CN101528254A - Construction of recombinant virus vaccines by direct transposon-mediated insertion of foreign immunologic determinants into vector virus proteins - Google Patents

Construction of recombinant virus vaccines by direct transposon-mediated insertion of foreign immunologic determinants into vector virus proteins Download PDF

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CN101528254A
CN101528254A CNA2007800342740A CN200780034274A CN101528254A CN 101528254 A CN101528254 A CN 101528254A CN A2007800342740 A CNA2007800342740 A CN A2007800342740A CN 200780034274 A CN200780034274 A CN 200780034274A CN 101528254 A CN101528254 A CN 101528254A
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virus
viral
carrier
peptide
banzi virus
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K·V·普加乔夫
A·A·鲁缅采夫
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Sanofi Pasteur Biologics LLC
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Acambis Inc
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Abstract

The invention provides viral vectors, such as chimeric flavivirus vectors, including foreign peptides inserted into the target proteins of the vectors, methods of making and using these vectors, and compositions including the vectors.

Description

By direct transposon-mediated insertion of foreign immunologic determinants construction of recombinant virus vaccine in vector virus albumen
Invention field
The present invention relates to by direct transposon-mediated insertion of foreign immunologic determinants construction of recombinant virus vaccine and corresponding compositions and method in vector virus albumen.
Background of invention
Inoculation is one of achievement of medical domain maximum, makes the millions of people avoid the effect of crushing disease.Before vaccine is extensive use of, only in the U.S. every year have thousands of child and adult to die from infectious disease, more in the world wide.Inoculation is widely used in preventing and treating the infection that antibacterial, virus and other pathogen cause.In inoculation, use several diverse ways comprise to the pathogen of killing, attenuated live pathogen and inactive pathogen subunit.In viral infection, found that live vaccine can bring potent and the most lasting protective immune response.
Researched and developed the attenuated live vaccine of anti-flavivirus, they are general by infected mosquito and the little tunicary positive chain RNA virus of tick-borne volume.The jaundice seed culture of viruses of flaviviridae comprises about 70 kinds of viruses, major part wherein for example yellow fever (YF), dengue fever (DEN), Japanese encephalitis (JE) and tick encephalitis (TBE) is that main human pathogen is (with reference to Burke and Monath, Fields Virology, the 4th edition: 1043-1126,2001).
In the vaccine of research and development banzi virus, used diverse ways.For example two kinds of vaccines (the nerve vaccine is had a liking for by yellow hot 17D and France) (Monath, " Yellow Fever, " Plotkin and Orenstein have been researched and developed by continuous passage for yellow fever virus, Vaccines, the 3rd edition, Saunders, Philadelphia, 815-879 page or leaf, 1999).The banzi virus method of attenuating that another kind is used to inoculate relates to the structure chimeric flavirirus, and it comprises the component of two kinds of (or more) different banzi virus.How understanding makes up these chimeras need be explained the genomic structure of banzi virus.
Banzi virus albumen generates sophisticated virus protein generation (Amberg etc. by the posttranslational protein hydrolytic rupture that host and virus protease make up a series of complexity of this polyprotein by translating the single long frame of reading then to generate polyprotein, J.Virol.73:8083-8094,1999; Rice, " Flaviviridae, " In Virology, Fields (editor), Raven-Lippincott, NewYork, 1995, the I volume, the 937th page).In polyprotein, wherein " C " is capsid to these viral structural protein with the sequence arrangement of C-prM-E, and " prM " is the proteic precursor of peplos binding film (M), and " E " is envelope protein.These protein are present in the N end regions of polyprotein and non-structural protein (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) is positioned at the C end regions of this polyprotein.
Prepared and comprised from the structure of different banzi virus and the chimeric flavirirus of non-structural protein.For example so-called ChimeriVax TMTechnology used the capsid of yellow fever virus 17D and envelope protein (prM and E) that non-structural protein transmits other banzi virus (referring to, for example, Chambers etc., J.Virol.73:3095-3101,1999).This technology be used to research and develop anti-dengue, Japanese encephalitis (JE), Xi Niluo (WN) and St. Louis encephalitis (SLE) virus candidate vaccine (referring to, for example, Pugachev etc., New Generation Vaccines, the third edition, editors such as Levine, Marcel Dekker, New York, Basel, the 559-571 page or leaf, 2004; Chambers etc., J.Virol.73:3095-3101,1999; Guirakhoo etc., Virology 257:363-372,1999; Monath etc., Vaccine 17:1869-1882,1999; Guirakhoo etc., J.Virol.74:5477-5485,2000; Arroyo etc., Trends Mol.Med.7:350-354,2001; Guirakhoo etc., J.Virol.78:4761-4775,2004; Guirakhoo etc., J.Virol.78:9998-10008,2004; Monath etc., J.Infect.Dis.188:1213-1230,2003; Arroyo etc., J.Virol.78:12497-12507,2004; With Pugachev etc., Am.J.Trop.Med.Hyg.71:639-645,2004).
Based on ChimeriVax TMVaccine demonstrated more favorable properties, for example with regard to following character: in the substrate cell, duplicate, in mouse model low neurotoxicity, can in mice, monkey and people, induce potent protective immunity after high heredity and phenotypic stability, invalid the duplicating in mosquito (the uncontrollable propagation for the prevention occurring in nature is extremely important) and single dose give in high attenuation, the external and body in the monkey model and can not cause immunity back side effect.Really, ChimeriVax TM-JE vaccine virus comprises the prM-E gene from SA14-14-2 JE virus (the attenuated live JE that uses in China), has successfully carried out clinical preceding and I phase and II clinical trial phase (Monath etc., Vaccine 20:1004-1018,2002; Monath etc., J.Infect.Dis.188:1213-1230,2003).Similarly, to ChimeriVax TM-WN candidate vaccine has carried out successful I clinical trial phase, and it comprises the prM-E sequence from west nile virus, has mixed 3 special amino acid changes to strengthen attenuation (Arroyo etc., J.Virol.78:12497-12507,2004) in E albumen.
Carried out other method of attenuating, for example banzi virus mutation comprises chimeric flavirirus.These methods for example comprise in envelope protein, introduce replace, (example of these sudden changes is referring to below with reference to document: Men etc., J.Virol.70:3930-3937,1996 for the disappearance in 3 ' the untranslated district and the disappearance in the capsid protein; Mandl etc., J.Virol.72:2132-2140,1998; Durbin etc., AJTMH 65:405-413,2001; Pletnev, Virology 282:288-300,2001; Markoff etc., J.Virol.76:3318-3328,2002; Kofler etc., J.Virol.76:3534-3543,2002; Whitehead etc., J.Virol.77:1653-1657,2003; Pletnev etc., Virology 314:190-195,2003; Pugachev etc., Int.J.Parasitol.33:567-582,2003; Bredenbeek etc., J.Gen.Virol.84:1261-1268,2003; United States Patent (USP) the 6184024th B1 number; WO 02/095075; WO 03/059384; WO03/092592; WO 03/103571; WO 2004/045529; With WO 2006/044857).In other method, use transposon to detect this ChimeriVax TMThe site is inserted in the permission of-JE envelope protein E.According to this method, the insertion transposon of the alternative mutated viruses of living of usefulness expection exogenous peptide (referring to, for example WO 02/102828).
Mason and its colleague have delivered flavirirus vaccines (RepliVax) construction method (Mason etc., Virology 351:432-443,2006) based on false infectious viral particle (PIV) recently.In banzi virus PIVs (having described YF 17D and WN virus so far), this capsid protein gene disappearance is except occupying 5 ' cyclisation signal sequence of about 20 N end of C codon.In with the proteic cell of the trans C of providing, breed PIV.It is essential that C albumen is packaged in progeny virus (PIV) granule for PIV.The PIVs that has packed in the collecting cell culture supernatant also duplicates vaccine as the monocycle, and it can induce potent antibody response (because secretion of empty virion) and almost complete t cell response arm store.The potent part of this method is because banzi virus (for example, YF 17D) and PIV infect dendritic cell and activate ability (Palmer etc., J.Gen.Virol.88:148-156,2007 that multiple TLR approach enhance immunity is replied; Querec etc., J.E.M.203:413-424,2006).
Except the vaccine that infects as anti-flavivirus, proposed to use banzi virus for example chimeric flavirirus as transmitting the antigenic carrier of other non-banzi virus.In an embodiment of this use, the rational method that inserts exogenous peptide in YF17D envelope protein E has been described, this is based on the understanding to banzi virus granule quarternary structure, go into (Rey etc. in the electron density map as freezing electron-microscopic analysis and with the match of known E albumen dimer x-ray structure, Nature375:291-298,1995; Kuhn etc., Cell 108:717-725,2002).The trimerical three dimensional structure of E albumen (Modis etc., Nature 427:313-319,2004 of merging the back conformation have been analyzed; Bressanelli etc., EMBO J.23:728-738,2004).Galler and its colleague detected E albumen dimer and trimerical 3D structure and the Dimerized domain II of inference the fg ring should with dimer and trimer conformation expose with solvent in.They use and insert the body fluid and the t cell epitope of malaria in the E albumen of this hoop YF17D virus and reclaim some live mutant (Bonaldo etc., J.Virol.79:8602-8613,2005; Bonaldo etc., J.Mol.Biol.315:873-885,2002; WO 02/072835).Yet use this method can not guarantee that selected site allow/is suitable for inserting the exogenous peptide (some data such as Galler prove) of each expection most with regard to effective virus replication, immunogenicity and stability.And this method is not useable for the virus protein (for example, the prM/M of banzi virus, NS1 and other NS albumen of great majority) of 3D structure the unknown.
In additive method, if in virus O RF, insert between gene then can in the banzi virus carrier, express the former albumen/peptide of foreign immunologic.For example, Andino and colleague thereof attempt expressing in several sites of YE17D virus polyprotein the model 8-aminoacid antitumor CTL epitope of side joint virus N S2B/NS3 protease cracking site, for example NS2B/NS1 connects (McAllister etc., J.Virol.74:9197-9205,2000).Other people have used the immundominance t cell epitope (Barba-Spaeth etc., J.Exp.Med.202:1179-1184,2005) of NS2B/NS1 site expression of influenza virus.NS2B-NS3 connection in YF 17D virus such as Tao has been expressed the 10-aminoacid CTL epi-position of malarial parasite and has been demonstrated attacked the good protection (Tao etc., J.Exp.Med.201:201-209,2005) of mice by parasite.We connect the M2e peptide (United States serial 60/900672) of having expressed influenza virus at E/NS1 recently, and Bredenbeek also success connects the glycoprotein precursor (Bredenbeek etc., Virology345:299-304,2006) of having expressed Lassa virus at E/NS1.Also can use other genes to connect.In additive method, bicistronic mRNA is expressed exogenous peptide (for example, in 3 ' UTR).In additive method, the monocycle banzi virus has been developed to candidate's recombiant vaccine of anti-various pathogen, and has verified immunogenicity potential quality (Jones etc., Virology 331:247-259,2005 that recombinant replication; Molenkamp etc., J.Virol.77:1644-1648,2003; Westaway etc., Adv.Virus.Res.59:99-140,2003; Herd etc., Virology 319:237-248,2004; Harvey etc., J.Virol.77:7796-7803,2003; Anraku etc., J.Virol.76:3791-3799,2002; Varnavski etc., J.Virol.74:4394-4403,2000).In replicon, disappearance prM and E envelope protein gene or C-prM-E gene.Therefore, it can be in time multiplexed cell system but can not generate daughter of virus (therefore duplicating for the monocycle).It can be packaged in the virion when with trans prM-E of providing or C-prM-E gene.Interested exogenous antigen can suitably insert the disappearance position.With regard to RepliVax, duplicated for the monocycle immunity back, can further not diffuse to peripheral cell/tissue, obtains the immunne response to the heteroantigen of having expressed.Perhaps can carry out immunity by the replicon of inoculation naked DNA or rna form.It is former to express foreign immunologic in additive method in RepliVaxPIVs, for example replaces the C gene (Mason etc., Virology351:432-443,2006) of disappearance.
Stream emits background knowledgeStream emits immunogen to be used as model antigen in this application.Influenza virus is the main inducing of whole world acute respiration disease.Only can cause more than 100000 people and be in hospital and 20000 to 40000 people's death (Brammer etc., MMWRSurveill.Summ.51:1-10,2002:Lui etc., Am.J.Public Health 77:712-6,1987 in the outburst in U.S. every year; Simonsen, Vaccine 17:S3-10,1999; Thompson etc., JAMA289:179-186,2003).Because the child of annual influenza world wide interior about 20% and 5% adult are sick.There is the influenza A virus of three kinds of hypotypes in the crowd, to propagate in history, H1N1, H2N2 and H3N2.Since nineteen sixty-eight, almost had only H1N1 and H3N2 to propagate (Hilleman, Vaccine 20:3068-3087,2002; Nicholson etc., Lancet 362:1733-1745,2003; Palese etc., J.Clin.Invest.110:9-13,2002).Influenza B virus, it has only a kind of definite hypotype, also propagates in the mankind, but only causes the disease slighter than influenza A virus usually.Present inactivated vaccine comprises three kinds of components, based on H1N1 and the A strain of H3N2 influenza and the influenza B strain (Palese etc., J.Clin.Invest.110:9-13,2002) of screening.The periodicity pandemic disease, for example H1N1 pandemic disease in 1918 can cause millions of people's death.Influenza expert thinks that another kind of flu outbreak disease is inevitable and possibility coming (Webby and Webster, Science 302:1519-1522,2003).At present outburst-the Shi of H5N1 bird flu last maximum once, by the high killer strain of the mankind is caused-may (by making a variation and/or genetic recombination) become the diseased plant of being very popular that can cause serious consequence.In Holland thrilling situation again taking place in 2003, has broken out highly pathogenic H7N7 bird flu in the aviculture worker.Other hypotypes that can cause pandemic disease are H9 and H6 virus.Though the virulence than H5 and H7 is little, the two is transmitted to poultry from aquatic bird in 10 years in the past.In addition, in pig and people, detected H9N2 virus (Webby and Webster, Science302:1519-1522,2003).Although in the past in the several years to a large amount of concerns of avian viruses, people still feel worry to traditional H1, H2 and H3 subtype virus because since new antigenicity not the introducing of homophyletic high virulence may appear.For example, H2 virus belongs to the excessive risk classification, because they are inducements of nineteen fifty-seven " Asia " influenza pandemic, and circulates in wild duck and tame duck constantly.
The strategy of prevention at present and control influenza disease carries out immunity for annual at the Strain that may propagate then.The most approved influenza vaccines are formed by chicken embryo production and by the complete virus particle or the partially purified viral sub-units (" fragment " vaccine) of deactivation.These vaccines are 70 to 90% effective (Beyer etc., Vaccine 20:1340-1353,2002) in the normal health adult.Yet the effectiveness of the antagonism disease in the old people is lower.There are the attenuated live vaccine of intranasal administration in the U.S. and the former Soviet Union, and also (the 3rd edition .Levine edits, M.M.New York, Basel:Marcel Dekker for Treanor etc., In:New GenerationVaccines by chicken embryo production for it; The 537-557 page or leaf, 2004).U.S.'s vaccine (
Figure A20078003427400121
) approval be used for child below 5 years old and the people more than 55 years old, they but are the main target crowds of influenza vaccines.Because the main influenza hemagglutinin of immune system recognition and neuraminic acid pheron are through sudden change and reset constantly and change, vaccine component also needs to change the antigenicity feature that is about to popular Strain with reflection every year.Therefore existing vaccine must annually prepare, and prepares and can not store before season in influenza to be used for pandemic disease.In addition, use the efficient of Embryo Gallus domesticus protein production very low.Every egg can only be produced the inactivated vaccine of 1 to 2 people's dosage.It is the bottleneck of producing traditional vaccine at present that enough bioclean eggs are provided.Even the annual influenza vaccines (Gerdil, Vaccine 21:1776-1779,2003) that during pandemic disease, need to produce in 6 months q.s usually.Just making great efforts to carry out multiple research and development in cell culture, to produce influenza vaccines.Yet also there are many challenges in this method, especially uses unauthorized cell line.No matter being to use egg or cell culture to carry out production of vaccine all must use reverse genetics and hereditary rearrangement method and will expect that the new epidemic diseases strain of producing vaccine changes into the production strain that reproducible enough titres are used to produce.All these characteristics influenzas relevant with the conventional flow influenza vaccine are all inapplicable under the situation of pandemic disease.
Based on reorganization hemagglutinin (HA) or research and develop novel influenza vaccines by the HA of adenovirus or α viral and can enhance productivity, but can not solve the problem and the annual requirement that rebuilds vaccine of annual genetic shift.
Generally speaking, generally acknowledged present influenza vaccines need be faced following challenge:
1. the poor efficiency with regard to vaccine and Strain coupling difference; The restriction of the suitable cold mould virus of living to the range of age.
2. need annual tissue regeneration promoting vaccine to change with the antigen that solves virus.
3. vaccine yields poorly.
4. make up the time that suitable rearrangement virus is used to produce needs.
5. inefficiency of production is to satisfy the needs of pandemic disease.
6. the bio-safety problem in the large-scale production inactivation antigen virus.
7. to suitable cold mould viral vaccine attenuation deficiency causes untoward reaction (Treanor etc. among the egg product vaccinate hypersensitive or because some are lived, In:New Generation Vaccines, the 3rd edition .Levine edits, M.M.New York, Basel:Marcel Dekker; The 537-557 page or leaf, 2004).
" Holy grail " that influenza vaccines are learned single product for causing that but the wide spectrum to all influenza strains, long-term protective immunity and high yield production, cost are low, can store.
The virucidin that all effective conventional flow influenza vaccines can cause anti-HA, it has represented immune relevant protection at present.But the immunogenicity of HA all can change every year.In recent years, other influenza virus proteins also attract people's attention to become vaccine targets.M2 albumen is that the extracellular region domain (M2e) of M2 is high conservative in influenza A virus by it.Fig. 9 A shown we to before with the nearest people and the comparison (from http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU/html) of fowl M2e sequence.The M2e conservative domain between the human influenza virus not only, avian viruses M2e sequence are also closely arranged (closely aligned).The conservative proteic N end parts of M2e that is positioned at of the sequence of top level.Therefore it should be noted that very that N 13 aminoacid of end (dash area in the comparison) that shown the M2e peptide are main cause (Liu etc., FEMS Immunol.Med.Microbiol.35:141-146,2003 of inducing protection antibody; Liu etc., Immunol.Lett.93:131-136,2004; Liu etc., Microbes.Infect.7:171-177,2005).This causes the notion and the hope of general influenza A virus vaccine.
M2e has represented the outer 23-amino acid moiety of M2, and it is the sub-fraction of virus protein.Although not remarkable in influenza virus particles, M2 is abundant at the surface expression of virus-infection cell.But, when in common influenza infection or with traditional vaccine, inoculating, very little to the antibody response of M2 or M22 determinant.And, be presented at for the proteic non-viral neutralizing monoclonal antibody of M2 in the dead mouse model of influenza of passive transfer and have protectiveness (Fan etc., Vaccine 22:2993-3003,2004; Mozdzanowska etc., Vaccine 21:2616-2626,2003; Treanor etc., J.Virol.64:1375-1377,1990).Based on these results, many vaccine developers have great interest to M2 and its high conservative M2e domain as influenza A vaccine component.
Do not neutralize virus but fully reduce effective virus replication of M2 or M2e antibody to be protected from symptomatic disease.Generally acknowledge that the protection mechanism that is caused by M2 relates to the cell-mediated antibody dependent cellular cytotoxicity of NK (ADCC).The antibody of M2e extracellular region domain (mainly being the IgG2a subclass) can be discerned the epi-position of being showed on the virus infected cell, and it has determined to eliminate infection cell (Jegerlehner etc., J.Immunol.172:5598-1605,2004) by the NK cell.Because the immunity that is caused by M2 is the property killed not, there is limited virus replication after infecting, it can be used for activating the broad spectrum influenza immunne response.Theoretically speaking, this can cause to after run into identical virus or the longer stronger immunological memory of allos strain and better protection (third edition .Levine edit for Treanor etc., In:New Generation Vaccines, M.M.New York, Basel:Marcel Dekker; The 537-557 page or leaf, 2004).
Walter Fiers and colleague thereof (Ghent University, Belgium) are the pioneer of checking based on the vaccine potential quality of M2e.They to hepatitis B virus core albumen, can obtain M2e submission (HBc) (Fiers etc., Virus Res.103:173-176,2004 on the hepatitis B virus core protein surface with M2e determinant gene fusion when expressing in antibacterial; Neirynck etc., Nat.Med.5:1157-1163,1999).These HBc-M2e granules demonstrate immunogenicity and have protectiveness in the influenza viruse attack models of each species in mice and ferret.
Another kind of conservative influenza virus domain is the ripe cracking site HA of HA precursor protein 0Its high conservative (Macken etc., Osterhaus, A.D.M.E., Cox, N., Hampson A.W. edits, Options for the control of influenza IV.ElsevierScience, Amsterdam, The Netherlands, the 103-106 page or leaf, 2001) cause by two functional restraints.At first, this sequence must keep the suitable substrate of host protein enzyme to discharge two sophisticated HA subunits, HA 1And HA 2Secondly, HA 2N end comprise for infect very important fusogenic peptide (Lamb and Krug, the 4th edition .Knipe of In:Fields Virology. edits D.M., Howley, P.M., Griffin, D.E., wait Philadelphia:LippincottWilliams and Wilkins; The 1043-1126 page or leaf, 2001).Fusogenic peptide is all guarded in influenza A and B virus.In nearest report, Bianchi and colleague thereof (Bianchi etc., J.Virol.79:7380-7388,2005) verified influenza B virus in conjunction with HA 0Cleavage of peptide can cause the protective immunity of the different influenza B virus of antigenicity pedigree deadly attack in mice.It should be noted that in conjunction with A/H3/HA 0Peptide also can be protected the attack of being avoided influenza B virus by immune mouse.Arginine (last HA before the cracking site is guarded in-1 strictness 1Residue) and+3 and+9 phenylalanine residue (HA 2The the 3rd and the 9th residue) for monoclonal antibody in conjunction with extremely important.Therefore, the conservative C end parts of peptide shows relevant with protection, shows that preparation is based on HA 0The probability of the universal A of cracking domain and B human influenza virus vaccine.We are to people (H1, H2, H3 and B) HA 0With all available bird flu HA 0Sequence ( Http:// www.ncbi.nlm.nih.gov/genomes/FLU/FLU/html) comparison obtain the consensus sequence shown in Fig. 9 B (adding shade) for antibodies and the important zone of immunogenicity.
Recently, applicable comprehensive online database (The Immune Epitope Database and Analysis Resources (IEDB)) of catching various epi-position data (www.immuneepitope.org).These data of analysis-by-synthesis also identify many cross protection influenza epi-positions, its applicable to make up general vaccine (Bui etc., Proc.Natl.Acad.Sci.U.S.A.104:246-251,2007 and supplementary tables).Identified the two possible epi-position (the HA protective epitope who comprises the H3N2 that we use below) of B cell and T cell.Most B cell epitopes have conformation, so bulky.Yet, can by the application describe directly at random insertion identify the permission site of these longer epi-positions, for example in the secretory protein of ChimeriVax virus.We find is used for the more segmental site that highly allows of shorter insertion and can allows the long fragment (for example, in the prM of ChimeriVax-JE albumen, seeing below) of inserting.
People are studying various M2e subunit vaccine methods, comprise peptide conjugate and epitope display granule.Yet these methods need potent adjuvant to strengthen immunogenic immunogenicity a little less than these.This is with regard to M2e (and HA probably 0) especially it is important.Because the protection mechanism (ADCC) that is proposed needs high-caliber specific antibody to reach effectiveness.Generally believe that normal serum IgG competes the Fc receptor with special (anti-M2e) IgG on the NK cell, it is main mediation of protection.Therefore the alternative method that needs the general pandemic influenza vaccine of research and development.Suitable epi-position/antigenic availability of the medical importance of above-mentioned influenza, the needs that improve general influenza vaccines and influenza virus provides an example of important pathogen, and the method that can use the application to describe is produced the novel vaccine of anti-this pathogen.The method that the application describes can be applied to the structure of the new/improvement vaccine of anti-other pathogen with being equal to, and is as mentioned below.
Summary of the invention
The invention provides the virus genomic method that comprises the one or more heterologous peptides of one or more codings that generates.These methods may further comprise the steps: one or more target viral genes (for example, in one or more shuttle vectors or in intact virus genome background (context)) (i) are provided; (ii) mutation target viral gene is to insert the site at random; And the nucleic acid molecules of the heterologous peptides of (iii) will encoding is connected in the site at random of target viral gene mutation.This method can further comprise step (iv) with genomic nucleic acids library transfectional cell to start virus replication, (v) screening (effectively duplicating) the viral recon of living makes the effectively peptide of submission insertion then.When carrying out under the background at one or more shuttle vectors, this method can further comprise introduces in the viral genome of this target viral gene of can deriving the target viral gene that comprises the nucleic acid molecules storehouse of the heterologous peptides of encoding to substitute the corresponding step of inserting segmental viral gene of not having.
The inventive method also comprises by viral genome being introduced cell (for example Vero cell) and generates viral vector and isolated viral carrier from cell or its supernatant from viral genome.In addition, the target viral gene that is used for the inventive method can or not have from the virus that is used for this method (or comprise insert fragment by additive method) before and inserts segmental virus and obtain.
The inventive method also comprise mutation two or more (for example, 2,3,4 or more a plurality of) comprise that two or more (for example, 2,3, the 4 or more a plurality of) shuttle vector of target viral gene and in viral genome, introduce the target viral gene substitution (in theplace of) that two or more (for example, 2,3,4 or more a plurality of) comprise the nucleic acid molecules of one or more heterologous peptides of encoding and do not have to insert segmental corresponding viral gene.
The mutation step of the inventive method can relate to by transposon mutagenesis in the target viral gene simultaneously or introduce one or more transfer primers (transprimers) successively.These shift primer and can remove by endonuclease digestion, then can be by introduce the nucleic acid molecules of coding heterologous peptides in the target viral gene in the connection in restriction endonuclease digestion site.And the inventive method can relate to the library that generates the sudden change target gene.
The viral genome that is applied to the inventive method can be the banzi virus genome, and chimeric flavirirus for example for example comprises the chimeric flavirirus of (pre-membrane) and envelope protein before the film of a kind of capsid of banzi virus and non-structural protein and second kind of different banzi virus.In these examples, first and second kinds of banzi virus can independently be selected from for example Japanese encephalitis, dengue fever-1, dengue fever-2, dengue fever-3, dengue fever-4, yellow fever, Murray Valley encephalitis, St. Louis encephalitis, Xi Niluo, Kun Jin, rocio encephalitis, Erie's this encephalitis of crow, tick encephalitis, RSSE, KFD, Omsk hemorrhagic fever, ramaninjana, Bo Washeng, root bank, A Busaitaluofu, Hansom Lip river watt (Hansalova), A Boyi (Apoi) and extra large general virus.In addition, the present invention can use complete banzi virus genome (for example yellow fever virus genome, for example YF17D).
Before the target viral gene that is applied to the inventive method can for example be selected from coding peplos, capsid, film, NS1, NS2A, NS2B, NS3, NS4A, NS4B and the proteic gene of NS5.
Introduce virus genomic heterologous peptides according to the inventive method and can comprise one or more vaccine epi-positions (for example, B cell epitope and/or t cell epitope).This epi-position can be derived from the antigen of virus, antibacterial or parasitic disease substance.For example, this epi-position can be derived from influenza virus (for example people or bird flu virus).With regard to the influenza virus epi-position, this heterologous peptides can comprise influenza m 2 e peptide for example or comprise the peptide of influenza hemagglutinin precursor protein cracking site (HA0).In another example, this epi-position is derived from tumor associated antigen or allergen.The example of other sources that can obtain heterologous peptides (for example pathogen) and these peptides and epi-position hereinafter is provided.
The present invention also comprises viral genome or its combination that generates by any method described herein.And, the present invention includes viral vector by these viral gene group codings, comprise pharmaceutical composition and the pharmaceutically acceptable carrier or the diluent of these viral vector, and the method that gives patient's peptide, it relates to and gives the patient these pharmaceutical compositions.In an example of these class methods, described peptide is an antigen, carry out administration with induce at this antigen derived from pathogen or the immunne response of tumor.
No matter the present invention also comprises the banzi virus carrier of whether being produced by methods described herein, it comprises one or more one or more proteic heterologous peptides of capsid, cephacoria, peplos NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 that are selected from that are inserted into.Banzi virus can be yellow fever virus for example (for example, YF17D) or chimeric flavirirus (for example, comprising before the film of the capsid of first kind of banzi virus and non-structural protein and second kind of different banzi virus and the chimeric flavirirus of envelope protein).First and second kinds of banzi virus for example can independently be selected from Japanese encephalitis, dengue fever-1, dengue fever-2, dengue fever-3, dengue fever-4, yellow fever, Murray Valley encephalitis, St. Louis encephalitis, Xi Niluo, Kun Jin, rocio encephalitis, Erie's this encephalitis of crow, tick encephalitis, CEE, KFD, RSSE, Omsk hemorrhagic fever, ramaninjana, Bo Washeng, root bank, A Busaitaluofu, Hansom Lip river watt, A Boyi and Hai Pu virus.
The present invention further comprises nucleic acid molecules or its complement corresponding to the banzi virus carrier of above describing with other parts of this paper; The pharmaceutical composition and pharmaceutically acceptable carrier or the diluent that comprise these viral vector; And by giving these compositionss are transmitted method from peptide to the patient.In an example of these methods, described peptide is an antigen, carries out administration to induce the immunne response at derive this antigenic pathogen or tumor.
In specific embodiment, the present invention includes banzi virus carrier described herein, it is included in the heterologous peptides that inserts between the aminoacid 236 and 237 of non-structural protein 1 (NS1).Another example, but its individualism or insert fragment combination combination (for example NS1 inserts fragment) with other are the heterologous peptides that comprises the preceding proteic aminoterminal zone of film of inserting carrier.This insertion fragment can be positioned at-4 ,-2 or-1 before the cracking site before capsid/film for example, or before the film proteic 26 (or its combination).And albumen inserts to choose wantonly and comprises and can assist the proteolytic cleavage site that removes peptide before film the albumen before this film.
The instantiation that can be included in the peptide in the carrier of the present invention comprises influenza (for example people or fowl) M2e peptide or comprises influenza (for example people or influenza) hemagglutinin precursor protein cracking site (HA 0) peptide.Hereinafter other examples are provided with other parts of this paper.In addition, this carrier can comprise more than a kind of heterologous peptides, for example people and bird flu M2e peptide.In addition, carrier of the present invention can comprise that one or more second sites described herein adapt to (adaptations), and it can provide the carrier character (for example, the growth characteristics of improvement) of improvement
The present invention also comprises corresponding to the nucleic acid molecules of banzi virus vector gene group described herein or its complement.And, the present invention includes the medical composition that comprises viral vector.Said composition can be chosen wantonly and comprise one or more pharmaceutically acceptable carriers or diluent.In addition, said composition can be chosen wantonly and comprise adjuvant (for example, aluminium compound, for example Alumen (alum)).Compositions also can be lyophilized form.
The present invention also comprises method from peptide to experimenter's (for example human patients or animal for example domestic animal or domestic animal) that transmit, and it relates to and gives compositions as herein described.In an example, carry out administration to induce immunne response at derive this antigenic pathogen or tumor.In other examples, this method relates to and gives subunit vaccine.In these examples, can give banzi virus carrier and subunit vaccine simultaneously, the banzi virus carrier can be used as initial dose and gives, and subunit vaccine can be used as that booster dose gives or subunit vaccine given as initial dose and the banzi virus carrier gives as booster dose.This subunit vaccine for example can comprise the heterologous peptides that merges with hepatitis B virus core albumen (for example, influenza M2e peptide or comprise influenza hemagglutinin precursor protein cracking site (HA 0) peptide) the hepatitis B virus core granule.These peptides can be natural or consensus sequence as described herein.
The present invention also comprises the method for producing carrier described herein, relates to the sequence of the interested peptide of coding is inserted the site (using for example methods described herein) that allows this insertion through identifying.These carriers can be banzi virus carrier (for example, banzi virus carrier or chimeric flavirirus (for example, ChimeriVax as described herein TM-JE or ChimeriVax TM-WN)).The exemplary site that is used to insert comprises-4 ,-2 or-1 before the cracking site before NS1-236 and the capsid/film, or before the film proteic 26.
In addition, the present invention includes by for example any carrier described herein being mixed the method for producing pharmaceutical composition with pharmaceutically acceptable carrier or diluent, one or more adjuvants and/or one or more other active agents (for example subunit vaccine).
The present invention is also included within medicine use all viral vector described herein, nucleic acid molecules and the peptide that preparation is used for prevention described herein and Therapeutic Method.
The present invention has several advantages.For example, virus of live vaccine (ChimeriVax for example as used in the present invention TM, yellow fever virus or other virus of live vaccine) and can transmit micromolecule polypeptide antigen molecule (for example influenza M2e or HA 0The cracking site peptide) aspect provides significant benefits.Increase antigenic quality after using live vector for example to comprise (i) vaccination based on the advantage of the carrier of banzi virus; (ii) do not need adjuvant; (iii) to congenital intense stimulus of replying (for example YF17D is the most potent known immunogen) with acquired immunity; (iv) owing to for example ChimeriVax TM(YF17D) infect antigen presenting cell for example the ability of dendritic cell and macrophage make more favourable probability of carrying out antigen presentation; (v) acquisition can provide the probability of the single-shot vaccine of semelincident immunization; (vi) ChimeriVax TMThe peplos of vaccine virus is easy to exchange, makes to select different recombiant vaccinies, and some of them are more suitable for (produce dual vaccine, comprise anti-local popular yellow fever virus or avoid anti--carrier immunity in the colony) than other zones in different ground; (vii) complete banzi virus carrier modification alive is become packaged, monocycle duplicate replicon or above-mentioned PIVs with eliminate the untoward reaction probability or minimize use successively in the probability of anti--carrier immunization; (viii) will use between epi-position that direct random mutagenesis method described herein inserts and other genes or two along anti-express alternative replicon in the antigen of disappearance or PIVs in conjunction with reducing production costs to the more potent immunne response of a kind of pathogen (if epi-position and other antigens of having expressed belong to identical pathogen) or two or more pathogen (if the antigen of epi-position and other expression belongs to different pathogen) and (ix) obtaining.
Other advantages provided by the invention are relevant with the following fact: chimeric flavirirus carrier of the present invention also still can be induced the protective immunity of protein in the chimera of deriving especially being inserted the banzi virus of this chimeric peptide by abundant deactivation to reach safety.Some carriers that other safeties are used from the present invention are chimeric this fact of probability that returns back to wild type of therefore having eliminated.The present invention uses other advantages of carrier to duplicate in the cytoplasm of cell as banzi virus, and Bing Du replication strategy does not relate to viral genome is integrated into host cell like this, and important safety measure is provided.In addition, as hereinafter further describing, single carrier of the present invention can be used for transmitting single antigenic a plurality of epi-positions or derived from more than a kind of antigenic epi-position.
But attendant advantages be described herein directly at random the wide spectrum in the insertion identifying virus albumen allow the site, it can be directly used in and insert various other epi-positions (as the insertion site among the NS1 of illustration hereinafter) and longer insertion fragment.Attendant advantages is that some allow in other banzi virus equally in the insertion site that a kind of banzi virus camber allows, and this is because the proteic structure of different banzi virus/function conservative.Attendant advantages is that the recombinant flavivirus of load epi-position can be used as the Booster of subunit vaccine for example or the collaborative component of the mixed vaccine be made up of for example subunit or killed vaccine component, and it makes immunne response significantly improve (mixing with the ACAM-Flu-A subunit vaccine as the A25 vaccine of illustration hereinafter) with the recombinant virus component.In addition, described insertion at random can be applicable to any banzi virus of having reset (or deficiency banzi virus) genome, for example in modified TBE virus wherein structural protein gene be transferred to genomic 3 ' end and under the control of IRES element NS5 after expression (Orlinger etc., J Virol.80:12197-208,2006).
From the following detailed description, accompanying drawing and claim, may be obvious that other characteristics of the present invention and advantage.
The accompanying drawing summary
Fig. 1 is for extremely prM, E and/or the NS1 albumen of virus make up ChimeriVax by the total M2e peptide of transposon-mediated insertion at random TMThe diagram of-JE-flu.Use the method shown in this figure also can be with C and NS2A-NS5 targeting in inserting exogenous peptide (for example, t cell epitope)
Fig. 2 is the diagram that is structured in the ChimeriVaxTM-JE-flu plasmid library that comprises the M2e peptide that inserts at random in prM/M, E and the NS1 gene.
Fig. 3 has shown at ChimeriVax TMExpress the total M2e protective epitope of influenza A virus in the NS1 albumen of-JE virus, as shown in virus plaque dyeing.Infect back 4 days with the virus plaque in anti-JE polyclonal antibody (A) or the anti-M2e monoclonal antibody dyeing 35-mm hole.With the M2e-positive-virus plaque in anti-M2e monoclonal antibody (C) the dyeing 100-mm culture dish (comprising hundreds of virus plaques).
Fig. 4 is a form and a photo, has shown by M2e MAb or the JE polyclonal antibody (form on the left side; The clone that titre is the highest represents with runic) ChimeriVax of the screening purification measured TMThe titre analysis result of-JE-NS1/M2e virus clone deposit of P2 level (the last purification step after), and one of clone's dyeing example (photo on the right).These results show clone's purity and the evidence of high hereditary stability are provided.
Fig. 5 is ChimeriVax TM-JE vector virus is the particular location and the diagram of inserting segmental nucleotide and aminoacid sequence by the M2e that virus clone A11-A92 (comprising the clone A25 that is used for hereinafter described testing) order-checking is identified in the NS1 gene.Complete 105-nucleotide inserts the fragment highlight and represents.The M2e peptide that side joint GG residue (add to increase flexibility) is all contained on both sides adds frame table and shows.The BstBI restriction enzyme site underlines.Because the effect of transposon is duplicated insertion fragment (SV) two viral aminoacid before at the end that inserts fragment (adding double underline).
Fig. 6 A is ChimeriVax TMThe diagram of the clone A25 of-JE-NS1/M2e, it has shown that M2e inserts the position of fragment in viral genome.Fig. 6 B is for showing with M2e and JE-specific antibody 10 times the photo of A25 virus plaque of going down to posterity in the Vero cell that dyes.Fig. 6 C is and ChimeriVax TM-JE vector virus is compared the growth curve picture of A25 virus when P2 and P12 go down to posterity.The D picture group is for infecting A25 virus or ChimeriVax-JE carrier and with the immunofluorescence example of the cell of anti-JE or anti-M2e antibody staining, also having shown the viral effective expression M2e of A25 peptide.
Fig. 7 is for showing the figure of the 54th day M2e specific IgG total amount: from the ELISA OD of the serial dilution sample of the mice serum of 2 and 3 immune group 450Value sees Table 5.
Fig. 8 is by the figure of the survival curve of immune mouse after adapting to the reassortant influenza virus attack with 20 LD50 mices on the 55th day shown in the table 5.
Fig. 9 is the general M2e (A) and the HA of influenza A virus 0(B) the comparison diagram of epi-position.Most important sequence part (for example antibodies) adds shade.
Figure 10 is the example that can use the former construct of multi-resistance of the generation of insertion at random described herein: express the ChimeriVax-JE replicon of a plurality of influenza A virus immunogens as mechanism and multiple pandemic disease vaccine, for example express NA or HA and substitute the prM-E gene, insert the M2e epi-position among the NS1 for example at random, for example the immundominance t cell epitope among the NS3 and insert the additional immunogen in one of site (or more) between gene.2A autologous protein enzyme (from EMCV or FMDV) can be from the residue position cracking NA of glycoprotein.Perhaps, can use this IRES element to substitute 2A autologous protein enzyme with the proteic translation of initial NS again.Can use a plurality of elements (for example, 2A autologous protein enzyme, ubiquitin, IRES, be used for spontaneous (autonomous) AUG of NA gene) produce the N end of NA around the site.Similarly, can use the vaccine constructs that produces anti-several pathogen derived from the antigen of different pathogens.
Figure 11 is for ChimeriVax-JE/NS1-M2e RNA library transfection and cover example with the M2e antibody staining culture dish of the Vero cell competed between the elimination virus clone with agar immediately.The RNA that is used for transfection is synthetic on the dna profiling of external connection, and this dna profiling obtains by NS1-M2e gene library to the pBSA-AR3-stop carrier that connects from plasmid pUC-AR03-rM2e.
Figure 12. shown successful expression M2e peptide in the E of ChimeriVax-JE virus albumen: with the accumulation point (foci) of the painted insertion fragment of M2e MAb mutant.(A) the 6th day painted segmental variant of insertion (experiment 2) that comprises initial 35-aminoacid M2e.(B and C) the 4th day painted variant (experiment 3) that contains the 17-aminoacid M2e of 17-aminoacid M2e and 2 glycine residues of side joint respectively.
Figure 13 has shown that series connection insertion ChimeriVax-JE NS1-236 inserts the people M2e+ fowl M2e epi-position in site.Inserting segmental total size is 56 aminoacid.(A) diagram of the fowl M2e epi-position of adding A25 virus.(B) the concrete sequence of two variants of this virus: go up picture group and show the M2e that uses fowl M2e to insert segmental natural codon structure The people/ M2e FowlThe sequence (people M2e underlines, and fowl M2e underlines with hacures) of virus; The bottom picture group shows identical content, obtains higher hereditary stability except fowl M2e codon is changed over degenerate code.(C) M2e of usefulness JE and M2e antibody staining The people/ M2e FowlVirus plaque.
Figure 14 is presented at the NS1-236 that uses the M2e epi-position to identify and inserts ChimeriVax-JE virus tolerance HAtag (influenza H3) B/T cell epitope on the site.(A) insertion sequence of the live virus that reclaims.(B) with the virus plaque on the anti-HAtag MAb 12CA5 dyeing Vero cell.
Figure 15 shows foreign epitope expression multi-form in banzi virus prM, E and the NS1 albumen.
Figure 16 has shown that the ChimeriVax-JE that contains M2e in the prM albumen inserts the fragment variant.(A) compare M1, the M2, M3, M6 and the M8 clone's that in experiment once, measure plaque example with ChimeriVax-JE.(B) growth curve of prM-M2e clone contrast ChimeriVax-JE carrier.
Figure 17 contains the diagram that Me inserts segmental ChimeriVax-JE cloned sequence among the prM.Shown the most probable signal peptidase cracking site of SignalP 3.0 in the sequence of threads prediction.
Figure 18 has shown the ChimeriVax-WN02 analog of A25 (ChimeriVax-JE/M2eNS1-236) virus: make up and formation and with M2eMAb painted plaque under agar covers in the 6th day.
Figure 19 has shown ChimeriVax-WN02/A25 and ChimeriVax-WN02/A25adapt virus.(A) compare the plaque of the plaque purified virus reserve that under methylcellulose covers, formed in the 5th day with ChimeriVax-WN02 with the A25 prototype virus.(B) growth curve in the Vero cell, MOI 0.001.
Detailed Description Of The Invention
The invention provides the method that generates the viral vectors comprise heterologous peptides, the viral vectors that comprises these peptides, by give this live vector for example with induce be introduced into deriving peptide the immune response of the pathogen composition that transmits the method for these peptides and comprise these viral vectors. These viral vectors, peptide, holding in detail mutually of method and composition see below.
Central feature of the present invention relate to by the immunogenic peptide that inserts at random Various Diseases originality organism in the attenuated live vaccine virus protein make up the recombinant vaccine of living with these peptides of effective expression and submission in infected cell to immune system, purpose is to induce potent, the permanent immunity to the target pathogen. For effective submission, in the exogenous peptide radom insertion virus protein with typical example such as B cell epitope, for example only be that albumen in infected emiocytosis (for example, the amino terminal part of NS1 and flavivirus prM) or the virion (M of flavivirus and E envelope protein) is to stimulate potent anti-peptide antibody to reply. Peptide, but for example comprise in the peptide radom insertion non-structural viral protein of t cell epitope that it can be synthetic at infected cell interior, cause by MHC I/II compound to immune system submission exogenous peptide to induce potent cellular immunity. In structural proteins, insert the submission that also can cause effective MHC mediation.
As hereinafter describing in detail, the insertion of random fashion allows the most effective recombinant virus variant that copies of screening in the viral gene according to the present invention, and maximum immunogenicity (best peptide conformation) and the most stable expression of the peptide that inserts are provided. Also as mentioned below, that can commercially buy comprises that for example the transposon-mediated insertion system of removable transfer primer can be used as the instrument that makes up recon of the present invention. EXPERIMENTAL EXAMPLE is hereinafter partly described the inventive method in detail. Simple, in these embodiments, will in A type strains of influenza viruses, insert ChimeriVax by the total B cell epitope M2e of influenza virus (also comprising the T cell epitope) the M2 albumen of high conservativeTMIn the NS1 of-JE vaccine virus, prM/M and the E gene. In NS1, prM/M and E albumen, observe a plurality of virus clones of expressing the M2e peptide through anti-M2e antibody recognition, some of them purifying step of going forward side by side is carried out external/body internal characteristic research. In addition, as discussed below, the NS1 Insert Fragment is transferred to ChimeriVax from the ChimeriVaxTM-JE backgroundTM-WN。
An element of the inventive method is that transposons only is used for to expection gene (a more gene) this fact of the one or more restriction enzyme sites of radom insertion. Then the dna fragmentation of required exogenous peptide of will encoding is integrated into this gene at the restriction enzyme site place. Then the mutator library can be integrated into (cDNA of RNA virus) in the complete viral genome, then transfectional cell and gather in the crops the allos daughter of virus. Virus is oneself " selection " optimal insertion point, and does not affect its vigor and effectively copy. Then the mutated viruses clone of rapid screening sufficient amount uses inserting the special high antigenicity of antibody test of peptide; detect high immunogenicity (suitable peptide conformation and to the immunocyte submission) and detect anti-peptide immune response and/or protection and genetic stability to attacking by immune animal; for example existence and the expression by this peptide in the process of monitoring the mutated viruses that repeatedly goes down to posterity in external or the body; and gene order-checking shows the valuable any adaptation of biology table shape tool to recombinant vaccine virus (for example, output is higher in the production, genetic stability is stronger and immunogenicity higher). The result identifies the vaccine virus variant of " best ". This method that " allows virus determine " therefore can provide many benefits.
Provide the principle of the radom insertion method on the inventive method basis to be illustrated in Fig. 1. In this embodiment, the M2e peptide of influenza A is introduced in structure prM/M and E albumen and the non-structure NS1 albumen. These structural proteins discharge from cell with virion (also may secrete prM N end), and NS1 is transported to the surface of infection cell, and its part is separated and in born of the same parents' outer circulation. The outer submission of born of the same parents is the prerequisite of potent antibody response. Therefore use NS1 albumen submission M2e especially noticeable, because this peptide is passed to cell surface, imitated the natural situation of influenza m 2, its some aspects for the M2 mediated immunity are important; And prM and E albumen submission can cause higher immune response, because a plurality of peptide copy is being assumed to stronger immunogenic virion surface submission.
At first use can the commercial kit of buying New England Biolabs (Beverly for example, MA) GPS-LS Tn7 mediation mutagenesis kit enters restriction enzyme site (for example PmeI site) random integration in the target gene of subclone (most ofly to be site of each gene molecule, although can change as required this frequency, for example mutagenesis by additional cycle). Then remove the transfer primer part of transposons and use the M2eDNA Insert Fragment to substitute by restriction endonuclease (for example PmeI) digestion, thereby generate the mutator plasmid library. The gene molecule of sudden change is connected to ChimeriVaxTMThe full-length cDNA of-JE. Then the in-vitro transcription dna profiling uses rna transcription thing transfectional cell. Separate the single clone of the progeny virus of living and existence, immunogenicity and the genetic stability of detection M2e peptide. This embodiment is described in further detail in hereinafter EXPERIMENTAL EXAMPLE part.
In the variant of said method, mutagenesis occurs in whole intact virus genomes (full-length cDNA of the virus that comprises RNA of for example cloning or the complete genome component of dna virus) or comprises in the dna fragmentation of several viral genes in plasmid, then reclaim the insertion mutation body of living. In these embodiments, this virus is " selection " optimal exogenous peptide insertion point in special target protein not only, and it also is chosen in the optimal target protein of encoding in complete genome group or the large segment of genome. In another variant, the suitable gene of other carrier organism styles such as bacterium (such as salmonella etc.) also can similarly carry out the restructuring variant that then Random insertion mutagenesis screens this organism that can be used as vaccine.
In another variant of methods described herein; use successively or simultaneously more than the identical gene of one transposon mutagenesis with the immunogene peptide of radom insertion more than one, then the live virus clone of the different exogenous antigen determinants of a kind of pathogen (for example to strengthen immunogenicity/protectiveness) or several pathogen (for example to produce recombinant vaccine) is carried in screening. This radom insertion method also can be expressed platform (for example, McAllister etc., J.Virol.74:9197-205,2000 with aforesaid other in virus/carrier organism body; Bredenbeek etc., Virology 345:299-304,2006) combination expresses a kind of antigen or the candidate vaccine of several antibody of synantigen not to generate. In addition, additional protein of expressing can be molecules of immunization stimulus, various known cell factors for example, and it can stimulate suitable immune response branch to strengthen the immunogenicity/efficient of recombinant vaccine. In addition, the method can be used for identifying that wide spectrum allows insertion point (for example N end regions of NS1-236 and prM (for example, amino acid/11-5)). Furthermore, the candidate's recon that screens itself can be used as vaccine or with other (for example, subunit or deactivation or other are lived) vaccine group cooperations be initial agent or reinforcing agent (if using successively different components) or as collaborative vaccine component (if inoculating simultaneously different components).
The noticeable characteristics of methods described herein comprise following content: (i) use the generation (Fig. 2) in antibiotics resistance gene (with the epi-position Insert Fragment) the helper plasmid library of cleavable; (ii) introduce the probability (Fig. 2) that terminator codon or frameshit occur to minimize the few virus of Insert Fragment in the total length plasmid clone (viral cDNA); (iii) process the plasmid library comprise the radom insertion fragment to eliminate the few dna profiling of any Insert Fragment or transfectional cell or the RNA that is used for transfection are carried out gradient dilution to minimize the competition between the few virus of insertion mutation body and Insert Fragment (E protein expression part) with the PmeI enzyme; And (iv) use the easy virus clone that comprises Insert Fragment that separates of plaque purifying and cell monolayer immunostaining combination.
Viral vectors
Can be used for embedded virus of the present invention can be based on ChimeriVaxTMVirus, it is comprised of by the first flavivirus (for example, skeleton flavivirus) that structural proteins corresponding to the second virus replace structural proteins as mentioned above. For example, chimera can be comprised of by the first flavivirus of the prM of the second flavivirus and the replacement of E albumen prM and E albumen wherein.
Being used for embedded virus of the present invention can be by any virus combination preparation. Can be used for the present invention and comprise that as the example of the concrete flavivirus of first and second kinds of viruses mosquito passes flavivirus, for example encephalitis B, dengue fever (serotype 1-4), yellow fever, Murray Valley encephalitis, St. Louis encephalitis, Xi Niluo, Kun Jin, sieve Theo encephalitis and ilheus encephalitis virus; TBE, for example CEE, Siberia encephalitis, RSSE, KFD, Omsk hemorrhagic fever, ramaninjana, Bo Washeng, root bank, A Busaitaluofu, Hansom Lip river watt, A Boyi and Hai Pu virus; And the virus of hepatitis seed culture of viruses (for example HCV).
The instantiation behaviour yellow fever virus vaccine strain YF17D that can be used for embedded virus of the present invention, prM wherein and E albumen are replaced by the prM of another kind of virus and E albumen, for example japanese encephalitis virus, west nile virus, Saint Louis virus, Murray Valley encephalitis are viral, dengue fever virus or any other flavivirus, and be for example above one of listed. For example following approving and forwarding date saved is that the chimeric flavirirus that was stored in Virginia, USA Manassas American type culture collection (ATCC) on January 6th, 1998 under budapest treaty can be used for the present invention: chimeric yellow fever 17D/ encephalitis B SA14-14-2 virus (YF/JE A1.3; ATCC accession number ATCC VR-2594) and chimeric yellow fever 17D/ dengue fever 2 types virus (YF/DEN-2; ATCC accession number ATCC VR-2593).
For example United States Patent (USP) the 6962708th and No. 6696281; International Application No. WO 98/37911 and WO 01/39802; And Chambers etc., J.Virol.73:3095-3101 provides preparation to can be used for the details of embedded virus of the present invention in 1999, and its complete content is all with the reference form and in this paper. In addition, these embedded viruses can comprise attenuation sudden change, and the list of references of for example above quoting with this paper (also can be referring to for example WO 2003/103571; WO 2005/082020; WO 2004/045529; WO 2006/044857; WO 2006/116182) described in the person. For example in U.S. Patent number 6962708, provide the virus sequence information that can be used for preparing virus of the present invention (also can be referring to, Genbank accession number NP_041726; CAA27332; AAK11279; P17763; Annotate: these sequences only are exemplary; Many other flavivirus sequences known in the art also can be used for the present invention). Additional example comprises Genbank accession number NC_002031, it is provided in this paper with sequence appendix 3 (YF17D), Genbank accession number AF315119, it is provided in this paper with sequence appendix 4 (JE-SA-14-14-2), and Genbank accession number AF196835, it is provided in this paper with sequence appendix 5 (west nile virus). These sequence informations only are exemplary, and many other flavivirus sequences of the present invention that can be used for are arranged. In addition, these sequences can comprise the described sudden change of this paper (and institute's citing document), are contained in as in the described chimera of this paper (with the document of quoting) and/or comprise Insert Fragment as herein described.
In using the advantage of ChimeriVaxTM vaccine as carrier, a major advantage is that (it is the major antigen determinant of anti-flavivirus immunity to envelope protein, be in this case anti-carrier immunity) be easy to exchange, so that can use identical YF17D skeleton (it can be applied to identical individuality successively) to make up several different vaccines. In addition, different recombinant C himeriVaxTMThe insertion vaccine can be determined as and more be applicable to the popular particular geographic area of different flavivirus, as the dual vaccine of the popular flavivirus in anti-place and another kind of target pathogen. For example, ChimeriVaxTM-JE-influenza virus more is applicable to the popular Asia of JE to be protected from the attack of JE and influenza, and the YF17D influenza vaccines more are applicable to the popular Africa of YE and South America, ChimeriVaxTM-WN-influenza more is applicable to the popular U.S. of WN and part Europe and the Middle East, ChimeriVaxTM-dengue fever-influenza more is applicable to exist the torrid zone of dengue fever virus.
Except chimeric flavirirus, other flavivirus for example non-chimeric flavirirus can be used for support according to the present invention. Can be used for these viral examples of the present invention and comprise attenuated live vaccine, YF17D and derived from YF17D strain person for example, it obtained (Smithburn etc. by attenuation wild type Asibi strain originally, " Yellow Fever Vaccination; " World Health Organization, the 238th page, 1956; Freestone, (editors) such as in Plotkin, Vaccines, second edition, W.B. Saunders, Philadelphia, U.S.A., 1995). The example of the YF17D strain that can be used for virus of the present invention of can deriving be YF17D-204 (
Figure A20078003427400271
Sanofi-Pasteur，Swiftwater， PA，USA； ，Sanofi-Pasteur，Marcy-L’Etoile，France； ARILVAX TM，Chiron，Speke，Liverpool，UK；
Figure A20078003427400273
, Berna Biotech, Bern, Switzerland; YF17D-204 France (X15067, X15062); YF17D-204,234 US (Ric etc., Science 229:726-733,1985)), and other examples of spendable these Strain are YF17DD strain (GenBank accession number U 17066), YF17D-213 (GenBank accession number U17067) and the Galler etc. that are closely related, Vaccines 16 (9/10): 1024-1028, the 1998 flavivirus 17DD strains of describing. Except these Strain, the mankind for example in the human patients flavirirus vaccines strain of any other suitable attenuation all can be used for the present invention.
Except chimeric flavirirus and complete flavivirus for example the yellow fever virus (for example YE17D vaccine), the inventive method also can be used with other non-yellow fever virus, attenuation or vaccine virus (RNA and comprise the virus of DNA). The example of these vaccine viruses comprises the deactivation strain (for example, vaccinia virus, varicella virus etc.) for measles, rubella, Venezuelan equine encephalomyelitis (VEE), sub-thread anti-chain virales (rhabdovirus, parainfluenza virus etc.) and dna virus.
In addition, except above-mentioned live virus, the packing replicon of expressing exogenous peptide in replicon skelemin (for example, NS1 and other NS albumen and C) can be used for the present invention. The method can be used for for example needing to strengthen security or minimizes in the situation of anti-carrier immunity (antibody response of the anti-envelope protein that neutralizes) to use identical carrier preparation can be applied to the different vaccines of same individual or express several antigens in the sub-construct of identical copy. Figure 10 is seen in the diagram of this construct. Set up the technology that makes up the monocycle replicon, also verified immunogene potential quality (Jones etc., Virology 331:247-259,2005 of replicon; Molenkamp etc., J.Virol. 77:1644-1648,2003; Westaway etc., Adv.Virus.Res.59:99-140,2003). In the example of these replicons, most prM and E envelope protein gene have been lacked. Therefore, it can in time multiplexed cell system, still can not produce daughter of virus (therefore copying for the monocycle). It can be packed in the virion when the trans prM-E of providing gene. And, when cell is infected by the replicon of these packings (for example, after the inoculation), carry out afterwards the monocycle and copy, can further not diffuse to peripheral cell/tissue. In addition, the immunogenic peptide of radom insertion can be at PIVs (for example, Mason etc., Virology 351:432-443,2006) under the background with other antigens and any other defective virus vaccine constructs, complete vector virus, (for example reset virus, Orlinger etc., J.Virol.80:12197-12208,2006) by between gene, bicistronic mRNA expresses additional antigen and substitutes the mode such as PIV disappearance and make up.
Can in a kind of virus, make up from the protective epitope of different pathogens and to obtain trivalent, tetravalent vaccine etc. And, can use the ChimeriVax that comprises from non-endemicity flavivirus envelopeTMVariant is avoided otherwise can be limited natural anti-carrier immunity in the colony of the validity that is seeded in a certain geographic area (ChimeriVax for exampleTM-JE carrier can be used for not existing the U.S. of JE).
Furthermore, the present invention includes virus, flavivirus (for example yellow fever virus such as YF17D and chimeric flavirirus for example, person as described herein), it comprises one or more heterologous peptides Insert Fragments as described herein in the albumen that is selected from C, prM, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 albumen, no matter whether adopt the method for the invention preparation. Being used for of describing in this paper test example accurately instructed the how albumen of other flavivirus albumen of mutagenesis (C and NS2A-NS5) and other vector virus, bacterium etc. of those skilled in the art to the method that prM, E and NS1 insert. Because C and NS2A-NS5 flavivirus albumen are mainly cell inner expression (except C, it also is the part of virion), these albumen are best suited for and insert T cell external source immunogene epi-position; But also can insert the B cell epitope, because the in vivo anti-overwhelming majority (if not all) born of the same parents' inner virus albumen generation of some antibody responses.
Heterologous peptides
Viral vector of the present invention can be used for transmitting any peptide or albumen with prevention or therapeutic value.For example, carrier of the present invention can be used for inducing in any insertion virus protein (for example before the peplos of banzi virus, the film, capsid and non-structural protein) based on proteinic antigenic immunne response (prevention or treatment).
But each self-contained single epi-position of carrier of the present invention.Perhaps, can single site (for example, with the form of multi-epitope, wherein can separate different epi-positions by flexible joint, for example amino acid whose polyglycine extends), different loci or its combination in any in carrier insert a plurality of epi-positions.These different epi-positions can belong to or derived from not belonging to together and/or not of the same race derived from single pathogen.This carrier can comprise a plurality of peptides, for example as a plurality of copies of the listed peptide of this paper or as the combination of the listed peptide of this paper.As an example, this carrier can comprise people or fowl M2e peptide (and/or its conserved sequence)
Can be used for antigen of the present invention can be derived from infectant for example as virus, antibacterial and parasite.The instantiation of this infectant is an influenza virus.Antigenic instantiation from influenza virus comprises derived from hemagglutinin (HA; For example one of H1-H6 or its subunit) (or subunit HA1 and HA2), neuraminidase (NA; One of N1-N9 for example), M2, M1, nucleoprotein (NP) and B albumen.For example, can use the peptide (NIPSIQSRGLFGAIAGFIE of A/H1 strain, the PAKLLKERGFFGAIAGFLE of the NVPEKQTRGIFGAIAGFIE of A/H3 strain and influenza B strain) or the M2e (SLLTEVETPIRNEWGCRCNDSSD) that comprise hemagglutinin precursor protein cracking site (HA0).Other examples of the peptide of guarding in the influenza virus can be used for the present invention and comprise: the NBe peptide (consensus sequence MNNATFNYTNVNPISHIRGS) that influenza virus B is conservative; The proteic ectodomain of the BM2 of influenza virus B (consensus sequence MLEPFQ) and from the M2e peptide (MSLLTEVETLTRNGWGCRCSDSSD) of HAN1 bird flu virus.Can be used for other examples of influenza peptides of the present invention and the protein of (for example through fragmentation) such peptide of can deriving is described in US2002/0165176, US 2003/0175290, US 2004/0055024, US 2004/0116664, US 2004/0219170, US 2004/0223976, US 2005/0042229, US2005/0003349, US 2005/0009008, US 2005/0186621, No. the 4752473rd, United States Patent (USP), No. the 5374717th, United States Patent (USP), No. the 6169175th, United States Patent (USP), No. the 6720409th, United States Patent (USP), No. the 6750325th, United States Patent (USP), No. the 6872395th, United States Patent (USP), WO 93/15763, WO 94/06468, WO 94/17826, WO96/10631, WO99/07839, WO 99/58658, WO 02/14478, WO 2003/102165, WO2004/053091, WO 2005/055957, and the sequence appendix 1 and 2 that comprises of this paper (and this paper quote list of references), its content is all with the reference form and in this paper.And; can select the conservative immunity/protectiveness T and the B cell epitope of influenza from the www.immuneepitope.org data base; but candidate's epi-position (Bui etc. of many cross protections have wherein been identified; Proc.Natl.Acad.Sci.U.S.A 104:246-251; 2007 and supplementary tables), comprise the HA epi-position of the H3N3 virus that we describe hereinafter.The present invention can use any peptide from online IEDB source, for example, comprises the conservative B that Bui above etc. describes and the influenza virus epi-position of t cell epitope.
From people/animal disease substance for example epi-position, the herpes simplex virus (HSV) of parasite (for example malaria), other pathogen viruses (for example human papillomavirus (HPV)), the epi-position of human immunodeficiency virus (HIV, for example gag) and hepatitis C virus (HCV) and antibacterial (for example mycobacterium tuberculosis, clostridium difficile and helicobacter pylori) also can be used for carrier of the present invention.Can from document, find the various suitable epi-position of these and other pathogen easily.For example Schiller and its colleague identify the proteic intersecting protective epi-position/peptide of mastoid process tumor virus L2, and it can induce the wide spectrum cross-neutralization antibody that can protect different HPV phenotypes to attack, for example HPV16 virus (WO2006/083984 A1; QLYKTCKQAGTCPPDIIPKV) for example L2 proteic amino acid/11-88, or amino acid/11-200, or amino acid/11 7-36.WO 2004/053091, WO03/102165, WO 02/14478 and US 2003/0185854 provide and can be used for other pathogen of the present invention and from the antigen of these pathogen and the example of epi-position, its content is with the reference form and in this paper.
The additional example that can obtain antigenic pathogen is listed in hereinafter table 1, and such antigenic instantiation comprises the person that lists in table 2.In addition, the instantiation that can insert the epi-position of carrier of the present invention is provided in table 3.As shown in table 3, the epi-position that is used for carrier of the present invention can be B cell epitope (for example, neutralizing epitope) or t cell epitope (being t helper cell and cytotoxic T cell specificity epitope).
Carrier of the present invention can be used for transmitting except that derive antigen the antigen of pathogen.For example, such carrier can be used for transmitting the immunotherapy that tumor associated antigen is used for cancer.Kinds of tumors related antigen known in the art also can give according to the present invention.The example of cancer (and corresponding tumor associated antigen) is: melanoma (NY-ESO-1 albumen (especially being positioned at the CTL epi-position of amino acid/11 57-165 position), CAMEL, MART 1, gp100, tyrosine associated protein TRP1 and 2 and MUC1); Adenocarcinoma (ErbB2 albumen); Colorectal cancer (17-1A, 791Tgp72 and carcinoembryonic antigen); Carcinoma of prostate (PSA1 and PSA3).Heat shock protein (hsp110) also can be used as antigen.
In other examples of the present invention, can use the coding expection that it is produced the foreign protein of the irritated inducing antigen epi-position of immunne response.In addition, carrier of the present invention can comprise be used for targeting vector with transmit peptide for example antigen to the part of the experimenter's who gives this carrier cell (cell that for example, comprises ligand receptor).
Peptide or the proteic size of inserting carrier of the present invention can be 3-1000 amino acid whose length, and 5-500,10-100,20-55,25-45 or 35-40 amino acid whose length is for example determined the length of thinking fit as those skilled in the art.As described in other parts of this paper, aminoterminal cephacoria insertion fragment described herein can provide inserts the more probability of long segment (seeing below).Furthermore, peptide described herein can comprise that appended sequence maybe can reduce length, also can determine the person of thinking fit as those skilled in the art.The peptide that this paper lists can as shown herely be present in the carrier of the present invention or modify by for example replacing or lack one or more aminoacid (for example, 1,2,3,4,5,6,7,8,9,10 or more aminoacid).In addition, this peptide can be bigger peptide and is present in the carrier.
The present invention also comprises and identifies and use the wide spectrum that can insert a plurality of different peptides to allow to insert the site, and NS1-236 for example is shown under the situation of two different chimeras (seeing below).Other wide spectrums allow the site to comprise that embedded virus (comprises ChimeriVax TM-JE and ChimeriVax TM-WN (seeing below)) prM aminoterminal zone.Can be in these viral 1-50 positions, for example insert in one or more sites of 1-25,1-15,1-10 or 1-5.
Further, (for example, Vero) cultivating second site that obtains adapts to from cell to the present invention includes discriminating and use.These adaptations can provide benefit, for example strengthen to duplicate etc.The instantiation that can be used for these adaptations under other situations is shown in hereinafter EXPERIMENTAL EXAMPLE.
Produce and administration
Can use the standard method of this area to prepare above-mentioned virus.For example, can will introduce therefrom primary cell, Embryo Gallus domesticus or the diploid cell of (or in its supernatant) purification progeny virus corresponding to virus genomic RNA molecule.Can produce the additive method of this viroid and use heteroploid cell, Vero cell (Yasumura etc. for example, Nihon Rinsho 21:1201-1215,1963) in the example of these class methods, can will introduce heteroploid cell corresponding to virus genomic nucleic acid molecules (for example, RNA molecule), results virus from the culture medium of cell culture, with nuclease (the two endonuclease of degradable DNA and RNA for example, for example, Benzonase TMNo. the 5173418th, United States Patent (USP)) handles the virus of gathering in the crops, concentrate (for example, for example can holding back by use, the filter of 500kDa molecule carries out ultrafiltration) and, then spissated virus is configured to vaccine through the virus that nuclease is handled.The details of this method is provided in WO 03/060088 A2, and it is with the reference form and in this paper.Furthermore, the above-mentioned document of quoting provides the method for producing embedded virus when mentioning the structure of embedded virus construct.
Amount and the method afford carrier of the present invention that can conveniently determine with those skilled in the art.With regard to chimeric flavirirus and yellow fever virus for regard to the carrier on basis, such carrier can the mode identical with the yellow fever 17D Vaccine gives and disposes, for example the fluid of the cell culture harvest that infects with the clarification suspension of infected Embryo Gallus domesticus tissue or from chimeric yellow fever virus.Therefore carrier of the present invention can (for example be configured to comprise 100 to 1000000 infectious units, plaque forming unit or TCID) sterile aqueous solution with intraperitoneal for example, intramuscular, subcutaneous or intradermal routes administration (about the details of intradermal vaccination method referring to, for example, WO 2004/0120964).In addition, because banzi virus can infect human host by mucosal route, oral cavity route (Gresikova etc. for example, " Tick-borne Encephalitis, " The Arboviruses, Ecology andEpidemiology, Monath (ed.), CRC Press, Boca Raton, Florida, 1988, Volume IV, 177-203), so can give these carriers by mucosal route.
When being used for immunization method, this carrier can be used as can be by the adult of concrete pathogenic infection and child's the first preventive of exempting from.This carrier also can be used as the Booster by the immunne response treatment patient who stimulates the anti-antigenic virus of this peptide of deriving.For example, the recon of the expression proteic epi-position of E6/E7 or complete E6/E7 albumen or HPV can be used as therapeutic HPV vaccine.
For using vaccine, optional use adjuvant well known by persons skilled in the art.The immunogenicity that adjuvant can be used for strengthening chimeric vector is Liposomal formulation, synthetic adjuvant, for example (as QS21), muramyldipeptide, monophosphoryl lipid A or poly-phosphorus piperazine for example.Though these adjuvants are generally used for strengthening the immunne response to inactivated vaccine, they also can be used for live vaccine.For example with regard to the chimeric vector of oral cavity administration, the sudden change derivant of for example colibacillary heat-stable toxin of mucosal adjuvants (LT) or LT can be used as adjuvant with regard to mucosal route.In addition, coding can be had in the gene insertion vector of cytokine of adjuvanticity.Therefore, the Codocyte factor for example the gene of GM-CSF, IL-2, IL-12, IL-13 or IL-5 can insert to produce with the exogenous antigen gene and can obtain the enhanced vaccine of immunne response or to regulate more being specific to the immunity that cell, body fluid or mucosa are replied.Perhaps, can be respectively simultaneously or give cytokine successively from recombinant vaccine virus by known method (for example direct inoculation, naked DNA, with viral vector etc.).
Virus of the present invention can be used in combination with other immunization methods.For example virus can be given with comprising identical or different anti-antigenic subunit vaccine combination.Combined method of the present invention can comprise and gives the present invention virus and other forms of antigen (for example, subunit form or comprise that the hepatitis core protein (for example is included in the hepatitis core protein (HBc-M2e of the lip-deep M2e peptide of escherichia coli generation simultaneously; Fiers etc., Virus Res.103:173-176,2004)) doser).Perhaps, carrier of the present invention can just exempt from-strengthen to be used in combination in the strategy with additive method (for example subunit or HBc method), and carrier wherein of the present invention or additive method all can be used as just exempts from, and uses another kind of method as reinforcement or opposite then.Further, the present invention includes and use carrier of the present invention as first the exempting from of just exempting from and strengthen the two-strengthens tactful.
Except vaccine is used, can understand as those skilled in the art, carrier of the present invention can be used for introducing the gene therapy method and the cancer therapy of therapeutic genes product in patient's cell.Furthermore, the recombinant virus chimeric or complete banzi virus for example described herein that comprises the immunogen epi-position can be used in first exempting from/strengthened scheme to strengthen the drug effect of subunit or complete organism killed vaccine, similar to recombinant alpha virus replication (US 2005/0208020 A1).And hereinafter our some data prove that also comprising exogenous peptide when the two being mixed and inoculate simultaneously (for example, exists potent synergism between banzi virus ChimeriVax-JE/NS1-M2e) and the subunit vaccine (for example HBc-M2e).The latter can obtain new effective combination-vaccine preparation, and it does not need adjuvant and new expectation characteristics can be provided, and for example the Th1 in the immunne response moves (shift).In addition, can as described hereinly express foreign epitope, yet not be to use recombinant virus, can for example use formalin with its deactivation and as killed vaccine as live vaccine at virion surface (at prM-E).If this method was especially suitable when vector virus was wild-type virus (it can have immunogenicity to people/animal).
EXPERIMENTAL EXAMPLE
Following EXPERIMENTAL EXAMPLE has shown to ChimeriVax TMInsert M2e sequence and HA epi-position among-the JE.Also sequence is inserted ChimeriVax TM-WN construct.The method of describing among this embodiment also can be used for other viruses, for example chimeric flavirirus and based on the carrier (for example, replicon and PIVs) of virus and other carrier organism bodies mentioned above sequence inserted other protein and to be inserted other peptides.
Yellow fever 17D (YF17D) attenuated live vaccine was applied to the mankind always in past 60 years, has extraordinary safety records and permanent immunity can be provided behind single dose administration.As indicated above, ChimeriVax TM-JE is the attenuated live recombinant vaccine strain, and the gene of some structural protein (PrME) of the YF17D that wherein encodes is by the corresponding gene substitution from the viral SA14-14-2 of gene attenuation Japanese encephalitis (JE).With duplicate relevant capsid and all non-structure (NS) genes in these chimeric born of the same parents derived from the YF17D vaccine strain.Similarly, ChimeriVax TM-WN is the attenuated live recombinant vaccine strain, and the PrM of the YF17D that wherein encodes and the proteic gene of E are by the corresponding gene substitution of west nile virus strain.This chimeric exemplary application the sequence of west nile virus strain NY99-flamingo 382-99 (GenBank accession number AF196835).In a further embodiment, be called ChimeriVax herein TM-WN02, the lysine that NY99-flamingo382-99 peplos sequence is 107 is replaced by phenylalanine, and 316 alanine is replaced by valine, and 440 lysine is replaced by arginine.
The plasmid construction step that Fig. 2 shows is described in this part.Structure serves as that the basis is from comprising ChimeriVax with pBeloBac11 low copy number carrier TMThe pBSA simple substance grain construct of the global cDNA of-JE virus begins.By ChimeriVax with YFM5 ' 3 ' SA14-14-2 TMThe special cDNA part of-JE-(with the SP6 promoter) and YF5.2SA14-14-2 plasmid (ChimeriVax TMTwo initial plasmids of-JE) be assemblied in a low copy number carrier pBeloBac11 (New England Biolabs, Beverly, MA) middle this carrier that makes up.This carrier comprises the restriction enzyme site that is suitable for gene sub-clone (shown above the viral genome among the upper right corner plasmid figure among Fig. 2) of several uniquenesses.Added limitations restriction enzyme site SphI, NsiI and the EagI that will be used for sub-clone prM, E and NS1 gene by silent site orthomutation (the step 1-3 among Fig. 2) introduce the pBSA plasmid.
(introduce three target genes in the step 6) and use the Tn7 transposon that (step 7) is with gained plasmid random mutation to the PUC18 plasmid vector.The escherichia coli that transformed are grown in the presence of chloromycetin (chloramphenicol resistance gene is by the removable transfer primer coding of transposon), and preparation is by three mutant plasmid libraries of a large amount of bacterial clump representatives.When preparation mutant plasmid library, the quantity of each library bacterium colony should be higher than three times of nucleotide quantity in the mutant DNA sequence at least to guarantee then to have mixed relevant external source insertion fragment (encoded peptide is M2e for example) the nucleotide of each target gene after.The colony counts in each library is shown in Fig. 2.(step 8) is by PI digestion with reconnect to remove and shift primer (step 9), the only remaining insertion fragment at random that comprises 15 nucleotide in special PmeI site in each gene molecule to the pUC18 carrier by sub-clone for prM, the E of sudden change and NS1 gene library.For the insertion of auxiliary M2e, at first assembling comprises the SmaI-SmaI box (step 4-5) of M2e and kalamycin resistance gene.By removing Kan in the digestion of genetic engineering side joint BstBI site rGene.Since the PmeI site of the 9th step, antibacterial is grown in the presence of kanamycin screen the library (step 12) that comprises M2e this box insertion library.The natural human influenza AM2e consensus sequence SLLTEVETPIRNEWGCRCNDSSD that is used for this structure is modified to two cysteine residues and replaces to serine to avoid unwanted S-S bridging, it can not influence the antigenicity/immunogenicity of this peptide, and all adds glycine residue to increase flexibility (GGSLLTEVETPIRNEWGSRSNDSSDGG) on both sides.By from gained comprises the gene library of M2e at random, removing kalamycin resistance gene (step 13) with BstBI digestion.
In a method (the method A of Fig. 2), in order to produce ChimeriVax with the M2e that inserts viral prM, E and NS1 gene at random TM-JE-flu template cDNA library (comprising PmeI site at random) is cloned into the mutant gene library of step 9 the modified pBSA plasmid of step 1-3.Yet, when we attempt for the first time with M2e/Kan rLower (the step 11) of quantity of the bacterial clump in the pBSA-AR3-rM2e/Kan library that gained is grown in the presence of kanamycin during the pBSA-AR3-rPmeI library of box inserting step 10.However, this method allows to make up fast the library (for example, from malarial parasite, TB, viral pathogen etc.) that comprises any immunogen epi-position.
In another method (method B), (step 14) is introduced pBSA-AR1-3 plasmid (step 15) with this modified gene that comprises viral lethal mutation at first termination codon/frameshift mutation to be modified the prM, the E that introduce sub-clone and NS1 gene.Can eliminate like this because whole ChimeriVax TMThe ChimeriVax that do not suddenly change behind the transfectional cell that exists that comprises not mutated template part in the-JE-flu template library TMThe probability that-JE occurs.At last, comprise at random M2e in the step 13 and insert fragment person and obtain ChimeriVax by the target gene fragment in step 15 library is replaced to TMThe total length template library (step 16) of-JE-flu virus.
In order to produce ChimeriVax with the total M2e sequence of inserting NS1 TM-JE-flu virus, with XhoI (being positioned at the XhoI site of viral cDNA end) with the linearisation of pBSA-AR3-rM2e plasmid library and with SP6RNA polymerase (the SP6 promoter is positioned at the upstream of viral cDNA) in vitro transcription, transfection Vero cell then.At 3-6 after the transfection days results daughter of virus when cytopathic effect can detect or be obvious.Measure the virus titer of the sample of gathering in the crops by plaque algoscopy (methylcellulose covering), wherein use the anti-JE ascites of mice hyperimmune (ATCC) dyeing methanol fixedly monolayer maybe can use monoclonal antibody (Mab) 14C2 of the commercial influenza M2e epi-position of buying only to detect the plaque of the M2e peptide that expression discerned by Mab to detect all plaques.Overall titre surpasses 7 log 10Pfu/ml.The positive plaque of M2e is easy to detect and reach 0.4% (Fig. 3 A and 3B) of total plaque.The positive plaque of some M2e is the same big with the negative plaque of M2e, shows effective virus replication.The total plaques of great majority are the M2e feminine gender, and this is because some insertion fragment instabilities in site at random of NS1, make to occur not suddenling change ChimeriVax immediately after the transfection TM-JE virus.Perhaps, exist this insertion fragment but can't be near antibody.
Can use few techniques to separate one positive-virus clone.We are with MAb dyeing (immune focus measurement method) and the combination of plaque purification.In this algoscopy, covered by agar with the Vero cell of gradient dilution virus transfection.Remove agarose in the time of the 5th day and dye with methanol fixed cell monolayer (for example, in culture dish, Fig. 3 C) and with MAb.Arrange and reclaim corresponding to the gel section of positive M2e plaque agarose and culture dish also freezing.Perhaps still fix without methanol with Mab staining cell monolayer.Scrape cell the positive plaque and freezing from plastics carefully.Use this step to separate about 80 candidate's virus clones and the plaque purification by 1-2 wheel is further purified.The another kind of method of our use makes up with the positive hole of identifying the highest may the dilution (preferably being infected by single positive-virus granule) with MAb staining cell monolayer in 96 orifice plates cleer and peaceful on the whole dilution of virus, the harvesting.This method obtains 37 candidate clones.Further the analysis showed that these one of show as pure clone, and other still mixes with the negative virus of M2e.
The M2e positive colony that next can detect enough big quantity (for example; immunogenicity 50-100) and in the mice (and/or ferret) to the protection efficient (comprising long-term protection) of wild type influenza viruse attack; use obtainable animal model and method (for example with the anti-M2e antibody titer in the measurement mice serum; use ELISA (using synthetic M2e peptide) to measure IgG/IgM or homotype IgG1/IgG2 antibody) and the activity (or in body) in external ADCC detects, carry out immune focus detection and/or order-checking then.
In further research and development, after last 3-4 the plaque purification step that results virus after transfection is carried out by go down to posterity 13 clones' of production virus deposit of twice amplification the Vero cell.These are through sample called after P2 of amplification research virus deposit (going down to posterity 2 behind the purification).The titer determination of deposit is 2.6 * 10 6-1.0 * 10 7Pfu/mL.Importantly, show that the virus deposit is pure with M2eMAB and JEHIAF dyeing generation titre (Fig. 4) much at one.In addition, this structure is first evidence of recombinant virus hereditary stability.If the impure or unsettled words of virus, ChimeriVax does not suddenly change TM-JE virus will be grown and be surpassed the recon of expressing M2e, and this is not existing situation.In addition, fix effective M2e dyeing that (only detecting surface protein) all observes virus plaque with methanol fixed cell (detecting in the born of the same parents and surface protein) with without methanol.Therefore, the NS1 albumen that comprises the M2e peptide normally is transported to the surface of infection cell and is secreted probably as expecting.But NS1 is the outer submission peptide of active surface/born of the same parents therefore, and it is very important for anti-M2e antibody response potent in the inductor.
13 clones' (A11-92 among Fig. 4) NS1 gene checked order insert segmental site to measure their M2e.Surprisingly, find that this 35 aminoacid insertion fragment is arranged in all 13 sites that the clone is identical, holds half at the proteic C of NS1, at ChimeriVax TMAfter the virus genomic nucleotide 3190 of-JE, between virus N S1 amino acid residue 236 and 237.This inserts segmental exact nucleotide sequence and on every side NS1 nucleotide and amino acid residue are shown in Fig. 5.
For the identical most probable viral isolating plaque of these clones when observing CPE that be interpreted as in the site of this insertion fragment in all 13 clones for gathering in the crops after 6 days from vero cells infection.In the virus replication before competition between the different initial variants (containing the insertion fragment in different loci) occurs in results, a kind of variant becomes and preponderates in viral colony.Therefore, the clone of 13 screenings represents a kind of insertion variant.
In order to solve this problem of competition between the variant, can prepare other clones (for example, if necessary seeking more immunogen candidate vaccine) by carrying out plaque select immediately after the transfection.In one method of back, with synthetic RNA transfection Vero cell in the body and cover with agar immediately, then with M2e antibody staining cell and from agar, gather in the crops positive colony.We carry out transfection with the rna transcription thing and carry out this trial, itself or will be connected to pBSA-AR3-in pBSA-AR3 plasmid library (Fig. 2) or the body from the NS1-M2e gene library of plasmid pUC-AR03-rM2e (it has more representativeness than pBSA-AR3 library) and stop producing in the carrier (Fig. 2) by transcribing in the body.Removed agar in 4-5 days and cover, with M2eMAb staining cell monolayer.Observe a plurality of positive-virus accumulation points of different sizes.The example of the dyeing culture dish of the Vero cell of the RNA transfection that obtains with DNA Connection Step in the body as shown in figure 11.Collection is further purified corresponding to the agarose of several bigger positive plaques part and the plaque purification by additional cycle.What is interesting is that when the new variant of order-checking, they have identical M2e insertion with A25 virus.This identifies NS1-236 is that height in the NS1 albumen allows the site, and it can produce and highly effectively duplicate the insertion mutant, produces maximum plaque.Yet, judge that by the accumulation point of different sizes among Figure 11 clearly this M2e inserts fragment and interleaves (intercalate) different loci at NS1.The poor efficiency of some formation intermediate or small plaque duplicates variant and has practical value.
We also use the BstBI restriction enzyme site that is positioned at A25 clone (Fig. 5) M2e insertion fragment end to add second influenza protective epitope in this NS1 site.For example, we mixed from the H5N1 bird flu by the M2e epi-position of two glycine joint side joints to increase flexibility (as Figure 13 A diagram) and to have obtained live virus.Therefore, back one is inserted mutant and is comprised series connection human influenza M2e, is thereafter bird flu M2e.This virus can be general vaccine, and it can protect colony to avoid the attack of human influenza A strain and bird flu.In this construct, at first will insert segmental NS1 gene and be cloned into ChimeriVax-JE through the method for reverse genetics and infect among the clone from the people M2e that contains of A25 virus.Then by adding fowl M2e sequence at double chain DNA fragment of forming by two annealed phosphoric acid oligonucleotide of BstBI site clone.Made up the M2e of two versions The people/ M2e Fowl, the natural M2e sequence with H5N1 influenza virus is (except the penult cysteine replaces to serine; Sequence is shown in the group that goes up of Figure 13 B), the natural H5N1 codon in another is replaced to minimize the nucleotide sequence similarity (sequence is shown in following group of Figure 13 B) with last visitor M2e sequence by degenerate codon.The latter is in order to reduce the probability of people M2e and fowl M2e homologous recombination in the recombinant virus, and it should obtain higher viral hereditary stability.Dye with JE and M2e specific antibody (Figure 13) plaque to constructed virus.The plaque size is compared to some extent with A25 parent virus and is reduced.The higher (～5log of P1 virus titer that gathers in the crops immediately after the transfection 10Pfu/ml).The virus though in the Vero cell, further do not go down to posterity, but estimate that P2 and the follow-up titre that goes down to posterity are higher, because compare the titre of P2 ChimeriVax construct with P1 higher usually.This experiment obviously shows and longer insertion fragment (with regard to 56 amino acid lengths and from regard to the additional residue of the minority of transposon) can be mixed and use the short insertion site that fragment (35 aminoacid M2e epi-positions of A25 virus) is identified of inserting.Another important conclusions is to insert fowl M2e sequence to people M2e sequence to have changed whole insertion sequences of comparing the NS1-236 site with A25 virus (may and structure).This is first experimental evidence of the wide spectrum permissibility in this insertion site.
In addition, HA 0Influenza A epi-position can be similar series system and M2e combination.Other influenza virus epi-positions, for example from the proteic viral neutralizing epitope of HA, or the CTL epi-position can be separately or insert this site analog of some other sites in NS1 or other virus proteins (or by) with various combinations.
In order to verify that further NS1-236 inserts the wide spectrum permissibility in site; after the N1-236 residue, make up the SKAFSNCYPYDVPDYASL linear protection epi-position (being also referred to as the HAtag epi-position) of influenza H3 virus; it can provide the protection (Bui etc. for the strain of various H3 influenza; Proc.Natl.Acad.Sci.U.S.A.104:246-251; 2007), use two plasmid methods of standard to generate recombinant virus.Two glycine in this epi-position of both sides side joint to increase flexibility and its cysteine residues becomes serine.The insertion sequence of the live virus that reclaims is shown in Figure 17 A.With anti-HAtag MAb 12CA5 dyeing virus plaque (Vero cell) (Figure 14 B).Therefore, insert the site by the NS1-236 that inserts the discovery of M2e epi-position at random and allow to have not homotactic fully epi-position (for example, HAtag).To insert fragment similar to M2e, this case verification the insertion of B cell epitope and t cell epitope because HAtag represents B cell and T cell influenza virus epi-position the two (Bui etc., Proc.Natl.Acad.Sci.U.S.A.104:246-251,2007).
ChimeriVax TMThe hereditary stability of-JE-NS1/M2e virus and the growth kinetics of cell culture
Will be in second pass for having the highest titre 7log 10The NS1 gene of the A25 virus clone of pfu/mL (Fig. 6, A group) is used for further biological feature study.Among Fig. 6 by the immunofluorescence additional identification of the A25 infection cell of M2e MAb (and JE antibody) specific staining the effective expression of M2e.
In order to measure whether inheritance stability of this virus, in can be used for the Vero cell of production of vaccine, it is gone down to posterity 10 times to the P12 level with about MOI 0.001pfu/mL.When P12 virus in immune focus measurement method during by M2eMAB or JE HIAF dyeing, all plaques are all by two kinds of antibody stainings and produce identical titre 8log 10Pfu/mL (Fig 6B).This shows that 12 the virus of going down to posterity can be stablized and keeps it to insert fragment.
Because the virus cytopathy degeneration that becomes is stronger, some Vero are cell adapted to be occurred in and goes down to posterity, and the plaque of P12 level is greater than the plaque of P2 virus.The average diameter of P12 virus plaque becomes and ChimeriVax TM-JE vector virus is comparable.When the full gene group of order-checking P12 virus, detect 8 nucleotide and change (table 4).Four changes cause that aminoacid replaces: the proteic residue E357 of E place valine becomes alanine, and NS4B-95 place methionine becomes valine, and and then (N1-235 place serine is to leucine and residue 1 from 2 replacements of M2e peptide in the upstream InsPlace's phenylalanine is to leucine).Some of back adapt to necessarily to be increased with better virus replication (as follows) is relevant with the plaque size.These do not have ChimeriVax in changing TMThe back mutation of-JE vaccine attenuation labelling (as shown in table 4, other three the stable M2e insertion fragments that kept of sudden change of cloning among A11, A79 and the A88 that also are passaged to P12 and carried out order-checking).
In the Vero cell, compare horizontal A25 clone of P2 and P12 and ChimeriVax TMThe growth kinetics of-JE parent vector virus.Fig. 6 C has shown the result of a representative experiment (MOI 0.001).P2 virus can effectively be grown, but is slower than ChimeriVax TM-JE reached peak value on the 6th day, than vector virus slow one day.Opposite P12 virus at the 5th day to be higher than ChimeriVax TMThe titre of-JE reaches peak value, is higher than 7log 10Pfu/mL.P12 virus more effective duplicates more obvious during MOI 0.1.Therefore, A25 is cloned in and can more effectively duplicates after going down to posterity for 10 times in the Vero cell.Some sequences of finding when P12 change the high yield production that helps recombinant vaccine virus.
Use ChimeriVax TM-JE-NS1/M2e A25 virus is set up and is used to analyze ChimeriVax TMThe pilot scale research of the mouse model of the immunogenicity of-JE/flu recon and protection efficient
For any viral vaccine carrier, the especially rodent person's (for example, the natural host of YF (the wild type prototype of YF17D) is monkey and people) that is not the natural host, set up relevant and available small animal model is full of challenges.With such model should the various conformations of more a plurality of expression the relative immunity originality of recombinant virus construct of exogenous peptide.In order to determine best route of administration and to obtain ChimeriVax TMThe immunogenic preliminary data of-JE-NS1/M2e is used 5log 10A25 clone subcutaneous (SC) or abdominal cavity (IP) immunity of pfu/ dosage (1 and 2 group respectively, table 5) array 5 all big Balb/c mices (N=10).Matched group 3 is accepted the hepatitis B core granule (HBc-M2e that escherichia coli produce lip-deep M2e peptide that is included in of subcutaneous 10 μ g dosage; Fiers etc., Virus Res.103:173-176,2004) and adsorbed onto alum adjuvant; Similarly should group at the 20th day booster immunization.Use ChimeriVax TM-JE carrier (5log 10Pfu) immune negative control group 4 and 5 or simulation immunity (diluent).
In the serum of collecting in the 1st, 3,7,9 and 11 day, measure the viremia in the single animal that has inoculated virus.A25 does not all cause detectable viremia under two kinds of approach.Inoculation ChimeriVax TMIn 10 animals of-JE virus 2 only had low-level viremia (50 and 275pfu/mL) at the 1st day, it represents the virus inoculated probably.Therefore, A25 virus can not cause that tangible system infects by these two kinds of approach.
The 38th day, get the blood sample of all animals and measure the anti-M2e antibody response respectively organize plasma sample with ELISA.In the virus immunity group, only in 2 groups (A25IP), detect low-level replying, its total IgG and IgG2a titre are 100 (not having detectable IgG1), and 3 groups titre is higher as expecting: total IgG, IgG1 and IgG2a are respectively 218700,218700 and 24300.Owing to this reason, used 5log on the 40th day 101,2 and 4 groups of the viral separately reinforced immunologicals of pfu: 1 group of subcutaneous reinforcement and (IP) strengthened in other group abdominal cavities.(5 groups of diluent of also accepting abdominal cavity dosage).Two week backs (the 54th day) are got the animal blood sample once more and detect M2e antibody response (table 5) in blood serum samples.A25 virus is strengthened the remarkable increase that causes antibody titer in 2 groups (A25IP/IP).The total IgG titre of this group increased about 30 times to 2700.The total IgG titre of 3 groups (HBc-M2e) is 72900.The 450nmOD reading of the total IgG of 2 groups and 3 groups is shown in Fig. 7.Importantly, reply although the HBc-M2e immunity causes tangible IgG1, the antibody of nearly all A25 virus induction is IgG2a hypotype (table 5).IgG2a antibody is main mediation of ADCC, and its M2e-that is considered to influenza infection induces the main mechanism of protection.Therefore, be used to measure ChimeriVax TMIP strengthened setting up after the immunogenic effective mouse model of-JE/flu recon relied on the IP immunity.
The 55th day, use high dose 20LD 50Mice adapt to reassortant influenza virus intranasal (IN) immune animal.This dosage is than the 4LD that is used for HBc-M2e research 50Immunizing dose is high 5 times, and we painstakingly select high dose to compare when passing through ChimeriVax to answer with the HBc-M2e immunity TMEven lower this problem of more effective protection that whether may produce of-JE viral vector transmission immunity back M2e antibody titer.In theory, this can stimulate owing to the non-specific virus of antigen presenting cell, CTL replys (the M2e peptide comprises the CTL epi-position), induce auxiliary and some the inherent immunity mechanism of potent T cell.Immunity back survival curve is seen Fig. 8.Give challenge dose as expection, the survival of HBc-M2e immune animal is (50%) not exclusively.With two animals survived in 2 groups of A25 virus IP/IP immunity, this group has the highest M2e antibody titer in two A25-immune group (20% survives).A survival animal (A25SC/SC) in 1 group.All animals in the negative control group 4 and 5 are all dead.Can show obvious dependency between protection level and the M2e antibody titer from these data, no matter with recombinant virus or subunit vaccine immune animal.Yet, it should be noted that some above-mentioned mechanism may play a role in the A25 immunity, because it is unknown and may be because limited the duplicating of virus in this model and very low to give the microgram amount of actual M2e of mice.This can explain in the hamster model on the one hand, wherein expects more effective periphery virus replication.In primates/mankind, ChimeriVax TM-JE (and ChimeriVax TMWith the YF 17D Vaccine) cause that relative efficient system infects, peak viremia titre is about 2log 10Pfu/Ml.Therefore, can expect behind the relative low dosage single virus inoculation that potent M2e to influenza replys and protects.
Use the mouse experiment 2 of A25 virus
Add mouse experiment with more young big mice (from two suppliers) of 4 weeks with A25 virus, use higher A25IP dosage (7log 10Pfu/ml).Experimental design sees Table 6.In overwhelming majority's group, use A25P2 virus deposit (it also is used for experiment before); This experiment also comprises the group (#5) of the cell adapted A25P12 virus of the above-mentioned Vero of inoculation.Negative control is ChimeriVax-JE and diluent (2,4 and 7 groups).Positive control Taconic mice SC inoculation and the blended HBc-M2e granule of adsorbed onto alum adjuvant (being called Acam-Flu-A).In Jackson mice group, produces two groups and be used to detect synergism between A25 virus and the Acam-Flu-A: only accept Acam-Flu-A and do not need adjuvant through the IP approach for 8 groups, 9 groups of IP accept and the blended Acam-Flu-A of A29.The all mices of reinforced immunological after inoculating 1 month were measured independent serum (being used for total IgG) at the 59th day with ELISA) or respectively organize the specific antibody titre (total IgG and IgG1, IgG2a, IgG2b and IgG3 type) that serum mixes (being used for the IgG homotype); Also measure M2e specificity total IgG titre the 30th day (before the reinforcement).The ELISA titre sees Table 7; Provide the GMT value of the total IgG of measuring in the independent serum.These data and before mouse experiment data consistent are except the animal of A25 immunity has significantly higher M2r specific antibody titre.Seroconversion took place on the 30th day in the animal that the overwhelming majority has inoculated A25 and Acam-Flu-A after first time dosage.The 59th day (strengthen back about 1 month) A25 and all animals in the Acam-Flu-A group be seropositivity and with compared the total IgG titre on the 30th day and significantly increase.As expection, Acam-Flu-A/ adsorbed onto alum adjuvant immunity (3 groups) causes that dominant Th2 type replys, and to compare the IgG1 titre the highest with other IgG homotypes.A25 (1,5 with 6 groups) immunity the causing relevant Th1 type of dominant and higher IgG2a titre is replied, and IgG2a is the type that needs through the M2e of ADCC mechanism mediate protection; Detect also and relevant IgG2b and the IgG3 antibody (Jegerlehner etc., J.Immunol.172:5598-5605,2004) of ADCC mechanism.This at one-time authentication insert the high immunogenicity of the M2e epi-position in ChimeriVax-JE NS1-236 site.
Of the present invention one important be found to be to compare with independent inoculation Acam-Flu-A or A25 virus inoculate Acam-Flu-A and A25 simultaneously and can significantly strengthen anti-M2e antibody response (in the comparison sheet 79 groups and 8 and 6 groups).The 59th day, the total IgG GMTs of 9 groups and 8 groups was respectively 95940 and 35050 (the 30th day proportional differences even more obvious).Therefore, observe the potent synergism of inoculation simultaneously.And, although Acam-Flu-A can induce most Th1 types to reply (titre of IgG1, IgG2a, IgG2b and IgG3 is respectively 72900,8100,300 and 900) separately, inoculate Acam-Flu-A and A25 simultaneously and can cause tangible Th2 to move, can be by the lower and significantly higher proof of other antibody morphism ratios (titre of IgG1, IgG2a, IgG2b and IgG3 is respectively 72900,72900,8100 and 8100) of IgG1 ratio.This synergism is not only to increase owing to A25 virus in the animal that is inoculated simultaneously causes antigen (M2e) quality, because inoculate the immunne response (in Jackson balb/c mice, seeing Table 6 groups in 7) that A25 can cause moderate separately.These effects also may be owing to the effect of adjuvant to virus replication in the dendritic cell of for example inoculation site.Be reported in and observed such adjuvant effect (Thompson etc., Proc.Natl.Acad.Sci.U.S.A.103:3722-3727,2006 in α virus replication; Hidmark etc., J.Virol.80:7100-7110,2006).
Insert the expression of the M2e in the ChimeriVax-JE E albumen at random
Whether carry out three measurings can insert M2e at random and be expressed in the virion surface in the E of ChimeriVax-JE carrier albumen.In first experiment, on the pBSA-AR2-rM2e plasmid library, synthesize RNA (the 16th step of Fig. 2) with the SP6RNA polymerase.Use lipofectamine RNA transfection Vero cell.Only observe not mutated viruses plaque in the cell conditioned medium of results, it can not be dyeed by M2e MAb.On supposing, with regard to inserting NS1 at random, so owing to insert fragment and do not carry M2e in the unstable site of E and insert segmental virus and can occur fast and become preponderating.In second experiment, from pUCAR02-rM2e, extract E-M2e gene library (the 13rd step of Figure 12) and the external pBSA-AR2 termination carrier (the 15th step of Fig. 2) that is connected to NsiI and KasI.To connect product linearisation and in vitro transcription with XhoI.With synthetic RNA electroporation Vero cell, serial dilution infection cell suspension (to reduce the interference between not sudden change and the M2e positive-virus) places culture dish with cell diluent then.Add the Vero cell of untransfected to guarantee the cell monolayer fusion to having inoculated more in the culture dish of the liquid of transfectional cell of highly diluted.In conjunction with after, cover cell monolayer with agar.(after removing the agarose covering) observes several positive accumulation points in high transfectional cell dilution (1: 4 and 1: 8) when using M2e Mab staining cell monolayer after 6 days.Figure 12 A has shown an example of this accumulation point.Compare with the more observed accumulation points of NS1-M2e library transfection, the quantity of these accumulation points and size are less, show to compare the more difficult 35-aminoacid insertion fragment (being used for pUC-AR02-rM2e, same as shown in Figure 5) of inserting with NS1 in E albumen.In the 3rd experiment, insert fragment (SLLTEVETPIRNEWGSR) by the M2e that the production of two complementary phosphorylation primer annealings is short.It is as follows that this inserts segmental nucleotide sequence: 5 '-P-AGC CTT CTA ACC GAG GTC GAA ACGCCT ATC AGA AAC GAA TGG GGG AGC AGA-3 '
Similarly produce identical still two ends and comprise the insertion fragment (21 aminoacid of total length) that two additional glycine joint residues are used to increase flexibility.Second segmental nucleotide sequence of insertion is as follows:
5’-P-GGA GGA AGC CTT CTA ACC GAG GTC GAA ACG CCTATC AGA AAC GAA TGG GGG AGC AGA GGC GGC-3’
With two insert flush end PmeI site that fragments are connected to the pUC-AR02-rTn7enr library replace shift primer (step 8, Fig. 2).Before connecting with this vector plasmid DNA dephosphorylation.Prepare two new plasmid libraries respectively, pUC-AR2-17M2e and pUC-AR2-17gM2e.The NsiI-KasI insertion fragment in two libraries is transferred to pBSA-AR2 termination carrier, obtains pBSA-AR2-17M2e and pBSA-AR2-17gM2e total length library, it then can be used in vitro transcription.At first anyly do not comprise this and insert segmental total length template DNA molecule (and comprising insert in the segmental molecule excision insert the PmeI cloning site at fragment two ends) to remove with two libraries of PmeI digestion back.With XhoI the SP6 rna polymerase transcribe is also used in their linearisations then.Cover with agarose in conjunction with the back to culture dish and at cell with transcript electroporation Vero cell and inoculation (not diluting).For fear of the interference of inserting the few virus of fragment, be in early days after the transfection the 4th day with M2e Mab staining cell monolayer.Observe about 100 the little accumulation points of as many as in two transfections.The example of these accumulation points is seen Figure 12 A and B, is respectively to comprise in the E albumen that 17-aminoacid M2e inserts segmental ChimeriVax-JE virus and GG-17 aminoacid-GG inserts fragment.Therefore, foreshorten to the recovery that 17 or 21 aminoacid can significantly increase recombinant virus with inserting fragment from 35 aminoacid.More viewed M2e are positive, and variant is in a single day separated can duplicate well.If necessary, can from additional transfection, separate more effective variant that duplicates.In addition, can some second site mutations occur with hope at the variant that continuous passage in the Vero cell is for example duplicated at a slow speed and improve growth.This embodiment has obviously verified the probability of inserting the former epi-position of foreign immunologic in E albumen at random.
In the prM of ChimeriVax-JE vector virus albumen, insert the M2e epi-position at random
Different expression waies is seen Figure 15 in the viral glycoprotein (prM, E or NS1).It is the strongest that therefore the proteic epi-position of insertion E also can be contemplated to immunogenicity with submission in virion (180 copy) surface.Expression in the NS1 albumen is passed to infected cell surface with the epi-position of inserting and is passed to outside the born of the same parents in secreting type NS1 oligomer.Though above-mentioned EXPERIMENTAL EXAMPLE has been verified the high immunogenicity of a back mode; but the expression that is lower than in this case among the E is (still enough high for some epi-positions; virucidin's epi-position for example, its with non-neutralizing epitope for example the M2e of influenza compare the protection that can provide more potent).So since known in the particulate maturation process of banzi virus furin will cause the lip-deep part submission of virion to the expression among the incomplete cracking prM of prM and outside the additional born of the same parents of the secreting type prM N end parts that generates by the furin cracking, express.This expression way also expects to have high immunogenicity, and is higher than the immunogenicity of expressing among the NS1.If can insert epi-position in ripe M albumen (the C end parts of prM), but the equal submission of all epi-position molecules is similar to the expression among the E in the surface of virion (180 copy).
In order between SphI and NsiI site (SphI is positioned at the initial upstream of prM gene), M2e epi-position (inserting 35 aminoacid of fragment length overall) to be inserted the prM of ChimeriVax-JE, make up pBSA-AR1-rM2e plasmid library (Fig. 2).The representativeness in this library is about 10 5The clone.Used as the template of in vitro transcription, gained rna transcription thing is used to use lipofectamine transfection Vero cell monolayer.With agarose cover transfectional cell and at 5-6 days with M2e MAb dyeing.Observe the positive plaque of M2e.Results are cloned and are being further purified in several plaque purification of taking turns in addition corresponding to the M2e positive-virus of positive plaque from agarose covers, and amplification is gone down to posterity 2 times to prepare 5 pure virus deposits, called after M1, M2, M3, M6 and M8 then.
With M2e and JE antibody all new recombinant clones that effectively dye, and with JE antibody staining ChimeriVax-JE vector virus plaque.The 5th day painted plaque example seen Figure 16 A in standard plaque algoscopy (methylcellulose covering).Compare with ChimeriVax-JE, the plaque that M1, M2 and M3 insert the fragment mutant is bigger, and M6 and M8 clone's plaque is littler.(difference of this plaque size is more obvious under agarose covers).Therefore, but compare M1-3 with vector virus and be cloned in and externally duplicate better.This point in growth curve experiment, be confirmed (Figure 16 B).The M1-3 clonal growth is faster and produce the titre higher than ChimeriVax-JE, and the titre of M6 and M7 is lower a little.
Measure the segmental position of insertion by order-checking.The results are shown in Figure 17.What is interesting is that M2e inserts the N end that fragment is added into the JE-specificity prM of ChimeriVax-JE virus in M1, M2 and M3, although at different aminoacid places.Clone M6 is identical with the position among the M8 (after the Pro of virus O RF residue 147; Or prM-26).In ChimeriVax-JE virus, the N of prM end (MKLS...) forms (Figure 17) by the cracking of host cell signal peptidase.In clone M1, M2 and M3, insert fragment and mixed 4,1 and 2 amino acid residue place, the initial upstream of JE prM respectively.Therefore the N end of sudden change prM comprises the M2e peptide sequence in these viruses, is thereafter 4,1 and 2 viral residues before the natural prM sequence, is the prM sequence then.Use two kinds of signal peptidase cracking sites (as shown in figure 17) that different algorithm predicts is new with common SignalP 3.0 at sequence of threads.In the M1 clone, the N terminal amino acid of two removable one or three M2e of possible cracking site.Predict strongly that in M2 it is complete M2e sequence of N end glycine that single cracking can obtain the back.In M3, perhaps this N end in M2 or three of M2 residue fallen by other possible cracking cracking.Three clones' plaque can be shown in M1 and the M3 cracking that the loss of M2e residue is minimum by M2e MAb this fact that effectively dyes.Importantly, the signal peptidase cracking M1-3 that predicted clone's probability greater than ChimeriVax-JE (for example, the M2 clone be 0.387 relative ChimeriVax-JE 0.073).This may be interpreted as what M1-3 virus good than ChimeriVax-JE parent growth.
Therefore, prM albumen height allows the insertion of different loci, especially N end residue.Based on stating result's (more effectively in the bigger plaque, Vero cell duplicate, the M1-3 clone of higher prediction in signal peptidase cracking probability), we think that the N end (looking inessential to the particulate assembling of banzi virus) of prM allows to insert the site for wide spectrum and can tolerate various other insertion fragments, comprise the long fragment (for example, 50,100,200,400 aminoacid etc.) of inserting.Therefore we insert HIV gag, peptide, the influenza HA of about 200 the first residues of HPV16 L2 albumen of as many as in this site 1With total length HA (about 550 aminoacid of length).These are designed to comprise before vector virus prM sequence and heterologous sequence (as the situation of M2e among the M1-3 clone) that N end or prM merge, or by mixing other signals from the prM cracking, or suitable protease cracking site or autologous protein enzyme.
Make up the ChimeriVax-WN analog (comprise M2e at NS1-236 and insert segmental ChimeriVax-JE) of A25 virus
ChimeriVax-JE and above-mentioned A25 virus can not effectively be duplicated (for example, not having detectable inoculation back viremia) in mice.Yet ChimeriVax-JE can duplicate (～2 log better in the mankind 10The pfu/ml viremia) (Monath etc., J.Infect.Dis.188:1213-1230,2003), so A25 virus can be induced high M2e antibody response in human body and protect them to avoid influenza infection.We have verified ChimeriVax-WN virus (WN02 people's vaccine version recently; WO 2004/045529) can in hamster, duplicate (～3log well 10The pfu/ml viremia) (WO 2006/116182) also can duplicate (～2 log well in the people 10The pfu/ml viremia) (Monath etc., Proc.Natl.Acad.Sci.U.S.A.103:6694-6699,2006).In order to use more potent model (ChimeriVax-WN02 contrasts ChimeriVax-JE in the mice in the hamster) to obtain the additional evidence of protecting in the M2e epi-position that express in the NS1-236 site of ChimeriVax virus, make up the ChimeriVax-WN02/M2e of A25 virus NS1-236Analog.Use standard clone technology replaces the special prM-E gene of JE-among the pBSA that comprises total length ChimeriVax-JEcDNA with the prM-E gene of ChimeriVax-WN02 virus.Obtain pBWN02 plasmid (Figure 18) like this.To comprise and insert segmental NS1 gene (Vero that contains or do not contain M2e sequence upstream is cell adapted) from A25 virus M2e and be cloned into pBWN02.Two plasmids that in vitro transcription obtains cover with rna transcription thing transfection Vero cell and with agar.Observed very large plaque at the 6th day, with M2e MAb dyeing (Figure 18, bottom picture group).
With ChimeriVax-WN02/M2e NS1-236Two version WN02/A25 and WN02/A25adapt carry out a plaque purification and cloned the deposit of virus by amplification preparation in the Vero cell.Be shown in Figure 19 A with ChimeriVax-WN02 and the correlated plaque example of ChimeriVax-JE.The growth curve of new virus is seen Figure 19 B in the Vero cell.(the peak titre is respectively～7.5log the WN02/A25 viral growth inferior to ChimeriVax-WN02 10Pfu/ml contrast～8.7log 10Pfu/ml).A25 virus (seeing Fig. 6 C) is similar to adapting to, and WN02/A25adapt version (comprising 2 amino acid changes in M2e sequence upstream) is grown better, and is almost the same with ChimeriVax-WN02 good.Therefore, originally introduce the proteic insertion fragment of ChimeriVax-JENS1 and successfully be transferred to the ChimeriVax-WN02 vaccine virus.Originally observed two cell culture adapt to the growth that has strengthened WN02/A25 virus in A25 virus.
Conclusion
In a word, we have successfully carried out transposon-mediated mutation ChimeriVax TMThe prM/M of-JE vaccine virus, E and NS1 gene are to insert the total M2e protective epitope of influenza A virus at random, and purpose is to generate the general efficiently vaccine of influenza A.Comprise this and insert segmental virus mutant (can by anti-M2e antibody recognition) and the M2e peptide is inserted the feasibility of having verified this method in the E albumen by in prM and NS1 albumen, introducing fast some.We also show ChimeriVax TMThe A25 clone and the ChimeriVax of-JE-NS1/M2e virus TMThe M2e that several clones of-JE-prM/M2e virus can effectively duplicate in the Vero cell and identify in these viruses inserts the site.And we have shown A25 virus inheritance stability, because it can keep M2e to insert fragment in external 10 low MOI go down to posterity.Some insertion sites of being identified by direct random mutagenesis method of the present invention are that wide spectrum allows with regard to inserting clip size and sequence with regard to the two, as using NS1-236 site illustration.The permission found in a kind of banzi virus is inserted the site and be can be used for other banzi virus, as in our experiment by comprising the NS1 gene transfer of M2e to ChimeriVax-WN institute illustration from ChimeriVax-JE.Furthermore, having set up successfully that immunogenic effective IP just exempts from the analysis mice/IP strengthens model and proves that one is inserted the fragment variant and has high immunogenicity.Duplicate (comprising IP inoculation back) although can not detect the periphery of mice, this virus has high immunogenicity and induces dominant IgG2a M2e antibody, and its ADCC mediate protection for the M2e immunity is very necessary.Another novel discovery in our experiment be the viral recon of inoculation expression M2e peptide simultaneously and the subunit synergism based on the candidate vaccine of M2e.
As mentioned above, methods described herein are applicable to every other ChimeriVax TMTarget protein and other virus of live vaccine as carrier comprise YF17D or non-banzi virus live vaccine, or non-live vector organism.This method can be used for making up the recombiant vaccine that human publilc health and beasts are had the pathogen on a large scale of importance.
Order (Fig. 2) that it should be noted that listed construction step can change and still belong to category of the present invention.And transposon can be used for directly inserting at random restriction enzyme site or foreign epitope at random except that Tn7.The latter and use restriction enzyme site rather than PmeI insert at random, or the different selection markers of any this construction step, or use any distinct methods to separate to live in mutated viruses (for example used, or cell sorting is to separate positive cell etc.) or external or the body and virus to be carried out characteristic research etc. do not change implication of the present invention by the ELISA of the cell conditioned medium of viral infection.
Can the derive example list of pathogen of epi-position/antigen/peptide of table 1-
Virus:
Banzi virus
Yellow fever virus
Japanese encephalitis virus
Dengue virus, 1,2,3 and 4 types
West nile virus
Tick-brone encephalitis virus
Hepatitis C virus (for example, genotype 1a, 1b, 2a, 2b, 2c, 3a, 4a, 4b, 4c and 4d)
Papovavirus section
Human papillomavirus
Retroviridae
The human immunodeficiency virus, the I type
The human immunodeficiency virus, the II type
Simian immunodeficiency virus
The mankind have a liking for T lymphocyte virus I and II type
Hepatovirus section
Hepatitis B virus
Picornaviridae
Hepatitis A virus (HAV)
Rhinovirus
Poliovirus
Herpetoviridae
Herpes simplex virus, the I type
Herpes simplex virus, the II type
Cytomegalovirus
The Epstein epstein-Barr virus
Varicella zoster virus
Togaviridae
α virus
Rubella virus
Paramyxoviridae
Respiratory syncytial virus
Parainfluenza virus
Measles virus
Mumps virus
Orthomyxoviridae family
Influenza virus
Filoviridae
Marburg virus
Ebola virus
Rotavirus section:
Rotavirus
Coronaviridae
Coronavirus
Adenoviridae
Adenovirus
Rhabdoviridae
Rhabdovirus
Antibacterial:
Enterotoxigenic escherichia coli
Enteropathogenic Escherichia coli
Campylobacter jejuni
Helicobacter pylori
Salmonella typhi
Vibrio cholera
Clostridium difficile
Clostridium tetani
Micrococcus scarlatinae
Bordetella pertussis
Neisseria meningitidis
Neisser's coccus
Legionella neumophilus
The coating Pseudomonas
Haemophilus
Shigella
Parasite
Plasmodium
Schistosoma
Trypanosoma
Toxoplasma
Cryptosporidium
Pneumocystis belongs to
Leishmaniasis
Table 2-institute influenza virus screens antigenic example
Virus Antigen
Banzi virus
Yellow fever virus Nucleocapsid, M and E glycoprotein
Japanese encephalitis virus “
Dengue virus, 1,2,3 and 4 types “
West nile virus “
Tick-brone encephalitis virus “
Hepatitis C virus Nucleocapsid, E1 and E2 glycoprotein
Papovavirus section:
Human papillomavirus L1 and L2 capsid protein, E6 and E7 transforming protein (oncogene)
Retroviridae
The human immunodeficiency virus, the I type Gag、pol、vif、tat、vpu、env、 nef
The human immunodeficiency virus, the II type “
Simian immunodeficiency virus “
The mankind have a liking for T lymphocyte virus I and II type Gag、pol、env
Table 3-institute influenza virus/antigenic B and t cell epitope example
Figure A20078003427400531
Figure A20078003427400541
The hereditary stability of table 4 ChimeriVax-JE-NS1/M2e virus and clone A11, A79 and A88.Order-checking intact virus genome when the P12 hereditary stability goes down to posterity.Show nucleotide change/heterogeneity and amino acid change.
Figure A20078003427400542
Figure A20078003427400551
1NS1 inserts segmental site and nucleotide/aminoacid. and numbering is seen Fig. 5.
The M2e antibody response of table 5 Balb/c mice the 54th day (strengthening 2 weeks of back for 1,2,4 and 5 group)
1For virus, immunity and booster dose are 5 log 10Pfu; For HBc-M2e, dosage is 10g granule+Alumen.
2Strengthened 3 groups at the 20th day, and strengthened 1,2,4 and 5 group at the 40th day.
The mouse experiment #2 (from two suppliers' big female balb/c mice of 4 weeks) of A25 virus is used in table 6. design.Measured ELISA antibody titer (strengthening approximately back month) on the 59th day.
Figure A20078003427400561
The M2e-specific antibody of mice is replied in table 7. experiment 2
Figure A20078003427400571
The content of all lists of references of above quoting is with the reference form and in this paper.Herein singulative for example the use of " " and " being somebody's turn to do " do not get rid of the implication of corresponding plural form, unless indicate opposite situation in the article.Therefore, for example, give " a kind of " banzi virus if a claim indicates, its also can be regarded as comprise give unnecessary in a kind of banzi virus, except as otherwise noted.Other embodiments are in following claim.

Claims (57)

1. generate the virus genomic method of the nucleic acid molecules that comprises the heterologous peptides of encoding, this method may further comprise the steps:
(i) provide the target viral gene;
(ii) mutation target viral gene; And
The nucleic acid molecules of heterologous peptides of (iii) will encoding is connected to the mutation site of target viral gene.
2. the method for claim 1 further may further comprise the steps:
(iv) use genomic nucleic acids library transfectional cell with initial virus replication; And
(v) screen the live virus recon.
3. the method for claim 1, described target viral gene wherein is provided in shuttle vector, and described method further may further comprise the steps after the nucleic acid molecules of the heterologous peptides of will encoding is connected to the mutation site of target viral gene: the viral genome of the target viral gene of sudden change being introduced the derivative goal viral gene, substitute the corresponding viral gene of this insertion of disappearance, comprise with generation and insert segmental viral genome.
4. the method for claim 3, wherein said shuttle vector comprises two or more target viral genes.
5. the process of claim 1 wherein provides described target viral gene in intact virus genome background, and the generation of described method comprises the segmental viral genome of insertion.
6. each method among the claim 1-5, it further comprises by introducing to comprise in cell and inserts segmental viral genome, inserts segmental viral genome and generates viral vector from comprising.
7. the method for claim 6, it further comprises isolated viral carrier from cell or its supernatant.
8. the method for claim 3, it comprises that mutation comprises two or the two or more shuttle vectors of multiple target viral gene more, and two or more target viral genes that comprise the nucleic acid molecules of one or more heterologous peptides of encoding are introduced is substituted disappearance in the viral genome of this target gene of deriving and insert segmental corresponding viral gene and comprise with generation and insert segmental viral genome.
9. the process of claim 1 wherein that described mutation step comprises by transposon mutagenesis introduces one or more transfer primers in the target viral gene.
10. the method for claim 9 wherein removes one or more transfer primers by endonuclease digestion, and introduces the target viral gene by the nucleic acid molecules of the heterologous peptides of will encoding in the connection in restriction endonuclease digestion site.
11. the method for claim 1, it further comprises the library that generates sudden change target viral gene.
12. each method among the claim 1-5, wherein said target viral gene are included in and are applied to the insertion fragment that this method is introduced by the method for claim 1 before once more.
13. the process of claim 1 wherein that described viral genome is the banzi virus genome.
14. the method for claim 13, wherein said banzi virus are chimeric flavirirus.
15. the method for claim 14, wherein said chimeric flavirirus comprise the capsid of first kind of banzi virus and the preceding and envelope protein of film of non-structural protein and second kind of different banzi virus.
16. the method for claim 15, wherein said first kind and second kind of banzi virus independently are selected from Japanese encephalitis, dengue fever-1, dengue fever-2, dengue fever-3, dengue fever-4, yellow fever, Murray Valley encephalitis, St. Louis encephalitis, Xi Niluo, Kun Jin, sieve Theo encephalitis, Erie's this encephalitis of crow, tick encephalitis, CEE, Siberia encephalitis, RSSE, KFD, Omsk hemorrhagic fever, ramaninjana, Bo Washeng, root bank, A Busaitaluofu, Hansom Lip river watt, A Boyi and Hai Pu virus.
17. the process of claim 1 wherein described target viral gene be selected from coding peplos, capsid, film before, NS1, NS2A, NS2B, NS3, NS4A, NS4B and the proteic gene of NS5.
18. the process of claim 1 wherein that described heterologous peptides comprises the vaccine epi-position.
19. the method for claim 18, wherein said vaccine epi-position is B cell epitope or t cell epitope.
20. the method for claim 18, wherein said epi-position is derived from viral pathogen, bacterial pathogens, parasitic disease substance, allergen antigen, or tumor associated antigen.
21. the method for claim 20, wherein said viral pathogen are influenza virus.
22. the method for claim 21, wherein said heterologous peptides comprise influenza M2e peptide or comprise the peptide of influenza hemagglutinin precursor protein cracking site (HA0).
23. the method for claim 21, wherein said influenza virus are fowl or human influenza virus.
24. the process of claim 1 wherein that described viral genome comprises the nucleic acid molecules of coding more than a heterologous peptides, and described heterologous peptides comprises people and bird flu M2e peptide.
25. viral genome or its complement by each method generation among the claim 1-24.
26. viral vector by the viral gene group coding of claim 25.
27. comprise insert be selected from capsid, film before, the banzi virus carrier of the heterologous peptides in peplos, NS1, NS2A, NS2B, NS3, NS4A, NS4B and the NS5 albumen.
28. the banzi virus carrier of claim 27, wherein said banzi virus are yellow fever virus.
29. the banzi virus carrier of claim 27, wherein said banzi virus are chimeric flavirirus.
30. the banzi virus carrier of claim 29, wherein said chimeric flavirirus comprise the capsid of first kind of banzi virus and the preceding and envelope protein of film of non-structural protein and second kind of different banzi virus.
31. the banzi virus carrier of claim 30, wherein first and second kinds of banzi virus independently are selected from Japanese encephalitis, dengue fever-1, dengue fever-2, dengue fever-3, dengue fever-4, yellow fever, Murray Valley encephalitis, St. Louis encephalitis, Xi Niluo, Kun Jin, sieve Theo encephalitis, Erie's this encephalitis of crow, tick encephalitis, CEE, Siberia encephalitis, RSSE, KFD, Omsk hemorrhagic fever, ramaninjana, Bo Washeng, root bank, A Busaitaluofu, Hansom Lip river watt, A Boyi and Hai Pu virus.
32. each banzi virus carrier among the claim 27-31, wherein said banzi virus carrier comprise the heterologous peptides between the aminoacid 236 and 237 that inserts non-structural protein 1 (NS1).
33. each banzi virus carrier among the claim 27-32, wherein said banzi virus carrier comprise the heterologous peptides that inserts the preceding proteic aminoterminal zone of carrier film.
34. the banzi virus carrier of claim 33, wherein said heterologous peptides are inserted in before capsid/film before-4 ,-2 or-1 before the cracking site or the film proteic 26.
35. the banzi virus carrier of claim 33 or 34, its further comprise auxiliary before the film albumen remove the molten protein cleavage site of described peptide.
36. each banzi virus carrier among the claim 27-35, wherein said heterologous peptides comprise influenza M2e peptide or comprise the peptide of influenza hemagglutinin precursor protein cracking site (HA0).
37. the banzi virus carrier of claim 36, wherein said influenza virus are bird flu virus or human influenza virus.
38. each banzi virus carrier among the claim 27-37, wherein said viral genome comprises the nucleic acid molecules of coding more than a kind of heterologous peptides, and described heterologous peptides comprises people and bird flu M2e peptide.
39. each banzi virus carrier among the claim 27-38, it further comprises one or more second sites and adapts to.
40. nucleic acid molecules or its complement corresponding to each banzi virus vector gene group among the claim 27-39.
41. comprise the pharmaceutical composition of each viral vector among the claim 27-39.
42. the pharmaceutical composition of claim 41, it further comprises pharmaceutically acceptable carrier or diluent.
43. the pharmaceutical composition of claim 41 or 42, it further comprises adjuvant.
44. the pharmaceutical composition of claim 43, wherein said adjuvant comprises aluminium compound.
45. the pharmaceutical composition of claim 44, wherein said aluminium compound are Alumen.
46. give the method for patient's peptide, described method comprises the compositions that gives among patient's claim 41-45 each.
47. the method for claim 46, wherein said peptide are antigen and carry out administration to induce the immunne response to the described pathogen or the described antigenic tumor of deriving.
48. the method for claim 47, it further comprises and gives subunit vaccine.
49. the method for claim 48, wherein give described banzi virus carrier and subunit vaccine simultaneously, give described banzi virus carrier, give described subunit vaccine to strengthen dosage just to exempt from dosage, or give described subunit vaccine just to exempt from dosage, give described banzi virus carrier to strengthen dosage.
50. the method for claim 48 or 49, wherein said subunit vaccine comprises the hepatitis B virus core granule, and it comprises the heterologous peptides that merges with hepatitis B virus core albumen.
51. the method for claim 50, the heterologous peptides of wherein said fusion comprise influenza M2e peptide or comprise the peptide of influenza hemagglutinin precursor protein cracking site (HA0).
52. each method among the viral vector of the viral genome of each pharmaceutical composition, claim 25, claim 26 or claim 1-24 or the 46-51 among the nucleic acid molecules of each banzi virus carrier, claim 40, the claim 41-45 among the claim 27-39, wherein said heterologous peptides or peptide source select other parts of this paper to comprise what form and sequence appendix were listed.
53. prepare the method for viral vector, described method comprises sequence from the coding related peptides to the site of the described insertion of permission of banzi virus that insert.
54. the method for claim 53, wherein said viral vector comprises banzi virus or chimeric flavirirus.
55. the method for claim 54, wherein said NS1-236 position or the preceding proteic aminoterminal of described carrier film that is inserted in described viral vector partly carries out.
56. inserting, the method for claim 55, wherein said aminoterminal be positioned at-4 ,-2 or-1 before the cracking site before capsid/film, or before the film proteic 26.
57. the method for pharmaceutical compositions, described method comprise each banzi virus carrier among the claim 27-39 is mixed with pharmaceutically acceptable carrier or diluent, adjuvant and/or additional active agents.
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CN103328647A (en) * 2010-09-21 2013-09-25 廖经伦 Recombinant flaviviral constructs and uses thereof
WO2017107219A1 (en) * 2015-12-22 2017-06-29 成都生物制品研究所有限责任公司 Japanese encephalitis/yellow fever chimeric virus and preparation method and use thereof
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CN101850116A (en) * 2010-06-18 2010-10-06 中国人民解放军军事医学科学院军事兽医研究所 Method for constructing virus live vector recombinant vaccine by utilizing transposon
CN101850116B (en) * 2010-06-18 2012-07-04 中国人民解放军军事医学科学院军事兽医研究所 Method for constructing virus live vector recombinant vaccine by utilizing transposon
CN103328647A (en) * 2010-09-21 2013-09-25 廖经伦 Recombinant flaviviral constructs and uses thereof
CN103328647B (en) * 2010-09-21 2016-05-04 廖经伦 Flavivirus recombinant and application thereof
CN107074913A (en) * 2014-06-23 2017-08-18 巴斯德研究院 The dengue fever virus of fragment comprising non-structural protein is fitted together to multi-epitope and its purposes in the immunogenic composition that anti-dengue virus infects
CN107074913B (en) * 2014-06-23 2022-02-25 巴斯德研究院 Dengue virus chimeric polyepitope comprising fragments of non-structural proteins and uses thereof
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