PIV (BPIV), Sendai virus (SeV), Avian pneumo-encephalitis virus (NDV), SV 41 virus (SV5), mumps virus (MuV), Measles virus (MeV), canine distemper virus (CDV), rabies virus (RaV) or vesicular stomatitis virus (VSV).

5. method as claimed in claim 4 is characterized in that described allos sudden change minus-stranded rna virus is RSV cpts248 (ATCC VR 2450), RSV cpts248/404 (ATCC VR2454), RSV cpts248/955 (ATCC VR 2453), RSV cpts530 (ATCC VR2452), RSV cpts530/1009 (ATCC VR 2451), RSV cpts 530/1030 (ATCCVR 2455), RSV B-1 cp52/2B5 (ATCC VR 2542) or RSV B-1 cp-23 (ATCC VR 2579).

6. method as claimed in claim 2, it is characterized in that described recombination group or anti-genome are modified, replace, lack or insert with the amino acid that is coded in RSV NS1, NS2, N, P, M, SH, M2 (ORF1), M2 (ORF2), L, F or the G albumen.

7. method as claimed in claim 2 is characterized in that the described sudden change of introducing gives the phenotype of described recombinant negative strand rna virus temperature sensitivity (ts) in described recombinant protein.

8. the method for claim 1 is characterized in that the described sudden change of introducing is included in the amino acid replacement in the RSV L albumen in described recombinant protein.

9. method as claimed in claim 8 is characterized in that the attenuation sudden change of being differentiated comprises that the amino acid on proteic 521 phenylalanines of L of human RSV cpts530 (ATCC VR 2452) replaces in described allos sudden change minus-stranded rna virus.

10. the method for claim 1 is characterized in that described recombinant negative strand rna virus is parainfluenza virus (PIV).

11. method as claimed in claim 10 is characterized in that described PIV is human PIV1 (HPIV1), human PIV2 (HPW2), human PIV3 (HPIV3), ox PIV (BPIV) or mouse PIV (MPIV).

12. method as claimed in claim 10 is characterized in that described allos mutated viruses is respiratory syncytial virus (RSV), human parainfluenza virus (HPIV) 1, HPIV2, HPIV3, ox PIV (BPIV), Sendai virus (SeV), Avian pneumo-encephalitis virus (NDV), SV 41 virus (SV5), mumps virus (MuV), Measles virus (MeV), canine distemper virus (CDV), rabies virus (RaV) or vesicular stomatitis virus (VSV).

13. method as claimed in claim 10 is characterized in that described recombination group or anti-genome are modified, and replaces, lacks or insert with the amino acid that is coded in PIV N, P, C, D, V, M, F, HN or the L albumen.

14. method as claimed in claim 10 is characterized in that the described sudden change of introducing gives described recombinant negative strand rna virus temperature sensitivity (ts) phenotype in described recombinant protein.

15. method as claimed in claim 10 is characterized in that described allos sudden change minus-stranded rna virus is HPIV3 JS cp45.

16. method as claimed in claim 15 is characterized in that the attenuation sudden change of being differentiated includes the amino acid replacement in HPIV3 JS cp45 L albumen in described allos sudden change minus-stranded rna virus.

17. method as claimed in claim 16 is characterized in that the amino acid that the attenuation sudden change of being differentiated includes on 942 tyrosine in HPIV3 JS cp45 L albumen replaces in described allos sudden change minus-stranded rna virus.

18. method as claimed in claim 16 is characterized in that the amino acid that the attenuation sudden change of being differentiated includes on 992 leucines in HPIV3 JS cp45 L albumen replaces in described allos sudden change minus-stranded rna virus.

19. method as claimed in claim 16 is characterized in that the amino acid that the attenuation sudden change of being differentiated includes on amino acid sites 1558 place's Threonines in HPIV3 JS cp45 L albumen replaces in described allos sudden change minus-stranded rna virus.

20. method as claimed in claim 15 is characterized in that the attenuation sudden change of being differentiated includes the amino acid in HPIV3 JS cp45 F albumen and replaces in described allos sudden change minus-stranded rna virus.

21. method as claimed in claim 20 is characterized in that the amino acid that the attenuation sudden change of being differentiated includes on 420 Isoleucines in HPIV3 JS cp45 F albumen replaces in described allos sudden change minus-stranded rna virus.

22. method as claimed in claim 20 is characterized in that the amino acid that the attenuation sudden change of being differentiated includes on 450 L-Ala in HPIV3 JS cp45 F albumen replaces in described allos sudden change minus-stranded rna virus.

23. method as claimed in claim 10 is characterized in that the amino acid that the attenuation sudden change of being differentiated includes on 521 phenylalanines in human RSV cpts530 (ATCC VR2452) L albumen replaces in described allos sudden change minus-stranded rna virus.

24. method as claimed in claim 23, it is characterized in that described recombinant negative strand rna virus is human PIV3 (HPIV3), and the amino acid that the described sudden change of introducing includes on proteic 456 phenylalanines of described HPIV3 L is replaced in described recombinant protein.

25. method as claimed in claim 10 is characterized in that the attenuation sudden change of being differentiated includes the amino acid on proteic 170 phenylalanines of SeV C and replaces in described allos sudden change minus-stranded rna virus.

26. method as claimed in claim 25, it is characterized in that described recombinant negative strand rna virus is human PIV3 (HPIV3), and the amino acid that the described sudden change of introducing includes on proteic 164 phenylalanines of described HPIV3 C is replaced in described recombinant protein.

27. the method for claim 1 is characterized in that described recombinant negative strand rna virus is Measles virus (MeV).

28. method as claimed in claim 27 is characterized in that described allos mutated viruses is respiratory syncytial virus (RSV), human parainfluenza virus (HPIV) 1, HPIV2, HPIV3, ox PIV (BPIV), Sendai virus (SeV), Avian pneumo-encephalitis virus (NDV), SV 41 virus (SV5), mumps virus (MuV), Measles virus (MeV), canine distemper virus (CDV), rabies virus (RaV) or vesicular stomatitis virus (VSV).

29. method as claimed in claim 27 is characterized in that described allos sudden change minus-stranded rna virus is HPIV3 JS cp45.

30. method as claimed in claim 29, it is characterized in that the attenuation sudden change of in described allos sudden change minus-stranded rna virus, being differentiated include one in HPIV3 JS cp45 L albumen 942 tyrosine or the amino acid on 992 leucines replace.

31. the method for claim 1, it is characterized in that recombinant negative strand rna virus is to have recombination group or anti-genomic embedded virus, described recombination group or anti-genome comprise and the heterologous gene of another minus-stranded rna virus kind, hypotype or strain or the part or all of genome or the anti-genome of gene fragment bonded minus-stranded rna virus kind, hypotype or a strain.

32. method as claimed in claim 31 is characterized in that described attenuation mutated viruses is incorporated as in described heterologous gene or gene fragment encoded protein or the albumen zone.

33. method as claimed in claim 31, it is characterized in that described embedded virus is to have recombination group or anti-genomic parainfluenza virus (PIV), described recombination group or anti-genome contain HN and at least one gene of F glycoprotein gene or the part or all of genome or the anti-genome of gene fragment bonded PIV kind, hypotype or a strain with allos PIV kind, hypotype or strain.

34. method as claimed in claim 33 is characterized in that the HN of described human PIV3 and F gene are replaced by HN and the F gene of human PIV1.

35. method as claimed in claim 31, it is characterized in that described embedded virus is to have recombination group or anti-genomic respiratory syncytial virus (RSV), described recombination group or anti-genome include F, G and at least one gene of SH glycoprotein gene or the part or all of genome or the anti-genome of gene fragment bonded RSV kind, hypotype or a strain with allogenic RSV kind, hypotype or strain.

36. method as claimed in claim 35 is characterized in that the F of described human RSV hypotype A and G glycoprotein are replaced by F and the G glycoprotein of human RSV hypotype B.

37. the method for claim 1 is characterized in that described recombination group or anti-genome are further modified, with one or more other attenuation sudden changes available from the sudden change minus-stranded rna virus that biologically obtains of encoding.

38. method as claimed in claim 37, it is characterized in that described recombinant negative strand rna virus is respiratory syncytial virus (RSV), and described recombination group or anti-genome encoding are present in one group of sudden change RSV strain that biologically obtains at least one until the sudden change of whole attenuation, and described group comprises cpts RSV 248 (ATCC VR 2450), cpts RSV 248/404 (ATCC VR 2454), cpts RSV 248/955 (ATCC VR 2453), cpts RSV 530 (ATCC VR 2452), cpts RSV 530/1009 (ATCC VR 2451), cpts RSV530/1030 (ATCC VR 2455), RSV B-1 cp52/2B5 (ATCC VR 2542) and RSV B-1 cp-23 (ATCC VR 2579).

39. method as claimed in claim 37 is characterized in that described recombinant negative strand rna virus is that parainfluenza virus (PIV) and described recombination group or anti-genome encoding are present among the HPIV3JS cp45 at least one until whole attenuation sudden changes.

40. method as claimed in claim 37 is characterized in that described recombination group or anti-genome comprise at least a attenuation sudden change, its change by a plurality of Nucleotide in the codon of this sudden change of special coding is stablized.

41. the method for claim 1, it is characterized in that described recombination group or anti-genome are further modified at the nucleotide modification of phenotypic alternation by carrying out specially, described phenotypic alternation is selected from growth characteristics, attenuation, temperature sensitivity, acclimatization to cold, little plaque size, host range is restricted or immunogenic change.

42. method as claimed in claim 41 is characterized in that described recombinant negative strand rna virus is respiratory syncytial virus (RSV), and described recombination group or anti-genome comprise the modification of SH, NS1, NS2 or G gene.

43. method as claimed in claim 42 is characterized in that described SH, NS1 or NS2 genetically deficient or this expression of gene are eliminated.

44. method as claimed in claim 42 is characterized in that described nucleotide modification comprises nucleotide deletion, insertion, increase or the rearrangement of the cis regulating and controlling sequence of selected RSV gene in recombination group or the anti-genome.

45. the method for claim 1 is characterized in that described recombinant negative strand rna virus is subviral particle.

46. a separated attenuation recombinant negative strand rna virus, it comprises:

Recombination group or anti-genome and produce the necessary viral protein of described recombinant negative strand rna viral infection particle, described recombination group or anti-genome are modified, with in the recombinant protein of the described recombinant virus of encoding corresponding to the sudden change on the amino acid position of the amino acid position of the attenuation sudden change of in allos sudden change minus-stranded rna virus, being differentiated, described sudden change is given described recombinant virus attenuation phenotype by introducing in described recombinant protein.

47. attenuation recombinant negative strand rna virus as claimed in claim 46 is characterized in that described recombinant negative strand rna virus is respiratory syncytial virus (RSV).

48. attenuation recombinant negative strand rna virus as claimed in claim 47 is characterized in that described RSV is human RSV hypotype A, human RSV hypotype B, ox RSV, mouse RSV or bird RSV.

49. attenuation recombinant negative strand rna virus as claimed in claim 47 is characterized in that described allos sudden change minus-stranded rna virus is respiratory syncytial virus (RSV), human parainfluenza virus (HPIV) 1, HPIV2, HPIV3, ox PIV (BPIV), Sendai virus (SeV), Avian pneumo-encephalitis virus (NDV), SV 41 virus (SV5), mumps virus (MuV), Measles virus (MeV), canine distemper virus (CDV), rabies virus (RaV) or vesicular stomatitis virus (VSV).

50. attenuation recombinant negative strand rna virus as claimed in claim 49 is characterized in that described allos sudden change minus-stranded rna virus is RSV cpts248 (ATCC VR 2450), RSVcpts248/404 (ATCC VR 2454), RSV cpts248/955 (ATCC VR 2453), RSV cpts530 (ATCC VR 2452), RSV cpts530/1009 (ATCC VR 2451), RSV cpts530/1030 (ATCC VR 2455), RSV B-1 cp52/2B5 (ATCC VR2542) or RSV B-1 cp-23 (ATCC VR 2579).

51. attenuation recombinant negative strand rna virus as claimed in claim 47, it is characterized in that described recombination group or anti-genome are modified, replace, lack or insert with the amino acid in coding RSV NS1, NS2, N, P, M, SH, M2 (ORF1), M2 (ORF2), L, F or the G gene.

52. attenuation recombinant negative strand rna virus as claimed in claim 47 is characterized in that the sudden change of introducing gives the phenotype of described recombinant negative strand rna virus temperature sensitivity (ts) in described recombinant protein.

53. attenuation recombinant negative strand rna virus as claimed in claim 47 is characterized in that the sudden change of introducing is included in the amino acid replacement in the RSV L albumen in described recombinant protein.

54. attenuation recombinant negative strand rna virus as claimed in claim 47 is characterized in that the amino acid that the attenuation sudden change of being differentiated is included on proteic 521 phenylalanines of L of human RSVcpts530 (ATCC VR 2452) replaces in described allos sudden change minus-stranded rna virus.

55. attenuation recombinant negative strand rna virus as claimed in claim 46 is characterized in that described recombinant negative strand rna virus is parainfluenza virus (PIV).

56. attenuation recombinant negative strand rna virus as claimed in claim 55 is characterized in that described PIV is human PIV1 (HPIV1), human PIV2 (HPIV2), human PIV3 (HPIV3), ox PIV (BPIV) or mouse PIV (MPIV).

57. attenuation recombinant negative strand rna virus as claimed in claim 55 is characterized in that described allos mutated viruses is respiratory syncytial virus (RSV), human parainfluenza virus (HPIV) 1, HPIV2, HPIV3, ox PIV (BPIV), Sendai virus (SeV), Avian pneumo-encephalitis virus (NDV), SV 41 virus (SV5), mumps virus (MuV), Measles virus (MeV), canine distemper virus (CDV), rabies virus (RaV) or vesicular stomatitis virus (VSV).

58. attenuation recombinant negative strand rna virus as claimed in claim 55 is characterized in that described recombination group or anti-genome are modified, and replaces, lacks or insert with the amino acid in coding PIV N, P, C, D, V, M, F, HN or the L albumen.

59. attenuation recombinant negative strand rna virus as claimed in claim 55 is characterized in that the sudden change of introducing gives described recombinant negative strand rna virus ts phenotype in described recombinant protein.

60. attenuation recombinant negative strand rna virus as claimed in claim 55 is characterized in that described allos sudden change minus-stranded rna virus is HPIV3 JS cp45.

61. attenuation recombinant negative strand rna virus as claimed in claim 60 is characterized in that the attenuation sudden change of being differentiated comprises the amino acid replacement that contains in the HPIV3 JScp45 L albumen in described allos sudden change minus-stranded rna virus.

62. attenuation recombinant negative strand rna virus as claimed in claim 60 is characterized in that the attenuation sudden change of being differentiated includes the amino acid replacement on proteic 942 tyrosine of HPIV3 JS cp45L in described allos sudden change minus-stranded rna virus.

63. attenuation recombinant negative strand rna virus as claimed in claim 60 is characterized in that the attenuation sudden change of being differentiated includes the amino acid replacement on 992 leucines of HPIV3 JS cp45L albumen in described allos sudden change minus-stranded rna virus.

64. attenuation recombinant negative strand rna virus as claimed in claim 60 is characterized in that the attenuation sudden change of being differentiated includes the amino acid replacement on 58 Threonines of HPIV3 JS cp45L protein 15 in described allos sudden change minus-stranded rna virus.

65. attenuation recombinant negative strand rna virus as claimed in claim 60 is characterized in that the attenuation sudden change of being differentiated includes the amino acid replacement in the HPIV3 JS cp45F albumen in described allos sudden change minus-stranded rna virus.

66. attenuation recombinant negative strand rna virus as claimed in claim 60 is characterized in that the attenuation sudden change of being differentiated includes the amino acid replacement on 0 Isoleucine of HPIV3 JS cp45F protein 42 in described allos sudden change minus-stranded rna virus.

67. attenuation recombinant negative strand rna virus as claimed in claim 60 is characterized in that the attenuation sudden change of being differentiated includes the amino acid replacement on 450 L-Ala of HPIV3 JS cp45F albumen in described allos sudden change minus-stranded rna virus.

68. attenuation recombinant negative strand rna virus as claimed in claim 55 is characterized in that the attenuation sudden change of being differentiated includes the replacement of 521 phenylalanines of human RSVcpts530 (ATCC VR 2452) L albumen in described allos sudden change minus-stranded rna virus.

69. as the described attenuation recombinant negative strand rna of claim 68 virus, it is characterized in that described recombinant negative strand rna virus is human PIV3 (HPIV3), and the amino acid that the described sudden change of introducing includes on proteic 456 phenylalanines of described HPIV3 L is replaced in described recombinant protein.

70. attenuation recombinant negative strand rna virus as claimed in claim 55 is characterized in that the attenuation sudden change of being differentiated includes the amino acid replacement on proteic 170 phenylalanines of SeV C in described allos sudden change minus-stranded rna virus.

71. as the described attenuation recombinant negative strand rna of claim 70 virus, it is characterized in that described recombinant negative strand rna virus is human PIV3 (HPIV3), and the amino acid that the described sudden change of introducing includes on proteic 164 phenylalanines of described HPIV3 C is replaced in described recombinant protein.

72. attenuation recombinant negative strand rna virus as claimed in claim 46 is characterized in that described recombinant negative strand rna virus is Measles virus (MeV).

73., it is characterized in that described allos mutated viruses is respiratory syncytial virus (RSV), human parainfluenza virus (HPIV) 1, HPIV2, HPIV3, ox PIV (BPIV), Sendai virus (SeV), Avian pneumo-encephalitis virus (NDV), SV 41 virus (SV5), mumps virus (MuV), Measles virus (MeV), canine distemper virus (CDV), rabies virus (RaV) or vesicular stomatitis virus (VSV) as the described attenuation recombinant negative strand rna of claim 72 virus.

74., it is characterized in that described allos sudden change minus-stranded rna virus is HPIV3 JS cp45 as the described attenuation recombinant negative strand rna of claim 72 virus.

75., it is characterized in that the attenuation sudden change of being differentiated includes the amino acid replacement on 2 tyrosine of HPIV3 JS cp45L protein 94 in described allos sudden change minus-stranded rna virus as the described attenuation recombinant negative strand rna of claim 74 virus.

76., it is characterized in that the attenuation sudden change of being differentiated includes the amino acid replacement on 992 leucines of HPIV3 JS cp45L albumen in described allos sudden change minus-stranded rna virus as the described attenuation recombinant negative strand rna of claim 74 virus.

77. the recombination group of a separated coding recombinant negative strand rna virus or anti-genomic polynucleotide molecule, described recombination group or anti-genome are modified, with the sudden change on the amino acid position of the amino acid position that suddenlys change corresponding to the attenuation of being differentiated in allos sudden change minus-stranded rna virus in the recombinant protein of the described virus of encoding, described sudden change is given described recombinant virus attenuation phenotype by introducing in described recombinant protein.

78. separated polynucleotide as claimed in claim 1 is characterized in that described recombinant negative strand rna virus is respiratory syncytial virus (RSV).

79., it is characterized in that described RSV is a human RSV hypotype A, human RSV hypotype B, ox RSV, mouse RSV or bird Pneumovirinae RSV as the described separated polynucleotide of claim 78.

80., it is characterized in that described allos sudden change minus-stranded rna virus is allogenic RSV, human parainfluenza virus (HPIV) 1, HPIV2, HPIV3, ox PIV (BPIV), Sendai virus (SeV), Avian pneumo-encephalitis virus (NDV), SV 41 virus (SV5), mumps virus (MuV), Measles virus (MeV), canine distemper virus (CDV), rabies virus (RaV) or vesicular stomatitis virus (VSV) as the described method of claim 78.

81. as the described separated polynucleotide molecule of claim 78, it is characterized in that described recombination group or anti-genome are modified, replace, lack or insert with the amino acid in coding RSV NS1, NS2, N, P, M, SH, M2 (ORF1), M2 (ORF2), L, F or the G albumen.

82., it is characterized in that the sudden change of introducing gives described recombinant negative strand rna virus temperature sensitivity (ts) phenotype in described recombinant protein as the described separated polynucleotide molecule of claim 78.

83., it is characterized in that the sudden change of introducing comprises the amino acid replacement in the RSV L albumen in described recombinant protein as the described separated polynucleotide molecule of claim 77.

84., it is characterized in that the amino acid that the attenuation sudden change of differentiating is included on proteic 521 phenylalanines of L of human RSV cpts530 (ATCC VR 2452) replaces in described allos sudden change minus-stranded rna virus as the described separated polynucleotide molecule of claim 83.

85., it is characterized in that described recombinant negative strand rna virus is parainfluenza virus (PIV) as the described separated polynucleotide molecule of claim 77.

86., it is characterized in that described PIV is human PIV1 (HPIV1), human PIV2 (HPIV2), human PIV3 (HPIV3), ox PIV (BPIV) or Sendai virus (SeV) as the described separated polynucleotide molecule of claim 85.

87., it is characterized in that described allos mutated viruses is respiratory syncytial virus (RSV), human parainfluenza virus (HPIV) 1, HPIV2, HPIV3, ox PIV (BPIV), Sendai virus (SeV), Avian pneumo-encephalitis virus (NDV), SV 41 virus (SV5), mumps virus (MuV), Measles virus (MeV), canine distemper virus (CDV), rabies virus (RaV) or vesicular stomatitis virus (VSV) as the described separated polynucleotide molecule of claim 85.

88. as the described separated polynucleotide molecule of claim 85, it is characterized in that described recombination group or anti-genome are modified, replace, lack or insert with the amino acid in coding PIV N, P, C, D, V, M, F, HN or the L albumen.

89., it is characterized in that the sudden change of introducing gives described recombinant negative strand rna virus temperature sensitivity (ts) phenotype in described recombinant protein as the described separated polynucleotide molecule of claim 85.

90., it is characterized in that described allos sudden change minus-stranded rna virus is HPIV3 JS cp45 as the described separated polynucleotide molecule of claim 85.

91., it is characterized in that the attenuation sudden change of being differentiated includes the amino acid replacement in the HPIV3 JS cp45L albumen in described allos sudden change minus-stranded rna virus as the described separated polynucleotide molecule of claim 90.

92., it is characterized in that the attenuation sudden change of being differentiated includes the amino acid replacement on 2 tyrosine of HPIV3 JS cp45L protein 94 in described allos sudden change minus-stranded rna virus as the described separated polynucleotide molecule of claim 91.

93., it is characterized in that the attenuation sudden change of being differentiated includes the amino acid replacement on 992 leucines of HPIV3 JS cp45L albumen in described allos sudden change minus-stranded rna virus as the described separated polynucleotide molecule of claim 91.

94., it is characterized in that the attenuation sudden change of being differentiated includes the amino acid replacement on 58 Threonines of HPIV3 JS cp45L protein 15 in described allos sudden change minus-stranded rna virus as the described separated polynucleotide molecule of claim 91.

95., it is characterized in that the attenuation sudden change of being differentiated includes the amino acid replacement in the HPIV3 JS cp45F albumen in described allos sudden change minus-stranded rna virus as the described separated polynucleotide molecule of claim 90.

96., it is characterized in that the attenuation sudden change of being differentiated includes the amino acid replacement on 0 Isoleucine of HPIV3 JS cp45F protein 42 in described allos sudden change minus-stranded rna virus as the described separated polynucleotide molecule of claim 95.

97., it is characterized in that the attenuation sudden change of being differentiated includes the amino acid replacement on 450 L-Ala of HPIV3 JS cp45F albumen in described allos sudden change minus-stranded rna virus as the described separated polynucleotide molecule of claim 95.

98., it is characterized in that the attenuation sudden change of being differentiated includes the amino acid replacement on 521 phenylalanines of human RSVcpts530 (ATCC VR 2452) L albumen in described allos sudden change minus-stranded rna virus as the described separated polynucleotide molecule of claim 85.

99. as the described separated polynucleotide molecule of claim 98, it is characterized in that described recombinant negative strand rna virus is human PIV3 (HPIV3), and the amino acid that the described sudden change of introducing at described recombinant protein is included on proteic 456 phenylalanines of L of described HPIV3 is replaced.

100., it is characterized in that the attenuation sudden change of being differentiated includes the replacement of 0 phenylalanine of SeV C protein 17 in described allos sudden change minus-stranded rna virus as the described separated polynucleotide molecule of claim 85.

101. as the described separated polynucleotide molecule of claim 100, it is characterized in that described recombinant negative strand rna virus is human PIV3 (HPIV3), and the amino acid that the sudden change of introducing is included on 4 phenylalanines of C protein 16 of described HPIV3 is replaced in this recombinant protein.

102., it is characterized in that described recombinant negative strand rna virus is Measles virus (MeV) as the described separated polynucleotide molecule of claim 77.

103., it is characterized in that described allos mutated viruses is respiratory syncytial virus (RSV), human parainfluenza virus (HPIV) 1, HPIV2, HPIV3, ox PIV (BPIV), Sendai virus (SeV), Avian pneumo-encephalitis virus (NDV), SV 41 virus (SV5), mumps virus (MuV), Measles virus (MeV), canine distemper virus (CDV), rabies virus (RaV) or vesicular stomatitis virus (VSV) as the described separated polynucleotide molecule of claim 102.

104., it is characterized in that described allos sudden change minus-stranded rna virus is HPIV3 JS cp45 as the described separated polynucleotide molecule of claim 102.

105., it is characterized in that the attenuation sudden change of being differentiated includes the amino acid replacement on 2 tyrosine of HPIV3JS cp45 L protein 94 in described allos sudden change minus-stranded rna virus as the described separated polynucleotide molecule of claim 103.

106., it is characterized in that the attenuation sudden change of being differentiated includes the amino acid replacement on 992 leucines of HPIV3 JScp45 L albumen in described allos sudden change minus-stranded rna virus as the described separated polynucleotide molecule of claim 103.

107. as the described separated polynucleotide molecule of claim 77, it is characterized in that described recombinant negative strand rna virus is to have recombination group or anti-genomic embedded virus, described recombination group or anti-genome comprise and the heterologous gene of another minus-stranded rna virus kind, hypotype or strain or the part or all of genome or the anti-genome of gene fragment bonded minus-stranded rna virus kind, hypotype or a strain.

108., it is characterized in that described attenuation sudden change is introduced in in described heterologous gene or gene fragment encoded protein or the albumen zone as the described separated polynucleotide molecule of claim 107.

109. as the described separated polynucleotide molecule of claim 107, it is characterized in that described embedded virus is to have recombination group or anti-genomic parainfluenza virus (PIV), described recombination group or anti-genome contain HN and at least one gene of F glycoprotein gene or the part or all of genome or the anti-genome of gene fragment bonded PIV kind, hypotype or a strain with allogenic PIV kind, hypotype or strain.

110., it is characterized in that the HN of described human PIV3 and F gene are replaced by HN and the F gene of human PIV1 as the described separated polynucleotide molecule of claim 109.

111. as the described separated polynucleotide molecule of claim 107, it is characterized in that described embedded virus is to have recombination group or anti-genomic respiratory syncytial virus (RSV), described recombination group or anti-genome contain F, G and at least one gene of SH glycoprotein gene or the part or all of genome or the anti-genome of gene fragment bonded RSV kind, hypotype or a strain with allos RSV kind, hypotype or strain.

112., it is characterized in that the F of described human RSV hypotype A and G glycoprotein are replaced by F and the G glycoprotein gene of human RSV hypotype B as the described separated polynucleotide molecule of claim 111.

113. as the described separated polynucleotide molecule of claim 77, it is characterized in that described recombination group or anti-genome are further modified, pick up from the one or more additional attenuation sudden change of the sudden change minus-stranded rna virus that biologically obtains with coding.

114. as the described separated polynucleotide molecule of claim 113, it is characterized in that described recombinant negative strand rna virus is respiratory syncytial virus (RSV), and described recombination group or anti-genome encoding are present in one group of sudden change RSV strain that biologically obtains at least one until the sudden change of whole attenuation, and described group comprises cpts RSV 248 (ATCC VR 2450), cpts RSV 248/404 (ATCC VR 2454), cpts RSV 248/955 (ATCC VR2453), cpts RSV 530 (ATCC VR 2452), cpts RSV 530/1009 (ATCCVR 2451), cpts RSV 530/1030 (ATCC VR 2455), RSV B-1 cp52/2B5 (ATCC VR 2542) and RSV B-1 cp-23 (ATCC VR 2579).

115. as the described separated polynucleotide molecule of claim 113, it is characterized in that described recombinant negative strand rna virus is parainfluenza virus (PIV), and described recombination group or anti-genome encoding are present among the HPIV JS cp45 at least one until whole attenuation sudden changes.

116. as the described separated polynucleotide molecule of claim 77, it is characterized in that described recombination group or anti-genome comprise at least a attenuation sudden change, its change by a plurality of Nucleotide in the codon of special this sudden change of coding is stabilized.

117. as the described separated polynucleotide molecule of claim 77, it is characterized in that described recombination group or anti-genome by further being modified at the nucleotide modification of phenotypic alternation specially, described phenotypic alternation is selected from growth characteristics, attenuation, temperature sensitivity, acclimatization to cold, little plaque size, host range is restricted or immunogenic change.

118. as the described separated polynucleotide molecule of claim 117, it is characterized in that described recombinant negative strand rna virus is respiratory syncytial virus (RSV), and described recombination group or anti-genome contain the modification of SH, NS1, NS2 or G gene.

119., it is characterized in that described SH, NS1 or NS2 genetically deficient or this expression of gene are eliminated as the described separated polynucleotide molecule of claim 117.

120., it is characterized in that described nucleotide modification comprises nucleotide deletion, insertion, increase or the rearrangement of the cis regulating and controlling sequence of selected RSV gene in described recombination group or the anti-genome as the described separated polynucleotide molecule of claim 117.

121. expression vector, it contains recombination group or anti-genomic polynucleotide molecule and a transcription terminator of a transcripting promoter that can be operatively connected, a coding recombinant negative strand rna virus, it is characterized in that described recombination group or anti-genome are modified, with the sudden change on the amino acid position of the amino acid position that suddenlys change corresponding to the attenuation of being differentiated in the allos minus-stranded rna virus in the recombinant protein that is coded in described virus, described sudden change is given described recombinant virus attenuation phenotype by being incorporated in the described recombinant protein.

122. a method that stimulates the individual immunity system with the protective reaction of the sick plain poison of reactance strand RNA, it comprises and gives on individual and the physiology attenuation recombinant negative strand rna virus as claimed in claim 46 of q.s in the immunity of acceptable carrier bonded.

123., it is characterized in that the dosed administration of described attenuation recombinant negative strand rna virus with 103 to 106 PFU as the described method of claim 122.

124., it is characterized in that described attenuation recombinant negative strand rna virus is respiratory syncytial virus (RSV) or parainfluenza virus (PIV) as the described method of claim 122.

125. an immunoreactive immunogenic composition that brings out anti-minus-stranded rna virus, comprise with physiology on the attenuation recombinant negative strand rna virus as claimed in claim 46 of q.s in the immunity of acceptable carrier bonded.

126., it is characterized in that attenuation reorganization negative strand viruses is respiratory syncytial virus (RSV) or parainfluenza virus (PIV) as the described immunogenic composition of claim 122.

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